Reduced Sirtuin1 expression at the femoral neck in women who sustained an osteoporotic hip fracture.

UNLABELLED: To investigate the role of Sirtuin1 in osteoporosis, Sirtuin1 was determined at the femoral neck in female patients undergoing hip operation for fractured hip or osteoarthritis. Reduced Sirtuin1 was found in osteoporotic patients. Pharmacologic activation of Sirtuin1 reduced sclerostin, an inhibitor of bone formation. Activation of Sirtuin1 may be a new direction to generate therapies for osteoporosis. INTRODUCTION: The aim of the study are to investigate the role of Sirtuin1 (Sirt1), an anti-aging factor and a player in age-associated diseases, in osteoporotic hip fractures, and test the hypothesis that Sirt1 is a negative regulator of sclerostin, a bone formation inhibitor, in human femoral bone marrow mesenchymal cells (BM-MSCs). METHODS: Sirt1 and sclerostin were determined by western blot in bone samples obtained intra-operatively from the inferior medial cortex of the femoral neck (calcar region) in female patients undergoing partial hip replacement for fractured neck of femur (N = 10) or hip replacement for osteoarthritis (N = 8) (mean ± SD age 81 ± 8.1 vs. 68 ± 9.3 years; BMI 26.2 ± 3.6 vs. 25.9 ± 7.1 kg/m(2) in osteoporotic and osteoarthritis patients). Calcar thickness and femoral bone mineral density (BMD) were determined preoperatively by X-ray using a digital TraumaCad(™) software and DEXA. Femoral BM-MSCs were collected intra-operatively and treated with SRT3025, a Sirt1 activator. Sclerostin and dentin matrix acidic phosphoprotein (DMP1) were determined by western blot and messenger RNA (mRNA) expression of Lef1 and DMP1 was evaluated by quantitative real-time PCR. RESULTS: Osteoporotic (OP) patients had reduced cortical thickness, femoral neck, and total hip BMD compared to osteoarthritis (OA) patients. Calcar Sirt1 expression was significantly reduced, while sclerostin was markedly increased in OP compared to OA patients. Sirt1 and sclersotin expressions were inversely correlated (r = -0.49, P = 0.047). SRT3025 administration down-regulated sclerostin and up-regulated DMP1 protein level and increased LEF1 and DMP1 mRNA expressions in OP patient-derived BM-MSCs. CONCLUSIONS: Reduced femoral neck Sirt1 may play a role in osteoporotic hip fractures in part via influencing local sclerostin expression. The therapeutic potential of Sirt1 activation in osteoporosis warrants further investigation.

Effect of different dress style on vitamin D level in healthy young Orthodox and ultra-Orthodox students in Israel.

UNLABELLED: Ultra-orthodox Jewish lifestyle, which encourages modest dress and indoor scholarly activity, represents a risk factor for vitamin-D deficiency. Our study in healthy young males from higher education religious institutions located in the same geographical area showed frequent and severe vitamin D deficiency, strongly correlated with the degree of sun exposure. However, PTH level was usually normal. INTRODUCTION: Ultra-orthodox Jewish lifestyle encourages modest dress and indoor scholarly activity. As such, it represents a risk factor for vitamin-D deficiency, a worldwide problem previously underestimated in sunny countries. Our aim was to characterize the vitamin-D status of religious Jewish males according to sun exposure and outdoor activity, and study the correlation between serum 25-hydroxyvitamin D (25(OH) D) and PTH level. METHODS: Seventy-four young adult males were recruited from three Jewish higher education institutions (Yeshiva) in Jerusalem. Yeshiva-A ultra-Orthodox students (aged 20.1 ± 0.6) wear traditional clothing, live in dormitories and stay mostly indoor. Yeshiva-B ultra-Orthodox students (aged 33.0 ± 4.2) dress similarly but have regular outdoor activities. Yeshiva-C religious students (aged 19 ± 2.0) participate in a mixed army/Yeshiva program. Weekly outdoor activity time and degree of sun exposure were estimated by questionnaire. RESULTS: 25(OH)D was 8.9 ± 3.6, 10.2 ± 5.7 and 21.7 ± 10.4 ng/ml (mean ± SD) in Yeshiva A, B and C. 25(OH)D was correlated with degree of sun exposure (r = 0.54, p < 0.0001) and inversely correlated with PTH (r = -0.3, p = 0.01). Levels below 20 ng/ml were considered as vitamin D deficiency. PTH was normal in 87% of vitamin D-deficient subjects from Yeshiva-A and Yeshiva-C (mean age 20), compared to 52% of Yeshiva-B students (mean age 33). Bone mineral density studied in a random subset (n = 14) of vitamin D-deficient subjects showed Z-scores of -1.5 ± 1.0, -1.8 ± 0.8, -2.1 ± 0.4 in femoral neck, spine and radius. CONCLUSIONS: Severe vitamin-D deficiency is extremely prevalent in ultra-Orthodox males. Despite rare secondary hyperparathyroidism, they represent an important previously unrecognized high-risk group for metabolic bone disease.

