Serum Levels of Free 1,25-Dihydroxyvitamin D in Vitamin D Toxicity

  1. John M. Pettifor, MBBCH, PhD;
  2. Daniel D. Bikle, MD, PhD;
  3. Meropi Cavaleros, MT;
  4. Dianne Zachen, MT;
  5. Mahomed C. Kamdar, MBBS; and
  6. Frederick P. Ross, PhD
  1. From the University of the Witwatersand, Johannesburg, South Africa. The University of California, San Francisco, California. The University of Natal, Durban, South Africa. Requests for Reprints: John M. Pettifor MBBCh, Mineral Metabolism Research Unit, Department of Pediatrics, Baragwanath Hospital, P.O. Bertsham 2013, South Africa. Acknowledgments: The authors thank Dr. I. Jialal, Dr. M.C. Rajput, and Dr. A.R. Seebaran for their assistance and Dr. P.K. Naidoo, medical superintendent of the R.K. Khan Hospital, Chatsworth, South Africa, for permission to publish. Grant Support: By a grant from the South African Medical Research Council.
     
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    Abstract

    Objective: To determine the serum level of free 1,25-dihydroxyvitamin D (1,25-[OH]2D) in patients with vitamin D toxicity and to assess the in vitro effect of differing concentrations of vitamin D metabolites on the free serum levels of 1,25-(OH)2D.

    Design: 1] A case study of patients hospitalized with vitamin D toxicity after accidentally ingesting a veterinary vitamin D concentrate and 2) an in vitro experiment in which vitamin D metabolites in various concentrations were added to normal serum and their effect was noted on percentage of free 1,25-(OH)2D.

    Patients: 11 patients (age range, 8 to 69 years) were studied 10 to 40 days after hospitalization for hypercalcemia.

    Measurements: Serum total 25-hydroxyvitamin D (25-OHD) and 1,25-(OH) (2D) levels were measured by radioreceptor assays. The percentage of free 1,25-(OH)2D was measured by centrifugal ultrafiltration isodialysis and was used to calculate actual free 1,25-(OH)2D levels. In the in vitro studies, vitamin D metabolites (25-OHD; 24,25-[OH]2D; 25,26-[OH]2D; and 25-OHD-26,23 lactone) were added to normal serum in concentrations expected to occur with vitamin D toxicity. The percentage of free 1,25-(OH) (2D) was measured by isodialysis.

    Results: All patients presented with marked hypercalcemia (mean calcium level, 3.99 ±0.33 mmol/L). Serum 25-OHD levels ranged from 847 to 1652 nmol/L, and total 1,25-(OH)2D levels (mean, 106 ±86 pmol/L) were elevated in only three patients. The percentage of free 1,25-(OH)2D (mean, 1.023% ±0.366%) was elevated in all nine patients in whom it was measured. Actual free 1,25-(OH)2D levels (mean, 856 ±600 fmol/L) were elevated in six of the nine patients. Total 1,25-(OH)2D levels were correlated with 25-OHD levels (r = 0.66; P = 0.03), whereas total and free 1,25-(OH)2D levels were highly correlated (r = 0.957; P < 0.001). In the in vitro studies, the percentage of free 1,25-(OH)2D increased after 25-OHD or 24,25-(OH)2D was added.

    Conclusions: Although the patients had normal or near-normal total 1,25-(OH)2D values, most patients had elevated free 1,25-(OH)2D levels. These findings suggest that elevated free 1,25-(OH)2D levels might play a role in the pathogenesis of hypercalcemia in vitamin D toxicity.

    Vitamin D toxicity in humans is characterized by markedly elevated serum levels of 25-hydroxyvitamin D (25-OHD) [1-5]. However, levels of the active metabolite 1,25-dihydroxyvitamin D (1,25-[OH]2D) have been reported to be either normal to decreased [4, 5] or elevated [1, 2, 6]. In rats, experimentally induced vitamin D toxicity leads to a more than 10-fold increase in circulating 25-OHD levels but to a 37% decrease in 1,25-(OH)2D levels [7].

    The absence of consistently elevated levels of 1,25-(OH)2D in vitamin D toxicity has led to the suggestion that metabolites other than 1,25-(OH)2D might be responsible for the hypercalcemia and hypercalciuria [8, 9]. The high 25-OHD levels might directly interact with the vitamin D receptor in the intestine and bone [7]. Vieth [8] has suggested that the high levels of 25-OHD could cause an increase in free levels of 1,25-(OH)2D (despite normal total levels), which might be responsible for the changes in calcium homeostasis.

