Effect of external abdominal irradiation on intestinal morphology and brush border membrane enzyme and lipid composition

Previous studies have shown that external abdominal irradiation is associated with alterations in intestinal morphology and function. The activity of the jejunal brush border membrane (BBM) enzyme markers sucrase (S) and alkaline phosphate (AP) were not altered by 600 rad irradiation in the rat. In contrast, ileal BBM, AP, and AP/S were increased 3, 7/8, and 28 days postirradiation. The total lipid composition of the jejunal BBM was lower than in control animals only at 3 days postirradiation; this was due to a decrease in the total free fatty acid content. In addition to a lower total free fatty acid content, the ileal BBM contained an increased amount of total phospholipid (PL) which resulted in an increased phospholipid/cholesterol ratio at 3 days following irradiation. Variations in the BBM phospholipid composition occurred in both jejunum and ileum. In the jejunal BBM, the phospholipid composition changes did not alter the choline or amine phospholipid content; therefore, the choline/amine phospholipid ratio was unaffected by irradiation at 600 rad. In the ileal BBM, the phosphatidyl ethanolamine was increased at 3, 7/8, 14, and 28 days following irradiation. The choline/amine phospholipid ratio was not altered in the ileal BBM due to concomitant increases in lecithin content. Jejunal villus height, villus surface area, and the number of cells per villus were decreased at 3 days postirradiation, but increased by day 7/8 and 14 postirradiation to levels much higher than observed in control jejunal villi. The mucosal surface area was decreased at 3 and 7/8 days following irradiation but returned to control values by Day 14. Jejunal microvillus morphology was unaffected by irradiation. Few significant changes were observed in ileal villus morphology following irradiation at 600 rad. Ileal villus height, villus surface area, and mucosal surface area did not change, but the number of cells per villus initially decreased at 3 days and then increased beyond control values at 7/8 and 14 days postirradiation. Ileal microvillus height was significantly decreased only at 7 days postirradiation, while the number of microvilli per micron was increased only at 3 days postirradiation. This study suggests that changes in intestinal morphology and brush border composition may contribute to the altered passive permeation toward lipids which has been reported following abdominal radiation.     

Postnatal development of intestinal bile salt transport. Relationship to membrane physico-chemical changes

The postnatal development of intestinal bile salt transport in the rat was examined using the villus technique. Jejunal uptake of taurocholate was linear with respect to incubation concentration at all study ages. Ileal uptake was linear with taurocholate concentration during the first 2 postnatal weeks; a curvilinear relationship indicating the presence of saturable transport appeared during the third week. With the appearance of ileal active transport at age 3 weeks, the Km (app) was constant at 0.49 mM, 0.59 mM, and 0.50 mM in 3-week, 4-week, and adult animals, respectively. The V(app) was 14.65 nmol X mg-1 (dry wt) X min-1 at 3 weeks and declined with age to 11.40 and 10.51 nmol X mg-1 (dry wt) X min-1 in 4-week and adult animals, respectively. The role of physico-chemical changes in the microvillus membrane in the development of ileal active transport was examined. With increasing postnatal age, microvillus membrane cholesterol content rose while the phospholipid content remained unchanged in both ileum and jejunum. Corresponding rises in the cholesterol/phospholipid ratio were observed in both sites. Simultaneously, the microvillus membrane fatty acid composition was changing from predominantly saturated to unsaturated species in both ileum and jejunum. The microvillus membrane fluorescence anisotropy (r) increased with postnatal age in jejunum when measured at 25 degrees C and 37 degrees C and ileum when measured at 25 degrees C; however, no change was noted in ileum when measured at 37 degrees C. Ileal active bile salt transport develops during the third postnatal week, and is associated with concurrent changes in membrane lipid composition and fluidity when measured at 25 degrees C.(ABSTRACT TRUNCATED AT 250 WORDS)     

Intestinal brush border membrane marker enzymes, lipid composition and villus morphology: effect of fasting and diabetes mellitus in rats

