Haem Biosynthesis and Antioxidant Enzymes in Circulating Cells of Acute Intermittent Porphyria Patients.

The aims of the present study were to explore the expression pattern of haem biosynthesis enzymes in circulating cells of patients affected by two types of porphyria (acute intermittent, AIP, and variegate porphyria, VP), together with the antioxidant enzyme pattern in AIP in order to identify a possible situation of oxidative stress. Sixteen and twelve patients affected by AIP and VP, respectively, were analysed with the same numbers of healthy matched controls. Erythrocytes, neutrophils and peripheral blood mononuclear cells (PBMCs) were purified from blood, and RNA and proteins were extracted for quantitative real time PCR (qRT-PCR) and Western-blot analysis, respectively. Porhobilinogen deaminase (PBGD) and protoporphyrinogen oxidase (PPOX) gene and protein expression was analysed. Antioxidant enzyme activity and gene expression were additionally determined in blood cells, together with protein carbonyl content in plasma. PBMCs isolated from AIP patients presented low mRNA levels of PBGD when compared to controls, while PBMCs isolated from VP patients presented a decrease in PPOX mRNA. PPOX protein content was higher in AIP patients and lower in VP patients, compared to healthy controls. Regarding antioxidant enzymes, PBMCs and erythrocyte superoxide dismutase (SOD) presented statistically significant higher activity in AIP patients compared to controls, while catalase activity tended to be lower in these patients. No differences were observed regarding antioxidant gene expression in white blood cells. Circulating cells in AIP and VP patients present altered expression of haem biosynthetic enzymes, which could be useful for the differential diagnosis of these two types of porphyria in certain difficult cases. AIP patients present a condition of potential oxidative stress similar to VP patients, evidenced by the post-transcriptional activation of SOD and possible catalase impairment.

Antioxidants restore protoporphyrinogen oxidase in variegate porphyria patients.

BACKGROUND: Variegate porphyria (VP) is the result of decreased protoporphyrinogen oxidase (PPOX) activity and results in the accumulation of porphyrins and porphyrin precursors. Our aims were to analyse the basal antioxidant defences and oxidative damage markers and the effects of a diet supplementation with vitamins E and C on the oxidant/antioxidant status and PPOX gene expression in lymphocytes of variegate porphyria (VP) patients. MATERIALS AND METHODS: Twelve women affected by VP and 12 control women participated in a randomized and double-blind crossover study. Each participant took either 50 mg/day vitamin E and 150 mg/day vitamin C or a placebo for 6 months. RESULTS: Lymphocyte PPOX gene expression, together with catalase and glutathione peroxidase activities, was reduced in VP women. No differences were observed in the levels of malondialdehyde and protein carbonyl derivatives. Stimulated lymphocyte H2 O2 production was higher in porphyric women. Supplementation with antioxidant vitamins increased PPOX expression in VP patients. Glutathione reductase (GRd) and superoxide dismutase (SOD) activities were higher in the treatment groups. CONCLUSIONS: Lymphocytes from VP patients show reduced PPOX expression and present a greater susceptibility to producing H2 O2 and impaired H2 O2 detoxifying mechanisms. Supplementation with vitamins E and C restores PPOX expression in VP patients and enhances GRd and SOD activity, suggesting the potential benefits of a diet rich in vitamins E and C in these patients.

Impaired lymphocyte mitochondrial antioxidant defences in variegate porphyria are accompanied by more inducible reactive oxygen species production and DNA damage.

This study aimed to analyse lymphocyte reactive oxygen species (ROS) production and detoxification mechanisms and the appearance of oxidative damage in variegate porphyria (VP) patients. Twelve women affected by VP and 12 pair-matched healthy control women participated in the study. VP women presented impaired expression of the mitochondrial proteins protoporphyrinogen oxidase, uncoupling protein-3, Bcl-2 and sirtuin 3. Lymphocytes from VP women presented higher H(2)O(2) production than controls after stimulation with phorbol myristate acetate. The inhibition of H(2)O(2) production after in vitro lymphocyte treatment with myxothiazol pointed towards complex III of the mitochondrial respiratory chain as the main contributor of the higher ROS production in porphyric subjects. No differences were observed between VP and control subjects in the levels of DNA damage, assessed by the comet assay method in un-treated lymphocytes. However, DNA damage, expressed both as a percentage of DNA in tail and as the tail moment, was greater in VP women than controls after lymphocyte treatment with H(2)O(2). In conclusion, lymphocytes from VP women showed impaired expression of mitochondrial antioxidant defences but no significant signs of oxidative stress were evidenced in basal, non-stressing conditions; however, lymphocytes of VP women were more susceptible to producing mitochondrial ROS and to suffering oxidative damage when submitted to stressful situations.

Variegate porphyria induces plasma and neutrophil oxidative stress: effects of dietary supplementation with vitamins E and C.

