A thromboxane effect of a hydroxytyrosol-rich olive oil wastewater extract in patients with uncomplicated type I diabetes.

OBJECTIVE: To assess the antioxidant/non-antioxidant effects of a hydroxytyrosol (HT)-rich phenolic extract from olive mill wastewaters administered with a breakfast. DESIGN, SETTING AND SUBJECTS: Five type I diabetic patients received 25 mg of HT the first day and 12.5 mg/day the following 3 days. Blood sampling was carried out at T(0) (baseline) and T(4d) just before the breakfast + HT administration and at time points 1, 2, 3 and 4 h after T(0). Urines (24-h) were collected from T(0) to T(4d). Baseline HbA1c was generally inferior to 10%, glycemia was within the range 6-24 mmol/l, whereas total cholesterol, HDL-chol and triglycerides were normal. RESULTS: The major finding was the 46% decrease in the serum TXB(2) production after blood clotting at T(4d). Plasma vitamin A, E, beta-carotene were not changed. Vitamin C tended to increase (P = 0.075). Plasma antioxidant capacity was enhanced at T(0)+1 h only, whereas its main determinants (albumin, bilirubin, uric acid) were not modified. Urinary 8-isoPGF(2alpha) levels were highly variable and were not affected significantly by HT administration. CONCLUSION: The major effect of HT accounts for an antiaggregating platelet action, leading to a possible prevention of thrombotic and microthrombotic processes.

Determination of beta2-microglobulin in biological samples using an immunoenzymometric assay (chemiluminescence detection) or an immunoturbidimetric assay: comparison with a radioimmunoassay.

Monitoring beta2-microglobulin (beta2M) in biological fluids has gained considerable interest in pathologies such as haematologic malignancies, renal diseases, and chronic inflammatory diseases. Due to limitations of the RIA in the routine laboratory, we measure beta2M with non-isotopic methods. 189 patients suffering from myeloma (n=66), end stage renal failure (n=54) or inflammation (n=69) were included in this study. beta2M was determined in serum, urine and dialysate using an immunoenzymometric assay with chemiluminescence detection [Immulite Diagnostic Products Corporation (DPC), La Garenne Colombes, France] and an immunoturbidimetric assay (Olympus, Rungis, France). The data were compared with a radioimmunoassay (Immunotech, Marseille, France) taken as a reference. Using serum samples, the immunoenzymometric assay with chemiluminescence detection and the immunoturbidimetric assay have reliable analytical performances. Values obtained with serum samples are highly correlated with the radioimmunoassay (DPC/RIA r2=0.84; Olympus/RIA r2=0.94) whatever the type of pathology; however an over-estimation which could be related to cross reactivity with beta2M fragments was observed with the RIA method as suggested by crossover calibration and recovery studies. Values obtained with urinary samples (n=96) are closely related to those obtained with the RIA (DPC/RIA r2 = 0.98; Olympus/RIA: r2=0.99). Despite the low levels observed in dialysate (n=57) good correlations were observed between Olympus vs DPC (r2=0.85). By contrast, the two non-isotope methods are poorly related with the RIA method (DPC vs RIA r2=0.47 and Olympus vs RIA r2=0.54). In conclusion, the immunoenzymometric assay with chemiluminescence detection or the immunoturbidimetric assay could be used in the routine laboratory in order to determine beta2M in plasma, urine and dialysate.

Mechanism of in vitro heme-induced LDL oxidation: effects of antioxidants.

