Evaluation of Metabolic Changes in Acute Intermittent Porphyria Patients by Targeted Metabolomics.

Acute intermittent porphyria (AIP) is an inherited rare hepatic disorder due to mutations within the hydroxymethylbilane gene. AIP patients with active disease overproduce aminolevulinic acid (ALA) and porphobilinogen (PBG) in the liver which are exported inducing severe neurological attacks. Different hepatic metabolic abnormalities have been described to be associated with this condition. The goal of this research was to explore the metabolome of symptomatic AIP patients by state-of-the art liquid chromatography-tandem mass spectrometry (LC-MS/MS). A case versus control study including 18 symptomatic AIP patients and 33 healthy controls was performed. Plasmatic levels of 51 metabolites and 16 ratios belonging to four metabolic pathways were determined. The results showed that the AIP patients presented significant changes in the two main areas of the metabolome under study: (a) the tryptophan/kynurenine pathway with an increase of tryptophan in plasma together with increase of the kynurenine/tryptophan ratio; and (b) changes in the tricarboxylic acid cycle (TCA) including increase of succinic acid and decrease of the fumaric acid/succinic acid ratio. We performed a complementary in vitro study adding ALA to hepatocytes media that showed some of the effects on the TCA cycle were parallel to those observed in vivo. Our study confirms in plasma previous results obtained in urine showing that AIP patients present a moderate increase of the kynurenine/tryptophan ratio possibly associated with inflammation. In addition, it also reports changes in the mitochondrial TCA cycle that, despite requiring further research, could be associated with an energy misbalance due to sustained overproduction of heme-precursors in the liver.

Role of delta-aminolevulinic acid in the symptoms of acute porphyria.

BACKGROUND: Attacks of neuropathic pain, usually abdominal, are characteristic of the acute porphyrias and accompanied by overproduction of heme-precursor molecules, specifically delta-aminolevulinic acid and porphobilinogen. The basis for the acute symptoms in these diseases has been speculative. METHODS: We review genetic acute porphyria, hereditary tyrosinemia, and an acquired condition, lead poisoning. All perturb heme synthesis and present with a similar pain syndrome. RESULTS: Although each of these conditions has characteristic urine biochemistry, all exhibit excess delta-aminolevulinic acid. Moreover, in all, treatment with hemin reduces delta-aminolevulinic acid and relieves symptoms. In contrast, use of recombinant porphobilinogen deaminase to knock down porphobilinogen in acute porphyria was ineffective. CONCLUSIONS: There is now convincing evidence that delta-aminolevulinic acid is the cause of pain in the acute porphyrias. The efficacy of hemin infusion is due mainly, if not entirely, to its inhibition of hepatic delta-aminolevulinic acid synthase-1, the enzyme that catalyzes delta-aminolevulinic acid formation. Delta-aminolevulinic acid synthase-1 is a rational target for additional therapies to control symptoms in acute porphyria.

Imported occupational lead poisoning: report of four cases.

BACKGROUND: In most industrialized countries, occupational lead poisoning has become increasingly rare, however this metal remains a serious health hazard in the rest of the world. REPORT OF CASES: We observedfour male patients (aged 35 / 54 years) who had suffered recurrent abdominal pain due to recent lead exposure (for 7 to 13 months) in two Chinese battery recycling plants. On their return to Italy, three of them presented normocytic, normochromic anaemia. The diagnosis was confirmed by high lead levels in the blood and urine, decreased erythrocyte delta-aminolevulinic acid dehydratase (ALA-D), raised erythrocyte zinc protoporphyrin (ZP), and elevated urinary excretion of b-aminolevulinic acid (ALA-U) and porphyrins. Chelation with EDTA resulted in increased urinary lead excretion, improvement of the clinical picture, decreased ZP, and progressive normalization of the other lead biomarkers (Pb-B, ALA-D, ALA-U, urinary porphyrins). CONCLUSIONS: Temporary work in developing countries may result in imported lead poisoning. Differential diagnosis of this unusual condition requires careful medical history collection and specific toxicological analysis. Preventive measures for workers going abroad are needed.

Oral supplementation of gossypin during lead exposure protects alteration in heme synthesis pathway and brain oxidative stress in rats.

