Relative Value of 25(OH)D and 1,25(OH)2D Measurements

Vitamin D status and metabolism is characterized by many circulating metabolites. Serum 25-hydroxyvitamin D [25(OH)D], 1,25-dihydroxyvitamin D [1,25(OH)2D], and 24,25-dihydroxyvitamin D [24,25(OH)2D] have been assessed in many epidemiological studies.1 The first two metabolites are of major interest for the assessment of vitamin D status and evaluation of vitamin D metabolism. The major circulating metabolite is 25(OH)D, whereas the active metabolite is 1,25(OH)2D, which is responsible for most actions of vitamin D. The production of 25(OH)D may be limited in severe liver disease. The hydroxylation to 1,25(OH)2D is more often restricted by kidney disease or decrease of renal function, as occurs with aging. The production of 24,25(OH)2D is induced by 1,25(OH)2D and is part of the catabolic pathway. The characteristics of both metabolites are presented in Table 1. The concentration of 25(OH)D is almost 1000-fold of that of 1,25(OH)2D, and the half-life of 25(OH)D is much longer, implying that its concentration is more stable. Serum 25(OH)D is decreased by thyroid hormone, anticonvulsants, cholestyramine, and orlistat, but otherwise stable.2 The concentration of 1,25(OH)2D is directly influenced by serum calcium and phosphate, calcium intake, and immobility.3 Serum 1,25(OH)2D is increased by PTH and PTH-related peptide, prolactin, estradiol, testosterone, prostaglandins, and bisphosphonates and decreased by corticosteroids, phosphatonin, ketoconazole, heparin, and thiazides.2 The stimulation by other hormones and negative feedback loops classify it as a hormone. Epidemiological studies in older persons where both metabolites have been assessed have shown that 1,25(OH)2D is better kept within reference limits than 25(OH)D.1 A study in patients with hip fracture and controls of similar age4 showed that very low levels of 25(OH)D were very common in the patients and even in controls, whereas subnormal levels of 1,25(OH)2D were only observed in part of the patients and not in the controls (Fig. 1). The low levels of 1,25(OH)2D in these patients were partly caused by decreased renal function but also to severe vitamin D deficiency, because the synthesis of 1,25(OH)2D becomes substrate-dependent, as can be concluded from the positive correlation between serum 25(OH)D and 1,25(OH)2D in the patients (r = 0.49, p < 0.001) and not the controls.4 This indicates that serum 25(OH)D is the measurement of choice to diagnose vitamin D deficiency and to assess vitamin D status. On the other side, serum 1,25(OH)2D is not suitable to assess vitamin D status because it is kept within reference limits as long as possible by hormonal mechanisms, especially PTH for stimulation and serum calcium and phosphate for suppression. Serum 25(OH)D should not be considered in isolation but should be accompanied by clinical information and by serum calcium, phosphate, albumin, creatinine, and alkaline phosphatase levels and, when feasible, by a 24-h urine calcium excretion. On the other hand, a 24-h urine collection is a nuisance to the patient and often inaccurate. Serum 25(OH)D and 1,25(OH)2D concentrations in 125 patients with hip fracture and 74 controls of similar age. The measurements were done by high-performance liquid chromatography followed by competitive protein binding assays for 25(OH)D and 1,25(OH)2D, respectively. The solid line for 25(OH)D indicates the threshold for deficiency, and below the dashed line, serum 25(OH)D levels are insufficient. The solid line for 1,25(OH)2D is the lower reference limit (adapted from Lips et al.4; thresholds for deficiency and insufficiency of serum 25(OH)D and for deficiency of serum 1,25(OH)2D adapted from references 1 and 33). Serum 25(OH)D is relatively stable and not directly influenced by the diet (e.g., calcium intake) and life style (e.g., mobility). Serum 25(OH)D should be assessed in persons at risk for vitamin D deficiency or insufficiency, including nutritional causes, malabsorption (e.g., celiac disease or inflammatory bowel disease), and patients with renal disease (e.g., the nephrotic syndrome when it is lost with the urine) (Table 2). Older persons and patients with osteoporosis1, 5 are at risk, as well as small children and pregnant women. A special risk group are immigrants from southern countries to more temperate regions. Very low serum 25(OH)D levels were observed in pregnant immigrant women from the Middle East and