Modification of enzyme activities in experimental atherosclerosis in the rabbit

Atherosclerosis Elsevier Publishing

MODIFICATION SCLEROSIS

Company,

Amsterdam

OF ENZYME

IN THE

41

- Printed in The Netherlands

ACTIVITIES

IN EXPERIMENTAL

ATHERO-

RABBIT

J. PATELSKI*, D. E. BOWYER, G. A. GRESHAM

A. N. HOWARD,

I. W. JENNINGS,

C. J. R. THORNE

AND

Departments of Pathology, Cambridge (Great Britain) (Revised,

Investigative

Medicine

and Biochemistry,

University

of Cambridge,

received January 29th. 1970)

SUMMARY

In rabbits fed an atherogenic semi-synthetic diet, the following were found: in the aortic wall, decreased cholesterol esterase and increased lipase and phospholipase A activities, no changes in malate and lactate dehydrogenase activities, enhanced incorporation of free fatty acid into cholesterol esters, and increased accumulation of mainly esterified and also free cholesterol; increase in plasma cholesterol and serum phospholipase A activity but no change in liver phospholipase A and serum and liver lipase activities. Compared with control animals, those fed the same diet and injected with Lipostabil

(a drug containing

polyunsaturated

lecithin)

showed no alterations

in the

aortic enzyme activities and serum phospholipase A and in the incorporation of free fatty acid into aortic cholesterol esters; serum and liver lipase activities were increased. Compared with atherogenic diet-fed animals injected with saline, the severity of atherosclerosis and the incorporation of free fatty acids into the aortic wall were reduced; there was no change in the elevated plasma cholesterol levels. The mechanism of cholesterol ester accumulation in the arterial

wall is dis-

cussed in the light of these observations.

Key words:

Cholesterol

esters - Lipolytic

enzymes

- Lipostabil

- Rabbit

atherosclerosis

INTRODUCTION

The cholesterol * Permanent (Poland).

relationship between the metabolism of phospholipids, glycerides esters in the arterial wall and the development of atherosclerosis address:

Department

of

Physiological

Chemistry,

Medical

Academy,

Atherosclerosis,

and has Poznan

1970, 12: 41-53

42

J. PATELSKI et al.

become a subject of considerable interest in recent years. In vitro studies on the biochemistry of enzymes involved in metabolism of these fatty acid esters have been performedr, but not much is known about the factors affecting the enzyme activities irz viva. In a previous study, it was shown that in experimental atherosclerosis in rats and in rabbits, there is an increase in lipase* and a decrease in cholesterol esterase activity in the arterial wall. It was suggested that these might be contributing factors in the production of the arterial lesionss. The present experiments were carried out in order to test whether the activities of the arterial enzymes involved in fatty acid ester metabolism could be modified and how this might affect the arterial

wall. Measurements

were made in rabbits

fed

control and atherogenic diets and injected with a drug containing polyunsaturated lecithin, of the following: (1) the specific activities of phospholipase A, lipase and cholesterol esterase, and lactate and malate dehydrogenases in aortic, liver and serum extracts, (2) [ I-r%]oleic acid incorporation into aortic lipids, (3) concentration of aortic phospholipids, free and esterified cholesterol, and free fatty acids. The aortas were also examined macroscopically

and microscopically.

MATERIALS AND METHODS

Reagents and substrates Commercial reagents of reagent grade were used and organic solvents were redistilled before use. The purity of substrates liquid chromatographys.

was checked by thin-layer and gas-

The substrates were prepared as hydrosolsl as follows: both

glyceryl trioleate (Calbiochem, U.S.) and cholesteryl

oleate (Koch-Light,

England)

were dissolved in boiling absolute ethanol (50 pmoles/5 ml). 5 ml of solution was then added to 15 ml of water (for glyceryl trioleate) or 15 ml of 2.135% (w/v) sodium taurocholate

solution (Calbiochem) (for cholesteryl oleate) in 25 ml glass vessels, at

approximately 80°C and the mixture sonicated for 20 min (MSE Ultrasonic disintegrator, output 60 W, Measuring and Scientific Equipment LTD, England, for 5 min). These hydrosols were kept at room temperature.

Lecithin

(from egg yolk,

Koch-Light), and polyunsatureated lecithin (EPL, Nattermann) were prepared by sonicating the phospholipid in distilled water and stored at 4°C. Total4 and frees fatty acid concentrations were measured in the hydrosols. Lipostabil (Nattermann, Cologne, Germany) was an intravenous injection of EPL commercial product did not contain nicotine acid or AMP.

