Trace elements


Trace elements


Trace elements


Trace elements

10.1 Laboratory investigation of trace elements

Lothar Thomas

10.1.1 Essential trace elements

Trace elements are defined as inorganic substances accounting for less than 0.01% of the dry weight of the human body (Fig. 10.1-1 – Minerals and trace elements in the periodic table). Trace elements are categorized into three groups /1/:

  • Essential trace elements. An element is considered essential to the organism when reduction of its exposure below a certain limit results consistently in a reduction in a physiologically important function, or when the element is an integral part of an organic structure performing a vital function in the organism /2/. Essential trace elements are chromium (Cr), cobalt (Co), copper (Cu), iron (Fe), manganese (Mn), molybdenum (Mo), selenium (Se), zinc (Zn) and iodine (I). The criteria for a trace element to fulfill the “essential” definition are shown in Tab. 10.1-1 – Criteria of an essential trace element and biochemical functions are shown in Tab. 10.1-2 – Biochemical functions of essential trace elements. The essential trace element iron and its metabolism are described in Chapter 7 – Iron metabolism.
  • Trace elements that are probably essential. This group comprises nickel (Ni), silicon (Si), tin (Sn), lead (Pb), vanadium (V) and fluorine (F). However, there are numerous reasons why fluorine, vanadium and silicon should be classified under essential trace elements.
  • Elements toxic to man. This group comprises: Aluminum (Al), silver (Ag), arsenic (As), gold (Au), barium (Ba), bismuth (Bi), cadmium (Cd), cesium (Cs), mercury (Hg), platinum (Pt), titanium (Ti) and thallium (Tl). However, since all elements are toxic depending on the dose, this group is better designated as trace elements of unknown physiological function.

10.1.2 Intake and health effects of trace elements

The daily requirement of trace elements is in the milligram or microgram range. Food is the essential source of trace element supply. Only a few trace elements are supplied by water or air. Good sources are meat and fish products; fruits and vegetables also ensure an adequate supply /12/. Gastrointestinal absorption provides high bio availability. Organometallic substances are better absorbed than the purely inorganic forms of a trace element, and absorption is better from animal than from plant products. As a rule, a balanced Central-European mixed diet guarantees adequate supply of essential trace elements /3/.

The absorption of trace elements is reduced in diseases like fibrocystic enteral disease, short-bowel syndrome, Crohn’s disease or impaired by intraluminal factors. The absorption of zinc, for example, is impaired by a high dietary phytate content or under high calcium supplementation. High zinc supplementation impairs the absorption of copper.

Uptake is controlled via the intestinal mucosa cell receptors. Magnesium and iron, for example, are taken up via the divalent metal ion transporter.

The trace elements are transported in the circulation bound to plasma proteins, such as albumin, or to specific proteins, for example copper to ceruloplasmin or form complexes with to amino acids and other small molecules. Consequently, dysproteinemia can cause a significant change in trace element concentration. In systemic inflammation, the formation of /12/:

  • Negative acute phase proteins such as transferrin and albumin is reduced and, therefore, the concentration of bound iron, chromium and zinc also decreases.
  • Positive acute phase proteins such as ceruloplasmin is enhanced and, therefore, the copper concentration also increases.

Most trace elements are ubiquitous in all tissues, but some are more concentrated in specific tissues than others, for example zinc in the eyes. The key locations of trace elements are the liver, muscles and kidneys; however, the trace element content in these tissues is not representative of the overall body trace element stores.

Trace elements are excreted in very different ways; some are preferably excreted with the urine, others with the stool and still others with sweat.

Imbalance in essential trace elements is caused by /12/:

  • Deficiency occurring in inflammatory bowel disease and chronic diarrhea and for dietary reasons. Dietary deficiency states are common in developing countries and occur less frequently in industrial countries and, if so, preferably in older individuals. In a Viennese study /4/, the essential trace elements were measured in a non-selected cohort of 1750 individuals. In all, 91.5% had a deficiency in one or several of the three essential trace elements zinc, selenium and molybdenum. Increased consumption is thought to be the cause of this phenomenon because the essential trace elements are dislodged from their activity and binding sites by competing toxic elements taken in from the environment.
  • Imbalance because the trace element enters a third compartment (pleural effusion, ascites) or enteral absorption is impaired by another trace element present in much greater amounts. High zinc supplementation, for example, impairs the absorption of copper.
  • Toxic concentrations. Trace elements can accumulate in severe renal insufficiency or, depending on the galenics, be taken up in increased amounts upon administration as a drug.

10.1.3 Indication

The indication for determination is not difficult where trace element deficiency or burden is suspected based on clinical symptoms.

Clinical symptoms and diseases with suspected:

Under supply of trace elements is generally suspected in the following diseases /5/:

  • Enteral absorption disorders (malabsorption syndrome, pancreatic insufficiency, bowel resection, ulcerative colitis, chronic diarrhea, active liver disease, alcohol abuse, vegetarian diet)
  • Chronic renal insufficiency
  • Pregnancy, lactation period, growth phase, re convalescence, high-performance sport
  • Parenteral nutrition
  • Uncontrolled supplementation of elements (e.g. due to interaction during resorption)
  • Impaired wound healing, immunodeficiency, recurrent dermatitis, insulin resistance.

Body burden is generally suspected under the following conditions /2/:

  • Chronic supplementation of trace elements at high dosage
  • Genetic diseases (Wilson’s disease, Menkes syndrome, Whipple’s disease, acrodermatitis enteropathica, phenylketonuria, maple syrup urine disease).

10.1.4 Method of determination

Examples of methods used in routine laboratories:

  • Spectrophotometry
  • Atomic emission spectrophotometry (AES) with electrothermal (ET-AES) or flame technology (F-AES)
  • Flame atomic emission spectrophotometry (F-AES)
  • Flame atomic emission spectrophotometry with inductively coupled plasma (ICP-AES)
  • Optical emission spectrophotometry with inductively coupled plasma (ICP-OES)
  • Voltammetry (VOLT).

Examples of methods used in research applications:

  • Neutron activation analysis (NAA)
  • Inductively coupled plasma-mass spectrometry (ICP-MS)
  • Differential pulse cathodic stripping voltammetry (DPCSV) or differential pulse anodic stripping voltammetry (DPASV)
  • Proton induced X-ray emission spectrometry (PIXE)
  • X-ray fluorescence analytic (RFA)
  • Wave length disperse X-ray fluorescence spectrometry.

10.1.5 Specimen

Serum, EDTA plasma, EDTA blood: 5 mL

Samples should be taken in colorless plastic tubes or containers. Sampling from the patient should be performed under fasting conditions in the morning; the use of a plastic blood collection tube is optimal.

24-hour urine sample; the entire volume to be handed in to the laboratory. The inner surface of the plastic sampling container should be cleaned with concentrated hydrochloric acid or nitric acid (50 mL in a 2–3 liter container) and the acid rinsed off with distilled water. The patient should urinate directly into the container.

10.1.6 Clinical significance

As a rule, the status of trace elements is assessed based on their concentration in serum, plasma and whole blood. The results should be evaluated in consideration of biological influence factors, such as /6/:

  • Hormonal influences of different nature during childhood, puberty, pregnancy, adulthood and old age
  • Inflammation and tissue damage that may lead to the redistribution of trace elements between plasma and tissue /7/
  • Renal failure or diabetes mellitus that may lead to concentration changes of trace elements between plasma and blood cells /8/.

Essential trace elements are involved in various metabolic pathways. More than 300 enzymes from all six categories of the classification of enzymes need zinc (Zn), copper (Cu) or selenium (Se).

  • Zinc is an essential element in mitochondrial enzymes and is involved in the synthesis of ATP, for oxidizing Fe2+ to Fe3+, and for the formatin of connective tissue
  • Selen is an essential component of the antioxidative system and has an important role in thyroid functions.

Intensified metabolism can cause short-term trace element changes in the blood and tissues. Rapid decrease in zinc and iron concentrations can occur in many cases following burns or surgery, whereas increased metabolism leads to high concentrations in tissue /10/.

Unlike clinical chemical biomarkers, the blood concentration of trace elements reflects the biochemical processes in the tissue only to a limited extent. This is because the concentration is homeostatically controlled over large parts and subject to numerous complex influences. Changes in blood concentration are usually not detected until the onset of initial deficiency symptoms. Trace element deficiency

In deficiency, numerous trace elements are increasingly absorbed and decreasingly excreted. Such a homeostasis regulation seems to be more pronounced for cationic trace elements like iron, copper, chromium and zinc than for anionic ones like fluorine, iodine, arsenic and selenium /11/.

Due to the numerous functions of trace elements in all aspects of metabolism, deficiency usually causes unspecific general symptoms, especially because inadequate intake or loss via the kidneys or intestines in many cases affect all trace elements. Specific clinical deficiency symptoms and disorders do not become evident until the trace element deficit is high (Tab. 10.1-3 – Symptoms indicating specific chronic trace element deficiency/1/. Definitions for trace element requirement are shown in Tab. 10.1-5 – Definitions for trace element requirement. Trace element toxicity

All trace elements can cause tissue injury if excessively accumulated in the body /3/. Because the balance of essential trace elements is kept constant by adaptation of the absorption and elimination rate, chronic or acute intoxication are rarely induced physiologically. Most causes are genetic or based on accidental, homicidal or suicidal ingestion or administration. The symptoms of chronic body burden such as nausea, vomiting, seizures and diarrhea are also vague; element-specific symptoms only occur in acute intoxication (Tab. 10.1-4 – Symptoms indicating a specific chronic trace element burden). Determination of important trace elements

Many essential trace elements are constituents of functional groups of key enzymes. Although the plasma level does not accurately reflect the body status of trace elements, their concentration is measured in many cases, mostly in combination with other indices such as enzyme activity or tissue content, to determine deficiency or toxicity, especially in the setting of congenital disorders.

Copper, zinc and selenium are important components of antioxidant enzymes, such as glutathione peroxidase, super oxide dis mutase and catalase. The three trace elements play an important role in the pathogenesis of malignant tumors and cardiovascular disease and are also involved in lipid per oxidation.

10.1.7 Comments and problems


Contamination or analyte loss during sampling, processing and storage can cause differences in trace element concentration of up to one decimal power. The lower the concentrations of a trace element in the specimen compared to its environmental concentration, the stronger the effect of contamination is on the result. There is a high contamination risk, for example, for Zn, Cr, Mn and Ni /12/.

Blood sampling

In order to avoid contamination during blood sampling, commercially available, trace element-free blood collection assemblies and safety tubes containing EDTA or lithium heparin should be used. If metal-free systems are unavailable, the use of serum is to be preferred for concentration determination because anticoagulants for plasma production can contain high amounts of trace elements. The blood collection assembly (monovettes, indwelling venous catheters or coated needles) is to be checked for contamination with trace elements /13/, steel needles cannot be used for blood collection. The first 5 mL of blood must under no circumstances be used for trace element analysis.



There are not differences in the concentration of essential trace elements between serum and plasma.


Urine specimens are unsuitable for diagnosing an under supply of essential trace elements because it is not possible to distinguish between existing under supply and reduced current intake if excretion is low /14/. For example, high urinary zinc excretion is also possible in the setting of zinc deficiency. Within the scope of occupational health checks, however, renal excretion can characterize heavy metal toxic burden because risk assessment does not distinguish between current and chronic exposure.

Hair and nails

The element concentration in hair and nail samples is informative to some extent in toxicological or forensic analyses, but unusable for determining the essential trace element supply status /15/. Factors influencing the assessment are the variability in hair structure and metabolism, external deposit of elements and contamination of the sample during processing.

Lacrimal fluid, cerebrospinal fluid, saliva, breast milk

These specimens are only to a limited degree suitable as biomarkers of the trace element status. Numerous biological influences on the element concentration of these specimens have been found besides analysis interference factors, and no reliable relation with the supply status has been determined.

Sample processing

Treat all equipment for the processing and storing of samples taken for determining trace elements with a high environmental concentration, such as containers, cups and pipette tips, with 1% nitric acid over night, then rinse it three times with trace element-free water and dry it contamination-free. It may be necessary in some cases to work under extremely complex clean room conditions. Besides dust and smoke in the environment, cosmetics and sweat entering the samples following contact with pipette tips are sources of sample or reagent contamination. Contamination-free handling and thorough internal quality assurance are important.

Method of determination

Neutron activation analysis (NAA) is the most sensitive and reliable method of element determination down to the sub-ppb level and, therefore, indispensable as a reference method. A high sample throughput is possible; moreover, NAA provides the widest measuring range of all procedures. However, the procedure is very time-consuming and costly as well as stationary.

Atomic emission spectrometry with its variants (ET-AES and F-AES) and various background correction techniques (deuterium lamp or Zeeman effect) are the leading methodologies for clinical routine determination. In most cases, these procedures allow easy sample processing:

  • Whole blood, plasma or urine can be used for measurement immediately following dilution without sample preparation.

Biological variation

According to a systematic review of within-subject and between-subject biological variation estimates of Zn, Cu and Se using inductively-coupled-plasma mass-spectrometry, should be encouraged /9/.

Interference factors

The analysis of trace elements can be affected by chemical, physical and spectral interferences /16/. The main problems are matrix effects and matrix differences between reference or calibration materials and patient specimens. Spectral interferences can be easily corrected by applying continuum source or Zeeman effect background correction (components of the atomic emission spectrometer). Another option for the compensation of impurity signals is to select an adequate calibration procedure /17/. Interferences from physical and chemical properties of the sample matrix can be minimized by adding tensides or modifiers.

10.1.8 Physiology

Trace elements are transported in the blood by metalloproteins that either function as transport proteins with different specificity for trace elements (e.g., albumin for Cu, Ni and Zn, and transferrin for Cr, Fe, Mn and Zn) or as control proteins with high-specificity. Metalloproteins regulate the binding and/or release of trace elements and in this way control trace element-dependent processes. Metallothionein, the best-known metalloprotein, mainly binds zinc besides small amounts of Cu and Cd and has a central position in the metabolism of various organs /18/. Another function of metalloproteins is to bind toxic metals and, thus, protect the body against their toxic effect.

Trace elements have the following biochemical functions:

  • Structure-stabilizing function: this includes the maintenance of the quaternary structure of enzymes and the integrity control of biomembranes and structures through mercaptide formation. Trace elements are coordinately bound in the metalloenzymes. Their release causes the enzymes to lose their catalytic function. Metalloenzymes have important functions in the protection of cells against free oxygen radicals (e.g., the copper/zinc-dependent or manganese-dependent super oxide dis mutase or the selenium-dependent glutathione peroxidase).
  • Catalytic function (Tab. 10.1-6 – Trace elements acting as catalysts). Trace elements play an important role in enzyme activation as coenzymes or prosthetic group. They reversibly bind to an apoprotein as cofactors and thereby create the structural prerequisites for the detection, binding and chemical reaction of the substrate. Hydrolases are examples of this mode of catalytic effect in the presence of Co, Cu, Fe, Mn, Se or Zn.
  • Regulatory effect. This effect refers to the release of insulin, sex and growth hormones and processes in the immune system, such as the triggering of cellular immunity, antibody reaction, phagocyte activity and activation of the complement system /19/.


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6. Kruse-Jarres JD, Rükgauer M. Fortschritte und Grenzen der Analytik von Spurenelementen. Mta 1997; 12: 707–14.

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8. Schmitt Y. Copper and zinc determination in plasma and corpuscular components of peripheral blood of patients with preterminal and terminal renal failure J Trace Elements Med Biol 1997; 11: 210–4.

9. Coskun A, Aarsand AK, Braga F, Carobene A, Diaz-Garzon, et al. On behalf of the European Federation of Clinical Chemistry and Laboratory Medicine Working Group on Biological Variation and Task group for The Biological Variation Database. Clin Chem Lab Med 2022; 60 (4): 479–82.

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10.2 Chromium (Cr)

Lothar Thomas

Chromium is a transition element and exists in various valence states. The most strongly oxidized valence state is hexavalent Cr, which is unstable and has a toxic effect on the body. It is this form of chromium that is known for toxic damage in occupational medicine. Trivalent Cr is the biologically active and stable form. It is a constituent part of food and also occurs in organic complexes, for example, with niacin. It is debatable whether Cr should be on the list of essential trace elements /1/.

10.2.1 Indication

Chromium deficiency is suspected:

  • In impaired glucose tolerance (e.g., poor adjustability of insulin-dependent diabetics)
  • During pregnancy and lactation
  • Under diets with a high proportion of fiber.

Chromium intoxication and/or burden is suspected:

  • At occupational exposure to hexavalent Cr
  • Following metallic hip prosthesis implantation
  • Under long-term total parenteral nutrition.

10.2.2 Method of determination

Refer to Section 10.1.4 – Method of determination.

10.2.3 Specimen

Blood sampling with metal-free blood collection assembly:

  • Tube with lithium heparinate for plasma: 2 mL
  • Nonadditive tube for serum: 2 mL
  • Whole blood (lithium heparinate) in suspected hexavalent Cr intoxication: 5 mL

24 h urine following glucose and/or insulin challenge. The entire urine sample is to be handed in to the laboratory.

10.2.4 Reference interval


< 10 nmol/L

< 0.5 μg/L*

Whole blood

10–75 nmol/L

0.5–3.7 μg/L


< 13 nmol/24 h

< 0.7 μg/24 h

* Detection limit; conversion: μg/L × 19.2 = nmol/L

Refer to references /234/ in addition.

10.2.5 Clinical significance

The presence of chromium in the organism is important because it coordinates the normal function of the body through proper metabolic transformations e.g., maintaining blood sugar levels, normalizes cholesterol concentration, and takes part in the metabolism of fats, proteins and carbohydrates. Chromium deficiency

It is only the determination of trivalent Cr that is significant for the diagnosis and therapy of Cr deficiency. According to the US-American National Academy of Sciences, there are no clinical indications of deficiency if the daily Cr content in food does not fall below 35 μg, corresponding to an amount of 0.5 μg/kg of body weight. Only 0.4–2.5% of this amount are enterally absorbed. Lower intake leads to an increased absorption rate, while higher intake reduces the absorption rate. The pathogenic effect of Cr and Co is described in Causes with a deficiency of chromium

Diabetes mellitus

Studies have shown that pathological glucose levels in patients with Cr deficiency improved by Cr substitution. The proposal of treating diabetics with a chromium nicotine (3-carboxypyridine) complex (chromium picolinate), 50% of which are enterally absorbed, was rejected by the US Food and Drug Administration for lack of reliable evidence.

Cr-containing traditional Chinese medicine Tianmai tablet (TMXKT) is approved for treating newly diagnosed type 2 diabetes. In a study /5/ TMXKT combined with conventional Western medicine was beneficial for improving fasting plasma glucose, 2 h plasma glucose, HbA1c, and the body mass index for newly diagnosed type 2 diabetes.


Women with several pregnancies and breast feeding women have an increased need for chromium.

Acute and chronically active infections

Cr deficiency in tissues can result from a Cr transport disorder. Cr is transported to the tissues bound to transferrin, which is a negative acute phase protein.

Metabolic stress

Glucose burden leads to increased renal excretion of Cr within the following 2 hours. Toxicity of chromium

The toxicity of Cr depends on its valence state. Tetravalent, pentavalent and especially hexavalent Cr are carcinogens, have a caustic effect and cause contact sensitivity. Primary target organs are the respiratory tract, lungs and kidneys. Hexavalent Cr in dust is associated with contact dermatitis and lung carcinoma. Orally ingested trivalent Cr has almost no toxic effect because it is poorly absorbed. The carcinogenic effect of hexavalent Cr on the gastrointestinal tract is low. According to a metaanalysis /6/ evaluating data from 1950 to 2009, the following mortality ratios compared to non-exposed individuals were calculated for cancer of: oral cavity 1.02, esophagus 1.17, stomach 1.09, colon 0.89 and rectum 1.17. The toxicity of Cr in total parenteral nutrition and metal-on-metal hip prostheses is described in Tab. 10.2-1 – Toxicity of chromium.

10.2.6 Comments and problems


The high environmental concentrations of Cr pose a significant risk of sample contamination during venipuncture procedure, 24 h urine collection, storage and sample processing.


If tightly sealed, samples can be stored at 4 °C for 2 weeks.

10.2.7 Pathophysiology

The daily intake of Cr recommended by the US-American Food Nutrition Board should be 15 μg/day in children up to 8 years, 25 μg/day in children up to 13 years, 35 μg/day in adolescents 14–18 years and 35 μg/day in adults. The body’s Cr content is 10–20 mg, primarily distributed over bones, liver and spleen. Cr is transported in the blood bound to transferrin and albumin and competes with iron for binding sites at transferrin. In acute phase response, transferrin synthesis is down regulated by the liver, resulting in low Cr and Fe in plasma. In hemochromatosis, excessive loading of transferrin with iron impedes Cr transport, a potential cause of impaired glucose tolerance in this disease.

Some patients with trivalent Cr deficiency develop diabetes-like symptoms with elevated levels of glucose, insulin, triglycerides and cholesterol that can be reversed by Cr substitution /7/. According to recent suggestions /8/, Cr has a stimulating effect at the insulin receptor. If the insulin receptor is activated by its ligand, Cr enters the cell and binds to a low-molecular peptide forming a complex. This complex increases the receptor activity, is released by the cell and inactivated when normal plasma glucose concentrations have been established.

Hexavalent Cr is a respiratory carcinogen causing a broad spectrum of DNA breaks, thus having both genotoxic and mutagenic effects. It remains in the lungs for an extended period of time and is still detectable, especially in the superior lobe, even if Cr exposure has ceased. Therefore, it is important that worker exposure to chromic acid in electroplating plants be reduced to a minimum /9/.

Hexavalent Cr enters the cell through non-specific anion channels and is reduced to lower valence states down to trivalent Cr by ascorbate, glutathione and cysteine. Trivalent Cr has a weak membrane permeability capacity and is trapped in the cell, where it forms DNA adducts causing DNA-strand breaks, deregulated DNA repair mechanisms, micro satellite instability, inflammatory responses and leading to the disruption of regulatory gene networks responsible for the balance of cell survival and cell death /10/.

Cr exerts an effect on bones by modulating their biochemical parameters. With considerable accumulation of Cr in the skeleton, the activity of alkaline phosphatase decreases, which affects the bone formation. Analysis of the content of Cr in certain parts of the knee joint found the highest levels in the femoral part of the knee joint and the lowest in the tibia /13/.


1. Vincent JB. Chromium: celebrating 50 years as an essential element? Dalton Transactions 2010;39: 3787–94.

2. Meissner D. Chrom. In: Biesalski HK, Köhrle J, Schümann K (eds). Vitamine, Spurenelemente und Mineralstoffe. Stuttgart; Thieme 2002: 235– 6.

3. Davies S, McLaren-Howard J, Hunnisett A, Howard M. Age-related decreases in chromium levels in 51,665 hair, sweat, and serum samples from 40,872 patients – implications for the prevention of cardiovascular disease and type II diabetes mellitus. Metabolism 1997; 46: 469–73.

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5. Gu Y, Xu X, Wang Z, Xu Y, Liu X, et al. Chromium-containing traditional Chines medicine, Tianmai Xiaoke tablet, for newly diagnosed type 2 diabetes mellitus: a meta-analysis and sensitive review of randomized clinical trials. Evidence-Based Comlementary and alternative Medicine 2018, article ID 3708637.

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7. Anderson RA, Polansky MM, Bryden NA, Canary JJ. Supplemental chromium effects on glucose, insulin, glucagon, and urinary chromium losses in subjects consuming controlled low-chromium diets. Am J Clin Nutr 1991; 54: 909–16.

8. Vincent JB. Recent advances in the nutritional biochemistry of trivalent chromium. Proc Nutr Soc 2004; 63: 41–7.

9. Wiwanitkit V. Minor heavy metal: a review on occupational and environmental intoxication. Indian J Occup Environ Med 2008; 12: 116–21.

