Jaffar Nourooz-Zadeh

Plasma 8‐epi‐PGF2α levels are elevated in individuals with non‐insulin dependent diabetes mellitus

This study reports plasma levels of a specific nonenzymatic peroxidation product of arachidonic acid, esterified 8‐epi‐PGF2α, from healthy‐ and NIDDM individuals as an index of oxidative stress in vivo. Plasma 8‐epi‐PGF2α was isolated by solid‐phase extraction on a C,8 followed by an NH2 cartridge and analyzed by GC‐MS/NICI as PFB‐ester/TMS‐ether derivative. We found that the average concentration of esterified 8‐epi‐PGF2α among NIDDM subjects (0.93 ± 0.07 nM, n = 39) was higher (P < 0.0001, Mann‐Whitney test) than in healthy individuals (0.28 ± 0.04 nM, n = 15). These data indicate that NIDDM is associated with increased plasma lipid peroxidation.

Elevated Levels of Authentic Plasma Hydroperoxides in NIDDM

Using a precise technique for measuring authentic plasma lipid hydroperoxides (ROOHs), we show that individuals with non-insulin-dependent diabetes mellitus (NIDDM) have higher levels of ROOH than do control subjects. ROOHs were measured by the ferrous oxidation with xylenol orange assay coupled with the selective ROOH reductant triphenylphosphine. Formation of the ferric xylenol orange complex was determined at 560 nm and calibrated against H2O2. For 22 individuals with NIDDM, a concentration of 9.04 ± 4.3 μmol/l (mean ± SD) ROOH was recorded. This concentration was higher (P < 0.0005 by separate-variance t test) than that of plasma ROOHs from control subjects (3.76 ± 2.48 μmol/l). There was no difference between concentrations of plasma malondialdehyde measured as thiobarbituric acid–reactive material (TBARM) in NIDDM or control subjects (1.00 ± 0.70 vs. 1.21 ± 0.62 μmol/l, respectively; P > 0.1). A trend to lower vitamin E levels in the NIDDM group (9.03 ± 3.31 vs. 10.31 ± 5.02 μg/ml in control subjects) failed to achieve significance at the 95% confidence level. Plasma ROOHs in the diabetic group did not correlate with total plasma cholesterol, triglyceride, fasting glucose, HbA1, vitamin E, or TBARM levels. These data indicate that measurement of authentic ROOHs shows NIDDM to be associated with oxidative stress, which may be unrelated to abnormalities in lipid metabolism and glycemic control.

F4 ‐ Isoprostanes as Specific Marker of Docosahexaenoic Acid Peroxidation in Alzheimer's Disease

Abstract : F2‐isoprostanes are prostaglandin‐like compounds derived from free radical‐catalysed peroxidation of arachidonic acid. Peroxidation of eicosapentaenoic acid produces F3‐isoprostanes, whereas peroxidation of docosahexaenoic acid would give F4‐isoprostanes. This study demonstrates the presence of esterified F4‐isoprostanes in human brain and shows that levels are elevated in certain brain cortex regions in Alzheimers disease. Our data with Alzheimers disease suggest that analysis of F4‐isoprostanes will provide new opportunities to study lipid peroxidation in the neurodegenerative diseases.

α-lipoic acid decreases oxidative stress even in diabetic patients with poor glycemic control and albuminuria

