Paola Manini

Comparison between exhaled and sputum oxidative stress biomarkers in chronic airway inflammation

The aim of the present study was to compare aldehyde levels resulting from lipid peroxidation in exhaled breath condensate (EBC) and induced sputum (IS) supernatant of subjects with asthma and chronic obstructive pulmonary disease (COPD). Aldehydes (malondialdehyde (MDA), acrolein, n-hexanal (C6), n-heptanal (C7), n-nonanal (C9), 4-hydroxynonenal (HNE) and 4-hydroxyhexenal (HHE)) in both biological fluids were measured by liquid chromatography-tandem mass spectrometry. MDA concentrations in sputum were 132.5 nM (82.5–268.8) and 23.7 nM (9–53.7) in EBC. Similarly, C6, C7 and C9 concentrations in IS were 1.5–4.7-fold higher than in EBC. Acrolein levels were 131.1 nM (55.6–264.6) in IS and 45.3 nM (14.4–127.1) in EBC. The concentrations of HNE and HHE in IS were not significantly different from the levels in EBC. Aldehyde levels in EBC did not show any correlation with aldehyde levels in IS or with differential sputum cellular count. In COPD, MDA in EBC, but not its IS counterpart, was negatively correlated with the severity of disease. In conclusion, the data presented here show that aldehydes can be detected in both exhaled breath condensate and supernatant of induced sputum, but that their relative concentrations are different and not correlated with each other. Therefore, with regard to lipid peroxidation products, exhaled breath condensate and induced sputum must be considered as independent techniques.

Exhaled breath condensate as a suitable matrix to assess lung dose and effects in workers exposed to cobalt and tungsten.

The aim of the present study was to investigate whether exhaled breath condensate (EBC), a fluid formed by cooling exhaled air, can be used as a suitable matrix to assess target tissue dose and effects of inhaled cobalt and tungsten, using EBC malondialdehyde (MDA) as a biomarker of pulmonary oxidative stress. Thirty-three workers exposed to Co and W in workshops producing either diamond tools or hard-metal mechanical parts participated in this study. Two EBC and urinary samples were collected: one before and one at the end of the work shift. Controls were selected among nonexposed workers. Co, W, and MDA in EBC were analyzed with analytical methods based on mass spectrometric reference techniques. In the EBC from controls, Co was detectable at ultratrace levels, whereas W was undetectable. In exposed workers, EBC Co ranged from a few to several hundred nanomoles per liter. Corresponding W levels ranged from undetectable to several tens of nanomoles per liter. A parallel trend was observed for much higher urinary levels. Both Co and W in biological media were higher at the end of the work shift in comparison with preexposure values. In EBC, MDA levels were increased depending on Co concentration and were enhanced by coexposure to W. Such a correlation between EBC MDA and both Co and W levels was not observed with urinary concentration of either element. These results suggest the potential usefulness of EBC to complete and integrate biomonitoring and health surveillance procedures among workers exposed to mixtures of transition elements and hard metals.

Interest of genotyping and phenotyping of drug-metabolizing enzymes for the interpretation of biological monitoring of exposure to styrene

In the field of occupational and/or environmental toxicology, the measurement of specific metabolites in urine may serve to assess exposure to the parent compounds (biological monitoring of exposure). Styrene is one of the chemicals for which biological monitoring programs have been validated and implemented in environmental and occupational medicine. However, inter-individual differences in the urinary excretion exist both for the main end-products (mandelic acid and phenylglyoxylic acid) and for its specific mercapturic acids (phenylhydroxyethylmercapturic acids, PHEMA). This limits to a certain extent the use of these metabolites for an accurate assessment of styrene exposure. In a group of 26 volunteers selected with relevant genotypes, and exposed to styrene vapours (50 mg/m3, 8 h) in an inhalation chamber, we evaluated whether genotyping or phenotyping relevant drug-metabolizing enzymes (CYP2E1, EPHX1, GSTM1, GSTT1 and GSTP1) may help to explain the observed inter-individual variability in the urinary metabolite excretion. Peripheral blood lymphocytes were used for genotyping and as reporter cells for the phenotyping of CYP2E1 and EPHX1. The GSTM1 genotype was clearly the most significant parameter explaining the variance in urinary PHEMA excretion (6-fold lower in GSTM1 null subjects; P < 0.0001) so that systematic GSTM1 genotyping should be recommended routinely for a correct interpretation of PHEMA urinary levels. Variant alleles CYP2E1*6 (7632T>A) and His113EPHX1 were associated with a significant reduction of, respectively, the expression (P = 0.047) and activity (P = 0.022) of the enzyme in peripheral blood lymphocytes. In combination with GSTM1 genotyping, the phenotyping approach also contributed to improve the interpretation of urinary results, as illustrated by the combined effect of CYP2E1 expression and GSTM1 allelic status that explained 77% of the variance in PHEMA excretion and allows the recommendation of mercapturates as specific and reliable biomarkers of exposure to styrene.