10. Nickens KP, Patierno SR, Ceryak S. Chromium genotoxicity. a double-edged sword. Chem Biol Iteract 2010; 188: 276–88.

11. Moukarzel A. Chromium in parenteral nutrition: too little or too much. Gastroenterology 2009; 137: S18–S28.

12. Van der Straeten C, Tower SS, Hart AJ, Moyer TP. Cobalt and chromium measurement in patients with metal hip prostheses. Clin Chem 2013; 59: 880–6.

13. Roczniak W, Brodziak-Dopierala B, Cipora E, Jakobik-Kolon A, Konieczny M, Babuska-Roczniak M. Analysis of the content of chromium in certain parts of the human knee joint. Int J Envir Res Public Health 2018; 15; 1013. doi: 10.3390%2Fijerph15051013.

10.3 Cobalt (Co)

Lothar Thomas

Co is a steel-gray, shiny, hard metal mainly used in alloys and for varnish and paint production. The abundance of Co in the earth’s crust is 3.7 × 10–3%. In most of its compounds, Co is divalent and trivalent. Co chemistry is dominated by Co (II) oxidation state in the aqueous phase of terrestrial environments primarily due to the extremely low solubility of Co (III).

10.3.1 Indication

  • Suspected Co deficiency in macrocytic anemia and neuropathy
  • Suspected intoxication due to occupational exposure
  • Following metallic hip prosthesis implantation (refer to Section 10.2 – Chromium (Cr)).

10.3.2 Method of determination

Refer to Section 10.1.4 – Method of determination.

10.3.3 Specimen

Blood sampling with metal-free blood collection assembly:

  • Tube with lithium heparinate for plasma: 2 mL
  • Tube without anticoagulant for serum: 2 mL
  • Whole blood (lithium heparinate) in suspected hexavalent Cr intoxication: 5 mL

24 h urine following glucose and/or insulin challenge. The entire urine sample is to be handed in to the laboratory.

10.3.4 Reference interval /1/

Serum, plasma

< 10 nmol/L

< 0.6 μg/L1

Whole blood

8.5–66 nmol/L

0.5–3.9 μg/L


< 26 nmol/24 h2

< 1,5 μg/24 h2

1 Detection limit; 2 Related to 1.5 L daily excretion

Conversion: μg/L × 17.0 = nmol/L

Refer to reference /1/ in addition.

10.3.5 Clinical significance

Co is a constituent part of the essential vitamin B12 and vital for the human body. The Co requirement is met by a normal (balanced) diet; need 1.5 μg vitamin B12 a day. Cobalt deficiency

A deficiency in Co is almost always associated with a deficiency in vitamin B12 /2/. Deficiency exists if plasma levels are below 5 nmol/L /3/. Co deficiency is caused by inadequate intake of vitamin B12 from food. Vitamin B12 is mainly present in meat, fish, eggs, innards, milk and milk products. Plant-based food only contains small amounts of vitamin B12. Therefore, it is not possible to meet the Co and vitamin B12 requirement on a strictly plant-based diet. In industrial countries, dietary deficiency is a more common cause of vitamin B12 deficiency than malnutrition. This is due to the exclusion of animal source foods in vegan diets, eating disorders, alcoholism, homelessness and retirement home meals. Other causes include intrinsic factor deficiency due to atrophic gastritis or partial intestinal resection. Vitamin B12 deficiency due to primary Co deficiency is rare. Clinical symptoms of vitamin B12 deficiency are macrocytic anemia and neuropathy. For further information, see Section 13.3 – Vitamin B12 (cobalamin). Toxicity of cobalt

Traditionally, nickel, Co and Cr have been the most important contact allergens, and 1–3% of the population are allergic to Co or Cr /4/.

Co poisoning occurs in the enamel, glass and ceramics industry. Symptoms are:

  • In acute poisoning: spasmic abdominal pain, nausea and vomiting
  • In chronic poisoning: polycythemia, thyroid hypofunction and cardiac and pulmonary insufficiency.

In dyshidrotic eczema metal hypersensitivity does not play a role. High oral ingestion of nickel and/or Co should be considered, regardless of patch test results /5/.

For Co following implantation of metallic hip prosthesis, see Tab. 10.2-1 – Toxicity of chromium.

10.3.6 Comments and problems

Preanalytic phase

The high environmental concentrations of Co pose a significant risk of contamination during sampling, sample processing and storage.

Reference interval

As a rule, the reference interval values of Co range near the lower detection limit of the methods in all specimens. Therefore, under supply is very difficult to diagnose for technical reasons and also because of the great risk of sample contamination.

Stability in serum/plasma

If tightly sealed, samples can be stored at 4 °C for 2 weeks.

10.3.7 Pathophysiology

Following absorption in the ileum, cobalamin (vitamin B12) binds to the transport protein transcobalamin and is thus transported to all metabolically active cells /6/. When passing through the liver, 80–90% of the cobalamin bind to the protein haptocorrin and are then released again to the plasma as cobalamin-transcobalamin complex or secreted to the bile as cobalamin-haptocorrin complex.

Cellular uptake of the cobalamin-transcobalamin complex is effected by binding to a cell membrane receptor and subsequent transfer into the cytoplasm by endocytosis /7/. Transcobalamin is degraded in the lysosomes and cobalamin is converted to the following cofactors (see also Chapter 13 – Homocysteine, vitamin B12, folates, vitamin B6, choline, betaine):

  • Methyl cobalamin, a cofactor for the cytosolic enzyme methionine synthase. The enzyme catalyzes the transformation of homocysteine into methionine using N5-methyltetrahydrofolate; therefore, homocysteine is elevated in cobalamin deficiency.
  • The mitochondrial enzyme methyl malonyl-CoA mutase. The enzyme converts methyl malonyl CoA to succhinyl-CoA and requires 5’desoxyadenosylcobalamin as cofactor. The elevated methyl malonic acid in cobalamin deficiency is a direct consequence of a block of this pathway.

Due to the described cofactor effect, cobalamin is important for the transfer of C1 fragments in the methylation cycle. See Chapter 13 – Homocysteine, vitamin B12, folates, vitamin B6, choline, betaine. Clinical symptoms of cobalamin deficiency are macrocytic anemia and neuropathy. Cobalamin can only be synthesized by bacteria and not by the human body and, therefore, is essential for humans.

The body’s Co content is 2–5 mg, primarily distributed over liver, kidneys, muscles and bone marrow /1/. In order to maintain vitamin B12 storage in the liver sufficient for 5–10 years /8/, a daily intake of 3 μg of vitamin B12 is recommended /9/.

Approximately 90% of the Co from dietary intake are excreted again with the stool. Interactions with iron and manganese in enteral absorption can lead to reduced supply. Excessive intake is renally excreted again.


1. Thunus L, Lejeune R. Cobalt. In: Seiler HG, Sigel A, Sigel H (eds). Metals in clinical and analytical chemistry. New York; Marcel Dekker Inc 1994: 333–8.

2. Snow CF. Laboratory diagnosis of vitamin B12 and folate deficiency: a guide for the primary care physician. Arch Int Med 1999; 159: 1289–98.

3. Taylor A. Detection and monitoring of disorders of essential trace elements. Ann Clin Biochem 1996; 33: 486–510.

4. Thyssen JP, Menne T. Metal allergy – a review on exposures, penetration, genetics, prevalence, and clinical implications. Chem Res Toxicol 2010; 23: 309–18.

5. Stuckert J, Nedorost S. Low-cobalt diet for dyshidrotic eczema patients. Contact Dermatitis 2008; 59: 361–5.

6. van Asselt D, Thomas CMG, Segers MFG, Blom HJ, Wevers RA, Hoefnagels WHL. Cobalamin-binding proteins in normal and cobalamin-deficient older subjects. Ann Clin Biochem 2003; 40: 65–9.

7. Quadros EV, Nakayama Y, Sequeira M. The protein and the gene encoding the receptor of the cellular uptake of transcobalamin-bound cobalamin. Blood 2009; 113: 186–92.

8. Reiss J, Anke M. Cobalt. In: Biesalski HK, Köhrle J, Schümann K (eds). Vitamine, Spurenelemente und Mineralstoffe. Stuttgart; Thieme 2002: 222–3.

9. Deutsche Gesellschaft für Ernährung (DGE), Österreichische Gesellschaft für Ernährung (ÖGE), Schweizerische Gesellschaft für Ernährungsforschung (SGE). Referenzwerte für die Nährstoffzufuhr. Frankfurt; Umschau Brauns GmbH, 2000.

10.4 Copper (Cu)

Lothar Thomas

Behind Fe and Zn, Cu is the third most common trace element in the human body. It occurs cuprous copper (Cu+) and cupric copper (Cu2+). Cuprous copper is sparingly soluble in water; the form predominantly present in biological systems is cupric copper. Cu is an integral part of a number of enzymes. Cu facilitates electron transfer reactions when incorporated into specific cuproproteins and is utilized for such important processes as mitochondrial respiration, melanin biosynthesis, dopamine metabolism, iron homeostasis, oxidative defense, connective tissue formation, and peptide amidation /1/. As transition metal, Cu plays an important role in the anti-oxidative system of the cell.

10.4.1 Indication

Dietary Cu deficiency is suspected in /2/:

  • Iron refractory anemia with neutropenia
  • Total parenteral nutrition
  • Menkes syndrome.

Body Cu burden is suspected in /2/:

  • Cu intoxication
  • Wilson’s disease.

10.4.2 Method of determination

  • Electrothermal atomic emission spectrometry (ET-AES) /3/.
  • Flame atomic emission spectrometry (F-AES)
  • Photometric measurement of sulfonated bathocuproine /4/.

Principle of the bathocuproine method

Serum copper is released from its protein binding, reduced to Cu (I) and reacts with bathocuproine disulfonate to form a colored complex (not suitable for Cu determination in urine).

10.4.3 Specimen

  • Plasma or serum: 1 mL each
  • 24 h urine (collection container pre-filled with 10 mL of concentrated hydrochloric acid): 5 mL
  • 24 h urine following the administration of 4 × 250 mg D-penicillamine for Wilson’s disease diagnosis: 5 mL

10.4.4 Reference interval

The reference intervals for copper in serum and urine are shown in Tab. 10.4-1 – Copper reference intervals in serum and urine. Refer to references /567/ in addition.

10.4.5 Clinical significance

The adult organism contains 80–100 mg Cu. In industrial nations, the average daily adult diet contains about 5 mg of Cu, approximately 40% of which is absorbed via the upper gastrointestinal tract /1/. According to the US-American Third National Health and Nutrition Survey, the intake of Cu is 1–1.6 mg/day /8/. Daily Cu intake should be 20 μg/kg of body weight in adults and 50 μg/kg of body weight in children /2/. The mean daily requirement is 0.70 mg in adults and 0.26–0.68 mg (depending on age) in children /9/. Cu absorption decreases with increasing supply levels. Increasing the supply from 0.8 mg/day to 7.5 mg/day will no more than double the absorbed Cu quantity /2/. Main dietary Cu sources are legumes, nuts, haddock, liver and chocolate /8/. Divalent Cu ingested with food is reduced to monovalent Cu for absorption. Excess Cu is excreted via the bile and to a small extent via the kidneys. Laboratory findings

Cu level in plasma or serum should always be assessed in conjunction with the concentration of the plasma protein ceruloplasmin and, if acquired Cu deficiency is suspected, the superoxide dismutase (SOD) activity in erythrocytes should be determined additionally /2/. In plasma, 95% of the Cu is bound to ceruloplasmin; plasma Cu level increases in inflammation because ceruloplasmin is an acute-phase protein.

The plasma Cu level shows circadian changes with a peak in the morning, which is higher in women than in men and increases with age. Plasma Cu concentration increases due to estrogen intake, pregnancy, inflammation and stress and shows a tendency to decrease under corticosteroid therapy or at increased endogenous corticosteroid synthesis /2/.

Urinary Cu excretion is an important criterion for the diagnosis and therapy monitoring of Wilson’s disease (see also Section 18.7 – Ceruloplasmin (Cp)). Copper deficiency

Cu deficiency is either congenital or acquired. Congenital deficiency is rare and primarily relates to the Menkes syndrome. Wilson’s disease is a congenital ceruloplasmin deficiency. Acquired forms of Cu deficiency can occur in the malabsorption syndrome and under long-term parenteral nutrition and are rarely caused by reduced dietary Cu intake. In the setting of acquired Cu deficiency, supplementation with zinc, iron, fructose or chelating agents is the reason in many cases /9/.

Cu deficiency may also occur in loss of ceruloplasmin due to nephrotic syndrome or severe skin burns. Other causes are short bowel syndrome, celiac disease, tropical and non-tropical sprue, ileo-jejunal bypass and cystic fibrosis. The main diseases and symptoms of Cu deficiency are shown in Tab. 10.4-2 – Diseases and conditions with copper deficiency and copper overload.

Clinical symptoms of Cu deficiency are of hematological nature (anemia, neutropenia, rarely thrombocytopenia) and increased bone fragility.

The activity of superoxide dismutase (SOD) in erythrocytes is a better parameter for evaluation of Cu status. SOD is reduced in copper depletion and increases again in repletion /10/. SOD activity is not affected by biological influence factors governing ceruloplasmin concentration; it is reduced in Cu deficiency and increases under Cu substitution.

Metallothionein-induced Cu malabsorption may occur due to months of self-medication with zinc at a dosage ≥ 50 mg/day. This mechanism is utilized for Wilson’s disease therapy by oral administration of high doses of zinc. Copper toxicity

In most cases, elevated plasma Cu levels are non-specific and of no differential diagnostic and therapeutic significance. They are measured:

  • In the last trimester of pregnancy and under the intake of estrogens (oral contraceptives or postmenopausal hormone substitution) due to increased ceruloplasmin synthesis
  • In inflammation with acute- phase response such as acute and chronically active inflammations, tissue damage, metastasized tumors and infections
  • In diabetes mellitus type 1, pancreatic insufficiency and chronic hepatitis
  • In acute severe poisoning from soluble Cu in gram quantities (lethal dose 10 g), leading to hemolysis, liver and kidney damage /2/
  • In chronic intoxication from Cu-contaminated water or food and drink stored in Cu-containing containers.

Diseases with body Cu overload are shown in Tab. 10.4-2 – Diseases and conditions with copper deficiency and copper overload.

10.4.6 Comments and problems

Blood sampling

The tourniquet should not stop blood flow in the veins for more than a minute before blood is drawn. Compression increases the Cu-binding protein ceruloplasmin and elevated copper levels are measured.


More than 90% of the Cu in plasma is bound to ceruloplasmin. In Wilson’s disease, the free, non-bound Cu proportion is higher.

The calculation of ceruloplasmin bound Cu (Cp-Cu) /11/ is performed according to the equations:

  • Cp [mg/L] × 0.34 = Cp-Cu [μg/dL]
  • Cp [mg/L] × 0.054 = Cp-Cu [μmol/L]

Stability in serum/plasma

If tightly sealed, samples can be stored at 4 °C for 2 weeks.

Intraindividual variability

Biological variability: CV 5.6% /12/. Minimal quality specification developed from the biological intra- and inter-individual variability are in serum and plasma for Cu ± 0.84 μmol/L or 12% of the assigned target concentration /13/.

10.4.7 Pathophysiology

Cu is a critical functional component of a number of essential enzymes such as superoxide dismutase in the cytosol, cytochrom C oxidase in the mitochondria, and tyrosinase and β-hydroxylase in the secretory compartments /14/. Copper absorption

A number of proteins play an important role in the absorption, distribution and removal of Cu from the cells /1516/.

These proteins are (Tab. 10.4-3 – Proteins of copper metabolism):

  • The high-affinity Cu transporter CTR1
  • The low-affinity Cu transporter CTR2
  • The Cu chaperones CCS (Cu chaperone for SOD1) and ATOX1 (antioxidant protein 1)
  • The Cu efflux transporters ATP7A (Cu transporting alpha polypeptide) and ATP7B (Cu transporting beta polypeptide).

Dietary Cu2+ needs to be reduced to Cu+ before uptake across the apical membrane by copper transporter 1 (CTR1). The reduction is mediated by several reductases. The cell surface expression of CTR1 is regulated by cellular copper levels. In the intestinal epithelial cells, the copper chaperone, antioxidant-1 (ATOX1) shuttles copper to the copper transporting ATPase (ATP7A), which exports copper into the portal blood. After outward transfer, the intracellular Cu1+ is oxidized to become Cu2+, bound to α2-macroglobulin and albumin and delivered to the liver via the portal circulation.

The intestinal copper transport is shown in Fig. 10.4-1 – Duodenal enterocytes absorb dietary Cu+ and Fe3+ via transporters located at the luminal membrane).

Mutations in gene ATP7A are associated with Menkes disease, an X-linked recessive copper deficiency disorder.

Dietary Cu in excess is bound to metallothionein and excreted with the stool, for example following enterocyte apoptosis. Handling of copper in the liver

Cu2+ transported to the hepatocyte via albumin and α2-macroglobulin is transferred to the Cu transporter CTR1 in the reduced form Cu+ /17/. CTR1 releases Cu+ to chaperones for distribution to Cu-dependent enzymes. ATP7B pumps Cu into the trans-Golgi network, where it is incorporated into ceruloplasmin and other Cu proteins. Excess copper stimulates the translocation of ATP7B from the trans-Golgi network to the canalicular membrane of the hepatocyte, facilitating secretion into the bile. Biliary secretion of Cu appears to require the interaction between ATP7B and COMMDI (Copper metabolism MURR1 domain).

Four hours following the administration of Cu, more than 95% are removed from the circulation by the liver, and 6–9% appear incorporated in ceruloplasmin after 24 hours. Ceruloplasmin plays no essential role in Cu transport, and it is assumed that Cu bound to histidine and other amino acids provides for the delivery of Cu to various tissues. Cu burden and increased absorption lead to overload of the hepatocyte’s Cu pool, triggering increased excretion via the bile.

This is performed by the abundantly synthesized ATP7B that is preferably localized in the trans-Golgi network and, at high intracellular Cu concentrations, cycles between the trans-Golgi network and the canalicular membrane, thus effecting outward Cu transfer from the hepatocyte /17/. Copper and erythropoiesis

Erythrocytes contain 2.5 μg of Cu per gram. Cu uptake takes place once Cu (II) is reduced to Cu (I), catalyzed by the metalloreductases Steap 2–4, via the receptor CTR1 /16/. In cytoplasm, the CCS chaperones transport Cu (I) to SOD1. Cu is also delivered to the erythroblast mitochondria for the synthesis of Cu-containing enzymes such as cytochrome c oxidase. The occurrence of anemia in Cu deficiency is thought to be due to a disorder of the iron metabolism affecting the steps of cellular uptake, intracellular mobilization, uptake into the mitochondria and hemoglobin synthesis. Copper and enzymes

Cu is the cofactor of key metabolic enzymes with a broad range of activity. Tab. 10.4-4 – Copper-containing enzymes and their biochemical function shows some of these enzymes and explains their function.


1. Tao TJ, Gitlin JD. Hepatic copper metabolism: insights from genetic disease. Hepatology 2003; 37: 1241–7.

2. Schünemann K, Classen HG, Dieter HH, König J, Multhaup G, Rükgauer M, et al. Hohenheim Consensus Workshop: Copper. Eur J Clin Nutr 2002; 56: 469–83.

3. Rükgauer M, Kruse-Jarres JD. Analytik von Kupfer in Körperflüssigkeiten. In: Günzler H, Bahadir AM, Borsdorf R, Danzer K, Fresenius W, Galensa R, Hubder W, Lüderwald I, Schwedt G, Tölg G, Wisser H (eds). Analytiker-Taschenbuch, Band 14. Heidelberg; Springer 1996: 283–300.

4. Landers JW, Zak B. Determination of serum copper and iron in a single small sample. Amer J Clin Path 1958; 29: 590.

5. Milne DB, Johnson PE. Assessment of copper status: effect of age and gender on reference ranges in healthy adults. Clin Chem 1993; 39: 883–7.

6. Lokitch G. Trace elements in pediatrics. JIFFC 1996; 9: 46–51.

7. Brätter P, Heseker H, Kruse-Jarres JD, Liesen H, Negretti de Brätter V, Pietrzik K, Schümann K. Mineralstoffe, Spurenelemente und Vitamine: Leitfaden für die ärztliche Praxis. Gütersloh: Bertelsmann Stiftung 2002: 63–125.

8. Institute of Medicine. Copper. In: Food and Nutrition Board, ed. Dietary reference intakes: vitamin A, vitamin K, arsenic, boron, chromium, copper, iodine, iron, manganese, molybdenum, nickel, silicon, vanadium and zinc. Washington, DC: National Academy Press, 2002; 224–57.

9. Turnland JR. Copper. In: Shils M, Shike M, Ross AC, et al, eds. Modern nutrition in health and disease. 10th ed. Philadelphia PA. Lippincott, Williams and Wilkins, 2005; 286–99.

10. Milne DB. Copper intake and assessment of copper status. Am J Clin Nutr Suppl 1998; 67: 1041–5.

11. Houwen RHJ, Hattun van J, Hoogenraad TU. Wilson disease. Netherlands J Med 1993; 43: 26.

12. Gonzalez-Revalderia J, Garcia-Bermejo S, Menchén-Herreros A, Fernandez-Rodriguez E. Biological variation of Zn, Cu and Mg in serum of healthy subjects. Clin Chem 1990; 36: 2140–1.

13. Arnaud J, Weber JP, Weykamp CW, Parsons PJ, Angerer J, Mairiaux E, et al. Quality specifications for the determination of copper, zinc, and selenium in human serum and plasma: evaluation of an approach based on biological and analytical variation. Clin Chem 2008; 54: 1892–9.

14. Palumaa P. Copper chaperons, the concept of conformational control in the metabolism of copper. FEBS Lett 2013; 587; 1902–10.

15. Nishito Y, Kambe T. Absorption mechanisms of iron, copper, and zinc: an overview. J Nutr Sci Vitaminol 2018; 64: 1–7.

16. Collins JF, Prohaska JR, Knutson MD. Metabolic crossroads of iron and copper. Nutrition Reviews 2010; 68: 133–47.

17. Tao TY, Gitlin JD. Hepatic copper metabolism: insights from genetic disease. Hepatology 2003; 1241–7.

18. Kaler SG, Holmes CS, Goldstein DS, et al. Neonatal diagnosis and treatment of Menkes disease. N Engl J Med 2008; 358: 605–14.

19. Fuhrman MP, Hermann V, Masidonski P, Eby C. Pancytopenia after removal of copper from total parenteral nutrition. J Parenter Enteral Nutr 2000; 24: 361–6.

20. Valurupalli M, Divakaran S, Parnes A, Levy BD, Loscalzo J. The element of surprise. N Engl J Med 2019; 181: 1365–71.

21. Copper supplementation in parenteral nutrition of cholestatic infants. J Pediatr Gastroenterol Nutr 2010; 50: 650–4.

22. Igic PG, Lee E, Harper W, Roach KW. Toxic effects associated with consumption of zinc. Mayo Clin Proc 2002; 77: 713–6.

23. Aggett PJ. Aspects of neonatal metabolism of trace elements. Acta Pediatr Suppl 1994; 402: 75.

24. Bandmann O, Weiss KH, Kaler SG. Wilsons disease and other neurological copper disorders. Lancet Neurol 2015; 14: 103–13.

25. Jaiser SR, Winston GP. Copper deficiency myelopathy. J Neurol 2010; 257: 869–81.

26. Ernst B, Turnheer M, Schmid SM, Schultes B. Evidence for the necessity to systematically assess micronutitient status prior to bariatric surgery. Obes Surg 2009; 19: 66–73.

27. Gupta A, Lutsenko S. Human copper transporters: mechanisms, role in human diseases and therapeutic potential. Future Med Chem 2009; 1: 125–42.

