Atherosclerosis,
21 (1975) 337-347
337
0 Elsevier Scientific Publishing Company, Amsterdam
LYSOLECITHIN-DEPENDENT PLASMA
MILADA
- Printed In The Netherlands
RELEASE OF CHOLESTEROL
DOBIASOVzk, EVA FALTOVA,
JOSEF
MAREK
AND
FROM RAT
JIRf OBENBERGER
Isotope Laboratory of Biological Institutes and Institute of Physiology, Czechoslovak Academy of Sciences, Institute of Pathological Anatomy, Charles University, and the Eye Research Laboratory, Czechoslovak Academy of Sciences, Prague (Czechoslovakia)
(Received September 2nd, 1974) (Accepted December 17th, 1974)
SUMMARY
We studied the effect of lysolecithin on the clearing of plasma cholesterol. The immediate and maximal conversion of plasma lecithin to lysolecithin was produced in rats by intravenous injection of phospholipase A. The changes which took place in the converted lysolecithin and of cholesterol were followed in rats which had previously received [ssP]phosphate and [14C]cholesterol. We followed simultaneously the in vitro changes in blood removed immediately after the in vivo administration of phospholipase A. The experiments showed that a substantial part of the plasma lecithin was converted to lysolecithin within the first minute after intravenous administration of phospholipase A. In the course of 60 min of blood incubation, the ratio of plasmatic lysolecithin in the closed system continued to increase. At the same time the content of cholesterol also increased. Zn vivo, the converted lysolecithin was quickly released from the plasma, so that within 10 min the original lecithin content dropped to 15-5 % depending on the dose of phospholipase A that had been administered. The content of sphingomyelin and lysolecithin, which increased only temporarily shortly after injection, did not alter during the experiment. The level of plasma cholesterol esters, however, dropped significantly, whereas the free cholesterol content increased. The molar ratio of the drop in lipid phosphorus and cholesterol esters in plasma after the administration of phospholipase A was similar. A significantly higher cholesterol content was found in the liver of animals treated with phospholipase A.
Key words:
[14C]Cholesterol - [14C]Cholesterol esters - Liver - [32P]Lysolecithin - Phospholipase A - [32P]Phospholipids - Plasma - Rats
338
M. DOBIhSOVii,
E. FALTOVA,
J. KAREK,
J. OBENBERGER
INTRODUCTION
The significance
of phospholipids
in the development
of atherosclerosis
has
been known for a long time. But its active effect on the bond1 and transport of plasma cholesterol2 is only apparent from recent papers. As demonstrated recently3,4, the turnover of the lipid component of plasma lipoproteins is faster than the turnover of their protein component, and consequently the concept of phospholipids as a potential vehicle for plasma cholesterol seems to be more plausible. In this connection we were interested in the role of lysolecithin which has some characteristics suitable for the assumed transporting function, although, except in rat@, it represents a minority component of plasma phospholipids. A substantial part of lysolecithin originates in the plasma itself through the action of lecithin-cholesterol acyltransferase6 and from there passes into the organs, where it is very rapidly metabolised7s. Since lysolecithin is the component of plasma phospholipids with the highest metabolic rateg, it is not significantly retained in the plasma, even at a maximum LCAT activitylO. On the other hand, it is deposited in a tenfold quantity in the aortae of hypercholesterolaemic squirrel monkeyslo. It was demonstrated in in vitro experiments that the affinity of lysolecithin
to the tissue membranes
is much
higher than other phospholipidsi1J2, while its bond in plasma lipoproteins is very loose isJ1. Cholesterol in mixed micelles with lysolecithin has the same affinity to the membranes and to lipoproteins as lysolecithinrs.
In the present paper, we tried to verify the hypothesis that lysolecithin originating in the plasma may affect the movement of plasma cholesterol. For this purpose, a model situation was selected in which, under in viva conditions a sudden and extensive conversion of plasma lecithin to lysolecithin (a-acylglycerylphosphoryl choline16) takes place after an intravenous administration of phospholipase A, as described by Klibanskyl:. We followed the changes over a period of time in the content esterified cholesterol in plasma and in the content of phospholipids and in the liver. To ascertain the effect of applied phospholipase A on plasma also observed the changes in a blood sample taken immediately after an injection MATERIALS
of phospholipase
of free and cholesterol lecithin, we intravenous
A.
