188
Biochimica et Biophysics Acta, 530 (1978) 188-196 0 Elsevier/North-Holland Biomedical Press
BBA 57221
METABOLIC FATE OF THE PHOSPHATIDYLCHOLINE COMPONENT OF VERY LOW DENSITY LIPOPROTEINS DURING CATABOLISM BY THE PERFUSED RAT HEART
P.H.E. GROOT and A. VAN TOL Department of Bioc~e~is~y i, Faculty of Medicine, Erasmus U~iuersity Rotte~da~, P.0. Box 1738, Rotterdan (The Netherlands) (Received February 6th, 1978)
Summary The fate of the phosphatidylcholine component of very low density lipoproteins was studied during degradation by the isolated perfused rat heart, using 32P-labelled phospholipid and 3H-labelled triglycerides on rat very low density lipoprotein. After hydrolysis of 90-95% of very low density lipoprotein triglyceride, only 30-40s of the initial phosphatidyl[32P]choline was catabolized to lysophosphatidylcholine and to a water-soluble 32P-containing metabolite, both of which accumulated in the perfusion medium. Analysis of the density of products of very low density lipoprotein catabolism showed that phosphatidylcholine also disappe~ed from very low density lipoprotein (p < 1.006 g/ml) during its catabolism by transfer to lipoproteins of 1.019 < p < 1.05 g/ml and p > 1.21 g/ml density ranges. --____. _ _--Introduction Recently, it was demonstrated that purified lipoprotein lipase (glycerol ester hydrolase, EC 3.1.1.3) is able to hydrolyze l-acyl ester bonds of phosphatidylethanolamine and phosphatidylcholine [l--3]. Although a decade ago a similar conclusion was reached by Vogel and co-workers [4-6] with lipoprotein lipase purified from postheparin plasma, less attention was paid to this work when it became evident that most of the postheparin plasma phospholipase Al activity could be attributed to an enzyme of hepatic origin, different from lipoprotein lipase [ 71. We have studied the substrate specficity of rat heart lipoprotein lipase and showed that its phospholipase Al activity was dependent on the presence of apolipoprotein C-II, as is the case with its triacylglycerol hydrolase activity [ 31. Whether phospholipase A1 activity of lipoprotein lipase is involved in the in vivo degradation of triglyceride-rich lipoproteins has not been elucidated [ 81.
189
We studied the catabolism of 32P-labelled phospholipid of rat very low density lipoprotein (VLDL *) by the perfused rat heart, a model in which lipopro~in lipase is present at its natural locus. For comparison, triglyceride hydrolysis in VLDL was studied simultaneously using 3H-labelled triglyceride on rat VLDL. Materials and Methods HJ32P04 and [9,10-3H]oleic acid were purchased from the Radiochemical Centre {Amersh~, U.K.). Silica gel G plates, 0.25 mm thick, were obtained from Merck (M~nheim, G.F.R.). Bovine serum albumin, fraction V, was a product of Sigma (St. Louis, U.S.A.) and defatted according to Chen [9]. Male Wistar rats were used for all our experiments. VLDL was labelled biosynthetitally by injection of either H332P04 or [9,10-3H]oleic acid into rats as described by Rubenstein and Rubenstein [lo] and by Fielding and Higgins [ 111, respectively. Unlabelled VLDL was obtained from overnight fasted rats. The animals were bled under ether anaesthesia and their blood was collected and allowed to clot at 0°C. The serum was isolated by low speed cent~fugation. On the same day a density gradient cent~figation was started to isolate VLDL. The experimental procedure was adapted from Redgrave et al. [ 121 and modified as described before [13,14). The label distribution in the isolated VLDL is described in Results. In our perfusion studies, labelled and unlabelled VLDL were mixed resulting in specific activities of about 450 dpm/nmol and 150 dpm/nmol for 3H-labelled triglycerides and [ 32P]phosphatidylcholine, respectively . Rat hearts were obtained from animals (body weight 220-300 g) after an overnight fasting period and perfused backwards according to the Langendorff technique at 37°C. The apparatus for the recirculating perfusion was adapted from Morgan et al. [15] and all glass surfaces were siliconized with Siliclad (Clay Adams, New York, U.S.A.). The basic medium consisted of Krebs-Ringer bicarbonate solution supplemented