Vitamin Assays (A, E, K, B1, B6, C)

Vitamin assays measure the circulating or functional level of individual vitamins in patients with suspected deficiency or toxicity. This article covers the vitamins that are tested less often than vitamin D, B12 and folate: fat soluble vitamins A, E and K, and water soluble vitamins B1 (thiamine), B6 (pyridoxine) and C (ascorbic acid).

Each has a specific preferred test, because plasma concentration alone does not reliably reflect tissue status for several of them. Deficiency usually follows malabsorption, alcohol use disorder, bariatric surgery, restrictive diets, prolonged parenteral nutrition, chronic illness and drug effects.

Alcohol use disorder, bariatric surgery and malabsorptive disease (coeliac disease, cystic fibrosis, cholestasis, pancreatic insufficiency) account for most deficiencies in high income settings, while dietary insufficiency and infection dominate in low income settings. Vitamin A deficiency is the leading cause of preventable childhood blindness, affecting about 250 million preschool children worldwide. Thiamine deficiency causes Wernicke encephalopathy, a medical emergency. Scurvy has re-emerged in people with restricted diets.

Indications. Malabsorption or steatorrhoea. Bariatric surgery follow up. Alcohol use disorder. Unexplained neuropathy, encephalopathy, ataxia or ophthalmoplegia. Night blindness, xerophthalmia or poor wound healing. Easy bruising or bleeding with a normal platelet count. Gingival bleeding and perifollicular haemorrhage. Long term parenteral nutrition. Anticonvulsant, isoniazid or hydralazine therapy. Monitoring supplementation and suspected toxicity.

Specimen. Fasting venous blood. Vitamins A, E, B6 and B1 are light sensitive, so samples are protected from light, kept cool, and separated promptly. Vitamin A and E: serum or plasma (heparin or EDTA), 1 mL. Vitamin K: citrate plasma for prothrombin time and PIVKA-II, and serum for phylloquinone by chromatography. Vitamin B1: whole blood in EDTA for thiamine diphosphate. Vitamin B6: EDTA plasma for pyridoxal 5-phosphate. Vitamin C: heparin plasma on ice, stabilised with metaphosphoric acid, and frozen quickly.

Vitamin A (retinol).

• Plasma retinol: adults 1.05 to 2.8 micromol/L (30 to 80 microg/dL). Deficiency under 0.7 micromol/L, and severe deficiency under 0.35 micromol/L.
• Retinol is carried by retinol binding protein and falls in protein malnutrition, liver disease, zinc deficiency, inflammation and infection (negative acute phase reactant), so it is interpreted with C-reactive protein. Retinol binding protein is measured alongside, and the retinol to retinol binding protein ratio is about 0.8 to 1.0 in health.
• Deficiency: night blindness, xerophthalmia, Bitot spots, keratomalacia, follicular hyperkeratosis, and increased infection and mortality in children.
• Toxicity: over 3.5 micromol/L, with headache, raised intracranial pressure, hepatotoxicity, bone pain and teratogenicity.

Vitamin E (alpha tocopherol).

• Plasma alpha tocopherol 12 to 42 micromol/L in adults. Deficiency under 12 micromol/L, interpreted as the ratio to total lipids (under 0.8 mg/g lipid suggests deficiency), because it is carried in lipoproteins and rises with hyperlipidaemia.
• Deficiency: spinocerebellar ataxia, peripheral neuropathy, retinopathy, haemolytic anaemia in premature infants. Causes include abetalipoproteinaemia, cholestasis, cystic fibrosis, short bowel syndrome and familial isolated vitamin E deficiency.

Vitamin K.

• Phylloquinone by liquid chromatography: 0.3 to 2.6 nmol/L. Direct levels are rarely used.
• Prothrombin time prolongation that corrects with vitamin K is the functional test. PIVKA-II (protein induced by vitamin K absence, des-gamma-carboxy prothrombin) is raised in deficiency (over 2 ng/mL) but also in hepatocellular carcinoma.
• Deficiency: newborns, malabsorption, antibiotics, warfarin, cholestasis, liver disease. Presents with bruising, mucosal bleeding and intracranial haemorrhage in infants.

Vitamin B1 (thiamine).