AND METHODS
Animals Male Wistar rats, body weight 430470 g, maintained on the standard laboratory diet (23 % protein, 44 % carbohydrate, 10 % fat - wet weight), were used for these experiments. The animals were subdivided into 2 experimental groups and were labelled with radioactive isotopes. Labelling with [saP]orthophosphate was first carried out: the rats received an intraperitoneal dose of 300 &i without a carrier in 0.3 ml physiological saline, pH 7.4. 24 hr later, when the specific activity of plasma phospholipids was equals, this group was injected with phospholipase A. Phospho-
LYSOLECITHIN-DEPENDENT
lipids
were determined
RELEASE
in plasma
OF CHOLESTEROL
FROM RAT PLASMA
and liver as a function
of time after enzyme
339 ad-
ministration (O-72 hr) and of the administered dose (25, 50, 100 pg per animal). Another group of [s2P]labelled rats was injected with various doses (25, 50, 100 ,ug per animal) of phospholipase A. These animals were bled within 1 min of injection and the changes induced by enzyme were studied further in blood in vitro. Labelling with [14C]4-cholesterol was then carried out: rats were injected into the tail vein with 15-17 ,uCi of [W]4-cholesterol of 58 mCi/mol specific activity (Radiochemical Centre, Amersham) sonication with 3.5 physiological saline. cholesterol in plasma
in 0.3 ml physiological saline. The solution was prepared by ,ug cholesterol, 35 ,ug lysolecithin and 0.1 ml ethanol per 1 ml Five days later, when the specific activity of free and esterified was equal 18, this group also received phospholipase A. Transfers
of [r4C]cholesterol and [r4C]cholesterol esters after a constant A (100 ,ug per animal) were followed after 15 min and 4 hr.
dose of phospholipase
Administration of phospholipase A Naja Naja venom from Sigma Chemical Corporation was used as a source of phospholipase A (EC 3.1.1.4). The venom was dissolved in physiological saline (330 ,ug/ml) and proteases were inactivated by exposure to 90 “C for 10 minlg. The weight of venom was used as value of administered dose of phospholipase A. Animals were injected into the tail vein with different doses of phospholipase A in a constant volume of 0.3 ml. Sampling of blood and tissue Blood samples (0.5 ml) were drawn from the tail at various time intervals and placed in 2 ml heparinised test-tubes. For the final sampling the animals were lightly anaesthetised with ether, a larger volume of blood was removed from the left ventricle and the animals were then bled to death. Liver was washed with physiological saline and a 200 mg sample from a constant site was removed and frozen in liquid nitrogen and stored in a deep-freeze for further processing. Blood was centrifuged at 1000 rpm for 5 min within 5 min of removal. Blood withdrawn within 1 min after intravenous administration of phospholipase A was immediately centrifuged at 4 “C and an aliquot was transferred to a Dubnoff incubator for 1 hr at 37 “C. These samples were removed at various time intervals, cooled in running cold water and centrifuged within 10 min of removal. Methods for lipid determination and measurement of radioactivity Total lipids from plasma and liver were extracted in chloroform-methanol (2: 1, v/v) according to the method of Folch et aZ.20. Lipid phosphorus was determined according to the method of Chen et al.21 and Bartlettaa. The composition ofphospholipids in lipid extracts was determined by TLC on silica gel covered by aluminium folia, Silufol 150 x 150 mm (Kavalier, Czechoslovakia) in chloroform-methanol-water (24:7:1, v/v). Total cholesterol was determined by the method of Franey and Amador23. The
340
M. DOBIASOVA,
content
of free and esterified
cholesterol
E. FALTOVA,
was determined
J. MAREK,
J. OBENBERGER
in the isotopic
experiments
by measurement of radioactivity of the appropriate samples after separation of the lipid extract by TLC. For the latter technique we used Silufol in hexane-diethyletherethylacetate
(50:50:1.5.
v/v).
Sample radioactivity
was measured
by liquid scintillation
(100 mg POPOP
and
4 g PPO per 1 toluene) on the Isocap instrument (Nuclear Corp., Chicago). Lipid extracts from plasma were first evaporated to dryness in the measurement vials and then the scintillation fluid was added. After TLC separation the Silufol was cut according to spots identified by autoradiography. Spots containing [i%]cholesterol or [14C]cholesterol esters were scratched, extracted in chloroform-methanol (2: 1, v/v) and measured after evaporation of the extract. Spots corresponding to [32P]phospholipids were measured directly in scintillation fluid. Autoradiography was carried out with ORWO films R 3 (VEB Filmfabrik, Wolfen, G.D.R.). RESULTS
In vitro changes of [32P]lipids and cholesterol in rat plasma after the in viva administration of phospholipase A Blood was withdrawn within one min of an intravenous injection of different doses (25, 50, 100 lug) of phospholipase A. Part of the blood was immediately centrifuged at low temperature; another part was incubated at 37 “C. Table 1 shows changes in the activity of plasma [a2P]lipid phosphorus, which had already occurred within the first min of intravenous application of the enzyme. When compared with the activity of [ssP]lipids in plasma sampled immediately before the injection, [32P]lipid phosphorus decreased by 17-32 %, depending on the dose administered. Fig. 1 shows that even in the course of this first min lysolecithin had increased as compared with the controls. The changes are expressed in percentage [ssP]lysolecithin of the sum of [ssP]lecithin and [ssP]lysolecithin. The maximum conversion of lecithin to lysolecithin had already been reached 5 min after incubation. Also in control samples, the proportion of lysolecithin increased in the course of incubation, obviously under
I
TABLE
RAT PLASMA PASE
A (cpm
[32P]LIPID
ACTIVITY
[38P]lipid/25
BEFORE
AND
1 MIN AFTER
INJECTION
OF VARIOUS
DOSES OF
PHOSPHOLI-
1’1 plasma)
Before
I min after
y(, decrease
Controls
1242 & 37a
1211 & 26
2.5 xt 1.3
Dose per animal: 25 lLg
1317 i
23
1083 & 29
1223 i 40 1315 % 19
898 f 32 889 rt 16
50 pg lc@Ecg & Mean
+ S.E.M.
