LC-MS/MS method for simultaneous quantification of heparan sulfate and dermatan sulfate in urine by butanolysis derivatization.

Mucopolysaccharidoses are a group of lysosomal storage disorders (LSDs) characterized by the accumulation of glycosaminoglycans (GAGs). Recently, LC-MS/MS has been widely applied in GAGs analysis combined with different sample preparations for cleaving GAGs to disaccharide units. The aim of the present is paper is to present a new method for the simultaneous quantification of urinary dermatan sulfate (DS) and heparan sulfate (HS) by LC-MS/MS, after butanolysis reaction. Chromatographic separation was achieved with a gradient of acetonitrile and water in 0.1% formic acid on a Kinetex Biphenyl analytical column in 21 min. Calibration curves ranging from 0.78 to 50 µg/mL for HS and from 1.56 to 100 µg/mL for DS were prepared and the coefficient of determination (r2) was higher than 0.99 for both analytes. Intra-day and inter-day imprecisions and the bias for both compounds were <10.0%. Up to now, most analytical procedures for quantifying GAGs have not had a high level of reproducibility among laboratories, despite the availability of various techniques. The adoption of a new protocol incorporating the methods outlined in this paper could significantly improve the quality and reproducibility of MS results. A procedure using simple steps for preparing samples and reagents that are easily available on the market could promote the standardization of analytical procedures and increase the use of these measurements in clinical practice.

Development of a fast LC-MS/MS protocol for combined measurement of six LSDs on dried blood spot in a newborn screening program.

New treatment options and improved strategies for Lysosomal Storage Disorders (LSDs) diagnosis on dried blood spot (DBS) have led to the development of several pilot newborn screening programs. Building on a previously published protocol, we devised a new 6-plex assay based on a single DBS punch incubated into a buffer containing a combination of substrates (GAA, GLA, ASM, GALC, ABG and IDUA). This new protocol incorporates a new trapping and clean-up procedure using perfusion chromatography connected on-line with an analytical column for analyte separation, after enzymatic reaction. Results are available after 4.5 min. Several incubation times were tested in order to reduce sample preparation times and to improve accuracy and reproducibility, also regarding the quenching of the reaction within the time window of linear product accumulation. The collected data demonstrate that an incubation time of 4 h is enough to achieve good reaction efficiency without any impact on sensitivity. The method proved versatile and robust for various instrument configurations. The fast sample preparation and running times allow a high sample throughput; an advantage in newborn screening procedures. This method can also be used for diagnostic purposes, allowing a rapid diagnosis in a few hours.

Data in support for the measurement of heparan sulfate and dermatan sulfate by LC-MS/MS analysis.

This article provides supplementary data for the paper "LC-MS/MS method for simultaneous quantification of heparan sulfate and dermatan sulfate in urine by butanolysis derivatization" (Forni et al., 2018). Several parameters were tested to optimize sample preparation by butanolysis in order to carry out simultaneous quantifications of HS and DS by tandem mass spectrometry. Here we describe step-by-step instructions to perform HS and DS analysis in urine samples using external calibration curves of standards of known concentration. Sample are quantified by interpolation from the calibration curve and reported in µg/mL. Then, HS and DS are normalized to creatinine concentration and reported as mg/g uCr.

2'-C-Methyl branched pyrimidine ribonucleoside analogues: potent inhibitors of RNA virus replication.

RNA viruses are the agents of numerous widespread and often severe diseases. Their unique RNA-dependent RNA polymerase (RDRP) is essential for replication and, thus, constitutes a valid target for the development of selective chemotherapeutic agents. In this regard, we have investigated sugar-modified ribonucleoside analogues as potential inhibitors of the RDRP. Title compounds retain 'natural' pyrimidine bases, but possess a beta-methyl substituent at the 2'-position of the D- or L-ribose moiety. Evaluation against a broad range of RNA viruses, either single-stranded positive (ssRNA+), single-stranded negative (ssRNA-) or double-stranded (dsRNA), revealed potent activities for D-2'-C-methyl-cytidine and -uridine against ssRNA+, and dsRNA viruses. None of the L-enantiomers were active. Moreover, the 5'-triphosphates of the active D-enantiomers were found to inhibit the bovine virus diarrhoea virus polymerase. Thus, the 2'-methyl branching of natural pyrimidine ribonucleosides transforms physiological molecules into potent, broad-spectrum antiviral agents that merit further development.

Metabolomics profiling reveals different patterns in an animal model of asphyxial and dysrhythmic cardiac arrest.

