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The pathogenesis of liver disease in protoporphyria has been presumed to result from the hepatic deposition of protoporphyrin. To examine the effects of protoporphyrin on hepatic bile flow and histopathology, studies were performed employing an isolated, in situ, rat liver perfusion system. Rat livers in the control group were perfused with 0-80 μmol sodium taurocholate/h. Rat livers in the experimental group were perfused with sodium taurocholate and (a) sufficient quantities of protoporphyrin to produce maximal canalicular secretion and (b) perfusate protoporphyrin concentrations of 0.01, 0.1, and 1 μM. The administration of protoporphyrin sufficient to achieve maximal canalicular secretion was found to significantly reduce bile flow in rats infused with 0, 40, and 80 μmol sodium taurocholate/h. Linear regression analysis defined the relationship between bile flow and biliary bile acid secretion and showed that the bile acid-independent fraction of bile flow was reduced (P < 0.01). Bile acid-dependent flow was unaffected and there was no significant difference in biliary bile acid secretion rates between control and protoporphyrin-perfused livers. Perfusion of rat livers with varying concentrations of protoporphyrin demonstrated the reduction of bile flow was dose-related. Analysis of perfusate enzyme activity did not reveal abnormalities that could account for the cholestasis. Studies to evaluate the effect of protoporphyrin on regional hepatic hemodynamics were inconclusive. Histopathological studies of control and protoporphyrin-perfused rat livers did not show abnormalities on light microscopy. However, canalicular dilatation, distortion, and loss of microvilli were present in the protoporphyrin-perfused livers examined by transmission electron microscopy. Although ultraviolet microscopy showed diffuse fluorescence of the hepatocytes and canaliculi of protoporphyrin-perfused livers, the deposition of protoporphyrin in amorphous or crystalline forms was notably absent in studies with polarizing and transmission electron microscopy. These studies provide evidence that protoporphyrin has hepatotoxic properties that affect the canalicular secretory apparatus. The mechanism(s) responsible for the injury require further clarification.

To determine the effect of bile acids on hepatic protoporphyrin metabolism, balance studies were performed in isolated perfused rat livers. Hepatic protoporphyrin metabolism was found to increase linearly as a function of protoporphyrin dose in livers infused with and without taurocholate (0.7 μmol/min), but their rates differed significantly. Employing a standard 1500-nmol protoporphyrin bolus dose, infusions (0.7 μmol/min) of taurocholate, glycocholate, deoxycholate, and chenodeoxycholate, but not tauroursodeoxycholate or ursodeoxycholate, increased protoporphyrin metabolism 1.7-to 2.7-fold over control (0 bile acid) values. Bile acid infusion ranging from 0.175 to 1.4 μmol/min confirmed that both taurocholate and chenodeoxycholate increased protoporphyrin disposal significantly more than ursodeoxycholate. For all bile acids, the increase of protoporphyrin metabolism was most pronounced between biliary bile acid excretion rates of 10–50 nmol/min · g liver. These data indicate that (a) bile acids facilitated protoporphyrin metabolism, (b) bile acid structure influenced the effect, and (c) ursodeoxycholate may not be a prime candidate to study the role of bile acids in the treatment of protoporphyria.

To clarify the pathogenesis of protoporphyrin-induced cholestasis, liver surface membrane enzyme activities were determined after (a) isolated rat liver perfusion with protoporphyrin administered by bolus (1.0 mumol) or bolus plus constant infusion (1.0 mumol + 0.05 mumol/min) and (b) combination of liver surface membrane with protoporphyrin (0.9-53.4 nmol/ml) in vitro. The perfusion studies showed that protoporphyrin significantly inhibited Na+, K+- and Mg+2-adenosine triphosphatase activities. In vitro, these adenosine triphosphatase activities and the 5'-nucleotidase activity were inhibited linearly by protoporphyrin up to a concentration of approximately 18 mumol/ml; thereafter, enzyme activity was maintained. Greater inhibition of the adenosine triphosphatase activities occurred with protoporphyrin than was reported for chlorpromazine at similar molar concentrations. This effect was independent of the quantity of membrane protein analyzed and was not reversible with a 50:1 dilution. The inhibitory effect of protoporphyrin on surface membrane enzyme activities was also nonselective. Although the hepatotoxic effects of protoporphyrin may be more generalized, the present data underscore protoporphyrin's toxic interaction with liver surface membranes.