Liquid chromatography-multistage tandem mass spectrometry for the quantification of dihydroxynonene mercapturic acid (DHN-MA), a urinary end-metabolite of 4-hydroxynonenal.

The mercapturic acid conjugate of 1,4-dihydroxynonene (DHN-MA) is a urinary metabolite of 4-hydroxynonenal (4-HNE), one of the main lipid peroxidation products occurring in vivo. To determine its level in urine, a combination of liquid chromatography with positive electrospray-multistage tandem mass spectrometry has been developed. A deuterated analog of the target compound (DHN-MA) with six deuterium atoms was synthesized and used as the internal standard. Three-stage tandem mass spectrometry was used, providing good selectivity for the detection of DHN-MA. The response of the system to DHN-MA was linear in the 5-100 ng range. Urine samples spiked with different levels of standard DHN-MA were used to evaluate the influence of matrix effects on the linearity. The repeatability of the method was also determined by using repeated 5 ng injections of DHN-MA, providing a RSD of 10%. The method was then applied to the determination of DHN-MA in rat urine samples; increased levels of urinary DHN-MA in urine from rats treated with BrCCl3 indicates that lipid peroxidation processes take place in such rats.

Pyrene degradation by two fungi in a freshwater sediment and evaluation of fungal biomass by ergosterol content.

Mucor racemosus var. sphaerosporus and Phialophora alba were investigated for their abilities to degrade pyrene in a freshwater sediment, with or without glucose supply as nutrient or carbon source, during 90 days. The ergosterol contents in sediment were quantified to estimate fungal biomass and to assess the correlation between fungal activity and biodegradation of pyrene. Results showed that, in an heterogeneous environment, these fungi presented different abilities to degrade pyrene. P. alba increased the degree of pyrene degradation by 9%, compared to the native micro-organisms, but a supply of glucose acted as an inhibitor to pyrene disappearance. M. racemosus var. sphaerosporus was not efficient at sediment bioremediation (with or without glucose added), because it reduced the rate of pyrene degradation by the native microflora. In any case, there was no increase of ergosterol in boxes during bioremediation experiments. In our experimental conditions, ergosterol content could not be correlated to pyrene degradation.

Liquid chromatography study of pyrene degradation by two micromycetes in a freshwater sediment.

Pyrene biodegradation in a freshwater sediment without fungi supply, or inoculated with two sediment micromycetes, Mucor racemosus var. sphaerosporus and Phialophora alba was studied after 0, 5, 13, 28, 60 and 90 days. The influence of glucose addition was estimated, and a liquid chromatographic method for simultaneous quantitative determination of residual anthracene, fluoranthene and pyrene in the sediment was developed. Samples with PAHs were extracted in Soxhlet with ethyl acetate, and LC analysis was performed on a 5 microm Supelcosil column (150 x 4.6 mm I.D.) with gradient elution (2 ml min(-1)) of acetonitrile-water and UV detection at 254 nm. Recoveries of anthracene, fluoranthene and pyrene were 90.3%+/-1.1%, 93.2%+/-0.9% and 90.42%+/-1.9%, respectively, without interference. The native sediment microorganisms (with or without glucose added) have shown 35% pyrene degradation and sediment with glucose inoculated by the strains revealed 40%.

Dansyl amino acid enantiomer separation on a teicoplanin chiral stationary phase: effect of eluent pH.

The retention and separation of a series of D,L dansyl amino acids (used as test solutes) on a teicoplanin stationary phase were investigated over a wide range of mobile phase (citrate buffer-methanol, 90:10, v/v) pH. An approach based on the development of various equilibria was carried out in order to describe the retention behavior of the solute in the chromatographic system. The equilibrium constants corresponding to the transfer of the anionic and zwitterionic forms of the dansyl amino acids from the mobile to the stationary phase were determined. These values allowed one to explain the decrease in the retention factor and the associated increase in the separation factor as the eluent pH was increased. Thermodynamic parameter variations were calculated so that the driving forces of the solute association with the teicoplanin phase were derived. This approach indicated that the chiral discrimination was principally controlled by the interaction between the anionic form of the solute and the stationary phase.

