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In nonalcoholic steatohepatitis animal models, an increased lipid droplet size in hepatocytes is associated with fibrogenesis. Hepatocytes with large droplet (Ld-MaS) or small droplet (Sd-MaS) macrovesicular steatosis may coexist in the human liver, but the factors associated with the predominance of one type over the other, including hepatic fibrogenic capacity, are unknown. In pre-ischemic liver biopsies from 225 consecutive liver transplant donors, we retrospectively counted hepatocytes with Ld-MaS and Sd-MaS and defined the predominant type of steatosis as involving ≥50% of steatotic hepatocytes. We analyzed a donor Patatin-like phospholipase domain-containing protein 3 (PNPLA3) rs738409 polymorphism, hepatic expression of proteins involved in lipid metabolism by RT-PCR, hepatic stellate cell (HSC) activation by α-SMA immunohistochemistry and, one year after transplantation, histological progression of fibrosis due to Hepatitis C Virus (HCV) recurrence. Seventy-four livers had no steatosis, and there were 98 and 53 with predominant Ld-MaS and Sd-MaS, respectively. In linear regression models, adjusted for many donor variables, the percentage of steatotic hepatocytes affected by Ld-MaS was inversely associated with hepatic expression of Insulin Induced Gene 1 (INSIG-1) and Niemann-Pick C1-Like 1 gene (NPC1L1) and directly with donor PNPLA3 variant M, HSC activation and progression of post-transplant fibrosis. In humans, Ld-MaS formation by hepatocytes is associated with abnormal PNPLA3-mediated lipolysis, downregulation of both the intracellular cholesterol sensor and cholesterol reabsorption from bile and increased hepatic fibrogenesis.

Background & Aims We analysed for the first time whether recipient perioperative serum total cholesterol (sTC) concentration is associated with liver transplantation outcome. Methods We studied noncholestatic cirrhotics submitted to primary deceased-donor liver transplantation in a prospective group (n = 140) from Rome and in a validation retrospective cohort (n = 157) from Udine, Italy. Pre-ischaemia and post-reperfusion cholesterol metabolism gene mRNA was measured by RT-PCR in 74 grafts of the study group. Results At Cox regression analysis, independently from confounders including recipient MELD score, the recipient pre-operative sTC pooled quintiles 2–5, compared with the lowest quintile showed HR (95% CI) and significances for overall graft loss (GL) of 0.215 (0.104–0.444) P

The aim of this investigation was to compare different indices for evaluating nutritional conditions of red deer (Cervus elaphus) in an alpine environment during the autumn in order to detect the most convenient ones for management purposes in our specific situation. Body conditions of 274 red deer were evaluated using kidney fat index, back fat index and body condition scores. Body Condition Scores was the easiest but the least reliable method. Both kidney fat index and back fat index were significantly affected by age and sex class (always lower in younger animals) and, in females, also by lactation status. In stags, a negative regression effect of culling date on both kidney fat index and back fat index was observed. A significant positive correlation between kidney fat index and back fat index was recorded. Both kidney fat index and back fat index were objective indicators of nutritional status and sensitive to changes in physical conditions, but back fat index was both quicker and easier to be measured. As a direct implication, we suggest that back fat index can be a practical and reliable indicator for monitoring red deer conditions in alpine areas during the autumn, provided that the effects of sex, age and date of culling are taken into account.

Background/Aims We evaluated the role and mechanisms by which the GH/IGF1 axis modulates cholangiocyte proliferation. Methods GH-receptors (GH-R), IGF1, IGFBP3 (binding protein 3), IGF1-R and receptor substrates (IRS) were evaluated in cholangiocytes of normal or bile duct-ligated (BDL) rat livers. The effects of GH and IGF1 on proliferation of normal quiescent cholangiocytes and the transduction pathways involved were investigated. Results IGF1, GH-R, IGF1-R, IRS-1/2 were expressed in normal cholangiocytes and overexpressed in cholangiocytes proliferating after BDL which also secrete IGF1 in a higher amount than normal cells. IGFBP3, which may counter-regulate IGF1 effects, was decreased in BDL cholangiocytes. IGF1 promoted cholangiocyte proliferation in association with overexpression of p-IGF1R, IRS1, IRS-2, p-ERK1/2 and p-AKT. GH induced IGF1 expression and release in isolated cholangiocytes, and reproduced the effects of IGF1 but GH effects were abolished by IGF1-R blocking antibody, suggesting IGF1 as a mediator of GH. Finally, IGF1 and 17β-estradiol reciprocally potentiated their proliferative effects on cholangiocytes, and by interacting at both receptor and post-receptor levels. Conclusions Cholangiocytes respond to GH with production and release of IGF1 that modulates cell proliferation by transduction pathways involving IGF1-R, IRS1/2 and both ERK and PI3-kinase pathways. The biliary epithelium is a target of GH/IGF1 liver axis.

The proband is a 50 year-old woman born from a consanguineous marriage. She has been suffering from angina pectoris since the age of 38 and underwent coronary bypass surgery for three-vessel disease at 48. The presence of low plasma levels of total cholesterol and high density lipoprotein (HDL) cholesterol (2.4 and 0.1 mmol/l) and apo AI (15 mg/dl), associated with corneal lesions and a mild splenomegaly suggested the diagnosis of Tangier disease. However, none of the other features of Tangier disease, including hepatomegaly, anemia and peripheral neuropathy, were present. The analysis of the dinucleotide microsatellites located in chromosome 9q31 region demonstrated that the proband was homozygous for the alleles of D9S53, D9S1784 and D9S1832. The mother and son of the proband, both with low levels of HDL cholesterol, shared one of the proband's haplotypes, whereas neither of these haplotypes was present in the normolipidemic proband's sister. The sequence of ATP-binding cassette transporter 1 (ABC1-1) cDNA obtained by reverse transcription-PCR (RT-PCR) of total RNA isolated from cultured fibroblasts showed that the proband was homozygous for a CT transition in exon 13, which caused a tryptophane for arginine substitution (R527W). This mutation was confirmed by direct sequencing of exon 13 amplified from genomic DNA. It can be easily screened, as the nucleotide change introduces a restriction site for the enzyme Afl III. R527W substitution occurs in a highly conserved region of the NH2 cytoplasmic domain of ABC1 protein. R527W co-segregates with the low HDL phenotype in the family and was not found in 200 chromosomes from normolipidemic individuals.

Both estrogen and dietary n-3 polyunsaturated fatty acids are known to be hypocholesterolemic, but appear to exert their effects by different mechanisms. In this study, the interaction between dietary fish oil (rich in n-3 polyunsaturated fatty acids) and estrogen in the regulation of hepatic cholesterol metabolism and biliary lipid secretion in rats was studied. Rats fed a low fat or a fish oil-supplemented diet for 21 days were injected with 17alpha-ethinyl estradiol (5 mg/kg body weight) or the vehicle only (control rats) once per day for 3 consecutive days. Estrogen-treatment led to a marked reduction in plasma cholesterol levels in fish oil-fed rats, which was greater than that observed with either estrogen or dietary fish oil alone. The expression of mRNA for cholesterol 7alpha-hydroxylase was decreased by estrogen in rats fed a low fat or a fish oil-supplemented diet, while the output of cholesterol (micromol/h/kg b.wt.) in the bile was unchanged in both groups. Cholesterol levels in the liver were increased by estrogen in rats given either diet, but there was a significant shift from cholesterol esterification to cholesteryl ester hydrolysis only in the fish oil-fed animals. Estrogen increased the concentration of cholesterol (micromol/ml) in the bile in rats fed the fish oil, but not the low fat diet. However, the cholesterol saturation index was unaffected. The output and concentration of total bile acid was also unaffected, but changes in the distribution of the individual bile acids were observed with estrogen treatment in both low fat and fish oil-fed groups. These results show that interaction between estrogen-treatment and dietary n-3 polyunsaturated fatty acids causes changes in hepatic cholesterol metabolism and biliary lipid secretion in rats, but does not increase the excretion of cholesterol from the body.

