Is 5-aminolevulinic acid involved in the hepatocellular carcinogenesis of acute intermittent porphyria?

5-Aminolevulinic acid (ALA) is a heme precursor that accumulates in acute intermittent porphyria (AIP) due to enzymatic deficiencies in the heme biosynthetic pathway Its accumulation has been associated with several symptoms, such as abdominal pain attacks, neuromuscular weaknesses, neuropsychiatric alterations and increased hepatocellular carcinoma (HCC) incidence. The use of exogenous ALA to elevate porphyrin levels in tumor photodynamic therapy, adds further significance to ALA toxicology. Under ferritin mediated and metal catalyzed oxidation, ALA produces reactive oxygen species that can damage plasmid and isolated DNA in vitro, and increases the steady-state level of 8-oxo-7,8-dihydro-2'-deoxyguanosine in liver, spleen and kidney DNA and 5-hydroxy-2'-deoxycytidine in liver DNA of ALA-treated rats. The in vitro DNA damage could be partially inhibited by SOD, catalase, DTPA, mannitol and melatonin. ALA also promotes the formation of radical-induced base degradation products in isolated DNA. 4,5-Dioxovaleric acid, the final oxidation product of ALA, alkylates guanine moieties within both nucleoside and isolated DNA, producing two diastereoisomeric adducts. Dihydropyrazine derivatives of ALA generated by its dimerization, promote DNA strand-breaks and 8-oxodGuo formation in the presence of Cu2+. Together these results reinforce the hypothesis that the DNA damage induced by ALA may be associated with the development of HCC in individuals suffering from AIP.     

Biosynthesis of delta-Aminolevulinic Acid in Chlamydomonas reinhardtii: Study of the Transamination Mechanism Using Specifically Labeled Glutamate

The first committed intermediate of chlorophyll biosynthesis, δ-aminolevulinic acid (ALA), is synthesized from glutamate in the plant cell. The last step of ALA synthesis is a transamination reaction which converts glutamate-1-semialdehyde (GSA) to ALA. The mechanism of the transamination was examined by using glutamate, specifically labeled with either 1-¹³C or ¹⁵N, as substrate for ALA synthesis. After incubating with crude enzymes extracted from Chlamydomonas reinhardtii, the distribution of labels in purified ALA molecules was examined by nuclear magnetic resonance spectroscopy and gas chromatography-mass spectrometry. We found that both isotopes were present in the same ALA molecule. We interpret the results to mean that intermolecular transamination occurs during the conversion of GSA to ALA.     

Chlorophyll biosynthesis in Chlamydomonas starts with the formation of glutamyl-tRNA

An RNA moiety has been shown to be involved in the conversion of Glu to delta-aminolevulinic acid (ALA), the first committed intermediate of the chlorophyll pathway. We now have evidence suggesting that in Chlamydomonas, the first reaction for converting Glu to ALA is the aminoacylation of Glu to a Glu-specific tRNA. The Glu-tRNA thus formed could be the substrate for Glu-1-semialdehyde synthesis catalyzed by a postulated dehydrogenase. Glu-1-semialdehyde can be converted to ALA by an aminotransferase. Of the three reactions converting Glu to ALA, only the second reaction, catalyzed by a postulated dehydrogenase, is sensitive to inhibition by heme (a known inhibitor of ALA synthesis). We think the regulated enzyme of ALA synthesis is the postulated dehydrogenase. It is postulated that in the chloroplast of Chlamydomonas, the synthesis of ALA and the synthesis of proteins may share a common pool of glutamyl-tRNA.     

Altered regulation of hepatic heme metabolism in cadmium exposed chick embryo

An investigation on the process of heme metabolism with special emphasis on ALA synthetase, heme synthetase and heme oxygenase was studied in cadmium exposed chick embryo to enlighten the mechanism of cadmium embryotoxicity. Cadmium chloride injection (2.5-10 mumole/kg) to chick embryo increases the activity of ALA synthetase by 5-7 folds, however, it inhibits the activity of heme synthetase significantly. The activity of heme oxygenase is further shown to be enhanced by cadmium chloride treatment. These changes are accompanied by a marked reduction in hepatic heme content. The induction of ALA synthetase and heme oxygenase was dependent on the initial concentration of exogenous cadmium. Pretreatment with actinomycin D completely blocks the cadmium mediated induction of both ALA synthetase and heme oxygenase. Time course studies on the stimulation of these two enzymes show that cadmium enhances the activity of heme oxygenase to its maximum level after 24 h. of injection, whereas ALA synthetase activity reaches its highest value only by 48 h. and both the enzymes remain elevated at least upto 96 h. This observation can be correlated with the hepatic heme level at different time intervals after cadmium exposure. These observations suggest the presence of regulatory process for heme metabolism which is susceptible to alteration of 'regulatory heme pool' caused by cadmium.     

