Porfiria hepato eritropoyética asociada a diabetes mellitus tipo 1: a propósito de un caso clínico / A clinical case of hepato-erythropoietic porphyria associated with type 1 diabetes mellitus / Relato de caso de porfiria hepato-eritropoiética associada a diabetes mellitus tipo 1

Las porfirias son un grupo complejo y heterogéneo de defectos en la vía de la síntesis del hemo. La porfiria hepato eritropoyética es un subtipo muy poco frecuente y de presentación en la infancia, con compromiso cutáneo predominante. Describimos el caso clínico de una paciente de 5 años, que se presenta con lesiones cutáneas e hipertricosis, se confirma el diagnóstico por elevación de uroporfirinas en orina y secuenciación del gen UROD.

Porphyria is a complex and heterogeneous group of heme synthesis disorder. Hepato-erythropoietic porphyria is a very rare subtype that onsets in childhood, and shows predominant skin involvement. We describe the clinical case of a 5-year-old patient who showed skin lesions and hypertrichosis and whose diagnosis was confirmed due to increased uroporphyrins in urine and UROD gene sequencing

A porfiria é um grupo complexo e heterogêneo de distúrbios da síntese do grupo heme. A porfiria hepato-eritropoiética é um subtipo muito raro que se inicia na infância e mostra envolvimento predominante da pele. Descrevemos o caso clínico de uma paciente de 5 anos que apresentou lesões cutâneas e hipertricose e cujo diagnóstico foi confirmado por aumento de uroporfirinas na urina e sequenciamento do gene UROD.

Direct assay of uroporphyrin and coproporphyrin in human urine by reverse-mode field amplified sample injection-sweeping and micellar electrokinetic chromatography.

In this study, an on-line stacking capillary electrophoresis (CE) method, reverse-mode field amplified sample injection equipped with sweeping micellar electrokinetic chromatography (RMFASI-sweeping MEKC), was established for direct determination of uroporphyrin and coproporphyrin in human urine. Porphyrins playing a very important role in the biosynthesis of heme, chlorophyll and other important enzymes are a series of important molecules in organism. Therefore, determination of porphyrin metabolites, uroporphyrin and coproporphyrin, was very important for clinical survey of some diseases. In this study, the urine sample after simple dilution could be directly analyzed by this on-line stacking CE method. The optimal CE separation buffer was 70 mM phosphate buffer at pH 3. Before sample injection, a water plug was introduced (2.5 psi for 10s), and then the samples were loaded by electrokinetic injection (-10 kV, 200 s). Finally, the phosphate buffer (70 mM, pH 3) containing 100mM SDS was served as the sweeping buffer to stack and separate the analytes at -20 kV. The calibration curves were linear over a range of 15-200 ng/ml for uroporphyrin, and 300-1000 ng/ml for coproporphyrin. The coefficient of correlation (r) in intra-batch (n=5) and inter-batch (n=5) analysis was above 0.983. The LODs (S/N=3) were 5 ng/ml for uroporphyrin, and 100ng/ml for coproporphyrin. The absolute values of relative standard deviation (RSD) and relative error (RE) in intra-batch (n=5) and inter-batch (n=5) assays were less than 8.6% showing the good precision and accuracy. The stacking method was successfully applied in real urine sample and feasible for serving as a tool for detection of uroporphyrin and coproporphyrin in clinical.

A novel, selective, and rapid fluorimetric method for the simultaneous analysis of coproporphyrin and uroporphyrin in urine.

A simple and rapid spectrofluorimetric method is described for the determination of the closely overlapping mixture of coproporphyrin (CP) and uroporphyrin (UP) in urine samples. Matrix Isopotential Synchronous Fluorescence Spectrometry (MISFS) was applied to improve the spectral resolution for the severely overlapped spectra of the urinary porphyrins. First-order derivative technique eliminates the background interference of each component on the other. Using these two techniques together, selectivity was improved, while maintaining a high sensitivity, and time-consuming separation processes and multiple scanning processes were avoided. The limits of detection were 0.15 nmol L(-1) and 0.1 nmol L(-1) for CP and UP, respectively. The concentrations of CP and UP were determined from the peak amplitudes of the Derivative Matrix Isopotential Synchronous Fluorescence (DMISF) spectra, at their detection points where the interference was suppressed. Porphyrins excretion in urine samples, collected from normal subjects, was studied. A comparison between the new method and the anion-exchange chromatographic method of Martinez and Mills was established using Bland-Altman method and the results indicate that these two methods are in a good agreement with each other.

