Enzymes

1.1 Diagnostic enzymology

Lothar Thomas

1.1.1 Enzymes in serum

The enzymes measured in serum, plasma and extravascular fluids for diagnostic purposes are biocatalysts. Even minor quantities are sufficient to attain the equilibrium of a chemical reaction by lowering the activation energy. Enzymes have a reaction specificity meaning that a chemical reaction can only be catalyzed by an enzyme specifically required for this reaction. Furthermore, enzymes have a substrate specificity meaning that only a specific substance or substance group functions as a reactant and is converted to the product.

Factors influencing enzyme activity

The enzymes in serum come from tissue cells or result from secretory enzymes entering the blood. The tissue enzymes mainly originate from the cells’ main metabolic chains. In the cells, they are either dissolved in the cytoplasm or bound to cell structures such as the mitochondria. Secretory enzymes such as peptidases and hydrolases are usually secreted in an inactive form, while only a few enzymes such as cholinesterases pass into the plasma in an active form. Enzyme activity in the plasma depends on factors governing the extent of release. The release of cell-specific enzymes is regulated by the extent of cell damage. Increased enzyme production of individual cells or the proliferation of enzyme-producing tissue are decisive for the release of secretory enzymes /1/.

Enzyme release: Low activity of cell-specific enzymes in the blood of healthy individuals is based on the impermeability of the metabolically active cell membrane. Any pathological process affecting the cell membrane’s energy supply, for example inadequate supply with ATP or other high-energy substrates due to ischemia or anoxia, can cause disintegration of the cell membrane and consecutive release of enzymes. First, the membrane potential is upset. K⁺ leaves the cell and Na⁺ and water enter the cell causing swelling. Subsequent Ca²⁺ entry activates hydrolases and peptidases which results in the destruction of intracellular structures and leakage of the cell membrane. Cytoplasmic enzymes are the first to appear in the blood, followed by mitochondrial and membrane-bound enzymes. The extent and rate of enzyme release depend on the size and the concentration gradient of a given enzyme between cytoplasm and extracellular space. In healthy individuals, intracellular enzyme concentration is very much higher than the concentration in plasma. For example, the AST and ALT activities in the liver, kidney, heart and skeletal muscle are higher than in the plasma of a healthy individual as follows:

• AST 7000 fold, 4500 fold, 8000 fold and 5000 fold
• ALT 2800 fold, 1200 fold, 400 fold and 300 fold.

Enzymes pass from the interstitium to the blood either via direct transfer across the capillary wall or indirectly via the lymphatic pathways. Direct transfer is the case in well-vascularized tissue such as the liver parenchyma, and indirect transfer takes places in tissue with a less permeable capillary membrane such as the muscles. The extent of enzyme release depends on the enzyme’s intracellular localization. Enzymes dissolved in cytoplasm appear in the blood relatively soon after cell damage with an easily measurable activity. Enzymes bound to subcellular structures such as the mitochondria take longer. As a rule, an enzyme pattern corresponding to the intracellular enzyme distribution appears in the blood within 24 hours after cell damage including necrosis /1/.

Changes in enzyme production: Changes in enzyme production can manifest as reduced or elevated activity in the blood compared to the reference interval without the presence of cell damage. Reduced activity is less in the focus of diagnostic attention as it is mainly based on genetic dysfunction of the tissue enzymes such as ALP. In contrast, reduced activity in secretory enzymes is often based on a reduction in the relevant enzyme-producing tissue, for example reduced CHE in liver cirrhosis. Increased enzyme release into the blood without the presence of tissue damage can have the following causes:

• An increase in the number and/or biochemical activity of tissue cells. The proliferation and increased activity of osteoblasts in adolescents, for example, causes elevated ALP due to the increased production of the bone-specific isoenzyme.
• Enzyme induction. Tissue cells increasingly produce enzymes, for example, chemical stimulation of the hepatocytes by alcohol, barbiturates or phenytoin enhances the production of GGT by these cells. Moreover, obstruction of the bile ducts, for example, stimulates the synthesis of the liver isoenzyme ALP.
• Development of new tissue. During the last trimenon of pregnancy, for example, the placenta synthesizes ALP determined as placental isoenzyme in the blood of the pregnant woman. A similar isoenzyme can also be produced by malignant germ cell tumors of the testes.

Tissue damage and enzyme elevation

The most common enzyme elevations are caused by damage of the liver, myocardium, skeletal muscle and erythrocytes. Liver-induced enzyme elevations result from direct damage of the cell membrane by viruses, the toxic effect of drugs and poisons or tissue hypoxia. The latter usually causes centrilobular necrosis of hepatocytes and can result from acute right heart failure, portal hypertension or arterial hypoxia, for example, in cases of shock. In contrast, hepatic infarctions are rare because the liver has a dual blood supply from the hepatic arteries and the portal vein system.

The situation is different for the heart. As a rule, occlusion of the end arteries results in hypoxemic necrosis of myocytes due to the segmental blood supply of the myocardium. An enzyme pattern corresponding to that of the myocardial cell appears in the blood within 24 hours.

Damage to the skeletal muscles associated with enzyme release is manifold, the most important being injuries, hypoxic necroses, inflammations, infections, degenerative diseases, toxic damage (alcohol), uremia and neurogenic myopathies.

The lysis of erythrocytes causes the release of LD, the enzyme with the highest activity in these cells. A distinction is made between in-vivo and in-vitro hemolysis. In-vivo hemolysis occurs within the blood vessel (intravascular) and is immune-mediated in most cases, while in-vitro hemolysis is based on the destruction of the erythrocytes during blood sampling or several days of storing of whole blood prior to analysis.

In kinetic techniques used in clinical chemical routine diagnostics to determine the enzyme activity, concentrations of up to 10^–10 mol/L have been identified. This high analytical sensitivity allows the detection of even minor tissue damage by enzymatic analysis. Even if only 1 in approx. 750 hepatocytes is damaged, this induces elevated ALT levels to beyond the upper reference limit.

Clearance of serum enzymes

It is important to know about the clearance of enzymes in order to asses the enzyme activity as a diagnostic biomarker of organic diseases. Low-molecular enzymes such as the α-amylase are partly eliminated renally. Primarily, however, enzymes passing from the tissues into the blood are internalized in the cells of the reticuloendothelial system via receptor-mediated endocytosis.

There are, for example, specific receptors on the hepatocyte cell membrane that react with the N-galactosyl residues of the intestinal isoenzyme of ALP. This results in intracellular degradation into peptides available for participation in metabolic processes. The half-life of many enzymes in the blood is 4–24 hours (Tab. 1.1-1 – Half-lives of serum enzymes). It can be extended as a result of complexing of the enzyme with immunoglobulins or impaired function of the tissues or organs responsible for the clearance. In liver cirrhosis, for example, the clearance of enzymes is lowered due to a reduction in liver tissue. The half-life of α-amylase can be extended as a result of renal insufficiency or complexing with immunoglobulin as, for example, in cases of macroamylasemia.

1.1.1.1 Determination of enzymatic activity

The essential characteristic of an enzyme (E) is its ability to catalyze the conversion of a substance, also referred to as substrate (S), into a product (P). This happens according to the following reaction process /2/:

E+S → ES → E+P

In a first reaction step, the substrate binds to the enzyme forming the enzyme-substrate complex; the substrate part of this complex is converted to a product and the product is released. The released enzyme immediately binds another substrate molecule. The turnover number of most enzymes is at least 100 substrate molecules per enzyme molecule per second.

The determination of the conversion rate, measured as change of a substance (decrease in substrate or increase in product) per time unit, provides the basis for quantitative enzyme analysis. In enzyme analysis, the conversion rate is also referred to as reaction rate.

The concentration of an enzyme is determined by measuring the enzyme’s catalytic activity. The activity is preferably determined by kinetic assay.

Kinetic assay /2/: Under conditions where the concentration of the enzyme is much lower in the assay medium than that of its substrate, many enzymes behave according to the Michaelis-Menten model that correlates the velocity of an enzymatic reaction with the molar concentration of the enzyme and its substrate. Thus, the following correlation exists between the reaction velocity V of the enzyme reaction, its maximum velocity (V_max) and the substrate concentration [S]:

The Michaelis-Menten constant (K_m) is the substrate concentration, at which half of V_max of an enzyme-catalyzed reaction is reached. As a rule, it is 10^–2 to 10^–5 mol/L. The lower the value of K_m, the higher is the affinity of the substrate to the enzyme. Under defined conditions such as temperature, pH value, ionic strength etc and at constant enzyme concentration, the correlation between V and [S] follows a hyperbola that approaches the maximum V_max asymptotically. If [S] is much more abundant compared to K_m, V is independent of [S]. In this case, we say that the enzyme is saturated with substrate. Here, the amount of substrate converted by the enzyme is proportional to the amount of enzyme in the assay medium and the duration of the reaction.

The Michaelis-Menten model is inadequate to describe enzymatic reactions where individual participants show cooperative behavior, enzymes with allosteric reactions and multi-substrate reactions.

Photometric measurement of enzyme reaction /3/: The photometric measurement of an enzyme reaction is based on a change in light absorption (ΔA) during a time interval (Δt) due to the decrease in substrate concentration or increase in product concentration. The change in absorption (ΔA/Δt) is converted to a change in substrate or product concentration over time. This is done by multiplication with the factor V/ε x l, where V is the volume of the assay medium, ε is the molar absorption coefficient and l is the length of the light path through the cuvette. ΔA is non-dimensional; the dimensions of the other variables are T = Δt, L³ = volume V, L² × N^–1 = ε, N = substrate concentration and L= Liter. The reaction rate is calculated according to the following equation:

N × T^–1 is the amount of substrate turned over per time unit or the amount of product yielded per time unit and indicates the catalytic activity of an enzyme. The catalytic activity is given in katal.

The photometric analysis of the amount of turned-over substrate or the resulting amount of product is based on the principle of light absorption by an absorbing substance in a non-absorbing solvent according to the Bouguer-Lambert-Beer law.

A = ε × c × d

A is the absorption, c the substrate or product concentration, d the length of the light path through the cuvette and ε the molar decadic absorption coefficient of the substrate or product measured. The dimension of ε follows from:

ε = A/c × d = 1 × mol^–1 × mm^–1

The physiological substrates and products of most enzymes are colorless. Therefore, synthetic (chromogenic) substrates are used where a colored reaction product is obtained or an indicator reaction is arranged after the actual measurement reaction. In some enzymes that react directly with the co-substrates (co-enzymes) NADH₂ and NADPH₂ resulting in the oxidized co-enzyme forms NAD or NADP, the decline in NADH₂ and NADPH₂ is determined at 344 nm, 334 nm or 365 nm as indicator of enzymatic activity (simple optical assay). The reaction for the determination of LD is a typical example of a simple optical assay (see Section 1.11 – Lactate dehydrogenase (LD)). If the substrate produced in the measurement reaction cannot be immediately determined by photometry, it is associated with the indicator reaction through an auxiliary reaction in a reaction sequence involving the co-enzymes NADP/NADPH₂. The reaction for the determination of CK is an example of this (see Section 1.8 – Creatine kinase (CK)).

Comparable results can only be obtained if enzyme activities are measured at the same conditions. Therefore, the International Federation of Clinical Chemistry (IFCC) established a reference system for the measurement of the catalytic activity of many enzymes /4/. The measurement temperature is 37 °C. The reference system comprises primary reference methods, certified reference preparations and a worldwide network of reference laboratories.

The enzymatic activity is given in kinetic units because the enzymes are determined quantitatively based on their catalytic activity. The following units are defined:

• International unit (IU) by the Commission of Enzymes of the International Union of Biochemistry (IUB). 1 IU is the amount of enzyme that catalyzes a substrate turnover of 1 μmol per minute. The catalytic enzyme concentration is given in U/L, kU/L or mU/L.
• Katal by the International Union of Pure and Applied Chemistry and the IUB. A katal expresses the catalyzation of the substrate turnover of 1 mol per second. Enzyme activity is given in katal/L or μkatal/L. The katal is in line with the Systeme Internationale (SI) where the Mol is the unit of the turned-over substrate and the second is the unit of time. Hence, 1 U = 1 μmol/60 s = 0.0167 μmol/s or 1.0 μkatal/L = 60 U/L.

1.1.1.2 Isoenzymes and isoforms

Isoenzymes

According to the IUPAC-IUB Commission on Biochemical Nomenclature, isoenzymes are defined as follows: Proteins with similar enzymatic activity encoded by different genes. Besides different amino acid sequence and different catalytic properties, they can differ in regards to affinity to substrates, activators and inhibitors, optimal pH and temperature and heat stability. The following categories of isoenzymes are distinguished /5/:

• Genetically independent enzymes encoded by various gene loci such as the mitochondrial and cytoplasmic forms of malat-dehydrogenase and AST.
• Alloenzymes – these are enzyme variants encoded by allelic genes of a single gene locus, for example the alloenzymes of glucose-6-phosphate dehydrogenase.
• Heteropolymeric enzymes. These are non-covalent hybrid molecules of two or more different polypeptide chains, for example the intermediate isoenzymes of lactate dehydrogenase.

Isoforms

Enzyme isoforms result from post translational modification of an enzyme, for example the tissue-nonspecific ALP from which the isoforms of liver ALP, bone ALP and kidney ALP are formed post translationally.

Isoenzyme and isoform analysis

Determination of activity: An isoenzyme family can have similar, but not identical, catalytic activities. They mostly differ slightly in optimal pH, affinity constants for the substrate and sensitivity to inhibitors or denaturing reagents. Alloenzymes can, but do not necessarily have to, differ in their catalytic activity. Severe or even terminal illness can result if it is due to the mutant allele that an enzyme, which plays an important role in metabolism, is not produced or the produced form is not activatable.

Isoenzymes are distinguished by determining their enzymatic activity. This is done by measuring the differences in catalytic activity against substrate analogs or in the presence of inhibitors. Moreover, the catalytic activity of isoenzymes differs in regards to the stability toward denaturing reagents. As a rule, multiple enzymes do not show any differences in stability toward denaturing reagents.

Separative methods: A change in a small number of amino acid residues causes no, or only very minor, changes in the molecular weight of the isoenzymes. Therefore, chromatographic methods are not very useful in the differentiation of alloenzymes, but all the more so in the differentiation of multiple enzyme forms. If structural changes in isoenzymes cause a change in electric charge, agarose gel electrophoresis or isoelectrofocusing can be suitable separation methods. If there are variations in the carbohydrate side chains, lectin affinity chromatography can be useful.

Immunological analysis: Isoenzymes or multiple enzyme forms can differ in their antigenic determinants and be identified based on the binding of specific antibodies.

Clinical significance of isoenzymes and isoforms

The presence of isoenzymes and isoforms in serum increases the diagnostic sensitivity and specificity for the detection of organic diseases. The clinical conclusions are is described in the relevant articles on enzymes in this chapter.

1.1.1.3 Macroenzymes

Isoenzymes and isoforms are differentiated from enzymes characterized by multiplicity (multiple enzymes) that form based on post translational changes as a result of /6, 7/:

• Conjugation with other molecules or binding to other molecules after degradation
• Polymerization of single subunits to form a major complex
• Complex formation between enzyme and immunoglobulin
• Allosteric modification of enzymes.

The most common macroenzymes are immunoglobulin-associated (type 1 macroenzymes); all others are referred to as type 2 macroenzymes.

Immunoglobulin-associated enzymes: They are the most common form of macroenzymes and result from the formation of an immune complex between enzyme and autoantibody. This is a normal antigen-antibody reaction. The antigenic determinant of the enzyme binds to the Fab fragment of the antibody forming a macromolecular enzyme-immunoglobulin complex. The higher molecular weight with subsequently delayed clearance results in an accumulation in serum and elevated enzyme activity.

Some antibodies only react with a specific isoform of the enzyme, others react with all isoforms. The binding of the enzyme to the Fab fragment of the antibody stabilizes the enzyme’s activity in regards to thermal effects, influences the rate of elimination from the bloodstream and also has an effect on the enzyme kinetic characteristics. A slight inhibitory effect can be determined in many cases, whereas strongly inhibiting antibodies are rare.

Enzymes bound to other molecules: Complex formation is not only possible with autoantibodies, but also with molecules such as hydroxy ethyl starch, lipoproteins and α₂-macroglobulin. This classification also includes oligomeric forms such as mitochondrial CK that passes into plasma in cellular necrosis. The biliary tract ALP, an alkaline phosphatase, is a different form of macroenzyme. It is bound to membrane fragments and detectable in serum in cholestasis.

Macroenzymes often persist for a long time, can cause elevated enzyme activity and thus simulate disease.

Clinical significance of the macroenzymes

Macroenzymes have a higher molecular weight than the free enzymes and therefore circulate in the plasma longer. Macroenzymes do not function as biomarkers of specific diseases. Elevated enzyme levels in serum caused by macroenzymes often lead to confusion among the treating physician and the laboratory and result in a multitude of further cost-intensive, unnecessary procedures. The significance of clinically important macroenzymes is shown in Tab. 1.1-2 – Macroenzymes: Characterization, clinical significance and laboratory findings.

Immunoglobulin-bound macroenzymes: macroforms have been described for the following diagnostically important enzymes: ALT, ALP, α-amylase, AST, CK, GGT, LD, lipase. Macroenzymes are rare events in healthy individuals, but if an enzyme level is pathologically elevated, it persists for a long time. The persistence of activity can remain almost unchanged for years. It is true that the concurrent occurrence of macroenzymes and autoimmune disorders has been described in individual cases. If detected, however, it must not be assessed as a specific and sensitive indication of a specific, manifested disease. There are no reliable indications that immunoglobulin-bound macroenzymes express an autoimmune disorder or that these circulating enzyme immune complexes themselves have a detrimental impact. The prevalence of macroenzymes generally increases with increasing age.

The fact that there are often more antibodies than the corresponding enzyme is another special characteristic of immunoglobulin-bound macroenzymes. Enzyme molecules released due to acute tissue damage can bind immediately to the free binding sites and be converted to the macroform. Thus, they may escape electrophoretic determination of the isoenzymes. On the other hand, the excess of antibodies results in competition for the enzyme molecule in the immunological enzymatic and isoenzymatic analysis; this may cause falsely low concentrations, especially in cases with short incubation periods.

Immunoglobulin-bound macroenzymes that are detected by coincidence in many cases only represent the tip of an iceberg; a much higher prevalence can easily be determined if they are selectively searched for. The detectability of macroenzymes in the blood of patients and healthy individuals increases as a function of the sensitivity of the detection procedure used. In these cases, however, the enzyme activities range within the reference interval. On this account, the prevalence given in the references often varies and seems contradictory at first glance.

Enzymes bound to other molecules: Macroforms have been described for the following diagnostically important enzymes: ALP, amylase, CK and GGT. The activity-time curve of this type of macroenzymes in many cases reflects the course of a disease. These macroenzymes can also be temporarily observed after therapeutic measures. Therefore, they may no longer be detectable if the disease improves or is cured. The determination of the prevalences for non-immunoglobulin-bound macroenzymes is even more strongly dependent on the detection procedure used and the selection of examined patients than for immunoglobulin-bound macroenzymes.

Unclear constellation of enzymes: It is important in all cases where the constellation of the enzymes does not match the clinical aspects to confirm or exclude the presence of macroenzymes. It often happens that elevated activities are prematurely classified as laboratory errors if no corresponding clinical correlate can be found. The verified presence of a macroenzyme can spare the patient from wrong diagnostic and therapeutic decisions. In particular, this applies to macro CK, which relatively often interferes with the determination of CK-MB.

1.1.1.4 Antibodies to tissue-specific enzymes

These autoantibodies circulate in blood and are directed against tissue-specific enzymes or regulators of specific enzyme activities /8/. The enzymes are expressed only in one tissue, for example against thyroid peroxidase, or in several tissues, for example against pyruvate dehydrogenase (Tab. 1.1-3 – Antibodies with regulating effects to the activity of tissue-specific enzymes). Macroenzymes often occur in older individuals and have no clinical significance in most cases, while antibodies to tissue-specific enzymes or their regulators show significant disease association.

1.1.2 Diagnostic information of enzymes

Indications of enzyme determination in serum or plasma include:

• Detection of tissue damage
• Organ-specific localization of the damage
• Determination of the extent of damage
• Determination of the severity of cell damage (reparable or irreparable)
• Laboratory tests on the underlying disease
• Differential diagnosis of the disease of an organ (localization of the single cell damage in the affected tissue).

Information is gained from:

• The level of enzyme activity in serum
• The determination of enzyme patterns (total of a spectrum of enzyme activities concurrently determined in serum)
• The assessment of enzyme activities in relation to one another, for example the calculation of enzyme ratios
• The monitoring of enzyme activities
• The determination of isoenzymes.

The level of enzyme activity is the resultant of several processes characterized by a typical time course. The following questions should be asked in the interpretation of an elevated enzyme level:

• Is there an increased release of the enzyme from an organ (e.g., tissue damage?)
• Is there a disturbance of the standard elimination mechanisms that usually remove the enzyme from circulation (e.g., renal insufficiency or liver cirrhosis?)
• Can the enzyme have bound to a serum component (e.g., is there a macroenzyme?)
• Is the elevated enzyme activity caused by increased enzyme production (e.g., enzyme induction?).

Location of the disease (organ localization)

The damaged tissue or organ can be localized by:

• The determination of specific enzymes of important organs
• The differentiation of isoenzymes
• The assessment of symptom-oriented enzyme patterns.

Tissue-specific enzymes: These are enzymes that occur exclusively in specific organs/tissues or occur there at very high activity compared to other organs/tissues. Increased presence in serum indicates the origin of the enzyme (Tab. 1.1-4 – Specific enzymes of important organs).

Isoenzymes: The tissue distribution of isoenzymes is genetically determined. Differentiation allows to determine the tissue where the elevated enzyme activity originated (e.g., pancreatic amylase, salivary gland amylase, myocardium-specific CK-MB or erythrocytic LD-1).

Enzyme pattern: Enzyme patterns can provide information regarding organ diagnosis based on the interrelation of enzyme activities. Aminotransferases are the basic enzymes of most enzyme patterns; the enzyme ratio is an indicatory criterion. More than 90% of all enzyme elevations are caused by the tissues of the liver, myocardium, skeletal muscles and erythrocytes. These tissues are significant for the differential diagnosis. The differentiation of the damage of one of the three tissues from liver injury is possible based on the CK/AST and LD/AST ratios (Tab. 1.1-5 – Differentiation of liver disease from other tissue damage).

Stage of the pathological process

Each mechanism that causes enzyme release to the blood and enzyme elimination from the blood shows a typical time course. The interaction of the different time courses results in characteristic activity-time curves. These curves can be used to derive a diagnostic time frame within which elevated enzyme activities are expected if the relevant disease is present. Moreover, the stage of the disease can be assessed based on this time frame.

If the organ is known, enzyme activities are usually higher in acute processes than in chronic ones. In acute organic diseases, the stage of the disease can also be determined from the ratio between enzymes with a short half-life and those with a long half-life. Differences in half-lives distort the organ-specific enzyme profile in serum and thus provide important information on the course of the disease. In acute hepatitis, for example, a declining AST/ALT ratio indicates remission of the hepatitis because the half-life of ALT is longer than that of AST.

Severity of cell damage

The severity is determined based on the ratio between structure-bound enzymes and enzymes dissolved in the cytoplasm (Tab. 1.1-6 – Enzyme pattern in acute severe tissue damage with cell necrosis). In milder damage, enzymes of the cytoplasm such as ALT and cytoplasmic AST are released. In severe damage with cell necrosis, the mitochondrial enzymes such as AST and GLD also pass into the plasma.

In liver disease, the extent of single cell damage is indicated by the AST/ALT ratio and the (AST + ALT)/GLD ratio. Values of the AST/ALT ratio above 1 or the (AST + ALT)/GLD ratio below 20 indicate acute severe hepatocellular damage. The enzyme pattern in serum in acute cell damage is similar to that of the tissue of origin.

Extension of cell damage

The level of enzyme activity and the integral below the activity-time curve, determined by 2–3 measurements within 24 hours for several days, correlate with the amount of tissue acutely damaged. High enzyme elevations indicate damage to major organs such as the liver or skeletal muscles.

Diagnosis of the disease

The enzyme pattern can provide decisive clues for diagnosis in patients with ambiguous acute clinical symptoms. If the pattern of CK, AST, ALT and lipase is determined in thoracic and/or abdominal pain, for example, it is with high probability that normal CK rules out myocardial infarction, normal ALT rules out acute liver disease and normal lipase rules out pancreatitis within a diagnostic time frame of 3–12 hours.

Differential diagnosis of the disease of an organ

In differential diagnosis, the ratio between the serum values of enzymes that are exclusively localized in defined structures or tissues of an organ and enzymes that occur in all cells of the organ with roughly the same activity is assessed, for example the behavior of GGT, ALP or GLD with reference to the aminotransferases in liver disease. The calculation of the ratio allows to differentiate between the following acute liver diseases:

1.1.3 Enzymes in the differential diagnosis of acute diseases

In acute situations, the enzyme pattern of aminotransferases, CK, α-amylase/lipase, ALP and GGT provides information on the presence of important organic diseases. Several examples are listed below.

1.1.3.1 Differential diagnosis in acute thoracic and abdominal pain

Enzyme patterns for differential diagnosis in acute thoracic pain are shown in Tab. 1.1-7 – Enzyme pattern in acute thoracic or abdominal pain.

1.1.3.2 Enzyme patterns in liver disease

Enzyme patterns in acute and chronic liver diseases are shown in Tab. 1.1-8 – Enzyme pattern in acute liver diseases.

1.1.3.3 Enzyme activities in intensive care patients

Multi morbid patients and patients with sepsis, pneumonia, peritonitis, acute pancreatitis, postoperative or posttraumatic conditions, severe gastrointestinal complications, cardiac diseases including failure of the cardiac pumping function, persistent shock conditions or hematological diseases can show changed enzyme activities of these organs of origin despite primarily healthy liver and pancreas. Lack of perfusion of these organs is the cause in many cases (Tab. 1.1-9 – Median peak value of 100 critically ill patients with no primary liver disease, but with bilirubin values > 3 mg/dL (51 μmol/L)).

The mortality risk of critically ill patients is correlated with the presence of elevated liver enzyme activities on admission to the intensive care unit. Patients with elevated ALT, GGT or ALP up to twofold the upper reference limit on admission to the ICU have a lower chance of survival within 30 days (mean odds ratios: 2.7, 2.8, 3.9) than those without pathological levels. Episodes of artificial respiration or hemofiltration result in pathological ALT activities after three days (mean odds ratio: 2.7) /26/.

1.1.3.4 Enzyme activities in surgical interventions

This involves muscle enzymes in the first place and liver enzymes in the second (Tab. 1.1-10 – Incidence of elevated enzyme values after abdominal surgery). The maximum enzyme activity in interventions without complications is reached after 24–36 hours. The activity and duration of enzyme elevation are dependent on the nature and extent of intervention.

In courses without complications, the levels return to normal within 1 week. In abdominal interventions, elevated activities were measured for CK in 76% and for AST in 50% of the cases. The activities of CK were elevated up to seven-fold the upper reference limit (median: 2.1-fold) and those of AST were elevated up to 3.5-fold (median: 1.2-fold) /27/.

1.1.3.5 Enzymes in cerebral seizures

Grand mal seizures are regularly associated with elevated CK. The activity in idiopathic grand mal seizure is up to 6-fold the upper reference limit; much higher levels are observed in grand mal seizure after alcohol withdrawal, and 50–100-fold elevated levels are measured in the epileptic state. Peak values are reached within 1–3 days; levels decline to within the reference interval after 4–10 days. After the epileptic state, the activity pattern of CK, LD and AST corresponds to that in myocardial infarction; the ALT can also be elevated. Patients suffering idiopathic grand mal seizure do not have a uniform enzyme pattern /28/. The CK isoenzymes CK-MB and CK-BB do not show pathological levels.

1.1.3.6 Enzymes in cancer

Elevations of enzyme activities in serum measured in tumor diseases are dependent on the stage of the tumor. However, the enzymes are not suited for use as a screening method for malignant tumors. Organ-specific enzymes such as GGT or the so-called ubiquitous enzymes can be elevated /29/. The latter belong to the large group of glycolytic enzymes such as LD. They are involved in the metabolism of the cell and ubiquitous in all organs. Enzyme elevations occurring in tumor diseases can be based on:

• Increased synthesis due to the tumor such as ALP in osteogenic tumors
• Blocked duct systems (e.g. elevated ALP due to the regurgitation of the enzyme to the blood) for example, because of obstruction of the bile ducts in metastatic hepatocellular carcinoma
• Induction of the enzyme by the tumor (e.g., ALP and GGT in metastatic hepatocellular carcinoma)
• Change in permeability of the tumor cell and resulting leakage of the enzyme into circulation (e.g., acidic phosphatase in prostate carcinoma).

The behavior of enzymes in carcinoma patients is shown in Tab. 1.1-11 – Enzymes in malignant diseases.

1.1.4 Biological influence factors and interference factors

Elevated or low enzyme activities can be due to biological influence factors and interference factors affecting the relevant assay /24/. Biological influence factors lead to changes in the enzyme activity in serum in vivo, i.e., already before blood sampling, while interference factors change the result in vitro (Tab. 1.1-12 – Factors interfering with the serum enzyme determination).

1.1.4.1 Biological influence factors

Important biological influence factors causing changes in enzyme activity include diagnostic and therapeutic measures, food intake, alcohol, physical exertion, pregnancy, body position and constriction method during blood sampling as well as post translational changes in the enzyme such as macroenzyme formation.

Food intake

Elevations of the ALT activity by 10% and the AST activity by 20% compared to the initial level can occur two hours after a generous lunch; the ALP activity can also be elevated significantly. The elevation of ALP is especially pronounced in individuals with blood groups 0 and B and Lewis-positive.

Blood sampling

Posture and tourniquet application have a clinical relevant effect on the serum enzyme activity when the levels are in the upper reference interval. If the blood samples are collected with the patient in a sitting position after seating for at least 15 min. (the normal office situation), the enzyme values are 5–10% higher. Tourniquet application for more than 2 min. has the same effect so that the addition of both biological factors can lead to an increase in enzyme activity of 10–20% /32/. Compression of the vein for 6 min. leads to an increase of 8–10% in lipase, ALT, CK, GGT and LD by 8–10% /33/.

Intraindividual variation

No significant clinically relevance in diurnal variations of the serum enzymes have been found.

In a study /34/, the following values for the coefficient of variation (CV) were determined for the intraindividual variance within 6 months: CK 22.8%, ALT 30%, AST 12.2%, LD 10.3%, GGT 12.9%, ALP 7.4%.

Age dependency

The following age-related differences have been described for some enzymes /35/:

• ALP is about 3-fold higher in children and adolescents than in adults and increases mildly in women after menopause
• ALT decreases in men in older age and remains constant in women
• AST increases in older age, especially in women
• CK decreases pronouncedly in men in older age.

Physical exertion

Muscle activity causes elevated enzyme activities, especially for CK, AST and LD. The level, duration and frequency of the elevation are dependent on the physical condition. The increase follows the pattern CK > AST > LD; levels usually return to normal within a week /36/.

In bodybuilders, for example, 5-fold elevated CK and 2-fold elevated AST activities have been reported. The ALT elevates further upon the intake of anabolics.

On the third day during an ultra-long distance run of 50 klometers per day for 20 days, the athletes show an average elevation of CK to 20-fold, CK-MB to 2-fold and AST to 3-fold the upper reference interval value. Afterwards, until the end of the run, the CK decreases to 8-fold the upper reference interval value and CK-MB and AST return to levels within the reference interval /37/.

Nutrition

Prolonged fasting as well as high protein intake can result in elevated aminotransferases. The LD can increase after high-fat diet and decrease after low-fat diet. Individuals with blood groups 0 and B and Lewis-positive show elevated ALP after a high-fat meal due to the pronounced increase in intestinal ALP.

