Biomedical subjects
R Fanelli
Publications and source records attributed to R Fanelli.
Comparative studies of the leachate of an industrial landfill by gas chromatography-mass spectrometry, liquid chromatography-nuclear magnetic resonance and liquid chromatography-mass spectrometry.
Nowadays, the need to have a realistic characterization of industrial effluents in the environment has become more and more recognized. A palette of different analytical methods both for sample extraction and instrumental analysis are available today, some older, others introduced more recently. The aim of this research is to compare a number of these techniques. To do this we studied a real leachate from an industrial landfill and carried out chemical analyses for organic pollutants, using different extraction methods based on solid-phase extraction and solid-phase microextraction and different instrumental techniques such as GC-MS, LC-MS, NMR and LC-NMR. Results show the performances of the different techniques, which are complementary.
Identification and measurement of endogenous beta-oxidation metabolites of 8-epi-Prostaglandin F2alpha.
F2-isoprostanes are prostaglandin-like compounds derived from nonenzymatic free radical-catalyzed peroxidation of arachidonic acid. 8-epi-Prostaglandin (PG) F2alpha, a major component of the F2-isoprostane family, can be conveniently measured in urine to assess noninvasively lipid peroxidation in vivo. Measurement of major metabolites of endogenous 8-epi-PGF2alpha, in addition to the parent compound, may be useful to better define its formation in vivo. 2,3-Dinor-5,6-dihydro-8-epi-PGF2alpha is the only identified metabolite of 8-epi-PGF2alpha in man, but its endogenous levels are unknown. In addition to this metabolite, we have identified another major endogenous metabolite, 2,3-dinor-8-epi-PGF2alpha, in human and rat urine. The identity of these compounds, present at the pg/ml level in urine, was proven by a number of complementary approaches, based on: (a) immunoaffinity chromatography for selective extraction; (b) gas chromatography-mass spectrometry for structural analysis; (c) in vitro metabolism in isolated rat hepatocytes; and (d) chemical synthesis of the enantiomer of 2,3-dinor-5, 6-dihydro-8-epi-PGF2alpha as a reference standard. In humans, the urinary excretion rate of both dinor metabolites is comparable with that of 8-epi-PGF2alpha. Both metabolites increase in parallel with the parent compound in cigarette smokers, and they are not reduced during cyclooxygenase inhibition. Another beta-oxidation product, 2, 3,4,5-tetranor-8-epi-PGF2alpha, was identified as a major product of rat hepatocyte metabolism. In conclusion, at least two major beta-oxidation products of 8-epi-PGF2alpha are present in urine, which may be considered as additional analytical targets to evaluate 8-epi-PGF2alpha formation and degradation in vivo.
Carcinogen-DNA adducts as tools in risk assessment.
Genotoxic chemicals are known to react with DNA either directly or after metabolic activation to form adducts, a step thought to be relevant with respect to chemical carcinogenesis. Evaluation of cancer risk due to exposure to chemicals requires information about the internal dose which depends on individual variation in rates of metabolic activation and detoxification. The presence and the amount of specific DNA adducts provide a good indication of chemical exposure and genetic damage resulting from exposure to carcinogens and account for some of the factors affecting individual susceptibility to cancer. Analysis of DNA adducts requires that the sensitivity of the methods be sufficiently high to allow the detection of about 1 adduct/109 normal nucleotides. Most suitable methods are based on 32P-postlabelling, immunoassays or physico-chemical techniques such as HPLC coupled to synchronous fluorescence spectroscopy or gas chromatography-mass spectrometry. These methods have been used to assess human exposure to a variety of chemical carcinogens including polycyclic aromatic hydrocarbons, aromatic amines, heterocyclic aromatic amines or aflatoxins. In some instances, the use of DNA-adducts has given accurate estimates of risk.
Level, sources and toxicity of polychlorinated biphenyls in the Italian diet.
