High-Protein Supplementation and Neuromuscular Electric Stimulation after Aneurysmal Subarachnoid Hemorrhage Increases Systemic Amino Acid and Oxidative Metabolism: A Plasma Metabolomics Approach

Background The INSPIRE randomized clinical trial demonstrated that a high protein diet (HPRO) combined with neuromuscular electrical stimulation (NMES) attenuates muscle atrophy and may improve functional outcomes after aSAH. Using an untargeted metabolomics approach, we sought to identify specific metabolites mediating these effects. Methods Blood samples were collected from subjects on admission prior to randomization to either standard of care (SOC; N=12) or HPRO+NMES (N=12) and at 7 days as part of the INSPIRE protocol. Untargeted metabolomics were performed for each plasma sample. Paired fold changes were calculated for each metabolite among subjects in the HPRO+NMES group at baseline and 7 days after intervention. Changes in metabolites from baseline to 7 days were compared for the HPRO+NMES and SOC groups. Sparse partial least squared discriminant analysis (sPLS-DA) identified metabolites discriminating each group. Pearson’s correlation coefficients were calculated between each metabolite and total protein per day, nitrogen balance, and muscle volume Multivariable models were developed to determine associations between each metabolite and muscle volume. Results A total of 18 unique metabolites were identified including pre and post treatment and differentiating SOC vs HPRO+NMES. Of these, 9 had significant positive correlations with protein intake: N-acetylserine (ρ=0.61, P =1.56x10 (-3) ), N-acetylleucine (ρ=0.58, P =2.97x10 (-3) ), β-hydroxyisovaleroylcarnitine (ρ=0.53, P =8.35x10 (-3) ), tiglyl carnitine (ρ=0.48, P =0.0168), N-acetylisoleucine (ρ=0.48, P =0.0183), N-acetylthreonine (ρ=0.47, P =0.0218), N-acetylkynurenine (ρ=0.45, P =0.0263), N-acetylvaline (ρ=0.44, P =0.0306), and urea (ρ=0.43, P =0.0381). In multivariable regression models, N-acetylleucine was significantly associated with preserved temporalis [OR 1.08 (95%CI 1.01, 1.16)] and quadricep [OR 1.08 (95%CI 1.02, 1.15)] muscle volume. Quinolinate was also significantly associated with preserved temporalis [OR 1.05 (95%CI 1.01, 1.09)] and quadricep [OR 1.04 (95%CI 1.00, 1.07)] muscle volume. N-acetylserine, N-acetylcitrulline, and b-hydroxyisovaleroylcarnitine were also associated with preserved temporalis or quadricep volume. Conclusions Metabolites defining the HPRO+NMES intervention mainly consisted of amino acid derivatives. These metabolites had strong correlations with protein intake and were associated with preserved muscle volume.