C-Metabolic flux analysis detected a hyperoxemia-induced reduction of tricarboxylic acid cycle metabolism in granulocytes during two models of porcine acute subdural hematoma and hemorrhagic shock.
Eva-Maria Wolfschmitt, Josef Albert Vogt, Melanie Hogg, Ulrich Wachter, Nicole Stadler, Thomas Kapapa, Thomas Datzmann, David Alexander Christian Messerer, Andrea Hoffmann, Michael Gröger, Franziska Münz, René Mathieu, Simon Mayer, Tamara Merz, Pierre Asfar, Enrico Calzia, Peter Radermacher, Fabian Zink
Author Information
Eva-Maria Wolfschmitt: Institute for Anesthesiological Pathophysiology and Process Engineering, University Hospital Ulm, Ulm, Germany.
Josef Albert Vogt: Institute for Anesthesiological Pathophysiology and Process Engineering, University Hospital Ulm, Ulm, Germany.
Melanie Hogg: Institute for Anesthesiological Pathophysiology and Process Engineering, University Hospital Ulm, Ulm, Germany.
Ulrich Wachter: Institute for Anesthesiological Pathophysiology and Process Engineering, University Hospital Ulm, Ulm, Germany.
Nicole Stadler: Institute for Anesthesiological Pathophysiology and Process Engineering, University Hospital Ulm, Ulm, Germany.
Thomas Kapapa: Clinic for Neurosurgery, University Hospital Ulm, Ulm, Germany.
Thomas Datzmann: Clinic for Anesthesia and Intensive Care, University Hospital Ulm, Ulm, Germany.
David Alexander Christian Messerer: Institute for Anesthesiological Pathophysiology and Process Engineering, University Hospital Ulm, Ulm, Germany.
Andrea Hoffmann: Institute for Anesthesiological Pathophysiology and Process Engineering, University Hospital Ulm, Ulm, Germany.
Michael Gröger: Institute for Anesthesiological Pathophysiology and Process Engineering, University Hospital Ulm, Ulm, Germany.
Franziska Münz: Institute for Anesthesiological Pathophysiology and Process Engineering, University Hospital Ulm, Ulm, Germany.
René Mathieu: Clinic for Neurosurgery, Bundeswehrkrankenhaus, Ulm, Germany.
Simon Mayer: Clinic for Neurosurgery, Bundeswehrkrankenhaus, Ulm, Germany.
Tamara Merz: Institute for Anesthesiological Pathophysiology and Process Engineering, University Hospital Ulm, Ulm, Germany.
Pierre Asfar: Département de Médecine Intensive - Réanimation et Médecine Hyperbare, Centre Hospitalier Universitaire, Angers, France.
Enrico Calzia: Institute for Anesthesiological Pathophysiology and Process Engineering, University Hospital Ulm, Ulm, Germany.
Peter Radermacher: Institute for Anesthesiological Pathophysiology and Process Engineering, University Hospital Ulm, Ulm, Germany.
Fabian Zink: Institute for Anesthesiological Pathophysiology and Process Engineering, University Hospital Ulm, Ulm, Germany.
Introduction: Supplementation with increased inspired oxygen fractions has been suggested to alleviate the harmful effects of tissue hypoxia during hemorrhagic shock (HS) and traumatic brain injury. However, the utility of therapeutic hyperoxia in critical care is disputed to this day as controversial evidence is available regarding its efficacy. Furthermore, in contrast to its hypoxic counterpart, the effect of hyperoxia on the metabolism of circulating immune cells remains ambiguous. Both stimulating and detrimental effects are possible; the former by providing necessary oxygen supply, the latter by generation of excessive amounts of reactive oxygen species (ROS). To uncover the potential impact of increased oxygen fractions on circulating immune cells during intensive care, we have performed a C-metabolic flux analysis (MFA) on PBMCs and granulocytes isolated from two long-term, resuscitated models of combined acute subdural hematoma (ASDH) and HS in pigs with and without cardiovascular comorbidity. Methods: Swine underwent resuscitation after 2 h of ASDH and HS up to a maximum of 48 h after HS. Animals received normoxemia (PO = 80 - 120 mmHg) or targeted hyperoxemia (PO = 200 - 250 mmHg for 24 h after treatment initiation, thereafter PO as in the control group). Blood was drawn at time points T1 = after instrumentation, T2 = 24 h post ASDH and HS, and T3 = 48 h post ASDH and HS. PBMCs and granulocytes were isolated from whole blood to perform electron spin resonance spectroscopy, high resolution respirometry and C-MFA. For the latter, we utilized a parallel tracer approach with 1,2-C glucose, U-C glucose, and U-C glutamine, which covered essential pathways of glucose and glutamine metabolism and supplied redundant data for robust Bayesian estimation. Gas chromatography-mass spectrometry further provided multiple fragments of metabolites which yielded additional labeling information. We obtained precise estimations of the fluxes, their joint credibility intervals, and their relations, and characterized common metabolic patterns with principal component analysis (PCA). Results: C-MFA indicated a hyperoxia-mediated reduction in tricarboxylic acid (TCA) cycle activity in circulating granulocytes which encompassed fluxes of glutamine uptake, TCA cycle, and oxaloacetate/aspartate supply for biosynthetic processes. We further detected elevated superoxide levels in the swine strain characterized by a hypercholesterolemic phenotype. PCA revealed cell type-specific behavioral patterns of metabolic adaptation in response to ASDH and HS that acted irrespective of swine strains or treatment group. Conclusion: In a model of resuscitated porcine ASDH and HS, we saw that ventilation with increased inspiratory O concentrations (PO = 200 - 250 mmHg for 24 h after treatment initiation) did not impact mitochondrial respiration of PBMCs or granulocytes. However, Bayesian C-MFA results indicated a reduction in TCA cycle activity in granulocytes compared to cells exposed to normoxemia in the same time period. This change in metabolism did not seem to affect granulocytes' ability to perform phagocytosis or produce superoxide radicals.