Quantitation of ergosterol in river sediment by liquid chromatography.

The ergosterol content of a river sediment can be used as an indicator of fungal activity. A method is developed for the extraction and determination of ergosterol in river sediment as part of a study to assess the correlation between fungal activity and biodegradation of pyrene, which is an environmental pollutant. This method is based on saponification and the liquid-liquid extraction of ergosterol by ethyl acetate. Quantitation and detection are performed isocratically by liquid chromatography on a 5-microm Hypersil C18 column with methanol-acetonitrile (80:20, v/v) as the mobile phase and detection at 282 nm. The detection limit is 50 ng/mL ergosterol, which is equivalent to 0.1 microg/g. The recovery of ergosterol at a concentration level in the range of 2 to 12 microg/mL is 91.7% +/- 3.1% without interferences. This method is applied in order to successfully quantitate the ergosterol content in a river sediment with or without a fungus supply.

Mobile-phase-viscosity dependence on DNA separation in slalom chromatography.

Slalom chromatography (SC) is an alternative chromatographic procedure for the separation of relatively large double-stranded DNA molecules and is based on a new principle. The retardation of the DNA fragments from the cleavage of the Lambda DNA by the KpnI restriction enzyme was studied using an acetonitrile-phosphate buffer as a mobile phase with various concentrations of viscosity modifier (i.e. glycerol) and a C1 column as a stationary phase. The DNA molecule retention was accurately described over the glycerol concentration range using a model previously established. It was shown that the eluent viscosity increase enhanced the slalom chromatographic capacity to separate the DNA fragments. A connection between SC and 'hydrodynamic chromatography' processes was predicted to link the two processes in a global separation mechanism based on a non-equilibrium principle.

Metabolism of 4-hydroxynonenal, a cytotoxic product of lipid peroxidation, in rat precision-cut liver slices.

4-Hydroxy-2-nonenal (HNE) is a major aldehydic product of lipid peroxidation known to exert several biological and cytotoxic effects. The in vitro metabolism of [4-(3)H]-HNE by rat precision-cut liver slices was investigated. Liver slices rapidly metabolize HNE - about 85% of 0.1 microM [4-(3)H]-HNE was degraded within 5 min of incubation. The main metabolites of HNE identified were 4-hydroxynonenoic acid (HNA), glutathione-HNE-conjugate (HNE-GSH), glutathione-1,4-dihydroxynonene-conjugate (DHN-GSH) and cysteine-HNE-conjugate (HNE-CYS). Whereas glutathione conjugation demonstrated saturation kinetics (K(m)=412.2+/-152.7 microM and V(max)=12.3+/-2.5 nmol h(-1) per milligram protein), HNA formation was linear up to 500 microM HNE in liver slices. In contrast to previous reports, no trace of the corresponding alcohol of the HNE, 1,4-dihydroxynon-2-ene was detected in the present study. Furthermore, the beta-oxidation of HNA including the formation of tritiated water was demonstrated. The identification of 4-hydroxy-9-carboxy-2-nonenoic acid and 4,9-dihydroxynonanoic acid demonstrated that omega-oxidation significantly contributes to the biotransformation of HNE in liver slices.

Effect of temperature on DNA fractionation in slalom chromatography.

Slalom chromatography is an alternative chromatographic procedure for the analysis of relatively large double-stranded DNA molecules and is based on a hydrodynamic principle. The retardation of the DNA fragments from the cleavage of the Lambda DNA by the KpnI restriction enzyme was studied using an acetonitrile-phosphate buffer as a mobile phase (flow rate equal to 0.3 ml/min) and a C1 column as a stationary phase at various temperatures. It was shown that the temperature constituted an important parameter for the separation of the DNA fragments in slalom chromatography. The DNA hydrodynamic behavior with the temperature was related to the variation in the fluid viscosity and the modification of the elastic properties of the biopolyrner.