BACKGROUND: Heme protein toxicity, owing to generation of reactive oxygen species most likely by direct interaction between heme iron and hydrogen peroxide (H2O2), may be involved in various pathologies, including atherogenesis and pigmentary acute renal failure. The aim of this study was to investigate the mechanism of heme cytotoxicity and the effects of antioxidant therapies in an in vitro model of heme-induced low-density lipoprotein (LDL) oxidation. MATERIALS AND METHODS: Human LDLs were exposed to heme, iron (Fe), protoporphyrin (PPIX) and PPIX-Zinc (Zn) with or without H2O2. Lipid peroxidation was monitored by measurement of conjugated diene formation (at the 234-nm absorbance). The effect of various antioxidants, such as vitamin E and vitamin C, reduced glutathione (GSH), and oxidized glutathione (GSSG), mannitol and desferoxamine (DFO) was further investigated in the established in vitro model of LDL oxidation. RESULTS: Incubation of LDLs in the presence of heme/H2O2 induced lipid peroxidation with the optimal oxidation rate being at 5 microm heme/100 microm H2O2 doses. By contrast, incubation of LDL with H2O2, Fe, Fe/H2O2, PPIX, PPIX/H2O2, heme or PPIX-Zn did not initiate any LDL oxidation. In vitro, the vitamin E load protected native LDLs against heme/H2O2 oxidative modifications. Incubation of LDLs with increasing doses of vitamin C, GSH and DFO conferred a dose-dependent protection, while mannitol and GSSG had no effect. CONCLUSIONS: Initiation and propagation of heme-induced lipid peroxidation is not mediated by a Fenton reaction but depends on specific interactions between heme and H2O2. It may result from the generation of ferryl and perferryl radicals derived from hemic Fe and H2O2 interactions. A protective effect of vitamins E, C, GSH and DFO was demonstrated in this model.

Comparison between the enzymatic vitros assay for creatinine determination and three other methods adapted on the Olympus analyzer.

To evaluate the relationship between the enzymatic Vitros assay for creatinine determination and other methods, we determined creatinine concentration in 400 heparin samples. Plasma creatinine level was successively determined on the Vitros 750 analyzer (Johnson & Johnson Co., Rochester, NY) and on the Olympus AU2700 analyzer (Olympus France, Rungis, France), using three reagents in the same assay: Olympus-Jaffé and two enzymatic commercial kits-Crea Plus (Roche Diagnostics, Meylan, France) and Enzymatic Creatinine (Randox, Mauguio, France). Comparison of Jaffé and enzymatic measurements of plasma creatinine levels revealed a high correlation when considering all values ranged from 20-1000 micromol/l (r > 0.99). However, for values <60 micromol/l, enzymatic reagents provided best results. Enzymatic methodology is a better clinical choice for the accurate measurement of creatinine, especially for neonates, pediatrics, and hematology units. Because analytical performance and the costs of Randox creatinine were satisfactory for our laboratory, this method, adapted on the Olympus analyzer, was chosen for routine determination of creatinine levels. According to the Valtec protocol (8), no interferences such as hemolysis, lipemia, or bilirubin were detected for Enzymatic Creatinine Randox.

Exchanging carbohydrates for monounsaturated fats in energy-restricted diets: effects on metabolic profile and other cardiovascular risk factors.

OBJECTIVE: To investigate whether improvements in cardiovascular risk factors, as observed in energy-balance conditions after exchanging carbohydrates (CHO) for monounsaturated (MUFA) fats, are also observed in energy-restricted conditions. DESIGN: Longitudinal, clinical intervention study using two types of energy-restricted diets (-30% of initial energy intake) with similar levels of saturated and polyunsaturated fats: a high CHO diet (55% of energy from CHOs, 10% from MUFAs) and a high MUFA diet (40% of energy from CHOs, 25% from MUFAs). SUBJECTS: A total of 32 overweight subjects (nine males, 23 females, BMI: 26-45 kg/m(2)). MEASUREMENTS: Body weight, serum lipids, fasting plasma insulin and phospholipid fatty acid composition of red blood cells were measured at baseline and after 8 weeks. Various oxidative status parameters (plasma lipid hydroperoxides, total plasma antioxidant capacity, plasma uric acid and vitamin E) and serum-induced smooth muscular cell (SMC) proliferation were also measured at these time points. RESULTS: Weight loss (1.1 kg/week over the first 4 weeks and 6.7 kg at week 8) was not significantly affected by the diet composition. Both diets reduced significantly total serum cholesterol, but the MUFA-rich diet showed better effects on fasting serum triacylglycerol (TG) than the CHO-rich diet: 1.18 vs 1.51 mmol/l for the MUFA-rich diet (after vs before, P<0.05) and 1.42 vs 1.62 for the CHO-rich diet. After 8 weeks, plasma vitamin E concentrations were positively associated with the oleic acid level of red blood cell phospholipids and showed opposite variations in both diets (increase with the MUFA-rich diet and decrease with the CHO-rich diet). Relative changes in SMC proliferation induced by sera were negatively associated with the ratio oleic:linoleic acid of red blood cell phospholipids and were significantly higher with the CHO-rich diet. CONCLUSIONS: The MUFA-rich diet showed better effects on serum TG than the CHO-rich diet, even with energy restriction and weight loss. The results suggest also a protective effect of oleic acid on oxidative stress and SMC proliferation, two other important cardiovascular risk factors.

