Heme synthesis in Trypanosoma cruzi: influence of the strain and culture medium

The activity of the following enzymes involved in the biosynthesis of porphyrins was determined in two strains of Trypanosoma cruzi (Y and CL) grown in two culture media (LIT and Warren): succinyl coenzyme A synthetase (Suc.CoA-S), 5-aminolevulinate synthetase (ALA-S), 4,5-dioxovaleric acid transaminase (DOVA-T), 5-aminolevulinate dehydratase (ALA-D), porphobilinogenase (PBGase), deaminase and heme synthetase (Heme-S). The amount of 5-aminolevulinic acid (ALA) and porphobilinogen, porphyrins and heme was also determined. ALA and PGB were detected in both strains of T. cruzi. However, ALA was not detected in epimastigotes of the Y strain grown in the LIT medium. The content of ALA and PBG varied according to the strain and the growth medium. No free porphyrins and heme were detected in both strains of T. cruzi. The activity of Suc.CoA-S and DOVA-T was markedly influenced by the strains of the parasite and the growth medium. No significant DOVA-T activity was detected in epimastigotes of the CL strain grown in the Warren's medium. No significant activity of ALA-D, PBGase and deaminase was detected in T. cruzi. Activity of Heme-S was detected in both strains of T. cruzi when mesoporphyrin, protoporphyrin or deuteroporphyrin was used as substrate. The enzyme activity was influenced by the strain of the parasite, the growth medium and the substrate used.     

Heme synthesis in Crithidia deanei: influence of the endosymbiote

The activity of the following enzymes involved in the biosynthesis of porphyrins was determined in endosymbiote-free and endosymbiote-containing Crithidia deanei grown in a chemically defined medium: succinyl Coenzyme A synthetase (Suc.CoA-S), 5-aminolevulinate synthetase (ALA-S), 4,5-dioxovaleric acid transaminase (DOVA-T), 5-aminolevulinate dehydratase (ALA-D), porphobilinogenase (PBGase), deaminase and heme synthetase (Heme-S). The amount of 5-aminolevulinic acid (ALA) and porphobilinogen, porphyrins and heme was also determined. ALA and PBG were detected in C. deanei. The levels of free porphyrins was low. Heme concentration was nil. The activity of ALA-D, deaminase and PBGase was not detected in C. deanei. The activity of Suc.CoA-S and ALA-S were twice higher in symbiote-containing than in aposymbiotic C. deanei. Aposymbiotic cells had a higher activity of DOVA-T than symbiote-containing cells. The level of Heme-S, measured using protoporphyrin as substrate, was twice as high in symbiote-containing than in symbiote-free cells.     

The source of heme for vascular heme oxygenase II: de novo heme biosynthesis in rat aorta

Heme is an essential prosthetic group or substrate for many proteins, including hemoglobin, and hemo enzymes such as nitric oxide synthase, soluble guanylyl cyclase, and heme oxygenase (HO). HO is responsible for the breakdown of heme into equimolar amounts of biliverdin, iron, and carbon monoxide, the latter of which is thought to play a role in the regulation of vascular tone. It is not clear whether the source of heme for cardiovascular functions is derived from uptake from the extracellular milieu or synthesis. In this study, we tested the hypothesis that blood vessels obtain their supply of heme for HO through de novo synthesis. Adult male Sprague-Dawley rat aorta was incubated at 37 degrees C in Krebs' solution with 1 micro M [14C]delta-aminolevulinic acid (ALA). [14C]ALA uptake was linear for about 30 min and reached a plateau at approximately 100 min. The radioactivity was incorporated into porphyrins and heme as determined by esterification of 14C-labelled metabolites and thin-layer chromatography. The first and rate-limiting step of heme biosynthesis is catalyzed by ALA synthase (ALA-S), the activity of which was determined in rat aorta using a radiometric assay, approximately 250 nmol x (g wet mass)(-1) x h(-1). Inducing HO-1 in rat aorta with S-nitroso-N-acetylpenicillamine (500 micro M) did not increase ALA-S activity as compared with basal activity levels of the enzyme. It appears that there is a sufficient amount of heme available under basal ALA-S activity conditions to meet the increased demand for heme resulting from HO-1 induction. These observations indicate that the complete enzymatic pathway for de novo heme biosynthesis resides in rat aorta and furthermore indicate that de novo heme synthesis is capable of supplying a substantial portion of the heme substrate for HO in the aorta.     

