Delta-aminolevulinic Acid Synthetase Activity Research Articles

Effect of Cholestasis Produced by Bile Duct Ligation on Hepatic Heme and Hemoprotein Metabolism in Rats

Cholestasis produced by bile duct ligation has been associated with decreased concentrations of hepatic microsomal cytochrome P450 and decreased hepatic microsomal oxidative drug metabolism. Bile duct ligation producing cholestasis results in a marked increase in hepatic microsomal heme oxygenase activity, with corresponding decreases in hepatic microsomal cytochrome P450 concentration, reduced form of nicotinamide adenine dinucleotide phosphate-cytochrome c reductase activity, and hepatic delta-aminolevulinic acid synthetase activity. As sham-operated rats also demonstrate a less prolonged decrease in cytochrome P450 concentration and reduced form of nicotinamide adenine dinucleotide phosphate-cytochrome c reductase activity, the metabolic effects of surgery and anesthesia must also be involved in these alterations in microsomal oxidative drug metabolism. The relative rate of hepatic cytochrome P450 synthesis and of degradation are both decreased after bile duct ligation. These data suggest that decreased hepatic microsomal cytochrome P450 concentrations in cholestasis are partly the result of decreased cytochrome P450 synthesis. Increased levels of heme oxygenase activity are not related to increased cytochrome P450 turnover, but may instead reflect enlargement and increased catabolism of a free heme pool resulting from decreased hemoprotein (cytochrome P450) synthesis.

Influences of maternal ethanol intake on maternal and perinatal hepatic heme and drug metabolizing enzymes in rats.

The effect of ethanol on maternal and neonatal hepatic heme and drug metabolizing systems was determined. Ethanol (16%, w/v) was administered orally as drinking solution to pregnant or lactating rats at different pre- and post-natal stages. The dams and pups were sacrificed on days 7, 14 and 21 after parturition, respectively. Ethanol administration to lactating rats from just after birth caused an appreciable decrease in the maternal and neonatal body and liver weights. In addition, the activities of nicotinamide adenine dinucleotide phosphate-cytochrome c reductase, nicotinamide adenine dinucleotide-cytochrome b5 reductase and heme oxygenase were significantly enhanced in the livers of neonates whose mothers were exposed to the ethanol during only first week of lactation, but those activities were not altered in the maternal livers. However, no remarkable alterations were observed in the contents of cytochrome P-450 and b5, and the activities of aminopyrine demethylase, aniline hydroxylase and delta-aminolevulinic acid synthetase in the livers of neonates from mothers who had received ethanol during lactation period or last week of gestation, although the activities of aminopyrine demethylase and aniline hydroxylase were enhanced significantly in lactating dams by ethanol consumption for 14 d after parturition.

Inhibition of ferrochelatase by N-methylprotoporphyrin IX is not accompanied by delta-aminolevulinic acid synthetase induction in chick embryo liver cell culture.

N-Methylprotoporphyrin has been shown to markedly inhibit ferrochelatase activity in chick embryo liver cell culture without inducing delta-aminolevulinic acid (ALA) synthetase activity. This result supports the idea that the effects of 3,5-diethoxycarbonyl-1,4-dihydro-2,4,6-trimethylpyridine (DDC) on ALA synthetase activity and ferrochelatase activity are dissociated and that inhibition of ferrochelatase alone is not sufficient to cause induction of ALA synthetase. We conclude that the porphyrinogenic activity of DDC can be explained only in part by the actions of N-methylprotoporphyrin.

A study of a female with congenital sideroblastic anemia

A female infant with congenital refractory sideroblastic anemia is described. A marked reduction of delta-aminolevulinic acid (ALA) synthetase activity of erythroblasts was noticed with and without treatment of pyridoxal phosphate. Mitochondrial neutral protease activity of erythroblasts, which inactivates specifically the apo form of ALA synthetase, was normal and the sensitivity of apo form of ALA synthetase to the neutral protease was also normal. It was speculated that the reduction of ALA synthetase activity is not due to the high speed of destruction, but rather due to the impairment in production of ALA synthetase, which could explain the unresponsiveness to pyridoxine therapy in this case.

