The origin of chylomicron phosphatidylcholine in the rat.

This study investigates the pathways of origin of chylomicron phosphatidylcholine (PC) using a lymph- and bile-fistulated rat infused with a stabilized triolein emulsion. [(14)C-glycerol]PC was used to evaluate chylomicron PC generated by lyso PC acyltransferase. The percentage of chylomicron PC derived from the PC infused was directly proportional to the PC concentration in the infusate. When the infusate PC concentration was 10 mM, essentially all the chylomicron PC was derived therefrom at 4-6 h of infusion. Incorporation of the radiolabel was not found to be as great in the lymph subnatant PC as in chylomicron PC, suggesting that chylomicron and lymph subnatant PC might be supplied from different PC precursor pools.(32)P(i) was infused into similarly prepared rats to judge chylomicron PC synthesized from de novo sources. In these experiments it was found that the percentage of chylomicron PC derived from de novo synthesis was inversely related to the PC concentration of the infusate. This suggests that exogenously infused PC inhibits de novo PC synthesis. When [(32)P]rat bile PC was infused with [(14)C-glycerol]potato PC, the bile PC was preferred as a chylomicron precursor despite the greater similarity of the saturated fatty acids of potato PC to those of chylomicron PC. When the saturated fatty acids of bile and chylomicron PC were compared, chylomicron PC was significantly richer in stearate, suggesting extensive enterocyte modification of the saturated fatty acids of bile PC.

Mechanism of lipid mobilization by the small intestine after transport blockade.

The nonionic detergent, Pluronic L-81 (L-81) has been shown to block the transport of intestinal mucosal triacylglycerol (TG) in chylomicrons. This results in large lipid masses within the enterocyte that are greater in diameter than chylomicrons. On removal of L-81, mucosal TG is rapidly mobilized and appears in the lymph. We questioned whether the blocked TG requires partial or complete hydrolysis before its transport. Rats were infused intraduodenally with [3H]glyceryl, [14C]oleoyl trioleate (TO) and 0.5 mg L-81/h for 8 h, followed by 120 mumol/h linoleate for 18 h. Mesenteric lymph was collected and analyzed for TG content and radioactivity. An HPLC method was developed to separate TG on the basis of its acyl group species. The assumed acyl group composition was confirmed by gas liquid chromatography analysis. TG lymphatic output was low for the first 8 h but increased to 52 mumol/h at the 11th h of infusion (3 h after stopping L-81). 38% of the infused TO was retained in the mucosa after the 8-h infusion. 95% of mucosal TG was TO, 92% of the radioactivity was in TG, and 2.4% of the 14C disintegrations per minute was in fatty acid. HPLC analysis of lymph at 6, 10, 12, and 14.5 h of infusion showed a progressive rise in TG composed of one linoleate and two oleates, to 39%; and in TG composed of two linoleates and one oleate to 20% at 14.5 h of infusion. On a mass basis, however, 80% of the TG acyl groups were oleate. 3H/14C ratios in the various TG acyl group species reflected the decrease in oleate. We conclude that first, unlike liver, most mucosal TG is not hydrolyzed before transport. The mechanism of how the large lipid masses present in mucosal cells after L-81 infusion are converted to the much smaller chylomicrons is unknown. Second, the concomitant infusion of linoleate did not impair lymph TG delivery after L-81 blockade.

Intestinal phospholipase, a novel enzyme.

We evaluated phospholipase activity in the intestine of rats and other species. Phospholipase activity was assayed by a surface barostat technique or an egg yolk titration system. Mucosal activity was found only by the surface barostat technique with phosphatidylglycerol as substrate; it was not found with phosphatidylcholine as substrate in assays by either technique. In gut luminal fluid activity was found when both phosphatidylcholine and phosphatidylglycerol were used as substrate in assays by the surface barostat technique, and phosphatidylcholine as substrate yielded activity in egg yolk titration. In rats in which pancreatic juice had been diverted, mucosal and gut luminal phospholipase activity was greater than in controls, thus demonstrating that enzyme activity was not due to pancreatic phospholipase. Bacterial origin of phospholipase activity was excluded in that phospholipase activity was found in germ-free rats; gastric and salivary gland origins were excluded in that continued phospholipase activity was found in rats with gastric fistula. The physiological importance of the enzyme was established by the finding that rats with pancreatic fistula absorbed 111 mumol of phosphatidylcholine and that controls absorbed 119 mumol of a 135-mumol load. Activity was found to be three times greater in the distal than in the proximal intestine; in cryptal cells it was 10 times greater than in villus tip cells. 65% of the activity in the gut lumen was tightly bound to particulate matter. We propose that intestinal phospholipase may be important in gut bacterial control, in the digestion of vegetable matter (phosphatidylglycerol is a major phospholipid in both plants and bacteria), and in the digestion of phospholipids in the gut lumen.

