Subclinical hypervitaminosis A in rat: Measurements of bone mineral density (BMD) do not reveal adverse skeletal changes

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Abstract

We have previously shown that subclinical hypervitaminosis A in rats causes fragile bones. To begin to investigate possible mechanisms for Vitamin A action we extended our previous study. Forty-five mature female Sprague-Dawley rats were divided into three groups, each with 15 animals. They were fed a standard diet containing 12 IU Vitamin A per g pellet (control, C), or a standard diet supplemented with 120 IU (“10 × C”) or 600 IU (“50 × C”) Vitamin A/g pellet for 12 weeks. At the end of the study, serum retinyl esters were elevated 4- and 20-fold. Although neither average food intake nor final body weights were significantly different between groups, a dose-dependent reduction in serum levels of Vitamin D and E, but not Vitamin K, was found. In the 50 × C-group the length of the humerus was the same as in controls, but the diameter was reduced (−4.1%, p < 0.05). Peripheral quantitative computed tomography (pQCT) at the diaphysis showed that bone mineral density (BMD) was unchanged and that periosteal circumference had decreased significantly (−3.7%, p < 0.05). Ash weight of the humerus was not affected, but since bone volume decreased, volumetric BMD, as measured by the bone ash method, even increased (+2.5%, p < 0.05).

In conclusion, interference with other fat-soluble Vitamins is a possible indirect mechanism of Vitamin A action. Moreover, BMD measurements do not reveal early adverse skeletal changes induced by moderate excesses of Vitamin A in rats. Since the WHO criterium for osteoporosis is based on BMD, further studies are warranted to examine whether this is also true in humans.

Introduction

It has long been known that large doses of retinol (Vitamin A) are severely toxic to the rat appendicular skeleton [1], [2], [3]. In humans, a high dietary intake of Vitamin A is associated with an increased risk of hip fracture [4], [5]. The active metabolite retinoic acid binds and activates the retinoid acid receptors (RARs) which, like the Vitamin D receptor belong to the family of steroid hormone receptors. Upon ligand binding, these receptors bind to specific sequences in the genome, thus functioning as transcription factors in the nucleus [6]. The different RAR subtypes are all expressed in osteoblasts [7], [8], [9] and a bone-resorptive effect of Vitamin A has been reported from in vitro studies [10], [11], [3], [12]. Therefore, it is likely that Vitamin A can exert a direct toxic effect on bone tissue via its active metabolite retinoic acid. However, also indirect mechanisms, via interaction with other agents of importance for bone metabolism, such as Vitamin D, have been proposed. We have previously shown that the acute serum-calcium-increasing effect of a single dose of Vitamin D can be antagonized by retinyl palmitate in man [13], but the relationship is still hardly studied in humans. In rat, an excessive intake of Vitamin A diminishes the curative effect of Vitamin D on rickets [14] and an increased intake of Vitamin D has been shown to alleviate the symptoms of hypervitaminosis A [15]. An antagonism of Vitamin D action on bone and intestine by Vitamin A has been confirmed in vivo in rat [16].

However, the relevant mechanisms for the action of Vitamin A on the skeleton, especially at moderately increased dietary intake levels, are still poorly defined. We recently demonstrated a negative effect on bone strength, evident as decreased three-point bending breaking force of the femur, in rats with subclinical hypervitaminosis A [17]. To begin to investigate possible mechanisms for Vitamin A action, we extended our study on rats.

Section snippets

Animals and experimental design

Animals and experimental design have been described previously [17]. All animal experiments were approved by the local ethical committee for animal experiments. Briefly, 45 female Sprague-Dawley rats, 3 months of age, were obtained from Möllegaards Breeding Centre, Ltd. (Skensved, Denmark). They were kept in groups of five animals in polycarbonate cages with hardwood chips as bedding in a room maintained at 21 ± 1 °C (relative humidity 50 ± 5%), using a 12 h light:dark cycle with the light cycle

Evaluation of retinoid uptake and Vitamin A intoxication

To verify Vitamin A uptake and storage in the rats, retinoids in liver, kidney and serum were analyzed using high-performance liquid chromatography (HPLC) as presented in detail previously [17].

Analyses of Vitamin D, E and K

All serum samples were analyzed within the same assay to avoid interassay variations. Analyses of fat-soluble vitamins were done by AS Vitas (Oslo, Norway, www.vitas.no) using high-performance liquid chromatography (HPLC), which was performed with a HP 1100 liquid chromatograph (Agilent Technologies,

Evaluation of retinoid uptake and Vitamin A intoxication

Serum retinyl esters were elevated 4- and 20-fold and the total amount of liver retinoid increased 3- and 7-fold in the 10 × C and 50 × C group, as reported previously [17]. Neither average food intake nor final body weights were significantly different between groups; 0.05, 0.05, and 0.06 g food/g rat and 321.5 ± 6.4, 328 ± 5.7 and 319.5 ± 4.3 g/rat among C, 10 × C and 50 × C rats, respectively.

Effects on bone geometry

Bone dimensions of humerus are presented in Table 1. In the 50 × C group the diameter was decreased (−4.1%, p < 0.05)

Discussion

In the present study, we have investigated the mechanisms of Vitamin A action on bone tissue in rats with subclinical hypervitaminosis A. We hereby show that in the rats with the highest Vitamin A intake (50 × C group), the volume and diameter, but not the BMD, as measured at the diaphysis with pQCT, of the long bones are affected. Moreover, we show for the first time that subclinical hypervitaminosis A in rats leads to a reduction in serum levels of both Vitamins D and E.

In a previous study on

Acknowledgments

This work was supported by grants from the Swedish Medical Research Council, Torsten and Ragnar Söderberg Foundation, and Agricultural Sciences and Spatial Planning (grant 216/2004-2088).

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    P.M. Lind. and S. Johansson contributed equally to this study.

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