Comparison of Different Lipid Substrates on Intestinal Adaptation in The

Gut 1993; 34:1069-1074 1069

Comparison ofdifferent lipid substrates on intestinal adaptation in the rat Gut: first published as 10.1136/gut.34.8.1069 on 1 August 1993. Downloaded from

M Galluser, B Czernichow, H Dreyfus, F Gosse, B Guerold, J Kachelhoffer, M Doffoel, F Raul

Abstract phology, epithelial cell renewal, brush border The relative effects of medium chain (MCT) membrane lipid composition, and hydrolase and long chain triglycerides (LCT) on activities in the jejunum and ileum of adult rats. intestinal morphology and functions were As a frame of reference, we compared the results compared. Adult rats received intragastrically of the enterally nourished rats with those from for 10 days an isoenergetic mixture containing sham operated animals fed orally. either 50% MCT/50% LCT or 100% LCT. The other constituents of the diets were identical, and animals fed a standard diet orally were Methods used as a reference group. Animals who were given the MCT/LCT diet showed a higher ANIMALS AND DIETS mucosal mass and protein content and Adult Wistar rats (n= 30) with a mean body increased villus length and crypt depth in the weight of340 g were randomly divided into three proximal part of the small intestine compared groups. The reference group (n= 10) was sham with the LCT and control diet groups. operated and fed orally with the standard Administration of [3H] thymidine 12 hours pelleted diet containing (in terms of energy) 24% before death resulted in a significant increase protein, 67% carbohydrate, and 8% corn oil in the incorporation of the precursor into (UAR, Villemoisson sur Orge, France). cellular DNA in the jejunum of rats given The other two groups were fed intragastrically MCT. In rats given LCT as the only fat, the via gastrostomy tubes. They received either a free fatty acid content of the microvillus mem- mixture of 50% MCT/50% LCT (39 kcal/day; brane showed a 20 fold increase and at the soya oil 10% containing 31% of essential fatty

same time there was a significant drop in the acids, MCT 10% and lecithin 1 2% containing http://gut.bmj.com/ cholesterol content and in the cholesterol/ less than 1-5% cholesterol; Medialipide 20%, protein ratio. Differences in the lipid com- Bruneau, Boulogne-Billancourt, France, n= 10) position of enterol diet or in the microvillus or 100% LCT (39 kcal/day; soya oil 20% contain- membrane did not effect adversely membrane ing 62% of essential fatty acids, lecithin 1-2% bound hydrolase activities. These findings containing less than 1-5% cholesterol; Endo- suggest that MCT in the diet confers lipide 20%, Bruneau, Boulogne-Billancourt,

advantages in addition to the provision of France, n= 10). The amino acid and carbo- on September 28, 2021 by guest. Protected copyright. rapidly available energy. hydrate contents of both mixtures were (Gut 1993; 34: 1069-1074) identical. The L-amino acid mixture (18 kcal/ day) contained (per 1); isoleucine 7 5g, leucine 13-0 g, lysine 10-3 g, methionine 4-7 g, phenyl- Medium chain triglycerides (MCT) are known to alanine 10-6 g, threonine 7 5 g, tryptophan 1 9 g, have several physiological advantages over long valine 9-4 g, arginine 32-4 g, histidine 8 5 g, chain triglycerides (LCT). ' MCT are more alanine 11-3 g, aspartic acid 17-6 g, cysteine rapidly hydrolysed by pancreatic lipase than 1-9 g, glutamic acid 17-6 g, glycine 14-9 g, LCT. The medium chain fatty acids (MCFA) proline 6 2 g, serine 3 0 g, tyrosine 0 6 g. The that originate from MCT hydrolysis are less carbohydrate was glucose 50% (30 kcal/day). Department of Celi Biology and Digestive hydrophobic than their long chain counterparts. The enteral mixtures contained the same Physiopathology, The MCFA are transported principally by the amounts of electrolytes and were given con- INSERM U61, 3 avenue portal vein to the liver for oxidation rather than tinuously over 24 hours at a flow rate of Moliere, 67200 which 3 All animals had free access to drink- Strasbourg, France through the intestinal lymphatic vessels ml/hour. M Galluser, are used by LCT.2 In addition, MCFA are ing water. All diets were isoenergetic (87 kcal/ B Czernichow, independent of carnitine for entry into the day) and were given for 10 days. F Gosse of all tissues. MCT a J Kachelhoffer, mitochondria represent M Doffoel, rapidly available and a high energy fuel that is F Raul less likely to be desposited and more likely to be TISSUE COLLECTION oxidized in the tissues.' These properties have The entire jejunoileum from the ligament of Centre for Neurochemistry of the led us to consider MCT as an appropriate energy Treitz to the ileocaecal junction was excised Central Nervous System, substrate in the nutritional support of patients and flushed with cold saline. The proximal Cronenburg, Strasbourg with malabsorption or decreased pancreatic and part of the jejunum, corresponding to the first H Dreyfus, the B Guerold bile acid secretions. However, little is known 10 cm from the ligament of Treitz, and about the effect of MCT on intestinal mor- proximal part of the ileum, corresponding to Correspondence to: Dr F Raul, U61 INSERM, 3 phology and function.4 The aim of our study was the first 10 cm from the middle of the Avenue Mofiere, 67200 to compare the relative effects on intestinal jejunoileum were resected. The length of Strasbourg, France. function of MCT and LCT given by enteral the segments was measured by stretching the Accepted for publication 22 December 1992 route. We studied mucosal mass and mor- intestine with a 3 g weight. 1070 Galluser, Czernichow, Dreyfus, Gosse, Guirold, Kachelhoffer, Doffoel, Raul

