14 research outputs found
Bioanalysis of Pteroyl Derivatives in Various Aspects of Human Health
This dissertation describes the development of bioanalytical strategies for a group of experimental and known therapeutic agents and chemically related essential substances. The array of analytes include the antifolate methotrexate, its various (in)active metabolites and related bio-conjugates, plus several forms of folic acid family, a group of essential substances not synthesized by mammalian species, but endogenously required in numerous biochemical processes. These analytes all share a common structural feature, the heterocyclic pteridine ring system. Analyte identity results from variations of the ptereridine-ring system redox state, a result of minor structural variations of the heterocyclic ring and changes in conjugation status (i.e. polyglutamated or nanoparticle conjugated). Specific and sensitive detection of the individual species was of high interest, as the various chemical forms of these substances may constitute biomarkers for individualizing low dose MTX therapy in autoimmune diseases such as JIA, but is also required for the (pre)clinical studies of novel nano-device MTX drug delivery systems. The dissertation presents state-of-the-art bioanalytical methodology suitable for the specific determination of any specific analyte in the presence of all other analytes. The strategy for each particular analyte possesses its own unique strength and weakness, with the final strategy collectively being based on the specific structure and associated physical-chemical property of a given analyte, the biological environment, and the clinical question to be answered
LC-MS/MS Method for the determination of carbamathione in human plasma
Liquid chromatography-tandem mass spectrometry methodology is described for the determination of S-(N,N-diethylcarbamoyl)glutathione (carbamathione) in human plasma samples. Sample preparation consisted of a straightforward perchloric acid medicated protein precipitation, with the resulting supernatant containing the carbamathione (recovery ∼98%). For optimized chromatography/mass spec detection a carbamathione analog, S-(N,N-di-i-propylcarbamoyl)glutathione, was synthesized and used as the internal standard. Carbamathione was found to be stable over the pH 1-8 region over the timeframe necessary for the various operations of the analytical method. Separation was accomplished via reversed-phase gradient elution chromatography with analyte elution and re-equilibration accomplished within 8 minutes. Calibration was established and validated over the concentration range of 0.5-50 nM, which is adequate to support clinical investigations. Intra- and inter-day accuracy and precision determined and found to be < 4% and < 10%, respectively. The methodology was utilized to demonstrate the carbamathione plasma-time profile of a human volunteer dosed with disulfiram (250 mg/d). Interestingly, an unknown but apparently related metabolite was observed with each human plasma sample analyzed
Folate determination in human health: UPLC–MS/MS is the emerging methodology of choice
Urinary Biomarkers of Trimethoprim Bioactivation in Vivo Following Therapeutic Dosing in Children
The
antimicrobial trimethoprim-sulfamethoxazole (TMP-SMX) is widely
used for the treatment of skin and soft-tissue infections in the outpatient
setting. Despite its therapeutic benefits, TMP-SMX has been associated
with a number of adverse drug reactions, which have been primarily
attributed to the formation of reactive metabolites from SMX. Recently,
in vitro experiments have demonstrated that TMP may form reactive
intermediates as well. However, evidence of TMP bioactivation in patients
has not yet been demonstrated. In this study, we performed in vitro
trapping experiments with <i>N</i>-acetyl-l-cysteine
(NAC) to determine stable markers of reactive TMP intermediates, focusing
on eight potential markers (NAC-TMP adducts), some of which were previously
identified in vitro. We developed a specific and sensitive assay involving
liquid chromatography followed by tandem mass spectrometry for measurement
of these adducts in human liver microsomal samples and expanded the
methodology toward the detection of these analytes in human urine.
Urine samples from four patients receiving TMP-SMX treatment were
analyzed, and all samples demonstrated the presence of six NAC-TMP
adducts, which were also detected in vitro. These adducts are consistent
with the formation of imino-quinone-methide and para-quinone-methide
reactive intermediates in vivo. As a result, the TMP component of
TMP-SMX should be considered as well when evaluating adverse drug
reactions to TMP-SMX
Measurement of methotrexate polyglutamates in human erythrocytes by ion-pair UPLC–MS/MS
Metabolic and Molecular Insights into an Essential Role of Nicotinamide Phosphoribosyltransferase
Nicotinamide phosphoribosyltransferase (NAMPT) is a pleiotropic protein implicated in the pathogenesis of acute respiratory distress syndrome, aging, cancer, coronary heart diseases, diabetes, nonalcoholic fatty liver disease, obesity, rheumatoid arthritis, and sepsis. However, the underlying molecular mechanisms of NAMPT in these physiological and pathological processes are not fully understood. Here, we provide experimental evidence that a Nampt gene homozygous knockout (Nampt−/−) resulted in lethality at an early stage of mouse embryonic development and death within 5–10 days in adult mice accompanied by a 25.24 ± 2.22% body weight loss, after the tamoxifen induction of NamptF/F × Cre mice. These results substantiate that Nampt is an essential gene for life. In Nampt−/− mice versus Nampt+/+ mice, biochemical assays indicated that liver and intestinal tissue NAD levels were decreased significantly; histological examination showed that mouse intestinal villi were atrophic and disrupted, and visceral fat was depleted; mass spectrometry detected unusual higher serum polyunsaturated fatty acid containing triglycerides. RNA-seq analyses of both mouse and human pediatric liver transcriptomes have convergently revealed that NAMPT is involved in key basic cellular functions such as transcription, translation, cell signaling, and fundamental metabolism. Notably, the expression of all eight enzymes in the tricarboxylic acid cycle were decreased significantly in the Nampt−/− mice. These findings prompt us to posit that adult Nampt−/− mouse lethality is a result of a short supply of ATP from compromised intestinal absorption of nutrients from digested food, which leads to the exhaustion of body fat stores