Denis Blache

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)

Acute methionine load-induced hyperhomocysteinemia enhances platelet aggregation, thromboxane biosynthesis, and macrophage-derived tissue factor activity in rats.

A moderate elevation of plasma homocysteine is a risk factor for atherosclerosis and arterial and veinous thrombosis. However, the mechanisms leading to vascular disorders are poorly understood because studies that have investigated the potential atherothrombogenicity of hyperhomocysteinemia in vivo are scarce. Using a rat model, we were the first to show that dietary folic acid deficiency, a major cause of basal hyperhomocysteinemia, is associated with enhanced macrophage‐derived tissue factor and platelet activities. We proposed that an homocysteine‐induced oxidative stress may account for this hypercoagulable state. To determine the true thrombogenicity of moderate hyperhomocysteinemia and better understand its etiology, we have carried out an acute methionine load in control and folate‐deficient animals. When rats were fed the control diet, a transient fourfold increase in plasma homocysteine levels was observed 2 h after the methionine administration. As with prolonged dietary folic acid deficiency, this methionine load potentiated the platelet aggregation in response to thrombin and ADP as well as the thrombin‐induced thromboxane synthesis. It also stimulated the basal and lipopolysaccharide‐induced tissue factor activity of peritoneal macrophages. These prothrombotic effects were associated with an increased lipid peroxidation characterized by an elevation of plasma conjugated dienes, lipid hydroperoxides, and thiobarbituric acid‐reactive substances. When rats were fed a folic acid‐deficient diet, the methionine load did not cause any further increase in plasma homocysteine concentration, platelet activation, macrophage tissue factor‐dependent coagulation, or lipoperoxidation. Altogether, our data showed that the prethrombotic state due to both the altered remethylation and transsulfuration pathways resulted from the moderate elevation of circulating homocysteine. We conclude that moderate hyperhomocysteinemia plays a role in the development of a thrombogenic state that might be mediated by the occurrence of oxidative stress.—Durand, P., Lussier‐Cacan, S., Blache, D. Acute methionine load‐induced hyperhomocysteinemia enhances platelet aggregation, thromboxane biosynthesis, and macrophage‐derived tissue factor activity in rats. FASEB J. 11, 1157–1168 (1997)

Impaired Homocysteine Metabolism and Atherothrombotic Disease

Based on recent retrospective, prospective, and experimental studies, mild to moderate elevation of fasting or postmethionine-load plasma homocysteine is accepted as an independent risk factor for cardiovascular disease and thrombosis in both men and women. Hyperhomocysteinemia results from an inhibition of the remethylation pathway or from an inhibition or a saturation of the transsulfuration pathway of homocysteine metabolism. The involvement of a high dietary intake of methionine-rich animal proteins has not yet been investigated and cannot be ruled out. However, folate deficiency, either associated or not associated with the thermolabile mutation of the N5,10-methylenetetrahydrofolate reductase, and vitamin B6 deficiency, perhaps associated with cystathionine β-synthase defects or with methionine excess, are believed to be major determinants of the increased risk of cardiovascular disease related to hyperhomocysteinemia. Recent experimental studies have suggested that moderately elevated homocysteine levels are a causal risk factor for atherothrombotic disease because they affect both the vascular wall structure and the blood coagulation system. The oxidant stress that results from impaired homocysteine metabolism, which modifies the intracellular redox status, might play a central role in the molecular mechanisms underlying moderate hyperhomocysteinemia-mediated vascular disorders. Because folate supplementation can efficiently reduce plasma homocysteine levels, both in the fasting state and after methionine loading, results from further prospective cohort studies and from on-going interventional trials will determine whether homocysteine-lowering therapies can contribute to the prevention and reduction of cardiovascular risk. Additionally, these studies will provide unequivocal arguments for the independent and causal relationship between hyperhomocysteinemia and atherothrombotic disease.

The importance of proteins in defense against oxidation.

Free radicals are a normal feature of cellular oxygen metabolism. However, free radical-associated damage is an important factor in many pathological and toxicological processes. For a long time, lipid peroxidation, mediated by oxygen-derived free radicals, was probably the most extensively investigated process. From more recent studies, it has become evident that proteins are also the targets of free radicals, and this has important implication for their activity, unfolding, and degradation, as well as in cell functioning. After giving a brief overview of the key role of proteins in the overall antioxidant defense, this review examines their role as targets of oxidation reactions, taking into account the reactivity of amino acid residues and some of their oxidation products. In light of recent data, we then consider the specific role of sulfur-containing amino acids in protein degradation and their possible interplay with the reversal of limited oxidative lesions. The participation of proteins in the overall antioxidant defense is also discussed, specifically the role of metallothionein as an intracellular antioxidant and that of albumin as a circulating antioxidant.

Pro-thrombotic effects of a folic acid deficient diet in rat platelets and macrophages related to elevated homocysteine and decreased n-3 polyunsaturated fatty acids

