Robert F. Raicht

Sterol balance studies in the rat. Effects of dietary cholesterol and β-sitosterol on sterol balance and rate-limiting enzymes of sterol metabolism

Sterol balance measurements using isotopic and chromatographic techniques were carried out in rats fed diets containing beta-sitosterol (0.8%) and cholesterol (1.2%). The activities of the rate-limiting enzymes of cholesterol synthesis (beta-hydroxy-beta-methylglutaryl-CoA reductase, EC 1.1.1.34) and bile acid synthesis (cholesterol 7 alpha-hydroxylase) were determined in the same animals. Cholesterol feeding increased cholesterol absorption from 1.2 to 70 mg/day. The increased absorption was compensated for by inhibition of hepatic cholesterol synthesis, enhanced conversion of cholesterol to bile acids (from 13.7 to 27.3 mg/day) and a slight increase in the excretion of endogenous neutral steroids (from 7.7 to 11.2 mg/day). Despite the adaptation there was accumulation of cholesterol in the liver (from 2.2 to 9.2 mg/g). Beta-Sitosterol feeding inhibited cholesterol absorption (calculated absorption was zero). In these rats there was enhanced cholesterol synthesis (from 20.0 to 28.8 mg/day, but no change in the rates of bile acid formation. Measurements of the activities of the rate-limiting enzymes showed fair correlation with cholesterol-bile acid balance. In cholesterol fed animals, beta-hydroxy-beta-methylglutaryl-CoA reductase was inhibited 80% and cholesterol 7 alpha-hydroxylase was enhanced 61%. In beta-sitosterol-fed animals, the reductase was increased 2-fold and cholesterol 7 alpha-hydroxylase was not significantly different from controls.

Hemorrhoids or Rectal Varices: Defining the Cause of Massive Rectal Hemorrhage in Patients With Portal Hypertension

Identifying the source of lower gastrointestinal hemorrhage in patients with chronic liver disease and portal hypertension can be challenging. We present 2 cases of hemorrhage from rectal varices and a discussion on the differences between simple hemorrhoids and rectal varices. Evaluation of rectal bleeding in patients with portal hypertension is discussed and possible therapeutic options are described.

Effects of clofibrate on sterol metabolism in the rat.

Abstract Sterol balance measurements using isotopic and Chromatographie techniques were carried out in rats on a stock diet and a stock diet plus 0.3% clofibrate (α-p-chlorophenoxyisobutyryl ethyl ester (CPIB)). At the end of the feeding period the activity of hepatic microsomal β-hydroxy-β-methylglutaryl-CoA reductase (EC 1.1.1.34), the rate limiting enzyme of cholesterol synthesis was determined in each animal. Administration of the drug caused a significant decrease in fecal bile acid output (10.5–7.6 mg/day), no change in fecal neutral steroid output (11–10 mg/day) and a decrease in cholesterol synthesis (16.6–12.6 mg/day). Biliary cholesterol concentration was reduced from 0.23 mg/ml in the controls to 0.11 mg/ml in the clofibrate treated group. A decrease in both liver cholesterol and plasma cholesterol was also observed in response to the drug. Measurement of the activity of the rate limiting enzyme of cholesterol synthesis showed a 2-fold decrease of activity when cholesterol synthesis was reduced 25%.

Influence of Bile on Kinetic Behavior of Colonic Epithelial Cells of the Rat

The acute effect of bile deprivation on colonic epithelial cell proliferation in Sprague-Dawley rats was investigated and compared with its effect on jejunum and ileum. 2. days after the creation of bile fistula, tritiated thymidine was injected and animals sacrificed 1 and 24 h later. Compared with control untreated animals, sham-operated restrained rats had a reduced labeling and mitotic index of the colonic epithelial cell population as well as a slower migration of cells to the lumen. Colonic cell proliferation in animals deprived of bile flow was reduced a further 50%. Moreover, no evidence of cell migration or appreciable decline in grain density was seen over 24 h in bile fistula rats. Alterations in cell proliferation in both sham and bile fistula treated rats became less marked as one proceeded proximally to the small bowel. Therefore, significant alterations in cell kinetics result when normal bile flow is interrupted, suggesting its importance in the regulatory control of colonic cell proliferation.