HISTOLOGICAL TECHNIQUES and charring with 10% CuSO4 in 8% ortho- The first 2 cm of the jejunal and ileal segments phosphoric acid by densitometry on a Camag

were removed for morphometrical studies. The densitometer.9 Gut: first published as 10.1136/gut.34.8.1069 on 1 August 1993. Downloaded from samples were fixed by Bouin fixative then dehydrated and embedded in paraffin. Sections (5 rim) were stained with haematoxylin and ENZYME ASSAYS eosin. The villus length and crypt depth were Sucrase activity was determined according to the measured with a semiautomatic image analyser method of Dahlqvist.'0 Lactase activity was (MOP/AM 01 System, Munich, Germany). Only assayed in the presence of p-chloromercuriben- villi sectioned from their base to the top with a zoate." Aminopeptidase was measured using single epithelial layer at their tip and crypts with L-alanine-p-nitroanilide as substrate.'2 a visible lumen along their entire depth were Enzyme activities were expressed either as considered suitable. specific activities (mU per mg of protein) or as segmental activities (mU per cm of intestinal length). One unit of enzyme activity equals one LABELLED PRECURSOR ADMINISTRATION ,tmol ofproduct formed per minute at 37°C. Four animals in each group fed by the enteral route were injected with [3H]-thymidine (80 Ci/ mmol, Amersham, France) as an indicator of OTHER BIOCHEMICAL ANALYSIS crypt cell renewal in the jejunum and ileum. For Protein content was determined in homogenates this purpose 0-3 [tCi/g body weight of [3H]- and brush border membranes according to the thymnidine was administered intraperitoneally 16 method of Lowry et al.'3 DNA was measured hours before the end of the feeding period. in homogenates with the bisbenzimidazole Incorporation of [3H]-thymidine into DNA was method'4 using calf thymus DNA as a standard. determined in mucosal homogenates. Radio- activity was measured after extraction with ice cold trichloroacetic acid followed by washing STATISTICS with ethanol. Numerical data are reported as mean (SEM) values. Statistical differences between dietary groups for a given intestinal segment were MEMBRANE PREPARATION AND LIPID EXTRACTION evaluated by analysis of variance (ANOVA); The mucosa of each segment was scraped off, specific differences were identified using weighed, and homogenised in 50 mM mannitol Duncan's multiple range test. and 2 mM Tris-HCl (pH 7-1). Brush border http://gut.bmj.com/ membranes were isolated from mucosal homogenates as described by Schmitz et al.S Lipids Results were extracted according to the method of Folch et a16 modified by Suzuki.7 Lipids were then MUCOSAL MASS AND PROTEIN CONTENT purified through a Sephadex G-25 superfine The body weight gain of the rats after 10 days of column.8 Elutions were performed with chloro- enteral feeding was similar to that of animals fed

