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Effects of folic acid supplementation on bone turnover markers in male rats subjected to experimental hyperhomocysteinemia

Al-Saeed, Hanan F.

doi: 10.1097/01.MJX.0000421842.06688.02
ORIGINAL ARTICLES
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Objective The objective of this study was to examine whether lowering homocysteine (HCY) with folate has beneficial effects on altered structural and biomechanical properties of bone, induced by experimental hyperhomocysteinemia (HHCY).

Methods Thirty adult male Wistar albino rats were randomized into three equal groups: a control group, an HHCY group, and an HHCY+folate group. After 8 weeks of treatment, blood was collected to measure the plasma concentrations of HCY and some bone markers including osteocalcin, bone-specific alkaline phosphatase, and tartrate-resistant acid phosphatase (TRAP5b). Also, histopathological examination of bone was carried out.

Results TRAP5b showed higher plasma concentrations, whereas plasma concentrations of osteocalcin and bone-specific alkaline phosphatase were significantly lower in HHCY rats compared with the control rats. Feeding of rats with a folate-enriched diet showed a significant reduction in the plasma HCY level and induced a significant improvement in the disturbances resulting from HHCY on the bone structure and turnover markers.

Conclusion The results of this study indicate that HHCY is associated with altered bone turnover. Also, folate supplementation may have beneficial effects to avoid osteoporosis and fractures.

Department of Physiology, Faculty of Medicine for Girls, Al-Azhar University, Cairo, Egypt

Correspondence to Hanan F. Al-Saeed, Department of Physiology, Faculty of Medicine for Girls, Al-Azhar University, 11849 Cairo, Egypt Tel: +20 111 334 4638; fax: +20 264 34989; e-mail: hananfathy_1972@yahoo.com

Received July 24, 2012

Accepted September 1, 2012

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Background

Osteoporosis is a systemic bone disease characterized by low bone mass and microarchitectural deterioration of bone tissue, leading to enhanced bone fragility and a resultant increase in the risk of fracture 1. Epidemiologic data have indicated that among other common age-related diseases, osteoporosis ranks number 1 in women and number 2 in men 2. Others have reported that the total number of fractures, and hence the cost to society, will increase considerably as a result of demographic changes in the number of elderly individuals 1. Thus, prevention of osteoporosis by identification of risk factors or risk indicators and the development of new treatment strategies are major issues.

Besides classical osteoporotic risk factors such as vitamin D deficiency and low calcium intake 3, it has been proposed that hyperhomocysteinemia (HHCY) is a novel and independent risk factor for osteoporosis 4. Homocysteine (HCY) is the final degradation product of the methionine pathway and is elevated if folate, vitamin B6 and B12, important coenzymes of the HCY degrading remethylation and trans-sulfuration pathway, are deficient 5. It is well known that folic acid supplementation can reduce the elevated HCY levels. Therefore, folic acid supplementation may also help reverse the problems associated with HHCY 6. Consequently, the present study aimed to evaluate the effects of experimental HHCY and folic acid supplementation on bone metabolism of rats by measuring indices of bone resorption and bone formation (bone turnover markers) and histopathological examination.

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Materials and methods

Induction of hyperhomocysteinemia

After 7 days of acclimatization, 30 adult male Wistar albino rats, weighing 180–200 g, were randomized into three groups: a control group (n=10), an HHCY group (n=10), and an HHCY group (n=10) that received folic acid powder at a moderate dose 8 mg/kg diet 7. The animals were kept in plastic cages at room temperature (23°C) under a day/night cycle in our institutional facility, with free access to food and water. In accordance with previous studies, HHCY was induced by the administration of L-methionine at 24 g/kg diet 8. All diets had identical compositions that did not contain HCY or methionine. The treatment period for both groups was 8 weeks.

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Sample collection and biochemical assays

The animals were anesthetized at the end of the experiments and blood samples were obtained from the orbital sinus of overnight fasted rats. Blood was immediately centrifuged at 3000 rpm for 20 min. Plasma was separated and stored at −80°C until the day of analysis. In the blood specimens, we measured HCY and biochemical bone turnover markers [osteocalcin (OC), bone-specific alkaline phosphatase (BAP), and tartrate-resistant acid phosphatase (TRAP5b)] using enzyme-linked immunoassay kits (Formosa Biomedical kits, Taipei, Taiwan). All analyses were carried out in a blinded manner. Also, histopathological examination of bone was carried out.

