5-Aminolevulinate dehydratase porphyria: Update on hepatic 5-aminolevulinic acid synthase induction and long-term response to hemin.

BACKGROUND: 5-Aminolevulinic acid dehydratase (ALAD) porphyria (ADP) is an ultrarare autosomal recessive disease, with only eight documented cases, all of whom were males. Although classified as an acute hepatic porphyria (AHP), induction of the rate limiting hepatic enzyme 5-aminolevulinic acid synthase-1 (ALAS1) has not been demonstrated, and the marrow may also contribute excess 5-aminolevulinic acid (ALA). Two patients have died and reported follow up for the others is limited, so the natural history of this disease is poorly understood and treatment experience limited. METHODS: We report new molecular findings and update the clinical course and treatment of the sixth reported ADP patient, now 31 years old and the only known case in the Americas, and review published data regarding genotype-phenotype correlation and treatment. RESULTS: Circulating hepatic 5-aminolevulinic acid synthase-1 (ALAS1) mRNA was elevated in this case, as in other AHPs. Gain of function mutation of erythroid specific ALAS2 - an X-linked modifying gene in some other porphyrias - was not found. Seven reported ADP cases had compound heterozygous ALAD mutations resulting in very low residual ALAD activity and symptoms early in life or adolescence. One adult with a germline ALAD mutant allele developed ADP in association with a clonal myeloproliferative disorder, polycythemia vera. CONCLUSIONS: Elevation in circulating hepatic ALAS1 and response to treatment with hemin indicate that the liver is an important source of excess ALA in ADP, although the marrow may also contribute. Intravenous hemin was effective in most reported cases for treatment and prevention of acute attacks of neurological symptoms.

Detection of Stimulated Erythropoiesis by the RNA-Based 5'-Aminolevulinate Synthase 2 Biomarker in Dried Blood Spot Samples.

BACKGROUND: Despite implementation of the Athlete Biological Passport 10 years ago, blood doping remains difficult to detect. Thus, there is a need for new biomarkers to increase the sensitivity of the adaptive model. Transcriptomic biomarkers originating from immature reticulocytes may be reliable indicators of blood manipulations. Furthermore, the use of dried blood spots (DBSs) for antidoping purposes constitutes a complementary approach to venous blood collection. Here, we developed a method of quantifying the RNA-based 5'-aminolevulinate synthase 2 (ALAS2) biomarker in DBS. MATERIALS: The technical, interindividual, and intraindividual variabilities of the method, and the effects of storage conditions on the production levels of ALAS2 RNA were assessed. The method was used to monitor erythropoiesis stimulated endogenously (blood withdrawal) or exogenously (injection of recombinant human erythropoietin). RESULTS: When measured over a 7-week period, the intra- and interindividual variabilities of ALAS2 expression in DBS were 12.5%-42.4% and 49%, respectively. Following withdrawal of 1 unit of blood, the ALAS2 RNA in DBS increased significantly for up to 15 days. Variations in the expression level of this biomarker in DBS samples were more marked than those of the conventional hematological parameters, reticulocyte percentage and immature reticulocyte fraction. After exogenous stimulation of erythropoiesis via recombinant human erythropoietin injection, ALAS2 expression in DBS increased by a mean 8-fold. CONCLUSIONS: Monitoring of transcriptomic biomarkers in DBS could complement the measurement of hematological parameters in the Athlete Biological Passport and aid the detection of blood manipulations.

Recurrent attacks of acute hepatic porphyria: major role of the chronic inflammatory response in the liver.

BACKGROUND: Acute intermittent porphyria (AIP) is an inherited disorder of haem metabolism characterized by life-threatening acute neurovisceral attacks due to the induction of hepatic δ-aminolevulinic acid synthase 1 (ALAS1) associated with hydroxymethylbilane synthase (HMBS) deficiency. So far, the treatment of choice is hemin which represses ALAS1. The main issue in the medical care of AIP patients is the occurrence of debilitating recurrent attacks. OBJECTIVE: The aim of this study was to determine whether chronic hemin administration contributes to the recurrence of acute attacks. METHODS: A follow-up study was conducted between 1974 and 2015 and included 602 French AIP patients, of whom 46 had recurrent AIP. Moreover, we studied the hepatic transcriptome, serum proteome, liver macrophage polarization and oxidative and inflammatory profiles of Hmbs-/- mice chronically treated by hemin and extended the investigations to five explanted livers from recurrent AIP patients. RESULTS: The introduction of hemin into the pharmacopeia has coincided with a 4.4-fold increase in the prevalence of chronic patients. Moreover, we showed that both in animal model and in human liver, frequent hemin infusions generate a chronic inflammatory hepatic disease which induces HO1 remotely to hemin treatment and maintains a high ALAS1 level responsible for recurrence. CONCLUSION: Altogether, this study has important impacts on AIP care underlying that hemin needs to be restricted to severe neurovisceral crisis and suggests that alternative treatment targeting the liver such as ALAS1 and HO1 inhibitors, and anti-inflammatory therapies should be considered in patients with recurrent AIP.