with 2 mg/ml glucose and saturated with 95% O2 and 5% CO2 at 37°C (pH 7.4). The hearts were preperfused for 10 min (without recirculation) at a constant pressure of 55 mm Hg. Degradation of VLDL was studied subsequently during a rec~culating perfusion with basic medium supplemented with VLDL and 3% (w/v) defatted bovine serum albumin (25-30 ml, 8-12 ml/min, 55 mm Hg). Every 15 min, 1.2 ml samples of the perfusion medium were taken and analyzed for total radioactivity and label distribution. The lipids in the medium were extracted according to Bligh and Dyer [16] and the water phase was saved for radioactivity determinations. Neutral lipids in the CHC13 phase were separated on Silica gel G plates developed in hept~e/ether/aceti~ acid (60 : 40 : 1, v/v). Phospholipids present in the CHC13 phase were analyzed on a second plate, developed in CHCl3/ CH,OH/acetic acid/H20 (100 : 50 : 18 : 8, v/v). After identification, the spots were scraped off the plates, sonicated in 5 ml Hz0 and the associated radioactivity was determined by liquid scintillation counting, using 10 ml In&gel as scintillation fluid. * VLDL, very low density lipoproteins @ < 1.006 g/ml): LDL, low density lipoproteins 1.05 g/ml); HDL, high density lipoproteins (1.05 < p < 1.13 g/ml).
(1.006 < p
1.13 g/ml *). The distribution of the associated radioactivity among lipid classes and water phase in each fraction was studied as described above. Triglycerides and phospholipids in VLDL were determined according to Laurel1 [18] and Barlett [ 191, respectively. Radioactivity was determined in a Packard model 544 liquid scintillation counter, using Instagel as counting fluid. Determinations of densities of salt solutions were performed at 20°C using an Anton Paar digital precision density meter model DMA 40. Results Fig. 1 shows data from a representative experiment from a group of 5 (compare Table I). During 95 min perfusion, more than 90% VLDL-triglycerides are hydrolyzed and most of the 3H label is recovered as 3H-labelled fatty acids and 3Hz0. During this period, 30-40% VLDL-phosphatidylcholine is hydrolyzed. Most of the radioactivity lost from phosphatidylcholine is recovered as lysophosphatidylcholine; a smaller percentage is recovered in a water-soluble compound, probably glycerophosphorylcholine. No 32P radioactivity is lost from the medium during perfusion. 4-20s (dependent on the perfusion time) of the total 3H radioactivity present in the medium at the beginning of the perfusion is gradually lost and recovered in the heart. This indicates that part of 3H-labelled fatty acids, formed by VLDL-triglyceride hydrolysis, are incorportated into heart lipids. Trapping of intact VLDL particles in the heart can be excluded as 32P-labelled phospholipid radioactivity is not lost from the perfusion medium at any time. In all our experiments, phosphatidylcholine hydrolysis was linear with time, while the rate of triglyceride hydrolysis declined rapidly (Fig. 1). The latter result was expected, since the initial VLDL concentration (about 0.15 mM triglycerides) only exceeds the apparent K, for rat heart lipoprotein lipase by a factor of 2 [ 71. Moreover, Higgins and Fielding [ 201 have demonstrated that as the level of triglycerides in partially degraded VLDL decreases, the V of rat heart lipoprotein lipase for this VLDL decreases, Absolute rates of triglyceride and phosphatidylcholine hydrolysis in VLDL by the perfused heart are given in Table I. Results obtained in five studies using two different preparations of labelled VLDL were very reproducible. The absolute rate of triglyceride hydrolysis is 40 times the rate of phosphatidylcholine hydrolysis. Comparable results have been obtained with purified rat heart lipoprotein lipase and artificial emulsions of phosphatidylcholine and triglycerides [3]. The absolute rate of phos‘phatidylcholine hydrolysis (Table I) may be slightly underestimated, as lyso* HDL was isolated from this narrow density range in order to obtain uncontaminated HDL [141. It was found that the recovery of HDL-cholesterol and HDL-phospholipids was only slightly increased by using the density range 1.05-l .21 g/ml; compare also ref. 17.
