Biomedical subjects
Publications and source records attributed to N Sannolo.
Recent studies suggest that professional exposure to Extremely Low Frequency-Electro Magnetic Field (ELF-EMF) can increase the risk of sudden cardiac death. Aim of our work was to find predictive parameters of arrhythmic risk in a population of 28 railways drivers exposed to ELF-EMF. Our findings were that the exposure did not reduce HRV and did not increase the risk of arrhythmias.
Hospital personnel involved in antineoplastic drug preparation and administration to patients are exposed to large amounts of these drugs. Labour legislation indicates the necessity of planning monitoring strategies aimed at prevention and/or reduction of drug exposure. Monitoring strategies consist of quantitative determinations of indicators, present in environmental and biological matrices. Among the antineoplastic drugs widely used, cyclophosphamide (CP) has been identified as a suitable indicator of potential exposure to mixtures of antineoplastic drugs. Many literature methods for quantitative analysis of CP involve either liquid (LC) or gas chromatography (GC) with mass spectrometry (MS), both of which require use of a suitable internal standard. The present work focuses on the synthesis of mono- and diiodocyclophosphamide (CPI and CPI(2)) to be used as internal standard. These compounds were analyzed by GC/EI-MS/MS and LC/ESI-MS(n) using ion trap mass spectrometry. The product ion mass spectra are interpreted in terms of proposed structures of fragment ions. Iodine-chlorine substitution resulted in a weakening of the carbon-halogen bond with a noteworthy influence on the ion fragmentation processes. The proposed suitability of CPI and CPI(2) as internal standards was based on similarities to CP as regards ionization and fragmentation processes. The results obtained suggest that CPI could be used as internal standard for CP quantification by LC/ESI-MS/MS, and CPI(2) for GC/EI-MS/MS analyses.
Urinary S-phenylmercapturic acid (S-PMA) is considered a useful biomarker for the measurement of low levels of benzene exposure, related to occupational exposure, smoking habits or environmental pollution. S-PMA quantitative analysis requires highly sensitive and specific techniques and purification procedures, mainly based on liquid-liquid or solid-phase extraction, which result in time expensive analyses. A method was developed for the quantitative determination of S-PMA in urine by using a simple, reproducible and easily automatizable HPLC purification followed by LC/ESI-NI/MS2 analysis. In order to reduce the cost of the analysis, related to the use of expensive labeled standards, p-bromo-S-phenylmercapturic acid (p-Br-S-PMA) was synthesized, characterized and used as internal standard. The feasibility and efficacy of the proposed method were examined by constructing calibration curves in the range from 6.2 to 200 microg/l and data were analyzed in terms of linearity and statistical parameters. The detection limit, related to the purification of 1 ml urine sample is 5 microg/l. The method was applied to the analysis of 12 urine samples from smoker subjects non-occupationally exposed to benzene. S-PMA urinary levels ranged from 13.6 to >200 microg/l, suggesting a high influence of life style in the S-PMA excretion. The proposed analytical method is suitable for the biological monitoring of both smoker and non-smoker workers, occupationally exposed to benzene. By processing at least 2 ml of urine samples, the method appears to be also useful for the evaluation of benzene uptake due to the environmental pollution.
The reaction products of epichlorohydrin with human alpha- and beta- globins, obtained through in vitro incubation of these compounds and red blood cells, were determined by using reversed-phase high-performance liquid chromatography (RP-HPLC), electrospray ionization mass spectrometry and matrix-assisted laser desorption/ionization tandem mass spectrometry. The alpha-globin was much more reactive than the beta-globin. At low incubation ratios, approximating the order of magnitude of epichlorohydrin concentration as found in workplaces, the only modified peptide still detectable was the 62-90 belonging to the alpha-chain and carrying an incremental mass of 92 u on either His72 or His89. Given that the two peptides co-eluted in a single chromatographic peak during RP-HPLC separation, they could be chosen as suitable biomarkers for quantification in the setting up of a new methodology for the biological monitoring of persons occupationally exposed, replacing currently known procedures.
