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Publisher Summary The enzyme uroporphyrinogen III synthase catalyzes a remarkable reaction in which preuroporphyrinogen is rearranged and cyclized to yield uroporphyrinogen III. Uroporphyrinogen III is the common precursor for hemes, chlorophylls, vitamin B 12 , and all other tetrapyrroles. This chapter presents coupled assay for uroporphyrinogen III synthase. In the method presented in this chapter, preuroporphyrinogen is generated in situ from porphobilinogen by the action of purified porphobilinogen deaminase and the amount of uroporphyrinogen III formed by the synthase from the preuroporphyrinogen is then quantified, against the unavoidable formation of uroporphyrinogen. This is achieved by spectrophotometric or fluorometric analysis of uroporphyrins I and III, the formation of which is validated by high-performance liquid chromatography separation of the isomers. The method is particularly useful during enzyme purifications in which large numbers of samples need to be assayed rapidly. The assay method has also been adapted to measure enzyme levels in blood and other tissues.

When humans or plants are exposed to certain chemicals which interfere with heme biosynthetic enzymes, porphyrinogen intermediates accumulate and are oxidized to cytotoxic porphyrins. Here we have investigated the role of peroxidases in porphyrinogen oxidation. Horseradish peroxidase (HRP) rapidly oxidizes uroporphyrinogen to uroporphyrin and this is inhibited by ascorbic acid. HRP also oxidizes deuteroporphyrinogen (a synthetic porphyrin similar to protoporphyrinogen), but the yield of porphyrin is lower than with uroporphyrinogen as substrate. This low yield is in part due to a rapid, HRP-dependent conversion of deuteroporphyrin (but not uroporphyrin) to a green compound with spectral characteristics of a chlorin with a large peak at 638 nm. This reaction requires addition of a sulfhydryl reductant such as glutathione and is inhibited by ascorbic acid. These findings suggest that cellular peroxidases and ascorbic acid levels may play a role in modifying the phototoxic tetrapyrroles which accumulate in plants and humans after certain environmental exposures.

Alterations of specific metabolic pathways can be used as sensitive indicators of toxicity by chemicals and can give valuable information on the mechanism(s) involved. Short-term effects of lead on hepatic haem biosynthesis were studied in an in vitro system. Primary cultures of adult rat hepatocytes were exposed for 24–48 hr to lead (0.024–3.6 m m ), and excreted and intracellular porphyrins were measured in untreated and lead-treated cultures. Cytotoxicity, as estimated by enzyme leakage, and morphological alterations were also evaluated. Control hepatocytes produced porphyrins at a rate of 387 pmol/mg cellular protein/day. Most of the released and intracellular porphyrins were protoporphyrins, although uro- and coproporphyrins were also detected in lower amounts. After 24 hr of exposure to 0.1–3.6 m m Pb 2+ , excreted porphyrins decreased by 24–92% and intracellular porphyrins by 36–60%, while 48 hr of exposure to 0.024–3.6 m m Pb 2+ caused a progressive reduction of 77–97% in porphyrin excretion and of 49–67% in intracellular porphyrins. Lead exposure also produced a differential decrease of proto-, copro- and uro-porphyrin excretion. These lead effects can be explained mainly by inhibition of the enzyme 5-aminolaevulinate dehydratase, resulting in a decreased monopyrrole supply for porphyrin biosynthesis, and probably by inhibition of the enzyme uroporphyrinogen decar☐ylase. Morphological alterations and enzyme leakage were detected only after 24 hr of exposure to 2.4 m m and 48 hr of exposure to 3.6 m m Pb 2+ , respectively. The results show that changes in porphyrin production, and particularly in their excretion, in cultured rat hepatocytes are useful indicators of lead toxicity, since they are more sensitive than enzyme leakage and can give preliminary information on the enzyme(s) that could be affected. They also suggest the potential benefits of the use of this method for the evaluation of compounds that alter haem biosynthesis.

A 4-hydroxybenzoate decarboxylase from the anaerobe Clostridium hydroxybenzoicum strain JW/Z-1T was purified and partially characterized. It had an apparent molecular mass of 350 kDa and consisted of six identical subunits of 57 kDa each. The temperature optimum for the decarboxylation was approximately 50 degrees C, the optimum pH 5.6-6.2. The pI of the enzyme was 5.1. The activation energy for decarboxylation of 4-hydroxybenzoate was 65 kJ.mol-1 (20-37 degrees C). The enzyme also catalyzed decarboxylation of 3,4-dihydroxybenzoate. The apparent Km and kcat values obtained for 4-hydroxybenzoate were 0.40 mM and 3.3 x 10(3) min-1, and for 3,4-dihydroxybenzoate 1.2 mM and 1.1 x 10(3) min-1, respectively, at pH 6.0 and 25 degrees C. The enzyme activity was not influenced by the addition of biotin or avidin to either the crude cell extracts or the purified enzyme. The p-hydroxyl group of hydroxybenzoate appears to be essential for binding by the enzyme. The N-terminal amino acid sequence shows some similarity to the uroporphyrinogen decarboxylases from Synechococcus and Saccharomyces. The enzyme catalyzed the reverse reactions, that is, the carboxylation of phenol to 4-hydroxybenzoate and of catechol to 3,4-dihydroxybenzoate. The carboxylation did not require ATP.

Raised plasma uroporphyrin levels were found in all the 14 patients with end-stage renal disease studied, 7 of whom were on continuous ambulatory peritoneal dialysis (CAPD) and 7 on hemodialysis (HD). The elevation observed in the HD group was higher than that noted in the CAPD group; 18-fold in the former and 13-fold in the latter. The difference in uroporphyrin plasma levels between the two dialysis populations might be explained, at least partially, by the reduced activity of uroporphyrinogen decarboxylase (UROD), the enzyme which converts uroporphyrinogen to coproporphyrinogen. A decrease of 48% was noted in the HD group, whereas no change was observed in the CAPD group. A significant negative correlation (r = -0.37, p0.01) was found between the concentration of uroporphyrin in plasma and the activity of UROD. In view of the data shown, it is suggested that erythrocyte UROD activity should be interpreted with caution in HD patients suspected of having porphyria cutanea tarda.

The extant biosynthesis of tetrapyrrole macrocycles has been considered a paradigm for the prebiotic formation of such molecules, yet only a few analogous non-enzymic reactions along the overall process have been demonstrated. In a prior study, the aqueous non-enzymic reaction of a dione and an aminoketone (δ-aminolevulinic acid) afforded uroporphyrinogen, Nature's last universal precursor to all extant tetrapyrroles. Here, in one flask the non-enzymic combinatorial reaction of two diones (substituents = acetic acid and methyl) and two aminoketones (substituents = propionic acid and ethyl) yields four pyrroles, which upon subsequent combinatorial reaction afford a distribution of porphyrinogens. A software program for virtual library generation predicts 538 porphyrinogens from this [2 × 2] reaction (owing to combinations and permutations) of which there are 25 sets of isomers based on condensed formulas of substituents. The collection spans the entire range of polarity enabled by the biosynthesis including uro- (number of carboxylic acids = 8), copro- (4), meso- (2), and etio-porphyrinogen (0). The first two are successive intermediates in the extant biosynthesis, the latter two resemble in polarity the advanced biosynthetic products protoporphyrin and chlorophyll. Upon consideration of substituent patterns, the porphyrins (obtained by oxidation of the porphyrinogens) can be grouped into one of four polarity categories (predicted percentage): hydrophilic (0.4%), uncertain (83.6%), amphiphilic (15.6%), and hydrophobic (0.4%). HPLC and mass spectrometry data are consistent with expectations to the limit of analytical capabilities. Thus, in terms of the polarity of the tetrapyrrole macrocycles formed, an all-at-once non-enzymic combinatorial process recapitulates features of the stepwise biosynthetic pathway.

