Vitamins - Deficiency and Toxicity

Indications for Testing

Laboratory testing for vitamins is used to:

• Assess nutritional status (deficiency, sufficiency, or toxicity)
• Monitor therapeutic supplementation

Vitamin A

Vitamin A refers to a group of fat-soluble retinoids with the biologic activity of all-trans retinol, including retinol, retinal, retinoic acid, and retinyl esters.  Carotenoids are metabolized into vitamin A within the body. Deficiency may arise from fat malabsorption disorders (e.g., celiac disease, Crohn disease), cystic fibrosis, liver disease, and bariatric surgery. ^,  Excessive preformed vitamin A in body stores (hypervitaminosis A) can occur as a result of chronic or acute excess intake, such as from consumption of supplements, and may lead to adverse consequences before symptoms become apparent.  Patients with a fat malabsorption disease who are receiving high-dose vitamin A supplementation should be monitored to verify proper dosing and avoid toxicity. Regular monitoring is recommended for patients who have had bariatric surgery. Refer to the ARUP Consult Bariatric Surgery - Nutritional Assessment topic for specific recommendations.

Serum or plasma concentrations of retinol (the main circulating form of vitamin A) and/or the proteins that circulate with it (particularly retinol-binding protein) are widely used and considered sufficient to determine vitamin A status. ^,  Because retinol-binding protein is a negative acute-phase reactant, retinol concentration will decrease if inflammation is present; consider measurement of C-reactive protein (CRP) to distinguish between nutritional and inflammatory causes of reduced concentration.  Retinol-binding protein is also affected by liver disease, zinc deficiency, and protein malnutrition, all of which may lead to decreased plasma retinol concentration. 

Vitamin A concentration is tightly regulated in the circulation and may not decrease until liver stores are nearly depleted. ^,  Hepatic reserves may be indirectly assessed with plasma dose-response tests that involve evaluation of retinol in serum before and after administration of a small amount of vitamin A.  The definitive assessment of vitamin A status when indicated is evaluation of hepatic reserves via biopsy. ^, 

Vitamin B₁ (Thiamine)

Vitamin B₁, or thiamine (thiamin), is a water-soluble vitamin that is important in energy metabolism.  The primary active form of thiamine in the body is thiamine diphosphate (TDP, also known as thiamine pyrophosphate).  Risk factors for thiamine deficiency include alcohol dependence, bariatric surgery, malabsorption, older age, and HIV/AIDS.  TDP can be measured in erythrocytes or whole blood and is the preferred analyte for assessment of thiamine status.  Erythrocyte thiamine concentration reflects thiamine status, and whole blood concentration correlates well with erythrocyte concentration, especially when corrected for hemoglobin.  Because plasma thiamine concentration does not directly reflect thiamine concentration in tissue, plasma testing is not recommended for nutritional assessment; TDP should be measured in whole blood or erythrocytes instead.

Serial measurements of thiamine (e.g., in patients with Wernicke-Korsakoff syndrome) should be performed using the same assay, preferably using a whole blood specimen. Regular monitoring is recommended for patients who have had bariatric surgery and have risk factors for thiamine deficiency. Refer to the ARUP Consult Bariatric Surgery - Nutritional Assessment topic for specific recommendations.

Vitamin B₂ (Riboflavin)

Vitamin B­₂ (riboflavin) is a water-soluble vitamin that is a critical part of the flavin adenine dinucleotide (FAD) and flavin mononucleotide (FMN) coenzymes, which are involved in energy production and numerous other metabolic and cellular functions.  Testing of riboflavin status is not routinely performed in healthy individuals.  Risk factors for riboflavin deficiency include endocrine disorders (e.g., thyroid hormone insufficiency), vegan diet, vegetarian diet (in athletes), low consumption of meat or dairy products during pregnancy, and riboflavin transporter deficiency neuropathy.  Riboflavin deficiency is often associated with deficiency in other B vitamins because riboflavin plays a role in the activation of vitamins B₃ and B₆. 

Direct measurement of riboflavin and its metabolites (FAD and FMN) can be performed in erythrocytes or plasma.  FAD measurements in plasma are affected by inflammation; therefore, measurement of FAD concentration in erythrocytes is recommended in critically ill patients. 

