Metabolic parameters


Metabolic parameters


Metabolic parameters


Metabolic parameters

5.1 Ammonia

Lothar Thomas

In physiological body fluids ammonia/ammonium has a pKa of 9.05 at 37 °C. At physiological pH approximately 99% of ammonia is present in blood as NH4+ cation in the circulation. An alkaline blood pH favours the formation non-dissociated NH3 gas, more acidic pH the formation of NH4+. The NH4+ cation diffuses or is transported more efficiently than NH3 into the tissues /1/. Ammonia is derived mainly from deamination of the α-amino nitrogen of amino acids and is toxic at elevated concentrations. Most of the daily ammonia load comes from the digestion of dietary proteins and the bacterial synthesis of ammonia in the intestine. Under normal conditions, deamination of glutamine to glutamate in the liver releases ammonia, which then is converted to the non-toxic nitrogen-rich compound urea, which is subsequently excreted into the urine.

5.1.1 Indication

Neurological manifestations in patients:

  • With liver cirrhosis
  • With liver failure of different origins (alcohol-toxic, infectious)
  • On aggressive chemotherapy
  • On treatment with valproic acid
  • With severe gastrointestinal hemorrhage
  • With a portocaval/portosystemic shunt.

Suspected congenital metabolic disorder in newborns, children and, less commonly, adults.

5.1.2 Method of determination

Enzymatically without prior deproteinization /2/

Principle: in the presence of NADPH2, ammonia is transferred to 2-oxoglutarate by glutamate dehydrogenase (GLD), forming glutamate and NADP. The decrease in NADPH2 is measured. The decrease in absorbance at 334 or 340 nm is proportional to the ammonia concentration in the reaction mixture.

2-oxoglutarate + NH 4 + + NADPH 2 GLD glutamate + NADP + H 2 O

Ammonia-specific electrode /3/

Principle: NH4+ cations of the sample are converted to NH3 (gas) in an alkaline buffer. The released gas diffuses through the pores of a membrane into the inner NH4Cl containing solution of a pH electrode. Ammonia is directly measured via the pH increase of the inner electrode.

5.1.3 Specimen

EDTA or heparin plasma from venous or arterial blood (ideally dipotassium EDTA plasma with added sodium borate) /4/: 1 mL

5.1.4 Reference interval

Refer to Tab. 5.1-1 – Ammonia reference intervals.

5.1.5 Clinical significance

Hyperammonemias are classified into hereditary and acquired forms (Tab. 5.1-2 – Congenital and acquired (secondary) causes of hyperammonemia).

In adults, hyperammonemias are usually associated with liver failure, while in children they mostly result from hereditary defects of enzymes of the urea cycle /7/. Hyperammonemias cause neurological manifestations, which are characterized by hepatic encephalopathy and cerebral edemas of varying severity. The edemas are usually localized in the cerebral cortex, but also affect the white matter to a certain extent. In these patients, diminished urea synthetic capacity causes impairment of the physiological route of ammonia detoxification, leading to elevated ammonia levels with neurotoxic effects to the central nervous system. Hepatic encephalopathy is, however, not caused by the diminished urea synthetic capacity, but acts synergistically with systemic inflammation, hypoxia due to reduced cerebral blood circulation, and metabolic disorders that are not associated with ammonia genesis. Initial clinical symptoms are loss of appetite, vomiting and hyperventilation, later followed by lethargy, encephalopathy, and seizures. A progressive increase in ammonia to 5–6 times the upper reference interval value leads to cerebral edema, coma, and possibly death. In patients with hyperammonemia and hepatic encephalopathy, the duration of the hyperammonemia determines the reversibility of the cerebral pathology as well as the prognosis.

The rate of ammonia synthesis and the concentration in arterial blood are determined by two factors:

  • The balance of protein anabolism and catabolism
  • The integrity of the urea cycle.

The ammonia derived from the deamination of amino acids is detoxified through the synthesis of glutamine and urea. Glutamine is produced in all tissues, in particular muscle, by the transfer of ammonia to the α-ketoglutarate formed in the citric acid cycle. The α-ketoglutarate transports ammonia from the periphery to the liver by the incorporation of potentially two ammonia molecules. In the liver, the ammonia molecules are involved in the synthesis of urea or amino acids. Due to the large capacity of the urea cycle, liver diseases usually have no impact on this process. Hereditary hyperammonemias

Hereditary hyperammonemias are caused by disorders classified as neurometabolic diseases, a large group of heterogeneous diseases which primarily affect the central nervous system (Tab. 5.1-4 – Hereditary hyperammonemias/9/. Their cumulative incidence is 1 in 500 individuals. Neurometabolic diseases can be caused by an enzyme defect of the urea cycle or can occur secondary to organic aciduria or fatty acid oxidation disorders. In children aged up to 2 years, clinical symptoms of hereditary hyperammonemias often appear as early as in the first weeks of life. A second peak of incidence occurs at age 12 to 15 years, at the end of puberty, when the growth rate declines.

Suspected manifest encephalopathy in hereditary hyperammonemia is diagnosed based on clinical symptoms. In children these are nausea, vomiting, disorientation, seizures, and lethargy. Newborns refuse food, and infants and older children have anorexia and ataxia. It must be noted that genetic urea cycle disorders can occur at any age.

There are also documented cases of neurometabolic diseases in older age groups. The classification of hereditary hyperammonemias is based on the biochemical substances produced or metabolized, or the enzyme defect that is present.

A distinction is made between the following groups (Tab. 5.1-3– Enzyme defects and substrates accumulating in hereditary hyperammonemias):

  • Urea cycle disorders (cumulative incidence 1 : 20,000; the most common defect is ornithine transcarbamylase deficiency) /10/.
  • Amino acid transport disorders such as hyper ornithinemia-hyperammonemia-hypercitrullinemia (HHH) syndrome and lysin uric protein intolerance (LPI)
  • Organic acidurias (cumulative incidence 1 : 10,000) such as propionic aciduria and methyl malonic aciduria
  • Fatty acid oxidation disorders (e.g., acyl-CoA dehydrogenase deficiency)
  • Mitochondriocytopathies with disorders of energy metabolism (e.g., respiratory chain defects, pyruvate carboxylase deficiency)
  • Hyperinsulinism-hyperammonemia (HI/HA) syndrome.

Before considering a diagnosis of a hereditary disorder in newborns or infants, acquired hyperammonemias must be excluded (e.g., liver insufficiency, portosystemic shunt, liver bypass, Reye’s syndrome, valproic acid therapy, hypovolemic shock, transient neonatal hyperammonemia, neonatal Herpes simplex virus infection, perinatal asphyxia, cardiac insufficiency, sepsis, infection with urea-splitting bacteria). Biomarkers in the diagnosis of hereditary hyperammonemia

General investigations /11/

Ammonia, glucose, lactate, ketone bodies, anion gap (Tab. 5.1-5 – Differential diagnosis of acute hereditary hyperammonemias).

It is important to determine several biomarkers, since ammonia levels can be below 100 μg/dL (60 μmol/L) or only slightly elevated during the asymptomatic interval of neurometabolic disorders. Increased protein intake or extreme physical stress can also lead to a physiologically elevated concentration of ammonia. The following assays are recommended:

  • Blood gas analysis; hyperammonemia with metabolic acidosis is more likely to be due to organic aciduria than an urea cycle disorder, although acidosis can also develop in the latter. However, urea cycle disorders are much more frequently associated with alkalosis than with acidosis.
  • Blood glucose; if levels are normal, HI/HA syndrome can be excluded
  • Ketone bodies in urine; ketonuria is more suggestive of organic aciduria
  • Serum urea; although the serum urea concentration is often low in urea cycle disorders, it is not a sensitive and specific parameter
  • ALT, prothrombin time and albumin are important for excluding acute liver failure.

Special investigations in suspicion of hyperammonemia and neurometabolic disorders

In many cases, a general differentiation of disorders is possible by measuring amino acids and organic acids in urine and/or, if necessary, plasma (Tab. 5.1-5 – Differential diagnosis of acute hereditary hyperammonemias). For final differentiation, the activity of the enzyme suspected to be deficient is measured and molecular genetic assays are carried out. Urea cycle defects

Genetic urea cycle defects can occur in every age. In all defects, alanine and glutamine are elevated.

The incidence of ornithine transcarbamylase (OTC) deficiency with an estimated incidence of approximately 1:40,000 is the most common disorder in urea genesis. The plasma ammonia concentration frequently exceeds 1,700 μg/dL (1,000 μmol/l). The chromatogram of organic acids shows a large peak of orotic acid. Children with severe OTC deficiency typically present after 24 h with irritability, poor feeding, progressive lethargy, and seizures progressing to hyperammonemic coma /35/.

In citrullinemia (argininosuccinate synthetase deficiency), the citrulline concentration is above 1,500 μmol/L. In ornithine transcarbamoylase (OTC) and carbamoyl phosphate synthetase (CPS) deficiency, the citrulline and arginine concentrations are reduced.

Argininosuccinase deficiency is characterized by high urinary clearance of argininosuccinate with only slightly elevated levels in plasma.

In arginase deficiency, the concentration of arginine in plasma and urine is significantly elevated.

It is not possible to differentiate OTC, CPS and N-acetyl glutamate synthetase (NAGS) deficiencies from other urea cycle defects based on the amino acid profile. It can, however, be done using the allopurinol loading test, because in all urea cycle enzyme defects, except CPS and NAGS deficiency, there is increased formation of orotic acid from carbamoyl phosphate due to impaired metabolism of the latter in the urea cycle (Fig. 5.1-1 – Structural and functional organization and regulation of the hepatic and renal ammonia metabolism). Allopurinol, or its metabolite oxipurinol ribonucleotide, inhibit orotidine mono phosphate decarboxylase, resulting in increased orotic acid in urea cycle enzyme defects. This is, however, not the case in NAGS or CPS deficiency. The allopurinol loading test is especially useful for differentiating between OTC, CPS and NAGS deficiency /12/.

Allopurinol loading test

Allopurinol is administered as a single dose (adults 300 mg), and after 6 hours urine is collected over 24 hours. Increased excretion is indicative of OTC deficiency and rules out CPS- or NAGS deficiency. The test has a high specificity for urea cycle defects and is also used for identifying asymptomatic heterozygous carriers of OTC deficiency. Disorders of amino acid transport

Increased clearance of the respective amino acids is found in HHH and LPI syndrome (Tab. 5.1-4 – Hereditary hyperammonemias). Excretion of orotic acid may also be increased. In HHH syndrome, hyperornithinemia may be absent if protein intake is low. In LPI syndrome, the plasma concentration of the dibasic amino acids, except cysteine, may be only slightly elevated or even normal. Therefore, measurements in spontaneous morning urine are important in the diagnosis of these disorders /10/. Organic aciduria

The measurement of organic acids in urine and plasma is performed using gas chromatography mass spectrometry. Relatively common organic acidurias/acidemias include methylmalonic aciduria, propionic acidemia, and isovaleric acidemia. Disorders of fatty acid oxidation

In disorders of fatty acid oxidation, β-oxidation is activated in the catabolism of fatty acids, producing dicarboxylic acids such as adipic acid, sebacic acid and suberic acid, which are excreted in urine /13/. High excretion of dicarboxylic acids in combination with diminished or absent excretion of ketone bodies indicates a disorder of fatty acid oxidation. The dicarboxylic acid pattern also provides information as to whether the acyl-CoA dehydrogenases which metabolize short-chain or long-chain fatty acids are affected. For example, the presence of hydroxy dicarboxylic acids indicates a deficiency of 3-hydroxy-acyl-CoA dehydrogenase, which metabolizes long-chain fatty acids. Mitochondriocytopathies with disorders of energy metabolism

For further information see Ref. /14/. Hyperinsulinism-hyperammonemia syndrome

Molecular genetic detection of mutant glutamate dehydrogenase. For further information see Ref. /15/. Acquired hyperammonemias

Acquired hyperammonemias are predominantly of hepatic origin and caused by severe liver disease, usually liver cirrhosis. The latter leads to reduced synthesis of urea and glutamine, which is further compromised by the increased production of ammonia induced by the catabolic state. In liver cirrhosis, there is a combination of metabolic (reduction of the liver parenchyma) and hemodynamic components (portocaval/portosystemic shunts). Ammonia-containing blood reaches the systemic circulation directly from the intestine, bypassing the liver.

Acquired hyperammonemias can cause hepatic encephalopathy. About 5–6 fold elevations of blood ammonia [concentrations above 300 μg/dL (176 μmol/L)] will produce coma, however the brain will not develop tolerance for repeated exposure to ammonia. The persistent or episodic hyperammonemia can be expected to exact a permanent toll on brain function /8/. Hyperammonemic encephalopathy is a clinical syndrome characterized by abnormal mental status and neuromuscular manifestations, such as asteriks, tremor, opthalmoplegia, incoordination, and incontinence. Latent encephalopathy is diagnosed by psychomotor tests such as linking numbers and arithmetic exercises and measuring reaction time /15/. Other causes of acquired hyperammonemia are rare. They are listed in Tab. 5.1-6 – Acquired hyperammonemias. Hepatic portosystemic encephalopathy

The term hepatic encephalopathy (HE) covers a multitude of potentially reversible symptoms ranging from discrete neuropsychiatric abnormalities to coma /16/. HE is a secondary, metabolic, cerebral and neuromuscular disorder which is caused by chronic liver disease or acute, severe failure of the liver parenchyma. To differentiate HE from other encephalopathies, the following conditions must be present /16/:

  • Fulminant hepatic failure, also known as acute HE or type A HE. In this acute type of HE, a cerebral edema develops, with the associated symptom of intracranial pressure.
  • Surgical or spontaneous portocaval shunt without underlying liver disease (type B HE).
  • Liver cirrhosis with the signs of functional impairment or portal hypertension (type C HE). This type of HE is subcategorized into an episodic (triggered by precipitating factors), a persistent, and a minimal form.

The hyperammonemic HE results from the reduced capacity of the liver to synthesize urea and glutamine. Ammonia from endogenous protein metabolism and from enteral bacterial breakdown of protein is insufficiently metabolized by the liver. This occurs in liver diseases with extensive reduction of functional liver parenchyma such as liver cirrhosis and acute liver failure with severe impaired function of parenchymal tissue.

With a portocaval shunt, intestinal blood bypasses the liver and reaches directly the systemic circulation. In shunt-operated patients, this occurs via portocaval or splenorenal shunts. In acute liver failure intrahepatic shunts allow blood to circulate unchanged from the portal vein to the hepatic vein.

Triggering factors of HE in patients with liver cirrhosis and portal hypertension include:

  • Gastrointestinal hemorrhages (esophageal varices)
  • Alkalosis, hypophosphatemia
  • Dietary problems (high protein intake, alcohol, obstipation, vomiting, diarrhea, surgery)
  • Sedatives, hypnotics, diuretics
  • Acute and chronic infections, in particular in conjunction with long-term treatment with corticosteroids
  • Insufficiently controlled use of diuretics (carbonic anhydrase inhibitors).

Causes of acute liver failure with HE include:

  • Acute viral hepatitides
  • Toxic hepatitides (e.g., from amanita phalloides, acetaminophen, industrial solvents, other hepatotoxic substances)
  • Acute drug-induced toxic hepatitis (tricyclic antidepressants, NSAID, INH, halothane)
  • Hypoxic liver failure (acute right heart failure)
  • Other causes such as Budd-Chiari syndrome, acute gestational fatty liver, severe malignant liver infiltration, autoimmune hepatitis. Acquired hyperammonemias are listed in Tab. 5.1-6 – Acquired hyperammonemias.

5.1.6 Comments and problems

Blood sampling

Increased GGT activity can increase the rate of ammonia formation in plasma manyfold the mean value of healthy individuals. In samples with elevated GGT the glutamate is cleaved, producing NH3. With a GGT level of 1,000 U/L, glutamate cleavage activity is 35 times higher compared to activity in the reference range /4/. Blood specimens anticoagulated with dipotassium EDTA should contain 5 μL of sodium borate (0.4 mol/L) (pH 7) and 50 μL of L-serine solution (0.1 mol/L) per 1 mL blood, for prevention of glutamate cleavage.

Capillary values are higher than the corresponding arterial concentration in the same individuals /29/.

Interference factors

The formation of ammonia following blood collection increases with the number of erythrocytes and thrombocytes and the level of GGT /30/.

Hemolysis causes falsely elevated levels, since the ammonia concentration is approx. 3 times higher in the erythrocytes than in plasma. Approximately 75% of the ammonia in whole blood is found in erythrocytes.

Reference interval

Levels in capillary blood are higher than those in arterial blood. No differences are observed in arterial and venous plasma of resting patients without liver disease. Blood collection after physical work leads to elevated levels in venous plasma /29/.


For transportation to the laboratory blood samples must be cooled in ice-water and transported within 15 minutes. If it can be ensured that the transport temperature will not exceed 20 °C, the specimen may be transported without ice after being briefly cooled in ice beforehand. A temperature of –30 °C allows long-term storage of the plasma without an increase in ammonia levels /30/.

5.1.7 Pathophysiology

Oxidation of fat and carbohydrates yields as the only end products CO2 and H2O, both of which are eliminated via the lungs and kidneys. Oxidation of proteins additionally leads to a formation of HCO3 and NH4+ /31/. Complete oxidation of protein, however, yields HCO3 and NH4+ in stoichiometric amounts. In man, ingestion of an average of 100 g protein per day results in the daily formation of about 1 mole each of HCO3 and NH4+. Such high amount of HCO3 cannot be excreted via the kidneys in view of the limited urine volume. The major pathway for metabolically generated HCO3 is hepatic urea synthesis in the liver consuming HCO3 and NH4+ in the same stoichiometry as they are produced during protein breakdown:

2 NH 4 + + 2 HCO 3 urea + CO 2 + H 2 O

Thus, the formation of approx. 30 g of urea per day leads to the elimination of 1 mole of the strong base HCO3 (pK 6.1) and 1 mole of the weak acid NH4+ (pK 8.9). This mechanism contributes significantly to stabilizing the body’s pH level. NH4+ can also be disposed by glutamine formation that occurs in the liver and other organs, and NH4+ can be excreted into urine after renal hydrolysis of glutamine /31/. Therefore, the route of nitrogen disposal by either urea or glutamine synthesis determines the rate of HCO3 removal and the liver becomes an important organ in acid-base homeostasis. Transport of ammonia

Ammonia is formed in all tissues and is toxic in particular for the nerve tissue. Physiologically, most of it is eliminated via the liver and a small amount of it via the kidneys /32/. The ammonia released in the tissues is detoxified by converting it to glutamine for transport to the liver. Glutamine is produced in the tissues from glutamate and ammonia in a reaction catalyzed by the enzyme glutamine synthetase.

Glutamate + NH 4 + + ATP glutamine glutamine + ADP + P synthetase

The plasma concentration of glutamine is approximately 700 μmol/L, which is high compared to the other amino acids. Another form of transport for ammonia is alanine. In this process alanine transaminase and glutamate transaminase catalyze the transfer of amino groups from most amino acids to form L-alanine from pyruvate or L-glutamate from α-ketoglutarate (see Section 1.6 – Alanine aminotransferase (ALT), Aspartate aminotransferase (AST)). Since L-alanine is also the substrate for glutamate transaminase, all the amino nitrogen from amino acids that undergo transamination can be concentrated in glutamate. Glutamate metabolism

Ammonia incorporated in glutamine or glutamate is metabolized in the liver /24/. Glutamate is the only amino acid that undergoes oxidative deamination to ammonia. The reaction is catalyzed by GLD:

Glutamate + NADP + + H 2 O GLD α-ketoglutarate + NADPH + H + + NH 3

The glutaminase- and GLD-mediated reactions occur in the mitochondria of the hepatocytes. The intramitochondrial concentration of glutamate determines the rate of formation of N-acetyl glutamate and urea. The first step in urea synthesis, the formation of carbamoyl phosphate, takes place close to the oxidative deamination of glutamate and the citric acid cycle. Both generate the substrates for the synthesis of carbamoyl phosphate (Fig. 5.1-1 – Structural and functional organization and regulation of the hepatic and renal ammonia metabolism). The formation of urea, which starts with the formation of carbamoyl phosphate, occurs in a four-step cycle which ends with the cleavage of arginine into ornithine and urea. Urea is water-soluble, non-toxic, well permeable and is eliminated via the kidneys.

The two major ammonia detoxifying systems, urea and glutamine synthesis are heterogeneously distributed in the liver acinus. Whereas periportal hepatocytes contain urea cycle enzymes, only a small perivenous cell population at the acinar outflow is able to eliminate ammonia by glutamine synthesis. Thus, the two major ammonia detoxification systems, urea and glutamine synthesis, are anatomically organized in sequence. Perivenous glutamine synthetase acts as a high affinity scavenger for NH4+ ions not extracted by upstream urea synthesis and the latter pathway is a periportal low affinity system for ammonia detoxification /33/.

In summary, glutamate metabolism is linked with (Fig. 5.1-2 – Metabolic pathways of glutamate/24/:

  • Aminotransferase reactions. In protein metabolism, aminotransferases transfer amino groups to and from glutamate for the breakdown and resynthesis of amino acids. The glutamate produced in the breakdown of amino acids is converted to α-ketoglutarate by GLD. The α-ketoglutarate is then metabolized in the citric acid cycle.
  • Completion of amino acid degradation. Glutamate completes amino acid degradation through oxidative deamination by GLD to α-ketoglutarate and NH4+.
  • Urea genesis. In the liver the intramitochondrial concentration of glutamate determines the production of N-acetyl glutamate by N-acetyl glutamate synthetase and, thus, governs the rate of urea genesis.
  • Glutamine synthetase which adds ammonia to glutamate to form glutamine.
  • Production of glutathione. Glutamate, cysteine and glycine are synthesized into glutathione, which protects the cells from oxidation.
  • The central nervous system (CNS). In the CNS a glutamate-glutamine shuttle is essential for the non-toxic recycling of glutamate from astrocytes to neurons. Glutamate is the major excitatory neurotransmitter of the CNS. Astrocyte uptake of glutamate is crucial for preventing toxic extracellular uptake of glutamate. Glutamate taken up by astrocytes is converted to glutamine. This glutamine can then be released for re-uptake by neurons and used to regenerate glutamate for neurotransmission. In hyperammonemia, increased formation of glutamine and glutamate may act as a sump for ammonia, leading to accumulation of glutamine in the brain and causing brain swelling through shifts in intracellular osmoles. Renal ammoniogenesis 

Renal ammoniogenesis is the direct elimination of ammonia via the kidneys /33/. When ammonia is produced in the kidneys, glomerular filtered luminal glutamine as well as contra luminal plasma glutamine is metabolized to glutamate and ammonia by GLD of the tubular cells. NH3 is released into the tubular lumen and binds with H+ to form NH4+. Ammoniogenesis thus serves the elimination of protons and is excreted at a rate of approximately 35 mmol/24 h. About one third of the ammonia formed in the kidneys is released into the urine and two thirds into the renal veins.

The detoxification of ammonia via the kidneys and liver is influenced by the acid-base balance. For example, a decrease in the extracellular pH from 7.4 to 7.3 causes a 70% decrease in GLD activity in the periportal hepatocytes. As a result, in acidosis, urea synthesis in the periportal hepatocytes is reduced in favor of increased glutamine production in the perivenous cells. The urea cycle is down regulated in order to conserve HCO3 which would otherwise be used up in the formation of carbamoyl phosphate. The increased amounts of glutamine produced are transported to the kidneys and help compensate the acidosis. By formation of ammonia, which binds with H+ to form NH4+ , the latter are eliminated and not reabsorbed by the tubules.

The NH4+ and HCO3 homeostasis is also regulated by the concerted action of the liver and kidneys. In renal insufficiency with reduced renal elimination of NH4+, the urea cycle is stimulated, since the increase in NH4+ in the periportal hepatocytes activates GLD. This leads to increased consumption of HCO3 with consecutive compensation of the alkalosis by metabolic acidosis. Elimination of ammonia in liver cirrhosis

In patients with liver cirrhosis, urea synthesis is reduced by approx. 80% due to the reduced liver parenchyma. This leads to deficient consumption of HCO3 and consequently metabolic alkalosis /34/. The alkalosis activates hepatic glutamine synthetase and stimulates the production of glutamine approx. 5-fold. Thus, the flux of ammonia through the urea cycle is increased despite decreased capacity of the cycle, and the cirrhotic patient excretes near-normal amounts of urea. If acidosis develops due to sepsis, cardiac insufficiency or drugs, this compensatory stimulation is lost due to inhibition of glutamine synthetase. Hepatic, portosystemic hyperammonemia

The ammonia concentration in portal vein blood is approximately 154 μg/dL (90 μmol/L). The ammonia is produced from amino acids and urea by bacteria in the intestinal tract. Ammonia transported into the liver is normally eliminated as follows:

  • 70% via the urea cycle
  • 30% via the formation of glutamine with subsequent recirculation and introduction into the urea cycle. In liver cirrhosis, the number of glutamine-producing cells around the central vein is diminished to such an extent that the capacity for synthesis decreases significantly so that the NH4+ can no longer be taken up, resulting in hyperammonemia. Acquired hyperammonemia

In newborns, the enzymes of the urea cycle have approximately 50% activity and reach adult capacities within 6 months. Genetic defects of enzymes 1–4 of the urea cycle lead to inhibition of the formation of urea, resulting in hyperammonemia. Deficiencies of the 5th enzyme, arginase, are less commonly associated with hyperammonemia.


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5.2 Bilirubin

Lothar Thomas

Total bilirubin in serum (BT) is composed of the following four fractions /1/:

  • Unconjugated bilirubin (Bu), the bilirubin fraction that is present in the first few days of life. Bu is extremely apolar and practically insoluble in water at physiological pH and normal body temperature. In plasma it is present in a folded structure, the so-called ZZ conformation, loosely bound to albumin. This type of bilirubin is also referred to as ZZ bilirubin. There are three metabolic and excretory pathways for the elimination of ZZ bilirubin: conjugation with glucuronic acid, photo isomerization, and oxidation.
  • Conjugated bilirubin (Bc) bound to sugar; the glucuronidation products are bilirubin mono glucuronide (C-8), bilirubin mono glucuronide (C-12), and bilirubin diglucuronide. The conjugates are water-soluble and are secreted into bile by hepatocytes against a concentration gradient. In icteric sera with a high proportion of conjugated bilirubin, the main fraction consists of bilirubin mono glucuronide.
  • δ-bilirubin (Bδ); bilirubin is covalently bound to albumin via an amide bond between its propionic acid side chain and the ε-amino group of a lysine residue on albumin.
  • Unbound unconjugated bilirubin (BF) also referred to as free bilirubin (BF).

Due to the different reactions of Bu, Bc and Bδ with diazo reagent, the following bilirubin fractions are differentiated in routine clinical diagnostics:

  • Total bilirubin: diazo reagent reacts with Bu, Bc and Bδ in the presence of an accelerator
  • Direct bilirubin: diazo reagent reacts immediately without the presence of an accelerator. The main fraction of Bc and Bδ as well as a small but variable fraction of Bu are measured. The serum concentration of direct-reacting bilirubin is therefore only a limited indicator of the concentration of Bc.
  • Indirect bilirubin: is the difference of total bilirubin minus direct bilirubin.

Since Bc is a better criterion for the differential diagnosis of jaundice than direct bilirubin, the latter should no longer be assayed.

5.2.1 Indication

Diagnosis, differential diagnosis and monitoring of jaundice.

5.2.2 Method of determination Total bilirubin

Bu, Bc and Bδ are measured.

Method based on the Jendrassik-Gróf principle /2/, suggested reference method recommended as per NCCLS /3/

Principle: in the presence of caffeine reagent, Bu, Bc and Bδ react with diazotized sulfanilic acid producing azobilirubin of red color in neutral solution. Addition of ascorbic acid, alkaline tartrate and dilute HCl changes the color of the azodye to blue and shifts the absorption maximum from 530 to 598 nm. Bc and Bδ react rapidly with diazotized sulfanilic acid, Bu does so slowly, but quickly after displacement from albumin by caffeine reagent the reaction proceeds rapidly. For the sample blank, the diazotized sulfanilic acid is replaced by sulfanilic acid.

DPD method /4/

Principle: Bu, Bc and Bδ react with 2.5-dichlorobenzene- diazonium salt in 0.1 mol/L HCl to form azodyes, which are measured quantitatively at 540–560 nm. For the determination of BT, Bu is released from albumin by the detergent Triton X-100. The reaction mixture of the sample blank contains only 0.1 mol/L HCl; therefore no color reaction occurs.

Direct spectrophotometry (bilimeter) /5/

Used for the bilirubin determination in neonatal plasma containing mainly Bu.

Principle: the absorbance of the plasma within a capillary tube is measured spectrophotometrically at approximately 460 nm. The spectral interference by hemoglobin is compensated by an additional measurement at 550 nm. The absorbance values at both wavelengths represent the sum of absorbencies of hemoglobin and bilirubin present in the specimen. The difference of absorbance A460–A550 represents the absorbance of bilirubin only, because the absorbance of hemoglobin is identical at both wavelengths.

Multilayer film-slide technology /6/

Principle: the film slide contains three layers:

  • The upper spreading layer containing caffeine and sodium benzoate to separate Bu from albumin
  • The second layer retains proteins
  • The third layer is the reaction zone. Bu and Bc interact with a specific charged polymer called a mordant. The concentrations of Bu and Bc are calculated from the measured reflection densities and the predetermined molar reflectivities of the two bilirubin species at two wavelengths (400, 460 nm) and use of simultaneous equations.

Enzymatic method /7/

Principle: BT, in the presence of bilirubin oxidase, is oxidized to biliverdin by molecular oxygen.

Bilirubin + ½ O 2 bilirubin biliverdin + H 2 O oxidase Biliverdin + O 2 purple colored pigments

The purple compound is measured at 450 nm. Bu, Bc and Bδ are measured at pH 8.2 in the presence of sodium dodecyl sulfate and sodium cholate.

Transcutaneous bilirubinometry (TcB) /8/

The method is used to determine total bilirubin in newborns.

Principle: the meters work by directing light (380–760 nm) into the skin of the neonate and measuring the intensity of specific wavelength that is returned. The meter analyzes the spectrum of optical signal reflected from subcutaneous tissues. The optical signals are converted to electrical signals by a photocell. These are analyzed by a microprocessor to generate a serum bilirubin value. The light absorption of interfering factors, such as hemoglobin, melanin, and dermal thickness, is mathematically subtracted to estimate the bilirubin concentration in the capillary beds and subcutaneous tissue. Bilirubin fractions

Direct reacting bilirubin

Bilirubin immediately reacts with diazo reagent in the absence of an accelerator such as caffeine reagent. Determination is of Bc, Bδ and partially of Bu. This method is still used as part of many mechanized analysis systems, but should be replaced by the specific determination of Bc.

Unconjugated bilirubin (Bu)

Is calculated based on the difference of total bilirubin minus direct bilirubin. The goal is the determination of Bu. The calculation is meaningful only in the case of hemolytic jaundice and hereditary hyperbilirubinemia since little Bδ accumulates, but not in obstructive hepatobiliary jaundice, because of the associated almost proportional increase in both Bc and Bδ. Bδ enters into the determination of direct reacting bilirubin and leads to a falsely low value for Bu.

Conjugated bilirubin (Bc/1/

Diazo method: keeping Bu from reacting requires dilution of the serum with HCl and incubating for at last 5 min. before adding diazo reagent. Bδ enters the reaction only to a limited extent.

Enzymatic method: at a pH of approximately 10, bilirubin oxidase oxidizes Bc, but not Bu or Bδ, to biliverdin /7/.

Multilayer film-slide technology: different methods based on film slide technology are available allowing separate determination of Bu, Bc and Bδ.

Unbound unconjugated bilirubin (free bilirubin; BF)

Peroxidase method: non albumin bound bilirubin is oxidized to a color-less product by ethyl hydrogen peroxide in a reaction catalyzed by horseradish peroxidase, while albumin-bound bilirubin is protected from oxidation /9/. The rate of decrease in absorbance of bilirubin is measured at 440 nm.

5.2.3 Specimen

  • Serum, plasma: 1 mL
  • Capillary plasma (Heparin, EDTA): 0.05 mL

5.2.4 Reference interval

Refer to Tab. 5.2-1 – Bilirubin reference intervals and references /91011/.

5.2.5 Clinical significance

Hyperbilirubinemia is a symptom and clinically causes jaundice if bilirubin levels are ≥ 4 mg/dL (68 μmol/L) in neonates and infants and ≥ 3 mg/dL (51 μmol/L) in older children and adults. Classification of jaundice

Prehepatic jaundice

There is overproduction of bilirubin, which is most commonly caused by hemolytic anemias, neonatal jaundice, ineffective erythropoiesis, infections such as malaria, transfusion reactions, burns, resorption of large hematomas, and hereditary hyperbilirubinemia.

Intrahepatic jaundice

The most frequent causes are infectious or toxic injury of the liver parenchyma. Generally, possible causes include acute and chronic viral hepatitides, bacterial and parasitic liver diseases, liver metastases, drug-induced parenchymatous and cholestatic liver injury as well as involvement of the liver in other underlying diseases. Another significant group are hereditary hyperbilirubinemias.

Post hepatic jaundice

This is caused by mechanical obstruction of the bile ducts (bile duct stones, carcinoma of the pancreatic head, biliary atresia, primary sclerosing cholangitis). Differentiation of jaundice

Differential diagnostic information is provided by:

  • Total bilirubin (BT = Bu + Bc + Bδ)
  • The concentration of conjugated bilirubin (Bc)
  • The Bc to BT ratio
  • The LD to AST ratio
  • The levels of ALT, GGT and ALP activities. Prehepatic jaundice

Non-conjugated bilirubin (Bu) is elevated. Prehepatic hyperbilirubinemias have a negligible proportion of Bδ, so that Bu is calculated from the difference of BT minus Bc. Refer to Tab. 5.2-2 – Prehepatic hyperbilirubinemias.

Prehepatic jaundice due to hemolysis or ineffective erythropoiesis can be ruled out if BT is above 6 mg/dL (103 μmol/L). Significantly elevated bilirubin levels are only seen in transfusion incidents in the AB0 system, hemolytic crises (e.g., in sickle-cell anemia, neonatal jaundice, and certain hereditary hyperbilirubinemias).

The determination of direct reacting bilirubin and the direct reacting bilirubin/total bilirubin ratio are useful in the differentiation of hemolytic from hepatobiliary jaundice only up to a BT concentration of 3 mg/dL (51 μmol/L) /12/. Using a ratio of 0.33 as a discriminator, values below predict hemolytic jaundice with a diagnostic sensitivity of 80% and values above predict hepatobiliary jaundice with a sensitivity of 86%.

In differentiation to hepatic jaundice, an LD/AST ratio of ≥ 5 is suggestive of hemolytic jaundice. Other findings pointing to hemolytic jaundice include:

  • In urine: bilirubin negative and urobilinogen positive
  • Decrease in serum haptoglobin, and reticulocytosis. Intrahepatic jaundice

If hyperbilirubinemia is accompanied by elevated liver enzymes, then the jaundice is likely to be hepatic. In this case, differentiation from post hepatic jaundice is required.

Hyperbilirubinemia has low diagnostic sensitivity for liver diseases and therefore is not a screening parameter. More than 40% of patients with clinical liver disease have a BT concentration below 1.2 mg/dL (20 μmol/L) and another 25% have subicteric levels i.e., bilirubin concentrations in the range of 1.2–2.9 mg/dL (20–50 μmol/L). Many liver patients present with sub icterus as the main symptom of concern Tab. 5.2-3 – Intrahepatic hyperbilirubinemias.

In hepatic jaundice, Bu, Bc and Bδ are elevated, with Bc accounting for over 50%. The main causes are viral hepatitides and conditions with reduced bilirubin excretion due to lack of energy, such as sepsis, total parenteral nutrition, or serious surgical interventions. The proportions of Bc and Bδ of BT are of prognostic value. For example, a decrease in Bc is a sensitive indicator of improvement while an increase in Bδ predicts prolonged illness. In the case of decreasing or slightly to moderately elevated BT levels, direct bilirubin can account for 80% of total bilirubin, since Bδ, which enters into the determination of direct reacting bilirubin, has a half-life of 18 days.

In newborns and infants, Bδ is low in non-hepatic jaundice (e.g., neonatal jaundice, septic shock, hemolysis). An increase of Bδ over 10% of BT, suggests a hepatogenic cause (e.g., Cytomegalovirus infection, biliary atresia, hepatitis B infection) /13/.

Other findings are:

  • In urine: bilirubin positive, urobilinogen positive. Post hepatic jaundice

In obstructive jaundice, ALT activities are rarely higher than 10-fold the upper reference interval value. The elevation in BT is due to the parallel increase in Bc + Bδ, with Bc accounting for the largest proportion (Tab. 5.2-4 – Post hepatic hyperbilirubinemia). In acute disorders with jaundice, fresh obstructive jaundice can be ruled out if ALT is normal or elevated more than 25-fold /11/.

