Journal: Proceedings of the National Academy of Sciences of the United States of America
Article Title: Ethionamide activation and sensitivity in multidrug-resistant Mycobacterium tuberculosis
Figure Lengend Snippet: In vivo production of (2-ethyl-pyridin-4-yl)methanol ( 5 ) from ETA by whole cells of MTb. A culture of MTb strain H37Rv at OD 650 of 1.0 was concentrated 10-fold in 7H9 media, and [1- 14 C]ETA at 0.01 μg/ml was added. ( A ) After incubation at 37°C metabolism of [1- 14 C]ETA was visualized by TLC and autoradiography. Lanes a–h correspond to sequential filtered culture samples taken at 0.2, 0.25, 0.75, 1.5, 2.5, 5.0, 8.5, and 25 h, respectively. Lane i represents media autooxidation after 25 h of incubation without bacterial cells. The metabolites observed cochromatographed with commercial and characterized synthetic samples of ETA ( 1 ), ETA S -oxide ( 2 ), ETA nitrile ( 3 ), and ETA amide ( 4 ). ( B ) Mycobacteria from the same sequential culture aliquots (500 μl) were collected by filtration onto 0.22-μm filter disks under vacuum, they were washed twice with PBS (500 μl), and the cell-associated radioactivity was measured. ( C ) The reversed-phase HPLC retention time of the unknown major metabolite ( 5 ) was used to guide cold large-scale ETA feeding experiments where we isolated unlabeled metabolite that gave a mass of 137 (137.9 MH + ). We assigned this as (2-ethyl-pyridin-4-yl)methanol and confirmed the identity of ( 5 ) by cochromatography with a synthetic characterized alcohol standard. The upper HPLC continuous radiodetector spectrum corresponds to A lane i, media control and the lower spectrum; lane d, time point 1.5 h, where the UV 254 trace of (2-ethyl-pyridin-4-yl)methanol is superimposed in gray.
Article Snippet: HPLC separation of the [14 C]ETA metabolite mixture was achieved by using a reverse-phase LUNA column [5 μm, C18(2), 250 × 4.6 mm, Phenomenex, Torrence, CA] with a gradient of (0–5 min) 0% acetonitrile, 100% water; then (5–65 min) to 70% acetonitrile; then (65–80 min) to 100% acetonitrile (all solvents contained 0.1% trifluoroacetic acid).
Techniques: In Vivo, Incubation, Thin Layer Chromatography, Autoradiography, Filtration, Radioactivity, High Performance Liquid Chromatography, Isolation