7I

/VO, 6 S

STUDIES OF THE INTERACTION OF LCAT WITH

LIPOPROTEIN SUBSTRATES IN HDL

DEFICIENT PLASMA SYSTEMS

THESIS

Presented to the Graduate Council of the

University of North Texas in Partial

Fulfillment of the Requirements

For the Degree of

MASTER OF SCIENCE

By

Sulabha Paranjape, B. S. , M. S.

Denton, Texas

August, 1989

Paranjape, Sulabha A., Studies of the interaction of LOAT with

lipoprotein substrates in HDL deficient plasma systems.

Master of Science (Biochemistry), August 1989, 62 pp., 5 tables, 7

figures, References, 67 titles.

Enzymatic and lipid transfer reactions involved in reverse

cholesterol transport were studied in HDL deficient plasma systems.

Fasting plasma samples were obtained from control and cholesterol

fed guinea pigs as well as from a fish eye disease patient and were

used to localize the enzyme LCAT among plasma lipoproteins (VLDL,

LDL, and HDL). In both guinea pig and fish eye disease patient

plasma, the LCAT activity was found in association with the HDL type

particles. Cholesterol feeding in guinea pigs altered the properties of

lipoprotein substrates for LCAT resulting in some changes,

specifically: 1) decreased fractional rate of plasma cholesterol

esterification and, 2) lower transfer of free cholesterol (FC) and

esterified cholesterol (CE) within the lipoprotein fractions.

TABLE OF CONTENTS

Page

LIST OF TABLES...... . .... ...... vi

LIST OF ILLUSTRATIONS .. ............ vii

Chapter

I. INTRODUCTION........... ...... 1

Cholesterol Metabolism and Transport.

Role of LCAT in reverse cholesterol transport.

Comparison of plasma lipoproteins of Guinea pigs and

humans.

Purpose of the Study.

II. MATERIALS AND METHOD. . ............ 18

Materials

Plasma

Chemicals

Methods

Gel filtration chromatography.

Density gradient ultracentrifugation.

Poly ethylene glycol precipitation.

iv

Chapter

Preparation of human HDL substrate

Lecithin cholesterol acyl transferase (LCAT) activity

Plasma incubation study.

Analytical methods.

Free and total cholesterol determination

Protein determination.

Analytical gel electrophoresis of HDL fraction.

III. RESULTS........._.,.....0..........26

Distribution of Lecithin Cholesterol acyltransferase

activity among plasma lipoproteins.

Guinea pigs.

fish eye disease.

Effect of cholesterol feeding on endogeneous

LCAT activity in guinea pigs.

Plasma incubation Studies.

Origin of lipid substrates and the fate of the

product cholesteryl esters of the LCAT reaction

in guinea pigs.

Protein composition of lipoproteins.

IV. DISCUSSION..o-.-.......................45

V. REFERENCES,-..-..- . . .. . . ........ 57

V

Page

LIST OF TABLES

Page

Table

I. Classification and composition of human

plasma lipoproteins .............. .....2

II. Effect of cholesterol feeding on the plasma

cholesterol esters turnover determined in vitro. . . . 35

III. Contribution of lipoproteins to the free cholesterol pool

and the fate of cholesteryl esters following the

incubation of plasma samples in control animals. . . . 38

IV. Contribution of lipoproteins to the free cholesterol pool

and the fate of cholesteryl esters following the

incubation of plasma samples in cholesterol fed

animals.................. . .............. 39

V. Determination of total apoprotein of lipoprotein

fractions,..............................42

vi

LIST OF ILLUSTRATIONS

Figure

1. Sequential steps in the LDL pathway in cultured humanfibroblasts............. .... . . . . ......

2. Principal reaction catalyzed by plasma Lecithin :cholesterol acyltransferase... .. . . .0. .0...

3. Reverse cholesterol transport mechanism. .

4A. Distribution of LCAT activity in control guinea pig

plasma by gel chromatography.. ......

4B. Distribution of LCAT activity in cholesterol fed

guinea pig plasma by gel chromatography ...

Page

. 4

6

. .7

. 27

.28

5A. Distribution of LCAT activity in control guinea pig plasma

by ultracentrifugation..............0...........31

5B. Distribution of LCAT activity in cholesterol fed guineapig plasma by ultracentrifugation... ... ........ 32

vii

Figure Page

6. Distribution of LCAT activity in fish eye disease plasma. 33

7. Gel electrophoresis pattern of HDL from control andcholesterol fed guinea pigs. .... . .. ...... .43

viii

CHAPTER I

INTRODUCTION

Cholesterol metabolism and transport.

Elevated plasma cholesterol is a major risk factor for

premature atherosclerosis and coronary heart disease (1). Because of

its insolubility in aqueous media, cholesterol circulates as a

component of complex macromolecules called plasma lipoproteins.

Despite the efficiency of this transport system, undesirable

accumulation of cholesterol in tissue can occur. For example,

prolonged exposure of arterial walls to high concentrations of

cholesterol containing lipoproteins is a major contributing factor to

atherosclerosis, and the excess of cholesterol in the bile generally

leads to the formation of gall stones (2).

Human plasma lipoproteins have been classified into four major

classes : chylomicrons, very low density lipoproteins (VLDL), low

density lipoproteins and high density lipoproteins (HDL) (3,4). Some

of the basic properties of these lipoproteins are shown in Table I.

Each groupof lipoproteins performs separate and specific

functions in the body. Chylomicrons transport mainly dietary lipids

from the intestine to adipose tissue and to the liver. Cholesterol and

1

z -

OH,

zUM-

Z- w

o l E-4 0-

000 q 0

N C

W- 0 -00

z

00

0CIi 0O

2

004

V0co0

C)

- )

3

triglycerides are incorporated in the intestine into chylomicrons

which are subsequently secreted into the lymph and then into the

blood stream via the thoracic duct. As they pass into capillary beds,

the triglyceride core is hydrolyzed by the enzyme lipoprotein lipase

(LPL) on the outer surface of endothelial cells. The hydrolysis of

chylomicron triglycerides by LPL requires apoprotein (APO) CII as a

cofactor (5). Once most of the triglycerides in the chylomicrons are

hydrolyzed, the remaining particles, called chylomicron remnants,

are released into the plasma. These remnant particles are rich in

cholesterol and are rapidly taken up by the liver (6).

The liver is the major organ of cholesterol biosynthesis as well

as catabolism. Hepatic cholesterol can be excreted into the bile or

secreted into the plasma as a component of VLDL. This lipoprotein

possesses an outer monolayer of specific apoproteins, phospholipids

and free cholesterol, and a core of triglycerides and a small amount

of cholesteryl esters (6). Degradation of VLDL begins through

interaction with HDL and lipolysis of its triglycerides. This reaction

is also catalysed by the enzyme lipoprotein lipase, with the aid of APO

CII (acquired from HDL) (5). After removal of triglycerides from

VLDL, a smaller and denser particle called intermediate density

lipoprotein (IDL) is formed. IDL is further degraded by hepatic

triglyceride lipase, and it is eventually transformed into low density

4

lipoprotein (6).

Low density lipoproteins transport cholesterol to the tissues.

The uptake of LDL by cells is accomplished via the LDL receptor

pathway (7). This system was originally described in cultured

fibroblasts by Goldstein and Brown and is known to play a major role

in maintaining body cholesterol homeostasis (8). The sequential

steps in the LDL receptor pathway are summarized in Figure 1.

HMGCoA4,Reductase

LDL Receptors --

LDL ACAT

" cholesterolV 0

Ch lesteryl Lchol terolLineolate ole ate

........... ..--- LDL

Protein amino Receptorsacids --

LDL Internalization -.- Lysosomal ___Regulatory

Binding Hydrolysis Actions

FIGURE 1. Sequential steps in the LDL receptor pathway in culturedhuman fibroblasts adapted from (7).

