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This study was designed to evaluate the effect of high concentrations of melatonin on the peroxidation of human low density lipoproteins (LDLs) initiated by O(2)(*-) and ethanol-derived peroxyl radicals (RO(2)(*)) from water gamma radiolysis in the presence of ethanol. LDL (3 g/l; total LDL concentration) was oxidized in the absence of melatonin or in its presence at three concentrations (50 x 10(-6), 100 x 10(-6) or 250 x 10(-6) mol/l) in ethanol. Radiolytic yields (i.e. number of mole consumed or produced per Joule) of the markers of lipid peroxidation were determined (i.e. decrease in the endogenous antioxidants alpha-tocopherol and beta-carotene, formation of conjugated dienes and of thiobarbituric acid-reactive substances [TBARS]). Melatonin decreased the yields of lipid peroxidation products and delayed the onset of the propagation phase for conjugated dienes and TBARS in a concentration-dependent manner. Nevertheless, melatonin did not protect endogenous alpha-tocopherol against peroxyl-induced oxidation (probably due to a lower scavenging capacity than that of alpha-tocopherol towards peroxyl radicals), but delayed the consumption of LDL endogenous beta-carotene and decreased its rate of disappearance. The effect of melatonin seemed to be the highest for a melatonin concentration of 250 x 10(-6) mol/l.

This study was designed to evaluate the effect of ethanol on the peroxidation of human low-density lipoprotein (LDL) initiated by oxygen free radicals (O(2)(.-) and (.)OH in the absence of ethanol; O(2)(.-) and ethanol-derived peroxyl radicals, RO(2)(.), in the presence of ethanol) generated by gamma radiolysis. Initial radiolytic yields as determined by several markers of lipid peroxidation [i.e. decrease in endogenous antioxidants alpha-tocopherol and beta-carotene, formation of conjugated dienes and of thiobarbituric acid-reactive substances (TBARS)] were determined in 3 g liter(-1) LDLs (expressed as total LDL concentration) in the absence of ethanol or its presence at six different concentrations (0.42-17 x 10(-2) mol liter(-1)). Ethanol acted as an antioxidant by decreasing the rate of consumption of LDL endogenous antioxidants and the yields of formation of lipid peroxidation products, and by delaying the onset of the propagation phase for conjugated dienes and TBARS. With regard to the different markers studied, except for alpha-tocopherol and beta-carotene consumption, the effect of ethanol did not appear to be dependent on its concentration. Indeed, (.)OH were scavenged by ethanol at the lowest ethanol concentration (0.42 x 10(-2) mol liter(-1)), leading to RO(2)(.). These RO(2)(.) resulted in lower radiation-induced yields related to endogenous antioxidant consumption or to formation of lipid peroxidation products (for example, approximately 10% of RO(2)(.) oxidized LDLs from TBARS). Thus, under our in vitro conditions, ethanol behaved as an antioxidant when added to the LDL solutions. This should be taken into account in the reported antioxidant activity of wine. This is also of interest when lipophilic compounds have to be added as ethanolic solutions to LDLs to evaluate in vitro their antioxidant activity toward LDL peroxidation.

This study was designed to evaluate the antioxidant effect of aminoguanidine toward human low-density lipoproteins (LDLs) initiated by oxygenated free radicals (*OH/O(2)*-) generated by gamma radiolysis. Initial radiolytic yields related to the markers of lipid peroxidation [i.e. decrease in endogenous alpha-tocopherol and beta-carotene, formation of thiobarbituric acid-reactive substances (TBARS) and conjugated dienes] were determined in 3 g liter(-1) LDLs (expressed as total LDL concentration) in the absence and presence of 10 different concentrations of aminoguanidine (from 0.04 to 5 mmol liter(-1)). Fluorescence and relative electrophoretic mobility of oxidized LDLs were also studied as markers that indirectly reflect the attack of the protein moiety of LDLs (namely apolipoprotein B). Our data clearly showed the inhibitory effect of aminoguanidine on lipid peroxidation induced in LDLs by *OH/O(2)*- in a concentration-dependent manner. This effect probably resulted from a scavenging activity of aminoguanidine toward *OH. In contrast, aminoguanidine did not appear to react significantly with O(2)*-, which resulted in a poor residual lipid peroxidation. Our data led us to determine an optimum [aminoguanidine]/[LDL] ratio ranging from 250 to 500 to obtain the best in vitro protection of LDLs under our experimental conditions. It is also of great interest that aminoguanidine was able to protect endogenous alpha-tocopherol and beta-carotene of LDLs upon *OH/O(2)*(-)-induced oxidation.

