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Activities of lipid reesterifying enzymes in jejunal microsomes of bile fistula rats attempts to correlate enzyme activities with microsomal phospholipid content
Biochimica et Biophysics Acta, 326 (1973) 345-354 0 Elsevier Scientific Publishing Company, Amsterdam
- Printed in The Netherlands
BBA 56357
ACTIVITIES OF LIPID REESTERIFYING ENZYMES IN JEJUNAL MICROSOMES OF BILE FISTULA RATS ATTEMPTS TO CORRELATE ENZYME ACTIVITIES WITH MICROSOMAL PHOSPHOLIPID CONTENT
J. B. RODGERS,
R. TANDON
and R. J. O’BRIEN
Department of Medicine, Albany Medical College, Albany, IV.Y. (Received July 26th,
12208
(U.S.A.)
1973)
SUMMARY
I. Microsomes from control and bile fistula rat jejuna were assayed for specific and total activities of acyl-CoA synthetase (acid: CoA ligase (AMP) EC 6.2.1.3) and acyl-CoA : monoglyceride acyltransferase. In addition, measurements of microsomal phospholipid content were performed. In certain groups of rats the classes of microsomal phospholipid were quantitated. 2. Specific and total enzyme activities were found to be reduced in bile fistula rats as previously observed. Enzyme activities were restored to normal levels in bile fistula rats by infusing sodium taurocholate and lecithin at concentrations of 7.5 and 2 mM, respectively. Infusion of 7.5 mM sodium taurocholate alone resulted in some increase in enzyme specific activities toward the normal range. Values for total enzyme activities were actually normal under these conditions. Infusion of greater concentrations of bile salts and/or lecithin was not as effective in maintaining normal enzyme activities. 3. Phospholipid content of microsomes was decreased in bile fistula rats. This change was found to be the result of reduced recoveries of lecithin. Intraduodenal infusion of moderate amounts of sodium taurocholate (7.5 mM) and lecithin (2 mM) restored microsomal phospholipid content to normal. It was also observed, however, that this could be accomplished by infusing 7.5 mM sodium taurocholate without lecithin into bile fistula rats. It is concluded, therefore, that under the conditions of this study the presence of lecithin in the intestinal lumen is not essential for the maintenance of normal amounts of phospholipid of jejunal microsomes. 4. Microsomal enzyme activity was generally normal when microsomal phospholipid content was found to be normal and was abnormal when phospholipid levels were depressed. Statistical analysis showed a correlation existed between enzyme specific activities and phospholipid content. It was not definitely established, however, whether changes in microsomal phospholipid composition directly influenced microsomal enzyme activities or whether changes in enzyme activities and phospholipid _ Abbreviation:
DTNB, 5,5’-dithiobis-Q-nitrobenzoic
acid).
346
J. B. RODGERS et al.
levels merely vary in the same direction in response to changes in the amount of fat being absorbed by the intestine under different experimental conditions.
INTRODUCTION
During the process of lipid absorption by the small bowel absorbed free fatty acids and monoglyceride are reesterified to triglyceride prior to the transport of lipid out of the intestine and into the lymphatic system. Recent studies’ reported from this laboratory demonstrated that diversion of bile away from the intestine in rats produced a significant reduction of activity of the lipid reesterifying enzymes in the intestinal mucosa. These changes, furthermore, were associated with a significant decrease of esterification of fatty acids by everted jejunal sacs from bile fistula rats incubated in a micellar solution of bile salts and oleic acid. Thus, bile has an effect on the intracellular process of lipid reesterification in addition to its well described function in the intestinal lumen where it is required to form mixed micelles for efficient absorption of lipid. Phospholipids are an essential component of microsomes, the subcellular compartment of the mucosa where lipid reesterification takes place2. Bile is rich in lecithin and it therefore seems possible that abnormalities of intracellular function caused by bile diversion might be produced by changes in phospholipid metabolism of the small intestine. Others have demonstrated that lecithin present in the intestinal lumen is hydrolyzed to I-acyl-lysolecithin3 and that after absorption the small intestine can reacylate this material to lecithin 4,J. Thus, it is very likely that bile contributes significantly to the phospholipid pool in the small bowel. If so then bile diversion might be expected to interfere with mucosal phospholipid production which in turn, could adversely alter microsomai function causing reduced enzyme activities. The present studies were done to determine what components in bile are essential for maintaining activities of the mucosal lipid reesterifying enzymes at normal levels. Also phospholipid content of jejunal microsomes was measured to demonstrate whether bile diversion significantly alters phospholipid metabolism of mucosal microsomes and if so, whether this change could be correlated with the abnormalities of mucosal enzyme activities observed -under these conditions. METHODS
Materials
Oleic acid was purchased from Mann Research Laboratories, New York, N.Y. and was stated to be 99 % pure by the supplier. Monoolein was purchased from Horme1 Institute, University of Minnesota, Austin, Minn., and was also stated to be 99 % pure. Palmitoyl-CoA, Grade II, CoA and ATP were obtained from Sigma Corp., St. Louis, MO. Bovine albumin, Fraction V, was purchased from Pentex, Kankakee, Ill. Cleland’s reagent (dithiothreitol) was obtained from Calbiochem, Los Angeles, Calif., and 5,5’-dithiobis-(a-nitrobenzoic acid) (DTNB) from Aldrich Chemical Co., Milwaukee, Wise. Sodium taurocholate was purchased from K and K Laboratories, Inc., Plainview, N.Y. Lecithin, used for preparation of artificial bile in these studies, was plant lecithin, practical grade, purchased from Eastman Kodak, Rochester, N.Y.