Cardiac and hepatic effects of pre- and postnatal exposure to polybrominated biphenyls in rats.

Body weight gain and hepatic concentrations of vitamin A were reduced in Sprague-Dawley rats by pre- and postnatal exposure to 100 ppm polybrominated biphenyls (PBBs). The ratio of liver weight to body weight, activity of hepatic delta-aminolevulinic acid (ALA) synthetase, and urinary excretion of uro- and coproporphyrins were increased by PBBs. Treatment with PBBs also increased the left atrial inotropic response to calcium. However, PBBs had no effect on development of the adrenergic neuronal transport system in heart, left atrial baselike peak tension, or inotropic response to ouabain. Thus PBBs retarded body weight gain and produced a variety of alterations in liver, but had little effect on cardiac contractile function.

Treatment of the Porphyrias: Mechanisms of Action

The porphyrias are diseases that result from inherited or acquired abnormalities of porphyrin-heme synthesis in the liver and the bone marrow. Only the hepatic porphyrias are known to be aggravated by exposure to a variety of exogenous drugs and chemicals. Simple avoidance of these agents will reduce the risk of developing hepatic porphyria and may lead to clinical improvement in patients with active disease. Some types of therapy of the hepatic porphyrias are effective because of their ability to modulate the activity of delta-aminolevulinic acid synthetase, the rate-limiting enzyme for heme synthesis. Most of the porphyrias are associated with cutaneous photosensitivity, the treatment of which centers about either reducing the excessive production of porphyrins or of inhibiting the photobiological response to these photosensitizing chemicals in the skin.

Acute effect of methamphetamine on heme oxygenase activity and on cytochrome P-450 content in the liver of female rats.

After a single treatment with d-methamphetamine (philopon) at a dose of 8 mg/kg in 10 weeks old female Wistar rats, heme oxygenase activity was enhanced at 3 hr and reached it peak level at 12 hr, whereas cytochrome P-450 content showed the lowest level at 12 hr. However, activity of delta-aminolevulinic acid synthetase was not affected. Twenty-four hr later, all measured parameters returned to nearly initial levels at zero time. The pretreatment with cycloheximide blocked the increase of heme oxygenase activity in philopon-treated animals.

Rifampicin-induced porphyria cutanea tarda

Summary A patient who developed porphyria cutanea tarda and disturbed liver function tests following treatment with rifampicin and isoniazid for a tuberculous psoas abscess is reported. The patient had normal liver function tests prior to receiving anti-tuberculosis chemotherapy, but was subsequently demonstrated to have cholelithiasis. Challenge testing with both drugs incriminated rifampicin as the agent precipitating porphyria cutanea tarda and disturbance of the liver function tests particularly the serum bilirubin. The association between rifampicin and porphyria cutanea tarda has not previously been described but might be expected because of the ability of rifampicin to induce various liver enzyme systems including delta aminolevulinic acid synthetase activity. This further illustration of rifampicin hepatotoxicity emphasizes the need for regular monitoring of liver function when rifampicin is prescribed. Rifampicin should be used with extreme caution in any patient with a previous history of porphyria cutanea tarda.

STUDIES ON THE INHIBITORY ACTION OF CHLORAMPHENICOL AND THIAMPHENICOL UPON ERYTHROPOIESIS

In order to elucidate the mechanism by which both chloramphenicol (CP) and thiamphenicol (TP) induce reversible erythroid suppression, an inhibitory effect of these drugs on heme synthesis was investigated in the patients having CP and/or TP-induced acute bone marrow failure.The patients consisted of five TP cases and the case which had been given both drugs. All cases but a case of polycythemia vera had reversible erythroid suppression. The case of polycythemia vera died of cerebral thrombosis. The anemia observed at the occurrence of acute bone marrow failure were normocytic and normochromic in all cases but one of polycythemia vera, which already had low value of MCV, MCH and MCHC prior to TP administration. Among six patients one had pancytopenia, three had only anemia and two had anemia with thrombocytopenia. Normal to slightly elevated level of serum iron was observed in all cases which were measured one to two days after the occurrence of acute bone marrow failure. Morever, the appearance of ringed sideroblasts were noted in two of six cases which bone marrow examination was performed.Out of five cases examined, two showed a decrease in deltaaminolevulinic acid synthetase (δ-A-LA-s) activity while the other two did the normal values. Out of three cases two showed an increase in both free erythrocyte copro- and protoporphyrin, and the third did the normal value. Neither inhibitory effect of CP succinate nor TP on δ-ALA-s was observed in the in vitro study examined at a concentration of less than 10-3 mol of CP or TP.

