Rapid reduction of intestinal cytochrome a,a3 during lethal endotoxemia.

In vivo near-infrared spectrophotometry was used to determine whether lethal endotoxemia impairs small intestinal oxidative phosphorylation as reflected by the redox state of mitochondrial cytochrome a,a3 (AA3). Adult male Sprague-Dawley rats were anesthetized with 2.1% isoflurane in 30% O2:70% N2O, and the small intestine was partially exteriorized for spectrophotometric monitoring (OMNI-3). By 5 min after an iv bolus of Escherichia coli endotoxin (40 mg/kg, LD90, n = 7) a significant shift toward reduction in intestinal AA3 had occurred in association with hypotension and a marked fall in both superior mesenteric artery blood flow (SMAF) and cardiac output. In a separate group (n = 7) SMAF was kept at the baseline level by periodic infusions of donor rat plasma begun 1 min after endotoxin injection, and the reduction in AA3 was again found despite the fluid loading intervention which successfully maintained not only organ blood flow, but also cardiac output and mean arterial pressure in their normal ranges. Further experiments (n = 34) measuring SM vascular bed oxygen consumption indicated that intestinal VO2 remained unchanged during early endotoxemia. These findings suggest a rapid impairment of oxidative phosphorylation by endotoxin which seems to occur through direct (and/or indirect) toxic cellular effects rather than through impaired tissue perfusion.

Near-infrared monitoring of cerebral oxygen sufficiency. I. Spectra of cytochrome c oxidase.

Near-infrared (NIR) difference spectra were obtained for oxidized cytochrome c oxidase of isolated mitochondria in vitro and of cerebral tissue in situ observed through scalp and skull. The broad peaks of maximal absorption observed in both were not inconsistent with the customary assignment of an 830 nm peak. However, the ratios of the intensity of the NIR band to that of the visible peak (605 nm), which we found to be identical for in-vitro and in-situ spectra, were consistently and significantly higher than those of the various purified enzyme preparations reported in the literature. In addition the half-band widths of our in-vitro and in-situ preparations were narrower. Haemoglobin spectra in the NIR obtained in clear and in highly light-scattering media showed almost total absence of band distortion in this spectral region, suggesting that the differences observed are not due to scattering effects. Anoxia and the specific oxidase inhibitors, cyanide and carbon monoxide, caused the expected disappearance of the band in both the mitochondria in vitro and the cerebrum in situ. The 830 nm band observed in intact, well-oxygenated animal preparations was therefore identified with the NIR absorption band of oxidized cytochrome c oxidase, notwithstanding the differences with the observations on purified preparations. This points to the possibility of developing instrumentation and techniques for the non-invasive monitoring of the redox state of cytochrome c oxidase as an index to cerebral oxygen sufficiency, i.e. adequate delivery and utilization of oxygen to and by brain tissue.

A near-infrared spectrophotometric method for studying brain O2 sufficiency in man during +Gz acceleration.

A technique for the noninvasive monitoring of cerebral oxygen status was evaluated on volunteer subjects on the USAF School of Aerospace Medicine centrifuge. By using multiwavelength near-infrared spectrophotometry, the instrumentation measured changes in the quantities of reduced and oxygenated hemoglobin (and their sum, an indicator of cerebral blood volume), and the quantity of oxidized cytochrome c oxidase within the forebrain. Tests used acceleration of up to 9 G with onset rates from 0.1 to 5.0 G.s-1, anti-G suits and straining maneuvers, and hyperoxic and hypoxic breathing mixtures. In general, +Gz acceleration produced a fall in blood volume within the cerebral microcirculation with a relative increase in the content of reduced hemoglobin and a tendency towards reduction of cytochrome c oxidase. These findings are discussed in relation to accepted changes in arterial blood pressure, cerebral blood flow, and arterial oxygen saturation caused by acceleration exposure.

Skeletal muscle oxygen availability during respiratory acid-base disturbances in cats.

Respiratory acid-base disorders elicit physiological responses that alter O2 delivery to various tissues. We have used a near infrared (NIR) optical technique to monitor cytochrome a,a3 oxidation state, tissue O2 store (relative hemoglobin plus myoglobin oxygenation), and regional blood volume in intact resting skeletal muscle during respiratory acid-base disturbances in anesthetized cats. Hypercapnic acidosis and hypocapnic alkalosis were produced in separate groups of animals by ventilation with increasing concentrations of CO2 (n = 13) or hyperventilation (n = 8). Respiratory acidosis decreased oxygen availability to hindlimb muscle while respiratory alkalosis did not change tissue oxygenation. Inspired CO2 progressively decreased muscle blood volume, cytochrome a,a3 oxidation level, and muscle oxygen store. These optical responses were greatly attenuated both by pre-treatment with bretylium and by hemorrhagic hypotension, suggesting mediation through sympathetic vasoconstriction. Metabolic acidosis, produced by intravenous HCl infusion (n = 8), did not reproduce the hindlimb optical responses mediated by CO2. These experiments demonstrate that hypercapnic acidosis significantly decreases oxygen supply to resting skeletal muscle in the anesthetized cat, probably via neuroregulatory responses to CO2 which do not depend on changes in arterial [H+] in the tested pH range.

Differences in brain cytochrome responses to carbon monoxide and cyanide in vivo.

