Osteoporosis and obesity are chronic disorders that are increasing in prevalence. The pathophysiology of these diseases is multifactorial and includes genetic, environmental and hormonal determinants. Long considered as distinct disorders that rarely are found in the same individual, emerging evidence from basic and clinical studies support an important interaction between adipose tissue and the skeleton. Adiposity can influence bone remodeling through three possible mechanisms including secretion of cytokines that directly target bone, adipokines that influence the central nervous system thereby changing sympathetic impulses to bone, and paracrine influences on adjacent skeletal cells. This review will focus on our current understanding of bone-fat interactions and the clinical implications of recent studies linking obesity to osteoporosis.
Bone has classically been viewed as an inert structure that is necessary for mobility, calcium homeostasis, and maintenance of the hematopoietic niche. Recent advances in bone biology using complex genetic manipulations in mice have highlighted the importance of bone not only as a structural scaffold to support the human body, but also as a regulator of a number of metabolic processes that are independent of mineral metabolism. These advances point to the skeleton as an endocrine organ that modulates glucose tolerance and testosterone production by secretion of a bone-specific protein, osteocalcin. This review will detail how bone has emerged as a bona-fide endocrine “gland”, and with that, the potential therapeutic implications that could be realized for this hormone-secreting tissue.
Vitamin D an ancient secosteroid is essential for mineral homeostasis, bone remodeling, immune modulation, and energy metabolism. Recently, debates have emerged about the daily vitamin D requirements for healthy and elderly adults, the safety and efficacy of long term supplementation and the role of vitamin D deficiency in several chronic disease states. Since this molecule acts as both a vitamin and a hormone, it should not be surprising that the effects of supplementation are multi-faceted and complex. Yet despite significant progress in the last decade, our understanding of vitamin D physiology and the clinical relevance of low circulating levels of this vitamin remains incomplete. The present review provides the reader with a comprehensive and up-to-date understanding of vitamin D requirements and safety. It also raises some provocative research questions.
Vitamin D; diagnosis; treatment
Caloric restriction is associated with a reduction in body weight and temperature but a reduction in trabecular bone volume and paradoxically an increase in adipocytes within the bone marrow. The nature of these adipocytes is uncertain, although there is emerging evidence of a direct relationship between bone remodeling and brown adipocytes. For example, in heterotropic ossification, brown adipocytes set up a hypoxic gradient that leads to vascular invasion, chondrocyte differentiation and subsequent bone formation. Additionally, deletion of retinoblastoma protein in an osteosarcoma model leads to increased hibernosarcoma (brown fat tumor). Interestingly, brown adipose tissue (BAT) senescences with age at a time when thermoregulation becomes altered,, bone loss becomes apparent and sympathetic activity increases. Interestingly, heart rate is an unexpected but good predictor of fracture risk in elderly individuals, pointing to a key role for the sympathetic nervous system in senile osteoporosis. Hence the possibility exists that BAT could play an indirect role in age-related bone loss. However, evidence of an indirect effect from thermogenic dysfunction on bone loss is currently limited. Here, we present current evidence for a relationship between brown adipose tissue and bone as well as provide novel insights into the effects of thermoregulation on bone mineral density.
The role of circadian proteins in regulating whole body metabolism and bone turnover has been studied in detail and has led to the discovery of an elemental system for timekeeping involving the core genes Clock, Bmal1, Per, and Cry. Nocturnin, a peripheral circadian-regulated gene has been shown to play a very important role in regulating adipogenesis by deadenylation of key mRNAs and intra-cytoplasmic transport of PPARγ. The role that it plays in osteogenesis has previously not been studied in detail. In this report we examined in vitro and in vivo osteogenesis in the presence and absence of Nocturnin and show that loss of Nocturnin enhances bone formation and can rescue Rosiglitazone induced bone loss in mice. The circadian rhythm of Nocturnin is likely to be an essential element of marrow stromal cell fate.