28. Ghayour-Mobarhan M, Taylor A, New SA, Lamb DJ, Ferns GAA. Determinants of serum copper, zinc and selenium in healthy subjects. Ann Clin Biochem 2005; 42: 364–75.

29. Rükgauer M, Schmitt Y, Zeyfang A. Bedeutung von Chrom, Kupfer, Selen und Zink bei Diabetes mellitus Typ 1 und Typ 2 mit Folgeerkrankungen. J Lab Med 2006; 30: 192–200.

10.5 Magnesium (Mg)

Lothar Thomas

Magnesium ion is the fourth most common cation in the body and the second most common intracellular cation after potassium ion. Only 1% of magnesium is present in the extracellular fluid. The Mg2+ is an important electrolyte serving as cofactor in > 600 enzymatic processes in the organism /34/.

The magnesium fractions in plasma are in equilibrium and comprise:

  • Magnesium ions (Mg2+)
  • Protein-bound Mg, primarily bound to albumin
  • Complex-bound Mg in the salt form as magnesium bicarbonate, magnesium carbonate, magnesium acetate, magnesium phosphate and magnesium citrate.

10.5.1 Indication

Magnesium deficiency is suspected in:

  • Hypocalcemia, hypocalciuria and hypercalciuria
  • Neuromuscular hyperexcitability (tremor, increased tendon reflexes, tics, tetany, severe spasms)
  • Gastrointestinal diseases with malabsorption
  • Heart failure (congestive, tachycardia, arrhythmia, ventricular fibrillation)
  • Renal diseases.

Regular magnesium monitoring is recommended:

  • Under long-term diuretics therapy
  • In poorly stabilized diabetes mellitus
  • In chronic intestinal malabsorption
  • In alcohol withdrawal
  • In intensive care patients
  • Under long-term total parenteral nutrition
  • In renal insufficiency.

Magnesium intoxication is suspected in:

  • Hyporeflexia
  • Hypotension due to hypovolemia
  • Respiratory depression
  • Coma.

Ionized magnesium (Mg2+)

Main indication for the determination of Mg2+ is hypoproteinemia, mimicking hypomagnesemia.

10.5.2 Method of determination

Total magnesium

Flame atomic emission spectrometry (F-AES).

Xylidyl blue method

Principle: magnesium forms a soluble red colored compound with blue-colored alcoholic xylidyl blue solution at alkaline pH (pH 9–10). The color intensity of the reaction mixture is directly correlated to the magnesium concentration in the sample /1/.

Camalgite method

Principle: magnesium reacts with camalgite at alkaline pH forming a pink-colored compound. The intensity of the color, measured at 546 nm, is directly correlated to the magnesium concentration in the sample.

Urine samples should not be analyzed using the xylidyl blue method or camalgite method.

Ionized magnesium (Mg2+)

The potentiometric measurement is the method of choice in clinical chemistry. The equilibrium of protein-bound magnesium and Mg2+ is influenced by ionic strength, temperature and by calcium ions. Ca2+ interfere with all Mg2+. Thus it is necessary to determine both ions simultaneously in each sample and correct the result for Ca2+ interference according the known sensitivities of Mg2+ or Ca2+. The concentration of Mg2+ in plasma depends on pH, mainly because binding of magnesium to albumin increases with pH. For this reason the ion-selective electrode system should measure pH simultaneously to allow adjustment to pH 7.4. Per 0.1 increase in pH a 2–3% decrease in Mg2+ is measured. Samples exceeding pH 7.8 should not be readjusted by CO2 changes or acidification, because of a risk of irreversible side reactions of Mg2+ /31/.

Ion-selective electrode system (ISE) for potentiometric measurement /2/

Principle: the measurement system basically comprises an Mg2+ ion-selective electrode system (ISE) and a reference electrode. On the sample side, these two electrodes are bridged by the sample (e.g., calibrator, plasma or blood). Two electrical leads connect the electrodes to a voltmeter, which measures the potential difference (E). E is formally equal to the electrical potential difference between the Mg-ISE and the reference electrode and a salt bridge. After the system has been calibrated, ISE potential difference of the sample is compared to the values for the calibrators. Using an algorithm related to the Eisenman-Nikolsky equation, the substance concentration of Mg2+ in the sample is then calculated.

10.5.3 Specimen

  • Plasma or serum: 1 mL
  • 24 h urine specifying the collected volume: 5 mL

10.5.4 Reference interval

Refer to references /34/ and Tab. 10.5-1 – Reference intervals for magnesium.

10.5.5 Clinical significance

Total body stores are 24 grams or 1988 mEq elemental magnesium. About 99% of the magnesium present in the human body are found in the intracellular compartment and are distributed over the skeletal system (53%), muscles (27%) and non-muscular soft tissue (19%) and only 1% is in the extracellular fluid. The skeletal system, the gastrointestinal tract and the kidneys regulate the plasma magnesium concentration. About 0.5% of the magnesium is in the erythrocytes and 0.3% is in plasma. Under physiological conditions the fractions in plasma are Mg2+ 59–72%, complexed magnesium 5–11%, and protein-bound magnesium 23–31% /2/. Magnesium is ubiquitous in nature and occurs primarily in greens because chlorophyll contains a high amount of magnesium. However, nuts, cereals, seafood and meat also contain magnesium. Drinking water, especially hard water, contains about 30 mg magnesium per liter /5/.

The US-American Food and Nutrition Board determined a daily magnesium requirement of 250–265 mg for women and 330–350 mg for men. The tolerable upper intake level was determined as 310–320 mg for women and 400–420 mg for men. The daily intake of approximately 10% of the US-American population above 19 years of age is only 50% of these amounts /6/.

The plasma/serum magnesium level inadequately reflects the body’s magnesium content and also depends on the albumin concentration. A reduction in the body’s magnesium content can be associated with normal plasma concentrations. The serum Mg2+ level is dependent on the pH and lower in alkalosis due to increase in protein binding. Hypomagnesemia

Hypomagnesemia is diagnosed if the serum magnesium concentration is below 0.7 mmol/L (1.7 mg/dL) with or without body depletion. It is a common electrolyte disorder and detected in up to 10% of hospitalized patients. The incidence of hypomagnesemia in intensive care patients is up to 50% /7/.

The symptoms of moderate hypomagnesemia include mild tremors, apathy, generalized weakness to cardiac ischemia (widening of the QRS, peaking of T waves) /34/.

More than 12 genes are directly or indirectly involved in magnesium transport and can be classified in the following groups /35/:

  • hypercalciuric hypomagnesemia (mutations in the genes CLDN 16, CLDN 19, CASR, CLCNKB, and CASR)
  • Gitelman-like hypomagnesemia (mutations in the genes CLCNKB, SLC12A3, BSND, KCNJ10,FYXD2, HNF1B, and PCBD1)
  • mitochondrial hypomagnesemia (SARS2, MT-TI, and Kearns-Sayre Syndrom)
  • other hypomagnesemias (TRPMG, 2 CNMM2, EGFR, EGF, KCNA1, and FAM111A).

Patients with magnesium concentrations < 0.5 mmol/L (1.2 mg/dL) develop cardiac symptoms, especially in the presence of hypokalemia, manifested as long PR and QT intervals in the ECG. Hypomagnesemia predisposes individuals to cardiac glycoside toxicity, but digoxin toxicity-induced ectopic cardiac arrhythmia can also be terminated by magnesium administration /8/.

Often, the clinical symptoms of hypomagnesemia are similar to those of hypocalcemia and there are concomitant Mg2+ and Ca2+ homeostasis disorders. Moreover, hypomagnesemia can be the cause of hypocalcemia.

There are two major pathways leading to hypomagnesemia, namely gastrointestinal and/or renal losses. Diseases and conditions with magnesium deficiency are shown in Tab. 10.5-2 – Diseases and conditions with magnesium deficiency.

The following markers are important for the differential diagnosis of hypomagnesemia:

  • Calcium in plasma/serum
  • Excretion of Mg2+ and Ca2+ in 24-hour urine
  • Molecular biological assays for finalizing the diagnosis. Hereditary hypomagnesemia

Patients with hypomagnesemia and secondary hypocalcemia due to dysfunction in the Mg2+ channel TRPM6 suffer from reduced renal and intestinal magnesium absorption /9/. Hereditary renal hypomagnesemia is differentiated based on the genes encoding the renal Mg2+ transport (Tab. 10.5-2 – Diseases and conditions with magnesium deficiency and Fig. 10.5-1 – Renal magnesium transport). Often hereditary hypomagnesemia is associated with disturbances in renal Ca2+ excretion.

Hypomagnesemia and hypercalciuria

This combination is found in various forms of the Bartter syndrome, autosomal dominant hypocalcemia (ADH) and familial hypomagnesemia with hypercalciuria and nephrocalcinosis (FHHNC).

Hypomagnesemia and normocalciuria

This combination is found in autosomal dominant hypomagnesemia (AD hypo Mg2+) due to mutations in the gene Kv1.1 and in isolated recessive renal hypomagnesemia (IRH) due to mutations in the pro-EGF gene.

Hypomagnesemia and hypocalcuria

This combination of hypomagnesemia and hypocalcuria occurs in:

  • Gitelman syndrome due to mutations in the gene NCC
  • SeSAME syndrome due to mutations in the gene Kir4.1.
  • Mature-onset diabetes of the young type 5 (MODY5) due to mutations in the gene HNF1B
  • Isolated dominant hypomagnesemia due to mutations in the gene FXYD2. Hypermagnesemia

Hypermagnesemia is most common in patients with renal insufficiency who take magnesium-containing antacids and laxatives. Clinical signs correlate with increasing serum levels of Mg2+ /10/:

  • Hyporeflexia and drowsiness occur at serum levels > 2.0 mmol/L. Hyporeflexia indicates the severity of hypermagnesemia and predicts cardiac and respiratory toxicity.
  • PR, QRS, and QT interval prolongation may occur with concentrations as low as 2.5 mmol/L
  • Somnolence and hypotension occur at 3.0 to 3.5 mmol/L
  • Paralysis of voluntary muscles and areflexia occur at 5.0 mmol/L.

Intravenous therapy requires monitoring of serum Mg2+ concentration and of renal function. Hypermagnesemia occurs in acute renal failure and in the final stage of chronic renal insufficiency. The effects of hypermagnesemia can be interpreted as Ca2+ antagonism (muscle weakness, paralysis and even cardiac arrest). In cases of poisoning, the Mg2+ effect is mitigated by parenteral administration of equimolar amounts of Ca2+. Pregnant women and neonates

It has been suggested that hypomagnesemia during pregnancy may be associated with preeclampsia, impaired growth and pre-term delivery. Preeclampsia is the main cause of maternal-fetal morbidity and mortality, with an incidence of up to 3–5% in pregnant women. In a study /33/ a new upper Mg2+ reference value of 3.5 mmol/L was established for pregnant women. In neonates hypermagnesemia was diagnosed at concentrations of 2.3 ± 0.3 mmol/L and hypomagnesemia at concentrations of 0.53 ± 0.08 mmol/L.

10.5.6 Comments and problems

Total magnesium

Measurement of elevated concentrations:

  • If congestion of the vein takes several minutes for sampling of blood, because 33% of magnesium is bound to plasma proteins
  • Hemolysis increases the serum magnesium level because the magnesium concentration in erythrocytes is almost three times as high as in plasma.

Intraindividual variance: CV 3.4%.

Stability in serum/plasma: If tightly sealed samples can be stored at 4 °C for 2 weeks.

Ionized magnesium

Blood sampling: If venous blood is used, sampling of Mg2+ should take place without a torniquet and with the patient sitting at rest to assure a state of equilibrium.

Determination of Mg2+: Ca2+ interfere with the determination of Mg2+, and thus it is necessary to determine both cations simultaneously. Na+ interfere also, but to a lesser extent than Ca2+. Compensation is achieved by using adequate calibration solutions /2/.

10.5.7 Pathophysiology

The majority of dietary magnesium is absorbed in the small intestine /9/:

  • By passive para cellular transport; Mg2+ follows a concentration gradient, 90% of the Mg2+ take this way.
  • By active movement in a trans cellular saturable process along the trans epithelial Mg2+ channels TRPM6 and TRPM7. These channels direct the divalent cations Mg2+ and Ca2+ along an electrochemical gradient into the cells of the gastrointestinal tract and the renal distal tubules. Mutations of TPRM6 are the cause of primary hypomagnesemia with secondary hypocalcemia (HSH).

About 30% of magnesium in plasma are bound to proteins, the rest is available for free glomerular filtration. The filtered amount of Mg2+ is handled as follows (Fig. 10.5-1 – Renal magnesium transport):

  • The proximal tubule reclaims only 10–20% of the filtered Mg2+, clearly in contrast to the other cations, such as Na+, K+ and Ca2+. The para cellular reabsorption of Mg2+ is achievable due to water removal along the length of the proximal tubule, resulting in an increased luminal Mg2+ concentration.
  • In the thick ascending limb of the loop of Henle 60–70% are reabsorbed. The lumen-positive trans epithelial voltage gradient established by Na+-K+-ATPase is the most important driving force.
  • The remainder (10%) of the filtered Mg2+ is re-absorbed in the distal convoluted tubule via an active trans cellular process. Reabsorption of Mg2+ is active trans cellular through Mg2+ channels formed by magnesiotropic proteins, such as TRPM6. The function of these channels is significantly influenced by stimulating factors, such as the epidermal growth factor (EGF) and the K+ channels Kv1.1 and Kir4.1. Diseases induced by dysfunctions of these proteins are described in Tab. 10.5-2 – Diseases and conditions with magnesium deficiency.

Impaired secretion of parathyroid hormone (PTH) is one of the major mechanism of hypocalcemia in magnesium deficiency /11/. Many hypomagnesemic patients have low or normal PTH concentrations despite the presence of hypocalcemia. These findings along with the fact that serum PTH concentrations increase after magnesium supplementation indicate that PTH secretion is impaired in these patients. The most important regulator of PTH secretion is the Ca2+ sensing receptor (CaSR) in parathyroid cells. Binding of Ca2+ with the CaSR cause an increase in intracellular Ca2+ content and a reduction in PTH secretion. Mg2+ competes with Ca2+ for the CaSR with an affinity 2–3-fold less than that for Ca2+ causing a rise in the intracellular Ca2+ concentration. Thus, Mg2+ acts as a Ca2+ agonist at very high concentrations but serves as an antagonist at lower concentrations. Based upon these observations, the reduced PTH secretion in hypomagnesemia is based on a reduction in the competitive inhibition by Mg2+ of Ca2+ binding with its receptor. Such an alteration in Ca2+ binding by the CaSR enables parathyroid cells to respond to serum Ca2+ with higher sensitivity and causes a reduction in PTH secretion at lower serum Ca2+ concentrations /11/.


1. Mann CR, Yoe JH. Spectrophotometric determination of magnesium with sodium-1-azo-2-hydroxy-3-(2,4-dimethylcarboxanilido)-naphthalene-1-(2-hydroxy-benzene-5 sulfonate). Analyt Chem 1956; 28: 202.

2. Rayana MCB, Burnett RW, Covington Ak, D’Orazio P, Fogh-Andersen N, Jacobs E, et al. IFCC Guideline for sampling, measuring and reporting ionized magnesium in plasma. Clin Chem Lab Med 2008; 46: 21–6.

3. Meites S, ed. Pediatric Clinical chemistry, 3rd ed. Washington DC; AACC Press 1989: 191.

4. Külpmann WR, Rössler J, Brunkhorst R, Schüler A. Ionised and total magnesium serum concentrations in renal and hepatic disease. Eur J Clin Chem Clin Biochem 1996; 34: 257–64.

5. Al-Ghamdi SMG, Cameron EC, Sutton RAL. Magnesium deficiency: pathophysiologic and clinical overview. Am J Kidney Dis 1994; 24: 737–52.

6. Moshfegh A, Goldman J, Ahuja J, Rodes D, LaComb R. What we eat in America, NHANES 2005–2006. US Department of Agriculture. www.ars.usda.gov/ARSUserFiles/80400530/pdf/0102/usualintaketables2001-02.pdf. November 2009.

7. Buckley MS, LeBlanc JM, Cawley MJ. Electrolyte disturbances associated with commonly prescribed medications in the intensive care unit. Crit Care Med 2010; 38: S253–S264.

8. Arsenian MA.Magnesium and cardiovascular disease.Progr in Cardiovasc Dis 1993;35: 271–309.

9. San-Cristobal P, Dimke H, Hoenderop JGJ, Bindels RJM. Novel molecular pathways in renal Mg2+ transport: a guided tour along the nephron. Curr Opin Nephrol Hypertens 2010; 19: 456–62.

10. Razavi B, Somers D. Hypermagnesemia-induced multiorgan failure. Am J Med 2000; 108: 686–7.

11. Matsumoto T. Magnesium deficiency and parathyroid function. Internal Medicine 1995; 34: 603–4.

12. Buckley MS, LeBlanc JM, Cawley MJ. Electrolyte disturbances associated with commonly prescribed medications in the intensive care unit. Crit Care Med 2010; 38, 6 Suppl: S253–S264.

13. Nielsen FH. Magnesium, inflammation, and obesity in chronic disease. Nutr Rev 2010; 68: 333–40.

14. Ford ES. Body mass index, diabetes, and C-reactive protein among US adults. Diabetes Care 1999; 12: 1971–7.

15. Paecock JM, Ohira T, Post W, Sotoodehnia N, Rosamond W, Folsom AR. Serum magnesium and risk of sudden cardiac death in the Atherosclerosis RISK in Communities (ARIC) Study. Am Heart J 2010; 160: 464–70.

16. Milionis H, Alexandrides GE, Liberopoulos EN, Bairaktari ET, Goudevenos J, Elisaf MS. Hypomagnesemia and concurrent acid-base and electrolyte abnormalities in patients with congestive heart failure. Eur J Heart Failure 2002; 4: 167–73.

17. Van Laecke S, Nagler EV, Verbeke F, Van Biesen W, Vanholder R. Hypomagnesemia and the risk of GFR decline in chronic kidney disease. Am J Med 2013; 126: 825–31

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

19. Martini LA, Catania AS, Ferreira SRG. Role of vitamins and minerals in prevention and management of type 2 diabetes. Nutr Rev 2010; 68: 341–54.

20. Montagnana M, Lippi G, Tagher G, Salvagno GL, Guidi GC. Relationship between hypomagnesemia and glucose homeostasis. Clin Lab 2008; 54: 169–72.

21. Schnepp M, Koall W, Deuber HJ, Osten B. Einfluss der immunsuppressiven Behandlung mit cyclosporin A auf den Magnesiumhaushalt von Patienten nach Nierentransplantation. Nieren- und Hochdruckkrankheiten 2000; 29: 29–33.

22. Viljoen A, Batchelor B, Ghuran A. Woman with hypomagnesemia and hypocalcemia. Clin Chem 2015; 61: 699–703.

23. Kuipers T, Thang sHD, Arntzenius AB. Hypomagnesemia due to use of proton pump inhibitors. The Netherlands J of Med 2009; 67: 169–72.

24. Hou J, Renigunta A, Gomes AS, et al. Claudin 16 and claudin 19 interactions is required for their assembly into tight junctions and for renal absorption of magnesium. Proc Ntl Acad Sci USA 2009; 106: 15350–5.

25. Walder RY, Landau D, Meyer P, et al. Mutation of TRPM6 causes familial hypomagnesemia with secondary hypocalcemia. Nat Genet 2002; 31: 171–4.

26. Gronestege WM, Thebault S, van der Wijst J, et al. Impaired basolateral sorting of pro-EGF causes isolated recessive renal hypomagnesemia. J Clin Invest 2007; 117: 2260–7.

27. Nijenhuis T, Vallon V, van der Kemp AW, et al. Enhanced passive Ca2+ reabsorption and reduced Mg2+ channel abundance explains thiazide-induced hypocalciuria and hypomagnesemia. J Clin Invest 2005; 115: 1651–8.

28. Gaudemanns B, van der Wijst J, Scola RH, et al. A missense mutation in the Kv1.1 voltage gated potassium channel-encoding gene KCNA1 is linked to human autosomal dominant hypomagnesemia. J Clin Invest 2009; 119: 936–42.

29. Meij IC, Koenderink JB, van Bokhoven H, et al. Dominant isolated renal magnesium loss is caused by misrouting of the Na+–K+-ATPase gamma-subunit. Nat Genet 2000; 26: 265–6.

30. Scholl UI, Choi M, Liu T, et al. Seizures, sensineural deafness ataxia, mental retardation, and electrolyte imbalance (SeSAME syndrome) caused by mutations in KCNJ10. Proc Natl Acad Sci 2009; 106: 5842–7.

31. Rayana MCB, Burnett RW, Covington AK, D’Orazio P, Fogh-Andersen N, Jacobs E, et al. IFCC guideline for sampling, measuring and reporting ionized magnesium in plasma. Clin Chem Lab Med 2008; 46 (1): 21–6.

32. Thomassen JQ, Tolstrup JS, Nordestgaard BG, Tybjerg-Hansen A, Frikke-Schmidt R. Plasma concentrations of magnesium and risk of dementia: a general population study of 102648 individuals. Clin Chem 2021; 67 (6): 899–911.

33. Laguna J, Bonifacio RF, Munoz LM, Bedini JL, Roiges DS, Rico N. Reporting magnesium critical results: clinical impact on pregnant women and neonates. Clin Chem Lab Med 2022; 60 (11): e253–e255.

34. Salinas M, Lopez-Garrigos M, Flores E, Leiva-Salinas C. Improving diagnosis and treatment of hypomagnesemia. Clin Chem Lab Med 2024; 62 (2): 234–48.

35. Viering DHHM, deBaaij JHF, Walsh SB, Kleta R, Bockenhauer D. Genetic causes of hypomagnesemia, a clinical overview. Pediatr Nephrol 2017; 32: 1123–35.

10.6 Manganese (Mn)

Lothar Thomas

Mn is the 5th most abundant metal and the 12th most abundant element on earth. Mn exists in 11 oxidation states, as well as salt and chelate forms. It is only found as Mn(I) and Mn (II) in biological systems of humans and animals and as Mn (II), Mn (III) and Mn (IV) in plants.

10.6.1 Indication

If the following is suspected:

  • Inhalative Mn intoxication
  • Mn intoxication in chronic liver disease.

10.6.2 Method of determination

Refer to Section 10.1.4 – Method of determination.

10.6.3 Specimen

  • Plasma or serum: 1 mL
  • 24 h urine specifying the collected volume: 5 mL

10.6.4 Reference interval

Refer to references /12/ and Tab. 10.6-1 – Reference intervals for manganese.

10.6.5 Clinical significance

Mn is considered to be an essential trace element required as a catalytic cofactor for a variety of important enzyme reactions. An average adult has 10–12 mg Mn incorporated into the active centers of the various metalloenzymes (e.g., arginase, glutamine synthetase and Mn super oxide dis mutase) which are located mostly in the mitochondria /3/. Mn is enough in diet one imagines.

Mn is necessary for bone mineralization, is involved in the mediation of the immune response, the eradication of reactive oxygen species, and the metabolism of carbohydrates, proteins and lipids /4/.

The concentration of Mn in whole blood is 10–15-fold higher than those in plasma/serum because the Mn concentration in the erythrocytes is much higher than in plasma. Therefore, mild hemolysis during blood sampling will already lead to elevated levels. The Mn concentration is dependent on age. Levels are significantly higher in the first year of life than later. The concentration is also higher during pregnancy /5/.