OBJECTIVE: The objective was to study the efficacy of oral supplementation of gossypin, a flavonoid, during lead exposure in preventive alterations in the heme synthesis pathway, brain oxidation, and tissue lead uptake in rats. METHODS: Male rats were used for the experiment and were exposed to lead (0.5% in drinking water) or lead plus oral supplementation of gossypin (25 or 100mg/kg) for 3 wk to determine the preventive effect of gossypin against lead toxicity. Animals were sacrificed after 3 wk for various biochemical variables suggestive of oxidative stress and heme synthesis pathway in addition to the concentration of lead in the blood and brain. RESULTS: Exposure to lead produced significant inhibition in the activity of blood delta-aminolevulinic acid dehydratase accompanied by an increase in urinary delta-aminolevulinic acid and the levels of reactive oxygen species. There were significant alterations in the levels of glutathione, thiobarbituric acid-reactive substances, reactive oxygen species, and superoxide dismutase activity on lead exposure. Most of these alterations were significantly prevented by oral coadministration of gossypin, particularly at the dose of 100mg/kg. CONCLUSION: The antioxidant and moderate chelating properties of oral gossypin suggest a promising role in use either as a nutritional supplement during lead exposure or as a complementary chelating agent during chelation therapy.

Toxic effects of arsenic (III) on some hematopoietic and central nervous system variables in rats and guinea pigs.

OBJECTIVE: To evaluate the effects of arsenic (III) exposure on porphyrin metabolism and the central nervous system supplemented with data on the effect of hepatic and renal tissues of rats and guinea pigs. METHODS: Rats and guinea pigs were exposed to 10 or 25 ppm arsenic in drinking water for 16 weeks. RESULTS: Following chronic arsenic (III) exposure, delta-aminolevulinic acid dehydratase activity in blood showed a significant reduction as did the total cell counts (RBC and WBC) and reduced glutathione with increased urinary delta-aminolevulinic acid. Zinc protoporphyrin, a sensitive indicator of iron deficiency and impairment of heme biosynthesis, showed a significant increase in arsenic exposure. The hepatic delta-aminolevulinic acid dehydratase and delta-aminolevulinic acid synthetase activity increased in chronic arsenic (III) exposure in rats and guinea pigs. Significant changes in the steady-state level of three major neurotransmitters, dopamine, norepinephrine, and 5-hydroxytryptamine, and monoamine oxidase were observed following chronic arsenic (III) exposure. CONCLUSION: At low doses (10 and 25 ppm in drinking water), the effects of arsenic on hematopoietic indices and whole-brain neurotransmitter concentrations were more prominent in guinea pigs than in rats with some variability in the dose response.

[The problem of lead exposure in the Piedmont: from the workplace to the general environment]. / Il problema dell'esposizione a piombo in Piemonte: dal posto di lavoro all'ambiente di vita.

In the first part of this study, the most frequently professional lead exposure observed since 1984 at the Occupational Health Clinic of Turin University are summarized and compared with exposure observed in the past. The second part analyzes the present blood lead levels in the general (non-occupationally exposed) population observed in a screening campaign carried out in Piedmont in 1993-94 within the framework of the national programme by biology surveillance prescribed by Presidential Decree no. 496 of 1982. Results agree with literature data and show a reduction in blood lead levels markedly below the Italian legal limits. Nevertheless, lead pollution in non-exposed subjects still represents a major hygienistic problem, because a relevant number of children have blood lead concentrations above 10 micrograms/ 100 ml, which is considered a safe level for avoiding irreversible injury to nervous system.

Compared effects of high oral Mg supplements and of EDTA chelating agent on chronic lead intoxication in rabbits.

Our previous experiments showed that excessive oral Mg intake has beneficial effects in chronic Pb poisoning, inducing decrease of Pb body burden and its increased elimination via urine. The aim of this work was to investigate and to compare the effect of Mg on urinary Pb elimination with the effect of calcium-disodium edetate (CaNa2EDTA)--chelating agent currently used in therapy of chronic Pb intoxication. Besides, under the same experimental conditions, biochemical parameters protoporphyrin IX (ppIX), zinc protoporphyrin (Znpp) and delta-aminolevulinic acid (ALA) were determined. Experiments were carried out on rabbits previously intoxicated for 4 weeks with 20 mg Pb/kg b.w. per day. After the period of intoxication, one group of animals was given per os, for 28 days, 40 mg Mg/kg, the other one was i.v. treated for 7 days with 15 mg CaNa2EDTA/kg (therapeutic doses), while the third one (rabbits without therapy) served as a control. During the period of detoxication, lead was determined in blood and urine samples, and ppIX and Znpp were determined in blood and ALA in urine. Results suggest that oral treatment with magnesium, although inducing later Pb elimination than EDTA, has even better effect on investigated biochemical parameters than chelating therapy.

