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Bile acids and lipid metabolism
ARCHIVES
OF
BIOCHEMISTRY
AND
BIOPHYSICS
Bile Acid’s II. Essential CECIL
for
EKTENMAl?,-,‘j
Lipid
and
Lipid
Metabolism
Role of Bile Acids in Bile Phospholipid
AND
The Znstitute
253-256 (1969)
130,
* ROY J. HOLLOWAY, GEORGE F. LEONG
Excretion
M. I,. ALBRIGHT,
Research, Berkeley, California 94702, and the U.S. Naval Laboratory, San Francisco 94124, California Received
September
30, 19G8; accepted
November
&adiological
Defense
26, 1968
The isolated rat liver perfusion system has been used to study the release of bile phospholipid P32 following the injection of inorganic P3* into the perfusate. It was demonsrtated that in the absence of added bile salt very little PLP3* was excreted into bile, but when bile salt was added to the perfusate the PLP3* excretion increased markedly. At the start of bile salt infusion, the specific activity of the bile phospholipids was lower than that of the liver, but at later times the specific activity of bile phospholipids was greater than liver PL specific activity. It is suggested that bile acids play an essential role in the transport of phospholipid from liver to bile.
Previous studies using the isolated perfused rat liver (1, 2) have indicated that bile acids may play an important role in the excretion of bile lipids, particularly phospholipids (PL) and free cholesterol (FC). Biliary levels of both FC and PL were markedly increased following addition of bile salts to the perfusate. It had also been observed by one of US (G.F.L.) that essentially no Ps2activity was found in bile following injection of inorganic P3* into the rat liver perfusion system unless bile salt was also added to the perfusate. Since nearly all of the P32 activity in bile after addition of Pa and bile salt to the perfusion system was insoluble in trichloroacetic acid, it seemed probable that the P3* activity in bile was present as phospholipid P3* V’LW and that transport of PLP32 into bile, therefore, required the presence of added bile salts. Consequently, experiments designed to test this possibility were carried out. The results obtained strongly suggest that bile salts are 1 Institute for Lipid Research, 2127 Bonar St., Berkeley, California 94702. 2 Consultant to the Biological and Medical Sciences Division, U.S.N.R.D.L. 253
obligatory in the transport of most, if not all, of the phospholipids from liver into bile. METHODS Liver perfusion techniques. The apparatus and general techniques used for rat liver perfusions were those described by Brauer et al. (3). Livers were taken from ether-anesthetized donor-fed male Sprague-Dawley rats weighing 300-350 g. The time from the tying of the portal vein to reestablishment of circulation in the perfusion systern never exceeded 4 min. The perfusion medium was approximately 130 ml of whole rat blood containing about 1 mg heparin. An initial perfusion period of 60-90 min were used to establish good blood and bile flow rates and to eliminate the vasoconstrictor factor present in fresh rat blood (4). Perfusions were carried out in a chamber maintained at 37” and a relative humidity of 50-60s. Blood flow rates ranged from 1.27 to 2.40 ml/min/g liver after the initial equilibration period. Bile flow rates, usually about 1 ml/hr at the end of the equilibration period usually fell very gradually throughout the remainder of the perfusion period, but sometimes fell to values as low as 0.5 ml/hr. Materials. Two bile salts were used: Sodium cholate, special enzyme grade (Mann Research Laboratories) and chenodeoxycholic acid, Grade A (Calbiochem). Sodium cholate was dissolved
254
ENTENMAN
INFLUENCE
ET AL.
TABLE I OF TAUROCHOLATE INFUSION ON EXCR~JTION OF PHOSPHOLIPID Pa2 BY THE ISOLATED PKRFUSF,D R.~T LIVER
-
Exp. no.