Adequate Mn intake is approximately 2 mg/day and should not exceed 11 mg/day /6/. Food containing ample amounts of Mn are meat, fish, nuts, blueberries and dried fruit. Hypomanganesemia

Clinically recorded Mn deficiency is extremely rare, except in experimental studies. Only a small number of cases have been reported to date, for example, hypomanganesemia was detected in patients with short bowel syndrome. Fetal intrauterine growth retardation has been reported in pregnant women with hypomanganesemia /5/. Patients under long-term parenteral nutrition /3/ and chronic hemodialysis patients /7/ do not develop Mn deficiency. The clinical findings in experimentally induced deficiency were reduced glucose tolerance, bone growth disorder, skin alterations and reduced HDL cholesterol. Hypermanganesemia

The body is protected from accumulating excess dietary Mn because only a small fraction of dietary Mn is absorbed /5/. Mn absorption can also be inhibited by dietary constituents, such as plant fibers, phytates, ascorbic acid, iron, calcium and phosphor. Body burden results primarily from inhalation of manganese dusts and, less commonly, from consumption of water with an Mn concentration of approximately 100 μg/L, normally 10 μg/L /4/. Under usual circumstances the amount of Mn in the air is below 0.05 μg Mn/m3. Excess Mn accumulation in the brain may occur when significant atmospheric Mn contamination is associated with certain industrial processes. In addition, exhaust gasoline-burning engines contribute to atmospheric Mn contamination. The source is a consequence of the use of Mn in a common gasoline additive (methylcyclopentadienyl manganese tricabonyl) as anti-knock additive.

Diseases and disorders with hypermanganesemia are shown in Tab. 10.6-2 – Diseases and conditions with manganese body burden.

10.6.6 Comments and problems

Pre analytic phase

High Mn concentrations in airborne dust pose a significant risk of contamination during sampling, storage and sample processing. Therefore, it is recommended to work under clean room conditions, if possible.

Blood sampling

It is recommended to use metal-free blood collection assemblies for blood sampling. The high Mn content in erythrocytes simulates elevated plasma levels already in mild hemolysis.

Stability in serum/plasma

If tightly sealed, samples can be stored at 4 °C for 2 weeks.

10.6.7 Pathophysiology

Mn is ubiquitous in the environment. It makes up 0.1% of the earth’s crust, and the Mn content in soil is 40–900 mg/kg. Mn and Fe have many properties in common. Both are divalent and trivalent in biological systems, have almost the same ionic radius and are intestinally absorbed via the divalent metal ion transporter (DMT) and, in blood, also transported by plasma proteins. Mn3+ is transported by transferrin, while Mn2+ binds to other plasma proteins. Mn also occurs as free ion in plasma. The adult body’s Mn content is 10–20 mg. Different fractions of the daily dietary intake of 1.8–2.6 mg are absorbed in the small intestine. The fraction is 5% in young women with iron deficiency and only 1% in those without iron deficiency /8/. This is presumably due to competition between Mn and Fe for the DMT. The enterally absorbed Mn is subject to homeostatic regulation. Excessive amounts of dietary Mn are eliminated by the liver through excretion via the bile.

Mn intake by inhalation of dusts and vapors circumvents this protective mechanism enabling Mn to directly enter the central nervous system, especially the basal ganglia. This affects the neurotransmitter system, especially the dopaminergic system, which is important for motor coordination, attention and perception. According to a theory /9/, neurotoxicity is caused by deregulation of the glutamine-glutamate cycle in the astrocytes due to Mn-induced oxidative stress.

A substantial part of the Mn content is incorporated in the active center of metalloenzymes, such as /4/:

  • Arginase. This enzyme catalyzes the last step in the urea cycle:

Arginine + H2O Ornithine + urea

  • Glutamine synthetase (GS). The enzyme converts glutate to glutamine, catalyzes the NH3 removal from the body and keeps the pH constant. GS is associated with 80% of Mn in brain and mostly concentrated in the globus pallidus.

Glutamate + ATP + NH3 Glutamine + ADP + phosphate + H2O

  • Pyruvate carboxylase. The enzyme catalyzes the conversion of pyruvate to oxaloacetate in the mitochondria and plays an important role in gluconeogenesis, lipogenesis, neurotransmitter synthesis and glucose-induced insulin secretion in pancreatic beta cells.

Pyruvate + CO2 + ATP + H2O Oxaloacetate + ADP + phosphate + 2 H+

  • Mn super oxide dis mutase. This mitochondrial enzyme catalyzes the dis mutation of the super oxide (O2)radical into either ordinary O2 or H2O2 and protects against the effects of free radicals.

2O2–. + 2 H+ O2 + H2O

Acute and chronic intoxication, especially by inhalation of Mn vapors or manganese dioxide dust in manganese ore mining and processing, are the main manifestations of hypermanganesemia. Manganism, a condition resembling Parkinson’s disease, primarily develops following increased exposure to Mn in combination with a liver-related reduction in Mn excretion.


1. Chiswell B, Johnson D. Manganese. In: Seiler HG, Sigel A, Sigel H (eds). Metals in clinical and analytical chemistry. New York; Marcel Dekker 1994: 467–78.

2. Burguera JL, Burguera M, Alarcon OM. Blood levels of zinc, cobalt, copper, iron and manganese in children from Merida, Venezuela. Trace Elem Med 1992; 4: 194–7.

3. Hardy G. Manganese in parenteral nutrition: who, when, and why should we supplement? Gastroenterology 2009; 137: S29–S35.

4. Menezes-Filho JA, Bouchard M, Sarcinelli P, Moreira JC. Manganese exposure and the neuropsychological effect on children and adolescents: a review. Rev Panam Salud Publica/Pan AM Public Health 2009; 26: 541–8.

5. Wood R. Manganese and birth outcome. Nutr Rev 2009; 67: 416–20.

6. Standing Committee on the Scientific Evaluation of Dietary Reference Intakes, Food and Nutrition Board. Dietary reference intakes for vitamin A, vitamin K arsenic, boron, chromium, copper, iodine, iron, manganese, molybdenum, nickel, silicon, vanadium, and zinc. Institute of Medicine. Washington, DC: National Academy of Sciences; 2001.

7. Rucker D, Thadhani R, Tonelli M. Trace element status in hemodialysis patients. Semin Dial 2010; 23: 389–95.

8. Finley JW. Manganese absorption and retention by young women is associated with serum ferritin concentration. Am J Clin Nutr 1999; 70: 37–43.

9. Aschner J, Aschner M. Nutritional aspects of manganese homeostasis. Mol Aspects Med 2005; 26: 352–62.

10. Guilarte TR. Manganese and Parkinson’s disease: a critical review and new findings. Environ Health Perspect 2010; 118: 1071–80.

11. Lucchini RG, Aschner M, Landrigan PJ, Cranmer JM. Neurotoxicity of manganese: inducations for future research and public health intervention from manganese 2016 conference. Neurotoxicology 2018; 64: 1–4.

12. HO CSH, Ho RCM, Quek AML. Chronic manganese toxicity associated with voltage-gated potassium channel complex antibodies in a relapsing neuropsychiatric disorder. Int j Environ Res Public Health 2018; !5. doi: 10.3390/ijerph15040783.

13. Burton NC, Guilarte TR. Manganese neurotoxicity: lessons learned from longitudinal studies in nonhuman primates. Environ Health Perspect 2009; 117: 325–32.

14. Menezes-Filho JA, Bouchard M, Sarcinelli P, Moreira JC. Manganese exposure and the neuropsychological effect on children and adolescents: a review. Rev Panam Salud Publica/Pan AM Public Health 2009; 26: 541–8.

15. Takser L, Mergler D, Hellier G, Sahuquillo J, Huel G. Manganese, monoamine metabolite levels at birth, and child psychomotor development. Neurotoxicology 2003; 24: 667–74.

16. Dickerson RN. Manganese intoxication and parenteral nutrition. Nutrition 2001; 17: 689–93.

17. McMillan MB, Mulroy C, MacKay NW, et al. Correlation of cholestasis with serum copper and whole blood manganese levels in pediatric patients. Nutr Clin Pract 2008; 23: 161–5.

10.7 Molybdenum (Mo)

Lothar Thomas

Molybdenum is a transition element and can be present in the oxidation states Mo (II) to Mo (VI). The most important oxidation state Mo (VI) is present, for example, in salts of molybdic acids (molybdates). Mo (V) and Mo (IV) are other important oxidation states. The oxidation states Mo (III) and Mo (II) are expressed in clusters of molybdenum (III) chloride and molybdenum (II) chloride. Mo is very abundant in the oceans in the form of [MoO2]4– anion. The abundance of Mo in the earth’s crust is 1.1 × 10–4 percent.

10.7.1 Indication

None because no clinical symptoms of deficiency or body Mo burden have been identified. Mo was only included in this chapter because it is an essential trace element.

10.7.2 Method of determination

Refer to Section 10.1.4 – Method of determination.

10.7.3 Specimen

  • Plasma or serum: 1 mL
  • 24 h urine specifying the collected volume: 5 mL

10.7.4 Reference interval

Refer to Ref /1/ and Tab. 10.7-1 – Reference intervals for molybdenum.

10.7.5 Clinical significance

Mo is an essential element for plants, animals and microorganisms. Its anionic form is available for the organisms in the soil and in sea water. Thus, because of its abundant availability in food for man, nutritional deficiency conditions are not to be expected.

Mo exposure can be detected based on urinary Mo excretion /2/. Specific clinical symptoms of increased body Mo burden have not been identified to date.

10.7.6 Comments and problems

Pre analytic phase

The high environmental concentration of Mo pose a significant risk of contamination during sampling, storage and sample processing.

Blood sampling

Plasma/serum Mo concentration increases already in mild hemolysis because of the high Mo content in erythrocytes.

Stability in serum/plasma

If tightly sealed, samples can be stored at 4 °C for 2 weeks.

10.7.7 Pathophysiology

The body Mo stores in adults are 8–10 mg and mostly reside in the skeleton (60%) and liver (20%) /3/. The daily recommended intake is 75–250 μg for adults and 2 μg/kg of body weight for children /4/.

Mo is enterally absorbed in the anion form via a transport system that also handles other metal ions. In the circulation, Mo is bound to erythrocytes and in plasma, it is primarily bound to α2-globulins. Mo is to a large extent excreted via the kidneys.

Mo is biologically inactive in the cell unless it is complexed by the molybdenum cofactor (Moco), a tricyclic pterin compound /5/. The pterin structure of Moco is unique in nature and has probably been evolved to control and maintain the specific redox properties of Mo. The task of Moco is to position the catalytic element Mo correctly within the active center of enzymes, to control its redox behavior and to participate with its pterin ring system in the electron transfer to or from the Mo atom. A mutational block of Moco biosynthesis leads to essential loss of metabolic body functions.

The human body has four Mo-enzymes /5/:

  • Aldehyde oxidase (AO). The AO catalyzes the oxidation of a variety of aromatic and non-aromatic heterocycles and aldehydes, thereby converting them to the respective carboxylic acid.
  • Xanthine dehydrogenase (XDH). XDH is a key enzyme for purine degradation and oxidizes hypoxanthine to xanthine and xanthine to uric acid by simultaneous release of electrons from the substrate (see Section 5.4 – Uric acid). The congenital absence of xanthine oxidase leads to xanthinuria concomitant with hypouricemia and elevated xanthine and hypoxanthine concentrations in the blood and urine triggering the development of xanthine stones and myopathy.
  • Sulfite oxidase (SO). The enzyme catalyzes the oxidation from sulfite to sulfate, the final step in the degradation of sulfur-containing amino acids.
  • Nitrate reductase (NR), a key enzyme of nitrate assimilation. NR does not occur in animals and catalyzes the reduction of nitrate to nitrite in the cytosol. Sulfite oxidase deficiency

The sulfite oxidase (SO), an enzyme containing the Mo cofactor (Moco), catalyzes the oxidation of sulfite to sulfate, the final step in the degradation of sulfur-containing amino acids /6/. SO resides in the inter membrane space of mitochondria, where it exists as a homo dimer. Upon oxidation of sulfite, Mo(VI) is reduced to Mo(IV) by two electrons. The electrons are subsequently transferred to the heme Fe(III) in the cytochrome b5 domain in a two-step reaction, which is followed by a transfer of the electrons from Fe(II) to cytochrome c. The SO-catalyzed reaction is vital for humans, because the deficiency of this enzyme leads to severe neurological abnormalities and premature death. Clinical symptoms of SO deficiency include dislocation of ocular lenses, mental retardation and attenuated growth of the brain. Those affected usually die at 1–2 years of age. SO deficiency can result from:

  • Mutation of the MOCS1 gene (type A deficiency), MOCS2 gene (type B deficiency) or GPHN gene. All of these genes encode the cofactor Moco /7/.
  • Mutation in the SO-encoding gene. Significant changes in the substrate-binding pocket of SO have been detected /6/.


1. Brätter P, Forth W, Fresenius W, Holtmeier HJ, Hoyer S, Kruse-Jarres JD, Liesen H, Mohn L, Negretti de Brätter V, Reichlmayr-Lais AM, Sitzer G, Tölg G. Mineralstoffe und Spurenelemente: Leitfaden für die ärztliche Praxis. Gütersloh; Bertelsmann Stiftung 1992: 151.

2. Bertram HP. Spurenelemente. Analytik, ökotoxikologische und medizinisch-klinische Bedeutung. München; Urban & Schwarzenberg 1992: 12–59.

3. Novotny JA, Pterson CA. Molybdenum. Adv Nutr 2018; 9: 272–3.

4. Deutsche Gesellschaft für Ernährung (DGE), Österreichische Gesellschaft für Ernährung (ÖGE), Schweizerische Gesellschaft für Ernährungsforschung (SGE). Referenzwerte für die Nährstoffzufuhr. Frankfurt; Umschau Brauns GmbH, 2000.

5. Mendel RR, Bittner F. Cell biology of molybdenum. Biochim Biophys Acta 2006; 1763: 621–35.

6. Karakas E, Wilson HL, Graf TN, Xiang S, Jaramillo-Busquets S, Rajagopalan KV, Kisker C. Structural insights into sulfite oxidase deficiency. J Biol Chem 2005; 280: 33506–15.

7. Reiss J, Hahnewald R. Molybdenum cofactor deficiency: mutations in GPHN, MOCS1 and MOCS2. Hum Mutat 2010; Oct 28.

10.8 Nickel (Ni)

Lothar Thomas

Ni occurs in the oxidation states Ni(I), Ni(II) and Ni(III). Ni(II) is the most common form in biosystems. Ni is the 24th most abundant element in the earth’s crust. Dissolved Ni2+ are hydrated in aqueous solution at neutral pH to green hexahydrate [Ni(H2O)6]2+. The fact that no Ni-containing enzymes or cofactors have been found in higher organisms has raised more doubt about Ni being an essential trace element.

10.8.1 Indication

Suspected Ni intoxication.

10.8.2 Method of determination

Refer to Section 10.1.4 – Method of determination.

10.8.3 Specimen

  • Plasma or serum: 1 mL
  • 24 h urine specifying the collected volume: 5 mL

10.8.4 Reference interval

Refer references /12/ and to Tab. 10.8-1 – Reference intervals for nickel.

10.8.5 Clinical significance

Ni is found widely in the environment and is an essential micro element but toxic. Ni is enough in diet one imagines. Nickel and contact allergy

Ni is the most common contact allergen /3/. Ni is everywhere. It is present in drinking water, food, jewelry, coins, spectacle frames, dental fillings, prostheses, buttons, zippers, tools, alkaline batteries, insecticides, dyes, pigments, nickel-plated items and fuel additives. The prevalence of Ni allergy is 8–28% in women and 1–5% in men. Prolonged contact with Ni-containing objects leads to localized dermatitis. Piercing is also an important risk factor for Ni allergy.

According to the Nickel Directive of the European Union, Ni containing products coming into prolonged contact with the skin may not release more than 0.2 μg/cm2/week of Ni.

As a rule, contact allergies are not associated with increased Ni concentrations in serum or increased urinary Ni excretion. For example, population based studies in a Russian refinery found a median urinary Ni concentration of 3.4 μg/L, while a Norwegian population 10 kilometers away only had a concentration of 0.6 μg/L. However, the incidence of Ni allergy was higher in the Norwegian population /4/. Nickel deficiency

There is no Ni deficiency in humans because of the wide distribution of Ni in the soil, water, air and food. Daily dietary Ni intake has been reported differently as 35 μg, 100–300 μg or 25–35 μg /5/. In general, the daily Ni intake is more than triple the daily requirement. Toxicity of Nickel

Epidemiological studies have provided evidence of an increased risk of respiratory and nasal cancer in miners and workers at nickel smelters /5/. Therefore, the International Committee on Nickel Carcinogenesis in Man suggested that respiratory cancer risks are primarily related to exposure to soluble Ni concentrations above 1 mg/m3 and to exposure to less soluble forms at concentrations above 10 mg/m3 /6/. About 2% of the work force in Ni-related industries is exposed to airborne Ni-containing particles in concentrations ranging from 0.1–1 mg/m3. Ni intoxication results mainly from exposure to Ni(CO)4.

The lung has the general tendency of storing Ni irrespective of the way it enters the body. Inhaled Ni particles taken in via the respiratory tract, for example, remain in the lungs for an extended period of time. Transfer to the blood is slow; hence, elevated concentrations in blood are only measured under high body Ni burden. Urinary excretion depends on the body burden. Excretions above 30 μg/L (510 nmol/L) in workers at Ni-processing plants indicate significant occupational exposure /7/. The half-life of renal Ni excretion is 20–60 hours.

10.8.6 Comments and problems

Pre analytic phase

The relatively high environmental concentrations of Ni pose a significant risk of contamination during sampling, storage and sample processing.


The urinary excretion of Ni is rapid and elimination appears to follow first-order kinetics without evidence of dose-dependent excretion. The half-life of urinary removal of Ni range from 20 to 60 hours /5/.

Stability in serum/plasma

If tightly sealed, samples can be stored at 4 °C for 2 weeks.

10.8.7 Pathophysiology

Ni is an essential catalytic cofactor for enzymes in eubacteria, archibacteria, fungi and plants. These enzymes catalyze a diverse array of reactions that include both redox and non-redox chemistries and allow organisms to inhabit a diverse range of environmental niches /8/. Ni proteins are key factors in the one-carbon metabolism of methanotrophs and methanogens, thus playing an important role in global carbon cycle /8/. Seven of the eight known Ni enzymes involve the use and/or production of gases(CO, CO2, methane, H2, ammonia and O2) that play important roles in the global biological carbon, nitrogen, and oxygen cycles /9/. However, no Ni deficiency has been identified except in Ni-hyper accumulating plants and possibly oceanic plankton.

The amount of Ni in individuals not exposed to Ni is approximately 7.3 μg/kg of body weight. Intake is by inhalation, ingestion and, to a small extent, via the skin. Enteral intake depends on the form of Ni in the food. Usually, 1–2% of the Ni contained in food are absorbed. Soluble Ni enters the cell by diffusion and presumably through Ca2+ channels, and insoluble Ni enters by phagocytosis. Ni is retained by the lungs, kidneys, brain and pancreas and excreted renally or with the stool /5/.

Sources of environmental Ni burden relate to Ni production and processing and recycling of Ni-containing products. Ni also occurs naturally in the soil and air and is emitted as oxide, sulfide, silicate, in soluble form and, to a lesser extent, in elemental form. Airborne Ni concentration in industrial areas is 120–170 ng/m3 and in non-industrial areas 6–17 ng/m3.

Acute Ni intoxication is rare. Toxic and carcinogenic effects of Ni are significant in occupational medicine:

  • If inhaled, Ni dusts and salts acutely cause interstitial pneumonia, pulmonary edema and hemorrhage, later possibly followed by hepatic and renal failure and hematological complications /10/.
  • Ni compounds are carcinogenic. Ni is thought to cause gene inactivation by inducing DNA methylation. A proposed mechanism of Ni-induced DNA hyper methylation includes the ability of Ni to substitute for magnesium in the DNA backbone. In support of epigenetic mechanisms, it has been reported that Ni inhibits histone H3K9 methylases that depend on Fe and 2-oxoglutarate for their enzymatic activity and, as a result, cause an increase in global H3K9 methylation /11/.


1. Schaller KH, Raithel HJ, Angerer J. Nickel. In: Seiler HG, Sigel A, Sigel H (eds). Metals in clinical and analytical chemistry. New York; Marcel Dekker 1994: 505–18.

2. Mückter H. Nickel. In: Biesalski HK, Köhrle J, Schümann K (eds). Vitamine, Spurenelemente und Mineralstoffe. Stuttgart; Thieme 2002: 194–8.

3. Schram SE, Warshaw EM, Laumann A. Nickel hypersensitivity: a clinical review and call to action. Int J Dermatol 2010; 49: 115–25.

4. Smith-Sivertsen T, Tchachtchine V, Lund E, et al. Urinary nickel excretion in populations living in the proximity of two Russian nickel refineries: a Norwegian-Russian population based study. Environ Health Perspect 1998; 106: 503–11.

5. Denkhaus E, Salnikow K. Nickel essentiality, toxicity, and carcinogenicity. Crit Rev Oncol/Hematol 2002; 42: 35–56.

6. International Committee on Nickel Carcinogenesis in Man. Report of the International Committee on Nickel Carcinogenesis in Man. Scand J Work Environ Health 1990; 6: 1–82.

7. Ulrich L, Sulcova M, Spacek L, Neumanova E, Vladar M. Investigation of professional nickel exposure in nickel refinery workers. Sci Total Environ 1991; 101: 91–6.

8. Li Y, Zamble DB. Nickel homeostasis and nickel regulation: an overview. Chem Rev 2009; 109: 4617–43.

9. Ragsdale SW. Nickel-based enzyme systems. J Biol Chem 2009; 284: 18571–5.

10. Sunderman Jr FW, Dingle B, Hopfer SM, Swift T. Acute nickel toxicity in electroplating workers who accidently ingested a solution of nickel sulfate and nickel chloride. Amer J Industr Med 1988; 14: 257–66.

11. Arita A, Costa M. Epigenetics in metal carcinogenesis: nickel, arsenic, chromium and cadmium. Metallomics 2009; 1: 222–8.

10.9 Selenium (Se)

Lothar Thomas

Se is a metalloid element preferably occurring in the oxidation states –2, +4 and +6 and found in the biologically and technically important species listed in Tab. 10.9-1 – Biologically and technically important selenium species. The abundance of Se in the earth’s crust is 9 × 10–6 percent. Selenides have the oxidation state –2, selenites have +4 and selenates have +6 (Tab. 10.9-1).

10.9.1 Indication

Se deficiency is suspected:

  • Under diets or long-term parenteral nutrition
  • In malabsorption (Crohn’s disease, celiac disease, intestinal resection)
  • In vegetarians, alcoholics
  • In non-specific symptoms such as fatigue, low performance, hair loss, arthritis, whitening of fingernails.

Chronic Se intoxication is suspected:

  • In garlic-like breath, spotted streaked fingernails, gastrointestinal complaints, hyperreflexia, pain in the extremities.

Acute Se intoxication is suspected:

  • In vomiting, diarrhea, garlic-like breath, muscle spasms, metabolic acidosis.

10.9.2 Method of determination

Methodologies used for assessing the Se status comprise direct determination of Se or determination of the glutathione peroxidase activity. Direct determination of Se

Currently, most clinical analyses for Se continue to be performed using graphite furnace atomic absorption spectrometry and molecular fluorescence spectrometry /1/. Determination of glutathione peroxidase activity

Principle: the glutathione peroxidase (GP) catalyzes the glutathione (GSH) dependent reduction of organic peroxides and H2O2. Hydrogen is transferred from GSH to H2O2 and GSH is oxidized to GSSG. The catalytic center of GP contains L-selenocystein.

2 GSH + H 2 O 2 GP GSSG + 2 H 2 O

In the next step GSSG is regenerated to GSH by glutathione reductase (GR).


NADPH consumption is the indicator of GR activity. The Se-free GP only reacts with organic hydro peroxides such as cumolhydroperoxide. The total GP activity is determined first using H2O2 and then the Se-free GP is determined using the substrate cumolhydroperoxide /2/.

10.9.3 Specimen

  • Serum/plasma, whole blood, erythrocytes: 1 mL each
  • 24 h urine specifying the collected volume: 5 mL

10.9.4 Reference interval

Refer to Ref. /3/ and Tab. 10.9-2 – Reference intervals for selenium.

