Biomedical subjects

L B Fay

Publications and source records attributed to L B Fay.

At least 37 records · Page 2Linked to original sources

PubMed1999

Urinary isoprostane excretion is not confounded by the lipid content of the diet.

This study aims to determine if isoprostanes accurately reflect in vivo lipid peroxidation or whether they are influenced by the lipid content of the diet. Isoprostanes were measured in urine of healthy subjects under different conditions of lipid intake and under conditions of oxidative stress (fasting). We found that isoprostanes were not influenced by the lipid content of the diet: the urinary level remained constant over 24 h as well as over 4 consecutive days when switching from high to low lipid intake. Urinary isoprostane excretion was increased by 40% following a 24 h fast. We concluded that urinary isoprostane excretion reflects endogenous lipid peroxidation in vivo.

Adult↗

PubMed1999

Interspecies differences in metabolism of heterocyclic aromatic amines by rat and human P450 1A2.

The catalytic efficiences of cytochrome P450 (P450)-mediated N-oxidation of the 2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline (MeIQx) and 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) by recombinant human P450 1A2 were 10-19-fold higher than rat P450 1A2, while methoxyresorufin O-demethylation activity was comparable for both P450s. Similar findings were observed with rat and human liver microsomal samples. Interspecies differences in P450 enzyme expression and catalytic activities may be significant and must be considered when assessing human health risk.

Animals↗

PubMed1999

Stability of cyclopropane and conjugated linoleic acids during fatty acid quantification in lactic acid bacteria.

Seven methods commonly used for fatty acid analysis of microorganisms and foods were compared to establish the best for the analysis of lyophilized lactic acid bacteria. One of these methods involves fat extraction followed by methylation of fatty acids, while the other methods use a direct methylation of the samples, under different operating conditions (e.g., reaction temperature and time, reagents, and pH). Fatty acid methyl esters were identified by gas chromatography-mass spectrometry and quantified by on-column capillary gas chromatography. Two reliable methods for the analysis of fatty acids in bacteria were selected and further improved. They guarantee high recovery of classes of fragile fatty acids, such as cyclopropane and conjugated acids, and a high degree of methylation for all types of fatty acid esters. These two direct methylation methods have already been successfully applied to the analysis of fatty acids in foods. They represent a rapid and highly reliable alternative to classical time- and solvent-consuming methods and they give the fatty acid profile and the amount of each fatty acid. Using these methods, conjugated linoleic acids were identified and quantified in lactic acid bacteria.

Bacteria↗

PubMed1999

Synthesis of trans-4,5-epoxy-(E)-2-decenal and its deuterated analog used for the development of a sensitive and selective quantification method based on isotope dilution assay with negative chemical ionization.

The volatile compound trans-4,5-epoxy-(E)-2-decenal (1) was synthesized in two steps with good overall yields. The newly developed method is based on trans-epoxidation of (E)-2-octenal with alkaline hydrogen peroxide followed by a Wittig-type chain elongation with the ylide formylmethylene triphenylphosphorane. For the synthesis of [4,5-2H2]-trans-4,5-epoxy-(E)-2-decenal (d-1), [2,3-2H2]-(E)-2-octenal was prepared by reduction of 2-octyn-1-ol with lithium aluminum deuteride and subsequent oxidation of [2,3-2H2]-(E)-2-octen-1-ol with manganese oxide. Compound d1 was used as internal standard for the quantification of 1 by isotope dilution assay. Among various mass spectrometry (MS) ionization techniques tested, negative chemical ionization with ammonia as reagent gas gave best results with respect to both sensitivity and selectivity. The detection limit was found to be at about 1 pg of the analyte introduced into the gas chromatography-MS system.

Aldehydes↗

PubMed1999

Synthesis of deuterated volatile lipid degradation products to be used as internal standards in isotope dilution assays. 1. Aldehydes.

