Estimation of lactate threshold by near infrared spectroscopy.
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Adult↗
Biomedical subjects
C E Cooper
Publications and source records attributed to C E Cooper.
In vivo measurements of mitochondrial function and cell death following hypoxic/ischaemic damage to the new-born brain.
Critically impaired gas exchange to the brain due to decreased oxygen (hypoxia) or reduced blood flow (ischaemia) is a major cause of brain injury in the perinatal period. There is an accumulating body of evidence suggesting that the irreversible cellular damage in the neonatal brain that occurs subsequent to an hypoxic/ischaemic insult is at the level of the mitochondria. Much of this evidence has been obtained by novel non-invasive measurements of mitochondrial function in vivo. This review focuses on four techniques: near-infrared spectroscopy, magnetic resonance spectroscopy, magnetic resonance imaging and electron paramagnetic resonance spectroscopy. The advantages and disadvantages of these in vivo methods are described in patients and animal models. The picture that emerges is of a slow (1-2 day) energy failure, occurring at the level of the brain mitochondria and leading to a primarily apoptotic cell death. Moderate post-insult hypothermia prevents this damage by an unknown mechanism. It is stressed that isolated cell studies alone are not sufficient to understand the processes occurring at the biochemical and physiological levels. The use of the non-invasive techniques described to understand the biochemistry occurring in vivo is therefore an invaluable aid in integrating cellular and organismal studies of the role of mitochondria in cell death.
Apoptosis↗
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.
Animals↗
Noninvasive assessment of changes in cytochrome-c oxidase oxidation in human subjects during visual stimulation.
In this study the authors used a whole-spectrum near-infrared spectroscopy approach to noninvasively assess changes in hemoglobin oxygenation and cytochrome-c oxidase redox state (Cyt-Ox) in the occipital cortex during visual stimulation. The system uses a white light source (halogen lamp). The light reflected from the subject's head is spectrally resolved by a spectrograph and dispersed on a cooled charge-coupled device camera. The authors showed the following using this approach: (1) Changes in cerebral hemoglobin oxygenation (increase in concentration of oxygenated hemoglobin, decrease in concentration of deoxygenated hemoglobin) in the human occipital cortex during visual stimulation can be assessed quantitatively. (2) The spectral changes during functional activation cannot be completely explained by changes in hemoglobin oxygenation solely; Cyt-Ox has to be included in the analysis. Only if Cyt-Ox is considered can the spectral changes in response to increased brain activity be explained. (3) Cytochrome-c oxidase in the occipital cortex of human subjects is transiently oxidized during visual stimulation. This allows us to measure vascular and intracellular energy status simultaneously.
Adult↗
A causative role for redox cycling of myoglobin and its inhibition by alkalinization in the pathogenesis and treatment of rhabdomyolysis-induced renal failure.
Muscle injury (rhabdomyolysis) and subsequent deposition of myoglobin in the kidney causes renal vasoconstriction and renal failure. We tested the hypothesis that myoglobin induces oxidant injury to the kidney and the formation of F2-isoprostanes, potent renal vasoconstrictors formed during lipid peroxidation. In low density lipoprotein (LDL), myoglobin induced a 30-fold increase in the formation of F2-isoprostanes by a mechanism involving redox cycling between ferric and ferryl forms of myoglobin. In an animal model of rhabdomyolysis, urinary excretion of F2-isoprostanes increased by 7.3-fold compared with controls. Administration of alkali, a treatment for rhabdomyolysis, improved renal function and significantly reduced the urinary excretion of F2-isoprostanes by approximately 80%. EPR and UV spectroscopy demonstrated that myoglobin was deposited in the kidneys as the redox competent ferric myoglobin and that it's concentration was not decreased by alkalinization. Kinetic studies demonstrated that the reactivity of ferryl myoglobin, which is responsible for inducing lipid peroxidation, is markedly attenuated at alkaline pH. This was further supported by demonstrating that myoglobin-induced oxidation of LDL was inhibited at alkaline pH. These data strongly support a causative role for oxidative injury in the renal failure of rhabdomyolysis and suggest that the protective effect of alkalinization may be attributed to inhibition of myoglobin-induced lipid peroxidation.
