Surfactant disaturated-phosphatidylcholine kinetics in acute respiratory distress syndrome by stable isotopes and a two compartment model

BACKGROUND: In patients with acute respiratory distress syndrome (ARDS), it is well known that only part of the lungs is aerated and surfactant function is impaired, but the extent of lung damage and changes in surfactant turnover remain unclear. The objective of the study was to evaluate surfactant disaturated-phosphatidylcholine turnover in patients with ARDS using stable isotopes. METHODS: We studied 12 patients with ARDS and 7 subjects with normal lungs. After the tracheal instillation of a trace dose of (13)C-dipalmitoyl-phosphatidylcholine, we measured the (13)C enrichment over time of palmitate residues of disaturated-phosphatidylcholine isolated from tracheal aspirates. Data were interpreted using a model with two compartments, alveoli and lung tissue, and kinetic parameters were derived assuming that, in controls, alveolar macrophages may degrade between 5 and 50% of disaturated-phosphatidylcholine, the rest being lost from tissue. In ARDS we assumed that 5–100% of disaturated-phosphatidylcholine is degraded in the alveolar space, due to release of hydrolytic enzymes. Some of the kinetic parameters were uniquely determined, while others were identified as lower and upper bounds. RESULTS: In ARDS, the alveolar pool of disaturated-phosphatidylcholine was significantly lower than in controls (0.16 ± 0.04 vs. 1.31 ± 0.40 mg/kg, p < 0.05). Fluxes between tissue and alveoli and de novo synthesis of disaturated-phosphatidylcholine were also significantly lower, while mean resident time in lung tissue was significantly higher in ARDS than in controls. Recycling was 16.2 ± 3.5 in ARDS and 31.9 ± 7.3 in controls (p = 0.08). CONCLUSION: In ARDS the alveolar pool of surfactant is reduced and disaturated-phosphatidylcholine turnover is altered

Surfactant disaturated-phosphatidylcholine kinetics in acute respiratory distress syndrome by stable isotopes and a two compartment model

BACKGROUND: In patients with acute respiratory distress syndrome (ARDS), it is well known that only part of the lungs is aerated and surfactant function is impaired, but the extent of lung damage and changes in surfactant turnover remain unclear. The objective of the study was to evaluate surfactant disaturated-phosphatidylcholine turnover in patients with ARDS using stable isotopes. METHODS: We studied 12 patients with ARDS and 7 subjects with normal lungs. After the tracheal instillation of a trace dose of (13)C-dipalmitoyl-phosphatidylcholine, we measured the (13)C enrichment over time of palmitate residues of disaturated-phosphatidylcholine isolated from tracheal aspirates. Data were interpreted using a model with two compartments, alveoli and lung tissue, and kinetic parameters were derived assuming that, in controls, alveolar macrophages may degrade between 5 and 50% of disaturated-phosphatidylcholine, the rest being lost from tissue. In ARDS we assumed that 5–100% of disaturated-phosphatidylcholine is degraded in the alveolar space, due to release of hydrolytic enzymes. Some of the kinetic parameters were uniquely determined, while others were identified as lower and upper bounds. RESULTS: In ARDS, the alveolar pool of disaturated-phosphatidylcholine was significantly lower than in controls (0.16 ± 0.04 vs. 1.31 ± 0.40 mg/kg, p < 0.05). Fluxes between tissue and alveoli and de novo synthesis of disaturated-phosphatidylcholine were also significantly lower, while mean resident time in lung tissue was significantly higher in ARDS than in controls. Recycling was 16.2 ± 3.5 in ARDS and 31.9 ± 7.3 in controls (p = 0.08). CONCLUSION: In ARDS the alveolar pool of surfactant is reduced and disaturated-phosphatidylcholine turnover is altered

Pilot Investigation of SARS-CoV-2 Variants in the Island of Sicily Prior to and in the Second Wave of the COVID-19 Pandemic