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Histopathological examinations

Autopsy samples were taken from the femur bone of rats in different groups and fixed as well as decalcified in formalin formic acid. Then, the usual procedures of histological examination were carried out. Sections were stained with hematoxylin and eosin and then examined under an electric light microscope 9.

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Statistical analysis

Data are expressed as mean±SD. Statistical comparisons between different groups were carried out using the Kruskal–Wallis test, followed by the Mann–Whitney test to compare between two means. Pearson’s correlation analysis was also carried out in order to test the association between HCY and bone markers. All calculations were carried out using the SPSS 16.0 software package (IBM Corporation, United states). Significance was considered at P less than 0.05.

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Results

As expected, the plasma concentrations of HCY were significantly higher in the HHCY group compared with the control group. Feeding of rats with a folate-enriched diet led to a significant reduction in the plasma HCY level compared with the HHCY group, but still significantly higher than that of the control group (Table 1).

Table 1

Table 1

The bone resorption marker TRAP5b showed higher plasma concentrations in HHCY than in the control rats. However, the plasma concentrations of the bone formation markers OC and BAP were significantly lower in HHCY rats compared with those in the control rats. Daily oral supplementation of rats with a folate-enriched diet induced a significant improvement in the disturbances resulting from HHCY on the plasma turnover markers. This was manifested by a significant reduction in TRAP5b levels and a significant increase in OC and BAP levels compared with those in the HHCY group. All the measured markers almost returned to their original levels in the control group.

In addition, the total plasma HCY levels were strongly correlated to TRAP5b and moderately correlated to OC levels (Figs 1 and 2).

Fig. 1

Fig. 1

Fig. 2

Fig. 2

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Histopathological findings

The normal histological structures of the cartilaginous structure and bone epiphysis as well as the trabecullae and bone marrow were observed in the control group. Bone resorption was observed in the epiphysis and trabecullae of the HHCY rats (Fig. 3). However, normal histological structure was observed in the HHCY+folate group (Fig. 4).

Fig. 3

Fig. 3

Fig. 4

Fig. 4

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Discussion

The present study found a consistent relationship between HHCY and disturbed bone turnover, as indicated by increased bone resorption marker (TRAP5b) and decreased bone formation markers (OC and BAP) concentrations as well as altered histological bone structure. Feeding of HHCY rats with a folate-enriched diet led to a reduction in the HCY concentration, returned these biomarkers to their control levels, and improved the bone morphology.

In the present study, a significant reduction in bone formation markers (OC and BAP) in HHCY rats was observed. Moreover, the total plasma HCY levels were strongly correlated to TRAP5b and moderately correlated to OC levels.

OC is a noncollagenous bone matrix protein that is considered a specific osteoblast activity marker 10. Herrmann et al. 8 have reported that OC showed a strong decrease of up to 34% in HHCY rats. Furthermore, Sakamoto et al. 11 have reported impaired function and reduced OC production with an increase in the HCY concentration in cultured osteoblasts. Others have reported that HHCY might interfere with the activity of osteoblasts as indicated by decreased bone formation and a significant reduction in serum OC concentrations in HHCY rats 12,13.

BAP is synthesized by the osteoblasts and is believed to be involved in the calcification of bone matrix. Although its precise role in the bone formation process is unknown, BAP is considered to be a highly specific marker of the bone-forming activity of osteoblasts 14. Carmel et al. 15 have reported reductions in BAP and OC concentrations in vitamin B12-deficient individuals, a result further supported by the finding of a vitamin B12 dependency of human osteoblast progenitor cells 16. Therefore, although not measured in the present study, a possible HHCY-induced decrease in the serum vitamin B12 concentration may also play a role in the observed alterations in bone metabolism. In contrast to our finding, normal indices of bone formation were observed in HHCY developing chicken 17. However, it is questionable whether the results of developing chicken can be transferred to the situation in adult mammals.