The uptake of LDL by cell surface receptors is mediated by the

protein component of LDL, APO B. The extent of binding of LDL is a

function of the number of receptors which in turn is regulated by the

cell's need for cholesterol (7). LDL binds to the receptor and enters

the cell by absorptive endocytosis. Within the cell APO B is degraded

5

to aminoacids and the cholesteryl esters are hydrolyzed by lysosomal

cholesterol esterase. The resulting free cholesterol is discharged

into the cytoplasm where it can be utilized for membrane

biosynthesis or for re-esterification for storage by enzyme Acyl CoA

cholesterol acyl transferase (ACAT). The rate of receptor synthesis

requires the uptake of LDL particles. This system is designed to

keep the intracellular cholesterol at the optimum level and protect

the cell from accumulating large excesses of cholesterol.

In order to keep the cholesterol level of extrahepatic tissues

constant, cholesterol has to be transported to the liver for catabolism

and excretion from the body. This process is referred to as reverse

cholesterol transport. Although the physiological role of the Lecithin

: cholesterol acyl transferase (LCAT) reaction has not been precisely

established, it has been proposed that the enzyme plays a key role in

reverse cholesterol transport (15,17).

Role of Lecithin : Cholesterol Acyl Transferase (LCAT) in reverse

cholesterol transport.

LCAT is an essential component of human blood plasma, and it

is responsible for the production of plasma cholesteryl esters. LCAT

is synthesized in the liver and then released in the plasma where the

enzyme acts mainly on HDL cholesterol (9,10,11). APO AI is the

major polypeptide component of HDL and has been found to be an

activator for the LCAT reaction in vitro (12). By catalyzing the

6

transfer of a fatty acyl chain from the C:2 position of phosphatidyl

choline to unesterifled cholesterol, LCAT promotes the formation of

lysophosphatidyl choline and cholesteryl esters (13). (Figure 2)

LECITHIN CHOLESTEROL

CH2 0 - saturated fatty acid

HO - unsaturated fatty acid

CH. 0- P - choline HO2 I

OH ILECITHIN:CHOLESTEROL

ACYLTRANSFERASE

LYSOLECITHIN

CH2 0 - saturated fatty acid

CHOH

0CH2 0 - P - choline

OH

FIGURE 2.

jr

+

CHOLESTERYL ESTER

unsaturatedfatty acid

Principal reaction catalyzed by plasma lecithin cholesterolacyltransferase.

A role for LCAT in reverse cholesterol transport was originally

proposed by Glomset in 1968 (11). He suggested that the action of

LCAT on HDL promotes the transfer of cholesterol from peripheral

cells to the liver. Fielding and Fielding (14) have suggested that

LCAT, cholesterol ester transfer protein (CETP) and specific

components of HDL constitute a discrete cholesteryl ester transfer

I I

7

complex, (HDL/LCAT complex) that facilitates reverse cholesterol

transport. The postulated function of this transfer complex is shown

in Figure 3 (15).

EMVDL

FC A -1 PL

FC HLCAT __CA-1

AA--1+ + D

FCN ARANSFERPLL

FC

JA-i :PL:CFCHDL LCATHD-

SEFFLUX --------- 0-- ESTERIFICATION - TRANSFER -

FIGURE 3. The coupling of efflux, esterification, and transfer inmaintaining the free-cholesterol potential gradient between cellmembranes and plasma in the presence of lecithin: cholesterolacyltransferase (LCAT) activity. FC, free cholesterol; PL,phospholipid; CE, cholesteryl ester; LL, lysolecithin. Adapted from(15).

It has been proposed that the HDL accepts free cholesterol at

the surface of the cell membrane followed by the esterification of

cholesterol via the LCAT reaction. Some of the cholesteryl esters are

then transferred to VLDL and LDL by CETP while another portion

becomes the HDL core. The substrate lipids for the LCAT reaction

have been shown to originate from VLDL and LDL because the LCAT

8

catalysed cholesteryl esters stay mainly on the surface of HDL. These

lipids reach the HDL complex presumably by nonenzymatic transfer

or exchange (16). The HDL particle is then regenerated to accept

more cholesterol from the surface of the cell membrane and go

through the plasma cholesteryl ester cycle until it is removed from

the circulation. The biproduct of LCAT reaction, lysolecithin, is

accepted by serum albumin and transported to the liver for

catabolism (15).

Although the substrate specificity of the LCAT reaction has not

yet been fully elucidated, the enzyme has a high affinity for the

smaller size HDL particles to form specific enzyme substrate

complexes (17). This esterification reaction is the major source of

plasma cholesterol esters in humans.

In addition to its role in reverse cholesterol transport, LCAT

has been postulated to have a role in the maintenance of the proper

composition of plasma membranes and lipoproteins and in the

maintenance of plasma/tissue cholesterol equilibrium. Schumaker

and Adams (18) suggested that LCAT might play a role in preserving

the overall spherical structure of chylomicron and VLDL particles by

eliminating excess surface components during their catabolism. The

action of lipoprotein lipase, results in the removal of the core

triglycerides and the production of smaller diameter remnant

9

particles. However, the surface components (phospholipids and

unesterified cholesterol) of these remnants remain essentially intact.

LCAT activity is required for removing excess phospholipid and free

cholesterol from the remnants of triglyceride rich lipoproteins in

order to maintain the proper surface to volume ratio during the

catabolism of these particles.

A great deal of information is available concerning the

mechanism of the action of LCAT. However, very little is known

about the nature and the composition of the substrates for LCAT in

vivo. Similarly, little definitive information is currently available on

the respective roles of lipoproteins regarding the origin of lipid

substrates and the fate of the product (CE) of the LCAT reaction.

Most of the available evidence suggests that HDL particles are the

best substrates for LCAT in vivo (19).

The hypothesis of Glomset, proposed in 1968, was revived

when it was suggested that increased HDL level protects against

premature development of atherosclerotic disease (19). Since then a

number of studies have shown a negative correlation between HDL

levels and accelerated vascular diseases in humans (20,21). In spite

of considerable investigation, the tissue sites and mechanism of

degradation of HDL are still obscure.

One of the difficulties in studying HDL isolated by

ultracentrifugation is its high degree of heterogeneity (22). HDL has

10

been separated into at least three sub-populations: HDL1 , HDL2 , and

HDL3. Some of the studies show that there is an interconversion of

HDL subclasses under physiological conditions. HDL3 accepts free

cholesterol from cells and becomes a substrate for the LCAT reaction.

The HDL3 to HDL2 conversion occurs only after the free cholesterol

is esterified by LCAT. The phospholipid and free cholesterol

enriched HDL3 is considered as an intermediate in the path of HDL3

-- > HDL2 (23). HDL1 is an APO E rich lipoprotein of lesser density

and larger diameter than HDL2. Some of the studies show that the

APO E rich HDLj particles are formed in the plasma from APO Al

rich HDL2 or even HDL3 particles (22). If the assumption that APO

AI containing HDL2 is converted to APO E containing HDLj is

correct, then during this conversion some APO AI must be displaced

from the HDL2 and replaced by APO E (22).

The hypothesized action of LCAT on plasma lipoproteins is

predicated on the assumption that the enzyme associates with a

small number of specific (HDL-type) macromolecules (11,24). These

lipoproteins apparently represent the key elements of reverse

cholesterol transport and have been referred to as the cholesteryl

ester transfer complex (24,25). In the plasma of LCAT deficient

patients, the LCAT activity derived from in vitro supplementation

with purified LCAT, located at or near the elution volume of the LCAT

activity of control sample upon gel chromatography (26). These data

11

indicate that the LCAT molecules in normal (endogeneous) as well as

in LCAT deficient patients assembles lipoprotein complexes that

resemble HDL in size. These observations are similar to those seen

in conditions where HDL levels are very low e.g. Tangier disease (27).

These data strongly support the notion that the enzyme/substrate

complex for the LCAT reaction consists of a specific sub-fraction of

HDL and LCAT. This complex may thus function as a unit until it is

removed from circulation.

Comparision of the plasma lipoproteins of guinea pigs and humans.

The main model system used for this study was guinea pig

plasma. These animals have remarkably low concentrations of HDL in

their blood plasma and, as is the case in humans, LDL is the major

lipid bearing fraction (28). Cholesterol feeding of guinea pigs results

in a rapid expansion of the plasma cholesterol pool and subsequent

development of atherosclerosis (28).

As mentioned earlier, LDL is the major lipid carrier lipoprotein

in the plasma of guinea pigs. The LDL fractions of guinea pigs are

denser (1.005-1.10 gm/ml) than those of most other species (1.006-

1.063 gms/ml). In addition, guinea pig LDL has a smaller diameter

than normal human LDL. (28).

