Regulation and tissue-specific expression of δ-aminolevulinic acid synthases in non-syndromic sideroblastic anemias and porphyrias.

Recently, new genes and molecular mechanisms have been identified in patients with porphyrias and sideroblastic anemias (SA). They all modulate either directly or indirectly the δ-aminolevulinic acid synthase (ALAS) activity. ALAS, is encoded by two genes: the erythroid-specific (ALAS2), and the ubiquitously expressed (ALAS1). In the liver, ALAS1 controls the rate-limiting step in the production of heme and hemoproteins that are rapidly turned over in response to metabolic needs. Several heme regulatory targets have been identified as regulators of ALAS1 activity: 1) transcriptional repression via a heme-responsive element, 2) post-transcriptional destabilization of ALAS1 mRNA, 3) post-translational inhibition via a heme regulatory motif, 4) direct inhibition of the activity of the enzyme and 5) breakdown of ALAS1 protein via heme-mediated induction of the protease Lon peptidase 1. In erythroid cells, ALAS2 is a gatekeeper of production of very large amounts of heme necessary for hemoglobin synthesis. The rate of ALAS2 synthesis is transiently increased during the period of active heme synthesis. Its gene expression is determined by trans-activation of nuclear factor GATA1, CACC box and NF-E2-binding sites in the promoter areas. ALAS2 mRNA translation is also regulated by the iron-responsive element (IRE)/iron regulatory proteins (IRP) binding system. In patients, ALAS enzyme activity is affected in most of the mutations causing non-syndromic SA and in several porphyrias. Decreased ALAS2 activity results either directly from loss-of-function ALAS2 mutations as seen in X-linked sideroblastic anemia (XLSA) or from defect in the availability of one of its two mitochondrial substrates: glycine in SLC25A38 mutations and succinyl CoA in GLRX5 mutations. Moreover, ALAS2 gain of function mutations is responsible for X-linked protoporphyria and increased ALAS1 activity lead to acute attacks of hepatic porphyrias. A missense dominant mutation in the Walker A motif of the ATPase binding site in the gene coding for the mitochondrial protein unfoldase CLPX also contributes to increasing ALAS and subsequently protoporphyrinemia. Altogether, these recent data on human ALAS have informed our understanding of porphyrias and sideroblastic anemias pathogeneses and may contribute to new therapeutic strategies.

Clinical, Biochemical and Molecular Characteristics of the Main Types of Porphyria.

Porphyrias are diverse disorders that arise from various inherited enzyme defects in the heme biosynthesis pathway, except for porphyria cutanea tarda (PCT), in which the enzyme deficiency in most cases is acquired. The biosynthetic blocks resulting from the defective enzymes are largely expressed either in the liver or bone marrow, the sites where the majority of heme is produced. Although the pathophysiologic mechanisms of the clinical manifestations of the porphyrias are not fully understood, two cardinal features prevail: skin photosensitivity and neurologic symptoms of intermittent autonomic neuropathy, acute neurovisceral attacks, and disorders of the nervous system. The primary diagnosis of the proband is based on biochemical testing, which is not always able to identify acute porphyrias, especially in asymptomatic family carriers when heme precursors and porphyrins excretion is normal, low-normal and high-reduced values of enzyme activity overlap, and hematological diseases responsible for abnormal blood cells distribution coexist. Molecular analysis of gene mutations responsible for each type of porphyria is the best diagnostic approach for symptomatic as well as presymptomatic gene carriers.

[Strategies for diagnosis and biochemical control of porphyrias]. / Diagnostik og biokemisk kontrol af porfyrisygdomme.

Porphyrias are rare, distinct and well characterized diseases due to impairment of one of the eight steps in the biosynthesis of haem, which is the functional group of haemoglobin, myoglobin and cytochromes, including the cytochrome P-450 family. The actual strategies for diagnosis and biochemical control of the five most common porphyrias are described.

[Anaesthetics and porphyria].

