Search

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.

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.

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.

Background & Aims: Hepatic iron toxicity may be mediated by free radical species and lipid peroxidation of biological membranes. The antioxidant property of silybin, a main constituent of natural flavonoids, was investigated in vivo during experimental iron overload., Methods: Rats were fed a 2.5% carbonyl-iron diet and 100 mg.kg body wt-1.day-1 silybin for 4 months and were assayed for accumulation of hepatic lipid peroxidation by-products by immunocytochemistry, mitochondrial energy-dependent functions, and mitochondrial malondialdehyde content., Results: Iron overload caused a dramatic accumulation of malondialdehyde-protein adducts into iron-filled periportal hepatocytes that was decreased appreciably by silybin treatment. The same beneficial effect of silybin was found on the iron-induced accumulation of malondialdehyde in mitochondria. As to the liver functional efficiency, mitochondrial energy wasting and tissue adenosine triphosphate depletion induced by iron overload were successfully counteracted by silybin., Conclusions: Oral administration of silybin protects against iron-induced hepatic toxicity in vivo. This effect seems to be caused by the prominent antioxidant activity of this compound.

Background/aims: The molecular defect of genetic hemochromatosis (GH) is unknown. It is believed that low expression of duodenal ferritin in GH is caused by tissue or cell specific defect of ferritin synthesis. Our study was designed to ascertain whether the control of duodenal ferritin synthesis in GH was defective., Methods: Expression at the single cell level of H and L ferritin messenger RNAs and protein and activity of the iron regulatory factor, which controls the translation of ferritin messenger RNA, were assessed in 43 duodenal biopsy specimens from individuals with GH, secondary hemochromatosis (SH), anemia, or normal iron balance., Results: Signal for ferritin H and L subunit messenger RNAs was detected in both absorptive and nonabsorptive cells by in situ hybridization, but in 10 of 14 patients with untreated GH, the signal was lower than in patients with SH or normal subjects. However, immunostaining for ferritin protein documented a diffuse/cytoplasmic pattern, whereas a supranuclear/granular staining was found in normal subjects or patients with SH. The spontaneous activity of duodenal iron regulatory factor was consistently higher in patients with GH than in normal subjects or subjects with anemia or SH., Conclusions: In patients with GH, ferritin gene transcription is preserved in both absorptive and nonabsorptive intestinal cells. Low accumulation of ferritin is not caused by a defective control of ferritin synthesis but by low expression of ferritin messenger RNA and sustained activity of iron regulatory factor.

Background/aims: Liver fibrosis and cirrhosis represent common pathological findings in humans with iron overload. This study was undertaken to assess whether in vivo targeting of iron to liver parenchymal or nonparenchymal cells would differently affect collagen gene activity., Methods: Rats were treated with an iron diet or intramuscular injections of iron dextran, and in situ hybridization analyses on liver samples were performed., Results: These iron treatments determined parenchymal or reticuloendothelial cell iron overload, respectively. The typical distribution of iron into different liver cells was documented by histochemistry and confirmed by in situ hybridization analysis with a ferritin L complementary RNA probe. In iron-fed rats, in situ hybridization analysis identified a signal for collagen type I messenger RNA into nonparenchymal cells in zones I and II. In rats with nonparenchymal cell iron overload, no activation of collagen gene expression was detected into or near iron-laden nonparenchymal cells. These findings were also confirmed by quantitative Northern blot analysis., Conclusions: The results of this study indicate that, regardless of the total hepatic iron burden, selective localization of iron into liver cells (i.e., parenchymal cells) is required for the activation of collagen gene during long-term iron overload in rodents.

In recent years, identifying the hepatic cell type responsible for collagen synthesis in experimental models of postnecrotic or inflammatory fibrosis has been the subject of active investigation. In primary iron overload states, however, hepatic fibrosis and cirrhosis occur without accompanying necroinflammatory phenomena. In this study, we combined morphological, immunological, cell isolation and purification and molecular biological techniques to identify the hepatic cell responsible for enhanced collagen type I gene expression during chronic enteral iron overload in the rat. Ultrastructural analysis of liver tissue sections from iron-loaded rats specifically revealed an altered appearance of fat-storing cells, which showed few if any fat droplets left and increased rough endoplasmic reticulum. In situ hybridization analysis with specific complementary RNA probes identified enhanced signal for collagen type I into nonparenchymal cells in zones 1 and 2, without signal over the background onto iron-laden hepatocytes. Immunocytochemistry with desmin antibodies combined with in situ hybridization on the same tissue sections identified the cells expressing high level of collagen type I transcripts as fat-storing cells. Northern-blot analysis on RNA extracted from various purified cell isolates, confirmed the presence of collagen type I mRNA signal only into the fat-storing cells isolate. Our study shows that in an experimental model of metabolic fibrosis in which the hepatotoxin selectively accumulates into parenchymal cells, fat-storing cells are the main source of enhanced collagen type I gene expression.

