[Show abstract][Hide abstract] ABSTRACT: A dynamic surface tension detector (DSTD) has been equipped with an additional pressure sensor for simultaneous viscosity measurements, as a detector for flow injection analysis. The viscosity measurement is based on a single capillary viscometer (SCV) placed in parallel configuration with the DSTD. The viscometer in the optimized conditions consists of a PEEK capillary (i.d.=0.25 mm, L=75 cm) kept at constant temperature using a thermostatic bath, which leads on the two sides to the two arms of a differential piezoelectric pressure transducer with a range of 0-35 psi. The DSTD, described previously, measures the changing pressure across the liquid/air interface of 2 μL drops repeatedly forming at the end of a capillary. SCV performance has been evaluated by measuring dynamic viscosity of water/glycerol mixtures analysed in flow injection and comparing the results with the values reported in the literature. The detection limits of SCV and DSTD, calculated as 3σ of the blank, were 0.012 cP and 0.6 dyn cm(-1), respectively. The FI-SCV-DSTD system has been applied to the study of temperature-induced denaturation/aggregation process in bovine serum albumin (BSA). The results have been supported and discussed with respect to BSA conformational analysis performed using Fourier Transform infrared spectroscopy.
[Show abstract][Hide abstract] ABSTRACT: We developed a photochemical method for the online oxidation of p-hydroxymercurybenzoate (PHMB), an organic mercury species widely used for mercaptan and thiolic compound labeling. The method is based on a fully integrated online UV/microwave (MW) photochemical reactor for the digestion of PHMB, followed by cold vapor generation atomic fluorescence spectrometry (CVG-AFS) detection. The MW/UV process led to the quantitative conversion of PHMB and thiol-PHMB complexes to Hg(II), with a yield between 91% and 98%, without using chemical oxidizing reagents and avoiding the use of toxic carcinogenic compounds. This reaction was followed by the reduction of Hg(II) to Hg(0), performed in a knitted reaction coil with NaBH(4) solution, and AFS detection in an Ar/H(2) miniaturized flame. The low MW power applied (18 W) allowed us to keep constant the temperature of the photochemical reactor (21 ± 1 °C), using a flowing water bath. This avoided peak widening due to diffusion processes generally occurring at high temperatures and in the additional cooling coil. This method has been applied to the determination of thiols in human plasma, blood, and wine.
[Show abstract][Hide abstract] ABSTRACT: Monomethylmercury and ethylmercury were determined on line using flow injection-chemical vapor generation atomic fluorescence spectrometry without neither requiring a pre-treatment with chemical oxidants, nor UV/MW additional post column interface, nor organic solvents, nor complexing agents, such as cysteine. Inorganic mercury, monomethylmercury and ethylmercury were detected by atomic fluorescence spectrometry in an Ar/H2 miniaturized flame after sodium borohydride reduction to Hg0, monomethylmercury hydride and ethylmercury hydride, respectively. The effect of mercury complexing agent such as cysteine, ethylendiaminotetracetic acid and HCl with respect to water and Ar/H2 microflame was investigated.The behavior of inorganic mercury, monomethylmercury and ethylmercury and their cysteine-complexes was also studied by continuous flow-chemical vapor generation atomic fluorescence spectrometry in order to characterize the reduction reaction with tetrahydroborate. When complexed with cysteine, inorganic mercury, monomethylmercury and ethylmercury cannot be separately quantified varying tetrahydroborate concentration due to a lack of selectivity, and their speciation requires a pre-separation stage (e.g. a chromatographic separation). If not complexed with cysteine, monomethylmercury and ethylmercury cannot be separated, as well, but their sum can be quantified separately with respect to inorganic mercury choosing a suitable concentration of tetrahydroborate (e.g. 10−5molL−1), thus allowing the organic/inorganic mercury speciation.The detection limits of the flow injection-chemical vapor generation atomic fluorescence spectrometry method were about 45nmolL−1 (as mercury) for all the species considered, a relative standard deviation ranging between 1.8 and 2.9% and a linear dynamic range between 0.1 and 5μmolL−1 were obtained. Recoveries of monomethylmercury and ethylmercury with respect to inorganic mercury were never less than 91%. Flow injection-chemical vapor generation atomic fluorescence spectrometry method was validated by analyzing the TORT-1 certificate reference material, which contains only monomethylmercury, and obtaining 83±5% of monomethylmercury recovered, respectively. This method was also applied to the determination of monomethylmercury in saliva samples.
