LIPOLYSIS AND ABSORPTION OF FAT IN THE RAT STOMACH

Transcription

1 GASTROENTEROLOGY Copyright 1969 by T he Williams & Wilkins Co. Vol. 56, No. 2 Printed in U.S.A. LIPOLYSIS AND ABSORPTION OF FAT IN THE RAT STOMACH SUSANNE BENNETI CLARK, PH.D., BARRY BRAUSE, AND PETER R. HOLT, M.D. Gastrointestinal Unit, Department of Medicine, St. Luke's Hospital Center, New York, New York The triglyceride lipolytic activity of gastric mucosal homogenates was studied in Wistar strain rats with exclusion of pancreatic enzyme contamination by prior prolonged pancreatic flow diversion. Considerable hydrolysis of a medium chain triglyceride, trioctanoin, occurred, but enzyme activity using triolein as substrate was barely detectable. Although the ph optimum for lipolytic activity was 6.5, significant hydrolysis occurred at ph 3.5 and the enzyme was very stable at this low ph. Taurocholate at concentration of 2 to 20 mm did not alter the rate of lipolysis. In pyloric ligated rats in vivo, considerable hydrolysis of both trioctanoin and triolein was measured when the ph was as low as 2. In addition, absorption of octanoic acid from the gastric lumen was readily detectable. Gastric lipolytic activity may be important in the digestion of fat, particularly of medium chain length, both in the stomach and in the intestinal lumen in patients with pancreatic exocrine insufficiency and steatorrhea. Hydrolysis of long chain triglycerides (fatty acids over 14 carbons) is a necessary prerequisite for the normal intestinal absorption of fat. The pancreas is the major source of lipolytic enzymes for the hydrolysis of these dietary triglycerides. Lipolysis by one or perhaps more pancreatic lipases 1 occurs in the intestinal lumen at oil-water interfaces resulting in Received May 24, Accepted August 16, Address requests for reprints to: Dr. Peter R. Holt, St. Luke's Hospital Center, Amsterdam and 114th Streets, New York, New York This work was supported in part by Grants HE and AM from the National Institutes of Health, United States Public Health Service. Mr. Mohammed Tajuddin assisted in the early stages of this study in partial fulfillment of the requirements for the degree of Master of Sciences in the Institute of Nutritional Sciences, Columbia University. The excellent independent technical work of Miss Michele Moore is also gratefully acknowledged. 214 the formation of monoglycerides and fatty acids. The existence of a gastric lipase was first postulated by Volhard 2 but the evidence for such an enzyme in gastric secretions or gastric mucosa has been disputed. 3, 4 In most previous studies insufficient care was taken to avoid contamination of gastric contents or mucosa by intestinal contents containing pancreatic enzymes. In addition the emphasis in these experiments has been on the ability to hydrolyze long chain triglycerides. Recently there has been much interest in the clinical use of semisynthetic triglycerides of medium chain length (MCT) (consisting of fatty acids of 6 to 10 carbons chain length) for the therapy of disorders of intestinal absorption including pancreatic insufficiency. It has been shown that significant quantities of unhydrolyzed M CT may be absorbed by the intestinal mucosa intact in the

2 February 1969 GASTRIC LIPOLYSIS AND ABSORPTION OF FAT 215 absence of pancreatic lipase in the lumen. However, under these conditions the maximal absorption rate for MeT was found to be 4-fold less than that for previously hydrolyzed triglyceride. 5 An active lipase from the stomach might therefore increase the capacity of the small intestine to absorb this fat. The purpose of the present study was to reinvestigate whether a gastric mucosal lipase existed using a rat preparation in which contamination by pancreatic enzymes was excluded by prior pancreatic flow diversion. Test substances used were a typical long chain triglyceride, triolein, and one of medium chain length, trioctanoin. In addition, the hydrolysis and absorption of these test lipids from the stomach was studied in vivo in pyloric ligated rats with pancreatic flow diversion to the ileum. Materials and Methods Trioctanoin-l-14C was obtained from New England Nuclear Corporation and was found to be over 99% pure by silicic thin layer chromatography. Triolein-l-HC, sodium octanoate I_HC, and sodium oleate-l-hc were obtained from Nuclear-Chicago, and were over 99% 98%, and 98% pure by thin layer chromatog raphy, respectively. These compounds were not purified further prior to use. Trioctanoin, octanoic, and oleic acids were obtained from Applied Science Laboratories, State College, Pa., and were over 99% pure. Triolein was obtained from Mann Research