We used the duplicate portion method to measure the daily dietary intake of total and congener-specific polychlorinated biphenyls (PCB) and to assess their potential toxicity in a group of 20 subjects consuming a typical Italian diet. The mean +/- SD intake of total PCB, measured by GC-MS, was 3.72 +/- 1.51 micrograms/person/day, comparable to values reported in similar studies world-wide, with individual intakes varying within one order of magnitude, from 0.97 to 10.59 micrograms/person/day. The di-ortho congeners 153, 18 and 138 were the PCB found in the highest concentrations (respectively 13.8%, 11.4% and 10.9% of the total) while the non-ortho coplanar congeners (77, 126 and 169) amounted to 0.5% of the total. The corresponding levels of toxicity (TCDD-like TEQ values ascribable to PCB) ranged from 4.6 up to 119 pg/person/day of TCDD-equivalents in 18 subjects, i.e. presumed no-risk levels, but with peaks of 2109 and 4553 pg/person/day in two subjects with significant intakes of the congener 126. Principal components analysis and redundancy analysis showed dairy products, meat and fish were the principal sources of PCB, and vegetables those with the highest toxicity index in the Italian diet.
Perceived risk of cancer in Italy.
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AV node ablation and pacemaker implantation after withdrawal of effective rate-control medications for chronic atrial fibrillation: effect on quality of life and exercise performance.
We assess whether AV node ablation and pacemaker implantation after discontinuation of effective rate-control medical therapy for chronic atrial fibrillation had a positive impact on quality of life and exercise performance. To assess the possibility of a placebo effect following pacemaker implantation, the study included three groups of patients. Group 1 underwent an echocardiogram, treadmill exercise, and quality of life measurement 1 month prior to and 6 months following AV node ablation and pacemaker implantation associated with discontinuation of rate-control medications. Group 2 underwent AV node ablation and pacemaker implantation without discontinuation of antiarrhythmic rate-control drugs. Group 3 underwent pacemaker implantation without performing AV node ablation and continuing rate-control medical therapy. At the 1- and 6-month evaluation, the patients in group 1 showed a significant improvement of left ventricular ejection fraction, quality of life, and activity scores. The exercise duration and the maximal VO2 consumption, however, did not change significantly. A slight improvement of the quality of life and physical activity scores was observed in the group undergoing AV node ablation without withdrawal of medications. However, no significant changes were observed in the group receiving only the pacemaker without modification of medical therapy and with intact AV node conduction. In conclusion, in patients with chronic atrial fibrillation, discontinuation of effective rate-control medical therapy followed by AV node ablation and permanent pacing appeared to improve quality of life and activity scores despite no change in exercise duration. The improvement observed does not seem to reflect a placebo effect.
Seasonal effect on airborne pyrene, urinary 1-hydroxypyrene, and benzo(a)pyrene diol epoxide-hemoglobin adducts in the general population.
Exposure to airborne polycyclic aromatic hydrocarbons (PAHs) in 65 employees (40 sampled both in summer and winter, 15 sampled in summer only, and 10 sampled in winter only) with no occupational exposure to PAHs was assessed by measuring: personal exposure to pyrene, urinary excretion of 1-hydroxypyrene (1-OHP), and benzo(a)pyrene diol epoxide adducts to hemoglobin (BPDE-Hb). Overall, office employees were exposed to significantly higher levels of pyrene in winter (4.54 +/- 2.35 ng/m3, mean +/- SD) than in summer (1.67 +/- 1.92 ng/m3, mean +/- SD; P < 0.001), but no such seasonal variability was observed in 1-OHP excretion. Tobacco smoking was the major determinant of 1-OHP excretion. BPDE-Hb adducts were measured by gas chromatography-mass spectrometry as benzo(a)pyrene tetrols (BPT) released from adducted hemoglobin. In the 65 employees analyzed, mean BPT levels +/- SD were higher in winter (0.14 +/- 0.38 fmol/mg Hb) than summer (0.031 +/- 0.022 fmol/mg Hb). This difference was not statistically significant, probably because of the small proportion of subjects with detectable adducts (11% in summer and 16% in winter). BPDE-Hb adducts were not significantly associated with sex, age, diet, smoking habits, or with pyrene levels and 1-OHP excretion. This is the first report providing reference BPDE-Hb adduct values for the general population not occupationally exposed to environmental PAHs and shows a tendency to seasonal variability, with higher BPT levels in winter when environmental PAHs are also high.
