Heme Demand Research Articles

Mechanisms of Neuronal Damage in Acute Hepatic Porphyrias.

Porphyrias are a group of congenital and acquired diseases caused by an enzymatic impairment in the biosynthesis of heme. Depending on the specific enzyme involved, different types of porphyrias (i.e., chronic vs. acute, cutaneous vs. neurovisceral, hepatic vs. erythropoietic) are described, with different clinical presentations. Acute hepatic porphyrias (AHPs) are characterized by life-threatening acute neuro-visceral crises (acute porphyric attacks, APAs), featuring a wide range of neuropathic (central, peripheral, autonomic) manifestations. APAs are usually unleashed by external “porphyrinogenic” triggers, which are thought to cause an increased metabolic demand for heme. During APAs, the heme precursors δ-aminolevulinic acid (ALA) and porphobilinogen (PBG) accumulate in the bloodstream and urine. Even though several hypotheses have been developed to explain the protean clinical picture of APAs, the exact mechanism of neuronal damage in AHPs is still a matter of debate. In recent decades, a role has been proposed for oxidative damage caused by ALA, mitochondrial and synaptic ALA toxicity, dysfunction induced by relative heme deficiency on cytochromes and other hemeproteins (i.e., nitric oxide synthases), pyridoxal phosphate functional deficiency, derangements in the metabolic pathways of tryptophan, and other factors. Since the pathway leading to the biosynthesis of heme is inscribed into a complex network of interactions, which also includes some fundamental processes of basal metabolism, a disruption in any of the steps of this pathway is likely to have multiple pathogenic effects. Here, we aim to provide a comprehensive review of the current evidence regarding the mechanisms of neuronal damage in AHPs.

Complex response to physiological and drug-induced hepatic heme demand in monoallelic ALAS1 mice

Regulation of 5-aminolevulinate synthase 1 (ALAS1) for nonerythroid heme is critical for respiration, cell signaling mechanisms and steroid/drug metabolism. ALAS1 is induced in some genetic disorders but unlike other genes in the heme pathway, a gene variant of ALAS1 associated with inherited disease has not been reported. BALB/c mice carrying a null ALAS1 allele caused by a βGEO insert were developed and used to determine the consequences of heme demand of a semi gene copy number. Homozygous disruption of ALAS1 (−/−) was lethal for embryo development post day 6.5 but expression in heterozygotes (+/−) was sufficient for the number of offspring and survival. In both wild type (WT +/+) and +/− mice expression of ALAS1 RNA was greatest in liver and harderian gland and much lower in kidney, lung, heart, brain and spleen. The effects of one WT ALAS1 allele in +/− mice on mRNA levels in liver and harderian gland were less marked compared to brain and other organs that were examined. Many other genes were up-regulated by heterozygosity in liver and brain but to a minimal extent. Hepatic heme oxygenase 1 (HMOX1) mRNA expression was significantly lower in +/− mice but not in brain. No elevated translation of WT allele ALAS1 mRNA was detected in +/− liver as a compensatory mechanism for the disabled allele. Fasting induced ALAS1 mRNA in both WT and +/− mice but only in +/− was this manifest as increased ALAS1 protein. The hepatic protoporphyria-inducing drug 4-ethyl-DDC caused induction of hepatic ALAS1 mRNA and protein levels in both WT and +/− mice but markedly less in the mice with only one intact allele. The findings illustrate the complex response of ALAS1 expression for heme demand but limited evidence that upregulation of a wild type allele can compensate for a null allele.

PGRMC2 is an intracellular haem chaperone critical for adipocyte function.

