Erythrocyte ALAD Activity Research Articles

Correlation between lead exposure indicators and sister chromatid exchange (SCE) frequencies in lymphocytes from inorganic lead exposed workers.

Inorganic lead exposure was studied in 31 volunteers employed in storage battery plant. The genotoxicity of lead was measured in terms of sister chromatid exchange (SCE). Erythrocyte delta-aminolevulinic acid dehydrogenase (ALAD) activity, urinary delta-aminolevulinic acid (U-ALA), and blood lead levels (PbBs) were also determined to evaluate some possible relations between these lead exposure indicators and the observed SCE frequencies. Blood lead concentration of 36.31 microg/dl was determined as an average level in the workers. Consequently decreased ALAD activity in erythrocytes and increased U-ALA excretion was observed in statistically higher PbBs when compared with the control group. A statistically significant correlation was observed between the PbBs and SCE frequencies (p < 0.05). Moreover, the correlation between U-ALA excretion and SCE frequencies (p < 0.01) was relatively higher than the correlation between PbBs and SCE frequencies. These results might indicate a possible mechanism of ALA mediation in the genotoxic effects of lead.

Molecular analysis of δ-aminolevulinate dehydratase deficiency in a patient with an unusual late-onset porphyria

Cloning, expression, and genotype studies of the defective gene for delta-aminolevulinate dehydratase (ALAD) in a patient with an unusual late onset of ALAD deficiency porphyria (ADP) were carried out. This patient was unique in that he developed the inherited disease, together with polycythemia, at the age of 63. ALAD activity in erythrocytes of the patient was less than 1% of the normal control level. ALAD complementary DNA (cDNA) isolated from the patient's Epstein-Barr virus (EBV)-transformed lymphoblastoid cells had 2 base transitions in the same allele, G(177) to C and G(397) to A, resulting in amino acid substitutions K59N and G133R, respectively. It has been verified that the patient had no other ALAD mutations in this and in the other allele. By restriction fragment length polymorphism (RFLP) analysis, all family members of the proband who had one-half ALAD activity compared with the ALAD activity of the healthy control were shown to have the same set of base transitions. Expression of ALAD cDNA in CHO cells revealed that K59N cDNA produced a protein with normal ALAD activity, while G133R and K59N/G133R cDNA produced proteins with 8% and 16% ALAD activity, respectively, compared with that expressed by the wild type cDNA. These findings indicate that while the proband was heterozygous for ALAD deficiency, the G(397) to A transition resulting in the G133R substitution is responsible for ADP, and the clinical porphyria developed presumably due to an expansion of the polycythemic clone in erythrocytes that carried the mutant alad allele.

Molecular analysis of δ-aminolevulinate dehydratase deficiency in a patient with an unusual late-onset porphyria

AbstractCloning, expression, and genotype studies of the defective gene for δ-aminolevulinate dehydratase (ALAD) in a patient with an unusual late onset of ALAD deficiency porphyria (ADP) were carried out. This patient was unique in that he developed the inherited disease, together with polycythemia, at the age of 63. ALAD activity in erythrocytes of the patient was less than 1% of the normal control level. ALAD complementary DNA (cDNA) isolated from the patient's Epstein-Barr virus (EBV)–transformed lymphoblastoid cells had 2 base transitions in the same allele, G177 to C and G397 to A, resulting in amino acid substitutions K59N and G133R, respectively. It has been verified that the patient had no other ALAD mutations in this and in the other allele. By restriction fragment length polymorphism (RFLP) analysis, all family members of the proband who had one-half ALAD activity compared with the ALAD activity of the healthy control were shown to have the same set of base transitions. Expression of ALAD cDNA in CHO cells revealed that K59N cDNA produced a protein with normal ALAD activity, while G133R and K59N/G133R cDNA produced proteins with 8% and 16% ALAD activity, respectively, compared with that expressed by the wild type cDNA. These findings indicate that while the proband was heterozygous for ALAD deficiency, the G397 to A transition resulting in the G133R substitution is responsible for ADP, and the clinical porphyria developed presumably due to an expansion of the polycythemic clone in erythrocytes that carried the mutant aladallele.