(mg/ml) but unlike the

Alzimals Male New Zealand White rabbits, which were 24 weeks of age at the start of the experiment, were used. Control animals were fed a laboratory stock diet of commercial * Lipase-glycerol-ester hydrolase (EC 3.1.1.3)) phospholipase A-phosphatide-acyl hydrolase (EC 3.1.1.4), cholesterol esterase-sterol-ester hydrolase (EC 3.1.1.13)) malate dehydrogenase-Lmalate:NAD oxidoreductase (EC 1.1.1.37), lactate dehydrogenase-L-1actate:NAD oxidoreductase (EC 1.1.1.27). Atherosclerosis,

1970, 12: 41-53

MODIFiCATION

pellets.

OF ENZYME

ACTIVITIES

The experimental

animals

IN EXPERIMENTAL

43

ATHEROSCLEROSIS

were all fed a semi-synthetic

diet which produced

hyperlipaemia and atherosclerosis6. They were injected every 2nd day via the marginal ear vein as follows: Group A: 1.0 ml saline for 10 weeks, Group B: 1.0 ml Lipostabil for 10 weeks, Group C: 0.5 ml saline for 18 weeks, Group D: 0.5 ml Lipostabil

for 18

weeks. Preparation

of enzyme

extracts

After 24 h fasting,

the animals

drawn from the inferior

were killed by asphyxiation

vena cava. Samples

and frozen in solid COz. The abdominal

of liver were taken

aortas were prepared

with COz and blood (approximately

for perfusion

0.5 g)

and estima-

tion of lipid concentrations, as described below. The thoracic aortas were removed, the adventitia and loose connective tissue carefully stripped off, the aortas opened and divided

longitudinally

into two equal parts.

used for gross staining

and for microscopical

with

water,

ice-cold

Frozen

distilled

aortas

lyophilised

dried

with

blotting

and frozen livers were pulverised

for 48 h. Acetone-butanol

One part was put into formalin

examination.

and

The other part was rinsed

paper

and

frozen

in solid COz.

in a freeze press and the sera were

powders were then prepared

at -20°C

accord-

ing to MORTON~. For the assay of the activity of aortic enzymes, the powders were extracted with 50 o/0 (v/v) glycerol in water (7.515 mg/ml) for 30 min at 4°C using a Dacie

electric

cell suspension

mixer

(Surgical

and Scientific

Apparatus,

Matburn,

England). The extracts were decanted and filtered through Whatman No. 1 paper. The solid residue was re-extracted twice in half the original volume of glycerol-water by homogenising

in an Ultra-Turrax

(Janke and Kunkel)

for 5 sec. The second extracts

were filtered and mixed with the first. Liver and serum powders were extracted water (20 mg/ml) for 30 min at 4°C using the cell suspension determined according to Lowry et al. as described by LEGGETT Assay

of enzyme The

Protein

with was

BAILEY~.

activities

lijolytic

enzymes.

earlier’.

The reaction

distilled

water as follows:

(ii) Glyceryl

mixer.

trioleate

mixtures

The assay contained

was carried

out by the method

1 mM concentrations

(i) Egg yolk lecithin

or polyunsaturated

at pH 8.3 for the aortic enzyme,

of substrates lecithin

described in glass at pH 8.0.

pH 8.4 for the liver enzyme and

pH 8.9 for the serum enzyme and reduced glutathione (0.1 mAI). (iii) Cholesteryl oleate at pH 8.6 and reduced glutathione (0.1 mM) and sodium taurocholate (4 mm). Reactions were carried out by continuous titration in a pH-stat (Radiometer, Copenhagen) at 30°C under nitrogen, using 0.002 N KOH solution as the titrating reagent. The pH of the reaction mixture was adjusted to the appropriate value in the pH-stat and the enzyme extract (0.03-0.06 mg protein in 0.2-0.4 ml of aortic extract, 0.200.40 ml of protein in 0.05-O. 1 ml of liver extract and 0.50-1.00 mg of protein in 0.050.10 ml of serum extract) was added to give a total reaction mixture of 1 ml. Enzyme activity was measured by calculating the rate of release of fatty acid after the reaction had proceeded for 5 min, and expressed in munits of specific activity, i.c. in nequivalents of titrateable acidity released per min per mg of protein. Atherosclerosis,

1970, 12: 41-53

44

J. PATELSKI

Malate and lactate dehydrogenases.

