Effects of diet composition on mutagenic activity in urine

The effects of dietary habits on mutagenic activity in urine were investigated using the umu test based on the use of the genetically engineered bacteria Salmonella typhimurium TA 1535 pSK1002. Genotoxic effects in sample urine were detected by measuring the activation of the SOS response in the bacteria and recording the beta- galactosidase activity. Human subjects consisted of smokers and non-smokers. Urine from subjects who consumed fish showed the highest mutagenic activity, followed by the urine samples from subjects who ate pork or beef. Chicken induced a low level of mutagenic activity. When the subjects ate fried or roasted animal foods, the urine samples gave higher mutagenicity than the urine samples from the subject who consumed non-fried or non-roasted animal foods. When the subject ate vegetables along with a diet rich in animal foods, the activity in urine decreased. Herbs and spices gave the same tendency toward decline as vegetables. Non-smoker urine shower mutagenic activity than samples from smokers.     

Meat meal in the diet of the early-weaned pig IV. The supplementation of diets with tryptophane,lysine and methionine

Two experiments were conducted with 72 pigs between 28 and 56 days of age to study the effect of tryptophane supplementation on their performance when fed on diets containing wheat and meat meal.In the first experiment, pigs were fed on a basal diet (Diet 1) or on the same diet supplemented with calcium dihydrogen phosphate (Diet 2), bone meal (Diet 3) or bone meal plus tryptophane (Diet 4), all to 3.1% calcium. The weight gains of the pigs (315 g day^?1) fed on Diet 3 were significantly lower than that of the pigs fed on the other three diets (363 g day^?1). The feed conversion ratios showed a similar trend. Diet 3 contained 0.16% tryptophane while the other diets contained 0.18–0.19% tryptophane. The crude protein, lysine and methionine contents of all diets were similar.In the second experiment, a basal diet containing meat meal and bone meal was supplemented with tryptophane, lysine plus methionine or all three amino acids. Feed intake was increased by all amino acid supplements. Weight gains were improved significantly (57%) by the addition of all three amino acids to the diets, but the improvements due to tryptophane alone (28%) or methionine plus lysine (35%) were not significant. Tryptophane supplementation alone or with lysine plus methionine increased the nitrogen retention of the pigs.It was concluded that the requirement for tryptophane of pigs between 28 and 56 days of age was greater than 0.16% of diets containing wheat and meat meal.     

Determination of urinary and salivary cotinine using gas and liquid chromatography and enzyme-linked immunosorbent assay

The objective of this study was to compare cotinine concentrations in urine and saliva using gas chromatography (GC), high-performance liquid chromatography (HPLC) and enzyme-linked immunosorbent assay (ELISA). Ninety-four subjects were selected (27 smokers and 67 non-smokers) and interviewed using questionnaire. Of the non-smokers, 39 had been exposed to environmental tobacco smoke (ETS) and 28 had not been exposed to ETS. Cotinine levels among smokers were highest using all three measurements, followed by ETS exposed subjects and non-smokers. Cotinine levels in urine, using HPLC, correlated significantly with levels measured using ELISA (r=0.92) and GC-nitrogen-phosphorus detection (NPD) (r=0.92). Salivary cotinine levels measured using ELISA did not correlate significantly with either HPLC (r=0.37) or GC-NPD (r=0.33) measurements. Multiple regression models were used to adjust for age, gender, drug use and health status, and it was found that cotinine levels in urine and saliva were significantly correlated with smoking pack-year. The authors conclude that urinary cotinine concentration is a more accurate biomarker for ETS than salivary cotinine concentration.     

Urinary mutagenicity in nonsmokers following exposure to fresh diluted sidestream cigarette smoke

Ten healthy male and 10 healthy female 'never-smoking' subjects (ages 21-50) participated in a 5-day environmental room study to determine if an acute exposure to a high level of fresh diluted sidestream smoke (FDSS) would alter urinary mutagenicity. On Monday, Tuesday, Thursday and Friday, the 20 subjects sat in environmental rooms for 7.33h and were exposed to filtered and humidified air. On Wednesday, the 20 subjects were exposed in the environmental rooms for 7.33h to an average respirable suspended particle (RSP) concentration of 179 microg/m(3) of FDSS generated by machine smoking 1R4F Kentucky reference cigarettes. This level of FDSS is approximately three times the ETS level seen in the top 5% of US workplaces which allow smoking. A cumulative 7.33h air sample from each environmental room was collected and determined to be mutagenic by Ames Salmonella assay. Subjects' urinary mutagenicity was measured on Wednesday as compared with Tuesday or Thursday by assaying concentrates of 24h urine samples in Ames Salmonella bacterial strains TA98 and YG1024. Diet was strictly controlled on all study days, with broiled and pan-fried meat not served to minimize ingestion of mutagenic protein pyrolysis products. Although all the urinary mutagenicity values were within the range reported for minor changes in diet, the subjects experienced a small but statistically significant increase (p<0.05) in urinary mutagenicity in strain YG1024, but not in the less sensitive strain TA98 on the day of FDSS exposure.     