Erythrocyte delta-aminolevulinic acid dehydratase genotype and other mechanisms affecting workers' susceptibility to lead.

In this study, the role of delta-aminolevulinic acid dehydratase (ALAD) variants in lead susceptibility was examined. The study subjects comprised 223 male workers, and the relationship between their blood lead level and erythrocyte ALAD activity or plasma/urine delta-aminolevulinic acid level was studied. Leukocyte specimens from 11 workers, whose erythrocyte ALAD activities were as low as one-fifth that of the other normal workers, were subjected to analyses of their ALAD and ALAD alleles. Further, the entire exon fragment of the ALAD gene was analyzed by polymerase chain reaction, and the reaction product was used as a target for direct DNA sequencing. Genomic DNA analysis revealed that all 11 workers had the ALAD allele, whereas the entire ALAD gene analysis failed to indicate other variants, except for the Rsa I site. The depletion in erythrocyte ALAD activity was not found to be caused by the ALAD allele.

Erythrocyte ALA-d activity in experimentally lead-poisoned ducks and its change during treatment with disodium calcium EDTA

To determine useful procedures for the diagnosis and prognosis of lead poisoning in waterfowl caused by ingestion of lead pellets, erythrocyte delta-aminolevulinic acid dehydratase ( ALA-d) was investigated in experimentally lead-poisoned ducks. A highly positive correlation was observed between the concentration of blood lead and the ALA-d activity ratio (the ratio of activated:non-activated enzyme activity) in those birds given seven lead pellets (3 mm diameter). The ALA-d activity ratio rapidly increased after the administration of lead pellets, but began to fall immediately after the initiation of disodium calcium ethylenediamine tetra-acetate ( CaEDTA) therapy which resulted in a rapid decrease in the concentration of lead in the blood of these birds. In contrast, the ALA-d activity remained inhibited even after blood lead levels began to decrease following treatment. These results demonstrated that the ALA-d activity ratio is a very useful and sensitive indicator for the diagnosis and evaluation of therapeutic effects after lead poisoning in waterfowl.

The prevalence and retention of lead pellets in Japanese quail

Thirty-six Japanese quail (18 control birds, 18 lead-dosed birds) were used. The 18 quail were dosed with #4 lead weight that were orally inserted into the proventriculus. Delta-aminolevulunic acid dehydrase (ALAD) activity in erythrocytes in the dosed quail decreased 90% (p < 0.01) after one week as compared with the undosed quail. This inhibition of ALAD activity in erythrocytes indicates lead exposure. Radiographics were obtained at 0, 1, 4, 9, 22, and 32 days. The lead pellets remained in the gizzard and became smaller in 4 days. At day 22, after the lead treatment, in 8 quail of the treated quail 12, the lead pellets disappeared. At day 32, all lead pellets disappeared. These findings indicate that the ingested lead pellets are absorbed gradually in the intestine. The lead concentrations in the blood, liver, kidney, and femur of the lead-dosed quail were significantly higher than in the undosed quail until the 6th week. At week 2, the lead concentration of the proventriculus, gizzard, gizzard contents, duodenum, small intestine, and cecum in the dosed quail was significantly higher. Lead concentration of feces was significantly higher at weeks 2 and 4 (p < 0.01). Throughout this study, no lead pellets were found in the feces.

Sex difference in free erythrocyte protoporphyrin (FEP) level. IV. Sex difference in FEP level in rabbits exposed to lead.

