Mdr P-glycoproteins are not essential for biliary excretion of the hydrophobic heme precursor protoporphyrin in a griseofulvin-induced mouse model of erythropoietic protoporphyria

Abstract

Hepatic complications in erythropoietic protoporphyria (EPP) have been attributed to toxic actions of accumulated protoporphyrin (PP). PP can only be removed via the bile but transport systems involved have not been defined. The aim of this study was to gain insight in the mode of biliary PP excretion, with emphasis on the potential contribution of the Mdr1 P-glycoprotein export pump and biliary lipids as PP carriers. Control mice and mice homozygous for Mdr1a/b (Abcb1) or Mdr2 (Abcb4) gene disruption, the latter unable to secrete phospholipids and cholesterol into bile, were treated with griseofulvin to chemically induce protoporphyria. All groups showed dramatically increased PP levels in erythrocytes and liver after griseofulvin treatment. Histologically, massive PP deposits were found in livers of control and Mdr1a/b ؊/؊ mice but not in those of Mdr2 ؊/؊ mice. Serum unesterified cholesterol and phospholipids were increased by griseofulvin because of formation of lipoprotein-X in control and Mdr1a/b ؊/؊ mice only. Yet, bile flow was not impaired in griseofulvintreated mice, and biliary bile salt, phospholipid, and cholesterol secretion rates were significantly increased. Surprisingly, biliary PP excretion was similar in all 3 groups of griseofulvin-treated mice: the observed linear relationship between hepatic and biliary PP concentrations and identical liver-to-bile concentration ratios in treated and untreated mice suggest a passive mode of excretion. In conclusion, the data show that Mdr P-glycoproteins are not critically involved in biliary removal of excess PP and indicate that the presence of biliary lipids is required for formation of intrahepatic PP deposits. (HEPATOLOGY 2002;35: 299-306.) F errochelatase (EC 4.99.1.1) is a mitochondrial enzyme that transfers iron into protoporphyrin (PP) during the formation of heme. 1 In erythropoietic protoporphyria (EPP), an inherited disease caused by a mutated ferrochelatase gene, ferrochelatase activity is markedly reduced, i.e., in severe cases to 4% to 20% of control values. 2 Impaired ferrochelatase activity leads to a reduced heme synthesis rate and, via induction of the preceding steps in the biosynthetic cascade, to accumulation of the highly hydrophobic heme precursor PP in erythrocytes and liver. 1 Common clinical signs of EPP are erythema and edema caused by PP phototoxicity in the skin. 3 In the liver, PP deposits can be found both in cells and in small bile channels. 4 Severe hepatic complications in EPP are relatively rare, but indicative for transplantation in up to 10% of all cases. 5 PP is not enzymatically altered nor conjugated in the liver. 4 It is highly hydrophobic and is therefore removed from the body exclusively via the bile, followed by fecal excretion. 6 Mechanisms responsible for PP secretion into bile are currently not known. Previous studies performed by our laboratory showed association of PP with cholesterol/phospholipid vesicles in bile, 7 suggesting that these may act as PP carriers in this aqueous solution. Reduction of bilia...

Evaluation of the Contribution of the ATP Binding Cassette Transporter, P-glycoprotein, to in Vivo Cholesterol Homeostasis

“…The mole fraction of gallbladder cholesterol, phospholipid, and bile salts was calculated for each mouse and the mean values presented in Figure 6. Consistent with previous studies, the gallbladder bile in this study was unaffected by Pgp deletion when the mice were fed a normal-fat, normal-cholesterol diet 23,35,36 . Pgp deletion affected gallbladder bile composition when mice were fed diets containing elevated fat (45%kcal vs. 25%kcal) or elevated cholesterol (0.20% vs. 0.02% w/w).…”

Lyophilic properties of protoporphyrin and bilirubin

“…." 1 From these dual assertions they speculate on the mechanism by which PP is excreted across the canalicular membrane into bile. Although the first assertion is literally true, there is no evidence for the second and together they give an inaccurate impression of those properties of PP and bilirubin (BR) that control their hepatobiliary transport and metabolism.…”

“…This may have led to the common notion that PP is "hydrophobic," even "highly hydrophobic." 1,4 However, PP is not only insoluble in water but is insoluble in many nonpolar solvents. Describing it as hydrophobic is like describing a zebra as either black or white.…”

“…That may account for the formation of PP crystals in protoporphyric livers. 1 Because BR and PP have such diametrically different structures and lipophilicities it is unlikely that they cross biomembranes by identical mechanisms and probably unwise to consider one a model for the other. 1 The mechanisms of hepatic uptake of PP and BR and canalicular excretion of PP are uncertain.…”

“…1 Because BR and PP have such diametrically different structures and lipophilicities it is unlikely that they cross biomembranes by identical mechanisms and probably unwise to consider one a model for the other. 1 The mechanisms of hepatic uptake of PP and BR and canalicular excretion of PP are uncertain. But, being chemical processes, they are dependent (inter alia) on the stereochemistry and lyophilic properties of the two pigments.…”