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.

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 nitrated and hydroxylated aromatic compounds from benzene and peroxynitrite, a possible mechanism of benzene genotoxicity.

Peroxynitrite, the reaction product of nitric oxide (NO.) and superoxide anion (O2.-) produced during immune activation by a variety of inflammatory cells, may contribute to genotoxicity of benzene through its ability to carry out hydroxylation and nitration. After exposure of benzene to synthesised peroxynitrite, phenol, nitrophenols (p-nitrophenol, o-nitrophenol and m-nitrophenol) and nitrobenzene were identified in the reaction mixture by HPLC separation and single UV wavelength and diode array detection. The formation of phenol, nitrophenols and nitrobenzene showed a linear relationship with both benzene and peroxynitrite concentrations. The molar ratio for phenol/(nitrobenzene and nitrophenols) was approximately 9/5 with a total product yield of 14% hydroxylated and nitrated products as based on peroxynitrite. The physiological relevance of the chemical reaction between benzene and peroxynitrite was tested by detecting the reaction products in human neutrophils (2.5 x 10(7)cells/ml) incubated with 10 mM benzene for 25 min. The concentration of phenol and p-nitrophenol were found to be 1.29+/-0.22 and 1.56+/-0.61 microM (mean+/-SD) in the incubation medium of the neutrophils pretreated with phorbol myristate acetate (500 nM) for 5 min, respectively, whereas no metabolites were detected if the neutrophils were not pretreated. Nitrated aromatic compounds are known to be more carcinogenic than the parent compounds. It is reported that acute and chronic infection increases the risk of cancer at various sites; and that anti-inflammatory agents decrease benzene myelotoxicity. We suggest that the increased production of peroxynitrite during chronic inflammation combined with benzene exposure may increase the carcinogenicity of benzene by a mechanism that includes the formation of metabolites from the chemical reaction between benzene and peroxynitrite. Thus, peroxynitrite mediated hydroxylation and nitration of benzene during immune activation represent a novel in vivo mechanism for generation of proximal carcinogens of benzene.

Cyanate-mediated inhibition of neutrophil myeloperoxidase activity.

Cyanate (CNO-) forms spontaneously in solutions containing urea, and is present in urine and the body fluids of uraemic patients. We have explored the possibility that CNO- might be one of the unknown substances responsible for the reported impairment, by urine and uraemic plasma, of neutrophil oxidative metabolism (especially as measured by luminol-enhanced chemiluminescence). Luminol-enhanced chemiluminescence generated by human neutrophils derives predominantly from the activity of myeloperoxidase (MPO) which produces hypochlorous acid from H2O2 and Cl-. We hypothesized that CNO- (which resembles the 'pseudohalide' thiocyanate, an alternative substrate for MPO) might somehow interfere with the activity of MPO. In support of this, we find: (i) CNO- inhibits both peroxidative and halogenating activities of MPO and also inhibits the enzyme within intact human neutrophils; (ii) the inhibition is H2O2-dependent, irreversible, accompanied by covalent addition of [14C]CNO- (or a carbon-containing fragment thereof) to the enzyme; (iii) CNO- also inhibits Cl-/H2O2/MPO-mediated bacterial killing. Impairment of this arm of neutrophil bactericidal activity by CNO- formed from urea may be one factor in the risk of urinary-tract infection associated with urinary stasis and perhaps in the generalized increase in susceptibility to infection in uraemic patients.

Low-density lipoprotein is the major carrier of lipid hydroperoxides in plasma. Relevance to determination of total plasma lipid hydroperoxide concentrations.

High-density lipoprotein (HDL) has been proposed as the principal carrier of hydroperoxides in plasma, based upon data gathered with an HPLC-chemiluminescence technique. To test this hypothesis we have measured total lipid hydroperoxides in native plasma using the ferrous oxidation in Xylenol Orange (FOX) assay and then fractionated plasma into very-low-density lipoprotein (LDL) and HDL fractions. Hydroperoxides were found to accumulate principally (more than 65%) in LDL, as judged by hydroperoxide content per amount of protein or cholesterol, or expressed as a proportion of total hydroperoxide in plasma. Plasma was also incubated at 37 degrees C in the presence and absence of 2,2'-azo-bis-(2-amidinopropane) hydrochloride (AAPH), an azo-initiator of lipid peroxidation. The majority of hydroperoxides generated in plasma were recovered in the LDL fraction. Furthermore, when isolated lipoproteins were subject to oxidation initiated by AAPH, very-low-density lipoprotein and LDL showed the greatest propensity for hydroperoxide accumulation, whereas HDL seemed relatively resistant. Estimates for plasma and LDL peroxidation based upon techniques which measure total lipid hydroperoxides suggest that levels of hydroperoxides in plasma and LDL are far higher than that those estimates generated by ostensibly more selective techniques. Higher levels of hydroperoxides in LDL than those reported by HPLC-chemiluminescence also seem in greater accordance with other available data concerning LDL oxidation.

A discontinuous method for catalase determination at 'near physiological' concentrations of H2O2 and its application to the study of H2O2 fluxes within cells.

We describe a discontinuous method for the measurement of catalase using the Ferrous Oxidation in Xylenol orange (FOX) assay. Samples containing catalase are incubated with H2O2 for varying time intervals prior to rapid mixing of aliquots of the incubation mixtures with FOX reagent, which measures residual H2O2. Absorbance is then read at 560 nm after 30-min incubation at room temperature. Decay of H2O2 is proportional to catalase activity in the original sample. An adaptation of the method involves exposure of intact cells to H2O2-generating systems in the presence of aminotriazole. The extent of catalase inactivation allows estimates of total H2O2 accumulation within the cell.