Chemicals, N. Y. Sodium taurocholate (99.5% pure) was a gift of Eli Lilly and Company and Tween 80 (polyoxyethylene sorbitan mono oleate) was obtained from Atlas Powder Company, Wilmington, Delaware. For in vitro homogenate studies, male or female Wistar strain rats weighing approximately 250 to 300 g were anesthetized with ether and the common bile duct was cannulated with P.E. 50 polyethylene tubing (O.D , I.D ) close to the duodenum, the other end being brought through the skin by a separate stab wound in the flank. The animals were kept in restraint cages and were allowed only 0.45% NaCI in 0.45% NaHC03 solution to drink during the 65-hr period before sacrifice. Control animals were allowed only tap water to drink and were kept in restraint for the same period of time. Sham operations were performed on some control animals under ether anesthesia and the animals handled. in a similar manner. In several studies only pancreatic flow was diverted by cannulating the bile duct above and below the entrance of the pancreatic duct and returning the flow of bile to the duodenum while draining pancreatic secrl;ltions to the exterior. The adequacy of pancreatic diversion was tested by assaying the proteolytic activity of intestinal contents at the time of sacrifice using a casein substrate (10 ml of a 0.1% buffered casein solution ph 7.6 incubated at 37.5 C for 15 min with 1 to 10 ml of proximal intestinal washings). Acceptable animals had less than 10% of normal proteolytic activity. Sixty-five hours after surgery the animals were stunned by a blow on e hea, the stomach removed, opened, rmsed WIth ice cold saline, and the mucosa of the whole stomach scraped off with a spatula onto a glass plate that was kept on ice. Histological sections revealed that most of the mucosa was stripped by this process, although islands of mucosa were occasionally not removed. The mucosal scrapings were diluted (1: 30, weight to volume) with the modified Kreb's Ringer phosphate buffer used for incubation and homogenized in the cold at a standard speed and time in a Potter Elvejem homogenizer. The resulting homogenate was strained through a double layer of gauze and used for incubations within 15 min of preparation. Homogenates of gastric mucosa for subcellular fractions were prepared as described above but M mannitol in 0.01 M Tris maleate buffer, ph 6.5, was used. The homogenate was centrifuged at 1400 g for 10 min to separate a "nuclear" fraction containing nuclei and unbroken cells. The resulting supernatant was centrifuged at 5900 g for 15 min to separate a "mitochondrial" fraction, and then at 105,000 g for 60 min to separate a "microsomal" and supernatant "cell sap" fraction. The mitochondrial and microsomal fractions were resuspended in mannitol buffer and again centrifuged. The washed pellets of subcellular fractions were then suspended in the incubation medium while the 105,000 g supernatant was used directly. Subcellular fractions and initial homogenate were kept at 4 C until the tissue preparations were complete, when all fractions were incubated simultaneously. The incubation medium consisted of 0.5 to 1.0 ml of homogenate, 20!lIlloles of taurocholate, 120!lIlloles of triglyceride-hc, made up

3 216 CLARK ET AL. Vol. 56, No.2 to 2 ml with a Krebs Ringer phosphate buffer (ph 6.5) modified to contain one-half of the usual concentration of calcium unless otherwise stated. Incubations were carried out in stoppered, center-well 25-ml Erlenmeyer flasks, for 60 min at 37 C in duplicate or triplicate. Duplicate control flasks containing no homogenate were carried through the whole procedure. When 14C02 collections were required, dissolved gas was liberated by the addition of 0.1 ml of 5 N HCI to the outer chamber and trapped in the center well in hyamine hydroxide. The hyamine hydroxide was counted with appropriate internal standards. To study the ph optimum, a 0.12 M phosphate buffer was used for incubation between ph 5.7 and 7.8, 0.10 M glycine buffer for ph 1.8, 0.10 M citrate for ph 3.5, and 0.10 M borate for ph 8.5. The anion concentrations were kept constant for these studies. To study the stability of enzyme action after exposure at low ph, homogenates were first incubated at 37 C in a 0.1 M glycine buffer, ph 3.0. At the end of 30 min, 25 Jlliters of 2 M sodium phosphate buffer were added to half of the incubation flasks to change the ph to 6.5. At this point the HC-Iabeled triglyceride was added and the incubation continued for 1 hr. A parallel set of control preincubations and incubations were made with the ph kept at 6.5 throughout. Studies of gastric hydrolysis of triglycerides in vivo were performed in