191 I
I
3
I
I
B
dp
32~
dP’3/,l
Xl
x102 *
*
_
-60
l
-20
0
20
10 60 min. perfusion
80
0
20
time
40 60 min perfusion
80 time
Fig. 1. Triglyceride and phosphatidylcholine hydrolysis in VLDL during recirculating perfusion in the isolated perfused rat heart. A rat heart (wet weight 1.13 g) was perfused with medium supplemented with double labelled VLDL. as described in Materials and Methods. The starting volume was 30 ml. At t = 0. the following label distributions were observed. 3H-labelled VLDL: triglycerides 68.696, monoacylglycerol plus phospholipids 8.3%. dlacylglycerol 7.2% free fatty acids 4.2%. cholesterolester 6.1% and watersoluble radioactivity l,O%. 32P-labelled VLDL: phosp~tidylcho~e 8&l%, lysophosphatidylcho~e 4.2%. sphingomyelin 8.6% and water soluble radioactivity 2.2%. The radioactivity assoelated with other phospholipids was insignificant. The total VLDL triglyceride concentration at t = 0 was 0.16 mM. Samples of 1.2 ml were taken at t = 5 and then every 15 mln and were analyzed for label distribution. At t = 50 an extra sample of 8 ml was taken for density gradient analysis (see legend Table II). Data are plotted in dpm/ml perfusion medium. Values for L3Hl-labelled diacylglycerol. 3H-labelled monoacylglycerol plus phosphollpids. 3H-labelled cholesterolester and 32P-labelled sphingomyelin remain constant during the perfusion and are not plotted in this figure. 19.9% of 3H- and 1.8% of 32P radioactivity, present in the medium at zero time were recovered in the heart at the end of the perfusion, after 5 min flushing with nonradioactive preperfuslon medium, A: l-. total 3H-radioactivlty; n-, 3Htotal 32~ radiolabelled triglycerides; v----v. 3H-labelled fatty acids; A-A 1 3H~0. B: .A, activity; n-------x . phosphatidyl[3*Plcholine; v -r, lysophosphatidyl[32Plcholine: A-A water-soluble 32P radioactivity.
l
phosphatidylcho~ne is converted to some extent into a water-soluble 32P-tontaining metabolite, probably by an unidentified lysophospholipase present in accumulated in the our system (Fig. 1 and Table IIB). As no 32P radioactivity
TABLE I ABSOLUTE RATES OF VLR~TRIGLYCERIDE AND VLDL-PHOSPHATIDYL~HOLINE YSIS DURING VLDL DEGRADATION BY THE ISOLATED PERFUSED RAT HEART
HYDROL-
Rat hearts (wet weight 0.89-1.7 g) wei% perfused with double labelled VLDL as described in Materials and Methods, during 50-85 min. Initial VLDL-triglyceride and VLDL-phosphatidylcholine concentrations varied between 0.12 and 0.16 mM and 0.026 and 0.034 mM, respectively. Absolute rates of triglyceride hydrolysis were calculated from the triglyceride disappearance during the first 15 min of perfusion. As phosphatidylcholine hydrolysis was linear in time, its rate was calculated from lysophosphatidylcholine formation during the whole perfusion period. Rates are given in run01 triglyceride hydrolyzed/~n per g heart (wet weight). Triglyceride hydrolysis (mean t SE.)
Lysophosphatidylcholine (mean f S.E.)
