Transcardial gradient of adiponectin, interleukin-6 and tumor necrosis factor-α in overweight coronary artery disease patients.

BACKGROUND: Obesity is associated with coronary artery disease (CAD), where epicardial adipose tissue (EAT) express proatherogenic cytokines (i.e., interleukin-6 (IL-6), tumor necrosis factor-α (TNF-α)) and decreases production of beneficial adiponectin. Studies on endocrine role of EAT are mostly based on assessing cytokines' mRNAs, whereas cytokine blood levels might not readily correlate. In order to get better insight into the endocrine role of thickened EAT in CAD, we assessed transcardial gradient of adiponectin, IL-6 and TNF-α. METHODS: We assessed anthropometric and ultrasound measures in cohort of fifty nondiabetic subjects (21 CAD and 29 non-CAD). Blood sampled from aortic root and coronary sinus was assayed for adiponectin, IL-6 and TNF-α, using ELISA. RESULTS: Except thicker EAT in CAD subjects, anthropometric measures were similar (overweight), with higher adiponectin in coronary sinus than in aortic root (p<0.001) in both groups. CAD subjects had lower adiponectin in aortic root (p<0.001) and higher levels of TNF-α in coronary sinus than in aortic root (p=0.05). EAT thickness positively correlated with hip circumference (p=0.038) and negatively correlated with adiponectin levels (for both p<0.05). CONCLUSIONS: Transcardial gradient of adiponectin in non-CAD and CAD overweight subjects was similar, while latter had lower systemic adiponectin level and thicker EAT. EAT with thickening reaches the threshold level of near-maximal down-regulation of adiponectin and its further thickening is not associated with continued decrease of adiponectin production. In CAD patients levels of TNF-α were higher, but IL-6 were not, and these cytokines might be flush out by lymphatic route.

Perforating corneal injury in rat and pentadecapeptide BPC 157.

Based on its healing effects in various tissues, we hypothesized that the stable gastric pentadecapeptide BPC 157 heals corneal ulcerations in rats and effects corneal transparency. We made a penetrant linear 2-mm incision in the paralimbal region of the left cornea at the 5 o'clock position with a 20-gauge MVR incision knife at 45° under an operating microscope. Medication was BPC 157 (2 pg/mL, 2 ng/mL, and 2 µg/mL distilled water, two eye drops/left rat eye) immediately after injury induction and then every 8 h up to 120 h; controls received an equal volume of distilled water. In contrast to the poor healing response in controls, BPC 157 significantly accelerated the healing process in 2 µg and 2 ng BPC 157-treated eyes, starting 24 h after the injury, and the fluorescein and Seidel tests became negative. The epithelial defects were completely healed at 72 h (2 µg BPC 157-treated group) and at 96 h (2 ng BPC 157-treated group) after injury. Aqueous cells were absent at 96 h and 120 h after injury in the 2 µg and 2 ng BPC 157-treated groups, respectively. In conclusion, BPC 157 effects the rapid regaining of corneal transparency. Whereas controls developed new vessels that grew from the limbus to the penetrated area, BPC 157-treated rats generally had no new vessels, and those that did form in the limbus did not make contact with the penetrated area. Thus, BPC 157 eye drops successfully close perforating corneal incisions in rats.

Extended Eversion Carotid Endarterectomy: Computation of Hemodynamics.

BACKGROUND: Stroke prevention includes surgery for significant stenosis of internal carotid artery (ICA). Consensus on a standard approach lacks and one alternative approach is eversion carotid endarterectomy (eCEA). To overcome disadvantages of eCEA, we developed extended-eversion carotid endarterectomy (exeCEA). Aiming to investigate hemodynamics after different surgical approaches, we created computational fluid dynamics (CFD) models of exeCEA and eCEA with included progressing lumen narrowing, representation of artery restenosis at the incision line. METHODS: Blood flow velocities, volume flow rates, and wall shear stress (WSS) were established in carotid arteries from models of eCEA and exeCEA with included increasing groove (1, 1.5, 2, and 2.5 mm) at the "incision line", under input pressure of 120 and 150 mm Hg. RESULTS: For the corresponding restenosis grade, models of exeCEA had a larger orifice toward ICA, lower blood flow velocities and higher volume flow rates in ICA, with lower volume flow rates in external carotid artery. WSS values in ICA of exeCEA models were lower than in eCEA models, later reaching the thrombotic range. CONCLUSIONS: CFD showed better hemodynamic properties in exeCEA models, indicating presented approach might be better at preserving brain perfusion.

