Broadband near-infrared spectroscopy can detect cyanide-induced cytochrome aa₃ inhibition in rats: a proof of concept study

Clinically available near-infrared spectroscopy (NIRS) devices use two to five wavelengths of light to measure the relative amounts of oxyhemoglobin and deoxyhemoglobin in tissue to determine tissue hemoglobin oxygen saturation (StO₂). In addition to StO₂, broadband NIRS devices (using hundreds of wavelengths of light) may be able to measure the oxidation state of mitochondrial cytochrome aa₃ (Cyt_ox) which reflects the subcellular energetic state. We hypothesize that broadband NIRS devices can measure Cyt_ox independent of changes in hemoglobin saturation.

In this prospective non-randomized study, 20 male Sprague-Dawley rats (300 g) were anesthetized with isoflurane, tracheally intubated, and ventilated with 100% O₂ containing 2% isoflurane. They were subsequently instrumented with a broadband NIRS device that used a halogen light source coupled to an emitting fibreoptic cable. Three receiving fibreoptic cables were utilized; one analyzed the light source and the other two were directed at the base of the skull. Each receiving fibre was connected to a spectrometer to measure light intensity. Sodium cyanide (NaCN) 5 mg·kg⁻¹ iv was injected in order to produce cytochrome aa₃ reduction. Two to three minutes after injection, oxygen was eliminated and 100% nitrogen (i.e., anoxia) was used for ventilation in order to induce a reduction in both cytochrome aa₃ and hemoglobin desaturation. Changes in the cytochrome oxidation state and hemoglobin oxygenation were calculated using a broadband algorithm and compared before and after both the NaCN and anoxia interventions.

The NaCN injection resulted in a decrease in median [interquartile range (IQR)] deoxyhemoglobin (−0.014 [−0.29 to −0.005] arbitrary units [AU]; P < 0.001), an increase in oxyhemoglobin (0.013 [−0.011 to 0.031] AU; P < 0.001), and a reduction in cytochrome aa₃ (−0.015 [−0.020 to −0.011] AU; P < 0.001). Anoxia resulted in an increase in median [IQR] deoxyhemoglobin (0.13 [0.11 to 0.18] AU; P < 0.001), a decrease in oxyhemoglobin (−0.17 [−0.22 to −0.15] AU; P < 0.001), and a reduction in cytochrome aa₃ (−0.04 [−0.06 to −0.03] AU; P < 0.001).

Broadband NIRS can effectively measure the directionality of changes in both Cyt_ox and StO₂ by uncoupling the cytochrome and hemoglobin signals through inhibition of the electron transport chain and anoxia.