-
Pharmacological Research Mar 2024Chronic interstitial fibrosis presents a significant challenge to the long-term survival of transplanted kidneys. Our research has shown that reduced expression of...
Chronic interstitial fibrosis presents a significant challenge to the long-term survival of transplanted kidneys. Our research has shown that reduced expression of acyl-coenzyme A oxidase 1 (ACOX1), which is the rate-limiting enzyme in the peroxisomal fatty acid β-oxidation pathway, contributes to the development of fibrosis in renal allografts. ACOX1 deficiency leads to lipid accumulation and excessive oxidation of polyunsaturated fatty acids (PUFAs), which mediate epithelial-mesenchymal transition (EMT) and extracellular matrix (ECM) reorganization respectively, thus causing fibrosis in renal allografts. Furthermore, activation of Toll-like receptor 4 (TLR4)-nuclear factor kappa-B (NF-κB) signaling induced ACOX1 downregulation in a DNA methyltransferase 1 (DNMT1)-dependent manner. Overconsumption of PUFA resulted in endoplasmic reticulum (ER) stress, which played a vital role in facilitating ECM reorganization. Supplementation with PUFAs contributed to delayed fibrosis in a rat model of renal transplantation. The study provides a novel therapeutic approach that can delay chronic interstitial fibrosis in renal allografts by targeting the disorder of lipid metabolism.
Topics: Animals; Rats; Acyl-CoA Oxidase; Allografts; Fibrosis; Kidney; Kidney Transplantation; Lipids; Metabolic Diseases
PubMed: 38367917
DOI: 10.1016/j.phrs.2024.107105 -
Cureus Feb 2022While a large proportion of ST-segment elevation on EKG is related to myocardial ischemia, the differential diagnosis must include pericarditis, channelopathies, and...
While a large proportion of ST-segment elevation on EKG is related to myocardial ischemia, the differential diagnosis must include pericarditis, channelopathies, and various genetic conditions. Identifying and working up such abnormalities present a challenge to primary care providers (PCPs). We present two clinical cases of young male patients with ST-segment elevation in anteroseptal leads suspicious for Brugada syndrome and show how to risk stratify and manage them. Our first case presents a 23-year-old male with no past medical history with acute onset substernal chest pain, shortness of breath, and palpitations. Initial workup revealed negative serial troponins and normal B-type natriuretic peptide (BNP). The EKG revealed ST elevation in lead V2. An evaluation for Brugada syndrome was pursued. Upon completion of a procainamide challenge, it was determined that he did not have Brugada syndrome and was shortly discharged. Our second case presents a 33-year-old male with no pertinent cardiac medical history who presented to an outpatient cardiology clinic after discovering an incidental ST elevation in V2 on EKG. His family history was negative for early atherosclerotic cardiovascular events or sudden cardiac death. The patient's initial workup was negative. Suspicion for Brugada syndrome leads to performing a procainamide challenge, which was significant for ST changes in the anterolateral leads. He was asymptomatic during the challenge and initial presentation, and no further intervention was indicated. He was advised to avoid sodium channel blocking medications and treat any fevers and was sent for genetic testing. These cases illustrate the importance of maintaining an appropriate suspicion for Brugada syndrome in young patients with minimal ischemic risk factors. We discuss a guideline-directed algorithmic workup for PCPs in suspicious individuals. Stratifying patients based on the presence of symptoms, history of tachyarrhythmias, and EKG findings before and after drug challenge allows physicians to guide further management of these patients.
PubMed: 35273866
DOI: 10.7759/cureus.21921 -
Journal of Chromatography. B,... Jan 2023Therapeutic drug monitoring (TDM) of cardiovascular drugs is essential to improve treatment efficacy and minimize toxicity because of the usage of multiple drugs with a...
Development and validation of an analytical method using liquid chromatography-tandem mass spectrometry for the therapeutic drug monitoring of seven cardiovascular drugs in clinical usage.
