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 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.
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.
There are no conflicts of interest.
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