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The purpose of this study was to evaluate a new pharmacological strategy using a first-generation succinate prodrug, NV118, in peripheral blood mononuclear cells (PBMCs) obtained from subjects with carbon monoxide (CO) poisoning and healthy controls. We obtained human blood cells from subjects with CO poisoning and healthy control subjects. Intact PBMCs from subjects in the CO and Control group were analyzed with high-resolution respirometry measured in pmol O 2 per second per 10 -6 PBMCs. In addition to obtaining baseline respiration, NV118 (100 μM) was injected, and the same parameters of respiration were obtained for comparison in PBMCs. We measured mitochondrial dynamics with microscopy with the same conditions. We enrolled 37 patients (17 in the CO group and 20 in the Control group for comparison) in the study. PMBCs obtained from subjects in the CO group had overall significantly lower respiration compared with the Control group ( P < 0.0001). There was a significant increase in respiration with NV118, specifically with an increase in maximum respiration and respiration from complex II and complex IV ( P < 0.0001). The mitochondria in PBMCs demonstrated an overall increase in net movement compared with the Control group. Our results of this study suggest that the therapeutic compound, NV118, increases respiration at complex II and IV as well as restoration of mitochondrial movement in PBMCs obtained from subjects with CO poisoning. Mitochondrial-directed therapy offers a potential future strategy with further exploration in vivo.

Tissue P CO values have not been previously estimated under conditions where the blood carboxyhemoglobin % saturation ([COHb]) is at a normal level or increased. Tissue P CO values are not known for conditions when [COHb] is increased during CO therapy or during CO poisoning. Using a modified Krogh parallel capillary-tissue model, mean tissue P CO was calculated for when [COHb] was 1, 5, 10, and 15% saturation, relevant to CO therapy, and 20, 30, and 40% saturation, relevant to CO poisoning. Calculations were made for the time during which CO was being inhaled, after cessation of CO uptake, and for different O 2 extractions from blood flowing in the model capillary. The T 1/2 of relevant CO reactions was used in these calculations. When the [COHb] increased to 5 to 10% saturation, mean tissue P CO values increased to 500 to 1,100% of values when the [COHb] was 1% saturation. When the [COHb] increased to 20 to 40% saturation, mean tissue P CO values increased to 2,300 to 5,700% of the 1% saturation value. Results indicate the utility of the modified Krogh model in furthering understanding the physiology of determinants of tissue P CO and should facilitate future studies of in vivo CO binding to different extravascular heme proteins during CO therapy and during CO poisoning. NEW & NOTEWORTHY Tissue P CO levels resulting from carboxyhemoglobin concentrations achieved during CO therapy or during CO poisoning have not been previously estimated. Results published here show that at carboxyhemoglobin levels achieved during CO therapy there are 500 to 1,100% increases in mean tissue P CO values. With carboxyhemoglobin increases associated with toxic effects, there are 2,300 to 5,700% increases in the mean tissue P CO . These differences suggest a basis for understanding the therapeutic and toxic effects of CO.

To better understand factors that influence carbon monoxide (CO) washout rates, we utilized a multicompartment mathematical model to predict rates of CO uptake, distribution in vascular and extravascular (muscle vs. other soft tissue) compartments, and washout over a range of exposure and washout conditions with varied subject-specific parameters. We fitted this model to experimental data from 15 human subjects, for whom subject-specific parameters were known, multiple washout carboxyhemoglobin (COHb) levels were available, and CO exposure conditions were identical, to investigate the contributions of exposure conditions and individual variability to CO washout from blood. We found that CO washout from venous blood was biphasic and that postexposure times at which COHb samples were obtained significantly influenced the calculated CO half times (P < 0.0001). The first, more rapid, phase of CO washout from the blood reflected the loss of CO to the expired air and to a slow uptake by the muscle compartment, whereas the second, slower washout phase was attributable to CO flow from the muscle compartment back to the blood and removal from blood via the expired air. When the model was used to predict the effects of varying exposure conditions for these subjects, the CO exposure duration, concentration, peak COHb levels, and subject-specific parameters each influenced washout half times. Blood volume divided by ventilation correlated better with half-time predictions than did cardiac output, muscle mass, or ventilation, but it explained only approximately 50% of half-time variability. Thus exposure conditions, COHb sampling times, and individual parameters should be considered when estimating CO washout rates for poisoning victims.