Efficient transfer of genes maintaining a correct hormonal control in transfected cells is the prerequisite for gene regulation studies and for gene therapy. Differentiated cells, like adipocytes or hepatocytes, are difficult to transfect. In an attempt to improve gene transfer, we first transiently transfected cultured 3T3-F442A adipocytes with a construct containing the simian virus 40 (SV40) promoter fused to the chloramphenicol acetyltransferase (CAT) gene (pSV2-CAT), using various cationic liposomes. Among these, only lipofectAMINE was five times more efficient than the standard calcium phosphate procedure. To further augment efficiency, we transfected 3T3-F442A adipocytes and FAO hepatoma cells with the lipofectAMINE/pSV2-CAT complex in the presence of replication-deficient recombinant type-5 adenovirus at 200 pfu/cell. CAT activity of transiently transfected cells was increased about 50-fold when compared to the calcium phosphate procedure. To determine whether this methodology would be useful for obtaining stable transfectants and would not interfere with correct gene regulation, we used a construct containing -2100 to +69 bp of the phosphoenolpyruvate carboxykinase gene fused to the CAT gene (pPL1-CAT). This construct was shown previously to be cAMP-responsive after calcium-phosphate-mediated transfection of adipocytes and hepatoma cells. 3T3-F442A or FAO cells in which pPL1-CAT was either transiently or stably transferred by lipofectAMINE and adenovirus responded to isoproterenol or cAMP, respectively, with a 2-3-fold increase in CAT activity. Therefore the association of liposomes and adenovirus is an efficient method for transient or stable transfer of regulated genes in adipocytes and hepatoma cells.

Background: Oxidative modification of low-density lipoprotein (LDL) plays a key role in the pathophysiology of atherosclerosis. LDL-apheresis, which involves direct removal of plasma LDL from circulating blood, is an efficient treatment of homozygous familial hypercholes terolemia (FH). Methods: We evaluated impact of long-term LDL apheresis treatment on the atherogenicity of the major LDL subclasses (light, LDL1, and LDL2, density [d] 1.018-1.030 g/mL; inter mediate, LDL3, d 1.030-1.040 g/mL, and dense LDL, LDL4 and LDL5, d 1.040-1.065 g/mL) separated by density gradient ultracentrifugation in severe FH patients. Therefore, we compared the oxidative resistance as well as the chemical and physical properties of each LDL subpopulation in the FH group with those in the corresponding LDL subfractions from normocholesterolemic control subjects. Results: Both intermediate and dense LDL subfractions were significantly more resistant to copper-mediated oxidation in FH patients treated regularly by LDL-apheresis than their counterpart controls. The lag phases for LDL3, LDL4, and LDL5: 63.9 ± 1 1.6, 55.8 ± 1.2, and 47.2 ± 6.5 min. in FH patients were significantly longer than those of the corresponding subfractions in normocholesterolemic controls (P < .01 for LDL3 and LDL5, P < .005 for LDL4). This protective effect was reflected in the delayed formation of biologically active lipid oxidation products such as oxysterols, lipid hydroperoxides, dienes, and dienals in the intermediate and dense LDL subfractions of FH patients. These findings may result from lower "seed" contents of lipid hydroperoxide (LOOH) detected as dienes in plasma LDL from apheresis-treated FH patients; indeed, baseline LOOH/diene contents in all 5 LDL subclasses from FH patients were significantly lower than those of the corresponding subclasses in normolipidemic subjects (P < .0005). On the other hand, the enhanced oxidative resistance of both intermediate (LDL3) and dense (LDL4 and LDL5) LDL subpopulations in FH patients could not be accounted for by any consistent modification in chemical composition or in lipophilic antioxidant content, although minor differences were observed between patients and controls in unsaturated fatty acid profile. In contrast, sphingomyelin content was enriched in FH LDL subclasses, potentially resulting in reduced penetration of the hydrophilic surface layer of LDL by oxygen radicals. Conclusion: We conclude that low concentrations of preformed lipid hydroperoxides and dienes, together with surface sphingomyelin enrichment, can account for the enhanced oxi dative resistance of intermediate (LDL3) and atherogenic dense LDL (LDL4, LDL5) induced by long-term LDL apheresis in severe FH patients.