The isotopically labeled compounds [5,6-(2)H(2)]hexanal (d-I), [2, 3-(2)H(2)]-(E)-2-nonenal (d-II), [3,4-(2)H(2)]-(E,E)-2,4-nonadienal (d-III), and [3,4-(2)H(2)]-(E,E)-2,4-decadienal (d-IV) were prepared in good yields using new or improved synthesis procedures. Labeling position, chemical purity, and isotopic distribution of the compounds were characterized by various MS and NMR techniques. These molecules are used as internal standards in quantification experiments based on isotope dilution assay. Synthesis of d-I, d-III, and d-IV has not yet been reported in the literature.

Aldehydes↗

PubMed1999

Synthesis of deuterated volatile lipid degradation products to be used as internal standards in isotope dilution assays. 2. Vinyl ketones.

The isotopically labeled compounds [5,6-(2)H(2)]-(Z)-1, 5-octadien-3-one (d-I) and [1-(2)H(1;2),2-(2)H(1;1)]-1-octen-3-one (d-II), as well as the unlabeled reference compound (Z)-1, 5-octadien-3-one (I) were prepared by improved synthesis procedures. Labeling position, chemical purity, and isotopic distribution of the compounds were characterized by various MS and NMR techniques. These molecules are used as internal standards in quantification experiments based on isotope dilution assay. The newly prepared compound d-II was synthesized in a simple two-step procedure, and formation of the main isotopomers was studied in model systems.

Deuterium↗

PubMed1998

Morphological transformation of C3H/M2 mouse fibroblasts by extracts of human mammary lipid.

Mammary lipid may act as a reservoir for genotoxins. Mammary lipid extracts (MLEs), obtained from eight UK women (21-41 years) undergoing reduction mammoplasty, were examined for their abilities to morphologically transform C3H/M2 mouse fibroblasts. Resultant transformation rates were 0.27, 0.33, 0.07, 0.29, 0.21, 0.00, 0.07, and 0.13 transformed foci/treated dish, respectively. Although the lipid-extraction procedure used was originally designed to extract heterocyclic aromatic amines (HAAs), liquid chromatography/mass spectroscopy (LC/MS) with selective ion monitoring has failed to detect HAAs in any of the lipid extracts so far examined. Genotoxicities were also assessed in S. typhimurium TA98 and in metabolically competent human (MCL-5) cells by the micronucleus and by the alkaline single-cell gel ("comet") assays. The MLEs induced bacterial mutagenicity rates ranging from 0 to 498 revertants/plate/g-lipid equivalent and micronucleus-formation rates from 0 to 20 micronuclei/500 binucleate cells/g-lipid. Median comet tail lengths (induced with MLEs of 8.0 g-lipid equivalent) ranged from 6.0 to 74.0 micrometer. The results demonstrate the presence of as-yet-unidentified transforming agents in mammary lipid.

Adult↗

PubMed1998

Determination of selenium stable isotopes by gas chromatography-mass spectrometry with negative chemical ionisation.

A gas chromatography mass spectrometric method using negative chemical ionisation was developed for the determination of stable isotopes of selenium for evaluation of selenium absorption and retention from foods in humans. The method involves an acid digestion to convert all selenium into selenite, which subsequently reacts with 4-nitro-o-phenylene-diamine to form a volatile piazselenole. The piazselenole, after extraction into an organic solvent, was analysed for its isotopic selenium composition by gas chromatography mass spectrometry. Negative chemical ionisation is reported for the first time for the determination of selenium stable isotopes and its analytical characteristics were compared to those of electron impact mass spectrometric ionisation, classically used for the determination of selenium. The negative chemical ionisation technique allowed accurate determination of total selenium by isotope dilution and of selenium isotope ratios in biological samples. The repeatability for total selenium and for stable isotope ratios was good (R.S.D.< or =10%) within the range of 50 to 250 ng selenium. The detection limit for the investigated selenium isotopes was approximately 1 pg (signal to noise ratio at 3). The applicability of the developed stable isotope methodology was demonstrated by the determination of the selenium absorption and retention from foods in a pilot study using one human adult.

