Laboratory Diagnostics

Calcium distribution in the body

• 99% skeleton-bound (approx. 1 kg)
• 1% extracellular space
• daily dynamic exchange of both compartments

The total calcium in serum is divided as follows:

• approx. 40% is bound to protein, predominantly albumin
• approx. 10% is present in the form of inorganic complexes, e.g. calcium bound to phosphate
• approx. 50% is free: ionized calcium

Reference ranges for adults: Total calcium and ionized calcium

Assessment Diagnostics: Total calcium and ionized calcium⁷

The total calcium concentration in serum is strongly influenced by the protein content, especially by albumin. A decrease in albumin by 1g/dl leads to a decrease in total calcium by about 1 mg/dl (0.25 mmol/l).

Ionized calcium is considered a better indicator of calcium status as it represents the biologically active form. With higher specificity and sensitivity, ionized calcium is generally increased or decreased in the same diseases as total calcium. To determine the true calcium situation, the determination of ionized calcium is particularly important in the following cases: in newborns and premature infants (often acidosis and/or hypoproteinemia), after massive transfusions (calcium complexes due to citrate), in cardiopulmonary bypass, cardiac incidents during hemodialysis, in total protein values below 60 and above 85 g/l, mild degrees of hyperparathyroidism are occasionally only detected by repeated determinations of ionized calcium.

However, ionized calcium is also influenced by numerous factors such as haemolysis, physical activity, blood pH, diurnal fluctuations or transient after food intake.

In addition, sample collection is very complex and therefore generally not yet suitable for practical use: close the sample container immediately (free of air bubbles), transport ice-cooled. With good hyperaemia and good circulatory conditions, capillary puncture (earlobe, fingertip, in infants the lateral areas of the heel) gives comparable values to arterial blood sampling: heparinized glass capillary, fill capillary completely, insert wire pins for later mixing, close capillary on both sides with caps, store between cooling elements, analysis within 1h.

Reference ranges for adults: Calcium excretion in urine

Assessment Diagnostics: Calcium excretion in urine

Assessment of calcium metabolism with increased or decreased serum calcium. Bone pain, kidney stones, renal insufficiency, cortisol therapy.

Differential diagnosis: familial hypocalcuric hypercalcemia and primary hyperparathyroidism⁸

Measurement of calcium excretion in 24-hour urine is considered a useful additional parameter for assessing calcium status. However, this should only be evaluated in conjunction with the serum concentrations of calcium, phosphate, parathyroid hormone and vitamin D. In addition, the calcium urine value is dependent on oral calcium intake.⁹

Reference ranges Iron in serum^10*

Even if the concentration of iron is regularly measured in the blood serum, the values are not very meaningful for assessing total body iron. In addition to slight variations from laboratory to laboratory, the serum value itself is subject to strong fluctuations: On the one hand, the value is subject to hourly fluctuations and, on the other, to a circadian rhythm with higher serum levels in the afternoon. It is also influenced by whether and what the patient has eaten.

It is therefore best to take a blood sample on an empty stomach and in the morning.

In addition, serum iron is sensitive to hemolysis and the serum level is increased by iron released from erythrocytes - both in the blood sample and in the case of pathological hemolysis.

Instead of measuring serum iron alone, a combination of the following laboratory parameters has therefore proven effective in determining iron metabolism:¹¹

Although a low Hb status indicates anemia, the value says nothing about the filling status of the iron stores. The depot iron, the serum ferritin, is responsible for this; it is the central laboratory value as a measure of the filling status of the iron stores. This shows whether the iron depot in the body is full, reduced or depleted. If this value is too low, there is an iron deficiency: 

• Serum ferritin ≤ 30 ng/ml indicates depletion of the total body iron reserves available for hemoglobin synthesis.
• Between 12 and 30 ng/ml, the first symptoms of iron deficiency appear.
• Serum ferritin ≤ 40 ng/ml can already lead to diffuse hair loss in women.
• As serum ferritin correlates well with tissue iron, the value is part of the diagnostic standard: 1 μg/l serum ferritin corresponds to 8 to 10 mg of storage iron.

Caution: In the case of infections or inflammation, the ferritin value is distorted and can be normal or elevated, even though the iron stores are empty. And whether there is an infection or inflammation in the body can be detected with the help of the CRP value. If elevated CRP is detected in the blood test, transferrin saturation provides a more reliable indication of iron availability.

