Determination of dimethylarginine levels in rats using HILIC-MS/MS: an in vivo microdialysis study

Nitric oxide (NO) is one of the most important mediators and neurotransmitters and its levels change under pathological conditions. NO production may be regulated by endogenous nitric oxide synthase (NOS) inhibitors, in particular asymmetric dimethylarginine (ADMA). Most of the interest is focused on ADMA, since this compound is present in plasma and urine and accumulation of ADMA has been described in many disease states but little is known about cerebrospinal fluid (CSF) concentrations of this compound and of its structural isomer symmetric dimethylarginine (SDMA). To determine the levels of methylarginines, we here present a new hydrophilic interaction chromatography (HILIC)-MS/MS method for the precise determination of these substances in CSF from microdialysis samples of rat prefrontal cortex (PFC). The method requires only minimal sample preparation and features isotope-labelled internal standards.     

The N-terminal 1-16 peptide derived in vivo from protein seminal vesicle protein IV modulates alpha-thrombin activity: potential clinical implications

We have previously shown that seminal vesicle protein IV (SV-IV) and its 1-70 N-terminal fragment have anti-inflammatory activity and modulate anti-thrombin III (AT) activity. Moreover, mass spectrometry analysis of purified SV-IV has shown that the protein was found to be highly heterogeneous and 14% of the total SV-IV molecules are truncated forms, of particular interest the 1-16, 1-17, and 1-18 peptides. In this work we report experimental data which demonstrate that the 1-16 peptide (P1-16) possesses a marked effect on the AT activity by preventing the formation of the thrombin-AT complex. We found that the formation of thrombin-AT complex is markedly decreased in the presence of P1-16 used at equimolar concentration with thrombin as evaluated with SDS-PAGE. We also monitored the conformational changes of thrombin in the presence of different P1-16 concentrations, and calculated the K(d) of thrombin/P1-16 system by circular dichroism technique. The probable interaction sites of P1-16 with thrombin have been also evaluated by molecular graphics and computational analyses. These results have potential implications in the treatment of sterility and thrombotic diseases.     

Mass spectrometric approaches for elucidation of antigenantibody recognition structures in molecular immunology

Mass spectrometric approaches have recently gained increasing access to molecular immunology and several methods have been developed that enable detailed chemical structure identification of antigen-antibody interactions. Selective proteolytic digestion and MS-peptide mapping (epitope excision) has been successfully employed for epitope identification of protein antigens. In addition, "affinity proteomics" using partial epitope excision has been developed as an approach with unprecedented selectivity for direct protein identification from biological material. The potential of these methods is illustrated by the elucidation of a beta-amyloid plaque-specific epitope recognized by therapeutic antibodies from transgenic mouse models of Alzheimer's disease. Using an immobilized antigen and antibody-proteolytic digestion and analysis by high resolution Fourier transform ion cyclotron resonance mass spectrometry has lead to a new approach for the identification of antibody paratope structures (paratope-excision; "parex-prot"). In this method, high resolution MS-peptide data at the low ppm level are required for direct identification of paratopes using protein databases. Mass spectrometric epitope mapping and determination of "molecular antibody-recognition signatures" offer high potential, especially for the development of new molecular diagnostics and the evaluation of new vaccine lead structures.     

A novel LIF-CE method for the separation of hyaluronan- and chondroitin sulfate-derived disaccharides: Application to structural and quantitative analyses of human plasma low- and high-charged chondroitin sulfate isomers

The report describes a rapid and simple CE method using LIF detection for the analysis of unsaturated disaccharides obtained from enzymatic depolymerization of plasma chondroitin sulfate (CS) isomers. The disaccharide reducing groups were labeled with 2-aminoacridone (AMAC). The fluorotagged products can be separated by reversed-polarity CE using a sodium acetate buffer, pH 3.8, in the presence of 0.05% methylcellulose. The choice of the appropriate electrophoretic conditions was performed after a deep analysis of the most important parameters affecting analyte separation. In particular, the effect of both run buffer concentration and pH on resolution, efficiency, migration times, and peak area was evaluated. The selected electrophoretic conditions allowed us to separate the CS isomers-derived Delta-disaccharides in less than 12 min, also resolving the nonsulfated disaccharides released from CS isomers from those released from hyaluronan (HA). Moreover, these conditions gave a good reproducibility of both the migration times (CV%, 0.25) and the peak areas (CV%, 1.4). Intra- and interassay CV were 5.37 and 7.23%, respectively, and analytical recovery was about 86%. The applicability of the above method to the quantitative and structural disaccharide analyses of plasma CS isomers was investigated. Data obtained from 44 healthy human subjects were compared with those obtained by a fluorophore-assisted carbohydrate electrophoresis (FACE) reference assay, by using the Passing and Bablok regression and Bland-Altman tests. The developed method could represent a good tool for an ultrasensitive analysis of CS isomers in biological samples from different sources, particularly when samples are available in very low amounts.     

