T H E E F F E C T S O F P U R E A N D MICELLAR SOLUTIONS O F D I F F E R E N T BILE SALTS ON MUCOSAL MORPHOLOGY I N R A T J E J U N U M I N VIVO
JEAN W. KEELING*, S. P. LAMABADUSURIYA~ AND J. T. HARRIES Institute of Child Health, Guirford Street, London WClN IEH
I N patients with bacterial overgrowth of the small intestine the unconjugated dihydroxy bile salt deoxycholate appears in easily detectable amounts following intraluminal deconjugation and 7a dehydroxylation of trihydroxy bile salts (TabaqchaliandBooth, 1966 ; Rosenberg, Hardison andBull, 1967 ; Tabaqchali, Hatzioannou and Booth, 1968; Ament et al., 1972; Mallory, Kern and Smith, 1973). In such patients the mechanism of malabsorption is controversial (Dietschy, 1967; Donaldson, 1967). Donaldson (1965), Kim et al. (1966), Tabaqchali et al. (1968) and Shiner (1969) were unable to demonstrate dehitive morphological abnormalities in small intestinal mucosa obtained from patients with blind loop syndromes, but more recently Ament et al. (1972) using amultiple biopsy technique have shown unequivocal patchy abnormalities in the jejunal mucosa of such patients. These workers found micellar solubilisation of free fatty acids to be unimpaired in their patients and suggested that mucosal abnormalities induced by deoxycholate may play a role in the pathophysiology of blind loop steatorrhoea. The in-vivo effects of deoxycholate on small intestinal mucosal morphology have been reported in a variety of animal species (Shiner, 1969; Low-Beer, Schneider and Dobbins, 1970; Harries and Sladen, 1972; Sladen and Harries, 1972; Teem and Phillips, 1972; Gracey et al., 1973), but none of these studies has compared the effects of pure solutions of deoxycholate with solutions containing mixtures of deoxycholate, conjugated bile salts and lipid. Since the intraluminal environment in normal or disease states contains mixtures of bile salts, lipids and other solutes, interactions between substances within the intestinal lumen may modify the biological effects of any one given species. In this study we have compared the effects of deoxycholate on jejunal mucosal morphology with a variety of other bile salts, and with a structurally related steroid antibiotic, fusidic acid. In addition, the influence of pure and mixed micellar solutions of taurocholate on deoxycholate-inducedabnormalities of the mucosa has been investigated. Received 17 Mar. 1975; Accepted 3 July 1975. Present addresses: * Department of Pathology, The John Radcliffe Hospital, Headington, Oxford. t 47,42nd Lane, Colombo 6, Sri Lanka. 1. PA%-VOL.
118 (1976)
157
158
JEAN W. KEELING, S. P. LAMABADUSURIYA AND J. T. HARRIES
METHODS All experiments were performed on male Wistar rats weighing 250-300 g following a 12-18-hr fast when tap water was allowed ab libitum, using a closed loop technique which has previously been described and validated (Harries and Sladen, 1972; Sladen and Harries, 1972). Under ether anaesthesia a 20-30-cm proximal jejunal loop was prepared and 2-3 ml of the solution under study was instilled into the loop and the abdominal cavity closed. Body temperature was maintained at 37°C and following a 25-min period the animal was killed and the jejunal loop removed. All solutions were isotonic (285 mOsm/kg), phosphate buffered (2.88 mM NaH2P04; 12.12 m NaZHP04) to pH 7.4 and, contained 4 mM KC1 and 20 m glucose. Following addition of bile salts residual tonicity was made up with NaCl, the final concentration of Na+ and C1- varying from 135 to 162 m and 111-138 r m respectively. The sodium salts of deoxycholic, cholic, taurodeoxycholic (Sigma London Chemical Co. Ltd, Surrey), taurocholic (Koch-Light Laboratories Ltd, Bucks.), chenodeoxycholic, glycochenodeoxycholic and glycohyodeoxycholic acids (Maybridge Research Chemicals, Launceston, Cornwall) were checked for their purity by thin-layer chromatography. Sodium fusidate (Leo Pharmaceutical