Adult↗

PubMed1998

Evaluation of taurine metabolism in cats by dual stable isotope analysis.

Taurine kinetics in cats was investigated using a bolus dose of [15N]- and [1,2-13C2]taurine. The comparison of [15N]- and [13C2]taurine kinetics permitted an evaluation of the extent of taurine transamination. A methodology which involves N-pentafluorobenzoyl di-n-butylamine derivatization of taurine and GC/MS measurements of the 15N- and 13C-enrichments in cat urine was developed. Accuracy of the measurements was determined using pure standard compounds and the results showed that [13C2]taurine does not interfere with [15N]taurine. In cats, no differences were observed between both tracers. Therefore, we conclude that taurine reversible transamination does not occur at a significant level in cats.

Animals↗

PubMed1998

Covalent modifications of aminophospholipids by 4-hydroxynonenal.

Lipid oxidation is implicated in a wide range of pathophysiological disorders, which leads to reactive compounds such as aldehydes. Among them 4-hydroxynonenal (4-HNE) reacts strongly with the NH2 groups of amino acids and forms mainly Michael adducts and minor Schiff-base adducts. Such reactions occur also with compounds containing thiol groups. No data are available describing 4-HNE interactions with amino-phospholipids. To investigate such a possibility, 4-HNE was incubated with either phosphatidylethanolamine (PE) or phosphatidylserine (PS) in an aqueous-organic biphasic system and the resulting products were identified by liquid chromatography-mass spectrometry (LC-MS). Our study points out the potential capacity of 4-HNE to react with phospholipids containing amino groups and particularly PE. The main resulting compounds found were a Michael adduct plus a minor Schiff base adduct, which was partly cyclized as a pyrrole derivative via a loss of water. Its stabilization as a pyrrole derivative allows to differentiate 4-HNE from the other aldehydes generated via lipid oxidation (e.g., malondialdehyde, 2-nonenal) that lack the 4-hydroxyl group. Their formation seems not to be affected when the pH varies from 6.5 to 8.5. Surprisingly, PS reacted poorly producing only a small amount of Michael adduct, the Schiff-base adduct being nondetectable. We conclude that such adducts, if they are formed in cell membranes, could alter the phospholipase-dependent cell signaling.

Aldehydes↗

PubMed1998

Metabolism of the food-borne mutagen 2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline in humans.

The metabolism of 2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline (MeIQx) was investigated in five human volunteers given a dietary equivalent of 14C-labeled MeIQx. The amount of the dose excreted in urine ranged from 20.2% to 58.6%, with unmetabolized MeIQx accounting for 0.7-2.8% of the dose. Five principal metabolites were detected in urine, and four of the derivatives were characterized by on-line UV spectroscopy and by HPLC-MS following immunoaffinity chromatography. Two metabolites were identified as the phase II conjugates N2-(3,8-dimethylimidazo[4,5-f]quinoxalin-2-yl)sulfamic acid (MeIQx-N2-SO3(-)) and N2-(beta-1-glucosiduronyl)-2-amino-3,8-dimethylimidazo[4,5-f ]quinoxaline (MeIQx-N2-Gl). Two other metabolites were the cytochrome P450-mediated (P450) oxidation products 2-amino-8-(hydroxymethyl)-3-methylimidazo[4,5-f]quinoxaline (8-CH2OH-MeIQx), and N2-(beta-1-glucosiduronyl)-N-hydroxy-2-amino-3,8-dimethylimidaz o[4,5-f]quinoxaline (NOH-MeIQx-N2-Gl). The latter product is a conjugate of the genotoxic metabolite 2-(hydroxyamino)-3,8-dimethylimidazo-[4,5-f]quinoxaline (NHOH-MeIQx). A large interindividual variation was observed in the metabolism and disposition of MeIQx; these four metabolites and unchanged MeIQx combined accounted for 6.3-26.7% of the total dose. The remaining principal metabolite found in all subjects accounted for 7.6-28% of the dose. It has not been previously identified in rodents or nonhuman primates, and its structure remains unknown. P450-mediated ring oxidation of MeIQx at the C-5 position, a major pathway of detoxication in rodents, was not detected in humans. Both 8-CH2OH-MeIQx formation and NHOH-MeIQx formation are catalyzed by P450 1A2 and may be useful biomarkers of P450 1A2 activity in humans. The levels of NHOH-MeIQx-N2-Gl found in human urine ranged from 1.4% to 10.0% of the dose, which is significantly higher than that formed in rodents and nonhuman primates undergoing cancer bioassays. Thus, bioactivation of MeIQx by P450-mediated N-oxidation is extensive in humans.