Our aim was to analyse the influence of variegate porphyria (VP) on the antioxidant defenses and markers of oxidative damage and inflammation in plasma and neutrophils and the effects of dietary supplementation with vitamins E and C on these parameters in plasma, neutrophils and erythrocytes. Twelve women affected by VP and twelve pair-matched healthy control women participated in a double-blind crossover study. Each participant took 50 mg/d of vitamin E and 150 mg/d of vitamin C, or a placebo, for 6 months, by consuming an almond-based beverage as the vehicle. Women affected by VP presented higher C-reactive protein and malondialdehyde (MDA) circulating levels. Plasma antioxidant defenses were not different between porphyric and control women. Neutrophils from VP women presented decreased catalase (CAT) and glutathione reductase (GR) activities together with increased protein carbonyl levels. Reactive oxygen species (ROS) production from stimulated neutrophils was also higher in porphyric women than their controls. Dietary supplementation was effective in increasing alpha-tocopherol levels in neutrophils and in reducing MDA levels in plasma. Erythrocyte CAT and GR activities were enhanced by the enriched beverage only in the control subjects. In conclusion, women affected by VP present a situation of inflammation, plasma oxidative damage and neutrophils more primed to the oxidative burst, with decreased antioxidant activities and increased ROS production capabilities and protein oxidative damage. Dietary supplementation with vitamin E (50 mg/d) and vitamin C (150 mg/d) for 6 months decreased plasma oxidative damage and enhanced the erythrocyte activities of CAT and GR.

montalcino, A zebrafish model for variegate porphyria.

OBJECTIVE: Inherited or acquired mutations in the heme biosynthetic pathway leads to a debilitating class of diseases collectively known as porphyrias, with symptoms that can include anemia, cutaneous photosensitivity, and neurovisceral dysfunction. In a genetic screen for hematopoietic mutants, we isolated a zebrafish mutant, montalcino (mno), which displays hypochromic anemia and porphyria. The objective of this study was to identify the defective gene and characterize the phenotype of the zebrafish mutant. MATERIALS AND METHODS: Genetic linkage analysis was utilized to identify the region harboring the mno mutation. Candidate gene analysis together with reverse transcriptase polymerase chain reaction was utilized to identify the genetic mutation, which was confirmed via allele-specific oligo hybridizations. Whole mount in situ hybridizations and o-dianisidine staining were used to characterize the phenotype of the mno mutant. mRNA and morpholino microinjections were performed to phenocopy and/or rescue the mutant phenotype. RESULTS: Homozygous mno mutant embryos have a defect in the protoporphyrinogen oxidase (ppox) gene, which encodes the enzyme that catalyzes the oxidation of protoporphyrinogen. Homozygous mutant embryos are deficient in hemoglobin, and by 36 hours post-fertilization are visibly anemic and porphyric. The hypochromic anemia of mno embryos was partially rescued by human ppox, providing evidence for the conservation of function between human and zebrafish ppox. CONCLUSION: In humans, mutations in ppox result in variegate porphyria. At present, effective treatment for acute attacks requires the administration intravenous hemin and/or glucose. Thus, mno represents a powerful model for investigation, and a tool for future screens aimed at identifying chemical modifiers of variegate porphyria.

Utility of plasma fluorometric emission scanning for diagnosis of the first 2 cases reports of variegate porphyria: a very rare type of porphyrias in Thai.

Two Thai women who are siblings presented with a history of recurrent pruritic vesicles on dorsum of both hands and extensor surface of forearms where the sun-exposed areas are. The excoriated vesicles were healed with depressed scars. They had no previous history of intense abdominal pain, seizure, or psychiatric disorder Urinary porphyrins were analyzed by High Performance Liquid Chromatography (HPLC). The level of coproporphyrin III was detected to be higher than the uroporphyrin level. Fluorescence emission scanning of both patients' plasma was performed and demonstrated typical emission peak at 626 nm, that confirmed the diagnosis of variegate porphyria.

Overrepresentation of the founder PPOX gene mutation R59W in a South African patient with severe clinical manifestation of porphyria.

A patient, who presented with abdominal pain and severe photosensitivity that resulted in scarring and mutilation of the fingers, nose and ears, was referred for biochemical assessment of porphyria and DNA screening. Although these clinical manifestations were suggestive of both acute porphyria and congenital erythropoietic porphyria, the biochemical profile was consistent with variegate porphyria (VP). Analysis of the protoporphyrinogen oxidase (PPOX) gene underlying VP resulted in the identification of the founder mutation R59W in a heterozygous state in this patient. Despite extensive mutation analysis, no other potential disease-causing genetic alterations could be detected in the PPOX gene or the uroporphyrinogen III synthase gene. Slight overrepresentation of the mutant PPOX allele was however, observed repeatedly in DNA of the proband compared to other R59W heterozygotes, including his mother who also tested positive for mutation R59W using restriction enzyme analysis and direct DNA sequencing. Confirmation of this phenomenon by real-time polymerase chain reaction analysis and microsatellite analysis, using highly informative markers flanking the PPOX gene, raised the possibility of partial homozygosity for VP in this patient. This study represents the first report of overrepresentation of mutation R59W in a patient with a severe form of VP. A homozygote for the R59W mutation has never been detected, and the severe clinical manifestation observed in our patient is consistent with the hypothesis that such a genotype will not be compatible with life.