The success of an invasive procedure for the correction of cholestasis can be assessed quickly by monitoring Bc. Due to its short half-life, Bc decreases faster than BT /3/. The determination of Bc however, may not be performed as direct bilirubin, because Bδ, which still remains elevated for several weeks post obstruction, is measured as part of it /1/.

Other findings in post hepatic jaundice:

  • In urine: bilirubin positive, urobilinogen negative. Hereditary hyperbilirubinemia

Hyperbilirubinemia in neonates, infants, older children and young adults presents a differential diagnostic problem. In neonates, the condition can be benign, as in breast milk jaundice, or potentially fatal, as in hereditary fructose intolerance. The jaundice can originate primarily in the liver, as in acute viral hepatitis, or outside the liver, as in biliary atresia, or can be secondary to a non-hepatic cause, such as hemolysis (e.g., due to glucose-6-phosphate dehydrogenase (G6-PD) deficiency, or sepsis). Hereditary hyperbilirubinemias must always be included in the differential diagnosis /14/.

Hereditary hyperbilirubinemia is characterized by liver dysfunction without hepatocellular damage (Tab. 5.2-5 – Hereditary hyperbilirubinemias). One of the first steps in the diagnosis is the measurement of BT and the quantitative determination of Bc and Bu. Conjugated hyperbilirubinemias are always caused by a hepatobiliary disorder, and conjugated bilirubin is harmless. Chronic, more severe unconjugated hyperbilirubinemias can cause bilirubin encephalopathy, while mildly elevated concentrations of Bu have an anti oxidative effect and counteract the development of oxidative stress.

Hereditary hyperbilirubinemias /14/ are classified into (Fig. 5.2-1 – Differential diagnosis of hereditary hyperbilirubinemia):

  • Disorders of bilirubin conjugation and elimination. Non-conjugated hyperbilirubinemias include type I and type II Crigler-Najjar syndrome (Arias syndrome) and Gilbert’s syndrome.
  • Hereditary cholestasis with predominantly conjugated hyperbilirubinemia. These include Dubin-Johnson syndrome, Rotor syndrome, benign recurrent intrahepatic cholestasis (BRIC), progressive familial intrahepatic cholestasis (PFIC), and Alagille syndrome.

In theory, the measurement of Bc is a good indicator for differentiating between the two hereditary types of hyperbilirubinemia. In practice, however, this is not the case, since most mechanized analytical systems measure direct bilirubin but not Bc. This is too unspecific, because direct bilirubin is also measured in healthy individuals where physiologically it should not be detectable. Neonatal hyperbilirubinemia

Neonates have a reduced erythrocyte life span compared to adults. The conversion of hemoglobin (Hb) to unconjugated bilirubin and the conjugation of the latter are reduced in the postnatal period, leading to increased serum levels of unconjugated bilirubin. In addition, the Hb from hematomas also has to be metabolized in the postnatal period. Moreover, an acute-phase reaction activates hem oxygenase-1, leading to further accumulation of Hb.

Approximately 60% of term and 85% of pre-term newborns will develop clinically apparent jaundice because of an increase in unconjugated bilirubin /15/. This physiological jaundice becomes clinically apparent on day 3, peaks on day 5–7 when most newborns are already at home, and resolves by day 14. Physiological jaundice is usually benign. However, if unconjugated bilirubin levels get too high, bilirubin can cross the blood brain barrier where it is neurotoxic. Therefore it is useful to classify newborn jaundice according to the age when the baby becomes visibly jaundiced.

The incidence and risk factors of jaundice in newborns are /16/:

  • Uncommon on days 1–2. The underlying disease for jaundice is antibody mediated hemolysis e.g., Rhesus, ABO, and others. Newborns of Rh(D) negative mothers, or newborns of mothers with a positive antibody screen, should routinely have cord blood sent for blood group and direct antibody test (Coomb's test). Any newborn who is Coomb's test positive should have a total bilirubin determination in the first 24 hours. Any newborn who is clinically jaundiced within the first 24 hours requires urgent testing to exclude hemolysis.
  • Normal incidence on days 3–10, mostly uncomplicated, in rare cases complicated (e.g., G6PDH deficiency) or increased incidence of premature newborns
  • Prolonged jaundice is defined as a duration of more than 14 days in full-term infants and more than 21 days in premature infants. It is clinically useful to classify (i) Predominantly unconjugated prolonged jaundice usually found in breast milk nutrition. Breast fed neonates are four times more likely to have hyperbilirubinemia with levels above 10 mg/dL (172 μmol/L) than those on a formula diet. However, they do not have higher bilirubin peak levels /16/. (ii) Predominantly conjugated prolonged jaundice usually always pathological. The newborn should be investigated for intra-hepatic (e.g., hepatitis) and obstructive (e.g., biliary atresia) causes of prolonged jaundice. See Section

Physiological transient unconjugated jaundice in the first week of life must be differentiated from hyperbilirubinemia which exceeds a time-dependent physiological upper limit on the bilirubin nomogram and can lead to bilirubin-induced neurologic dysfunction (BIND). Refer to Fig. 5.2-2 – Hour-specific bilirubin nomogram showing the increase in bilirubin in term or near-term neonates.

In most cases, total bilirubin is determined in neonates. If the upper limit in the bilirubin nomogram is exceeded or if there is prolonged jaundice, unconjugated and conjugated bilirubin has to be determined in addition to total bilirubin.

Hereditary disorders of bilirubin clearance (Gilbert’s syndrome, Crigler-Najjar syndrome, Dubin-Johnson syndrome), G6-PD deficiency and hereditary spherocytosis alone do not cause neonatal hyperbilirubinemia. Frequently they are not diagnosed until jaundice is provoked by an extrinsic event.

Discharge before 72 hours is a risk factor for the development of severe hyperbilirubinemia, mainly because outpatient surveillance cannot be as close as that provided in the postnatal ward. Unconjugated unbound hyperbilirubinemias in premature infants

Total bilirubin concentrations increase with perinatal age and higher bilirubin levels are tolerated at older age. However, studies on bilirubin induced neurological damage only provide limited evidence on harmful total serum bilirubin levels, because most factors that increase the risk of neurodevelopmental delay (e.g., asphyxia, intracranial hemorrhage, prematurity) also increase total serum bilirubin levels and bilirubin induced neurotoxicity (BIND), especially in pre-term infants /17/.

Most pre-term infants less than 35 weeks gestational age have elevated total serum bilirubin concentrations which often present as jaundice. When left unmonitored or untreated in these infants, an elevated total bilirubin level can progress to silent symptomatic neurologic manifestations. Acute bilirubin encephalopathy is acute progressive, and often reversible with aggressive intervention, whereas kernicterus (or chronic bilirubin encephalopathy) is the syndrome of chronic, post-icteric and permanent neurologic sequelae that is associated with more serious and usually irreversible manifestations /18/. Dysmyelination and degeneration in the globus pallidus, subthalamic nucleus, and cerebellum are neuropathologic findings of kernicterus in neonates, albeit characteristically late (> 10 days) in onset /19/.

Low bilirubin kernicterus in pre-term neonates, though rare, remains an unpredictable and refractory form of brain injury. Low bilirubin kernicterus is defined as the occurrence of kernicterus at total bilirubin levels below commonly recommended exchange transfusion thresholds (Tab. 5.2-6 – Guidelines for exchange transfusion in low birth weight infants). The exchange transfusion treatment thresholds reflect both the total bilirubin level and the presence of neurotoxicity risk factors.

The low bilirubin icterus appears to result only when a rare constellation of co-morbid conditions and CNS findings that are frequently, albeit not invariably, observed /19/:

  • Hypoalbuminemia < 2.5 g/dL. Mean serum albumin levels reported for pre-term infant below 30 weeks` gestation are approximately 1.9 g/dL (90% CI 1.2–2.8 g/dL) and do not approach 2.5 g/dL until 36–37 weeks’ gestation
  • Co-morbid CNS findings of intraventricular hemorrhage and periventricular white matter injury
  • Perinatal and early postnatal infection/inflammation is an important contributer to brain structural and functional abnormalities later in life. Notable contributers are chorioamnionitis, sepsis and necrotizing enterocolitis
  • Chronic bilirubin-induced neuroinflammation. Persistent inflammation is recognized as an important risk factor for central nervous system injury in pre-term neonates.

Risk stratification based on clinical factors and bilirubin measurement have been integral parts of neonatal hyperbilirubinemia. Recommended bilirubin measurements are /19/:

  • Total bilirubin to gauge the size of the neonate's bilirubin
  • Unconjugated unbound bilirubin (no commercial assay is available)
  • Bilirubin/albumin ratio can serve as a proxy for unbound bilirubin but has a limited and conflicting track record in predicting adverse neurodevelopmental outcome. Japanese investigators /19, 20/ found that a bilirubin-albumin ratio of ≥ 0.50 μmol/L/μmol/L (≥ 4.25 mg/dL/g/dL) for infants of 30–34 weeks gestation and ≥ 0.40 μmol/L/μmol/L (≥ 3.4 mg/dL/g/dL) for infants of below 30 weeks' gestation predicted putative neurotoxic unbound bilirubin levels of ≥ 1.0 μg/dL.
  • Unconjugated unbound bilirubin/total bilirubin ratio (Bf/TBC; μg/mg). In a study /21/ the ratio was tested in neonates who were admitted to the neonatal intensive care unit. One group had normal and the other had abnormal auditory brainstem response. In normal newborns the Bf/TBC was mean + SD = 0.062 ± 0.034 (range 0.009–0.200) in the abnormal group mean + SD = 0.109 ± 0.039 (range 0.034–0.167). Conjugated hyperbilirubinemia of the newborn

The presence of conjugated bilirubin indicates a pathologic process and usually prolonged jaundice. Conjugated hyperbilirubinemia can be of infectious, endocrine or genetic etiology. The most common surgically correctable cause is extrahepatic biliary atresia.

If conjugated hyperbilirubinemia is present, the following laboratory tests must be performed within 24 h according to the suspected diagnosis:

  • Sodium, potassium, creatinine, urea
  • Blood count including reticulocytes
  • Blood group typing (ABO, Rh)
  • Blood gas analysis
  • Glucose, lactate, ammonia
  • Cholesterol, triglycerides
  • Blood and urine culture tests for pathogenic bacteria
  • Stool color analysis
  • PT and aPTT
  • Serology for hepatitis A, B, C
  • IgM antibodies for rubella, toxoplasmosis, Herpesvirus, Cytomegalovirus.
  • TSH, free T4, cortisol and α1-antitrypsin
  • Organic acids and amino acids in urine
  • Enzyme tests in red blood cells for galactosemia.

Tables describing scores and staging of liver diseases:

5.2.6 Comments and problems

Pre analytic factors

Neonatal bilirubin is often measured in a capillary blood sample obtained by heel puncture. Common pre analytical errors in blood collection include /22/:

  • Use of “adult-size” lancets that are too long for blood collection in neonates. There is a risk of puncture wound osteomyelitis.
  • In vitro hemolysis, in particular when using micro containers for blood collection. Hemolysis occurs in 0.2% of specimens and is the cause of specimen rejection by the laboratory in 39.7% of cases.
  • Exposure of the specimen to light.

Method of determination /123/

Calibration: the recommended standard reference material for the calibration of bilirubin assays is the SRM 916a preparation by the National Institute for Standards and Technology (NIST).

Total bilirubin: so far it is not known whether the reference method recommended by the NCCLS /3/ measures Bδ correctly. The linear range of the assay is up to 27 mg/dL (462 μmol/L). Interference by hemoglobin is found only at concentrations ≥ 2 g/L.

Direct reacting bilirubin: the extent to which Bu is simultaneously measured depends on the pH of the reaction mixture: the lower the pH, the less Bu is measured. Bδ is always included in the measurement. Laboratories should ensure that the assays they use do not measure direct reacting bilirubin above 0.1 mg/dL (1.7 μmol/L) in healthy individuals /24/.

Conjugated bilirubin: the method used for the determination of Bc does not detect Bu. However it has interference by the presence of hemoglobin, since during preincubation of the specimen with HCl hemoglobin is oxidized to methemoglobin, producing H2O2. The latter causes destruction of the azobilirubin formed in the main reaction resulting in falsely low results.

Enzymatic method for BT: at concentrations below 1.1 mg/dL (19 μmol/L), the values are on average 0.1 mg/dL (1.7 μmol/L) lower than those measured with the reference method. Direct bilirubin concentrations in the sera of newborns with non-conjugated hyperbilirubinemia are higher when measured using the bilirubin oxidase method than with the diazo method. This is also the case in neonates after light therapy /25/. Hemoglobin concentrations ≥ 2 g/L lead to a 5–18% decrease in BT.

Spectrophotometric determination of bilirubin: Bilirubinometers are used to measure neonatal bilirubin. Measurements are carried out at two wavelengths i.e., of 460 nm and 550 nm. Although hemoglobin is compensated by the measurement at two wavelengths, a free hemoglobin concentration of 4 g/L causes a reduction in bilirubin within the threshold range for phototherapy from 16.3 mg/dL (279 μmol/L) to 15.6 mg/dL (265 μmol/L) /5/. Carotenes which often can cause interference are only in very small quantities in the serum of neonates. The most common interference mimicking falsely high values is the plasma turbidity (lipemia), because the light dispersion it causes is greater at 460 nm than at 550 nm. The results obtained with bilirubinometers are highly dependent on the protein matrix. The calibration should therefore be performed with commercial sera specifically designed for this type of assay, or adult sera whose concentrations were determined with the reference method. Levels above 17.5 mg/dL (300 μmol/L) should not be measured spectrophotometrically, since absorbance values are obtained that deviate from the theoretical linearity of Lambert-Beer’s law. For the decision on whether to proceed with exchange transfusion, the direct spectrophotometric bilirubin determination must not be used as the sole method of analysis. Additional bilirubin determination based on the diazo reaction (Jendrassik/Gróf, DPD) is recommended /26/.

Neonatal hyperbilirubinemia

There are clininical significant method-dependent differences in total serum bilirubin results from neonatal samples. In a study /76/ measured on 7 commercial platforms 24 to 30% difference in results for individual samples were measured, largely due to calibration differences between assays.

Influencing factors /24/

  • Day-to-day variation: BT 30%.
  • Fasting: BT elevation by 100 to 200% after 48 h.
  • Physical stress: 30% increase in BT.
  • Pregnancy: 33% decrease in BT during the last trimester.
  • Oral contraceptives: 15% decrease in BT /27/.

Interfering factors

Exposure of the specimen to light: up to 30% decrease in BT after 1 h.

Sample stability

When kept at room temperature in a light-proof container, BT is stable for 3 days /28/.


During phototherapy in hyperbilirubinemic neonates non-conjugated bilirubin is converted to photo isomers which are more water-soluble. The diazo method measures higher levels in neonates receiving phototherapy than the bilirubin oxidase method /29/. Neonatal bilirubin levels measured with the multi layer film-slide technique are higher than the determination based on the diazo reaction /30/.

5.2.7 Biochemistry

Approximately 80–85% of the bilirubin produced daily originates from the degradation of hemoglobin released by the breakdown of senescent erythrocytes. At the end of their life span of approximately 120 days, the erythrocytes are taken up by macrophages of the reticuloendothelial system, in particular in the spleen. The degradation of hemoglobin occurs in two steps. The Fe3+ heme binds to the membrane-bound enzyme hem oxygenase and is reduced to Fe2+ heme in a reaction catalyzed by NADPH-cytochrome P450 reductase (Fig. 5.2-3 – Breakdown of the heme molecule). The Fe2+ heme is then broken down by oxidation into equimolar amounts of CO and biliverdin. The hemoglobin molecule degrades, globin is metabolized, iron binds to transferrin, and the biliverdin is reduced to bilirubin by cytosolic biliverdin reductase. The CO produced binds to hemoglobin to form carboxyhemoglobin and is then eliminated via the pulmonary gas exchange.

The breakdown of 1 g of hemoglobin yields 34 mg of bilirubin. Approx. 250 mg of bilirubin is produced daily in the physiological breakdown of hemoglobin. The remaining 15–20% of the bilirubin produced daily is derived from the breakdown of heme-containing proteins such as myoglobin, cytochromes and catalases. Bilirubin accumulates in the bone marrow in the case of ineffective erythropoiesis that is associated with impaired maturation processes, such as in megaloblastic anemia, thalassemia, porphyria, and myelodysplastic syndrome. In these conditions, up to 80% of the bilirubin can result from ineffective erythropoiesis.

Bu binds to albumin in the blood for transport to the liver. Upon reaching the liver, bilirubin dissociates from albumin in the space of Disse and is taken up by the hepatocyte across the sinusoidal part of the cell’s plasma membrane. The uptake is mediated by a transport system and occurs actively against a concentration gradient. The sinusoidal membrane’s transport system for organic ions and possibly bilirubin, but not for bile acids, is believed to consist of bilitranslocase, bromosulfophthalein/bilirubin binding protein (BBBP), and organic anion-binding protein (OABP). Organic anion transport peptides (OATPs) may also play a role. The transport of unconjugated bilirubin across the sinusoidal part of the plasma membrane is, however, not yet well understood /31/.

In the cytosol of the hepatocyte, Bu binds to ligandin and Z-protein. Ligandin has a higher affinity and transports Bu to the endoplasmic reticulum where it is glucuronidated by the action of uridine-5’-diphosphate glucuronyltransferases (UGTs) /32/. Essential cosubstrates of the UGTs are uridine diphosphate glucuronic acid or other UDP sugars. Bu is glucuronidated as follows (Fig. 5.2-4 – Structure of unconjugated bilirubin):

  • At the propionic acid side chain located at C8 of the two central pyrole rings, forming bilirubin mono glucuronide (C8)
  • At the propionic acid side chain located at C12 of the two central pyrole rings, forming bilirubin mono glucuronide (C12)
  • Both isomers can then be further glucuronidated to diglucuronides.

By its conversion to glucuronide bilirubin becomes water-soluble. The transport of the glucuronide across the canalicular domain of the hepatocyte’s plasma membrane is mediated by multi drug resistance proteins (MRPs), of which six are known.

Approximately 80% of the bilirubin secreted into bile is bilirubin diglucuronide, 15% is bilirubin mono glucuronide, and 5% is mixed conjugates with sugars such as glucose and xylose. About 1–2% of the bilirubin is secreted into bile non-conjugated.

The excretion of the Bc from the hepatocyte into the bile capillaries depends on the energy available and is the slowest process in bilirubin metabolism. A healthy liver can eliminate approximately 1 g of conjugated bilirubin per day i.e., 2–5 times the amount of bilirubin produced by the body. Inhibition of the excretion mechanism by drugs such as digoxin or by bromosulfophthalein causes Bc to accumulate in the hepatocyte from where it is then released into plasma.

Bc reaches the intestines via the biliary tract. Due to the impact of intestinal bacteria urobilinogen is formed. Approximately 70% of the urobilinogen is reabsorbed in the intestine, transported to the liver via the portal vein, and excreted again via the biliary tract (enterohepatic circulation). Approximately 2–4 mg of urobilinogen is excreted in urine daily. This amount is 2–3 times higher if bilirubin levels are increased (e.g., due to hemolytic anemia) and 4–10 higher if the liver parenchyma is damaged (e.g., in acute viral hepatitis or in the presence of a portocaval shunt). If the bile ducts are obstructed, the enterohepatic urobilinogen circulation is interrupted, and urobilinogen is not detectable when bilirubinuria is present.

In conjugated hyperbilirubinemias, in particular obstructive jaundice, a rapid decrease in Bc (half-time of only hours) is the most sensitive indicator for complete removal of the occlusion. Assays measuring BT are, however, unsuitable for monitoring the decline of conjugated hyperbilirubinemia, since they also measure Bδ with a half-time of 18 days. Bc can, however, be monitored using methods that specifically measure Bc.

In acute hepatitides, parenchymal cell damage leads to jaundice. It results from impaired excretion of biliary excreted substances into the bile capillaries and regurgation of Bc back into the bloodstream.

UDP-glucuronyltransferase (UGT1A1) glucuronidates bilirubin and is encoded by the UGT1A1 gene. A polymorphism in the promoter of the gene, which contains repeats of thymidine and adenine (TA) (TATA box), is responsible for the amount of UGT1A1 in the cell. If the TATA box is

  • homozygous for 7 TA repeats (genotype 7/7), the cell contains low UGT1A1 activity
  • homozygous for 6 TA repeats (genotype 6/6, wild type), the cell contains high UGT1A1 activity
  • heterozygous (genotype 6/7), the cell contains moderate UGT1A1 activity.

In Europe, 24% of individuals have genotype 6/6, 39% genotype 6/7, and 8% genotype 7/7. A variation in nucleotide 211 (G to A, heterozygous and homozygous) causes hyperbilirubinemia in neonates.

Bu is highly apolar and practically insoluble in water. This is due to its folded configuration, which is stabilized by six bridging hydrogen bonds yielding the so-called ZZ conformation (Fig. 5.2-4 – Structure of unconjugated bilirubin). Under the effect of phototherapy in the neonates with unconjugated hyperbilirubinemia, the bridging hydrogen bonds rupture, resulting in isomers with a partially open (ZE or EZ) or completely open (EE) conformation. These conformations are more water soluble and can therefore be better eliminated.

The cause of bilirubin encephalopathy is free bilirubin, whose concentration increases with a BT concentration above 17 mg/dL (291 μmol/L). Free bilirubin dissolves readily in fat and is neurotoxic in that it damages the mitochondria in the astrocytes. Since the binding of Bu to albumin decreases in acidosis as well as being impaired by certain drugs such as salicylic acid, both factors also promote an increase in BF.

Bδ has a half-life of 18 days due to its covalent linkage with albumin. It is not detected in healthy individuals, in Gilbert’s syndrome, and in neonatal hyperbilirubinemia. In diseases with conjugated hyperbilirubinemia (e.g., parenchymatous and obstructive jaundice, Bδ can account for 20–50% of BT in the acute phase). As the clinical condition improves and BT decreases, the Bδ fraction can increase to 50–90% so that hyperbilirubinemia can persist for a relatively long period of time.


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5.3 L-Carnitine

Hermann Seim, Lothar Thomas

Carnitine (3-hydroxy-4-N-trimethylammonium butyrate) occurs in nature as the L-(–) stereoisomer, either in free form or esterified through its hydroxyl group with fatty acids. A range of endogenous carnitine can be measured in plasma /1/:

  • Total L-carnitine: the sum of free carnitine and esterified carnitine
  • Free L-carnitine: the proportion of the unesterified carnitine in plasma is about 90% of total carnitine.
  • Total acylcarnitine: L-carnitine is esterified through its hydroxyl group with long-chain (C12–C18) or shorter-chain fatty acyl groups (C2–C10). The proportion of total acylcarnitine in plasma is about 5% of total carnitine. Acyl compounds contain the acyl group (R-CO-)
  • Acetylcarnitine: the proportion of L-carnitine trans esterified to acetyl carnitine is about 5% of total carnitine.

Carnitines are low molecular weight endogenous compounds present in all mammalian species. The biological activities of carnitines are:

  • To function as an essential carrier of acyl groups from the cytoplasma into the mitochondria and hence to the site of fatty acid β-oxidation and consequently to produce energy
  • To maintain inner mitochondrial concentrations of free coenzyme A by accepting short-chain acyl groups for the corresponding acyl-CoA and transporting them out of the mitochondria.

Mitochondrial fatty acid oxidation is a fundamental source of cellular energy, particularly in cardiac and skeletal muscle. Approximately 98% of the total carnitine pool within the body is located in these tissues. Despite the fact that only 1% of the body’s carnitine pools is present in blood, the plasma concentration is used as an indicator of the total body carnitine content.

L-Carnitine body pool is achieved by absorption from dietary source, in particular red meat, and by endogenous biosynthesis requiring lysine and methionine.

5.3.1 Indication

Suspected carnitine deficiency in conjunction with:

  • Symptoms such as myasthenia, myalgia, cardiomyopathy, hypo ketotic hypoglycemia, failure to thrive in neonates and infants
  • Malnutrition including kwashiorkor and cachexia
  • Long-term carnitine-free parenteral nutrition
  • Drug-induced deficiency caused by treatment with (e.g., valproic acid or pivalic acid)
  • Congenital enzyme defects (e.g., medium-chain acyl-CoA dehydrogenase deficiency (MCAD))
  • Organic acidurias (e.g., propionic aciduria)
  • Lipid storage myopathies
  • Pregnancy, hemodialysis (situational).

5.3.2 Method of determination

There are two main principles for the determination of free carnitine and total carnitine which is often determined concomitantly. One is a colorimetric assay the other is a radioisotopic assay.

Colorimetric assay /23/

L-Carnitine reacts with acetyl CoA catalyzed by carnitine acetyl transferase (CAT, EC to form acetyl L-carnitine and CoASH. CoASH reacts nonenzymatically with 5,5’-dithiobis-2-nitro benzoate (DTNB) to form 5-thio-2-nitro benzoate (TNB). The concentration of TNB is measured spectrophotometrically. Two sets of test tubes are used. In the first set free carnitine is determined. To the second set 2 mol/L KOH is added used for hydrolyzing the sample for total carnitine determination. The values thus obtained have to be multiplied by factors corresponding to dilution of the samples and other variables. The CAT is highly specific for L-(–) carnitine.

L-carnitine + acetyl-CoA CAT acetyl-L-carnitine + CoASH CoASH + DTNB CoA-S-NB + TNB + H +

Radioisotopic assay /4/

The radioisotopic assay is based on the stoichiometric acetyl-transfer from 14C-labelled acetyl coenzyme A to carnitine catalyzed by CAT, and measurement of isotope content of the 14C acetyl carnitine formed.

Chromatographic methods

The following methods are used for determining L-carnitine and its esters: gas chromatography and high-pressure liquid chromatography (HPLC), often coupled with tandem mass spectrometry or HPLC electrospray mass spectrometry /5/.

5.3.3 Specimen

  • Serum (EDTA plasma), urine: 1 mL
  • Biopsy material, seminal plasma

5.3.4 Reference interval

Refer to Tab. 5.3-1 – Carnitine reference intervals.

5.3.5 Clinical significance

Low serum free L-carnitine and/or high acylcarnitine/free L-carnitine ratio generally result in diagnosis of L-carnitine deficiency. In the healthy population the frequency of L-carnitine deficiency is very low because L-carnitine is supplied satisfactorily through diet and renal and hepatic biosynthesis. Typically L-carnitine deficiency is associated with various disorders such as erythropoietin resistant anemia, muscle weakness, cardiac dysfunction and dialysis-related symptoms. L-carnitine supplementation improves such disorders /16/.

L-carnitine is synthesized in the liver from methionine and protein-bound lysine. However, most of the L-carnitine required daily comes from dietary sources, in particular red meat. No hereditary defect of L-carnitine synthesis is currently known, although the synthetic pathway is not yet fully developed in premature infants /14/.

L-carnitine deficiency is a condition in which free L-carnitine is reduced. Often, the L-carnitine/acylcarnitine ratio is decreased due to the latter being elevated. Cellular L-carnitine deficiencies are of clinical significance. They cannot always be detected by measuring the serum L-carnitine concentration.

L-carnitine deficiencies are classified as follows based on clinical and etiopathogenic criteria:

  • Muscular; only the muscles are affected
  • Systemic; L-carnitine is reduced in all tissues
  • Primary; this type of deficiency is a disorder of L-carnitine metabolism, which results in a low concentration of free L-carnitine. It is associated with cardiomyopathy, encephalopathy and myasthenia and can lead to premature death if untreated. The disorder is due to a defect of L-carnitine uptake into the cell or a defect of L-carnitine transport to the mitochondrion.
  • Secondary; this type of L-carnitine deficiency is milder than the primary type and results from diminished renal or hepatic function, extreme malnutrition, or use of medications, such as valproic acid or pivampicillin.

Primary L-carnitine deficiency is a rare disease, whereas the secondary type is clinically common. The deficiency can be efficiently treated by supplementation with L-carnitine (approximately 10–100 mg/kg per day). The response to L-carnitine application in muscular L-carnitine deficiency varies, in the systemic form it is vital.

The L-carnitine concentration in the tissues is 10–100-fold higher than that in blood or extracellular fluid. What is important clinically is that:

  • Reduced tissue L-carnitine levels indicate L-carnitine deficiency syndrome
  • A persistently reduced serum L-carnitine concentration over weeks or months suggests L-carnitine deficiency in the tissues
  • A normal serum L-carnitine concentration does not rule out L-carnitine deficiency in individual organs, in particular the heart and skeletal muscles, since there may be isolated dysfunction of the L-carnitine transport system of the cell membrane (e.g., muscular L-carnitine deficiency). Muscular L-carnitine deficiency

Myogenous primary L-carnitine deficiency

The deficiency is confined to the muscles /15/. The diagnosis can be made by determining L-carnitine in muscle tissue, since the serum L-carnitine concentration is within the reference range. Even though 99% of the L-carnitine pool resides in the skeletal muscle, nearly 90% of this pool must be depleted before the serum level falls below the reference interval /16/. Clinical symptoms include episodes of myasthenia and myalgia, in particular of the extremities and neck muscles; fasting or a high-fat diet induce an adjusted form of ketogenesis. Histochemically, a form of lipid storage myopathy is present, in which lipid containing vacuoles are stored in type I muscle fibers. This form of the deficiency can manifest from infancy to adulthood and is less progressive than the systemic form. Muscular L-carnitine deficiency is inherited in an autosomal recessive pattern, or acquired.

Myogenous secondary L-carnitine deficiency

Secondary deficiency is confined to the muscles, but associated with other muscular diseases (e.g., Duchenne muscular dystrophy or metabolic myopathies (mitochondriopathies). Systemic primary L-carnitine deficiency

Primary L-carnitine deficiency is hereditary and can be due to the following defects (Fig. 5.3-1 – Mitochondrial carnitine system):

  • Defect of the L-carnitine transporter of the cell membrane (L-carnitine uptake defect, CUD). In CUD, the resorption of L-carnitine from the intestine and the reabsorption of L-carnitine in the proximal renal tubules are inhibited, and in other tissues the accumulation of L-carnitine within the cell is reduced.
  • Defects of enzymes which transport long-chain fatty acids to the mitochondria i.e., carnitine palmitoyltransferase I (CPT I), carnitine palmitoyltransferase II (CPT II), and the carnitine carrier, also known as carnitine-acylcarnitine translocase.

Hereditary causes are shown in Tab. 5.3-2 – Hereditary causes of systemic secondary carnitine deficiency.

L-carnitine uptake defect (CUD) /17/

Clinical findings: there are two forms of CUD: a cardiomyopathic form which begins in early childhood, and a hepatic form with recurrent symptoms similar to Reye’s syndrome.

Laboratory findings: free and total L-carnitine are very low, acylcarnitine is normal. Fasting glucose and ketone bodies are reduced. Further results: ammonia elevated, metabolic acidosis, creatine kinase elevated, myoglobin elevated, aminotransferases elevated.

CPT-1 deficiency /17/

Clinical findings: only the liver is affected. Episodic liver dysfunction, often accompanied by hypoglycemia. Some patients develop renal tubular acidosis. The mothers of these patients may have a history of acute hepatic steatosis in pregnancy.

Laboratory findings: total L-carnitine reduced, acylcarnitine reduced, free L-carnitine normal or elevated. Fasting hypoglycemia and hypo ketonemia. Metabolic acidosis, ammonia normal, aminotransferases and creatine kinase normal.

Carnitine-acylcarnitine translocase deficiency /17/

Clinical findings: this deficiency can result in sudden infant death or can be of a milder nature. Both cases are characterized by cardiomyopathic and hepatic dysfunction of different severity.

Laboratory findings: acylcarnitine elevated, free L-carnitine reduced. Fasting hypoglycemia and hypo ketonemia. Metabolic acidosis, ammonia, lactate, uric acid, aminotransferases and creatine kinase elevated.

CPT-2-deficiency /17/

Clinical findings: there are two forms:

  • Neonatal form, which is usually lethal and may be accompanied by cystic kidneys
  • Childhood-onset form that manifests in adulthood. This deficiency is one of the most common biochemically defined causes of myoglobinuria in adults /18/. CPT-2 deficiency becomes evident in particular after intense physical stress. It is associated with myalgias with myoglobinuria and rhabdomyolysis. Other precipitating factors include fasting with and without physical exercise, cold exposure, and recurrent infections, all of which ultimately are conditions of increased fatty acid oxidation. There is little or no lipid storage in the skeletal muscle /181920/.

Laboratory findings: total L-carnitine normal or elevated, acylcarnitine elevated, free L-carnitine reduced. Fasting glucose and ketone bodies reduced. Liver enzymes, creatine kinase and myoglobin elevated. Systemic secondary L-carnitine deficiency

This group includes L-carnitine deficiencies that are associated with defined genetic defects outside the carnitine system, complex diseases, or extreme malnutrition. Genetic defects include organic acidurias, enzyme defects of mitochondrial β-oxidation and of the respiratory chain (Tab. 5.3-3 – Acquired causes of systemic secondary carnitine deficiency). In serum there is a marked deficiency of free L-carnitine (below 18 μmol/L).

Secondary L-carnitine deficiency often presents as a mere functional deficiency in which free L-carnitine is reduced, but total L-carnitine is still within the reference range due to elevated acylcarnitine /21/. Thus L-carnitine is lacking as a transfer recipient for excessive pathogenic acyl groups from coenzyme A.

5.3.6 Comments and problems

Interference factors

Since in colorimetric assays the sulfhydryl group of free coenzyme A is determined using dithiobisnitrobenzoate, endogenous SH groups interfere with the assay. This can be prevented by prior oxidation with H2O2 and subsequent decomposition of the latter by catalase /2/.

Influence factors

The concentration of total L-carnitine in erythrocytes is comparable to that in serum, but with a different degree of acylation /1229/. The concentration of L-carnitine in leukocytes is higher than that in serum and dependent on the degree of activation /31/.


Total L-carnitine can be stored for up to 1 week at 4–6 °C. Changes in the free L-carnitine and acylcarnitine fractions can occur proportionally to the duration of storage due to hydrolysis, in particular of the short-chain acylcarnitines. For long-term stability of total L-carnitine and its fractions, deep-frozen storage is required /2/.

5.3.7 Pathophysiology

Carnitine is a water-soluble amino acid derivative with a molecular weight of 161 Da, and it has an important role in fatty acid metabolism in skeletal muscle. Carnitine exists in different forms in serum, including free carnitine and acyl carnitine. Before a fatty acid (FA) liberated from triglycerides can enter the metabolic pathway it must be activated to form an acyl-CoA. This activation step is catalyzed by acyl-CoA synthetase via a two-step reaction /32/:

1) the formation of an intermediate fatty acyl-AMP with the release of pyrophosphate

FA + ATP acyl-AMP + PPi

2) the formation of a fatty acyl-CoA with the release of AMP

Acyl-AMP + CoA acyl-CoA + AMP

Activated fatty acids are unable to cross the inner mitochondrial membrane in a free form. An L-carnitine acyl transferase (CPT I) that is located at the outer face of the inner mitochondrial membrane catalyzes the formation of acyl carnitine esters and free coenzyme A from L-carnitine and acyl CoA esters. The free coenzyme is then available to the cell for further use and the carnitine ester is passed to the inner surface of the membrane by a carnitine-acylcarnitine translocase that is located within the membrane. At the inner face, a second acyl transferase (CPT II) acts on the acylcarnitine and coenzyme A within the mitochondrion, to release L-carnitine and to reform the fatty acid CoA ester, the substrate of β-oxidation. The L-carnitine, in turn, is recycled back to the outer surface of the membrane by the translocase, where it can participate in another round of the trans esterification/translocation system (Fig. 5.3-1 – Mitochondrial carnitine system/16/.

The total L-carnitine pool in adults is 15–20 g, 95% of which resides in the skeletal muscle and is replaced relatively slowly.

Most of the L-carnitine required daily comes from dietary sources. L-carnitine is absorbed in the duodenum and jejunum by a Na+-dependent active transport mechanism or by diffusion and comes from animal products (meat, milk) (Fig. 5.3-2 – The role of carnitine). Plant sources of L-carnitine are limited. The bioavailability of orally applied L-carnitine is approximately 5–20%.

The smaller proportion of the L-carnitine supply is synthesized in the body from the essential amino acids lysine and methionine (Fig. 5.3-3 – Endogenous synthesis of carnitine). In addition, vitamins C, B6 and niacin as well as Fe2+ are required. The last step in the biosynthesis, the hydroxylation of γ-butyrobetaine, occurs in the liver and kidneys.

