Determination of ghrelin and desacyl ghrelin in human plasma and urine by means of LC-MS/MS for doping controls.

The hunger hormone ghrelin (G) is classified as prohibited substance in professional sport by the World Anti-Doping Agency (WADA), due to its known growth hormone releasing properties. The endogenous bioactive peptide consists of 28 amino acids with a caprylic acid attached to serine at position 3. Within this study, it was aimed to develop methods to determine G and desacyl ghrelin (DAG) in plasma and urine by means of LC-MS/MS. Two strategies were applied with a bottom-up approach for plasma and top-down analyses for urine. Both sample preparation procedures were based on solid-phase extraction for enrichment and sample clean-up. Method validation showed good results for plasma and urine with limits of detection (LODs) for G and DAG between 30 and 50 pg/ml, recoveries between 45-50%, and imprecisions (intra- and inter-day) between 3% and 24%. Plasma analysis was also valid for quantification with accuracies determined with ~100% for G and ~106% for DAG. The minimum required performance level for doping control laboratories is set to 2 ng/ml in urine, and the herein established method yielded acceptable results even at 5% of this level. As proof-of-concept, plasma levels (G and DAG) of healthy volunteers were determined and ranged between 30 and 100 pg/ml for G and 100-1200 pg/ml for DAG. In contrast to earlier reported studies using ligand binding assays for urinary G and DAG, in this mass spectrometry-based study, no endogenous urinary G and DAG were found, although the LODs should enable this.

Vaspin in Serum and Urine of Post-Partum Women with Excessive Gestational Weight Gain.

Background and objectives: Data concerning vaspin in obstetric aspects are limited and conflicting. The aim of the study was to evaluate vaspin concentrations in the serum and urine of women with excessive gestational weight gain (EGWG) in the early post-partum period (i.e., 48 h after delivery), when placental function no longer influences the results. Materials and Methods: The study subjects were divided into two groups of 28 healthy controls and 38 mothers with EGWG. Maternal body composition and hydration status were evaluated by the bioelectrical impedance analysis (BIA) method. Concentrations of vaspin, fatty acid-binding protein 4 (FABP4), leptin, and ghrelin were determined via enzyme-linked immunosorbent assay (ELISA). Results: Serum vaspin levels were lower in the EGWG group, whereas no significant differences were noted between the groups, with regard to the urine vaspin concentrations. In both studied groups, the serum vaspin concentrations correlated positively with the urine FABP4 levels and negatively with gestational weight gain, body mass index gain in the period from pre-pregnancy to 48 h after delivery (ΔBMI), and fat tissue index (FTI). In the multiple linear regression models, the serum vaspin concentrations were positively dependent on the serum FABP4 levels, as well as negatively dependent on triglycerides, FTI, and ΔBMI. Conclusions: Our study revealed that the EGWG mothers were characterized by significantly lower serum vaspin concentrations in the early post-partum period compared with the subjects that had appropriate gestational weight gain. Our observation supports previous hypotheses that vaspin might be used as a marker of lipid metabolism in pregnancy and maternal adipose tissue. Considering the fact that FABP4 is widely referred to as a pro-inflammatory adipokine, further research on the protective role of vaspin seems crucial, especially in the context of its relationship to FABP4.

Ghrelin in Serum and Urine of Post-Partum Women with Gestational Diabetes Mellitus.

Women with a previous history of gestational diabetes mellitus (GDM) have a significantly increased risk of developing type 2 diabetes, obesity, and cardiovascular diseases in the future. The aim of the study was to evaluate ghrelin concentrations in serum and urine in the GDM group in the early post-partum period, with reference to laboratory results, body composition, and hydration status. The study subjects were divided into two groups, that is, 28 healthy controls and 26 patients with diagnosed GDM. The maternal body composition and hydration status were evaluated by the bioelectrical impedance analysis (BIA) method. The concentrations of ghrelin in the maternal serum and urine were determined via enzyme-linked immunosorbent assay (ELISA). The laboratory and BIA results of the mothers with GDM were different from those without GDM. Urine ghrelin positively correlated with serum ghrelin and high-density lipoprotein cholesterol (HDL) levels in healthy mothers. There were direct correlations between urine ghrelin and HDL as well as triglycerides levels in the GDM group. Neither the lean tissue index nor body cell mass index were related to the serum ghrelin concentrations in this group. Only the urine ghrelin of healthy mothers correlated with the fat tissue index. Our results draw attention to urine as an easily available and appropriable biological material for further studies.