Diabetes mellitus is associated with enhanced passive intestinal uptake of cholesterol and fatty acids. In order to determine the basis for these changes in intestinal permeability, the jejunal morphology and the lipid content of purified brush border membranes (BBM) were measured in fasted and fed control (C) and streptozotocin diabetic (DM) rats. There was no difference between C and DM in BBM sucrase or alkaline phosphatase; fasting had no effect on BBM enzymes in C, but in DM fasting was associated with increased sucrase activity per length of jejunum. In C fasting was associated with higher levels of BBM total phospholipid, lecithin, choline and amine phospholipids, whereas fasting in DM was associated with higher BBM cholesterol and lower free fatty acids. In the fasting DM, there was a greater villus and mucosal surface area than in the fasting C. A previous study demonstrated that with fasting in DM versus C, cholesterol uptake was unchanged, but when animals were fed, cholesterol and fatty acid uptake were greater into the jejunum of fed DM as compared with fed C. In the BBM of fed DM as compared with C, there was a significant increase in total phospholipid, lecithin, phosphatidyl ethanolamine, choline and amine phospholipids, and phospholipid/cholesterol ratio. Thus, (1) fasting is associated with changes in intestinal morphology, BBM lipids; (2) the effect of fasting is different in DM and C; (3) the enhanced uptake of lipids into the jejunum of fed diabetic rats is not due to changes in villus morphology, but may be due to alterations in the BBM phospholipids.     

Localization of acyl-coenzyme A: cholesterol acyltransferase in villus and crypt cells of chick intestine

Endogenous cholesterol esterification by acyl-CoA:cholesterol acyltransferase (EC 2.3.1.26) was studied in isolated enterocytes obtained from chick duodenal, jejunal, and ileal villi and crypts, using [14C]oleoyl-CoA as substrate. The maximal specific activity in each cell fraction was found in chick jejunum, followed by duodenum and ileum. Jejunal upper and mid villi showed higher specific activities than lower villi and crypts. Epithelial cells isolated from chick intestine also incorporated oleoyl-CoA into different lipids using the endogenous substrates. Upper and mid villus cells showed the maximal incorporation of oleoyl-CoA into triglycerides in duodenum and jejunum. Levels of oleoyl-CoA incorporation into phospholipids were higher than those found in the synthesis of triglycerides or cholesterol esters, whatever may be the cell fraction considered. Upper villus cells also showed the highest specific activity in the incorporation of oleoyl-CoA into phospholipids. The acyl-CoA hydrolase specific activity was practically similar in all the cell fractions obtained from chick duodenum, jejunum, and ileum.     

Diets alter jejunal morphology and brush border membrane composition in streptozotocin-diabetic rats

Previous studies have demonstrated enhanced active and passive uptake of many nutrients in animals with experimental diabetes. These changes in absorption cannot be explained by differences in intestinal morphology, although the brush border membrane (BBM) phospholipids do change in diabetes. Manipulation of diet produces alterations in intestinal uptake of lipids and glucose. This study was undertaken to determine the effect of diet and diabetes on jejunal morphology and BBM lipid composition. Rats were rendered hyperglycemic with streptozotocin and were fed for 2 weeks on a diet that was high or low in carbohydrate, essential fatty acids, cholesterol, or protein. In both control and diabetic rats, these diets produced changes in villus height and BBM sucrase and alkaline phosphatase activities. In both control and diabetic rats, BBM phospholipids were unaffected by changes in the dietary content of essential fatty acids, cholesterol, or protein, but total BBM phospholipid content was reduced in animals fed low as compared with high carbohydrate diet. Total BBM phospholipid content was higher in diabetic than in control animals fed the low protein diet, whereas BBM phospholipid content was lower in diabetic than in control animals fed the high carbohydrate diet, and was even lower in diabetic animals fed the low as compared with the high carbohydrate diet. These changes in total phospholipids were due to alterations in the BBM content of phospholipids containing choline. In control animals, BBM cholesterol was higher in rats fed the low as compared with the high cholesterol diet, or the low as compared with the high protein diet.(ABSTRACT TRUNCATED AT 250 WORDS)     

