Heme synthesis in hereditary hemolytic anemias: decreased delta-aminolevulinic acid synthetase in hemoglobin K?ln disease

The activities of delta-aminolevulinic acid (ALA) synthetase and ALA dehydratase in cord blood erythrocytes of newborn infants and peripheral blood red cells of patients with beta-thalassemia major, beta-thalassemia intermedia, hemoglobin K?ln (Hb K?ln) disease, sickle cell anemia, and pyruvate kinase deficiency were studied. The activity of ALA dehydratase did not vary appreciably with the number of immature RBC (reticulocytes and nucleated red blood cells) or the severity of the hemolytic anemia except in pyruvate kinase deficiency. The activity of ALA synthetase was linearly correlated with the number of immature RBC (r=0.974, p is less than 0.001). The ALA synthetase activity was significantly decreased in the RBC of Hb K?ln (p is less than 0.01) when compared with the activity in immature RBC of newborns and of patients with pyruvate kinase deficiency, sickle cell anemia, and thalassemia intermedia.     

The effects of anemia on heme synthesis parameters during lead exposure

The effects of anemia during lead exposures were studied using an infant baboon animal model. When the hemoglobin concentration was reduced to less than 70% of normal, a marked blood lead increase was observed and the free erythrocyte porphyrin value, aminolevulinic acid dehydratase activity and reticulocyte counts increased. Special emphasis should be placed on nutritional effects in lead exposures.     

Changes of serum hemolytic activity and the number of reticulocytes in canine Babesia gibsoni-infection

When the serum hemolytic activity in Babesia gibsoni-infected dogs was determined with self red blood cells from the infected animals, the decrease in the activity is paralleled with the increase in the number of reticulocytes. The activity determined with red blood cells from phenylhydrazine-induced anemia also decreased parallel with the increase in the number of reticulocytes. These results suggest that the rapid decrease in the serum hemolytic activity after reaching the peak is due to the increase in reticulocytes, which are probably unsusceptible to the hemolytic factor(s).     

A unique pulmonary venous abnormality: with associated pulmonary arterial anomaly and arteriovenous malformation

Lead and cadmium were administered intraperitoneally, singly and jointly, to the mice. The levels of cadmium, copper, manganese, lead and zinc were determined in liver, kidney and brain by atomic absorption spectrophotometric technique and delta-aminolevulinic acid dehydratase (ALA-D) activity was determined in erythrocytes. The tissue levels of some of these metals were found significantly altered by cadmium and lead both, but cadmium was found to have no effect on blood on ALA-D activity.     

Propanediol utilization genes (pdu) of Salmonella typhimurium: three genes for the propanediol dehydratase

The propanediol utilization (pdu) operon of Salmonella typhimurium encodes proteins required for the catabolism of propanediol, including a coenzyme B12-dependent propanediol dehydratase. A clone that expresses propanediol dehydratase activity was isolated from a Salmonella genomic library. DNA sequence analysis showed that the clone included part of the pduF gene, the pduABCDE genes, and a long partial open reading frame (ORF1). The clone included 3.9 kbp of pdu DNA which had not been previously sequenced. Complementation and expression studies with subclones constructed via PCR showed that three genes (pduCDE) are necessary and sufficient for propanediol dehydratase activity. The function of ORF1 was not determined. Analyses showed that the S. typhimurium propanediol dehydratase was related to coenzyme B12-dependent glycerol dehydratases from Citrobacter freundii and Klebsiella pneumoniae. Unexpectedly, the S. typhimurium propanediol dehydratase was found to be 98% identical in amino acid sequence to the Klebsiella oxytoca propanediol dehydratase; this is a much higher identity than expected, given the relationship between these organisms. DNA sequence analyses also supported previous studies indicating that the pdu operon was inherited along with the adjacent cobalamin biosynthesis operon by a single horizontal gene transfer.     

HLA antigens in three Siberian populations

The biological responses of the heme biosynthesis pathway in male workers moderately exposed to lead are discussed in relation to the concentration of lead in the blood. The level of erythrocyte delta-aminolevulinic acid dehydratase (ALA-D) activity in the group of lead-exposed workers was remarkably reduced while the level of erythrocyte protoporphyrin (Proto) in them was strikingly increased, compared to normal levels. On the other hand, the amounts of hemoglobin (Hb) and urinary delta-aminolevulinic acid (ALA) in the group of lead-exposed workers kept the normal levels. In the workers moderately exposed to lead, the log of erythrocyte Proto level was closely correlated to the blood lead level and the sensitivity of the Proto test was almost equal to that of erythrocyte ALA-D test. It was observed that the erythrocyte Proto was remarkably increased even in lead-exposed workers whose ALA excretion into the urine was in the range of normal level.     