Therapeutic drug monitoring (TDM) of cardiovascular drugs is essential to improve treatment efficacy and minimize toxicity because of the usage of multiple drugs with a very limited therapeutic range and the high pharmacokinetic variation in patients. We developed and validated a reliable and economical liquid chromatography/tandem mass spectrometry (LC-MS/MS) method for the determination of seven cardiovascular drugs-procainamide, lidocaine, quinidine, deslanoside, digoxin, atorvastatin, and digitoxin-for clinical usage. Serum samples were prepared by simple protein precipitation with an organic solvent consisting of acetonitrile and methanol (2:1 v/v) and analyzed under optimized LC-MS/MS conditions. The chromatographic separations were accomplished within 15 min on a reversed-phase C18 column with a gradient elution of aqueous solvent and acetonitrile while maintaining 0.1 (v/v) % formic acid and 2 mM ammonium formate. The optimized MS/MS conditions in ESI-positive mode offered sufficient sensitivity for the seven cardiovascular drugs (LOQs between 0.5 and 1 ng/mL). This method was fully validated including linearity, selectivity, accuracy, precision, carry-over, and matrix effects. Additionally, stability under several conditions was tested to determine how to handle the standard solutions and serum samples. The seven cardiovascular drugs, simultaneously, were precisely and accurately analyzed in intra- and inter-day assays (RSD < 6 % and recovery between 96.3 and 102.8 %) using only two isotope-labeled internal standards (lidocaine-(diethyl-d10) and digoxin-21, 21, 22-d3). The presented method also showed good accuracy in analyzing the seven drugs in hyperlipidemia, hyperalbuminemia, and hyperglycemia serum, allowing it to be recommended as a common and routine analysis method for cardiovascular drugs in clinical practice.
Topics: Humans; Chromatography, Liquid; Cardiovascular Agents; Tandem Mass Spectrometry; Drug Monitoring; Solvents; Digoxin; Lidocaine; Chromatography, High Pressure Liquid; Reproducibility of Results
PubMed: 36469961
DOI: 10.1016/j.jchromb.2022.123552 -
Methods in Molecular Biology (Clifton,... 2021The use of sequential exoglycosidase digestion of oligosaccharides followed by LC-FLD, LC-MS or CE analysis provides detailed carbohydrate structural information. Highly...
The use of sequential exoglycosidase digestion of oligosaccharides followed by LC-FLD, LC-MS or CE analysis provides detailed carbohydrate structural information. Highly specific exoglycosidases cleave monosaccharides from the nonreducing end of an oligosaccharide and yield information about the linkage, stereochemistry and configuration of the anomeric carbon. Here we use combinations of exoglycosidases to precisely characterize glycans on the Fc domain of therapeutic antibodies and dimeric fusion proteins. The workflow described includes glycan release with Rapid™ PNGase F (NEB #P0710), direct labeling of released glycans with procainamide (PCA) or 2-aminobenzamide (2AB), cleanup of labeled glycans and a 3 h enzymatic digestion with exoglycosidases. This protocol is designed for completion within an 8 h time frame to allow for subsequent LC-FLD, LC-MS, or CE analysis overnight.
Topics: Antibodies, Monoclonal; Carbohydrate Conformation; Chromatography, High Pressure Liquid; Fluorescent Dyes; Fluorometry; Glycoproteins; Glycoside Hydrolases; Glycosylation; Hydrolysis; Mass Spectrometry; Peptide-N4-(N-acetyl-beta-glucosaminyl) Asparagine Amidase; Polysaccharides; Procainamide; Protein Processing, Post-Translational; Recombinant Fusion Proteins; Research Design; Substrate Specificity; Workflow; ortho-Aminobenzoates
PubMed: 33908014
DOI: 10.1007/978-1-0716-1241-5_19 -
Heart Rhythm Feb 2024More than a hundred genetic loci have been associated with atrial fibrillation (AF). But the exact mechanism remains unclear and the treatment needs to be improved.
BACKGROUND
More than a hundred genetic loci have been associated with atrial fibrillation (AF). But the exact mechanism remains unclear and the treatment needs to be improved.
OBJECTIVE
This study aimed to investigate the mechanism and potential treatment of NPPA mutation-associated AF.
METHODS
Nppa knock-in (KI, p.I137T) rats were generated, and cardiac function was evaluated. Blood pressure was recorded using a tail-cuff system. The expression levels were measured using real-time polymerase chain reaction, enzyme-linked immunosorbent assay or Western blot analysis, and RNA-sequence analysis. Programmed electrical stimulation, patch clamp, and multielectrode array were used to record the electrophysical characteristics.
