Annie Perromat

Lipoperoxidation in plasma and red blood cells of patients undergoing haemodialysis: Vitamins A, E, and iron status

In 14 patients undergoing haemodialysis, lipoperoxidation (LPO) processes were determined in plasma and red blood cells (RBC) before and after a dialysis session by determining (a) the direct substrate, polyunsaturated fatty acids (PUFA); (b) the end product of LPO, malondialdehyde (MDA); and (c) the hydrophobic antioxidant systems, vitamins A and E. In plasma before dialysis, linoleic and arachidonic acid, and the antioxidant vitamin E, were significantly lowered as compared to the healthy controls (p < 0.05). On the contrary, the free MDA level was enhanced (p < 0.05). These results were emphasized by a dialysis session. In RBC of these patients, no difference in linoleic acid, free MDA, or vitamin E level were observed before or after dialysis when compared to controls. However, only vitamin A was significantly higher in haemodialysis patients (before and after dialysis) and in renal failure patients (p < 0.05) than in the healthy control group. The present results suggest that increased RBC vitamin A may offer some degree of protection against oxidative stress in erythrocytes, but not in plasma where LPO is demonstrated.

Short-Term Insulin Therapy and Normoglycemia: Effects on erythrocyte lipid peroxidation in NIDDM patients

OBJECTIVE To evaluate erythrocyte lipid peroxidation (LPO) before and after an adaptive short-term insulin therapy in NIDDM patients who were chronically hyperglycemic. RESEARCH DESIGN AND METHODS Twenty-six patients with NIDDM (mean HbA1c, 11.28%) aged 53.04 ± 2.03 years were submitted for 3 days to constant intravenous glucose and continuous insulin perfusion at an adaptable rate to maintain glycemia within the normal range. An evaluation of LPO at baseline and after euglycemic insulin therapy was determined by erythrocyte free and total malondialdehyde (MDA) levels, polyunsaturated fatty acid (PUFA) percentage, vitamin E and glutathione content, and the following antioxidant enzymatic activity determinations: glutathione peroxidase (GPX), superoxide dismutase (SOD) and catalase (CAT). Fasting serum glucose, HbA1c, triglycerides, cholesterol, and HDL cholesterol levels were also determined at these time points. RESULTS At baseline, erythrocyte free and total MDA were significantly higher in NIDDM patients than in control subjects (11.14 ± 0.80 vs. 1.74 ± 0.11 nmol/g Hb [P < 0.0001] for free MDA; 18.04 ± 1.79 vs. 7.85 ± 0.55 nmol/g Hb [P < 0.0001] for total MDA). PUFAs, particularly C20:4 and C22:5, were increased (14.69 ± 0.34 vs. 12.03 ± 0.31 and 2.31 ± 0.04 vs. 1.71 ± 0.03% of total fatty acids, respectively). Vitamin E and glutathione were reduced significantly (6.16 ± 0.61 vs. 14.84 ± 0.64 nmol/g Hb and 0.42 ± 0.04 vs. 0.97 ± 0.06 mmol/l, respectively). No difference was observed for the enzymatic activities. After euglycemic insulin therapy, triglycerides significantly decreased compared with baseline concentrations (1.55 ± 0.13 vs. 2.42 ± 0.22 mmol/l; P < 0.001), whereas other lipidic parameters were unchanged. Free MDA significantly decreased (8.60 ± 0.76 vs. 11.14 ± 0.80 nmol/g Hb [P < 0.01]), while vitamin E increased (7.93 ± 0.73 vs. 6.16 ± 0.61 nmol/g Hb [P < 0.05]). No difference was observed for PUFAs, glutathione, or total MDA. CONCLUSIONS The observed erythrocyte LPO in NIDDM decreased after a short-term adaptive insulin therapy. This decrease could be principally attributed to the normalized glycemia that reduces reactive oxygen species (ROS) production, which in turn may explain the increase in erythrocyte membrane vitamin E and the decrease in MDA. This study shows the value of a euglycemic environment in NIDDM to reduce LPO and, at long range, to minimize clinical diabetes complications.