Analysis in the rat of 4-hydroxynonenal metabolites excreted in bile: evidence of enterohepatic circulation of these byproducts of lipid peroxidation.

4-Hydroxynonenal (HNE) is a cytotoxic product resulting from the lipid peroxidation of membrane polyunsaturated fatty acids. In vitro, metabolism mainly leads to the corresponding alcohol (DHN), carboxylic acid (HNA), and the glutathione conjugate, whereas in vivo, mercapturic acid conjugates of HNE, DHN, HNA, and HNA-lactone and, more recently, dicarboxylic acids and related mercapturate conjugates were identified in urine of rats. In the study presented here, the identity of the HNE biotransformation products in the bile of rats following a single iv administration of [4-(3)H]HNE and the potential for enterohepatic recycling of HNE metabolites were investigated. The identity of metabolites was assessed by comparison of their HPLC retention times with those of the corresponding synthesized standards and by mass spectrometry analysis. Five metabolites were present in the bile; two of them corresponded to HNE- and DHN-glutathione conjugates. Two others metabolites were identified as DHN- and HNA-lactone mercapturic acid conjugates. The fifth metabolite was isolated but remained unidentified. As previously observed for urinary elimination, the kinetic excretion of biliary metabolites exhibited a rapid metabolism of HNE in rats. Within 4 h of injection, the bile accounted for 19.5% (+/-2.8%) of the injected radioactivity, whereas only 3% was found in the feces within 48 h [Alary, J., et al. (1995) Chem. Res. Toxicol. 8, 34-39]. The extent of HNE enterohepatic recycling was estimated utilizing a modified version of the linked rat model in three animals. All rat recipients were found to have measurable levels of HNE metabolites in bile, confirming that HNE is likely to undergo enterohepatic recirculation in the rat. The extent of recycling was approximatly 7. 7% of the total dose in this model. Two unknown metabolites were present in the bile of recipient rats and not found in the bile of donors rats, suggesting that intestinal microflora and/or intestinal mucosa could biotransform HNE-related compounds before or during the reabsorption process.

Blood cholesterol and walnut consumption: a cross-sectional survey in France.

BACKGROUND: The preventive role of polyunsaturated fatty acids in cardiovascular disease has been recognized. We conducted a cross-sectional study to assess the association between walnut consumption (oil and kernel) as a source of polyunsaturated fatty acids and blood lipid levels. METHODS: Seven hundred ninety-three persons, males and females, ages 18-65 years, living in a walnut production area (Dauphiné, France) attended health screening visits organized by the Agriculture Social Security. Past diet (1-year recall, including walnut and animal fat consumption) and cardiovascular risk factors were ascertained using food frequency questionnaires. For each participant a blood sample was taken to measure HDL, LDL, and total cholesterol; apo A1; and apo B. RESULTS: A high level of HDL cholesterol and apo A1 was associated with a high amount of walnut consumption (oil and kernel) in the regular diet, with a positive trend with increasing degree of walnut consumption. This association did not appear to be confounded by dietary animal fat and alcohol as measured in this study. Other blood lipids did not show significant associations with walnut consumption. CONCLUSION: The positive effect of walnut consumption on blood HDL cholesterol and apo A1 is of special interest since these lipid parameters have been shown to be negatively correlated with cardiovascular morbidity.

In vivo involvement of cytochrome P450 4A family in the oxidative metabolism of the lipid peroxidation product trans-4-hydroxy-2-nonenal, using PPARalpha-deficient mice.

