Staffan Ewerth

Hepatic Uptake of Bile Acids in Man: FASTING AND POSTPRANDIAL CONCENTRATIONS OF INDIVIDUAL BILE ACIDS IN PORTAL VENOUS AND SYSTEMIC BLOOD SERUM

This investigation was undertaken in order to (a) characterize the postprandial inflow of individual bile acids to the liver and (b) determine if peripheral venous bile acid levels always adequately reflect the portal venous concentration, or if saturation of hepatic bile acid uptake can occur under physiological conditions. In five patients with uncomplicated cholesterol gallstone disease, the umbilical cord was cannulated during cholecystectomy, and a catheter was left in the left portal branch for 5 to 7 d. The serum concentrations of cholic acid, chenodeoxycholic acid, and deoxycholic acid in portal venous and systemic circulation were then determined at intervals of 15 to 30 min before and after a standardized meal. A highly accurate and specific gas chromatographic/mass spectrometric technique was used. The sum of the fasting concentrations of the three bile acids averaged 14.04+/-4.13 mumol/liter in portal venous serum, and 2.44+/-0.31 mumol/liter in peripheral venous serum. The estimated hepatic fractional uptake of cholic acid was approximately 90%, and those of chenodeoxycholic acid and deoxycholic acid were 70-80%. This resulted in an enrichment of systemic bile acids in the dihydroxy bile acid species. In response to a standardized meal, portal venous bile acid concentrations increased two- to sixfold, with a peak seen 15-60 min after the meal. The maximum postprandial portal venous bile acid concentration averaged 43.04+/-6.12 mumol/liter, and the corresponding concentration in peripheral serum was 5.22+/-0.74 mumol/liter. The estimated fractional uptakes of the individual bile acids were not affected by the increased inflow to the liver. The peripheral venous concentrations of individual as well as total bile acids were well correlated with those in portal venous serum. The results (a) give a quantitation of postprandial bile acid inflow to the liver and (b) indicate that the hepatic uptake system for bile acids in healthy man cannot be saturated during maximal inflow of endogenous bile acids. Measurement of peripheral serum bile acids can thus give important information on the status of the enterohepatic circulation.

Bile acid sequestrants: mechanisms of action on bile acid and cholesterol metabolism

SummaryInterruption of the enterohepatic circulation of bile acids by cholestyramine or colestipol influences the hepatic metabolism of cholesterol in many ways. The synthesis of bile acids is increased, as reflected by a several-fold increase in the activity of the cholesterol 7a hydroxylase, the rate-determining enzyme in bile acid synthesis. The increased metabolism of cholesterol to bile acids causes an enhanced demand of cholesterol in the hepatocytes, which respond with both new synthesis of cholesterol, as reflected in a several-fold increase of the HMG-CoA reductase activity, and increased expression of LDL receptors. As a consequence, the plasma level of LDL-cholesterol is lowered. The hepatic secretion rate of VLDL particles is increased. Cholestyramine therapy does not affect the output of biliary lipids or the cholesterol saturation of bile, indicating that treatment with bile acid sequestrants should not be associated with any increased risk of gallstone formation.

Bile acid synthesis in humans: Regulation of hepatic microsomal cholesterol 7α-hydroxylase activity

The present work tested the hypothesis that portal venous bile acids regulate the activity of the cholesterol 7 alpha-hydroxylase and studied the influence of hepatic microsomal free cholesterol concentration on the enzyme activity. Operative liver biopsies and samples of portal venous blood were obtained from a total of 61 patients with gallstones who were undergoing cholecystectomy. Fifteen of the patients were treated with cholestyramine (16 g/day) for 2-3 wk before operation and 23 patients with chenodeoxycholic acid (15 mg/kg.day) or ursodeoxycholic acid (15 mg/kg.day) for 3-4 wk before operation. Highly accurate methods based on isotope dilution-mass spectrometry were used for assay of the cholesterol 7 alpha-hydroxylase activity, the concentration of free cholesterol in the microsomes, and the levels of individual bile acids in portal venous blood. Cholestyramine treatment increased the cholesterol 7 alpha-hydroxylase activity about sixfold, from 7.6 +/- 1.1 (mean +/- SEM) to 45.7 +/- 6.7 pmol/min.mg protein. Administration of chenodeoxycholic acid reduced the enzyme activity considerably to 1.0 +/- 0.3 pmol/min.mg protein, whereas ursodeoxycholic acid did not significantly affect the enzyme activity (7.9 +/- 2.2 pmol/min.mg protein). The concentration of microsomal free cholesterol remained essentially unchanged in spite of a 45-fold variation in enzyme activity. There was a negative correlation between the absolute as well as the relative concentration of chenodeoxycholic acid in portal blood and the activity of the cholesterol 7 alpha-hydroxylase, whereas there was no correlation between the total concentration of bile acids and the enzyme activity. It is concluded that the composition of individual bile acids may be more important than the total concentration of bile acids in the portal vein for the regulation of the cholesterol 7 alpha-hydroxylase activity in humans. It is further concluded that chenodeoxycholic acid is a considerably stronger suppressor of bile acid synthesis than ursodeoxycholic acid.

