Plasma leptin values in postmenopausal women with osteoporosis

Obesity has a protective eff ect against osteoporosis and this eff ect has been attributed to a high body fat content. It has been shown that the leptin concentration is higher in obese patients. Leptin, the protein product of obesity gene, is a hormone produced in adipose tissue. Some studies suggest that endogenous leptin might infl uence bone metabolism in postmenopausal women. In this study, we investigated plasma leptin concentrations in postmenopausal women with osteoporosis and also analyzed the relationship between plasma leptin levels and bone mineral density (BMD) in order to understand the potential role of leptin in maintaining bone mass. Forty-two postmenopausal women with osteoporosis and thirty seven age and BMI-matched healthy postmenopausal women were included in the study. Th e mean femoral neck BMD value in the patient group was signifi cantly lower than that in the control group (.±. g/cm and .±. g/cm, respectively; p<.). Th e mean plasma leptin concentration in the patient group was not signifi cantly diff erent from that in the control group (p>.). Plasma leptin levels were correlated with BMI in both groups (p<. in the patient group and p=. in controls). Th ere was also a strong positive correlation between plasma leptin levels and fat in both groups (p<. in the patient group and p<. in controls). But there was no correlation between plasma leptin levels and femoral neck BMD values in both groups. Our results do not support the hypothesis that leptin itself plays an important role in maintaining bone mass in postmenopausal women. ©  Association of Basic Medical Sciences of FB&H. All rights reserved


INTRODUCTION
Obesity is considered as one of the protecting factors against osteoporosis.Obese women usually show an increased bone mineral density (BMD) [].Although the exact mechanism is unclear, bone protective eff ects of obesity may involve increased aromatization of androgen to estrogen in adipose tissue [,], lowered sex hormone binding globulin levels [], direct eff ects of high circulating levels of insulin on bone formation [,] or mechanical loading on bone tissue [,].Leptin, the protein product of obesity (ob) gene, produced in adipose tissue and plays an important role in regulating of food intake and energy expenditure [].It has been shown that the leptin concentration is higher in obese patients, and also, reduction in body weight results in a signifi cant decrease in leptin concentration [].Th omas et al. [] showed that leptin acts on human bone marrow stromal cells to enhance osteoblastic diff erentiation in their in vitro study.Recent basic and clinical studies suggest that endogenous leptin might play some physiological role in maintaining bone mass and bone quality in postmenopausal women [, ].On the other hand, some studies do not emphasize such an association between serum leptin and bone mineral density in postmenopausal women [,].However, very little is known about the effects of leptin on bone metabolism in adult women.In this study we assessed plasma leptin concentrations,  fat and BMD values in either osteoporotic and healthy postmenopausal women.We also analyzed the relationship between plasma leptin levels and BMD in order to understand the potential role of leptin to determine bone mass.

Patients
Forty-two postmenopausal women with osteoporosis (mean ages of . ± . years and body mass index (BMI) of . ± . kg/m  ) who admitted to our outpatient clinic were included in the study.Th irty seven age and BMI-matched healthy postmenopausal women (mean ages of . ± . years and BMI of . ± . kg/m  ) who were selected from the same clinic served as controls.All subjects who had been natural postmenopausal for at least  year, gave written informed consent to participate in the study which was carried out in accordance with the Helsinki declaration.We excluded the subjects who had diabetes mellitus, cardiovascular disease, thyroid disorder or metabolic bone diseases and a history of trauma or smoking habits.None were taking medication infl uencing bone metabolism.Subjects were also excluded if they had vertebral compression fractures on lateral spine radiographs.Height and weight of all patients and controls were measured by standard procedure.BMI was calculated as the weight in kg per height in m  .

Biochemical measurements
Blood sampling Specimens were collected using standard venipuncture technique as - h fasting blood and allowed to clot for  minutes.Then all samples were separated by centrifugation for  minutes at  g and stored at - o C until analysis day for leptin determinations.

Enzyme-Linked Immunosorbent Assay (ELISA) for quantitative measurement of leptin
The procedure (Diagnostic Systems Laboratories, Inc., Webster, Texas, USA) is an enzymatically amplified 'two-step' sandwich-type immunoassay.Intra-assay coefficient of variation is . for . ± . ng/ml.

BMD and body composition measurements
The diagnosis of osteoporosis was based on femoral neck BMD measurements.Patients with femoral neck BMD . standard deviations below a reference range established using our own data obtained from a Turkish population of normal healthy women using dual energy X-ray absorptiometry (T score less than -.) were accepted as having osteoporosis.BMD values at the femoral neck and percentage body fat (fat mass as percentage of body weight) were measured by dual energy X-ray absorptiometry using Norland XR -WBL (Fort Atkinson, WI, USA).Coefficient of variation of fat mass measurement was ..

Statistical analysis
Associations are given as Pearson's correlation coefficients.Student t-tests were used for comparisons between groups.All tests were two-tailed, and a  significance level was maintained.These calculations were performed using the SPSS software, version . for Windows.

RESULTS
Th e clinical characteristics of the patient and control groups at baseline are shown in Table .Th e mean femoral neck BMD value in the patient group (. ± . g/cm  , mean ± SD) was signifi cantly lower than that in the control group (. ± . g/cm  , mean ± SD; p<.).Th ere was no signifi cant diff erence in plasma leptin concentrations of both groups (p=.).
Plasma leptin levels were correlated with BMI in both groups (r=.,p<. in the patient group and r=., p=. in controls) (Figure ).Th ere was also a strong positive correlation between plasma leptin levels and percentage body fat in both groups (r=.,p<. in the patient group and r=., p<. in controls) (Figure ).But there was no correlation between plasma leptin levels and femoral neck BMD values in both groups (r=-.,p=. in the patient group and r=., p=. in controls).A significant correlation was not observed between percentage body fat and femoral neck BMD values in both groups (r=-.,p=. in the patient group and r=., p=. in controls).

