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 mumol/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 mumol/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 mumol/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 micrograms/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.

Hydroperoxides in plasma are reduced by intensified insulin treatment. A randomized controlled study of IDDM patients with microalbuminuria.

OBJECTIVE: An association between reactive oxygen species and diabetic micro- and macrovascular complications has been proposed. In the present study, we have examined the effect of an improved blood glucose control on plasma levels of hydroperoxides in patients with IDDM. RESEARCH DESIGN AND METHODS: Subjects included 30 young IDDM patients with microalbuminuria who were randomized to receive either continuous subcutaneous insulin infusion (CSII) by a portable insulin pump (n = 15) or conventional insulin treatment (CIT) (n = 15) for 24 months. Plasma levels of hydroperoxides were measured by the ferrous oxidation with Xylenol Orange, version 2 (FOX2) assay. This method measures total lipid hydroperoxides and, unlike other methods, does not suffer from extraction losses. RESULTS: The mean HbA1c level was lower in the CSII group at the end of the study than in the CIT group: (mean [95% CI]) 8.6 (8.1-9.1) vs. 9.6 (9.0-10.3)%, respectively (P < 0.002). The level of plasma hydroperoxides was very similar at the start of the study but was significantly lower in the CSII group compared with the CIT group at the end of the study: 2.9 (2.1-3.7) vs. 4.3 (3.2-5.4) mumol/l, respectively (P < 0.02). In the CSII group, hydroperoxides were reduced by 31% from baseline (P < 0.001), whereas there was no change in levels of hydroperoxides in the CIT group. Mean hydroperoxide levels correlated with mean HbA1c during the study (r = 0.39, P < 0.04). Hydroperoxide levels were associated with the levels of microalbuminuria (r = 0.45, P < 0.02). CONCLUSIONS: This study provides support for the hypothesis that hyperglycemia is an important factor in the generation of hydroperoxides, and, thus, reactive oxygen species, in the circulation of IDDM patients.

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-PGF2 alpha 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.

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.

Plasma 8-epi-PGF2 alpha 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-PGF2 alpha, from healthy- and NIDDM individuals as an index of oxidative stress in vivo. Plasma 8-epi-PGF2 alpha was isolated by solid-phase extraction on a C18 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-PGF2 alpha 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.

alpha-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.

Measures of oxidative stress in heterozygous familial hypercholesterolaemia.

Familial hypercholesterolaemia (FH) may be associated with increased oxidative stress which may contribute to atherogenesis. Plasma lipid hydroperoxides (ROOHs), 8-epi PGF(2alpha) and alpha-tocopherol were measured in normal subjects and in newly referred heterozygous FH patients and used as indices of oxidative stress. ROOH levels were higher (+16%), albeit non-significantly, in FH patients than in controls subjects (4.4+/-0.3 vs. 3.8+/-0.3 micromol/l; n=51 and 40, respectively). 8-epi PGF(2alpha) levels were significantly greater (+56%) in the FH patients than in controls (0.43+/-0.06 vs. 0.27+/-0.05 nmol/l; P<0.05; n=14 and 16, respectively). FH patients with vascular disease had significantly higher (+32%) levels of ROOH compared with patients without vascular disease (4.9+/-0.40 vs. 3.7+/-0.33 micromol/l; P<0.05; n=27 and 24, respectively). Similarly, 8-epi PGF(2alpha) concentrations were higher (+100%) in the FH patients with vascular disease than in those without it (0.6+/-0.08 vs. 0.3+/-0.10 nmol/l; P<0.05; n=6 and 8, respectively). Absolute alpha-tocopherol levels in FH patients were similar to those in controls (21.0+/-0.70 vs. 23.8+/-1.30 micromol/l). When alpha-tocopherol levels were expressed relative to cholesterol, however, the concentrations were found to be significantly lower (-43%) in FH patients than in controls (2.9+/-0.10 vs. 5.1+/-0.40 micromol/mmol, P<0.0005). There were no differences in absolute or cholesterol standardised alpha-tocopherol levels in patients with and without vascular disease. These data suggest that oxidative stress is increased in FH-patients and is particularly pronounced in those patients with vascular disease. It is possible that increased oxidative stress may precede the development of vascular disease.

