Variegate Porphyria (VP) is an autosomal dominant disorder found worldwide but is rare in Italy. In this study we provide an overview of clinical, biochemical and genetic background of 33 Italian VP patients diagnosed in the last fifteen years. About 70% of patients had experienced clinical symptoms: 43.4% had photosensivity, 8.7% acute attacks and 47.8% both. Among the 33 patients, 14 different mutations were identified. Of these only 6 defects have been previously described in other countries and 8 are unique having been identified for the first time in Italy. Two of these, the c.851G>T and the c.1013C>G, were found in two and four unrelated families respectively. No mutation has been found in homozygosis and no significant correlation has been observed between specific clinical and biochemical manifestations and the type of mutation. In contrast, normal faecal protoporphyrin excretion was high predictive of silent phenotype. Normal urinary excretion of PBG and ALA, predicted absence of neurovisceral symptoms. This paper represents the first compilation of data on genotype-phenotype relation in Italian patients with VP.

Coproporphyrin (CP) is one of the main by-products of heme biosynthesis and its abnormal accumulation is associated with different forms of porphyria. Indirect data obtained from animal and human models have suggested a possible role for Multidrug Resistance-associated Protein 2 (MRP2) and other MRPs in hepatocyte excretion of CP. Using normal, MRP2-deficient and a cholestatic rat model, we have assessed the role of MRPs in CP disposition. MRP levels were assayed using immunofluorescence. Biliary and urinary excretion patterns of CP and conjugate bilirubin were measured during equimolar infusions of CP isomers with and without phenoldibromopthalein sulfonate (BSP), a well-known MRP2 substrate. Our results suggest a role for the MRP system as a possible regulator of CP traffic and accumulation in normal and pathological conditions. Alteration in this systems (as observed in cholestatic disease) may play an important role in triggering clinical expression of porphyria in individuals with underlying mutations leading to porphyrin accumulation and may help explain the phenotypic heterogeneity in patients affected by different forms of porphyrias.

Background: Endothelial dysfunction in cigarette smokers has been ascribed to increased oxidative damage. The aims of the present study were to compare the endothelial function of normotensive smokers with that of non-smokers and to examine its relation to some parameters representative of oxidative damage and of antioxidant capacity.Methods: We investigated 32 chronic smokers (15-30 cigarettes daily) affected by coronary heart disease, ranging from acute myocardial infarction to instable angina pectoris, and 28 matched non-smokers without any definite risk factors. All subjects underwent assessment of nitric oxide (NO)-dependent endothelial function, measured as brachial artery vasodilatation in response to reactive ischemia, using a standardized echographic method. Plasma and urinary levels of NO were also measured in all subjects, as were urinary 15-isoprostane F-2t, plasma serum lipids, homocysteine (Hcy), ascorbic acid, retinol, tocopherol, and alpha- and beta-carotene (by high-performance liquid chromatography).Results: Smokers showed a significantly lower NO-mediated vasodilatation response (3.50% vs. 6.18%, p<0.001) and higher levels of urinary NO metabolites and 15-isoprostane F-2t. They also had higher levels of Hey (p<0.001); these values were significantly and inversely related to NO serum levels (r=-0.512, p<0.001). Moreover, smokers had a significant and corresponding reduction in circulating levels of ascorbic acid, tocopherol, and alpha- and beta-carotene.Conclusions: The present study shows a clear relation between endothelial dysfunction (NO production impairment) and cigarette smoking, especially in the presence of high levels of LDL-cholesterol. It also defines some markers of both oxidative damage and antioxidant protective capacity in this condition. The monitoring of these factors may be advisable in order to assess the amount of endothelial damage. (c) 2007 European Federation of Internal Medicine. Published by Elsevier B.V. All rights reserved.

