EXPLORE: A Prospective, Multinational, Natural History Study of Patients with Acute Hepatic Porphyria with Recurrent Attacks.

BACKGROUND AND AIMS: Acute hepatic porphyria comprises a group of rare genetic diseases caused by mutations in genes involved in heme biosynthesis. Patients can experience acute neurovisceral attacks, debilitating chronic symptoms, and long-term complications. There is a lack of multinational, prospective data characterizing the disease and current treatment practices in severely affected patients. APPROACH AND RESULTS: EXPLORE is a prospective, multinational, natural history study characterizing disease activity and clinical management in patients with acute hepatic porphyria who experience recurrent attacks. Eligible patients had a confirmed acute hepatic porphyria diagnosis and had experienced ≥3 attacks in the prior 12 months or were receiving prophylactic treatment. A total of 112 patients were enrolled and followed for at least 6 months. In the 12 months before the study, patients reported a median (range) of 6 (0-52) acute attacks, with 52 (46%) patients receiving hemin prophylaxis. Chronic symptoms were reported by 73 (65%) patients, with 52 (46%) patients experiencing these daily. During the study, 98 (88%) patients experienced a total of 483 attacks, 77% of which required treatment at a health care facility and/or hemin administration (median [range] annualized attack rate 2.0 [0.0-37.0]). Elevated levels of hepatic δ-aminolevulinic acid synthase 1 messenger ribonucleic acid levels, δ-aminolevulinic acid, and porphobilinogen compared with the upper limit of normal in healthy individuals were observed at baseline and increased further during attacks. Patients had impaired quality of life and increased health care utilization. CONCLUSIONS: Patients experienced attacks often requiring treatment in a health care facility and/or with hemin, as well as chronic symptoms that adversely influenced day-to-day functioning. In this patient group, the high disease burden and diminished quality of life highlight the need for novel therapies.

Recurrent porphyria attacks in a Chinese patient with a heterozygous PBGD mutation.

We report here the case of a 32-year-old Chinese Han woman who presented with frequent severe abdominal pain, convulsion, numbness and confusion. She also had hypertension, hyponatremia, chronic renal failure, anemia and a high urinary δ-aminolevulinic acid concentration. We identified a heterozygous splicing mutation in intron 11 (IVS11-2A→G) of the porphobilinogen (PBG) deaminase gene (PBGD) in her genomic DNA. This mutation had previously been reported in a North American patient, but was absent from 50 healthy Chinese controls.

Involvement of some low-molecular thiols in the peroxidative mechanisms of lead and ethanol action on rat liver and kidney.

The involvement of low-molecular thiols, such as reduced glutathione (GSH) and metallothionein (Mt), in the mechanisms of the peroxidative action of lead (Pb) and ethanol (EtOH) in liver and kidney was investigated on rats treated with 500 mg Pb/l (in drinking water) and 5 g EtOH/kg body wt./24h (p.o.), alone and in conjunction with each other for 12 weeks. Beside of GSH and Mt, concentration of total and non-protein SH groups (TSH and NPSH, respectively) in these organs as well as the blood activity of dehydratase of delta-aminolevulinic acid (delta-ALAD) and the urinary concentration of delta-aminolevulinic acid (delta-ALA) were determined. The exposure to Pb and EtOH alone and in conjunction with each other led to a decrease in the blood delta-ALAD activity and an increase in the urinary delta-ALA concentration, and these effects were more markedly advanced at co-exposure. In the liver and kidney of rats treated with Pb and/or EtOH, a decrease in concentrations of GSH and NPSH was noted, compared to control. However, in the Pb+EtOH group, only the liver concentrations of NPSH and GSH were lower also compared to the Pb and EtOH groups. The liver concentration of TSH decreased in the rats exposed to EtOH alone and in conjunction with Pb, whereas the kidney concentration of TSH decreased only at co-exposure to Pb and EtOH. Mt concentration was unchanged except for an increase in the liver in the Pb and Pb+EtOH groups. Two-way analysis of variance (ANOVA/MANOVA) revealed that the changes noted at the co-exposure to Pb and EtOH resulted from an independent action of the two xenobiotics as well as from their interactive action. Negative correlations noted between the liver and kidney concentrations of GSH and/or NPSH and recently reported malondialdehyde (MDA, an indicator of lipid peroxidation) concentration in both organs of those rats indicate the relationship between the content of SH groups and the intensity of the Pb and/or EtOH-induced lipid peroxidation. The results allow for the conclusion that the decrease in the liver and kidney concentrations of GSH and NPSH are involved in the mechanisms of the peroxidative action of Pb and EtOH alone and at co-exposure in these organs.