Isolation and properties of the mixed lipid micelles present in intestinal content during fat digestion in man.

To evaluate better the physicochemical characteristics of human fat digestion, a method was developed which allowed characterization of the bile acid-lipid mixed micelles of the aqueous phase of post-prandial duodenal fluid. Duodenal fluid was collected after a 36-g fat breakfast for two 90-min periods and for 60 min after i.v. cholecystokinin and was ultracentrifuged at 15,400,000 g-min. The aqueous phase was isolated, passed through a 200-nm filter, and the mixed micelles were concentrated by an ultrafiltration procedure using a 1.5-nm filter. The 1.5-nm retentate was eluted from Sepharose 6B columns with 1.5-nm filtrate for both preequilibration fluid and eluent. 1.5-nm filtrate approximated the monomer concentrations. Each sample was assayed for bile acid, fatty acid, lecithin, lysolecithin, protein, cholesterol, and counterions (pH, Na+, K+, Ca2+). Constituents were concentrated only on the 1.5-nm filter. On gel permeation chromatography, coincident peaks were observed for bile acid, fatty acid, lysolecithin, and cholesterol; and were eluted with a Kav range of 0.50-0.68 (corresponding to a Stokes radius of 2.3-3.5 nm). An average density of 1.25 and coincident peaks of bile acid and fatty acid were found for the mixed micelles on sucrose density gradients. The regression lines of micellar fatty acid, lysolecithin, and cholesterol vs. bile acid gave a stoichiometry of 1.4 mol fatty acid, 0.15 mol lysolecithin, and 0.06 mol cholesterol for each mole of bile acid. Mixed micelles were homogeneous in composition. These results provide direct evidence for the existence of the postprandial mixed micelle and describe several of its physicochemical properties.

A GPI-anchored co-receptor for tissue factor pathway inhibitor controls its intracellular trafficking and cell surface expression.

BACKGROUND: Tissue factor pathway inhibitor (TFPI) lacks a membrane attachment signal but it remains associated with the endothelial surface via its association with an, as yet, unidentified glycosyl phosphatidylinositol (GPI)-anchored co-receptor. OBJECTIVES/METHODS: Cellular trafficking of TFPI within aerolysin-resistant ECV304 and EA.hy926 cells, which do not express GPI-anchored proteins on their surface, was compared with their wild-type counterparts. RESULTS AND CONCLUSIONS: Although aerolysin-resistant cells produce normal amounts of TFPI mRNA, TFPI is not expressed on the cell surface and total cellular TFPI is greatly decreased compared with wild-type cells. Additionally, normal, not increased, amounts of TFPI are secreted into conditioned media indicating that TFPI is degraded within the aerolysin-resistant cells. Confocal microscopy and studies using metabolic inhibitors demonstrate that aerolysin-resistant cells produce TFPI and transport it into the Golgi with subsequent degradation in lysosomes. The experimental results provide no evidence that cell surface TFPI originates from secreted TFPI that binds back to a GPI-anchored protein. Instead, the data suggest that TFPI tightly, but reversibly, binds to a GPI anchored co-receptor in the ER/Golgi. The co-receptor then acts as a molecular chaperone for TFPI by trafficking it to the cell surface of wild-type cells or to lysosomes of aerolysin-resistant cells. TFPI that escapes co-receptor binding is secreted through the same pathway in both wild-type and aerolysin-resistant cells. The data provide a framework for understanding how TFPI is expressed on endothelium.

Ontogeny of intestinal phospholipase A2 in the rat.

The ontogenic development of intestinal phospholipase A2 in the rat was studied. Enzyme activity was first observed at 11 days after birth; it peaked at 24 days and fell to adult levels at 30 days. Weaning did not affect activity; fasting and the feeding of a fat-free diet lowered activity.