Therapeutic efficacy of combined meso 2,3-dimercaptosuccinic acid and calcium disodium edetate treatment during acute lead intoxication in rats.

1. The therapeutic ability of Ca disodium EDTA and meso 2,3-dimercaptosuccinic acid (DMSA) was studied, both individually and when given in combination, in reducing lead concentration in blood and other soft tissues, and in restoring lead induced altered biochemical variables in acute lead intoxicated rats. 2. Combined treatment with the above two chelating agents was more beneficial in reducing blood and hepatic lead compared to treatment with these drugs alone. Kidney lead concentration however, remained high following combined treatment, indicating the possibility of extra renal burden following treatment. 3. Lead sensitive biochemical variables also responded more favourably to combined treatment than treatment with these drugs alone. However, clinical biochemical indices indicate caution regarding the use of this new treatment regimen, and further investigation is required.

[Evaluation of initial results of treatment of lead poisoning with EDTA]. / Valutazione dei primi risultati della terapia del saturnismo con EDTA.

The results of EDTA therapy were studied in 37 workers of a battery factory consisting of males with varying degrees of occupational lead poisoning (low exposure: 10 subjects, blood lead levels (PbB) lower than 400 micrograms/l with slight alterations in heme biosynthesis; beyond limit of effect: 5 subjects, PbB > 400 micrograms/l; slight intoxication: 19 subjects, with marked alterations in heme synthesis and preclinical signs of intoxication; average degree of intoxication: 3 subjects with clinical signs of intoxication. Clinical symptoms and the following parameters were investigated: blood lead (PbB), delta-aminolevulinic acid dehydratase in erythrocytes (ALA-D), zinc protoporphyrin (PP) in erythrocytes and delta-aminolevulinic acid (ALA) in 24-hour urine before and after EDTA chelating therapy. Simultaneous measurement of ALA-D and PP showed high diagnostic sensitivity in detecting lead poisoning in occupationally exposed subjects. In view of the high interindividual variability of the results, these indices did not, however, permit a useful differentiation to be made of the different degrees of intoxication at individual level, even though a good correlation was observed between PbB and porphyrin metabolism indices. From the alterations observed in ALA-D and PP values it was not possible to establish an association between degree of alteration and types of clinical symptoms in the different intoxication studies. At the end of EDTA treatment, a clinical improvement was observed in all cases studied but only in 5 cases was a reduction in PbB observed, to levels below 1.20 mol/l, which is accepted as a permissible limit for the general population; in 17 cases PbB remained at levels above the critical value for occupational lead poisoning (400 micrograms/l), although there was a decrease after treatment. The improvement observed in the indices of porphyrin metabolism at the end of treatment was only slight: significant variations were measured only for PbB. After treatment no association was observed between ALA-D and PP variations in erythrocytes and improvement in clinical symptoms; measurement of these indices therefore seems to be of little use in assessing the efficacy of the treatment. In spite of its limited diagnostic sensitivity during intoxication, measurement of ALA in urine could be useful to assess the efficacy of chelating therapy in subjects in whom the values are initially altered.

Influence of methionine and zinc supplementation during chelation of lead in rats.

The influence of methionine and Zn supplementation on the therapeutic efficacy of calcium disodium ethylenediamine tetraacetic acid (CaNa2 EDTA) and 2,3-dimercaptopropane 1-sulphonate (DMPS) in lead intoxication was investigated in rats. The combined treatment with CaNa2 EDTA and methionine +Zn or DMPS and methionine +Zn was more effective than the respective chelator alone in decreasing the blood and tissue burden of Pb and increasing urinary excretion of Pb, with the former combination being more effective than the later. However, simultaneous supplementation of the amino acid and essential trace element did not improve upon the efficacy of the chelator in reversing lead-induced biochemical alterations.

Biochemical diagnosis of a fatal case of Günther's disease in a newborn with hydrops foetalis.