Decrease of hepatic delta-aminolevulinate dehydratase activity in an animal model of fatigue

Fatigue can be defined physiologically as inability to maintain the expected power output. At present, no standard of fatigue are yet available. In order to find biomarkers of fatigue, we investigated the level of delta-aminolevulinic acid (ALA), the first intermediate metabolite in the heme biosynthetic pathway, in the plasma and urine of an animal model of fatigue. To prepare fatigued animals, we kept rats for 5 days in a cage filled with water to a height of 1.5 cm. As a result, the plasma and urinary ALA levels were increased in the fatigued animals as compared with those in the control animals. One day after the rats had been returned to their normal cages, these increased levels were restored to the control ones. We also examined the activity of the enzyme ALA dehydratase (ALAD), which is the second enzyme in the heme biosynthetic pathway, and ALAD gene expression during the fatigue and its recovery sessions. The ALAD activity, as well as its gene expression, in the liver of the fatigued animals was decreased as compared with those of the control animals. Both activity and gene expression of ALAD were recovered to their respective control levels after the rats had been allowed to rest in their normal cages for 1 day. Furthermore, the activity of ALA synthase (ALAS), the rate-limiting enzyme in the heme biosynthesis, in the liver was increased after the fatigue session for 5 days. Although this level of increase in the plasma concentration of ALA may not induce fatigue, increase in plasma and urinary ALA levels can be biomarkers of fatigue.     

The Bacillus subtilis hemAXCDBL gene cluster, which encodes enzymes of the biosynthetic pathway from glutamate to uroporphyrinogen III.

We have recently reported (M. Petricek, L. Rutberg, I. Schr?der, and L. Hederstedt, J. Bacteriol. 172: 2250-2258, 1990) the cloning and sequence of a Bacillus subtilis chromosomal DNA fragment containing hemA proposed to encode the NAD(P)H-dependent glutamyl-tRNA reductase of the C5 pathway for 5-aminolevulinic acid (ALA) synthesis, hemX encoding a hydrophobic protein of unknown function, and hemC encoding hydroxymethylbilane synthase. In the present communication, we report the sequences and identities of three additional hem genes located immediately downstreatm of hemC, namely, hemD encoding uroporphyrinogen III synthase, hemB encoding porphobilinogen synthase, and hemL encoding glutamate-1-semialdehyde 2,1-aminotransferase. The six genes are proposed to constitute a hem operon encoding enzymes required for the synthesis of uroporphyrinogen III from glutamyl-tRNA. hemA, hemB, hemC, and hemD have all been shown to be essential for heme synthesis. However, deletion of an internal 427-bp fragment of hemL did not create a growth requirement for ALA or heme, indicating that formation of ALA from glutamate-1-semialdehyde can occur spontaneously in vivo or that this reaction may also be catalyzed by other enzymes. An analysis of B. subtilis carrying integrated plasmids or deletions-substitutions in or downstream of hemL indicates that no further genes in heme synthesis are part of the proposed hem operon.     

Effect of endogenous heme generation on delta-aminolevulinic acid synthase activity in rat liver mitochondria.

This study examined the possibility that generation of heme within mitochondria may provide a local concentration sufficient to inhibit the activity of delta-aminolevulinic acid (ALA) synthase, the enzyme that catalyzes the rate-limiting step in hepatic heme biosynthesis. This was accomplished by simultaneously running ALA synthase and heme synthase activities in intact mitochondria isolated from rat liver. Radiochemical assays were used to measure the enzyme activities. ALA synthase activity did not decrease as the rate of heme formation was increased by varying the concentration of substrates for heme synthase. Even at a rate of heme generation estimated to be at least 75 times the rate occuring in vivo, ALA synthase activity was unchanged. We conclude that end product inhibition of ALA synthase activity by heme is not an important physiological mechanism for regulation of hepatic heme biosynthesis.     