delta-Aminolevulinic Acid Formation from gamma,delta-Dioxovaleric Acid in Extracts of Euglena gracilis

γ,δ-Dioxovaleric acid (DOVA) has been proposed as a precursor to heme and chlorophyll in plants and algae. DOVA transaminase activity was found in extracts of the unicellular green alga Euglena gracilis Klebs strain Z Pringsheim. Optimum conversion of DOVA to δ-aminolevulinic acid (ALA) occurred at pH 6.8. ALA formation was linear with time for at least 30 minutes at 37° C and was proportional to amount of cell extract in the incubation mixture. Boiled cell extract was inactive. DOVA transaminase from either wild-type or aplastidic derivative strain W₁₄ZNaIL ran as a single band in agarose gel permeation chromatography, with a calculated molecular weight of 98,000 ± 3,000. l-Glutamic acid was the most effective amino donor. d-Glutamic acid was inactive. K_m values for l-glutamic acid and DOVA were 11 and 1.1 millimolar, respectively. Pyridoxal phosphate stimulated activity maximally at 30 micromolar, and (aminooxy)acetate was strongly inhibitory. Glyoxylic acid was a competitive inhibitor with respect to DOVA, with an inhibition constant of 0.62 millimolar. Wild-type and aplastidic cells vielded equal activity, 31 ± 1 nanomoles ALA per 30 minutes per 10⁷ cells, whether grown in light or dark. DOVA transaminase could not be separated from glyoxylate transaminase activity by agarose gel permeation or diethylaminoethyl-cellulose column chromatography. In all fractions, glyoxylate transaminase activity was at least 75 times greater than DOVA transaminase activity. DOVA transamination appears to be catalyzed by glyoxylate transaminase, and not to be of physiological significance with respect to chlorophyll synthesis in Euglena.     

Production of delta-aminolevulinate: subcellular localization and purification of murine hepatic L-alanine: 4,5-dioxovaleric acid aminotransferase

1. L-Alanine: 4,5-dioxovaleric acid aminotransferase (DOVA transaminase) activity was measured in murine liver, kidney and spleen homogenates. 2. Among the organs examined, the specific activity of the enzyme was highest in kidney, followed by liver then spleen. 3. No differences in DOVA transaminase activity in kidney, liver and spleen homogenates were detected between mouse strains C57BL/6J and DBA/2J. 4. Based on enzyme activity, the capacity of DOVA transaminase to catalyze the formation of delta-aminolevulinic acid (ALA) in liver appeared much greater than the capacity of ALA synthase. 5. In DBA/2J animals, DOVA transaminase activity in liver mitochondrial fractions prepared by differential centrifugation was 24 nmol ALA formed/hr/mg protein compared with 0.63 nmol ALA formed/hr/mg protein for ALA synthase. 6. Cell fractionation analyses indicated that liver DOVA transaminase is located in the mitochondrial matrix. 7. The liver enzyme was purified from mitoplasts by chromatography on DEAE-Sephacel followed by affinity chromatography on L-alanine-AH-Sepharose. 8. The specific activity of the purified DOVA transaminase was 1600 nmol ALA formed/hr/mg protein. 9. The yield of the purification was ca 90 micrograms of protein per gram liver wet weight. 10. The purified enzyme had a subunit mol. wt of 146,000 +/- 5000 as determined by electrophoresis under denaturing conditions.     

Characterization of chicken liver L-alanine:4,5-dioxovaleric acid aminotransferase

