Porphobilinogen Deaminase Activity Research Articles

An Overview of Acute Hepatic Porphyrias: Clinical Implications, Diagnostic Approaches, and Management Strategies.

Porphyrias are inborn errors of heme biosynthesis pathway that result in neurovisceral and/ or cutaneous manifestations which occur with episodic attacks, usually accompanied by a multisystemic involvement. Acute hepatic porphyrias include acute intermittent porphyria, variegate porphyria, hereditary coproporphyria, and aminolevulinic acid dehydratase deficiency porphyria. Acute hepatic porphyrias may present with symptoms of an affected central, peripheral, and autonomic nervous system and are generally diagnosed in time of an acute neurovisceral attack. In children, clinical picture is more complicated and presents with neurological findings predominantly. First-line investigation should be the urinary porphobilinogen and aminolevulinic acid performance when acute hepatic porphyria is clinically suspected. Comprehensive testing including urine porphyrin separation, fluorescence scanning of diluted plasma at neutral pH, evaluation of fecal porphyrins, and measurement of erythrocyte porphobilinogen deaminase activity is indicated for confirmation or exclusion of the porphyria and define the type of acute hepatic porphyrias. The main aim of the treatment is to decrease aminolevulinic acid, porphobilinogen, and porphyrins by reducing hepatic ALAS1 activity. The first measure should always be the avoidance of any porphyrinogenic drugs. Hemin therapy should not be delayed in the treatment of a severe acute attack. Gonadotropin-releasing hormone analogs and prophylactic hemin protocols can be used for selected cases with more than 4 attacks per year. Givosiran is a promising treatment option for severe cases.

Explore: A Prospective, Multinational Natural History Study of Patients with Acute Hepatic Porphyrias (AHP) with Recurrent Attacks

Introduction: The acute hepatic porphyrias (AHP) are inherited metabolic disorders caused by deficiencies of specific enzymes in the heme biosynthetic pathway. Of the four AHP, the most common is acute intermittent porphyria (AIP), an autosomal dominant disease due to the half-normal activity of porphobilinogen deaminase (PBGD). These diseases are characterized by life-threatening acute neurovisceral attacks that involve the autonomic, peripheral and central nervous systems, and often require urgent medical care and/or hospitalization. Acute attacks are triggered by a number of factors, including cytochrome P450-inducing drugs, dieting, and hormonal fluctuations, all of which result in the increased expression of hepatic 5-aminolevulinic acid synthase (ALAS1), the first and rate-limiting enzyme of the heme biosynthetic pathway. When ALAS1 activity is induced, the inherited enzyme deficiency becomes limiting and results in the increased hepatic production of the upstream neurotoxic porphyrin precursors, 5-aminolevulinic acid (ALA) and porphobilinogen (PBG), which accumulate in the plasma and urine.Methods: EXPLORE (ClinicalTrials.gov Identifier: NCT02240784) is an on-going, prospective, international, observational study characterizing the natural history and clinical management of patients with AHP with recurrent attacks (> 3 attacks/year) or who receive hemin prophylaxis to prevent attacks. Patient medical history, physical examination and porphyrin precursors, along with questionnaires on porphyria activity, quality of life and healthcare utilization were collected.Results: A total of 112 patients with AHP were enrolled from 13 countries, and have been followed for 6 to 12 months. The mean patient age is 39 years, with 89% female, and 93% with AIP, 4% variegate porphyria (VP), and 3% hereditary coproporphyria (HCP). The most common associated medical conditions were hypertension (23%), headaches/migraine (11%), depression (17%) and insomnia (12%). Patients reported a mean of 9.3 attacks in the 12 months prior to enrolling in the study, with pain being the most common symptom in 99% of attacks. Chronic symptoms in between attacks were also reported by 65% of patients, with pain being the most frequent symptom. Overall, 46% of patients experienced symptoms daily. While on study, the annualized attack rate was 4.9 attacks/person, with a mean duration of 7 days. In patients that were not being treated with hemin prophylactically, attack rate was 5.5 attacks/person and in patients being treated with hemin prophylactically, attack rate was 4.0 attacks/person. Seventy-seven percent of attacks required treatment at a healthcare facility or with intravenous hemin. Patients reported diminished quality of life (EQ-5D-5L), with the greatest impact seen in the domains of pain/discomfort, usual activities and anxiety/depression. Asymptomatic patients have induced ALAS1 and high urinary ALA/PBG compared to normal healthy individuals, which increase further during attacks.Conclusions: EXPLORE is the first international, natural history study in patients with AHP. Results from this study demonstrate that patients suffer from both acute and chronic symptoms (most commonly pain), during and in between attacks, which collectively result in a diminished quality of life. Furthermore, in patients treated with hemin prophylactically, attacks continued to occur. Given the continued morbidity and mortality, there remains an unmet need for an efficacious, simple, fast-acting and better tolerated treatment for patients with AIP. DisclosuresWindyga:Baxalta: Honoraria, Research Funding; Biogen Idec: Honoraria, Research Funding; Baxter Healthcare: Honoraria, Research Funding; Bayer: Honoraria, Research Funding; CSL Behring: Honoraria, Research Funding; Novo Nordisk: Honoraria, Research Funding; Octapharma: Honoraria, Research Funding; Roche: Honoraria, Research Funding; Sanofi: Honoraria, Research Funding; Pfizer: Honoraria, Research Funding; Shire: Honoraria, Research Funding. Cappellini:Sanofi-Genzyme: Honoraria, Research Funding, Speakers Bureau; Novartis: Speakers Bureau; Celgene: Honoraria; Vifor: Honoraria. Rock:Alnylam: Employment. Chan:Alnylam: Employment. Soh:Alnylam: Employment. McCarthy:Alnylam: Employment. Querbes:Alnylam: Employment. Penz:Alnylam: Employment. Simon:Alnylam: Employment.