The effects of cholesteryl ester transfer protein (CETP) on the direct uptake of HDL cholesteryl ester by the liver was investigated using the rat in vivo and the isolated perfused rat liver as experimental models. Rat plasma was incubated with [3H]cholesterol in the presence or absence of partially purified human CETP for 18 hr and [3H] cholesteryl ester-labeled HDL was then isolated by ultracentrifugation. The CETP-treated as compared to untreated HDL showed a small shift toward a lower density in the peak of lipoprotein cholesterol, suggesting that the HDL particle size was increased. After injection of the labeled HDL into rats in vivo, more radioactivity remained in the plasma after 60 min when the CETP-treated preparation was used, but the amounts found in the liver and secreted in the bile were not significantly different from those obtained with the untreated HDL. The distribution of the label remaining in the plasma after 60 min between different density fractions corresponding to HDL subclasses suggested that the uptake of HDL2 and HDL3 was delayed by CETP treatment. Radioactivity from CETP-treated HDL was also removed from the perfusate of isolated perfused rat livers more slowly than that from untreated HDL, and in this case the amount found in the liver after 60 min was significantly lower. These findings indicate that treatment with CETP has a direct inhibitory effect on the clearance of rat HDL cholesteryl ester from the blood and its uptake by the liver.

We investigated whether S-adenosyl-L-methionine (SAMe), dilinoleoylphosphatidylcholine (DLPC), or SAMe + DLPC influence liver lipid composition as well as acute ethanol hepatotoxicity in the isolated perfused rat liver (IPRL). SAMe (25 mg/kg intramuscularly three times a day) was administered for five consecutive days, while DLPC was administered intraperitoneally for five days. The liver was then isolated, perfused with taurocholate to stabilize bile secretion, and exposed to 0.5% ethanol for 70 min. SAMe, without changing total phospholipid (PL) content, induced an increase in the phosphatidylcholine/phosphatidylethanolamine (PC/PE) molar ratio in both liver homogenate and microsomes and a significant enrichment of 16:0-20:4 and 18:0-20:4 PC molecular species. DLPC induced a significant enrichment of PL in liver homogenate and microsomes due to a contemporary increase in PC and PE. The PC enrichment specifically involved 16:0-20:4 and 18:0-20:4 PC molecular species besides the HPLC peak containing the administered 18:2-18:2 PC species. DLPC + SAMe increased the concentration of PC in liver homogenate and microsomes due to a specific enrichment of 16:0-22:6, 16:0-20:4, and 18:0-20:4 PC molecular species, and the HPLC peak containing the administered 18:2-18:2 PC species. Ethanol acute exposure in the control IPRLs for 70 min induced a depletion of cholesterol in both liver homogenate and microsomes without significant changes in the composition of PL classes and PC molecular species. SAMe, DLPC, or SAMe + DLPC counteracted the cholesterol depletion induced by ethanol, indicating that phospholipid changes promoted by these treatments all induce a major resistance of liver membranes to the effect of ethanol. Ethanol administration in control IPRLs induced a fivefold increase of AST and LDH release in the perfusate, depletion of glutathione in homogenates and mitochondria, decreased oxygen liver consumption, and inhibition of bile flow. These effects of ethanol were significantly antagonized by SAMe. In contrast, DLPC alone only minimally attenuated enzyme release in the perfusate and the inhibitory effect of ethanol on bile flow, but it failed to influence the depletion of total and mitochondrial glutathione or the depressed oxygen consumption induced by ethanol. DLPC, administered together with SAMe, added nothing to the protective effect of SAMe against ethanol hepatotoxicity and cholestasis. In conclusion, this study demonstrates that both SAMe and DLPC induced marked modifications in the lipid composition of liver membranes with a similar enrichment of polyunsaturated PC molecular species. Only SAMe, however, significantly protected against the hepatotoxic and cholestatic effect of acute ethanol administration, an effect associated with maintained normal glutathione mitochondrial levels and oxygen liver consumption. This indicates that the protective effect of SAMe against ethanol toxicity is linked to multiple mechanisms, the maintenance of glutathione levels probably being one of the most important.

The clinical expression of heterozygous familial hypercholesterolemia (FH) is highly variable even in patients carrying the same LDL receptor (LDL-R) gene mutation. This variability might be due to environmental factors as well as to modifying genes affecting lipoprotein metabolism. We investigated Apo E (2, 3, 4), MTP (-493G/T), Apo B (-516C/T), Apo A-V (-1131T/C), HL (-514C/T and -250G/A), FABP-2 (A54T), LPL (D9N, N291S, S447X) and ABCA1 (R219K) polymorphisms in 221 unrelated FH index cases and 349 FH relatives with defined LDL-R gene mutations. We found a significant and independent effect of the following polymorphisms on: (i) plasma LDL-C (Apo E, MTP and Apo B); (ii) plasma HDL-C (HL, FABP-2 and LPL S447X); (iii) plasma triglycerides (Apo E and Apo A-V). In subjects with coronary artery disease (CAD+), the prevalence of FABP-2 54TT genotype was higher (16.5% versus 5.2%) and that of ABCA1 219RK and KK genotypes lower (33.0% versus 51.5%) than in subjects with no CAD. Independent predictors of increased risk of CAD were male sex, age, arterial hypertension, LDL-C level and FABP-2 54TT genotype, and of decreased risk the 219RK and KK genotypes of ABCA1. These findings show that several common genetic variants influence the lipid phenotype and the CAD risk in FH heterozygotes.

Mutations in ABCA1 have been shown to be the cause of Tangier disease (TD) and some forms of familial hypoalphalipoproteinemia (HA), two genetic disorders characterized by low plasma HDL levels. Here we report six subjects with low HDL, carrying seven ABCA1 mutations, six of which are previously unreported. Two mutations (R557X and H160FsX173) were predicted to generate short truncated proteins; two mutations (E284K and Y482C) were located in the first extracellular loop and two (R1901S and Q2196H) in the C-terminal cytoplasmic domain of ABCA1. Two subjects found to be compound heterozygotes for ABCA1 mutations did not have overt clinical manifestations of TD. Three subjects, all with premature coronary artery disease (pCAD), had a combination of genetic defects. Besides being heterozygotes for ABCA1 mutations, two of them were also carriers of the R3500Q substitution in apolipoprotein B and the third was a carrier of N291S substitution in lipoprotein lipase. By extending family studies we identified 17 heterozygotes for ABCA1 mutations. Plasma HDL-C and Apo A-I values in these subjects were 38.3 and 36.9% lower than in unaffected family members and similar to the values found in heterozygotes for Apo A-I gene mutations which prevent Apo A-I synthesis. This survey underlines the allelic heterogeneity of ABCA1 mutations and suggests that: (i) TD subjects, if asymptomatic, may be overlooked and (ii) there may be a selection bias in genotyping towards carriers of ABCA1 mutations who have pCAD possibly related to a combination of genetic and environmental cardiovascular risk factors.