Although, HDL levels are very low in these animals, their

density (1.10-1.21 gm/ml.) and composition are similar to human

HDL. The major apoprotein of guinea pig HDL is APO Al. Another

12

apoprotein similar to human APO All is also present.

Fatty acid composition of lipoproteins in guinea pigs.

Linoleate is the major fatty component of LDL and VLDL

accounting for 70% of the fatty acyl moiety of cholesteryl esters. The

fatty acid composition of the plasma phospholipids is identical in

VLDL and LDL. The major fatty acids present in phospholipids are

stearate and linoleate. Linoleate is also a major fatty acid component

of the triglycerides in both LDL and VLDL (29).

Guinea pig serum apoproteins

APO B is the major apoprotein of VLDL and LDL. The molecular

weight of this apoprotein on SDS-PAGE is higher than 400,000 Da.

Based on amino acid composition analysis, guinea pig APO B appears

analogous to human APO B. A number of isoforms of APO B have also

shown to be present in guinea pigs (30).

Half of the protein moiety of guinea pig HDL is APO AI which

can be resolved into two bands on gel electrophoresis. Another

apoprotein identified in guinea pig HDL resembles human APO AII.

A number of other low molecular weight apoproteins are also

detectable on gel electrophoresis of guinea pig VLDL and LDL (31).

One of these resembles the human APO CI. Guinea pigs do not have

an apoprotein analogous to the human APO CII, the activator of

lipoprotein lipase (29). Although an APO CII type LPL activator is

absent, guinea pigs have an efficient triglyceride clearing system as

13

evidenced by their resistance to hypertriglyceridemia even when fed

a high fat diet (32)

Characteristics of fish eve disease plasma lipoproteins.

The second model system used in this study was plasma from

fish eye disease patients. Fish eye disease is a familial condition

clinically characterized by severe corneal opacities, appearing at an

early age and causing visual impairment (32). A second

characteristic feature is a pronounced dyslipoproteinaemia. The

most striking abnormality is a 90% reduction in circulating HDL and

HDL cholesterol as compared to normal subjects. HDL isolated from

the plasma of fish eye disease patients constitute a homogeneous

population of abnormally small but spherical particles, in which only

about 20% of the cholesterol is esterified as compared to 75-80% in

control HDL (33). The extremely low cholesteryl ester content of

HDL in fish eye disease is remarkable because the plasma cholesterol

esterification proceeds at an apparently normal rate in vivo resulting

in normal relative cholesteryl ester contents of the two other plasma

lipoproteins, VLDL and LDL. The plasma pool of APO AI is also

reduced by 90% in these patients (33).

Purpose of this study.

As has earlier been described already, HDL is a major

lipoprotein involved in the LCAT reaction and reverse cholesterol

14

transport. A significant amount of information is now available

concerning the structure and mechanism of LCAT, but only limited

information is available regarding the nature of the substrate for LCAT

in vivo. Equally little or no information is available concerning the

mechanism(s) whereby substrate lipids are transferred from lower

density lipoproteins to HDL. We have yet to learn about the

mechanisms involved in the delivery of substrates to the enzyme

surface, the nature of the enzyme/substrate complex.and the mode of

product removal in this reaction. HDL deficient plasma, (such as fish

eye disease and guinea pig plasma) were chosen as model systems for

this study because they represent attractive models for the

investigation of enzyme-lipoprotein interactions.-

The overall goal of this study was to investigate the relationship

between LCAT and circulating lipoproteins, specifically HDL, in order

to understand their respective contributions to reverse cholesterol

transport. The primary objective was to gain additional insights into

the origin of lipid substrates and the fate of the product cholesteryl

esters of the LCAT in guinea pig plasma. The second objective was to

study the HDL/LCAT complex in a system where the HDL levels are

very low and the LCAT activity is normal.

15

REFERENCE LIST

1. Paul S. Roheim (1986) Am. J. Cardiol. 57, 3C-10C.

2. Lipids in human nutrition. Germain J. Brisson. University of Laval,

Quebec, Canada.

3. Lindgren, F.T., Jensen, L.C., and Hatch, R.T. (1972) Blood Lipids

and lipoproteins pp. 181, Wiley (Interscience) N.Y.

4. Lees, R.S. and Hatch, R.T. (1963) J. Lab. Clin. Med. 61, 518 - 527.

5. Fielding, C.J. and Fielding, P.E. (1971) FEBS Letters 15, 355.

6. Lipoiproteins and corona heart disease. New aspects in the

diagnosis and therapy of disorders of lipid metabolism.

7. Brown, M.S. and Goldstein, J.L. (1976) Science 191 , 150.

8. Goldstein, J.L. and Brown, M.S. (1973) Proc. Natl. Acad. Sci. USA

70 , 2804 - 2808.

9. Glomset, J.A., Janssen, E.T., Kennedy, R. and Dobbins, J. (1966)

J. Lipid. Res. 7, 639 - 648.

10. Glomset, J.A., (1970) Am. J. Clin. Nutr. 23 , 1129 - 1136.

11. Glomset J.A. (1968) J. Lipid Res. 9, 155.

12. Fielding, C.J., Shore, V.G. and Fielding, P.E. (1972) Biochim.

Biophys. Acta 270 , 513.

13. Glomset, J.A., Parker, F.T., Jaden, M. and Williams, R.H. (1962)

Biochim. Biophys. Acta 58, 396 - 406.

14. Fielding C.J. and Fielding P.E. (1981) Proc. Natl. Acad. Sci. USA

16

78, 3911.

15. Fielding C.J. and Fielding P.E. (1980) Proc. Natl. Acad. Sci. USA

77, 3327 - 3330.

16. Nichols, A.V., and Smith, J.J. (1965) J. Lipid Res. 6 , 206 - 210.

17. Jahani, M. and Lacko, A.G., (1981) J. Lipid Res. 22, 1102.

18. Scumaker, V.N., and Adams, G.H. (1969) Annual Rev. Biochem

38, 113 - 136.

19. Akanuma, Y. and Glomset, J.A. (1968) J. Lipid Res. 9, 620.

20. Assmann, G. (1976) Lipoprotein Metabolism edited by Greten,

H. 106 - 110.

21. Carew, T.E., Koschensky, T, Hayes, S.B., and Steinberg, D.

(1976) Lancet 2 , 1315 - 1317.

22. Eisenberg S. (1984) J. Lipid Res. 25 , 1017.

23. Anderson,D.W.., Nichols, A.V. et. al. (1977) Biochim. Biophys.

Acta. 493 , 55 - 68.

24. Fielding C.J, and Fielding P.E. (1985) Biochemistry of lipids and

membranes (Vance D.E. and Vance J.E. eds) 1st edition pp 404 -

474. The Benjamin Cummings Publishing Company, CA.

25. Fielding P.E. and Fielding C.J. (1980) Proc. Natl. Acad. Sci.U.S.A.

78, 3911.

26. M. C. Park, B.J. Kudchodkar, J. Frohlich and A.G. Lacko (1987)

Archives of Biochem. and Biophys. 258, 545 - 554.

27. P. Haydn Pritchard, Roger McLeod, Jiri Frohlich, M.C. Park, B.J.

17

Kudchodkar and A.G. Lacko (1988) Biochim. Biophys. Acta. 958, 227.

28. P. Barter, 0. Faergeman and R.J. Havel (1977) Metabolism 26

No. 6 , 615- 621.

29. Chapman M.J. (1980) J. Lipid Res. 21 , Animal lipoproteins

review. pp. 827-828.

30. Chapman M.J., G.L. Mills and J.H. Ledford (1975) The

Biochemical Journal. 149 , 423 - 436.

31. Luck S.S. Guo, Robert L. Hamilton, Rosemarie Oswald and R.J.

Haval. (1982) J. Lipid Res. 23, 548-555

32. Homquist, L. and Carlson, K. (1987) Lipids 22 No. 5, 305 - 311.

33. Carlson and Holmquist, L. (1885) Acta Med. Scand.218, 197-

205

CHAPTER II

MATERIALS AND METHODS

Animals

Hartley strain male guinea pigs were used for this study.

