Organ-specific metabolic pathways, including those related to mitochondrial metabolism, could provide insight into mechanisms underlying sepsis-induced organ dysfunction. However, it remains unclear if metabolic changes result from or precede clinical organ dysfunction. To determine if blood concentrations of the mitochondrial metabolites acetylcarnitine and l-carnitine correlate with organ-specific signals of sepsis-induced dysfunction, we performed a series of translational analyses of two cohorts of human sepsis and experiments using a murine model of polymicrobial sepsis. We evaluated the association between mitochondrial metabolites and clinical indices of organ function. In the blood of patients with sepsis or septic shock, we found metabolic signals of dysfunctional mitochondrial β-oxidation that were correlated with clinical measures of renal and liver dysfunction. The relevance of these findings was corroborated in an experimental model that showed distinct patterns of change in organ metabolism that correlated with the blood acetylcarnitine to l-carnitine ratio. In addition, sepsis-induced changes in organ metabolism were distinct in the liver and kidney, highlighting the unique energy economies of each organ. Importantly, metabolic changes preceded changes in clinical indices of organ function and histological evidence of cellular apoptosis. On the basis of these findings, sepsis-induced disruption in blood concentrations of specific metabolites could serve as more physiologically relevant indicators of early organ dysfunction than those we presently use. These early metabolite signals provide mechanistic insights into altered metabolism that may hold the key to timely identification of impending organ dysfunction. This could lead to strategies directed at the interruption of sepsis-induced organ failure.

Secondary bacterial pneumonia is a frequent complication of acute lung injury (ALI) and the acute respiratory distress syndrome (ARDS). Prior efforts to explain increased pneumonia risk in ALI/ARDS have focused on impaired immunity and bacterial virulence, but have overlooked the potential contribution of ecological factors within acutely injured lungs. Here, we show that lung injury profoundly alters the alveolar metabolic microenvironment, a change that can be exploited by a common pneumonia pathogen. In mice and humans, we found that growth of Pseudomonas aeruginosa (the most prominent respiratory pathogen in a retrospective cohort of patients with ARDS) is enhanced by increased alveolar concentrations of multiple ALI/ARDS-associated metabolites, which are derived from the blood and cross the compromised alveolar-capillary barrier during alveolar leak. Collectively, this work reveals an ecological mechanism by which pathogens may survive in the injured lung microenvironment and identifies new potential targets for secondary pneumonia prevention and treatment. ### Competing Interest Statement The authors have declared no competing interest.

Study Objective: Levocarnitine (L-carnitine) has shown promise as a metabolic-therapeutic for septic shock, where mortality approaches 40%. However, high-dose (>= 6 grams) intravenous supplementation results in a broad range of serum concentrations. We sought to describe the population pharmacokinetics (PK) of high-dose L-carnitine, test various estimates of kidney function, and assess the correlation of PK parameters with pre-treatment metabolites in describing drug response for patients with septic shock.Design: Population PK analysis was done with baseline normalized concentrations using nonlinear mixed effect models in the modeling platform Monolix. Various estimates of kidney function, patient demographics, dose received, and organ dysfunction were tested as population covariates.Data Source: We leveraged serum samples and metabolomics data from a phase II trial of L-carnitine in vasopressor-dependent septic shock. Serum was collected at baseline (T0); end-of-infusion (T12); and 24, 48, and 72 h after treatment initiation.Patients and InterventionPatients were adaptively randomized to receive intravenous L-carnitine (6 grams, 12 grams, or 18 grams) or placebo.Measurements and Main Results: The final dataset included 542 serum samples from 130 patients randomized to L-carnitine. A two-compartment model with linear elimination and a fixed volume of distribution (17.1 liters) best described the data and served as a base structural model. Kidney function estimates as a covariate on the elimination rate constant (k) reliably improved model fit. Estimated glomerular filtration rate (eGFR), based on the 2021 Chronic Kidney Disease Epidemiology collaboration (CKD-EPI) equation with creatinine and cystatin C, outperformed creatinine clearance (Cockcroft-Gault) and older CKD-EPI equations that use an adjustment for self-identified race.Conclusions: High-dose L-carnitine supplementation is well-described by a two-compartment population PK model in patients with septic shock. Kidney function estimates that leverage cystatin C provided superior model fit. Future investigations into high-dose L-carnitine supplementation should consider baseline metabolic status and dose adjustments based on renal function over a fixed or weight-based dosing paradigm.