The erythrocyte glutathione reductase activity coefficient is an indirect but sensitive measure of riboflavin status.  Erythrocyte glutathione reductase activity is measured as the ratio of erythrocyte glutathione reductase in freshly lysed erythrocytes without and with stimulation with exogenous FAD. ^,  Thresholds for diagnosing deficiency have not been firmly established,  however, and variation between methods may lead to differences in results.  Erythrocyte glutathione reductase activity is not useful in patients with glucose-6-phosphate dehydrogenase (G6PD) deficiency. 

Vitamin B₃ (Niacin)

Vitamin B₃ (niacin) is a collective term for nicotinic acid, nicotinamide, and derivatives with the same biologic activity.  The main active form of niacin is the coenzyme nicotinamide adenine dinucleotide (NAD), which is converted into nicotinamide adenine dinucleotide phosphate (NADP); both NAD and NADP are required for numerous enzymatic reactions within the body.  Risk factors for niacin deficiency include malabsorption; inflammatory bowel disease; vitamin B₂, B₆, or iron deficiency; alcohol dependence; anorexia; cirrhosis of the liver; HIV/AIDS; carcinoid syndrome; and Hartnup disease.  Niacin status is assessed by measuring the metabolites N1-methyl-nicotinamide and N1-methyl-2-pyridone-5-carboxamide in urine. ^,  Niacin may be directly measured in plasma, although plasma concentration is not a reliable indicator of niacin status. ^, 

Very-high-dose niacin is used to treat hyperlipidemia and other conditions.  Niacin may lead to adverse side effects when administered in pharmacologic doses; therefore, multiple organizations recommend testing hepatic transaminase, fasting blood glucose or hemoglobin A1c, and uric acid in supplement users before initiating therapy, when dosage is increased, and every 6 months after reaching a maintenance dose. 

Vitamin B₅

Vitamin B₅, or pantothenic acid, is a water-soluble vitamin used in the synthesis of coenzyme A and acyl carrier protein. ^,  Pantothenic acid deficiency is rare outside of severe malnutrition; healthy individuals are not routinely assessed for pantothenic acid status. ^,  Pantothenic acid status can be directly measured in urine and whole blood. ^, 

Vitamin B₆

Vitamin B₆ is a general term for pyridoxine, pyridoxal, pyridoxamine, and their esters. Pyridoxal 5’ phosphate (PLP) and pyridoxamine 5’ phosphate are the active forms and are involved in numerous enzymatic reactions; 4-pyridoxic acid is a catabolic product of vitamin B₆ metabolism. ^,  Although studies have shown that almost a quarter of people in the United States who do not take a vitamin B₆ supplement have low plasma PLP concentrations,  clinically relevant vitamin B₆ deficiency is rare in otherwise healthy individuals and usually occurs in conjunction with other B vitamin deficiencies. Risk factors for deficiency include obesity, malabsorption (e.g., due to celiac disease or Crohn disease), hemodialysis, alcohol dependence, chronic renal insufficiency, pyridoxine-dependent epilepsy, and pregnancy, especially in cases of eclampsia or preeclampsia.  Vitamin B₆ supplements may be used to treat nausea and vomiting in pregnancy. 

No single marker reflects vitamin B₆ status; therefore, use of a combination of tests is recommended.  PLP concentration in plasma is the most common measure of vitamin B₆ status ^,  and may be the best indicator of tissue stores, given that it is thought to reflect the concentration of PLP in the liver.  Reduced concentration of 4-pyridoxic acid, a product of vitamin B₆, may be observed in vitamin B₆ deficiency; 4-pyridoxic acid concentration can be measured in plasma or urine.  Vitamin B₆ status also can be indirectly assessed by tryptophan metabolite concentrations and by measuring erythrocyte aminotransferase saturation by PLP (aspartate and alanine). ^,  Erythrocyte aminotransferase results may be less useful when assessing vitamin B₆ status. 