The glycoprotein transferrin, which is produced in the liver, acts as an iron transporter from cell to cell in the organism. The transferrin saturation (TSAT) shows how much iron is actually transported. The reference range is defined as 20 to 45 %. If the transferrin saturation is below the lower limit of 20 %, the body has too little iron available for metabolism and iron deficiency-related erythropoiesis occurs. For this reason, transferrin saturation is considered a more suitable laboratory value for the detection of iron deficiency in inflammatory processes in the organism and the resulting falsified serum ferritin.

Other parameters for determining iron status

• soluble transferrin receptor

Soluble transferrin receptors are transferrin receptors (TfR) that form a complex with the iron transporter transferrin and are found freely in the blood plasma (sTfR, s = soluble).

They have the task of absorbing the iron transported to the cell by transferrin and bringing it into the cell. Over 80 % of transferrin receptors are localized on the precursor cells of erythropoiesis, with the exception of erythrocytes. Therefore, the sTfR concentration reflects both the iron requirement and the number of erythropoiesis cells. In iron deficiency, the sTfR concentration in the serum increases as the erythropoiesis cells produce more transferrin receptors. This is relevant for iron diagnostics because the increase in serum sTfR occurs before the haemoglobin level drops. The sTfR status reflects the current iron requirement, while ferritin gives an indication of how full the iron stores are. A combination of these two values provides a good picture of the iron status. ¹²

Erythrocyte indices:  Volume and hemoglobin concentration of the erythrocytes¹⁴

• MCV: mean cell volume of the erythrocytes, calculated from the erythrocyte and haematocrit value, standard value: 81-100 fL

• MCH: mean haemoglobin content of the erythrocytes, calculated from the erythrocyte and Hb value. Standard value approx. 30 pg

• MCHC: average hemoglobin concentration of the erythrocytes, calculated from the Hb and hematocrit value. Normal value: 30-36 g/dl

  The erythrocyte indices provide indications of possible causes of anemia:

• MCV low: microcytic anemia, e.g. due to iron deficiency

• MCV high: macrocytic or pernicious anemia, e.g. due to vitamin B12 deficiency

• MCV normal: normocytic anemia, especially in the case of bleeding

• MCH low: hypochromic anemia, e.g. due to iron deficiency e.g. due to iron deficiency
• MCH high: hyperchromic anemia, e.g. due to chronic alcohol abuse
• MCH normal: normochromic anemia, e.g. in anemia due to infections, inflammation or tumor diseases

Iron in urine¹⁵

Urine samples are unsuitable for the diagnosis of iron deficiency, because in the case of low excretion it is not possible to distinguish between an existing deficit of the biofactor iron and a reduced current absorption.

Iron content in hair

The iron content in hair and nail samples has a certain significance in toxicological aspects, but cannot be used to prove the iron status. Factors influencing the evaluation are the different structure and metabolism of the hair, external depositsand contamination of the sample.

The above-mentioned physiological correlations make the laboratory diagnosis of magnesium deficiency more difficult, as the determination of serum values is not always conclusive. Despite serum values in the normal range, a magnesium deficiency may be present intracellularly. The serum magnesium concentration only drops when the magnesium store is exhausted, i.e. hypomagnesemia is considered an indication of a massive magnesium deficiency. However, hypomagnesemia can also be masked by a previous release of magnesium from the intracellular space.¹³

Nevertheless, according to scientific studies and recommendations of the Society for Magnesium Research, the minimum target value for magnesium in serum is 0.85 mmol/L.^14,15,16

More helpful is the measurement of ionized magnesium, the actually active magnesium, which can already be reduced while the serum levels are still within the normal range. The measurement of ionized magnesium on magnesium-sensitive electrodes in serum measures the physiologically active fraction of magnesium, namely the fraction to which tissue reacts. Ionized magnesium is therefore considered a more sensitive indicator of the actual magnesium available at the cellular level.

However, the sampling procedure is very complex and therefore generally not yet suitable for practical use: close the sample container immediately (free of air bubbles), transport ice-cold. With good hyperaemia and good circulation conditions, capillary puncture (earlobe, fingertip, in infants the lateral areas of the heel) gives comparable values to arterial blood sampling: heparinized glass capillary, fill capillary completely, insert wire pins for later mixing, close capillary on both sides with caps, store between cooling elements, analysis within 1h.