Trans-4-hydroxy-2-nonenal (HNE) is a potent cytotoxic and genotoxic compound originating from the peroxidation of n-6 polyunsaturated fatty acids. Its metabolism has been previously studied in the rat (Alary et al. 1995. Chem. Res. Toxicol., 8: 35-39). In addition to major urinary mercapturic derivatives, some polar urinary metabolites were isolated and could correspond to hydroxylated compounds. 4-Hydroxynonenoic acid (HNA), resulting from the oxidation of the HNE carbonyl group, is a medium chain fatty acid and its omega-hydroxylation might be hypothesized. Therefore, the involvement of the CYP 4A family isoenzymes in the metabolism of [3H]HNE has been investigated in vivo using inducer treatments (fibrates) in wild-type or in peroxisome proliferator-activated receptor alpha (PPARalpha)-deficient mice. In wild-type mice, but not in PPARalpha (-/-) mice, fibrate treatments resulted in an increase of two urinary metabolites characterized, after HPLC purifications and mass spectrometry analyses, as the omega-hydroxylated metabolite of HNA, i.e., 4,9-dihydroxy-2-nonenoic acid, and its oxidized form, 4-hydroxy-2-nonene-1,9-dicarboxylic acid. The formation of the latter is correlated accurately to laurate hydroxylase activity studied concurrently in microsomes prepared from the liver of these animals. Basal levels of these two metabolites were measured in urine of normal and PPARalpha-deficient mice. These results are in accord with an implication of the P450 4A family in the extended oxidative metabolism of 4-HNE.

Identification of novel urinary metabolites of the lipid peroxidation product 4-hydroxy-2-nonenal in rats.

Following iv administration of 4-hydroxy-2-nonenal (HNE) and [4-3H]HNE to rats, 15 polar urinary metabolites accounting for about 50% of the urinary radioactivity were separated by HPLC. Among them, eight major compounds and tritiated water were quantified. The metabolites were unequivocally characterized using GC/MS and ESI/MS/MS/MS. Most of "HNE polar metabolites" originate from omega-oxidation of 4-hydroxy-2-nonenoic acid (HNA): 9-hydroxy-HNA, its mercapturic acid conjugate, and two diastereoisomers of the corresponding lactone. The oxidation of 9-hydroxy-HNA by alcohol and aldehyde dehydrogenases leads to the excretion of 9-carboxy-HNA and of the corresponding lactone mercapturic acid conjugate. 1, 4-Dihydroxy-2-nonene (DHN) originating from the reduction of HNE by alcohol dehydrogenase was to a lesser extent omega-hydroxylated, leading to 9-hydroxy-DHN which was excreted as a mercapturic acid conjugate (two diastereoisomers).

Fatty acid nutriture in hospitalized elderly women.

OBJECTIVE: The aim of this study was to measure the fatty acid (FA) dietary intakes and the FA composition of plasma total lipids in a selected group of hospitalized elderly patients. METHODS: Twenty-three women aged 76 to 99 years were recruited. FA were analyzed in 5-day duplicate portions and in plasma by gas liquid chromatography. RESULTS: The hospitalized elderly women ingested an average of 5.22 megajoules (MJ) and 45.9 g of lipids per day. Polyunsaturated fatty acids (PUFA) represented 11.0% and saturated fatty acids (SFA) 53.6% of the lipid intake. Minimal recommendations for linoleic acid intake were reached in average, but 32% of the patients ingested less than 3 g of linoleic acid/d. Eighty-six percent received less than 0.5% of energy for alpha-linolenic acid and 64% had low intakes in very long-chain n-3 FA. In parallel, these patients presented several biochemical signs of essential fatty acids (EFA) insufficiency (decrease in linoleic acid, increase in monounsaturated fatty acids (MUFA), in n-7 FA and in indexes of delta-6 and delta-9 desaturase activities). CONCLUSIONS: Hospitalized elderly patients have low PUFA intakes and show biochemical indices of EFA insufficiency. These patients might benefit from a nutritional supplementation providing both EFA and antioxidant micronutrients to limit the risk of skin troubles, immune system impairment and vascular disease often observed in institutionalized elderly subjects.

Metabolism and cytotoxicity of chlorpropham (CIPC) and its essential metabolites in isolated rat hepatocytes during a partial inhibition of sulphation and glucuronidation reactions: a comparative study.

