Agostino Gaudio

Effects of Genistein and Hormone-Replacement Therapy on Bone Loss in Early Postmenopausal Women: A Randomized Double-Blind Placebo-Controlled Study

The natural isoflavone phytoestrogen genistein has been shown to stimulate osteoblastic bone formation, inhibit osteoclastic bone resorption, and prevent bone loss in ovariectomized rats. However, no controlled clinical trial has been performed so far to evaluate the effects of the phytoestrogen on bone loss in postmenopausal women. We performed a randomized double‐blind placebo‐controlled study to evaluate and compare with hormone‐replacement therapy (HRT) the effect of the phytoestrogen genistein on bone metabolism and bone mineral density (BMD) in postmenopausal women. Participants were 90 healthy ambulatory women who were 47–57 years of age, with a BMD at the femoral neck of <0.795 g/cm2. After a 4‐week stabilization on a standard fat‐reduced diet, participants of the study were randomly assigned to receive continuous HRT for 1 year (n = 30; 1 mg of 17β‐estradiol [E2] combined with 0.5 mg of norethisterone acetate), the phytoestrogen genistein (n = 30; 54 mg/day), or placebo (n = 30). Urinary excretion of pyridinoline (PYR) and deoxypyridinoline (DPYR) was not significantly modified by placebo administration either at 6 months or at 12 months. Genistein treatment significantly reduced the excretion of pyridinium cross‐links at 6 months (PYR = −54 ± 10%; DPYR = −55 ± 13%; p < 0.001) and 12 months (PYR = −42 ± 12%; DPYR = −44 ± 16%; p < 0.001). A similar and not statistically different decrease in excretion of pyridinium cross‐links was also observed in the postmenopausal women randomized to receive HRT. Placebo administration did not change the serum levels of the bone‐specific ALP (B‐ALP) and osteocalcin (bone Gla protein [BGP]). In contrast, administration of genistein markedly increased serum B‐ALP and BGP either at 6 months (B‐ALP = 23 ± 4%; BGP = 29 ± 11%; p < 0.005) or at 12 months (B‐ALP = 25 ± 7%; BGP = 37 ± 16%; p < 0.05). Postmenopausal women treated with HRT had, in contrast, decreased serum B‐ALP and BGP levels either at 6 months (B‐ALP = −17 ± 6%; BGP = −20 ± 9%; p < 0.001) or 12 months (B‐ALP = −20 ± 5%; BGP = −22 ± 10%; p < 0.001). Furthermore, at the end of the experimental period, genistein and HRT significantly increased BMD in the femur (femoral neck: genistein = 3.6 ± 3%, HRT = 2.4 ± 2%, placebo = −0.65 ± 0.1%, and p < 0.001) and lumbar spine (genistein = 3 ± 2%, HRT = 3.8 ± 2.7%, placebo = −1.6 ± 0.3%, and p < 0.001). This study confirms the genistein‐positive effects on bone loss already observed in the experimental models of osteoporosis and indicates that the phytoestrogen reduces bone resorption and increases bone formation in postmenopausal women.

Effects of the Phytoestrogen Genistein on Bone Metabolism in Osteopenic Postmenopausal Women: A Randomized Trial