Patients with acute porphyria are at risk of life-threatening attacks when exposed to stress, fast, infection, alcohol and especially some drugs, including older anaesthetics. Acute porphyrias are rare inherited diseases caused by inefficient enzymatic activity within the haem synthesis. During attacks the patient suffers from severe abdominal pain, cardiovascular instability, neurological symptoms etc. Preventive measures and treatment should be known to anaesthesiologists and surgeons in particular and known to other clinicians in general. In order to assist the clinicians, drug databases are available online.

Lon peptidase 1 (LONP1)-dependent breakdown of mitochondrial 5-aminolevulinic acid synthase protein by heme in human liver cells.

5-Aminolevulinic acid synthase (ALAS-1) is the first rate controlling enzyme that controls cellular heme biosynthesis. Negative feedback regulation of ALAS-1 by the end product heme is well documented and provides the foundation for heme treatment of acute porphyrias, a group of diseases caused by genetic defects in the heme biosynthesis pathway and exacerbated by controlled up-regulation of ALAS-1. Heme is known to affect ALAS-1 activity by repressing gene transcription, accelerating mRNA degradation, and impeding pre-ALAS-1 mitochondrial translocation. In the current study, we examined the effect of heme on the rate of mature ALAS-1 protein turnover in human cells and tissues and explored the mediator involved in this new regulatory mechanism. We found that heme and other metalloporphyrins such as CoPP and CrPP decreased mitochondrial ALAS-1 protein through proteolysis. This degradative effect cannot be emulated by iron or free protoporphyrin, two major chemical components of the heme ring, and is independent of oxidative stress. Down-regulating the activity of mitochondrial LONP1, an ATP-dependent protease that controls the selective turnover of mitochondrial matrix proteins, with potent inhibitors and specific siRNA diminished the negative effect of heme on mitochondrial ALAS-1. Therefore, our data support the existence of a conserved heme feedback regulatory mechanism that functions on the mature form of ALAS-1 protein through the activity of a mitochondrial proteolytic system.

Mice brain nitric oxide synthase response induced by anaesthetics and other porphyrinogenic drugs.

Porphyrias neuropathophysiology could be related to low levels of heme as a cofactor for nitric oxide synthase (NOS). We examined how anaesthetics and other porphyrinogenic agents affect mice NOS activity and expression. Brain response was differential depending on the cellular fraction analyzed. Most of the drugs diminished cytosolic activity. Instead, isoflurane, enflurane and ethanol increased mitochondrial activity. NOS expression also depended on the drug tested. A comparative study was performed in liver. Our present and previous results indicate the widespread action of porphyrinogenic agents in brain, which could be the reason why it is difficult to establish the onset of acute porphyria neurological manifestations.

The porphyrias: pathophysiology.

Porphyrias are a group of inherited and acquired metabolic disorders due to a defect in haem biosynthesis. An enzymatic defect at different steps of haem synthesis leads to tissue accumulation and excessive excretion of porphyrins and/or their toxic precursors. The specific patterns of accumulation determine the variety of clinical manifestations, ranging from acute neurovisceral attacks to skin lesions and liver disease. Most enzyme defects represent partial deficiencies, while familial cases are linked to autosomal or recessive traits. The incomplete penetrance of the genetic defects often requires the triggering or aggravating effect of host-related or environmental factors. While genetics has a role in confirming clinical suspicion and in family screening, biochemical and clinical studies are still central in the diagnosis.

Allosteric inhibition of human porphobilinogen synthase.