Nocturnin; rosiglitazone; PPARγ
Insulin-like growth factor (IGF)-I is important in the acquisition and maintenance of both soft and hard tissues. Skeletal remodeling requires energy and recent work has demonstrated that bone can influence insulin sensitivity and thereby regulate metabolic processes. New insights from mouse models into the role of IGF-binding proteins (IGFBPs) as more than mere depots for the IGFs has reignited investigations into the metabolic targets influenced by the IGF regulatory system and the pathways that link bone to adipose tissue. Although there remains continued uncertainty about the relative balance between the effects of circulating vs tissue IGF-I actions, the role of the IGFBPs has been redefined both as modulators of IGF-I action and as independent signaling factors. This review highlights several recent findings that shed new light on the physiologic role of the IGF regulatory system and its influence on skeletal and fat metabolism.
Osteoporosis, a syndrome characterized by thin bones and fractures, has become more prevalent in both women and men. Established therapies for this disorder consist primarily of drugs that prevent bone loss, such as the bisphosphonates and selective estrogen receptor modulators. Although these drugs have been shown to reduce fractures in randomized trials, there is an urgent need for treatments that could lower fracture risk further without additional adverse effects. The introduction of parathyroid hormone (teriparatide), which significantly increases bone mineral density, albeit for a relatively short duration, raised expectations that drugs which stimulate bone formation might cure osteoporosis. After outlining current approaches to treating osteoporosis, this review focuses on emerging therapeutic opportunities for osteoporosis that are based on recent insights into skeletal physiology. Such novel strategies offer promise for not only reducing age-related bone loss and the associated risk of fractures, but restoring bone mineral density to healthy levels.
Only three decades ago adipose tissue was considered inert with little relationship to insulin resistance. Similarly bone has long been thought purely in its structural context. In the last decade, emerging evidence has revealed important endocrine roles for both bone and adipose tissue. The interaction between these two tissues is remarkable. Bone marrow mesenchymal stem cells give rise to both osteoblasts and adipocytes. Leptin and adiponectin, two adipokines secreted by fat tissue, control energy homeostasis, but also have complex actions on the skeleton. In turn, the activities of bone cells are not limited to their bone remodeling activities, but also to modulation of adipose sensitivity and insulin secretion. This review will discuss these new insights linking bone remodeling to the control of fat metabolism and the association between diabetes mellitus and osteoporosis.
Osteoporosis; diabetes mellitus; obesity; leptin; osteocalcin
Aging is associated with profound changes in bone mass and body composition. Emerging evidence supports the hypothesis that alterations in mesenchymal stromal cell fate are a critical etiologic factor. In addition, timekeeping at the cellular level is affected as aging progresses, particularly in the adipocyte. In this Extra View we discuss the interactive role of three molecules, PPARγ, nocturnin and IGF-I, in regulating stem cell fate in the marrow and the potential implications of this network for understanding cellular aging.
aging; bone; PPARγ; nocturnin; IGF-I
Recent studies suggest a link between brown adipose tissue (BAT) and bone. The purpose of our study was to investigate the effects of BAT on femoral bone structure.
Materials and Methods
We studied 105 patients (19 m, 86 f, mean age 45.5±16.1 y) who underwent F18-FDG positron emission tomography/computed tomography (PET/CT) for benign etiologies (n=20) or follow-up of successfully treated malignancies (n=85); mean time between PET/CT and last form of treatment was 14.8±18.0 months. BAT volume by PET/CT; femoral bone structure by CT (total femoral cross-sectional area (CSA), cortical CSA); thigh muscle CSA and thigh subcutaneous fat CSA by CT were assessed.
There were positive correlations between BAT volume and total femoral CSA and cortical CSA, independent of age, BMI and history of malignancy (P<0.05). BAT volume correlated positively with thigh muscle CSA and thigh fat CSA (p<0.05). When total femoral CSA was entered as a dependent variable and BAT volume, age and BMI as independent variables in a forward stepwise regression model, BAT volume was the only predictor of total femoral CSA. When femoral cortical CSA was entered as a dependent variable and BAT volume, age and BMI as independent variables, BAT volume was the only predictor of femoral cortical CSA.
