Skip to main content
PMC home page
Philosophical Transactions of the Royal Society B: Biological Sciences logoLink to Philosophical Transactions of the Royal Society B: Biological Sciences
. 1997 Jun 29;352(1354):669–676. doi: 10.1098/rstb.1997.0048

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

C E Cooper 1, R Springett 1
PMCID: PMC1691958  PMID: 9232854

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.

Full Text

The Full Text of this article is available as a PDF (343.1 KB).

Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Bashford C. L., Barlow C. H., Chance B., Haselgrove J. The oxidation--reduction state of cytochrome oxidase in freeze trapped gerbil brains. FEBS Lett. 1980 Apr 21;113(1):78–80. doi: 10.1016/0014-5793(80)80499-6. [DOI] [PubMed] [Google Scholar]
  2. Bolaños J. P., Peuchen S., Heales S. J., Land J. M., Clark J. B. Nitric oxide-mediated inhibition of the mitochondrial respiratory chain in cultured astrocytes. J Neurochem. 1994 Sep;63(3):910–916. doi: 10.1046/j.1471-4159.1994.63030910.x. [DOI] [PubMed] [Google Scholar]
  3. Brown G. C., Cooper C. E. Nanomolar concentrations of nitric oxide reversibly inhibit synaptosomal respiration by competing with oxygen at cytochrome oxidase. FEBS Lett. 1994 Dec 19;356(2-3):295–298. doi: 10.1016/0014-5793(94)01290-3. [DOI] [PubMed] [Google Scholar]
  4. Brown G. C., Crompton M., Wray S. Cytochrome oxidase content of rat brain during development. Biochim Biophys Acta. 1991 Mar 29;1057(2):273–275. doi: 10.1016/s0005-2728(05)80109-4. [DOI] [PubMed] [Google Scholar]
  5. Cady E. B., Lorek A., Penrice J., Wylezinska M., Cooper C. E., Brown G. C., Owen-Reece H., Kirkbride V., Wyatt J. S., Osmund E. Brain-metabolite transverse relaxation times in magnetic resonance spectroscopy increase as adenosine triphosphate depletes during secondary energy failure following acute hypoxia-ischaemia in the newborn piglet. Neurosci Lett. 1994 Dec 5;182(2):201–204. doi: 10.1016/0304-3940(94)90797-8. [DOI] [PubMed] [Google Scholar]
  6. Castro L., Rodriguez M., Radi R. Aconitase is readily inactivated by peroxynitrite, but not by its precursor, nitric oxide. J Biol Chem. 1994 Nov 25;269(47):29409–29415. [PubMed] [Google Scholar]
  7. Chandel N., Budinger G. R., Kemp R. A., Schumacker P. T. Inhibition of cytochrome-c oxidase activity during prolonged hypoxia. Am J Physiol. 1995 Jun;268(6 Pt 1):L918–L925. doi: 10.1152/ajplung.1995.268.6.L918. [DOI] [PubMed] [Google Scholar]
  8. Cleeter M. W., Cooper J. M., Darley-Usmar V. M., Moncada S., Schapira A. H. Reversible inhibition of cytochrome c oxidase, the terminal enzyme of the mitochondrial respiratory chain, by nitric oxide. Implications for neurodegenerative diseases. FEBS Lett. 1994 May 23;345(1):50–54. doi: 10.1016/0014-5793(94)00424-2. [DOI] [PubMed] [Google Scholar]
  9. Cooper C. E., Matcher S. J., Wyatt J. S., Cope M., Brown G. C., Nemoto E. M., Delpy D. T. Near-infrared spectroscopy of the brain: relevance to cytochrome oxidase bioenergetics. Biochem Soc Trans. 1994 Nov;22(4):974–980. doi: 10.1042/bst0220974. [DOI] [PubMed] [Google Scholar]