Hepatic cholesterol metabolism in estrogen-treated men

Operative liver biopsies were obtained from two male patients who developed gallstone disease during estrogen treatment of metastatic prostatic carcinoma. The heparin-sensitive binding of 125I-low-density lipoprotein (LDL) to liver homogenates (reflecting the expression of the LDL receptor) was determined, together with the activities of the rate-limiting enzymes in cholesterol synthesis [3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) reductase], bile acid production (cholesterol 7 alpha-hydroxylase), and cholesterol esterification (acyl CoA:cholesterol acyl transferase). The results were related to data available in 18 patients (5 male, 13 female) who underwent cholecystectomy because of gallstone disease. The hepatic 125I-LDL-binding activity was increased threefold compared with five controls, and the activity of HMG-CoA reductase was increased twofold. There was no major difference in the activities of cholesterol 7 alpha-hydroxylase or acyl CoA:cholesterol acyl transferase. The concentration of free and total cholesterol in liver microsomes was approximately 30% lower in the estrogen-treated men than in 11 controls. The results indicate that estrogen at pharmacological doses stimulates hepatic LDL-receptor expression and HMG-CoA reductase activity in men. The increased LDL-receptor expression could in part explain the enhanced plasma clearance of injected 125I-LDL and hence the reduction in plasma LDL cholesterol previously shown to occur in estrogen-treated men.

25-Hydroxylase activity in subcellular fractions from human liver. Evidence for different rates of mitochondrial hydroxylation of vitamin D2 and D3

25-Hydroxylation of vitamin D2 and D3 was studied in subcellular fractions from human liver, using a technique based on isotope dilution-mass spectrometry. The mitochondrial fraction fortified with isocitrate catalysed 25-hydroxylation of vitamin D3 at a rate of about 10 pmol/mg protein X min. Under the same conditions, the rate of 25-hydroxylation of vitamin D2 was less than 2 pmol/mg protein X min. Crude microsomes fortified with NADPH catalysed 25-hydroxylation of vitamin D3 to a very low extent, and this activity was not linear with the amount of microsomal protein. A higher rate of conversion was obtained with a partially purified cytochrome P-450 fraction in the presence of NADPH-cytochrome P-450 reductase and NADPH. This fraction also catalysed 25-hydroxylation of 1 alpha-hydroxyvitamin D3 and 5 beta-cholestane-3 alpha, 7 alpha, 12 alpha-triol. 25-Hydroxylation of vitamin D2 could not be detected, neither with crude microsomes, nor with the microsomal cytochrome P-450 fraction. Since the assay for 25-hydroxyvitamin D2 was less sensitive than that for 25-hydroxyvitamin D3, these experiments do not rule out the presence of some 25-hydroxylase activity towards vitamin D2 in the microsomes. The results are discussed in relation to previous work in which a lower toxicity has been reported for vitamin D2 than for vitamin D3 in some mammalian species.

Biliary excretion of iron and ferritin in idiopathic hemochromatosis

The role of biliary excretion of iron and ferritin in iron overload was studied and evaluated. Ten patients with idiopathic hemochromatosis and two groups of controls (14 gallstone patients and 16 healthy subjects) were included. Liver tissue (obtained by percutaneous or operative biopsy) was investigated with light microscopy and transmission electron microscopy in combination with x-ray microanalysis. Fasting bile samples were obtained through duodenal aspiration or at cholecystectomy. Iron was determined in liver tissue and bile using atomic absorption spectroscopy, and ferritin was determined in serum and bile with a radioimmunoassay technique. All patients with hemochromatosis had iron-positive staining as seen in light microscopy. Electron microscopy showed iron-containing proteins in the lysosomes and cytosol of liver parenchymal cells, and this observation was supported by x-ray microanalysis. Hepatic iron concentration was increased about eightfold in the patients with hemochromatosis (p less than 0.001). Biliary iron concentration, expressed per millimole of bile acid, was increased about twofold (p less than 0.05) and biliary ferritin concentration about fivefold (p less than 0.001) in hemochromatosis. Four of the patients with hemochromatosis were reexamined after completed treatment with venesection; this resulted in normalized biliary concentrations of iron and ferritin. We conclude that biliary secretion of ferritin occurs in humans and that both iron and ferritin excretion are enhanced in hepatic iron overload. The apparently limited capacity of biliary iron excretion may be of importance for the hepatic iron accumulation in hemochromatosis.