MRNA-based therapy in a rabbit model of variegate porphyria offers new insights into the pathogenesis of acute attacks

Variegate porphyria (VP) results from haploinsufficiency of protoporphyrinogen oxidase (PPOX), the seventh enzyme in the heme synthesis pathway. There is no VP model that recapitulates the clinical manifestations of acute attacks. Combined administrations of 2-allyl-2-isopropylacetamide and rifampicin in rabbits halved hepatic PPOX activity, resulting in increased accumulation of a potentially neurotoxic heme precursor, lipid peroxidation, inflammation, and hepatocyte cytoplasmic stress. Rabbits also showed hypertension, motor impairment, reduced activity of critical mitochondrial hemoprotein functions, and altered glucose homeostasis. Hemin treatment only resulted in a slight drop in heme precursor accumulation but further increased hepatic heme catabolism, inflammation, and cytoplasmic stress. Hemin replenishment did protect against hypertension, but it failed to restore action potentials in the sciatic nerve or glucose homeostasis. Systemic porphobilinogen deaminase (PBGD) mRNA administration increased hepatic PBGD activity, the third enzyme of the pathway, and rapidly normalized serum and urine porphyrin precursor levels. All features studied were improved, including those related to critical hemoprotein functions. In conclusion, the VP model recapitulates the biochemical characteristics and some clinical manifestations associated with severe acute attacks in humans. Systemic PBGD mRNA provided successful protection against the acute attack, indicating that PBGD, and not PPOX, was the critical enzyme for hepatic heme synthesis in VP rabbits.

AbhemC encoding porphobilinogen deaminase plays an important role in chlorophyll biosynthesis and function in albino Ananas comosus var. bracteatus leaves.