The structural characterisation of the adducts formed by in vitro interaction of hemoglobin (Hb) with 1,2,3,4-diepoxybutane (DEB), the most reactive 1,3-butadiene (BD) metabolite, was obtained by liquid chromatography/electrospray ionisation mass spectrometry (LC/ES-MS) analysis of modified tryptic peptides of human hemoglobin chains. The reactive sites of human hemoglobin towards DEB and its hydroxylated derivatives (trihydroxybutyl (THB)-derivatives) were identified through the characterisation of alkylated tryptic peptides by matrix-assisted laser desorption/ionisation tandem mass spectrometry (MALDI-MS/MS). Based on this characterisation, a procedure was set up to measure the Hb-adducts of THB-derivatives by isotope dilution mass spectrometry with the use of a deuterated peptide standard. The results obtained here could permit optimisation of molecular dosimetry of BD-adducts, and extension of the analysis to the biological monitoring of occupational exposure to butadiene.
The present study demonstrates that NO produced in vitro by inducible nitric oxide synthase in red cells can convert hemoglobin contained in the red cells to S-nitrosohemoglobin. Experiments carried out either in the absence or in the presence of a low molecular weight thiol, such as cysteine, showed that in the first case the target of NO is heme-Fe2+. On the contrary, in the presence of cysteine, the first step is the formation of S-nitrosocysteine, followed by transfer of the NO group to a particular cysteine residue of beta-globin, cysteine 93. These results confirm previous data indicating the preferential formation of S-nitrosohemoglobin at that site by chemical methods [Ferranti et al. (1997) FEBS Lett. 400, 17-24], and the existence of a physiological mechanism of inactivation for NO circulating in blood. The analysis of S-nitrosohemoglobin can also allow the quantification of the NO levels in blood to be applied for in vitro and in vivo measurements.
The use of aspecific methylation reaction in combination with MS procedures has been employed for the characterization of the nucleophilic residues present on the molecular surface of the human 2,3-diphosphoglycerate/deoxy-hemoglobin complex. In particular, direct molecular weight determinations by ESMS allowed to control the reaction conditions, limiting the number of methyl groups introduced in the modified globin chains. A combined LCESMS-Edman degradation approach for the analysis of the tryptic peptide mixtures yielded to the exact identification of methylation sites together with the quantitative estimation of their degree of modification. The reactivities observed were directly correlated with the pKa and the relative surface accessibility of the nucleophilic residues, calculated from the X-ray crystallographic structure of the protein. The results here described indicate that this methodology can be efficiently used in aspecific modification experiments directed to the molecular characterization of the surface topology in proteins and protein complexes.
A specific and sensitive method based on gas chromatography/tandem mass spectrometry with on-column injection was developed to quantify simultaneously cyclophosphamide and ifosphamide in urine by using trophosphamide as an internal standard. The urine samples were extracted with diethyl ether and derivatization was performed with heptafluorobutyric anhydride. The detection limits of cyclophosphamide and ifosphamide in urine samples were 0.1 and 0.5 ng ml(-1), respectively, with a signal-to-noise ratio of 3 : 1. The sensitivity, the specificity and the low cost of the instrumentation involved make this method suitable for economical analysis on a large scale, such as for the biological monitoring of occupational exposure to cyclophosphamide and ifosphamide in production plants and in hospitals during their pharmacological use.
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The present study reports a procedure developed for the identification and quantitative analysis of the adducts formed by interaction of methyl bromide with human hemoglobin, based on combined analysis by electrospray mass spectrometry and automated Edman degradation of either intact globin chains or tryptic peptides of globin chains. The procedure has allowed identification of the reactive sites in human hemoglobin, and has been applied to the analysis of samples modified in vitro by methyl bromide. The results obtained represent the basis for the complete structural characterization of the modified hemoglobin and demonstrate the usefulness of the proposed analytical approach for the evaluation of the degree of alkylation and the identification of modified amino acids in proteins.
