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A patient with erythropoietic protoporphyria was studied to determine the sites of excess protoporphyrin formation. The patient's protoporphyrin was pulse labeled by the simultaneous administration of the precursors 2-glycine-(14)C and 3,5-delta-aminolevulinic acid-(3)H; delta-aminolevulinic acid preferentially labels the hepatic pool. Blood and feces were studied at intervals for the next 14 days. Protoporphyrin was extracted from erythrocytes, plasma, and feces, identified by thin-layer chromatography, and quantitated spectrophotometrically, and its specific activity was determined by liquid scintillation spectrometry. Analysis of the kinetic and isotopic data indicated at least two sources of protoporphyrin, one localized in the erythroid cells, a second in the liver. The liver was responsible for the majority of the excess protoporphyrin. This report thus provides evidence of a genetic porphyria exhibiting an abnormality of porphyrin biosynthesis in at least two tissues. We propose that the disease, erythropoietic protoporphyria, be renamed erythrohepatic protoporphyria.

We have recently identified and characterized NADPH-dependent microsomal heme oxygenase as the major enzymatic mechanism for the conversion of hemoglobin-heme to bilirubin-IXalpha in vivo. Enzyme activity is highest in tissues normally involved in red cell breakdown, that is, spleen, liver, and bone marrow, but it usually is negligible in the kidney. However, renal heme oxygenase activity may be transiently increased 30- to 100-fold following hemoglobinemia that exceeded the plasma haptoglobin-binding capacity and consequently resulted in hemoglobinuria. Maximal stimulation of enzyme activity in rats is reached 6-16 hr following a single intravenous injection of 30 mg of hemoglobin per 100 g body weight; activity returns to basal levels after about 48 hr. At peak level, total enzyme activity in the kidneys exceeds that of the spleen or liver. Cyclohexamide, puromycin, or actinomycin D, given just before, or within a few hours after, a single intravenous injection of hemoglobin minimizes or prevents the rise in renal enzyme activity; this suggests that the increase in enzyme activity is dependent on continued synthesis of ribonucleic acid and protein. The apparent biological half-life of renal heme oxygenase is about 6 hr. These observations indicate that functional adaptation of renal heme oxygenase activity reflects enzyme induction either directly or indirectly by the substrate, hemoglobin. Filtered rather than plasma hemoglobin appears to regulate renal heme oxygenase activity. Thus, stabilization of plasma hemoglobin in its tetrameric form with bis (N-maleimidomethyl) ether, which diminishes its glomerular filtration and retards it plasma clearance, results in reduced enzyme stimulation in the kidney, but enhances its activity in the liver. These findings suggest that the enzyme is localized in the tubular epithelial cells rather than in the glomeruli and is activated by luminal hemoglobin. Direct support for this concept was obtained by the demonstration of heme oxygenase activity in renal tubules isolated from rabbits that had been injected with hemoglobin.

Intermittent acute porphyria has recently been distinguished biochemically from other genetic hepatic porphyrias by the observation of diminished hepatic uroporphyrinogen I synthetase activity and increased delta-aminolevulinic acid synthetase activity. Since deficient uroporphyrinogen I synthetase may be reflected in nonhepatic tissues, we have assayed this enzyme in red cell hemolysates from nonporphyric subjects and from patients with genetic hepatic porphyria. Only patients with intermittent acute porphyria had decreased erythrocyte uroporphyrinogen I synthetase activity which was approximately 50% of normal. The apparent K(m) of partially purified uroporphyrinogen I synthetase was 6 x 10(-6)m in both nonporphyrics and patients with intermittent acute porphyria. These data provide further evidence for a primary mutation affecting uroporphyrinogen I synthetase in intermittent acute porphyria. Further-more, results of assay of red cell uroporphyrinogen I synthetase activity in a large family with intermittent acute porphyria suggest that this test may be a reliable indicator of the heterozygous state.