BackgroundThe chimeric leaves of Ananas comosus var. bracteatus are composed of normal green parts (Grs) and albino white parts (Whs). Although the underlying mechanism of albinism in A. comosus var. bracteatus leaves is not fully understood, it is likely associated with the chlorophyll (Chl) biosynthesis. In this biosynthetic process, porphobilinogen deaminase (PBGD) plays a crucial role by catalyzing the conversion of porphobilinogen (PBG) to uroporphyrinogen III (Urogen III). Therefore, its encoding gene AbhemC was investigated here in association with Chl biosynthesis and albinism in chimeric A. comosus var. bracteatus leaves.MethodsThe Chl content, main Chl biosynthesis precursor content, and main enzyme activity were determined and compared between the Whs and Grs of A. comosus var. bracteatus leaves. In addition, AbhemC was cloned and its transcriptional expression and prokaryotic protein expression were analyzed. Furthermore, RNAi-mediated silencing of AbhemC was produced and assessed in tobacco plants.ResultsThe concentration of Chl a and Chl b in the Grs was significantly higher than that in the Whs, respectively. Additionally, the content of the Chl biosynthesis precursor Urogen III decreased significantly in the Whs compared with the Grs. Thus, the transition of PBG to Urogen III may be the first rate-limiting step leading to albinism in the chimeric leaves of A. comosus var. bracteatus. The gene AbhemC comprised 1,135 bp and was encoded into a protein with 371 amino acids; phylogenetically, AbhemC was most closely related to hemC of pineapple. Prokaryotic expression and in vitro enzyme activity analysis showed that the cloned mRNA sequence of AbhemC was successfully integrated and had PBGD activity. Compared with control plants, transgenic tobacco leaves with pFGC5941-AbhemC-RNAi vector were substantially less green with significantly reduced hemC expression and Chl content, as well as reduced PBGD enzyme activity and significantly decreased content of Chl biosynthesis precursors from Urogen III onwards. Our results suggest that the absence of hemC expression reduces the enzyme activity of PBGD, which blocks the transition of PBG to Urogen III, and in turn suppresses Chl synthesis leading to the pale-green leaf color. Therefore, we suggest that AbhemC plays an important role in Chl synthesis and may be an important factor in the albinism of A. comosus var. bracteatus leaves.

Early Physiotherapy Intervention of a Patient with Acute Intermittent Porphyria-A Single Case Study

Background: The Porphyrias are metabolic disorders, each resulting from the deficiency of a specific enzymein the heme biosynthetic pathway. Acute intermittent porphyria (AIP) is an autosomal dominant inbornerror characterized by decreased activity of porphobilinogen (PBG) deaminase leading to increased levelsof haem precursors, namely amino levulinic acid (ALA) and PBG. The major manifestations of theacutehepatic porphyrias are neurologic- neuropathic abdominal pain, peripheral motor neuropathy, and mentaldisturbanceswith attacks often precipitated by steroid, hormones, certain drugs, and nutrition influences.Purpose: Intensive care measures and a multidisciplinary therapeutic approach are essentialbut thereis lessdocumented evidence are available.If this aspect is incorporated early in the rehabilitation programme, thenthe functional recovery in such will be better.so the aim of the study is to find how Early Physiotherapyintervention using Comprehensive need based Approach to improve the functional status of porphyriapatient.Key Points of Case: A 17 years old male patient wasdiagnosed with acute intermittent Porphyria. Havingthe history of abdominal pain since last 3 months with family history of AIP which was diagnosed very late.A detailed Physical and Neurological examination of this patient was taken. Along with medical treatment,early physiotherapy intervention using comprehensive need base approach was being given to the patient.The Barthel Index was taken to assess the functional status of the patient and Berg Balance Scale was takento assess balance on admission and subsequent follow-ups. Post-intervention follow-ups were taken at 6weeks and 12 weeks period.Conclusion: A clinically significant improvement was found on Barthel Index and Berg Balance Scale.Early Physiotherapy intervention using a Comprehensive need based Approach is highly recommended toimprove the functional status of a patient with porphyria

Host Porphobilinogen Deaminase Deficiency Confers Malaria Resistance in Plasmodium chabaudi but Not in Plasmodium berghei or Plasmodium falciparum During Intraerythrocytic Growth.