    We recently tested this hypothesis by measuring the free levels of 1,25-(OH)2D in patients who were evaluated for vitamin D toxicity after inadvertently using a vitamin D concentrate as a cooking oil. We also did in vitro studies using mixtures of various vitamin D metabolites to simulate the vitamin D toxic state so that we could assess their effect on the percentage of free 1,25-(OH)2D in normal serum.

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    Methods

    Patients

    Ten members of a family and their domestic maid (age range, 8 to 69 years) were hospitalized during a 10-day period after unintentionally using a veterinary vitamin D concentrate (cholecalciferol in peanut oil; 2 million U/g) as a cooking oil.

    All patients presented with abdominal cramps, vomiting, and neurologic symptoms and were found to have severe hypercalcemia (Table 1). Although they were treated with a combination of intravenous fluids, diuretics, corticosteroids, and calcitonin, four patients died from related complications.

    View this table:
    Table 1. Serum Biochemical Values in the 11 Patients Hospitalized with Vitamin D Toxicity*

    Laboratory Evaluation

    Blood samples for the estimation of vitamin D metabolite concentrations were obtained between 10 and 40 days after admission while the patients were receiving therapy for hypercalcemia. Free levels of 1,25-(OH)2D were not measured in two patients because of insufficient serum volumes. Serum total 25-OHD and 1,25-(OH)2D levels were measured by the kidney cytosol [10] and calf thymus receptor [11] assays, respectively. All the specimens were measured in one assay. The intra-assay coefficients of variation were 1.1% for 25-OHD and 16.0% for 1,25-(OH)2D.

    The percentage of free 1,25-(OH)2D was measured using centrifugal ultrafiltration isodialysis [12, 13]. The intra-assay variation was 13%. We calculated the free levels of 1,25-(OH)2D using the following formula:

    Free 1,25-(OH)2D (fmol/L) = percentage of free

    1,25-(OH)2D x total 1,25-(OH)2D level (pmol/L) × 10.

    We measured serum levels of vitamin D-binding protein (DBP) by radial immunodiffusion [14] and used a rabbit antihuman DBP antibody and purified human DBP as the standard.

    In the in vitro studies, we determined the ability of exogenously added vitamin D metabolites to alter the percentage of free 1,25-(OH)2D by adding the desired amount of metabolites with the tracers Hydrogen-3-1,25-(OH)2D and Carbon-14-glucose to the incubation tube, removing the vehicle by drying it under a nitrogen stream, and then adding serum (0.45 mL) for a 45-minute incubation period at 37 °C. Duplicate 0.2-mL aliquots from each incubate were then subjected to centrifugal ultrafiltration as previously described [12, 13]. Each incubation was done in duplicate, and the results are reported as the mean ±range. The serum sample used in the in vitro study was obtained from a normal control; it contained 100 nM of 25-OHD and 85 pM of 1,25-(OH)2D. The 25-OHD3-26,23 lactone was provided by Dr. Ronald Horst (Ames, Iowa); the other vitamin D metabolites were provided by Dr. Milan Uskokovic (Hoffman-LaRoche, Nutley, New Jersey).

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    Results

    The results of the relevant investigations are shown in Table 1. At hospital admission, all patients had markedly elevated serum calcium values. In keeping with the typical presentation of vitamin D toxicity, most patients also had increased serum inorganic phosphate levels and urea concentrations. Levels of 25-OHD ranged from 847 to 1652 nmol/L, which are 8 to 15 times greater than the upper limit of normal. Total 1,25-(OH)2D levels were elevated in 3 of the 11 patients, and the percentages of free 1,25-(OH)2D were more than two standard deviations above the reference mean in all 9 patients in whom it was measured. The free levels of 1,25-(OH) (2D) were also more than two standard deviations above the reference mean in 6 of the 9 patients.

    The free levels of 1,25-(OH)2D correlated with the total 1,25-(OH)2D levels (r = 0.957; P < 0.001), which in turn correlated with 25-OHD levels (r = 0.660; P = 0.027). However, neither the free levels of 1,25-(OH)2D nor the percentage of free 1,25-(OH)2D were correlated with DBP or albumin concentrations. Serum calcium values measured when blood samples were obtained for vitamin D metabolites did not correlate with the free 1,25-(OH)2D; total 1,25-(OH)2D; or 25-OHD levels.