pyloric ligated rats. Twenty-four hours before the experiments, rats weighing 230 to 270 g were prepared by cannulating the common bile duct close to the duodenum and implanting the distal end of the silicone tubing into the distal ileum by means of a purse-string suture. In this way, bile and pancreatic juice were diverted from the upper intestine while electrolyte balance was adequately maintained. The animals were kept unrestrained without food, but were allowed tap water ad libitum for 24 hr. On the day of the study, the pylorus was tied off and a purse-string suture was made in the fundus of the stomach. The labeled test meal was introduced into the stomach by means of a syringe, the purse-string suture was drawn tight, and the needle was withdrawn. No leakage of the test meal occurred. At the end of the study period, the stomach was tied and removed and the gastric contents were flushed out using 10 ml of chloroform methanol (2: 1). The ph was determined and the washings then extracted and analyzed as described below. A test meal of triolein (approximately 290 /illloies as fatty acid) in 0.4 ml or trioctanoin (830 /illloies as fatty acid) in 0.5 ml of emulsified solution was used. It consisted of: trioctanoin (20 mi) or triolein (25 ml) or octanoic acid (8 mi), lecithin (2.4 g), pluronic F 68 (0.6 g), and dextrose (4.15 g) to 100 ml with water sonicated to form a stable emulsion. Histological examination of the stomach by light microscopy following a 2-hr exposure to.a triolein or trioctanoin test meal revealed normal mucosa and submucosa. Mild inflammatory changes were present on the serosal surface probably secondary to operative handling. Analytical methods. The incubation reaction was stopped by cooling the flasks at -15 C for 1 min when 10 ml of cold chloroform methanol (2: 1) were added. The mixture was transferred to stoppered tubes and acidified, and 210!lmoles of carrier octanoic acid and 24!lmoles of carrier trioctanoin were added. The tubes were shaken for 2 min and the layers were allowed to separate. An aliquot of the methanol layer was kept for counting. The chloroform layer was extracted twice with 0.2 N NaOH saturated with NaC!. The aqueous layer was acidified with HCI and washed three times with a total of 8 ml of hexane, of which a 5-ml aliquot was counted. The chloroform layer was reduced in volume under a stream of nitrogen to about 50!lliters and counted. For some studies an aliquot of the chloroform phase was analyzed by silicic acid thin layer chromatography by methods previously described from this laboratory. b 14C radioactivity counting was performed in 15 ml of Bray solution ' in a scintillation spectrometer at 4 C with appropriate internal standards. The large amounts of carrier trioctanoin and octanoic TABLE 1. Effect of carrier lipid on separation of octanoic acid from trioctanoin during extraction. Amount of carrier lipid added to extraction mixture '?CO:r He tri.- cts in chlor-.octanoln in oform In hexane methanol layer layer layers % % % Trioctanoin, 6 J.lmoles Octanoic acid, 42 J.lmoles Trioctanoin, 24 J.lmoles Octanoic acid, 210 J.lmoles

4 February 1969 GASTRIC LIPOLYSIS AND ABSORPTION OF FAT 217 EFFECT OF ph ON LIPOLYSIS OF TRIOCTANOIN BY GASTRIC MUCOSAL HOMOGENATES Results In vitro studies. In a series of preliminary studies in vitro it was shown that trioctanoin hydrolysis by gastric mucosal homogenates was linear for at least a 60- min incubation period. The amount of octanoic acid liberated during incubation was usually about 20% and in no experiment exceeded 50% of the quantity of trioctanoin initially present, suggesting that there was product inhibition of the enzyme reaction. The amount of gastric homogenate used during subsequent studies was therefore usually kept lower than that which would result in 35% hydrolysis. A broad ph optimum for gastric trioctanoin lipolysis between ph 6.3 and 7.0 was found and activity diminished significantly at higher and lower ph values (fig. TABLE 2. Effect of pancreatic diversion on lipolytic activity of rat gastric homogenates ph FIG. 1. Effect of ph on lipolysis of trioctanoin by gastric mucosal homogenates. Incubations performed in various buffers for 1 hr at 37 C. Mean of two to six determinations. Vertical bars represent SE of the mean. F.A., fatty acid. acid were added as smaller amounts previously used' did not seem to assure adequate extraction of the I C trioctanoin and octanoic acid (table 1). Duplicate control flasks without tissue or with boiled homogenate were carried through each experiment to check reproducibility. Homogenate protein concentration was assayed by the Biuret methods and subcellular fraction protein concentration by the method of Lowry. 