10.9.5 Clinical significance

Se, like many other trace elements, has a bimodal effect; its beneficial effects occur in a limited range of daily intake, below which it cannot perform its essential function and above which its effects are toxic. The Se status is best diagnosed by determining the Se level in plasma/serum, whole blood or erythrocytes (with reference to hemoglobin).

The following determinations are indicated /13/:

  • Se level in plasma/serum for assessment of the current status
  • Se concentration in whole blood and urine as long-term parameters in selenosis
  • Urinary Se excretion and plasma Se concentration in suspected acute Se intoxication. Selenium concentration in plasma/serum

Se is both an essential and toxic element /3/. Therefore, the two extremes of distribution are of medical interest. The lowest plasma Se levels have been reported from Se-depleted regions in China and lead to Keshan disease. The highest concentrations associated with intoxication symptoms have been reported from Enshi, likewise a region in China, and result from excessive dietary Se intake. Mean plasma Se concentrations differ between the continents; they are 197 μg/L (2.5 μmol/L) in the USA and 41 μg/L (0.52 μmol/L) in Serbia. There are also great regional differences, for example 90 to 197 μg/L (1.15 to 2.5 μmol/L) in the USA. In Europe, the lowest concentrations have been measured in Eastern Europe and the highest ones in Norway at 114–131 μg/L (1.45–1.67 μmol/L) /5/. In Germany, the mean levels are 70–80 μg/L (0.89–1.0 μmol/L) /3/.

The Se concentration reflects the amount of Se bound to selenoproteins, especially selenoprotein P that binds more than 50% of the Se in plasma. At a concentration of up to 70 μg/L (0.89 μmol/L), the plasma Se concentration correlates to the Se intake. At higher concentrations, the Se-binding proteins reach a saturation plateau indicating that the Se requirement is met /6/.

Plasma measurements of Se need to be treated with some caution because organic Se in the form of selenomethionine may be incorporated nonspecifically into proteins in place of methionine, without reflecting a change in the Se loading of the transport proteins and without ensuring that the Se requirement of the tissues is met /7/.

Trauma and systemic inflammation cause a decrease in Se concentration because Se is bound to negative acute phase proteins. Therefore, the serum Se level does not reflect the Se status in such conditions /7/. Glutathione peroxidase activity in plasma

Glutathione peroxidase activity in plasma (GSH Px-3) is a good biomarker for assessing optimal Se supply, although it only represents 20% of the plasma Se concentration /8/. Its activity changes quickly as a function of changed Se intake. Normal levels reflect a serum Se concentration above 90 μg/L (1.1 μmol/L), corresponding to a daily Se intake of at least 200 μg /9/. Selenium supply

Se is provided to the body almost exclusively by dietary intake. In Germany, the requirement is met mainly through the consumption of sausage and meat products. According to an estimate of the German Nutrition Society, daily Se intake is 50 μg in men and 45 μg in women. Most Se ingested through dietary intake is present as selenomethionine or selenocysteine. Both are 90% absorbed. Inorganic Se is present as selenite or selenate and 50–90% absorbed.

Minimum Se supply is 20 μg/day to prevent severe deficiency such as Keshan disease. In the USA, a Se intake of 55 μg/day is stipulated to ensure optimal functioning of selenoproteins, and in New Zealand, an intake of 68 μg/day is stipulated to ensure optimal GSHPx-3 activity /7/. The supplementation of Se in chronic metabolic diseases and the anti oxidative role of nutritional supplementation of Se is presented in Ref. /19/

A scheme for diagnosing Se deficiency is shown in Tab. 10.9-3 – Diagnosis of selenium deficiency.

The substitution of Se as a function of plasma concentration is presented in Tab. 10.9-4 – Selenium status and indication of selenium administration. Selenium deficiency diseases

Typical diseases for nutritional Se deficiency with a Se intake below 20 μg/day include juvenile cardiomyopathy (Keshan disease) and destructive osteoarthropathy (Keshan-Beck disease). Se under supply can occur under parenteral nutrition, under diets without adequate Se supplementation, in malabsorption in the setting of Crohn’s disease, celiac disease, intestinal resection and in vegetarians and alcoholics. Besides these Se deficiency diseases, there are diseases and situations with increased Se requirement where the clinical situation can be improved by Se substitution (Tab. 10.9-5 – Diseases and conditions associated with selenium deficiency or toxicity). However, some diseases associated with Se deficiency in single studies have not been confirmed by meta analysis. Toxicity of selenium

The toxicity of selenomethionine is lower than that of inorganic Se compounds. Because of the relative narrow therapeutic range of Se, excessive supplementation should also be taken into account. High plasma Se concentrations are helpful in the detection of potential toxicity, but there is lack of evidence regarding direct correlation between plasma concentration and intake. For Se toxicity, see Tab. 10.9-5 – Diseases and conditions associated with selenium deficiency or toxicity.

10.9.6 Comments and problems

Pre analytic phase

The risk of contamination during sampling and sample processing is low.

Reference interval

Due to significant geographical variation of dietary Se intake, one universal reference interval for plasma/serum Se concentration was not feasible because the concentration primarily depends on nutritional intake, geographical region and regional span of life /5/.

Reliability of determination

The analytical coefficient of variation should not exceed 12% /10/.

Stability in serum/plasma

If tightly sealed, samples can be stored at 4 °C for 2 weeks.

10.9.7 Pathophysiology

Se is essential for human beings and mammals in the biosynthesis of selenocysteine, also referred to as amino acid 21. The essential role of this amino acid is reflected by the existence of a specific transfer RNA for the incorporation of selenocysteine into mammal proteins /11/. At least 25 selenoproteins have been identified to date (Tab. 10.9-6 – Selenoproteins and their function).

The adult body’s Se content is 10–15 mg; endocrine organs, gonads, brain and red meat have the highest Se concentration. Human dietary Se intake is in the form of Se amino acids (selenomethionine, selenocysteine, selenocystine) and less in methylated or inorganic forms. Se containing amino acids, especially selenomethionine, have a high bioavailability. The bioavailability of Se contained in cereal products, wheat and vegetables is 85–100% and higher than in meat and dairy products. The Se level in fish is high and the bioavailability is 20–50%.

Selenates are reduced to selenites; these are reduced to selenium hydrogen in the blood and by erythrocytes and converted into selenides (Tab. 10.9-1 – Biologically and technically important selenium species).

Selenides are the key metabolic Se species and the precursor of selenocysteine. Selenides are transferred to mono-, di- and trimethylated compounds. Trimethylated compounds can be eliminated via urinary excretion, dimethylated ones via respiration and monomethylated ones are released in the course of the methylmethionine metabolism /12/.

Se from vegetal food is mainly absorbed in the form of selenomethionine and, after distribution, unsystematically incorporated into Se-containing proteins (albumin, hemoglobin) instead of methionine. It is accumulated for long-term (months) storage. Selenomethionine not used for protein biosynthesis is degraded to methionine producing selenocysteine.

Selenocysteine is not incorporated into proteins instead of cysteine. It is produced from selenides from Se transfer to the amino acid serine. Selenocysteine is then incorporated into Se-dependent proteins by mediation of a specific t-RNA. These proteins are responsible for redox system homeostasis, regulation of transcription factors and the formation and inactivation of peripheral thyroid hormones (Tab. 10.9-5 – Diseases and conditions associated with selenium deficiency or toxicity).

Four isoenzymes have been identified to date as components of glutathione peroxidase (GPX). They catalyze the reduction of H2O2 and lipid peroxides (ROOH) to water and the corresponding alcohols forming oxidized glutathione. There is a functional relation with super oxide dis mutase. The latter reduces the O2 radicals to peroxides that are then deactivated by the GPX (see Section 19.2 – Oxidative stress).

The thioredoxin reductases reduce, and thus regenerate, oxidized thiol groups and other oxidant systems such as vitamin C and vitamin E. They have an important function in the protection of the cells against oxygen and its reactive species.


1. Sheehan TMT, Halls DJ. Measurement of selenium in clinical specimens. Ann Clin Biochem 1999; 36: 301–15.

2. Günzler W, Kremers W, Flohe L. Hoppe Seyler’s Z Physiol Chem 1972; 353: 987–99.

3. Mitteilung der Kommission Methoden und Qualitätssicherung in der Umweltmedizin. Selen in der Umwelt. Bundesgesundheitsbl-Gesundheitsforsch-Gesundheitsschutz 2006; 49: 88–102.

4. Muntau AC, Streiter M, Kappler M, Röschinger W, Schmid I, Rehnert A, et al. Age related reference values for serum selenium concentrations in infants and children. Clin Chem 2002; 48: 555–60.

5. Alfthan G, Neve J. Reference values for serum selenium in various areas-evaluated according to the TRACY protocol. J Trace Elem Med Biol 1996; 10: 77–87.

6. Food and Nutrition Board IoM. Dietary reference intakes for vitamin C, vitamin E, selenium, and carorenoids. Washington DC: National Academic Press: 2000.

7. Shenkin A. Selenium in intravenous nutrition. Gastroenterology 2009; 137: S61–S69.

8. Cohen HJ, Chovaniec ME, Mistretta D, et al. Selenium repletion and glutathione peroxidase-differential effects on plasma and red cell activity. Am J Clin Nutr 1985; 41: 735–47.

9. Rayman M. The importance of selenium to human health. The Lancet 2000; 356: 233–41.

10. Arnaud J, Weber JP, Weykamp CW, Parsons PJ, Angerer J, Mairiaux E, et al. Quality specifications for the determination of copper, zinc, and selenium in human serum and plasma: evaluation of an approach based on biological and analytical variation. Clin Chem 2008; 54: 1892–9.

11. Kryukov GV, Castellano S, Novoselov SV, et al. Characterization of mammalian selenoproteins. Science 2003; 300: 1439–43.

12. Alaejos MS, Diaz-Romero FJ, Diaz-Romero C. Selenium and cancer: some nutritional aspects. Nutrition 2000; 16: 376–83.

13. Yang GQ. Keshan Disease: An endemic selenium-related deficiency disease. In: Chandra, RK. Trace elements in nutrition of children, Nestle Nutrition Workshop Series, Vol. 8. New York; Raven Press 1985: 273.

14. Lippman SM, Klein EA, Goodman PJ, et al. Effect of selenium and vitamin E on risk of prostate cancer and other cancers: the Selenium and Vitamin E Cancer Prevention Trial (SELECT). JAMA 2009; 276; 1957–63.

15. Baum MK, Shor-Posner G, Lai S, Quesada JA, Campa A, Jose-Burbano M, et al. High risk of HIV-related mortality is associated with selenium deficiency. J Acquir Immune Defic Syndr 1997; 15: 370–4.

16. Gärtner R. Die medizinische Bedeutung von Selen. J Lab Med 2006; 30: 201–8.

17. Moncayo R, Moncayo H, Kapelari K. Nutritional treatment of incipient thyroid autoimmune disease. Influence of selenium supplementation on thyroid function and morphology in children and young adults. Clin Nutr 2005; 24: 530–1.

18. Yang G, Zhou R. Further observations on the human maximum safe dietary selenium intake in a seleniferous area of China. J Trace Elem Electrolytes Health Dis 1994; 8: 159–65.

19. Wang N, Tan HY, Li S, Xu Y, Guoo W, Feng Y. Supplementation of micronutrient selenium in metabolic diseases: ist role as an antioxidant. Oxidative Medicind and Cellular Longlevity 2017. doi: 10.1155/2017/7478523.

20. Kawai M, Shoji Y, Onima S, Etani Y, Ida S. Thyroid hormone status in patients with severe selenium deficiency. Clin Pediatr Endocrinol 2018; 27: 67–74.

10.10 Zinc (Zn)

Lothar Thomas

Zn is a heavy metal, occurs in the divalent form and is one of the most important trace elements in the body. Zn is the 23rd most abundant element in the earth’s crust. The Zn2+level in sea-water about 10–8 mol/L and is about 10–9 molar in mammalian cells and 10–3 molar in cell organelles /1/. The risk of free radical binding is low because of a highly conserved and non-variable valence. Zn2+ is a good electron acceptor. The most important zinc compound used in smelting is zinc sulfide, which occurs as cubic sphalerite (zinc blend) and hexagonal wurtzite.

10.10.1 Indication

Zinc deficiency is suspected in:

  • Under supply of trace elements in general
  • Diarrhea
  • Hemodialysis
  • Therapy-resistant dermatosis.

Suspected intoxication due to occupational exposure.

10.10.2 Method of determination

Refer to Ref. /1/ and Section 10.1.4 – Method of determination.

10.10.3 Specimen

  • Plasma or serum: 1 mL
  • 24 h urine specifying the collected volume: 5 mL

10.10.4 Reference interval

Refer to Ref. /23/ and Tab. 10.10-1 – Reference intervals for zinc.

10.10.5 Clinical significance

Zn is ubiquitous in the tissues. About 86% reside in the muscles and high contents are found in the prostate, pancreas, cortex of the kidney and hippocampus. Zn is a constituent part of about 120 enzymes such as carbonic anhydrases, carboxypeptidases, alkaline phosphatases, oxidoreductases, transferases, ligases, hydrolases and isomerases /4/. Furthermore, Zn is incorporated in the zinc fingers that control genes via transcriptional activation and repression. Thus, Zn influences the amino acid and protein metabolism. Although an alteration in one of these functions usually does not result in specific disease, Zn is of systemic significance. Zinc deficiency

Zn is an important part of DNA polymerase, reverse transcriptase, RNA polymerase, t-RNA synthetase and the protein elongation factor /4/. Zn deficiency leads to growth delay and teratogenic effects such as occurring in acrodermatitis enteropathica. Since dysfunctions in protein biosynthesis and cellular mechanisms also occur in the presence of Zn deficiency, global functions such as growth, cellular immunity, fertility and wound healing are affected.

Under nutritional deficiency and catabolic conditions with gradual negative Zn balance, the serum concentration is kept relatively constant by Zn release from muscle tissue, but decrease in diarrhea because of rapid losses. Therefore, Zn supplementation is required in increased gastrointestinal losses, hyper catabolism and under amino acid infusions.

The regulation of plasma Zn levels usually ranges within 0.8–1.2 mg/L (12–18 μmol/L). In many cases, Zn deficiency in the blood is not detected for a long time because the muscles and bones have ample reserves, although they do not actually function as storage tissues for this element. Therefore, there is no correlation between the serum Zn level and the concentration in muscle tissue /5/. Accordingly, the plasma concentration depends on numerous factors and is neither correlated with alimentary depletion nor with tissue Zn concentration. Thus, plasma and serum are only to a limited extent suitable for determination of the Zn status. Moreover, the serum Zn level decreases in acute phase reaction.

Diseases and conditions associated with Zn deficiency are shown in Tab. 10.10-2 – Diseases and conditions associated with zinc deficiency or toxicity. Zinc intoxication

Compared to several other metal ions with similar chemical properties, zinc intoxication is relatively harmless because efficient regulatory mechanisms prevent the uptake of cytotoxic doses of exogenous zinc /6/. For further information, see Tab. 10.10-2 – Diseases and conditions associated with zinc deficiency or toxicity.

10.10.6 Comments and problems

Pre analytic phase

The high environmental concentrations of Zn pose a risk of contamination during sampling, storage and sample processing. If the blood cells are not separated following centrifugation, the serum Zn level increases by approximately 6% per hour.

Blood sampling

It is recommended to use metal-free blood collection assemblies for blood sampling and to pre-rinse all equipment (containers, pipette tips, measuring cups) coming into contact with the sample with acid /7/. The tourniquet should not stop blood flow in the veins for more than a minute before blood is drawn. Compression increases the Zn-binding proteins and elevated Zn concentrations are measured.


Hemolysis leads to the release of Zn from the erythrocytes that contain more than 10 times the concentration of Zn than plasma /8/. The blood should be collected under fasting conditions because the plasma Zn level decreases following food intake.

Reliability of determination

The analytical coefficient of variation should not exceed 12% /9/.

Stability in serum/plasma

If tightly sealed, samples can be stored at 4 °C for 2 weeks.

10.10.7 Pathophysiology

Zn is subject to efficient homeostatic control. The human body Zn content is 2–3 g and resides primarily in the muscles, bones, liver, kidneys, hematopoietic system, skin and thymus. On a cellular level, 30–40% are localized in the nucleus, 50% in the cytoplasm and the rest in the membranes. Cellular Zn homeostasis is regulated by two transporter families, the Zn importers (Zip) transporting Zn into the cytosol and the Zn transporters (ZnT) handling outward transport /6/.

Oral uptake of Zn leads to absorption throughout the enterocytes of the small intestine, presumably via the divalent metal ion transporter, and bound to metallothionein (MT). Absorption is regulated by MT and influenced by other essential elements such as Ca, Cu, Mn, Fe and Ni /10/. The MTs play an important role in Zn homeostasis because they are able to form complexes with 20% of the intracellular Zn. MTs have a molecular weight of 6–7 kDa, are cysteine-rich and 1 MT molecule can bind up to 7 Zn2+. Cu ions are bound by MTs with a higher affinity than Zn2+. Under long-term oral Zn substitution in doses above 180 mg/day, Cu deficiency can occur due to competition between Cu and Zn. However, this is only relevant to absorption if the amount of Zn in the nutrition is more than 5-fold the amount of Cu.

In plasma, Zn is bound to the negative acute-phase proteins albumin, transferrin and α2-macroglobulin and is transported to the liver. Zn that is not released to these proteins from the enterocytes remains bound to MTs and is lost with the feces in enterocyte apoptosis. Thus, non-required absorbed Zn is eliminated in this way and a small fraction is excreted renally.

Zn plays an important role in the regulation of cell apoptosis and can either be pro- or anti-apoptotic. Accumulation of intracellular Zn, either as a result of exogenous administration or release from intracellular stores by reactive oxygen species or NO activates pro-apoptotic molecules like p38 or K+ channels leading to cell death /6/.

The anti-apoptotic effect is thought to be based on interaction between Zn and apoptosis-regulating proteins; Zn is, for example, a caspase inhibitor.

Zn is a constituent part of the zinc finger proteins /11/. The expression reservoir of all the genes is governed by transcriptional activators and repressors that bind to specific sites in the genome. There are several protein folds that can elicit sequence specific DNA binding, including helix-turn-helix, leucine zipper and zinc finger domains. The C2H2 zinc finger motif comprises 20–30 amino acids containing two cysteine and histidine residues coordinated by a zinc atom /11/.

Zn is present as a divalent cation and does not require redox reaction during the intestinal membrane transport process as observed for iron and copper. Enterocyte Zn influx and efflux are controlled by two Zn transporter families, Zn transporter (ZNT) and Zrt- and Irt-related protein (ZIP) /24/.


1. Fabris N, Moccnegiani E. Zinc, human disease and aging. Aging Clin Exp Res 1995; 7: 77–93.

2. Brätter P, Heseker H, Kruse-Jarres JD, Liesen H, Negretti de Brätter V, Pietrzik K, Schümann K. Mineralstoffe, Spurenelemente und Vitamine: Leitfaden für die ärztliche Praxis. Gütersloh; Bertelsmann Stiftung 2002: 63–125.

3. Elsenhans B. Zink. In: Biesalski HK, Köhrle J, Schümann K (eds). Vitamine, Spurenelemente und Mineralstoffe. Stuttgart; Thieme, 2002: 151–60.

4. Kimura E, Kikuta E. Why zinc in zinc enzymes? From biological roles to DNA-base-selective recognition. JBIC 2000; 5: 139–55.

5. Ladefoget K, Hagen K. Correlation between concentrations of magnesium, zinc and potassium in plasma, erythrocytes, and muscles. Clin Chim Acta 1988; 177: 157–66.

6. Plum LM, Rink L, Haase H. The essential toxin: impact of zinc on human health. Int J Environ Res Public Health 2010; 7: 1342–65.

7. Rükgauer M, Schmitt Y, Schneider H, Kruse-Jarres JD. Trace element balance in patients undergoing hemodialysis. Trace Elem Electrolytes 1994; 11: 155–68.

8. Oster O, Huesgen G, Prellwitz W. Präanalytische und analytische Probleme einer teilmechanisierten photometrischen Serumzinkbestimmung. Ärztl Lab 1987; 33: 177–85.

9. Arnaud J, Weber JP, Weykamp CW, Parsons PJ, Angerer J, Mairiaux E, et al. Quality specifications for the determination of copper, zinc, and selenium in human serum and plasma: evaluation of an approach based on biological and analytical variation. Clin Chem 2008; 54: 1892–9.

10. Sanstead HH. Understanding zinc: Recent observations and interpretations. J Lab Clin Med 1994; 124: 322–7.

11. Davis D, Stokoe D. Zinc finger nucleases as tools to understand and treat human diseases. BMC Med 2010: 8: 42 (published online)

12. Wolman SL, Anderson GH, Marliss EB, et al. Zinc in parenteral nutrition. Requirements and metabolic effects. Gastroenterology 1979; 76: 458–67.

13. Briefel RR, Bialostosky K, Kennedy-Stephenson J, McDowell MA, Wright JD. Intake of the US population: findings from the third National Health and Nutrition Examination Survey 1988–1994. J Nutr 2000; 1367S–1373S.

14. JeejeebHoy K. Zinc: an essential trace element for parenteral nutrition. Gastroenterology 2009; 137: S7–S12.

15. Widdowson EM, Dauncey J, Shaw JCL. Trace elements in foetal and early postnatal development. Proc Nutr Soc 1974; 33: 275–84.

16. Fischer Walker CL, Black RE. Zinc for the treatment of diarrhoe: effect on diarrhoea morbidity, mortality and incidence of future episodes. Int J Epidemiol 2010; 39 suppl 1: i63–i69.

17. Prasad AS. Zinc deficiency and effects of zinc supplementation on sickle cell anemia subjects. Clin Biol Res 1981; 55: 99–122.

18. Aggett PJ. Acrodermatitis enteropathica. J Inherit Metab Dis 1983; 6: 39–43.

19. Cope EC, Levenson CW. Role of zinc in the development and treatment of mood disorders. Curr Opin Clin Nutr Metab Care 2010; 13: 686–9.

20. Rucker D, Thadhani R, Tonelli M. Trace element status in hemodialysis patients. Seminars in Dialysis 2010: 23: 389–95.

21. Proksch E, Kölmel K. Zinkmangelsyndrom als Nebenwirkung von Chelatbildnern. Dtsch Med Wschr 1985; 110: 1001–3.

22. Himoto T, Masaki T. Associations between zinc deficiency and metabolic abnormalities in patients with chronic liver disease. Nutrients 2018; 10. doi: 10.3390%2Fnu10010088.

23. Dekker LH, Villamor E. Zinc supplementation in children is not associated with decreases in hemoglobin concentrations. J Nutr 2010; 140: 1035–40.

24. Nishito Y, Kambe T. Absorption mechanisms of iron, copper and zinc: anoverview. J Nutr Sci Vitaminol 2018; 64: 1–7.

10.11 Iodine (I)

Lothar Thomas

Iodine is a non-metal and belongs to the halogen group. The abundance of iodine in the earth’s crust is 6.1 × 10–5  percent, however, with a very uneven distribution. Iodine occurs in nature exclusively as iodide (I) in the bound form.

Iodides are metal salts of hydroiodic acid and also include covalent iodides of non-metals such as alkyl or aryl iodides. The concentration of iodide in sea water is about 50 μg/L. Iodide ions in sea water are oxidized to elemental iodine, which volatilizes into the atmosphere and is returned to the soil by rain, completing the cycle.

Iodates are metal salts of iodic acid. Iodate is widely used in salt iodization.

10.11.1 Indication

Iodine under supply is suspected in:

  • Pregnant women and neonates in iodine deficiency areas
  • Developmental disorders in infants
  • Goiter.

