Demonstration and some properties of the phospholipase a, lipase and cholesterol esterase from the aortic wall

Journal of A therosclerosis Research

Elsevier Publishing Company, Amsterdam - Printed in The Netherlands

D E M O N S T R A T I O N A N D SOME P R O P E R T I E S OF T H E P H O S P H O L I P A S E A, L I P A S E A N D C H O L E S T E R O L E S T E R A S E FROM T H E A O R T I C W A L L

j. PATELSKI, Z. WALIGORA AND S. SZULC Department of Physiological Chemistry, Medical Academy, Pozna~ (Poland)

(Received October 18th, 1966)

SUMMARY Phospholipase A, lipase and cholesterol esterase have been d e m o n s t r a t e d in the aortic wall. Some properties of these enzymes are described and discussed.

INTRODUCTION Different metabolic activities of the arterial wall and the importance of their alterations in the development of atherosclerosis have become a subject of considerable interest in recent years 1. Among them, the lipolytic or esterolytic activity, i.e. the ability to c a r r y out hydrolysis of carboxylic acid esters, is supposed to p r e v e n t the accumulation of f a t t y acid esters in the aortic wallg',3. However, little is known about specific esterases of the aorta as regards hydrolysis of glycerides, phospholipids and cholesterol esters. Esterase activities of a m m o n i a extracts from rat aortas in the presence of either coconut oil emulsion or chylomicrons were demonstrated b y KORN4. Higher enzyme activity values of different tissues observed using lipoproteins instead of entirely lipid substrates h a v e been ascribed to the specific action of "lipoprotein lipase ''5-s. When these observations are considered, however, the possibility t h a t there m a y act as m a n y different esterases as there are different f a t t y acid esters in chylomicrons and other lipoproteins cannot be overlooked. Thus, beside lipase also phospholipase and cholesterol esterase activities of crude extracts from the aortic wall h a v e to be t a k e n into account. A m m o n i a extracts from the aortic wall reveal cholesterol esterase activity. This e n z y m e as well as cholesterol and f a t t y acid esterifying e n z y m e from pig aortas h a v e been d e m o n s t r a t e d b y our l a b o r a t o r y previously 9. This p a p e r provides evidence for the existence of phospholipase A (phosphatide acyl-hydrolase, E.C 3.1.1.4), lipase (glycerol-ester hydrolase, E.C 3.1.1.3) and cholesterol esterase (sterol-ester hydrolase, E.C 3.1.1.13) in the aortic wall and describes some properties of these enzymes in crude extracts from pig aortas. J. Atheroscler. Res., 7 (1967) 453-461

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J. PATELSKI, Z. WALIG(~RA, S. SZULC

MATERIAL AND METHODS

Reagents and substrates All the commercial reagents used were of analytical grade: sodium, potassium and calcium chlorides (Merck, Germany), reduced glutathione, protamine sulphate (Light, England), sodium taurocholate (British Drug Houses) and heparin (HofmannLaRoche, Switzerland). Aqueous solutions were prepared in glass-distilled water. Organic solvents were purified, dehydrated and stored according to the methods in c o m m o n use. Hyperlipaemic serum of h u m a n blood, obtained 3 hours after a f a t t y meal consisting of 500 ml of cream, 50 g of bread and 100 g of butter, has been used in estimations of the lipolytic activity of the aorta. As substrates for particular enzyme examinations lecithin from egg yolk (Merck, Germany), purified after HANAHAN 10, glycerol trioleate (British Drug Houses) and cholesteryl oleate synthetized according to PAGE AND RUDY 11, have been used. Lecithin was prepared as a 0.5 % hydrosol and stored at 4 ~ Lysolecithin was proved absent b y thin-layer c h r o m a t o g r a p h y according to MARINETT112. Both, the glycerol trioleate and cholesteryl oleate were prepared as follows9: the esters were dissolved in hot 96 % ethanol and rapidly put into hot (approx. 80 ~ w a t e r at constant mixing by m e a n s of an electric vibrator (50 cycles/sec, 20 W, perforated disk 0 4 cm and a beaker 500 ml of volume and 0 10 cm). The proportions generally used a m o u n t e d to: 20/zmoles of glycerol trioleate or 40/zmoles of cholesteryl oleate in 2 ml of ethanol added to 3 or 6 ml of water, respectively. Larger volumes (400 ml) of the substrates were prepared and used in particular groups of investigations. Partial dispersion of the esters was obtained and the undispersed r e m n a n t s were r e m o v e d b y filtration ( W h a t m a n No. 1). The concentration of esterified f a t t y acids in the substrate hydrosols has been estimated b y titration of non-esterified 13 and total 14 f a t t y acids. The substrate hydrosols prepared in this way were stable at room t e m p e r a t u r e for several weeks.