Acute intermittent porphyria (AIP) is an autosomal disorder caused by molecular abnormalities in the gene coding for hydroxymethylbilane synthase (HMBS), the third enzyme in the heme biosynthetic pathway. So far, more than 242 different mutations responsible for AIP have been identified in this gene. In an Italian family with typical clinical and biochemical signs of AIP, no mutation was found by direct sequencing of the entire hydroxymethylbilane synthase gene (HMBS). All the symptomatic patients showed apparent homozygosity and absence of mendelian segregation for eleven common polymorphisms along the gene. Excluding interference of polymorphisms in the primer sites, we assumed the presence of a complete HMBS gene deletion. In order to identify the size of this deletion, single nucleotide polymorphisms (SNPs) analysis was extended to flanking genes, H2A Histone Family member X (H2AFX) and Dolichyl-Phosphate N-Acetylglucosamine Phosphotransferase 1 (DPAGT1), downstream and Vacuolar protein sorting 11 (VPS11), upstream. Heterozygous polymorphisms in the VPS11 and DPAGT1 genes were found. Thus, we performed a Long-PCR with primers situated in regions outside the homozygous polymorphisms and we identified a double deletion with inversion on chromosome 11 (g22516974_22524062del7088, g22524062_22524278inv216, g22524278_22531093del6815). Even if the deletions include the entire HMBS and H2AFX genes and 1463 bp of the final portion of DPAGT1 gene, our patients had no other symptoms than AIP.

Summary: Given the crucial role of iron and porphyrins in oxidative cellular damage in the chronic porphyrias, we undertook an extensive study in families with acute porphyrias to evaluate the possible role of similar oxidative damage in these diseases, whose natural history is often also complicated by neoplastic evolution. Four unrelated patients with acute intermittent porphyria (AIP) were studied together with 37 members of four different families. Aminolevulinic acid and porphobilinogen were measured in urine, and porphyrins in urine, plasma and stools. The activity of the congenitally deficient enzyme, porphobilinogen deaminase, and the concentrations of plasma iron, transferrin, ferritin, and various antioxidants (ascorbic acid, retinol, tocopherol, α- and β-carotene, by a personal HPLC method) and the urinary and plasma metabolites of nitrous oxide were also assayed. The results showed no relationship between the observed increase of porphyrin metabolites and the presence of markers of oxidative damage or the decrease of circulating antioxidants: however, when such a decrease was registered, it depended on spontaneous or iatrogenic iron accumulation. We conclude that family screening, recommended for the identification of AIP carriers, must also include evaluation of iron stores with a view to preventing the oxidative damage and in order to forestall the neoplastic evolution of the disease.

Background: Recent trials in digestive-tract cancer have produced conflicting results regarding the protective role of liposoluble vitamins. Accordingly, we have undertaken an extensive appraisal of the behaviour of retinol and tocopherol in both human upper and lower digestive neoplasms. Materials and Methods: The subjects comprised six healthy controls, 10 patients with symptomatic cholelithiasis, 13 with gastric neoplasms, 12 with colo-rectal neoplasms and 13 with digestive neoplasms and liver metastases. Retinol and tocopherol in the plasma, liver, as well as from secondary malignant nodules of the liver, were determined following a high performance liquid chromatographic technique. Results: The plasma concentration of retinol was significantly reduced only in some neoplastic groups with respect to the non-neoplastic groups. Plasma tocopherol levels remained almost unchanged and, surprisingly, they were higher even in the neoplastic patients and in relation to total serum lipids. There was a sharp decrease in the liver tissue levels of total, mainly esterified, retinol in both cholelithiasis and neoplastic groups; tocopherol, on the other hand, remained more or less unchanged, except in the liver metastatic nodules, where it increased. Conclusion: Levels of plasma vitamin and tissue retinol and tocopherol appear to be unpredictable on the basis of the corresponding circulating levels, and this must be taken into account when prescribing dietary and therapeutic regimes.

