Enhancement of fatty acid and cholesterol synthesis accompanied by enhanced biliary but not very-low-density lipoprotein lipid secretion following sustained pravastatin blockade of hydroxymethyl glutaryl coenzyme A reductase in rat liver.

A 3-week treatment of rats with pravastatin (PV) augmented biliary cholesterol and phospholipid output 3.6- and 2.2-fold over controls, while bile acid (BA) output and kinetics were unchanged. No major changes were detected in hepatic and serum cholesterol concentrations despite the PV inhibitory property on hydroxymethyl glutaryl coenzyme A (HMG CoA) reductase. To evaluate the mechanisms of this adaptive phenomenon, several parameters of hepatic lipid homeostasis were assessed. Biliary cholesterol changes could not be attributed to an increased influx of lipoprotein cholesterol to the liver and bile. Hepatic low-density lipoprotein (LDL) receptor content, as inferred from Western blot analysis, was unchanged, as was the biliary excretion of labeled cholesterol derived from chylomicron remnants. In vivo 3H2O-incorporation studies showed an 80% increase in hepatic cholesterol synthesis, evidence for bypass of the PV block. Remarkably, fatty acid synthesis was also stimulated twofold, providing substrate for hepatic triglycerides, which were slightly enhanced. However, serum triglycerides decreased 52% associated with a 22% decrease in hepatic very-low-density lipoprotein (VLDL) secretion. Thus, the biochemical adaptation following PV treatment produces complex alterations in hepatic lipid metabolism. An enhanced supply of newly synthesized cholesterol and fatty acids in association with a limited VLDL secretion rate augments the biliary lipid secretion pathway in this experimental model.

Capillary blood flow to the skin of forearm in cirrhosis.

In liver cirrhosis a hyperkinetic circulatory state is frequently observed as a consequence of an arterial or even a venous peripheral vasodilatation with secondary increase in cardiac output. Indirect evidence suggests that, in liver disease, the manifestation of warm hands, capillary pulsation, or palmar erythema may also relate to such a state by way of an increased skin blood flow. The purpose of the present study has been to directly assess capillary skin blood flow in liver disease through the clearance of a locally injected radioactive substance. The study was performed in 24 patients with different liver diseases, including 14 Child class II cirrhotics, and in 9 control subjects. A small volume of Na 131I solution was injected at the volar surface of the forearm, and radioactive counts were recorded continuously for ten seconds every minute for up to twenty minutes. The best fit line of the disappearance rate was determined by the least square method, and both its T/2 and an estimated blood flow parameter were calculated. The T/2 of isotope disappearance rate was 4.54 +/- 0.71 and 4.38 +/- 0.68 minutes in cirrhotics and controls, respectively. Similarly, estimates of skin blood flow (mL/min/100 g tissue) were 7.82 +/- 1.28 in the cirrhotic patients, not significantly different from those in both patients with mixed liver diseases (7.6 +/- 2.86) and control subjects (8.06 +/- 1.04). Parameters of skin blood flow were also invariant in respect to the various etiologies of liver disease. Thus, the present findings indicate that capillary skin blood flow is not affected by the hyperdynamic circulatory changes occurring in liver cirrhosis.(ABSTRACT TRUNCATED AT 250 WORDS)

[Physiopathology of iron metabolism and hemochromatosis: interrelationships, assessment and pathogenesis of iron overload, screening]. / Fisiopatologia del metabolismo del ferro ed emocromatosi: interrelazioni, inquadramento e patogenesi del sovraccarico di ferro, screening.