Hypomethylation in cervical tissue: is there a correlation with folate status?

5-Aminolevulinic acid (ALA) is the first intermediate substrate in the heme synthetic pathway and is the substrate of aminolevulinic acid dehydratase (ALAD, porphobilinogen synthase). Because lead effectively inhibits ALAD activity, resulting in accumulation of ALA in urine and blood, urinary ALA (ALAU) has been used as a biomarker for lead exposure or early biologic effect of lead. Intraindividual variation in urinary excretion of ALA requires the use of 24-hour urine samples or adjustment of single urine samples by other normalizing variables, such as urinary creatinine concentration. Previous studies of ALAU concentration have used various adjustment methods; however, few have compared creatinine-adjusted ALAU concentration with ALA concentration in plasma (ALAP) from subjects with low (< 30 micrograms/dL) to moderate (< 60 micrograms/dL) levels of blood lead. To determine if creatinine-adjusted ALAU is associated with ALAP, we measured ALAU, ALAP, and urinary creatinine in 65 Korean lead workers with blood lead concentrations in the range of 14-60 micrograms/dL. ALAU, ALAU/creatinine, or ALAU/log creatinine all correlated with ALAP. However, ALAU/creatinine correlated more closely with ALAP based on Spearman's r (rs = 0.40, P, = 0.0009), supporting the use of ALA/creatinine in single urine samples as a surrogate for ALAP.     

delta-Aminolevulinic acid in plasma or whole blood as a sensitive indicator of lead effects, and its relation to the other heme-related parameters

To evaluate the subclinical effect of lead exposure, we determined delta-aminolevulinic acid (ALA) levels in plasma (ALA-P), blood (ALA-B), and urine (ALA-U) and the activity of delta-aminolevulinic acid dehydratase (ALAD) in lead workers. Almost all of the ALA molecules in blood were present in plasma and not in blood cells, irrespective of the blood lead concentration (Pb-B). ALA-P or ALA-B levels increased slowly at Pb-B levels below 40 micrograms/dl (slow phase) and rapidly at levels above 40 micrograms/dl (rapid phase). In both phases, ALA-P and ALA-B were well correlated with Pb-B and ALAD activity. The threshold value (no-effect level) of Pb-B for elevation of the ALA-P or ALA-B level was coincident with that for ALAD inhibition; the value was around 5 micrograms/dl. In the rapid phase, ALA-P increased continuously up to 100 micrograms/dl of Pb-B, while ALAD activity reached a plateau. Receiver operative characteristic (ROC) plot analyses indicated that ALA-P and ALAD activity [ALAD(u)] had a similar diagnostic value at Pb-B levels between 10 and 40 micrograms/dl, although ALAD(%), the remaining ALAD activity as a percentage of the whole activity restored by zinc and dithiothreitol, had the most powerful diagnostic efficiency at these Pb-B levels. By contrast, ALA-U and zinc protoporphyrin were less effective for the diagnosis of lead exposure than ALAD and ALA-P. These findings indicate that ALA-P is the best discriminators of lead exposure form baseline to high levels of exposure.     

Active potassium transport in reticulocytes of high-K+ and low-K+ sheep

The kinetics of active K+ transport were studied in immature red blood cells cells from high-K+ and low-K+ sheep particulary with respect to the effects of varying intracellular K+ concentration, [K]i. Comparison was made with active transport, or pump, activity in mature high-K+ and low-K+ red cells. Reticulocytes from both types of sheep had much higher maximal active K+ influxes than did mature cells. In both types of reticulocytes, and in mature high-K+ cells as well, the pump was relatively insensitive to increasing [K]i. In contrast, intracellular K+ markedly inhibited the pump in mature low-K+ cells. Active K+ transport in low-K+ reticulocytes, however, as in mature low-K+ cells, is stimulated by specific isoimmune anti-L serum. Therefore the K+ pumps of high-K+ and low-K+ reticulocytes have similar kinetic properties. Maturation of the red cells, involving inactivation of most of the pump activity in both cell types, results in mature high-K+ and low-K+ cells with K+ pumps of very different kinetic characteristics.     