In this study, perfused livers from Yoshida rats, either on a normal diet or on a diet with 0.3% probucol, were examined. The analysis of liver lipid content and of bile and lipoprotein secretion changes showed that probucol had a relevant effect on liver lipid biosynthesis. In particular, it reduced the production of triacylglycerols and, to a much greater extent that of cholesterol. In addition, probucol reduced plasma cholesterol concentration by decreasing esterified cholesterol in HDL1 and HDL2 fractions. Furthermore, HDL1 composition of both hepatic neosynthetized and circulating particles was strongly modified by probucol. Finally, probucol did not appear to induce significant differences in lipid bile secretion while phospholipid secretion from perfused livers was increased. These facts suggest that the hypolipidemic action of probucol is not mediated by an increase in bile steroid secretion, but rather by a direct reduction in hepatic lipoprotein cholesterol secretion. This secretion induces a modified plasma profile of HDL particles such that these variations are advantageous in terms of reverse cholesterol transport.

Lipoprotein and liver lipids of spontaneously hyperHpidemic Yoshida rats were compared with those of normolipidemic Wistar animals for studying their age- and strain-related differences. Both strains showed an age-related increase in the total plasma cholesterol concentration. However, the Yoshida strain had a higher content of cholesteryl esters and triglycerides than the Wistar strain in both young and adult animals (2- and 8-month-old animals, respectively). The free cholesterol content was also higher, but only in the 8-month-old animals. Both strains showed an age-related increase in the proportion of HDL1 and a symmetrical decrease in both the HDL2 and HDL3 subfractions, but the variations were more evident in the Yoshida strain. The study of strain-related differences suggested that the spontaneous hypertriglyceridemia of the Yoshida strain was not only related to the higher amount and proportion of the VLDL fraction, but also to the higher content of triglycerides in the LDL fraction. The livers of Yoshida rats accumulated more triglycerides (with an age-related progression) than those of Wistar rats. The major lipid classes in the liver of Yoshida rats contained a significantly higher proportion of monounsaturated fatty acyls. Furthermore, this proportion showed an age-related increase in all the lipid classes, but in cholesteryl esters. This suggested that liver desaturases had a relevant role in the development of hyperlipidemia, and of its age-related variations, in the Yoshida strain. In conclusion, Yoshida rats appeared a better model for the study of age-related alterations in the liver lipid metabolism since the alterations are stronger and earlier than in Wistar animals and are not complicated by the development of degenerative pathologies typical of old rats.

The aim of this study was to determine whether the common forms of dyslipidemia could affect either the lipid composition or insulin receptor processing (down-regulation) of erythrocytes. The study included 22 patients with type IIa hypercholesterolemia, 15 patients with type IV hypertriglyceridemia and 12 patients with type IIb hyperlipidemia. Ten normolipidemic subjects were used as controls. Their erythrocyte membranes were analyzed for lipid composition and insulin receptor down-regulation. The results show that all the hyperlipidemias investigated were characterized by significant increases in the cholesterol to phospholipid molar ratio (0.56 +/- 0.08 in controls and 1.11 +/- 0.13, 1.09 +/- 0.14, 1.04 +/- 0.15, p < 0.001, in types IIa, IIb and IV, respectively). Surface insulin receptors of type IIa and IIb patients did not appear to down-regulate when compared to normal subjects, but rather up-regulated (+65.2% in controls, -1.0% and -8.7%, p < 0.001, in type IIa and IIb patients, respectively). Patients with type IV hypertriglyceridemia showed a residual capacity for insulin receptor internalization (10.7% down-regulation). Membranes of all the patients contained a higher proportion of phosphatidylethanolamine; the molar ratio of sphingomyelin to phosphatidylcholine was significantly higher in types IIb than in controls (1.22 +/- 0.11 and 1.12 +/- 0.10, p < 0.05, respectively); all the patients showed a lower content of polyunsaturated fatty acids in the major glycerophospholipid classes. However, type IV hypertriglyceridemics showed less variations, especially in the phosphatidylserine fraction. These results indicate that the alterations in lipoprotein pattern may affect both the lipid membrane equilibria and the processing ability of surface insulin receptors. Copyright 1995 S. Karger AG, Basel

Background & Aims Biliary tree, liver, and pancreas share a common embryological origin. We previously demonstrated the presence of stem/progenitor cells of endodermal origin in the adult human extrahepatic biliary tree. This study evaluated the human foetal biliary trees as sources of stem/progenitor cells of multiple endodermal-derived mature fates. Methods Human foetal intrahepatic and extrahepatic biliary tree tissues and isolated cells were tested for cytoplasmic and surface markers of stem cells and committed progenitors, as well as endodermal transcription factors requisite for a liver versus pancreatic fate. In vitro and in vivo experiments were conducted to evaluate the potential mature fates of differentiation. Results Foetal biliary tree cells proliferated clonogenically for more than 1month on plastic in a serum-free Kubota medium. After culture expansion, cells exhibited multipotency and could be restricted to certain lineages under defined microenvironments, including hepatocytes, cholangiocytes, and pancreatic islet cells. Transplantation of foetal biliary tree cells into the livers of immunodeficient mice resulted in effective engraftment and differentiation into mature hepatocytes and cholangiocytes. Conclusions Foetal biliary trees contain multipotent stem/progenitor cells comparable with those in adults. These cells can be easily expanded and induced in vitro to differentiate into liver and pancreatic mature fates, and engrafted and differentiated into mature cells when transplanted in vivo . These findings further characterise the development of these stem/progenitor cell populations from foetuses to adults, which are thought to contribute to liver and pancreas organogenesis throughout life.

As part of a study of the effects of aging on lipoprotein metabolism, the uptake and processing in vivo of cholesterol from high density lipoprotein (HDL) was compared in young (3 months of age) and mature (10-12 months of age) rats by studying the fate of HDL [3H] unesterified cholesterol or [3H] cholesteryl ester after intravenous administration. Radioactivity from [3H] unesterified cholesterol was cleared from the blood more slowly in older rats, and this difference was accounted for by decreased uptake by the liver. Uptake by other tissues were unaffected. In addition, a shift in the distribution of radioactivity across the plasma lipoprotein density range from the d = 1.125-1.250 g/ml (HDL3) to the d = 1.050-1.085 g/ml (HDL1) fraction was observed in the mature as compared to the young rat group. The secretion of radioactivity from [3H] unesterified cholesterol into bile was also decreased in the older animals, particularly in the first hour after injection of the label. In the case of HDL labeled with [3H] cholesteryl ester, clearance from the blood was similar in both age groups in the first 30 min after injection, but was significantly lower in older rats at later time points. After 180 min, less radioactivity was found in the VLDL density fraction in mature as compared to young rats, suggesting that hepatic secretion of VLDL cholesterol originating from HDL cholesteryl ester is less efficient in the older animals. The amount of radioactivity from HDL [3H] cholesteryl ester secreted in bile was less in the mature rat group at all time points measured.(ABSTRACT TRUNCATED AT 250 WORDS)

We have shown previously that the age-dependent increase in plasma cholesterol levels observed in male Wistar rats is associated with relevant changes in the lipoprotein pattern (in particular, with a much higher proportion of the HDL1 class) that are evident in animals from the age of 9 months. In this study, the possibility that a decreased catabolism of HDL1 cholesterol may cause this is evaluated by infusing this lipoprotein fraction labeled with [14C]cholesterol into both young (3.5 +/- 0.5 months) and adult (13.0 +/- 1.0 months) male Wistar rats with a permanent biliary drainage. The clearance of radioactivity from the blood compartment was slower in the older animals than in the younger ones. Conversely, the incorporation of radioactivity into plasma cholesteryl esters and the secretion of radioactivity into bile was higher in the younger animals. These results support the hypothesis that the age-related increase in HDL1 proportion is due, at least in part, to a slower liver catabolism of HDL1-cholesterol.