JEJUNAL LIPID ENZYMES
347
Gas-liquid chromatography of this commercially available phospholipid showed that it resembled biliary lecithin with regards to its fatty acid composition. Both contain over 50% polyunsaturated long-chain fatty acids with the majority of this type of fatty acid being supplied by linoleic acid. The synthetic liquid diet used for intraduodenal perfusion was an ammo acid diet purchased from Nutritional Biochemicals Co., Cleveland, Ohio. A slurry of this diet was made by adding distilled water to 250 g of diet to make a total of I 1. Lipid in the diet was supplied by corn oil and the diet as finally prepared contained 2.5 g of lipid per IOOml. Preparation of animals Male Sprague-Dawley rats weighing zoo-250 g were used. Animals were operated upon under ether anesthesia. Control animals had a polyethylene tube secured into the first part of the duodenum with the other end brought to the outside through the abdominal incision, as previously described’. Bile iistula rats also had cannulation of the duodenumpZu.s cannulation of the common bile duct as described’. The other ends of both cannulas were brought to the outside,through the abdominal incision. All animals were then placed in restraining cages and 0.9 y0 saline was infused for the first 24 h post-operatively at a rate of 1.2 ml/h via the duodenal cannula by means of a Harvard infusion pump. During the next 48 h various materials were infused at a rate of 2.4 ml/h. Animals were allowed water ad Zibitumwhile in the restraining cages. Two control groups were used, one infused with diet (CI) and one with 0.9 % saline (Cz). Two similar bile fistula groups were included (BFI and BF2). Additionally, four other groups of bile fistula rats were studied. Two of these were infused with diet plus various concentrations of bile salts and lecithin (BF3 infused with diet, 7.5 mM sodium taurocholate, and 2 mM lecithin; BF4 infused with diet, 7.5 mM sodium taurocholate, and 4 mM lecithin). The molar relationships between bile salts and lecithin selected for these studies are fairly similar to that of rat biliary lecithin reported by Balint et al. 6. This is especially the case for the BF3 group. The other two groups of bile fistula rats studied were infused with only diet and bile salts (BF5 infused with diet and 7.5 mM sodium taurocholate; BF6 infused with diet and 20 mM sodium taurocholate). Eight rats were studied in each group. Bile salts and lecithin were added directly to the liquid diets prior to use and the entire mixture was homogenized using a Potter-Elvehjem homogenizer. After completion of the infusion period animals were sacrificed by a blow on the head and the upper half of the small bowel distal to the ligament of Treitz (jejunum) was removed and flushed with ice-cold saline. The mucosa was scraped on a cold glass plate and microsomal fractions, used as the source of enzymes, were prepared as previously described’. Protein determinations on microsomal fractions were performed according to the method of Lowry et al. *. Assays of acyl-CoA synthetase using oleic acid as substrate were performed spectrophotometrically by the hydroxamate-trapping method’ as previously modified”. Assays of acyl-CoA : monoglyceride acyltransferase were performed using pahnitoyl-CoA and monoolein as substrates and measuring the rate of CoA liberated with DTNB by continuous recording spectrophotometry as previously described ‘I. Specific activity of both enzymes was expressed as nmoles of product formed per min per mg microsomal protein. Total enzyme activity was obtained by multiplying specific activity by the amount of microsomal protein recovered per jejunal sample and was expressed as nmoles per min per jejunal sample.
348
J. B. RODGERS et al.
Following the enzyme assays microsomes were extracted for lipid in 20 vol. of chloroform-methanol (2 : I, v/v) as described by Folch et ~1.‘~. After partitioning with 0.9 % saline the chloroform layer was removed, brought to volume and a phosphate determination was performed by the Fiske-SubbaRow method as described by Balint et a1.13. Lipid extracts of microsomes of some groups of rats were further analyzed by thin-layer chromatography as previously described by Balint et aI.l4 to identify and quantitate the classes of microsomal phospholipids. The quantitative results of microsomal phospholipid determinations were expressed per unit of microsomal protein (mg/mg). As amounts of both phospholipid and protein present in microsomes vary under different conditions, it was felt that this ratio would best reflect significant changes of microsomal composition. All mean values of enzyme assays and chemical determinations on microsomes for each group of rats were compared to results obtained from diet-infused controls (0) using Student’s t-test of analysis of independent variables. Results from three groups of rats were further analyzed statistically by obtaining the Pearson productmoment correlation coefficient’ 5 to determine whether values observed for enzyme specific activities correlated with results of the microsomal phospholipid : protein ratios. RESULTS
Values for specific activities of jejunal lipid reesterifying enzyme activities are given in Fig. I. The greatest activities were observed in both control groups (CI and C2). One bile fistula group infused with diet plus moderate concentrations of sodium taurocholate and lecithin (BF3) also had high specific enzyme activities. Values observed in this group were nearly identical to those of the diet-infused controls (CI). In all other bile fistula groups specific enzyme activities were signiticantly reduced compared to the values observed for Cr. This included the BFq group which was
Cl
c2
WI
BF2
BF3
BF4
BF5
BF6
mACYlSc4: MONCGLYCEKEAOLTAANSEFGE mKfL-CoA SYNTHETASE Fig. I. Specific enzyme activity of acyl-CoA: monoglyceride acyltransferase and acyl-CoA synthetase in jejunal microsomes from control and bile fistula rats. The height of the bars represents the mean for each group. The line at the top of the bar indicates f S.E. The number above the bar is the P value comparing the mean of a group with the mean for diet-infiistd controls (Cr). NS, not significant. See Methods for the composition of the perfusate infused into the duodenum in the control (C) and bile fistula (BF) groups for the 48-h period prior to sacrifice.
JEJUNAL LIPID ENZYMES
349
infused with the same materials as the BF3 group but the lecithin concentration in the infusate given to BFq rats was twice as great. Enzyme specific activities were lowest in bile fistula rats infused with saline alone (BF2). Infusing diet alone (BFI) or diet plus bile salts without added lecithin (BFg, BF6) produced some improvement in enzyme specific activities when results were compared to the BF2 group. Values observed in these experimental groups, however, were still significantly less than those determined for diet-infused controls (CI).
Cl
c2
EFI
BF2
EF3
BF4
BF5
BF6
Fig. 2. Total enzyme activity of acyl-CoA:monoglyceride acyltransferase and acyl-CoA synthetase in jejunal microsomes from control and bile fistula rats. The height of the bars represents the mean for each group. The line at the top of the bar indicates f S.E. The number above the bar is the P value comparing the mean of a group with the mean for diet-infused controh (CI). NS, not significant.
Results of total enzymatic activites are found in Fig. 2. Relative results of total activities among the various groups were not quite the same as those observed for specific enzyme activities. As might be expected, values for diet-infused controls (CI) were relatively great and were only exceeded by bile fistula rats infused with diet pZus moderate concentrations of bile salts and lecithin (BF3). The difference of total activities between these two groups was not significant. Lowest values were again noted in bile fistula rats infused with saline alone (BF2). Total enzyme activities for C2, BFI and BFq were also significantly reduced compared to the mean values observed for Cr. Results in these groups, however, were not quite as abnormal as values determined for saline-infused bile fistula rats. In bile fistula rats infused with a mixture of diet and bile salts without lecithin (BFg, BF6) the total enzyme activities were essentially in the normal range. The only significant difference observed between values determined for these two groups and the CI group was that for acyl-CoA: monoglyceride-acyltransferase of rats infused with 20 mM bile salts (BF6). The amounts of jejunal microsomal protein recovered from the BFg and BF6 groups were somewhat greater than that recovered from diet-infused controls. Thus, while specific activities were obviously reduced in these two bile fistula groups, total enzyme activities (spec. act. x microsomal protein) were much closer to the values found in the dietinfused controls. Table I gives the results for the amounts of several phospholipids recovered from jejunal microsomes. The total phospholipid:protein ratio was determined for
3. B. RODGERS et al.
350 TABLE I
PHOSPHOLIPID TO PROTEIN RATIOS OF JEJUNAL MICROSOMES OF CONTROL AND BILE FISTULA RATS Animals studied were the same as indicated in Methods. Chemical determinations done as described in Methods. Results expressed as mean f SE. The P value compares mean value of the group with the mean of the diet-infused controls (CI). NS, not significant.