9 Calculation of enzyme activity in vitro was' based on the quantity of fatty acid liberated per milligram of protein per hour during incubation. Calculation of hydrolysis of triglyceride in pyloric ligated animals in vivo was made both from amount of triglyceride remaining in gastric contents and amount of fatty acid released assuming that any lipid absorbed from the stomach had undergone prior hydrolysis. 10 Animal No. of Trioctanoina No. of preparation studies studies Triolein mp,mole/ F.A.b/mg/hr mf,lmole/ mg/h, Control ± <5.0 Pancreatic and bile diversion ± <5.0 Pancreatic diversion ± 4.7 a Results ± SE. b F.A., fatty acid. TABLE 3. Distribution of trioctanoin lipolytic activity in subcellular fractions of rat gastric mucosaa Subcellular fraction F.A.b released mf,lmole/ mg/hr Control Animal preparation % Pancreatic and bile diverted mf,lmole/ mg/h, Fraction of total homogenate activity F.A. released Fraction of total homo- genate activity "Nuclear" Mitochondrial Microsomal Supernatant Whole homogenate a All fractions kept at 4 C after preparation and incubated at the same time for 60 min at 37 C. Studies performed in 3 control and 4 diverted animals. Mean of all studies. b F.A., fatty acid. %

5 218 CLARK ET AL. Vol. 56, No.2 1). All subsequent trioctanoin incubations were performed atph 6.5. The hydrolysis of trioctanoin by gastric homogenates of undiverted control rats was at least 30-fold greater than that of triolein which was barely measurable (table 2). Prior diversion of pancreatic secretions reduced the rate of trioctanoin hydrolysis from about 180 to about 50 mj.lmoles fatty acid released per milligram of protein per hour. Diversion of only pancreatic flow and returning bile to the intestinal lumen did not enhance this hydrolytic activity. The striking difference in trioctanoin hydrolysis between pancreatic diverted animals and animals in whom pancreatic secretions were not diverted but whose gastric mucosa was thoroughly washed prior to homogenization suggested the presence of adherent pancreatic lipase and led us to study the distribution of the enzyme activity in subcellular fractions of gastric mucosa in the two groups of animals. It is evident (table 3) that the highest specific activity was found in the microsomal fraction of both groups of animals. In the pancreatic flow-diverted animals, over 90% of the enzyme activity of the whole homogenate was present in the supernatant fraction. In contrast, the enzyme activity was distributed throughout all of the subcellular fractions in undiverted control animals. To compare the capacity of the stomach to hydrolyze trioctanoin with that of other tissues, studies were performed using colonic and jejunal mucosal homogenates in pancreatic diverted animals. The mucosa of the colon hydrolyzed little trioctanoin whereas the jejunal mucosal activity was over lo-fold greater than in the stomach (table 4). No triolein hydrolysis could be detected in the jejunum. In addition, only minimal metabolism of trioctanoin to 14C02 was found in these organs, insufficient to alter the calculation of enzyme hydrolytic activity by the homogenates. In order to study whether the gastric mucosal homogenates could synthesize triglycerides, two incubation studies were performed with 120 mj.lmoles of octanoic acid-i- 14C. No l4c-iabeled glycerides were recovered after I-hr incubation. Sodium taurocholate at concentrations of 2 to 20 mm did not significantly change the rate of hydrolysis of trioctanoin by gastric homogenates. Tween 80 (0.5%) strongly inhibited trioctanoin hydrolysis but this inhibition was overcome by the addition of 20 mm sodium taurocholate to the incubation medium. Since the ph optimum for gastric trioctanoin hydrolytic activity was found to TABLE 4. Triglyceride lipolysis and CO 2 production by gastric, colonic, and j ejunal mucosal homogenatesa Substrate Tissue Trioctanoin Triolein F.A.b released "CO, F.A. "CO, evolved released evolved "'I'mole/mg/hr % F.A. ml'mole/ % F.A. released mg/llr released Stomache ± 13.8 <5.0 <5.0 <5.0 Colone ± 1.4 Jejunumd ± 38.6 < a Figures represent mean of two to six studies on each tissue from pancreatic diverted animals. Results ± SE. b F.A., fatty acid. e 60-min incubation. d 10-min incubation. T ABLE 5. Effect of preincubation at differing ph on ttioctanoin lipolysis of rat gasttic homogenatesa Preincubationb (ph) Incubatione (ph) SE. Fatty acid released ml'mole/ mg/hr ± ± ± 7.6 a Mean of six studies in 3 animals. Results ± b Preincubation without added substrate for 30 min at 37 C. e Incubation with added trioctanoin-1-14c for 60 min at 37 C.

