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Assigning the correct configuration at C2 in sialosides is a standing problem because of the absence of an anomeric hydrogen. All different empirical rules that have been proposed over the years lack general applicability. In particular, the correct configuration of several 3,4-unsaturated derivatives of N-acetylneuraminic acid (Neu5Ac), which have been recently shown to be novel sialidase/neuraminidase inhibitors, could only be tentatively assigned by similarity with the reported 3,4-unsaturated 2O-methyl sialosides. In this work, we overcome this problem as we devised a rapid synthetic method to unequivocally resolve the anomeric configuration of the 3,4-unsaturated Neu5Ac derivatives through the synthesis of the corresponding unreported unsaturated 1,7-lactones. Moreover, we discovered a diagnostic 13C nuclear magnetic resonance signal that allows the formulation of a new empirical rule for the direct assignment of the C2 stereochemistry of these molecules, even when only one of the two C2 epimers is available.

In this work we made an attempt to assess the effect of drug-induced changes of flexibility on the penetration of deformable vesicles into the human skin. Eight cationic liposomes with different degrees of flexibility were obtained by entrapping unfractionated heparin, enoxaparin, and nadroparin. The deformability was studied by a novel, facile, and reliable extrusion assay appositely developed and validated by means of quantitative nanoscale mechanical AFM measurements of vesicle elastic modulus (log10(YM)). The proposed extrusion assay, determining the forces involved in vesicles deformation, resulted very sensitive to evidence of minimal changes in bilayer rigidity (σ) and vesicle deformation (K). The drug loading caused a reduction of liposome flexibility with respect to the reference plain liposomes and in accordance to the heparin type, drug to cationic lipid (DOTAP) ratio, and drug distribution within the vesicles. Interestingly, the σ and log10(YM) values perfectly correlated (R2= 0.935), demonstrating the reliability of the deformability data obtained with both approaches. The combination of TEM and LC–MS/MS spectrometry allowed the pattern of the penetration of the entire vesicles into the skin to be followed. In all cases, intact liposomes in the epidermis layers were observed and a relationship between the depth of penetration and the liposome flexibility was found, supporting the hypothesis of the whole vesicle penetration mechanism. Moreover, the results of the extent (R24) of vesicle penetration in the human skin samples showed a direct relation to the flexibility values (σ1= 0.65 ± 0.10 MPa → R24= 3.33 ± 0.02 μg/mg; σ2= 0.95 ± 0.04 MPa → R24= 1.18 ± 0.26 μg/mg; σ3= 1.89 ± 0.30 MPa → R24= 0.53 ± 0.33 μg/mg).

We developed a liquid chromatography electrospray ionisation multistage mass spectrometry LCESIMSMS approach based on precursorion scanning and evaluated it to characterize the covalent modifications of Cys34 human serum albumin HSA caused by oxidative stress and reactive carbonyl species RCS adduction. HSA was isolated and digested enzymatically to generate a suitablelength peptide LQQCPF containing the modified tag residue. The resulting LQQCPF peptides were identified by LCESIMSMS in precursorion scan mode and further characterized in production scan mode. The product ions for precursorion scanning were selected by studying the MSMS fragmentation of a series of LQQCPF derivatives containing Cys34 modified with different α,βunsaturated aldehydes and di and ketoaldehydes. We used a Boolean logic to enhance the specificity of the method: this reconstitutes a virtual current trace vCT showing the peaks in the three precursorion scans, marked by the same parent ion. The method was first evaluated to identify and characterize the Cys34 covalent adducts of HSA incubated with 4hydroxyhexenal, 4hydroxytrans2nonenal HNE and acrolein ACR. Then we studied the Cys34 modification of human plasma incubated with mildly oxidized lowdensity lipoproteins LDL, and the method easily identified the LQQCPF adducts with HNE and ACR. In other experiments, plasma was oxidized by 2,2azobis2amidinopropane HCl AAPH or by Fe2H2O2. In both conditions, the sulfinic derivative of LQQCPF was identified and characterized, indicating that the method is suitable not only for studying RCSmodified albumin, but also to check the oxidative state of Cys34 as a marker of oxidative damage. Copyright © 2008 John Wiley & Sons, Ltd.