Rapid genotyping of CYP2D6, CYP2C19 and TPMT polymorphisms by primer extension reaction in a dipstick format

In recent years an increasing amount of interest has been directed at the study and routine testing of polymorphisms responsible for variations in drug metabolism. Most of the current methods involve either time-consuming electrophoresis steps or specialized and expensive equipment. In this context, we have developed a rapid, simple and robust method for genotyping of CYP2D6*3, CYP2D6*4, CYP2C19*2, CYP2C19*3 and TPMT*2 single nucleotide polymorphisms (SNP). Genomic DNA is isolated from whole blood and the segments that span the SNP of interest are amplified by PCR. The products are subjected directly (without purification) to two primer extension (PEXT) reactions (three cycles each) using normal and mutant primers in the presence of biotin-dUTP. The PEXT primers contain a (dA)₃₀ segment at the 5′ end. The PEXT products are detected visually by a dry-reagent dipstick-type assay in which the biotinylated extension products are captured from immobilized streptavidin on the test zone of the strip and detected by hybridization with oligo(dT)-functionalized gold nanoparticles. Patient samples (76 variants in total) were genotyped and the results were fully concordant with those obtained by direct DNA sequencing.     

Multivariate calibration on NIR data: development of a model for the rapid evaluation of ethanol content in bakery products

A new NIR method based on multivariate calibration for determination of ethanol in industrially packed wholemeal bread was developed and validated. GC-FID was used as reference method for the determination of actual ethanol concentration of different samples of wholemeal bread with proper content of added ethanol, ranging from 0 to 3.5% (w/w). Stepwise discriminant analysis was carried out on the NIR dataset, in order to reduce the number of original variables by selecting those that were able to discriminate between the samples of different ethanol concentrations. With the so selected variables a multivariate calibration model was then obtained by multiple linear regression. The prediction power of the linear model was optimized by a new “leave one out” method, so that the number of original variables resulted further reduced.     

Synthesis of beta‐proline like derivatives and their evaluation as sodium channel blockers

A simple and convenient procedure for the preparation of beta‐proline like derivatives in their racemic and optically active forms has been reported. The compounds have been screened for their potential activity as sodium channel blockers.     

4,2-Ribbon like ferromagnetic cyano-bridged Fe(III)2Ni(II) chains: a magneto-structural study

The low-spin iron(III) complex AsPh(4)[Fe(III)(bpy)(CN)(4)].CH(3)CN (1) [AsPh(4) = tetraphenylarsonium cation] and the heterobimetallic chains [{Fe(III)(L)(CN)(4)}(2)Ni(II)(H(2)O)(2)].4H(2)O with L = bpy (2) and phen (3) [bpy = 2,2'-bipyridine and phen = 1,10-phenanthroline] have been prepared and their structures determined by X-ray diffraction methods. The structure of 1 consists of mononuclear [Fe(bpy)(CN)(4)](-) anions, tetraphenylarsonium cations and acetonitrile molecules of crystallization. The iron(III) is hexacoordinated with two nitrogen atoms of the bidentate bpy and four carbon atoms of four terminal cyanide groups building a distorted octahedral surrounding around the metal atom. 2 and 3 are isomorphous compounds whose structure is made up of neutral 4,2-ribbon like bimetallic chains of formula [{Fe(III)(L)(CN)(4)}(2)Ni(II)(H(2)O)(2)] where the [Fe(III)(L)(CN)(4)](-) unit acts as a bis-monodentate bridging ligand toward the trans-diaquanickel(II) units through two of its four cyanide groups in cis positions. The chains exhibit two orientations in the unit cell and they interact with each other through hydrogen bonds involving the coordination and crystallization water molecules together with the uncoordinated cyanide nitrogen atoms of the [Fe(L)(CN)(4)](-) units. Compounds 2 and 3 behave as ferromagnetic Fe(III)(2)Ni(II) chains which interact ferromagnetically at very low temperatures in the case of 2, whereas metamagnetic-like behaviour is observed for with a critical field (H(c)) around 200 G. For H > H(c) the ferromagnetic Fe(III)(2)Ni(II) chains of 3 exhibit a frequency dependence of the out-of-phase ac susceptibility signal at T < 3.5 K.