Studies were conducted with rats to investigate whether platelet activating factor (PAF) and nitric oxide (*NO)-derived oxidants played roles in the initial adherence of neutrophils to vasculature in the brain after carbon monoxide (CO) poisoning. Before CO poisoning, rats were treated with the competitive PAF receptor antagonist WEB-2170 or with the peroxynitrite scavenger selenomethionine. Both agents caused significantly lower concentrations of myeloperoxidase in the brain after poisoning, indicating fewer sequestered neutrophils. Similarly, both agents reduced the concentration of nitrotyrosine, indicating less oxidative stress due to *NO-derived oxidants. There were no alterations in whole brain homogenate PAF concentration measured by immunoassay and bioassay, nor were there changes in phosphatidylcholine concentration. Immunohistochemical imaging showed PAF to be more heavily localized within perivascular zones after CO poisoning. Neutrophils colocalized with both PAF and nitrotyrosine in brains of rats killed immediately after CO poisoning. We conclude that qualitative changes in brain PAF are responsible for neutrophil adherence immediately after CO poisoning and that activated neutrophils trigger the initial rise in brain nitrotyrosine. Persistent PAF-mediated neutrophil adherence required production of *NO-derived oxidants because when oxidants were scavenged, neutrophil adherence was not maintained.

We have investigated at ambient temperatures (Tam) of 25 and 5 degrees C the effects of ambient hypoxia (Hxam; fractional inspired O2 = 0.14) and hypercapnia (fractional inspired CO2 = 0.04) on ventilation (V), O2 uptake (VO2), and colonic temperature (Tc) in 12 conscious rats before and after carotid body denervation (CBD). The rats were concomitantly exposed to CO hypoxia (HxCO; fractional inspired CO = 0.03-0.05%), which decreases arterial O2 saturation by approximately 25-40%. The results demonstrate the following. 1) At Tam of 5 degrees C, in both intact and CBD rats, V/VO2 is larger when Hxam or CO2 is associated with HxCO than with normoxia. At Tam of 25 degrees C, this is also the case except for CO2 in CBD rats. 2) At Tam of 5 degrees C, the changes in VO2 and Tc seem to result from additive effects of the separate changes induced by Hxam, CO2, and HxCO. It is concluded that, in conscious rats, central hypoxia does not depress respiratory activity. On the contrary, particularly when VO2 is augmented during a cold stress, both V/VO2 during HxCO and the ventilatory responses to Hxam and CO2 are increased. The mechanisms involved in this relative hyperventilation are likely to involve diencephalic integrative structures.

Rats exposed to CO in a manner known to cause oxidative stress in brain exhibited a twofold increase in plasma levels of oxidized proteins, thiobarbituric acid-reactive substances (TBARS), oxidized glutathione (GSSG), and reduced glutathione (GSH). Changes were neither directly related to hypoxic stress from carboxyhemoglobin nor significantly influenced by circulating platelets or neutrophils. Treatment with the nitric oxide synthase inhibitor N omega-nitro-L-arginine methyl ester inhibited elevations in GSH and GSSG but not changes in oxidized proteins or TBARS, suggesting that two oxidative mechanisms may be operating in this model and that GSH and GSSG elevations involved nitric oxide-derived oxidants. Elevations of blood GSH and GSSG occurred at different anatomic sites, indicating that no single organ was the source of the increased peptides. Animals that underwent exchange transfusion with a hemoglobin-containing saline solution did not exhibit elevations in GSH and GSSG, suggesting that blood-borne cells released these peptides in response to oxidative stress. In in vitro studies, erythrocytes, but not platelets and leukocytes, responded to oxidative stress from peroxynitrite by releasing GSH, whereas no release was observed in response to nitric oxide or superoxide. Glucose, maltose, and cytochalasin B, agents that protect extracellular components of the hexose transport protein complex from oxidative stress, prevented GSH release. The data indicate that nitric oxide-derived oxidants are involved in CO-mediated oxidative stress within the vascular compartment and that elevations of several compounds may be useful for identifying exposures to CO likely to precipitate brain injury.