Hepatic metabolism of cholesterol in Crohn's disease: Effect of partial resection of ileum

To study cholesterol metabolism in Crohns disease and especially the effect of ileum resection, liver biopsy specimens were obtained from patients undergoing partial ileal resection because of Crohns disease (n = 17) and patients with Crohns colitis undergoing colectomy (n = 3). Gallstone-free patients (n = 16) undergoing cholecystectomy because of adenomyomas or polyps of the gallbladder served as controls. The mean levels of cholesterol 7 alpha-hydroxylase activity and 3-hydroxy-3-methylglutaryl coenzyme A reductase activity, rate-determining enzymes in bile acid, and cholesterol synthesis, respectively, were twofold to threefold higher in the ileum-resected patients than in the controls. Significant positive correlations were obtained between length of resected ileum and cholesterol 7 alpha-hydroxylase activity. Provided patients who had received total parenteral nutrition preoperatively were excluded from analysis, a significant correlation was also observed between length of resected ileum and 3-hydroxy-3-methylglutaryl coenzyme A reductase activity. Significant positive correlations were also obtained between length of resected ileum and serum levels of 7 alpha-hydroxycholesterol (a marker for bile acid biosynthesis) and lathosterol (a marker for cholesterol synthesis). The plasma levels of total and low-density lipoprotein cholesterol were negatively correlated to the length of resected ileum. The expression of hepatic low-density lipoprotein-receptor binding activity was determined in five of the patients and in three of the controls. A significant positive correlation was observed between 3-hydroxy-3-methylglutaryl coenzyme A reductase activity and low-density lipoprotein-receptor binding activity. The results show that malabsorption of bile acids leads to parallel stimulation of cholesterol synthesis, cholesterol degradation, and low-density lipoprotein-receptor expression in human liver. The resulting effect in the present patients was a significant reduction in low-density lipoprotein cholesterol.

Cholestyramine Treatment Reduces Postprandial but not Fasting Serum Bile Acid Levels in Humans

Fasting serum concentrations of cholic acid, chenodeoxycholic acid, and deoxycholic acid were determined in healthy subjects and in patients with familial hypercholesterolemia before and during treatment with cholestyramine. The bile acids were analyzed by a specific isotope-dilution technique by using gas chromatography-mass spectrometry. Cholestyramine treatment did not change the fasting concentration of total bile acids, but the contribution of cholic acid was increased; those of chenodeoxycholic acid and deoxycholic acid were decreased. No decrease of fasting bile-acid concentrations in portal venous serum was seen in 2 cholestyramine-treated gallstone patients. The postprandial total bile-acid concentration was about 40% lower during cholestyramine treatment in healthy subjects, reflecting a reduced postprandial inflow of bile acids to the liver. This degree of interruption of the postprandial enterohepatic circulation may be sufficient to produce a near maximal bile-acid biosynthesis rate and to promote lowering of plasma cholesterol also in the fasting state. It is concluded that the postprandial bile-acid inflow to the liver may be more important as a regulator of bile-acid biosynthesis than is the fasting level of bile acids.

Glucagon receptor of human liver. Studies of its molecular weight and binding properties, and its ability to activate hepatic adenylyl cyclase of non-obese and obese subjects.

The glucagon receptor and the adenylyl cyclase system of human liver membranes were studied in six non-obese and six obese subjects who had elevated insulin and plasma glucagon levels. Analysis of specific glucagon binding by the method of Scatchard demonstrated a linear (monocomponent) plot with a dissociation constant of 2-3 nM, and the binding at low hormone concentrations was sensitive to guanosine triphosphate (GTP). The molecular weight of the glucagon receptor was 63,000 D as determined by an affinity labeling procedure and sodium dodecyl sulfate gel electrophoresis. Affinity labeling of this structure was specific for glucagon and inhibited by GTP. Glucagon stimulated the production of cyclic adenosine monophosphate (cAMP) by human membranes with half-maximal activation elicited by 6 nM hormone. The human cyclase system required GTP to facilitate an optimal glucagon response. NaF (10 mM) also activated the cyclase system and produced the same magnitude of response as maximum glucagon activation. A comparison of the liver adenylyl cyclase system of non-obese and obese subjects was made using glucagon (5 nM and 1 microM) and NaF (10 mM). No significant differences in cAMP production were noted between the two groups, regardless of the agent used to activate the enzyme. These findings agree with the glucagon binding studies that showed similar amounts of binding activity in the membranes from the two groups. Also, there was no influence of either age or sex of the subjects on the adenylyl cyclase response. In conclusion, human liver membranes contain a glucagon receptor and an adenylyl cyclase system that correspond closely to the well-studied system in animal liver. This system in human obesity is not altered by the approximately twofold elevation in plasma glucagon that occurs in this metabolic disorder.

Studies of the human liver insulin receptor in noninsulin-dependent diabetes mellitus

The insulin binding characteristics and the structural components of the insulin receptor were studied in the purified liver plasma membranes from seven patients with noninsulin-dependent diabetes (NIDDM) and seven control subjects. In comparison to the controls, diabetic subjects had a 65% reduction in plasma insulin levels in response to an oral glucose load. Specific insulin binding by liver membranes from diabetic patients was, however, twofold greater than the binding activity by membranes from control subjects. This alteration resulted largely from an increase in the number of insulin receptors and a modest increase in receptor binding affinity. Holo (nonreduced) receptor species of similar molecular weights were detected by an affinity labeling technique in the two membrane preparations and sulfhydryl reduction demonstrated an insulin binding subunit of 125,000 mol wt. Overall, these results show that the hepatic insulin resistance of NIDDM cannot be explained by a deficiency in insulin binding.