Spinning more yarn: endozepine.
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Rapid solid-phase extraction method for automated gas chromatographic-mass spectrometric determination of nicotine in plasma.
The aim of this study was to develop a simple, rapid and sensitive assay of nicotine in plasma for automated gas chromatographic-mass spectrometric analysis. Biological samples were extracted using pre-packed Extrelut-1 columns with 5 ml of ethyl acetate. Quantitative analysis was done using deuterium-labelled nicotine as internal standard. The limit of quantitation was 0.5 ng in 1-ml plasma samples. Precision was ranging from 13.3% to 1.64% (R.S.D.) depending on the concentration, while the deviation was 4.16%. This method has been used for determination of nicotine bioavailability from new, low-dosage, nicotine chewing gum strips.
Comparison of steerable continuous-wave versus pulsed-wave Doppler ultrasonography to renal artery angiography in diagnosing renal artery stenosis.
Pulsed-wave Doppler ultrasonography is widely used to noninvasively diagnose renal artery stenosis. The use of steerable continuous-wave Doppler has never been tested. We compared pulsed and steerable continuous-wave Doppler ultrasonography, demonstrating that although both methods are highly sensitive for severe stenoses, continuous-wave Doppler shows a better sensitivity for mild to moderate stenoses.
Impact of inherited polymorphisms in glutathione S-transferase M1, microsomal epoxide hydrolase, cytochrome P450 enzymes on DNA, and blood protein adducts of benzo(a)pyrene-diolepoxide.
The benzo(a)pyrene (BaP) metabolite benzo(a)pyrenediolepoxide (BPDE) is strongly implicated as a causative agent of lung cancer. To assess the risk of exposure to BaP, we made a combined analysis of levels of BPDE adducts to hemoglobin (Hb), serum albumin (SA), and lymphocyte DNA in 44 patients with incident lung cancer, as a prototype of a population mainly exposed to tobacco-derived BaP. We also investigated whether genetic polymorphisms of cytochrome P450IA1 (CYPIA1), microsomal epoxide hydrolase (mEH), and glutathione S-transferase M1 (GSTM1), which are involved in BaP metabolism, can be determinants of adduct formation. BPDE-Hb, BPDE-SA, and BPDE-DNA adducts were quantified as BaP tetrols released from hydrolysis of macromolecules and measured by high-resolution gas chromatography-negative ion chemical ionization-mass spectrometry to achieve high specificity and sensitivity. Individuals with detectable Hb adducts were positive for SA adducts but not vice versa, suggesting that BPDE-Hb adducts are less informative indicators of BaP exposure. Using PCR methods on DNA, we characterized GSTM1 deletion, CYPIA1 MspI and exon 7 valine variants, and mEH polymorphisms at amino acid positions 113 (EH3) and 139 (EH4). Levels of BPDE adducts were no different among CYPIA1, mEH, and GSTM1 genotypes. However, individuals with measurable BPDE-SA adducts were CYPIA1 variant carriers more frequently (P = 0.03). There was a slightly higher percentage of DNA detectable adducts in subjects with CYPIA1 exon 7 valine polymorphism. When subjects were classified by both polymorphisms on the mEH gene, those with two slow alleles (EH3 homozygous mutated) and no fast alleles (EH4 homozygous wild type) had a lower frequency of BPDE-SA adducts and no DNA adducts (P = 0.06). These results are based on a small number of observations thus far, but this exploratory study suggests that CYPIA1 and mEH variants might have an impact on BPDE exposure markers such as BPDE-SA adducts. Chemical specificity in adduct measurements is important to identify the biomarkers that reflect BaP exposure more accurately.
Structural characterization of mono- and dihydroxylated metabolites of paclitaxel in rat bile using liquid chromatography/ion spray tandem mass spectrometry.
