A zebrafish model for hepatoerythropoietic porphyria.

Defects in the enzymes involved in the haem biosynthetic pathway can lead to a group of human diseases known as the porphyrias. yquem (yqe(tp61)) is a zebrafish mutant with a photosensitive porphyria syndrome. Here we show that the porphyric phenotype is due to an inherited homozygous mutation in the gene encoding uroporphyrinogen decarboxylase (UROD); a homozygous deficiency of this enzyme causes hepatoerythropoietic porphyria (HEP) in humans. The zebrafish mutant represents the first genetically 'accurate' animal model of HEP, and should be useful for studying the pathogenesis of UROD deficiency and evaluating gene therapy vectors. We rescued the mutant phenotype by transient and germline expression of the wild-type allele.

Positional cloning of the zebrafish sauternes gene: a model for congenital sideroblastic anaemia.

Many human anaemias are caused by defects in haemoglobin synthesis. The zebrafish mutant sauternes (sau) has a microcytic, hypochromic anaemia, suggesting that haemoglobin production is perturbed. During embryogenesis, sau mutants have delayed erythroid maturation and abnormal globin gene expression. Using positional cloning techniques, we show that sau encodes the erythroid-specific isoform of delta-aminolevulinate synthase (ALAS2; also known as ALAS-E), the enzyme required for the first step in haem biosynthesis. As mutations in ALAS2 cause congenital sideroblastic anaemia (CSA) in humans, sau represents the first animal model of this disease.

Sequential induction of heme pathway enzymes during erythroid differentiation of mouse Friend leukemia virus-infected cells.

The process of erythroid differentiation in mouse Friend leukemia virus transformed cells (T3-C1-2) was examined by following changes in several enzyme activities of the heme biosynthetic pathway and in heme concentration while the cells were undergoing erythroid differentiation after treatment with dimethylsulfoxide. Untreated cells on the one hand, have a limited capacity for spontaneous differentiation. On the other hand, dimethylsulfoxide(DMSO)-treated cells showed an increase in the activities of delta-aminolevulinic acid (ALA) synthetase, ALA dehydratase, uroporphyrinogen-I synthetase, ferrochelatase, and heme concentration by days 1, 1.5, 2, and 4, respectively. The increase of the heme pathway enzymes and heme concentration followed the order of these enzymes or products as they are arranged in the heme biosynthetic pathway. These changes induced by DMSO were effectively inhibited by treatment with actinomycin D, suggesting that continued RNA synthesis is required for the differentiation process. 5-bromo-2'-deoxyuridine (BrdU) (10(-5) M) inhibited the DMSO-induced changes of the heme pathway enzymes. BrdU was most effective when it was present during the first 2 days of cell culture. It gradually lost its inhibitory effect when added after the 3rd day or later. The BrdU-mediated inhibition was completely overcome by the addition of thymidine (7 x 10(-5) M), but not by uridine (7 x 10(-5) M). All these data suggest that a sequential induction of the heme pathway enzyme takes place during erythroid differentiation of Friend leukemia cells, and that the sequential induction of the enzymes may be due to a sequential activation of genes coding for these enzyme activities.

The influence of steroid hormone metabolites on the in vitro development of erythroid colonies derived from human bone marrow.

Certain C19 and C21 steroid metabolites, when incubated with normal human bone marrow cells in culture, increased the number of erythroid colonies in the presence of erythropoietin. Among a number of pairs of C5 epimeric steroids tested, most 5beta (A:B cis) steroids stimulated the growth of both early erythroid progenitor cells (BFU-E) and late erythroid progenitor cells (CFU-E), whereas only a few 5alpha-(A:B trans) steroids stimulated the growth of CFU-E. No 5alpha-compounds of six pairs of steroids studied were found to stimulate BFU-E formation. This structure-activity relationship conforms with that previously observed in studies of steroid induction of ALA-synthase in avian embryo liver cells and hemoglobin synthesis in the cultured avian blastoderm. When human bone marrow cells were preincubated with the steroids for 2 d, followed by incubation with erythropoietin, only the 5 beta-compounds stimulated the growth of BFU-E. Similarly, when addition of steroids was delayed in relation to erythropoietin in the culture, only the 5 beta-derivative of a pair of C5 epimeric compounds displayed an enhancing effect on the growth of BFU-E. This effect required that the steroid addition be made no later than 48 h after initiation of the culture. These data demonstrate that certain natural steroid metabolites significantly stimulate erythropoiesis in normal human bone marrow cells in culture. They also indicate that 5 beta-compounds are more stimulatory than their 5 alpha-epimers, and they suggest that these 5 beta-steroids act preferentially on very primitive erythroid progenitor cells, probably on BFU-E.