6 February 1969 GASTRIC LIPOLYSIS AND ABSORPTION OF FAT 219 TRIOCTANOIN TRIOLEIN 900 ' ' TG IOd I MIG t:o 6 6 =t <.t) " TIME (minutes) FIG. 2. Trioctanoin and triolein hydrolysis in pyloric ligated rats in vivo. Analysis of lipid recovered from gastric contents at timed intervals. Data are presented as fatty acid equivalents of fat measured, vertical bars represent SD of the mean (thus, 300 /tmoles of trioctanoin are represented by 900 /tmoles as fatty acid equivalent). Measurement of ph performed when gastric contents were analyzed at the various time periods. Groups contained 2 to 9 rats. T.G., triglyceride; F.A., fatty acid, M.G., monoglyceride. be above 6 and the hydrogen ion concentration of gastric juice is high, it was of importance to determine whether the enzyme is stable when exposed to a low ph. Preincubation of gastric homogenates for 30 min at ph 3 and subsequent incubation at the same ph resulted in 75% inhibition in enzyme activity. In contrast, incubation at ph 6.5 after a 30-min exposure at ph 3.0 resulted in no significant reduction in trioctanoin hydrolysis (table 5). In vivo studies. In pyloric ligated animals where pancreatic flow had been previously diverted, the hydrolysis of trioctanoin (approximately 300 /-tmoles == 900 /-tmoles calculated as fatty acid equivalent) and triolein (approximately 100 /-tmoles == 300 /-tmoles calculated as fatty acid equivalent) was studied in vivo for periods of up to 120 min. After an initial delay of 15 min, the recovery of trioctanoin in gastric contents fell steadily, reaching about 210 J.tffioles after 90 min (fig. 2). There was a parallel increase in the recovery of octanoic acid from the stomach. Essentially, no diglycerides or monoglycerides were present. Significant triolein hydrolysis also occurred although at a rate some 8- to lo-fold less than that for trioctanoin. Again the recovery of

7 220 CLARK ET AL. Vol. 56, No.2 monoglyceride was small representing at all times less than 2% of the lipid recovered from the lumen. It should be noted that the final ph of gastric contents was unusually below 3.5 in these studies. The rate of in vivo hydrolysis of trioctanoin was calculated at various time periods following introduction of fat into the pyloric ligated stomach. It 1S ev1-300 =t "- 200 II) i;:! Q: RATE OF GASTRIC LIPOLYSIS OF TRIGLYCERIDE IN VIVO TRIOCTANOIN / TR 10LEIN J TIME (MINUTES) FIG. 3. Calculated rates of gastric lipolysis of trioctanoin and triolein in pyloric l igated rats in vivo at timed intervals following introduction of fat into the stomach. Analyses performed in the same groups of animals shown in figure 2. Rates of enzyme activity calculated per hour (vertical bars represent SD of the mean). TOTAL LIPID RECOVERED... FROM LIGATED STOMACH II) 100 \I) c:. <t l!j TRIOCTANOI N TRIOLEIN \.> 100% M FA 100%' MFA II; TIME (MINUTES) FIG. 4. Gastric recoveries of total lipid at timed intervals following introduction into the stomach. Analyses performed in the same group of animals shown in figure 2. Administered dose of fat = 100%. (Vertical bars represent SD of the mean). dent (fig. ' 3) that hydrolysis of trioctanoin is not detectable for the first 15 min, but that subsequently hydrolytic rate remains constant for 90 min at a rate of about 160 moles per hr. Triolein hydrolytic rate was calculated at less than 20 moles per hr. The total amount of 14C lipid recovered from the stomach in studies in which trioctanoin hydrolysis was being measured appeared to decrease after 60 min of incubation (fig. 4) but this was not statistically significant because of some variation in recoveries between animals. As gastric absorption of octanoic acid might have been expected, a series of experiments was performed in pyloric ligated rats, introducing octanoic acid-ll4c (approximately 300 moles into the stomach). Significant absorption of this fatty acid was