Cardiac arrest (CA) is not a uniform condition and its pathophysiology strongly depends on its cause. In this work we have used a metabolomics approach to study the dynamic metabolic changes occurring in the plasma samples of a swine model following two different causes of CA, namely asphyxia (ACA) and ventricular fibrillation (VFCA). Plasma samples were collected at baseline and every minute during the experimental phases. In order to identify the metabolomics profiles characterizing the two pathological entities, all samples were analysed by 1H NMR spectroscopy and LC-MS/MS spectrometry.The metabolomics fingerprints of ACA and VFCA significantly differed during the peri-arrest period and the resuscitation phase. Major alterations were observed in plasma concentrations of metabolites related to tricarboxylic acid (TCA) cycle, urea cycle, and anaplerotic replenishing of TCA. ACA animals showed significant metabolic disturbances during the asphyxial and CA phases, while for VFCA animals this phenomenon resulted shifted at the resuscitation phase. Interestingly, starting from the asphyxial phase, the ACA animals were stratified in two groups based on their metabolomics profiles that resulted to be correlated with the clinical outcome. Succinate overproduction was observed in the animals with the worse outcome, suggesting a potential prognostic role for this metabolite.

Computer-aided design, synthesis, and anti-HIV-1 activity in vitro of 2-alkylamino-6-[1-(2,6-difluorophenyl)alkyl]-3,4-dihydro-5-alkylpyrimidin-4(3H)-ones as novel potent non-nucleoside reverse transcriptase inhibitors, also active against the Y181C variant.

Dihydro-alkoxy-benzyl-oxopyrimidines (DABOs) are a family of potent NNRTIs developed in the past decade. Attempts to improve their potency and selectivity led to thio-DABOs (S-DABOs), DATNOs, and difluoro-thio-DABOs (F(2)-S-DABOs). More recently, we reported the synthesis and molecular modeling studies of a novel conformationally constrained subtype of the S-DABO series characterized by the presence of substituents on the methylene linkage connecting the pyrimidine ring to the aryl moiety (Mai, A., et al. J. Med. Chem. 2001, 44, 2544-2554). Now we report the computer-aided design, synthesis, and biological evaluation of four new DABO prototypes (5-alkyl-2-cyclopentylamino-6-[1-(2,6-difluorophenyl)alkyl]-3,4-dihydropyrimidin-4(3H)-ones, F(2)-NH-DABOs) in which the sulfur atom of the related F(2)-S-DABOs is replaced by an amino group. For these studies, we used as a reference model the cocrystallized MKC-442/RT complex. Docking studies with Autodock of the newly designed F(2)-NH-DABOs on the ligand-derived RT confirmed the findings previously described for the F(2)-S-DABOs. The F(2)-NH-DABO binding mode resembles that reported for F(2)-S-DABOs, with the difference that the NH moiety at the C-2 position represents a new anchor site for ligand/enzyme complexation. The predicted inhibition constant (K(i)) values by the internal scoring function of Autodock, and the predicted IC(50) values by the application of a VALIDATE II/HIV-RT model strongly suggested the synthesis of the designed amino-DABOs. F(2)-NH-DABOs were shown to be highly active in both anti-RT and anti-HIV biological assays with IC(50) and EC(50) comparable with that of the reference compound MKC-442. Interestingly, 2-cyclopentylamino-6-[1-(2,6-difluorophenyl)ethyl]-3,4-dihydro-5-methyl pyrimidin-4(3H)-one (9d) was active against the Y181C HIV-1 mutant strain at submicromolar concentration, with a resistance value similar to that of efavirenz, the last FDA-approved NNRTI for AIDS therapy, and 2-fold lower than that of its 2-cyclopentylthio counterpart 8d. The introduction in 9d of a new anchor point (pyrimidine C-2 NH group), with the formation of a new hydrogen bond with Lys101, could compensate for the lack of positive hydrophobic ligand/NNBP interactions due to the Tyr181 to Cys181 mutation.

Design, synthesis, SAR, and molecular modeling studies of acylthiocarbamates: a novel series of potent non-nucleoside HIV-1 reverse transcriptase inhibitors structurally related to phenethylthiazolylthiourea derivatives.

A novel series of potent, selective HIV-1 N-acylthiocarbamate (ATC) nonnucleoside reverse transcriptase inhibitors (NNRTIs) is described. The title compounds were synthesized through a highly convergent, one-pot procedure. In cell-based assays, the lead compound (17c) prevented the HIV-1 multiplication with an EC(50) of 8 microM. The lead optimization strategy was developed by single or multiple modifications of the three molecular portions, in which 17c was notionally divided. Molecular modeling studies led to the synthesis of O-(2-phthalimidoethyl)-N-(p-substituted phenyl)-N-acylthiocarbamates, which showed in vitro activities against HIV-1 in the low nanomolar range. Nevertheless, the title compounds retained low potency against HIV-1 strains carrying mutations (K103R, Y181C, and K103N/Y181C) responsible for NNRTI resistance. The hypothetical docking model of RT/17c and RT/25c, derived from X-ray crystallographic structure of a PETT derivative in complex with HIV-1 RT, revealed that the model structures of ATCs do not approximate the NNRTI butterfly-like conformation. Analysis of these hypotetical complexes helps to rationalize some SARs and resistance data.