Accumulation of protoporphyrin IX from delta-aminolevulinic acid in bovine skin fibroblasts with hereditary erythropoietic protoporphyria. A gene-dosage effect.

Bovine skin fibroblasts accumulated protoporphyrin IX when incubated in culture with the porphyrin-heme precursor, delta-aminolevulinic acid (ALA). Fibroblasts from cattle homozygous for erythropoietic protoporphyria (EPP) and with the clinical symptoms of the disease accumulated approximately sixfold greater amounts of protoporphyrin IX than cells from normal control animals. Cells from obligatory heterozygous animals, which are clinically normal, accumulated an intermediate level of protoporphyrin IX. When these cells were incubated with ALA and CaMg EDTA, all types of cells accumulated approximately the same amount of protoporphyrin IX (approximately 500 nmol/mg protein), suggesting that ferrochelatase activity was equally low after inhibition by treatment with CaMg EDTA in all cells. Thus the ratio of protoporphyrin IX accumulation from ALA in cultures treated with CaMg EDTA compared with controls treated with ALA alone was greatest in normal cells, least in EPP cells, and intermediate in the heterozygote cells. These findings suggest that the amount of protoporphyrin IX accumulation from ALA reflects the extent of deficiency of ferrochelatase and is proportional to the dosage of abnormal EPP gene in cultured fibroblasts. Similarly, stimulation of porphyrin accumulation by CaMg EDTA reflects diminished ferrochelatase activity in these cells. Thus, the results of this study demonstrate the usefulness of estimating protoporphyrin IX formation from ALA for the detection of an EPP gene defect in cultured bovine skin fibroblasts.

Hereditary tyrosinemia. Formation of succinylacetone-amino acid adducts.

Succinylacetone (SA) (4,6-dioxoheptanoic acid) is an abnormal metabolite produced in patients with hereditary tyrosinemia as a consequence of an inherited deficiency of fumaryl acetoacetate hydrolase activity. Patients with this disease are associated with a number of abnormalities, including aminoaciduria, proteinuria, liver failure, commonly hepatoma, and decreased GSH concentration in the liver. In the course of our studies of tyrosinemia, we found that the urine of patients with this disorder contains material(s) that absorbs light at 315 nm. We investigated the nature of the 315 nm material in detail. SA was found to react with amino acids and protein nonenzymatically, to form stable adducts at physiological temperature and pH. All SA adducts with amino acids and/or proteins exhibited an absorption peak at 315 nm. Although all amino acids reacted with SA, the most reactive amino acid was lysine (Lys), followed, in order, by glycine, methionine, phenylalanine, serine, alanine, and glutamine. SA-adducts were unstable at pH below 6, while they were made considerably more stable after reduction with NaBH4, suggesting that SA forms an adduct via Schiff base formation. High-performance liquid chromatography (HPLC) analysis of urines from patients with tyrosinemia revealed the existence of SA-glycine, SA-methionine, SA-tyrosine, and SA-phenylalanine. After digestion of urines with proteinase K, three more HPLC peaks appeared, which all corresponded to SA-Lys adducts. TLC analysis of SA-Lys showed that SA-Lys could form as many as seven different adducts. No SA-adduct peaks were observed in HPLC in urines from normal subjects, patients with other forms of aminoaciduria, or patients with the nephrotic syndrome. In addition to amino acids and proteins, SA reacted with reduced glutathione (GSH) and formed a stable adduct. These findings suggest that SA adduct formation with amino acids, GSH, and proteins is a significant process occurring in tyrosinemia, and may account for certain of the pathologic findings in this hereditary disorder.