CI c2 BFI BF2 BF3 BF4 BF5
BF6
16.9 12.6 15.5 12.0
-_t 0.6 & 0.8 & 0.9 f 0.6
19.7
i.
I.0
18.3 k 1.1 19.6 & 1.0 18.5 f 1.0
< 0.001 NS < 0.001 <
0.05
NS < 0.05 NS
0.218 0.201 0.149 0.106
f 0.02 & 0.02 f 0.01 rfi 0.01
0.319
rir: 0.01
0.158 & 0.02 0.195 i: 0.01 0.186 i: 0.02
NS
0.160 & 0.02
-
0.095 f 0.01
<
0.232 f
<
0.05
<
0.001
<
0.005
0.01
0.01
0.059 & 0.007
-
0.054 -f 0.006
NS
0.068 f 0.004
NS
NS NS NS
every group. Results of this ratio were abnorm~ in groups BFI and BFz compared to the CI group. The ratios in bile fistula rats infused with diet and moderate concentrations of bile salts and lecithin (BF3) and in bile fistula rats infused with diet and bile salts alone (BF5, BF6) were within or above the normal range. The ratio was also determined not to be significantly reduced in bile fistula rats infused with 7.5 mM bile salts and 4 mM lecithin (BF4). However, the value for the ratio in this group was closer to that observed for BFI and BF2. Significance from the CI group was not achieved, in part, due to greater variation of the results in the BF4 group. Values for this ratio generally were noted to be greater in groups with high specific enzyme activities. This was especially apparent when relative values of acylCoA synthetase and phospho~pid:protein ratios were reviewed. Values of this enzyme activity and the phospholipid:protein ratio were both greatest in groups CI, C2 and BF3 and were lowest in BF2. The other groups had values for enzyme activities and ratios in between these two extremes. In three groups the classes of microsomal phospholipid were quantitatively determined. Lecithin and phosphatidyl ethanolamine accounted for at least 95% of microsomal phospholipids. In diet-infused controls (CI) and in diet-infused bile fistula rats (BFI) the lecithin fraction of the phospholipids was about 75 and 6d%, respectively. The l~i~n:protein ratio was signifi~ntly lower (P < 0.01) in the BFI group compared to that found in the CI group while this ratio was sibilantly greater (P < 0.005) in the bile fistula group infused with diet and moderate concentrations of bile salts and lecithin (BF3). On the other hand, there were no significant differences in the phosphatidyl ethanolamine:protein ratios between these two bile fistula groups and the controls. These data suggested that less microsomal lecithin had been synthesized by the diet-infused bile fistula rats (BFI) compared to that synthesized by rats in groups CI and BF3. As specific enzyme activities were also lower in BFI rats compared to the other groups it appeared that microsomal lipid reesterifying enzyme activities and microsomal l~~n:protein ratios were both affected adversely by diversion of bile.
JEJUNAL
LIPID ENZYMES
351
It also seemed likely that these two changes were related. This question was further analyzed by statistical methods obtaining the Pearson product-moment correlation coefficient relating enzyme specific activities to three variables; total phospholipid: protein, lecithin: protein, and phosphatidyl ethanolamine: protein ratios. All scores for these three groups were combined for this analysis. As eight rats were studied in each group, 24 pairs of scores were analyzed by this method. Results are shown in Table II. Significant correlations were achieved for each enzyme specific activity and TABLE II PEARSON PRODUCT-MOMENT CORRELATION COEFFICIENT COMPARING SPECIFIC ACTIVITIES TO VARIOUS MICROSOMAL PHOSPHOLIPID:PROTEIN
ENZYME RATIOS
The following groups of rats were studied; diet-infused controls (Cr), diet-infused bile flstula rats (BFI) and bile fistula rats infused with diet plus 7.5 mM sodium taurocholate and z mM lecithin (BF3). All materials were infused intraduodenally at a constant rate according to the conditions described in Methods. Preparation of microsomes, enzyme assays, and chemical determinations as described in Methods. The upper set of figures is the RHO matrix derived from 24 pairs of scores. The lower set of figures is the probability matrix. Values underlined in the probability matrix are those that have achieved statistical significance. I
2 3 4 5
I .oo 0.69 0.59 0.65 0.23
0.65 0.69 0.29
1.00 0.99 0.69
I.00 0.59
I.ocl
I
2
3
4
5
1.00
I 2
0.01
1.00
3
0.01 -
0.01 --
1.00
4
0.01 --0.27
0.01
0.01
1.00
0.17
0.01 --
0.01
I .oo
1
2
3
4
5
5
I, 2, 3, 4, 5,
I.00
specific activity of monoglyceride acyltransferase. specific activity of acyl-CoA synthetase. total phospholipid:protein ratio. lecithin: protein ratio. phosphatidyl ethanolamine:protein ratio.
the total phospholipid : protein and lecithin : protein ratios but not for the phosphatidyl ethanolamine: protein ratio. DISCUSSION
Results of the present studies confirm that diversion of bile decreases the activities of the lipid reesterifying enzymes in the jejunum. This can be best corrected in bile tistula rats by infusing intraduodenally moderate amounts of bile salts and lecithin along with the liquid diet. Thus specific (Fig. I) and total (Fig. 2) enzyme activities were well within the normal range in the group of bile fistula rats that received this mixture (BF3). It will be noted, however, that infusion of diet and moderate concentrations of bile salts without added lecithin (BFg) also maintained the jejunal lipid
352
J. B. RODGERS
et al.
