Vitamins and Nutrition
Vitamins and Nutrition
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Vitamins act mainly as enzymatic cofactors, so deficiency assessment takes one of three routes: measuring the active cofactor or precursor directly, measuring a dependent enzymatic activity, or evaluating urinary metabolites or excretion after a test load. A normal serum level does not guarantee adequate cellular function, and vice versa. Interpretation requires correlation with dietary history and physical findings.1
The vitamins
| Vitamin | Function | Deficiency syndrome | Key laboratory notes |
|---|---|---|---|
| A (retinol) | Vision through rhodopsin, cell differentiation, immunity | Night blindness, growth retardation, dermatitis, recurrent infection | Carried by retinol-binding protein with transthyretin; toxicity from supplement or liver-organ overdose; carotenoids spare preformed-vitamin-A toxicity, but high-dose beta-carotene supplements can be harmful in smokers or asbestos-exposed people. Retinol is light-sensitive; follow the performing laboratory’s handling requirements. |
| D (calciferol) | Calcium and phosphate absorption, bone mineralization | Rickets in children, osteomalacia in adults | Synthesis and regulation are covered in the PTH-vitamin D axis; 25-OH-D is the status marker, because 1,25(OH)2D’s half-life is hours and its concentration is tightly regulated. |
| E (tocopherol) | Membrane antioxidant protecting against lipid peroxidation | Hemolytic anemia, especially premature infants; ataxia | Deficiency risk with fat malabsorption in cystic fibrosis and abetalipoproteinemia |
| K | Cofactor for hepatic γ-carboxylation of factors II, VII, IX, X, and proteins C and S | Bleeding, easy bruising | Intestinal bacteria synthesize menaquinones that may supply part of the requirement, with an uncertain contribution; warfarin blocks the same cycle; prothrombin time is the functional assay. |
| B1 (thiamine) | Coenzyme, TPP, in carbohydrate and branched-chain amino acid decarboxylation | Beriberi, cardiac failure and neuropathy; seen with chronic alcoholism | |
| B2 (riboflavin) | Flavin coenzymes FMN and FAD in redox reactions | Angular stomatitis, dermatitis, photophobia | Body stores last about 5 months; no known toxicity |
| B3 (niacin) | NAD+ and NADP+ coenzymes in respiration, lipid and fatty-acid metabolism, glycolysis | Pellagra: dermatitis, diarrhea, dementia, death | Synthesized in part from tryptophan; pharmacologic doses lower lipids but cause flushing |
| B5 (pantothenic acid) | Precursor of coenzyme A | Fatigue, headache, muscle cramps, rare | |
| B6 (pyridoxine) | Coenzyme in amino acid metabolism | Seizures, anemia, dermatitis, depression; associated with hyperhomocysteinemia | Antagonized by isoniazid, steroids, penicillamine. Light-sensitive in some procedures; follow the performing laboratory’s requirements. |
| B7 (biotin) | Carboxylase coenzyme in gluconeogenesis and lipogenesis | Alopecia, dermatitis, seizures | Raw egg-white avidin binds biotin and can induce deficiency; parenteral nutrition is a risk. High-dose biotin supplements also interfere with streptavidin-biotin immunoassays, as the endocrine methods module covers. |
| B9 (folate) | One-carbon transfer coenzyme for DNA synthesis | Megaloblastic anemia, neural tube defects | Erythrocyte folate reflects tissue stores; supplementation reduces neural tube defect risk; antagonized by phenytoin, methotrexate, alcohol, trimethoprim |
| B12 (cobalamin) | Coenzyme for hematopoiesis and fatty-acid metabolism | Pernicious and megaloblastic anemia, neurologic abnormality | Absorption requires gastric intrinsic factor; deficiency overlaps hematologically with folate deficiency |
| C (ascorbic acid) | Collagen cross-linking through proline and lysine hydroxylation; antioxidant | Scurvy: hemorrhage, poor wound healing | Increases iron absorption; urinary ascorbate is a poor status marker |
Vitamin D testing
Isotope-dilution LC-MS/MS is the reference measurement procedure for 25-hydroxyvitamin D, as practiced in the CDC’s higher-order reference method and traceable through NIST standard reference materials, within the CDC/NIST Vitamin D Standardization Program. It resolves and separately quantifies 25-OH-D2, from ergocalciferol and plant sources and supplements, and 25-OH-D3, from cholecalciferol, skin synthesis, and animal sources, using chromatography, historically on pentafluorophenylpropyl phases, that separates the C3-epimer of 25-OH-D3 so the epimer does not contribute to the 25-OH-D3 result; the epimer is abundant in infants and can otherwise inflate a result. Immunoassays report a single total 25-OH-D value from antibody binding. Different antibodies show different relative affinity for 25-OH-D2 versus 25-OH-D3, and that unequal cross-reactivity is the main source of immunoassay-to-immunoassay and immunoassay-to-LC-MS/MS discordance, mattering most in patients taking ergocalciferol, D2, supplementation. C3-epimer interference is generally low for most immunoassays.2
25-OH-D is the analyte of choice for assessing vitamin D status. Its half-life runs in weeks and it reflects total body stores. 1,25-dihydroxyvitamin D has a half-life in hours, is tightly regulated by PTH, phosphate, and calcium, and can be normal or even elevated in frank deficiency. Deficiency cutoffs are decision limits that differ among guidelines and laboratories: the 2011 Endocrine Society guideline defined deficiency below 20 ng/mL, a still-commonly used laboratory cutoff, while the 2024 guideline abandoned universal sufficiency and insufficiency thresholds for generally healthy adults and recommends against routine screening in that population. Interpret a 25-OH-D result against the requesting laboratory’s stated cutoffs and the patient’s clinical setting.3
Light-sensitive vitamins
Vitamin A, carotene, vitamin B6, and folate degrade on light exposure. The performing laboratory specifies protection requirements for these assays, typically foil-wrapping or amber containers from collection through analysis. Without that protection, results can be falsely low. Each assay’s procedure defines which analytes require protection and what protection its method requires. General handling for fat-soluble vitamins follows each laboratory’s procedure for prompt separation and storage; prolonged room-temperature storage and hemolysis can degrade several of these analytes before testing.