Enzyme preparations Thoracic parts of aortas were obtained from several hundreds of pigs killed in slaughter house, cleaned in the cold room (0-2 ~ and sliced b y means of a freezing microtome. Wet and dried slices (acetone- and a c e t o n e - b u t a n o l " p o w d e r " prepared after MORTON15 a t - - 1 0 ~ were extracted with water, w a t e r containing electrolytes (sodium chloride 0.90 %, potassium chloride 1.22 %) and a g l y c e r o l - - w a t e r mixture (1 : 1, v/v). The extraction (9 g of wet or 3 g of dried tissue slices per 30 ml of a solvent) was carried out in Dacie electric cell suspension mixer at 4 ~ for 30 min. E x t r a c t s were stored at 4 ~ and the total protein concentration estimated b y the biuret reaction according to GLEISS AND HINSBERG9,16.

A ssay of enzyme activities Lipolytic activity of the aorta has been estimated using the sample-type j . Atheroscler. Res., 7 (1967) 453-46I

P H O S P H O L I P A S E A, L I P A S E A N D C H O L E S T E R O L E S T E R A S E IN T H E AORTIC W A L L

455

method according to ZEMPL~NYI et al. 2. Samples containing 5 ml of hyperlipaemic serum or 5 ml of glycerol trioleate hydrosol + 5 ml of buffer solution of Tris(hydrox y m e t h y l ) a m i n o m e t h a n e - H C 1 (0.05 M, pI-I 8.3) and either 50 mg of wet tissue slices or 5 ml of water e x t r a c t from acetone-butanol powder (12 m g of protein) were incubated for 150 rain at 37 ~ Non-esterified f a t t y acids have been extracted from three I ml specimens, before and after incubation, and titrated according to DOLE 13. The increase in their concentration was compared with the corresponding substrate controls and the lipolytic activity values were expressed in 10 -3 # m o l e s / m g of wet tissue or extract protein per 150 min. Final results represent mean values of three or more experiments. The activities of particular enzymes were assayed b y the continuous titration of f a t t y acid released into the reaction mixture at constant p H and 25 ~ The following reaction mixtures were usually used, unless otherwise stated. (1) Lecithin 2 5 / , m o l e s 4 ml of hydrosol (800 g accepted as molar weight) + water 20 ml + glycerol-water extract from a c e t o n e - b u t a n o l powder, 2.5-3.5 mg of protein/1 ml, p H 8.0. (2) Glycerol trioleate 8 #moles/5 ml of hydrosol + water 13 ml + w a t e r extract, 6.0-8.0 mg of protein/2 ml, pFI 8.3. (3) Cholesteryl oleate 20/zmoles/8 ml of hydrosol + sodium taurocholate 80 #moles/0.5 m l of aqueous solution + w a t e r 9.5 ml + glycerol-water extract, 5.0-7.0 mg of protein/2 ml, p H 8.6. The desired p H of b o t h the substrate solutions and e n z y m e e x t r a c t s were adjusted separately, before and after mixing. Determinations were carried out with 0.02 N solution of sodium hydroxide b y means of a p H - s t a t (Radiometer, Titrator lc-type and Titrigraph S B R 2c-type), with nitrogen flow. The increase in f a t t y acid concentration was read at the 5th minute of the reaction. The titration values of substrate controls (without enzymes) and of enzyme controls (without substrates) were subtracted from those of the whole system. Results obtained h a v e been expressed in milliunits of specific activity, i.e. in 10 -a /~moles/mg of protein/rain. Measurements of enzyme activities in particular groups of investigations were carried out and repeated three times or more in a short series on freshly prepared extracts. Specific activity values for the lipase and cholesterol esterase were corrected m a t h e m a t i c a l l y for spontaneous loss of the enzyme activities in extracts stored at 4 ~. RESULTS

Table 1 shows the results of the lipolytic activity obtained b y means of the sample-type method. The activity value found using w a t e r extract from acetonebutanol powder was approx, four times higher than t h a t obtained with wet tissue slices when incubated in hyperlipaemic serum. Activity observed following incubation of the extract with a hydrosol of glycerol trioleate was approx. 7 times lower, b u t it reached almost the same value when calcium chloride was added to the reaction mixture (Table 1). Table 2 shows the results obtained using the continuous titration procedure. I t can be seen t h a t the specific activity values of enzymes examined in extracts prepared j . Atheroscler. Res., 7 (1967) 453--461

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TABLE 1 THE

LIPOLYTIC

ACTIVITY

VALUES

OF THE

Experiment No.