Probucol inhibits mononuclear cell adhesion to vascular endothelium in the cholesterol-fed rabbit.

Mononuclear cells, isolated from the blood of hyperlipidaemic patients, are hyper-reactive and possess an increased propensity to adhere to vascular endothelial cells. Hyperlipidaemia is also associated with a dysfunctional endothelium, to which mononuclear cells stick with greater avidity. In order to assess the importance of lipid peroxidation and free-radical generation in these processes, we have investigated the effects of probucol on mononuclear cell adhesion to vascular endothelial cells in vivo and in vitro in the cholesterol-fed rabbit. New Zealand White rabbits were fed either: (i) control chow (n = 15), (ii) 2% cholesterol (n = 11), or (iii) 2% cholesterol with 1% probucol (n = 11). Mononuclear cell adherence to endothelium in the common carotid artery was assessed 5 weeks after the start of the experimental diet using the Hoechst 33342 staining technique. The 2% cholesterol diet caused a more than 6-fold increase in mean mononuclear cell adherence (P < 0.001). Concurrent probucol therapy abrogated the effects of cholesterol feeding, and in animals in this group, in vivo mononuclear cell adherence did not differ significantly from control animals. In vivo mononuclear cell adherence was directly related to serum cholesterol levels (r = 0.68, P < 0.0001) and inversely related to serum probucol concentrations (r = -0.63, P < 0.002). Concurrent probucol therapy also reduced the in vitro binding of mononuclear cells, isolated from hypercholesterolaemic animals, to endothelial cell monolayers (P < 0.01). These data suggest that the increased binding of mononuclear cells to vascular endothelium of cholesterol-fed rabbits may be a free radical mediated process that is inhibited by antioxidants.

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.

The steady-state levels of oxidative DNA damage and of lipid peroxidation (F2-isoprostanes) are not correlated in healthy human subjects.

Oxidative damage to DNA in human tissues can be determined by measuring multiple products of oxidative damage to the purine and pyrimidine bases using gas chromatography-mass spectrometry (GC-MS). Oxidative damage to lipids (lipid peroxidation) can be quantitated by the mass spectrometry-based determination of F2-isoprostanes, specific end-products of the peroxidation of arachidonic acid residues in lipids. For both DNA base damage products and 8-epi prostaglandin F2alpha (PGF2alpha), there is a wide variation in levels between different healthy human subjects. We measured multiple products of oxidative damage to DNA bases in white cells, and 8-epi PGF2alpha in plasma, from blood samples obtained from healthy human subjects in the UK and in Portugal. No correlation of 8-epi PGF2alpha levels with levels of any modified DNA base (including 8-hydroxyguanine) was observed. We conclude that no single parameter can be measured as an index of "oxidative stress" or "oxidative damage" in vivo.

Effect of dialysis on oxidative stress in uraemia.