Effects of Two Highly Monounsaturated Oils on Lipid Composition and Enzyme Activities in Rat Jejunum

The effects of two monounsaturated fatty acid (MUFA) oils, olive oil (OO)and high-oleic sunflower oil (HOSO), with high content in oleic acid butdiffering in their non-fatty acid fraction, on brush-border membrane(BBM) lipid composition and fluidity and on mucosal enzyme activitiesof rat jejunum were studied. Animals were given semipurified diet withlinoleic acid to prevent essential fatty acid deficiency (control group)or semipurified diet containing 10% of either OO or HOSO for 12weeks. There was a significant decrease in the content of jejunalBBM phospholipids together with an increase in the level of freecholesterol in both oil-fed rats, when compared to controlgroup. Although the increase in the BBM free cholesterol levelwas not statistically significant in HOSO-fed rats, a significantdecrease in the phospholipid/free cholesterol ratio was found inboth OO and HOSO-fed animals compared to control group. Rat jejunalBBM had a high level of free fatty acids which was increased in BBMisolated from OO and HOSO-fed animals. There was no statisticalsignificant difference in the phospholipid distribution between thecontrol and the OO group. However, HOSO-fed animals showed the lowestlevel of phosphatidylethanolamine together with the highestphosphatidylcholine content and the phosphatidylcholine/sphingomyelinratio. The fatty acid pattern of jejunal BBM lipids was modifiedaccording to the major fatty acids in the oils. There was a decreasein both stearic acid (18:0) and linoleic acid (18:2 n-6), togetherwith an increase in oleic acid (18:1 n-9) in jenunal BBM isolatedfrom both oil experimental groups. All these results were accompaniedby a significant increase in the BBM fluidity (as assessed bysteady-state fluorescence polarization of diphenylhexatriene) isolatedfrom oil-fed rat, when compared to control group. OO and HOSO-fedanimals had the lowest activities of sucrase and maltase, whilealkaline phosphatase activity only was decreased in HOSO-fedanimals. The specific activity of maltase was not modified in anyexperimental rats. In summary, both MUFA oils induced similar effectson jejunal BBM lipid composition, fluidity, sucrase, maltase andlactase activities. Furthermore, HOSO intake resulted in a lowestalkaline phosphatase activity which was accompanied by changes inindividual phospholipid composition. All these results suggest thateffects of MUFA oils on jejunal BBM lipid composition and hydrolaseactivities are most likely due to the presence of high content ofoleic acid rather than other components contained in the non-fattyacid of olive oil.     

Effects of ethinyl estradiol on intestinal membrane structure and function in the rabbit

Structural and functional properties of the small intestinal microvillus membrane were evaluated in the rabbit after administration of ethinyl estradiol, a synthetic estrogen with a demonstrated propensity to alter hepatic membrane lipid fluidity, and promote cholestasis. In the jejunum, no estrogen-induced changes in microvillus membrane total lipid, cholesterol or phospholipid content were observed. However, the ileal microvillus membrane in estradiol-treated animals demonstrates significant reductions vs. controls (per mg protein) in total lipid (0.55 milligrams vs. 0.89 milligrams) [corrected] and phospholipid (206.7 micrograms vs. 304.91 micrograms) (p less than 0.001) content, as well as modifications in specific phospholipid species. The increase in the ileal microvillus membrane cholesterol: phospholipid molar ratio (0.65 vs. 0.51, p less than 0.05) was associated with a significant decrease in membrane lipid fluidity reflected by an increase in fluorescence anisotropy measurements utilizing diphenyl hexatriene as the fluorophore (r at 25 degrees C = 0.306 vs. 0.282, p less than 0.05). Thermotropic lipid phase transitions, assessed by Arrhenius plots of both fluorescence data and ileal microvillus membrane p-nitrophenylphosphatase activity demonstrate that phase changes occur between and 24 and 28 degrees C in both treated and untreated groups. Within the temperature range studied (40-10 degrees C) no differences from control were observed in microvillus membrane alkaline phosphatase activity following estrogen treatment. These data therefore indicate that ethinyl estradiol-induced effects on microvillus membrane lipid composition and physical properties occur predominantly in the ileum and appear to be related, in part, to specific alterations in the availability of phospholipid following estrogen treatment.     

