Δ-aminolevulinic Acid Synthase Research Articles

Effect of alcohol on δ-aminolevulinic acid dehydratase and porphyrin metabolism in man

The influence of alcohol on porphyrin metabolism was investigated in 6 healthy non-alcoholics and 19 patients with chronic alcohol abuse. In the healthy subjects, blood and urine samples were obtained before and after acute alcohol exposure, whereas in the chronic alcoholics only one examination was performed. In both groups a significant inhibition of δ-aminolevulinic acid dehydratase was demonstrated. The activity was partially restored in vitro by addition of zinc ions or dithiothreitol. A combination of both activators produced reactivation to normal levels. Coproporphyrinuria was more prominent in chronic alcoholics (373 nmol/24 h on average, upper norm 119 nmol/24 h) compared to non-alcoholics (140 nmol/24 h). Urinary porphobilinogen and δ-amino-levulinic acid were normal except for a moderately increased δ-aminolevulinic acid in four healthy individuals. In conclusion, alcohol causes reversible inhibition of δ-aminolevulinic acid dehydratase. The metabolic changes reflect both an inhibition of δ-aminolevulinic acid dehydratase and coproporphyrinogen oxidase; a simultaneous, moderate induction of hepatic δ-aminolevulinic acid synthase is suggested. Erythrocyte δ-aminolevulinic acid dehydratase activity could serve as a sensitive indicator for both acute and chronic alcohol consumption even better than Coproporphyrinuria.

Oxygen tension regulated expression of the hemA gene of Rhodobacter capsulatus

The promoter of the Rhodobacter capsulatus hemA gene, coding for the enzyme δ-aminolevulinic acid synthase (ALAS), was identified by trans-complementation of a δ-aminolevulinic acid (ALA)-dependent mutant and found to be located within a 170 bp region proximal to the hemA gene. The activity of the hemA promoter was demonstrated by lacZ fusion and in vitro transcription-translation. An open reading frame (ORFX) was found downstream of hemA. The activity of the hemA promoter, but not that of the ORFX promoter, increased when oxygen tension was lowered in the culture. Deletions upstream of the hemA promoter region did not affect ALAS activity and formation of pigment-protein complexes in R. capsulatus.

Elevation of δ-aminolevulinic acid synthase and cytochrome PB 1 P450 messenger RNA levels by dihydropyridines, dihydroquinolines, sydnones, and N-ethylprotoporphyrin IX

A series of compounds that increase the activity of δ-aminolevulinic acid synthase (ALAS) in chick embryo hepatocyte cultures were studied for their effects on steady-state levels of mRNA for ALAS and phenobarbital-inducible cytochrome PB, P450. N-Ethylprotoporphyrin IX (N-EtPP), which is believed to lower heme levels by inhibition of ferrochelatase (FC), had little effect on steady-state ALAS mRNA levels. 3,5-Diethoxycarbonyl-l,4-dihydro-2,6-dimethyl-4-isobutylpyridine (4-isobutyl DDC), which is believed to lower heme levels by repetitive destruction of the heme moiety of cytochrome P450, increased steady-state levels of ALAS mRNA by approximately 2-fold. 3,5-Diethoxycarbonyl-1, 4-dihydro-2,6-dimethyl-4-ethylpyridine (4-ethyl DDC) which inhibits FC activity and destroys the heme moiety of cytochrome P450, increased ALAS mRNA levels approximately 4-fold. A combination of N-EtPP and 4-isobutyl DDC produced a synergistic increase in ALAS mRNA levels to approximately 6-fold over control levels. The synergistic increase in ALAS activity observed previously with this combination can be explained, at least in part, by a synergistic increase in ALAS mRNA levels. Other porphyrinogenic agents, which function as mechanism-based inactivators of cytochrome P450 and elevate ALAS activity, were found to elevate ALAS mRNA. These compounds included 3-[2-(2,4,6-trimethylphenyl)thioethyl]-4-methylsydnone (TTMS), 2,4-diethyl-2-methyl-1,2-dihydroquinoline (DMDQ), and 2,2,4-trimethyl-1,2,dihydroquinoline (TMDQ). The elevation of ALAS mRNA by these porphyrinogenic agents is probably due to their lowering of cellular heme levels by a combination of ferrochelatase inhibition and repetitive destruction of the heme moiety of cytochrome P450. The lowering of heme levels should result in an enhancement of ALAS mRNA half-life as it has been demonstrated by others that heme shortens the half-life of ALAS mRNA. It was of interest that some of these drug treatments also caused an elevation in steady-state levels of cytochrome PB 1 P450 mRNA; the exception was TTMS, which along with its analogue 3-(2-phenylethyl)-4-methylsydnone (PEMS), did not alter cytochrome PB 1 P450 mRNA levels. Increases in steady-state levels of cytochrome pb 1 P450 mRNA subsequent to increases in steady-state levels of ALAS mRNA were observed with 4-ethyl DDC, 4-isobutyl DDC, DMDQ, and TMDQ. The data obtained with N-EtPP and a combination of N-EtPP and 4-isobutyl DDC on cytochrome PB 1 P450 mRNA levels do not support the contention that heme functions as a positive regulator of cytochrome P450 gene expression.

