Analysis of 8-hydroxydeoxyguanosine among workers exposed to diesel particulate exhaust: comparison with urinary metabolites and PAH air monitoring

Oxidative DNA damage and repair, as measured by 8-hydroxy-2'-deoxyguanosine (8-OHdG) in urine and DNA samples were studied in association with work-related diesel exhaust exposure among garage and waste collection workers. Seasonal variations of the urinary 8-OHdG levels in pre- and two post-workshift urine samples of 29 exposed workers and 36 control persons were evaluated. The mean±SE levels of post-workshift 8-OHdG (μmol/mol crea) were 1.52±0.44 in winter and 1.61±0.33 in summer for the exposed workers, and 1.56±0.61 in winter and 1.43±0.49 in summer for the controls, respectively. No significant difference in the urinary 8-OHdG levels between exposed workers and control subjects in winter (p=0.923) and summer (p=0.350) was observed. A linear mixed model, adjusted for years of employment, age, ex/non-smoking and BMI, indicated no significant dose exposure-relationships between the urinary 8-OHdG and 15 PAH air concentrations nor between the 8-OHdG and 7 PAH monohydroxy-metabolites analyzed in the same workers. 8-OHdG was also analyzed in the mononuclear cell DNA of 19 exposed and 18 control subjects. The mean value of 8-OHdG/non-modified 2'-deoxyguanosine (8-OHdG/105 dG±SE) were 4.89±0.17 for the exposed and 4.11±0.16 for the control persons, which showed no correlation with the urinary 8-OHdG levels (r=0.01, n=28, P=0.96). The PAH exposure at workplaces was mainly composed of volatile compounds, particularly naphthalene, suggesting low exposure through the respiratory tract and a low effect of PAH in ROS induction.     

Urinary excretion of 8-hydroxy-2'-deoxyguanosine measured by high-performance liquid chromatography with electrochemical detection

There is good evidence that oxidative DNA damage permanently occurs in living cells. The oxidative DNA damage product 8-hydroxy-2'-deoxyguanosine (8-OHdG) is one of the predominant forms of radical-induced lesions to DNA, and has therefore been widely used as a biomarker for oxidative stress, either in cellular DNA or as DNA repair product in urine. In this paper we describe the use of a high-performance liquid chromatographic procedure with electrochemical detection for the measurement of urinary 8-OHdG. Our study has addressed the questions (i) of baseline urinary levels of 8-OHdG in spot urine and 24-h urine, (ii) of inter- and intra-individual variation of this biomarker, and (iii) of confounding factors for the excretion of 8-OHdG. No significant difference between the mean group levels of 8-OHdG/creatinine in spot urine (2.03+/-1.21 micromol/mol, n=148) and in 24-h urine (1.86+/-1.09 micromol/mol, n=67) was observed. However, when only 24-h urine was used for analysis, 8-OHdG was found to be statistically significantly higher in smokers. By multiple linear regression analysis, urinary creatinine was identified as the only predictor of 8-OHdG/24 h (r(p)=0.33, P=0.007). High intra-individual coefficients of variation of 8-OHdG/24 h were observed in two healthy subjects over a period of 10 consecutive days (37 and 57%, respectively), indicating that the intra-individual fluctuation of urinary 8-OHdG has so far been underestimated. Therefore, we suggest that single values of 8-OHdG should be considered with caution, in particular in small study groups and when spot urine is used.     

Urinary levels of 8-hydroxydeoxyguanosine as a marker of oxidative damage in road cycling

We have determined the urinary 8-hydroxydeoxyguanosine (8-OHdG) levels of eight professional cyclists during a 4-day and a 3-week stage races. Monitoring of heart rates was used to establish zones corresponding to different intensities of exercise. The urinary 8-OHdG excretion, expressed by body weight, increased significantly in the first day or the first week of each race, respectively, and did not show further increases thereafter. Maximum 8-OHdG levels were reached in parallel to longer times spent at high intensities of exercise. Urinary excretion of creatinine increased with exercise, and changes in 8-OHdG levels were not detected when corrected by creatinine excretion. Serum glutathione concentrations did not change significantly at any point during exercise. We conclude that road cycling courses with an oxidative damage to DNA, which is sustained as long as the exercise is repeated. Both adaptation of antioxidant defenses and a decreased capacity to maintain a high intensity of effort may contribute to explain the absence of progressive increases in 8-OHdG excretion. The results of this study also confirm that the correction procedure using the amount of creatinine excreted should not be used when studying effects of exercise on urinary 8-OHdG.     

