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Carbon Monoxide Toxicity
Abstract
The sections in this article are:
Body CO Stores
Location
Carbon Monoxide Exchanges Between Lung and Body Stores and Processes That Determine Hb CO and Body CO Stores
Endogenous CO Production
Carbon Monoxide Metabolism as a CO Sink
Carbon Monoxide Binding to Proteins Found in Mammalian Tissues
Structure and Reactivity of CO
Carbon Monoxide and O 2 Competition
Carbon Monoxide Binding to Hb
Effects of CO Binding to Hb on Oxygenation in Peripheral Tissues
Extravascular CO ‐Binding Proteins
... They attributed the gender difference due to the fact that females have less muscle mass, and therefore less myoglobin mass, than males. However, while males may have more myoglobin mass than females, about 10-15% of total body CO is bound to myoglobin in skeletal and cardiac muscle, while the rest is chemically bound to Hb (Coburn and Forman 1987). Thus, Deller and colleagues did not consider a difference in HbTOT between males and females. ...
... At steady state, the CO distribution between the blood and tissues are similar for COHb between 0 and 60% (Coburn 1970). At higher [COHb], myoglobin may bind more CO due to the relative hypoxia in the myocytes (Coburn and Forman 1987), and since males have more myoglobin, at high [COHb], they may have prolonged t ½ due to increased myoglobin binding of CO. In addition, despite the prolonged rate of CO exposure in our subjects, no steady state was achieved (Longo and Hill 1977). ...
... There is a vertical asymptote of 0 (suggesting an infinitely large t ½ with a D L CO of 0); however, the horizontal asymptote is not 0, implying that other factors limit t ½ at infinite D L CO. One of these factors is _ V A , because with increasing _ V A the horizontal asymptote approaches 0. Total myoglobin body stores are considered a significant reservoir for CO with 10-15% of total body CO being bound to myoglobin (Coburn and Forman 1987). Males, by virtue of their greater muscle mass, have more myoglobin than females. ...
The purpose of this study was to verify the previously reported shorter half-time of elimination (t ½) of carbon monoxide (CO) in females com-pared to males. Seventeen healthy subjects (nine men) completed three ses-sions each, on separate days. For each session, subjects were exposed to CO to raise the carboxyhemoglobin percentage (COHb) to ~10%; then breathed in random order, either (a) 100% O 2 at poikilocapnia (no CO 2 added), or (b) hyperoxia while maintaining normocapnia using sequential gas delivery, or (c) voluntary hyperpnea at~4x the resting minute ventilation. We mea-sured minute ventilation, hemoglobin concentration [Hb] and COHb at 5 min intervals. The half-time of reduction of COHb (t ½) was calculated from serial blood samples. The total hemoglobin mass (Hb TOT) was calcu-lated from [Hb] and estimated blood volume from a nomogram based on gender, height, and weight. The t ½ in the females was consistently shorter than in males in all protocols. This relationship was sustained even after controlling for alveolar ventilation (P < 0.05), with the largest differences in t ½ between the genders occurring at low alveolar ventilation rates. However, when t ½ was further normalized for Hb TOT , there was no significant difference in t ½ between genders at alveolar ventilation rates between 4 and 40 L/min (P = 0.24). We conclude that alveolar ventilation and Hb TOT are sufficient to account for a major difference in CO clearance between genders under resting (nonexercising) conditions.
... It is important to note that the evidence supporting Haldane's HbCO theory of CO poisoning is indirect. "The argument that CO toxicity is due only to reaction with Hb is supported only by negative evidence; i.e., no one has shown that CO binding to compounds other than Hb occurs in amounts sufficient to cause deleterious effects" (Coburn and Forman, 2011). Today we now know that CO has a number of different action sites, many of which will be discussed later in the manuscript, but the commonly explained pathophysiology for this process erroneously remains focused on the interaction of Hb and CO. ...
... This binding requires both a low intracellular oxygen tension, and a very high intracellular CO tension. CO binding also requires the cytochromes to be in a reduced state (Coburn and Forman, 2011). Whether these conditions occur in vivo and, if they do occur, whether they are clinically relevant has yet to be demonstrated. ...
... The end result is lipid peroxidation of the brain, which starts during recovery from carbon monoxide poisoning. With reperfusion of the brain, leukocyte adhesion and the subsequent release of destructive enzymes and excitatory amino acids all amplify the initial oxidative injury [10][11][12]. The net result is cognitive defects, particularly in memory and learning with movement disorders that may not appear for days following the initial poisoning. ...
... A year later Lahiri et al. (1993) showed that CO at low concentrations inhibited the activity of the CSN elicited by hypoxia while at high concentrations (4-5× prevailing PO 2 ) stimulated the activity in the CSN, being this last excitatory, but not the inhibitory effect, sensitive to light, an unequivocal indication that excitation was due to cytochrome oxidase inhibition, while the inhibitory effect was independent of CO actions at the mitochondrial level. Third, a survey to general biology books indicated that all proteins handling O 2 are metallo-or hemoproteins, being also known that in biological systems CO is very unreactive, and as O 2 , CO is handled by metallo-or hemoproteins proteins (Coburn and Forman, 1987). Fourth, the notion of channel gating implied molecular rearrangements of the channel protein as to open the channel pore and allow the ion to pass through the selectivity filter. ...
Oxygen-sensing and transduction in purposeful responses in cells and organisms is of great physiological and medical interest. All animals, including humans, encounter in their lifespan many situations in which oxygen availability might be insufficient, whether acutely or chronically, physiologically or pathologically. Therefore to trace at the molecular level the sequence of events or steps connecting the oxygen deficit with the cell responses is of interest in itself as an achievement of science. In addition, it is also of great medical interest as such knowledge might facilitate the therapeutical approach to patients and to design strategies to minimize hypoxic damage. In our article we define the concepts of sensors and transducers, the steps of the hypoxic transduction cascade in the carotid body chemoreceptor cells and also discuss current models of oxygen- sensing (bioenergetic, biosynthetic and conformational) with their supportive and unsupportive data from updated literature. We envision oxygen-sensing in carotid body chemoreceptor cells as a process initiated at the level of plasma membrane and performed by a hemoprotein, which might be NOX4 or a hemoprotein not yet chemically identified. Upon oxygen-desaturation, the sensor would experience conformational changes allosterically transmitted to oxygen regulated K+ channels, the initial effectors in the transduction cascade. A decrease in their opening probability would produce cell depolarization, activation of voltage dependent calcium channels and release of neurotransmitters. Neurotransmitters would activate the nerve endings of the carotid body sensory nerve to convey the information of the hypoxic situation to the central nervous system that would command ventilation to fight hypoxia.
... Although the HO reaction represents the major enzymatic source of CO, a minor component of endogenous CO may also arise from poorly defined nonheme sources. CO may arise as a by-product of lipid oxidation or as the product of cytochrome p450-dependent metabolism of xenobiotics (e.g., methylene chloride) [82]. ...
