Assay of hydroxyl radicals generated by focused ultrasound.

Water sonolysis leads to the formation of hydroxyl radicals (OH*). Various techniques are used to detect the OH* production and thus to assess the level of ultrasound-mediated cavitation generated in vitro. In this study, we used terephthalic acid (TA) as an OH* trap. This method is based on the fluorescent properties of hydroxyterephthalic acid (HTA) formed by the reaction of TA with OH* and used as an indicator of the degree of inertial cavitation caused. The experimental system is comprised mainly of a focused piezoelectric ultrasound transmitter and a measurement cell containing 1X PBS/TA diluted solution. In the first part, we aimed to characterize the most appropriate experimental conditions (TA dosimeter solution, irradiation time) in order to optimize the resulting HTA fluorescence values. Then, we could determine that the HTA production increased with the level of the cavitation phenomenon caused by the acoustic power from which OH* production may be estimated.

[Fatty acids regulate NOX activity]. / Régulation de l'activité des NADPH oxydases en présence d'acides gras.

NADPH oxidase (NOX) is a multimeric enzyme including a catalytic unit, gp91(phox), and several regulating subunits: p22(phox), p40(phox), p47(phox), p67(phox). This enzyme, also known as flavocytochrome b(588), is responsible for a deliberate production of superoxyde anion (O2*-). This enzyme, initially described in polynuclear neutrophils (NOX 2), belongs to a complex family of multimeric isoenzymes whose members are present in many cell types. NOXs are generally associated to cell signaling and they seem involved in physiological phenomena (vascular reactivity, proliferation and cellular migration...) as well as in many diseases. Lipids in general and poly unsaturated fatty acids (PUFA) in particular are able to modulate the activity of NOX in many models. With our fibroblastic model, we show that only arachidonic acid (AA) is able to activate the enzyme directly whereas many PUFA are able to induce a production of reactive oxygen species (ERO). Moreover the decrease of ERO production and NOX activity in fibroblasts triggered by PUFA does not depend on SOD activity but the time course of this decrease is associated with the expression of heme oxygenase 1 (HO-1). Besides a regulation by protein subunits, we propose, according to this model, a loop of regulation of NOX activity including a stimulation by lipids associated with an inhibition by HO-1. Thus, lipids, by interaction with phospholipase A2, release arachidonic acid which stimulates NOX, amplifying superoxyde anion production. This oxygen species may induce redox-sensitive gene transcription such as HO-1. Consequently this enzyme inhibits NOX activity and limits superoxyde anion production by heme degradation and CO production.

Plasma fatty acids and lipid hydroperoxides increase after antibiotic therapy in cystic fibrosis.

The present authors investigated whether cystic fibrosis is linked to a defect in fatty acids and assessed the impact of the main patients' characteristics on the levels of several fatty acids, mostly during respiratory exacerbation and after antibiotic therapy. Fatty acid phospholipid and cholesteryl ester levels were measured in stable-state patients and controls. No differences were found concerning either the fractions of palmitic and oleic acids or the cholesteryl esters of alpha-linolenic and arachidonic acids. However, phospholipids of alpha-linolenic and arachidonic acids, as well as cholesteryl esters and phospholipids of stearic and linoleic acids, were lower in patients than in controls, but fractions of dihomo-gamma-linolenic, docosatetraenoic, docosapentaenoic, palmitoleic and eicosatrienoic acids were higher. Fatty acid levels, oxidative stress markers, nutrients, body mass index and forced expiratory volume in one second (FEV(1)) were measured in patients before and after antibiotic courses for bronchial exacerbation. After adjustments, palmitic, stearic, alpha-linolenic, linoleic, arachidonic, palmitoleic and oleic acids generally decreased during exacerbation but almost all increased after antibiotic courses. Nearly all fractions increased along with FEV(1) and a positive relationship linked fatty acids to lipid hydroperoxides. There was no general drop in fatty acids. Patients' fatty acid profiles depended on the pulmonary function and the inflammation state.