Animals↗
Interaction of peroxynitrite with mitochondrial cytochrome oxidase. Catalytic production of nitric oxide and irreversible inhibition of enzyme activity.
Purified mitochondrial cytochrome c oxidase catalyzes the conversion of peroxynitrite to nitric oxide (NO). This reaction is cyanide-sensitive, indicating that the binuclear heme a3/CuB center is the catalytic site. NO production causes a reversible inhibition of turnover, characterized by formation of the cytochrome a3 nitrosyl complex. In addition, peroxynitrite causes irreversible inhibition of cytochrome oxidase, characterized by a decreased Vmax and a raised Km for oxygen. Under these conditions, the redox state of cytochrome a is elevated, indicating inhibition of electron transfer and/or oxygen reduction reactions subsequent to this center. The lipid bilayer is no barrier to these peroxynitrite effects, as NO production and irreversible enzyme inhibition were also observed in cytochrome oxidase proteoliposomes. Addition of 50 microM peroxynitrite to 10 microM fully oxidized enzyme induced spectral changes characteristic of the formation of ferryl cytochrome a3, partial reduction of cytochrome a, and irreversible damage to the CuA site. Higher concentrations of peroxynitrite (250 microM) cause heme degradation. In the fully reduced enzyme, peroxynitrite causes a red shift in the optical spectrum of both cytochromes a and a3, resulting in a symmetrical peak in the visible region. Therefore, peroxynitrite can both modify and degrade the metal centers of cytochrome oxidase.
Animals↗
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.
Animals↗
The relationship of oxygen delivery to absolute haemoglobin oxygenation and mitochondrial cytochrome oxidase redox state in the adult brain: a near-infrared spectroscopy study.
Near-infrared spectroscopy was used to determine the effect of changes in the rate of oxygen delivery to the adult rat brain on the absolute concentrations of oxyhaemoglobin, deoxyhaemoglobin and the redox state of the CuA centre in mitochondrial cytochrome oxidase. The cytochrome oxidase detection algorithm was determined to be robust to large changes in haemoglobin oxygenation and concentration. By assuming complete haemoglobin deoxygenation and CuA reduction following mechanical ventilation on 100% N2O, the absolute concentration of oxyhaemoglobin (35 microM), deoxyhaemoglobin (27 microM) and the redox state of CuA (82% oxidized) were calculated in the normal adult brain. The mean arterial blood pressure was decreased by exsanguination. When the pressure reached 100 mmHg, haemoglobin oxygenation started to fall, but the total haemoglobin concentration and oxidized CuA levels only fell when cerebral blood volume autoregulation mechanisms failed at 50 mmHg. Haemoglobin oxygenation fell linearly with decreases in the rate of oxygen delivery to the brain, but the oxidized CuA concentration did not start to fall until this rate was 50% of normal. The results suggest that the brain maintains more than adequate oxygen delivery to mitochondria and that near-infrared spectroscopy may be a good measure of oxygen insufficiency in vivo.
Animals↗
Reactions of nitric oxide with mitochondrial cytochrome c: a novel mechanism for the formation of nitroxyl anion and peroxynitrite.
The aerobic reactions of nitric oxide with cytochrome c were analysed. Nitric oxide (NO) reacts with ferrocytochrome c at a rate of 200 M-1 s-1 to form ferricytochrome c and nitroxyl anion (NO-). Ferricytochrome c was detected by optical spectroscopy; NO- was detected by trapping with metmyoglobin (Mb3+) to form the EPR-detectable Mb-nitrosyl complex, and by the formation of dimers in yeast ferrocytochrome c via cross-linking of the free cysteine residue. The NO- formed subsequently reacted with oxygen to form peroxynitrite, as measured by the oxidation of dihydrorhodamine 123. NO binds to ferricytochrome c to form the ferricytochrome c-NO complex. The on-rate for this reaction is 1.3+/-0.4x10(3) M-1.s-1, and the off-rate is 0.087+/-0.054 s-1. The dissociation constant (Kd) of the complex is 22+/-7 microM. These reactions of NO with cytochrome c are likely to be relevant to mitochondrial metabolism of NO. Ferricytochrome c can act as a reversible sink for excess NO in the mitochondria. The reduction of NO to NO- by ferrocytochrome c may play a role in the irreversible inhibition of mitochondrial oxygen consumption by peroxynitrite. It is generally assumed that peroxynitrite would be formed in mitochondria via the reaction of NO with superoxide. The finding that NO- is formed from the reaction of NO and ferrocytochrome c provides a means of producing peroxynitrite in the absence of superoxide, via the reaction of NO- with oxygen.