Prevalence of the palmaris longus in relation to the hand dominance.

PURPOSE: The incidence of left-handedness in the general population is between 8 and 15%. There is a presumption that the prevalence of palmaris longus muscle differ between right-handed and left-handed people. This prospective study was conducted to determine the prevalence of the palmaris longus in relation to the hand dominance. METHODS: The study included 542 subjects (216 male and 326 female). They were initially tested to hand dominance and after that they were asked to do the standard test (Schaeffer's test) for the assessment of the palmaris longus tendon. If the tendon was not visualized or palpable, four additional tests (Thompson's test, Mishra's tests I and II, Pushpakumar's "two-finger sign" method) were done to confirm its absence. RESULTS: Right hand dominance was recorded in 452 (83.4%) subjects while the left hand dominance was recorded in 90 (16.6%) subjects. In right-handed subjects, palmaris longus tendon was absent on the right side in 24 (5.3%) and on the left side in 50 (11.1%) cases. In left-handed subjects, it was absent on the right side in 18 (20%) and on the left side in 2 (2.2%) cases. These differences were statistically significant. Bilateral absence of palmaris longus tendon was similar in both examined groups (25.1% in the overall series, 24.3% in right-handed subjects, 28.9% in left-handed subjects). CONCLUSIONS: The results of our study show that a right-sided absence was more common in left-handed persons while the left-sided absence was more common in right-handed persons. Unilateral tendon absence was more common on the non-dominant hand.

Low adiponectin serum level--reduced protective effect on the left ventricular wall thickness.

Adiponectin, secreted by fat tissue, is down - regulated in obesity and may be involved in obesity-related disorders. It has anti-inflammatory, antiatherosclerotic and antidiabetic effect. Obesity is a strong predictor for hypertension and cardiovascular diseases. Recent studies showed that adiponectin level has important role in metabolic disorders, arterial hypertension and ischemic heart disease but its effect on left ventricular hypertrophy (LVH) has not been fully clarified. The aim of this research is to determine whether the protective effect of adiponectin against development of left ventricular hypertrophy is decreased in hypertensive overweight patients. The study included 61 adult, overweight hypertensive patients, with body mass index in range 25-30 kg/m2. Patients had regular morning glucose serum values and regular creatinine level. They were divided into four groups, according to sex and the presence of LVH. There were 16 female and 15 male hypertensive patients with LVH and 15 female and 15 male hypertensive patients without LVH, who were a control group. Glucose profile, lipidogram, creatinine clearance and anthropometric measures were determined in all patients. Cardiovascular measurements were taken applying two-dimensional ultrasound. Adiponectin serum level was measured using enzyme immunoassay (ELISA). Results showed that adiponectin serum level was significantly lower in hypertensive, overweight females and males with LVH than in the control groups without LVH. Adiponectin serum level did not correlate significant with intraventricular or with posterior wall thickness of left ventricle. Hypoadiponectinemia presents part of neurohumoral, non-haemodynamic system who contributes to obesity-related hypertension and left ventricular hypertrophy development. Low adiponectin level together with others adipokines, cytokines and chemokines secreted by fat tissue could contribute to pathophysiologic changes of the myocardium via unknown molecular mechanisms yet.

Numerical analysis of standard and modified osteosynthesis in long bone fractures treatment.

The fundamental problem in osteoporotic fracture treatment is significant decrease in bone mass and bone tissue density resulting in decreased firmness and elasticity of osteoporotic bone. Application of standard implants and standard surgical techniques in osteoporotic bone fracture treatment makes it almost impossible to achieve stable osteosynthesis sufficient for early mobility, verticalization and load. Taking into account the form and the size of the contact surface as well as distribution of forces between the osteosynthetic materials and the bone tissue numerical analysis showed advantages of modified osteosynthesis with bone cement filling in the screw bed. The applied numerical model consisted of three sub-models: 3D model from solid elements, 3D cross section of the contact between the plate and the bone and the part of 3D cross section of the screw head and body. We have reached the conclusion that modified osteosynthesis with bone cement resulted in weaker strain in the part of the plate above the fracture fissure, more even strain on the screws, plate and bone, more even strain distribution along all the screws' bodies, significantly greater strain in the part of the screw head opposite to the fracture fissure, firm connection of the screw head and neck and the plate hole with the whole plate and more even bone strain around the screw.