Preterm infant formula supplementation with alpha linolenic acid and docosahexaenoic acid.

OBJECTIVES: To investigate if supplementation of preterm infant formula with a high docosahexaenoic acid/eicosapentaenoic acid (DHA/EPA) ratio together with alpha-linolenic acid (ALA) was able to maintain plasma and red blood cell DHA levels similar to that obtained with breast milk feeding without altering n-6 fatty acid status. DESIGN AND SUBJECTS: Preterm infants of mothers who elected not to breast feed (n=13) were assigned to ALA- and DHA-enriched formula (DHA group: DHA/EPA=5/l). Infants fed breast milk (n=25) constituted a reference group (BM group). Anthropometric and fatty acid parameters (plasma phospholipids, cholesterol esters, triglycerides and red blood cell phosphatidylethanolamine, PL, CE, TG, RBC-PE, respectively) were obtained after 2 days (D2) and 15 days (D15) of enteral feeding and at the 37th week (W37) of post-conception age and 1 month later (W37+30) in the DHA group. Mean DHA intake ranged between 16.5+/-1.6 and 17.9+/-2.9 mg/kg/day between D2 and W37+30. RESULTS: At W37, infant weights, heights, and head circumferences were similar in DHA and BM groups. PL DHA was maintained in the DHA group at the same level as in the BM group and the same for DHA in PE at W37. In RBC-PE and at W37, AA status was the same in both groups. In PL, AA levels remained very stable throughout the study; however, in the DHA group AA levels in PL remained in the range observed with standard formulas. CONCLUSION: The combined 18:3 n-3 and DHA supplementation of infant formula with DHA/EPA ratio 5/l is compatible with growth and n-3 fatty acid metabolism similar to that of preterm infants fed human milk.

Wine phenolic antioxidants inhibit AP-1 transcriptional activity.

Some of the beneficial effects of moderate wine consumption may be related to the antioxidant properties of polyphenolic compounds containing tannins, flavonoids, and phenolic acids. Cellular actions have recently been reported and may involve the modulation of transcriptional factors such as AP-1 (activator protein-1), which controls the expression of various genes implicated in inflammation processes, cell differentiation, and proliferation. The aim of this study was to evaluate the modulation of AP-1 activity by the phenolic acids (gallic, caffeic, protocatechic, paracoumaric, sinapic, and ferulic acids) that are present in wine and to compare their modulating pathways to those of lipophilic or hydrophilic "chain-breaking" antioxidants (such as DL-alpha-tocopherol or trolox) vitamin C, nitric oxide, and reduced glutathione. AP-1 response was studied on a cell line (MTLN) derived from MCF-7 cells transfected with luciferase gene under TRE sequence control. After stimulation by phorbol 12-myristate 13-acetate (PMA; 100 nM, 6 h, 10(-7) M), luciferase activity was determined by a luminescence method in the presence of luciferine/coenzyme A solution using a luminometer (LKB 1251, Finland). Antioxidants to be tested were incubated with cells in the presence or absence of PMA. Stimulation with PMA resulted in an AP-1-mediated increase in luciferase gene expression corresponding to an 8-fold increase in luciferase activity. After stimulation by PMA, a dose-dependent inhibition of AP-1 was observed with the six phenolic acids in the 20 nM-20 microM concentration range: gallic acid > caffeic > protocatechic, paracoumaric, sinapic acids > ferulic acid. Inhibition was more pronounced with phenolic acids than with DL-alpha-tocopherol (IC(50) = 5 +/- 4.5 microM for gallic acid vs 85 +/- 11 microM for vitamin E). None of the hydrophilic antioxidants inhibited PMA-induced AP-1 activation. None of the antioxidants tested in the absence of PMA stimulation induced any activation or inhibition of AP-1. Our results suggest that phenolic acids may act directly on cell signaling via inhibition of AP-1 transcriptional activity. In addition to preventing LDL oxidation in the arterial wall, our observations indicate that phenolic acids have a cell-mediated capacity to prevent some of the processes involved in atherosclerosis in a plasma concentration range compatible with nutritional intakes.