Spectrochimica Acta Part B Atomic Spectroscopy 01/2011; · 3.14 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Despite the considerable number of published studies in the field of S-nitrosothiols (RSNO), the determination of these compounds in biological samples still represents an analytical challenge, due to several technical obstacles and often long sample preparation procedures. Other problems derive from the intrinsic lability of RSNO and the absence of certified reference material, analytically validated methods or suitable internal standards. Also, thiols and nitrites are usually present at high concentrations in biological matrices, and all precautions must be adopted in order to prevent artifactual formation of RSNO. Preanalytical steps (sampling, preservation and pre-treatment of samples) are particularly critical for the obtainment of reliable measurements. Three main mechanisms have been identified capable of compromising the assays: metal-catalyzed RSNO decomposition, reduction of the S-NO bond by thiols (transnitrosylation reactions) and enzymatic degradation of S-nitroso-glutathione (GSNO) by endogenous γ-glutamyltransferase (GGT) activity possibly present in the sample. If not adequately controlled, these factors likely contribute to the wide dispersion of values reported in the literature for RSNO and GSNO concentration in biological fluids, blood in the first place. The use of metal chelators, thiol reagents and GGT inhibitors appears therefore mandatory.
Life sciences 10/2010; 88(3-4):126-9. · 2.56 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Chromatographic determination of glutathione disulfide (GSSG) without any preliminary reduction has been presented using GSSG derivatization by p-hydroxymercuribenzoate (pHMB) in strong alkaline medium followed by the determination of GS-pHMB complex by reversed phase chromatography coupled to chemical vapour generation and atomic fluorescence detector (RPC-CVGAFS). A detection limit of 35 nM for GSSG (corresponding to 1.8 pmol) detected as GS-pHMB species was achieved based on a signal-to-noise ratio of 3 in buffer and in blood. The proposed method was applied to the determination of GSSG in whole blood and validated by the classical determination of GSSG by derivatization after reduction with dithiothreitol (DTT).
[Show abstract][Hide abstract] ABSTRACT: In this work we compared the results of the GSNO determination in human plasma by two independent methods. The first method is based on the pre-column derivatization of GSNO thiolic part by p-hydroxymercury benzoate (PHMB) and followed by the determination of GS-PHMB product by reversed phase chromatography coupled to chemical vapour generation atomic fluorescence spectrometry (RPC-CVGAFS). The second method is based on RPC separation of GSNO from interfering compounds and the post-column, on-line enzymatic hydrolysis of GSNO by commercial gamma-glutamyl transferase (GGT) and fluorescence detection. Endogenous GSNO was determined only in plasma from blood sampled by syringe (not by Vacutainers) and ranged between 157 and 257nM on the basis of RPC-CVGAFS method, and between 90 and 225nM by RPC-FD method. There was a good correlation between the two methods (slope=1.06+/-0.09, R(2)=0.9543). RPC-CVGAFS method based on PHMB derivatization determined a GSNO concentration 60+/-20nM in excess with respect to RPC-FD method. Sampling issues connected with common blood sampling procedures like venipuncture and sampling in syringe or Vacutainers still introduce in GSNO analysis unknown factors, which require further investigations.
[Show abstract][Hide abstract] ABSTRACT: The determination of S-nitrosoglutathione (GSNO) levels in biological fluids is controversial, partly due to the laborious sample handling and multiple pretreatment steps required by current techniques. GSNO decomposition can be effected by the enzyme gamma-glutamyltransferase (GGT), whose involvement in GSNO metabolism has been suggested. We have set up a novel analytical method for the selective determination and speciation of GSNO and its metabolite S-nitrosocysteinylglycine, based on liquid chromatography separation coupled to on-line enzymatic hydrolysis of GSNO by commercial GGT. In a post-column reaction coil, GGT allows the specific hydrolysis of the gamma-glutamyl moiety of GSNO, and the S-nitrosocysteinylglycine (GCNO) thus formed is decomposed by copper ions originating oxidized cysteinylglycine and nitric oxide (NO). NO immediately reacts with 4,5-diaminofluorescein (DAF-2) forming a triazole derivative, which is detected fluorimetrically. The limit of quantitation (LOQc) for GSNO and GCNO in plasma ultrafiltrate was 5 nM, with a precision (CV) of 1-6% within the 5-1500 nM dynamic linear range. The method was applied to evaluate the recovery of exogenous GSNO after addition of aliquots to human plasma samples presenting with different total GGT activities. By inhibiting GGT activity in a time dependent manner, it was thus observed that the recovery of GSNO is inversely correlated with plasmatic levels of endogenous GGT, which indicates the need for adequate inhibition of endogenous GGT activity for the reliable determination of endogenous GSNO.