DISCUSSION
An in vitro study, it has been discovered that human bone marrow stromal cells also express high-affinity receptors for leptin and that leptin induces differentiation of stromal cells toward the osteoblastic lineage [].On the contrary, Ducy et al. [] did not found any leptin receptors among osteoblast cells and intracerebroventricular infusion of leptin in ob/ob, db/db, and wild mice led to a rapid and massive decrease in bone mass, leading to the suggestion that leptin may inhibit bone formation through its binding to leptin receptors in the hypothalamus and to the hypothesis that bone remodeling is under central control.
A number of investigators have addressed the role of leptin in human bone physiology.Most of them have found leptin to have no eff ect.Our present results are in accord with the previous fi ndings by Rauch et al. [], that none of the indices of bone density was signifi cantly related to leptin serum concentrations before or after adjustment for body mass index.In postmenopausal women, Rauch et al. [], and Goulding and Taylor [] found no correlation between circulating leptin levels and markers of bone turnover and thus speculated that leptin played no signifi cant role in the regulation of bone cell activity.Ruhl and Everhart [] found that increasing levels of leptin were associated with higher BMD levels in  pre-and postmenopausal women from NHANES III (Third U.S. National Health and Nutrition Examination Survey) but the association was no longer significant after adjusting for BMI.In the study of Shaarawy et al. [], no relationships were found between BMD and serum leptin levels.A study in children has also shown that plasma leptin levels were not related to BMD [].
There are also some studies suggesting a possible role for leptin in the regulation of human BMD.In a study of  postmenopausal women, aged - years, Martini et al. [] showed signifi cant positive associations between leptin and BMI (stronger with fat mass than lean mass), as well as with bone turnover markers and bone mass.In a study of  healthy nonobese Australian women aged - years, leptin was positively associated with bone mineral content (BMC) and BMD [].Yamauchi et al. [] also demonstrated an effect of leptin on bone mass in  postmenopausal women, independent of percentage fat.In this group, plasma leptin (but not percentage fat) was signifi cantly lower in women with vertebral fractures than in those without fractures.Th omas et al. [] demonstrated that serum leptin levels were signifi cantly related to BMD in the pre-and postmenopausal women, but not in the men.Blain et al. [] reported that leptin was signifi cantly correlated with whole body and femoral neck BMD in a sample of postmenopausal, non-obese women and that this association was independent of the infl uence years after menopause, fat mass, creatinine clearance, calcium intake, and other hormonal factors exert on BMD.
In this study, no correlation with BMDL-L was found.Our data show that there was no significant difference between plasma leptin levels of healthy and osteoporotic postmenopausal women.Besides no significant correlation was observed between plasma leptin levels and bone mineral density values of the femoral neck.Th ese fi ndings suggest that circulating plasma leptin does not have a direct influence on bone mass in postmenopausal women.
It is generally accepted that obesity is a major factor protecting against osteoporosis in women.Whether the protective eff ect is due to the increased weight load or enlarged adipose tissue mass exerting specifi c eff ects or to other possibilities, remain an open question.Leptin is an important regulator of the mass of adipose tissue and of body weight; it operates by inhibiting food intake and stimulating energy expenditure.It is suggested that plasma leptin concentrations directly correlate with BMI and the amount of body fat; obese subjects show higher levels than normal subjects and underweight subjects have extremely reduced leptin levels, which rise after partial weight recovery [, , ].In similar, we found a strong positive correlation between percentage body fat and plasma leptin levels in both groups.However, we failed to show such an relation between percentage body fat and BMD values.In contrast to our results, as Reid et al. [] reported that BMD is strongly related to fat mass in postmenopausal women.Chen et al. [] and Kandeel et al. [] concluded that bone mineral mass is more closely related to lean tissue mass than to fat tissue mass in postmenopausal women and premenopausal women, respectively.Bedogni et al. [] concluded that total bone mineral content (BMC) is more associated with lean tissue mass than with fat mass.
According to the results of not only in vivo but also in vitro studies, the main effect of leptin on BMD remains controversial.These contradictory results might result from the different population groups.Further studies including testing of multiple genes in both obese and lean subjects, with epidemiologic data on dietary habits in different ethnic groups, are necessary to better understand the role of leptin in regulating weight in human populations.In summary, our results do not support the hypothesis that leptin itself does not have an important role in maintaining bone mass in postmenopausal women.

CONCLUSION
We have investigated plasma leptin concentrations in postmenopausal women with osteoporosis and also analyzed the relationship between plasma leptin levels and bone mineral density in order to understand the potential role of leptin in maintaining bone mass.The results of the present study do not support the hypothesis that leptin itself plays an important role in maintaining bone mass in postmenopausal women.However, future studies on the current topic are recommended.

DECLARATION OF INTEREST
Th e authors declare no confl ict of interest.

FIGURE 1 .
FIGURE 1. Correlation between plasma leptin levels and Body Mass Index in the patient and control groups.

FIGURE 2 .
FIGURE 2. A strong positive correlation between percentage body fat and plasma leptin levels in the patient and control groups.

TABLE 1 .
Baseline clinical characteristics of the patient and control groups.
Note.Values are the mean + SD.Student t test; p< 0.05