25-Hydroxyvitamin D

Vitamin D is made in the skin from 7-dehydrocholesterol under ultraviolet B light, and also comes from the diet and supplements as cholecalciferol (D3) and ergocalciferol (D2). The liver converts it to 25-hydroxyvitamin D (25-OH D), the main circulating form with a half life of 2 to 3 weeks, and the kidney then converts it to the active hormone 1,25-dihydroxyvitamin D (calcitriol) under the control of parathyroid hormone, phosphate and FGF23.

Serum 25-OH D reflects total vitamin D supply and is the test of vitamin D status. It is measured by immunoassay or by liquid chromatography with mass spectrometry, which is the most accurate method.

The test identifies deficiency in patients at risk and in those with bone or calcium disorders, and it confirms vitamin D excess in unexplained hypercalcaemia. It is not a routine screening test in well people.

Vitamin D deficiency is common, affecting an estimated 20 to 40% of adults in many regions and more than 1 billion people worldwide. It is more frequent at high latitude, in people with dark skin, in those who cover their skin or stay indoors, in older adults, in obesity, and in malabsorption, chronic kidney disease and liver disease. Nutritional rickets remains frequent in infants and children in some regions.

Indications. Rickets or osteomalacia, bone pain or proximal muscle weakness, osteoporosis and fragility fracture, hypocalcaemia, raised alkaline phosphatase or parathyroid hormone, hypophosphataemia, malabsorption (coeliac disease, inflammatory bowel disease, pancreatic insufficiency, bariatric surgery), chronic kidney or liver disease, long term anticonvulsant, glucocorticoid or antiretroviral therapy, hypercalcaemia, and granulomatous disease.

Specimen. Serum or heparin plasma. Fasting is not needed. Stable for days at 2 to 8 °C. Levels are lower in winter by about 20 to 30%.

Conversion: ng/mL multiplied by 2.5 gives nmol/L. Different cut offs are used in different settings, and local ranges take precedence.

25-hydroxyvitamin D. Interpretation bands commonly used:

• Under 25 nmol/L (under 10 ng/mL): deficiency, with high risk of rickets and osteomalacia.
• 25 to 50 nmol/L (10 to 20 ng/mL): insufficiency, with risk of secondary hyperparathyroidism in people with low calcium intake.
• 50 to 125 nmol/L (20 to 50 ng/mL): adequate for most people. Some use 75 nmol/L as a target in osteoporosis and malabsorption.
• Over 125 nmol/L (over 50 ng/mL): beyond the range needed for bone health, with no added benefit.
• Over 250 nmol/L (over 100 ng/mL): potentially toxic. Hypercalcaemia usually appears over 375 nmol/L (150 ng/mL).

Raised: supplement excess, tanning, rarely contaminated foods, and (with normal results for 1,25-dihydroxyvitamin D) no disease. Lowered: reduced sun exposure, dark skin, covering, age, obesity, malabsorption, nephrotic syndrome (loss of vitamin D binding protein), liver disease, enzyme inducing drugs (phenytoin, carbamazepine, rifampicin), glucocorticoids, and acute illness.

1,25-dihydroxyvitamin D. Range: about 40 to 160 pmol/L. It is not a measure of status, since the level is held up by parathyroid hormone even in deficiency. It is requested when the cause of hypercalcaemia or hypocalcaemia needs clarifying. Raised: sarcoidosis and other granulomatous disease, lymphoma (extrarenal conversion), primary hyperparathyroidism, hypophosphataemia, and hereditary vitamin D-resistant rickets type 2 (very high).

Lowered: chronic kidney disease, hypoparathyroidism, vitamin D-dependent rickets type 1A, and tumour induced and X-linked hypophosphataemia (inappropriately normal).

Vitamin D deficiency with its effects.

• Low or low normal calcium, low phosphate, raised alkaline phosphatase and raised parathyroid hormone indicate osteomalacia or rickets.
• Raised parathyroid hormone with normal calcium and low 25-OH D indicates secondary hyperparathyroidism.
• Bone pain, proximal weakness, waddling gait, pseudofractures (Looser zones) and fragility fractures in adults.
• Rickets in children: delayed walking, bowed legs, widened wrists, rachitic rosary, craniotabes, hypocalcaemic seizures, and dilated cardiomyopathy in infants.

Vitamin D excess. 25-OH D over 375 nmol/L with hypercalcaemia, suppressed parathyroid hormone and hypercalciuria. Symptoms are those of hypercalcaemia: polyuria, thirst, constipation, confusion, nephrocalcinosis, and kidney injury. Levels fall slowly over weeks because of fat storage.

Hypercalcaemia with normal 25-OH D and raised 1,25-dihydroxyvitamin D. Sarcoidosis, tuberculosis, lymphoma, or inactivating CYP24A1 mutations (infantile and adult hypercalcaemia with raised 1,25-dihydroxyvitamin D and low parathyroid hormone).

Response to treatment. After starting replacement, 25-OH D is rechecked after at least 3 months, since earlier tests do not show the new steady state. Persistent low levels suggest non-adherence or malabsorption. Calcium, phosphate and alkaline phosphatase normalise within weeks to months in osteomalacia.

Critical values and findings needing immediate action.

• Corrected calcium under 1.9 mmol/L or ionised calcium under 0.9 mmol/L with tetany, seizures, laryngospasm, QT prolongation or cardiomyopathy.
• Corrected calcium over 3.0 mmol/L with raised or suppressed parathyroid hormone and a high 25-OH D, a signal of vitamin D toxicity needing hospital treatment.
• An infant with low 25-OH D and seizures or heart failure.

Interferences and pitfalls.

• Assays vary in accuracy, and immunoassays can read differently for D2 and D3 and differ from mass spectrometry by 10 to 20%. Serial results are best compared on the same method.
• Levels fall in acute illness, after major surgery and with inflammation, so a single value in a sick patient understates status.
• Vitamin D binding protein varies with ancestry, pregnancy, liver disease and nephrotic syndrome, which alters total 25-OH D without changing free hormone to the same degree.
• Seasonal variation can place the same person in two categories.
• Biotin can interfere with some immunoassays.
• Routine repeat testing in a patient on stable supplement is not needed.
• Obese patients need higher doses to reach the same level.

Special groups.

• Neonates and infants: cord 25-OH D is about two thirds of the maternal level, and exclusively breastfed infants of deficient mothers are at risk of rickets.
• Children: deficiency causes rickets, hypocalcaemic seizures and delayed growth.
• Pregnancy: deficiency is common, and severe deficiency causes neonatal hypocalcaemia.
• Older adults: reduced skin synthesis and sun exposure, and a higher risk of falls and fractures.
• People with dark skin or covering clothing at high latitude: deficiency is frequent.
• Chronic kidney disease: 25-OH D is still measured, and active vitamin D analogues are used in addition.
• Regions: deficiency also occurs in sunny settings because of covering and indoor life.

Follow up tests. Corrected calcium, albumin, phosphate, alkaline phosphatase, parathyroid hormone, magnesium, creatinine and eGFR, urinary calcium and creatinine, coeliac serology, faecal elastase, 1,25-dihydroxyvitamin D, FGF23 in hypophosphataemia, radiographs of wrists and knees in children, bone density scan, and CYP24A1 genetic testing for unexplained hypercalcaemia.