An important component in host resistance to malaria infection are inherited mutations that give rise to abnormalities and deficiencies in erythrocyte proteins and enzymes. Understanding how such mutations confer protection against the disease may be useful for developing new treatment strategies. A mouse ENU-induced mutagenesis screen for novel malaria resistance-conferring mutations identified a novel non-sense mutation in the gene encoding porphobilinogen deaminase (PBGD) in mice, denoted here as PbgdMRI58155. Heterozygote PbgdMRI58155 mice exhibited ~50% reduction in cellular PBGD activity in both mature erythrocytes and reticulocytes, although enzyme activity was ~10 times higher in reticulocytes than erythrocytes. When challenged with blood-stage P. chabaudi, which preferentially infects erythrocytes, heterozygote mice showed a modest but significant resistance to infection, including reduced parasite growth. A series of assays conducted to investigate the mechanism of resistance indicated that mutant erythrocyte invasion by P. chabaudi was normal, but that following intraerythrocytic establishment a significantly greater proportions of parasites died and therefore, affected their ability to propagate. The Plasmodium resistance phenotype was not recapitulated in Pbgd-deficient mice infected with P. berghei, which prefers reticulocytes, or when P. falciparum was cultured in erythrocytes from patients with acute intermittent porphyria (AIP), which had modest (20–50%) reduced levels of PBGD. Furthermore, the growth of Pbgd-null P. falciparum and Pbgd-null P. berghei parasites, which grew at the same rate as their wild-type counterparts in normal cells, were not affected by the PBGD-deficient background of the AIP erythrocytes or Pbgd-deficient mice. Our results confirm the dispensability of parasite PBGD for P. berghei infection and intraerythrocytic growth of P. falciparum, but for the first time identify a requirement for host erythrocyte PBGD by P. chabaudi during in vivo blood stage infection.

Acute Intermittent Porphyria, Posterior Reversible Encephalopathy Syndrome and Hypertensive Emergency, A Review of The Literature

Acute intermittent porphyria is an autosomal dominant inborn error characterized by decreased activity of porphobilinogen deaminase leading to increased levels of haem precursors, namely aminolevulinic acid and porphobilinogen. Posterior reversible encephalopathy syndrome is a clinical radiographic syndrome of heterogeneous etiologies characterized by headache, seizures, altered consciousness, and visual disorder associated with potentially reversible neuroradiological abnormalities predominantly in the parieto-occipital lobes. There are few previous reports of acute intermittent porphyria associated with posterior reversible encephalopathy syndrome, which are thought to result from peaks in hypertension.

Disease pharmacokinetic-pharmacodynamic modelling in acute intermittent porphyria to support the development of mRNA-based therapies.

Acute intermittent porphyria (AIP) results from haplo-insufficiency of the porphobilinogen deaminase (PBGD) gene encoding the third enzyme in the haem biosynthesis pathway. As liver is the main organ of pathology for AIP, emerging therapies that restore enzyme hepatic levels are appealing. The objective of this work was to develop a mechanistic-based computational framework to describe the effects of novel PBGD mRNA therapy on the accumulation of neurotoxic haem precursors in small and large animal models. Liver PBGD activity data and/or 24-hr urinary haem precursors were obtained from genetic AIP mice and wild-type mice, rats, rabbits, and macaques. To mimic acute attacks, porphyrogenic drugs were administered over one or multiple challenges, and animals were used as controls or treated with different PBGD mRNA products. Available experimental data were sequentially used to build and validate a semi-mechanistic mathematical model using non-linear mixed-effects approach. The developed framework accounts for the different biological processes involved (i.e., mRNA sequence, release from lipid nanoparticle and degradation, mRNA translation, increased PBGD activity in liver, and haem precursor metabolism) in a simplified mechanistic fashion. The model, validated using external data, shows robustness in the extrapolation of PBGD activity data in rat, rabbit, and non-human primate species. This quantitative framework provides a valuable tool to compare PBGD mRNA drug products during early preclinical stages, optimize the amount of experimental data required, and project results to humans, thus supporting drug development and clinical dose and dosing regimen selection.

Sex differences in vascular reactivity in mesenteric arteries from a mouse model of acute intermittent porphyria

Background and aimsAcute intermittent porphyria (AIP) results from a partial deficiency of porphobilinogen deaminase (PBGD). Symptomatic AIP patients, most of whom are women, experience acute attacks characterized by severe abdominal pain and abrupt increases in blood pressure. Here, we characterized the reactivity of mesenteric arteries from male and female AIP mice with ~30% of normal PBGD activity and wild type C57BL/6 mice. MethodsAn acute porphyric attack was induced in AIP mice by treatment with phenobarbital. Vascular responses to K+, phenylephrine (PE), acetylcholine (ACh), and hemin were determined (Wire Multi Myograph). ResultsMaximal contraction to PE was increased in arteries from male and female AIP mice (p < .05) during an induced attack of acute porphyria. Female AIP arteries had increased sensitivity to PE (p < .05) even after nitric oxide (NO) blockade with Nω-nitro-L-arginine methyl ester (L-NAME) (p < .05). Maximal relaxation to ACh was similar in males and females with lower sensitivity in female AIP arteries (p < .05). Hemin induced greater relaxation in AIP arteries in both males and females (p < .05). Summary/conclusionsSex differences in this AIP mouse model include a pro-contractile response in females. These alterations may contribute to the increased blood pressure during an acute attack and provide a novel mechanism of action whereby heme ameliorates the attacks.

Effect of 5-aminolevulinic acid on the expression of carcinogenesis-related proteins in cultured primary hepatocytes.

Acute intermittent porphyria (AIP) is a heme pathway disorder caused by a decrease in the activity and synthesis of porphobilinogen deaminase. Thus, the first heme precursor 5-aminolevulinic acid (ALA) accumulates in the liver. Reactive oxygen species (ROS) resulting from ALA oxidation may be correlated to a higher incidence of hepatocellular carcinoma (HCC) in AIP patients. However, the molecular mechanisms of this relationship have not been thoroughly elucidated to date. In this study, we investigated the effect of increasing levels of ALA on the expression of proteins related to DNA repair, oxidative stress, apoptosis, proliferation and lipid metabolism. Primary rat hepatocytes were isolated by the collagenase perfusion method, lipoperoxidation was evaluated by a TBA fluorimetric assay and Western blotting was used to assess protein abundance. The data showed that ALA treatment promoted a dose-dependent increase of p53 expression, downregulation of Bcl-2, HMG-CoA reductase and OGG1 and an increase in lipoperoxidation. There was no alteration in the expression of the transcription factor NF-κB, catalase and superoxide dismutase. ALA oxidation products induced protein regulation patterns, suggesting the interconnection of cellular processes, such as the intrinsic pathway of apoptosis, redox homeostasis, cell proliferation, lipid metabolism and DNA repair. This study helps to elucidate the molecular mechanisms of hepatotoxicity mediated by ALA pro-oxidant effects and supports the hypothesis that ALA accumulation correlates with a higher incidence of hepatic carcinogenic events.

Hereditary Coproporphyria Presenting with Weakness and Photosensitive Rash: A case Report and Literature Review

Hereditary coproporphyria (HCP) is a form of porphyria arising from a deficiency of the enzyme, coproporphyrinogen oxidase, which results in the accumulation of coproporphyrin in the heme biosynthetic pathway. In the current study, we report a case of a 22-year-old male with a history of six recurrent intestinal obstructions, mainly presenting with reduced upper limb strength, photosensitive rash, and abdominal pain this admission. The laboratory tests revealed increased levels of coproporphyrin, uroporphyrinogen and porphyrin in stool sample and porphobilinogen deaminase activity was within the normal range. All these led to the diagnosis for HCP. Single intravenous glucose therapy improved the patient’s condition and no relapse was observed during the 3-month follow-up period. Since then, no other pathological or genomic manifestations were observed.

Effects of volatile anaesthetics on heme metabolism in a murine genetic model of Acute Intermittent Porphyria. A comparative study with other porphyrinogenic drugs

BackgroundAcute Intermittent Porphyria (AIP) is an inherited disease produced by a deficiency of Porphobilinogen deaminase (PBG-D). The aim of this work was to evaluate the effects of Isoflurane and Sevoflurane on heme metabolism in a mouse genetic model of AIP to further support our previous proposal for avoiding their use in porphyric patients. A comparative study was performed administering the porphyrinogenic drugs allylisopropylacetamide (AIA), barbital and ethanol, and also between sex and mutation using AIP (PBG-D activity 70% reduced) and T1 (PBG-D activity 50% diminished) mice. MethodsThe activities of 5-Aminolevulinic synthetase (ALA-S), PBG-D, Heme oxygenase (HO) and CYP2E1; the expression of ALA-S and the levels of 5-aminolevulinic acid (ALA) were measured in different tissues of mice treated with the drugs mentioned. ResultsIsoflurane increased liver, kidney and brain ALA-S activity of AIP females but only affected kidney AIP males. Sevoflurane induced ALA-S activity in kidney and brain of female AIP group. PBG-D activity was further reduced by Isoflurane in liver male T1; in AIP male mice activity remained in its low basal levels. Ethanol and barbital also caused biochemical alterations. Only AIA triggered neurological signs similar to those observed during human acute attacks in male AIP being the symptoms less pronounced in females although ALA-S induction was greater. Heme degradation was affected. DiscussionBiochemical alterations caused by the porphyrinogenic drugs assayed were different in male and female mice and also between T1 and AIP being more affected the females of AIP group. General significanceThis is the first study using volatile anaesthetics in an AIP genetic model confirming Isoflurane and Sevoflurane porphyrinogenicity.

Heme Binding to Porphobilinogen Deaminase from Vibrio cholerae Decelerates the Formation of 1-Hydroxymethylbilane.

Porphobilinogen deaminase (PBGD) is an enzyme that catalyzes the formation of hydroxymethylbilane, a tetrapyrrole intermediate, during heme biosynthesis through the stepwise polymerization of four molecules of porphobilinogen. PBGD from Vibrio cholerae was expressed in Escherichia coli and characterized in this study. Unexpectedly, spectroscopic measurements revealed that PBGD bound one equivalent of heme with a dissociation constant of 0.33 ± 0.01 μM. The absorption and resonance Raman spectra suggested that heme is a mixture of the 5-coordinate and 6-coordinate hemes. Mutational studies indicated that the 5-coordinate heme possessed Cys105 as a heme axial ligand, and His227 was coordinated to form the 6-coordinate heme. Upon heme binding, the deamination activity decreased by approximately 15%. The crystal structure of PBGD revealed that His227 was located near Cys105, but the side chain of His227 did not point toward Cys105. The addition of the cyanide ion to heme-PBGD abolished the effect of heme binding on the enzymatic activity. Therefore, coordination of His227 to heme appeared to induce reorientation of the domains containing Cys105, leading to a decrease in the enzymatic activity. This is the first report indicating that the PBGD activity is controlled by heme, the final product of heme biosynthesis. This finding improves our understanding of the mechanism by which heme biosynthesis is regulated.

Validation and evaluation of two porphobilinogen deaminase activity assays for diagnosis of acute intermittent porphyria

BackgroundAcute intermittent porphyria (AIP) is caused by diminished activity of porphobilinogen deaminase (PBGD). The purpose of this study was to validate and compare two assays for PBGD activity. The clinical sensitivity of the PBGD activity assays in AIP diagnosis was also evaluated. MethodsThis study included 74 subjects from 18 Taiwanese families including symptomatic patients with AIP, asymptomatic carriers, and healthy family members. The specific mutations in AIP patients were identified by DNA sequencing. PBGD activity was measured in erythrocytes by quantifying formation of coproporphyrin or uroporphyrin by the enzyme using porphobilinogen (PBG) as a substrate and fluorimetry for detection. ResultsThe calibration curves obtained with pure coproporphyrin or uroporphyrin were linear with correlation coefficients >0.99 in the range of 0–200nM for coproporphyrin and 0–150nM for uroporphyrin. The coefficients of variation for within-run and between-day imprecision were <9.8% for both assays. The three groups of subjects were used to establish the best cut-off of PBGD activity for identifying symptomatic AIP patients by using area under receiver operating characteristic curve analysis. The symptomatic AIP patients and asymptomatic carriers had significantly lower PBGD activity compared with the healthy family members (all p<.001). ConclusionTwo different PBGD activity assays were validated. The best cut-off for coproporphyrin was derived as 46.4nmol/h/mL RBC with corresponding sensitivity of 100% and specificity of 100% and the best cut-off for uroporphyrin was derived as 43.7nkat/L RBC with corresponding sensitivity of 100% and specificity of 97.4%.

Physiological Characterization and Comparative Transcriptome Analysis of White and Green Leaves of Ananas comosus var. bracteatus.

Leaf coloration is one of the most important and attractive characteristics of Ananas comosus var. bracteatus. The chimeric character is not stable during the in vitro tissue culturing. Many regenerated plants lost economic values for the loss of the chimeric character of leaves. In order to reveal the molecular mechanisms involved in the albino phenotype of the leaf cells, the physiological and transcriptional differences between complete white (CWh) and green (CGr) leaf cells of A. comosus var. bracteatus were analyzed. A total of 1,431 differentially expressed unigenes (DEGs) in CGr and CWh leaves were identified using RNA-seq. A comparison to the COG, GO and KEGG annotations revealed DEGs involved in chlorophyll biosynthesis, chloroplast development and photosynthesis. Furthermore, the measurement of main precursors of chlorophyll in the CWh leaves confirmed that the rate-limiting step in chlorophyll biosynthesis, and thus the cause of the albino phenotype of the white cells, was the conversion of pyrrole porphobilinogen (PBG) to uroporphyrinogen III (Uro III). The enzyme activity of porphobilinogen deaminase (PBGD) and uroporporphyrinogn III synthase (UROS), which catalyze the transition of PBG to Uro III, was significantly decreased in the CWh leaves. Our data showed the transcriptional differences between the CWh and CGr plants and characterized key steps in chlorophyll biosynthesis of the CWh leaves. These results contribute to our understanding of the mechanisms and regulation of pigment biosynthesis in the CWh leaf cells of A. comosus var. bracteatus.

Phenanthrene-triggered Chlorosis is caused by elevated Chlorophyll degradation and leaf moisture

Leaf is an important organ in responding to environmental stresses. To date, chlorophyll metabolism under polycyclic aromatic hydrocarbon (PAH) stress is still unclear. Here we reveal, for the first time, the chlorophyll metabolism of wheat seedling leaves in response to phenanthrene (a model PAH) exposure. In this study, the hydroponic experiment was employed, and the wheat seedlings were exposed to phenanthrene to observe the response at day 1, 3, 5, 7 and 9. Over the exposure time, wheat leaf color turns light. With the accumulation of phenanthrene, the concentrations of glutamate, 5-aminolevulinic acid, uroporphyrinogen III, protoporphyrin IX, Mg-protoporphyrin IX and protochlorophyllide increase while the concentrations of porphobilinogen and Chlorophyll b decrease. Also chlorophyll a content rises initially and then declines. Uroporphyrinogen III synthase and chlorophyllase are activated and porphobilinogen deaminase activity declines in the treatments. Both chlorophyll synthesis and degradation are enhanced, but the degradation rate is faster. Phenanthrene accumulation has significant and positive effects on increase of glutamate, 5-aminolevulinic acid, uroporphyrinogen III, protoporphyrin IX, Mg-protoporphyrin IX and protochlorophyllide concentrations. There is a negative correlation between phenanthrene accumulation and total chlorophyll. Additionally, the leaf moisture increases. Therefore, it is concluded that wheat leaf chlorosis results from a combination of accelerated chlorophyll degradation and elevated leaf moisture under phenanthrene exposure. Our results are helpful not only for better understanding the toxicity of PAHs to plants and crop PAH-adaptive mechanism in the environment, but also for potentially employing the changes of the chlorophyll-synthesizing precursors and enzyme activities in plant leaves as indicators of plant response to PAH pollution.

Late Acute Intermittent Porphyria Attack In A Patient With Type 2 Diabetes

Abstract Background. Acute intermittent porphyria (AIP) is a hereditary metabolic aberration resulting from a partial defect in the activity of the enzyme porphobilinogen deaminase (PBDG) during the course of haeme synthesis. Diabetic metabolism may attenuate the episodes of porphyria related symptoms. Case report. Our subject (male; age 75) was hospitalized one week after onset of diffuse abdominal pain and constipation and overt type 2 diabetes mellitus (DM). The patient’s long history of alcohol intake with acute alcohol consumption 12 days before admission, in the presence of abdominal pain with spectacular remission after oral administration of 5% glucose solution, accompanied by a 2.5 fold increase of urinary porphobilinogen with normal values for porphyrins and urinary lead, and normal full blood count establishes the diagnosis of AIP. Conclusion We describe a case of AIP probably triggered by acute alcohol consumption, with neurovisceral dominant clinical picture mimicking an acute abdomen. Late disease occurrence as first acute episode at older age accompanied by overt type 2 DM, suggests a latent type of AIP in our patient. The appropriate recognition of latent AIP cases in proband’s offspring prevents unnecessary blind surgery when repeated episodes of unexplained abdominal pain occur.

