Roelf Vonk

Enterohepatic circulation in the rat

Several compounds, in particular bile acids, undergo enterohepatic circulation (EHC). Limited data are available on the pathophysiologic aspects of this circulation. In the present study we describe a surgical technique in rats that allows a long-term, reversible interruption of the EHC and monitoring of peripheral blood levels without direct surgical intervention. This technique excludes the effects of anesthesia and surgical trauma. The models validity has been tested extensively. We used this animal model to investigate acute and chronic effects of interruption and subsequent restoration of the EHC on (a) nutritional status, (b) plasma cholesterol levels and hepatic cholesterol synthesis, and (c) biliary bile acid, phospholipid, and cholesterol excretion. Interruption of the EHC resulted in an increased food intake and enhanced fecal energy loss, caused by a less efficient intestinal absorption. Plasma cholesterol concentrations declined immediately after the interruption of the EHC, but returned to almost control values during bile diversion. A marked overshoot followed the subsequent restoration of the EHC. Hepatic cholesterol synthesis showed a five-fold increase after 8 days of bile diversion but returned to control values within 2 days after restoration of the EHC. After interruption of the EHC, bile acid, phospholipid, and cholesterol excretion decreased sharply but stabilized after 3 h at 7.6%, 20%, and 23%, respectively, of their initial values. Bile acid output, representing hepatic synthesis, slowly increased over 4 days, but never exceeded 13% of its value during intact EHC. Subsequent restoration of the EHC could rapidly reverse the observed effects of the interruption. The animal model described in the present study is an excellent tool in studying the acute and chronic effects of disturbances of the EHC.

Incomplete intestinal absorption of fructose.

Intestinal D-fructose absorption in 31 children was investigated using measurements of breath hydrogen. Twenty five children had no abdominal symptoms and six had functional bowel disorders. After ingestion of fructose (2 g/kg bodyweight), 22 children (71%) showed a breath hydrogen increase of more than 10 ppm over basal values, indicating incomplete absorption: the increase averaged 53 ppm, range 12 to 250 ppm. Four of these children experienced abdominal symptoms. Three of the six children with bowel disorders showed incomplete absorption. Seven children were tested again with an equal amount of glucose, and in three of them also of galactose, added to the fructose. The mean maximum breath hydrogen increases were 5 and 10 ppm, respectively, compared with 103 ppm after fructose alone. In one boy several tests were performed with various sugars; fructose was the only sugar incompletely absorbed, and the effect of glucose on fructose absorption was shown to be dependent on the amount added. It is concluded that children have a limited absorptive capacity for fructose. We speculate that the enhancing effect of glucose and galactose on fructose absorption may be due to activation of the fructose carrier. Apple juice in particular contains fructose in excess of glucose and could lead to abdominal symptoms in susceptible children.

Dietary fish oil-induced changes in intrahepatic cholesterol transport and bile acid synthesis in rats.

Hepatic cholesterol metabolism was studied in rats fed purified diets supplemented (9% wt/wt) with either fish oil (FO) (n-3 fatty acids) or corn oil (CO) (n-6 fatty acids) for 4 wk. Rats were equipped with permanent catheters in heart, bile duct, and duodenum to allow studies under normal feeding conditions. [3H]-cholesteryl oleate-labeled small unilamellar liposomes, which are rapidly endocytosed by hepatocytes, were intravenously injected to label intrahepatic cholesterol pools, and plasma and bile were collected. FO as compared to CO induced a lowering of plasma cholesterol levels by 38% and of triglyceride levels by 69%. This reduction in plasma lipids in FO rats was accompanied by: (a) an increased bile acid pool size (28%); (b) a fourfold increase in the ratio cholic acid/chenodeoxycholic acid in bile; (c) increased biliary excretion of cholesterol (51%); (d) accelerated excretion of endocytosed free cholesterol into bile; (e) accelerated incorporation of endocytosed cholesterol in bile acids; (f) a significant increase in the bile acid-independent fraction of bile flow; and (g) a threefold increase in hepatic alkaline phosphatase activity. The results show that FO induces changes in transport and metabolic pathways of cholesterol in the rat liver, which result in a more rapid disposition of plasma-derived cholesterol into the bile.