Novel indolyl aryl sulfones active against HIV-1 carrying NNRTI resistance mutations: synthesis and SAR studies.

The potent anti-HIV-1 activities of L-737,126 (2) and PAS sulfones prompted us to design and test against HIV-1 in acutely infected MT-4 cells a number of novel 1- and 3-benzenesulfonylindoles. Indoles belonging to the 1-benzenesulfonyl series were found poorly or totally inactive. On the contrary, some of the 3-benzenesulfonyl derivatives turned out to be as potent as 2, being endowed with potencies in the low nanomolar concentration range. In particular, (2-methylphenyl)sulfonyl (72) and (3-methylphenyl)sulfonyl (73) derivatives showed EC(50) values of 1 nM. Introduction of two methyl groups at positions 3 and 5 of the phenyl ring of 2 furnished derivatives (80 and 83) which showed very potent and selective anti-HIV-1 activity not only against the wt strain, but also against mutants carrying NNRTI-resistant mutations at positions 103 and 181 of the reverse transcriptase gene.

Simple, short peptide derivatives of a sulfonylindolecarboxamide (L-737,126) active in vitro against HIV-1 wild type and variants carrying non-nucleoside reverse transcriptase inhibitor resistance mutations.

Non-nucleoside reverse transcriptase inhibitors (NNRTIs) active against NNRTI-resistant mutants were obtained by introducing two methyl groups at positions 3 and 5 of the benzenesulfonyl moiety of L-737,126 (1) and coupling one to three glycinamide/alaninamide units to its carboxyamide function. In cell-based assays, the new derivatives showed activities against HIV-1 wild type and NNRTI-resistant mutants [Y181C, K103N-Y181C, and triple mutant (K103R, V179D, P225H) highly resistant to efavirenz] superior to that of the parent indole derivative 1.

Reducing the False-Positive Rate for Isovalerylcarnitine in Expanded Newborn Screening: The Application of a Second-Tier Test

Abstract The isodecyl neopentanoate is an ingredient used in the cosmetic industry to prepare a nipple fissure balm. We report on 12 newborns that showed elevated C5-acylcarnitine levels upon newborn screening following treatment with balm. The first 3 neonates were immediately recalled for confirmatory tests and resulted negative for both isovaleric acidemia and short/branched chain acyl-CoA dehydrogenase deficiency. In the other 9 cases, the immediate recall was avoided by applying a new second-tier test able to confirm the presence of pivaloylcarnitine. The concentration of C5-acylcarnitine was measured in the days following the suspension of balm application. Abnormal concentrations of C5-acylcarnitine did not seem to be associated with free carnitine deficiency, probably due to the short time of exposure. A direct correlation between balm ingestion and the elevation in pivaloylcarnitine has been demonstrated in 10 adult volunteers. The commercial balm containing a pivalic acid derivative is causal of false-positive results during newborn screening, and it could have the potential to cause secondary carnitine deficiency when used chronically.

5-Alkyl-2-alkylamino-6-(2,6-difluorophenylalkyl)-3,4-dihydropyrimidin-4(3H)-ones, a new series of potent, broad-spectrum non-nucleoside reverse transcriptase inhibitors belonging to the DABO family.

2-Alkylamino-6-[1-(2,6-difluorophenyl)alkyl]-3,4-dihydro-5-alkylpyrimidin-4(3H)-ones (F(2)-NH-DABOs) 4, 5 belonging to the dihydro-alkoxy-benzyl-oxopyrimidine (DABO) family and bearing different alkyl- and arylamino side chains at the C(2)-position of the pyrimidine ring were designed as active against wild type (wt) human immunodeficiency virus type 1 (HIV-1) and some relevant HIV-1 mutants. Biological evaluation indicated the importance of the further anchor point of compounds 4, 5 into the non-nucleoside binding site (NNBS): newly synthesized compounds were highly active against both wild type and the Y181C HIV-1 strains. In anti-wt HIV-1 assay the potency of amino derivatives did not depend on the size or shape of the C(2)-amino side chain, but it associated with the presence of one or two methyl groups (one at the pyrimidine C(5)-position and the other at the benzylic carbon), being thymine, alpha-methyluracil or alpha-methylthymine derivatives almost equally active in reducing wt HIV-1-induced cytopathogenicity in MT-4 cells. Against the Y181C mutant strain, 2,6-difluorobenzyl-alpha-methylthymine derivatives 4d, 5h'-n' showed the highest potency and selectivity among tested compounds, both a properly sized C(2)-NH side chain and the presence of two methyl groups (at C(5) and benzylic positions) being crucial for high antiviral action.