Category Archives: Supplements

DHA More Potent Inhibitor of Osteoclasts than EPA

Abstract

Docosahexaenoic acid is more potent inhibitor of osteoclast differentiation in RAW 264.7 cells than eicosapentaenoic acid.

Fish oil rich in n-3 polyunsaturated fatty acids, especially eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) protects inflammation induced bone loss in chronic inflammatory diseases like rheumatoid arthritis, periodontitis, and osteoporosis. EPA and DHA differentially regulate functional parameters and gene expression in different cell types. One of the risk factors for bone loss in inflammatory bone diseases is the elevation of bone-resorbing osteoclasts and a very few studies so far have indicated that attenuation of osteoclastogenesis might be one of the mechanisms by which n-3 PUFA exert its effect on bone loss protection. However, the precise mechanism underlying this process remains unclear. Receptor activator of NF-kappaB ligand (RANKL) is known to be the most critical mediator of osteoclastogenesis. Therefore, in this study, we examined the differential effect of EPA and DHA on RANKL-stimulated osteoclastogenesis and RANKL signaling using a murine monocytic cell line RAW 264.7. DHA was found to inhibit osteoclast differentiation, activation and function more potently than EPA. The differential potential also closely correlated with the inhibition of osteoclast-specific genes like tartrate resistant acid phosphatase, cathepsin K, calcitonin receptor, matrix metalloproteinase-9 expression and osteoclast-specific transcription factor, c-Fos, as well as osteotropic proinflammatory cytokine, TNF-alpha to a greater extent with DHA than EPA. Further, pretreatment of RAW 264.7 cells with DHA also showed significantly reduced activation of NF-kappaB and p38MAPK than EPA. Our findings suggest that DHA may be much more effective than EPA in alleviating RANKL induced proinflammatory cytokine production, intracellular signaling activation, thereby decreasing osteoclast activation and bone resorption.

Rahman MM, Bhattacharya A, Fernandes G
J. Cell. Physiol. Jan 2008
PMID: 17929247

DHA prevents, and GLA Exacerbates, Bone Loss in Ovariectomized Rats

Abstract

Specific effects of gamma-linolenic, eicosapentaenoic, and docosahexaenoic ethyl esters on bone post-ovariectomy in rats.

Long chain polyunsaturated fatty acids (LCPUFAs) are involved in the regulation of bone metabolism. Increased dietary consumption of n-3, and possibly some n-6, LCPUFAs may limit postmenopausal bone loss. The aim of this study was to determine the effects on bone of specific fatty acids within the n-3 and n-6 LCPUFA families in ovariectomized (OVX) rats. Rats were OVX or sham-operated and fed either a control diet (OVX and sham) or a diet supplemented with 0.5 g/kg body weight/day of gamma-linolenic (GLA), eicosapentaenoic (EPA), docosahexaenoic (DHA) ethyl esters or a mixture of all three (MIX) for 16 weeks. Bone mineral content (BMC), area, and density and plasma concentrations of insulin-like growth factor-I, vitamin D, selected biochemical markers of bone metabolism, and parathyroid hormone (PTH) were determined. The OVX-induced decrease in lumbar spine BMC was significantly attenuated by DHA but not by EPA or GLA supplementation or supplementation with a mixture of all three LCPUFAs. Endosteal circumferences of tibiae were significantly greater in DHA and EPA compared to OVX. Plasma C-terminal telopeptide of type I collagen and osteocalcin concentrations were not significantly different in the DHA group compared to OVX. Femur BMC decreased by a significantly greater amount in GLA than OVX, and final plasma PTH concentrations were significantly higher in GLA compared to all other groups. In conclusion, DHA ameliorated OVX-induced bone mineral loss. GLA exacerbated post-OVX bone mineral loss, possibly as a result of PTH-induced bone catabolism.

Poulsen RC, Firth EC, Rogers CW, Moughan PJ…
Calcif. Tissue Int. Dec 2007
PMID: 18008020

Japanese Apricot Shows Potential to Prevent Osteoporosis

Abstract

A Prunus mume extract stimulated the proliferation and differentiation of osteoblastic MC3T3-E1 cells.

Osteoporosis is a serious disease caused by decreased bone mass. There is constant matrix remodeling in bones, by which bone formation is performed by osteoblastic cells, whereas bone resorption is accomplished by osteoclast cells. We investigated the effect of a Japanese apricot (Prunus mume SIBE. et ZUCC.) extract on the proliferation and osteoblastic differentiation in pre-osteoblastic MC3T3-E1 cells. An alkaline phosphatase (ALP) activity assay, cell proliferation assay, alizarin red staining and expression analysis of osteoblastic genes were carried out to assess the proliferation and osteoblastic differentiation. The water-soluble fraction of Prunus mume (PWF) increased the ALP activity, cell proliferation and mineralization. The gene expression of osteopontin and bone morphogenetic protein-2, which are markers in the early period of osteoblastic differentiation, were significantly enhanced by the PWF treatment. PWF therefore stimulated the proliferation and osteoblastic differentiation of cells and may have potential to prevent osteoporosis.