Vitamin B₇ (Biotin)

Vitamin B₇, or biotin, is a cofactor for enzymes involved in metabolism and is also involved in histone modification, cell signaling, and gene regulation.  Dietary biotin deficiency is rare but can occur in individuals with biotinidase deficiency.  Other risk factors include malabsorption, pregnancy, breastfeeding, and alcohol dependence.  Biotin supplements, many containing more than 500 times the recommended daily intake of biotin, are promoted to improve hair, skin, and nail health. Such high-dose supplementation may interfere with laboratory tests. Measurement of biotin in plasma or serum enables assessment of biotin concentration but does not indicate biotin status ; low biotin concentrations in plasma and serum are not sensitive markers of inadequate biotin intake because they do not decrease substantially with mild biotin deficiency. 

Vitamin B₉ (Folate)

Vitamin B₉ (folate, folic acid, or folacin) is essential for gene expression and cell division.  Risk factors for folate deficiency and corresponding megaloblastic anemia include malabsorptive disorders (celiac disease, tropical sprue, Crohn disease), alcohol dependence, SLC46A1 (protein-coupled folate transporter) variants, and the 677C>T MTHFR gene variant. ^,  Folate deficiency is usually accompanied by deficiency in multiple other nutrients, especially vitamin B₁₂, and importantly, folate supplementation is thought to mask the symptoms of vitamin B₁₂ deficiency.  Pregnant individuals require increased folate intake to reduce the risk of neural tube defects in their infants. 

Folate deficiency can be assessed using direct measurement in whole blood or a combination of serum and erythrocytes. ^,  Serum concentrations may be maintained even when tissue stores are depleted; thus, although a low concentration may indicate a long-term abnormality or low intake, an adequate concentration does not necessarily indicate adequate tissue stores. ^, ,  Additionally, plasma homocysteine can be used as a functional indicator of folate status but is not a specific marker for folate status and can be dependent on factors such as age and sex. ^, 

Vitamin B₁₂

Vitamin B₁₂, or cobalamin, is the name for several forms of cobalt-containing compounds with vitamin B₁₂ activity.  Cobalamins, specifically the active forms methylcobalamin and 5-deoxyadenosylcobalamin, are essential coenzymes for methyl transfer reactions in the body.  Vitamin B₁₂ is also necessary for proper blood formation, DNA synthesis, and neurologic health.  Although most people in the U.S. consume more than the recommended amounts of vitamin B₁₂, vitamin B₁₂ deficiency is still a common condition and may lead to megaloblastic anemia.  Risk factors for vitamin B₁₂ deficiency include vegetarian diet, gastrointestinal disorders (e.g., celiac disease), older age, pernicious anemia, and low stomach acidity; maternal vegan diet is a risk factor for vitamin B₁₂ deficiency in infants. ^,  Monitoring for vitamin B₁₂ deficiency should be considered in individuals using proton pump inhibitors for prolonged periods,  individuals prescribed metformin, particularly those with anemia or peripheral neuropathy,  and patients who have had bariatric surgery; refer to the ARUP Consult Bariatric Surgery - Nutritional Assessment topic for specific recommendations.

Serum and plasma B₁₂ concentrations reflect both intake and stores.  However, serum concentrations may be maintained even when tissue stores are depleted; thus, although a low concentration may indicate a long-term abnormality or low intake, an adequate concentration does not necessarily indicate adequate tissue stores.  Insufficient vitamin B₁₂ causes the concentration of methylmalonic acid (MMA) to increase.  Because an elevated MMA concentration is tightly correlated with vitamin B₁₂ status, MMA concentration might be a more sensitive, more reliable, and earlier indicator of vitamin B₁₂ deficiency than vitamin B₁₂ plasma concentration. ^,  Furthermore, MMA concentration is not affected by folate deficiency and is therefore more specific than other indirect measures of vitamin B₁₂ status.  MMA concentration is increased in individuals with impaired renal function and in older individuals (>65 years of age) in the absence of B₁₂ deficiency. ^,  Plasma homocysteine concentration may increase in the case of vitamin B₁₂ deficiency, although it may also be elevated due to vitamin B₆ or folate deficiency or other factors (e.g., impaired renal function). ^,  Thus, plasma homocysteine can be used as a nonspecific indicator of B₁₂ status, particularly in combination with other tests.