Archives of Biochemistry and Biophysics 06/2009; 487(2):146-52. · 3.37 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: S-nitrosoglutathione (GSNO) is a nitric oxide (NO) donor compound which has been postulated to be involved in transport of NO in vivo. It is known that gamma-glutamyl transpeptidase (GGT) is one of the enzymes involved in the enzyme-mediated decomposition of GSNO, but no kinetics studies of the reaction GSNO-GGT are reported in literature. In this study we directly investigated the kinetics of GGT with respect to GSNO as a substrate and glycyl-glycine (GG) as acceptor co-substrate by spectrophotometry at 334 nm. GGT hydrolyses the gamma-glutamyl moiety of GSNO to give S-nitroso-cysteinylglycine (CGNO) and gamma-glutamyl-GG. However, as both the substrate GSNO and the first product CGNO absorb at 334 nm, we optimized an ancillary reaction coupled to the enzymatic reaction, based on the copper-mediated decomposition of CGNO yielding oxidized cysteinyl-glycine and NO. The ancillary reaction allowed us to study directly the GSNO/GGT kinetics by following the decrease of the characteristic absorbance of nitrosothiols at 334 nm. A K(m) of GGT for GSNO of 0.398+/-31 mM was thus found, comparable with K(m) values reported for other gamma-glutamyl substrates of GGT.
Archives of Biochemistry and Biophysics 11/2008; 481(2):191-6. · 3.37 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: L'S-nitrosilazione e' un processo di comunicazione cellulare ubiquitario nei sistemi biologici. Tuttavia, la ricerca sul signaling richiede metodi analitici per la determinazione delle specie dell'ossido nitrico (RSNOs) sensibili e specifici. La determinazione diretta delle RSNOs (metodi UV ed elettrochimici) e' limitata a concentrazioni micromolari. Approcci alternativi non rivelano RSNOs nella loro forma intatta, ma si basano sulla rivelazione indiretta dei loro metaboliti dopo fotolisi o riduzione chimica del legame S-NO: il radicale dell'ossido nitrico (NO), o il nitrito, suo prodotto di ossidazione e il prodotto tiolico. Il nitrito rilasciato e' stato rivelato mediante varie tecniche come la reazione di Griess, fluorimetria o rivelazione elettrochimica accoppiata o no a cromatografia liquida, o la gas cromatografia accoppiata a spettrometria di massa (GC-MS). Il radicale NO viene rivelato generalmente mediante chemiluminescenza o fluorimetria. Notoriamente, la misura di questi metaboliti in matrici biologiche e' difficile e richiede procedure complicate di manipolazione del campione, che coinvolgono l'interazione dei metaboliti degli RSNOs con i reattivi impiegati e con i costituenti della matrice biologica. Il tipo di matrice, la scelta della procedura di preparazione del campione, del sistema impiegato per la rottura del legame S-NO (fotolisi, HgCl2, HgCl2/V(III), KI/I2, Cys/KI/Cu(I), Cu(I)/Cys, Cu(I)/KI/I2, CO/Cu(I)/Cys, DTT), dell'analita rivelato (NO, nitrito o tioli), del reagente per la rivelazione (cromoforo, fluoroforo o ozono), della tecnica di rivelazione, possono pesantemente influenzare i risultati dell'analisi. Altri saggi per gli RSNOs ragionevolmente sensibili e accurati sono quelli basati sull'immunostaining. Ciascun saggio, tuttavia, ha dei limiti e dovrebbe essere complementato in maniera quantitativa da altri saggi. Il progresso continuo nell'ambito dei metodi analitici per il dosaggio degli RSNOs, del controllo delle procedure di campionamento e della fase preanalitica e' fondamentale per la comprensione del ruolo fisio-patologico degli RSNOs. Parole chiave: Ossido nitrico; S-nitrosotioli; Metodi Methods of assay of nitric oxide and its derivates. A review. S-Nitrosylation is a ubiquitous signaling process in biological systems. However, research regarding this signaling requires sensitive and specific analytical methods for the determination of nitric oxide species (RSNOs). Their direct quantitative UV or electrochemical detection has been limited to micromolar concentrations. Alternative approaches do not detect RSNOs in their intact form, but are based on indirect detection of their metabolites after photolysis or chemical reduction of S-NO bond: nitric oxide (NO) radical or its end oxidation product nitrite (NO2 - ) and the thiolic product. Releasable nitrite has been detected by various techniques such as the Griess reaction, fluorimetry and electrochemical detection, coupled or not with liquid chromatography, or gas chromatography-mass spectrometry (GC-MS). NO radical is generally detected by chemiluminescence or fluorimetry. Notoriously, the measurement of these metabolites in biological matrices is difficult and it requires complex sample handling, involving the interaction of these metabolites with various reactants and biological constituents. The sample matrix, the choice of sample preparation, of the system employed for the cleavage of S-NO bond (photolysis, HgCl2, HgCl2/V(III), KI/I2, Cys/KI/Cu(I), Cu(I)/Cys, Cu(I)/KI/I2, CO/Cu(I)/Cys, DTT), of the detected analyte (NO, nitrite, or thiol), of the detection reactant (chromophore, fluorophore or ozone), of the detection technique may strongly affect the results of the analysis. Other assays that provide reasonably sensitive and accurate data regarding biological S-nitrosothiols include immunostaining. Each assay, however, has limitations and should be quantitatively complemented by separate assays. Continued improvement in assays, in the control of sampling and pre-analytical procedures is fundamental to understand RSNO physio-pathological role.