Measurement of the mitochondrial membrane potential and pH gradient from the redox poise of the hemes of the bc1 complex.

The redox potentials of the hemes of the mitochondrial bc(1) complex are dependent on the proton-motive force due to the energy transduction. This allows the membrane potential and pH gradient components to be calculated from the oxidation state of the hemes measured with multi-wavelength cell spectroscopy. Oxidation states were measured in living RAW 264.7 cells under varying electron flux and membrane potential obtained by a combination of oligomycin and titration with a proton ionophore. A stochastic model of bc(1) turnover was used to confirm that the membrane potential and redox potential of the ubiquinone pool could be measured from the redox poise of the b-hemes under physiological conditions assuming the redox couples are in equilibrium. The pH gradient was then calculated from the difference in redox potentials of cytochrome c and ubiquinone pool using the stochastic model to evaluate the ΔG of the bc(1) complex. The technique allows absolute quantification of the membrane potential, pH gradient, and proton-motive force without the need for genetic manipulation or exogenous compounds.

Spectral components of the α-band of cytochrome oxidase.

Oxidative redox titrations of the mitochondrial cytochromes were performed in near-anoxic RAW 264.7 cells by inhibiting complex I. Cytochrome oxidation changes were measured with multi-wavelength spectroscopy and the ambient redox potential was calculated from the oxidation state of endogenous cytochrome c. Two spectral components were separated in the α-band range of cytochrome oxidase and they were identified as the difference spectrum of heme a when it has a high (a(H)) or low (a(L)) midpoint potential (E(m)) by comparing their occupancy during redox titrations carried out when the membrane potential (ΔΨ) was dissipated with a protonophore to that predicted by the neoclassical model of redox cooperativity. The difference spectrum of a(L) has a maximum at 605nm whereas the spectrum of a(H) has a maximum at 602nm. The ΔΨ-dependent shift in the E(m) of a(H) was too great to be accounted for by electron transfer from cytochrome c to heme a against ΔΨ but was consistent with a model in which a(H) is formed after proton uptake against ΔΨ suggesting that the spectral changes are the result of protonation. A stochastic simulation was implemented to model oxidation states, proton uptake and E(m) changes during redox titrations. The redox anti-cooperativity between heme a and heme a(3), and proton binding, could be simulated with a model where the pump proton interacted with heme a and the substrate proton interacted with heme a(3) with anti-cooperativity between proton binding sites, but not with a single proton binding site coupled to both hemes.

Oxidation and reduction of cytochrome oxidase in the neonatal brain observed by in vivo near-infrared spectroscopy.

Near-infrared spectroscopy was used to determine the relationship between the redox state of mitochondrial cytochrome oxidase CuA and haemoglobin oxygenation in the isoflurane-anaesthetized neonatal pig brain. Adding 7% CO2 to the inspired gases increased the total haemoglobin concentration by 8 microM and oxidized CuA by 0.2 microM. Decreasing the inspired oxygen fraction to zero for 90 s dropped the oxyhaemoglobin concentration by 27 microM and reduced CuA by 1.8 microM. However, no change in the CuA redox state was observed until oxyhaemoglobin had decreased by more than 10 microM. The response of the CuA redox state to these stimuli was very similar following 80% replacement of the haemoglobin by a perfluorocarbon blood substitute; this demonstrates that the results in the normal haematocrit were not a spectral artefact due to the high haemoglobin/cytochrome oxidase ratio. We conclude that the large reductions in the CuA redox state during anoxia are caused by a decrease in the rate of oxygen delivery to the cytochrome oxidase oxygen binding site; the small oxidations, however, are likely to reflect the effects of metabolic changes on the redox state of CuA, rather than increases in the rate of oxygen delivery.

Oxygen dependency of cerebral oxidative phosphorylation in newborn piglets.

Changes in hemoglobin oxygenation and oxidation state of the CuA centre of cytochrome oxidase were measured with full spectral near infrared spectroscopy simultaneously with phosphorus metabolites using nuclear magnetic resonance 31P spectroscopy at high time resolution (10 seconds) during transient anoxia (FiO2 = 0.0 for 105 seconds) in the newborn piglet brain. During the onset of anoxia, there was no change in either phosphocreatine (PCr) concentration or the oxidation state of the CuA centre of cytochrome oxidase until there was a substantial fall in cerebral hemoglobin oxygenation, at which point the CuA centre reduced simultaneously with the decline in PCr. At a later time during the anoxia, intracellular pH decreased rapidly, consistent with a fall in cerebral metabolic rate for O2 and reduced flux through the tricarboxylic acid cycle. The simultaneous reduction of CuA and decline in PCr can be explained in terms of the effects of the falling mitochondrial electrochemical potential. From these observations, it is concluded that, at normoxia, oxidative phosphorylation and the oxidation state of the components of the electron transport chain are independent of cerebral oxygenation and that the reduction in the CuA signal occurs when oxygen tension limits the capacity of oxidative phosphorylation to maintain the phosphorylation potential.