Iodine intoxication is suspected in:

  • Iodine-Basedow phenomenon
  • Wolff-Chaikoff effect
  • Monitoring of iodine intake (measurement of urinary iodine excretion).

10.11.2 Method of determination

  • HPLC with electrochemical detection
  • Plasma mass spectrometry (ICP-MS)
  • Colorimetric assay (cerium-arsenite method), where yellow cerium (IV) is converted to colorless cerium (III) in a iodide-catalyzed redox reaction
  • Quick color test for semi-quantitative determination of the urinary iodide concentration. Iodide-catalyzed oxidation of tetra methyl benzidine with peracetic acid results in blue-green color.

10.11.3 Specimen

  • Random urine specimen (determination of iodide and creatinine): 5 mL
  • 24 h urine: 5 mL
  • Serum (no preferred analyte): 1 mL

10.11.4 Reference interval

Refer to References /12/ and Tab. 10.11-1 – Reference intervals for iodine.

10.11.5 Clinical significance

Iodine is an important element for the tyroid hormone production, and its deficiency or excessive intake is associated with various thyroid diseases. Iodine (as iodide) is widely but unevenly distributed in the Earth’s environment. Leaching from glaciations, flooding and erosions have depleted surface soils of iodide. Iodine-deficient soils are common in the mountainous areas and areas of frequent flooding, especially in South and Southeast Asia. Many island areas, including the Midwestern region of North America, central Asia and Africa, and central and Eastern Europe, are iodine deficient. The most iodide is found in the oceans /3/. Iodine deficiency

About 2.2 billion people (one third of the world population) live in countries affected by iodine deficiency and are at risk of the resulting diseases.

A population’s dietary habits and its access to sources of iodine (seafood, milk, dairy products, ionized salt, and iodine containing supplements) are important for securing sufficient iodine intake.

Iodine deficiency diseases affect individuals of any age, but pregnant women, their fetus, neonates, children and adolescents are affected the most. The most common anomalies are endemic goiter, spontaneous abortion, stillbirth, congenital anomalies and increased perinatal mortality. As endocrine disorder, iodine deficiency causes hypothyroidism, which, in its most extreme form, results in cretinism. Severe hypothyroidism leads to impaired mental development, impaired growth and premature puberty. The consequences of mild hypothyroidism are not as known in detail /4/.

Iodine deficiency is caused by soils low in iodine. In plant foods grown in iodine-deficient soils, for example, iodine concentration is 10 μg/kg dry weight, compared with approximately 1 mg/kg dry weight in plants from iodine-sufficient soils. The native iodine content of most foods and beverages is 3–80 μg per serving. Major dietary sources of iodine in Europe and the United States are bread and milk. Foods of marine origin have higher iodine content than milk and bread /3/.

For adequate iodine intake of the population, institutions make recommendations according to specific definitions (see Tab. 10.1-5 – Definitions for trace element requirement).

Recommendations for daily iodine intake regarding age and population group are listed in Tab. 10.11-2 – Daily iodine intake recommendations.

During pregnancy, daily iodine requirement increases by nearly 50%, because of an increase in renal iodine excretion, increased thyroid hormone production and fetal requirement. Studies show that the diet of the pregnant women did not necessarily secure a sufficient iodine intake. Median urinary iodine excretion in Norwegian /16/ pregnant women was 85 μg/l and in pregnant women living in Brazil /17/ the excretion was 146 μg/l.

The following recommendations are made for iodine intake under total parenteral nutrition:

  • Infants 1 μg/kg of body weight /5/.
  • Children 1 μg/kg of body weight /5/.
  • Adults 70–140 μg/day /6/.

Four methods are generally recommended for assessment of iodine nutrition in populations /3/:

  • Urinary iodine concentration and/or excretion, an indicator of recent iodine intake (days)
  • Goiter rate, changes reflect long-term iodine nutrition (months to years)
  • Thyroid-stimulating hormone (TSH) in plasma/serum. This biomarker is correspondent to urinary iodine.
  • Thyroglobulin (Tg) in plasma/serum. This biomarker shows an intermediate response (weeks to months).

Increased iodine intake in an iodine-deficient population is associated with a small increase in the prevalence of subclinical hypothyroidism and thyroid autoimmunity. Variations in population iodine intake do not affect risk for Graves’ disease or thyroid cancer, but correction of iodine deficiency might shift thyroid cancer subtypes toward less malignant forms /7/. Urinary iodine excretion

More than 90% of dietary iodine eventually appears in the urine; therefore, the urinary iodine expressed in μg/L and/or μg/24 h or in relationship to creatinine (μg iodine/g creatinine) is an excellent biomarker of the current iodine intake /3/. The determination in 24 h urine is impractical. Hence, the determination in a spot urine and expression of the excretion in μg/L or in relationship to creatinine excretion are preferred for population studies. Variations in hydration among individuals even out in a large amount of samples, so that the median urinary iodine in spot samples correlates well with that of 24-h samples. The urinary iodine to creatinine ratio can be misleading for estimating daily iodine excretion from spot urine, especially in malnourished individuals where creatinine excretion is low. Daily iodine intake for population estimates can be calculated from urinary iodine, using estimates of mean 24-h urine volume and assuming an average iodine bio availability of 92% using the formula /8/:

Daily iodine intake (μg) = Urinary iodine (μg/L) × 0,0235 × body weight (kg)

A urinary iodine concentration of 100 μg/L roughly corresponds to a daily intake of 150 μg. The assessment of iodine intake based on urinary iodine excretion and the resulting evaluation are shown in Tab. 10.11-3 – Assessment of iodine intake based on urinary iodine excretion.

In a study /9/, school-age children aged 6–16 years with goiter had a median iodine excretion of 36 μg/L (0.28 μmol/L) at normal TSH, T4 and T3 concentrations. After 4 years of therapy with iodized oil, median excretion was 188 μg/L (1.46 μmol/L).

Association between urinary sodium levels and iodine status was shown in Korea. Low sodium and iodine excretion were most common in younger adults (age 19) while higher excretions were most common in elderly people (age, 60 to 75 years) /18/. Estimation of iodine intake

Determination of thyroid volume

The thyroid volume determined by ultrasonography in Dutch individuals is 12.7 ± 4.6 mL in men and 8.7 ± 3.9 mL in women /10/. Higher values indicate the presence of goiter. However, inter assay variability is 26%.


TSH can be used as a biomarker of iodine nutrition. However, in older children and adults, although the TSH concentration may be slightly increased by iodine deficiency, values often remain within the reference interval and TSH is therefore an insensitive biomarker /3/.

In contrast, TSH is a sensitive biomarker of the iodine status in the newborn period. Compared with the adult, the newborn thyroid contains less iodine but has higher rates of iodine turnover. Particularly when iodine supply is low, maintaining high iodine turnover requires increased TSH stimulation. Serum TSH concentrations are therefore increased to more than 5 mU/L in iodine-deficient newborns for the first few week of life, a condition termed transient newborn hypothyroidism. In areas of iodine deficiency, an increase in transient newborn hypothyroidism, indicated by more than 3% of newborn TSH values above the threshold of 5 mU/L whole blood collected 3 to 4 days after birth, suggests iodine deficiency in the population /311/.

Thyroglobulin (Tg)

Tg is synthesized in the thyroid and is the most abundant intrathyroidal protein. In iodine sufficiency, small amounts are secreted into the plasma, where concentration is < 10 μg/L /3/. In areas with endemic goiter, serum Tg level increases due greater thyroid cell mass and TSH stimulation. Plasma Tg concentration is correlated with urinary iodine excretion. Tg decreases under potassium iodide and iodized oil substitution and reaches normal concentrations after several months. Tg is a more sensitive biomarker of iodine repletion than TSH and T4.

Thyroperoxidase antibodies (anti-TPO)

The prevalence of subclinical hypothyroidism in children and adolescents was significantly higher in iodine deficiency and the iodine excessive groups compared to those with normal excretion (100–300 μg/L). In the excessive group the excretion was higher than 1,000 μg/L because of excessive iodine intake /19/.

Pattern of circulating thyroid hormones

Thyroid hormone concentrations are poor indicators of iodine status. In populations with moderate to severe iodine deficiency T3 increases or remains unchanged and T4 usually increases. These changes are often within the reference range or overlap with iodine sufficient populations /3/.

Cretinism and thyroid insufficiency develop in regions of chronic severe iodine deficiency, where individuals show dramatically elevated TSH and very low T4 and T3. Iodine intoxication

Iodine intakes of up to 1 mg/day are well tolerated by adults because the thyroid is able to adjust to a wide range of intakes and regulate the biosynthesis and release of thyroid hormones /12/. Some individuals with increased iodine intake, especially those with previous deficiency, develop hyperthyroidism (the Iodine-Basedow phenomenon). However, excessive intakes may inhibit iodine uptake by the thyroid and consequently inhibit the biosynthesis of thyroid hormones (Wolff-Chaikoff effect). In children, daily intakes above 500 μg are associated with increased thyroid volume.

Parenteral administration of iodine can cause acute hypersensitivity response such as cutaneous and mucosal hemorrhages, angioneurotic edema, fever, arthralgia, eosinophilia and lymphadenopathy.

Chronic iodine intoxication causes metallic taste, increased salivation, headache, pulmonary edema and gastrointestinal complaints.

10.11.6 Comments and problems

Urinary iodine excretion

Iodine intake and renal iodine secretion are well correlated, but there is only a mildly positive correlation between thyroidal iodine content and iodine excretion /13/.

Method of determination

A micromethod of the cerium-arsenite method shows adequate reliability for the determination of low urinary iodine concentrations /14/.

10.11.7 Pathophysiology

Iodine is ingested in several chemical forms. Iodide is nearly completely absorbed in the stomach and duodenum. Iodate, widely used in salt iodization, is reduced to iodide in the gut before enteral absorption. The absorption rate of iodide is greater than 90%. Absorption by the intestinal enterocytes is mediated by the sodium/iodine symporter (NIS) localized in the apical membrane of the enterocytes. NIS cotransports two Na+ together with one I, with the Na+ gradient being the driving force for I transport. The required energy is supplied by the quabain-sensitive Na+-K+-ATPase /3/.

The distribution space of absorbed iodine is nearly equal to the extracellular fluid volume. Iodine is cleared from the circulation mainly by the thyroid and kidneys. Under physiological conditions, only 10% of the absorbed iodine are taken up by the thyroid; the rest is eliminated renally. In iodide deficiency, the thyroid takes up about 80% of the enterally absorbed iodine.

Iodide is transported into the thyrocytes and thyroid hormones are synthesized as follows (Fig. 10.11-1 – Iodide transport from the thyrocyte to the thyroid follicle) /315/:

  • Basolateral uptake of iodide is mediated by the Na+/I symporter (NIS)
  • The iodide is transported from the thyrocyte to the colloid follicle at the apical membrane by pendrin, a Cl/I transporter
  • At the transition from the thyrocyte to the colloid follicle, thyroxine peroxidase (TPO) and H2O2 oxidize iodine and attach it to tyrosyl residues on thyroglobulin to produce monoiodotyrosine (MIT) and diiodotyrosine (DIT), the precursors of thyroid hormone
  • TPO then catalyzes the coupling of the phenyl groups of the iodotyrosines through a diether bridge to form the thyroid hormones
  • Linkage of two DIT molecules produces T4, and linkage of a DIT and a MIT produces T3
  • Iodine comprises 65% and 59% of the weights of T4 and T3, respectively
  • Following thyroglobulin endocytosis, endosomal and lysosomal proteases digest thyroglobulin and release T4 and T3 into the circulation.

The adult body’s iodine content is 15–20 mg. The half-life of iodine in plasma is approximately 10 hours. The intrathyroidal iodine store theoretically ensures sufficient supply for half a year of thyroid hormone production; however, a significant part is bound in non-homogeneous tyrosine residues. Iodine in the liver predominantly resides in lysosomes, presumably from the degradation of iodine-containing proteins.

Dietary substances can interfere with thyroid metabolism and aggravate the effect of iodine deficiency. Such substances, also referred to as goitrogens, contain glucosinolates; their metabolites compete with iodine for thyroidal uptake. Cruciferous vegetables, such as broccoli, kale and white cabbage, contain glucosinolates. Cigarette smoking is also associated with higher thiocyanate levels that may inhibit iodine uptake by the thyrocytes, as is perchlorate. Soybeans and millet contain flavonoids that may impair TPO activity. Selenium deficiency exacerbates the effects of iodine deficiency because deiodinases and glutathione peroxidase are selenium-dependent enzymes.


1. World Health Organization, United Nations Children’s Fund, International Council for the Control of Iodine Deficiency Disorders 2007: Assessment of iodine deficiency disorders and monitoring their elimination, 3rd edition. Geneva: WHO.

2. Gutekunst R, Magiera U, Teichert HM. Jodmangel in der Bundesrepublik Deutschland. Med Klinik 1993; 88: 525–8.

3. Zimmermann MB. Iodine deficiency. Endocrine Reviews 2009; 30: 376–408.

4. WHO/UNICEF/ICCIDD 2001 assessment of iodine deficiency disorders and monitoring their elimination. A guide for programme managers. Second edition. Geneva.

5. Koletzko B, Goulet O, Hunt J, et al. Guidelines on pediatric parenteral nutrition of the European Society of Pediatric Gastroenterology, Hepatology and Nutrition (ESPGHAN) and the European Society for Clinical Nutrition and Metabolism (ESPEN). J Pediatr Gastroenterol Nutr 2005; 41 (Suppl 2): S1–S87.

6. Koretz RL, Lipman TO, Klein S, et al. AGA technical review on parenteral nutrition. Gastroenterology 2001; 212: 970–1001.

7. Zimmermann MB, Boelaert K. Iodine deficiency and thyroid disorders. Lancet Diabetes Endocrinol 2015; Apr; 3 (4) 286–95.

8. Institute of Medicine, Academy of Sciences 2001. Dietary reference intakes for vitamin A, vitamin K, arsenic, boron, chromium, copper, iodine, iron, manganese, molybdenum, nickel, silicon, vanadium, and zinc. Washington, DC: National Academy Press.

9. Markou KB, Tsekouras A, Anastasiou E, Vlassopoulou B, Koukkou E, Vagenakis GA, et al. Treating iodine deficiency: long-term effects of iodine repletion on growth and pubertal development in school-age children. Thyroid 2008; 18: 449–54.

10. Berghout A, Wiersinga WM, Smits NJ, et al. Determinants of thyroid volume by ultrasonography in healthy adults in a non-iodine deficient area. Clin Endocrinol (Oxf) 1987; 26: 273–80.

11. Alexander EK, Pearce EN, Brent GA, Brown RS, Chen H, Dosiou C, et al. 2017 guidelines of the American Thyroid Association for the diagnosis and management of thyroid disease during pregnancy and the postpartum. Thyroid 2017; 27: 315–89.

12. Zimmermann B. Iodine: it’s important in patients that require parenteral nutrition. Gastroenterology 2009; 137: S36–S46.

13. Reiners C, Ugur T, Yavus A. In vivo-Bestimmung des Jodgehaltes der menschlichen Schilddrüse – Korrelation mit der Urin-Jodexkretion. In: Körle J (ed.) Mineralstoffe und Spurenelemente. Stuttgart 1998; Wiss. Verlagsgesellschaft: 93–101.

14. Hussain H, Khalid NM, Selamat S, Nazaimoon WMW. Evaluation of the performance of a micromethod for measuring urinary iodine by using six sigma quality metrics. Ann Lab Med 2013; 33: 319–25.

15. Spitzweg C, Heufelder AE, Morris JC. Thyroid iodine transport. Thyroid 2000; 10: 321–30.

16. Dahl L, Markhus MW, Sanchez PVR, Moe V, Smith L, Meltzer HM, Kjellevord M. Iodine deficiency in a study population of Norwegian pregnant women – results from the Little in Norway study. Nutrients 2018, 10. doi: 10.3390%2Fnu10040513.

17. Mioto V, de Castro Nassif Gomez Mosteiro, de Camargo, RYA, Borel AR, Catarino RM, Kobayashi S, et al. High prevalence of iodine deficiency in pregnant women living inadequate iodine area. Endocrine Connections 2018; 7. doi: 10.1530/ec-18-0131.

18. Ahn J, Lee Hj, Lee J, Baek jY, Song E, Oh HS, et al. Association between urinary sodium levels and iodine status in Korea. Korean J Intern Med 2017. doi: 10.3904/kjim.2017.375.

19. Kang MJ, Hwang IT, Chung HR. Excessive iodine intake and subclinical hypothroidism in children and adolescents aged 6–19 years: results of the sixth Korean National Health Nutrition Examination Survey. Thyroid 2018. doi: 10.1089/thy.2017.0507.

Table 10.1-1 Criteria of an essential trace element /3/

  • Integral part of the natural environment
  • Present in food in physiological quantities
  • Occurs in tissues at relatively constant concentration
  • Already present in neonates and also in breast milk
  • Deprivation leads to similar physiological and structural changes in man and mammal. The changes regress upon intake of the trace element
  • A biological function in the body is associated with the essential trace element.

Table 10.1-2 Biochemical functions of essential trace elements /3/

  • Enzymatic activity as part of the prosthetic group or as cofactor
  • Transport of oxygen (iron, copper)
  • Organization and structure of macromolecules (e.g., copper in keratin, silicon in connective tissue, zinc in transcription factors (zinc finger))
  • Vitamin activity (cobalt in vitamin B12)
  • Hormonal activity (iodine in thyroid hormones)

Table 10.1-3 Symptoms indicating specific chronic trace element deficiency /3/

Clinical symptoms/diagnosis


Susceptibility to infection, immunodeficiency


Wound healing disorder, persistent skin disease


Sensory function disorder (sense of touch, sense of smell)


Anemia, feeling of faintness

Iron, copper

Frequent bone fractures




Retardation of growth and sexual development


Impaired glucose tolerance


Inadequate behavior and individual development disorder

Chromium, iron, iodine, manganese, selenium, molybdenum

Myopathy, cardiac and skeletal


Table 10.1-4 Symptoms indicating a specific chronic trace element burden /3/

Disease/clinical symptoms


Wilson’s disease

Cholestasis-lymphedema syndrome


Hereditary hemochromatosis



Bantu siderosis


Beer drinker’s cardiomyopathy


Indian childhood cirrhosis


Enshi County selenosis


Aboriginal manganism


Endemic fluorosis


Chronic renal insufficiency

Chromium, nickel

Total parenteral nutrition

Chromium, nickel, manganese


Copper, zinc

Prosthetic implants

Cobalt, iron, manganese, molybdenum, nickel, vanadium, silicon

Excessive supplementation

Copper, chromium, selenium, zinc

Hepatobiliary stasis

Copper, manganese


Copper, iron, selenium

Table 10.1-5 Definitions for trace element requirement



EAR (Definition of the US Institute of Medicine)

Estimated average requirement (EAR); refers to the daily intake of trace elements concerning the requirement of half of the population in a specific life situation. The EAR does not apply to the single individual but to specific groups.

RDA (Definition of the US Institute of Medicine)

Recommended dietary allowance (RDA); refers to the daily intake quantity covering the requirement of 97–98% of the population. The RDA can be applied as the goal of trace element intake of a single individual.

AI (Definition of the US Institute of Medicine)

Adequate nutrient intake (AI). Recommendation given if there is inadequate evidence on EAR calculation.

RNI (WHO definition)

Recommended nutrient intake (RNI). Comprises the daily requirement of trace elements of each healthy individual.

Table 10.1-6 Trace elements acting as catalysts, according to Ref. /20/












Enzymes activated by trace elements



































































Metalloenzymes with tight trace element binding

Aldehyde oxidase











Alkaline phosphatase











Alcohol dehydrogenase











Carbonic anhydrase











Carboxypeptidase A











Carboxypeptidase B











Cytochrome C-oxidase











Type I iodothyronine-5’-deiodase











Glutathione peroxidase











Glutamate dehydrogenase











Lactate dehydrogenase











Malate dehydrogenase











NADP-cytochrome reductase











Nucleoside phosphorylase











Succinate dehydrogenase











Superoxide dismutase












































Xanthine oxidase











Table 10.2-1 Toxicity of chromium

Clinical and laboratory findings

Total parenteral nutrition (PN)

Under PN considerable amounts of Cr are administered for a short or long term and are found in high concentrations. According to a study /11/, 50% of patients on short-term PN had serum levels > 10-fold of normal (upper reference interval value of 3.8 nmol/L), 18% were > 20-fold, and 2% were > 40-fold. Urinary Cr excretion was elevated up to 20-fold /7/. This is caused by the substitution of parenteral nutrients by Cr, their natural Cr content and Cr contamination of amino acids.

Metal-on-metal (MoM) hip prostheses /13/

Approximately 1 million MoM hip prostheses have been implanted worldwide. These implants contain femoral and acetabular bearing surfaces that are composed predominantly of Cr and Co. Characteristic wear pattern of MoM hip-resurfing arthroplasty is initially characterized by a running-in period of increased wear with metal debris formation (Cr ions and Co ions and particles containing both metals), followed by a lower-wear steady state. The duration of the running-in period varies, but is thought to be up to 1 million joint cycles and is usually over 9–12 months in younger, more active resurfacing patients. With well-positioned prostheses, this is followed by the bedding-in phase with minimized wear and reduced Cr and Co release and decreased concentrations in the synovial fluid. If a hip prosthesis is removed, Co tends to be cleared quickly, about a 5-fold reduction within 2 months in whole blood; Cr tends to get bound periprosthetically, so it clears more slowly. In cases of mal positioning of the acetabular component of the hip prosthesis continuous or increasing amounts of Cr and Co are generated and released. The reactions are manifested either as inflammatory fluid collections or as cystic or solid non-infectious soft-tissue masses around the hip or osteolytic lesions. The immunological reactions are subdivided in two categories:

  • Metal reactivity, an immunological response to Cr and Co containing particles and Co ions and Cr ions. This is a normal immunologic response to a large amount of metal debris.
  • Metal allergy manifested as a type IV hypersensitivity reaction which occurs in patients with a genetic allergic pre deposition.

MoM implanted patients are at no greater risk for solid tumors, but at a 2–3-fold higher risk for hematological malignancy, particularly lymphoma.

Laboratory findings: individuals without Cr or Co exposure and with no metal prostheses have Cr and Co concentrations < 1 μg/L in serum. Patients with well-functioning MoM prostheses, reach a steady state after 3 years and have:

  • Acceptable upper limits of 4.6 μg/L for Cr and 4.0 μg/L for Co for unilateral prostheses. A different author quotes < 10 μg/L for Co. For bilateral prostheses upper limits are 7.4 μg/L for Cr and 5.0 μg/L for Co.

In the absence of clinical and radiographic symptoms, a routine follow-up regimen is 1, 2, 3, 5, 7 and 10 years postoperatively.

  • Concentrations between 4–10 μg/L (unilateral prosthesis) and 10–20 μg/L (bilateral prosthesis) represent a moderate increase and additional investigations are advocated
  • Concentrations above 20 μg/L are concerning, because they are a sign of high wear and my be associated with systemic toxicity. Synovial fluid Cr and Co values above 5000 μg/L are associated with adverse reaction to metal debris.

Table 10.4-1 Copper reference intervals in serum and urine /567/










0–4 mo.



4–6 mo.



7–12 mo.



1–5 yrs



6–9 yrs



10–13 yrs



14–19 yrs




No estrogen



5.7–119 μmol/mol creatinine (3.2–67 μg/g creatinine)







0.16–0.94 μmol (10–60 μg)/24 h

Conversion: μg/dL × 0.157 = μmol/L, μg/g creatinine × 1.781 = μmol/mol creatinine

Table 10.4-2 Diseases and conditions with copper deficiency and copper overload

Clinical and laboratory findings

Wilson’s disease (hepatolenticular degeneration)

Wilson’s disease is an autosomal recessive disorder caused by genetic inactivation of the ATP-driven transporter ATP7B (Tab. 10.4-3 – Proteins of copper metabolism). This ATPase is localized in the trans-Golgi network and, in the hepatocyte, transports Cu to the secretory pathways bound to apoceruloplasmin for subsequent excretion via the bile (for further information, see Section 18.7 – Ceruloplasmin (Cp)).