Daher steht die Messung des ionisierten Magnesiums im Serum in der Standarddiagnostik – ebenso wie die Messung des Lymphozyten- oder Muskel-Magnesiums – noch nicht zur Verfügung.

In general, therefore, explicit attention should be paid to the patient's medical history, particularly in the case of the biofactor magnesium, especially in risk groups such as cardiovascular patients, diabetics and the elderly and those with deficiency symptoms.

Magnesium standard values^17,18

Magnesium im Serum

Magnesium in serum

• Material: Serum
• Amount of material: 1 ml
• Reference range

• Zielgruppe	Norm
  Neugeborene	0,48-1,05 mmol/L
  Kinder	0,6-0,95 mmol/L
  Frauen	0,77-1,03 mmol/L
  Männer	0,73-1,06 mmol/L

Target group Standard

Newborns 0.48-1.05 mmol/L

Children 0.6-0.95 mmol/L

Women 0.77-1.03 mmol/L

Men 0.73-1.06 mmol/L

Magnesium in whole blood

• Material: whole blood (intra- and extracellular), also possible from heparin blood
• Material quantity: 2 ml
• Reference range: 30-40 mg/L
• Measuring method: ICP-MS

Magnesium in erythrocytes

• Material: EDTA blood
• Material quantity: 5 ml
• Reference range: 1.65 - 2.65 mmol/l erythrocytes
• Measuring method: AAS

There are indications that the detection of the magnesium content in erythrocytes only reflects the current body magnesium content to a limited extent. Erythrocytes do not have all magnesium transport systems and can only absorb the biofactor poorly. The erythrocyte magnesium concentration gives no indication of the current magnesium supply, but represents the supply in the last few weeks, i.e. for the time in which the erythrocytes were formed.¹⁹

Magnesium in urine

• Material: 24-hour urine
• Amount of material: 10 ml urine
• Reference range: 2.05 - 8.5 mmol/24 hours.
• Method: Photometry

With the magnesium retention test (magnesium loading test) it is possible in principle to detect a chronic magnesium deficiency. For this purpose, the magnesium concentration in the 24-hour urine collection is determined and compared with the concentration after an intravenous magnesium load. If less than 60 % of the infused magnesium is excreted, this indicates a deficiency.

However, the magnesium retention test is not part of standard diagnostics either. In addition, its significance is influenced by kidney function. Increased renal magnesium losses due to diabetes, alcohol abuse or medication such as diuretics falsify the test.

Reference ranges zinc in serum

• adults: 0.6 - 1.2 mg/dl or 9 - 18 µmol/l

• Altersgruppe	mg/dl	µmol/l
  bis 60 J	0,7 – 1,5	10,7 – 23,0
  60 - 90 J	0,6 – 1,1	9,6 – 16,4
  > 90 J	0,5 – 1,0	8,0 – 15,1

Age group

up to 60 y

60 - 90 y

> 90 y

Reference ranges for zinc in plasma

• women: 9 - 22 µmol/l or 0.6 - 1.45 mg/dl
• men: 12 - 26 µmol/l or 0.8 - 1.7 mg/dl

Reference ranges for zinc in whole blood

• 61 - 115 µmol/l or 4.0 - 7.5 mg/l

The reference ranges may vary slightly depending on the laboratory and measurement method. However, a tendency for the values to shift towards lower reference ranges with age is comparable.

Age causes slight changes in the standard values, but these are not necessarily synchronous for serum and whole blood. Gender-specific differences are also recognizable.

Caution: Incorrectly high zinc values are possible due to the use of glass tubes. Small amounts of zinc continuously diffuse out of the glass, which falsify the zinc concentrations. However, various types of Teflon and polyethylene also contain zinc. It is best to use polypropylene.

Determination methods

• AAS (atomic absorption spectrophotometry)
• photometric PAPS method (PAPS = pyridylazo dye).

The biofactor zinc is so concentrated in almost all compartments that AAS is used as a method. Photometry does not adequately detect low zinc plasma levels.

Assessment of zinc diagnostics

Unlike iron, for example, there is currently no reliable biomarker for zinc for the routine assessment of a deficiency. The measurement of the zinc concentration in serum or the activity of zinc-containing enzymes and, in particular, the determination of the zinc content in hair and urine have shown unconvincing results in scientific studies.^20,21

Around 95% of the total body zinc is found intracellularly. Serum contains significantly less than 1% of the body's zinc.