Animals were housed individually in stainless steel cages in a

controlled temperature environment. They were fed regular Purina

Mills Chow diet containing 4% fat, 18% protein, 16% fiber, and 3.5%

minerals.

The animals were divided into two groups. The first group of

animals were continued on the regular chow diet, while the second

group was switched to an experimental diet, similar in composition

to the regular diet except containing additional cholesterol and fat.

The experimental diet was prepared by coating the regular chow

with 1% cholesterol dissolved in 5% melted butter. The

experimental animals were fed with this 1% cholesterol and an

additional 5% fat diet for 6 weeks.

Collection of blood

At the end of 6 weeks four cholesterol fed animals and four

control animals were anesthesized with ether and blood was

18

19

withdrawn from the heart and transferred to chilled tubes containing

0.5 M EDTA (ethylenediamine tetracetic acid). Plasma was obtained

immediately after blood collection by low speed centrifugation at 40 C

for 30 minutes. An aliquot of plasma from each animal (2-3 ml) was

kept frozen and the remainder was used for the determination of

lecithin: cholesterol acyl transferase (LCAT) activity, isolation of

lipoproteins, and determination of cholesterol. Plasma from one fish

eye disease patient was supplied by Dr. J. Frohlich, University of

British Columbia, Vancouver, B.C. Canada.

Materials

CQhemicals

Triethylamine, cholesterol and triglyceride aqueous standards

were purchased from Sigma Chemical Co., St. Louis, Missouri.

Acrylamide, bisacrylamide, sodium dodecyl sulfate (SDS), ammonium

per sulfate, i3-mercaptoethanol, glycine, N,N,N',N'-

tetramethylethylenediamine (TEMED), Coomassie blue G-250, Tris

base and Bio-Gel A 5M for gel filtration were obtained from Bio-Rad

Laboratories, Richmond, California. Triglyceride and cholesterol

assay kits and DTNB (5,5' dithiobis nitrobenzoic acid) were

purchased from Boehringer-Mannheim Co., Indianapolis, Indiana.

Molecular weight standards for gel electrophoresis were obtained

from Pharmacia Fine Chemicals., Piscataway, New Jersey. [7a-3 H]

Cholesterol was obtained from ICN Biomedicals Inc., Radiochemicals

20

division., Irvine, California. Polyethylene glycol-6000 was obtained

from Matheson, Colman and Bell, East Rutherford, New Jersey. All

other chemicals were obtained from Fisher Scientific Company., Fair

Lawn, New Jersey.

Methods

Distribution of LCAT activity among plasma lipoproteins.

Gel chromatography

The plasma from guinea pigs and a fish eye disease patient was

fractionated by gel filtration chromatography as described by Rudel

and et.al.(1). Plasma samples from control and cholesterol fed guinea

pigs and the fish eye disease subject (2.5 ml) were labeled with 25 gl

(0.1 mCi/ml) of [3 H] cholesterol as a marker by incubating for 16 hrs

at 40 C. The samples were then applied to a column (1.5 x 90 cm)

packed with Biogel A 5M. The column was equilibrated with 0.0 1%

EDTA, 0.15M NaCl, pH 7.0. Lipoproteins were isolated by eluting the

column with the same buffer at a flow rate of 15 ml/hr. Two ml

fractions were collected and the radioactivity and LCAT activity were

determined.

Density gradient ultracentrifugation.

The plasma samples from guinea pigs were separated by

density gradient ultracentrifugation (2). Two ml of plasma were

transferred to ultracentrifuge tubes with 1 ml markings. The density

of the plasma was adjusted to 1.25 gm/ml by dissolving solid KBr

21

(388 mg/ml). The plasma was then sequentially layered under NaCl -

KBr salt solutions of densities 1.225 gm/ml (2 ml) and 1.10 gm/mi

(4 ml) and the tube was filled with distilled water. The tubes were

placed in a Sorvall SW 40, Ti rotor and centrifuged at 39,000 xg for

22 hrs in a Sorvall OTD-B ultracentrifuge at 150 C. One ml fractions

were removed from the top of the tubes by a narrow bore Pasteur

pipette. The cholesterol concentration and LCAT activity were

determined in each fraction.

Polyethylene glycol precipitation

Fish eye disease plasma was precipitated by using a method

described by Vikari to isolate the HDL/LCAT complex (3). The fish

eye disease plasma was treated with a 50% (W/V) solution of

polyethylene glycol (PEG-6000) to achieve a final concentration of

6% (W/V). The mixture was stirred for 30 minutes at 40 C and

centrifuged at 7000 xg. The supernatant solution was carefully

removed. The LCAT activity was measured in the supernatant as well

as in the precipitates by the Glomset and Wright method.

Preparation of human HDL substrate

HDL substrate was prepared for the measurement of exogenous

LCAT activity (4). HDL solution, containing 50 mg of protein, was

mixed with 10 ml of an emulsion of [1,2-3 H] - cholesterol (5 gci/ml)

in 10 percent (w/v) fatty acid free bovine serum albumin (BSA) (5).

To this emulsion, a solution of BSA in buffer was added to achieve a

22

final volume of 100 ml, a final BSA concentration of 2 percent (w/v)

and a final buffer concentration of 0.01 M Tris, 0.005 M EDTA, 0.15

M NaCl and 0.005 M P-mercaptoethanol and a final pH of 7.2.

Lecithin: cholesterol acyl transferase LCAT) activity

The LCAT assay was conducted on alternate fractions by the

Glomset and Wright method (6). HDL substrate (200gl) was

incubated overnight at 370C with 250. of each fraction as the LCAT

source. The reaction was stopped by adding 2 ml of isopropanol.

Lipids were extracted from lipoprotein and the lipid mixture was

evaporated under a nitrogen stream. The dry residues were

dissolved in 100 gl of n-heptane and applied to plastic backing silica

gel sheets for thin layer chromatography (TLC). TLC was carried out

in petroleum ether: diethyl ether: acetic acid (90:10:1, V/V/V). The

lipid bands were visualized by exposing the air dried TLC plate to

iodine vapors. After evaporation of iodine, the zones containing

cholesterol and cholesteryl esters were cut out of and placed in

scintillation fluid for radioactivity counting. The zones containing the

radioactive cholesterol and cholesteryl esters were counted and the

percent esterification was calculated for each assay.

% of cholesterol esterified = Radioactivity in CE (CPM) x 100.

(LCAT activity) Radioactivity in TC (CPM)

where CE = cholesteryl esters; TC = total cholesterol. (FC + CE)

23

The initial rate of endogenous cholesterol esterification was

measured by the modified method of Stokke and Norum (7). The

Stokke and Norum type of assay is known to reflect parameters other

than endogenous enzyme levels, such as the concentration and

composition of endogenous substrates. The net cholesterol

esterification (mg/day) rate was calculated as a product of the plasma

FC pool (mg) and fractional rate of esterification (% cholesterol

esterified per day). The size of the plasma FC pool was calculated as

the product of the FC concentration (mg/ml) and the plasma volume

(ml), which was assumed to represent 4% of body weight. (8)

Plasma Incubation Study

Plasma isolated from control and cholesterol fed guinea pigs

was incubated for 0 and 8 hrs at 370C. to determine the lipoprotein

cholesterol changes during the incubation periods. After incubation,

the individual lipoproteins were isolated by density gradient

ultracentrifugation as described above (2). The following three

fractions of different volumes and densities were pooled as VLDL,

LDL and HDL for the determination of the distribution of cholesterol.

Fraction 1 VLDL d<1.01 gm/ml (1 ml)

Fraction 2 LDL d=1.01-1.065 gm/ml (4 ml)

Fraction 3 HDL d=1.065-1.19 gm/ml (4 ml)

24

Analytical methods

Cholesterol determination :

Total and free cholesterol were measured enzymatically in the

fractions as described above, using enzymatic kits from Bohringer

Mannheim. The concentration of cholesteryl ester fraction was

calculated as the difference between total and free cholesterol.

Protein determination :

Total protein was determined in the lipoprotein fractions

shown above by the modified Lowry method (9).