The liver and kidneys play a key role in the homeostasis of L-carnitine metabolism. Following its intestinal absorption, L-carnitine travels to the hepatocytes via the portal vein blood and is partially esterified with fatty acids to form acylcarnitines. Free L-carnitine and the acylcarnitines are distributed to the organs via the blood circulation. In the kidneys, 95% of the filtered L-carnitine is reabsorbed. Clearance of acylcarnitine is significantly higher than that of free L-carnitine.

Fatty acids are metabolized to CO2 and H2O in all organs, except the brain and the erythrocytes. This process occurs in the mitochondria in the intermediate vicinity of the citric acid cycle and the respiratory chain. The hydrogen produced in the breakdown of fatty acids is transferred to FAD and NAD and metabolized for energy.

The metabolism of the fatty acids from triglycerides involves the following steps (Fig. 5.3-1 – Mitochondrial carnitine system):

  • Fatty acids are activated in the cytoplasm of the cells by linking of their carboxyl group with acetyl-CoA. This step is catalyzed by thiokinases (acyl-CoA synthetases).
  • For transport to the mitochondrion, the fatty acids must be converted to L-carnitine ester (acylcarnitine). This reaction is catalyzed by carnitine acyltransferases (CAT), the most important ones being the carnitine palmitoyltransferases (CPT), which catalyze the transport of long-chain fatty acids. The CoA residue is exchanged for L-carnitine by the action of CPT I on the outer mitochondrial membrane (Fig. 5.3-1 – Mitochondrial carnitine system). The fatty acid residue is transferred back to CoA by the action of CPT II on the inner mitochondrial membrane.
  • Fatty acid metabolism begins in the mitochondrial matrix, where, catalyzed by acyl-CoA dehydrogenase, the fatty acids undergo oxidization to form unsaturated compounds.

Three types of acyl-CoA synthetases, carnitine acyltransferases and acyl-CoA dehydrogenases are known:

  • With a substrate specificity for long-chain fatty acids (C14 to C20; palmitoyl)
  • With a specificity for medium-chain fatty acids (C8 and C10; octanyl)
  • and with a specificity for acetate and propionate only.

Carnitine acyltransferase (CAT, EC

The CAT resides in the mitochondria and peroxisomes and catalyzes the reversible transfer of short-chain acyl groups from coenzyme A to carnitine. In the numerous secondary L-carnitine deficiencies with elevated acylcarnitine, the CAT is responsible for removing toxic acyl groups from the mitochondria. The activity of this enzyme determines the availability of free CoA.

Carnitine octanoyltransferase (EC

This enzyme catalyzes the reversible transfer of medium-chain acyl residues (C8 and C10 with highest affinity) from coenzyme A to L-carnitine. Due to the localization in the mitochondria and peroxisomes, this enzyme accounts for the transport of medium-chain acyl residues from the peroxisomes to the mitochondria as the site of energy production (carnitine shuttle).

Carnitine palmitoyltransferase (CPT, EC

This enzyme is required for transporting the long-chain fatty acids into the mitochondria. CPT consists of two enzymes, CPT I and CPT II, which differ in their location (CPT I is located in the outer mitochondrial membrane, CPT II on the matrix side of the inner mitochondrial membrane), specific regulation, and physiological function (Fig. 5.3-1 – Mitochondrial carnitine system). The physiological inhibitor of CPT I is malonyl-CoA, the first intermediate in the synthesis of fatty acids (Fig. 5.3-4 – Regulatory role of carnitine palmitoyl transferase I (CPT I) in the liver). Malonyl-CoA regulates fatty acid oxidation by inhibiting CPT I.

The effects are as follows /33/:

  • When carbohydrates are consumed (high insulin/glucagon ratio), hepatic lipogenesis is increased, malonyl-CoA increases, CPT I is inhibited, and newly formed long-chain acyl-CoA fatty acids are converted to triglycerides instead of being oxidized. The triglycerides are released from the liver as very-low-density lipoproteins (VLDL) and stored in adipose tissue.
  • In the fasting state (low insulin/glucagon ratio), the concentration of malonyl-CoA is low due to the low substrate flow through glycolysis, CPT I is active, and the free fatty acids supplied by adipose tissue undergo β-oxidation, producing ketone bodies in the process.

Carnitine-acylcarnitine translocase

The translocase is located in the inner mitochondrial membrane and catalyzes the transport of acylcarnitines of any chain length and L-carnitine across the mitochondrial membrane (both directions) in stoichiometric exchange for the relevant substance according to concentration gradient. The long-chain acylcarnitines are transported into the mitochondria for β-oxidation in exchange for mitochondrial L-carnitine (Fig. 5.3-1 – Mitochondrial carnitine system).


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5.4 Uric acid

Lothar Thomas

Uric acid and its ionized form, monosodium urate, are the end products of purine metabolism in humans. The size of the body’s uric acid pool is the result of production and elimination. Hyper- and hypouricemia are not diseases. While hyperuricemia is primarily a symptom of gout, it also occurs secondarily in association with risk factors of cardiovascular disease and in association with metabolic syndrome and conditions with increased cell turnover. Hypouricemia is of limited pathological significance. Urinary uric acid excretion can be a useful marker in the workup of hyper- and hypouricemia.

5.4.1 Indication

Serum, plasma

  • Together with physical examination in internistic medical check-up
  • Family history of gout or history of renal calculi
  • Clinical symptoms indicative of an acute gout attack
  • Monitoring of gout therapy
  • In patients with hypertension, hyperlipidemia, overweight, prediabetes, diabetes mellitus, chronic kidney disease
  • Cardiovascular disease and stroke
  • Suspected gestosis
  • Diseases, conditions and therapies possibly causing secondary hyperuricemia (e.g., polycythemia vera, starvation diets, alcohol consumption, cytostatic therapy and tumor radiotherapy, cyclosporine therapy in transplant recipients)
  • In children with hypo ketonemic hypoglycemia.


Detection of increased endogenous uric acid synthesis:

  • Gout in childhood and adolescence
  • Nephrolithiasis associated with the formation of uric acid- or calcium-containing kidney stones with normal or borderline serum uric acid levels
  • Conditions associated with hypouricemia.

5.4.2 Method of determination

Methods for the determination of uric acid in biological fluids rely on the use of the enzyme uricase /1/.

Kinetic UV-method /2/

Principle: uric acid is degraded by uricase to allantoin and H2O2. The resulting H2O2 is quantitated by use of catalase and aldehyde dehydrogenase (ADH). The increase in NADPH concentration, measured by change in absorbance at 340 or Hg 334 nm is proportional to the amount of uric acid (Tab. 5.4-1 – Kinetic UV-method for the determination of uric acid).

Colorimetric method /3/

In the first step uric acid is oxidized by oxygen and uricase form allantoin, carbon dioxide and H2O2. For routine diagnostic purposes most assays involve a peroxidase or catalase system coupled with an oxigen acceptor to produce a chromogen.

  • Trinder’s reaction: in the presence of peroxidase, H2O2 reacts with a chromogen system consisting of phenol and 4-aminophenazon to form a red chinonimine whose absorption is followed at approximately 500 nm. There are a number of modifications for this method which all involve the phenol component. Either chlorinated phenols or chlorinated benzene sulfonic acids are used /4/.
  • Kageyama’s reaction: catalase, in the presence of H2O2, converts methanol to formaldehyde. The latter reacts with acetyl acetone in the presence of ammonium ions, forming a yellow dye whose absorbance is measured at 410 nm /5/.

5.4.3 Specimen

  • Serum, plasma (no EDTA, citrate, oxalate): 1 mL
  • Urine: 24 h-collection without additive
  • Uric acid/creatinine ratio: spontaneously voided urine sample without additives.

5.4.4 Reference interval

Refer to Tab. 5.4-2 – Uric acid reference intervals.

5.4.5 Clinical significance

By definition, hyperuricemia is a condition when the plasma uric acid concentration exceeds the solubility limit of monosodium urate at 37 °C of 6.8 mg/dL (400 μmol/L). At higher concentrations the plasma is supersaturated, leading to the precipitation of monosodium urate under certain physical conditions. Hypouricemia is defined as a uric acid level ≤ 2 mg/dL (119 μmol/L).

In the period around the 1920s, uric acid levels in the population of industrial nations were below 3.5 mg/dL (210 μmol/L), increasing continuously up to 2-fold over the next 50 years. Levels in women are about 0.5 to 1 mg/dL (30–60 μmol/L) lower than those in men due to the uricosuric effect of estrogens. After menopause, uric acid levels in women increase and eventually approximate those in men (Fig. 5.4-1 – Serum uric acid concentration as a function of age, sex, and race). The intraindividual and inter individual variation in serum uric acid is multifactorial and influenced by genetic and environmental factors. Approximately two-thirds of the uric acid produced daily is excreted via the kidneys, the remainder via the intestine in stool. Hyperuricemia

Hyperuricemia has a high prevalence in the population. The Framingham study found that 9.2% of men and 0.4% of women had hyperuricemia, and 19% of those with hyperuricemia had gout /10/. A German study /6/ found that 2.6% of the female blood donors and 28.6% of the male donors were hyperuricemic. In women hyperuricemia often does not occur until after menopause.

Hyperuricemia is associated with other metabolic disorders, such as insulin resistance, diabetes mellitus, metabolic syndrome, obesity, hyperlipoproteinemia, excessive alcohol consumption and diseases such as hypertension and chronic renal insufficiency, and is an independent risk factor for cardiovascular disease /1112/.

Hyperuricemia is the result of (Tab. 5.4-3 – Causes of hyperuricemia) /1314/:

  • Reduced renal uric acid excretion due to genetic defects (URAT1 transporter) or multiple causes (congestive heart failure, volume depletion, diuretics) that lead to increased renal tubular reabsorption of uric acid. 90% of individuals with gout-related hyperuricemia have reduced uric acid excretion. The extent of hyperuricemia is also influenced by environmental factors.
  • Overproduction of uric acid due to a diet high in meat and fish, or excessive alcohol consumption.

The differentiation between uric acid overproduction and reduced renal elimination is evaluated by determining the uric acid excretion in a urine sample collected over a period of 24 h or calculating the uric acid/creatinine ratio in a spontaneously voided urine sample.

Hyperuricemia is classified into /14/:

  • Primary forms. These are most commonly caused by genetic variations of the urate transport molecules in the renal tubules which, in combination with a protein-rich diet, lead to increased renal reabsorption of uric acid (underexcretors). These patients require a 1–2 mg/dL (59–119 μmol/L) higher plasma uric acid concentration compared to healthy individuals to excrete the same amount of uric acid. In rare cases, the condition may be caused by hereditary enzyme defects which lead to increased uric acid production (over producers). Both inherited disorders often lead to gout, which is then classified as primary.
  • Secondary forms. These include all cases in which hyperuricemia or gout is due to diseases that do not primarily affect purine metabolism. Causes include increased production of uric acid from exogenous or endogenous purines, reduced renal excretion of uric acid, or physiologically unspecific causes (Tab. 5.4-4 – Classification of hyperuricemia/13/. From hyperuricemia to gout

At a pH of 7.4, approximately 90% of uric acid exists in the form of monosodium urate. Concentrations of 8 mg/dL (476 μmol/L) and higher can cause the urate to precipitate in the tissues. Gout occurs in 0.5–7% of men and 0.1% of women. There is no clear correlation between the serum uric acid concentration and the precipitation of a gout attack. According to one study /15/, the annual incidence of gout is 0.5% for uric acid levels of 7.0–8.9 mg/dL (416–529 μmol/L) and 4.9% for levels above 9.0 mg/dL (535 μmol/L), with gout attacks usually only occurring after 20–40 years of persistent hyperuricemia. In patients with a congenital enzyme defect, the first gout attack usually occurs as early as in adolescence. Laboratory diagnosis of hyperuricemia and gout

Hyperuricemia is diagnosed if uric acid levels in morning fasting serum are elevated two to three times on different days to concentrations above 6.0 mg/dL (357 μmol/L) in women and above 7.0 mg/dL (416 μmol/L) in men. To allow a general assessment, patients should remain on their usual diet, medication and alcohol intake as in the weeks/months prior to the test /14/. For influences of drugs refer to Tab. 5.4-5 – Influence of drugs on the serum uric acid concentration.

Measurement of uric acid in urine

Once gout has been diagnosed, its possible causes must be investigated. The determination of urinary uric acid allows to answer the following questions:

  • If hyperuricemia is present, is it of endogenous origin i.e., caused by disease, or of exogenous origin i.e., caused by diet?
  • If nephrolithiasis is present, is the patient an over producer or underexcretor?
  • If uric acid levels are borderline or elevated, is renal function impaired?

Uric acid excretion in the 24 h-urine sample and/or the uric acid/creatine ratio in spot urine allow the following diagnostic conclusions:

  • A uric acid excretion up to 600 mg (3.57 mmol) on a low-purine diet and up to 800 mg (4.76 mmol) on a normal diet in hyperuricemic patients indicate that the hyperuricemia is due to impaired tubular uric acid secretion and is therefore primary. In these underexcretors, the fractional excretion of uric acid (FEUA) is less than 4.0% /16/.
  • An increased FEUA or uric acid/creatinine ratio is indicative of uric acid overproduction, regardless of whether serum uric acid is elevated. Normalization of uric acid excretion on a low-purine diet indicates that the uric acid is synthesized from exogenous purines. If this is not the case, then the uric acid is mainly a breakdown product of endogenous purines.

The following should be taken into account when interpreting uric acid excretion:

  • Is the glomerular filtration rate (GFR) reduced? If this is the case, overproduction may be missed in the presence of normal uric acid excretion.
  • In patients with impaired GFR, elevated uric acid excretion confirms overproduction of uric acid
  • The uric acid/creatinine ratio in spot urine which, if above 0.80, is used an indicator for uric acid overproduction, correlates only moderately with uric acid excretion in 24 h-urine due to considerable diurnal fluctuation in uric acid excretion
  • A free uric acid clearance rate above 10% indicates increased excretion, a rate below 4% indicates decreased excretion (see calculation in Tab. 5.4-6 – Calculation of fractional uric acid clearance.
  • Has the patient been taking any drugs that can influence the production and elimination of uric acid (Tab. 5.4-5 – Influence of drugs on the serum uric acid concentration)? Clinical manifestations of hyperuricemia

Although hyperuricemia has a high prevalence, most people affected by it do not develop any clinical symptoms. However, the incidence of complications correlates with the level of serum uric acid (Tab. 5.4-7 – Frequency of gout as a function of the serum uric acid concentration) and the level of uric acid excretion.

Clinical manifestations of hyperuricemia include /17/:

  • Acute gouty arthritis
  • Inter critical gout
  • Tophaceous, often primary chronic gout, accompanied by soft tissue and bone tophi
  • Renal involvement in gout (e.g., nephrolithiasis, interstitial urate nephropathy (gouty kidney) with hypertension, and obstructive uric acid nephropathy
  • Association with hypertension, metabolic syndrome and cardiovascular disease.

Acute gouty arthritis

Acute gouty arthritis occurs as an unprovoked acute attack after 20–40 years of hyperuricemia or can be due to one of the factors causing increased uric acid that are listed in Tab. 5.4-4 – Classification of hyperuricemias.

Acute gouty arthritis clinically presents as severely painful mono arthritis, predominantly of the first metatarsal phalangeal joint (podagra). The pain often lasts for a week and is self-limiting. Uric acid levels can be normal during an acute attack and are therefore best measured 2–3 weeks after the attack. Since uricemias are often detected early during a checkup, timely medically prescribed treatment has led to a decline in the incidence of acute gouty arthritis.

Inter critical gout

This is the condition that occurs after the acute gout attack has resolved and the patient has become asymptomatic. If no prophylactic therapy is initiated, the intervals between attacks can become shorter and the attacks longer. More joints can become involved in the disease and tophaceous gout can develop. Clinically, inter critical gout rarely presents with tophi, but radiographic examinations often reveal bone destruction.

Tophaceous gout

Tophi are nodular masses of monosodium urate crystals which deposit in bones, usually near joints, in cartilage, bursae and synovial tendon sheaths. This form also occurs as primary chronic gout i.e., without previous acute gouty arthritis. It is associated with older age of onset, manifests as polyarticular gout, especially in the lower joints, and commonly also affects women /18/.


About 30–40% of patients with an acute gout attack have a history of renal calculi. Approximately 40% of patients with myeloproliferative diseases also develop renal calculi. However, not all kidney stones in hyperuricemic patients are urate calculi. In gout patients, the prevalence of nephrolithiasis correlates with the level of serum uric acid and urinary uric acid excretion (Tab. 5.4-8 – Frequency of nephrolithiasis as a function of serum uric acid level and uric acid clearance). Approximately 85% of renal calculi in hyperuricemic patients contain uric acid.

Urate calculi form when urine becomes oversaturated with undissociated uric acid. Although hyperuricosuria may be present, the main causes of the formation of urate calculi are low urine pH and low urine volume, not hyperuricosuria /19/. About 90% of patients with urate calculi have a first morning urine pH below 5.7, and many even have an average pH of 5.5 /20/. All patients with gouty diathesis have an increased filtered uric acid load and acidic urine, but only 28% develop urate calculi on a low-purine diet /21/. Besides gout, there are other causes associated with the formation of acidic urine, such as physical exercise and dehydration, which lead to the formation of urate calculi. In patients with an ileostomy or Crohn’s disease, dehydration is thought to be the main factor /19/.

Urate nephropathy /22/

This nephropathy, which is also known as gouty kidney, is a manifestation of chronic gout. Here, precipitation of mono urate crystals in the medullary interstitium and renal pyramids has led to inflammatory changes. Urate nephropathy is associated with a limited glomerular filtration rate, proteinuria and hypertension.

Acute uric acid nephropathy /23/

Acute uric acid nephropathy is acute post renal failure caused by the precipitation of urate crystals in the tubules and collecting ducts due to acute severe overproduction of uric acid. It is promoted by dehydration and acidosis. Findings include hyperuricemia above 12 mg/dL (714 μmol/L) and a random spot urine uric acid/creatinine ratio above 1.0. In other forms of acute renal failure the ratio is below 1.0.

Acute uric acid nephropathy occurs in blast crises in leukemias before or during cytostatic therapy. It can also occur at the beginning of uricosuric therapy, if therapy concepts such as sufficient fluid intake, neutralization of urine, and up titration of uricosuric medication are disregarded. Treatment of hyperuricemia

The treatment goals in patients with hyperuricemia and gout are as follows /1324/:

  • Resolve the acute gout attack. Most patients with acute attacks, in whom a diagnosis of crystal arthropathy was confirmed by joint puncture, are treated with non-steroidal anti-inflammatory drugs (NSAID), which have fewer side effects and a longer-lasting effect than colchicine. Alternatives are corticosteroids or ACTH. While all these drugs help eliminate the acute pain, they do not correct the cause of the hyperuricemia nor the deposition of urate crystals in the tissues. Colchicine is the drug of choice in patients in whom the diagnosis of crystal arthropathy is not confirmed.
  • Prevent further gout attacks and reverse the complications of gout. The likelihood of a recurrent gout attack within a year is 78%, and within 5 years 89%. During this period, the gouty tophi continuously become larger, trigger a destructive inflammatory response in the tissues and lead to the destruction of cartilage and bones. The body’s uric acid pool increases continuously. To correct these complications, the uric acid pool must be normalized again. This requires serum uric acid levels to be reduced to below 6.8 mg/dL (404 μmol/L), or better, to below 5.0 mg/dL (297 μmol/L). Reducing the concentration only to 8 mg/dL (476 μmol/L), for example, is insufficient, because although the rate at which gouty tophi develop is slowed down, there is no reduction in the uric acid pool. Therapy with specific uric acid-lowering drugs containing xanthine oxidase inhibitors, such as allopurinol, begins 2–3 weeks after the acute gout attack. Patients treated with uricosuric drugs, such as sulfinpyrazone and probenecid, are typically under 60 years of age, have a creatinine clearance above 80 [ml × min.–1 × (1.73 m2)–1], uric acid excretion below 800 mg (4.76 mmol)/24 h, are on a specific diet (avoidance of offal, seafood, and fructose-containing drinks) and have no renal calculi.
  • Eliminate associated conditions that contribute to the hyperuricemia and gout. These include criteria of the metabolic syndrome, such as obesity, hypertension, hypertriglyceridemia, and insulin resistance. In addition, excessive alcohol consumption must be avoided.

Tab. 5.4-9 – Diseases and conditions associated with hyperuricemia lists diseases associated with hyperuricemia, and Tab. 5.4-10 – Association of hyperuricemia with other diseases associates hyperuricemia with other diseases. Hypouricemia

Hypouricemia is defined as a condition with serum uric acid concentration ≤ 2 mg/dL (119 μmol/L). It has a prevalence of 0.2–0.5% in outpatients and of about 1% in clinical patients. Hypouricemias usually occur without clinical symptoms and are coincidental findings /26/. Hypouricemias can be caused by:

  • Reduced uric acid formation. This form of metabolic hypouricemia is found in hereditary xanthinuria, hereditary purine nucleoside phosphorylase deficiency, and allopurinol therapy.
  • Increased renal uric acid excretion. Causes include uricosuric drugs, syndrome of inappropriate secretion of antidiuretic hormone (SIADH), Fanconi syndrome, malignant diseases, AIDS, severe liver injury, severe burns, diabetes mellitus, and hyper eosinophilic syndrome.
  • A combination of metabolic and renal hypouricemia.

Diseases associated with hypouricemia are listed in Tab. 5.4-11 – Diseases and conditions associated with hypouricemia.

In most cases, hypouricemia is caused by drugs that interfere with renal tubular uric acid transport /27/. These include acetohexamide, allopurinol, azathioprine, bishydroxycoumarin, clofibrate, contrast agent, fenofibrate, fenoprofen, guaifenesin, halogenates, losartan, phenylbutazone, probenecid, salicylates, and tienilic acid. Once these drugs are discontinued, uric acid levels generally normalize within 14 days.

5.4.6 Comments and problems

Blood sampling

Blood should preferably be collected in the morning and after fasting, not after intense physical work or after excessive exposure to the sun, which can lead to considerable increases. A normal continental breakfast does not significantly increase uric acid levels. Uric acid is not subject to circadian variation, but undergoes daily fluctuations.

Anticoagulants and stabilizers

EDTA, citrate, oxalate, sodium fluoride, cyanide, formaldehyde and oxonic acid cause reduced levels by inhibiting uricase.

Method of determination

Methods that measure the H2O2 produced in a coupled reaction, may generally experience interference from light scattering due to turbidity of the sample, spectral interference by bilirubin and hemoglobin, or interference by reducing substances such as ascorbic acid or phenol-type substances that are similar to those of the O2 acceptor /1/. Trinder’s reaction produces falsely low readings in the presence of calcium dobelisate and α-methyldopa. Homogentisic acid at levels above 50 mg/L leads to elevated values in the aldehyde dehydrogenase method and causes pseudo hypouricosuria in the uricase-peroxidase reaction /48/.

Increased excretion of homogentisic acid occurs in alkaptonuria, a hereditary disease caused by a deficiency of the enzyme homogentisic acid oxidase (EC, which results in insufficient conversion of homogentisic acid to 4-maleylacetoacetate.

Reference interval

In men, uric acid levels reach a plateau at 20–24 years of age and subsequently remain constant through life provided that weight remains the same. In women, levels increase between the ages of 15 to 19 years, then plateau until menopause before rising again /49/.

The lower uric acid concentration in women compared to men is due to higher uric acid clearance. A metabolic balance study of 19- to 32-year-old individuals on an isoenergetic formula diet showed uric acid levels of 3.0 ± 0.5 mg/dL (178 ± 30 μmol/L) for women and 4.1 ± 0.7 mg/dL (244 ± 42 μmol/L) for men /49/. Women who use oral contraceptives have lower uric acid levels than women of the same age group who do not /6/. Caucasians have higher uric acid levels than blacks /50/.


IgM-monoclonal gammopathies can interfere with the measurement by precipitation of immunoglobulins in H2O2-coupled reactions /51/.

To measure uric acid in urine, refrigerated samples must be warmed up for 60 min. at 37 °C or for 10 min. at 60 °C. Otherwise readings will be falsely low by 20%, since part of the precipitated sodium mono urate does not dissolve /52/.


Approximately 3 days at room temperature.

5.4.7 Pathophysiology

The total amount of uric acid (C5H4N4O3) present in the body is referred to as uric acid pool and is approximately 1 g. Although purines are synthesized and metabolized in all tissues, uric acid synthesis only occurs in tissues that contain xanthine oxidase, mainly the liver and the small intestine (Fig. 5.4-2 – Important enzymatic steps in the regulation of purine metabolism).

The uric acid pool is derived from endogenously synthesized uric acid (approximately 350 mg/day) and dietary purines (more than 300 mg/day) /44/.

Approximately 80% of uric acid is eliminated through the kidneys and less than 20% through the intestine. The physiological renal excretion is up to 800 mg (4.76 mmol) per day. Uric acid reaches the large intestine through saliva, bile, gastric and pancreatic juices and is broken down into CO2 and NH3.

At pH 7.4, most uric acid circulates in plasma in an ionized form as monosodium urate and mono- potassium urate, only a small amount of it is present in the form of free acid. The solubility product of the salts is 8.4 mg/dL (500 μmol/L) and that of the free acid 6.8 mg/dL (400 μmol/L). Precipitation of urates, in particular in the less perfused tissues, occurs as a result of an increase in uric acid, cooling, or a pH shift towards acidosis.

The presence of hyperuricemia is the indicator of an increased uric acid pool, which can result from uric acid overproduction, reduced excretion of uric acid, or a combination of both. The uric acid pool can be elevated up to 30 g /11/.

In 99% of cases, primary hyperuricemia is due to selective impairment of renal uric acid elimination. In healthy individuals, uric acid clearance is 8–10 mL/min., with approximately 6–12% of the filtered uric acid appearing in urine. The filtered uric acid is almost completely reabsorbed in the proximal tubule, subsequently secreted in the distal tubule, and partly reabsorbed. It is believed that, in hyperuricemia, tubular secretion of uric acid is impaired.

In less than 1% of cases, primary hyperuricemia is due to endogenous overproduction of uric acid. Possible causes include deficiencies of enzymes in uric acid metabolism or increased activity of enzymes (e.g., PRPP synthetase) (Fig. 5.4-2 – Important enzymatic steps in the regulation of purine metabolism).

Lesch-Nyhan syndrome, an X-chromosomal disorder with a recessive inheritance pattern, is caused by deficient activity of hypoxanthine-guanine phospho-ribosyl transferase (HGPRT). This enzyme is responsible for the resynthesis of adenosine mono phosphate (AMP) and guanosine mono phosphate (GMP) from purine bases released in the endogenous degradation of DNA and RNA. HGPRT deficiency is associated with a reduced intracellular concentration of AMP and GMP. As a result, adenosine phospho ribosyl transferase (APRT), the enzyme which catalyzes the de novo synthesis of nucleotides, is not feedback-inhibited by the two nucleotides, thus promoting the de novo synthesis of purines from 5-phospho ribosyl-1-pyrophosphate. As a result, the de novo synthesis can be increased up to 20-fold, which leads to a significant increase in the pool of uric acid and to a 3–4 fold higher excretion of uric acid /47/.

Acute urate nephropathy, as seen in tumor lysis syndrome, is caused by urate crystal-dependent and urate crystal-independent mechanisms /53/:

  • The result of the urate crystal-dependent mechanism is tubular obstruction by uric acid precipitation. Uric acid has a pKa of 5.75 and is thus a weak acid. At pH 5.0, urine is saturated with uric acid at a concentration as low as 15 mg/dL (892 μmol/L), while at pH 7.0 saturation is reached at a concentration of 200 mg/dL (11.9 mmol/L). In the presence of acidic urine, uric acid can precipitate in the distal tubule and the collecting duct, causing tubular obstruction. Granulocytes migrate into the tissue and interstitial nephritis develops as a late effect of sterile inflammation. Depending on the interstitial uric acid concentration, which mirrors the serum concentration, needle-shaped monosodium urate crystals are deposited in the interstitium, resulting in the formation of urate micro tophi.
  • The urate crystal-independent mechanisms are activated by acute changes in the autoregulation of renal blood flow. Reduced renal perfusion leads to tissue hypoxia, and the subsequent re perfusion injury triggers an inflammatory response. The damaged cells release cytokines and chemokines, and adhesion molecules are expressed, causing granulocytes and monocytes to accumulate in the peri tubular capillaries. This further reduces renal perfusion, leading to vascular and tubular injuries and the development of renal insufficiency.

Increased intake of dietary purines is not a significant factor in enzyme deficiency-induced hyperuricemia, but it does promote the manifestation of gout.

Secondary hyperuricemia is the result of purine overload. They can be due to endogenous or exogenous causes.

It is not always possible to clearly differentiate between primary and secondary hyperuricemia, because often an hereditary disposition to gout becomes manifest only through other influences (e.g., high intake of purines, or in the course of other underlying diseases).

Increasing nutritional purine intake leads to an increase in serum uric acid levels, where the magnitude of the increase depends on the type of purines consumed. AMP and GMP lead to a greater increase in uric acid when consumed as nucleotides than when consumed in the form of RNA and DNA. Due to its higher susceptibility to hydrolysis, RNA leads to a two-fold increase in uric acid compared to the same intake of DNA. Approximately 60% of RNA compared to only 30% of DNA is reabsorbed by the intestine.

Administration of sugars, such as fructose, sorbitol and xylitol, leads to an increase in serum uric acid levels. This is thought to be caused by an increase in the de novo synthesis of purine and increased formation of uric acid from preformed purines.

Fasting causes a rapid increase in uric acid as a result of increased endogenous production of purines due to the metabolism of endogenous substances on the one hand and reduced renal uric acid secretion due to acidosis on the other hand.

All conditions associated with acidosis lead to reduced renal uric acid excretion.

Urate has antioxidant properties in vivo and inhibits the destructive effect of reactive oxygen species (ROS) on proteins, lipids and deoxyribonucleic acids. An increased uric acid concentration is also thought to have an oxidative effect under certain conditions.

The enzyme xanthine oxidase, which catalyzes the conversion of the purines xanthine and hypoxanthine to uric acid, has two isoforms:

  • The dehydrogenase form, which forms uric acid and NADH2
  • The oxidase form, which generates uric acid and H2O2.

In the setting of ischemia and tissue hypoxia there is increased conversion of the dehydrogenase form to the oxidase form, promoting production of ROS /54/.

Uric acid excretion requires special transporters, which are located in the proximal tubular cells of the kidneys, in the intestinal enterocytes and the vascular smooth muscle cells. Their role in the homeostasis of uric acid is not yet well understood /55/.


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5.5 Ketone bodies

Lothar Thomas

Acetoacetate (AcAc), β-hydroxy butyrate (β-HB) and acetone are ketone bodies. They are produced by ketogenesis, a mitochondrial process in which acetyl-CoA from the β-oxidation of fatty acids is converted to AcAc. A small amount of AcAc is converted to acetone by spontaneous decarboxylation. The greater portion is converted to β-HB by the enzyme β-hydroxy butyrate dehydrogenase (β-HBD) (Fig. 5.5-1 – Conversion of acetoacetate to β-hydroxy- butyrate and acetone). The ratio of ketone bodies in the blood depends on the redox state of the cells. In healthy individuals on a normal diet, AcAc and β-HB are present in approximately equimolar amounts. Acetone accounts for less than 5%.

Ketone bodies can be measured in blood and urine. In individuals on a normal diet, the capacity of the tissues to oxidize ketone bodies is sufficient to metabolize the amounts released by the liver. This is not the case in conditions of insufficient carbohydrate intake, fasting, and poorly controlled diabetes mellitus.

5.5.1 Indication

Differentiation of hyperglycemias with and without metabolic acidosis from other acidoses with a high anion gap such as:

  • Diabetic ketoacidosis (diabetic coma)
  • Hyperglycemic hyper osmolar non-ketotic syndrome
  • Alcoholic ketoacidosis
  • In combination with lactate measurement, differentiation from lactic acidosis and acidoses associated with sepsis, shock, intoxication (CO, cyanide, salicylates), severe hypoxemias, convulsions, malignancies (lymphomas)
  • Intoxication with ethyl substitutes (glycols, methanol)
  • Uremia.

In combination with glucose and lactate, as a screening test for suspected congenital metabolic disorder in neonates and infants.

5.5.2 Method of determination

Although all three ketone bodies can be quantified in serum and urine, the following methods have become established for practical reasons:

  • Quantitative measurement of β-HB in serum
  • Combined measurement of β-HB and AcAc, also sequentially to differentiate between the two
  • Rapid assays for determining ketone bodies in urine, which can also be used in diluted serum.

Quantitative determination of β-hydroxy butyrate (β-HB)

Principle: initially, the serum sample is deproteinized thus avoiding the spontaneous decarboxylation of AcAc into acetone. β-HB is determined by its quantitative conversion into AcAc and NADH catalyzed by the enzyme β-HBD. AcAc can also be determined according this principle. Since the reaction equilibrium is on the side of the β-HB, the AcAc is bound to hydrazone (e.g., by using hydrazine) thus removing it from the reaction. The increase of NADH2 is measured spectrophotometrically /1/.

β-HB + NAD β-HBD AcAc + NADH + H +

In a commercial card test, diaphorase catalyzes the reduction of nitro blue tetrazolium (NBT) by NADH2 to form a blue product whose absorbance is measured.

Quantitative measurement of total ketone bodies

Principle: β-HB and AcAc are determined as total ketone bodies in a recycling reaction. The serum/plasma sample is incubated with thio-NAD and β-HBD. AcAc is converted to β-HB and NADH2 to NAD (Fig. 5.5-2 – Principle of total ketone body measurement using a recycling method). Then β-HB is converted to AcAc again and thio-NADH is formed from thio-NAD. The increase in thio-NADH is measured spectrophotometrically and depends on the concentration of total ketone bodies in the sample /2/. Separate measurement of β-HB is also possible. For this, AcAc is converted to acetone in a prior reaction.

Separate measurement of β-hydroxy butyrate and acetoacetate

Principle: AcAc and β-HB are measured in separate assay samples in a reaction catalyzed by β-HBDH. In the first assay sample, AcAc is converted to β-HB and the total amount of ketone bodies is determined, while in a second sample only β-HB is measured. The concentration of AcAc is determined from the difference in absorbance in assay sample 1–2. In an indicator reaction catalyzed by NADH oxidase, the resulting NADH is converted to H2O2. The latter oxidizes a chromogen in a reaction catalyzed by peroxidase, and the absorbance of this chromogen is measured photometrically /3/.

Semi quantitative determination of ketone bodies in urine or serum (rapid assay)

Principle: in an alkaline environment AcAc and acetone react with sodium nitroprusside and glycine producing a purple-colored complex. Only AcAc and acetone are measured in the reaction, but not β-HB. The analytical sensitivity for AcAc is 50 mg/L and for acetone 500 mg/L.

Test strips allow a distinction to be made between several ketone body concentration ranges. The test may also be used for semi quantitative measurements in serum and plasma. The sample should be diluted with physiological saline solution at a ratio of 1 : 2 /4/.

5.5.3 Specimen

  • Whole blood: 0.1–1 mL
  • Serum, plasma (if only β-HB is measured): 1 mL

5.5.4 Reference interval

Refer to Tab. 5.5-1 – Reference intervals of ketone bodies.

5.5.5 Clinical significance

Ketone bodies and the determination of the anion gap allow the differentiation between different types of metabolic acidoses (e.g., those with a normal and those with an increased anion gap) /6/.

Calculation of the anion gap

  • Anion gap (mmol/L) = [Na+] – ([Cl] + [HCO3])
  • Reference interval 8–16 mmol/L.

Normal anion gap metabolic acidoses

These acidoses are associated with hyperchloremia and can be caused by systemic infections, renal tubular acidosis, medication of carbo anhydrase inhibitors, and hyperkalemic acidosis. Increased anion gap metabolic acidosis

These types of acidoses are associated with normochloremia or occasionally with hypochloremia and may result from:

  • Ketoacidosis; AcAc and β-HB are elevated (e.g., in diabetic and alcoholic ketoacidosis and congenital metabolic disorders such as organic acidurias)
  • Lactic acidosis; lactate is elevated (e.g., in impaired tissue perfusion and congenital lactic acidoses) (see Section 5.6 – Lactate)
  • Uremia; reduced excretion of acids such as phosphates and sulfates
  • Rhabdomyolysis; increased release of sulfur-containing amino acids
  • Intoxication with salicylates and ethyl substitutes such as ethylene glycol, formaldehyde, toluol, methanol. Ketoacidosis

At physiologic pH, the ketone bodies β-HB and AcAc circulate in plasma as anions. The H+-ions of the ketone bodies are buffered by HCO3, and reduce the HCO3concentration resulting in metabolic acidosis. Since HCO3 as a measured anion is replaced by the not-measured keto anions, the anion gap increases. The retention of keto anions, which results in an increase in anion gap is quantitatively similar to the decrease in plasma HCO3-concentration. The fractional reabsorption of AcAc and β-HB in the kidney is only 75–85%. Therefore, ketonuria occurs during periods of enhanced keto acid production because of the difference between the quantity of keto anions filtered compared with the amount reabsorbed. The absolute quantity of glucose and keto anions in diabetic ketoacidosis excreted in the urine is directly related to the glomerular filtration rate /7/.