Increased urinary N-telopeptide cross-linked type 1 collagen predicts bone loss in patients with inflammatory bowel disease.

OBJECTIVE: Reduced bone mineral density (BMD) is common in patients with inflammatory bowel disease (IBD), but the factors associated with its longitudinal rate of change have not been established. We prospectively assessed the rate of change in BMD, and its association with biochemical markers of bone turnover. METHODS: Twenty-two patients with Crohn's disease and 14 ulcerative colitis patients age 37.1 +/- 11.6 yr were followed for 2 yr. Lumbar spine (L2-L4) and femoral neck BMD were measured by dual x-ray absorptiometry at baseline and 24 months. Bone-specific alkaline phosphatase, osteocalcin, urinary N-telopeptide crosslinked type 1 collagen (NTx), parathyroid hormone, and 25-hydroxyvitamin-D were determined at baseline. RESULTS: At baseline, 59% of Crohn's patients and 43% of ulcerative colitis patients were osteoporotic, with spine or femoral neck BMD T-score < -2.5. Spine BMD, and spine and femoral neck T-scores were lower and disease duration was longer in nine patients with ileal resection compared with nonoperated patients (0.84 +/- 0.15 g/cm2 vs 0.96 +/- 0.11 g/cm2, -3.0 +/- 1.5 vs -1.7 +/- 1.3, -3.2 +/- 1.5 vs -2.2 +/- 1.0, respectively; all p < 0.05). At 24 months, 13/36 (36%) and 14/36 (39%) patients experienced spinal and femoral neck bone loss, respectively, with mean annual percent BMD changes of -2.0% and -1.5%, respectively. NTx, a bone resorption marker, inversely correlated with spinal BMD rate of change (r = -0.4, p < 0.05). Using quartiles analysis, patients with the highest NTx (Q4) experienced the greatest decrease in spine BMD compared with patients with the lowest NTx (Q1). CONCLUSIONS: Spine and femoral neck bone loss continues over time in more than one-third of IBD patients. Increased NTx level predicts spinal bone loss in IBD patients.

The decrease in serum bone-specific alkaline phosphatase predicts bone mineral density response to hormone replacement therapy in early postmenopausal women.

Hormone replacement therapy (HRT) prevents bone loss in postmenopausal women. Up to 20% of women demonstrate no increase in bone mineral density (BMD) on HRT. We examined whether early changes in serum bone alkaline phosphatase (B-ALP) predict long-term BMD changes in postmenopausal women on HRT. Ninety women within 1 year of menopause were randomly assigned to continuous or sequential estrogen/progestin (beta estradiol/norethisterone acetate) if naturally postmenopausal, or beta estradiol if within 1 month of surgical menopause. Spine, femoral neck BMD (DXA), and B-ALP were determined over 2 years. The mean percent BMD changes were 3.8%, 2.9%, 1.6% in the spine and 2.4%, 4.0%, 1.1% in the femoral neck in sequential, continuous, and estrogen alone treatment groups, respectively, significantly different from zero except for femoral neck BMD change in the estrogen alone group. HRT was associated with spine and femoral neck BMD loss in 17.4% and 25.3% of women, respectively. In estrogen/progestin-treated women, baseline B-ALP correlated with spine BMD change (r = 0.42, P < 0.01). At 3 months, B-ALP dropped significantly in the estrogen/progestin-groups with a maximal decrease at 12 months, but no change from baseline in the estrogen alone group. Using quartile analysis, women with the greatest drop in B-ALP (> or = 50%) at 6 months demonstrated the greatest gain in spine BMD at 2 years. A 40% decrease at 6 months in B-ALP had a 56% sensitivity, 83% specificity, 95% positive predictive value for spine BMD gain at 2 years. The decrease in B-ALP can be used to monitor BMD response to HRT.

