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Philos Trans R Soc Lond B Biol Sci. 1997 June 29; 352(1354): 669–676.
PMCID: PMC1691958

Measurement of cytochrome oxidase and mitochondrial energetics by near-infrared spectroscopy.

Abstract

Cytochrome 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 CuA centre, has a strong absorbance in the near-infrared that enables it to be detectable in vivo 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 near-infrared 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 CuA redox state in vivo (Cooper et al. 1994), but most significant is likely to be the molecular oxygen concentration (at low oxygen tensions, electrons build up on CuA 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 CuA 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 vivo 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 vivo. 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 near-infrared 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 make 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 cytochrome near-infrared spectroscopy signal in vivo (Cooper et al. 1997) and the factors affecting the oxidation state of cytochrome oxidase CuA (Cooper et al. 1994). In this article we would like to strike a somewhat more optimistic note--we will stress the usefulness this measurement may have in the clinical environment, as well as describing conditions under which we can have confidence that we are measuring real changes in the CuA redox state.

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Selected References

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