Fluorimetric determination of hepatic δ-aminolevulinic acid synthase activity by high-performance liquid chromatography

A fluorimetric method for measuring the activity of δ-aminolevulinic acid synthase (ALAS) in the liver of mice has been developed. The liver homogenate was used as the enzyme source. The final concentration of glycine (substrate) used for the assay was 100 m M. The δ-aminolevulinic acid (ALA) formed during incubation was converted into a highly fluorescent derivative by condensation with acetylacetone and formaldehyde (application of the Hantzsch reaction). This derivative was completely separated from other fluorescent substances in the reaction medium, and it was determined using a high-performance liquid chromatograph equipped with a fluorescence monitor ( 370 460 nm). The activity of ALAS was expressed as nmol ALA formed per gram liver per hour.

Molecular regulation—Biological role of heme in hematopoiesis

Heme synthesis and degradation play pivotal roles in the regulation of growth and differentiation of erythroid and non-erythroid cells. Heme synthesis in mammalian cells involves eight enzymes which are localized in mitochondrial and cytoplasmic compartments. These enzymes have been well-characterized and cDNAs for six of the enzymes has been cloned. Two enzymes in the enzymes of the heme biosynthetic pathway, δ-aminolevulinic acid synthase (ALAS) and porphobilinogen deaminase (PBG-D) have special features and may have regulatory functions in heme synthesis by hematopoietic cells. ALAS exists as two isozymes which are encoded by non-erythroid and erythroid-specific genes, respectively. By contrast, PBG-D, which also exists as two isozymes, arises from a single gene comprised of two overlapping transcriptional units, each with its own promoter. Transcription from one or the other of these promoters gives rise through differential splicing to two distinct mRNA species which encode the distinct nonerythroid and erythroid isoforms. On the other hand, heme catabolism is determined by the levels of the heme oxygenase system. The enzyme has been purified and the cDNA for heme oxygenase has been cloned. Repression of heme oxygenase in erythroid progenitor cells may initiate differentiation. In addition, recent evidence has suggested that heme may have a broader role in hematopoiesis and in the network of cytokine production by adherent stromal cells.

Studies on induction of δ-aminolevulinic acid synthase, ferrochelatase, cytochrome P-450 and cyclic AMP by phenformin: Chlorpropamide, allylisopropylacetamide and lead in hepatocytes from normal and experimental diabetic rats