Vasculometry of upper and lower extremities in correlation with development of pathologic conditions like the diabetic foot.

We assume that the vascular apparatus of the lower limb did not evolutionary adapt to leg mass and volume. The lower limb is greater in length and volume that the upper limb, and therefore the arteries should have a bigger diameter and cross-sectional area. During pathoanatomic autopsies at the Department of Pathology of University Hospital Center Osijek we have taken segments of 1 cm of length from the subclavian, femoral, radial and tibial artery. Our sample contained segments from 51 bodies, 24 female and 27 male. We have measured leg and arm length and circumference. From these data the idealized limbs volume was calculated by geometric approximations to a cone fragment. The relation between idealized leg and arm volume and arterial cross-sectional area were calculated. For statistical analysis, Student's t-test was used. At the Department of Radiology of the University Hospital Center Osijek we measured the diameter of subclavian and femoral artery in systole and diastole in 41 patients (21 female and 20 male) by Color Doppler ultrasound, and the circumference and length of upper and lower limb was measured. There is a slightly difference between the diameter and cross-sectional area of subclavian and femoral artery. Leg length was for 48.5% bigger than arm length and the difference in volume between upper and lower limb is significantly different. The foot has four to five times greater volume than the arm, and is vascularised by an arterial tree of similar diameter. This fact proves our hypothesis that the blood supply to the lower limbs compared to the mass of tissue is smaller.

Real mineral density of the sternum.

Mineral density of the sternum is insufficiently known. The aim of this research was to investigate mineralisation of the sternum and collect normative data on mineral density of the standard male and female sternum in elderly people (average age of female samples was 64 and male's was 62 years). The research was conducted on 93 cadaveric sternums, 56 male and 37 female samples. To determine regional mineral density of the sternum each sample was cut into six bony segments (Figure 1). Mineral density of every segment was determined using the method of ashing. Male sternums were on average denser than female ones in all segments. Average mineral density of the manubrium in women was 0.169 g/cm3 and 0.220 g/cm3 in men. Average mineral density of the body of the sternum also showed existence of sex difference; it was 0.160 g/cm3 in women and 0.227 g/cm3 in men. Both male and female sternums showed identical mineral density distribution. Mineral density of the manubrium and the body was roughly equal, while the analysis of longitudinal segments showed that the central part of both the manubrium and the body of the sternum was denser than lateral parts. Complex determination of the real mineral density for defined segments of the sternum and analysis of the obtained results were used to create the map of mineral density of the sternum in men and women (Figure 2). Maximum density values were four times greater than minimum density values for analysed samples. These data showed that osteoporosis also occurs on the sternum. Loss of structure and lower mineral density decrease the sternum quality and increase the risk of sternal dehiscence after median sternotomy.

Morphometric characteristics of thyroid cartilage in people of Eastern Croatia.

The aim of this research was to describe thyroid cartilage morphometry in the population of Eastern Slavonia in detail. The research was carried out on 68 samples of adult thyroid cartilages. There was statistically significant difference between all analysed parameters in male and female samples, except for the distance between the superior horns tips. All parameters had greater values in men, except for the thyroid angle, which was greater in women. There was significant correlation between most of the measured parameters. The strongest correlation was noticed in the distance between the horns at all measured levels and between the pairs of parameters measured for the left and right side respectively. The difference of our results in comparison to the results obtained by other researchers is probably caused by the fact that the measurements were taken on the samples obtained from different populations.

Advantages of modified osteosynthesis in treatment of osteoporotic long bones fractures--experimental model.