1. A procedure is described for purifying the enzyme L-alanine:4,5-dioxovaleric acid aminotransferase (DOVA transaminase) from chicken liver. The enzyme catalyzes a transamination reaction between L-alanine and 4,5-dioxovaleric acid (DOVA), yielding delta-aminolevulinic acid (ALA). 2. In cell fractionation studies, DOVA transaminase activities were detected in mitochondria and in the post-mitochondrial supernatant fraction from liver homogenates. 3. For the mitochondrial enzyme, any of most L-amino acids could serve as a source for the amino group transferred to DOVA, but L-alanine appeared the preferred substrate. At pH 7.0, the enzyme had an apparent Km of 60 microM for DOVA and of 400 microM for L-alanine. 4. The enzyme was purified from disrupted mitoplasts in three steps: chromatography on DEAE-Sephacel, gel filtration through Sephadex G-150, and chromatography on hydroxyapatite. The yield was approx. 100 micrograms of enzyme protein per 10 g wet wt of liver. 5. The purified enzyme had a subunit mol. wt of 63,000 as determined by gel electrophoresis under denaturing conditions. 6. The activity of DOVA transaminase was also measured in embryonic chicken liver, and based on activity, the enzyme's capacity to produce ALA was significantly greater than that of ALA synthase. Unlike ALA synthase, however, DOVA transaminase activity did not increase in liver mitochondria of chicken embryos exposed for 18 hr to two potent porphyrogenic agents.     

Alternative Routes for the Synthesis of 5-Aminolevulinic Acid in Maize Leaves : I. Formation from 2-Ketoglutarate via 4,5-Dioxovaleric Acid

Cell-free extracts from greening maize (Zea mays L.) leaves catalyze the conversion of [¹⁴C]2-ketoglutarate (KG) to [¹⁴C]5-aminolevulinic acid (ALA) in a reaction which requires NADH and an amino donor and shows maximal activity around pH 6.5. The enzymic system is located in the cytosol. This cell fraction contains a low level of `KG dehydrogenase' activity and a transaminase which catalyzes the conversion of 4,5-dioxovaleric acid (DOVA) to ALA. The transaminase can use glutamate, aspartate, or alanine as amino donor. It is effectively inhibited by aminooxyacetate and ethylenediamine tetraacetate and shows maximal activity at pH 6.7. The activity of DOVA transaminase is only slightly affected by preillumination of leaves and can also be detected in green leaves and in roots.

DOVA was isolated from leaves and roots and determined as its benzoquinoxaline derivative. Significant amounts were found only in tissues in which ALA had accumulated or after it was exogenously supplied. DOVA was labeled in vivo by both [¹⁴C]ALA and [¹⁴C]KG. Small amounts were also formed from ALA in a cell-free system.

It is suggested that DOVA may be an intermediate in the diversion of ALA to respiratory metabolism and that it is not involved in the biosynthesis of this porphyrin precursor.

     

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-induced ROS in Trypanosoma cruzi activates CaMKII-like that triggers epimastigote proliferation. One helpful effect of ROS

Heme is a ubiquitous molecule that has a number of physiological roles. The toxic effects of this molecule have been demonstrated in various models, based on both its pro-oxidant nature and through a detergent mechanism. It is estimated that about 10 mM of heme is released during blood digestion in the blood-sucking bug's midgut. The parasite Trypanosoma cruzi, the agent of Chagas' disease, proliferates in the midgut of the insect vector; however, heme metabolism in trypanosomatids remains to be elucidated. Here we provide a mechanistic explanation for the proliferative effects of heme on trypanosomatids. Heme, but not other porphyrins, induced T. cruzi proliferation, and this phenomenon was accompanied by a marked increase in reactive oxygen species (ROS) formation in epimastigotes when monitored by ROS-sensitive fluorescent probes. Heme-induced ROS production was time- and concentration-dependent. In addition, lipid peroxidation and the formation of 4-hydroxy-2-nonenal (4-HNE) adducts with parasite proteins were increased in epimastigotes in the presence of heme. Conversely, the antioxidants urate and GSH reversed the heme-induced ROS. Urate also decreased parasite proliferation. Among several protein kinase inhibitors tested only specific inhibitors of CaMKII, KN93 and Myr-AIP, were able to abolish heme-induced ROS formation in epimastigotes leading to parasite growth impairment. Taken together, these data provide new insight into T. cruzi- insect vector interactions: heme, a molecule from the blood digestion, triggers epimastigote proliferation through a redox-sensitive signalling mechanism.     

