Mechanism of cadmium-decreased glucuronidation in the rat.

In isolated rat hepatocytes, cadmium (0-200 microM) decreased the overall glucuronidation of both isopropyl N-(3-chloro-4 hydroxyphenyl)carbamate (4-hydroxychlorpropham, 4-OHCIPC) and 4-nitrophenol in a concentration-dependent manner. In contrast, in native rat liver microsomes, glucuronidation of 4-OHCIPC was increased by cadmium through activation of microsomal 4-OHCIPC glucuronosyl transferase. In addition, in rat microsome incubations, the net amount of 4-OHCIPC glucuronide was also indirectly increased by cadmium through a reduction in the activity of beta-glucuronidase. As the effect of cadmium on the activity of 4-OHCIPC glucuronosyl transferase could not account for the decrease in glucuronide formation in intact hepatocytes, the influence of cadmium on the availability of UDP-glucuronic acid (UDPGA) was investigated further. In isolated rat hepatocytes, cadmium depleted the UDPGA content in a dose-dependent manner without a change in the UDP glucose (UDPG) content. Cadmium did not increase the breakdown of UDPGA by microsomal UDPGA pyrophosphatase but strongly decreased (30-66%) the synthesis of the cofactor in the cytosol by inhibiting UDP-glucose dehydrogenase (UDPGDH). Cadmium (10-50 microM) was found to inhibit the purified enzyme from bovine liver (EC 1.1.1.22) non-competitively. In vivo in the absence of a substrate undergoing glucuronidation, cadmium administration, 1.5 and 2.5 mg Cd/kg i.v., to normally fed rats resulted in a 15 and 30% decrease of hepatic UDPGA, respectively. However, in the liver, neither the NAD+/NADH ratio nor the UDPG content was significantly changed following cadmium treatment. Both in vitro and in vivo results support the conclusion that in intact cells the reduction in overall 4-OHCIPC glucuronidation caused by cadmium was due to a decrease in UDPGA availability which results from the inhibiting effect of cadmium on UDPGDH.

Effects of varying dietary proteins on plasma lipids and rabbit platelet function.

The aim of this paper was to study the effect of dietary proteins on rabbit platelet aggregation induced by ADP, collagen and thrombin. In addition, blood coagulation tests (Stypven, Stypven cephalin and cephalin kaolin plasma clotting times) were performed on these rabbits fed semi-synthetic diets containing animal protein (casein) or vegetable proteins (walnut or soybean). As part of this study, total cholesterol, HDL cholesterol, phospholipid and triglyceride plasma levels were determined and platelet fatty acid composition was analysed. The results show that the addition of walnut meal in the diet decreases platelet aggregability and total plasma cholesterol. The relative effects of casein, soybean and walnut protein on platelet aggregability best correlated with the ratio of lysine to arginine in the protein and the ratio of HDL to LDL. An increase in the ratio C20:5 n-3/C20:4 n-6 in platelet fatty acids was also seen in walnut and soybean protein-fed animals, but the changes were greatest in soybean protein-fed animals while inhibition of platelet aggregation was greatest in walnut protein-fed animals.

Cadmium-induced alterations of chlorpropham metabolism in isolated rat hepatocytes.

In order to investigate the various steps of chlorpropham (CIPC) metabolism which could be influenced by cadmium, isolated rat hepatocytes were incubated in the presence of CIPC (0.1 mM) and of increasing Cd concentrations (0-180 microM). The results showed that Cd accumulation in hepatocytes was in good correlation to its concentration in the incubation medium. At 90 microM Cd, hydroxylation of CIPC was only slightly decreased by 30%, while CIPC hydrolysis into 3-chloraniline was unaffected by the presence of Cd. Accordingly, unchanged CIPC increased in hepatocytes. At 27 microM Cd, free 4-hydroxychlorpropham (4-OHCIPC) increased in the intracellular medium as a consequence of a strong suppression of both sulfation and glucuronidation which was related to the strong depletion of the intracellular ATP level under the combined influences of both cadmium and free 4-OHCIPC. Acetylation of 3-chloroaniline, which represents a minor pathway of CIPC metabolism, was already markedly suppressed (43%) with the lowest Cd concentration (27 microM). These in vitro results suggest that Phase II reactions are more sensitive to Cd than Phase I processes and that Cd enhanced the CIPC cytotoxicity as shown by alterations of the membrane integrity.