Porphobilinogen synthase (PBGS) catalyzes the first common step in tetrapyrrole (e.g. heme, chlorophyll) biosynthesis. Human PBGS exists as an equilibrium of high activity octamers, low activity hexamers, and alternate dimer configurations that dictate the stoichiometry and architecture of further assembly. It is posited that small molecules can be found that inhibit human PBGS activity by stabilizing the hexamer. Such molecules, if present in the environment, could potentiate disease states associated with reduced PBGS activity, such as lead poisoning and ALAD porphyria, the latter of which is associated with human PBGS variants whose quaternary structure equilibrium is shifted toward the hexamer (Jaffe, E. K., and Stith, L. (2007) Am. J. Hum. Genet. 80, 329-337). Hexamer-stabilizing inhibitors of human PBGS were identified using in silico prescreening (docking) of approximately 111,000 structures to a hexamer-specific surface cavity of a human PBGS crystal structure. Seventy-seven compounds were evaluated in vitro; three provided 90-100% conversion of octamer to hexamer in a native PAGE mobility shift assay. Based on chemical purity, two (ML-3A9 and ML-3H2) were subjected to further evaluation of their effect on the quaternary structure equilibrium and enzymatic activity. Naturally occurring ALAD porphyria-associated human PBGS variants are shown to have an increased susceptibility to inhibition by both ML-3A9 and ML-3H2. ML-3H2 is a structural analog of amebicidal drugs, which have porphyria-like side effects. Data support the hypothesis that human PBGS hexamer stabilization may explain these side effects. The current work identifies allosteric ligands of human PBGS and, thus, identifies human PBGS as a medically relevant allosteric enzyme.

A plant porphyria related to defects in plastid import of protochlorophyllide oxidoreductase A.

The plastid envelope of higher plant chloroplasts is a focal point of plant metabolism. It is involved in numerous pathways, including tetrapyrrole biosynthesis and protein translocation. Chloroplasts need to import a large number of proteins from the cytosol because most are encoded in the nucleus. Here we report that a loss-of-function mutation in the outer plastid envelope 16-kDa protein (oep16) gene causes a conditional seedling lethal phenotype related to defects in import and assembly of NADPH:protochlorophyllide (Pchlide) oxidoreductase A. In the isolated knockout mutant of Arabidopsis thaliana, excess Pchlide accumulated in the dark operated as photosensitizer and provoked cell death during greening. Our results highlight the essential role of the substrate-dependent plastid import pathway of precursor Pchlide oxidoreductase A for seedling survival and the avoidance of developmentally programmed porphyria in higher plants.

delta-Aminolevulinate dehydratase (ALAD) porphyria: the first case in North America with two novel ALAD mutations.

The molecular basis of the enzymatic defect responsible for delta-aminolevulinate dehydratase (ALAD) porphyria (ADP) was investigated in a 14-year-old male who presented clinical and laboratory findings typical of ADP. Nucleotide sequence analysis of ALAD cDNAs from the proband revealed two novel mutations, a 265G to A base transition (C1) and a 394C to T base transition (C2), resulting in amino acid substitutions, Glu89Lys and Cys132Arg, respectively. Both mutations were present within exon 5 of the ALAD gene, and appeared to influence the binding of zinc to the enzyme which is essential for enzyme activity. It was found that the C1 mutation was inherited from his father, while the C2 mutation was from his mother. Expression of these mutant ALAD cDNAs in Chinese hamster ovary cells produced normal ALAD mRNA levels, but markedly decreased ALAD protein and enzyme activity. These results suggest that the combination of the two aberrant ALADs with little enzyme activity accounts for the markedly decreased ALAD activity observed in the proband. This case represents the molecular analysis of the ALAD gene defects in the first case of ADP identified in North America, who is a compound heterozygote for two novel ALAD gene defects.

Nutritional regulation of hepatic heme biosynthesis and porphyria through PGC-1alpha.

Inducible hepatic porphyrias are inherited genetic disorders of enzymes of heme biosynthesis. The main clinical manifestations are acute attacks of neuropsychiatric symptoms frequently precipitated by drugs, hormones, or fasting, associated with increased urinary excretion of delta-aminolevulinic acid (ALA). Acute attacks are treated by heme infusion and glucose administration, but the mechanisms underlying the precipitating effects of fasting and the beneficial effects of glucose are unknown. We show that the rate-limiting enzyme in hepatic heme biosynthesis, 5-aminolevulinate synthase (ALAS-1), is regulated by the peroxisome proliferator-activated receptor gamma coactivator 1alpha (PGC-1alpha). Elevation of PGC-1alpha in mice via adenoviral vectors increases the levels of heme precursors in vivo as observed in acute attacks. The induction of ALAS-1 by fasting is lost in liver-specific PGC-1alpha knockout animals, as is the ability of porphyrogenic drugs to dysregulate heme biosynthesis. These data show that PGC-1alpha links nutritional status to heme biosynthesis and acute hepatic porphyria.