Cell migration and cortisone induction of sucrase activity in jejunum and ileum

The increase of sucrase activity in homogenates of jejunum and ileum of suckling rats after cortisone administration has been investigated. Serial tissue sections of villi and crypts were also assayed for sucrase activity and these results were compared with the migration of cells labelled with [(3)H]thymidine along the villus. By using a low dose of cortisone (0.5mg/day per 100g body wt.) it was found that the sensitivity of the small intestine producing system to cortisone stimulation increased during the suckling period. On the other hand, 5mg of cortisone/day per 100g body wt. produced practically the same increase of sucrase during the entire suckling period. Sucrase activity in homogenates of the entire small-intestinal wall was first detected 24h after the first injection of cortisone (5mg/day per 100g body weight) to 9-day-old animals and maximum activity both in the jejunum and ileum was reached by 120h. Jejunal activity was greater than ileal activity, but the rate of the increase was similar. The half-time of the increase was 23-27h, whereas enterocytes migrate from the base to the tip of the villi in approximately 72h. Comparison of sucrase activity in serial tissue sections of villi and crypts at various times after cortisone treatment showed that the leading edge of sucrase activity proceeds toward the tip of the villi at the same rate as the advancing edge of newly formed cells. Sucrase activity increased in the newly induced cells as they migrated to the tip of the villi. It was concluded that the increase of sucrase activity in suckling rats after cortisone stimulation is due to at least three factors: (1) increase of activity in newly differentiating cells, (2) increased percentage of villus cells with sucrase activity and (3) continued production or activation of sucrase activity as the cells migrate along the villi.     

Regional differences in lipid composition and incorporation of saturated and unsaturated fatty acids into microsomal membranes of rat small intestine

Incorporation of [1-14C]palmitic (16:0) and [1-14C]linoleic (18:2 omega 6) acids into microsomal membranes of proximal (jejunum) and distal (ileum) regions of rat small intestine was investigated, and the lipid composition, including fatty acid profiles of membrane phospholipids, was determined. Jejunal microsomes contained significantly higher amounts of total phospholipids, phosphatidylcholine, and phosphatidylinositol, and lower amounts of cholesterol and sphingomyelin when compared with ileal microsomes. Jejunal microsomal phospholipids contained higher levels of stearic (18:0), 18:2 omega 6, and eicosapentaenoic (20:5 omega 3) acids followed by reduced levels of oleic (18:1 omega 9), arachidonic (20:4 omega 6), and docosahexaenoic (22:6 omega 3) acids when compared with those from the ileum, except for phosphatidylinositol where no significant difference between 20:4 omega 6 content of each site was observed. In both jejunal and ileal microsomes, incorporation of [1-14C]18:2 omega 6 was significantly higher than that of [1-14C]16:0. Incorporation of both [1-14C]16:0 and [1-14C]18:2 omega 6 was significantly higher in jejunal microsomal lipid fractions (phospholipids, diacylglycerols, triacylglycerols) when compared with the ileal microsomal fraction. These data suggest that (1) jejunal and ileal microsomal membranes differ from each other in terms of lipid composition and lipid synthesis, (2) site variations in the specificity of acyltransferases for different fatty acids exist, and (3) higher delta 9-, delta 6-, delta 5-, and delta 4-desaturase activities exist in ileal compared with jejunal enterocytes.     