In the present cross-sectional study, the influence of alpha-lipoic acid on markers of oxidative stress, assessed by measurement of plasma lipid hydroperoxides (ROOHs), and on the balance between oxidative stress and antioxidant defence, determined by the ratio ROOH/(alpha-tocopherol/cholesterol), was examined in 107 patients with diabetes mellitus. Patients receiving alpha-lipoic acid (600 mg/day for > 3 months) had significant lower ROOHs and a lower ROOH/(alpha-tocopherol/cholesterol) ratio than those without alpha-lipoic acid treatment [ROOH: 4.76 +/- 2.49 vs. 7.16 +/- 3.22 mumol/l; p < .0001] and [ROOH/(alpha-tocopherol/cholesterol): 1.37 +/- 0.72 vs. 2.16 +/- 1.17; p < 0.0001]. In addition, the influence of glycemic control and albuminuria on ROOHs and on the ratio of ROOH/(alpha-tocopherol/cholesterol) was examined in the presence and absence of alpha-lipoic acid treatment. Patients were subdivided into three groups based on (1) their HbA1 levels (< 7.5, 7.5-9.5, and > 9.5%) and (2) their urinary albumin concentrations (< 20, 20-200, and > 200 mg/l). Neither poor glycemic control, nor the presence of micro- or macroalbuminuria prevented the antioxidant effect of alpha-lipoic acid. Using stepwise multiple regression analysis, alpha-lipoic acid was found to be the only factor significantly predicting low ROOHs and a low ratio of ROOH/(alpha-tocopherol/cholesterol). These data provide evidence that treatment with alpha-lipoic acid improves significantly the imbalance between increased oxidative stress and depleted antioxidant defence even in patients with poor glycemic control and albuminuria.

Analysis of F2-isoprostanes as indicators of non-enzymatic lipid peroxidation in vivo by gas chromatography-mass spectrometry: development of a solid-phase extraction procedure

Recently, it has been reported that a series of prostaglandin F2-like compounds (F2-isoprostanes) are produced in vivo during peroxidation of arachidonic acid by a mechanism independent of the cyclooxygenase pathway. Of these, 8-epi-PGF2 alpha is shown to be a potent vasoconstrictor. We describe an improved method for analysing F2-isoprostanes in biological fluids. The method involves solid-phase extraction on an octadecylsilane (C18) and an aminopropyl (NH2) cartridge. After conversion to pentafluorobenzyl ester and trimethylsilyl ether derivatives, F2-isoprostanes are analysed by negative-ion chemical ionization mass spectrometry using tetradeuterated PGF2 alpha as the internal standard. The limit of detection of the assay was 10 pg/ml, with a coefficient of variation ranging from 9.4 to 15.1%. Analysis of plasma samples from healthy volunteers (n = 7) revealed no quantifiable levels of free (unesterified) 8-epi-PGF 2 alpha. However, the plasma samples contained 58 to 166 pg/ml of 8-epi-PGF2 alpha when analyzed for the total (sum of free and esterified) F2-isoprostanes. The main advantages of the method lie in the improved recovery, gas chromatographic separation and speed compared to existing techniques.

Early oxidative stress in the diabetic kidney: Effect of DL-α-lipoic acid

Oxidative stress is implicated in the pathogenesis of diabetic nephropathy. The attempts to identify early markers of diabetes-induced renal oxidative injury resulted in contradictory findings. We characterized early oxidative stress in renal cortex of diabetic rats, and evaluated whether it can be prevented by the potent antioxidant, DL-α-lipoic acid. The experiments were performed on control rats and streptozotocin-diabetic rats treated with/without DL-α-lipoic acid (100 mg/kg i.p., for 3 weeks from induction of diabetes). Malondialdehyde plus 4-hydroxyalkenal concentration was increased in diabetic rats vs. controls (p < .01) and this increase was partially prevented by DL-α-lipoic acid. F2 isoprostane concentrations (measured by GCMS) expressed per either mg protein or arachidonic acid content were not different in control and diabetic rats but were decreased several-fold with DL-α-lipoic acid treatment. Both GSH and ascorbate (AA) levels were decreased and GSSG/GSH and dehydroascorbate/AA ratios increased in diabetic rats vs. controls (p < .01 for all comparisons), and these changes were completely or partially (AA) prevented by DL-α-lipoic acid. Superoxide dismutase, glutathione peroxidase, glutathione reductase, glutathione transferase, and NADH oxidase, but not catalase, were upregulated in diabetic rats vs. controls, and these activities, except glutathione peroxidase, were decreased by DL-α-lipoic acid. In conclusion, enhanced oxidative stress is present in rat renal cortex in early diabetes, and is prevented by DL-α-lipoic acid.