The porphyrias are disorders associated with inherited or acquired enzyme deficiencies in the heme biosynthetic pathway. The differential diagnosis is often difficult since the phenotype is very similar in some forms and the biochemical tests are not commonly available. Here we provide an update on the molecular diagnosis of porphyrias in Italy and a flow-chart to facilitate the identification of mutations in heme biosynthetic genes. The molecular analysis has allowed us to identify the molecular defect underlying the disease in 66 probands with different porphyrias [acute intermittent porphyria (AIP), variegate porphyria (VP), porphyria cutanea tarda (PCT), erythropoietic protoporphyria (EPP)]. No Italian patients with defects in coproporphyrinogen oxidise (CPOX) gene, responsible for hereditary coproporphyria (HCP), have been detected. The molecular characterization has been extended to 115 relatives with the identification of 55 asymptomatic mutation carriers and 60 normal subjects. We have so far identified 50 different mutations among 4 genes associated with the most common porphyrias showing a high molecular heterogeneity: 22 in the hydroxymethylbilane synthase (HMBS) gene (AIP), 7 in the protoporphyrinogen oxidase (PPOX) gene (VP), 16 in the uroporphyrinogen decarboxylase (UROD) gene (PCT) and 5 in the ferrochelatase (FECH) gene (EPP). Among the 50 molecular defects, 29 seem to be restricted to the Italian population.

We investigated through a population-based case-control study the hypothesis that disturbances in the chemistry of copper and zinc and in activity of the antioxidant enzyme copper/zinc superoxide-dismutase (SOD1) are involved in the etiopathogenesis of sporadic amyotrophic lateral sclerosis (ALS). We recruited 20 patients with sporadic ALS and 22 population controls from three northern Italian provinces, and we analyzed zinc and copper content and SOD1 activity in erythrocytes. These variables were unrelated to disease progression as evaluated through a disability score; zinc concentrations inversely correlated with copper in referents but not in patients. SOD1 activity was lower and erythrocyte zinc and copper levels were slightly higher in patients than in referents. Comparing the second to the bottom tertile of erythrocyte SOD1 activity, relative risk of ALS was 0.4 (95% confidence interval 0.1–2.0); the risk further decreased to 0.1 (95% confidence interval 0–0.9) for comparison of highest to lowest tertile (P for trend 0.027). Copper and zinc levels were not associated with disease risk. Our findings indicate that a lower SOD1 activity is associated with ALS, but we cannot be sure whether this association is a marker of causal action or is secondary to a confounder, or to disease onset itself.

The aim of this study was to evaluate the prognostic factors at presentation and survival in Italian patients with hepatocellular carcinoma (HCC). Clinical and demographic data of 176 patients consecutively observed from 1993 to 1997 were evaluated by univariate and multivariate analyses. Overall median survival was 18 months. At univariate analysis, low albumin, high bilirubin, high alkaline phosphatase, high alpha-fetoprotein (AFP); high platelet count, hepatitis B surface antigen (HBsAg)-positivity, the presence of ascites, of encephalopathy, of portal vein thrombosis (PVT), male sex, no treatment, poor differentiation, untreatable tumours and incidental diagnosis were each associated with shorter survival. HBsAg-positive subjects more often presented with untreatable lesions or diffuse tumours (P=0.001 and P=0.007, respectively) and had significantly worse survival (P=0.0057). By multiple regression analysis, low albumin, high bilirubin, abnormal AFP, presence of PVT and of untreatable lesions were independent risk factors for worse survival. Thus, the most important factors influencing survival are the degree of functional impairment of the liver, the presence of hepatitis B viral (HBV) infection, the type of diagnosis and the aggressiveness of the tumour.