To the Editor: We read with interest the editorial by Tracy1 on inflammation in cardiovascular disease (CVD). Serum concentrations of C-reactive protein (CRP) were demonstrated to be related to increased risk of CVD, which underlines the potential inflammatory nature of human atherosclerosis.2 3 Interestingly, an unexpected association between CRP and body mass index (BMI) was found in several population studies2 3 4 without any explanation.1 The production of CRP is regulated by cytokines, …

In clinical research, the definition of the upper limit of normal (ULN) alanine transaminase (ALT) is never detailed. However, such a definition can vary and may have practical consequences. Our aim was to assess factors associated with serum ALT activity in apparently healthy subjects and then to apply seven different definitions of ULN in three different populations so as to assess the prevalence of subjects with normal ALT among blood donors and among hepatitis C patients before (normal ALT hepatitis C patients) and after treatment (interferon [IFN] responders). ALT measurements were performed in the same laboratory using the same technique; 1,033 donors were prospectively investigated, 186 patients with hepatitis C never treated and 40 patients treated with 3 MU three times per week of IFN-alpha for at least 6 months. The seven definitions (D) of ULN were: D1: 95th percentile of ALT; D2: 95th percentile after separating males and females; D3: males and females separately, ULN=10 (mean of log10 ALT + 1.96 SD); D4: ULN=45 IU/L given by the manufacturer; D5: mean + 1 SD after exclusion of the 5% extreme values; D6: 95th percentile after separating subjects with body mass index (BMI) under or equal to the median (23); and D7: 95th percentile after separating subjects according to BMI and sex. BMI and male sex were independently associated (P.0001; logistic regression) with ALT, without an association with alcohol. The range of ULN varied from 26 IU/L in females (D5) to 66 IU/L in males with BMI23 (D7). Depending on the definition, the prevalence of blood donors with normal ALT varied from 82% to 96%, i.e., a range of 14%; that of hepatitis C patients with normal ALT varied from 16% to 27%, i.e., a range of 11%; the prevalence of IFN responders varied from 25% to 42%, i.e., a range of 17%. Definitions of normal ALT values should be adjusted for sex and BMI to reduce artificial heterogeneity in blood donor selection and in hepatitis C clinical studies.

patients, evidenced by a decrease in plasma seleniumBackground. An oxidative stress has been reported in levels and in P-GSH-Px activity. However, plasmapatients with chronic renal failure (CRF) treated by TBARS were not higher in CAPD patients than inhaemodialysis. To our knowledge, only scant informa- age-matched non-CRF control subjects, probably ontion is available concerning CRF patients treated by account on the patients’ ages.continuous ambulatory peritoneal dialysis (CAPD)with regard to their redox and nutritional status. Key words: Antioxidants; chronic renal failure; continu-Methods. The oxidative stress and the biological nutri- ous ambulatory peritoneal dialysis; denutrition; retinoltional status were evaluated in 20 elderly CRF patients binding proteintreated by CAPD, compared with a control group of30 elderly non-CRF patients. Plasma peroxidationproducts were assayed as thiobarbituric acid-reactivesubstances (TBARS), and two enzymatic antioxidant Introduction systems were determined: erythrocyte superoxide dis-mutase (SOD), glutathione peroxidase activity inplasma (P-GSH-Px) and in erythrocytes (E-GSH-Px). Oxidative stress, which occurs when there is excessiveSelenium, vitamin E, b-carotene and vitamin A were free radical production or low antioxidant levels, hasevaluated as plasma non-enzymatic antioxidants. been reported in patients with chronic renal failureNutritional status and iron status were assessed by (CRF) treated by haemodialysis [1,2]. In addition, thedetermining serum albumin, prealbumin, iron, ferritin incidence of atherosclerosis is increased in suchpatientsand transferrin concentrations. [3]. The oxidative stress could be involved in theseResults. Plasma TBARS concentration was high in cardiovascular complications, which include athero-sclerosis, accelerated ageing, cataract, b2both groups (CAPD: 1.37±0.06 mmol/l versus non- -microglobulinCRF: 1.41±0.06 mmol/l; P=NS), compared with arthropathy, impaired erythrocyte deformability,usual values (0.60 to 1.20 mmol/l), on account of the increased haemolysis and platelet dysfunction.patients’ ages. SOD and E-GSH-Px activities were Numerous studies in haemodialysed patients investi-normal in both groups. A significant lowering in gated markers of oxidative stress, such as malon-P-GSH-Px activity was observed only in CAPD dialdehyde (MDA) or thiobarbituric acid reactivepatients (211±14 U/l, usual values: 480 to 650 U/l). substances (TBARS), or antioxidant defence systemsPlasma selenium concentration, decreased in both such as erythrocyte superoxide dismutase (SOD),groups, was significantly lower in CAPD than in non- glutathione peroxidase activity in erythrocytesCRF patients (P

