Delta-aminolevulinic Acid Dehydrase Research Articles

Hemorheology in occupational lead exposure

The purpose of this investigation was to study hemorheological parameters in occupationally lead exposed men. For 15 clinically healthy lead-exposed male subjects [age 34.6 (SD 8) years] the viscous (epsilon') and elastic (epsilon") components of whole blood viscosity corrected for 45% hematocrit were measured for shear rates between 1.s-1-100.s-1 at 37 degrees C. Moreover, lead concentrations in blood and urine and the delta-aminolevulinic acid dehydrase level were determined. Fifteen unexposed age-matched men were used as referents. The mean lead concentration in the blood and urine of the lead-exposed men was 48.7 (SD 16.2) micrograms. dl-1 and 38.8 (SD 17.1) micrograms.g creatinine-1, respectively. While epsilon" was significantly increased only at low shear rates, epsilon' was elevated throughout the investigated shear range in comparison with the values of the unexposed referents. These results are characteristic of erythrocytes with enhanced rigidity. The aggregation and filtration index of erythrocytes were significantly elevated for the lead-exposed workers. A positive correlation existed between lead in blood and epsilon' or epsilon", whereas delta -aminolevulinic acid dehydrase and epsilon' or epsilon" were inversely correlated. No differences in plasma viscosity or in lipid status could be detected. Increased blood lead concentrations can be considered an additional risk factor for vascular diseases predisposing towards microvascular occlusion.

Treatment of the porphyrias.

There are seven porphyrias which are caused by defective functions of the enzymes in the haem biosynthesis. Pathogenic mechanisms and symptoms differ greatly in individual porphyrias and, consequently, most of them require a specific therapy. Clinically, the three most important entities are acute porphyric attack, porphyria cutanea tarda and protoporphyria. For an acute porphyric attack the treatment of choice is administration of haem; the other measures are elimination of precipitating factors and symptomatic therapy for many associated symptoms. Porphyria cutanea tarda is controlled by removal of iron by phlebotomies or with low-dose chloroquine. Skin symptoms in protoporphyria can be alleviated with betacaroten but there is no effective procedure to normalize disturbed porphyrin metabolism; hepatic failure seen in some patients may need a liver transplantation. The only effective treatment in congenital erythropoietic porphyria is probably a bone marrow transplantation. No satisfactory treatment is available for very rare delta-aminolevulinic acid dehydrase deficiency porphyria.

Iron distribution in Belgrade rat reticulocytes after inhibition of heme synthesis with succinylacetone

We have used succinylacetone (4,6-dioxoheptanoic acid), a specific inhibitor of delta-aminolevulinic acid dehydrase, to gain insight into the defect in iron metabolism in the Belgrade anemia. The Belgrade rat has an inherited microcytic, hypochromic anemia associated with poor iron uptake into developing erythroid cells. Succinylacetone inhibits heme synthesis, leading to nonheme iron accumulation in mitochondria and cytosol of normal reticulocytes. When succinylacetone is used to inhibit Belgrade heme synthesis, iron from diferric transferrin does not accumulate in the stromal fraction that contains mitochondria, nor does 59Fe accumulate in the nonheme cytosolic fraction. Hence, the defect in the Belgrade rat reticulocyte occurs in the endocytic vesicle or in a step subsequent to iron transit from the vesicle but before the nonheme cytosolic or mitochondrial iron fractions. Therefore, the mutation affects either the release of iron from transferrin or iron transport from the vesicle to the mitochondrion.

The heme biosynthetic pathway in lymphocytes of patients with malignant lymphoproliferative disorders

The metabolism of heme is impaired in lymphocytes of patients with malignant lymphoproliferative disorders (MLPO). Two of the enzymes of the heme biosynthetic pathway, delta-aminolevulinic acid dehydrase (ALAD) (EC 4.2.1.24) and ferrochelatase (FC) (EC 4.99.1.1) are markedly reduced. The activity of porphobilinogen deaminase (PBGD) (EC 4.3.1.8) is increased. The rate-limiting enzyme of heme biosynthesis in the liver, aminolevulinate synthase (ALAS) (EC 2.3.1.37) remains unchanged although the concentration of total heme in the lymphocytes is markedly reduced. This might reflect a lack of negative feedback inhibition by heme on ALAS activity in this system.

Delta-Aminolevulinic acid dehydrase (porphobilinogen synthase) in two families with inherited enzyme deficiency.