form:methanol:water (60:35:4 5, v/v/v) and the isoenergetic pelleted diet and reached 10% of on September 28, 2021 by guest. Protected copyright. chloroform:methanol (2:1, v/v). The dried lipids the initial body weight. Variations in intestinal were resuspended in chloroform and passed mucosal mass and in mucosal brush border through a Kieselgel 60 (Merck, France) column, membrane protein content are shown in Table I. and neutral lipids were eluted with chloroform. The mucosal weight and protein content were Thin layer chromatography of the neutral lipids significantly higher in the jejunum of rats given was performed in chloroform:acetone (96:4, MCT/LCT than in the controls or the animals v/v). Cholesterol was detected by primulin spray given LCT as the only fat source. In this and extracted with diethylether. The spots segment, the brush border protein content was corresponding to other neutral lipids were also significantly increased in rats given the enteral scraped from the plate and chromatographed diet compared with the orally fed animals. In the again on high performance thin layer plates ileum, no diet related changes or group differ- (Merck 5641) in hexane:diethylether:acetic acid ences were observed with regard to the mucosal (70:30:1, v/v/v). They were then quantified by mass or mucosal and brush border protein comparison with known amounts of standards content. The brush border membrane purification rate corresponding to the amount of membrane protein obtained from mucosal homogenates was similar in the various groups TABLE I Effect ofdiet composition on intestinal weight andprotein content. (Values are mean (SEM)) and was not related to changes in the lipid composition of the diet. Protein content (mg) Dietgroup Mucosal weight (g) Mucosa Brush border (no of MUCOSAL MORPHOMETRY animals) Jtejunum Ileum Jrejunum Ileum 3rejunum Ileum The effects of the various diets on jejunal and Orallyfed(6) 0-51(0-02)* 0-51(0-03)* 58(2)* 58(2)* 1-9(0-05)* 1-7(0-1)* ileal morphology are shown in Figure 1. Animals MCT/LCT (6) 0 64 (0-02)t 0 47 (0 03)* 82 (2)t 59 (3)* 2-6 (0.2)t 1-7 (0-1)* fed enterally showed a significant increase in the LCT(6) 052(002)* 044(002)* 65(2)t 54(3)* 2-3(0-I)t 1-6(0-1)* height ofthe jejunal and ileal villi compared with Orally fed=rats fed the standard diet. MCT/LCT=rats fed enterally with a mixture of 50% medium orally fed control rats. The fat composition ofthe chain/50% long chain triglycerides as the fat source. LCT=rats fed enterally with 100% long chain triglyceride as the fat source. All diets were isoenergetic and were given for 10 days. For a given enteral diet did not change the height of the villi intestinal segment */t/1, p<0-05. in either intestinal segment. The crypts were Different lipidsubstrates in rat intestine 1071

Jejunum Ileum compared with rats given LCT as the only source Villus height of v 110 7001 b b lipid (205 (15) (12) [3H] dpm/4g DNA, p

100 LIPID COMPOSITION OF BRUSH BORDER MEMBRANE In the jejunal brush border membranes the 0- a composition ofthe neutral lipids was very similar in the rats fed a standard diet enterally and those -300 given the mixture containing MCT enterally (Fig 3). In both groups, cholesterol represented 45% -200 E a' a' a' and fatty acids 3 to 5% of the total neutral lipid at cr content of the brush border membrane. The -10 Jb' Crypt depth pattern of the neutral lipid composition was changed in the brush border membrane of rats Figure 1: Effect ofdietaryfat on villus height and crypt depth in thejejunum and ileum. Ratsfed orally with the standard fed enterally with LCT as the only source of fat. diet ( O), animalsfed enterally with 50% medium chain The free fatty acid content of the microvillus triglycerides/50% long chain triglycerides (U) or with 100% membrane showed a 20 fold increase in this long chain triglycerides (0) asfat source. Results are expressed as mean (SEM) ofat least 30 villi. The number of group compared with the other groups, and an animals used isgiven in Table 1. For thejejunum or the ileum, important drop in the cholesterol content was columns that do not share a common superscript letter differ observed at the same time. significantly: p<005. The effects ofthe LCT diet on the neutral lipid composition of the ileal brush border membrane were similar to those reported previously in the significantly deeper in the jejunum of rats fed jejunum (Fig 3). The MCT diet also led to a five

enterally than in controls. It is worthy of note fold increase in the fatty acids and a decrease in http://gut.bmj.com/ that the diet containing MCT led to a greater the cholesterol content of the membrane but increase in the crypt depth in the jejunum only. these changes were less important than those The crypt depth in the ileum was not changed by observed in the LCT group. As shown in Table the fat composition ofthe various diets. II, the membrane cholesterol/protein ratios of the jejunal and ileal microvilli were lower in the rats of the LCT group. However, there were no