DK2 recording

spectrophotometer.

glycine-sodium sodium

hydroxide

buffer

Assays were carried out at 30°C in a Beckman

The reaction

In_-lactate

was measured

(BDH).

by following

Aortic perfusion

aortas

were ligated

sions were carried

L-malate

0.1 ml of extract the increase

(Calbiochem)

was added

in absorption

to start

or 0.1 ml of 1.0 M the reaction

which

at 340 nm (ref. 9).

were perfused

essentially

and the aorta suspensed

as described in a perfusion

out for 1 h at 37°C with Krebs bicarbonate

out the experiment albumin

2.7 ml of 0.1 M

contained

estimation of ,%$&I!composition

ad

The abdominal major branches

cuvettes

(pH 10.0); 0.1 ml of 11.7 mM NAD (Boehringer,

and 0.1 ml of 1.0 M sodium

Germany)

et d.

with Os/5 o/o COs, containing

(Cohn fraction

5, Armour)

beforelo. chamber.

buffer, gassed through-

4 mg/lOO ml defatted

and 0.1 ,uequiv/ml

The Perfu-

bovine

serum

of [ 1-r4C]oleic acid (Amersham,

England). Following the perfusion, the aortas were rinsed with cold 0.9 y. saline and the intima, plus approximately one-third of the inner media peeled off, frozen and disintegrated by freeze pressing. The lipids were then extracted with chloroformmethanol

(2:1, v/v) and aliquots

of the extract

separated

raphy into the major neutral lipids and phospholipids, (Merck) run with light petroleum (b.p. 40-60”C)-diethyl v/v/v).

The separated

lipids were located

by spraying

on thin-layer

chromatog-

using silica gel H plates ether-acetic acid (85:15:1,

plates with a fluorescent

dyerr.

Radioactivity was measured in separated fractions in a Packard series 4000 scintillation counter. The concentrations of lipids were measured by chemical assay; phospholipid cholesterol

by assay of phosphorus, by

the

method

of BABSON

by the method of USHERI~, free and esterified et al.13 and free fatty acids by the method of

MARSH AND WEINSTEIN14.

Macroscopic

and microscopic

examination

of the aortas

The aortas were examined macroscopically for intimal lesions after gross staining with Sudan IV according to the method of HOLMAN et a1.15~16and microscopically after staining sections by a variety of conventional methods. RESULTS

Data on fatty acid composition and concentrations of the substrates used for lecithin contained enzyme activity assays are listed in Table 1. Polyunsaturated 79.3% polyunsaturated fatty acids, mainly linoleic (77.4%) with less than 15% saturated fatty acids. Lecithin from egg yolk contained 24.8% oleic and 55.9 y. saturated fatty acids, mainly palmitic (45.8%). All of the fatty acids in glyceryl trioleate, and 93.7 o/o of the fatty acid in cholesteryl oleate was oleic acid. Not less than 98.5 y. of the total fatty acids of the substrates were present in an esterified form in the hydrosols. The measured concentrations of the substrates in the disperse phase of the hydrosols amounted to 100 y. for polyunsaturated lecithin, approximately 90 y. Atherosclerosis,

1970, 12: 41-53

1

ACID

Lecithin Polyunsaturated lecithin Glyceryl trioleate Cholesteryl oleate

Substrate

FATTY

TABLE

AND

4.2

0.3

1.9

45.8

11.3

1611

76:O

2.6

10.1

18:O

77.4

5.9

93.7

1.4

11.5

24.8

100.0

1812

1.9

1813

OF SUBSTRATES

18x1

CONCENTRATION

Fatty acid composition

COMPOSITION

0.2

2013

100

0.6

24

48

7,002 2,206

0.7 1.1

0

13,300

0

0.9

92

12,215

1.5

190

2.9

2.8

nmolesl ml

%

hydvosol

88

70

y0

esterified (found)

Fatty acid concentration free (found)

others

20~4

2,500

10,000

13,300

2,230

7,050

13,300

12,405

92.m0kl

ml ml 13,300

found

theoretical nmolesl

total

w

G e 1,

;;

5

5 5

5

5

animals

of

No.