The biosynthesis of uroporphyrinogen III, the precursor of hemes, chlorophylls, corrins and related structures, is catalyzed by the porphobilinogenase system (PBGase), a complex of two enzymes, PBG-Deaminase (PBG-D) and Isomerase. Although the separate enzymes have been studied in some detail less work has been performed on the properties of the complex. In this study the kinetic behaviour of the enzyme PBGase in a normal yeast strain, D273-10B, and its derivative B231 has been investigated. Uroporphyrinogen formation was linear with time up to 2 hr at 37 degrees C. The enzyme complex shows classical Michaelis-Menten kinetics. From the double reciprocal plots kinetic parameters were estimated for PBGase and PBG-D. Porphyrins were found to be competitive inhibitors with respect to porphobilinogen (PBG) and these compounds appeared to act as inhibitors by forming dead-end complexes with the free enzyme. 5-Aminolevulinic acid (ALA) also inhibited PBGase and this inhibition was overcome by addition of levulinic acid (2 microM). These results indicate that ALA, is not an inhibitor but acts through its conversion into porphyrins which are the true inhibitors.

1. The effect of the administration of several doses of Enflurane and Isoflurane (2 ml/kg, i.p., daily) on heme metabolism and glucose levels was studied. 2. Liver and kidney delta-Aminolevulinic acid synthetase (ALA-S) activities were 85% (P0.01) induced after the third dose of Enflurane, instead induction of this enzyme was only detected, in animals receiving one dose of Isoflurane. 3. Blood Porphobilinogenase and deaminase (50%, P0.01) inhibition was produced only when animals received a single dose of the anesthetics. 5. ALA-S induction observed after the third dose of anesthetics could be a consequence of long lasting depletion in heme synthesis produced by blocking at uroporphyrinogen level.

Two alternatives for the treatment of lead intoxication, administration of zinc or a thiol donor, S-adenosyl-L-methionine (SAM), were analysed. Rats were exposed to lead (Pb)-acetate (60 mg/l) in drinking water during 90 days; one group also received SO4Zn in water (40 mg/l), while another received both Pb and SAM (5 mg/24 hr intraperitoneally. Erythrocytic delta-aminolaevulinic dehydratase (ALA-D) activity was significantly reduced (P < 0.001) both in rats receiving Pb alone and in rats receiving Pb and each of the other two treatments. The high erythrocytic uroporphyrinogen synthetase (URO-S) activity noticed in Pb administered rats, was significantly (P < 0.001) reduced in animals treated either with zinc or with SAM. Hepatic ALA-D activity tended to decrease while renal enzyme activity was not modified by the low level Pb exposure used in this work. Interestingly, SAM treated rats in both tissues exhibited significantly (P < 0.01) higher activities of the enzyme. It is argued that SAM treatment causes a surplus of thiols that allows the full expression of ALA-D catalytic activity.

Previous studies on the mechanism of the uroporphyria caused by polyhalogenated aromatic hydrocarbons have indicated a key role of cytochrome P450 of the 1A subfamily in catalyzing uroporphyrinogen (UROgen) oxidation. Here we report that ascorbic acid (ASC) inhibits UROgen oxidation in primary cultures of chick embryo hepatocytes and hepatic microsomes from chickens and mice. In hepatocyte cultures, 0.15 mM ASC totally prevented the accumulation of uroporphyrin (URO) induced by treatment of cells with the combination of 3,4,3',4'-tetrachlorobiphenyl (TCB) and 2-propyl-2-isopropylacetamide (PIA), but had no effect on the induction of protoporphyrin accumulation by PIA and desferrioxamine. However, addition of 5-aminolevulinic acid (ALA) to cultures treated with PIA plus TCB decreased the ability of ASC to prevent URO accumulation, suggesting that the effectiveness of ASC was dependent on the intracellular concentration of ALA or its metabolites. Similarly, when chick hepatocyte cultures were treated with TCB plus exogenous ALA to produce URO accumulation, the effectiveness of ASC was also less than when ALA was produced endogenously. Under this condition, addition of piperonyl butoxide, a P450 inhibitor, increased ASC inhibition of URO accumulation. ASC competitively inhibited the oxidation of UROgen by hepatic microsomes from chicks or mice treated with 3-methylcholanthrene (MC) with Ki for ASC being about 0.1 mM. ASC prevented formation of a 500-nm absorbing compound, probably tetrahydrouroporphyrin, the first intermediate in UROgen oxidation. These results are consistent with ASC preventing URO accumulation in hepatocytes by competitive inhibition of the first step of UROgen oxidation and suggest a new physiological role of ASC, that of maintaining UROgen in the reduced state.

Uroporphyrinogen decarboxylase (EC 4.1.1.37) was purified 600-fold from Rhodobacter sphaeroides grown anaerobically in the light. The enzyme, under both denaturing and non-denaturing conditions, is a monomer of M(r) 41,000. The Km values are 1.8 microM and 6.0 microM for the conversion of uroporphyrinogen I and III to coproporphyrinogen I and III respectively. The enzyme is susceptible to inhibition by both uroporphyrinogen and uroporphyrin. The pH optimum is 6.8 and the isoelectric point is 4.4. The importance of cysteine and arginine residues is implicated from studies with inhibitors. The sequence of the first 29 amino acids of the N-terminus shows a high degree of similarity to the primary structures of other uroporphyrinogen decarboxylases. Studies on the order of decarboxylation of the four acetic acid side chains of uroporphyrinogen III suggest that at high substrate levels a random route is preferred.

Both major inducible forms of liver cytochrome P450 can interact with polyhalogenated biphenyls (PBCS) of the appropriate configuration to produce oxidizing species, as judged by the degradation of bilirubin, a readily oxidizable compound, by isolated microsomes in vitro. Planar PCBs are active with cytochrome P450 IA1, while non-planar di-orthosubstituted compounds are effective with cytochrome P450 IIB1.Evidence for uroporphyrinogen oxidation under similar conditions has also been obtained with 3-methylcholanthrene-induced rat liver microsomes, where the stimulatory effect of a planar biphenyl, though not as great and consistent as with bilirubin, could also be demonstrated. The nature of the oxidizing species produced by the microsomes is still uncertain, but bilirubin undergoing enzymic oxidation shows similar spectral changes as in a chemical oxidation system. we suggest that a PCB of the appropriate steric configuration may first induce the relevant cytochrome in vivo, then become bound to the active site of the induced cytochrome and lead to production of oxidative species. These may then attack not only bilirubin, but also other key targets in the cell, including hexahydroporphyrins (porphyrinogens) and DNA bases.