Context Women seeking alternative treatments to preserve bone often use phytoestrogens, but evidence of their effectiveness is lacking. Phytoestrogens are found in soy products. Genistein is a phytoestrogen with a structure similar to that of 17-estradiol. Contribution This randomized trial compared genistein, 54 mg/d, with placebo for 24 months in 389 osteopenic postmenopausal women. Increases in bone mineral density were greater with genistein than with placebo. Genistein also had favorable effects on markers of bone metabolism. Genistein did not increase endometrial thickness, but it did cause gastrointestinal side effects. Implications Genistein appears to have a favorable effect on markers of bone health in osteopenic postmenopausal women. Studies of its effect on fractures are needed. The Editors Postmenopausal osteoporosis is caused by a sharp decrease in estrogen levels that leads to an increased rate of bone remodeling (13). Currently available treatments for postmenopausal osteoporosis include hormone replacement therapy; calcitonin; bisphosphonates; and selective estrogen receptor modulators, such as raloxifene (4, 5). Although hormone replacement therapy is effective in reducing postmenopausal bone loss (68), it is associated with a higher risk for breast, endometrial, and ovarian cancer; cardiovascular disease; venous thromboembolism; and stroke (810). Epidemiologic data indicate that women who ingest high amounts of phytoestrogens, particularly isoflavones in soy products, have less risk for osteoporosis than do those who consume a typical Western diet (1113). Consequently, many women use phytoestrogens to maintain bone density. Genistein, an isoflavone phytoestrogen that is abundant in soybean products, structurally resembles 17-estradiol (14). As a natural selective estrogen receptor modulator, genistein may positively regulate bone cell metabolism without harmful estrogenic activity in the breast and uterus. This safe profile results from the greater affinity of genistein for estrogen receptor-, which is more abundant in bone, than for estrogen receptor-, which is abundant in reproductive tissue. Observational studies suggest that postmenopausal Asian women who consume diets high in isoflavones have a lower rate of fracture than that in other groups (15, 16). However, the mechanism of action of genistein on bone is not yet fully understood. In postmenopausal women, treatment with genistein (54 mg/d) increased bone mineral density (BMD) at the lumbar spine and femoral neck with no clinically significant adverse effects on the breast and uterus (17). In the same cohort, genistein decreased the ratio of soluble receptor activator of nuclear factor-B ligand to osteoprotegerin, which may partly account for its positive effects on BMD (18). These investigations were the first to evaluate the effects of purified, standardized genistein on bone health, but they were short in duration and included only 90 patients. One published trial assessed the effect of isoflavones on BMD in postmenopausal women, but it compared isoflavone-rich soy milk (containing only a small amount of genistein) with natural transdermal progesterone (19). Other studies of pure genistein from our research group have focused on cardiovascular outcomes or menopausal symptoms rather than on bone health (2022). We performed a randomized, placebo-controlled trial of the effects of pure genistein on bone density and bone metabolism over 24 months in a larger cohort of osteopenic postmenopausal women. Methods Design and Setting The study protocol is consistent with the principles of the Declaration of Helsinki, and participants gave written informed consent. Participants were recruited from women reporting to the Center for Osteoporosis in the Department of Internal Medicine and the Center for Menopause in the Department of Obstetrical and Gynecological Sciences, University of Messina (Messina, Italy), and to the Department of Medical Physiopathology, University La Sapienza (Rome, Italy). Three hundred eighty-nine women met the inclusion criteria and agreed to participate (Figure 1). Figure 1. Study flow diagram. Participants Participants were women 49 to 67 years of age who had been postmenopausal for at least 12 months at baseline, were in good general health, had not had a menstrual period in the preceding year, had not undergone surgically induced menopause, and had a follicle-stimulating hormone level greater than 50 IU/L and a serum 17-estradiol level of 100 pmol/L or less (27 pg/mL). At the start of the study, a complete family history was obtained, physical examination and laboratory evaluation (chemical analytes and hematologic measurements) were performed, and BMD was measured at the lumbar spine and femoral neck. Exclusion criteria were clinical or laboratory evidence of confounding systemic diseases, such as cardiovascular, hepatic, or renal disorders; coagulopathy; use of oral or transdermal estrogen, progestin, androgens, selective estrogen receptor modulators, or other steroids; use of biphosphonates, cholesterol-lowering therapy, or cardiovascular medications (including antihypertensive drugs) in the preceding 6 months; smoking habit of more than 2 cigarettes daily; treatment in the preceding year with any drug that could have affected the skeleton; family history of estrogen-dependent cancer; and BMD at the femoral neck greater than 0.795 g/cm2 (which corresponds to a T-score of 1.0 SD). Randomization and Intervention We assigned patients to groups by using a computer-generated randomization sequence with a permuted block size of 4, stratified by center. After a 4-week stabilization period during which participants received a standard low-soy, reduced-fat diet, participants were assigned to receive genistein (n= 198), 54 mg/d in 2 tablets (Laboratori Plants, Messina, Italy), or placebo (n= 191) (Figure 1). The biological effects of phytoestrogen intake are described elsewhere (23). No patient withdrew from the study during the stabilization period. The purity of genistein was 98%. Placebo and genistein tablets were identical in appearance and taste. Both genistein and placebo tablets contained calcium carbonate (500 mg) and vitamin D (400 IU). All participants were counseled on an isocaloric, reduced-fat diet composed of 25% to 30% energy from fat, less than 10% energy from saturated fatty acids, 55% to 60% energy from carbohydrates, and 15% energy from protein, with a cholesterol intake less than 300 mg/d and fiber intake of 35 g/d or greater. Recommended daily caloric intake was based on body size and was calculated by using the HarrisBenedict equation. We used this diet during the stabilization period to ensure that all participants had the same energy intake and to avoid interference with the lipid profile. The intake of soy products, legumes, or other nutrient supplements was prohibited. The isoflavone intake before randomization, as assessed by using a food-frequency questionnaire, was 1 to 2 mg/d. This intake has been shown to be typical in Western populations (24). Participants used this diet throughout the study, and adherence was reinforced by a nutritionist. Diet and body mass index were evaluated in all participants during follow-up. Primary Outcome The BMD at the anteroposterior lumbar spine and femoral neck was measured by using dual-energy x-ray absorptiometry (Hologic QDR 4500 W, Technologic, Turin, Italy) at baseline and after 12 and 24 months of treatment. The instrument was calibrated daily according to the manufacturers instructions. Reproducibility was calculated as a coefficient of variation obtained by weekly measurements of a standard phantom on the instrument and by repeated measurements obtained in 3 patients of different ages. The coefficient of variation of our instrument is 0.5% with the standard phantom; in vivo, we calculated a coefficient of variation of 1.1% for the lumbar spine and 1.5% for the femoral neck. Secondary Outcomes Bone Resorption Markers At baseline, 12 months, and 24 months, a 2-hour fasting morning urine sample was collected at the same time of day to assess urinary excretion of pyridinium crosslinks (pyridinoline and deoxypyridinoline). Pyridinoline (normal range, 26 to 91 pmol/mol creatinine) and deoxypyridinoline (normal range, 3 to 21 pmol/mol creatinine) were measured by using high-performance liquid chromatography (Bio-Rad Laboratories, Hercules, California). Bone Formation and Bone Growth Markers and Other Variables After an overnight fast, venous blood samples were collected between 8 a.m. and 9 a.m. through a polyethylene catheter inserted in a forearm vein. The serum was separated from the blood corpuscles by centrifugation and kept frozen at 70C until analysis for bone formation and bone growth markers, calcium, intact parathyroid hormone, 25-hydroxyvitamin D3, 17-estradiol, follicle-stimulating hormone, total cholesterol, high-density lipoprotein cholesterol, low-density lipoprotein cholesterol, and triglycerides. Serum bone-specific alkaline phosphatase (normal range, 8.5 to 17.9 g/L) and insulin-like growth factor I (normal range, 9.6 to 21.2 nmol/L) were measured by using an immunoenzymatic assay (Pantec, Turin, Italy). Serum calcium (normal range, 2.25 to 2.75 mmol/L [9 to 11 mg/dL]), serum phosphorus (normal range, 1.13 to 1.45 mmol/L [3.5 to 4.5 mg/dL]), and urinary creatinine (130 to 220 molkg1d1 [14.71 to 24.89 mg/kg of body weight per day]) were measured by using automated routine procedures. Parathyroid hormone (normal range, 12 to 100 pg/dL), 25-hydroxyvitamin D3 (normal range, 1.25 to 7.5 nmol/L), and follicle-stimulating hormone (normal range, 21 to 153 IU/L in the postmenopausal phase) were measured by using high-performance liquid chromatography (Bio-Rad Laboratories). 17-Estradiol (normal range, 37 to 110 pmol/L in the postmenopausal phase) was evaluated by using a solid-phase immunoassay (Roche Diagnostics, Monza, Italy). Total cholesterol,

