The Journal of Steroid Biochemistry and Molecular Biology
25-Hydroxyvitamin D requirement for maintaining skeletal health utilising a Sprague-Dawley rat model
Introduction
Vitamin D bioactivity is determined by the synthesis of the active metabolite, calcitriol (1,25D), a vitamin D specific nuclear transcription factor within target tissues. Vitamin D is activated by sequential hydroxylation reactions firstly to form 25-hydroxyvitamin D (25D) and secondly 1,25D. Activation of circulating 25D to 1,25D is catalysed by the renal cytochrome P450 enzyme 25D-1α-hydroxylase (CYP27B1) which is up-regulated by the action of parathyroid hormone (PTH) particularly in association with hypocalcaemia [1], [2], [3]. 1,25D production up-regulates transcription of the enzyme 25D-24-hydroxylase (CYP24) which catalyses the clearance of vitamin D metabolites. The regulation of both synthesis and catabolism of 1,25D in the kidney have equally important roles in modulating circulatory 25D levels [4].
To study the effects of vitamin D-depletion on bone health, we have developed an animal model to investigate the effects of frank vitamin D-deficiency as well as graded vitamin D-depletion on the skeleton. It is generally accepted that serum 25D levels between 100 and 150 nmol/L provide sufficient vitamin D to maintain healthy bone tissue. Some authors have suggested that levels above 50 nmol/L are adequate. Biochemical data provide clinical evidence that early stage vitamin D depletion occurs when circulating 25D levels fall below 50–75 nmol/L characterised by secondary hyperparathyroidism and increased bone turnover markers [5], [6], [7]. Frank vitamin D-deficiency gives rise to osteomalacia in adults and has been reported with circulating 25D levels of less than 10 nmol/L [8]. Under these conditions administration of intravenous calcium and oral phosphate can ameliorate most measures of bone health suggesting that vitamin D is required to normalise blood calcium and phosphate levels to maintain healthy bone [9], [10]. However, the ‘double knock-out’ VDR-null/CYP27B1-null mice demonstrated persistent bone growth deficiencies and decreased bone mineral even when normocalcemia was restored [11]. The mechanism by which vitamin D-depletion gives rise to osteoporosis remains controversial. By altering the dietary vitamin D intake in Sprague-Dawley rats, we can create suitable models of vitamin D depletion to investigate the roles of vitamin D in the maintenance of bone architecture and bone cell activity.
Section snippets
Animals
Male Sprague-Dawley pups (n = 41) were raised in an incandescent-lighted environment, and were maintained on a 1% calcium, 0.3% phosphorus semi-synthetic diet deficient in vitamin D (AIN Special vitamin D-deficient mixture, ICN, CA, USA) [12]. Animals raised to be vitamin D-replete (n = 6) were exposed to normal fluorescent lighting and were maintained on standard 1% calcium supplemented semi-synthetic diet (AIN-93-VX, ICN, CA, USA) containing 1000 IU (25 μg)/kg diet of vitamin D. All animals were
Serum biochemistry
Total serum calcium levels ranged from 2.45 nmol/L in animals fed on 50 ng of vitamin D/day to 2.96 nmol/L in animals fed on 500 ng of vitamin D/day in animals fed on 0.4% calcium (Table 1). No hypocalcemia was detected in any of the dietary treatment groups and serum PTH levels were not significantly induced in any of the groups. Serum phosphate levels ranged between 1.35 and 1.93 in these animals and showed no relationship with vitamin D intake. Serum ALP levels decreased with increasing dietary
Discussion
The procedure reported here describes the development of animals with circulating 25D levels strongly related to dietary vitamin D intake over a range of concentrations from severely depleted to replete levels. The dietary calcium level of 0.4% is sufficient to maintain normocalcemia without severe secondary hyperparathyroidism even at the zero vitamin D intake. Under these conditions serum 25D levels were determined by the dietary intake. Serum 1,25D levels were dependent on 25D levels until
References (17)
- et al.
Regulation of 25-hydroxyvitamin D3 1alpha-hydroxylase gene expression by parathyroid hormone and 1,25-dihydroxyvitamin D3
Arch. Biochem. Biophys.
(2000) Basal and parathyroid hormone induced expression of the human 25-hydroxyvitamin D 1alpha-hydroxylase gene promoter in kidney AOK-B50 cells: role of Sp1, Ets and CCAAT box protein binding sites
Int. J. Biochem. Cell Biol.
(2002)Determinants of circulating 1,25-dihydroxyvitamin D(3) levels: the role of renal synthesis and catabolism of vitamin D
J. Steroid Biochem. Mol. Biol.
(2004)Vitamin D status: effects on parathyroid hormone and 1,25-dihydroxyvitamin D in postmenopausal women
Am. J. Clin. Nutr.