Proof that Sn-protoporphyrin inhibits the enzymatic catabolism of heme in vivo. Suppression of 14CO generation from radiolabeled endogenous and exogenous heme sources.

Sn-protoporphyrin (SnPP) suppresses generation of 14CO from hepatic heme labeled with delta-aminolevulinic acid (5-[14C]ALA) or from infused [14C]hemin in rats. SnPP administered 1 h before administration of 5-[14C]ALA virtually abolished the peak output of 14CO occurring 2-3 h after injection of this heme precursor, and during the succeeding 12 h reduced 14CO excretion by approximately 61% compared with controls. When [14C]hemin was infused, SnPP diminished 14CO excretion by approximately 50%. These findings, derived from experiments using radiolabeled endogenous and exogenous heme sources, establish conclusively that the synthetic metalloporphyrin SnPP inhibits the oxidative degradation of heme in the intact animal.

Studies in porphyria. IV. Expression of the gene defect of acute intermittent porphyria in cultured human skin fibroblasts and amniotic cells: prenatal diagnosis of the porphyric trait.

The gene lesion of the porphyrin-heme synthetic pathway in acute intermittent porphyria (AIP) is reflected in a deficient level of activity of the cytosol enzyme uroporphyrinogen I synthetase (URO-S). A marked URO-S deficiency has been demonstrated in the liver and in circulating erythrocytes of individuals with both active and latent AIP. This enzymic abnormality accounts for the excessive production and excretion into urine of the porphyrin precursors, lamda-aminolevulinic acid (ALA) and porphobilinogen (PBG) in AIP subjects. In this study, utilizing cell culture techniques, a marked URO-S deficiency has also been demonstrated in skin fibroblasts from AIP patients and in cells derived through aminocentesis from an approximately 17-wk old fetus. The prenatal diagnosis of the AIP trait in this fetus was confirmed postnatally by the demonstration in the child of a deficient level of erythrocyte URO-S activity which was comparable to those found in her AIP mother and affected sibling and which was approximately one-half the levels characterizing her normal father and aunt and a second unaffected sibling. The identification of the URO-S deficiency in cultured human fibroblasts from AIP patients was facilitated by a newly developed, sensitive assay for the enzyme activity. In this assay, the ability of such cells to convert ALA to protoporphyrin was quantitated; in the sequence of reactions involved in this transformation, URO-S is limiting so that the gene defect of AIP could be simply and precisely determined by appropriate spectrofluorometry of cell extracts. The technique described has distinct advantages over the direct enzymatic assay for URO-S activity in cultured human skin fibroblasts and permits clear differentiation of AIP carrier from normal individuals.

Long-term administration of massive doses of Sn-protoporphyrin in anemic mutant mice (sphha/sphha).

The effects of long-term administration of very large doses of Sn-protoporphyrin on hematological indices, histological changes, plasma bilirubin levels, tissue heme oxygenase activity, and activities of heme biosynthetic enzymes, were examined in genetically anemic mutant mice with hemolytic anemia (sphha/sphha). Long-term weekly treatment with Sn-protoporphyrin (100 mumol/kg body weight for 32 wk) did not alter hematological indices, histological findings, or enzyme activities related to heme biosynthesis, even though it resulted in sustained decreases in microsomal heme oxygenase activity in the liver, kidney, and spleen, and a prolonged decrease in plasma bilirubin concentration. Inhibition of heme oxygenase did not alter the level of cytochrome P-450 in the liver and the kidney. The results indicate that long-term treatment with massive doses of Sn-protoporphyrin suppresses bilirubin formation but does not produce significant histopathological changes or appreciably interfere with heme synthesis, in this strain of genetically anemic mice. These findings provide further support for the idea that suppression of heme degradation to bile pigment by the inhibition of heme oxygenase may prove useful to the prevention of severe hyperbilirubinemia in humans.