OF THE

1.1e

3.1 * 0.7

3.6 & 0.8 3.6 -+ 1.3

3.2 + 0.5

3.4 *

( % wlw)

Extracted proteinb

AORTA

(+) 137 y0 34 f 8.8

42 f 5.2 48 + 8.3

35 * 4.1 100% 41 + 10.8

Pkospholipase A ‘1

18 i

5.7

27 * 8.5

19 * 0.9

Phospholipase AC2

Cholesterol esterasec4

\go; +’ 37 *?1.4 51 * 16.4

30 + 2.4 100 y0 57 f 12.8 + +)

+ +)

19 1O4.4

(t3j;

18 1’6.8 8 + 2.8

(Z5$

24 + 5.8 100 y0 6 & 5.3

(nequivlminlmg)

Lipaseca

361 f

90

419 f 149 422 + 137

381 + 117

Malate dehydrogenasedl

213 f

210 f 271 i

218 &

78

57

84 132

Lactate dehydrogenased2

alIntraperitonea1 injection of 1.0 ml 6 times a week. ~(2,s) Intravenous injections of (2) 0.5 ml and (3) 1 ml 3 times a week. b Extraction from acetone-butanol powder with 50 o/o (v/v) glycerol in water (7.5-15 mg/ml) using a Dacie electric cell suspension mixer, followed by second extraction (7.5-15 mg/0.5 ml) using an Ultra-Turrax homogenizer at 4’C, for 30 min and 10 set respectively. c Reaction mixtures (total volume 1 ml) contained: (1) lecithin 1 mM; (2) polyenephosphatidyl choline 1 mM; (3) glyceryl trioleate 1 mM, reduced glutathioneo .I mM; (4) cholestery loleate 1 mM; reduced glutathione 0.1 mM, sodium taurocholate 4 mM and enzyme extract 0.03-0.06 mg protein/ potassium 0.2-0.4 ml. The pH values were (1,2) 8.0 (3) 8.3 (4) 8.6. The reaction temperature was 30% The titration reagent was 0.002 N hydroxide solution (using pH-Stat, Radiometer). d Reaction mixtures (total volume 3 ml) used were as follows: glycine 0.09 M pH 10, NAD 3.9 * 10-4 M and sodium L-malate 3.3 * 10-s M (1) or sodiumDL-lactate 3.3 * 10-e M (2) and 0.1 ml enzyme extract. The reaction was followed by measuring the increase in absorption at 340 nm at 30°C Beckman DK2 recording spectrophotometer). e Means k standard deviations. The means were compared by Analysis of Variance. Where the values for means were different from control, the statistical significance is indicated by: (+) P < 0.05; (+ +)P < 0.02; (f + +)P < 0.01; (+ + + +) P < 0.001. The percentage changes of significantly altered means are tabulated. Glycerol-ester hydrolase and sterol-ester hydrolase were significantly negatively correlated, Y = -0.96, P < 0.01.

Lipostabilaa

0.9 y0

Lipostabilas NaClas

10 18

18

NaCPl 0.9 %

10

None

Injection

ACTIVITIES

Atherogenic semisynthetic diet containing 20 y0 beef tallow

get (weeks)

Time

DEHYDROGENASE

18

Diet

3

4. P

AND

Control

ESTERASE

0

:

y

Lipostabil

18

114& 11.7 485& 217 (++) 578 f 139 (Jr-k-t) 5

5

5

0

0

5

3

2

0

7

2

1

0

2

3

0

2

0

5.9 f 1.14

4.9 * 1.56d 5.6 -f 1.06 11.4 & 3.60

8.0 & 3.20 15.8 & 6.90

Let

8.1 i 2.85

5.9 f 1.92 7.2 & 1.56

Sph

0.4 * 0.11

1.1 * 1.26 0.5 5 0.38 3.7 + 1.16

4.6 i 0.75 4.1 & 2.08

(ng/mg protein)

LL

free fatty acids

(+)

(-t)

significance

k;+) 1.92

2.4 & 0.91 6.6 + 2.40

free

0.4 & 0.26 4.8 + 1.33 (+++I 3.9 -J= 2.41

ester

cholesterol

For explanation, see Table 2. Grade 0 = no &ions; grade 1 = < 1 %; grade 2 = 1-5 7;; grade 3 = > 5 0h diseased aorta. PE = phosphatidyl ethanolamine. Let = lecithin. Sph = sphingomyelin. LL = lysolecithin. Means f SD. The means were compared by Analysis of Variance. Where the values for means were different from control, the statistical is indicated by: (+) P < 0.05; (+ +) P 6 0.02; (+ + +) P G 0.01.