57.6 f 2.4 01 = 5)
1.46 C 0.14 (n = 5)
formation
TABLE
II
was sampled
(A) AND 32P-LABELLED VLDL by density
DEGRADATION
shown in Fig. 1, and subfractionated
LIPIDS (B) DURING
at t = 0, 50 and 95 min during the experiment
OF 3H-LABELLED gradient
ultracentrifugation
95
50
0
A. Time
1.006 1.019 1.05
1.006 1.019 1.05
1.006 1.019 1.05
< < < < > >
P< < P<
Biochimica et Biophysics Acta, 530 (1978) 188-196 0 Elsevier/North-Holland Biomedical Press
BBA 57221
METABOLIC FATE OF THE PHOSPHATIDYLCHOLINE COMPONENT OF VERY LOW DENSITY LIPOPROTEINS DURING CATABOLISM BY THE PERFUSED RAT HEART
P.H.E. GROOT and A. VAN TOL Department of Bioc~e~is~y i, Faculty of Medicine, Erasmus U~iuersity Rotte~da~, P.0. Box 1738, Rotterdan (The Netherlands) (Received February 6th, 1978)
Summary The fate of the phosphatidylcholine component of very low density lipoproteins was studied during degradation by the isolated perfused rat heart, using 32P-labelled phospholipid and 3H-labelled triglycerides on rat very low density lipoprotein. After hydrolysis of 90-95% of very low density lipoprotein triglyceride, only 30-40s of the initial phosphatidyl[32P]choline was catabolized to lysophosphatidylcholine and to a water-soluble 32P-containing metabolite, both of which accumulated in the perfusion medium. Analysis of the density of products of very low density lipoprotein catabolism showed that phosphatidylcholine also disappe~ed from very low density lipoprotein (p < 1.006 g/ml) during its catabolism by transfer to lipoproteins of 1.019 < p < 1.05 g/ml and p > 1.21 g/ml density ranges. --____. _ _--Introduction Recently, it was demonstrated that purified lipoprotein lipase (glycerol ester hydrolase, EC 3.1.1.3) is able to hydrolyze l-acyl ester bonds of phosphatidylethanolamine and phosphatidylcholine [l--3]. Although a decade ago a similar conclusion was reached by Vogel and co-workers [4-6] with lipoprotein lipase purified from postheparin plasma, less attention was paid to this work when it became evident that most of the postheparin plasma phospholipase Al activity could be attributed to an enzyme of hepatic origin, different from lipoprotein lipase [ 71. We have studied the substrate specficity of rat heart lipoprotein lipase and showed that its phospholipase Al activity was dependent on the presence of apolipoprotein C-II, as is the case with its triacylglycerol hydrolase activity [ 31. Whether phospholipase A1 activity of lipoprotein lipase is involved in the in vivo degradation of triglyceride-rich lipoproteins has not been elucidated [ 81.
189
We studied the catabolism of 32P-labelled phospholipid of rat very low density lipoprotein (VLDL *) by the perfused rat heart, a model in which lipopro~in lipase is present at its natural locus. For comparison, triglyceride hydrolysis in VLDL was studied simultaneously using 3H-labelled triglyceride on rat VLDL. Materials and Methods HJ32P04 and [9,10-3H]oleic acid were purchased from the Radiochemical Centre {Amersh~, U.K.). Silica gel G plates, 0.25 mm thick, were obtained from Merck (M~nheim, G.F.R.). Bovine serum albumin, fraction V, was a product of Sigma (St. Louis, U.S.A.) and defatted according to Chen [9]. Male Wistar rats were used for all our experiments. VLDL was labelled biosynthetitally by injection of either H332P04 or [9,10-3H]oleic acid into rats as described by Rubenstein and Rubenstein [lo] and by Fielding and Higgins [ 111, respectively. Unlabelled VLDL was obtained from overnight fasted rats. The animals were bled under ether anaesthesia and their blood was collected and allowed to clot at 0°C. The serum was isolated by low speed cent~fugation. On the same day a density gradient cent~figation was started to isolate VLDL. The experimental procedure was adapted from Redgrave et al. [ 121 and modified as described before [13,14). The label distribution in the isolated VLDL is described in Results. In our perfusion studies, labelled and unlabelled VLDL were mixed resulting in specific activities of about 450 dpm/nmol and 150 dpm/nmol for 3H-labelled triglycerides and [ 32P]phosphatidylcholine, respectively . Rat hearts were obtained from animals (body weight 220-300 g) after an overnight fasting period and perfused backwards according to the Langendorff technique at 37°C. The apparatus for the recirculating perfusion was adapted from Morgan et