Urinary Levels of 8-Hydroxydeoxyguanosine as a Marker of Oxidative Damage in Road Cycling

We have determined the urinary 8-hydroxydeoxyguanosine (8-OHdG) levels of eight professional cyclists during a 4-day and a 3-week stage races. Monitoring of heart rates was used to establish zones corresponding to different intensities of exercise. The urinary 8-OHdG excretion, expressed by body weight, increased significantly in the first day or the first week of each race, respectively, and did not show further increases thereafter. Maximum 8-OHdG levels were reached in parallel to longer times spent at high intensities of exercise. Urinary excretion of creatinine increased with exercise, and changes in 8-OHdG levels were not detected when corrected by creatinine excretion. Serum glutathione concentrations did not change significantly at any point during exercise. We conclude that road cycling courses with an oxidative damage to DNA, which is sustained as long as the exercise is repeated. Both adaptation of antioxidant defenses and a decreased capacity to maintain a high intensity of effort may contribute to explain the absence of progressive increases in 8-OHdG excretion. The results of this study also confirm that the correction procedure using the amount of creatinine excreted should not be used when studying effects of exercise on urinary 8-OHdG.     

Production d'NH4 par la crevette Crangon crangon L. dans deux écosystémes côtiers. approche expérimentale et étude de l'influence du sédiment sur le taux d'excrétion

Estimation of the ammonia production of the shrimp C. crangon in two littoral ecosystems (oligotrophic sand and eutrophic mud) was determined in winter and summer conditions from laboratory observations in experimental microcosms. The ammonia excretion rate of C. crangon was not influenced by either the sediment type or the ammonia concentration of the overlying water; on the other hand, the mean excretion rate and the response to initial handling stress increased markedly as shrimp were deprived of soft substratum.

The daily ammonia production of C. crangon was 16 μmol NH₃ · g ⁻¹ wet wt · day ⁻¹ in winter and 40 μmol in summer. A gross production of 12 μmol NH₃ · m⁻² · day ⁻¹ and 300–700 μmol μ m⁻² · day⁻¹, respectively, could be expected in the two ecosystems studied. This would account for 5% (winter) and 2–4% (summer) of the total NH⁺₄ flux at the sediment-water interface. The contribution of the excretion of all macrofauna to the NH⁺₄ flux from the sediment is discussed.     

Validation of urinary thiocyanate as a biomarker of tobacco smoking

Thiocyanate ion (SCN) is the major detoxication product of cyanide, which is converted to SCN by a thiosulphate sulphurtransferase, mainly in hepatic mitochondria. Low-level cyanide exposure for man is caused by factors such as dietary intake of cyanogenic glucosides, tobacco smoking, drug administration and occupational exposure to organic nitriles. Urinary SCN concentration was determined through a commercial kit for the analysis of cyanide in water. Spot urine samples were collected at 7:30 h and 12:30 h, from 99 healthy male white-collar office workers (non-smokers n=72, smokers n=27). Comparison of SCN excretion values did not show any difference between the morning and midday samples. The SCN median value of non-smokers was 24 μmol l^-1 (range 9-24 μmol l^-1) and was statistically different from that of smokers (SCN = 92 μmol l^-1, range 33-275 μmol l^-1) (     

Urinary 8-oxo-7,8-dihydro-2'-deoxyguanosine and 5-(hydroxymethyl) uracil in smokers