CTP:phosphocholine cytidylyltransferase in intestinal mucosa.

CTP:phosphocholine cytidylyltransferase is thought to be a rate-limiting enzyme in phosphatidylcholine synthesis. This enzyme has not been well studied in intestine. We found that activity was greater in the non-lipid stimulated state (cytosolic form of the enzyme) than any previous tissue investigated (2.7 nM/min per mg protein). On addition of lysophosphatidylethanolamine, the enzyme only increased in activity 2.4-fold which is less than any previously reported tissue on lipid stimulation. As compared to liver, the enzyme was resistant to inhibition by chlorpromazine (gut, 100% activity remaining at 80 microM; 14% in liver). Tetracaine and propranolol were found to be impotent as inhibitors of the intestinal enzyme. Octanol-water partitioning showed that both chlorpromazine and tetracaine were hydrophobic, propranolol was not. pKa studies demonstrated that at the reaction pH, chlorpromazine would be uncharged. Physiologic experiments in which de novo phosphatidylcholine synthesis was either stimulated by bile duct fistulization and triacylglycerol infusion or suppressed by including phosphatidylcholine in a lipid infusion demonstrated that the enzyme (cytosolic enzyme) responded by decreasing Vmax but that the Km remained the same. In sum, these studies suggest that CTP:phosphocholine cytidylyltransferase in intestine is unique as compared to other tissues and that its response to a physiological stimulus is counter to that which would be adaptive.

Purification and partial characterization of intestinal acid lipase.

Intestinal acid lipase is an enzyme whose greatest specific activity is localized to the villus tips of the proximal intestine (Rao, R.H. and Mansbach, C.M. (1990) Biochim. Biophys. Acta 1043, 273-280). This suggests that it plays a role in the processing of dietary lipids. We purified the enzyme in order to better characterize it. Acid lipase was isolated from intestinal mucosa of rats by a combination of ammonium sulfate precipitation, butanol extraction and chromatography on DEAE Bio-Gel, CM Bio-Gel and Sephadex G-75. This resulted in a single protein of Mr 53,700 on SDS-polyacrylamide gel electrophoresis. The isolation scheme produced a 3344-fold purification resulting in an enzyme whose specific activity was 801 mumols/min per mg protein. The yield was 50%. The purified enzyme was stimulated (20-fold) by the addition of tauro- or glycocholate but no other conjugated bile acid. A sharp peak in activity occurred at pH 5.6. The pI of the enzyme was 6.2. The reaction products produced under prolonged incubation suggested that monoacylglycerol was not hydrolyzed since an overabundance of monoacylglycerol was found with respect to the amount of fatty acid produced. These results suggested that intestinal acid lipase is potentially important in the metabolism of dietary lipids. Its proportionate role awaits further documentation.

Acid lipase in rat intestinal mucosa: physiological parameters.

Previous studies have shown that up to a half of infused triacylglycerol does not exit the intestine via lymphatics. This suggests the presence of a mucosal lipase which could provide fatty acids for potential transport via the portal vein. The present study describes an acid-active lipase in rat intestinal mucosa. Acid lipase was assayed using a glyceryl tri[14C]oleate emulsion (pH 5.8). Mucosal homogenates were differentially centrifuged to yield cellular organelles and cytosol. Cells were sequentially released from villi using citrate and EDTA. The enzyme was found to be most active in the proximal quarter intestine and in the upper third of villi. Its greatest activity was in the lysosomal fraction. Esophageal diversion demonstrated that lingual lipase was not the precursor of the mucosal acid lipase. Bile salts stimulated activity 3- to 5-fold, but other neutral or anionic detergents were inhibitory. Of the detergents tested, taurocholate at super critical micellar concentrations could restore activity only with SDS. Sepharose 6B chromatography suggested that the enzyme partitioned into an SDS and taurocholate mixed micelle. We conclude that mucosal acid lipase is a distinct, intrinsic enzyme of the intestinal mucosa. It is predominantly lysosomal in origin. The location of its greatest activity in the villus tips of the proximal intestine suggests that it is potentially involved in mucosal triacylglycerol disposal.

The contribution of serum phosphatidylcholine and lysophosphatidylcholine to lymph phosphatidylcholine.