al. [15] and all glass surfaces were siliconized with Siliclad (Clay Adams, New York, U.S.A.). The basic medium consisted of Krebs-Ringer bicarbonate solution supplemented with 2 mg/ml glucose and saturated with 95% O2 and 5% CO2 at 37°C (pH 7.4). The hearts were preperfused for 10 min (without recirculation) at a constant pressure of 55 mm Hg. Degradation of VLDL was studied subsequently during a rec~culating perfusion with basic medium supplemented with VLDL and 3% (w/v) defatted bovine serum albumin (25-30 ml, 8-12 ml/min, 55 mm Hg). Every 15 min, 1.2 ml samples of the perfusion medium were taken and analyzed for total radioactivity and label distribution. The lipids in the medium were extracted according to Bligh and Dyer [16] and the water phase was saved for radioactivity determinations. Neutral lipids in the CHC13 phase were separated on Silica gel G plates developed in hept~e/ether/aceti~ acid (60 : 40 : 1, v/v). Phospholipids present in the CHC13 phase were analyzed on a second plate, developed in CHCl3/ CH,OH/acetic acid/H20 (100 : 50 : 18 : 8, v/v). After identification, the spots were scraped off the plates, sonicated in 5 ml Hz0 and the associated radioactivity was determined by liquid scintillation counting, using 10 ml In&gel as scintillation fluid. * VLDL, very low density lipoproteins @ < 1.006 g/ml): LDL, low density lipoproteins 1.05 g/ml); HDL, high density lipoproteins (1.05 < p < 1.13 g/ml).
(1.006 < p
1.13 g/ml *). The distribution of the associated radioactivity among lipid classes and water phase in each fraction was studied as described above. Triglycerides and phospholipids in VLDL were determined according to Laurel1 [18] and Barlett [ 191, respectively. Radioactivity was determined in a Packard model 544 liquid scintillation counter, using Instagel as counting fluid. Determinations of densities of salt solutions were performed at 20°C using an Anton Paar digital precision density meter model DMA 40. Results Fig. 1 shows data from a representative experiment from a group of 5 (compare Table I). During 95 min perfusion, more than 90% VLDL-triglycerides are hydrolyzed and most of the 3H label is recovered as 3H-labelled fatty acids and 3Hz0. During this period, 30-40% VLDL-phosphatidylcholine is hydrolyzed. Most of the radioactivity lost from phosphatidylcholine is recovered as lysophosphatidylcholine; a smaller percentage is recovered in a water-soluble compound, probably glycerophosphorylcholine. No 32P radioactivity is lost from the medium during perfusion. 4-20s (dependent on the perfusion time) of the total 3H radioactivity present in the medium at the beginning of the perfusion is gradually lost and recovered in the heart. This indicates that part of 3H-labelled fatty acids, formed by VLDL-triglyceride hydrolysis, are incorportated into heart lipids. Trapping of intact VLDL particles in the heart can be excluded as 32P-labelled phospholipid radioactivity is not lost from the perfusion medium at any time. In all our experiments, phosphatidylcholine hydrolysis was linear with time, while the rate of triglyceride hydrolysis declined rapidly (Fig. 1). The latter result was expected, since the initial VLDL concentration (about 0.15 mM triglycerides) only exceeds the apparent K, for rat heart lipoprotein lipase by a factor of 2 [ 71. Moreover, Higgins and Fielding [ 201 have demonstrated that as the level of triglycerides in partially degraded VLDL decreases, the V of rat heart lipoprotein lipase for this VLDL decreases, Absolute rates of triglyceride and phosphatidylcholine hydrolysis in VLDL by the perfused heart are given in Table I. Results obtained in five studies using two different preparations of labelled VLDL were very reproducible. The absolute rate of triglyceride hydrolysis is 40 times the rate of phosphatidylcholine hydrolysis. Comparable results have been obtained with purified rat heart lipoprotein lipase and artificial emulsions of phosphatidylcholine and triglycerides [3]. The absolute rate of phos‘phatidylcholine hydrolysis (Table I) may be slightly underestimated, as lyso* HDL was isolated from this narrow density range in order to obtain uncontaminated HDL [141. It was found that the recovery of HDL-cholesterol and HDL-phospholipids was only slightly increased by using the density range 1.05-l .21 g/ml; compare also ref. 17.