The birth of a male baby was induced at 32 weeks. In utero, the child presented, inter alia, signs of hydrops, hepatosplenomegaly and anaemia. Two in utero transfusions for correction of the anaemia were performed at 28 and 29 weeks, respectively. The baby rapidly presented respiratory distress with mixed acidosis. Three hours after birth, pink urine was excreted. Signs of icterus necessitated phototherapy, after which photosensitivity occurred. Erythrocytes were fluorescent under long-wavelength UV light. The baby died 24 hours after birth, displaying severe acidosis, a diffuse haemorrhagic syndrome, and repeated brady-cardia which did not respond to isoprenaline. The analysis of porphyrins in urine, blood and faeces of the baby gave the following results: 1) uroporphyrin (I and III isomeric series) was increased in urine and faeces, with traces in erythrocytes and plasma; 2) heptacarboxyporphyrin I was found mainly in urine and much less in erythrocytes, plasma and faeces; 3) coproporphyrin I was increased in urine, erythrocytes, plasma and faeces, and 4) 5-aminolaevulinic acid and porphobilinogen in urine and plasma were within the reference ranges. Determination of the enzymes of haem biosynthesis in erythrocytes and lymphocytes showed that both parents possessed only 50% of the normal activity of cosynthase. A previously described point mutation in codon 73 was observed in one parent. Fatal cases of neonatal Günther's disease are extremely rare and such an observation, according to our knowledge, is probably one of the first described.

Controlled study of meso-2,3-dimercaptosuccinic acid for the management of childhood lead intoxication.

We examined the efficacy and safety of meso-2,3-dimercaptosuccinic acid (DMSA) in children with markedly elevated blood lead (BPb) concentrations. Among 19 children with BPb concentrations of 50 to 69 micrograms/dl (2.41 to 3.33 mumol/L) who received a 5-day inpatient oral course of DMSA (1050 mg/m2 per day), the mean BPb concentration decreased by 61%; in four who received calcium disodium ethylenediaminetetraacetic acid (CaNa2EDTA) (1000 mg/m2 per day intravenously), it decreased by 45% (p less than 0.0007). Urinary lead excretion was comparable in both groups. Treatment with DMSA was more effective than treatment with CaNa2EDTA in restoring metabolic activity to the heme pathway and was well tolerated even among nine patients who received concomitant iron supplementation and two who had homozygous deficiency of glucose-6-phosphate dehydrogenase. On discharge, these 19 children received either no chelation therapy or DMSA, 350 or 700 mg/m2 per day for 14 days on an outpatient basis. After 14 days the mean BPb values for the no-chelation, low-DMSA, and high-DMSA groups were 73%, 66%, and 50% of the pretreatment values, respectively. We conclude that a 5-day oral course of DMSA is effective in the treatment of children with severe lead poisoning. In addition, on an outpatient basis the administration of DMSA, 700 mg/m2 per day, is capable of delaying the typical rebound in BPb values and should ultimately reduce the need for repeated hospitalizations.

Influence of simultaneous supplementation of zinc and copper during chelation of lead in rats.

The influence of zinc and copper supplementation during chelation therapy to reduce zinc and copper imbalance and promote lead elimination from the body, was investigated in rats poisoned with lead. The simultaneous supplementation of zinc and copper increased urinary lead excretion by calcium disodium ethylenediamine tetraacetic acid (CaNa2EDTA) compared to treatment with CaNa2EDTA alone. Combination therapy was effective in potentiating the depletion of blood and renal lead by CaNa2EDTA and meso 2,3-dimercapto succinic acid (DMSA). Combination therapy was also more effective in reducing hepatic lead by CaNa2EDTA and blood lead by 2,3-dimercapto propane sulphonate (DMPS). Zinc and copper supplementation produced a more effective reversal of inhibited blood delta-aminolevulinic acid dehydratase (ALAD) activity, urinary delta-aminolevulinic acid excretion and depleted body zinc and copper status.

Beneficial effects of zinc supplementation during chelation treatment of lead intoxication in rats.

The ability of zinc to enhance the efficacy of commonly used chelating drugs in lead intoxication and to reduce the resulting zinc imbalance, was investigated in rats. The simultaneous zinc supplementation increased urinary lead elimination by calcium disodium ethylenediaminetetraacetate (Ca disodium EDTA) and 2,3 dimercaptosuccinic acid (DMSA). Combination therapy was also effective in potentiating the depletion of blood, hepatic and renal lead by calcium disodium EDTA and D-penicillamine (DPA), renal lead by DMSA and reversal of inhibited blood delta-aminolevulinic acid dehydratase (ALAD) activity by calcium disodium EDTA and DPA. The body zinc status was also maintained as reflected by urinary, blood and tissue levels of zinc.

Preventive and therapeutic role of vitamin E in chronic plumbism.