Total perfusion time bin)
Time after Pa2 injection (min)
CPM PLPa2in
‘e&d of bile salt infusiona (min)
PlasmaC
a Sodium b Counts c Counts
90 135 175 205 235 265
0 45 85 115 145 175
125 155 185 215 245 275 305
0 30 60 90 120 150 180
100 145 190 220 260
0 45 90 120 160
0 30 60 90
413 586 9,712 23,140 29,190
16 76 106 187 310 403
50 255 330 4,978 14,045 17,923
194 269 426 1,170
349 823 34,154 57,838
50,175
0 30 60 90
53,960
0
21,200
5,800
3o 70
1
Taurocholate was infused into the perfusate per minute per gram of liver. per minute per milliliter of plasma or bile.
in isotonic saline and chenodeoxycholic acid in 20 mM sodium bicarbonate. The carrier-free NaHzP320a used was obtained from Volk Radiochemical Co. Experiments 1-S. In these experiments three rat livers were perfused as described above for periods (90-125 min) sufficient to reach equilibration and to eliminate from the perfusion system essentially all of the preformed bile acids. At that time approximately 250 &i of carrier-free NaH2. P3204 was injected into the blood reservoir and rapidly mixed by swirling. This time is designated as “zero” time for the P32 injection in Table I. Perfusion was continued for an additional period (85-90 min) to permit synthesis of P3* labeled phospholipids by the liver and collection of plasma and bile samples in the absence of added bile salt. Sodium cholate solution was then infused into the perfusion system at a constant rate of 10 mg/hr using a Harvard Apparatus double syringe pump and a calibrated syringe. The time at which the infusion began is designated as “zero” time for bile salt perfusion in Table I. Perfusion was continued for an additional period (70-90 min, Table I). Liver biopsy samples were taken at the
ll 165 129 285 404
4,300
175,700
~
at the rate of 10 mg/hr.
time bile salt infusions were started and again at the end of the experiment. To obtain the biopsy specimens, a small lobe of the liver was tied near the hilum using 4.0 black braided silk thread. The right lateral lobe was taken for the first biopsy and caudate lobes for the second sample. At the intervals indicated in Table I, bile samples were collected. Plasma samples were taken at the end of the intervals as shown in Table I. Experiment 4. This experiment was carried out essentially as in Exps. l-3 except that chenodeoxycholic acid was used instead of sodium cholate. Liver, plasma and bile samples were taken after P32 injection first during a period prior to infusion of bile salt and, again, 1 hr after beginning the bile salt infusion. The data are shown in Table II. Experiments 5-7. These experiments were designed to serve as “controls” for Exps. 14 in that the perfusion period after Pa2 injection but before bile salt infusion was extended to approximate the total perfusion time in Exps. 14. After these intervals in Exps. 5 and 6, sodium cholate was infused as before in order to demonstrate that the liver was still capable of responding in the same manner as in Exps. l-4. In Exp. 7, however, bile salts were
BILE
ACIDS
AND
BILE
LIP111
TABLE CH.~NG:~S
ESCltETION
II
P32 IN LIVER, PLASMI AND BILE AS .\ FUNCTION OF TIME .~FTER INJECTION OF P32 AND BILE SALTS INTO ISOL~TI~:D PERFUSION SYSTEMS Act ratios Bile Liver Plasma