Gaseous molecules continue to hold new promise in molecular medicine as experimental and clinical therapeutics. The low molecular weight gas carbon monoxide (CO), and similar gaseous molecules (e.g., H2S, nitric oxide) have been implicated as potential inhalation therapies in inflammatory diseases. At high concentration, CO represents a toxic inhalation hazard, and is a common component of air pollution. CO is also produced endogenously as a product of heme degradation catalyzed by heme oxygenase enzymes. CO binds avidly to hemoglobin, causing hypoxemia and decreased oxygen delivery to tissues at high concentrations. At physiological concentrations, CO may have endogenous roles as a signal transduction molecule in the regulation of neural and vascular function and cellular homeostasis. CO has been demonstrated to act as an effective anti-inflammatory agent in preclinical animal models of inflammation, acute lung injury, sepsis, ischemia/reperfusion injury, and organ transplantation. Additional experimental indications for this gas include pulmonary fibrosis, pulmonary hypertension, metabolic diseases, and preeclampsia. The development of chemical CO releasing compounds constitutes a novel pharmaceutical approach to CO delivery with demonstrated effectiveness in sepsis models. Current and pending clinical evaluation will determine the usefulness of this gas as a therapeutic in human disease.
Understanding of the mechanisms behind carbon monoxide intoxication and brain functions in vivo has a significant value in optimizing the therapeutic approach in patients (Thom and Keim, J Toxicol Clin Toxicol 27:141–156, 1989). A multiparametric monitoring assembly (MPA) connected to the brain of unanesthetized rats (Mayevsky et al. SPIE 1431:303–313, 1991) was used to assess the toxic effects of CO on various brain functions. Energy metabolism was evaluated by optical monitoring of relative CBF (using Laser Doppler flowmeter) and intramitochondrial redox state (using NADH surface fluorometry). Ionic homeostasis was assessed by measurements of K+, Ca2+, and H+ Levels in the extracellular space (using surface mini electrodes). The electrical parameters were ECoG and DC steady potential. Exposing the awake rat to 1000 ppm CO (40 min) followed by 3000 ppm (20 min) led to an increase in CBF followed by the development of brain depolarization characterized by changes in ionic homeostasis similar to those recorded under cortical spreading depression. Exposing the CO-intoxicated brain to external stimulation (KCl solution) led to metabolic and hemodynamic responses typical to an ischemic brain, namely, that due to limited O2 delivery to the cells, the increase in energy demand led to a decrease in CBF and an increase in NADH redox state. Also in most animals tested, oscillations in CBF and NADH redסx state were recorded after exposure to 3000 ppm CO as described in the ischemic brain (Mayevsky and Ziv Neurol Res 13:39–47, 1991). Therefore, we tried to improve brain oxygenation and interfere with the development of CO intoxication by exposing rats to 1–3 ATA 24 h before (Thom, Toxicol Appl Pharmacol 105:340–344, 1990a) as well as immediately after CO exposure. The results also indicated that suppression of brain energy metabolism is a characteristic manifestation of CO poisoning in rats. Restoration of cerebral energy metabolism by adequate dosage of HBO2 may become an important factor for recovery of brain activities after CO poisoning.
Carbon monoxide (CO) is capable of binding to some proteins containing other transition metals at their active sites, for instance, cobalt, nickel, and copper, thereby interfering with their functions. This chapter provides a summary of the biochemistry of CO as it pertains to heme proteins in mitochondria and their relationships to changes in mitochondrial and cellular function. In the cell, cytochrome c oxidase, the terminal enzyme of the mitochondrial electron transport system, reduces molecular O 2 to water in a four‐electron reaction. The proclivity of CO to enhance mitochondrial oxidant production has been shown for endogenous CO in intact hepatic blood vessels, resulting in increased susceptibility to further oxidant stress. At low CO concentrations, the initial CO‐mediated redox changes in the electron transport chain are sensed as oxidant stress, generating a mitochondrial stress response that includes mitochondrial biogenesis and other elements of mitochondrial quality control.
Cetacea is a clade well-adapted to the aquatic lifestyle, with diverse adaptations and physiological responses, as well as a robust antioxidant defense system. Serious injuries caused by boats and fishing nets are common in bottlenose dolphins (Tursiops truncatus); however, these animals do not show signs of serious infections. Evidence suggests an adaptive response to tissue damage and associated infections in cetaceans. Heme oxygenase (HO) is a cytoprotective protein that participates in the anti-inflammatory response. HO catalyzes the first step in the oxidative degradation of the heme group. Various stimuli, including inflammatory mediators, regulate the inducible HO-1 isoform. This study aims to characterize HO-1 of the bottlenose dolphin in silico and compare its structure to the terrestrial mammal protein. Upstream HO-1 sequence of the bottlenose dolphin was obtained from NCBI and Ensemble databases, and the gene structure was determined using bioinformatics tools. Five exons and four introns were identified, and proximal regulatory elements were detected in the upstream region. The presence of 10 α-helices, three 310 helices, the heme group lodged between the proximal and distal helices, and a histidine-25 in the proximal helix serving as a ligand to the heme group were inferred for T. truncatus. Amino acid sequence alignment suggests HO-1 is a conserved protein. The HO-1 “fingerprint” and histidine-25 appear to be fully conserved among all species analyzed. Evidence of positive selection within an α-helix configuration without changes in protein configuration and evidence of purifying selection were found, indicating evolutionary conservation of the coding sequence structure.
It is estimated that 10% of carbon throughout the cosmos is in the form of carbon monoxide (CO) [1]. Earth’s earliest prebiotic atmosphere included the trinity of gasotransmitters CO, nitric oxide (NO), and hydrogen sulfide (H2S), for which all of life has co-evolved with [2]. Speculation for extraterrestrial life in harsh environments is similarly based on harnessing atmospheric CO as a primary energy source [3]. The history of CO can be loosely traced to mythological and prehistoric origins with fundamental understanding emerging in the middle ages. Ancient literature is focused on CO’s deadly toxicity which is understandable in the context of our primitive relationship with coal and fire. Scientific inquiry into CO appears to have emerged throughout the 1700s followed by chemical and toxicological profiling throughout the 1800s. Despite CO’s ghastly reputation, several of the 18th and 19th century scientists suggested a therapeutic application of CO. Since 2000, the fundamental understanding of CO as a deadly nuisance has undergone a paradigm shift such that CO is now recognized as a neurotransmitter and viable pharmaceutical candidate. This review is intended to provide a brief history on the trace origins pertaining to endogenous formation and therapeutic application of CO.
Objective:
In this study, the aim was to compare the rate of decrease in carboxyhemoglobin (COHb) values at consecutive time points and calculate the half-life of COHb (COHb t½) in patients admitted to the emergency department (ED) with carbon monoxide (CO) poisoning and treated with either high flow nasal cannula oxygen therapy (HFNC) or non-rebreather face mask (NRFM).