Abnormal kinetic behavior of uroporphyrinogen decarboxylase obtained from rats with hexachlorobenzene-induced porphyria.

Uroporphyrinogen decarboxylase is an essential enzyme in all organisms and functions in the heme biosynthetic pathway, catalyzing the decarboxylation of the four acetate groups of uroporphyrinogen to form coproporphyrinogen. This work examines whether the four sequential decarboxylations occur at the same active site, and explores whether hexachlorobenzene-induced porphyria affects the behavior of the enzyme. For this purpose, kinetic competition studies were done with mixtures of uroporphyrinogen III and pentacarboxyporphyrinogen III. With the enzyme from normal rats, a constant velocity was obtained with all the mixtures, indicating that uroporphyrinogen and pentacarboxy-porphyrinogen react at the same active site, i.e. the first and fourth decarboxylations occur at the same site. In contrast, in experiments with enzyme from rats with hexachlorobenzene-induced porphyria, the total rate for mixtures was always lower than the reference rate; and a curve with a deep minimum was obtained, indicating that the two reactions occur at functionally different sites, but with cross-inhibition. This suggests that the modifications induced in the enzyme by hexachlorobenzene cause the two active sites to become nonequivalent and functionally different. The question is discussed how the hexachlorobenzene treatment may produce this abnormal kinetic behavior, and alternative hypotheses are considered.

Glycogen metabolism and glucose transport in experimental porphyria.

Hexachlorobenzene (HCB) is a fungicide of well-known porphyrinogenic ability, which induces an experimental porphyria that resembles human porphyria cutanea tarda (PCT) in several animal species. It has been demonstrated that high glucose ingestion prevents porphyria development, and high-fat/high-protein diets enhance HCB porphyrinogenic ability. On the contrary, a diet rich in carbohydrates reduces HCB effects. The aim of this work was to study HCB effects on glycogen synthesis and degradation, as well as on glucose synthesis and transport, in order to elucidate whether would justify the beneficial use of carbohydrates in this porphyria. Rats were treated with HCB dissolved in corn oil (five daily doses 100mg/kg body weight). Results showed that: (1) HCB caused an increase in glycogen content; (2) glycogen synthase activity increased three times, and phosphorylase activity decreased about 40% due to fungicide intoxication. The effect of HCB on these two activities accounted for the higher glycogen content observed in treated animals; (3) three gluconeogenic enzymes were reduced 30-50%; (4) glucose uptake in the liver decreased in all weeks studied. The alterations found in glucose synthesis, its uptake in liver and other tissues, and its release from glycogen might contribute to the biochemical porphyria picture and would account for the effect of glucose above mentioned.

[Treatment of acute porphyrias. The importance of follow-up of patients and carriers]. / Akut porphyriák kezelése. A betegek és hordozók gondozásának jelentósége.