Background: It is known that antioxidant liposoluble vitamins and carotenoids are reduced in liver cirrhosis, but little is known about chronic viral hepatitis, where oxidative damage has to be taken into account. Methods: Fifty-five patients with chronic hepatitis, mainly C virus-related, were matched with 16 patients with biliary stones and 20 healthy controls. Plasma and liver analyses were carried out using a well-tried HPLC technique that affords an accurate quantification of retinol, tocopherol, alpha- and beta-carotene, cryptoxanthin, and lycopene. Results: Plasma concentration of retinol, tocopherol, beta-carotene, and lycopene was significantly decreased in both patient groups, particularly in those with chronic hepatitis. In contrast, liver concentration of both esterified and free retinol, tocopherol, and some carotenoids was better preserved in the hepatitis group than in the cholelithiasis group. A strict correspondence between aminotransferases and the amount of liver-stored retinol was documented. Conclusions: Plasma vitamin and carotenoid depletion co-existing with preserved liver storage may indicate a functional defect in liver pool mobilization or even a real depletion of the antioxidant defenses, which play a key role in averting cellular damage. The implications for nutrition and therapy need to be taken into account.

Acute intermittent porphyria (AIP) is an autosomal disorder caused by molecular abnormalities in the gene coding for hydroxymethylbilane synthase (HMBS), the third enzyme in the heme biosynthetic pathway. So far, more than 170 different mutations responsible for AIP have been identified worldwide in the HMBS gene. In this study we have performed molecular characterization in 14 patients with suspected diagnosis of AIP and in 29 family members of Italian ancestry. Molecular analysis of the HMBS gene allowed us to identify 13 different mutations among 14 patients with reduced HMBS activity: 5 splicing defects (IVS9+22 G>A, 612 G>T, IVS11-2 delA, IVS12+2 T>C, and IVS13-1 G>A), 1 small insertion (182 insGA), 1 small deletion (730-731 delCT), and 6 missense/nonsense mutations (76 C>T, 295 G>A, 331 G>A, 580 C>T, 673 C>T, and 874 C>T), resulting in single-amino-acid substitutions or protein truncations. Six of these molecular abnormalities had already been described and 7 are new findings. In a previous work on an Italian population we detected 7 different mutations among 8 AIP patients. This study has raised to 18 the number of different mutations so far found among the Italian AIP population, 11 of which are new findings. We can conclude that the mutation screening in the Italian population contributes to improvement of the diagnostic approach of AIP and to establishing possible clustering of mutations in the Mediterranean area.

A female child with peculiar facies, obesity, cleft lip and palate, growth hormone deficiency and mental retardation is described. The present case does not appear to fit any of the known syndromes.

We analyzed the 1986-1997 mortality in a cohort of 2065 residents of an Italian municipality which had been exposed to drinking water with a high content of inorganic selenium over a long period of time, and compared it with mortality in the remainder of the municipal population. Mortality from malignant neoplasms increased [standardized mortality ratio (SMR) 1.17, 95% confidence interval (CI) 0.96-1.42], mainly due to an excess mortality from melanoma and colorectal cancer in both sexes, kidney cancer in men, and lymphoid malignancies in women. Overall cardiovascular mortality changed little (SMR 1.05, 95% CI 0.89-1.23), despite the higher cerebrovascular mortality (SMR 1.43, 95% CI 1.03-1.93). Coronary disease mortality slightly decreased (SMR 0.87, 95% CI 0.63-1.16), due to a low mortality among women. We also noted an excess mortality from Parkinson's disease in men and from motor neuron disease in women. Evaluation of these findings is, however, hampered by the lack of information about potential lifestyle confounders, the fact that the exposure could only be characterized by a simple dichotomization, and the inconsistencies of most estimates between the two sexes.

