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Urate is the terminal product of purine metabolism in primates, including humans. Urate is also an efficient scavenger of oxidizing species and is thought to be an important antioxidant in human body fluids. Allantoin, the major oxidation product of urate, has been suggested as a candidate biomarker of oxidative stress because it is not produced metabolically. Although urate is converted to allantoin under strongly alkaline pH, such conditions have been used in the past to facilitate extraction of allantoin. We evolved a method for the determination of allantoin concentrations in human plasma and serum by gas chromatography-mass spectrometry without such artifact. With this method, we show that alkaline conditions do indeed cause breakdown of urate, leading to significant overestimation of allantoin concentration in human samples. By using our alternative method, serum samples from 98 volunteers were analyzed, and allantoin levels were found to be significantly lower than was previously reported. The in vivo utility and sensitivity of our method was further evaluated in human nasal-lining fluids. We were able to demonstrate an ozone-induced increase in allantoin, in the absence of increases in either ascorbate or glutathione oxidation products.

Rabbits were fed a diet containing 1% cholesterol for 8 weeks and the levels of iron and oxidized lipids in liver analysed using atomic absorption spectroscopy and gas chromatography-mass spectrometry. A non-significant trend to an increase in iron level, but significant increases in the lipid peroxidation products, F(2)-isoprostanes and the cholesterol oxidation products 7 beta hydroxycholesterol, 7 ketocholesterol and cholesterol 5,6-alpha epoxide were detected in the liver of the cholesterol-fed rabbits. Histological analysis showed greater accumulation of lipids by Sudan red labelling in hepatocytes of zone I than zones II and III of the liver acinus. The increase in lipids coincided with an increase in iron staining in macrophages around liver microvessels and increased immunostaining to melanotransferrin and the lipid peroxidation product, 4-hydroxynonenal (4-HNE), in zone 1. The results are suggestive of microvascular damage associated with iron accumulation and oxidative stress in the liver during hypercholesterolemia.

Findings that antioxidant treatment may be beneficial in Alzheimer's disease indicate that oxidative stress is an important factor in its pathogenesis. Studies have also suggested that cholesterol imbalance in the brain might be related to the development of neurological disorders. Previously, we have reported that U18666A, a cholesterol transport-inhibiting agent, leads to apoptosis and intracellular cholesterol accumulation in primary cortical neurons. In this study, we found that neuronal apoptosis mediated by U18666A is associated with oxidative stress in the treated cortical neurons. Cortical neurons treated with U18666A also showed decreased secretion and increased intraneuronal accumulation of beta-amyloid. The association of neuronal apoptosis with oxidative stress and Abeta accumulation may provide clues to the pathogenesis of Alzheimer's disease, as well as the role oxidative stress plays in other neurodegenerative diseases.

Dark soy sauce (DSS) is a powerful antioxidant in vitro. We investigated whether this effect could occur in vivo and improve vascular function. Healthy human subjects were given DSS or placebo meals in a randomized, crossover study. Blood and urine were sampled before and 1, 2, 3, and 4h after the meal for F(2)-isoprostanes (total, free, and esterified) and 8OHdG measurements. Blood pressure, vascular augmentation index (AIx), and heart rate (HR) were also measured. Plasma total F(2)-isoprostanes significantly decreased 3h after placebo and the decrease was greater for DSS. Plasma free and esterified F(2)-isoprostanes were also significantly decreased after DSS. Both placebo and DSS meals increased urinary F(2)-isoprostanes at 1h but not thereafter, and lowered urinary 8OHdG levels, DBP and AIx, and increased HR. We conclude that DSS decreases lipid peroxidation in vivo. However, oxidative damage biomarkers changed after the placebo meal, a phenomenon to consider when designing interventional studies.