Re-evaluation of the ferrous oxidation in xylenol orange assay for the measurement of plasma lipid hydroperoxides

The ferrous oxidation in xylenol orange version 2 (FOX2) assay coupled with triphenylphosphine has recently been employed for the measurement of total plasma hydroperoxides (ROOHs). In this study, we have evaluated sample handling and the effect of storage conditions on ROOH levels in human plasma (n = 32). Mean level of ROOHs in fresh plasma was 8.35 +/- 3.09 mumol/l (range 4.03-19.5 mumol/l). Addition of butylated hydroxytoluene (BHT) immediately after sample collection had no effect on the concentration of ROOHs. Storage of samples at -70 degrees C for 6 weeks was associated with a variable degree of loss of detectable ROOHs. A mean ROOH level of 6.00 +/- 2.23 mumol/l (range 2.88-13.5 mumol/l) was recorded after 6 weeks of storage at -70 degrees C. There was no difference in the mean level of ROOHs between samples stored for 6 and 60 weeks at -70 degrees C. Inclusion of BHT had no effect on the stability of plasma ROOHs during prolonged storage. Intra-assay coefficients of variation were < 12%, with the lowest variation in fresh samples (7.6%). In conclusion, these results suggest that the FOX2 assay should be a useful tool for measurement of ROOHs in fresh plasma samples but not in stored samples.

Increased oxidative damage to all DNA bases in patients with type II diabetes mellitus

Gas chromatography‐mass spectrometry was used to measure the oxidative DNA damage in diabetic subjects and controls. Levels of multiple DNA base oxidation products, but not DNA base de‐amination or chlorination products, were found to be elevated in white blood cell DNA from patients with type II diabetes as compared with age‐matched controls. The chemical pattern of base damage is characteristic of that caused by an attack on DNA by hydroxyl radical. An increased formation of the highly reactive hydroxyl radical could account for many of the reports of oxidative stress in diabetic subjects. There was no evidence of an increased DNA damage by reactive nitrogen or chlorine species.

Formation of F2-isoprostanes during aortic endothelial cell-mediated oxidation of low density lipoprotein

We investigated the formation of F2‐isoprostanes produced by non‐enzymatic peroxidation of arachidonic acid during rabbit aortic endothelial cell‐mediated oxidation of low density lipoprotein (LDL). Free and total (sum of free and esterified) levels of F2‐isoprostanes were measured using a solid‐phase extraction procedure and gas chromatography‐mass spectrometry. Free levels of F2‐isoprostanes in native LDL were 0.06 ± 0.03 ng/mg protein (n = 4), whereas total levels were 0.28 ± 0.09 ng/mg protein (n = 4). Both free and total levels of the isoprostanes were found to increase during the oxidation. 8‐epi‐PGF2α was the major isoprostane formed (free and total concentrations after 24 h, 2.50 ± 0.24 and 6.42 ± 1.36 ng/mg protein (n = 4), respectively). The release of F2‐isoprostanes during aortic endothelial cell‐induced oxidation of LDL could be a contributory factor in the development of atherosclerosis.

Activation of Aldose Reductase in Rat Lens and Metal-Ion Chelation by Aldose Reductase Inhibitors and Lipoic Acid

Sorbitol formation in rat lenses incubated with high levels of glucose was related to activation of aldose reductase (AR). The hyperglycaemia-activated aldose reductase was inhibited by alpha-lipoic (thioctic) acid, O-phenanthroline and aldose reductase inhibitors (ARIs) including Zeopolastat (ZPLS), Sorbinil (SBN) and AL-1576. This study also examined ARIs for the ability to chelate metal ions. We found that ARIs suppress copper-dependent ascorbate oxidation, lipid peroxidation and hydrogen peroxide production in erythrocytes. ARIs also increased partition of copper ions into noctanol, which indicates formation of lipophilic complexes. Our data support the hypothesis that transition metals may be involved in activation of the polyol (aldose reductase) pathway. Also, ARIs function as metal-chelating antioxidants that may contribute to their therapeutic role for diabetic complications.