Abstract 165 Porphyria cutanea tarda (PCT), the most common porphyria in humans, results from subnormal activity of uroporphyrinogen decarboxylase (URO-D) in hepatocytes. The enzymatic defects results in cellular accumulation of the highly carboxylated uroporphyrinogen and heptacarboxyl porphyrinogen. The excess porphyrinogens are oxidized to porphyrins, exported to plasma and eventually eliminated in the urine. The mechanism by which uroporphyrin and heptacarboxyl porphyrin are transported across cellular membranes is not known. We generated a mouse model of the familial form of PCT 1. Mice heterozygous for a deletion of the Uro-d gene and homozygous for deletion of the hemochromatosis gene Hfe (genotype Uro-d+/−;Hfe−/−) accumulate hepatic porphyrins in a pattern similar to humans with PCT. Hepatocytes were isolated from porphyric mice using an in situ two-step EGTA/collagenase perfusion followed by an iso-density Percoll centrifugation step to remove damaged cells. Confocal microscopy was used to examine cells for porphyrin fluorescence by illuminating cells at 405 nm and monitoring the emission between 600 and 650 nm. Fluorescent porphyrins were present as punctate bodies within the cytosol of the hepatocytes. Co-staining with fluorescent probes (mitotracker and lysotracker) demonstrated co-localization of porphyrins with lysotracker, indicating that porphyrins were present in lysosomes. We utilized a yeast model to determine the molecular mechanism by which porphyrins are transferred from the cytosol to the lysosome. In S. cerevisiae the vacuole is analogous to the mammalian lysosome. The yeast homolog of Uro-d is HEM12. In a hem12 deletion strain, uroporphyrin accumulates in the vacuole. ABC transporters have previously been shown to transport tetrapyrroles across membranes. We used a candidate approach to identify the ABC transporter responsible for vacuolar transport of uroporphyrin by creating deletion strains of the five known vacuolar ABC transporters BPT1, YBT1, YCF1, VMR1 and NFT1. Vacuolar membranes were prepared from these strains by spheroplasting yeast with oxalyticase, osmotically lysing the cells and floating the vacuoles on a Ficoll gradient. Isolated vacuoles were assayed for tetrapyrrole uptake by incubating vacuoles (200 ug total protein), substrate (20 uM) and Mg2+-ATP (50 uM) at 30 °C for 1, 2, 4 and 8 min in 100 uL of import buffer (300 mM sorbitol, pH 6.8). As controls, reactions were carried out at 4 °C and in the absence of ATP. Vacuolar import of uroporphyrin was temperature-dependent (no import occurred at 4 °C) and was dependent on the presence of ATP. Uroporphyrinogen was imported but, the rate was approximately 10% that of uroporphyrin. No import of protoporphyrin IX was observed. Import of uroporphyrin into vacuoles from each of the ABC transporter mutants was essentially the same as wild-type vacuoles with the exception of the ycf1 deletion strain. There was no import of uroporphyrin or uroporphyrinogen in vacuoles from the ycf1 deletion stain. The mammalian ABC-type transporter with the highest homology to yeast YCF1 is ABCC1. ABCC1 has recently been shown to transport a tetrapyrrole (cobalamin) 2 making ABCC1 a candidate transporter responsible for moving uroporphyrin across cellular membranes in humans. 1. Phillips JD, Jackson LK, Bunting M, et al. A mouse model of familial porphyria cutanea tarda. Proc Natl Acad Sci U S A. 2001;98:259-264. 2. Beedholm-Ebsen R, van de Wetering K, Hardlei T, Nexo E, Borst P, Moestrup SK. Identification of multidrug resistance protein 1 (MRP1/ABCC1) as a molecular gate for cellular export of cobalamin. Blood. 2010;115:1632-1639. Disclosures: No relevant conflicts of interest to declare.

Porphobilinogen-deaminase (PBG-D) and PBG-D-isomerase complex (PBG-D-I) from pig liver were isolated and partially purified. Uroporphyrinogen I and III formation was found to be linear with time and protein concentration. Optimal pH was about 7.4 and 7.6-7.8 for PBG-D and PBG-D-I complex, respectively. Some properties of the isolated enzymes were studied. Molecular mass determination gave a value of 40,000 Da for PBG-D and 50,000 Da for the complex. Both enzymes exhibited classical Michaelis-Menten kinetics. Km and Fmax parameters were estimated. The effect of several divalent cations, ammonia and thiol reagents was also investigated. The differential action of some of these chemicals on PBG-D and PBG-D-I system would suggest that PBG-D and isomerase may not be only physically adjacent but actually associated.

Uroporphyrinogen decarboxylase (URO-D) is a cytosolic heme-biosynthetic enzyme that converts uroporphyrinogen to coproporphyrinogen. Defects at the uroporphyrinogen decarboxylase locus cause the human genetic disease familial porphyria cutanea tarda. A splice site mutation has been found in a pedigree with familial porphyria cutanea tarda that causes exon 6 to be deleted from the mRNA. The intron/exon junctions on either side of exon 6 fall between codons, so the resulting protein is shorter than the normal protein, missing only the amino acids coded by exon 6. The shortened protein lacks catalytic activity, is rapidly degraded when exposed to human lymphocyte lysates, and is not detectable by Western blot analysis in lymphocyte lysates derived from affected individuals. The mutation was detected in five of 22 unrelated familial porphyria cutanea tarda pedigrees tested, so it appears to be common. This is the first splice site mutation to be found at the URO-D locus, and the first mutation that causes familial porphyria cutanea tarda to be found in more than one pedigree.

Human porphyria cutanea tarda is an unusual consequence of common hepatic disorders such as alcoholic liver disease. Hepatic iron plays a key role in the expression of the metabolic lesions, i.e., defective hepatic decarboxylation of porphyrinogens, catalyzed by uroporphyrinogen decarboxylase. This prompted the present study to determine the in vitro effects of iron on the uroporphyrinogen substrate in the absence and presence of atmospheric oxygen. We observed that (i) unless oxygen is the limiting reactant, autoxidation of ferrous iron and iron-catalyzed oxidation of uroporphyrinogen occurred soon after initiating the reaction at pH 7.4 and 30 degrees C in buffers which are non- or poor chelators of iron; (ii) the rates of uroporphyrinogen oxidation were proportional to the initial concentration of ferrous ion; (iii) about 70% of the oxidations of uroporphyrinogen were accountable due to a free-radical chain reaction pathway involving superoxide radical and hence inhibitable by superoxide dismutase; (iv) uroporphyrinogen could be further oxidized to completion by the hydroxyl radical since the reaction was partially inhibited by both mannitol and catalase which prevent hydroxyl radical production; (v) the oxidizing effects of ferric ion on uroporphyrinogen were none or negligible as compared to those of ferrous ion. Ferric was reduced to ferrous ion in the presence of dithiothreitol. When the ferrous ion thus formed was reoxidized in the presence of atmospheric oxygen, minor but definite oxidations of both uroporphyrinogen and dithiothreitol were observed. The oxidations of Fe2+ and uroporphyrinogen could be blocked by 1,10-phenanthroline, a ferrous iron chelator. The data suggest that ferrous is the reactive form of iron that may contribute to pathogenic development of the disease by irreversibly oxidizing the porphyrinogen substrates to nonmetabolizable porphyrins, which accumulate in porphyric liver.