It has been postulated that dialysis of patients with chronic renal failure (CRF) is associated with increased lipid peroxidation which may contribute to vascular and other complications of the syndrome. In the present study, a specific and precise technique [ferrous oxidation in xylenol orange (FOX) assay] was used to measure plasma lipid hydroperoxides (ROOHs) in three groups of uraemic patients. Patients were either studied before starting dialysis (n = 12) or on continuous ambulatory peritoneal dialysis (CAPD, n = 12) or haemodialysis (HD, n = 36) and compared to healthy controls (n = 20). Plasma ROOHs were markedly elevated in HD patients compared with the controls (7.01 +/- 2.9 microM versus 4.25 +/- 2.05 microM; P < 0.005, Mann-Whitney test). Plasma ROOH concentrations in the CAPD patients were increased but not significantly higher than controls (5.36 +/- 3.56 microM versus 4.25 +/- 2.05 microM). By contrast, no differences in ROOH levels were found between controls and predialysis patients. There was no difference in plasma thiobarbituric acid reactive substances (TBARS) between control and the three CRF groups. Absolute and cholesterol standardised plasma alpha-tocopherol levels were lower in the patients (whether they were on dialysis or not) than in the controls (18.62 +/- 6.88 microM versus 22.73 +/- 5.33 microM; P < 0.01 and 1.99 +/- 1.88 microM/mM versus 5.25 +/- 1.0 microM/mM; P < 0.0005, respectively). This study provides direct evidence that enhanced oxidative stress in CRF patients is related to the dialysis treatment rather than the disease itself. Further studies will be necessary to establish the relationships between plasma measures of oxidative stress and cardiovascular complications in CRF patients under dialysis and whether treatment with antioxidants may reduce oxidative stress or reverse adverse effects associated with dialysis.

Increased levels of plasma 8-EPI-PGF2α in patients with end stage renal failure.

F2-isoprostanes are a series of prostaglandin-F2 like compounds specifically derived from peroxidation of arachidonic acid by a mechanism independent of the cyclooxygenase pathway. Of these, 8-epi PGF2α is shown to be a potent vasoconstrictor. In this study, we have analysed plasma 8-epi PGF2α as a marker of oxidative stress in patients with end stage renal failure (ESRF) undergoing haemodialysis (HD) or continuous ambulatory peritoneal dialysis (CAPD). Plasma F2-isoprostanes were isolated by solid-phase extraction on a C18 followed by an NH2 cartridge. Quantitative analysis of the F2-isoprostanes as pentafluorobenzyl (PFB) ester/trimethylsilyl (TMS) ether derivatives was carried out by gas chromatography-electron capture mass spectrometry. For 34 individuals with ESRF, the mean level of esterified 8-epi PGF2α was 0.58 ± 0.22 M; range 0.21-1.16 nM. 8-epi PGF2α concentration in the patient groups was markedly higher (P<0.0005 by separate variance t-test) than that of control subjects (n=15) 0.28 ± 0.17 nM; range 0.02-0.63 nM. There was no difference in levels of 8-epi PGF2α in plasma from patients undergoing HD or CAPD, nor was there any association with age, plasma lipids or plasma creatinine. These data provide direct evidence of increased oxidative stress in individuals with ESRF. This marker should be useful in clinical studies examining the degree of oxidative stress in vivo and the therapeutic impact of antioxidants.

A diet containing rapeseed oil-based fats does not increase lipid peroxidation in humans when compared to a diet rich in saturated fatty acids.

OBJECTIVE: To compare the effects of a rapeseed oil-based diet containing an increased proportion of easily oxidised polyunsaturated fatty acids such as alpha-linolenic acid with a diet rich in saturated fatty acids on the degree of lipid peroxidation in the human body. DESIGN: A randomised cross-over study. SUBJECTS AND INTERVENTIONS: Nineteen healthy moderately hyperlipidemic subjects (six women and 13 men, age 50+/-8 y and body mass index (BMI) 24.5+/-2.6 kg/m(2)) were given a rapeseed oil-based diet (RO) and a control diet (SAT) rich in saturated fatty acids during two consecutive 4 week periods separated by a 4 week wash-out period. Biomarkers of lipid peroxidation and antioxidants were analysed in plasma and urine. RESULTS: No significant differences in plasma or urinary levels of free 8-iso-prostaglandin F(2alpha), plasma total 8-iso-prostaglandin F(2alpha) plasma hydroperoxides or plasma malondialdehyde were observed between the RO and SAT diets (P=0.14-0.95). A higher concentration of serum gamma-tocopherol was detected after the RO diet compared to the SAT diet (P<0.001), whereas the serum alpha-tocopherol concentration and plasma antioxidative capacity did not differ between the two test diets. The total cholesterol, LDL cholesterol and LDL/HDL ratio were lower after the RO diet compared to the SAT diet (P<0.001), while HDL cholesterol and total triglyceride levels were similar after the two diets. CONCLUSION: These results suggest that a rapeseed oil-based diet rich in alpha-linolenic acid does not seem to increase the degree of lipid peroxidation in plasma and urine compared to a diet rich in saturated fats. This is possibly due to a sufficient content of antioxidants in the rapeseed oil diet to increase circulating concentrations of antioxidants that may protect unsaturated fatty acids from oxidation. SPONSORSHIP: Swedish Council for Forestry and Agricultural Research and Foundation for Geriatric Research.