In patients with intact kidney function, there is a quantitative relationship between ketonemia and ketonuria. In the presence of ketonemia (β-HB and AcAc), urine assays show a positive 1+ positive result for levels ≥ 8 mg/dL (0.8 mmol/L), and a 3 + positive result for levels ≥ 13 mg/dL (1.3 mmol/L) /8/. Using rapid tests for detecting ketosis can be problematic. The tests only respond to AcAc and acetone, but not to β-HB. The ratio of ketone bodies in the blood depends on the redox state of the cell. In severe ketoacidosis, for example, the ratio of β-HB to AcAc is shifted towards β-HB (e.g., 6 : 1) due to an enormous excess of NADH.

The concentration of AcAc in urine may be only just above the detection limit, even though the patient has severe clinical symptoms. Under treatment, clinical symptoms improve, the formation of excess NADH is reduced, and less AcAc is converted to β-HB, leading to increased AcAc in urine. This results in the paradoxical situation that the improvement of clinical symptoms is accompanied by apparently worsened ketonuria. Therefore, only the quantitative measurement of serum β-HB, or better β-HB and AcAc, reflects the progress of ketosis.

The main ketoacidoses are: Diabetic ketoacidosis (DKA)

DKA is a metabolic derangement consisting of three concurrent abnormalities: high blood glucose, high ketone bodies, and metabolic acidosis /79/. Approximately 25–40% of children with type 1 diabetes present with DKA at initial manifestation, while in adult diabetics DKA develops as a result of a comorbidity due to poor compliance or incorrect insulin administration (insulin pump). Another type of diabetes associated with the production of ketone bodies besides type 1 is ketosis prone diabetes, which is an intermediate form between type 1 and type 2. About half of all type 1 patients have no autoantibodies at initial manifestation, but a lack of β-cell reserve and ketosis /10/.

DKA is differentiated from hyperglycemic hyper osmolar non-ketotic syndrome (HHNS). The hallmark of HHNS is severe hyperglycemia, elevated serum osmolality, and extensive dehydration with absence of ketoacidosis. Approximately 20% of patients with DKA and HHNS are admitted to hospital in a comatose state /10/.

Both HHNS and DKA are characterized by insulinopenia and clinically they differ in:

  • Age of onset (DKA in adolescent type 1 diabetics, HHNS in older type 2 diabetics)
  • Extent of dehydration (mild to moderate in DKA, severe in HHNS)
  • Severity of ketosis (moderate to severe in DKA, lack of ketogenesis in HHNS).

Laboratory findings differentiating DKA and HHNS are listed in Tab. 5.5-4 – Differential diagnosis of diabetic coma, HHNS, and acidoses. Alcoholic ketoacidosis (AKA)

The main finding in AKA is metabolic acidosis with an increased anion gap, which is generally accompanied by a compensatory respiratory alkalosis with low PCO2 /12/. Acidosis in glycogen storage disease

In glycogen storage diseases, in particular type I glycogenosis (von Gierke disease), there is reduced hepatic synthesis of glucose, leading to hypoglycemia, lactic acidosis and ketonemia even after short periods of fasting. Hypoglycemia and ketonemia occur rarely in type IV and occasionally in type VI, in which they are milder in nature /13/.

5.5.6 Comments and problems

Detection limit of rapid assays

Rapid urine assays are based on the sodium nitroprusside method. The detection limit is 50 mg/L for AcAc and 500 mg/L for acetone; β-HB is not detected.


AcAc is unstable and quickly decarboxylated to β-HB and acetone. At a storage temperature of –20 °C the concentration decreases by about 40% and at a temperature of –80 °C it decreases by 15% within 40 days /21/. Therefore, the serum proteins must be precipitated with perchloric acid immediately after collection if AcAc is to be measured. β-HB is stable for up to 4 h at 4 °C in whole blood and for up to 48 h in serum and plasma /23/.

5.5.7 Pathophysiology


Ketogenesis is a mitochondrial process that involves the following reactions (Fig. 5.5-3 – Pathophysiology of diabetic ketoacidosis):

  • Two acetyl-CoA molecules generated from the β-oxidation of fatty acids are condensed by the enzyme acetoacetyl-CoA thiolase to form the intermediate acetoacetyl-CoA
  • A third acetyl-CoA molecule is subsequently condensed by HMG-CoA synthase to form the key intermediate β-hydroxy-β-methylglutary-CoA (HMG-CoA)
  • HMG-CoA is then split into acetoacetate and acetyl-CoA by the enzyme HMG-CoA lyase
  • Part of the acetoacetate is reduced to β-HB by β-HBD in the presence of NADH. The ratio of AcAc to β-HB depends on the intramitochondrial NADH/NAD ratio.

Ketosis develops as a result of excess production of acetyl-CoA. For example, β-oxidation of 1 mol of C16 fatty acid is converted to 8 moles of acetyl-CoA. This is enzymatically condensed with oxaloacetate derived from carbohydrate metabolism, to give citrate, which is the major component of the tricarboxylic acid cycle. An increase in the glucagon/insulin ratio increases fatty acid oxidation and thus the formation of ketone bodies. The increased formation of ketone bodies results from a reduction in the:

  • Availability of carbohydrates (e.g., due to fasting, frequent vomiting, alcoholism, glycogen storage disease)
  • Reduced glucose uptake in the cells due to insulin deficiency (e.g., in diabetic ketoacidosis).

Diabetic ketoacidosis /181920/

DKA is the result of absolute or relative insulin deficiency in combination with increased activity of counter regulatory hormones (glucagon, catecholamines, cortisol, growth hormone). Absolute insulin deficiency is often present in children with undiagnosed diabetes type 1 or in insulin-dependent diabetics who fail to inject insulin. Relative insulin deficiency is due to elevated levels of counter regulatory hormones such as occur with vomiting, gastrointestinal disease with diarrhea, trauma, or sepsis.

A low concentration of insulin and high concentrations of counter regulatory hormones accelerate the development of a catabolic state with the following effects (Fig. 5.5-3 – Pathophysiology of diabetic ketoacidosis):

  • Increased production of glucose in the liver and kidneys by glycogenolysis and gluconeogenesis
  • Reduced glucose uptake in muscle and fat tissues due to insulin deficiency-induced reduced translocation of (GLUT)4-glucose transporters of the cell membrane, resulting in hypoglycemia and hyper osmolality. Hyperglycemia above the renal glucose threshold of 180 mg/dL (10 mmol/L) in combination with hyper ketonemia results in osmotic diuresis with dehydration and electrolyte loss, which are often exacerbated by vomiting.
  • Increased lipolysis and ketogenesis, resulting in ketonemia and metabolic acidosis
  • Inhibition of hepatic glycolysis in which glucose is converted to pyruvate, which then is used for the synthesis of amino acids and lipids, the generation of ATP in the citric acid cycle, and the formation of NAD by conversion of pyruvate to lactate
  • A low concentration of insulin triggers the mobilization of fatty acids from adipose tissue by stimulating hormone-sensitive lipase. The free fatty acids are transported to the liver where they are metabolized by β-oxidation, thus producing ketone bodies.

The main factor in the development of ketoacidosis in DKA is the excess of glucagon and its effect on the hepatocyte, since glucagon inhibits lipogenesis and stimulates fatty acid oxidation. The first step in the synthesis of free fatty acids is the conversion of acetyl-CoA to malonyl-CoA by the action of acetyl-CoA carboxylase. The free fatty acids are either used for lipogenesis or undergo mitochondrial fatty acid oxidation and ketogenesis (Fig. 5.5-3 – Pathophysiology of diabetic ketoacidosis).

Glucagon inhibits acetyl-CoA carboxylase, resulting in less malonyl-CoA. Malonyl-CoA is a strong inhibitor of fatty acid oxidation since it inhibits carnitine-palmitoyl transferase I (CPT I), which mediates the transport of free fatty acids to the mitochondrion (Fig. 5.5-3). Due to the decrease in malonyl-CoA in diabetic ketoacidosis there is increased activity of CPT I, leading to increased fatty acid oxidation. The resulting excess acetyl-CoA is not fed into the mitochondrial citric acid cyle, but takes the alternative pathway, forming AcAc and β-HB (Fig. 5.5-3 – Pathophysiology of diabetic ketoacidosis).

The pathway of ketone body production can be inhibited by activation of carbohydrate metabolism. In ketoacidosis induced by fasting this occurs by consuming an adequate amount of carbohydrates, in DKA by administering insulin, which promotes glucose uptake into the cells. Both cases lead to increased production of oxaloacetate, an acceptor for acetyl-CoA which feeds the latter into the citric acid cycle.

The insulin-antagonistic effect of catecholamines, cortisol and growth hormone in DKA leads to reduced peripheral glucose uptake. In the absence of insulin, catecholamines also promote the breakdown of triglycerides in adipocytes and increase the release of fatty acids.

DKA develops in the following four stages /1820/:

  • Stage I: lipoprotein lipase of the vascular endothelium fails to be activated by insulin. Triglycerides are not cleaved off by very-low-density lipoproteins (VLDL) and transported into the fat cells, resulting in the hypertriglyceridemia in DKA. In the tissue cells, lipase is elevated. As a result, increasing amounts of fatty acids are released into the circulation.
  • Stage II: the counter regulatory hormones glucagon, catecholamines and cortisol stimulate hepatic gluconeogenesis and glycogenolysis in the skeletal muscle and inhibit glucose uptake and oxidation by cells. The result is excess glucose production relative to glucose use and hyperglycemia.
  • Stage III: osmotic diuresis and dehydration. Glucosuria results in an osmotic diuresis with resultant polyuria and polydipsia. If fluid intake is maintained dehydration is minimal, and blood glucose will stabilize at about 300–400 mg/dL (16.7–22.2 mmol/L). If fluid intake cannot be maintained, as would occur during severe DKA or illness associated with vomiting, dehydration results. With severe dehydration the glomerular filtration rate (GFR) and the filtered glucose decrease, resulting in marked hyperglycemia. The GFR is reduced by approximately 25%. Blood glucose levels are near 600 mg/dL (33.3 mmol/L). Blood glucose concentrations above 800 mg/dL (44.4 mmol/l) usually indicate a GFR that is reduced by about 50% /18/.
  • Stage IV: the accumulation of glucose in the extracellular space causes an osmotic shift of cellular water to the extracellular compartment. This results in a dilutional hyponatremia. In the early phase of DKA, serum potassium and phosphate levels are normal or elevated, since the acidosis causes the potassium to shift to the extracellular compartment. Potassium losses are caused by urinary excretion of potassium along with keto acids and the effect of the increased aldosterone secretion as response of dehydration. As a result there is a total body potassium deficiency of 5–10 mmol/kg of body weight. Acidosis and hyperglycemia also cause a loss of phosphate.
  • Brain edema

Brain edema

Brain edema is the most serious complication of childhood DKA /26/. The incidence is about 1% with mortality rates between 21–50%. Osmotic activity of particles in brain cells during consistent hyperglycemia prevent cellular dehydration. As glycemia rapidly diminishes with onset of therapy, osmolytes remain within the brain cells causing an osmotic gradient that drives water from the extracellular compartment into the cytoplasm, causing intracellular swelling. This is due to the fact that osmolytes are eliminated from within the cell through osmolyte channels at a slow rate. Therefore water flow may be an issue of timing and cell membrane osmolyte channel number of the cell, rather than just an issue of osmotic gradient.

Hyperglycemic hyper osmolar non-ketotic syndrome (HHNS)

HHNS is characterized by hyperglycemia, increased serum osmolality, and prolonged dehydration in the absence of ketoacidosis (Fig. 5.5-4 – Pathophysiology of hyperglycemic hyper osmolar non-ketotic syndrome). Glucosuria reduces the renal concentration capacity, thus increasing the loss of water. The reduction of the intravascular volume in combination with the possible presence of renal insufficiency reduce the GFR and lead to a rise in blood glucose. Since patients with HHNS have a higher concentration of insulin in portal vein blood than those with DKA, the liver is able to metabolize fatty acids via a non-ketogenic pathway. Hyper osmolality and dehydration inhibit lipolysis, resulting in a reduced fatty acid supply for the liver. Patients with HHNS therefore do not have significant ketosis or acidosis. However, due to the higher glucose levels, they have greater volume depletion than DKA patients, leading to the development of pre renal azotemia /14/.

Alcoholic ketoacidosis

Alcoholic ketoacidosis results when mobilization of fatty acids occurs in conjunction with a ketogenic state in the liver. This condition is caused by a decreased ratio of insulin to glucagon. Reduced insulin levels result from glycogen depletion from starvation, decreased gluconeogenesis, and suppression of insulin release due to activation of sympathetic nerves /21/. Activation of the sympathetic nervous system and increased levels of growth hormone, cortisol, and ethanol account for the increased magnitude of fatty acid mobilization, as compared with simple starvation. Ethanol metabolism leads to an increased ratio of NAD/NADH that contributes to decreased gluconeogenesis and facilitates production of ketone bodies, specifically β-hydroxybutyric acid (Fig. 5.5-5 – Pathophysiology of alcoholic ketoacidosis) /1221/.

When keto acids enter the extracellular fluid, the dissociated H+ reacts with bicarbonate to generate CO2 and water. As a consequence, the bicarbonate level increases; this accounts for the increase in the anion gap /21/.


1. Kientsch-Engel RI, Sies EA. D-(–)-3-Hydroxybutyrate and acetoacetate. In: Bergmeyer HU (ed). Methods of enzymatic analysis. Weinheim; VCH Verlagsgesellschaft 1985, Vol VIII: 60–8.

2. Hirano T. Sensitive and simplified method for the differential determination of serum levels of ketone bodies. Modern Med Lab 1991; 19: 1113–7.

3. Uno S, Takehiro O, Tabata R, Ozawa K. Enzymatic method for determining ketone body ratio in arterial blood. Clin Chem 1995; 41: 1745–50.

4. Buttery JE, Adamek KJ. Evaluation of Ketostix for plasma acetoacetate. Am J Clin Pathol 1984; 82: 441–3.

5. Young DS, Huth EJ (eds). SI units for clinical measurement. Philadelphia; American College of Physicians 1998; 62, 157.

6. Fleckman AM. Diabetic ketoacidosis. Endocrinol Metab Clin North Am 1993; 22: 181–207.

7. Kitabchi AE. Diabetic ketoacidosis. Med Clin North Am 1995; 79: 9–37.

8. Heinemann L, Asche W, Withold W, Berger M. Quantitative Beziehung zwischen der mit vier Schnelltests bestimmten Ketonurie und der gleichzeitig vorliegenden Ketonämie. Diab Stoffw 1994; 3: 339–42.

9. Trachtenbarg DE. Diabetic ketoacidosis. Amer Family Phys 2005; 71: 1705–14.

10. Balasubramanyam A, Nalini R, Hampe CS, Maldonado M. Syndromes of ketosis-prone diabetes. Endocrine Reviews 2008; 29: 292–302.

11. Stoner GD. Hyperosmolar hyperglycemic state. Amer Family Phys 2005; 71: 1723–30.

12. Caspar CB, Risti B, Iten BX, Jost R, Russi EW, Speich R. Alkoholische Ketoazidose. Schweiz Med Wochenschr 1993; 123: 1929–34.

13. Burchell A. Glycogen storage disease and the liver. Bailliere’s Clinical Gastroenterology 1998; 12: 337–54.

14. Delaney MF, Zisman A, Kettyle WA. Diabetic ketoacidosis and hyperglycemic hyperosmolar nonketotic syndrome. Endocrinol Metab Clin North Am 2000; 683–705.

15. Agus MSD, Wolfsdorf JI. Diabetic ketoacidosis in children. Pediatr Clin N Am 2005; 52: 1147–63.

16. Eurodiap study group. Microvascular and acute complications in IDDM patients: the Eurodiap complications study. Diabetologia 1994; 37: 278–85.

17. Ghetti S, Lee JK, Sims CE, DeMaster DM, Glaser NS. Diabetic ketoacidosis and memory dysfunction in children with type 1 diabetes. J Pediatr 2010; 156: 109–14.

18. Dhatariya K. The use of point-of-care blood ketone monitors in the management of diabetic keoacidosis in adults. Ann Clin Biochem 2014; 51: 525–7.

19. Glaser N. Pediatric diabetic ketoacidosis and hyperglycemic hyperosmolar state. Pediatr Clin N Am 2005; 52: 1611–35.

20. Umpierrez GE, Khajavi M, Kitabchi AE. Review: diabetic ketoacidosis and hyperglycemic hyperosmolar nonketotic syndrome. Am J Med Sci 1996; 311: 225–33.

21. Palmer BF, Clegg DJ. Electrolyte disturbances in patients with chronic alcohol-use disorder. N Engl J Med 2017; 377: 1368–77.

22. Rosenbloom AL. Hyperglycemic hyperosmolar state: an emerging pediatric problem. J Pediatr 2010; 156: 180–4.

23. Saudubray JM, Ogier H, Bonnefont JP, et al. Clinical approach to inherited metabolic diseases in the neonatal period: a 20-year survey. J Inherited Metab Dis 1989; 12, Suppl 1: 25–41.

24. Schlump JU, Mayatepek E, Spiekerkötter U. Significant increase of succinylacetone within the first 12 h of life in hereditary tyrosinemia type 1. Eur J Pediatr 2010; 169: 569–72.

25. Kabadi UM. Pancreatic ketoacidosis: ketonemia associated with acute pancreatitis. Postgrad Med J 1995; 71: 32–5.

26. Toledo JD, Modesto V, Peinador M, Alvarez P, Lopez-Prats JL, Sanchis R, Vento M. Sodium concentrations in rehydration fluids for children with ketoacidotic diabetes: effect on sodium concentration. J Pediatr 2009; 154: 895–900.

5.6 Lactate

Lothar Thomas

Lactate is the final product of anaerobic glucose metabolism. It is oxidized in the citric acid cycle in the presence of oxygen, or undergoes gluconeogenesis within the Cori cycle. Lactate is one of the intermediates of metabolism, whose concentration increases as metabolism is altered during hypoxia. Lactate is therefore considered a marker of tissue hypoxia.

5.6.1 Indication

Plasma, whole blood

  • Prognosis and monitoring of sepsis, septic shock and intoxications
  • Diagnosis of occult tissue hypoxias with a normal arterial PO2 and monitoring of treatment results
  • Evaluation of unclear cases of metabolic acidoses, especially those associated with increased anion gap and comatose patients
  • Diagnosis of acute intestinal vascular occlusion
  • Fetal distress during labor and delivery
  • Primary test in children with suspected congenital metabolic disorders (e.g., in combination with glucose, ketone bodies and ammonia).

Cerebrospinal fluid

Acute inflammation in the central nervous system.

5.6.2 Method of determination

The measurement of L-lactate is based on the conversion to pyruvate. This reaction is either catalyzed by lactate dehydrogenase (LD) to form NADH + H+ or by lactate oxidase (LOD) to form H2O2.

LD-catalyzed reaction

Principle: lactate is oxidized to pyruvate by LD in the presence of NAD /1/. The NADH formed is measured spectrophotometrically at 340 or 366 nm as a measure of lactate. The balance of the reaction is such that lactate formation is greatly favored. In order that quantitative turnover of lactate occurs, certain reaction conditions have to be met:

  • Alkaline reaction environment; this causes the reaction equilibrium to shift towards pyruvate
  • Pyruvate must be removed at equilibrium; accomplished by the transamination reaction
  • The H+ ions must be captured; achieved by the presence of alkaline buffer environment.
L-lactate + NAD + LD pyruvate + NADH + H + Pyruvate + L-glutamate ALT L-alanine + α-ketoglutarate

Lactate oxidase dehydrogenase (LOD) catalyzed reaction

With clinical chemistry analyzers, the H2O2 obtained is measured colorimetrically. With point-of-care (POCT) analyzers and blood gas analyzers, H2O2 is measured amperometrically /2/.

Principle of amperometric measurement: this method uses a lactate-sensitive electrode on which the enzyme LOD is immobilized by attachment to a membrane surrounding an amperometric electrode. LOD generates H2O2 from lactate and O2. The H2O2 diffuses towards a platinum electrode, which is maintained at a certain potential relative to a silver reference cathode. At the platinum electrode, H2O2 is oxidized to O2, resulting in a change of potential, which is directly related to the lactate concentration.

5.6.3 Specimen

  • Capillary blood: mix 1 volume part of capillary blood into 1 volume part of 0.6 mol/L perchloric acid (approximately 7%)
  • Arterial or venous whole blood: collect in 5 mL tube containing 12.5 mg of sodium fluoride and 10 mg of potassium oxalate /3/
  • Arterial or venous plasma, obtained from stabilized whole blood (see above)
  • Cerebrospinal fluid, centrifuged.

5.6.4 Reference interval

Refer to Tab. 5.6-1 – Reference interval for lactate and References /245/

5.6.5 Clinical significance

In many pathological processes an increase in blood lactate reflects impaired tissue oxigenation. The lactate concentration in blood reflects the ratio of lactate production to lactate consumption by the different organs. Lactate is produced by muscle, in particular during intense physical exercise, as well as by the brain, intestine, and red cells. It is metabolized by the liver, the kidneys, and the heart. Lactate assays therefore do not measure the lactate turnover of a specific organ, but the measured concentration is the net result of the production and consumption of the whole organism. The baseline lactate concentration is kept constant within a narrow range. The quantitative contribution of an organ to the entry into or removal of lactate from the blood depends on influencing factors such as rest, exercise, hypoxia, diet, alcohol, and drugs. Critically ill and malnourished patients may have severe tissue hypo perfusion, but only slightly elevated lactate levels due to the lack of availability of metabolizable glucose /6/. Lactate is included in the guidelines of the Third International Consensus Definitions for Sepsis and Septic shock, emphazising the need for repeated measurements in patients of initial hyperlactatemia /47/.

Elevated blood lactate levels can be due to increased production, reduced clearance, or a combination of both, depending on clinical circumstances. Severe hyper lactatemia only develops in the setting of increased peripheral production together with reduced hepatic metabolic capacity due to a liver disease /7/.

Lactate is the product of glycolysis. The reversible reaction expressed by the equation promotes lactate synthesis at a normal lactat/pyruvate ratio of 10 : 1.

[Lactate] = K × [pyruvate] × [NADH] × H + [NAD + ]

If blood lactate is elevated, a differential diagnosis is required to distinguish between hyper lactatemia and lactic acidosis. Lactate levels of 18–45 mg/dL (2–5 mmol/L) are defined as hyper lactatemia, higher levels as lactic acidosis.

To assess the pathological quality of elevated lactate concentrations, the following biomarkers allow the evaluation of pathology:

  • Blood pH, HCO3, PCO2, PO2
  • Calculation of the anion gap
  • Ketone body concentration in serum or urine
  • Monitoring of the lactate concentration
  • Creatinine and urea
  • Aminotransferases
  • Toxicological investigations
  • Organic acids in urine. Hyper lactatemia

Hyper lactatemia represents a condition characterized by /8/:

  • Moderately elevated lactate levels (18–45 mg/dL; 2.0–5.0 mmol/L)
  • Absence of metabolic acidosis (pH < 7.30)
  • Absence of major perfusion defects of organs.

Hyper lactatemia without acidosis occurs in the setting of increased glycolysis of glucose to lactate (e.g., intense physical work, catecholamine infusion, or alkalosis). Causes and diseases associated with hyper lactatemia without acidosis are shown in Tab. 5.6-2 – Causes and diseases leading to hyper lactatemia and, less frequently, lactic acidosis. Lactic acidosis

In contrast to hyper lactatemia, in lactic acidosis the homeostatic regulation of lactate metabolism has failed. This can be due to excessive stress on the existing regulation or due to mitochondrial dysfunction.

Lactic acidosis is classified into two categories /910/:

  • Type A is caused by an imbalance of the requirement and supply of organs with oxygen. Due to hypo perfusion induced tissue hypoxia, glucose metabolism switches from aerobic mitochondrial to anaerobic cytoplasmic glycolysis. This results in reduced oxidation of pyruvate in the citric acid cycle, accumulation of lactate, and reduced synthesis of ATP.
  • Type B is caused by an existing organic or systemic disease without primary indication of hypo perfusion and hypoxia of organs (e.g., sepsis, diabetes mellitus, acute or chronic liver disease, renal failure, malignant tumor, medication, drugs and toxins, as well as congenital metabolic disorders).

Refer to (Tab. 5.6-3 – Classification of lactic acidosis)

Hyper lactatemias with progression to lactic acidosis are listed in Tab. 5.6-4 – Hyper lactatemias with frequent progression to lactic acidosis. Lactic acidosis is the most common type of metabolic acidosis. Like diabetic and alcoholic acidosis, it is associated with an increased anion gap. In contrast to ketoacidosis, which is also associated with elevated lactate, although usually at levels below 45 mg/dL (5.0 mmol/L), ketone bodies are not elevated in lactic acidosis.

In lactic acidosis, it initially seems easier to determine the severity of metabolic acidosis by measuring the pH rather than lactate, since the formation of lactate and H+ ions is a stoichiometric process. The H+ ions are formed during the hydrolysis of ATP and immediately used for oxidative phosphorylation under aerobic conditions. Under anaerobic conditions this step is inhibited, and for each molecule of lactate one H+ ion is produced. In pure lactic acidoses, such as hemorrhagic shock, there is thus a calculable relationship between the blood pH, the measured PCO2 and the concentration of HCO3 (Tab. 5.6-5 – Prediction equations for metabolic acidosis).

This does not apply for complex conditions, and these can be predicted. They are:

  • Concomitant renal insufficiency
  • Preexisting disorders of acid-base-balance such as metabolic alkalosis in the presence of chronic obstructive pulmonary disease.

Even the increased anion gap that is present in hyper lactatemia does not provide any further information, since it is influenced by ketone bodies and other non-measured anions. This is the case (e.g., with organic acidurias and fatty acid oxidation defects) where the anion gap is not so much due to the lactate but rather due to the organic acids, and is often above 25 mmol/L. This is also the case with exogenously supplied organic acids such as salicylates.

Patients with lactic acidosis do not present with a clear clinical picture. Common symptoms are tachypnea, hypotension, and impaired mental state. In many cases, a combination of type A and type B lactic acidosis is present such as increased synthesis and decreased elimination of lactate and H+ ions /6/. Hereditary lactic acidosis

The etiology of hereditary lactic acidoses can be of primary or secondary nature (Tab. 5.6-6 – Hereditary metabolic disorders causing lactic acidosis). They are a primary phenomenon in disorders of pyruvate and hepatic glycogen metabolism, gluconeogenesis defects, disorders in the citric acid cycle, and respiratory chain disorders. They occur as a secondary phenomenon in disorders of acetyl-CoA metabolism, if pyruvate cannot enter the citric acid cycle.

Lactic acidosis in children primarily suggests a hereditary metabolic disorder, although it is much more frequently due to other causes such as shock, sepsis, cardiopulmonary diseases or drug poisoning.

Forearm ischemic work test

The test is used for the detection of some metabolic myopathies. When performed under ischemic conditions, the forearm ischemic work test according to McArdle (Tab. 5.6-7 – Ischemic forearm work test (McArdle)) may induce exercise intolerance (muscle cramps, pain, rhabdomyolysis and compartment syndrome) in patients with glycogenosis /11/.

A new non-ischemic forearm work test (Tab. 5.6-8 – Non-ischemic forearm ischemic work test) avoids the exercise intolerance associated with McArdle’s test. The new test measures lactate and ammonia during grip exercise /12/. Fig. 5.6-1 – Mean lactate and ammonia levels in the non-ischemic forearm exercise test shows the behavior of lactate and ammonia during the test for different myopathies. Cerebrospinal fluid lactate

Many inflammatory, vascular, metabolic and neoplastic diseases of the brain and meninges present with an elevated cerebrospinal fluid (CSF) lactate. The determination of lactate is of diagnostic importance in differentiating /5/:

  • Between bacterial and viral meningitis
  • Between transitory ischemic attacks and generalized seizures in cases lacking history and clinical findings
  • Between artificial blood contamination of CSF and cerebral or subarachnoidal hemorrhage.

The lactate concentration is of prognostic value in vascular and traumatic brain diseases and in intoxications, but not in inflammatory diseases. The behavior of CSF lactate in meningeal and cerebral diseases is shown in Tab. 5.6-9 – Meningeal and cerebral diseases in which the cerebrospinal fluid (CSF) lactate is relevant.

5.6.6 Comments and problems

Blood sampling

Arterial or capillary blood should be collected. Venous specimens should be obtained without the use of a tourniquet. The lactate concentration is generally 4.5–9.0 mg/dL (0.5–1.0 mmol/L) higher in venous blood than in arterial blood. Influencing factors such as venous stasis, problems with vascular puncture, or crying in children also cause elevations.

To prevent cellular glycolysis after sampling, different alternatives are suggested for specimen handling /40/:

  • Mix 1 mL of blood with 1 mL of ice-cold 7% perchloric acid immediately after collection, leave for 10 min., centrifuge and measure in the supernatant. If this cannot be done on the ward, place heparinized blood on ice and immediately transport it to the laboratory.
  • Collect blood with a syringe containing 2 mg of sodium fluoride and 2 mg of potassium oxalate per mL of blood collected.


Lactate measurements in venous blood specimens or venous blood gas specimens (VBGs) are influenced by tube additives, time and temperature of transport or storage and hemolysis. In blood specimens lactate levels increase over time, because the continuing glycolysis requires:

  • stabilizing reagents (sodium fluoride)
  • an anticoagulant (potassium oxalate or K3EDTA).

In a study /48/ stability of lactate concentrations and dependence on turnaround time were compared in VBGs to those assayed in venous sodium fluoride/potassium oxalate plasma. In conclusion:

  • Lactate concentrations were higher in VBGs for concentrations below 4 mmol/l, but difference remained within the intraindividual variation.
  • Lactate concentrations measured in sodium fluoride/potassium oxalate plasma remained stable and within the analytical variation for a turnaround time at least 2 hours.
  • VBG lactate can be considered as stable for up to 45 min from an analytical standpoint, and for up to 1 hour considering the intra-individual variation.

Assay-dependency of results

The enzymatic measurement in plasma and the amperometric analysis in whole blood produce similar results /2/. Whole blood and plasma values measured with the enzymatic method are not comparable. The higher the hematocrit, the more they differ.

Interfering factors

Leukocytosis increases the lactate concentration in stabilized whole blood by no more than 2.7 mg/dL (0.3 mmol/L) within 8 h /3/. Glycolates and glyoxylic acid lead to falsely high lactate values in POCT analyzers that use aperometric measurement /41/.

Biological variability

The intraindividual variation (VK) is 16.7% with an analytical variation of 3.3% /42/.

5.6.7 Pathophysiology

In most tissues dietary carbohydrates are metabolized to pyruvate via glycolysis in the cytoplasm of the cells. One molecule of glucose is converted into two molecules of pyruvate, generating energy for the cell in the form of two molecules of ATP without consuming O2 /2943/.

Pyruvate follows the following metabolic pathways (Fig. 5.6-2 – Energy metabolism of the cell):

  • Oxidative decarboxylation. Pyruvate is decarboxylated to acetyl-CoA in the mitochondrion by pyruvate dehydrogenase, fed into the citric acid cycle and metabolized to CO2 and H2O. In combination with the oxidative phosphorylation of the respiratory chain, a total of 18 molecules of ATP are produced per decarboxylated molecule of pyruvate.
  • Conversion to lactate by LD. Two molecules of ATP per molecule of pyruvate are produced. A resting person produces 1300 mmol of lactate per day, 40–60% of which is taken up by the liver and converted to glucose via gluconeogenesis (Cori cycle). Some gluconeogenesis also occurs in the skeletal muscles, heart and kidneys /44/. Lactate can be converted back to pyruvate. This process uses four molecules of ATP. Since most pyruvate is fed into the citric acid cycle, its plasma concentration is low and the ratio of lactate to pyruvate is 10 : 1.
  • Carboxylation to malate and oxalacetate.
  • Transamination to alanine, catalyzed by alanine aminotransferase.

Mitochondrial oxidative phosphorylation

In normal functioning mitochondria, mitochondrial respiration is tightly coupled to the formation of the H+ motive force, and the decrease of H+ motive force is closely linked to the synthesis of ATP. Mitochondrial oxidative phosphorylation is the main pathway for ATP synthesis in the cells and works as follows /47/:

  • Redox energy from the breakdown of food is converted to a H+-gradient across the inner mitochondrial membrane, which is dissipated to catalyze the inner mitochondrial membrane while consuming oxygen.
  • Respiratory chain complexes I, III, and IV pump protons across the inner mitochondrial membrane while consumimg oxygen.
  • Complex V, the central enzyme in energy conversion, dissipates the H+-gradient across the membrane, causing phosphorylation of ADP to ATP.

In tissues such as the red blood cells, brain, skeletal muscle, intestinal mucosa or the adrenal cortex, the pyruvate produced by glycolysis does not enter the mitochondrion, but is mostly converted to lactate. This is then transported to the liver, converted back to pyruvate and then to glucose through gluconeogenesis, or it is used in the synthesis of fatty acids.

The liver is the main organ involved in the production of glucose and clearance of lactate. In a fasting state, lactate is primarily used for gluconeogenesis due to increased activity of mitochondrial pyruvate carboxylase. In a non-fasting state, the oxidation of pyruvate to acetyl-CoA is the preferred pathway. It is stimulated by the pyruvate dehydrogenase complex in the mitochondrion.

Tissue hypoxia

Whether pyruvate is oxidized to acetyl-CoA or reduced to lactate depends on the NADH/NAD ratio. Adequate availability of NAD promotes the conversion to acetyl-CoA and thus the synthesis of ATP by oxidative phosphorylation. A decrease in the O2 supply to the vessels triggers a series of reactions:

  • More O2 is extracted from capillary blood by expansion of the capillary bed per tissue volume. This especially affects organs that have sudden high energy consumption, such as the skeletal muscle and, less frequently, the heart muscle.
  • If oxygen supply is inadequate, glycolysis is increased, causing lactate to accumulate in the cell. Lactate leaves the cell in exchange for OH ions, mediated by a membrane-associated antiport system that is dependent on the blood pH. While alkalemia promotes the removal, acidemia increases the uptake of lactate into the cells.

Together with lactate, H+ ions leave the cell. However, hyper lactatemia does not necessarily lead to acidosis. Whether or not it develops depends on the lactate concentration, the body’s buffering capacity, and the coexistence of pathological conditions, such as sepsis or liver disease, which predispose the patient to tachypnea and alkalosis. In the presence of ischemic injury, for example, the liver produces lactate instead of utilizing it.

In hypovolemic and cardiogenic shock, blood lactate levels do not always correlate with the severity of tissue hypo perfusion. This is the case, for example, with hypo perfusion in the innervation area of the splanchnic nerve. In some cases there is no hyper lactatemia, while in other cases large amounts of lactate can be produced in the intestine, resulting in hyper lactatemia despite the absence of marked hypo perfusion. The lactate concentration is an indicator of the severity of shock and is of prognostic value only if the diagnosis of hypo perfusion is confirmed and other causes of hyper lactatemia can be excluded /6/.

The severity of hyper lactatemia depends on the substrate availability. For example, severely ill or malnourished patients can have mild hyper lactatemia despite severe hypo perfusion, but a high mortality probability /6/.

If anaerobic glycolysis prevails in hypo perfusion, the pH decreases. Clinical symptoms include dilatation of the smooth vascular muscles, vasodilatation and hypotension.


In sepsis, unforeseeable metabolic changes occur that lead to elevated glucose consumption and accumulation of lactate despite adequate oxygen supply to the organs. Endotoxinemia reduces the arterial glucose concentration and increases the glucose content in the muscles with normal plasma insulin levels. It has been suggested that one cause of the hyper lactatemia may be the conversion of a proportion of the enzyme activity of pyruvate dehydrogenase to an inactive isoform by endotoxin. As a result, more pyruvate is converted to lactate instead of entering the citric acid cycle. The accumulation of lactate in sepsis probably results from increased glucose turnover in combination with inadequate pyruvate metabolism in the citric acid cycle /10/.

Hereditary hyper lactatemia

Congenital hyper lactatemia is primarily the result of disorders of energy metabolism and are due to defects in the nuclear or mitochondrial genome. The incidence is approximately 1 in 5,000 neonates, for glycogenoses it is approximately 1 in 25,000 births in Europe. Even though the individual disorders are rare, the hyper lactatemias they cause pose diagnostic problems, since hyper lactatemia is much more likely to be due to secondary causes than primary defects. Patients with primary hyper lactatemia usually have severe metabolic acidosis, hyperventilation and ataxia or an altered neurological state /43/.