Heme is an essential prosthetic group of numerous proteins and a central signaling molecule in many physiologic processes1,2. The chemical reactivity of heme requires that a network of intracellular chaperone proteins exist to avert the cytotoxic effects of free heme, but the constituents of such trafficking pathways are unknown3,4. Heme synthesis is completed in mitochondria, with ferrochelatase (FECH) adding iron to protoporphyrin IX. How this vital but highly reactive metabolite is delivered from mitochondria to hemoproteins throughout the cell remains poorly defined3,4. Here, we show that PGRMC2 is required for delivery of labile, or signaling heme, to the nucleus. Deletion of PGMRC2 in brown fat, which has a high demand for heme, reduced labile heme in the nucleus and increased stability of the heme-responsive transcriptional repressors Rev-Erbα and BACH1. Ensuing alterations in gene expression spawn severe mitochondrial defects that rendered adipose-specific PGRMC2-null mice unable to activate adaptive thermogenesis and prone to greater metabolic deterioration when fed a high-fat diet. In contrast, obese-diabetic mice treated with a small-molecule PGRMC2 activator showed substantial improvement of diabetic features. These studies uncover a role for PGRMC2 in intracellular heme transport, reveal the impact of adipose tissue heme dynamics on physiology, and suggest that modulation of PGRMC2 may revert obesity-linked defects in adipocytes.

Systemic messenger RNA as an etiological treatment for acute intermittent porphyria.

Acute intermittent porphyria (AIP) results from haploinsufficiency of porphobilinogen deaminase (PBGD), the third enzyme in the heme biosynthesis pathway. Patients with AIP have neurovisceral attacks associated with increased hepatic heme demand. Phenobarbital-challenged mice with AIP recapitulate the biochemical and clinical characteristics of patients with AIP, including hepatic overproduction of the potentially neurotoxic porphyrin precursors. Here we show that intravenous administration of human PBGD (hPBGD) mRNA (encoded by the gene HMBS) encapsulated in lipid nanoparticles induces dose-dependent protein expression in mouse hepatocytes, rapidly normalizing urine porphyrin precursor excretion in ongoing attacks. Furthermore, hPBGD mRNA protected against mitochondrial dysfunction, hypertension, pain and motor impairment. Repeat dosing in AIP mice showed sustained efficacy and therapeutic improvement without evidence of hepatotoxicity. Finally, multiple administrations to nonhuman primates confirmed safety and translatability. These data provide proof-of-concept for systemic hPBGD mRNA as a potential therapy for AIP.

Polycyclic Aromatic Hydrocarbons (PAHs) Mediate Transcriptional Activation of the ATP Binding Cassette Transporter ABCB6 Gene via the Aryl Hydrocarbon Receptor (AhR)

Liver is endowed with a mechanism to induce hepatic cytochromes P450 (CYP450s) in response to therapeutic drugs and environmental contaminants, leading to increased detoxification and elimination of the xenobiotics. Each CYP450 is composed of an apoprotein moiety and a heme prosthetic group, which is required for CYP450 activity. Thus, under conditions of CYP450 induction, there is a coordinate increase in heme biosynthesis to compensate for the increased expression of CYP450s. ABCB6, a mitochondrial ATP binding cassette transporter, which regulates coproporphyrinogen transport from the cytoplasm into the mitochondria to complete heme biosynthesis, represents a previously unrecognized rate-limiting step in heme biosynthesis. However, it is not known if exposure to drugs and environmental contaminants induces ABCB6 expression, to assure an adequate and apparently coordinated supply of heme for the generation of functional cytochrome holoprotein. In the present study, we demonstrate that polycyclic aromatic hydrocarbons (PAHs), the widely distributed environmental toxicants shown to induce porphyrin accumulation causing hepatic porphyria, up-regulate ABCB6 expression in both mice and humans. Using siRNA technology and Abcb6 knock-out mice, we demonstrate that PAH-mediated increase in hepatic porphyrins is compromised in the absence of ABCB6. Moreover, in vivo studies in aryl hydrocarbon receptor (AhR) knock-out mice demonstrate that PAH induction of ABCB6 is mediated by AhR. Promoter activation studies combined with electrophoretic mobility shift assay and chromatin immunoprecipitation assay demonstrate direct interactions between the AhR binding sites in the ABCB6 promoter and the AhR receptor, implicating drug activation mechanisms for ABCB6 similar to those found in inducible cytochrome P450s. These studies are the first to describe direct transcriptional activation of both mouse and human ABCB6 by xenobiotics.