Fundamental aspects of iron metabolism relate to the dynamic processes of metal plasma transport as well as cell storage and efflux. Transferrin not only carries iron in the plasma but also delivers it to the various cells by binding to a diffuse specific cell receptor; it also acts by chelating cell iron. Ferritin co-operates by storing iron in the cell. By a still unknown regulatory mechanism, iron, from the ferritin pool, is redistributed in the cell to a cytosolic, easily chelatable, "transit" pool or to a degradative lysosomal hemosiderin pool from which it is slowly released outside the cell. Iron overload, such as that typical of hyperhemolysis or hemochromatosis, profoundly impairs its metabolism by saturating and/or altering transferrin and ferritin, by freeing iron from any regulated transport, thus allowing parenchymal deposition and damage. An important aspect still awaiting clarification relates to the different storage of excess iron in the parenchymal cells, as in hemochromatosis, or in the reticuloendothelial system such as in hemosiderosis. Studies using cellular models attempt to evaluate such differences in terms of altered properties of the iron proteins or their cell receptors, and of the different cell responsivity to non-transferrin iron. In the expectation of better knowledge, attention should be concentrated, from a clinical standpoint, on precise assessment of iron deposits in the tissues with the aim of preventing its excessive accumulation and parenchymal damage. In hemochromatosis, the risk of iron overload is evaluated by HLA typing.

Evolving concepts in the pathophysiology of biliary lipid secretion.

Secretion of biliary cholesterol and phosphatidylcholine is a complex process essentially involving lipid supply to the canalicular membrane from either preformed or neosynthetic hepatic sources, and the detergent action of bile salts. Previous research has shown that an altered secretion of biliary lipids and/or bile salts firmly disposes to gallstone formation, and may also be involved in the pathogenesis of cholestasis. Recently, attention has been turned to the molecular and genetic factors underlying biliary lipid secretion, and this approach has provided a significant body of new data among which: 1. The biochemical and genetic characterization of glycoproteins sP-gp and mdr2-Pgp functioning in the canalicular transport of bile salts and phosphatidylcholine, and the evaluation of their role in experimental and human cholestasis; 2. The identification of genetic patterns determining susceptibility to gallstone formation via an increased secretion of biliary lipids. It is likely that an expansion of these research lines and methodology will contribute to a better biochemical characterization of bile lipid secretion with expected benefits upon the diagnosis and treatment of related diseases; 3. A more defined appreciation of the coordinate roles played by the hepatocyte lipid synthesis and canalicular transport in the activation of the biliary lipid secretion pathway.

High affinity binding of chylomicron remnants to rat liver plasma membranes.

The binding of chylomicron remnants rat liver plasma membranes was studied. Liver membranes bound up to 8 times more remnants than they bound chylomicrons. The remnant particle appeared to bind to the membrane as a unit. Remnant binding was greatest to liver plasma membrane; only one third as much binding was observed with whole liver homogenate, and virtually no binding occurred to erythrocyte membranes or glass. Binding was saturable and had kinetics compatible with the existence of a high affinity site. Half-maximal binding occurred at 27 micron. Competitive binding studies revealed no competition with albumin, a triglyceride dispersion, cholesterol/lecithin vesicles, very low density lipoprotein, or low density lipoprotein. Some displacement of remnant binding was observed with chylomicrons and high density lipoprotein. Binding was decreased by treatment of the membranes with trypsin or the presence of heparin in the incubation medium. These studies suggest that there is a high affinity receptor for the chylomicron remnant on the surface of the hepatocyte.

Comparison of the effects of cholic acid and chenic acid feeding on rates of cholesterol synthesis in the liver of the rat.

To compare the ability of cholic acid and chenic acid to suppress cholesterol synthesis in the liver, these two bile acids were fed in varying amounts to rats for either 66 hr or 6 weeks. In both instances there were significant changes in the bile acid pool in the small intestine and suppression of the rate of cholesterol synthesis in the liver. The administration of cholic acid, however, consistently produced greater suppression of the rate of cholesterol synthesis from octanoate or of microsomal HMG CoA reductase activity than did the administration of a similar amount of chenic acid. Furthermore, this difference was present whether the rates of cholesterogenesis were measured at high-substrate concentrations, ie, under conditions where Vmax was apparently achieved, or under circumstances where there was essentially no extracellular substrate present. These findings do not support the view that the superiority of chenic acid for dissolution of gallstones is secondary to its greater effect as an inhibitor of hepatic cholesterol synthesis.

Urinary excretion of D-glucaric acid, total glucuronic acid and total porphyrins in porphyria cutanea tarda.