OBJECTIVES: Perturbed host metabolism is increasingly recognized as a pillar of sepsis pathogenesis, yet the dynamic alterations in metabolism and its relationship to other components of the host response remain incompletely understood. We sought to identify the early host-metabolic response in patients with septic shock and to explore biophysiological phenotyping and differences in clinical outcomes among metabolic subgroups. DESIGN: We measured serum metabolites and proteins reflective of the host-immune and endothelial response in patients with septic shock. SETTING: We considered patients from the placebo arm of a completed phase II, randomized controlled trial conducted at 16 U.S. medical centers. Serum was collected at baseline (within 24 hr of the identification of septic shock), 24-hour, and 48-hour postenrollment. Linear mixed models were built to assess the early trajectory of protein analytes and metabolites stratified by 28-day mortality status. Unsupervised clustering of baseline metabolomics data was conducted to identify subgroups of patients. PATIENTS: Patients with vasopressor-dependent septic shock and moderate organ dysfunction that were enrolled in the placebo arm of a clinical trial. INTERVENTIONS: None. MEASUREMENTS AND MAIN RESULTS: Fifty-one metabolites and 10 protein analytes were measured longitudinally in 72 patients with septic shock. In the 30 patients (41.7%) who died prior to 28 days, systemic concentrations of acylcarnitines and interleukin (IL)-8 were elevated at baseline and persisted at T24 and T48 throughout early resuscitation. Concentrations of pyruvate, IL-6, tumor necrosis factor-α, and angiopoietin-2 decreased at a slower rate in patients who died. Two groups emerged from clustering of baseline metabolites. Group 1 was characterized by higher levels of acylcarnitines, greater organ dysfunction at baseline and postresuscitation (p < 0.05), and greater mortality over 1 year (p < 0.001). CONCLUSIONS: Among patients with septic shock, nonsurvivors exhibited a more profound and persistent dysregulation in protein analytes attributable to neutrophil activation and disruption of mitochondrial-related metabolism than survivors.

Understanding of lung-specific metabolic changes to ARDS development is limited due to the paucity of longitudinal studies and that the lung is difficult to repeatedly sample. We obtained a "metabolic lung biopsy" by simultaneous acquisition of trans-pulmonary whole blood (WB) from the pulmonary (PA) and carotid (CA) arteries every 4h and hourly volatile organic compounds (VOCs) in exhaled breath from a swine model that develops both radiographic and histo-morphologic evidence of ARDS. WB was assayed by quantitative proton nuclear magnetic resonance spectroscopy and exhaled breath by gas chromatography. Data were analyzed by a mixed effect model with random intercept. P/F ratio was used as the fixed effect to determine the average slope for each metabolite. 37 WB metabolites and 55 named VOCs were detected from 4 of 5 and 5 of 5 swine, respectively. With declining P/F ratio, there was increased excretion/production (PA<CA) of key WB energy metabolites including (A) ATP (p=0.007), (B) glucose (p=0.03), (C) ADP (p=0.12) and (D) lysine (p=0.14). In exhaled breath, as ARDS develops, (E) 3-methylheptane (p<0.001), which may be a by-product of lipid peroxidation and (F) a-pinene (p<0.001), a terpene with anti-inflammatory activity, increased and decreased, respectively. These findings suggest that a "metabolic lung biopsy" is feasible and could aid in informing lung-specific metabolic processes that participate in ARDS pathogenesis.