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25. van der Beek A, de Meijer PH, Meinders AE. Lactic acidosis: pathophysiology, diagnosis and treatment. Neth J Med 2001; 58: 128–36.

26. Lange CM, Bojunga J, Hofmann WP, Wunder K, Mihm U, et al. Severe lactic acidosis during treat-ment of chronic hepatitis B with entecavir in patients with impaired liver function. Hepatology 2009; 50: 2001–6.

27. John M, Mallal S. Hyperlactatemia syndromes in people with HIV infection. Current Opinion Infect Dis 2002; 15: 23–9.

28. Raeven GM, Hollenbeck C, Geng CY, et al. Measurement of plasma glucose, free fatty acid, lactate, and insulin for 24 hours in patients with NIDDM. Diabetes 1988; 37: 1020–4.

29. de Backer D. Lactic acidosis. Intensive Care Med 2003; 29: 699–702.

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36. Kakiuchi T, Nosho T, Oka M, Tashiro K. Hyperammonemia in a carbamoyl-phosphate-1-deficiency recipient after living-donor liver transplantation from a carrier donor: a case report. Front Med 2023. doi: 10.3389/fmed.2023.1327854.

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43. Robinson BH. Lactic acidemia. Biochem Biophys Acta 1993; 1182: 231–44.

44. Cohen RD. Roles of liver and kidney in acid-base regulations and its disorders. Br J Anaesth 1991; 67–73.

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Table 5.1-1 Ammonia reference intervals


  • Umbilical artery /5/*

109 ± 122

  • Umbilical vein /5/*

84 ± 94

  • Premature infants /6/


  • Full-term infants /6/


  • Children (1 month to > 14 y) /7/




Data expressed in μg/dL, values are x ± 2 s or * median ± s; values for venous plasma; conversion: μg/dL × 0.5872 = μmol/L

Table 5.1-2 Congenital (primary) and acquired (secondary) causes of hyperammonemia /8/

Congenital (primary) etiologies

  • N-acetylglutamate synthetase deficiency
  • Carbamoyl phosphate synthetase deficiency
  • Ornithine transcarbamylase deficiency
  • Argininosuccinate synthetase deficiency (citrullinemia)
  • Argininosuccinate lyase deficiency (argininosuccinate aciduria)
  • Arginase deficiency (argininemia)

Secondary etiologies

  • Liver cirrhosis (all causes)
  • Fatty acid oxidation disorders (e.g., MCAD defect)
  • Organic acidurias (e.g., propionic aciduria, methylmalonic aciduria, isovaleric aciduria)
  • Mitochondrial toxins (e.g., valproic acid)
  • Acute liver failure (e.g. tylenol toxicity, Wilson’s disease)

MCAD, medium-chain acyl-CoA dehydrogenase deficiency

Table 5.1-3 Enzyme defect and substrates accumulating in hereditary hyperammonemias /1112/


Enzyme defect

Reaction affected,
accumulating substrates

N-acetylglutamate synthetase (NAGS) deficiency

N-acetyl-glutamate synthetase

The synthesis of N-acetylglutamate from glutamate and thus the channeling of glutamate into the urea cycle is impaired

Carbonic anhydrase deficiency (OMIM 615751) /36/

Carbonic anhydrase

An autosomal recessive disorder with increased ammonia (> 500 umol/L), increased lactate and decreased glucose

Urea cycle defects

Carbamoyl phosphate synthetase 1 (CPS) deficiency

Carbamyl phosphate synthetase 1

No carbamyl phosphate is formed from the substrates glutamate, NH3, CO2 and ATP.

Ornithine transcarbamylase deficiency

Ornithine transcarbamylase (OTC)

OTC catalyzes the condensation of ornithine with carbamoyl phosphate to form citrulline


Argininosuccinate synthetase (AS)

The synthesis of argininosuccinate from citrulline and aspartate is inhibited

Argininosuccinic aciduria

Argininosuccinase (AL)

The conversion of argininosuccinate to arginine and fumarate is inhibited



The cleavage of arginine to urea and ornithine is inhibited

Organic acidurias

Propionic aciduria

Propionyl-CoA carboxylase

Threonine, isoleucine, methionine, valine

Methylmalonic aciduria

Methylmalonyl- CoA mutase

Threonine, isoleucine, methionine, valine

Isovaleric aciduria

Isovaleryl-CoA dehydrogenase


3-hydroxy-3-methylglutaric aciduria

3-hydroxy-3-methylglutaryl- CoA lyase

1. The last step of leucine oxidation is stopped

2. The formation of ketone bodies from fatty acids is inhibited

Disorders of fatty acid oxidation

Acyl-CoA dehydrogenase defects

Acyl-CoA dehydrogenase defects for:

short-chain (C4–C6), medium-chain (C4–C14) and long-chain (C12–C18) fatty acids

Fatty acids are not metabolized in the mitochondria

Carnitine-related disorder

Carnitine palmitoyl transferase deficiency

Fatty acids are not transported into the mitochondria

Disorder of pyruvate metabolism

Pyruvate dehydrogenase complex

The formation of acetyl-CoA from pyruvate is impaired

Disorder of pyruvate metabolism

Pyruvate carboxylase

Gluconeogenesis is inhibited, since pyruvate is not converted to oxaloacetate and then phosphoenolpyruvate

Table 5.1-4 Hereditary hyperammonemias /1920/

Clinical and laboratory findings

Urea cycle defect

All urea cycle defects have an autosomal recessive inheritance, with the exception of the most common defect, ornithine transcarbamylase (OTC) deficiency, which is X-linked. A distinction is made between neonatal-onset, often lethal forms of the defect in hemizygous male infants, and childhood- or adult-onset forms with less severe progression. Causes can include change to a higher protein diet, or infectious diseases /17/. Findings include hepatomegaly, which is usually found during acute hyperammonemic episodes, and jaundice in newborns. The individual enzyme deficiencies cannot be differentiated based on clinical symptoms, but only by performing biomarkers. For OTC in particular, there are over 100 known mutations with varying effects on substrate turnover and thus on clinical symptoms /11/.

Laboratory findings: hyperammonemia with maximum levels of 850–3400 μg/dL (500–2000 μmol/L). Urea cycle defects do not usually cause hypoglycemia, ketonemia/-uria or hyper lactatemia; the anion gap is normal /18/. ALT can be elevated up to 10-fold the upper reference interval value.

Organic aciduria

These are autosomal recessive diseases which are characterized by deficiencies of enzymes that are responsible for the metabolism of the branched-chain amino acids leucine, isoleucine, and valine. Defects of isoleucine and valine metabolism lead to methyl malonic acidemia and propionic acidemia; isovaleric acidemia is a disorder of leucine metabolism. The spectrum of clinical symptoms in seriously ill neonates is rather unspecific. Food refusal, failure to thrive, lethargy, apathy, hypotension and cerebral seizures are more suggestive of sepsis or cerebral hemorrhage. In its early stages, metabolic acidosis is often misinterpreted as being caused by sepsis or hypoxia /12/.

Laboratory findings: typical findings include metabolic acidosis, ketonuria, and hyperammonemia with max. levels of 170–340 μg/dL (100–200 μmol/L) /18/. Due to the unmeasurable anions, the anion gap is increased. In newborns, the pH is 6.9–7.1, and hyper lactatemia and hypoglycemia may be present. Episodic granulocytopenia and thrombocytopenia can be misinterpreted as being related to sepsis.

Disorder of fatty acid oxidation

Disorders of fatty acid oxidation are either due to enzyme deficiencies of the mitochondrial β-oxidation of fatty acids or due to a defect of the carnitine palmitoyl transferase complex, which transports long-chain fatty acids across the mitochondrial membrane. Clinical symptoms often manifest only during fasting or infections. In infants, the main manifestations are central nervous symptoms such as impaired consciousness, coma, and unexpected death. Defects of the carnitine palmitoyl transferase complex and long-chain acyl-CoA dehydrogenases are accompanied by cardiac symptoms, myopathy, and liver disease /2122/.

Laboratory findings: common findings associated with acute symptoms include hypo ketotic hypoglycemia, elevated ALT and CK, reduced carnitine concentration, and moderately elevated ammonia levels. In the non-acute condition it is important that children fast and reach blood glucose levels well below 60 mg/dL (3.3 mmol/L) to allow significant excretion of dicarboxylic acids without the associated ketonuria. In the fasting state, ammonia levels are elevated above 170 μg/dL (100 μmol/L), urea and dicarboxylic acid are elevated without concomitant increase in ketone bodies /18/. Additional findings, in particular in older children, can include hyperuricemia, lactate elevations of varying degrees, myoglobinuria.

Hyperornithinemia-hyperammonemia, hyper citrullinemia (HHH syndrome)

This is an autosomal recessive disease caused by a defect in the transport of ornithine into the mitochondria, which results in ornithine accumulating in the cytoplasm, reduced mitochondrial synthesis of citrulline, and reduced capability to eliminate carbamoyl phosphate and ammonia. Clinically, children with HHH syndrome present with episodic vomiting, irritability and seizures from age 1–2 due to increased protein intake /18/.

Laboratory findings: increased excretion of orotic acid in urine as a result of the accumulation of carbamoyl phosphate in the mitochondria, which also contributes to hypercitrullinuria by increased transcarbamylation of lysine. The hyperammonemia associated with increased excretion of orotic acid can lead to confusion with OTC deficiency.

Lysinuric protein intolerance (LPI)

LPI is an autosomal recessive disease caused by a defect in the transport of dibasic amino acids across the basolateral cell membrane of the tubular cell of the kidney and the intestinal enterocytes. The deficient renal and intestinal absorption of arginine and ornithine leads to a lack of substrate availability for OTC, impaired ammonia metabolism, reduced synthesis of citrulline, increased levels of carbamoyl phosphate in the mitochondria, and increased excretion of orotic acid in urine. Clinical symptoms first appear in early childhood as dietary protein intake increases. They include insufficient food intake, vomiting, lethargy, and episodes of diarrhea. Symptoms in older children include aversion against high-protein foods, hepatomegaly, myasthenia, osteoporosis, and thinning hair.

Laboratory findings: significantly increased excretion of lysine with moderately elevated arginine, ornithine and citrulline in urine. Episodic elevation of ammonia. Other findings may include elevated ALT, LD and thyroxine-binding globulin, moderate anemia, leukopenia, and thrombocytopenia /18/.

The HI-HA syndrome is an unusual form of congenital hyperinsulinism (HI) and associated with dominantly-expressed mutations of the mitochondrial glutamate dehydrogenase (GLD) which is encoded by the GLUD1 gene. As a result, GLD exhibits reduced sensitivity to inhibition by guanosin-5`triphosphate. This defect results in abnormally high rates of glutmate oxidation, leading to excessive insulin secretion and impaired detoxification of ammonia by the liver /23/. Affected patients suffer from recurrent hypoglycemias. Clinical symptoms differ significantly from those present in classic hyperinsulinism syndromes in children. The latter, which is due to a defect of the sulfonylurea receptor/potassium channel complex, causes persistent hypoglycemias as early as in the first days of life. Affected children are larger than normal and overweight for their gestational age. Children with HI-HA syndrome are of normal weight at birth, and hypoglycemia may not manifest until years later. In some cases, HI-HA syndrome is not diagnosed until adulthood. The hypoglycemia can be mild to severe. Ammonia levels in blood are persistently 2–10 times higher than normal. Due to the moderate nature of the hyperammonemia, affected children do not exhibit the classic symptoms of hyperammonemia such as lethargy and vomiting /24/.

Carbamoyl-phosphate synthetase 1 (CPS1)

CPS1 deficiecy is an autosomal recessive congenital urea cycle disorder characterized by hyperammonemia with a morbidity rate of 1/50.000 to 1/800.000.In some cases intensive care is provided due to hyperammonitic encephalopathy.

Laboratory findings: in plasma ammonia concentrations > 400 μg/dL, elevated glutamine and alanine concentrations, and decreased arginine and and citrulline. No increase in urinary orotic acid excretion. The genetic test shows a compound heterozygous pathogenic variant in the CPS1 gene /36/.

Table 5.1-5 Differential diagnosis of acute hereditary hyperammonemias, modified from Ref. /1718/








Amino acids, organic acids



Urea cycle defect






Alanine + glutamine

Citrulline + arginine

Orotic acid n






Alanine + glutamine

Citrulline + arginine

Orotic acid ↑↑






Citrulline ↑↑

Glutamine Arginine

Orotic acid






Citrulline + glutamine + argininosuccinate


Orotic acid






Arginine ↑↑ Glutamine

Orotic acid

Organic aciduria





Propionic acid and methylmalonic acid and isovaleric acid

Fatty acid oxidation



Dicarboxylic acids

Alanine and glutamine and proline






Ornithine and citrulline

Orotic acid ↑↑






Lysine ↑↑








, elevated; n, normal; , reduced; +, present; AS, argininosuccinate synthetase; AL, argininosuccinase; CPS, carbamoyl phosphate synthetase; OTC, ornithine transcarbamylase; HHH, hyperornithinemia-hyperammonemia-hypercitrullinemia syndrome; LPI, lysinuric protein intolerance; HI/HA, hyperinsulinism-hyperammonemia syndrome; Kb, ketone bodies

Table 5.1-6 Aquired hyperammonemias

Clinical and laboratory findings

Hepatic encephalopathy /16/

Clinical findings: clinical symptoms range from mildly impaired intellectual function, anxiety and personality changes to coma. Initial manifestations are motor restlessness and nervousness.

Laboratory findings: clinical signs of hepatic encephalopathy can be expected with venous plasma ammonia above 150 μg/dL (88 μmol/L). The ammonia level correlates with the severity of hepatic encephalopathy. In comatose states concentrations above 300 μg/dL (176 μmol/L) are measured. Arterial plasma levels correlate better with the severity of hepatic encephalopathy than venous plasma levels.

Significant elevations of ammonia are found in shunt-operated patients, where the portal vein ammonia concentration is about 300 μg/dL (176 μmol/L) due to the intestinal absorption of ammonia produced by bacteria. In approximately 10% of patients with hepatic encephalopathy ammonia levels are not elevated.

High-dose chemotherapy

High-dose chemotherapy can lead to hyperammonemia with normal liver function.

Clinical findings: symptoms are comparable to those in hepatic encephalopathy.

Laboratory findings: hyperammonemia, respiratory alkalosis due to hyperventilation. In a study /25/ of patients receiving treatment for acute leukemia, ammonia levels were 29–119 μg/dL (17–70 μmol/L) prior to therapy and 123–590 μg/dL (72–347 μmol/L) after therapy. 14% of the patients had levels above 340 μg/dL (200 μmol/L) and encephalopathy.


Multiple myelomas, in particular those with meningeal involvement, can be associated with hyperammonemia. Here, concentrations of 195–330 μg/dL (114–194 μmol/L) have been measured /26/.

Reye’s syndrome

Disease that has been described in children up to 15 years /27/. The cause is unknown but is thought to be mitochondrial dysfunction. Morphological analysis shows lipid vacuoles in the hepatocytes and renal tubules.

Clinical findings: vomiting, dysfunction of the central nervous system and liver. It is believed that viral infections or toxic factors (salicylates) may be involved in triggering the disease.

Laboratory findings: elevated aminotransferases, prolonged prothrombin time, metabolic acidosis, hypoglycemia, reduced ketone bodies in plasma. Ammonia levels are in the range of 170–600 μg/dL (100–350 μmol/L). There is a negative correlation between maximum ammonia levels and the probability of survival.

Valproic acid (VPA) therapy /28/

The therapeutic range of VPA is 63–133 mg/L. Hyperammonemia can develop during prolonged therapy with VPA. This usually occurs in the case of polymedication that influences the VPA concentration. Hyperammonemic encephalopathies have been described in epileptic and psychiatric patients. Ammonia levels are 170–340 μg/dL (100–200 μmol/L). In tumor patients receiving chemotherapy with high doses of valproic acid, ammonia levels can be elevated 10-fold. The concentrations of ammonia and VPA are generally poor predictors of encephalopathy. The disease is thought to be caused by accumulation of VPA metabolites, which lead to reduced availability of N-acetyl glutamate and thus inhibition of mitochondrial carbamoyl phosphate synthase-1, the first step in the urea cycle.

Urinary tract infection

Patients with anomalies of the urinary system and bladder have frequent or chronic urinary tract infections. If there is extensive colonization by urea-hydrolyzing bacteria such as Proteus mirabilis, these bacteria convert urea to NH4+, 50% of which are present as NH3 at a pH of 9. The ammonia is absorbed, leading to ammonia concentrations of up to 700 μg/dL (411 μmol/L) in children /19/.

Newborn with a low birth weight

Asymptomatic hyperammonemia with levels 2-fold the upper reference interval value is found in some newborns with a birth weight < 2500 g. After 4 weeks the concentration decreases to adult levels. In a study /5/ of newborns with a low birth weight, ammonia levels were 121 ± 45 μg/dL (71 ± 26 μmol/L), expressed as x ± s.

Transient neonatal hyperammonemia

Rare condition in newborns during the first 2 days of life. It is often accompanied by pulmonary dysfunction. The hyperammonemia can be more severe than in children with hereditary urea cycle defects /20/.

Table 5.2-1 Bilirubin reference intervals

Total bilirubin



Neonates /10/

24 h

≤ 8.7

≤ 150

2. day



3. day



4.–6. day




> 1 month



Adults /1/



Population in the USA* /11/

≤ 1.4

≤ 24

Direct bilirubin

Adults and children

≤ 0.1

≤ 2

Unbound unconjugated bilirubin

Neonates /9/



Conversion: mg/dL × 17.104 = μmol/L, * Values expressed as 2.5 th and 97.5th percentiles

Table 5.2-2 Diseases with prehepatic, predominantly non-conjugated hyperbilirubinemia

Clinical and laboratory findings

Corpuscular hemolytic anemia, e.g.,

  • thalassemias
  • sickle-cell anemia
  • hereditary spherocytosis
  • G6-PD deficiency

Typical findings in corpuscular hemolytic anemias include reticulocytosis, morphologically altered erythrocytes, direct Coombs test-positive result, decreased haptoglobin and possibly hemopexin levels, hemoglobinuria, hemosiderinuria, possibly urobilinogenuria, absence of bilirubinuria. The erythrocyte life span is reduced. Hyperbilirubinemia only occurs when the rate of hemolysis is above 5% (normal: 0.8%); levels above 6 mg/dL (103 μmol/L) are rare, Bu accounts for a large proportion of BT. In chronic hemolytic anemias with BT above 6 mg/dL (103 μmol/L), hepatic function is likely to be impaired as well /33/.

Extra corpuscular hemolytic anemia, e.g.,

  • transfusion reaction
  • autoimmune hemolysis
  • dyserythropoetic anemia
  • drug-induced

Acute hemolytic transfusion reactions: they are usually related to AB0 blood group incompatibility, less frequently to incompatibilities in the Rh system or other blood group systems. Hemolytic reactions due to AB0 incompatibility occur within minutes of the transfusion, are severe and intravascular (immediate reaction). Hemolytic reactions caused by antibodies against other blood group systems (e.g., Rh, are delayed and are milder in nature (early reaction)). Late reactions, which take place after the development of an antibody, do not become manifest until the second week after the transfusion. Hyperbilirubinemia associated with immediate reactions occurs after 6–12 h. The first manifestations are clinical symptoms and signs of hemolysis such as hemoglobinemia, hemoglobinuria, and a decline in haptoglobin. Hemosiderinuria is a late symptom (from day 2).

Autoimmune hemolytic anemias /34/: in these anemias, Hb is reduced, BT is moderately elevated up to 6 mg/dL (103 μmol/L), LD is elevated, reticulocytosis is present, and haptoglobin is below 30 mg/dL.

Dis erythropoietic anemias: these anemias are mild and are characterized by ineffective erythropoiesis and morphologic abnormalities of the red cell precursors. Bilirubin elevations are mild and usually accompanied by splenomegaly.

Drug-induced hemolytic anemias: they are predominantly caused by autoantibodies. BT is moderately elevated up to 6 mg/dL (103 μmol/L).

Neonatal jaundice /1535/

Neonatal jaundice is a physiological, transient, non-conjugated type of hyperbilirubinemia that occurs during the neonatal period. The concentration of Bu, measured as BT or skin bilirubin, rises continuously during the first days of life, peaks at age 3–5 days in the state of equilibrium between bilirubin production and clearance, and then normalizes at age 7–10 days. Several studies reported a 95th percentile of the peak level in breast fed newborns of 15.1–17.6 mg/dL (259–301 μmol/L) /15/. If BT exceeds 17 mg/dL (291 μmol/L) in term and pre-term infants, phototherapy is recommended. If levels are above 25 mg/dL (428 μmol/L), exchange transfusion is considered. A more frequently used method is the measurement of the rates of rise in bilirubin in the first 5 days of life (Fig. 5.2-2 – Hour-specific bilirubin nomogram showing the increase in bilirubin in term or near-term neonates). Appropriate therapeutic measures are then taken according to the thresholds.

Hyperbilirubinemia is classified as follows:

  • Low, medium or high risk for acute bilirubin encephalopathy depending on the 95th percentile for age in hours (Fig. 5.2-2).
  • Extreme, if bilirubin is above 25 mg/dL (428 μmol/L) i.e.,above the 99.9th percentile of the maximum level of healthy newborns. If phototherapy is performed on infants older than 48 h with BT levels in the range of 17–22.9 mg/dL (291–392 μmol/L), elevations above 25 mg/dL (428 μmol/L) can be avoided in 85% of cases. Predictors of elevations above this level include gestational age, a rapid increase in bilirubin, family history, and hematomas /36/.
  • Dangerous, if bilirubin is above 30 mg/dL (513 μmol/L). According to a British study, the incidence of such levels is 7.1 in 100,000 births. In most cases, infants had already been discharged, and 80% were being breast-fed /37/.

Clinically, half of all term infants are jaundiced. A bilirubin rate of rise greater than 0.1 mg × dL–1 × h–1 within the first 48 hours of life is indicative of medium risk, a rate greater than 0.2 mg × dL–1 × h–1 of high risk /38/. In these cases, other causes of the jaundice must be investigated, because a rise of this magnitude is not physiologic. Prolonged non-conjugated hyperbilirubinemia with BT levels remaining above 8.8 mg/dL (150 μmol/L) for more than 14 days and above 6 mg/dL (100 μmol/L) for more than 28 days is common in Asian infants. The prolonged jaundice is thought to be partly due to a variation in nucleotide 211 of the UGT1A1 gene of uridine diphosphate glucuronyltransferase /39/. Physiologic causes contributing to the rise in bilirubin within the first few days after birth include hematomas and persistent enterohepatic bilirubin circulation, which is increased by breast-feeding. Pathologic causes of the displacement of bilirubin from albumin include acidosis, ketosis, and kidney failure.

Hyperbilirubinemia is the most common cause of readmission to hospital in neonates who are discharged relatively early after birth. It is therefore recommended that neonates with a predischarge bilirubin rate of rise greater than 0.1 mg × dL–1 × h–1 be retested 2–3 days later. One study /40/ has shown that a bilirubin level in the low-risk zone (predischarge rate of rise below 0.1 mg × dL–1 × h–1) is not a reliable criterion, since half of all neonates readmitted for jaundice and bilirubin levels above 17 mg/dL (291 μmol/L) had predischarge levels in the low-risk zone.

BIND /41/

Bilirubin-induced neurological dysfunction (BIND) comprises classic acute bilirubin encephalopathy (kernicterus) and milder forms. Free bilirubin (BF) is neurotoxic. Only BF can pass the intact blood-brain barrier. Ill neonates who have blood concentrations of metabolites and drugs that displace bilirubin from albumin, or in whom bilirubin is released more readily due to the presence of ketosis or acidosis, as well as neonates with blood group incompatibility and direct Coombs test-positive results have higher concentrations of BT and BF and thus a higher risk of neurotoxicity. This is also the case with pre-term neonates. Their immature blood-brain barrier allows albumin-bound bilirubin to pass into the central nervous system where it is released. Jaundice with BT levels > 25 mg/dL (428 μmol/L) can lead to injury of the cerebral basal ganglia with long-term damage such as choreoathetosis, gaze palsy, loss of hearing, and development retardation. Four clinical phases of kernicterus have been identified:

  • The first phase is characterized by muscular hypotension, lethargy, and poor suck
  • The main symptom of the second phase is muscular hypertension, which can manifest as opisthotonos and fever
  • In the third phase, which manifests at the end of the first week of life, spasticity decreases or resolves completely
  • The fourth phase occurs in the second month of life or later and is characterized by extrapyramidal symptoms.

In a study /42/ of children with BT levels > 30 mg/dL (513 μmol/L) and advanced-phase acute bilirubin encephalopathy, the majority of subjects showed reversibility of the neurological symptoms in response to aggressive therapy.

Without intervention by phototherapy or exchange transfusion, 1% of pre-term neonates and 15% of mature neonates with hemolytic disease developed acute bilirubin encephalopathy /43/. The measurement of BT and BF therefore appears an acceptable marker for the presence of neurotoxic bilirubin. However, the concentration of BT correlates poorly with BIND and acute bilirubin encephalopathy, and there are many false-positive indications of the risk of bilirubin encephalopathy. As a result, exchange transfusions are performed quite frequently to prevent few cases of potential encephalopathy. There is evidence to suggest that the measurement of Bf can improve the situation /44/.

Hemolytic disease of the fetus and newborn – Generally

If the maternal immune system comes into contact with fetal erythrocytes of an incompatible blood group, this can lead to sensitization and the formation of antibodies in the mother. After the initial contact, antibodies are formed very gradually. For example, when a Rh-negative gravida comes into contact with Rh-positive fetal blood, antibodies will not be detectable until 16 weeks later. In most cases, antibody titers become easily detectable only after new contact with the antigen due to boosting. The maternal antibodies attack the fetal erythrocytes and cause hemolytic anemia which, in severe cases, can lead to hydrops fetalis and fetal death and, in mild cases, to increased jaundice in the neonate /45/.

– Rhesus incompatibility

The mother has produced antibodies to one of the Rhesus antigens D, C, c, E, e, which are fully developed as early as at day 30–45 post conception. Feto-maternal transfusions are observed from week 4 post conception and increase during the course of the pregnancy. Incompatibility, including miscarriage, only occurs with the second child. The mother has a positive result in the indirect Coombs test, the child a positive result in the direct Coombs test. If the mother and the child are both D-positive or D-negative, a test for incompatibility of other Rhesus antigens must be performed. In the child the blood type determination may result in a D-negative result despite having the D antigen. This may be due to complete occupation of the D-antigen of the infant’s erythrocytes with maternal anti-D-antibodies. Specific postnatal therapy of the newborn focuses on the anemia and hyperbilirubinemia. Common practice suggests that bilirubin levels should be kept below 20 mg/dL (342 μmol/L) /46/.

– AB0 incompatibility

AB0 incompatibility occurs in 15–25% of pregnancies, and hemolytic disease of the neonate in 1–4% of cases, depending on the ethnic constellation of the population. AB0 incompatibility may already occur during the first pregnancy. Children frequently affected are those of A or B type and an 0 type mother. Because AB0 antigens are not fully developed until the end of the fetal period, incompatibilities are not observed in premature neonates. Full-term neonates develop rapidly increasing, non-conjugated hyperbilirubinemia within the first 72 h, in which BT levels can rise above 20 mg/dL (342 μmol/L). The titer of the anti-A- and anti-B antibodies of the IgG class in the maternal serum is an indicator of whether a severe or mild hemolytic anemia can be expected in the neonate. Titers ≥ 512 predict a severe anemia with a diagnostic sensitivity of 90% and a specificity of 72%, and at titers ≥ 2048 phototherapy is always required /47/.

– Blood transfusion in premature neonate

Packed red blood cells (erythrocyte concentrate) of adult donors are used for correction of anemia in premature neonates. This leads to an increase in bilirubin. According to one study /48/ premature neonates with a birth weight < 1,250 g showed a daily rise in bilirubin of 1.4 mg/dL (24 μmol/L) during the first 10 days after blood transfusion. According to another investigation premature neonates with a birth weight > 1,250 g revealed a daily increase of 0.6 mg/dL (10 μmol/L).

Lucey-Driscoll syndrome

Intensive, temporary, non-conjugated hyperbilirubinemia of the neonate.

Primary shunt hyperbilirubinemia

Rare disorder caused by increased bilirubin production in the bone marrow. The course of the condition varies, with first clinical symptoms usually appearing not before puberty.

Hyperbilirubinemia after thoracic surgery

Hyperbilirubinemia develops in 51% of open-heart surgery patients and in 64% of patients who have undergone esophagectomy /49/. Average BT levels are 3 mg/dL (51 μmol/L). In these cases the hyperbilirubinemia is caused by hemolysis and is non-conjugated.

Table 5.2-3 Diseases with hepatic, predominantly conjugated hyperbilirubinemia

Clinical and laboratory findings

Acute hepatitis caused by hepatotropic viruses – Generalized

The incidence of elevated BT depends on the virus and age of the patient. In adults, jaundice develops in 70% of cases of hepatitis A, 33–50% of cases of hepatitis B and 22–30% of cases of hepatitis C. Children are less likely to develop jaundice than adults; patients with icteric hepatitis generally recover better than those with anicteric hepatitis. There is a direct correlation between age and peak serum bilirubin in children; an increase of 10 years in age is associated with an average increase of 5 mg/dL (85 μmol/L) in bilirubin /50/. The different degrees of severity of acute viral hepatitis are associated with the following average bilirubin levels /12/: Anicteric 1 mg/dl (17 μmol/L), typical icteric 8 mg/dL (137 μmol/L), cholestatic 18 mg/dL (308 μmol/L), necrotizing 18 mg/dL (308 μmol/L). In the case of a fatal outcome and development of a necrotic pattern of the liver enzymes (AST, ALT, LD, GLD activity of the same magnitude), bilirubin levels will continue to rise. If there are no complications during the course of the viral hepatitis, BT levels remain elevated for an average of 3 weeks, with peak levels generally occurring in the second week or, in mild cases, as early as in the first week after the onset of jaundice.

– Acute hepatitis A, acute hepatitis B

Rapid increase in aminotransferases and BT, rarely above 20 mg/dL (342 μmol/L). Aminotransferases reach their peak levels approximately 1 week after the onset of jaundice, while BT levels do not peak until the second week.

In two-thirds of patients, BT is in the range of 1–10 mg/dL (17–170 μmol/L); in mild forms of the disease levels rise up to 5 mg/dL (85 μmol/L). Concentrations rarely exceed 20 mg/dL (342 μmol/L). In severe forms of the disease, levels can be as high as 60 mg/dL (1030 μmol/L). BT decreases gradually during the course of the disease and normalizes within 6 weeks /51/.

– Acute hepatitis C, acute hepatitis D, acute hepatitis E

Jaundice occurs in only 25% of cases, BT 5–15 mg/dL (85–257 μmol/L). In individuals with concurrent hepatitis B infection, BT levels are similar to those in hepatitis B. Bilirubin levels behave similarly to those in hepatitis A. In some cases, the hepatitis is cholestatic in nature, in particular in pregnant women with BT persistently above 20 mg/dL (342 μmol/L) and high ALP and GGT activity.

– Involvement of the liver in systemic viral diseases

Approximately 50% of cases are found to have mild hyperbilirubinemia with BT levels below 5 mg/dL (85 μmol/L). In isolated cases of Epstein-Barr virus associated liver involvement and infectious mononucleosis, BT levels as high as 20 mg/dL (342 μmol/L) and above have been reported. Also refer to the section on aminotransferases.

Chronic hepatitis caused by hepatotropic viruses

The low-activity form is associated with normal BT levels; in the medium-activity form, discretely elevated subicteric levels up to 3 mg/dL (51 μmol/L) are possible. In the high-activity form, concentrations can be similar to those seen in acute viral hepatitis /52/.

Autoimmune hepatitis

Total bilirubin (BT) ranges between 2 and 10 mg/dL (34–171 μmol/L).

Alcoholic hepatitis

In asymptomatic alcoholic hepatitis, BT levels are within the reference interval, in the persisting form they are in the upper reference interval, in the chronic progressive form up to 2 mg/dL (34 μmol/L), and in acute hepatitis levels can exceed 10 mg/dL (170 μmol/L). Patients with severe hepatitis and a decrease in BT of at least 25% after 6 to 9 days of treatment with corticosteroids had a better outcome than those with a smaller decrease /53/.


Acute intoxication with aliphatic halogenated hydrocarbons (carbon tetrachloride, chloroform) and aromatic halogenated hydrocarbons (bromo-benzene, polychlorinated biphenyls) initially presents with non-specific general gastrointestinal symptoms, followed by a dramatic increase in aminotransferases and BT within 48 h. Drugs such as acetaminophen (paracetamol) and halothane induce acute hepatitis, with BT above 20 mg/dL (342 μmol/L) in fatal cases. In 40% of halothane-induced hepatitis cases, BT concentrations are below 10 mg/dL (170 μmol/L).

Drugs which cause mild liver injury with only slightly elevated liver enzymes do not lead to an increase in bilirubin. In cases of more severe liver injury presenting as viral hepatitis, aminotransferases are elevated approximately 10-fold and BT up to 5 mg/dL (85 μmol/L). Drugs causing cholestasis lead to an elevation in BT above 5 mg/dL (85 μmol/L) and to elevated ALP. Aminotransferases are slightly increased or not elevated, GGT is not elevated proportionately to ALP /53/.

Liver cirrhosis – Posthepatitic

Laboratory tests are an important tool in the assessment of the severity of cirrhosis. One evaluation system is the Child-Pugh score (Tab. 5.2-7 – Classification of liver function based on the Child-Pugh score). Child A score patients undergoing a surgical portosystemic shunt procedure due to esophageal variceal bleeding have a good prognosis for the next three years /54/.

– Primary biliary cirrhosis (PBC)

Hyperbilirubinemia does not develop until the symptomatic phase of PBC i.e., relatively late. In one study /55/, 36% of patients with PBC had hyperbilirubinemia at admission. 60% of patients developed hyperbilirubinemia during the course of the disease, with elevated levels of Bu and Bc /56/. Increasing or persistently elevated levels above 2 mg/dL (34 μmol/L) were a poor prognostic sign /57/.

– Alcoholic liver cirrhosis

In one study /58/, patients with alcoholic liver cirrhosis had an average BT concentration of 1.4 mg/dL (23 μmol/L) with a range of 0.2–7.5 mg/dL (3–128 μmol/L).

– Hepatocellular carcinoma (HCC)

Most patients with HCC are subicteric with BT levels below 3 mg/dL (51 μmol/L). The BT concentration is used as a prognostic criterion in the Okuda staging system (Tab. 5.2-7 – Okuda staging system for primary hepatocellular carcinoma). 40% of patients with liver metastases have hyperbilirubinemia /59/.

Jaundice in pregnancy /60/ –Hyperemesis gravidarum

BT can rise up to levels of 7 mg/dL (120 μmol/L) associated with a slight increase in aminotransferases and ALP.

– Benign recurrent cholestasis of pregnancy

This is a reversible form of cholestasis that occurs in the last trimester of pregnancy. The main symptom is pruritus, followed by jaundice 2 weeks later, with BT rarely exceeding 6.4 mg/dL (109 μmol/L). Pruritus and hyperbilirubinemia disappear within 2 weeks after delivery. These patients have a mutation of the Multi-drug resistance gene 3 (MDR3), which encodes canalicular phosphatidyl translocase. Also refer to the Section 1.9 – Gamma-glutamyl transferase (GGT).

– HELLP syndrom

Patients are typically Caucasian females who are over 25 years of age and multipara. The syndrome develops during weeks 24–28 of pregnancy. Elevations in BT can be as high as 2.6–16.6 mg/dL (44–284 μmol/L) and reflect the hemolysis and hepatocellular dysfunction.

– Eclampsia

Jaundice is relatively mild, BT is below 5 mg/dL (85 μmol/L) with a concomitant slight increase in aminotransferases in 40% of gravidae.

Hypoxic hepatitis

Hypoxic hepatitis, also known as ischemic hepatitis or shock liver, results from reduced hepatic blood flow. BT concentration can rise above 20 mg/dL (342 μmol/L) /61/. Also refer to the Section 1.6 – Alanine aminotransferase (ALT), Aspartate aminotransferase (AST).

Partial hepatectomy

The preoperative concentration of BT as well as the number of resected segments are important criteria of postoperative hyperbilirubinemia. The parenchymal ischemia and sepsis have synergistic impacts. Prior to the surgery, the range was 0.3–1.6 mg/dL (5–27 μmol/L), while post surgery it was 0.3–51 mg/dL (5–872 μmol/L), but in most patients it did not exceed 4.4 mg/dL (75 μmol/L) /62/. ALP was slightly elevated and cholesterol reduced.