Lactose intolerance: analysis of underlying factors

Background We studied the degree of lactose digestion and orocecal transit time (OCTT) as possible causes for the variability of symptoms of lactose intolerance (LI) in a sample of a population with genetically determined low lactase activity.

Non-Invasive Detection of Low-Intestinal Lactase Activity in Children by Use of a Combined 13CO2/H2 Breath Test

BACKGROUND The aim of the study was to diagnose hypolactasia with a higher accuracy than with the traditional H2 breath test. METHODS We used a combined 13C-lactose 13CO2/H2 breath test, which was performed in 33 patients in whom lactase activity was measured. RESULTS Lactase activity was reduced in 13 cases. The sensitivity and specificity of the H2 test were 54% and 90%; those of the 13CO2 test 69% and 70%. False-negative results did not always occur in the same patients. In five of six patients with both test results abnormal, lactase activity was low. In 13 of 15 patients with both test results normal, lactase activity was normal. In 6 of 12 cases with only 1 test abnormal, lactase activity was low. CONCLUSION The combined H2/13CO2 breath test (sensitivity, 85%; specificity, 65%) is more adequate for diagnosis of hypolactasia than the H2 breath test alone.

The 13C/2H-glucose test for determination of small intestinal lactase activity

To diagnose hypolactasia, determination of lactase enzyme activity in small intestinal biopsy material is considered to be the golden standard. Because of its strongly invasive character and the sampling problems, alternative methods have been looked for.

Ketoconazole blocks bile acid synthesis in hepatocyte monolayer cultures and in vivo in rat by inhibiting cholesterol 7 alpha-hydroxylase.

In cultured hepatocytes conversion of [4-14C]cholesterol into bile acids was dose dependently reduced by the antimycotic drug ketoconazole, giving half-maximal inhibition at 10 microM ketoconazole in rat hepatocytes and at 1 microM in human hepatocytes. No change was observed in the ratio of produced cholic, beta-muricholic, and chenodeoxycholic acid with increasing amounts of the drug. Conversion of [4-14C]7 alpha-hydroxycholesterol, an intermediate of bile acid pathway, to bile acids was not affected by ketoconazole. These results together with kinetic studies with rat liver microsomes, demonstrating noncompetitive inhibition (Ki = 0.4 microM), indicate that cholesterol 7 alpha-hydroxylase is the main site of inhibition. In bile-diverted rats a single dose of ketoconazole (50 mg/kg) dramatically impaired bile flow and biliary bile acid output (92% inhibition). A similar blockade was observed using [4-14C]cholesterol as precursor for bile acid synthesis. Therefore, treatment of patients with this drug may inhibit bile acid synthesis, resulting in a reduction of the bile acid pool size after long-term ketoconazole therapy.