191 I
I
3
I
I
B
dp
32~
dP’3/,l
Xl
x102 *
*
_
-60
l
-20
0
20
10 60 min. perfusion
80
0
20
time
40 60 min perfusion
80 time
Fig. 1. Triglyceride and phosphatidylcholine hydrolysis in VLDL during recirculating perfusion in the isolated perfused rat heart. A rat heart (wet weight 1.13 g) was perfused with medium supplemented with double labelled VLDL. as described in Materials and Methods. The starting volume was 30 ml. At t = 0. the following label distributions were observed. 3H-labelled VLDL: triglycerides 68.696, monoacylglycerol plus phospholipids 8.3%. dlacylglycerol 7.2% free fatty acids 4.2%. cholesterolester 6.1% and watersoluble radioactivity l,O%. 32P-labelled VLDL: phosp~tidylcho~e 8&l%, lysophosphatidylcho~e 4.2%. sphingomyelin 8.6% and water soluble radioactivity 2.2%. The radioactivity assoelated with other phospholipids was insignificant. The total VLDL triglyceride concentration at t = 0 was 0.16 mM. Samples of 1.2 ml were taken at t = 5 and then every 15 mln and were analyzed for label distribution. At t = 50 an extra sample of 8 ml was taken for density gradient analysis (see legend Table II). Data are plotted in dpm/ml perfusion medium. Values for L3Hl-labelled diacylglycerol. 3H-labelled monoacylglycerol plus phosphollpids. 3H-labelled cholesterolester and 32P-labelled sphingomyelin remain constant during the perfusion and are not plotted in this figure. 19.9% of 3H- and 1.8% of 32P radioactivity, present in the medium at zero time were recovered in the heart at the end of the perfusion, after 5 min flushing with nonradioactive preperfuslon medium, A: l-. total 3H-radioactivlty; n-, 3Htotal 32~ radiolabelled triglycerides; v----v. 3H-labelled fatty acids; A-A 1 3H~0. B: .A, activity; n-------x . phosphatidyl[3*Plcholine; v -r, lysophosphatidyl[32Plcholine: A-A water-soluble 32P radioactivity.
l
phosphatidylcho~ne is converted to some extent into a water-soluble 32P-tontaining metabolite, probably by an unidentified lysophospholipase present in accumulated in the our system (Fig. 1 and Table IIB). As no 32P radioactivity
TABLE I ABSOLUTE RATES OF VLR~TRIGLYCERIDE AND VLDL-PHOSPHATIDYL~HOLINE YSIS DURING VLDL DEGRADATION BY THE ISOLATED PERFUSED RAT HEART
HYDROL-
Rat hearts (wet weight 0.89-1.7 g) wei% perfused with double labelled VLDL as described in Materials and Methods, during 50-85 min. Initial VLDL-triglyceride and VLDL-phosphatidylcholine concentrations varied between 0.12 and 0.16 mM and 0.026 and 0.034 mM, respectively. Absolute rates of triglyceride hydrolysis were calculated from the triglyceride disappearance during the first 15 min of perfusion. As phosphatidylcholine hydrolysis was linear in time, its rate was calculated from lysophosphatidylcholine formation during the whole perfusion period. Rates are given in run01 triglyceride hydrolyzed/~n per g heart (wet weight). Triglyceride hydrolysis (mean t SE.)
Lysophosphatidylcholine (mean f S.E.)
57.6 f 2.4 01 = 5)
1.46 C 0.14 (n = 5)
formation
TABLE
II
was sampled
(A) AND 32P-LABELLED VLDL by density
DEGRADATION
shown in Fig. 1, and subfractionated
LIPIDS (B) DURING
at t = 0, 50 and 95 min during the experiment
OF 3H-LABELLED gradient
ultracentrifugation
95
50
0
A. Time
1.006 1.019 1.05
1.006 1.019 1.05
1.006 1.019 1.05
< < < < > >
P< < P<
P < P < P
>
1.006 1.019 1.05 1.13 1.13 1.21) *
1.006 1.019 1.05 1.13 1.13 1.21) *
1.006 1.019 1.05 1.13 1.13 1.21) *
range
P