Liver transplantation

Measured daily, Bc and Bδ are good markers for the detection of transplant rejection and more suitable than the enzymes AST and ALP. Transplant rejection is suggested by the following bilirubin profiles in relation to BT /63/:

  • Rapid decrease in Bδ or its persistence at a 30% proportion of total bilirubin
  • Steep increase in Bc or its persistence at greater than 50% proportion of total bilirubin.

Table 5.2-4 Diseases associated with posthepatic, predominantly conjugated hyperbilirubinemia

Clinical and laboratory findings

Extrahepatic cholestasis

In 40% of cases, extrahepatic cholestasis is caused by bile duct stones. Usually there is incomplete obstruction of the choledochus, as seen in pancreatitis, in early-stage tumors and in lymph node swelling. In these cases, the jaundice is acute, with BT above 10 mg/dL (170 μmol/L) in two-thirds of cases. The complete obstruction of the Papilla Vateri is usually due to a carcinoma of the papilla or of the head of pancreas. The associated jaundice develops slowly and is progressive. Patients often present with subicteric levels (below 3 mg/dL; 51 μmol/L) /64/.

Bile duct obstruction (stone, tumor, liver fluke), Bile duct tumors (cholangiocarcinoma, papillary carcinoma), common Bile compression (pancreatic carcinoma, lymphomas of the porta hepatis region, pancreatitis), anatomic variant

Obstruction of the bile duct leads to an increase in serum bilirubin after a latency period of 3 days to 1 week, followed by a phase of continuous increase in bilirubin levels of 1–3 mg/dL (17–51 μmol/L) per day until a plateau of approximately 15–25 mg/dL (257–428 μmol/L) is reached at the end of the second week. The rise is slower than in acute hepatitis, with plateau levels rarely exceeding 30 mg/dL (513 μmol/L). Higher levels indicate complications (e.g., renal failure, infection, hemolysis).

Cholestases with pathogenic etiologies in the choledochus include purulent cholangitis, primary sclerosing cholangitis, and primary biliary cirrhosis.

In purulent cholangitis with the three symptoms of fever, jaundice and pain in the upper right abdomen, leukocytosis, significantly elevated CRP and an increase in cholestasis enzymes is present. The severity of hyperbilirubinemia depends on whether incomplete or, less frequently, complete obstruction syndrome develops. Urine urobilinogen is positive in acute cholangitis, even in the absence of jaundice.

Primary sclerosing cholangitis (PSC)

Serum bilirubin levels are usually normal in early stages, but rise gradually as the disease progresses. Occasionally, BT levels are seen to fluctuate in early stages. They reflect transient blockages caused by inflammation, strictures, or small gallstones. Also refer to the Section 1.6 – Alanine aminotransferase (ALT), Aspartate aminotransferase (AST).


With increased synthesis of bilirubin there is a risk of gallstones developing. Non-conjugated bilirubin binds to calcium in the bile ducts, forming calcium bilirubin salts, which in turn are the nidus for the formation of cholesterol stones. One study /65/ correlated the prospective risk of developing gallstones with the bilirubin concentration. Individuals with the homozygous genotype 7/7 of UGT glucuronyltransferase A1 had an increased risk (average hazard ratio 1.18) and an average bilirubin concentration of 1.34 mg/dL, which was, on average, 0.5 mg/dL (8 μmol/L) higher compared to the wild type (genotype 6/6).

Biliary atresia (BA) /66/

Biliary atresia occurs with an incidence of approximately 1 in 15,000 live births and is the most common indication for a liver transplant in children. It is caused by an inflammatory process of unknown etiology, which originates in the extrahepatic bile ducts. The inflammation causes necrosis and obliteration of the bile ducts, which are progressively destroyed, leading ultimately to cirrhosis of the liver. The neonates have prolonged jaundice with BT concentrations in the range of 10–20 mg/dL (171–342 μmol/L), hepatomegaly with GGT levels of 200–600 U/L, and elevated lipoprotein X of 2–9 g/L. Their stool may be of normal color during the first few days, but later becomes colorless. Urine is dark, and bilirubinemia is present. Except in a small number of breast-fed newborns, prolonged jaundice is often caused by neonatal hepatitis, immunologically-induced hemolysis, and biliary atresia. Once hemolyis has been excluded as the cause, biliary atresia must be differentiated from neonatal hepatitis due to (e.g., hepatitis B, cytomegalic or rubella infection). Other possible causes include α1-antitrypsin deficiency, cystic fibrosis, and metabolic diseases such as fructosemia and galactosemia.

Table 5.2-5 Hereditary hyperbilirubinemias

Clinical and laboratory findings

Gilbert’s syndrome (GS), also known as familial non hemolytic hyperbilirubinemia or Meulengracht’s disease /14/

GS is caused by a 70–80% reduction in the uridine diphosphate glucuronyltransferase isoform 1A1 (UGT1A1) encoded by the UGT1A1 gene, which is associated with a TATA box promoter region /67/. The latter usually has 6 thymidine-adenine repeats. In many populations, GS is associated with a homozygous expansion of 7 TA repeats, also called UGT1A1*28. In 94% of cases GS is also associated with isoforms UGT1A6 and UGT1A7, which cause a 50% and 70% reduction in activity, respectively. Especially in Asian regions, GS can also be the result of heterozygous missense mutations such as Gly71Arg (UGT1A1*6) or Tyr486Asp (UGT1A1*7), without affecting the TATA box.

GS is characterized by normal liver enzymes, normal liver histology, reduced clearance of bilirubin from blood, and mild jaundice with a tendency to fluctuate. GS is diagnosed in up to 5% of the population. In these cases non-conjugated hyperbilirubinemia is present. Symptoms often do not appear until adolescence or adulthood. Some patients have unspecific symptoms such as fatigue, weakness, or abdominal problems. Significant increases in bilirubin are often only observed under stress conditions (e.g., fasting due to weight reduction, vomiting during pregnancy, infections, stress situations). Bilirubin levels rarely exceed 4–5 mg/dL (68–85 μmol/L), although concentrations of up to 8 mg/dL (137 μmol/L) have been reported. Hematologic diseases and hepatic parenchymal cell injury must be excluded. Helpful tests include blood count, differential blood count, reticulocyte count, and normal ALT, GGT and ALP activities. GS is caused by a 25–30% reduction in bilirubin clearance due to defective glucuronidation of bilirubin in the hepatocytes as a result of UDP-glucuronyltransferase A1 (EC reduction to about 30%. Clinically suspected GS is confirmed by molecular genetic analysis. This requires 5 mL of EDTA blood.

The enzyme UDP-glucuronyltransferase A1 also plays a key role in the metabolism of drugs (e.g., the chemotherapeutic drug CPT-11/irinotecan). In patients with dinucleotidase expansion of 7 repeats the chemotherapeutic drug is metabolized slower, which leads to severe side effects. While there are no severe side effects in genotype 6/6, their incidence is up to 50% or above in genotypes 6/7 and 7/7, respectively.

Crigler-Najjar syndrome (CNS) /14/

CNS is an autosomal recessive, inherited disease which manifests as non-hemolytic jaundice with severe non-conjugated hyperbilirubinemia. CNS is characterized by complete deficiency or reduced activity of the enzyme uridine 5’-diphosphate (UDP) glucuronyltransferase (EC (UGT), which catalyzes the glucuronidation of bilirubin. The non-conjugated hyperbilirubinemia develops during the first few days of life. Causes such as hemolysis or sepsis must be excluded. There are two types of CNS:

  • Type I CNS; characterized by complete deficiency of the enzyme bilirubin-UDP-glucuronyltransferase, which is encoded by the UGT1A1 gene and localized on chromosome 2q37.1. Over 90 genetic alterations, such as mutations, small insertions or small deletions in the 5 exons of the UGT1A1 gene have been described. The prevalence is 1 : 1,000,000. The Bu concentration is 20–35 mg/dL (342–599 μmol/L); even levels above 50 mg/dL (855 μmol/L) have been reported. Only few patients reach adulthood. All patients have a damaged central nervous system and many die from bilirubin encephalopathy. The Bu concentration can be temporarily reduced by phototherapy and plasmapheresis. The only cure is a liver transplant.
  • Type II CNS; characterized by a defect in the variable region of exon A1 of UDP-glucuronyltransferase A1. The activity of UDP glucuronyltransferase A1 is reduced to below 10%. Bu usually does not exceed 20 mg/dL (342 μmol/L). In approximately 50% of patients jaundice occurs during the first year of life, but it also may not develop until 30 years of age or later. In type II, the hyperbilirubinemia can be reduced by treatment with phenobarbital (maintenance dose 0.6 mg/kg/day). This is not possible in type I.

The diagnosis of CNS is confirmed by molecular genetic analysis or by measuring UGT activity in a liver biopsy sample.

Dubin-Johnson syndrome (DJS)

Autosomal recessive disease which does not become clinically manifest until age 10–25. It has a particularly high prevalence (1 : 300) in Iranian Jews.

The liver tissue is macroscopically dark due to deposition of brown pigment, which is caused by a defect in the excretion of bilirubin and other organic ions (e.g., bromosulfophthalein, X-ray contrast medium, rose bengal) /67/. There is also a defect in coproporphyrin metabolism with a reduction in isomer III and increase in isomer I of usually less than 45% to approximately 80%. Hyperbilirubinemia is usually in the range of 2–5 mg/dL (34–85 μmol/L), but levels can rise as high as 20 mg/dL (342 μmol/L), in particular in the neonatal type. Bc makes up approximately 50%. The jaundice is discrete, may be temporarily absent, and becomes more severe in the presence of clinical symptoms such as pain or pressure in the upper abdomen, fatigability, weakness, lack of appetite. Episodes can be triggered by stress, menstruation, alcohol and 19-nor-steroids with a methyl or ethyl group in position 17-α. A characteristic feature is the kinetics of the bromosulfophthalein test. Mild elevations in ALP may occur. An important diagnostic criterion is the coproporphyrin III/coproporphyrin I ratio. In healthy individuals it is 3–4, in DJS it is below 0.5 /68/.

Recent studies suggest that, in DJS, biliary elimination of leukotrienes is defective and compensated by renal elimination. In one study /69/, the urinary leukotriene/creatinine (nmol/mol) ratio for LTE4 was 9–25 in healthy individuals and 69–201 in DJS; for ω-carboxy-LTE4 it was 13–33 in healthy individuals and 267–353 in DJS, and for ω-carboxy-tetranor-LTE3 10–36 in healthy individuals and 439–587 in DJS.

Rotor syndrome

Familial hyperbilirubinemia with elevated Bc /67/. Bc rarely exceeds 5 mg/dL (85 μmol/L). Bromosulfophthalein retention is markedly increased but, unlike with the Dubin-Johnson syndrome, there is no renewed increase after 90 minutes. The liver is macroscopically normal. Coproporphyrin excretion is elevated 3–5-fold compared to that of healthy individuals and patients with Dubin-Johnson syndrome. In Rotor syndrome, the coproporphyrin III/coproporphyrin I ratio is lower than that in healthy individuals, but higher than in Dubin-Johnson syndrome.

Benign recurrent intrahepatic cholestasis (BRiC)

Episodic intrahepatic cholestatis, which predominantly occurs in spring and autumn. BRC clinically presents as obstructive jaundice with severe pruritus and gastrointestinal problems. Laboratory findings show the presence of conjugated hyperbilirubinemia with levels above 20 mg/dL (342 μmol/L) and 4–5-fold elevation of ALP without concurrent increase in aminotransferases and GGT. In 75% of cases, the onset of the disease occurs before age 20, the icteric phases last 4–5 months with intervals of 1 month up to 20 years /70/.

This type of cholestasis shows parallels with benign recurrent intrahepatic cholestasis, but differs from it in its rapid progression and its laboratory findings. Lipoprotein X (LPX) is not detectable, serum cholesterol is low, GGT activity is normal in the presence of hyperbilirubinemia. PFIC is classified into three types /71/:

  • Type I PFIC, also known as Byler’s disease. It develops during the first year of life and occurs in episodes. Children with this condition develop liver cirrhosis. GGT is normal. The FIC1 gene, which is defective in this disease, encodes a P-type ATPase, which is believed to act as amino phospholipid translocase. It is located in the ileum and in cholangiocytes.
  • Type II PFIC. The clinical manifestation and laboratory findings are the same as in type I. There is a bile acid translocase defect in the hepatic bile capillaries.
  • Type III PFIC. This familial disease is similar to primary sclerosing cholangitis. In terms of laboratory findings it differs from the other two types by high cholesterol, high GGT, and the presence of LPX. There is a mutation in the Multidrug resistance gene 3 (MDR3), which encodes the phosphatidyl translocase of the bile capillaries. Thus, no phosphatidylcholine is transported into the gallbladder, and as a result the capillaries are damaged and GGT is elevated.

Table 5.2-6 Guidelines for exchange transfusion in low birth weight infants based on total bilirubin and bilirubin/albumin ratio, modified according to Ref. /72/

Birth weight (g)

< 1,250







Standard risk and bilirubin (mg/dL)





Standard risk and bilirubin/albumin ratio (mg/mg) x 1,000





High risk and bilirubin (mg/dL)





High risk and bilirubin/albumin ratio (mg/mg) x 1,000





Risk factors: Apgar < 3 at 5 min; PaO2 ≤ 40 mmHg at > 2 h, pH ≤ 7.15 at ≥ 1 h,

Birth weight < 1,000 g, hemolysis; clinical or central nervous system deterioration;

total protein ≤ 4 g/dL or albumin ≤ 2.5 g/dL

Table 5.2-7 Classification of liver function based on the Child-Pugh score /73/









< 2 (34)

> 3.5

> 70




2–3 (34–51)






> 3 (51)

< 2.8

< 40



PTT, prothrombin time

Table 5.2-8 Okuda staging system for primary hepatocellular carcinoma /74/


0 points

1 point

A: Liver involvement

≤ 50%

> 50%

B: Ascites



C: Bilirubin

≤ 3 mg/dL

(51 μmol/L)

> 3 mg/dL

(51 μmol/L)

D: Albumin

≥ 3 g/dL

< 3 g/dL

A + B + C + D = 0

A + B + C + D = 1–2

A + B + C + D = 3–4

points: stage 1

points: stage 2

points: stage 3

The median survival time is 11 months in stage 1, three months in stage 2 and one month in stage 3. The 1-year survival rates in stages 1–3 are 39%, 12% and 3%, respectively.

Table 5.3-1 L-Carnitine reference intervals


Adults /6/

Free L-carnitine

Total L-carnitine





Data in μmol/L

Neonates/infants /7/

Age group

Free L-carnitine

Total L-carnitine

1. day



2.–7. day



8.–28. day



29. day – 1 yr



No further significant changes after 1st year of life. Data in μmol/L.


Renal L-carnitine clearance varies greatly between individuals, depending, inter alia, on food intake and diet.

Adults /89/

60–600 μmol/day

Neonates /10/

1.4–16 μmol/day

Infants /10/

50–250 μmol/day

Free L-carnitine

Total L-carnitine


Clearance [ml/min.] /11/






Free L-carnitine [μmol/g NCP]

Total L-carnitine [μmol/g NCP*]

Muscle /12/





Liver /13/





* NCP, non-collagen protein, specified as x ± 2 s

Seminal plasma /13/


Free L-carnitine





Data expressed in μmol/L

Table 5.3-2 Hereditary causes of systemic secondary L-carnitine deficiency

Diseases, defects, metabolic situations

Clinical and laboratory findings

Organic acidurias /22/

Isovaleric aciduria (isovaleryl-CoA dehydrogenase)

Propionic aciduria (propionyl-CoA carboxylase)

Methyl malonic aciduria (methyl malonyl-CoA mutase apoenzyme, vitamin B12 deficiency)

Glutaric aciduria I (glutaryl-CoA dehydrogenase)

The accumulating acyl-CoA compounds have a detrimental, partly toxic effect. The CoA pool in the mitochondria is occupied and energy-providing CoA-dependent processes are inhibited, resulting in energy deficiency. L-Carnitine is the transfer recipient of acyl groups from CoA as catalyzed by carnitine acyl transferases; L-Carnitine also transports these groups out of the mitochondria and cells. Thus CoA is released again for participation in physiological reactions. In the kidneys, acylcarnitine is more poorly reabsorbed than free L-Carnitine, thus resulting in secondary serum tissue L-Carnitine deficiency due to excretion of acylcarnitines.

Enzyme defects of mitochondrial β-oxidation and the respiratory chain /23/

Medium-chain acyl-CoA dehydrogenase (MCAD)

Long-chain acyl-CoA dehydrogenase (LCAD)

Short-chain acyl-CoA dehydrogenase (SCAD)

Long-chain L-3-hydroxyacyl-CoA dehydrogenase (LCHAD)

Electron transfer flavoprotein (ETF, FAD)

Electron transfer flavoprotein dehydrogenase

The direct reduction in the β-oxidation of fatty acids leads to fat accumulation in tissues as well as fasting intolerance with hypoglycemia and hypo ketonemia. ω-oxidation produces dicarboxylic acid CoA or dicarboxylic acid carnitine esters. The clearance of acylcarnitines from the cells and their increased excretion via the kidneys lead to secondary L-Carnitine deficiency. Defect-specific acylcarnitines are detectable in urine.

Table 5.3-3 Acquired causes of systemic secondary L-carnitine deficiency

Clinical and laboratory findings

Long-term L-carnitine-free diet /2425/– Total parenteral nutrition (TPN), Cachexia and extremely restrictive vegetarian diets

Even though as part of tube feeding and TPN protocols special consideration is given to appropriately adjusting the intake of essential amino acids, fats, minerals, vitamins and carbohydrates, the endogenous L-carnitine biosynthesis alone is not sufficient to maintain L-carnitine levels for an extended period of time. L-carnitine balance is also influenced by the excretion of L-carnitine, in particular long-chain acylcarnitine, via the gallbladder. Dietary L-carnitine or L-carnitine applied in the form of oral medication can be metabolized by bacteria in the gastrointestinal tract. Patients on TPN who are not deficient in L-carnitine should be administered 2–5 mg/kg of body weight of L-carnitine daily in 3–4 applications. Pharmacological dosages are between 50 and 100 mg/kg of body weight per day and should be reserved for the removal of toxic metabolic products (e.g., in the case of congenital metabolic disorders).

Medication /26/ – Valproic acid, pivalic acid

By activation of the acids, acyl-CoA compounds are formed and CoA-dependent metabolic pathways are inhibited. Valproyl- and pivaloyl-L-carnitine excretions lead to iatrogenic L-carnitine deficiency.


Decrease of the L-carnitine pool occur in patients who are on hemodialysis. They manifest as skeletal muscle myopathy, impaired exercise capacity, cardiomyopathy and complications during dialysis such as hypotension, seizures, weakness, and fatigue. In hemodialysis patients, the pool of free L-carnitine is reduced compared to normal individuals. Despite significantly reduced free L-carnitine in plasma, the total L-carnitine concentration remains relatively stable. This is due to an increase in acylcarnitines to levels between 33% and 47.6% in hemodialysis patients compared to levels between 11.6% and 17.4% in normal individuals. In one study /27/, the composition of the L-carnitine pool was as follows: patients on dialysis: 57.2% of free L-carnitine and 42.8% of acylcarnitine; healthy controls: 82.6% of free L-carnitine and 17.4% of acylcarnitine. The accumulation of acylcarnitine in chronic hemodialysis patients depends on the chain length of fatty acid carnitine esters, the associated increased molecular weight, and the lipophilicity, which also depends on the fatty acid chain length.

There is a correlation between the concentration of free L-carnitine in plasma and erythrocyte osmotic resistance: the higher the L-carnitine concentration, the higher the resistance. Dialysis patients who are supplemented with L-carnitine have been shown to require approximately 20% less erythropoietin for the treatment of renal anemia than patients who are not supplemented.

Pregnancy /28/

The serum concentration of free L-carnitine is decreased as early as in the 12th week of pregnancy. By the time of delivery, it has decreased to a level consistent with systemic primary L-carnitine deficiency (10.0 ± 5.1 μmol/L). The acylcarnitine concentration, however, has remained stable so that the esterified fraction accounts for over 50% of total L-carnitine. The high fraction of acylcarnitine results from increased fatty acid metabolism in pregnancy; the secondary L-carnitine deficiency is due to increased renal clearance of L-carnitine (over 3-fold) and the L-carnitine requirement of the fetus.

Diabetes type 1

In children and adolescents with diabetes type 1, the plasma levels of total L-carnitine and free L-carnitine are reduced. The reduction depends on the duration of the diabetes and may be related to the complications of the disease. In one study /29/, the concentrations of total L-carnitine, free L-carnitine and acylcarnitine in μmol/L were 30.1 ± 7.26, 20.0 ± 4.5, and 10.2 ± 6.47 respectively. The ratio of acylcarnitine to free carnitine was 0.544 ± 0.369.

Table 5.4-1 Kinetic UV-method for the determination of uric acid /2/

Uric acid + 2 H 2 O + O 2 uricase allantoin + CO 2 + H 2 O 2 H 2 O 2 + ethanol catalase acetaldehyde + 2 H 2 O Acetaldehyde + NADP + ADH acetate + NADP + + H +

Table 5.4-2 Uric acid reference intervals

Serum, plasma

Adults /6/:

2.3–6.1 (137–363)

3.6–8.2 (214–488)

Clinically, the upper reference interval values are too high. The defined upper thresholds are as follows:


≤ 6.0 (357)

≤ 7.0 (416)

Children /7/:

1–30 days

1.0–4.6 (59–271)

1.2–3.9 (71–230)

31–365 days

1.1–5.4 (65–319)

1.2–5.6 (71–330)

> 1–3 years

1.8–5.0 (106–295)

2.1–5.6 (124–330)

4–6 years

2.0–5.1 (118–301)

1.8–5.5 (106–325)

7–9 years

1.8–5.5 (106–325)

1.8–5.4 (106–319)

10–12 years

2.5–5.9 (148–348)

2.2–5.8 (130–342)

13–15 years

2.2–6.4 (130–378)

3.1–7.0 (183–413)

16–18 years

2.4–6.6 (142–389)

2.1–7.6 (124–448)

Data expressed in mg/dL (μmol/L). For children values are the 2.5th and 97.5th percentiles, for adults x ± 2s. Conversion: mg/dL × 59.485 = μmol/L

Uric acid clearance in adults /8/:

+ up to 800 mg (4.76 mmol) on normal diet, up to 600 mg (3.57 mmol) on low-purine diet

Uric acid clearance in adults /6/: + 5–12 ml/min.

Uric acid/creatinine ratio in adults /8/:

+ < 0.80 (mg/mg) on normal diet

Fractionated excretion of uric acid (FEUA): 4–10%

Uric acid clearance in male infants in the first year of life /9/:

0.14–2.02 mmol/L; 0.03–0.58 mmol/24 h; 3.8–111 μmol/kg; uric acid/creatinine ratio 0.03–1.03 mmol/mmol; clearance 0.09–1.79 ml/min. or 0.41–10.3 ml/min. × 1.73 m2; fractional excretion 0.84–21.5%

Conversion of uric acid: mg × 59.48 = μmol

Table 5.4-3 Causes of hyperuricemia /11/

  • Diet: increased consumption of purines from animal products rich in cells (offal); alcohol consumption
  • Increased consumption of dietary fructose
  • Reduced elimination due to renal insufficiency
  • Increased cell turnover (polycythemia vera, hemolytic anemia), chemotherapy of malignant tumors
  • Diuretics, low-dose aspirin therapy

Mutation of genes such as:

  • XO, encodes xanthine oxidase
  • URAT1, encodes the urate transporter which transports uric acid into the tubular lumen
  • OAT1 and OAT3, encode organic anion transporters, which take up uric acid from the tubular lumen in exchange for anions
  • SCL2A9 , encodes the fructose transporter

Table 5.4-4 Classification of hyperuricemias /1416/



Reduced renal elimination


Idiopathic HGPRT deficiency

  • Total (Lesch-Nyhan syndrome)
  • Partial (Kelley-Seegmiller syndrome)

Increased PRPP synthase activity

Genetic polymorphisms in the genes of the uric acid transporters (Tab. 5.4-3 – Causes of hyperuricemia) lead to increased renal tubular reabsorption of uric acid with consecutive accumulation of uric acid in plasma and gouty diathesis


Myeloproliferative diseases

Lymphoproliferative diseases

Hemolytic diseases

Polycythemia vera


Psoriasis, severe type

Glucose-6-phosphatase deficiency (type I glycogenosis)

Excessive use of alcohol

Purine-rich diet

Cytostatic therapy and radiotherapy

Remission of anemias (pernicious, hemolytic)

Physical exercise


Reduced renal clearance due to chronic nephropathy, decreased renal perfusion or tubular dysfunction, e.g.,

  • Fasting
  • Ketoacidosis (diabetic, alcoholic)
  • Lactic acidosis (for causes see section 5.6)
  • Drugs e.g., salicylates (< 2 g/day), pyrazinamide, diuretics, levodopa, carbidopa, ethambutol, nicotinic acid, cyclosporine, alcohol
  • Hyperparathyroidism, hypothyroidism
  • EPH gestosis
  • Lead nephropathy
  • Bartter syndrome
  • Down syndrome


Increased uric acid clearance

Decreased/normal uric acid clearance

HGPRT, hypoxanthine-guanine phosphoribosyltransferase; PRPP, phosphoribosyl pyrophosphate

Table 5.4-6 Calculation of fractional uric acid clearance (FEUA)

FE (%) = 100 × Urinary UA (mg/dL) × Serum Cr (mg/dL) Serum UA (mg/dL) × Urinary Cr (mg/dL)

Cr, creatinine; UA, uric acid; S, serum; U, urine

Table 5.4-5 Influence of drugs on the serum uric acid concentration /26/



Salicylates < 2 g/day



Cyclosporine A


Nicotinic acid






Sorbit, xylitol infusions







Salicylates > 2 g/day

Uricosuric drugs







* In low doses, ** In higher doses

Table 5.4-7 Frequency of gout as a function of the serum uric acid level /25/


Frequency (%)

< 6












≥ 9,0



Values expressed in mg/dL (μmol/L)

Table 5.4-8 Frequency of nephrolithiasis as a function of serum uric acid level and urinary uric acid clearance /25/


Frequency (%)

Serum uric acid concentration*










> 11.0



Urinary uric acid clearance**

< 300















> 1100



* Values expressed in mg/dL (μmol/L); ** Data in mg (mmol/L)

Table 5.4-9 Diseases and conditions associated with hyperuricemia

Clinical and laboratory findings

Primary hyperuricemia – Acute gouty arthritis /1314/

Acute gouty arthritis usually initially manifests as nocturnal-onset acute attacks of mono arthritis accompanied by the clinical signs of pain, inflammation, swelling, and redness, with the metatarsophalangeal joint and other joints of the lower extremities being affected first. Triggers of an acute attack are: excessively large meal, heavy alcohol consumption, physical or mental stress, change in temperature and climate. Adequate treatment with an NSAID leads to resolution of pain within 24 h of an acute attack.

Laboratory findings: the inflammation manifests as mild neutrophilia with a possible left shift, and an elevated erythrocyte sedimentation rate. While the presence of acute arthritis and uric acid levels above 9 mg/dL (535 μmol/L) are highly suggestive of acute gouty arthritis, they do not prove it. Similarly, normal levels do not exclude gouty arthritis. For example, 43% of patients with an acute gout attack have serum uric acid levels within the reference range while in approximately 70% of patients levels are an average 2.0 mg/dL (119 μmol/L) lower than during the pain-free interval /28/. In uncertain cases a joint puncture must be performed to exclude other crystal arthropathies such as acute pseudo gout (chondrocalcinosis). The presence of birefringent uric acid crystals, which were phagocytized by polymorphonuclear neutrophil granulocytes (PMN) and whose tips project from the PMN, is a reliable indicator of gout (also see Section 49 – Synovial fluid). Further investigations for determining the cause of the gout include blood count, LD activity, serum protein electrophoresis, creatinine, and fractional uric acid clearance.

– Lesch-Nyhan syndrome

Lesch-Nyhan syndrome is a hereditary X-linked recessive condition which leads to gout in childhood. It is due to a severe or complete deficiency of hypoxanthine-guanine phospho ribosyl transferase (HGPRT) activity. The main clinical manifestations are uric acid deposition in the kidneys and urinary tract, joint symptoms, changes in personality, and megaloblastic anemia /29/. In heterozygous mutation carriers with HGPRT deficiency, hyperuricemia, renal calculi, and gout attacks may be the only symptoms.

Secondary hyperuricemia – Generalized

Secondary hyperuricemias are less common than the primary forms and result from different disorders and diseases.

– Renal insufficiency

The majority of patients with renal insufficiency have elevated uric acid levels, although these rarely exceed 10 mg/dL (595 μmol/L). There is no direct relationship between increased uric acid concentration and progression of renal insufficiency.

The incidence of hospital-acquired acute kidney injury (AKI) is estimated to be 3–7% from epidemiological studies, but increases to 20–30% in the intensive care unit setting. Apart from the established crystal precipitation with profound hyperuricemia, various non-crystal mechanisms have also been proposed in the pathogenesis of AKI. In a retrospective study /55/ evaluating the risk of AKI serum uric levels > 9.4 mg/dL (564 μmol/l) had the highest incidence of AKI, Odds ratio 1.79 (95% CI 1.13–2.82)

– Preeclampsia (PE) /30/

PE is diagnosed based on the symptoms of hyperuricemia, hypertension and proteinuria which occur from pregnancy week 20. In an uncomplicated pregnancy, uric acid levels fall by 25–35% during the first trimester before increasing steadily to levels of same-age non-pregnant women at delivery. The hyperuricemia in PE is of multifactorial origin. Underlying conditions include increased uric acid production from maternal, fetal or placental tissues due to ischemia or re perfusion injury, as well as reduced antioxidant capacity. The uric acid level prior to pregnancy week 25 is not a reliable predictor for PE. In preeclamptic pregnancies, elevated uric acid usually predates the development of hypertension and proteinuria. In some cases, creatinine, cholesterol and triglycerides are also elevated, but the highest correlation is observed between uric acid and PE; the odds ratios of hyperuricemia, hypercholesterolemia and hypertriglyceridemia are 35.3, 6.9 and 5.6 respectively. While in complication-free pregnancies uric acid levels are in the range of 5.1–5.8 mg/dL (297–345 μmol/L) in pregnancy week 38, they are between 6.6 and 8.3 mg/dL (393–494 μmol/L) in preeclamptic pregnancies.

– Tumor lysis syndrome (TLS) /31/

The main diagnostic symptoms of TLS are hyperuricemia, hyper phosphatemia, hyperkalemia, and hypocalcemia. They occur when the kidneys cannot eliminate the large amounts of uric acid, phosphate and potassium that are released as a result of therapy-induced cell lysis. If the renal load becomes too much, acute renal failure can occur. Predisposing factors include large tumor bulk, rapid tumor growth, high sensitivity of the tumor to a chemotherapeutic, pre-existing dehydration, hyperuricemia or impaired kidney function. B- and T-cell leukemias have the highest proportion of rapidly proliferating tumor cells and lead to a significant increase in uric acid even without chemotherapy. In these malignant diseases LD is high, and patients with pre-existing high LD activity prior to chemotherapy are generally more likely to develop therapy-induced TLS.

Laboratory findings: TLS should be suspected if uric acid is above 10 mg/dL (595 μmol/L), potassium above 6 mmol/L and phosphate above 5 mg/dL (1.62 mmol/L). Once chemotherapy has started, potassium levels usually rise after 6–72 h, phosphate levels after 24–48 h and uric acid levels after 48–72 h. In acute myeloic and acute lymphatic leukemia, levels can be as high as 20 mg/dL (1190 μmol/L) and higher. Severe hyperuricemias are also regularly observed in chronic myeloic leukemia, but rarely in chronic lymphatic leukemia. A multivariate analysis /32/ of patients with acute myeloid leukemia has shown that the following laboratory test results prior to therapy are indicative of the risk of TLS: elevated LD, uric acid above 7.5 mg/dL (446 μmol/L) and a leukocyte count above 25 × 109/L. During radiation therapy for malignant tumors (e.g., malignant non-Hodgkin lymphomas) serum uric acid levels can be as high as 50 mg/dL (2,970 μmol/L).

– Cyanotic congenital heart disease (CCHD)

Patients with CCHD develop polycythemia and impaired kidney function with increasing age. They also develop hyperuricemia. For example, 10 out of 59 patients aged 1 month to 30 years had uric acid levels > 8.0 mg/dL (476 μmol/L) /33/. In decompensated cardiac failure, the hyperuricemia is caused by reduced renal clearance of uric acid due to reduced tubular secretion.

– Transplant-associated hyperuricemia

Patients receiving treatment with cyclosporine develop hyperuricemia following heart transplant and kidney transplant surgery. In one study /34/, 81% of women and 72% of men with normal uric acid levels prior to transplant surgery had elevations above 7.5 mg/dL (446 μmol/L) and 8.5 mg/dL (506 μmol/L), respectively, 35 months after the transplant. Various factors contribute to hyperuricemia, including hypertension, reduced transplant function, use of diuretics, and use of cyclosporine.

– Calorie-free diet

Calorie-free diets lead to significant changes in the metabolic parameters: within 2 weeks, uric acid increases by an average 35% to 8.7 mg/dL (514 μmol/L), serum creatinine increases by approximately 11%, and urea levels fall by 26% /35/.

– Poisoning

Substances which cause primary damage to the renal tubules, such as lead, cadmium, and beryllium, lead to hyperuricemia due to reduced uric acid secretion by the renal tubules.

– Disorders of thyroid function

There is a clear association between hypothyroidism and hyperuricemia, but not between hyperthyreosis and hyperuricemia /36/. Hypothyroid hyperuricemia is believed to be due to a reduction in renal plasma flow and glomerular filtration rate.

– Alcohol

Consumption of alcohol leads to an increase of lactate and thus inhibition of renal uric acid excretion. In addition, alcoholic beverages, in particular beer, contain significant amounts of purines. Alcoholics are on average more likely to develop gout.

– Diuretics

In approximately 30% of patients receiving diuretic treatment, in particular thiazide treatment, the uric acid concentration increases by a maximum of 1 mg/dL (59 μmol/L). Up to 5% of hypertensive patients on long-term treatment with thiazide develop gout. Diuretics reduce renal elimination of uric acid due to competitive inhibition of uric acid secretion by the tubules and increased tubular reabsorption as a result of diuretic-induced volume depletion /37/.

– Peritoneal dialysis

Hyperuricemia with levels above 7.0 mg/dL (416 μmol/L) is diagnosed in approximately 30% of patients with peritoneal dialysis. It becomes more severe with decreasing residual renal function. Hypertensive patients have higher levels than normotensive individuals /38/.

– Congenital metabolic disorder

Medium-chain acyl-CoA dehydrogenase (MCAD) deficiency: with an incidence of 1 : 23,000, MCAD is one of the most common disorders of fatty acid metabolism in Northern Europe. It is a defect of acyl-CoA dehydrogenase in the breakdown of medium-chain fatty acids. Approximately 80–90% of individuals with this condition have a point mutation at position 985 A>G, which leads to protein misfolding. The defect is inherited in an autosomal recessive manner. Clinical symptoms usually first appear in infancy and include non-acidotic, hypo ketonemic hypoglycemia and slightly elevated amino­trans­ferases /39/. Approximately 85% of patients have hyperuricemia with levels in the range of 10–25 mg/dL (595–1487 μmol/L).

Other metabolic disorders associated with the concurrent presence of hypoglycemia, hyperuricemia and lactic acidosis, but without hypo ketonemia, include glycogen storage diseases, Reye’s syndrome, glucose-6-phosphatase deficiency, and hereditary fructose intolerance /40/.

Table 5.4-10 Association of hyperuricemia with other diseases /1112/

Clinical and laboratory findings

Hyperuricemia and associations – Generalized

There is evidence to suggest that hyperuricemia plays an important role in the development of hypertension, renal disease, and cardiovascular disease. Mechanisms involved in the development of these disorders are the renin-angiotensin-aldosterone system and endothelial dysfunction with the resulting reduced formation of NO.

– High blood pressure

Numerous studies have reported that hyperuricemia is associated with an increased risk for hypertension developing within 5 years of diagnosis. The association is stronger with primary than with secondary hypertension. Approximately 90% of adolescents with initial manifestation of hypertension have a uric acid concentration above 5.5 mg/dL (330 μmol/L). Their blood pressure correlates linearly with the uric acid concentration. Pathophysiologically, the process occurs in two steps: first, there is renal vasoconstriction due to hyperuricemia, resulting in reduced endothelial formation of NO. This activates the renin-angiotensin-aldosterone system, resulting in increased blood pressure. Histological studies describe an arteriolosclerosis consistent with that classically occurring in essential hypertension. Another cause of the increasing incidence of hypertension across the world is seen in the increased consumption of fructose in recent decades, which reduces the ATP pool and promotes the synthesis of uric acid. It is thought that there is a correlation between fructose consumption, hypertension and hyperuricemia.