Sustained Enzymatic Correction by rAAV-Mediated Liver Gene Therapy Protects Against Induced Motor Neuropathy in Acute Porphyria Mice

Acute intermittent porphyria (AIP) is characterized by a hereditary deficiency of hepatic porphobilinogen deaminase (PBGD) activity. Clinical features are acute neurovisceral attacks accompanied by overproduction of porphyrin precursors in the liver. Recurrent life-threatening attacks can be cured only by liver transplantation. We developed recombinant adeno-associated virus (rAAV) vectors expressing human PBGD protein driven by a liver-specific promoter to provide sustained protection against induced attacks in a predictive model for AIP. Phenobarbital injections in AIP mice induced porphyrin precursor accumulation, functional block of nerve conduction, and progressive loss of large-caliber axons in the sciatic nerve. Hepatocyte transduction showed no gender variation after rAAV2/8 injection, while rAAV2/5 showed lower transduction efficiency in females than males. Full protection against induced phenobarbital-attacks was achieved in animals showing over 10% of hepatocytes expressing high amounts of PBGD. More importantly, sustained hepatic expression of hPBGD protected against loss of large-caliber axons in the sciatic nerve and disturbances in nerve conduction velocity as induced by recurrent phenobarbital administrations. These data show for the first time that porphyrin precursors generated in the liver interfere with motor function. rAAV2/5-hPBGD vector can be produced in sufficient quantity for an intended gene therapy trial in patients with recurrent life-threatening porphyria attacks.

Functional Assignments for the Carboxyl-Terminal Domains of the Ferrochelatase from Synechocystis PCC 6803: The CAB Domain Plays a Regulatory Role, and Region II Is Essential for Catalysis

Ferrochelatase (FeCH) catalyzes the insertion of Fe(2+) into protoporphyrin, forming protoheme. In photosynthetic organisms, FeCH and magnesium chelatase lie at a biosynthetic branch point where partitioning down the heme and chlorophyll (Chl) pathways occurs. Unlike their mammalian, yeast, and other bacterial counterparts, cyanobacterial and algal FeCHs as well as FeCH2 isoform from plants possess a carboxyl-terminal Chl a/b-binding (CAB) domain with a conserved Chl-binding motif. The CAB domain is connected to the FeCH catalytic core by a proline-rich linker sequence (region II). In order to dissect the regulatory, catalytic, and structural roles of the region II and CAB domains, we analyzed a FeCH ΔH347 mutant that retains region II but lacks the CAB domain and compared it with the ΔH324-FeCH mutant that lacks both these domains. We found that the CAB domain is not required for catalytic activity but is essential for dimerization of FeCH; its absence causes aberrant accumulation of Chl-protein complexes under high light accompanied by high levels of the Chl precursor chlorophyllide. Thus, the CAB domain appears to serve mainly a regulatory function, possibly in balancing Chl biosynthesis with the synthesis of cognate apoproteins. Region II is essential for the catalytic function of the plastid-type FeCH enzyme, although the low residual activity of the ΔH324-FeCH is more than sufficient to furnish the cellular demand for heme. We propose that the apparent surplus of FeCH activity in the wild type is critical for cell viability under high light due to a regulatory role of FeCH in the distribution of Chl into apoproteins.