RESULTS
Mutant rats displayed downregulated expression of atrial natriuretic peptide but elevated blood pressure and enlarged left atrial end-diastolic diameter. Further, gene topology analysis suggested that the majority of differently expressed genes in Nppa KI rats were related to inflammation, electrical remodeling, and structural remodeling. The expression levels of C-C chemokine ligand 5 and galectin-3 involved in remodeling were higher, while there were declined levels of Na1.5, Ca1.2, and connexin 40. AF was more easily induced in KI rats. Electrical remodeling included abbreviated action potentials, effective refractory period, increased late sodium current, and reduced calcium current, giving rise to conduction abnormalities. These electrophysiological changes could be reversed by the late sodium current blocker ranolazine and the Na1.8 blocker A-803467.
CONCLUSION
Our findings suggest that structural remodeling related to inflammation and fibrosis and electrical remodeling involved in late sodium current underly the major effects of the Nppa (p.I137T) variant to induce AF, which can be attenuated by the late sodium current blocker and Na1.8 blocker.
Topics: Animals; Rats; Action Potentials; Atrial Fibrillation; Atrial Natriuretic Factor; Atrial Remodeling; Heart Atria; Inflammation; Mutation; Myocytes, Cardiac; Procainamide; Sodium
PubMed: 37924963
DOI: 10.1016/j.hrthm.2023.10.025 -
Pharmaceutics Jul 2021In this study, possible changes in the expression of rat organic cationic transporters (rOCTs) and rat multidrug and toxin extrusion proteins (rMATEs) following...
Effects of 1α,25-Dihydroxyvitamin D on the Pharmacokinetics of Procainamide and Its Metabolite N-Acetylprocainamide, Organic Cation Transporter Substrates, in Rats with PBPK Modeling Approach.
In this study, possible changes in the expression of rat organic cationic transporters (rOCTs) and rat multidrug and toxin extrusion proteins (rMATEs) following treatment with 1α,25-dihydroxyvitamin D (1,25(OH)D) were investigated. Rats received intraperitoneal administrations of 1,25(OH)D for four consecutive days, and the tissues of interest were collected. The mRNA expression of rOCT1 in the kidneys was significantly increased in 1,25(OH)D-treated rats compared with the control rats, while the mRNA expressions of rOCT2 and rMATE1 in the kidneys, rOCT1 and N-acetyltransferase-II (NAT-II) in the liver, and rOCT3 in the heart were significantly decreased. Changes in the protein expression of hepatic rOCT1 and renal rOCT2 and rMATE1 were confirmed by western blot analysis. We further evaluated the pharmacokinetics of procainamide (PA) hydrochloride and its major metabolite N-acetyl procainamide (NAPA) in the presence of 1,25(OH)D. When PA hydrochloride was administered intravenously at a dose 10 mg/kg to 1,25(OH)D-treated rats, a significant decrease in renal and/or non-renal clearance of PA and NAPA was observed. A physiological model for the pharmacokinetics of PA and NAPA in rats was useful for linking changes in the transcriptional and translational expressions of rOCTs and rMATE1 transporters to the altered pharmacokinetics of the drugs.
PubMed: 34452094
DOI: 10.3390/pharmaceutics13081133 -
Journal of Chromatography. A May 2023Organophosphorus nerve agent (OPNA) adducts to butyrylcholinesterase (BChE) can be applied to confirm exposure in humans. A sensitive method for generic detection of G-...
Generic detection of organophosphorus nerve agent adducts to butyrylcholinesterase in plasma using liquid chromatography-tandem mass spectrometry combined with an improved procainamide-gel separation and pepsin digestion method.