African countries in The Hague in The Netherlands.6 More than 80% of Turkish and Moroccan women had levels <25 nM, and in ∼10%, serum 25(OH)D was not detectable.6 A low serum 25(OH)D is usually accompanied by an increase of serum PTH, but the latter may still be in the normal range. On the other hand, immobility can lower serum PTH, even in case of vitamin D deficiency. Another reason to measure serum 25(OH)D is in hypercalcemic patients when there is a suspicion of vitamin D intoxication. This may occur with over-the-counter drugs, fortification errors, or too high doses for a prolonged period.7-10 During vitamin D intoxication, serum 25(OH)D is very high, typically >200 nM, and serum 1,25(OH)2D usually is normal. The commercial immunoassays for 25(OH)D show interassay CVs of ∼10%, but the interassay CV may be considerably higher (up to 25%) in the low range (e.g., <15 nM). This should be kept in mind when following an individual patient over time. The assessment of serum 25(OH)D may still show a considerable interlaboratory variation,11 even when programs have been established for standardization and quality control. Another problem is the difference between assays in affinity for vitamin D3 and vitamin D2 metabolites.12 Some assays may detect both metabolites to a similar degree, but other assays may only detect vitamin D3 metabolites. This is not a problem in countries where vitamin D2 is very uncommon (e.g., The Netherlands), but it may lead to the false diagnosis of vitamin D deficiency when food is fortified with vitamin D2 or supplements contain this variant. Whereas most clinicians agree that a serum 25(OH)D < 25 nM (10 ng/ml) represents vitamin D deficiency, opinions vary on what should be considered an adequate serum 25(OH)D level. A consensus panel in 2005 concluded that an adequate serum 25(OH)D should be >50–80 nM according to different opinions.13 In this review, serum 25(OH)D <25 nM is considered vitamin D deficiency, and a level between 25 and 50 nM is considered vitamin D insufficiency (Fig. 1).1, 14 More recently, serum 25(OH)D levels >80–100 nM were advocated. These high levels are based on the observations that serum PTH is still decreasing when serum 25(OH)D increases to 100 nM. However, these observations are from population-based studies, and the assessment of serum PTH is not helpful in the individual patient. When serum 25(OH)D is low, serum PTH is relatively high, but often still in the normal range, and the increase of serum PTH is blunted in many patients (i.e., functional hypoparathyroidism). The increase of serum PTH in vitamin D-deficient patients depends on renal function, and the increase is greater when glomerular filtration rate is lower.15 Another factor causing functional hypoparathyroidism in patients with vitamin D deficiency is magnesium deficiency.16 In that case, serum PTH did increase in patients with vitamin D deficiency after a magnesium loading test. In case of immobile patients with vitamin D deficiency, serum PTH may be low because of bone loss and an increase of serum calcium.17 The sensitivity to the action of vitamin D may differ between individuals depending on the genetic profile (e.g., vitamin D receptor [VDR] polymorphisms).18 Vitamin D supplementation and serum 25(OH)D show a dose-response curve, but the increase of serum 25(OH)D depends on baseline serum 25(OH)D and on the vitamin D dose. With a baseline serum 25(OH)D of 25 nM, a daily vitamin D supplement of 400 IU (10 μg) increases mean serum 25(OH)D to >60 nM, and a dose of 800 IU (20 μg) increases mean serum 25(OH)D to ∼80 nM.19 The increase is less when baseline serum 25(OH)D is higher. Adequate serum 25(OH)D levels are particularly important in risk groups (e.g., the elderly and patients with chronic kidney disease).1 Whereas mean serum 25(OH)D levels in independent elderly are ∼40–50 nM, serum 25(OH)D in institutionalized elderly is much lower, ∼20–30 nM.1 The active metabolite, serum 1,25(OH)2D, should be measured in case of disorders of 1α-hydroxylation in the kidney. This is most common in chronic renal failure, and the average serum 1,25(OH)2D level drops gradually with the decrease of the glomerular filtration rate.20 Other compelling reasons to assess serum 1,25(OH)2D are absence of 1α-hydroxylase in the kidney in vitamin D-dependent rickets type 121 or decrease (or sometimes increase) of this enzyme in hypophosphatemic rickets (Table 3).22 Treatment with physiological doses of 