Effects of colloidal iron overload on renal and hepatic siderosis and the femur in male rats.

Genetic hemochromatosis is an iron overload disorder, and osteopenic and osteoporotic. Femoral neck bone mineral density (BMD) appears to fall with rising hepatic iron concentrations. A critical role for iron in mediating tissue injury is played via hydroxyl radical formation in nephrotoxicity. We investigated the effects of a colloidal iron overload on renal function, organ siderosis, and femoral bone in male rats. Iron overload reduced body growth, and increased the weights of the liver and spleen. Marked deposition of iron was noted in liver and kidney. Activities of lactate dehydrogenase and alkaline phosphatase were decreased, and the concentrations of blood urea nitrogen and creatinine were increased with the reduction in plasma calcium and inorganic phosphorus levels, i.e. functions of the liver and kidney might be affected by reactive oxygen species such as the superoxide radical, H2O2, and the hydroxyl radical produced by overloaded iron. Damage to the proximal tubular epithelial cells of the kidney and a loss of connectivity of cancellous bone in the epiphysis and of trabecular bone in the metaphysis of the distal femur were observed in iron-overloaded rats with a reduction of femoral bone mineral density, i.e. reabsorption of calcium from the proximal tubular epithelial cells of the kidney might be affected and urinary discharge of calcium might be elevated. It was suggested that iron overload gave rise to osteoporosis combined with renal dysfunction and liver iron overload syndrome.

Hereditary tyrosinemia and the heme biosynthetic pathway. Profound inhibition of delta-aminolevulinic acid dehydratase activity by succinylacetone.

Succinylacetone (4,6-dioxoheptanoic acid) is an abnormal metabolite produced in patients with hereditary tyrosinemia as a consequence of an inherited deficiency of fumarylacetoacetate hydrolase. It is known that patients with this hereditary disease excrete excessive amounts of delta-aminolevulinic acid (ALA) in urine and that certain patients have an accompanying clinical syndrome resembling that of acute intermittent porphyria (AIP). In order to elucidate the relation of succinylacetone to the heme biosynthetic pathway, we have examined the effects of this metabolite on the cellular heme content of cultured avian hepatocytes and on the activity of purified ALA dehydratase from normal human erythrocytes and from mouse and bovine liver. Our data indicate that succinylacetone is an extremely potent competitive inhibitor of ALA dehydratase in human as well as in animal tissues. By using purified preparations of the enzyme from human erythrocytes and mouse and bovine liver, an inhibitor constant ranging from 2 x 10(-7) M to 3 x 10(-7) M was obtained. In cultured hepatocytes, succinylacetone also inhibited ALA dehydratase activity, decreased the cellular content of heme and cytochrome P-450, and greatly potentiated the induction response of ALA synthase to drugs such as phenobarbital, chemicals such as allylisopropylacetamide and 3,5-dicarbethoxy-1,4-dihydrocollidine, and natural steroids such as etiocholanolone. Four patients with hereditary tyrosinemia have been studied and all were found to have greatly depressed levels of erythrocyte ALA dehydratase activity and elevated concentrations of this inhibitor in urine. These findings indicate that tyrosinemia is a disorder of special pharmacogenetic interest because succinylacetone, an abnormal product of the tyrosine metabolic pathway, resulting from the primary gene defect of the disease, profoundly inhibits heme biosynthesis in normal cells through a blockade at the ALA dehydratase level, leading to clinical and metabolic consequences that mimic another genetic disease, AIP.

Interaction between succinyl CoA synthetase and the heme-biosynthetic enzyme ALAS-E is disrupted in sideroblastic anemia.