4 animals
in each group.
17.8 f
1.2
26.6 h 2.1 32.4 zt 0.1
LYSOLECITHIN-DEPENDENT
RELEASE OF CHOLESTEROL
341
FROM RAT PLASMA
60%
10 Minutes
Cl
Contro&
?? 25Kl
of
30
incubation
El
of
5OPTJ
60
blood
Ia
10089
Fig. 1. [32P]lysolecithin content (%) in rat plasma after injection of various doses of phospholipase A. The content of [32P]lysolecithin is expressed in % from the sum of [32P]lecithin and [32P]lysolecithin. Time 0 is the value in ,plasma of non-incubated blood which was removed immediately after in vivu injection of phospholipase A.
the effect of LCAT. Fig. 2 shows the plasma cholesterol values of incubated blood in control and treated animals. The values of plasma cholesterol did not alter in controls, whereas the plasma cholesterol the beginning, continued to increase.
in the treated
animals,
In vivo changes of [32P]lipids in rat plasma after the administration
increased
already
at
of phospholipase
A
Changes in the content of [ssP]lipid phosphorus and in the composition of phospholipids in plasma were observed in vivo over a 10 min-48 hr period after intravenous administration of various doses (25, 50, 100 ,ug) of phospholipase A. In control animals the levels of [asP]lipid phosphorus activity hardly altered during the 24-48 hr period after administration of [s2P]orthophosphate, so that the effect of phospholipase A on plasma phospholipids could be clearly observed at this time. After administration of phospholipase A the activity of plasma [asP]phospholipids was clearly dose and time dependent (Table 2). Within 10 min the decrease of activity was 46-54 % of the original value. The marked effect of dosage appeared predominantly in the return rate of activity to original values. Parallel with the decrease of activity of [saP]lipid phosphorus in the plasma there was also a decline in the content of lipid phosphorus (Table 3). As opposed to the radioactivity, however, the latter value decreased only to 68 % of the original levels by 4 hr after the maximal dose. This indicated that after administration of phospholipase A the specific activity of phospholipids in plasma decreased. The decrease in lipid phosphorus was mainly brought about by a change in lecithin, as shown in Table 4. Ten min after injection of phospholipase A there was a
342
M. DOBIASOVA,
E. FALTOVA,
J. MAREK, J. OBENBERGER
r
10
5
30
Minutes Of Incubation
of
60
blood
Fig. 2. The cholesterol content in plasma @g/ml) after injection of various doses of phospholipase Conditions as in Fig. 1.
A.
TABLE 2 RAT PLASMA
[32P]~l~~~ACTIVITY AFTER TREATMENT
WITH
A (cpm
VARIOUS DOSES OF PHOSPHOLIPASE
[32P]lipid/10 ~1 plasma) Time after phospholipase A injection 0
IO tnin
I hr
4 hr
24 hr
48 hr
1006k39
6611k25
Controls
876 * 59” 825 4 21
900 & 29
92.5 k 47
Treated 25 /% 50 Pg lOOPug
759 i 39 752 i_ 27 748 f 39
482 & 51 349 * 22 288 i 13
558 f 40 428 & 51 283 + 58
411 + 29 359 f 24 343 & 24
790 * 58 598 + 34 612 + 37
571 + 51 557% 55 582+ 29
a Mean % S.E.M. 8 animals in each group. TABLE 3 RAT PLASMA
LIPID P CONTENT
AFTER TREATMENT
WITH VARIOUS DOSES OF PHOSPHOLIPASE
A (pg lipid
P/ml plasma) Time after phospholipase A injection: 0
10 min
4hr
24 hr
48 hr
Controls
51.7 * 2.3&
49.3 & 2.5
51.5 + 3.4
-
52.0 f
Treated 25 pg 50 Pg 100 L&g
50.0 * 1.7 48.3 * 2.6 50.2 + 2.1
41.5 i_ 3.6 32.8 f 3.8
43.2 & 3.7 40.5 Ik 4.1 34.0 + 3.3
50.2 i 2.8 43.5 It 3.7 43.5 + 2.8
50.6 i_ 3.1 48.4 f 2.2
& Mean & S.E.M. 8 animals in each group.