Analytical gel electrophoresis

HDL fractions from both control and cholesterol fed animals

were analysed for protein composition by SDS-Polyacrylamide gel

electrophoresis (10). HDL fractions from both of these animal groups

were pooled separately. They were dialyzed against 0.01 M Tris

buffer containing 0.15 M sodium chloride and 0.005 M EDTA, pH

7.4, and gel electrophoresis was carried out in the presence of 0.1%

W/V sodium dodecyl sulfate.

25

REFERENCE LIST

1. Rudel, L.L., Lee, J.A., Morris, M.D. and Felts,J.M. (1974)

Biochem J. 139 , 85-89.

2. P. N. Demacker et. al. (1983) Clin. Chem. 29149, 656-663.

3. Vikari, J. (1976) Scand. J. Clin. Lab. Invest. 36 , 265-270.

4. Jahani, M., Huttash R, and Lacko A. G. (1980) Prep. Biochem.10,

431-444.

5. Lacko, A. G., Rutenberg, H. L., and Solott, L. A. (1973) Biochem.

Med. 7 , 178.

6. Glomset, J.A., and Wright, J.L. (1963) Biochim. Biophys. Acta,70,

389.

7. K. T. Stokke and K. R. Norum (1971) J. Clin. Lab. Invest, 27, 21-

27.

8. I. S. Edelman and J. Liebman, Anatomy of body water and

electrolytes. (1959) Am. J. Med., 27 , 256-277.

9. Lowry, O.H., Rosebrough, N.J., Farr, A.L. et. al. (1951) J. Bio.

Chem. 193 , 265.

10. Laemmli, U.K. (1970) Nature 227, 680.

CHAPTER III

RESULTS

Distribution of Lecithin : Cholesterol acyl transferase activity among

plasma lipoproteins.

A) Guinea pigs.

i) Bio-gel A 5M column chromatography : In order to determine the

distribution of the LCAT activity among plasma lipoproteins, 3 H

cholesterol labeled plasma samples from control and cholesterol fed

animals were fractionated on a Bio-gel A 5M column (1). Figures 4[A]

and 4[B] show the radioactivity and the LCAT activity patterns of

control and cholesterol fed guinea pig plasma, respectively. The

radioactivity pattern shows three main radioactive peaks

corresponding to the elution volumes of VLDL, LDL and HDL isolated

from normal human plasma by sequential ultracentrifugation. The

first two peaks corresponded to VLDL and LDL, respectively and the

third small peak corresponded to HDL. The LCAT activity was

measured in alternate fraction by using exogeneous human HDL as

substrate (2). The data presented here (Fig 4A and 4B) show that

even though the HDL levels were very low in these animals, the LCAT

26

27

CONTROL PLASMA

UL1L LOL HDL- Plasma

Proteins 1800[W 12

U 600-+ %CEW4 8

400 -06

W4

0 200-

-2

0 00 20 40 60 80 100

FRACTION NUMBER

FIGURE 4fA]

The distribution of LCAT activity in the plasma of a control guinea pig.Aliquots of plasma from a control animal were labeled with [ 3 H]cholesterol and chromatographed on a Bio-Gel A 5M column(1.5 x 90 cm). Fractions (2 ml) were collected and analyzed forradioactivity (CPM) and LCAT activity (% CE) as described undermethods. The elution volumes of standard lipoproteins (VLDL, LDL,HDL) isolated by ultracentrifugation are indicated.

28

CHOLESTEROL FED PLASMA

ULDL LDL HOLPlasmaProteins

40 60 80 100

20

15

101

0

FRACTION NUMBER

FIGURE 4FBI

The distribution of LCAT activity in the plasma of a cholesterol fedguinea pig. Aliquots of plasma from a cholesterol fed animal werelabeled with [3 H] cholesterol and chromatographed on a Bio-GelA 5M column (1.5 x 90 cm). Fractions(2 ml) were collected andanalyzed for radioactivity (CPM) and LCAT activity (% CE) as describedunder methods. The elution volumes of standard lipoproteins(VLDL, LDL, HDL) isolated by ultracentrifugation are indicated.

2000-

15004

%NOW1000

0

500.

0

CE

- %

0

-

20

29

activity was associated with this lipoprotein fraction both in control

and in cholesterol fed guinea pigs. These data also show that there is

a shift in the LCAT activity pattern towards larger molecular size

particles in cholesterol fed animals as compared to the controls.

ii) Density gradient ultracentrifugation : In order to confirm the

results obtained by gel filtration chromatography, the distribution of

LCAT among lipoprotein fractions was also determined after

fractionation of plasma lipoproteins by density gradient

ultracentrifugation as described in methods (3). The data presented

in Figure 5[A] and 5[B] show the cholesterol concentration and the

distribution of LCAT activity in the plasma lipoprotein fractions. The

Cholesterol concentration patterns show that fractions in the LDL

density range contained most of the cholesterol found in plasma both

in control and cholesterol fed guinea pigs. LCAT activity on the other

hand was associated with the HDL3 type molecules. The results from

this experiment also show a shift of LCAT activity towards lower

density (HDL2 density range) fractions in cholesterol fed animals

(Figure 5B). Enrichment of free cholesterol in HDL2 fractions of

cholesterol fed goinea pigs, could shift the LCAT activity towards this

density range.

30

B) Fish eye disease.

i) Bio-gel A 5M column chromatography:

The chromatography pattern shown in Figure 6 was obtained

with plasma from fish eye disease patient. The plasma sample was

labeled with [3H] cholesterol and then fractionated on a gel filtration

column. Gel chromatography (1) and LCAT activity measurement (2)

was performed as described above for guinea pigs. The radioactivity

pattern shows three peaks corresponding to the elution volumes of

three human lipoprotein standards. In the Fish eye disease patient's

plasma LCAT was also associated only with HDL size particles even

though this plasma was abnormally low in these types of lipoproteins.

ii) Polyethylene glycol precipitation :

In order to confirm that LCAT is indeed associated with HDL

type lipoprotein particles, Polyethylene glycol (PEG) precipitation

was used to separate HDL from VLDL and LDL. PEG is a neutral

polymer which selectively precipitates VLDL and LDL from the

plasma without altering the enzyme activity (4). Absence of APO B

containing lipoproteins (VLDL and LDL) in the PEG supernatant

(containing HDL/LCAT complex) was confirmed by polyacrylamide

gel electrophoresis. Our data show that after PEG precipitation all of

the LCAT activity originally present in the plasma (3.1 %

esterification/hr) was recovered in the PEG 6000 supernatant (3.5 %

esterification/hr), indicating that in FED plasma LCAT is associated

31

CONTROL PLASMA

VLDL < 1.006 gm/mlLDL = 1.006-1.063 gm/mlHDI2 = 1.063-1.125 gm/mlHDL3 = 1.125-1.21 gm/ml

LOL

% C

HOLPlasmaProtein 2

ml I 41 444E

44444

I~4444~4

4

444I4 44

44

4 4,4 4

444

44 444 4

4, 44 44 4

4 44 4

41-mRaom in04m- -- S -. 0 . . 0 .- .--

1.03 1.06 1.09 1.12 1.15 1.18 1.21 1.24

DENSITY FRACTIONS

FIGUR E 5 [A'

The distribution of LCAT activity in the plasma of control guinea pig.Aliquots of plasma from a control animal were labeled with [3 HIcholesterol and lipoproteins were separated by density gradientultracentrifugation. Fractions (1 ml) were collected and analyzed fortotal cholesterol concentration (TC) by using the enzymatic method.

ULDL

280

240

200

160

120 -

80-

40

z

IQz00

n

10

.Q

0

1.00

I . .I I wommom

32

CHOLESTEROL FED PLASMA

VLDL < 1.006 gm/mlLDL = 1.006-1.063 gm/mlHDL2 = 1.063-1.125 gm/mlHDL3 =1.125-1.21 gm/mil

ULDL LDL HDL Plasma

1200# i4 Proteins 6

1000

z 800

600

4 400

0 200

4~

* j.tg/rnl .9/~

0/o CE/

_____________________________________________________ 4~'9

9*99

~9

99

'99

99

5

25

CM U0 01 A-1.00 1.03 1.06 1.09 1.12 1.15 1.18 1.21 1.24

DENSITY FRACTIONS

FIGURE 5 [B]

The distribution of LCAT activity in the plasma of cholesterol fedguinea pig. Aliquots of plasma from a cholesterol fed animal werelabeled with [3 H] cholesterol and lipoproteins were separated bydensity gradient ultracentrifugation. Fractions (1 ml) were collectedand analyzed for total cholesterol concentration (TC) by using theenzymatic method.