Clinical presentation and electrophysiological findings of porphyric neuropathies: A follow‐up study

A case series of acute intermittent porphyria (AIP) is described that focuses on the clinical course of the disease with regard to neurological manifestations of the peripheral nervous system. Eight patients were diagnosed with AIP on the basis of characteristic clinical findings, erythrocyte porphobilinogendeaminase activity, neuropathic patterns, serial changes in nerve conduction studies (NCS), and temporal relationship of central nervous system involvement. Six patients diagnosed with AIP<2 months after symptom onset had neuropathy that was predominantly upper extremity, motor, and proximal. NCS recovery rates were slower in the lower than the upper limbs. Two patients diagnosed >2 months after symptom onset had distal sensorimotor polyneuropathy. The findings from this case series suggest that the peripheral nerves may be differentially and selectively involved in different diagnostic stages of porphyric neuropathy.

Porphobilinogen deaminase gene mutations in Polish patients with non-erythroid acute intermittent porphyria.

Acute intermittent porphyria (AIP) is an metabolic disorder characterized by a partial deficiency of the porphobilinogen deaminase, the enzyme of heme biosynthesis. The metabolic defect in AIP involves an approximately half-normal activity of porphobilinogen deaminase (PBGD, EC 4.3.1.8), the enzyme catalyzing condensation of four porphobilinogen molecules to hydroxymethylbilane. Due to tissue-specific alternative transcript splicing, the PBGD gene mutations within the range of exons 3-15 may lead to classical AIP involving erythrocytes and all the other tissues. Mutations within intron and exon 1 may result in the so-called non-erythroid AIP in which the PBGD activity is normal in erythrocytes and diminished in other tissues. The aim of the present study was to characterise molecular errors in the PBGD gene in Polish patients with non-erythroid AIP and to evaluate the efficacy of the DNA sequencing method in the early diagnosis of this disorder. Twenty five members of nine non-erythroid AIP families were assessed. In each of them DNA sequencing was performed using the Big Dye Terminator Cycle Sequencing Kit v.1.1 on the Hitachi 3730 Analyzer (Applied Biosystem, USA). Four mutations were detected in intron 1 of the PBGD gene, including one unreported novel mutation, 33+(4-12) del AGTGCTGAG, of an unknown biological mechanism, and three previously described mutations, i.e. 33+1 G > A, 33+2 T > C, 33+5 G > C, responsible for abnormal transcript splicing in the area of exon 1. Of 14 asymptomatic members of proband families in 6 subjects were diagnosed with AIP, and in 8 the AIP was excluded based on the DNA sequencing method. DNA sequencing based analysis is the only reliable method for correct diagnosis of asymptomatic non-erythroid AIP patients with normal urinary excretion of heme precursors. The mutations found in Polish patients with non-erythroid AIP represented those of splice defect and resulted in abnormal exon 1 splicing.

Preclinical Development of a Subcutaneous ALAS1 RNAi Therapeutic for Treatment of Hepatic Porphyrias Using Circulating RNA Quantification.

The acute hepatic porphyrias are caused by inherited enzymatic deficiencies in the heme biosynthesis pathway. Induction of the first enzyme 5-aminolevulinic acid synthase 1 (ALAS1) by triggers such as fasting or drug exposure can lead to accumulation of neurotoxic heme intermediates that cause disease symptoms. We have demonstrated that hepatic ALAS1 silencing using siRNA in a lipid nanoparticle effectively prevents and treats induced attacks in a mouse model of acute intermittent porphyria. Herein, we report the development of ALN-AS1, an investigational GalNAc-conjugated RNAi therapeutic targeting ALAS1. One challenge in advancing ALN-AS1 to patients is the inability to detect liver ALAS1 mRNA in the absence of liver biopsies. We here describe a less invasive circulating extracellular RNA detection assay to monitor RNAi drug activity in serum and urine. A striking correlation in ALAS1 mRNA was observed across liver, serum, and urine in both rodents and nonhuman primates (NHPs) following treatment with ALN-AS1. Moreover, in donor-matched human urine and serum, we demonstrate a notable correspondence in ALAS1 levels, minimal interday assay variability, low interpatient variability from serial sample collections, and the ability to distinguish between healthy volunteers and porphyria patients with induced ALAS1 levels. The collective data highlight the potential utility of this assay in the clinical development of ALN-AS1, and in broadening our understanding of acute hepatic porphyrias disease pathophysiology.