  10. Edwards A. D., Yue X., Squier M. V., Thoresen M., Cady E. B., Penrice J., Cooper C. E., Wyatt J. S., Reynolds E. O., Mehmet H. Specific inhibition of apoptosis after cerebral hypoxia-ischaemia by moderate post-insult hypothermia. Biochem Biophys Res Commun. 1995 Dec 26;217(3):1193–1199. doi: 10.1006/bbrc.1995.2895. [DOI] [PubMed] [Google Scholar]
  11. Ferrari M., Williams M. A., Wilson D. A., Thakor N. V., Traystman R. J., Hanley D. F. Cat brain cytochrome-c oxidase redox changes induced by hypoxia after blood-fluorocarbon exchange transfusion. Am J Physiol. 1995 Aug;269(2 Pt 2):H417–H424. doi: 10.1152/ajpheart.1995.269.2.H417. [DOI] [PubMed] [Google Scholar]
  12. Hall D. M., Buettner G. R., Matthes R. D., Gisolfi C. V. Hyperthermia stimulates nitric oxide formation: electron paramagnetic resonance detection of .NO-heme in blood. J Appl Physiol (1985) 1994 Aug;77(2):548–553. doi: 10.1152/jappl.1994.77.2.548. [DOI] [PubMed] [Google Scholar]
  13. Hampson N. B., Camporesi E. M., Stolp B. W., Moon R. E., Shook J. E., Griebel J. A., Piantadosi C. A. Cerebral oxygen availability by NIR spectroscopy during transient hypoxia in humans. J Appl Physiol (1985) 1990 Sep;69(3):907–913. doi: 10.1152/jappl.1990.69.3.907. [DOI] [PubMed] [Google Scholar]
  14. Hantraye P., Brouillet E., Ferrante R., Palfi S., Dolan R., Matthews R. T., Beal M. F. Inhibition of neuronal nitric oxide synthase prevents MPTP-induced parkinsonism in baboons. Nat Med. 1996 Sep;2(9):1017–1021. doi: 10.1038/nm0996-1017. [DOI] [PubMed] [Google Scholar]
  15. Hausladen A., Fridovich I. Superoxide and peroxynitrite inactivate aconitases, but nitric oxide does not. J Biol Chem. 1994 Nov 25;269(47):29405–29408. [PubMed] [Google Scholar]
  16. Hazeki O., Seiyama A., Tamura M. Near-infrared spectrophotometric monitoring of haemoglobin and cytochrome a, a3 in situ. Adv Exp Med Biol. 1987;215:283–289. doi: 10.1007/978-1-4684-7433-6_31. [DOI] [PubMed] [Google Scholar]
  17. Heiss L. N., Lancaster J. R., Jr, Corbett J. A., Goldman W. E. Epithelial autotoxicity of nitric oxide: role in the respiratory cytopathology of pertussis. Proc Natl Acad Sci U S A. 1994 Jan 4;91(1):267–270. doi: 10.1073/pnas.91.1.267. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Henry Y., Ducrocq C., Drapier J. C., Servent D., Pellat C., Guissani A. Nitric oxide, a biological effector. Electron paramagnetic resonance detection of nitrosyl-iron-protein complexes in whole cells. Eur Biophys J. 1991;20(1):1–15. doi: 10.1007/BF00183275. [DOI] [PubMed] [Google Scholar]
  19. Hope P. L., Costello A. M., Cady E. B., Delpy D. T., Tofts P. S., Chu A., Hamilton P. A., Reynolds E. O., Wilkie D. R. Cerebral energy metabolism studied with phosphorus NMR spectroscopy in normal and birth-asphyxiated infants. Lancet. 1984 Aug 18;2(8399):366–370. doi: 10.1016/s0140-6736(84)90539-7. [DOI] [PubMed] [Google Scholar]
  20. Hoshi Y., Hazeki O., Tamura M. Oxygen dependence of redox state of copper in cytochrome oxidase in vitro. J Appl Physiol (1985) 1993 Apr;74(4):1622–1627. doi: 10.1152/jappl.1993.74.4.1622. [DOI] [PubMed] [Google Scholar]