8 páginas, 2 tablas, 2 figuras. The datasets presented in this study can be found in online repositories. The names of the repository/repositories and accession number(s) can be found below: https://www.ncbi.nlm.nih.gov/genbank/, OM510944; https://www.ncbi.nlm.nih.gov/genbank/, OM510945; https://www.ncbi. nlm.nih.gov/genbank/, OM510946; https://www.ncbi.nlm.nih. gov/genbank/, OM510947; https://www.ncbi.nlm.nih.gov/genbank/, OM510948; https://www.ncbi.nlm.nih.gov/genbank/, OM510949; https://www.ncbi.nlm.nih.gov/genbank/, OM51 0950; https://www.ncbi.nlm.nih.gov/genbank/, OM510951; and https://www.ncbi.nlm.nih.gov/genbank/, OM510952.After 2 years of the COVID-19 pandemic, we continue to face vital challenges stemming from SARS-CoV-2 variation, causing changes in disease transmission and severity, viral adaptation to animal hosts, and antibody/vaccine evasion. Since the monitoring, characterization, and cataloging of viral variants are important and the existing information on this was scant for Sicily, this pilot study explored viral variants circulation on this island before and in the growth phase of the second wave of COVID-19 (September and October 2020), and in the downslope of that wave (early December 2020) through sequence analysis of 54 SARS-CoV-2-positive samples. The samples were nasopharyngeal swabs collected from Sicilian residents by a state-run one-health surveillance laboratory in Palermo. Variant characterization was based on RT-PCR amplification and sequencing of four regions of the viral genome. The B.1.177 variant was the most prevalent one, strongly predominating before the second wave and also as the wave downsized, although its relative prevalence decreased as other viral variants, particularly B.1.160, contributed to virus circulation. The occurrence of the B.1.160 variant may have been driven by the spread of that variant in continental Europe and by the relaxation of travel restrictions in the summer of 2020. No novel variants were identified. As sequencing of the entire viral genome in Sicily for the period covered here was restricted to seven deposited viral genome sequences, our results shed some light on SARS-CoV-2 variant circulation during that wave in this insular region of Italy which combines its partial insular isolation with being a major entry point for the African immigration.This research received external funding to CR-G and EM from “Agencia Valenciana de Innovación: COVID-19, Ayudas de Concesión Directa a Soluciones Científico-Innovadoras Directamente Relacionadas Con La Lucha Contra La COVID-19,” (Ref COVID-19-203) and from “Conselleria de Innovación Universidades, Ciencia y Sociedad Digital: Subvenciones a Grupos de Investigación Emergentes” (Ref, GV/2021/163); to VR from the Agencia Estatal de Investigación of the Spanish Government (Ref PID2020-120322RB-C21) and from the European Commission–NextGeneration EU CSIC Global Health Platform, Spanish Ministry of Science and Innovation (Ref MCIN/AEI/10.13039/501100011033); and to the Istituto Zooprofilattico Sperimentale della Sicilia “A. Mirri” by the Project “COVID-19: Traiettorie Evolutive di SARS-CoV-2 ed Indagine Sul Ruolo Degli Animali” (IZS SI 03/20 RC), funded by the Italian Ministry of Health.Peer reviewe

Quantification of Underivatised Amino Acids on Dry Blood Spot, Plasma, and Urine by HPLC\u2013ESI\u2013MS/MSAmino Acid Analysis

Enzyme deficiencies in amino acid (AA) metabolism affecting the levels of amino acids and their derivatives in physiological fluids may serve as diagnostically significant biomarkers for one or a group of metabolic disorders. Therefore, it is important to monitor a wide range of free amino acids simultaneously and to quantify them. This is time consuming if we use the classical methods and more than ever now that many laboratories have introduced Newborn Screening Programs for the semiquantitative analysis, detection, and quantification of some amino acids needed to be performed in a short time to reduce the rate of false positives.We have modified the stable isotope dilution HPLC-electrospray ionization (ESI)-MS/MS method previously described by Qu et al. (Anal Chem 74: 2034-2040, 2002) for a more rapid, robust, sensitive, and specific detection and quantification of underivatised amino acids. The modified method reduces the time of analysis to 10 min with very good reproducibility of retention times and a better separation of the metabolites and their isomers.The omission of the derivatization step allowed us to achieve some important advantages: fast and simple sample preparation and exclusion of artefacts and interferences. The use of this technique is highly sensitive, specific, and allows monitoring of 40 underivatized amino acids, including the key isomers and quantification of some of them, in order to cover many diagnostically important intermediates of metabolic pathways.We propose this HPLC-ESI-MS/MS method for underivatized amino acids as a support for the Newborn Screening as secondary test using the same dried blood spots for a more accurate and specific examination in case of suspected metabolic diseases. In this way, we avoid plasma collection from the patient as it normally occurs, reducing anxiety for the parents and further costs for analysis.The same method was validated and applied also to plasma and urine samples with good reproducibility, accuracy, and precision. The fast run time, feasibility of high sample throughput, and small amount of sample required make this method very suitable for routine analysis in the clinical setting