EFFECTS OF MCT AND LCT ON INTESTINAL CELL significant changes in the cholesterol/protein on September 28, 2021 by guest. Protected copyright. RENEWAL ratios in rats fed MCT enterally and those fed [3H]-thymidine (0-3 [iCi/g body weight) was orally. administered intraperitoneally 16 hours before killing the rats fed enterally. As illustrated in Figure 2, the animals given MCT showed a EFFECT OF DIETARY FAT ON INTESTINAL significant increase in the uptake of [3H]- HYDROLASE ACTIVITIES thymidine into DNA of the jejunal mucosa The effects of the various diets on jejunal and ileal hydrolase activities are shown in Figures 4 and 5. Elemental diets (amino acids and glucose) given by the enteral route and which contained Jejunum Ileum MCT and/or LCT did not significantly change the total activity ofsucrase in the jejunal segment (Fig 4). In the cell, however, the specific activity Z 200 a ofsucrase was significantly lower (1-5 fold) in the CD jejunal brush border membrane of animals fed -- 150 enterally than in rats fed a standard diet orally -o (Fig 5). In the ileum, enteral feeding caused a

E TABLE II Effect ofdiet composition on the cholesterollprotein .C 50 ratio injejunal and ileal brush border membrane ofrats It Diet group Jejunum Ileum (no ofrats) (jig cholesterol/mg protein) Figure 2: Effect ofthefat composition ofthe an enterallyfed Orally fed (6) 61-2 (5 1)* 625 (5.9)* diet on['HJ thymidine incorporation into cellular DNA in the MCT/LCT (6) 59 3 (5 0)* 58 2 (5-2)* jejunal and ileal mucosa. Results are expressed as mean LCT (6) 51 9 (4-8)* 45 1 (3 7)t (SEM) offour rats per group. Animals werefed enterally with 50% medium chain triglycerides/50% long chain triglycerides Cholesterol was extracted and analysed as described in methods. (V21) or with 100% long chain triglycerides (01) as thefat For details of groups see Table I. For a given intestinal segment source. For thejejunum: *, p<001. *t, p<0o05. 1072 Galluser, Czernichow, Dreyfus, Gosse, Guerold, Kachelhoffer, Doffoel, Raul

Jejunum Ileum dramatic drop (about three fold) in the total and specific sucrase activities when compared with the reference group. Gut: first published as 10.1136/gut.34.8.1069 on 1 August 1993. Downloaded from Total and specific lactase activities remained at their reference value in the jejunum and ileum when the diet given enterally contained MCT. The rats fed with the LCT diet showed a significant increase in the total activity in both intestinal segments (Fig 4). The specific activity of lactase in the brush border membranes was not changed, however, when compared with the other groups (Fig 5). The total and specific activities of aminopeptidase were significantly enhanced by enteral feeding, especially in the ileum (1-5 fold).

Discussion These results show that dietary fats affected intestinal mucosal mass and morphology, cell renewal in the crypts, the lipid composition of ,, G) 2 i ,,, 0 0) 2 2 . ) the microvillus membrane of the enterocytes, 4-~ t.4-V and membrane bound enzyme activities in the 0) 0 ~~~~~~~a5 ~~~~~3 0) 0) ~~~~~~~~)oi5 0 . >. 0 >-_> 0 L- > jejunum and ileum. The effects varied with the ClC) -~~~ L C)CC) - LL of fat in the diet. The that 0 0 type diet contained 0 ~~~~~~~~~050% MCT/50% LCT led to an increase in the mucosal mass and protein content compared Figure 3: Effect ofdietaryfat on the neutral lipid composition ofthe microvillus membrane in with the isoenergetic diet containing LCT as the thejejunum and ileum. Lipids were extracted and analysed as reported in methods. Ratsfed orally with the standard diet ([), animalsfed by enteral route with 50% medium chain triglycerides/50% long chain triglycerides (@) or with 100% long chain triglycerides (W) as the fat source. Jejunum Ileum 800 Sucrase a 700. ,-I- http://gut.bmj.com/ 600- a 0 500. Jejunum Ileum C) 400- 300 Sucrase 300. 250F a E 200- b b a