  21. Iadecola C., Pelligrino D. A., Moskowitz M. A., Lassen N. A. Nitric oxide synthase inhibition and cerebrovascular regulation. J Cereb Blood Flow Metab. 1994 Mar;14(2):175–192. doi: 10.1038/jcbfm.1994.25. [DOI] [PubMed] [Google Scholar]
  22. Iadecola C., Zhang F., Xu S., Casey R., Ross M. E. Inducible nitric oxide synthase gene expression in brain following cerebral ischemia. J Cereb Blood Flow Metab. 1995 May;15(3):378–384. doi: 10.1038/jcbfm.1995.47. [DOI] [PubMed] [Google Scholar]
  23. Inagaki M., Tamura M. Preparation and optical characteristics of hemoglobin-free isolated perfused rat head in situ. J Biochem. 1993 Jun;113(6):650–657. doi: 10.1093/oxfordjournals.jbchem.a124098. [DOI] [PubMed] [Google Scholar]
  24. Jenkins B. G., Koroshetz W. J., Beal M. F., Rosen B. R. Evidence for impairment of energy metabolism in vivo in Huntington's disease using localized 1H NMR spectroscopy. Neurology. 1993 Dec;43(12):2689–2695. doi: 10.1212/wnl.43.12.2689. [DOI] [PubMed] [Google Scholar]
  25. Knowles R. G., Moncada S. Nitric oxide synthases in mammals. Biochem J. 1994 Mar 1;298(Pt 2):249–258. doi: 10.1042/bj2980249. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Lipton S. A., Choi Y. B., Pan Z. H., Lei S. Z., Chen H. S., Sucher N. J., Loscalzo J., Singel D. J., Stamler J. S. A redox-based mechanism for the neuroprotective and neurodestructive effects of nitric oxide and related nitroso-compounds. Nature. 1993 Aug 12;364(6438):626–632. doi: 10.1038/364626a0. [DOI] [PubMed] [Google Scholar]
  27. Liu X., Kim C. N., Yang J., Jemmerson R., Wang X. Induction of apoptotic program in cell-free extracts: requirement for dATP and cytochrome c. Cell. 1996 Jul 12;86(1):147–157. doi: 10.1016/s0092-8674(00)80085-9. [DOI] [PubMed] [Google Scholar]
  28. Lorek A., Takei Y., Cady E. B., Wyatt J. S., Penrice J., Edwards A. D., Peebles D., Wylezinska M., Owen-Reece H., Kirkbride V. Delayed ("secondary") cerebral energy failure after acute hypoxia-ischemia in the newborn piglet: continuous 48-hour studies by phosphorus magnetic resonance spectroscopy. Pediatr Res. 1994 Dec;36(6):699–706. doi: 10.1203/00006450-199412000-00003. [DOI] [PubMed] [Google Scholar]
  29. Macnab A. J., Gagnon R. E. Potential sources of discrepancies between living tissue near infrared spectroscopy algorithms. Anal Biochem. 1996 May 1;236(2):375–377. doi: 10.1006/abio.1996.0189. [DOI] [PubMed] [Google Scholar]
  30. Marks K. A., Mallard C. E., Roberts I., Williams C. E., Gluckman P. D., Edwards A. D. Nitric oxide synthase inhibition attenuates delayed vasodilation and increases injury after cerebral ischemia in fetal sheep. Pediatr Res. 1996 Aug;40(2):185–191. doi: 10.1203/00006450-199608000-00002. [DOI] [PubMed] [Google Scholar]
  31. Marks K. A., Mallard E. C., Roberts I., Williams C. E., Sirimanne E. S., Johnston B., Gluckman P. D., Edwards A. D. Delayed vasodilation and altered oxygenation after cerebral ischemia in fetal sheep. Pediatr Res. 1996 Jan;39(1):48–54. doi: 10.1203/00006450-199601000-00007. [DOI] [PubMed] [Google Scholar]
  32. Matcher S. J., Cooper C. E. Absolute quantification of deoxyhaemoglobin concentration in tissue near infrared spectroscopy. Phys Med Biol. 1994 Aug;39(8):1295–1312. doi: 10.1088/0031-9155/39/8/008. [DOI] [PubMed] [Google Scholar]