Discrepancies between apolipoprotein E phenotyping and genotyping in the elderly.

We estimated the frequencies of phenotype (isoelectric focusing; IEF) vs. genotype (PCR/Hhal) discordance in a sample of an aged population (> 65 years). Both phenotype and genotype techniques have been used in the study of apolipoprotein E (apoE) polymorphism in 125 elderly subjects. The discordance between phenotype and genotype was unresolved in 11 (8.8%) of the 125 unrelated subjects studied. We observed a significant association between the presence of the E4 allele and both Alzheimer's disease (chi2 = 13, p < 0.001) and increased cholesterol concentration (Mann Whitney, p < 0.03). These relationships were not affected by the techniques used. Our results indicate that transcriptional modulation and post-transductional modifications in normal ageing and in aged-related diseases may explain in part discrepancies between gene analysis and protein characterisation.

The relationship between apolipoprotein E4 and lipid metabolism is impaired in Alzheimer's disease.

BACKGROUND: Human apolipoprotein (Apo) E4 (ApoE4) is an important determinant of lipid metabolism and cell-to-cell cholesterol transport. It is also a major genetic risk factor for both vascular disease and familial and sporadic late-onset Alzheimer's disease (AD). Since vascular pathology could dramatically reduce neuronal reserve capacity, a biological chain of events between ApoE4, hypercholesterolemia and AD has been postulated. The aim of the present study is to explore the relationship between lipid metabolism and ApoE isoforms in a large series of elderly subjects in relation to the presence or absence of AD. METHODS: Of 332 referrals to a neurology clinic specializing in memory disorders, 146 were given a diagnosis of AD (age, mean +/- SD 76 +/- 13.1 years, 64.4% women) according to DSM-IIIR criteria. One hundred and seventy-six subjects were included as controls (age 80 +/- 5.6 years, 58% women). The ApoE phenotype was determined by the isoelectrofocalization method, cholesterol, triglycerides, phospholipids, ApoA and ApoB were determined by routine chemistry. FINDINGS: A significant association was observed between the E4 allele and AD (chi2 = 13, p < 0.001) only below age 80. In the control group, cholesterol levels were found to be significantly higher in men but not in women with an E4 allele (6.35 +/- 1.3 mmol/l) as opposed to those without (5.8 +/- 1.2 mmol/l). ApoB levels were also found to be higher in the presence of ApoE4, with no gender effect. Within the AD group no significant relationship was found between ApoE4 and cholesterol levels (mean 6.05 +/- 0.9 mmol/l in E4-AD subjects versus 5.8 +/- 1.21 mmol/l in non-E4-AD subjects). Similar observations were made in relation to triglycerides and phospholipids. INTERPRETATION: Our results demonstrate the disappearance of the ApoE4-raising effect on serum cholesterol, triglyceride and phospholipid levels in AD suggesting a more complex relationship between AD and lipid metabolism than has previously been supposed. This lipid abnormality may further contribute to the progression of AD.

Specific antioxidant activity of caffeoyl derivatives and other natural phenolic compounds: LDL protection against oxidation and decrease in the proinflammatory lysophosphatidylcholine production.

Specific antioxidant activity (SAA) (i.e., activity related to the molar or gallic acid equivalent amount of antioxidant) of natural polyphenolic mixtures or pure phenolic compounds was studied using their capacity to delay the conjugated diene production brought about by in vitro LDL copper-mediated or AAPH-mediated oxidation. The cinnamic acid series (caffeic, sinapic, ferulic acids) displayed a constant SAA over a large range of concentrations, whereas the benzoic acid series (gallic and protocatechuic acids) showed much higher SAA at low concentrations. The natural phenolic mixtures had a constant SAA. The highest SAA was obtained with caffeoyl esters (caffeoylquinic, rosmarinic, and caffeoyltartaric acids) and catechin for the copper-oxidation and the AAPH-oxidation system, respectively. Phenolic mixtures and acids delayed vitamin E depletion and decreased proinflammatory lysophosphatidylcholine production. As with polyphenols, probucol delayed lysophosphatidylcholine and conjugated dienes production, at higher concentrations, but was not effective at preventing vitamin E depletion. Polyphenols prevent the oxidation of LDL and its constituents (vitamin E, phosphatidylcholine), which is compatible with an antiinflammatory and antiatherosclerotic role in pathophysiological conditions.

