Ethnic differences in osteocalcin gamma-carboxylation, plasma phylloquinone (vitamin K1) and apolipoprotein E genotype.

OBJECTIVE: To investigate plasma osteocalcin gamma-carboxylation and its relationship to plasma phylloquinone concentration and apolipoprotein E (apoE) genotype in women from three ethnic groups with differing osteoporotic fracture risk. DESIGN AND SUBJECTS: Fasted blood samples were collected from postmenopausal Gambian (n=50), British (n=31) and Chinese women (n=23), and 11 premenopausal women in each group from three cross-sectional studies. RESULTS: After adjustment for total osteocalcin, plasma undercarboxylated osteocalcin (adjusted ucOC) was lowest in Chinese and highest in British women postmenopause (British vs Chinese 103% higher, P<0.0001; Gambian vs Chinese 66% higher, P<0.01). No differences were observed premenopause. Within each ethnic group, adjusted ucOC was similar pre- and postmenopause. Postmenopause, plasma phylloquinone was higher in Chinese women (1.0 ng/ml) than in British (0.31 ng/ml) and Gambian women (0.36 ng/ml) (P<0.0001). Premenopause, plasma phylloquinone was higher in Gambian and Chinese women (0.6 ng/ml) than in British women (0.3 ng/ml; P=0.01). Plasma phylloquinone and adjusted ucOC were inversely related in postmenopausal British women (R2=32.4%; P=0.0008). ApoE4 frequency was Gambian 32.6%, British 13.8% and Chinese 6%. A lower adjusted ucOC was associated with apoE2 genotype in British and Chinese women. Ethnic differences in adjusted ucOC persisted after adjustment for phylloquinone and apoE genotype. CONCLUSION: These preliminary data indicate suboptimal vitamin K status in postmenopausal British compared to Chinese and Gambian women. Ethnic differences in apoE genotype may also influence osteocalcin gamma-carboxylation status. The study highlights the need for larger epidemiological investigations of ethnic differences in vitamin K status and the possible implications to bone health.

The effects of estrogen on osteoprotegerin, RANKL, and estrogen receptor expression in human osteoblasts.

Estrogen is essential for bone growth and development and for the maintenance of bone health in adulthood. The cellular responses of osteoblasts and osteoclasts to estrogen are initiated via two high-affinity receptors (ERs). Osteoblasts synthesize RANKL (receptor activator of NF-kappaB ligand), necessary for osteoclast formation and function, and osteoprotegerin (OPG), its decoy receptor. To investigate the effects of estrogen on the expression of OPG, RANKL, and ERs in human osteoblasts, cells were cultured with physiological (10(-10) M) and high-dose (10(-7) M) 17beta-estradiol for 24 and 48 h. Proteins and corresponding mRNA levels were quantitatively determined by immunocytochemistry and RT-PCR. OPG expression was significantly increased three- and sevenfold at 24 h with 10(-10) M (P < 0.05) and 10(-7) M (P < 0.01) estradiol, respectively, compared to untreated cells. Similar but smaller increases were seen at 48 h (P < 0.05). Osteoblasts treated with estradiol demonstrated increased RANKL protein expression at 24 h (P < 0.05), but this was not maintained at 48 h. ERalpha expression was significantly increased by high-dose estradiol (P < 0.01) at 24 h and dose-dependently increased at 48 h (P < 0.01), while ERbeta was only increased at 24 h (P < 0.01). The estrogen-induced protein expression of ER, OPG, and RANKL was abrogated when cells were cultured in the presence of the estrogen antagonist ICI 182780. mRNA levels at 24 h demonstrated a significant suppression of RANKL with the low-dose but not the high dose. ERalpha mRNA but not ERbeta expression was up-regulated by estrogen. Our results suggest that estrogen may exert its anti-resorptive effects on bone, at least in part, by stimulating ER and OPG expression in osteoblasts.

Megakaryocyte population in human bone marrow increases with estrogen treatment: a role in bone remodeling?

Skeletal effects of conventional hormone replacement therapy (HRT) are predominately antiresorptive, while high doses of estrogen have anabolic effects. The mechanisms mediating these effects are unclear but may involve cells in the bone marrow. We have investigated the in vivo effects of estrogen on the megakaryocyte (MK) population in bone marrow in 10 postmenopausal women before and after 2 years of conventional HRT, in 11 women after long-term, high-dose estradiol therapy, and in 2 premenopausal and 4 postmenopausal women who had received no previous estrogen treatment. Transiliac crest biopsies were halved and either decalcified and paraffin wax embedded for immunolocalization studies or dehydrated and embedded in LR White resin for histology. MKs were identified morphologically, and the bone marrow cell population and MK number quantified by cell counting in a defined area of view (1 mm(2)) from 5 randomly selected fields of bone marrow. Compared with pretreatment values, significantly higher MK numbers were found after conventional HRT treatment (before treatment, mean +/- SEM; 7.3 +/- 1.1 vs. after treatment, 18.0 +/- 1.6/5 mm(2); p < 0.0001), while the greatest MK number was associated with long-term, high-dose estradiol treatment (32.8 +/- 2.1/5 mm(2); p < 0.0001). Total bone marrow cell number did not differ significantly between groups. Immunolocalization studies revealed more intense estrogen receptor (ER)beta expression in MKs in the high-dose estradiol-treated group but similar levels of weak ERalpha staining in MKs in the control and high-dose estrogen-treated groups. Positive immunoreactivity for transforming growth factor (TGF)beta1, 2, and 3 and TGFbeta receptor I, II, and III was detected in MKs, with more intense staining being demonstrated in the high-dose estradiol-treated group, particularly for TGFbeta2 and TGFbetaRI and II. Our results demonstrate an increase in the MK population in bone marrow from women treated with estrogen. The ability of MKs to express ERs and synthesise TGFbeta, a potent mitogen in osteoblast differentiation, suggests that these cells may play a role in mediating estrogen-induced effects on bone.

Bone changes after 3 mo of lactation: influence of calcium intake, breast-milk output, and vitamin D-receptor genotype.

Factors influencing the change in bone mineral after 3 mo of lactation were investigated in 47 breast-feeding mothers, 11 formula-feeding mothers, and 22 nonpregnant, nonlactating control subjects. At 6-8 wk postpartum, the breast-feeding group had a mean (+/-SD) calcium intake of 34.8+/-13.2 mmol/d and breast-milk volume, calcium concentration, and calcium output of 0.865+/-0.230 L/d, 7.41+/-1.25 mmol/L, and 6.41+/-2.00 mmol/d, respectively. There was no relation between calcium intake and any breast-milk variable. Dual-energy X-ray absorptiometry of the whole body, spine, hip, and forearm was performed at 0.5 and 3 mo. There were significant decreases in bone mineral content at the spine (3.96%; 95% CI: 4.86%, 3.06%), femoral neck (2.39%; 95% CI: 3.61%, 1.17%), total hip (1.51%; 95% CI: 2.45%, 0.60%), and whole body (0.86%; 95% CI: 1.29%, 0.43%) in breast-feeding mothers but not in formula-feeding mothers or nonpregnant, nonlactating women. These changes were not related to calcium intake, breast-milk calcium concentration, vitamin D-receptor genotype, postpartum weight change, or use of the progesterone-only contraceptive pill. After adjustment for bone area, breast-milk volume and height were identified as significant predictors at the spine, such that greater decreases were associated with taller mothers (P = 0.007) and those with greater breast-milk volume (P = 0.001). This finding suggests that the marked bone mineral changes observed in breast-feeding mothers represented a physiologic response to lactation that was independent of dietary calcium supply.