Delta-aminolevulinic acid dehydratase activity in the general population of southern Minas Gerais, Brazil.

Blood samples were collected from 113 subjects (56 males and 57 females) living in the district of Alfenas, in southern Minas Gerais state, Brazil, to establish reference values for delta-aminolevulinic acid dehydratase activity (ALA-D, EC 4.2.1.24). The state of health of the population was confirmed by hematological and biochemical parameters analyzed in blood and urine samples. ALA-D determination was performed according to the Berlin & Schaller spectrophotometric method. Distribution may be regarded as according to normal distribution and reference values obtained, in micromol x min(-1) x L(-1) erythrocytes, were: mean (+/- SD) = 54.5 (+/- 9.8); 95% confidence interval = 52.7-56.4; lower reference value (mean-2 SD) = 34.9. Mean ALA-D activity was higher than any other published elsewhere and the reference values established are useful as a baseline for evaluating ALA-D activity when monitoring persons exposed to lead. Age, gender, drinking, or smoking did not significantly alter (Student t-test, p < or = 0.05) the reference values for ALA-D.

Influence of the delta-aminolevulinic acid dehydratase (ALAD) polymorphism on biomarkers of lead exposure in Turkish storage battery manufacturing workers.

BACKGROUND: The relationship between delta-aminolevulinic acid dehydratase polymorphism (ALAD) and biomarkers of exposure was investigated in Turkish lead workers in this study. METHODS: Seventy two male lead battery manufacturing workers were selected for the study. Blood lead (BPb) and urinary lead (UPb) concentrations were determined by atomic absorption spectrometry. Erythrocyte ALAD activity and urinary 5-aminolevulinic acid (UALA) were measured spectrophotometrically. The polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) technique was used to determine the genotype of the ALAD gene. RESULTS: In total, 51 workers (70.8%) had the ALAD 1-1 genotype, whereas 21 workers (29.2%) had the ALAD 1-2 genotype. No significant relationships were found between the two genotypes and BPb, UPb, and ALAD activity. ALAD1 homozygotes showed significantly higher levels of UALA in comparison with those ALAD2 carriers. CONCLUSIONS: ALAD 1-1 individuals might be an increased risk compared to ALAD2 carriers to disturbance in heme biosynthetic pathway in high lead exposure.

The decrease in uroporphyrinogen decarboxylase activity induced by ethanol predisposes rats to the development of porphyria and accelerates xenobiotic-triggered porphyria, regardless of hepatic damage.

We evaluated the porphyrinogenic ability of ethanol (20% in drinking water) per se, its effect on the development of sporadic porphyria cutanea tarda induced by hexachlorobenzene in female Wistar rats (170-190 g, N = 8/group), and the relationship with hepatic damage. Twenty-five percent of the animals receiving ethanol increased up to 14-, 25-, and 4.5-fold the urinary excretion of delta-aminolevulinate, porphobilinogen, and porphyrins, respectively. Ethanol exacerbated the precursor excretions elicited by hexachlorobenzene. Hepatic porphyrin levels increased by hexachlorobenzene treatment, while this parameter only increased (up to 90-fold) in some of the animals that received ethanol alone. Ethanol reduced the activities of uroporphyrinogen decarboxylase, delta-aminolevulinate dehydrase and ferrochelatase. In the ethanol group, many of the animals showed a 30% decrease in uroporphyrinogen activity; in the ethanol + hexachlorobenzene group, this decrease occurred before the one caused by hexachlorobenzene alone. Ethanol exacerbated the effects of hexachlorobenzene, among others, on the rate-limiting enzyme delta-aminolevulinate synthetase. The plasma activities of enzymes that are markers of hepatic damage were similar in all drug-treated groups. These results indicate that 1) ethanol exacerbates the biochemical manifestation of sporadic hexachlorobenzene-induced porphyria cutanea tarda; 2) ethanol per se affects several enzymatic and excretion parameters of the heme metabolic pathway; 3) since not all the animals were affected to the same extent, ethanol seems to be a porphyrinogenic agent only when there is a predisposition, and 4) hepatic damage showed no correlation with the development of porphyria cutanea tarda.