Plasma lipoproteins in human and rodent species show marked differences in their distribution patterns. However, more analogies are evident with hamsters than with rats. Plasma lipoproteins of golden Syrian hamster comprise significant amounts of the VLDL, LDL and HDL classes, and the HDL fraction does not appear to be divided into subclasses. HDL is the main lipoprotein class and shows an age-related reduction from 73% to 65% of total lipoproteins in young and old animals, respectively. The LDL class represents about the 12% of total lipoprotein throughout the life-span examined by us (1.5–9-month-old animals). The VLDL fraction shows an age-related increase from 15% in younger animals to 24% in older ones. We observed also that in the hamsters the distribution of cholesterol between free and esterified forms, and the distribution of phospholipid classes in the different lipoprotein fractions are more similar to those found in man than to those in the Wistar rat. This suggests that the golden Syrian hamster, particularly when older than 6 months, is a better model for experimental studies on lipoprotein metabolism than Wistar rats.

Multipotent stem/progenitors are present in peribiliary glands of extrahepatic biliary trees from humans of all ages and in high numbers in hepato-pancreatic common duct, cystic duct, and hilum. They express endodermal transcription factors (e.g., Sox9, SOX17, FOXA2, PDX1, HES1, NGN3, PROX1) intranuclearly, stem/progenitor surface markers (EpCAM, NCAM, CD133, CXCR4), and sometimes weakly adult liver, bile duct, and pancreatic genes (albumin, cystic fibrosis transmembrane conductance regulator [CFTR], and insulin). They clonogenically expand on plastic and in serum-free medium, tailored for endodermal progenitors, remaining phenotypically stable as undifferentiated cells for months with a cell division initially every � 36 hours and slowing to one every 2-3 days. Transfer into distinct culture conditions, each comprised of a specific mix of hormones and matrix components, yields either cords of hepatocytes (express albumin, CYP3A4, and transferrin), branching ducts of cholangiocytes (expressing anion exchanger-2-AE2 and CFTR), or regulatable C-peptide secreting neoislet-like clusters (expressing glucagon, insulin) and accompanied by changes in gene expression correlating with the adult fate. Transplantation into quiescent livers of immunocompromised mice results in functional human hepatocytes and cholangiocytes, whereas if into fat pads of streptozocin-induced diabetic mice, results in functional islets secreting glucose-regulatable human C-peptide. Conclusion: The phenotypes and availability from all age donors suggest that these stem/ progenitors have considerable potential for regenerative therapies of liver, bile duct, and pancreatic diseases including diabetes. (HEPATOLOGY 2011;54:2159-2172)

Both Wistar and Sprague-Dawley rat strains have been used for experimental studies on lipoprotein metabolism, although the lipoprotein characteristics of the former strain are less well known than those of the latter. We have defined more precise conditions for separating by density gradient ultracentrifugation the different lipoprotein classes from plasma of young male Wistar rats. Present results confirm that Wistar rats, like other rat strains, have negligible amounts of low density lipoproteins and levels of very low density lipoproteins much lower than high density lipoproteins. The differences between rat strains appeared in the profile of lipoprotein denser than 1.08 g/ml. In fact, we showed Wistar rats have considerable amounts of high density lipoprotein-3 subfraction with a density range between 1.17 and 1.21 g/ml, a mean diameter of 4.9 nm, and a low cholesterol content. In analogy with the Sprague-Dawley strain, Wistar rats had a small proportion of high density lipoprotein-1 subfraction (about 17% of total lipoproteins) and, at variance, in Wistar rats, we did not observe the presence of the so-called very high density lipoprotein fraction described previously in the Sprague-Dawley strain.

The effects of HDL1 lipoprotein infusion on biliary lipid secretion were studied in the in vitro model of rat perfused liver. A strong increase in bile flow was observed during and after lipoprotein infusion. This caused a significant rise in cholesterol, phospholipid and bile salt secretions. However, only the percentage of cholesterol increased with respect to the other bile lipids. The changes observed in the cholesterol/phospholipid molar ratio values of liver membrane subfractions (i.e., liver plasma membrane, mitochondria plus lysosomes and microsomes) isolated from the perfused rat liver after HDL1 administration were not significant.

Cholesteryl Ester Storage Disease (CESD) is a rare recessive disorder due to mutations in LIPA gene encoding the lysosomal acidic lipase (LAL). CESD patients have liver disease associated with mixed hyperlipidemia and low plasma levels of high-density lipoproteins (HDL). The aim of this study was the molecular characterization of three patients with CESD. LAL activity was measured in blood leukocytes. In two patients (twin sisters) the clinical diagnosis of CESD was made at 9years of age, following the fortuitous discovery of elevated serum liver enzymes in apparently healthy children. They had mixed hyperlipidemia, hepatosplenomegaly, reduced LAL activity (∼5% of control) and heteroalleic mutations in LIPA gene coding sequence: (i) the common c.894 G>A mutation and (ii) a novel nonsense mutation c.652 C>T (p.R218X). The other patient was an 80year-old female who for several years had been treated with simvastatin because of severe hyperlipidemia associated with low plasma HDL. In this patient the sequence of major candidate genes for monogenic hypercholesterolemia and hypoalphalipoproteinemia was negative. She was found to be a compound heterozygote for two LIPA gene mutations resulting in 5% LAL activity: (i) c.894 G>A and (ii) a novel complex insertion/deletion leading to a premature termination codon at position 82. These findings suggest that, in view of the variable severity of its phenotypic expression, CESD may sometimes be difficult to diagnose, but it should be considered in patients with severe type IIb hyperlipidemia associated with low HDL, mildly elevated serum liver enzymes and hepatomegaly.

Lysophosphatidylcholine is a major metabolic product in the plasma and cellular turnover of phospholipids, with well-known membrane-toxic and proinflammatory properties. Because the liver plays a key role in plasma lysophosphatidylcholine removal and biotransformation and because virtually nothing is known of these processes in a diseased organ, the hepatobiliary metabolism of lysophosphatidylcholine was investigated in rats with carbon tetrachloride-induced liver cirrhosis. Twelve adult male Wistar rats with histologically confirmed cirrhosis and 8 control animals were fitted with jugular and biliary catheters and allowed to recover. The animals were kept under constant IV infusion of taurocholate (1 mumol/min). Two microcuries of sn-1[14C]palmitoyl-lysophosphatidylcholine was administered as a single bolus. The fate of the injected radioactivity, including removal from plasma, uptake, and subcellular location in the liver and molecular and aggregative forms, was studied by combined chromatographic and radiochemical methods. Major findings were (a) that lysophosphatidylcholine has a prolonged permanence in plasma of cirrhotic rats, due both to decreased hepatic clearance and to depressed conversion into phosphatidylcholine; (b) that the rate of lysophosphatidylcholine acylation is much slower in the cirrhotic than in the normal liver, both at the microsomal and at the cytosolic level; (c) that cytosolic lysophosphatidylcholine in the cirrhotic liver, but not in the normal liver, is predominantly non-protein bound; (d) that the strict molecular selectivity of lysophosphatidylcholine acylation observed in controls is partially lost in cirrhosis; and (e) that a consistent fraction of lysophosphatidylcholine is converted into triacylglycerols in cirrhotics but not in controls. These findings show a profound derangment of lysophosphatidylcholine handling and processing in the cirrhotic liver, which is of potential pathogenetic significance.