The present work demonstrates that phenformin exerted an inducing effect on δ-amino-levulinic acid synthase (ALA-S) and ferrochelatase activities and on cytochrome P-450 content in isolated hepatocytes from rats with experimental diabetes. Similar results were obtained with respect to ALA-S activity and cytochrome P-450 content when chlorpropamide was used. The inducing effect exerted by allylisopropylacetamide (AIA) on ALA-S and ferrochelatase activities in diabetic hepatic cells was markedly greater than that observed in normal hepatocytes. This stimulatory response was not enhanced by adding dibutyryl cyclic AMP (cAMP). When phenformin was added to isolated rat hepatocytes of normal rats, induction of ALA-S and ferrochelatase activities and cytochrome P-450 content was observed only in the presence of added dibutyryl cAMP. Addition of chlorpropamide to this in vitro system did not exert an inducing effect on the same enzymes even in the presence of dibutyryl cAMP. The present results add more experimental evidence about the lability of the heme pathway of diabetic hepatocytes.

Inhibition of ferrochelatase by N-ethylprotoporphyrin does not alter steady state mRNA levels of δ-aminolevulinic acid synthase

Inhibition of ferrochelatase by N-ethylprotoporphyrin does not alter steady state mRNA levels of δ-aminolevulinic acid synthase

Effects of 3-(2-phenylethyl)-4-methylsydnone and related sydnones on heme biosynthesis

3-[2-(2,4,6-Trimethylphenyl)thioethyl]-4-methylsydnone (TTMS) and 3-(2-phenylethyl)-4-methylsydnone (PEMS) cause mechanism-based inactivation of rat hepatic microsomal cytochrome P-450 and the formation of N-alkylprotoporphyrins in rat liver. In the present study, we have shown that both TTMS and PEMS cause mechanism-based inactivation of chick embryo hepatic microsomal cytochrome P-450. TTMS also caused the inhibition of ferrochelatase activity, the accumulation of protoporphyrin IX, and an increase in the activity of δ-aminolevulinic acid synthase in chick embryo liver cell culture. PEMS was devoid of effect on ferrochelatase activity, porphyrin accumulation, and δ-aminolevulinic acid synthase activity. There are two possible explanations for the lack of effect of PEMS on heme biosynthesis: (1) the ring-A- and/or ring-B-substituted regioisomers of the N-phenylethyl-and N-phenylethenylprotoporphyrins which are produced during the mechanism-based inactivation of cytochrome P-450 by PEMS are too bulky to fit into the active site of ferrochelatase to inhibit its activity, in contrast to the N-vinylprotoporphyrin formed from TTMS; and (2) the N-alkylprotoporphyrins produced consist of the ring-C- and/or ring-D-substituted regioisomers, which are not inhibitors of ferrochelatase activity.

Chronic inhalation effects of ethylene oxide on porphyrin-heme metabolism

The effects of chronic ethylene oxide (EtO) inhalation on porphyrin-heme metabolism were investigated. When Wistar male rats were exposed to 500 ppm EtO for 6 h a day, 3 times a week for 13 weeks, hemoglobin content significantly decreased, and a normocytic and normochromic anemia was found. In the liver, cytochrome P-450 and protoheme significantly decreased but wet weight, microsomal protein and cytochrome b 5 were not affected. The activity of δ-aminolevulinic acid (ALA) synthase increased while ALA dehydratase did not change. The activity of hepatic ferrochelatase decreased time-dependently. Uroporphyrin increased 37% and coproporphyrin tended to increase in the liver. The concentration of protoporphyrin in the liver and erythrocytes tended to increase. Coproporphyrin excretion in the urine showed a 5–6-fold increase while there was no significant increase in urinary ALA excretion. These results indicate that chronic inhalation of EtO causes alterations of hepatic porphyrin-heme metabolism as well as anemia and may affect mechanisms of adaptation to xenobiotics.

