The application of gas chromatography and mass spectrometry to the study of human diseases: the identification and quantitation of the urinary volatiles associated with a number of genetic defects

The past 20 years have seen a rapid growth in studies of inborn errors of metabolism. One of the main aids to this growth has been the concommitant development in organic analytical techniques, especially those based on gas chromatography (GC) and combined gas chromatography-mass spectrometry (GC-MS). The identification of the organic acids present in body fluids using GC-MS techniques has been remarkably successful in discovering new and diagnosing known inborn errors of metabolism. This type of analysis requires the separation of the organic acid fraction from the body fluid, followed by derivatisation of the acids to increase their volatility thus enabling characterization of the acids by GC and GC-MS. The organic acid profile so produced is now used routinely in some laboratories to screen patients suspected of suffering from a metabolic disorder. The success of this technique has led to the further development of the concept of metabolic profiles of specific classes of metabolites in body fluids. Workers investigating perfumes and food aromas and flavours have developed specialized techniques for identifying odorous volatile organic compounds. These methods are based on headspace extraction and concentration of volatile organics followed by analysis of the underivatised extract by capillary GC and capillary GC-MS. These methods have been applied to the analysis of volatile metabolites in human body fluids. The profile of volatile metabolites in normal human urine is well characterized. This thesis describes the application of volatile metabolite profiling to a number of inborn errors of metabolism which are characterized by peculiar body odours. It is shown that the urinary volatiles profile for patients with Phenylketonuria, Maple Syrup Urine Disease, Isovaleric Acidemia and Trimethylaminuria (fishy odour syndrome) is in each case surprisingly different to the normal urinary volatiles profile. In the Maple Syrup Urine Disease case we found metabolites which occur distal to the metabolic block. Since we had not observed these compounds in the organic acids profile further investigation was needed to ensure that they were not artifacts introduced by the analytical technique. This led to our development of an improved method for analysing volatile short chain fatty acids. We found a new major metabolite in Isovaleric Acidemia. This compound had not been previously observed because its volatility resulted in excessive losses during sample work up. We developed a quantitative method for estimating trimethylamine in urine. The technique was based on isotope dilution methodology and used headspace trapping for the extraction of trimethylamine from urine. We used this determination to study some proposed therapies for patients with the fishy odour syndrome. We concluded that because the volatiles profiles of patients with inborn errors of metabolism mentioned above were strikingly abnormal these profiles could be used as an aid to the diagnosis of the disorders. In addition, by using this technique new and unexpected metabolites which may help clarify the basis of the metabolic distress can be found. Moreover, by adjusting the headspace conditions specific analytes of interest in these cases can be quantified