The intravenous administration of dimethylethanolamine in the rat promotes a selective enrichment of liver membranes with polyunsaturated phosphatidylcholines. The effect of dimethylethanolamine pretreatment on cholestasis induced by estradiol 17 beta-D-glucuronide, a potent cholestatic agent, was assessed in this study. Dimethylethanolamine, dissolved in sodium-taurocholate was infused intravenously (0.3 mg/kg/min) for 15 hr. One group of control rats (estradiol 17 beta-D-glucuronide controls) received the bile salt only. An estradiol 17 beta-D-glucuronide bolus was then injected intravenously (10.4 mg/kg) into dimethylethanolamine-pretreated and estradiol 17 beta-D-control rats, and its effect on bile flow and biliary lipid secretion was compared for 3 hr. The estradiol 17 beta-D-glucuronide inhibitory effect on bile flow and biliary lipid secretion was significantly antagonized by dimethylethanolamine pretreatment. The maximum inhibition of bile flow was found 30 min after estradiol 17 beta-D-glucuronide administration, when it decreased from 3.5 +/- 0.4 microliters/min/100 gm (basal) to 0.9 +/- 0.3 microliters/min/100 gm in estradiol 17 beta-D-glucuronide controls, whereas in dimethylethanolamine-pretreated rats this decreased only from 3.2 +/- 0.4 (basal) to 2.3 +/- 0.4 microliters/min/100 gm. Bile flow and the biliary secretion of cholesterol, phosphatidylcholine and bile salts were significantly higher in the dimethylethanolamine-pretreated rats than in estradiol 17 beta-D-glucuronide controls (p less than 0.02) during the cholestatic phase. The inhibitory effect of estradiol 17 beta-D-glucuronide on bile flow was associated with a marked decrease of membrane fluidity (p less than 0.001) assessed by 1,6-diphenyl-1,3,5-hexatriene fluorescence anisotropy and with a cholesterol enrichment of microsomes, sinusoidal and canalicular liver plasma membranes and inhibition of sinusoidal Na+,K(+)-ATPase activity (p less than 0.05). These membrane alterations persisted 180 min after estradiol 17 beta-D-glucuronide administration despite complete normalization of bile flow. Dimethylethanolamine pretreatment significantly counteracted the reduction of membrane fluidity (p less than 0.001), the cholesterol enrichment and the inhibition of Na+,K(+)-ATPase (p less than 0.05) promoted by estradiol 17 beta-D-glucuronide administration in all membrane subfractions 30 and 180 min after administration. In addition, dimethylethanolamine-pretreated rats had more polyunsaturated fatty acids in membrane phosphatidylcholine with respect to the control groups. Dilatation of canaliculi and loss of microvilli were evident in estradiol 17 beta-D-glucuronide controls 180 min after estradiol 17 beta-D-glucuronide administration.(ABSTRACT TRUNCATED AT 400 WORDS)

The metabolic fate of [14C]cholesterol carried in the VLDL, LDL, HDL2 and HDL3 lipoprotein fractions has been investigated in bile-fistula rats with jugular vein cannulation. After the depletion of bile salt pool, the labelled lipoprotein fraction was administered either with or without the continuous infusion of sodium taurocholate. The proportion of labelled steroids secreted in the bile within 3 h after the lipoprotein administration was generally higher when the exogenous bile salt was infused. Specifically, the HDL2 fraction induced the secretion of relatively high proportions of labelled steroids, mainly bile salts, either with or without the taurocholate infusion. The other lipoprotein fractions increased the proportion of free cholesterol in steroid bile secretion under taurocholate administration. The label associated to the LDL fraction showed a higher biliary secretion and a more steady clearance from plasma if the exogenous bile salt was not administered. In this same condition, the administration of HDL3 fraction gave the highest values of radioactivity recovered in the liver (mainly as free cholesterol) and a significant increase of cholesterol in the biliary secretion. The present results suggest that each lipoprotein fraction tested may contribute in a peculiar manner to bile salt and cholesterol biliary secretion. Both the expression of apo-E and apo B,E receptors and the levels of circulating bile salts appear to have a role in this process.

The noradrenergic nucleus Locus Coeruleus (LC) densely innervates limbic structures. In rats, the damage to LC by the neurotoxin DSP-4, converts episodic limbic seizures induced by bicuculline infusion in the anterior piriform cortex (APC) into self-sustaining status epilepticus (SE). SE induced by this approach is similar to SE induced by co-infusing cyclothiazide and bicuculline into APC in rats bearing an intact LC. As opposed to other commonly used rat SE models (e.g. systemic kainate or pilocarpine), this approach allows one to analyze the effects of SE on brain regions which are solely due to spreading of seizure activity, rather than to direct effect of systemic chemoconvulsant. We evaluated the expression of Fos protein (an immediate early gene product), and the local cerebral metabolic rates for [14C] 2-deoxyglucose (lCMRglc), in rats following SE induced either by cyclothiazide+bicuculline or by DSP-4+bicuculline. We demonstrated that regional Fos expression after SE does not parallel the increase in lCMRglc, in LC-lesioned rats. In DSP-4+bicuculline rats there is an overall lower expression of the protein as compared with the cyclothiazide+bicuculline or bicuculline alone groups; even more, such a difference co-exists with an higher lCMRglc in the DSP-4+bicuculline-treated rats in some regions, as compared with the other groups. These data show that LC neurons play an important role in determining immediate early genes expression even in conditions of strong pathological activation, such as limbic SE. This might have relevant effects in the plastic mechanisms related with epileptogenesis.

We investigated the effect of statins and statins plus ezetimibe in 65 FH heterozygotes carrying LDLR-defective or LDLR-negative mutations as well as the effect of ezetimibe monotherapy in 50 hypercholesterolemic (HCH) patients intolerant to statins. PCSK9 and NPC1L1 genes were analysed to assess the role of genetic variants in response to therapy. In FH patients combined therapy reduced LDL-C by 57%, irrespective of the type of LDLR mutation. The additional decrease of plasma LDL-C induced by ezetimibe showed wide inter-individual variability (from -39% to -4.7%) and was negatively correlated with percent LDL-C decrease due to statin alone (r=-0.713, P0.001). The variable response to statins was not due to PCSK9 gene variants associated with statin hyper-sensitivity. The highest response to ezetimibe was observed in a carrier of R174H substitution in NPC1L1, which had been found to be associated with high cholesterol absorption. In HCH patients, ezetimibe monotherapy induced a variable decrease of plasma LDL-C (from -47.7% to -13.4%). To investigate this variability, we sequenced NPC1L1 gene in patients with the highest and the lowest response to ezetimibe. This analysis showed a higher prevalence of the G allele of the c.816 CG polymorphism (L272L) in hyper-responders, an observation confirmed also in FH patients hyper-responders to ezetimibe. In both FH and HCH patients, the G allele carriers tended to have a higher LDL-C reduction in response to ezetimibe. These observations suggest that in FH heterozygotes LDL-C reduction following combined therapy reflects a complex interplay between hepatic synthesis and intestinal absorption of cholesterol.

The diagnosis of familial hypercholesterolemia (FH) in unselected children is difficult due to the frequent overlap of cholesterol values in affected and non-affected and the paucity of physical signs. Nevertheless, detection and treatment of FH in childhood has been advocated to prevent atherosclerosis in these patients. Here, we report the results of a screening program in a cohort of 157 unrelated, hypercholesterolemic (HC) children (age range 2-15 years; mean 8.3+/-3.4 years) carried out by a combination of family study and molecular analysis of the LDLR gene. On the basis of the familial phenotype, 27 (17.2%) were classified as probable FH and 49 (31.2%) as affected by FCHL. Among probable FH children, 14 (51.8%) carried mutant LDLR alleles, giving an overall 8.9% prevalence of FH. Most of LDLR variants were already reported, but three new mutations G266C, T368M, and D451Y were identified. Beside increased TC and LDL-C (p0.001), FH children showed decreased HDL-C (p0.05) and higher prevalence of family history of CAD when compared to non-FH children. None presented tendon xanthomas. We estimated that LDL-C3.9 mmol/L was the best cut off value for diagnosing FH in these children, showing 79% sensitivity and 71.0% specificity. We propose the use of a LDL-C cut off level associated with a family study to identify FH among HC children.