IN PHOSPHOLIPID
i
T
Total Exp. Bile salt infusecI ’p$IhSlcm no. time ,:min)*
Time Time after sii1tbile per P= fused bin) (min)
__-
--
PLP@
PLP3Sd PLP32 g; PLPX’
_. Cheno-deoxycholate
4
Sodium taurocholate
Sodium tauroeholate
i
30 57 SO 110 140 170 200 230
0 30 60 90 120 150
50 80 110 140 170 190 235
0 30 60 90 120 140 185
45 85 135 145 205 265 305 325
,I1
1Plasma Bile SA SA PLp31 ’ PLP32 __ Liver Liver 1 PLPII .IS “,“/&z’ 1:,E? I 7) 7) I
PLP32
‘/
0
I
I liob
103,700
7i
2.9
164,000
98
17.0
246 22,190 52,100
3.5 ’ 48.6 3.5 70.3 0.038 98 226 I 157 333 1 0.17
0.91 3.40
I
51,800 48 / 178.500 1 Ii0 ,
0 50 60 120 180 503.000
50b 6ib
10.2 15.0
I 413 / 370 I 19.0
/
4lh 43 50 26,100
.5;82 .8 ~ 54 2.2 30.9
213” 1 300 1 ii4 1,270 i 18,020 lR.950
4.2 6.1 I 35 5.6 54 4.8 160 5.1 250 “8.0 4i5 40’3 I 470
I i / 0.0
! 0.75
0.037 1 o.i9 ~ 0.046 ~ 1.14 ~
iTone I
712 1 8.4 ! x5
! 0.041
0.09
a Bfter an initial perfusion period for equilibration inorganic PSzwas injected into the perfusste at “zero” time. At a later period sodium taurooholate was infused into the perfusate at the rate of 10 mg/hr. b PLPn was not detected in samples taken 60 min prior to thia sample. c PLP32activity is expressedaa CPM/gram liver or CPM/milliliter plasma or bile. d Specific activity is the ratio of PLPJ*/PLPal in lg liver or 1 ml of plasma or bile. not infused at any time during the perfusion period. The data are shown in Table II. Analytical methods. Lipids were extracted from bile and plasma samples using 20 vol of chloroform-methanol (2:1, v/v) per volume of fluid at room temperature (5). After filtration, the extract was shaken with 0.2 vol of 0.057, aqueous calcium chloride solution, allowed to stand for 30 min, then centrifuged. After removing most of the methanol-water supernate, the lower layer was washed twice with pure solvents upper phase containing 570 NaCl to insure complete removal of any Paz-labeled nonlipid contaminants. Aliquots of the lower chloroform phase were used for determination of PLPa2 and PLPS1. The liver samples (approximately 1 g) was
added to a 30-ml capacity Tenbrock tissue grinder containing 7.0 ml methanol and finely subdivided. Exactly 14.0 ml of freshly distilled chloroform was then added, mixed well and transferred to a 20 X 150.mmscrew-cap tube. The tube was capped and heated at 60” for 1 hr. After cooling the mixture was filtered and the filtrate was collected in a 20 X 150-mm screw-cap tube. The extract was partitioned with 0.2 vol 0.05y0 CaCl* and then washed using the techniques described above for plasma and bile. Aliquots of the washed chloroform phase were used for PLP3l and PLP3L analyses. Phospholipid Pa1 was determined by the method of Bartlett (6). For PLPa2 determinations, appropriate aliquots were pipetted into counting
256
ENTENMAN
vials and the solvent of toluene:2-ethoxyethanol(2:1, v/v) containing 5 g 2,5-diphenyloxazole (PPO) per liter. Pa2activity was determined using a Packard Tricarb scintillation counter. Specific activity of the phospholipids was expressed as the ratio of total PLPaZ to total PLP31 per milliliter of plasma or bile or per gram of liver.
ET AL.
