Nermin Kilic

A comparison of leptin and ghrelin levels in plasma and saliva of young healthy subjects

In the last 10 years, saliva has been increasingly used as a diagnostic fluid and in predictions of disease progression. Leptin and ghrelin are synthesized in several tissues including the salivary glands. The action of ghrelin is antagonistic to that of leptin. This study was undertaken to measure and compare the saliva ghrelin-leptin and plasma ghrelin-leptin levels in healthy young subjects. In 30 healthy subjects, after an overnight fast, saliva and plasma leptin levels were measured using the ELISA method while saliva and plasma immunoreactive ghrelin levels were measured using a commercial radioimmunoassay (RIA). The latter uses 125I-labeled bioactive ghrelin as a tracer and a rabbit polyclonal antibody raised against full-length octanoylated human ghrelin (Phoenix, Europe, Karlsruhe, Germany). The results of this investigation revealed that saliva leptin levels (6.19+/-2.10 microg/l) were lower than plasma levels (7.39+/-3.23 microg/l) while saliva ghrelin levels (188.5+/-84.7 pg/ml) were higher than plasma levels (126.4+/-38.5 pg/ml), when male and female subjects were considered together. Saliva leptin levels (5.93+/-1.94 microg/l) were lower than plasma levels (6.22+/-2.92 pg/ml) while saliva ghrelin levels (190.3+/-80.2 pg/ml) were higher than plasma levels (120.4+/-35.7 pg/ml) in young males. Saliva leptin levels (6.47+/-2.29 microg/l) were lower than plasma levels (8.73+/-3.14 microg/l) while saliva ghrelin levels (183.2+/-90.2 pg/ml) were higher than plasma levels (129.3+/-42.8 pg/ml) in young females, and both saliva and plasma leptin levels were slightly lower in male subjects in comparison with female subjects. Also, Immunohistochemistry study indicated that ghrelin positivity was found in ductus epithelium of salivary gland. We have demonstrated for the first time that saliva ghrelin levels were higher than in plasma while saliva leptin levels were almost the same as in plasma. Measurements of ghrelin and leptin in saliva is non-invasive, simple, and generally much preferred by patients and thus may be an acceptable alternative to plasma sampling.

Antioxidant properties of chromium and zinc: in vivo effects on digestibility, lipid peroxidation, antioxidant vitamins, and some minerals under a low ambient temperature.

The effects of chromium (chromium picolinate, CrPic) and zinc (ZnSO4H2O) supplementation on serum concentrations of malondialdehyde (MDA) (an indicator of lipid peroxidation) and serum status of some antioxidant vitamins and minerals of laying hens (Hy-Line) reared at a low ambient temperature (6.8°C) were evaluated. One hundred twenty laying hens (Hy-Line; 32 wk old) were divided into 4 groups, 30 hens per group. The hens were fed either a basal diet or the basal diet supplemented with either 0.4 mg Cr/kg of diet, 30 mg Zn/kg of diet, or 0.4 mg Cr plus 30 mg Zn/kg of diet. Digestibility of nutrients (dry matter [DM], organic matter [OM], crude protein [CP], and ether extract [EE]) increased by supplementation of chromium and zinc (p<0.05). Supplemental chromium and zinc increased serum vitamins C and E but decreased MDA concentrations (p<0.05). Additionally, supplemental chromium and zinc caused an increase in the serum concentrations of Fe, Zn, Mn, and Cr (p < 0.05). The present study showed that low ambient temperature causes detrimental effects on the digestibility of nutrients and antioxidant status and that such detrimental effects caused by low ambient temperature can be alleviated by chromium and zinc supplementation, particularly when Cr and Zn were simultaneously included into the diet. Data obtained in the present study suggest that such supplementation can be considered as a protective management practice in a diet of laying hens for alleviating negative effects of cold stress.