Urine and serum ghrelin, sCD80 and sCTLA-4 levels in doxorubicin-induced experimental nephrotic syndrome.

BACKGROUND: Nephrotic syndrome (NS) is an immune-mediated disorder associated with hyperlipidemia. NS has been proposed to be mediated through CD80-related T cell immune response, which could be blocked using soluble cytotoxic T lymphocyte-associated s(CTLA)-4. Although ghrelin is a hormone-modulating lipid metabolism and suppressing immune system, the precise role of ghrelin in NS is not well established. METHODS: We evaluated the levels of ghrelin, soluble CD80 (sCD80) and sCTLA4 in serum and urine in doxorubicin-induced NS in rats. We also investigated the relation between their levels and the levels of serum total cholesterol (TC), triglyceride, albumin and urine protein. RESULTS: While urinary ghrelin levels were significantly lower in the nephrotic rats compared to the control group, serum ghrelin levels were comparable in the nephrotic and control rats. In contrast, serum and urinary sCD80 and sCTLA4 levels were higher in the nephrotic rats than the controls. The urinary ghrelin levels were negatively correlated with the levels of serum triglyceride, TC and urine protein, sCD80 and sCTLA4. The urine sCD80 levels were positively correlated with the TC, urine protein and urine sCTLA4 levels, and negatively correlated with the serum albumin. The urine sCTLA4 levels were positively correlated with the TC and urine protein levels and negatively correlated with the serum albumin levels. In regression analysis, the urine ghrelin levels significantly relate to urine sCD80 levels. Besides, hyperlipidemia in NS did not appear to be related to serum ghrelin levels. CONCLUSION: Low urine ghrelin levels might be relevant to pathogenesis of doxorubicin-induced NS. The reduction in urine ghrelin levels might also be associated with increased levels of urine sCTLA4 and sCD80 which reflect proteinuria.

Active ghrelin and the postpartum.

Postpartum depression (PPD) occurs in 10-15 % of women. The appetite hormone ghrelin, which fluctuates during pregnancy, is associated with depression in nonpregnant samples. Here, we examine the association between PPD and active ghrelin from pregnancy to postpartum. We additionally examine whether ghrelin changes from pregnancy to postpartum and differs between breastfeeding and non-breastfeeding women. Sixty women who participated in a survey examining PPD and had information in regard to ghrelin concentrations were included in the study. The Edinburgh Postnatal Depression Scale was used to assess symptoms of PPD. Raw ghrelin levels and ghrelin levels adjusted for creatinine were included as outcomes. Women screening positive for PPD at 12 weeks postpartum had higher pregnancy ghrelin concentrations. Ghrelin concentrations significantly decreased from pregnancy to 6 weeks postpartum and this change differed based on pregnancy depression status. Finally, ghrelin levels were lower in women who breastfed compared with women who were bottle-feeding. No significant findings remained once ghrelin levels were adjusted for creatinine. Although results do not suggest an association between PPD and ghrelin after adjusting for creatinine, future research should continue to explore this possibility extending further across the postpartum period with larger sample sizes.

Elevated circulating ghrelin, but not peptide YY(3-36) levels, in term neonates with infection.

BACKGROUND: Early diagnosis and treatment of neonatal infection is important to prevent morbidity and mortality. The gastrointestinal tract-derived hormones ghrelin and peptide YY (PYY), which participate in the regulation of food intake and energy balance, may also play roles in the inflammatory response. Their involvement in neonatal infection is not known. METHODS: Plasma ghrelin and PYY(3-36) levels were serially measured (by ELISA) on Days 0, 1, 2, 3 and 7 following admission in 36-term neonates with febrile infection (22 of them were septic) and once in 20 healthy term neonates of similar postnatal age and gender distribution, as controls. Associations of ghrelin and PYY(3-36) levels with clinical and laboratory parameters, including anthropometrics, fever, leukocyte and platelet counts, serum glucose, C-reactive protein (CRP) and serum amyloid A levels, were assessed. RESULTS: Plasma ghrelin levels were significantly higher in infected neonates than in controls at each study day (p=0.009), whereas PYY(3-36) levels did not differ significantly between patients and controls at any day. In infected neonates, ghrelin levels on admission correlated negatively with serum glucose levels (p=0.003), whereas fever change during the course of infection was significantly associated with change of ghrelin levels (p=0.01). Receiver operating characteristic analysis of ghrelin levels resulted in significant areas under the curve (AUC) for detecting infected neonates on admission (AUC=0.728, p=0.005). CONCLUSIONS: Circulating ghrelin, but not PYY(3-36), levels are increased in neonates with infection, possibly reflecting and/or participating in the inflammatory process.