1. Some polyhalogenated aromatic chemicals such as 2,3,7,8-tetrachloro-p-dioxin, brominated and chlorinated biphenyls, and hexachlorobenzene cause in humans, animals and hepatocyte systems a partial block in haem biosynthesis leading to accumulation and excretion of uroporphyrin, the oxidation product of the unstable biosynthetic intermediate uroporphyrinogen. 2. The involvement of reactive toxic metabolites of the halogenated chemicals has previously been suggested. The evidence presented in this paper supports a different mechanism involving chronic induction of the microsomal cytochrome P-450 system, mobilization of hepatocellular iron and associated oxidative stress. Besides oxidation of uroporphyrinogen to uroporphyrin, an inhibitor of uroporphyrinogen decarboxylase may also be formed. 3. Studies with iron-loaded mice and chicken embryo hepatocytes show that under appropriate conditions iron alone, or chemicals such as beta-naphthoflavone which induce the same cytochromes P-450 isozymes as do the chlorinated aromatics, will cause a similar uroporphyria. These findings provide an experimental model for the human disease porphyria cutanea tarda, sometimes occurring in patients with liver damage. 4. Experiments with rats and iron-loaded mice indicate that there may also be an association between the induction of uroporphyria and the development of liver tumours after administration of polyhalogenated aromatic chemicals.

Uroporphyrinogen decarboxylase (EC 4.1.1.37) was purified about 14000-fold to homogeneity from the yeast Saccharomyces cerevisiae with a 70% overall yield. The purification included affinity chromatography on uroporphyrin-I-Affi-Gel 102. The specific activity of the final preparation was 1750 nmol coproporphyrinogen formed.h-1.(mg protein)-1 at pH 7.5 and 37 degrees C using 4 microM uroporphyrinogen I as substrate. The purified enzyme has a minimum molecular mass of 38 kDa by sodium dodecyl sulfate/polyacrylamide gel electrophoresis and 46 kDa by gel filtration, suggesting that yeast uroporphyrinogen decarboxylase is a monomer. Chromatofocusing gave a pI of 6.0. Enzyme activity was inhibited by metals, such as Cu2+, Zn2+, Fe2+, Fe3+ and by sulfhydryl-specific reagents, but no cofactor requirement could be demonstrated. The optimum pH was pH 5.7 for uroporphyrinogens I and III and heptacarboxylate porphyrinogen I as estimated by coproporphyrinogen formation. The optimum pH for substrate decarboxylation was pH 5.7 for uroporphyrinogen I, but pH 6.8 for the two other substrates. The Km values at pH 5.7 were 10 nM for uroporphyrinogen I, 6 nM for uroporphyrinogen III and 7 nM for heptacarboxylate porphyrinogen I as measured by coproporphyrinogen formation. The pattern of accumulation of intermediate and final decarboxylation products and the rates of the successive decarboxylations were determined for the three substrates at different concentrations at pH 5.7 and pH 6.8. The rate-limiting step at 4 microM substrate concentration was the elimination of the second carboxyl group of uroporphyrinogen III and the fourth carboxyl of uroporphyrinogen I. An antiserum to purified yeast uroporphyrinogen decarboxylase was used to characterize the protein in several mutants.

The effects of hexachlorobenzene (HCB) administration on the development of porphyria and on changes in thyroid function and thyroid hormone metabolism were examined. Female Wistar rats were treated with HCB for 1 or 8 weeks. At both treatment times liver weight was notably increased with a slight change in thyroid weight at 8 weeks. Serum thyroxine (T4) levels were depressed, whereas levels of triiodothyronine (T3) were not depressed significantly at both treatment times. One or eight weeks of HCB treatment did not alter the incorporation and distribution of [125I] into intrathyroidal aminoacids. A 50% reduction in protein bound iodine (PB[125I]) was seen in both groups of animals. HCB altered [125I]T4 metabolism in rat liver slices, increasing T4 dehalogenation. HCB administration for 1 week did not affect urinary excretion of porphyrins or their precursors, or hepatic porphyrin content. The activity of aminolaevulinate synthase was not affected, but there was a 25% and 51% inhibition in porphyrinogen carboxy-lyase (PCL) activity for the uroporphyrinogen disappearance or the coproporphyrinogen formation respectively. After 8 weeks of HCB administration the rats showed a characteristic porphyria. Our results show that HCB treatment increased hepatic thyroxine metabolism, without alterations in thyroid hormone synthesis. Serum T4 and PCL activity behaved differently in both time- and dose-dependent studies, with serum T4 being the more sensitive parameter which responded at earlier times and lower doses.

Effects of erythropoietin treatment on haem synthesis in peripheral blood were evaluated in 11 patients on haemodialysis. After 2 weeks of erythropoietin, mean (SEM) uroporphyrinogen-I synthase activity increased significantly from 88 (10) to 116 (9) pmol/h per mg protein. Haem synthase activity, thought to be the rate-limiting step in erythroid haem synthesis, also showed a significant increase from 4·5 (0·8) to 8·4 (1·8) pmol/h per 10 6 reticulocytes. 4 patients, who showed only a partial response to erythropoietin, had significantly higher serum aluminium concentrations than the 7 who responded fully (225 [44] vs 55 [23] μg/l); erythrocyte protoporphyrin concentrations in partial responders were also much higher than in responders (973 [120] vs 388 [29] nmol/l). Aluminium intoxication may cause resistance to erythropoietin by interference with haem synthesis, with accumulation of protoporphyrin.

The oxidation of uroporphyrinogen, an intermediate of the heme biosynthetic pathway, by methylcholanthrene-inducible isozymes(s) of cytochrome P-450 has been proposed to play a role in the development of chemically induced uroporphyria. Prior work from this laboratory indicated that although addition of 3,4,3',4'-tetrachlorobiphenyl is required for uroporphyrinogen oxidation by methylcholanthrene-induced chick embryo liver microsomes, this biphenyl is not required for the oxidation catalyzed by hepatic microsomes from methylcholanthrene-induced rodents. Here we investigated whether rodent microsomes catalyze uroporphyrinogen oxidation without addition of 3,4,3',4'-tetrachlorobiphenyl because the chemical used as an inducer remains bound to cytochrome P-450. Hepatic microsomes containing almost no residual inducer were isolated from rats treated with a low dose of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). These microsomes oxidized uroporphyrinogen at high rates without addition of 3,4,3',4'-tetrachlorobiphenyl. Inducer-free microsomal cytochrome P-450 was also obtained by inducing cytochrome P-450 in rats and mice with isosafrole, which was then removed from the isolated microsomes by butanol treatment. This procedure resulted in microsomes with high activity for uroporphyrinogen oxidation. Furthermore, addition of chlorobiphenyl to these inducer-free microsomes was inhibitory. Hepatic microsomes from isosafrole-induced C57BL/6 and DBA mice, rendered inducer-free by butanol treatment, oxidized uroporphyrinogen at the same rate even though these two strains differ markedly in their susceptibility to chemically induced uroporphyria. We conclude that uroporphyrinogen oxidation is catalyzed by cytochrome P-450 that is free of inducer.

Routes are developed for synthesis of the tripyrrolic macrocyclic spiro-lactam 39. A minor product from the synthesis, thought earlier to be an atropisomer, has been shown by molecular mechanics calculations and re-investigation to be a dimer.The octa-acid derived from 39 closely resembles the spiro-pyrrolenine 2 proposed as a biosynthetic intermediate for uroporphyrinogen Ill. This octa-acid acts as a strong inhibitor of cosynthetase (uroporphyrinogen Ill synthase) whilst other similar systems which lack some of its functionality do not. These results strongly support the view that the spiro system 2 is indeed the biosynthetic intermediate for formation of uroporphyrinogen III 3 from hydroxymethylbilane 1.

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.

Uroporphyrinogen (URO) decarboxylase was measured in hemoglobin-free erythrocytes from subjects with familial porphyria cutanea: the mean activity was about 50% of that found in erythrocytes from normal subjects. Asymptomatic carriers were always found in the family. No enzyme deficiency was found in erythrocytes from subjects with sporadic porphyria cutanea. The measurement of URO decarboxylase in erythrocytes seems to allow an easy distinction between these two groups of porphyria cutanea.