- 11 1- 11 - ! ., , - - , , It " k m - - , 4 w .

ONSW^

33

i l II

0M0m

000

0F

(NWdO) 'Io-a S3Z1OHD Hv

QO

0=

-J0-J

-J0-J

-4

0f

I II I

Cd

00 U 0

ta

8 o

PC

0 0

Q cu

*U.

. .

...0 .. , -w%o PC

0 d

10 !

a I -F

34

with HDL type lipoprotein particles.

Thus, the results obtained both in guinea pigs and in fish eye

disease patients, show that even in the presence of abnormally low

HDL, LCAT is associated with HDL size particles.

Effect of cholesterol feeding on endogeneous LOAT activity.

The fractional rates and net rates of plasma cholesterol

esterification determined as described in methods are shown in

Table 11(5). The fractional rate of endogeneous cholesterol

esterification was 10.6 + 0.88%/hr for the control animals. This

value is significantly higher (p<0.0005) than the fractional rate in

animals that were fed cholesterol (2.6 0.60%/hr). The net rate, on

the other hand, showed no significant differences between the

control and cholesterol fed animals. Control animals produced 31.9

+ 7.5 mg of cholesterol esters per day while cholesterol fed animals

produced 28.6 + 5.4 mg of cholesteryl esters per day.

Plasma LCAT activity as measured with an exogenous substrate

[indicative of LCAT mass (6)] was not affected by cholesterol feeding

(Figure 4A and 4B), (7). However the decreased fractional rate of

cholesterol esterification in cholesterol fed animals (control 10.6 +

0.88 %/hr; cholesterol fed 2.62 + 0.60 %/hr) could indicate some

other changes, specifically:

1. Transfer of substrate lipids from the site of origin to the site of

35

TABLE II

EFFECT OF CHOLESTEROL FEEDING ON THE PLASMACHOLESTERYL ESTERS TURNOVER DETERMINED IN VITRO

GROUP ICONTROL ANIMALS

Animal BODY UNESTERIFIED LCAT ACTIVTYnumber WEIGHT CHOLESTEROL

gms. Ig. %/lhr. mg / day

1) 824 11.7 11.1 31.1

2) 1136 11.5 11.5 31.8

3) 1136 17.2 10.1 41.5

4) 1023 10.1 9.6 23.3

1030 64 12.6 1.4 10.6 0.88 31.9 7.5

GROUP IICHOLESTEROL FED ANIMALS

Animal BODY UNESTERIFIED LCAT ACTIVITYnumber WEIGHT CHOLESTEROL

gMs. mg. %/hr. mg/day

1) 1046 68.1 2.2 35.8

2) 859 42.6 2.8 28.4

3) 945 33.6 3.4 27.6

4) 1050 44.8 2.1 22.6

975 39 47.3 6.4* 2.6 0.60* 28.6 5.4

The plasma samples from four control and four cholesterol fedguinea pigs were assayed by the method of Stokke and Norumto evaluate the endogeneous enzyme activity (%/ hr LCAT activity).Unesterified cholesterol (mg) was calculated assuming the plasmavolume to be 4% of the body weight (gms).mg/day is net rate of esterification calculated as the product ofunesterified cholesterol pool and fractional rate.

* denotes values significantly different (p<0.0005) from controlanimals.

OWN ".4-- ,

36

esterification : Although plasma VLDL and LDL are poor substrates for

the LCAT reaction, most of the FC used for the esterification reaction

originates from these lipoproteins (8). Therefore the FC from VLDL

and LDL is transferred to HDL (the site of location of LCAT) for

esterification. As can be seen from figures 4 A&B and 5 A&B, most of

the cholesterol, both in control (figs 4A, 5A) and cholesterol fed (figs

4B,5B) guinea pigs, was found in VLDL and especially in LDL. The

rate of decrease in the transfer of the FC from these lipoprotein

could decrease the fractional rate of esterification.

2. Transfer of product cholesteryl esters from the site of LCAT

reaction to the acceptor lipoproteins: Similar to humans, guinea pig

lipoprotein contains cholesteryl ester transfer protein (CETP) which

transfers the cholesteryl esters from the site of formation (HDL or its

subpopulation) to the acceptor lipoproteins (VLDL, LDL). The HDL

particle is thus regenerated to accept more free cholesterol from

donor lipoproteins and peripheral tissues. A decrease in the transfer

of CE from HDL to VLDL and LDL could decrease the fractional rate of

esterification through the inhibition of enzyme reaction by the

product ( product inhibition).

3. Changes in the composition of substrate lipoprotein(s): Plasma

HDL is the preferred substrate for the LCAT reaction. However, HDL

is composed of different subpopulations (9). Data obtained from

37

studies of human plasma suggest that the substrate potential of APO E

rich HDL is lower than that of APO A rich HDL (10). Therefore an

increase in the APO E containing larger HDL could result in lower

fractional rates of esterification.

The following experiments were therefore performed to

distinguish between these possibilities.

Origin of lipid substrates and the fate of the product cholesteryl

esters of the LCAT reaction in guinea pigs.

The following experiments were performed to investigate the

source of free cholesterol(FC) in the LCAT reaction. The plasma

samples from both control and cholesterol fed guinea pigs were

incubated at 370 C for 0 and 8 hrs. After incubation, the lipoprotein

fractions corresponding to VLDL, LDL and HDL were isolated by

density gradient ultracentrifugation, dialyzed and their free and total

cholesterol contents were determined. The cholesteryl ester

concentration was calculated as the difference between total and free

cholesterol values. The data in Tables IV and V show that during the

8 hr incubation period, the free cholesterol content of the plasma

decreases while the cholesteryl ester concentration increases. In

control animals VLDL and LDL fractions loose 5.9 and 5.1 mg/dl of

free cholesterol, respectively while in cholesterol fed animals VLDL

and LDL loose 5.7 and 9.5 mg/dl of free cholesterol, respectively.

These data show that the free cholesterol derived from both VLDL

38

TABLE IIICONTROL ANIMALS

Contribution of lipoproteins to the free cholesterol pool utilizedfor the LCAT reaction and the fate of cholesteryl esters followingthe incubation of plasma samples in control animals.

FC mg/dl.

O0hr 8 hrMean S.D. Mean S.D. 8 hr - Ohr

VLDL 11.8 0.5 5.9 0.1 -5.9

LDL 13.7 0.6 8.6 0.3 -5.1

HDL 1.0 0.3 2.9 0.4 +1.9

-9.1

CE mg/dl.

O0hr 8 hrMean S.D. Mean S.D. 8 hr - Ohr

VLDL 3.2 0.4 11.0 0.5 +7.9

LDL 68.1 5.5 80.0 7.3 +11.9

HDL 14.0 1.7 3.0 0.8 - 11.0

+8.8

Aliquots (2 ml) of plasm from four control guinea pigs wereincubated at 00 C and 3 C for 8 hr. Following incubation, thelipoproteins were seperated by density gradient ultracentrifugationas described under methods. Lipoprotein fractions were pooled andtotal (TC) and free cholesterol (FC) contents were determinedenzymatically; esterified cholesterol (CE) was obtained as thedifference between TC and FC values. Shown above are contributionsto the FC pool for LCAT reaction removed from VLDL and LDL(8hr-Ohr FC). The total CE accepted from HDL by VLDL and LDL isalso shown above (8hr-Ohr CE).

39

TABLE IV

CHOLESTEROL FED ANIMALS

Contribution of lipoproteins to the free cholesterol pool utilizedfor the LCAT reaction and the fate of cholesteryl esters followingthe incubation of plasma samples in cholesterol fed animals.