Cytochrome oxidation-reduction responses to two mitochondrial electron transport inhibitors, carbon monoxide (CO) and cyanide (CN), were studied in the intact brains of fluorocarbon-circulated rats. In vivo reflectance spectrophotometry indicated that cortical b-type cytochromes (564 nm) were highly resistant to reduction by CN in the presence of O2 but showed reduction responses to the administration of 1-5% CO in 90% O2. In contrast, cyanide-sensitive cytochromes aa3 (605 nm) and c + c1 (551 nm) did not increase their reduction levels during exposure to 5% CO in 90% O2. The in vivo CO-mediated b-cytochrome reduction responses did not occur after pretreatment with the cytochrome b inhibitor, antimycin A. Transmission spectrophotometry of superfused hemoglobin-free rat brain slices confirmed cortical b-type cytochromes to be CN-resistant in the presence of O2. Another cytochrome absorbing at 445 nm also was resistant to reduction by 1-mM cyanide in vitro, but it could be reduced anaerobically. The reduced 445-nm cytochrome bound CO in the presence of cyanide. We postulate that this CN-resistant CO binding component might account for in vivo cytochrome aa3-CO interactions and directly or indirectly modulate cytochrome b reduction responses to CO. In any event, the spectral data indicate different primary tissue target sites for CO and CN in living rat brain and also suggest different bioenergetic consequences of exposure to the two agents.

Near-infrared spectrophotometric monitoring of oxygen distribution to intact brain and skeletal muscle tissues.

During continuous near-infrared optical monitoring of brain cortex and hindlimb skeletal muscles, anesthetized, ventilated cats were exposed either to progressive alveolar hypoxia, or to acute hemorrhage followed in some cases by resuscitation. Hypoxia decreased cytochrome a,a3 oxidation state in muscle more than in brain, while tissue blood volume increased in brain and decreased in muscle. At a PaO2 of 25 torr, cytochrome a,a3 oxidation level in the brain was sufficient to support EEG activity, but the cytochrome a,a,3 oxidation state in resting, innervated hindlimb muscle was near zero. Hemorrhagic hypotension invariably decreased cytochrome a,a3 oxidation state and tissue blood volume more in muscle than in brain, and muscle cytochrome a,a3 was completely reduced at about a 25-ml/kg blood loss. These observations, supported by noninvasively measured changes in near-infrared absorption in the tissues during serial intravascular injections of indocyanine green dye, indicate that different cytochrome responses to hypoxia and oligemia in muscle vs. brain tissue are attributable to different regional circulatory adjustments.

Energy metabolism and in vivo cytochrome c oxidase redox relationships in hypoxic rat brain.

Rats were subjected to graded arterial hypoxia while we measured changes in the oxidation level of cytochrome c oxidase (cytochrome aa3) in the brain by a non-invasive, optical technique. The experiments were terminated at different arterial oxygen tensions (hypoxic levels) and the in vivo observations were compared with in vitro measured changes in metabolites known to reflect limitations in cellular aerobic energy production, e.g. glucose, pyruvate, lactate, phosphocreatine, ATP and ADP. Using absorption changes at 605 nm, in vivo cytochrome aa3 was 46% reduced in normoxia as determined by the range between the maximal oxidation level attained with animals breathing 85% O2 + 15% CO2 and maximal reduction with anoxia (100% N2). Hypoxia reduced cytochrome aa3 to levels of 52, 67, 76, and 84% at mean PaO2 values of 53, 39, 35 and 28 mm Hg, respectively. These increases in reduced cytochrome correlated significantly (r = 0.94) with cortical phosphocreatine depletion, lactate production, and increases in the lactate/pyruvate ratio. However, there were no significant changes in ATP or ADP. Rats did not survive below an FIO2 of 7% because of a precipitous fall in arterial blood pressure. Hypoxically-induced cerebral isoelectricity was coincident with a 50% increase in the cytochrome reduction level (to 73% of the total range defined above). Our results indicate that in vivo monitoring of the reduction level of cytochrome aa3 provides an early, continuous, and direct measure of intracellular oxygen insufficiency at levels which adversely affect aerobic energy production.

Carbon monoxide-cytochrome interactions in the brain of the fluorocarbon-perfused rat.

Reflectance spectrophotometry through the skull was used to investigate carbon monoxide (CO) binding by tissue hemoproteins in the brains of barbiturate-anesthetized Sprague-Dawley rats. After splenectomy and extensive perfluorotributylamine exchange transfusion, steady-state spectral scans were obtained in Soret and visible wave-length regions during O2 ventilation, during subsequent exposure to O2-enriched gases containing 1, 3, or 5% CO, and finally after N2 anoxia. These CO exposures were well-tolerated and electroencephalograph (EEG) activity continued to be present. Initial difference spectra were influenced by CO binding to residual hemoglobin, but spectral evidence of CO-mediated b-type cytochrome reduction was obtained in the visible region as CO concentration was increased to 3 or 5%. This was associated with Soret spectra compatible with formation of the reduced cytochrome a3-CO complex. Reduction of cytochrome a at 605 nm and cytochrome c + c1 at 550 nm was absent. These findings may indicate respiratory chain branching through b cytochromes, either to a separate a3-like oxidase independent of the classical cytochrome aa3 or to an unidentified alternative CO-sensitive oxidase.

Spectrophotometry of cerebral cytochrome a, a3 in bloodless rats.

Bloodless, anesthetized Sprague-Dawley rats produced by perfluorochemical exchange transfusion and splenectomy were used to obtain cerebral mitochondrial difference spectra directly through the skull by both reflectance and transmission spectrophotometry. Reflectance peaks were about one-third in amplitude compared to transmission peaks, however percentage reduction levels of cytochrome a, a3 in animals breathing 100% oxygen were comparable. Cytochrome a, a3 was 13-14% reduced under these conditions as determined by the ratio of the optical density difference at 605 minus 620 nm to the optical density difference at the same wavelength pair with maximal cytochrome a, a3 oxidation during respiration on 95% oxygen + 5% carbon dioxide at 3 and 4 atm. absolute pressure.