– Acute and chronic renal insufficiency

The majority of patients with renal insufficiency have elevated uric acid levels, although these rarely exceed 10 mg/dL (595 μmol/L). There is no direct relationship between increased uric acid concentration and progression of renal insufficiency.

The incidence of hospital-acquired acute kidney injury (AKI) is estimated to be 3–7% from epidemiological studies, but increases to 20–30% in the intensive care unit setting. Apart from the established crystal precipitation with profound hyperuricemia, various non-crystal mechanisms have also been proposed in the pathogenesis of AKI. In a retrospective study /41/ evaluating the risk of AKI serum uric levels > 9.4 mg/dL (564 μmol/l) had the highest incidence of AKI, Odds ratio 1.79 (95% CI 1.13–2.82).

– Metabolic syndrome

Epidemiological studies have shown a relationship between the uric acid concentration and metabolic syndrome. The uric acid concentration correlates positively with blood pressure, obesity, the HOMA index (measure of insulin resistance), fasting glucose, and the amount of insulin and triglycerides, and negatively with HDL cholesterol. Hyperuricemia can, however, also develop in non-obese individuals with metabolic syndrome. Only 5.9% of patients with a normal body mass index and uric acid concentrations below 6 mg/dL (360 μmol/L), but 59% of patients with a concentration above 10 mg/dL (595 μmol/L) have metabolic syndrome.

The hyperinsulinism in metabolic syndrome is thought to promote hyperuricemia, since insulin increases the renal tubular reabsorption of Na+, thus reducing the secretion of uric acid.

In the Finnish Diabetes Prevention Study /42/, individuals with impaired glucose tolerance were twice as likely to develop type 2 diabetes within 4.1 years if the baseline uric acid concentration was 6.4–10.5 mg/dL (381–622 μmol/L) compared to individuals with a concentration of 1.6–5.2 mg/dL (99–310 μmol/L). Approximately 31.9 % of type 2 diabetics had uric acid concentrations above 5.7 mg/dL (340 μmol/L) for women and above 7.0 mg/dL (420 μmol/L) for men.

– Type I diabetes mellitus

Above a certain level, serum uric acid causes endothelial dysfunction and renal disease in certain patient groups. For example, patients with type 1 diabetes and uric acid concentrations in the upper reference interval have a reduced glomerular filtration rate. Uric acid levels predict the development of albuminuria over 6 years in patients with type 1 diabetes. For example, in the Coronary Artery Calcification in Type 1 Diabetes study /43/, patients who developed micro- or macro albuminuria had levels of 5.3 ± 1.2 mg/dL, (315 ± 54 μmol/L), while those who did not had levels of 4.8 ± 0.9 mg/dL (286 ± 71 μmol/L). The risk increased by 80% with each 1 mg/dL (59 μmol/L) increase in uric acid.

– Cardiovascular disease (CVD)

The role of elevated uric acid as a risk factor for CVD has been confirmed in several studies. The Rotterdam study investigated 4385 women and men with uric acid levels of 2.3–7.6 mg/dL (198–453 μmol/L) and 4.1–8.0 mg/dL (245–476 μmol/L), respectively, over a period of 8.6 years /44/. Women with a uric acid concentration above 5.4 mg/dL (321 μmol/L) and men with a level above 6.3 mg/dL (375 μmol/L) had hazard ratios of 1.68 for CVD and 1.87 for myocardial infarction. Women and men with a hazard ratio of 1.0 had uric acid levels of ≤ 4.4 mg/dL (263 μmol/L) and ≤ 5.2 mg/dL (310 μmol/L), respectively.

Another study /45/, which investigated 83,683 individuals, showed that those with uric acid levels above 6.7 mg/dL (399 μmol/L) had a higher cardiovascular mortality with a hazard ratio of 1.51 compared to those with levels below 4.6 mg/dL (274 μmol/L).

– Stroke

In the ARIC study /46/, the 10-year stroke risk in individuals with a mean age of 54 years and uric acid levels of ≤ 4.8 mg/dL (286 μmol/L), 4.9–5.8 mg/dL (291–345 μmol/L), 5.9–6.8 mg/dL (351–404 μmol/L) and ≥ 6.9 mg/dL (410 μmol/L) was 2.0%, 2.7%, 3.4% and 4.6%, respectively.

Another study /45/ showed that individuals with uric acid levels above 6.7 mg/dL (399 μmol/L) had a higher stroke mortality with a hazard ratio of 1.59 compared to those with a concentration below 4.6 mg/dL (274 μmol/L).

Table 5.4-11 Diseases and conditions associated with hypouricemia

Clinical and laboratory findings

Reduced uric acid synthesis – Xanthinuria

Decreased uric acid concentration and urinary excretion of uric acid due to reduced activity of xanthine oxidase. Concurrent increase in serum and urinary oxypurines. Uric acid is usually below 1 mg/dL (59 μmol/L), although it can vary between 0.05 and 1.6 mg/dL (3 to 95 μmol/L). Many patients with this disease are asymptomatic /47/.

– Over medication with allopurinol

A decline in uric acid levels to below 2 mg/dL (119 μmol/L) is not uncommon. Allopurinol inhibits xanthine oxidase and reduces the biosynthesis of purine by forming allopurinol phospho ribosyl pyrophosphate.

– Serious liver disease

Likely reduction of the xanthine oxidase content in the liver in diseases with hepatic decomposition.

– Increased renal clearance

Increased renal clearance of uric acid can occur in isolated or generalized tubular defects and can be of idiopathic or symptomatic nature.

Isolated cases of patients with a uric acid concentration below 2 mg/dL (119 μmol/L) and elevated (idiopathic) uric acid elimination have been reported. In a study /39/ of 64 patients with hypouricemia, nine patients were identified as having a malignant disease. The cause was unclear, as does the reason for the increased uric acid clearance in some patients with serious liver disease.

Hypophosphatemia, renal tubular acidosis, renal glucosuria, aminoaciduria and increased uric acid clearance with hypouricemia are indicative of Fanconi syndrome. In incomplete forms, the various abnormalities occur in different combinations.

Increased renal uric acid clearance with hypouricemia is also found in Wilson’s disease, cystinosis, and heavy metal poisoning.

– Drugs

Two-thirds of hypouricemias are caused by drugs, mainly salicylates (above 2 g/day), X-ray contrast agents (in particular those used to visualize the gallbladder and kidneys), expectorants containing glycerol guaiacolate, uricosuric drugs, phenylbutazone, antiepileptics, and estrogens.

Table 5.5-1 Reference intervals of ketone bodies

After overnight fast /5/:

  • β-hydroxy butyrate in serum/plasma: < 3.5 mg/dL (340 μmol/L)
  • Acetoacetate in serum/plasma: < 0.67 mg/dL (66 μmol/L)
  • Acetoacetate in urine: < 50 mg/dL (4.9 mmol/L)
  • Acetone in urine: < 0.25 mg/dL (43 μmol/L)

Conversion of β-hydroxy butyrate: mg/dL × 96.1 = μmol/L

Conversion of acetoacetate: mg/dL × 98.0 = μmol/L

Conversion of acetone: mg/dL × 172.4 = μmol/L

Table 5.5-2 Behavior of ketone bodies in hyperglycemia

Clinical and laboratory findings

Diabetic ketoacidosis (DKA)

In the USA and Europe, 15–67% of patients with newly diagnosed type 1 diabetes present with DKA, with the latter accounting for 65% of all hospital admissions up to age 19. The mortality rate of DKA in children is 0.1–0.31% /15/. The risk of DKA in established type 1 diabetes is 3–30 episodes per patient and year in children and 4.6–8 episodes per patient and year in adults, with DKA accounting for 2–8% of all hospital admissions of diabetic patients /14/. According to the EURODIAB study /16/, the incidence of diabetic ketoacidosis requiring hospitalization is 8.5%. DKA is due to absolute or relative insulin deficiency and occurs in type 1 diabetes, in ketosis-prone diabetes and, much less frequently, in type 2 diabetes. The main triggers of DKA include: primary diagnosis of diabetes, forgetting to inject insulin in diagnosed diabetes, infections (pneumonia, urinary tract infection, gastroenteritis, otitis, appendicitis), pancreatitis, myocardial infarction, trauma, psychological and emotional stress, corticosteroid therapy /914/.

DKA develops within 24 h, with metabolic changes appearing 1–2 h earlier in patients on short-acting insulin therapy than in patients using other insulin such as lispro.

Clinical findings: patients with DKA typically have polyuria, polydipsia, polyphagia, weakness, and Kussmaul breathing. Approximately 50–80% of patients suffer from nausea and vomiting, and 30% from abdominal pain /9/. In children, DKA has a symptom-free window of 4–12 h. About 1% of children with DKA may develop cerebral edemas with impaired neurological function. The mortality rate in these cases can be as high as 21–50%. Besides vasogenic mechanisms with ischemia, DKA is considered to be due to secondary causes such as dehydration, acidosis, hypocapnia, and therapeutic use of bicarbonate. It is believed that DKA causes mild cerebral damage in all children, even if there are no detectable changes in the mental state /17/.

Laboratory findings: the diagnostic criteria advocated by the Joint British Diabetes Society Inpatient Care Group are /18/: ketonemia > 3.0 mmol/L or significant ketonuria (more than 2+ on standard urine sticks), blood glucose > 11 mmol/L or known diabetes mellitus, bicarbonate < 15 mmol/L and or venous pH < 7.3. Additional findings are increased anion gap, hyper osmolality, hyponatremia, hypophosphatemia, hyperamylasemia, and leukocytosis.

  • Blood glucose: the levels are above 200 mg/dL (11.1 mmol/L) in children and above 250 mg/dL (13.9 mmol/L), usually 400–500 mg/dL (22.2–27.8 mmol/L), in adults. Glucose levels above 600 mg/dL (33.3 mmol/L) are rare, unless the GFR is reduced by about 25% due to dehydration. If the GFR is reduced to 50% due to dehydration, glucose levels can be above 800 mg/dL (44.4 mmol/L). Approximately 15% of patients have glucose levels below 350 mg/dL (19.4 mmol/L) /14/. This is seen in cases where gluconeogenesis is reduced (alcohol abuse, liver disease, food deprivation, prolonged fasting), in pregnancy, in patients treated with insulin prior to hospital admission, and after frequent vomiting. The glucose levels influence the GFR. It is reduced by 30–40% with glucose levels in the range of 500–600 mg/dL (27.8–33.3 mmol/L), and by 50% if glucose is above 800 mg/dL (44.4 mmol/L) /19/. The glucose concentration should, however, not be an important factor in the assessment of the severity of DKA /20/.
  • Ketone bodies: the concentration of serum ketone bodies is an important criterion for the diagnosis and assessment of the severity of DKA. Ketonemia is present if the concentrations of AcAc and β-HB are above 3 mmol/L. The main metabolic product in diabetic ketoacidosis is β-HB, although rapid urine tests, which are based on the principle of the sodium nitroprusside reaction, will only detect AcAc. This is of no relevance in typical DKA, as the ratio of β-HB to AcAc is 3 : 1. Only the severity of ketonemia cannot be detected by rapid assays. This is different if there also is excessive alcohol consumption or lactic acidosis. Here, the ratio is shifted strongly in favor of β-HB and the extent of the ketonemia is not detected /14/. In such cases, β-HB must be measured directly.
  • Acidosis: is diagnosed based on reduced bicarbonate, PCO2 and pH and is caused by the accumulation of β-HB and AcAc. In mild DKA, the pH is below 7.3 and plasma bicarbonate below 15 mmol/L, in moderate DKA, the pH is below 7.2 and plasma bicarbonate below 10 mmol/L, and in severe DKA, the pH is below 7.1 and bicarbonate below 5 mmol/L. It should, however, be taken into account that DKA can occur in combination with other disorders of acid-base balance. For example, depending on respiratory compensation, the pH and bicarbonate can be normal or even elevated in the presence of metabolic alkalosis due to frequent vomiting, abuse of diuretics, severe volume contraction, or use of alkali. It is important that mixed acid-base disorders are diagnosed, because they indicate that there are other problems besides DKA /20/.
  • Anion gap: can be used as an indicator of the concentration of ketone bodies in plasma in DKA. The reference interval is 8–16 mmol/L. In uncomplicated DKA, a 1 mmol/L increase in the anion gap corresponds to a 1 mmol/L decrease in bicarbonate (Δgap = Δanion gap – Δbicarbonate). If Δgap is increased (above +6), then metabolic alkalosis is present in addition, since the increase in the anion gap is greater than the decrease in bicarbonate. If Δgap is negative (below –6), then hyperchloremic acidosis is present in addition, since the increase in the anion gap is less than the decrease in bicarbonate /20/. Under effective DKA treatment, the anion gap decreases.
  • Plasma osmolality: is usually elevated. If it is less than 320 mmol/kg, other etiologies besides DKA should be considered. Each 100 mg/dL (5.6 mmol/L) increase or decrease in blood glucose changes plasma osmolality by 5.6 mmol/kg. Effective osmolality is calculated as follows: mmol/L = 2 × Na+ (mmol/L) + glucose (mmol/L) /21/.
  • Sodium: DKA causes a fluid loss of 30–100 mL and a loss of Na+ of 5–13 mmol per kg of body weight. At the same time, however, the hyperglycemia causes dilutional hyponatremia of 1.6 mmol/L for each 100 mg/dL (5.6 mmol/L) increase in glucose above normal, despite the presence of dehydration. The cause of this is the hyperglycemia-associated osmolar gradient, which causes a fluid shift from the intracellular to the extracellular space. Normal or even hypernatremic levels in DKA with marked hyperglycemia indicate significant hyperosmolality and severe fluid loss /1418/.
  • Potassium: patients with DKA have a total body potassium (K+) deficit of 3–6 mmol/kg of body weight and a general K+ deficiency. Most have normokalemia or mild hyperkalemia. The hyperkalemia results from the combined presence of acidosis, insulin deficiency and increased osmolality, which induces a shift of K+ from the intracellular to the extracellular space. If there is vomiting, hypokalemia can result. If serum K+ levels are below 5.0 mmol/L, potassium must be replaced prior to starting DKA treatment /1418/.
  • Phosphate: acidosis and hyperglycemia cause the body to lose phosphate through the kidneys due to the phosphate being shifted from the intracellular to the extracellular space. Like potassium, the phosphate concentration in serum is not an indicator of the phosphate deficit /14/.
  • Leukocytes: adults may have leukocytosis with leukocyte counts of (10–15) × 109/L; counts of 25 × 109/L are rare. Children may have leukocytosis with leukocyte counts of (40–60) × 109/L. Stress and dehydration lead to demarcation of the polymorphonuclear granulocytes and monocytes. Under effective treatment, leukocytes decline rapidly /1418/.

Hyperglycemic hyperosmolar non-ketotic syndrome (HHNS) /11/

HHNS is mainly characterized by hyperosmolality, hyperglycemia, severe dehydration, absence of ketosis or mild ketosis, and mild neurological changes. HHNS usually develops after prolonged polyuria and polydypsia which result in severe dehydration with a fluid loss approximately twice as high as in DKA. Due to the hypertonicity of the hyperosmolar state, the intravascular volume is maintained for an extended period of time, so that the clinical symptoms of dehydration are masked. While DKA predominantly occurs in insulin-dependent diabetics, HHNS usually develops in non-insulin-dependent diabetics. It is also seen in children. In a study /22/ of children with the syndrome, the mean age was 14.8, and slim children had type 1 diabetes while obese children had type 2. The mortality rate of the latter was 53%. In North America, the incidence of HHNS is 17.5 per 100,000 individuals per year. Patients are typically aged 55–70 and have known type 2 diabetes. Approximately 39% of patients have acute infections, 18% have dementia, and 28% live in nursing homes. The mortality rates are 12–46% /14/. The development of HHNS from poor glucose control to severe hyperglycemia with extreme hyper osmolality and dehydration takes between 2 days and 2 weeks /13/.

  • Blood glucose: levels are usually higher in HHNS than in DKA. An arbitrary level of 600 mg/dL (33.3 mmol/L) has been defined as a diagnostic criterion /20/.
  • Ketone bodies: may be detectable in some patients by a rapid urine test.
  • Metabolic acidosis: absent. The pH is usually above 7.3 and bicarbonate above 20 mmol/L. If the pH is below 7.3, ketone bodies and lactate should also be assayed, since DKA and HHNS may be present in combination.
  • Anion gap: is normal or slightly increased. If it is greater than 12, a differential diagnosis for lactic acidosis or other causes should be considered.
  • Plasma osmolality: by definition, the osmolality must be greater than 320 mmol/kg to be diagnostic of HHNS. The calculation of effective plasma osmolality is described under DKA. The mortality rate depends on the plasma osmolality. It is 7% for levels below 350 mmol/kg, 14% for 350–374 mmol/kg, 32% for 375–399 mmol/kg, and 37% for levels above 400 mmol/kg /14/.
  • Other parameters: creatinine, urea, total protein and hematocrit are elevated.

Table 5.5-3 Diseases and conditions with ketonuria and/or ketonemia in non diabetics

Clinical and laboratory findings

Alcoholic ketoacidosis (AKA) /1421/

The syndrome of a wide-gap metabolic acidosis, malnutrition, and binge drinking superimposed on chronic alcohol abuse is most commonly referred to as alcoholic ketoacidosis. AKA is an important differential diagnosis of the metabolic acidoses with an increased anion gap. It generally occurs in patients with years of alcohol abuse who, due to alcohol-induced gastritis, stop eating for 2–3 days and suffer from vomiting.

Clinical findings: the main symptoms are nausea, vomiting, abdominal pain, tachycardia, and Kussmaul breathing.

Laboratory findings: increased anion gap; metabolic acidosis, which is generally accompanied by compensatory respiratory alkalosis with reduced PO2. Approximately 10% of patients have hypoglycemia, 90% are test positive for ketone bodies in urine. If no ketone bodies are detected, the following may be present:

  • Predominant formation of β-HB. Due to the high NADH/NAD ratio in alcoholics, 5–7 times more β-HB than AcAc is produced. Rapid assays can only detect the AcAc, provided it is not below the detection limit.
  • A milder form of alcoholic acidosis in which only serum β-HB is elevated.
  • A metabolic acidosis of different origin, with increased anion gap (e.g., uremia, intoxication with methanol, glycol or salicylates).

In some patients, alcohol is no longer detectable. In one study /21/, it was below 0.5 ‰ (11 mmol/L) in two out of three patients, and its concentration was inversely correlated with that of β-HB. Electrolyte imbalances with reduced serum Na+, K+, phosphate, Ca++ and Mg2+ levels as well as moderately elevated aminotransferases at admission are common. Lactate is normal or only slightly elevated.

Congenital metabolic disorders

Ketonuria in the newborn is always an important indicator of a metabolic disorder. It is seen in organic aciduria, lactic acidosis, gluconeogenesis defect, fructosemia, maple syrup urine disease, sometimes in galactosemia and type-I-tyrosinemia /23/. Significantly elevated succinylacetone levels (above 5 μmol/L) in the first 12 hours of life are found in hereditary type-I-tyrosinemia /24/.

Pancreatic ketoacidosis

In acute pancreatitis, cases with metabolic acidosis, increased anion gap, ketonemia, and ketonuria have been described. The ketoacidosis is thought to be caused by elevated levels of ketone bodies which are due to the mobilization and metabolism of fatty acids from fat depots by elevated lipase in the circulation /25/.

Extreme physical stress, fasting

Following hypoglycemia due to deficient insulin and increased glucagon action, the formation of ketone bodies results from increased lipolysis. In the fasting state, ketonuria usually occurs after 48 h.

Table 5.5-4 Differential diagnosis of diabetic coma, HHNS, and acidoses /691114/






Blood glucose
mg/dL (mmol/L)

> 250

> 600

< 200

< 200






Anion gap

> 16

< 16

> 16

> 16







< 320

> 320

< 320

< 320


< 15

> 20

< 15

< 15


< 7.30


< 7.30

< 7.25

PCO2 mmHg

< 35


< 35

< 35

Lactate mg/dL

< 35

< 35

< 35

> 45

C-peptide μg/L

< 0.7

> 1.8

> 1.8

> 1.8

* Hyperglycemic hyperosmolar non-ketotic syndrome; pos., positive; neg., negative

Table 5.6-1 Reference intervals for lactate

Neonates (capillary blood) /4/

2.4–20 (0.27–2.2)

Adults and children:

  • Arterial whole blood or plasma

< 16 (1.8)

  • Venous whole blood or plasma /2/

4.5–20 (0.5–2.2)

Cerebrospinal fluid /5/:

  • 0–15 yrs

9.9–16.2 (1.1–1.8)

  • 16–50 yrs

13.5–18.9 (1.5–2.1)

  • Over 50 yrs

15.3–23.4 (1.7–2.6)

Data expressed in mg/dL (mmol/L); values are 5th and 95th percentiles; conversion: mg/dL × 0.11 = mmol/L.

Table 5.6-2 Causes and diseases leading to hyper lactatemia and, less frequently, lactic acidosis /8/

Clinical and laboratory findings

Physical work, e.g. competitive sports, grand mal seizure

The increased release of lactate by the muscle tissue during physical work is usually compensated by increased oxidation of lactate. Despite increased lactate turnover, lactate levels in plasma are increased only up to about 3-fold compared to levels measured at rest (i.e., mild hyper lactatemia is present). During physical exercise, such as a one-hour run, lactate values rise to approximately 55 mg/dL (6 mmol/L) after 10 min. before falling to about 18 mg/dL (2 mmol/L) after 30 min. and then remaining roughly constant until the end of the run due to the increased supply of aerobic energy. During intense exercise, when physical work requires 50–80% of the maximum O2 consumption, lactate concentrations increase significantly, leading to lactic acidosis with levels above 135 mg/dL (15 mmol/L). Arm activity causes a greater increase in lactate levels than leg activity /13/. Following intense activity, lactic acidosis normalizes faster under light physical activity than at rest.


Excessive intake of alkali (sodium bicarbonate, sodium pyruvate, sodium lactate) and carbohydrate infusions (glucose, fructose, sorbitol, xylitol) cause hyper lactatemia without lactic acidosis.

Administration of high doses of insulin

In diabetics, high doses of insulin can lead to hyper lactatemia with levels up to 72 mg/dL (8 mmol/L) and a short-term decrease in the pH due to the increased peripheral production of lactate and inhibition of gluconeogenesis.


Hyperventilation (e.g., in intensive care patients or diseases of the central nervous system) leads to respiratory alkalosis. This is compensated for by increased production of lactate.

Intra- and postoperative

Hyper lactatemias with lactate concentrations up to 45 mg/dL (5 mmol/L) can occur within 48 h after or during surgery such as coronary bypass operations /14/. They can usually be compensated by the body’s own regulatory mechanisms without lactic acidosis occurring.

Catecholamine infusion

Infusion of catecholamines or other sympathicomimetic drugs as well as ingestion or administration of catecholamine-releasing substances, such as theophylline, cocaine, or ether, can cause hyper lactatemia. In this case, the hyperlactatemia results from the constriction of vessels, which leads to hypoperfusion in the liver and skeletal muscles. The hyper lactatemia is the result of increased release of lactate by the muscles and reduced uptake by the liver /8/.


Lactic acidosis is one of the contraindications for the administration of metformin and renal insufficiency. In a study /49/, there was no lactic acidosis in patients with moderate renal insufficiency and an eGFR of 30–60 [ml × min-1 × (1.73 m2)-1].


Individuals with fire smoke inhalation suffer from monoxide as well as cyanide poisoning. Cyanide poisoning results from inhalation of hydrogen cyanide gas. Cyanide inhibits oxidative phosphorylation by binding to ferric iron of cytochrome a3. The lactate concentration is the most useful test to assess the severity of the state. The concentration of lactate can rise above 10 mmol/l. The signs and symptoms of cyanide poisoning include headache, confusion, lethargy, dyspnea, nausea, and metallic taste. Consequences are seizures or coma, cardiovascular collapse, and quickly to death at higher concentra­tions /4950/.

Antidotal strategy is focused on the intravenous administration of hydroxocobalamin(the FDA approved the commercial drug Cyanokit®). The antidotal strategy is based on the affinity of cyanide to cobalt (refer to Fig. 13.3-1 – Basic structure of cobalamin forms). Hydroxocobalamin combines with cyanide to form cyanocobalamin (vitamin B12). Cyanide presents higher affinity for this antidote than for cytochrome oxidase.

Table 5.6-3 Classification of lactic acidosis, modified from Ref. /8/

Acquired forms, type A: tissue hypoxia

Tissue hypoperfusion

  • Reduced vascular tone, increased tissue permeability
  • Left heart failure, reduced cardiac output
  • Septic shock

Reduced arterial O2 saturation

  • Asphyxia
  • Hypoxemia (PO2 below 35 mm Hg)
  • CO poisoning
  • Severe, life-threatening anemia

Acquired forms, type B: no tissue hypoxia

  • Sepsis, infections such as malaria, cholera
  • Renal insufficiency, severe hepatic function impairment, cancer, diabetic ketoacidosis
  • Drugs and toxins such as biguanides, alcohol, salicylates, methanol, ethylene glycol, cyanides, niacin, catecholamines, papaverine, diethyl ether, paracetamol, nalidixic acid, isoniazid, streptozotocin, sorbitol, parenteral nutrition, lactulose, theophylline, cocaine, vitamin B deficiency, paraldehyde, reverse transcriptase inhibitors
  • Various (e.g., epileptic seizure, D-lactic acidosis)

Table 5.6-4 Hyperlactatemias with frequent progression to lactic acidosis /6/

Clinical and laboratory findings

Shock /9/

Pathophysiological causes of shock can be:

Reduced cardiac output and reduced O2 supply of the tissues: hypovolemia (internal or external loss of fluids), cardiac in nature (myocardial infarction, severe cardiomyopathy, myocarditis, cardiac arrhythmia, severe valvular heart disease), obstructive disease (pulmonary embolism, tension pneumothorax, cardiac tamponade).

Circulation disorder with systemic reduction of vascular resistance and reduced O2 extraction by the tissues due to inflammatory cytokines in severe sepsis or anaphylactic shock. Cardiac output is usually increased.

In intensive care units, septic shock dominates, followed by cardiogenic and hypovolemic shock. Elevated lactate levels indicate an abnormal cellular metabolism. In the setting of reduced cardiac output, the hyper lactatemia is due to a hypoxia-induced anaerobic metabolism. In the case of a circulation disorder, increased glycolysis and inhibition of pyruvate dehydrogenase are also thought to play a role. In liver cirrhosis, lactate elimination is reduced, and the blood pH does not correlate well with the lactate concentration /6/.

Hyper lactatemia is an independent prognostic parameter for assessing the odds of survival. Patients with circulatory shock and lactate levels < 18 mg/dL (2.0 mmol/L), 18–36 mg/dL (2–4 mmol/L) and 37–72 mg/dL (4–8 mmol/L) have mortality rates of 10%, 50% and 90%, respectively /15/. Lactate levels > 36 mg/dL (4 mmol/L), which are rare in postoperative patients, are associated with an increased rate of need for intensive care in normotensive patients with systemic inflammatory response syndrome (SIRS). Levels of this magnitude that persist over 24 hours are associated with a high rate of mortality /16/. The lactate concentration is a better indicator of tissue hypoxia than the anion gap and the blood pH, since individuals with diseases such as acute liver failure or septic shock tend to develop alkalosis. In patients with severe shock, lactate levels increase by 7.2 mg/dL (0.8 mmol/L) every 10 minutes. In the case of lactate levels > 27 mg/dL (3 mmol/L), a decrease of ≥ 20% within 2 h is associated with reduced in-hospital mortality.

Lactate clearance (LC): to allow an early assessment of the situation in severe sepsis and septic shock, it is recommended that LC be determined. It is calculated based on the lactate level at admission to intensive care (LC0) and the lactate level after 6 h (LC6) using the following formula: LC(%)=(LC0 – LC6) × 100/LC0. For each 10% decrease in lactate clearance, mortality is reduced by approximately 11%. Patients with a lactate clearance ≥ 10% had a greater decrease of the APACHE score over 72 h and a lower 60-day mortality than patients with an LC below 10% /17/.

Intensive care patients

For intensive care patients a prognostic marker is required so that aggressive treatment can be started if needed. Besides the APACHE, SOFA and SAPS scores, the lactate concentration is also recommended. Although the patient groups with the worst outcome have the highest lactate concentrations, there is no threshold that predicts who will develop multiple-organ failure or die within 28 days.

Biguanide-associated lactic acidosis

Unlike the other biguanides, metformin is not associated with significant lactic acidosis. When treated with the now-withdrawn biguanides, diabetics typically had complications (inflammations, heart, kidney, liver) and symptoms such as abdominal complaints, tachypnea and somnolence.

Laboratory findings: metabolic non-ketotic acidosis, pH below 7.25, lactate concentration 90–180 mg/dL (10–20 mmol/L), creatinine elevated.

Detection of acute intraabdominal vascular occlusions

In patients with acute abdomen and intestinal vascular occlusion, the lactate concentration is 68 ± 26 mg/dL (7.5 ± 2.9 mmol/L) compared to 18 ± 10 mg/dL (2.0 ± 1.1 mmol/L) in patients without vascular occlusion. In patients who had undergone reconstruction of the aorta and mesenteric arteries, the postoperative lactate concentrations measured over 24 h were 39 ± 9 mg/dL (4.3 ± 1.0 mmol/L) if there were no complications, but as high as 500 mg/dL (60 mmol/L) in the presence of acute mesenteric occlusion /19/.

Organ transplantation

Approximately 20% of heart transplant patients develop elevated lactate levels within the first hour after surgery. Two-thirds had concentrations less than 45 mg/dL (5 mmol/L), the remaining patients had levels up to 133 mg/dL (14.6 mmol/L). Normalization occurred within 24 h /20/. In liver transplant patients, the lactate concentrations measured 24 h after re perfusion is a prognostic indicator. Patients with a functioning transplant had levels below 18 mg/dL (2.0 mmol/L) while those with a failed transplant had levels above 36 mg/dL (4.0 mmol/L) /21/.

Cardiac surgery

A critical goal of intraoperative management is optimizing systemic perfusion to maintain organ function. During cardiac surgery, both hemodynamic changes inherent to surgery and management stategies while on cardiopulmonary bypass can reduce systemic perfusion.

Intraoperative lactate concentration is an important predictive feature to measure overall systemic perfusion. Hyperlactatemia after cardiopulmonary bypass is associated with postoperative morbidity, increased duration of intensive care stay, and mortality. In a study of 2187 patients the maximum lactate concentration in the 24 h after surgery was a median of 4.6 mmol/L (interquartile range 2.8 to 7.3 mmol/L) /48/.

Intrapartum fetal distress

Lactate and fetal scalp blood pH are important parameters for discriminating between normal and distressed fetuses during labor and delivery. PH levels above 7.25 and lactate concentrations below 18 mg/dL (2.0 mmol/L) indicate that the fetus is normal (not distressed) according to one study /22/. The postpartum lactate concentrations in the umbilical cord artery were below 25 mg/dL (2.8 mmol/L) in these cases.

Alcohol intoxication, CO poisoning, severe acute anemia

In acute alcohol intoxication, the lactic acidosis is thought to be due to the oxidation of alcohol to acetaldehyde. Both reactions lead to the formation of NADH. As a result, the ratio of NADH to NAD in the cytoplasm increases, and if the mechanism responsible for transporting NADH into the mitochondria is impaired, more lactate is produced. In addition, alcohol is believed to inhibit the synthesis of glucose from pyruvate via the Cori cycle /8/.

In the case of CO poisoning, oxygen exchange is thought to be impaired, since CO, similar to cyanide, reacts with the cytochromes of the respiratory chain /8/.

Severe anemias with Hb levels below 50 g/L, such as are seen in African children with Plasmodium falciparum malaria, cause an increase in the lactate concentration up to 135 mg/dL (15 mmol/L), which normalizes within hours following blood transfusion /23/.

Intoxication, e.g., methanol, ethylene glycol, acetaminophen, salicylate

Intoxication with these substances causes an anion gap of often more than 20 mmol/L, and lactic acidosis. Clinically, patients present with a variety of neurological symptoms, even coma. Methanol is metabolized to formaldehyde and formic acid, ethylene glycol to glycolic acid and oxalic acid /8/. These metabolites or their starting substances, both of which, as unmeasurable anions, significantly increase the anion gap, inhibit the mitochondrial oxidation of pyruvate, leading to the development of lactic acidosis. Salicylate intoxications are associated with salicylate concentrations above 300 mg/L. In children, metabolic acidosis dominates, while adults have mixed respiratory and metabolic acidosis.

Acute liver failure, caused by the severe hepatotoxic effect of paracetamol, is a short and acute disease of rapid progression. It starts with acute failure of the liver and other organ systems, such as the kidneys, and leads to hemodynamic instability and encephalopathy. The severity of the hyper lactatemia is an indicator of the hepatic damage, systemic perfusion impairment, and hypoxia. Patients with higher lactate levels have a lower probability of survival. For example, a lactate value of ≥ 31.5 mg/dL (3.5 mmol/L) measured shortly after admission has a diagnostic sensitivity for a lower likelihood of survival of 67% at a specificity of 95%. The positive likelihood ratio is 13, the negative likelihood ratio 0.35 /7/.

Thiamine (vitamin B1) deficiency (intensive care patients, chronic alcoholics, beriberi)

Thiamine is the cofactor for pyruvate decarboxylase, which channels pyruvate into the citric acid cycle and thus the aerobic metabolism. If thiamine is deficient, less pyruvate is consumed, causing lactate levels to rise. Thiamine deficiency occurs in intensive care patients and chronic alcoholics. In the latter it manifests as Wernicke’s encephalopathy or neuritis. The marked thiamine deficiency causes a lactic acidosis similar to beriberi. Thiamine replacement therapy causes lactate levels to decrease by approximately 90 mg/dL (10 mmol/L) to 18 mg/dL (2.0 mmol/L) 40 min. following supplementation in intensive care patients with lactic acidosis and confirmed thiamine deficiency /24/.

Treatment with isoniazid, niacin, lactulose

In the setting of preexisting illnesses such as hepatopathy, hepatic encephalopathy, or isoniazid-induced acute hepatic damage, these drugs can cause lactic acidosis with a lactate concentration up to 90 mg/dL (10 mmol/L) /8/.

Malignant tumors, tumor lysis syndrome

Hyper lactatemias occur predominantly with malignant tumors of the hematopoietic system, such as leukoses and malignant Hodgkin's and non-Hodgkin lymphomas, as well as with large liver tumors and liver metastases. Lactic acidosis often develops when there are additional complications due to impaired renal function or sepsis /8/.

Patients with tumor lysis syndrome, which is characterized by rapid development of hyperuricemia, hyperkalemia, hyper phosphatemia, hypocalcemia and azotemia, can develop lactic acidosis with lactate levels up to 135 mg/dL (15 mmol/L) /25/.

Hepatitis B treatment with entecavir /26/

In patients with chronic hepatitis B, treatment with entecavir is considered if HBV-DNA is above 2000 IU/mL, ALT is elevated, and hepatic histology shows moderate necroinflammation or fibrosis. In a study of 16 patients with advanced liver cirrhosis and chronic hepatitis and a MELD (Model for End-Stage Liver Disease) score ≥ 20. 5 patients developed lactic acidosis after 4 to 240 days of treatment with entecavir. Laboratory findings were: lactate levels 26–200 mg/dL (2.6–22 mmol/L), pH 7.02–7.4, base excess –5 to –18 mmol/L.

HIV infection

HIV infected patients who are treated with nucleoside analog reverse transcriptase inhibitors (NRTIs) can develop mitochondrial toxicity syndrome, which is characterized by lactic acidosis and hepatic steatosis, as a result of damage caused to the mitochondrial DNA (mtDNA). The rank order of potency of the NRTIs in the reduction of the mtDNA is as follows: zalcitabine > didanosine > stavudine > zidovudine > abacavir.

A distinction is made between /27/:

  • Severe cases with lactic acidosis and lactate levels above 45 mg/dL (5 mmol/L). They occur with an incidence of 1.7–25.2 cases per 1,000 patient-years and are associated with a mortality rate of 30–60%. They present primarily with gastrointestinal complaints or unspecific symptoms such as nausea, vomiting, anorexia, abdominal pain, and mild hepatomegaly, which then progress to metabolic acidosis with hyperventilation, arrhythmia, and organ failure. In addition, these patients develop pancreatitis, neuropathy, and high CK activities. Symptoms are more common in women and in individuals with AIDS and preexisting liver diseases such as hepatitis B or hepatitis C co-infection.
  • Milder symptomatic cases with hyper lactatemia and levels of 26–56 mg/dL (2.9–6.2 mmol/L) with mild to moderate symptoms as described above, and without acidosis. The incidence of symptomatic hyper lactatemia is 14.5 per 1,000 patient-years.
  • Compensated, asymptomatic hyper lactatemia. In this third type, mild to moderate, sometimes only intermittent lactate elevations of up to about 135 mg/dL (15 mmol/L) are observed. There are none of the clinical symptoms as described above. Often, lactate is only elevated during the first 6 to 12 months.