200 100 on September 28, 2021 by guest. Protected copyright. a 150 E Lactase 100 a ////. ..-...... b b 500 50 n...... a :::::: a a 01 SSSi-6zI I o/J4o _ 2 ...-.... g...la ;; 0. 3 Lactase C) 10 31 0 b b a E 2 2! E -a a *-ab . -- 21 11CO -- E Figure 4: Effect ofdietary Aminopeptidase fat on sucrase, lactase, and 900 b aminopeptidase total b activities (mU per cm o 800 intestinal length) in the c 700 a) jejunum and ileum. Results + 600 are expressed as mean b a C.) 500. (SEM) ofsix rats per group. Aminopeptidase a For a given intestinal 400- E 400 a segment, columns that do not 350- b b D 300 share in a common ...... 1 superscript letter differ 300- b -f 200 significantly, p<0 05. Rats b 100 250- a fed orally with the standard E I .,,,zz I.. ... , c;;;m, diet ( D), animalsfed by 200 C . ... enteral route with 50% b...... Figure 5: Effect ofdietaryfat on sucrase, lactase, and medium chain triglyceridesl E 150...... aminopeptidase specific activities (mU per mgprotein) in the 50% long chain ,...... triglycerides 100 *...... brush border membranes ofthejejunum and ileum. Results are (1) or with 100% long chain expressed as mean (SEM) ofsix rats per group. For a given triglycerides (Pr) as thefat 50- intestinal segment, columns that do not show a common I source. O a I I I superscript letter differ significantly, p<0 05. Different lipid substrates in rat intestine 1073

only fat source. The effects of the MCT/LCT phlorizin hydrolase." The bulk of the enzymatic mixture were prominent in the proximal part of molecule carrying the catalytic sites is located

the small intestine and caused an increase in outside the membrane, directly in contact with Gut: first published as 10.1136/gut.34.8.1069 on 1 August 1993. Downloaded from crypt cell renewal in this segment, as shown the intraluminal content. In this case it seems by the histomorphological studies and by the quite clear that the lipid environment of the determination of [3H]-thymidine incorporation anchor may have little influence on the catalytic into cellular DNA. In contrast, both enteral activity ofthe enzyme. mixtures, independently of their fat composi- The present report shows that the changes in tion, showed similar effects on the ileal mucosal the lipid composition of the diet given by the mass and morphology. The specific action of enteral route did not have any deleterious effect MCT on the proximal part of the small intestine on the activities of sucrase, lactase, and amino- might be directly related to the fact that MCT are peptidase. Sucrase activity is known to be more rapidly absorbed than LCT, and this may stimulated by intraluminal polysaccharides and explain the major trophic effect on this segment. sucrose.28 The drop observed for sucrase specific Indeed, the increased trophic response of the activity in animals fed enterally might therefore intestinal mucosa to MCT might be related be the consequence of the presence of glucose as to enhanced calcium and amino acid uptake the only carbohydrate source when compared and also to increased intracellular protein with controls which were given a diet rich in synthesis. 15-18 The positive effect of the MCT on polysaccharides. crypt cell renewal might be advantageous in the As shown previously,29 the maintenance of case of a hypotrophic villus, which is present in sucrase total activity and the higher sucrase many physiological states such as severe mal- specific activity in the jejunal compared with the nutrition, aging, etc. On the other hand, this ileal mucosa and microvillus membrane of rats effect could be deleterious in pathological states fed enterally seems to be related to the prefer- that are characterised by a villus hypotrophy and ential absorption by the jejunum of intraluminal crypt hypertrophy.'9 When rats were given the amino acids that are used by the enterocytes for MCT/LCT diet, the effects on the distal part of the synthesis ofmembrane proteins.'" the small intestine were very similar to those In contrast to LCT, MCT is rapidly absorbed described for the LCT diet, probably as a and hydrolysed by the intestinal mucosa in cases consequence of the lower MCT and higher LCT of bile salt and pancreatic lipase deficiency. The concentration in the lumen of the ileum. main interest in providing a diet containing a When elemental diets with a high lipid content mixture of MCT/LCT is to combine the physio- were given to rats, the diet with 50% MCT/50% logical advantages ofMCT with the maintenance