Cigarette smoke is known to generate free radicals by various mechanisms. In this study involving 30 non-smokers and 30 smokers, we show that urinary excretion of 5-(hydroxymethyl) uracil (HMUra) was not different in the two groups (6.54±2.07 vs. 6.70±1.68 nmol/mmol creatinine). In contrast, 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxo-dGuo) excretion increased by 16% (1.16±0.35 vs. 1.35±0.50 nmol/mmol creatinine, p=0.039). Results concerning 8-oxo-dGuo are in agreement with those of previous studies. We observed significant multiple correlations between HMUra and creatinine (r_p=0.44), BMI (r_p=-0.27) and nicotine derivatives (r_p=0.26). Multiple correlation analysis showed relations between 8-oxo-dGuo on the one hand, and: creatinine (r_p=0.36), nicotine derivatives (r_p=0.29), BMI (r_p=-0.24) on the other.     

Evaluation of enzyme-linked immunosorbent assay and liquid chromatography-tandem mass spectrometry methodology for the analysis of 8-oxo-7,8-dihydro-2'-deoxyguanosine in saliva and urine

While ELISA is a frequently used means of assessing 8-oxo-7,8-dihydro-2-deoxyguanosine (8-oxodG) in biological fluids, differences in baseline urinary 8-oxodG levels, compared to chromatographic techniques, have raised questions regarding the specificity of immunoassays. Recently, ELISA of salivary 8-oxodG has been used to report on periodontal disease. We compared salivary 8-oxodG levels, determined by two commercial ELISA kits, to liquid chromatography-tandem mass spectrometry (LC-MS/MS) with prior purification using solid-phase extraction. While values were obtained with both ELISA kits, salivary 8-oxodG values were below or around the limit of detection of our LC-MS/MS assay. As the limit of detection for the LC-MS/MS procedure is much lower than ELISA, we concluded that the assessment of salivary 8-oxodG by ELISA is not accurate. In contrast to previous studies, ELISA levels of urinary 8-oxodG (1.67 ± 0.53 pmol/μmol creatinine) were within the range reported previously only for chromatographic assays, although still significantly different than LC-MS/MS (0.41 ± 0.39 pmol/μmol creatinine; p = 0.002). Furthermore, no correlation with LC-MS/MS was seen. These results question the ability of ELISA approaches, at present, to specifically determine absolute levels of 8-oxodG in saliva and urine. Ongoing investigation in our laboratories aims to identify the basis of the discrepancy between ELISA and LC-MS/MS.     

Urinary 8-Hydroxydeoxyguanosine in Infants and Children

Several diseases of prematurity are thought to be related to oxidative injury and many of the available markers are unsatisfactory. An assay was developed using HPLC with electrochemical detection for the quantitation of urinary 8-hydroxy-2′-deoxyguanosine (8-OHdG) as a proposed indicator for oxygen-derived free radical injury to DNA in preterm infants.

A median value of 3.79 pmol/mol creatinine was obtained for normal children (2–15 years old, n = 14). Urinary 8-OHdG excretion in neonates ranged from 0–99μmol/mol creatinine. There were no gestation or birthweight related differences in urinary 8-OHdG, and no correlation with urinary malondialdehyde. Mean 8-OHdG excretion increased with postnatal age (r= 0.80, p < 0.0001, n = 15), mirroring the growth velocity curve. These changes could also be due to changes in the activity of the enzyme responsible for 8-OHdG excision.

Urinary 8-OHdG levels are unlikely to accurately reflect oxygen derived free radical activity given the strength of the relationship with growth.     