Pregnancy and oral contraceptives

Working pregnant women can have elevated CK, especially CK-BB. Oral contraceptives in doses corresponding to those of the micro pill usually do not cause elevated enzyme levels. Enzyme induction has been described for ALP and GGT if the micro pill contains ethinylestradiol /38/.

Alcohol

Depending on the consumed quantity and duration of consumption, alcohol causes elevated GGT levels and with additional liver injury also elevated ALT, AST and GLD. The elevation can be considerable if the alcohol is consumed during or after physical exertion. The enzyme increase in alcoholism follows the pattern GGT > AST, ALT > GLD.

Drugs

Drugs can cause elevated enzyme activities due to enzyme induction or tissue damage (Tab. 1.1-13 – Drugs governing the rate of enzyme-catalyzed reactions).

1.1.4.2 Interference factors

Drugs, hemolysis, hyperbilirubinemia, hyperlipidemia, metabolites of the sample and anticoagulants are key interference factors that can cause a change in enzyme activity.

Metabolite-induced interferences

Pyruvate concentrations of the sample higher than 1,100 μmol/L inhibit the optical assay through NADH₂ consumption in the preincubation step /39/. Falsely normal activities of AST, ALT and GLD are measured. The pyruvate passes from the erythrocytes into the serum if the erythrocytes are separated many hours later.

Drugs

Drugs have little effect on in-vitro kinetic enzymatic analysis. A detailed round-up of references is presented in Ref. /40, 41, 42/.

Hyperbilirubinemia

Hyperbilirubinemia usually does not interfere with kinetic enzymatic analysis. In a study /43/, in which the bilirubin concentration in samples was 29.2 mg/dL (500 μmol/L), the levels of ALP, ALT, AST, α-amylase, CK, GGT and LD were determined on 16 automatic analyzers. The measurement of AST was interfered on three and that of ALT on two automatic analyzers and that of LD on one automatic analyzer.

Hemolysis

Hemolysis interferes with kinetic enzymatic analysis depending on the concentration. In a study /43/, in which the concentration of free hemoglobin in samples was 2.4 g/L (240 μmol/L), the activities of ALP, α-amylase, CK and GGT were determined on 16 clinical chemical analyzers. Interferences with the measurement of ALP was found in 8 test systems, with the CK in 9 and with the measurement of GGT in 3.

Hypertriglyceridemia

Hypertriglyceridemia usually does not interfere with kinetic enzymatic analysis on automatic analyzers up to a concentration of 610 mg/dL (700 μmol/L) /43/.

Anticoagulants in diagnostic samples

All enzymatic analyses can be performed in serum and in heparin-anticoagulated plasma. The determination of ALT, AST, CK, GLD and LD is also possible in EDTA plasma. Citrate plasma should not be used for enzymatic analysis because there is only insufficient data available on interference factors /44/.

Enzyme stability during storage /45/

In sera separated from the clot, ALP, α-amylase, ALT, AST, CK, CHE, GGT and LD remain stable for at least 4 days at 9 °C and, at a temperature of 20 °C, ALP, α-amylase, ALT, AST and CHE remain stable for 3 days.

References

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3. Haar HP, Netheler H, Ziegenhorn J. Absorption photometry, nephelometry, turbidimetry. In: Bergmeyer HU, ed. Methods in enzymology, vol 1. Weinheim, VCH 1981: 280–305.

4. IFCC primary reference procedures for the measurement of catalytic activity concentrations of enzymes at 37 °C. Part 1–7. Clin Chem Lab Med 2002; 40: 631–745.

5. IUPAC-IUB Commission on Biochemical Nomenclature. Nomenclature of Multiple Forms of Enzymes. Recommendations 1976. J Biol Chem 1977; 252: 5939-41.

6. Remaley AT, Wilding P. Macroenzymes: Biochemical characterization, clinical significance, and laboratory detection. Clin Chem 1989; 35: 2261–70.

7. Sturk A, Sanders GTB. Macroenzymes: prevalence, composition, detection and clinical relevance. J Clin Chem Clin Biochem 1990; 28: 65–81.

8. Kiechle FL, Quattrociocci-Longe TM, Brinton D, Gordon S, Sykes E, Elkhalifa MY. Autoantibodies to specific enzymes: a review. Ann Clin Lab Sci 1996; 26: 195–207.

9. Wenham PR, Chapman B, Smith AF. Two macromolecular complexes between alkaline phosphatase and immunoglobulin A in a patient’s serum. Clin Chem 1983; 29: 1845–9.

10. Mifflin E, Bruns DE. University of Virginia Case Conference. Macroamylase, macro creatine kinase, and other macroenzymes. Clin Chem 1985; 31: 1743–8.

11. Zaman Z, van Orshoven A, Marien G, Fevery J, Blanckaert N. Simultaneous macroamylasemia and macrolipasemia. Clin Chem 1994; 40: 939–42.

12. Stasia MJ, Suria A, Renversez JC, Pene F, Morel-Fermiez A, Morel F. Aspartate aminotransferase macroenzyme complex in serum identified and characterized. Clin Chem 1994; 40: 1340–3.

13. Krishnamurthy S, Korenblat KM, Scott MG. Persistent increase in aspartate aminotransferase in an asymptomatic patient. Clin Chem 2009; 55: 1573–7.

14. Tameda M, Shiraki K, Ooi K, Takase K, Kosaka Y, Nobori T, et al. Aspartate aminotransferase-immunoglobulin complexes in patients with chronic liver disease. World J Gastroenterol 2005; 11: 1529–31.

15. Caropreso M, Fortunato G, Lenta S, Palmieri D, Esposito M, Vitale DF, et al. Prevalence and long-term course of macro-aspartate aminotransferase in children. J Pediatr 2009; 154: 744–8.

16. Laureys M, Sion JP, Slabbynck H, Steenssens L, Cobbaert C, Derde MB, et al. Macromolecular creatine kinase type 1: a serum marker associated with disease. Clin Chem 1991; 37: 430–4.

17. Davidson DF, Scott JG. Detection of creatine kinase isoenzymes. Ann Clin Biochem 2012; 49: 482–5.

18. Wenham PR, Horn DB, Smith AF. Multiple forms of γ-glutamyltransferase: a clinical study. Clin Chem 1985; 31: 569–73.

19. Nemesanszky E, Lott AJ. Gamma-glutamyltransferase and its isoenzymes: progress and problems. Clin Chem 1985; 31: 797–803.

20. Klonoff DC. Macroamylasemia and other immunoglobulin-complex disorders. West J Med 1980; 133: 392–407.

21. Weijers RNM, Mulder J, Kruijswijk H. Partial characterization, properties and clinical significance of a lactate dehydrogenase immunoglobulin A kappa complex in serum. Clin Chem 1983; 29: 272–8.

22. Bode C, Riederer J, Brauner B, Bode JC. Macrolipasemia: a rare case of persistently elevated serum lipase. Am J Gastroenterol 1990; 85: 412–6.

23. Frank B, Gottlieb K. Amylase normal, lipase elevated: is it pancreatitis? A case series and review of the literature. Am J Gastroenterol 1999; 94: 463–9.

24. Schmidt E, Schmidt FW. Enzym-Muster. Diagnostik 1975; 8: 427–32.

25. Kleinberger G. Leberfunktionsstörungen und Leberschäden bei kritisch kranken Intensivpatienten. Leber, Magen, Darm 1985; 15: 175–7.

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1.2 Assessment of the diagnostic ability of enzymes and scores

Lothar Thomas

Elevated liver enzymes can have a multitude of causes and causative factors. As a rule, a disease at hand can be diagnosed based on the medical and medication history, clinical examinations with upper abdominal sonography and laboratory diagnostic findings.

Besides diagnosing, biomarkers are useful to differentiate between acute and chronic hepatopathy, assess the severity, assist etiological verification, draw prognostic conclusions and perform therapy monitoring.

General clinical liver-associated tests

The following important general tests and analyses primarily give a direction to further procedure in many cases, based on their constellation and taking into account the medical and medication history /1/:

• The aminotransferases ALT and AST. Elevated levels act as markers of liver inflammation (e.g., viral hepatitis, autoimmune hepatitis, non-alcoholic fatty liver) and are indicative of a relevant liver disease. The non-alcoholic fatty liver has become the most commonly diagnosed cause of chronic liver disease. Levels within the reference interval do not rule out liver disease, especially in chronic infections with hepatitis viruses. Aminotransferases are not only an indicator of liver disease, but also a biomarker of the general morbidity and mortality risk.
• Gamma-glutamyl transferase (GGT). This is a cholestatic metabolic biomarker. It is elevated in alcoholic or non-alcoholic fatty liver disease. Patients with elevated GGT also have the risk of increased cardiovascular mortality.
• The alkaline phosphatase (ALP). It acts as a biomarker of cholestasis (e.g., primary biliary cirrhosis, primary sclerosing cholangitis).
• Hepatitis serology (HB_SAg and anti-HBc) is part of the general clinical tests in elevated aminotransferase and suspected viral hepatitis.
• Glutamate dehydrogenase (GLD) and the bilirubin are significant for assessing the tissue damage in acute severe liver disease. However, the GLD level only allows limited conclusions as to the extent of tissue damage and prognosis.

1.2.1 Determination of extrahepatic factors

The enzymes analyzed in general clinical tests can also be elevated in hepatic co-reactions within the scope of extrahepatic and systemic diseases. This can be the case, for example, in pancreatitis, exogenous-toxic, exogenous-allergic, autoimmune, vascular and metabolic diseases.

Assays: Blood count, albumin, serum protein electrophoresis, cholesterol, triglycerides, HbA_1c, ferritin /1/.

Complementary investigations /1/

If the general clinical liver-associated tests indicate a liver disease, further assays are required for differentiation.

Assays: HBsAg, anti-HCV, IgG, IgA, IgM, ANA, AMA, SMA, LKM-Ak, SLA, pANCA (see Chapter 25 – Autoimmunity and autoantibody testing), ceruloplasmin, Cu in 24 h urine, HFE mutation, α₁-antitrypsin genotype.

Chronic liver disease

Chronic liver diseases are usually asymptomatic. ALT and GGT are moderately elevated to 2–5-fold the upper reference limit. One should keep in mind in differential diagnosis that, according to investigations in the U.S.A., elevated ALT occurs in 8% of the normal population and 70% of these cases are primarily unclear. The main cause seems to be fatty liver in the form of non-alcoholic steatohepatitis (NASH) in adiposity and metabolic syndrome, and not the non-alcoholic fatty liver (NAFL) /2/.

Inflammation and fibrosis are the essential characteristics of chronic liver disease. Classification into stages is based on the assessment of the progressive deposition of fibrotic extracellular matrix (ECM), also referred to as fibrogenesis. The qualitative composition of ECM (various types of collagen, proteoglycans, structural glycoproteins, hyaluronic acid) and their spatial distribution within the liver are subject to significant variation.

In addition, the stage is determined based on compensatory regenerative processes causing changes in the anatomic structure.

Liver biopsy is the gold standard for the assessment of fibrosis. Information regarding fibrotic transformation can also be obtained from biomarkers (increase in De Ritis ratio, thrombocytopenia, hypergammaglobulinemia, cytokeratin-18 fragments). Fibrosis staging scores have been developed because of the low diagnostic value of the individual parameters (Tab. 1.2-1 – Asessment of the diagnostic ability of scoring systems in acute kidney failure and acute liver failure).

Fatty liver

The following are distinguished from alcoholic fatty liver:

• Non-alcoholic fatty liver disease (NAFLD)
• Non-alcoholic fatty liver (NAFL), a benign form of NAFLD
• Non-alcoholic steatohepatitis (NASH). NASH is characterized by microvascular steatosis, enlargement of the hepatocytes with lipid accumulation and lobular or portal inflammation with and without fibrosis.

Liver cirrhosis

Liver cirrhosis is the final stage of many chronic liver diseases progressing for years or decades /3/. It is characterized by lobular transformation of the parenchyma with septal fibrosis, infiltration of inflammatory cells and a change in the vascular bed. The progression of liver cirrhosis is governed by its etiology. The main causes of liver cirrhosis are alcoholic and non-alcoholic fatty liver disease, hepatitis B, hepatitis C and the combination of hepatitis C and alcohol abuse. Increasing structural changes of the tissue lead to a loss of function and portal hypertension. Vascular dysfunction results in the increased release of vasoconstrictive hormones. Imminent complications include: intestinal hemorrhage, ascites, encephalopathy and hepatocellular carcinoma /5/.

Hepatocellular carcinoma (HCC)

The majority of hepatocellular carcinomas arise in a background of cirrhosis. The risk of carcinoma depends on the present liver disease and is /3/:

• 19.8% in 13 years in chronic hepatitis B with a viral load > 10⁷ copies/mL
• 2.9% in 10 years in autoimmune hepatitis.

Laboratory findings

According to laboratory test results in alcoholic fatty liver, GGT is elevated higher than ALT. These ratios are inverted in NASH associated with elevated enzyme levels. Liver enzymes are usually not elevated in NAFL. Findings in chronic liver disease with transition into liver cirrhosis include mildly elevated aminotransferases, hypoalbuminemia, plasma thrombine time extension, reduced cholinesterase and hyperbilirubinemia. Advanced liver disease with transition into cirrhosis is indicated by thrombocytopenia.

Cytokeratin-18 fragments (CK-18) are detectable in serum and act as a fibrosis marker of NASH. In a study /4/, the mean CK-18 values of controls were 145 U/L (25th to 75th percentile 126–190) and those of patients with NASH were 244 (161–427) U/L. Expressed as odds ratio, the risk of NASH increased by 30% with every increase by 50 U/L.

1.2.2 Scores: Liver failure, non-alcoholic fatty liver disease

Liver failure can be acute or based on irreversible chronic liver disease. Orthotopic liver transplantation is the only causal therapy of liver failure /6/.

Chronic diseases causing liver failure include: alcohol-toxic liver cirrhosis, viral and autoimmune hepatitides, cholestatic liver diseases, malignomas, metabolic disorders and genetic disorders (Wilson’s disease, hemochromatosis, α₁-antitrypsin deficiency, urea cycle disorders, storage diseases).

Acute liver failure is potentially reversible. Acute paracetamol intoxication and acute hepatitis B infection are the main causes of acute liver failure. Orthotoptic liver transplantation is the therapy option for the prognostic assessment and selection of patients with irreversible liver failure. Scoring systems are available to identify indications (Tab. 1.2-1 – Assessment of the diagnostic ability of scores in acute kidney failure and acute liver failure).

The Model for Endstage Liver Disease (MELD) score has been established in the Eurotransplant region to regulate organ allocation. It is based on the determination of bilirubin and creatinine in serum and International Normalized Ration (INR) in citrated blood.

MELD/PELD score /7/

The current version Model for End-stage Liver Disease (MELD) consisting of the international ratio (INR) serum bilirubin, and creatinine, had been used to determine organ allocation priorities for liver transplantation. The final mutivariable model (MELD 3.0) was characterized by (1) the additional variables of female sex and serum albumin, (2) interactions between bilirubin and sodium and between albumin and creatinine, and (3) upper bound for creatinine at 3.0 mg/dL. MELD 3.0 had better discrimination

The Pediatric End-stage Liver Disease (PELD) score for children serve as indicators for the urgency of orthotopic liver transplantation. An estimate of the three-month mortality in the end stage of chronic liver disease is possible.

Calculation of the MELD score

Calculation of the MELD 3.0 score

Calculation of the PELD score

Assessment of MELD score: Scores are between 6 and 40, the higher the score, the higher the probability to die without transplantation.

Assessment of MELD 3.0 score: Scores are between 13 and 26, the higher the score, the higher the probability to die without transplantation /56/.

Assessment of MELD score: The validity of the MELD score for assessing the prognosis of liver failure and the prognosis after transplant is shown in Tab. 1.2-3 – Mortality rate (%) after 3 months as a function of the MELD score. For assessment, the PELD score is transformed into the MELD score using an equation /7/.

Prognosis of paracetamol-induced liver injury /8/

Determine the α-fetoprotein (AFP) on a daily basis for 7 days from the point in time at which the ALT exceeds 1,000 U/L. Survivors show elevated AFP in the median on day 1, non-survivors only after 4.1 days. An AFP level of not more than 3.9 μg/L on day 1 identifies non-survival (diagnostic sensitivity of 100% at 74% specificity, positive predictive value: 45%, negative predictive value: 100%). Refer to Tab. 1.2-4 – Modified Child-pugh score.

Non-alcoholic fatty liver disease (NAFLD) fibrosis score

The term non-alcoholic fatty liver disease covers cases of a wide spectrum of severity, ranging from bland fatty liver without any inflammation and with little or no tendency to progress all the way to non-alcoholic steatohepatitis (NASH) with inflammatory reactions and hepatocyte damage, with or without fibrosis. Between 5 and 20% of patients with fatty liver develop NASH over the clinical course; in some 10-20% this develops into higher-grade fibrosis, in 5% this is progressive to full-blown cirrhosis. The NAFLD fibrosis score should be performed for diagnosis. The score is computed on the basis of the parameters age, body-mass index, diabetes status , ASAT ALAT, platelet count and albumin level. The positive predictive value is 82% to 90% and a negative predictive value is 88% to 93% /9, 10/.

Hepatopathies and biomarkers

For the clinical and laboratory findings in acute and chronic hepatopathies refer to

• Tab. 1.2-5 – Biomarkers in hepatopathies
• Tab. 1.2-6 – Hepatitis B states
• Tab. 1.2-7 – Assessment of patients with chronic hepatitis B.

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56. Ricci Z, Cruz D, Ronco C. The RIFLE criteria and mortality in acute kidney injury: a systematic review. Kidney Int 2008; 73: 538–46.

57. Hoste EAJ, Clermont G, Kersten A, Venkataraman R, Angus DC, De Bacquer D, Kellum JA. Crit Care 2006. doi: 10.1186/cc4915.

58. Uchino S, Kellum JA, Bellomo R, Doig GS, Morimatsu H, Morgera S, et al. Acute renal failure in critically ill patients: a multinational, multicenter study. JAMA 2005; 294: 813–8.

1.3 Alkaline phosphatase (ALP)

Lothar Thomas

Alkaline phosphatase (orthophosphoric-monoester hydrolase) is a cell membrane-bound enzyme expressed in all tissues. ALP in serum represents the activity of multiple forms of the enzyme. More than 17 isoforms are of ALP are detectable by use of an isoelectric focusing technique. Four genetically encoded variants of ALP have been identified /1/:

The three tissue-specific isoenzymes intestinal ALP, placental ALP and germ cell ALP

• The tissue-nonspecific ALP.

The genes of the tissue-specific isoenzymes primarily occur in the tissues after which they are named. The tissue-nonspecific gene is expressed by various tissues. The products of this gene are subject to post translational modification in glycosylation as a result of which isoforms such as the liver ALP, bone ALP and kidney ALP are formed. In all, at least 15 ALP isoforms have been identified. The gene of the placental ALP exists in numerous allelic modifications.

In every tissue, ALP shows a certain extent of microheterogeneity in terms of molecular structure and size that depends on the glycation pattern. Moreover, ALP can be present as macroenzyme or as particulate ALP bound to membrane fragments.

The liver ALP, bone ALP and intestinal ALP can be detected in the serum of healthy individuals by using conventional assay methods. The high performance liquid chromatography with post-column reaction detection allows to determine six ALP isoforms in the serum of healthy individuals: bone/intestinal ALP, two bone ALPs and three liver-ALPs /2/. They mainly differ in the sialic acid content of their carbohydrate side chains.

The determination of total ALP is performed routinely. Even if the total ALP is normal, ALP isoenzymes are determined to enhance diagnostic sensitivity and specificity at specific diagnostic requirements. The most common requirement is to differentiate between the isoforms liver ALP and bone ALP in elevated total ALP.

Alkaline phosphatase in malignant tumors

The tissue-specific isoenzymes placental ALP and germ cell ALP can be increasingly expressed in malignant tumors. They are also known as “Regan-type ALP". Their expression in tumor cells suggests that they are oncofetal proteins involved in tumor genesis. Tumors expressing these isoenzymes are differentiated as follows:

• Eutopic expression; a physiologically present ALP is increasingly synthesized
• Ectopic expression; an ALP that is not physiologically present in the tissue, but occurs, for example, in malignant tumors, is synthesized.

The placental ALP produced by cells of the syncytiotrophoblasts can be ectopically expressed in carcinomas. It is also referred to as Regan-type ALP because it was primarily detected in a lung carcinoma patient named Regan. The biochemical behavior of the Regan ALP is similar to that of the placental ALP with a few exceptions. It is detected in seminomas, in ovarian, uterine and pulmonary cancer, in malignant tumors of the gastrointestinal tract, hypophysis and thymus.

1.3.1 Indication

Total ALP

• Diagnosis and monitoring of cholestasis in hepatobiliary diseases such as obstructive jaundice, biliary cirrhosis, cholangitis, cholestatic form of viral hepatitis, drug-induced and alcoholic hepatitis, primary liver tumors, liver metastases.
• Diagnosis and monitoring of skeletal diseases such as Paget’ s disease, rickets, osteomalacia, vitamin D deficiency-induced bone disease, renal-induced osteopathy, primary bone tumors, bone metastases, multiple myeloma, hyper parathyroid disorder, acromegaly, hyperthyreosis, ectopic ossification, sarcoidosis, bone tuberculosis.
• Familial hypo phosphatasemia, adynamic bone disease, hypothyroidim.

ALP isoenzymes

The diagnostic value of total ALP is sufficient in numerous clinical requirements. If the total ALP is elevated, the determination of isoenzymes, especially the bone ALP, allows conclusions as to the tissue or organ of origin.

The determination of the bone ALP is indicated /3/:

• If osteopathy is suspected, for example in renal insufficiency or tumor patient monitoring
• For the monitoring and therapeutic assessment of osteopathy because bone ALP responds more sensitively to changes in the bone metabolism than total ALP
• Differentiation between bone ALP and liver ALP.

1.3.2 Method of determination

1.3.2.1 Total ALP (orthophosphoric- monoester phospho hydrolase, EC 3.1.3.1)

Method of the International Federation of Clinical Chemistry (IFCC) /4/

Principle: The determination of the catalytic concentration of ALP in serum depends on the chosen measurement parameters. Especially the choice of the buffer is of great importance. The phosphomonoesterase activity of ALP is determined using 4-nitro phenyl phosphate (NPP) as a substrate. In the presence of 2-amino-2-methyl-1-propanol (AMP), ALP acts as phospho transferase and transfers a phosphate group of NPP to AMP, which, in turn, catalyzes the dephosphorylation of the substrate. The amount of colored product (NP) formed per time unit, measured as absorption increase at 405 nm, is an indicator of the catalytic activity of ALP (Tab. 1.3-1 – Principle of ALP determination). The reaction process is started by adding the substrate.

1.3.2.2 ALP isoenzymes and isoforms

Numerous methods for the determination of isoenzymes and isoforms of the ALP have been described in the past 30 years. More than 15 isoenzymes and isoforms are differentiated by different methods /2/. The differentiation of elevated total ALP by liver ALP and bone ALP is important in clinical routine diagnostics and can be performed by electrophoresis. The quantitative determination of bone ALP is performed by immunoassays or lectin precipitation. The placental isoenzyme is determined quantitatively by immunoassay.

Electrophoretic differentiation

Separation following neuraminidase treatment: Before electrophoretic separation, serum is incubated with neuraminidase. This enzyme removes negatively charged sialic acid residues from the surface of the bone ALP faster than from that of the liver ALP.

The fractionation of the ALP isoforms toward the anode on the carrier medium polyacrylamide or agarose under alkaline buffer conditions slows down the electrophoretic mobility of bone ALP in relation to liver ALP. This allows to differentiate between liver ALP and bone ALP. Separation by electrophoresis in untreated serum yields insufficient results. Semi quantitative evaluation is performed densitometrically based on the isoform activities visualized on the carrier medium.

Lectin affinity electrophoresis /5/: Using carrier medium containing wheat germ lectin (cellulose acetate sheet, agarose gel), serum is fractionated under alkaline buffer conditions to migrate toward the anode. Wheat germ lectin binds the bone ALP and thus makes it less mobile. As a result, it remains near the location where it was applied and is separated from the other ALP isoforms, especially liver ALP. Semi quantitative evaluation is performed reflectometrically based on the isoform activities visualized on the separation sheet.

Quantitative determination of bone ALP

Lectin precipitation /6/: First, the total ALP is determined. Then the bone ALP is precipitated using a precipitating agent, a wheat germ lectin, and the residual activity is subsequently measured in the supernatant. The activity of bone ALP is obtained by calculating the difference.

ELISA for bone ALP determination /7/: The serum sample is incubated with a buffer in the well of a micro titer plate coated with monoclonal antibodies to bone ALP. After removal of the non-bound material, the substrate p-nitrophenyl phosphate is added and the antibody-bound enzyme activity is determined by photometry.

Immunometric assay for bone ALP determination /8/: This is a solid-phase assay in a two-step process. A specific determinant of the bone ALP in the sample reacts with the monoclonal antibody on a sphere (solid phase). At the same time, a second specific determinant of the bone ALP reacts with a second, radioactively labeled or enzyme-labeled monoclonal antibody forming a sandwich of solid phase, bone ALP and labeled antibody. After washing the sphere, the solid phase-bound radioactivity or enzyme activity are measured. Lower detection limit: 2 μg/L.

Quantitative determination of placental ALP

ELISA for activity determination /9/: The serum sample is incubated with a buffer for 3 hours at 37 °C in the well of a micro titer plate coated with monoclonal antibodies to human placental ALP. After removal of the antibody-unbound sample parts in a washing step, the serum is incubated with substrate solution (p-nitrophenyl phosphate) and the enzyme activity is subsequently determined by photometric measurement of the formed p-nitro phenolate at 405 nm. Lower detection limit: 30 mU/L.

1.3.3 Specimen

Serum or heparin anticoagulated blood; no EDTA, citrate or oxalate plasma: 1 mL

1.3.4 Reference interval

See Tab. 1.3-2 – Reference interval of ALP.

1.3.5 Clinical significance

1.3.5.1 Elevated activity of total ALP

In healthy adults, the ALP measurable in serum or plasma consists of approximately equal proportions of liver ALP and bone ALP. In children up to 15 years of age, the proportion of the bone isoform in the ALP activity is as high as 80%. About 25% of healthy individuals also have intestinal ALP that accounts for approximately 10% of the ALP activity under fasting conditions. The proportion of other ALP isoenzymes or isoforms in the ALP activity is below 5% /14/.

Elevated ALP can have physiological causes or be based on diseases of the liver or skeletal system. The leukocytes and kidneys also release ALP into circulation, but not in quantities that lead to ALP elevations above the upper reference limit.

Rising or elevated levels of total ALP and its isoenzymes and isoforms can have the following physiological causes:

• In pregnancy. The ALP activity increases significantly in the second trimenon and, in the last trimenon, reaches a peak level 2–3-fold the level in the first trimenon. In the last trimenon, the ALP activity consists of 51% placental ALP, 37% bone ALP, 9% liver ALP and 3% intestinal ALP /15/. The increase in ALP activity during pregnancy is correlated with the increase in cholesterol and triglycerides /16/. Levels return to initial values 4–6 weeks post partum /16/.
• In children during the period of growth (bone ALP). The median levels of total ALP and bone ALP are relatively constant up to 10 years of age, rise until 14 years (2–3-fold) and decline again afterwards /17/.
• Postprandial in individuals with blood groups 0 and B, Lewis-positive, who are secretors of the H blood group substance (intestinal ALP). The activity of intestinal ALP increases after food intake, especially after a high-fat meal, because the enzyme is transported lymphogenically into the blood via the thoracic duct. The intestinal ALP can be elevated if the blood sample is taken earlier than 12 hours after food intake /18/.
• In women in late menopause. Individuals with normal premenopausal total ALP and bone ALP can show postmenopausal elevations of 30–60%, although the levels remain within the reference interval in many cases /19/. If the total ALP is in the upper third of the reference interval, a bone density measurement should be performed and the bone ALP and parathyroid hormone determined. Moreover, a biomarker indicating increased bone resorption should be determined, for example N-terminal pro peptide (PINP) or carboxy-terminal cross-linked telopeptides (β-crosslaps). See also Section 6.10, Section 6.11 and Section 6.12.

Pathologically elevated total ALP and/or ALP isoenzymes or isoforms can have the following causes:

• Hepatobiliary diseases (Tab. 1.3-3 – Hepatic and biliary tract diseases possibly associated with elevated total ALP)
• Diseases of the skeletal system and vitamin D metabolism disorders (Tab. 1.3-4 – Diseases of the skeletal system associated with elevated total ALP)
• Malignant tumors (Tab. 1.3-5 – ALP as biomarker in malignant tumors and bone metastases)
• Systemic diseases without primary liver or bone diseases (Tab. 1.3-6 – Elevated ALP without recognizable hepatobiliary disease or bone disease).

The determination of isoenzymes and isoforms of ALP in cases of elevated total ALP or activities still within the reference interval can provide the following information:

• The origin of the ALP; it is clinically significant to clarify whether the elevated total ALP is liver-induced or skeleton-induced. The two canalicular enzymes liver ALP and GGT have very similar characteristics; therefore, an elevated GGT usually indicates that the liver is the organ of origin. However, this does not exclude the concurrent presence of elevated bone ALP, especially in tumor patients. The semi quantitative differentiation of isoenzymes by electrophoresis is sufficient in such cases. The cross-reactivity with liver ALP in assays for the quantitative determination of bone ALP can be as high as 5–16%. Therefore, these assays are not suited to determine bone ALP elevations if total ALP is 2–3 fold elevated and GGT levels are also elevated many times over.
• The metabolic activity of the skeletal system. The bone ALP is moderately to significantly elevated in osteoblastic processes such as metastasizing prostate carcinoma, mildly to moderately elevated in osteoclastic processes such as metastasizing breast carcinoma or multiple myeloma and not or only mildly elevated in osteoporosis. For the assessment of these processes based on quantitative determination, the diagnostic sensitivity and specificity of the bone ALP are higher than those of the total ALP.
• The growth of malignant tissue. Neoplastic ALP, also known as “Regan-type ALP", may be detectable. Since the Regan-type ALP and the placental ALP show the same electrophoretic behavior and only differ in some biochemical characteristics, the Regan-type ALP is recorded as placental ALP. It is determined in serum in patients with testicular, ovarian, pulmonary, bladder and gastrointestinal tumors /20/.

Diseases of the liver and bile ducts

Diseases of the liver and bile ducts are the most common causes of elevated total ALP. The enzyme is pathologically elevated in about 60% of diseases of the liver and bile ducts. In a pattern together with AST, ALT and GGT, ALP is of differential diagnostic significance for the detection of cholestatic conditions. Compared to aminotransferase activity, the ALP is high in cholestasis and normal or mildly elevated in the absence of the cholestatic component. The diagnostic sensitivity of ALP is 80–100% in cholestatic liver disease and only 25% in alcoholic liver injury /21/. Therefore, in liver diseases with high GGT, the level of total ALP is a good diagnostic criterion for differentiating between alcoholic damage and cholestatic processes. No or relatively mild elevations of total ALP compared to GGT are indicative of alcoholic damage. Elevated ALP is also detected in metastatic and infiltrative liver diseases such as leukemia, lymphoma or sarcoidosis.

Drug-induced toxic liver injury can imitate almost the entire spectrum of liver diseases. However, acute hepatitis is the most common form with an incidence of about 90%. Drug-induced toxic hepatitis is subdivided into three forms based on the determination of ALT and ALP /22/:

• Acute hepatocellular course. The ALT is higher than 2-fold the upper reference limit with an ALT/ALP ratio above 5. This is usually an immuno-allergic hepatitis that can be induced by many drugs and is usually cured within 1–3 months.
• Acute cholestatic course. This course is characterized by an isolated elevation of the total ALP to more than 2-fold the upper reference limit. The pure cholestatic form manifests with pruritus and jaundice, the levels of conjugated bilirubin and GGT are elevated and aminotransferases are normal. It is caused by hormone preparations in most cases. The acute cholestatic hepatitic form is associated with fever and shivers, and the ALT/ALP ratio is below 2.
• Mixed pattern acute hepatitis. The ALT/ALP ratio is 2–5. Clinical pathological manifestations are similar to a combination of hepatocellular and cholestatic hepatitis. The prognosis is more favorable than that for the hepatocellular course of the disease.