LCT determined the most appropriate lipid ofa sufficient supply ofessential fatty acid which http://gut.bmj.com/ content and composition in the brush border corresponded to halfthe amount provided by the membrane. The LCT diet, however, caused LCT diet. Indeed, changes in intestinal profound changes in the neutral lipid composi- morphology and membrane lipid composition tion and cholesterol content of the membranes. have been reported in cases where the level of The lower cholesterol content of the microvillus essential fatty acid in the diet is insufficient.303' membrane observed with the LCT diet might We have shown that an enteral elemental diet

affect membrane fluidity, which might then be containing MCT increases the intestinal mucosal on September 28, 2021 by guest. Protected copyright. higher than in other dietary conditions.20 mass, exerts a major trophic effect on the jejunal Changes in the lipid composition of the mem- mucosa, does not affect brush border lipid brane might have important consequences for composition, and does not affect adversely nutrient and electrolyte transport by the several hydrolase activities, especially those in enterocytes. These processes are known to be the proximal part of the small intestine. These dependent on the membrane lipid composition findings suggest that dietary MCT may have and fluidity, which could change the density or further advantages for the small intestine in activity, or both of specific carriers.2' It has addition to providing rapidly available energy. been reported that Na+ dependent D-glucose transport seems to be influenced by the lipid The authors are grateful to B Lafleuriel for the illustrations and composition and the fluidity of the I Gillot for typing the manuscript. 23 membrane.22 1 Bach AC, Babayan VK. Medium-chain triglycerides: an The excess of free fatty acids observed in the update. AmJ Clin Nutr 1982; 36: 950-62. 2 Greenberger NJ, Rodgers JB, Isselbacher KJ. Absorption of brush border membranes of rats given the LCT medium and long-chain triglycerides: factors influencing diet might originate from the increased intra- their hydrolysis and transport. Jf Clin Invest 1966; 45: 217-27. luminal concentration of free fatty acid resulting 3 Mascioli EA, Bistrian BR, Babayan VK, Blackburn GL. from pancreatic lipase activity. The high amount Medium-chain triglycerides and structured lipids as unique nonglucose energy sources in hyperalimentation. Lipids offree fatty acid in the microvillus might modify 1987; 22: 421-23. the levels of membrane peroxides, and changes 4 Takase S, Goda T. Effects of medium-chain triglycerides on brush border membrane-bound enzyme activity in rat small in lipid peroxides could affect regulation of cell intestine. J Nutr 1990; 120: 969-76. metabolism and function.24 The present data 5 Schmitz J, Preiser H, Maestracci D, Ghosh BK, Cerda JJ, Crane RK. Purification ofthe human intestinal brush border indicate that there is no direct relationship membrane. Biochim Biophys Acta 1973- 92: 305-11. between the lipid composition of the membrane 6 Folch J, Lees M, Sloane-Stanley GH. A simple method for the isolation and purification of total lipids from animal tissues. and the activities of disaccharidases and amino- J BiolChem 1957;* 226: 497-509. peptidase. These enzymes are anchored in the 7 Suzuki K. Pattern of mammalian brain gangliosides. J Neurochem 1965; 12: 604-11. membrane by a short hydrophobic segment 8 Dreyfus H, Louis JC, Harth 5, Mandel P. Gangliosides in which correspond to the N-terminal of the cultured neurons. Neuroscience, 1980; 5: 1647-55. 9 Baron BG, Coburn R. Comparison of two copper reagents for molecule for sucrase-isomaltase and amino- detections of saturated neutral lipids by charring densito- peptidase25 26 and to the C-terminal for lactase- metry.JZ Liquid Chromatog 1984; 7: 2793-801. 1074 Galluser, Czernichow, Dreyfus, Gosse, Guerold, Kachelhoffer, Doffoel, Raul