In surgery of fractured long bones, a patient suffering from osteoporosis represents constant challenge to a surgeon and applied material and instruments that need to destroy as little as possible of an already damaged bone. One potential way of increasing the contact surface between the implants and osteoporotic bone is injection of bone cement (methyl-metacrilat, Palakos) into a prepared screw bed. This method of osteosynthesis was therefore subjected to experimental research to prove that application of modified osteosynthesis using bone cement in treatment of fractures in osteoporotic patients has advantage over the standard method of osteosynthesis because this modified method enables significantly greater firmness and stability of the osteosynthesis, which is the essential precondition of a successful fracture healing. The research was carried out on six macerated cadaveric preparations of a shin bone from the osteological collection from Institute for Anatomy, School of Medicine, University "J. J. Strossmayer". All samples of long bones were artificially broken in the middle part of the diaphysis and then standard osteosynthesis and modified osteosynthesis with screws filled with bone cement were performed on the samples. Results show that under identical static action of the moment of torsion in the modified osteosynthesis torsion angle deviation is lower than in the standard osteosynthesis. In modified osteosynthesis with bone cement the first results for angle of torsion deviation greater than 0.2 degrees were noticed after 120 minutes, while in the standard method of osteosynthesis they were noticed already in the first minute.

Effect of different combination of maternal and postnatal diet on adipose tissue morphology in male rat offspring.

PURPOSE: Adipose tissue expansion can occur through several different ways and, under certain conditions, can be connected with chronic inflammation. TNF-α is one of the important cytokines involved in this process. Prolonged inflammation in obesity can lead to obesity-related insulin resistance and tissue dysfunction. The aim of our study was to investigate how different combination of maternal and postnatal diet affects offspring adipose tissue morphology and adipose tissue TNF-α expression. METHODS: Ten female Sprague Dawley rats, 9 weeks old, were randomly divided into two groups and fed either standard laboratory chow or food rich in saturated fatty acids during 6 weeks and then mated with the same male rat. After birth and lactation male rat offspring from both groups were divided into four subgroups depending on the diet they were fed until 22 weeks old. Samples of white adipose tissue were taken from the subcutaneous, epididymal, and perirenal fat pad. On tissue sections, histomorphometric analysis was conducted using CellProfiler program v 2.1.1, and immunohistochemical staining for TNF-α was performed. RESULTS: Greater mean surface area of subcutaneous and epididymal adipocytes was found in groups of male rat offspring with altered diet. In perirenal adipose tissue, the highest number of adipocytes was measured in the group where both mother and offspring were fed a high-fat diet. Adipocyte staining intensity for TNF-α did not differ significantly between the groups. CONCLUSIONS: Together with our previously published data, our results lead to the conclusion that alteration of postnatal diet can lead to TNF-α and adipocyte morphology changes.

Hyperleptinemia--non-haemodynamic risk factor for the left ventricular hypertrophy development in hypertensive overweight females.

Obesity is directly and strongly associated with hypertension and left ventricular hypertrophy (LVH). Development of LVH is multifactorial, caused both by haemodynamic and non-haemodynamic factors. Hypertension is the main haemodynamic factor. Humoral mechanisms, as a non-haemodynamic factor for LVH development, have not been completely explained. The aim of this study is to determine whether hyperleptinemia can be one of humoral--non-haemodynamic factor inducing LVH together with haemodynamic factors in overweight females. The study was done on thirty six adult, overweight female patients, body mass index in range 25-30 kg/m2. Patients are nondiabetic with regular renal function. Twenty one female patients were hypertensive with left ventricular hypertrophy. Control group included fifteen hypertensive female patients without left ventricular hypertrophy. In all patients was determined glucose profile and creatinine clearance, cholesterol, triglycerides, LDL, HDL. Weight, high, circumference of the waist and hips was taken. Cardiovascular determination was done applying two-dimensional ultrasound. Serum leptin level was measured using radioimmunoassay method (RIA). Results showed that serum leptin level was significantly higher in hypertensive, overweight females with LVH. This suggests that non-haemodynamic factors, such as hyperleptinemia, participate in left ventricular hypertrophy development together with haemodynamic factors in adult hypertonic, overweight females.

Functional status of hip joint after surgical and conservative treatment of acetabular fracture.

Both conservative and surgical treatment of acetabular fractures alter biomechanical conditions in the hip joint resulting in various degenerative changes in the acetabulum and the femur head. These changes may progress to secondary coxarthrosis, causing dysfunction of the hip joint. The aim of this paper is to compare the outcomes of both conservative and surgical treatments in different types of acetabular fractures, so that clear indications for either conservative or surgical treatment could be determined. This paper is based on retrospective study of 103 patients with acetabular fracture (21 surgically treated and 82 conservatively treated). In this study the incidence of particular types of acetabulum fractures, the treatment period, the incidence of complications and the functional status of the hip after the treatment were analyzed. In patients with anterior fracture surgical treatment lasted three times less than conservative treatment and resulted in far better functional status of the hip joint compared to conservatively treated patients. However, in patients with transverse fractures the functional status was better and the treatment period shorter after the conservative treatment. We believe that the present treatment indications should be corrected so that in anterior column fracture the surgical method should be preferred, whereas the transverse fracture should be treated conservatively. In other types of acetabular fracture, with the radiographic roof arc angle of 45 degrees or less, the surgical method should be preferred to conservative method.