Autosomal recessive hypercholesterolemia (ARH) is a rare disorder, due to complete loss of function of an adaptor protein (ARH protein) required for receptor-mediated hepatic uptake of LDL. ARH is a phenocopy of homozygous familial hypercholesterolemia (HoFH) due to mutations in LDL receptor (LDLR) gene; however, previous studies suggested that ARH phenotype is less severe than that of HoFH. To test this hypothesis we compared 42 HoFH and 42 ARH patients. LDLR and ARH genes were analysed by Southern blotting and sequencing. LDLR activity was measured in cultured fibroblasts. In ARH plasma LDL cholestrol (LDL-C) level (14.25+/-2.29 mmol/L) was lower than in receptor-negative HoFH (21.38+/-3.56 mmol/L) but similar to that found in receptor-defective HoFH (15.52+/-2.39 mmol/L). The risk of coronary artery disease (CAD) was 9-fold lower in ARH patients. No ARH patients

We studied a three generation family with co-dominant monogenic hypercholesterolemia and hypoalphalipoproteinemia. The proband, a 48 year-old male, was found to be heterozygous for a previously reported mutation in LDL receptor (LDL-R) gene (IVS15-3 ca) and a novel mutation in exon 6 of lecithin cholesterol acyltransferase (LCAT) gene (c.803 GA) causing a non-synonymous amino acid substitution (p.R244H). These mutations segregated independently in the family. The LDL-R mutation was associated with high levels of LDL-C (6.20-9.85 mmol/L) and apo B (170-255 mg/dL), comparable to those previously reported in carriers of the same mutation. The LCAT mutation was associated with low levels of HDL-C (0.67-0.80 mmol/L) and apo A-I (96-110 mg/dL). The proband had reduced LCAT function, as measured by cholesterol esterification rate (29 nmol/(mL/h) versus 30-60 nmol/(mL/h)), LCAT activity (10 nmol/(mL/h) versus 20-55 nmol/(mL/h)) and LCAT mass (2.87 microg/mL versus 3.1-6.7 microg/mL). Carriers of LCAT mutation had lower LCAT activity and a tendency to reduced cholesterol esterification rate (CER) and LCAT mass as compared to non-carrier family members. The LCAT mutation was not found in 80 control subjects and 60 patients with primary hypoalphalipoproteinemia. Despite the unfavourable lipoprotein profile, the proband had only mild clinical signs of atherosclerosis. This unexpected finding is probably due to the intensive lipid lowering treatment the patient has been on over the last decade.

Objective— To better understand the role of lecithin:cholesterol acyltransferase (LCAT) in lipoprotein metabolism through the genetic and biochemical characterization of families carrying mutations in the LCAT gene. Methods and Results— Thirteen families carrying 17 different mutations in the LCAT gene were identified by Lipid Clinics and Departments of Nephrology throughout Italy. DNA analysis of 82 family members identified 15 carriers of 2 mutant LCAT alleles, 11 with familial LCAT deficiency (FLD) and 4 with fish-eye disease (FED). Forty-four individuals carried 1 mutant LCAT allele, and 23 had a normal genotype. Plasma unesterified cholesterol, unesterified/total cholesterol ratio, triglycerides, very-low-density lipoprotein cholesterol, and pre-β high-density lipoprotein (LDL) were elevated, and high-density lipoprotein (HDL) cholesterol, apolipoprotein A-I, apolipoprotein A-II, apolipoprotein B, LpA-I, LpA-I:A-II, cholesterol esterification rate, LCAT activity and concentration, and LDL and HDL 3 particle size were reduced in a gene–dose-dependent manner in carriers of mutant LCAT alleles. No differences were found in the lipid/lipoprotein profile of FLD and FED cases, except for higher plasma unesterified cholesterol and unesterified/total cholesterol ratio in the former. Conclusion— In a large series of subjects carrying mutations in the LCAT gene, the inheritance of a mutated LCAT genotype causes a gene–dose-dependent alteration in the plasma lipid/lipoprotein profile, which is remarkably similar between subjects classified as FLD or FED.

Objective— Mutations in LPL or APOC2 genes are recognized causes of inherited forms of severe hypertriglyceridemia. However, some hypertrigliceridemic patients do not have mutations in either of these genes. Because inactivation or hyperexpression of APOA5 gene, encoding apolipoprotein A-V (apoA-V), causes a marked increase or decrease of plasma triglycerides in mice, and because some common polymorphisms of this gene affect plasma triglycerides in humans, we have hypothesized that loss of function mutations in APOA5 gene might cause hypertriglyceridemia. Methods and Results— We sequenced APOA5 gene in 10 hypertriglyceridemic patients in whom mutations in LPL and APOC2 genes had been excluded. One of them was found to be homozygous for a mutation in APOA5 gene (c.433 C>T, Q145X), predicted to generate a truncated apoA-V devoid of key functional domains. The plasma of this patient was found to activate LPL in vitro less efficiently than control plasma, thus suggesting that apoA-V might be an activator of LPL. Ten carriers of Q145X mutation were found in the patient’s family; 5 of them had mild hypertriglyceridemia. Conclusions— As predicted from animal studies, apoA-V deficiency is associated with severe hypertriglyceridemia in humans. This observation suggests that apoA-V regulates the secretion and/or catabolism of triglyceride-rich lipoproteins.

Background and Aim: Several genetic polymorphisms have been found to be involved in cardiovascular risk, and many studies have documented the beneficial effect of systematic physical activity (PA) on the cardiovascular system. Our aim was to investigate the interactive effects of PA and genetic background on plasma lipids and homocysteine (tHcy) levels. Methods and Results: Clinical and metabolic parameters, dietary intakes and some polymorphisms of the genes involved in cardiovascular risk (Apo E, fatty acid binding protein-2, Apo AII, hepatic lipase and methylene tetrahydrofolate reductase) were determined in 100 men aged over 40 years who cycle 120–150 Km/week and 100 age-matched sedentary controls. The physically active subjects had lower concentrations of plasma LDL cholesterol (LDL-C), triglyceride (TG), Apo B, glucose and tHcy, and higher concentrations of plasma HDL cholesterol (HDL-C) and Apo AI than the sedentary men; they also had larger LDL particle sizes (LDLs). The LDL-C and Apo B raising effect of the Apo E ɛ4 allele detectable in the sedentary subjects was totally absent in the cyclists, in whom the LDL-C and Apo B lowering effect of the ɛ2 allele was observed. PA blunted the TG-raising effect of the Apo AII-265TT genotype, and amplified the HDL-C raising effect of the HL -250AA genotype. PA had a small but significant lowering effect on plasma tHcy adjusted for folate levels in subjects with the 677TT genotype of the MTHFR gene. Conclusions: Extended high-intensity PA in men aged over 40 years may modify their metabolic cardiovascular risk factors even in the presence of some unfavourable genotypes.

Two siblings with high density lipoprotein (HDL) deficiency and no plasma apolipoprotein A-I (Apo A-I) were found to be homozygous for a cytosine deletion in exon 3 of Apo A-I gene (c.85 del C, Q5FsX11). This mutation causes a frameshift leading to a premature stop codon and abolishes the synthesis of Apo A-I. Although both siblings had corneal opacifications and planar xanthomas, only one of them had premature coronary artery disease, probably as the result of mildly elevated LDL levels. In two other unrelated subjects HDL deficiency was due to heterozygosity for a nucleotide substitution in exon 4 of Apo A-I gene (c.494 T>G, L141R). Both Apo A-I mutations were reported previously in an Italian kindred which included compound heterozygotes and simple heterozygotes. We investigated all carriers of these mutations in the three kindreds and in the one previously reported. Plasma Apo A-I and HDL-C levels were lower in the mutation carriers than in non-carrier family members. These levels, however, were lower in L141R carriers than in carriers of c.85 del C. Haplotype analysis performed using several polymorphisms suggested that both the c.85 del C and L141R are likely to be recurrent mutations.

