25-Hydroxyvitamin D requirement for maintaining skeletal health utilising a Sprague-Dawley rat model

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Abstract

To study the role of vitamin D to optimise bone architecture, we have developed an animal model to investigate the effects of frank vitamin D-deficiency as well as graded depletion of circulating 25-hydroxyvitamin D3 (25D) levels on the skeleton. Rats fed on dietary vitamin D levels from 0 to 500 ng/day achieved diet-dependent circulating levels of 25D ranging from 11 to 115 nmol/L. Levels of serum 1,25-dihydroxyvitamin D3 (1,25D) increased as dietary vitamin D increased between 0 and 200 ng/day at which point a maximum level was achieved and retained with higher vitamin D intakes. The renal levels of 25-hydroxyvitamin D-1α-hydroxylase (CYP27B1) mRNA were highest in animal groups fed on vitamin D between 0 and 300 ng/day. In contrast, renal 25-hydroxyvitamin D 24-hydroxylase (CYP24) mRNA levels increased as dietary vitamin D increased achieving maximum levels in animals receiving 500 ng vitamin D/day. This animal model of vitamin D depletion is suitable to provide invaluable information on the serum levels of 25D and dietary calcium intake necessary for optimal bone structure. Such information is essential for developing nutritional recommendations to reduce the incidence of osteoporotic hip fractures.

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

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