The changes in metabolism and cytotoxicity of chlorpropham (CIPC) and its major metabolites, 4-hydroxychlorpropham (4-OH CIPC), 3-chloroaniline, and 3-chloroacetanilide were investigated in isolated rat hepatocyte suspensions after a partial inhibition of sulphation and glucuronidation and the two reactions combined in an attempt to assess the part of each of them in the enhanced CIPC toxicity observed in vivo after D-galactosamine treatment. With sulphation and glucuronidation effective, CIPC has a cytolytic effect and reduces intracellular ATP and K+ level while 4-OH CIPC has a weak cytolytic effect but modifies ATP and K+ level in a greater extent than CIPC. Inhibition of sulphation does not affect the cytotoxicity of CIPC or 4-OH CIPC because there is a compensatory increase in the amount of 4-OH CIPC glucuronide formed and the level of free 4-OH CIPC always remain low. In contrast, when incubations are carried out with either CIPC or 4-OH CIPC, the presence of D-galactosamine leads to a decrease of glucuronide and sulphate conjugates accompanied, respectively, by a 3.6-fold and 6. 9-fold increase of the free 4-OH CIPC level in the culture medium. This alteration of the metabolism is followed by a marked reduction of ATP synthesis with a concomitant modification of cell permeability. The cytolytic effect is due to CIPC itself, whereas the effect on energy supply was attributed to free 4-OH CIPC. The results demonstrate a combined effect of free 4-OH CIPC and D-galactosamine on intracellular ATP level that could account for the partial inhibition of sulphation. This change in the CIPC metabolism could explain the increased CIPC toxicity observed in vivo after D-galactosamine pretreatment.

1,4-Dihydroxynonene mercapturic acid, the major end metabolite of exogenous 4-hydroxy-2-nonenal, is a physiological component of rat and human urine.

In the present study 1,4-dihydroxynonene mercapturic acid (DHN-MA), previously shown to be the major urinary metabolite of 4-hydroxy-2-nonenal (HNE) administered to the rat, was characterized and determined to be a normal constituent of rat and human urine. DHN-MA was excreted as a mixture of at least two stereoisomers as determined by ion trap LC-MS/MS/MS after solid-phase extraction and HPLC purification. The 24-h urinary excretion of this compound was about 10 ng and 5 microg for rat and human, respectively. This end metabolite of the lipid peroxidation product HNE could represent a specific and noninvasive biomarker.

Fish oil effects on tissular fatty acids and plasma lipid peroxidation in zinc deficient rats.

Zinc has been reported to play a key role in lipid metabolism as well as in defences against oxidative stress. The aim of this work was to investigate the fatty acid distribution (plasma, heart, kidney, liver) and peroxidation (plasma) in zinc-deficient rats fed with n-6 fatty acids (10% corn oil) or n3 fatty acid fish oil (10% Maxepa). Zinc deficiency led to a decreased tissular and plasma n-6/n-3 ratio both in triglycerides and phospholipids. This effect was more marked in the Maxepa group than in the corn oil group. In plasma, the TBARs/TG + PL ratio was significantly enhanced in zinc-deficient animals, especially in rats receiving Maxepa. With regard to these results, zinc deficiency could appear as an aggravating factor of oxidative risk when associated with a n-3 fatty acid-rich diet. This work draws attention to the harmful oxidative risk associated with patients' intake of fish oil concentrate, without taking into account their antioxidant dietary intakes and status.

Antioxidant vitamins in hospitalized elderly patients: analysed dietary intakes and biochemical status.

