6. TOXICOKINETICS AND METABOLISM 6.1 Introduction The binding of carbon monoxide to haemoglobin, producing car- boxyhaemoglobin, decreases the oxygen carrying capacity of blood and interferes with oxygen release at the tissue level; these two main mechanisms of action underlie the potentially toxic effects of low- level carbon monoxide exposure. Impaired delivery of oxygen can interfere with cellular respiration and result in tissue hypoxia. Hypoxia of sensitive tissues, in turn, can affect the function of many organs, including the lungs. The effects would be expected to be more pronounced under conditions of stress, as with exercise, for example. Although the principal cause of carbon monoxide-induced toxicity at low exposure levels is thought to be increased carboxyhaemoglobin formation, the physiological response to carbon monoxide at the cellu- lar level and its related biochemical effects are still not fully under- stood. Other mechanisms of carbon monoxide-induced toxicity have been hypothesized and assessed, such as hydroxyl radical production (Piantadosi et al., 1997) and lipid peroxidation (Thom, 1990, 1992, 1993) in the brain of carbon monoxide-poisoned rats, but none has been demonstrated to operate at relatively low (near-ambient) carbon monoxide exposure levels. 6.2 Endogenous carbon monoxide production In addition to exogenous sources, humans are also exposed to small amounts of carbon monoxide produced endogenously. In the process of natural degradation of haemoglobin to bile pigments, in concert with the microsomal reduced nicotinamide adenine dinucleo- tide phosphate (NADPH) cytochrome P-450 reductase, two haem oxygenase isoenzymes, HO-1 and HO-2, catalyse the oxidative break- down of the "-methene bridge of the tetrapyrrol ring of haem, leading to the formation of biliverdin and carbon monoxide. The major site of haem breakdown, and therefore the major organ for production of endogenous carbon monoxide, is the liver (Berk et al., 1976). The spleen and the erythropoietic system are other important catabolic generators of carbon monoxide. Because the amount of porphyrin breakdown is stoichiometrically related to the amount of endogenously formed carbon monoxide, the blood level of carboxy- haemoglobin and the concentration of carbon monoxide in the 134 Toxicokinetics and Metabolism alveolar air have been used with mixed success as quantitative indices of the rate of haem catabolism (Landaw et al., 1970; Solanki et al., 1988). Not all endogenous carbon monoxide comes from haemoglobin degradation. Other haemoproteins, such as myoglobin, cytochromes, peroxidases and catalase, contribute approximately 20–25% to the total amount of carbon monoxide generated (Berk et al., 1976). Approximately 0.4 ml carbon monoxide/h is formed by haemoglobin catabolism, and about 0.1 ml/h originates from non-haemoglobin sources (Coburn et al., 1964). Metabolic processes other than haem catabolism contribute only a very small amount of carbon monoxide (Miyahara & Takahashi, 1971). In both males and females, week-to- week variations in carbon monoxide production are greater than day- to-day or within-day variations. Moreover, in females, carboxyhaemo- globin levels fluctuate with the menstrual cycle; the mean rate of carbon monoxide production in the premenstrual, progesterone phase almost doubles (Delivoria-Papadopoulos et al., 1970; Lynch & Moede, 1972). Neonates and pregnant women also showed a significant increase in endogenous carbon monoxide production related to increased breakdown of red blood cells. Any disturbance leading to increased destruction of red blood cells and accelerated breakdown of other haemoproteins would lead to increased production of carbon monoxide. Haematomas, intra- vascular haemolysis of red blood cells, blood transfusion and ineffec- tive erythropoiesis will all elevate the carbon monoxide concentration in the blood. Degradation of red blood cells under pathological conditions such as anaemias (haemolytic, sideroblastic, sickle cell), thalassaemia, Gilbert’s syndrome with haemolysis and other haema- tological diseases will also accelerate carbon monoxide production (Berk et al., 1974; Solanki et al., 1988). In patients with haemolytic anaemia, the carbon monoxide production rate was 2–8 times higher and blood carboxyhaemoglobin concentration was 2–3 times higher than in normals (Coburn et al., 1966). Increased carbon monoxide production rates have been reported after administration of pheno- barbital, progesterone (Delivoria-Papadopoulos et al., 1970) and diphenylhydantoin (Coburn, 1970). The biological significance of the haem oxygenases appears to be not limited to the above-mentioned role. Evidence is accumulating to suggest that the formed carbon monoxide functions as a gaseous