Antioxidant Effects of a Supplemented Very Low Protein Diet in Chronic Renal Failure

Increased peroxidation of lipids in red blood cells (RBC) in patients with advanced chronic renal failure (CRF) reflects increased generation of reactive oxygen species (ROS), which may contribute to the metabolic damage induced by CRF and to its progression. We have evaluated parameters indicative of lipoperoxidation (LPO) of RBC at baseline in patients with CRF compared to controls, and the effects of a very low protein diet supplemented with amino and keto acids and vitamins A, C, E (VLPD) over a 6-month period. The presence of peroxidation damage in CRF patients before the administration VLPD was demonstrated by elevated levels of free malondialdehyde (MDA) (p < .0003) and decreased levels of polyunsaturated fatty acids (PUFA), particularly C20:4 (p < .001), C22:4 (p < .0001) and C22:5 (p < .0001) when compared to controls. Similarly, RBC vitamin E content was significantly decreased (p < .0001) while enzymatic activities were unalterated. VLPD reduced erythrocyte LPO as suggested by (a) decreased levels of free and total RBC MDA (p < .003 and p < .03, respectively), (b) increased levels of PUFA, particularly C22:4 and C22:5 (p < .003 and p < .03, respectively), and (c) increased levels of vitamins A and E (p < .001 and p < .04, respectively) as compared to prediet results. Antioxidant enzyme activities were not modified. These results suggest that VLPD has a protective role against LPO of erythrocytes in patients with CRF.

IN VITRO INFLUENCE OF ASCORBATE ON LIPID PEROXIDATION IN RAT TESTIS AND HEART MICROSOMES

Lipid peroxidation (LPO) in rat testis and heart microsomes was compared using the ADP/Fe2+ as initiator with and without ascorbate at different concentrations. The extent of LPO was estimated by the levels of TBARS and PUFA. Without ascorbate, LPO was higher in heart than in testis despite elevated levels of catalase in heart. With increased ascorbate concentrations, a biphasic effect of LPO was observed. For a concentration ≤ 0.2 mM, ascorbate acted as pro-oxidant and increased TBARS correlated with decreased PUFA were observed both in testis and heart. Above 0.2 mM, ascorbate acts as antioxidant but differences in the rate of LPO were observed. In heart decreased TBARS correlated with increased PUFA whereas in testis TBARS only decreased, PUFA were not significantly modified. These results suggest different mechanisms in LPO initiation in the two organs. Increasing concentrations of H2O2 produced directly elevated TBARS levels in testis while a lag phase was observed in heart before the increase, suggesting that H2O2 was the essential ROS produced by ascorbate-ADP/Fe2+. The effects of scavengers such as catalase and ethanol showed an inhibitory effect on TBARS production only in testis, suggesting the role of H2O2/OH⋅ as an initiator of LPO. In heart, catalase produced a slight increase in TBARS levels whereas no modification was observed with ethanol, suggesting a possible direct activation by ADP/Fe2+ through a metal-oxo intermediate.

Relationship between dietary retinol and α‐tocopherol and lipid peroxidation in rat liver cytosol

The effects of retinol and α‐tocopherol‐deficient and supplemented diets on the cytosolic concentration of thiobarbituric acid reactive substances (TBARS) in rat liver have been studied. Physiological lipoperoxidation (LPO) was observed in liver cytosol of control rats (TBARS = 0.315 ± 0.034 nmol of MDA equivalents/mg of liver cytosolic proteins). In retinol‐deficient diets there was a decrease in retinolaemia and the absence of retinol in liver cytosol while cytosolic TBARS increased significantly (P < 0.001). Vitamin E was not found in cytosolic fractions, except in α‐tocopherol‐supplemented diet rats. α‐Tocopherol‐deficient diets induced an absence of vitamin E in the serum and cytosolic TBARS were increased compared to controls (P < 0.001). Supplementation of the diet with retinol and α‐tocopherol or both in combination induced a significant decrease in liver cytosolic TBARS (P < 0.001). Finally the combination of low dietary supplementation with retinol and α‐tocopherol (ten times the normal diet eac...

Free radical inhibitor effect of retinol after carbon tetrachloride intoxication in the rat

A study was conducted to explore the free radical inhibitor effect of retinol in Male Wistar rats. When retinol-deprived animals were considered retinol-depleted (after a period of 8 weeks), rats of each group, control and depleted, received an intraperitoneal injection of mineral oil (5 ml/kg body weight) or an equivalent volume of 20% carbon tetrachloride (CCl4) dissolved in mineral oil. The animals were killed by decapitation 4 h after administration of CCl4 and liver, heart, spleen, brain and testes were quickly removed. Minced tissues were homogenized and microsomes were prepared; vitamins A and E were monitored and malondialdehyde (MDA) content was estimated. Retinol-depleted rats showed an hepatic vitamin A level less than 10 pmol/mg protein, compared to control rats (15-45 pmol). In all hepatic preparations, we found low vitamin E levels (100-1300 pmol/mg protein). MDA production increased significantly in livers and hearts of retinol-depleted rats but not in brains, spleens and testes. Hearts contain less lipids and vitamin E than these latter organs, which could correlate with the highest production of MDA.