Validity and reliability of a new food frequency questionnaire compared to 24 h recalls and biochemical measurements: pilot phase of Golestan cohort study of esophageal cancer.

BACKGROUND: A pilot study was carried out to evaluate validity and reproducibility of a food frequency questionnaire (FFQ), which was designed to be used in a prospective cohort study in a population at high risk for esophageal cancer in northern Iran. METHODS: The FFQ was administered four times to 131 subjects, aged 35-65 years, of both sexes. Twelve 24-h dietary recalls for two consecutive days were administered monthly during 1 year and used as a reference method. The excretion of nitrogen was measured on four 24-h urine samples, and plasma levels of beta-carotene, retinol, vitamin C and alpha-tocopherol was measured from two time points. Relative validity of FFQ and 24-h diet recall was assessed by comparing nutrient intake derived from both methods with the urinary nitrogen and plasma levels of beta-carotene, retinol, vitamin C and alpha-tocopherol. RESULTS: Correlation coefficients comparing energy and nutrients intake based on the mean of the four FFQ and the mean of twelve 24-h diet recalls were 0.75 for total energy, 0.75 for carbohydrates, 0.76 for proteins and 0.65 for fat. Correlation coefficients between the FFQ-based intake and serum levels of beta-carotene, retinol, vitamin C and vitamin E/alpha-tocopherol were 0.37, 0.32, 0.35 and 0.06, respectively. Correlation coefficients between urinary nitrogen and FFQ-based protein intake ranged from 0.23 to 0.35. Intraclass correlation coefficients used to measure reproducibility of FFQ ranged from 0.66 to 0.89. CONCLUSION: We found that the FFQ provides valid and reliable measurements of habitual intake for energy and most of the nutrients studied.

Plasma carotenoids as biomarkers of intake of fruits and vegetables: ecological-level correlations in the European Prospective Investigation into Cancer and Nutrition (EPIC).

OBJECTIVE: The aim of this study was to assess the ability of a single 24-h dietary recall (24HDR) and food questionnaires (FQ) to predict plasma carotenoid levels at the ecological level by assessing the relationship between mean plasma carotenoid levels and mean intake of fruit and vegetables measured by 24HDR and FQ across 16 European regions. DESIGN: A random subsample of 3089 subjects was included, stratified by age and gender. They provided blood samples and dietary information between 1992 and 2000 as part of the European Prospective Investigation into Cancer and Nutrition. RESULTS: Using Spearman's correlation coefficients, the correlations between mean regional 24HDR fruit and vegetable variables and corresponding mean plasma carotenoid levels were generally higher than the correlations using FQ means. The highest correlation was between the 24HDR citrus fruit variable and beta-cryptoxanthin (r = 0.90). For 24HDR, total fruits and vegetables were highly correlated with lutein, zeaxanthin, and beta-cryptoxanthin (r = 0.83-0.87), while vegetables were more closely related with lutein (r = 0.69) and zeaxanthin (r = 0.68), and fruits correlated with zeaxanthin (r = 0.87) and beta-cryptoxanthin (r = 0.84). Root vegetables (r = 0.81) and total carrots (r = 0.71) were well correlated with alpha-carotene. In the multivariate models adjusting for age, body mass index, and season, and using observations of means stratified by sex and region, the association was generally higher for 24HDR compared to FQ. CONCLUSION: Mean regional intakes of fruits and vegetables in several European countries were closely correlated with corresponding mean plasma levels of individual carotenoids. Fruits and vegetables measured by 24HDR were generally better able to predict plasma carotenoids at the ecological level.

Plasma carotenoids as biomarkers of intake of fruits and vegetables: individual-level correlations in the European Prospective Investigation into Cancer and Nutrition (EPIC).