The inheritance of a deficient delta-aminolevulinic acid dehydrase (ALA-D; synonym: porphobilinogen synthase; EC 4.2.1.24) was studied in blood samples of two families over three generations. The propositus in each family was a young male acute hepatic porphyria patient with an almost complete ALA-D deficiency in the homozygous state (ALA-D activity less than 2% of controls). Heterozygotes are clinically non-affected (mean ALA-D 36% of controls). The mode of transmission could be traced by enzyme activity and electrophoretic polymorphism studies. Heterozygotes are detected by the demonstration of enzyme activity in the gel. The notation D was used for the gene expressing the defective enzyme. The "phenotype" D-1 was observed in six, the "phenotype" D-2 in three of all heterozygotes studied. These results are compatible with a single normal allele in heterozygotes responsible for enzyme activity. Quantitative assays and the segregation pattern in both families suggest a 3-allele-system for the inheritance of ALA-D deficiency.

Augmentation of Hematoporphyrin Uptake and In Vitro-Growth Inhibition of L1210 Leukemia Cells by Succinylacetone

Succinylacetone (SA; 4,6-dioxoheptanoic acid), a specific inhibitor of delta-aminolevulinic acid dehydrase (ALAD) (the second enzyme of the heme biosynthetic pathway), was tested for its effect in L1210 cells from inbred DBA/2 mice. ALAD from broken L1210 cells was completely inhibited by 1 microM SA, but in whole cells activity was decreased only 83% after incubation of the cells with 2.5 mM SA for 3 days. When incubated with hematoporphyrin (HP), L1210 cells rapidly took up porphyrin from the medium, and this uptake could be augmented by pretreatment of the cells with SA; but this enhancement of porphyrin uptake occurred gradually over a period of days. When SA-treated and untreated L1210 cells were incubated with increasing concentrations of HP in the medium, SA-treated cells reached the saturation concentration of cellular porphyrin at lower medium HP concentrations than did untreated cells. Growth of L1210 cells could be inhibited by 2 mM SA or more. Addition of increasing amounts of serum to cultures of cells containing SA did not reverse the growth inhibition due to SA. Porphyrin uptake from HP in the medium in nonmalignant fibroblast line 3T3 was much lower than in L1210 cells and could not be enhanced by incubation of the cells with SA.

Augmentation of Hematoporphyrin Uptake and in Vitro-Growth Inhibition of L1210 Leukemia Cells by Succinylacetone

Succinylacetone (SA; 4,6-dioxoheptanoic acid), a specific inhibitor of delta-aminolevulinic acid dehydrase (ALAD) (the second enzyme of the heme biosynthetic pathway), was tested for its effect in L1210 cells from inbred DBA/2 mice. ALAD from broken L1210 cells was completely inhibited by 1 microM SA, but in whole cells activity was decreased only 83% after incubation of the cells with 2.5 mM SA for 3 days. When incubated with hematoporphyrin (HP), L1210 cells rapidly took up porphyrin from the medium, and this uptake could be augmented by pretreatment of the cells with SA; but this enhancement of porphyrin uptake occurred gradually over a period of days. When SA-treated and untreated L1210 cells were incubated with increasing concentrations of HP in the medium, SA-treated cells reached the saturation concentration of cellular porphyrin at lower medium HP concentrations than did untreated cells. Growth of L1210 cells could be inhibited by 2 mM SA or more. Addition of increasing amounts of serum to cultures of cells containing SA did not reverse the growth inhibition due to SA. Porphyrin uptake from HP in the medium in nonmalignant fibroblast line 3T3 was much lower than in L1210 cells and could not be enhanced by incubation of the cells with SA.

Gas sniffing as a form of substance abuse.

The authors review the existing literature on inhalation of gasoline fumes, highlighting the acute and chronic physical and psychological effects. The clinical picture of gas sniffing includes visual hallucinations, changes in consciousness, euphoria, nystagmus, dizziness, weakness and tremors. There is the possibility of rapid recovery, sudden death or brain damage with chronic abuse. When leaded gasoline is abused then blood and urine lead levels and erythrocytic delta-aminolevulinic acid dehydrase (ALAD) levels may be helpful. Although the treatment of acute and chronic gas sniffing syndromes is often supportive and non-specific, when lead levels are high chelated therapy is indicated including British anti-lewisite, calcium disodium versenate or D-penicillamine. We also report our findings on an isolated native Indian population where intentional gas sniffing has reached epidemic proportions. Ten percent of the total population and 25% of the children between 5 and 15 years of age had been identified as gasoline inhalation abusers. In this population, the most important etiological factors included environmental, family and cultural components. The authors emphasize the need to correct the family and social deficiencies in such communities if the incidence of gas sniffing is to be decreased.

Effects of iron deficiency on heme biosynthesis in Rhizobium japonicum.