Methods:
This retrospective, cohort study with historical controls was performed over a 2-year period and included adult patients with CO poisoning, whose COHb values were checked. The HFNC group consisted of patients admitted to the ED with CO poisoning when HFNC was available in the hospital, while the NRFM group consisted of patients who presented to the ED with CO poisoning before the availability of HFNC. The primary outcome of the study was to detect the COHb t½.
Results:
A total of 71 patients were enrolled in the study. While 35 patients received oxygen with NRFM, 36 patients received HFNC. The mean COHb t½ in the HFNC group was 41.1 min (95% CI: 31.0-58.4) and 64.0 min (95% CI: 43.5-114.4) in the NRFM group. We did not find a significant difference in the COHb t½ between the HFNC group and NRFM group (p = 0.099). COHb levels between treatment arms at serial time points showed a statistically significant difference at 60 min (p = 0.048). We compared the decay constant and half-life of COHb between groups according to gender. In both genders, COHb t½ was significantly different between groups, and COHb t½ was lower in the patients treated with HFNC.
Conclusion:
HFNC was effective in reducing the half-life of COHb values in patients with carbon monoxide poisoning. Prospective studies to be conducted in larger groups are needed to fully understand the effect of HFNC on carbon monoxide poisoning.
Absorbance measurements via transmitting light spectroscopy in microtiter plates are established for high throughput screening of biological systems. These measurements allow for the determination of important process parameters within a short time. However, absorbance determination via transmitted light measurements is not always feasible. As for carbon monoxide difference absorbance spectroscopy, used for concentration measurements of active P450 monooxygenases (P450s), security standards and consistent gassing have to be addressed. In this study, a non-invasive online measuring principle for absorbance via scattered light is proposed. Based on optical fiber measurements, a decrease in scattered light signals at 450 nm wavelength of reflecting polymer particles is observed, and P450 concentrations are calculated. In this way, high throughput determination of P450 concentrations in a secure, gas-tight environment is realized. The designed method was successfully applied to concentration measurements and carbon monoxide saturation kinetics ranging from 0.3 to 5.0 µM P450 BM3 achieving a measurement accuracy of ± 0.05 µM P450. This article is protected by copyright. All rights reserved
Oxygen chemoreception by the peripheral chemoreceptors is expressed by an increased chemosensory discharge, but the mechanism and cellular locus of oxygen chemoreception are not established unequivocally. An approach to understanding these unknowns is to study structure and function of the chemoreceptors in chronic stimulus change, e.g., hypoxia and hyperoxia. Chronic hypoxia is known to stimulate carotid body cellular growth and to increase catecholamine content and metabolism (1–4). However, it is not clear whether these consequences are due to local effects of low tissue Po2 or are secondary to responses of other tissues, elaborating a growth factor such as erythropoietin during systemic hypoxic hypoxia. One way to answer the question is to use an agent that would stimulate erythropoietic but not carotid body response. Chronic CO inhalation is known to stimulate erythropoiesis (5) and acute CO inhalation during normoxia does not stimulate carotid chemoreceptor activity (6), presumably because of a high blood flow to its tissue (7,8). A high blood flow and, hence, a high O2 delivery would prevent a lowering of tissue Po2, which usually occurs in tissues with an average blood flow due to carboxyhemoglobinemia (3). Therefore, we compared the effects of chronic CO inhalation and hypoxic hypoxia on carotid body structure and catecholamine content.
As one of the leading causes of poisonings worldwide, it is imperative that prehospital specialists are aware of carbon monoxide (CO) poisoning and its management. Awareness of the epidemiology, and the common presentations of CO poisoning may lead to prompt evaluation and early initiation of life-saving therapy. Children under 5 years of age have the highest incidence of CO-related ER visits and are at greatest risk of CO toxicity. The clinical features are nonspecific and misdiagnoses are common. Therefore, prehospital providers should have a high index of suspicion for CO intoxication in patients that experience headache, vomiting, or altered level of consciousness following exposure to hydrocarbon combustion within an enclosed space. A carboxyhaemoglobin level is a quick and reliable way to diagnose CO exposure. To prevent complications such as altered cerebellar function, seizures, rhabdomyolysis and dysrhythmias, early recognition and treatment is imperative. Removal from the source of exposure and the provision of 100% oxygen form the cornerstone of management. Preventive strategies should also be explored in susceptible populations.
Carbon monoxide (CO) may inhibit mitochondrial electron transport in the brain and increase the toxic effects of the gas. This hypothesis was investigated in anesthetized rats during CO exposure and recovery at either normobaric or hyperbaric O2 concentrations. During exposure and recovery, we measured the oxidation level of cerebrocortical cytochrome c oxidase by differential spectroscopy and biochemical metabolites known to reflect aerobic energy provision in the brain. CO exposure (HbCO = 71 +/- 1%) significantly decreased blood pressure and cytochrome oxidation level. Cerebral ATP was maintained while lactate/pyruvate, glucose, and succinate rose, and phosphocreatine (PCr) fell, relative to control (P less than 0.05). Intracellular pH (pHi) calculated from the PCr equilibrium also declined during the exposures. During recovery, HbCO fell more rapidly at hyperbaric than at normobaric O2 levels, but returned to 10% or less in both groups by 45 min. Cytochrome oxidation state improved to 80% of control after 90 min at normobaric O2, but recovered completely after hyperbaric O2 (P less than 0.05). In normobaric O2, PCr and pHi continued to fall for 45 min after CO exposure and did not recover completely by 90 min. PCr and pHi in animals after hyperbaric O2 improved within 45 min, but also remained below control at 90 min. These data indicate that intracellular uptake of CO can impair cerebral energy metabolism, despite the elimination of HbCO from the blood.
The method that Haldane used in 1911, when he claimed that O2 comes to be secreted into the pulmonary capillary blood after a few days at altitude, required that CO be an indifferent gas except for one property: it combines with Hb. But that is now known not to be true. CO is formed when Hb is katabolised, it reacts with many substances in the body, and it is involved in transmissions between cells. If, instead of supposing that CO is an indifferent gas, one proceeds from Barcroft's observation that PaO2 remains equal to PAO2 on going to altitude, the conclusion from Haldane's observations must be that he showed that CO has come to be actively excreted from the body after a few days at altitude. And that presumably happened because, as Killick (J. Physiol., London, 107: 27-44, 1948) suggested, Haldane's method required that the subject be repeatedly exposed to CO. The transport of CO around the body by Hb, and the possible effects on this, and on DLCO, of active excretion of CO, have to be considered.
Carbon monoxide (CO) has many effects in biology due to its complex biochemical activities. These actions of CO depend primarily on its ability to bind heme proteins (Hp) and to inhibit or alter their biochemical functions. Whether CO is derived from exogenous or endogenous sources, its cellular activity is related to its concentration and the concentration of molecular O(2), as well as to the availability of reduced transition metals such as Fe(II). In this respect, the CO/O(2) ratio and O(2)-dependent changes in local oxidation-reduction state assume critical importance in determining the physiological effects of CO by affecting the functions of specific Hp. By interacting with Hp, CO influences electron-transport reactions in a variety of ways, which can produce either prooxidant or antioxidant effects. Similarly, Hp relationships also govern how changes in CO concentration influence the physiological and pathological effects of nitric oxide and the relationships of the two biologically active gases to metal-catalyzed oxidations. This article provides a brief update on the biochemistry of CO as it relates to Hp binding, chemical oxidative processes, and cellular function.