An overview of the laboratory diagnosis of lead poisoning

Four tests for the evaluation of lead poisoning are reviewed from both the clinical and methodological aspects. Whole blood or erythrocyte lead measurements appear to provide the best means of assessing the bodily burden of lead with electrothermal and Delves cup flame atomic absorption spectorphotometric techniques providing accurate and precise results. Urine lead is less reliable as a screening test for lead poisoning but is excellent for monitoring the course of ethylenediamine tetraacetic acid (EDTA) therapy. Atomic absorption methods for urine are made difficult by the variable matrix of urine but satisfactory electrothermal and flame procedures have been described. Erythrocyte delta-aminolevulinic acid dehydratase activity is a very sensitive index of lead exposure,--perhaps too sensitive. Analytical procedures for measuring this enzyme are subject to errors and many complicating factors such as lack of stability of the specimen limit the usefulness of the test. Urine delta-aminolevulinic acid is of questionable value as a screening procedure and also is subject to analytical problems.     

The activity of certain hydrolases of rat erythrocytes in experimental magnesium deficiency

In rats with induced syndrome of chronic magnesium deficiency, occurrence of haemolytic anaemia in conjuction with a shortening of the erythrocyte survival time and marked reticulocytosis among other symptoms became noticeable. In the present experiment the authors undertook to study the behaviour of the lysosomal enzymes in the erythrocytes of magnesium-deficient rats. In these animals anaemia and reticulocytosis as well as a marked increase in the percentage of the erythrocytes showing positive reactions to the acid phosphatase and beta-glucuronidase tests developed. It seems likely that the positive lysosomal reactions were obtained with younger blood cells which did not stain with brilliant cresyl blue but still retained single lysosomes in their cytoplasm. This assumption was confirmed by ultrastructural studies which demonstrated the presence of siderosomes inside both the reticulocytes and the mature erythrocytes. The changes in the percentages of reticulocytes and enzyme-positive erythrocytes were independent of the histological structure of the experimental rat's thymus. In the reticulocytes as well as in the mature erythrocytes, the noteworthy presence of degenerated mitochondria containing electron-dense material seems to be a morphological sign of impairment of the magnesium-deficient rat's erythrocytes.     

The human placenta's lead level as a parameter of the ecological lead exposure. Its validity in comparison to the lead level in blood, the activity of the delta-aminolevulinic acid dehydratase and the concentration of the free erythrocyte porphyrins of newborns and their mothers (author's transl)

In order to estimate the ecological exposure of lead, placenta- and blood-investigations were made at four collectives from variously industrialized regions (Ruhrregion, Middle Frankonia Centre, Bavarian Forest). 148 normal births and 19 premature births (in each case mothers and newborns) were listed as well as twelve abortions. We investigated the lead-level in blood, the activity of delta-aminolaevulinic acid dehydratase (ALA-D) the concentration of free erythrocyte porphyrine (FEP) and the placentas' lead concentration. Though in the Ruhrregion (Dortmund) significantly higher lead levels in blood were found compared to the Bavarian Forest, the results together, were in the normal range, less than 35 mug%. In an average the mothers' lead level in blood was around 1.4 times (ca. 5 mug%) above that of their newborns; analysing this statistically, highly significant correlations were found. However for the ALA-D activity and the FEP-results no direct dependence of the lead levels in blood could be found. In the placentas mean lead concentrations between 1.94 mug and 2.23 mug per gram dry-weight (30.6 mug-38.9 mug/100 g wet-weight) were gained. In the contrary to the measured results of lead in blood the average placentas' lead level of the most and least industrialized regions were almost identical. A correlation between the mothers' respectively their children's lead levels in blood and the placental lead concentrations could not be proved. No relation could be found between the results and the gestation ages. As final results: 1. The placenta is no ideal investigation object concerning the environment's lead exposure. 2. It has no special barriere - or depot - function in lead metabolism. 3. In order to estimate the environment's lead exposure the determination of the lead level in blood will also be in future the optimal method. This investigation is of special value because of its validity of the results and the practicability of winning the samples compared to other parameters and biological materials.     