Synergistic induction of δ-aminolevulinic acid synthase activity by N-ethylprotoporphyrin IX and 3,5-diethoxycarbonyl-1,4-dihydro-2,6-dimethyl-4-isobutylpyridine

The levels of the cellular free heme pool in chick embryo hepatocyte culture were lowered using N-ethylprotoporphyrin IX ( N-ethylPP) and analogues of 3,5-diethoxycarbonyl-1,4-dihydro-2,4,6-trimethylpyridine (DDC), and the effect on δ-aminolevulinic acid synthase (ALAS) was examined. N-EthylPP, which lowers cellular heme levels by inhibiting ferrochelatase activity, produced an induction of ALAS activity to 444% of control at 3 hr after its administration. 4-Ethyl DDC, which lowers heme levels by destroying the heme moiety of cytochrome P-450 and lowering ferrochelatase activity, caused an induction of ALAS to 565% of control at 12 hr after administration. 4-Isobutyl DDC, which lowers heme levels by destroying the heme moiety of cytochrome P-450, induced the activity of ALAS to 289% of control at 3 hr after administration. This indicates that ferrochelatase inhibition is a more important mechanism of heme lowering than alkylation of cytochrome P-450 heme when both heme-depleting mechanisms are acting in chick embryo liver cells. It was anticipated that administration of a combination of 4-isobutyl DDC plus N-ethylPP would mimic the effect of 4-ethyl DDC. However, this combination induced ALAS activity to levels that were much greater than those observed after 4-ethyl DDC (1257% of control at 12 hr). This synergistic induction may be attributable to lowering of free heme levels to the point where transcription, translation, and translocation of ALAS are all derepressed.

Decreased estrogen hydroxylation in male rat liver following cimetidine treatment

Administration of cimetidine (600 μmol/kg × 5) to adult male rats resulted in 55 and 25% decreases, respectively, in estradiol 2- and 16α-hydroxylation. The same treatment also decreased the activities of ethylmorphine demethylase, aryl hydrocarbon hydroxylase, aniline hydroxylase and heme oxygenase but did not inhibit the activities of 7-ethoxycoumarin de-ethylase and δ-aminolevulinic acid synthase or decrease cytochrome P-450 content. in vitro addition of cimetidine (10–300 μM) also inhibited estradiol hydroxylations, and the effect was additive in rats pretreated with cimetidine in vivo; the other enzymic activities studied were completely unaffected by in vitro addition of cimetidine. In contrast, there was no effect of cimetidine either in vivo or in vitro on any of these activities in female rats. The results point to a wide variation in the susceptibilities of different isozymes of cytochrome P-450 to inhibition by cimetidine and suggest that such differential susceptibilities are also highly dependent on the sex of the animal.

Screening costramid libraries for chromosomal genes: an alternative interspecific hybridization method

Broad host-range RK2-based cosmid vectors (‘costramids’) are increasingly used in molecular genetic studies of Gram-negative soil bacteria such as Rhizobium spp. we describe a simple modification of existing methods, whereby a genomic library constructed in a stringently replicated vector can be screened for genes which are undetectable by colony hybridization due to background cross-hybridization. This method allows the use of ‘heterologous’ probes (interspecies hybridization) to isolate several presumptive genes of interest from a gene bank of Rhizobium sp. NGR234 made in the costramid pRK7813. These are a gene with homology to the citrate synthase gene (gltA) or Escherichia coli, the gene encoding δ-aminol evulinic acid synthase (hemA), and a gene or genes regulating dicarboxylate transport.

Influence of chronic alcohol consumption on hepatic heme and porphyrin metabolism

To study the effect of prolonged alcohol consumption on hepatic heme and porphyrin metabolism, female Wistar rats were fed for 60 days a nutritionally adequate liquid diet containing 36% of total calories as ethanol, whereas the control diet was isocaloric and contained no alcohol. Compared to pair-fed controls, the administration of the alcohol diet resulted in an increased hepatic activity of δ-aminolevulinic acid synthase by 223% (112.3 ± 19.6 nmoles/hr/100 g b.wt. vs. 362.8 ± 42.5; P < 0.01), an enhanced urinary excretion of δ-aminolevulinic acid by 101% (64.8 ± 11.8 nmoles/day vs. 130.8 ± 22.4; P < 0.05), and an augmented urinary output of total porphyrins by 142% (1.2 ± 0.2 nmoles/day vs. 2.9 ± 0.5; P < 0.05). Concomitantly, the hepatic content of cytochrome P-450 was significantly enhanced and that of hepatic catalase activity marginally increased, whereas the hepatic iron content remained unaltered. In summary, the feeding of rats with a liquid alcohol diet for 60 days results in changes of hepatic heme and porphyrin metabolism which are associated and may be causally related with an induction of hepatic hemoproteins and subsequent derepression of hepatic δ-aminolevulinic acid synthase, whereas hepatic iron appears to play no pathogenic role.