OBJECTIVE: The aim in this study was to assess the association between individual plasma carotenoid levels (alpha-carotene, beta-carotene, lycopene, beta-cryptoxanthin, lutein, zeaxanthin) and fruit and vegetable intakes recorded by a calibrated food questionnaire (FQ) and 24-h dietary recall records (24HDR) in nine different European countries with diverse populations and widely varying intakes of plant foods. DESIGN: A stratified random subsample of 3089 men and women from nine countries participating in the European Prospective Investigation into Cancer and Nutrition (EPIC), who had provided blood samples and dietary and other lifestyle information between 1992 and 2000, were included. RESULTS: beta-Cryptoxanthin was most strongly correlated with total fruits (FQ r = 0.52, 24HDR r = 0.39), lycopene with tomato and tomato products (FQ r = 0.38, 24HDR r = 0.25), and alpha-carotene with intake of root vegetables (r = 0.39) and of total carrots (r = 0.38) for FQ only. Based on diet measured by FQ and adjusting for possible confounding by body mass index (BMI), age, gender, smoking status, alcohol intake, and energy intake, the strongest predictors of individual plasma carotenoid levels were fruits (R(partial)(2) = 17.2%) for beta-cryptoxanthin, total carrots ((partial)(2) = 13.4%) and root vegetables (R(partial)(2) = 13.3%) for alpha-carotene, and tomato products (R(partial)(2) = 13.8%) for lycopene. For 24HDR, the highest R(partial)(2) was for fruits in relation to beta-cryptoxanthin (7.9%). CONCLUSIONS: Intakes of specific fruits and vegetables as measured by food questionnaires are good predictors of certain individual plasma carotenoid levels in our multicentre European study. At individual subject levels, FQ measurements of fruits, root vegetables and carrots, and tomato products are, respectively, good predictors of beta-cryptoxanthin, alpha-carotene, and lycopene in plasma.

[Redox status in HIV+ patients under HAART]. / Statut oxydatif de patients VIH positifs sous trithérapie.

Oxidative stress decreases immune defences and is also suggested to participate in the activation of HIV virus replication. That is why we decided to explore some biomarkers of oxidative stress (reduced glutathione, lipoperoxides, true malondialdehyde and vitamin C) in 20 HIV positive patients whose HIV replication was determined by measurement of RNA viral load. Reduced glutathione is decreased in HIV positive patients, without correlation with the viral load. The patients mean content of lipoperoxides is twice that of controls but with such a large range that there is no statistical difference.

[Plasma ascorbic acid: preanalytical factors and new HPLC method]. / Acide ascorbique plasmatique: facteurs préanalytiques et nouvelle méthode de mesure.

We describe a fast ED-HPLC method for ascorbic acid analysis, carried out on a new mixed mode chromatographic column. Due to its functional dodecylsilane group associating a quaternary ammonium, this column is simpler and easier to handle than previous ones because without any counter-ion in the mobile phase. As ascorbic acid and glutathione are very often measured on the same blood sample, it was efficient to validate a common pretreatment solution for ascorbic acid in plasma and glutathione in whole blood. This pretreatment is based on the use of a sulfosalycilic acid, N-ethylmaleimide, EDTA mix recently described. Plasma samples, stored after deproteinisation and without centrifugation, are stable for three weeks at -20 or -80 degrees C.

Lipid peroxidation assessment by malondialdehyde measurement in parenteral nutrition solutions for newborn infants: a pilot study.