Spectrum of effects of dietary long-chain fatty acids on rat intestinal glucose and lipid uptake

Isocaloric modification in the ratio of dietary polyunsaturated-to-saturated fatty acids influences intestinal uptake of actively and passively transported nutrients. This study was undertaken to determine which dietary fatty acid was responsible for these alterations in absorption. Adult female rats were fed isocaloric semisynthetic diets high in palmitic and stearic acids (SFA), oleic acid (OA), linoleic acid (LA), or linolenic acid (LNA). An in vitro technique was used to measure the uptake of varying concentrations of glucose as well as a series of fatty acids and cholesterol. Jejunal uptake of 40 mM glucose was highest in rats fed SFA and lowest in those fed LA; ileal glucose uptake was similar in OA, LA, and LNA, but was lowest in SFA. Jejunal uptake of medium-chain fatty acids (8:0-12:0) was higher in OA than in other diet groups; ileal uptake of medium-chain fatty acids was unaffected by diet. Jejunal and ileal uptake of 18:2 was higher in LNA than in SFA or OA; the uptake of the other long-chain saturated or unsaturated fatty acids was unchanged by diet. The ileal but not the jejunal uptake of cholesterol was increased in LA as compared with SFA or OA, and reduced in LNA as compared with LA. These transport changes were not explained by differences in the animals' food consumption, body weight gain, intestinal mass, or mucosal surface area. We postulate that these diet-induced transport alterations may be mediated via changes in brush border membrane phospholipid fatty acyl composition. Thus, intestinal transport of nutrients may be varied by isocaloric changes in the dietary content of individual fatty acids.     

Importance of jejunum for bile acid absorption in pigeons: effect of jejunal resection on plasma cholesterol and cholesterol excretion

Previous studies from our laboratory suggested that the jejunum could be the major site of bile acid absorption in the pigeon. To confirm this observation directly, the effects of jejunal resection on the fecal excretion of bile acids and neutral sterols and on the level of plasma cholesterol were examined in the pigeon. This surgical procedure specifically increased (p less than 0.05) the excretion of bile acids, with no change in the excretion of neutral sterols. The total fecal steroid excretion in the pigeons with jejunal resection was significantly (P less than 0.01) higher than that in the control group. Jejunal resection also decreased the plasma cholesterol level in the pigeon. These results confirm our earlier suggestion that the jejunum is the major site of bile acid absorption in pigeons, unlike that in mammals, in which the ileum is the major site.     

Resection of rabbit ileum: effect on brush border membrane enzyme markers and lipids

Alterations in transport function have been described 6 weeks after surgical resection of 50% of the distal small intestine. Previous studies demonstrated a modest increase in the jejunal uptake of medium chain length fatty acids following resection, while the uptake of many other lipids (cholesterol, bile acids, fatty alcohols, short and long chain length fatty acids) appears to be unaffected. Marked changes in the kinetic constants for the carrier-mediated uptake of four sugars and leucine were observed following resection, but the changes in transport were not associated with changes in the mucosal surface area. This study was undertaken to examine the possible adaptive mechanisms that occur with ileal resection in the rabbit. A 29% increase in the wet weight of jejunal mucosal scrapings and a 53% increase in jejunal brush border membrane (BBM) protein was observed following resection. The jejunal BBM sucrase (S) was unchanged following ileal resection, but alkaline phosphatase (AP) total activities were increased in the resected rabbits. This resulted in a 45% increase in the ratio of AP/S with resection. The lipid composition (total free fatty acids, total bile acids, total cholesterol, total phospholipids, individual phospholipids, and the ratio of total phospholipids/total cholesterol) of BBM was similar in control and resected rabbits. This suggests that quantitative rather than qualitative changes in the membrane composition may be responsible for the transport changes observed in resected animals.     

Adaptation of intestinal glucose transport in rats with diabetes mellitus occurs independent of hyperphagia.