BAT volume is a positive predictor of femoral bone structure and correlates positively with thigh muscle and subcutaneous fat, possibly mediated by muscle. These results provide further evidence of a positive effect of BAT on bone.
brown adipose tissue (BAT); bone; structure; muscle; fat
Adipocytes reside in discrete, well-defined depots throughout the body. In addition to mature adipocytes, white adipose tissue depots are composed of many cell types, including macrophages, endothelial cells, fibroblasts, and stromal cells, which together are referred to as the stromal vascular fraction (SVF). The SVF also contains adipocyte progenitors that give rise to mature adipocytes in those depots. Marrow adipose tissue (MAT) or marrow fat has long been known to be present in bone marrow (BM) but its origin, development, and function remain largely unknown. Clinically, increased MAT is associated with age, metabolic diseases, drug treatment, and marrow recovery in children receiving radiation and chemotherapy. In contrast to the other depots, MAT is unevenly distributed in the BM of long bones. Conventional quantitation relies on sectioning of the bone to overcome issues with distribution but is time-consuming, resource intensive, inconsistent between laboratories and may be unreliable as it may miss changes in MAT volume. Thus, the inability to quantitate MAT in a rapid, systematic, and reproducible manner has hampered a full understanding of its development and function. In this chapter, we describe a new technique that couples histochemical staining of lipid using osmium tetroxide with microcomputerized tomography to visualize and quantitate MAT within the medullary canal in three dimensions. Imaging of osmium staining provides a high-resolution map of existing and developing MAT in the BM. Because this method is simple, reproducible, and quantitative, we expect it will become a useful tool for the precise characterization of MAT.
Bone marrow adipogenesis is a normal physiologic process in all mammals. However, its function is unknown. The mesenchymal stem cell is the marrow precursor for adipocytes as well as osteoblasts, and PPARγ is an essential differentiation factor for entrance into the fat lineage. Mouse models have provided significant insight into the molecular cues that define stromal cell fate. In humans, accelerated marrow adipogenesis has been associated with aging and several chronic conditions including diabetes mellitus and osteoporosis. Newer imaging techniques have been used to determine the developmental time course of fat generation in bone marrow. However, more studies are needed to understand the interrelationship among hematopoietic, osteoblastic, and adipogenic cells within the marrow niche.
adipogenesis; marrow stromal cells; osteoblastogenesis; bone mass; aging
Insulin-like growth factor 1 (IGF-1) is an unique peptide that functions in an endocrine/paracrine and autocrine manner in most tissues. Although it was postulated initially that liver-derived IGF-1 was the major source of IGF-1 (that is, the somatomedin hypothesis), it is also produced in a wide variety of tissues and can function in numerous ways as both a proliferative and differentiative factor. One such tissue is bone and all cell lineages in the skeleton have been shown to not only require IGF-1 for normal development and function but also to respond to IGF-1 via the IGF-1 receptor. Ligand-receptor activation leads to several distinct downstream signaling cascades, which have significant implications for cell survival, protein synthesis and energy utilization. The novel role of IGF-1 in regulating metabolic demands of the bone remodeling unit is currently under investigation. More studies are likely to shed new light on various aspects of skeletal physiology and potentially may lead to new therapeutics.
Despite a wealth of clinical data showing an association between inflammation and degenerative disorders in elderly, the immune sensors that causally link systemic inflammation to aging remain unclear. Here we detail a mechanism that the Nlrp3 inflammasome controls systemic low grade age-related ‘sterile’ inflammation in both periphery and brain independently of the non-canonical caspase-11 inflammasome. Ablation of Nlrp3 inflammasome protected mice from age-related increases in the innate immune activation, alterations in CNS transcriptome and astrogliosis. Consistent with the hypothesis that systemic low grade inflammation promotes age-related degenerative changes, the deficient Nlrp3 inflammasome mediated caspase-1 activity improved glycemic control and attenuated bone loss and thymic demise. Notably, IL-1 mediated only Nlrp3 inflammasome dependent improvement in cognitive function and motor performance in aged mice. These studies reveal Nlrp3 inflammasome as an upstream target that controls age-related inflammation and offer innovative therapeutic strategy to lower Nlrp3 activity to delay multiple age-related chronic diseases.