Evaluation of the inclusion of circular RNAs in mRNA profiling in forensic body fluid identification.

The use of messenger RNA (mRNA) profiling is considered a promising method in the identification of forensically relevant body fluids which can provide crucial information for reconstructing a potential crime. However, casework samples are usually of limited quantity or have been subjected to degradation, which requires improvement of body fluid identification. Circular RNAs (circRNAs), a class of products from the backsplicing of pre-mRNAs, are shown to have high abundance, remarkable stability, and cell type-specific expression in human cells. In this study, we investigated whether the inclusion of circRNAs in mRNA profiling improve the detection of biomarkers including δ-aminolevulinate synthase 2 (ALAS2) and matrix metallopeptidase 7 (MMP7) in body fluid identification. The major circRNAs of ALAS2 and MMP7 were first identified and primer sets for the simultaneous detection of linear and circular transcripts were developed. The inclusion of circRNAs in mRNA profiling showed improved detection sensitivity and stability of biomarkers revealed by using serial dilutions, mixed samples, and menstrual bloodstains as well as degraded and aged samples. Therefore, the inclusion of circRNAs in mRNA profiling should facilitate the detection of mRNA markers in forensic body fluid identification.

Isoniazid inhibits human erythroid 5-aminolevulinate synthase: Molecular mechanism and tolerance study with four X-linked protoporphyria patients.

Mutations in the C-terminus of human erythroid 5-aminolevulinate synthase (hALAS2), a pyridoxal 5'-phosphate (PLP)-dependent enzyme, are associated with two different blood disorders, X-linked sideroblastic anemia (XLSA) and X-linked protoporphyria (XLPP). XLSA-causing mutations yield hALAS2 variants with decreased activity, while XLPP-causing mutations result in a gain-of-function of hALAS2. There are no specific treatments for XLPP. Isonicotinic acid hydrazide (isoniazid, INH), an antituberculosis agent, can cause sideroblastic anemia as a side-effect, by limiting PLP availability to hALAS2, via inhibition of pyridoxal kinase or reaction with pyridoxal to form pyridoxal isonicotinoyl hydrazone. We hypothesized that INH also binds and directly inhibits hALAS2. Using fluorescence-activated cell sorting and confocal fluorescence microscopy, we demonstrate that INH reduces protoporphyrin IX levels in HeLa cells expressing either wild-type hALAS2 or XLPP variants. In addition, PLP and pyridoxamine 5'-phosphate (PMP) reversed the cellular inhibition of hALAS2 activity by INH. Steady-state kinetic analyses with purified hALAS2 indicated that INH directly inhibits the enzyme, noncompetitively or uncompetitively, with an apparent Ki of 1.2µM. Circular dichroism spectroscopy revealed that INH triggered tertiary structural changes in hALAS2 that altered the microenvironment of the PLP cofactor and hampered the association of PLP with apo-hALAS2. Treatment of four XLPP patients with INH (5mg·kg-1·day-1) over a six-month period was well tolerated but without statistically significant modification of PPIX levels. These results, taken together, permit us to further an INH inhibition kinetic mechanism for ALAS, which suggests the possible use of INH-derived drugs in treating patients with XLPP and potentially other protoporphyrin-accumulating porphyrias.

Circadian rhythms in acute intermittent porphyria--a pilot study.

BACKGROUND: Acute intermittent porphyria (AIP) is an inherited disorder of haem synthesis wherein a partial deficiency of porphobilinogen (PBG) deaminase (PBGD) with other factors may give rise to biochemical and clinical manifestations of disease. The biochemical hallmarks of active AIP are relative hepatic haem deficiency and uncontrolled up-regulation of hepatic 5-aminolevulinic acid (ALA) synthase-1 (ALAS1) with over-production of ALA and PBG. The treatment of choice is intravenous haem, which restores the deficient regulatory haem pool of the liver and represses ALAS1. Recently, haem has been shown to influence circadian rhythms by controlling their negative feedback loops. We evaluated whether subjects with AIP exhibited an altered circadian profile. MATERIALS AND METHODS: Over a 21-h period, we measured levels of serum cortisol, melatonin, ALA, PBG and mRNA levels (in peripheral blood mononuclear cells) of selected clock-controlled genes and genes involved in haem synthesis in 10 Caucasian (European-American) women who were either postmenopausal or had been receiving female hormone therapy, six of whom have AIP and four do not and are considered controls. RESULTS: Four AIP subjects with biochemical activity exhibited higher levels of PBG and lower levels and dampened oscillation of serum cortisol, and a trend for lower levels of serum melatonin, than controls or AIP subjects without biochemical activity. Levels of clock-controlled gene mRNAs showed significant increases over baseline in all subjects at 5 a.m. and 11 p.m., whereas mRNA levels of ALAS1, ALAS2 and PBGD were increased only at 11 p.m. in subjects with active AIP. CONCLUSIONS: This pilot study provides evidence for disturbances of circadian markers in women with active AIP that may trigger or sustain some common clinical features of AIP.