The effects of iron deficiency on heme biosynthesis in Rhizobium japonicum were examined. Iron-deficient cells had a decreased maximum cell yield and a decreased cytochrome content and excreted protoporphyrin into the growth medium. The activities of the first two enzymes of heme biosynthesis, delta-aminolevulinic acid synthase (EC 2.3.1.37) and delta-aminolevulinic acid dehydrase (EC 4.2.1.24), were diminished in iron-deficient cells, but were returned to normal levels upon addition of iron to the cultures. The addition of iron salts, iron chelators, hemin, or protoporphyrin to cell-free extracts did not affect the activity of these enzymes. The addition of levulinic acid to iron-deficient cultures blocked protoporphyrin excretion and also resulted in high delta-aminolevulinic acid synthase and delta-aminolevulinic acid dehydrase activities. These results suggest the possibility that rhizobial heme biosynthesis in the legume root nodule may be affected by the release of iron from the host plant to the bacteroids.

Inhibition of delta-aminolevulinic acid dehydrase by 4,6-dioxoheptanoic acid.

Inhibition of delta-aminolevulinic acid dehydrase by 4,6-dioxoheptanoic acid.

Degradation and excretion after tetraethyllead injection and erythrocyte delta-aminolevulinic acid dehydrase activity inhibition in rabbit (author's transl)

Rabbits were injected tetraethyllead (Et4Pb) i.v. only once; total lead and triethyllead (Et3Pb) levels in organs were determined 24 hours, and 30, 60, and 90 days later; and the lead clearance rates in the organs were compared with each other. Total lead and Et3Pb outputs in the feces and urine of these rabbits were sequentially determined after the injection. Other rabbits were injected Et3Pb i.v. only once, and Et3Pb and total lead clearances from organs were determined 24 hours and 20 days after the injection. In order to compare the clearances of Et4Pb and inorganic lead from organs, rabbits were injected lead acetate i.v. only once; lead acetate levels in various organs were determined 24 hours and 30 days after the injection; and the in vivo distribution patterns of this compound were compared with the distribution patterns of lead in the aforemetioned animals injected Et4Pb. Furthermore, lead outputs in the feces and urine of these animals were sequentially determined after the injection, to observe the differences in the output patterns between these animals and the aforementioned rabbits injected Et4Pb. 1) The rabbits 24 hours after the i.v. injection of 12 mg/kg of Et4Pb (7.7 mg/kg as lead) gave such a pattern of total lead distribution into various organs that total lead was distributed into the liver>kidney»brain>skeletal muscle>cardiac muscle>spinal cord in the decreasing sequence of level. The total lead levels in the organs showed such a pattern that the level in the liver was 43 times higher, the level in the kidney was 19 times higher, the level in the brain was 4.5 times higher, the level in the skeletal muscle was 4 times higher, the level in the cardiac muscle was 4 times higher, and the level in the spinal cord was 3 times higher than the blood total lead level. The Et3Pb levels in various organs were of such a pattern that the compound was distributed into the liver>kidney»brain>skeletal muscle>cardiac muscle>spinal cord in the decreasing sequence of level; and the Et3Pb levels in the organs showed that the level in the liver was 100 times as high, the level in the kidney was 40 times as high, the level in the brain was 10 times as high, the level in the skeletal muscle was 9 times as high, the level in the cardiac muscle was 8 times as high, and the level in the spinal cord was 5.5 times as high as that in the blood. The Et3Pb/ total lead level ratio was 86% in the liver, 77% in the kidney, 85% in the brain, 79% in the skeletal muscle, 78% in the cardiac muscle, and 37% in the blood. The total lead levels in various organs of rabbits 30 days after the i.v. injection of Et4Pb showed such a pattern that the level in the liver was 1/20; the level in the kidney, 1/50; the level in the brain, 1/40, the level in the skeletal muscle, 1/40; the level in the cardiac muscle, 1/130, and the level in the blood, 1/8, compared with the levels 24 hours after the injection. The Et3Pb/total lead level ratio 30 days after the injection of Et4Pb was 96% in the liver, 6l% in the kidney, 38% in the brain, and 2% in the blood. Sixty days after the injection of Et4Pb, Et3Pb was demonstrated in the liver, kidney and blood, but 90 days after the injection, it was demonstrated in minute amounts only in the liver and kidney. 2) In the rabbits 24 hours after the i.v. jnjection of 6 mg/kg of Et3Pb, total lead was distributed into the liver>kidney»brain>skeletal muscle>spinal cord>cardiac muscle>blood in the decreasing sequence of level. This group showed such Et3Pb/total lead ratios as 100% in the brain, spinal cord and skeletal muscle, 80-90% in the liver and kidney, and 28% in the blood, Twenty days after the injection of Et3Pb, the total lead levels in the organs were decreased in a similar pattern to those in the aforementioned rabbits injected Et4Pb, with the Et3Pb/total lead level ratio being 7l% in the brain, 89% in the liver and 2% in the blood. 3) The rabbits

Influence of middle molecules on the anemia of uremic patients.

