Fig. 5

Combinatorial Glu + NA + NAC therapy partially restored altered intermediary metabolism of gas-1(fc21) mutant C. elegans. Whole worm concentrations and stable isotopic enrichment in free amino and organic acids of gas-1(fc21) worms were evaluated in synchronous young adult populations treated for 24 h with Glu + NA + NAC (glucose, nicotinic acid, and n-acetylcysteine) triple combination therapy or its single components and their pairwise combinations. (A) Whole worm free amino acid concentrations were measured by HPLC and normalized to overall protein concentration (nmol/mL per mg protein) for glutamic acid (GLU), glutamine (GLN) and alanine (ALA). Symbol color denotes experimental condition as defined in key, with separate symbols shown to indicate results of three independent replicate experiments performed. Random effects ANOVA statistical analysis was used to account for experimental batch effects. * P < 0.05 when compared to buffer (water) only exposed gas-1(fc21) worms. Results for additional HPLC amino acid levels are shown in Supplementary Material, Fig. S6. (B) Absolute isotopic enrichment of each amino acid metabolite in all molecular species for glutamate (GLU +2, +3, +4 and + 5), glutamine (GLN +2, +3, +4 and + 5), and alanine (ALA+2 and + 3) (indicating the number of carbon atoms on which ¹³C enrichment was present from U-¹³C6-glucose fed to the worms) was quantified by GC/MS in synchronous adult worm populations following U-¹³C6-glucose feeding to N2 (WT) or gas-1(fc21) worms for 24 h on the first day of adulthood. gas-1(fc21) worms were maintained in buffer (water)-only conditions or with Glu + NA + NAC as triple, pairwise, or single component therapies for 24 h, and compared to untreated N2 (WT) worms. Three biological replicate experiments were performed * P < 0.05 and ** P < 0.01 when compared to buffer (water) only exposed gas-1(fc21) worms. Results for additional GC/MS analytes are shown in Supplementary Material, Figs. S7–S8.