Heme-deficient mutants of Saccharomyces cerevisiae have been isolated from two isogenic strains with the use of an enrichment method based on photodynamic properties of Zn-protoporphyrin. They defined seven non-overlapping complementation groups. A mutant representative of each group was further analysed. Genetic analysis showed that each mutant carried a single nuclear recessive mutations. Biochemical studies showed that the observed accumulation and/or excretion of the different heme synthesis precursors by the mutant cells correlated well with the enzymatic deficiencies measured in acellular extracts. Six of the seven mutants were blocked in a different enzyme activity: 5-aminolevulinate synthase, porphobilinogen synthase, uroporphyrinogen I synthase, uroporphyrinogen decarboxylase, coproporphyrinogen III oxidase and ferrochelatase. The other mutant had the same phenotype as the mutant deficient in ferrochelatase activity. However, it possessed a normal ferrochelatase activity when measured in vitro, so this mutant was assumed to be deficient in protoporphyrinogen oxidase activity or in the transport and/or reduction of iron. The absence of PBG synthesis led to a total lack of uroporphyrinogen I synthase activity. The absence of heme, the end product, led to an important increase of coproporphyrinogen III oxidase activity, while the activity of 5-aminolevulinate synthase, the first enzyme of the pathway, was not changed. These results are discussed in terms of possible modes of regulation of heme synthesis pathway in yeast.

Cell-free extracts from Clostridium tetanomorphum, a microorganism which synthesizes corrins but no heme, are capable of converting both 5-aminolevulinate and uroporphyrinogen III into cobyrinic acid. Comparative examinations with (14C)5-aminolevulinate and (14C)uroporphyrinogen yielded corresponding results. Cell-free extracts from Clostridium tetanomorphum contain uroporphyrinogen III. To obtain good radiochemical yields it is therefore necessary to use substrates of high specific radioactivity. A method for the preparation of 14C-labelled uroporphyrin I-IV with high specific radioactivity is described.

A hydroxyl radical-generating system, hypoxanthine/xanthine oxidase/Fe-EDTA, oxidises uroporphyrinogens to uroporphyrins and to more polar nonporphyrin products. The evidence suggests that the nonporphyrin products have inhibitory activity towards mouse liver uroporphyrinogen decar☐ylase which could explain some forms of human and experimental porphyrias.

1. 1. The effect of light and growing time on chlorophyll, protein content and the activity of porphobilinogenase (PBGase), in Euglena gracilis was studied. 2. 2. It was found that neither protein nor chlorophyll content or PBGase activity were dependent upon the days of growing or the light conditions of growth. 3. 3. Some properties of PBGase were studied. The addition of sodium and magnesium salts to the assay system did not modify enzyme activity. Uroporphyrinogen formation was linear with protein over a wide range of enzyme concentration. 4. 4. When activity was expressed on the basis of the amount of porphyrins formed an optimum pH of 7.4 was obtained but when it was expressed in terms of PBG consumed two pH maxima at 7.0 and 8.5 were observed. 5. 5. Direct plots of velocity against PBG concentration, when activity was measured as PBG consumed, were not hyperbolic; double-reciprocal plots were not linear and Hill plots gave a n value of 2, indicating the existence of positive co-operative effects between two binding sites for PBG per molecule of PBGase.

Patients with active lymphoproliferative diseases were shown to have high activity of erythrocyte uroporphyrinogen synthetase (URO-S), the enzyme which converts porphobilinogen to uroporphyrinogen. In a few patients examined the lymphocyte URO-S was markedly increased. No correlation was found between the high URO-S activity and the degree of anemia, reticulocytosis, or the presence of hemolysis. Patients with epithelial malignancies and with some common viral diseases had normal erythrocyte URO-S values. Three patients with nonalcoholic cirrhosis also had high erythrocyte URO-S activities. The determination of erythrocyte and lymphocyte URO-S activity may be of aid in the diagnosis of lymphoproliferative diseases. It may also indicate whether remission has been achieved and whether treatment should be continued or reinstituted. These preliminary observations justify the investigation of a larger patient and control material.

δ-Aminolevulinic acid (ALA) synthetase is considered the rate-limiting enzyme in heme biosynthesis in mature mammalian liver. However, under various physiologic or toxicologic conditions, other enzymes of the heme biosynthetic pathway may become rate-limiting in this process. In the present studies, the ontogenic development of uroporphyrinogen (uro) decarboxylase was measured in rat liver, and the properties and potential influence of this enzyme on heme biosynthetic capability in adult and fetal liver were assessed. In addition, a quantitative comparison of the activity of uro decarboxylase with that of ALA synthetase was made as a means of estimating the relative effects of specific inhibitors of uro decarboxylase on hepatic heme biosynthetic capability at each stage of development. The results indicate that fetal uro decarboxylase activity is over three times that of the adult and that enzyme activity declines to the adult levels concomitant with a decrease in the hematopoietic cell composition of the liver near the time of birth. Moreover, fetal uro decarboxylase may be substantially more susceptible to physiologic or toxicologic alteration than is the adult enzyme. The fetus may, therefore, be at greater risk with respect to compromise of heme biosynthetic capability by agents which alter uro decarboxylase activity.

Effects of iron were studied in cultured chick embryo liver cells to help elucidate the effect of hepatic iron in the human disease porphyria cutanea tarda and in toxic porphyria caused by chemicals. These cultures have proven useful because (a) phenobarbital and phenobarbital-like drugs induce a common form(s) of cytochrome P-450 (P-450-phenobarbital) in these cultures; (b) 20-methylcholanthrene and certain other polycyclic hydrocarbons induce a different form(s) (P-450-methylchol-anthrene), and (c) uroporphyria can be produced rapidly by exposure to suitable chemicals. In these cultures, treatment with iron alone did not produce porphyrin accumulation, and treatment with iron + 5-aminolevulinate caused accumulation of protoporphyrin, as did treatment with 5-aminolevulinate alone. However, treatment with phenobarbital-like drugs and iron, the latter at a concentration as low as 0.2 microM, led to accumulation of uro- and heptacarboxylporphyrins. Potentiation of uroporphyrin accumulation by iron began before there was a detectable synergistic increase in activity of 5-aminolevulinate synthase, the rate-controlling enzyme of heme synthesis. In contrast, treatment of cultures with 20-methylcholanthrene, in the presence or absence of iron, did not result in uroporphyrin accumulation or an increase in the activity of 5-aminolevulinate synthase. Uroporphyrinogen decarboxylase activity was unchanged by drug and iron treatments. Inhibitors of P-450-phenobarbital, SKF525A and piperonyl butoxide, as well as cadmium and cycloheximide prevented the porphyrin accumulation produced by glutethimide + iron, even though, except with cycloheximide, these substances further increased 5-aminolevulinate synthase activity. In vitro, uroporphyrin was oxidized autocatalytically by iron. In intact hepatocytes, even low concentrations of iron (0.2 to 20 microM), in the presence of a form of cytochrome P-450 induced by phenobarbital-like chemicals, produces uroporphyria primarily by enhancing uroporphyrinogen oxidation, not by inhibition of the decarboxylase. Induction of 5-aminolevulinate synthase amplifies the porphyrin overproduction.

