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Previous studies examining the involvement of oxidative stress in the substantia nigra in Parkinson's disease have measured terminal products of lipid peroxidation or the function of antioxidant defense systems. We report a more specific early marker of lipid peroxidation, lipid hydroperoxides, in a high-performance liquid chromatography (HPLC) and electron spin resonance (ESR) investigation. HPLC-chemiluminescent detection revealed two classes of lipid hydroperoxides in brain tissue extracts--free fatty acid hydroperoxides and cholesterol lipid hydroperoxides. Only cholesterol lipid hydroperoxides were consistently detected in all tissue extracts. Cholesterol lipid hydroperoxides had a 10-fold increase in the Parkinson's disease substantia nigra compared to control subjects. ESR detection of radical degradation products, including those of lipid hydroperoxides, in nigral homogenates incubated with the spin trap N-t-butyl-alpha-phenyl nitrone (PBN) showed a marked variation in ESR signal between tissues. Despite the increased levels of lipid hydroperoxides in parkinsonian substantia nigra, there was no overall difference in ESR signal intensity between nigral tissues from controls and from patients with Parkinson's disease. The increased levels of an early component of the peroxidation chain in substantia nigra in Parkinson's disease support the hypothesis of a continuous toxic process involving oxygen radical activity. However, using previously frozen tissue, ESR evidence for increased radical formation could not be demonstrated.

The increasing interest in the role of free radicals in the pathogenesis of human disease has led to an increased need for techniques to measure free radicals and their reactions in vivo and, most importantly, in the clinical situation. Free radicals are extremely reactive and thus short lived. Consequently, free radicals are not amenable to direct assay and free radical activity is usually assessed by indirect methods such as measurement of the various end products of reactions with lipids, proteins and DNA. A vast array of analytical techniques has been developed to measure these end products though not all of them are applicable to the clinical situation where the only samples normally available are blood, urine and expired breath. Lipid peroxidation is the most intensively studied process and provides a number of possibilities for assays. Protein and nucleic acid oxidation are attracting increasing attention at the present time. The techniques currently available, however, are limited to semi-quantitative assays of damage to broad classes of biomolecules and there is an urgent need for more specific and informative methods.

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Patients undergoing percutaneous transluminal coronary angioplasty (PTCA) were investigated for the production of free radicals and cholesterol hydroperoxides during reperfusion. Fifteen patients were studied. Ischaemia during balloon inflation was assessed by serial coronary sinus lactate analysis (mean maximal increase in anterior descending artery dilation was 130%), and by the demonstration of reperfusion hyperaemia (mean increase of coronary sinus oxygen saturation 74%). Free radicals were detected by electron spin resonance (ESR) spin trapping using the spin trap PBN (N-t-Butyl-alpha-phenylnitrone). Radical adducts were detected in up to 50% of samples taken during reperfusion after anterior descending lesion angioplasty. No radicals were detected in control samples or during the ischaemic phase. Radical detection was positively correlated with the change in coronary sinus lactate (p less than 0.025). Coronary sinus cholesterol hydroperoxide analysis did not show a significant increase over control during reperfusion, due in part to unexpectedly high pre angioplasty levels. This study provides clear evidence for the production of a burst of free radicals and evidence for lipid peroxidation in the minutes following myocardial reperfusion during angioplasty. A relationship between the severity of the ischaemic insult and the detection of radical adducts has also been found.

A modification of a method using high-performance liquid chromatography (HPLC) with chemiluminescence (CL) detection for the measurement of lipid hydroperoxides (LOOH) in human blood plasma has been developed. The system involves separation of different classes of LOOH using reverse-phase HPLC, and post-column detection of CL produced by isoluminol oxidation during the reaction of LOOH with microperoxidase. Complete ultra-violet absorption spectra are collected with an in-line diode-array detector and used to confirm a positive CL response due to LOOH, or other compounds, by the presence or absence, respectively, of the LOOH conjugated diene chromophore. We have used the method to investigate the stability of exogenous 15(S)-HPETE (a hydroperoxide of eicosatetraenoic acid) and conjugated dienes (of both 15(S)-HPETE and its reduced metabolite, 15(S)-HETE) in human plasma stored at various temperatures. A large and rapid loss of the hydroperoxide occurred in plasma incubated at 0 degrees C or 27 degrees C, whereas only a small reduction in the level of conjugated dienes was found. 15(S)-HPETE in PBS was stable under the same conditions, and zero time recovery of the hydroperoxide from denatured plasma and from buffer containing albumin was identical to that of fresh plasma. Our data suggest that the observed temperature-dependent loss of exogenous hydroperoxide from fresh plasma results from a combination of enzymatic degradation to the hydroxy derivative and binding to plasma albumin. 15(S)-HPETE was found to be stable in plasma stored at -70 degrees C for up to 2 weeks and in liquid nitrogen for 3 months in the presence of the antioxidants butylated hydroxytoluene (BHT) and desferal, with no significant loss of conjugated dienes.

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