22 (1975) 287-29 I (‘8 Elsevier Scientific Publishing Company, Amsterdam - Printed in The Netherlands

Athwosdwosis,

THE ARTERIAL CHOLESTEROL IN

VITRO

LENGTH

ACYL-CoA:CHOLESTEROL ACYLTRANSFERASE ESTER HYDROLASE ACTIVlTIES

EFFECT OF SUBSTRATES AND SATURATION

J. PATELSKI,

B. PNIEWSKA,

WITH FATTY AClD OF DIFFERENT

M. PIORUfiSKA

AlLV

287

AND

CHAIN

OBREjBSKA*

Dc~pcrrtmwt c?f’ Biochemistry,, Medicrrl Acu&II~_I~, Pozrruti (Polutd) (Received August 27th, 1974) (Revised received March 4th, 1975) (Accepted

March 4th, 1975)

AHSTRACT

Cholesterol acyltransferase and cholesterol esterase activities of protein extracts from pig aortas have been examined and the ratios of synthesis/hydrolysis rates

in the presence

of substrates

with different

obtained were in the numerical linolenate ;, staerate. The results are discussed

order:

cholesterol

wall.

Key words:

esters in the arterial

cholesteryl

in relation

Aorta - Ac),l-CoA:cllol~.stcrol asp - DifJrrewt fatty

fatty acids estimated. oleate

;- palmitate

The values ’

3 linoleate

to the known

different

accumulation

ot

acyltransferase

- Cl~ol~~stwol ester ii~drol-

acid substrates

INTRODUCTION

The question of different rates of synthesis and hydrolysis of cholesterol esters in the arterial wall attracts attention1p3 because of the different accumulation of these esters”,j and the importance ascribed to their increased synthesis and decreased hydrolysis”. In this report, investigations of the in vitro effect of substrates with different

* Permanent address: Institute of Occupational

Medicine, t6di,

Poland.

J. PATELSKI,

288 length and saturation cholesterol MATERIAL

B. PNIEWSKA,

M. PIORUtiSKA,

of the fatty acid chain on the cholesterol

esterase activities

M. OBR&BSKA

acyltransferase”

and

of the aortic wall are presented.

AND METHODS

Reagents and substrates Commercial used. Substrates linoleate

reagents

of reagent

were checked

and linolenate

grade

for purity,

(Sigma,

U.S.A.)

and redistilled

organic

palmitate,

were prepared

as hydrosols

previously6 but without the addition of sodium taurocholate. acid (Sigma) were prepared in the same way. Aqueous solutions of palmitic, Goodman7.

stearic,

oleic, linoleic

and linolenic

powders,

and the powders

stearate,

acid were obtained

were extracted

were oleate,

as described

Cholesterol and oleic of the potassium salts

Enzyme preparation Thoracic aortas from several pigs were pooled and processed butanol

solvents

and cholesteryl

according

to

to obtain acetone-

with water for cholesterol

acyl-

transferase and with 0.25 mol/l sucrose for cholesterol esterase, as described elsewherels. The mixtures were spun at 1500 x g (Servall, RC-2, U.S.A.) at 4°C for 10 min, the clear supernatants decanted and used for enzyme estimations. Protein was determined

by the biuret method

according

to Gleiss and Hinsbergs.

Enzyme estimations Enzyme activities were examined using the sample-type measuring a change in concentration of free fatty acids extracted

method based on from reaction mixt-

ures before and after incubation. Reaction mixtures for cholesterol esterase contained one of the cholesterol esters: palmitate (1.5 x 1O-3 mol/l), stearate, oleate (10d3 mol/l), linoleate (1.5 x 10-s mol/l)

and linolenate

(1.25

x

10-a mol/l),

reduced

glutathione

( 1O-4 mol/l),

calcium chloride (5 x 10-J mol/l), tris(hydroxymethyl)aminomethane (1OW moljl), and enzyme extract (approximately 1.8 mg of protein/l.4 ml) in a total volume of 7 ml, at pH 8.6. The various concentrations of substrates used were found to be optimallO and substrate inhibition of the enzyme has previously been demonstrated11 ll. The reaction mixtures were incubated in glass-stoppered test tubes placed in a Dacie electric cell suspension mixer (Surgical and Scientific Apparatus, Matburn, England) at 30°C for 30 min. Fatty acids were extracted from 1 ml samples with 2.5 ml of the extraction mixture according to Dole12 and were then spectrophotometritally determined in 2 ml of heptane phase according to Mosingerls, in triplicate. The activity of cholesterol acyltransferase was assayed under substrate satura-

