Indications for Testing

  • Vitamin B12 – at-risk populations
    • Malabsorption, use of H2 receptor antagonists, proton-pump inhibitors, vegetarian diet, aged adults
  • Vitamin D – at-risk populations
    • Poor nutritional intake, limited sun exposure, malabsorption, liver failure, or renal insufficiency
    • Aged adults
    • Certain medications (eg, antiseizure and AIDS medications, glucocorticoids)
  • Vitamin K – patient with elevated INR and unresponsive to vitamin K supplementation (Choosing Wisely: 5 Things Physicians and Patients Should Question; American Society of Clinical Pathology, 2015)
  • Other vitamins – symptoms consistent with deficiency plus risk factor for deficiency

Laboratory Testing

  • Order specific testing based on symptoms and risk assessment
  • For B12 deficiency – consider concurrent testing for folate level; refer to the following for more information
  • For vitamin D deficiency
    • Order test for 25-hydroxy vitamin D by chemiluminescence if available
    • Recommended values – no consensus on optimal concentrations (Institute of Medicine, 2011)
      • 20-30 ng/mL – defined as insufficiency
      • <20 ng/mL – defined as deficiency
    • Do not order 1,25-dihydroxy test unless patient has hypercalcemia or decreased renal function (ASCP's Pathology-Related Choosing Wisely Recommendations, 2015 [Endocrine Society and American Association of Clinical Endocrinologists])
    • Fractionation testing for D2 and D3 not necessary for initial evaluation of vitamin D deficiency
      • Method used should ideally measure D2 and D3 metabolites equally


  • Consider vitamin D deficiency screening for patients at risk
    • Use 25-hydroxy vitamin D test for 
      • Institutionalized refugees
      • Celiac disease
      • Patients with osteoporosis
      • Patients from areas with high vitamin D deficiency
      • Older adults (age is a risk factor)
  • Insufficient evidence to assess harms or benefits of screening for vitamin D deficiency (USPSTF, 2015)
  • Do not perform as population-based screening (Choosing Wisely: 5 Things Physicians and Patients Should Question, 2015)


  • Vitamin D
    • Monitor therapy response – check 25-hydroxy vitamin D 10-12 weeks after initiating therapy
    • Patients who do not appear to be responding to therapy – vitamin D2 or D3 testing may be helpful

Clinical Background

Vitamins are required in the diet because they are not adequately synthesized in the human body.

  • Only small amounts are necessary to catalyze essential biochemical reactions
  • Most deficiencies are rare in healthy persons in the U.S.
  • Disease states generally result from poor diet (eg, elderly, alcoholism)
  • Body stores vary by vitamin
    • Thiamine (B1) and folate stores are small and rapidly depleted
    • Cobalamin (B12) stores are large
  • Vitamins play several roles in disease processes
    • Diseases can cause vitamin deficiency
    • Vitamin deficiency or excess can cause disease
    • High doses of certain vitamins can be used to manage some diseases
  • Water-soluble vitamins

    Water-Soluble Vitamins


    Disease States

    B1 (thiamine)
    Catalyzes reactions that produce energy

    Sources – legumes, nuts, whole grains
    Inhibitors – alcohol, coffee, loop diuretics, raw fish, shellfish, tea

    Vitamin B1 deficiency
    Found in U.S. primarily in alcoholics
    Wet beriberi – high-output cardiac failure
    Dry beriberi – symmetrical peripheral neuropathy
    Wernicke encephalopathy – beriberi combined with alcoholism; horizontal nystagmus, ophthalmoplegia, cerebellar ataxia, mental impairment
    Wernicke-Korsakoff syndrome – coexistence of additional loss of memory and confabulatory psychosis

    Vitamin B1 toxicity – rare reports of anaphylaxis

    B2 (riboflavin)
    Catalyzes reactions that produce energy; coenzyme in the flavoproteins that participate in tissue oxidation and respiration processes