Delta-Aminolevulinic acid synthetase in erythroblasts of patients with pyridoxine-responsive anemia. Hypercatabolism caused by the increased susceptibility to the controlling protease.

Properties of delta-aminolevulinic acid synthetase in erythroblasts of patients with pyridoxine-responsive anemia were investigated with special reference to the protease in mitochondria of erythroblasts. delta-Aminolevulinic acid synthetase activity in erythroblasts of patients with this disease before treatment was extremely decreased, whereas it gradually increased in parallel with the improvement of anemia by the therapy with pyridoxal phosphate. The amount of apo-delta-aminolevulinic acid synthetase in erythroblasts before treatment was also extremely diminished. Apparent affinity to pyridoxal phosphate of the apo-delta-aminolevulinic acid synthetase obtained from erythroblasts of the patients was almost the same as that of normal controls. The activity of a new protease which is considered to be engaged in the regulation of delta-aminolevulinic acid synthetase levels in mitochondria of erythroblasts was shown to be in normal range in erythroblasts of the patients. On the other hand, apo-delta-aminolevulinic acid synthetase obtained from the patients was extremely sensitive to the protease. These results indicate that disturbance of heme synthesis characteristic to pyridoxine-responsive anemia could be ascribed to the hypercatabolism of delta-aminolevulinic acid synthetase caused by the increased susceptibility to the controlling protease in erythroblasts.

Developmental and convalescent changes of the anemia caused by excess methionine in the rat.

An experiment was performed to investigate the progressive changes of the anemia caused by excess methionine in rats, during the developmental and convalescent periods. Hemoglobin concentration, hematocrit value and red blood cell count were elevated in the initial stage of excess methionine feeding, they then gradually decreased. During the convalescent period, after ceasing administration of the excess methionine diet, symptoms of anemia gradually disappeared. The nitrogen efficiency ratio was markedly decreased by excess methionine feeding, however, it rapidly recovered after ceasing administration of the excess methionine diet. Iron deposited in the spleen also disappeared quickly during the convalescent period. Of the symptoms of anemia caused by excess methionine, the activity of delta-aminolevulinic acid synthetase (ALAS) in the bone marrow showed the most acute changes. As mentioned previously (1), although lowered globin biosynthesis due to amino acid imbalance could be considered as one of the important causes of the anemia and also would be one of the causes of the compensative elevation of ALAS activity, the possibility still remain that there are other unknown effects of excess methionine on ALAS activity in the bone marrow in this case.

Influence of cycloheximide on normal and induced δ-aminolevulinie acid synthetase (aminoketone synthesis) of rat liver in vivo and in vitro

2 and 5 mg/kg cycloheximide given i.p. 30 minutes prior to the inducer allylisopropylacetamide (AIA) diminished or prevented the enhancement of delta-aminolevulinic acid synthetase (ALAS) activity in newborn rats. In adult rats the small AIA effect 24 hours after pretreatment was enhanced by 2 mg/kg cycloheximide i.p., when the inhibitor was injected 2 hours prior to the inducer. The basic aminoketone synthesis was inhibited by cycloheximide alone. 150-600 microgram/ml cycloheximide had no effect on the aminoketone synthesis of rat liver slices in vitro during 4 hours of incubation. The in vitro induction by 0.6 mg/ml AIA was enhanced by cycloheximide, if the inducer was added 1 hour after preincubation with cycloheximide. It is supposed, that the cytoplasmically localized precursor-ALAS is activated at the beginning of the induction process prior to the initiation of de novo enzyme synthesis, at least in vitro.

Increased delta-aminolevulinic acid synthetase activity in rat ventricle after acute exercise.

Increased delta-aminolevulinic acid synthetase activity in rat ventricle after acute exercise.

Study of factors causing excess protoporphyrin accumulation in cultured skin fibroblasts from patients with protoporphyria.