Increased sclerostin serum levels associated with bone formation and resorption markers in patients with immobilization-induced bone loss.

CONTEXTnSclerostin, a Wnt signaling antagonist on the osteoblasts produced by osteocytes, is regulated by mechanical strain and is implicated in the pathogenesis of disuse bone loss. There are no data on sclerostin in humans.nnnOBJECTIVEnThe aim of the study was to evaluate sclerostin in patients immobilized after stroke, compared with control subjects, and to analyze its relationship with markers of bone formation and resorption.nnnDESIGNnThis was a cross-sectional study.nnnSETTING AND PATIENTSnWe studied 40 postmenopausal women immobilized after a single episode of stroke 6 months or longer after onset, and 40 postmenopausal women from the general community. Bone status was assessed by quantitative ultrasound measurements at the calcaneus. Bone alkaline phosphatase (b-AP), carboxy-terminal telopeptide of type I collagen (CrossLaps), and sclerostin were evaluated by ELISA. We also used ELISA to measure serum levels of Dickkopf-1, another soluble inhibitor of Wnt/beta-catenin signaling, highly expressed by osteocytes.nnnRESULTSnImmobilized patients had higher sclerostin serum levels (median 0.975 ng/ml; 25th to 75th percentiles 0.662-1.490) than controls (median 0.300 ng/ml; 25th to 75th percentiles 0.165-0.400: P < 0.0001) and an increased bone turnover with a more significant rise in bone resorption (CrossLaps) than formation (b-AP) markers. Sclerostin correlated negatively with b-AP (r = -0.911; P < 0.0001) and positively with CrossLaps (r = 0.391; P = 0.012). Dickkopf-1 did not significantly differ between the groups. Patients also had quantitative ultrasound measurements index lower than controls (P < 0.001).nnnCONCLUSIONSnThis study shows for the first time that long-term immobilized patients present hypersclerostinemia associated with reduced bone formation, and suggests that sclerostin could be a link between mechanical unloading and disuse osteoporosis in humans.