Presence of obestatin in breast milk : Relationship among obestatin, ghrelin, and leptin in lactating women

OBJECTIVE The peptide hormones ghrelin and leptin have been found in blood and breast milk. This study was undertaken to investigate whether breast milk also contains obestatin, which is derived from the same gene as ghrelin but has opposite actions, and to characterize the relations among serum and milk ghrelin, obestatin, and leptin levels in lactating mothers. METHODS Venous blood, colostrum, and mature milk were obtained from healthy lactating women (n = 31) just before suckling. The ghrelin and obestatin concentrations were determined by radioimmunoassay. Leptin levels were measured by enzyme-amplified sensitivity immunoassay. RESULTS Obestatin levels in colostrum (538.9 pg/mL) and mature milk (528.5 pg/mL) were more than twice the corresponding blood levels (270.3 and 289.4 pg/mL, respectively). In contrast, leptin levels in colostrum (2.01 ng/mL) and mature milk (2.04 ng/mL) were more than five-fold lower than the corresponding blood levels (11.54 ng/mL). There was no correlation between breast milk ghrelin levels and leptin (r = -0.18, P > 0.05). However, there was a positive correlation between leptin levels in breast milk and blood (r = 0.369, P < 0.05). CONCLUSION The origin of milk obestatin is not currently known, but it comes from the blood or breast and may drain through the mammary glands into the milk. Ghrelin, obestatin, and leptin in the milk may directly affect appetite and their levels may be related to the regulation of energy balance and the pathogenesis of obesity.

Relationship among levels of leptin and zinc, copper, and zinc/copper ratio in plasma of patients with essential hypertension and healthy normotensive subjects

Obesity is among the main contributing factors in the etiology of essential hypertension (EHT). Leptin, the product of the ob gene, is expressed mainly in adipose tissue. We examined the relationship between two trace elements, zinc (Zn) and copper (Cu), and leptin in patients with EHT (n=35) and normotensive (NT) controls (n=50) because leptin as well as Zn and Cu were reported to be associated with the pathophysiology of EHT. Plasma leptin levels were determined with a commercial enzyme-linked immunosorbent assay (ELISA) kit. Atomic absorption spectrophotometry was utilized to determine plasma Zn and Cu levels. There was a negative correlation between leptin and Zn, and the Zn/Cu ratio (r=−0.359, p<0.05; r=0.361, p<0.05, respectively) in pooled subjects. When subjects were divided based on the presence or absence of hypertension, there was a negative correlation between leptin and Zn (r=−0.375, p<0.05) as well as leptin and Zn/Cu ratio (r=−0.398, p<0.05) in NT subjects. Similar trends were observed when leptin/BMI (body mass index) levels were utilized. There was no significant correlations between levels of Cu and leptin or leptin/BMI. In conclusion, in addition to high leptin levels, Zn and the Zn/Cu ratio were lower in patients with EHT compared to NT controls.

Ghrelin and obestatin levels in end-stage renal disease.

Malnutrition is fairly common in end-stage renal disease (ESRD) patients, persistent lack of appetite being a major symptom. Ghrelin and obestatin are two hormones that are involved in appetite and energy homeostasis. The present study examined ghrelin and obestatin levels in 24 ESRD patients undergoing haemodialysis and 24 age-matched healthy controls. Serum and saliva ghrelin and obestatin levels in the ESRD patients were significantly higher compared with controls, while saliva ghrelin and obestatin levels in all study participants were significantly higher than serum levels. Saliva ghrelin correlated with serum ghrelin and saliva obestatin correlated with serum obestatin in all study participants, although there was no correlation between ghrelin and obestatin levels. In conclusion, the results suggest that the kidneys may have a role in the metabolism and/or clearance of obestatin, as they do for ghrelin. Further studies are needed to determine if elevated levels of these hormones in ESRD patients contribute to the malnutrition that is common in these patients.

Effects of tibolone on plasma homocysteine levels in postmenopausal women

OBJECTIVE To investigate the effects of tibolone on levels of plasma homocysteine, an independent risk factor for cardiovascular disorders, in postmenopausal women. DESIGN Prospective, randomized clinical study. SETTING University hospital. PATIENT(S) Postmenopausal healthy women. INTERVENTION(S) Tibolone (2.5 mg/d) or calcium (1250 mg/d) and conjugated equine estrogens (0.625 mg/d) plus medroxyprogesterone acetate (5 mg/d) were administered orally for 6 months. Blood samples were collected at the start and the end of therapy. MAIN OUTCOME MEASURE(S) Plasma homocysteine levels. RESULT(S) Administration of tibolone and calcium caused only a 4% decrease in plasma homocysteine levels compared with initial levels. In contrast, conjugated equine estrogens plus medroxyprogesterone acetate caused a 29% decrease in plasma homocysteine levels. CONCLUSION(S) Despite the reported beneficial effect of tibolone on the serum lipid profile, tibolone had no statistically significant effect on serum homocysteine levels in postmenopausal women. The possible cardiovascular protective role of tibolone might be unrelated to its effects on homocysteine levels.