This study was designed to evaluate the antioxidant effect of probucol on peroxidation of low-density lipoproteins (LDLs) initiated by oxygenated free radicals ( O2· -) and ethanol-derived peroxyl radicals ( ${\rm RO}_{2}{}^{\cdot}$ ) generated by ? radiolysis. Initial radiolytic yields related to the markers of lipid peroxidation [i.e. decrease in endogenous ?-tocopherol, formation of thiobarbituric acid-reactive substances (TBARS) and conjugated dienes] were determined as a function of LDL concentration (1.5 and $3\ {\rm g}\ {\rm l}^{-1}$ , expressed as total LDL) and in the absence or the presence of probucol at different concentrations (2.3× 10-6, $3.5\times 10^{-6}$ , 9× 10-6 and $20.5\times 10^{-6}\ {\rm mol}\ {\rm l}^{-1}$ ). Our results showed that probucol was able to decrease not only the yields of TBARS and conjugated dienes but also the levels of these peroxidation products obtained at high doses (2500 Gy) compared ...

The aim of this work was to specify the mechanisms involved in the radical oxidation of human high-density lipoprotein (HDL) and to compare these mechanisms with those described previously for the oxidation of low-density lipoprotein (LDL) under the same experimental conditions (Bonnefont-Rousselot et al., Radiat, Res. 134, 271-282, 1993). The oxidation of HDL, initiated by .OH or .OH/O(.-)2 free radicals from gamma radiolysis of water, was evaluated as a function of increasing radiation dose by analyzing quantitatively the decrease of endogenous alpha-tocopherol and the formation of oxidation products (thiobarbituric acid-reactive substances and conjugated dienes). All qualitative conclusions were supported by quantitative data (radiation yields and concentrations of the oxidation markers at high radiation doses) and by the mechanisms of the kinetics, .OH free radicals in the absence of oxygen were less efficient in initiating HDL oxidation than in the presence of oxygen (action of .OH/O(.-)2 free radicals), which was in agreement with the enhancement of the action of .OH free radicals by oxygen. The remaining significant level of vitamin E in HDLs at high radiation doses in the absence of oxygen could be explained by a regeneration of vitamin E by an oxidation product that was able to reduce the alpha-tocopheroxyl radical. The yields related to the decrease in the vitamin E content of HDLs after exposure to radiation with .OH or .OH/O(.-)2 free radicals were slightly higher than those obtained previously in LDLs under similar experimental conditions. Moreover, in the presence of oxygen, .OH free radicals led to a lower formation of thiobarbituric acid-reactive substances in HDLs than in LDLs. Such discrepancies in the behavior of these two lipoprotein fractions could be related to the differences in the chemical composition of HDLs and LDLs.

The aim of this study was to analyze quantitatively the oxidative modification of low-density lipoproteins (LDLs) induced by OH· free radicals produced by gamma radiolysis, in the absence or in the presence of oxygen (action of OH· free radicals, or simultaneous action of OH· and ${\rm O}_{2}^{\overline{\cdot}}$ free radicals, respectively). The effects of increasing radiation doses on aqueous LDL solutions have been monitored by several parameters: a decrease in endogenous vitamin E, the formation of thiobarbituric acidreactive substances (TBARS) and conjugated dienes, the appearance of a differential fluorescence (excitation wavelength = 360 nm), and an increase in the relative electrophoretic mobility. Initial radiation yields (decrease in vitamin E, formation of TBARS) have been determined at pH 7 as a function of LDL concentration (from 0.75 to 9 g liter-1). From the comparison of these yields with those of OH· and ${\rm O}_{2}^{\overline{\cdot}}$ free radicals produced by water radiolysis, we have d...

This study aimed to analyze quantitatively the initiation and the consequences of low-density lipoprotein (LDL) peroxidation by O2·−/HO2· free radicals produced by gamma radiolysis. The action of i...