reesterifying capacity within the normal range. While specific enzyme activities (Fig. I) were not as great as in the diet-infused controls (CI), the amounts of microsomal protein recovered from jejunal samples from this experimental group were somewhat greater than those for the CI group resulting in values for total enzyme activities that were not significantly different from those for the diet-infused controls (Fig. 2). When the concentrations of either lecithin or bile salts were increased in the infusate above optimal levels, the results of the jejunal enzyme assays were abnormal (BFq and BF6). This was especially the case when the infusate contained an apparent excess of lecithin (BFq). The reason for this adverse effect of infusing excessive lecithin or sodium taurocholate is not known. It is possible, however, that excessive amounts of lecithin and/or bile salts in the intestinal lumen produce unsaturated mixed micelles resulting in inhibition of lipid absorption. If this explanation were correct, then enzyme activity in the mucosa might be expected to decrease as previous studies’,” suggest these enzymes respond to the substrate load. Rampone16 studying the effects of bile salts and raw bile on fat absorption by everted intestinal sacs from rats noted that addition of raw bile to a micellar solution of free fatty acid and bile salts inhibited uptake of the lipid by these sacs. These observations also suggest that making the luminal environment very favorable for the formation of unsaturated mixed micelles may inhibit lipid absorption. With bile diversion both the mucosal lipid reesterifying enzyme activities and the phospholipid content of mucosal microsomes are reduced. Bile is rich in lecithin. As others have demonstrated that lysolecithin derived from digestion of biliary lecithin in the intestinal lumen can be absorbed and reacylated to lecithin in the mucosa4”, it is likely that biliary lecithin is a major source of the phospholipid required by the intestinal mucosa for normal microsomal structure and function. From the results in groups BFI and BF2 it appeared likely that the adverse effect on intracellular function of the intestine produced by bile diversion was due at least in part, to the absence of biliary lecithin producing abnormalities of the mucosal microsomes. The observations on group BF3 infused with an artificial bile containing 7.5 mM sodium taurocholate and 2 mM lecithin supported this concept as both microsomal structure and function were normal when both bile salts and lecithin were replaced. The studies on bile fistula rats infused with diet and 7.5 mM sodium taurocholate alone (BFg), however, indicate that compensatory mechanisms in the mucosa can meet the requirements for microsomal phospholipid in the absence of biliary lecithin, suggesting that phospholipid in bile is not essential for normal intracellular function of the small bowel. Both the microsomal total phospholipid:protein ratio (Table I) and values for total microsomal enzyme activities were within normal limits for this group of animals, Thus it appears that if adequate concentrations of bile salts are present within the small bowel lumen to form a micellar solution during infusion of diet, the mucosal cells, despite the deficiency of biliary lecithin, are capable of achieving maximal lipid reesterifying capacity for efficient absorption and subsequent transport of the lipid in the diet. In addition to obtaining preliminary information on the source of microsomal phospholipid of the mucosa, studies were also done to demonstrate whether there was any relationship between the amount of phospholipid in mucosal microsomes and the specific activities of the membrane-bound lipid reesterifying enzymes. Previous studies on a variety of enzymes present in hepatic microsomes have indicated by others”-”
JEJUNAL
LIPID ENZYMES
353
that activities of these enzymes were affected by the microsomal phospholipid content. Observations in diet- and saline-infused controls indicate that there is normally a fairly definite amount of phospho~pid present in jejunal ~~osorn~ relative to the amount of microsomal proiein recovered. In these two groups the total phospholipid : protein ratios were very similar (Table I). With infusion of the diet in the Cr group an increased amount of jejunal microsomal protein was observed. The amount of microsomal phospholipid recovered in this group was proportionally increased thus keeping the ratio similar to the ratio for saline-infused controls. In general it was observed that groups of rats with high microsomal total phospholipid:protein ratios also had relatively high specific enzyme activities. Attempts to demonstrate a relationship between microsomal composition and enzyme activity by statistical methods indicated that such a correlation could be described (Table II). Furthermore, when classes of phospholipid were quantitated, the correlation was found to exist for lecithin but not for the phosphatidyl ethanolamine content. This statistical analysis suggests a requirement for lecithin in mucosal microsomes for maintenance of maximal lipid reesterifying enzyme activity. This possibility is far from definitely established, however, as this relationship may be only a reflection of the increased requirements for lecithin by the small bowel for other intracellular processes that are carried out while the intestine is eBciently absorbing lipid such as for lipoprotein production for chylomicron formation. Whether a specific requirement for lecithin or a specific species of lecithin definitely exists for maintaining maximal lipid reesterifying capacity of mucosal microsomes will require further study. The present investigations demonstrate, however, that in response to a lipid load being actively absorbed by the small intestine, the amount of protein and phospholipid present in jejunal microsomes increases. Under normal conditions a major source of this phospholipid is likely to be derived from acylation of absorbed biliary lysolecithin. When biliary lecithin is not present in the intestinal lumen but lipid absorption is proceeding efficiently due to the infusion of adequate amounts of bile salts, the mucosa can apparently maintain normal amounts of microsomal phospholipid by alternate means. ACKNOWLEDGMENTS
The authors wish to express their gratitude to Mrs Suzanne Kokkinis for expert technical assistance. This work was supported by U.S. Public Health Service Grants AMI 1979 and AMr5281. REFERENCES Tandon, R., Edmonds, R. H. and Rodgers, J. B. (Ig72) Gustroenterology 63, ggc-Too3 Senior, J. R. (1964) J. Lipid Res. 5,4g5-521 Borgstrom, 3. (1957) Acta Chem. Sc~nd. II, 749-751 Scow, R. O., Stein, Y. and Stein, 0. (1967) J. Biol. Chem. 242,4grg+g24 Nilsson, A. (1968) Biocidm. Bioplrys. Actu 152, 379-390 Bafint, J. A., Beeler, D. A., Kyriakides, E. C. and Treble, D. i-I. (1971) J. Lab. Clfn. Med. 77, 122133 Rodgers, J. B., Riley, E. M., Drummey,G. D. and Isselbacher,K. J. (1967)Gust*oenterology 53, 547-556
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et al.
8 Lowry, 0. H., Rosebrough, N. J., Farr, A. L. and Randall, R. J. (1951) J. Biol. Chem. 193, 265-275 9 Komberg, A. and Pricer, W. E. (1953) J. Biol. Chem. 204, 329-343 IO Rodgers, J. B., Tandon, R. and Fromm, H. (1972) Biochim. Biophys. Acta 270,453-462 II Rodgers, J. B. (1969) J. LipidRes. IO, 427-432 12 Folch, J., Lees, M. and Sloane-Stanley, G. H. (1957) J. Biol. Chem. 226, 497-509 13 Balint, J. A., Kyriakides, E. C., Spitzer, H. L. and Morrison, E. S. (1965) J. Lipid Res. 6, 96-99 14 Balint, J. A., Beeler, D. A., Treble, D. H. and Spitzer, H. L. (1967) J. Lipid Res. 8, 486-493 15 Bruning, J. L. and Kintz, B. L. (1968) Computational Han&ook of Statistics, pp. 152-155, Scott, Foresman and Co., Glenview, III. 16 Rampone, A. J. (1972) J. Physiol. London 222, 679-690 17 Jones, P. D., Holloway, P. W., Peluffo, R. 0. and Wakil, S. J. (1969) J. Biol. Chem. 244, 744-754 18 Trump, B. F., Duttera, S. M., Byrne, W. L. and Arstila, A. V. (1970) Proc. Natl. Acad. Sci. U.S. 66, 433-440 19 Zakim, D. (1970) J. Biol. Chem. 245,4953-4961 20 Vessey, D. A. and Zakim, D. (1971) J. Biol. Chem. 246, 4649-4656 21 Rogers, M. J. and Strittmatter, P. (1973) J. Biol. Chem. 248, 800-806
- Printed in The Netherlands
BBA 56357
ACTIVITIES OF LIPID REESTERIFYING ENZYMES IN JEJUNAL MICROSOMES OF BILE FISTULA RATS ATTEMPTS TO CORRELATE ENZYME ACTIVITIES WITH MICROSOMAL PHOSPHOLIPID CONTENT
J. B. RODGERS,
R. TANDON
and R. J. O’BRIEN
Department of Medicine, Albany Medical College, Albany, IV.Y. (Received July 26th,
12208
(U.S.A.)