The ability of vitamin E to prevent or treat experimental lead intoxication was investigated in rats. Lead ingestion (10 mg/kg, lead as lead acetate, orally for 6 weeks) significantly inhibited the activity of blood delta-aminolevulinic acid dehydratase (ALAD), reduced the brain dopamine (DA) contents, enhanced the blood zinc protoporphyrin, and enhanced the urinary excretion of delta-aminolevulinic acid (ALA). Lead exposure also elevated brain norepinephrine, homovanillic acid, and 5-hydroxyindole acetic acid (5-HIAA) levels and concentration of lead in blood and tissue. Simultaneous supplementation of vitamin E along with lead significantly reduced the inhibition of blood ALAD activity, brain DA and 5-HIAA levels, and elevation of urinary ALA excretion. Blood and liver lead concentrations were also significantly reduced by simultaneous supplementation with vitamin E. Postlead exposure treatment with vitamin E was ineffective in reducing the lead-induced effects, except that the inhibition of blood ALAD activity was slightly reduced. The present results suggest that vitamin E given simultaneously with lead is effective in reducing the severity of lead intoxication.

Influence of methionine supplementation in chelation of lead in rats.

The influence of methionine supplementation on the efficacy of common antidotes to lead poisoning, calcium disodium ethylenediaminetetraacetate (CaNa2EDTA) and D-penicillamine (DPA), was investigated in rats. The animals were given lead acetate (0.1% in drinking water) for 12 weeks and thereafter treated with CaNa2EDTA. DPA (0.3 mmol/kg, intraperitoneally), DL-methionine (1.34 mmol/kg, intragastrically), or the combination of a chelating agent and methionine for 3 days. While chelating agents enhanced the urinary excretion of Pb, methionine increased the fecal excretion of Pb significantly. Treatment with the combination of a chelating agent and methionine did not potentiate the effect of each antidote. However, methionine supplementation increased the efficacy of both chelating agents in reducing the hepatic and renal Pb burden but not the blood Pb level. The Pb-induced inhibition of blood delta-aminolevulinic acid dehydratase activity and the increase in urinary excretion of delta-aminolevulinic acid were reversed to a certain extent by CaNa2EDTA, DPA, and methionine but the combination did not improve their individual performances. The beneficial effects of methionine may be attributed to its ability to increase the bioavailability of glutathione (GSH), useful in chelating Pb and counter-acting the toxic effects, as evidenced by restoration of the Pb-induced decrease in hepatic GSH level by treatment with methionine. Methionine may be useful as a supportive therapy in chelation of Pb.

Influence of dietary protein deficiency on lead-copper interaction in rats.

The influence of dietary protein deficiency on the effects of exposure to lead or its combination with copper was investigated in rats. The administration of lead (100 ppm in drinking water) inhibited the activity of blood delta-aminolevulinic acid dehydratase; decreased hemoglobin, brain dopamine, and 5-hydroxytryptamine; and increased urinary excretion of delta-aminolevulinic acid, blood zinc protoporphyrin, and tissue accumulation of lead more markedly in animals fed a protein-deficient diet (10% casein) than in those fed a normal diet (21% casein). The simultaneous supplementation of copper (100 ppm in diet) reduced some of the lead-induced alterations and body uptake of lead more efficiently in animals fed a normal diet than in those fed a protein-deficient diet, which shows that the beneficial effects of copper in lead toxicity are adversely affected by low dietary protein.

Thiamine and zinc in prevention or therapy of lead intoxication.

Thiamine, zinc or their combination given through gastric gavage were investigated for their ability to prevent or treat experimental lead toxicity in rats. Simultaneous dietary supplementation with thiamine plus zinc was found to be the most effective way of reducing the lead-induced inhibition of delta-aminolevulinic acid dehydratase activity in blood, urinary, excretion of delta-aminolevulinic acid and accumulation of lead in blood, liver and kidney. Prevention was more effective than post-lead exposure treatment which may be due mainly to the decrease in the absorption of lead in the gastro-intestinal tract in the presence of thiamine and/or zinc.

Chelation in metal intoxication. XXIV: Influence of various components of vitamin B complex on the therapeutic efficacy of disodium calcium versenate in lead intoxication.

The supplementation of vitamin-B complex reduces lead intoxication. With a view to identify the components of vitamin-B complex responsible for such protection, riboflavin, calcium pentothenate, pyridoxine, nicotinamide, folic acid and cyanocobalamine were investigated for their ability, and their influence on the efficacy of disodium calcium versenate (Na2CaEDTA), to enhance the urinary excretion of lead, mobilize tissue lead and restore lead induced biological alterations in lead intoxicated rats. Folic acid and pyridoxine besides thiamine may be the responsible factors in prophylaxis of lead poisoning by vitamin B complex or in enhancing the antidotal properties of Na2CaEDTA.