The conversion of xanthine dehydrogenase to xanthine oxidase and lipid peroxidation were measured in brain from carbon monoxide- (CO) poisoned rats. Sulfhydryl-irreversible xanthine oxidase increased from a control level of 15% to a peak of 36% over the 90 min after CO poisoning, while the conjugated diene level doubled. Reversible xanthine oxidase was 3-6% of the total enzyme activity over this span of time but increased to 31% between 90 and 120 min after poisoning. Overall, reversible and irreversible xanthine oxidase represented 66% of total enzyme activity at 120 min after poisoning. Rats depleted of this enzyme by a tungsten diet and those treated with allopurinol before CO poisoning to inhibit enzyme activity exhibited no lipid peroxidation. Treatment immediately after poisoning with superoxide dismutase or deferoxamine inhibited lipid peroxidation but had no effect on irreversible oxidase formation. Biochemical changes only occurred after removal from CO, and changes could be delayed for hours by continuous exposure to 1,000 ppm CO. These results are consistent with the view that CO-mediated brain injury is a type of postischemic reperfusion phenomenon and indicate that xanthine oxidase-derived reactive oxygen species are responsible for lipid peroxidation.

Clinical and animal data suggest that the pathogenesis of CO poisoning extends beyond the inhibition of hemoglobin function, but no mechanism has been identified. Evidence of neurological compromise, particularly loss of consciousness, has been implicated as a marker for increased mortality and morbidity in clinical reports. Experiments were carried out with rats to assess whether CO exposure may cause brain lipid peroxidation. With the use of two methods, measurement of conjugated dienes and thiobarbituric acid reactivity, brain lipid peroxidation could be documented as a result of exposure to CO at a concentration sufficient to cause unconsciousness. Products of lipid peroxidation were increased by 75% over the base-line values 90 min after CO exposure. Unconsciousness was associated with a brief period of hypotension, so brief that in itself it caused no apparent insult. Lipid peroxidation occurred only after the animals were returned to CO-free air, and there was no direct correlation with the carboxyhemoglobin level. This work may provide an explanation for a number of currently poorly understood clinical observations regarding CO poisoning.

Isolated perfused dog hearts were made hypoxic by respiring the support dog with low oxygen (hypoxic hypoxia) or with carbon monoxide (CO hypoxia). Each heart was exposed to both types of hypoxia, separately. Effects on coronary flow (Qt), coronary vascular resistance, cardiac oxygen consumption (Vo2), and contractility (%deltadP/dt) were studied. Two series of experiments were done. Series I: At constant perfusion pressure. As oxygen content (Cao2) was lowered from 20 to 5 vol%, Qt doubled with hypoxic hypoxia and almost tripled with CO hypoxia (P less than 0.01). Vo2 and contractility increased with both types of hypoxia. Beta-adrenergic blockade eliminated the increase in VO2 and contractility but not the difference in Qt increase between hypoxic and CO hypoxia. Series II: At constant Qt (with beta-blockade), vascular resistance decreased more with CO than hypoxic hypoxia. Finally, alpha-blockade eliminated the difference in vascular resistance and thus with complete (alpha and beta) blockade, the two types of hypoxia have the same effect and are indistinguishable.

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.

A new method has been developed for the determination of maximal reduction of NAD in the rat cerebral cortex. NADH fluorescence (450 nm) induced by 366-nm light and UV reflectance were measured by a time-sharing light pipe fluorometer. The redox state of the cortical surface was altered by perfusion of oxygen or carbon monoxide through a Teflon chamber adjacent to the dura. This study examines changes caused by local perfusion with the two gases in normoxia, hypoxia, and anoxia. Alternation of topical carbon monoxide and oxygen becomes effective in altering the intracellular redox state at 15% inspired oxygen and caused 20% changes at zero inspired oxygen. Conversely, topical application of oxygen to the systemically anoxic tissue causes oxidation of reduced NAPH in the cells within the field of fluorometric observation equivalent to that caused by breathing approximately 8% oxygen systemically.