In this study, the HHCY animals showed significantly higher levels of TRAP5b than the control ones. TRAP5b, the active isoform of TRAP, is specifically synthesized by bone-resorbing osteoclasts. Reactive oxygen species generated by TRAP5b have been suggested to participate in the degradation of endocytosed bone matrix products in resorbing osteoclasts 18. Elevated serum TRAP5b levels have been detected in diseases characterized by increased bone resorption such as Paget’s disease of the bone, hemodialysis, primary hyperparathyroidism, and metastatic malignancies involving bone resorption 19. Untreated postmenopausal women have higher levels of serum TRAP5b than postmenopausal women on estrogen replacement therapy 20. Specific determination of TRAP5b enzyme has, therefore, been considered potentially useful as a marker of the bone resorption rate for many years 19. Several clinical studies have indicated that HHCY is associated with elevated serologic bone resorption markers 21,22. Herrmann et al. 23 have reported a significant correlation between HCY and urinary desoxypyridinoline cross-links (DPD) in 143 perimenopausal and postmenopausal women. Dhonukshe-Rutten et al. 24 have also found increased urinary excretion of DPD in women with high plasma HCY and a low serum vitamin B12 concentration compared with those with normal HCY and vitamin B12 concentrations. Similarly, a positive correlation between HCY and another bone resorption marker, carboxyterminal telopeptide of human collagen I, has been reported by Bode et al. 25. In addition, others have reported that at physiologic concentrations, HCY may increase osteoclast formation and activity 26. Cell culture experiments also support the hypothesis of an HCY-induced stimulation of bone resorption and decreased bone formation. Herrmann et al. 27 have reported that HCY induced a dose-dependent increase in osteoclast activity. Also, Koh et al. 28 have reported a direct activation of osteoclast formation and activity by HCY.

However, the mechanism by which high HCY could affect bone is still not clear. It has been proposed that HCY might interfere with the synthesis and cross-linking of collagen. Impaired collagen cross-linking would decrease the stability and strength of the collagen network, which would decrease bone strength and increase the risk of fracture 29. Moreover, HCY may directly activate osteoclast formation and activity through increased generation of intracellular reactive oxygen species 30.

However, little is known about the effect of HCY-lowering therapy on markers of bone turnover. This study tested the hypothesis that reducing HCY with folate would favorably affect biomarkers of bone turnover. We found that daily oral supplementation of HHCY rats with a folate-enriched diet induced a significant improvement in the disturbances resulting from HHCY on the plasma turnover markers.

In agreement with our results, Sato et al. 31 have reported that stroke patients in Japan who received folate and vitamin B12 supplements over 2 years had a significantly lower rate of hip fracture than did a placebo group. Others have reported a positive association between nutrient dense foods (intake of fruits, vegetable, and whole grain) and bone health 32. However, Holstein et al. 22 have found that low serum folate and vitamin B6 concentrations are associated with an altered morphology of human bone. The effects of folate on bone health may be attributed to its HCY-lowering effect. Folate acts as a substrate in the remethylation of HCY to methionine 33. Because HCY levels were not directly associated with bone mineral density as folic acid was, researchers have pointed to previous studies suggesting that the role of folic acid in bone health goes beyond reducing HCY levels. It includes roles in DNA methylation 34, as well as nitric oxide synthesis 35, which can be ‘particularly interesting’ for bone metabolism.

In this study, the HHCY rats showed bone resorption in the epiphysis and trabecullae, whereas folate supplementation improved the histopathological alteration observed in the HHCY group. Our findings are in agreement with those of Herrmann et al. 8, who found a marked loss of cancellous bone, which was most pronounced at the distal femur, through the histomorphometric analyses and biomechanical testing of the rats subjected to moderate HHCY. They also found a strong increase in bone fragility, by up to 36%, after 12 weeks of HHCY. Herrmann et al. 29 have reported a strong accumulation of HCY in bone tissue of hyperhomocysteinemic animals because of trapping of circulating HCY in the extracellular bone matrix that was accompanied by significant bone loss and a reduction in bone strength. Others have found that in HHCY rats, the relation between bone resorption and formation indicates a shift toward bone resorption 12. In a double-blind placebo-controlled trial, which involved supplementation of 628 patients for 2 years with a placebo or 5 mg/day of folate and 1500 µg/day vitamin B12, the folate-treated group showed a 75% reduction in femoral neck fractures, and the overall fracture rate was strongly decreased 36. In addition, Holstein et al. 22 have provided the first evidence that low serum folate and vitamin B6 are related to an altered cancellous bone structure in humans. They suggested that these alterations in cancellous bone structure may be explained by an impaired osteoblast activity as indicated by a low serum OC concentration in individuals with low serum folate and vitamin B6 concentrations.

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Conclusion

The present study provides strong evidence that HHCY is a possible causal osteoporotic factor and supplementation of folate has beneficial effects on bone health. Future studies will be required to clarify the mechanisms of these effects.

Table

Table

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Acknowledgements

Conflicts of interest

There are no conflicts of interest.

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