The biochemical paradigm for carbon monoxide (CO) is driven by the century-old Warburg hypothesis: CO alters O(2)-dependent functions by binding heme proteins in competitive relation to 1/oxygen partial pressure (PO(2)). High PO(2) thus hastens CO elimination and toxicity resolution, but with more O(2), CO-exposed tissues paradoxically experience less oxidative stress. To help resolve this paradox we tested the Warburg hypothesis using a highly sensitive gas-reduction method to track CO uptake and elimination in brain, heart, and skeletal muscle in situ during and after exogenous CO administration. We found that CO administration does increase tissue CO concentration, but not in strict relation to 1/PO(2). Tissue gas uptake and elimination lag behind blood CO as predicted, but 1/PO(2) vs. [CO] fails even at hyperbaric PO(2). Mechanistically, we established in the brain that cytosol heme concentration increases 10-fold after CO exposure, which sustains intracellular CO content by providing substrate for heme oxygenase (HO) activated after hypoxia when O(2) is resupplied to cells rich in reduced pyridine nucleotides. We further demonstrate by analysis of CO production rates that this heme stress is not due to HO inhibition and that heme accumulation is facilitated by low brain PO(2). The latter becomes rate limiting for HO activity even at physiological PO(2), and the heme stress leads to doubling of brain HO-1 protein. We thus reveal novel biochemical actions of both CO and O(2) that must be accounted for when evaluating oxidative stress and biological signaling by these gases.
Over the last decade, studies have unraveled many aspects of endogenous production and physiological functions of carbon monoxide (CO). The majority of endogenous CO is produced in a reaction catalyzed by the enzyme heme oxygenase (HO). Inducible HO (HO-1) and constitutive HO (HO-2) are mostly recognized for their roles in the oxidation of heme and production of CO and biliverdin, whereas the biological function of the third HO isoform, HO-3, is still unclear. The tissue type-specific distribution of these HO isoforms is largely linked to the specific biological actions of CO on different systems. CO functions as a signaling molecule in the neuronal system, involving the regulation of neurotransmitters and neuropeptide release, learning and memory, and odor response adaptation and many other neuronal activities. The vasorelaxant property and cardiac protection effect of CO have been documented. A plethora of studies have also shown the importance of the roles of CO in the immune, respiratory, reproductive, gastrointestinal, kidney, and liver systems. Our understanding of the cellular and molecular mechanisms that regulate the production and mediate the physiological actions of CO has greatly advanced. Many diseases, including neurodegenerations, hypertension, heart failure, and inflammation, have been linked to the abnormality in CO metabolism and function. Enhancement of endogenous CO production and direct delivery of exogenous CO have found their applications in many health research fields and clinical settings. Future studies will further clarify the gasotransmitter role of CO, provide insight into the pathogenic mechanisms of many CO abnormality-related diseases, and pave the way for innovative preventive and therapeutic strategies based on the physiologic effects of CO.
This study characterizes microsomal heme oxygenase, a previously undescribed enzyme which catalyzes the oxidation of heme at the α-methene bridge to form biliverdin. This step is then coupled with soluble NADPH-dependent biliverdin reductase to form bilirubin; microsomal heme oxygenase is rate-limiting in this pathway. By all analytical criteria, the product of this reaction is bilirubin. Most, if not all, of the bilirubin is of the IX α configuration, which is the sole isomeric form of bilirubin occurring physiologically. Heme oxygenase is localized specifically to the microsomal fraction, has an absolute and stoichiometric requirement for NADPH and molecular oxygen, generates carbon monoxide in amounts equimolar to bilirubin, and is inhibited by carbon monoxide. These and other data suggest that this enzyme is a mixed function oxygenase. The enzyme is most active with protohemin IX or methemalbumin; substrates with less activity are methemoglobin, the α and β chains of hemoglobin, deuterohemin IX, coprohemin I, and the hemoglobin-haptoglobin complex, in this order. Oxyhemoglobin, carboxyhemoglobin, myoglobin, and free porphyrins are not acted upon by the enzyme. The apparent Km for protohemin IX is 5.0 µm, and for the other substrates ranges from 4.5 to 5.1 µm. Sodium dodecyl sulfate, lipase, phospholipase, trypsin, potassium cyanide, sodium azide, and p-hydroxymercuribenzoate inhibit the enzyme. The kinetics and tissue distribution of this enzyme suggest that it is of major importance in the physiological degradation of hemoglobin and other hemoproteins to bile pigment.
In this paper we present probable structures for derivatives of horseradish and cytochrome c peroxidases and also propose a simple scheme which encompasses the molecular structures and electronic configurations of the derivatives of these and other protoheme proteins in the oxidation states from II to VI. Four lines of evidence are used to define the structures of the heme groups and their associated ligands. These are: low temperature optical spectra in the visible region, electron paramagnetic resonance spectra taken at 1.5°K, the oxidation state of the compounds, and proof of the identity of the ligands in position 6 of the heme iron atom. Only the ferrous and ferric forms of heme iron are needed to describe all of these compounds, and higher oxidation states of the iron atom need not be invoked. A nomenclature describing the electronic configuration of the heme and its associated ligands is presented, which specifies for each compound, the d electron configuration of the heme, the structure of the sixth ligand of the heme iron atom, and the oxidation state of the compound. The electronic configuration of the iron, ferrous or ferric, is not necessarily related to the oxidation state of the compound.
An extension of our reasoning leads us to conclude that the electronic configurations of oxyhemoglobin, oxymyoglobin, and oxyperoxidase are the same, and that the oxygenation reaction involves the migration of an electron from the ferrous heme iron to the oxygen molecule. In consequence, the heme iron atoms of these compounds are formally in the ferric low spin state, and the oxygen molecules in the sixth ligand positions each carry 3 oxidizing eq.
The reaction between carboxyhemoglobin and reduced microperoxidase (MP): Hb4(CO)4 + 4MP=Hb4 + 4MPCO, recently reported by us, has been further studied. By generating species Hb4(CO), Hb4(CO)2, and Hb(CO)3 in the stopped flow cuvette by the reaction of dithionite with the species of the general formula Hb4(O2)x(CO)y(x + y=4) in the presence of microperoxidase it has been possible to determine the stepwise CO dissociation rate constants l4, l3, l2, and l1. The overall CO dissociation rate constant l, which is the same in this system as l4, is not affected by 2,3-diphosphoglyceric acid. The activation energy of the reaction is 21,400 cal in 15-25 degrees range. The ratio deltal/deltapH is approximately 3 in 6.5 to 7.5 pH range. The kinetic data indicate that, compared to HbO2, the contribution to the cooperativity of the dissociation rate constants of carboxyhemoglobin is greatly reduced. The ligand-dependent differences in the reactions of Hb with CO, O2, and NO suggest that in the combination reactions the ligand plays an active role in the rate-limiting step.