We describe the methodology used for quantifying and characterizing porphyrins in various tissues and in excreta, in the diagnosis and monitoring of the therapeutic modulation of biochemical disease activity in a 53-year-old white man who has a rare form of familial porphyria cutanea tarda with bone marrow rather than hepatic expression of the disease. Liquid-chromatographic and thin-layer chromatographic analyses of the patients's urine and skin showed predominantly heptacarboxylic porphyrin and uroporphyrin, whereas his stool and bile contained isocoproporphyrin and coproporphyrin as the major products. The data reflect defective uroporphyrinogen decarboxylation. Both analytical methods gave quantitatively similar results for urinary and fecal porphyrins. A triple-lumen perfusion study of samples procured both at the ampulla of Vater and 15 cm downstream provided data for porphyrins excreted in the bile and their reabsorption in the small intestine. We evaluated: suppression by hypertransfusion of bone marrow overproduction of porphyrins and reduction of enteral absorption of porphyrins by orally administered charcoal (Acta Char) and cholestyramine.

To determine if clinically observed disorders in heme biosynthetic enzymes, known as sporadic porphyria cutanea tarda (PCT), could be reproduced in experimental animals, male Fischer rats were treated with ethanol, estrogen and hexachlorobenzene (HCB). A series of heme biosynthetic enzymes were assayed. In the rats given free access to 8% ethanol-drinking water for 15 weeks, delta-aminolevulinate (ALA) dehydratase was significantly reduced in erythrocytes. In the liver, ALA synthetase and uroporphyrinogen (UROgen) decarboxylase activities remained unchanged. In bone marrow cells, these activities did not change markedly. In the rats treated with estrogen (1 mg estrioltripropionate/rat/week, IM), no body weight gain was observed during the treatment for 15 weeks and urinary ALA excretion increased to 1.7 fold over normal level. In the liver, a significant increase was observed in the activity of ALA dehydratase, but other enzymes remained within the normal level. In bone marrow cells and erythrocytes, ALA dehydratase was also increased. ALA synthetase increased only in bone marrow cells to 2.1 times higher than the control level. In rats fed 0.3% HCB-diet for 8 weeks, urinary excretion of ALA, coproporphyrin and uroporphyrin increased to 2.4, 3.3 and 3.8 times higher than the controls, respectively. In the liver, an increase was observed in ALA synthetase, while a decrease was observed in ALA dehydratase and UROgen decarboxylase. In bone marrow cells and erythrocytes, ALA dehydratase was reduced and activities of other enzymes did not show any changes. These results indicate that alcohol, estrogen and HCB do not produce phenomena similar to those observed clinically in PCT.

Purified S-adenosyl-L-methionine: uroporphyrinogen (uro'gen) III methyl transferase (SUMT), the first methylase of the vitamin B12 pathway (from Pseudomonas denitrificans), synthesizes a type I isomer of sirohydrochlorin from uro'gen I (1).

The preparation of the aminomethyl-bilinogen which results from formal "head to tail" condensation of porphobilinogen is described. The chemical cyclocondensation of this compound at pH 7.4 yields uroporphyrinogen I. Enzymatic studies with enzyme preparations from Propionibacterium shermanii, which synthesize uroporphyrinogens from porphobilinogen, show that the rate of cyclisation is increased by these enzymes and indicate that the bilinogen also might be used for uroporphyrinogen III formation. This is also suggested by studies on the formation of cobyrinic acid from [4-14C]5-aminolevulinate via uroporphyrinogen III in the presence of the aminomethylbilinogen by cell-free extracts from Clostridium tetanomorphum.

Rapid and reproducible assays for uroporphyrinogen III synthase (URO-S; EC 4.2.1.75) have been developed and used to determine the enzymatic activity in human erythrocytes and cultured lymphoid cells. In the coupled-enzyme assay, porphobilinogen was first converted to hydroxymethylbilane, the natural substrate for URO-S, by hydroxymethylbilane synthase which was conveniently obtained from heat-treated erythrocyte lysates. In the direct assay, synthetic hydroxymethylbilane was used as substrate. In both assays, the uroporphyrinogen reaction products were oxidized to their respective uroporphyrin isomers, which were then resolved and quantitated by reversed-phase high-pressure liquid chromatography. Both assays were optimized for pH, substrate concentration, and linearity with time and protein concentration. The mean URO-S activities in normal human erythrocyte lysates determined by the coupled-enzyme and direct assays were 7.41 ± 1.35 and 7.64 ± 1.73 units/mg protein, respectively. In normal human cultured lymphoid cells, the mean activities were 13.7 ± 1.39 and 17.6 ± 1.15 units/mg protein for the coupled-enzyme and direct assays, respectively. In four families with congenital erythropoietic porphyria, both assays reliably identified the markedly decreased URO-S activities in erythrocytes and cultured lymphoid cells from affected homozygotes and the half-normal activities in these sources from obligate heterozygotes. The coupled-enzyme assay was easier to perform and was suited for clinical diagnostic assays and for monitoring enzyme purification procedures, while the direct assay, which required substrate preparation and technical dexterity, was best for kinetic studies of URO-S.

Porphyrin biosynthesis in mammalian skin and in skin obtained from patients with selected types of porphyria has been studied. Cutaneous porphyrinogenesis required the precursor δ-aminolevulinic acid (ALA) which, when added to murine, rat, and human skin in vitro, was rapidly converted to porphyrins. Total porphyrin content was quantitated by fluorescence assay, and spectral studies indicated that more than 80% of the porphyrin produced was protoporphyrin. The majority of skin porphyrinogenesis occurred in epidermis or in epidermal derivatives such as hair roots. Known inducers of hepatic δ-aminolevulinic acid synthetase (ALAS), the rate-limiting enzyme for heme biosynthesis, were not inducers when added to skin in vitro. Skin from patients with acute intermittent porphyria demonstrated a 43% decrease in cutaneous porphyrin production as compared to unaffected normals. This is consistent with the known deficiency of uroporphyrinogen synthetase that has been previously demonstrated in the liver and red blood cells of these patients. Porphyrinogenesis in skin of patients with porphyria cutanea tarda was not different from controls. These studies demonstrate that skin has the enzymatic capacity to synthesize porphyrins from added ALA and that cutaneous porphyrinogenesis from ALA is deficient in patients with acute intermittent porphyria.

A cytochrome oxidase-deficient mutant ( pop4 ) of Saccharomyces cerevisiae which accumulates porphyrins has been characterized. The pop4 mutation is recessive and affects a single nuclear gene. The bulk of the accumulated porphyrins consists of uroporphyrin and its partial decar☐ylation products, suggesting that the mutation causes a block at the level of uroporphyrinogen decar☐ylase. However, pop4 is not allelic to hem6 or pop3 , putative decar☐ylase structural-gene mutants. These results suggest that there may be a uroporphyrinogen decar☐ylase isoenzyme specifically involved in heme a production.