Use of mitochondrial inhibitors to demonstrate that cytochrome oxidase near-infrared spectroscopy can measure mitochondrial dysfunction noninvasively in the brain.

The use of near-infrared spectroscopy to measure noninvasively changes in the redox state of cerebral cytochrome oxidase in vivo is controversial. We therefore tested these measurements using a multiwavelength detector in the neonatal pig brain. Exchange transfusion with perfluorocarbons revealed that the spectrum of cytochrome oxidase in the near-infrared was identical in the neonatal pig, the adult rat, and in the purified enzyme. Under normoxic conditions, the neonatal pig brain contained 15 micromol/L deoxyhemoglobin, 29 micromol/L oxyhemoglobin, and 1.2 micromol/L oxidized cytochrome oxidase. The mitochondrial inhibitor cyanide was used to determine whether redox changes in cytochrome oxidase could be detected in the presence of the larger cerebral hemoglobin concentration. Addition of cyanide induced full reduction of cytochrome oxidase in both blooded and bloodless animals. In the blooded animals, subsequent anoxia caused large changes in hemoglobin oxygenation and concentration but did not affect the cytochrome oxidase near-infrared signal. Simultaneous blood oxygenation level-dependent magnetic resonance imaging measurements showed a good correlation with near-infrared measurements of deoxyhemoglobin concentration. Possible interference in the near-infrared measurements from light scattering changes was discounted by simultaneous measurements of the optical pathlength using the cerebral water absorbance as a standard chromophore. We conclude that, under these conditions, near-infrared spectroscopy can accurately measure changes in the cerebral cytochrome oxidase redox state.

Hemodynamic and metabolic responses to moderate asphyxia in brain and skeletal muscle of late-gestation fetal sheep.

The purpose of this study was to investigate metabolic and hemodynamic responses in two fetal tissues, hindlimb muscle and brain, to an episode of acute moderate asphyxia. Near-infrared spectroscopy was used to measure changes in total hemoglobin concentration ([tHb]) and the redox state of cytochrome oxidase (COX) simultaneously in the brain and hindlimb of near-term unanesthetized fetal sheep in utero. Oxygen delivery (DO(2)) to, and consumption (VO(2)) by, each tissue was derived from the arteriovenous difference in oxygen content and blood flow, measured by implanted flow probes. One hour of moderate asphyxia (n = 11), caused by occlusion of the maternal common internal iliac artery, led to a significant fall in DO(2) to both tissues and to a significant drop in VO(2) by the head. This was associated with an initial fall in redox state COX in the leg but an increase in the brain. [tHb], and therefore blood volume, fell in the leg and increased in the brain. These data suggest the presence of a fetal metabolic response to hypoxia, which, in the brain, occurs rapidly and could be neuroprotective.

Precise measurement of cerebral blood flow in newborn piglets from the bolus passage of indocyanine green.

Indocyanine green (ICG) is a near-infrared dye that has the potential to be used as a tracer for the minimally invasive measurement of cerebral blood flow (CBF). In order to examine the technique, the arterial and cerebral concentrations of ICG were measured in newborn piglets during the bolus passage of ICG at normocapnia and two levels of mild hypercapnia. The results were analysed by applying the Fick principle in both integral and differential forms using a linear regression technique to improve the precision of calculated values of CBF. It was found that the integral method, which has been used previously, is particularly sensitive to errors in the time registration between the arterial and tissue signals whereas the differential method is less so. In addition, the differential method allows the venous outflow to be calculated which gives further information on the state of the capillary bed. CBF was 39.7 +/- 4.6 ml 100 g(-1) min(-1) at an arterial carbon dioxide tension (PaCO2) of 33.0+/-2.2 mmHg and increased to 53.7+/-9.1 and 75.4+/-15.2 ml 100 g(-1) min(-1) at a PaCO2 of 42.1 +/- 2.6 and 54.2 +/- 3.1 mmHg respectively (mean +/- SD, n = 7). There was no significant change in cerebral metabolic rate for oxygen, validating the value of blood flow to an arbitrary scaling factor. When the inspired CO2 fraction was returned to zero, calculated CBF returned to baseline with a variation of 7% of the mean, indicating that this technique is highly precise.

Measurement of the oxidation state of mitochondrial cytochrome c from the neocortex of the mammalian brain.

Diffuse optical remission spectra from the mammalian neocortex at visible wavelengths contain spectral features originating from the mitochondria. A new algorithm is presented, based on analytically relating the first differential of the attenuation spectrum to the first differential of the chromophore spectra, that can separate and calculate the oxidation state of cytochrome c as well as the absolute concentration and saturation of hemoglobin. The algorithm is validated in phantoms and then tested on the neocortex of the rat during an anoxic challenge. Implementation of the algorithm will provide detailed information of mitochondrial oxygenation and mitochondrial function in physiological studies of the mammalian brain.

Starting young? Children's experiences of trying smoking during pre-adolescence.