effect of bile salts on excretion of phospholipid into bile. In Exp. 7, where bile acid was not added to the perfusate at any time, the liver PLP32 increased markedly after Pa2 injection, but essentially no PLPa appeared in the bile. The finding of higher specific activity RESULTS AND DISCUSSION values for bile PL than for liver PL or plasma From the data obtained in the above ex- PL is in agreement with the studies of periments (Table I), it can be seen that per- Zilversmit et al. (7) and suggests that bile PLP32 is not derived from a common pool of fusion of rat liver with whole blood to which liver phospholipids but may come from a bile salts had not been added led to very smaller separate pool within the liver. It low levels of phospholipids in the bile. Upon injection of inorganic P32 during this per- should be noted, however, that bile PLP3l fusion period, the liver incorporated P32 levels never reach zero and that even into the phospholipids but released only rel- without added bile salts in the perfusate some PLP32 appears in bile. The specific atively small amounts of PLPs2 into the bile. activities of the bile PL in the absence of However, immediately after the beginning of bile salt infusion (Exp. 14) biliary PLP32 bile salts is much lower than the specific increased markedly and continued to rise activities of the PL after bile salt infusion. This suggests that this phospholipid may be throughout the remainder of the experimenderived from plasma or from a slowly turntal periods. In Exps. 5-6, the perfusion time after the injection of P32 was extended to ing over pool of liver phospholipids. Addi140-180 min in the absence of added bile tional experiments are needed to provide the salts. These periods equaled or exceeded the solution to these questions. perfusion times after P32 injection in Exps. REFERENCES l-4 in which added bile salts were also present during the last half of the periods. 1. KAY, R. E. AND ENTENMAN, C., Am. J. Physiol. Again, the livers in Exps. 5-6 incorporated ZOO,855 (1961). Pa into phospholipids but excreted very 2. ENTENMAN, C., HOLLOWAY, R. J., ALBRIGHT, little into bile. Thus, the marked increase in M. L., AND LEONG, G. F., Proc. Sot. Exptl. Biol. Med. 137, 1093 (1968). biliary PLP32 excretion noted in Exps. 14 were due to the stimulatory effects of added 3. BRMJER, R. W., PESSOTTI, R. L., AND PIZZOLATO, P., Proc. Sot. Exptl. Biol. Med. 73, bile salts and not due simply to a delayed 174 (1951). response. Then, to show that these livers, 4. BRAUER, R. W., LF.oNG,G. F., AND PESSOTTI, even after the extended periods with P32 R. L.. Am. J. Physiol. 174,394 (1953). alone, could still exhibit increased PLP32 5. FOLCH, J., LEES, M., AND SLOANE STANLEY, release upon addition of bile salts to the G. H., J. Biol. Chem. 226,497 (1957). perfusate, sodium cholate was infused for 6. BARTLETT, G. R., J. Biol. Chem. 234,466 (1959). 45-60 min. The increase in biliary PLP32 7. ZILVERSMIT, D.B. AND VAN HANDEL, E., Arch. observed again demonstrates the marked Biochem. Biophys. 73, 224 (1958).
OF
BIOCHEMISTRY
AND
BIOPHYSICS
Bile Acid’s II. Essential CECIL
for
EKTENMAl?,-,‘j
Lipid
and
Lipid
Metabolism
Role of Bile Acids in Bile Phospholipid
AND
The Znstitute
253-256 (1969)
130,
* ROY J. HOLLOWAY, GEORGE F. LEONG
Excretion
M. I,. ALBRIGHT,
Research, Berkeley, California 94702, and the U.S. Naval Laboratory, San Francisco 94124, California Received
September
30, 19G8; accepted
November
&adiological
Defense
26, 1968
The isolated rat liver perfusion system has been used to study the release of bile phospholipid P32 following the injection of inorganic P3* into the perfusate. It was demonsrtated that in the absence of added bile salt very little PLP3* was excreted into bile, but when bile salt was added to the perfusate the PLP3* excretion increased markedly. At the start of bile salt infusion, the specific activity of the bile phospholipids was lower than that of the liver, but at later times the specific activity of bile phospholipids was greater than liver PL specific activity. It is suggested that bile acids play an essential role in the transport of phospholipid from liver to bile.