Diseases of the skeletal system

Total ALP is commonly used as a biomarker in suspected disease of the skeletal system. However, the lack of diagnostic sensitivity and specificity is a disadvantage of this method. Hence, total ALP is only elevated in significant involvement of the skeletal system, as for example in Paget’s disease, vitamin D deficiency rickets, metastatic bone formation or lytic lesions and primary hyperparathyroid disorder. Total ALP is elevated only occasionally in osteopenic and osteoporotic osteopathies. In these cases, the determination of bone ALP, especially in monitoring, is of diagnostic significance /23/. This is because the bone ALP, like osteocalcin, indicates bone formation, whereas N-terminal pro peptide (PINP) and carboxy-terminal cross-linked telopeptides (β-crosslaps) are products of bone resorption. However, since bone resorption and bone formation are linked, the biomarkers of both processes behave concordantly and assume similar in many cases.

Exceptions from this rule can be found in glucocorticoid treatment during which bone formation is acutely inhibited and bone resorption is enhanced. In anabolic therapy, bone formation is enhanced and bone resorption remains unaffected /24, 25/.

Malignant tumors

Significant elevations of total ALP can be measured in patients with malignant tumors. In many cases, the determination of the ALP isoenzymes or isoforms confirms the metastatic spread of the tumor to specific tissues. For example, elevated bone ALP in prostate carcinomas indicates metastatic spread to the skeletal system, elevated liver ALP in colon carcinoma indicates hepatogenic metastasis and elevated levels of both isoforms in lung carcinoma indicate metastasis in both organs.

The ALP isoenzymes placental ALP and Regan-type ALP fulfill the essential criteria of a tumor marker in patients with germ cell tumors of the testes. The homology of the two isoenzymes is 98%; therefore, they are often referred to as being synonymous. The Regan-type ALP is detected in serum in testicular, ovarian, pulmonary, bladder and gastrointestinal tumors /20/.

Kasahara ALP is a multiple ALP, which – in biochemical terms – is a heterodimer of placental ALP and intestinal ALP and cannot be determined with commercially available test kits. It is detected in hepatocellular carcinoma and renal cell carcinoma.

Tumor patients occasionally show unknown variants of ALP isoforms that cannot be determined in detail.

Elevated ALP may also be caused by ALP isoenzymes complexed with immunoglobulins (see also Tab. 1.1-2 – Macroenzymes: Characterization, clinical significance and laboratory findings) /26/.

Cardiometabolic diseases

In the United States National Health and Nutrition Examination Survey (NHANES) 2005–2006, the level of total ALP was significantly correlated with age, hip circumference, body mass index, blood pressure, physical exertion, ethnic origin and triglyceride levels. Compared to the lowest quartile of ALP, individuals with the highest quartile increasingly showed cardiovascular diseases (odds ratio: 1.9) as well as high blood pressure, hypercholesterolemia and diabetes mellitus (odds ratio: 1.7). It is assumed that the ALP, similarly to CRP, acts as a cardiometabolic biomarker and has higher levels in atherosclerosis and peripheral vascular disease /27/.

1.3.5.2 Reduced ALP activity

Low ALP levels in serum are rare and have an incidence of about 0.2% in clinical patients (Tab. 1.3-7 – Diseases associated with low ALP levels).

Hereditary hypophosphemia, a significant form of reduced ALP activity, can be associated with low total ALP and skeletal diseases due to lack of expression of tissue-nonspecific ALP /28/.

1.3.6 Comments and problems

Complexing substances such as citrate, EDTA or oxalate bind cations such as zinc and magnesium, which are important cofactors for ALP activity. The ALP activities measured in such anticoagulated plasma samples are falsely low /73/. This is also the case in samples taken after administration of blood transfusions because infused citrate causes decrease of enzyme activity.

Twelve-hour fasting is necessary prior to blood sampling because elevated total ALP levels of 30 U/L on average can occur 2–4 hours after food intake due to intestinal ALP entering the circulation. The fasting intestinal ALP in diabetics was 11–79 U/L and postprandially increased to 41–106 U/L /74/. The activity of intestinal ALP is elevated after high-fat meals.

Hemolysis

Hemoglobin inhibits the activity by about 3% /73/.

Half-lives

• Total ALP: 3–7 days
• Liver ALP: 3 days
• Bone ALP: 40 hours
• Intestinal ALP: under 1 hour
• Placental ALP: 4–7 days.

Drugs

Drugs can have an elevating or lowering effect on ALP activity /75/.

Elevating effect: Allopurinol, amsacrine, carbamazepine, cotrimoxazole, cyclophosphamide, disopyramide, erythromycin, gold salts, isoniazid, ketoconazole, mercaptopurine, methotrexate, methoxyflurane, α-methyldopa, methyltestosterone, oxacillin, oxyphenisatine, papaverine, penicillamine, perhexiline, phenobarbital, phenylbutazone, phenytoin, primidone, propylthiouracil, ranitidine, trimethoprim/sulfamethoxazole, sulfasalazine, valproate, verapamil.

Lowering effect: Clofibrate, oral contraceptives.

Stability in serum

At 20 °C activity reduced by 3% elevated after 3 days; at 4 °C no reduced activity within 1 week /76/.

Quality assurance

The ALP level can increase within a period of several hours after the lyophilized control sera are dissolved, depending on the lyophilized ALP isoenzyme. The reconstitution times specified by the manufacturer must be adhered to.

Method of determination of ALP isoenzymes

ELISA for activity determination: Cross-reactivity of 3–8% if the total ALP is pathological /8, 55/.

Immunometric assay: The cross-reactivity of bone ALP and liver ALP can be as high as 16%. Only the liver ALP should be elevated if the disease is hepatobiliary and not skeletal. Under this condition, the immunometric assay does not yield a pathological bone ALP result until the total ALP is 2.6-fold elevated /77/.

Electrophoretic separation in carrier medium containing wheat germ lectin: This method shows the same precision and accuracy for the bone ALP and liver ALP as the precipitation method. Smearing of the bone ALP band toward the anode occurs in high bone ALP levels.

Age dependency

The total ALP remains approximately constant up the 10 years of age, then increases continuously and reaches the highest median level at 14–16 years of age. This peak level can be up to 4-fold the upper reference limit of adults in girls and up to 5-fold the upper reference limit of adults in boys /17/. After 20–25 years of age, the levels of the two genders converge and remain constant in men until death /73/.

Women show menopausal elevations of the total ALP by 30–60% due to increasing bone ALP; the upper reference limit is usually not exceeded /78/.

1.3.7 Pathophysiology

The gene ALP encodes activity of total ALP. The enzyme consists of the isoenzymes liver ALP, bone-ALP and tissue-non-specific ALP.

• Liver ALP, kidney ALP, and tissue-non-specific ALP are the tissue-specific isoenzymes of ALP (TSALP)
• The tissue-non-specific isoenzyme of ALP (TNSALP) differs due to different post-translational glycolization into the isoforms bone ALP, intestinal ALP, placental ALP and germline ALP.

The TNSALP is a membrane-bound enzyme localized in all tissues. It binds to the outer surface of the cell membrane through a carboxy-terminal phosphatidylinositol glycan anchor. The membrane-bound form is a tetramer that is detached by the phospholipases C and D and then enters the blood circulation. Here, the ALP occurs as a dimer with two active centers, containing two zinc atoms and one magnesium atom each.

The TNSALP gene is expressed in many tissues The enzymes produced by these organs/tissues are isoforms differing due to post translational modification /14/. The isoforms have an identical primary protein structure, but differ in regards to their extent of sialylation. This results in changes in electrophoretic mobility, stability to heat and the inhibitory effect of certain chemicals. For example, the placental isoform can be differentiated from the liver-specific isoenzyme, bone-specific isoformand kidney-specific isoenzyme based on activity, inhibition by L-phenylalanine and heat stability at 65 °C for 10 minutes.

The TNSALP consists of a group of isoenzymes encoded by four gene loci. The genes encoding the enzymes bone ALP, intestinal ALP, placenta ALP and germline ALP are localized on the short arm of the chromosome 2.

The specific activity of the ALP measured in U/g of tissue is 3,214 in the placenta, 2,524 in the small intestine, 619 in the kidney, 571 in bone and 100 in the liver.

Artificial, high-concentration substrates are used for determining the ALP. However, pyrophosphate, phospho ethanolamine and pyridoxal-phosphate are the natural substrates of plasma ALP The concentration of inorganic phosphate is a natural regulator of ALP activity in plasma. At normal phosphate concentrations, the ALP activity is inhibited by 50% compared to pyridoxal-phosphate, decreases as a function of increasing phosphate concentrations and rises as a function of decreasing concentrations /79/.

The main function of TNSALP is to cleave compounds of phosphor i.e., inorganic pyrophosphate, phospho-ethanolamine and pyridoxal-5-phosphate. Defects of TNSALP cause extracellular accumulation of phosphates causing bone mineralization defects. Accumulation of pyrophosphate inhibits the synthesis of hydroxylapatite cristals and growth of the bone. Less growth is the cardinal clinical feature of hereditary hypophosphatasia (HPP) /28/.

Pyridoxal-5-phosphate is the phosphorylated form of pyridoxine (vitamin B6) which is dephosphorylated by TNSALP. This is a necessary step for crossing the blood-brain barrier and neuronal uptake of vitamin B6. Within the cell pyridoxal is phosphorylated to pyridoxal-5-phosphate a cofactor in gamma-aminobutyric acid synthesis. In hereditary hypophosphatasia (HPP), inability to cleave pyridoxal-5-phosphate may lead to vitamin B6 deficiency in the central nervous system and seizures can arise /28/.

1.3.7.1 Liver ALP

Liver ALP is produced on all levels of the biliary tract system from the bile canaliculi in the liver to the mucosa of the gall bladder and the large bile ducts. Any obstruction of the bile flow, be it in the bile canaliculi or in the minor duodenal papilla, causes the induction of liver ALP in the bile ducts. The mediator of this enzyme induction is unknown. The ALP and bilirubin usually behave concordantly in serum if, for example, the bile duct is obstructed as in occlusion by a biliary calculus or in carcinoma of the head of pancreas. However, if only the right hepatic bile duct is obstructed, bilirubin and ALP do not behave concordantly. In this case, ALP is elevated, but bilirubin is normal. This is due to the presence of intrahepatic anastomoses of the biliary ducts that allow bilirubin excretion through the left hepatic duct. Isolated elevations of ALP with normal aminotransferases also occur in infiltrative liver diseases such as tumors and granulomas, but macro ALP should also be considered (see Section 1.1.2 – Diagnostic information of enzymes). Elevated ALP occurs if a bile duct is compressed and the bile flow is obstructed due to metastases, tumors or granulomas.

1.3.7.2 Bone ALP

Bone ALP is localized on the osteoblast cell membrane. Increased osteoblast activity causes an increase in the enzyme level. Bone ALP is immediately involved in bone mineralization as demonstrated by findings on hereditary hypophosphatasia. It was shown that genetically modified knock-out mice lacking the TNSALP gene had progressive osteopathy but no secondary hyperparathyroid disorder. The function of bone ALP in the mineralization process has not been clearly determined. It is assumed to elevate the local concentration of inorganic phosphate, locally inhibit mineralization or act as calcium-binding protein.

Patients with prostate, breast or lung carcinoma often develop skeletal metastases in the course of the disease. In this case, bone is resorbed by osteoclasts and newly formed by osteoblasts. Elevated bone ALP is only to be expected if the osteoblast activity is not lower than the osteoclast activity. Hence, elevated ALP is common in tumor diseases associated with osteoblastic metastasis, such as prostate carcinoma; in contrast, ALP levels in diseases associated with osteolytic metastasis are dependent on compensatory osteoblast activity. If bone metastasis is present, biphosphonates are used to inhibit bone resorption and reduce bone pain. The resulting increase in bone ALP about one month after start of therapy is thought to be caused by increased recruiting of osteoblasts. The decrease in bone ALP occurring after 2–3 months is thought to indicate the return to the coupling of bone resorption and bone formation.

It has been generally accepted that osteoporosis represents increased postmenopausal bone loss and is attributed to increased bone remodeling. The bone mass of women suffering from osteoporosis is negatively correlated with the biomarkers of bone formation and resorption. However, the predictive value of the individual bone marker is low. The combination of bone ALP, osteocalcin, N-terminal propeptide (PINP), carboxy-terminal cross-linked telopeptides and calcium excretion is thought to have a predictive value of 60–70%.

1.3.7.3 Intestinal ALP

The intestinal ALP is expressed by the enterocytes and can be easily measured in the blood of secretors of the H blood group substance (blood groups 0 and B). The proportion of intestinal ALP in total ALP in such individuals is about 10–20%. The activity of intestinal ALP increases after food intake, especially after high-fat meals.

The intestinal ALP can be elevated in patients with liver cirrhosis and other severe hepatopathies. The elevations are caused by an absolute reduction in asialoglycoprotein receptors. Intestinal ALP is bound to the surface of the hepatocyte membrane by these receptors and then disintegrated. Moreover, in autoimmune hepatitides, antibodies to the asialoglycoprotein receptor can occur and inhibit the receptor. Congestion in the small intestine in cases of right heart failure can induce increased synthesis of intestinal ALP.

1.3.7.4 Placental ALP

The placental ALP is a heat-stable fetal isoenzyme. There is homology with the Regan-type ALP. Placental ALP is produced by placental syncytiotrophoblast from fetal week 12 on and is present in serum as a tetramer with a molecular weight of 300 kDa. Significant amounts of Regan-type ALP are detected in seminomas and ovarian tumors. However, this enzyme is not specific to such tumors, but is also detected in the plasma membrane of type 1 pneumocytes and on the basal membrane between these cells. A high percentage of the Regan-type ALP, an isoenzyme usually not detectable in healthy individuals, can be measured in smokers. There is a linear relationship between nicotine intake and the level of Regan-type ALP. The concentration declines 1–2 months after smoking has been given up.

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35. George GO, Spiegelman GA, Barkin JS. Normal serum alkaline phosphatase: an unusual finding in early suppurative biliary obstruction. Am J Gastroenterol 1993; 88: 771–3.

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45. Sjöden G, Rosenquist M, Kriegholm E, et al. Verapamil increases serum alkaline phosphatase in hypertensive patients. J Int Med 1990; 228: 339–42.

46. Renal osteodystrophy symposium: calcium metabolism in health and uremia. Amer J Med Sci 1999; 317: 355–435.

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51. Nanke J, Kotake S, Akama H, Kamatani N. Alkaline phosphatase in rheumatoid arthritis patients: possible contribution of bone-type ALP to the raised activities of ALP in rheumatoid arthritis patients. Clin Rheumatol 2002; 21: 198–202.

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1.4 α-Amylase

Klaus Lorentz

Human α-amylases (1,4-α-D-glucan 4-glucanohydrolase, EC 3.2.1.1) are monomeric proteins with 97% homologous amino acid sequences in the pancreatic enzyme (P-amylase) and salivary gland enzyme (S-amylase). The isoenzymes from the pancreas and salivary gland have roughly the same catalytic activities in the serum and urine of healthy individuals. Lipase is another enzyme available for determination besides α-amylase in suspected acute pancreatitis and is given preference by many clinicians (see also Section 1.12 – Lipase).

1.4.1 Indication

• Evidence and exclusion of acute pancreatitis (in acute epigastralgia)
• Evidence of chronic pancreatitis (in recurrence)
• Exclusion of pancreas involvement in abdominal disease and surgical intervention
• Monitoring after endoscopic retrograde choledochopancreatography
• Parotitis (epidemic, marantic, postoperative, alcohol-induced).

1.4.2 Method of determination

Principle: The enzyme activity is measured continuously by the breakdown of defined oligosaccharides carrying an aromatic residue at the reducing group of the first glucose molecule (G1). Upon attack of 2-chloro-4-nitrophenyl-α-D-maltotrioside by α-amylase, the aromatic residue is directly liberated and measured. Using the IFCC method with a longer-chain substrate (EPS), the aromatic residue 4-nitro phenyl-G (7 – x) is dissociated by α-glucosidase and measured as yellow chromophore. Substitution at the carbon atom 6 of the glucose (G7) of the non-reducing end protects the oligosaccharides from attack by auxiliary enzymes.

Method of the International Federation of Clinical Chemistry and Laboratory Medicine (IFCC) /1/

Abbreviations: EPS, 4.6-ethylidene(G1)-4-nitrophenyl(G7)-α-(1–4)-maltoheptaoside; G, α-(1–4)-D-glucopyranosyl

1.4.3 Specimen

• Serum, heparin anticoagulated blood, extravascular fluid: 1 mL
• Urine (12 hour collected urine or random specimen): 1 mL

1.4.4 Reference interval

Refer to Tab. 1.4-1 – Reference intervals of amylase.

1.4.5 Clinical significance

1.4.5.1 α-Amylase in serum

An increase in enzyme activity in serum is the only uncontroversial diagnostic finding in the detection of pancreatic disease. In acute and recurrent inflammations, it is most reliably detected within the optimal diagnostic interval of 5–10 hours after the onset of pain in the upper abdomen. The quality of diagnostic information is defined by the upper reference limit besides the stage of the disease. Pancreatic amylase is considered superior to α-amylase (total) /6/, but the time of analysis is more important than the selection of the enzyme. α-Amylase and lipase regularly show parallel behavior with delayed decline of lipase to normal levels. The activities in serum in no way reflect the severity of the disease because α-amylase and lipase activities below 3 × the upper reference limit are also measured in severe courses /7/.

Levels within the reference interval (especially for α-amylase) occasionally occur in acute, preferably alcohol-induced, pancreatitis /7/. On the other hand, elevated α-amylase is observed in 8% of hospitalized patients without the presence of pancreatic disease. Hence, determination is not indicated if pancreatic disease is not suspected /8/.

Elevated levels indicate the following diseases:

• Acute pancreatitis: On the day of disease onset, the diagnostic sensitivity of α-amylase is 80% and remains below that of lipase and pancreatic amylase (Tab. 1.4-2 – Causes of pancreatogenic hyperamylasemia); the interval grows until day 5; the diagnostic sensitivity decreases to below 70% afterwards /9/.
• Recurrence of chronic pancreatitis: The time profile and relationship of the two enzymes during an acute episode correspond to those in acute inflammation. However, at a diagnostic specificity of over 90%, the sensitivity prevailing in acute inflammation is reached only occasionally. Low α-amylase/lipase ratios are considered to be typical of advanced stages /10/.
• Pancreatitis after endoscopic retrograde choledochopancreatography (ERCP). After surgery, the lipase activity rises higher than that of α-amylase, reaches a peak level after 6 hours (like α-amylase), but remains elevated for more than 3 days /11/. In acute pancreatitis, levels remain at peak values for up to 24 hours /12/; if, after 2 hours, α-amylase remains below the upper reference limit of 2.4 × and lipase remains below 4.2, × the absence of acute pancreatitis can be expected at a negative predictive value of 0.98 /13/.

Chronic asymptomatic hyperamylasemias are based on pancreatic or extra pancreatic diseases in about half of the cases; macroamylasemia and/or familial hyperamylasemia are diagnosed in about 5% of the cases, each, and no cause is found in 40% of the cases /14/. The existence of the ubiquitous isoform X-amylase is of intestinal origin, and P-amylase and S-amylase can originate from neoplasms and hepatic tissues /15/. Therefore, elevated α-amylase and lipase can be expected in liver diseases /16/, but also following cardiac circulatory failure with liver congestion /17/ that generally induces hyperamylasemia. Moreover, ascending parotitis should also be considered in postoperative stomatitis.

Elevated α-amylase and lipase by 10–15% have been described in fructose malabsorption /18/ and anticonvulsant therapy /19/. Pancreatic carcinomas cause hyperenzymemia in pancreatic duct occlusion. In bulimia and anorexia, amylasemias occasionally reach levels as high as 2 × the upper reference limit /20/, mostly due to parotitis following stomatitis. Other causes of extrapancreatic hyperamylasemias are shown in Tab. 1.4-3 – Causes of extra pancreatic hyperamylasemias.

Low levels in obese individuals have little significance. Hypoamylasemia as a symptom of pancreas failure or as missing response to a secretion stimulus (negative evocation test) is not found except in terminal stages.

1.4.5.2 α-Amylase in urine

A urine analysis is indicated in hyperamylasemia if macroamylasemia or renal insufficiency are suspected (see also Tab. 1.1-2 – Macroenzymes: characterization, clinical significance and laboratory findings).

1.4.5.3 α-Amylase in extravascular fluids

Postoperative analysis of drainage secretion is performed to determine the presence of a pancreatic fistula. High concentrations in pleural effusions (in basal pleuritis and thoracic duct injury) indicate pancreatitis. S-amylase is reported to be elevated in leukemias, lung carcinoma and lung metastasis /21/.

1.4.6 Comments and problems

Macroamylasemia (see also Section 1.1 – Diagnostic enzymology)

Macroamylases are non-uniform complexes (molecular weight above 400 kDa), in which α-amylase adheres to the Fab region of immunoglobulins (IgA in most cases, IgG under 30%, other Ig under 5%). In addition, albumin and α₁-antitrypsin are found occasionally. Escaping glomerular filtration due to their size, they remain in the serum and cause up to 4-fold elevated activities. S-amylase is involved in most cases, but often not detected because the epitopes of the enzyme are covered by immunoglobulins. Characteristic findings include chronic hyperamylasemia without clinical correlate, normal or low amylase in urine and normal lipase activity. This harmless anomaly is found in 0.1% of the population. However, monoclonal gammopathies should be searched for in macroamylasemia.

Macro forms are more common following infusion of hydroxy ethyl starch (HAES) that forms high-molecular complexes with amylase. The supply of 500 mL 6% HAES causes elevated α-amylase concentrations for 3–5 days because glomerular filtration of the enzyme is only possible after HAES breakdown. This hyperamylasemia can be misdiagnosed as postoperative pancreatitis. The lipase level is normal.

Method of determination

All procedures measure up to 10 × the upper reference limit as a function of activity, and their substrates keep well in solution. Glucose concentrations above 10 g/L only slightly reduce the measured signal; pyruvate and lactate do not interfere.

Tests that release 4-nitro phenol or 2-chloro-4-nitro phenol as chromophore tolerate higher concentrations of bilirubin, triglycerides and hemoglobin, but the enzyme activity in samples with recent hemolysis (due to reduced hemoglobin absorption) is falsely indicated as too low /22/. Lipoproteins can cause low levels in lipemic sera.

Amylase in urine

The secretion and clearance of α-amylase allow certain diagnostic conclusions in suspected macroamylasemia and renal insufficiency, whereas determination in urine and the clearance ratio have no significance in the diagnosis of acute pancreatitis.

Anticoagulants

EDTA and oxalate are not recommended because of the binding of Ca²⁺, heparin does not interfere. Some tests include magnesium in the reagent to reactivate amylase in EDTA plasmas.

Reference interval

The α-amylase has a typical age profile with high individual fluctuations. Pregnancy has no effect. The concentration of salivary enzyme in neonates is only 25–50% of the final level reached after 12 months. Pancreatic amylase only appears after 1–2 months of life and increases continuously up to 10 years of age; after this, the reference interval for adults applies.

Stability

Stable in serum for 1 week at 4 °C or 25 °C or for at least 1 year at –28 °C (also for evidence of macroamylasemia). Unchanged activity for at least one day at 4 °C in urine and even up to one month in sterile storage; do not deep-freeze.

1.4.7 Pathophysiology

Human α-amylases [1,4-α-D-glucanglucanohydrolases, EC 3.2.1.1] represent three almost identical monomeric proteins. After dissociation of a signal peptide of 15 amino acids, they pass from the endoplasmic reticulum into the cytoplasm as active enzymes with uniformly 496 amino acids /23/. Their gene loci in the chromosome 1p21 encode as follows for three isoenzymes with molecular weights around 54 kDa: AMY2A for the pancreatic enzyme, AMY2B for the ubiquitous enzyme and AMY1 for the enzyme of the salivary glands. It has 97% homology with pancreatic amylase, with 15 deviating amino acids; the ubiquitous isoenzyme even has 98% homology due to only 5 substituted amino acids. Hence, salivary gland amylase can be inhibited to residual activity below 3% by concurrently using two monoclonal antibodies; however, this does not apply to the ubiquitous isoenzyme. There exist glycosylated forms of the two isoenzymes with a molecular weight of 57–62 kDa. Their glucan residues can be dissociated enzymatically in vivo with no loss of activity.

Amylases consist of three domains. Domain A includes the active center with activating Cl^– that can be substituted by anions of similar size. A protective Ca²⁺ in domain B aligns against the catalytic center and stabilizes the enzyme molecule, which therefore is inactivated by calcium-complexing anticoagulants and heavy metals. Domain C carries the glycan residue in glycosylated forms and varies strongly depending on the species. All isoenzymes reduce polymeric carbohydrates into disaccharides by random hydrolysis of 1,4-α-glucosidic bonds. Oligosaccharides are cleaved at preferred bonds which can result in glucose and maltose being transferred to the substrate.

α-Amylase is synthesized in the secretory epithelium of the salivary glands and pancreas and, in low concentration, also in hepatic and cancerous tissue /15/. As in lipase, a proenzyme can be detected in the acinus epithelium but does not appear extracellularly. In healthy individuals, more than 99% of the enzyme are released into the gastrointestinal tract, whereas impaired flow and inflammations of the organ/tissue result in increased amounts entering the circulation. Substantial destruction of secretory parenchyma has little effect on the activity in serum.

Otherwise, the enzyme behaves like lipase, but has a longer half-life (9.3–17.7 hours). Complete glomerular filtration and 50% tubular reabsorption of the α-amylase is thought to be the cause of this difference. Reabsorption is restricted due to tubular damage (in burns, ketoacidosis, acute pancreatitis) and proteinuria /24/, and amylase clearance (normally 2.8–4.6 mL/min.) increases as a result. This also increases the clearance ratio [(amylase clearance/creatinine clearance) × 100] from 1.8–3.2% in healthy individuals to more than 10%. Mostly glomerular damage (chronic glomerulonephritis, nephrosclerosis) slightly increases the ratio to as high as 9% due to a major decline in creatinine filtration. A relative increase in the pancreas fraction and a pronounced decrease in α-amylase excretion are observed.

References

1. Schumann G, Aoki R, Ferrero CA, Ehlers G, Ferard G, Gella FJ, et al. IFCC primary reference procedures for the measurement of catalytic activity concentrations of enzymes at 370 C. Reference procedure for the measurement of catalytic concentration of α-amylase. Clin Chem Lab Med 2006; 44: 1146–55.

2. Lorentz K, Gütschow B, Renner F. Evaluation of a direct α-amylase assay using 2-chloro-4-nitrophenyl-α-D-maltotrioside. Clin Chem Lab Med 1999; 37: 1053–62.

3. Genzyme Diagnostics. Amylase dual reagent (instructions for use) 1994.

4. Junge W, Wortmann W, Wilke B, Waldenström J, Kurrle-Weittenhiller A, Finke J, Klein G. Development and evaluation of assays for the determination of total and pancreatic amylase at 37 °C according to the principle recommended by the IFCC. Clin Biochem 2001; 34: 607–15.

5. Abicht K, Heiduk M, Körn S, Klein G. Lipae, p-amylase, CRP-hs and creatinine: reference intervals from infancy to childhood. Abstract 15th IFCC-FESC, Barcelona 2003.

6. Melzi d’Eril GV, Bosoni T, Lesi C. Pancreatic amylase in serum for differential diagnosis of acute pancreatitis and acute abdominal diseases. Clin Chem 1989; 35: 2142–3.

7. Lankisch PG, Burchard-Reckert S, Lehnick D. Underestimation of acute pancreatitis: patients with only a small increase in amylase/lipase levels can also have or develop severe acute pancreatits. Gut 1999; 44: 542–4.

8. Lankisch PG, Doobe C, Finger T, Lübbers H, Mahlke R, Brinkmann G, et al. Hyperamylasemia and/or hyperlipasemia: incidence and underlying cause in hospitlized patients with non-pancreatic diseases. Scand J Gastroenterol 2009; 44: 237–41.

9. Gwozdz GP, Steinberg WM, Werner W, Henry JP, Pauley C. Comparative evaluation of the diagnosis of acute pancreatitis based on serum and urine assays. Clin Chim Acta 1990; 187: 243–54.

10. Dominguez-Muñoz JE, Pieramico O, Büchler M, Malfertheiner P. Ratios of different serum pancreatic enzymes in the diagnosis and staging of chronic pancreatitis. Digestion 1993; 54: 231–6.

11. Müller-Hansen J, Müller-Plathe O, Pröpper H. Untersuchungen zur diagnostischen Sensitivität von Lipase- und Amylase-Bestimmungen. Einfluß der ERCP auf die Aktivität von Serum-Lipase und Amylase. Ärztl Lab 1986; 32: 17–23.

12. Conn M, Goldenberg A, Concepcion L, Mandeli J. The effect of ERCP on circulating pancreatic enzymes and pancreatic protease inhibitors. Am J Gastroenterol 1991; 86: 1011–4.

13. Gottlieb K, Sherman S, Pezzi J, Esber E, Lehman GA. Early recognition of post-ERCP pancreatitis by clinical assessment and serum pancreatic enzymes. Am J Gastroenterol 1996; 91: 1553–7.

14. Pezzilli R, Morselli-Labate M, Casadei R, Campana D, Rega D, Santini D, et al. Chronic asymptomatic pancreatic heperenzymemia is a benign condition in only half of the cases: a prospective study. Scand J Gastroenterol 2009; 44: 888–93.

15. Nakamura Y, Tomita N, Nishide T, Emi M, Horii A, Ogawa M, Mori T, Kosaki G, Okabe T, Fujisawa M, Ohsawa N, Kameya T, Matsubara K. Production of salivary type α-amylase in human lung cancer. Gene 1989; 77: 107–12.

16. Tsuzuki T, Shimizu S, Takahashi S, Iio H. Hyperamylasemia after hepatic resection. Am J Gastroent 1993; 88: 734–6.

17. Góth L, Mészáros I, Scheller G. Hyperamylasemia and α-amylase isoenzymes in acute liver congestion due to cardiac circulatory failure. Clin Chem 1989; 35: 1793–4.

18. Ledochowski M, Murr C, Lass-Flörl C, Fuchs D. Increased serum amylase and lipase in fructose malabsorbers. Clin Chim Acta 2001; 311: 119–23.

19. Hermida J, Tutor JC. Serum amylase and lipase activities in epileptic patients treated with enzyme-inducing anticonvulsant drugs. Clin Chim Acta 2001; 303: 181–3.

20. Hempen I, Lehnert P, Fichter M, Teufel J. Hyperamylasämie bei Anorexia nervosa und bulimia nervosa. Dtsch Med Wschr 1989; 114: 1913–6.

21. Joseph J, Viney S, Beck P, Strange C, Sahn SA, Basran GS. A prospective study of amylase-rich pleural effusions with special reference to amylase isoenzyme analysis. Chest 1992; 102: 1455–9.

22. Gosling P, Zareian M. Fresh haemolysis interferes with blocked p-nitrophenyl-maltoheptaoside amylase methods. Ann Clin Biochem 1994; 31: 371–3.

23. Nishide T, Emi M, Nakamura Y, Matsubara K. Corrected sequences of cDNAs for human salivary and pancreatic α-amylases. Gene 1986; 50: 371–2.

24. Andreulli A, Bergia R, Masoero G, Biardi P, Pellegrino S, Tondolo M. Amylase to creatinine clearance ratio in renal diseases. Gastrenterology 1979; 77: 86–90.

