Richard W. Browne

The Analysis of Free Radicals, Lipid Peroxides, Antioxidant Enzymes and Compounds Related to Oxidative Stress as Applied to the Clinical Chemistry Laboratory

It is almost impossible to read through a medical journal, or even the newspaper and not encounter an article that deals with oxidative stress, or with antioxidant involvement in a disease process. Indeed, free radicals, their reactive intermediates, low molecular weight aldehyde byproducts derived from lipid peroxidation and antioxidant status are important measurements we can utilize to provide a more comprehensive understanding of pathologic mechanisms (1–8). All subcellular organelles normally generate superoxide (O2·-), hydrogen peroxide and a variety of free radicals ie; hydroyl (OH·), perhydroxy(HO2·), carbon and nitrogen centered. It has been estimated that 10 billion of these radicals are produced daily via autoxidation and metabolic reactions. In cellular injury, increased amounts of O2·- radicals and peroxides can arise from the mitochondrial electron-transport system during hypoxia and following reperfusion, they can arise primarily through the activation of NADPH oxidase in phagocyte plasma membranes or from platelet derived endoperoxides of arachidonic acid, from the conversion of xanthine dehydrogenase to xanthine oxidase in tissue and from the generation of OH· radicals in iron-catalyzed reactions involving hemoproteins (9). The most current review by Chaudiere covers theoretical and factual site-specific formation and damage (10).

characterization Of Peroxidized Lipids In Bruch's Membrane

PURPOSE To determine if peroxidized lipids occur in Bruchs membrane isolates and to characterize the type present in human necropsy specimens. METHODS Bruchs membrane isolates from eye bank eyes obtained from 13 white donors were homogenized. Measurement of peroxidized lipids was done with the fluorometric thiobarbituric acid assay and high pressure liquid chromatography. RESULTS Bruchs membrane isolate homogenates contained native unsaturated fatty acids and peroxidized lipids in a ratio of about 200:1. The amount of thiobarbituric acid reacting substances increased exponentially with age. The peroxidized lipids identified in Bruchs membrane isolates were derived from long chain polyunsaturated fatty acids, particularly docosahexaenoic acid and linolenic acid, which are normally found in the photoreceptor outer segments. CONCLUSIONS Lipids are known to accumulate in Bruchs membrane, an acellular layer with no known intrinsic mechanisms to combat lipid peroxidation. In related studies, lipid peroxides have been shown to induce neovascularization by inducing expression of a cascade of angiogenic cytokines. This is the first study to show that lipid peroxides, biological molecules that have the potential to incite new vessel growth, occur in Bruchs membrane. The increase in amount of peroxidized lipids with age, combined with their vasogenic potential, suggests that peroxidized lipids may play a role in the etiology of age-related macular degeneration, particularly choroidal neovascularization.

BioCycle study: design of the longitudinal study of the oxidative stress and hormone variation during the menstrual cycle.

Studies in both human and animal species have suggested that oxidative stress may be associated with health outcomes, including the risk of infertility in both males and females. Sex hormones have been shown to have antioxidant properties. The difficulty in studying the role of oxidative stress in females is partly due to fluctuation in these endogenous sex hormones across the menstrual cycle. The aim of this study was to determine the association of oxidative stress levels with endogenous reproductive hormone levels and antioxidants, including vitamin levels, across the menstrual cycle in a prospective cohort of premenopausal women. The goal was to enroll 250 healthy, regularly menstruating premenopausal women for two menstrual cycles. Participants visited the clinic up to 8 times per cycle, at which time blood and urine were collected. The visits occurred at key hormonally defined phases of the menstrual cycle, with the help of an algorithm based on cycle length and data from a fertility monitor. In addition, participants were administered standardised questionnaires, had various physical measures taken, and had other pertinent data collected. A total of 259 women were enrolled in this study, with 250 completing two cycles, despite a demanding study protocol which participants were required to follow. This report describes the study design, baseline characteristics and visit completion rate for the BioCycle study.

Antioxidants, oxidative stress, and pulmonary function in individuals diagnosed with asthma or COPD.