1. The contribution of serum phosphatidylcholine and 1-acyl lysophosphatidylcholine to chylomicron and mesenteric lymph lipoproteins of heavier buoyant density was studied in rats with catheters placed in the jugular vein, duodenum, common bile duct and mesenteric lymph duct. The effect of including 10 mM phosphatidylcholine in the triolein emulsion infused into the duodenum was also studied. 2. The intravenous infusion of phosphatidylcholine did not affect delivery of phosphatidylcholine into the lymph when phosphatidylcholine was included in the duodenal infusion. However, on intravenous lysophosphatidylcholine infusion, phosphatidylcholine transport into the lymph was increased both in chylomicrons and the other lipoproteins found in the lymph when phosphatidylcholine was included in the duodenal infusion. 3. The incorporation of serum phosphatidylcholine into chylomicron phosphatidylcholine was minimal and decreased further by intraduodenal phosphatidylcholine infusion. Incorporation into the heavier lymph lipoproteins was less than 20%. 4. The incorporation of serum lysophosphatidylcholine into chylomicrons was 17% at 4--6 h of infusion which was decreased by intraduodenal phosphatidylcholine as was the incorporation into the heavier lipoproteins of lymph. 5. It is concluded that serum phosphatidylcholine is a poor precursor of chylomicron phosphatidylcholine and that while lysophosphatidylcholine is a somewhat better precursor, its contribution to chylomicron phosphatidylcholine is limited by its serum concentration.

Detection of free radicals as a consequence of rat intestinal cellular drug metabolism.

Because the intestine is the first pass organ for orally administered drugs and because some of these drugs are known to undergo oxidative metabolism leading to the formation of free radicals, we investigated the potential for this to occur in cell suspensions of rat enterocytes. As part of our study, the effect of intracellularly produced superoxide on cellular metabolism was investigated. The drugs chosen were the quinone, menadione and the aromatic nitro-containing compound, nitrazepam. On incubation of both drugs with isolated enterocytes and the spin trap, 5,5-dimethyl-1-pyrroline N-oxide (DMPO), rapid appearance of an electron paramagnetic resonance (EPR) spectrum was recorded which was characteristic of hydroxyl radicals being spin trapped by DMPO giving 2,2-dimethyl-5-hydroxy-1-pyrrolidenyloxyl (DMPO-OH). Experiments were conducted which determined that the EPR spectrum of DMPO-OH resulted from the initial spin trapping of superoxide by DMPO to yield the corresponding nitroxide, 2,2-dimethyl-5-hydroxyl-1-pyrrolidenyloxyl (DMPO-OOH). Bioreduction of DMPO-OOH by glutathione peroxidase led to the rapid formation of DMPO-OH. We believe this enzymic pathway accounted for the EPR spectrum noted in incubations with either drug in the presence of the spin trap, DMPO. The incubation of enterocytes with both drugs did not mediate release of 51Cr nor lactate dehydrogenase. However, production of 14CO2 from [14C]glucose was severely inhibited (4-5-fold) in the presence of both drugs, while the incorporation of [14C]leucine into trichloroacetic acid precipitable protein was antagonized by menadione only. We conclude that superoxide can be demonstrated to arise as the result of enterocyte metabolism of menadione or nitrazepam. The consequence of oxidative metabolism of these drugs results in cellular dysfunction.

Effect of fat pre-feeding on bile flow and composition in the rat.

Studies were conducted in rats to determine if the increase in lymph triacylglycerol output on pre-feeding a 20% glyceryltrioleate diet (Mansbach, C.M., II and Arnold, A. (1986) Am. J. Physiol. 251, G263-269) was due to an increase in phosphatidylcholine output into bile. Rats who were fed chow or pre-fed the 20% fat diet were equipped with biliary and duodenal cannulas and infused with glucose-saline while bile was collected hourly. The next day a taurocholate-glyceryltrioleate infusion was given and bile collected for 5 h. Bile flow, bile acid, phosphatidylcholine and cholesterol output were greater in the chow fed group than controls during the 6 h of the glucose saline period. Outputs were low overnight. During the taurocholate-glyceryltrioleate infusion, bile flow, bile acid, phosphatidylcholine and cholesterol output were all greater in the fat pre-fed group than the chow fed controls. We conclude that fat pre-feeding profoundly influences biliary composition and flow. The 2-fold increase in biliary phosphatidylcholine output during duodenal lipid infusion offers a potential explanation for the increased delivery of triacylglycerol into the lymph in rats on a similar fat pre-feeding program.