A. Torrice ∗ ,2, G. Carpino 1, A. Fraveto 2, A. Renzi 3, M.C. Bragazzi 2, F. Giuliante 4, A.M. Derose 2, V. Cardinale 2, R. Gentile 2, P. Onori 3, C. Napoletano 5, A. Franchitto 3, A. Cantafora 2, G. Grazi 2, E. Gaudio 2, D. Alvaro 2 1Health Science, University of Rome “Foro Italico”, Rome, Italy; 2Medico-Surgical Sciences and Biotechnologies, Sapienza University of Rome, Rome, Italy; 3SAIMLAL, Sapienza, University of Rome, Rome, Italy; 4Surgery, Hepatobiliary Unit, Catholic University of The Sacred Heart School of Medicine, Rome, Italy; 5Experimental Medicine, Sapienza University of Rome, Rome, Italy

Several studies have suggested that the oxidative modification of low-density lipoprotein (LDL) could play a key role in the early stages of atherosclerosis. The susceptibility of LDL to oxidation has been found to be greater in patients with coronary heart disease. Familial hypercholesterolaemia (FH) is a powerful clinical model in which to study the predictive role of LDL in atherogenesis. LDL-apheresis is a treatment that is able to decrease lipid levels in plasma. This study was aimed at investigating the reducing capacity of erythrocytes and the in vitro susceptibility to oxidation of LDL isolated from patients with homozygous, heterozygous and double-heterozygous FH, who were treated fortnightly with LDL-apheresis or left untreated. In 14 FH patients, at baseline and after a cycle of treatment, the susceptibility of LDL to oxidative modification was analysed by studying the kinetics of conjugate diene formation. Plasma hydroperoxides, polyunsaturated fatty acid content, LDL electrophoretic mobility on agarose, the titre of auto-antibodies against oxidized LDL and serum paraoxonase activity were also measured. Furthermore, in order to evaluate a potential relationship between LDL oxidation and redox status, erythrocyte GSH and ATP levels were determined in FH patients treated regularly or never treated previously by LDL-apheresis. Unlike in the control group, the oxidative status of LDL in all FH patients was modified by LDL-apheresis, as revealed by the higher negative charge and the increase in levels of hydroperoxides and antibodies against oxidized LDL in the plasma. Our findings suggest both an acute effect and a long-term effect of LDL-apheresis in FH patients treated with dextran sulphate cellulose apheresis. The acute effect of LDL-apheresis on the susceptibility to oxidation of plasma and LDL was demonstrated by significant decreases in plasma hydroperoxide content, total LDL concentration and polyunsaturated fatty acid content. The increased resistance of LDL to oxidation was shown by prolongation of the lag time (P < 0.05) in samples after a single cycle of treatment. The long-term effect of LDL-apheresis was demonstrated by the comparable values for lag phases (obtained from the kinetics of conjugate diene formation) in patients under active treatment and controls. Compared with healthy controls and untreated patients, the erythrocyte GSH content was significantly higher (P⩽ 0.001) in the treated group, suggesting the activation of reducing mechanisms.

23 RECIPIENT MTHFR C677T POLYMORPHISM PREDICTS PRE-TRANSPLANT CIRRHOSIS PROGRESSION AND SURVIVAL AFTER LIVER TRANSPLANTATION S. Ginanni Corradini1, M.A. Burza1, F. Ferri1, A. Molinaro1, A. Cantafora1, I. Blotta1, M. Mordenti1, M.G. Mazzucconi2, G. Ferretti1, V. Giannelli1, M. Merli1, A.F. Attili1, P.B. Berloco1, M. Rossi1. Liver Transplant Unit and Dep of Cell. Biotechnology and Hematology, Sapienza Univ. of Rome, Italy

The role of the gallbladder in gallstone pathogenesis is still unclear. We examined the effects of gallbladder mucosal lipid absorption on lipid composition and cholesterol crystallization in bile.The in vitro-isolated, intra-arterially perfused gallbladder model was used (1) to compare the absorption rates of lipids from standard bile by gallbladders obtained from 7 patients with cholesterol gallstones and 6 controls; and (2) to measure the microscopic cholesterol crystal detection time in cholesterol-enriched pig bile before and after lipid absorption by the pig gallbladder.Control gallbladders, but not cholesterol gallstone gallbladders, significantly reduced cholesterol (P0.02) and phospholipid (P0.01) and increased bile salt (P0.01) molar percentages in bile over a 5-hour period by efficient and selective cholesterol and phospholipid absorption. A histomorphometric study of the epithelial cells showed significantly higher values for nuclear density (P0.01) and nuclear (P0.05) and cytoplasmic (P0.05) areas in the cholesterol gallstone than the control group. Sequential microscopy of cholesterol-enriched pig bile showed significantly shorter cholesterol filament (P0.01) and typical cholesterol plate (P0. 02) detection times before than after exposure of bile to the gallbladder lipid absorption.In cholesterol gallstone disease, the human gallbladder epithelium loses its capacity to selectively and efficiently absorb cholesterol and phospholipids from bile, even if it is hyperplastic and hypertrophic. This epithelial dysfunction eliminates the positive effect that the normal gallbladder exerts on cholesterol solubility in bile and might be a pathogenetic cofactor for cholesterol gallstone formation.

Abstract —One of the genetic features of the Sardinian population is the high prevalence of hemoglobin disorders. It has been estimated that 13% to 33% of Sardinians carry a mutant allele of the α-globin gene (α-thalassemia trait) and that 6% to 17% are β-thalassemia carriers. In this population, a single mutation of β-globin gene (Q39X, β 0 39) accounts for >95% of β-thalassemia cases. Because previous studies have shown that Sardinian β-thalassemia carriers have lower total and low density lipoprotein (LDL) cholesterol than noncarriers, we wondered whether this LDL-lowering effect of the β-thalassemia trait was also present in subjects with familial hypercholesterolemia (FH). In a group of 63 Sardinian patients with the clinical diagnosis of FH, we identified 21 unrelated probands carrying 7 different mutations of the LDL receptor gene, 2 already known (313+1 g>a and C95R) and 5 not previously reported (D118N, C255W, A378T, T413R, and Fs572). The 313+1 g>a and Fs572 mutations were found in several families. In cluster Fs572, the plasma LDL cholesterol level was 5.76±1.08 mmol/L in subjects with β 0 -thalassemia trait and 8.25±1.66 mmol/L in subjects without this trait ( P 0 -thalassemia major and whose LDL cholesterol level was below the 50th percentile of the distribution in the normal Sardinian population. The hypocholesterolemic effect of β 0 -thalassemia trait emerged also when we pooled the data from all FH subjects with and without β 0 -thalassemia trait, regardless of the type of mutation in the LDL receptor gene. The LDL-lowering effect of β 0 -thalassemia may be related to (1) the mild erythroid hyperplasia, which would increase the LDL removal by the bone marrow, and (2) the chronic activation of the monocyte-macrophage system, causing an increased secretion of some cytokines (interleukin-1, interleukin-6, and tumor necrosis factor-α) known to affect the hepatic secretion and the receptor-mediated removal of apolipoprotein B–containing lipoproteins. The observation that our FH subjects with β 0 -thalassemia trait (compared with noncarriers) have an increase of blood reticulocytes (40%) and plasma levels of interleukin-6 (+60%) supports these hypotheses. The lifelong LDL-lowering effect of β 0 -thalassemia trait might slow the development and progression of coronary atherosclerosis in FH.

