Fig. 8. Intermediary metabolites decreased in ethanol treated myotubes and skeletal muscle in mice with ALD reversed by HMB. (A). Pyruvate and TCA cycle intermediates were quantified by mass spectrometry in C2C12 myotubes that were either untreated or treated with 100 mM EtOH with/without 50 µM HMB for 6hours (B) Pyruvate and TCA cycle intermediates were quantified by mass spectrometry in gastrocnemius muscle from PF mice and mALD treated with and without HMB. (C) Concentration of HMB in medium from myotubes treated with/without HMB. (D) Concentration of KIC in medium and myotube lysates treated with and without HMB. (E) Concentration of KIC in gastrocnemius muscle from mALD and PF mice treated with/without HMB. (F) Representative chromatograms and mass spectra of HMB in medium. (G) Representative chromatograms and mass spectra of KIC in gastrocnemius muscle from mALD. All data expressed as mean±SD from at least 6 biological replicates for experiments in myotubes and n=4 for PF and n=6 for mALD in each group. * p<0.05; ** p<0.01, *** p<0.001. EtOH: ethanol; HMB b-hydroxy-b-methyl butyrate; UnT: KIC a-keto isocaproic acid; untreated controls; PF: pair-fed; mALD: mouse model of ALD; TCA:Tricarboxylic acid.