An experimental study on Japanese White (JW) rabbits was conducted to clarify the sex difference in FEP level. Male and female rabbits (n = 14 of each sex) were used. The animals of the same sex were divided into 3 groups; i.e., control group (5% glucose solution only, n = 4), low lead dose group (Pb 0.4 mg/kg.BW, n = 5) and high lead dose group (Pb 2 mg/kg.BW, n = 5). Lead was injected intravenously twice a week for 5 wk. The following parameters were determined once a week for 5 wk: blood lead (Pb-B), FEP, Ht, Hb, erythrocyte ALA-D activity, erythrocyte pyrimidine 5'-nucleotidase (P5N), urinary coproporphyrin (CP-U), urinary delta-aminolevulinic acid (ALA-U), iron in serum (Fe-S), and serum GOT and GTP. Average levels of FEP in female rabbits were higher than those in males between the 1st and 3rd week after the lead injection in the low lead dose groups, and in the final week in the high lead dose groups. In the periods without lead injection, the average levels of FEP in the female groups were not significantly higher than the corresponding levels in the male groups in every week except in the first week in the control. However, the mean of FEP levels in all female rabbits without Pb treatment was higher than that in male rabbits (t-test). By the analysis of variance for the gains of FEP from the initial value, only the low lead dose group showed a significant sex difference (female > male); that is, the female group tended to increase when compared with the male group. Furthermore, the week when FEP began to increase in the female groups was earlier than that in the male groups in the low lead dose group. In the high lead dose group, both sexes reacted to the lead exposure from the same early week. As for the parameters of anemia, the average levels of Ht and Hb tended to be lower in females than in males, but Fe-S levels were not affected by lead in both sexes and no consistent sex difference could be observed. By lead exposure, ALA-D and P5N were inhibited, and ALA-U was increased, but these parameters showed no evident sex difference. The average levels of CP-U tended to be higher in females than in males in the administration of low lead dose and to be inversely higher in males than in females in the administration of high lead dose.(ABSTRACT TRUNCATED AT 400 WORDS)

Sex difference in free erythrocyte protoporphyrin (FEP) level. I. Sex difference in FEP level in healthy rural residents

FEP and other hematological parameters were measured in 157 healthy rural residents (98 females and 59 males). The mean FEP in females was higher than in males (p less than 0.05); mean +/- SD = 62.98 +/- 19.36 and 54.57 +/- 21.20 micrograms/dl.pcv, respectively, although both of the means were within normal limits. There was no significant sex difference in erythrocyte ALA-D activity. Whole blood lead (Pb-B) level in females showed a tendency to be lower than in males, but there was no significant sex difference in erythrocyte lead level. The mean value of hematocrit (Ht), hemoglobin (Hb) and iron in serum (Fe-S) were lower in females than in males (p less than 0.01). Serum GOT and GPT level tended to be lower in females than in males (0.05 less than p less than 0.1). There was hardly any significant relationship between Pb-B and each parameter of lead exposure, because the subjects in this study were only rural residents with no occupational lead exposure and with their Pb-B levels being extremely low. As for the parameters of anemia, Fe-S was positively correlated with Ht and Hb level and negatively correlated with FEP level. By sex, Fe-S was correlated with Ht and FEP level only in females. As for the possible reasons why FEP level in females in higher than in male, women tended to have iron-deficiency induced by blood loss due to menstruation, pregnancy, and difference in dietary pattern from males.

Toxicity of lead acetate to female rabbits after chronic subcutaneous administration. 1. Biochemical and clinical effects

The effect of chronic subcutaneous administration of lead acetate was studied in female rabbits. The low-dose group (15 animals) received three times a week 0.10-0.20 microgram/kg body weight and the high-dose group (15 animals) 0.80-1.20 micrograms/kg. The control group received the vehicle only. Concentrations of lead in blood in the low-dose group increased to ca. 400 micrograms/l after 70 days and in the high-dose group to ca. 900 micrograms/l after 110 days. After 7.5 months eight animals of each group were sacrificed. The remaining rabbits were kept for an additional 4 months without treatment. Blood lead concentrations decreased with a half-time of 60-70 days. During exposure the gain in body weight was lower in the high-dose group than in the control group and the low-dose group. The high-dose group developed slight anaemia and low MCV, MCH and MCHC, and basophilic stippling of erythrocytes. These effects disappeared during recovery. ALAD activity in erythrocytes was very low during exposure in both exposed groups and did not reach control values during recovery. During exposure the concentrations of ZPP and ALA-U increased, but only ALA-U returned to normal during recovery. No other effects of lead on the composition of the urine were observed.(ABSTRACT TRUNCATED AT 250 WORDS)