Expression of growth factor receptor-encoded mRNA by colonic epithelial cells is altered in inflammatory bowel diseas

A link between inflammation of the colon in inflammatory bowel disease (IBD) and the increased risk of colon cancer in ulcerative colitis (UC) may be provided by growth factor receptor genes. Their expression may be altered in response to growth factors present in the mucosa, and this, in turn, may induce further genetic changes, linked to carcinogenesis, in the cells of the colonic epithelium. To test this hypothesis, we assayed steady-state levels of eight growth factor receptor mRNAs in colonic epithelial cells of IBD patients and controls. Four of these genes (EGF-R, IGFI-R, CSF1-R, andPDGF-R-β) were expressed in epithelial cells, whereas four (erbB-2, erbB-3, NGF-R, andmet) were not. The level of the former in involved or uninvolved IBD was considerably lower than in normal epithelial cells from either sporadic colon cancer or diverticulitis patients. In contrast, expression was much higher in IBD patients with colon tumors than in active chronic IBD. The level of PDGF-R-β mRNA was two- to fourfold higher in involved than in uninvolved areas of the colons of two UC patients, but not in one Crohns disease patient. Message abundance of its ligand,PDGF-β, however, was the same in paired UC samples. The pattern of expression ofPDGF-β andcripto was identical to that ofEGF-R, whereas the level of mRNA of amphiregulin was the same in active chronic IBD and IBD patients with tumors. A fourth growth factor,Kfgf, was not expressed. Increased levels of PDGF-R-β mRNA in involved UC relative to uninvolved UC may be related to the disease process in UC. Decreased expression of growth factor- and growth factor receptor-encoded mRNA in active chronic IBD may be related to the disease process, or it may be an effect of steroid therapy undergone by these patients. Enhanced expression of these genes in IBD patients with tumors compared to those without tumors suggests that this may be a marker for development of colon cancer in IBD.

Purification of total RNA from human stool samples

While colonoscopy may detect early-stage colontumors, a less invasive and more costeffective techniquewould be beneficial. Stool, which picks up sloughed-offcolonic epithelial cells, would be ideal for sampling the mucosa; shed tumor cells maydisplay alterations in gene expression observed inintact tumors. It is first necessary, however, to showthat RNA can be isolated from human feces and that this RNA contains human gene transcripts. We havetherefore developed a method for the isolation of totalRNA from freshly passed human stool, consisting of lysisin chaotropic agents, repeated extraction with phenol and phenolchloroform, and absorptionwith an RNA-binding resin. After treatment withRNase-free DNase I, we assayed these preparations forthe presence of human RNA by quantitative slot blotting, northern blotting, and reversetranscription-polymerase chain reaction (RT-PCR). Weobtained 5-30 μg RNA per gram of stool from cancerpatients, and about 5 μg RNA per gram of controlstool. Quantitative slot blotting showed that about 10% of this RNAwas of human origin. Both northern blotting and RT-PCRdemonstrated the presence of human RNA in these samples.To unambiguously demonstrate the isolation of RNA from stool, we incubated a mixture ofrat cells and control human stool at 37°C for up to24 hr. RT-PCR of the RNA isolated from this sampleclearly revealed the presence of rat-specific mRNA.These experiments indicate that RNA can be isolatedfrom human stool and that message encoded by human genescan be assayed in these preparations. This procedure mayprovide a powerful tool to identify patients at risk for colon cancer.

Kinetic and morphologic alterations in the colon of a patient with multiple polyposis.

The histologic and proliferative characteristics of 16 colonic biopsies taken from an operative specimen of a 38‐year‐old female patient with multiple polyposis are presented. Kinetic measurements are based on 3HTdR incorporation accomplished in vitro, using both single and double labelling techniques. Epithelial cells in adenomatous tissue were more actively engaged in DNA synthesis than those normal‐appearing mucosa (L.I. 13.0 ± 6.9 versus 9.3 ± 1.6); however, because of extreme variability between biopsies, the difference was not significant. No difference in S phase duration was found, but a faster turnover time (Tg) than that in the normal appearing colonic mucosa was estimated (Tg 52.6 hours versus 74.2 hours).