Yeast 5-aminolevulinate synthase provides additional chlorophyll precursor in transgenic tobacco

Synthesis of the tetrapyrrole precursor 5-aminolevulinate (ALA) in plants starts with glutamate and is a tRNA-dependent pathway consisting of three enzymatic steps localized in plastids. In animals and yeast, ALA is formed in a single step from succinyl CoA and glycine by aminolevulinate synthase (ALA-S) in mitochondria. A gene encoding a fusion protein of yeast ALA-S with an amino-terminal transit sequence for the small subunit of ribulose bisphosphate carboxylase was introduced into the genome of wild-type tobacco and a chlorophyll-deficient transgenic line expressing glutamate 1-semi-aldehyde aminotransferase (GSA-AT) antisense RNA. Expression of ALA-S in the GSA-AT antisense transgenic line provided green-pigmented co-transformants similar to wild-type in chlorophyll content, while transformants derived from wild-type plants did not show phenotypical changes. The capacity to synthesize ALA and chlorophyll was increased in transformed plants, indicating a contribution of ALA-S to the ALA supply for chlorophyll synthesis. ALA-S activity was detected in plastids of the transformants. Preliminary evidence is presented that succinyl CoA, the substrate for ALA-S, can be synthesized and metabolized in plastids. The transgenic plants formed chlorophyll in the presence of gabaculine, an inhibitor of GSA-AT. Steady-state RNA and protein levels and, consequently, the enzyme activity of GSA-AT were reduced in plants expressing ALA-S. In analogy to the light-dependent ALA synthesis attributed to feedback regulation, a mechanism at the level of intermediates or tetrapyrrole end-products is proposed, which co-ordinates the need for heme and chlorophyll precursors and restricts synthesis of ALA by regulating GSA-AT gene expression. The genetically engineered tobacco plants containing the yeast ALA-S activity demonstrate functional complementation of the catalytic activity of the plant ALA-synthesizing pathway and open strategies for producing tolerance against inhibitors of the C5 pathway.     

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.     

Regulation of heme pathway in regenerating mouse liver.

1. delta-Aminolevulinic acid synthetase (ALA-S), rhodanese and microsomal heme oxygenase (MHO), were quantitated in Cl4C induced regenerating mouse liver. 2. Maximal hepatomegalia was observed at 48 hr after i.p. injection of a single dose of the toxin. 3. ALA-S activity decreased on day 2, and then significantly increased (50%) between days 3 and 7, returning afterwards to control values. 4. Cytoplasmic rhodanese, as well as MHO activities, exhibited a clear correlation as compared with the ALA-S activity profile. 5. Porphyrin biosynthesis from precursor delta-aminolevulinic acid (ALA) was significantly increased even after 15 days of intoxication. 6. Present results would indicate that Cl4C is acting in a dual fashion.     

Inhibition of δ-aminolevulinic acid synthetase and δ-aminolevulinic acid dehydrase by L-2-amino-4-methoxy-trans-3-butenoic acid in the rat

The rate limiting enzyme of heme biosynthesis, δ-aminolevulinic acid synthetase (ALA synthetase), and the second enzyme in the heme biosynthetic pathway, δ-aminolevulinic acid dehydrase (ALA dehydrase), were inhibited by the olefinic amino acid L-2-amino-4-methoxy - $\text{trans}$-3-butenoic acid (AMTB). Administration of AMTB (20 mg/kg; i.p.) to rats inhibited ALA synthetase and ALA dehydrase in control animals and in animals with markedly elevated activity of ALA synthetase which resulted from the administration of 3,5-dicarbethoxy-1,4-dimethyl-collidine (DDC, 200 mg/kg, i.p.) or allylisopropylacetamide (200 mg/kg, s.c.). AMTB also blocked the synthesis of rat hepatic porphyrins and inhibited the increase in the urinary excretion of δ-aminolevulinic acid and porphobilinogen following DDC (150 mg/kg, p.o.) administration. Preincubation of AMTB with liver mitochondria or a soluble fraction of liver decreased the activity of mitochondrial ALA synthetase and soluble ALA dehydrase, respectively.     

The Trypanosoma cruzi proteins TcCox10 and TcCox15 catalyze the formation of heme A in the yeast Saccharomyces cerevisiae

Trypanosoma cruzi, the etiologic agent for Chagas’ disease, has requirements for several cofactors, one of which is heme. Because this organism is unable to synthesize heme, which serves as a prosthetic group for several heme proteins (including the respiratory chain complexes), it therefore must be acquired from the environment. Considering this deficiency, it is an open question as to how heme A, the essential cofactor for eukaryotic CcO enzymes, is acquired by this parasite. In the present work, we provide evidence for the presence and functionality of genes coding for heme O and heme A synthases, which catalyze the synthesis of heme O and its conversion into heme A, respectively. The functions of these T. cruzi proteins were evaluated using yeast complementation assays, and the mRNA levels of their respective genes were analyzed at the different T. cruzi life stages. It was observed that the amount of mRNA coding for these proteins changes during the parasite life cycle, suggesting that this variation could reflect different respiratory requirements in the different parasite life stages.     

