Lipid analysis of mycolic acids from M. tuberculosis.

M. tuberculosis cultures were grown until the OD₆₀₀ was 0.3 to 0.4, and 5-ml cultures were treated with the various drugs. At 1 h postincubation, M. tuberculosis was labeled using 1 μCi/ml of [¹⁴C]acetate (56 mCi/mmol; PerkinElmer, USA) for 4 h. The labeled bacilli were harvested, and the pellets were hydrolyzed by addition of 2 ml tetra-n-butyl ammonium hydroxide (TBAH) and incubated overnight at 100°C. The fatty acids were esterified by addition of 4 ml of dichloromethane, 300 μl of methyl iodide, and 2 ml of water, followed by end-to-end mixing for 1 h at room temperature. The phases were separated by centrifugation, and the lower phase was washed twice with distilled water, dried under a stream of air, resuspended in 3 ml of diethyl ether, and centrifuged. The material insoluble in the solvent was transferred to new glass tubes, dried, and resuspended in 200 μl dichloromethane. Equal volumes were loaded on silica gel 60 F₂₅₄ thin-layer chromatography (TLC) plates and resolved using hexane-ethyl acetate (19:1, vol/vol; two runs). The bands corresponding to FAMEs and MAMEs were visualized by phosphorimaging and quantified using ImageQuant (version 5.2) software (GE Healthcare Life Science, UK).

For analysis of TDM and TMM levels, M. tuberculosis cells were treated and labeled as described above. The labeled bacilli were harvested, resuspended in chloroform-methanol (2:1, vol/vol), and incubated overnight at 55°C. The samples were centrifuged, and the supernatants were dried in new glass tubes and resuspended in 200 μl of chloroform-methanol (2:1, vol/vol). Equal volumes of the various samples were spotted on normal-phase TLC plates and resolved using chloroform-methanol-water (62:24:4, vol/vol/vol), and radioactive spots were visualized by phosphorimaging. The radioactive spots corresponding to TDM and TMM were quantified using ImageQuant (version 5.2) software (GE Healthcare Life Sciences, UK).