Serum and urinary leptin and ghrelin in children with nephrotic syndrome.

OBJECTIVE: The aim of the present study was to evaluate the serum and urinary levels of leptin and ghrelin in children with primary idiopathic nephrotic syndrome (NS), to compare these results between patients during the relapse and remission phase and to evaluate the possible role of leptin and ghrelin in the pathogenesis of NS. PATIENTS AND METHODS: Forty-nine children with primary idiopathic NS (25 children with relapse and 24 children in remission), who were followed up at the Pediatric Nephrology Unit, enrolled. Twenty-eight age- and sex-matched healthy children served as controls. Serum and urinary leptin levels were determined by immunoenzymatic ELISA, and serum and urinary ghrelin levels were determined by the RIA method. RESULTS: The serum leptin levels were significantly lower in the children with NS during the relapse phase than in the children with NS during remission or in the controls (1.42±0.34 ng/dl and 3.60±0.70 ng/ml; p<0.01, 1.42±0.34 ng/ml and 5.27±4.67 ng/ml; p<0.001, respectively). The urinary leptin excretion levels were significantly higher in the relapse group than in the controls (0.40±0.11 ng/ml and 0.12±0.06 ng/ml, p<0.01, respectively). The serum ghrelin levels were similar between the study groups (p>0.05). The urinary ghrelin excretion levels were significantly higher in the relapse group than in the remission group and the controls (965.0 pg/ml [93-3711] and 679.7 pg/ml [93-3783], p<0.05; 965.0 pg/ml [93-3711] and 387.7 pg/ml [114-1214], p<0.001, respectively). The urinary ghrelin levels were also significantly higher in the remission group than in the controls (679.7 pg/ml [93-3783] and 387.7 pg/ml [114-1214]), p<0.01, respectively). The serum leptin levels were positively correlated with the serum albumin levels (r=0.440, p<0.05) and were negatively correlated with the serum triglyceride levels during the relapse phase. The urinary leptin and ghrelin levels were positively correlated with proteinuria in the relapse group. CONCLUSIONS: We propose that leptin plays a role in the pathophysiology of NS and is associated with proteinuria, hypoproteinemia and hyperlipidemia. The significant urinary excretion of ghrelin in children with NS is possibly due to underlying pathophysiological changes, and normal serum ghrelin levels might be associated with an unknown compensatory mechanism.

Simultaneous quantification of acylated and desacylated ghrelin in biological fluids.

This study reports simultaneous quantification of both acylated and desacylated forms of ghrelin in biological samples, utilizing a reverse-phase high-performance liquid chromatography (HPLC) system. The HPLC assay was also compared with RIA assays in use. Biological samples (serum, saliva, urine, milk) known for the presence of ghrelin were collected from a total of eight post-partum women and eight male volunteers. Analysis of ghrelin with HPLC was also validated for linearity, precision, detection limit and accuracy. An elution time of 6 min was observed for pure (commercial) desacylated human ghrelin and for the same form of the hormone from all body fluids studied. The elution time for acylated pure human ghrelin and that in body fluids, however, was around 16 min. The mean recovery rate was over 90% for both forms with no significant interference. The lowest detectable levels for acylated and desacylated ghrelin with the method used here were 11 (+/-2) and 14 (+/-3) pg mL(-1), respectively. Given its simplicity, accuracy, time and cost-effectiveness, the HPLC method described here for determination of two forms of ghrelin (active and inactive) might prove useful for certain diagnostic purposes.