* Cholesterol acyltransferase = Acyl-CoA:cholesterol = sterol ester hydrolase (EC 3.1.1.13).

acyltransferase

(EC 2.3.1), cholesterol esterase

ARTERIAL

ACYL-COA:CHOLESTEROL

tion, where no inhibition mixtures mol/l),

consisted

ACYLTRANSFERASE

of the enzyme

of an appropriate

ATP (1O-4 mol/l),

CoA*SH

occurred,

ACTIVITIES

as described

289 previouslya.

fatty acid (2 x 10-a mol/l), (IO-” mol/l),

magnesium

sodium taurocholate (10-S mol/l), reduced glutathione (5 x lO-‘L moljl), and enzyme extract (approximately

Reaction

cholesterol

chloride

(10-a

(IO-” mol/l),

(IO-’ moljl), phosphate buffer 2.2 mg protein/ml) in a total

volume of 10 ml, at pH 6.5. Incubation followed as previously described, for 60 min. Fatty acids were extracted and titrated according to Dolel’, using 0.01 N NaOH and Nile blue indicator14, in triplicate. Results are expressed in milliunits of enzyme specific activity, i.e. in nequiv. of fatty acid esterified or released per min per mg of protein. Means obtained from 5 series of determinations for substrates with oleic acid were compared using Student’s t-test. Ratios of synthesis/hydrolysis rates for these and substrates with other fatty acids (3 series of determinations) were calculated. The mean values were considered as significantly different when distinct from each other by one range of magnitude (0.1-1.0

or 1.0-10.0).

(without

substrates)

Substrate were taken

controls

(without

enzymes)

and enzyme

controls

into account.

RESULTS

The mean specific activities ase obtained

in the presence

of cholesterol

of different

acyltransferase

substrates

and cholesterol

are presented

ester-

in Fig. 1.

A higher activity was found for cholesterol oleyltransferase (14.0 * 1.6 mlJ/ mg, N = 5) than for cholesteryl oleate hydrolase (7.6 * 1.1 mU/mg, N = 5; P < O.OOl), at different ranges of means f double standard deviations (P < 0.05) and the resulting synthesis/hydrolysis ratio was 1.84. The ratios for cholesteryl stearate

and linolenate

Actwity

are lower by one range of magnitude,

amounting

to 0.25 and

[mUI mg]

M 2 I/II

Activity

ratio

Fig. I. Arterial enzyme activities in the presence of substrates with different fatty acids. I: acylCoA:cholesterol acyltransferase; II: cholesterol ester hydrolase. C with subscript indicates the number of carbon atoms in the fatty acid chain and the number of double bonds. For details see text.

290

J. PATELSKI,

0.52 respectively.

The appropriate

within the same range, reaching

B. PNIEWSKA,

M. PIORUP;ISKA,

values for cholesteryl almost

palmitate

M. OBREBSKA

and linoleate

are

1.

DISCUSSION

The preferential hydrolysis

esterification

of other cholesterol

in the arterial

of cholesterol

esters may favour

wall. It is known

that cholesteryl

with oleic acid and preferential accumulation

of cholesteryl

oleate predominantly

the fatty streak in the rabbit15 and man4$5. However, there may be objections to a comparison

oleate

accumulates

of the absolute

in

rates of

synthesis and hydrolysis of cholesterol esters. This is because the possibility that the enzyme activities are modified by various factors, such as different extractability of the enzyme proteins and composition of the reaction systems used, cannot be excluded. Therefore, not so much the absolute values of synthesis/hydrolysis ratio as the differences between the ratio coefficients for the particular cholesterol esters are important. High and significant

correlations

between

the ratios

of the reaction

rates and

contents

of appropriate cholesterol esters found in fatty aortic intima4,5 and media” No significant correlation is found for may be calculated (v > 0.96, P < 0.05). normal intima and media4 and intima with perifibrous lipids, and also not when only absolute

rates of either synthesis

or hydrolysis

and the contents

of cholesterol

esters

are compared. Also other enzyme-catalyzed cholesterol16,17 or from cholesterol

reactions, such as transacylation from lecithin to ester to lysolecithinlo, may be important for the

arterial

of cholesterol

synthesis

and degradation

esters. Nevertheless,

the differences

between the ratios of the rates of the reactions catalyzed by the acyl-CoA :cholesterol acyltransferase and cholesterol ester hydrolase seem to be significant for the different intracellular accumulation of cholesterol esters in the arterial wall.