Increased rat testicular heme oxygenase activity associated with depressed microsomal heme and cytochrome P-450 levels after repeated administration of human chorionic gonadotropin

Repeated administration of human chorionic gonadotropin to rats results in a maximal depression of testicular microsomal heme and cytochrome P-450 levels at 24 h, followed by increases that plateau at pretreatment levels by day six. Associated with the depressed levels of microsomal heme and cytochrome P-450 is an increase of testicular microsomal heme oxygenase activity at 12–24 h. Testicular mitochondrial δ-aminolevulinic acid synthase activity was increased at 24 h, and remained elevated throughout the 9-day treatment period. Pretreatment with 1,4,6-androstatrien-3,17-dione, an aromatase inhibitor, failed to prevent the depression of testicular microsomal heme or cytochrome P-450 or increased heme oxygenase activity caused by repeated administration of human chorionic gonadotropin, and administration of estradiol benzoate failed to alter testicular microsomal heme oxygenase activity suggesting that these parameters were not related to altered testicular estrogen content caused by increased aromatase activity. These results suggest that increased testicular heme oxygenase activity is associated with decreased microsomal heme and cytochrome P-450 content during human chorionic gonadotropin-induced desensitization.

Inhibition of the biosynthesis of uroporphyrinogen and heme in rat liver during obstructive jaundice produced by bile duct ligation

Altered hepatic microsomal drug metabolism has been reported to occur in patients 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 ( K i ) for bilirubin was 1.5 μ m. 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 δ-[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 δ-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 δ-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.

Bacterial δ-aminolevulinic acid synthase activity is not essential for leghemoglobin formation in the soybean/ Bradyrhizobium japonicum symbiosis

Previous studies of legume nodules have indicated that formation of the heme moiety of leghemoglobin is a function of the bacterial symbiont. We now show that a hemA mutant of Bradyrhizobium japonicum that cannot carry out the first step in heme biosynthesis forms fully effective nodules on soybeans. The bacterial mutant strain was constructed by first isolating the wild-type hemA gene encoding delta-aminolevulinic acid synthase (EC 2.3.1.37) from a cosmid library, using a fragment of the Rhizobium meliloti hemA gene as a hybridization probe. A deletion of the hemA gene region, generated in vitro, then was used to construct the analogous chromosomal mutation by gene-directed mutagenesis. The mutant strain had no delta-aminolevulinic acid synthase activity and was unable to grow in minimal medium unless delta-aminolevulinic acid was added. Despite its auxotrophy, the mutant strain incited nodules that appeared normal, contained heme, and were capable of high levels of acetylene reduction. These results rule out bacterial delta-aminolevulinic acid synthase activity as the exclusive source of delta-aminolevulinic acid for heme formation in soybean nodules.

Effects of iron deficiency and chronic iron overloading on mitochondrial heme biosynthetic enzymes in rat liver