The first and the rate-limiting enzyme of heme biosynthesis is delta-aminolevulinate synthase (ALAS), which is localized in mitochondria. There are 2 tissue-specific isoforms of ALAS, erythroid-specific (ALAS-E) and nonspecific ALAS (ALAS-N). To identify possible mitochondrial factors that modulate ALAS-E function, we screened a human bone marrow cDNA library, using the mitochondrial form of human ALAS-E as a bait protein in the yeast 2-hybrid system. Our screening led to the isolation of the beta subunit of human ATP-specific succinyl CoA synthetase (SCS-betaA). Using transient expression and coimmunoprecipitation, we verified that mitochodrially expressed SCS-betaA associates specifically with ALAS-E and not with ALAS-N. Furthermore, the ALAS-E mutants R411C and M426V associated with SCS-betaA, but the D190V mutant did not. Because the D190V mutant was identified in a patient with pyridoxine-refractory X-linked sideroblastic anemia, our findings suggest that appropriate association of SCS-betaA and ALAS-E promotes efficient use of succinyl CoA by ALAS-E or helps translocate ALAS-E into mitochondria.

Metabolism of estradiol in liver cell culture. Differential responses of C-2 and C-16 oxidations to drugs and other chemicals that induce selective species of cytochrome P-450.

The oxidative metabolism of estradiol (the natural estrogen 2,3,5(10)-estratriene-3,17 beta-diol) at positions C-2 and C-16 was examined in primary cultures of chick embryo liver cells using estradiol which was labeled with 3H specifically at either the C-2 or C-16 position as the substrate. Oxidation of estradiol by the cultured liver cells was assessed by the release of 3H which accumulated as 3H2O in the culture medium; both C-2 and C-16 oxidative reactions were detectable in the liver cell cultures by this technique. When incubated with a concentration of estradiol substrate close to the Michaelis constant (Km), approximately 45.8 pmol [2-3H]estradiol and 5.0 pmol [16-3H]estradiol/mg protein per minute underwent oxidative metabolism in untreated cells. Total amounts of oxidized product formation after 2 h of incubation were 28 and 5 pmol/mg protein for C-2 and C-16 oxidation, respectively. Treatment of cultures with phenobarbital or 2-propyl-2-isopropylacetamide significantly increased oxidation at C-16 (1.9-fold and 2.6-fold greater than control values, respectively), whereas no significant change in C-16 oxidation was observed after treatment of the cultures with 3-methylcholanthrene, benzo[a]pyrene, or benz[a]anthracene. The latter chemicals, however, were found to increase the extent of oxidation at C-2 significantly (i.e., 1.5-2.2-fold increases over control values). The increase in C-2 oxidation after treatment of cultures with phenobarbital or 2-propyl-2-isopropylacetamide was significantly less than that observed for oxidation at C-16. The apparent Km values for these oxidations in control cultures were 23.5 and 30.3 microM for C-2 and C-16 oxidation, respectively; corresponding maximum velocity (Vmax) values were 119 and 11.7 pmol/mg protein per minute, respectively. These data indicate that the C-2 and C-16 oxidations of estradiol take place in cultured avian hepatocytes and that the extent of metabolism at these positions on the hormone molecule can be altered by chemicals, such as drugs and polycyclic aromatic hydrocarbons, which induce distinctive species of cytochrome P-450 in the liver.

Studies in porphyria. VII. Induction of uroporphyrinogen-I synthase and expression of the gene defect of acute intermittent porphyria in mitogen-stimulated human lymphocytes.