Vitamin C

Vitamin C (ascorbic acid), a key antioxidant within the body, is involved in a variety of biosynthetic reactions and plays a role in immunity and iron absorption. ^,  Vitamin C deficiency and toxicity are rare in the U.S. Insufficiency, however, may be present in infants not receiving breast milk or formula and in individuals who smoke, consume a diet with limited food variety, or have certain medical conditions (e.g., cancer). ^, 

There are currently no widely used functional indicators of vitamin C status; direct measurements are used instead.  Measurements of vitamin C in plasma and serum indicate the availability of circulating vitamin C and are considered reliable indicators of intake. ^,  Ascorbic acid may also be measured in the buffy coat or leukocytes.  Vitamin C in leukocytes may reflect the tissue vitamin C concentration more accurately than plasma measurements.  However, the performance of these assays is difficult; thus, they are not widely used.  Urine ascorbic acid concentration following a vitamin C loading dose may be useful in the diagnosis of scurvy.  Vitamin C is a light-, temperature-, and oxidant-sensitive vitamin and is unstable in plasma.  Addition of a preservative should be considered,  and samples should be carefully prepared and rapidly frozen to minimize exposure to light and heat.

Vitamin D

Vitamin D (calciferol) is the collective name for a group of fat-soluble compounds that includes ergocalciferol (vitamin D₂) and cholecalciferol (vitamin D₃). The body converts vitamin D into 25-hydroxyvitamin D [25(OH)D], its major circulating form, and 1,25-dihydroxyvitamin D [1,25(OH)₂D], its major active form.  Vitamin D status is assessed with testing for 25-hydroxyvitamin D (the preferred test for nutritional status), as well as measurement of 1,25-dihydroxyvitamin D [25(OH)₂D] if the patient has hypercalcemia or decreased kidney function.  Refer to the ARUP Consult Hypercalcemia and Hypocalcemia - Vitamin D Testing topic for specific recommendations.

For specific recommendations on vitamin D monitoring following bariatric surgery, refer to the ARUP Consult Bariatric Surgery - Nutritional Assessment topic.

Vitamin E

Vitamin E is a term for the group of antioxidant tocopherols and tocotrienols.  Vitamin E occurs in eight forms, including alpha (α)-tocopherol and gamma (γ)-tocopherol. Risk factors for deficiency include fat malabsorption disorders (e.g., Crohn disease, cystic fibrosis), and abetalipoproteinemia; those with cystic fibrosis are particularly prone to malabsorption of vitamin E and other fat-soluble vitamins. ^,  Alpha- and γ-tocopherol are the vitamers most commonly used to assess vitamin E status.  High-performance liquid chromatography is the preferred method for measuring vitamin E in serum or plasma.  Testing for vitamin E status in patients who have had bariatric surgery should be considered if signs or symptoms of deficiency are present, regardless of how much time has passed since the surgery was performed. Refer to the ARUP Consult Bariatric Surgery - Nutritional Assessment topic for specific recommendations.

Vitamin K

Vitamin K refers to fat-soluble compounds with a 2-methyl-1,4-naphthoquinone structure, including phylloquinone (K₁) and menaquinone (K₂).  Vitamin K deficiency is rare in adults, generally a result of malabsorption, cystic fibrosis, or liver disease, and is only clinically problematic when clotting time is increased. ^,  Laboratory testing for vitamin K status is not routinely performed except in patients receiving anticoagulant medication or who have a bleeding disorder.  Many cystic fibrosis organizations recommend vitamin K supplementation and careful monitoring of vitamin K status.  Testing for vitamin K status in patients who have had bariatric surgery should be considered if signs or symptoms of deficiency are present, regardless of how much time has passed since the surgery was performed. Refer to the ARUP Consult Bariatric Surgery - Nutritional Assessment topic for specific recommendations.

Vitamin K status is generally determined indirectly by measuring the function of vitamin K-dependent proteins.  Plasma prothrombin time (PT) and the international normalized ratio (INR, an expression of the prothrombin time as a fraction of control time) are the most commonly used tests. ^,  PT and INR increase in the case of vitamin K deficiency, reflecting clinically significant reduced clotting ability.  Functional vitamin K tests are commonly used for monitoring in warfarin dosing and bleeding disorders (e.g., hemophilia, uncommon factor deficiencies). 

Fasting serum or plasma phylloquinone concentration correlates with intake and is considered the best indicator of vitamin K₁ status.  However, this test does not assess menaquinone (K₂), and patients with low phylloquinone concentration may not exhibit clinical symptoms of vitamin K deficiency.