Sepsis‐induced metabolic dysfunction contributes to organ failure and death. L‐carnitine has shown promise for septic shock, but a recent phase II study of patients with vasopressor‐dependent septic shock demonstrated a non‐significant reduction in mortality. We undertook a pharmacometabolomics study of these patients (n = 250) to identify metabolic profiles predictive of a 90‐day mortality benefit from L‐carnitine. The independent predictive value of each pretreatment metabolite concentration, adjusted for L‐carnitine dose, on 90‐day mortality was determined by logistic regression. A grid‐search analysis maximizing the Z‐statistic from a binomial proportion test identified specific metabolite threshold levels that discriminated L‐carnitine responsive patients. Threshold concentrations were further assessed by hazard ratio and Kaplan‐Meier estimate. Accounting for L‐carnitine treatment and dose, 11 1H‐NMR metabolites and 12 acylcarnitines were independent predictors of 90‐day mortality. Based on the grid‐search analysis numerous acylcarnitines and valine were identified as candidate metabolites of drug response. Acetylcarnitine emerged as highly viable for the prediction of an L‐carnitine mortality benefit due to its abundance and biological relevance. Using its most statistically significant threshold concentration, patients with pretreatment acetylcarnitine greater than or equal to 35 µM were less likely to die at 90 days if treated with L‐carnitine (18 g) versus placebo (p = 0.01 by log rank test). Metabolomics also identified independent predictors of 90‐day sepsis mortality. Our proof‐of‐concept approach shows how pharmacometabolomics could be useful for tackling the heterogeneity of sepsis and informing clinical trial design. In addition, metabolomics can help understand mechanisms of sepsis heterogeneity and variable drug response, because sepsis induces alterations in numerous metabolite concentrations.

Background: Sepsis shifts cardiac metabolic fuel preference and this disruption may have implications for cardiovascular function. A greater understanding of the role of metabolism in the development and persistence of cardiovascular failure in sepsis could serve to identify novel pharmacotherapeutic approaches. Methods: Secondary analysis of prospective quantitative proton nuclear magnetic resonance (H-1-NMR) metabolomic data from patients enrolled in a phase II randomized control trial of L-carnitine in septic shock. Participants with a sequential organ failure assessment (SOFA) score of > = 5, lactate > = 2, and requiring vasopressor support for at least 4 h were eligible for enrollment. The independent prognostic value of metabolites to predict survival with shock resolution within 48 h and vasopressor free days were assessed. Concentrations of predictive metabolites were compared between participants with and without shock resolution at 48 h. Results: Serum H-1-NMR metabolomics data from 228 patients were analyzed. Eighty-one (36%) patients met the primary outcome; 33 (14%) died prior to 48 h. The branched chain amino acids (BCAA), valine, leucine, and isoleucine were univariate predictors of the primary outcome after adjusting for multiple hypothesis testing, while valine remained significant after controlling for SOFA score. Similar results were observed when analyzed based on vasopressor free days, and persisted after controlling for confounding variables and excluding non-survivors. BCAA concentrations at 48 h significantly discriminated between those with shock resolution versus persistent shock. Conclusions: Among patients with septic shock, BCAA concentrations independently predict time to shock resolution. This study provides hypothesis generating data into the potential contribution of BCAAs to the pathophysiology of cardiovascular failure in sepsis, opening areas for future investigations.