Influence of fried meat and fiber on cytochrome P-450 mediated activity and excretion of mutagens in rats

Fried meat was included in the diet of Sprague-Dawley rats during one week. Ingested and excreted amounts of mutagenic activity were determined daily by the use of the Ames' Salmonella/mammalian microsome test on extracts of the diet, urine and feces. In addition, the effect of the fried meat on ethoxyresorufin-O-deethylase activity in the small intestine and in the liver was measured. The results were compared to those from a control group of rats receiving boiled instead of fried meat as their source of protein. The diet containing boiled meat was not mutagenic and none of the samples from the control group, neither urine nor feces, contained any mutagenicity. The activity of ethoxyresorufin O-deethylase in the small intestine was increased by fried meat whereas this enzyme activity in the liver was unaffected. O-deethylation of ethoxyresorufin is catalyzed by cytochrome P-450 dependent enzyme(s) and induction of the enzyme activity might indicate an increased metabolic activation of premutagens and precarcinogens in the intestine. It is not yet known whether the mutagens excreted by the animals receiving the fried meat diet represent unmetabolized dietary mutagens or are formed as a result of metabolic activation of compounds in fried food. Dietary fibers i.e. wheat bran, pectin, cellulose or ViSiblin were included in the above-described diets and were shown to lower the mutagenic activity in urine and feces of the rats. Pectin significantly increased the hepatic ethoxyresorufin-O-deethylase and decreased the 16 alpha-hydroxylation of 4-androstene-3,17-dione.     

Relationship between FTC 'tar' and urine mutagenicity in smokers of tobacco-burning or Eclipse cigarettes

The US Federal Trade Commission (FTC) classifies domestic cigarettes into one of three 'tar' categories based on 'tar' and nicotine levels. The objective of the present study was to determine urine mutagenicity in groups of smokers of ultra-low 'tar' (ULT), full-flavor low 'tar' (FFLT) and full-flavor 'tar' (FF) filtered cigarettes after switching to primarily tobacco-heating Eclipse cigarettes. Sixty-seven smokers maintained a specified diet and consumed ad libitum their usual brands of cigarettes, switched to Eclipse, and switched back to their usual brands. Twenty-four hour urine samples were collected weekly, concentrated on XAD-2 resin, and tested in the Ames mutagenicity assay using bacterial strains TA98 and YG1024 with S9 metabolic activation. Daily consumption of cigarettes was not significantly different (at P<0.05) between FTC 'tar' categories and average daily cigarette consumption did not change significantly in any smoker group after switching to Eclipse cigarettes. Average urine mutagenicity was 47% less (P<0.05) for ULT than for FFLT usual brand smokers as measured by the more sensitive strain YG1024, although no significant differences (P<0.05) were observed in urine mutagenicity between usual brand FTC 'tar' categories as measured by strain TA98. The reduction in urinary mutagens in the more sensitive strain, YG1024, observed in ULT smokers as compared with higher 'tar' categories suggest reduced exposure to mutagens. Usual brand salivary cotinine in the ULT group was significantly lower (P<0.05) than the FF group and the FFLT group. Salivary cotinine did not differ significantly (at P<0.05) among the smoker groups when smoking Eclipse compared to usual brand. After switching to Eclipse, the following reductions in urinary mutagenicity were observed: ULT, 70.1+/-6.4% (TA98), 70.9+/-6.2% (YG1024); FFLT, 77.1+/-2.4% (TA98), 73.6+/-2.0% (YG1024); and FF, 76.1+/-3.5% (TA98), 71.4+/-4.0% (YG1024). Across all 'tar' categories, cigarette smokers experienced significant reductions (P<0.05) in urine mutagenicity, but not salivary cotinine, upon switching to Eclipse. The reduction in urine mutagenicity when smoking Eclipse provides supporting evidence that Eclipse may present less risk of cancer compared to cigarettes currently in the market.     