Changes induced in measurements of endogenous carbon monoxide (CO) production by blood in the lumen of the gut were studied in five normal volunteers. The study was undertaken because exogenous heme is absorbed by intestinal mucosal cells where the porphyrin ring is split with the release of CO that could contribute to blood CO levels and lead to a fallacious diagnosis of hemolytic disease. Volunteers who consumed 200 ml of their own blood doubled their endogenous production of CO (0.69 versus 0.34 mumoles/kg/hr). This suggested that at least 3% of the ingested heme was degraded and recovered as CO within 2 1/2 hr. Measurements of serum bilirubin also showed a significant increase after ingestion of blood. These data indicate that blood in the gastrointestinal tract can interfere with quantification of heme and bilirubin turnover from measurements of either endogenous CO production or bilirubin and suggest that this might occur with the ingestion of meat.
The effects of CO and hypoxic hypoxia on catecholamine steady-state levels and turnover were studied in several areas of the rat brain. The depletion of norepinephrine (NE) or dopamine (DA) after AMPT was employed as an index of turnover. Both CO (0.17% or 0.15%) and hypoxia (7.5% or 8.0% O2) exposures caused increased NE turnover and decreased NE steady-state levels in the hypothalamus. The exposures did not affect NE levels or turnover in the hippocampus or olfactory tubercles. The effects of the exposures on DA were different from those on NE. Both CO and hypoxia caused decreased DA turnover in the olfactory tubercles and caudate nucleus and increased DA steady-state levels in the hypothalamus. Steady-state levels of NE in the heart were decreased in the CO exposed rats. Thus, the effects of CO and hypoxia are specific both in terms of neurotransmitter and brain region.
Changes in intracellular Po2 in myoglobin containing skeletal muscle during exercise were estimated in normal nonathlete subjects from measurements of shifts of CO between blood and muscle under conditions where the total body CO stores remained constant. Exercise was performed on a bicycle ergometer. In 1.5–2 and 6–7 min runs at Vo2 max with the subject breathing 21% O2, mean MbCO/HbCO increased 146 +/- 7 and 163 +/- 11% of resting values, respectively (P less than 0.05). With the subjects breathing 13–14% O2, in 1.5–2 and 6–7 min runs, Vo2 max fell an average of 4.3 +/- 5.1% and 12.0 +/- 5.2%, respectively, and mean MbCO/HbCO increased to 233 +/- 18% and 210 +/- 52% of resting value, respectively (P less than 0.05). These findings suggest that mean myoglobin Po2 fell during exercise at Vo2 max, with the subjects breathing 21% O2 and the decrease in mean myoglobin Po2 was greater with the subject breathing 13–14% O2. There was considerable variability in different subjects and in some, the data were not consistent with intracellular O2 availability limiting aerobic metabolism. The data support a postulate that there are several limiting factors for the aerobic capacity, including intracellular O2 availability.
The effects of glutethimide and phenobarbital on the plasma unconjugated bilirubin concentration, hepatic bilirubin clearance (CBR) and plasma bilirubin turnover (BRT) were determined in 19 patients with mild chronic unconjugated hyperbilirubinemia (Gilbert's syndrome) and 11 normal volunteers. CBR and BRT were calculated from plasma radio-bilirubin disappearance curves obtained both before and during drug administration. The response to both drugs was essentially identical. During drug administration, the patients with Gilbert's syndrome attained a new steady state in which the plasma unconjugated bilirubin concentration ) and CBR 314 ± 25% of baseline. In the normal volunteers, fell to 65 ± 6%, while CBR increased to 135 ± 9% of baseline during the period of drug administration. Although neither total red cell volume nor the half-life of 51Cr-labeled erythrocytes was altered by either agent, daily plasma bilirubin turnover fell significantly (P < 0.05) to 87 ± 4% of baseline in the 30 subjects studied, and endogenous carbon monoxide production, which provides an independent estimate of the rate of heme degradation and bilirubin formation, was 93 ± 5 % of control (0.2 > P > 0.1). These studies indicate that both accelerated hepatic bilirubin clearance and reduced plasma bilirubin turnover contribute to the reduction in bilirubin concentration observed during administration of phenobarbital and glutethimide. The methods employed provided no evidence that these agents produce an increase in the rates of heme catabolism, bilirubin production or carbon monoxide formation.
Haloforms are metabolized to carbon monoxide by hepatic microsomal mixed function oxidases and this reaction is markedly stimulated by sulfhydryl compounds. Maximal stimulation occurred at 0.5 mM glutathione (GSH). Formation of 1 mole of carbon monoxide (CO) resulted in the disappearance of 2 moles of GSH and the production of 1 mole of oxidized glutathione (GSSG). Incubation of 13CHBr3, or 12CHBr3 in the presence of 18O2, resulted in the formation of similarly enriched 13CO or C18O respectively. Furthermore, a primary isotope effect was observed when CDBr3 served as the substrate. Dibromocarbonyl is an intermediate in the reaction since 2-oxothiazolidine-4-carboxylic acid (OTZ) was detected when CHBr3 was incubated in the presence of cysteine. In addition, when 13CHBr3 was used, a similarly enriched [13C]OTZ was formed. Based on these observations, the following mechanism for the conversion of haloforms to CO is proposed: CHX → COHX3 → X2CO; X2CO + GSH → GS(C = O)X; GS(C = O)X + GSH → GSSG + :C = O.
Anesthetized albino rats were exposed to 500 and 1000 ppm carbon monoxide in air for a period of 2.5 hours, during which time the surface oxygen tension of brain and liver were monitored continuously. At the end of the exposure period the brain and liver were frozen rapidly (liquid nitrogen and freeze-clamping, respectively), and after suitable preparation were analyzed for their concentration of ATP, creatine phosphate, lactate, and pyruvate. The changes in surface oxygen tension were more drastic than the chemical changes in the two organs, but by both criteria the metabolic effect of carbon monoxide was more severe in liver than in brain. The metabolic significance of surface oxygen tension measurements is questioned.
The structure of human carbonmonoxy haemoglobin has been determined to 2.7 Å resolution using X-ray data for the native protein only. The atomic co-ordinates were refined from those of an initial model based on the co-ordinates of the closely related protein horse methaemoglobin whose structure is known at high resolution (Ladner et al., 1977). The space group of the new unit cell (P41212) is such that the location of the haemoglobin molecules is specified by two parameters only, and the values of these were found through a search for the best initial R-factor. The refined structure of human carbonmonoxy haemoglobin is, as expected, generally similar to that of horse methaemoglobin. The root-meansquared shift for all atoms between the initial model and the final co-ordinates was 1.35Å.The new structure confirms that the CO ligand lies off the normal to the haem plane in both α and β subunits, as indicated previously by a difference Fourier map of CO versus horse methaemoglobin (Heidner et al., 1976). The Fe-C-O group, assumed to be linear, makes an angle of about 13 ° with the haem normal and it points towards the inside of the haem pocket. In the α subunit the iron atom lies in the mean plane of the haem within experimental error. In the β subunit the situation is less clear in that unconstrained refinement put the iron atom 0.22 Å from the haem plane, a distance that is times the expected error. The new structure also confirms that in carbonmonoxy haemoglobin the side chain of cysteine β93(F9) points away from the surface of the molecule into the pocket between helices F, G and H that, in deoxyhaemoglobin, is occupied by the side chain of tyrosine β145(HC2). A detailed comparison of the structures of the deoxy and liganded forms from the same species is now possible and the conclusions drawn from this comparison are given in a separate publication (Baldwin & Chothia, 1979).
The enxymatic characteristics of mitochondrial 26-hydroxylase system for 5β-cholestane-3α,7α,12α-triol were studied with rat liver mitochondria and the inner membrane-matrix fractions with special reference to its intramitochondrial localization.1Conditions for the assay of enzymatic activity of the hydroxylase system were established with rat liver mitochondrial preparations and inner membrane-matrix fractions derived by digitonin treatment.2It was established that rat liver mitochondria, as well as microsomes, possessed the hydroxylase system for 5β-cholestane-3α,7α,12α-triol as an intrinsic constituent. Product analyses revealed that only the mitochondrial hydroxylase system was specifically active for C-26 position. In contrast, the microsomal system was found to be apparently unspecific on C-26 position but rather more active for other adjacent positions.3Among the tested citric acid cycle intermediates and nicotinamide coenzymes, isocitrate and NADPH exerted the most remarkable enhancement of the 26-hydroxylase activity. NADP and NADH were totally inert under the present assay conditions.4Partial rupture of the mitochondrial structure by hypotonic or digitonin treatment was essential for the reactivity of 26-hydroxylase system under the present experimental conditions. Accessibility to the intramitochondrial reacting site of the exogenous reactants such as NADPH, isocitrate and 5β-cholestane-3α,7α,12α-triol in particular was apparently improved by the partial rupture.5The NADPH-dependent or isocitrate-dependent 26-hydroxylase system was totally insensitive to inhibitors of the respiratory electron transfer such as potassium cyanide, antimycin A, rotenone and amytal. However, it was specifically inhibited by phenyl isocyanide.6Remarkable sensitivity to carbon monoxide of the 26-hydroxylase system was demonstrated. Apparent Michaelis constant for oxygen and partition coefficient (between carbon monoxide and oxygen) of the 26-hydroxylase system were estimated to be approximately 10–20 μM and 0.1, respectively. Thus, the possible functioning of a “cytochrome P-450”-like entity in intramitochondrial 26-hydroxlase system was proposed.7The localization of 26-hydroxylase system was assigned to be in the inner membranematrix region, on the basis of the distribution of intramitochondrial marker enzymes during the course of serial solubilization by digitonin.
The structure of human oxyhaemoglobin was determined by single crystal X-ray analysis at 2·1resolution. Data were collected on an Arndt-Wonacott camera at −2°C. The structure was refined to an R factor of 0·223 by the Jack-Levitt method, starting from Baldwin's model of human carbon monoxide haemoglobin.
The active sites in the α and β subunit are distinct. The iron atoms are 0·16(8)and 0·00(8)from the mean plane of the porphyrin carbons and nitrogens (0·12(8)and −0·11(8)from the mean plane of the porphyrin nitrogens) in the α and β subunit, respectively, in correlation with the orientation of HisF8 relative to the porphyrin nitrogens. The haem group appears to be nearly planar in the α subunit but ruffled in the β subunit. The Fe-O(1)-O(2) angles are 153(7)° and 159(12)° in the α and β subunit, respectively. The oxygen molecule forms a hydrogen bond to Nε of HisE7 in the α, but either none or a weak one in the β subunit. The following bond lengths were found: Fe-Nε(HisF8)=1·94(9)(α) and 2·07(9)(β); Fe-O(1)=1·66(8)(α) and 1·87(13)(β); Fe-Nporph (mean=1·99(5)(α) and 1·96(6)(β). These dimensions agree with the values obtained in oxymyoglobin and model compounds.
The C-terminal residues, ArgHC3(141α) and HisHC3(146β), are relatively delocalized, and their positions do not enable them to form the intersubunit salt bridges in which they are involved in deoxyhaemoglobin. The penultimate tyrosine residues, TyrHC2 140α and 145β, are relatively localized and maintain the hydrogen bonds to the carbonyl oxygens of ValFG5 (93α and 98β), with only minor variations compared to their geometry in deoxyhaemoglobin. TyrHC2(145β), however, alternates between a major and a minor site, in conjunction with CysF9(93β), both sharing the internal pocket between the F and H helices while in the major conformation. This suggests that the role of the penultimate tyrosines in the allosteric mechanism may differ from that previously proposed by Perutz.
The overall quaternary structure of oxyhaemoglobin is identical, within experimental error, to that of carbon monoxide haemoglobin, and thus confirms the applicability of the allosteric mechanisms proposed by Perutz and Baldwin & Chothia to the process of oxygen binding.
The endogenous production of carbon monoxide (VCO) and total serum bilirubin (SB) have been followed in five healthy male volunteers during one baseline day and one day with no caloric intake. VCO in the morning studies was 11.2±1.7 (mean ±1 S.E.M.) on the baseline day and 10.1±2.3 μmol/mmol total body heme (TBH) and day on the fasting day, respectively. In studies before noon, VCO increased significantly on both days, to values of 17.8±1.6 and 19.6±2.2 μmol/mmol TBH and day, respectively. In the first study in the afternoon, VCO differed significantly between the two days, amounting to 12.1±3.0 and 23.7 ±3.5 on the baseline and the fasting day, respectively. The difference was still significant in the evening, when VCO was 11.6±3.1 and 22.1±4.9 μmol/mmol TBH and day. SB followed the same pattern, with mean values of 4.0±0.3, 4.9±0.3, 4.2±0.9 and 3.0±0.3 μg/ml during the baseline day and 4.5±0.6, 5.4±1.2, 7.0±0.5 and 8.5±1.0 μg/ml, respectively, during fasting day. Only insignificant amounts of conjugated bilirubin were found. The studies confirm earlier reports on the effect of caloric restriction on VCO. Since this effect is simultaneous with an increase in SB, it is concluded that the changes are secondary to an increase in total heme catabolism. They might be due to an increase in intracellular hepatic heme turnover but it cannot be excluded that starvation affects erythropoiesis and/or red cell catabolism, thereby causing an increase in VCO and SB.
The kinetic constants for the reactions of Aplysia myoglobin with oxygen and carbon monoxide are essentially the same as those of horse myoglobin except for the “off” constant for oxygen. In the case of Aplysia myoglobin, this constant has a value of about 70 sec−1 at 20 ° as compared with a value of about 10 sec−1 for horse myoglobin. This difference accounts for the difference of oxygen affinity for the two proteins.