AIM: To determine malondialdehyde (MDA) concentrations in parenteral nutrition admixtures exposed to ambient room light, and in the serum of neonates. METHODS: Using a new method to measure MDA specifically, this study analysed MDA of lipid-containing all-in-one admixtures provided by the pharmacy, with a composition identical to that used in routine clinical conditions. First, 12 admixtures were exposed to ambient light for 24 h, in the neonatal intensive care unit. Secondly, 18 solutions were either exposed to (n = 9) or protected from ambient light (n = 9) during the same period. Samples of admixtures were collected at baseline and 24 h later, for MDA measurement. Serum MDA was also randomly measured in orally fed healthy neonates. RESULTS: After 24 h exposure to ambient room light, MDA concentrations in parenteral nutrition admixtures increased from 179 (129, 348) nmol l(-1) to 5800 (1632, 14679) nmol l(-1) (p = 0.0002) [50th (10th, 90th) centiles]. When admixtures were protected from light, the increase in MDA was significantly lower than without protection: 187 (60, 429)nmol l(-1) versus 13 696 (3472, 26 049)nmol l(-1) (p = 0.0003). In 54 infants with a gestational age of 33 (28, 39) wk and a birthweight of 1750 (960, 3388) g, plasma MDA concentrations were 173 (98, 315) nmol l(-1). CONCLUSION: In solutions protected from light, MDA concentrations were low and were close to the serum MDA concentrations observed in orally fed neonates. Administration of all-in-one admixtures containing lipids in ambient lighting results in intravenous infusion of high levels of MDA which may present an additional source of morbidity in immature infants. This study confirms the need to protect parenteral admixtures from light.

Synthesis of 9,9,9-trideutero-1,4-dihydroxynonane mercapturic acid (d3-DHN-MA), a useful internal standard for DHN-MA urinalysis.

Racemic 1,4-dihydroxynonane mercapturic acid (DHN-MA) and 9,9,9-trideutero-1,4-dihydroxynonane mercapturic acid (d3-DHN-MA) are synthesized on a 400-mg scale (overall yield approximately 40%) by a two-step sequence involving Michael addition of N-acetyl-L-cysteine to methyl 4-hydroxynon-2(E)-enoate or methyl 9,9,9-trideutero-4-hydroxynon-2 (E)-enoate, followed by reduction of the intermediate adducts with lithium borohydride. The requisite starting methyl esters are obtained, respectively, from heptanal or 7,7,7-trideuteroheptanal and methyl 4-chlorophenylsulfinylacetate via a sulfoxide piperidine and carbonyl reaction described in the literature. The 7,7,7-trideuteroheptanal is easily prepared by classical methods in four steps from 6-bromo-1-hexanol. 13C NMR data indicate that DHN-MA as well as d3-DHN-MA are obtained as mixtures of four diastereomers. Preliminary results show that d3-DHN-MA could be used as an internal standard for mass spectrometric quantification of DHN-MA in human urine.

Dietary and blood antioxidants in patients with chronic heart failure. Insights into the potential importance of selenium in heart failure.

BACKGROUND: Chronic heart failure (CHF) seems to be associated with increased oxidative stress. However, the hypothesis that antioxidant nutrients may contribute to the clinical severity of the disease has never been investigated. AIMS: To examine whether antioxidant nutrients influence the exercise capacity and left ventricular function in patients with CHF. METHODS: Dietary intake and blood levels of major antioxidant nutrients were evaluated in 21 consecutive CHF patients and in healthy age- and sex-matched controls. Two indexes of the severity of CHF, peak exercise oxygen consumption (peak VO2) and left ventricular ejection fraction (LVEF), were measured and their relations with antioxidants were analysed. RESULTS: Whereas plasma alpha-tocopherol and retinol were in the normal range, vitamin C (P=0.005) and beta-carotene (P=0.01) were lower in CHF. However, there was no significant association between vitamins and either peak VO2 or LVEF. Dietary intake (P<0.05) and blood levels of selenium (P<0.0005) were lower in CHF. Peak VO2 (but not LVEF) was strongly correlated with blood selenium: r=0.76 by univariate analysis (polynomial regression) and r=0.87 (P<0.0005) after adjustment for age, sex and LVEF. CONCLUSIONS: Antioxidant defences are altered in patients with CHF. Selenium may play a role in the clinical severity of the disease, rather than in the degree of left ventricular dysfunction. Further studies are warranted to confirm the data in a large sample size and to investigate the mechanisms by which selenium and other antioxidant nutrients are involved in CHF.