2.8
LYSOLECITHIN-DEPENDENT
RELEASE
OF CHOLESTEROL
343
FROM RAT PLASMA
TABLE 4 RAT PLASMA
[32P]LECITHIN
AND [32P]LYSOLECITHIN
CONTENTS AFTER TREATMENT
WITH
PHOSPHOLIPASE
A
[aaP]lecithin, [32P]lysolecithin cpm/50 p&land % of PC (lecithin). Time after phospholipase A injection
Controls Treated 25 1% 50 M
100 {cg
24 hr
4 hr
IO niifl
LPC
PC
LPC
y;,
PC
LPC
“b
2872
1376
67.6
2524
1340
65.3
347
2064 1368
14.4 II.7
809 416
1370 1439
37.1 22.4
1810 1548
1290 1126
1312
8.3
150
1390
9.4
1424
1023
181 119
PC
48 hr y;
PC
LPC
y0
1214
760
61.5
58.9 57.9
922 970
549 589
62.7 62.2
58.2
874
539
61.8 _
Eight animals in each group. Samples for TLC were collected in twos. Percentage of PC was calculated from the sum of [3*P]lecithin and [32P]lysolecithin.
Fig. 3. Autoradiogram (TLC) of plasma [32P]phospholipids of control and phospholipase A treated (10 min, 100 /ig) rats. C, control; T, treated; PC, lecithin: LPC, lysolecithin; Sph, sphingomyelin.
decrease in [32P]lecithin content. Only 10 min after a dose of 25 ,ug enzyme was there an increase in lysolecithin content as a result of lecithin conversion, while at other doses and time periods the lysolecithin content remained unchanged. The percentage of [32P]lecithin content expresses the effect of the dose on its decrease and subsequent increase in the course of time. The content of [a2P]sphingomyelin, which represents about I1 % of the total activity of plasma phospholipids, did not change after the administration of phospholipase A (Fig. 3). From these and results given earlier, it appears that lysolecithin which in rat
344 TABLE
M. DOBI~~OV~,
E. FALTOVA,
J. MAREK. J. OBENBERGER
5
RAT PLASMA ESTEROL AND
CONTENTS
OF LIPID
P,
TOTAL CHOLESTEROL, TOTAL
[I-'C]CHOLESTEROL ESTERS AFTER TREATMENT
[laC]~~~~~~~~~~,
WITH
PHOSPHOLIPASE
FREE [14C]~~~~-
A (100 pg/animal)
Lipid P (!47lml)
Total CH (Mlml)
Total [14C]CH
Free [14C]CH
[14C]CHE
(CPmlmli
(wmlmll
(cpmlml)
Controls
43.0 + 1.48
874 & 19
24,455 i_ 1770
4500 * 315
19,800 III 1530
After treatment: 15 min 4 hr
26.6 & 2.1d 23.5 xk l.2d
750 & 36c 571 i 316
21,OOOi 1060h 14,740 * 7746
663Oi 678 4110 f 416
14,520 ??1810h 10,160 & 2200c
a Mean k S.E.M. 8 animals in each group. b P < 0.05. c P < 0.01. d P < 0.001. CH: cholesterol; CHE: cholesterol esters. TABLE 6 DECREASE
IN RAT
PLASMA
LIPID P AND
CHOLESTEROL
AFTER
TREATMENT
WlTH
A
PHOSPHOLIPASE
( 100 pg/animal) (Difference (A) between controls and treated groups in pmol. Lipid P (pmoljml)
Cholesterol ester (clmollml)
Controls
1.39 f 0.11”
1.82 i 0.14
Afier treatment 15 min 4 hr
0.87 A 0.06 0.76 % 0.04
0.52 0.63
1.34 f 0.16 1.02 * 0.22
A (pmoliml)
0.48 0.80
a Mean * S.E.M.
plasma
was converted
from lecithin
in an extremely
short time after the injection
of
phospholipase A, is cleared from the plasma; the results of these changes, therefore, are a nearly complete disappearance of lecithin. Cholesterol
in rat plasma after administration
of phospholipase
A
Table 5 gives the results of experiments, in which the content of lipid phosphorus and cholesterol, the activity of total cholesterol and the activity of free and esterified cholesterol were all investigated after separation by TLC. The data show that total cholesterol significantly decreased 15 min after injection of phospholipase A, but that in fact this decrease was due to cholesterol esters. The content of free cholesterol either did not change, or increased somewhat in the first phase after injection of the enzyme. The molar ratio of the decrease in lipid phosphorus and cholesterol esters was calculated from the experimental data (Table 6). Fifteen min after administration of
LYSOLECITHIN-DEPENDENT
RELEASE OF CHOLESTEROL
345
FROM RAT PLASMA
TABLE I
Total cholesterol
Controls
content
Cm&?)