    Sources – broccoli, eggs, enriched breads, fish, lean meats, legumes, milk, other dairy products
    Inhibitors – sunlight rapidly degrades vitamin B2 in foodstuffs

    Vitamin B2 deficiency
    Mucocutaneous lesions including magenta tongue, angular stomatosis, seborrhea, cheilosis

    Vitamin B2 toxicity
    None reported; gastrointestinal tract can absorb only limited quantities

    B3 (niacin)
    Catalyzes the metabolism of fatty acids, amino acids and carbohydrates

    Sources – beans, eggs, meat, milk
    Inhibitors – B2 or B6 deficiency reduces conversion of tryptophan to niacin; drugs that may interfere with metabolism include alcohol, amitriptyline, chlorpromazine, imipramine

    Vitamin B3 deficiency
    Found in U.S. primarily in alcoholics
    Can occur in carcinoid and Hartnup diseases
    Pellagra – pigmented rash in sun-exposed areas (Casal necklace), bright-red tongue, diarrhea, apathy
    When used as drug therapy, flushing and headache frequently accompany treatment doses

    Vitamin B3 toxicity
    Hepatotoxicity is most serious problem
    Glucose intolerance
    , macular edema, macular cysts

    B5 (pantothenic acid)
    Functions in the metabolism and biosynthesis of many compounds

    Sources – broccoli, egg yolk, liver, yeast
    Inhibitors – none

    Vitamin B5 deficiency
    Deficiency has been demonstrated only experimentally (postulated cause of burning-feet syndrome in prisoners)
    Gastrointestinal disturbances, depression, paresthesias, ataxia, hypoglycemia

    Vitamin B5 toxicity – none reported

    B6 (pyridoxal 5’-phosphate)
    Coenzyme in transaminase reactions

    Sources – legumes, meats, nuts, wheat bran
    Inhibitors – drugs such as cycloserine, isoniazid,  L-dopa, penicillamine

    Vitamin B6 deficiency
    Seborrhea, glossitis, seizures, neuropathy, depression, confusion, microcytic anemia

    Vitamin B6 toxicity – severe sensory neuropathy

    B7 (biotin)
    Coenzyme in transfer of carbonyl groups

    Sources – beans, egg yolks, liver, soy, yeast
    Inhibitors – egg whites

    Biotin deficiency
    Previously demonstrated in patients with short-bowel syndrome receiving total parenteral nutrition
    Adults – mental status changes, anorexia, nausea, seborrheic rash
    Infants – hypotonia, lethargy, apathy, alopecia and rash on ears

    Biotin toxicity – None reported

    Folate (B9, folic acid, folacin)
    Coenzyme in metabolic reactions

    Sources – fortified breads, cereals and grain products, fruits, leafy vegetables, organ meats, yeast
    Inhibitors – anticonvulsants, chemotherapy agents, malabsorptive disorders (sprue), methotrexate

    Folate deficiency
    Megaloblastic anemia without neurologic symptoms
    Fetal open neural tube defects
    May be related to increased development of certain cancers

    Folate toxicity – none reported

    B12 (cobalamin)
    Cofactor for enzymatic reactions, metabolism of odd-chain fatty acids, and methylation of homocysteine

    Sources – animal products, dairy products
    Inhibitors – achlorhydria, H2 receptor antagonists, overgrowth of intestinal organisms (eg, short-bowel syndrome), proton-pump inhibitor drugs

    Vitamin B12 deficiency
    Deficiency found in ~30% of people >60 yrs
    Megaloblastic (macrocytic) anemia
    Neurologic manifestations – loss of vibratory and position sense, abnormal gait, dementia, depression, loss of bowel and bladder control

    Vitamin B12 toxicity – none reported

    C (ascorbic acid)
    Coenzyme in formation of collagen and synthetic reactions
    Free radical scavenger with antioxidant activity

    Sources – citrus fruits, green vegetables, potatoes, tomatoes
    Inhibitors – smoking, hemodialysis

