1,25-Dihydroxyvitamin D is not responsible for toxicity caused by vitamin D or 25-hydroxyvitamin D
Research highlights
► 1,25-Dihydroxyvitamin D is not responsible for vitamin D intoxication. ► 25-Hydroxyvitamin D is likely responsible for vitamin D3 intoxication. ► CYP27B1 deletion does not reduce or enhance vitamin D intoxication.
Introduction
Vitamin D intoxication first appeared following the preparation of synthetic vitamin D2 [1] and vitamin D3 [2]. Although vitamin D is normally produced in skin by ultraviolet (UV)1 photolysis of 7-dehydrocholesterol to produce vitamin D3, there is no report of vitamin D intoxication by UV irradiation. Clearly, the production of vitamin D3 in skin by UV is limited by one of several suggested mechanisms [3]. Certainly toxicity by vitamin D occurs with the administration of fish liver oils, a natural source with sufficient concentrations to produce intoxication [4]. The symptoms of vitamin D intoxication are well known and are the result of hypercalcemia, hyperphosphatemia, and a high calcium/phosphorus product in the serum [5], [6]. The symptoms of vitamin D intoxication are thirst, itchiness, diarrhea, malaise, wasting, polyuria and diminished appetite resulting from primarily renal failure [5], [6], [7], [8]. Calcification following vitamin D intoxication has been found in a large number of tissues but especially kidney, aorta, heart, lung, and subcutaneous tissue [4], [5], [6]. Toxicity of vitamin D beyond hypercalcemia and mineralization is unknown and high levels of vitamin D are tolerated by patients who are resistant to vitamin D, as for example in vitamin D-dependent rickets type 2 or X-linked hypophosphatemic rickets, where high levels of vitamin D compounds are used [9], [10]. These findings suggest that vitamin D toxicity results not from the vitamin D molecule itself but from the elevations of plasma calcium and phosphorus to un-physiological, high levels.
Physiologically, vitamin D is not biologically active itself and must first be hydroxylated in the liver very likely by the CYP2R1 to produce the plasma form of vitamin D, 25-hydroxyvitamin D (25(OH)D) [11]. This compound at physiologic concentrations is biologically inactive and must be converted further to an active hormonal form, 1,25-dihydroxyvitamin D (1,25(OH)2D) to carry out the physiologic functions of vitamin D [12]. Final hydroxylation of vitamin D is carried out in the proximal convoluted tubule cells of the kidney by CYP27B1, an enzyme that is tightly regulated by the need for calcium and phosphorus [12]. The need for calcium is translated by the parathyroid glands that secrete parathyroid hormone (PTH) in response to the need for calcium. The PTH, in a mechanism not fully understood, induces the CYP27B1 gene to produce the active enzyme that carries out the 1α-hydroxylation [13]. As plasma calcium rises, the production of the vitamin D hormone is shut down by halting the secretion of PTH and by feedback suppression of the preproparathyroid gene [14], [15], [16]. Although low blood phosphate also stimulates expression of the CYP27B1 gene, its effect is not as dramatic as the serum calcium/parathyroid axis [17], [18]. On the other hand, hydroxylation of 25(OH)D appears to be relatively unregulated in which large doses of vitamin D are readily converted to the 25(OH)D as illustrated by the work of Shephard & DeLuca [19]. In those experiments and others, plasma levels of 1,25(OH)2D appeared to fall with increasing doses of vitamin D presumably because the 1-hydroxylase system is shut down as PTH secretion is suppressed [12]. Vitamin D toxicity has been claimed to be the result of high levels of 25(OH)D dislodging the plasma-bound 1,25(OH)2D giving rise to high levels of free 1,25(OH)2D3 that are in turn toxic [5], [20]. The possible mechanisms of vitamin D toxicity have been suggested and have not been resolved [5], [6].
Recently, several groups have produced CYP27B1 knockout (KO) mice [21], [22], [23]. In our group, the CYP27B1 KO mouse was produced by substituting the galactosidase gene for the CYP27B1 coding sequence [23]. This mouse has no CYP27B1 activity and is unable to produce 1,25(OH)2D. Using this tool, we have now been able to demonstrate that intoxication with vitamin D3 occurs equally well in the CYP27B1 KO mouse as it does in the wild type. This allows the conclusion that the real toxicant in vitamin D intoxication is not 1,25(OH)2D. It also makes untenable the “free” 1,25(OH)2D hypothesis of vitamin D intoxication. This paper presents the experiments leading to that conclusion.
Section snippets
Animals
The development of the CYP27B1 (−/−) mice has been described [19]. Heterozygote pairs were used to generate the CYP27B1 −/− homozygotes. PCR analysis was used to confirm the genotype. Breeders were maintained on Purina chow #5015. Weanlings were maintained on the chow for 1 week as the genotyping was performed. Confirmed homozygotes were then grown for 3 weeks on a purified diet 11 (0.47% calcium, 0.3% phosphorus) [24] supplemented with 4 ng 1,25(OH)2D3 per mouse/day dissolved in the Wesson oil of
Results
Both wild-type and the mutant mice tolerated doses of up to 0.625 mg vitamin D3/kg body weight since they continued to grow throughout the 5-week period (Fig. 1). However, doses of 10.0 and 5.0 mg/kg were clearly toxic in both the wild-type and mutant mice. Since they had lost more than 20% of their body weight by 3 weeks, they were terminated. The intermediate dose levels inhibited growth but the animals were able to maintain weight throughout the 5-week period. The serum calcium data shown in
Discussion
The most potent form of vitamin D is 1,25(OH)2D3, the hormone produced by the CYP27B1 in the proximal convoluted tubule cells of the kidney [27]. It is, therefore, possible that this compound could be responsible for vitamin D intoxication especially since it is very toxic when directly administered [28]. However, the activity of CYP27B1 is tightly regulated and as serum calcium rises, parathyroid hormone is suppressed and parathyroid hormone is the chief activator of the CYP27B1 system [12].
Acknowledgment
This work was funded by the Wisconsin Alumni Research Foundation.
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