• Whole blood thiamine diphosphate (TDP): 70 to 180 nmol/L by chromatography. Under 70 nmol/L indicates deficiency. Erythrocyte transketolase activity with thiamine pyrophosphate effect (activation coefficient over 1.25 means deficiency) is the older functional test.
• Plasma thiamine is unreliable. Urinary thiamine reflects recent intake.
• Deficiency: dry beriberi (neuropathy), wet beriberi (high output heart failure, lactic acidosis), and Wernicke encephalopathy (confusion, ataxia, ophthalmoplegia). The diagnosis is clinical, and treatment is not delayed to await results.

Vitamin B6 (pyridoxine).

• Plasma pyridoxal 5-phosphate (PLP): 20 to 125 nmol/L. Under 20 nmol/L indicates deficiency. PLP falls in inflammation and in pregnancy. The erythrocyte aspartate aminotransferase activation coefficient is a functional measure.
• Deficiency: peripheral neuropathy, seborrhoeic dermatitis, glossitis, cheilosis, seizures in infants, sideroblastic anaemia. Causes: isoniazid, hydralazine, penicillamine, cycloserine, alcohol, renal failure and dialysis.
• Toxicity: sensory neuropathy at doses over 100 mg a day, sometimes at lower doses, with plasma PLP over 500 nmol/L.

Vitamin C (ascorbic acid).

• Plasma ascorbate: 23 to 85 micromol/L (0.4 to 1.5 mg/dL). Deficiency under 11 micromol/L (0.2 mg/dL), and depletion 11 to 23 micromol/L. Leucocyte ascorbate reflects tissue stores but is rarely available.
• Plasma levels fall with acute illness, smoking, inflammation and sepsis, and rise within hours of ingestion.
• Scurvy: perifollicular haemorrhage, corkscrew hairs, gingival bleeding, poor wound healing, bone pain and anaemia, and diagnosis is clinical with levels under 11 micromol/L.
• Interferes with glucose dipsticks and creatinine assays at high doses.

Critical values and findings needing immediate action.

• Suspected Wernicke encephalopathy (confusion, ataxia, ophthalmoplegia in an at risk patient): immediate intravenous thiamine, before glucose, without waiting for the assay.
• Prolonged prothrombin time with bleeding and suspected vitamin K deficiency: intravenous vitamin K and, if life threatening bleeding, prothrombin complex concentrate.
• Newborn with bleeding: prophylactic or therapeutic vitamin K.
• Xerophthalmia or corneal ulceration: urgent high dose vitamin A, and ophthalmic care to prevent blindness.
• Vitamin A toxicity in pregnancy or papilloedema.

Interferences and pitfalls.

• Light exposure and delay degrade vitamins A, E, B6, B1 and C, so samples are shielded and processed quickly.
• Acute phase response lowers plasma vitamin A, C and PLP and raises ferritin and caeruloplasmin, so levels are read with C-reactive protein.
• Vitamin A and E are lipid bound, and lipid adjusted results avoid false deficiency or normality.
• Recent supplementation normalises plasma concentration without correcting tissue stores.
• Folate and B12 deficiency, alcohol, magnesium deficiency (which impairs thiamine dependent enzymes) and refeeding syndrome coexist with thiamine deficiency, and thiamine is given before refeeding.
• Reference ranges vary by method, age and laboratory.
• Multiple deficiencies are common after bariatric surgery and in malabsorption, and screening a panel is more productive than single tests.

Special groups.

• Children: vitamin A deficiency in low income regions, vitamin K deficiency bleeding in newborns who have no prophylaxis, and vitamin C deficiency with restricted diets in autism.
• Pregnancy: vitamin A is limited because of teratogenicity at doses over 3000 microg (10 000 IU) retinol a day, while B6 is used for nausea.
• Older adults: low intake, drug effects and malabsorption cause multiple deficiencies.
• Bariatric surgery patients: lifelong monitoring of vitamins A, E, K, B1 and B6 along with B12, folate, iron, zinc and copper.
• Regions: vitamin A deficiency is endemic in parts of sub-Saharan Africa and South Asia, with supplementation programmes.

Follow up tests. Prothrombin time and INR, retinol binding protein, lipid profile, liver function tests, full blood count and film, B12, folate, ferritin, zinc and copper, magnesium, calcium and vitamin D, faecal elastase, coeliac serology, nerve conduction studies and neuroimaging in suspected Wernicke encephalopathy, and eye examination.