The aim of this study was to optimize methods for quantifying 13 uridine 59-diphosphate-glucuronosyltransferase (UGT) isoforms (UGT1A1, 1A3, 1A4, 1A6, 1A7, 1A8, 1A9, 1A10, 2B4, 2B7, 2B10, 2B15, and 2B17) in human liver, intestinal, and kidney microsomes, and in recombinant human UGT-expressing insect cell membranes (rhUGTs) by targeted peptide-based quantification using liquid chromatography-tandem mass spectrometry. Production of targeted peptides was compared by combining three denaturing agents (urea, sodium deoxycholate, and octyl glucoside) and three denaturing temperatures (37°C, 60°C, and 95°C) followed by tryptic digestion for 2-20 hours. Denaturing conditions and digestion times yielding high production efficiency varied markedly among isoforms and specimens, indicating the importance of specific optimization. Each UGT isoform was quantified using the methods found to be optimal.

Thrombin-catalyzed factor VIII activation is an essential positive feedback mechanism regulating intrinsic blood coagulation. A factor VIII human antibody, A-FF, with C2 epitope, exclusively inhibited factor VIII activation and cleavage at Arg 1689 by thrombin. The results suggested that A-FF prevented the interaction of thrombin with factor VIII and that the C2 domain was involved in the interaction with thrombin. We performed direct binding assays using anhydro-thrombin, a catalytically inactive derivative of thrombin in which the active-site serine is converted to dehydroalanine. Blood clotting factor VIII (FVIII) 1 is a crucial glycoprotein that accelerates the intrinsic coagulation cascade by acting as a cofactor of factor IXa in the tenase complex (1), and a deficiency of FVIII results in the common hereditary bleeding disorder, hemophilia A. FVIII circulates in plasma as a noncovalent complex with von Willebrand factor (vWF) that stabilizes the synthesis and cofactor activity of FVIII (2-4). Mature FVIII is synthesized as a single chain polypeptide of approximately 300 kDa consisting of 2,332 amino acid residues with a mosaic domain structure arranged in the order of A1-A2-B-A3-C1-C2 (5-7) and secreted into plasma as variable series of heterodimers consisting of the heavy chain (HCh) and the light chain (LCh). The HCh is composed of the A1, A2, and parts of the B domain and exhibits size heterogeneity (90 -210 kDa) due to proteolysis within the B domain. The 90-kDa HCh reflects the absence of the B domain, whereas the 210-kDa species includes the intact B domain. The LCh (80 kDa) corresponds to the A3, C1, and C2 domains. The two chains are noncovalently linked by metal ions between the A1 and A3 domains (6, 7).FVIII is transformed into an active form by limited proteolysis by two essential serine proteases, thrombin and FXa (8, 9). This procoagulant activity is more pronounced after thrombin activation than after FXa activation (10). Cleavage at Arg 740 removes the size-heterogeneous B domain from the HCh, producing a 90-kDa HCh fragment, and further cleavage of the 90-kDa HCh fragment (A1-A2) at Arg 372 between the A1 and A2 domains produces 54-(A1) and 44-kDa (A2) species. Cleavage of the 80-kDa LCh fragment (A3-C1-C2) at Arg 1689 between the B and A3 domains removes 40 amino-terminal acidic peptides from the A3 domain and produces a 72-kDa fragment. A unique cleavage by FXa at Arg 1721 produces a 67-kDa LCh fragment. The active form of FVIII is a metal-linked heterotrimer consisting of the A1/A2/A3-C1-C2 domains, lacking the middle B domain (6). Cleavage at Arg 740 between the A2 and B domains does not contribute to the generation of the FVIII activity. In contrast, cleavages at Arg 372 and Arg 1689 are essential for optimal FVIII activity (11-13). Activation of human FVIII at its physiological concentration and at pH 7.4 is followed by a reduction in activity (14). This loss of activity is not caused by further proteolysis but by dissociation of the A2 domain from the active form of FVIII heterotrime...

The chemical reaction of a hydrogenated diamond surface with the radical initiator benzoyl peroxide was performed in order to investigate the reactivity of the diamond surface. It was found that the hydrogen atoms on the diamond surface are abstracted by the radical species generated from benzoyl peroxide. The reaction rate was calculated from the experimental data. The hydrogen abstracted diamond surface reacts with radical species generated from the benzoyl peroxide, not with the solvent.

ABSTRACT:The mass balance and metabolite profiles of 2-(2- total of 10 metabolites were found in urine. On the basis of the metabolites found in urine, major primary metabolic reactions of mirabegron were estimated to be amide hydrolysis (M5, M16, and M17), accounting for 48% of the identified metabolites in urine, followed by glucuronidation (M11, M12, M13, and M14) and N-dealkylation or oxidation of the secondary amine (M8, M9, and M15), accounting for 34 and 18% of the identified metabolites, respectively. In feces, the radioactivity was recovered almost entirely as the unchanged form. Eight of the metabolites characterized in urine were also observed in plasma. These findings indicate that mirabegron, administered as a solution, is rapidly absorbed after oral administration, circulates in plasma as the unchanged form and metabolites, and is recovered in urine and feces mainly as the unchanged form.amino