Effects of ethyl alcohol ingestion on the disturbance of porphyrin metabolism by lead

A study was made on the possibility of synergistic effects of ethyl alcohol and lead on porphyrin metabolism in rabbits. Experimental rabbits were divided into 4 groups. Group A was the control group not given any treatment, and the other 3 groups (Groups B, C and D) were treated with ethyl alcohol, lead, and ethyl alcohol and lead respectively, for 2 months. Ethyl alcohol solution (5%) was administered to rabbits in Groups B and D as drinking water on every weekday. The average dose of alcohol was 6 ml/kg/day (18 ml/cap/day). Lead was injected intravenously to rabbits in Groups C and D at a dose of 0.5 mg Pb/kg on alternate days (3 times per week). Furthermore, a large dose of Pb was administered to other rabbits (Group C'). In rabbits treated with alcohol alone (Group B), no effect was observed in the biochemical indicators related to porphyrin metabolism. In the groups treated with lead (Groups C and C') and with lead and alcohol combined (Group D), some biochemical changes in porphyrin metabolism developed with increase of Pb-B, i.e. increase of ALA-S activity and total porphyrin content in the bone marrow, elevation of FEP level, increase of ALA-U and CP-U, and decrease of ALA-D activity in erythrocytes. Comparison of Groups C and D showed that CP-U and ALA-U increased significantly in Group D, but no significant difference was observed between both groups in FEP and in ALA-S activity in the bone marrow and liver. The other laboratory measurements, such as total porphyrin contents in the liver and plasma, and GOT or GPT level in serum, showed no significant change in all the groups. In the present study, the biochemical changes suggesting synergism of lead and ethyl alcohol were observed slightly in ALA-U and CP-U but not in ALA-S and FEP. These results suggest that these changes are essentially due to lead rather than mutual enhancement of the direct effects of these two toxins on porphyrin metabolism. However, it still remains to be determined whether or not ethyl alcohol affects the liver and kidney functions which may be related to ALA and CP excretion.

Metal-induced alterations of delta-aminolevulinic acid dehydratase.

ALAD is a cytoplasmic enzyme that catalyzes the second step of the heme biosynthesis pathway, that is, the condensation of two molecules of delta-aminolevulinic acid into porphobilinogen. ALAD is a zinc-dependent enzyme; thiol groups are essential for its activity; and in vitro experiments show that ALAD can be activated or inhibited by several metal ions including A;3+, Pb2+, Cd2+, Hg2+, Ag2+, and Cu2+. To explain these effects, it has been postulated that metals bind to thiol groups of allosteric sites and, according to their structure, provoke allosteric transitions to the active or inactive form of the enzyme. Under current environmental and occupational exposure levels, lead is practically the only metal that can affect ALAD activity. Erythrocyte ALAD is the most sensitive indicator of lead exposure: effects of exposure are detectable even when blood lead levels are within the "normal" range. Zinc protects ALAD in vitro and in vivo from the inhibitory effect of lead. There is also some suggestion that aluminum could be responsible for the decreased erythrocyte ALAD activity observed in patients on chronic hemodialysis.