NaCl 0.9 y0

18

None

0

PE

$hospholi$idsc

total No. of animals

No. of animals at each grade of lesionb

A orta

Aortic atherosclerosis

OF AORTAS

Plasma cholesterol (mgl 100 ml)

COMPOSITION

Injectiona

LIPID

Atherogenic semi-synthetic diet containing 20 y0 beef tallow

zet (wks)

Time

AND

18

OF LESIONS

3

Control

Diet

SEVERITY

TABLE

48

J.

for egg yolk lecithin and cholesteryl amount taken for preparation Specific activities

oleate and 70%

PATELSKI

for glyceryl trioleate

-et ai!.

of the

of the hydrosols.

of the aortic phospholipase

A, lipase, cholesterol

esterase,

malate and lactate dehydrogenases in control and experimental rabbits are presented in Table 2. The mean values of protein extracted from acetone-butanol aortas were not significantly

powders from

different from each other. Animals fed atherogenic

diets and injected with saline showed increased phospholipase A (after 18 weeks) and lipase activities

and decreased cholesterol esterase activities

(both after 10 and 18

weeks) compared with normal. The arterial lipolytic enzyme activities

of animals fed

the atherogenic diet and injected with Lipostabil were not significantly different from normal. No alterations were observed in the activities of malate and lactate dehydrogenases. Atherosclerotic

lesions could be seen macroscopically

in experimental

animals

fed for 18 weeks but not in those fed for 10 weeks. Plasma cholesterol, the extent and severity of atherosclerosis and the lipid composition of aortas is presented in Table 3. Plasma cholesterol

was elevated 4-fold in animals fed the semi-synthetic

diet but

there was no significant difference between those injected with saline or Lipostabil. The lesions in the saline group were well advanced and consisted of collections of lipid-filled cells with pyknotic nuclei, interspersed with fine elastic fibrils, separating the endothelium from the inner elastic lamina. The lipid was in globular form; there were no cholesterol clefts. The inner elastic lamina in all cases was fragmented and has lost its normal refractility.

There was duplication and even triplication of this layer in

some cases. Smooth muscle cells immediately underlying the lesions showed accumulation of lipid in their cytoplasm. Endothelial

cells overlying the lesions were swollen

and presented a rough surface to the circulating blood. Basically, the lesions in animals treated with Lipostabil were similar to those of the saline group, but on a reduced scale. No animals in the group treated with Lipostabil

had severe atherosclerotic

lesions (Grade 3) whilst in contrast, in the groups injected with saline, 2 animals out of 5 showed Grade 3 lesions. There was no significant difference between groups in the concentrations of any of the phospholipid classes. Mean concentrations of esterified and free cholesterol in the aorta were 12 and 2.8 times higher respectively in animals injected with saline and 9.8 and 2.3 times higher in those injected with Lipostabil, compared with normal. Table 4 summarises the results for the incorporation of [1-r%]oleic acid into the aortic phospholipids, free fatty acids, triglycerides and cholesteryl esters. Within each lipid class, a large spread in individual values was observed. Comparison of mean values by Student’s t test revealed only a significant difference in incorporation into cholesterol esters in the controls fed the atherogenic diet. The values of the activities of lipolytic enzymes extracted from liver and serum are shown in Table 5. The atherogenic diet with injection of saline caused a significant increase only in serum phospholipase A activity, compared with normal. The injection of Lipostabil prevented this increase and, furthermore, stimulated the lipase activity of both liver and serum. Cholesterol esterase activity could not be demonAtherosclerosis,

1970, 12: 41-53

4

NaCl 0.9 %

Lipostabil

18

18

5

5

5

No.