1.5 Angiotensin-converting enzyme (ACE)

Lothar Thomas

Angiotensin-I-converting enzyme (peptidyl-dipeptidase A; EC 3.4.15.1) was originally described as dipeptidyl carboxypeptidase /1/. ACE is primarily localized in the endothelial cells of the pulmonary capillaries and the kidney cortex.

Tissue-specific ACE is involved physiologically (Fig. 1.5-1 – Biochemical effects of ACE):

• As key enzyme in the renin-angiotensin aldosterone system; ACE cleaves the dipeptide L-histidyl-L-leucine from angiotensin I and converts angiotensin I to the potent vasopressor angiotensin II.
• In the degradation of the vasodilatative nonapeptide bradykinin through the sequential release of L-phenylanalyl-L-arginine and L-seryl-L-proline.

Serum ACE (SACE) is not involved in these reactions; its pathophysiological significance remains to be clarified. Elevated SACE activities have been described for a number of diseases, especially sarcoidosis (Besnier-Boeck-Schaumann disease).

1.5.1 Indication

• Suspected sarcoidosis
• Assessment of granuloma load and monitoring respiratory disease severity in sarcoidosis
• Monitoring the treatment of sarcoidosis.

1.5.2 Method of determination

Routine assays cleave synthetic N-terminal blocked tripeptides, which release hippuric acid (benzoyl-glycine) upon splitting. Many synthetic substrates have been developed for ACE determination. The substrates are aryl-oligopeptides shorter than the natural substrates, usually tripeptides with blocked N-terminus, mainly hippuryl-glycyl-glycine (hippuryl as benzoylglycyl), hippuryl-histidyl-leucine (HHL) and furylacryloyl-phenylalanyl-glycyl-glycine (FAPGG). The substrates are used for the spectrophotometric, fluorimetric, radiometric and chromatographic methods /1/.

Method according to Lieberman /2/

Principle: the release rate of hippuric acid from HHL is measured by spectrophotometry at 228 nm after ethyl acetate extraction.

Method according to Neels et al. /3/

Principle: the release rate of glycyl-glycine from hippuryl-glycyl-glycine (hippuryl as benzoylglycyl) is determined in a chromogenic reaction. Photometric measurement at 420 nm.

Method according to Ryan et al. /4/

Principle: ³H-hippuryl-glycyl-glycine is used as a substrate and the release rate of ³H-hippuric acid is measured.

Method according to Friedland and Silverstein /5/

Principle: HHL is used as a substrate. The release rate of histidyl-leucine is determined spectrofluorimetrically after formation of a fluorescence adduct with orthophenylenediamine.

Method according to Holmquist et al. /6/

Principle: ACE catalyzes the hydrolysis of FAPGG to furylacryloyl-phenylalanine and glycyl-glycine. The hydrolysis causes a blue shift in the absorption of the assay medium. The reduction in absorption is measured at 340 nm. It is directly proportional to the ACE activity of the sample.

1.5.3 Specimen

Serum, heparin anticoagulated blood (no EDTA plasma): 1 mL

1.5.4 Reference interval

Refer to Tab. 1.5-1 – Angiotensin-converting enzyme reference intervals.

1.5.5 Clinical significance

The serum ACE (SACE) activity in healthy individuals presumably involves released enzyme anchored as ectoenzyme in macrophages in the lumen-facing vessel wall /1/. Elevated SACE activities originate from granulomas. Granuloma is a feature of many chronic interstitial lung diseases such as sarcoidosis, hypersensitivity pneumonitis, berylliosis, histiocytosis X and are structured masses composed of activated macrophages and their derivatives, i.e. epithelioid cells and giant cells /8/. A few disease states, in addition to sarcoidosis, are consistently associated with elevated ACE in about 25% of the cases, for example Gaucher’s disease, hyperthyreosis, diabetes mellitus with retinopathy, liver cirrhosis, silicosis, asbestosis, lymphangiomyomatosis, chronic fatigue syndrome /9/.

Low ACE is thought to be a marker of endothelial dysfunction of the vascular bed, for example in pulmonary damage of toxic origin, deep vein thrombosis, hypothyroidism or after chemotherapy and radiotherapy of malignant tumors /1/. The validity of low SACE activities is unknown.

1.5.5.1 Sarcoidosis

Clinical aspects /8, 10/

The goal in the diagnosis of pulmonary sarcoidosis is to rule out all diagnoses other than sarcoidosis that are consistent with the clinical situation /20/. Sarcoidosis is a granulomatous multi-systemic disease of unknown etiology. It mainly affects the lungs and intrathoracic lymph nodes featuring noncaseating granulomas. The typical histological findings are well-formed epitheloid-cell granulomas whose compact core is composed of macrophages and macrophage-derived cells (epitheloid and giant cells), closely associated with CD4⁺T lymphocytes The peripheral component contains CD8⁺ lymphocytes, CD4⁺ FOXP3⁺ Treg cells, Th17 cells, B lypmhocytes, and IgA-producing plasma cells.

The probability that other organs are included in sarcoidosis are:

• Liver, spleen in 25–70% of the cases.
• Skin involvement 10–60%, eye involvement 10–25%.
• Peripheral lymph nodes and skeletal muscles < 30%.
• Salivary glands, central nervous system, myocardium < 5%.

The following forms of the disease are distinguished:

• Acute form. This form develops abruptly within a few weeks and accounts for 20–40% of all sarcoidosis cases. The disease is manifested by respiratory symptoms, retrosternal chest pain, fever, arthralgia, erythema nodosum, hilus adenopathy, as well as uveitis (Löfgren’s syndrome) and severe maladies. Oligosymptomatic forms also occur.
• Chronic form. This form is symptom-free; about one third of the patients suffer from cough, dyspnea and – rarely – hemoptysis. In contrast to the findings, these patients appear to be remarkably healthy. Extra pulmonary manifestations are often the leading symptom in this disease, pulmonary symptoms can be absent.

Prevalence: Germany 40–50/100,000 inhabitants, Spain, Italy < 10/100,000; there is a high prevalence in blacks and Puerto Ricans.

Prognosis

The natural course of sarcoidosis is not predictable. Patients with advanced pulmonary infiltrates and splenomegaly can have spontaneous remission, while others with asymptomatic hilar adenopathy can develop a severe clinical picture. Spontaneous remission occurs in 70% of patients with bi-hilar lymphadenopathy (type I), in 50% of patients with pulmonary infiltrates and bi-hilar lymphadenopathy (type II) and in 50% of patients with pulmonary infiltrates without bi-hilar lymphadenopathy (type III). Mortality is 40% in those with pulmonary fibrosis (type IV). Generally, the more severe the clinical findings at the time of diagnosis and the more organ systems are involved by the disease, the more frequent adverse courses are observed /8/.

Neurosarcoidosis /11, 12/

Neurological symptoms due to involvement of the central nervous system (CNS) occur in about 5% of patients with systemic sarcoidosis, 10–30% of the patients show initial neurological symptoms when presenting for the first time; the incidence of isolated CNS sarcoidosis is reported to be below 0.2 related to 100,000 Caucasians. Neurosarcoidosis can affect the meninges, brain parenchyma, spinal chord and peripheral nerves. The differentiation between intracranial sarcoidosis and other neurological disorders can be difficult, especially if there are no symptoms of extra cranial manifestation.

Diagnosis and management

No specific and sufficiently accurate biomarker has yet been identified for the diagnosis and clinical management of sarcoidosis. Among diagnostic markers serum angiotensin converting enzyme (SACE) has a controversial role in diagnosing and managing sarcoidosis as it is not sensitive enough for the diagnosis of systemic sarcoidosis and shows normal concentrations at disease onset. Lung imaging and measurement of SACE concentration are recommended at the initial evaluation of sarcoidosis. A major complication of hepatic sarcoidosis is portal hypertension.

Serum ACE (SACE)

The rate of false positives is about 50%, despite SACE concentration is used as a test for /27/:

• Confirming a diagnosis of sarcoidosis
• Estimating the organism’s granuloma load
• Following the course of the disease
• Assessing the course under corticosteroid treatment.

Diagnostics: In acute pulmonary sarcoidosis, the positive predictive value of SACE is 75–90% and the negative is 70–80% /13/. The diagnostic sensitivity of elevated SACE activity in combination with a radiographic finding of type II or type II in the ⁶⁷Ga scan is reported to be 100%. A normal SACE in combination with a negative ⁶⁷Ga scan excludes pulmonary sarcoidosis. Chronic sarcoidosis is often associated with normal SACE levels; elevated activities also occur in other granulomatous diseases.

A normal SACE does not rule out sarcoidosis. This is because the SACE reference interval is dependent on the genetic polymorphism, and the reference values were defined without taking the polymorphism into account /14/. For example, acute sarcoidosis associated with erythema nodosum can show values within the reference interval /13/. In sarcoidosis of the liver manifested as sclerosing cholangitis, extrahepatic cholestasis or Budd-Chiari syndrome, the SACE is elevated in about 15% of the cases, and the ALP is always 2–5-fold elevated /15/.

Estimation of the granuloma load /13/: The ACE level reflects the granuloma load of the entire organism, independently of the affected organ. This especially applies to systemic sarcoidosis. Isolated sarcoidosis, for example sarcoidosis of the CNS or cardiac sarcoidosis, does not cause elevated ACE. This can also be the case in less vascularized, sometimes large mediastinal lymphomas.

Course of disease /13/: Initially low SACE activity is indicative of a good prognosis; the prognosis is less favorable if the activity is 2–3-fold elevated. The SACE activity increases along with the progression of the disease and reaches peak levels in chronic active disease with a high granuloma load.

Course under corticosteroid treatment /16/: The SACE level is generally not the criterion for a decision in favor of this treatment, but pronouncedly elevated activities above the upper reference limit can be considered an indication of need for treatment and good responsiveness. The higher the initial level, the longer the corticosteroid treatment must be to attain normal SACE levels and improvement of the clinical symptoms. An effective dosage usually results in decreasing SACE levels already after 1–2 weeks. In sarcoidosis of the lungs, the decrease precedes radiological improvement. The absence of a decrease is indicative of inadequate dosage or lack of compliance.

The SACE can increase again after the end of treatment and normalization of the SACE. This is the earliest sign of recurrence where clinical symptoms do not necessarily have to be present. Resumption of treatment is only indicated if clinical and radiological changes are detected.

Some cured patients can show SACE re-elevation without clinical recurrence. In this case, however, the enzyme activities are usually lower than the levels before start of treatment.

Spontaneous remissions of sarcoidosis are associated with a gradual decrease in SACE. The level does not decline as abruptly as under corticosteroid treatment.

ACE in neurosarcoidosis

Elevated activities of SACE and ACE in cerebrospinal fluid (CSF) are not specific of neurosarcoidosis and can also be measured in other neurological diseases such as CNS infections, brain tumors and Guillain-Barré syndrome /11/. Elevated ACE in CSF are detected in 55% of neurosarcoidosis cases, 5% of sarcoidosis cases without CNS involvement and 13% in other neurological diseases /17/. Other authors only see a significance of the ACE in CSF in the assessment of neurosarcoidosis under corticosteroid treatment /18/.

Other biomarkers in sarcoidosis /8/

• The concentration of the soluble tumor necrosis factor receptor II (sTNF-R II) is elevated in serum. Its concentration is an indicator of the inflammatory activity of sarcoidosis /19/.
• The sIL-2R is a biomarker of T-cell activity and reveals an intimate relationship between this parameter and the clinical activity of disease. Elevated sIL-2R concentrations are a good progression parameter in sarcoidosis.
• Patients without indications for therapy but with high sIL-2R serum levels indicating T-cell activation in the course of sarcoidosis tend towards a progressing course with subsequent indications for corticoid therapy /8, 19/.
• Immunoglobulin concentrations are elevated, especially IgG and IgA
• Circulating immune complexes are detectable
• Lymphopenia is present in many cases
• Ionized calcium in blood and its excretion in urine are elevated in some cases because of the formation of 1,25(OH)₂D in the epithelioid cells, causing an increase in intestinal calcium absorption (see also Section 6.6).
• HLA characteristics: Constitutive expression of HLA-B13 is detected in chronic sarcoidosis, while constitutive expression of HLA-B8, A1, Cw 7, DR 3n are detected in sarcoid arthritis and erythema nodosum.
• Bronchoalveolar lavage (BAL): The percentage of lavage lymphocytes, which is usually below 20%, can increase to more than 50%; elevated counts of CD4⁺T cells and a higher CD4⁺/CD8⁺ ratio from normally 1–3 to more than 5, partly even more than 12, especially in acute sarcoidosis and Löfgren’s syndrome, can also occur (see also Chapter 48). The percentage of the lymphocytes in BAL and the CD4⁺/CD8⁺ ratio to a certain extent correlate to the spontaneous course of sarcoidosis. Patients with acute disease and a good prognosis have high numbers of lymphocytes with an elevated CD4⁺/CD8⁺ T cell ratio. Those with more chronic disease and risk of deterioration exhibit only moderately elevated values /8/.

1.5.5.2 Differential diagnosis of pulmonary sarcoidosis

Pathological features in alternative diseases are shown in Tab. 1.5-2 – Elevated ACE at other diseases and conditions.

1.5.6 Comments and problems

Sampling

Serum or heparin anticoagulated blood should be used as specimen. Metal chelators such as EDTA are not suited as anticoagulant because they reduce the SACE activity. ACE is a metallopeptidase with a zinc atom in the active center; the binding of the zinc atom by the chelate former significantly reduces the enzyme activity.

ACE inhibitors such as captopril, enalapril must have been discontinued 4 weeks beforehand because otherwise the measured values are too low due to ACE inhibition /23/.

Method of determination

About 25% of the sera of patients, especially of sarcoidosis patients, contain an internal ACE inhibitor that significantly lowers the ACE activity. The inhibitory effect is mediated if the serum is diluted 1 : 10 /23/.

Tests using hippuryl-glycyl-glycine as a substrate are more suited than those using HHL because they are less susceptible to hydrolysis by carboxypeptidases /13/. Tests using FAPGG as a substrate have advantages because the hydrolysis rate is greater by a factor of 3. The reference interval is also higher as a result. Moreover, kinetic measurement is possible at 340–345 nm. Thus, measurements can be performed on mechanized automatic analyzers /7/. The effect of both substrates (HHL and FAPGG) is dependent on Cl^–. Therefore, the assay medium must have an NaCl concentration of 250–350 mmol/L /1/.

Reference interval

The intraindividual variation of SACE is small, while the inter individual variation is high. The factor 6, for example, ranges between the lower and upper reference limits in adults. Infants and boys in puberty have higher SACE activities than adults.

The following reference intervals have been determined for children based on the substrate FAPPG /24/:

Values expressed as x ± s and in U/L.

Drugs /23/

Captopril inhibits the ACE activity at a half-life of 1–4 days in some patients and 10–17 days in others. The effect of captopril on the SACE activity can vary:

• It decreases as a function of storage time: However, 10 days of deep-freezing at –70 °C increases the activity by 15–50%.
• The effect is dependent on the patient’s individual metabolization rate.
• The inhibitory effect is lost under dialysis therapy.
• It is independent of the serum dilution in the reaction medium.

Enalapril inhibits the SACE activity almost completely; the effect cannot be reversed through storage, serum dilution or dialysis. Therefore, the administration of enalapril should be considered if the SACE levels are very low.

Stability

ACE is stable in serum and plasma for at least 1 week at +4 °C and for 3 months at –20 °C /7/.

1.5.7 Pathophysiology

ACE is an ubiquitous enzyme expressed by vascular endothelial cells, macrophages, proximal tubule kidney cells, Leydig cells and chorioid plexus cells. Therefore, it is present in many organs and especially abundant in the lungs and kidneys. It is an ectoenzyme bound to the outside of the cell membrane through a hydrophobic peptide residue. The SACE activity is released by enzymatic cleavage, especially from vascular endothelium of the lungs /1/.

ACE is a zinc metalloprotease with a molecular weight of 150–170 kDa. The difference in molecular weight is based on different glycosylation in the individual tissue; for example, the carbohydrate portion of ACE of the lung is 30% and contains fucose, mannose, N-acetylglucosamin, glucose and sialic acid /25/. ACE cleaves the last two amino acids of peptide substrates; each peptide serves as a substrate provided that proline is not the last but one amino acid.

ACE is a key enzyme of the renin-angiotensin system (RAS) and kallikrein system due to the following reactions:

• In the RAS (Fig. 1.5-2 – Effect of ACE on the renin-angiotensin system (RAS) and kinin-kallikrein system), ACE dissociates the C-terminal His-Leu dipeptide from angiotensin I and forms the vasoactive octapeptide angiotensin II. In another possible step, the aspartic acid at position 1 can be dissociated from angiotensin II forming angiotensin III. The latter is a less potent vasoconstrictor than angiotensin II /1/.
• In the kallikrein system, ACE dissociates the C-terminal dipeptide Phe-Arg from bradykinin. This inactivates the vasodilatator.

Based on these reactions, the ACE has the following effects:

• Activation of vasoconstriction through angiotensin II
• Inactivation of the vasodilatory effect by cleavage of bradykinin
• Stimulation of aldosterone synthesis in the adrenal gland through angiotensin III resulting in Na⁺ and water retention and K⁺ elimination.

The dual role of ACE in blood pressure maintenance (Fig. 1.5-2 – Effect of ACE on the renin-angiotensin system (RAS) and kinin-kallikrein system) and homeostasis of the electrolyte and water balance have made it an ideal working point for medical treatment (ACE inhibitors) of high blood pressure and congestive heart failure.

Various polymorphisms of the ACE gene and possible disease associations have been identified /26/. They refer to both the ACE activity of the tissues and to SACE. The polymorphisms consist of the presence (insertion allele I) or absence (deletion allele D) of a 287 bp DNA fragment in an ALU repetitive sequence in intron 16 of the ACE gene /1/. The genotypes II, ID and DD are distinguished. The SACE activity in individuals of genotype DD is about twice as high as that in individuals of genotype II. However, there are no differences in ACE kinetics, in angiotensin II and aldosterone levels, and no significant differences in the blood pressure.

Sarcoidosis is a granulomatous disease of unknown genesis, characterized by an accumulation of activated T-cells and macrophages in the affected organ (the lungs in most cases). These release interferon-γ, TNF-α and other pro inflammatory cytokines as mediators of an inflammation and cellular immune reaction, causing the subsequent formation of non-caseating epithelioid cell granulomas. In the inflamed regions, there is a high number of CD4⁺T-cells. The inflammatory process is the same in all organs affected, including the CNS. The persistence of the inflammation induces fibrotic changes with irreversible tissue damage.

The sarcoid granulomas consist of lymphocytes, macrophages, epithelioid cells, mast cells, eosinophil granulocytes and fibroblasts. There is a strong influx of immune cells and a strong proliferation and apoptosis of cells in the granuloma. ACE is released during the conversion of macrophages in epithelioid cells. Alveolar macrophages stimulated by T-lymphocytes also release ACE into the circulation. Therefore, the SACE activity is an indicator of the organism’s granuloma load, while the concentration of sIL-2R, neopterin and sTNF-R II indicates the extent of immune cell activation in sarcoidosis /8, 13/.

References

1. Baudin B. New aspects of angiotensin-converting enzyme: from gene to disease. Clin Chem Lab Med 2002; 40: 256–65.

2. Lieberman J. Elevation of serum angiotensin-converting enzyme (ACE) level in sarcoidosis. Am J Med 1975; 59: 36–72.

3. Neels HM, van Sande ME, Sharpé SL. Sensitive colorimetric assay for angiotensin-converting enzyme in serum. Clin Chem 1983; 29: 1399–403.

4. Ryan J W, Chung A, Ammons C, Carlton ML. A simple radioassay for angiotensin-converting enzyme. Biochem J 1977; 167: 501–5.

5. Friedland J, Silverstein E. A sensitive fluorometric assay for serum angiotensin-converting enzyme. Am J Clin Pathol 1976; 60: 416–20.

6. Holmquist B, Bunning B, Riordan JF. A continuous spectrophotometric assay for angiotensin-converting enzyme. Anal Biochem 1979; 95: 540–3.

7. Bénéteau B, Baudin B, Morgant G, Giboudeau J, Baumann F CH. Automated kinetic assay of angiotensin-converting enzyme in serum. Clin Chem 1986; 32: 884–6.

8. Müller-Quernheim J. Sarcoidosis: clinical manifestations, staging and therapy (part II). Respiratory Medicine 1998; 92: 140–9.

9. Lieberman J. Angiotensin-converting enzyme in nonpulmonary sarcoidosis. Sem Resp Med 1992, 13: 399–401.

10. Schmidt M. Sarkoidose. Allergologie 1996; 19: 212–5.

11. Nowak DA, Widenka DC. Neurosarcoidosis: a review of its intracranial manifestation. J Neurol 2001; 248: 363–72.

12. Gullapalli D, Phillips LH. Neurologic manifestations of sarcoidosis. Neurologic Clinics 2002; 20: 59–81.

13. Bénéteau-Burnat B, Baudin B. Angiotensin-converting enzyme: clinical applications and laboratory investigations on serum and other biological fluids. CRC Rev Clin Lab Sci 1991; 28: 337–56.

14. Muller BR. Analysis of serum angiotensin-converting enzyme. Ann Clin Biochem 2002; 39: 456–43.

15. Blich M, Edoute Y. Clinical manifestation of sarcoid liver disease. J Gastroenterol Hepatol 2004; 19: 732–7.

16. Wurm K. Die Bedeutung des ACE-Tests bei Sarkoidose. Z Allgemeinmed 1987; 63: 1004–6.

17. Oksanen V. New cerebrospinal fluid, neurophysiological and neuroradiological examinations in the follow-up of neurosarcoidosis. Sarcoidosis 1987; 4: 105–10.

18. Dale JC, O’Brien JF. Determination of angiotensin-converting enzyme levels in cerebrospinal fluid is not a useful test for the diagnosis of neurosarcoidosis. Mayo Clin Proc 1999; 74: 535–9.

19. Rothkranz-Kos S, v Dieijen-Visser MP, Mulder PGH, Drent M. Potential usefulness of inflammatory markers to monitor respiratoy functional impairement in sarcoidosis. Clin Chem 2003; 49: 1510–7.

20. Valeyre D, Brauner M, Benaudin JF, Carbonnelle E, Duchemann B, Rotenberg C, et al. Differential diagnosis of pulmonary sarcoidosis: a review. Front Med 2023; doi: 10.3389/fmed.2023.1150751.

21. Ozawa T, Ninomiya J, Honma T, et al. Increased serum angiotensin-converting enzyme activity in patients with mixed connective tissue disease and pulmonary hypertension. Scand J Rheumatol 1995; 24: 38–43.

22. Quellete DR, Kelly JW, Anders GT. Serum angiotensin-converting enzyme level is elevated in patients with human immunodeficiency virus infection. Arch Intern Med 1992; 152: 321–4.

23. Lieberman J, Zakria F. Effect of captopril and enalapril medication on the serum ACE test for sarcoidosis. Sarcoidosis 1989; 6: 118–23.

24. Bénéteau-Burnat B, Baudin B, Morgant G, Baumann F Ch, Giboudeau J. Serum angiotensin-converting enzyme in healthy and sarcoidotic children: comparison with the reference range for adults. Clin Chem 1990; 36: 344–6.

25. Das M, Soffer RL. Pulmonary angiotensin-converting enzyme: structural and catalytic properties. J Biol Chem 1975; 250: 6762–8.

26. Crisan D, Carr J. Angiotensin I-converting enzyme. Genotype and disease associations. J Molecular Diagn 2000; 2: 105–15.

27. D’Alessandro M. Editorial: sarcoidosis and autoimmunity: From bench to bedside. Frontiers in Medicine 2023. doi: 10.3389/fmed2023.1147529.

1.6 Alanine aminotransferase (ALT), Aspartate aminotransferase (AST)

Lothar Thomas

The aminotransferases, also referred to as transaminases, are a group of enzymes catalyzing a reversible conversion of α-keto acids into amino acids by transferring an amino group. The ALT is located in the cytosol of the cells and can be found mainly in the liver and kidneys and, to a small extent, also in the myocardium and skeletal muscle. The main concentration of ALT occurs in the liver; hence, elevated activity in serum is a specific marker of liver disease. The AST is located in the cytosol and in the mitochondria of the cells. This ubiquitous enzyme is found in high concentrations in liver, nervous tissue, skeletal and cardiac muscle.

1.6.1 Indication

ALT

Serves as key parameter of hepatocellular injury and in monitoring and therapy assessment:

• Verification of jaundice and sub jaundice
• Liver disease due to hepatotropic viruses
• Liver involvement in systemic viral diseases, bacterial and parasitic infections
• For the diagnosis of chronic liver disease
• In autoimmune liver disease
• For the detection of liver injury due to alcohol, drugs, hepatotoxins, intoxicants, toxic chemicals at the workplace and in the environment, overnutrition (non-alcoholic steatosis of the liver) and parenteral nutrition
• In suspected mass in the liver
• In liver disease during pregnancy
• Suspected hereditary metabolic disorder (hemochromatosis, Wilson’s disease, α₁-antitrypsin deficiency, cystic fibrosis)
• Indication and monitoring of antiviral therapy in chronic hepatitis and therapeutic assessment.

AST

Supplements the ALT in the diagnosis of liver disease:

• For differential diagnosis
• In etiological verification and for severity assessment and staging of the disease
• For the prognostic assessment of myocardial injury in myocardial infarction.

1.6.2 Method of determination /1, 2/

AST (EC 2.6.1.1)

IFCC Primary Reference Procedure for the Measurement of Catalytic Activity Concentration of Aspartate Aminotransferase at 37 °C

Principle: AST catalyzes the reaction between L-aspartate and 2-oxoglutarate and oxaloacetate formed is reduced by NADH catalyzed by malate dehydrogenase (MDH) (Fig. 1.6-1 – Principle of AST and ALT determination according to IFCC). Pyridoxal phosphate is added to activate apo-AST which may be present in the specimen. LD is added to reduce pyruvate and shorten the duration of preincubation needed in order to obtain a stable initial absorbance. The measured rate of NADH₂ decrease is proportional to the AST activity.

ALT (EC 2.6.1.2)

IFCC Primary Reference Procedure for the Measurement of Catalytic Activity Concentration of Alanine Aminotransferase at 37 °C

Principle: ALT catalyzes the reaction between L-alanine and 2-oxoglutarate, and the pyruvate formed is reduced by NADH in a reaction catalyzed by LD (Fig. 1.6-1 – Principle of AST and ALT determination according to IFCC). Pyridoxal phosphate is added to activate apo-ALT which may be present in the specimen. The measured rate of NADH₂ decrease is proportional to the ALT activity.

1.6.3 Specimen

Serum, plasma (heparin, EDTA, citrate, oxalate): 1 mL

1.6.4 Reference interval

See Tab. 1.6-1 – Reference interval

1.6.5 Clinical significance

Elevated aminotransferases indicate the presence of liver disease; normal levels, however, do not exclude such a disease, especially in chronic hepatitis. In the USA, the prevalence of elevated aminotransferase levels is 7.9% and correlated to fatty liver disease. Aminotransferases are also a predictor of the metabolic syndrome (MetS), diabetes mellitus (DM) and cardiovascular disease. According to the Framingham Offspring Heart Study /6/, among individuals at baseline, per 1 standard deviation increase in log ALT level, there were increased odds of the development of MetS [odds ratio (OR) 1,21] and DM (OR, 1.48) over 20 years of follow-up.

In pediatric hospitals /7/, about 12% of isolated aminotransferase elevations are due to genetic disorders. In particular cases of muscular dystrophy masquerading as liver disease and false diagnosis of cryptogenic liver disease in patients with cystic fibrosis, celiac disease, glycogen storage disease and other congenital metabolic disorders.

The AST is no longer significant in the diagnostics of acute myocardial infarction (AMI) and much less sensitive in the diagnostics of skeletal muscle diseases than the CK. Therefore, the AST in AMI and myopathies is only analyzed for differential diagnostic reasons.

1.6.5.1 Liver diseases

The ALT is a key biomarker for the inflammatory damage of the liver parenchyma. It is the basic marker in laboratory staging of liver disease and requested in the following cases:

• Screening for liver disease in the absence of clinical symptoms.
• Selectively, if clinical symptoms such as epigastralgia, jaundice, hepatomegaly or coma indicate a liver disease. In many cases, AST, GGT, GLD, LD and ALP are also requested out of differential diagnostic considerations. For differential diagnosis, see also Section 1.2 – Biomarkers of liver disease.
• In known liver disease for severity assessment of the liver disease, for etiological verification and prognostic assessment. AST, GGT, ALP, CHE and GLD in combination with ALT provide diagnostically conclusive patterns.

The frequency (%) of elevated ALT levels in liver disease is shown in Tab. 1.6-2 – ALT activities in serum and their frequency in hepatobiliary diseases. The increase of ALT and AST in hepatopathies is assessed in Tab. 1.6-3 – Aminotransferases in patients with hepatopathies.

1.6.5.1.1 Screening for liver disease

The aminotransferases, GGT, ALP and CHE are recommended as screening pattern for liver disease. For this purpose, elevated aminotransferase levels serve as a biomarker of parenchymal injury, elevated GGT serves as an indicator of metabolic toxic damage, the ALP serves as a biomarker of cholestasis, and decreased CHE serves as an indicator of reduced functional hepatocyte mass. Up to 90% of patients with liver injury are diagnosed based on this enzyme pattern. The laboratory findings do not indicate functional hyperbilirubinemias, cases with non-alcoholic fatty liver disease, chronic hepatitis C and residual stages of hepatitides without inflammatory activity of the liver parenchyma. In a study /8/ on 1,154 patients with hepatobiliary diseases, 15% did not show elevated ALT. The majority of the patients suffered from liver metastases, extrahepatic bile duct obstruction, liver cirrhosis and drug-induced liver injury. The cirrhosis patients showed lower CHE levels, the others mainly elevated GGT levels. Since there is no indication of increased ALT production in the hepatocytes in liver injury, any level exceeding the upper reference limit is primarily considered an indication of liver injury. Unexpected elevations of ALT or AST should be verified by determination using a new sample. This is because the intraindividual variation of the aminotransferases is 10–30% from one day to the next; moreover, elevated activities can also be measured after heavy physical exertion.

1.6.5.1.2 Medical decision-making approach in symptomatic patients

Acute abdominal pain: The enzyme pattern should be selected such as to allow the detection of other diseases such as pancreatitis, ileus, tubal pregnancy or myocardial infarction besides acute liver injury due to acute hepatitis, acute bile duct obstruction, acute cholecystitis and acute impaired perfusion disorder. Therefore, besides ALT, AST and GGT, the diagnostic pattern should include GLD, lipase or α-amylase, cardiac troponin and hCG as well as the blood count and C-reactive protein as supplementary parameters. ALT levels within the reference interval exclude acute liver disease or liver involvement. This also applies to acute obstructive jaundice, which can also be ruled out if the ALT level is more than 20-fold the upper reference limit. In acute alcoholic hepatitis, one of the few acute liver diseases besides the Reye syndrome where the AST is initially higher than the ALT level, the aminotransferase activities are 10–20-fold the upper reference limit.

If the aminotransferases are elevated more than 20-fold the upper reference limit, differential diagnosis has to decide between acute viral hepatitis, acute impaired perfusion and acute toxic liver injury. The GLD is about as high as the aminotransferase levels in impaired perfusion, about half as high in toxic injury and not higher than 10% of the aminotransferase activity in viral hepatitis. The determination of the CHE allows to differentiate between acute intoxication and acute impaired perfusion. Whereas the CHE level decreases to below 50% of the lower reference limit in intoxication, this does not apply to impaired perfusion /7/.