A novel mutation of delta-aminolaevulinate dehydratase in a healthy child with 12% erythrocyte enzyme activity.

Cloning, expression and phenotype studies of the defective gene for delta-aminolaevulinate dehydratase (ALAD) in a family with an asymptomatic girl who had ALAD deficiency were carried out. The proband was identified by neonatal ALAD screening, and had erythrocyte ALAD activity at 12% of the normal control. She was heterozygous for ALAD deficiency, which was inherited from her father. Nucleotide sequence analysis of the cloned ALAD cDNA revealed C36 to G and T168 to C mutations on the same allele. The former mutation resulted in F12L substitution, whereas the latter was a silent mutation. All family members who had decreased ALAD activity had the same mutation. Expression of the mutant ALAD cDNA in Chinese hamster ovary cells produced an ALAD protein without significant enzyme activity. Additionally, the mutant ALAD cDNA which encodes F12L substitution produced an aberrant migration pattern in polyacrylamide gel electrophoresis under denaturing conditions. This finding probably reflects an abnormal folding of the F12L protein, since the mutation occurred in the alpha1 helix of the N-terminal arm of the enzyme, which is involved in the extensive quaternary interactions among the subunits. This is also the first report of ALAD gene mutation in an asymptomatic subject.

Urinary 5-aminolevulinic acid (ALA) adjusted by creatinine: a surrogate for plasma ALA?

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.

Lack of relationship between hair lead levels and some usual markers (blood lead levels, ZPP, urinary ALA-D) in occupationally exposed workers.

Blood and hair samples collected from 54 male workers occupationally exposed to lead were assayed for this metal by graphite furnace atomic absorption spectrophotometry. Blood ZPP and urinary ALA-D were also determined for most subjects tested. Blood and hair lead concentrations (PbB and PbH) ranged from 100 to 770 ng/ml (10 to 77 micrograms/100 ml) (mean +/- SD: 384.6 +/- 143.4 ng/ml (38.46 +/- 14.34 micrograms/100 ml)), and from 3 to 243 ng/mg (mean +/- SD: 102.4 +/- 72.6 ng/mg), respectively. No correlation was observed between the PbH and PbB values, nor between PbH and ZPP or ALA-D values. Neither hair coloration nor subjects' age were related to PbH levels. Results are discussed in the light of the existing literature.

Liver delta-aminolevulinate dehydratase activity in amitriptyline- or chlorpromazine-treated rats.

Amitriptyline (AMT) and chlorpromazine (CPZ) (0.5 mg per animal, i.p.) were injected into rats separately for 30 days and their effects on heme metabolism in liver were examined. Significant decreases in the delta-aminolevulinate dehydratase activity were observed following the administration of both drugs (mean value of AMT-group: 6.58 U/g tissue; and CPZ-group: 7.04 U/g tissue) in comparison to that of controls (11.71 U/g tissue); however total liver heme content was not altered. When 24-h urinary excretions of delta-aminolevulinate (ALA) and porphobilinogen (PBG) were measured on the last day of the experiment, a slight (AMT-group: 38.40 micrograms/day) to distinct (CPZ-group: 59.11 micrograms/day) increase of urinary ALA was observed, while PBG excretion tended to decline only moderately under CPZ (3.52 micrograms/day), but significantly in presence of AMT (2.16 micrograms/day). Mean values obtained from control group were 32.12 micrograms/day for ALA and 4.25 micrograms/day for PBG.

Deficiency of porphobilinogen synthase associated with acute crisis. Diagnosis of the first two cases in Chile by laboratory methods.

Erythrocyte porphobilinogen synthase deficiency was confirmed by the determination of its activity in blood and also by the high levels of both porphyrins and 5-aminolaevulinic acid in the urine of two siblings. They presented with a picture of porphyric attack characterized by abdominal colic pain, high blood pressure, tachycardia and severe constipation. The profile of both porphyrins and their precursors in urine and blood resembled lead poisoning. However, this was ruled out because both patients had normal blood levels of lead. Furthermore, porphobilinogen synthase activity did not normalize when it was determined in the presence of dithiothreitol or dithiothreitol plus zinc chloride. No other causes to account for a deficiency in porphobilinogen synthase activity were identified. The simultaneous occurrence of similar clinical and biochemical symptoms suggests that the same triggering factor was present. Because the activity of porphobilinogen synthase was less than 4% of normal values, it is possible that these patients were homozygotes with respect to this defect, which could explain the presence of clinical symptoms. We propose that this metabolic defect is not uncommon and it should be kept in mind when diagnosing of porphyrias or heavy metal intoxications.