Feline acute intermittent porphyria: a phenocopy masquerading as an erythropoietic porphyria due to dominant and recessive hydroxymethylbilane synthase mutations.

Human acute intermittent porphyria (AIP), the most common acute hepatic porphyria, is an autosomal dominant inborn error of heme biosynthesis due to the half-normal activity of hydroxymethylbilane synthase (HMB-synthase). Here, we describe the first naturally occurring animal model of AIP in four unrelated cat lines who presented phenotypically as congenital erythropoietic porphyria (CEP). Affected cats had erythrodontia, brownish urine, fluorescent bones, and markedly elevated urinary uroporphyrin (URO) and coproporphyrin (COPRO) consistent with CEP. However, their uroporphyrinogen-III-synthase (URO-synthase) activities (deficient in CEP) were normal. Notably, affected cats had half-normal HMB-synthase activities and elevated urinary 5-aminolevulinic acid (ALA) and porphobilinogen (PBG), the deficient enzyme and accumulated metabolites in human AIP. Sequencing the feline HMB-synthase gene revealed different mutations in each line: a duplication (c.189dupT), an in-frame 3 bp deletion (c.842_844delGAG) identical to that causing human AIP and two missense mutations, c.250G>A (p.A84T) and c.445C>T (p.R149W). Prokaryotic expression of mutations c.842_844delGAG and c.445C>T resulted in mutant enzymes with <1% wild-type activity, whereas c.250G>A expressed a stable enzyme with approximately 35% of wild-type activity. The discolored teeth from the affected cats contained markedly elevated URO I and III, accounting for the CEP-like phenocopy. In three lines, the phenotype was an autosomal dominant trait, while affected cats with the c.250G>A (p.A84T) mutation were homozygous, a unique recessive form of AIP. These animal models may permit further investigation of the pathogenesis of the acute, life-threatening neurological attacks in human AIP and the evaluation of therapeutic strategies. GenBank Accession Numbers: GQ850461-GQ850464.

Spot urine porphyrins/creatinine ratio profile of healthy Brazilian individuals adjusted for personal habits

Changes in urinary porphyrin excretion may be the result of hereditary causes and/or from environmental or occupational exposure. The objective of this study was to measure the amount of some porphyrins in spot urine samples obtained from volunteers randomly selected from a healthy adult population of São Paulo with a sensitive HPLC method and to estimate normal ranges for a non-exposed population. Spot urine samples were collected from 126 subjects (both genders, 18 to 65 years old) not occupationally exposed to porphyrinogenic agents. Porphyrin fractions were separated on RP-18 HPLC column eluted with a methanol/ammonium acetate buffer gradient, pH 4.0, and measured fluorometrically (excitation 405 nm/emission 620 nm). The amount of porphyrins was corrected for urinary creatinine excretion. Only 8-carboxyl (uro) and 4-carboxyl (copro) porphyrins were quantified as µg/g creatinine. Data regarding age, gender, occupational activities, smoking and drinking habits were analyzed by Mann-Whitney and Kruskal-Wallis tests. Uroporphyrin results did not differ significantly between the subgroups studied. Copro and uro + copro porphyrins were significantly different for smokers (P = 0.008) and occupational activities (P = 0.004). With respect to alcohol consumption, only men drinking >20 g/week showed significant differences in the levels of copro (P = 0.022) and uro + copro porphyrins (P = 0.012). The 2.5-97.5th percentile limit values, excluding those for subjects with an alcohol drinking habit >20 g/week, were 0-20.8, 11.7-93.1, and 15.9-102.9 µg/g creatinine for uro, copro and uro + copro porphyrins, respectively. These percentile limit values can be proposed as a first attempt to provide urinary porphyrin reference values for our population, serving for an early diagnosis of porphyrinopathies or as biomarkers of exposure to porphyrinogenic agents.