Recent studies have demonstrated the biological importance of the interaction of S-nitrosothiols, which can be considered as nitric oxide (NO) protein donors, especially haemoglobin, at the level of Cys residues. It was recently proposed that S-nitrosohaemoglobin is formed within red blood cells and serves as a regulatory function. In human haemoglobin the alpha-subunit contains one Cys residue and the beta-subunit contains two Cys residues, one of which (beta-Cys93) is highly reactive and conserved among species, although its function has remained unknown. Electrospray ionization mass spectrometry was used to monitor the results of exposure of haemolysates to S-nitrosocysteine under different conditions and thus addressed some aspects of NO-haemoglobin interaction. When an equimolar ratio of S-nitrosothiol was added to haemoglobin, only a single NO molecule was added. Peptide mapping by liquid chromatography-mass spectrometry located the nitrosyl group at the level of beta-Cys93 demonstrating that this was the preferred site of formation of S-nitrosohaemoglobin. The present data also suggest that electrospray mass spectrometry can allow quantification and characterisation of S-nitrosoproteins in blood.
The in vitro interaction of the antineoplastic drug 1,3-bis-(2-chloroethyl)-1-nitrosourea (BCNU) and acrolein with model peptides has been investigated in order to provide a detailed description of their electrophilic reactivity towards biological macromolecules. Following incubation with these substances, the modified species were separated by HPLC and identified by fast atom bombardment mass spectrometry, whereas the reactive amino acids within the peptide structure were assigned by tandem mass spectrometry. Incubation with BCNU led essentially to the formation of an N-terminal carbamoyl derivative that slowly decomposed to form three isomeric structures and a very minor ethylated adduct. Alkylation with acrolein gave rise to a mixture of different adducts due to the reaction of both the double bond and the carbonyl group. Two species containing intramolecular cross-links were also observed. These results constitute the prerequisite for in vitro and in vivo studies on the modification of haemoglobin in patients following treatment with antineoplastic drugs.
Nitroarenes are environmental contaminants produced during incomplete combustion processes. Nitroreduction, the most important pathway of nitroarene toxification, occurs mainly in the liver and intestine. In the present study, we show that human red cells may also possess the metabolic competence to reduce 1-nitropyrene (NP) and 3-nitrofluoranthene (NF), the nitroarenes chosen as model compounds, to their corresponding amino derivatives, 1-aminopyrene (AP) and 3-aminofluoranthene (AF). The requirement of the cofactor couple NADH/FMN suggests that erythrocyte nitroreductase activity occurs via one electron transfer. The presence of oxygen strongly inhibited the haemolysate-catalyzed nitroarene reduction, whether measured as amine formation or nitroarene disappearance. Intermediate reactive species, that bind covalently to haemoglobin and/or other erythrocyte proteins, are formed during nitroreduction catalyzed by human haemolysate. In fact, the reduced metabolites AP and AF were released after mild acid hydrolysis of red cell proteins exposed to NP and NF, thus suggesting that sulphinamide adducts have been formed.