1,25(OH)2D leads to normalization of the serum 1,25(OH)2D levels in these patients. Inborn errors of the VDR in vitamin D-dependent rickets type 2 cause a postreceptor defect and are associated with very high serum 1,25(OH)2D levels.23 Hypercalcemia may be caused by an inappropriate increase of serum 1,25(OH)2D in granulomatous diseases, such as sarcoidosis,24, 25 tuberculosis, and inflammatory bowel disease. In these diseases, 1α-hydroxylation occurs in extrarenal tissues, in macrophages, and in granulomas. Hypercalcemia and increased serum 1,25(OH)2D have also been described in a HIV-infected patient because of disseminated Mycobacterium avium infection.26 On the other hand, HIV-protease inhibitors may impair 1α-hydroxylation, leading to low BMD in HIV patients.27 Extrarenal formation of 1,25(OH)2D also occurs in rheumatoid arthritis and in lymphoproliferative diseases.28 Whereas the renal hydroxylation of 25(OH)D into 1,25(OH)2D is tightly regulated by feedback control, the extrarenal hydroxylation by activated macrophages is not. In these diseases, a positive correlation is observed between serum 25(OH)D and 1,25(OH)2D,28 whereas in normal circumstances, it is not. Treatment with surgery, chemotherapy, or glucocorticoids leads to a quick fall of serum 1,25(OH)2D and resolution of the hypercalcemia.29, 30 In patients with severe vitamin D deficiency, the formation of 1,25(OH)2D may be limited because of lack of substrate. In such a population, a positive correlation between serum 25(OH)D and 1,25(OH)2D may also be observed.4, 5, 31 A seasonal increase of serum 1,25(OH)2D may accompany the increase of serum 25(OH)D in spring in older vitamin D-deficient persons.31 When serum 1,25(OH)2D is low, in case of severe vitamin D deficiency, hypocalcemia, hypophosphatemia and an elevated alkaline phosphatase are usually observed. In case of severe vitamin D deficiency, vitamin D therapy may cause an increase of serum 1,25(OH)2D.19, 32 The threshold serum 25(OH)D where vitamin D therapy still may increase 1,25(OH)2D may be identified by repeated measurements of serum 1,25(OH)2D. This may play a role in the establishment of the minimally required serum level of 25(OH)D. For the diagnosis of hypercalcemia, the measurement of both metabolites may be necessary, when vitamin D intoxication or extrarenal conversion of 25(OH)D into 1,25(OH)2D are considered. The commercial immunoassays for 1,25(OH)2D vary with regard to specificity and may measure 1α-hydroxylated inactive metabolites apart from 1,25(OH)2D.12 This may be a problem in various metabolic diseases besides chronic kidney disease. The results of measurement of serum 25(OH)D in the same sample may vary up to 30% or more between different laboratories. Standardization of the commercial assays for serum 25(OH)D is necessary. Some assays may measure 25(OH)D3 better than 25(OH)D2.12 This is not a problem when the only source of vitamin D is sunshine or when only vitamin D3 is available as addition to nutrition or as vitamin D supplement, as is the case in The Netherlands. However, when vitamin D2 supplements are an important vitamin D source, the assay should measure both metabolites to a similar degree. A discussion is ongoing on what level of 25(OH)D should be required for bone health and the prevention of other vitamin D-related diseases. A gold standard for the 25(OH)D assay is needed to establish consensus on the required level for serum 25(OH)D. The routine assays for 1,25(OH)2D vary even more than those for 25(OH)D, and these assays should also be standardized. Serum 25(OH)D should be measured to assess nutritional vitamin D status and to diagnose vitamin D deficiency and insufficiency and to check the effect of treatment. Risk groups include young children, pregnant women, older persons, immigrants, and persons with specific diseases causing decreased absorption or increased loss of vitamin D. Serum 25(OH)D should also be measured to diagnose vitamin D intoxication. Serum 1,25(OH)2D should be measured in disorders of 1α-hydroxylation in the kidney or in extrarenal tissues causing decreased or increased production of 1,25(OH)2D. In the latter case, it plays a role in the diagnosis of hypercalcemia. Serum 1,25(OH)2D should also be assessed when vitamin D resistance or insensitivity is suspected as in case of genetic defects of the VDR. The measurement of serum 1,25(OH)2D also plays an important role in research [e.g., when assessing effects of 1,25(OH)2D].