Changes in the activities of the protoheme-synthesizing system during the growth of yeast under different conditions.

The levels of some enzymatic activities involved in protoheme synthesis have been measured in subcellular fractions obtained at different stages of the growth of the yeast Saccharomyces cerevisiae grown anaerobically and aerobically with glucose (50 or 6 g/ liter), and ethanol (20 g/liter) as the carbon source. The degree of repression of the respiratory system is estimated by the respiratory capacity of whole cells, by the activities of succinate-cytochrome c reductase and cytochrome c oxidase of the mitochondrial particles, and by the cytochrome spectra. The results show that (i) the more porphyrins (cytochromes) that are synthesized by the cells, the lower is the specific activity of δ-aminolevulinic acid (ALA) synthetase and the higher is the specific activity of ALA dehydratase, the activity ratio ALA synthetase/ALA dehydratase decreasing at least 10-fold compared to the repressed cells; (ii) the amount of intracellular ALA found under all conditions tested (from 0.05 to 1.5 mm in the cell sap) correlates well with the measured ALA synthetase activity; its presence argues against a rate-limiting function for ALA synthetase and rather favors such a role for the ALA dehydratase in the formation of heme in yeast; (iii) the rate of porphyrin synthesis measured in vitro is higher in the case of cells with high cytochrome contents; and (iv) the specific activities of succinyl CoA synthetase and protoheme ferrolyase are always present in nonlimiting amounts. Some experiments are described showing that the values of the activities which are calculated from these in situ and in vivo experiments compare well with the values measured in vitro in the acellular extracts. The results concerning the enzymatic activities, together with (i) the excretion of coproporphyrin(ogen) and the accumulation of protoporphyrin + Zn-protoporphyrin in anaerobiosis, (ii) the presence of protoporpho(di)methene (P503) in anaerobic and repressed cells, and (iii) the presence of intracellular ALA under all growth conditions, are discussed in terms of possible control(s) of heme synthesis in yeast.     

Production of delta-aminolevulinic acid: characterization of murine liver 4,5-dioxovaleric acid: L-alanine aminotransferase.

1. We report on the kinetic properties of murine liver 4,5-dioxovaleric acid:L-alanine aminotransferase (DOVA transaminase). 2. The transamination of 4,5-dioxovaleric acid (DOVA) led to the production of delta-aminolevulinic acid. 3. L-Alanine was the preferred amino group donor among the common 20 amino acids. 4. The optimum pH of the reaction was 7-8. 5. A Km of 220 microM for DOVA and a Km of 970 microM for L-alanine were obtained. 6. The reaction was inhibited by each of the following: glyoxylate, beta-chloroalanine, methylglyoxal, delta-aminolevulinate, pyruvate, heme, and gabaculine. 7. None of several xenobiotic inducers of microsomal mixed function oxidases tested had a significant effect on DOVA transaminase activity in studies performed with murine primary hepatocyte cultures.     