The aim of this study was to elucidate the vasodilating mechanism of sodium nitroprusside (SNP). To do this, SNP was intravenously infused in pigs (1.67 µmol/kg), and the following paramagnetic metabolites were identified by electron spin resonance: 1) nitrosylhemoglobin [HbFe(II)NO] as an index of the bioconservative pathway; 2) transferrin; 3) [Fe(II)(CN)5 NO]3- and [Fe(II)(CN)4 NO]2-, the reduced penta- and tetracoordinated intermediates of SNP, respectively; and 4) methemoglobin (met-Hb). The results indicate the following: 1) ≈17% of the dose is converted to HbFe(II)NO at the end of infusion; 2) NO administered as SNP does not undergo bioinactivation (oxidative metabolism), because no significant increase of met-Hb was observed; 3) the equilibrium involving the paramagnetic species of SNP is shifted toward HbFe(II)NO, because a significant increase of transferrin but no detection of the reduced paramagnetic intermediates of SNP was observed. The results obtained indicate that the hemodynamic effect induced by SNP is not mediated by HbFe(II)NO, at least under physiological conditions; hence, a direct release of NO from SNP in the vascular target should be considered. To demonstrate this mechanism, endothelial cells were incubated with SNP, and the release of NO was determined by a novel chemiluminescence method. The results indicate that the endothelium is able to metabolize SNP, with the formation of stoichiometric amounts of NO. In conclusion, SNP is rapidly metabolized to HbFe(II)NO, but the pharmacological response is mediated by a direct mechanism of NO release of the parent compound at the cellular target.

Several pieces of evidence indicate that albumin modified by HNE is a promising biomarker of systemic oxidative stress and that HNE‐modified albumin may contribute to the immune reactions triggered by lipid peroxidation‐derived antigens. In this study, we found by HPLC analysis that HNE is rapidly quenched by human serum albumin (HSA) because of the covalent adduction to the different accessible nucleophilic residues of the protein, as demonstrated by electrospray ionization mass spectrometry (ESI‐MS) direct infusion experiments (one to nine HNE adducts, depending on the molar ratio used, from 1 : 0.25 to 1 : 5 HSA : HNE). An LC‐ESI‐MS/MS approach was then applied to enzymatically digested HNE‐modified albumin, which permitted the identification of 11 different HNE adducts, 8 Michael adducts (MA) and 3 Schiff bases (SB), involving nine nucleophilic sites, namely: His67 (MA), His146 (MA), His242 (MA), His288 (MA), His510 (MA), Lys 195 (SB), Lys 199 (MA, SB), Lys525 (MA, SB) and Cys34 (MA). The most reactive HNE‐adduction site was found to be Cys34 (MA) followed by Lys199, which primarily reacts through the formation of a Schiff base, and His146, giving the corresponding HNE Michael adduct. These albumin modifications are suitable tags of HNE‐adducted albumin and could be useful biomarkers of oxidative and carbonylation damage in humans. Copyright © 2006 John Wiley & Sons, Ltd.

The aim of this paper is to provide an overview on Electron Paramagnetic Resonance (EPR) spectroscopy [or equivalently Electron Spin Resonance (ESR)], indicating the areas of consolidated and potential applications in the pharmaceutical, biomedical and clinical fields, and the instrumental and conceptual developments that are involved. The unique capability of EPR to detect and characterise free radicals and other species with unpaired electrons has been successfully applied to measure both in vitro and in vivo free radical intermediates from drugs and oxidative processes, to study the free radical/antioxidant properties of drugs, to study drug-membrane interactions or to monitor pharmaceuticals radio sterilisation. Although EPR has been mainly devoted in the past to qualitative analysis, following development of software and high-sensitivity instrumentation, applications of the technique have been extended more recently to the quantitative analysis. The potential value of in vivo EPR includes: drug detection in body fluids, pharmacokinetic and metabolism studies, direct detection or measurement of free radicals (especially nitric oxide, the critical physiological mediator of vascular homeostasis and cardiac function). In addition, because EPR almost uniquely can make repeated and accurate measurements of pO2 in tissues, another interesting area in the pharmaceutical field is oximetry (now emerging also for clinical use), and some examples of its application in drug/formulation development, and in preclinical studies are reported.