BACKGROUND:Porphyria cutanea tarda and haemochromatosis are taken to be spontaneous human models of oxidative cellular damage, with an increased risk of fibrosis and cancer evolution.AIM:To define the relative pro-oxidant roles of porphyrin and iron, in their different molecular forms, and their effects on antioxidant biological systems.PATIENTS:A group of 17 patients with porphyria cutanea tarda and a group of 14 patients with primary and secondary haemochromatosis, were compared with 21 healthy controls.METHODS:Plasma retinol, tocopherol, alpha- and beta-carotene, ascorbic acid, glutathione, malonyldialdehyde and red blood cell free iron were determined using high performance liquid chromatography.RESULTS:Only a modest increase in iron stores was demonstrated in the porphyria cutanea tarda group; in the haemochromatosis patients ferritin levels were almost seven times higher. By contrast, there was a sharp and virtually identical increase in red blood cell free iron and malonyldialdehyde in both the patient groups. A significant reduction was observed in retinol, alpha-, beta-carotene and red blood cell glutathione levels being more marked in porphyria cutanea tarda than in haemochromatosis patients.CONCLUSIONS:The study confirms the strong pro-oxidant effects of porphyrins in vivo, through an induction of the free toxic iron form, even though the total iron pool is not greatly expanded. The additional free-iron and porphyrin oxidant effects are documented both in red blood cell and plasma in the porphyria cutanea tarda group. It confirmed that aging exerts a negative influence in terms of pro- and antioxidant balance in all cases, but particularly in the haemochromatosis group.

The high incidence of hepatocellular carcinoma (HCC) in cirrhosis, where previous studies have indicated a severe reduction in several antioxidant vitamin factors, prompted us to compare plasma liposoluble vitamins with tocopherol content in healthy and neoplastic liver tissue in humans. This, with a view to a more positive preventive dietary approach, given the conflicting results obtained by liposoluble vitamin dietary supplementation in different malignancies. Eleven patients with cirrhosis, 18 patients affected by cirrhosis with HCC, and 10 patients with liver metastases (LM) from digestive tract adenocarcinomas were compared with controls who had undergone perlaparoscopic cholecistectomy. Plasma alpha- and beta-carotene, retinol and tocopherol, together with liver tocopherol, from both nonmalignant portions and malignant nodules of the same organ, were determined by high-performance liquid chromatography following a well-assessed technique. The results confirm a trend towards a reduction in circulating carotenoids and tocopherol in cirrhosis and in patients affected by cirrhosis with HCC. Tocopherol content in liver tissue is significantly decreased in cirrhosis (0.26 + 0.03 micromol/g prot., mean + SEM, P < .001) and in cirrhotic areas of the HCC group (0.31 + 0.02, P < .002), with respect to its content in liver specimens of healthy controls (0.46 + 0.03) and in healthy areas of the same organ in patients with LM (0.41 + 0.03). Tocopherol concentration is further reduced by 50% in malignant liver nodules of HCC, with respect to surrounding cirrhotic tissue, whereas in metastatic liver nodules from digestive neoplasms the tocopherol content is almost twice that of healthy surrounding areas. This unpredictable tocopherol behavior in liver specimens, of secondary as opposed to primary malignancies of the liver, affords further insight into the conflicting effects of liposoluble vitamins employed in the chemopreventive treatment of different malignant diseases, where hepatic tocopherol concentration show opposite trends: halved in primary HCC and doubled in LM of digestive adenocarcinomas, with respect to healthy controls.

AbstractWe assessed total body water (TBW) and extracellular water (ECW) in thirty-four non-ascitic cirrhotics and twenty healthy controls by 2H2O and Br dilution. In the same subjects, bioelectric impedance (BI) was recorded at multiple frequencies. Body hydration was similar for controls (mean 55·6 (SD 6·7)), less-severe cirrhotics (Child-Pugh classification A; CPA; n 21, mean 56·2 (SD 6·2)) and moderately-severe cirrhotics (Child-Pugh classification B; CPB; n 13, mean 57·2 (SD 5·4)). However, intracellular water standardized per litre TBW was significantly higher in CPB subjects (mean 27·0 (SD 7·5); P<0·01) compared with CPA (mean 21·3 (SD 10·6)) and control subjects (mean 18·0 (SD 9·8)). Published formulas for predicting TBW and ECW from BI at multiple frequencies were applied to the cirrhotics. These formulas gave accurate predictions of TBW and ECW, although standard errors of estimates were higher for CPB subjects (TBW ≤ 2·5 and ECW ≤ 2·11) than those for CPA (TBW ≤ 2·0 and ECW ≤ 1·81) and control (TBW 1·4 and ECW 0·9 1) subjects

