Chapter 21 Laboratory Tests for the Determination of Vitamin Status
Introduction
Assessment of Vitamin Status
See Table 21-1 for laboratory tests and optimal ranges for common vitamins.1–5
TABLE 21-1 Laboratory Tests and Optimal Ranges for Common Vitamins
| NUTRIENT | TEST | ACCEPTABLE LEVEL |
|---|---|---|
| Water-Soluble | ||
| Ascorbic acid | Serum Leukocyte Load test |
>0.3 mg/dL 30 mcg/108 WBCs 0.3-2.0 mg/h in control 24-49 mg/h after 500 mg |
| Biotin | 3-hydroxyisovalerate | <20 mcg/mg creatinine (overnight urine) |
| Folate | Erythrocyte folate | >160-650 ng/mL (~350 nmol/L) |
| Serum homocysteine | <10 mcmol/L | |
| Niacin | Urinary N-methylnicotinamide 2-pyridone 5-carboxamide (2-PYR) |
>1.6 mg/g creatinine
>1.6 mg/g creatinine |
| RBC NAD/NADP | >1.0 | |
| Pantothenic acid | Urinary pantothenic acid | >1 mg/day |
| Pyridoxine | Serum level | >50 ng/mL |
| Tryptophan load | <35 mg/24 h xanthurenic acid | |
| AST | <1.5 (ratio) | |
| ALT | <1.25 | |
| Plasma pyridoxal 5-phosphate | >30 nmol/L | |
| Urinary 4-PASerum homocysteine | >3.0 mol/d<10 µmol/L | |
| Riboflavin | EGRAC | <1.3 |
| Thiamine | RBC transketolase Whole blood thiamine (HPLC) |
<15% increas e>16ng/mL |
| Vitamin B12 | Serum B12 Urinary methylmalonic acid Serum merhylmalonic acid Serum homocysteine Holotranscobalamin |
>150 pg/mL <5 mcg/mg creatinine <0.45 mcmol/L <0.10 mcmol/L |
| Fat-Soluble | ||
| Vitamin A | Plasma retinol: | 15-60 mcg/dL: |
| 0-5 mo | >20 | |
| 6 mo-17 yr | >30 | |
| Adult | >20 | |
| Vitamin D | 25 (OH) vitamin D | 40-80 ng/mL |
| Vitamin E | Plasma α-tocopherol | >16.2 mcmol/L |
| α-tocopherol:cholestrol | >5.2 mcmol/L | |
| Vitamin K | % serum uncarboxylated osteocalcin | <20 ? (optimal not yet determined) |
ALT, alanine aminotranferase; AST, aspartate aminotransferase; EGOT, erythrocyte glutamic oxaloacetic transaminase; EGPT, erythrocyte glutamic pyruvic transaminase: FAD, flavin adenine dinucleotide; H2O2, hydrogen perioxide; NAD, nicotinamide adenine dinucleotide; NADP, nicotinamide adenine dinucleotide phosphate; RBC, red blood cell; WBC white blood cell.
Data from references 1-5.
Water-Soluble Vitamins
Ascorbic Acid (Vitamin C)
Assessment of vitamin C is particularly difficult because ascorbate readily oxidizes in assay samples. In addition, serum levels reflect recent dietary uptake rather than actual tissue levels. Recent research in an animal model of vitamin C deficiency (the Gulo mouse) clearly demonstrated that a dietary intake that does not lead to serum saturation of vitamin C results in tissue deficits.6 Serum saturation of vitamin C was required to achieve tissue concentrations similar to wild-type animals, which can synthesize ascorbate. In humans, maximum serum saturation from oral dosing was predicted to be roughly 1/60th of that achieved with intravenous administration, highlighting the inability of serum levels to predict optimal physiologic function.7,8 Leukocyte levels are not as susceptible to dietary intake but are also readily affected by infection, hypoglycemia, and many common prescription and over-the-counter drugs. The popular lingual ascorbate test does not appear to be reliable because it does not correlate well with leukocyte or serum levels. The loading test, if carefully controlled, is probably most accurate, although good standard ranges have yet to be determined. Finally, discovery of ascorbate-dependent enzymes involved in cell signaling pathways and epigenetic modulation offer the possibility for more functional analysis in the future, although unfortunately no definitive analysis is currently available.9
Biotin
Biotin is a vitamin B complex especially affected by oral antibiotics. Food is a poor source of this vitamin, making humans more dependent on gut flora sources. Elevation of 3-hydroxyisovalerate (3-HIA) due to deficiency of the biotin-dependent enzyme appears a useful measure. Additionally, two recent studies in which healthy volunteers were intentionally made biotin deficient suggested that elevated urinary levels of 3-HIA-carnitine might be a sensitive indicator, even for a marginal deficiency. This offers greater accuracy and is less prone to laboratory error than the traditional urinary 3-HIA.10,11 Plasma levels of 3-HIA-carnitine may also prove to be a sensitive marker and reduce the dependency on renal function for an accurate determination, an important consideration during pregnancy.12
Folate
Serum folate is too greatly affected by recent consumption to be clinically useful. Homocysteine levels may be elevated because of deficiency of vitamins B6 and/or B12 as well as folate. Erythrocyte folate is more accurate and considered a more reliable indicator of tissue status. Evaluation of other B vitamin status, particularly B12, may be necessary to rule out a folate deficiency. Although the presence of neutrophil hypersegmentation has been used to identify folate deficiency, this also occurs with B12 and iron deficiency, making it a very nonspecific marker.13
Niacin
Although measurement of nicotinic acid in the blood is not very reliable, measurement of its metabolites provides a clinically useful function assessment. Several metabolite tests are now available, including urinary levels of 2-pyridone 5-carboxymide and n-methylnicotinamide.14,15
Pantothenic Acid
Serum pantothenic acid does not correspond well with dietary intake, although erythrocyte levels are more closely correlated.16 Measurement of urinary excretion of pantothenic acid appears reliable.