The need for accurate and noninvasive evaluation of liver iron stores prompted us to evaluate the reliability of high-field magnetic resonance imaging equipment in liver patients with low or moderate siderosis, given the poor results obtained using systems operating at low field strength in such cases. Twenty patients with sporadic porphyria cutanea tarda and 28 with comparable chronic liver diseases (chronic hepatitis or cirrhosis) and moderate siderosis were compared with 10 patients with idiopathic or secondary hemochromatosis and 10 healthy controls. Plasma iron profile, ferritin concentration and liver iron concentration, determined with atomic absorption spectroscopy, were matched with the magnetic resonance parameters-namely, transverse relaxation time and the signal intensity for a given proton amount, obtained with equipment operating at a field strength of 1.5 T. Hemochromatosis patients with mean liver iron concentrations of 550 mumol/gm dry wt (vs. 10 mumol of controls) exhibited an impressive reduction in the signal intensity with respect to the other three groups, and this reduction prevented any further comparison with the same porphyria cutanea tarda and chronic liver disease groups, whose liver iron level was twice that of the controls. The signal intensity remained almost unchanged in the latter groups, whereas the transverse relaxation time was significantly reduced. Moreover, correlation with liver iron was significantly inverse in the case of the transverse relaxation time (n = 17, r = 0.62, p = 0.008) and direct in the case of the transverse relaxation rate. The transverse relaxation time values returned to normal in five patients who had completed an iron-depletion program.(ABSTRACT TRUNCATED AT 250 WORDS)

The ingestion of an amino acid mixture lacking tryptophan causes a rapid fall of plasma tryptophan in healthy subjects. This is because amino acids elicit protein synthesis and endogenous tryptophan is incorporated into new proteins. If protein synthesis is the mechanism through which tryptophan-free solution decrease blood tryptophan, it may be interesting to study tryptophan levels after a tryptophan-free mixture in subjects with impaired protein synthesis. In the present paper we show that in 27 cirrhotics the administration of a tryptophan-free solution caused a fall of total plasma tryptophan that began significantly later than in 14 control subjects, the delay being significantly proportional to the severity of the disease. The difference between control and cirrhotic subjects was due to the bound fraction of plasma tryptophan. The diagnostic and clinical usefulness of our findings are discussed.

To gain insights at the molecular level into the expression of iron-regulated genes [transferrin (Tf), transferrin receptor (TfR), and ferritin H and L subunits] in human intestinal areas relevant to iron absorption, the steady-state levels of specific messenger RNAs (mRNAs) were analyzed in gastric and duodenal samples obtained from 6 normal subjects, or 10 patients with anemia, 14 patients with untreated iron overload, and 8 patients with various gastrointestinal disorders. No Tf mRNA was detected in human gastroduodenal tissue, confirming earlier findings in the rat. In normal subjects, although higher levels of ferritin H- and L-subunit mRNAs were consistently found in duodenal than in gastric samples, no differences in the content of TfR transcripts were detected. However, a dramatic increase in TfR mRNA levels was specifically found in duodenal samples from subjects with mild iron deficiency but severe anemia. This response of the TfR gene is presumably secondary to decreased cellular iron content due to its accelerated transfer into the bloodstream, as also indicated by the low levels of ferritin subunit mRNAs found in the same tissue samples, and is not linked to faster growth rate of mucosal cells because no changes in duodenal expression of histone, a growth-related gene, were detected. In patients with secondary iron overload, a down-regulation of duodenal TfR gene expression and a concomitant increase in ferritin mRNA content were documented. On the contrary, a lack of TfR gene down-regulation and an abnormally low accumulation of ferritin H- and L-subunit mRNAs were detected in the duodenums of subjects with idiopathic hemochromatosis. Whether these molecular abnormalities in idiopathic hemochromatosis are relevant to the metabolic defect(s) of the disease is presently unknown.

The role played by carotenoids, retinol and tocopherol in quencing oxidative cellular damage and combatting tumor growth is well documented, but little is known about their activity in human liver cirrhosis (LC), where oxidative damage and tumoral complications are common-place. We investigated 59 patients with LC of different etiology on admission to hospital and compared them with 32 healthy controls, matched for age and sex. Nutritional (cutaneous skinfolds, creatinine-height index) and serum parameters were determined; of these, alpha- and beta-carotene, cryptoxanthin, lycopene, retinol and alpha-tocopherol were detected by an high-performance liquid chromatographic (HPLC) technique, devised in our laboratory, which afforded an accurate and simultaneous resolution of all six compounds. The results point to a significant reduction in almost all the vitamin factors in LC, as well as in total serum lipids. In consequence, the ratio tocopherol/total serum lipids remains almost unchanged: 2.45 +/- 0.08 (m +/- se) in controls and 2.34 +/- 0.16 in patients. The effects of age, sex, nutritional habits, alcohol, malnutrition and the severity of the disease were also evaluated in relation to the vitamin-factor levels. It is suggested that the reduced levels observed in LC patients are due to a number of factors including portal hypertension and lymphatic circulation impairment, and it is concluded that thorough screening and improved diet are beneficial in the follow-up of LC.