Impairment of mitochondrial respiration following ex vivo cyanide exposure in peripheral blood mononuclear cells

ABSTRACT

Objectives: The objective of this study is to measure mitochondrial respiration using intact cells from whole blood exposed to cyanide as a new biomarker for mitochondrial inhibition. Methods: A single nontourniqueted venous blood sample was collected from 10 healthy volunteers after informed consent. Venous lactate was measured immediately following blood collection. Half of the remaining blood sample was then incubated with 100 mM of potassium cyanide (KCN) for 5 min, and half of the sample remained unexposed. Repeat lactate measurements were performed from blood exposed and not exposed to KCN. Measurement of mitochondrial respiration: intact PBMCs were placed in a 2-mL chamber at a final concentration of 2–3 × 10⁶ cells/mL. Measurements of oxygen consumption were performed at 37°C in a high-resolution oxygraph (Oxygraph-2k Oroboros Instruments, Innsbruck, Austria). Oxygen flux (in pmol O₂/s/10⁶ cells), which is directly proportional to oxygen consumption, was recorded continuously using DatLab software 6 (Oroboros Instruments). Results: There were significance differences in the relevant key parameters of mitochondrial respiration: Of the parameters measuring mitochondrial respiration, four of the six demonstrated a statistically significant mean difference between control and cyanide: for routine respiration (mean difference [control-cyanide]: 8.9 pmol O₂/s/10⁶ cells; 95% CI: 5.6–12.2, p < 0.0001); Proton Leak (mean difference: 0.73 pmol O₂/s/10⁶ cells; 95% CI: −0.33–1.79, p = 0.157); Maximal respiration (mean difference: 21.7 pmol O₂/s/10⁶ cells; 95% CI: 16.0–27.6, p < 0.0001); Residual oxygen consumption (mean difference 0.25 pmol O₂/s/10⁶ cells; 95% CI: −0.68–1.18, p = 0.557). There was a significant difference in spare respiratory capacity (SRC) and adenosine triphosphate (ATP)-linked respiration with the control samples demonstrating a higher SRC and ATP-linked respiration. Finally, there is a statistically significant difference in lactate (mean difference −0.32, 95% CI: −0.41 to −0.23, p < 0.0001), though clinically similar level, with a higher lactate concentration in the cyanide samples. Conclusions: In this ex vivo model, the measurements of key parameters in mitochondrial respiration may be a more sensitive measure of cellular function when compared to lactate.

Keywords:

• Cardiovascular
• metabolic
• pharmaceuticals
• complications of poisoning

Acknowledgements

We thank Prasanth Potluri, PhD (The Children’s Hospital of Philadelphia-Center of Mitochondrial and Epigenomic Medicine) in assisting with the preparation of the cyanide used in this study.

Disclosure statement

The authors report no conflicts of interest. The authors alone are responsible for the content and writing of this article.

Funding information

The project described was supported by award K12 HL109009 from the National Heart, Lung, and Blood Institute.