1973)
SUMMARY
I. Microsomes from control and bile fistula rat jejuna were assayed for specific and total activities of acyl-CoA synthetase (acid: CoA ligase (AMP) EC 6.2.1.3) and acyl-CoA : monoglyceride acyltransferase. In addition, measurements of microsomal phospholipid content were performed. In certain groups of rats the classes of microsomal phospholipid were quantitated. 2. Specific and total enzyme activities were found to be reduced in bile fistula rats as previously observed. Enzyme activities were restored to normal levels in bile fistula rats by infusing sodium taurocholate and lecithin at concentrations of 7.5 and 2 mM, respectively. Infusion of 7.5 mM sodium taurocholate alone resulted in some increase in enzyme specific activities toward the normal range. Values for total enzyme activities were actually normal under these conditions. Infusion of greater concentrations of bile salts and/or lecithin was not as effective in maintaining normal enzyme activities. 3. Phospholipid content of microsomes was decreased in bile fistula rats. This change was found to be the result of reduced recoveries of lecithin. Intraduodenal infusion of moderate amounts of sodium taurocholate (7.5 mM) and lecithin (2 mM) restored microsomal phospholipid content to normal. It was also observed, however, that this could be accomplished by infusing 7.5 mM sodium taurocholate without lecithin into bile fistula rats. It is concluded, therefore, that under the conditions of this study the presence of lecithin in the intestinal lumen is not essential for the maintenance of normal amounts of phospholipid of jejunal microsomes. 4. Microsomal enzyme activity was generally normal when microsomal phospholipid content was found to be normal and was abnormal when phospholipid levels were depressed. Statistical analysis showed a correlation existed between enzyme specific activities and phospholipid content. It was not definitely established, however, whether changes in microsomal phospholipid composition directly influenced microsomal enzyme activities or whether changes in enzyme activities and phospholipid _ Abbreviation:
DTNB, 5,5’-dithiobis-Q-nitrobenzoic
acid).
346
J. B. RODGERS et al.
levels merely vary in the same direction in response to changes in the amount of fat being absorbed by the intestine under different experimental conditions.
INTRODUCTION
During the process of lipid absorption by the small bowel absorbed free fatty acids and monoglyceride are reesterified to triglyceride prior to the transport of lipid out of the intestine and into the lymphatic system. Recent studies’ reported from this laboratory demonstrated that diversion of bile away from the intestine in rats produced a significant reduction of activity of the lipid reesterifying enzymes in the intestinal mucosa. These changes, furthermore, were associated with a significant decrease of esterification of fatty acids by everted jejunal sacs from bile fistula rats incubated in a micellar solution of bile salts and oleic acid. Thus, bile has an effect on the intracellular process of lipid reesterification in addition to its well described function in the intestinal lumen where it is required to form mixed micelles for efficient absorption of lipid. Phospholipids are an essential component of microsomes, the subcellular compartment of the mucosa where lipid reesterification takes place2. Bile is rich in lecithin and it therefore seems possible that abnormalities of intracellular function caused by bile diversion might be produced by changes in phospholipid metabolism of the small intestine. Others have demonstrated that lecithin present in the intestinal lumen is hydrolyzed to I-acyl-lysolecithin3 and that after absorption the small intestine can reacylate this material to lecithin 4,J. Thus, it is very likely that bile contributes significantly to the phospholipid pool in the small bowel. If so then bile diversion might be expected to interfere with mucosal phospholipid production which in turn, could adversely alter microsomai function causing reduced enzyme activities. The present studies were done to determine what components in bile are essential for maintaining activities of the mucosal lipid reesterifying enzymes at normal levels. Also phospholipid content of jejunal microsomes was measured to demonstrate whether bile diversion significantly alters phospholipid metabolism of mucosal microsomes and if so, whether this change could be correlated with the abnormalities of mucosal enzyme activities observed -under these conditions. METHODS
Materials
Oleic acid was purchased from Mann Research Laboratories, New York, N.Y. and was stated to be 99 % pure by the supplier. Monoolein was purchased from Horme1 Institute, University of Minnesota, Austin, Minn., and was also stated to be 99 % pure. Palmitoyl-CoA, Grade II, CoA and ATP were obtained from Sigma Corp., St. Louis, MO. Bovine albumin, Fraction V, was purchased from Pentex, Kankakee, Ill. Cleland’s reagent (dithiothreitol) was obtained from Calbiochem, Los Angeles, Calif., and 5,5’-dithiobis-(a-nitrobenzoic acid) (DTNB) from Aldrich Chemical Co., Milwaukee, Wise. Sodium taurocholate was purchased from K and K Laboratories, Inc., Plainview, N.Y. Lecithin, used for preparation of artificial bile in these studies, was plant lecithin, practical grade, purchased from Eastman Kodak, Rochester, N.Y.