B12 and folate
Laboratories measure serum B12 and folate by competitive protein-binding assay or immunoassay. Chemiluminescent formats have largely replaced radioassay. Hemolysis falsely raises serum folate because erythrocytes concentrate folate several-fold over plasma, so even mild hemolysis invalidates the result. RBC folate requires a whole-blood specimen, EDTA, lysed, plus a concurrent hematocrit to express folate per unit of packed cells, and it reflects folate stores over roughly the preceding 2 to 3 months, while the serum level tracks recent intake. RBC folate was historically the tissue-stores test, but its current role varies among laboratories. Serum folate is the preferred routine test in some laboratories because of RBC-folate analytic variability. Interpretation follows the requesting laboratory’s approach. Recent dietary intake, recent hematinic supplementation, and recent blood transfusion can all confound a result; a blood transfusion introduces donor B12 and folate. Note these factors at collection.1
When serum B12 is borderline but clinical suspicion persists, methylmalonic acid is the preferred confirmatory measurement: it rises in intracellular B12 deficiency, stays normal in isolated folate deficiency, and rises with renal impairment, age, and bacterial overgrowth, so interpretation uses the reporting laboratory’s interval. Homocysteine rises in both B12 and folate deficiency and is less specific. NICE guidance treats a total B12 in a borderline zone as inconclusive pending further testing; measurement before supplementation, when clinically safe, preserves the result’s meaning.4
Vitamin K testing
Vitamin K itself is rarely measured directly. The functional index of vitamin K-dependent γ-carboxylation of factors II, VII, IX, X and proteins C and S consists of PIVKA-II, protein induced by vitamin K absence or antagonism-II, an undercarboxylated prothrombin also called des-gamma-carboxyprothrombin, and the prothrombin time. PIVKA-II is a functional marker of defective carboxylation with recognized non-nutritional causes, including warfarin, cholestasis, liver disease, and hepatocellular carcinoma, so an elevated value requires interpretation against medication history and hepatic function as well as vitamin K status.5
Nutritional markers
Prealbumin is the clinical name for transthyretin, TTR, a tetrameric transport protein with a short half-life of about 2 days. It binds thyroxine and, through retinol-binding protein, vitamin A. Inflammation and illness strongly affect its concentration, so prealbumin is not used alone as a marker of nutritional status. ASPEN guidance uses it only as a corroborating measure alongside clinical assessment. The protein module covers transthyretin as the prealbumin band in CSF electrophoresis.6
References
- Rifai N, Chiu RWK, Young I, Burnham CAD, Wittwer CT, eds. Tietz Textbook of Laboratory Medicine. 7th ed. Elsevier; 2023.
- Centers for Disease Control and Prevention. Vitamin D reference laboratory: CDC clinical standardization programs. Accessed August 31, 2026. The CDC higher-order ID-LC-MS/MS procedure separates 25(OH)D3 from 3-epi-25(OH)D3 and is traceable to NIST standards through the Vitamin D Standardization Program.
- Demay MB, Pittas AG, Bikle DD, et al. Vitamin D for the prevention of disease: an Endocrine Society clinical practice guideline. J Clin Endocrinol Metab. 2024;109(8):1907-1947. doi:10.1210/clinem/dgae290. The 2024 guideline does not set universal sufficiency thresholds; laboratory deficiency cutoffs such as 20 ng/mL are decision limits, not reference intervals.
- National Institute for Health and Care Excellence. Vitamin B12 deficiency in over 16s: diagnosis and management. NICE guideline NG239. 2023. Accessed August 31, 2026. NICE
- Fauci AT, Locarnini F, Foschi M, Vitolo A. Protein induced by vitamin K absence or antagonist II: experience to date and future directions. Crit Rev Oncol Hematol. 2024;193:104215. doi:10.1016/j.critrevonc.2023.104215
- American Society for Parenteral and Enteral Nutrition. Prealbumin monitoring in nutrition support. ASPEN guidance. Accessed August 31, 2026. ASPEN. Use as a corroborative nutrition marker only, with inflammation and illness as major confounders.