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.

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.

Thioctic (lipoic) acid: a therapeutic metal-chelating antioxidant?

Thioctic (alpha-lipoic) acid (TA) is a drug used for the treatment of diabetic polyneuropathy in Germany. It has been proposed that TA acts as an antioxidant and interferes with the pathogenesis of diabetic polyneuropathy. We suggest that one component of its antioxidant activity requiring study is the direct transition metal-chelating activity of the drug. We found that TA had a profound dose-dependent inhibitory effect upon Cu(2+)-catalysed ascorbic acid oxidation (monitored by O2 uptake and spectrophotometrically at 265 nm) and also increased the partition of Cu2+ into n-octanol from an aqueous solution suggesting that TA forms a lipophilic complex with Cu2+. TA also inhibited Cu(2+)-catalysed liposomal peroxidation. Furthermore, TA inhibited intracellular H2O2 production in erythrocytes challenged with ascorbate, a process thought to be mediated by loosely chelated Cu2+ within the erythrocyte. These data, taken together, suggest that prior intracellular reduction of TA to dihydrolipoic acid is not an obligatory mechanism for an antioxidant effect of the drug, which may also operate via Cu(2+)-chelation. The R-enantiomer and racemic mixture of the drug (alpha-TA) generally seemed more effective than the S-enantiomer in these assays of metal chelation.

Erythrocyte catalase inactivation (H2O2 production) by ascorbic acid and glucose in the presence of aminotriazole: role of transition metals and relevance to diabetes.

Erythrocytes exposed to ascorbic acid in the presence of aminotriazole undergo a dose- and time-dependent inactivation of endogenous catalase which is proportional to environmental hydrogen peroxide (H2O2) concentrations. The production of H2O2 seems to be dependent upon the availability of transition metal chelatable by o-phenanthroline (OPT), although the kinetics of catalase inactivation and H2O2 production by externally added copper ions in the presence of OPT is complex. Furthermore, although glucose is also able to undergo a transition-metal-catalysed oxidation yielding H2O2, the production of H2O2 by glucose seems to be a minor process by comparison with ascorbic acid oxidation. Indeed, on the basis of these data, transition-metal-catalysed ascorbic acid oxidation is likely to be a more important source of oxidative stress in the diabetic state than hyperglycaemia.

Measurement of plasma hydroperoxide concentrations by the ferrous oxidation-xylenol orange assay in conjunction with triphenylphosphine.

We describe the application of the FOX2 (ferrous oxidation in xylenol orange, version 2) method to the measurement of hydroperoxides in plasma. Authentic plasma hydroperoxides can be determined by a strategy in which the hydroperoxide reductant, triphenylphosphine, is used to discriminate between the background signal generated by ferric ions present in plasma and that generated by hydroperoxide in plasma. The approach was validated by extraction of total lipids from plasma using ethyl acetate prior to assay with the FOX2 reagent. Plasma from 23 normal individuals contained hydroperoxide in the range of 0.22 to 7.8 microM with a mean of 3.02 microM and a population standard deviation of 1.85 microM. After partitioning with ethyl acetate, plasma hydroperoxide levels ranged from 0.22 to 6.22 microM, with a mean value of 2.52 microM and a population standard deviation of 1.65 microM.

Peroxide accumulation in detergents.

It is known that polyether detergents have a propensity for peroxide accumulation but the rate of this accumulation and the conditions under which it occurs are not well-appreciated. We here describe the use of the FOX (ferrous oxidation in Xylenol orange) assay as a screen for detergent peroxide accumulation. We also show that a chain-breaking antioxidant, but not a metal-chelating agent block peroxide accumulation in detergent stored in the light at room temperature.

Cataract and UV radiation.

The idea that UV radiation causes cataract has superficial biological plausibility based upon studies of UV-B on lens components, whole lenses and experimental animals. However, epidemiological studies on man, particularly geographical studies, are highly confounded by socioeconomic factors and yield little information. Population-based exposure-cataract studies are preferable since UV incidence versus UV exposure can be weighted with respect to risk and protective factors. However, the cataractogenic potency of UV seems to be low and certainly lower than the cataractogenic potency of factors such as repeated diarrhoea so that it is doubtful whether a clear answer to the question 'Does UV cause cataract' will ever be provided.

Aminoguanidine: a drug proposed for prophylaxis in diabetes inhibits catalase and generates hydrogen peroxide in vitro.

Aminoguanidine (AG) has been proposed as a drug of potential benefit in prophylaxis of the complications of diabetes. We show here that AG irreversibly inhibits catalase with an efficacy similar to aminotriazole. AG also produces hydrogen peroxide, in a transition metal-catalysed process which may be partially dependent upon prior hydrolysis of AG to semicarbazide and hydrazine. These observations may be of importance in proposals for the long term administration of AG in diabetes.

Diabetes mellitus and free radicals. Free radicals, transition metals and oxidative stress in the aetiology of diabetes mellitus and complications.

Diabetes mellitus is a syndrome initially characterized by a loss of glucose homeostasis. The disease is progressive and is associated with high risk of atherosclerosis, kidney and nerve damage as well as blindness. Abnormalities in the regulation of peroxide and transition metal metabolism are postulated to result in establishment of the disease as well as its longer term complications. Diabetes mellitus is associated with oxidative reactions, particularly those which are catalyzed by decompartmentalized transition metals, but their causative significance in diabetic tissue damage remains to be established.