The activity of heme synthetase, which catalyzes the chelation of ferrous iron to protoporphyrin to form heme, is deficient in sonicates of skin fibroblasts cultured from patients with protoporphyria. During culture in Eagle's medium supplemented with fetal calf serum, these cells do not accumulate protoporphyrin, however. This may be due to a minimal requirement for heme synthesis, since glycine is incorporated into heme at a low rate which is similar to that in normal fibroblasts. In addition, the activity of delta-aminolevulinic acid (ALA) synthetase, the first and rate-limiting enzyme of heme biosynthesis which catalyzes the formation of ALA from glycine, is normal in lysates of the fibroblasts. Cultured fibroblasts were therefore incubated with ALA in order to bypass the rate-limiting step of heme biosynthesis. In the presence of 25 muM iron, protoporphyrin was detected in protoporphyria cell lines when the concentration of ALA in the medium reached 50 muM, but not in normal lines. As the concentration of ALA was increased above 50 muM, all lines accumulated protoporphyrin. However, the amount was 2-3 times more in cultured fibroblasts from patients with protoporphyria, reflecting their deficiency of heme synthetase activity. When iron was not added to the medium, protoporphyrin accumulated to a similar degree in normal and protoporphyria fibroblasts; this was significantly more than that in the presence of iron. These studies indicate that excessive protoporphyrin accumulation in protoporphyria, which is due principally to deficient heme synthetase activity, may be modified by the rate of ALA formation in heme-producing tissues, and by the availability of iron.

Bone marrow delta-aminolevulinic acid synthetase activity in hematological disorders.

An improved radiochemical method to measure the o-aminoJevulinic acid (ALA) synthetase activity in human bone marrow erythroblasts by incubating bone marrow cell homogenate with 14C-succinate and succinyl CoA generating system has been reported. Studies were made to ob­ tain the optimal conditions for the enzyme assay and to confirm the reliability of the assay pro­ cedures. ALA-synthetase in erythroblasts was found to be decreased in all 3 cases of iron deficiency anemia and 3 cases of l3-thalassemia minor. In 5 cases of idiopathic sideroblastic anemia, the enzyme activity was markedly decreased. A case of sideroblastic anemia, who restored normal erythropoiesis following the administration of pyridoxal phosphate (PLP) showed low PLP level in serum & blood as well as in vivo evidence of PLP deficiency and an in vitro stimulation of ALA­ synthetase activity by PLP. Erythroblasts of 4 patients with erythroleukemia showed diminished bone marrow ALA-synthetase activity.

Delta-Aminolevulinic acid synthetase in the heart.

The regultion of cardiac delta-aminolevulinic acid synthetase activity was studied in rat heart homogenates. Optimal conditions were determined for the measurement of delta-aminolevulinic acid and an appropriate assay was established for the heart. The activity of cardiac delta-aminolevulinic acid synthetase was determined in rats either fed ad libitum or starved for 24 or 48 h. Marked decreases in delta-aminolevulinic acid synthetase activity were observed in homogenates or mitochondrial fractions prepared from hearts of fasted animals and an explanation for previous findings that the enzyme is undetectable in heart tissue is provided. Dexamethasone treatment was effective in reversing the decreases brought about by fasting but had no effect on the delta-aminolevulinic acid synthetase activity in heart homogenates from fed rats. ACTH treatment had no effect in fed or starved rats. Decreases in delta-aminolevulinic acid synthetase activity induced by fasting were not reversed in homogenates or mitochondrial preparations by succinyl-CoA-generating systems or when alpha-ketoglutarate was substituted for succinate in homogenate preparations. Cardiac delta-aminolevulinic acid dehydratase levels are not altered by fasting. Agents such as allylisopropylacetamide or 3,5-dicarbethoxy-1,4-dihydrocollidine, which produce marked increases in hepatic delta-aminolevulinic acid synthetase activity, have no effects on the activity of this enzyme in the heart.

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

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

Heme synthetase deficiency in human protoporphyria. Demonstration of the defect in liver and cultured skin fibroblasts.