Substrate solution

1 2 3

hyperlipaemic serum

Enzyme preparation

Lipolytic activity 10-3 #moles/ rag~ 150 rain

glycerol trioleate hydrosol+ calcium

4

AORTA

%

wet tissue slices 28 ~ 108 I water extract from 15 ~ a c e t o n e - b u t a n o l powder 106

100 14 99

chloride solution E x p e r i m e n t a l conditions: 5 mi of substrate solution + 50 m g (1) or 5 ml ( 12 mg of protein; 2, 3, 4) of enzyme p r e p a r a t i o n + 5 ml of Tris(hydroxymethyl)aminomethane-MC1 buffer solution (0.05 M , p H 8.3) alone (2, 3) or with 2.2% calcium chloride (4). Corresponding controls consisted of hyperlipaemic serum (1) a n d glycerol trioleate hydrosol w i t h all t h e other reagents except for w a t e r instead of enzyme e x t r a c t (2, 3, 4). The increase in non-esterified f a t t y acid concentrations has been estimated b y extraction and t i t r a t i o n of f a t t y acids according to DOLE in three parallel specimens of 1 ml each, before and 150 rain after i n c u b a t i o n of t h e reaction m i x t u r e s a t 37 ~ TABLE 2 SPECIFIC

ACTIVITY

VALUES

OF ESTERASES

OF THE

AORTA

Substrate lecithin Material extracted

glycerol trioleate

cholesteryl oleate

glycerol-H20 (1 : 1, v/v)

H20

glycerol-H20 (1 : 1, v/v)

p H 8.0

p H 8.3

p H 8.6

370 100 69 23

30 100 43 0

10 (25) b 100 37 25

Extract

A-B-(acetone-butanol)powder (mU) W e t tissue slices (%) A-powder from extracted tissue (%) A-B-powder from e x t r a c t e d tissue (%)

(260)a (100) (86) (27)

31 ( 3 3 )

25

34

Reaction mixtures: 25/*moles of lecithin or 8/*moles of glycerol trioleate or 20 #moles of cholesteryl oleate + 2 ml of t h e extract + water up to 20 ml 25 ~ T i t r a t i o n reagent 0.02 N N a O H using pH-stat. a Specific a c t i v i t y values (in parentheses) of e x t r a c t s i n a c t i v a t e d for 10 rain a t 60 ~ b The a c t i v i t y value obtained with sodium t a u r o c h o l a t e present in the reaction m i x t u r e in 4 • 10 -3 molar concentration.

from acetone-butanolpowder

were distinctly higher than those in extracts from wet

t i s s u e s l i c e s ( T a b l e 2). Highest phospholipase and cholesterol esterase activities were found in glycerolwater extracts.

Highest

lipase activity

j . Atheroscler. Res., 7 (1967) 453-461

appeared

in water

extracts.

These extracts

PHOSPHOLIPASE A, LIPASE AND CHOLESTEROL ESTERASE IN THE AORTIC W A L L TABLE

457

3

ESTERASE

ACTIVITIES

OF

EXTRACTS

PREPARED

Solvent

ACETONE--BUTANOL

POWDER

FROM

AORTAS

g/lO0 ml

protein (mg/ml)

Phosphatide Glycerol-ester Sterol-ester acylhydrolase (2) hydrolase (3) hydrolase(1) ( 10-3 #moles/mg/min)

SO (v/v)

3.50 3.00

185 260

30 18

0 25

5.00 5.00

147 --

16 0

10 1

Extract

Water Glycerolwater KC1 NaC1

FROM

1.22 0.90

R e a c t i o n m i x t u r e s : l e c i t h i n 25 # m o l e s (1), glycerol t r i o l e a t e 8 # m o l e s (2), c h o l e s t e r y l o l e a t e 20 # m o l e s (3) a n d a p p r o p r i a t e e n z y m e e x t r a c t s 2 m l a n d w a t e r u p to 20 m l ; p H 8.0 (1), 8.3 (2) a n d 8.6 (3); 25 ~ T i t r a t i o n r e a g e n t 0.02 N N a O H u s i n g p H - s t a t .