Organophosphorus nerve agent (OPNA) adducts to butyrylcholinesterase (BChE) can be applied to confirm exposure in humans. A sensitive method for generic detection of G- and V-series OPNA adducts to BChE in plasma was developed by combining an improved procainamide-gel separation (PGS) and pepsin digestion protocol with ultra-high-pressure liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS). Residual matrix interferences from prior PGS purification of OPNA-BChE adducts from plasma were found to be a critical cause of significantly reduced UHPLC-MS/MS detection sensitivity. In our developed on-column PGS approach, the matrix interference was successfully removed by adding an appropriate concentration of NaCl to the washing buffer, and it could capture ≥92.5% of the BChE in plasma. The lower pH value and the longer digestion time in all previous pepsin digestion methods were found to be a key accelerated aging factor of several adducts such as tabun (GA)-, cyclohexylsarin (GF)-, and soman (GD)-BChE nonapeptide adducts, making them difficult to detect. The aging event of several OPNA-BChE nonapeptide adducts was so successfully addressed that the formic acid level in enzymatic buffer and digestion time were lowered to 0.05% (pH 2.67) and 0.5 h, respectively, and the post-digestion reaction was immediately terminated. The improved condition parameters were optimal for pepsin digestion of all types of OPNA-BChE adducts into their individual unaged nonapeptide adducts with the highest yields, expanding the applicability of the method. The method had a nearly one-fold decrease in sample preparation time through the reduction of digestion time and removal of ultrafiltration procedure after digestion. The limit of identification (LOI) were determined respectively as 0.13 ng mL, 0.28 ng mL, 0.50 ng mL, 0.41 ng mL and 0.91 ng mL for VX-, sarin (GB)-, GA-, GF-, and GD-exposed human plasma, being low exposure value compared to previously documented approaches. The approach was utilized to fully characterize the adducted (aged and unaged) BChE levels of five OPNAs in a series of their individual exposed concentration (1.00-400 nM) of plasma sample, and successfully detect OPNA exposure from all unknown plasma samples from OPCW's second and third biomedical proficiency tests. The OPNA-BChE adducts, their aged adducts, and unadducted BChE from OPNA-exposed plasma can simultaneously be measured using the method. The study provides a recommended diagnostic tool for generic verification of any OPNA exposure with high confidence by detecting its corresponding BChE adduct.
Topics: Humans; Aged; Nerve Agents; Butyrylcholinesterase; Tandem Mass Spectrometry; Procainamide; Pepsin A; Organophosphorus Compounds; Chromatography, Liquid; Digestion
PubMed: 37075496
DOI: 10.1016/j.chroma.2023.463990 -
Journal of Pharmaceutical Analysis Mar 2023Sialylated -glycan isomers with α2-3 or α2-6 linkage(s) have distinctive roles in glycoproteins, but are difficult to distinguish. Wild-type (WT) and glycoengineered...
Sialylated -glycan isomers with α2-3 or α2-6 linkage(s) have distinctive roles in glycoproteins, but are difficult to distinguish. Wild-type (WT) and glycoengineered (mutant) therapeutic glycoproteins, cytotoxic T lymphocyte-associated antigen-4-immunoglobulin (CTLA4-Ig), were produced in Chinese hamster ovary cell lines; however, their linkage isomers have not been reported. In this study, -glycans of CTLA4-Igs were released, labeled with procainamide, and analyzed by liquid chromatography-tandem mass spectrometry (MS/MS) to identify and quantify sialylated -glycan linkage isomers. The linkage isomers were distinguished by comparison of 1) intensity of the -acetylglucosamine ion to the sialic acid ion (Ln/Nn) using different fragmentation stability in MS/MS spectra and 2) retention time-shift for a selective value in the extracted ion chromatogram. Each isomer was distinctively identified, and each quantity (>0.1%) was obtained relative to the total -glycans (100%) for all observed ionization states. Twenty sialylated -glycan isomers with only α2-3 linkage(s) in WT were identified, and each isomer's sum of quantities was 50.4%. Furthermore, 39 sialylated -glycan isomers (58.8%) in mono- (3 -glycans; 0.9%), bi- (18; 48.3%), tri- (14; 8.9%), and tetra- (4; 0.7%) antennary structures of mutant were obtained, which comprised mono- (15 -glycans; 25.4%), di- (15; 28.4%), tri- (8; 4.8%), and tetra- (1; 0.2%) sialylation, respectively, with only α2-3 (10 -glycans; 4.8%), both α2-3 and α2-6 (14; 18.4%), and only α2-6 (15; 35.6%) linkage(s). These results are consistent with those for α2-3 neuraminidase-treated -glycans. This study generated a novel plot of Ln/Nn versus retention time to distinguish sialylated -glycan linkage isomers in glycoprotein.