Inflammation; T cells; brain; macrophages; Pycard; IL-1β; aging; caspase-1; ASC; LPS; dementia; Lipotoxicity; complement; interferon; dentate gyrus
Fat mass may be modulated by the number of brown-like adipocytes in white adipose tissue (WAT) in humans and rodents. Bone remodeling is dependent on systemic energy metabolism and, with age, bone remodeling becomes uncoupled and brown adipose tissue (BAT) function declines. To test the interaction between BAT and bone, we employed Misty (m/m) mice, which were reported be deficient in BAT. We found that Misty mice have accelerated age-related trabecular bone loss and impaired brown fat function (including reduced temperature, lower expression of Pgc1a and less sympathetic innervation compared to wildtype (+/+)). Despite reduced BAT function, Misty mice had normal core body temperature, suggesting heat is produced from other sources. Indeed, upon acute cold exposure (4°C for 6 hr), inguinal WAT from Misty mice compensated for BAT dysfunction by increasing expression of Acadl, Pgc1a, Dio2 and other thermogenic genes. Interestingly, acute cold exposure also decreased Runx2 and increased Rankl expression in Misty bone, but only Runx2 was decreased in wildtype. Browning of WAT is under the control of the sympathetic nervous system (SNS) and, if present at room temperature, could impact bone metabolism. To test whether SNS activity could be responsible for accelerated trabecular bone loss, we treated wildtype and Misty mice with the β-blocker, propranolol. As predicted, propranolol slowed trabecular BV/TV loss in the distal femur of Misty mice without affecting wildtype. Finally, the Misty mutation (a truncation of DOCK7) also has a significant cell-autonomous role. We found DOCK7 expression in whole bone and osteoblasts. Primary osteoblast differentiation from Misty calvaria was impaired, demonstrating a novel role for DOCK7 in bone remodeling. Despite the multifaceted effects of the Misty mutation, we have shown that impaired brown fat function leads to altered SNS activity and bone loss, and for the first time that cold exposure negatively affects bone remodeling.
bone; brown adipose tissue; DOCK7; Misty; thermogenesis
Adolescents with anorexia nervosa (AN) are amenorrheic and have decreased bone mass accrual and low bone mineral density (BMD). The regulation of mesenchymal stem cell differentiation is an important factor governing bone formation. Preadipocyte factor 1 (Pref-1), an inhibitor of adipocyte and osteoblast differentiation, is elevated in states of estrogen deficiency. In this study, we aim to (i) investigate effects of transdermal estradiol on Pref-1 in adolescent girls with AN, and (ii) examine associations of changes in Pref-1 with changes in lumbar BMD and bone turnover markers.
Adolescent girls with AN and normal-weight controls were studied cross-sectionally. Girls with AN were examined longitudinally in a double-blind study and received transdermal estradiol (plus cyclic medroxyprogesterone) or placebo for twelve months.
69 girls (44 with AN, 25 normal-weight controls) 13–18 years were studied at baseline; 22 AN girls were followed prospectively.
Pref-1 levels, bone formation and resorption markers, and BMD.
Pref-1 levels decreased in girls with AN after treatment with transdermal estradiol compared with placebo (−0.015±0.016 vs. 0.060±0.026 ng/ml, p=0.01), although at baseline, levels did not differ in AN versus controls (0.246±0.015 vs. 0.267±0.022 ng/ml). Changes in Pref-1 over twelve months correlated inversely with changes in lumbar BMD (r=−0.48, p=0.02) and positively with changes in CTX (r=0.73, p=0.006).
For the first time, we show that Pref-1 is negatively regulated by estradiol in adolescent girls with AN. Inhibition of Pref-1 may mediate the beneficial effects of transdermal estradiol replacement on BMD in girls with AN.
