Pharmacometabolomics of l-carnitine treatment response phenotypes in patients with septic shock

Abstract

Rationale: Sepsis therapeutics have a poor history of success in clinical trials, due in part to the heterogeneity of enrolled patients. Pharmacometabolomics could differentiate drug response phenotypes and permit a precision medicine approach to sepsis.

Objectives: To use existing serum samples from the phase 1 clinical trial of l-carnitine treatment for severe sepsis to metabolically phenotype l-carnitine responders and nonresponders.

Methods: Serum samples collected before (T0) and after completion of the infusion (T24, T48) from patients randomized to either l-carnitine (12 g) or placebo for the treatment of vasopressor-dependent septic shock were assayed by untargeted (1)H-nuclear magnetic resonance metabolomics. The normalized, quantified metabolite data sets of l-carnitine- and placebo-treated patients at each time point were compared by analysis of variance with post-hoc testing for multiple comparisons. Pathway analysis was performed to statistically rank metabolic networks.

Measurements and main results: Thirty-eight metabolites were identified in all samples. Concentrations of 3-hydroxybutyrate, acetoacetate, and 3-hydroxyisovalerate were different at T0 and over time in l-carnitine-treated survivors versus nonsurvivors. Pathway analysis of pretreatment metabolites revealed that synthesis and degradation of ketone bodies had the greatest impact in differentiating l-carnitine treatment response. Analysis of all patients based on pretreatment 3-hydroxybutyrate concentration yielded distinct phenotypes. Using the T0 median 3-hydroxybutyrate level (153 μM), patients were categorized as either high or low ketone. l-Carnitine-treated low-ketone patients had greater use of carnitine as evidenced by lower post-treatment l-carnitine levels. The l-carnitine responders also had faster resolution of vasopressor requirement and a trend toward a greater improvement in mortality at 1 year (P = 0.038) compared with patients with higher 3-hydroxybutyrate.

Conclusions: The results of this preliminary study, which were not readily apparent from the parent clinical trial, show a unique metabolite profile of l-carnitine responders and introduce pharmacometabolomics as a viable strategy for informing l-carnitine responsiveness. The approach taken in this study represents a concrete example for the application of precision medicine to sepsis therapeutics that warrants further study.

Keywords: 3-hydroxybutyric acid; individualized medicine; ketone bodies; nuclear magnetic resonance; sepsis.

Figure 1.

Metabolic pathways of carnitine, showing the relationship of several of the differentiating metabolites of l-carnitine treatment response (47). Lysine and methionine are precursors of carnitine, which is required for the transport of long-chain fatty acids (LCFAs) into the mitochondria. Acetylcarnitine is formed by the acetylation of carnitine. Subsequently, it is hydrolyzed by plasma esterases to form carnitine. Ketone bodies can be produced via acetyl-coenzyme A (acetyl-CoA) and β-oxidation of LCFAs in the mitochondria. Phenylalanine is metabolized to tyrosine, which participates in the synthesis of adrenaline. TCA = tricarboxylic acid cycle.

Figure 2.

Differences in aqueous serum metabolite concentrations between l-carnitine- and placebo-treated patients with septic shock. (A) Carnitine (*P < 0.001), (B) acetylcarnitine (*P < 0.001; ⁺P = 0.001), and (C) creatinine (*P = 0.008; ⁺P = 0.050) levels were different at 24 and 48 hours and (D) malonate (*P = 0.002) and (E) methionine (*P = 0.032) levels were different at 24 hours. There were no differences in metabolite concentrations between the two groups at T0 (before treatment). Data represent medians ± the interquartile range of 11–16 l-carnitine- and placebo-treated patients at each time point.

Figure 3.