Previous studies using the isolated perfused rat liver (1, 2) have indicated that bile acids may play an important role in the excretion of bile lipids, particularly phospholipids (PL) and free cholesterol (FC). Biliary levels of both FC and PL were markedly increased following addition of bile salts to the perfusate. It had also been observed by one of US (G.F.L.) that essentially no Ps2activity was found in bile following injection of inorganic P3* into the rat liver perfusion system unless bile salt was also added to the perfusate. Since nearly all of the P32 activity in bile after addition of Pa and bile salt to the perfusion system was insoluble in trichloroacetic acid, it seemed probable that the P3* activity in bile was present as phospholipid P3* V’LW and that transport of PLP32 into bile, therefore, required the presence of added bile salts. Consequently, experiments designed to test this possibility were carried out. The results obtained strongly suggest that bile salts are 1 Institute for Lipid Research, 2127 Bonar St., Berkeley, California 94702. 2 Consultant to the Biological and Medical Sciences Division, U.S.N.R.D.L. 253
obligatory in the transport of most, if not all, of the phospholipids from liver into bile. METHODS Liver perfusion techniques. The apparatus and general techniques used for rat liver perfusions were those described by Brauer et al. (3). Livers were taken from ether-anesthetized donor-fed male Sprague-Dawley rats weighing 300-350 g. The time from the tying of the portal vein to reestablishment of circulation in the perfusion systern never exceeded 4 min. The perfusion medium was approximately 130 ml of whole rat blood containing about 1 mg heparin. An initial perfusion period of 60-90 min were used to establish good blood and bile flow rates and to eliminate the vasoconstrictor factor present in fresh rat blood (4). Perfusions were carried out in a chamber maintained at 37” and a relative humidity of 50-60s. Blood flow rates ranged from 1.27 to 2.40 ml/min/g liver after the initial equilibration period. Bile flow rates, usually about 1 ml/hr at the end of the equilibration period usually fell very gradually throughout the remainder of the perfusion period, but sometimes fell to values as low as 0.5 ml/hr. Materials. Two bile salts were used: Sodium cholate, special enzyme grade (Mann Research Laboratories) and chenodeoxycholic acid, Grade A (Calbiochem). Sodium cholate was dissolved
254
ENTENMAN
INFLUENCE
ET AL.
TABLE I OF TAUROCHOLATE INFUSION ON EXCR~JTION OF PHOSPHOLIPID Pa2 BY THE ISOLATED PKRFUSF,D R.~T LIVER
-
Exp. no.
Total perfusion time bin)
Time after Pa2 injection (min)
CPM PLPa2in
‘e&d of bile salt infusiona (min)
PlasmaC
a Sodium b Counts c Counts
90 135 175 205 235 265
0 45 85 115 145 175
125 155 185 215 245 275 305
0 30 60 90 120 150 180
100 145 190 220 260
0 45 90 120 160
0 30 60 90
413 586 9,712 23,140 29,190
16 76 106 187 310 403
50 255 330 4,978 14,045 17,923
194 269 426 1,170
349 823 34,154 57,838
50,175
0 30 60 90
53,960
0
21,200
5,800
3o 70
1
Taurocholate was infused into the perfusate per minute per gram of liver. per minute per milliliter of plasma or bile.
in isotonic saline and chenodeoxycholic acid in 20 mM sodium bicarbonate. The carrier-free NaHzP320a used was obtained from Volk Radiochemical Co. Experiments 1-S. In these experiments three rat livers were perfused as described above for periods (90-125 min) sufficient to reach equilibration and to eliminate from the perfusion system essentially all of the preformed bile acids. At that time approximately 250 &i of carrier-free NaH2. P3204 was injected into the blood reservoir and rapidly mixed by swirling. This time is designated as “zero” time for the P32 injection in Table I. Perfusion was continued for an additional period (85-90 min) to permit synthesis of P3* labeled phospholipids by the liver and collection of plasma and bile samples in the absence of added bile salt. Sodium cholate solution was then infused into the perfusion system at a constant rate of 10 mg/hr using a Harvard Apparatus double syringe pump and a calibrated syringe. The time at which the infusion began is designated as “zero” time for bile salt perfusion in Table I. Perfusion was continued for an additional period (70-90 min, Table I). Liver biopsy samples were taken at the
ll 165 129 285 404
4,300
175,700
~
at the rate of 10 mg/hr.