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.

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.

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.

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.

Cadmium accumulation and cytotoxicity in rat hepatocytes co-cultured with a liver epithelial cell line.

Cadmium accumulation and cytotoxicity were compared in hepatocytes in primary culture (HPC) or co-cultured (COC) with a rat liver epithelial cell line (RLEC). Cells were exposed for a 15-day period at 0, 0.045, 0.45 and 0.9 mum-Cd in the incubation medium. Cadmium uptake in COC was always linearly Cd dose and time dependent. In contrast, at the two highest doses for RLEC and only at the highest dose for HPC, a plateau in Cd uptake was observed. In viable cells 95% of the intracellular Cd was found in the cytosol, entirely protein bound. In the three types of culture the amount of Cd-metallothionein (MT) complex was proportional to Cd uptake and was in the order: COC > HPC > RLEC. Lactate dehydrogenase release in the medium and the DNA content of the plated cells at the end of exposure accounted for a marked Cd-induced cytotoxic effect in RLEC and a lesser effect in HPC. In contrast, no significant cytolysis was observed in COC. This suggests that although greater Cd accumulation was observed in COC than in HPC and RLEC, the detoxification procedure, dependent on Cd-MT complex formation, was more efficient and longer preserved in COC owing to a greater maintenance of hepatocyte specific functions, as evidenced by albumin secretion. This COC model offers a powerful tool for studying long-term accumulation and cytotoxicity of Cd in vitro at low exposure levels.

[Not Available]. / Separation chromatographique des azepines Nanochromatographie sur couche mince et chromatographie en phase liquide sous pression.

Two chromatographic methods are proposed for separation, identification and determination of dibenzoazepines and benzodiazepines. A micro thin-layer method has been developed and transposed to give an HPLC system, which requires a slight modilfication of the solvent. Retention data are given, and two quantitative applications arc described.

[Not Available]. / Dosage des xanthiques naturels par CLHP Comparaison des methodes et applications.

The three natural xanthines, caffeine, theobromine and theophylline, have been determined by HPLC on columns of silica gel or a C(18)-bonded phase. For the determination in beverages, HPLC on silica gel was used. The method was applied to coffee, tea, maté, in extracts or in the drinks themselves. For the extracts a comparison was made with other methods for determining caffeine, HPLC seems a method well suited to analysis of complex matrices for the xanthines.

HPLC analysis of antioxidants.

Qualitative and quantitative HPLC methods are described for the analysis of mixtures of twelve antioxidants. For identification of the components present, gradient elution with a convex profile from 35:65 v/v water-methanol to pure methanol is used, on a Waters 5 mum C(18) Resolve column, with an ultraviolet detector. Propyl gallate and methyl p-hydroxybenzoate cannot be separated, however. For quantitative analysis, with ultraviolet and electrochemical detectors in series, the 35:65 water-methanol mixture or pure methanol is used as the eluent, under isocratic conditions, with lithium perchlorate as supporting electrolyte. An applied potential ranging from +0.8 to + 1.7 V allows detection of all the antioxidants tested. Both modes of detection are very sensitive, with limits of detection as low as 61 pg (UV, methyl p-hydroxybenzoate) and 360 pg (electrochemistry, butylhydroxyanisole).

Determination of trace amounts of nickel by atomic-absorption spectrometry after carbonyl generation.