    The results of the in vitro studies on the effect of adding various metabolites to a control serum sample on the percentage of free 1,25-(OH)2D are shown in Table 2. When 25-OHD was added in concentrations similar to those found in the patients, the percentage of free 1,25-(OH)2D increased from 0.312% to 0.557%. A smaller but consistent increase in the percentage of free 1,25-(OH)2D was noted when 24(R),25-(OH)2D was added to the serum sample. No effect was noted when 25-OHD-26,23 lactone or 25(R),26-(OH)2D was added. The addition of a combination of 25-OHD; 25-OHD-26,23 lactone; 24(R),25-(OH)2D; and 25,26-(OH)2D in concentrations that might be found in vitamin D toxicity did not increase the percentage of free 1,25-(OH)2D more than did the addition of 25-OHD alone.

    View this table:
    Table 2. Effect of Adding Various Concentrations of Vitamin D Metabolites on the Percentage of Free 1,25-(OH)2D*
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    Discussion

    As in previous studies [1-4, 6], 25-OHD levels were consistently and markedly elevated in the patients with vitamin D toxicity. In our study, most total 1,25-(OH)2D levels were within the normal range. Elevated values were only marginally increased, particularly if the ages of the patients are considered. These findings are in agreement with those of several studies [2, 4, 5, 7] in which vitamin D toxicity was associated with a normal or low 1,25-(OH)2D level, but they do not support the findings of Mawer and colleagues [1], who reported elevated 1,25-(OH) (2D) levels.

    The inability to document elevated 1,25-(OH)2D levels in most patients with vitamin D toxicity has led to many hypotheses about the cause of the hypercalcemia, such as a direct action of 25-OHD on the 1,25-(OH)2D receptor [1, 7] and an increase in free levels of 1,25-(OH)2D caused by the displacement of 1,25-(OH)2D from DBP by the high 25-OHD levels [8]. The elevated free 1,25-(OH)2D levels in most patients in our study might support the latter hypothesis.

    The presence of elevated free 1,25-(OH)2D levels despite normal total 1,25-(OH)2D levels suggests that 1,25-(OH)2D is displaced from DBP by the micromolar concentrations of 25-OHD and other unmeasured metabolites (such as 25-OHD-26,23 lactone; 24,25-[OH]2D; and 25,26-[OH]2D) [7]. Concentrations of DBP are approximately 5 × 10 (−6) M, and the vitamin D metabolites appear to bind to DBP in a 1:1 molar ratio and to compete for the same site. However, this hypothesis has not been rigorously tested. Thus, as the vitamin D metabolite concentrations approach those of DBP, the level of saturation of DBP binding reaches a point where the percentage of bound metabolites decreases in a clinically significant manner [13, 15].

    The above hypothesis is supported in part by our in vitro studies. When 25-OHD was added to normal serum in the same concentrations (800 nM) as those recorded in the patients, the percentage of free 1,25-(OH) (2D) increased by 78%. Surprisingly, doubling the concentration of added 25-OHD did not further increase the percentage of free 1,25-(OH)2D. The addition of 24,25-(OH)2D also increased the percentage in a dose-dependent manner, but the percentage was not affected when we added 25-OHD-26,23 lactone or 25,26-(OH)2D in the concentrations reported by Horst and colleagues [16, 17] to occur in pigs given large doses of vitamin D. In our in vitro experiments, a combination of various metabolites did not increase the percentage of free 1,25-(OH)2D more than did the addition of 25-OHD alone, and it did not increase the percentage to the levels found in the patients. An explanation for these discrepancies is unclear, although it is possible that in vitamin D toxicity, other metabolites such as vitamin D itself might influence the binding of 1,25-(OH)2D to DBP.

    In conclusion, we found that patients with vitamin D toxicity had elevated free 1,25-(OH)2D levels despite normal or only marginally elevated total 1,25-(OH)2D levels. The increased free levels of 1,25-(OH)2D might contribute to the pathogenesis of hypercalcemia in vitamin D toxicity.

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    1. Ann Intern Med April 1, 1995 vol. 122 no. 7 511-513
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