Abdominal pain in a patient with acute lymphoblastic leukaemia

Dear Editor, A 57-year-old man was admitted to our hospital with acute lower abdominal pain and diarrhoea. The patient had been diagnosed with a CD20 and bcr-abl positive B-cell acute lymphoblastic leukaemia 15 months ago and was treated with four cycles rituximab-Hyper-CVAD. He was healthy with a complete molecular remission at his last routine examination 4 weeks ago. A CT scan of the abdomen revealed colitis of the colon descendens with perifocal oedema. Antibacterial therapy with piperazillin/tazobactam was initiated resulting in a normalisation of the initially elevated inflammatory blood markers. In contrast, the abdominal pain worsened although the analgesic therapy was substantially intensified. Due to increased pain, the patient’s oral food intake was poor. Additionally, he developed tachycardia, hypertension as well as episodes with aggressiveness and loss of orientation. Further investigations, including extensive blood and bacterial stool analysis as well as a gastroand colonoscopy, were unremarkable except for the urine, which turned from a clear yellow to a dark red colour (Fig. 1), highly suggestive for an acute porphyria. Indeed, increased levels of δ-aminolevulinic acid and porphobilinogen were found in the urine. All porphyrinogenic drugs were halted, an adequate caloric intake was guaranteed by nasogastric feeding and the patient was treated with exogenous hemearginate for 4 days. The patient recovered completely from his symptoms within a few days and was discharged after 2 weeks of hospitalisation. A molecular analysis was positive for the presence of the W283X-mutation in the porphobilinogen deaminase gene, a common mutation in Swiss residents with acute intermittent porphyria (AIP). He was completely asymptomatic when last seen in April 2009. AIP is an autosomal-dominant inherited disease of the heme biosynthetic pathway with a prevalence of symptomatic disease of one to two per 100,000 [1]. In AIP, the activity of the porphobilinogen deaminase (PBG) is reduced, resulting in an accumulation of upstream porphyrin precursors δ-aminolevulinic acid and porphobilinogen. At the moment, over 250 mutations in the PBG gene are known. Nearly 60% of all Swiss patients with an AIP carry the nonsense mutation W283X [2]. The majority of the mutation carriers are clinically healthy and exhibit symptoms only in situations when precipitating factors are present [3]. These include various drugs, poor energy intake during fasting as well as stress from illness and fever. Under these circumstances, the heme biosynthetic pathway gets activated and results in an excess accumulation of the porphyrin precursors δ-aminolevulinic acid and porphobilinogen due to the PGB enzyme deficiency. The key issues for acute therapy are to avoid drugs known to be porphyrinogenic, to promptly treat infections and to maintain an adequate caloric intake. Furthermore, intravenous heme derivates supporting the increased heme demand are the most effective treatment modalities for acute porphyria attacks [4]. A. Breitenstein (*) Department of Internal Medicine, University Hospital Zurich, Ramistrasse 100, 8091 Zurich, Switzerland e-mail: alexander.breitenstein@usz.ch

A tRNA Glu that uncouples protein and tetrapyrrole biosynthesis

Glu-tRNA is either bound to elongation factor Tu to enter protein synthesis or is reduced by glutamyl-tRNA reductase (GluTR) in the first step of tetrapyrrole biosynthesis in most bacteria, archaea and in chloroplasts. Acidithiobacillus ferrooxidans, a bacterium that synthesizes a vast amount of heme, contains three genes encoding tRNA Glu. All tRNA Glu species are substrates in vitro of GluRS1 from A. ferrooxidans. Glu - tRNA 3 Glu , that fulfills the requirements for protein synthesis, is not substrate of GluTR. Therefore, aminoacylation of tRNA 3 Glu might contribute to ensure protein synthesis upon high heme demand by an uncoupling of protein and heme biosynthesis.

The source of heme for vascular heme oxygenase II: de novo heme biosynthesis in rat aorta.

Heme is an essential prosthetic group or substrate for many proteins, including hemoglobin, and hemo enzymes such as nitric oxide synthase, soluble guanylyl cyclase, and heme oxygenase (HO). HO is responsible for the breakdown of heme into equimolar amounts of biliverdin, iron, and carbon monoxide, the latter of which is thought to play a role in the regulation of vascular tone. It is not clear whether the source of heme for cardiovascular functions is derived from uptake from the extracellular milieu or synthesis. In this study, we tested the hypothesis that blood vessels obtain their supply of heme for HO through de novo synthesis. Adult male Sprague-Dawley rat aorta was incubated at 37 degrees C in Krebs' solution with 1 micro M [14C]delta-aminolevulinic acid (ALA). [14C]ALA uptake was linear for about 30 min and reached a plateau at approximately 100 min. The radioactivity was incorporated into porphyrins and heme as determined by esterification of 14C-labelled metabolites and thin-layer chromatography. The first and rate-limiting step of heme biosynthesis is catalyzed by ALA synthase (ALA-S), the activity of which was determined in rat aorta using a radiometric assay, approximately 250 nmol x (g wet mass)(-1) x h(-1). Inducing HO-1 in rat aorta with S-nitroso-N-acetylpenicillamine (500 micro M) did not increase ALA-S activity as compared with basal activity levels of the enzyme. It appears that there is a sufficient amount of heme available under basal ALA-S activity conditions to meet the increased demand for heme resulting from HO-1 induction. These observations indicate that the complete enzymatic pathway for de novo heme biosynthesis resides in rat aorta and furthermore indicate that de novo heme synthesis is capable of supplying a substantial portion of the heme substrate for HO in the aorta.