Diurnal variation of bone mineral turnover in elderly men and women.

The diurnal variation of markers of bone mineral metabolism have been documented in pre- and early postmenopausal women. Such rhythms have clinical implications for timing of sample collection and assessment of therapeutic intervention. To examine the diurnal variation of bone turnover in the elderly, we examined markers of bone formation [serum osteocalcin (OC) and bone-specific alkaline phosphatase (B-ALP)]; a marker of bone resorption (urinary N-telopeptide cross-linked collagen type 1 [NTX]); and serum calcium and parathyroid hormone (PTH) over 24 hours. Subjects were healthy community-dwelling elderly who were on no medications known to significantly alter bone mineral metabolism. Subjects included 14 women [74 +/- 6 years (mean +/- SD)] and 14 men (80 +/- 5 years). Over the 24-hour sampling period, mean serum OC, B-ALP, and calcium values were similar in elderly men and women. However, mean serum PTH was significantly higher in elderly men compared with women (P < 0.05). The magnitude of the diurnal variation of urinary NTX was significantly higher in women compared with men (P < 0.05). There was a significant diurnal variation for serum OC, B-ALP, calcium, PTH, and urinary NTX in both elderly men and women. The magnitude of the diurnal variation was approximately 10-20% of mean value for OC and B-ALP, 30% for PTH, and up to 40% for urinary NTX. We conclude that there is significant diurnal variation in the markers of bone mineral metabolism for elderly men and women. The peak value, which on average would be 20% higher than the mean value for urinary NTX, highlights the importance of the timing of sample collection for appropriate interpretation of therapeutic response. In addition, gender-related differences, including relatively higher levels of serum PTH and lower levels of urinary NTX in elderly men, may help explain differences in rates of bone loss in this age group.

Biochemical markers of bone turnover reflect femoral bone loss in elderly women.

Although over 90% of hip fractures occur in patients over age 70, few data are available on femoral bone loss in this age group. To examine the relationship between biochemical markers of bone turnover and femoral bone loss in the elderly, 36 female and 17 male, healthy, community-dwelling elderly over age 65 (mean +/- SD age: women 71 +/- 4 years, men 75 +/- 5 years) were followed for 3 years. Annual bone mineral density measurements of the hip and lumbar spine by dual-energy x-ray absorptiometry (DXA) were obtained and biochemical markers of bone resorption (urinary N-telopeptide crosslinks, free pyridinoline, total pyridinoline, total deoxypyridinoline, and hydroxyproline) and bone formation (serum osteocalcin, bone-specific alkaline phosphatase) were obtained at the end of year 3. In elderly women, longitudinal bone loss at the total hip was negatively correlated with markers of bone resorption (r = -0.39 to -0.52, P < 0.05), bone formation (r = -0.38, P < 0.05), and age (r = -0.39, P < 0.05). Markers of bone resorption were correlated with markers of bone formation (r = 0.63 to 0.74, P < 0. 01). In multiple regression analysis, urinary N-telopeptide crosslinks (marker of resorption), serum osteocalcin (marker of formation), and serum parathyroid hormone explained 43% of the variability of bone loss at the total hip in women. These parameters were not related to bone loss in men. We conclude that femoral bone loss increases with age in women over 65. Measurements of specific biochemical markers of bone turnover are correlated with longitudinal bone loss in elderly women. These markers may help identify women at greatest risk for bone loss who would benefit most from therapeutic interventions.

Characteristics of falls in 70 year olds in Jerusalem.