Actigraphic sleep-wake patterns and urinary 6-sulfatoxymelatonin excretion in patients with Alzheimer's disease

Recent studies suggest melatonin, due to its antioxidant and free-radical- scavenging actions, may play a role in the neuroprotection against amyloid, which is implicated in the pathogenesis of Alzheimer's disease (AD). In this study, we determined urinary 6-sulfatoxymelatonin (aMT6s) excretion together with actigraphic sleep-wake patterns of untreated male patients with AD who lived at home. Results were compared with those obtained from normal age-matched elderly and normal young male subjects. Similar measurements were also performed in another group of patients with AD who were treated with a cholinesterase inhibitor (Donepezil, Aricept). Total 24h aMT6s values were significantly reduced in elderly controls (19.9h ± 5.2 μg/24h), in those with untreated AD (12.7 ± 4.4 μg/24h), and in patients treated for AD (12.4 ± 4.4 μ g/24h) compared with normal young men (32.8 ± 3.1 μ g/24h). A day-night difference in aMT6s was evident in all young controls, in 50% of elderly controls, in only 20% of patients with untreated AD, and in 67% of those with AD receiving Aricept. Sleep quality (expressed as sleep efficiency, wake time, and long undisturbed sleep duration) was better in young and elderly controls compared with the two groups of patients with AD. There was no significant correlation between aMT6s values or sleep patterns and the severity of cognitive impairment in patients with AD. Taken together, these data suggest that disrupted sleep, decreased melatonin production, and partial lack of day-night difference in melatonin secretion were observed equally in normal elderly and in patients with AD. Our results do not permit drawing any conclusion as to whether changes in urinary aMT6s excretion is correlated with disturbed sleep in patients with AD. (Chronobiology International, 18(3), 513-524, 2001)     

Urinary excretion of immunoreactive prostaglandin F2 alpha in healthy children and adults

The 24-hours urinary excretion of immunoreactive prostaglandin F2 alpha (U-iPGF2 alpha) in normal children on a free diet was not significantly different in 30 boys (aged 3-15 years; geometric mean 589 ng/24 h) compared to 27 girls (aged 4-14 years; mean 473 ng/24 h). In both sexes this excretion rose with age until adolescence where it reached a plateau. In normal adults the men had significantly higher (p less than 0.001) excretions of U-iPGF2 alpha than the women; also body weight and urinary creatinine excretion were higher in men (p less than 0.001). In the children, as well as in the total population, U-iPGF2 alpha correlated best with body weight (r = 0.44 and r = 0.48 respectively; p less than 0.001) and the urinary creatinine excretion (r = 0.53 and 0.57 respectively; p less than 0.001); both body weight and urinary creatinine excretion are reflections of total body development. After the correction for urinary creatinine excretion or for body weight, the sex difference in the adult U-iPGF2 alpha totally disappeared.     

Gas chromatographic determination of inorganic tin in rat urine after a single oral administration of stannous chloride and mono-, di-, and triphenyltin chloride

A method is described for the determination of inorganic tin by gas chromatography with flame photometric detection. The inorganic tins, stannous and stannic, were extracted with hydrochloric acid and n-hexane—benzene in the presence of 0.05% tropolone, and both inorganic tins were pentylated to tetrapentyltin with a Grignard reagent prior to gas chromatography. The absolute limit of detection for tetrapentyltin was 3 pg as tin. The recovery of stannous chloride added to rat urine samples was 80.2 ± 2.4% (mean ± S.D., n = 8). The application of this method to the study of urinary excretion of inorganic tin and organotin compounds in rats following oral administration of tin compounds is presented. The urinary excretion of tin compounds was observed over a period of 96 h following administration of stannous chloride or phenyltin compounds. Most of the inorganic tin was excreted into urine within 24 h after administration of stannous chloride. In the experiments on organotin administration, the level of the excretion as total tin for monophenyltin reached a maximum ca. 0–24 h after administration, whereas the maxima for di- and triphenyltin were found after 24–48 h and 48–72 h, respectively. The predominant excretion product of these tin compounds found in urine was monophenyltin.     