LIPIDS

0-f animals

0.79

11.22 + 8.53

5.93 f

7.29 & 4.46~

PL

Incorporation

f- 3.15

9.21 i

6.35

6.14 & 3.62

6.43

FFA

of jl-14C]oleic

acid

(+,P

< 0.05.

a See Table 2. b PL = phospholipids. FFA = free fatty acids. TG = triglycerides. CE = cholesterol esters. c Mean & SD. The means were compared by Student’s t test. Where the means were different from control,

None

Atherogenic semisynthetic diet containing 20 y0 beef tallow

AORTIC

Injectiona

INTO

18

ACID

Control

[1-14’40LEIC

Time on diet (weeks)

OF

Diet

THE INCORPORATION

TABLE

& 6.12

the significance

2.24 & 2.38

5 0.06

tissue)

level is indicated

(+) 304 y0 0.19 & 0.27

100 y0 0.46 & 0.25

0.14

CEb

dry defatted

1.08 + 0.53

4.70

TG

(~r,umoleslmg

by

%

z

5

b

ri .&. 7 E

3

G

2. P

:: F a

2

D

Lipostabil

18

SERUM

5

5

5

No. of animals

BLOOD

2.3

21 & 2.1

20 f

22 & 3.26

16.6 f

6.5

16.9 + 6.1

15.8 & 6.1 3.0

31.4 + 8.7

6.7 f

9.3 * 3.4 100 y0

42 f

44 f 4.0

8.5

39 & 6.4

extracted protein ( % (w/w))

extracted proteinb (% (wlw)) lipasec2 phospholipase A ‘1 (nequiv/min/mg)

Serum

Livev

8.2 f 2.2 (++++) 273 y0 2.6 f 1.6

3.0 & 0.9 100 y0

3.1

(1z7+, + +) 0

26.4 & 3.0

19.2 f

16.8 f 3.6 100% m

phospholipasec4 lipase AC3 (nequiv[minlmg)

8 Intravenous injections of 0.5 ml 3 times a week. b Extraction from acetone-butanol powder with water (20 mg/ml) using a Dacie electric cell suspension mixer at 30°C for 15 min. C Reaction mixtures (total volume 1 ml) contained: (1,3) lecithin 1 mM, (2.4) glycerol trioleate 1 m&I, reduced glutathione 0.1 mM, and liver extract 0.20-0.40 mg protein/O.OS-0.10 ml, or serum extract 0.5-1.0 mg protein/O.OS-0.10 ml. The pH values were (1,3) 8,0, (2) 8.4, and (4) 8.9. The reaction temperature was 30°C. The titraton reagent was 0.002 N potassium hydroxide solution (using pH-Stat Radiometer). * For explanation, see Table 2.

NACl 0.9 %

none

Injections

AND

18

Time on diet (weeks)

OF THE LIVER

Atherogenic semisynthetic diet containing 20 y0 beef tallow

ACTIVITIES

18

5

c0lltr01

Diet

ESTERASE

TABLE

8

MODIFICATION

strated

OF ENZYME

ACTIVITIES

in liver or serum under

No further

experiments

IN EXPERIMENTAL

the conditions

to determine

no significant difference powders in each group.

51

used for the assay of aortic

the optimal

in the amount

ATHEROSCLEROSIS

parameters

of protein

enzymes.

were done. There was

extracted

from acetone-butanol

DISCUSSION

As we have noted beforea, the atherogenic semi-synthetic lipase activity and decreased the cholesterol esterase activity rabbit.

In addition,

pholipase

in this experiment,

A, which was active

there was an increase

against

both

diet increased the of the aorta in the

in the activity

egg yolk lecithin

lecithin. The observation of an increased phospholipase A activity by the improved extraction of enzyme by the double-extraction acetone-butanol

powders.

polyunsaturated

lecithin

The absolute

The relative moreover

lecithin.

Changes in the lipolytic of lesions

and

of the substrate

enzyme

activities

could be demonstrated.

was higher than the rate

due to the different

fatty acid composi-

which influences

the enzyme for the b-position of the lecithins 173. the kind of fatty acid present at the a-positioni7. formation

of egg yolk lecithin

of the egg yolk lecithin

This is probably

tion (see Table 1) and configuration

of hydrolysis

can be explained procedure on the

were the same in all groups.

rate of hydrolysis

for polyunsaturated

rates

of phos-

and polyunsaturated

the specificity

of

The enzyme may also be affected by

of the arterial

wall occurred

There was no change,

before the

however,

in the

aortic malate and lactate dehydrogenases. This observation suggests that although there was a change in enzymes of specific function, there was no change in general metabolic activity. The enzyme activities of serum, and liver, on the other hand, showed a different picture of alteration. The only change was the marked increase in the serum phospholipase

activity.