(cpmig)
5.34 rt 0.12a
87,720 f
5.90 f 0.14” 6.02 & 0.08c
93,960 * 3940 99,230 * 7520
8940
After treatment:
15 min 4 hr
a Mean + S.E.M. 8 animals in each group. b P < 0.01. c P
21 (1975) 337-347
337
0 Elsevier Scientific Publishing Company, Amsterdam
LYSOLECITHIN-DEPENDENT PLASMA
MILADA
- Printed In The Netherlands
RELEASE OF CHOLESTEROL
DOBIASOVzk, EVA FALTOVA,
JOSEF
MAREK
AND
FROM RAT
JIRf OBENBERGER
Isotope Laboratory of Biological Institutes and Institute of Physiology, Czechoslovak Academy of Sciences, Institute of Pathological Anatomy, Charles University, and the Eye Research Laboratory, Czechoslovak Academy of Sciences, Prague (Czechoslovakia)
(Received September 2nd, 1974) (Accepted December 17th, 1974)
SUMMARY
We studied the effect of lysolecithin on the clearing of plasma cholesterol. The immediate and maximal conversion of plasma lecithin to lysolecithin was produced in rats by intravenous injection of phospholipase A. The changes which took place in the converted lysolecithin and of cholesterol were followed in rats which had previously received [ssP]phosphate and [14C]cholesterol. We followed simultaneously the in vitro changes in blood removed immediately after the in vivo administration of phospholipase A. The experiments showed that a substantial part of the plasma lecithin was converted to lysolecithin within the first minute after intravenous administration of phospholipase A. In the course of 60 min of blood incubation, the ratio of plasmatic lysolecithin in the closed system continued to increase. At the same time the content of cholesterol also increased. Zn vivo, the converted lysolecithin was quickly released from the plasma, so that within 10 min the original lecithin content dropped to 15-5 % depending on the dose of phospholipase A that had been administered. The content of sphingomyelin and lysolecithin, which increased only temporarily shortly after injection, did not alter during the experiment. The level of plasma cholesterol esters, however, dropped significantly, whereas the free cholesterol content increased. The molar ratio of the drop in lipid phosphorus and cholesterol esters in plasma after the administration of phospholipase A was similar. A significantly higher cholesterol content was found in the liver of animals treated with phospholipase A.
Key words:
[14C]Cholesterol - [14C]Cholesterol esters - Liver - [32P]Lysolecithin - Phospholipase A - [32P]Phospholipids - Plasma - Rats
338
M. DOBIhSOVii,
E. FALTOVA,
J. KAREK,
J. OBENBERGER
INTRODUCTION
The significance
of phospholipids
in the development
of atherosclerosis
has
been known for a long time. But its active effect on the bond1 and transport of plasma cholesterol2 is only apparent from recent papers. As demonstrated recently3,4, the turnover of the lipid component of plasma lipoproteins is faster than the turnover of their protein component, and consequently the concept of phospholipids as a potential vehicle for plasma cholesterol seems to be more plausible. In this connection we were interested in the role of lysolecithin which has some characteristics suitable for the assumed transporting function, although, except in rat@, it represents a minority component of plasma phospholipids. A substantial part of lysolecithin originates in the plasma itself through the action of lecithin-cholesterol acyltransferase6 and from there passes into the organs, where it is very rapidly metabolised7s. Since lysolecithin is the component of plasma phospholipids with the highest metabolic rateg, it is not significantly retained in the plasma, even at a maximum LCAT activitylO. On the other hand, it is deposited in a tenfold quantity in the aortae of hypercholesterolaemic squirrel monkeyslo. It was demonstrated in in vitro experiments that the affinity of lysolecithin
to the tissue membranes
is much
higher than other phospholipidsi1J2, while its bond in plasma lipoproteins is very loose isJ1. Cholesterol in mixed micelles with lysolecithin has the same affinity to the membranes and to lipoproteins as lysolecithinrs.
In the present paper, we tried to verify the hypothesis that lysolecithin originating in the plasma may affect the movement of plasma cholesterol. For this purpose, a model situation was selected in which, under in viva conditions a sudden and extensive conversion of plasma lecithin to lysolecithin (a-acylglycerylphosphoryl choline16) takes place after an intravenous administration of phospholipase A, as described by Klibanskyl:. We followed the changes over a period of time in the content esterified cholesterol in plasma and in the content of phospholipids and in the liver. To ascertain the effect of applied phospholipase A on plasma also observed the changes in a blood sample taken immediately after an injection MATERIALS
of phospholipase
of free and cholesterol lecithin, we intravenous
A.