Isolation of the early phase of chylomicron formation in intestinal epithelial cells of rats.

Lipid is first observed electron microscopically in the rough endoplasmic reticulum of intestinal epithelial cells during active lipid absorption. We have been able to isolate this subcellular fraction by using discontinuous sucrose gradients of 0.25/0.86/1.11 M sucrose. A preliminary low speed centrifugation of mucosal homogenate removed the heavier subcellular organelles. The resulting supernatant was centrifuged at 5.25 x 10(6) x g.min. The pellet from this centrifugation was placed on top of the gradient and the fractions isolated at the density interfaces after centrifugation at 25.5 x 10(6) x g.min. The isolated fractions were characterized enzymatically and electron microscopically. Electron microscopically, the fractions were predominantly composed of rounded vesicles decorated with ribosomes. Most contained lipid droplets whose diameters were 453 nm in the lighter membranes and 245 nm in the membranes isolated from the heavier density region. The vesicles contained NADPH cytochrome c reductase and glucose-6-phosphatase activity indicative of the presence of microsomes. Contamination with other subcellular organelles was minimal. These studies demonstrate a method which enables the isolation of vesicles containing chylomicron-sized particles which are from the earliest phase of chylomicron formation. Isolation of chylomicrons from these vesicles will enable a better understanding of the maturation process of chylomicrons as they traverse the intestinal epithelial cell.

beta-Sitosterol as a nonabsorbable marker of dietary lipid absorption in man.

beta-[14C]Sitosterol was evaluated for its usefulness as a nonabsorbable marker of lipid absorption in intubation studies. A liquid meal, including [3H]triolein as a marker of absorbable lipid, was used. The beta-sitosterol was shown to remain well mixed with triolein in the gastric antrum, validating its suitability for this type of study. In small bowel fluid, the beta-sitosterol was shown to partition into the aqueous phase in proportion to triolein and its hydrolysis products. With this system, lipid absorption was shown to take place chiefly in the jejunum in normal individuals.

Advances in nutrition and gastroenterology: summary of the 1997 A.S.P.E.N. Research Workshop.

BACKGROUND: The 1997 A.S.P.E.N. Research Workshop was held at the annual meeting in San Francisco, on January 26, 1997. The workshop focused on advances in clinical and basic research involving the interface between nutrient and luminal gastroenterology. METHODS: Presentations on the genetic regulation of gastrointestinal development, the molecular biology of small intestinal adaptation, the effect of nutrition support on intestinal mucosal mass, the relationship between nutrition and gastrointestinal motility, nutrient absorption, and gastrointestinal tract substrate metabolism were made by the preeminent leaders in the field. RESULTS: The investigators presented an insightful analysis of each topic by reviewing data from their own laboratories and the published literature. CONCLUSIONS: This workshop underscored the important interactions between nutrition and luminal gastroenterology at the basic science, metabolic/physiologic, and clinical levels. The integration of presentations from the different disciplines provided a unique interaction of information and ideas to advance our understanding of nutrition and gastrointestinal tract.

Effect of acute dietary alteration upon intestinal lipid synthesis.

The specific activities of three enzymes engaged in complex lipid synthesis, diglyceride acyltransferase, cholinephosphotransferase, and lysolecithin acyltransferase were studied in intestinal mucosa of hamsters fed either saline, hydrolyzed casein, or corn oil for 9 1/2 hr. In the most proximal intestine, saline feeding was associated with a reduced specific activity in villous tips with all three enzymes studied when compared with the two caloric supplemented groups. In the most distal intestine, oil feeding increased the activity of lysolecithin acyltransferase and choline phosphotransferase twofold as compared to casein fed hamsters; diglyceride acyltransferase was increased one- and one-half-fold. The response of lysolecithin acyltransferase and diglyceride acyltransferase to fat feeding was incomplete when compared to hamsters fed a fat supplemented diet for 7 days, suggesting that their pattern of response to dietary substrate was similar to the disaccharidases. By contrast, the response of cholinephosphotransferase to fat feeding was complete at 24 hr, suggesting that it responds in a manner similar to the glycolytic enzymes.