Heme metabolism of Plasmodium is a major antimalarial target

The malarial parasite manifests unique features of heme metabolism. In the intraerythrocyte stage it utilizes the host hemoglobin to generate amino acids for its own protein synthesis, but polymerizes the acquired heme as a mechanism for detoxification. At the same time the parasite synthesizes heme de novo for metabolic use. The heme biosynthetic pathway of the parasite is similar to that of hepatocytes and erythrocytes. However, while the parasite makes its own delta-aminolevulinate (ALA) synthase that is immunochemically different from that of the host, it imports ALA dehydrase and perhaps the subsequent enzymes of the pathway from the host red cell. Many schizonticidal drugs such as chloroquine and artemisinin act by interfering with the heme metabolism of the parasite and there is scope to design new molecules based on the unique features of this metabolic machinery in the parasite.     

Occurrence of two 5-aminolevulinate biosynthetic pathways in Streptomyces nodosus subsp. asukaensis is linked with the production of asukamycin

We report the results of cloning genes for two key biosynthetic enzymes of different 5-aminolevulinic acid (ALA) biosynthetic routes from Streptomyces. The genes encode the glutamyl-tRNAGlu reductase (GluTR) of the C5 pathway and the ALA synthase (ALAS) of the Shemin pathway. While Streptomyces coelicolor A3(2) synthesizes ALA via the C5 route, both pathways are operational in Streptomyces nodosus subsp. asukaensis, a producer of asukamycin. In this strain, the C5 route produces ALA for tetrapyrrole biosynthesis; the ALA formed by the Shemin pathway serves as a precursor of the 2-amino-3-hydroxycyclopent-2-enone moiety (C5N unit), an antibiotic component. The growth of S. nodosus and S. coelicolor strains deficient in the GluTR genes (gtr) is strictly dependent on ALA or heme supplementation, whereas the defect in the ALAS-encoding gene (hemA-asuA) abolishes the asukamycin production in S. nodosus. The recombinant hemA-asuA gene was expressed in Escherichia coli and in Streptomyces, and the encoded enzyme activity was demonstrated both in vivo and in vitro. The hemA-asuA gene is situated within a putative cluster of asukamycin biosynthetic genes. This is the first report about the cloning of genes for two different ALA biosynthetic routes from a single bacterium.     

Free radical generation during delta-aminolevulinic acid autoxidation: induction by hemoglobin and connections with porphyrinpathies

delta-Aminolevulinic acid (ALA), a heme precursor accumulated in acute intermittent porphyria and saturnism, undergoes autoxidation leading to ammonium ion and probably the corresponding alpha-ketoaldehyde. This reaction is accelerated by addition of oxyhemoglobin (oxyHb) and other iron complexes. OxyHb is concomitantly oxidized to metHb; the apparent second-order rate constant of oxyHb/ALA coupled oxidation is ca. 10 M-1 min-1.1H NMR and uv spectral studies suggest that ALA undergoes enolization before consuming the dissolved oxygen. Spin-trapping experiments demonstrate formation of both the hydroxyl radical and a substrate-derived carbon-centered radical during ALA oxidation. Generation of active oxygen species by ALA might be related to the neuropathy associated to some acquired and inherited porphyrinpathies.     

Alterations in hepatic heme metabolism in fish exposed to sublethal cadmium levels

A study on hepatic heme metabolism with special emphasis to ALA synthetase, ALA dehydratase and heme oxygenase was carried out in cadmium exposed freshwater fish Channa punctatus to enlighten the mechanism of cadmium induced toxicity. Cadmium exposure (0.5-5.0 mg/1) for 7 days increased the hepatic level of ALA, along with the depletion in heme content, which are characteristic to chemical porphyria. The resultant enhancement in the activities of ALA synthetase and heme oxygenase were further shown to be dose dependent. ALA dehydratase activity on the other hand was enhanced only at higher exposure. Time course studies on the enzyme activities and heme content showed that ALA synthetase started to increase after 24 hrs., reached maximum at 7 days and came back nearly to normal level after 30 days of exposure. Simultaneously maximum depletion in heme level occurred on 7 days of exposure, tending to return to normal on 30 day. In addition, attempt has been made to correlate alterations in heme metabolism due to cadmium with the histopathological manifestations in liver.     

Role of the hemA gene product and delta-aminolevulinic acid in regulation of Escherichia coli heme synthesis.