Laboratory findings: low serum/plasma Cu, increase in phases of acute hemolysis, mostly very low ceruloplasmin. Urinary Cu excretion below 40 μg/24 h, following the administration of D-penicillamine (4 × 250 mg orally): above 100 μg/24 h. Increased Cu content in the liver above 250 μg/g dry weight. See also Section 18.7.

Menkes disease (Kinky hair or steely hair syndrome)

Menkes disease is an X-linked disorder. Cu efflux from the enterocytes into the blood and, thus, into the brain is reduced following Cu absorption from the intestinal lumen. This is caused by mutations in the Menkes ATPase (ATP7A)-encoding gene (Tab. 10.4-3 – Proteins of copper metabolism). Patients show retarted growth development, disturbed temperature control, connective tissue abnormalities, convulsions, mental retardation and die in early childhood /18/.

Laboratory findings: low plasma Cu, very low ceruloplasmin. Age-dependent low Cu content in the liver, increased Cu content in the duodenal mucosa.

Anemia, granulocytopenia

Hematological findings in Cu deficiency are anemia, leukocytopenia and, rarely, thrombocytopenia. In many cases, these are the first symptoms in children with Cu deficiency. The erythrocytes are usually microcytic, but show multiple morphology including microcytic, normocytic and macrocytic cells /19/. The erythrocyte life span is reduced due to increased lipid peroxidation of the erythrocyte membrane. In many cases, the precursor cells in the bone marrow undergo cytoplasmic vacuolization as seen in alcohol intoxication, drug intoxication, chemotherapy and myeloproliferative syndrome. The mechanisms causing Cu deficiency-induced anemia are based on Cu involvement in iron transport and heme synthesis. Ceruloplasmin, for example, is a ferrioxidase that binds Cu and, similarly to hephaestin, transforms divalent iron to the trivalent form that can bind to transferrin following release at the basolateral membrane of the enterocyte (see also Fig. 7.1-1 – Iron transport in the small intestine by the enterocytes). Cytochrome oxidase is also Cu-dependent and required for the reduction of trivalent iron to the divalent form for integration into the heme molecule.

Nutritional copper deficiency

Nutritional Cu deficiency is rare and usually results from severe prolonged malabsorption (e.g., in neonates, children with severe malabsorption and patients on parenteral nutrition without Cu substitution).

Parenteral nutrition: reports are limited to a few cases and refer to infants with short bowel syndrome and adults with scleroderma and severe malabsorption /20/. Cu supplementation in parenteral nutrition is also recommended for cholestatic infants who accumulate Cu due to cholestasis /21/.

Zinc-induced Cu deficiency: prolonged zinc intake leads to reduced metallothionein (MT)-induced Cu absorption due to a competition between zinc and Cu. MT expression is up regulated under zinc burden, and MT binds Cu with a higher affinity than zinc. The MT-Cu complex is excreted via the bile /22/.

Pregnancy: Cu deficiency is very rare. Enteral absorption of Cu is increased in pregnant women due to Cu consumption by the fetus /2/.

Infancy: the fetal liver accumulates large amounts of Cu, with the Cu storage depending in the neonate’s gestational age /23/. If dietary Cu intake is reduced in neonates with reduced Cu accumulation, due to parenteral nutrition, formula preparations or feeding of high-iron milk (competitive inhibition of Cu absorption), there is the risk of nutritional Cu deficiency during the first months of life.

Laboratory findings: in severe Cu deficiency, the Cu level in plasma/serum is low, and ceruloplasmin and erythrocytic super oxide dismutase are also low.

Nephrotic syndrome

Reduced serum Cu level due to renal loss of ceruloplasmin.

Neurodegenerative disease /24/

Neurodegenerative diseases associated with disruption of brain metal homeostasis include Alzheimer’s disease (AD), Parkinson’s disease (PD) and Huntington’s disease (HD).

AD: these patients are said to have a tendency to elevated Cu concentrations, low ceruloplasmin and reduced SOD activity in the cerebrospinal fluid (CSF).

PD: the Cu content in the reticular formations is said to be higher in these patients than in healthy individuals, also concerning concentrations in the CSF. Neuronal Fe uptake is thought to be disturbed as a result. The significance of Cu in PD is generally unclear.

HD: elevated and low Cu concentrations have been measured in these patients. The pathophysiology is unclear.


Myelopathies associated with Cu deficiency occur in patients in their 5th and 6th decades of life and are more common in women than in men. The etiologies of Cu deficiency are gastrointestinal surgery, high zinc supplementation and malabsorption syndrome /25/.

Bariatric surgery

Bariatric surgery is performed to reduce the body weight in severe obesity. While the body mass index, glucose tolerance and cholesterol concentration improve postoperatively, it is not uncommon that these individuals suffer from Cu deficiency. In many cases, the deficiency is not diagnosed until the onset of anemia and leukopenia /26/.

Cisplatin resistance

Cisplatin is delivered into the cell via the receptor CTR1 and transported intracellularly by the Cu transporter ATP7B. Cu and cisplatin interact with ATP7B at various locations. Cu deficiency caused by CTR1 down regulation and ATP7A up regulation is thought to be the cause of cisplatin resistance /27/.

Cardiovascular disease

There is a positive correlation between serum Cu level and HDL concentration. Reduced Cu intake promotes hypercholesterolemia: studies have shown that mild Cu deficiency in association with zinc substitution may increase the risk of atherosclerosis /28/.

Diabetes mellitus

Type-1 diabetics have higher Cu concentrations in whole blood, plasma and mononuclear cells. This does not apply to type 2 diabetics. Moreover, the serum Cu level in patients with more than three markers of metabolic syndrome manifestation is reported to be 30% higher than in healthy individuals /29/.

Table 10.4-3 Proteins of copper metabolism /1727/




The high-affinity Cu transporter 1 (CTR1) has the following functions:

  • Cu crosses the apical membrane of enterocytes via a process that is largely independent of CTR1. The release of Cu from the cytoplasm into the blood across the basolateral membrane is mediated by CTR1.
  • Uptake of transport protein-bound Cu into the hepatocyte. CTR1 is also responsible for the uptake of cisplatin, a drug used for treating various carcinomas.


Human cells express the Cu-ATPases ATP7A and ATP7B. The transporters use the energy of ATP hydrolysis to transport copper in the reduced Cu(I) form from the cytosol across cellular membranes. The transported Cu (I) is either released into the blood stream for further distribution to tissues (in case of ATP7A) or it is exported into the bile for eventual removal from the body (in case of ATP7B). They do this indirectly because the Cu-ATPases are not constitutively present at the plasma membrane /17/. Mutations in the ATP7A gene cause the Menkes syndrome; hence, ATP7A is also referred to as Menkes ATPase.

  • ATP7A

The Cu transporter Menkes ATPase (ATP7A) delivers Cu to the secretory pathway of multiple cell types. Atox1 delivers Cu to the Menkes ATPase. Mutations in the ATP7A gene cause Menkes disease.

  • ATP7B

In hepatocytes this ATPase resides in the trans-Golgi network, transporting Cu into the secretory pathway for incorporation into apoceruloplasmin and excretion into the bile /17/. Inherited loss-of-function mutations in the ATP7B gene are associated with Wilson’s disease; hence, ATP7B is also referred to as Wilson ATPase. Frequent H1069Q mutation causes temperature-dependent disorder in ATP7B protein folding, which leads to incorrect localization of the protein in the endoplasmic reticulum.


The cytoplasmic Cu chaperone Atox1 transports intracellular Cu to specific proteins. Atox1 is required for Cu delivery to the ATPases ATP7A and ATP7B.


Metallothioneins are evolutionarily conserved, cysteine-rich intracellular proteins capable of binding Cu, Cd and Zn. Some of the different metallothioneins are present in all cells. Their function is to bind and store excessive Cu, especially in the hepatocytes.


Metallochaperones are low-molecular proteins in the cytoplasm and bind Cu in the hepatocytes. Each metallochaperone has a specific function regarding the transport of, for example, Cu into specific cell organelles.

Ceruloplasmin (Cp), hephaestin

Cp and hephaestin are essential ferrooxidases containing several Cu atoms. Cp is synthesized in hepatocytes and secreted into the plasma following the incorporation of 6 Cu atoms. Since Cp contains 95% of the Cu in plasma, its concentration is an indicator of hepatic Cu metabolism. In Cu deficiency, as hepatic Cu stores decrease, there is minimal transport of Cu into the hepatocyte secretory pathway, and the biliary Cu content and serum Cp concentration are diminished. In patients with Wilson disease the lack of functional ATP7B also results in secretion of apoceruloplasmin and the resulting decrease in serum Cp concentration is a diagnostic hallmark of this disorder /17/.

Table 10.4-4 Copper-containing enzymes and their biochemical function /20/




Ferrooxidases such as ceruloplasmin and hephaestin catalyze the oxidation of divalent to trivalent iron. Divalent iron enters the blood through the basolateral membrane of the enterocyte, is oxidized and trivalent iron binds to transferrin. Iron occurs intracellularly in the divalent form and extracellularly in the trivalent form. In general, ferrooxidases oxidize iron during transition from the intracellular to the extracellular compartment.


These enzymes participate in the crosslinking of collagen and elastin in connective tissues. Cu deficiency leads to reduced activity with impaired bone formation and defects of the connective tissue and blood vessels.

Dopamine hydroxylase

This enzyme catalyzes the transformation of dopamine to norepinephrine in the brain. Reduced activity in Cu deficiency leads to neurological disorders.

Superoxide dismutase (SOD)

The copper/zinc SOD acts as free radical scavenger and deactivates free oxygen radicals increasingly generated under oxidative stress (see Section 19.2 – Oxidative stress). In Cu deficiency, free radical production increases due to reduced SOD activity and oxidative stress can occur as a result.

Monoamine oxidase

Monoamine oxidase catalyzes the formation of serotonin.


This enzyme participates in the synthesis of melanin. In Cu deficiency, hypopigmentation can occur.

Table 10.5-1 Reference intervals for magnesium





mmol/24 h
(mg/24 h)­



children /3/


Adults /4/



ionized Mg


Conversion: mg/dL × 0.4114 = mmol/L

Table 10.5-2 Diseases and conditions with magnesium deficiency

Clinical and laboratory findings

Gastrointestinal disease

Daily magnesium intake is approximately 15 mmol (360 mg). Of this, 24–75% are absorbed. Persistent diarrhea, short bowel syndrome, fistulas, magnesium-poor nutrition and, rarely, malabsorption syndrome can result in reduced serum magnesium concentration.

Intensive care patients /12/

The prevalence of hypomagnesemia in intensive care patients is up to 50%. Besides gastrointestinal causes, drugs inducing a disturbance of renal reabsorption and increased magnesium excretion play an important role. The following drugs are significant:

  • Loop diuretics and thiazides that increase magnesium excretion
  • Aminoglycosides; the more they accumulate, the severer the hypomagnesemia
  • Digoxin; is thought to inhibit the Na+-K+-ATPase activity and, as a result, trans cellular tubular transport of Mg2+.

Hypermagnesemia above 2.0 mmol/L can be caused by increased intake of Mg-containing products like laxatives, antacids or enemas. Lithium intoxication also leads to elevated magnesium levels because high lithium doses presumably interfere with renal magnesium excretion.

Decreased Intake /34/

The 2001–2008 National Health and Nutrition Examination survey (NHANES) showed that > 50% of the adult population of US did not consume the recommended magnesium intake. The richest dietary sources of magnesium are whole grain cereals, green vegetables, beans, nuts, and seafood.

Approximately 99% of total body magnesium is stored in muscles, bones, and non-muscular soft tissue.

Intestinal absorption occurs predominantly in the small intestine (24–76% is absorbed in the gut).

Gastrointestinal losses (acute or chronic diarrhea, malabsorption, and steatorrhea, and small bypass surgery) are common settings of hypo­mag­ne­semia /32/.

Renal excretion is the most important factor in magnesium homeostasis.

Inflammation /13/

Elevated serum C-reactive protein (CRP) levels are inversely related to the magnesium intake. A magnesium intake < 10% of the recommended daily allowance can lead to inflammation manifested as activation of leukocytes and macrophages, secretion of inflammatory cytokines and increase in CRP. The 1999–2002 NHANES study found that children consuming less than 75% of the recommended daily allowance (RDA) of magnesium were 1.94 times more likely to have elevated serum CRP levels than children consuming more than the RDA for magnesium. Increase in CRP was 1.75-fold more common in adults ingesting only 50% of the RDA.

Obesity and Inflammation: a low magnesium status in obesity is associated with chronic inflammation. In the NHANES study of 1988–1994, for example, the odds ratios in individuals with a body mass index (BMI) of > 25–< 30, 30–< 35, 35–< 40 and ≥ 40 were 1.51, 3.9, 6.1 and 9.3, respectively, compared with individuals with a BMI ≤ 25 kg/m2 /14/.

In general, an increase in CRP at marginal-to-moderate dietary magnesium deficiency indicates the tendency toward low-grade inflammation and concurrently the tendency toward the development of atherosclerosis, hypertension, diabetes mellitus and osteoporosis.

Sudden cardiac death (SCD) /15/

In the Atherosclerosis Risk in Communities (ARIC) Study, 264 cases of SCD were observed in a cohort of 14,232 examined individuals aged 45–60 years over the course of 12 years. Their magnesium levels were categorized by the quartiles < 0.75 mmol/L; 0.78–0.80 mmol/L; 0.83–0.85 mmol/L and ≥ 0.87 mmol/L. The hazard ratios of SCD in the magnesium quartiles were 1.00, 0.97, 0.70, 0.62, with an almost 40% reduced risk of SCD in quartile 4 versus 1 of serum magnesium.

Congestive heart failure (CHF) /16/

Magnesium deficit is frequently observed in patients with CHF either as an isolated disorder or in the context of other electrolyte and acid-base abnormalities. These disorders are principally attributed to the activation of neurohumeral mechanims as well as drugs (diuretics) commonly used in this patients. In respiratory alkalosis Mg2+ is shifted into the intracellular compartment.

Chronic kidney disease (CKD) /17/

CKD patients with a serum magnesium concentration < 1.8 mg/dL (0.74 mmol/L) had a mortality hazard ratio of 1.6 compared to those with concentrations > 2.2 mg/dL (0.91 mmol/L). Moreover, the annual decrease in eGFR is more pronounced in patients with CKD and hypomagnesemia than in those with normal magnesium levels.


Diuretics can cause hypomagnesemia due to increased renal Mg2+ excretion. Loop diuretics (azosemide, bumetanide, furosemide, piretanide, torasemide) lead to reduced reabsorption of Mg2+ in the loop of Henle; thiazides (hydrochlorothiazide, xipamide, chlortalidone) reduce reabsorption in the distal tubule.

Chronic alcoholism /18/

Hypomagnesemia was the most common electrolyte disturbance observed in 30% of chronic alcoholic patients. Causes were assumed to be reduced magnesium intake, the occurrence of diarrhea under alcohol withdrawal and, in one third of the patients, respiratory alkalosis. In respiratory alkalosis Mg2+ is shifted into the intracellular compartment.

Diabetes mellitus /19/

The association of reduced magnesium intake and diabetes type 2 has been shown in the Nurses Health Study. The odds ratio for diabetes type 2 was 0.62 in individuals in the highest magnesium intake quintile compared to 1.00 in those in the lowest quintile. It was concluded that adequate magnesium supplementation reduced the relative risk of diabetes by 40%. In the Health Professionals Follow-Up-Study in men, the relative risk of diabetes type 2 was 0.67 in the group with the highest magnesium intake.

Hypomagnesemia is more common in patients with a fasting plasma glucose (FPG) > 126 mg/dL (7.0 mmol/L). In a multi variable linear regression analysis, magnesium was significantly associated with FPG only in patients with FPG > 7.0 mmol/L /20/.

Cyclosporin therapy in kidney transplanted patients

Many kidney transplanted patients under cyclosporin therapy exhibit hypomagnesemia. For example, the serum level was < 0.70 mmol/L in 20% of the patients not receiving azathioprine and prednisolone, while it was higher in those receiving azathioprine and prednisolone. Possible renal Mg2+ loss is assumed /21/.

Proton pump inhibitors (PPI)

PPI are H+-K+-ATPase inhibitors and represent the most efficient drugs for treatment of acid-related gastrointestinal disease. It is important in peptic ulcer therapy to increase the pH to values above 4. Such values are attained after 1 week of therapy with a daily dose of 40 mg omeprazole or 60 mg lanzoprazole. At normal gastric H+ concentration, the H+ compete with Mg2+ for ligand binding sites in the food. The PPI induce reduced H+ secretion; consequently, less Mg2+ are released from the binding sites and are therefore not available for intestinal absorption. Gastrointestinal Mg2+ absorption occurs via passive para cellular diffusion and active transport via apical membrane Mg2+ channels (TRPM-6/7) in enterocytes. Disruption of the TRPM-6/7-mediated active transport of dietary Mg2+ across the apical membrane of the intestine due to a decrease in luminal H+ concentration as a result proton pump inhibition, or impaired passive Mg2+ absorption across the intercellular junctions causes magnesium deficiency /22/.

Laboratory findings: in a case description /23/, patients, who had been administered a PPI for more than 1 year and were admitted to hospital with lethargy and muscle cramps, had serum levels of 0.18 mmol/L magnesium and 1.26 mmol/L calcium, and Mg2+ excretion was 0.11 mmol/L.


There is no magnesium deficiency in preeclampsia. However, parenteral magnesium therapy reduces systemic and cerebral vasospams in this disease (1 g of magnesium sulfate contains 89 mg = 4.0 mmol = 8.12 mEq). The effect of magnesium is thought to be based on calcium antagonism either at the calcium channels or in the intracellular compartment.

Hereditary disorders in magnesium homeostasis – Generalized /9/

Hereditary hypomagnesemia is diagnosed by identifying the genes encoding the mechanisms of renal tubular Mg2+ transport. The proximal thick ascending limb of the loop of Henle reabsorbs 65–70% of the glomerularly filtered Mg2+, whereas the distal convoluted tube reabsorbs 10–20%. A differentiation is made between proximal and distal disorders in Mg2+ transport.

– Bartter syndrome

Bartter syndrome is caused by mutations in genes encoding the proteins NKCC2 (SLC12A1), ROMK (KCNJ1) CLC-Kb (CLCNKB), Barttin (BSND) and the calcium-sensitive receptor (CaSR). It is a disorder of the thick ascending limb of Henle. Patients have severe renal Na+ and Cl wasting with a normotensive or hypotensive phenotype.

Laboratory findings: hypokalemia, metabolic alkalosis, high renin, hyperaldosteronism, high prostaglandin E2. Only some patients develop hypomagnesemia and hypercalciuria

– FHHNC /24/

Patients suffering from familial hypomagnesemia with hypercalciuria and nephrocalcinosis (FHHNC) develop severe hypomagnesemia due to renal Mg2+ wasting. Mutations in the genes CLDN 16 and CLDN 19 have been identified as the cause of disturbed Mg2+ reabsorption in the ascending limb of the loop of Henle. The genes encode the tight junction proteins claudin-16 and claudin-19. The change in the claudin-16/19 complex results in impaired cation selectivity which reduces the lumen-positive voltage potential and, thus, the electrochemical gradient responsible for Mg2+ reabsorption.

Laboratory findings: pronounced renal loss of Mg2+ with hypomagnesemia and also hypercalcuria. Not all disorders with partial loss of the tight junction function lead to hypomagnesemia.

– HSH /25/

Hypomagnesemia with secondary hypocalcemia (HSH) is an autosomal recessive disorder that manifests clinically 2–4 weeks after birth with symptoms of hypomagnesemia. Clinical symptoms are disturbed neuromuscular excitability, muscle spasms, tetany and generalized convulsions. The HSH is caused by a mutation in the gene TRPM6 encoding the transient receptor potential channel melastenin member 6 (TRPM6). TRPM6 is an epithelial Mg2+ channel belonging to the super family of TRP channels. TRPM6 mediates the Mg2+ transport across cellular membranes. The protein is primarily expressed in the luminal membrane of the distal convoluted tubule of the kidney and in the luminal membrane of the intestine, predominantly of the colon.

Laboratory findings: serum magnesium levels of 0.1–0.4 mmol/L due to impaired intestinal Mg2+ absorption and renal reabsorption, hypomagnesemia induced hypocalcemia.

– IRH /26/

The underlying cause of isolated renal hypomagnesemia (IRH) is a mutation in the pro epidermal growth factor gene, the precursor of the epidermal growth factor (EGF). EGF activates TRPM6 by shuttling it from intracellular compartments to the plasma membrane. The mutated form of pro-EGF is processed incorrectly and is, therefore, unable to stimulate Mg2+ re-absorption via TRPM6, explaining the renal Mg2+ wasting.

Laboratory findings: serum magnesium levels of 0.53–0.66 mmol/L were measured in twin sisters; calcium excretion was normal. Inappropriately high fractional renal excretion of Mg2+.

– Gitelman syndrome /27/

A renal salt-wasting disorder caused by mutations in the SLC12A3 gene, encoding the thiazide-sensitive Na+–Cl co transporter in the distal tubule. Hypomagnesemia is due to a reduced abundance of TRPM6, leading to renal Mg2+ wasting.

Laboratory findings: hypomagnesemia, hypokalemic metabolic alkalosis, hypocalcuria. The latter results from increased proximal tubular reabsorption of Ca2+ due to mild volume depletion.

– Autosomal dominant hypomagnesemia /28/

Defects are in the KCNA1 gene encoding the voltage-gated K+ channel Kv 1.1. The channel co localizes with TRPM6 along the luminal membrane of the distal convoluted tubule. Clinical symptoms are recurrent muscle cramps, tetany, tremor, muscle weakness cerebellar atrophy, and myokymia. The absence of Kv1.1 depolarizes the apical cell membrane and, thereby, prevents the establishment of a favorable electrochemical driving force for Mg2+ entry across the plasma membrane /9/.

Laboratory findings: serum magnesium levels of 0.28–0.37 mmol/L were measured in the setting of normocalcuria.,

– Isolated dominant hypomagnesemia /29/

A mutation in the FXYD2 gene is the underlying defect. The affected patients present with renal Mg2+, wasting accompanied by hypocalcuria. The FXYD2 gene encodes the γ-subunit of Na+-K+-ATPase. Mutations in the FXYD2 alter the affinity for Na+ and K+ which is caused by misrouting of the Na+-K+-ATPase γ-subunit. The lower electrochemical potential resulting from reduced activity of the plasma membrane is inadequate to drive the Mg2+ across the membrane. Clinically, the patients suffer from convulsions.

Laboratory findings: serum magnesium levels below 0.4 mmol/L because of renal wasting, accompanied by hypocalcuria.

– SeSAME syndrome /30/

The tubulopathy is caused by a defect in the KCNJ10 gene, encoding an inwardly rectifying K+ channel 10 (Kir4.1) expressed in kidney, brain and inner ear. The mutations cause a pronounced decrease in K+ influx into the cells of these organs. This is thought to impair Mg2+ transport via TRPM6. Clinical symptoms are sensineural deafness, ataxia, mental retardation and electrolyte imbalance (SeSAME).

Laboratory findings: patients exhibit the phenotype of Gitelman syndrome in the setting of hypocalciuria.


Mg2+ affect both neuronal and vascular functions. In a study /32/ low and high concentrations were associated with high risk of vascular-related non-Alzheimer dementia. Multifactorially adjusted hazard ratios for non-Alzheimer dementia were 1.50 (95% CI 1.21–1.87) for the lowest and 1.34 (1.07–1.69) for the highest versus the fourth quantile (reference) of plasma Mg2+ concentrations. The lowest risk was observed at a concentration of 2.07 mg/dL (0.85 mmol/L).