A 50% reduction in the activity of uroporphyrinogen-I (URO) synthase in liver, erythrocytes, and cultured skin fibroblasts characterizes all patients with clinically active acute intermittent porphyria (AIP). The same enzyme defect has also been demonstrated in the erythrocytes and skin fibroblasts of completely latent gene carriers of this disorder and presumably exists in the liver as well. In this study, we examined whether or not the formation of URO-synthase is impaired in AIP cells using lymphocytes treated with mitogens or infected with Epstein-Barr virus. Both mitogens (phytohemagglutinin and pokeweed mitogen) and Epstein-Barr virus induced the synthesis of URO-synthase in lymphocytes, but the induction of URO-synthase in AIP lymphocytes was only 50% as compared with that in normal lymphocytes. The impaired induction of URO-synthase in AIP lymphocytes reflects a specific gene defect because AIP lymphocytes showed normal [(3)H] thymidine uptake into DNA, [(3)H] uridine uptake into RNA, and normal delta-aminolevulinic acid (ALA) synthase, ALA-dehydratase, catalase activities, and heme content. Utilizing the same methodology, the ferrochelatase deficiency of hereditary erythropoietic protoporphyria could also be identified. The K(m) of the induced URO-synthase in AIP cells was identical to that of the enzyme in normal cells. The induced URO-synthase of mitogen-treated AIP lymphocytes was not accompanied by a concurrent enhanced level of ALA-synthase. Moreover, the URO-synthase deficiency in lymphocytes from actively ill AIP patients was not different from the level of enzyme activity when they were in clinical remission, or when compared with the enzyme activity of cells from completely latent AIP gene carriers. The results of this study indicate that the URO-synthase deficiency in AIP may be the result of a gene mutation regulating the rate of synthesis of a normal enzyme rather than a mutation causing a structural abnormality of this enzyme protein.

Porphyrin-heme biosynthesis in organotypic cultures of mouse dorsal root ganglia. Effects of heme and lead on porphyrin synthesis and peripheral myelin.

Well-myelinated cultures of mouse dorsal root ganglia incubated for 48 h with sigma-aminolevulinic acid (ALA) showed intense porphyrin fluorescence localized in myelin sheaths but not in axons or neuronal somata. When the cultures were continuously incubated with a high concentration of lead, focal swelling and segmental degeneration of myelin began to develop within 2 wk. Incubation of cultures with ALA after 3 wk of lead treatment revealed markedly decreased porphyrin fluorescence in myelin sheaths compared with untreated controls. After 6 wk of lead treatment, myelin showed severe segmental degeneration. Porphyrin fluorescence from ALA at this time was barely detectable in these cultures. No fluorescence was visible in the demyelinated axons; however, silver-impregnation staining after fixation demonstrated continuity of the axon despite the severe loss of myelin. When cultures were continuously incubated with lead and heme together for 6 wk, the segmental demyelination seen in cultures treated with lead alone did not occur. These findings suggest that the lead-induced segmental demyelination in cultured mouse dorsal root ganglia may be due to toxic effects of the metal on the heme biosynthetic pathway in myelinating cells and that exogenous heme may counteract this toxic effect of lead.

Immunochemical study of uroporphyrinogen decarboxylase in a patient with mild hepatoerythropoietic porphyria.

Hepatoerythropoietic porphyria (HEP) is due to a marked deficiency of uroporphyrinogen (URO) decarboxylase, a cytosolic enzyme in the heme biosynthetic pathway. Using a radioimmunoassay method, we determined the concentration of URO decarboxylase protein in erythrocytes from a patient with mild HEP and found that the enzyme protein concentration had markedly decreased to less than 7% of the normal controls. This finding, however, was in contrast to the enzyme activity in the patient's erythrocytes, which was 16% of normal control levels and different from previously reported HEP cases in that erythrocytes in our patient contained disproportionately elevated URO decarboxylase activity in comparison to its immunoreactive material. Our findings suggests the possibility of a mutant isozyme in this patient that is not immunoreactive with an antibody raised against the normal enzyme.

Cloning and expression of the defective genes from a patient with delta-aminolevulinate dehydratase porphyria.

Cloning and expression of the defective genes for delta-aminolevulinate dehydratase (ALAD) from a patient with inherited ALAD deficiency porphyria (ADP) were carried out. Cloning of cDNAs for the defective ALAD were performed from EBV-transformed lymphoblastoid cells of the proband, and nucleotide sequences were determined. Two separate point mutations resulting in a single amino acid change in each ALAD allele were identified. One, C718----T, termed 'G1', occurred in the allele within the substrate-binding site, producing an Arg240----Trp substitution; the other, G820----A, termed 'G2', occurred downstream of this site in the other allele, resulting in an Ala274----Thr substitution. Using the reverse transcription-polymerase chain reaction, the mother, the brother, and the sister were shown to have the G1 defect. Expression of the G1 cDNA in Chinese hamster ovary cells produced ALAD protein with little activity; the G2 cDNA produced the enzyme with approximately 50% normal activity. Pulse-labeling studies demonstrated that the G1 enzyme had a normal half life, while the G2 enzyme had a markedly decreased half life. These data thus define the separate point mutations in each ALAD allele, as well as the altered properties of the two enzymic proteins encoded by the mutant genes in a patient with ADP.