%2o01 E

--~20 40

~ 2O

111

10

7.5

8.0

8.5 pH

9.0

Fig. 1. E f f e c t of p H of t h e r e a c t i o n m i x t u r e s o n t h e p h o s p h o l i p a s e (I), lipase (II) a n d c h o l e s t e x o l e s t e r a s e (III) a c t i v i t i e s . R e a c t i o n m i x t u r e s : l e c i t h i n 25 # m o l e s (I), glycerol t r i o l e a t e 8 # m o l e s (II), c h o l e s t e r y l o l e a t e 20 # m o l e s + s o d i u m t a u r o c h o l a t e 80 # m o l e s (III) a n d g l y c e r o l - w a t e r e x t r a c t 10 m i n u t e s i n a c t i v a t e d a t 60 ~ (I), w a t e r e x t r a c t (II), g l y c e r o l - w a t e r e x t r a c t (III) f r o m a c e t o n e b u t a n o l p o w d e r , 2 re_l, r e s p e c t i v e l y , a n d w a t e r u p to 20 re_l; 25 ~ u s i n g p H - s t a t , a n d t i t r a t i o n r e a g e n t 0.02 N N a O H .

contained more protein and showed only approx. 70 % of phospholipase activity of glycerol-water extracts and no cholesterol esterase activity. In extracts prepared with potassium chloride solution only 40-55 % of the enzyme activities of the appropriate richest extracts have been found. Extracts prepared with sodium chloride solution were poorly active (Table 3). Further results were obtained using extracts from acetone-butanol powder, J. Atheroscler. Res., 7 (1967) 4 5 3 - 4 6 1

458

1. PATELSKI, Z. WALIGORA, S. SZULC

100-

~80~< 60"

2O

Time (h)

Fig. 2. Phospholipase (I), lipase (II) and cholesterol esterase (III) activities in extracts stored at 4 ~ Experimental conditions as given in Fig. 1. p H 8.0 (I), 8.3 (II) and 8.6 (III). TABLE 4 :EFFECT

OF ELECTROLYTES

ON ESTERASE

Inhibition ( % )

(1) Phosphatide acyl hydrolase

(2)

Glycerol ester hydrolase

ACTIVITIES

OF THE

AORTA

KCl NaC1 concentration (10 -3 M )

50

200 pIs0

CaCl~

13.0 1.7

0.1 1.9

100

4.0 0.8

50

6.5 pIs0

0.5-20.0 a

1.5 2.2

100

2.8 4.0

(3) Sterol ester hydrolase

50

6.3 pIso

100

1.9 2.2

25.0

0.5 2.7

10.0

3.3 2.5

]Experimental conditions as given in Fig. 1 and appropriate electrolyte concentrations, pH 8.0 (1),

8.3 (2) ~nd 8.6 (31.

pIs0, -log M concentration of the compound at which 50 % of t h e a c t i v i t y was decreased. a 9-13 % inhibition 9

viz., glycerol-water extracts for the phospholipase and cholesterol esterase and water extracts for the lipase estimations. Fig. 1 illustrates the effect of p H of the reaction mixture, with substrate saturation, on the phospholipase, lipase and cholesterol esterase activities. Storage of the enzyme extracts at 4 ~ results in a decrease in the cholesterol exterase and lipase activities of 7 % and 11 ~ respectively. B o t h enzymes are inactivated b y heating for 10 min at 60 ~ There is a fall in total phospholipase activity of 20-30 % of the initial values during the first hour of storage at 4 ~ (or after 10 min heating of the extract at 60~ F u r t h e r storage brings a b o u t only a further small dej . Atheroscler. Res., 7 (1967) 453-461

PHOSPHOLIFASE A, LIPASE AND CHOLESTEROLESTERASE IN THE AORTIC WALL

459

ooI 400 -

300220[

10(

o

~

t

~

~

C o n c e n t r Q t i o n ( r n g l l O 0 ml )

Fig. 3. Susceptibility of t h e aortic lipase to heparin (H) and p r o t a m i n e sulphate (PS). E x p e r i m e n t a l conditions see Fig. 1 (n) + corresponding concentrations of h e p a r i n and p r o t a m i n e sulphate in t h e reaction mixtures, p H 8.3.