Medroxyprogesterone acetate, enoxaparin and pentoxyfylline cause alterations in lipid peroxidation, paraoxonase (PON1) activities and homocysteine levels in the acute oxidative stress in an experimental model of spinal cord injury.

Summary. Background: Effects of medroxyprogesterone acetate, enoxaparin and pentoxyfylline on lipid peroxidation, antioxidant defence system, paraoxonase activities, and homocysteine levels in an experimental model of spinal cord injury were investigated. Method: Sixty-three male albino Wistar rats were anaesthetized by 400 mg/kg chloral hydrate and divided into 5 groups. G1 (n 7) = control group provided the baseline levels. G2–G5 underwent T3–6 total laminectomies and spinal cord injuries by clip compression at T4–5 levels. Medications were applied to G3–G5 right after the injury. Hence, G2 constituted laminectomy + injury (lam+I); G3 = lam + I + medroxyprogesterone acetate (MPA), G4 = lam + I + enoxaparin (E), and G5 = lam+I+pentoxyfylline (P) groups. Animals were decapitated either at the 1st or 4th hour after injury. Tissue and blood malonyldialdehyde (MDA) and plasma homocysteine and erythrocyte superoxide dismutase (SOD) levels, and erythrocyte glutathione peroxidase (GSH-Px) and plasma paraoxonase (PON1) activities were assayed. SPSS 9.0 program was used for statistical analysis and graphics. Intergroup comparisons were made by Bonferroni corrected Mann Whitney U test (P<0.025), and intragroups comparisons by Wilcoxon Rank test (P<0.03). Findings: In intergroup comparison, G1–G2, G1–G3, G1–G5, G2–G3, G2–G4, and G4–5 groups differed from each other for all parameters (P<0.025, MWU) except for G4–G5 4th hour MDA levels. G1–G4 was similar for all 1st hour parameters (P>0.025, MWU), but different for 4th hour (P<0.025, MWU) except for GSH-Px and SOD levels. For G2–G5, all parameters for 1st and 4th hour were similar except for 4th PON1, Hcy and SOD levels. For G3–G4, all 1st hour parameters were different from each other (P<0.025, MWU); whereas all 4th hour parameters were similar except for SOD level. For G3–G5, all parameters at 1st and 4th hour were similar except for 4th hour GSH-Px, PON1, and Hcy. In intragroup comparison, all parameters differed from each other at all times (P<0.03, WRT) except for 1st hour G4 MDA, Hcy and SOD levels compared to basal levels. Interpretation: In injury groups, plasma Hcy levels decreased and PON1 activities increased as erythrocyte SOD level and GSH-Px activities decreased in parallel to increases of tissue and blood MDA levels. These changes were relatively suppressed by MPA, enoxaparin and pentoxyfylline administrations at varying degrees. Enoxaparin was the most powerful agent, particularly at 1st hour. MPA was also effective, particularly at 4th hour. Pentoxyfylline despite having slight effect at 4th hour, was not effective according to both control and injury groups. Enoxaparin and MPA can be used in the treatment of spinal cord injuries. PON1 and Hcy are helpful in monitoring the antioxidant defence system as well as SOD and GSH-Px, both in injury and medically treated groups.

Ghrelin and obestatin expression in oral squamous cell carcinoma: an immunohistochemical and biochemical study.