AND METHODS
Animals Male Wistar rats, body weight 430470 g, maintained on the standard laboratory diet (23 % protein, 44 % carbohydrate, 10 % fat - wet weight), were used for these experiments. The animals were subdivided into 2 experimental groups and were labelled with radioactive isotopes. Labelling with [saP]orthophosphate was first carried out: the rats received an intraperitoneal dose of 300 &i without a carrier in 0.3 ml physiological saline, pH 7.4. 24 hr later, when the specific activity of plasma phospholipids was equals, this group was injected with phospholipase A. Phospho-
LYSOLECITHIN-DEPENDENT
lipids
were determined
RELEASE
in plasma
OF CHOLESTEROL
FROM RAT PLASMA
and liver as a function
of time after enzyme
339 ad-
ministration (O-72 hr) and of the administered dose (25, 50, 100 pg per animal). Another group of [s2P]labelled rats was injected with various doses (25, 50, 100 ,ug per animal) of phospholipase A. These animals were bled within 1 min of injection and the changes induced by enzyme were studied further in blood in vitro. Labelling with [14C]4-cholesterol was then carried out: rats were injected into the tail vein with 15-17 ,uCi of [W]4-cholesterol of 58 mCi/mol specific activity (Radiochemical Centre, Amersham) sonication with 3.5 physiological saline. cholesterol in plasma
in 0.3 ml physiological saline. The solution was prepared by ,ug cholesterol, 35 ,ug lysolecithin and 0.1 ml ethanol per 1 ml Five days later, when the specific activity of free and esterified was equal 18, this group also received phospholipase A. Transfers
of [r4C]cholesterol and [r4C]cholesterol esters after a constant A (100 ,ug per animal) were followed after 15 min and 4 hr.
dose of phospholipase
Administration of phospholipase A Naja Naja venom from Sigma Chemical Corporation was used as a source of phospholipase A (EC 3.1.1.4). The venom was dissolved in physiological saline (330 ,ug/ml) and proteases were inactivated by exposure to 90 “C for 10 minlg. The weight of venom was used as value of administered dose of phospholipase A. Animals were injected into the tail vein with different doses of phospholipase A in a constant volume of 0.3 ml. Sampling of blood and tissue Blood samples (0.5 ml) were drawn from the tail at various time intervals and placed in 2 ml heparinised test-tubes. For the final sampling the animals were lightly anaesthetised with ether, a larger volume of blood was removed from the left ventricle and the animals were then bled to death. Liver was washed with physiological saline and a 200 mg sample from a constant site was removed and frozen in liquid nitrogen and stored in a deep-freeze for further processing. Blood was centrifuged at 1000 rpm for 5 min within 5 min of removal. Blood withdrawn within 1 min after intravenous administration of phospholipase A was immediately centrifuged at 4 “C and an aliquot was transferred to a Dubnoff incubator for 1 hr at 37 “C. These samples were removed at various time intervals, cooled in running cold water and centrifuged within 10 min of removal. Methods for lipid determination and measurement of radioactivity Total lipids from plasma and liver were extracted in chloroform-methanol (2: 1, v/v) according to the method of Folch et aZ.20. Lipid phosphorus was determined according to the method of Chen et al.21 and Bartlettaa. The composition ofphospholipids in lipid extracts was determined by TLC on silica gel covered by aluminium folia, Silufol 150 x 150 mm (Kavalier, Czechoslovakia) in chloroform-methanol-water (24:7:1, v/v). Total cholesterol was determined by the method of Franey and Amador23. The
340
M. DOBIASOVA,
content
of free and esterified
cholesterol
E. FALTOVA,
was determined
J. MAREK,
J. OBENBERGER
in the isotopic
experiments
by measurement of radioactivity of the appropriate samples after separation of the lipid extract by TLC. For the latter technique we used Silufol in hexane-diethyletherethylacetate
(50:50:1.5.
v/v).
Sample radioactivity
was measured
by liquid scintillation
(100 mg POPOP
and
4 g PPO per 1 toluene) on the Isocap instrument (Nuclear Corp., Chicago). Lipid extracts from plasma were first evaporated to dryness in the measurement vials and then the scintillation fluid was added. After TLC separation the Silufol was cut according to spots identified by autoradiography. Spots containing [i%]cholesterol or [14C]cholesterol esters were scratched, extracted in chloroform-methanol (2: 1, v/v) and measured after evaporation of the extract. Spots corresponding to [32P]phospholipids were measured directly in scintillation fluid. Autoradiography was carried out with ORWO films R 3 (VEB Filmfabrik, Wolfen, G.D.R.). RESULTS
In vitro changes of [32P]lipids and cholesterol in rat plasma after the in viva administration of phospholipase A Blood was withdrawn within one min of an intravenous injection of different doses (25, 50, 100 lug) of phospholipase A. Part of the blood was immediately centrifuged at low temperature; another part was incubated at 37 “C. Table 1 shows changes in the activity of plasma [a2P]lipid phosphorus, which had already occurred within the first min of intravenous application of the enzyme. When compared with the activity of [ssP]lipids in plasma sampled immediately before the injection, [32P]lipid phosphorus decreased by 17-32 %, depending on the dose administered. Fig. 1 shows that even in the course of this first min lysolecithin had increased as compared with the controls. The changes are expressed in percentage [ssP]lysolecithin of the sum of [ssP]lecithin and [ssP]lysolecithin. The maximum conversion of lecithin to lysolecithin had already been reached 5 min after incubation. Also in control samples, the proportion of lysolecithin increased in the course of incubation, obviously under
I
TABLE
RAT PLASMA PASE
A (cpm
[32P]LIPID
ACTIVITY
[38P]lipid/25
BEFORE
AND
1 MIN AFTER
INJECTION
OF VARIOUS
DOSES OF
PHOSPHOLI-
1’1 plasma)
Before
I min after
y(, decrease
Controls
1242 & 37a
1211 & 26
2.5 xt 1.3
Dose per animal: 25 lLg
1317 i
23
1083 & 29
1223 i 40 1315 % 19
898 f 32 889 rt 16
50 pg lc@Ecg & Mean
+ S.E.M.