The present findings, along with the observation between the activities of the arterial lipase and cholesterol in the consideration the arterial

of the mechanism

enzymes

thin, but favours

the accumulation

The cholesterol decreased question

to hyperlipaemia

hydrolysis of whether

of arterial

lipid accumulation.

acts against of cholesterol

esters may accumulate

of a negative correlation esterase, are of importance

retention

The response

of triglyceride

of

and leci-

esters.

in the arterial

wall as a result

of both

and increased synthesis. In fact, it is difficult to answer in cholesterol esterase is primarily or secondarily

a decrease

the in-

volved in this process. It may be that the enzyme is inhibited by some toxic factors, but this has not yet been investigated. It is possible, however, that there is substrate inhibition, which has been demonstrated in vitro19 or, more likely, classical product inhibition of the enzyme. With respect to cholesterol ester synthesis, esterification of cholesterol with fatty acid.+-21 and the enzyme system catalyzing this reaction22 have been demonstrated in the aortic wall. The rate of cholesterol ester formation is clearly influenced by the concentrations of free cholesterol and fatty acids. Cholesterol is synthesised slowly, if at all, by the arterial wall and most is derived from the plasma. Atherosclerosis,

1970, 12: 41-53

et al.

J. PATELSKI

52 On the other hand, fatty acids may be derived result of de nova synthesissepzs, transacylations4 An enhanced aortas of animals

incorporation fed atherogenic

with the concomitant

from the plasma albumin or local lipolysis.

of free fatty

acids into

diet indicates

decrease in the cholesterol

acids will lead to enhanced

accumulation

would be a role of enhanced

lipolysis

cholesterol

a higher turnover esterase,

esters

esters

of the

of these esters. Thus

any excess supply

of cholesterol

in the arterial

and also as a

of fatty

by synthesis.

Such

wall.

On the other hand, an inadequate lipolysis in plasma and liver would favour the maintenance of hyperlipaemia and provide substrates for the arterial enzymes. Prevention

of an increase

in lipase and a decrease

arterial wall could be expected to act favourably lation. An increase in lipolysis in the circulating way by removing Lipostabil

triglyceride

was seen to alter

accumulation

of cholesterol

which

in cholesterol

is the main

substrate

the enzyme

activities

esters.

is in agreement

This

ments,

Lipostabil

did not cause a depression

aortic enzyme

activities.

for the arterial

and, furthermore, with

ADAMS et al.25 on decreased aortic cholesterol accumulation cholesteroldiet and injected with a higher dosage of Lipostabil.

aortic atherosclerosis which we observed basis of decreased hypercholesterolaemia,

of plasma

the

lipase.

to reduce

the

observation

of

cholesterol.

The decrease

in

with Lipostabil could not be explained on the but was more likely due to the changes in

The mechanism

environment

in the

in rabbits fed a high However, in our experi-

of action

remains

to be elucidated.

A better understanding of the mechanism of lipid accumulation wall will depend upon the measurement of relative rates of synthesis of lipids in a physiological

esterase

in reducing cholesterol ester accumublood and liver would act in a similar

and under

the influence

in the arterial and catabolism

of different

agents.

ACKNOWLEDGEMENTS

The authors Council, London, assistance

thank Nattermann for their support.

and Co. of Cologne and the Tobacco They gratefully acknowledge the

Research technical

of Mrs. M. Apps and Mrs. J. King.

REFERENCES PATELSKI, J., 2. WALIGORA AND S. SZULC, Demonstration and some properties of the phosphoRes., 1967, 7: 453. lipase A, lipase and cholesterol esterase from the aortic wall, J. Atherosclev. PATELSKI, J., D. E. BOWYER, A. N. HOWARD AND G. A. GRESHAM, Changes in phospholipase A, lipase and cholesterol esterase activity in the aorta in experimental atherosclerosis in the rabbit and rat, J. Atheroscler. Res., 1968, 8: 221. BOWYER, D. E., W. M. F. LEAT, A. N. HOWARD AND G. A. GRESHAM, The determination of the fatty acid composition of serum lipids separated by thin-layer chromatography; and a comparison with column chromatography, Biochim. biophys. Acta (Am-t.), 1963, 70: 423. ALBRINK, M. J., The microtitration of total fatty acids of serum with notes on the estimation of triglycerides, J. Lipid Res., 1959, 1: 53. DOLE, V. P., A relation between non-esterified fatty acids in plasma and the metabolism of glucose, J. din. Invest., 1955, 35: 150. HOWARD, A. N., G. A. GRESHAM, D. JONES AND I. W. JENNINGS, The prevention of rabbit atherosclerosis by soya bean meal, J. Atheroscler. Res., 1965, 5: 330.