    Vitamin C deficiency
    Scurvy – bleeding into skin, inflamed and bleeding gums, bleeding into joints, impaired bone growth

    Vitamin C toxicity – elevated liver enzymes, abdominal pain, diarrhea

  • Fat-soluble vitamins

    Fat-Soluble Vitamins


    Disease States

    A (retinol)
    Required for normal vision, growth, and differentiation of epithelial tissue as well as bone growth, immunity, reproduction, and embryonic development

    Sources – beef, egg yolk, fish, liver, vegetables
    Inhibitors – ethanol, mineral oil, neomycin, cholestyramine

    Vitamin A deficiency
    Ophthalmic – xerophthalmia, Bitot spots, corneal ulcers
    Dermatologic – hyperkeratotic skin lesions

    Vitamin A toxicity
    Acute – increased intracranial pressure, vertigo, diplopia, seizures, headaches
    Chronic – cheilosis, glossitis, alopecia, bone pain, hyperlipidemia, liver fibrosis

    D (calcitriol)
    Hormone precursor
    Required for calcium absorption, bone metabolism, regulation of cell development and the immune system

    Sources – dairy, egg yolks, fish oils, fortified foods; also synthesized in the body in response to ultraviolet radiation
    Inhibitors – barbiturates, isoniazid, phenobarbital, phenytoin, rifampin, sunblock

    Vitamin D deficiency
    Children – rickets and rachitic rosaries (expansion of growth plate)
    Adults – osteomalacia, osteoporosis; hypocalcemia and hypophosphatemia with impaired mineralization of bone matrix

    Vitamin D toxicity
    Rare; associated with ≥10,000 IU/day intake (not seen until 25(OH)D ≥150 ng/mL)

    E (tocopherol)
    Coenzyme in formation of collagen, synthetic reactions, antioxidant activity and free radical scavenger

    Sources – sunflower oil, safflower oil, wheat germ, soybean
    Inhibitors – none

    Vitamin E deficiency
    Almost exclusively in severe and prolonged malabsorptive disorders
    Peripheral neuropathy – areflexia, ataxia, ophthalmoplegia, skeletal myopathy

    Vitamin E toxicity
    Reduced platelet aggregation, interference with warfarin treatment

    K (phylloquinone [K1], menaquinone [K2])
    Essential for carboxylation of glutamic acid residues in proteins required for coagulation

    Sources – butter, coffee, egg yolk, green leafy vegetables, ground beef, milk, pears
    Inhibitors – broad spectrum antibiotics, warfarin

    Vitamin K deficiency
    Hemorrhage of mucous membranes and gastrointestinal tract

    Vitamin K toxicity
    Infants – hemolytic anemia and hyperbilirubinemia

Indications for Laboratory Testing

  • Tests generally appear in the order most useful for common clinical situations
  • Click on number for test-specific information in the ARUP Laboratory Test Directory
Test Name and Number Recommended Use Limitations Follow Up
Vitamin B1 (Thiamine), Whole Blood 0080388
Method: Quantitative High Performance Liquid Chromatography

Preferred specimen for thiamine assessment in patients with suspected deficiency

Do not use to monitor thiamine supplementation

Vitamin B2 (Riboflavin) 0081123
Method: Quantitative High Performance Liquid Chromatography

Assesses riboflavin concentration in serum or plasma

Niacin (Vitamin B30092168
Method: Quantitative High Performance Liquid Chromatography

Assesses niacin concentration in plasma

Vitamin B5 (Pantothenic Acid), Serum 2006982
Method: Quantitative Cell Based Assay

Assesses pantothenic acid concentration in serum

Vitamin B6 (Pyridoxal 5-Phosphate) 0080111
Method: Quantitative High Performance Liquid Chromatography

Assesses pyridoxine concentration in serum or plasma

Specimen collected following an 8-hour or overnight fast accurately indicates vitamin B6 nutritional status; non-fasting specimen concentration reflects recent vitamin intake