Measurement of pulmonary surfactant disaturated-phosphatidylcholine synthesis in human infants using deuterium incorporation from body water

The aim of the study was to determine surfactant palmitate disaturated-phosphatidylcholine (DSPC-PA) synthesis in vivo in humans by the incorporation of deuterium from total body water into DSPC-PA under steady state condition. We studied three newborns and one infant (body weight (BW) 4.6 \ub1 2.9 kg, gestational age 37.5 \ub1 2 weeks, age 9 \ub1 9 days) and four preterm newborns (BW 1.3 \ub1 0.6 kg, gestational age 30.3 \ub1 2.5 weeks, postnatal age 8.8 \ub1 9.2 h). All infants were mechanically ventilated during the study and the four preterm infants received exogenous surfactant at the start of the study. We administered 0.44 g 2H 2O/kg BW as a bolus intravenously, followed by 0.0125 g 2H2O/kg BW every 6 h to maintain deuterium enrichment at plateau over 2 days. Urine samples and tracheal aspirates (TA) were obtained prior to dosing and every 6 h thereafter. Isotopic enrichment curves of DSPC-PA from sequential TA and urine deuterium enrichments were analyzed by Gas Chromatography-Isotope Ratio-Mass Spectrometry (GC-IRMS) and normalized for Vienna Standard Mean Ocean Water. Enrichment data were used to measure DSPC-PA fractional synthesis rate (FSR) from the linear portion of the DSPC-PA enrichment rise over time, relative to plateau enrichment of urine deuterium. Secretion time (ST) was defined as the time lag between the start of the study and the appearance of DSPC-PA deuterium enrichment in TA. Data were given as mean \ub1 SD. All study infants reached deuterium-steady state in urine. DSPC-PA FSR was 6.5 \ub1 2.8%/day (range 2.6-10.2). FSR for infants who did not receive exogenous surfactant was 5.7 \ub1 3.5%/day (range 2.6-9.9%/day) and 7.3 \ub1 2.1%/day (range 5.1-10.2%/day) in the preterms, whereas DSPC-PA ST was 10 \ub1 10 h and 31 \ub1 10 h respectively. Surfactant DSPC-PA synthesis can be measured in humans by the incorporation of deuterium from body water. This study is a simpler and less invasive method compared to previously published methods on surfactant kinetics by means of stable isotope

Simultaneous quantitative determination of NG,NG-dimethyl-l-arginine or asymmetric dimethylarginine and related pathway's metabolites in biological fluids by ultrahigh-performance liquid chromatography/electrospray ionization-tandem mass spectrometry

BACKGROUND: Asymmetric dimethylarginine (ADMA), an endogenous nitric oxide (NO) formation inhibitor, has emerged as a promising biomarker of NO-associated endothelial dysfunction in cardiovascular diseases as well in chronic renal failure. The interest in potentially fundamental role of this metabolite, in basic and clinical research, led to the development of numerous analytical methods for the quantitative determination of ADMA and dimethylarginines in biological systems, notably plasma, serum and urine. OBJECTIVES: The aim of this work was to present a simple, fast and accurate UPLC-tandem-MS-based method for the simultaneous determination and quantification of arginine, ADMA, SDMA, NMMA, homo-arginine and citrulline. This method is designed for high sample throughput of only 10 \u3bcL of human plasma, serum or urine. METHODS: The analysis time is reduced to 1.9 min by an ultrahigh-performance liquid chromatography run coupled with electrospray ionization (ESI) in the positive mode tandem mass spectrometry detection. RESULTS: The method was validated in plasma, serum and urine. Correlation coefficients (r(2)) of the calibration curves in all matrices considered ranged from 0.9810 to 0.9993. Inter- and intra-assay precision, accuracy, recovery and carry-over were evaluated for validation. The LOD was 0.01 \u3bcM for all compounds in water, plasma and serum and 0.1 \u3bcM in urine. The LOQ was 0.05 \u3bcM for ADMA, SDMA, NMMA and H-Arg and 0.5 \u3bcM for Arg and Cit in water, plasma and serum; while in urine was 0.1 \u3bcM for ADMA, SDMA, NMMA and H-Arg and 0.5 \u3bcM for Arg and Cit. The precision was ranged from 1% to 15% expressed as CV% and the accuracy (bias %) was <\ub17% for all added concentrations with the exception of NMMA (-10%). ADMA mean plasma levels, measured in healthy adults and newborns, were in accord with literature data published: (M\ub1SD) 0.56\ub10.10 \u3bcM and 0.84\ub10.21 \u3bcM, respectively, showing that ADMA levels in plasma decreased with age. In serum we have similar data (0.54\ub10.18 \u3bcM and 1.14\ub10.36 \u3bcM), while in neonatal urine ADMA was 11.98\ub17.13 \u3bcmol mmol(-1) creatinine. CONCLUSIONS: Data from calibration curves and method validation reveal that the method is accurate and precise. The fast run time, the feasibility of high sample throughput and the small amount of sample required make this method very suitable for routine analysis in the clinical setting