Acute porphyrias are caused by the inherited decreased activity of the enzymes of the heme biosynthesis pathway. Depending on the affected enzyme there are 4 types of them: acute intermittent porphyria, porphyria variegata, coproporphyria and delta-aminolevulinic acid dehydratase deficient porphyria, listed in order of their frequency. Basically the clinical picture is the same in the four types of acute porphyria. The most frequent complaints and symptoms are: cramping abdominal pain, nausea, vomiting, muscle weakness of the limbs then, in the advanced phase, there is a red-colored urine, hyponatremia, subileus, acute psychosis and Landry-type paralysis. Without proper treatment death is caused by respiratory paralysis or serious arrhythmia. In case of suspicion of acute porphyria it is mandatory to identify the type of the acute porphyria and the actual status of the patient. The later indicates what kind of treatment should be used. In the acute phase the early therapy with heme arginate is the treatment of choice. Since the clinical symptoms are precipitated by endogenous or exogenous inducing factors--most often by drugs-, the drugs negatively affecting the heme biosynthesis should be omitted at once even in the suspicion of acute porphyria. The role of the inducing factors in the manifestation of the clinical symptoms makes possible the prevention. It is possible to avoid the inducing factors and this way to prevent the acute attack if the acute porphyrias are recognized in time and the patients and the carriers are under regular control. The patients receive special identification card and the up-to-date list of safe drugs. They can use only these drugs in any kind of illness. Other drugs should be considered as porphyrinogenic since it is impossible to predict based on their chemical structure if they negatively affect the heme biosynthesis.

[Acute porphyrias in differential diagnosis]. / Akut porphyriák a differenciáldiagnosztikában.

The characteristic symptoms for acute porphyrias are caused by the inherited decreased activity of the enzymes of the heme biosynthesis pathway. Usually there is an exogenous or endogenous factor inhibiting the heme biosynthesis or increasing the consumption of heme produced in already decreased amount. The most important precipitating factors are the therapeutic drugs. Therefore, certain therapeutic drugs ordered for carriers or patients with acute porphyria are serious risk factors. It is very important to identify patients and carriers with acute porphyria as early as possible and to make a close follow-up so the development of the symptoms of the life threatening acute attack could be prevented. It is very difficult to suspect the diagnosis of acute porphyria. There is a very characteristic discrepancy between the serious complaints and the actual clinical findings. The severe cramping abdominal pain, nausea, vomiting, muscle weakness of the limbs and sensory loss are the main signs at the beginning. The specific symptoms which help to establish the diagnosis--red-colored urine, hyponatremia, tachycardia, hypertension, subileus, acute psychosis, gradually developing paresis of the lower and then the upper limbs--are characteristic for the later phase of the acute attack. Very often there is a rapid progression with Landry-type paralysis developing in days or even in hours, following respiratory paralysis or serious arrhythmia is the cause of the death. In case of suspicion of acute porphyria the patient should be directed to a department where the specific laboratory methods--measurement of the porphyrin precursors, porphyrins and their isomers in urine and feces, quantitation of protoporphyrin in red blood cells, measurement of the plasma porphyrin and enzyme activity--to diagnose the different types of the disease and the immediate specific treatment with heme arginate are possible if needed. All of these are available in the National Porphyria Center.

Microsomal enzyme induction and clinical aggravation of porphyria: the evaluation of human urinary 6beta-hydroxycortisol/cortisol ratio as the index of hepatic CYP3A4 activity.

The clinical aspect of porphyria has been investigated, and it is well known that porphyrinogens such as estrogens and alcohol or other inducers of P450 isoenzymes exacerbate the porphyric state. However, there can be a delay in diagnosing porphyria and a difficulty in selecting safe medicine for it even today. A 21-year-old woman developed epilepsy, disturbance of mental state, and spastic tetraparesis during the convalescent period after acute viral encephalitis. She was diagnosed with porphyria after the fifth hospitalization. In the course of modifying her anticonvulsant regimen, the authors examined the 6beta-hydroxycortisol/cortisol ratio (6beta-OHF/F) in her urine, which can be the index of hepatic CYP3A4 activity, with electrospray ionization/mass spectrometry/mass spectrometry (ESI/MS/MS). Generalized and partial complex seizures, other neurological signs and symptoms, and laboratory data were improved after modification of her anticonvulsant regimen. This is the first report of evaluating the urinary 6beta-hydroxycortisol/cortisol ratio in a case of porphyria.

[Nephrologists and porphyrias]. / I nefrologi e le porfirie.