Vitamin B7 (Biotin)  2003184
Method: Bioassay

Assesses biotin concentration in serum

Vitamin B12 and Folate 0070160
Method: Quantitative Chemiluminescent Immunoassay

Assesses vitamin B12 and folate concentration in serum or plasma

Aids in detection of vitamin B12 and folate deficiency in individuals with macrocytic or unexplained anemia, or unexplained neurologic disease

Vitamin C (Ascorbic Acid), Plasma 0080380
Method: Quantitative High Performance Liquid Chromatography

Assesses vitamin C concentration in plasma

Vitamin A (Retinol), Serum or Plasma 0080525
Method: Quantitative High Performance Liquid Chromatography

Assesses vitamin A concentration in serum or plasma

Includes measurement of retinol and retinyl palmitate concentration

This assay does not measure other vitamin A metabolites such as retinaldehyde or retinoic acid

Vitamin D, 25-Hydroxy 0080379
Method: Quantitative Chemiluminescent Immunoassay

Preferred screening and monitoring test for vitamin D deficiency

Preferred test to monitor response to supplementation

Vitamin E, Serum or Plasma 0080521
Method: Quantitative High Performance Liquid Chromatography

Assesses vitamin E concentration in plasma

Includes measurement of alpha tocopherol and gamma tocopherol concentrations

Vitamin K1, Serum 0099225
Method: Quantitative High Performance Liquid Chromatography

Assesses vitamin K1 concentration in serum

Additional Tests Available
Click the plus sign to expand the table of additional tests.
Test Name and NumberComments
Vitamin B1 (Thiamine), Plasma 0080389
Method: Quantitative High Performance Liquid Chromatography

Assesses thiamine concentration in plasma to monitor vitamin B1 concentration in patients receiving supplementation

Do not use to determine thiamine deficiency

25-Hydroxyvitamin D2 and D3 by Tandem Mass Spectrometry, Serum 2002348
Method: Quantitative High Performance Liquid Chromatography-Tandem Mass Spectrometry

Nonpreferred screening test for vitamin D deficiency

Alternative test for monitoring response in individuals who are not responding to supplementation

Vitamin B12  0070150
Method: Quantitative Chemiluminescent Immunoassay

Assesses vitamin B12 concentration in serum

Aids in detection of vitamin B12 deficiency in individuals with macrocytic or unexplained anemia, or unexplained neurologic disease

Folate, Serum 0070070
Method: Quantitative Chemiluminescent Immunoassay

Assesses folate concentration in serum

Aids in detection of folate deficiency

Vitamin B12 with Reflex to Methylmalonic Acid, Serum (Vitamin B12 Status) 0055662
Method: Quantitative Chemiluminescent Immunoassay/Quantitative High Performance Liquid Chromatography-Tandem Mass Spectrometry

Preferred reflex test for detection of vitamin B12 deficiency in individuals with macrocytic or unexplained anemia, or unexplained neurologic disease

Assesses vitamin B12 concentration in serum

Rules out pernicious anemia

Reflex pattern – if Vitamin B12 is less than 300 pg/mL, then methylmalonic acid, serum (Vitamin B12 Status) will be added

Folate, RBC 0070385
Method: Quantitative Chemiluminescent Immunoassay

Aids in detection of folate deficiency

Preferred specimen for assessment of folate concentration in whole blood

Vitamin D, 1, 25-Dihydroxy 0080385
Method: Quantitative Chemiluminescent Immunoassay

Preferred test for individuals with hypercalcemia or renal failure in addition to Vitamin D, 25-Hydroxy testing

Normal result does not rule deficiency

Vitamin B12 Deficiency Panel 2012276
Method: Quantitative Gas Chromatography/Mass Spectrometry 

Not recommended for initial testing in suspected B12 deficiency; may be useful when B12 and MMA results alone are equivocal

Panel includes methylmalonic acid, 2-methylcitric acid, homocysteine, and cystathionine