Spot urine porphyrins/creatinine ratio profile of healthy Brazilian individuals adjusted for personal habits.

Changes in urinary porphyrin excretion may be the result of hereditary causes and/or from environmental or occupational exposure. The objective of this study was to measure the amount of some porphyrins in spot urine samples obtained from volunteers randomly selected from a healthy adult population of São Paulo with a sensitive HPLC method and to estimate normal ranges for a non-exposed population. Spot urine samples were collected from 126 subjects (both genders, 18 to 65 years old) not occupationally exposed to porphyrinogenic agents. Porphyrin fractions were separated on RP-18 HPLC column eluted with a methanol/ammonium acetate buffer gradient, pH 4.0, and measured fluorometrically (excitation 405 nm/emission 620 nm). The amount of porphyrins was corrected for urinary creatinine excretion. Only 8-carboxyl (uro) and 4-carboxyl (copro) porphyrins were quantified as microg/g creatinine. Data regarding age, gender, occupational activities, smoking and drinking habits were analyzed by Mann-Whitney and Kruskal-Wallis tests. Uroporphyrin results did not differ significantly between the subgroups studied. Copro and uro + copro porphyrins were significantly different for smokers (P = 0.008) and occupational activities (P = 0.004). With respect to alcohol consumption, only men drinking >20 g/week showed significant differences in the levels of copro (P = 0.022) and uro + copro porphyrins (P = 0.012). The 2.5-97.5th percentile limit values, excluding those for subjects with an alcohol drinking habit >20 g/week, were 0-20.8, 11.7-93.1, and 15.9-102.9 microg/g creatinine for uro, copro and uro + copro porphyrins, respectively. These percentile limit values can be proposed as a first attempt to provide urinary porphyrin reference values for our population, serving for an early diagnosis of porphyrinopathies or as biomarkers of exposure to porphyrinogenic agents.

Porphyria cutanea tarda and liver disease. A retrospective analysis of 17 cases from a single centre and review of the literature.

BACKGROUND/AIMS: Sporadic Porphyria Cutanea Tarda (sPCT) is associated with liver disease, e.g. HCV infection, haemochromatosis and especially alcoholic liver disease. We conducted a retrospective analysis on the prevalence of liver disorders in association with Porphyria Cutanea Tarda (PCT), in a university referral centre. METHODS: The PCT cases were retrieved from computerized databases. Patient files lacking information on the presence of concomitant liver disease were excluded from further analysis. RESULTS: 29 PCT patients were retrieved from our databases, of which 17 patients with sPCT were retained for further analysis. Patients were middle aged (mean age: 43 +/- 3) and there was no gender difference (10 males vs. 7 females). Almost all patients had iron overload (14/17). 5 patients had chronic HCV, with type 1b in 3 of them, 7 abused alcohol, 4 patients had hereditary haemochromatosis (3 homozygous C282Y--1 heterozygous H63D/C282Y). In 3 patients sPCT was associated with medication intake and one patient had chronic hepatitis B (HBV). 13 patients were treated with phlebotomies, with success in 11/13. 4 patients were treated with chloroquine, 3 of which also underwent phlebotomies. Of the 5 patients with HCV, 3 were successfully treated with combined antiviral therapy; one of them is planned to be treated; one patient never received therapy and was lost from follow-up. One patient developed hepatocellular carcinoma (HCC) during a median follow-up of 24 years. CONCLUSIONS: We found a significant association between sPCT and liver disorders, such as chronic HCV infection, alcohol abuse, iron overload and hereditary haemochromatosis. Therefore, patients presenting with PCT should be screened for concomitant liver disease. Iron overload is present in a majority of patients, the majority of patients can be successfully treated with phlebotomies. The risk of developing HCC in our sPCT patients and in literature is low.