Modification of reperfusion-induced ionic imbalance by free radical scavengers in spontaneously hypertensive rat retina

We studied the effects of free radical scavengers, superoxide dismutase (SOD), vitamin E, and EGB 761, on ion shifts (Na⁺, K⁺, and Ca²⁺) induced by ischemia reperfusion in rat retina obtained from spontaneously hypertensive rats. Eyes were subjected to 90 min of retinal ischemia followed by 24 h of reperfusion. Two basic protocols were used: (1) chronic application, in which rats received SOD (7500, 15,000, and 30,000 U/kg, i.v.), vitamin E (50, 100, and 200 mg/kg, i.v.), and EGB 671 (50, 100, and 200 mg/kg, orally) for 10 d, respectively; and (2) acute administration, in which 7500, 15,000, and 30,000 U/kg of SOD, 50, 100, and 200 mg/kg of vitamin E, and 50, 100, and 200 mg/kg of EGB 761 were administered after an ischemic episode, at the onset of reperfusion, respectively. In the drug-free control group, 90 min ischemia followed by 24 h of reperfusion resulted in an accumulation of retinal sodium and calcium from their nonischemic control values of 76 ± 4 and 3.2 ± 0.1 μmol/g dry weight to 112 ± 6 (p < .001) and 6.2 (p < .001) μmol/g dry weight, respectively. Tissue potassium loss was also observed in this model of retinal ischemia reperfusion, and after 90 min ischemia followed by 24 h of reperfusion potassium content was significantly reduced from its nonischemic control value of 266 ± 5 to 207 ± 6 (p < .001) μmol/g dry weight. The chronic administration of SOD, vitamin E, and EGB 761 dose dependently reduced the reperfusion-induced ionic imbalance and improved the recovery of retinal ion contents. When these drugs were administered at the onset of reperfusion (acute administration), SOD and EGB 761 still significantly improved the recovery of retinal ion contents, but vitamin E failed to protect the ischemic reperfused retina. Our results indicate that the elimination of oxygen free radicals by free radicals scavengers may reduce the reperfusion-induced ionic imbalance and improve the ionic homeostasis in the injured retinal cells obtained from spontaneously hypertensive rats.     

Effects of supplemental oxygen on urinary 8-hydroxy-2’-deoxyguanosine levels in extremely low birth weight infants

Abstract

As the effects of supplementary oxygen on urinary excretion of 8-hydroxy-2’-deoxyguanosine (8-OHdG) are poorly understood, urinary 8-OHdG levels (ng/mg creatinine) were determined longitudinally on the postnatal day (PND) 1, 3, and 30 in 16 neonates with birth weight < 1000 g. No supplementary oxygen was required in 9 neonates during the first 24 h of life. Urinary 8-OHdG level on PND 1 was inversely correlated with birth weight in these 9 neonates (P = 0.0323) and was higher in four with birth weight < 750 g than five with birth weight > 750 g (41.0 ± 6.9 vs. 5.6 ± 2.7, respectively, P = 0.0200). Median urinary 8-OHdG on PND 1 of these 9 neonates was significantly lower than that of 7 neonates with oxygen (9.3 vs. 60.2, respectively), although there were no significant differences in clinical background, such as birth weight, between the two groups. Five of the 9 did not require supplemental oxygen at all during the first 30 days of life. Median urinary 8-OHdG levels were consistently significantly lower in the 5 neonates than in 11 neonates with oxygen transiently or persistently (9.3 vs. 54.6, 19.1 vs. 61.4, and 28.3 vs. 145 on PND 1, 3, and 30, respectively), although there were no differences in clinical background, such as birth weight, between the two groups. Urinary 8-OHdG on PND 30 was significantly positively correlated with supplemental oxygen dose on PND 30 (P < 0.0001), but not with birth weight in the 16 neonates. These results suggest that higher supplemental oxygen tension caused higher urinary 8-OHdG in this population.     

Urinary excretion of immunoreactive prostaglandin F2α in healthy children and adults

The 24-hours urinary excretion of immunoreactive prostaglandin F_2α (U-iPGF_2α) in normal children on a free diet was not significantly different in 30 boys (aged 3–15 years; geometric mean 589 ng/24 h) compared to 27 girls (aged 4–14 years; mean 473 ng/24 h). In both sexes this excretion rose with age until adolescence where it reached a plateau.In normal adults the men had significantly higher (p < 0.001) excretions of U-iPGF_2α than the women; also body weight and urinary creatinine excretion were higher in men (p < 0.001).In the children, as well as in the total population, U-iPGF_2α correlated best with body weight (r = 0.44 and r = 0.48 respectively; p < 0.001) and the urinary creatinine excretion (r = 0.53 and 0.57 respectively; p < 0.001); both body weight and urinary creatinine excretion are reflections of total body development. After the correction for urinary creatinine excretion or for body weight, the sex difference in the adult U-iPGF_2α totally disappeared.     