Products, Ballerup, Denmark) and oleic acid (Sigma London Chemical Co. Ltd) were 99-100 per cent. pure. The mixed micellar solutions were prepared on the same day as the experiments and kept at 37°C until used. The determined amount of oleic acid in hexane was aliquoted into a testtube and the hexane evaporated to dryness under nitrogen, and then solubilised with ether; this solution was allowed to evaporate under nitrogen leaving a thin film of lipid on the testtube wall. The taurocholate containing test solution (20 rm taurocholate; 2.5 rm deoxycholate; 4 mM KC1; 20 m glucose; 100 mM NaCl; phosphate buffered to p H 7.4) was then added to the test-tube and agitated for a few minutes; this process produced a crystal clear solution with an optical density of zero compared with the phosphate buffer mixture; NaCl was then added in amounts to give the final solution an osmolality of 285 mOsm/kg, and the pH checked. Immediately after killing the animal the jejunal segment was removed with minimum handling, placed on a piece of cork, and opened longitudinally along the antimesenteric border with round-ended scissors. The opened intestine was then pinned out straight without stretching, with the mucosal surface upwards, and the cork floated in a large volume of buffered formalin with the intestine on the underside, and left for 2 days to ensure adequate fixation. After hation, the intestine was removed from the cork and blocks were taken from the whole of the specimen cutting against a straight edge to ensure that sectioning was at right-angles to the mucosal surface. The surface to be sectioned was notched so that each section could be correctly orientated in relation to adjacent sections. The blocks were embedded in paraffin, sectioned and stained with haematoxylin and eosin and periodic acidSchiff with an haematoxylin nuclear stain. Sections were examined using a Zeiss photoscope Mk 1, and the surface to volume ratio of the mucosa determined by the method of Dunnill and Whitehead (1971) after Risdon and Keeling (1974). The surface to volume ratio was determined at several sites of each jejunal segment examined. The significanceof differences between mean values was assessed by the Student t test, and all values are expressed as meanf 1 SE throughout the paper.
RESULTS Following exposure to control solution, the jejunal mucosa was composed of narrow villi of approximately equal length covered by regularly arranged columnar epithelial cells with nuclei sited at the base of the cells. The lamina propria contained mixtures of lymphocytes and plasma cells, and there was 110 luminal debris (fig. 1).
159
BILE SALTS AND THE JHUNUM
Except for slight luminal debris, mucosa exposed to 1 m deoxycholate was no different to controls (fig, 2). In contrast, 2.5 mM deoxycholate produced several histological abnormalities (fig. 3) ; villi were shorter and broader, there was desquamation of villous tip cells, the lamina propria showed an increase in cellular infiltration, polymorphonuclear leucocytes were present beneath the epithelium of many villous tips, and luminal debris was increased. Similar but more marked abnormalities were produced by 5 m~ deoxycholate (fig. 4). Chenodeoxycholate,another unconjugated dihydroxy bile salt, at a concentration of 5 MM produced similar changes to an equimolar concentration of deoxycholate. Sodium fusidate at a concentration of 1 mM produced no histological TABLE Efects of pure and mixed micellar solutions of different bile salts on surface to volume (cllh) ratios in closed loops Surface to volume ratios (mean values& 1 SE)
:Instilledsolutions I
Controls (15) 1 r n deoxycholate ~ (10) 2.5 rn deoxycholate (10) 5 m~ deoxycholate (5) 20 m~ taurocholate (3) 2.5 m~ deoxycholate 20 m~ taurocholate (5) 2.5 m~ deoxycholate 20 m~ taurocholate 10 m~ oleic acid (8) 1 m~ fusidate (2) 5 m~ fusidate (3)
I
Difference from control
1
...