Pyridoxine
Several procedures are available for assessing vitamin B6 status. Unfortunately, substantial agreement on the best methodology has not been established, because variations in phenotypes significantly alter the results of functional and loading tests. The active form of pyridoxine (pyridoxal 5′-phosphate [P5P]) is involved in some 60 enzymes, so deficient activity of these enzymes can be measured as a functional assessment of pyridoxine. Plasma levels of P5P appear to be a better functional indicator than erythrocyte levels, at least in patients with rheumatoid arthritis.17 Plasma levels below 30 nmol/L (considered borderline deficient) have independently been associated with an increased risk for coronary artery disease, with a particularly high risk when combined with high-sensitivity C-reactive protein.18,19 Although suitable for most circumstances, plasma P5P does not appear to be reliable during pregnancy or the acute phase of myocardial infarction, and alternatives should be used. Urinary 4-pyridoxic acid is a useful marker of recent intake only, whereas erythrocyte aminotransferase (EAST) and erythrocyte alanine aminotransferase (EALT) activation by pyridoxal phosphate may be a better indicator of long-term status.20 Elevated homocysteine may also be a sign of deficiency, at least in some populations.21
Riboflavin
The most common measure of riboflavin is red blood cell (RBC) glutathione reductase activity. The enzyme is stimulated in vitro by the addition of flavin adenine dinucleotide, and expressed as erythrocyte glutathione reductase activation coefficient (EGRAC). An EGRAC greater than 1.30 is typically used as a cutoff (with higher values indicative of suboptimal status), although one recent study suggested this threshold for deficiency might need to be raised. This same study also demonstrated that riboflavin supplementation caused an increase in hemoglobin status among women with the lowest riboflavin intakes, despite no change in iron intake or absorption.22 EGRAC is not reliable in individuals with glucose-6-phosphate dehydrogenase deficiency. In this population, pyridoxamine phosphate oxidase may be more appropriate.23 Blood riboflavin levels are not reliable because of technical difficulties in measurement. Lastly, homocysteine levels may be relevant for those with genetic variation in the ethylenetetrahydrofolate reductase gene. Individuals with the TT genotype of the C677 T polymorphism may have increased riboflavin needs.24
Thiamine
The most common measure of thiamin is erythrocyte transketolase activity. The enzyme is stimulated in vitro by the addition of thiamine pyrophosphate. Elevation in activity greater than 15% indicates a functional deficiency. The test is not reliable in patients with diabetes mellitus, pernicious anemia, or a significant negative nitrogen balance. For example, growing evidence has indicated that a tissue deficiency of thiamine exists in patients with diabetes, and may increase the risk for vascular and neurologic complications. Despite markedly reduced plasma levels of thiamine among individuals with type 1 and type 2 diabetes, erythrocyte transketolase activity remained normal, largely because of an upregulation of RBC thiamine transporter levels.25,26 Advances in high-performance liquid chromatography technology now allow for direct measurement of either whole blood or erythrocyte thiamine, which may be a valuable alternative.27,28
Additionally, measurement of amino acids and their ketoacid analogues that are excreted in thiamine deficiency is being used. (See Brally and Lord4 for a more complete discussion.)
