Dityrosine (dityrosine), an oxidation product of tyrosine produced by reaction between tyrosyl radicals, is becoming established as a biomarker of free radical oxidative protein damage in vivo. Attempts to measure dityrosine concentrations in various physiological and pathological systems have produced varied and often contradictory results. Dityrosine concentrations in urine, plasma, cerebrospinal fluid (CSF) and brain tissue varying over three orders of magnitude have been reported, together with inconsistent claims of significant dityrosine elevation in several ageing-related pathologies. Some of these findings have contributed to the implication of free radical activity in the pathology of several neurodegenerative disorders, vascular and ocular abnormalities and in phagocyte response to infection.

The aim of this study was to test the hypothesis that dityrosine levels are elevated in ageing and ageing-related disease. The study also aims to determine the utility of dityrosine measurement as an index of oxidative damage, and elucidate possible explanations for the inconsistent levels reported.

An assay for the quantification of dityrosine was developed using capillary HPLC with electrospray tandem quadrupole mass spectrometry (HPLC-MS/MS). The assay was highly specific for dityrosine and has the highest absolute sensitivity for dityrosine of any method reported to date, with a detection limit of 3 femtomoles of dityrosine on-column. Urine samples from volunteers of different age and from hospital patients with various pathologies were analysed. Plasma protein hydrolysates from control, Alzheimer’s and stroke subjects were analysed, together with hydrolysates of post mortem brain tissue from Alzheimer’s and control subjects.

Urinary dityrosine level is elevated in states of acute infection and inflammation, but does not correlate with age or chronic disease. Protein dityrosine in four sections of Alzheimer’s brain was not significantly different from control sections. Dityrosine was present in human plasma and tissue proteins at approximately 5-35 residues per million tyrosine residues, and in normal urine at 5-25 micromol/mol creatinine or 20-200 nM. Most of the discrepancies in the literature relate to inadequate specificity of the analytical method. Interpretation of published data with critical appraisal of measurement technology specificity is essential in developing an accurate understanding of the role of free radicals in ageing and disease.