The analysis of the unstable tibia fracture treatment applying internal stabilization method.

The study included 51 patients with tibia fractures, who underwent percutaneous bone reposition and stabilization with unrimed tibial locking nail. The results obtained using this method were compared with those obtained by standard fracture treatment where flat and anatomic plates were applied (n = 64). In patients who had osteosynthetic material implanted percutaneously (using unrimed tibial locking nail) there was no incidence of post surgical osteitis or any pseudarthrosis. The healing callus of the fracture was of lesser quality and spindle shaped, suggesting that fracture stabilization using this method was less efficient. In patients with fractures stabilized by the open method using flat and anatomic plates (n = 64), we noticed 3.1% (n = 2) cases of osteitis and 4.7% (n = 3) cases of pseudarthrosis. Due to lesser incidence of postoperative osteitis, our method of choice in tibia fractures would be percutaneous stabilization with unrimed tibial locking nail. However, this treatment method has its disadvantages, too. Fracture callus is of lesser quality and it is spindle shaped. Furthermore, there are problems with adequate percutaneous reposition in some cases, as well as necessity for radiological checking.

Chemerin Blood Levels are Associated with MRI Measured Volumes of Abdominal Adipose Tissue Compartments and Lifestyle Choices.

Obesity is a low-grade chronic inflammatory state, in which a cytokine chemerin with its immunometabolic modulation has an important role. We aimed to study in a healthy population relationships between serum chemerin levels, lifestyle choices and magnetic resonance imaging (MRI) assessed volumes of abdominal visceral (VAT) and subcutaneous (SAT) adipose tissues, which have different cytokine expression profiles. Overall, 73 healthy participants undertook lifestyle questionnaire and underwent anthropometric measurements along with MRI scanning of abdominal SAT and VAT. Furthermore, complete blood count was determined along with chemerin and fibrinogen serum levels. Regression model for prediction of chemerin serum levels was built after controlling for sex, age and anthropometric measures. Median serum chemerin was 141 (125-195) ng/mL. Serum chemerin had a moderate positive correlation with SAT and VAT volumes. The two most important predictors of chemerin levels were MRI detected SAT and thigh circumference. Independently, chemerin levels were associated with smoking, preference of salty food, and favoring flavor/simplicity of preparation over nutritional value of the food. Serum chemerin seems to be more strongly correlated with the volume of abdominal SAT than VAT, with certain lifestyle choices influencing chemerin levels independently of abdominal fat.

Different combinations of maternal and postnatal diet are reflected in changes of hepatic parenchyma and hepatic TNF-alpha expression in male rat offspring.

Obesity is related to increased TNF-alpha production in different tissues. TNF-alpha is connected to mitochondrial dysfunction in the liver and also development of fatty infiltration of the liver. Also, postnatal change from normal to high-fat diet causes a significant increase in TNF-alpha serum levels. The aim of this research was to determine how maternal diet and switching male offspring to a different dietary regime after lactation influences rat liver. Ten female Sprague Dawley rats at nine weeks of age were randomly divided in two groups and fed either standard laboratory chow or high-fat diet during six weeks, and then mated with the same male subject. After birth and lactation male offspring from both groups were further divided into four subgroups depending on their subsequent diet. At 22 weeks of age, the animals were weighted, sacrificed and major organs were collected and weighted. Immunohistochemistry for TNF-alpha was performed on liver, and liver samples were analyzed for pathohistological changes. The group in which mothers were fed standard chow and offspring high-fat diet had the most pronounced changes: heaviest liver, poorest histopathological findings and strongest TNF-alpha immunohistochemical staining of liver parenchyma. High-fat diet during pregnancy and lactation and switching to high-fat diet postnatally affects liver weight, histological structure and TNF-alpha expression in male offspring.

