Sharma, G.; (2012) Methods for the measurement of urinary biomarkers of oxidative stressapplication to type 1 diabetes mellitus. Doctoral thesis , UCL (University College London).

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Abstract

The principal aim of the study was to develop methods for the measurement of potential urinary biomarkers of oxidative stress using liquid chromatography/tandem mass spectrometry with minimum sample preparation to avoid artefact formation. Initially the development of an assay to measure the urinary concentrations of isoprostanes (8- isoPGF2α) was attempted but this did not prove to be sufficiently sensitive and gave nonreproducible results. An assay to measure the intact sulphate and glucuronide conjugates of urinary metabolites of vitamin E [α-tocopheronolactone (α-TLHQ) and α-carboxy-ethylhydroxychroman (α-CEHC)] was then developed, as it has been suggested that α-TLHQ with an oxidised chroman ring might be an indicator of oxidative stress. A novel method was also developed to quantitate urinary amino acids associated with NO• metabolism (Larginine - precursor, L-citrulline - product, L-ADMA –inhibitor of nitric oxide synthase and L-homocysteine – reduces bioavailability of nitric oxide). This method was extended to quantitate seven additional amino acids. The latter two methods were applied to 32 children with type 1 diabetes and compared with age and sex matched controls. The mean concentrations of all the α-THLQ conjugates were highly significantly increased in the diabetic subjects (p<0.002). The concentrations of the α-CEHC conjugates were also increased but not to the same degree of significance (p<0.05). When the diabetic children were divided into those who were poorly (n=24) and adequately (n=8) controlled, the α- THLQ conjugates remained highly significantly increased (p<0.002) in the poorly controlled group compared to controls. However, the concentrations of the α-CEHC conjugates were not significantly different. The diabetic subjects had a highly significantly increased concentration (p<0.0001) of all the urinary amino acids studied compared to controls. These results suggest that the measurement of urinary α-TLHQ conjugates may provide a useful biomarker of oxidative stress. The clinical relevance of the increased concentrations of urinary amino acids in children with type 1 diabetes requires further investigation.

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