Nadine Loreau

Glucose and free radicals impair the antioxidant properties of serum albumin

Epidemiological data consistently show that reduced levels of serum albumin, which is the most abundant protein in plasma, are associated with an increased mortality risk. Various biological properties evidenced by direct effects of the albumin molecule may explain its beneficial effects. The present work aimed to investigate in vitro whether glycation or free radicals or both factors would affect the antioxidant properties of bovine serum albumin (BSA). Glycation was performed by long‐term incubations (60 days) of BSA with increasing concentrations of glucose (up to 500 mmol/l) at 37°C. Minimally oxidized BSA was obtained after controlled incubations of dialyzed BSA samples with a water‐soluble free radical generator [2,2′ azo‐bis(2‐amidinopropane) HCl]. The glycation‐mediated modifications and the free radical‐induced conformational changes of BSA were monitored using intrinsic fluorescence measurements of the tryptophan residues and acrylamide as a quenching agent. Thiol groups, Amadori glycophore contents, and boronate binding were also measured. We found that the changes observed in the conformation of the BSA molecule were associated with modifications of its antioxidant properties. The latter were studied by the copper‐mediated oxidation of human low density lipoproteins and the free radical‐induced blood hemolysis test. Our data support the concept that oxidative‐induced BSA modifications are important determinants in the antioxidant properties of BSA. Glycated BSA still behaved as an antioxidant but became pro‐oxidant in the presence of copper, probably by generating oxygenated species. These data confirm the key role of metals ions in this process. Although these results warrant further in vivo investigations, we propose that, considering the poor glucose control found in diabetics as well as the key role of oxidative stress in vascular complications, glycation‐mediated and free radical‐induced impairment of the antioxidant properties of albumin might be important parameters in vascular complications encountered in diabetes.—Bourdon, E., Loreau, N., Blache, D. Glucose and free radicals impair the antioxidant properties of serum albumin. FASEB J. 13, 233–244 (1999)

Differential effects of cysteine and methionine residues in the antioxidant activity of human serum albumin

Antioxidant properties of human serum albumin (HSA) may explain part of its beneficial role in various diseases related to free radical attack. In the present study, the antioxidant role of Cys and Met was studied by copper-mediated oxidation of human low density lipoproteins and by free radical-induced blood hemolysis which essentially assessed metal-chelating and free radical scavenging activities, respectively. Mild conditions were set up to specifically modify Cys and Met residues by N-ethylmaleimide (NEM) and chloramine T treatments, respectively. We found that Met and Cys accounted for 40–80% of total antioxidant activity of HSA. Copper binding to HSA was decreased by about 50% with chloramine T treatment of Met whereas no change was observed after NEM treatment of Cys. Although other amino acid residues are likely to be involved in anti-/prooxidant properties of HSA, from our data, we propose that Cys chiefly works as a free radical scavenger whereas Met mainly acts as a metal chelator.

Oxidative stress leads to cholesterol accumulation in vascular smooth muscle cells

The transformation of macrophages and smooth muscle cells into foam cells by modified low-density lipoproteins (LDL) is one of the key events of atherogenesis. Effects of free radicals have mainly been studied in LDL, and other than toxicity, data dealing with direct action of free radicals on cells are scarce. This study focused on the direct effects of free radicals on cholesterol metabolism of smooth muscle cells. A free radical generator, azobis-amidinopropane dihydrochloride, was used, and conditions for a standardized oxidative stress were set up in vascular smooth muscle cells. After free radical action, the cells presented an accumulation of cholesterol that appeared to be the result of: (i) an increase in cholesterol biosynthesis and esterification; (ii) a decrease in cell cholesteryl ester hydrolysis; and (iii) a reduced cholesterol efflux. All these parameters were opposed by antioxidants. In addition, oxidant stress induced an increased degradation of acetyl-LDL, whereas no change was noted for native LDL. From this data, it was concluded that cholesterol metabolism of vascular smooth muscle cells was markedly altered by in vitro treatment with free radicals, although cell viability was unaffected. The resulting disturbance in cholesterol metabolism favors accumulation of cholesterol and cholesteryl esters in vascular cells, and thus may contribute to the formation of smooth muscle foam cells.

Gas chromatographic analysis of resveratrol in plasma, lipoproteins and cells after in vitro incubations

Resveratrol is a trihydroxystilbene present in certain red wines. It may play a role in the inhibition of lipoprotein oxidation and platelet activity. We have developed the first method to measure resveratrol in animal and human samples and to study its incorporation in vitro. After adding epicoprostanol as an internal standard, samples are subjected to lipid extraction in the presence of antioxidant and under dim light to minimize both denaturation and isomerization of the trans-resveratrol to the cis-form. Extracts were purified by cold acetone precipitation and the resveratrol-containing acetone phase was evaporated under nitrogen. The resveratrol was analyzed as a trimethylsilyl derivative by capillary gas chromatography which resolved the cis- and trans-resveratrol (6.6 and 12.9 min, respectively). Analyses of samples spiked with pure trans-resveratrol (0.1 to 10 microg) indicated that the method was specific and gave excellent linearity and recovery (96.8%) with a high reproducibility (coefficient of variation: 3.3%). The detection limit was about 50 ng/ml. Applications show that resveratrol was incorporated into blood cells and lipoproteins after in vitro incubations with plasma, lipoproteins and cells.