We have previously shown that uroporphyrinogen is oxidized to uroporphyrin by microsomes (microsomal fractions) from 3-methylcholanthrene-pretreated chick embryo liver [Sinclair, Lambrecht & Sinclair (1987) Biochem. Biophys. Res. Commun. 146, 1324-1329]. We report here that a specific antibody to chick liver methylcholanthrene-induced cytochrome P-450 (P-450) inhibited both uroporphyrinogen oxidation and ethoxyresorufin O-de-ethylation in chick-embryo liver microsomes. 3-Methylcholanthrene-pretreatment of rats and mice markedly increased uroporphyrinogen oxidation in hepatic microsomes as well as P-450-mediated ethoxyresorufin de-ethylation. In rodent microsomes, uroporphyrinogen oxidation required the addition of NADPH, whereas chick liver microsomes required both NADPH and 3,3',4,4'-tetrachlorobiphenyl. Treatment of rats with methylcholanthrene, hexachlorobenzene and o-aminoazotoluene increased uroporphyrinogen oxidation and P-450d, whereas phenobarbital did not increase either. The contribution of hepatic P-450c and P-450d to uroporphyrinogen oxidation and ethoxyresorufin O-de-ethylation in methylcholanthrene-induced microsomes was assessed by using specific antibodies to P-450c and P-450d. Uroporphyrinogen oxidation by methylcholanthrene-induced rat liver microsomes was inhibited up to 75% by specific antibodies to P-450d, but not by specific antibodies to P-450c. In contrast, ethoxyresorufin de-ethylation was inhibited only 20% by anti-P450d but 70% by anti-P450c. Methylcholanthrene-induced kidney microsomes which contain P-450c but non P-450d did not oxidize uroporphyrinogen. These data indicate that hepatic P-450d catalyses uroporphyrinogen oxidation. We suggest that the P-450d-catalysed oxidation of uroporphyrinogen has a role in the uroporphyria caused by hexachlorobenzene and other compounds.

Normal or increased amounts of series III porphyrins with greater amounts of series I were observed on incubation of PBG in hemolysates of congenital erythropoietic porphyria vs. normal erythrocytes, human or bovine. Correlation with reticulocyte percentage was poor, in the aggregate a general trend toward increased values of both isomers I and III was noted with increasing reticulocytes. When the percent of type III was low the net amount was increased as compared with normal. Hemolysates of non-porphyric, reticulocyte-rich red cells (hemolytic or posthemorrhagic anemia) formed only minute amounts of type I porphyrin but at the same time no more, or even less type III than the porphyric hemolysates, although representing red cells of greater reticulocyte content. No evidence of deficient heme synthesis was observed in porphyric hemolysates incubated with [14 C] -porphobilinogen or 59Fe. Other studies of porphyric hemolysates incubated with and without added mouse spleen synthetase failed to reveal evidence of an absolute UPG-III cosynthetase (Co-S) deficiency. The large increases of type I porphyrin with normal or increased formation of type III, both in the disease and in the hemolysates, are believed due to a primary increase of ALA-S or UPG-S activity rather than a decrease of Co-S. Possible mutations which might be responsible for this increase are considered.

1. 1. Clinical and biochemical analogies and progressions exist among the hereditary acute hepatic porphyrias (AHP), acute intermittent porphyria (AIP). variegate porphyria (PV) and hereditary coproporphyria (HCP). 2. 2. The AHP are essentially regulatory disorders at the molecular level, and have several features in common: drug idiosyncracy, enhanced excretion of porphyrins and their precursors (ALA and PBG) in typical constellations in the urine, elevated porphyrin excretion in the stool (in PV and HCP, and occasionally in AIP). enhanced inducibility of hepatic ALA synthase due to partial enzyme defects in the biosynthetic sequence favorable response to treatment with glucose, propranolol, and hematin, and development in a four-stage progression: phase of the genetic defect→ latency phase (compensated → decompensated) → clinical manifestation. 3. 3. Cutaneous signs can be fully lacking not only in AIP, but also in HCP and PV. 4. 4. The absolute extent of the enzyme defect can vary considerably (overlapping with normal). 5. 5. Gradual transitions between AIP and PV, and probably among AIP, HCP and PV as well. are suggested by several observations: PV can occur without cutaneous signs, thus imitating AIP: in AIP, elevated excretion of porphyrins in the stool is occasionally seen in both the manifest and latent phases, and coproporphyrin can be the dominant urinary porphyrin in AIP: both of these findings can cause confusion of AIP with PV. 6. 6. In HCP fecal excretion of protoporphyrin can reach the same level as that of coproporphyrin. If in AIP patients fecal porphyrin excretion is increased, one obtains the first complete overlapping between AIP and PV. 7. 7. In both AIP and in normals PBG in elevated concentrations can not enzymatically be converted to porphyrinogens in equimolar amounts by liver tissue (“physiologic URO'gen synthase defect”). Thus the step catalysed by URO'gen synthase apparently is the bottleneck or limiting step in porphyrin formation. 8. 8. Similar excretion profiles of porphyrin precursors and porphyrins (COPRO > URO >PENTA or URO >COPRO >PENTA) in AIP indicate limitation of uroporphyrinogen synthesis which due to the kinetics of URO'gen synthase (very low activity in human liver) becomes apparent when there is a massive oversupply of PBG (e.g. oral loading with ALA in normals or induction of ALA synthase in AHP). 9. 9. The increased porphyrin synthesis in AIP can therefore be interpreted as part of a compensatory mechanism or regulatory adaptive process to insure formation of adequate amounts of heme. 10. 10. The elevated substrate supply is due to counterregulation. and compensates the partial defect: elevated excretion of metabolites distal to URO'gen synthase masks the deficiency of this enzyme; elevated porphyrin excretion in a clinical-biochemical sign of compensation in AIP. 11. 11. The enzyme defect in the red cells has no regulatory consequence for hemoglobin synthesis.

The ability of rat hepatic uroporphyrinogen cosynthase to direct formation of uroporphyrinogen III and the synthesis of uroporphyrinogen in vitro was impaired by sulfamerazine. Inhibition was reversed by the addition of folic acid. Administration of a single, oral dose (1 g/kg) of sulfamerazine to rats was associated with elevated levels of hepatic uroporphyrin I isomer. These results suggest that sulfonamides may interfere with the biosynthesis of uroporphyrinogen III.

Altered hepatic microsomal drug metabolism has been reported to occur in afflicted with hyperbilirubinemia. Similarities of the chemical structures of hydroxymethylbilane, an intermediate in the biosynthesis of uroporphyrinogen, to bilirubin prompted investigations of the effect of bilirubin on the activity of uroporphyrinogen I synthase (porphobilinogen deaminase, EC 4.3.1.8) and the biosynthesis of heme. Bilirubin was found to be a reversible, noncompetitive inhibitor of uroporphyrinogen I synthase. The inhibition constant (Ki) for bilirubin was 1.5 microM. Bile acids had no effect on rat hepatic uroporphyrinogen I synthase activity. Hyperbilirubinemia was achieved in rats by biliary ligation in order to investigate whether elevated levels of bilirubin impair the biosynthesis of hepatic heme in vivo. The relative rate of heme biosynthesis, as measured by the rate of incorporation of delta-[4-14C]aminolevulinic acid into heme, was decreased 59% 24 h after biliary obstruction. The levels of hepatic microsomal heme and cytochrome P-450 were decreased by 43 and 40%, respectively, 72 h after biliary obstruction. The activities of hepatic delta-aminolevulinic acid synthase and uroporphyrinogen I synthase were increased by 39 and 46%, respectively, 72 h after biliary obstruction. During the 48- to 72-h period following biliary obstruction, the urinary excretion of porphobilinogen and uroporphyrin was increased 3.0- and 3.5-fold, respectively, whereas, the urinary excretion of delta-aminolevulinic acid was not altered. During this 48-to 72-h time interval following biliary obstruction, 100% of the uroporphyrin was excreted as isomer I. These results indicate that bilirubin is capable of depressing the biosynthesis of rat hepatic heme and thus cytochrome P-450-mediated drug metabolism by inhibition of the formation of uroporphyrinogen. These findings are a plausible mechanism for reports of impaired clearance of various drugs in patients afflicted with hyperbilirubinemic disease states.