JEJUNAL LIPID ENZYMES
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Gas-liquid chromatography of this commercially available phospholipid showed that it resembled biliary lecithin with regards to its fatty acid composition. Both contain over 50% polyunsaturated long-chain fatty acids with the majority of this type of fatty acid being supplied by linoleic acid. The synthetic liquid diet used for intraduodenal perfusion was an ammo acid diet purchased from Nutritional Biochemicals Co., Cleveland, Ohio. A slurry of this diet was made by adding distilled water to 250 g of diet to make a total of I 1. Lipid in the diet was supplied by corn oil and the diet as finally prepared contained 2.5 g of lipid per IOOml. Preparation of animals Male Sprague-Dawley rats weighing zoo-250 g were used. Animals were operated upon under ether anesthesia. Control animals had a polyethylene tube secured into the first part of the duodenum with the other end brought to the outside through the abdominal incision, as previously described’. Bile iistula rats also had cannulation of the duodenumpZu.s cannulation of the common bile duct as described’. The other ends of both cannulas were brought to the outside,through the abdominal incision. All animals were then placed in restraining cages and 0.9 y0 saline was infused for the first 24 h post-operatively at a rate of 1.2 ml/h via the duodenal cannula by means of a Harvard infusion pump. During the next 48 h various materials were infused at a rate of 2.4 ml/h. Animals were allowed water ad Zibitumwhile in the restraining cages. Two control groups were used, one infused with diet (CI) and one with 0.9 % saline (Cz). Two similar bile fistula groups were included (BFI and BF2). Additionally, four other groups of bile fistula rats were studied. Two of these were infused with diet plus various concentrations of bile salts and lecithin (BF3 infused with diet, 7.5 mM sodium taurocholate, and 2 mM lecithin; BF4 infused with diet, 7.5 mM sodium taurocholate, and 4 mM lecithin). The molar relationships between bile salts and lecithin selected for these studies are fairly similar to that of rat biliary lecithin reported by Balint et al. 6. This is especially the case for the BF3 group. The other two groups of bile fistula rats studied were infused with only diet and bile salts (BF5 infused with diet and 7.5 mM sodium taurocholate; BF6 infused with diet and 20 mM sodium taurocholate). Eight rats were studied in each group. Bile salts and lecithin were added directly to the liquid diets prior to use and the entire mixture was homogenized using a Potter-Elvehjem homogenizer. After completion of the infusion period animals were sacrificed by a blow on the head and the upper half of the small bowel distal to the ligament of Treitz (jejunum) was removed and flushed with ice-cold saline. The mucosa was scraped on a cold glass plate and microsomal fractions, used as the source of enzymes, were prepared as previously described’. Protein determinations on microsomal fractions were performed according to the method of Lowry et al. *. Assays of acyl-CoA synthetase using oleic acid as substrate were performed spectrophotometrically by the hydroxamate-trapping method’ as previously modified”. Assays of acyl-CoA : monoglyceride acyltransferase were performed using pahnitoyl-CoA and monoolein as substrates and measuring the rate of CoA liberated with DTNB by continuous recording spectrophotometry as previously described ‘I. Specific activity of both enzymes was expressed as nmoles of product formed per min per mg microsomal protein. Total enzyme activity was obtained by multiplying specific activity by the amount of microsomal protein recovered per jejunal sample and was expressed as nmoles per min per jejunal sample.
348
J. B. RODGERS et al.
Following the enzyme assays microsomes were extracted for lipid in 20 vol. of chloroform-methanol (2 : I, v/v) as described by Folch et ~1.‘~. After partitioning with 0.9 % saline the chloroform layer was removed, brought to volume and a phosphate determination was performed by the Fiske-SubbaRow method as described by Balint et a1.13. Lipid extracts of microsomes of some groups of rats were further analyzed by thin-layer chromatography as previously described by Balint et aI.l4 to identify and quantitate the classes of microsomal phospholipids. The quantitative results of microsomal phospholipid determinations were expressed per unit of microsomal protein (mg/mg). As amounts of both phospholipid and protein present in microsomes vary under different conditions, it was felt that this ratio would best reflect significant changes of microsomal composition. All mean values of enzyme assays and chemical determinations on microsomes for each group of rats were compared to results obtained from diet-infused controls (0) using Student’s t-test of analysis of independent variables. Results from three groups of rats were further analyzed statistically by obtaining the Pearson productmoment correlation coefficient’ 5 to determine whether values observed for enzyme specific activities correlated with results of the microsomal phospholipid : protein ratios. RESULTS
Values for specific activities of jejunal lipid reesterifying enzyme activities are given in Fig. I. The greatest activities were observed in both control groups (CI and C2). One bile fistula group infused with diet plus moderate concentrations of sodium taurocholate and lecithin (BF3) also had high specific enzyme activities. Values observed in this group were nearly identical to those of the diet-infused controls (CI). In all other bile fistula groups specific enzyme activities were signiticantly reduced compared to the values observed for Cr. This included the BFq group which was
Cl
c2
WI
BF2
BF3
BF4
BF5
BF6
mACYlSc4: MONCGLYCEKEAOLTAANSEFGE mKfL-CoA SYNTHETASE Fig. I. Specific enzyme activity of acyl-CoA: monoglyceride acyltransferase and acyl-CoA synthetase in jejunal microsomes from control and bile fistula rats. The height of the bars represents the mean for each group. The line at the top of the bar indicates f S.E. The number above the bar is the P value comparing the mean of a group with the mean for diet-infiistd controls (Cr). NS, not significant. See Methods for the composition of the perfusate infused into the duodenum in the control (C) and bile fistula (BF) groups for the 48-h period prior to sacrifice.
JEJUNAL LIPID ENZYMES
349
infused with the same materials as the BF3 group but the lecithin concentration in the infusate given to BFq rats was twice as great. Enzyme specific activities were lowest in bile fistula rats infused with saline alone (BF2). Infusing diet alone (BFI) or diet plus bile salts without added lecithin (BFg, BF6) produced some improvement in enzyme specific activities when results were compared to the BF2 group. Values observed in these experimental groups, however, were still significantly less than those determined for diet-infused controls (CI).
Cl
c2
EFI
BF2
EF3
BF4
BF5
BF6
Fig. 2. Total enzyme activity of acyl-CoA:monoglyceride acyltransferase and acyl-CoA synthetase in jejunal microsomes from control and bile fistula rats. The height of the bars represents the mean for each group. The line at the top of the bar indicates f S.E. The number above the bar is the P value comparing the mean of a group with the mean for diet-infused controh (CI). NS, not significant.
Results of total enzymatic activites are found in Fig. 2. Relative results of total activities among the various groups were not quite the same as those observed for specific enzyme activities. As might be expected, values for diet-infused controls (CI) were relatively great and were only exceeded by bile fistula rats infused with diet pZus moderate concentrations of bile salts and lecithin (BF3). The difference of total activities between these two groups was not significant. Lowest values were again noted in bile fistula rats infused with saline alone (BF2). Total enzyme activities for C2, BFI and BFq were also significantly reduced compared to the mean values observed for Cr. Results in these groups, however, were not quite as abnormal as values determined for saline-infused bile fistula rats. In bile fistula rats infused with a mixture of diet and bile salts without lecithin (BFg, BF6) the total enzyme activities were essentially in the normal range. The only significant difference observed between values determined for these two groups and the CI group was that for acyl-CoA: monoglyceride-acyltransferase of rats infused with 20 mM bile salts (BF6). The amounts of jejunal microsomal protein recovered from the BFg and BF6 groups were somewhat greater than that recovered from diet-infused controls. Thus, while specific activities were obviously reduced in these two bile fistula groups, total enzyme activities (spec. act. x microsomal protein) were much closer to the values found in the dietinfused controls. Table I gives the results for the amounts of several phospholipids recovered from jejunal microsomes. The total phospholipid:protein ratio was determined for
3. B. RODGERS et al.
350 TABLE I
PHOSPHOLIPID TO PROTEIN RATIOS OF JEJUNAL MICROSOMES OF CONTROL AND BILE FISTULA RATS Animals studied were the same as indicated in Methods. Chemical determinations done as described in Methods. Results expressed as mean f SE. The P value compares mean value of the group with the mean of the diet-infused controls (CI). NS, not significant.