found to occur from the gastric lumen at a rate of about 45 oles per hr. Discussion The present studies demonstrate the presence of enzyme(s) in the rat stomach that hydrolyze triglycerides of long and medium chain length with the formation of free fatty acids. The experiments were performed in animals whose pancreatic flow had previously been diverted away from the upper small intestine. This experimental procedure was adopted since contamination of gastric mucosa of washed stomach was likely in undiverted animals for the following reasons: (1) trioctanoin hydrolysis by gastric and intestinal homogenates was 2- to 3-fold greater in control than diverted animals; (2) variable triolein hydrolysis was present in washed jejunal mucosa in control animals but absent in diverted animals; and (3) the presence of large quantities of enzyme activity distributed in many subcellular fractions of gastric mucosa of control animals suggested contamination by extraneous enzyme attached to cell membranes. Similar contamination of washed small intestinal segments by pancreatic enzymes has been suggested previouslylo during studies of intestinal cholesterol esterase activity in rats. It is clear that

8 February 1969 GASTRIC LIPOLYSIS AND ABSORPTION OF FAT 221 many earlier studies of gastric lipases failed to eliminate the possibility of adherent pancreatic enzyme, and that future studies of gastric and intestinal enzymes must take this problem into consideration. Gastric lipase appears to differ from pancreatic lipase in several respects. Significant gastric trioctanoin hydrolysis occurred below ph 3.5 although the ph optimum for enzyme activity was around ph 6.5. In. addition, in vitro studies demonstrated that the enzyme was quite stable at low ph and regained full activity when incubated subsequently at optimal ph. Pancreatic lipase activity, on the other hand, is very low at ph levels below 4 and is quite unstable at acid ph (1). In pyloric ligated rats the ph of gastric contents was usually below 3.5, yet rapid lipolysis of both trioctanoin and triolein did occur. In vivo hydrolysis of long chain triglycerides by pancreatic enzyme in the intestinal lumen is virtually absent when the ph is low. Furthermore, a very recent preliminary report indicates that human gastric and pancreatic lipase can be separated chromatographically.1l The studies of Morgan et al. 12 suggest that two lipolytic enzymes can be separated chromatographically from pancreatic juice that differ principally in their ability to hydrolyze glycerides in emulsified or in micellar (water soluble) form. The present studies have not attempted to define whether gastric hydrolysis of long chain and medium chain triglycerides occurs by identical or differing enzymes. Sodium taurocholate at concentrations of 2 to 20 mm did not alter the rate of trioctanoin hydrolysis by gastric homogenates in vitro but did prevent loss of activity induced by Tween 80. In this respect taurocholate may be protecting the enzyme in a manner similar to the action of the bile salt in preventing proteolytic inactivation of cholesterol ester hydrolase. 13 From the maximal rates of hydrolysis of homogenates measured in vitro it can be calculated that the enzyme present in the whole gastric mucosa should hydrolyze no more than 0.5 mole of trioctanoin per hr, yet rates of 160 moles per hr were measured in vivo. Although it is hazardous to compare in vitro enzyme activity with rates in vivo, the difference was quite remarkable. Furthermore, in all studies of gastric triglyceride lipolysis, fatty acid formation did not begin until about 15 min after introduction of fat into the lumen. Thereafter, lipolysis occurred at a constant rate for at least 90 min. The gastric contents were not washed out prior to the introduction of the fat so that basal lipolytic activity should have been present in the stomach. The results suggest that the presence of fat in the stomach, and perhaps the first traces of fatty acid formed, stimulated synthesis of new enzymes. Alternatively, secretion of an enzyme present in the mucosa in an inactive form may have occurred. The production of active pepsin from pepsinogen is stimulated in a similar fashion by the presence of food in the stomach. 