Mutagen levels in urine from snuff users, cigarette smokers and non tobacco users--a comparison

The mutagenic activity of concentrates of urine from snuff users, cigarette smokers and non tobacco users has been investigated. A concentration procedure involving use of Sep-Pak C18 columns and elution with methylene chloride was used. The concentrates were assayed for mutagenicity towards strain TA98 of Salmonella typhimurium, both in the presence and absence of a metabolic activation system, the post-mitochondrial liver fraction (S9) from Aroclor 1254 induced rats. The mean mutagenic activity of smokers' urine concentrates was 8.6 X 10(3) revertants per 24 h and significantly higher than the corresponding values for snuff users, abstinent snuff users and non tobacco users, which were (1.3, 1.3 and 0.9) X 10(3), respectively. No significant difference in mutagenic activity was found between urine from snuff users, whether using or abstaining from snuff, and urine from non tobacco users. It could thus be concluded that the level of urinary mutagens, isolated by adsorption on Sep-Pak C18 columns, is not elevated by habitual usage of Swedish wet snuff.     

Mutagenicity of food pellets from human diets in The Netherlands

Food pellets from human diets, prepared according to mean consumption figures in The Netherlands, were assessed on mutagenicity and mutagens were identified. Three types of human meals were compared: raw (C), heated (D) and heated with vegetables and fruit (E, a complete meal). In addition 2 animal diets were tested: commercial control diet (A), and a control diet to which vegetables and fruit had been added (B). All human diets contained: 40.6 energy (E)% fat, 13.2 E% protein, 46.2 E% carbohydrate and 5.2% (w/w) fibre. For animal diets these figures were 21.6, 26.0, 52.4 and 10.7% respectively. After extraction samples were tested in the Salmonella-microsome test, tester strains TA1538, TA98 and TA100. Human diets with heated products (D, E) were both clearly mutagenic with approximately 300-500 revertants per gram. Food pellets from animal diets (A, B) displayed no mutagenic activity. HPLC-derived chromatographic fractions of diets D and E showed 3 large mutagenic areas identified as IQ (2-amino-3 methyl-imidazo-[4,5-f]quinoline) and MeIQx (2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline, DiMeIQx (2-amino-3,4,8-trimethylimidazo[4,5-f]quinoxaline and PhIP (2-amino-6-phenylimidazo[4,5-b]pyridine) and other mutagens not completely defined. This mutagen profile was similar to that found previously for fried beef. Mass estimates for these potent mutagens amounted to 15-20 micrograms/kg. Health implications of these findings are discussed. As IQ, MeIOx and DiMeIQx have been found to be weakly carcinogenic in rodents and many other initiating and modulating factors may be present in a complex human diet, a chronic toxicity study is indicated.     

Stress reactivity and its relationship to beef quality in Nguni steers supplemented with Acacia karroo leaves

The objective of this study was to determine the levels of catecholamines and their relationship to beef quality in Nguni steers fed on Acacia karroo leaves. A total of 30 19-month-old steers were randomly assigned to A. karroo leaves (AK), sunflower cake (SF) and the control with no supplement (CN) diets. The AK and SF diets provided the steers with an additional 150 g of protein per day for 60 days. Catecholamine levels were determined from urine samples collected from each steer before and after slaughter. The Musculus longissimus thoracis et lumborum was sampled for selected meat quality measurements. Nguni steers on the CN diet had higher (P < 0.05) concentrations of post-mortem urinary norepinephrine and dopamine compared with those that received the AK and SF diets. Norepinephrine was negatively linearly related (P < 0.05) to the Warner-Bratzler shear force value of meat aged for 21 days and cooking loss of meat aged for 2 days (CL2) in steers that were given the SF diet. Meat pH and drip loss values were inversely related (P < 0.05) to epinephrine concentration in steers that received the AK diet. Dopamine concentration was negatively linearly related (P < 0.05) to water holding capacity and CL2 for steers on the CN diet. For steers on the CN diet, lightness (L*) values increased (P < 0.05) with increase in dopamine concentration. It was concluded that stress responsiveness and its relationship to certain beef quality attributes could be positively manipulated by supplementation with A. karroo leaves.     