CI c2 BFI BF2 BF3 BF4 BF5
BF6
16.9 12.6 15.5 12.0
-_t 0.6 & 0.8 & 0.9 f 0.6
19.7
i.
I.0
18.3 k 1.1 19.6 & 1.0 18.5 f 1.0
< 0.001 NS < 0.001 <
0.05
NS < 0.05 NS
0.218 0.201 0.149 0.106
f 0.02 & 0.02 f 0.01 rfi 0.01
0.319
rir: 0.01
0.158 & 0.02 0.195 i: 0.01 0.186 i: 0.02
NS
0.160 & 0.02
-
0.095 f 0.01
<
0.232 f
<
0.05
<
0.001
<
0.005
0.01
0.01
0.059 & 0.007
-
0.054 -f 0.006
NS
0.068 f 0.004
NS
NS NS NS
every group. Results of this ratio were abnorm~ in groups BFI and BFz compared to the CI group. The ratios in bile fistula rats infused with diet and moderate concentrations of bile salts and lecithin (BF3) and in bile fistula rats infused with diet and bile salts alone (BF5, BF6) were within or above the normal range. The ratio was also determined not to be significantly reduced in bile fistula rats infused with 7.5 mM bile salts and 4 mM lecithin (BF4). However, the value for the ratio in this group was closer to that observed for BFI and BF2. Significance from the CI group was not achieved, in part, due to greater variation of the results in the BF4 group. Values for this ratio generally were noted to be greater in groups with high specific enzyme activities. This was especially apparent when relative values of acylCoA synthetase and phospho~pid:protein ratios were reviewed. Values of this enzyme activity and the phospholipid:protein ratio were both greatest in groups CI, C2 and BF3 and were lowest in BF2. The other groups had values for enzyme activities and ratios in between these two extremes. In three groups the classes of microsomal phospholipid were quantitatively determined. Lecithin and phosphatidyl ethanolamine accounted for at least 95% of microsomal phospholipids. In diet-infused controls (CI) and in diet-infused bile fistula rats (BFI) the lecithin fraction of the phospholipids was about 75 and 6d%, respectively. The l~i~n:protein ratio was signifi~ntly lower (P < 0.01) in the BFI group compared to that found in the CI group while this ratio was sibilantly greater (P < 0.005) in the bile fistula group infused with diet and moderate concentrations of bile salts and lecithin (BF3). On the other hand, there were no significant differences in the phosphatidyl ethanolamine:protein ratios between these two bile fistula groups and the controls. These data suggested that less microsomal lecithin had been synthesized by the diet-infused bile fistula rats (BFI) compared to that synthesized by rats in groups CI and BF3. As specific enzyme activities were also lower in BFI rats compared to the other groups it appeared that microsomal lipid reesterifying enzyme activities and microsomal l~~n:protein ratios were both affected adversely by diversion of bile.
JEJUNAL
LIPID ENZYMES
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It also seemed likely that these two changes were related. This question was further analyzed by statistical methods obtaining the Pearson product-moment correlation coefficient relating enzyme specific activities to three variables; total phospholipid: protein, lecithin: protein, and phosphatidyl ethanolamine: protein ratios. All scores for these three groups were combined for this analysis. As eight rats were studied in each group, 24 pairs of scores were analyzed by this method. Results are shown in Table II. Significant correlations were achieved for each enzyme specific activity and TABLE II PEARSON PRODUCT-MOMENT CORRELATION COEFFICIENT COMPARING SPECIFIC ACTIVITIES TO VARIOUS MICROSOMAL PHOSPHOLIPID:PROTEIN
ENZYME RATIOS
The following groups of rats were studied; diet-infused controls (Cr), diet-infused bile flstula rats (BFI) and bile fistula rats infused with diet plus 7.5 mM sodium taurocholate and z mM lecithin (BF3). All materials were infused intraduodenally at a constant rate according to the conditions described in Methods. Preparation of microsomes, enzyme assays, and chemical determinations as described in Methods. The upper set of figures is the RHO matrix derived from 24 pairs of scores. The lower set of figures is the probability matrix. Values underlined in the probability matrix are those that have achieved statistical significance. I
2 3 4 5
I .oo 0.69 0.59 0.65 0.23
0.65 0.69 0.29
1.00 0.99 0.69
I.00 0.59
I.ocl
I
2
3
4
5
1.00
I 2
0.01
1.00
3
0.01 -
0.01 --
1.00
4
0.01 --0.27
0.01
0.01
1.00
0.17
0.01 --
0.01
I .oo
1
2
3
4
5
5
I, 2, 3, 4, 5,
I.00
specific activity of monoglyceride acyltransferase. specific activity of acyl-CoA synthetase. total phospholipid:protein ratio. lecithin: protein ratio. phosphatidyl ethanolamine:protein ratio.
the total phospholipid : protein and lecithin : protein ratios but not for the phosphatidyl ethanolamine: protein ratio. DISCUSSION
Results of the present studies confirm that diversion of bile decreases the activities of the lipid reesterifying enzymes in the jejunum. This can be best corrected in bile tistula rats by infusing intraduodenally moderate amounts of bile salts and lecithin along with the liquid diet. Thus specific (Fig. I) and total (Fig. 2) enzyme activities were well within the normal range in the group of bile fistula rats that received this mixture (BF3). It will be noted, however, that infusion of diet and moderate concentrations of bile salts without added lecithin (BFg) also maintained the jejunal lipid
352
J. B. RODGERS
et al.