Effect of acetaminophen on heme metabolism in rat liver

BACKGROUND AND AIMS: Acetaminophen (APAP) or paracetamol is a hepatotoxic drug through mechanisms involving oxidative stress. To know whether mammalian cells possess inducible pathways for antioxidant defense, we have to study the relationship between heme metabolism and oxidative stress. METHODS: fasted female Wistar rats received a single injection of APAP (3.3 mmol kg(-1) body weight) and then were killed at different times. Heme oxygenase-1 (HO), delta-aminolevulinic acid (ALA) synthase, ALA dehydratase, and porphobilinogenase activities, lipid peroxidation, GSH, catalase and glutathione peroxidase, were measured in liver homogenates. The antioxidant properties of bilirubin and S-adenosyl-L-methionine were also evaluated. RESULTS: APAP increased lipid peroxidation (115% +/- 6; S.E.M., n=12 over control values) 1 h after treatment. GSH reached a minimum at 3 h (38% +/- 5) increasing thereafter. At the same time antioxidant enzymes reached minimum values (catalase, 5. 6 +/- 0.4 pmol mg(-1) protein, glutathione peroxidase, 0.101 +/- 0.006 U mg(-1) protein). HO induction was observed 6 h after treatment reaching a maximum value of 2.56 +/- 0.12 U mg(-1) protein 15 after injection. ALA synthase (ALA-S) induction occurred after enhancement of HO, reaching a maximum at 18 h (three-fold the control). ALA dehydratase activity was first inhibited (31 +/- 3%) showing a profile similar to that of GSH, while porphobilinogenase activity was not modified along the whole period of the assay. Administration of bilirubin (5 micromol kg(-1) body weight) or S-adenosyl L-methionine (46 micromol kg(-1) body weight) 2 h before APAP treatment entirely prevented the increase in malondialdehyde (MDA) content, the decrease in GSH levels as well as HO and ALA-S induction. CONCLUSION: This study shows that oxidative stress produced by APAP leads to increase in ALA-S and HO activities, indicating that toxic doses of APAP affect both heme biosynthesis and degradation.     

Bilirubin is highly effective in preventing in vivo delta-aminolevulinic acid-induced oxidative cell damage

Delta-aminolevulinic acid (ALA), precursor of heme, accumulates in a number of organs, particularly in liver of patients with acute porphyrias or lead intoxication. This study characterizes the involvement of bilirubin as an antioxidant in a chronic intoxication with ALA. Female Wistar rats were injected intraperitoneally a daily dose of 40 mg ALA/body wt., during 10 days. A marked increase in lipid peroxidation and a decrease in GSH content were observed 24 h after the last injection of ALA. The activities of liver antioxidant enzymes, superoxide dismutase (SOD), catalase (CAT) and glutathione peroxidase were also diminished. ALA synthase (ALA-S) and heme oxygenase-1 were induced. Both ALA dehydratase (ALA-D) and porphobilinogenase (PBG-ase) activities were inhibited. Administration of bilirubin (5 mmol/kg body wt.) 2 h before ALA treatment entirely prevented the effects of ALA. Co-administration of ALA and Sn-protoporphyrin IX (Sn-PPIX; 100 microg/body wt., i.p.), a potent inhibitor of heme oxygenase, completely abolished its induction and provoked a marked decrease in liver GSH levels as well as an increase in lipid peroxidation. These results add further support to the proposal assigning bilirubin a key protective role against oxidative damage here induced by ALA.     

Distribution of delta-aminolevulinic acid synthetase and delta-aminolevulinic acid dehydratase in liver and kidney of rainbow trout (Salmo gairdnerii)

1. Activities of delta-aminolevulinic acid synthetase (ALA-S) and delta-aminolevulinic acid dehydratase (ALA-D) in trout liver and kidney were compared with those in the mouse. 2. ALA-S activity (per unit tissue fresh weight) exceeded ALA-D activity in trout liver and kidney. 3. In trout kidney, ALA-S activity slightly exceeded, and ALA-D activity far exceeded, their activities in trout liver. 4. In trout, heme synthesis differs from that in mammals in that appreciable synthesis occurs in the kidney, and in that ALA-S activity is not rate limiting.     

The effect of aluminum chloride on some steps of heme biosynthesis in rats after oral exposure

Certain disturbances in heme biosynthesis induced by aluminum chloride were examined. The experiment was performed on female rats that received AlCl₃ orally at the dose 100 mg Al/kg daily for 21 d. The effects of aluminum on the activity of delta-aminolevulinic acid synthetase (ALA-S), dehydratase (ALA-D), and heme oxygenase (O.H.) were observed on 3, 7, 14, and 21 d in liver and kidneys of rats. Also the activity of ALA-D in blood and the concentration of delta-aminolevulinic acid (ALA-U) in urine were observed. Orally administered aluminum caused increase in the activity of ALA-D in the liver and blood, and parallel decrease of ALA-U in urine (r=−0.85) of rats. Aluminum chloride also induced an increase of ALA-S and O.H. in the liver but not in the kidneys. The changes of the enzymes activity participating in heme biosynthesis after administration of aluminum may be correlated with anemia and iron metabolism in rats.