The zinc content in hair and nail samples has a certain significance in toxicological aspects, but cannot be used to prove the zinc status. Factors influencing the evaluation are the different structure and metabolism of the hair, external deposits and contamination of the sample.

Urine samples are unsuitable for diagnosing a zinc deficiency, as it is not possible to differentiate between an existing deficit of the biofactor zinc and a reduced current absorption in the case of low excretion.

An alternative, but time-consuming, is zinc analysis in whole blood, in which the erythrocytes are taken into account in addition to the serum. As the majority of zinc is bound to erythrocytes (approx. 85%), whole blood diagnostics are subject to fewer interfering influences.

It would be possible to examine tissue samples, but this is not very suitable for therapeutic practice. In addition, the activity of alkaline phosphatase and the zinc binding capacity are meaningful parameters, although they are also of little practical use.²²

The determination of zinc in plasma is the most frequently used laboratory method. However, there are also indications that determination in serum is preferable to plasma, as anticoagulants sometimes contain measurable amounts of zinc.

It should also be borne in mind that the zinc plasma concentration is kept constant over a wide intake range by adjusting intake and excretion. Normal plasma levels do not rule out a zinc deficiency. Conversely, low zinc levels in plasma do not necessarily indicate a deficiency, as stress, heart attacks, infections and inflammation as well as hypalbuminemia in liver diseases and hemodilution, e.g. during pregnancy, can also lower the levels. Burns or surgical interventions also often lead to a rapid drop in the zinc concentration in plasma.

The simplest and most reliable method of zinc diagnostics, according to the recommendation of the German Nutrition Society (DGE), is based on the anamnesis of possible causes or risk factors and the reduction of deficiency symptoms after zinc supplementation.^23,24

Vitamin B₁ (thiamine) is water-soluble and is one of the essential vitamins, as the human organism cannot produce it but must absorb it with food. The term vitamin B₁ refers to the compounds thiamine (T) and its metabolites in the form of esters - designated as mono-, di- and triphosphate and abbreviated as TMP, TDP and TTP.

Thiamine diposphate is also known as thiamine pyrophosphate (TPP). TPP is the coenzyme form (cocarboxylase) of thiamine and dominates in comparison to thiamine, as it is formed from the latter by the enzyme thiamine pyrophosphokinase. While thiamine is usually the usual form of administration through food or supplements, TDP/TPP is the physiologically active and therefore most important component. TPP is a component of enzymes that play an important role in carbohydrate and amino acid metabolism. Free thiamine is only found in very low concentrations in plasma. Most of it is bound to the erythrocytes as TPP: About 75 % of the total thiamine in whole blood is found in erythrocytes, about 15 % in leukocytes and about 10 % in plasma.

Reference ranges (laboratory-dependent):

• Thiamine, free (T): 1-10 nmol/l
• Thiamine monophosphate (TMP): 3-15 nmol/l
• Thiamine diphosphate or pyrophosphate (TDP/TPP): 100-270 nmol/l²⁴ or 66.5-200 nmol/l²⁵

• Material: EDTA blood or heparin blood, store and transport refrigerated/frozen and protected from light and wrapped in aluminum foil
• Amount of material: 2 ml
• Measurement method: HPLC

Vitamin-B₁-status:

• Measurement of vitamin B₁ vitamers in EDTA blood using HPLC including measurement of the supplemented form to exclude recent vitamin B₁ intake.

In addition, if chronic vitamin B₁ deficiency is suspected

• TPP effect (in vitro function test):

This measures the enzyme activity of the vitamin B₁-dependent transketolase in the erythrocytes. This decreases with increasing vitamin B₁ deficiency. If an excess of the coenzyme TPP is added in a parallel sample, the enzyme activity is stimulated. If the vitamin B₁ status of the initial sample is sufficient, little or no stimulation of the transketolase is to be expected. The higher the activability of the erythrocyte transketolase, the worse the vitamin B₁ status. 

• Findings:

An in vitro stimulation of the transketolase enzyme by TPP > 20 % indicates a vitamin B₁ deficiency.

• Caution: not suitable as the sole test:

Transketolase is only one of several vitamin B₁-dependent functions, and the TPP effect is also influenced by other factors, e.g. liver disease or diabetes mellitus. In addition, a recent intake of vitamin B₁ cannot be diagnosed with this functional test.