reesterifying capacity within the normal range. While specific enzyme activities (Fig. I) were not as great as in the diet-infused controls (CI), the amounts of microsomal protein recovered from jejunal samples from this experimental group were somewhat greater than those for the CI group resulting in values for total enzyme activities that were not significantly different from those for the diet-infused controls (Fig. 2). When the concentrations of either lecithin or bile salts were increased in the infusate above optimal levels, the results of the jejunal enzyme assays were abnormal (BFq and BF6). This was especially the case when the infusate contained an apparent excess of lecithin (BFq). The reason for this adverse effect of infusing excessive lecithin or sodium taurocholate is not known. It is possible, however, that excessive amounts of lecithin and/or bile salts in the intestinal lumen produce unsaturated mixed micelles resulting in inhibition of lipid absorption. If this explanation were correct, then enzyme activity in the mucosa might be expected to decrease as previous studies’,” suggest these enzymes respond to the substrate load. Rampone16 studying the effects of bile salts and raw bile on fat absorption by everted intestinal sacs from rats noted that addition of raw bile to a micellar solution of free fatty acid and bile salts inhibited uptake of the lipid by these sacs. These observations also suggest that making the luminal environment very favorable for the formation of unsaturated mixed micelles may inhibit lipid absorption. With bile diversion both the mucosal lipid reesterifying enzyme activities and the phospholipid content of mucosal microsomes are reduced. Bile is rich in lecithin. As others have demonstrated that lysolecithin derived from digestion of biliary lecithin in the intestinal lumen can be absorbed and reacylated to lecithin in the mucosa4”, it is likely that biliary lecithin is a major source of the phospholipid required by the intestinal mucosa for normal microsomal structure and function. From the results in groups BFI and BF2 it appeared likely that the adverse effect on intracellular function of the intestine produced by bile diversion was due at least in part, to the absence of biliary lecithin producing abnormalities of the mucosal microsomes. The observations on group BF3 infused with an artificial bile containing 7.5 mM sodium taurocholate and 2 mM lecithin supported this concept as both microsomal structure and function were normal when both bile salts and lecithin were replaced. The studies on bile fistula rats infused with diet and 7.5 mM sodium taurocholate alone (BFg), however, indicate that compensatory mechanisms in the mucosa can meet the requirements for microsomal phospholipid in the absence of biliary lecithin, suggesting that phospholipid in bile is not essential for normal intracellular function of the small bowel. Both the microsomal total phospholipid:protein ratio (Table I) and values for total microsomal enzyme activities were within normal limits for this group of animals, Thus it appears that if adequate concentrations of bile salts are present within the small bowel lumen to form a micellar solution during infusion of diet, the mucosal cells, despite the deficiency of biliary lecithin, are capable of achieving maximal lipid reesterifying capacity for efficient absorption and subsequent transport of the lipid in the diet. In addition to obtaining preliminary information on the source of microsomal phospholipid of the mucosa, studies were also done to demonstrate whether there was any relationship between the amount of phospholipid in mucosal microsomes and the specific activities of the membrane-bound lipid reesterifying enzymes. Previous studies on a variety of enzymes present in hepatic microsomes have indicated by others”-”
JEJUNAL
LIPID ENZYMES
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that activities of these enzymes were affected by the microsomal phospholipid content. Observations in diet- and saline-infused controls indicate that there is normally a fairly definite amount of phospho~pid present in jejunal ~~osorn~ relative to the amount of microsomal proiein recovered. In these two groups the total phospholipid : protein ratios were very similar (Table I). With infusion of the diet in the Cr group an increased amount of jejunal microsomal protein was observed. The amount of microsomal phospholipid recovered in this group was proportionally increased thus keeping the ratio similar to the ratio for saline-infused controls. In general it was observed that groups of rats with high microsomal total phospholipid:protein ratios also had relatively high specific enzyme activities. Attempts to demonstrate a relationship between microsomal composition and enzyme activity by statistical methods indicated that such a correlation could be described (Table II). Furthermore, when classes of phospholipid were quantitated, the correlation was found to exist for lecithin but not for the phosphatidyl ethanolamine content. This statistical analysis suggests a requirement for lecithin in mucosal microsomes for maintenance of maximal lipid reesterifying enzyme activity. This possibility is far from definitely established, however, as this relationship may be only a reflection of the increased requirements for lecithin by the small bowel for other intracellular processes that are carried out while the intestine is eBciently absorbing lipid such as for lipoprotein production for chylomicron formation. Whether a specific requirement for lecithin or a specific species of lecithin definitely exists for maintaining maximal lipid reesterifying capacity of mucosal microsomes will require further study. The present investigations demonstrate, however, that in response to a lipid load being actively absorbed by the small intestine, the amount of protein and phospholipid present in jejunal microsomes increases. Under normal conditions a major source of this phospholipid is likely to be derived from acylation of absorbed biliary lysolecithin. When biliary lecithin is not present in the intestinal lumen but lipid absorption is proceeding efficiently due to the infusion of adequate amounts of bile salts, the mucosa can apparently maintain normal amounts of microsomal phospholipid by alternate means. ACKNOWLEDGMENTS
The authors wish to express their gratitude to Mrs Suzanne Kokkinis for expert technical assistance. This work was supported by U.S. Public Health Service Grants AMI 1979 and AMr5281. REFERENCES Tandon, R., Edmonds, R. H. and Rodgers, J. B. (Ig72) Gustroenterology 63, ggc-Too3 Senior, J. R. (1964) J. Lipid Res. 5,4g5-521 Borgstrom, 3. (1957) Acta Chem. Sc~nd. II, 749-751 Scow, R. O., Stein, Y. and Stein, 0. (1967) J. Biol. Chem. 242,4grg+g24 Nilsson, A. (1968) Biocidm. Bioplrys. Actu 152, 379-390 Bafint, J. A., Beeler, D. A., Kyriakides, E. C. and Treble, D. i-I. (1971) J. Lab. Clfn. Med. 77, 122133 Rodgers, J. B., Riley, E. M., Drummey,G. D. and Isselbacher,K. J. (1967)Gust*oenterology 53, 547-556
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et al.
8 Lowry, 0. H., Rosebrough, N. J., Farr, A. L. and Randall, R. J. (1951) J. Biol. Chem. 193, 265-275 9 Komberg, A. and Pricer, W. E. (1953) J. Biol. Chem. 204, 329-343 IO Rodgers, J. B., Tandon, R. and Fromm, H. (1972) Biochim. Biophys. Acta 270,453-462 II Rodgers, J. B. (1969) J. LipidRes. IO, 427-432 12 Folch, J., Lees, M. and Sloane-Stanley, G. H. (1957) J. Biol. Chem. 226, 497-509 13 Balint, J. A., Kyriakides, E. C., Spitzer, H. L. and Morrison, E. S. (1965) J. Lipid Res. 6, 96-99 14 Balint, J. A., Beeler, D. A., Treble, D. H. and Spitzer, H. L. (1967) J. Lipid Res. 8, 486-493 15 Bruning, J. L. and Kintz, B. L. (1968) Computational Han&ook of Statistics, pp. 152-155, Scott, Foresman and Co., Glenview, III. 16 Rampone, A. J. (1972) J. Physiol. London 222, 679-690 17 Jones, P. D., Holloway, P. W., Peluffo, R. 0. and Wakil, S. J. (1969) J. Biol. Chem. 244, 744-754 18 Trump, B. F., Duttera, S. M., Byrne, W. L. and Arstila, A. V. (1970) Proc. Natl. Acad. Sci. U.S. 66, 433-440 19 Zakim, D. (1970) J. Biol. Chem. 245,4953-4961 20 Vessey, D. A. and Zakim, D. (1971) J. Biol. Chem. 246, 4649-4656 21 Rogers, M. J. and Strittmatter, P. (1973) J. Biol. Chem. 248, 800-806