Objective:The objective of this study was to investigate the association between antioxidant nutrients and markers of oxidative stress with pulmonary function in persons with chronic airflow limitation.Design:Cross-sectional study exploring the association of antioxidant nutrients and markers of oxidative stress with forced expiratory volume in the first second (FEV1%) and forced vital capacity (FVC%).Setting/Subjects:The study data included 218 persons with chronic airflow limitation recruited randomly from the general population of Erie and Niagara counties, New York State, USA.Results:After adjustment for covariates, multiple linear regression analysis showed that serum β-cryptoxanthin, lutein/zeaxanthin, and retinol, and dietary β-carotene, β-cryptoxanthin, lutein/zeaxanthin, vitamin C, and lycopene were positively associated with FEV1% (P<0.05, all associations). Serum vitamins β-cryptoxanthin, lutein/zeaxanthin, and lycopene, and dietary β-cryptoxanthin, β-carotene, vitamin C, and lutein/zeaxanthin were positively associated with FVC% (P<0.05, all associations). Erythrocytic glutathione was negatively associated with FEV1%, while plasma thiobarbituric acid-reactive substances (TBARS) were negatively associated with FVC% (P<0.05).Conclusion:These results support the hypothesis that an imbalance in antioxidant/oxidant status is associated with chronic airflow limitation, and that dietary habits and/or oxidative stress play contributing roles.

Continuing medical education review: choroidal neovascularization in age-related macular degeneration--what is the cause?

After completion of this article, the reader will be able to: 1) describe, define, and discuss various theories concerning the pathogenesis of choroidal neovascularization in age-related macular degeneration, 2) identify and classify risk factors for the development of age-related macular degeneration, 3) outline and specify how stimuli for neovascularization may arise, and 4) critique proposed etiologic theories.

Manganese‐induced rat pheochromocytoma (PC12) cell death is independent of caspase activation

Manganese (Mn) is an essential mineral that at high concentrations can produce an irreversible syndrome resembling Parkinsons disease. To examine the mechanism by which Mn elicits its toxic response, we have selected the rat pheochromocytoma cells (PC12) as our model system because it possesses much of the biochemical machinery associated with dopaminergic neurons. Mn‐induced PC12 cell death is both time and concentration dependent with approximately 50% cell survival at 48 hr in the presence of 0.3 mM Mn. To determine whether oxidative stress contributed to cytotoxicity induced by Mn, lipid peroxidation was assessed in Mn‐treated in PC12 cells. The highly sensitive HPLC assay that measures the lipid peroxide product, 9‐HODE, was used and results of these experiments demonstrate there was no increase in the lipid peroxidation in cells exposed to 0.3 mM Mn for 24 hr. Mn was found to stimulate the activation of the apoptotic marker proteins, p38 and caspase‐3 within the first 24 hr of treatment. The selective inhibitor of caspase‐3, DEVD‐CHO, and the nonselective caspase inhibitor, Z‐VAD‐FMK, however, fail to prevent Mn‐induced PC12 cell death. Studies were performed to determine the role of mitochondria in initiating or supporting Mn cytotoxicity, because Mn has been reported to cause changes in membrane permeability. Mn caused a decrease in ATP levels in PC12 cells in both a time and concentration dependent manner. We hypothesize that both apoptosis and necrosis contribute to PC12 cell death although the necrotic events prevail even when the apoptotic signaling is inhibited. J. Neurosci. Res. 61:162–171, 2000.

Clozapine N-Oxide Administration Produces Behavioral Effects in Long–Evans Rats: Implications for Designing DREADD Experiments

Abstract Clozapine N-oxide (CNO) is a ligand for a powerful chemogenetic system that can selectively inhibit or activate neurons; the so-called Designer Receptors Exclusively Activated by Designer Drugs (DREADD) system. This system consists of synthetic G-protein-coupled receptors, which are not believed to be activated by any endogenous ligand, but are activated by the otherwise inert CNO. However, it has previously been shown that the administration of CNO in humans and rats leads to detectable levels of the bioactive compounds clozapine and N-desmethylclozapine (N-Des). As a follow-up, experiments were conducted to investigate the effects of CNO in male Long–Evans rats. It was found that 1 mg/kg CNO reduced the acoustic startle reflex but had no effect on prepulse inhibition (PPI; a measure of sensorimotor gating). CNO (2 and 5 mg/kg) had no effect on the disruption to PPI induced by the NMDA antagonist phencyclidine or the muscarinic antagonist scopolamine. In locomotor studies, CNO alone (at 1, 2, and 5 mg/kg) had no effect on spontaneous locomotion, but 5 mg/kg CNO pretreatment significantly attenuated d-amphetamine-induced hyperlocomotion. In line with the behavioral results, fast-scan cyclic voltammetry found that 5 mg/kg CNO significantly attenuated the d-amphetamine-induced increase in evoked dopamine. However, the effects seen after CNO administration cannot be definitively ascribed to CNO because biologically relevant levels of clozapine and N-Des were found in plasma after CNO injection. Our results show that CNO has multiple dose-dependent effects in vivo and is converted to clozapine and N-Des emphasizing the need for a CNO-only DREADD-free control group when designing DREADD-based experiments.

