Metabolic trajectories for fold-changes of identified biomarkers (lactate, uric acid, fatty acids, glutamine, methionine and citrate) in the time course of the first OLTx1 (failed live donor graft, red circles and red dash lines) and second OLTx2 (functional deceased donor graft, blue triangles and blue solid lines) liver transplantations. Normal levels are referred to 0 (“normal”, black dash lines). Z-test was performed to validate identified markers and find significant fold-changes in their concentrations between patient and healthy controls. Due to a large concentration range of endogenous metabolites in the blood detected by NMR (from 10 μM to 10 mM and above), the absolute concentrations of high-expressed metabolites (lactate, uric acid, fatty acids, and glutamine) were used for the statistical analysis. For low-abounded metabolites (citrate and methionine), normalized concentrations (as a percentage of healthy subject concentrations [change = ([patient − normal]/normal)]) were used.

Principle component analysis (PCA): (A) PCA scores (t_i) on global metabolic pattern to visualize group clustering between the first failed and second successful OLTx compared to the normal blood profile; (B) PCA plots (p_i) on absolute metabolite concentrations and (C) on normalized metabolite concentrations to distinguish single putative biomarkers responsible for cloistering pattern on the loading plot in (A). In the first step, the PCA scores (t_i,) described the variation in the sample direction, i.e. similarity/dissimilarity between samples. This step allowed for pattern recognition (normal/abnormal group assignment in A). Each point (in circles or triangles) represent a metabolic profile from each samples. In the next step, the PCA loading plots (p_i) described the variation in the variable direction, i.e. similarity/dissimilarity between variables. This step explained the patterns in previous scores (t_i) and identified metabolites which were responsible for the group clustering (B, C). The metabolites condensed in the center do not contribute to group clustering in (A). The “outliner” metabolites differ among the group. All mathematical models were built with R package (2.00). First OLTx (graft failure, big red circles) time points: T1-pretransplant; T1-2hr after first transplantation; T1-24hr; and T1-48-hr; and second OLTx (functional graft, blue small circles) time points: T2-pre; T2-2hr after second transplantation; T2-24hr; and T2-48hr. Four blood profiles from healthy volunteers (normal 1, 2, 3, and 4, black triangles) are clustering with the data points from the second transplant. All data are based on PCA of blood metabolic profiles.