The underlying molecular mechanism of carcinogenesis in oral squamous cell carcinoma (OSCC) is poorly understood and appears to be controlled on many genetic, environmental, and hormonal factors. Obestatin and ghrelin, two recently discovered hormones, are co-expressed in endocrine cells. The purpose of this investigation was to examine the immunohistochemical features of OSCCs in relation to the tissue concentration of ghrelin and obestatin. The association between OSCC and Epstein Barr Virus (EBV) status was also explored. The expression of ghrelin and obestatin was examined by immunohistochemistry and immunoassay in oral biopsy specimens: 10 benign squamous epithelial cell samples, 10 microinvasive squamous cell carcinomas, and seven well-differentiated and seven poorly differentiated OSCCs. The presence of EBV was evaluated in these samples using immunohistochemistry. The concentrations of ghrelin and obestatin in tissue homogenates were measured by RIA and ELISA, respectively. Squamous cell carcinomas and benign tissue samples were positive for anti-EBV antibody, and obestatin and ghrelin were shown to be co-expressed in all stratified squamous epithelium samples. Expression of ghrelin and obestatin was decreased or absent in OSCCs in relation to the invasiveness of the carcinoma; ghrelin and obestatin levels in cancerous tissue homogenates were lower than in benign tissue homogenates. These results indicate that the concentrations and distribution of immunoreactive obestatin and ghrelin might be helpful in distinguishing OSCC from benign tumors. Maintaining normal levels of these hormones might be required for regulation of normal cell division. However, detailed studies will be required for better understanding of the complex mechanism of carcinogenesis relating to OSCCs.

Saliva/serum ghrelin, obestatin and homocysteine levels in patients with ischaemic heart disease

Summary Background: We aimed to compare ghrelin, obestatin, homocysteine (Hcy), vitamin B12 and folate levels in the serum and saliva of ischaemic heart disease patients. Methods: Serum and saliva were collected from 33 ischaemic heart disease (IHD) patients and 28 age- and body mass index-matched healthy individuals. Levels of acylated and desacylated ghrelin, obestatin and Hcy were determined using the ELISA method. Results: Acylated ghrelin, desacylated ghrelin and obestatin levels in the saliva were found to be higher than those in the serum of the control group, while acylated and desacylated ghrelin levels in the saliva were significantly lower than those in the serum. Obestatin levels were higher in IHD patients (p = 0.001). Saliva and serum vitamin B12 and folate levels in IHD patients were significantly lower than in the control group (p = 0.001). Conclusions: It was determined that serum ghrelin levels increased in ischaemic heart disease patients, while serum levels of obestatin decreased.

Des-Acylated Ghrelin, Rather Than Acylated Ghrelin, Might Be More Valuable in Inflammatory Bowel Diseases

To the Editor We read with interest the article entitled ‘‘Serum Ghrelin Levels in Inflammatory Bowel Disease with Relation to Disease Activity and Nutritional Status’’ by Ates et al. that was recently published in Dig Dis Sci [1]. They reported the increase in acylated ghrelin in serum from patients with active ulcerative colitis (UC) and Crohn’s disease (CD) compared with those in corresponding serum samples of patients in remission. Even though the data seem interesting and very novel, and the outcomes are clear in their study, we strongly believe that there are some important points that need to be addressed and clarified. First of all, it is unclear whether they investigated the serum or plasma ghrelin level from patients with UC and CD even though serum ghrelin levels are mentioned in their title. In our opinion, they investigated the plasma not serum ghrelin level since in their Methods section they state that blood was directly drawn into a tube bearing ethylenediaminetetraacetic acid (EDTA), and EDTA or another blood anticoagulant is used for the preparation of plasma not serum samples. As is known, serum is a clear liquid without fibrinogen and other clotting factors, so that serum is different from plasma [2]. The second point we would like to address is that in such a comparative study the measuring acylated ghrelin alone cannot go to the right address. As is known, ghrelin 28-amino acid peptid has a modification on its third residue, which is a serine. The hydroxyl group of Ser3 is post-translationally octanoylated (acyl addition). This modification seems to be essential for growth hormone release and appetite [3]. It was also known that the des-acylated form of ghrelin stimulated cell proliferation, adipogenesis [4], and counteracted the metabolic action of acylated ghrelin [5] as well as circulated in far greater amounts than the acylated form. If so, in term of inflammatory bowel diseases (ulcerative colitis and Crohn’s disease), the des-acylated form of ghrelin might be more physiologically important because inflammation in the gut is associated with increased epithelial cell proliferation [6]. Finally, to clarify a link with ghrelin and inflammatory bowel diseases, we would like to suggest that two forms of ghrelin should be tested in future experiments to obtain more reliable results.