time bile salt infusions were started and again at the end of the experiment. To obtain the biopsy specimens, a small lobe of the liver was tied near the hilum using 4.0 black braided silk thread. The right lateral lobe was taken for the first biopsy and caudate lobes for the second sample. At the intervals indicated in Table I, bile samples were collected. Plasma samples were taken at the end of the intervals as shown in Table I. Experiment 4. This experiment was carried out essentially as in Exps. l-3 except that chenodeoxycholic acid was used instead of sodium cholate. Liver, plasma and bile samples were taken after P32 injection first during a period prior to infusion of bile salt and, again, 1 hr after beginning the bile salt infusion. The data are shown in Table II. Experiments 5-7. These experiments were designed to serve as “controls” for Exps. 14 in that the perfusion period after Pa2 injection but before bile salt infusion was extended to approximate the total perfusion time in Exps. 14. After these intervals in Exps. 5 and 6, sodium cholate was infused as before in order to demonstrate that the liver was still capable of responding in the same manner as in Exps. l-4. In Exp. 7, however, bile salts were
BILE
ACIDS
AND
BILE
LIP111
TABLE CH.~NG:~S
ESCltETION
II
P32 IN LIVER, PLASMI AND BILE AS .\ FUNCTION OF TIME .~FTER INJECTION OF P32 AND BILE SALTS INTO ISOL~TI~:D PERFUSION SYSTEMS Act ratios Bile Liver Plasma
IN PHOSPHOLIPID
i
T
Total Exp. Bile salt infusecI ’p$IhSlcm no. time ,:min)*
Time Time after sii1tbile per P= fused bin) (min)
__-
--
PLP@
PLP3Sd PLP32 g; PLPX’
_. Cheno-deoxycholate
4
Sodium taurocholate
Sodium tauroeholate
i
30 57 SO 110 140 170 200 230
0 30 60 90 120 150
50 80 110 140 170 190 235
0 30 60 90 120 140 185
45 85 135 145 205 265 305 325
,I1
1Plasma Bile SA SA PLp31 ’ PLP32 __ Liver Liver 1 PLPII .IS “,“/&z’ 1:,E? I 7) 7) I
PLP32
‘/
0
I
I liob
103,700
7i
2.9
164,000
98
17.0
246 22,190 52,100
3.5 ’ 48.6 3.5 70.3 0.038 98 226 I 157 333 1 0.17
0.91 3.40
I
51,800 48 / 178.500 1 Ii0 ,
0 50 60 120 180 503.000
50b 6ib
10.2 15.0
I 413 / 370 I 19.0
/
4lh 43 50 26,100
.5;82 .8 ~ 54 2.2 30.9
213” 1 300 1 ii4 1,270 i 18,020 lR.950
4.2 6.1 I 35 5.6 54 4.8 160 5.1 250 “8.0 4i5 40’3 I 470
I i / 0.0
! 0.75
0.037 1 o.i9 ~ 0.046 ~ 1.14 ~
iTone I
712 1 8.4 ! x5
! 0.041
0.09
a Bfter an initial perfusion period for equilibration inorganic PSzwas injected into the perfusste at “zero” time. At a later period sodium taurooholate was infused into the perfusate at the rate of 10 mg/hr. b PLPn was not detected in samples taken 60 min prior to thia sample. c PLP32activity is expressedaa CPM/gram liver or CPM/milliliter plasma or bile. d Specific activity is the ratio of PLPJ*/PLPal in lg liver or 1 ml of plasma or bile. not infused at any time during the perfusion period. The data are shown in Table II. Analytical methods. Lipids were extracted from bile and plasma samples using 20 vol of chloroform-methanol (2:1, v/v) per volume of fluid at room temperature (5). After filtration, the extract was shaken with 0.2 vol of 0.057, aqueous calcium chloride solution, allowed to stand for 30 min, then centrifuged. After removing most of the methanol-water supernate, the lower layer was washed twice with pure solvents upper phase containing 570 NaCl to insure complete removal of any Paz-labeled nonlipid contaminants. Aliquots of the lower chloroform phase were used for determination of PLPa2 and PLPS1. The liver samples (approximately 1 g) was
added to a 30-ml capacity Tenbrock tissue grinder containing 7.0 ml methanol and finely subdivided. Exactly 14.0 ml of freshly distilled chloroform was then added, mixed well and transferred to a 20 X 150.mmscrew-cap tube. The tube was capped and heated at 60” for 1 hr. After cooling the mixture was filtered and the filtrate was collected in a 20 X 150-mm screw-cap tube. The extract was partitioned with 0.2 vol 0.05y0 CaCl* and then washed using the techniques described above for plasma and bile. Aliquots of the washed chloroform phase were used for PLP3l and PLP3L analyses. Phospholipid Pa1 was determined by the method of Bartlett (6). For PLPa2 determinations, appropriate aliquots were pipetted into counting
256
ENTENMAN
vials and the solvent of toluene:2-ethoxyethanol(2:1, v/v) containing 5 g 2,5-diphenyloxazole (PPO) per liter. Pa2activity was determined using a Packard Tricarb scintillation counter. Specific activity of the phospholipids was expressed as the ratio of total PLPaZ to total PLP31 per milliliter of plasma or bile or per gram of liver.