The capability of high performance liquid chromatography/ion spray mass spectrometry (HPLC/ISP-MS) and HPLC/ISP-tandem mass spectrometry (HPLC/ISP-MS/MS) were investigated to achieve mass separation as well as structural characterization of taxol metabolites directly in rat bile, without their previous isolation. HPLC/ISP-MS yielded information on the molecular weights of several hydroxylated derivatives while HPLC/ISP-MS/MS allowed the on-line structural characterization of all metabolites present in different ratios in rat bile. The approach led to the extraction of nine metabolites and their distinction from the other endogenous contaminants. These metabolites were recognized as three dihydroxytaxols, four monohydroxytaxols, one deacetyltaxol and one containing the taxane ring. Among the derivatives, we were able to identify four new metabolites of paclitaxel belonging to the dihydroxy and monohydroxy series, never previously detected. HPLC/ISP-MS/MS enabled the classification of all di- and monohydroxy isomers. These results demonstrate that the high sensitivity of this method, based on the combined use of tandem mass spectrometry with chromatographic separation, can be considered as offering a valid approach to the detection of new taxol derivatives directly in biological fluids.
Reduction of urinary 8-epi-prostaglandin F2 alpha during cyclo-oxygenase inhibition in rats but not in man.
1. 8-epi-prostaglandin (PG) F2 alpha, a major F2 isoprostane, is produced in vivo by free radical-dependent peroxidation of lipid-esterified arachidonic acid. Both cyclo-oxygenase isoforms (COX-1 and COX-2) may also form free 8-epi-PGF2 alpha as a minor product. It has been recently seen in human volunteers that the overall basal formation of 8-epi-PGF2 alpha in vivo is mostly COX-independent and urinary 8-epi-PGF2 alpha is therefore an accurate marker of 'basal' oxidative stress in vivo. 2. To test the validity of this marker in the rat, we evaluated in vivo the effect of COX inhibition on the formation of 8-epi-PGF2 alpha vs prostanoids. Two structurally unrelated COX inhibitors (naproxen: 30 mg kg-1 day-1; indomethacin: 4 mg kg-1 day-1) were given i.p. to rats kept in metabolic cages. In vivo formation of 8-epi-PGF2 alpha was assessed by measuring its urinary excretion. Prostanoid biosynthesis was assessed by measuring urinary excretion of major metabolites of thromboxane (TX) and prostacyclin (2,3-dinor-TXB1 and 2,3-dinor-6-keto-PGF1 alpha). All compounds were selectively measured by immunopurification/gas chromatography-mass spectrometry. 3. Naproxen reduced urinary excretion of 2,3-dinor-TXB1 and 2,3-dinor-6-keto-PGF1 alpha but, unexpectedly, also that of 8-epi-PGF2 alpha (82, 49 and 52% inhibition, respectively). Indomethacin had a similar effect (77, 69 and 55% inhibition). Esterified 8-epi-PGF2 alpha in liver and plasma remained unchanged after indomethacin. 4. These findings prompted us to re-assess the contribution of COX activity to the systemic production of 8-epi-PGF2 alpha in man. We gave naproxen (1 g day-1) to healthy subjects (four nonsmokers and four smokers). Urinary 8-epi-PGF2 alpha remained unchanged in the two groups (9.63 +/- 0.99 before vs 10.24 +/- 1.01 after and 20.14 +/- 3.00 vs 19.03 +/- 2.45 ng h-1 1.73 m-2), whereas there was a marked reduction of major urinary metabolites of thromboxane and prostacyclin (about 90% for both 11-dehydro-TXB2 and 2,3-dinor-TXB2; > 50% for 2,3-dinor-6-keto-PGF1 alpha). 5. To investigate whether rat COX-1 produces 8-epi-PGF2 alpha more efficiently than human COX-1, we measured the ex vivo formation of 8-epi-PGF2 alpha and TXB2 simultaneously in whole clotting blood. Serum levels of 8-epi-PGF2 alpha and TXB2 were similar in rats and man. 6. We conclude that a significant amount of COX-dependent 8-epi-PGF2 alpha is present in rat but not in human urine under normal conditions. This implies that urinary 8-epi-PGF2 alpha cannot be used as an index of near-basal oxidant stress in rats. On the other hand, our data further confirm the validity of this marker in man.
Salivary nitrate, nitrite and N-nitroso compounds in patients with cancer of the upper aerodigestive tract.
