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1-Phenyl-2-(pyrrolidin-1-yl)octan-1-one (PV9) and 16 metabolites, including diastereomers and conjugates, were identified or tentatively detected in human urine by gas chromatography-mass spectrometry and liquid chromatography-high-resolution tandem mass spectrometry. These urinary metabolites indicated that the metabolic pathways of PV9 include: (1) the reduction of ketone groups to their corresponding alcohols; (2) oxidation of the pyrrolidine ring to the corresponding pyrrolidone; (3) aliphatic oxidation of the terminal carbon atom to the corresponding carboxylate form, possibly through an alcohol intermediate (not detected); and (4) hydroxylation at the penultimate carbon atom to the corresponding alcohols followed by further oxidation to ketones, and combinations of these steps. In addition, results from the quantitative analyses of five phase-I metabolites using newly synthesized authentic standards suggested that the main metabolic pathway includes the aliphatic oxidation of terminal and/or penultimate carbons. Human metabolism of PV9 differed significantly from those of a-pyrrolidinovalerophenone and a-pyrrolidinobutiophenone, suggesting that the main metabolic pathways of a-pyrrolidinophenones significantly change depending on the alkyl chain length of the parent molecule.

Many cellular proteins with reactive thiols form covalent bonds with electrophiles, thereby modifying their structures and activities. Here, we describe the recovery of a glycolytic protein, glyceraldehyde-3-phosphate dehydrogenase (GAPDH), from such an electrophilic attack by 1,2-napthoquinone (1,2-NQ). GAPDH readily formed a covalent bond with 1,2-NQ through Cys152 at a low concentration (0.2 μM) in a cell-free system, but when human epithelial A549 cells were exposed to this quinone at 20 μM, only minimal binding was observed although extensive binding to numerous other cellular proteins occurred. Depletion of cellular glutathione (GSH) with buthionine sulfoximine (BSO) resulted in some covalent modification of cellular GAPDH by 1,2-NQ and a significant reduction of GAPDH activity in the cells. Incubation of native, but not boiled, human GAPDH that had been modified by 1,2-NQ with GSH resulted in a concentration-dependent removal of 1,2-NQ from the GAPDH conjugate, accompanied by partial recovery of lost catalytic activity and formation of a 1,2-NQ-GSH adduct (1,2-NQ-SG). While GAPDH is recognized as a multifunctional protein, our results show that GAPDH also has a unique ability to recover from electrophilic modification by 1,2-NQ through a GSH-dependent S-transarylation reaction.

To obtain fundamental information on the drug incorporation into hair, time-course changes in drug distribution along single-strand hair were observed after a single oral administration of zolpidem (ZP), one of the most frequently used hypnotic agents. Quantitative sectional hair analyses of 1-mm segments were performed for each single-strand hair using a validated LC-MS/MS procedure. ZP was detected in all specimens plucked at 10 and 24 hours after a single dose, and the distribution ranged over the whole hair root (4-5 mm in length). A significantly high concentration of ZP was detected in the hair bulb region, whereas much lower concentrations were widely observed in the upper part of the hair root of those samples; this suggested that the incorporation of ZP occurred in two regions, mainly in the hair bulb and to a lesser extent in the upper dermis zone. The ZP-positive area formed lengths of up to 10-12 mm after a single administration, indicating that its incorporation from the hair bulb would continue for about 2 weeks. Time-course changes in the ZP concentration in the hair root additionally revealed that only a small portion of ZP that initially concentrated in the bulb was successively incorporated into the hair matrix and moved toward the keratinized region as hair grew. These findings should be taken into account upon discussing individual drug-use history based on hair analysis. The matrix-assisted laser desorption/ionization mass spectrometry imaging of ZP in the same kinds of hair specimens was also successfully achieved.