  33. Matcher S. J., Cope M., Delpy D. T. Use of the water absorption spectrum to quantify tissue chromophore concentration changes in near-infrared spectroscopy. Phys Med Biol. 1994 Jan;39(1):177–196. doi: 10.1088/0031-9155/39/1/011. [DOI] [PubMed] [Google Scholar]
  34. Matcher S. J., Elwell C. E., Cooper C. E., Cope M., Delpy D. T. Performance comparison of several published tissue near-infrared spectroscopy algorithms. Anal Biochem. 1995 May 1;227(1):54–68. doi: 10.1006/abio.1995.1252. [DOI] [PubMed] [Google Scholar]
  35. McCormick D. C., Edwards A. D., Brown G. C., Wyatt J. S., Potter A., Cope M., Delpy D. T., Reynolds E. O. Effect of indomethacin on cerebral oxidized cytochrome oxidase in preterm infants. Pediatr Res. 1993 Jun;33(6):603–608. doi: 10.1203/00006450-199306000-00015. [DOI] [PubMed] [Google Scholar]
  36. Mehmet H., Yue X., Squier M. V., Lorek A., Cady E., Penrice J., Sarraf C., Wylezinska M., Kirkbride V., Cooper C. Increased apoptosis in the cingulate sulcus of newborn piglets following transient hypoxia-ischaemia is related to the degree of high energy phosphate depletion during the insult. Neurosci Lett. 1994 Nov 7;181(1-2):121–125. doi: 10.1016/0304-3940(94)90574-6. [DOI] [PubMed] [Google Scholar]
  37. Miyake H., Nioka S., Zaman A., Smith D. S., Chance B. The detection of cytochrome oxidase heme iron and copper absorption in the blood-perfused and blood-free brain in normoxia and hypoxia. Anal Biochem. 1991 Jan;192(1):149–155. doi: 10.1016/0003-2697(91)90200-d. [DOI] [PubMed] [Google Scholar]
  38. Nicholls P., Chanady G. A. Titration and steady-state behaviour of the 830 nm chromophore in cytochrome c oxidase. Biochem J. 1982 Jun 1;203(3):541–549. doi: 10.1042/bj2030541. [DOI] [PMC free article] [PubMed] [Google Scholar]
  39. Nollert G., Möhnle P., Tassani-Prell P., Uttner I., Borasio G. D., Schmoeckel M., Reichart B. Postoperative neuropsychological dysfunction and cerebral oxygenation during cardiac surgery. Thorac Cardiovasc Surg. 1995 Oct;43(5):260–264. doi: 10.1055/s-2007-1013224. [DOI] [PubMed] [Google Scholar]
  40. Pantopoulos K., Weiss G., Hentze M. W. Nitric oxide and the post-transcriptional control of cellular iron traffic. Trends Cell Biol. 1994 Mar;4(3):82–86. doi: 10.1016/0962-8924(94)90179-1. [DOI] [PubMed] [Google Scholar]
  41. Patel J., Marks K. A., Roberts I., Azzopardi D., Edwards A. D. Ibuprofen treatment of patent ductus arteriosus. Lancet. 1995 Jul 22;346(8969):255–255. doi: 10.1016/s0140-6736(95)91304-1. [DOI] [PubMed] [Google Scholar]
  42. Roth S. C., Edwards A. D., Cady E. B., Delpy D. T., Wyatt J. S., Azzopardi D., Baudin J., Townsend J., Stewart A. L., Reynolds E. O. Relation between cerebral oxidative metabolism following birth asphyxia, and neurodevelopmental outcome and brain growth at one year. Dev Med Child Neurol. 1992 Apr;34(4):285–295. doi: 10.1111/j.1469-8749.1992.tb11432.x. [DOI] [PubMed] [Google Scholar]
  43. Shimaoka M., Iida T., Ohara A., Taenaka N., Mashimo T., Honda T., Yoshiya I. NOC, a nitric-oxide-releasing compound, induces dose dependent apoptosis in macrophages. Biochem Biophys Res Commun. 1995 Apr 17;209(2):519–526. doi: 10.1006/bbrc.1995.1532. [DOI] [PubMed] [Google Scholar]
