Recent studies have identified and characterized the enzymatic mechanism by which hemoglobin-heme is converted to bilirubin. Under physiologic conditions the enzyme system, microsomal heme-oxygenase, is most active in the spleen followed by the liver and bone marrow, all of which are tissues that normally are involved in the sequestration and metabolism of red cells. Indirect evidence suggested that the reticuloendothelial system is important in this process. To test this hypothesis, conversion of heme to bilirubin was studied in macrophages obtained by chemical or immunological means from the peritoneal cavity or from the lungs of rodents. Homogenates of pure populations of these cells were devoid of heme-oxygenase activity, unless before harvesting the macrophages had been exposed to methemalbumin, microcrystalline hemin, or hemoglobin in vivo. In macrophages exposed to heme pigments, the specific activity of heme-oxygenase was far in excess of that in the spleen or liver. Enzyme activity was also present in the granulomatous tissue surrounding subcutaneous hematomas.
The heme-oxygenase system in macrophages resembles that in the spleen and liver in that it is localized in the microsomal fraction, has an absolute requirement for molecular oxygen and NADPH, is inhibited by carbon monoxide, and has a similar Km. These findings indicate that cells of the reticuloendothelial system, presumably including the Kupffer cells of the liver and the macrophages of the spleen, possess the enzymatic machinery for converting hemoglobin-heme to bilirubin. The reaction is a mixed function oxidation, probably involving cytochrome P450 as the terminal oxidase. Enzyme activity in macrophages is capable of regulatory adaptation in response to substrate loads. In the standard assay system for the enzyme, disappearance of heme always was in excess of the amount of bilirubin formed, suggesting the simultaneous presence of alternate routes of heme degradation not involving bilirubin as an end product or intermediate.
Sequestration and degradation of red blood cells (RBC) are believed to occur in part in the liver, but the magnitude and cellular localization of this process remain uncertain. This problem was studied in rats by investigating isolated parenehymal and sinusoidal cell populations of the liver. After digesting the perfused liver with pronase, hepatic sinusoidal cells were isolated free of RBC and debris. Of the isolated cells, 90 % were phagocytic, as judged by their uptake of colloidal ~gSAu or of aggregated albumin-lalI administered in vivo After administration of spherocytic (heat-treated) RBC, however, only about one quarter of the isolated cells were found to contain phagocytized RBC. This apparently distinct popula~ tion of RBC-phagocydzing cells is designated as '°erythrophagocytic (EP)" ceils. The EP cell population was further characterized functionally by its specific phagocytosis of coltoidaI carbon and of 99mtechnetium-sulfur colloid and histochemically by its peroxidase activity. The role of the EP population in the catabolism of RBC-hemoglobin was studied in isolated hepatic sinusoidal ceils by assay of microsomal heme oxygenase (MHO), which is the inducible enzyme system that converts heine to bilirubin. The MHO activity of individual sinusoidal isolates was related directly to their content of EP ceils
The liver participates in the removal from the circulation of both damaged red blood cells (RBC) and plasma hemoglobin. The specific hepatic cell types involved in these processes have been identified by fractionation of rat liver into pure isolates of parenchymal and sinusoidal cells. After injection of 59Fe-labeled hemoglobin, 85%-95% of the radioactivity in the liver was associated with the parenchymal cells, regardless of whether the hemoglobin was bound to haptoglobin or was free in plasma. By contrast, 59Fe-labeled spherocytic RBC were sequestered entirely by the sinusoidal cell population. Stimulation of microsomal heme oxygenase by administered hemoglobin or RBC indicated that these cell fractions not only sequester but also degrade the ingested hemoglobin-heme. Infusion of doubly labeled 59Fe, 125-I-hemoglobin indicated that the hepatic parenchymal cells remove the intact hemoglobin molecule without exchange or transfer of the heme moiety to other carrier proteins. By contrast, heme bound to albumin was detached from the albumin before its uptake by the parenchymal cells. These findings suggest that, contrary to previous belief, hepatic parenchymal cells play a key role in the metabolism of plasma hemoglobin.
Mitochondria from the whole brain or cerebellum of newborn guinea pigs with experimental bilirubin encephalopathy failed to exhibit uncoupling of oxidative phosphorylation. The pigment concentrations required to initiate uncoupling in vitro are much higher than those found in the brain of neurotoxic animals.
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