4 animals
in each group.
17.8 f
1.2
26.6 h 2.1 32.4 zt 0.1
LYSOLECITHIN-DEPENDENT
RELEASE OF CHOLESTEROL
341
FROM RAT PLASMA
60%
10 Minutes
Cl
Contro&
?? 25Kl
of
30
incubation
El
of
5OPTJ
60
blood
Ia
10089
Fig. 1. [32P]lysolecithin content (%) in rat plasma after injection of various doses of phospholipase A. The content of [32P]lysolecithin is expressed in % from the sum of [32P]lecithin and [32P]lysolecithin. Time 0 is the value in ,plasma of non-incubated blood which was removed immediately after in vivu injection of phospholipase A.
the effect of LCAT. Fig. 2 shows the plasma cholesterol values of incubated blood in control and treated animals. The values of plasma cholesterol did not alter in controls, whereas the plasma cholesterol the beginning, continued to increase.
in the treated
animals,
In vivo changes of [32P]lipids in rat plasma after the administration
increased
already
at
of phospholipase
A
Changes in the content of [ssP]lipid phosphorus and in the composition of phospholipids in plasma were observed in vivo over a 10 min-48 hr period after intravenous administration of various doses (25, 50, 100 ,ug) of phospholipase A. In control animals the levels of [asP]lipid phosphorus activity hardly altered during the 24-48 hr period after administration of [s2P]orthophosphate, so that the effect of phospholipase A on plasma phospholipids could be clearly observed at this time. After administration of phospholipase A the activity of plasma [asP]phospholipids was clearly dose and time dependent (Table 2). Within 10 min the decrease of activity was 46-54 % of the original value. The marked effect of dosage appeared predominantly in the return rate of activity to original values. Parallel with the decrease of activity of [saP]lipid phosphorus in the plasma there was also a decline in the content of lipid phosphorus (Table 3). As opposed to the radioactivity, however, the latter value decreased only to 68 % of the original levels by 4 hr after the maximal dose. This indicated that after administration of phospholipase A the specific activity of phospholipids in plasma decreased. The decrease in lipid phosphorus was mainly brought about by a change in lecithin, as shown in Table 4. Ten min after injection of phospholipase A there was a
342
M. DOBIASOVA,
E. FALTOVA,
J. MAREK, J. OBENBERGER
r
10
5
30
Minutes Of Incubation
of
60
blood
Fig. 2. The cholesterol content in plasma @g/ml) after injection of various doses of phospholipase Conditions as in Fig. 1.
A.
TABLE 2 RAT PLASMA
[32P]~l~~~ACTIVITY AFTER TREATMENT
WITH
A (cpm
VARIOUS DOSES OF PHOSPHOLIPASE
[32P]lipid/10 ~1 plasma) Time after phospholipase A injection 0
IO tnin
I hr
4 hr
24 hr
48 hr
1006k39
6611k25
Controls
876 * 59” 825 4 21
900 & 29
92.5 k 47
Treated 25 /% 50 Pg lOOPug
759 i 39 752 i_ 27 748 f 39
482 & 51 349 * 22 288 i 13
558 f 40 428 & 51 283 + 58
411 + 29 359 f 24 343 & 24
790 * 58 598 + 34 612 + 37
571 + 51 557% 55 582+ 29
a Mean % S.E.M. 8 animals in each group. TABLE 3 RAT PLASMA
LIPID P CONTENT
AFTER TREATMENT
WITH VARIOUS DOSES OF PHOSPHOLIPASE
A (pg lipid
P/ml plasma) Time after phospholipase A injection: 0
10 min
4hr
24 hr
48 hr
Controls
51.7 * 2.3&
49.3 & 2.5
51.5 + 3.4
-
52.0 f
Treated 25 pg 50 Pg 100 L&g
50.0 * 1.7 48.3 * 2.6 50.2 + 2.1
41.5 i_ 3.6 32.8 f 3.8
43.2 & 3.7 40.5 Ik 4.1 34.0 + 3.3
50.2 i 2.8 43.5 It 3.7 43.5 + 2.8
50.6 i_ 3.1 48.4 f 2.2
& Mean & S.E.M. 8 animals in each group.