Stimulators and inhibitors of hepatic porphyrin formation in human sera.

Human sera were found to contain factors that stimulate and factors that inhibit porphyrin formation by cultured avian liver cells. The capacity of sera to stimulate or inhibit porphyrin formation varied in different hormonal states and in the porphyrias. Sera from 31 post partum women, eight of whom were not lactating, inhibited porphyrin formation to a mean level 30% below the level in control cultures and also inhibited drug and steroid stimulation of porphyrin formation. In contrast, mean porphyrin formation compared to control cultures was increased between 9 and 21% by sera from 52 normal subjects, 16 women on oral contraceptives, and 11 pregnant women. It was increased 193% by sera from nine subjects with acute intermittent porphyria and 172% by sera from 13 subjects with porphyria cutanea tarda. Heated sera or ethanol extracts of sera from all groups of subjects further increased the mean porphyrin stimulation by sera and, for the post partum subjects, eliminated the inhibitory effect. Ethanol extracts of sera from 28 oral contraceptive-treated women caused significantly greater mean stimulation of porphyrin formation than did extracts of sera from 30 normal women. While sera from 17 out of 22 porphyric subjects contained both stimulatory and inhibitory factors, 5 out of 22 had no evidence of an inhibitory component. There appeared to be heterogeneity in the occurrence of the factors among porphyrics.The factor(s) in sera responsible for porphyrin stimulation were heat-stable and insensitive to trypsin; were present in the supernates after ethanol precipitation of plasma proteins; were extractable in ethyl acetate and nondialyzable; and they migrated with the albumincontaining fraction of serum during electrophoresis. The factor(s) responsible for porphyrin inhibition were heat labile, sensitive to trypsin, and resistant to neuraminidase; were present in the ethanol precipitates of sera and were nondialyzable; and they migrated with the gamma globulin fraction of serum during electrophoresis. Inhibition of porphyrin formation was not attributable to heme, free or bound as hemoglobin, hemopexin, or hemalbumin.

Studies in porphyria: functional evidence for a partial deficiency of ferrochelatase activity in mitogen-stimulated lymphocytes from patients with erythropoietic protoporphyria.

In this paper we show that the ferrochelatase defect in erythropoietic protoporphyria (EPP) can readily be identified in mitogen-stimulated lymphocytes since such cells from patients with EPP accumulate approximately twice as much protoporphyrin IX as cells from normal subjects when incubated with a porphyrin precursor, gamma-aminolevulinic acid (ALA). Treatment of cultures with ALA and with the iron chelator, CaMgEDTA significantly increased the level of protoporphyrin IX in mitogen-stimulated lymphocytes from normal subjects, while the same treatment failed to produce an increase in protoporphyrin IX in cell preparations from EPP patients. In contrast to the results with the chelator treatment, supplementation of the cultures with iron and ALA reduced the level of protoporphyrin IX in normal cells, but not in EPP cells. These findings are compatible with a partial deficiency of ferrochelatase in EPP lymphocytes. The gene defects of acute intermittent porphyria and hereditary coproporphyria have previously been identified using lymphocyte preparations from the gene carriers of these diseases. The present study demonstrates that EPP represents another form of human porphyria in which the gene defect of the disease can now be identified in lymphocyte preparations.

Heme oxygenase-1: a fundamental guardian against oxidative tissue injuries in acute inflammation.

Free heme contributes as a major threat to the oxidative tissue injuries because it catalyzes the formation of reactive oxygen species. When free heme concentration is increased, it results in the induction of heme oxygenase-1 (HO-1), which then breaks free heme down. As such, HO-1 plays a pivotal role in the protection of tissues from oxidative injuries.