The application of gas chromatography and mass spectrometry for the diagnosis and study of genetic disorders - the use of stable isotope labelled substrates for the study of two patients with non-hepatic tyrosinaemia
Doctor of Philosophy
Gan, Ignatius Eng Tho, The application of gas chromatography and mass spectrometry for the diagnosis and study of genetic disorders - the use of stable isotope labelled substrates for the study of two patients with non-hepatic tyrosinaemia, Doctor of Philosophy thesis, , University of Wollongong, 1976. http://ro.uow.edu.au/theses/4475
Deuterated tyrosine has been used to study tyrosine metabolism in one normal and two patients with rare forms of non-hepatic tyrosinaemia. The labelled tyrosine was ingested orally, after which urine and serum samples were examined by GC-MS at regular intervals for residual labelled tyrosine and tyrosine metabolites.
The derivatives of choice for GC-MS were the O-methyl ether of the N-neopentylidene tyrosine methyl ester, the trimethyl silyl derivatives of the phenolic acids and the N-trifluoroacetyl O-trimethyl silyl derivatives of the catecholamines.
It was shown that loading rates of 220 μmoles of deuterated tyrosine/kg body weight were adequate for precise measurement of the stable isotope content of urinary and serum tyrosine and tyrosine metabolites. This load approximates to that provided by a protein containing meal, so these stable isotope load studies can be claimed to mimic physiological conditions.
The amount of deuterated substrate used in this study was not considered a health risk, as even complete isotope retention in vivo would only increase the natural abundance in the body by less than 20%.
Deuteration experiments were made with a combined GC-MS instrument operating under computer control using time averaging and mass fragmentometry. The studies on the two patients and a normal control were carried out under conditions of high and low tyrosine loads. The results of these studies defined the biochemical lesion in one patient at the site of p-hydroxyphenylpyruvic acid oxidase. Although the site of the primary defect in the other patient has not been established. It is clear that it is not at the site of p-hydroxyphenylpyruvic acid oxidase. This patient suffered from substrate inhibition of p-hydroxyphenylpyruvic acid oxidase when on a normal diet. Resolution of the clinical and biochemical symptoms with disappearance of the inhibition of p-hydroxyphenylpyruvic acid oxidase occurred with the implementation of a diet low in phenylalanine and tyrosine. Although the fundamental defect in this patient is not resolved, the findings appear compatible with a defect in hepatic soluble tyrosine aminotransferase. The rates of deuteration of tyrosine metabolites have allowed us to distinguish between individuals having different clinical manifestations but similar biochemical abnormalities.