Measurement of glutathione synthesis by isotope ratio mass spectrometry in systemic inflammation.
Doctoral thesis, UCL (University College London).
During sepsis and critical illness, a systemic inflammatory response can significantly produce reactive oxygen and nitrogen species, which can damage host tissues. Usually, several different antioxidant defences protect host cells from these reactive species. One of the important antioxidant defences is glutathione, a tripeptide synthesised from the precursor amino acids cysteine, glutamate and glycine. The aim of this study was to measure glutathione synthesis in vivo, using deuterated glycine as a tracer. Firstly, a new method for analysis of glutathione and glycine using both gas chromatography-mass spectrometry (GC-MS) and gas chromatography-isotope ratio mass spectrometry (GC-IRMS) was developed. The derivatives of both compounds were measured in a single chromatographic analysis, and the method was compatible with both GC-MS and GC-IRMS, and capable of measuring low carbon-13 or deuterium enrichment of glutathione in 50μl of erythrocytes. Erythrocyte glutathione synthesis was measured, using GC-IRMS, in critically ill infants and children who had been infused with deuterated glycine. Glutathione fractional synthesis rate (FSR) was not statistically different between septic and non-septic patients. In order to determine whether glutamine is able to increase GSH synthesis by acting as a glutamate precursor, erythrocyte GSH synthesis was measured using GC-IRMS in septic infants and children randomised to receive glutamine dipeptide or placebo. There was no significant difference in glutathione FSR between those given glutamine and those given placebo. Intestinal glutathione depletion is known to occur following intestinal ischaemia-reperfusion injury. I measured glutathione synthesis in a rat model of intestinal ischaemia-reperfusion injury, and found that hypothermic rats with ischaemia-reperfusion injury had a higher intestinal glutathione synthesis than normothermic rats, providing a potential mechanism by which hypothermia may maintain intestinal glutathione levels. In conclusion, the newly developed method of glutathione analysis using GC-IRMS was useful for compound specific isotope analysis of biological samples.
|Title:||Measurement of glutathione synthesis by isotope ratio mass spectrometry in systemic inflammation|
|Open access status:||An open access version is available from UCL Discovery|
|UCL classification:||UCL > School of Life and Medical Sciences > Faculty of Population Health Sciences > Institute of Child Health|
Archive Staff Only