PubMed: 37102108
DOI: 10.1016/j.jpha.2023.01.001 -
Scientific Reports May 2020Two unique housefly strains, PSS and N-PRS (near-isogenic line with the PSS), were used to clarify the mechanisms associated with propoxur resistance in the housefly,...
Two unique housefly strains, PSS and N-PRS (near-isogenic line with the PSS), were used to clarify the mechanisms associated with propoxur resistance in the housefly, Musca domestica. The propoxur-selected resistant (N-PRS) strain exhibited >1035-fold resistance to propoxur and 1.70-, 12.06-, 4.28-, 57.76-, and 57.54-fold cross-resistance to beta-cypermethrin, deltamethrin, bifenthrin, phoxim, and azamethiphos, respectively, compared to the susceptible (PSS) strain. We purified acetylcholinesterase (AChE) from the N-PRS and PSS strains using a procainamide affinity column and characterized the AChE. The sensitivity of AChE to propoxur based on the bimolecular rate constant (K) was approximately 100-fold higher in the PSS strain compared to the N-PRS strain. The cDNA encoding Mdace from both the N-PRS strain and the PSS strain were cloned and sequenced using RT-PCR. The cDNA was 2073 nucleotides long and encoded a protein of 691 amino acids. A total of four single nucleotide polymorphisms (SNPs), I162M, V260L, G342A, and F407Y, were present in the region of the active site of AChE from the N-PRS strain. The transcription level and DNA copy number of Mdace were significantly higher in the resistant strain than in the susceptible strain. These results indicated that mutations combined with the up-regulation of Mdace might be essential in the housefly resistance to propoxur.
Topics: Acetylcholinesterase; Animals; Cholinesterase Inhibitors; Gene Expression Regulation, Enzymologic; Houseflies; Insect Proteins; Insecticide Resistance; Insecticides; Mutation; Polymorphism, Single Nucleotide; Propoxur
PubMed: 32439946
DOI: 10.1038/s41598-020-65242-3 -
Analytical Chemistry May 2022Sialic acids have diverse biological roles, ranging from promoting up to preventing protein and cellular recognition in health and disease. The various functions of...
Sialic Acid Derivatization of Fluorescently Labeled -Glycans Allows Linkage Differentiation by Reversed-Phase Liquid Chromatography-Fluorescence Detection-Mass Spectrometry.
Sialic acids have diverse biological roles, ranging from promoting up to preventing protein and cellular recognition in health and disease. The various functions of these monosaccharides are owed, in part, to linkage variants, and as a result, linkage-specific analysis of sialic acids is an important aspect of glycomic studies. This has been addressed by derivatization strategies using matrix-assisted laser desorption/ionization mass spectrometry (MS) or sialidase digestion arrays followed by liquid chromatography (LC)-MS. Despite this, these approaches are unable to simultaneously provide unambiguous assignment of sialic acid linkages and assess further isomeric glycan features within a single measurement. Thus, for the first time, we present the combination of procainamide fluorescent labeling with sialic acid linkage-specific derivatization via ethyl esterification and amidation for the analysis of released plasma -glycans using reversed-phase (RP)LC-fluorescence detection (FD)-MS. As a result, α2,3- and α2,6-sialylated -glycans, with the same mass prior to derivatization, are differentiated based on retention time, precursor mass, and fragmentation spectra, and additional sialylated isomers were also separated. Furthermore, improved glycan coverage and protocol precision were found via the novel application using a combined FD-MS quantification approach. Overall, this platform achieved unambiguous assignment of -glycan sialic acid linkages within a single RPLC-FD-MS measurement, and by improving their retention on RPLC, this technique can be used for future investigations of released -glycans as an additional or orthogonal method to current analytical approaches.
Topics: Chromatography, Reverse-Phase; N-Acetylneuraminic Acid; Polysaccharides; Sialic Acids; Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
PubMed: 35482581
DOI: 10.1021/acs.analchem.1c02610