Measurement of cytochrome oxidase and mitochondrial energetics by near–infrared spectroscopy

Abstract

SUMMARYCytochrome oxidase is the terminal electron acceptor of the mitochondrial respiratory chain. It is responsible for the vast majority of oxygen consumption in the body and essential for the efficient generation of cellular ATP. The enzyme contains four redox active metal centres ; one of these, the binuclear Cu A centre, has a strong absorbance in the near-infrared that enables it to be detectable in i o by near-infrared spectroscopy. However, the fact that the concentration of this centre is less than 10 % of that of haemoglobin means that its detection is not a trivial matter.Unlike the case with deoxyhaemoglobin and oxyhaemoglobin, concentration changes of the total cytochrome oxidase protein occur very slowly (over days) and are therefore not easily detectable by nearinfrared spectroscopy. However, the copper centre rapidly accepts and donates an electron, and can thus change its redox state quickly ; this redox change is detectable by near-infrared spectroscopy. Many factors can affect the Cu A redox state in i o , but the most significant is likely to be the molecular oxygen concentration (at low oxygen tensions, electrons build up on Cu A as reduction of oxygen by the enzyme starts to limit the steady-state rate of electron transfer).The factors underlying haemoglobin oxygenation, deoxygenation and blood volume changes are, in general, well understood by the clinicians and physiologists who perform near-infrared spectroscopy measurements. In contrast, the factors that control the steady-state redox level of Cu A in cytochrome oxidase are still a matter of active debate, even amongst biochemists studying the isolated enzyme and mitochondria. Coupled with the difficulties of accurate in i o measurements it is perhaps not surprising that the field of cytochrome oxidase near-infrared spectroscopy has a somewhat chequered past. Too often papers have been written with insufficient information to enable the measurements to be repeated and few attempts have been made to test the algorithms in i o.In recent years a number of research groups and commercial spectrometer manufacturers have made a concerted attempt to not only say how they are attempting to measure cytochrome oxidase by nearinfrared spectroscopy but also to demonstrate that they are really doing so. We applaud these attempts, which in general fall into three areas : first, modelling of data can be performed to determine what problems are likely to derail cytochrome oxidase detection algorithms (Matcher et al. 1995) ; secondly haemoglobin concentration changes can be made by haemodilution (using saline or artificial blood substitutes) in animals (Tamura 1993) or patients (Skov & Greisen 1994) ; and thirdly, the cytochrome oxidase redox state can be fixed by the use of mitochondrial inhibitors and then attempts made to cause spurious cytochrome changes by dramatically varying haemoglobin oxygenation, haemoglobin concentration and light scattering (Cooper et al. 1997).We have previously written reviews covering the difficulties of measuring the cyt...

Cerebral Hemodynamics and Metabolism During Cardiac Arrest and Cardiopulmonary Resuscitation Using Hyperspectral Near Infrared Spectroscopy

“…30 Therefore, although hNIRS has shown to be a promising tool for the measurement of tissue oxygenation, the possibility of crosstalk between Cyt-ox and hemoglobin changes has questioned the reliability of the Cyt-ox measurements. 30, 31 Our novel hNIRS algorithm successfully avoided the crosstalk between the hemodynamics and metabolic changes. Compared to the MBLL algorithm, which was inconsistent in this study, the measured changes of Cyt-ox using our modified DA algorithm were consistent with the hemodynamic changes.…”

Regulation of cytochrome oxidase redox state during umbilical cord occlusion in preterm fetal sheep

“…NIRS also can provide information on the redox state of the copper A core of cytochrome oxidase, the terminal electron acceptor of the mitochondrial electron transport chain (12,54). Using the algorithm developed by Wray et al (55), changes in its redox state correlate closely with flow of reducing equivalents down the phosphorylation pathway and depletion of high-energy metabolites during hypoxia or intense oxidative activity in other tissues (8,37,47).…”

Can Peripheral Blood Mononuclear Cells Be Used as a Proxy for Mitochondrial Dysfunction in Vital Organs During Hemorrhagic Shock and Resuscitation?

“…Although promising, NIRS is technically challenging and not clinically practical. In addition to the potential difficulty in interpreting spectral overlap between hemoglobin, myoglobin, and mitochondrial cytochromes, significant artifact from the variability in depth and composition of subcutaneous tissues among patients as well as increasing tissue edema following trauma may make accurate measurements challenging (3,10). …”

“…While blood lactate, base deficit, and venous oxygen saturation can be used to assess impaired aerobic metabolism globally, these biomarkers are neither specific for mitochondrial dysfunction nor informative about the site of cellular injury (9). Although near infrared spectroscopy and electron paramagnetic resonance have been used to assess mitochondrial function following hemorrhagic shock (3), practical and technical challenges have limited their use to research settings (10). …”