Chronic diabetes enhances intestinal absorption of glucose and induces hyperphagia. We examined the enhanced intestinal absorption of glucose in ad libitum-fed rats with streptozocin-induced diabetes mellitus and compared these results with those obtained from pair-fed diabetic animals. Maximal transport capacity (Vmax) and carrier affinity (K0.5) were determined by measuring jejunal and ileal short circuit current (Isc) responses to varying concentrations of 3-O-methyl-D-glucopyranose and D-glucose. Pair-fed diabetic animals maintained the same body weight as animals fed ad libitum, although ad libitum-fed diabetic rats had an increased oral chow intake. Age-matched control rats maintained a constant jejunal and ileal Vmax and K0.5 throughout the study. Diabetic rats fed ad libitum demonstrated an enhanced Vmax and K0.5 in both jejunum and ileum. Pair feeding diabetic animals further enhanced jejunal Vmax while lowering jejunal K0.5 levels. In contrast, pair feeding diabetic animals delayed and blunted changes in ileal Vmax and prevented changes in ileal K0.5. In conclusion, signals other than those of hyperphagia regulate kinetic changes in glucose absorption during diabetes mellitus. Furthermore, these changes have differing effects on jejunum and ileum.     

Treatment of the enhanced intestinal uptake of glucose in diabetic rats with a polyunsaturated fatty acid diet

Intestinal absorption of most nutrients is enhanced in diabetic rats. We wished to test the hypothesis that manipulation of dietary fatty acids will modify enhanced uptake of glucose in rats with established streptozotocin-diabetes. Chow-fed control rats or animals with one week of streptozotocin-diabetes were continued on chow or were fed ad libitum for three weeks with semisynthetic isocaloric diets containing a high content of either essential polyunsaturated or non-essential saturated fatty acids. The jejunal and ileal in vitro uptake of varying concentrations of glucose was much higher in diabetic than control rats fed chow or the saturated fatty acid diet. In contrast, the enhanced uptake of this sugar was reduced or normalized in diabetic rats fed the polyunsaturated fatty acid diet. Feeding the polyunsaturated fatty acid diet was associated with increased brush-border membrane activity of alkaline phosphatase in diabetic jejunum and ileum, but neither the saturated fatty acid diet nor the polyunsaturated fatty acid diet altered brush-border membrane cholesterol or phospholipids in control or in diabetic rats. Mucosal surface area was similar in diabetic rats fed the saturated fatty acid diet or the polyunsaturated fatty acid diet. Thus, (1) feeding the polyunsaturated fatty acid diet diminishes the enhanced jejunal and ileal uptake of glucose in diabetic rats, and (2) the influence of the polyunsaturated fatty acid diet on uptake in diabetic rats was not explained by alterations in intestinal morphology or brush-border membrane content of cholesterol or phospholipids. This study suggests that manipulation of dietary lipids may play a role in the normalization of the enhanced intestinal glucose uptake in rats with established diabetes.     

Uptake of selenate and selenite by isolated intestinal brush border membrane vesicles from pig,sheep, and rat

Selenate and selenite uptakes by isolated intestinal brush border membrane vesicles (BBMV) from pig, sheep, and rat were investigated. Selenate uptake into jejunal and ileal, but not duodenal, BBMV from pig was stimulated by an inwardly directed transmembrane Na⁺ gradient (Na _out ⁺ >Na _in ⁺ ). Selenate transport into rat ileal and sheep jejunal BBMV was also enhanced in the presence of a Na⁺ gradient. Unlike selenate uptake, selenite uptake was not Na⁺ dependent, neither in pig small intestine nor in sheep jejunum and rat ileum. Uptake of selenate represented real uptake into the vesicular lumen, whereas selenite uptake was a result of an extensive binding of⁷⁵Se to the membranes. Thiosulfate at a 250-fold concentration of selenate completely inhibited Na⁺-dependent selenate uptake into pig jejunal BBMV. Furthermore, Na⁺-dependent sulfate uptake was totally inhibited in the presence of a 250-fold selenate concentration. The results clearly show that selenate transport across the BBM of pig jejunum and ileum, sheep jejunum, and rat ileum is partially energized by a transmembrane Na⁺ gradient. Moreover, it is concluded from the results that there exists a common transport mechanism for sulfate and selenate in the BBM. The extensive binding of⁷⁵Se from⁷⁵Se-labeled selenite to the membranes could be from a spontaneous reaction of selenite with membrane-associated SH groups.     