Quantitative determination of urinary 8-hydroxy-2'-deoxyguanosine level in healthy Japanese volunteers

8-hydroxy-2'-deoxyguanosine (8-OHdG), as a measure of oxidative stress, was measured in healthy Japanese volunteers using an ELISA (New 8-OHdG Check, JICA). Analysis of daytime spot urine of 83 healthy male subjects and smoking habit, exercise and age revealed significant correlation only between the urinary level of 8-OHdG and age. As the inter-individual variation of 8-OHdG of the daytime spot urine was relatively high, we next determined inter-and intra-individual variation of 5 healthy volunteers. The levels of 8-OHdG/creatinine in morning spot urine significantly correlated with 8-OHdG levels in 24-h pool urine. Thus, a morning spot urine sample can be used for the measurement of 8-OHdG instead of inconvenient 24-h sampling.     

Patients with dystrophinopathy show evidence of increased oxidative stress

Duchenne muscular dystrophy (DMD) is associated with an increase in oxidative stress. We measured 24 h 8-hydroxy-2'-deoxyguanosine (8-OHdG) excretion in 24 patients with MD (DMD + Becker's MD), 23 with myotonic dystrophy, and 34 healthy controls. The 8-OHdG/creatinine ratio was higher in patients with dystrophinopathy ( upward arrow 48%, p <.01) but not myotonic dystrophy, as compared to healthy controls. These results indicate that 8-OHdG excretion can be used as a marker of oxidative stress in clinical trials with dystrophinopathy.     

Evaluation of urinary 1-hydroxypyrene, S-phenylmercapturic acid, trans,trans-muconic acid, 3-methyladenine, 3-ethyladenine, 8-hydroxy-2'-deoxyguanosine and thioethers as biomarkers of exposure to cigarette smoke

The objective was to evaluate the utility of urinary 1-hydroxypyrene (1-OHP), S-phenylmercapturic acid (S-PMA), trans,trans-muconic acid (t,t-MA), 3-methyladenine (3-MeAd), 3-ethyladenine (3-EtAd), 8-hydroxy-2'-deoxyguanosine (8-OHdG) and thioethers as biomarkers for assessing the exposure in adult smokers who switched from smoking conventional cigarettes to candidate potential reduced exposure products (PREP) or who stopped smoking. Two electrically heated smoking systems (EHCSS) were used as prototype cigarettes that have significant reductions in a number of mainstream smoke constituents as measured by smoking machines relative to those from conventional cigarettes. Urine samples were collected from a randomized, controlled, forced-switching study in which 110 adult smokers of a conventional cigarette brand (CC1) were randomly assigned to five study groups. The groups included the CC1 smoking group, a lower-tar conventional cigarette (CC2) smoking group, EHCSS1 group, EHCSS2 group and a no smoking group that were monitored for 8 days. Biomarkers were measured at baseline and day 8. The daily excretion levels of these biomarkers were compared among the groups before and after switching, and the relationships between the daily excretion levels of these biomarkers and cigarette smoking-related exposure were investigated using Pearson product-moment correlation and multiple regression analyses. It was concluded that under controlled study conditions: (1) 1-OHP, S-PMA and t,t-MA are useful biomarkers that could differentiate exposure between smoking conventional and EHCSS cigarettes or between smoking conventional cigarettes and no smoking; between S-PMA and t,t-MA, the former appeared to be more sensitive; (2) 3-MeAd could only differentiate between smoking conventional cigarettes and no smoking; the results for 3-EtAd were not conclusive because contradictory results were observed; (3) 8-OHdG had a questionable association with smoking and therefore the utility of this biomarker for smoking-related exposure could not be established; and (4) urinary excretion of thioethers as a biomarker lacked sensitivity to demonstrate a clear dose-response relationship in conventional cigarette smokers, although it could differentiate the excretion levels between those subjects who smoked a conventional cigarette and those who stopped smoking.     

