Michel Clerc

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.

Relationship between red blood cell antioxidant enzymatic system status and lipoperoxidation during the acute phase of malaria

OBJECTIVE To investigate the relationship between oxidative stress and aggrevation of the disease in patients with malaria. METHODS AND RESULTS In the present study lipoperoxidation was demonstrated during the acute phase of malaria by a significant decrease in polyunsaturated fatty acids (PUFA). The lowest values of PUFA were obtained for C20:4 and C22:6, which were the main targets of reactive oxygen species (ROS) when parasitemia was higher than 1%. Similarly, plasma vitamins E and A were significantly reduced during the acute phase of malaria owing to their consumption in part as antioxidants. However, evaluation of the antioxidant enzymatic system in red blood cells of malaria patients indicated no significant difference from controls. Only superoxide dismutase activity tended to decrease when parasitemia increased. CONCLUSION The results suggest that superoxide radicals are the main ROS produced during the acute phase of malaria, and that rejuvenation of RBC during hemolysis involving increased enzyme activities interacts to protect RBC from excessive superoxide radical production.

DEINOXANTHIN : A NEW CAROTENOID ISOLATED FROM DEINOCOCCUS RADIODURANS

Abstract We have previously demonstrated that carotenoids which support the color of the red pigmented strains of Deinococcus radiodurans could be responsible for the antioxidant activity of these bacteria submitted to hydroxy radicals. Here we report on the isolation and identification of the major carotenoid of this bacterium. Based on UV/Vis-, 1 H NMR- and mass spectra as well as the chemical properties the carotenoid was identified as (all- E )-2,1′-dihydroxy-3′-4′-didehydro-1′,2′-dihydro-β,ψ-carotene-4-one 1 . This carotenoid has never been isolated before and we propose the name deinoxanthin for this new compound. The constitution of a new carotenoid 1 was determined by chemical and spectroscopic methods.

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.

Phosphorus-31 nuclear magnetic resonance of isolated rat liver during hypothermic ischemia and subsequent normothermic perfusion

The effects of prolonged hypothermic ischemia and subsequent normothermic perfusion on the energetic metabolism and intracellular pH (pHin) of isolated rat livers were studied by phosphorus-31 nuclear magnetic resonance spectroscopy. Nucleoside triphosphate (NTP) depletion and intracellular pH were studied within an 18-h-storage phase, by using the following preservation media: Eurocollins (EC), UW Lactobionate (UW) and Bretschneiders solution (HTK). Values obtained after 8-h ischemia were chosen to estimate the performance of the various media: NTP levels were 37 +/- 7%, 10 +/- 5% and 0% of control levels, respectively, in livers stored in UW, HTK and EC solutions. pHin reached values of 7.15 +/- 0.10 in UW and HTK, and 6.96 +/- 0.10 in EC-stored livers. Ischemic damage was assessed by reperfusing the stored organ with Krebs medium: NTP recovery was around 70 +/- 20% for the three solutions used. Recovery of pHin was near the control value (7.23 +/- 0.08), except for EC solution (7.05 +/- 0.20). The main results are that (i) the rates of NTP and pHin decrease are strongly dependent on the nature of the preservation solution, whereas (ii) NTP recovery is not significantly different during post-ischemic reperfusion. With regard to animal survival, UW solution is at present considered largely superior to EC medium for liver preservation. Thus, our data suggest that the rates of NTP depletion and pHin fall during cold preservation could be both considered as better indicators assessing liver injury than the post-ischemic NTP recovery.

The Place of Electron Spin Resonance Methods in the Detection of Oxidative Stress in Type 2 Diabetes with Poor Glycemic Control

OBJECTIVE Chronic production of reactive oxygen species (ROS) and/or deficiency in the antioxidant defense system are observed in non-insulin-dependent diabetes mellitus (NIDDM) patients. As an adjunct to the usual indirect parameters for evaluating oxidative stress, we assessed the feasibility of oxyradicals detection in venous blood by electron spin resonance spectroscopy (ESR). Detection of the ascorbate pool was also performed using the validated ESR analysis of the ascorbyl free radial (AFR)-dimethyl sulfoxide (DMSO) complex. METHODS AND RESULTS Plasma lipoperoxidation was characterized by higher levels of total MDA (1.50 +/- 0.08 nmol/L), lower levels of GSH (0.54 +/- 0.02 mmol/L) and of vitamin A (2.13 +/- 0.52 mumol/L) in the NIDDM group than in the controls (0.75 +/- 0.05 nmol/L, 0.90 +/- 0.05 mmol/L, 3.52 +/- 1.04 mumol/L, respectively). Improvement of the ESR measurement of oxyradical adducts has been previously obtained by addition of a new sensitive nitrone (DEPMPO), which acts as a spin-trap. However, in our experiment the ESR signal-to-noise ratio was too low to detect significative oxyradicals adducts in total venous blood of NIDDM patients having a weak production of ROS. A significant difference (p < 0.002) was observed in DMSO/AFR index between controls (24.00 +/- 4.10 nmol/L) and NIDDM patients (7.28 +/- 2.36 nmol/L) suggesting ascorbate depletion related to the free radical production. CONCLUSION The DMSO/AFR index could be an interesting additional marker of oxidative stress during a chronic production of ROS.

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...

A normal rate of cellular cholesterol removal can be mediated by plasma from a patient with familial lecithin-cholesterol acyltransferase (LCAT) deficiency.

Lecithin-cholesterol acyltransferase (LCAT) is the major enzyme involved in the esterification of cholesterol in circulating plasma lipoproteins. In the present study, we describe the molecular defects in the LCAT gene and in lipoprotein metabolism of a 34-year-old patient presenting with features of classic familial LCAT deficiency. DNA sequencing revealed two separate point mutations in exon 3 of the patients LCAT gene: a C to A substitution converting Tyr(83) to a Stop and a C to T transition converting an Arg(99) to a Cys. Digestion of patient PCR-amplified DNA with the restriction enzymes AccI and AciI established that the patient was a compound heterozygote for both mutations. In vitro expression of LCAT (Arg(99)-->Cys) in human embryonic kidney-293 cells demonstrated reduced expression, as well as reduced secretion and/or increased intracellular degradation of the mutant enzyme with significantly decreased alpha-LCAT specific activity, thus, establishing the functional significance of the LCAT (Arg(99)-->Cys) mutation. The plasma cholesterol esterification rate (CER, 2+/-0.3 nmol/ml/h), alpha-LCAT activity (2.9+/-0.1 nmol/ml/h) and LCAT concentration (0.3+/-0.1 microg/ml) were 2.9%, 2.3% and 6.1% that of normal subjects, respectively. Analysis of the patients plasma lipid profile revealed reduced plasma concentrations of total cholesterol (111+/-0.5 mg/dl), HDL cholesterol (1.6+/-0.2 mg/dl), apolipoprotein (apo) A-I (52+/-4 mg/dl) and apo A-II (11+/-0.5 mg/dl). Nevertheless, for the first time, we demonstrate that the LCAT-deficient plasma is as efficient as control plasma in cholesterol efflux experiments performed with [(3)H]-cholesterol loaded fibroblasts. This result could explain the absence of premature atherosclerosis in this LCAT-deficient patient.