Decreased erythrocyte δ-aminolevulinate dehydratase activity after styrene exposure

δ-Aminolevulinate dehydratase (ALA-D:porphobilinogen synthase, 5-aminolevulinate hydro-lyase, EC 4.2.1.24) activity was depressed markedly in red cells of rats exposed to 0.21 g/m 3 styrene, a chemical widely used in commercial products. The depression was not restored in vitro after treatment with dithiothreitol and zinc. Consistent with this finding, radioimmunoassay of the enzyme protein demonstrated reduction in the enzyme concentration by styrene exposure. There was a good correlation between the decrease in enzyme activity and its concentration in the styrene-treated animals, suggesting that the depression of the enzyme activity was essentially due to the reduction in the enzyme content. Decrease in the enzyme content in bone marrow cells to almost the same extent as that in erythrocytes seems to indicate the decreased synthesis of ALA-D in the bone marrow. In vitro studies showed that styrene 7,8-oxide, the major intermediate of styrene metabolism, decreased the activity of purified ALA-D but that styrene, the parent compound itself, had no inhibitory effect. The activity and concentration of erythrocyte ALA-D in workers chronically exposed to styrene were also depressed significantly. These findings indicate that the styrene exposure-mediated decrease of ALA-D activity in erythrocytes was a reflection of reduction in the enzyme protein, which may have been the result of styrene 7,8-oxide action, and they suggest that a similar process may also be involved in the reduction of erythrocyte ALA-D in styrene-exposed workers.

δ-Aminolevulinic acid dehydratase activity in erythrocytes of diabetic patients

Erythrocytes delta-aminolevulinate dehydratase activity was assayed in 41 diabetic patients and 33 normal controls. It was found that in diabetic patients the erythrocyte ALA-D activity was lower than in controls, and the difference of the mean values was statistically highly significant (P less than 0.001). We found a significant negative correlation (r = - 0.846, P less than 0.001) between ALA-D activity and blood glucose levels. For this reason, using normal adult human whole blood haemolysates, it was investigated the effects in vitro of glucose and insulin on normal erythrocytic ALA-D. No significant difference in ALA-D activity was found in the presence of insulin. On the other hand, there was considerable decrease in the enzyme activity in the blood samples after glucose addition.

Measurements of the Erythrocyte ALA-D Activity with and without Heart Treatment as an Indicator of Lead Exposures

Measurements of the Erythrocyte ALA-D Activity with and without Heart Treatment as an Indicator of Lead Exposures

Lead toxicity in the pregnant rat. II. Effects of low-level lead on delta-aminolevulinic acid dehydratase activity in maternal and fetal blood or tissue.

Lead was administered to pregnant rats in drinking water (5 ppm) from day 1 to day 18 or 21 of gestation, at which time the dams and fetuses were sacrificed. The red blood cell count and hemoglobin concentration were significantly lower in the lead-treated fetuses than in untreated controls on day 21 of gestation, but not in the lead-treated dams. The delta-aminolevulinic acid dehydratase (ALAD) activity of erythrocytes was significantly reduced in the lead-treated dams and their fetuses on days 18 and 21 of gestation. AL AD activity in the liver was significantly higher in the leadtreated fetuses than in the controls. There was no significant difference in placental AL AD activity between the lead-treated and control animals. Lead in tissues increased significantly in the liver of the lead-treated fetuses.There were significant positive correlations between the maternal and fetal blood lead level, liver lead level and erythrocyte ALAD activity. There was a significant negative correlation between the maternal and fetal liver ALAD activity.

Changes in serum aluminium, blood zinc, blood lead and erythrocyte δ-aminolaevulinic acid dehydratase activity during haemodialysis

In a study of 18 patients on haemodialysis, erythrocyte δ-aminolaevulinic acid dehydratase (ALAD), serum aluminium, blood zinc and blood lead concentrations were increased significantly following haemodialysis. The increase in erythrocyte ALAD activity was found to be significantly linearly correlated with the increase in blood zinc concentrations. In comparison with control subjects, the patients had significantly lower activities of ALAD and significantly increased serum aluminium concentrations. When zinc and lead concentrations were corrected for packed cell volume they were found to be significantly higher in the patient group than in the control group.