Metabolite profiles were different in l-carnitine-treated survivors and nonsurvivors. The ketone bodies (A) 3-hydroxybutyrate and (B) acetoacetate were higher at T0 (before treatment) and T24 (24 [±4] h after treatment) in nonsurvivors (*P < 0.001, ⁺P = 0.065, and ^#P = 0.019). The metabolites (C) 3-hydroxyisovalerate (*P < 0.001, ⁺P = 0.005) and (D) creatine (⁺P = 0.0496) were also elevated in nonsurvivors at T24 compared with survivors. (E) Betaine (*P = 0.005) was higher at T24 and (F) valine was higher (*P = 0.031) at T48 (48 [±4] h after treatment) in survivors. Notably, both (G) carnitine (*P = 0.001, ⁺P < 0.001) and (H) acetylcarnitine (*P = 0.004, ⁺P = 0.001) were increased in nonsurvivors, suggesting differences in l-carnitine metabolism between the two groups. (I) The acetylcarnitine–carnitine (AC:C) ratio, an indicator of carnitine homeostasis, was higher at T0 (*P = 0.001) and more variable over time in nonsurvivors compared with survivors, suggesting a greater disturbance in carnitine metabolism. There were no differences in the T0 concentrations of betaine, valine, carnitine, or acetylcarnitine between survivors and nonsurvivors. Data represent medians ± the interquartile range of seven or eight patients per group.

Figure 4.

(A) 3-Hydroxybutyrate (3-OHB) and (B) acetoacetate concentrations and (C) acetylcarnitine–carnitine (AC:C) ratios of placebo- and l-carnitine-treated patients who were categorized as either high or low ketone, based on the 3-OHB concentration at T0 (before treatment) (see text). The higher levels of ketone bodies and AC:C ratios suggest that high-ketone patients had a greater disruption in metabolic homeostasis than did low-ketone patients. This was not differentiated by (D) the T0 total SOFA scores, (E) point-of-care lactate concentrations, or (F) glucose concentrations as measured by nuclear magnetic resonance because they were not different across groups. SOFA = Sequential Organ Failure Assessment.

Figure 5.

Metabolite profiles of patients with septic shock and high or low ketones treated with l-carnitine were different from those of placebo-treated patients. (A) Methionine was increased in low-ketone, l-carnitine-treated patients compared with low-ketone, placebo-treated patients at T24 (24 [±4] h after treatment) (*P = 0.021). There was also a trend toward (B) increased lysine in the low-ketone, l-carnitine-treated patients (*P = 0.078 vs. low-ketone, placebo-treated patients). Methionine and lysine are precursors of endogenous carnitine synthesis. At T24, (C) phenylalanine trended higher (T24) in low-ketone, l-carnitine-treated patients than in high-ketone, l-carnitine-treated patients (*P = 0.051), low-ketone, placebo-treated patients (⁺P = 0.078), and high-ketone, placebo-treated patients (^#P = 0.010). (D) Tyrosine followed a similar trend to phenylalanine in which low-ketone, l-carnitine-treated patients had higher levels at T24 compared with high-ketone, l-carnitine-treated patients (*P = 0.008). Phenylalanine and tyrosine, which can be synthesized from phenylalanine, are proteinogenic amino acids and participate in the synthesis of catecholamines. High-ketone, l-carnitine-treated patients had higher carnitine (E) levels compared with low-ketone, L-carnitine-treated patients (*P = 0.003) and high- and low-ketone, placebo-treated patients (⁺P < 0.001) at T24 and low-ketone L-carnitine, low-ketone placebo, and high-ketone placebo at T48 (*^+#P < 0.0001). A similar pattern was evident in acetylcarnitine (F) concentration in which the T24 and T48 level in high-ketone, L-carnitine-treated patients was higher compared with low-ketone L-carnitine-treated patients (*P = 0.001 at T24; *P < 0.0001 at T48), low-ketone, placebo-treated patients (⁺P = 0.008 at T24; ⁺P < 0.0001 at T48) and high-ketone, placebo-treated patients (^#P < 0.001 at both T24 and T48). At no time were there differences between the low- and high-ketone, placebo-treated groups. There were also no differences in these metabolite levels between groups at T0 (before treatment). Data represent medians ± the interquartile range.

Figure 6.

Low-ketone, l-carnitine-treated patients had a trend toward lower 1-year mortality compared with the other groups of patients (chi square P = 0.038).