We initiated these studies to help clarify the roles of heme, delta-aminolevulinic acid (ALA), hemA, and hemM in Escherichia coli heme synthesis. Using recombinant human hemoglobin (rHb1.1) as a tool for increasing E. coli's heme requirements, we demonstrated that heme is a feedback inhibitor of heme synthesis. Cooverexpression of rHb1.1 and the hemA-encoded glutamyl-tRNA (GTR) reductase increased intracellular levels of ALA and heme and increased the rate of rHb1.1 formation. These results support the conclusion that heme synthesis is limited by ALA (S. Hino and A. Ishida, Enzyme 16:42-49, 1973; W. K. Philipp-Dormston and M. Doss, Enzyme 16:57-64, 1973) and that the hemA-encoded GTR reductase is a rate-limiting enzyme in the pathway (J.-M. Li, C. S. Russell, and S. D. Cosloy, Gene 82:2099-217, 1989). Increasing the copy number of hemM, whose product is believed to be required for efficient ALA formation (W. Chen, C. S. Russell, Y. Murooka, and S. D. Cosloy, J. Bacteriol. 176:2743-2746, 1994; M. Ikemi, K. Murakami, M. Hashimoto, and Y. Murooka, Gene 121:127-132, 1992), had no effect on either ALA pools or the rate of rHb1.1 accumulation. The hemA-encoded GTR reductase was found to be regulated by ALA. Some of our results differ from those reported by Hart and coworkers (R. A. Hart, P. T. Kallio, and J. E. Bailey, Appl. Environ. Microbiol. 60:2431-2437, 1994), who concluded that ALA formation is not the rate-limiting step in E. coli cells expressing Vitreoscilla hemoglobin.     

DNA damage by 5-aminolevulinic and 4,5-dioxovaleric acids in the presence of ferritin

Cellular accumulation of 5-aminolevulinic acid (ALA), the first specific intermediate of heme biosynthesis, is correlated in liver biopsy samples of acute intermittent porphyria affected patients with an increase in the occurrence of hepatic cancers and the formation of ferritin deposits in hepatocytes. 5-Aminolevulinic acid is able to undergo enolization and to be subsequently oxidized in a reaction catalyzed by iron complexes yielding 4,5-dioxovaleric acid (DOVA). The released superoxide radical (O(*-)(2)) is involved in the formation of reactive hydroxyl radical ((*)OH) or related species arising from a Fenton-type reaction mediated by Fe(II) and Cu(I). This leads to DNA oxidation. The metal catalyzed oxidation of ALA may be exalted by the O(*-)(2) and enoyl radical-mediated release of Fe(II) ions from ferritin. We report here the potentiating effect of ferritin on the ALA-mediated cleavage of plasmid DNA and the enhancement of the formation of 8-oxo-7, 8-dihydro-2'-deoxyguanosine (8-oxodGuo). Plasmid pBR322 was incubated with ALA and varying amounts of purified ferritin. DNA damage was assessed by gel electrophoresis analysis of the open and the linear forms of the plasmid from the native supercoiled structure. Addition of either the DNA compacting polyamine spermidine or the metal chelator ethylenediaminetetraacetic acid (EDTA) inhibited the damage. It was also shown that ALA in the presence of ferritin is able to increase the oxidation of the guanine moiety of monomeric 2'-deoxyguanosine (dGuo) and calf thymus DNA (CTDNA) to form 8-oxodGuo as inferred from high performance liquid chromatography (HPLC) measurements using electrochemical detection. The formation of the adduct dGuo-DOVA was detected in CTDNA upon incubation with ALA and ferritin. In a subsequent investigation, the aldehyde DOVA was also able to induces strand breaks in pBR322 DNA.     