Association of 1 hydroxypyrene glucuronide in human urine with cigarette smoking and broiled or roasted meat consumption

Humans are exposed to polycyclic aromatic hydrocarbons PAHs from various occupational, dietary, environmental and medicinal sources. We measured 1 hydroxypyrene glucuronide 1 OHP gluc concentration in urines from male non smokers n = 50 , smokers of blond tobacco n = 31 , smokers of black tobacco n = 16 , and pipe smokers n = 3 . Immunoaffinity chromatography was used as a preparative step and synchronous fluorescence spectroscopy as the quantitation method. The concentration of 1 OHP gluc in urine from smokers mean SE: 1.04 0.13 pmol ml-1 urine was significantly higher than in urine from non smokers 0.55 0.05 pmol ml-1 urine by the Wilcoxon rank sum test non smokers versus all smokers, p = 0.001; vs black tobacco smokers, p = 0.001; vs blond tobacco smokers, p = 0.007 . Urinary 1 OHP gluc concentration among subjects who had consumed roasted, grilled or broiled meat within the past 24 h was elevated compared with those who had not p = 0.025 . Multiple linear regression showed significant associations of urinary 1 OHP gluc with number of cigarettes smoked p = 0.002 and consumption of roasted, grilled or broiled meat p = 0.028 . Systemic CYP1A2 activity estimated by caffeine metabolism was significantly correlated with urinary 1 OHP gluc concentration. However, this association was probably due to cigarette smoking, since adjusting for cigarette smoking by multiple linear regression made the association between urinary 1 OHP gluc and CYP1A2 phenotype non significant. These results further support the use of urinary 1 OHP gluc as a biomarker of recent pyrene exposure through inhalation or diet.     

A column-switching liquid chromatography-tandem mass spectrometry method for quantitation of 2-cyanoethylmercapturic acid and 2-hydroxyethylmercapturic acid in Chinese smokers

The acrylonitrile metabolites 2-cyanoethylmercapturic acid (CEMA) and 2-hydroxyethylmercapturic acid (HEMA) have been determined in human urine using an automated column-switching procedure. A diluted sample was centrifuged just prior to being injected into a reusable precolumn packed with a restricted access material and coupled to a liquid chromatography-tandem mass spectrometry system. This method achieved satisfactory reproducibility and accuracy. Average intra- and interday variations (% relative standard deviations) ranged from 2.4 to 3.8% for CEMA and from 2.7 to 10.5% for HEMA. The limits of quantification were 0.003 and 0.099ng/ml for CEMA and HEMA, respectively. It was used to study the uptake of acrylonitrile from smoke constituents by both nonsmokers and smokers of different tar yield cigarettes under ISO 3308 smoking condition. Metabolite concentrations in smoker urine samples were approximately 12 times higher compared with those in nonsmokers for CEMA and 3 times higher for HEMA. Urinary CEMA levels show a clear dose-response relationship with daily cigarette consumption and urinary cotinine. CEMA can also discriminate between smokers of different ISO cigarettes. Because HEMA is not specific, it is only slightly related to smoking and acrylonitrile exposure. The validated biomarker CEMA will continue to be useful for studies of acrylonitrile uptake by smokers.     

Quantification of cotinine in dried blood spots as a biomarker of exposure to tobacco smoke

Objective: We present an ultra-sensitive, minimally-invasive method for quantifying cotinine in dried blood spot (DBS) samples as a biomarker of exposure to tobacco smoke that can be collected using a simple heel or finger prick to obtain blood samples.

Methods: Cotinine levels were measured in matched plasma and reconstituted DBS samples from smokers and nonsmokers to evaluate assay parameters. In addition, we applied this new method to finger-prick DBS samples that were collected from infants, children and young adults ages 1–21 to estimate exposure to tobacco smoke. Partitioning of cotinine across red blood cells and haematocrit effects were investigated.

Results: Cotinine levels measured in matched plasma and reconstituted DBS samples from smokers and nonsmokers were found to be highly correlated (R²=0.94), with 100% sensitivity and 94% specificity to differentiate reported smokers from nonsmokers. With this method, the LOQ is <0.25?ng/mL using a single 3.2?mm punch of a DBS, and haematocrit effects are negligible.

Conclusions: This sensitive, high-throughput and minimally-invasive method for quantifying cotinine in DBS samples provides a simple and cost effective means for estimating exposure to tobacco smoke in population based studies, and has particular advantages in studies involving infants and children.     