Falling is a serious and common problem among the elderly and may result in injury and functional deterioration. The characteristics and risk factors for falling were studied in a cross-sectional study of a cohort of 70-year-old community-dwelling Jewish elderly in Jerusalem. The cohort was found to be representative of the total 70-year-old population of Jerusalem by virtue of having similar hospitalization and mortality rates. Around 28.2% of the cohort reported falling during the past year. Women were more likely than men to have fallen during the past year (39 vs. 19%; P < 0.001). Falls occurred mainly outside of the home, resulting in fractures in 8.4% of persons who fell, all of them women. A female gender, low visual efficiency, poor self-reported general health status, low serum hemoglobin levels, and low lymphocyte counts were associated with a higher incidence of falls. There was no relationship between the pattern of pharmaceutic drug use or utilization of health care services and the risk of falling. We conclude that among the younger elderly, characteristics of falls and risk factors may be different from those in the older elderly.

Evaluation in vivo of a potent parathyroid hormone antagonist: [Nle8,18,D-Trp12,Tyr34]bPTH(7-34)NH2.

In an effort to design and select potent parathyroid hormone (PTH) antagonists suitable for clinical utility, a PTH analog was evaluated in vivo in an animal model to assess its properties in preparation for human studies. The previously described PTH antagonist, [Nle8,18,D-Trp12,Tyr34]bPTH(7-34)NH2, which is highly active in vitro, was documented in these studies to be an effective antagonist of the PTH-stimulated calcemic response in vivo. In thyroparathyroidectomized (TPTX) rats, the efficacy of the antagonist was demonstrated to be dose-dependent. Inhibition was demonstrated when intravenous administration of antagonist started 1 h prior to coinfusion with the PTH agonist [Nle8,18,Tyr34]bPTH(1-34)NH2. Maximal inhibition by antagonist (an 84% decline in serum calcium levels compared with agonist alone) of the calcemic response was observed when a 200-fold molar excess of antagonist (12 nmol/h) was administered. At dose ratios of antagonist:agonist as low as 10:1, a 40-50% inhibition of PTH-stimulated calcemic response is evident, provided a longer (2 h) lead time for antagonist infusion is allowed. Based on these and related studies, the antagonist [Nle8,18,D-Trp12,Tyr34]bPTH(7-34)NH2 has displayed sufficient potency to obtain approval from the appropriate institutional and regulatory agencies for clinical trials in hypercalcemic states of parathyroid and tumor origin.

Approach to discovering novel therapeutic agents for osteoporosis based on integrin receptor blockade.

On a global scale, osteoporosis is a major and growing public health problem. In the United States, osteoporosis is present in 24 million people (mostly women) and contributes to more than 1.3 million fractures/year. Serious morbidity and mortality result from these fractures. Current therapies for osteoporosis are few, efficacy is limited, and side effects problematic. Fundamental to the pathophysiology of osteoporosis is an imbalance between the tightly coupled processes of bone resorption and bone formation that characterize normal bone remodeling. Our laboratory is engaged in a research effort focused on elucidating the role of the osteoclast integrin in bone resorption, defining the nature of ligand-integrin interactions, and developing antagonists for cell surface adhesion molecules, particularly the alpha v beta 3 vitronectin-like integrin receptor present on the surface of human osteoclasts. Peptides containing the internal arginine-glycine-aspartic acid (RGD) motif have been shown to inhibit osteoclast-mediated bone resorption in vivo. We are now designing more potent and selective inhibitors of bone resorption as a potential new mechanism-based therapeutic approach to osteoporosis based on a novel mechanism. In an effort to rapidly identify the highest affinity ligands for the human alpha v beta 3 integrin, we have generated combinatorial peptide libraries containing substantial structural diversity. For instance, based on all possible sequence combinations of extracellular matrix proteins known to bind alpha v beta 3, we recently synthesized and chemically analyzed a library of 360,000 peptides, all of which contain RGD.(ABSTRACT TRUNCATED AT 250 WORDS)

Blockade of osteoclast-mediated bone resorption through occupancy of the integrin receptor: a potential approach to the therapy of osteoporosis.