DESIGN: Descriptive study. SETTING: Geriatric department of the Grenoble University Hospital. SUBJECTS: 24 hospitalized elderly women: 13 long-stay patients and 11 in rehabilitation after femoral neck fracture. MAIN OUTCOME MEASURES: Retinol, carotene, tocopherol and vitamin C dietary intakes were evaluated by 5-day duplicate portion analysis. Circulating levels of retinol, beta-carotene, alpha-tocopherol and vitamin C were determined in parallel (HPLC). RESULTS: Mean intake of vitamin C (21 mg/d), and vitamin E (3.1 mg alpha-tocopherol equivalents TE/d) were low compared to recommendations, in relation with poor energy intake (5.27 MJ/d) and nutrient densities. More than 85% of the patients exhibited vitamin C and vitamin E intakes below two-thirds the recommendations (60 mg/d and 10 mg TE/d, respectively) and 50% did not meet recommendations for vitamin A (800 micrograms retinol equivalents/d). With the exception of retinol, dietary vitamin intakes were positively correlated to corresponding blood concentrations. No values below cut-off levels were found concerning plasma retinol, plasma tocopherol or ratio of alpha-tocopherol to cholesterol. In contrast, 26% and 32% of the elderly patients had low circulating levels of beta-carotene and vitamin C, respectively. CONCLUSIONS: The present study highlights low antioxidant vitamin intakes, particularly concerning vitamin E and vitamin C, and an important proportion of low blood vitamin C and beta-carotene concentrations in hospitalized elderly women. Further studies are needed to determine the actual requirements of hospitalized elderly patients and to evaluate the potential benefits of providing micronutrient-enriched foods to this population.

A survey of nitrate contents in Indonesian milk by enzymic analysis.

In this paper, a rapid and simple enzymic method is described for the determination of nitrate in 32 fresh and five dry Indonesian milk samples, deproteinized by Carrez reagents. Interference from albumin, casein, lactose and chloride ions was controlled. The calibration graph was linear over the range l-12.5 micrograms/ml NO3-; r = 0.9998. The limits of detection and quantification were found to be 0.45 micrograms/ml NO3- and 1 microgram/ml NO3- respectively. Standard nitrate solutions (10 micrograms/ml NO3-) were used to evaluate the precision. The results showed an average of 10.1 micrograms/ml, a standard deviation of 0.3 and a relative standard deviation of 3.4%. Adequate agreement was found between results obtained by the enzymic method and those of the French official reduction/photometric reference method (AFNor). Good recoveries (100% +/- 5%) were found for nitrate added to milk. The nitrate levels were in the range 1-2.6 mg/kg NO3- for fresh milk and 1.1-18 mg/kg NO3- for dry milk. All the results are in good agreement with those previously published for UK and American milk.

Effects of modulation of sulphation and glucuronidation on chlorpropham metabolism and cytotoxicity in isolated rat hepatocytes.

After modulation of sulphation and glucuronidation, the relationship between the changes in metabolism and cytotoxicity of chloropropham (CIPC), a widely used herbicide, was investigated in isolated rat hepatocyte suspensions. Under physiological conditions, CIPC had a cytolytic effect, modified membrane permeability and reduced intracellular ATP level. CIPC was metabolized by hepatocytes mainly into 4-OH chlorpropham sulphate (37%) and glucuronide conjugates (18%). Inhibition of sulphation, by omitting sulphate from the isolation and incubation media, did not affect the cytotoxicity of CIPC, since there was a 2.5-fold compensatory increase in 4-OH CIPC glucuronide. Inhibition of glucuronidation by adding 4 mM D-galactosamine in the incubation medium led to a 66% decrease of glucuronide conjugate and simultaneously to a 32% decrease of sulphate conjugate. In that case, concentrations of free 4-OH CIPC in both hepatocytes and incubation medium were markedly increased, while those of 3-chloroaniline and 3-chloroacetanilide were slightly modified and remained low. This alteration of metabolism was accompanied by modification of cell permeability and reduction in ATP synthesis. The cytolytic effect was due to CIPC itself, whereas the effect on energetic metabolism was attributed to a metabolite. Results demonstrated for the first time a partial inhibition of sulphation by D-galactosamine (4 mM), probably due to the effect of D-galactosamine on intracellular ATP levels.