anorexia nervosa; Pref-1; osteoporosis
Rapamycin has been shown to extend lifespan in numerous model organisms including
mice, with the most dramatic longevity effects reported in females. However, little is
known about the functional ramifications of this longevity-enhancing paradigm in mammalian
tissues. We treated 24-month-old female C57BL/6J mice with rapamycin for 3 months and
determined health outcomes via a variety of noninvasive measures of cardiovascular,
skeletal, and metabolic health for individual mice. We determined that while rapamycin has
mild transient metabolic effects, there are significant benefits to late-life
cardiovascular function with a reversal or attenuation of age-related changes in the
heart. RNA-seq analysis of cardiac tissue after treatment indicated inflammatory,
metabolic, and antihypertrophic expression changes in cardiac tissue as potential
mechanisms mediating the functional improvement. Rapamycin treatment also resulted in
beneficial behavioral, skeletal, and motor changes in these mice compared with those fed a
control diet. From these findings, we propose that late-life rapamycin therapy not only
extends the lifespan of mammals, but also confers functional benefits to a number of
tissues and mechanistically implicates an improvement in contractile function and
antihypertrophic signaling in the aged heart with a reduction in age-related
aging; hypertrophy; strain echocardiography; rapamycin; RAD; mTOR; RNA-seq
States of growth hormone (GH) resistance, such those observed in Laron’s dwarf patients, are characterized by mutations in the GH receptor (GHR), decreased serum and tissue IGF-1 levels, impaired glucose tolerance, and impaired skeletal acquisition. IGF-1 replacement therapy in such patients increases growth velocity but does not normalize growth. Herein we combined the GH-resistant (GHR knockout, GHRKO) mouse model with mice expressing the hepatic Igf-1 transgene (HIT) to generate the GHRKO-HIT mouse model. In GHRKOHIT mice, serum IGF-1 levels were restored via transgenic expression of Igf-1 allowing us to study how endocrine IGF-1 affects growth, metabolic homeostasis, and skeletal integrity. We show that in a GH-resistant state, normalization of serum IGF-1 improved body adiposity and restored glucose tolerance but was insufficient to support normal skeletal growth, resulting in an osteopenic skeletal phenotype. The inability of serum IGF-1 to restore skeletal integrity in the total absence of GHR likely resulted from reduced skeletal Igf-1 gene expression, blunted GH-mediated effects on the skeleton that are independent of serum or tissue IGF-1, and from poor delivery of IGF-1 to the tissues. These findings are consistent with clinical data showing that IGF-I replacement therapy in patients with Laron’s syndrome does not achieve full skeletal growth.
IGF-1; growth hormone receptor; bone; micro-computed tomography; betaislet; glucose tolerance
Patient bone mineral density (BMD) predicts the likelihood of osteoporotic fracture.
While substantial progress has been made toward elucidating the genetic determinants of
BMD, our understanding of the factors involved remains incomplete. Here, using a systems
genetics approach in the mouse, we predicted that bicaudal C homolog 1
(Bicc1), which encodes an RNA-binding protein, is responsible for a BMD
quantitative trait locus (QTL) located on murine chromosome 10. Consistent with this
prediction, mice heterozygous for a null allele of Bicc1 had low BMD. We
used a coexpression network–based approach to determine how Bicc1
influences BMD. Based on this analysis, we inferred that Bicc1 was
involved in osteoblast differentiation and that polycystic kidney disease 2
(Pkd2) was a downstream target of Bicc1. Knock down of
Bicc1 and Pkd2 impaired osteoblastogenesis, and
Bicc1 deficiency–dependent osteoblast defects were rescued by
Pkd2 overexpression. Last, in 2 human BMD genome-wide association
(GWAS) meta-analyses, we identified SNPs in BICC1 and
PKD2 that were associated with BMD. These results, in both mice and
humans, identify Bicc1 as a genetic determinant of osteoblastogenesis and
BMD and suggest that it does so by regulating Pkd2 transcript levels.
Recent studies have shown a positive correlation between brown adipose tissue (BAT) and bone mineral density (BMD). However, mechanisms underlying this relationship are unknown. Insulin-like growth factor 1 (IGF-1) is an important regulator of stem cell differentiation promoting bone formation. IGF binding protein 2 (IGFBP-2) binds IGF-1 in the circulation and has been reported to inhibit bone formation in humans. IGF-1 is also a crucial regulator of brown adipocyte differentiation. We hypothesized that IGFBP-2 is a negative and IGF-1 a positive regulator of BAT-mediated osteoblastogenesis. We therefore investigated a cohort of 15 women (mean age 27.7±5.7 years): 5 with anorexia nervosa (AN) in whom IGF-1 levels were low due to starvation, 5 recovered AN (AN-R), and 5 women of normal weight. All subjects underwent assessment of cold-activated BAT by PET/CT, BMD of the spine, hip, femoral neck, and total body by DXA, thigh muscle area by MRI, IGF-1 and IGFBP-2. There was a positive correlation between BAT and BMD and an inverse association between IGFBP-2 and both BAT and BMD. There was no association between IGF-1 and BAT. We show for the first time that IGFBP-2 is a negative predictor of cold-induced BAT and BMD in young non-obese women, suggesting that IGFBP-2 may serve as a regulator of BAT-mediated osteoblastogenesis.