Effect of nutritional status on mutagenicity of urine excreted by rats treated with standard/experimental carcinogens

Urine samples, collected from Sprague Dawley rats treated with extracts of tobacco/masheri, benzo (a) pyrene, N'-nitrosonornicotine, N'-nitrosodiethylamine and maintained on semi-synthetic diets sufficient or deficient in Vitamin A, B and protein were tested for mutagenicity using Salmonella/microsome assay. The mutagenic activity of urine or various treated groups was in the order deficient diet greater than standard laboratory diet greater than nutritionally sufficient diet. Present results confirmed the earlier observations that nutritionally deficient animals are likely to have more exposure to mutagenic metabolites that are generated by increased phase I enzymes and decreased detoxification system.     

Efficacy of High-Fiber Diets in the Management of Type 2 Diabetes Mellitus

ObjectiveTo review outcomes of randomized controlled clinical trials exploring the efficacy of different types of diets containing various amounts of fiber in the management of type 2 diabetes mellitus.MethodsWe searched PubMed, Medline, and Google Scholar for published data from the past decade (through December 2009) on dietary patterns and risk of type 2 diabetes mellitus. Only randomized controlled trials investigating the effect of whole grains, fiber, or vegetarian diets on type 2 diabetes were included. Search criteria included whole grain, fruit, vegetable, fiber, and meat intake regarding insulin sensitivity and glycemic responses in healthy, prediabetic, and diabetic persons.ResultsA total of 14 randomized clinical trials were included. Addition of insoluble or soluble fiber to meals, increased consumption of diets rich in whole grains and vegetables, and vegan diets improve glucose metabolism and increase insulin sensitivity. The greatest improvement in blood lipids, body weight, and hemoglobin A_1c level occurred in participants following low-fat, plant-based diets.ConclusionsIncreased consumption of vegetables, whole grains, and soluble and insoluble fiber is associated with improved glucose metabolism in both diabetic and nondiabetic individuals. Improvements in insulin sensitivity and glucose homeostasis were more evident in participants following a plant-based diet compared with other commonly used diets. (Endocr Pract. 2011;17:132-142)     

Just eating healthier is not enough: studying the environmental impact of different diet scenarios for Dutch women (31–50 years old) by linear programming

Purpose

Eating healthier or vegetarian and vegan diets are suggested options to reduce the environmental impact of the current diet. In this paper, we demonstrate a method that is able to identify diets with reduced environmental impact and are more similar to the current diet than predetermined scenarios. All diets were adequate and consisted of commonly available foods.

Methods

We used linear programming to find solutions that are as close as possible to the current diet of an average woman with age 31–50, first without any food groups’ constraints and later by imposing constraints on meat, fish, dairy, and eggs. Finally, we use a similar technique to search for the closest diet that achieves the same environmental reduction as the most restricted option (no meat, fish, dairy, or eggs), without restrictions on products. In the optimization algorithm, we incorporate popularity of food products in order to design menus which are feasible for the Dutch population.

Results and discussion

The results show that simply eating according to guidelines does not guarantee a diet with an improved environmental profile. Removing meat and fish from the diet reduces the environmental impact by about 21 %. A healthy vegan diet reaches 30 % environmental impact reduction, but leads to a diet with many changes in comparison to a typical Dutch diet and without meeting one of the health constraints (EPA?+?DHA—Eicosapentaenoic acid?+?Docosahexaenoic acid). We show that it is possible to find less restrictive solutions than vegetarian or vegan diets that still satisfy all nutritional requirements and have less environmental impact than the current one.

Conclusions

Just eating healthier is not enough in order to reduce environmental impact. However, designing a diet that meets dietary requirements must be a prerequisite for sustainable diets. Simply removing products from a diet can have as consequence that other products have to be added to compensate for the nutritional imbalances. We show, by using linear programming, that it is possible to reach 30 % reduction in the environmental impact with a diet which is relatively similar to the current one and could be more likely to be accepted.

     

Metals content in placentas from moderate cigarette consumers: correlation with newborn birth weight

Cigarette consumption during pregnancy produces deleterious effects in both, mother and fetus, some of them due to the presence of toxic elements in cigarette smoke, such as cadmium. Placenta constitutes a dual-purpose specimen for evaluating the pollutant burden exerted on the mother as well as on the fetus. The main objective of this study was to establish a correlation between placental concentration and distribution of some metal elements and birth weight of neonates delivered by mothers, who were either moderate smokers or nonsmokers. Forty nonsmoking and moderate smoking pregnant women paired per age, parity, weight, height and body mass index were selected. Smoking was assessed by self-reported cigarette consumption during pregnancy and urine cotinine concentration before delivery. Placental metal concentrations were evaluated by atomic absorption spectrometry (copper and cadmium) and neutron activation analysis (zinc and iron). Newborns from smokers had lower birth weights compared to infants from nonsmokers. Birth weights were correlated with placental cadmium concentrations in both, smokers and nonsmokers. Placental zinc and cadmium of smokers were mainly located at the maternal side and their levels were higher than those found in nonsmokers placentas. In addition, all metal nutrient/pollutant ratios were decreased in the smoker group. In this first study performed in our region, we found that moderate smoking mothers deliver neonates with decreased birth weight and highly correlated to placental cadmium concentration. Decreased metal nutrient/pollutant ratios, a condition here found in smokers, may indicate a placental dysfunction, contributing to impair birth weight.     