[Oxygen and the superoxide anion. Modulation of NADPH oxidase?]. / De l'oxygène à l'anion superoxyde. Peut-on moduler la NADPH oxydase?

Oxidative stress which results from an imbalance between oxidant production and antioxidant defense mechanisms can promote modifications of lipids, proteins and nucleic acids. This review focuses on the different pathways leading to Reactive Oxygen Species (ROS) production in particular on NADPH oxidase activation. This enzyme is localized in numerous cells including phagocytes and vascular cells and composed of membrane and cytosolic sub-units. The activation of the NADPH oxidase is largely involved in inflammation associated diseases such as asthma, Systemic Inflammatory Response Syndrome and aging associated diseases such as atherosclerosis and neurodeneratives diseases. The modulation of NADPH oxidase could be a way to limit or prevent the development of these diseases.

Decreased superoxide anion production in cultured human promonocyte cells (THP-1) due to polyphenol mixtures from olive oil processing wastewaters.

The purpose of this study was to examine whether human monocytic line THP-1 after differentiation into adherent macrophages, taken as a model of human macrophages implicated in atheroma, is able to produce lower quantities of O(2)(*)(-) either in the presence of polyphenol-rich olive oil wastewater (OWW) fractions or after OWW preincubation and withdrawal from the medium. In these respective conditions, the purpose was to examine the scavenging activity and the cell action of OWW toward O(2)(*)(-) production. It was clearly seen that OWW fractions lowered the O(2)(*)(-) production in both conditions, leading to the conclusion that they were able to scavenge O(2)(*)(-) and to depress O(2)(*)(-) production in the cell. Given the role of O(2)(*)(-) in LDL oxidation and oxidized LDL in atheroma, these results support an antiatherogenic role of OWW and its potential utilization as a food complement.

Serum fatty acid imbalance in bone loss: example with periodontal disease.

Among the numerous factors of bone remodelling, the local action of arachidonic acid metabolites together with cytokines, is particularly important, especially that of prostaglandin PGE2. It has been suggested that the alveolar bone destruction in periodontal disease and osteoporosis can be treated by reducing the ratio of arachidonic acid in phospholipids, which would diminish prostaglandin production. The aim of this study was to evaluate the main serum polyunsaturated fatty acids and a possible alteration in the level of arachidonic acid in patients suffering from periodontal bone loss. Of the 105 patients who participated the study, 78 were suffering from periodontal bone loss and 27 served as a control group. The fatty acids were measured in serum by gas-chromatography. The results showed that the level of fatty acids of the n-6 pathway was higher in our patients with bone loss than in the control group, whereas the reverse was observed with fatty acids of the n-3 pathway. In conclusion, our patients' bone losses are linked with an imbalance between n-6 and n-3 fatty acids, which seems to justify a diet increase in 20- and 22-carbon fatty acids.

Protective effects of high-density lipoprotein against oxidative stress are impaired in haemodialysis patients.