Vitamin B12
A recent analysis found that serum levels of holotranscobalamin may be the optimal first line diagnostic procedure, at least among elderly patients. It has been suggested to be one of the earliest markers of negative B12 balance, and its diagnostic accuracy is not affected by renal insufficiency, a problem with total cobalamin levels and methylmalonic acid.29,30
Measurement of either urinary or serum methylmalonic acid is considered fairly sensitive and specific in those with healthy renal function, especially when expressed as a ratio with the urinary creatinine measurement.31,32 Elevated homocysteine concentrations may indicate deficiency, but it is not specific to B12.
Fat-Soluble Vitamins
Vitamin A
Although liver biopsy is the most accurate method of vitamin A assessment, other less invasive and less expensive methodologies are more appropriate. As with most other nutrients, serum levels of vitamin A fall significantly only after tissue reserves have been depleted, and are subject to numerous laboratory challenges as well as other factors, (e.g., infection, protein status). Serum retinol binding protein is sometimes used as an alternative to serum retinol, because it avoids many of these complications, although it is also susceptible to artificial decreases due to inflammation or protein malnutrition.33 Optimal cutoffs have not been clearly established for retinol binding protein, although levels greater than 0.825 µmol/L have been suggested for children and greater than 1.05 µmol/L for adults.34,35 The dark adaptation test detects early deficiency of this nutrient, and provides a useful alternative (see Chapter 26). Lastly, given the variation in the ability to convert β-carotene to retinol, the use of plasma carotene levels is probably not a useful marker of vitamin A status.36
Vitamin D
The widespread deficiency of vitamin D has gained a much greater degree of recognition, with most recommendations based upon 25(OH) vitamin D levels. Most functional indicators, such as levels of parathyroid hormone, point to a level of 25(OH) vitamin D greater than or equal to 80 nmol/L as being sufficient, although some evidence has suggested higher levels might be optimal, particularly for cancer prevention.37–39
Vitamin E
Despite dietary intake of γ-tocopherol being ten fold greater than α-tocopherol, the preference of the α-tocopherol transfer protein for α-tocopherol results in much higher serum levels, which is why the latter is often used as a biomarker. Emerging evidence suggests that despite the ease in measurement, and the widespread supplementation with α-tocopherol, mixed tocopherols (such as the γ form) is likely of greater physiologic importance.40,41 Unfortunately, optimal levels of other biomarkers have not yet been established, although one review reported a γ-tocopherol levels range between 2 and 5 µmol/L.42 It is also important to adjust vitamin E levels to lipid levels, such as triglycerides or cholesterol, with the latter more commonly used.
Adipose and platelet levels have been suggested to be better biomarkers, as have cells with low-density lipoprotein receptors, such as mononuclear leukocytes and buccal mucosal cells.43
Vitamin K
In recent years the importance of vitamin K for physiologic functions other than blood coagulation has been recognized, including functions related to both bone and vascular disease. For this reason, the traditional methods of assessment, such as prothrombin and clotting assays, may not be sufficient markers of vitamin K status, at least in regard to these other functions.44 Better functional markers may be serum uncarboxylated osteocalcin (ucOC) levels, or perhaps a serum carboxylated OC to serum total OC ratio. One review suggested that a percent of ucOC greater than 20 is probably optimal, although genetic variations may modify this to some degree.45–47
Conclusion
As described here, many procedures are now available for the assessment of functional vitamin status. Although research continues in this important area, the reader is advised to carefully study the discussion of urinary organic acids profiling (see Chapter 28). Utilizing metabolic products excreted in the urine now allows the clinician far greater specificity in recognizing dysfunctional enzyme systems, whether they are due to genetic deviations or nutritional deficiencies.
1. Tierney L.M., McPhee S.J., Papdakis M.A. Current medical diagnosis and treatment. Stamford, CT: Appleton & Lange; 2001. 1239-1244
2. Rubenstein E., Federman D.D. Scientific american medicine. New York: Scientific American; 2004. 1-15
3. Werbach M.R. Textbook of nutritional medicine. Tarzana, CA: Third Line Press; 1999.
4. Brally J., Lord R.S. Laboratory evaluations in molecular medicine. Norcross, GA: Institute for Advances in Molecular Medicine; 2001.
5. Homocysteine Lowering Trialists Collaboration. Lowering blood homocysteine with folic acid based supplements: meta-analysis of randomised trials. BMJ. 1998;316:894–898.
6. Vissers M.C., Bozonet S.M., Pearson J.F., et al. Dietary ascorbate intake affects steady state tissue concentrations in vitamin C-deficient mice: tissue deficiency after suboptimal intake and superior bioavailability from a food source (kiwifruit). Am J Clin Nutr. 2011;93(2):292–301.
