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

Primer sequences and conditions used in the quantitative real time PCR analysis.

<p>Primer sequences and conditions used in the quantitative real time PCR analysis.</p

Effects of acute intermittent porphyria (AIP) and variegate porphyria (VP) on erythrocyte porphobilinogen deaminase (PBGD) and protoporphyrinogen oxidase (PPOX) protein content.

<p>Total protein content in the control group was normalized to 100% (dashed line). Statistical analysis: Student’s t-test for unpaired data. (*) indicates statistically significant differences between porphyria and control groups (p < 0.05). C1: Control for AIP patients. C2: Control for VP patients.</p

Effects of acute intermittent porphyria (AIP) and variegate porphyria (VP) on PBMCs porphobilinogen deaminase (PBGD) and protoporphyrinogen oxidase (PPOX) gene expression (A) and protein content (B, C).

<p>(A) The relative quantification was performed by standard calculations considering 2^(-ΔΔCt). mRNA levels of control subjects were arbitrarily referred to as 1 (dashed line). (B) Total protein content in the control group was normalized to 100% (dashed line). Results are expressed as mean ± sem. Statistical analysis: Student’s t-test for unpaired data. (*) indicates statistically significant differences between porphyria and control groups (p < 0.05). C1: Control for AIP patients. C2: Control for VP patients.</p