Although the risks smoking poses to health are now well known, many young people continue to take up the habit. While numerous cross-sectional studies of adolescents have identified correlates of smoking initiation, much less prospective, longitudinal research has been conducted with young children to gather their accounts of early experiences of smoking, and this study fills that significant gap. Quantitative and qualitative data, collected using questionnaires, interviews and focus groups, are presented from the pre-adolescent phase of the Liverpool Longitudinal Study of Smoking. By age 11, 27% of the cohort had tried smoking, 13% had smoked repeatedly and 3% were smoking regularly. Rates of experimentation increased over time. Qualitative data revealed that curiosity and the role of peers were central to children's accounts of early smoking. By pre-adolescence, children are at different stages in their smoking careers, therefore interventions must be targeted to their varied experiences. Current prevention strategies often focus on restricting access to cigarettes, but a broad range of intervention measures is required which take account of the multifactorial nature of smoking onset. To be effective, policies that aim to prevent smoking must be grounded in children's lived experiences.

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

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.

Oxygen dependency and precision of cytochrome oxidase signal from full spectral NIRS of the piglet brain.

Oxidation changes of the copper A (Cu(A)) center of cytochrome oxidase in the brain were measured during brief anoxic swings at both normocapnia and hypercapnia (arterial PCO(2) approximately 55 mmHg). Hypercapnia increased total hemoglobin from 37.5 +/- 9.1 to 50.8 +/- 12.9 micromol/l (means +/- SD; n = 7), increased mean cerebral saturation (Smc(O(2))) from 65 +/- 4 to 77 +/- 3%, and oxidized Cu(A) by 0.43 +/- 0.23 micromol/l. During the onset of anoxia, there were no significant changes in the Cu(A) oxidation state until Smc(O(2)) had fallen to 43 +/- 5 and 21 +/- 6% at normocapnia and hypercapnia, respectively, and the maximum reduction during anoxia was not significantly different at hypercapnia (1.49 +/- 0.40 micromol/l) compared with normocapnia (1.53 +/- 0.44 micromol/l). Residuals of the least squares fitting algorithm used to convert near-infrared spectra to concentrations are presented and shown to be small compared with the component of attenuation attributed to the Cu(A) signal. From these observations, we conclude that there is minimal interference between the hemoglobin and Cu(A) signals in this model, the Cu(A) oxidation state is independent of cerebral oxygenation at normoxia, and the oxidation after hypercapnia is not the result of increased cerebral oxygenation.

Fluorescence Photodiagnostics and Photobleaching Studies of Cancerous Lesions using Ratio Imaging and Spectroscopic Techniques.

Topical or systemic administration of 5-aminolaevulinic acid results in biosynthesis of the photosensitiser protoporphyrin IX (PpIX) with some selectivity for malignant lesions. Excitation near 400 nm excites both intrinsic green tissue autofluorescence and red fluorescence from PpIX which may be exploited for the optical diagnosis of malignant and premalignant disease. In this work the utility of a cooled 12-bit single chip charge-coupled device (CCD) colour camera was investigated for photodiagnostic fluorescence ratio imaging. The red to green fluorescence intensity ratios were calculated for each pixel in real-time and fluorescence ratio images were displayed typically at a rate of 2 frames/s. Laboratory tests of fluorescence ratio imaging showed good contrast enhancement between control tissues and tissue phantoms and those containing porphyrin photosensitisers. In preliminary clinical tests, a clear demarcation between neoplastic/cancerous lesions and adjacent normal tissue was demonstrated. The extent of PpIX photobleaching during photodynamic therapy was also investigated using fluorescence ratio imaging.

Utility and limitations of near-infrared spectroscopy during cardiopulmonary bypass in a piglet model.

Near-infrared spectroscopy assessment of cytochrome oxygenation could be a valuable technique to monitor cerebral intraneuronal oxygen delivery during cardiopulmonary bypass. However, the validity of the cytochrome signal has been questioned as it could easily be overwhelmed by the Hb signal. Five- to six-week-old control piglets (n = 5) underwent cardiopulmonary bypass at 37 degrees C. Study animals (n = 10) received 100 mg/kg of sodium cyanide to uncouple cytochrome from HB: Hematocrit was then decreased in steps of 5% from 35 to 5% with crystalloid hemodilution. In study piglets, the initiation of cardiopulmonary bypass was associated with oxygenated Hb increasing from 0 to 62.9 +/- 25.6 microM times the differential path-length factor, and oxidized cytochrome a,a3 increasing to 1.9 +/- 1.8 microM times the differential path-length factor. Cyanide caused oxygenated Hb to increase further to 132.3 +/- 48.9 microM times the differential path-length factor, and oxidized cytochrome c decreased to -7.0 +/- 2.6 microM times the differential path-length factor as anticipated, confirming uncoupling of electron transport. However, hemodilution subsequently resulted in linear decreases in oxidized cytochrome a,a3 (F = 8.57, p < 0.001) suggesting important cross-talk between the Hb and cytochrome signals as cytochrome is only intracellular. In control piglets, tissue oxygenation index showed a positive correlation with pump flow (r = 0.986, p = 0.013). The cytochrome signal as presently measured by near-infrared spectroscopy is highly dependent on hematocrit.