The urinary levels of D-glucaric acid, which is an index of hepatic microsome induction, and the excretion of glucuronic acid and porphyrins were measured in nine patients with Porphyria Cutanea Tarda (PCT) and twelve normal controls. The excretion of D-glucaric acid and glucuronic acid were respectively 3.5 and two times higher in PCT patients compared to controls. A statistical correlation could be demonstrated between urinary excretion of total porphyrins with that of glucaric and glucuronic acids. These findings indicate that microsomal function and porphyrin metabolic derangement are strictly related in PCT.

A high recovery method for concentrating microgram quantities of protein from large volumes of solution.

A method is presented for the recovery of 40-80% of the protein from a 1 microgram/ml solution. The final protein pellet is free of detergent and other ionic compounds and is thus compatible with any denaturing solution. The primary structure of the protein is unaffected by the procedure, making the final pellet an ideal sample for any analytical procedure to determine protein structure.

Dissociation of beta-hydroxy-beta-methylglutaryl-CoA reductase activity from the overall rate of cholesterol synthesis in the liver following the intravenous administration of lipid.

After the intravenous administration to the intact rat of triglyceride carried in either intestinal lipoproteins or in an artificial fat emulsion, the enzymatic activities of microsomal beta-hydroxy-beta-methylglutaryl-CoA reductase activity in the liver assayed in vitro became markedly elevated. This elevation of enzyme activity was not associated with a corresponding change in the overall rate of cholesterol synthesis in the rat liver slice as measured by the incorporation of either [3H]water or [1-14C]octanoate into nonsaponifiable lipids or into digitonin-precipitable sterols. The degree of dissociation of hydroxymethylglutaryl-CoA reductase activity from the overall rate of cholesterol synthesis correlated closely with the amount of lipid administered to the animal, the level of circulating lipids, and the level of ketone synthesis manifest in the liver cell suggesting that this phenomenon might be the consequence of a detergent effect of elevated cellular levels of fatty acids. In any event, under these experimental circumstances hydroxymethylglutaryl-CoA redutase activity no longer reflects the rate at which the liver cell is synthesizing cholesterol.

Influence of fructose feeding on individual enzymic reactions in the formation and metabolism of bile acids in rat liver homogenates.

1. Rats were maintained for 10 d on a semi-synthetic diet containing 700 g glucose or 700 g fructose/kg. Individual enzymic reactions in bile acid synthesis and metabolism were studied by measuring the 7alpha-hydroxylation of [4-14C]cholesterol, and 12alpha-hydroxylation of 7alpha-[6beta-3H]hydroxy-4-cholesten-3-one, the 26-hydroxylation of 5beta-[7beta-3H]cholestane-3alpha,7alpha-diol and the 6beta-hydroxylation of [3H]lithocholic acid in liver homogenates. 2. The serum cholesterol level was approximately the same in both groups of animals, but the serum triglyceride level was almost twice as high in the fructose-fed rats compared to the glucose-fed rats. 3. The 6beta-hydroxylation of [3H]ithocholic acid was increased by about 20% in the fructose-fed rats compared to the glucose-fed animals. The activities of the other enzymic reactions studied did not differ significantly between the two groups of animals. The findings are discussed in relation to previous knowledge concerning mechanisms regulating triglyceride, pre-beta-lipoprotein and bile acid synthesis.

Modulation of biliary lipid secretion by forskolin and cyclic AMP analogues.

Exposure of isolated perfused rat livers to either 100 microM-forskolin, a potent activator of adenylate cyclase, or to 0.5 mM-concentrations of the cAMP analogues chlorophenylthio cAMP (CPTcAMP), dibutyryl cAMP (dbcAMP) and 8-bromo cAMP (8BrcAMP), to provoke increases in intracellular concentrations of cAMP, resulted in marked changes in bile volume and composition. Bile flow reached a peak after 10 min, before declining towards control levels, and an increase in several secretory parameters was also observed at this time. At 20 min, a substantial decrease in the output of both phospholipid and cholesterol was evident, and this suppression of secretion was maintained throughout the remainder of the experiment. The order of effectiveness of the cAMP-elevating agents at decreasing biliary lipid output was CPTcAMP greater than forskolin greater than dbcAMP greater than 8BrcAMP. Biliary output of bile acids was essentially unaltered compared with controls; similarly, no decrease in the secretion of protein and triacylglycerols into the perfusion medium was observed. This suggests that the elevation of intracellular levels of cAMP may cause a selective inhibition of biliary lipid output rather than a more general inhibition of hepatic secretion.