Serum Clara cell protein (CC16) in healthy young smokers

The CC16 microprotein is the main secretory product of Clara cells, which are epithelial cells lining lung airways. In crossing through the bronchoalveolar/blood barrier, CC16 diffuses passively into plasma. Serum CC16 (sCC16) has recently been proposed as a biomarker for detecting Clara cell impairments. The aim of this study was to assess if sCC16 concentrations are reduced in a group of healthy young smokers. A group of 118 healthy young males volunteered to take part in the study. Each subject answered a questionnaire, and provided blood and urine samples. Serum CC16, urinary cotinine and creatinine were measured. Median serum CC16 concentrations were lower in smokers than in non-smokers (11.3 mug l^-1 vs 14.6 mug l^-1; p = 0.005; N = 89 and 29, respectively) but did not correlate with either the daily or the life-time cigarette consumption, or with urinary cotinine concentrations. sCC16 did not correlate with age or body mass index in the whole study population or in the groups of smokers and non-smokers. These results suggest the reduction in sCC16 concentrations in a group of healthy young smokers may be an early effect of cigarette smoking.     

The effect of short-term dietary modification on human fecal mutagenic activity

To assess the effect of short-term modification of diet on human fecal mutagenic activity, 6 subjects consumed 2 dietary regimes hypothesized to affect risk of colorectal cancer. After a 7-day baseline period, a 'low-risk' non-meat diet was consumed for 2 weeks followed by 2 weeks on a 'higher risk' diet which emphasized beef and refined grains. Fecal samples were collected at the end of each diet period and assayed for direct-acting mutagens with the fluctuation test for weak mutagens using Salmonella typhimurium TA100 and TA98 as tester strains. Fecal mutagenic activity on TA100 was increased for all subjects during the 'higher risk' period compared to the 'low risk' period. The average mutagenicity on TA98 was also increased, but the trend was not consistent for all subjects. The baseline diet and non-meat diet resulted in approximately equal mean fecal mutagenicity levels. These findings indicate that a diet high in meat and refined grain, as characterized here, increases fecal mutagenic activity within a 2-week period.     

Quantitation of isoprostane isomers in human urine from smokers and nonsmokers by LC-MS/MS

A simple, rapid liquid chromatography-tandem mass spectrometry method was developed to identify and quantitate in human urine the isoprostanes iPF(2 alpha)-III, 15-epi-iPF(2 alpha)-III, iPF(2 alpha)-VI, and 8,12-iso-iPF(2 alpha)-VI along with the prostaglandin PGF(2 alpha) and 2,3-dinor-iPF(2 alpha)-III, a metabolite of iPF(2 alpha)-III. Assay specificity, linearity, precision, and accuracy met the required criteria for most analytes. The urine sample storage stability and standard solution stability were also tested. The methodology was applied to analyze 24 h urine samples collected from smokers and nonsmokers on controlled diets. The results for iPF(2 alpha)-III obtained by our method were significantly correlated with results by an ELISA, although an approximately 2-fold high bias was observed for the ELISA data. For iPF(2 alpha)-III and its metabolite 2,3-dinor-iPF(2 alpha)-III, smokers had significantly higher concentrations than nonsmokers (513 +/- 275 vs. 294 +/- 104 pg/mg creatinine; 3,030 +/- 1,546 vs. 2,046 +/- 836 pg/mg creatinine, respectively). The concentration of iPF(2 alpha)-VI tended to be higher in smokers than in nonsmokers; however, the increase was not statistically significant in this sample set. Concentrations of the other three isoprostane isomers showed no trends toward differences between smokers and nonsmokers. Among smokers, the daily output of two type VI isoprostanes showed a weak correlation with the amount of tobacco smoke exposure, as determined by urinary excretion of total nicotine equivalents.