Advanced glycation end product-induced activation of NF-kappaB is suppressed by alpha-lipoic acid in cultured endothelial cells.

Depletion of cellular antioxidant defense mechanisms and the generation of oxygen free radicals by advanced glycation end products (AGEs) have been proposed to play a major role in the pathogenesis of diabetic vascular complications. Here we demonstrate that incubation of cultured bovine aortic endothelial cells (BAECs) with AGE albumin (500 nmol/l) resulted in the impairment of reduced glutathione (GSH) and ascorbic acid levels. As a consequence, increased cellular oxidative stress led to the activation of the transcription factor NF-kappaB and thus promoted the upregulation of various NF-kappaB-controlled genes, including endothelial tissue factor. Supplementation of the cellular antioxidative defense with the natural occurring antioxidant alpha-lipoic acid before AGE albumin induction completely prevented the AGE albumin-dependent depletion of reduced glutathione and ascorbic acid. Electrophoretic mobility shift assays (EMSAs) revealed that AGE albumin-mediated NF-kappaB activation was also reduced in a time- and dose-dependent manner as long as alpha-lipoic acid was added at least 30 min before AGE albumin stimulation. Inhibition was not due to physical interactions with protein DNA binding, since alpha-lipoic acid, directly included into the binding reaction, did not prevent binding activity of recombinant NF-kappaB. Western blots further demonstrated that alpha-lipoic acid inhibited the release and translocation of NF-kappaB from the cytoplasm into the nucleus. As a consequence, alpha-lipoic acid reduced AGE albumin-induced NF-kappaB mediated transcription and expression of endothelial genes relevant in diabetes, such as tissue factor and endothelin-1. Thus, supplementation of cellular antioxidative defense mechanisms by extracellularly administered alpha-lipoic acid reduces AGE albumin-induced endothelial dysfunction in vitro.

The use of cyclic voltammetry for the evaluation of antioxidant capacity.

Low-molecular weight antioxidants (LMWAs) play a major role in protecting biological systems against reactive oxygen-derived species and reflect the antioxidant capacity of the system. Cyclic voltammetry (CV), shown to be convenient methodology, has been validated for quantitation of the LMWA capacity of blood plasma, tissue homogenates, and plant extracts. Analysis of the CV tracing yields the values of (i) the biological oxidation potential, E and E(1/2), which relate to the nature of the specific molecule(s); (ii) the intensity (Ia) of the anodic current; and (iii) the area of the anodic wave (S). Both Ia and S relate to the concentration of the molecule(s). LMWA components of human plasma and animal tissues were identified and further validated by reconstruction of the CV tracing and by high-performance liquid chromatography-electrochemical detection. To reflect the oxidative stress status, the use of an additional parameter, R, has been proposed. R represents the level (%) of oxidized ascorbate (compared with total ascorbate) and is measured by high-performance liquid chromatography-electrochemical detection. All these parameters were monitored in healthy human subjects as well as in chronic (diabetes mellitus) and acute care patients (subjected to total body irradiation before bone marrow transplantation). The electroanalytical methodologies presented here could be widely employed for rapid evaluation of the status of subjects (in health and disease) for monitoring of their response to treatment and/or nutritional supplementation as well as for screening of specific populations.

Evaluation of plasma low molecular weight antioxidant capacity by cyclic voltammetry.

The low molecular weight antioxidants (LMWA) of biological samples include many compounds and contribute to the total antioxidant capacity of the system. They act as direct chemical scavengers neutralizing, reactive oxygen-derived species (ROS), and contribute to the ability of the sample to cope with oxidative stress. We propose cyclic voltammetry (CV) as a new method for evaluating the antioxidant capacity of plasma-LMWA and the severity of oxidative stress exerted on the plasma. It is based on the reducing properties of these molecules. CV has been proven to be a simple, sensitive and reliable method. Its tracing does not change during storage of frozen plasma for up to six months. We analyzed the CV tracings by the oxidation potential E1/2, and the current heights Ia of its anodic wave(s). E1/2 indicates the specific component of the LMWA and its ability to donate electron(s); Ia indicates the concentration of this component. Two anodic waves have been identified in human plasma, at E1/2 = 420 +/- 25 and 920 +/- 25 mV. Ascorbate (AA) and urate (UA) were identified as major LMWA components of the first anodic wave, and were confirmed by HPLC-electrochemical detection. Ia was shown to depend linearly on the concentration of either of these LMWA, both in buffer and in plasma. Oxidative stress exerted by exposure to peroxyl radicals, copper ions and ionizing irradiation caused marked changes in the CV tracing. These changes represent corresponding alterations particularly in la, rather than in E1/2. The Ia and E1/2 values reflect the antioxidant capacity of the plasma, while the change of Ia value represents the severity of the oxidative stress induced.

Citrus fruit supplementation reduces lipoprotein oxidation in young men ingesting a diet high in saturated fat: presumptive evidence for an interaction between vitamins C and E in vivo.

To determine the effects of vitamin C on cardiovascular risk factors, we studied dietary vitamin C enrichment in 36 healthy male students consuming a diet high in saturated fatty acids. After a 1-mo run-in period during which the subjects consumed approximately 50 mg ascorbic acid/d (low-C diet), half of the subjects were randomly assigned to receive 500 mg ascorbic acid/d for an additional 2 mo (high-C diet). Plasma ascorbic acid increased from 13.5 micromol/L with the low-C diet to 51.7 micromol/L with the high-C diet. Plasma cholesterol increased slightly with the high-C diet, but not above baseline concentrations. This increase was offset by an increase in the lag period of in vitro LDL oxidation, which correlated with plasma ascorbic acid concentrations (r = 0.735, P = 0.0012). Lipoprotein vitamin E concentrations were unchanged with the two diets. There were no effects on concentrations of fibrinogen or factor VII. The fact that ascorbic acid reduced the in vitro susceptibility of lipoproteins to oxidation provides presumptive evidence for an interaction between aqueous and lipophilic antioxidants (vitamins C and E ) in maintaining the integrity of LDL particles.