Vitamin B₁ in urine²⁶

• Material: 24-hour urine, collected via 5-10 ml glacial acetic acid
• Amount of material: 5 ml urine
• Reference range: > 100 µg/24 hrs.
• Method: HPLC

Vitamin B₆ is the collective name for various chemical compounds - pyridoxine, pyridoxal, pyridoxamine and their phosphorylated derivatives - whose activated metabolite is pyridoxal-5-phosphate. All derivatives can be converted into each other by the metabolism and have the same biological activity (vitamers).

The biofactor vitamin B₆ is determined from whole blood or serum/plasma. The normal values for pyridoxyl-5-phosphate (PALP) are  

• PALP in serum/plasma: 20-30 nmol/l (400-600 ng/dl)
• PALP in whole blood: 24-88 nmol/l (500-1800 ng/dl)
• Material: Serum or EDTA plasma, store and transport frozen (approx. -20°C) and wrapped in aluminum foil to protect from light
• Amount of material: 1-2 ml, depending on the laboratory
• Measurement method: HPLC

Total vitamin B12 serum level: normal range approx. 200-1.000 ng/l

Serum level < 200 ng/l: Vitamin B12 deficiency confirmed

Serum level 200-400 ng/l: Measure holotranscobalamin (holo-TC) in serum

Holo-TC < 35 pmol/l: Vitamin B12 deficiency confirmed

Holo-TC > 55 pmol/l: Vitamin B12 deficiency unlikely

Holo-TC 36-55 pmol/l: Measure methylmalonic acid (MMA) and/or homocysteine in serum

MMA > 300 nmol/l or > 0.4 µmol/l and homocysteine > 10 µmol/: Vitamin B12 deficiency confirmed.

• Material: 1 serum tube (total vitamin B₁₂, holo-TC, MMA) and 1 tube of acid citrate plasma (homocysteine)
• Haemolytic samples influence the test result and should not be used.
• Amount of material: 1 ml, store material wrapped in aluminum foil, protected from light if possible, and transport at +2°C - +8°C, freeze if necessary (- 20°C)
• Measurement method: ECLIA (electrochemiluminescence immunoassay)

Diagnosis of vitamin B₁₂ deficiency: what needs to be considered^27,28

The biofactor vitamin B₁₂ is bound to the intrinsic factor IF in the intestinal lumen of the distal ileum and absorbed via special receptors in the intestinal mucosa. In the plasma, part of the vitamin B₁₂ then binds to transcobalamin to form holotranscobalamin (holo-TC). However, the largest proportion of 70 to 90 % is bound to haptocorrin - a biologically inactive complex, as only liver cells and no other body cells have receptors for absorption. Only holo-TC, as a metabolically active form of vitamin B₁₂, can be absorbed by all cells via corresponding receptors.

When determining total vitamin B₁₂, no differentiation is made between the metabolically active and inactive form. The sole measurement of this laboratory parameter in the so-called “gray area” between 200 and 400 ng/l therefore provides no clear indication of whether holo-TC is available in sufficient quantities as the sole metabolically active form of vitamin B₁₂. The additional measurement of holo-TC in serum is then recommended.

However, there is also a gray area between 35 and 55 pmol/l in the holo-TC, in which a possible vitamin B₁₂ deficiency must be clarified diagnostically by determining other laboratory parameters - homocysteine and methyl malonic acid (MMA). Vitamin B₁₂ is one of the cofactors in methionine/homocysteine metabolism; in intracellular vitamin B₁₂ deficiency, the concentrations of homocysteine and MMA increase (see above for measured values).

Based on these physiological correlations, it is clear that the measurement of holo-TC, homocysteine and MMA has a higher sensitivity and specificity than the measurement of total vitamin B₁₂ alone:

• Holo-TC is considered the earliest laboratory parameter of vitamin B₁₂ deficiency.
• Low holo-TC alone already indicates depletion of vitamin B₁₂ stores.
• In combination with elevated MMA and homocysteine, holo-TC is an indicator of a metabolically manifest vitamin B₁₂ deficiency. Clinical symptoms may still be absent.
• As the first clinical signs of a vitamin B₁₂ deficiency are non-specific, at-risk patients such as the elderly, cardiovascular patients or diabetics should be examined at least every two to three years.

Please note:

In patients with chronic renal insufficiency, the significance of vitamin B₁₂ and holo-TC serum levels is limited. A suspected vitamin B₁₂ deficiency in these patients is diagnosed by a reduction of MMA in the serum after administration of cobalamin.