A mass spectrometric procedure is described for the structural study of the adducts formed in human hemoglobin by in vitro exposure of erythrocytes to the alkylating agent methyl bromide using different protein to reagent ratios. Peptide mapping by HPLC and tandem mass spectrometry allowed location of methylated amino acids within the protein sequence. A prominent reactivity of several nucleophilic side chains in human hemoglobin subunits was observed, which was modulated by the concentration of the alkylating agent. Cysteine residues, the main reactive sites, were fully methylated in hemoglobin exposed to a 10-fold excess of methyl bromide, differently from other residues, including histidines, showing a heterogeneous pattern of methylation that was largely directed by their environment. No evidence of methylation was found at the heme proximal histidines beta92 and alpha87. A more selective methylation was obtained when the ratio methyl bromide: hemoglobin was lowered to about 1:1. In this last case only specific residues were reactive. Among them, the N-terminal amino group of both alpha- and beta-globins, cysteine 104 in the alpha-chain and cysteine 93 (not cysteine 112) in the beta-chain, indicating a different accessibility to reaction of the sulfhydryl groups on the protein chain. Thus hemoglobin side chains are selectively modified and the degree of modification at each site is a function of the position of the single amino acid residue within the protein quaternary structure, raising the possibility that alterations of structure and functional properties of human hemoglobin following exposure to alkylating agents may be mediated through such covalent protein modifications. The results obtained demonstrate the usefulness of the analytical approach for the characterization of hemoglobin adducts with methyl bromide or similar compounds, which can constitute the basis for biomonitoring of human exposure.
An analytical procedure is described for the identification of the adducts formed by interaction of methyl bromide and haemoglobin. The reaction products of in vitro incubation of haemoglobin with methyl bromide have been characterised by electrospray mass spectrometry and gas chromatography-mass spectrometry. A prominent reactivity of several potential nucleophilic sites of haemoglobin was observed. Analogous results were recorded on blood samples of workers exposed to methyl bromide. The results obtained represent the basis for the complete structural characterisation of the modified haemoglobin and demonstrate the usefulness of the proposed analytical approach for the evaluation of alkylation degree and the identification of modified amino acids in proteins.
1. The globin chain components of Sprague-Dawley rat hemoglobin were obtained by reverse-phase HPLC which showed the presence of two alpha-chain and four beta-chains. 2. The accurate molecular weight of each globin chain was determined by means of electrospray mass spectrometry. Extensive mass spectrometric analysis on several enzymatic digests by fast atom bombardment mass spectrometry (FAB-overlapping) meant to determine the complete sequence of the alpha-major and of the four beta-globins. 3. The primary structure of the alpha-major globin was found in agreement with literature data (Garrick et al., 1975 Biochem. J. 149, 245-258; Chua et al., 1987). 4. Sequence analysis of the four beta-globin chains showed that amino acid differences are restricted to two protein portions: the region 22-25 and 123-125, the remaining portions of the molecule being unchanged in the four globins. Furthermore, all the amino acid replacements correspond to single point DNA mutations and (with the exception of the substitution Asp 22-->Asn in the beta 2-globin) involve uncharged substitutions.
The present study describes how mass spectrometry was extensively applied to the characterization and quantification of modified amino acids within the polypeptide chain of Angiotensin I, chosen as model substrate, combining the use of fast atom bombardment mass spectrometry with gas chromatography--mass spectrometry. The reaction products after in vitro incubation of Angiotensin I with styrene oxide, a well known carcinogen, under different conditions, have been characterized: a prominent reactivity of several potential nucleophilic sites of Angiotensin I was shown, including two histidine residues and a tyrosine residue; it is worth noting that it has never been stated that tyrosine is highly reactive with styrene oxide. The results obtained demonstrate the usefulness of mass spectrometry for the structural determination of chemically modified amino acids in peptides and proteins, and the presence of a reliable relationship between reaction conditions and the production of alkylated amino acids. This characterization procedure offers the possibility of identifying reactive sites following exposure to unknown alkylating agents.
The aim of this study was to evaluate the extent of endogenous carbon monoxide production in subjects with ineffective erythropoiesis. Carboxyhaemoglobin concentrations were measured in eight transfused and eight nontransfused thalassemic patients. Measurements were made on venous blood collected under controlled conditions and were corrected by simultaneous measurement of HbF levels. Nontransfused thalassaemic patients with abnormal erythropoiesis were found to have elevated levels of carboxyhaemoglobin, significantly higher than those found in transfused subjects, as well as decreased cardiac performance. It is important to consider carboxyhaemoglobin levels in planning transfusion programmes.