Molecular cloning, sequencing and characterization of the genes for adenosylcobalamin-dependent diol dehydratase of Klebsiella pneumoniae

Klebsiella pneumoniae and some of the other Enterobacteriaceae form both diol dehydratase and glycerol dehydratase in response to growth substrates. To compare these enzymes produced by the same bacterium, the pdd genes of K. pneumoniae encoding adenosylcobalamin-dependent diol dehydratase were cloned and sequenced. The sequential three open reading frames (pddA, pddB, and pddC genes) encoded polypeptides of 554, 228, and 174 amino acid residues with predicted molecular weights of 60,379(alpha), 24,401(beta), and 19,489(gamma), respectively. The deduced amino acid sequences of the subunits were 84-100% and 54-71% identical with those reported for diol dehydratases and glycerol dehydratases, respectively.     

Cofactor identification of threonine-serine dehydratase from sheep liver

L-Threonine-serine dehydratase catalyzes the conversion of L-threonine and L-serine to alpha-ketobutyric acid and pyruvate, respectively. The enzyme has been purified to homogeneity from extracts of sheep liver. In the past, various cofactors have been suggested for threonine dehydratase from both prokaryotic and eukaryotic tissue. While some direct evidence for the presence of pyriodoxal 5'-phosphate in impure preparations is present in the literature no direct evidence for the cofactor in homogeneous dehydrogenase from mammalian tissue has been reported. The threonine dehydratase of sheep liver has been obtained in a homogeneous form and a spectral study provides clear evidence for the presence of pyridoxal 5'-phosphate. Both the physical properties of homogeneous threonine dehydratase and a study of spectral properties of its cofactor are reported in this communication.     

Purification and properties of an iron-sulfur and FAD-containing 4-hydroxybutyryl-CoA dehydratase/vinylacetyl-CoA delta 3-delta 2-isomerase from Clostridium aminobutyricum

4-Hydroxybutyryl-CoA dehydratase, the key enzyme in the metabolism of gamma-aminobutyrate in Clostridium aminobutyricum, represents approximately 15-25% of the soluble protein. The enzyme was purified to homogeneity under anaerobic conditions to a specific activity of 209 nkat mg-1. The dehydratase catalyses the reversible conversion of 4-hydroxybutyryl-CoA (Km = 50 microM) to crotonyl-CoA and possesses a probably intrinsic vinylacetyl-CoA delta 3-delta 2-isomerase with a specific activity of 223 nkat mg-1. The equilibrium of the reversible dehydration was determined from both sides as K = [crotonyl-CoA]/[4-hydroxybutyryl-CoA] = 4.2 +/- 0.3. Cyclopropylcarboxyl-CoA was not converted to crotonyl-CoA. The native enzyme has an apparent molecular mass of 232 kDa and is composed of four apparently identical subunits (molecular mass = 56 kDa), indicating a homotetrameric structure. Under anaerobic conditions the active enzyme revealed a brown colour and contained 2 +/- 0.2 mol FAD (64 +/- 5% oxidized), 16 +/- 0.8 mol Fe and 14.4 +/- 1.2 mol inorganic sulfur, which probably form iron-sulfur clusters. Exposure to air resulted initially in a slight activation followed by irreversible inactivation. Concomitantly the vinylacetyl-CoA delta-isomerase activity was lost and the colour of the enzyme changed to yellow. Reduction by sodium dithionite yielded inactive enzyme which could be completely reactivated by oxidation with potassium hexacyanoferrate(III). The data indicate that the active enzyme contains oxidized FAD despite its sensitivity towards oxygen. During the dehydration a non activated C-H bond at C-3 of 4-hydroxybutyryl-CoA has to be cleaved. A putative mechanism for 4-hydroxybutyryl-CoA dehydratase is proposed in which this cleavage is achieved by a FAD-dependent oxidation of 4-hydroxybutyryl-CoA to 4-hydroxycrotonyl-CoA. In a second step the hydroxyl group is substituted by a hydride derived from the now reduced FAD in an SN2' reaction leading to vinylacetyl-CoA. Finally isomerisation yields crotonyl-CoA. 4-Hydroxybutyryl-CoA dehydratase is quite distinct from 3-hydroxyacyl-CoA dehydratase (crotonase) and 2-hydroxyacyl-CoA dehydratases. Contrary to the latter enzyme [e.g. (R)-lactyl-CoA dehydratase and (R)-2-hydroxyglutaryl-CoA dehydratase] which are composed of three different subunits and similarly catalyse the cleavage of a non activated C-H bond at C-3, 4-hydroxybutyryl-CoA dehydratase does not require ATP, MgCl2 and Ti(III)citrate for activity. Furthermore 4-hydroxybutyryl-CoA dehydratase is not inactivated by oxidants such as 5 mM 4-nitrophenol, 5 mM chloramphenicol and 5 mM hydroxylamine.     