Influence of condensation temperature on selected exhaled breath parameters

BackgroundThe effects of changes in cooling temperature on biomarker levels in exhaled breath condensate have been little investigated. The aim of the study was to test the effect of condensation temperature on the parameters of exhaled breath condensate and the levels of selected biomarkers.MethodsExhaled breath condensate was collected from 24 healthy subjects at temperatures of -10, -5, 0 and +5 C degrees. Selected parameters (condensed volume and conductivity) and biomarkers (hydrogen peroxide, malondialdehyde) were measured.ResultsThere was a progressive increase in hydrogen peroxide and malondialdehyde concentrations, and condensate conductivity as the cooling temperature increased; total condensate volume increased as the cooling temperature decreased.ConclusionThe cooling temperature of exhaled breath condensate collection influenced selected biomarkers and potential normalizing factors (particularly conductivity) in different ways ex vivo. The temperature of exhaled breath condensate collection should be controlled and reported.

Comparative investigation of UV, electrochemical and particle beam mass spectrometric detection for the high-performance liquid chromatographic determination of benzoic and cinnamic acids and of their corresponding phenolic acids

Abstract The capabilities of different detection techniques, UV, controlled-potential coulometry and particle-beam electron-impact mass spectrometry (PB-EI-MS) for the HPLC analysis of phenolic acids were studied; fifteen benzoic and cinnamic acid derivatives were considered. For the electrochemical detector (ED) a reversed-phase LC method was set up, whereas normal-phase partition chromatography, on a CN column, was used for UV and MS. Library-searchable EI mass spectra were obtained using the PB-MS technique with flow-injection analysis. UV detection was performed at 280 nm, whereas measurements with the LC-coulometric system were carried out using a porous graphite electrode. The detector responses were compared in terms of linearity, precision and limits of detection; for this purpose, the mass spectrometer was operated under selected-ion monitoring conditions. A linear dynamic range of at least 10 3 was found for the HPLC method with electrochemical detection, with detection limits ranging from 1 to 5 pg injected; the relative standard deviation (R.S.D.) was typically 0.6–3.0% at the 0.1 ng level ( n =4). Using UV or PB-EI-MS detection, minimum amounts in the 5–50 and 2–5 ng ranges, respectively, could be detected. Calibration curves were linear from the limit of detection to at least 15 μg for most of the analytes detected by UV; the R.S.D. of the peak areas obtained in UV mode ranged from 1.2 to 3.1% at the 500 ng level ( n =4). Non-linear behaviour over the entire amount range studied (from 10 ng to 10 μg) was observed using the LC-PB-MS technique, so that two different calibration fittings at low and high levels were calculated. Precision of the LC-PB-MS system was generally good (R.S.D. between 0.5 and 1.8% at the 100 ng level, n =4) except for caffeic acid (R.S.D. 7.5% at the 50 μg level, n =4).

Biological monitoring of low benzene exposure in Italian traffic policemen.

A comparative evaluation of urinary biomarkers was carried out to characterize benzene exposure in a group of 100 traffic policemen of the city of Parma (Italy). All subjects were monitored once, in two consecutive days characterized by similar climatic conditions but preceded by two windy days. Benzene ambient concentration measured by municipal air monitoring stations was 1 microg/m(3) (Day 1) and 2 microg/m3 (Day 2). Personal exposure to ambient concentrations of benzene, toluene, ethylbenzene and xylene (BTEX) was assessed by using Radiello((R)) passive-diffusive samplers in a subgroup of 24 workers. Benzene metabolites, t,t-muconic acid (t,t-MA) and S-phenylmercapturic acid (S-PMA) were determined by isotopic dilution liquid chromatography-tandem mass spectrometry on spot urine samples collected at the end of the shift. Urinary benzene (U-B) was determined by solid-phase microextraction gas chromatography-mass spectrometry. Airborne benzene concentration expressed as median [and interquartile range] was 6.07 [0.28-9.53] microg/m(3), as assessed by personal sampling. Urinary concentrations of biomarkers in the whole group were 41.8 [34.1-89.8] microg/g creatinine for t,t-MA, 0.67 [0.23-1.32] microg/g creatinine for S-PMA, and 0.16 [0.13-0.26] microg/l for U-B. Smokers eliminated significantly higher concentrations of unchanged BTEX and benzene metabolites than non-smokers (p < 0.05). When traffic policemen were distinguished into indoor (n=31) and outdoor workers, no significant differences were observed for either airborne benzene or urinary biomarkers. Significantly lower concentrations of S-PMA and U-B were determined in samples collected at Day 1 as compared to Day 2 (p < 0.0001 and p = 0.003, respectively) suggesting that these biomarkers are enough sensitive and specific to detect changes in airborne benzene concentration even at few microg/m(3).