Administration, Oral↗

PubMed1997

Determination of heterocyclic aromatic amines in food products: automation of the sample preparation method prior to HPLC and HPLC-MS quantification.

Heat-processing protein-rich foods may cause the formation of heterocyclic aromatic amines (HAAs), all of which have mutagenic and some also carcinogenic potential. Accurately measuring HAA levels in food products is therefore a necessary to realistically assess this risk factor. A solid-phase extraction method for quantitative HAA analysis has been developed by us over the last few years. This method has recently been automated using a robotic workstation and now allows almost unattended sample preparation, a process which saves a human operator about five hours of benchwork. Cleaned-up samples were analyzed by high performance liquid chromatography (HPLC) and ultraviolet (UV) or mass spectrometric (MS) detection. While HPLC-UV remains the daily tool to quantify HAAs, we found HPLC-electrospray-MS to be an alternative detection method with unique advantages, suited for both HAA identification and quantification.

Amines↗

PubMed1997

Urinary excretion of 3-methyladenine after consumption of fish containing high levels of dimethylamine.

The urinary excretion of the DNA alkylation product, 3-methyladenine (3-MeAde), was measured in human volunteers who were on controlled diets and consumed fresh fish, or frozen-stored fish that contained 50-fold higher levels of dimethylamine (DMA), with or without ingested nitrate. DMA potentially could react with nitrosating agents in the diet or within the body, and produce the potent carcinogen N-nitrosodimethylamine (NDMA), which can then react with DNA to form several adducts including 3-MeAde. Our findings show that there was no increase in urinary levels of 3-MeAde after consumption of fish preserved by frozen storage relative to levels after consumption of fresh fish. Furthermore, consumption of 225 mg sodium nitrate (equal to the nitrate content in a large glass of beet juice) at 1 h prior to consumption of the frozen-stored fish did not increase urinary 3-MeAde levels as would be expected if nitrate enhanced endogenous nitrosation of DMA. In contrast, urinary excretion of 3-MeAde from a volunteer who was a moderate cigarette smoker (11 cigarettes per day) was approximately 3- to 8-fold higher than dietary 3-MeAde intake. These findings indicate that consumption of high levels of DMA in fish does not result in detectable levels of NDMA formation and genetic damage as measured by the urinary biomarker 3-MeAde.

Adenine↗

PubMed1996

Oxidation of caffeine and related methylxanthines in ascorbate and polyphenol-driven Fenton-type oxidations.

Caffeine and related methylxanthines were subjected to free radical mediated oxidation by incubation with Fe(3+)-EDTA/ascorbate and Fe(3+)-EDTA/polyphenolics. The reaction mixtures were analysed by reverse-phase HPLC, revealing the corresponding C-8 hydroxylated analogues as the major products of hydroxyl radical mediated attack. Further oxidation products of caffeine, analysed by liquid chromatography-mass spectrometry (LC-MS), were the N1-, N3- and N7-demethylated methylxanthine analogues theobromine, paraxanthine and theophylline, respectively. Isolable amounts of the imidazole ring operated 6-amino-5-(N-formylmethyl-amino)-1,3-dimethyl-uracil (1,3,7-DAU) derivative were also detected, which was characterised by 1H NMR and mass spectroscopy. The identified products indicate that the pertinent chemical reactions, i.e. C-8 hydroxylation, demethylations, and C8-N9 bond scission, are comparable to the primary metabolic pathways of caffeine in humans. The influence of pH, transition metals, hydrogen peroxide, free radical scavengers and metal chelators on caffeine oxidation was studied. This report illustrates that natural food-borne reactants can aid in identifying specific chemical markers of free radical induced damage. Furthermore, potentially anti-and pro-oxidative reactions can be elucidated which may be important in assessing the impact of nutrient additives and supplements on the shelf life and stability of foods and beverages.