The final step in heme biosynthesis is chelation of porphyrin with Fe++ catalyzed by the mitochondrial enzyme heme synthetase. We have employed a sensitive radiochemical assay for this enzyme, using 59Fe and deuteroporphyrin or protoporphyrin as substrates. In this method iron is maintained in the ferrous state, oxygen is excluded from the incubation system, and labeled heme product is extracted into ethyl acetate. This assay has been used to measure the activity of heme synthetase in homogenates of liver, obtained by needle biopsy, and in sonicates of human skin fibroblasts, cultured in vitro. In addition, activity of the first enzyme of the heme synthetic pathway, delta-aminolevulinic acid synthetase, has been measured in fibroblast lysates. Lysates of fibroblasts from eight patients with protoporphyria had activities of delta-aminolevulinic acid synthetase which did not differ significantly from those of eight normal fibroblast lines, whereas activity of heme synthetase, with either deuteroporphyrin or protoporphyrin as substrate, was markedly decreased in sonicates of skin fibroblasts from these patients, the mean being 8% of control with deuteroporphyrin and 14% with protoporphyrin as substrate. In homogenates of liver from five patients with protoporphyria, activity of heme synthetase was also significantly less than that found in six patients without prophyria, the mean being 13% of control with protoporphyrin as substrate. These results provide evidence that decreased activity of heme synthetase is the basic defect in the heme synthetic pathway in protoporphyria. This deficiency is probably responsible for protoporphyrin accumulation and hence the biochemical and clinical features observed in protoporphyria.

Studies of murine erythroid cell development. Synthesis of heme and hemoglobin.

Techniques of cell separation were used to isolate murine erythroid precursors at different states of maturation. Cells were studied before and after short-term incubation in the presence or absence of erythropoietin. Complementary results were obtained by direct examination of the cell fractions and by the short-term culture experiments. Indices of heme synthesis, including incorporation of 59Fe or [2-14C]glycine into heme and activity of delta-aminolevulinic acid synthetase, were already well developed in the least mature cells, chiefly pronormoblasts. Activity then rose moderately in the cell fractions consisting primarily of basophilic and polychromatophilic normoblasts, and fell off with further increases in cell maturity. On short-term culture in the presence of erythropoietin, activity declined with increasing cell maturation except in the least mature fraction where the original level of activity was maintained. By contrast, synthesis of labeled hemoglobin ([3H]leucine) was very low in the least mature cell fractions and rose progressively with increasing cell maturity. The rate of hemoglobin synthesis increase in cells at all stages of maturation when cultured in the presence of erythropoietin. Despite the different patterns observed for heme synthesis and hemoglobin synthesis, both synthetic activities were consistently higher in cells cultured with erythropoietin as compared to controls. These findings suggest that erythropoietin stimulates biochemical differentiation of erythroid precursors at various stages of maturation. They also demonstrate an asynchronism between heme synthesis and hemoglobin syhthesis; heme synthesis is already well developed in the least mature erythroid cells and begins to diminish as the capacity for hemoglobin synthesis continues to rise.

Induction of delta-aminolevulinic acid synthetase in isolated rat liver cells by steroids.

The role of steroids in regulation of delta-aminolevulinic acid synthetase has been studied in isolated rat liver cell suspensions under conditions previously shown to support inducation of the enzyme by drugs. Addition of a variety of C-19 and C-21 steroids to cell suspensions resulted, after 4 to 6 hours of incubation, in 2- to 5-fold increase in the activity of delta-aminolevulinic acid synthetase as measured in liver cell homogenates. The increase was prevented by cycloheximide. The most active steroid inducers tested were pregnene or pregnane derivatives with keto or hydroxyl groups at C-3 and C-20; in particular a beta-hydroxyl group at C-20 enhanced activity. These C-21 steroids at optimal initial concentrations caused 3- to 5-fold induction over 4 hours. A number of C-19 androstene and androstane compounds caused 2- to 3-fold inducation over the same period. Hydrocortisone had no effect. For a variety of androstane and pregnane derivatives, inducation by 5alphaH steroids was as great as or greater than that by 5betaH compounds, in contrast to previous findings in chick embryo liver. Induction of delta-aminolevulinic acid synthetase by steroids in isolated liver cells was shown to be subject to feedback repression by hemin.