Table 10.6-1 Reference intervals for manganese /12/

Serum, plasma

  • Adults

5–20 nmol/L

0.3–1.1 μg/L

  • Children

3–13 nmol/L

0.2–0.7 μg/L

Whole blood

110–200 nmol/L

6.0–11.0 μg/L


27–40 nmol/24 h*

1.25–2.25 μg/24 h

* Related to 1.5 L daily urine volume

Conversion: μg/L × 18.2 = nmol/L

Table 10.6-2 Diseases and conditions with manganese body burden

Clinical and laboratory findings

Neurotoxicity /10/

Increased accumulation of Mn in the brain results in neurological symptoms with cognitive, psychiatric and movement abnormalities. The highest Mn concentrations are found in basal ganglia. Some of the manifested clinical features correspond to those of Parkinson’s disease. Unlike the idiopathic Parkinson’s disease, however, Mn-induced parkinsonism does not lead to the degeneration of diencephalic dopaminergic neurons. It is associated with chronic liver disease, especially with advanced liver cirrhosis. Because Mn is excreted in the bile, Mn excretion is impaired in liver cirrhosis, with subsequent increased Mn release to the blood and accumulation in the brain. Besides chronic liver diseases, Mn intake via inhalation and abuse of the intravenously injected psychon stimulant ephedrine (Russian cocktail) play a role. In Russian cocktail, ephedrine is oxidized using potassium permanganate in acetic solution.

Strong evidence now exists for causal associations between Mn and both neurodevelopmental and neurodegenerative disorders /1112/.

Laboratory findings: the Mn values in blood are elevated in some of the patients; evidence of Mn in the brain is provided by imaging. Mn concentrations measured in addicts using the Russian cocktail are as high as 2–3 mg/L (i.e., almost 500-fold higher than normal).

Other neurotoxic diseases /13/

Mn is released to the environment as a product of industrial activities (melting of ferrous alloys, welding, glass-making, battery manufacturing, mining), through the use of the pesticide maneb and the use of antiknock agents (methyl cyclopentadienyl manganese tricarbonyl, MMT) in gasoline. Increased intake, especially unregulated intake via inhalation, exceeds the Mn excretion capacity of the liver and results in Mn accumulation in the CNS. Moreover, inhaled Mn can be transported directly into the brain via the olfactory tract. Clinical symptoms are of extra-pyramidal nature and include kinetic tremor, bradykinesia, rigidity, dystonia and gait disturbances. In addition to these motor abnormalities, neuropsychological symptoms such as deficits in working memory and spatial orientation occur.

Postnatal exposure of children and adolescents to Mn is associated with cognitive function disturbance and hyperactive behavior /14/.

Laboratory findings: at excessive Mn exposure, concentrations of 182–728 nmol/L (10–40 μg/L) in plasma are measured /15/.

Parenteral nutrition /6/

In parenteral nutrition, the food is enriched with Mn. More than 50% of patients on long-term parenteral nutrition have elevated Mn blood levels, often associated with cerebral and hepatic complications /16/. The American Society for Parenteral and Enteral Nutrition and the European Society for Clinical Nutrition and Metabolism recommend a daily Mn administration of 60–100 μg in adults or 1 μg/kg/day for children, with a maximum of 50 μg/day for children.

Chronic hemodialysis

There is no indication regarding Mn supplementation in these patients /7/.


Low Mn excretion with the bile can result in hyper manganesemia and accumulation of Mn in the brain. This does not apply to biliary atresia. In pediatric patients, however, cholestasis is not a predictor of the Mn status because 13 in 20 children with cholestasis had normal serum Mn levels /17/.

Table 10.7-1 Reference intervals for molybdenum /1/

Serum, plasma

< 10 nmol/L(1

< 1 μg/L

Whole blood

10–100 nmol/L

1–10 μg/L


150–240 nmol/24 h(2

15–24/24 h

1 Detection limit; 2 Related to 1.5 L daily excretion; conversion: μg/L × 10.4 = nmol/L

Table 10.8-1 Reference intervals for nickel /12/


< 20 nmol/L

< 1 μg/L(1

Whole blood

< 20 nmol/L

< 1 μg/L(1


51–128 nmol/24 h(2

3–7.5 μg/24 h(2

1) Detection limit; 2) Related to 1.5 L daily excretion; conversion: μg/L × 17.0 = nmol/L

Table 10.9-1 Biologically and technically important selenium species /3/





Hydrogen selenide



Metabolite, not free





Dimethyl selenide



Respiratory air

Trimethyl selenium ion








In selenoenzymes




In garlic, broccoli


CH3Se-(CH2)2-CH(NH2)- COOH


Selenoproteins (oxidation stage Se2–)

Heavy metal selenides

CuSe, AgSe, HgSe


Selenium minerals

Elementary selenium

3 modifications


Technical application

Selenium dioxide



Technical application

Selenious acid



Technical application




Metabolite, supplement

Selenic acid



Metabolite, supplement




Metabolite, supplement

Table 10.9-2 Reference intervals for selenium /34/

Se in serum/plasma



Children up to 1 yr



Children 2–5 yrs



Children 5–10 yrs



Children 10–16 yrs






Whole blood



Whole blood



Urine/24 h (adults)



(μg Se/g Hb)



* Related to 1.5 L daily excretion; conversion: μg/L × 0.0127 μmol/L

Glutathione peroxidase in serum /3/


Children up to 1 yr


Children 2–5 yrs


Children 5–10 yrs


Children 10–16 yrs­






Table 10.9-3 Diagnosis of selenium deficiency /3/


Whole blood

Red cells
(μg/g Hb)


Normal (> 50)

Normal (> 60)

Normal (> 0.2)

Within normal limits

Low (< 50)

Normal to low

Normal to low

Medium-term undersupply

Low (< 50)

Low (< 60)

Low (< 0.2)

Long-term undersupply

Normal (> 50)

Low (< 60)

Low (< 0.2)

Compensatory undersupply

Table 10.9-4 Selenium status and indication of selenium administration /3/


Selenium in plasma (μg/L)

Application and purpose


< 25

Substitution required



Optional substitution



Trial supplementation


> 120

Treatment of various diseases (indication not confirmed in most cases)


> 400

Table 10.9-5 Diseases and conditions associated with selenium deficiency or toxicity

Clinical and laboratory findings

Keshan disease /13/

An endemic dilative cardiomyopathy primarily affecting children and women of child-bearing age. Se deficiency is not the only cause.

Laboratory findings: Se in plasma/serum is below 20 μg/L (0.25 μmol/L), the activity of glutathione peroxidase in plasma is reduced to below one third.

Kashin-Beck disease /3/

Occurs in Se deficiency areas in China, Tibet, Korea and Siberia. It is a dystrophic osteoarthritis and spondylarthritis. Endochondral growth of the long and short bones is disturbed.

Laboratory findings: see Keshan disease.

Parenteral nutrition (PN)

Depending on the given situation, PN patients require Se substitution that should be given intravenously /7/.

  • In patients receiving PN at home, 63 μg/day of Se will meet the requirements, but is insufficient in 15%. In such cases, it is recommended to administer 85 μg/day.
  • Patients after surgery: a dosage of 60–100 μg/day is recommended
  • Critically ill in intensive care: patients with severe burns have a median Se requirement of 210 μg/day and, for compensation of the loss in the initial days, require 315–380 μg/day
  • Bone marrow transplantation: doses of 120 μg/day resulted in serum Se levels within the reference interval.
  • Sepsis: doses of 400 μg/day are beneficial in the patients during the first 9 days.

Malignant tumor

Various studies have shown that the plasma/serum Se concentrations in tumor patients are 5–35% lower than in healthy controls. Se levels in the upper reference interval are thought to be associated with lower cancer mortality and protect against the development of cancer, especially against prostate carcinoma /12/. However, the results of the Selenium and Vitamin E Cancer Prevention Trial (SELECT) have shown that the Se level is ineffective in cancer prevention /14/.


In HIV patients with a Se concentration below 85 μg/L (1.1 μmol/L), the probability of dying from this disease is approximately 20-fold higher than in patients with higher concentrations /15/.

Thyroid disorder

The thyroid is an organ with a high content of antioxidant selenoenzymes /16/. H2O2 is constantly generated for thyroid hormone production and any excess is reduced by the glutathione peroxidases.

Myxedema: combined Se and iodine deficiency in infants causes myxedema. Glutathione peroxidase activity is reduced and the formed oxygen radicals cannot be adequately detoxified, causing the inflammation and degradation of thyroid follicles.

Autoimmune thyroiditis (AIT): individuals with mild Se deficiency have increased prevalence of AIT. If Se was supplemented in the setting of AIT and an Se concentration of 68 μg/L (0.86 μmol/L), the Se concentration increased to 86 μg/L (1.1 μmol/L) and the AIT declined within 6 months /17/.

In a study /20/ severe Se deficiency (below 20 μg/L) was associated with increases in free T4 levels, but not with decreases and increases in free T3 and TSH levels, respectively

Various diseases

Suboptimal Se concentration is linked to inadequate immune function, infectious disease, dilative cardiomyopathy, acute myocardial infarction, reproductive disorder and numerous other diseases. The extent to which Se deficiency is an etiological factor or only an epiphenomenon needs to be further clarified.

Hemodialysis patients

These patients tend to have lower Se serum levels due to lower dietary protein intake.

Selenium intoxication

Chronic Se intoxication has been described following the intake of high Se doses with drinking water, food, upon consumption of Se-containing nuts or following misdosing of Se products. Intoxication mainly occurs in areas with a high Se content of the soil. Clinical symptoms occur after a daily intake of more than 1,000 μg for an extended period of time. A daily intake of up to 800 μg does not cause any symptoms. An intake of half of this amount, i.e. 400 μg/day, is considered tolerable.

Clinical symptoms /3/: garlic-like breath; spotted, brittle, streaked nails; brittle hair, hair loss, tooth decay, skin depigmentation, rashes, pain in the extremities, gastrointestinal complaints, fatigue, listlessness, exhaustion, apathy, low performance.

Laboratory findings: monitoring should be performed if the Se concentration in plasma/serum is above 160 μg/L (2.0 μmol/L) for a long period of time. In the presence of the above-mentioned clinical symptoms, the concentration is higher than 500–1,000 μg/L (6.4–12.7 μmol/L) /18/.

Table 10.9-6 Selenoproteins and their function /7/



Glutathione peroxidases (GPX)

Six different GPX isoforms (GPX1–GPX6) are distinguished. They are located differently in cells and tissues, have an antioxidant effect and remove hydroxyperoxides. The GPX are only optimally stimulated at a daily selenium intake of 90 μg /3/.

Thioredoxin reductases (TR)

Three different isoforms (TR1–TR3) are distinguished. They play multiple roles in the removal of peroxides, reduction of sulfur compounds such as thioredoxin and maintenance of the redox status of transcription factors.

Iodothyronine deiodases (D)

Three different isoforms (D1–D3) are distinguished. They contribute significantly to the formation and regulation of peripheral thyroid hormones:

D1 and D2 convert T4 into T3

D1 and D3 convert the bioactive T3 into the inactive reverse-T3.

The deiodases are optimally stimulated at a daily selenium intake of 40–55 μg /3/.

Selenoprotein P

This is the transport protein for Se in plasma; moreover, it has an antioxidant effect at endothelia. In the blood, 50–70% of Se is transported bound to selenoprotein P.

Selenoprotein W

This is an antioxidant protein in the myocardium and skeletal muscle and probably also in the brain.

Selenophosphate synthetase

This enzyme catalyzes the synthesis of selenophosphates that are required for the formation of selenocysteine and other selenoproteins.

15-kilodalton selenoprotein

This protein is present in the prostate at high concentration.

Table 10.10-1 Reference intervals for zinc /23/






0.6 –1,2



0.6 –1.45



0.8 –1.7

Whole blood


4.0 –7.5








μmol/24 h

mg/24 h*




* Related to 1.5 L urine volume; conversion: mg/L × 15.3 = μmol/L

Table 10.10-2 Diseases and conditions associated with zinc deficiency or toxicity

Clinical and laboratory findings

Reduced Zn intake

The daily Zn requirements are 2.5 mg to maintain Zn homeostasis in adults, requiring an enteral Zn supply of 8–11 mg/day /12/. The recommended intake is 2–3 mg/day for infants and 5–9 mg/day for older children. Reduced Zn intake can /1/:

  • Be dependent on the geographical region. This applies to regions with a low Zn content in the soil in Egypt or Iran, in many cases associated with nutritional deficiency or malnutrition.
  • At adequate calorie intake and normal Zn content of the soil be due to inappropriate food selection. For example, the prevalence of Zn deficiency is estimated to be 20% worldwide. The prevalence in individuals aged over 73 years in the USA is 42.5% /13/.
  • Occur in individuals with chronic alcoholism, chronic renal disease and enteral absorption disorders. Deficiency is moderate.
  • Be due to inadequate supply of the unborn child at pre term delivery.


The daily Zn requirement during pregnancy and lactation is 12–13 mg /14/. Zn is transferred from mother to child mainly during the last 10–12 weeks and a daily amount of 0.5–0.75 mg is taken up by the child during the last 3 weeks of gestation. Newborns need 0.3–0.5 mg per kg and day during the first weeks postpartum /15/.

Parenteral nutrition (PN) /14/

Depending on the given situation, patients on PN need Zn substitution as follows:

  • In patients without gastrointestinal losses, supplementation of 3–4 mg/day is recommended
  • In patients with fistula, diarrhea and intestinal drainage, 12 mg of zinc should be added for each liter of fluid loss
  • In patients with burns, addition of 36 mg/day may reduce infectious complications
  • Infants should be given 0.3 mg, older children should receive 0.05 mg, and in the growth phase 0.1 mg per kg of body weight and day in each case.


Diarrhea is the second most common cause of death in children under 5 years of age. Therefore, the WHO and the UNICEF recommend zinc supplementation for 10–14 days during and after the episode. According to a meta analysis /16/, Zn supplementation reduces the prevalence of diarrhea by 19%. The prevalence of severe acute lower respiratory infection (ALRI)/pneumonia episodes in the months following supplementation is reduced by 23%.

Sickle-cell anemia

These patients suffer from moderate Zn deficiency. Renal Zn excretion is increased due to high protein turnover resulting from hemolysis. Clinical symptoms are growth retardation, hypogonadism in men, poor dark adaptation, cellular immunodeficiency and hyperammonemia.

Acrodermatitis enteropathica /18/

This is a rare autosomal recessive metabolic disorder resulting from mutation of the intestinal Zn transporter Zip4. Clinical symptoms are alopecia, skin disorder, diarrhea, weight loss, reduced immune function, neuropsychological disorders and hypogonadism in men.

Depression /19/

Clinical studies and experimental work have revealed a link between reduced Zn status and neuropsychological disorders. Although not all depressions are due to Zn deficiency and the plasma Zn concentration is not an accurate indicator of the Zn status, it is recommended to consider Zn deficiency in patients symptomatic of depressive disorders.

Inflammation and infections

Zn is transported bound to albumin, α2-macroglobulin and transferrin. In systemic inflammation, the synthesis of albumin and transferrin as negative acute-phase proteins is reduced and their plasma concentration, and consequently that of Zn, decreases. In infections, Zn is increasingly retained in the liver thus causing reduced plasma concentration.

Chronic hemodialysis /20/

In many cases, patients with chronic kidney disease undergoing hemodialysis have plasma Zn concentrations below 0.6 mg/L (9 μmol/L). Therefore, oral substitution of 30–45 mg/day is recommended.

Zinc deficiency due to chelating agents /21/

In a case description, acute potassium permanganate poisoning (approximately 10 g absolute for 4 weeks) was treated with the chelating agents calcium disodium ethylenediamine tetra acetate and calcium trisodium pentetate. After 2 weeks of therapy, acrodermatitis enteropathica-like skin alterations due to Zn deficiency were found. The serum Zn level was 0.4 mg/L (6 μmol/L). Treatment with Zn led to significant improvement within 8 months.

Chronic liver disease

The liver mainly plays a crucial role in maintaining Zn homeostasis. Therefore, the occurrence of chronic liver diseases such as fatty liver, chronic hepatitis, and liver cirrhosis results in the impairment of Zn metabolism, and subsequently Zn deficiency. Zn deficiency causes plenty of metabolic abnormalities, including insulin resistance, hepatic steatosis and hepatic encephalopathy. Inversely, metabolic abnormalities like hypoalbuminemia in patients with liver cirrhosis often result in Zn deficiency /22/.

Anemia due to Zn burden

Zn, iron, copper (Cu) and vitamin A potentially interfere with each other in enteral absorption. Prolonged intake of high doses of Zn results in Cu deficiency. Both elements compete for binding to the metallothionein (MT) in the enterocytes. The MT expression in the enterocytes is up regulated by Zn supplementation, and MT binds Cu with a higher affinity than Zn. The MT-Cu complex remains in the enterocytes and together they are desquamated into the intestinal lumen. Cu deficiency causes anemia (see Section 10.4 – Copper (Cu)). Short-term Zn substitution in children with diarrhea does not lead to anemia /23/. Longer-term treatment with more than 150–180 mg Zn/day can trigger Cu deficiency (hypocupremia).

The LD50 has been estimated to be 27 g per day in humans. However, the uptake of such an amount is unlikely because 225–400 mg have been determined to be an emetic dose. Ingestion of toxic doses results in epigastric pain, nausea, vomiting, fatigue and lethargy. Oral intoxication can result from storage of food and drink of acidic nature in galvanized containers enabling the removal of zinc from the galvanized coating. Inhalation of zinc-containing smoke occurs during industrial processes like galvanization or during military action. Smoke bombs contain zinc oxide or zinc chloride. Soldiers inhaling this smoke may develop acute respiratory distress syndrome (ARDS). However, it is unclear whether Zn is the only cause of ARDS. The most widely known effect of inhaling zinc-containing smoke is the metal fume fever (MFF), which is mainly caused by inhalation of zinc oxide. In zinc smelting or welding, fresh metal fumes with a particle size below 1 μm are inhaled. Symptoms of this reversible syndrome begin a few hours after exposure and include fever, muscle inflammation, nausea, fatigue, cough, dyspnea and respiratory problems that improve after a few hours. In zinc intoxication, the amounts of Zn in air are approximately 320–580 mg/m3.

Table 10.11-1 Reference intervals for iodine /12/

Adults and children


0.31–0.61 μmol/L

40–80 μg/L


0.79–1.57 μmol/24 h

100–199 μg/24 h

0.94 μmol/g creatinine

120 μg/g creatinine

Conversion: μg/L × 0.0079 = μmol/L

Table 10.11-2 Daily iodine intake recommendations /3/

Age or population group


US Institute of Medicine (RDA values)

  • Infants 0–12 months (AI value)


  • Children 1–8 years


  • Children 9–13 years


  • Adults > 14 years


  • Pregnancy


  • Lactation


World Health Organization (RNI values)

  • Children 0–5 years


  • Children 6–12 years


  • Adults > 12 years


  • Pregnancy


  • Lactation


For abbreviations, see Tab. 10.1-5 – Definitions for trace element requirement

Table 10.11-3 Assessment of iodine intake based on urinary iodine excretion /3/



Iodine status

School children

Below 20


Severe iodine deficiency



Moderate iodine deficiency



Mild iodine deficiency





More than adequate

Risk of iodine-induced hyperthyreosis in sensitive groups

Above 300


Risk of iodine-induced hyperthyreosis or autoimmune thyroid disease

Pregnant women

Below 150





More than adequate

Above 499


Lactating women

Below 100


The values in lactating women are lower because of iodine clearance in breast milk

From 100


Children < 2 yrs

Below 100


≥ 100


Figure 10.1-1 Minerals and trace elements in the periodic table, according to Ref. /20/.

Li* Be* B F* Na Mg Al Si* P S Ci K Ca Ti V* Cr Mn Fe Co Ni* Cu Zn As Se Br Rb Sr Y Mo Ag Cd Sn* I Cs Ba Pt Au Hg Pb* Bi E Essential trace elements (gray fields) * Candidates for essential trace elements (fields with a star) Elements with known or unknown function

Figure 10.4-1 Duodenal enterocytes absorb dietary Cu+ and Fe3+ via transporters located at the luminal membrane. Absorption of the oxidized forms of the metals requires the action of a cell-surface reductase. In the cytosol, chaperone proteins (Atox1) deliver Cu+ to various proteins and organelles, excess Cu+ is sequestered by MT. Cu+ and Fe2+ are transported across the basolateral membrane by FPN and ATP7A, respectively. Modified from Ref. /16/. DMT, divalent metal ion transporter; FPN, ferroportin; HP, hephestin; CTR1, Cu transporter; CCS, Cu chaperone for super oxide dismutase 1 (SOD 1); ATOX1, antioxidant protein 1; MT, metallothionein; TGN, trans-Golgi network; ATP7A, Cu transporting ATPase.

Ferritin MT ATOX1 H P ? ? ? ? CCS Fe 2+ Fe 2+ Fe 3+ Nucleus Mitochondrion ATP7A ATP7A Cu 1+ Cu + Cu + ATP7A TNG SOD1 Cu 1+ Reductase DMT1 CTR1 2 × Fe 3+ Apo-TF FPN

Figure 10.5-1 Renal magnesium transport. With kind permission from Ref. /9/. The figure shows the Mg2+ transport mechanisms in the thick ascending limb (TAL) of the loop of Henle at the bottom and those in the distal convoluted tubule (DCT) at the top. Explanations: active transport of Na+, Cl and K+ in the TAL is driven by the furosemide sensitive co transporter NKCC2. The process is powered by basolateral Na+-K+-ATPase. Efflux of Cl occurs through CLC-Kb channels, but K+ is recycled back into the lumen via the renal outer medulla K+ (ROMK). The TAL is the main site of passive para cellular re-absorption of Mg2+. Back flux of Na+ in the upper TAL is mediated by the Claudin 16/19 complex. The Ca2+-sensitive receptor (CaSR) inhibits the cyclic AMP generation and hence NKCC2 activity, thereby reducing the lumen-positive voltage.

The DCT plays an important role in the fine-tuning of the systemic Mg2+ concentrations. The epithelial Mg2+ channel TRPM6 co localizes with the Na+-Cl co transporter NCC and the K+ channel Kv1.1 along the apical cell membrane. The epidermal growth factor receptor (EGFR), the Cl channel CLC-KB, the K+ channel Kir4.1, the Na+-K+-ATPase and its FXYD2-γ subunit are all located in the basolateral membrane. The hepatocyte nuclear factor (HNF1B) controls the extent of expression of the FXYD2 gene. It is currently not known how Mg2+ is extruded across the basolateral membrane.

Na,K ATPase γ-subunit K + Kir4.5/5.1 Mg 2+ ? Na,K ATPase γ-subunit K + Kir4.5/5.1 Mg 2+ ? Na,K ATPase γ-subunit Ca 2+ CaSR CIC-Kb Barttin Na + Maculadensa CD PT 10–20% CNT DCT 10% TAL 65–70% Cl K + Mg 2+ Mg 2+ Na + Cl K + HNF1B Barttin CIC-Kb Blood EGFR Urine Pro-urine ROMK NKCC2 Claudin 16–19 NCCKv1.1TRPM6 EGF Transcription

Figure 10.11-1 Iodide transport from the thyrocyte to the thyroid follicle. Modified according to Ref. /15/. For explanation, see section Pathophysiology. Abbreviations: TSHR, TSH receptor; NIS, Na+/J cotransporter, TPO, thyroxine peroxidase; TG, thyroglobulin; I, iodine ion

Tg Tg +I T3 + T4 T3 T4 T3 T4 TSH Na + 2 K + 3 Na + AMP cAMP ATP ADP + P 1 TPO I I I I I I Na + I Pendrin Nukleus NIS Apical Basolateral TG-Follikel Tg-T4/T3 TG TSHR ATPase
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