ET AL.
effect of bile salts on excretion of phospholipid into bile. In Exp. 7, where bile acid was not added to the perfusate at any time, the liver PLP32 increased markedly after Pa2 injection, but essentially no PLPa appeared in the bile. The finding of higher specific activity RESULTS AND DISCUSSION values for bile PL than for liver PL or plasma From the data obtained in the above ex- PL is in agreement with the studies of periments (Table I), it can be seen that per- Zilversmit et al. (7) and suggests that bile PLP32 is not derived from a common pool of fusion of rat liver with whole blood to which liver phospholipids but may come from a bile salts had not been added led to very smaller separate pool within the liver. It low levels of phospholipids in the bile. Upon injection of inorganic P32 during this per- should be noted, however, that bile PLP3l fusion period, the liver incorporated P32 levels never reach zero and that even into the phospholipids but released only rel- without added bile salts in the perfusate some PLP32 appears in bile. The specific atively small amounts of PLPs2 into the bile. activities of the bile PL in the absence of However, immediately after the beginning of bile salt infusion (Exp. 14) biliary PLP32 bile salts is much lower than the specific increased markedly and continued to rise activities of the PL after bile salt infusion. This suggests that this phospholipid may be throughout the remainder of the experimenderived from plasma or from a slowly turntal periods. In Exps. 5-6, the perfusion time after the injection of P32 was extended to ing over pool of liver phospholipids. Addi140-180 min in the absence of added bile tional experiments are needed to provide the salts. These periods equaled or exceeded the solution to these questions. perfusion times after P32 injection in Exps. REFERENCES l-4 in which added bile salts were also present during the last half of the periods. 1. KAY, R. E. AND ENTENMAN, C., Am. J. Physiol. Again, the livers in Exps. 5-6 incorporated ZOO,855 (1961). Pa into phospholipids but excreted very 2. ENTENMAN, C., HOLLOWAY, R. J., ALBRIGHT, little into bile. Thus, the marked increase in M. L., AND LEONG, G. F., Proc. Sot. Exptl. Biol. Med. 137, 1093 (1968). biliary PLP32 excretion noted in Exps. 14 were due to the stimulatory effects of added 3. BRMJER, R. W., PESSOTTI, R. L., AND PIZZOLATO, P., Proc. Sot. Exptl. Biol. Med. 73, bile salts and not due simply to a delayed 174 (1951). response. Then, to show that these livers, 4. BRAUER, R. W., LF.oNG,G. F., AND PESSOTTI, even after the extended periods with P32 R. L.. Am. J. Physiol. 174,394 (1953). alone, could still exhibit increased PLP32 5. FOLCH, J., LEES, M., AND SLOANE STANLEY, release upon addition of bile salts to the G. H., J. Biol. Chem. 226,497 (1957). perfusate, sodium cholate was infused for 6. BARTLETT, G. R., J. Biol. Chem. 234,466 (1959). 45-60 min. The increase in biliary PLP32 7. ZILVERSMIT, D.B. AND VAN HANDEL, E., Arch. observed again demonstrates the marked Biochem. Biophys. 73, 224 (1958).