98.45f. 1.07 85.28f 1.98 62.05h1.56 58.9 &2-16 105.93f6.54 60.86f2.11
t0.001
93.68f3.78
NS
92.45&0.75 56.6760.92
NS
JEAN W. KEELING*, S. P. LAMABADUSURIYA~ AND J. T. HARRIES Institute of Child Health, Guirford Street, London WClN IEH
I N patients with bacterial overgrowth of the small intestine the unconjugated dihydroxy bile salt deoxycholate appears in easily detectable amounts following intraluminal deconjugation and 7a dehydroxylation of trihydroxy bile salts (TabaqchaliandBooth, 1966 ; Rosenberg, Hardison andBull, 1967 ; Tabaqchali, Hatzioannou and Booth, 1968; Ament et al., 1972; Mallory, Kern and Smith, 1973). In such patients the mechanism of malabsorption is controversial (Dietschy, 1967; Donaldson, 1967). Donaldson (1965), Kim et al. (1966), Tabaqchali et al. (1968) and Shiner (1969) were unable to demonstrate dehitive morphological abnormalities in small intestinal mucosa obtained from patients with blind loop syndromes, but more recently Ament et al. (1972) using amultiple biopsy technique have shown unequivocal patchy abnormalities in the jejunal mucosa of such patients. These workers found micellar solubilisation of free fatty acids to be unimpaired in their patients and suggested that mucosal abnormalities induced by deoxycholate may play a role in the pathophysiology of blind loop steatorrhoea. The in-vivo effects of deoxycholate on small intestinal mucosal morphology have been reported in a variety of animal species (Shiner, 1969; Low-Beer, Schneider and Dobbins, 1970; Harries and Sladen, 1972; Sladen and Harries, 1972; Teem and Phillips, 1972; Gracey et al., 1973), but none of these studies has compared the effects of pure solutions of deoxycholate with solutions containing mixtures of deoxycholate, conjugated bile salts and lipid. Since the intraluminal environment in normal or disease states contains mixtures of bile salts, lipids and other solutes, interactions between substances within the intestinal lumen may modify the biological effects of any one given species. In this study we have compared the effects of deoxycholate on jejunal mucosal morphology with a variety of other bile salts, and with a structurally related steroid antibiotic, fusidic acid. In addition, the influence of pure and mixed micellar solutions of taurocholate on deoxycholate-inducedabnormalities of the mucosa has been investigated. Received 17 Mar. 1975; Accepted 3 July 1975. Present addresses: * Department of Pathology, The John Radcliffe Hospital, Headington, Oxford. t 47,42nd Lane, Colombo 6, Sri Lanka. 1. PA%-VOL.
118 (1976)
157
158
JEAN W. KEELING, S. P. LAMABADUSURIYA AND J. T. HARRIES
METHODS All experiments were performed on male Wistar rats weighing 250-300 g following a 12-18-hr fast when tap water was allowed ab libitum, using a closed loop technique which has previously been described and validated (Harries and Sladen, 1972; Sladen and Harries, 1972). Under ether anaesthesia a 20-30-cm proximal jejunal loop was prepared and 2-3 ml of the solution under study was instilled into the loop and the abdominal cavity closed. Body temperature was maintained at 37°C and following a 25-min period the animal was killed and the jejunal loop removed. All solutions were isotonic (285 mOsm/kg), phosphate buffered (2.88 mM NaH2P04; 12.12 m NaZHP04) to pH 7.4 and, contained 4 mM KC1 and 20 m glucose. Following addition of bile salts residual tonicity was made up with NaCl, the final concentration of Na+ and C1- varying from 135 to 162 m and 111-138 r m respectively. The sodium salts of deoxycholic, cholic, taurodeoxycholic (Sigma London Chemical Co. Ltd, Surrey), taurocholic (Koch-Light Laboratories Ltd, Bucks.), chenodeoxycholic, glycochenodeoxycholic and glycohyodeoxycholic acids (Maybridge Research Chemicals, Launceston, Cornwall) were checked for their purity by thin-layer chromatography. Sodium fusidate (Leo Pharmaceutical Products, Ballerup, Denmark) and oleic acid (Sigma London Chemical