Successful aging, characterized by little or no loss in physiological functions, should be the usual aging process in centenarians. It is known that well-preserved physiological functions depend on the proper functioning of cell systems. In this article we focus on cell membrane integrity and study the red blood cell membrane to evaluate the effect of physiological aging in centenarians. Fifteen healthy, self-sufficient centenarians, mean age 103 years, were examined by assessing hemocytometric values and some relevant characteristics of the erythrocyte membrane, i.e., the cholesterol/phospholipid molar ratio, the distribution of phospholipid classes and their fatty acid composition, the integral and skeletal protein profiles. The centenarians showed a significant decrease in the red blood cell count (p < 0.0002), hemoglobin (p < 0.0002), and hematocrit (p < 0.0005). The red blood cell membrane showed a significantly increased cholesterol/phospholipid molar ratio (p < 0.01), with a concomitant increase in polyunsaturated fatty acids in phosphatidylcholine (p < 0.001) and, to a lesser extent, in phosphatidylethanolamine. The electrophoretic pattern of membrane proteins was qualitatively normal compared to controls but the densitometric analysis showed a significant increase in the integral protein band 4.2 (p < 0.05) and in the skeletal protein actin (p < 0.001). Extreme longevity seems to be associated with a substantial integrity of the erythrocyte membrane. Moreover, the evident increase in polyunsaturated fatty acids and in actin are likely to improve the membrane fluidity and to strengthen the membrane structure.

The role of the excretion of dietary cholesterol in the hypocholesterolaemic effect of chronic fish oil feeding in rats was investigated. The hepatic uptake and processing of [ 3 H]cholesterol carried in chylomicrons derived from fish oil was studied in vivo in rats fed a low fat diet or a diet supplemented with fish oil for 21 days. In addition, the effects of the fish oil diet on cholesterol esterification, cholesteryl ester hydrolysis, bile acid synthesis and biliary lipid secretion were determined. In rats fed the fish oil as compared to the low fat diet, the uptake of [ 3 H]cholesterol from the blood and its secretion into bile as bile acids was significantly slower, and this was entirely due to a decrease in the bile acid fraction. Biliary bile acid mass secretion was unchanged by fish oil feeding, while biliary cholesterol and phospholipid secretion was increased. No significant differences were observed either in the expression of mRNA for cholesterol 7 α hydroxylase or the secretion of bile acids into bile after 20 h biliary drainage between the fish oil and low fat diet groups, suggesting that bile acid synthesis is not affected. These results indicate that the access of chylomicron cholesterol to the hepatic substrate pool for bile acid formation is decreased in the fish oil fed rats, and this, together with its slower uptake from the blood, accounts for the retardation of its excretion via the bile. Thus, the hypocholesterolemic effect of dietary fish oil in rats is not due to more rapid metabolism of cholesterol originating from the diet.

Background/Aims: The present study describes an embryonic-fetal liver culture system which allows morphogenetic interactions consistent with the development of the hepatocellular function. Methods: Intact livers from 8–12-weeks embryos were soaked in an extracellular matrix at 4°C and gently dissociated without any enzymatic treatment. The resulting spherical hepatic units were cultured in a chemically defined serum-free medium and seeded into an extracellular matrix layer. Adherent three-dimensional tissue specimens were examined at various times by light and electron microscopy to evaluate the maintenance of hepatocyte morphology. Results: The liver cells were viable for over 4 months; erythropoietic burst colonies were detected for longer than 6 weeks. Parallel detection of bile salt production in the medium by high performance liquid chromatography proved liver tissue functionality. Bile salt composition revealed predominance of taurine-conjugates rather than glycine. Maximum bile salt concentration (≈3 months) coincided with structural and ultrastructural observations indicating a marked decline in hematopoiesis, well-defined biliary canaliculi and formation of an organ-like structure. Conclusions: This three-dimensional culture system recapitulates fetal liver development with: (i) initial proliferation of both fetal erythropoietic and hepatic cells and (ii) subsequent shut-off of erythropoiesis and a shift to a more advanced stage of hepatocyte function, such as bile salt secretion.

In this study, we first developed and validated a new in vitro isolated, intra-arterially perfused, gallbladder model and then applied the method to investigate the absorption of biliary lipids by the gallbladder wall and the effect of this process on the composition of human bile. Oxygenated and glucose-added buffer was perfused through the cystic artery to maintain organ viability. A standard pooled natural bile, radiolabeled with H 3 -cholesterol and C 14 -palmitoyl-linoleoyl-phosphatidylcholine, was instilled in the lumen via a cystic duct catheter. Changes in bile volume and lipid concentrations were monitored at time intervals to evaluate the disappearance of lipids from bile caused by gallbladder absorptive function. Organ viability was demonstrated by stable lactate dehydrogenase (LDH) organ release and oxygen consumption throughout the experiments. In the pig, disappearance rates of lipids from bile were similar in vitro and in vivo, demonstrating the validity of the isolated in vitro model for functional studies. By applying our in vitro isolated preparation to the human gallbladder, we found that 23% of cholesterol and 32% of phosphatidylcholine, but only 9% of bile salts, disappeared from bile in 5 hours. As a consequence, at the end of the experiments, cholesterol (P

The uptake and processing of high-density lipoprotein (HDL) unesterified and esterified cholesterol were compared in vivo in the rat. HDL labelled with 3H in either unesterified cholesterol or cholesteryl ester was administered intravenously, and the clearance of radioactivity from the blood, its distribution in plasma lipoprotein density fractions, uptake by tissues, and appearance in bile were studied at intervals up to 180 min. 3H in HDL unesterified cholesterol was cleared more rapidly from the blood than that in HDL cholesteryl ester, and this difference was mainly due to rapid sequestration of [3H]unesterified cholesterol by the liver, with 58.2% of the administered dose found in this tissue after 10 min, compared to 6.8% of the [3H]cholesteryl ester dose. Non-hepatic tissues took up only a small proportion of the administered label from both HDL unesterified and esterified cholesterol, but on a per gram wet weight basis, the specific uptake of HDL cholesteryl ester in the adrenal glands and the spleen was higher than in the liver, particularly in the first 60 min. The distribution of radioactivity in the plasma lipoprotein density fractions remained constant between 10 and 180 min when [3H]unesterified cholesterol was used, but the proportion of plasma radioactivity from HDL labelled in esterified cholesterol in the very-low-density lipoprotein (VLDL) fraction increased from 0% to 26%, while in HDL there was a shift in the distribution of radioactivity from the most (d 1.125-1.250 g/ml) to the least (d 1.050-1.085 g/ml) dense sub-fractions. A greater percentage of the administered label from HDL unesterified cholesterol (8.8%) than from HDL cholesteryl ester (3.3%) was secreted into bile during 180 min, but the proportions secreted in bile acids and unesterified cholesterol were similar with both labels. These findings indicate that there are significant differences in the uptake and processing of HDL unesterified as compared to esterified cholesterol in the rat in vivo.

Our previous in vivo studies with guinea pigs, cats and rats showed that changes in the hepatocellular levels of taurine affect the liver lipid composition of these animal species (6,7,30). Specifically, we found that a taurine-supplemented diet lowered the liver concentrations of phospholipid, but had contrasting effects on neutral lipid content. Taurine supplementation increased the liver stores of triglyceride in guinea pigs and cats (species with a low endogenous synthesis of taurine), but depleted these liver stores in rats (species with an active endogenous synthesis of taurine). The changes were not only quantitative but also qualitative. Taurine supplement (but also its depletion) induced significant changes in the cholesterol proportion, in the ratio of phosphatidylethanolamine to phosphatidylcholine and in the fatty acid profile of the major phospholipid classes. The good agreement between our data and previous in vivo (4,18,29) and in vitro (25) studies, indicated that taurine has an important role not only in biliary lipid secretion, but also in liver lipid metabolism.