FC mg/dl

0hr 8 hrMean S.D. Mean S.D. 8 hr - 0 hr

VLDL 13.3 0.6 7.6 1.1 -5.7

LDL 74.0 3.9 64.5 3.6 - 9.5

HDL 4.5 0.8 9.0 0.8 +4.5

-10.7

CE mg/dlC E

mg/dl 0hr 8 hrMean S.D. Mean S.D. 8hr- Ohr

VLDL 3.8 0.2 17.0 1.2 +13.2

LDL 173 7.0 175 7.3 +2.0

HDL 8.3 0.6 4.5 0.3 -3.8

+11.4

Aliquots (2ml) of plasma from four cholesterol fed Guinea pigs wereincubated at 0 C and 370C for 8 hr. Following incubation, thelipoproteins were seperated by density gradient ultracentrifugationas described under methods. Lipoprotein fractions were pooled andtotal (TC) and free cholesterol (FC) contents were determinedenzymatically; esterified cholesterol (CE) was obtained as thedifference between TC and FC values. Shown above are contributionsto the FC pool for LCAT reaction removed from VLDL and LDL(8hr-Ohr FC). The total CE accepted from HDL by VLDL and LDL is alsoshown above (8hr-Ohr CE).

40

and LDL was used for esterification by LCAT. The changes in free

cholesterol were not proportional to the free cholesterol available in

the respective isolated lipoprotein fractions. For example, during

the 8 hr incubation period, 50% of the available free cholesterol from

VLDL (VLDL-FC) and 37% of the available free cholesterol from LDL

(LDL-FC) was esterified from control guinea pigs; while 43% of the

available VLDL-FC but only 13% of the available LDL-FC was esterified

from cholesterol fed guinea pigs. These data show that VLDL-FC and

LDL-FC are used approximately to an equal extent for esterification in

control guinea pigs whereas in the cholesterol fed guinea pigs VLDL-

FC becomes the major contributor of the FC substrate of the LCAT

reaction. The amount of cholesteryl esters formed during the 8 hr

incubation was 8.8 mg/dl in control animals and 10.9 mg/dl in

cholesterol fed animals.

In both control and cholesterol fed guinea pig plasma, the

esterified cholesterol generated by the LCAT reaction was aparantly

transferred back to VLDL and LDL along with the CE present in

lipoproteins (Tables IV and V). Data in Table IV and V suggest that

VLDL accepts 7.9 mg/dl cholesteryl esters and LDL accepts 11.9

mg/dl of cholesteryl esters in control animals while in cholesterol

fed animals VLDL accepts 13.2 mg/dl while LDL accepts only 2.0

mg/dl of cholesteryl esters. In control plasma, VLDL and LDL both

41

accept cholesteryl esters from HDL fraction, while in cholesterol fed

animals VLDL is a major recipient of cholesteryl esters.

Protein composition of lipoproteins.

The total protein present in each fraction of control and

cholesterol fed guinea pigs is given in Table VI (11). VLDL protein

concentrations were similar in both control and cholesterol fed

animals. LDL and HDL protein content increased significantly

(p<0.005) in cholesterol fed animals. Figure 7 shows the gel

electrophoresis pattern of HDL from control and cholesterol fed

guinea pigs. HDL from the control guinea pigs showed a single band

of APO AL, on the other hand, the HDL from cholesterol fed guinea

pigs showed the presence of both APO E and APO AI on SDS gel

electrophoresis.

42

TABLE V

PROTEIN DETERMINATION

Determination of total protein content of lipoprotein fractions.

CONTROL ANIMALSmg/dl

1) 2) 3) 4) Mean+S.D.

VLDL 10.8 12.4 13.6 10.3 11.8 1.5

LDL 12.2 13.6 15.2 11.9 13.2 1.5

HDL 13.4 10.3 12.6 12.4 12.2 1.3

CHOLESTEROL FED ANIMALSmg/dl

1) 2) 3) 4- Mean + S.D.

VLDL 13.8 15.9 12.4 15.3 14.4 1.6

LDL 26.8 30.4 25.7 29.8 28.2 2.3 *

HDL 27.6 22.8 23.1 26.3 25.0 2.4 *

Total protein content (mg/dl) of the each lipoprotein fraction wasdetermined by modified Lowery method.

* denotes values significantly different (p<0.0005) from controlanimals.

43

0.

0C1-4.

4- V4)."OO 4-A 0

At S. OO

I I '3 0

I I -C '3-W 0

" .S0 .-. 0 4-

--- .

SO.-L-*.-

4 4.

0

0= Or.

e;L- o._

orm4-

C 'V..- - 4.'.-. - .. C r

4J 4J-4-- (A

C Or-

0 00. C

r-(1)M

-- r 0orC

' I. . -. 2 P

(10.

Or . 0. %

0 aE -D

= - -r a =-0ol- t

-, -C . .- =-4

Or -> R M-~ *r % 0'-.

(C.0cLO *Y.--L-

- --- .C") 0 --dc 0 .'.-00 OC Co0'-

44

REFERENCE LIST

1. Rudel, L.L., Lee, J.A., Morris, M.D. and Felts,J.M. (1974) Biochem

J. 139 , 85-89.

2. Glomset, J.A., and Wright, J.L. (1963) Biochim. Biophys. Acta 70,

389.

3. P. N. Demacker et. al. (1983) Clin. Chem. 2914, 656-663.

4. Vikari, J. (1976) Scand. J. Clin. Lab. Invest. 36, 265-270.

5. K. T. Stokke and K. R. Norum (1971) J. Clin. Lab. Invest. 27, 21-

27.

6. Chen, C. H. and Albers, J. J. (1982) J. Lipid Res. 23 , 680-691

7. R. Oswald, M. Crim, M. Green and M. Meng (1979) Nutr. Meta.23,

42-50.

8. M. C. Park, B.J. Kudchodkar, J. Frohlich and A.G. Lacko (1987)

Archives of Biochem. and Biophys. 258 , 545-554.

9. Eisenberg S. (1984) J. Lipid Res. 25 , 1017.

10.Marcel Yves L., Camilla Vezina. et. al. (1980) Proc. Natl. Acad. Sci.

U.S.A. 77 , No 5 2969-2973.

11.Lowry, O.H., Rosebrough, N.J., Farr, A.L. et. al. (1951) J. Biol.

Chem. 193 , 265.

CHAPTER IV

DISCUSSION

It is now generally accepted that the LCAT reaction takes place

mainly on the surface of HDL molecules (1, 2, 3, 4) as the lipids

contained in HDL serve as the most suitable substrate for this

reaction. Fielding and Fielding (1977) suggested that LCAT, CETP

and specific components of HDL, constitute a discrete cholesteryl

ester transfer complex which facilitates reverse cholesterol transport

(5). Evidence available in the literature supports the notion that this

enzyme/substrate complex for the LCAT reaction consists of a

specific subfraction of HDL containing LCAT and a number of discrete

lipid and additional polypeptide components.

The studies presented here were designed to provide

information on specific phases of reverse cholesterol transport and

on the role of LCAT in this pathway regarding:

1. The site of esterification of cholesterol by LCAT in the plasma.

2. Transfer of free cholesterol between donor and acceptor

lipoproteins.

3. Transfer of cholesteryl esters from the substrate lipoprotein to

other lipoproteins.

45

46

other lipoproteins.

Earlier studies have shown that in the plasma of LCAT deficient

patients, the LCAT activity derived from the in vitro

supplementation of the plasma was located at or near the elution

volume of the LCAT activity of control plasma. These data indicate

that the LCAT molecules in normal (endogenous) as well as in LCAT

deficient patients (exogenous) associate with lipoprotein complexes

that resemble HDL in size (6). These observations are similar to

those seen in Tangier disease, where the endogenous LCAT activity

was also found associated with HDL size particles, despite the

absence of normal HDL in these patients (7). These observations

taken together with other findings obtained with liposome substrates

(8, 9) indicate that, the HDL/LCAT complex may be held together

such that the dissociation of this complex may be unlikely under

physiological conditions. Accordingly, this complex may function as a

unit from the time it is formed and until it is removed from the

circulation. If the HDL/LCAT complex was found to operate in this

manner in vivo, then the contributions of other lipoprotein classes

(LDL and VLDL) to the LCAT reaction would be to donate lipids to the

substrate/enzyme complex and to accept one of the products (CE) of

the reaction. The data presented here show that in both control and

cholesterol fed guinea pig plasma, LCAT molecules mainly associate

with HDL size lipoprotein particles even though the HDL level is very

47

low in these animals. Similar results were observed in fish eye

disease (FED) plasma. In addition, the results from polyethylene

glycol precipitation of the FED plasma also indicate the LCAT/HDL

interaction. After PEG precipitation of the LDL and VLDL, all the

LCAT activity was recovered in the supernatant solution. Therefore

the data obtained by this study support the model discussed above.