Antioxidant status and human health. Use of cyclic voltammetry for the evaluation of the antioxidant capacity of plasma and of edible plants.

The low molecular weight antioxidants (LMWA) play a major role in protecting biological systems against reactive oxygen-derived species (ROS), and reflect the antioxidant capacity of the system. The cyclic voltammetry (CV) has been conveniently used and validated for the quantitation of the antioxidant capacity of the LMWA of blood plasma, tissue homogenates, and plant extracts. The CV tracing provides the biological oxidation potential (E and E1/2 which relate to the nature of the molecule(s)), the intensity of the anodic current wave (Ia), and its area S (both relate to the concentration of the molecule(s)). The components of the first anodic wave of plasma were identified by comparison with HPLC-electrochemical detection. CV together with another plasma parameter R, which reflects the level of oxidized ascorbate, were used for the evaluation of the antioxidant status and the oxidative stress in healthy subjects and in chronic (diabetes mellitus) and acute patients (subjected to total body irradiation prior to bone marrow transplantation). These methodologies could be widely employed for rapid evaluation of subjects, in health and disease, for monitoring of their response to treatment and nutritional supplementation, and for screening of specific populations.

Antioxidant capacity of edible plants: extraction protocol and direct evaluation by cyclic voltammetry.

Reactive oxygen-derived species are produced in cells under physiological conditions and in response to stress. Among the various antioxidant systems responsible for protection against these species, the low-molecular-weight antioxidants (LMWA), such as ascorbate, play an important role. Cyclic voltammetry (CV) has been proposed as a tool for quantitation of the total antioxidant capacity of plasma. It has also been shown that biological oxidation potentials, as determined from the anodic current waves of the CV tracings, are specific characteristics of the various LMWA components, and that the amplitude of each wave can be used for quantitation of the specific component. The adaptation of CV for evaluation of the total antioxidant capacity of edible plants is demonstrated here. The area under the anodic current wave is proposed as a better indicator for the content of LMWA, compared with the amplitude. This distinction could prove valuable when more than a single molecule contributes toward a specific anodic wave and when the identities of the components of a wave are not known. Vegetables and fruits that are commonly consumed in the U.S. diet were used. They were extracted with either water, aqueous acetic acid (30%), or a mixture of water, acetic acid, and acetonitrile (40:30:30). The LMWA contents were evaluated by CV. In three to five steps the LMWAs were completely extracted from the edible foods, and their amounts were translated into equivalents of ascorbate.

The antioxidant status of patients subjected to total body irradiation.

BACKGROUND AND PURPOSE: Total body irradiation (TBI) is a routine preconditioning procedure for the treatment of leukemia and aplastic anemia, prior to bone marrow transplantation (BMT). Ionizing radiation generates reactive oxygen derived species (ROS) that can be removed by antioxidants. Our purpose is to determine the antioxidant status of patients undergoing TBI by evaluating the oxidant stress and their antioxidant capacity. MATERIAL AND METHODS: We evaluated by cyclic voltammetry (CV) the total antioxidant capacity (TAC) in plasma of 14 patients undergoing TBI prior to BMT. The levels of the antioxidants, ascorbic acid (AA) and uric acid (UA) were determined by HPLC-ECD. The oxidant stress level was calculated by the ratio [dehydro ascorbic acid]/total ascorbic acid]. RESULTS: TAC was reduced by 36% (p < 0.02) but after 4 months recovered to a level 22% higher than before the treatment (p < 0.05). Both, AA and UA, decreased following irradiation by 84% (p < 0.02) and 24% (p < 0.05) respectively, but returned to a level of 21% and 320% after 4 months compared to baseline values. The changes in [UA] were affected by Allopurinol (xanthine oxidase inhibitor), given as a routine pretransplant therapy until day -1. The [dehydroascorcbic acid]/[total ascorbic acid] (%) was 45% (range of normal controls = 13.2 +/- 1.5%) and increased by 69% following TBI. In order to obtain a decrease in the TAC of plasma in vitro, comparable to that in vivo, a 1000 fold higher dose of irradiation was required. CONCLUSIONS: TBI caused a pronounced decrease in antioxidant capacity and an excessive increase in oxidant stress. We assume that TBI alters antioxidant homeostasis greatly enhancing the stress damage. CV measurements may lead to a better understanding of the balance between oxidant stress and antioxidant utilization, and to a reconsideration of the routine use of Allopurinol as pretreatment for TBI, and antioxidant support before and/or after TBI.

The antioxidant properties of thioctic acid: characterization by cyclic voltammetry.

Thioctic acid (TA) level and its antioxidant capacity were monitored by cyclic voltammetry (CV), in solution and in human plasma. A linear correlation between the anodic wave current Ia at 900 mV and [TA] was obtained. This indicates that TA, commonly found in human plasma, is an antioxidant component characterized by a potential E1/2 = 900 mV and constitute, at least in part, the second anodic wave of the CV tracing. When plasma from diabetic patients was analyzed by CV, those patients under treatment with TA showed higher Ia than others not taking the drug. When the patients under treatment with TA were divided into groups according to the severity of their nephropathy, or according to the severity of their total complication count, Ia levels representing TA were significantly higher in those patients with more severe complications.