crease of 0.15 % / h (Fig. 2). The conversion of lecithin into lysolecithin in the reaction mixture containing the thermoresistant enzyme and lecithin, was found. Reduced glutathione added to the reaction m i x t u r e in aqueous solution, to achieve final concentration of 5 • 10 -5 M, more than doubled the lipase and cholesterol esterase activities a n d stabilized them for at feast 15 hours. I t had no activating effect on the phospholipase. Calcium, sodium and potassium ions in 10 -3 M concentration range examined decreased all the e n z y m e activities (Table 4). The activity of cholesterol esterase is enhanced approx. 2.5 times b y sodium taurocholate present in the reaction mixture in a molar concentration of 4" 10 -3 (see Table 2). The lipase and phospholipase activities are depressed b y this bile acid salt: appropriate pls0 coefficients (-log molar concentration of the c o m p o u n d at which 50 % of the activity is decreased) amount to 2.3 and 2.2, respectively. The lipase is a c t i v a t e d b y heparin in low concentrations and inhibited b y protamine sulphate as well as b y higher concentrations of heparin (Fig. 3). DISCUSSION

The higher lipolytic activity of a water extract from acetone-butanol powder as compared with t h a t of wet tissue slices, using the s a m p l e - t y p e m e t h o d and hyperlipaemic serum, was p r o b a b l y connected with the higher content of active proteins in the former. Undoubtedly, the use of the extract instead of tissue slices resulted in better substrate saturation of the enzymes. The enzymic activity of the extract incubated in glycerol trioleate hydrosol was much lower t h a n t h a t observed with hyperlipaemic serum. The e n h a n c e m e n t of glycerol trioleate hydrolyzing activity b y calcium chloride added to the reaction mixture indicates the requirement of a f a t t y acid j . Atheroscler. Res., 7 (1967) 453--461

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j. PATELSKI, Z. WALIG6RA, S. SZULC

acceptorS,17,18 in the sample-type method. For experiments carried out with hyperlipaemic serum the reaction mixture contained albumin which could act as the f a t t y acid acceptorS, 19. However, hyperlipaemic serum contains a mixture of different f a t t y acid esters and in the crude extracts different esterases are present, whereas the glycerol trioleate represents a specific substrate for one enzyme. Thus, the difference between enzyme activity values of extracts incubated either in hyperlipaemic serum or in glycerol trioleate hydrosol seems to be sufficiently explained b y the presence of a multi-enzyme system for hyperlipaemic serum. Results obtained b y the continuous titration of f a t t y acids released from pure hydrosol substrates provide evidence for the existence of different enzymes which catalyze hydrolysis of phospholipids, glycerides and cholesterol esters in the aortic wall. I t m a y be presumed t h a t all these enzymes act when chylomicrons or other lipoproteins are used for estimations of "lipoprotein lipase" in crude extracts. Regarding the extraction procedure, the higher enzyme activities of the extracts prepared from acetone-butanol powder than from wet tissue slices can be explained b y the action of butanol 15 and presumably of acetone in disrupting the lipoprotein complexes. I n the continuous titration of f a t t y acids released during substrate hydrolysis, calcium ions showed a depressing effect on the enzyme activities investigated. Sodium and potassium ions also showed this effect, but to a lesser degree. The described influence of electrolytes seems to depend on the known coagulating ability of ions on the substrate hydrosols20, 21. The total phospholipase activity of the a o r t a exceeded approx. 12 times t h a t of the lipase and over 8 times for the thermoresistant enzyme. The conversion of lecithin into lysolecithin with an extract inactivated at 60 ~ for 10 min indicates t h a t the thermoresistant enzyme is phospholipase A. Also its properties, such as p H o p t i m u m and thermoresistance, are similar to those of phospholipase A of other tissues 22-25. The observation t h a t heparin m a y activate or inhibit the aortic lipase in vitro, depending on the concentration, is in agreement with a similar one of KORN, using an a m m o n i a extract from rat heart acetone powderS. Excess of heparin, protamine sulphate, sodium taurocholate, sodium chloride and heating for 10 min at 60 ~ are known to inhibit "lipoprotein lipase", whether it is from tissues or postheparin and nonheparin plasmaS,26, 27. In our experiments the aortic lipase examined using glycerol trioleate hydrosol is also inhibited b y these factors. The effect of reduced glutathione indicates the i m p o r t a n c e of SH-enzyme groups in the lipase and cholesterol esterase activities. This compound m a y serve for stabilizing the two enzymes. The cholesterol esterase of the aorta is similar in its properties to those isolated from intestine and pancreas 28. The activating effect of sodium taurocholate on the cholesterol esterase seems to result from a coenzyme-like action, as was suggested b y MURTHu AND GANGULY 28, rather, than from its s u b s t r a t e emulsifying ability: both, the lipase and phospholipase were inhibited b y this salt. The different activity levels of arterial phospholipase A, lipase and cholesterol esterase m a y determine the different accumulation of the three main lipid esters in the arterial wall during ageing and in atherosclerosis. j . Atheroscler. Res., 7 (1967) 453.--461

