Branched-Chain Amino Acids Can Predict Mortality in ICU Sepsis Patients

Abstract

Sepsis biomarkers and potential therapeutic targets are urgently needed. With proton nuclear magnetic resonance (¹H NMR) spectroscopy, several metabolites can be assessed simultaneously. Fifty-three adult medical ICU sepsis patients and 25 ICU controls without sepsis were prospectively enrolled. ¹H NMR differences between groups and associations with 28-day and ICU mortality were investigated. In multivariate metabolomic analyses, we found separate clustering of ICU controls and sepsis patients, as well as septic shock survivors and non-survivors. Lipoproteins were significantly different between sepsis and control patients. Levels of the branched-chain amino acids (BCAA) valine (median 43.3 [29.0-53.7] vs. 64.3 [47.7-72.3] normalized signal intensity units; p = 0.005), leucine (57.0 [38.4-71.0] vs. 73.0 [54.3-86.3]; p = 0.034) and isoleucine (15.2 [10.9-21.6] vs. 17.9 [16.1-24.4]; p = 0.048) were lower in patients with septic shock compared to those without. Similarly, BCAA were lower in ICU non-survivors compared to survivors, and BCAA were good discriminators for ICU and 28-day mortality. In uni- and multivariable logistic regression analyses, higher BCAA levels were associated with decreased ICU- and 28-day mortality. In conclusion, metabolomics using ¹H NMR spectroscopy showed encouraging potential for personalized medicine in sepsis. BCAA was significantly lower in sepsis non-survivors and may be used as early biomarkers for outcome prediction.

Keywords: ICU; NMR spectroscopy; branched-chain amino acids; lipoproteins; metabolomics; sepsis.

Figure 1

Targeted metabolomic assessment of lipoproteins. (A) Multivariate data analyses of lipoprotein parameters with principal component analyses (PCA) and orthogonal partial least squares discriminant analysis (O-PLS-DA) for the differentiation between ICU controls (black) and sepsis patients (red). PCA: principal component (PC) 1 of 76.8% and PC2 of 11.0%. O-PLS-DA: correlation coefficient R²Y = 0.405 and cross-validation score Q² of 0.292 (p < 0.01). (B) Differences in lipoproteins between sepsis and control patients on a logarithmic scale (volcano plot). The vertical dashed lines mark the border to two-fold changes. The horizontal dashed line marks the level of significance at 0.05. (C) Boxplots of the most significant and most changed lipoproteins of sepsis (red color) and control (black color) patients. Abbreviations: HDFC = high density free cholesterol; H1FC = HDL-1 free cholesterol; VLDL = very-low-density lipoprotein; VLPN = VLDL particle number, V2FC = VLDL-2 free cholesterol; V3FC = VLDL-3 free cholesterol; V4FC = VLDL-4 free cholesterol; V4TG = VLDL-4 triglycerides; ABA1 = apolipoprotein B100 to apolipoprotein A-I ratio.

Figure 2

Untargeted metabolomic assessment of metabolites in sepsis. (A) Principal component analysis (PCA; left panel) and partial least squares discriminant analysis (PLS-DA; right panel) for the differentiation between the four groups of survivors and non-survivors, each for the shock and no-shock group. PCA: principal component (PC) 1 of 55% and PC2 of 14.3%. PLS-DA: Component 1 of 50.4%, Component 2 of 14.6%. (B) Pair-wise orthogonal partial least squares discriminant analysis (O-PLS-DA) showing acceptable clustering for survivors and non-survivor in the shock group (middle-left panel; Q² = 0.346, p < 0.01), as well as survivors of the no-shock and shock group (middle-right panel; Q² = 0.438, p < 0.01). No significant clustering was found for survivors and non-survivor in the no-shock group (left panel; p = 0.93) and for non-survivors of the no-shock and shock group (right panel; p = 0.62). (C) Differences in metabolites of all sepsis patients between survivors and non-survivors (volcano plot). The horizontal dashed line marks the level of significance at 0.05. The boxplots represent the most pronounced and most significant different metabolites. (D) Differences in metabolites of septic shock patients between survivors and non-survivors (volcano plot). The horizontal dashed line marks the level of significance at 0.05. The boxplots represent the most pronounced and most significant different metabolites. (E) Comparison between no-shock and septic shock patients. Boxplots of the most pronounced and most significant different metabolites. Abbreviations: BCAA = branched-chain amino acids (consisting of valine, leucine, and isoleucine). a.u. = arbitrary unit. * = unassigned metabolites.

Figure 3

Kaplan–Meier estimates for valine (A), leucine (B), and isoleucine (C). The Q1, i.e., the 25th percentile, corresponds to a cutoff value for valine, leucine, and isoleucine of 33.3, 44.1, and 13.7 NSI units, respectively. The 28-day Kaplan–Meier survival estimates for patients below or above the respective cutoff were 14% vs. 66% for valine, 21% vs. 64% for leucine, and 29% vs. 61% for isoleucine. Abbreviations: Q1 = 25th percentile of the respective branched-chain amino acid, ICU = intensive care unit.

Figure 4

Area under the receiver operating characteristics (AUROC) of branched-chain amino acids for ICU-mortality. (A): AUROC for valine (blue line, 0.75 [0.62–0.89]), leucine (red line, 0.73 (0.59–0.88)) and isoleucine (green line, 0.71 (0.57–0.85)). (B): AUROC for the sequential organ failure assessment (SOFA) score (blue line, 0.78 (0.65–0.91)) and the SOFA-score to branched-chain amino acid ratio (red line, 0.85 (0.73–0.96)). The orange line is the diagonal reference line (50% chance).

Figure 4

Area under the receiver operating characteristics (AUROC) of branched-chain amino acids for ICU-mortality. (A): AUROC for valine (blue line, 0.75 [0.62–0.89]), leucine (red line, 0.73 (0.59–0.88)) and isoleucine (green line, 0.71 (0.57–0.85)). (B): AUROC for the sequential organ failure assessment (SOFA) score (blue line, 0.78 (0.65–0.91)) and the SOFA-score to branched-chain amino acid ratio (red line, 0.85 (0.73–0.96)). The orange line is the diagonal reference line (50% chance).

Figure 5

Longitudinal data of branched-chain amino acid levels. Mean levels of branched-chain amino acids (top panel: valine; middle panel: leucine; bottom panel: isoleucine) over time in sepsis survivors (black line) and sepsis non-survivors (red line) from day 0 to day 3 and 7 after admission. Survivors compared to non-survivors had significantly higher levels of branched-chain amino acids on day 0. Note that in the non-survivor cohort, lower branched-chain amino acids were associated with decreased time until death.