14 Some gastric absorption of octanoic acid is not surprising because at the ph of gastric contents diffusion of this lipid soluble unionized compound should occur. Gastric absorption of acetic acid has been shown to be very rapid. 15 However, since the amount of octanoic acid absorbed from the stomach was only one-thirtieth of the maximal capacity of the small intestine to absorb hydrolyzed trioctanoin 5 this mechanism is unlikely to be clinically useful in the assimilation ofmct. In the presence of adequate amounts of pancreatic lipolytic enzymes, gastric enzymes are probably unnecessary for normal digestion and absorption of fat. However, absorption of long chain and medium chain triglycerides is impaired when insufficient pancreatic enzyme is present in the intestinal lumen and gastric lipolytic enzyme may then have a significant function. Thus in patients with pancreatic insufficiency or gastrojejunostomy, for example, fat digestion may be initiated by the gastric lipase at the ph of gastric contents and the rate of lipolysis may then be greatly increased in the intestinal lumen, where the ph is near optimum for enzyme activity. Medium chain triglycerides have been shown to

9 222 CLARK ET AL. Vol. 56, No.2 be particularly useful in the nutritional management of pancreatic insufficiency and steatorrhea 16 and gastric lipolytic enzymes may be of importance in hydrolyzing these triglycerides III such patients. REFERENCES 1. Mattson, F. H., and R. A. Volpenhein Carboxylic ester hydrolases of rat pancreatic juice. J. Lipid Res. 7: Volhard, F Ober Resorption und Fettspaltung im Magen. Munchen. Med. Wschr. 47: Oppenheimer, C Die Fermente und die Wirkungen, Vol. 1, p Junk, The Hague Sch nheyder, F., and K. Volqvartz The gastric lipase in man. Acta Physiol. Scand. 11: Bennett Clark, S., and P. R. Holt Ratelimiting steps in steady state intestinal absorption of trioctanoin-l-1'c. J. Clin. Invest. 47: Bray, G. A A simple efficient liquid scintillator for counting aqueous solutions in a liquid scintillation counter. Anal. Biochem. 1: Playoust, M. R., and K. J. Isselbacher Studies on the intestinal absorption and intramucosal lipolysis of a medium chain triglyceride. J. Clin. Invest. 43: Layne, E Spectrophotometric and turbidimetric methods for measuring protein. 2. Biuret method, p In S. P. Colowick and N. O. Nathan [eds.), Methods in enzymology, Vol. 3. Academic Press, New York. 9. Lowry, O. H., N. J. Rosebrough, A. L. Farr, and R. J. Randall Protein measurement with the folin phenol reagent. J. Bioi. Chem. 193: Borga, C. R., G. V. Vahouny, and C. R. Treadwell Role of bile salt and pancreatic juice in cholesterol absorption and esterification. Amer. J. Physiol. 206: Cohen, M., R. G. H. Morgan, and A. F. Hofmann Lipolytic activity of human gas Jricjuice. Fed. Proc. 27: Morgan, R. G. H., J. Barrowman, H. Filipek Wender, and B. Borgstrom The lipolytic enzymes of rat pancreatic juice. Biochim. Biophys. Acta 146: Vahouny, G. V., H. Kothari, and C. R. Treadwell Specificity of bile salt protection of cholesterol ester hydrolase from proteolytic inactivation. Arch. Biochem.121: Hirshowitz, B Secretion of pepsinogen, p In C. F. Code [ed.), Handbook on physiology, Section 6: Alimentary canal, Vol. 2. Williams & Wilkins Company, Baltimore. 15. Herting, D. C., N. D. Embree, and P. L. Harris Absorption of acetic acid and glycerol from the rat stomach. Amer. J. Physiol. 186: Holt, P. R Medium-chain triglycerides. A useful adjunct in nutritional therapy. Gastroenterology 53: