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Photodynamic therapy (PDT) treats nonmelanoma skin cancer. PDT kills cells through reactive oxygen species (ROS), generated by interaction among cellular O2, photosensitizer and specific light. Protoporphyrin IX (PpIX) is a photosensitizer produced from methyl aminolevulinate (MAL) by heme group synthesis (HGS) pathway. In PDT-resistant cells, PDT efficacy has been improved by addition of epigallocatechin gallate (EGCG). Therefore, the aim of this work is to evaluate the effect of EGCG properties over MAL-TFD and PpIX production on A-431 cell line. EGCG's role over cell proliferation (flow cytometry and wound healing assay) and clonogenic capability (clonogenic assay) was evaluated in A-431 cell line, while the effect of EGCG over MAL-PDT was determined by cell viability assay (MTT), PpIX and ROS detection (flow cytometry), intracellular iron quantification and gene expression of HGS enzymes (RT-qPCR). Low concentrations of EGCG (<50 µM) did not have an antiproliferative effect over A-431 cells; however, EGCG inhibited clonogenic cell capability. Furthermore, EGCG (<50 µM) improved MAL-PDT cytotoxicity, increasing PpIX and ROS levels, exerting a positive influence on PpIX synthesis, decreasing intracellular iron concentration and modifying HGS enzyme gene expression such as PGB (upregulated) and FECH (downregulated). EGCG inhibits clonogenic capability and modulates PpIX synthesis, enhancing PDT efficacy in resistant cells. [ABSTRACT FROM AUTHOR]

Acute intermitttent porphyria belongs to a rare group of diseases hallmarked by deficient biosynthesis of heme. It carries a significant symptom burden, both physical and emotional,and therefore palliative care has emerged as an essential tool in the armamentarium of porphyria management. It takes care of the patient as a whole and caters to all aspects that the disease process demands. There are many lacunae in the literature regarding the palliative management of porphyria. We are reporting a case of a 16-year-old female who presented with severe abdominal pain, lower backache and symmetrical bilateral lower limb pain to the palliative ward referred by the neurology department for supportive care. This case describes the palliative care aspects of porphyria management which was successfully provided in the palliative care unit right from referral till the last. A multidisciplinary palliative care team managed the patient, and the necessary interventions were provided to the patient and family. Palliative acre in AIP needs to be emphasized, and palliative care services need to be utilized in these cases. The unavailability of specific treatment measure, heme, in countries like India further emphasizes the need for long-term supportive care for the patient and family. The case shows the importance of palliative care throughout the disease course as it is a chronic disease with significant morbidity and carries a heavy symptom burden. This case provides the insight that rather than conventional management alone for such chronic diseases, palliative care should be incorportated. Early integration with palliative care helps in exploring all the domains of disease. This is one of the first cases reported highlighting palliative care in porphyria , bridging the gap in the literature. [ABSTRACT FROM AUTHOR]

Background and Purpose: Acute intermittent porphyria (AIP) is a rare disease caused by a genetic mutation in the hepatic activity of the porphobilinogen‐deaminase. We aimed to develop a mechanistic model of the enzymatic restoration effects of a novel therapy based on the administration of different formulations of recombinant human‐PBGD (rhPBGD) linked to the ApoAI lipoprotein. This fusion protein circulates in blood, incorporating into HDL and penetrating hepatocytes. Experimental Approach: Single i.v. dose of different formulations of rhPBGD linked to ApoAI were administered to AIP mice in which a porphyric attack was triggered by i.p. phenobarbital. Data consist on 24 h urine excreted amounts of heme precursors, 5‐aminolevulinic acid (ALA), PBG and total porphyrins that were analysed using non‐linear mixed‐effects analysis. Key Results: The mechanistic model successfully characterized over time the amounts excreted in urine of the three heme precursors for different formulations of rhPBGD and unravelled several mechanisms in the heme pathway, such as the regulation in ALA synthesis by heme. Treatment with rhPBGD formulations restored PBGD activity, increasing up to 51 times the value of the rate of tPOR formation estimated from baseline. Model‐based simulations showed that several formulation prototypes provided efficient protective effects when administered up to 1 week prior to the occurrence of the AIP attack. Conclusion and Implications: The model developed had excellent performance over a range of doses and formulation type. This mechanistic model warrants use beyond ApoAI‐conjugates and represents a useful tool towards more efficient drug treatments of other enzymopenias as well as for acute intermittent porphyria. [ABSTRACT FROM AUTHOR]

Acute hepatic porphyrias (AHPs) are inborn errors of hemebiosynthesis and its most common and severe type is the acute intermittent porphyria (AIP). AIP is an hereditary autosomal dominant disease caused by accumulated porphobilinogen deaminase (PBG) and delta aminolevulin acid (ALA) products. The main symptoms are severe abdominal pain, neuromuscular and psychiatric disturbances, nausea, vomiting, encephalopathy, tachycardia, seizures, tremors and hypertension, that usually are manifested by acute crises. The treatment is based on clinical management and in cases which the patient’s quality of life is affected liver transplantation (LT) may be an alternative choice. We report the case of a patient with AHP presenting recurrent crisis leading to chronic symptoms occurrence and poor quality of life with progressive unresponsiveness to hemin treatment. Patient was submitted to LT as curative therapy proposal, but patient still presents some clinical manifestations that may indicate the possibility of a secondary cause to explain persistence of her symptoms despite of biochemical normalization of ALA and PBG.

The enzyme hydroxymethylbilane synthase (HMBS; EC 4.3.1.8), also known as porphobilinogen deaminase, catalyses the stepwise addition of four molecules of porphobilinogen to form the linear tetrapyrrole 1‐hydroxymethylbilane. Thirty years of crystal structures are surveyed in this topical review. These crystal structures aim at the elucidation of the structural basis of the complex reaction mechanism involving the formation of tetrapyrrole from individual porphobilinogen units. The consistency between the various structures is assessed. This includes an evaluation of the precision of each molecular model and what was not modelled. A survey is also made of the crystallization conditions used in the context of the operational pH of the enzyme. The combination of 3D structural techniques, seeking accuracy, has also been a feature of this research effort. Thus, SAXS, NMR and computational molecular dynamics have also been applied. The general framework is also a considerable chemistry research effort to understand the function of the enzyme and its medical pathologies in acute intermittent porphyria (AIP). Mutational studies and their impact on the catalytic reaction provide insight into the basis of AIP and are also invaluable for guiding the understanding of the crystal structure results. Future directions for research on HMBS are described, including the need to determine the protonation states of key amino‐acid residues identified as being catalytically important. The question remains – what is the molecular engine for this complex reaction? Thermal fluctuations are the only suggestion thus far. [ABSTRACT FROM AUTHOR]

Pathogenic heterozygous variants in HMBS encoding the enzyme hydroxymethylbilane synthase (HMBS), also known as porphobilinogen deaminase, cause acute intermittent porphyria (AIP). Biallelic variants in HMBS have been reported in a small number of children with severe progressive neurological disease and in three adult siblings with a more slowly, progressive neurological disease and distinct leukoencephalopathy. We report three further adult individuals who share a distinct pattern of white matter abnormality on brain MRI in association with biallelic variants in HMBS, two individuals with homozygous variants, and one with compound‐heterozygous variants. We present their clinical and radiological features and compare these with the three adult siblings previously described with leukoencephalopathy and biallelic HMBS variants. All six affected individuals presented with slowly progressive spasticity, ataxia, peripheral neuropathy, with or without mild cognitive impairment, and/or ocular disease with onset in childhood or adolescence. Their brain MRIs show mainly confluent signal abnormalities in the periventricular and deep white matter and bilateral thalami. This recognizable pattern of MRI abnormalities is seen in all six adults described here. Biallelic variants in HMBS cause a phenotype that is distinct from AIP. It is not known whether AIP treatments benefit individuals with HMBS‐related leukoencephalopathy. One individual reported here had improved neurological function for 12 months following liver transplantation followed by decline and progression of disease. [ABSTRACT FROM AUTHOR]

BackgroundRecent descriptions of the clinical and laboratory features of subjects with acute porphyrias in the US are lacking. Our aim was to describe clinical, biochemical, and genetic features of 108 subjects.MethodsBetween September 2010 and December 2012, 108 subjects with acute porphyrias (90 acute intermittent porphyrias, 9 hereditary coproporphyrias, 9 variegate porphyrias) were enrolled into an observational study. Genetic testing was performed at a central genetic testing laboratory and clinical information entered into a central database. Selected features were compared with data for adults in the US.ResultsMost subjects (88/108, 81%) were female, with self-reported onset of symptoms in the second through fourth decades of life. The most common symptom was abdominal pain. Appendectomies and cholecystectomies were common before a diagnosis of porphyria. The diagnosis was delayed by a mean of 15 years. Anxiety and depression were common, and 18% complained of chronic symptoms, especially neuropathic and other pains. The incidences of systemic arterial hypertension, chronic kidney disease, seizure disorders, and psychiatric conditions were markedly increased. Mutations of the known causative genes were found in 102/105 of those tested, with novel mutations being found in 37, including in 7/8 subjects with hereditary coproporphyria. Therapy with intravenous hematin was the most effective therapy both for treatment of acute attacks and for prevention of recurrent attacks.ConclusionsAcute porphyrias often remain undiagnosed for more than a decade after first symptoms develop. Intravenous hematin is the treatment of choice, both for treatment of acute attacks and for prevention of recurrent attacks.