Determination of naphthalene metabolites in human urine by liquid chromatography-mass spectrometry with electrospray ionization

The use of a liquid chromatography-electrospray mass spectrometry system was investigated for the quantitative analysis of naphthalene metabolites (alpha-naphthol, alpha-naphthylglucuronide and beta-naphthylsulphate) in untreated urine samples. Chromatography was carried out under ion-suppressed reversed-phase conditions, by using high-speed (3 cm, 3 microns) columns and formic acid (2 mM) as a modifier in the mobile phase. The ionization was obtained in the negative-ion mode. Linearity, sensitivity and precision of the method were explored by operating in selected-ion monitoring mode. The method was applied to the quantitative analysis of naphthalene metabolites in untreated urine samples from workers in a naphthalene producing plant. Solid-phase extraction was used for sample clean-up and trace enrichment. Liquid chromatography-tandem mass spectrometry experiments were performed for confirmation purposes.

Simultaneous identification of different classes of hydrocarbons and determination of nitro-polycyclic aromatic hydrocarbons by means of particle beam liquid chromatography-mass spectrometry

Abstract LC-MS with a particle beam interface was used to separate and identify aliphatic hydrocarbons, polycyclic aromatic hydrocarbons (PAHs) and their nitrated derivatives (nitro-PAHs). In particular, the behaviour of nitro-PAHs and of two nitrated biphenyls was checked in terms of instrumental response. A normal-phase chromatographic system was used, with a silica column and heptane-tetrahydrofuran as mobile phase. Electron impact and positive- and negative-ion chemical ionization (NICI) modes were used; for nitro-PAHs, the best results were obtained under NICI conditions. In the flow-injection mode, detection limits as low as pg levels were obtained for 9-nitroanthracene, 3-nitro-9-fluorenone, 1-nitropyrene, 1,8-dinitronaphthalene, 2,7-dinitrofluorene and 2,7-dinitro-9-fluorenone, whereas the low-molecular-mass 1- and 2-nitronaphthalene were not detected even at μg levels; using the analytical column, a detection limit five times higher was determined for 1-nitropyrene. For most of the nitro-PAHs considered, the dependences of the intensities on the amounts injected were found to be linear in a range of more than two orders of magnitude.

Atmospheric pressure chemical ionization liquid chromatography/mass spectrometry in cholesterol oxide determination and characterization

An atmospheric pressure chemical ionization liquid chromatography/mass spectrometry (APCI-LC/MS) system was evaluated for the analysis of cholesterol oxidation products (COPs). High performance liquid chromatography separation of these compounds was performed under reversed-phase chromatographic conditions at 1 mL/min flow rate. Useful mass spectra for confirmation and quantitation purposes were obtained with positive-ion detection. The influence of several parameters, such as the temperature of the heated nebulizer interface, the voltage applied to the orifice and to the ring electrode on the mass spectra of the analytes, was studied and optimized in order to obtain the best sensitivity. Linearity, sensitivity and precision of the method were determined operating in the selected-ion monitoring mode. Detection limits were in the range 0.2–0.8 ng for all the compounds with linear calibration graphs in the 0.1–10 mg/L concentration range. The method was applied to the quantitative analysis of COPs in lyophilized beef preparations, and results were compared with those obtained using LC/UV and LC/MS with particle beam interface.

Liquid chromatography-electrospray tandem mass spectrometry of acidic monoamine metabolites

A new method based on liquid chromatography-tandem mass spectrometry has been developed for the determination of monoamine metabolites, i.e., homovanillic acid (HVA), vanilmandelic acid (VMA), 3,4-dihydroxyphenylacetic acid (DOPAC) and 5-hydroxyindoleacetic acid (5-HIAA) in human urine. Analytes were separated on a C16 amide (5 cm, 5 microm) column and ionized by negative ion electrospray. Operating in the selected-reaction monitoring mode, linearity was established over three-orders of magnitude and limits of detection were in the range 30-70 microg/l. Precision calculated as RSD was within 0.8-5.2% for all intra- and inter-day determinations. The method was applied to the quantitative analysis of monoamine metabolites in 700 urine samples from occupationally (adults) and environmentally (both children and adults) exposed people living in areas with different soil contamination from lead. The urinary excretion of monoamine metabolites was significantly higher (P<0.001) in the subgroup of children living in polluted areas as compared to the control group (HVA, 6.03 vs. 4.57 mg/g creatinine; VMA, 5.33 vs. 4.37 mg/g creatinine; 5-HIAA 3.24 vs. 2.45 mg/g creatinine). In adults belonging to both groups of subjects occupationally and environmentally exposed, no differences were detected in the urinary concentration of monoamine metabolites. However, adults showed lower values of HVA (2.57 mg/g creatinine), VMA (2.17 mg/g creatinine) and 5-HIAA (2.09 mg/g creatinine) as compared to children groups.