10 Dalhlqvist A. Method for assay of intestinal disaccharidases. 22 Brasitus TA, Schachter D, Mamouneas TG. Functional Anal Biochem 1964; 7: 18-25. interaction oflipids and proteins in rat intestinal microvillus 11 Koldovsky 0, Asp NG, Dahlqvist A. A method for the membranes. Biochemistry 18: 1979; 4136-44. Gut: first published as 10.1136/gut.34.8.1069 on 1 August 1993. Downloaded from separate assay of 'neutral' and 'acid' fI-galactosidase in 23 Brasitus TA, Schachter D. Cholesterol biosynthesis and homogenates of rat small intestinal mucosa. Anal Biochem modulation of membrane cholesterol and lipid dynamics in 1%9; 27: 409-18. rat intestinal microvillus membranes. Biochemistry 1982; 21: 12 Maroux S, Louvard D, Baratti J. The aminopeptidase from 2241-46. hogintestinal brush border. BiochimBiophysActa 1973; 321: 24 Turini M, Thomson ABR, Clandinin MT. Lipid composition 282-95. and peroxide levels on mucosal cells in rat large intestine in 13 Lowry OH, Rosebrough NJ, Farr AL, Randall RJ. Protein relation to dietary fat. Lipids 1991; 26: 431-40. measurement with the Folin phenol reagent. J Biol Chem 25 Semenza G. The insertion of stalked proteins of the brush 1951; 193:265-75. border membranes: the state of art in 1988. Biochem Int 14 Laberca C, Parigen K. A simple, rapid and sensitive DNA 1989; 18:15-33. assay procedure. Anal Biochem 1980; 102: 344-52. 26 Kenny AJ. Brush border peptidases. An overview. In: Lentze 15 Alpers DH. Protein synthesis in intestinal mucosa: the effect MJ, Sterchi EE, eds. Mammalian brush border membrane of route of administration of precursor amino-acids. J Clin proteins. Stuttgart, New York: Thieme, 1989: 130-38. Invest 1972; 51: 167-73. 27 ManteiN, VillaM, EnzlerT, Wacker H, Boll W, James Petal. 16 Agnew JE, Holdsworth C. The effect of fat on calcium Complete primary structure of human and rabbit lactase- absorption from a mixed meal in normal subjects, patients phlorizin hydrolase: implications for biosynthesis, mem- with malabsorptive disease, and patients with a partial brane anchoring and evolution of the enzyme. EMBO J gastrectomy. Gut 1971; 12: 973-7. 1988; 7:2705-13. 17 Tantibhedhyangkul P, Haskim SA. Medium-chain tri- 28 Koldolvsky 0. Developmental, dietary and hormonal control glyceride feeding in premature infants: effects on fat and of intestinal disaccharidases in mammals (including man). nitrogen absorption. Pediatrics 1975; 55: 359-69. In: Randle PJ, Whelan WJ, Steiner DF, eds. Carbohydrate 18 Tantibhedhyangkul P, Haskim SA. Medium-chain tri- metabolism and its disorders. London: Academic Press, 1981: glyceride feeding in premature infants: effects on calcium 482-522. and magensium 29 Czernichow B, Galluser M, Hasselmann M, Doffoel M, Raul absorption. Pediatric 1978; 61: 537-45. F. Effects of amino acids in mixtures given by enteral or 19 Bragg LE, Thompson JS, Rikkens LF. Influence of nutrient parenteral route on intestinal morphology and hydrolases in delivery on gut structure and function. Nutrition 1991; 7: rats. JPEN 1992; 16: 259-63. 237-43. 30 Snipes RL. The effects of essential fatty acid deficiency on 20 Proulx P. Structure-function relationships in intestinal brush the ultrastructure and functional capacity of the jejunal border membranes. Biochim Biophys Acta 1991; 1071: epithelium. Lab Invest 1%9; 18: 179-89. 255-71. 31 Christon R, Meslin JC, Thevenoux J, Linard A, Leger CL, 21 Clandinin MT, Cheema S, Field CJ, Garg ML, Venkatraman Delpal S. Effects of a low dietary linoleic acid level on J, Clandinin TR. Dietary fat: exogenous determination of intestinal morphology and enterocyte brush border mem- membrane structure and cell function. FASEB J 1991; 5: brane lipid composition. Reprod Nutr Dev 1991; 31: 2761-69. 691-701. http://gut.bmj.com/ on September 28, 2021 by guest. Protected copyright.