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REFERENCES 1 ZEMPLI~NYI, T., E n z y m e s of t h e arterial wall, J. Atheroscler. Res., 1962, 2: 2. 2 ZEMPL]ENYI, T., Z. LOJDA AND D. GRAFNETTER, R e l a t i o n s h i p of lipolytic a n d e s t e r o l y t i c a c t i v i t y of t h e a o r t a t o s u s c e p t i b i l i t y to e x p e r i m e n t a l a t h e r o s c l e r o s i s , Circulation Res., 1959, 7: 286. 3 ZEMPLI~NYI, T., T h e l i p o l y t i c a n d e s t e r o l y t i c a c t i v i t y of b l o o d a n d t i s s u e a n d p r o b l e m s of a t h e r o s c l e r o s i s . I n : R. PAOLETTI AND D. KRITCHEVSKY (Eds.) Advances in L i p i d Research, Vol. 2, A c a d e m i c P r e s s , N e w York, L o n d o n , 1964, p. 238. 4 KORN, E. D., C l e a r i n g factor, a h e p a r i n - a c t i v a t e d l i p o p r o t e i n lipase, P a r t 1 ( I s o l a t i o n a n d c h a r a c t e r i z a t i o n of t h e e n z y m e f r o m n o r m a l r a t h e a r t ) , J. biol. Chem., 1955, 215: 1. 5 KORN, E. D., P r o p e r t i e s of c l e a r i n g f a c t o r o b t a i n e d f r o m r a t h e a r t a c e t o n e p o w d e r , Science, 1954, 120: 399. 6 KORN, E. 3 . , C l e a r i n g f a c t o r , a h e p a r i n - a c t i v a t e d l i p o p r o t e i n lipase, P a r t 2 ( S u b s t r a t e specifi c i t y a n d a c t i v a t i o n of c o c o n u t oil), J. biol. Chem., 1955, 215: 1S. 7 WILLIAMS, G, R,, L i p o p r o t e i n t r a n s f o r m a t i o n s u n d e r t h e i n f l u e n c e of h e p a r i n a n d Clostridium welchii o - t o x i n , Biochim. Biophys. Acta, 1954, 13: 72. 8 KORN, E. D., T h e a s s a y of l i p o p r o t e i n lipase in vivo a n d in vitro. I n : D. GLICK (Ed.) Methods 6f Biochemical Analysis, Vol. 7, I n t e r s c i e n c e P u b l i s h e r s , N e w Y o r k , L o n d o n , 1959, p. 145. 9 PATELSKI, J., Esteraza Cholesterolowa Tetnicy Gl6wnej (Cholesterol e s t e r a s e of t h e a o r t a ) , P a f i s t w o w y Z a k l a d W y d a w n i c t w L e k a r s k i c h , W a r s a w , 1964. 10 HANAHAN, D, J., M. RODBELL AND L. D. TURNER, E n z y m a t i c f o r m a t i o n of m o n o p a l m i t o l e y l a n d m o n o p a l m i t o y l l e c t h i n (lysolecithins), J. biol. Chem., 1954, 206: 431. 11 PAGE, I. H. AND H . RUDY, 13bet die F e t t s / i u r e e s t e r d e s C h o l e s t e r i n s (On f a t t y a c i d e s t e r s of cholesterol), Biochem. Z., 1930, 220: 304. 12 MARINETTI, G. V., C h r o m a t o g r a p h i c s e p a r a t i o n , i d e n t i f i c a t i o n a n d a n a l y s i s of p h o s p h a t i d e s , J. L i p i d Res., 1962, 3: 1. 16 DOLE, V. P., A r e l a t i o n b e t w e e n n o n - e s t e r i f i e d f a t t y a c i d s in p l a s m a a n d t h e m e t a b o l i s m of glucose, J. clin. Invest., 1955, 35: 1S0. 14 ALBRINK, M. J., T h e m i c r o t i t r a t i o n of t o t a l f a t t y a c i d s of s e r u m w i t h n o t e s on t h e e s t i m a t i o n of t r i g l y c e r i d e s , J . L i p i d Res., 1959, 1: 53. 