Glucose 6 phosphate dehydrogenase g6pdh

DNJ (purity >98.0%) and SZ–A (lot number: 20130717-2), containing 37.5% of DNJ, 6.4% of FGM, and 4.8% of DAB, was kindly provided by the Department of Pharmaceutics (Institute of Materia Medica, Chinese Academy of Medical Sciences, Beijing, China). Miglitol (internal standard, IS) was purchased from J and K Scientific Ltd., (Beijing, China). FGM (purity >98.0%) was obtained from Medchem Express Co., Ltd., (Princeton, NJ, USA). DAB∙HCl (purity >98.0%), propranolol, atenolol, β-nicotinamide adenine dinucleotide phosphate (β-NADP) hydrate, d-glucose 6-phosphate (G-6-P) disodium salt hydrate, glucose-6-phosphate dehydrogenase (G-6-PDH), and ammonium hydroxide solution were products of Sigma-Aldrich Co., Ltd., (St. Louis, MO, USA). Tris(hydroxymethyl)aminomethane (Tris) was obtained from Sinopharm Chemical Reagent Co., Ltd., (Beijing, China). Anaerobic medium was purchased from Beijing Land Bridge Technology Co., Ltd., (Beijing, China). Acetonitrile and methanol were of HPLC grade (Merck KGaA, Darmstadt, Germany). Ultrapure water was prepared by a Milli-Q Reagent water system (Millipore, Billerica, MA, USA). All other chemicals were of analytical reagent grade and commercially available.

UniPR129 (N-(3α-hydroxy-5β-cholan-24-oyl)-L-β-homotryptophan), UniPR500 (N-(3-hydroxyimino-cholan-24-oyl)-L-β-homotryptophan), UniPR126, and UniPR141, employed as internal standards, were synthesized in our labs as previously described [8 (link),9 (link),11 (link)]. Mouse liver (MLM, from CD1 mice, pooled male) microsomes for phase I metabolism and human (pooled male and female) liver S9 fraction (HLS9) and mouse liver S9 fraction (MLS9 from CD1 mice, pooled male) for phase II metabolism were obtained from Xenotech, LLC (Cambridge, Kansas City, KS, USA). Glucose-6-phosphate (G6P), oxidized nicotinamide-adenine-dinucleotide phosphate (NADP⁺), Uridin-di-phosphoglucuronic acid (UDPGA), 3-phosphoadenosin-5-phosphate (PAPS), magnesium chloride (MgCl₂), dithiothreitol (DTT), and glucose-6-phosphate-dehydrogenase (G6PDH) were supplied by Sigma Aldrich (Milan, Italy). 85% v/v formic acid was provided by ACEF Spa (Piacenza, Italy); HPLC-grade acetonitrile (ACN) and dimethyl sulfoxide (DMSO), >99.9% purity, were supplied by Sigma Aldrich (Milan, Italy) and VWR Chemicals (Radnor, PE, USA), respectively. Ultra-pure Millipore water (Darmstadt, Germany) was employed for HPLC mobile phase and sample preparations.

For metabolization experiments, 1254 aroclor induced S9 rat liver extract was used (Moltox, NC, USA) and cofactors nicotinamide adenine dinucleotide phosphate (NADPH), Glucose-6-Phosphate (G6P) and MgCl₂ were purchased from Carl Roth (Karlsruhe, GER) and Glucose-6-Phosphate-Dehydrogenase (G6P-DH) from Sigma Aldrich (US). Two different S9 protocols were followed, with different S9 composition depending on the incubation time with the S9 mixture. Final concentration of the compounds in the wells were: 5 mM MgCl₂, 3 mM G6P, 0.2 mM NADPH, 0.3 units/mL G6P-DH and 330 µg/mL (3 h protocol) or 10 µg/mL (24 h protocol according to Mollergues et al. (2016) ) of S9 liver extract. Cells were either treated for 3 h with a higher concentrated S9 mix, then washed with Dulbecco’s phosphate buffered saline (DPBS) and further incubated with DMEM containing 5% FBS and 1% DMSO for another 21 h. Alternatively, treatment was done for 24 h with a lower concentrated mix, without a change of medium. Luciferase and resazurin measurements were conducted the same way as without S9 addition. The 1254 aroclor induced S9 rat liver extract was used simultaneously in the same laboratory for the Ames MPF™ assay to prove its functionality.