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1 April 1995 | Volume 122 Issue 7 | Pages 511-513
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.
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.
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. BRIEF COMMUNICATION
Serum Levels of Free 1,25-Dihydroxyvitamin D 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].
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Patients
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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) x 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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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.
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Discussion
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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 x 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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References
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1. Mawer EB, Hann JT, Berry JL, Davies M. Vitamin D metabolism in patients intoxicated with ergocalciferol. Clin Sci. 1985; 68:135-41.
2. Hughes MR, Baylink DJ, Jones PG, Haussler MR. Radioligand receptor assay for 25-hydroxyvitamin D2/D3 and 1 
, 25-dihydroxyvitamin D2/D3. J Clin Invest. 1976; 58:61-70.
3. Counts SJ, Baylink DJ, Shen FH, Sherrard DJ, Hickman RO. Vitamin D intoxication in an anephric child. Ann Intern Med. 1975; 82:196-200.
4. Mason RS, Lissner D, Grunstein HS, Posen S. A simplified assay for dihydroxylated vitamin D metabolites in human serum: application to hyper- and hypovitaminosis D. Clin Chem. 1980; 26:444-50.
5. Jacobus CH, Holick MF, Shao Q, Chen TC, Holm IA, Kolodny JM, et al. Hypervitaminosis D associated with drinking milk. N Engl J Med. 1992; 326:1173-7.
6. O'Riordan JLH, Adami S, Sandler LM, Clemens TL, Fraher LJ. Clinical application of radioimmunoassays for vitamin D metabolites. In: Norman AW, Schaefer K, Herrath D, Grigoleit HG, eds. Vitamin D: Chemical, Biochemical and Clinical Endocrinology of Calcium Metabolism. Berlin: Walter de Gruyter; 1982:751-6.
7. Shephard RM, Deluca HF. Plasma concentrations of vitamin D3 and its metabolites in the rat as influenced by vitamin D3 or 25-hydroxyvitamin D3 intakes. Arch Biochem Biophys. 1980; 202:43-53.
8. Vieth R. The mechanisms of vitamin D toxicity. Bone Miner. 1990; 11:267-72.
9. Chapuy MC, Meunier PJ. Metabolic basis of vitamin D intoxication. In: Cohen RD, Lewis B, Alberti KG, Denman AM, eds. The Metabolic and Molecular Basis of Acquired Disease. London: Bailliere Tindall; 1990:1824-34.
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11. Reinhardt TA, Horst RL, Orf JW, Hollis BW. A microassay for 1,25-dihydroxyvitamin D not requiring high performance liquid chromatography: application to clinical studies. J Clin Endocrinol Metab. 1984; 58:91-8.
12. Bikle DD, Gee E, Halloran B, Haddad JG. Free 1,25-dihydroxyvitamin D levels in serum from normal subjects, pregnant subjects, and subjects with liver disease. J Clin Invest. 1984; 74:1966-71.
13. Bikle DD, Siiteri PK, Ryzen E, Haddad JG. Serum protein binding of 1,25-dihydroxyvitamin D: a reevaluation by direct measurement of free metabolite levels. J Clin Endocrinol Metab. 1985; 61:969-75.
14. Bouillon R, van Baelen H, de Moor P. The measurement of vitamin D-binding protein in human serum. J Clin Endocrinol Metab. 1977; 45:225-31.
15. Bikle DD, Gee E, Halloran B, Kowalski MA, Ryzen E, Haddad JG. Assessment of the free fraction of 25-hydroxyvitamin D in serum and its regulation by albumin and the vitamin D-binding protein. J Clin Endocrinol Metab. 1986; 63:954-9.
16. Horst RL. 25-OHD3-26,23-lactone: a metabolite of vitamin D3 that is 5 times more potent than 25-OHD3 in the rat plasma competitive protein binding radioassay. Biochem Biophys Res Commun. 1979; 89:286-93.
17. Horst RL, Littledike ET, Gray RW, Napoli JL. Impaired 24,25-dihydroxyvitamin D production in anephric human and pig. J Clin Invest. 1981; 67:274-80.
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