Kono R, Okuno Y, Inada K, Tokuda A…
Biosci. Biotechnol. Biochem. 2011
PMID: 21979066 | Free Full Text

NAC Stimulates Osteoblastogenesis in Rats

Abstract

N-acetyl cysteine as an osteogenesis-enhancing molecule for bone regeneration.

Bone regeneration often requires cues from osteogenesis-inducing factors for successful outcome. N-acetyl cysteine (NAC), an anti-oxidant small molecule, possibly modulates osteoblastic differentiation. This study investigated the potential of NAC as an osteogenesis-enhancing molecule in vitro and in vivo. Various concentrations of NAC (0, 2.5, 5.0, and 10 mM) were added to rat bone marrow stromal cell or osteoblastic cell culture in media with or without dexamethasone. The results showed marked enhancement of alkaline phosphatase activity and mineralized matrix formation together with consistent upregulation of bone-related gene markers such as collagen I, osteopontin, and osteocalcin in the osteoblastic culture with addition of 2.5 or 5.0 mM NAC regardless of the presence of dexamethasone. Micro-CT-based analysis and histological observation revealed that addition of NAC to a collagenous sponge implanted in a critical size cortical bone defect (3.0 mm × 5.0 mm) in rat femur yielded acceleration and completion of defect closure, with thick, compact, and contiguous bone after 6 weeks of healing. In contrast, with sponge alone, only sparse and incomplete bone regeneration was observed during the matching healing period. These results indicate that NAC can function as an osteogenesis-enhancing molecule to accelerate bone regeneration by activating differentiation of osteogenic lineages.

Yamada M, Tsukimura N, Ikeda T, Sugita Y…
Biomaterials Aug 2013
PMID: 23711675

NAC Prevents Skeletal Aging in Transgenic Mice

Abstract

Swedish mutant APP suppresses osteoblast differentiation and causes osteoporotic deficit, which are ameliorated by N-acetyl-L-cysteine.

Reduced bone mineral density and hip fracture are frequently observed in patients with Alzheimer’s disease (AD). However, mechanisms underlying their association remain poorly understood. Amyloid precursor protein (APP) is a transmembrane protein that is ubiquitously expressed in bone marrow stromal cells (BMSCs), osteoblasts (OBs), macrophages (BMMs), and osteoclasts (OCs). Mutations in the APP gene identified in early-onset AD patients are believed to cause AD. But little is known about APP’s role in bone remodeling. Here, we present evidence for Swedish mutant APP (APPswe) in suppression of OB differentiation and function in culture and in mouse. APP expression in BMSCs increases during aging. Ubiquitous expression of APPswe in young adult Tg2576 transgenic mice (under the control of a prion promoter) recaptured skeletal “aging-like” deficits, including decreased OB genesis and bone formation, increased adipogenesis and bone marrow fat, and enhanced OC genesis and bone resorption. Remarkably, selective expression of APPswe in mature OB-lineage cells in TgAPPswe-Ocn mice (under the control of osteocalcin [Ocn] promoter-driven Cre) also decreased OB genesis and increased OC formation, resulting in a trabecular bone loss. These results thus suggest a cell-autonomous role for APPswe in suppressing OB formation and function, but a nonautonomous effect on OC genesis. Notably, increased adipogenesis and elevated bone marrow fat were detected in young adult Tg2576 mice, but not in TgAPPswe-Ocn mice, implying that APPswe in BMSCs and/or multicell types in bone marrow promotes bone marrow adipogenesis. Intriguingly, the skeletal aging-like deficits in young adult Tg2576 mice were prevented by treatment with N-acetyl-L-cysteine (NAC), an antioxidant, suggesting that reactive oxygen species (ROS) may underlie APPswe-induced osteoporotic deficits. Taken together, these results demonstrate a role for APPswe in suppressing OB differentiation and bone formation, implicate APPswe as a detrimental factor for AD-associated osteoporotic deficit, and reveal a potential clinical value of NAC in the treatment of osteoporotic deficits.

Xia WF, Jung JU, Shun C, Xiong S…
J. Bone Miner. Res. Oct 2013
PMID: 23649480

NAC Prevents Bone Loss in Diabetic Rats

Abstract

N-acetylcysteine decreases alveolar bone loss on experimental periodontitis in streptozotocin-induced diabetic rats.