Effects of the phytoestrogen genistein on hot flushes, endometrium, and vaginal epithelium in postmenopausal women: a 2-year randomized, double-blind, placebo-controlled study.

Objective: To evaluate in a 24-month, prospective, randomized, double-blind, placebo-controlled study whether pure administration of the phytoestrogen genistein (54 mg/d) might reduce the number and severity of hot flushes in postmenopausal women, with no adverse effect on the endometrium and vagina. Methods: A total of 389 participants met the parent study criteria and were randomly assigned to receive the phytoestrogen genistein (n = 198) or placebo (n = 191). About 40% of participants in both groups did not experience hot flushes, and the evaluation was performed in a subgroup of 236 participants (genistein, n = 119; placebo, n = 117). Reductions from the baseline in the frequency and severity of hot flushes were the principal criteria of efficacy. Endometrial thickness was evaluated by ultrasonography. The maturation value was also used to determine hormonal action on the vaginal cells. Results: There were no significant differences in vasomotor symptoms between groups at the baseline (4.4 ± 0.33 hot flushes per day in the genistein group and 4.2 ± 0.35 hot flushes per day in the control group). After 12 months of genistein therapy, there was a significant reduction (−56.4%) in the mean number of hot flushes, with a significant difference compared with the control group. After 24 months, there was no further decrease in the number of hot flushes in both groups. No significant difference was found in mean endometrial thickness and the maturation value score between the two groups, either at the baseline or after 24 months. Conclusions: The phytoestrogen genistein has been shown to be effective on vasomotor symptoms without an adverse effect on the endometrium and vagina, but after the first year, there was no further improvement in the decrease in hot flushes.

Sclerostin levels associated with inhibition of the Wnt/β-catenin signaling and reduced bone turnover in type 2 diabetes mellitus.

CONTEXTnPatients with type 2 diabetes (T2DM) have low bone turnover, poor bone quality, and circulating levels of sclerostin significantly higher than non-T2DM controls. There are no data on the possible association of sclerostin with β-catenin, a key component of the Wnt/β-catenin canonical signaling.nnnOBJECTIVESnThe aim of the study was to evaluate the circulating β-catenin levels in T2DM patients and to analyze their relationship with sclerostin and bone turnover markers.nnnDESIGNnThis was a cross-sectional study.nnnSETTING AND PATIENTSnThe study was conducted at a clinical research center. Forty T2DM postmenopausal women were studied and compared with 40 healthy controls. Bone status was assessed by dual-energy x-ray absorptiometry measurements (bone mineral density) and by measuring bone alkaline phosphatase and carboxy-terminal telopeptide of type 1 collagen. Sclerostin and β-catenin were evaluated by an immunoenzymetric assay.nnnRESULTSnConsistent with previous reports in T2DM subjects, we found sclerostin levels higher and bone turnover markers lower than controls. In our cohort of T2DM patients, β-catenin levels are significantly lower than in controls (median 1.22 pg/ml, 25th to 75th percentiles 0.50-2.80; and median 4.25 pg/ml, 25th to 75th percentiles 2.20-7.62, respectively; P=0.0002). β-Catenin correlated negatively with sclerostin (P<0.0001) and positively with bone alkaline phosphatase (P=0.0030) only in T2DM patients and negatively with age in both groups. Eight of the 40 T2DM patients had vertebral fractures.nnnCONCLUSIONSnThese results show for the first time that T2DM patients have serum concentrations of β-catenin lower than controls. The negative association of β-catenin with sclerostin suggests a biological effect of increased sclerostin on the Wnt signaling, which appears impaired in T2DM.