Folic acid is 95 % localized in the erythrocytes, only 5 % is found in the serum. A folic acid deficiency is therefore not usually measured by the folic acid concentration in the serum, but by erythrocyte analysis. A folic acid deficiency leads to megaloblastic, hyperchromic macrocytic anemia with increased MCV and MCH. The determination of folic acid in the erythrocytes is well suited for assessing the severity of a folic acid deficiency as, in contrast to the determination of serum folic acid, it is independent of short-term dietary influences. The determination of serum folic acid can be helpful in unclear cases.

Folic acid in the erythrocytes

• Reference range: 140-836 ng/ml or 400-1260 µg/l, depending on the age of the patient and laboratory
• Material: EDTA blood or heparin blood
• Amount of material: 1-2.7 ml, depending on the laboratory, store and transport refrigerated and wrapped in aluminum foil to protect from light
• Indications: Suspected intracellular folate deficiency in primary vitamin B₁₂ deficiency
• Measurement method: ILMA (immunoluminometric assay)

Folic acid in serum

• Reference range: 2.5-20 ng/ml, depending on the age of the patient and laboratory
• Material: serum or heparin plasma
• Amount of material: 1 ml, store and transport wrapped in aluminum foil to protect from light
• Indications: Suspected folic acid deficiency in macrocytic anemia, malabsorption, malnutrition, alcohol abuse, long-term hemodialysis, jejunum resection, liver insufficiency, psoriasis
• Measurement method: ILMA (immunoluminometric assay)

The detection of folic acid levels in erythrocytes together with folic acid in serum indicate the extent and severity of folic acid deficiency. Folic acid levels in the erythrocytes below 50 µg/l indicate a manifest folic acid deficiency. Reduction of intraerythrocytic folic acid with normal serum folic acid indicates a vitamin B₁₂ deficiency (see below).

Please note:

Folic acid deficiency is triggered by numerous drugs via inhibition of absorption or folic acid production or by folic acid antagonism. The following drugs should be considered in this regard: Aminopterin, amethopterin, Daraprim, pyrimethamine, hormonal contraceptives, sulfasalazine, antiepileptic drugs, aminosalicylic acid and phenacetin.

Not only a folic acid deficiency, but also a vitamin B₁₂ deficiency can lead to hematological disorders in the form of megaloblastic anemia. Megaloblastic anemia caused by folic acid deficiency cannot be distinguished clinically or microscopically from megaloblastic anemia caused by vitamin B₁₂ deficiency. If a folic acid deficiency is wrongly suspected in megaloblastic anemia due to a vitamin B₁₂ deficiency and only folic acid alone is supplemented, the neurological effects of the vitamin B₁₂ deficiency are intensified and irreversible neurological damage can occur as a result of this “folate trap”. Folic acid supplementation alone in the presence of a vitamin B₁₂ deficiency would therefore exacerbate vitamin B₁₂-dependent neurological disorders. For this reason, both biofactors should always be investigated in cases of suspected deficiency.

The Schilling test (1 µg of vitamin B12 radioactively labeled with ⁵⁷Co is administered orally and the excretion in 24-hour urine is monitored: In healthy patients, at least 5% of the radiolabeled vitamin B₁₂ is excreted renally) is obsolete and is now replaced by the reliable blood level tests mentioned above.

If a folic acid deficiency is detected and high-dose folic acid (daily dose: 5 mg) is prescribed, the therapist must ensure that the deficiency cannot be remedied by dietary means.

Folic acid preparations with a lower dosage (0.4-0.8 mg), which are recommended for the prevention of neural tube defects before and during pregnancy, are considered to be extremely useful from a medical point of view due to numerous positive study results, but are nevertheless not included in the standard benefits catalog of the SHI system. However, some health insurance companies reimburse folic acid preparations in the above-mentioned dosage via pregnancy programs or special statutory benefits.

Ascorbic acid is extremely sensitive to oxidation. An EGTA/GSH solution must therefore be added to the plasma to protect it from oxidation. The plasma stabilized in this way must be sent deep-frozen. Vitamin C is determined after this stabilization as the sum of ascorbic acid and dehydroascorbic acid.³⁰

• Reference range: 3-14 mg/l, corresponding to 18-114 μmol/l, depending on the age of the patient and laboratory
• Material: lithium heparin plasma, EGTA/GSH plasma or serum
• Amount of material: 0.5-2 ml. Store and transport protected from air and light, deep-frozen.
• Vitamin C is rapidly degraded in serum. It is therefore recommended to send lithium heparin plasma or EGTA/GSH plasma. Freeze the plasma and send it in immediately.
• Many laboratories also recommend the use of ready-made special tubes. Pipette 0.5 ml of plasma into the supplied EGTA/GSH solution within 30 minutes of blood collection, mix and freeze.
• Measuring method: HPLC, photometry

Please note:

A low vitamin C status can be assumed at values between 1.7 and below 3 mg/l, values below 1.7 mg/l indicate a vitamin C deficiency and at values below 1 mg/l there is an increased risk of scurvy.