16 MORTON, R. K . , M e t h o d s of e x t r a c t i o n of e n z y m e s f r o m a n i m a l t i s s u e s . In: S. P. COLOWICK AND N. O. KAPLAN (Eds.) Methods in Enzymology, Vol. l, A c a d e m i c Press, N e w Y o r k , 1955, p. 25. 16 HINSBERG, K . AND BLUT. I n : K. LANG, E. LEHNARTZ AND G. SIEBERT (Eds.), Hoppe-Seyler/Thierfelder Handbuch der physiologisch- und pathologisch-chemischen Analyse, Vol. 5, 10th ed. Springer, B e r l i n , G 6 t t i n g e n , Heidelberg, 1953, p. 1. 17 GROSSMAN, M., J. STADLER, A. CUSHING AND L. PALM, R e l a t i o n of lipolysis to d e t u r b i d i f i c a t i o n a n d r e t u r b i d i f i c a t i o n in h e p a r i n i n d u c e d l i p e m i c c l e a r i n g p h e n o m e n o n , Proc. Soc. exptl. Biol. ( N . Y . ) , 1955, 88: 132. 18 SEIFTER, J. ANn D. H . BAEDER, I n f l u e n c e of c a l c i u m a n d s e r u m a l b u m i n on c l e a r i n g of l i p e m i c dog p l a s m a " i n vitro", Proc. Soc. exptl. Biol. (N. Y . ) , 1955, 8 9 : 5 1 1 . 19 CAMPBELL, J., A. D. MARTUCCI AND G. R. GREEN, P l a s m a a l b u m i n s as all a c c e p t o r of free f a t t y acids, Biochem. J., 1964, 93: 183. 20 LEES, M. B., P r e p a r a t i o n a n d a n a l y s i s of p h o s p h a t i d e s . In: S. P. COLOWICK AND N. O. KAPLAN (Eds.) Methods in Enzymology, Vol. 3, A c a d e m i c Press, N e w York, 1958, p. 328. 21 ABRAMSON, M. B., R. KATZMAN AND H. P. GREGOR, A q u e o u s d i s p e r s i o n s of p h o s p h a t i d y l s e r i n e , J. Biol. Chem., 1964, 239: 70. 22 HAYAISHI, O. AND A. KORNBERG, M e t a b o l i s m of p h o s p h a t i d e s b y b a c t e r i a l e n z y m e s , or. Biol. Chem., 1954, 206: 647. 26 DAWSON, R. M. C., T h e p h o s p h o l i p a s e B of liver, Biochem. J., 1956, 64: 192. 24 DE GIER, J., G. M. DE HAAS AND L. L. VAN DEENEN, A c t i o n of p h o s p h o l i p a s e s f r o m Clostridium welchii a n d Bacillus cereus on red c e l l - m e m b r a n e s , Biochem. J., 1961, 81: 33P. 26 MAGEE, W . L., J. GALAI-HATCHARD, H. SANDERS AND R. H. S. THOMPSON, T h e p u r i f i c a t i o n a n d p r o p e r t i e s of p h o s p h o l i p a s e A f r o m h u m a n p a n c r e a s , Biochem. J., 1962, 83: 17. 26 ENGELBERG, H,, H u m a n e n d o g e n o u s l i p e m i a c l e a r i n g a c t i v i t y . S t u d i e s of lipolysis a n d effects of i n h i b i t o r s , J. Biol. Chem. 1956, 222: 601. 27 ZEMPLI~NYI, T. AND D. GRAFNETTER, F a t t y acid release o n i n c u b a t i o n of l i p a e m i c s e r u m w i t h r a t t i s s u e s . T h e i n f l u e n c e of h e p a r i n a n d f a s t i n g , Acta Int. Pharmacodyn., 1959, 122: 25. 28 MURTHY, S. K. AND J. GANGULY, S t u d i e s o n c h o l e s t e r o l e s t e r a s e s of t h e s m a l l i n t e s t i n e a n d p a n c r e a s of r a t s , Biochem. J., 1962, 83: 460.

]. Atheroscler. Res., 7 (1967) 4 5 3 - 4 6 1