Overgrowth of the intestine with anaerobic bacteria, long-term use of L. acidophilus

The bacteria produce D-lactate, which is absorbed by the intestine, leading to the development of D-lactic acidosis with an increased anion gap /8/. Since the commonly used laboratory assays only measure L-lactate, the possibility of the presence of D-lactic acidosis must be considered if the anion gap is increased for no explicable reason. D-lactate can be determined by gas chromatographic methods.

Diabetic ketoacidosis, alcoholic ketoacidosis

The lactate concentration can be a multiple of the upper reference interval value, but usually does not exceed 45 mg/dL (5.0 mmol/L). In this setting, the hyper lactatemia contributes to increasing the anion gap and aggravating the acidosis.

The hyper lactatemia is thought to be caused by inhibition of the hepatic absorption of lactate by the ketone bodies. Patients with non-insulin-dependent diabetes mellitus can have mildly elevated lactate levels /28/.

Liver cirrhosis

Under conditions of rest, 50% of the lactate is cleared by the liver. This is also the case in stabilized liver cirrhosis. If elevated levels of blood lactate are measured, the increase would have only occurred recently due to the deterioration of hepatic function /29/.

Neonatal hypoxia

Hypoxia in newborns is associated with a high degree of morbidity and mortality, in particular with neurodevelopmental disorders. In these cases, extra corporeal membrane oxygenation is a successful treatment option. The lactate concentration is a good predictor of therapeutic outcome. For example, in the case of hypoxemic neonates with a gestational age of 36–42 weeks and lactate ≥ 135 mg/dL (15 mmol/L) at admission, the following data were evaluated with regard to early death or neurodevelopmental disorders: diagnostic sensitivity 93% at a specificity of 100%, positive predictive value 100%, negative predictive value 90% /30/.

Table 5.6-5 Prediction equations for metabolic acidosis /45/

Equation 1: pCO2 = 7,..; PCO2 is equal to the two digits after the comma.

Equation 2: pCO2 = 1,5 × Plasma HCO3 + 8 ± 2

ad Equation 1: if the PCO2 measured is lower than predicted, the metabolic acidosis is compensated by respiratory alkalosis.

ad Equation 2: if the PCO2 measured is higher than predicted, then primary respiratory acidosis due to CO2 retention is present.

Table 5.6-6 Hereditary metabolic disorders causing lactic acidosis

Clinical and laboratory findings

Organic acidurias, fatty acid oxidations defects, urea cycle defects

In these defects, hyperlactatemia is a secondary phenomenon which does not occur regularly. For example, methyl malonic acidemia, propionic acid acidemia, isovaleric acidemia and fatty acid oxidation disorders cause lactic acidosis due to impaired acetyl-CoA metabolism, whereby the entry of pyruvate into the citric acid cycle is inhibited, resulting in increased production of lactate (Fig. 5.6-2 – Energy metabolism of the cell/31/.

Disorders of glycogen metabolism

Type III glycogenosis (Cori-Forbes disease): the disease is caused by a deficiency of glycogen debranching enzyme activity. The enzyme impairs the mobilization of glucose from glycogen, but not the production of glucose by gluconeogenesis (Fig. 5.6-2). Typical clinical symptoms include hepatomegaly, growth retardation, and distal myopathies with marked myasthenia, in particular under stress. Most patients have type IIIa of the disease, which affects the muscle and liver. Type IIIb only involves the liver. Liver symptoms decline with increasing age and disappear after puberty. The myasthenia is mild in childhood, but can become dominant in adulthood and lead to distal muscular dystrophy /32/.

Laboratory findings: in type III glycogen storage disease and glycogen synthase deficiency lactic acidosis occurs mainly in the fed state with lactic acidosis being moderate in severity and usually not exceeding 63 mg/dL (7.0 mmol/L) /32/. Depending on food intake and physical activity, patients can develop fasting hypoglycemia with ketonuria and hyper lactatemia. ALT can be elevated up to 5-fold, CK up to 200-fold. There is no increase in lactate following muscle contraction in the forearm working tests (Tab. 5.6-7 – Forearm ischemic work test (McArdle) and Tab. 5.6-8 – Non-ischemic work test).

Glycogen synthase deficiency: glycogen synthase (EC catalyzes glycogen synthesis by converting UDP glucose to glycogen. Deficiency in this enzyme leads to reduced glycogen synthesis. Patients typically present with childhood fasting hypoglycemia and hyper ketonemia and may develop postprandial hyperglycemia and hyper lactatemia, since glucose is preferably converted to lactate and therefore cannot be used for glycogen synthesis /32/.

Disorders of gluconeogenesis

Hereditary disorders of gluconeogenesis comprise deficiencies of the enzymes glucose-6-phosphatase, fructose 1,6 bisphosphatase (EC and phosphoenolpyruvate carboxykinase (EC Patients often present with fasting hypoglycemia and lactic acidosis secondary to the inability to recycle lactic acid to glucose via the Cori cycle (Fig. 5.6-2 – Energy mtabolism of the cell/31/.

Glucose-6-phosphatase: deficiency in this enzyme causes type Ia glycogen storage disease. Types Ib–Id are caused by deficiency in proteins in the endoplasmic reticulum which are responsible for the transport of glucose, glucose-6-phosphate and phosphate. Type I glycogenosis, also known as Von Gierke disease, is the most serious of the glycogen storage diseases because both glycogenolysis and gluconeogenesis are affected by the enzyme deficiency. Patients have massive liver enlargement, muscle weakness, a doll-like face and frequent episodes of hypoglycemia, lactic acidemia, hyperuricemia and hyperlipidemia /31/. The patients present either with hypoglycemia and lactic acidosis during the neonatal period or show hepatomegaly and/or hypoglycemic seizures at the age of 3–4 months. The hypoglycemia and hyperlactatemia occur in short episodes. Patients often have hyperlipidemia (LDL elevated, HDL reduced, apolipoprotein C-III elevated), and those with type I b may additionally have neutropenia /32/. The neutropenia is associated with impaired granulocyte function and bacterial infections /33/.

Fructose 1,6 bisphosphatase: deficiency in this enzyme produces significant hypoglycemia and lactic acidosis only during periods of fasting or infection /31/. Together with phosphofructokinase (EC, fructose 1,6 bisphosphatase plays a key role in the regulation of glycolysis and gluconeogenesis in the liver. During glycolysis, phosphofructokinase converts fructose-6-phosphate to fructose-1,6-bisphosphate, and during gluconeogenesis fructose-bisphosphatase catalyzes the reverse reaction (i.e., the conversion of fructose-1,6-bisphosphate back to fructose-6-phosphate). Deficiency in fructose-bisphosphatase impairs the balance, leading to the development of hypoglycemia, lactic acidosis and ketonemia in fasting patients /34/.

Phosphoenolpyruvate carboxykinase (PEPCK): this enzyme catalyzes the conversion of oxaloacetate to phosphoenolpyruvate in the cytoplasm. Phosphoenolpyruvate is then used for gluconeogenesis. Deficiency in PEPCK is a rare abnormality presenting clinically with hypoglycemia, lactic acidosis, hypotonia, hepatomegaly and failure to thrive /31/.

Disorders of pyruvate metabolism

Pyruvate represents the endpoint of glycolysis. From this point pyruvate has four metabolic pathways /31/: (i) conversion to lactate, catalyzed by LD; (ii) transamination to alanine, catalyzed by ALT; (iii) carboxylation to oxaloacetate, catalyzed by pyruvate carboxylase; and (iv) decarboxylation and activation to acetyl-CoA, catalyzed by the mitochondrial pyruvate dehydrogenase (PD) complex (Fig. 5.6-2 – Energy metabolism of the cell).

PD complex: the complex consists of three major catalytic components (E1–E3) and two regulatory components: PD (EC (E1), lipoate acetyl transferase (EC (E2), lipoamide dehydrogenase (EC (E3), PD phosphatase (EC, PD kinase (EC, and protein X. The PD complex (E1), which is a tetramer consisting of 2α (E1α) and 2β (E1β) subunits, is inactivated by phosphorylation catalyzed by PD kinase and activated by dephosphorylation catalyzed by PD phosphatase.

PD complex deficiencies: defects of the PD complex are the most frequently identified inherited metabolic causes of pediatric acidosis. The amount of residual enzyme activity often does not correlate with the severity of clinical symptoms /31/. One group of patients present with very low enzyme activity, severe lactic acidosis in the newborn period and die early on multiple organ system failure secondary to metabolic acidosis. In addition these patients show dysmorphic facial features similar to those of fetal alcohol syndrome. The second group of patients survive the neonatal period and live into the teenage years. They show delayed physical and cognitive development. Many patients have subacute necrotizing encephalomyelopathy (Leigh’s syndrome).

The laboratory evaluation shows blood lactate values of 27 to 54 mg/dL (3.0–6.0 mmol/L). The lactate/pyruvate ratio is normal, as both are equally elevated, unless the patient is in shock. The brain is a major producer of lactate. Therefore, the measurement of lactate in cerebrospinal fluid (CSF) may be more sensitive, CSF values greater than 18 to 27 mg/dL (2–3 mmol/L) are abnormal. In many patients with PD complex deficiencies the anion gap is increased and may have CSF lactate levels above 180 mg/dL (20 mmol/L) /31/. A diagnostic fast will improve the lactic acidosis and glucose challenge may produce worsening of symptoms.

Pyruvate carboxylase /31/: this biotin-requiring enzyme converts pyruvate to oxaloacetate in the presence of ATP and CO2. This is the first step in gluconeogenesis and is required for the function of the Krebs cycle. Patients with pyruvate carboxylase deficiency present clinically in two distinctive ways:

  • Predominantly European patients present in the neonatal period with severe lactic acidemia, hypotonia, seizures, failure to thrive, psychomotor retardation and hepatomegaly. The lactate/pyruvate ratio which is involved in maintaining the proper mitochondrial redox status, is increased due to a deficiency of aspartate. Deficiency of aspartate is responsible for the increased ammonia concentration. Hypoglycemia is not usually present.
  • Predominantly North American patients have mild hyper lactatemia with concentrations of 27 to 54 mg/dL (3–6 mmol/L) with severe episodes seen only during illness or fasting. Severe acidotic conditions occur predominantly in childhood and lead to neurodegenerative symptoms often seen in survivors. The laboratory evaluation shows increased alanine and proline, the lactate/pyruvate ratio may be normal /31/.

Disorders of the Krebs cycle

The citric acid cycle comprises the oxidative decarboxylation of citric acid to oxaloacetate, and the reducing equivalents NADH2 and FADH2 are generated (Fig. 5.6-2 – Energy metabolism of the cell). These are re oxidized in the electron transport chain, and the energy used to produce ATP for cellular needs. Because of the critical nature of these functions defects in the Krebs cycle are rare and complete absence of these functions is incompatible with life. Therefore most cases are partial deficiencies. The following defects have been well described /31/.

Fumarase deficiency: the deficiency causes an accumulation of fumaric acid and other proximal metabolites of the cycle. Patients present with childhood failure to thrive, encephalopathy, microencephaly, seizures, and hypotension. The laboratory evaluation shows lactic acidosis and increased urinary excretion of fumaric acid and succinic acid.

α-ketoglutarate dehydrogenase deficiency: the α-ketoglutarate dehydrogenase deficiency due to E3-deficiency occurs because this component is shared by three enzymes (pyruvate dehydrogenase, branched chain keto acid dehydrogenase and α-ketoglutarate dehydrogenase). In this rare deficiency patients develop an early onset encephalopathy with microcephaly and feeding difficulty. Children with this condition usually die early in life. The laboratory evaluation shows lactic acidosis, elevated plasma pyruvate and alanine, increased urinary excretion of α-ketoglutarate and the 2-oxo derivatives of the branched-chain amino acids /31/.

Mitochondrial respiratory chain defects – Generalized

The respiratory chain is located in the inner mitochondrial membrane. Here, the process of oxidative phosphorylation takes place. The energy generated from the redox reactions of the chain is used to generate ATP via complex V (ATP synthase). The respiratory chain is divided into five complexes /35/:

  • Complex I (NADH-coenzyme Q reductase) carries reducing equivalents from NADH to coenzyme Q and consists of different polypeptides, seven of which are encoded by mtDNA
  • Complex II (succinate-coenzyme Q reductase) carries the reducing equivalents from FADH2 to coenzyme Q and contains 5 polypeptides that are all encoded only by the nuclear DNA
  • Complex III (reduced coenzyme Q-cytochrome c reductase) carries reducing equivalents from coenzyme Q to cytochrome c and consists of 11 subunits, one of which is encoded by mtDNA
  • Complex IV (cytochrome c oxidase; COX) transfers reducing equivalents from cytochrome c to oxygen. This complex is composed of cytochrome a and a3, and 13 protein subunits, 3 of which are encoded by mtDNA.
  • The last complex, complex V, is ATP synthetase.

There are three classic pathogenic mtDNA mutations /36/:

1. mtDNA rearrangements, in which mtDNA is lost or duplicated.

2. mtDNA point mutations in tRNA genes or ribosomal RNA genes, which result in defects in mitochondrial protein synthesis.

3. Missense mutations, which change one amino acid, thus impairing an important function of the polypeptides in the respiratory chain.

– Various defects

In nearly all cases of mitochondrial disorders, the enzyme deficiency is partial since complete absence of activity is probably not compatible with life.

Disease-related length mutations usually occur sporadically and can affect large sections of the genome. The most common one is the common deletion mutation, which is approximately 5000 bp long. It affects about a third of the total genome and leads to the loss of genes that encode subunits of complexes I, IV and V as well as 5 tRNA.

Lethal infantile respiratory chain defects have been reported to occur with an incidence of 1 : 10,000. Depending on the organ manifestation, the following phenotypes can occur /3637/:

  • Chronic progressive external ophthalmopathy (CPEO), an ocular myopathy with retinal pigment degeneration and dysfunctions in the central nervous system
  • Kearns-Sayre syndrome (KSS) with CPEO, retinitis pigmentosa, cardiomyopathy, proximal myasthenia and sensorineural hearing loss
  • Pearson’s syndrome (sideroblastic anemia, panmyelophthisis, exocrine pancreatic insufficiency)
  • Alpers syndrome: progressive cerebral degeneration, severe seizures and liver damage
  • Myoclonic epilepsy and ragged-red fiber disease (MERRF), a disease that can develop at any age. Symptoms include epilepsy, cerebellar ataxia, and muscle ruptures.
  • Mitochondrial encephalomyopathy, lactic acidosis, and stroke-like episodes (MELAS): patients are usually under 45 years of age and present like young stroke patients. Approximately 80% of cases are caused by an A3243G mutation in the tRNA gene.
  • Leigh syndrome, also known as subacute necrotizing encephalopathy, is suspected in infants presenting with cranial nerve disorders and respiratory dysfunctions. COX deficiency is the most common biochemically detectable disorder in infants with systemic mitochondrial disease. Respiratory chain defects also play a causative role in hypertrophic childhood and adult cardiomyopathy /37/.

– Laboratory findings

An enzyme defect in the respiratory chain leads to an increase in the reducing equivalents NADH2 and FADH2 in the mitochondrion and cytoplasm and to functional impairment of the citric acid cycle. The excess NADH2 induces increased production of β-hydroxy butyrate from acetoacetate in the mitochondrion and increased production of lactate from pyruvate in the cytoplasm. These shifts are more pronounced after heavy physical work and after food intake, since both require increased amounts of NAD for the oxidation of substrates from glycolysis. Due to impaired function of the citric acid cycle, more acetyl-CoA undergoes ketogenesis, resulting in an increase in ketone bodies after meals. Therefore, hyper lactatemia, paradoxic ketonemia, and an increase in the lactate/pyruvate or NADH/NAD ratio to above 20 are suggestive of a respiratory chain defect /35/.

Table 5.6-7 Ischemic forearm exercise test (McArdle)

Principle: during ischemia and physical activity involving the forearm the maximal rise in lactate (5 to 25-fold of normal) occurs after 1–5 min. distal to the compression area, with normalization not only 20–30 minutes later. In Type V glycogen storage disease (Mc Ardle disease) the rise is minimal or does not occur at all /46/.


  • Wrap blood pressure cuff around the arm, do not inflate
  • Insert angiocatheter into anti cubital or forearm vein
  • Collect a blood sample 5 minutes after the catheter installation
  • Inflate blood pressure cuff beyond systolic pressure to cause ischemia
  • Instruct the patient to clench his hand into a tight fist (or to tightly squeeze an object) for 2 min.
  • Release the blood pressure cuff and draw blood at 1, 5, 10, 15 and 20 min. for lactate measurement

Table 5.6-8 Non-ischemic forearm work test

Principle: an angiocatheter is inserted into anti cubital or forearm vein of the exercising arm. The proband is instructed to squeeze a dynamometer grip for 30 sec. with at least 70% of maximum voluntary contraction force. Blood is sampled prior to the test and at 1, 2, 3, 4, 6 and 10 min. post exercise to measure lactate and ammonia levels. Detailed procedure see Ref. /12/.

Table 5.6-9 Meningeal and cerebral diseases in which the cerebrospinal fluid (CSF) lactate concentration is relevant for the differential diagnosis, therapy monitoring and prognosis

Clinical and laboratory findings

Bacterial meningitis

CSF findings are as follows: leukocyte count above (0.7–1.2) × 109/L with more than 60% granulocytes, total protein above 500 mg/L up to 800 mg/L, glucose below 45 mg/dL (2.5 mmol/L) or a CSF/serum ratio below 0.5; furthermore, detection of bacteria in a Gram-stained preparation.

The importance of lactate in bacterial meningitis results from the following data: lactate concentrations above 32 mg/dL (3.5 mmol/L) have a diagnostic sensitivity of 100% at a diagnostic specificity of 93% and a positive predictive value of 48%. If the combination of CSF cell count (above 0.8 × 109/L) and aforementioned lactate concentration is used, the specificity increases to 99% and the positive predictive value to 88%, whereas the diagnostic sensitivity decreases to 71% /5/.

The lactate concentration and leukocyte count normalize within 10 days of effective antibiotic therapy.

Viral meningitis

On the first day, both the leukocyte count and the granulocyte fraction may be increased. Lactate measurement does not allow clear differentiation from bacterial meningitis, since slightly to moderately elevated values may occur. It is not possible to differentiate patients with viral meningitis from patients without meningitis based on the lactate level, because there is a large overlap of the two groups /38/.

Tuberculous meningitis, fungal meningitis

Lactate concentration may be in the lower range of those found in patients with bacterial meningitis. In tuberculous meningitis normalization of lactate concentration may take several weeks under effective treatment.

Ischemic insult

The lactate concentration in CSF rises, there is a correlation between the size of the brain edema and the degree of mental impairment. An increase in lactate above 27 mg/dL (3.0 mmol/L) is associated with an unfavorable prognosis /38/.

Hemorrhagic insult

If there is a connection between the hemorrhagic site and the ventricular system, the CSF will be blood-tinged. If blood-tinged CSF is obtained from a patient with an acute event, the question arises whether a hemorrhagic insult or artificial blood contamination due to the tap has occurred. Normal lactate levels indicate artificial blood contamination /38/.

Epileptic seizure, transient ischemic attacks, syncopal episodes

In the case of inadequate history and missing clinical findings, generalized seizure activity can be differentiated from syncopal episodes or transient ischemic attacks by means of CSF lactate. Generalized epileptic seizures are associated with significantly elevated lactate as well as prolactin elevation for 6–12 h /38/.

Brain tumors, cerebral angiopathies

In brain tumors and cerebral angiopathies, lactate concentrations of up to 84 mg/dL (9.3 mmol/L) are measured /5/. In one study /5/, the determination of lactate alone would have led to the incorrect diagnosis of acute bacterial meningitis in 3% of cases. However, with a concomitant leukocyte count above 0.8 × 109/L, tumors and angiopathies could practically be ruled out.

Congenital metabolic disorders of the brain

Elevated lactate concentrations in the CSF are measured in congenital metabolic disorders with involvement of the central nervous system (e.g., pyruvate dehydrogenase deficiency). In a study of children with respiratory chain defects, 8 of 11 children had a CSF lactate concentration above 27 mg/dL (3.0 mmol/L). In serum, however, lactate was not elevated in two children. The determination of lactate in CSF is therefore considered an important primary test for the detection of these diseases /39/.

Figure 5.1-1 Structural and functional organization and regulation of the hepatic and renal ammonia metabolism, modified from Ref. /1317/. NH4+ ions produced during proteolysis in the liver cell are either detoxified via the urea cycle or temporarily stored in the mitochondria in the form of glutamine. NH4+ produced in different tissues are transported to the liver in the form of glutamine and channeled into the urea cycle like endogenous glutamine. If acidosis is present, HCO3 is conserved and the urea cycle is down regulated. Under these conditions, the kidneys maintain the homeostasis of NH4+ by increased absorption of glutamine and by excretion of NH4+ in urine.

In urea cycle defects, one of the following 5 enzymes of the cycle is deficient: carbamoyl phosphate synthetase (CPS), ornithine transcarbamoylase (OTC), argininosuccinate synthetase (AS), argininosuccinase (AL), arginase.

The function of the urea cycle depends on the availability of acetyl-CoA from fatty acid oxidation and pyruvate metabolism. Disorders of these metabolic pathways lead to the development of secondary hyperammonemias.

NAGS, N-acetyl-glutamate synthetase; GLD, glutamate dehydrogenase; AST, aspartate aminotransferase

Glutamine Tissue NH 4 + Cytoplasm Glutamine 2NH 4 + 2NH 4 + Urea Urea α-Keto-glutarate Glutaminase Glutamine Glutamate Glutaminesynthetase Glutami-nase Ketones Ketones Acylcarnitine Acyl-CoA Fatty acidoxidation Acetyl-CoA α-Ketoglutarate GLD NH 4 + ATP N-acetyl-glutamate CPS Urea Mitochondrium Acyl-CoA α-Ketoglutarate HCO 3 Citric acid cycle Citrate Carbamoyl- phosphate α-Ketoglutarate Malate Oxaloacetate Pyruvate Pyruvate Glucose Lactate Alanine Carbamoylphosphate Carbamoylaspartate OTC Citrulline Citrulline Aspartate AST α-Ketoglutarate Oxaloacetate ATP AS Arginine Fumarate Arginase Ornithine Ornithine Glutamate Argininesuccinate AL Carnitine Urea cycle Urine Renal tubular cell Liver cell NA GS

Figure 5.1-2 Metabolic pathways of glutamate, modified from Ref. /24/. The figure shows the pathways of urea synthesis, of energy synthesis in the citric acid cycle, of the formation of the neurotransmitter γ-aminobutyric acid, of the buffering of protons, and of the synthesis and degradation of amino acids. NAG, N-acetyl glutamate; α-KG, α-ketoglutarate; GAD, glutamate decarboxylase; GLD, glutamate dehydrogenase; GABA, γ-aminobutyric acid.

Ammoniabuffer andtransport system Glutamine Glutathione Creation of energy GABA Urea α-KG Amino acids N-acetylglutamate- synthetase Aminotransferasen GAD Glutaminase Glutamine- synthetase Glutamate Glutamate GLD Protein synthesis and degradation Ammonia-detoxification Neurotransmitter Citric acid cycle NAG + + NH3 NH 3 NH 2

Figure 5.2-1 Differential diagnosis of hereditary hyperbilirubinemia.

Jaundice Pathologic Acquired disorder of bilirubin metabolism Normal Congenital disorder of bilirubin metabolism likely Bilirubin predominantly Conjugated Unconjugated Gilbert's syndrome Crigler-Najjar ICrigler-Najjar II SBP retention (45 min.) NormalDubin-Johnson(SBP retention 90 min > 45 min) Pathological(Rotor syndrome) Bilirubin < 6 mg/dL (103 μmol/L) Bilirubin > 6 mg/dL (103 μmol/L) ALT, GGT, ChE Exclusion of bilirubin overproduction, e.g. determination of hemoglobin, reticulocytes, LD

Figure 5.2-2 Hour-specific bilirubin nomogram showing the increase in bilirubin in term or near-term neonates. The increase of the low-risk line (40th percentile) is 0.1 mg × dL–1 × h–1, that of the intermediate-risk line (75th percentile) 0.15 mg × dL–1 × h–1, and that of the line representing high risk of bilirubin encephalopathy (95th percentile) 0.20 mg × dL–1 × h–1. With kind permission from Ref. /75/.

Serum bilirubin (mg/dL) Age (hours) 0 10 15 20 5 0 12 24 36 48 60 72 84 96 108 120 Serum-Bilirubin (mmol/l) 85 170 255 340 High risk bilirubin track (age in hrs)Rate of bilirubin rise 0.2 mg/dL/hr Low risk bilirubin track (age in hrs). Rate of bilirubin rise 0.1 mg/dL/hr Increased rate0.2 mg/dl/h (high risk)0.15 mg/dl/h (middle risk)0.10 mg/dl/h(low risk)

Figure 5.2-3 Metabolism of the heme molecule.

Heme Hemoxygenase NADPH NADP CO COHb Biliverdin Bilirubin Biliverdin reductase InhaledCO Interactionwith otherenzyme(e.g. Guanylate cyclase) Pulmonary elimination NADPH cytochrome P450-reductase

Figure 5.2-4 Structure of unconjugated bilirubin (Bu). Due to its two propionic acid side chains, Bu primarily appears non-apolar; simplified structure at the top. The folded structure in the ZZ conformation (bottom) is apolar, since the propionic acid chains are firmly linked to the pyrrole nitrogens via bridging hydrogen bonds.

CO 2 H CO 2 H CH 2 CH 2 CH 2 CH 2 CH 2 CH 2 CH Me Me Me CH Me CH 2 CH CH N N N N H H H H O O C D Simplified structure Z-Z configuration A B C D A B

Figure 5.3-1 Mitochondrial carnitine system. Explanation see Section 5.3.7– Pathophysiology.

Carnitinepalmitoyl-trans-ferase I Carnitinepalmitoyl-transferase II Outer mitochondrialmembrane Inner mitochondrialmembrane Carnitineacetyl-transferase Free fatty acids Acyl-CoA carnitine Adeninenucleotidetranslocase Carnitine-acylcarnitinetranslocase Carnitine-acylcarnitinetranslocase CoA Acylcarnitine Carnitine Malonyl-CoA Acylcarnitine Carnitine Carnitine Carnitine Acetylcarnitine Acetyl-carnitine Matrix ATP ATP CoA Carnitine Carnitine CoA Acyl-CoA Acetyl-CoA β - oxidation O 2 Citricaciccycle

Figure 5.3-2 The role of L-carnitine in the metabolism include:

– Its vital role in the β-oxidation of long-chain fatty acids in the mitochondria, whereby the activated fatty acids are transported across the inner mitochondrial membrane in the form of acylcarnitines. This function can be impaired due to L-carnitine deficiency or reduced carnitine palmitoyl transferase (CPT) activity.

– Its influence on the degree of acetylation, particularly of coenzyme A, one of the key substances in intermediary metabolism. Many of the pleiotropic effects of L-carnitine are attributable to the regulation of the availability of coenzyme A for essential metabolic pathways.

Proteins Carbohydrates Fats Acetyl-CoA +L-carnitine Acetyl-L-carnitine + CoA Acetylations Fatty acid synthesis Ketogenesis Cholesterol synthesis Citric acid cycle Gluconeogenesis Degradation Glycolysis ß-oxidation 1. Carnitine deficiency 2. CPT activity Amino acids Glucose Fatty acids

Figure 5.3-3 Endogenous synthesis of L-carnitine. With kind permission from Ref. /30/. While most tissues are capable of synthesizing butyrobetaine from protein-bound trimethyllysine, most of the butyrobetaine is produced in skeletal muscle. The hydroxylation of butyrobetaine to L-carnitine only occurs in the liver and kidney. The rate-limiting step in the endogenous synthesis of L-carnitine is the hydrolysis of muscle protein.

S-Adenosyl-L-Methionine Protein-linked 6-N-Trimethyllysine Protein-linked L-Lysine 6-N-Trimethyllysine Protein hydrolysis 3-OH-6-N-Trimethyllysine 6-N-Trimethyllysine-hydroxylase 4-Trimethylaminobutyraldehyde 3-OH-6-N-Trimethyllysinealdolase 4-Trimethylaminobutyrate (Butyrobetaine) 4-Trimethylaminobutyral-dehyde dehydrogenase L-Carnitine Butyrobetaine-3-hydroxylase

Figure 5.3-4 Regulatory role of carnitine palmitoyl transferase I (CPT I) in the liver. With kind permission from Ref. /31/. The enzyme activity is controlled by malonyl-CoA. A rising concentration inhibits CPT I, which promotes the synthesis of triglycerides from fatty acid acyl-CoA and glycerate 3-phosphate. A low concentration activates CPT I, leading to increased fatty acid metabolism and formation of ketone bodies. ACC, acetyl-CoA carboxylase.

VLDL Triglycerides Acylcarnitines Ketone bodies Glycerate-3P Carnitine CPT I Glucose Pyruvate Fatty acidacyl-CoA Fatty acid ACC Insulin + Acetyl-CoA Acetyl-CoA Malonyl-CoA Citric acid

Figure 5.4-1 Serum uric acid concentration as a function of age, sex, and race. Data from Bogolusa Heart Study. With kind permission from Ref. /25/.  Caucasian and female;  Caucasian and male;  Black and female;  Black and male.

876543 476416357297238178 Uric acid level (mg/dL; mmol/L) 6 8 10 12 14 16 18 20 22 24 26 Years

Figure 5.4-2 Important enzymatic steps in the regulation of purine metabolism /20/: 1. Phosphoribosyl pyrophosphate synthetase (PRPP synthetase); 2. Glutamine phosphoribosyl pyrophosphate amidotransferase; 3. AMP deaminase, 4. Adenosine deaminase; 5. Hypoxanthine-guanine phosphoribosyl transferase (HGPRT); 6. Adenosine phosphoribosyl transferase (APRT); 7. Xanthine oxidase;  Feedback inhibition.

Ribose-5-phosphate + ATP 5-Phosphoribosyl-pyrophosphat Glutamine 5-phosphoribosyl-amine Guanylic acid Guanosine Guanine Inosinic acid Inosine Hypoxanthine Xanthine Uric acid Adenylic acid Adenine Adenosine 2 5 5 4 7 7 6 3 1

Figure 5.5-1 Conversion of acetoacetate to β-hydroxybutyrate and acetone. Reactions 1 and 2 are catalyzed by β-hydroxybutyrate dehydrogenase; reaction 3 is a spontaneous reaction.

Acetoacetate β-Hydroxybutyrate NADH + H + NAD + O = C - CH 3 CH 2 COO - - - H HO - C - CH 3 CH 2 COO - - - - CH 3 O = C CH 3 Acetone - - CO 2 (1) (2) (3)

Figure 5.5-2 Principle of total ketone body measurement using a recycling method. The rate of formation of thio-NADH is measured.


Figure 5.5-3 Pathophysiology of diabetic ketoacidosis. In insulin deficiency, glucagon stimulates the lipolysis in adipose tissue as well as hepatic glycogenolysis and gluconeogenesis. Inhibition of acetyl-CoA carboxylase occurs, resulting in malonyl-CoA deficiency. As a result, carnitine palmitoyl transferase is no longer inhibited, allowing more fatty acids to be transported into the mitochondria for oxidation and ketogenesis. promoting effect, inhibitory effect

Glycogen Glykogenolysis Glucose Hyperglycemia Ketonemia Insulin Glucagon Triglycerides Lipase Adipose tissue Hepatocyte Fatty acid Acyl-CoA Fatty acid Malonyl-CoA Acetoacetyl-CoA Acetyl-CoA-Carboxylase Lactat Pyruvate Oxalo- acetate Acetoacetate Acetoacetyl-CoA Fatty acid Acyl-CoA Fatty acid oxidation Acetyl-CoA Citrate Carnitine-Palmitoyl - transferase β-Hydroxy-butyrate Citric acid cycle M i t o c h o n d r i o Hyperglycemia Freefatty acids Glucose n Gluconeogenesis

Figure 5.5-4 Pathophysiology of hyperglycemic hyperosmolar non-ketotic syndrome (HHNS) (left) and diabetic ketoacidosis (DKA) (right). With kind permission from Ref. /20/. The absence of ketoacidosis in HHNS can be explained firstly by the fact that there is still sufficient endogenous insulin and, secondly, by the fact that there is little activity of the insulin counter regulatory hormones as compared with DKA.

Increased hepaticglucose production Reducedglucose consumption Released free fatty acids Increasedproduction of ketone bodies Hyperglycemia Ketoacidosis Liver Peripheral tissue Adipose tissue Liver Osmotic diuresis Reduced alkali reserve Volume depletion Metabolic azidosis

Figure 5.5-5 Pathophysiology of alcoholic ketoacidosis. Starvation, hypovolemia and relative insulin deficiency cause fatty acids to be mobilized from adipose tissue and to be metabolized to ketonic acids and lactate. Ethanol abuse leads to increased formation of NADH2 and depletion of NAD. Consequently, the citric acid cycle, gluconeogenesis, and the formation of pyruvate from lactate are inhibited. Modified from Ref. /12/. promoting effect, inhibitory effect

Volume depletion Fast Ethanol Acetaldehyde Acetate Acetyl-CoA Catecholamines Cortisol Growth hormone Glycogen-store Ketonemia Insulin Glucagon Triglycerides Lipase Adipose tissue Hepatocyte Free fatty acids fatty acid Acyl-CoA Fatty acid Malonyl-CoA Acetoacetyl-CoA Acetyl-CoA-Carboxylase NADH 2 Lactat Pyruvate Oxaloacetate Glukoneogenesis Acetoacetat Acetoacetyl-CoA Fatty acid Acyl-CoA Fatty acid oxidation Acetyl-CoA Citrate Carnitin palmitoyltransferase β-Hydroxy-butyrate M i t o c h o n d r i o Citric acid cycle n

Figure 5.6-1 Mean lactate (A–D) and ammonia (E–F) levels in the non-ischemic forearm work test in different myopathies. The bright areas correspond to x ± 2 s. With kind permission from Ref. /12/. C, healthy individuals; DC, patients with myasthenia, but without histological muscle damage; Glyc, patients with type 5 glycogen storage disease (myophosphorylase deficiency, McArdle’s disease) and type 3 glycogen storage disease (debranching enzyme deficiency); Mito, patients with mitochondrial myopathy.

C 0 2 4 6 8 10 Lactate (mmol/L) Time (min.) 7531 A DC 0 2 4 6 8 10 7531 B Glyc. 0 2 4 6 8 10 7531 C Mito. 0 2 4 6 8 10 7531 D 0 2 4 6 8 10 Ammonia (mmol/L) Time (min.) 3002001000 E F G H 0 2 4 6 8 10 3002001000 0 2 4 6 8 10 3002001000 0 2 4 6 8 10 3002001000

Figure 5.6-2 The cell obtains its energy aerobically via the citric acid cycle by oxidation of the substrates glucose, amino acids and fatty acids, and anaerobically via glycolysis. The reducing equivalents produced in this process, such as NADH2 and FADH2 , lead to the formation of ATP and the reduction of molecular O2 to H2O within the respiratory chain.

Lactate produced by glycolysis in the muscle cell is oxidized to pyruvate within the hepatocyte and then converted to glucose by gluconeogenesis. The muscle cell and hepatocyte thus participate in a metabolic cycle known as the Cori cycle. The glycolysis relies on the supply of NAD+, which is provided under aerobic conditions through the oxidation of NADH2 in the respiratory chain and under anaerobic conditions through the synthesis of lactate. In tissue hypoxia, the aerobic production of NAD is reduced, resulting in an increase in the NADH/NAD+ ratio, which promotes the production of lactate from pyruvate and thus the development of lactic acidosis.

NAD + NADH + H + Glycogen Anabolic Katabolic Fat Proteins Glucose Glycogenolysis Glycolysis Alanin Proteins Glycerinaldehyde-3-phosphate Pyruvate Gluconeogenesis Cori cycle Pyruvate carboxylase NAD + NADH + H + Pyruvate dehydrogenase NAD NADH + H + Proteolyse Acetyl-CoA Fatty acid oxidation Fat Lipolysis Freefatty acids Oxaloacetate Citrate Citric acidcycle NADH + H + NAD + NAD + NADH + H + NADH + + H + NAD + FADH 2 FAD A TP A TP AT P 1/2 O 2 H 2 O Lactat Respiratory chain 1,3-Diphosphoglycerat Fructose 1,6-biphosphatase Glucose-6-phosphatase
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