Fasting Upregulates npy, agrp, and ghsr Without Increasing Ghrelin Levels in Zebrafish (Danio rerio) Larvae.

Food intake in fish and mammals is orchestrated by hypothalamic crosstalk between orexigenic (food intake stimulation) and anorexigenic (food intake inhibition) signals. Some of these signals are released by peripheral tissues that are associated with energy homeostasis or nutrient availability. During the fish larva stage, orexigenic stimulation plays a critical role in individual viability. The goal of this study was to assess the mRNA levels of the main neuropeptides involved in food intake regulation (npy, agrp, carppt, and pomc), in concert with the mRNA levels and peptide levels of ghrelin, under a fasting intervention at the larval stage in zebrafish (Danio rerio). Prior to the fasting intervention, the zebrafish larva cohort was reared for 20 days post fertilization (dpf) and then randomly divided into two groups of 20 individuals. One group was subjected to a fasting intervention for 5 days (fasted group), and the other group was fed normally (fed group); this experimental protocol was performed twice independently. At the end of the fasting period, individuals from each experimental group were divided into different analysis groups, for evaluations such as relative gene expression, immunohistochemistry, and liquid chromatography coupled to nano high-resolution mass spectrometry (nLC-HRMS) analyses. The relative expression levels of the following genes were assessed: neuropeptide Y (npy), agouti-related peptide (agrp), proopiomelanocortin (pomc), cocaine and amphetamine-regulated transcript (cartpt), ghrelin (ghrl), ghrelin O-acyltransferase (mboat4), growth hormone secretagogue receptor (ghsr), and glucokinase (gck). In the fasted group, significant upregulation of orexigenic peptides (npy - agrp) and ghsr was observed, which was associated with significant downregulation of gck. The anorexigenic peptides (pomc and cartpt) did not show any significant modulation between the groups, similar to mboat4. Contrary to what was expected, the relative mRNA upregulation of the orexigenic peptides observed in the fasted experimental group could not be associated with significant ghrelin modulation as assessed by three different approaches: qPCR (relative gene expression of ghrelin), nLC-HRMS (des-acyl-ghrelin levels), and immunohistochemistry (integrated optical density of prepropeptides in intestinal and hepatopancreas tissues). Our results demonstrate that zebrafish larvae at 25 dpf exhibit suitable modulation of the relative mRNA levels of orexigenic peptides (npy and agrp) in response to fasting intervention; nevertheless, ghrelin was not coregulated by fasting. Therefore, it can be suggested that ghrelin is not an essential peptide for an increase in appetite in the zebrafish larva stage. These results give rise to new questions about food intake regulation factors in the early stages of fish.

Systematic analysis of glycerol: colourimetric screening and gas chromatography-mass spectrometric confirmation.

Glycerol is a naturally occurring polyol in the human body, essential for several metabolic processes. It is widely used in the food, pharmaceutical, and medical industries and in clinical practice as a plasma volume expander (PVE). Athletes, however, may use glycerol to mask the presence of forbidden substances or to enhance performance, inclusively through hyperhydration achieved by glycerol ingestion with added fluid. These practices are considered doping, and are prohibited by the World Anti-Doping Agency (WADA). Therefore, glycerol was introduced in the prohibited list. Doping through glycerol ingestion can readily be identified by detection of elevated glycerol concentrations in urine. In this paper, a protocol for the fast detection of glycerol in urine is proposed. It consists of a previous visual colourimetric screening, followed by a quantitative/qualitative confirmation analysis by mass spectrometry. The screening procedure involves a reaction in which polyhydric alcohols are oxidized by periodate to formic acid and formaldehyde, which is detected by the addition of a fuchsin solution. For the subsequent qualitative/quantitative confirmation analysis, a gas chromatography-mass spectrometry based approach with a non-deuterated internal standard and a drying step of only 10 min is proposed. The linear correlation was demonstrated within WADA´s threshold range. The calculated RSD were 2.1% for within-day precision and 2.8% for between-day precision. The uncertainty estimation was calculated, and a value of 2.7% was obtained. The procedure may also be used for the analysis of other polyols in urine, as for example the PVE mannitol.

Identification of sympathomimetic alkylamine agents in urine using liquid chromatography-mass spectrometry and comparison of derivatization methods for confirmation analyses by gas chromatography-mass spectrometry.

The detection of 11 sympathomimetic alkylamines in urine was presented with a focus on human doping control is proposed using liquid chromatography tandem mass spectrometry (LC-QqQ) and high resolution mass spectrometry (LC-HRMS) as a screening tool after a dilute-and-shoot (DS) approach. For the LC-HRMS analyses, several compounds exhibited better limits of detection (L.O.D.) than the LC-QqQ. However, due to their small differences in structure, co-elution among the alkylamines was observed. Therefore, the chemical conversion of the alkylamines into an appropriate derivative for the confirmation analyses using gas chromatography-mass spectrometry (GC-MS) was evaluated. Five derivatization approaches were evaluated in an attempt to increase the analytical response and the confidence of the identification. The choice of the appropriated derivative for each alkylamine makes their spectra more easily interpretable, fulfills the WADA's rather strict identification criteria and enables the unequivocal identification of alkylamines in urine.