Animals↗

PubMed1995

Influence of amino acids on the formation of mutagenic/carcinogenic heterocyclic amines in a model system.

Mixtures of creatinine, glucose and various single amino acids were heated at 180 degrees C for 10 min in an aqueous model system. The heated mixtures all showed mutagenic activity, ranging from 80 to 2400 TA98 revertant colonies/mumol creatinine with metabolic activation. Testing of HPLC fractions for mutagenic activity showed each mixture to contain several mutagenic components, some of which corresponded to known heterocyclic amines and others to unknown compounds. The presence of 2-amino-3-methyl-imidazo[4,5-f]quinoxaline, 2-amino-3,8-dimethylmidazo[4,5-f]quinoxaline and 2-amino-3,7,8-trimethylimidazo[4,5-f]quinoxaline in most of the samples was established using HPLC with photodiode array detection and liquid chromatography/mass spectrometry with electrospray interface and single ion monitoring. In addition, 2-amino-3,4,8-trimethylimidazo[4,5-f]quinoxaline, 2-amino-1-methyl-6-phenylimidazo[4,5-f]quinoxaline, 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine, 3-amino-1,4-dimethyl-5H-pyrido[4,3-b]indole and 3-amino-1-methyl-5H-pyrido[4,3-b]indole and the co-mutagenic compounds 9H-pyrido[3,4-b]indole and 1-methyl-9H-pyrido[3,4-b]indole were detected in some samples.

Amines↗

PubMed1995

Metabolism of the food-derived carcinogen 2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline (MeIQx) in nonhuman primates.

The metabolism and disposition of the food mutagen and rodent carcinogen 2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline was investigated in cynomolgus monkeys. Monkeys were administered a single dose of radiolabeled [14C]MeIQx (2.2 or 50 mumol/kg). Peak blood levels of radioactivity were observed within 1-3 h after dosing and declined rapidly thereafter. By 72 h after dosing, approximately 50% and 70% of the 2.2 mumol/kg, and 50 mumol/kg dose, respectively, was excreted in the urine. Approximately 15-20% of either dose was recovered in the feces. Eight metabolites and the parent compound were detected in urine by HPLC. The parent compound accounted for approximately 15-25% of the dose excreted in the urine. Seven MeIQx urinary metabolites were identified. Five metabolites were identical to MeIQx metabolites previously found in rats: MeIQx-N2-glucuronide, MeIQx-N2-sulfamate, MeIQx-5-sulfate, MeIQx-5-O-glucuronide, and 8-CH2OH-MeIQx-5-sulfate. Cynomolgus monkeys, however, metabolized MeIQx to a novel glucuronide conjugate of MeIQx not found in rats. Based upon mass spectroscopy and proton NMR analyses, the structure of this metabolite was consistent with an N1-glucuronide of MeIQx. This metabolite was the major urinary metabolite found in monkeys, accounting for 31-37% of the dose excreted in the urine over a 24 h period. One additional metabolite identified in urine and feces of MeIQx treated cynomolgus monkeys, that has not been found previously in any other animal model, was 7-oxo-MeIQx, a likely enteric bacterial metabolite of MeIQx. 7-Oxo-MeIQx accounted for 20-25% of the dose of MeIQx found in the urine and was the major fecal metabolite. The N2-glucuronide conjugate of the carcinogenic metabolite 2-hydroxyamino-3,8-dimethylimidazo[4,5-f]quinoxaline (NHOH-MeIQx) was not detected in urine or bile of monkeys, even after 10 daily doses of MeIQx (100 mumol/kg) were given. The results indicate that MeIQx is metabolically processed in monkeys via multiple pathways of detoxification. However, MeIQx is poorly metabolically activated via cytochrome P450 mediated N-oxidation. The in vivo metabolism of MeIQx in cynomolgus monkeys is different from that of the structurally related food-derived mutagen 2-amino-3-methylimidazo[4,5-f]quinoline (IQ), which is readily metabolically activated by this species and in contrast to MeIQx, has been shown to be a powerful hepatic carcinogen.

