Hepatic and bile porphyrins in patients with protoporphyria and liver failure.

The livers of patients who have protoporphyria and hepatic failure contain large amounts of pigment crystals. Two such patients underwent liver transplantation, providing the opportunity to identify the pigment crystals. Portions of liver were digested enzymically, sedimented through a sucrose gradient, treated with 1% sodium dodecylsulfate, and centrifuged to purify the crystals. Spectrophotometric and high-performance liquid chromatography analysis demonstrated them to be composed of protoporphyrin. Bile samples were obtained from the 2 patients, 4 other patients who did not have liver disease, and 10 control subjects. The porphyrin concentrations in bile from the 6 patients were significantly increased above controls (range 254-7884 micrograms/dl compared with 11-109 micrograms/dl). The ratio of protoporphyrin to bile acid in bile distinguished the 2 patients with advanced liver disease (3105 and 2756 micrograms/mmol) from the 4 patients without liver disease (range 61-926 micrograms/mmol). Thus, analysis of bile from patients with protoporphyria may help in evaluating their hepatobiliary status.

The enzyme defect in bovine protoporphyria. Studies with purified ferrochelatase.

Ferrochelatase was purified from the livers of normal and protoporphyria cattle by chromatography on Blue Sepharose CL-6B in order to investigate the enzyme defect in this disorder. The increase in specific activity (up to 2900-fold) indicated that the normal and protoporphyria enzymes were purified to a similar degree. The mutant enzyme had catalytic activity which was 10 to 15% of normal ferrochelatase, although the Michaelis constants for protoporphyrin and iron were similar. The molecular mass of the normal and protoporphyria enzyme protein was 40 kDa as evaluated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). In the presence of 15 mM sodium cholate, gel filtration demonstrated a similar size. However, at a lower concentration of sodium cholate (4 mM) the molecular mass was about 240 kDa, suggesting that the purified enzymes aggregate under this condition. Polyvalent antibodies were raised in rabbits using as antigens purified normal native enzyme and normal 40-kDa protein which had been further purified by preparative SDS-PAGE. In Western blots these antibodies complexed with both the normal and mutant 40-kDa proteins. The amount of 40-kDa protein in normal and protoporphyria mitochondrial fractions was also similar as evaluated by Western blots. These studies indicate that the ferrochelatase defect in bovine protoporphyria probably results from a point gene mutation that causes a minor change in enzyme structure.

Enzymatic formation of zinc-protoporphyrin by rat liver and its potential effect on hepatic heme metabolism.

The mitochondrial enzyme heme synthase (ferrochelatase) catalyzes the formation of heme from ferrous iron and protoporphyrin. Using a fluorometric assay, the enzymatic formation of zinc-protoporphyrin in rat liver tissue was compared with heme formation. With sonicated liver homogenate, the rate of zinc-protoporphyrin formation was similar to that of heme when each metal was present alone in the reaction mixture. When combined in the reaction mixture, the two metals competed for the enzyme. With intact mitochondria, zinc was a better substrate, as 90% of the product was zinc-protoporphyrin in the presence of 50 microM zinc and 50 microM iron. The effect of zinc-protoporphyrin on the induction of delta-aminolevulinic acid synthase, the rate-limiting enzyme in hepatic heme biosynthesis, was also examined in the rat. Intraperitoneal injection of allylisopropylacetamide (350 mumol) induced activity of the enzyme fourfold. Concomitant administration of zinc-protoporphyrin (4 mumol) suppressed the induction by 52%, nearly as effectively as the same amount of heme. These findings indicate that the formation of zinc-protoporphyrin in liver may (a) inhibit the synthesis of heme and (b) exert negative feedback regulation on delta-aminolevulinic acid synthase. This combination would reduce the hepatic heme level.

Bovine protoporphyria: documentation of autosomal recessive inheritance and comparison with the human disease through measurement of heme synthase activity.

Protoporphyria is an autosomal dominant disease in man in which protoporphyrin accumulated because of a defect in heme synthase (ferrochelatase) activity. A disease has been described in cattle that has the same manifestations as does the human disease. We measured heme synthase activity in sonicates of cultured skin fibroblasts and whole liver homogenates from animals with protoporphyria, their unaffected parents, and normal cattle in order to examine the mode of inheritance and compare it with human protoporphyria. The mean activity (+/- SEM) in fibroblasts from the three groups was 2.0 +/- 0.4, 47 +/- 12, and 149 +/- 10 pmol heme formed/mg protein per hr, respectively, consistent with autosomal recessive inheritance. Similarly, the levels of heme synthase activity in livers of the parents were intermediate to those of normal animals and of animals with protoporphyria. When compared with normal human fibroblasts and liver, the specific activity of heme synthase in normal bovine tissue was significantly higher. These studies indicate that manifestations of protoporphyria do not occur in cattle unless the animal is homozygous for the gene defect, whereas in humans, the heterozygous condition is sufficient. This is probably because the specific activity of heme synthase in cells of heterozygous animals is not reduced to a level that significantly alters heme metabolism.

A radiochemical assay for heme synthase activity.

A radiochemical assay for heme synthase (ferrochelatase) activity is described in this report. The principle of the assay is to measure the incorporation of 59Fe, added to the reaction flask with a known amount of iron, into heme. Iron is maintained in the ferrous state, and oxygen is excluded from the flask during the incubation of the substrates with enzyme. Labeled heme product is extracted by solvent partitioning and counted in a gamma scintillation spectrometer. The assay can detect picomole amounts of product and is most useful when low levels of heme synthase activity are present.