Grape Seeds Proanthocyanidins: Advanced Technological Preparation and Analytical Characterization

A “green” solvent-free industrial process (patent pending) is here described for a grape seed extract (GSE) preparation (Ecovitis™) obtained from selected seeds of Veneto region wineries, in the northeast of Italy, by water and selective tangential flow filtration at different porosity. Since a comprehensive, non-ambiguous characterization of GSE is still a difficult task, we resorted to using an integrated combination of gel permeation chromatography (GPC) and electrospray ionization high resolution mass spectrometry (ESI-HRMS). By calibration of retention time and spectroscopic quantification of catechin as chromophore, we succeeded in quantifying GPC polymers up to traces at n = 30. The MS analysis carried out by the ESI-HRMS method by direct-infusion allows the detection of more than 70 species, at different polymerization and galloylation, up to n = 13. This sensitivity took advantage of the nanoscale shotgun approach, although paying the limit of missed separation of stereoisomers. GPC and MS approaches were remarkably well cross-validated by overlapping results. This simple integrated analytical approach has been used for quality control of the production of Ecovitis™. The emerging feature of Ecovitis™ vs. a popular benchmark in the market, produced by a different technology, is the much lower content of species at low n and the corresponding increase of species at high n

Online trapping and enrichment ultra performance liquid chromatography-tandem mass spectrometry method for sensitive measurement of "arginine-asymmetric dimethylarginine cycle" biomarkers in human exhaled breath condensate.

BACKGROUND: Exhaled breath condensate (EBC) is a biofluid collected non invasively that, enabling the measurement of several biomarkers, has proven useful in the study of airway inflammatory diseases, including asthma, COPD and cystic fibrosis. To the best of our knowledge, there is no previous report of any analytical method to detect ADMA in EBC. OBJECTIVES: Aim of this work was to develop an online sample trapping and enrichment system, coupled with an UPLC-MS/MS method, for simultaneous quantification of seven metabolites related to "Arginine-ADMA cycle", using the isotopic dilution. METHODS: Butylated EBC samples were trapped in an online cartridge, washed before and after each injection with cleanup solution to remove matrix components and switched inline into the high pressure analytical column. Multiple reaction monitoring in positive mode was used for analyte quantification by tandem mass spectrometry. RESULTS: Validation studies were performed in EBC to examine accuracy, precision and robustness of the method. For each compound, the calibration curves showed a coefficient of correlation (r(2)) greater than 0.992. Accuracy (%Bias) was <3% except for NMMA and H-Arg (<20%), intra- and inter-assay precision (expressed as CV%) were within \ub120% and recovery ranged from 97.1 to 102.8% for all analytes. Inter-day variability analysis on 20 EBC of adult subjects did not demonstrate any significant variation of quantitative data for each metabolite. ADMA and SDMA mean concentrations (\u3bcmolL(-1)), measured in EBC samples of asthmatic adolescents are significantly increased (p<0.0001) than in normal controls (0.0040\ub10.0021 vs. 0.0012\ub10.0005 and 0.0020\ub10.0015 vs. 0.0002\ub10.0001, respectively), as well the ADMA/Tyr (0.34\ub10.09 vs. 0.12\ub10.02, p<0.0001) and the SDMA/Tyr ratio (0.10\ub10.04 vs. 0.015\ub10.004, p<0.0001). CONCLUSIONS: The proposed method features simple specimen preparation, maintenance of an excellent peak shape of all metabolites and reduced matrix effects as well mass spectrometer noise. Moreover, the possibility to perform different cycles of enrichment, using large injection volumes, compensated for the low concentration of analytes contained in EBC, leading to a good analytical sensitivity. Preliminary data obtained from asthmatic and healthy adolescents, demonstrated that the analytical method applied to EBC seems suitable not only for research purposes, but also for clinical routinely analysis