Ileal resection and dietary content of sucrose influence the intestinal uptake of monosaccharides

The intestinal uptake of 0.5 and 40 mM glucose, galactose, and 3-O-methyl glucose (3-O-MG) was examined in vitro in rabbits fed a high (HS) or a low (LS) sucrose diet. In animals with an intact intestinal tract, the jejunal uptake of 0.5 mM 3-O-MG was unaffected by the dietary content of sucrose, whereas the uptake of 40 mM 3-O-MG was lower in LS than HS. The uptake of 40 mM galactose was higher in LS than HS and the uptake of 0.5 mM galactose was similar in HS and LS, whereas the uptake of 0.5 mM but not 40 mM glucose was lower in LS than HS. In animals subjected 6 weeks previously to an ileal resection, the adaptive changes in the jejunal uptake of the hexoses in response to alterations in the dietary content of sucrose differed from the changes observed in rabbits with an intact intestinal tract. For example, feeding HS to ileal resected animals was associated with increased jejunal uptake of 40 mM galactose, decreased uptake of 40 mM glucose, and unchanged uptake of 40 mM 3-O-MG; whereas in control animals with an intact intestinal tract, feeding HS resulted in increased uptake of 40 mM 3-O-MG, decreased uptake of 40 mM galactose, and no change in the uptake of 40 mM glucose. A similar adaptive pattern was noted in the jejunum and ileum for the effect of dietary sucrose on the uptake of 0.5 and 40 mM glucose.(ABSTRACT TRUNCATED AT 250 WORDS)     

Defective brush-border expression of intrinsic factor-cobalamin receptor in canine inherited intestinal cobalamin malabsorption

Ligand binding activity of intrinsic factor-cobalamin receptor (IFCR) was determined in homogenates and isolated brush-border membranes (BBM) of ileum and kidney from dogs exhibiting simple autosomal recessive inheritance of selective cobalamin malabsorption (Fyfe, J. C., Giger, U., Hall, C. A., Jezyk, P. F., Klumpp, S. A., Levine, J. S., and Patterson, D. F. (1991) Pediatr. Res. 29, 24-31). IFCR activity of affected dog ileal homogenates was 3-4-fold higher than normal whereas IFCR activity in affected dog kidney homogenates was one-tenth of normal. The recovery of IFCR activity in the BBM of ileum and renal cortex of affected dogs was 30- and 20-fold less than normal, respectively. The dissociation constant (Kd) for intrinsic factor-cobalamin was similar in BBM of both tissues and was the same in affected and normal dogs. In the affected dog ileal BBM, activities of alkaline phosphatase and sucrase-isomaltase and vesicular transport of glucose and Na(+)-taurocholate were normal. Immunoblots showed no IFCR cross-reactive material in the ileal or renal BBM of affected dogs. IFCR purified by affinity chromatography from kidney of both normal and affected dogs had an Mr = 230,000. However, amino acid analysis revealed that the affected dog IFCR had more lysine than the normal, and protease cleavage of the purified IFCRs revealed different peptide maps. Asparagine-linked oligosaccharides of both proteins were sensitive to peptide N-glycosidase F cleavage, but only the affected dog IFCR was endoglycosidase H sensitive. These results suggest that cobalamin malabsorption in this canine family is caused by inefficient BBM expression of IFCR due to a mutation of IFCR and its retention in an early biosynthetic compartment.     

Biochemical analysis of jejunal brush border membrane of golden hamster: pathogenic modulations due to ancylostomiasis.