Only small amounts of vitamin D are ingested alimentarily; by far the largest proportion of 80 to 90 % is formed independently in the skin under the influence of UV-B radiation. The two most important vitamin D precursors are vitamin D3 (cholecalciferol), which is synthesized under UV radiation or absorbed through food of animal origin, and vitamin D2 (ergocalciferol), which comes from food of plant origin.

Both precursors are transported via the blood to the liver, primarily bound to the vitamin D-binding protein DBP, where they are converted by the enzyme cytochrome P450 2R1 to another intermediate form, 25(OH)D, calcidiol. Calcidiol is bound to DBP again in the liver and released into the blood. Calcidiol acts as a storage form of vitamin D and has the task of being able to balance out the breaks and peaks in the vitamin D supply via UV light.

Calcidiol, mainly bound to DBP, reaches the kidneys where it is converted into the physiologically active, i.e. actually effective, form of vitamin D, 1,25(OH)2D3 or calcitriol.

The determination of the vitamin D serum level merely reflects the vitamin D intake - alimentary and via the skin - over the last few days. In order to obtain an indication of the longer-term vitamin D supply, it is more useful to determine the calcidiol, whose half-life in the blood is given as one to two months.

Vitamin D status: reference values

The definition of vitamin D deficiency based on the 25-OH vitamin D level is still the subject of controversy. The US health organization Institute of Medicine IOM continues to name 50 nmol/l or 20 ng/ml as the lower limit.³¹ The vitamin D supply is therefore considered to be secure if the serum concentration of the storage form calcidiol = 25(OH)D is above 50 nmol/l or 20 ng/ml. (25(OH)D can be given in the units nmol/l or ng/ml, for the conversion the value is divided by 2.5).³²

25(OH)D in nmol/l 25(OH)D in ng/ml Interpretation

< 30 < 12 inadequate supply with increased risk of rickets, osteomalacia and osteoporosis

30 - < 50 12 - < 20 suboptimal supply

50 - < 75 20 - < 30 adequate supply

75 - < 125 30 - < 50 adequate supply without further additional benefit

≥ 125 ≥ 50 possible oversupply with increased risk of hypercalcemia, kidney stones or cardiac arrhythmia

Material: Serum³³

Amount of material: 1 ml

Measurement method: LIA

In contrast, an expert consensus from 2022 recommended slightly higher reference ranges. According to this, the lower calcidiol reference value should be 75 nmol/l or 30 ng/ml.³⁴

Free vitamin D - better assessment of the vitamin D supply³⁵

25(OH)D is lipophilic and therefore largely bound to carrier molecules and is thus transported in the blood. Albumin and 85 to 90 % of the vitamin D-binding protein DBP mentioned above serve as carrier molecules. Only a very small proportion of 25(OH)D, around 1 %, is free, i.e. unbound. However, only this free vitamin D is biologically active, as it can pass through the cell membrane and activate the intracellular nuclear vitamin D receptor, which belongs to the steroid receptor family (“free hormone hypothesis”).

However, the laboratory detection of 25(OH)D covers both the free, biologically available vitamin D and the part bound to carrier molecules. Differentiation is not possible. And since the DBP concentration is very susceptible to interference (hormones such as oestrogen, liver and kidney performance, genetics), the previously assumed correlation of free, biologically active vitamin D3 with total vitamin D - measured in the form of 25(OH)D - is considered unsatisfactory.

The determination of free vitamin D (fD) in serum is independent of the interfering variables mentioned and reflects the part of the biofactor that interacts with the vitamin D receptor. The measurement is routinely offered by some diagnostic laboratories.³⁶

Please note:

Being overweight increases the risk of vitamin D deficiency.³⁷  Even a 10 % weight gain leads to a drop in vitamin D3 levels of more than 4 %. A higher dosage of supplements may therefore be necessary for overweight people than for people of normal weight.