Circadian variation in oxidative stress markers in healthy and type II diabetic men

Seven clinically healthy, nondiabetic (ND) and four Type II diabetic (D) men were assessed for circadian rhythms in oxidative “stress markers.” Blood samples were collected at 3h intervals for ∼27 h beginning at 19:00h. Urine samples were collected every 3 h beginning with the 16:00h-19:00h sample. The dark (sleep) phase of the light-dark cycle extended from 22:30h to 06:30h, with brief awakening for sampling at 01:00h and 04:00h. Subjects were offered general hospital meals at 16:30h, 07:30h, and 13:30h (2400 cal in total/24 h). Serum samples were analyzed for uric acid (UA) and nitrite (NO) concentrations, and urine samples were assayed for 8-hydroxydeoxyguanosine (8-OHdG), malondialdehyde (MDA), and 8-isoprostane (ISP). Data were analyzed statistically both by the population multiple-components method and by the analysis of variance (ANOVA). The 24h mean level of UA and NO was greater in D than in ND subjects (424 vs. 338 μmol/L and 39.2 vs. 12.7 μM, respectively). A significant circadian rhythm in UA (p=0.001) and NO (p=0.048) was evident in ND but not in D (p=0.214 and 0.065). A circadian rhythm (p=0.004, amplitude=8.6 pmol/kgbw/3h urine vol.) was also evident in urine 8-OHdG of ND but not of D. The 24h mean levels of ND and D were comparable (76.8 vs. 65.7 pmol/kgbw/3h urine vol.). No circadian rhythm by population multiple-components was evident in MDA and ISP levels of ND subjects, or in 8-OHdG, MDA, and ISP in D. However, a significant time-effect was demonstrated by ANOVA in all variables and groups. The 24h mean of MDA and ISP in D was significantly greater than in ND (214 vs. 119 nmol/3h urine vol. and 622 vs. 465 ng/3h urine vol.). The peak concentrations of the three oxidative “stress markers” in urine, like those of serum NO, occurred early in the evening in both groups of men. This observation suggests a correlation between increased oxidative damage and increased rate of anabolic-catabolic events as evidenced by similarities in the timing of peak NO production and in parameters relevant to metabolic functions.     

Gas chromatographic determination of 3-butene-1,2-diol in urine samples after 1,3-butadiene exposure

1,3-Butadiene is an important industrial chemical and a common environmental contaminant. Because of its suspected carcinogenicity butadiene-related research has gained high activity. The obvious lack of knowledge so far has been that a biomonitoring method that can detect at least one of the metabolites of butadiene from body fluids or excretas does not exist. In this communication we describe a robust and simple analytical method which can be applied for biomonitoring purposes. We have developed a method that can detect 3-butene-1,2-diol in urine samples of rats inhalation-exposed to various concentrations of 1,3-butadiene. The method is based on liquid–liquid extraction and subsequent gas chromatographic analysis. The extraction efficiency of 3-butene-1,2-diol at a concentration of 2.2 μg/ml was 95% (SD=±3%, n=3) and was achieved by using sodium chloride saturation and isopropanol as an extracting solvent. The standard deviation of the gas chromatographic analysis was ±2% (n=12), the limit of detection was 0.08 μg/ml, the limit of quantitation was 0.11 μg/ml (SD=±4.8%, n=3) and the analysis was observed to be linear from 0.11 to 486 μg/ml (R=0.9987). Animals exposed to 1,3-butadiene showed a linear excretion of 3-butene-1,2-diol into urine as a function of butadiene exposure. During the exposure saturation of metabolism or accumulation of 1,3-butadiene or 3-butene-1,2-diol into the body was not observed in any exposure levels used.