The secondary-amine mono-oxygenase (EC 1.14.99.–) of Pseudomonas aminovorans is potently inhibited by carbon monoxide. The degree of inhibition of the purified enzyme was determined by the CO:O2 ratio rather than by the absolute concentration of carbon monoxide. The partition constant (the CO:O2 ratio causing 50% inhibition of activity) was 9.2 × 10−4. The inhibition could be reversed by light, and the extent of reversal was proportional to the light intensity. With monochromatic light of wavelength 417 nm, the light sensitivity, L, was determined to be 2.5 × 108 cm2 min/mol quantum. The photochemical action spectrum for the light reversal of inhibition showed a single maximum of effectiveness at about 420nm. The difference spectrum of the enzyme (reduced by NADH) on bubbling with CO (compared with an NADH-reduced reference sample) showed a peak at 426 nm. The preparation showed none of the spectral properties of cytochrome P-450 mono-oxygenase preparations, and was much more sensitive to carbon monoxide. The enzyme behaves as a typical o-type cytochrome (i.e. a carbon-monoxide-reactive b-type cytochrome), and in its sensitivity to carbon monoxide as well as in its spectral properties, shows close resemblances to haemoglobin.
Carbon monoxide production (VCO) total body heme (TBH) and serum bilirubin (SB) have been determined in healthy young men before and after 100 mg phenobarbital (10 subjects), 15 mg diazepam (7 subjects) and 75 mg oxazepam (7 subjects), respectively, daily for seven days. None of the drugs had any significant effect on VCO. SB and TBH were also unaffected. Baseline VCO (mean ±1 S.E.M.) was 12.6±0.6 μmol/mmol TBH and day. The postdrug VCO was 15.1 ± 1.8, 14.1 ± 1.4 and 14.7 ± 1.5 after phenobarbital, diazepam and oxazepam, respectively. The corresponding values for SB were 5.4±0.8, 6.0±1.5 and 6.2±1.0 μg/ml, compared to a baseline value of 6.6±0.8 μg/ml. However, when the pooled postdrug data were compared with the pooled baseline values, mean VCO showed a probably significant increase (from 12.6±0.6 to 14.7±1.0 μmol/mmol TBH and day (p<0.05). It is concluded that although phenobarbital is known to increase the hepatic heme turnover, this effect is not measurable in terms of total heme turnover, no more than 20% of which comes from the liver.
Earlier observations of an endogenous formation of carbon monoxide in man have been analysed with reference to the possibility that the carbon monoxide is formed in the blood by the breakdown of haemoglobin. The following results were obtained. The COHb concentration of blood calculated from determinations of the CO pressure in alveolar air, agrees with the COHb concentration determined directly from blood. The partial pressure of carbon monoxide in the alveolar air is, however, considerably greater than that of ordinary atmospheric air, which means that carbon monoxide is constantly exhaled during respiration.
If blood is incubated at 38 o C for 20–24 hours, an increase of 40–165 % can be shown in the COHb concentration, this increase being particularly pronounced after haemolysis. This apparent formation of carbon monoxide in blood is considerably increased at acid or alkaline pH's or by addition of sodium azide.
If the blood is shaken with carbon monoxide before incubation at 38 o C, the carboxyhaemoglobin concentration after 20 hours is decreased. If sodium azide is added to samples containing 5–30 % COHb. before incubation, the concentration is however increased, and at higher COHb‐concentrations there is a decrease, less than that observed without azide.
The formation of carbon monoxide was found to parallel the spontaneous formation of methaemoglobin on alteration of the pH or addition of sodium azide.
Addition of ascorbic acid and hydrogen peroxide greatly increases the CO formation. The amount of CO produced corresponds to a conversion of up to 15 per cent of the haemoglobin to COHb, calculated from the original haemoglobin concentrations.
The formation of CO seems to be parallel to the breakdown of haemoglobin to choleglobin (verdoglobin). This observation can be explained by the assumption that the opening of the tetra‐pyrrole ring occurs with the liberation of the a‐C‐atom after oxidation to CO.
It was possible to demonstrate in one subject that the amounts of CO produced and haemoglobin decomposed bear a quantitative relation of approximately 1 molecule CO: 1 haemin group.
Abstract Total heme catabolism has been studied through measurement of the endogenous production of carbon monoxide (V̇CO) in 8 patients with hemolysis, 7 with hypoproliferative anemia, 10 with refractory anemia and hypercellular bone marrow and 7 with splenomegaly, 6 of whom had myeloid metaplasia. Simultaneously, catabolism of circulating red cell hemoglobin heme (Vheme-c) was measured through labelling of the red cells with 51Cr, and the V̇CO/V̇heme-c ratio was calculated for each patient. From a control group it was calculated that this ratio should vary around 1.5. Since no isotope studies were performed in the control group, no range could be defined. Among patients with hemolysis the V̇CO/V̇heme-c ratio was found to vary between 1.3 and 1.8 except in 2 cases of paroxysmal nocturnal hemoglobinuria (PNH) and PNH?, respectively, in whom the ratios were found to be 0.6 and 0.7 suggesting some heme catabolism without corresponding CO formation. In the hypoproliferative group the ratio varied between 1.2 and 1.8 except in one patient treated with androgens, in whom the ratio was found to be 2.9, suggesting increased extraerythrocytic heme turnover. In patients with myeloid metaplasia the ratio varied between 1.3 and 1.8. On the other hand, the ratio varied between 2.4 and 3.0 among patients with refractory anemia and hypercellular bone marrow, thus confirming earlier findings that in this type of anemia turnover of bone marrow heme is markedly increased. A significant correlation was found between V̇CO and initial morning COHb% (r=0.84). The conclusions drawn are (a) that V̇heme-c sometimes represents less than 50% of total heme turnover and (b) that COHb and/or V̇CO reflect total heme turnover except in patients with blood loss or intravascular hemolysis with hemoglobinuria.
Rats were exposed to 14CH2Cl2 vapor in a closed rebreathing system. The total amount of respiratory 14CO2 and 14CO was accumulated for periods up to 15 hr after dosing. These two metabolic products accounted on the average for 76% of the radioactivity of the 14CH2Cl2 given. The major portion, 47%, was found as 14CO whose specific activity was close to that of the administered 14CH2Cl2. This suggests a nearly direct conversion of CH2Cl2 to CO. Approximately 29% of the radioactivity was recovered in the expired CO2. The oxidation of methylene chloride to CO and CO2 was similar qualitatively and quantitatively for male and female animals. The data suggest that methylene chloride acts as a direct substrate for metabolic formation of CO rather than by stimulating the conversion of endogenous carbon sources to CO.