The effects of iron deficiency and iron overloading on the mitochondrial enzymes involved in heme synthesis were studied in rat livers. The in vitro activities of several of the enzymes in this pathway were differentially influenced by the in vivo iron status of the animals. δ-Aminolevulinic acid synthase was slightly increased in iron-overloaded animals, but remained normal in iron-deficient animals (0.58 ± 0.09, 0.91 ± 0.19 and 0.61 ± 0.12 nmol δ-aminolevulinic acid/mg per h). Copro- and protoporphyrinogen oxidase activities were increased (20 and 60% above controls) in iron-deficient animals. In contrast, coproporphyrinogen oxidase was decreased by 20%, while protoporphyrinogen oxidase remained unchanged in iron-overloaded rats. These variations of activities were not due to changes in the affinity of these enzymes toward their substrates, as coporphyrinogen had the same Km in each case (0.62 ± 0.05 M) as did protoporphyrinogen (0.22 ± 0.035 M). Thus, the Km did not vary with the treatment received by the animals. Ferrochelatase activity was measured by both the pyridine hemochromogen method and by measurement of zinc protoporphyrin with endogenous zinc as substrate. In all cases, ferrochelatase was found to be able to synthesize zinc protoporphyrin with endogenous zinc as substrate. However, the apparent Km of zinc chelatase for protoporphyrin was significantly different in the three groups of animals with Km,appProto = 2.4 ± 0.1 10−7, 4 ± 0.3 10−7 and 9.10 ± 0.05 10−7 M in iron-overloaded, control and iron-deficient animals, respectively. When ferrochelatase activity was measured by pyridine hemochromogen, identical results were observed in iron-deficient and control animals but decreased by 45% in iron-overloaded animals. The mitochondrial heme content was also decreased by 40% in iron-overloaded rats but unchanged in either iron-deficient or control rats.

Studies on regulatory mechanisms of heme biosynthesis in hepatocytes from experimental-diabetic rats

Isolated hepatocytes from rats with experimental diabetes exhibit increased content of cytochrome P-450 and cyclic AMP and normal activities of the regulatory enzymes δ-aminolevulinic acid synthase and ferrochelatase. The inducing effect exerted by phenobarbital on cytochrome P-450, δ-aminolevulinic acid synthase and ferrochelatase biosynthesis and cyclic AMP content in diabetic hepatic cells is markedly greater than that observed in normal hepatocytes. This stimulatory response is neither enhanced by added dibutyryl cyclic AMP nor repressed by glucose. The present results suggest that the heme pathway of diabetic hepatocytes is more susceptible to porphyrinogenic factors.

Effect of glucose on induction of delta-aminolevulinic acid synthase, ferrochelatase and cytochrome P-450 hemoproteins in isolated rat hepatocytes by phenobarbital and lead.

In the present work we have been able to demonstrate that phenobarbital and lead exert an inducing effect on the biosynthesis of delta-aminolevulinic acid synthase, ferrochelatase and cytochrome P-450 hemoproteins in isolated rat hepatocytes of normal adult rats. Dibutyryl cyclic AMP enhances the induction effect produced by phenobarbital in this in vitro system. Glucose inhibits the induction of delta-aminolevulinic acid synthase and ferrochelatase. This repression effect can be reversed with increasing concentrations of dibutyryl cyclic AMP. No glucose effect was observed on the phenobarbital- and lead-mediated inductions of cytochrome P-450. he present results add more experimental evidence to support the concept that the last enzyme of the heme pathway is inducible, and as such may have a significant role in regulatory mechanisms of porphyrin and heme biosynthesis.

Impairment of induction of Δ-aminolevulinic acid synthase by gluconeogenic amino acids and carbohydrates in vitro

This study was undertaken in a system of chick embryo liver cells incubated in Earle's Basal Salt Solution with hormones. Impairment of induction of Δ-aminolevulinic acid synthase (ALAS) by allyl-isopropylacetamide (AIA) was observed in the presence of glucose. Fructose and various gluconeogenic substances including gluconeogenic amino acids had a similar effect. Leucine, which is purely ketogenic, did not influence induction of ALAS. SH-containing amino acids increased induction of ALAS by AIA. The glucose analogues 3-0-methylglucose and 2-deoxyglucose did not impair induction of ALAS by AIA. The inhibitory effect of glycerol, fructose, and glycine was not affected by 3-0-methylglucose but was reversed by 2-deoxyglucose. The results indicate that the salutory effects of proteins on acute attacks of hepatic porphyria are probably caused by their gluconeogenic properties and that glucose-6-phosphate, or a metabolite of glucose-6-phosphate that is not in the glycolytic pathway, is the active agent that leads to the glucose-like effect.