Studies on the Mechanism of Sn-Protoporphyrin Suppression of Hyperbilirubinemia Inhibition of Heme Oxidation and Bilirubin Production Creuza S. Simionatto, Karl E. Anderson, George S. Drummond, and Attallah Kappas The Rockefeller University Hospital, New York, New York 10021 Abstract The synthetic heme analogue Sn-protoporphyrin is a potent competitive inhibitor of heme oxygenase, the rate-limiting enzyme in heme degradation to bile pigment, and can entirely suppress hyperbilirubinemia in neonatal animals and signifi- cantly reduce plasma bilirubin levels in a variety of circum- stances in experimental animals and man. To further explore the mechanism by which this metalloporphyrin reduces bilirubin levels in vivo, we have examined its effects on bilirubin production in bile duct-cannulated rats, in which bilirubin derived from heme catabolism is known to be rapidly excreted in bile. The administration of Sn-protoporphyrin (10-50 ;Lmol/ kg body weight) was followed by prompt (within -1 h) and sustained (up to at least 18 h) decreases in bilirubin output, to levels 25-30 percent below the levels of bilirubin output in control bile fistula animals. The metalloporphyrin had no effect on bile flow or the biliary output of bile acids. Infusions of heme, which is taken up primarily in hepatocytes, or of heat- damaged erythrocytes, which are taken up in reticuloendothelial cells, resulted in marked increases in bilirubin output in bile in control animals; these increases were completely prevented or substantially diminished by Sn-protoporphyrin administra- tion. By contrast, the metalloporphyrin did not alter the high levels of bilirubin in plasma and bile that were achieved in separate experiments by the constant (16 h) infusion of uncon- jugated bilirubin to bile duct-cannulated rats. Thus, Sn-proto- porphyrin exerts no major effects on the metabolic disposition of preformed bilirubin. Heme oxygenase activities were mark- edly decreased in microsomal preparations from liver, spleen, and kidneys in these experiments, to a degree comparable to the decreases we have observed in the intact rat. We also demonstrated that a substantial proportion (19-35%) of a dose of Sn-protoporphyrin is promptly excreted in bile and that the time course of biliary excretion of this compound more closely reflects plasma concentrations of the metalloporphyrin, which decline rapidly, rather than concentrations in liver, which are considerably more persistent. These results indicate that Sn-protoporphyrin substantially reduces the in vivo production of bilirubin from the degradation of endogenous as well as exogenous heme in the rat. Moreover, this inhibitory effect of the synthetic metalloporphyrin on bilirubin production occurs in both hepatocytes and reticulo- endothelial cells, which are the major tissue sites for bilirubin Received for publication 17 July 1984 and in revised form IS October 1984. formation. In other studies, we have established that heme oxygenase blockade by Sn-protoporphyrin leads to a marked and rapid excretion of heme into bile presumably because the synthetic metalloporphyrin blocks heme from binding to the catalytic site of heme oxygenase, thereby preventing its metab- olism to bile pigment and making it available for excretion via the biliary system into the gut. These studies strongly suggest that Sn-protoporphyrin diminishes hyperbilirubinemia in ani- mals and man by inhibiting the production of the bile pigment in vivo, and that its principal mode of action involves a potent and sustained competitive inhibition of heme oxygenase. Introduction Sn-protoporphyrin is a synthetic heme analogue that potently inhibits the activity of heme oxygenase (1, 2), the rate-limiting enzyme for the degradation of heme to bile pigment (3). This inhibition is competitive in nature, and has been demonstrated in a number of human as well as animal tissues including liver, spleen, and kidney when the metalloporphyrin is admin- istered in vivo, or when it is added to microsomal preparations or to purified heme oxygenase in vitro (1, 2, 4-7). We have shown that administration of Sn-protoporphyrin shortly after birth prevents the development of neonatal hyperbilirubinemia in the rat (1, 2, 8), and this effect has been confirmed by others in the rhesus neonate (9). The compound also decreases plasma bilirubin levels in adult mice with congenital forms of severe hemolytic anemia (10), in the post-natal suckling rat with hyperbilirubinemia resulting from the administration of heme or the heme precursor 6-aminolevulinic acid ( 11), in the bile duct-ligated rat ( 12), and in patients with sustained jaundice due to primary biliary cirrhosis (12). It also evokes a marked increase in the excretion of heme in the bile of bile duct- cannulated rats (11). Using a fluorometric method for the measurement of Sn-protoporphyrin in adult rats (1 3), we have found that the metalloporphyrin is rapidly cleared from plasma, and persists in tissues where it can inhibit heme oxygenase activity promptly and for prolonged periods of time (14). The purpose of the present study was to examine the effect of Sn-protoporphyrin on bilirubin production in vivo, as assessed by measuring bilirubin output in the bile duct- cannulated (bile-fistula) animal. Bilirubin formed from endog- enous heme catabolism is normally quite rapidly and completely excreted in bile. Thus, a change in total biliary output of bilirubin in the absence of alterations in plasma bilirubin levels or bile flow represents good evidence of an altered rate of bilirubin production. In addition, we have examined the ability of Sn-protoporphyrin to influence the rate of conversion of exogenous heme to bilirubin in hepatocytes and reticuloen- dothelial cells utilizing both infused heme and infused heat- damaged erythrocytes in appropriate experiments. Finally, we Sn-Protoporphyrin Suppression of Bilirubin Production 513 J. Clin. Invest. © The American Society for Clinical Investigation, Inc. 0021-9738/85/02/0513/09 $ 1.00 Volume 75, February 1985, 513-521