Diaminonaphtalene, a new highly specific reagent for HPLC-UV measurement of total and free malondialdehyde in human plasma or serum.

We describe a new method to measure free and total malondialdehyde (MDA) in human plasma or serum, which is based on the derivatization of MDA with diaminonaphtalene (DAN) in an acidic medium at 37 degrees C. Derivatization is complete after 180 min at room temperature. By HPLC separation on a C18 column and diode array detection, the diazepinium thus formed exhibits a highly specific UV spectrum with a sharp maximum at 311 nm, which clearly distinguishes MDA from other short-chain aldehydes. Direct injection of deproteinized plasma avoids the use of an internal standard. The between-run imprecision is 9.1% (141 +/- 13 nM) for plasma and 6.6% (658 +/- 44 nM) for a commercial control. Typical within-day imprecision is 8% (93 +/- 7.5 nM) for total MDA, 3.2% (16 +/- 0.5 nM) for free MDA in plasma, and 1.6% (630 +/- 10 nM) for a commercial control. The recovery of MDA added to 10 different plasmas is 93-108% (mean = 100%). Plasma levels in healthy women (n = 79, 45-51 years) are 162 +/- 51 and 24 +/- 15 nM for total and free MDA, respectively. In younger men (n = 19, 21-37 years) total and free MDA are, respectively, 138 +/- 28 and 19 +/- 9 nM.

Serum carotenoids as biomarkers of fruit and vegetable consumption in the New York Women's Health Study.

OBJECTIVE: To investigate the usefulness of serum carotenoids as biomarkers of fruit and vegetable consumption. DESIGN: : Reproducibility study on three repeat measurements of serum carotenoids. Correlation analysis of carotenoids and dietary food intake, and regression analysis of potential predictive parameters for serum carotenoid levels. SETTING: New York, USA. SUBJECT: : Women participating in the New York Women's Health Study, a prospective study of sex hormones, diet and breast cancer. Forty-eight women with three repeat blood samples and 302 women having a blood sample and a dietary history questionnaire (including 47 subjects from the reproducibility study). RESULTS: Serum carotenoid concentrations were highly reproducible between one- and two-year repeat samples. Estimated fruit and vegetable consumption was positively correlated with serum carotenoid concentrations but correlation coefficients were low. Consumption of fruit was predictive for serum levels of beta-carotene, alpha-carotene and beta-cryptoxanthin, while vegetable consumption was predictive for serum beta-carotene, lutein, zeaxanthin and lycopene. Serum concentrations of cholesterol and triglycerides were predictive for serum carotenoids but adjustment for their levels had little or no influence on the correlation between fruit and vegetable consumption and serum carotenoid concentrations. CONCLUSIONS: One single serum measurement of alpha-carotene, beta-carotene and lutein can accurately rank subjects according to their usual serum level. Serum concentrations, however, correlate only moderately with estimated dietary intake of fruits or vegetables and should therefore be used with caution as biomarkers of fruit and vegetable intake.

Plasma carotenoids in relation to food consumption in Granada (southern Spain) and Malmö (southern Sweden).

We conducted a cross-sectional pilot study on healthy pre-menopausal women (aged 45-50 years) living in Granada, in the south of Spain (n = 39) and Malmö, in the south of Sweden (n = 38) in order to compare their plasma carotenoid levels and to investigate the relationship between the differences in food consumption. Plasma concentrations of six carotenoids were measured using high performance liquid chromatography, habitual diet (at individual level) was estimated by food frequency questionnaires and 24-hour diet recalls were used for standardised measurement of diet at group-level. We found that women in Granada consumed more fruit and vegetables than women in Malmö. Plasma concentrations of beta-cryptoxanthin, lycopene, zeaxanthin, total carotenoids and alpha-tocopherol were higher in Granada than in Malmö, although plasma concentrations of alpha-carotene and retinol were higher in Malmö. Both within and between study centres, consumption of fruit and vegetables correlated positively with plasma concentrations of different carotenoids. The study showed that differences in consumption of fruit and vegetables between the two European centres were reflected in plasma carotenoid concentrations.