Folic acid deficiency represents a vitamin deficiency that may be due either to an inadequacy of the dietary supply or to an increased requirement. It leads to a number of abnormalities including hematological, neurological and cardiovascular disorders. In this study, we investigated whether folic acid deficiency would influence platelet and macrophage activities. For 6 weeks, rats were fed a test diet containing a low amount of folic acid (250 mu g/kg) by comparison with a control diet (750 mu g/kg). We found 40 and 32 percent reductions (P < 0.05) of plasma and erythrocyte folates, respectively in the tested group. Peritoneal macrophages of the folic acid deficient animals exhibited greater (20 x) tissue factor (TF) activity than in the controls. We also found that folate depletion significantly enhanced the thrombin- and ADP-induced platelet aggregation (+64 and + 13 percent, respectively). Moreover, the results of incubations with radiolabeled arachidonic acid indicated that platelets of folic acid deficient animals incorporated more labeling than controls did. When stimulated with thrombin, the mobilization of arachidonate from platelet phospholipids and its subsequent formation of cyclooxygenase and lipoxygenase metabolites were enhanced in the deficient animals. In particular, thromboxane biosynthesis was markedly increased. The analysis of the plasma fatty acid composition showed a decrease in the plasma unsaturation index related to a marked fall of long chain (n-3) fatty acids which was also observed in platelets. These data suggested the occurrence of an oxidative stress in folic acid deficient animals which was confirmed by increases in plasma lipid peroxidation products (more than +20 percent) and an enhanced susceptibility of erythrocytes to free radicals (+23 percent). Altogether these data suggested that folic acid deficiency altered the circulating and cellular fatty acid composition and thus influenced the balance of the platelet eicosanoid synthesis. In addition, total homocysteine and glutathione concentrations were highly increased in plasma from folate-depleted rats. From these results, we conclude that folate deficiency can potentiate the coagulation pathway mediated by the macrophage TF as well as the platelet activation process. It is suggested that these dysfunctions might be related to the loss of (n-3) polyunsaturated fatty acids. The latter could result from an increased lipid peroxidation triggered by the folic acid deficiency-induced hyperhomocysteinemia.

Hyperhomocysteinemia induced by folic acid deficiency and methionine load — applications of a modified HPLC method

The increasing possibility that homocysteine might be involved in atherosclerosis in non-homocysteinuric subjects has required the measurement of low concentrations of this aminothiol in biological samples. The procedure described here represents an improvement of different HPLC methods. We utilized an isocratic HPLC system with fluorescence detection of plasma total homocysteine derivatized after reaction with ammonium 7-fluoro-benzo-2-oxa-1,3-diazole-4-sulphonate. With the help of the rapidly eluting internal standard N-acetyl-cysteine, the method ensures very good recovery (approximately 100%), reproducibility and precision (within-assay: 2.31%; day-to-day: 2.8%) in the physiological concentration range. This procedure allowed us to validate various animal models of hyperhomocysteinemia such as dietary folic acid deficiency in rat and acute methionine loads in rat and hamster. Using this method, we also confirmed that men have higher plasma total homocysteine levels than women. Due to its simplicity and reliability, our procedure is suitable for routine analysis of total homocysteine and other aminothiols (cysteine, cysteinyl-glycine and glutathione) in biological samples, as required in clinical and research laboratories.

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.

Phospholipid transfer protein (PLTP) deficiency reduces brain vitamin E content and increases anxiety in mice

Vitamin E supplementation constitutes a promising strategy in the prevention of neurodegenerative diseases. Here, we show that a phospholipid transfer protein (PLTP) is widely expressed in the brain where it appears to function as a transfer factor for α‐tocopherol, the main isomer of vitamin E. PLTP deficiency results in significant depletion of brain α‐tocopherol in both homozygous (−30.1%, P<0.0002) and heterozygous (−18.0%, P<0.05) PLTP knocked‐out mice. α‐tocopherol depletion in PLTP‐deficient homozygotes is associated with the elevation of lipofuscin (+25% and +450% increases in cortex and substantia nigra, respectively), cholesterol oxides (+54.5%, P<0.05), and cellular peroxides (+32.3%, P<0.01) in the brain. Complete PLTP deficiency in homozygotes is accompanied by increased anxiety as shown by fewer entries (8.3% vs. 44.4% in controls, P<0.01) and less time spent (1.7% vs. 41.3% in controls, P<0.05) in the open arms of an elevated plus‐maze, in the absence of locomotor deterioration. Thus, the vitamin E transfer activity of PLTP appears to be a key process in preventing oxidative damage in the brain, and PLTP‐deficient mice could be a new model of the contribution of oxidative brain injury in the etiology of neurodegenerative diseases.

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.

Folic Acid Deficiency Enhances Oral Contraceptive-Induced Platelet Hyperactivity

In previous studies conducted in female rats and in women, oral contraceptives (OC) were found to induce a platelet hyperactivity that was related to an oxidative stress. Because cases of megaloblastic anemia have been reported to occur in women taking OC, these treatments are suspected of depleting folate stores. In the study presented herein, which was conducted in rats, we sought to determine the influence of dietary folic acid deficiency (FD) on the thrombogenicity of OC. Animals were fed for 6 weeks with either a folic acid-deficient diet (250 micrograms/kg folic acid) or a control diet (750 micrograms/kg). One-half of the animals in each group were treated with OC (ethinyl estradiol plus lynestrenol). FD and OC individually potentiated platelet aggregation in response to thrombin and ADP and the release and metabolism of arachidonic acid, in particular, the biosynthesis of thromboxane. These platelet activities were further enhanced in animals given both the folic acid-deficient diet and the OC treatment. In addition, FD enhanced the pro-oxidant state in OC-treated rats characterized by (1) a fall in platelet and plasma n-3 fatty acids, (2) an increase in plasma lipid peroxidation products such as conjugated dienes, lipid peroxides, and thiobarbituric reactive substances, (3) a rise in ex vivo erythrocyte susceptibility to free radicals. Moreover, we found that OC treatment led to a reduction of plasma and erythrocyte folate concentrations associated with a moderate hyperhomocysteinemia. Under our experimental conditions, we did not find significant synergistic effects between OC and FD. We propose that, although the untoward effects associated with the OC treatment may not primarily be dependent on FD, the folic acid deficiency magnified OC-induced oxidative stress, which resulted in platelet hyperactivity by elevating the pro-oxidant homocysteine plasma concentration. Despite the limitations of this animal model, the data of the present study suggest that in addition to cigarette smoking, inadequate folic acid intake might predispose those taking OC to vascular thrombosis.