Animals↗

PubMed1995

Metabolism of heterocyclic aromatic amines and strategies of human biomonitoring.

The metabolism of 2-amino-3-methylimidazo[4,5-f]quinoline (IQ) and 2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline (MeIQx), two carcinogenic heterocyclic aromatic amines (HCAs) formed in cooked meats and fish, was studied in animal models and in vitro with human tissues to develop strategies for human biomonitoring. Both IQ and MeIQx are rapidly absorbed from the gastrointestinal tract of rodents and transformed into a number of products which are excreted in urine and feces. Detoxification occurs via cytochrome P450 mediated ring hydroxylation at the C-5 position followed by conjugation to sulfuric or beta-glucuronic acid. Other routes of detoxification include N2-glucuronidation and the uncommon pathway of sulfamate formation; N2-acetylation, however, appears to be a minor pathway. Metabolic activation by N-oxidation was demonstrated through formation of the metastable N2-glucuronide conjugate of the mutagenic N-hydroxy metabolites. These conjugates are excreted preferentially in urine rather than bile. Many of these metabolic pathways also exist in nonhuman primates. The binding of IQ and MeIQx to blood proteins is low and thus, human biomonitoring through protein adducts may be difficult. Human liver can metabolically activate HCAs by cytochrome P450 mediated N-oxidation and can also catalyze the detoxification of IQ and MeIQx through sulfamate formation; however, HCAs appear to be poor substrates for N-acetyltransferase. Preliminary data have shown that humans excrete both the N2-sulfamate and N2-glucuronide of MeIQx in urine following consumption of cooked meat. Thus, rodents, nonhuman primates and humans appear to have several common routes of HCA biotransformation.

Animals↗

PubMed1993

Determination of taurine metabolism by measurement of 15N-enriched taurine in cat urine by gas chromatography-mass spectrometry.

To understand the biological function of taurine, a study of taurine kinetics in the cat was undertaken. This paper describes a method developed for the accurate determination of 15N-taurine enrichment in cat urine by gas chromatography-mass spectrometry. 15N-Taurine was given to six animals as an oral bolus dose of 20 mg/kg body weight, and the urine was pooled on a daily basis. The hydrolysed or non-hydrolysed urine samples (for total and free taurine, respectively) were directly derivatized without further purification. The N-pentafluorobenzoyl di-n-butyl amide derivative obtained was analysed, and the fragment [M-(di-n-butyl amide)]+, carrier of the labelled nitrogen atom, was selectively recorded at m/z 302 (14N-taurine) and m/z 303 (15N-taurine). Calibration curves prepared in hydrolysed and non-hydrolysed urine samples spiked with 15N-taurine gave similar slopes to the calibration curve prepared in water. The average coefficient of variation observed for the mole percent excess in the non-hydrolysed samples was 1.22% (n = 92) and for the hydrolysed urine 1.00% (n = 98). There was no significant difference between free and total taurine enrichment. The half-life of taurine in cat body was found to be 29.3 +/- 2.9 h and 35.0 +/- 1.4 h for free and total taurine, respectively (non-significant). The taurine body pool, calculated by extrapolation of the curve to zero time, had a value of 137 +/- 22 ng/kg and 157 +/- 11 mg/kg for free and total taurine, respectively.

Animals↗