Sterol metabolism studies in the rat. Effects of dietary plant sterols and bile acids on sterol metabolism.

Abstract Sterol metabolism studies using a combination of thin-layer chromatography, gas-liquid chromatography and isotopic methods were carried out in rats fed diets containing: (a) control chow (group 1); (b) 0.8% β-sitosterol (group 2); (c) 0.8% β-sitosterol plus 0.5% sodium taurochenodeoxycholate (group 3); and (d) 0.8% β-sitosterol plus 0.5% sodium taurocholate (group 4). Feeding β-sitosterol led to an inhibition of cholesterol absorption, increased cholesterol output (cholesterol balance, 28.8 mg/day vs. 19.5 mg/day in controls) and no significant increase in bile acid synthesis. Rats receiving sodium taurochenodeoxycholate or sodium taurocholate in addition to β-sitosterol showed: (a) decreased levels of bile acid synthesis; (b) low cholesterol absorption; and (c) increased output of fecal and endogenous neutral sterols. Levels of cholesterol in plasma were not significantly different from those in the control animals. Liver cholesterol levels were significantly lower in the taurochenodeoxycholate plus β-sitosterol (group 3) vs. the taurocholate plus β-sitosterol animals (group 4). However, biliary cholesterol concentrations were elevated in both bile acid-plant sterol groups (0.38 mg/ml in group 3; 0.44 mg/ml in group 4 vs. 0.19 mg/ml in group 1). There were no significant differences in sterol metabolism between groups of rats fed sodium taurochenodeoxycholate plus β-sitosterol compared to rats fed sodium taurocholate plus β-sitosterol.

Ursodeoxycholic acid effects on sterol metabolism in rats

Sterol balance studies using isotopic and chromatographic techniques were performed in rats fed diets supplemented with ursodeoxycholic acid. Compared to controls, ursodeoxycholic acid dramatically altered sterol metabolism. Ursodeoxycholic acid was absorbed and circulated in the enterohepatic circulation. The biliary bile acid composition was significantly altered with ursodeoxycholic acid the predominant biliary bile acid (67%). Cholesterol absorption was depressed by 34%; bile acid synthesis was depressed by 30%; however, cholesterol balance was significantly increased. It is apparent that the effects of ursodeoxycholic acid on sterol metabolism are different in several respects from chenodeoxycholic acid.

Effects of dietary administration of chenodeoxycholic acid on N-methyl-N-nitrosourea-induced colon cancer in rats

Abstract This experiment examines the effects of chenodeoxycholic acid feeding on tumor incidence in rats treated with N -methyl- N -nitrosourea. Group 1 rats received control chow, group 2 control chow plus chenodeoxycholic acid (0.2%), group 3 control chow and N -methyl- N -nitrosourea, and group 4 control chow plus chenodeoxycholic acid (0.2%) and N -methyl- N -nitrosourea. After 28 weeks pathological and histological examination revealed no tumors in group 1 and group 2 rats. The N -methyl- N -nitrosourea group (group 3) had a tumor incidence of 49% (1.1 tumors/all animals) compared to the N -methyl- N -nitrosourea and chenodeoxycholic acid group (group 4) in which 62% of the animals had tumors (1.1 tumors/all animals). The number of tumors per tumor-bearing animal was slightly lower in group 4. The tumors were primarily adenomas, but 3 carcinomas in situ and 9 invasive carcinomas were also detected. Fecal analyses after 28 weeks showed a slight increase in neutral sterols in the N -methyl- N -nitrosourea group compared to controls (2.49 vs. 1.52 mg/g, respectively). In addition, fecal neutral sterols were slightly elevated in the chenodeoxycholic acid fed rats (groups 2 and 4). Conversion of cholesterol to coprostanol was similar in all groups. Total fecal bile acids were higher in chenodeoxycholic acid fed rats compared to control chow fed rats (groups 2 and 4 vs. groups 1 and 3). Major fecal bile acids in the chenodeoxycholic fed rats were chenodeoxycholic acid, lithocholic acid, α-muricholic acid, β-muricholic acid and ω-muricholic acid. The ability of rats to 6-hydroxylate and 7-hydroxylate bile acids may protect them from the increased tumor incidence observed in other carcinogen-bile acid experiments.