REFERENCES 1 PATELSKI, J.,

Estrraza

Cholesterolowa

Ternicy

Gldwnej lCholesterol esterasr of the aorta), Warszawa, 1964. BOWYER, D. E., HOWARD, A. N., GRESHAM, G. A., BATES, D. AND PALMER, B. V., Aorticperfusion in experimental animals. A system for the study of lipid synthesis and accumulation, Biochem. Pharmacol., 4 (1968) 235. KLIMI~SKA-PNIEWSKA, B., Enzymatyczna estryfikacja cholesterolu w tstnicy gl6wnej (Enzymatic esterification of cholesterol in the aorta), Poznariskie Towarzystwo Przyjacidl Nauk, Pvacr Kotnisji Medycyny DoSwiadczalnej, 42 (1970) 167. NELSON, W. R., WERTHESSEN, N. T., HOLMAN, R. T., HADAWAY, H. AND JAMES, A. T., Changes in fatty acid composition of human aorta associated with fatty streaking, Lancet, i (1961) 86. SMITH, E. B., EVANS, P. H., AND DOWNHAM, M. D., Lipid in the aortic intima. The correlation of morphological and chemical characteristics, J. Atheroscler. Res., 7 (1967) 171. PATELSKI, J., BOWYER, D. E., HOWARD, A. N., JENNINGS, 1.W., THORNE, C. J. R. AND GRESHAM, G. A., Modification of enzyme activities in experimental atherosclerosis in the rabbit, Atherosclerosis, 12 (1970) 41.

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2

3

4 5 6

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ACYL-COA:CHOLESTEROL

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ACYLTRANSFERASE

ACTIVITIES

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of human serum albumin with long-chain fatty acid anions. J. A/?icr. C/le/rr. Sot., 80 (1958) 3892. 8 PATELSKI,J., WALIG~RAAND SZULV, Z.S., Demonstrationandsomepropertiesof thephospholipase A, lipase and cholesterol esterase from the aortic wall, J. Athevoscier. Rrs., 7 (1967) 453. 9 HINSBERC;,K., Colorimetrische Bestimmung von Gesamteiweiss und seinen Fraktionen nach Gleiss und Hinsberg. In: LANG, K., LEHNARTZ, E. AND SIEBERT, G. (Eds.), Hopp~-S~,y/rr/Thic,~fklder Handbuch der physiologisch- WC/ patllologi.sc.h-c.llel)li.s~h~~t~ Ancrl,v.w, Vol. 5, 1Oth edit ion. Springer, Berlin, 1953, p. 25. IO PATELSKI,J. AND PIORU~SKA,M., Cholesterol ester degradation by transacylation with lysolecithin and hydrolysis in the arterial wall, To be published. I I PATELSKI,J., WALIG~)RA,Z. AND SZULC, S., Investigations of the aortic phospholipase A. lipase and cholesterol esterase, Enzy/>~e, 12 (1971) 299. I2 DOLE,V. P., A relation between non-esterified fatty acids in plasma and the metabolism of glucose, J. C/in. Invesf., 35 (1955) 150. 13 MOSINGER, F., Photometric adaptation of Dole’s microdetermination of free fatty acids. J. Lipid Rev., 6 (1965) 157. 14 ALBRINK, M. J., The microtitration of total fatty acids of seruni, with notes on the estimation of triglycerides, J. Lipid Rcs., 1 ( 1959) 53. 15 HOWARD,A. N., Experimental models for atherosclerosis and the study of arterial metabolism. In : COWGILL, C., ESTRICH, D. L., AND WOOD, P. D. (Eds.), Proceedings of’the 1968 Druc~l Confewnw WI The Turno,w qf’Lipids and Lipoproteins. U.S. Department of Health, Education and Welfare, Washington, D.C., 1968, p. 171. 16 ABDULLA,Y. H., ORTON, C. C. AND ADAMS,C. W. M., Cholesterol esterification by transacylation in human and experimental atheromatous lesions, J. Athrrosclw. Rex., 8 (1968) 967. 17 PATELSKI, J. AND TGRLII&KA. T., Lecithin:cholesterol acyltransferase and phospholipase A activities of the aortic wall. In: SAMCCHOWIE~,L. AND W~)JCICKI,J. (Eds.), Procerdings ofthr /972 Intw~ational S~n~po.cirm on fho.spho/ipid~, Internat. Sot. Biochem. Pharmacol. and Polish Pharmacol. Sot., Szczecin. 19?3, p. 91.