Cytochrome c Group↗
A common mechanism for the interaction of nitric oxide with the oxidized binuclear centre and oxygen intermediates of cytochrome c oxidase.
The reactions of nitric oxide (NO) with fully oxidized cytochrome c oxidase (O) and the intermediates P and F have been investigated by optical spectroscopy, using both static and kinetic methods. The reaction of NO with O leads to a rapid (approximately 100 s-1) electron ejection from the binuclear center to cytochrome a and CuA. The reaction with the intermediates P and F leads to the depletion of these species in slower reactions, yielding the fully oxidized enzyme. The fastest optical change, however, takes place within the dead time of the stopped-flow apparatus (approximately 1 ms), and corresponds to the formation of the F intermediate (580 nm) upon reaction of NO with a species that we postulate is at the peroxide oxidation level. This species can be formulated as either Fe5+ = O CuB2+ or Fe4+ = O CuB3+, and it is spectrally distinct from the P intermediate (607 nm). All of these reactions have been rationalized through a mechanism in which NO reacts with CuB2+, generating the nitrosonium species CuB1+ NO+, which upon hydration yields nitrous acid and CuB1+. This is followed by redox equilibration of CuB with Fea/CuA or Fea3 (in which Fea and Fea3 are the iron centers of cytochromes a and a3, respectively). In agreement with this hypothesis, our results indicate that nitrite is rapidly formed within the binuclear center following the addition of NO to the three species tested (O, P, and F). This work suggests that nitrosylation at CuB2+ instead of at Fea32+ is a key event in the fast inhibition of cytochrome c oxidase by NO.
Electron Transport Complex IV↗
MRI measurements of cerebral deoxyhaemoglobin concentration [dHb]--correlation with near infrared spectroscopy (NIRS).
Changes in physiological parameters such as cerebral blood flow, cerebral blood volume, oxygen extraction, and the size and distribution of cerebral blood vessels, result in changes in the local concentration of deoxyhaemoglobin ([dHb]). The purpose of this study was to quantitatively investigate the dependence of the R2* relaxation rate upon the [dHb] per voxel. Five neonatal piglets were studied in a 7 T/20 cm bore magnet. MRI was conducted using a 2.5 cm diameter surface coil placed over the parietal lobes. Four progressively T2*-weighted images were acquired, allowing the absolute quantitation of R2*. Simultaneous near infrared spectroscopy (NIRS) measurements were made from an area encompassing the MR imaging slice, and allowed the absolute quantitation of [dHb]. The arterial oxygen saturation (SaO2) of the piglet was lowered stepwise by decreasing the fractional inspired oxygen concentration (FiO2), which precipitated a change in [dHb]. NIRS and MRI measurements were made at each FiO2 step. The results demonstrate an extremely strong, linear relationship between R2* as determined by MRI and [dHb], as measured by NIRS. Whereas NIRS can only give us a global measure of [dHb], the results suggest the future use of MRI in producing high resolution relaxation rate maps related to the [dHb] distribution of the brain.
Animals↗
Peroxynitrite reacts with methemoglobin to generate globin-bound free radical species. Implications for vascular injury.
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Blood Vessels↗
Measurement of cerebral blood flow during cardiopulmonary bypass with near-infrared spectroscopy.