The common hookworm (Ancylostoma ceylanicum) infection of humans was studied in golden hamsters model system. Significant biochemical modulations were observed in hamster jejunal brush border membrane (BBM), the primary site of infection. Analysis of BBM at the peak of infection (3-weeks) revealed a marked decrease in the activities of sucrase, lactase and maltase, while activities of alkaline phosphatase, (Ca2+ + Mg2+)-ATPase and gamma-glutamyl transpeptidase were increased. Kinetic studies conducted with maltase, a superficially localised enzyme of jejunal BBM, revealed loss of enzyme active site during the infection. Among other constituents, the levels of cholesterol and triglycerides were significantly decreased with slight increase in phospholipid content in the infected animals. The hookworm infection also caused a decline in total hexose content indicating an altered membrane glycocalyx. Conversely, there was significant enhancement of hydroxyproline and sialic acid contents. SDS-PAGE analysis showed an enhancement in both low and high molecular weight proteins in jejunal BBM preparations of the infected group. Gel electrophoresis of glycoproteins further revealed the appearance of two additional peaks in the low molecular weight region and concomitant disappearance of a peak in the high molecular weight region. These results strongly support the view that the hookworm infection causes severe damage not to the site of attachment alone but also to the entire cell lining of the jejunum and therefore could influence overall digestion and absorption.     

Synthesis and coenzyme A-dependent esterification of cholesterol in rat intestinal epithelium. Differences in cellular localization and mechanisms of regulation

The current studies were undertaken to characterize the localization and regulation of cholesterol synthesis and acyl-CoA:cholesterol acyltransferase activity in rat intestinal crypt and villus cells. Both parameters were determined in groups of animals with widely varying sterol fluxes across the intestinal mucosa. In animals on control diet the rates of cholesterol synthesis, measured by the incorporation of [3H]water per mg of protein, were similar along the villus/crypt axis in the jejunum, whereas in the ileum, villus cells were significantly more active than crypt cells. In both areas, however, the majority of total synthetic activity was found in cells from the crypts and lower villi. In contrast, the highest specific and total acyl-CoA:cholesterol acyltransferase activity was recovered in the villus cells of the jejunum and ileum. Dietary cholesterol did not affect sterol synthesis in any of the cell fractions but increased acyl-CoA:cholesterol acyltransferase activity approximately 2-fold in jejunal cell fractions. Inhibition of cholesterol absorption or sequestration of intestinal bile acids stimulated sterol synthetic activity up to 7-fold, and this occurred mainly in the lower villus and crypt cells in both jejunum and ileum. An increased demand for lipoprotein cholesterol, generated by triglyceride feeding, similarly was associated with enhanced synthetic rates. However, unlike cholesterol feeding, these manipulations did not increase acyl-CoA:cholesterol acyltransferase activity in any of the villus cell fractions. These studies suggest, therefore, that the intracellular pools of cholesterol that regulate the rate of cholesterol synthesis and the rate of cholesterol esterification are functionally distinct.     

Influence of age on duodenal brush border membrane and specific activities of brush border membrane enzymes in Wistar rats.

To examine age-related changes in the morphology of intestinal brush border membrane (BBM; microvilli) and specific activities of intestinal BBM enzymes including alkaline phosphatase (ALP), gamma-glutamyl transpeptidase (gamma-GT), and disacchridase, four groups of Wistar rats were sacrificed at 2.5 wk, 5 wk, 5 mon and 23 mon. In an electron microscopic examination, morphologically a less dense BBM structure in the duodenum of rats aged 23 mon was observed than that of rats aged 5 mon. Specific activity of ALP in the duodenum from 5-mon-old rats was significantly higher than from rats aged 2.5 wk and 23 mon. The mucosal tissues from 5-wk-old rats had significantly higher specific activity of gamma-GT than did tissues from the other ages. In sucrase and maltase specific activities, 5-mon-old rats had higher activities of these enzymes than other age groups, especially 2.5-wk- and 23-mon-old rats. There was also a significant effect of site on intestinal BBM enzyme activities in post-weanling rats. Regional gradients of ALP and gamma-GT along the entire small intestine (duodenum > jejunum > ileum) were remarkable. Disaccharidase activities peaked in the jejunum and declined toward both the duodenum and ileum. Taken together the result obtained here suggested that 5-mon-old rats had the most elevated intestinal function. This result also strongly indicated that the structure of the intestinal BBM and development of intestinal BBM enzymes in Wistar rate were markedly influenced by age during the postnatal period.