Antibody-induced modulation of the leukocyte CD11b integrin prevents mild but not major renal ischaemic injury.

BACKGROUND: CD11/CD18 beta(2) integrins are involved in leukocyte adhesion to the activated endothelium, and therefore represent a possible therapeutic target in the prevention of ischaemic acute renal failure (ARF). METHODS: To assess the effect of an anti-CD11b monoclonal antibody (mAb) in ischaemic ARF, uninephrectomized Fischer rats were subjected to 45 or 60 min of warm renal ischaemia, then received 1 mg of anti-CD11b mAb 5 min before reperfusion. RESULTS: After 45 min of ischaemia, renal function tests at 24 and 48 h were less altered in mAb-treated than in control rats, but after 60 min of ischaemia the same level of renal insufficiency was observed in the two groups. In parallel, milder tubular necrosis and less leukocyte infiltration were observed in the treated group after 45 min of ischaemia, but no difference was seen after 60 min compared to the control group. The mAb was detected on blood neutrophils up to 48 h after infusion and a marked down-regulation of CD11b expression on neutrophil surfaces was documented by flow cytometry. CONCLUSION: These results indicate that anti-CD11b mAb administered prior to reperfusion decreases moderate ischaemic ARF but fails to prevent renal injury secondary to prolonged ischaemia in this model.

Time course of oxidative stress after major burns.

OBJECTIVE: To evaluate oxidative stress resulting from major burns in humans. DESIGN: Prospective clinical study with control group. SETTING: Mechanically ventilated adult patients admitted with more than 30% total burn surface area. PATIENTS AND PARTICIPANTS: 20 patients with a mean body surface burned area of 54%. MEASUREMENTS AND RESULTS: The oxidative stress evaluation was based on measurements of trace elements, vitamins, antioxidant enzymatic activity and end-products of lipid peroxidation. During the first 5 days after injury burn patients exhibit a decrease in selenium and antioxidant vitamins (C, beta-carotene, lycopene) and an increase in lipid peroxidation products (TBARS). CONCLUSION: Our results suggest that major burn is associated with oxidative stress during the 5 days after the initial injury, as demonstrated by a simultaneous decrease in antioxidant vitamins and a large increase in TBARS.

Measurement of oxidized glutathione by enzymatic recycling coupled to bioluminescent detection.

A new method is described for the quantification of oxidized glutathione (GSSG) in tissues by enzymatic recycling coupled to NADPH bioluminescent detection. Tissue samples are treated with metaphosphoric acid. In a first step, after derivatization of GSH with 4-chloro-7-trifluoromethyl-1-methylquinolinium (CFQ), GSSG is recycled in the presence of dithionitrobenzoic acid (DTNB) and NADPH by glutathione reductase. In a second step, the GSSG-dependent NADPH consumption is measured by luminescence with NADPH:FMN oxidoreductase-bacterial luciferase. The coefficient of variation for GSSG measurements on repeated assays (n = 5) is 2 and 3% for standards and tissue samples, respectively. The sensitivity of this method is at the picomole level and is convenient for determination of GSSG physiological concentrations in tissues: GSSG levels measured in rat liver and kidney ranged from 76 to 215 and 52 to 170 nmol/g wet weight, respectively.

[Measurement of carotenoids by high pressure liquid chromatography: from difficulties to solutions]. / Dosage des caroténoïdes par chromatographie liquide haute performance: des difficultés aux solutions.

The measurement of serum carotenoids by HPLC has been largely improved during the last 10 years. However these techniques still require much time and skills, and direct application of published methods is rarely satisfying. We report here the difficulties that we met to transfer some HPLC methods described in the literature to our laboratories. We propose some solution to overcome the problems that we have encountered, our experience will perhaps help out other biologists. We reported also some results obtained in healthy populations.