To evaluate their toxicity at the cellular level, middle molecules from uremic serum were incubated with erythrocytes from healthy subjects and the activity of the enzyme Delta-aminolevulinic acid dehydrase (D-ALA-D) and peroxidative hemolysis were investigated. Uremic middle molecules caused a significant decrease of the D-ALA-D activity of normal erythrocytes which was not due to differences in the concentrations of Pb, Cd or Zn. The decreased enzyme activity could be restored by adding reduced glutathione (GSH; 5 mmol/L) together with the middle molecules to the assay system. Uremic middle molecules caused a significant increase of peroxidative hemolysis in normal erythrocytes. Uremic middle molecules contribute to the anemia of uremic patients by impeding hemoglobin synthesis and by increasing peroxidative hemolysis, possibly by affecting SH-groups. H2O2-producing compounds should be avoided in uremic patients.

Studies of Delta-Aminolevulinic Acid Dehydrase from Skeletonema costatum, a Marine Plankton Diatom

The accumulation of delta-aminolevulinic acid and activities of delta-aminolevulinic acid dehydrase were examined in the marine diatom, Skeletonema costatum, grown in the presence of levulinic acid. Levulinic acid concentrations greater than 10 mm affect growth and morphology, and inhibit chlorophyll synthesis. The algae recover from the effects of levulinic acid after 48 hours of exposure. The recovery is characterized by increased cellular cholorphyll content, decreased delta-aminolevulinic acid accumulation, decreased 3-(3,4-dichlorophenyl)-1, 1-dimethylurea-enhanced in vivo fluorescence, and the induction of a levulinic acid-activated delta-aminolevulinic acid dehydrase which does not follow Michaelis-Menten kinetics. The data indicate that levulinic acid blocks may be ineffective in vivo, and that delta-aminolevulinic acid is metabolized to amino and dicarboxylic acids. delta-Aminolevulinic acid dehydrase activities are used to estimate the capacity for chlorophyll synthesis. Results suggest this diatom may be capable of rapid chlorophyll turnover, which would allow the plant to light-shade adapt on the time scales appropriate to vertical mixing rates in the sea.

Stimulation of tetrapyrrole formation in Rhizobium japonicum by restricted aeration.

Cultures of Rhizobium japonicum were grown with vigorous aeration to stationary phase and were then incubated under restricted aeration for several days. Under these "microaerobic" conditions, cellular heme content increased 10-fold, and visible amounts of porphyrins were released into the culture medium. The two predominant porphyrins produced were identified, on the basis of their spectrophotometric and chromatographic properties, as protoporphyrin and coproporphyrin. The cytochrome complement of microaerobic cells partially resembled that of the symbiotic bacteria in that cytochromes alpha-alpha3 were absent and a CO-binding cytochrome 552 was present. During the period of restricted aeration, at the time that the heme content was increasing, there was a similar 10-fold increase in the activities of the first two enzymes of heme biosynthesis, delta-aminolevulinic acid synthase and delta-aminolevulinic acid dehydrase. However, during the same period, the activity of succinyl thiokinase (an enzyme that is required in large amounts whether or not heme is being produced) increased only twofold. These results suggest that reduced oxygen tension may play a role in inducing heme synthesis necessary for leghemoglobin formation and bacterial differentiation in soybean root nodules.

Heme Synthesis in Soybean Root Nodules

During nodulation of soybean (Glycine max) by Rhizobium japonicum, variations in the activities of two enzymes of heme biosynthesis, delta-aminolevulinic acid synthase (ALAS) and delta-aminolevulinic acid dehydrase (ALAD) are described. delta-Aminolevulinic acid synthase activity is found in the bacteroid fraction of nodules, but is not detected in the plant fraction. Bacteroid ALAS activity parallels heme accumulation during nodule development. delta-Aminolevulinic acid dehydrase activity is found in both bacteroid and plant cytosol fractions. Bacteroid ALAD activity is constant or increases during nodulation while plant ALAD activity falls.Bacteroid ALAD activity is found in effective, not in inefficient nodules. Plant ALAD activity is found in both effective and inefficient nodules. Plant ALAD activity falls during development of both types of root nodules.These results support the contention that it is the bacteroid ALAS and ALAD activities, not those of the plant, that are directly involved in formation of leghemoglobin heme, suggesting that the bacteroid may be solely responsible for formation of leghemoglobin heme in the nodule symbiosis.

