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