A sensitive and reliable method for the atomic-absorption spectrometric determination of micro-amounts of Ni in organic and inorganic samples has been developed, with separation of nickel by carbonyl generation. Foreign anions and cations do not interfere. Only metallic iron hampers the generation of Ni(CO)(4) but this interference can be avoided if the latter is formed in nitric acid medium. In the range 0.02-0.1 mug, Ni can be determined with a precision of about 10%. The detection limit of the proposed method is 10 ng in the aliquot used.

[Not Available]. / Determination polarographique de facteurs vitaminiques P (rutine et ses derives).

Several flavonols of therapeutic interest on account of their vascular properties have been studied. They were all derived from the o-diphenol quercetol. Their electrochemical oxidation at a rotating solid electrode has been examined, and the choice of solvent and pH is discussed. Results obtained by d.c. and pulse polarography are presented. A relation between their structure and electrochemical behaviour has been established. Application to drug analysis is proposed, giving identification, determination and purity assay simultaneously. There is a linear response over the range 5 x 10(-6)-10(-4)M, a sufficient range for analysis of pharmaceuticals.

[Not Available]. / Détermination des médicaments dans les aliments.

Determination of drugs in foods. In foods, drugs can come from different sources: animal therapeutics, zootechnical feed supplementation or human food additives. The possible consequences arising from the presence of drugs in food are considered. The quantitative determination of drugs in feeds or in human foods must fulfil various requirements, including specificity, sensitivity, accuracy and rapidity. Spectroscopic, electrochemical and chromatographic techniques are discussed. Some examples reported are ipronidazole, pyrimethamine, diethylstilboestrol, decoquinate and chloramphenicol. The need for quality assurance in analysis is also discussed.

Determination of 2-carboxy thiazolidine-4-carboxylic acid in biological fluids by ion-exchange chromatography.

A column ion-exchange chromatographic method for the determination of 2-carboxy thiazolidine-4-carboxylic acid (TDCA) in blood and urine is described. After elimination of endogenous thiols, alkaline hydrolysis of the compound yields cysteine which is determined spectrophotometrically. The described method is specific for intact TDCA. Using a 3-ml aliquot of sample it allows the linear determination of this hepatoprotective drug from 5 micrograms TDCA ml-1 with a detection limit of 3 micrograms ml-1. The method was applied to study the time course of plasma levels and urinary excretion. An in vitro metabolism study showed that TDCA has a potential as a cysteine donor for glutathione synthesis.

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.

Determination of cobalt in forages by Zeeman electrothermal atomic absorption spectrophotometry without preliminary extraction.

A simple and rapid method, avoiding preliminary extraction, is described for the determination of Co in plant samples. The factors influencing the mineralization steps and the interference of major elements are investigated. The described method gives a recovery in the range 100-110%. Use of the standard addition method avoids interference from Ca and Fe. In the range 0.1-1 ppm (dry basis), Co can be determined with a precision of 5%. The detection limit of the method is 0.02 ppm of Co in forages.

A field study of the validity of static paper sampling in fluoride pollution surveys.

A field study of fluoride pollution and of its consequences was made over a period of five years in the vicinity of an alumina reduction plant. This study, based upon the use of a static soda impregnated paper sampler, shows that the results obtained are in good agreement with the atmospheric fluoride concentrations obtained with dynamic samplers, especially when fluoride is present in gaseous form (HF). The results suggest that vertically mounted paper samplers are less sensitive to the collection of particulate fluorides. Collection rate is strongly influenced and increases with speed. If this parameter is known, an accurate estimation of the absolute atmospheric concentration is possible; the values shown by this technique are in good correlation with the value (annual mean) of pasture fluoride content. As a correlation exists between pasture fluoride content and the symptoms shown by cattle, the static filter samplers can predict disease in the case of slow chronic fluorosis. The field study agrees with earlier laboratory experiments and shows the validity of these simple and inexpensive types of exposure methods.

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%.

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.