Jaundice and sub jaundice: In icteric patients or in the presence of hyperbilirubinemia, a differentiation between hepatic and hemolytic jaundice is required. The LD/AST ratio is significant besides the determination of the total and direct-reacting bilirubin; a ratio > 5 indicates the hemolytic genesis of jaundice (see also Section 1.11 – Lactate dehydrogenase (LD) and Section 5.2).

Coma: In a comatose condition, it is important to recognize the hepatic coma. In the setting of hepatogenic coma, a differentiation must be made between fulminant liver failure and hepatic encephalopathy. The aminotransferase levels in hepatic encephalopathy are similar to those of liver cirrhosis, i.e., they are mildly to moderately elevated, while those in fulminant liver failure due, for example, to paracetamol or death cap poisoning, or in halothane-induced cases show a characteristic necrosis pattern. The ALT and AST are more than 20-fold elevated, with the AST being higher than the ALT, and the GLD is on a similar level. In most cases of portosystemic encephalopathy, the CHE level is already low on admission of the patient, whereas in fulminant liver failure, the CHE decreases together with the albumin after the coagulation factors due to its long half-life.

Distinction and differentiation of cholestasis: The screening pattern by itself already indicates the presence of cholestasis. High GGT and ALP at only mildly to moderately elevated ALT levels indicate cholestasis, while much higher ALT levels compared to the GGT and ALP indicate hepatitis. A differentiation based on laboratory findings as to whether the cholestasis involves the large bile ducts, i.e., is of extrahepatic origin, or whether an intrahepatic obstruction of the small bile ducts and/or toxic accompanying cholestasis is present, can only be performed after extrahepatic cholestasis has been excluded by imaging.

1.6.5.1.3 Severity assessment of liver disease

The activity of the aminotransferases in serum is both correlated with the amount of damaged hepatocytes and the severity of the single cell damage and thus the acuteness of the liver disease. The damage of about 1 in 750 hepatocytes induces elevated ALT levels to beyond the upper reference limit. Information regarding the severity of cell damage is provided by the AST/ALT ratio (De-Ritis ratio). About 70% of the AST activity of the hepatocyte is located in the mitochondria and 30% is located in the cytoplasm. Ratios below 1.0 indicate a milder degree of liver injury, especially involving acute, reversible, inflammatory liver diseases such as acute viral hepatitis B. Ratios > 1.0 indicate a high degree of liver injury of the necrosis type /10/.

Acute hepatocellular injury: Acute liver disease can be due to viral hepatitis, alcoholic hepatitis, bile duct obstruction, toxic damage or acute impaired perfusion. Elevated ALT or AST levels higher than 10-fold the upper reference limit almost always indicate the presence of these diseases.

Chronic hepatocellular injury /10/: Chronic liver disease is defined as continuous hepatocellular necrosis and inflammation of the liver, in many cases associated with fibrosis. Chronic liver disease can proceed into liver cirrhosis and is the precondition for the development of hepatocellular carcinoma. In many cases, it results from hepatotropic viral infections, causes minimal symptoms and entails the risk of increased morbidity and mortality in the longer term. The persistence of ALT for more than 6 months after acute hepatitis B or elevated ALT levels measured several times within 6 months without a plausible explanation indicate chronic liver disease.

There is a correlation between progression and aminotransferase activity in chronic liver disease. Activities are high if cirrhosis develops quickly. In chronic liver disease, the ALT is usually higher than the AST level, or the AST is even normal. With advancing reduction of the liver parenchyma, the ALT deceases more strongly than the AST level and the De-Ritis ratio reaches values above 1. Chronic alcoholic hepatitis is an exception. Here, the De-Ritis ratio is above 1 (often even above 2) already from the very beginning. Drugs and chronic alcohol consumption are the essential causes of chronic liver disease in patients with negative virus markers. Other liver diseases associated with elevated ALT should not be forgotten, for example autoimmune hepatitis, cholestatic liver disease, α₁-antitrypsin deficiency and Wilson’s disease.

Liver cirrhosis /10/: Liver cirrhosis is characterized by fibrosis and the transformation of normal hepatic structure into abnormal lesions. It results from chronic liver disease with progressive necrosis of liver parenchyma. The developing regenerative nodules are not a fully adequate replacement for the necrotized parenchyma. This results in restricted liver function based on a reduced metabolic rate and reduced presence of enzymes in the hepatocytes. The advancing reduction of intact liver parenchyma is reflected by mildly elevated ALT and AST levels and an increased De-Ritis ratio. For the recognition of liver cirrhosis, the diagnostic sensitivity of a ratio > 1 is 32–83% and the diagnostic specificity is 75–100% /11, 12/.

The increasing reduction in liver function is indicated by the change in biomarkers. An extension of the prothrombin time, decreasing CHE, albumin and thrombocyte count are important indications of progression. These parameters should be determined every 3–6 months in liver cirrhosis monitoring.

1.6.5.1.4 Liver disease staging

The stage of acute liver disease can be assessed based on the changes of the aminotransferases over time and the correlation with the bilirubin concentration. The AST/ALT ratio, which decreases during curing, allows to assess the stage of the disease because the half-life of ALT (47 hours) is about three times as long as that of AST (17 hours). For example, the ratio is 0.6–0.8 in acute hepatitis as the ALT and AST reach peak levels, then decreases gradually and is 0.2–0.4 in the fourth week of the disease. The LD can be helpful in differentiating remittent icteric hepatitis from the early form of mild acute hepatitis with a low AST/ALT ratio. Whereas the LD activity is already normal again in the remittent form of the disease due to its half-life of only 10 hours, it ranges between the AST and ALT levels in the acute mild form /6/.

Aminotransferases in relation to bilirubin /10/: In acute viral hepatitis B, ALT and AST show peak levels at the onset of jaundice in the first week of the disease. The bilirubin reaches peak levels up to one week later. The aminotransferases decline continuously by about 10% each day at the onset of jaundice. The ALT remains elevated for 27 ± 16 days and the AST remains elevated for 22 ± 16 days. In acute toxic hepatitis and acute impaired liver perfusion, the peak levels of the aminotransferases are already reached within 24 hours after admission to hospital, where the AST is higher than the ALT. The aminotransferase levels decline again within the next 24 hours. The AST can decline by up to 50% and – due to its short half-life – decreases more pronouncedly than the ALT. The AST can return to within the reference interval already 7 days after acute injury.

1.6.5.1.5 Etiological verification of liver disease /9, 11/

The etiological verification of liver disease is part of the differential diagnosis and a prerequisite for causal therapy. The diagnostic value of the aminotransferases is small in this context. This especially applies to liver disease with mild injury-induced or approximately identical enzyme changes that occur, for example, in acute and chronic viral hepatitides and liver cirrhosis. Moreover, chronic active hepatitides caused by hepatotropic viruses can lead to enzyme changes to a similar degree as can acute hepatitides caused by non-hepatotropic viruses. Drug-induced toxic liver injury manifested as hepatitic or cholestatic forms, fatty liver or variable combinations can also be a problem.

The following assays are useful for etiological verification and ensure pathogenic evidence quality (see also Section 1.2 – Biomarkers of liver disease):

• Serologically and molecular biologically detectable biomarkers for differentiating between the different types of viral hepatitis
• The determination of autoantibodies in autoimmune liver disease
• An α-fetoprotein concentration providing evidence of the primary hepatocellular carcinoma
• Elevated carbohydrate deficient transferrin (CDT) indicating chronic alcohol abuse.

Etiological verification is possible to some extent in chronic hepatitis and liver cirrhosis by diagnostic enzymology in combination with serum protein electrophoresis and/or immunoglobulin determination. For example, autoimmune liver diseases and cirrhoses can be associated with hyperproteinemia and/or a high γ-globulin fraction in serum protein electrophoresis or – due to IgA increase – show a blended β- and γ-globulin fraction in alcohol-toxic etiology. Posthepatitic cirrhosis shows increased concentration of IgG, and primary biliary cirrhosis shows increased concentration of IgM relative to the other immunoglobulin classes.

1.6.5.1.6 Prognostic assessment of hepatitis

Prognostic assessment is focused on the following questions /8/:

• Will the acute hepatitis be cured or proceed into a chronic form?
• Will a necrotizing form develop and will liver failure occur?
• Is there evidence of therapeutic success?

Cured hepatitis: Acute viral hepatitis A and B are usually self-limiting; almost all cases of hepatitis A and 95% of the cases of hepatitis B are cured. About 85% of acute hepatitis C infections proceed into a chronic form. During the acute phase of hepatitis, the aminotransferases do not allow any conclusion as to whether hepatitis will be cured or develop into a chronic form in individual cases. The ALT and GGT are the last enzymes to return to normal levels. Therefore, monitoring is recommended including measurements every 2 weeks. If the enzyme levels have not normalized within 6 months or show recurrent elevations, a chronic form must be expected. This always applies if no antibodies against HBsAg and HBeAg are produced or if virus persistence is detected.

Transition into severe form: Enzyme analyses should detect patients with increased risk of liver failure. This applies to the necrotizing forms and affects 0.1% in hepatitis A and C, about 1% in hepatitis B, up to 20% in hepatitis D and up to 4% in hepatitis E (up to 20% in pregnant women). The level of aminotransferases is rather based on the etiology of acute liver injury than on severity. Therefore, the absolute level of aminotransferase activity cannot be used for prognostic assessment. However, a poor prognosis suggests itself if the necrosis pattern consists of decreasing levels of all hepatocyte enzymes or decreasing ALT and concurrently elevated AST, GLD and LD levels.

Antiviral hepatitis treatment /13, 14/: In virustatic treatment of chronic hepatitis B and C, the ALT is determined to assess treatment success. This is because the ALT is a biomarker for assessing the inflammatory activity of the liver, although it only allows limited conclusions regarding the extent of the inflammation and provides little information on the severity of fibrosis. For further information, see Section 1.2 – Biomarkers of liver disease.

1.6.5.2 Myopathies

The diagnostic sensitivity of the AST in acute myocardial infarction is 96% and the diagnostic specificity is 86% 12 hours after the acute event /15/. The ALT is only significant in the course of infarction if right heart failure is suspected. The sensitivity and specificity of the AST are less informative for the diagnosis of skeletal muscle diseases than those of the CK. Elevated AST and CK levels are indicative of muscle damage.

The levels of AST and ALT in myocardial disease are shown in Tab. 1.6-4 – Aminotransferases in cardiac diseases and diseases with similar symptoms and in disease of the skeletal muscle disease in Tab. 1.6-5 – Aminotransferase behavior in skeletal muscle damage.

1.6.6 Comments and problems

Method of determination

The IFCC method is optimized for 37 °C and contains pyridoxal-phosphate in the reagent mixture. Before the specific reaction is started by adding α-ketoglutarate, the ALT and/or AST are activated by saturation with pyridoxal-phosphate in a preincubation step. In addition, in the presence of NADH, pyruvate in the sample is converted to lactate /1, 2/. The addition of pyridoxal-phosphate has the advantage of stabilizing the enzymatic activity of the aminotransferases. Without the addition of pyridoxal-phosphate, falsely low activities are measured in samples with an insufficient concentration of endogenous pyridoxal-phosphate, for example in patients with myocardial infarction, liver disease or in intensive care patients.

A reduction in NADH would occur in the AST assay medium without the removal of pyruvate in the preincubation step /16/.

The ALT level and the upper reference limit are dependent on the analyzer. In a statewide study, for example, the inter laboratory variations were 69–83 U/L (variation of 14 U/L), but only 4–8 U/L if the measurements in the different laboratories were performed with an analyzer of the same diagnostics manufacturers /17/.

Specimen

Serum is recommended; heparin anticoagulated blood can cause turbidity of the assay medium /1/.

Reference interval

The upper reference levels (URLs) are optimized for the IFCC method and refer to adults at 20–60 years of age /1, 2/. The levels of Americans of African origin are 15% higher than those of Caucasians. In adults, the American College of Gastroenterology recommends ALT URLs of 33 U/L for males and 25 U/L for females, respectively. In Children the North American Society of Pediatric Gastroenterology, Hepatology and Nutrition states gender-specific URLs (26 U/L for boys and 22 U/L for girls). As a result of the inappropriate application of these URLs, there is a potential for over diagnosis /18/.

Macro AST

See Tab. 1.1-2 – Macroenzymes: characterization, clinical significance and laboratory findings.

Hemolysis

At ALT levels within the reference interval and a Hb value of 2.5 g/L and higher, hemolysis causes an elevation of the ALT by about 10%. The AST increases with increasing hemolysis starting from Hb values of 1.5 g/L. The activity of the ALT in the erythrocytes is 7-fold and that of the AST is 15-fold higher than in serum /19/. Hemolytic anemias cause mildly elevated AST levels at pronouncedly elevated LD; potassium is normal in in-vivo hemolysis and elevated in in-vitro hemolysis.

Biological factors

Half-life (hours) /20/: AST 17 ± 5, mitochondrial AST 87, ALT 47 ± 10.

Variation: The variation of aminotransferase levels in serum within day is 45%, with highest levels reached in the evening and lowest at night. Day-to-day variation is 5–10% /20/.

Body weight: The aminotransferase level in individuals with a high body mass index is 40% higher than in normal-weight individuals /20/.

Hemodialysis patients: Such patients may have low AST and ALT due to pyridoxal-phosphate deficiency /16/.

Stability

In serum ALT and AST are stable for 1 week at 9 °C; the AST decreases slightly continuously at 20 °C /21/.

1.6.7 Pathophysiology /6, 7, 8, 9/

The increase in serum activity of usually structure-committed liver enzymes is an indicator for architectural and functional disorders of the liver. The liver consists of parenchymal cells (60%) and sinusoidal cells (endothelial cells, Kupffer cells, fat storing cells and pit cells). The total number of cells of 1 mg of liver tissue is 202,000, including 171,000 parenchymal cells and 31,000 sinusoidal cells. The 300 billion parenchymal cells are in close contact with the circulating blood via the sinusoids. The sinusoidal cells are located in front of the parenchymal cells in the sinusoids and function as filters. For example, the Kupffer cells are highly mobile macrophages attached to the endothelium of the sinusoids. They remove cell debris, microorganisms and colloidal substances from the blood by phagocytosis.

The parenchymal cells do not represent a uniform cell population. They are adapted to the different supply of oxygen and substrates depending on their location in the hepatic lobule. This supply decreases on the way between the portal vein to the central vein, which results in different cell organelle and enzyme inventories. This is useful for diagnostic purposes because cell damage causes the release of soluble enzymes and the enzyme pattern measured in serum allows conclusions as to which region in the hepatic lobule was especially damaged. For example, peripheral damage causes a stronger release of the ALT from the cell than central damage because the ALT activity is higher in the periphery of the hepatic lobule than in the center.

The name “aminotransferase” describes the function of the enzymes AST and ALT, i.e. the transfer of the NH₂ group of amino acids to keto acids, preferably α-ketoglutarate. In this manner, the amino groups of the different amino acids are collected in a single amino acid, preferably glutamate, during the amino acid catabolism. In a subsequent series of reactions, for example oxidative desamination, the nitrogen is removed from the glutamate and converted to excretable nitrogenous compounds.

Liver, myocardium and skeletal muscle have a relatively high AST activity compared to other tissues. Therefore, they are almost always the tissues of origin of elevated AST activity.

The specific activity of ALT is about 10-fold higher in the liver than in the myocardium and skeletal muscle; hence, the ALT is considered a liver-specific enzyme. It is suited for use as screening enzyme for detecting liver diseases.

The AST and ALT are located in parenchymal and non-parenchymal cells of the liver. The ALT only dissolves in cytoplasm, has a molecular weight of about 110 kDa and its activity in the hepatocyte is 2800-fold higher than in serum. The AST is a dimeric molecule. The molecular weight of the dissolved form is 93 kDa and that of the mitochondrial form is 91 kDa. 30% of the AST is dissolved in cytoplasm and 70% binds to mitochondrial structures. The AST activity is 7,000-fold higher in the liver than in serum. Enzymes released from hepatocytes pass into the circulation easily and quickly because sinusoids have no basal membrane.

The enzyme pattern appearing in the plasma and the enzyme activities in liver disease are dependent on the nature of the damage.

In acute viral hepatitis, almost all cells of a hepatic lobule are affected. There is a mild inflammation of the hepatocytes associated with disturbance of cell membrane permeability, and cytoplasmic ALT and AST pass into the plasma. The AST/ALT ratio is usually below 1.0 because the amount of AST released into the plasma is smaller than that of ALT. Concerning the pathogenesis of viral hepatitis B, it is assumed that the virus itself causes little damage to the parenchymal cells. After recognition by T-lymphocytes, circulating viral antigens are thought to cause the proliferation of sessile T-lymphocytes that are directed against hepatocytes. The T-lymphocytes recognize virugenic proteins in the plasma membrane of the hepatocyte and cause their damage. This leads to increased permeability of the plasma membrane for ions resulting in colloid osmotic ballooning or even lysis of the cell.

In acute viral hepatitis, the level of the aminotransferases passing into the plasma is correlated with the amount of affected parenchymal tissue. If the hepatocellular injury is reversible, restitutio ad integrum is possible. More severe injury causes increased release of aminotransferases, especially mitochondrial AST, into the plasma. An AST/ALT ratio above 1.0 is an indicator of such processes and indicates hepatocyte necrosis.

Chronic inflammations of the liver are focal. In chronic active hepatitis, the cell necroses starting at the periportal fields only cause a moderate to medium increase in aminotransferases and LD. The AST/ALT is above 1. There is no direct correlation between the aminotransferase levels and the inflammatory activity.

The immune system of the sinusoidal cells can become insufficient in chronic liver diseases, especially in liver cirrhosis. In this case, antigens pass into the circulation. In particular, this occurs in impaired liver perfusion if the blood bypasses the liver by a spontaneous or surgical portocaval shunt. The immune response to antigens not removed by the liver causes poly clonal hypergammaglobulinemia in serum protein electrophoresis.

In alcoholic hepatitis, the serum AST is higher than the ALT. Whereas all other hepatitides cause a decrease in cytosolic and mitochondrial AST in the cells, the alcoholic form only results in a cytosolic decrease.

Obstructive jaundice and acute hypoxic hepatopathies due to impaired perfusion as well as toxic substances cause the necrosis of centroacinar parenchymal cells. This results in an over proportional increase in GLD compared to ALT.

In acute toxic liver injury with massive cell necrosis, the enzyme pattern in serum corresponds to that of the parenchymal cells: LD > AST > ALT > GLD.

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84. Mortier W. Muskelerkrankungen bei Kindern. Dtsch Ärztebl 1977; 74: 1081–4.

85. Schneider S. Maligne Hyperthermie. Dtsch Ärztebl 1983; 80: 41–4.

86. Berg A. Körperbelastung und Serumenzyme. Dtsch Zschr Sportmed 1979; 30: 128–30.

1.7 Cholinesterases (ChE)

Lothar Thomas

Vertebrates posses two Cholinesterase genes producing the enzymes acetylcholine acetylesterase (AChE)  and acylcholine acetylhydrolase (ChE) /1/.

The acetylcholine acetylhydrolase (EC 3.1.1.7), also referred to as acetylcholinesterase (AChE). This enzyme is present in the outer membrane of erythrocytes, in the gray matter of the central nervous system, in the sympathetic ganglia of the neuromuscular end plate, in the lung and spleen, but not in plasma. Under physiological conditions AChE performs the breakdown of acetylcholine, which is the chemical mediator responsible for the conduction of nerve impulses at the sites of cholinergic transmission. AChE hydrolyzes acetylcholine and is essentially inactive on butyrylcholine, arylesters and alcylesters.

Acylcholine acylhydrolase (EC 3.1.1.8), also referred to as cholinesterase (ChE). This enzyme is present in plasma, in the liver, intestinal mucosa, pancreas, spleen and the white matter of the central nervous system. Besides cholinesters, the ChE hydrolyzes benzoylcholine and butyrylthiocholine as well as arylesters and alcylesters. The function of this serum-specific enzyme is unknown. AChE and ChE are competitively inhibited by the alkaloids prostigmine and physostigmine. The following description only refers to ChE, i.e., the enzyme activity determined in plasma.

1.7.1 Indication

• Suspected impaired function of the liver
• Prior to the administration of succinylcholine-type muscle relaxants if there is evidence of a cholinesterase variant
• In extended apnea after surgery
• Pesticide poisoning
• Monitoring of pesticide-exposed workers
• Intensive care patients with pathological results of global blood coagulation tests or unexplainable hypoalbuminemia.

1.7.2 Method of determination

Determination of AChE (EC 3.1.1.7) /2/

Colorimetric method

Principle: acetylthiocholine is hydrolyzed by AChE to acetic acid and thiocholine. The catalytic activity of AChE is measured by following the increase of the yellow anion 5-thio-2-nitrobenzoate, produced from thiocholine when it reacts DTNB (DTNB, 5.5’-dithio-bis-2-nitro benzoic acid 10 mmol/L; NaHCO₃ 17.85 mmol/L = buffered Ellman’s reagent).

Benzoylcholine method /3/

Principle: benzoylcholine is hydrolyzed by AChE to benzoic acid and choline. The reduction in absorption of benzocholine is measured at 240 nm.

Proposal of Standard Method for the Determination of Cholinesterase (EC 3.1.1.8) at 37 °C /4/

Principle: butyrylthiocholine is hydrolyzed by CHE to butyric acid and thiocholine (Tab. 1.7-1 – Principle of CHE determination and calculation of dibucaine number). Thiocholine instantly reduces yellow hexacyanoferrat(III) to almost colorless hexacyanoferrat (II), thus allowing the direct spectrometric monitoring of the reaction (Tab. 1.7-2 – Chromogenic reactions for measuring the thiocholine produced).

1.7.2.1 Phenotyping of genetic variants

The synthesis of ChE in plasma is controlled by a gene locus on the long arm of chromosome 3.

The alleles U, A, S, F, H, J, K have been identified and are known to be responsible for the synthesis of potentially 28 phenotypes /1, 5/.

Identical genotypes may have different phenotypes because some individuals have more than one mutation in the same gene. The UA phenotype, for example, can correspond to the two genotypes UA and UAK.

Up to 45 different diploid genotypes, but only 11 different phenotypes are possible /6/. Some of the ChE variants (Tab. 1.7-8 – Biochemical characteristics of cholinesterase variants) cause lower ChE activity in plasma, do not hydrolyze succinylcholine and cause extended apnea after surgery during which succinylcholine-type muscle relaxants are administered.

Inhibition assays with dibucaine and fluoride are the classic method for biochemical phenotyping of ChE variants in plasma.

Dibucaine inhibition

Principle: In the presence of the local anesthetic dibucaine, the activity of normal ChE is inhibited more strongly than that of genetic ChE variants.

Based on the extent of inhibition, individuals are assigned to the following three groups /7/:

• Inhibition above 70%; the individual is homozygous for normal ChE in both genes
• Inhibition of 40–70%; the individual is heterozygous and has one gene for normal ChE and one for atypical ChE
• Inhibition below 30%; the individual is homozygous for an atypical variant in both genes

Using the benzoylcholine method, the ChE activity is determined in the presence and absence of 1 × 10^–5 mol/L dibucaine /7/. The approach using acetylthiocholine esters as a substrate is not as common /8/.

The dibucaine number is calculated using the equation shown in Tab. 1.7-1 – Principle of CHE determination and calculation of dibucaine number.

Fluoride inhibition

This assay is used to determine the fluoride-resistant ChE variants (Tab. 1.7-8 – Biochemical characteristics of cholinesterase variants). The determination is performed corresponding to the dibucaine number; the fluoride concentration in the assay medium is 5 × 10^–5 mol/L /9/.

Ro 2-0683 inhibition

Ro 2-0683 is a more selective inhibitor than dibucaine. Normal ChE (E₁^U) is inhibited by almost 100%, while the atypical variant (E₁^a) is not inhibited at all /5/.

1.7.3 Specimen

Serum, heparin anticoagulated blood: 1 mL

1.7.4 Reference interval

Refer to Tab. 1.7-3 – Cholinesterase reference intervals.

1.7.5 Clinical significance

ChE reductions are common in liver disease, can be drug-induced, occur in pesticide poisoning or, more rarely, are hereditary.

1.7.5.1 ChE in liver disease

ChE is synthesized in the liver and released into the plasma. The activity in serum depends on the adequate function and amount of liver parenchymal cells. Therefore, the ChE activity is a biomarker used to measure the global liver function. The ChE is only decreased below the lower reference limit in severe liver injury associated with reduced protein synthesis of the parenchymal cells or based on a pronounced reduction in parenchymal cell mass /12/.

The isolated determination of the ChE is generally not very efficient in the diagnosis of liver disease. For example, the positive predictive value of a pathological finding is only 21% at a prevalence of 8% of patients with liver disease in the clinical patient material, while the negative predictive value of the normal finding excluding liver disease is 97% /13/. Despite the low positive predictive value, the ChE has the following diagnostic significance (Tab. 1.7-4 – Liver diseases possibly associated with decreased ChE activity):

• Screening for liver disease in combination with GGT and ALT. Although the diagnostic sensitivity of the ChE for the detection of liver disease is lower than that of GGT and ALT, it is – in combination – important for screening. For in cases of advanced chronic liver disease, the GGT and ALT can be within the reference interval and the liver disease is only indicated by the reduced ChE level /12/.
• As an indicator of a co-reaction of the liver in systemic diseases. Reduced ChE not induced by a primary liver disease can be caused by severe disorders associated with a catabolic metabolic state, for example malignant diseases, autoimmune disorders, intensive care, protein malnutrition /14/.
• As a prognostic marker, especially during monitoring of liver cirrhosis, in fulminant hepatic failure or monitoring orthotopic liver transplantation. Declining or significantly low levels suggest a poor prognosis /12/.

ChE synthesis and albumin synthesis in the liver are coupled with each other. Therefore, changes in the ChE level not induced by the liver have no corresponding match in the behavior of the albumin concentration in serum. The diagnostic information shown in Tab. 1.7-5 – Differential diagnostic significance of combined determination of ChE and albumin can be derived from the behavior of albumin and ChE in serum.

The ChE activity of more than 50% of patients with alcoholic steatosis of the liver is within the upper reference range or slightly elevated /15/. Further diseases are shown in Tab. 1.7-6 – Other diseases possibly associated with reduced ChE activity.

1.7.5.2 Drug-induced ChE inhibition

The ChE is reversibly inhibited by the alkaloids prostigmine and physostigmine. The two alkaloids compete with the choline residue of acetylcholine for the binding site at the enzyme. Other drug and substances have an irreversible inhibitory effect on the ChE. A list of drugs is shown in Tab. 1.7-7 – Drug-induced inhibition of cholinesterase.

1.7.5.3 ChE in pesticide poisoning

Organophosphates and carbamates are used worldwide as pesticides. Poisoning with these substances is a problem and especially affects pesticide applicators and children. It is estimated that three million cases of poisoning and about 200,000 deaths occur worldwide every year /22/. Pesticides are also highly toxic for humans and can cause intended and unintended poisoning /23/.

Organophosphates: organophosphates are organic phosphoric acid esters, also referred to as alkyl phosphates, such as ethyl parathion, methyl parathion, demeton-S-methyl sulfoxide, carbophention, mevinphos, chlorpyrifos, dimethoate, naled, EPBP, phosalone. The inhibitory effect of the organophosphates is irreversible and can be different in vitro and in vivo. For example, malaoxon, the highly toxic form of malathione, only forms by oxidation after uptake in the body /24/.

Carbamates: formetanate HCl, methomyl, carbaryl. The inhibitory effect of the carbamates is reversible. Together with acetylcholine esterase, carbamates form an acetylcholine esterase-carbamate intermediate. Since this intermediate is soon subject to hydrolysis, the active enzyme is available again relatively quickly.

Mode of action of the organophosphates and carbamates: organophosphates and carbamates inhibit AChE in the neural tissue and erythrocytes and the CHE in plasma. The inhibition results in the accumulation of the acetylcholine. Concentrations of 20–30 μg/L in plasma are measured. The clinical symptoms of organophosphate poisoning are based on the stimulation of the muscarine- and nicotine-sensitive acetylcholine receptors of the muscles and CNS synapses. The inhibition of the ChE in serum is insignificant under toxicological aspects, but its declining activity in organophosphate poisoning allows conclusions by analogy regarding the remaining AChE activity.

Cholinergic poisoning is commonly treated by the administration of:

• Atropine. This competitive acetylcholine antagonist blocks the muscarine effect on the muscle.
• Nucleophilic antidotes such as pralidoxime or obidoxime for regenerating the AChE. Both substances have a chemical structure which fits the structure of the inhibited AChE.

Acute intoxication

The pesticides enter the body by gastrointestinal, respiratory and ocular uptake, via the skin, but most quickly by inhalation. The pesticides are quickly distributed throughout the body and accumulate in adipose tissue, liver and kidneys. Clinical symptoms occur within 12 hours unless the organophosphate is lipophilic (fenthione) or subject to metabolic activation (parathion). If the phosphoric acid esters are lipophilic, clinical symptoms start later and elimination takes several days. Clinical symptoms of organophosphate and carbamate poisoning are miosis, hyper salivation, nausea, vomiting, increased muscle tremor and sweating. The six most common symptoms in children are diarrhea, vomiting, miosis, bronchial hypersecretion, sweating and hypothermia /25/. The clinical symptoms occur if the ChE decreases to ≤ 60% of the lower reference limit following uptake of the pesticide.

Organophosphate intoxications are classified as follows based on the percentage of decrease in ChE activity /26/:

• Mild form (ChE 60–40%) with the above-mentioned symptoms in the clinical focus
• Moderately severe form (ChE 40–20%) with chest tightness and myalgia in addition to the above-mentioned symptoms
• Severe form (ChE below 20%) with the respiratory distress syndrome in the clinical focus.

In untreated cases, the ChE returns to activity within the reference interval 30–40 days after complete inhibition. This was shown by a study /25/ on children, who had ChE levels of 10–30% the lower reference limit on admission to hospital.

Acute carbamate poisoning is less severe than organophosphate poisoning. In many cases, it is self-limiting because active AChE is quickly available again due to spontaneous lysis of the acetylcholine esterase-carbamate intermediate.

Chronic exposure to pesticides

Chronic exposure can be asymptomatic or have non-specific symptoms such as diarrhea, weight loss, myasthenia and psychic symptoms.

Monitoring of exposed individuals

Monitoring of pesticide applicators can be performed by determining the ChE in serum or in the erythrocytes according to the following recommendation /27/: Before work with the pesticides starts, determine two basic levels at intervals of 3 to a maximum of 14 days if spraying is performed for more than 6 days a month. Then perform three determinations at monthly intervals. The relative risk of pesticide poisoning is increased in workers whose initial baseline serum levels are low or if there levels had already decreased to 60–80% of their baseline previously in the season.

1.7.5.4 ChE reduction by atypical variants

The biosynthesis of cholinesterase is controlled by 4 allelic genes at locus E1 and by rare genetic variants (Tab. 1.7-8 – Biochemical characteristics of cholinesterase variants).

The control is as follows /5/:

• The usual genotype E1U E1U controls the synthesis of normal cholinesterase activity in serum that is inhibitable by dibucaine by almost 80% and by Ro 2-0683 by almost 100%.
• The atypical genotype E1aE1a controls a ChE variant causing lower ChE activity in serum that is inhibited by dibucaine by less than 30% and by Ro 2-0683 almost not at all.
• The fluoride-sensitive genotype E1FE1F controls a variant that is inhibited by dibucaine to a small extent and by fluoride to a large extent.
• The silent genotype E1SE1S controls a ChE variant that lacks the structure required for hydrolyzing cholinester bonds and has no enzyme activity.

Besides the important genotypes mentioned, the four allelic genes can form further genotypes as shown in Tab. 1.7-7 – Drug-induced inhibition of cholinesterase.

Furthermore, there are the J, K and H genes. These three genes encode normal catalytic activity of the ChE molecule. However, there are fewer molecules in the plasma because of impaired synthesis or ChE instability. For example, the K variant is associated with a reduction in ChE activity by 33%, the J variant with a reduction by 66% and the H variant with a reduction by 90%.