Resorption of gas trapped in body cavities: comparison of alveolar and pleural space with inner ear and paranasal sinuses.

This paper describes our attempt to devise a short text aimed at improving students' understanding of gas resorption in body cavities. Students are expected to understand the mechanisms behind paranasal sinusitis, otitis media, closed pneumothorax, and atelectasis of collapsed lung tissue, all used as examples. On the basis of the interpretation that during pneumothorax resorption, gas diffuses down pressure gradients into the blood, students are encouraged to calculate tables of pressure gradients for the above-mentioned pathological conditions. After answering a few questions, students need to analyze and eventually accept the following conclusion: in cases of air trapping in collapsible body cavities, all gases will be fully reabsorbed without pain. Air trapping in bone cavities leads only to partial reabsorption of gases and results in subatmospheric intracavity pressure. Partial vacuum causes painful mucosal edema and free fluid secretion.

Duplex sonography of arteriovenous fistula in chronic hemodialysis patients.

Duplex sonography was used to assess functional features of arteriovenous fistula (AVF) for hemodialysis (HD). Internal diameter (ID), resistance index (RI) and blood flow (BF) velocity in feeding artery and in vein ofAVF, and venous BF volume were analyzed with purpose to determine the normal values. Presumed normal BF velocities are those of clinically well functioning shunts, allowing BF through HD lines of minimally 250 ml/min. Study included 66 nondiabetic HDpatients (30 women, 36 men), mean age 52-13 years, treated by HD for median 61 (4-252) months. Measurements in 47patients with clinically well functioning AVF were as followed: mean arterial ID 5.2 +/- 1.4 mm, median arterial RI 0.3 (0.3-0.9), median arterial BF velocity 1.5 (0.6-3.6) m/s, mean venous ID 7.6 +/- 2.2 mm, median venous RI 0.3 (0.3-0.9), mean venous BF velocity 1.6 +/- 0.7 m/s, and median venous BF volume 530 (120-1890) ml/min. Patients with poor functioning AVF had significantly less arterial ID, higher arterial RI, less venous ID, less venous BF velocity and volume. Duplex sonography findings obtained for clinically estimated well functioning shunt should be considered as normal Doppler values. Blood vessels' morphologic features depend upon age, and older patients have more pronounced changes.

Morphometric analysis of the sternum.

Sternum has a great clinical significance, considering that median sternotomy is the most common surgical approach used in cardiac surgery. The aim of this study is to standardize the sternum according to size, shape and sex and to obtain ranges of the "standard sternum". The study was done on 55 male and 35 female sterna of the average age of 65. Complex morphometric analysis of breadth, length and thickness of the sterna were performed on sternal segments which were defined by costal notches. Morphometric analysis shows that the general sternum structure in the females and in the males is equal. The standard dimensions of female and male sternum were determined. Standardization according to shape suggests that there is one standard sternum shape present in more than 2/3 of analysed samples of both sexes.

Effects of between generations changes in nutrition type on vaginal smear and serum lipids in Sprague-Dawley rats.

OBJECTIVE: Previous studies established that between generations changes in feeding protocol can have significant impact on reproductive physiology. The aim of the study was to determinate effects of mothers' nutrition and nutrition of the offspring on the characteristics of vaginal smear and serum lipid content. METHODS: Ten female rats were randomly divided in two groups; first group fed with food containing high content of saturated fatty acids (HFD) and the second with standard laboratory chow (CD). After coupling and lactation period their offspring were further randomly divided into two subgroups fed HFD or CD forming four study groups: (a) CD-CD, (b) CD-HFD, (c) HFD-CD and (d) HFD-HFD. The dams and offspring at the age of 37 and 18 weeks, respectively, were subjected to biochemical analysis of the blood and cytological analysis of the vaginal smears. Additionally body weight was recorded and body mass index (BMI) was calculated. RESULTS: The HFD-HFD group presented with highest levels of triglycerides and the CD-HFD with the highest levels of cholesterol. Therefore, triglyceride and cholesterol levels were significantly different among the groups (p = 0.001 and p = 0.002, respectively). Vaginal cytological smears analysis showed features of irregular phase interchanges or extended estrous phase in offspring of high-fat fed dams. CONCLUSION: Maternal HFD consumption predisposes offspring to increased risk of developing metabolic abnormalities and estrous disorders.