Dobutamine as a countermeasure for reduced exercise performance of rats exposed to simulated microgravity

Post-spaceflight results and findings from humans and rodents after conditions of bed rest or simulated microgravity indicate maximum exercise performance is significantly compromised. However, the chronic administration of dobutamine (a synthetic adrenomimetic) to humans in relevant experiments improves exercise performance by mechanisms that prevent the decline in peak O2 consumption (VO2peak) and reduce the concentration of lactic acid measured in the blood. Although dobutamine restores maximum VO2 values in animals participating in simulated microgravity studies, it is unknown whether injections of this alpha 1-, beta 1-, and beta 2-adrenoceptor agonist in rats will enhance exercise performance. To investigate this, adult male rats were assigned to three experimental groups: caged control receiving saline; head-down, tail-suspended (HDS) receiving saline (HDS-S); and an HDS group receiving dobutamine hydrochloride injections (1.8 mg/kg twice daily per rat). Treadmill tests were performed before suspension, at 14 days, and after 21 days. VO2peak, run time, and the rate of rise in colonic temperature (heating index) were evaluated after 14 days, whereas at 21 days, hemodynamic responses (heart rate, systolic blood pressure, and double product) were determined during submaximal exercise with blood pH, blood gases, and lactic acid concentration values obtained during maximal exercise. In contrast to the results for the HDS-S rats, dobutamine administration did restore VO2peak and "normalized" lactic acid concentrations during maximal exercise. However, daily injections were unable to enhance exercise performance aspects associated with treadmill run time, the mechanical efficiency of running, the heating index, or the retention of muscle and body mass. These simulated microgravity findings suggest that dobutamine's potential value as a countermeasure for postflight maximal performance or for egress emergencies is limited and that other countermeasures must be considered.     

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.     

Occult pneumococcal septicemia and pneumococcal pneumonia complicating pneumatoceles in a previously healthy child

1. The effect of in vitro glycation on delta-aminolevulinic dehydratase (ALA-D) under several experimental conditions was investigated. When preincubated with 500 mM glucose at 37 degrees C for 20 hr, ALA-D was 80% inactivated and glycated hemoglobin levels were increased more than fourfold. 2. Thiobarbituric acid species were not modified during glycation; therefore ALA-D inactivation cannot be attributed to glucose autoxidation. 3. Acetyl salicylic acid was effective in preventing both hemoglobin glycation and ALA-D inactivation by glucose. 4. A method has been developed for measuring protein glycation in vitro, in a crude preparation of red blood cells, which can also be applied to sugars other than glucose.     

Impact of air pollution by lead on the heme biosynthetic pathway in school-age children

Blood-lead level (Pb-B), erythrocyte delta-aminolevulinic acid dehydratase (ALAD) activity, free erythrocyte porphyrin (FEP) concentration, delta-aminolevulinic acid concentration in urine (ALAU), hematocrit value, and hemoglobin concentration were compared for groups of children 10-13 years old from areas differently polluted by lead (rural area and lead smelter area). The biological responses of the children were also compared with those observed in adults similarly exposed to lead (Pb-B: 10-40 mug/100 ml). Compared with the rural children, children living less than 1 km from the smelter exhibited a significant increase of Pb-B and FEP, a significant inhibition of ALAD, and a slight positive correlation of ALAU with Pb-B; however, they showed no biological signs of anemia. In children living approximately 1.5 km from the smelter, there was still a significant increase of Pb-B and a concomitant inhibition of ALAD, but no change in FEP concentration. Comparison of the dose-response curves between Pb-B and FEP in adult males, adult females, and children indicates that the sensitivity to lead is in the order of children larger than or equal to women greater than men. Based on the FEP response, it is proposed that 25 mug Pb/100 ml blood be regarded as the maximum biologically allowable concentration of lead in blood of school-age children.