Reduced ChE activities are clinically significant in patients treated with the neuromuscular blocker succinylcholine in surgical interventions. Upon administration of 1–1.5 mg succinylcholine per kg of body weight at normal ChE activity in plasma, this dose is hydrolyzed by AChE within 15 minutes. Pronouncedly reduced AChE or the presence of atypical ChE result in a significant relative overdose of succinylcholine and prolonged return to normal neuromuscular function. In a study /28/ analyzing 1,247 patients with abnormal response to succinylcholine, an explanation was found in 61.1% of the cases. The genotype was normal in 28.5%, abnormal in 46.5% and could not be determined in 24.9% of the cases.

The times elapsing until neuromuscular function is restored were as follows:

• 15–30 min. in patients who were heterozygous for an abnormal gene
• 35–45 min. in patients who were heterozygous for two abnormal genes
• 90–180 min. in patients with genotype E1aE1a
• 20 min. in patients with genotype E1aE1k
• 90 min. in patients with genotype E1aE1h (1 patient).

The presence of a single genetically variant allele does not necessarily lead to prolonged neuromuscular failure following the administration of succinylcholine. This is, however, the case in a heterozygous combination if /6/:

• There is a gene encoding for low ChE activity (genotypes E1UE1h, E1UE1J, E1UE1k, E1UE1S).
• Drugs are taken causing a reduction in ChE activity or if liver disease, for example cirrhosis, is present.

Instead of biochemical inhibition using dibucaine, fluoride or Ro 2-0683 for evaluation a ChE variant, it is recommended to perform a molecular genetic DNA analysis to precisely identify the mutation underlying the atypical ChE /29/.

1.7.5.5 Elevated ChE activity

Elevated ChE activities have no diagnostic significance. Tab. 1.7-9 – Diseases possibly associated with elevated ChE activity shows diseases that may be associated with elevated ChE.

1.7.6 Comments and problems

Method of determination

Butyrylthiocholine is the most common substrate used in ChE assays for liver function assessment. The benzoylcholine method is preferred for determining the dibucaine and fluoride numbers. The ChE concentration can also be determined using immunological methods.

Reference interval

The serum activity of the ChE during the neonatal period and the following weeks is only about 50% of that in adults. It then gradually rises, reaches adult levels at the age of 6, stabilizes until puberty and remains constant furthermore. Reported age-dependent changes remain within ranges of no clinical significance. The inter individual serum concentration depends on body weight, height and gender.

The serum concentration in postmenopausal women is reported to be about 15% higher than in premenopausal women.

The ChE activity decreases by 20–30% in the first trimenon of pregnancy, remains at this level throughout pregnancy and returns to normal a few weeks after delivery.

The intake of oral contraceptives containing ethinylestradiol can lower the ChE activity by 20%.

Half-life

Half-life is 10 (3.4–12) days in circulation /30/. Since the indication of changes in liver function is substantially delayed, the ChE is not an acute parameter.

Interference factors

Hemolysis: hemolysis simulates elevated ChE if acetylthiocholine is used as a substrate because the measurement also includes the AChE from erythrocytes. If the ChE assay is performed using the recommended standard method with butyrylthiocholine, there is no interference by hemolysis due to the small volume of the sample /10/.

Lipemia: No interference (small sample volume) /10/.

Hyperbilirubinemia: No interference at concentrations of up to 10 mg/dL (170 μmol/L); the sample must be diluted if concentrations are higher /10/.

Stability

The ChE is very stable. It remains stable for up to 1 year at room temperature (20 °C) or deep-frozen in serum /31/.

1.7.7 Pathophysiology

ChE in plasma is a tetrameric glycoprotein consisting of four identical subunits. Each subunit has 574 amino acids, 9 sugar chains and an active center. The four subunits are held together by disulfide bonds and hydrophobic, non-covalent forces /6/.

Vertebrates have two cholinesterase genes responsible for AChE and CHE enzyme synthesis. They differ in substrate specificity. The CHE hydrolyzes acetylcholine and butyrylthiolcholine, the AChE only hydrolyzes acetylcholine. The size of the acyl pocket in the active center of the two enzymes is the reason for this difference. The two bulky phenylalanine side chains of the butyrylthiocholine do not fit in the acyl pocket of the AChE.

The AChE has two main characteristics enabling it to hydrolyze acetylcholine soon after release by the cholinergic synapses:

• A high catalytic turnover rate
• Two main types of subunits, AChE_H and AChE_T that enable integration of the enzyme in the synaptic structures. Both main types have the same catalytic activity. The formation of the different AChE forms is tissue-specific and depends on whether the muscle is a slow-type or fast-type muscle /32/.

Drugs such as ecothiopate used for glaucoma treatment and cytotoxic drugs such as cyclophosphamide inhibit the AChE irreversibly by binding to an OH group of the serine in the active center of the enzyme. These substances are effective throughout the enzyme’s lifetime of several weeks and only become ineffective when new enzymes are synthesized in the liver.

Drugs binding to the active center via ionic or hydrogen bridging cause reversible inhibition. These are substances possessing a quarternary nitrogen atom, such as hexafluoronium.

Organophosphates and carbamates are a further group of ChE inhibitors. They are also referred to as anti cholinesterases. These substances are used as pesticides. Only organophosphates with a P = O bond have a potent ChE inhibiting effect. They are also referred to as direct inhibitors. Organophosphates with a P = S bond, for example malathion /23/ must first be metabolically converted to P = O. Therefore, they are referred to as indirect inhibitors. Clinical symptoms occur quickly in intoxication with direct inhibitors and are delayed and last longer in intoxication with indirect inhibitors. The following effects of ChE inhibitors are distinguished in clinical symptoms /21/:

• Muscarine-like effects such as nausea, vomiting, hyper salivation,sweating, bronchoconstriction
• Nicotine-like effects such as muscle fasciculation, tachycardia
• Central nervous effects such as drowsiness.

The ChE inhibitors cause physiological dysfunction of the AChE. The AChE blocks the effect of acetylcholine in the transmission of nerve impulses from cholinergic nerves to the postsynaptic side of the effector. ChE inhibitors cause the pathological elevation of acetylcholine at the motor end plate and at parasympathetic and preganglionic sympathetic nerve endings. This also results in hyper excitation in the sympathetic nervous system due to the release of adrenaline and noradrenaline. Since the behavior of the ChE in serum regarding ChE inhibitors is comparable to that of AChE of cholinergic nervous endings, the inhibition of the enzyme’s activity in serum reflects the degree of inhibition at the synapses.

References

1. Mosca A, Bonora R, Ceriotti F, Franzini C, Lando G, Patrosso MC, et al. Assay using succhinyldithiocholine as substrate: the method of choice for the measurement of cholinesterase catalytic activity in serum to diagnose succhinydicholine sensitivity. Clin Chem Lab Med 2003; 41: 317–22.

2. Whittaker M. Cholinesterases. In: Bergmeyer HU, ed. Methods of enzymatic analysis, vol IV. Weinheim; VCH 1984: 52–83.

3. Kalow W, Lindsay HA. A comparison of optical and manometric methods for the assay of human serum ChE. Can J Biochem Physiol 1955; 33: 568–75.

4. Proposal of standard methods for the determination of catalytic concentrations in serum and plasma at 37 °C. II. Cholinesterase (acylcholine acylhydrolase, EC 3.1.1.8). Eur J Clin Chem Clin Biochem 1992; 30: 163–70.

5. Pantuck EJ. Plasma cholinesterase: gene and variations. Anesth Analg 1993; 77: 380–6.

6. Jensen FS, Schwartz M, Viby-Mogensen J. Identification of human plasma cholinesterase variants using molecular biological techniques. Acta Anaesthesiol Scand 1995; 39: 142–9.

7. Kalow W, Staron N. On distribution and inheritance of atypical forms of human serum cholinesterase, as indicated by dibucaine numbers. Can J Biochem Physiol 1957; 35: 1305–17.

8. Holowina P, Newman DJ, Bruno C, La Gamba P, et al. Automated dibucaine number measurement with Du Pont Dimension ES and AR analyzers. Clin Chem 1995; 41: 644–7.

9. Harris H, Whittaker M. Differential inhibition of human serum cholinesterase with fluoride: recognition of two new phenotypes. Nature 1961; 191: 496–7.

10. German Society for Clinical Chemistry. Proposal of standard methods for the determination of enzyme catalytic concentrations in serum and plasma at 37 °C. II. Cholinesterase. Eur J Clin Chem Clin Biochem 1992; 30: 163–70.

11. Jensen FS, Skovgaard LT, Viby-Mogensen J. Identification of human plasma cholinesterase variants in 6688 individuals using biochemical analysis. Acta Anaesthesiol Scand 1995; 39: 157–62.

12. Schmidt E, Schmidt FW. Klinisch-chemische Untersuchungsmethoden. In: Schmidt E, Schmidt FW, Manns MP, eds. Lebererkrankungen. Stuttgart; Wissenschaftliche Verlagsgesellschaft 2000: 8–60.

13. Schmidt E, Schmidt FW. Klinik der Lebererkrankungen. In: Lang H, Rick W, Büttner H, eds. Validität klinisch chemischer Untersuchungen. Heidelberg: Springer, 1980: 92–112.

14. Guder WG. Modell Lebererkrankungen. In: Lang H, Rick W, Büttner H, eds. Validität klinisch-chemischer Untersuchungen. Heidelberg: Springer, 1980: 84–91.

15. Schenker S, Halff GA. Nutritional therapy in alcoholic liver disease. Semin Liver Dis 1993; 13: 196–209.

16. Toro FI, Deibis L, Machado IV, Colmenares C, Bianco NE, de Sanctis JB. Serum cholinesterase activity in viral hepatitis. Med Sci Res 1997; 25: 441–2.

17. Schmidt E, Schmidt FW. Klinische Diagnostik von Lebertumoren. Verh dt Krebs-Ges 1984; 5: 459–70.17.

18. Al-Kassab AS, Vijayakumar E. Profile of serum cholinesterase in systemic sepsis syndrome (septic shock) in intensive care units. Eur J Clin Chem Clin Biochem 1995; 33: 11–4.

19. Tromm A, Hüppe D, Than I, Schwegler U, Kuntz HD, Krieg M, May B. Die Serumcholinesterase als Aktivitätsparameter bei chronisch-entzündlichen Darmerkrankungen. Z Gastroenterol 1992; 30: 449–53.

20. Goedde HW, Benkmann HG, Das PK, Agarwal DP, Lang H, Würzburg U, Beckmann R. Activity of creatine kinase isoenzyme MB in serum and red cell acetylcholinesterase variants in patients with Duchenne muscular dytrophy. Klin Wschr 1977; 55: 215–7.

21. Jokanovic M, Maksimovic M. Abnormal cholinesterase activity: understanding and interpretation. Eur J Clin Chem Clin Biochem 1997; 35: 11–6.

22. O’Malley M. Clinical evaluation of pesticide exposure and poisoning. Lancet 1997; 349: 1161–6.

23. Stalikas CD, Konidari CN. Analytical methods to determine phosphonic and aminoacid group-containing pesticides. J Chromatogr A 2001; 907: 1–19.

24. Rodriguez OP, Muth GW, Berkman CE, Kim K, Thompson CM. Inhibition of various cholinesterases with the enantiomers of malaoxon. Bull Envir Contam Toxicol 1997; 58: 171–6.

25. El-Naggar AR, Abdalla MS, El-Sebaey AS, Badawy SM. Clinical findings and cholinesterase levels in children of organophosphates and carbamates poisoning. Eur J Pediatr 2009; 168: 951–6.

26. Okonek S. Aktuelle Gesichtspunkte zur Intoxikation durch Alkylphosphate. Internist 1975; 16: 123–30.

27. Fillmore CM, Lessenger JE. A cholinesterase testing program for pesticide applicators. JOM 1993; 35: 61–70.

28. Jensen FS, Viby-Mogensen J. Plasma cholinesterase and abnormal reaction to succinylcholine: twenty years experience with the Danish Cholinesterase Research Unit. Acta Anaesthesiol Scand 1995; 39: 150–5.

29. La Du BN. Butyrylcholinesterase variants and the new methods of molecular biology. Acta Anaesthesiol Scand 1995; 39: 139–41.

30. Huizenga JR, van de Belt K, Gips CH. The effect of storage at different temperatures on cholinesterase activity in human serum. J Clin Chem Clin Biochem 1985; 23: 283–5.

31. Ostergaard D, Viby-Mogensen J, Hanel HK, Skovgaard LT. Half-life of plasma cholinesterase. Acta Anaesth Scand 1988; 32: 266–9.

32. Massoulie J, Anselmet A, Bon S, Krejci E, Legay C, Morel N, Simon S. Acetylcholinesterase: C-terminal domains, molecular forms and functional localisation. J Physiol (Paris) 1998; 92: 183–90.

1.8 Creatine kinase (CK)

Lothar Thomas

The CK is a dimeric molecule encoded by genes whose products are CK-M, CK-B and CK-Mi. The CK-Mi is only located in the mitochondria. The activity of CK in serum comprises the cytoplasmic isoenzymes CK-MM, CK-MB, CK-BB. The CK activity in healthy individuals predominantly consists of CK-MM; the CK-MB and CK-BB only exist in traces or are not detectable. If the activity of the CK or one of the isoenyzmes is elevated, the isoenzyme pattern allows conclusions as to the underlying tissue damage.

1.8.1 Indication

If troponin is not available, as secondary biomarker in patients /1/:

• With clinical and ECC signs of acute myocardial infarction (indicated assay: CK-MB concentration)
• Monitoring acute myocardial infarction
• Myocarditis
• Suspected skeletal muscle disease, neurogenic myopathy or drug-induced myopathy
• Therapy monitoring of individual tumor patients.

1.8.2 Method of determination

IFCC Primary Reference Procedure for the Measurement of Catalytic Activity Concentrations of Creatine Kinase (CK) at 37 °C /2/

Principle: The CK (EC 2.7.3.2) catalyzes the reversible phosphorylation of creatine by ATP as shown in Tab. 1.8-1 – Principle of CK determination. Mg²⁺ is an obligate activating ion to form the ATP- and ADP-Mg²⁺ complexes. The hexokinase catalyzes the phosphorylation by ATP to form glucose-6-phosphate (G-6-P) and regenerates ADP for the CK reaction. The G-6-P is then oxidized with NADP⁺ to form 6-phospho gluconic acid and NADPH. The rate of NADPH formation is a measure of CK-activity of the phosphate group of phosphocreatine to Mg-ADP (Tab. 1.8-1 – Principle of CK determination).

Determination of the CK-MB concentration /3/

The CK-MB is determined by an immunochemical method.

Principle: Direct measurement of CK-B subunit at 37 °C by inhibiting M subunits with M subunit antibody.

1.8.3 Specimen

• Serum or heparin anticoagulated blood: 1 mL
• Anticoagulated whole blood: 0.02–0.05 mL

1.8.4 Reference interval

See Tab. 1.8-2 – Reference intervals of CK.

1.8.5 Clinical significance

CK in muscle injury

In most cases, the CK in the serum of healthy individuals corresponds to the activity of the muscle-specific isoenzyme CK-MM and therefore acts as a specific enzyme for the detection of muscle damage. However, activities within the reference interval do not exclude myopathy, and elevated activities can have physiological causes. Monitoring, and in unclear symptoms, the determination of the CK isoenzymes make it easier to determine whether the myocardium or skeletal muscle are affected. In hereditary skeletal muscle diseases the CK levels are higher than 1,000 U/L in neuropathic skeletal muscle diseases the levels are below 1,000 U/L.

CK in acute myocardial infarction (AMI)

The blood concentration of the CK increases following AMI (e.g., onset of infarction) and reaches pathological levels in 50% of the patients on average after 4–5 hours /9/. The CK is elevated on a regular basis within the diagnostic time frame of 8–24 hours following AMI and then – with great inter individual fluctuations – decline again to within the reference interval. The maximum CK activity rarely rises beyond 7500 U/L in AMI. Higher CK activities suggest concurrent disease of skeletal muscle. The recurrent elevation of CK and CK-MB indicates secondary trauma or reinfarction.

CK-MB concentration in acute myocardial infarction

In AMI, the profile of CK-MB is similar to that of the CK. The CK-MB reaches pathological concentrations in 50% of the patients after 3–4 hours /9, 10/, but declines earlier than the CK. AMI can be excluded if the CK-MB does not increase within the diagnostic time frame. The half-lives of the CK isoenzymes are shown in Tab. 1.8-3 – Half-lives and time of maximum activity in hours (h) after myocardial infarction.

Early recanalization (spontaneous recovery or therapeutic success) is recognized based on a rapid increase and early peak of the CK-MB concentration occurring within 16 hours. If samples are analyzed that were taken outside the narrow diagnostic time frame, the CK-MB can still, or already again, be within the reference interval.

The determination of CK-MB in diagnosing myocardial injury has the disadvantage that the CK-MB can also be released from non-cardiac muscle in some cases.

CK in myopathies

Myopathies are diseases of skeletal muscle. They can be congenital or acquired and occur already at birth or at a later age. Clinical symptoms are hypersensitivity, myalgia and myasthenia; the CK activity is normal or elevated /11/. The following distinctions are made:

• Myalgia; involves pain and weakness of the muscles without measurable cellular damage of muscle tissue. CK is not elevated.
• Myositis; has the same or more severe symptoms as myopathy; muscle cells are damaged and the CK is 3–10-fold elevated.
• Rhabdomyolysis; there is a strong damage of several muscles; the CK is elevated more than 10-fold the upper reference limit, renal function is impaired in many cases (elevated creatinine), the urine is brown and myoglobin is detectable.

The behavior of the CK and CK isoenzymes is shown for:

• Myocardial injury in Tab. 1.8-4 – CK and CK-MB activities in acute myocardial injury.
• Acute damage of skeletal muscle in Tab. 1.8-5 – CK and CK-MB activities in acute skeletal muscle damage.
• Chronic injury of skeletal muscle in Tab. 1.8-6 – CK activity in chronic skeletal muscle damage.
• Damage of other tissue in Tab. 1.8-7 – CK activity in other tissue damage.

1.8.6 Comments and problems

Determination of CK activity

The addition of N-acetyl cysteine to the reaction mixture reactivates the CK and protects it from oxidation processes. This requires a certain amount of time (lag-phase time). The reaction mixture also contains AMP and diadenosine pentaphosphate, thus suppressing interference by adenylate kinase (EC 2.7.4.3) from the erythrocytes, muscle, liver and platelets. EDTA stabilizes the CK and prevents possible inhibition by Ca²⁺ of the sample. Critical parameters of the determination are the (re)activation time, sample volume ratio and lag-phase time /2, 5/. An activation time of 180 sec. is sufficient for an exact measurement of new samples. An activation time of 300 sec. may be necessary for older samples and some quality assessment specimens. It is recommended to start the reaction process by addition of the substrate; start by addition of serum is possible /2, 5/.

CK-MB concentration

Interferences: besides interference typical of immunoassays, for example by human anti-mouse antibodies or high biotin doses, interference by CK-B-binding autoantibodies can occur /12/. The presence of macro CK does not interfere with the assay determination.

Reference interval

The re-evaluation of the reference interval for adults was obtained in hospitalized subjects /2, 4, 5/. This explains the lower levels compared to an outpatient cohort. The reference intervals for children were determined using a procedure adapted to the IFCC reference method /7/. In this method, the thiol compounds of the reagent show the strongest deviations. Hence, the values for younger children (higher proportion of CK-B) cannot be applied to the IFCC method without reservation.

Macro CK (see also Tab. 1.1-2 – Macroenzymes: Characterization, clinical significance and laboratory findings)

The prevalence of the macro CK in elevated serum CK activity is about 2% /13/. Macro CK type 1, a complex of immunoglobulin and CK, is the most common form. Exact evidence can only be provided by procedures where the molecular weight is determined, for example exclusion chromatography, gradient gel electrophoresis. Treatment of the serum with polyethylene glycol gives initial clues.

Stability

Serum samples and reference specimens not processed within 12 hours should be stored well sealed and dark. In that case, the activity will not change within 3 days at 4 °C and within 4 weeks at –20 °C /14/.

1.8.7 Pathophysiology

The enzymes CK and adenylate kinase are of vital importance for the synthesis of ATP, the immediate energy source of the tissues.

The CK participates in the energy supply to the muscle in two ways (Fig. 1.8-1 – Involvement of CK and adenylate kinase (AK) in energy production in the muscle). In the mitochondria, the site of energy production, the mitochondrial CK catalyzes the synthesis of phosphocreatine (PCr) from ATP. The high-energy PCr is then transported from the mitochondria to the cytoplasm by the PCr shuttle, where it is reconverted to ATP by CK at the sites of energy consumption (muscle contraction, ion channels of the membrane, syntheses) /15, 16/. The CK is present intracellularly in the free form or associated to corresponding cell structures.

Three cytoplasmic CK isoenzymes can be isolated from human tissue as dimers that can consist of the subunits M (M, muscle) and B (B, brain). Their gene loci are on chromosome 14 (B subunit) and the long arm of chromosome 19 (M subunit) /17/. The two mitochondrial CK isoenzymes are referred to as S-MTCK (sarcomeric) and U-MTCK (ubiquitous). In addition, there are inconsistent abbreviations for the dimeric form such as CK-MiMi, mCK, CK-MT or CKmito. The CK monomers with a molecular weight of 40 kDa consist of approximately 360 amino acids and contain SH groups. Hybridization between the M and B subunits that are already active as monomers results in CK-MB. There is no hybridization with the Mi subunit /18/.

Based on their distribution to specific tissues, the isoforms are referred to as muscle type (CK-MM), myocardial type (CK-MB), brain type (CK-BB) and mitochondrial type (CK-MiMi). Despite these designations, the tissue specificity of these isoenzymes is not very high. CK-BB is an ubiquitous isoenzyme that has its highest activity in the brain. Similarly, the predominant amount of CK-MB is found in skeletal muscle and only its highest concentration is located in the myocardium. There is very different data on the quantitative distribution of the CK isotypes in the tissues /19/. Tab. 1.8-8 – Tissue distribution of CK isoenzymes provides a rough overview of the distribution.

Since the CK enters the blood in the event of tissue injury, the isoenzyme pattern of the blood reflects the isoenzyme pattern of the damaged tissue. This statement is qualified by the following facts:

• The CK must not be prevented from entering the blood (blood-cerebrospinal fluid barrier, lymphatic pathways).
• A tissue must be able to release an amount of CK high enough to cause a measurable increase in activity in case of damage; no increase occurs in acute damage of the gall bladder, lung, liver, prostate, non-gravid uterus and veins.
• An increase in activity cannot be measured if the CK is inactivated or cleared from circulation too quickly. In most cases, this applies to the release of CK-BB in acute damage.
• Chronic myopathies can change the isoenzyme inventory of the muscle cell so that, besides the muscle-specific M subunit, the B subunit is synthesized again as in the fetal phase. This results in a higher concentration of CK-MB and possibly also CK-BB.

Having been released into the blood, the CK is subject to post synthetic modifications that lead to the CK isoforms. Isoforms with a normal molecular weight of about 80 kDa are created if the carboxypeptidase gradually removes the C-terminal lysin of both M chains.

Isoforms with an increased molecular weight higher than 200 kDa are created if CK is bound by specific immunoglobulins (macro CK type 1) or if the CK mito is present in the preferred oligomeric form (macro CK type 2).

References

1. Thygesen K, Alpert JS, White HD, Joint ESC/ACCF/AHA/WHF Task Force for the Redifinition of Myocardial Infarction. J Am Coll Cardiol 2007; 50: 2173–95.

2. IFCC Primary Reference Procedures for the Measurement of Catalytic Activity Concentrations of enzymes at 37 °C. Part 2. Reference procedure for the measurement of catalytic concentration of creatine kinase. Clin Chem Lab Med 2002: 40: 635–42.

3. Christenson RH, Vaidya H, Landt Y, Bauer RS, Green SF, Apple FA, et al. Standardization of creatine kinase-MB (CK-MB) mass assay: the use of recombinant CK-MB as a reference material. Clin Chem 1999; 45: 1414–23.

4. Thomas L, Müller M, Schumann G, Weidemann G, Klein G, Lunau S, Pick KH, Sonntag O. Consensus of DGKL and VDGH for interim reference intervals on enzymes in serum. J Lab Med 2005; 29: 301–8.

5. Schumann G, Klauke R. New IFCC reference procedures for the determination of catalytic activity concentrations of five enzymes in serum: preliminary upper reference limits obtained in hospitalized subjects. Clin Chim Acta 2003; 327: 69–79.

6. Stein W. Laboratory diagnosis of acute myocardial infarction. Darmstadt: GIT-Verlag, 1988.

7. Ghoshal AK, Soldin SJ. Evaluation of the Dade Behring Dimension RxL: integrated chemistry system-pediatric reference ranges. Clin Chim Acta 2003; 331: 135–45.

8. Apple FS, Quist HE, Doyle P, Otto AP, Murakami MM. Plasma 99th percentile reference limits for cardiac troponin and creatine kinase MB mass for use with European Society of Cardiology/American College of Cardiology consensus recommendations. Clin Chem 2003; 49: 1331–6.

9. Mair J, Smidt J, Lechleitner P, Dienst F, Puschendorf B. Equivalently early sensitivity of myoglobin, creatine kinase MB mass, creatine kinase and creatine kinase MB mass, creatine kinase isoform ratios, and cardiac troponins I and T for acute myocardial infarction. Clin Chem 1995; 41: 1266–72.

10. Zaninotto M, Altiner S, Lachin M, Carraro P, Plebani M. Fluoroenzymometric method to measure cardiac troponin I in sera of patients with myocardial infarction. Clin Chem 1996; 42: 1460–6.

11. Pasternak RC, Smith SC, Jr, Bairey-Merz CN, Grundy SM, Cleeman JI, Lenfant C. ACC/AHA/NHLBI clinical advisory on the use and safety of statins. J AM Coll Cardiol 2002; 40: 567–62.

12. Stein W, Bohner J. Influence of autoantibodies to creatine kinase-BB on assays for MB isoenzyme. Clin Chem 1985; 31: 1189–92.

13. Fahie-Wilson MN, Burrows S, Lawson GJ, Gordon T, Wong W, Dasgupta B. Prevalence of increased serum creatine kinase activity due to macro-creatine kinase and experience of screening programmes in district general hospitals. Ann Clin Biochem 2007; 44: 377–83.

14. ECCLS European Committee for Clinical Laboratory Standards. Standards for enzyme determination. Creatine kinase, aspartate aminotransferase, alanine aminotransferase, gamma-glutamyltransferase. Lund: ECCLS Central Office, document number 3–4, 1988.

15. Wallimann T. Bioenergetics: dissecting the role of creatine kinase. Curr Biol 1994; 4: 42–6.

16. Wallimann T, Hemmer W. Creatine kinase in non-muscle tissues and cells. Mol Cell Biochem 1994; 133–4: 193–220.

17. Stallings RL, Olson E, Strauss AW, Thompson LH, Badinski LL, Siciliano LJ. Human creatine kinase genes on chromosomes 15 and 19, and proximity of the gene for the muscle form to the genes for apolipoprotein C2 and excision repair. Am J Hum Genet 1988; 43: 144–51.

18. Payne RM, Strauss AW. Expression of the mitochondrial creatine kinase genes. Mol Cell Biochem 1994; 133–4: 235–43.

19. Lang H. Creatine kinase isoenzymes. Heidelberg: Springer, 1981.

20. Ravkilde J, Nissen H, Hørder M, Thygesen K. Independent prognostic value of serum creatine kinase isoenzyme MB mass, cardiac troponin T, and myosin light chain levels in suspected acute myocardial infarction. Analysis of 28 months of follow-up in 196 patients. J Am Coll Cardiol 1995; 25: 574–81.

21. Farah SY, Moss DW, Ribeiro P, Oakley CM, Sapsford RN. Interpretation of changes in the activity of creatine kinase MB isoenzyme in serum after coronary artery bypass grafting. Clin Chim Acta 1984; 141: 219–25.

22. Eltze C, Hildebrandt G, Johanson M. Über das Verhalten der Creatin-Kinase im Serum bei Muskelkater. Klin Wochenschr 1983; 61: 1147–51.

23. Manfredi TG, Fielding RA, O’Reilly KP, Meredith CN, Lee HY, Evans WJ. Plasma creatine kinase activity and exercise-induced muscle damage in older men. Med Sci Sports Exerc 1991; 23: 1028–34.

24. Lippi G, Schena F, Montagnana M, Salvagno GL, Guidi GC. Influence of acute physical exercise on emerging muscular biomarkers. Clin Chem Lab Med 2008; 46: 1314–8.

25. McMahon GM, Zeng X, Waikar SS. A risk prediction score for kidney failure or mortality in rhabdomyolysis. JAMA Internal Medicine 2013; 173: 1821–7.

26. Mohassel P, Mammen AL. The spectrum of statin myopathy. Curr Opin Rheumatol 2013; 25: 747–52.

27. Hackl W, Mauritz W, Schemper M, Winkler M, Sporn P, Steinbereithner K. Prediction of malignant hyperthermia susceptibility: statistical evaluation of clinical signs. Br J Anaesth 1990; 64: 425–9.

28. Arzneimittelkommission der deutschen Ärzteschaft. Akute Rhabdomyolyse unter Olanzapin. Dtsch Ärztebl 2005; 102: B1974–5.

29. Pearson CM, Rimer DG, Mommaerts WFHM. A metabolic myopathy due to absence of muscle phosphorylase. Am J Med 1961; 30: 502–17.

30. Stein W, Bohner J, Bahlinger M. Creatine kinases. In: Blaton V, van Steirteghem A. Plasma isoenzymes: the current status. Basel: Karger, 1986; 95–104.

31. Stein W, Bohner J, Schüch K. Creatine kinase-BB in blood cells: a marker of myeloproliferative disorders (MPD). Clin Chem 1986; 32: 1138.

32. Nordby HK, Urdal P. Creatine kinase BB in blood as index of prognosis and effect to treatment after severe head injury. Acta Neurochirurgica 1985; 76: 131–6.

33. Chemnitz G, Bartner J. Zur Bedeutung erhöhter Kreatinkinase-Aktivität im Serum nach apoplektischem Insult. Klinisches Labor 1994; 40: 529–33.

34. Stein W. Creatinkinase. In: Thomas L, ed. Labor und Diagnose. Frankfurt; TH-Books 2008: 89–97.

35. Maloney B, Park S, Sowizral M, Brackett I, Moslehi R, Chung WK, et al. Factors influencing creatin kinase MM concentrations in newborns and implications for newborn screening for Duchenne muscular dystrophy. Clin Biochem 2023; 118: 110614.

1.9 Gamma-glutamyl transferase (GGT)

Lothar Thomas

The GGT is a peptidase that transfers amino acids from one peptide to the next and thus functions as amino acid transferase. The GGT activity primarily is synthesized in the hepatobiliary system, and the enzyme is elevated in serum in many hepatobiliary diseases. Studies have shown strong positive associations between GGT and cardiovascular risk factors such as smoking, components of the metabolic syndrome (obesity hypertension, lipid metabolism, type 2 diabetes). Elevated GGT is also associated with chronic kidney disease independently of factors such as alcohol consumption /1/.

1.9.1 Indication

• Screening for hepatobiliary diseases in a pattern with the ALT and CHE
• Differential diagnosis and monitoring of hepatobiliary diseases
• Monitoring of chronic alcoholism in combination with other assays
• Risk marker for numerous chronic diseases.

1.9.2 Method of determination

IFCC Primary Reference Procedure for the Measurement of Catalytic Activity Concentration of γ-Glutamyltransferase at 37 °C /2/

Principle: The GGT (EC 2.3.2.2) catalyzes the transfer of the glutamyl residue from γ-glutamyl-3-carboxy-4-nitroanilide to glycylglycine releasing 5-amino-2-nitrobenzoate (Tab. 1.9-1 – Principle of GGT determination). The increase in concentration of this substance measured as absorption increase at 405 nm is proportional to the enzyme activity in the sample.