Lead and δ-aminolevulinic acid dehydratase in RBC's of urban mothers and fetuses

Environmental lead pollution may pose a health hazard to the mother and her fetus, but limited information concerning this problem is available. In this study, we examined erythrocyte δ-aminolevulinic acid dehydratase (ALAD) activity and erythrocyte lead levels in urban pregnant women and fetuses. The data show that the ratio of activated/nonactivated ALAD activity and erythrocyte lead are positively correlated in both the mothers and fetuses. The mean level of ALAD inhibition was found to be 28% in the mothers and 12% in the fetuses. The data also show that fetal erythrocytes have significantly higher levels of activated ALAD activity than maternal erythrocytes, and that a positive correlation exists between maternal and fetal erythrocyte lead levels. These results indicate that “normal” urban blood lead levels inhibit erythrocyte ALAD activity in the pregnant woman and fetus.

Blood lead and erythrocyte delta-aminolevulinic acid dehydratase levels in Manchester taxi drivers.

Among 40 Manchester taxi drivers the mean blood lead was 1.10 mumol/1 (22.8 mug per 100 ml). The mean erythrocyte delta-aminolevulinic acid dehydratase (ALAD) activity among 34 of them was 30.1 units. No significant association was found between the blood lead levels and erythrocyte ALAD activity in these 34 men. No significant association was found between either blood lead elvels or erythrocyte ALAD activity and duration of service or weekly mileage as a taxi driver or with drinking or smoking habits, or age. The mean blood lead of those with homes in the north east quadrant of the city was higher than of those living elsewhere but the difference was not statistically significant. Although there was no correlation between blood lead levels and the source of domestic water, the mean blood lead of those with lead domestic plumbing was appreciably higher than the level of those with copper plumbing. There was no indication that, by virtue of their occupation, the taxi drivers were liable to greater lead absorption than their fellow-citizens.

FLUOROMETRIC MICROMEASUREMENT OF DELTA-AMINOLEVULINIC ACID DEHYDRATASE ACTIVITY IN HUMAN ERYTHROCYTES AS AN INDEX OF LEAD EXPOSURE

I describe a fluorometric micromethod for determining delta-aminolevulinic acid dehydratase (ALA-D) activity in whole blood. In this method, the porpho-bilinogen formed under the incubation condition using 0.05ml of whole blood is easily converted to uroporphyrin by heating for 15 min at 100°C in an acidic medium in the presence of air. The uroporphyrin formed is a stable and highly fluorescent substance and its fluorescence intensity is measured using a spectrofluorophoto-meter.The ALA-D activity is expressed as nanomoles of uroporphyrin formed per hour per milliliter of erythrocytes. This method offers some advantages that it is much sensitive and is employed without Ehrlich's reagent.The erythrocyte ALA-D activity measured by the fluorometric method has a good correlation with that measured by the colorimetric method with Ehrlich's reagent (r=0.9484).

Interaction of zinc and lead on δ-aminolevulinate dehydratase

Rat erythrocyte δ-aminolevulinate dehydratase (ALA-D) has been confirmed to be an enzyme dependent on dietary zinc. Levels of blood ALA-D decreased substantially during the first five weeks after weaning and remained constant thereafter. Dietary lead (200 ppm) caused a dramatic fall in erythrocyte ALA-D activity and, following the removal of lead exposure, the return of the enzymatic activity toward normal values was quicker in the animals receiving higher zinc than in those on lower zinc diet. Furthermore, invitro addition of zinc to erythrocyte preparation obtained from rats exposed to dietary lead reactivated ALA-D to control values. Maximal enzymatic activity in both lead-fed and control animals occurred at 10−4M ZnCl2. There seems to be a competition between Pb++ and Zn++ in binding to the enzyme, with Pb++ as inhibitor and Zn++ as activator.