The rate at which CO displaced oxygen from combination with oxyhemoglobin solution has been measured at different oxygen tensions at 37 °C. The hemoglobins studied were human adult and fetal, horse, goat, dog, cat and rabbit. From these results we have determined (i) the velocity constant for the dissociation of oxygen from fully saturated hemoglobin (k4), (ii) the velocity constant for replacement of oxygen from fully saturated hemoglobin at low tensions of CO(m′∞), and (iii) the ratio of the velocity constants for the combination of CO and O2 with three-parts saturated hemoglobin (′l4/′k4). The values found for k4 and (m′∞) (each in units of sec-1) and for the ratio (′l4/′k4) were: human adult, 222, 16.9, and 0.30; human new-born, 300, 16.0, and 0.21; horse, 208, 13.3, and 0.26; goat, 214, 14.2, and 0.27; dog, 338, 21.1, and 0.25; cat, 286,17.4, and 0.24. The velocity of the displacement reaction varied among rabbits despite their having electrophoretically similar hemoglobins, and was slower than in the other species. The value of k4 for dog Hb, human Hb F, and cat Hb was significantly higher than for the hemoglobins of the human adult, horse, goat and rabbit. Goat hemoglobins A and B, whose α-chains differ, showed the same rate for the replacement reaction.
Oxygen dissociation curves of partially CO-saturated human whole blood drawn freshly or preserved more than 3 wk were studied. With increasing CO-hemoglobin concentrations, oxygen affinity of the blood increased and the Hill coefficient, n, fell and gradually approached unity. The changes induced by CO-hemoglobin showed practically no difference in the presence or absence of 2,3-diphosphoglycerate. The Bohr coefficient, deltalog P50/deltapH, was determined as a function of oxygen saturation for various concentrations of CO-hemoglobin. The coefficient remained essentially unchanged in the presence of CO-hemoglobin. In the presence of less than 50% CO-hemoglobin, a good agreement was observed between the observed oxygen dissociation curves and the curves calculated according to Roughton and Darling (Am. J. Physiol. 141: 17-31, 1944). Based on these results, physiological implications of carboxyhemoglobinemia are discussed quantitatively in comparison with methemoglobinemia.
The flow dependency of hepatic hexobarbital metabolism was examined in the isolated perfused rat liver. At low flow rates (0.5-1.0 ml/min/g of liver) hexobarbital clearance was found to depend on perfusion fluid flow, whereas at higher flow rates drug clearnace approached flow independence. Calculation of the in vivo hepatic blood flow rate suggested that hexobarbital metabolism in vivo should be highly dependent upon flow. Blood flow in the conscious rat was measured by use of radiolabeled microspheres during acute exposure to levels of hypoxic hypoxia (lowered pO2) or carbon monoxide which resulted in equal alterations in arterial oxyhemoglobin content (approximately 65% oxyhemoglobin). Hypoxic hypoxia (8% O2) caused a massive redistribution of flow away from the splanchnic area, resulting in a 45% decrease in hepatic blood flow. Carbon monoxide (500 ppm) was without significant effect on hepatic blood flow. These data would appear to explain the relatively greater inhibitory potency of hypoxic hypoxia on drug metabolism in vivo, since drug delivery to the liver is depressed by hypoxic hypoxia but unaffected by carbon monoxide exposure.
The oxygen dissociation curve and Bohr effect were measured in normal whole blood as a function of carboxyhemoglobin concentration [HbCO]. pH was changed by varying CO2 concentration (CO2 Bohr effect) or by addition of isotonic NaOH or HCl at constant PCO2 (fixed acid Bohr effect). As [HbCO] varied through the range of 2, 25, 50, and 75%, P50 was 26.3, 18.0, 11.6, and 6.5 mmHg, respectively. CO2 Bohr effect was highest at low oxygen saturations. This effect did not change as [HbCO] was increased. However, as [HbCO] was increased from 2 to 75%, the fixed acid Bohr factor increased in magnitude from -0.20 to -0.80 at very low oxygen saturations. The effect of molecular CO2 binding (carbamino) on oxygen affinity was eliminated at high [HbCO]. These results are consistent with the initial binding of O2 or CO to the alpha-chain of hemoglobin. The results also suggest that heme-heme interaction is different for oxygen than for carbon monoxide.
The cerebral metabolic effects of 2.5, 5, 7.5, 10, 20, 30 and 60 min exposure to 1% CO were studied in lightly anesthetized rats by measurement of cerebral cortical contents of selected glycolytic and citric acid cylce intermediates, as well as tissue energy phosphates. The initial change in the glycolytic sequence occurred at 2.5 min with decreases in tissue glucose and glucose-6-phosphate and increases in fructose-1-6-diphosphate which indicated an activation of phosphofructokinase and hexokinase. The "crossover" pattern between glucose-6-phosphate and fructose-1,6-diphosphate was present at 5, 7.5 and 10 min, but not at 20, 30 and 60 min and thus confirmed previous observations that detection of phosphofructokinase activation in acute unifactorial cerebral hypoxia requires tissue study during the early phases of the experimental exposure. The initial activation of phosphofructokinase occurred in the absence of detectable changes in the tissue content of ATP, ADP, AMP or phosphocreatine and therefore suggested that an imbalance of tissue energy homeostasis is not a prerequisite for the activation of glycolysis in CO intoxication. One percent CO resulted in an increasing malate/oxaloacetate ratio at 5 min, followed by a decrease in alpha-ketoglutarate and aspartate at 7.5 min which suggested a shift in the aspartate aminotransferase reaction towards the replenishment of oxaloacetate removed via the malate dehydrogenase reaction. Subsequent increases in alpha-ketoglutarate at 10, 20, 30 and 60 min were associated with increases in alanine, indicating a contributing role for a secondary shift of the alanine aminotransferase reaction in the replenishment of alpha-ketoglutarate. A comparison of the CO induced changes in the glycolytic and citric acid cycle pathways with those seen in acute hypoxemia indicates no basic qualitative differences in the metabolic responses of brain tissue to the two conditions.
Trihalomethanes (haloforms) were metabolized to carbon monoxide by a rat liver microsomal fraction requiring both NADPH and molecular oxygen for maximal activity. GSH alone did not serve as a cofactor; however, GSH in the presence of NADPH and oxygen produced an 8-fold increase in the metabolism of bromoform to CO. Similar results were obtained with other sulfhydryl compounds. The biotransformation of bromoform to CO was characterized with respect to time course, microsomal protein concentration, pH and temperature. The metabolism of haloforms to CO followed the halide order; thus, iodoform yielded the greatest amount of CO, whereas chloroform yielded the smallest amount. A KM of 6.78 +/- 2.71 mM was established for bromoform and the Vmax was 1.09 +/- 0.19 nmol of CO per mg of microsomal protein per min. The energy of activation for this reaction was 6.5 +/- 0.18 kcal/mol. Cytochrome P-450 was found to bind bromoform to produce a type I binding spectrum. Treatment of rats with phenobarbital or 3-methylcholanthrene increased the rate of conversion of bromoform to CO. Cobaltous chloride treatment of rats or storage of microsomal preparations at 4 degrees C reduced the rate of formation of CO from bromoform. SKF 525-A inhibited the conversion of bromoform to CO. These results suggest that haloforms are metabolized to CO via a cytochrome P-450-dependent mixed-function oxidase system.