N-nitroso compounds are carcinogens that can be ingested directly or synthesized from nitrites and nitrates. The possible role of N-nitroso compounds in the induction of upper aerodigestive tract tumours was considered in a case-control study conducted in the Valle d'Aosta, an Italian region with a high incidence of these neoplasms. Nitrate, nitrite, labile and stable N-nitroso compounds were analysed in the saliva of 36 patients with cancers of the upper aerodigestive tract and 23 healthy individuals. After allowing for tobacco, salivary nitrate, nitrite and N-nitroso compounds were not associated with an increased risk of upper aerodigestive tract cancers. The odds ratio for continuous units of total N-nitros compounds was 0.99 (95% confidence interval 0.9-1.1). Thus, salivary nitrate, nitrite and N-nitroso compounds might not be suitable markers for the assessment of the risk of cancer of the upper aerodigestive tract, although a role for N-nitroso compounds cannot be excluded.
Analysis of dexamethasone and betamethasone in bovine urine by purification with an "on-line" immunoaffinity chromatography-high-performance liquid chromatography system and determination by gas chromatography-mass spectrometry.
A method for the immunoaffinity extraction of dexamethasone and betamethasone in bovine urine, followed by high-pressure liquid chromatography (HPLC) fractionation and gas chromatography-mass spectrometry determination, is described. A commercial immunoaffinity gel, containing antibodies raised against dexamethasone, was used to prepare an immunoaffinity cartridge which was inserted in an automatic HPLC system for on-line extraction and purification. By injecting urine samples (spiked with flumethasone as internal standard) directly into the system, it was possible to collect purified fractions, containing the analytes of interest. The fractions were dried and derivatized to yield the tetra-trimethylsilyl derivatives of the three corticosteroids, which were analyzed by selected ion monitoring gas chromatography-mass spectrometry. The method allowed a very good purification of samples and reached a detection limit of 0.1 ng/ml for dexamethasone and 0.2 ng/ml for betamethasone. Several samples, coming from a steer treated with dexamethasone and from other bovines coming from breedings in northern Italy, were analyzed with the method described. Dexamethasone levels ranged from 0.12 to 146 ng/ml.
On-line monitoring of benzene air concentrations while driving in traffic by means of isotopic dilution gas chromatography/mass spectrometry.
There is no shortage of information about the average benzene concentrations in urban air, but there is very little about microenvironmental exposure, such as in-vehicle concentrations while driving in various traffic conditions, while refuelling, or while in a parking garage. The main reason for this lack of data is that no analytical instrumentation has been available to measure on-line trace amounts of benzene in such situations. We have recently proposed a highly accurate, high-speed cryofocusing gas chromatography/mass spectrometry (GC/MS) system for monitoring benzene concentrations in air. Accuracy of the analytical data is achieved by enrichment of the air sample before trapping, with a stable isotope permeation tube system. The same principles have been applied to a new instrument, specifically designed for operation on an electric vehicle (Ducato Elettra, Fiat). The zero emission vehicle and the fully transportable, battery-operated GC/MS system provide a unique possibility of monitoring benzene exposure in real everyday situations such as while driving, refuelling, or repairing a car. All power consumptions have been reduced so as to achieve a battery-operated GC/MS system. Liquid nitrogen cryofocusing has been replaced by a packed, inductively heated, graphitized charcoal microtrap. The instrument has been mounted on shock absorbers and installed in the van. The whole system has been tested in both fixed and mobile conditions. The maximum monitoring period without external power supply is 6 h. The full analytical cycle is 4 min, allowing close to real-time monitoring, and the minimum detectable level is 1 microgram/m3 for benzene. In-vehicle monitoring showed that, when recirculation was off and ventilation on, i.e., air from outside the vehicle was blown inside, concentrations varied widely in different driving conditions: moving from a parking lot into normal traffic on an urban traffic condition roadway yielded an increase in benzene concentration from 17 to 62.3 micrograms/m3 even if the actual distance was small. A larger increase was observed when a car was left with the engine running at a distance 2 m from the zero emission vehicle: We measured an increment of benzene concentrations from 15.2 to 174.4 micrograms/m3 with a car equipped with a catalytic converter, and from 19.1 to 386.3 micrograms/m3 with a car without such a converter.
Detection and quanification of trihalomethanes in drinking water from Alexandria, Egypt.
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