7. Padayatty S.J., Sun H., Wang Y. Vitamin C pharmacokinetics: implications for oral and intravenous use. Ann Intern Med. 2004;140(7):533–537.
8. Cullen J.J. Ascorbate induces autophagy in pancreatic cancer. Autophagy. 2010;6(3):421–422.
9. Pagé E.L., Chan D.A., Giaccia A.J., et al. Hypoxia-inducible factor-1alpha stabilization in nonhypoxic conditions: role of oxidation and intracellular ascorbate depletion. Mol Biol Cell. 2008;19(1):86–94.
10. Horvath T.D., Stratton S.L., Bogusiewicz A., et al. Quantitative measurement of urinary excretion of 3-hydroxyisovaleryl carnitine by LC-MS/MS as an indicator of biotin status in humans. Anal Chem. 2010;82(22):9543–9548.
11. Stratton S.L., Horvath T.D., Bogusiewicz A., et al. Urinary excretion of 3-hydroxyisovaleryl carnitine is an early and sensitive indicator of marginal biotin deficiency in humans. J Nutr. 2011;141(3):353–358.
12. Stratton S.L., Horvath T.D., Bogusiewicz A., et al. Plasma concentration of 3-hydroxyisovaleryl carnitine is an early and sensitive indicator of marginal biotin deficiency in humans. Am J Clin Nutr. 2010;92(6):1399–1405.
13. Sipahi T., Tavil B., Unver Y. Neutrophil hypersegmentation in children with iron deficiency anemia. Pediatr Hematol Oncol. 2002;19(4):235–238.
14. Seal A.J., Creeke P.I., Dibari F., et al. Low and deficient niacin status and pellagra are endemic in postwar Angola. Am J Clin Nutr. 2007;85(1):218–224.
15. Fu C.S., Swendseid M.E., Jacob R.A., et al. Biochemical markers for assessment of niacin status in young men: levels of erythrocyte niacin coenzymes and plasma tryptophan. J Nutr. 1989;119(12):1949–1955.
16. Eissenstat B.R., Wyse B.W., et al. Pantothenic acid status of adolescents. Am J Clin Nutr. 1986;44(6):931–937.
17. Chiang E.P., Bagley P.J., Roubenoff R., et al. Plasma pyridoxal 5’-phosphate concentration is correlated with functional vitamin B-6 indices in patients with rheumatoid arthritis and marginal vitamin B-6 status. J Nutr. 2003;133(4):1056–1059.
18. Lin P.T., Cheng C.H., Liaw Y.P., et al. Low pyridoxal 5’-phosphate is associated with increased risk of coronary artery disease. Nutrition. 2006;22(11-12):1146–1151.
19. Cheng C.H., Lin P.T., Liaw Y.P., et al. Plasma pyridoxal 5’-phosphate and high-sensitivity C-reactive protein are independently associated with an increased risk of coronary artery disease. Nutrition. 2008;24(3):239–244.
20. Hansen C.M., Shultz T.D., Kwak H.K., et al. Assessment of vitamin B-6 status in young women consuming a controlled diet containing four levels of vitamin B-6 provides an estimated average requirement and recommended dietary allowance. J Nutr. 2001;131(6):1777–1786.
21. Woolf K., Manore M.M. Elevated plasma homocysteine and low vitamin B-6 status in nonsupplementing older women with rheumatoid arthritis. J Am Diet Assoc. 2008;108(3):443–453. discussion 454
22. Powers H.J., Hill M.H., Mushtaq S., et al. Correcting a marginal riboflavin deficiency improves hematologic status in young women in the United Kingdom (RIBOFEM). Am J Clin Nutr. 2011;Jun;93(6):1274–1284.
23. Mushtaq S., Su H., Hill M.H., et al. Erythrocyte pyridoxamine phosphate oxidase activity: a potential biomarker of riboflavin status? Am J Clin Nutr. 2009;90(5):1151–1159.
24. Hoey L., McNulty H., Strain J.J. Studies of biomarker responses to intervention with riboflavin: a systematic review. Am J Clin Nutr. 2009;89(6):1960S–1980S.
25. Rabbani N., Thornalley P.J., et al. Emerging role of thiamine therapy for prevention and treatment of early stage diabetic nephropathy. Diabetes Obes Metab. 2011;Jul;13(7):577–583.
26. Thornalley P.J., Babaei-Jadidi R., et al. High prevalence of low plasma thiamine concentration in diabetes linked to a marker of vascular disease. Diabetologia. 2007;50(10):2164–2170.
27. Lima L.F., Leite H.P., Taddei J.A. Low blood thiamine concentrations in children upon admission to the intensive care unit: risk factors and prognostic significance. Am J Clin Nutr. 2011;93(1):57–61.