The purpose of this study was to evaluate the morphometric and histopathological changes associated with experimental periodontitis in diabetic rats in response to systemic administration of N-acetylcysteine (NAC), a sulfhydryl-containing thiol antioxidant.
Sixty Wistar rats were divided into six experimental groups: nonligated (NL) group; ligature-only (L) group; streptozotocin-only (STZ) group; STZ and ligature (STZ + L) group; and systemic administration of NAC and ligature (70 and 100 mg/kg body weight per day, respectively) (NAC70 and NAC100 groups). Diabetes mellitus was induced by 60 mg/kg of streptozotocin. Silk ligatures were placed at the gingival margin of the lower first molars of the mandibular quadrant. The study duration was 30 d and the animals were killed at the end of this period. Changes in alveolar bone levels were clinically measured and tissues were histopathologically examined to assess the differences among the study groups.
At the end of the 30-d study period, alveolar bone loss was significantly higher in the STZ + L group compared with the other groups (p < 0.05). Also, alveolar bone loss in all the NAC groups was significantly lower than in the STZ + L and L groups (p < 0.05). The osteoblastic activity in the NAC100 group was significantly higher than in the other groups (p < 0.05).
Within the limits of this study, it can be suggested that NAC, when administered systemically, prevents alveolar bone loss in the diabetic rat model.

Toker H, Ozdemir H, Balcı H, Ozer H
J. Periodont. Res. Dec 2012
PMID: 22712627

Folate + B12 Offer No Sensomotor Speed Benefit Despite Decreasing Homocysteine

Abstract

The association of betaine, homocysteine and related metabolites with cognitive function in Dutch elderly people.

The importance of the one-carbon metabolites, choline and homocysteine, to brain function is well known. However, the associations between the one-carbon metabolites choline, betaine, methionine and dimethylglycine with cognition in elderly are unclear. We therefore examined the associations of these metabolites with cognition in a double-blind, placebo-controlled trial. Individuals (n 195) were randomized to receive daily oral capsules with either 1000 microg cobalamin (vitamin B12), or 1000 microg cobalamin plus 400 microg folic acid, or placebo for 24 weeks. Concentrations of homocysteine, methionine, choline, betaine and dimethylglycine were assessed before and after 12 and 24 weeks of treatment. Cognitive function, including domains of attention, construction, sensomotor speed, memory and executive function, was assessed before and after 24 weeks of treatment. At baseline, elevated plasma homocysteine was associated with lower performance of attention, construction, sensomotor speed and executive function. In addition, betaine was positively associated with better performance of construction, sensomotor speed and executive function, whereas elevated concentrations of methionine were positively associated with sensomotor speed. Daily combined supplementation with cobalamin plus folic acid decreased total homocysteine concentrations by 36%, and increased betaine concentrations by 38%. Participants with the largest increases in betaine concentrations showed a borderline significant (P = 0.07) higher memory performance compared to those without it. Although this trial observed associations of homocysteine and betaine with cognitive domains prior to supplementation, decreased concentrations of homocysteine were not related to improved cognitive performance. There was a tendency of participants with the largest increases in betaine concentrations to show the greatest improvement in memory function.

Eussen SJ, Ueland PM, Clarke R, Blom HJ…
Br. J. Nutr. Nov 2007
PMID: 17537289

Folate, but Not Vitamin B2 or B12, Associated with Increased Done Density in Postmenopausal Women

Abstract

Dietary intake of folate, but not vitamin B2 or B12, is associated with increased bone mineral density 5 years after the menopause: results from a 10-year follow-up study in early postmenopausal women.

Folate, vitamin B2 (riboflavin), and vitamin B12 may affect bone directly or through an effect on plasma homocysteine levels. Previously, a positive association has been found between plasma levels and bone mineral density (BMD) as well as risk of fracture. However, there are limited data on whether dietary intakes affect bone. Our aim was to investigate whether intake of folate, vitamin B2) and vitamin B12, as assessed by food records affects BMD and fracture risk. In a population-based cohort including 1,869 perimenopausal women from the Danish Osteoporosis Prevention Study, associations between intakes and BMD were assessed at baseline and after 5 years of follow-up. Moreover, associations between intakes and 5- and 10-year changes in BMD as well as risk of fracture were studied. Intakes of folate, vitamin B2, and vitamin B12 were 417 (range 290-494) microg/day, 2.70 (range 1.70-3.16) mg/day, and 4.98 (range 3.83-6.62) microg/day, respectively, i.e., slightly above the intakes recommended by the United Nations Food and Agriculture Organization. At year 5, but not at baseline, cross-sectional analyses showed positive correlations between daily intake from diet and from diet plus supplements of folate and BMD at the femoral neck (P < 0.01). However, no associations were found between intakes and changes in BMD. During 10 years of follow-up, 360 subjects sustained a fracture. Compared with 1,440 controls, logistic regression analyses revealed no difference in intakes between cases and controls. A high dietary intake of folate, but not vitamin B2 or B12, exerts positive effects on BMD; but further studies are needed to confirm this association.