Lipid hydroperoxide stimulates retinal neovascularization in rabbit retina through expression of tumor necrosis factor-a, vascular endothelial growth factor and platelet-derived growth factor

To test the hypothesis that oxidative damage associated with tissue hypoxia plays a role in neovascularization, a lipid hydroperoxide (LHP) was injected into the vitreous of rabbits. Single injections of LHP (50–600μg) caused a sustained retinal neovascularization visualized clinically by ophthalmoscopy and confirmed by microscopy. Vasodilators, i.e. histamine and nitric oxide, peaked at 6h and 7 days, respectively. The levels of both tumor necrosis factor-α and interleukin-lα peaked at 12h and dropped to basal levels by 24h. Expression of vascular endothelial growth factor (VEGF) and transforming growth factor-β peaked at 24h and were sustained throughout the following 3 weeks, and platelet-derived growth factor was also elevated throughout the same period. Upregulation of these five angiogenic cytokines, but not basic fibroblast growth factor, occurred prior to the appearance of neovascularization. Leakage of fluorescein at the tips of new vessels was demonstrated by fluorescein angiography. Linoleic hydroperoxide induced neovascularization, but saturated or unsaturated native C-18 fatty acids had no effect. The cascade of multiple, angiogenic cytokines induced by LHP may interact to promote sustained neovascularization.

Endogenous Reproductive Hormones and C-reactive Protein Across the Menstrual Cycle The BioCycle Study

C-reactive protein (CRP) is one of the most commonly used markers of acute phase reaction in clinical settings and predictors of cardiovascular risk in healthy women; however, data on its physiologic regulation in premenopausal women are sparse. The objective of this study was to evaluate the association between endogenous reproductive hormones and CRP in the BioCycle Study (2005-2007). Women aged 18-44 years from western New York were followed prospectively for up to 2 menstrual cycles (n = 259). Serum levels of CRP, estradiol, progesterone, luteinizing hormone, and follicle-stimulating hormone were measured up to 8 times per cycle, timed by fertility monitors. CRP levels varied significantly across the cycle (P < 0.001). More women were classified as being at elevated risk of cardiovascular disease (CRP, >3 mg/L) during menses compared with other phases (12.3% vs. 7.4%; P < 0.001). A 10-fold increase in estradiol was associated with a 24.3% decrease in CRP (95% confidence interval: 19.3, 29.0). A 10-fold increase in luteal progesterone was associated with a 19.4% increase in CRP (95% confidence interval: 8.4, 31.5). These results support the hypothesis that endogenous estradiol might have antiinflammatory effects and highlight the need for standardization of CRP measurement to menstrual cycle phase in reproductive-aged women.

TBARS and cardiovascular disease in a population-based sample.

Background Oxygen radicals might play a crucial role in the pathogenesis of various diseases, including atherosclerosis. Thiobarbituric acid reaction substances (TBARS), a biomarker of oxidative stress, have been proposed as a summary measure of total circulating oxidation. However, there is no strong indication that circulating levels of TBARS are increased in patients with atherosclerosis. Design We evaluated the relation between TBARS and cardiovascular disease (CVD) in a cross-sectional random sample of white men and women from Buffalo, New York. Methods Logistic regression was used to estimate the risk associated with high levels of TBARS. The area under the ROC curve was used to evaluate the discriminating power of TBARS. Results After adjusting for age and gender, TBARS levels were significantly higher in those with prevalent CVD (OR=1.73, 95% CI=1.32–2.38), compared to those without a CVD diagnosis. These OR were almost 50% higher after correcting for measurement error (ME) (OR=1.93, 95% CI=1.07–3.40). The area under the ROC curve was 0.69 (95% CI=0.62–0.77) and when corrected for ME reached 0.80 (95% CI=0.65–0.89). Conclusions Our results indicate that elevated levels of TBARS were associated with increase risk of the prevalence of CVD, but this effect was no longer significant after adjusting for glucose.