Standard-Free Bioanalytical Approach for Absolute Quantitation of Drug Metabolites Utilizing Biosynthesis of Reciprocal Radio and Stable Isotopologues and Its Application

The following work describes a combined enzymatic and bioanalytical method that permits absolute quantitation of metabolites in biological samples without the requirement for reference metabolite standards. This technique was exemplified using a radio (¹⁴C) isotopologue and a stable (¹³C₆) isotopologue of acetaminophen as substrates for <i>in vitro</i> biosynthesis of the corresponding radio and stable isotope labeled metabolites, namely, ¹⁴C- and ¹³C₆-glucuronides and sulfates. By supplanting the use of authentic metabolite standards, traditionally used to calibrate ¹³C₆-metabolites via liquid chromatography-tandem mass spectrometry (LC-MS/MS), ¹³C₆-metabolites were radiocalibrated by their ¹⁴C-isotopologues via liquid chromatography coupled with radioactivity detection and mass spectrometry (LC-RAD/MS). The radiocalibrated ¹³C₆-isotopologues were in turn used to quantitate acetaminophen and its corresponding metabolites in rat plasma samples by LC-MS/MS. Variation between this and a conventional LC-MS/MS method using authentic standards for calibration was within ±17%, permitting its use in preclinical and clinical applications. Since authentic metabolite standards are not required under the concept of radio and stable isotopologues using adapted LC-RAD/MS protocols, significantly fewer resources are required to support accurate metabolite quantitation which in turn enables efficient analysis of simple and complex metabolite profiles

Data_Sheet_1_Immune cell early activation, apoptotic kinetic, and T-cell functional impairment in domestic pigs after ASFV CADC_HN09 strain infection.docx

African swine fever (ASF) caused by the African swine fever virus (ASFV) is a fatal and highly contagious disease of domestic pigs characterized by rapid disease progression and death within 2 weeks. How the immune cells respond to acute ASFV infection and contribute to the immunopathogenesis of ASFV has not been completely understood. In this study, we examined the activation, apoptosis, and functional changes of distinct immune cells in domestic pigs following acute infection with the ASFV CADC_HN09 strain using multicolor flow cytometry. We found that ASFV infection induced broad apoptosis of DCs, monocytes, neutrophils, and lymphocytes in the peripheral blood of pigs over time. The expression of MHC class II molecule (SLA-DR/DQ) on monocytes and conventional DCs as well as CD21 expression on B cells were downregulated after ASFV infection, implying a potential impairment of antigen presentation and humoral response. Further examination of CD69 and ex vivo expression of IFN-γ on immune cells showed that T cells were transiently activated and expressed IFN-γ as early as 5 days post-infection. However, the capability of T cells to produce cytokines was significantly impaired in the infected pigs when stimulated with mitogen. These results suggest that the adaptive cellular immunity to ASFV might be initiated but later overridden by ASFV-induced immunosuppression. Our study clarified the cell types that were affected by ASFV infection and contributed to lymphopenia, improving our understanding of the immunopathogenesis of ASFV.</p