Effect of cholestyramine treatment on biliary lipid secretion rates in normolipidaemic men.

This study was designed to clarify the effect of bile acid sequestrant treatment on the total biliary output rates of cholesterol, phospholipids and bile acids in man, and to correlate these changes with the alterations in plasma lipoprotein levels. For this purpose nine healthy, normolipidaemic men were treated with 16 g of cholestyramine daily over a period of 4 weeks, and the biliary secretion rates were measured by a duodenal perfusion technique. Resin therapy, which profoundly increases de novo synthesis of bile acids, resulted in a lowering of total plasma cholesterol levels, mainly due to a 35% reduction in low density lipoprotein (LDL) cholesterol, and in a 33% increase in plasma triglyceride levels, reflecting enhanced very low density lipoprotein (VLDL) triglyceride concentrations; high density lipoprotein (HDL) levels did not change. However, these lipoprotein changes did not correlate with any alterations in biliary lipid output. Total hepatic secretion rates of the biliary lipids remained generally unchanged during treatment, with a tendency towards lower cholesterol output, resulting in a lower molar percentage of cholesterol in hepatic bile, 3.4 +/- 0.4 vs. 2.9 +/- 0.2 mol %. This is probably due to an increased rate of conversion of cholesterol to bile acids in the hepatocyte. It is concluded that, in man, the liver may adapt well to changes in the enterohepatic circulation of bile acids, thereby maintaining output rates of biliary lipids at a relatively constant level.

ABO blood groups and serum gastrin.

Serum gastrin concentrations were measured in 75 subjects of blood group O and in 75 subjects of other blood groups. Gastrinaemia both basally and following stimulation by glycine drink or by insulin hypoglycaemia did not show any statistically significant differenc in blood group O people as compared to subjects of other blood groups. It is concluded that the claimed relationship between blood group O and parietal cell hyperplasia cannot be considered as secondary to a relationship between blood group O and increased gastrin-producting G cell mass.

Biliary excretion of chylomicron remnant cholesterol in the rat: responses to the expansion of their plasma pool and promoting role of stimulated bile-acid synthesis.

1. Chylomicron remnants, the intermediate intestinal lipoproteins carrying the bulk of dietary cholesterol, are actively taken up and degraded in the hepatocytes, releasing cholesterol which can be excreted in bile. To study this pathway, a mass of remnants, leading to a consistent rise in hepatic cholesterol, was administered as an intravenous bolus in rats with chronic bile fistula equilibrated by water, electrolyte and taurocholate infusions, and changes in biliary lipids and bile acids were evaluated for up to 24 h in comparison with baseline. 2. A mean 16% increase in the net output of bile acids was observed at each time interval after lipoprotein injection, accounting for a 24h cumulative excretion of approximately one-third of the administered cholesterol mass. These changes did not reach statistical significance however. The cholesterol output and concentrations of all biliary lipids did not vary either. Without taurocholate replacement, remnants injection was followed by a 15-20% decrease in bile acid and bile lipid secretion, presumably due to an insufficient hepatic bile-acid flux. 3. When [3H]cholesterol-labelled remnants were administered at the same mass in the chronic equilibrated bile fistula model, 21% of injected radioactivity was excreted in 24h, distributing mostly in acidic rather than neutral sterols (20.02 +/- 1.85 compared with 1.07 +/- 0.04), with an acidic to neutral sterol mean ratio of 16. 4. To exclude interfering effects from the administered cholesterol mass and chronic bile fistula, 3H-labelled remnants were also studied as a cholesterol trace injected in rats with acute bile fistula.(ABSTRACT TRUNCATED AT 250 WORDS)