OBJECTIVE: A novel noninvasive method for repeatedly measuring cerebral blood flow during cardiopulmonary bypass by near-infrared spectroscopy is described. The reproducibility of the method is investigated and a comparison is made with an established technique. METHODS AND RESULTS: The method is derived from the Fick principle and uses indocyanine green dye, injected into the bypass circuit, as an intravascular tracer. Cerebral blood flow was measured in nine children undergoing cardiopulmonary bypass on a total of 49 occasions. Results from this study suggest that an integrating period of 4 seconds provided a consistent measurement of global cerebral blood flow. The values obtained ranged from 3.2 to 32.4 (median 15.9) ml.100 gm-1.min-1. In an additional 10 children in whom repeated measurements were made, the coefficient of variation was 11% +/- 7% (mean +/- standard deviation). In a further study, the method was compared with microsphere injection in five piglets undergoing cardiopulmonary bypass. The comparison within each animal with the linear least squares method gave values for R2 in the range 0.91 to 0.99. The gradients of the fits ranged from 0.5 to 1.8 (median 1.0). The mean difference between the two techniques was 5.7 ml.100 gm-1.min-1 or 7%. The coefficient of variation for the piglets was 14% +/- 9% (mean +/- standard deviation). CONCLUSIONS: Indocyanine green and near-infrared spectroscopy allow frequent and repeated measurements of cerebral blood flow during cardiopulmonary bypass. The measurements are reproducible and accurately reflect changes in cerebral blood flow. It may be widely applicable both in research and clinical practice.
Animals↗
Reactivity of nitric oxide with cytochrome c oxidase: interactions with the binuclear centre and mechanism of inhibition.
Nitric oxide (NO) has recently been recognized as an important biological mediator that inhibits respiration at cytochrome c oxidase (CcO). This inhibition is reversible and shows competition with oxygen, the Ki being lower at low oxygen concentrations. Although the species that binds NO in turnover has been suggested to contain a partially reduced binuclear center, the exact mechanism of the inhibition is not clear. Recently, rapid (ms) redox reactions of NO with the binuclear center have been reported, e.g., the ejection of an electron to cytochrome a and the depletion of the intermediates P and F. These observations have been rationalized within a scheme in which NO reacts with oxidized CuB leading to the reduction of this metal center and formation of nitrite in a very fast reaction. Electron migration from CuB to other redox sites within the enzyme is proposed to explain the optical transitions observed. The relevance of these reactions to the inhibition of CcO and metabolism of NO are discussed.
Animals↗
Reactions of cross-linked ferric haemoglobins with hydrogen peroxide.
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Cross-Linking Reagents↗
Nitric oxide as an effector and a substrate for cytochrome c oxidase.
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Electron Transport Complex IV↗
Experimental and theoretical comparison of NIR spectroscopy measurements of cerebral hemoglobin changes.
Two near-infrared spectroscopy (NIRS) methods are available for measuring changes (Delta) in total cerebral hemoglobin concentration (CHC): 1) a continuous measurement of the changes in total hemoglobin concentration (Delta[Hb]tot) and 2) the difference between two absolute measurements of CHC, each derived from a small, controlled change in inspired O2 fraction. This paper investigates the internal consistency of these two methods by using an experimental and theoretical comparison. NIRS was used to measure [Hb]tot in five newborn piglets before and after a change in arterial PCO2. Delta[Hb]tot demonstrated a low coefficient of variation of 2.8 +/- 2.8 (SD) % which allowed changes in CO2-cerebral blood volume reactivity to be clearly discriminated. However, a high coefficient of variation of 22.8 +/- 3.5% on the DeltaCHC measurements obscured any CO2 reactivity changes. A theoretical analysis demonstrates the effects of optical pathlength, background absorption, scatter, and blood vessel diameter on both methods. For more accurate monitoring of CHC, individual measurements of optical pathlength and more accurate pulse oximetry are required.
Animals↗
Nitric oxide ejects electrons from the binuclear centre of cytochrome c oxidase by reacting with oxidised copper: a general mechanism for the interaction of copper proteins with nitric oxide?
Small increases in NO concentration can inhibit mitochondrial oxygen consumption by reacting at the binuclear haem a3/CuB oxygen reduction site of cytochrome c oxidase. Here we demonstrate that under normal turnover conditions NO reacts initially with the oxidised CuB rather than the haem a3. We propose that hydration of an initial Cu+/NO+ complex forms nitrite, a proton and CuB+; the latter ejects an electron from the binuclear centre and results in the observed (100 s(-1)) reduction of other electron transfer centres in the enzyme (haem a and CuA). These reactions may have implications for the interactions of NO with other copper proteins.
Binding Sites↗