1.9.3 Specimen

Serum, plasma (heparin, EDTA): 1 mL

1.9.4 Reference interval

See Tab. 1.9-2 – Reference intervals of GGT.

1.9.5 Clinical significance

The GGT is a liver-specific and bile duct-specific enzyme. Although other tissues also contain GGT, major elevations of the GGT not induced by the liver or bile ducts are rare.

However, the GGT is not only a biomarker for hepatobiliary diseases and excessive alcohol consumption, but also a metabolic toxic marker and an indicator of the presence of chronic diseases in asymptomatic patients. The activity of GGT in hepatobiliary diseases is shown in Tab. 1.9-4 – GGT in hepatic and biliary tract diseases and resulting from different etiologies in Tab. 1.9-5 – Various causes of elevated GGT levels.

1.9.5.1 Elevated GGT /5/

Drugs and alcohol: patho biochemically, both drugs and alcohol cause an induction of GGT synthesis. It must be kept in mind that there is great inter individual variation in enzyme induction.

Drugs, especially anticonvulsives (phenobarbital, phenytoin), psychotropic drugs, steroid hormones, anticoagulants, streptomycin, xenobiotics and carcinogens (nitrosamines), cause 1.5–3-fold elevations above the upper reference limit. Elevations due to other drugs such as streptokinase and oral contraceptives are not as high.

Alcohol in doses as low as 0.75 g/kg BW can cause elevated GGT within one day; the GGT is about 25% of the initial level after 2.5 days and returns to the initial level after 4 days. However, these elevations mainly remain within the reference interval.

Cholestasis syndromes: cholestasis is associated with structural changes of the cell membrane leading to the detachment of GGT located at the surface. In addition, GGT increasingly formed by induction no longer attaches to the cell membrane and immediately enters the circulation. Cholestasis can occur in acute and chronic hepatitis and in chronic liver diseases of other causes. In cholestasis, the GGT is elevated more than 5-fold the upper reference limit in many cases, while the GGT elevations found in acute and chronic hepatitides of viral genesis are not higher than 3–5-fold the upper reference limit.

Predictor for risk of disease: The GGT shows association with vascular and metabolic diseases and generally with morbidity and mortality. This association is independent of potentially concurrently present hepatic disease or alcohol abuse. The GGT is positively associated with /1/:

• Cardiovascular risk factors such as smoking
• Characteristics of the metabolic syndrome such as overweight, hypertension, lipometabolic disorders and type 2 diabetes.

The associations make the GGT a predictor for cardiovascular diseases and increased mortality. The GGT level is also associated with chronic renal disease, independently of alcohol consumption. The GGT levels are moderately elevated or range within the upper third of the reference interval. In a study /6/, GGT levels above 24 U/L were associated with increased myocardial infarction mortality. In a different study /7/, the incidence of diabetes mellitus type 2 was 3 times higher and that of a metabolic syndrome was 4 times higher in individuals with GGT levels of 36–50 U/L.

1.9.5.2 Diagnostic sensitivity and specificity of GGT for liver diseases

Since elevated GGT is triggered by numerous chronic diseases and also by primary liver injury, they have a good tissue specificity and high diagnostic sensitivity, but only a low specificity for hepatobiliary diseases. For example, the diagnostic sensitivity for hepatobiliary diseases is 95% at 96% specificity compared to healthy individuals and 74% compared to non-hepatobiliary disease patients. The GGT is the most frequently single elevated enzyme in hepatobiliary diseases with a proportion of 14% (6–20%); the proportion is even higher (22–30%) in individuals with isolated GGT elevations who have not been clinically diagnosed with a primary disease of the liver or bile ducts /8/. The GGT has a low diagnostic specificity for liver diseases if it is the only enzyme elevated. It does not gain in disease specificity and differential diagnostic significance until other enzymes such as the ALT and ALP are also determined.

Elevated aminotransferases at normal GGT are found in chronic liver disease.

1.9.5.3 Isolated elevation of GGT

Isolated elevations can be caused by /8/:

• Therapy-related induction of GGT synthesis, for example under anticonvulsive medication. Isolated GGT elevations by more than 3-fold the upper reference limit are no longer induced by therapy.
• Fatty liver, subclinical obstruction of the bile flow, space-occupying liver processes, chronic liver congestion in cardiac disease.
• Alcohol-induced liver disease, adiposity, diabetes mellitus.

1.9.5.4 Differential diagnostic significance of GGT

The GGT is significant under the following aspects:

• Differentiation of cholestasis from inflammatory liver diseases
• Differentiation of alcohol-induced disease from inflammatory liver disease
• Indication of hepatic steatosis (fatty liver).

Assessment criteria are:

• Behavior of the GGT in a pattern with the aminotransferases; the GGT/ALT ratio and/or GGT/AST ratio is assessed. In icteric patients, this ratio is a criterion for rating the degree of cholestasis in relation to cell membrane damage /9/.
• GGT level.
• Behavior of the GGT in relation to the cholestasis enzyme ALP.

1.9.5.5 Differentiation of cholestasis from inflammatory liver diseases /9/

In the setting of jaundice, the ALT activity and the GGT/ALT ratio allow a rough differentiation between hepatitic and cholestatic causes. For example, the ALT levels found in patients with obstructive jaundice are not higher than 25-fold the upper reference limit and the GGT/ALT ratio is always above 1 and even above 6 in many cases (Tab. 1.9-3 – Ratio GGT/ALT in hepatobiliary diseases) /9/. A more pronounced relative increase in the GGT compared to the ALP is another criterion in support of cholestatic syndrome. In intrahepatic cholestasis, the aminotransferases can be elevated with a profile approximately parallel to that of the elevated GGT and ALP so that the GGT/ALT ratio is only just above 1.

To a certain extent, the level of the GGT is diagnostically conclusive in intrahepatic cholestasis. For example /9/:

• Metastatic liver, cholangitis and primary biliary cirrhosis have GGT levels higher than 3-fold the upper reference limit in more than 98% of the cases.
• The increase in toxic liver injury is higher and/or lower than 3-fold the upper reference limit in equal shares.
• 85% of the cases of chronic hepatitis and 99% of the cases of alcoholic fatty liver have GGT levels below 3-fold the upper reference limit.

In the setting of jaundice, normal GGT and normal activity of other liver enzymes and LD suggest the presence of a bilirubin metabolic disorder.

In pediatric practice, the GGT has significant advantages over the ALP in the detection of cholestatic syndrome. The ALP is difficult to interpret in children due to age-dependent variations or possible vitamin D deficiency /10/.

1.9.5.6 Alcohol consumption

The effects of alcohol on health are a combination of positive and negative effects. Moderate drinking can have beneficial physical, mental and social effects, clinically significant drinking causes organ damage.

• Clinically significant alcohol consumption is the major contributer to several diseases, e.g. liver disease, pancreatitis, hypertension, diabetes mellitus, dementia, and several types of cancer. Often clinically significant alcohol consumption affects the liver and results in alcoholic liver disease. The National Institute on Alcohol Abuse and Alcoholism in the United States defines clinically significant alcohol consumption as consuming more than 14 standard drinks per week for men and more than 7 for women. One standard drink equals 14 g of alcohol. The European Association for the Study of the Liver and the Korean Association for the Study of the Liver indicate clinically significant alcohol consumption exceeds 210 g per week for men and 140 g per week for women.
• Safe consumption (moderate drinking). The National Institute on Alcohol Abuse and Alcoholism in the United States and the European Association for the Study of the Liver published no safe levels of alcohol consumption. A study /12/ showed that more than 11.5 ± 3.3 units of 80g per week according to 92 ± 26.4 g alcohol/week significantly increased the risk of developing liver-related diseases.
• Refer to:
• Tab. 1.2-3 – Biomarkers in hepatopathies
• Section 18.6 – Alcohol biomarkers
• Tab. 1.9-6 – Definition of a standard drink.

1.9.5.6.1 GGT as biomarker of alcoholism

GGT levels above the upper reference limit are measured in about 75% of alcohol addicts /11/. However, there is no correlation between the total quantity ingested in a certain period of time, the daily amount of intake or the duration of alcohol intake. Only 20–50% of the individuals, who daily consume large amounts of alcohol without being dependent, show elevated GGT. The GGT is a biomarker of chronic intake of larger amounts of alcohol and is not elevated in occasional drinkers after a “boozy evening” unless these individuals suffer from a liver disease /11/.

The daily consumption of more than 40 g of alcohol in habitual drinkers and at least 60 g of alcohol for at least 5 weeks in non-habitual drinkers is required before the GGT rises to pathological levels /13/. The GGT has a diagnostic sensitivity of 55–100% at 50–72% specificity in the diagnosis of chronic alcoholism /14/. Alcohol-induced elevation of the GGT in individuals under the age of 30 is rare. The diagnostic sensitivity of GGT is lower in women than in men /15/.

The GGT is suited for monitoring alcohol abstinence. Depending on the preexisting liver disease, the GGT declines with a half-life of 14–26 days and, after alcohol abstinence of 4–5 weeks, reaches the reference interval. The GLD is a good biomarker for monitoring alcohol withdrawal (see Section 1.10 – Glutamate dehydrogenase (GLD)).

1.9.6 Comments and problems

Blood sampling

GGT activity is lower in blood containing citrate, oxalate or fluoride than in serum.

Method of determination

The activities measured in heparin anticoagulated blood using the IFCC method (substrate γ-glutamyl-3-carboxy-4-nitroanilide) are too low /45/.

Reference interval

The upper reference limits of adults are too high because they are based on normal alcohol consumption. A presumably alcohol-induced, inter individual (but not generally intraindividual) increase in GGT levels occurs with increasing age /48/.

Hemolysis

Hemolysis leads to lower GGT levels at free Hb concentrations above 2 g/L /45/.

Half-life

The half-life is 7–10 days and, in alcoholics, up to 28 days /8/.

Macroenzymes

GGT macroenzymes are only present in hepatobiliary diseases, but have no diagnostic and differential diagnostic significance (for further information, see Tab. 1.1-2 – Macroenzymes: Characterization, clinical significance and laboratory findings).

Oral contraceptives

Elevated GGT occurs in oral contraceptives containing norgestrel or levonorgestrel as progestagen /47/.

Stability in serum

1 week at room temperature (20 °C) /49/.

1.9.7 Pathophysiology

The GGT is a heterodimeric protein each consisting of a single polypeptide chain. It is located on the cytoplasmic membrane of many somatic cells; the active center of the enzyme is directed outward. The luminal surfaces of cells with secretory or absorptive functions especially abound in GGT, but basolateral surfaces of renal tubule cells also contain GGT.

The GGT is the only enzyme that breaks down noteworthy amounts of glutathione (GSH) and GSH conjugates. In the γ-glutamate cycle, for example, it breaks down GSH (γ-glutamyl-cysteinyl-glycine), which is formed intracellularly and transported to the extracellular (luminal) side of the cell membrane, into cysteinylglycine and the γ-glutamyl residue. The transport of GSH into the extracellular compartment, the breaking down of its components by the GGT and their re-synthesis in GGT are referred to as γ-glutamate cycle (Fig. 1.9-1 – GGT and the glutamate cycle). In this way, the GGT supports the supply of GSH, the most important non-protein antioxidant, to the tissues. The reason why large amounts of GSH are secreted from the hepatocytes into the bile, from the proximal tubule cells into urine, from the type II pneumocytes into the alveoli and from the microvilli of the brush border into the intestine is to protect the cell membrane against oxidative destruction. Moreover, the GSH is the reservoir of cysteine, a relatively toxic amino acid, and its transport form in the organism; hence, the cysteine concentration is kept low /50/.

The GGT also plays an important role /50/:

• In the metabolism of inflammatory mediators (e.g., leukotrienes (LT). For example, the highly pro inflammatory and vasoconstrictive LTC₄ is formed by conjugation of GSH with LTA₄.
• In the metabolization of carcinogenic and toxic xenobiotics.

Under certain conditions, however, the degradation of GSH plays a pro-oxidant role. It is assumed that the oxidation of low density lipoprotein (LDL) by the GSG/GGT-dependent reduction of iron is a mechanism in the development of atherosclerosis. The GGT is detectable in foam cells of atherosclerotic lesions of the vascular intima. This is where the GGT forms cysteinylglycine. The cysteinylglycine reduces Fe (III) and thus promotes the Fe (II)-catalyzed oxidation of LDL. The increased synthesis of oxidized LDL promotes the progression of atherosclerosis. The GGT of atherosclerotic plaques is thought to come from the plasma or from macrophages with an up regulated GGT synthesis /51/.

The serum GGT originates from the liver and is present in a heterogeneous form. Most of it binds to lipoproteins, especially to HDL and also to LDL. A small proportion is soluble in water, has a molecular weight of 84 kDa and is similar to the GGT released from the hepatocyte membrane by proteases /52/.

The HDL-bound GGT is predominant in non-icteric liver diseases. The GGT bound to LDL is elevated in cholestasis, and the water-soluble form is elevated in all kinds of hepatopathies. The latter never exceeds a proportion of 20% in the total activity. It is assumed that the HDL-bound and LDL-bound GGT is released due to the solubilization of hepatocyte membranes by bile or as part of a membrane fragment following cell rupture. The water-soluble form is thought to be released from the cell membrane directly by proteases. The proteases dissociate the hydrophilic, enzymatically active part from the hydrophobic, membrane-bound domain of the enzyme /53/.

The GGT is cleared from the plasma mainly through the liver and secreted through the bile. The activity in the bile is about 10-fold higher than in plasma. A small part is catabolized by the kidneys and part is secreted with the urine.

In the fetal liver, the GGT is distributed evenly over the lobuli where it occurs dissolved in the hepatocyte and bound to the cell membrane. In the liver of adults, the GGT is located in the periphery of the lobuli and the activity of the dissolved form in the hepatocyte is low. The main proportion is located on the canalicular and sinusoidal membrane of the hepatocyte and the epithelial membrane of larger bile ducts /54/.

The synthesis of the GGT in the liver is induced by cholestasis, alcohol and drugs in therapeutic doses (e.g., phenytoin). The membrane-bound GGT propagates. The enzyme spreads mainly periportally from the canalicular membranes to the other parts of the cell membrane facing the Dissé space.

The elevated GGT measurable in serum after enzyme induction is dependent on the nature and extent of the noxa. Parenchymal damage must always be considered if the GGT is elevated more than 2-fold the upper reference limit or if the elevation occurs concurrently with an increase in other liver enzymes.

Elevated GGT in dysfunctional bile secretion can be caused by solubilization of the GGT by bile acids or an increase in GGT formation of the hepatocyte. The latter, in particular, is thought to be the reason for the GGT increase in hepatoma cells compressed by a liver tumor and in regenerating areas of the cirrhotic liver /55/. For the molecular pathogenesis of cholestasis, see Section 5.2 – Bilirubin.

References

1. IFCC Primary Reference Procedures for the Measurement of Catalytic Activity Concentrations of Enzymes at 37 °C. Part 6. Reference procedure for the measurement of catalytic concentration of γ-Glutamyltransferase. Clin Chem Lab Med 2002; 40: 734–8.

2. Abicht K, El-Samalouti V, Junge W, Kroll M, Luthe H, Treskes M, Klein G. Multicenter evaluation of new liquid GGT and ALP reagents with new reference standardization and determination of reference intervals (abstract). Clin Chem Lab Med 2001; 39: S346.

3. Ghoshal AK, Soldin SJ. Evaluation of the Dade Behring Dimension RxL: integrated chemistry system-pediatric reference ranges. Clin Chim Acta 2003; 331: 135–46.

4. von Herbay A, Strohmeyer G. Die erhöhte γ-GT (γ-Glutamyltransferase). Dtsch Med Wschr 1994; 119: 1041–4.

5. Haring R, Wallaschofski H, Nauck M, Dörr M, Baumeister SE, Völzke H. Ultrasonic hepatic steatosis increases prediction of mortality risk from elevated serum gamma-glutamyl transpeptidase levels. Hepatology 2009; 50: 1403–11.

6. Wannamethee G, Ebrahim S, Shaper AG. γ-glutamyltransferase: determinants and association with mortality from ischemic heart disease and all causes. Am J Epidemiol 1995; 142: 699–708.

7. Kim DJ, Noh JH, Cho NH, Lee BW, Choi JH, Jung JH et al. Serum γ-glutamyltransferase within its normal concentration range is related to the presence of diabetes and cardiovascular risk factors. Diabetic Medicine 2005; 22: 1134–40.

8. Schmidt E, Schmidt FW. Diagnostik des Ikterus. Dtsch Med Wschr 1984; 109: 139–44.

9. Schmidt E, Schmidt FW. γ-Glutamyl-Transpeptidase. Dtsch Med Wschr 1973; 98: 1572–8.

10. Cabrera-Abreu JC, Green A. Gamma-glutamyltransferase: value of its measurement in paediatrics. Ann Clin Biochem 2002; 39: 22–5.

11. Sharpe PC. Biochemical detection and monitoring of alcohol abuse and abstinence. Ann Clin Biochem 2001; 38: 652–64.

12. Moon SY, Son M, Kang YW, Koh M, Lee JY, Baek YH, Alcohol consumption and the risk of liver disease: a nationwide, population-based study. Frontiers in Medicine 2023. doi: 10.3389/fmed.2023.1290266.

13. Congrave KM, Saunders JB, Whitefield JB. Diagnostic tests for alcohol consumption. Alcohol Alcohol 1995; 30: 13–26.

14. Kristenson H, Trell E, Fex G, Hood B. Serum gamma glutamyl transferase: statistical distribution in a middle aged male population and evaluation of alcohol habits in individuals with elevated levels. Prev Med 1980; 9: 108–19.

15. Whitfield JB, Hensley WJ, Bryden D, Gallagher H. Effects of age and sex on biochemical responses to drinking habits. Med J Aust 1978; 2: 629–32.

16. Schmidt E. Normale γ-Glutamyltranspeptidase bei Hepatitis? Dtsch Med Wschr 1974; 99: 1696.

17. Berg PA, Klein R. Diagnose der primär biliären Zirrhose. Dtsch Med Wschr 1988; 113: 143–5.

18. Schmidt E, Schmidt FW. Klinisch-chemische Untersuchungsmethoden. In: Schmidt E, Schmidt FW, Chemnitz G, eds. Klinik der Gegenwart. München; Urban und Schwarzenberg 1984: E 381–421.

19. Saadeh S. The spectrum of nonalcoholic fatty liver disease: from steatosis to nonalcoholic steatohepatitis. Clev Clin J Med 2000; 67: 96–104.

20. Gastaldelli A, Kozakowa M, Hojlund K, Flyvberg A, Favuzzi A, Mitrakou A, et al. Fatty liver is associated with insulin resistance, risk of coronary heart disease, and early atherosclerosis in a large European population. Hepatology 2009; 49: 1357–44.

21. Loomba R, Sirlin CB, Schwimmer JB, Lavine JE. Advances in pedriatric nonalcoholic fatty liver disease. Hepatology 2009; 50: 1282–93.

22. Börsch G, Baier J, Glocke M, Nathusius W, Gerhardt W. Graphical analysis of laboratory data in the differential diagnosis of cholestasis: a computer-assisted prospective study. J Clin Chem Clin Biochem 1988; 26: 509–19.

23. Hunt CM, Sharara AI. Liver disease in pregnancy. Amer Fam Physic 1999; 59: 829–36.

24. Lammert F, Marschall HU, Glantz A, Matern S. Intrahepatic cholestasis in pregnancy: molecular pathogenesis, diagnosis and management. J Hepatol 2000; 33: 1012–21.

25. Kelly DA. Chronic hepatitis. In: Kelly DA, ed. Disease of the liver and biliary system of children. Oxford; Blackwell 1999: 97–123.

26. Jacquemin E. Progressive familial intrahepatic cholestasis and anomalies of hepatocellular metabolism of bile acids. Arch Pediatr 1998; 5: 59–61.

27. Penn R, Worthington DJ. Is serum γ-glutamyltransferase a misleading test? Brit Med J 1983; 286: 531–5.

28. Külling D, Sauter Chr. Die Bedeutung der γ-Glutamyltransferase zur Erfolgsbeurteilung einer Chemotherapie von Lebermetastasen. Schweiz Med Wschr 1990; 120: 1435–8.

29. Lewis JH. Drug-induced liver disease. Med Clin North Am 2000; 84: 1275–1311.

30. Farrell GF. Drugs and steatohepatitis. Sem Liver Dis 2002; 22: 185–94.

31. Chitturi S, George J. Hepatotoxicity of commonly used drugs: nonsteroidal antiinflammatory drugs, antihypertensives, antidiabetic agents, anticonvulsants, lipid-lowering agents, psychotropic drugs. Sem Liver Dis 2002; 22: 169–83.

32. Stedman C. Herbal hepatotoxicity. Sem Liver Dis 2002; 22: 195–206.

33. Strohm WD. Toxische Leberschädigung und Umwelt. Krankenhausarzt 1990; 63: 411–8.

34. Sillanaukee P, Olsson U. Improved diagnostic classification of alcohol abusers by combining carbohydrate-deficient transferrin and gamma glutamyltransferase. Clin Chem 2001; 47: 681–5.

35. Naveau S, Poynard T, Zourabichvili O, Hilpert G, Naveau S, Poitrine A, et al. Prognostic value of total serum bilirubin/gamma-glutamyltranspeptidase ratio in cirrhotic patients. Hepatology 1984; 4: 324–7.

36. Meeham JJ, Geogeson KE. Prevention of liver failure in parenteral nutrition-dependent children with short bowel syndrome. J Pediatr Surg 1997; 32: 473–5.

37. Sandock DS, Seftel AD, Resnick MI. The role of gamma-glutamyl transpeptidase in the preoperative metastatic evaluation of renal cell carcinoma. J Urol 1997; 157: 798–9.

38. Sealy CH, Vonbank A, Rein P, Woess M, Beer S, Aczel S, et al. Alanine aminotransferase and gamma-glutamyl transferase are associated with the metabolic syndrome but not with angiographically determined coronary atherosclerosis. Clin Chim Acta 2008; 397: 82–6.

39. Breitling LP, Raum E, Müller H, Rothenbacher D, Brenner H. Synergism between smoking and alcohol consumption with respect to serum gamma-glutamyltransferase. Hepatology 2009; 49: 802–8.

40. Meisinger C, Döring A, Schneider A, Löwel H. Serum γ-glutamyltransferase is a predictor of incident coronary events in apparently healthy men from general population. Atherosclerosis 2006; 189: 297–302.

41. Ruttmann E, Brant LJ, Concin H, Diem G, Rapp K, Ulmer H, et al. γ-Glutamyltransferase as a risk factor for cardiovascular disease mortality. Circulation 2005; 112: 2130–7.

42. Claessen H, Brenner H, Drath C, Arndt V. Gamma-Glutamyltransferase and disability pension: a cohort study of construction workers in Germany. Hepatology 2010; 51: 482–90.

43. Kazemi-Shirazi L, Endler G, Winkler S, Schickbauer T, Wagner O, Marsik C. Gamma glutamyltransferase and long-term survival: is it just the liver? Clin Chem 2007: 53: 940–6.

44. Claessen H, Brenner H, Frath C, Arndt V. Gamma-glutamyltransferase and disability pension: a cohort study of construction workers in Germany. Hepatology 2010; 51: 482–90

45. Stromme HJ, Theodorsen L. Heparin interference in the measurement of γ-GT with the Scandinavian and the IFCC recommended method. Scand J Clin Lab Invest 1985; 45: 437–42.

46. van der Meulen EA, van Sittert NJ, Koningh AGJ, Lugtenburg D, van Strick R. General approach to correction for bias in analytical performance in longitudinal studies illustrated by estimating the effect of age on γ-glutamyltransferase activity. Clin Chem 1993; 39: 1375–81.

47. Müller-Wiegand B. Bestimmung der γ-GT mit einem gut löslichen Substrat. Lab Med 1984; 8: 158–61.

48. Schiele F, Vincent-Viry M, Fournier B, Starck M, Siest G. Biological effects of eleven combined oral contraceptives on serum triglycerides, gamma glutamyltransferase, alkaline phosphatase, bilirubin and other biochemical variables. Clin Chem Lab Med 1998; 36: 871–8.

49. Heins M, Heil W, Withold W. Storage of serum or whole blood samples? Effects of time and temperature on 22 serum analytes. Eur J Clin Chem Clin Biochem 1995; 33: 231–8.

50. Lieberman MW, Barrios R, Carter BZ, Habib GM, et al. γ-glutamyl transpeptidase. What does the organization and expression of a multipromoter gene tell us about its functions? Am J Pathol 1995; 147: 1175–85.

51. Pompella A, Emdin M, Passino C, Paolicchi A. The significance of serum γ-glutamyltransferase in cardiovascular disease. Clin Chem Lab Med 2004; 42: 1085–91.

52. Wenham PR, Horn DB, Smith AF. Multiple forms of γ-GT: a clinical study. Clin Chem 1985; 31: 569–73.

53. Grostad M, Huseby NE. Clearance of different multiple forms of human γ-glutamyltransferase. Clin Chem 1990; 36: 1654–6.

54. Köttgen E, Reutter W, Gerok W. Two different γ-GT during development of liver and small intestine: a fetal (sialo-) and an adult (asialo) glycoprotein. Biochem Biophys Res Comm 1976; 72: 61–4.

55. Tsuchida S, Hoshino K, Sato T, et al. Purification of γ-GT from rat hepatomas and hyperplastic hepatic nodules, and comparison with the enzyme from rat kidney. Cancer Res 1979; 39: 4200–4.

1.10 Glutamate dehydrogenase (GLD)

Lothar Thomas

The GLD is an enzyme of the mitochondrial matrix and present in all tissues. Its activity in the liver is 10-fold higher than in other tissues; therefore, elevated activities in serum are exclusively caused by the liver. GLD has a mostly catabolic role in humans. It catalyzes the clearance of nitrogen from the organism by releasing ammonia from glutamate. Elevated GLD levels are a marker for liver diseases with cell necrosis.

1.10.1 Indication

• Assessment of the severity (necrosis) and degree of acute liver parenchymal injury
• Differential diagnosis of liver diseases
• Biomarker of alcohol withdrawal.

1.10.2 Method of determination

Proposal of Standard Methods for the Determination of Enzyme Catalytic Concentrations in serum and plasma at 37 °C. Glutamate dehydrogenase /1/

Principle: the GLD (EC 1.4.1.3) catalyzes the oxidative deamination of L-glutamate. Under assay conditions the decrease of NADH per unit of time corresponds to the catalytic concentration of the enzyme (Tab. 1.10-1 – Principle of GLD determination).

1.10.3 Specimen

Serum, plasma (heparin anticoagulated blood, EDTA, oxalate, citrate): 1 mL

1.10.4 Reference interval

See Tab. 1.10-2 – Reference intervals of GLD.

1.10.5 Clinical significance

The GLD is a liver-specific enzyme. However, it is not suited for use in screening for hepatobiliary diseases because its diagnostic sensitivity is only 47% /2/.

The practical value of GLD determination lies in the differential diagnosis of liver diseases. This is because elevated GLD in serum indicates severe parenchymal cell damage, and the activity related to the more readily released cytosolic aminotransferases is a criterion for rating the severity of acute liver injury. Compared to the ALT, which only occurs in the cytoplasm, and the AST, which is located in both the cytoplasm and the mitochondria, the GLD is:

• Sparsely released in generalized inflammatory diseases of the liver, for example viral hepatitides.
• Increasingly released in liver diseases in which necrosis of the hepatocytes in the centrilobular zone is the predominant event, such as obstructive liver disease, in hypoxic hepatopathy or toxic liver injury due, for example, to alcohol.

The (ALT + AST)/GLD ratio is a differential diagnostic criterion. It is useful in the situations listed in Tab. 1.10-3 – Differential diagnosis value of the ratio (ALT + AST)/GLD.

1.10.5.1 Differential diagnosis of jaundice

The GLD is important in the differential diagnosis of jaundice, especially in acute abdomen with possible liver involvement in the form of cholecystitis or acute bile duct obstruction. The lower the (ALT + AST)/GLD ratio, the higher the probability of intrahepatic or extrahepatic mechanical cholestasis; the higher the ratio, the higher the probability of acute hepatitis. For example, extrahepatic bile duct obstruction is excluded by the following findings:

• Elevated ALT to more than 25-fold the upper reference limit (Fig. 1.10-1 – Differential diagnosis of strongly elevated ALT activities based on GLD)
• (ALT + AST)/GLD ratio above 50
• ALP level within the reference interval.

The differential diagnosis remains open if the ratio is below 50 and, in particular, below 20.

1.10.5.2 Differential diagnosis of severe liver injury /3, 4/

The GLD is useful in differentiating between toxic and hypoxic liver injury and severe forms of acute hepatitis. In acute impaired hepatic perfusion, the activities of GLD are similar to those of the aminotransferases.

In acute endogenous or exogenous intoxication, the peak levels of the GLD are only half as high as those of the aminotransferases and, in the severe form of acute viral hepatitis, less than 5% of the aminotransferase levels. This also applies to the necrotizing form of acute viral hepatitis that may lead to acute hepatic failure. According to a study /4/, acute right heart failure, protracted septic-toxic circulatory failure and severe respiratory insufficiency are the most common causes of GLD activities higher than 25-fold the upper reference limit. The highest mean activity was found in patients with cor pulmonale following pulmonary embolism. The differentiation between the toxic and hypoxic genesis and the hepatitic genesis is possible. Toxic substances and hypoxia damage the central region of the hepatic lobule where the GLD is perivenously located.

The differential diagnosis of elevated GLD is shown in Tab. 1.10-4 – GLD in hepatic and biliary tract diseases.

1.10.5.3 Indicator of alcohol addiction

The GLD levels in alcohol-addicted individuals are substantially higher than those in individuals not addicted to alcohol. In a study /5/, healthy women and men had levels of 0.3 U/L (20 μkatal/L) and 0.6 U/L (40 μkatal/L), respectively, compared to alcoholics with levels of 4.9 U/L (296 μkatal/L) and 7.3 U/L (439 μkatal/L), respectively. The GLD is better suited for monitoring abstinence during alcohol withdrawal therapy than other biomarkers. The GLD declines more quickly than the AST and GGT during the first 7 days of abstinence, and a pronounced decrease can already be measured after 24 hours without alcohol intake.

1.10.6 Comments and problems

Method of determination

The method optimized for 37 °C is more robust than the previous one for 25 °C. It can be measured without preincubation with a longer temporal linear response. There are no blank values, and uncontrolled NADH consumption is avoided /1/.

Inhibition: Lower concentrations are measured in the presence of sodium fluoride.

Stability

GLD decreases by 10% within 24 hours at room temperature (20 °C) and by about 5% within 3 days at 4 °C.

Half-life

14–18 hours.

1.10.7 Pathophysiology

In plants and microorganisms, ammonia is incorporated directly or is produced by the metabolism of substances containing N₂, NO₃^– and NO₂^–. Ammonia is incorporated into L-glutamate mainly through reductive amination of 2-oxoglutarate. The physiological function of GLD is the oxidative deamination of glutamate, i.e. the enzyme catalyzes the removal of hydrogen from L-glutamate to form the corresponding ketimino acid that goes spontaneous hydrolysis to 2-oxoglutarate (the reverse reaction is used for the determination of GLD activity in vitro).

Through the release of NH₃ from glutamate, GLD catalyzes the clearance of nitrogen not needed for the re-synthesis of amino acids from the organism. Reduced NAD and NADP are regenerated by the respiratory chain. Oxalacetate acting as an acceptor is catalyzed by the AST and transaminated to aspartate whose nitrogen is incorporated in the urea synthesis /6/.