In vivo effect of diosmin on carrageenan and CCl4-induced lipid peroxidation in rat liver microsomes

The aim of this study was to compare the protective effect of a flavonoid, the 3,5,7-trihydroxy-4-methoxyflavone 7-rutinoside or diosmin, on liver microsomal lipid peroxidation induced in rats by either carbon tetrachloride or carrageenan. Thirty rats were divided into five groups. Group 1 received no chemical product and was considered as control. Groups 2 and 3 received either an intraperitoneal injection of carrageenan or carbon tetrachloride 48 or 24 hours before killing, respectively. Groups 4 and 5 were treated first with an intraperitoneal injection of diosmin and then by carrageenan (group 4) or carbon tetrachloride (group 5) 48 or 24 hours before killing, respectively. The lipoperoxidant effect of carrageenan and carbon tetrachloride was demonstrated by both significant decreases in polyunsaturated fatty acids, principally 20:4 (n - 6) (p < 0.05) and of vitamin A (p < 0.05) in groups 2 and 3. With diosmin treatment, only thiobarbituric acid reactive substances significantly decreased in group 4, whereas vitamin A level increased. These results could suggest that the effect of diosmin differs with the choice of chemical product used; it seems a better antioxidant against products inducing inflammation.

Free malondialdehyde determination by HPLC applied to microsomal studies

Malondialdehyde (MDA) is a product of lipid peroxidation in vivo. The most widely employed method for determination of free MDA is based on its reaction with thiobarbituric acid (TBA) which produces a pink pigment with an absorption maximum at 532-535 nm. However, quantitation of MDA is limited by its lack of specificity and a high performance liquid chromatographic (HPLC) method was recently developed in several laboratories. In the present study, free MDA levels were measured, after TBA reaction, spectrophotometrically and by HPLC in microsomes of different tissues from rats fed a vitamin A-deficient diet or not for 8 weeks, and treated or not with carbon tetrachloride. Incubation in vitro with NADPH (0.25 mM) or ascorbate (0.50 mM) in the presence of Fe2+ (5 microM)-ADP (0.5 mM), allowed us to estimate the total amount of enzymatic or non enzymatic lipoperoxidation. The MDA amount determined by HPLC is significantly lower than the TBA-reactive substances (TBA-RS) calculated spectrophotometrically as MDA equivalents. Moreover, HPLC separations performed on a mu Bondapack C18 column with a mobile phase of methanol/water 45/55 (v/v), containing 1% cetrimide revealed that three chromogens are present in microsomes incubated with ascorbate or NADPH. The TBA-RS visible spectra of microsomes incubated with activator are complex with an absorption maximum at 533 nm, which is specific for the MDA-TBA chromogen, and one at 450 nm. Identification of these TBA-RS, different from the MDA-TBA complex, is under investigation in our laboratory.

Lipid Peroxidation Studied by HPLC in Isolated Rat Liver: Comparative Efficiency of UW and HTK Preservation Solutions

Abstract Lipid peroxidation on preserved isolated rat liver. Lipoperoxidation (LPO) generated by activated oxygen species could be a cause of damage observed in isolated organs during normoxic reperfusion. Free and bound MDA, vitamin E and vitamin A analyses were performed by HPLC on extracts from livers preserved in two hepatic clinical preservation solutions; ATP level and intracellular pH were followed by 31P NMR. The data indicate that bound MDA values increased during ischemia and decreased upon cold and warm reperfusion. This rather unexpected MDA accumulation during ischemia supports the hypothesis that lipoperoxidation might be initiated by ADP-Fe+++-O2 − complex accumulating during this period. Furthermore, in lactobionate-solution preserved livers, we found during reperfusion a significant decrease in vitamin E content concomitantly with a decrease in bound MDA. This observation suggests a protective effect of vitamin E in lipoperoxide formation in livers stored in UW. This phenonenom is not obs...

Comparison of Methods Evaluating Lipoperoxidation in Plasma of Malaria Patients

Abstract Lipid peroxidation is believed to be involved in malaria and plasma of malaria patients may be damaged by reactive oxygen soecies This study compares determinations of malondialdehyde ifiDA), vitamins A and E by high performance liquid chromatography, thiobarbituric acid reactive substances (TBA-RS) by fluorometric analysis and fatty acids by gas chromatography. During malarial infection, polyunsaturated fatty acids (PUFA), vitamins A and E decreased significantly when TBA-RS increased significantly. However, there was no significant change >n total or MDA bound to amino-groups. On the other hand, there was no correlation between TBA-RS production and PUFA decrease or between TBA-RS production and vitamin A or E consumption. These results support the concept that other compounds such as proteins and cholesterol may be involved in the formation of these TBA-RS.