sig- nalling molecule (Verma et al., 1993; Sue-Matsu et al., 1994; Vincent 135 EHC 213: Carbon Monoxide et al., 1994). The discrete neuronal localization of HO-2 is essentially the same as that for soluble guanylate cyclase. Carbon monoxide acts by stimulating this enzyme in target cells. The soluble guanylate cyclase contains one haem per heterodimer. The haem moiety does not bind oxygen, but does bind carbon monoxide (or nitric oxide). The soluble guanylate cyclase activation by carbon monoxide results in an increase in cyclic guanosine monophosphate and subsequent relaxa- tion of smooth muscle cells, activation of nitric oxide synthase in endothelial cells and inhibition of platelet aggregation. Other minor sources of carbon monoxide include the oxidation of phenols, flavonoids and halomethanes, as well as the lipid peroxi- dation of membrane lipids (Rodgers et al., 1994). 6.3 Uptake of carbon monoxide and formation of carboxyhaemoglobin The rate of formation and elimination of carboxyhaemoglobin, its concentration in blood as well as its catabolism are controlled by numerous physical and physiological mechanisms (Figs. 4 and 5). The relative contribution of these mechanisms to the overall carboxy- haemoglobin kinetics will depend on the environmental conditions (ambient carbon monoxide concentration, altitude, etc.), physical activity of an individual and many other physiological processes, some of which are complex and still poorly understood. The mass transport of carbon monoxide between the airway opening (mouth and nose) and red blood cell haemoglobin is predomi- nantly controlled by physical processes. The carbon monoxide transfer to the haemoglobin binding sites is accomplished in two sequential steps: (1) transfer of carbon monoxide in a gas phase, between the airway opening and the alveoli; and (2) transfer in a “liquid” phase, across the air–blood interface, including the red blood cell. Although the mechanical action of the respiratory system and the molecular diffusion within the alveoli are the key mechanisms of transport in the gas phase, the diffusion of carbon monoxide across the alveoli– capillary barrier, plasma and red blood cell is the virtual mechanism in the liquid phase. Diffusion of gases across the alveolar air–haemoglobin barrier is an entirely passive process. In order to reach the haemoglobin binding 136 Toxicokinetics and Metabolism Fig. 4. Oxyhaemoglobin dissociation curves of normal human blood, of blood containing 50% carboxyhaemoglobin and of blood with a 50% normal haemoglobin concentration due to anaemia (adapted from Roughton & Darling, 1944; Rahn & Fenn, 1955; NRC, 1977). sites, carbon monoxide and other gas molecules have to pass across the alveoli–capillary membrane, diffuse through the plasma, pass across the red blood cell membrane and finally enter the red blood cell stroma before reaction between carbon monoxide and haemoglobin can take place. The molecular transfer across the membrane and the blood phase is governed by general physicochemical laws, particularly Fick’s first law of diffusion. The exchange and equilibration of gases between the two compartments (air and blood) are very rapid. The dominant driving force is a partial pressure differential of carbon monoxide across this membrane. For example, inhalation of a bolus of air containing high levels of carbon monoxide will rapidly increase blood carboxyhaemoglobin; by immediate and tight binding of carbon monoxide to haemoglobin, the partial pressure of carbon monoxide within the red blood cell is kept low, thus maintaining a high pressure differential between air and blood and consequent diffusion of carbon 137 Fig. 5. Transport, distribution and metabolism of carbon monoxide in body compartments. Toxicokinetics and Metabolism monoxide into blood. Subsequent inhalation of carbon monoxide-free air progressively decreases the gradient to the point of its reversal (higher carbon monoxide pressure on the blood side than in alveolar air), and carbon monoxide will be released into alveolar air. The air– blood pressure gradient for carbon monoxide is usually much higher than the blood–air gradient; therefore, the carbon monoxide uptake will be a proportionally faster process than carbon monoxide elimina- tion. The rate of carbon monoxide release will be further affected by the products of tissue metabolism. Under pathological conditions, where one or several components of the air–blood interface might be severely affected, as in emphysema, fibrosis or oedema, both the uptake and elimination of carbon monoxide will be affected. The rate of diffusion of gases might be altered considerably by many physiological factors acting concomitantly. Diurnal variations in carbon monoxide diffusion related to variations in haemoglobin