Rejnmark L, Vestergaard P, Hermann AP, Brot C…
Calcif. Tissue Int. Jan 2008
PMID: 18175033

Vitamins D + B Improve Bone Markers in Elderly

Abstract

One year B and D vitamins supplementation improves metabolic bone markers.

Vitamin D and vitamin B deficiency are common in elderly subjects and are important risk factors for osteoporosis and age-related diseases. Supplementation with these vitamins is a promising preventative strategy. The objective of this study was to evaluate the effects of vitamins D3 and B supplementation on bone turnover and metabolism in elderly people.
Healthy subjects (n=93; >54 years) were randomly assigned to receive either daily vitamin D3 (1200 IU), folic acid (0.5 mg), vitamin B12 (0.5 mg), vitamin B6 (50 mg), and calcium carbonate (456 mg) (group A) or only vitamin D3 plus calcium carbonate (group B) in a double blind trial. We measured at baseline and after 6 and 12 months of supplementation vitamins, metabolites, and bone turnover markers.
At baseline mean plasma 25-hydroxy vitamin D [25(OH)D] was low (40 or 30 nmol/L) and parathormone was high (63.7 or 77.9 pg/mL). 25(OH)D and parathormone correlated inversely. S-Adenosyl homocysteine and S-adenosyl methionine correlated with bone alkaline phosphatase, sclerostin, and parathormone. One year vitamin D3 or D3 and B supplementation increased plasma 25(OH)D by median 87.6% (group A) and 133.3% (group B). Parathormone was lowered by median 28.3% (A) and 41.2% (B), bone alkaline phosphatase decreased by 2.8% (A) and 16.2% (B), osteocalin by 37.5% (A) and 49.4% (B), and tartrate-resistant-acid-phosphatase 5b by 6.1% (A) and 36.0% (B). Median total homocysteine (tHcy) was high at baseline (group A: 12.6, group B: 12.3 µmol/L) and decreased by B vitamins (group A) to 8.9 µmol/L (29.4%). tHcy lowering had no additional effect on bone turnover.
One year vitamin D3 supplementation with or without B vitamins decreased the bone turnover significantly. Vitamin D3 lowered parathormone. The additional application of B vitamins did not further improve bone turnover. The marked tHcy lowering by B vitamins may modulate the osteoporotic risk.

Herrmann W, Kirsch SH, Kruse V, Eckert R…
Clin. Chem. Lab. Med. Mar 2013
PMID: 23183751

Homocysteine Decreases Bone Quality and is Antagonized by NAC

Abstract

Homocysteine alters the osteoprotegerin/RANKL system in the osteoblast to promote bone loss: pivotal role of the redox regulator forkhead O1.

In this study we determined the molecular mechanisms of how homocysteine differentially affects receptor activator of nuclear factor-κB ligand (RANKL) and osteoprotegerin (OPG) synthesis in the bone. The results showed that oxidative stress induced by homocysteine deranges insulin-sensitive FOXO1 and MAP kinase signaling cascades to decrease OPG and increase RANKL synthesis in osteoblast cultures. We observed that downregulation of insulin/FOXO1 and p38 MAP kinase signaling mechanisms due to phosphorylation of protein phosphatase 2A (PP2A) was the key event that inhibited OPG synthesis in homocysteine-treated osteoblast cultures. siRNA knockdown experiments confirmed that FOXO1 is integral to OPG and p38 synthesis. Conversely homocysteine increased RANKL synthesis in osteoblasts through c-Jun/JNK MAP kinase signaling mechanisms independent of FOXO1. In the rat bone milieu, high-methionine diet-induced hyperhomocysteinemia lowered FOXO1 and OPG expression and increased synthesis of proresorptive and inflammatory cytokines such as RANKL, M-CSF, IL-1α, IL-1β, G-CSF, GM-CSF, MIP-1α, IFN-γ, IL-17, and TNF-α. Such pathophysiological conditions were exacerbated by ovariectomy. Lowering the serum homocysteine level by a simultaneous supplementation with N-acetylcysteine improved OPG and FOXO1 expression and partially antagonized RANKL and proresorptive cytokine synthesis in the bone milieu. These results emphasize that hyperhomocysteinemia alters the redox regulatory mechanism in the osteoblast by activating PP2A and deranging FOXO1 and MAPK signaling cascades, eventually shifting the OPG:RANKL ratio toward increased osteoclast activity and decreased bone quality.

Vijayan V, Khandelwal M, Manglani K, Singh RR…
Free Radic. Biol. Med. Mar 2013
PMID: 23500899