BENIGN RECURRENT INTRAHEPATIC CHOLESTASIS - ALTERED BILE-ACID METABOLISM

Altered bile acid metabolism has been claimed to play a role in the etiology of benign recurrent intrahepatic cholestasis (BRIC). Therefore, we studied bile acid metabolism in detail in 10 patients with this syndrome. Pool sizes of both primary bile acids were estimated simultaneously, using deuterated cholic acid and chenodeoxycholic acid. The pool sizes of cholic acid and chenodeoxycholic acid, expressed in micromoles per kilogram body weight, were significantly contracted in BRIC patients during a cholestasis-free period: 8.0 +/- 4.2 and 11.7 +/- 4.7, respectively, versus 24.1 +/- 11.7 and 22.9 +/- 7.8 in controls. Fractional turnover rates (per day) for cholic acid and chenodeoxycholic acid were increased: 0.70 +/- 0.29 and 0.58 +/- 0.27, respectively, versus 0.29 +/- 0.12 and 0.23 +/- 0.10 in controls. Bile acid pool composition expressed as percentages in BRIC patients was cholic acid 34 +/- 17, chenodeoxycholic acid 38 +/- 9, deoxycholic acid 27 +/- 18, and lithocholic acid 1 +/- 1, with a glycine to taurine conjugation ratio of 6.7 +/- 4.9. Corresponding values for 32 controls were cholic acid 57 +/- 13, chenodeoxycholic acid 29 +/- 9, deoxycholic acid 14 +/- 9, and lithocholic acid less than 1, with a glycine to taurine conjugation ratio of 2.4 +/- 1.3. Fecal bile acid loss, in micromoles per kilogram body weight per day, was 11.2 +/- 9.0 in BRIC patients compared with 2.8 +/- 1.4 in controls. The serum 7 alpha-hydroxycholesterol level (nanomoles per liter) was significantly increased in BRIC patients: 326 +/- 179 versus 171 +/- 90 in controls. These results suggest that in BRIC patients spillover of bile acids into the colon occurs, which leads to increased fecal bile acid loss and a reduced bile acid pool size. Increased serum 7 alpha-hydroxycholesterol is probably indicative of an accelerated bile acid synthesis rate due to increased activity of cholesterol 7 alpha-hydroxylase, the enzyme catalyzing the first step in the major pathway of bile acid synthesis. The results of our study suggest that in BRIC patients a contracted bile acid pool increases the susceptibility of the liver for cholestatic agents.

Adenosine triphosphate-dependent copper transport in isolated rat liver plasma membranes.

The process of hepatobiliary copper (Cu) secretion is still poorly understood: Cu secretion as a complex with glutathione and transport via a lysosomal pathway have been proposed. The recent cloning and sequencing of the gene for Wilson disease indicates that Cu transport in liver cells may be mediated by a Cu transporting P-type ATPase. Biochemical evidence for ATP-dependent Cu transport in mammalian systems, however, has not been reported so far. We have investigated Cu transport in rat liver plasma membrane vesicles enriched in canalicular or basolateral membranes in the presence and absence of ATP (4 mM) and an ATP-regenerating system. The presence of ATP clearly stimulated uptake of radiolabeled Cu (64Cu, 10 microM) into canalicular plasma membrane vesicles and, to a lesser extent, also into basolateral plasma membrane vesicles. ATP-dependent Cu transport was dose-dependently inhibited by the P-type ATPase inhibitor vanadate, and showed saturation kinetics with an estimated Km of 8.6 microM and a Vmax of 6.9 nmol/min/mg protein. ATP-stimulated Cu uptake was similar in canalicular membrane vesicles of normal Wistar rats and those of mutant GY rats, expressing a congenital defect in the activity of the ATP-dependent canalicular glutathione-conjugate transporter (cMOAT). These studies demonstrate the presence of an ATP-dependent Cu transporting system in isolated plasma membrane fractions of rat liver distinct from cMOAT.

Bile diversion in rats leads to a decreased plasma concentration of linoleic acid which is not due to decreased net intestinal absorption of dietary linoleic acid

Decreased bile secretion into the intestine has been associated with low plasma concentrations of essential fatty acids (EFA) in humans. We studied the mechanism behind this relationship by determining the status and absorption of the major dietary EFA, linoleic acid (LA), in control and 1-week bile-diverted rats. The absorption of LA was quantified by a balance method and by measuring plasma concentrations of [13C]LA after its intraduodenal administration. Absolute and relative concentrations of LA in plasma were decreased in bile-diverted rats (P<0.01 and P<0.001, respectively). Fecal excretion of LA was increased at least 20-fold in bile-diverted rats (0.72+/-0.11 vs. 0.03+/-0.00 mmol/day; P<0.0001). Due to increased chow ingestion by bile-diverted rats, net intestinal absorption of LA was similar between bile-diverted and control rats (1.96+/-0.14 vs. 1.91+/-0.07 mmol/day, respectively; P>0.05). After intraduodenal administration of [13C]LA, plasma concentrations were approximately 3-4-fold lower in bile-diverted rats for at least 6 h (P<0.001). Plasma concentrations of both [12C]arachidonic acid and [13C]arachidonic acid were increased in bile-diverted rats (P<0.05). We conclude that decreased plasma concentrations of LA in 1-week bile-diverted rats are not due to decreased net intestinal absorption of LA, but may be related to increased metabolism of LA.