(2000)The relationship between vitamin D and parathyroid hormone: calcium homeostasis, bone turnover, and bone mineral density in postmenopausal women with established osteoporosis
Bone
(2004)- et al.
The mineralization index—a new approach to the histomorphometric appraisal of osteomalacia
Bone
(2004) Inactivation of the 25-hydroxyvitamin D-1alpha-hydroxylase and vitamin D receptor demonstrates independent and interdependent effects of calcium and vitamin D on skeletal and mineral homeostasis
J. Biol. Chem.
(2004)- et al.
AIN-93 purified diets for laboratory rodents: final report of the American Institute of Nutrition ad hoc writing committee on the reformulation of the AIN-76A rodent diet
J. Nutr.
(1993)
Cited by (35)
Vitamin D supplementation improves bone mineralisation independent of dietary phosphate in male X-linked hypophosphatemic (Hyp) mice
2021, BoneCitation Excerpt :The vitamin D3 supplemented diet, containing 20,000 IU vitamin D3/kg of diet, was able to significantly increase serum 25(OH)D levels in Hyp mice to 72.8 ± 4.9 nmol/L when fed alone and 118.8 ± 8.6 when combined with high phosphate diet. These levels of serum 25(OH)D has previously been demonstrated in rats to be associated with improved bone mineralisation when compared to animals with reduced serum 25(OH)D levels [11,12]. Diet containing 20,000 IU vitamin D3/kg fed to normal mice has also been shown to improve bone mass and strength in normal mice [9], and has also been shown to increase serum 25(OH)D levels as high as 500 nmol/L as measured by immunoassay when fed to normal mice [13].
Bone development in growing female mice fed calcium and vitamin D at lower levels than is present in the AIN-93G reference diet
2018, Bone ReportsCitation Excerpt :While the aforementioned studies have altered the level of vit D or Ca, the effect of lowering both the level of dietary vit D and Ca in combination on BMD and bone structure has not been thoroughly studied. Previous literature has demonstrated that no effect on bone health occurs when Ca is lowered to 2.5 g/kg diet, at REF vit D level, (Hunt et al., 2008) and no differences in serum Ca and PTH occurs in the absence of vit D and 4 g Ca/kg diet (Anderson et al., 2007). By reducing both the levels of vit D and Ca, it is possible to avoid or attenuate compensatory mechanisms that may mask the effect of either low vit D or Ca when the other nutrient is provided in excess.
Long-term vitamin D deficiency in older adult C57BL/6 mice does not affect bone structure, remodeling and mineralization
2016, Journal of Steroid Biochemistry and Molecular BiologyCitation Excerpt :Therefore, the mice used in our study may have had a lower demand for minerals than rodents used in other studies and passive calcium absorption may have met the demand for calcium. Nevertheless, it has been shown that rats fed on a diet containing 0.4% calcium were able to maintain normal calcium levels [45]. Thus, the concentrations of calcium and phosphate in our diet may have been prevented the development of mineralization defects in older adult C57BL/6 mice.
Comparison of the biological effects of exogenous and endogenous 1,25-dihydroxyvitamin D<inf>3</inf> on the mature osteoblast cell line MLO-A5
2016, Journal of Steroid Biochemistry and Molecular BiologyCitation Excerpt :Biologically, exogenous and endogenous sources of 1,25D were observed to have similar effects on the activities of proliferation, mineralisation and induction of a range of genes by MLO-A5 osteoblasts under osteogenic conditions at markedly reduced levels of 1,25D. Significant differences of pharmacokinetics and pharmacodynamics of 1,25D are evident between these two sources particularly in terms of modulating gene expression for Cyp24a1 and other genes largely expressed by embedded osteoblasts/osteocytes suggesting that endogenously synthesised 1,25D is more efficiently utilised by the differentiating osteoblast. These findings are consistent with numerous previous studies suggesting that autocrine synthesis of 1,25D in osteoblast lineage cells plays key roles in their activities [5,6,9,19–22]. Our findings also further indicate that the MLO-A5 cell line is a useful tool to further study the differential roles of endogenous and exogenous sources of 1,25D in bone homeostasis.
Vitamin D receptor overexpression in osteoblasts and osteocytes prevents bone loss during vitamin D-deficiency
2014, Journal of Steroid Biochemistry and Molecular BiologyCitation Excerpt :Our data indicate that a serum 25D levels greater than 26 nmol/L is required to maintain bone volume in WT mice under these circumstances. We have previously shown that serum 25D levels are an important determinant of trabecular bone mineral volume in rats when fed an adequate level of dietary calcium, and only when serum 25D levels were above 80 nmol/L was peak bone mineral volume achieved [4,5]. More recently, we also showed that in the presence of a low calcium diet and high 1,25D levels, peak bone mineral volume could not be achieved despite high serum 25D levels [24].
Target Genes: Bone proteins
2011, Vitamin D: Two-Volume Set