Mast cells and transforming growth factor-beta expression: a possible relationship in the development of porphyria cutanea tarda skin lesions.

BACKGROUND: Porphyria cutanea tarda (PCT) is a metabolic disease characterized by vesicles and blisters in sun-exposed areas and scleroderma-like lesions in sun-exposed and non-sun-exposed areas. Mast cells participate in the pathogenesis of bullous diseases and diseases that show sclerosis, including PCT. Moreover, transforming growth factor-beta (TGF-beta) is the main cytokine in the development of tissue sclerosis. The correlation of mast cells and TGF-beta with the lesions of PCT has not been examined, however. The possible role of mast cells and TGF-beta (and the relationship between them) in the development of PCT lesions is discussed. METHODS: To quantify mast cells and cells expressing TGF-beta in skin samples from patients with PCT and controls, immunohistochemical studies were performed in tissue sections allied to morphometric analyses. RESULTS: The numbers of mast cells and cells expressing TGF-beta per square millimeter were increased in the PCT group relative to controls, and there was a direct and significant correlation between the mast cell number and cells expressing TGF-beta in PCT. CONCLUSIONS: The results suggest that the increased number of mast cells and of cells expressing TGF-beta, as well as their direct correlation, may contribute to the pathogenesis of the skin lesions in PCT.

Urinary arsenic and porphyrin profile in C57BL/6J mice chronically exposed to monomethylarsonous acid (MMAIII) for two years.

Arsenicals are proven carcinogens in humans and it imposes significant health impacts on both humans and animals. Recently monomethylarsonous acid (MMA(III)), the toxic metabolite of arsenic has been identified in human urine and believed to be more acutely toxic than arsenite and arsenate. Arsenic also affects the activity of a number of haem biosynthesis enzymes. As a part of 2-year arsenic carcinogenicity study, young female C57BL/6J mice were given drinking water containing 0, 100, 250 and 500 microg/L arsenic as MMA(III)ad libitum. 24 h urine samples were collected at 0, 1, 2, 4, 8 weeks and every 8 weeks for up to 104 weeks. Urinary arsenic speciation and porphyrins were measured using HPLC-ICP-MS and HPLC with fluorescence detection respectively. DMA(V) was a major urinary metabolite detected. Significant dose-response relationship was observed between control and treatment groups after 1, 4, 24, 32, 48, 56, 88, 96 and 104 weeks. The level of uroporphyrin in 250 and 500 microg As/L group is significantly different from the control group after 4, 8, 16, 32, 56, 72, 80, 96 and 104 weeks. Coproporphyrin I level in 500 microAs/L group is significantly different from control group after 8, 24, 32, 40, 56, 72, 80, 88 and 104 weeks. After 4 weeks the level of coproporphyrin III concentration significantly increased in all the treatment groups compared to the control except week 16 and 48. Our results show urinary DMA(V) and porphyrin profile can be used as an early warning biomarker for chronic MMA(III) exposure before the onset of cancer.

[Porphyrins as the early biomarkers for arsenic exposure of human].

To investigate the effects of arsenic exposure on porphyrins excretion profiles in human, porphyrins were measured by HPLC and total arsenic by HG-AAS in urine samples collected from arsenicosis-endemic areas and control sites in Guizhou Province. Analytical data showed that urinary uroporphyrin-III and coproporphyrin-III were significantly elevated in arsenic-exposed group compared with those in control group, while urinary coproporphyrin-I was not significantly higher in arsenic-exposed group than that in control group. Not any significant difference was found in porphyrins between the male and female except for 20-40 years age group. As far as age was concerned, arsenic-exposed group of <20 years showed significant increases in uroporphyrin-III and coproporphyrin-III compared with the control group of <20 years. Similarly, arsenic-exposed groups of 20-40 years and >40 years also showed significant increases in coproporphyrin-III compared with corresponding age groups of control. Besides, there were positive correlations between the urinary arsenic and total coproporphyrin, and total porphyrin. The effects of arsenic exposure were associated with increased porphyrins excretion, which was suggested that porphyrins were possible to be used as biomarkers of early health effect due to arsenicosis.