Atherosclerosis,

1970, 12: 41-53

MODIFICATION

OF ENZYME

ACTIVITIES

IN EXPERIMENTAL

ATHEROSCLEROSIS

53

7 MORTON, R. K., Method of extraction of enzymes from animal tissues. In: S. P. COLOWICK AND N. 0. KAPLAN (Eds.), Methods in Enzymology, Vol. 7, Academic Press, New York, 1955, p. 25. * LECCETT BAILEY, J., Techniques in Protein Chemislvy, Elsevier, Amsterdam, 1962, p. 293. 9 NEILANDS, J. B., Lactic dehydrogenase of heart muscle. In: S. P. COLOWICK AND N. 0. KAPLAN (Eds.) Methods in Enzymology, Vol. 1, Academic Press, New York, 1955, p. 449. 10 BOWYER, D. E., A. N. HOWARD, G. A. GRESHAM, D. BATES AND B. V. PALMER, Aortic perfusion in experimental animals. A system for the study of lipid synthesis and accumulation, Biochem. Pharmacol., 1968, 4: 235. 11 JONES, D., D. E. BOWYER, G. A. GRESHA~~AND A. N. HOWARD, An improved spray reagent for detecting lipids on thin-layer chromatograms, J. Chromatog., 1966, 23: 172. 12 USHER, D. A., The estimation of phosphorus on paper chromatograms, J. Chromatog., 1963, 12: 262. 13 BABSON, A. L., P. 0. SHAPIRO AND 0. E. PHILLIPS, A new assay for cholesterol and cholesterol

ester in serum which is not affected by bilirubin, Cl&. chim. Acta, 1962, 7: 800. 14 MARSH, J. B. AND D. B. WEINSTEIN, Simple charring method for determination of lipids, ,I. Lipid Res., 1966, 7: 574. 15 HOLMAN, R. L., H. C. MCGILL, J. P. STRONG AND J. C. GEER, Macroscopic staining of aortas with Sudan, Lab. Invest., 1958, 7: 4. 16 HOWARD, A. N., G. A. GRESHAM, I. W. JENNINGS AND D. JONES, The effect of drugs on hypercholesterolaemia and atherosclerosis induced by semi-synthetic low cholesterol diets, Progr. biochem. Pharmacol., 1967, 2: 117. 17 MOORE, J. H. AND D. E. WILLIAMS, Some observations on the specificitv of phospholipasc A, Biochim. biophys. Acta (Amst.), 1964, 84: 41. 18 DE HAAS, G. H., F. J. M. DAEMEN AND L. L. M. VAN DEENEN, Positional specificity of phosphatide acyl hydrolase (phospholipase A), Nature (Lond.), 1962, 196: 68. 19 PATELSKI, J., Esteraza Cholesterolowa Tetnicv Gtownej (Cholesterol esterase of the aorta), Panstwowy Zaktad Wydawnictw Lekarskich,’ Warszawa, 1964. 20 LOFLAND, H. B., JR., D. M. MOURY, C. W. HOFFMAN AND T. B. CLARKSON, Lipid metabolism in pigeon aorta during atherogenesis, J. Lipid Res., 1965, 6: 112. 21 BOWYER, D. E., A. N. HOWARD AND G. A. GRESHAM, Lipid synthesis in perfused normal and atherosclerotic rabbit aortas, Biochem. J,, 1967, 103: 54 P. 22 PNIE~SKA, B., Enzymatyczna estryfikacja cholesterolu w tetnicy gtownej (Enzymatic esterification of cholesterol in the aorta), To be published. 23 WHEREAT, A. F., Fatty acid synthetis in cell-free system from rabbit aorta, J. Lipid Res., 1966, 7: 671. 24 ABDULLA, Y. H., C. C. ORTON AND C. W. M. ADAMS, Cholesterol esterification bv transacylation in human and experimental atheromatous lesions, J. Atheroscler. Res., 1968, 8: 967. 25 ADAMS, C. W. M.. Y. H. ABDULLA, 0. B. BAYLISS AND R. S. MORGAN, Modification of aortic atheroma and fatty liver in cholesterol-fed rabbits by intravenous injection of saturated and polyunsaturated lecithins, J. Path. Back, 1967, 94: 77.

Atherosclerosis,

1970, 12: 41-53