Long-term administration of massive doses of Sn-protoporphyrin in anemic mutant mice (sphha/sphha).

The effects of long-term administration of very large doses of Sn-protoporphyrin on hematological indices, histological changes, plasma bilirubin levels, tissue heme oxygenase activity, and activities of heme biosynthetic enzymes, were examined in genetically anemic mutant mice with hemolytic anemia (sphha/sphha). Long-term weekly treatment with Sn-protoporphyrin (100 mumol/kg body weight for 32 wk) did not alter hematological indices, histological findings, or enzyme activities related to heme biosynthesis, even though it resulted in sustained decreases in microsomal heme oxygenase activity in the liver, kidney, and spleen, and a prolonged decrease in plasma bilirubin concentration. Inhibition of heme oxygenase did not alter the level of cytochrome P-450 in the liver and the kidney. The results indicate that long-term treatment with massive doses of Sn-protoporphyrin suppresses bilirubin formation but does not produce significant histopathological changes or appreciably interfere with heme synthesis, in this strain of genetically anemic mice. These findings provide further support for the idea that suppression of heme degradation to bile pigment by the inhibition of heme oxygenase may prove useful to the prevention of severe hyperbilirubinemia in humans.

Identification of aged bloodstains through mRNA profiling: Experiments results on selected markers of 30- and 50-year-old samples.

Remarkable progress has been made in recent years on the research of body fluid identification through messenger RNA(mRNA) profiling. In order to examine the viability of mRNA profiling as a method to identify aged bloodstains, this study tested two groups of bloodstain samples, dated 30 years and 50 years back respectively, on seven blood specific markers, i.e. HBB, HBA, GYPA, CD93, ALAS2, SPTB (91bp and 247bp primers), and PBGD. Test results indicate that HBA and HBB are the most stable markers in aged bloodstains, returning positive results in over 80% of the 50-year-old samples and over 90% of the 30-year-old samples. This finding proves mRNA profiling an effective way of identifying aged bloodstains.

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

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

Myopathy, lactic acidosis, and sideroblastic anemia: a new syndrome.

We describe 2 sibs (brother and sister) with myopathy, sideroblastic anemia, lactic acidosis, mental retardation, microcephaly, high palate, high philtrum, distichiasis, and micrognathia. Very low levels of cytochromes a, b, and c were detected in the patients' muscle mitochondria. Deposition of iron within the mitochondria of bone marrow erythroblasts was observed on electron microscopy. Irregular and enlarged mitochondria with paracrystalline inclusions were also seen on electron microscopy of the patients' muscle specimen. Examination of DNA from the affected sibs showed no deletions in the mitochondrial DNA nor the mutations identified in the syndromes of mitochondrial myopathy, encephalopathy, lactic acidosis, and strokelike episodes (MELAS) or myoclonus, and epilepsy associated with rugged-red fibers (MERRF). Since the parents were first cousins and 2 of 6 sibs (male and female) were affected, we suggest that the syndrome expressed by our patients represents a previously unknown autosomal recessive disorder that includes mitochondrial myopathy, lactic acidosis, and sideroblastic anemia.

Delta-aminolaevulinic acid metabolism in normal and lead-exposed humans.

The activity of the haem biosynthetic enzymes delta-aminolaevulinic acid synthetase (ALA.S) and delta-aminolaevulinic acid dehydratase (ALA.D) were measured in the peripheral blood of a group of lead workers and control subjects. The haem precursor delta-aminolaevulinic acid (ALA) was measured in blood and urine, whilst lead levels were measured in whole blood. The inter-relationships between all these parameters were examined and quantified. The results demonstrate that above a blood lead concentration of 2 mumole/l and below an erythrocyte ALA.D activity of 18 nmole ALA utlized/min/ml red blood cells (R.B.C.), Haem synthesis is depressed to such an extent that the activity of leucocyte ALA.S, the rate-limiting enzyme of haem biosynthesis, is increased by negative feedback.

STZ-induced diabetes in mice and heme pathway enzymes. Effect of allylisopropylacetamide and alpha-tocopherol.