To date, existing animal models of the acute respiratory distress syndrome (ARDS) have failed to translate preclinical discoveries into effective pharmacotherapy or diagnostic biomarkers. To address this translational gap, we developed a high-fidelity swine model of ARDS utilizing clinically-relevant lung injury exposures. Fourteen male swine were anesthetized, mechanically ventilated, and surgically instrumented for hemodynamic monitoring, blood, and tissue sampling. Animals were allocated to one of three groups: 1) Indirect lung injury only : animals were inoculated by direct injection of E. coli into the kidney parenchyma, provoking systemic inflammation and distributive shock physiology; 2) Direct lung injury only : animals received volutrauma, hyperoxia, and bronchoscope-delivered gastric particles; 3) Combined indirect and direct lung injury: animals were administered both above-described indirect and direct lung injury exposures. Animals were monitored for up to 12 hours, with serial collection of physiologic data, blood samples, and radiographic imaging. Lung tissue was acquired post-mortem for pathological examination. In contrast to indirect lung injury only and direct lung injury only groups, animals in the combined indirect and direct lung injury group exhibited all of the physiological, radiographic, and histopathologic hallmarks of human ARDS: impaired gas exchange (mean PaO 2 /FiO 2 ratio 124.8 ± 63.8), diffuse bilateral opacities on chest radiographs, and extensive pathologic evidence of diffuse alveolar damage. Our novel porcine model of ARDS, built on clinically-relevant lung injury exposures, faithfully recapitulates the physiologic, radiographic, and histopathologic features of human ARDS, and fills a crucial gap in the translational study of human lung injury.

Background The systemic responses to infection and its progression to sepsis remains poorly understood. Progress in the field has been stifled by the shortcomings of experimental models which include poor replication of the human condition. To address these challenges, we developed and piloted a novel large animal model of severe infection that is capable of generating multi-system clinically relevant data. Methods Male swine (n = 5) were anesthetized, mechanically ventilated, and surgically instrumented for continuous hemodynamic monitoring and serial blood sampling. Animals were inoculated with uropathogenic E. coli by direct injection into the renal parenchyma and were maintained until a priori endpoints were met. The natural history of the infection was studied. Animals were not resuscitated. Multi-system data were collected hourly to 6 hours; all animals were euthanized at predetermined physiologic endpoints. Results Core body temperature progressively increased from mean (SD) 37.9(0.8)°C at baseline to 43.0(1.2)°C at experiment termination (p = 0.006). Mean arterial pressure did not begin to decline until 6h post inoculation, dropping from 86(9) mmHg at baseline to 28(5) mmHg (p = 0.005) at termination. Blood glucose progressively declined but lactate levels did not elevate until the last hours of the experiment. There were also temporal changes in whole blood concentrations of a number of metabolites including increases in the catecholamine precursors, tyrosine (p = 0.005) and phenylalanine (p = 0.005). Lung, liver, and kidney function parameters worsened as infection progressed and at study termination there was histopathological evidence of injury in these end-organs. Conclusion We demonstrate a versatile, multi-system, longitudinal, swine model of infection that could be used to further our understanding of the mechanisms that underlie infection-induced multi-organ dysfunction and failure, optimize resuscitation protocols and test therapeutic interventions. Such a model could improve translation of findings from the bench to the bedside, circumventing a significant obstacle in sepsis research.

Background: Though blood is an excellent biofluid for metabolomics, proteins and lipids present in blood can interfere with 1d-1H NMR spectra and disrupt quantification of metabolites. Here, we present effective macromolecule removal strategies for serum and whole blood (WB) samples. Methods: A variety of macromolecule removal strategies were compared in both WB and serum, along with tests of ultrafiltration alone and in combination with precipitation methods. Results: In healthy human serum, methanol:chloroform:water extraction with ultrafiltration was compared to methanol precipitation with and without ultrafiltration. Methods were tested in healthy pooled human serum, and in serum from patients with sepsis. Effects of long-term storage at −80 °C were tested to explore the impact of macromolecule removal strategy on serum from different conditions. In WB a variety of extraction strategies were tested in two types of WB (from pigs and baboons) to examine the impact of macromolecule removal strategies on different samples. Conclusions: In healthy human serum methanol precipitation of serum with ultrafiltration was superior, but was similar in recovery and variance to methanol:chloroform:water extraction with ultrafiltration in pooled serum from patients with sepsis. In WB, high quality, quantifiable spectra were obtained with the use of a methanol: chloroform precipitation.