The source of heme for vascular heme oxygenase I: heme uptake in rat aorta

During the last decade, heme oxygenase (HO) and carbon monoxide (CO) have garnered substantial research interest in terms of cell and organ regulation, especially as they bear on the central nervous system, organ transplantation, and the cardiovascular system. While the enzymatic mechanism, substrates, and products of HO are well known, it is not clear whether the cardiovascular system derives its supply of the heme substrate through de novo synthesis or uptake from the extracellular milieu. The objective of the present study was to test the latter possibility in rat aorta and to determine the influence of plasma proteins that bind heme in vivo, viz. hemopexin and albumin. Aortic tissue was exposed to [14C]heme in vitro, and the concentration and time dependence of heme uptake was assessed. The presence of hemopexin or albumin in the incubation medium dramatically decreased heme uptake by the aorta. Heme uptake by aortic tissue was not altered after induction of HO-1, which would be expected to increase tissue heme demand. In summary, the rat, isolated aorta was capable of obtaining heme from its external milieu, but this was obtunded in the presence of the plasma proteins hemopexin or albumin. For normal physiological situations, heme uptake may not be a usual source of substrate for vascular HO and hemoenzymes such as nitric oxide synthase, soluble guanylyl cyclase, and cyclooxygenase.

Rhodnius heme-binding protein (RHBP) is a heme source for embryonic development in the blood-sucking bug Rhodnius prolixus (Hemiptera, Reduviidae)

We have previously shown that Rhodnius prolixus’ eggs and hemolymph are pink due to the presence of the hemeprotein Rhodnius heme-binding protein (RHBP). In the hemolymph it functions as an antioxidant. Nevertheless, its function in eggs has not been determined. Here we present evidence that RHBP is a source of heme for embryonic development. RHBP content decreases during embryogenesis, but the total heme content of eggs remains unchanged. Biliverdin, the product of heme degradation, is not detectable in late embryos. The activity of the heme-synthesizing pathway is low throughout embryogenesis and rises sharply after nymphs’ hatching. Heme-radiolabeled eggs were produced and, at the day of hatching, nymphs were dissected. The presence of radiolabeled heme in their carcass is an indication that heme reutilization is occurring. The only animal known to reutilize heme in significant levels is the cattle tick Boophilus microplus, which cannot synthesize its own heme. Diversely, Rhodnius can synthesize its own heme but, in the context of embryogenesis, heme demand seems to be supplied by the programmed release of heme form RHBP. This behavior indicates that in Rhodnius, we might have a highly unusual profile: heme is both synthesized and reutilized.

Heme biosynthesis and oogenesis in the blood-sucking bug, Rhodnius prolixus

We have previously shown that the pathway of porphyrin synthesis operates in the blood feeding triatomine bug Rhodnius prolixus but not in the cattle tick Boophilus microplus. In the present paper we studied the correlation between heme synthesis and egg development in Rhodnius. There is a sharp increase heme biosynthetic capability in the fat body (160%) and in the ovaries (360%) in response to a blood meal, as evaluated from the activity of the enzyme δ-aminolevulinate dehydratase (EC 4.2.1.24). The in vivo inhibition of ALA-D by succinyl acetone results in a dose dependent decrease of oviposition. Oviposition is recovered when porphobilinogen, the product of the impaired reaction, is added to the succinyl acetone enriched blood. Taken together, these results show that heme biosynthesis is a fundamental event to vitellogenic females. The demand for heme in this metabolic juncture cannot be supplied by the heme eventually absorbed during blood digestion and associated with Rhodnius heme binding protein (RHBP), which is then incorporated into growing oocytes. Inhibition of heme biosynthesis results in lower levels of RHBP in the hemolymph, suggesting that the synthesis of this protein is controlled by heme availability.