A frequent coexistence of diabetes and porphyria disease has been reported. Under normal conditions, porphyrin biosynthesis is well regulated to only form the amount of heme required for the synthesis of the various hemoproteins. The activity of some heme enzymes and rhodanese in streptozotocin (STZ) induced diabetic mice and in allylisopropylacetamide (AIA) induced experimental acute porphyria mice has been examined. The role of alpha-tocopherol (alpha-T), reported to prevent protein glycation in vitro, has also been investigated. AIA induced hepatic delta-aminolevulinic acid synthetase (ALA-S) activity in control animals but was ineffective in the diabetic group. alpha-Tocopherol did not modify ALA-S activity in either group. delta-Aminolevulinic acid dehydratase (ALA-D) and deaminase activities were significantly diminished both in liver and blood of diabetic animals. alpha-Tocopherol prevented inhibition of ALA-D, deaminase and blood rhodanese activities in diabetic animals but alpha-tocopherol by itself did not affect the basal levels of the enzymes studied. The potential use of alpha-tocopherol to prevent late complications of diabetes, including the onset of a porphyria like syndrome is considered.

Occupational lead exposure in Finland. V. Shipyards and shipbreaking.

Three Finnish shipyards and two shipbreaking enterprises were studied for lead exposure. Blood lead (Pb-B) concentration was measured for 568 workers in the shipyards and 13 workers in the shipbreaking enterprises. Erythrocyte delta-aminolevulinic acid dehydratase (ALAD) activity was determined for 139 workers in one shipyard and 7 workers in one shipbreaking enterprise. Hemoglobin level was determined for 545 workers in the three shipyards and 7 workers in one shipbreaking enterprise. Lead exposure in the shipyards was relatively low. No Pb-B value exceeded 70mug/100 ml. The most exposed occupations were welders, plumbers, painters, repairmen, and sheet metal workers. The mean Pb-B did not exceed 40 mug/100 ml in any of these groups. In both shipbreaking enterprises one Pb-B value exceeded 70 mug/100 ml, the mean Pb-B values of all the workers in the two enterprises being 51 and 46 mug/100 ml. respectively. ALAD values corresponded well with the respective Pb-B values. All the hemoglobin mean values were normal, and there were no statistically significant differences between the hemoglobin values of different groups.

Disturbed iron metabolism among workers exposed to organic sulfides in a pulp plant.

The aim of this study was to investigate a possible relationship between exposure to sulfides and disturbances of the synthesis of heme and the erythrocytes. Eighteen workers exposed to sulfides at a pulp and paper plant were examined and compared with individually matched referents from a thermomechanical pulp plant without such exposure. The exposure levels of methylmercaptan, dimethylsulfide, and dimethyldisulfide were low. However, five subjects were exposed to high levels of short duration, and their data were analyzed separately. The activity of the enzymes delta-aminolevulinic acid synthase and heme synthase in reticulocytes, characteristics of the erythrocytes, and the iron status were analyzed. A minor decrease, not statistically significant, was observed for the enzymes among the five highly exposed subjects. However, the concentrations of iron and transferrin were elevated and the concentration of ferritin was low in comparison to the corresponding levels of the referents. This combination will not occur spontaneously. A previous study indicated that sulfides may inhibit heme synthesis, and the present study suggests that they may also disturb iron metabolism.

Tests available for assessing recent exposure to inorganic lead compounds and their use for screening purposes.

This paper indicates the tests available for measurement of inorganic lead uptake by the body. The tests are listed and then considered individually. In the light of recent great interest in lead as a health hazard, the best methods for screening are considered: this includes biological and biochemical tests. It is emphasized that the U.K. favours blood lead estimation, the EEC countries other than Britain use blood lead but are considering more widespread use of delta-amino laevulinic acid dehydratase estimation in blood. Some authorities in the United States of America, on the other hand, favour a free erythrocyte protoporphyrin test. The advantages and disadvantages are brought out and some tests are mentioned only in order to exclude them as unsuitable. Suitability of tests for industrial checks and general screening of the population are differentiated. The conclusion is that blood lead still provides the best overall check of lead uptake.

Decreased leukocyte ferrochelatase activity in erythropoietic protoporphyria.

We studied the porphyrin metabolism of a 7-year-old Japanese boy with erythropoietic protoporphyria (EPP) and his family members. Leukocyte ferrochelatase activity was markedly decreased in this patient, being approximately 12% of the mean value of normal controls (4 aged-matched healthy boys). In contrast, leukocyte delta-aminolevulinic acid (ALA) synthase activity was normal. The free protoporphyrin content of erythrocytes was greatly increased (4.3 mg/100 ml RBC), while erythrocyte ALA dehydratase and porphobilinogen (PBG) deaminase activities were 1.7- and 2.2-fold of respective control values. A survey of his family revealed that 12 of 19 members probably had manifest EPP or were EPP carriers. These results suggest that, in EPP, there might be an inherited impairement of ferrochelatase activity which gives rise to an elevation of erythroblast ALA dehydratase and PBG deaminase activities to compensate for a resultant decrease in heme production.