Preliminary Research: Application of Non-Invasive Measure of Cytochrome c Oxidase Redox States and Mitochondrial Function in a Porcine Model of Carbon Monoxide Poisoning

Abstract

Introduction: Carbon monoxide (CO) is a colorless and odorless gas that is a leading cause of environmental poisoning in the USA with substantial mortality and morbidity. The mechanism of CO poisoning is complex and includes hypoxia, inflammation, and leukocyte sequestration in brain microvessel segments leading to increased reactive oxygen species. Another important pathway is the effects of CO on the mitochondria, specifically at cytochrome c oxidase, also known as Complex IV (CIV). The purpose of this ongoing study is the preliminary development of a porcine model of CO poisoning for investigation of alterations in brain mitochondrial physiology.

Methods: Four pigs (10 kg) were divided into two groups: Sham (n = 2) and CO (n = 2). Administration of a dose of CO at 2000 ppm to the CO group over 120 minutes followed by 30 minutes of re-oxygenation at room air. The control group received room air for 150 minutes. Non-invasive optical monitoring was used to measure CIV redox states. Cerebral microdialysis was performed to obtain semi real-time measurements of cerebral metabolic status. At the end of the exposure, fresh brain tissue (cortical and hippocampal) was immediately harvested to measure mitochondrial respiration. Snap frozen cortical tissue was also used for ATP concentrations and western blotting.

Results: While a preliminary ongoing study, animals in the CO group showed possible early decreases in brain mitochondrial respiration, citrate synthase density, CIV redox changes measured with optics, and an increase in the lactate-to-pyruvate ratio.

Conclusions: There is a possible observable phenotype highlighting the important role of mitochondrial function in the injury of CO poisoning.

Keywords: Basic science; Biomarker; Carbon monoxide; Mitochondria; Optics.

Fig. 1

Isolated mitochondrial respiration of cortical and hippocampal tissue. Key respiration states of isolated mitochondria from cortical and hippocampal issue. Values expressed individually also seen in Table 2. OXPHOS, oxidative phosphorylation; ETS, electron transport chain; C, complex

Fig. 2

Non-invasive measure of change in oxidized complex IV concentrations. Temporal plots of optically measured changes in oxidized complex IV (CIV) in two pigs. One pig was exposed to 120 minutes of carbon monoxide (CO) at 2000 ppm (COHb of 45.5%; black curve), while the other pig was a sham (red curve). CO binds to CIV, limiting the oxygen coupling to CIV, which in turn leads to a decrease in the total amount of oxidized CIV (with a proportional increase in the reduced CIV concentration). The y-axis is a relative change from baseline: a −4 μM change in oxidized CIV (i.e., oxidized version of the cytochrome C oxidase) at the end of exposure means that the total concentration of the oxidized enzyme was reduced by 4 μM

Fig. 3

Cerebral microdialysis (cMD) measurements during CO exposure. Semi real-time measurements of cerebral metabolism taken every 30 minutes. Cerebral LPR between the two groups is shown at the specified time point (only LPR ratio shown). Values expressed individually. LPR, lactate/pyruvate ratio

Fig. 4

Adenosine triphosphate (ATP) concentrations. ATP concentrations were obtained using an ATP fluorometric assay in snap frozen brain tissue from all subject animals. Values expressed individually. ATP, adenosine triphosphate