As usually occurs for rare diseases, the word "PORPHYRIA" often recalls a confused topic with shaded boundaries, presenting "bullous" skin lesions, rare opportunity of diagnosis in clinical practice, unknown pathogenesis, and almost absent therapeutic options. The goal of this review is to draw attention to this topic, as new diagnostic and therapeutic tools might change the natural history of this disease. Porphyrias are disorders resulting from abnormalities of porphyrin metabolism. Porphyrins are molecules made up of four pyrrol rings, which constitute haeme-proteins, including haemoglobin. Following the "trigger" enzyme delta-aminolevulinic acid (ALA) synthase, which is capable of condensing succynil CoA and glycine, seven additional enzymes are involved in the process that eventually leads to haeme biosynthesis. Porphyrias are the result of total or partial deficiencies in these seven enzymes involved in haeme synthesis. Usually, the final haeme product exerts a negative feed-back on its synthesis. The enzyme deficiency that occurs in porphyrias is responsible for reduced haeme production, which, in turn, allows the cascade to be stimulated by increased activity of the trigger enzyme, ALA-synthase (ALA-s). However, due to the subsequent enzyme defects, notwithstanding increased ALA-s activity, haeme synthesis is blunted and intermediate metabolites accumulate. Clinical manifestations depend on which step the enzymatic defect occurs: if enzymatic defects are in the initial steps of the metabolic cascade, early metabolic intermediates will accumulate [i.e. ALA and porphobilinogen (PBG)] responsible for attacks of neurological dysfunction; if the enzymatic defects are in the final steps, sunlight-induced cutaneous lesions (phtotosensitivity) due to porphyrin accumulation in the skin will develop. The seven major human porphyrias may be classified into "hepatic or erythropoietic porphyrias" depending on the organ/tissue where metabolic alterations are more evident, or "acute or chronic porphyrias" depending on the prevalence of clinical symptoms, if neurologic (acute) or cutaneous (chronic). Only a small number of people with inherited enzyme deficiency will develop overt clinical disease, mainly because of the role of acquired aggravating and precipitating factors, such as drugs, hormonal causes, infection, caloric restriction, alcohol. The biochemical diagnosis of porphyrias relies on the detection of the consequences of increased ALA-s activity in the liver: overproduction, accumulation and increased excretion of early (ALA, PBG) or late (porphyrins) intermediate compounds in plasma, faeces and urine. A major difficulty arises from the knowledge that such abnormalities may be completely absent during the remission phases of the disease. Only in very specialised Centres it is now possible to measure specific haeme synthesis enzyme defects, and to perform molecular diagnosis by DNA analysis. The true prevalence of the diseases is unknown, ranging from 1:500 to 1:50,000. Therapeutic strategies include withdrawal of all common precipitants (drug, alcohol, fasting, infection), use of opiates and chlorpromazine, carbohydrates (300-400 g/day) and infusion of human haemine. Genetic therapies are being studied for the future.

A molecular, enzymatic and clinical study in a family with hereditary coproporphyria.

A 30-year-old woman suffered from acute crises with abdominal, neurological and psychiatric complaints. Urinary haem precursors and faecal porphyrins were excessively elevated compared to the upper level of the normal range. Urinary coproporphyrin isomer III was increased and faecal coproporphyrin isomers I and III showed a complete inversion of the normal ratio. Thus, hereditary coproporphyria was diagnosed in this woman. The father, one brother and a sister were shown to be gene carriers of hereditary coproporphyria by their urinary and faecal excretory constellations. The excretory patterns of the mother and a second brother were normal. Coproporphyrinogen oxidase activity was decreased to 49% and 58%, in the patient and her father, respectively. The mother's enzyme activity was normal (98%). Coproporphyrinogen oxidase concentration was enhanced 1.8-fold and 2.7-fold in the patient and her father, respectively. Mutation analysis revealed the insertion of an adenine at position 857 in exon 4 of the coproporphyrinogen oxidase gene. The gene defect was confirmed by denaturing gradient gel electrophoresis in the patient and her father. The patient was treated by intravenous interval therapy with haem arginate for 10 months, with good clinical and metabolic response.