The authors consider two groups of patients with overt sporadic porphyria cutanea tarda (PCT) from different continents, with the aim of evaluating the possible impairment of the liposoluble antioxidative system, given the possible synergic effect of porphyrins and iron in promoting oxidative cellular damage. Twenty-three Italian outpatients with overt sporadic PCT and 11 outpatients with PCT from Buenos Aires (Argentina) were matched with 60 patients with liver cirrhosis and 52 healthy Italian controls. Serum levels of alpha- and beta-carotene, cryptoxanthin, zeaxanthin, lutein, lycopene, retinol and alpha-tocopherol were detected by a high-performance liquid chromatographic technique devised in our laboratory, which afforded an accurate and simultaneous resolution of ah these compounds. The results point to a significant reduction in plasma levels of alpha- and beta-carotene in both the PCT populations with respect not only to controls, but also to the cirrhotic population, which had more severe liver damage. Moreover, other carotenoids with proven antioxidative properties, like cryptoxanthin and lycopene, are greatly reduced in our PCT populations. This confirms the suggested synergic effect of iron and porphyrins in the oxidative intracellular damage with consequent depletion of antioxidative liposoluble molecules.

The principles of generation of magnetic resonance imaging (MRI) are resumed by briefly explaining the effects of an external magnetic field (EMF) on hydrogen nuclei and of pulses of radiofrequency (RF) radiation. The latter creates a resonant effect, and the same nuclei, moved from the external field axis, when RF pulse is stopped, will "relax" to their original alignment in the magnetic field and in so doing radiate the absorbed energy to their surroundings. This energy provides a signal that can be detected and spatially resolved by the receiver coil wrapped around the patient, through a computerized system. After briefly explaining also the distinctive parameters T2 and T1, the author presents his experience in the MRI detection of different degrees of siderosis of the liver--ranging from idiopathic and secondary haemochromatosis to milder siderosis of alcoholic liver disease and porphyria cutanea tarda. The results were accomplished by employing an equipment operating at an enhanced field strength (1.5 Tesla). Previous reports have validated this technique in order to distinguish idiopathic from secondary haemochromatosis. Furthermore, the present study shows that even low to moderate degrees of liver iron deposition can be appreciated and roughly quantitated by the decrease of the transverse relaxation time (T2), which resulted proportional to the amount of liver iron, under these operating conditions. Thus, MRI is proposed as an useful and non-invasive way to detect iron deposition and to follow up iron depletion treatments.

The Authors determined zinc (Zn) and magnesium (Mg) in the plasma, urine, erythrocytes (RBCs), mono- and polymorphonuclear cells (MNCs and PMNs) of patients with overt alcoholic and nonalcoholic liver cirrhosis. In order to obtain a clearer clinical picture, biochemical and nutritional parameters (retinol, tocopherol, six different carotenoids, creatinine-height index and tricipital skinfold), as well as markers of portal hypertension (spleno-portal size and platelet count) were also evaluated.The plasma levels of Zn and Mg were found to be reduced, as were the urine levels of Mg. Urine Zn, on the other hand, was higher than normal. Plasma Zn correlated inversely, and urine Zn directly, with the severity of the disease, rather than with alcohol consumption or treatment with diuretics. Protein metabolism impairment would appear to affect the plasma transport of Zn rather than its overall availability in the organism; the opposite was found in the case of Mg, the availability of which appeared to be reduced. Determination of the two elements in RBCs, MNCs and PMNs suggested that a true nutritional deficit cannot be demonstrated. MNCs, rather than RBCs or PMNs seem better to reflect tissue status of trace elements.