5-Aminolevulinic acid synthesis in epimastigotes of Trypanosoma cruzi

BACKGROUND AND AIMS: Trypanosoma cruzi is the causative agent of Chagas disease or American trypanosomiasis. The parasite manifests a nutritional requirement for heme compounds because of its biosynthesis deficiency. The aim of this study has been to investigate the presence of metabolites and enzymes of porphyrin pathway, as well as ALA formation in epimastigotes of T. cruzi, Tulahuén strain, Tul 2 stock. METHODS: Succinyl CoA synthetase, 5-aminolevulinic acid (ALA) synthetase, 4,5-dioxovaleric (DOVA) transaminase, ALA dehydratase and porphobilinogenase activities, as well as ALA, porphobilinogen (PBG), free porphyrins and heme content were measured in a parasite cells-free extract. Extracellular content of these metabolites was also determined. RESULTS: DOVA, PBG, porphyrins and heme were not detected in acellular extracts of T. cruzi. However ALA was detected both intra- and extracellularly This is the first time that the presence of ALA (98% of intracellularly formed ALA) is demonstrated in the extracellular medium of a parasite culture. Regarding the ALA synthesizing enzymes, DOVA transaminase levels found were low (7.13+/-0.49EU/mg protein), whilst ALA synthetase (ALA-S) activity was undetectable. A compound of non-protein nature, low molecular weight, heat unstable, inhibiting bacterial ALA-S activity was detected in an acellular extract of T. cruzi. This inhibitor could not be identified with either ALA, DOVA or heme. CONCLUSIONS: ALA synthesis is functional in the parasite and it would be regulated by the heme levels, both directly and through the inhibitor factor detected. ALA formed can not be metabolized further, because the necessary enzymes are not active, therefore it should be excreted to avoid intracellular cytotoxicity.     

Kinemage of action - proposed reaction mechanism of glutamate-1-semialdehyde aminomutase at an atomic level

Glutamate-1-semialdehyde aminomutase (GSAM), a key enzyme in tetrapyrrole cofactor biosynthesis, performs a unique transamination on a single substrate. The substrate, glutamate-1-semialdehyde (GSA), undergoes a reaction that exchanges the position of an amine and a carbonyl group to produce 5-aminolevulinic acid (ALA). This transamination reaction is unique in the fact that is does not require an external cofactor to act as a nitrogen donor or acceptor in this transamination reaction. One of the other remarkable features of the catalytic mechanism is the release free in the enzyme active site of the intermediate 4,5-diaminovaleric acid (DAVA). The action of a gating loop prevents the escape of DAVA from the active site. In a MD simulation approach, using snapshots provided by X-ray crystallography and protein crystal absorption spectrometry data, the individual catalytic steps in this unique intramolecular transamination have been elucidated.     

Chemical synthesis of 4,5-dioxovaleric acid and its nonenzymatic transamination to 5-aminolevulinic acid

4,5-Dioxovaleric acid (DOVA) was synthesized from 5-bromolevulinic acid via formation of the pyridinium bromide of 5-bromolevulinic acid, followed by nitrone formation with p-nitrosodimethylaniline, and hydrolysis of the nitrone to yield DOVA. Partial purification of DOVA was obtained by passage of the reaction mixture through a cation exchange column. DOVA was identified by paper electrophoresis and by a specific fluorometric assay. DOVA was nonenzymatically transaminated to 5-aminolevulinic acid (ALA) with glycine serving as the amino donor. Other compounds tested were less effective amino donors. Glyoxylic acid was identified as a reaction product by paper electrophoresis and a specific calorimetric test. ALA was identified by paper electrophoresis, paper chromatography of a pyrrole derivative, reaction with Ehrlich reagent, and by its enzymatic conversion by a barley extract to porphobilinogen and uroporphyrin. The nonenzymatic transamination was inhibited by Tris and was stimulated by high pH. The existence of this nonenzymatic activity is discussed in relation to previous reports of dova transaminase activity in cell extracts.     

Heme biosynthesis in a chicken hepatoma cell line (LMH): comparison with primary chick embryo liver cells (CELC)

5-Aminolevulinic acid synthase (ALA synthase), the rate-controlling enzyme of hepatic heme biosynthesis, is feed-back repressed by heme. In the liver, chemicals such as barbiturates markedly induce ALA synthase, especially in the presence of partial defects of heme biosynthesis. The inducibility and regulation of ALA synthase have been investigated using a variety of models, including intact animals and liver cell culture systems. A widely used model that closely approximates what occurs in vivo and in humans is that of primary cultures of chick embryo liver cells (CELCs). However, CELCs have some limitations: the cells obtained are somewhat heterogeneous; isolation and culture must be repeated every week resulting in weekly variations; and cells are short-lived limiting the feasibility of time-course and transfection studies. The aim of this study was to determine if LMH cells, a chick hepatoma cell line, are a good model comparable to that of CELCs. In both cells similar patterns of response of, ALA synthase activities and mRNA levels, and of porphyrin accumulation were obtained following treatments known to affect heme biosynthesis. Similarly, heme repressed ALA synthase mRNA levels in both cell types and ALA synthase activities in LMH cells. We conclude that LMH cells are a useful model for the study of hepatic heme biosynthesis and regulation of ALA synthase.