28. Talwar D., Davidson H., Cooney J., et al. Vitamin B(1) status assessed by direct measurement of thiamin pyrophosphate in erythrocytes or whole blood by HPLC: comparison with erythrocyte transketolase activation assay. Clin Chem. 2000;46(5):704–710.
29. Valente E., Scott J.M., Ueland P.M., et al. Diagnostic accuracy of holotranscobalamin, methylmalonic acid, serum cobalamin, and other indicators of tissue vitamin B12 status in the elderly. Clin Chem. 2011;Jun;57(6):856–863.
30. Bamonti F., Moscato G.A., Novembrino C., et al. Determination of serum holotranscobalamin concentrations with the AxSYM active B(12) assay: cut-off point evaluation in the clinical laboratory. Clin Chem Lab Med. 2010;48(2):249–253.
31. Clarke R., Sherliker P., Hin H., et al. Detection of vitamin B12 deficiency in older people by measuring vitamin B12 or the active fraction of vitamin B12, holotranscobalamin. Clin Chem. 2007;53(5):963–970.
32. Vogiatzoglou A., Oulhaj A., Smith A.D. Determinants of plasma methylmalonic acid in a large population: implications for assessment of vitamin B12 status. Clin Chem. 2009;55(12):2198–2206.
33. de Pee S., Dary O. Biochemical indicators of vitamin A deficiency: serum retinol and serum retinol binding protein. J Nutr. 2002;132(suppl 9):2895S–2901S.
34. Gorstein J.L., Dary O., Pongtorn, et al. Feasibility of using retinol-binding protein from capillary blood specimens to estimate serum retinol concentrations and the prevalence of vitamin A deficiency in low-resource settings. Public Health Nutr. 2008;11(5):513–520.
35. Baeten J.M., Richardson B.A., Bankson D.D., et al. Use of serum retinol-binding protein for prediction of vitamin A deficiency: effects of HIV-1 infection, protein malnutrition, and the acute phase response. Am J Clin Nutr. 2004;79(2):218–225.
36. Leung W.C., Hessel S., Méplan C., et al. Two common single nucleotide polymorphisms in the gene encoding beta-carotene 15,15’-monoxygenase alter beta-carotene metabolism in female volunteers. FASEB J. 2009;23(4):1041–1053.
37. Souberbielle J.C., Body J.J., Lappe J.M., et al. Vitamin D and musculoskeletal health, cardiovascular disease, autoimmunity and cancer: recommendations for clinical practice. Autoimmun Rev. 2010;9(11):709–715.
38. Binkley N., Ramamurthy R., Krueger D. Low vitamin D status: definition, prevalence, consequences, and correction. Endocrinol Metab Clin North Am. 2010;39(2):287–301.
39. Garland C.F., French C.B., Baggerly L.L., et al. Vitamin D supplement doses and serum 25-hydroxyvitamin D in the range associated with cancer prevention. Anticancer Res. 2011;31(2):607–611.
40. Usoro O.B., Mousa S.A. Vitamin E forms in Alzheimer’s disease: a review of controversial and clinical experiences. Crit Rev Food Sci Nutr. 2010;50(5):414–419.
41. Devaraj S., Leonard S., Traber M.G., et al. Gamma-tocopherol supplementation alone and in combination with alpha-tocopherol alters biomarkers of oxidative stress and inflammation in subjects with metabolic syndrome. Free Radic Biol Med. 2008;44(6):1203–1208.
42. Traber M.G. Vitamin E regulatory mechanisms. Annu Rev Nutr. 2007;27:347–362.
43. Morrissey P.A., Sheehy P.J. Optimal nutrition: vitamin E. Proc Nutr Soc. 1999;58(2):459–468.
44. Cranenburg E.C., Schurgers L.J., Vermeer C., et al. The coagulation vitamin that became omnipotent. Thromb Haemost. 2007;98(1):120–125.
45. Iwamoto J., Sato Y., Takeda T., et al. High-dose vitamin K supplementation reduces fracture incidence in postmenopausal women: a review of the literature. Nutr Res. 2009;29(4):221–228.
46. McKeown N.M., Jacques P.F., Gundberg C.M. Dietary and nondietary determinants of vitamin K biochemical measures in men and women. J Nutr. 2002;132(6):1329–1334.
47. Nimptsch K., Nieters A., Hailer S., et al. The association between dietary vitamin K intake and serum undercarboxylated osteocalcin is modulated by vitamin K epoxide reductase genotype. Br J Nutr. 2009;101(12):1812–1820.