The GLD is located in the mitochondrial matrix. The specific activity in the liver is about 10-fold higher than in the kidney, brain and lung and about 80-fold higher than in skeletal muscle. In the hepatic lobules, the GLD activity is about twice as high in the centrilobular zone as in the periportal zone. Therefore, in relation to acute hepatitis, toxic alcohol-induced damage or acute impaired perfusion lead to a pronounced increase in GLD compared to the aminotransferases. The increases in activity in plasma are exclusively caused by the liver.

The molecular weight of GLD in serum is 336 kDa. The GLD consists six subunits. However, polymers with a molecular weight of up to 1,000 kDa can also be found in patients with liver injury.

The GLD is significant in the differential diagnosis of liver diseases because its concentration in the centrilobular zone of the hepatic lobule is 1.8-fold higher than peripherally /7/. Being at the end of the sinusiodal supply route the centrilobular zone is at greatest risk from hypoxia and is the first area where cell damage occurs if blood flow is disturbed.

The increase in GLD in obstructive jaundice is attributed to obstruction of bile acid drainage. It is thought that this leads to damage, particularly of the mitochondria in the centrilobular region, as a result of the detergent effect of the bile acids /8/.

References

1. German Society for Clinical Chemistry. Proposal of standard methods for the determination of enzyme catalytic concentrations in serum and plasma at 37 °C. III. Glutamate dehydrogenase. Eur J Clin Chem Clin Biochem 1992; 30: 493–502.

2. Schmidt E, Schmidt FW. Enzyme diagnosis in diseases of the liver and the biliary system. In: Advances in clinical enzymology. Basel: Karger, 1979: 239.

3. Schmidt E, Schmidt FW. Diagnostik des Ikterus. Dtsch Med Wschr 1984; 109: 139–46.

4. Chemnitz G, Schmidt E, Schmidt FW, Lobers J. Diagnostische und prognostische Bedeutung massiv erhöhter Glutamat-Dehydrogenase-Aktivität im Serum. Dtsch Med Wschr 1984; 109: 1789–93.

5. Kravos M, Malesic I. Glutamate dehydrogenase as a marker of alcoholism. Alcohol&Alcoholism 2010; 45:39–45

6. Schmidt E, Schmidt FW. Enzymdiagnostik von Lebererkrankungen in der Praxis. Diagnostik 1977; 10: 348–52.

7. Schmidt E, Schmidt FW. Alkoholschäden. In: Schmidt E, Schmidt FW, Manns MP, eds. Lebererkrankungen. Stuttgart; Wissenschaftliche Verlagsgesellschaft 2000: 214–404.

8. Assel H, Fedderke J, Schmidt E, Voges S. Correlations and factor analysis of enzymes in serum of patients with hepatic metastases. In: Goldberg DM, Werner M, eds. Selected topics in clinical enzymology. Berlin: de Gruyter, 1983.

9. Lamerz P, Stieber P, Borlinghaus P, Fateh-Moghadam A. Tumor markers in cancer of the liver. Diagn Oncol 1991; 1: 363–72.

10. Kommerell B. Hepatomegalie. Diagnostik 1980; 13: 78–93.

11. Groß W, Ring K, Lodemann E, eds. Physiologische Chemie. Weinheim; VCH-Verlagsgesellschaft 1989: 214–23.

12. Guder W, Habicht GA, Kleißl J, Schmidt U, Wieland OH. The diagnostic significance of liver cell inhomogeneity: serum enzymes in patients with central liver necrosis and the distribution of GLDH in normal human liver. Z Klin Chem Klin Biochem 1975; 13: 311–8.

13. Schmidt FW. Enzymes in cholestasis. In: Bianci L, Gerok W, Sickinger K, eds. Liver and bile. Lancaster: MTP-Press, 1977: 216.

1.11 Lactate dehydrogenase (LD)

Lothar Thomas

The LD is an NAD⁺-oxidoreductase and catalyzes the oxidation of lactate to pyruvate using NAD⁺ as an H⁺ acceptor. The reaction is reversible and at a physiological pH the equilibrium favours the reduction of pyruvate to lactate. The total LD (EC 1.1.1.27) measurable in serum consists of the activities of the five isoenzymes LD-1 to LD-5 (Tab. 1.11-1 – Isoenzymes of LD). The isoenzyme LD-1 converts the substrate 2-oxobutyrate to hydroxy butyrate at a higher rate than the other isoenzymes and can be measured separately as hydroxy butyrate dehydrogenase.

The LD is present in varying amounts in the cytoplasm of all cells in the body. Therefore, elevated levels of total LD are found in many pathological conditions but are as such of limited diagnostic and differential diagnostic value due to lack of organ specificity. However, if the total LD is elevated, quantitative differentiation of the isoenzymes can provide diagnostically useful organ-related information.

1.11.1 Indication

• Differentiation of jaundice
• Assessment of the degree of hemolysis in hemolytic and megaloblastic anemia
• In a pattern with the aminotransferases in suspected hypoxic or toxic liver injury
• Differentiation of tissue damage through isoenzyme determination in elevated LD
• Monitoring of the disease activity in Hodgkin’s and non-Hodgkin’s lymphomas and leukemias
• Monitoring and therapy control in ovarian dysgerminoma and germ cell tumor of the testes
• Late diagnosis of myocardial infarction (more than 36–48 hours after the acute event).

1.11.2 Method of determination

IFCC Primary Reference Procedure for the Measurement of Catalytic Activity Concentration of Lactate Dehydrogenase at 37 °C /1/

Principle: The LD activity is measured at a pH of 9.4 as the amount of lactate consumed, by continuous monitoring the increase in absorbance due to the reduction of NAD⁺ at 339 (Hg 334 or Hg 365). The equilibrium is far on the side of lactate and NADH (Tab. 1.11-2 – Principles of LD determination).

Selective determination of LD-1

Chemical inhibition of isoenzymes containing the M subunit

Principle: 1,6-hexanediol or sodium perchlorate are added to the reaction medium as selective inhibitors of the LD isoenzymes with M subunits so that only the LD-1 that has four H subunits is measured /2/.

Immunological inhibition of isoenzymes containing the M subunit

Principle: Antibodies against the M subunit are added to the sample and form immune complexes with isoenzymes possessing this subunit. The immune complexes are removed by centrifugation and LD-1 is determined in the supernatant /3/.

Differentiation of isoenzymes by electrophoresis

Principle: the serum is fractionated by electrophoresis on agarose gel or on cellulose acetate strips at an alkaline pH. The rate of migration to the anode depends on the subunit composition of the isoenzyme. Isoenzymes with the subunit H migrate quickly, those containing the subunit M migrate slowly; thus, LD-1 has the highest migration rate toward the anode and LD-5 the lowest. In agarose gel electrophoresis LD-5 migrates toward the cathode.

The isoenzyme fractions are visualized in cellulose acetate electrophoresis by coupling the enzymatically formed pyruvate with a tetrazolium salt /4/.

In agarose gel electrophoresis, the separation lane is covered with an overlay containing lactate and NAD⁺ and, after incubation at 37 °C, the fluorescence of NADH is measured at 410 nm, under excitation at 365 nm /2/.

1.11.3 Specimen

Serum, plasma, effusion fluid: 1 mL

1.11.4 Reference interval

See Tab. 1.11-3 – LD reference intervals.

1.11.5 Clinical significance

The LD is a cytoplasmic enzyme and present in all tissues. Leakage into the plasma can occur even after minor tissue damage, and levels are elevated in many pathological conditions. Diseases associated with increases in LD activity are shown in Tab. 1.11-4 – Diseases that may cause elevated LD in serum. The LD is diagnostically as unspecific as the erythrocyte sedimentation rate. Hence, it is of limited significance and should mainly be used /8/:

• In cardiology for the late diagnosis of myocardial infarction. The diagnostic sensitivity 24 hours after the acute event is 95% at 90% specificity. The diagnostic value of the LD-1/LD-2 ratio is higher; the diagnostic efficiency is 93–98%. The determination of troponin has super ceded the LD and its isoenzymes in cardiology.
• In hepatology for differentiating severe toxic liver injury and acute impaired hepatic perfusion from acute viral hepatitis. At the onset of clinical symptoms in acute impaired hepatic perfusion or toxic disorder, for example due to acetaminophen, the LD is higher than the aminotransferases in many cases. This is not the case in viral hepatitis /9/. The LD is generally the least specific enzyme in hepatopathy diagnosis /10/.
• In the monitoring of oncologic diseases such as malignant lymphomas, leukemias and several solid tumors such as germ cell tumors.
• In the differential diagnosis of jaundice, especially for differentiating between the hemolytic and the hepatic forms.

Important diagnostic indicators for tissue-specific differentiation of elevated LD levels are:

• The calculation of the LD/AST ratio
• The quantitative determination of the LD isoenzymes.

LD/AST ratio

The ratio is used to differentiate between prehepatic, hemolysis-induced or dyserythropoiesis-induced jaundice from hepatic jaundice (Fig. 1.11-1 – Differentiation between prehepatic and hepatic jaundice). Ratios above 5 are indicative of hemolytic jaundice lower values point to the hepatic form. Ratios above 5 can also occur in metastatic liver disease and infectious mononucleosis. In prehepatic jaundice, except in severe hemolytic crises (sickle-cell anemia), the bilirubin concentration is below 6 mg/dL (100 μmol/L) /10/.

Quantitative analysis of LD isoenzymes

The LD molecule consists of four polypeptide chains of the two types M and H, both of which are under separate genetic control. The five isoenzymes listed in Tab. 1.11-1 – Isoenzymes of LD are distinguished by the basis of their subunit composition. The assessment by electrophoresis distinguishes three LD patterns (Tab. 1.11-5 – Differential diagnostic information of electrophoretic LD patterns) /4/:

• Anodic pattern; LD 1 + 2
• Cathodic pattern; LD 4 + 5
• Intermediate group; LD-3 predominates.

1.11.6 Comments and problems

Sample processing

Plasma should be centrifuged at high speed (10 min. at 3,000 × g) because otherwise it will still contain platelets; these have a high LD concentration.

Reference interval

If the blood sample is collected after physical exertion (muscle activity), the LD levels can be higher than the upper reference limit. Capillary serum and plasma have higher LD levels. The levels in serum are higher than those in plasma due to hemolysis during the coagulation process.

Hemolysis

Hemolysis causes increased LD levels because the LD concentration in erythrocytes is 360-fold higher than in plasma. At a mean activity of 165 U/L, hemolysis of 0.8 g Hb/L causes an increase in LD by 58% /38/. The serum must have separated from the clot within 2 hours.

Platelet interference

Serum and platelet-free plasma have the same LD activity. The interference caused by platelet contamination in the LD activity of a plasma sample depends on the reaction conditions. If the sample is pipetted into a relatively large volume of hypotonic reagent, lysis will result in elevated LD levels. If the sample is added to an isotonic reagent, lysis will not occur, but optical interference will result. Falsely low LD levels are measured because the NADH-related absorbance during the LD reaction is masked by an increase in the absorbance caused by light absorption by the platelets /39/.

A stronger platelet contamination of heparin anticoagulated blood due to inadequate centrifugation only results in slightly increased imprecision in the IFCC method /40/.

Stability

LD in serum is stable for up to 7 days at room temperature (20 °C) /2/. For routine analysis, the serum should be stored at room temperature due to the instability of LD-4 and LD-5 under cold conditions.

Drugs

In vivo-elevations of the LD levels can be caused by allopurinol, amiodarone, androgenic/anabolic steroids, aspirin/salicylates, captopril, carbamazepine, chlorpromazine, cisplatin, clozapine, cumarin, dacarbazine, diltiazem, erythromycin, fluphenazine, gold salts, α-methyldopa, naproxen, paracetamol, papaverine, penicillamine, perhexillin, phenytoin, phenylbutazone, propylthiouracil, ranitidine, sulfasalazine, tienilic acid, valproate acid, verapamil /41/.

Half-life

LD-1, 4–5 days, LD-5, 10 hours.

Macro LD

For further information, see Tab. 1.1-2 – Macroenzymes: characterization, clinical significance and laboratory findings.

1.11.7 Pathophysiology

LD is a hydrogen-transferring enzyme that catalyzes the oxidation of L-lactate to pyruvate using the coenzyme NAD⁺ as a hydrogen acceptor. The reaction is reversible. The reduction of pyruvate to lactate is strongly promoted under physiological pH conditions.

Each LD molecule consists of 4 subunits with a molecular weight of 34 kDa. There are two types of subunits, the heart (H) type and the muscle (M) type, that are encoded by different gene loci. The H and M types are combined in the tissues to form five isoenzymes (LD-1–5). The H type is predominant in tissues with high O₂ consumption, while the M type is predominant in tissues with high glycolytic activity.

The LD is present in all somatic cells. It is dissolved in the cytoplasm and released in the event of cell damage. The enzyme activity differs in the individual tissues and is 147 U/g in skeletal muscle, 124 U/g in myocardium, 145 U/g in the liver and 106 U/g in the kidney. Erythrocytes contain 31 U/g of hemoglobin. The activity in the tissues is on average 500-fold higher than in serum, and even minor tissue injury can cause elevated LD levels. In addition, many tissues have a different isoenzyme inventory. LD-1 and LD-2 are predominant in the myocardium and erythrocytes, and LD-5 is predominant in the liver. LD-3 and LD-4 are present in the lung, lymphatic system, spleen, endocrine glands and platelets.

In diseases, the LD activity is dependent on the isoenzymes entering the plasma from the tissues, the elimination rate of the isoenzymes and their subunits. The half-life of the liver-specific LD-5 (M4) is 8–12 hours which is only about 1/10 of that of the heart-specific and erythrocyte-specific LD-1 (H4). Therefore, in many cases, a liver-specific LD enzyme pattern is only measured for a short time, while an enzyme pattern due to myocardial injury or hemolysis is measured for a longer time /11/.

The LD detectable in serum in progressive Duchenne muscular dystrophy mainly corresponds to the isoenzymes LD-1–3 and not to the LD-4 of the healthy skeletal muscle. Since this abnormality is also found in the carriers, these are thought to be genetically unable to form sufficient amounts of the M subunit /19/.

Concomitant hepatitis in infectious mononucleosis (IM) can be distinguished from the other viral hepatitides by the high activity of the LD in relation to the aminotransferases. It is not the hepatic isoenzyme LD-5 that is pathologically elevated, but LD-3 and LD-4 that originate from lymphoid cells.

Elevated LD levels in megaloblastic anemia are caused by ineffective erythropoiesis because red cell precursors do not mature in the medulla due to vitamin B₁₂ or folic acid deficiency and are subject to apoptosis.

References

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12. Werner M, Brooks SH, Mohrbacher RJ, Wasserman AG. Diagnostic performance of enzymes in the determination of myocardial infarction. Clin Chem 1982; 28:1297–302.

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14. Carstens V, Büscher J, Niehues B, Behrenbeck DW. Die intravasale Hämolyse bei neueren Herzklappenprothesen. Herz-Kreislauf 1981; 6: 261–5.

15. Meissner E, Fabel H. Akute Lungenembolie. Arzneimitteltherapie 1990; 8: 177–92.

16. Schmidt E, Schmidt FW. Enzyme diagnosis of diseases of the liver and the biliary system. In: Schmidt E, Schmidt FW, Trautschold I, Friedel R, eds. Advances in clinical enzymology. Basel; Karger 1979: 239–92.

17. Rotenberg DH, Weinberger I, Davidson E, et al. Total lactate dehydrogenase and its isoenzymes in serum of patients with infectious mononucleosis. Clin Chem 1991; 37: 116–7.

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19. Laudahn G, Heyck H, Feustel F. Enzyme im Serum bei Muskelkrankheiten. In: Praktische Enzymologie. Bern: Huber, 1968: 249.

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1.12 Lipase

Klaus Lorentz

Lipase measured in serum is synthesized in the acinar cells of the pancreas and stored in their granules. More than 99% is secreted into the pancreatic duct system via the apical pole of the cells. In acute pancreatitis, the enzyme is increasingly released into the blood due to increased permeability at the basal cell pole of lipase secreting cells.

1.12.1 Indication

• Evidence and exclusion of acute pancreatitis (in acute epigastralgia)
• Evidence of chronic pancreatitis (in recurrence)
• Exclusion of pancreas involvement in abdominal disease and surgical intervention
• Monitoring after endoscopic retrograde choledochopancreatography.

1.12.2 Method of determination

The hydrolysis of triglycerides, diglycerides and monoglycerides is the basic reaction of any determination. Lipase acts only when the substrate is in an emulsified form at the interface area between water and the substrate. For full catalytic activity, bile salts and a cofactor are required. The cofactor, called colipase, is a protein, forming a complex producing a conformational change for the enzyme, such that the enzyme more efficiently can bind to the substrate /1/.

Only the following two methods are on the market for application to automated analyzers:

• the enzymatic reaction rate diglyceride assay
• the 1,2-O-dilaurylrac-glycero-3-glutaric acid (4-methyl-resorufin) ester based assay.

In the titrimetric method, the release of fatty acid at the C atom 1 or 3 of glycerol is measured. Multistage photometric methods are based on the determination of glycerol following the enzymatic cleavage of remaining acyl residues. A single-step photometric procedure uses a triglyceride analog as a substrate /1/. The analytical specificity of all methods is limited because substrates that are soluble even in the presence of bile acids or detergent-dissolved substrates allow degradation by esterases.

Auto titration /2/: this method has the highest analytical specificity. During enzymatic hydrolysis, oleic acid released from a triolein or olive oil emulsion is continuously titrated with alkaline solution to pH 9.0 (pH-stat). Since a proton is neutralized with each cleaved ester bond, this method allows direct measurement, but is not suited for routine analysis due to its technical requirements. It is well standardized; all photometric methods refer to auto titration /2, 3/.

Turbidimetric assay /4/: the conditions are similar to those of auto titration; however, the increase in lipase after addition of heparin indicates a co-reaction of lipoprotein lipase. Measurement is performed by photometry near 550 nm referring to lipase standards calibrated by titrimetry. The start and duration of the measurement intervals are selected by analyzers such that temporal linear reaction rates are to be assumed and measuring ranges of 5–10-fold the upper reference limit are reached. There is the analytical shortcoming of positive interference by esterases, although all methods use esterases in their assay medium.

DGMRE method /1/: this is a single-step procedure that uses the substrate 1,2-dilauryl-rac-glycero-3-glutaric acid-(6-methylresorufin)-ester (DGMRE) as triglyceride analog. Two esters in this triglyceride analog are replaced by hydrolysis-resistant ether. Therefore, lipase cleaves an acromatic acyl residue only at C atom 1. The residue spontaneously decomposes into glutaric acid and red methylresorufin.

Diglyceride method /4/: in this color assay, lipase releases a 2-mono glyceride from a 1,2-diglyceride; the 2-mono glyceride is degraded to glycerol by mono glyceride lipase. After another three reactions (phosphorylation, oxidation, oxidative coupling), the glycerol is measured as benzochinonediimine dye. Besides lipoprotein lipase and cholesterol esterase, intestinal lipase and carboxyl esterase are also thought to react.

Multilayer film slide test (Vitros method) /5/: this method uses 1-oleoyl-2,3-diacetylglycerol as a substrate. Lipase cleaves it to oileic acid and 2,3-diacetylglycerol, from which glycerol is released through diacetinase and, after the reaction chain described above, measured as dye. Since dodecylbenzene sulfonate replaces bile acid as solubilizer, there is glycerol interference /6/ as well as interference from all enzymes mentioned /7/ so that the method is preferably applied for measuring gastrointestinal lipases /8/.

1.12.3 Specimen

• Serum, heparin anticoagulated blood: 1 mL
• Pleural effusion, ascites, drainage secretion, peritoneal irrigation fluid: 1 mL

1.12.4 Reference interval

Refer to Tab. 1.12-1 – Lipase reference intervals.

1.12.5 Clinical significance

The diagnostic assessment of the lipase activity in serum is dependent on the selected method and the clinical requirement. All methods are qualified for the monitoring of acute or chronic recurrent pancreatitis without involvement of other abdominal organs; only assays employing a triglyceride emulsion are suited for excluding these diseases.

The following rules should generally be adhered to when serum lipase activity is assessed:

• The lipase is more sensitive for detecting pancreatitis than the α-amylase. The increase of both enzymes proceeds synchronously, lipase leaves the reference interval not earlier, but higher and for longer (see Section 1.4 – α-amylase).
• The lower the cut-off level for pathological values, the more frequently extrahepatic diseases are diagnosed in the presence of elevated lipase activity.
• If the upper reference limit is defined as the decision limit, all methods have a diagnostic sensitivity of 90–100% at 60–97% specificity in acute pancreatitis or exacerbation of chronic inflammation. Refer to:
• Tab. 1.12-1 – Lipase reference intervals.
• Tab. 1.12-2 – Diagnostic sensitivity and specificity of lipase in acute pancreatitis independent on the thresholds.
• Under routine conditions, none of the methods described under "Method of determination” reaches the biochemical selectivity and analytical quality of the non-salivary isoamylase.

In contrast to the α-amylase, occasional subnormal lipase levels can be measured with the immunological assay /2/ in excretory pancreatic insufficiency /15/. In the course of pancreatic inflammations and following endoscopic retrograde choledochopancreatography (ERCP), lipase behaves like α-amylase and its non-salivary fraction. The original assumption of different lipase/α-amylase ratios in alcohol-induced and bile-induced pancreatitides did not stand critical verification. The detection of isolipases with the multi layer film slide test also did not find its way into clinical diagnostics because, compared to isoamylases, the significance of these (possible) isoenzymes is largely unknown.

Hyperlipasemias cause diagnostic problems in chronic inflammatory bowel diseases. Elevated α-amylase or lipase levels do not necessarily indicate pancreas involvement or pancreas injury by azathioprine and sulfasalazine /16/.

Hyperenzymemia is found in 14–21% of Crohn’s disease and ulcerative colitis, where lipase leaves the reference interval more often than α-amylase even in the absence of pancreatitis /17/.

Pancreatic carcinomas only cause hyperenzymemia (by occlusion of the pancreatic duct) in rare cases, while normal α-amylase but significantly elevated lipase levels are observed in undifferentiated malignoma and hepatocellular carcinomas and adenocarcinomas without autoptical evidence of pancreas involvement /18/. Lipase levels up to 20-fold the upper reference limit immunologically measured in cases of diabetic ketoacidosis are positively pancreatogenic. They occur in 50% of the patients with an increase in trypsin and α-amylase, but without clinical symptoms of pancreatitis /19/. The diseases associated with increased activity of lipase are shown in Tab. 1.12-3 – Causes of elevated lipase in serum.

1.12.6 Comments and problems

Anticoagulants

Due to complexing of Ca²⁺, the sample must not contain EDTA, oxalate, fluoride or citrate.

Method of determination

Except in the multi layer film slide test, sera with triglyceridemia above 870 mg/dL (10 mmol/L) should be diluted. Hemoglobin above 5 g/L and bilirubin above 47 mg/dL (800 μmol/L) decrease the measured values. Drugs in normal doses have no influence.

Using the assay with 1,2-diglyceride on random access analyzers carry-over of esterase from the cholesterol reagent into the reaction mixture has to be avoided. Glycerol interferes with the multi layer film slide test.

Comparable diagnostic sensitivity are measured using the enzymatic reaction rate diglyceride assay and the 1,2-O-dilaurylrac-glycero-3-glutaric acid (4-methyl-resorufin) ester based assay.

Macrolipasemia

Refer to Tab. 1.1-2 – Macroenzymes.

Stability

In serum at least 1 week at 4 °C or 25 °C or 1 year at –28 °C.

1.12.7 Pathophysiology

Human pancreatic lipase (triacylglycerol acyl hydrolase, EC 3.1.1.3) is a monomeric glycoprotein of 449 amino acids in two domains with a molecular weight of 47 kDa. Typically, it only hydrolyzes insoluble triglyceride esters of long-chain fatty acids at the water-substrate interface at pH 8.8–9.2 if the substrate includes bile acids in micellar complexes. Colipase (molecular weight 9.9 kDa) activates the catalytic center by opening an overlying lid of 12 amino acids, anchors it to the hydrophobic boundary and protects the enzyme against inactivation by bile acids.

Lipase is produced in the acinar cells of the pancreas and more than 99% is released to the duct system of the gland via the apical cell pole. Less than 1% enters the lymph and blood capillaries at the basal pole (exogenous/endogenous partition) resulting in a concentration gradient of 1 : 500 to 1 : 800 between serum and duodenal secretion.

In acute pancreatitis, the basal cell pole shows abnormal permeability associated with increased entry of the enzyme into the circulatory system. In addition, cell necrosis occurs in the hemorrhagic form. If normal drainage is impaired, for example by scar strictures in chronic inflammation, sialolithiasis, obstruction due to papillary tumor or papilledema, the secretion pressure causes isthmic dehiscences and drainage into pericapillary spaces.

In chronic pancreatitis, this process is associated with further parenchymal atrophy that is not detected by decreased lipase activity in serum because of the small endogenous enzyme fraction. On the contrary, the activity in serum rises on recurrence, as in acute inflammation of chronic pancreatitis, associated with pain due to impaired drainage of the congested organ. Therefore, functional insufficiency cannot be detected by lipase determination in serum, not even after secretin-pancreozymin stimulation, but only by measuring the intraduodenal effect of lipase.

The lipase level rises with increasing age. The activity measured in the serum of the umbilical cord is only 12% of that present between 3–50 years of age and increasing to 112% until 70 years of age /2/. The enzyme has a half-life of 6.9–13.7 hours. It undergoes glomerular filtration with a clearance of 6 mL/min, complete tubular re-absorption and is degraded so that lipase is detectable in urine only in pronounced proteinuria.

References

1. Pasqualetti S, Borrillo F, Rovegno L. Panteghini M. Pancreatic lipase: why laboratory community does not take enough care of this clinically important test? Clin Chem Lab Med 2021; 59 (12): 1914–20.

2. Tietz NW, Astles JR, Shuey DF. Lipase activity measured in serum by a continuous-monitoring pH-stat technique – an update. Clin Chem 1989; 35: 1688–93.

3. Ziegenhorn J, Neumann U, Knitsch KW, Zwez W. Determination of serum lipase. Clin Chem 1979: 25: 1067.

4. Imamura S, Misaki H. An enzymatic method using 1,2-diglyceride for pancreatic lipase test in serum. Clin Chem 1989; 35: 1126.

5. Mauck JC, Weaver MS, Stanton C. Development of a Kodak Ektachem clinical chemistry slide for serum lipase. Clin Chem 1984; 30: 1058–9.

6. Bilodeau L, Grotte DA, Preese LM, Apple FS. Glycerol interference in the serum lipase assay falsely indicates pancreas injury. Gastroenterology 1992; 103: 1066–7.

7. Demanet C, Goedhuys W. Haentjens M, Huyghens L, Blaton V, Gorus F. Two automated fully enzymatic assays for lipase activity in serum compared: positive interference from post-heparin lipase activity. Clin Chem 1992; 38: 288–92.

8. Kazmierczak SC, van Lente F. Effect of gastric lipase on turbidimetric and dry-film methods for measuring pancreatic lipase. Clin Chem 1992; 38: 2555–6.

9. Amodeo B, Schindler A, Schacht U, Wahl HG. Calculation of indirect reference intervals of plasma lipase activity of adults from existing laboratory data based on the reference limit estimator integrated in the OPUS: L information system. J Lab Med 2021; 45, 2: 131–4.

10. Fossati P, Ponti M, Paris P, Berti G, Tarenghi G. Kinetic colorimetric assay of lipase in serum. Clin Chem 1992; 38: 211–5.

11. Panteghini M, Pagani F, Bonora R. Clinical and analytical evaluation of a continuous enzymatic method for measuring pancreatic lipase activity. Clin Chem 1993; 39: 304–8.

12. Abicht K, Heiduk M, Körn S, Klein G. Lipase, p-amylase, CRP-hs, and creatinine: Reference intervals from infancy to childhood. Abstract European Congress of Clinical Chemistry and Laboratory Medicine. Barcelona 2003.

13. Tietz NW, Shuey DF. Lipase in serum – an elusive enzyme: an overview. Clin Chem 1993; 39: 746–56.

14. Soldin SJ, Brugnara C, Wong EC. Pediatric reference ranges. Washington DC; AACC Press 2003: 135.

15. Weiß T, Lorentz K. Vergleichende Lipasebestimmung mit turbidimetrischer Technik und Enzymimmunoassay. Lab Med 1984; 8: 63–7.

16. Katz S, Bank S, Greenberg RE, Lendvai S, Lesser M, Napolitano B. Hyperamylasemia in inflammatory bowel disease. J Clin Gastroenterol 1988; 10: 627–30.

17. Bokemeyer B. Asymptomatic elevation of serum lipase and amylase in conjunction with Crohn’s disease and ulcerative colitis. Z Gastroenterol 2002; 40: 5–10.

18. Møller-Petersen J, Andersen PT, Hjorne N, Ditzel J. Hyperamylasemia, specific pancreatic enzymes, and hypoxanthine during recovery from diabetic ketoacidosis. Clin Chem 1985; 31: 2001–4.

19. Müller-Hansen J, Müller-Plathe O, Pröpper H. Untersuchungen zur diagnostischen Sensitivität von Lipase- und Amylase-Bestimmungen. Einfluss der ERCP auf die Aktivität von Serum-Lipase und Amylase. Ärztl Lab 1986; 32: 17–23.

1.13 Acid phosphatase (ACP)

Lothar Thomas

1.13.1 Indication

• Suspected bone tumor and/or metastases
• Gaucher’s disease.

1.13.2 Method of determination /1/

Principle: Hydrolysis of 4-nitro phenyl phosphate by acid phosphatase at pH 4.9 liberates 4-nitro phenol. The reaction is stopped by raising the pH to 11 by addition of NaOH. At this pH the strongly-colored quinonoid 4-nitro phenolate ion is produced and its absorbance measured at 405 nm.

In the method modified by Hillmann enzymatic hydrolysis of 1-naphthyl phosphate at pH 5.6 liberates 1-naphthol. At this pH, 1-naphthol combines rapidly with the stabilized diazonium salt, Fast Red TR, present in the incubation mixture to form a red dye. The appearance of the dye is monitored continuously at 410 nm.

1.13.3 Specimen

Serum, plasma (no heparin and oxalate): 1 mL

1.13.4 Reference interval

Refer to Tab. 1.13-1 – Reference interval of acid phosphatase.

1.13.5 Clinical significance

The term acid phosphatase summarizes all phosphatases with their maximum enzymatic activity at a pH below 7.0. Accordingly, the ACP (EC 3.1.3.2) detectable in serum is a mixture of numerous enzymes predominantly coming from thrombocytes, erythrocytes, bones, cells of the reticuloendothelial system and the prostate. Activities from the prostate and thrombocytes, in particular, are tartrate-inhibitable. Elevated ACP levels can be indicators of prostate carcinoma, diseases of the skeletal system and the reticuloendothelial system (Tab. 1.13-2 – Diseases possibly associated with elevated ACP in serum).

References

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2. Andersch MA, Srzcypinski AJ. Use of p-nitrophenylphosphate as the substrate in the determination of serum acid phosphatase. J Clin Pathol 1947; 17: 571–4.

3. Kraus E, Sitzmann FC. Die saure Phosphatase im Serum bei Kindern. Pädiatr Prax 1973; 12: 321–6.

4. Seiler D, Nagel D, Tritschler W, Looser S. Saure Phosphatase im Serum (Substrat: α-Naphthylphosphat): Referenzwerte und diagnostische Aussage. J Clin Chem Clin Biochem 1983; 21: 519–25.

5. Pearson JC, Dombrovskis S, Dreyer J, et al. Radioimmunoassay of serum prostatic acid phosphatase after prostatic massage. Urology 1983; 21: 37–40.

6. Van Lente F. Alkaline and acid phosphatase determinations in bone disease. Orthoped Clin North Am 1979; 10: 437–50.

7. Mercer DW, Peters SP, Glew RH, Lee RE, Wenger DM. Acid phosphatase isoenzyme in Gaucher’s disease. Clin Chem 1977; 23: 631–5.