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.

Enhanced oxidative stress in Hashimoto's thyroiditis: inter-relationships to biomarkers of thyroid function.

OBJECTIVES: Oxidative stress has been implicated in the pathogenesis of several inflammatory and immune-mediated disorders including Hashimoto's thyroiditis (HT). The objectives of the present cross-sectional investigation were to estimate serum glutathione (GSH) status and the activities of its recycling enzymes in HT and to explore their interrelationships with biomarkers of autoimmunity and thyroid function. DESIGN AND METHODS: Newly diagnosed females with HT (n=44) and 58 matched control subjects were recruited. Thyroid hormone profile, anti-thyroperoxidase anti-body (TPO-AB), anti-thyroglublin antibody (Tg-AB), thyroid volume (Tvol), urinary iodine excretion (UIE), GSH and the activities of glutathione peroxidase (GPx), glutathione reductase and gamma-glutamyltransferase were assessed. RESULTS: Median UIE in HT was slightly but not significantly higher than that of controls. HT group exhibited higher levels of TSH, TPO-AB, Tg-AB and larger Tvol when compared with controls (P<0.001). The means of GSH and GPx in HT patients were significantly different from those of controls (P<0.001). In HT subjects, significant associations were seen between Tvol on TSH, GSH on TPO-AB, GSH on TSH and TPO-AB titers on TSH, respectively. CONCLUSIONS: This is the first study to demonstrate a substantial reduction in GSH status in HT subjects. Secondly, the interrelationship between the GSH contents and TPO-AB titers in HT provides a preliminary data to support the notion that GSH diminution is a hallmark of in the events leading to oxidative stress activation and the development of immunological intolerance in HT. Further studies are required to elucidate the role of GSH in the etiology of down-regulation of thyroid function.

Evaluation of Accessibility of Iodinated Salt and Nutritional Iodine Status during Pregnancy.

BACKGROUND: To assess accessibility of iodinated salt and urinary iodine concentrations (UIC) during pregnancy. This cross-sectional study was carried out between October and December, 2009 in Urmia County, West Azerbaijan (WA), Iran. METHODS: Data on demographic characteristics and iodinated salt accessibility were gathered through a questionnaire at 1st trimester. Household salt samples and urine samples (1st -and 3rd trimesters) were analyzed for iodine content. Pregnant women (n=490) at 1st trimester were interviewed. Of these, 490 subjects (12 prenatal care centers) were enrolled. RESULTS: All participants declared that they were exclusive users of iodinated salt. Segregation of the household salt samples according to iodine content (0, 8, 15 and 30 ppm) revealed that the respective distributions were 3.3%, 1.4%, 23.7% and 71.6%. Median UIC levels at 1st and 3rd trimesters were 73.5 µg/L and 114µg/L respectively. Accordingly, 86% and 70% of participants exhibited UIC < 150 µg/L. CONCLUSION: Median UIC during pregnancy in WA is markedly lower than those previously reported for regions with adequate iodine status in the country. Thus, extra iodine is needed to maintain adequate iodine store during gestation. In addition, this preliminary study reveals that a significant proportion (28%) of the household salt samples had low iodine content (≤ 15 ppm) although a level (>20 and <40 ppm) is mandatory in Iran. Further studies are deemed necessary to elucidate the cause(s) for manifestation iodine deficiency among pregnant women despite 20 years after iodine fortification strategy.