Impaired Cellular Cholesterol Efflux By Oxysterol-Enriched High Density Lipoproteins

One of the proposed antiatherogenicity role of high-density lipoproteins (HDL) is believed to stimulate removal of cholesterol from the peripheral cells back to the liver for excretion. We have investigated the effects of oxidation-related modifications of HDL on their ability to stimulate cholesterol efflux from cultured cells. Human HDL (HDL3, 1.13 < d < 1.21 g/ml) have been modified either by malondialdehyde or by copper-mediated oxidation (Ox-HDL3). Compared with native HDL3, the modified HDL3 resulted in a significantly reduced efflux of labeled cholesterol from preloaded macrophages (P388D1 cell line). Analysis of lipid composition of Ox-HDL3 by gas chromatography revealed the presence of oxysterols (OS). Enrichment of native HDL3 with oxysterols resulted in a reduced capacity to stimulate cholesterol efflux. The reduced ability of OS-enriched HDL3 to elicit cholesterol efflux may contribute to cellular cholesterol accumulation and subsequently to atherosclerosis.

Oxidant stress: the role of nutrients in cell-lipoprotein interactions.

Oxidant stress is increasingly becoming an important hypothesis to explain the genesis of several pathologies, including cancer, atherosclerosis and also ageing. Beside a few rare genetic defects, dietary factors are thought to play a key role in the regulation of the production of reactive oxygenated species. An imbalance between nutrients, and in particular those involved in antioxidant status, could explain the onset of an enhanced production of free radicals. We will briefly review information concerning oxidation of lipids and lipoproteins which lead to atherothrombosis. We also present new findings supporting a role for blood platelets in generating oxidant species. New data are also described concerning the role of oxygenated derivatives of cholesterol, oxysterols, in cellular cholesterol efflux and NO production. Also, new developments relating to the influence of direct effects of free radicals on cellular cholesterol homeostasis are presented. Finally, the in vitro effects of butyrate, a natural short-chain fatty acid produced by bacterial fermentation, in the protection against free radical-mediated cytotoxicity are discussed. These data provide information on the mechanisms of dietary antioxidants in preventing oxidant stress.

Role of the cyclic AMP-dependent pathway in free radical-induced cholesterol accumulation in vascular smooth muscle cells

We have previously reported that free radical-treated vascular smooth muscle cells (SMC) lead to cholesterol accumulation in vitro. In the current study, we investigated the effects of oxidative stress on cyclic AMP concentration and cAMP-dependent enzymes involved in cholesterol homeostasis in A7r5 cells. Under our conditions of a mild oxidative stress, namely with no change in cell viability, we found that free radicals, initiated using azobis-amidinopropane dihydrochloride (AAPH), resulted in a dose-dependent decrease in cellular cAMP which was opposed by vitamin E preincubation. Although the addition of adenylate cyclase activators (carbacyclin and forskolin) increased cAMP levels it did not succeed in restoring the AAPH-induced decrease. The oxidative stress-induced increase in activities of 3-hydroxy-3-methylglutaryl coenzyme A reductase and of acyl coenzyme A: cholesterol acyltransferase and the decrease in neutral cholesteryl ester hydrolase activity were suppressed by addition of dibutyryl cAMP. Taken together, these results strongly suggest that free radicals reduce cAMP concentrations by altering cell membrane adenylate cyclase activity. The changes of cAMP-dependent enzymes induced by oxidative stress resulting in cholesterol accumulation might be one of the processes leading to SMC-derived foam cells depicted in atheroma plaque. Moreover, if extrapolated to in vivo, these data may explain in part the beneficial effects of antioxidants in the reduction of cardiovascular diseases.

Involvement of Oxysterols and Lysophosphatidylcholine in the Oxidized LDL–Induced Impairment of Serum Albumin Synthesis by HEPG2 Cells

Abstract—Oxidized low density lipoproteins (Ox-LDLs) are increasingly thought to be a key element in atherogenesis. We have previously reported that serum albumin has important antioxidant properties and that a reduced synthesis of albumin may represent a crucial point in the overall antioxidant defense. In the present work, we aimed at determining whether Ox-LDL could modulate albumin synthesis in cultured human hepatocytes (HepG2 cells). With the use of enzyme immunoassay and radiolabeled leucine incorporation followed by specific immunoprecipitation, Ox-LDL was found to lead to a dose-dependent decrease in albumin secretion. Moreover, the protein synthesis and mRNA levels were decreased in the presence of Ox-LDL, as assessed by Northern blot analysis. Because oxysterols and lysophospholipids are key components of Ox-LDL, we tested the effects of oxysterols (7-ketocholesterol and 25-hydroxycholesterol) and lysophosphatidylcholine on albumin secretion and expression. In our experimental conditions, we found that incubations with oxysterols or lysophosphatidylcholine at pathophysiological concentrations similar to those measured in Ox-LDLs reproduced the above-mentioned inhibitory effects on albumin synthesis. On the basis of our in vitro data, we propose that this newly described biological effect of Ox-LDL might partly explain the findings of epidemiological studies indicating that reduced levels of serum albumin are associated with increased mortality.