This study aims to present the serum metabolite profiles of patients with acute intermittent porphyria (AIP) and identify specific metabolites that could potentially discriminate between AIP, asymptomatic HMBS mutation carriers, and healthy individuals. The study cohort included 46 female participants: 21 AIP patients, 5 asymptomatic carriers, and 20 ‘normal’ participants (without HMBS gene mutation). Serum samples were analyzed for 157 selected metabolites or clinical variables using an assay combining liquid chromatography MS/MS and direct flow injection. AUC analysis was used to distinguish unique variables between the three groups. A total of 15 variables differed significantly between the AIP and normal control group (VIP score > 1.0 and p < 0.05 with FDR correction). In AIP patients, the levels tyrosine, valine, and eGFR were significantly lower, and the levels of sphingomyelin C16:0, C24:0, C24:1, phosphatidylcholine diacyl C32:1, C36:1, C36:3, ornithine, sarcosine, citrulline, blood urea nitrogen AST, and ALT were significantly higher. The AUC of these 15 variables in discriminating between normal and AIP patients ranged between 0.73 and 0.94 (p < 0.05). In conclusion, serum metabolic profiles differ between normal individuals and patients carrying the HMBS mutation. The unique metabolites associated with AIP identified in this study may be useful for monitoring the development of AIP symptoms.

In most eukaryotic phototrophs, the entire heme synthesis is localized to the plastid, and enzymes of cyanobacterial origin dominate the pathway. Despite that, porphobilinogen deaminase (PBGD), the enzyme responsible for the synthesis of hydroxymethybilane in the plastid, shows phylogenetic affiliation to α-proteobacteria, the supposed ancestor of mitochondria. Surprisingly, no PBGD of such origin is found in the heme pathway of the supposed partners of the primary plastid endosymbiosis, a primarily heterotrophic eukaryote, and a cyanobacterium. It appears that α-proteobacterial PBGD is absent from glaucophytes but is present in rhodophytes, chlorophytes, plants, and most algae with complex plastids. This may suggest that in eukaryotic phototrophs, except for glaucophytes, either the gene from the mitochondrial ancestor was retained while the cyanobacterial and eukaryotic pseudoparalogs were lost in evolution, or the gene was acquired by non-endosymbiotic gene transfer from an unspecified α-proteobacterium and functionally replaced its cyanobacterial and eukaryotic counterparts.

Acute intermittent porphyria (AIP) is an autosomal dominant inherited disease with low clinical penetrance, caused by mutations in the hydroxymethylbilane synthase (HMBS) gene, which encodes the third enzyme in the haem biosynthesis pathway. In susceptible HMBS mutation carriers, triggering factors such as hormonal changes and commonly used drugs induce an overproduction and accumulation of toxic haem precursors in the liver. Clinically, this presents as acute attacks characterised by severe abdominal pain and a wide array of neurological and psychiatric symptoms, and, in the long-term setting, the development of primary liver cancer, hypertension and kidney failure. Treatment options are few, and therapies preventing the development of symptomatic disease and long-term complications are non-existent. Here, we provide an overview of the disorder and treatments already in use in clinical practice, in addition to other therapies under development or in the pipeline. We also introduce the pathomechanistic effects of HMBS mutations, and present and discuss emerging therapeutic options based on HMBS stabilisation and the regulation of proteostasis. These are novel mechanistic therapeutic approaches with the potential of prophylactic correction of the disease by totally or partially recovering the enzyme functionality. The present scenario appears promising for upcoming patient-tailored interventions in AIP.

Background Acute 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. Methods The 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. Results Isoflurane 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. Discussion Biochemical 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 significance This is the first study using volatile anaesthetics in an AIP genetic model confirming Isoflurane and Sevoflurane porphyrinogenicity. [ABSTRACT FROM AUTHOR]

The enzyme porphobilinogen deaminase (PBGD) is one of the key enzymes in tetrapyrrole biosynthesis. It catalyses the formation of a linear tetrapyrrole from four molecules of the substrate porphobilinogen (PBG). It has a dipyrromethane cofactor (DPM) in the active site which is covalently linked to a conserved cysteine residue through a thioether bridge. The substrate molecules are linked to the cofactor in a stepwise head-to-tail manner during the reaction, which is catalysed by a conserved aspartate residue: Asp82 in the B. megaterium enzyme. Three mutations have been made affecting Asp82 (D82A, D82E and D82N) and their crystal structures have been determined at resolutions of 2.7, 1.8 and 1.9 Å, respectively. These structures reveal that whilst the D82E mutant possesses the DPM cofactor, in the D82N and D82A mutants the cofactor is likely to be missing, incompletely assembled or disordered. Comparison of the mutant PBGD structures with that of the wild-type enzyme shows that there are significant domain movements and suggests that the enzyme adopts `open' and `closed' conformations, potentially in response to substrate binding. [ABSTRACT FROM AUTHOR]