2.8
LYSOLECITHIN-DEPENDENT
RELEASE
OF CHOLESTEROL
343
FROM RAT PLASMA
TABLE 4 RAT PLASMA
[32P]LECITHIN
AND [32P]LYSOLECITHIN
CONTENTS AFTER TREATMENT
WITH
PHOSPHOLIPASE
A
[aaP]lecithin, [32P]lysolecithin cpm/50 p&land % of PC (lecithin). Time after phospholipase A injection
Controls Treated 25 1% 50 M
100 {cg
24 hr
4 hr
IO niifl
LPC
PC
LPC
y;,
PC
LPC
“b
2872
1376
67.6
2524
1340
65.3
347
2064 1368
14.4 II.7
809 416
1370 1439
37.1 22.4
1810 1548
1290 1126
1312
8.3
150
1390
9.4
1424
1023
181 119
PC
48 hr y;
PC
LPC
y0
1214
760
61.5
58.9 57.9
922 970
549 589
62.7 62.2
58.2
874
539
61.8 _
Eight animals in each group. Samples for TLC were collected in twos. Percentage of PC was calculated from the sum of [3*P]lecithin and [32P]lysolecithin.
Fig. 3. Autoradiogram (TLC) of plasma [32P]phospholipids of control and phospholipase A treated (10 min, 100 /ig) rats. C, control; T, treated; PC, lecithin: LPC, lysolecithin; Sph, sphingomyelin.
decrease in [32P]lecithin content. Only 10 min after a dose of 25 ,ug enzyme was there an increase in lysolecithin content as a result of lecithin conversion, while at other doses and time periods the lysolecithin content remained unchanged. The percentage of [32P]lecithin content expresses the effect of the dose on its decrease and subsequent increase in the course of time. The content of [a2P]sphingomyelin, which represents about I1 % of the total activity of plasma phospholipids, did not change after the administration of phospholipase A (Fig. 3). From these and results given earlier, it appears that lysolecithin which in rat
344 TABLE
M. DOBI~~OV~,
E. FALTOVA,
J. MAREK. J. OBENBERGER
5
RAT PLASMA ESTEROL AND
CONTENTS
OF LIPID
P,
TOTAL CHOLESTEROL, TOTAL
[I-'C]CHOLESTEROL ESTERS AFTER TREATMENT
[laC]~~~~~~~~~~,
WITH
PHOSPHOLIPASE
FREE [14C]~~~~-
A (100 pg/animal)
Lipid P (!47lml)
Total CH (Mlml)
Total [14C]CH
Free [14C]CH
[14C]CHE
(CPmlmli
(wmlmll
(cpmlml)
Controls
43.0 + 1.48
874 & 19
24,455 i_ 1770
4500 * 315
19,800 III 1530
After treatment: 15 min 4 hr
26.6 & 2.1d 23.5 xk l.2d
750 & 36c 571 i 316
21,OOOi 1060h 14,740 * 7746
663Oi 678 4110 f 416
14,520 ??1810h 10,160 & 2200c
a Mean k S.E.M. 8 animals in each group. b P < 0.05. c P < 0.01. d P < 0.001. CH: cholesterol; CHE: cholesterol esters. TABLE 6 DECREASE
IN RAT
PLASMA
LIPID P AND
CHOLESTEROL
AFTER
TREATMENT
WlTH
A
PHOSPHOLIPASE
( 100 pg/animal) (Difference (A) between controls and treated groups in pmol. Lipid P (pmoljml)
Cholesterol ester (clmollml)
Controls
1.39 f 0.11”
1.82 i 0.14
Afier treatment 15 min 4 hr
0.87 A 0.06 0.76 % 0.04
0.52 0.63
1.34 f 0.16 1.02 * 0.22
A (pmoliml)
0.48 0.80
a Mean * S.E.M.
plasma
was converted
from lecithin
in an extremely
short time after the injection
of
phospholipase A, is cleared from the plasma; the results of these changes, therefore, are a nearly complete disappearance of lecithin. Cholesterol
in rat plasma after administration
of phospholipase
A
Table 5 gives the results of experiments, in which the content of lipid phosphorus and cholesterol, the activity of total cholesterol and the activity of free and esterified cholesterol were all investigated after separation by TLC. The data show that total cholesterol significantly decreased 15 min after injection of phospholipase A, but that in fact this decrease was due to cholesterol esters. The content of free cholesterol either did not change, or increased somewhat in the first phase after injection of the enzyme. The molar ratio of the decrease in lipid phosphorus and cholesterol esters was calculated from the experimental data (Table 6). Fifteen min after administration of
LYSOLECITHIN-DEPENDENT
RELEASE OF CHOLESTEROL
345
FROM RAT PLASMA
TABLE I
Total cholesterol
Controls
content
Cm&?)
(cpmig)
5.34 rt 0.12a
87,720 f
5.90 f 0.14” 6.02 & 0.08c
93,960 * 3940 99,230 * 7520
8940
After treatment:
15 min 4 hr
a Mean + S.E.M. 8 animals in each group. b P < 0.01. c P