  44. Skov L., Greisen G. Apparent cerebral cytochrome aa3 reduction during cardiopulmonary bypass in hypoxaemic children with congenital heart disease. A critical analysis of in vivo near-infrared spectrophotometric data. Physiol Meas. 1994 Nov;15(4):447–457. doi: 10.1088/0967-3334/15/4/006. [DOI] [PubMed] [Google Scholar]
  45. Stadler J., Billiar T. R., Curran R. D., Stuehr D. J., Ochoa J. B., Simmons R. L. Effect of exogenous and endogenous nitric oxide on mitochondrial respiration of rat hepatocytes. Am J Physiol. 1991 May;260(5 Pt 1):C910–C916. doi: 10.1152/ajpcell.1991.260.5.C910. [DOI] [PubMed] [Google Scholar]
  46. Stingele R., Wagner B., Kameneva M. V., Williams M. A., Wilson D. A., Thakor N. V., Traystman R. J., Hanley D. F. Reduction of cytochrome-c oxidase copper precedes failing cerebral O2 utilization in fluorocarbon-perfused cats. Am J Physiol. 1996 Aug;271(2 Pt 2):H579–H587. doi: 10.1152/ajpheart.1996.271.2.H579. [DOI] [PubMed] [Google Scholar]
  47. Tamura M. Non-invasive monitoring of the redox state of cytochrome oxidase in living tissue using near-infrared laser lights. Jpn Circ J. 1993 Aug;57(8):817–824. doi: 10.1253/jcj.57.817. [DOI] [PubMed] [Google Scholar]
  48. Thoresen M., Penrice J., Lorek A., Cady E. B., Wylezinska M., Kirkbride V., Cooper C. E., Brown G. C., Edwards A. D., Wyatt J. S. Mild hypothermia after severe transient hypoxia-ischemia ameliorates delayed cerebral energy failure in the newborn piglet. Pediatr Res. 1995 May;37(5):667–670. doi: 10.1203/00006450-199505000-00019. [DOI] [PubMed] [Google Scholar]
  49. Tsuji M., Naruse H., Volpe J., Holtzman D. Reduction of cytochrome aa3 measured by near-infrared spectroscopy predicts cerebral energy loss in hypoxic piglets. Pediatr Res. 1995 Mar;37(3):253–259. doi: 10.1203/00006450-199503000-00001. [DOI] [PubMed] [Google Scholar]
  50. Vanin A. F., Men'shikov G. B., Moroz I. A., Mordvintcev P. I., Serezhenkov V. A., Burbaev D. Sh. The source of non-heme iron that binds nitric oxide in cultivated macrophages. Biochim Biophys Acta. 1992 Jun 29;1135(3):275–279. doi: 10.1016/0167-4889(92)90231-y. [DOI] [PubMed] [Google Scholar]
  51. Wray S., Cope M., Delpy D. T., Wyatt J. S., Reynolds E. O. Characterization of the near infrared absorption spectra of cytochrome aa3 and haemoglobin for the non-invasive monitoring of cerebral oxygenation. Biochim Biophys Acta. 1988 Mar 30;933(1):184–192. doi: 10.1016/0005-2728(88)90069-2. [DOI] [PubMed] [Google Scholar]
  52. Wyatt J. S. Near-infrared spectroscopy in asphyxial brain injury. Clin Perinatol. 1993 Jun;20(2):369–378. [PubMed] [Google Scholar]
  53. van Bel F., Dorrepaal C. A., Benders M. J., Zeeuwe P. E., van de Bor M., Berger H. M. Changes in cerebral hemodynamics and oxygenation in the first 24 hours after birth asphyxia. Pediatrics. 1993 Sep;92(3):365–372. [PubMed] [Google Scholar]

Articles from Philosophical Transactions of the Royal Society B: Biological Sciences are provided here courtesy of The Royal Society

RESOURCES