1 A 423-fold purified fraction of uroporphyrinogen decarboxylase (EC 4.1.1.37) showing a specific activity of 770 units/mg protein has been employed in order to study some properties in etiolated Euglena gracilis Z. 2 Uroporphyrinogen decarboxylase has a relative molecular mass of 54000, an optimum pH of 7.2 and exhibits Michaelis-Menten kinetics, employing both uroporphyrinogen I and uroporphyrinogen III as substrates. 3 Anaerobic conditions seem not to be necessary for uroporphyrinogen decarboxylase activity. Neither EDTA nor cysteine affected enzyme activity, whereas dithiothreitol produced a remarkable activation of coproporphyrinogen formation. 4 Kinetic data employing both substrates showed an accumulation of porphyrinogen (i.e. hexa- and heptaporphyrin) containing six or seven COOH groups, depending on the uroporphyrinogen concentration used. 5 An unusual elution profile of the intermediates on Sephacryl S-200 was found.

Oral administration of a commercial PCB mixture to chickens caused a hepatic-type porphyria characterized by hepatic accumulation and urinary excretion of uroporphyrin. To clarify the mechanism of the porphyrinogenic activity of these PCBs, we studied the structural requirement of synthetic PCB for porphyrinogenic activities by using the cultured chick embryo liver cells and examined the relationship between induction of delta-aminolevulinic acid (ALA) synthetase and inhibition of uroporphyrinogen decarboxylase. We established that the porphyrinogenic effect of PCBs exhibits a sharply defined structure-activity relationship in that only 3,4,3',4'-tetrachlorobiphenyl and 3,4,5,3',4',5'-hexachlorobiphenyl produced a marked accumulation of uroporphyrin. We also demonstrated that in ALA-supplemented cultures, these same compounds lead to accumulation of a large amount of uroporphyrin III, whereas with other PCBs, which were weak inducers of porphyrin synthesis, the accumulated porphyrin was mostly protoporphyrin. was mostly protoporphyrin. Kinetic studies of the sequential decarboxylation of uroporphyrinogen with purified uroporphyrinogen decarboxylase were performed. The 3,4,3',4'-tetrachlorobiphenyl and 3,4,5,3',4',5'-hexachlorobiphenyl strongly inhibit uroporphyrinogen decarboxylase directly at two steps, i.e. first in the formation of hexacarboxylic porphyrinogen III from heptacarboxylic porphyrinogen III and second in the formation of heptacarboxylic porphyrinogen III from uroporphyrinogen III. The results confirmed that porphyrinogenic PCBs primarily inhibit uroporphyrinogen decarboxylase, leading to a depletion of heme as a result of which synthesis of ALA synthetase increased.

Red cell porphyrinogen carboxy-lyase activity was measured using uroporphyrinogen III as substrate in 18 normal persons, 7 male patients with porphyria cutanea tarda, 3 female patients with erythropoietic protoporphyria and 2 female patients with variegate porphyria. The mean values obtained in normal subjects were 0.151 nmol of uroporphyrinogen disappeared in 30 min per mg of protein, and 0.038 nmol of coproporphyrinogen formed in 30 min per mg of protein. We have not been able to detect significant differences between males and females. In porphyria cutanea tarda the enzyme activity was the same as in normal subjects considering either substrate disappearance or end product formation. The differences were not significant at the p < 0.05 level. Patients with variegate porphyria also exhibited normal erythrocyte porphyrinogen carboxy-lyase activity. The enzyme activity of erythrocytes from patients with erythropoietic protoporphyria was higher than in normals; mean values for specific activities being 0.204 nmol of uroporphyrinogen disappeared, and 0.071 nmol of coproporphyrinogen formed. The significance of the results with respect to the chemical picture of different porphyrias is discussed.

Cell-free preparations from liver of chick embryo treated with 3-methylcholanthrene catalyzed oxidation of uroporphyrinogen I in the presence of NADPH and 3,4,3′,4′-tetrachlorobiphenyl. Extracts of untreated embryo liver or liver from embryo treated with glutethimide, a phenobarbital-like inducer of cytochrome P450 in this system, did not catalyse the oxidation. Direct involvement of cytochrome P450 was demonstrated by inhibition of the oxidation by CO, piperonyl butoxide and specific antisera to the methylcholanthrene-induced cytochrome P450. 2,4,2′,4′-tetrachlorobiphenyl was inactive in the oxidation. These results may explain the role of induced cytochrome P450 in experimental uroporphyria. The oxidation may be useful as a simple assay for reactive O 2 species.

1. 1. The enzymes of haem biosynthesis have been measured in peripheral blood in two patients aged 6 and 20 yr with congenital (erythropoietic) porphyria. 2. 2. Excessive activity of leucocyte δ-aminolaevulinic acid synthase was found in both patients. 3. 3. The activity of erythrocyte porphobilinogen deaminase was elevated and coupled with a lowered activity of uroporphyrinogen cosynthase, which resulted in excessive production of Series l isomer porphyrin. 4. 4. This was confirmed by isomer analysis of urine which showed an excess in both of coproporphyrin 1. 5. 5. Faecal porphyrin analysis demonstrated excessive quantities of ‘X’ porphyrin in one patient in common with other photosensitizing porphyrias. 6. 6. These results confirm that the primary control of haem biosynthesis in congenital porphyria is at the level of δ-aminolaevulinic acid synthase. with a secondary control point at the level of porphobilinogen deaminase.

Glutamate 1-semialdehyde is formed in high yields by ozonolysis of 4-vinyl-4-aminobutyric acid and purification of the reaction products by cation exchange chromatography. The structure of the product and its pyrrole derivative have been confirmed by1H-NMR and13C-NMR spectroscopy, and by its conversion to σ-aminolevulinate by purified glutamate 1-semialdehyde aminotransferase, and to uroporphyrinogen with porphobilinogen and uroporphyrinogen synthetase. The NMR spectra provide evidence for the presence in solution of a hydrate of glutamate 1-semialdehyde. Concentration dependent formation of dimers of the hydrate was also shown. It is suggested that chemical conversion of glutamate 1-semialdehyde to σ-aminolevulinate proceeds through dimeric intermediate stages.

Acute intermittent porphyria (AIP) is a primary disorder of haem biosynthesis that is chemically characterised by raised urinary porphobilinogen (PBG). A defect in the biochemical pathway at the step of PBG conversion to uroporphyrinogen has been shown to be a result of a partial deficiency of the enzyme uroporphyrinogen I synthetase (uro I syn). The ascertainment rate of latent AIP (that is, chemically manifest but clinically asymptomatic) was examined in 185 individuals from 12 AIP kindreds using three parameters: red cell uro I syn, quantitative urinary PBG, and pedigree analysis with respect to uro I syn. Approximately 80% of individuals could be assigned as normal or latent AIP on the basis of the uro I syn assay alone. The remaining 20% could not be assigned because of an intermediate range of activity for the red cell assay in which the diagnosis cannot be certain. When the pedigree was used in the evaluation of the uro I syn data, the number of uncertain individuals, with respect to AIP, decreased to 10%. The enzyme method detected latent AIP in 37.5% of blood relatives, whereas quantitative urinary PBG alone detected only 15.2%. The pattern of inheritance for the uro I syn deficiency is consistent with Mendelian dominant inheritance, and it is likely that it is the basic inherited defect in AIP.