Effects of Selenium and Vitamin E Deficiencies in Lambs on Hepatic Microsomal Hemoproteins and Mitochondrial Respiration

Microsomal hemoprotein levels and delta-amino levulinic acid dehydrase activity were determined in livers and rate of oxygen uptake, ADP:O ratios, and respiration control index were determined on mitochondria from muscle, liver, and heart of normal and white muscle diseased (WMD) lambs. WMD lambs were produced by feeding their dams either low selenium purified or alfalfa hay diets. Vitamin E and/or selenium was injected in a 2 x 2 factorial treatment in the ewes fed purified diets. Hepatic microsomal cytochrome P 450 levels and total heme content were significantly lower in WMD lambs. Cytochrome b5 content was significantly lower in lambs on the -E-Se or -E + Se treatments than those on the +E--Se treatment, but the cytochrome b5 content was not different between WMD and normal lambs from ewes fed the hay diet. No differences were found in hepatic delta-amino levulinic acid dehydrase activity, or in the rate of oxygen uptake, ADP:O ratios or respiratory control index between mitochondria from normal and WMD lamb tissue on any of the treatments.

Responses of Young Calves to Low Doses of Lead

Twenty calves, week-old male Holsteins, 42.8 kg body weight, were assigned to four treatments by oral dose 0, 1.5, 3.0, and 6.0 mg lead carbonate/kg body weight given three times weekly until calf fetal hemoglobin had been converted to 100% adult hemoglobin for 2 consecutive wk. Analyses were for the first 7 wk when data were complete. Variables included lead in blood, body weight changes, erythrocyte delta-aminolevulinic acid dehydrase activity, packed cell volume, hemoglobin, conversion of fetal to adult hemoglobin, and erythrocyte 2,3-diphosphoglyceric acid. Early changes due to lead treatment were inhibition of delta-aminolevulinic acid dehydrase at all doses and increased loss of hemoglobin at 3.0 and 6.0 mg doses. Growth rate was not impaired seriously by lead treatment. The 2,3-diphosphoglyceric acid in erythrocytes reached peak value at 3 to 4 wk of age and then decreased with age, but 2,3-diphosphoglyceric acid of the treated calves was not changed by the low lead dosing. Low lead dosing will induce early metabolic changes in the young calf prior to inhibition of growth.

Influence of Dietary Zinc on Lead Toxicity in the Rat

An investigation of the influence of dietary zinc (8, 35, 200 ppm) on the toxicity of dietary lead (0, 50, 200 ppm) in the young male rat in a seven week period indicated that as dietary zinc increased, the severity of lead toxicity decreased. Evidence included decreased lead concentration in blood, liver, kidneys, and tibias; decreased excretion of urinary delta-aminolevulinic acid; decreased accumulation of free erythrocyte porphyrins; decreased inhibition of kidney delta-aminolevulinic acid dehydrase activity; and a decrease in apparent lead absorption. Infected zinc did not afford protection against lead toxicity. The data indicate that the protective effect of zinc on lead toxicity is largely mediated by an inhibition of lead absorption at the intestinal level.

Activity of erythrocyte delta-aminolevulinic acid dehydrase and its change by heat treatment as indices of lead exposure.

The effect of heat treatment on erythrocyte delta-aminolevulinic acid dehydrase (ALAD) activity in the blood of human subjects was examined in relation to their lead exposure.

Recovery of blood lead concentration and of red cell 8-aminolevulinic acid dehydrase activity in dogs following return to normal diets after 75 weeks of lead feeding.

Blood lead concentration and red cell delta-aminolevulinic acid dehydrase (ALAD) activity were determined at intervals for eight months after symptom-free dogs, which had received 100 ppm or 500-1000 ppm of lead, by weight, in their diets for 75 1/2 weeks, were returned to a lead-free diet. Statistical treatment of the data disclosed a linear relationship between the logarithm of blood lead concentration or of ALAD activity and the logarithm of time (experimental week) during the recovery period. The calculated regressions predict that recovery of ALAD will occur before recovery of the blood lead concentration. Predicted recovery times depend on the levels selected as those which must be attained for recovery to be complete, as well as on the dosage of lead. The logarithm of the slope of the ALAD recovery regression of the individual dog has a linear relation to the percentage reduction in ALAD due to the lead, the regression accounting for 92% of the variance in the recovery rates. The slope of the blood lead recovery regression has a linear relation to the increase in blood lead for the dogs which received the low lead diet, but not for those which received the high lead diet.