In FED, HDL is quantitatively and qualitatively abnormal. First,

HDL mass is reduced by 90%. Second, the HDL particles are

spherical but much smaller in diameter than normal HDL. Third, the

composition of HDL in FED is altered compared to normal HDL

having a lower relative cholesteryl esters content and a larger amount

of polar lipids (10). Even though HDL levels are reduced, APO AI is

normal in FED. However, evidence presented by Homlquist and

Carlson show that in FED plasma, almost all cholesteryl esters are

synthesized from VLDL FC and LDL FC by the action of LCAT activity

(3-LCAT) (10). These results show that in FED plasma, even though

LCAT is associated with HDL size particles, it is able to utilize free

cholesterol directly from LDL and VLDL.

The data presented in Tables IV and V show the net transfer of

free cholesterol from VLDL and LDL to HDL; and indice that VLDL

and LDL provide the free cholesterol to substrate lipoprotein for the

LCAT reaction in guinea pig plasma. Plasma HDL has been considered

48

the physiological substrate for the LCAT reaction. Although VLDL and

LDL are poor substrates for the LCAT reaction in vitro, they are

known to contribute lipid substrates by first transferring them to

HDL. Studies from our laboratory employing rats have shown that of

the two major circulating lipoproteins (VLDL and HDL), HDL is a

much better substrate than VLDL for the LCAT reaction (11). These

findings are similar to those reported for human plasma, where HDL

has been established as a far better substrate than either VLDL or LDL

(12, 13, 14). The substrate specificity of LCAT in hog plasma shows

that the enzyme activity obtained with HDL as a substrate was about

twice that obtained with LDL; the activity with VLDL as a substrate

was less than 20% of the activity with HDL (15). These data suggest

that in most animals LCAT is associated with HDL size particles and

uses HDL as a substrate for the esterification reaction.

The data presented in Tables IV and V indicate that VLDL and

LDL may both serve as indierct donors of free cholesterol to HDL for

the LCAT reaction. In cholesterol fed animals, the free cholesterol

donated to HDL by the LDL fraction is slightly higher than in control

animals. However, the percentage decrease in the free cholesterol

content of LDL is smaller than that of VLDL. In the cholesterol fed

animals, the free cholesterol available is four times larger than in the

control animals, yet the percentage of free cholesterol utilized for

esterification was low compared to the controls. These findings are

49

different from those obtained with human plasma where the free

cholesterol decrease occurred predominantly in the LDL fraction,

while only a small decrease in VLDL free cholesterol was observed

(16). Consequently, the data shown by this study suggest that the

decrease of free cholesterol in VLDL and LDL during incubation

period is likely to represent transfer of FC from these lipoproteins to

the LCAT/HDL complex prior to esterification by LCAT. The nature

of this transfer process and the factors that may catalyze it or

promote it are yet to be described.

The amount of cholesterol ester formed during the 8 hr

incubation was the same in control animals as in the cholesterol fed

guinea pigs, even though cholesterol fed animals had more free

cholesterol available in their plasma. The early finding of Nichols and

Smith (17) focused on the fate of cholesteryl ester generated by the

LCAT reaction. Since then, several lipid transfer proteins have been

identified that were able to catalyze the transfer of cholesteryl esters

from HDL to lower density lipoproteins to regenerate the LCAT

substrates (18, 5, 19). Our data show that in control guinea pig

plasma, VLDL and LDL both accept cholesteryl esters from HDL,

although LDL is a better acceptor of CE than VLDL. On the other

hand, in cholesterol fed animals, VLDL becomes a better acceptor of

CE than LDL. The basis for this difference in cholesteryl ester

accepting capacity of VLDL and LDL is currently not known.

50

Our results also show that in cholesterol fed guinea pigs, HDL is

enriched with free cholesterol compared to control animals. In

cholesterol fed animals, the amount of cholesteryl esters transferred

from HDL to VLDL and LDL decreased during the 8 hr incubation

period, compared to the controls. Cholesterol feeding in these

animals increases plasma HDL levels (20, 21) and leads to HDL

particles enrichment with unesterified cholesterol and APO E of

discoidal shape (21, 22), as compared to the discoidal HDL particles

found in the plasma of LCAT deficient patients (23, 24, 25, 26). The

HDL from cholesterol fed animals also has an unusually high molar

ratio of cholesterol to phospholipid (exceeds 0.5) and elevated levels

of APO AI and APO E. APO AI increases 2 fold while APO E increases

22 fold (27). As the APO E levels increase, the number of isoforms of

APO E also increase (27). This difference in the composition of HDL

particles in cholesterol fed animals may be the basis for the reduced

cholesteryl ester transfer from HDL to VLDL and LDL. Such

observations may be due to inhibition of CETP activity because of the

presence of abnormal lipoproteins. Our data show that upon

cholesterol feeding, the total protein content of LDL and HDL

increases in addition, the data from gel electrophoresis studies show

that HDL from cholesterol fed guinea pigs is enriched in APO E.

These observed changes may give rise to lipoproteins of abnormal

composition in these animals.

51

Eisenberg (28) reported recently that in VLDL subpopulations,

the higher molecular weight species are more efficient cholesteryl

ester acceptors presumably because of their low initial cholesteryl

ester content. Dullaart et.al (29), have shown that VLDL and LDL

have an approximately equal but wide ranging capacity to accept

cholesterol esters from HDL. They suggested that VLDL rich in

triglycerides and LDL with a lower free cholesterol to phospholipid

ratio may be a better acceptor of CE than other types of APO B

containing particles (29). The abnormal composition of LDL in

cholesterol fed guinea pigs, therefore may result in a lower capacity

of these lipoproteins for accepting CE from HDL.

Cholesterol fed and control guinea pigs had similar rates of

LCAT activity when human HDL was employed as an exogeneous

substrate (30). Furthermore, the human enzyme activity was reduced

when acting on cholesterol fed guinea pig substrate compared to the

activity obtained with human lipoprotein as substrates (30). These

finding suggest that LCAT secretion or LCAT activity is normal in

cholesterol fed guinea pigs and that under these conditions no

significant liver damage occured.

Our data also show that dietary cholesterol decreases the

fractional rate of plasma cholesterol esterification. The decreased

fractional rate may be indicative of changes in the nature of substrate

52

lipoproteins since the plasma LCAT activity (exogeneous activity) was

not affected by cholesterol feeding Plasma HDL is a preferred

substrate for the LCAT reaction in most animals (11,15). Because in

cholesterol fed guinea pigs, the HDL is rich in APO E and free

cholesterol and data obtained from studies with human plasma

suggest that the substrate potential of APO E rich HDL is lower than

that of APO AI rich HDL (31), the lower fractional esterification rates

in the cholesterol fed animals could be due to these changes in HDL

composition. The cholesterol fed guinea pigs develop fatty and

histologically abnormal livers (21). Therefore the hepatic uptake of

abnormal lipoproteins enriched in APO E may also be reduced in

these animals resulting in the accumulation of APO E and cholesterol

in the plasma.

On the other hand, the net rate of cholesterol esterification is

not affected by cholesterol feeding. In both group of animals, the

amount of cholesteryl esters formed per day is the same. These

observations indicate that the composition or nature of the

circulating lipoprotein substrate in cholesterol fed guinea pigs may

be the limiting factor for the LCAT reaction rather than a defect in

the secretion of the enzyme or some alterations in the enzyme

molecule itself.

The study of cholesterol esterification and transfer in control

and cholesterol fed guinea pigs has thus allowed a more detailed view

53

of the flux of lipoprotein cholesterol during in vitro incubation that

is likely to reflect the movement of free cholesterol and CE in vivo.

This study also has provided some evidence that the HDL fraction

with a higher amount of free cholesterol and APO E could lower the

endogeneous LCAT activity and play a significant role in reverse

cholesterol transport.

54

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