Bone resorption requires the tight attachment of the bone-resorbing cells, the osteoclasts, to the bone mineralized matrix. Integrins, a class of cell surface adhesion glycoproteins, play a key role in the attachment process. Most integrins bind to their ligands via the arginyl-glycyl-aspartyl (R-G-D) tripeptide present within the ligand sequence. The interaction between integrins and ligands results in bidirectional transfer of signals across the plasma membrane. Tyrosine phosphorylation occurs within cells as a result of integrin binding to ligands and probably plays a role in the formation of the osteoclast clear zone, a specialized region of the osteoclast membrane maintained by cytoskeletal structure and involved in bone resorption. Human osteoclasts express alpha 2 beta 1 and alpha v beta 3 integrins on their surface. Such signaling may also lead to "inside-out" effects, like increased expression of integrin receptors on the cell surface, or increased affinity of the integrin to its ligand. The alpha v beta 3 integrin, a vitronectin receptor, plays an essential role in bone resorption. Antibodies to this integrin and short synthetic RGD-containing peptides are able to block bone resorption in vitro. Echistatin, an RGD-containing protein from a snake venom, binds to the alpha v beta 3 integrin and blocks bone resorption both in vitro and in vivo. Peptides containing the RGD motif are potential competitive "antagonists" of the osteoclast integrins and may have utility in the blockade of bone resorption. Agonists may be identified by stimulation of intracellular signaling.(ABSTRACT TRUNCATED AT 250 WORDS)

Specificity of urinary excretion of cross-linked N-telopeptides of type I collagen as a marker of bone turnover.

Urinary excretion of cross-linked N-telopeptide of type I collagen (NTX) has been reported to be a specific indicator of bone resorption. We studied the utility of a new immunoassay for NTX as an indicator of changes in bone resorption caused by treatment with pamidronate (APD) followed by T3. Twenty-two male subjects received either placebo (Group 1) or APD on study days 1-2 (Group 2). One week later all subjects received T3 100 micrograms/day (days 8-15). Urinary NTX, pyridinoline (PYD), hydroxyproline (HYP), and creatinine (cr) were measured on 2-hour fasting urine samples at baseline (day 1), after APD/placebo (day 8), after T3 (day 16), and at days 30 and 58. NTX/cr excretion fell 85% after treatment with APD (P < 0.001 versus baseline), but not after placebo. The fall in mean urinary NTX after receiving APD was greater than the fall in PYD (25%) or HYP (31%) (P < 0.001 NTX versus PYD and HYP). After treatment with APD, NTX excretion remained suppressed below baseline until day 58, whereas PYD and HYP excretion returned to baseline by study day 16. Persistence of APD's effect on bone until day 58 was suggested by the fact that serum calcium and parathyroid hormone levels had not returned to baseline by day 58. On day 16, after all subjects were treated with T3, urinary NTX/cr rose significantly (P < 0.01) in Group 1 (-bisphosphonate) but not in Group 2 (+bisphosphonate).(ABSTRACT TRUNCATED AT 250 WORDS)

Parenteral pamidronate prevents thyroid hormone-induced bone loss in rats.

Pamidronate (APD) is a bisphosphonate that prevents bone loss from a variety of causes. We studied the role of APD in preventing thyroid hormone-induced bone loss. A total of 32 rats were assigned to one of four treatment groups: (1) -APD/triiodothyronine (-T3), (2) -APD/+T3, (3) +APD/-T3, or (4) +APD/+T3. In the first of two studies, the rats received APD for the first week and T3 for the second week, and then their blood was analyzed for alkaline phosphatase and osteocalcin. Alkaline phosphatase and osteocalcin were significantly higher (p < 0.05) in hyperthyroid rats (-APD/+T3, 3.9 +/- 0.25 mukat/liter and 23 +/- 1.6 nM, respectively) than in control animals (2.53 +/- 0.28 mukat/liter and 18.3 +/- 1.4 nM, respectively). Hyperthyroid rats pretreated with APD (+APD/+T3) had levels of alkaline phosphatase and osteocalcin no different from controls. In a second study, rats were divided into the same four groups, except they received APD/placebo and T3/placebo concomitantly for 3 weeks. At the end of the study, bone mineral density (BMD) of the femur, spine, and whole body was measured by dual-energy x-ray absorptiometry, and the calcium content of the femora was measured directly. In hyperthyroid rats (-APD/+T3) BMD was significantly lower than in controls in the spine (0.201 +/- 0.004 versus 0.214 +/- 0.002 g/cm2, p < 0.05) and femur (0.204 +/- 0.003 versus 0.218 +/- 0.002, p < 0.05).(ABSTRACT TRUNCATED AT 250 WORDS)