INTRODUCTION: Cardiovascular diseases represent the major cause of mortality in haemodialysis (HD) patients. Oxidized low-density lipoprotein (Ox-LDL) is a major cardiovascular risk factor, implicated in atherosclerotic plaque formation. It has been suggested that high-density lipoprotein (HD) has the capacity to reduce the oxidative modifications of LDL. The aim of this study is to analyse the protective effects of HDL in HD patients. METHODS: In vitro copper-induced LDL oxidation was evaluated in 12 patients with chronic renal failure (mean age 61.0+/-12.8 years) and compared to 25 healthy subjects (mean age 57.3+/-19.2 years). LDL were incubated in oxygen-saturated PBS, LDL oxidation was initiated by Cu (II) in the presence and absence of HDL and assessed by measuring the absorbance (abs) increase at 234 nm due to conjugated diene formation. Duration of lag time, maximum velocity (V(max.)) of lipid peroxidation, oxidation slope and half-time of maximum diene formation (T (1/2)) were obtained by kinetic modelling analysis. RESULTS: HDL (1.06+/-0.31 vs 1.23+/-0.39 mmol/l) and Apo AI (1. 17+/-0.39 vs 1.49+/-0.20 g/l) levels were decreased in HD patients. In the absence of HDL, LDL obtained from HD patients showed an enhanced susceptibility to oxidation in vitro as demonstrated by the significant decrease in lag time (54.5+/-22.2 vs 79.4+/-37.8 min) and a significant increase in V(max.) (0.026+/-0.006 vs 0.017+/-0. 005 abs/min). In all cases, HDL (from 0.1 to 2 microM) prevented LDL oxidation in vitro; however, this effect was significantly reduced in HD patients: increase in lag time 54.2% vs 150.4% in HD vs controls; increase in T (1/2) 52.2% vs 124.6% in HD vs controls; decrease in V(max). 13.5% vs 38.5% in HD vs controls. CONCLUSIONS: These results suggest that qualitative abnormalities such as an impairment of HDL-associated enzymes are associated with a decrease of HDL levels during HD. Hence, in addition to the known impairment of reverse cholesterol transport, the reduction of HDL protective capacity against oxidative stress could be involved in the development of HD-induced atherosclerosis.

Alpha linolenic acid in cholesterol esters: a marker of alphalinolenic acid intake in newborns.

OBJECTIVE: To evaluate alpha-linolenic acid (ALA) (18∶3 n-3) and linolenic acid (LA) (18∶2 n-6) in cholesterol esters (CE) as markers of ALA and LA dietary intakes in preterm infants. SUBJECTS: Forty-five preterm infants: two groups fed different formulas, the third fed human milk. DESIGN: ALA and LA dietary intakes were precisely recorded in each infant to accurately determine the cumulative amount of ingested ALA and LA during two intervals: (i) between the second day after the first significant formula intake (D0) and the fifteenth day (D15); and (ii) between D0 and the first day of the 37th week of post-conception age (W37). The corresponding amounts of ingested ALA and LA were related to ALA and LA levels determined by capillary column gas-liquid chromatography in plasma cholesterol esters at D15 and W37, respectively. RESULTS: ALA in CE was very significantly correlated to D0-D15 and D0-W37 ALA intakes (0.66; P=0.0001 and 0.70; P=0.0001), respectively. LA in CE was weakly correlated to D0-D15 LA intakes (0.03; P=0.01) and whatever the group (human milk or enriched formula) the correlation was lost at W37. CONCLUSION: In preterm infants, ALA in CE can be considered as representative of ALA dietary intakes, whereas LA in CE appears as a poor marker of LA intakes.

Effects of dietary crude palm oil, fish oil and their association on cholesterol and lipoprotein constants in rats which could be beneficial in humans.

The aim was first to examine the differential effects of crude and refined palm oil (CPO and RPO) on the lipid and lipoprotein constants of plasma in rats and to compare the effect of crude palm oil to that of fish oil. Secondarily, it was to know whether one can take advantage from the association of CPO with FO. Twenty-four-day-old weaning rats were divided into five experimental groups, each receiving a purified diet containing 10% oil as either a single oil or an equal amount of two oils. After a feeding period of 36 days, the main results were as follows. As compared to the rats fed the RPO diet, those fed the CPO diet had lower total cholesterol, LDL-C, VLDL-C and apoB and higher HDL-C/LDL-C and apoA1/apoB ratios. Those fed the FO diet had only lower VLDL-C and triglycerides and higher HDL-C and HDL-C/LDL-C ratio. Whereas FO associated with RPO in the same diet had the same effect as FO alone, FO associated with CPO tends to reinforce the effect of CPO. This is particularly true for the effects on apoB and apoA1 which were found to be synergistically depressed and enhanced, respectively. Given the role played by these biological constants as predictors of CVD in humans, and in spite of the fact that these predictors are not relevant in rats, these results would suggest the potential interest of CPO or the association of CPO with FO in human nutrition.