The histidine‐containing dipeptides carnosine (CAR) and structurally related anserine (ANS) and homocarnosine (HCAR), widely distributed in vertebrate organisms, have recently been proposed as endogenous quenchers for highly cytotoxic α,β‐unsaturated aldehydes generated by peroxidation. A sensitive, selective, specific and rapid liquid chromatographic/electrospray ionization tandem mass spectrometric assay was developed and validated for the simultaneous determination of these peptides in biological matrices in order to establish their plasma/tissue distribution. Samples (plasma or tissue homogenates from male rats) were prepared by protein precipitation with HClO4 (1 : 1, v/v) containing H‐Tyr‐His‐OH as internal standard. The supernatant was separated on a Phenomenex Sinergy polar‐RP column with a mobile phase of water–acetonitrile–heptafluorobutyric acid (9 : 1 : 0.01, v/v/v) at a flow‐rate of 0.2 ml min−1, with a run time of 10 min. Detection was effected on an ion trap mass spectrometer equipped with an electrospray ionization interface operating in positive ionization mode. The acquisitions were in the multiple reaction monitoring mode using the following precursor → product ion combinations: H‐Tyr‐His‐OH (internal standard) m/z 319 → 301; CAR m/z 227 → 210 + 209; ANS m/z 241 → 224 + 197 + 170; HCAR m/z 241 → 156. The method was validated over the concentration range 15–1000 nmol g−1 and the limit of quantification (LOQ) and limit of detection (LOD) were 12.5 and 4.2 pmol injected, respectively. The intra‐ and inter‐day precisions were <10% (≤17.47% at the LOQ) and the intra‐ and inter‐assay accuracies were within ±10% for all concentrations. The mapping profile in rat tissue gave the following results: the highest concentrations of CAR and ANS were found in skeletal muscles (soleus, gastrocnemius, tibialis), followed by the heart, cerebellum and brain (ANS below the LOQ). HCAR was found only in the brain and cerebellum. No histidine‐containing dipeptides were detectable in plasma, liver, kidney and lung. Copyright © 2004 John Wiley & Sons, Ltd.

Keratinocytes are potential targets of lipid peroxidation products (α,β-unsaturated aldehydes) generated in the skin following UV exposure, among which the most abundant and toxic product is 4-hydroxy-trans-2,3-nonenal (HNE). The aim of this study was to investigate the ability of keratinocytes (NCTC2544 cell lines) to detoxify HNE, through characterization of metabolites, until now never demonstrated, using a combined analytical approach (liquid chromatography (LC) and liquid chromatography/mass spectrometry (LC/MS)). Incubation of cells with HNE (up to 200 µM) was performed in order to evaluate the ability of the cells to detoxify this toxic aldehyde, and indicated that the cell viability was maintained under these conditions. LC analysis of the extracellular media from keratinocytes incubated with 100 µM HNE shows a time-dependent decrease of HNE, disappearance from the medium within 2 h and concomitant formation of two unconjugated (phase I) metabolites, 4-hydroxy-2-nonenoic acid (HNA) and 1,4-dihydroxy-2-nonene (DHN), which were both identified and quantified by LC and accounted for 48.8 ± 4.6% of the HNE dose. Four additional metabolites were identified in the extracellular medium by reversed-phase LC coupled with electrospray ionization tandem mass spectrometry (LC/ESI-MS/MS) with positive and negative ion detection as Michael adducts (phase II metabolites), arising by the addition of the nucleophilic sulfur of glutathione (GSH) to the electrophilic C-3 of HNE, followed by oxidation–reduction enzymatic processes. The GSH–HNE conjugates were (a) S-(4-hydroxynonanal-3-yl)glutathione, (b) S-(1,4-dihydroxy-nonane-3-yl)glutathione, (c) S-(4-oxononanal-3-yl)glutathione and (d) S-(4-oxo-nonan-1-ol-3-yl)glutathione, and accounted for 52.3 ± 5.8% of the HNE dose (35 nmol mg⁻¹ protein), as estimated indirectly by measuring the extent of cellular GSH consumption (18.7 ± 1.8 nmol mg⁻¹ protein). The time course of HNE biotransformation was then determined by monitoring the formation of metabolites inside and outside the cell at different times after HNE addition (5–120 min). A time-dependent and almost linear formation inside the cell was observed for all the metabolites (plateau after 15 min of incubation), followed by a rapid decay and a concomitant increase in the extracellular medium (plateau of formation after 60 min). This confirms that HNE diffuses into the cell where is totally metabolized through phase I and phase II reactions to unreactive products, which are then exported outside the cell. This is the first demonstration that skin epidermal cells are able to detoxify the cytotoxic products of lipid peroxidation. Copyright © 2003 John Wiley & Sons, Ltd.