brown adipose tissue (BAT); IGFBP-2; bone mineral density (BMD); anorexia nervosa (AN)
In tissues with complex architectures such as bone, it is often difficult to purify and characterize specific cell types via molecular profiling. Single cell gene expression profiling is an emerging technology useful for characterizing transcriptional profiles of individual cells isolated from heterogeneous populations. In this study we describe a novel procedure for the isolation and characterization of gene expression profiles of single osteoblast lineage cells derived from cortical bone. Mixed populations of different cell types were isolated from adult long bones of C57BL/6J mice by enzymatic digestion, and subsequently subjected to FACS to purify and characterize osteoblast lineage cells via a selection strategy using antibodies against CD31, CD45, and Alkaline Phosphatase (AP), specific for mature osteoblasts. The purified individual osteoblast lineage cells were then profiled at the single cell level via nanofluidic PCR. This method permits robust gene expression profiling on single osteoblast lineage cells derived from mature bone, potentially from anatomically distinct sites. In conjunction with this technique, we have also shown that it is possible to carry out single cell profiling on cells purified from fixed and frozen bone samples without compromising the gene expression signal. The latter finding means the technique can be extended to biopsies of bone from diseased individuals. Our approach for single cell expression profiling provides a new dimension to the transcriptional profile of the primary osteoblast lineage population in vivo, and has the capacity to greatly expand our understanding of how these cells may function in vivo under normal and diseased states.
This article presents one viewpoint on the issues surrounding placebo-controlled trials in osteoporosis. The other Sounding Board article in this issue presents an opposing view. At NEJM.org, in a related interactive feature, the authors of each article give their Point of View about the other article. At NEJM.org, readers can participate in forming community opinion by choosing one of the viewpoints and, if they like, providing their reasons.
Marrow adipose tissue (MAT) is functionally distinct from both white and brown adipose tissue and can contribute to systemic and skeletal metabolism. MAT formation is a spatially and temporally defined developmental event, suggesting that MAT is an organ that serves important functions and, like other organs, can undergo pathologic change. The well-documented inverse relationship between MAT and bone mineral density has been interpreted to mean that MAT removal is a possible therapeutic target for osteoporosis. However, the bone and metabolic phenotypes of patients with lipodystrophy argues that retention of MAT may actually be beneficial in some circumstances. Furthermore, MAT may exist in two forms, regulated and constitutive, with divergent responses to hematopoietic and nutritional demands. In this review, we discuss the role of MAT in lipodystrophy, bone loss, and metabolism, and highlight our current understanding of this unique adipose tissue depot.
lipodystrophy; marrow fat; osteoporosis; diabetes; marrow adipose tissue
Epidemiological studies indicate that higher bone mass is associated with moderate alcohol consumption in postmenopausal women. However, the underlying cellular mechanisms responsible for the putative beneficial effects of alcohol on bone are unknown. Excessive bone turnover, combined with an imbalance whereby bone resorption exceeds bone formation, is the principal cause for postmenopausal bone loss. This study investigated the hypothesis that moderate alcohol attenuates bone turnover following menopause.
Bone mineral density (BMD) was determined by dual energy x-ray absorptiometry in 40 healthy postmenopausal women (56.3 ± 0.5 years of age, mean ± SE) who consumed 19 ± 1 g alcohol/day. Serum levels of the bone formation marker osteocalcin and resorption marker C-terminal telopeptide (CTx) were measured by immunoassay at baseline (day 0) and following alcohol withdrawal for 14 days. Participants then consumed alcohol and were assayed the following morning.
BMD at the trochanter and total hip were positively correlated to level of alcohol consumption. Serum osteocalcin and CTx increased following abstinence (4.1 ± 1.6%; p = 0.01 and 5.8 ± 2.6%; p = 0.02 compared to baseline, respectively). Osteocalcin and CTx decreased following alcohol re-administration compared to the previous day (−3.4 ± 1.4%; p = 0.01 and −3.5 ± 2.1%; p = 0.05, respectively), to values that did not differ from baseline (p > 0.05).
Abstinence from alcohol resulted in increased markers of bone turnover whereas resumption of alcohol reduced bone turnover markers. These results suggest a cellular mechanism for the increased bone density observed in postmenopausal moderate alcohol consumers. Specifically, the inhibitory effect of alcohol on bone turnover attenuates the detrimental skeletal consequences of excessive bone turnover associated with menopause.
osteoporosis; DXA; osteocalcin; CTx; ethanol; serum estradiol