The effects of ethanol, estrogen, and hexachlorobenzene on the activities of hepatic delta-aminolevulinate synthetase, delta-aminolevulinate dehydratase, and uroporphyrinogen decarboxylase in male rats.

To determine if clinically observed disorders in heme biosynthetic enzymes, known as sporadic porphyria cutanea tarda (PCT), could be reproduced in experimental animals, male Fischer rats were treated with ethanol, estrogen and hexachlorobenzene (HCB). A series of heme biosynthetic enzymes were assayed. In the rats given free access to 8% ethanol-drinking water for 15 weeks, delta-aminolevulinate (ALA) dehydratase was significantly reduced in erythrocytes. In the liver, ALA synthetase and uroporphyrinogen (UROgen) decarboxylase activities remained unchanged. In bone marrow cells, these activities did not change markedly. In the rats treated with estrogen (1 mg estrioltripropionate/rat/week, IM), no body weight gain was observed during the treatment for 15 weeks and urinary ALA excretion increased to 1.7 fold over normal level. In the liver, a significant increase was observed in the activity of ALA dehydratase, but other enzymes remained within the normal level. In bone marrow cells and erythrocytes, ALA dehydratase was also increased. ALA synthetase increased only in bone marrow cells to 2.1 times higher than the control level. In rats fed 0.3% HCB-diet for 8 weeks, urinary excretion of ALA, coproporphyrin and uroporphyrin increased to 2.4, 3.3 and 3.8 times higher than the controls, respectively. In the liver, an increase was observed in ALA synthetase, while a decrease was observed in ALA dehydratase and UROgen decarboxylase. In bone marrow cells and erythrocytes, ALA dehydratase was reduced and activities of other enzymes did not show any changes. These results indicate that alcohol, estrogen and HCB do not produce phenomena similar to those observed clinically in PCT.

Lead exposure patterns and parameters for monitoring lead absorption among workers in Singapore.

A study was carried out among 1414 workers in 14 industries in Singapore to determine the extent of their exposure to lead and their lead absorption. High-level exposure situations found included secondary lead smelting, followed by mixing of lead salt stabilizers and lead storage battery manufacture. Other exposure situations included those relating to lead welding, print shop, wire splicing, leaded paint manufacture, fire-fighting and soldering. Early cases of lead absorption could not be detected by clinical signs alone. Other parameters such as blood lead and delta-aminolaevulinic acid dehydratase had to be used. There was good correlation between a rise in blood lead and a fall in delta-aminolaevulinic acid dehydratase levels.

Increase in the amount of erythrocyte delta-aminolevulinic acid dehydratase in workers with moderate lead exposure.

The amount of ALA-D in human erythrocytes was determined directly by radioimmunoassay or calculated from the restored activity assayed in the presence of zinc and dithiothreitol, and a good correlation was observed between the RIA-based and the restored activity-based amounts. The RIA-based amount of ALA-D in the blood of 10 normal individuals (blood lead levels of 5.6 +/- 2.3 micrograms/ml: mean +/- SD) and 19 lead-exposed workers (blood lead levels of 41.2 +/- 10.2 micrograms/100 ml) was 54.1 +/- 11.8 microgram/ml blood and 92.3 +/- 20.6 micrograms/ml blood, respectively, indicating an apparent increase of the enzyme amount in lead-exposed workers. A significant increase in the amount of erythrocyte ALA-D calculated from the restored activity in lead-exposed workers was observed even in the low blood lead level of 10-20 microgram/100 ml, resulting in the range of blood lead level 20-40 microgram/100 ml. No significant difference was observed in hematocrit and hemoglobin content between lead-exposed and non-exposed groups. These observations suggested that the increase of erythrocyte ALA-D in lead exposure was not due to anemia, which might result in the increase of young erythrocytes in peripheral blood. This increase in the amount of ALA-D in human erythrocytes might be a result of the function to overcome the inhibition of the enzyme in bone marrow cells during lead exposure, and these findings may throw light on the danger to human health of low-level lead toxicity.