Effects of Hormonal Replacement Therapy on Bone Metabolism in Young Adults with Beta-thalassemia Major

Abstract: The aim of our cross-sectional study was to evaluate the effects of hormonal replacement therapy (HRT) on a population of young thalassemics in order to understand better the role of hypogonadism in the balance of bone metabolism. Markers of bone turnover and bone mineral density (BMD) were measured in 40 young patients (mean age 19.8 ± 4.5 years) with beta-thalassemia major: 20 subjects were biochemically eugonadal, since they were undergoing HRT (group A, treated patients), and 20 were hypogonadic, having suspended HRT (group B, untreated patients). We also examined 20 healthy control subjects (group C) matched for age, anthropometric features and sex to the study groups. Our study shows that young thalassemic patients exhibit a significant loss of cortical and trabecular bone [aBMD L2–L4: 0.886 ± 0.052 g/cm2 (group A), 0.726 ± 0.040 g/cm2 (group B), 1.083 ± 0.090 g/cm2 (group C); aBMD femoral neck: 0.890 ± 0.071 g/cm2 (group A), 0.700 ± 0.065 g/cm2 (group B), 0.934 ± 0.076 g/cm2 (group C)]. Osteoporosis is only observed at the lumbar level in treated thalassemic patients, while in untreated patients it involves the femoral neck also. Bone turnover in thalassemic patients is higher in the resorptive phase, than in the neoformation phase and this is more marked in hypogonadicuntreated patients. In conclusion, our data demonstrate the important role played by hypogonadism in the development and deterioration of osteopenia/osteoporosis in thalassemia major. Consequently, sex hormone replacement therapy represents an appropriate tool in the prevention and treatment of osteoporosis in thalassemics, probably together with bisphosphonates in cases with severely increased bone resorption.

Osteoprotegerin and RANKL in the Pathogenesis of Thalassemia-Induced Osteoporosis: New Pieces of the Puzzle†

Osteoporosis represents an important cause of morbidity in adult thalassemic patients, and its pathogenesis has not, as yet, been completely clarified. In our study, we observed that thalassemic patients showed a significantly lower OPG/RANKL ratio than normal subjects. These data are extremely important for the possible therapeutic use of RANKL antagonists such as OPG in thalassemia‐induced osteoporosis.

Bisphosphonates in the treatment of thalassemia-induced osteoporosis

Abstract: The aim of our randomized, placebo-controlled study was to investigate the effects of 2 years’ daily oral administration of alendronate or intramuscular administration of clodronate every 10 days, on bone remodeling parameters and bone mineral density (BMD), safety and tolerability in a group of osteoporotic thalassemic patients. Twenty-five young patients (mean age 26.6 ± 7.1 years) with beta-thalassemia major were randomly divided to receive placebo or 100 mg of clodronate intramuscularly every 10 days or 10 mg of alendronate per os daily. All patients took 500 mg of elemental calcium and 400 IU cholecalciferol in the evening at meal time. After 2 years, pyridinium crosslinks, which are bone resorption markers, did not differ significantly from baseline values in the placebo group, whereas they had decreased significantly in the clodronate and alendronate groups. Osteocalcin, a bone formation marker, did not change significantly in the placebo group, whereas it decreased slightly, but not significantly, in the clodronate and alendronate groups after 12 and 24 months. At the end of the study, the lumbar spine BMD had decreased significantly in the placebo group; it did not change significantly in the clodronate group; in the alendronate group it had increased but not significantly, whereas the increase was significant with respect to the placebo group. Femoral neck BMD decreased significantly in the placebo group; it did not change significantly in the clodronate group, but increased significantly in the alendronate group. No relevant side effects were recorded during our study. In conclusion, in patients with thalassemia-induced osteoporosis, the daily administration of alendronate significantly increases BMD, the most important predictor of the risk of fracture at several sites. Clodronate treatment at our dosage is ineffective in this pathology in spite of the good compliance of patients.

OPG and sRANKL Serum Concentrations in Osteopenic, Postmenopausal Women After 2‐Year Genistein Administration

Introduction: RANKL and its decoy receptor osteoprotegerin (OPG) constitute a complex physiological mediator system involved in the regulation of bone resorption and may be responsible for the homeostatic mechanism of normal bone remodeling. Genistein, an isoflavone representing 1–5% of total phytoestrogen content in soybean products, may positively regulate cellular bone metabolism, but its mechanism of action on bone is not yet fully understood.

Neridronate Prevents Bone Loss in Patients Receiving Androgen Deprivation Therapy for Prostate Cancer

Today, androgen deprivation therapy is a cornerstone of treatment for advanced prostate cancer, although it presents important complications such as osteoporosis. Neridronate, a relatively new bisphosphonate, is able to prevent bone loss in patients with prostate cancer during androgen ablation.