Co. Ltd) were 99-100 per cent. pure. The mixed micellar solutions were prepared on the same day as the experiments and kept at 37°C until used. The determined amount of oleic acid in hexane was aliquoted into a testtube and the hexane evaporated to dryness under nitrogen, and then solubilised with ether; this solution was allowed to evaporate under nitrogen leaving a thin film of lipid on the testtube wall. The taurocholate containing test solution (20 rm taurocholate; 2.5 rm deoxycholate; 4 mM KC1; 20 m glucose; 100 mM NaCl; phosphate buffered to p H 7.4) was then added to the test-tube and agitated for a few minutes; this process produced a crystal clear solution with an optical density of zero compared with the phosphate buffer mixture; NaCl was then added in amounts to give the final solution an osmolality of 285 mOsm/kg, and the pH checked. Immediately after killing the animal the jejunal segment was removed with minimum handling, placed on a piece of cork, and opened longitudinally along the antimesenteric border with round-ended scissors. The opened intestine was then pinned out straight without stretching, with the mucosal surface upwards, and the cork floated in a large volume of buffered formalin with the intestine on the underside, and left for 2 days to ensure adequate fixation. After hation, the intestine was removed from the cork and blocks were taken from the whole of the specimen cutting against a straight edge to ensure that sectioning was at right-angles to the mucosal surface. The surface to be sectioned was notched so that each section could be correctly orientated in relation to adjacent sections. The blocks were embedded in paraffin, sectioned and stained with haematoxylin and eosin and periodic acidSchiff with an haematoxylin nuclear stain. Sections were examined using a Zeiss photoscope Mk 1, and the surface to volume ratio of the mucosa determined by the method of Dunnill and Whitehead (1971) after Risdon and Keeling (1974). The surface to volume ratio was determined at several sites of each jejunal segment examined. The significanceof differences between mean values was assessed by the Student t test, and all values are expressed as meanf 1 SE throughout the paper.
RESULTS Following exposure to control solution, the jejunal mucosa was composed of narrow villi of approximately equal length covered by regularly arranged columnar epithelial cells with nuclei sited at the base of the cells. The lamina propria contained mixtures of lymphocytes and plasma cells, and there was 110 luminal debris (fig. 1).
159
BILE SALTS AND THE JHUNUM
Except for slight luminal debris, mucosa exposed to 1 m deoxycholate was no different to controls (fig, 2). In contrast, 2.5 mM deoxycholate produced several histological abnormalities (fig. 3) ; villi were shorter and broader, there was desquamation of villous tip cells, the lamina propria showed an increase in cellular infiltration, polymorphonuclear leucocytes were present beneath the epithelium of many villous tips, and luminal debris was increased. Similar but more marked abnormalities were produced by 5 m~ deoxycholate (fig. 4). Chenodeoxycholate,another unconjugated dihydroxy bile salt, at a concentration of 5 MM produced similar changes to an equimolar concentration of deoxycholate. Sodium fusidate at a concentration of 1 mM produced no histological TABLE Efects of pure and mixed micellar solutions of different bile salts on surface to volume (cllh) ratios in closed loops Surface to volume ratios (mean values& 1 SE)
:Instilledsolutions I
Controls (15) 1 r n deoxycholate ~ (10) 2.5 rn deoxycholate (10) 5 m~ deoxycholate (5) 20 m~ taurocholate (3) 2.5 m~ deoxycholate 20 m~ taurocholate (5) 2.5 m~ deoxycholate 20 m~ taurocholate 10 m~ oleic acid (8) 1 m~ fusidate (2) 5 m~ fusidate (3)
I
Difference from control
1
...
98.45f. 1.07 85.28f 1.98 62.05h1.56 58.9 &2-16 105.93f6.54 60.86f2.11
t0.001
93.68f3.78
NS
92.45&0.75 56.6760.92
NS
