3.3 | Pathway enrichment analysis for variable metabolites in DCDA-C and DCDA-D twins.
The majority of metabolic pathways in amino acid, translation, and cofactor/vitamin metabolism were downregulated in both comparison 1 (DCDA-D-L/DCDA-C) and comparison 2 (DCDA-D-S/DCDA-C) (Figure 3A ). This included cysteine and methionine metabolism, glutathione metabolism, glycine, serine, and threonine metabolism, taurine and hypotaurine metabolism; one translation pathway was aminoacyl-tRNA biosynthesis; and three cofactor/vitamin metabolism included nicotinate and nicotinamide metabolism, pantothenate and CoA biosynthesis and thiamine metabolism. Intriguingly, two pathways associated with carbohydrate metabolism showed specific downregulation in DCDA-D-S twins compared to the DCDA-C twins (Figure 3A , comparison 2). No significant metabolic pathway change was detected in comparison 3 (DCDA-D-S/DCDA-D-L). The shortlisted pathways were linked to their shared metabolites and reconstructed into an in silico metabolic network (Figure 3B ). Cysteine, leucine, and threonine were directly linked to the aminoacyl-tRNA-related metabolites including cysteinyl-tRNA, leucyl-tRNA, and threonine tRNA respectively. The network showed that cysteine was converted into antioxidant glutathione through gamma-glutamyl-L-cysteine. Nicotinamide could lead to the production of coenzymes such as NADP+ and NAD+. Moreover, the network illustrated that aconitate hydratase catalyzes the isomerization of citrate to isocitrate through cis-aconitate in the TCA cycle (Figure 3B ). Notably, cysteine was the most interconnected metabolite that participated in seven significant pathways of glutathione metabolism, cofactors and vitamin metabolism, and four different amino acid metabolisms (Figure 3C ).