Triglyceride-poor very low density lipoprotein in human serum

23

CZinica Chimica Acta, 94 (1979) 23-29 0 EIsevier/North-Holland Biomedical Press

CCA 10039

TRIGLYCERIDE-POOR HUMAN SERUM

VERY LOW DENSITY LIPOPROTEIN IN

2. REINER and E. KOREN Laboratory for Experimental Medicine, Faculty of Pharmacy and Biochemistry, of Zagreb, A. Kovac’i&a 1, 4 1000 Zagreb ~Yugoslauia~

University

(Received September 22nd, 1978)

Summary The chemical and physical properties of very low density lipoproteins, isolated from the pool of the sera of 60 persons with high pre-fl and normal triglyceride and cholesterol concentrations, have been studied. These very low density lipoproteins, designated as triglyceride-poor very low density lipoproteins, consist of 20.5% phospholipids, 30.8% free cholesterol, 15% cholesterol esters and 33.7% triglycerides. Their protein content consists of 54.5% apo B, 26% apo A, 11.5% apo E and only 8% apo C, so they differ from any serum lipoprotein described until now. Triglyceride-poor very low density lipoproteins consist of spherical particles 300-450 d in diameter as revealed by electron microscopy.

Introduction According to Fredrickson’s classification of hyperlipoproteinaemis [l] which is widely used, an increased concentration of particular serum lipoprotein is always accompanied by an increase in the corresponding serum lipid concentration. Types II B, IV and V sera are, for example, characterized by the increased very low density lipoproteins (VLDL) and an elevated level of serum triglycerides because VLDL are normally lipoproteins with a very high triglyceride content. Occasionally sera with the increased VLDL and low triglyceride concen~ations have also been described [2,3]. However, neither the chemical nor physical characteristics of VLDL in such sera have been studied. In this paper we present the main characteristics of very low density lipoproteins isolated from the sera with normal triglyceride and cholesterol concentrations.

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Materials and methods Serum

sampling

and isolation of VLDL

A pool of sera (30 ml) was collected from 60 volunteers of both sexes with normal concentrations of serum triglycerides and cholesterol but with electrophoretically increased pre-fl (VLDL) concentrations. Blood was taken, after a 12 to 14 h overnight fast, from the cubital vein, allowed to clot at room temperature and serum was then separated by low speed centrifugation (3000 rpm for 15 min). From this pool VLDL were isolated by ultracentrifugation in a MSE-Super Speed 75 (Great Britain) with 6 X 14 ml Titanium Swing-Out rotor and polycarbonate tubes as follows. Serum was centrifuged at its own density of 1.028 g/ml for one hour at 4°C and 50 000 X g. The top 0.5 ml were then removed from each tube in order to ensure removal of chylomicrons. The rest of the serum was centrifuged at the density of 1.028 g/ml at 105 000 X g for 22 h at 4°C. After centrifugation the top 2 ml were carefully collected from each tube, diluted with 10 ml of Sorensen’s buffer pH 7.4, to a density of 1.006 g/ml, and recentrifuged for a further 22 h at 105 000 X g at 4°C. After the second centrifugation the top 2 ml (representing lipoproteins d < 1.006 g/ml) were taken for further analysis. Delipidation

and lipid analysis

Delipidation of VLDL was performed with ethanol-ether according to the method of Scanu and Granda [4]. Relative amounts of particular lipid fractions in ethanol-ether extracts were determined after their separation by thin-layer chromatography on Silicagel G (Merck, F.R.G.) with petrolether/glacial acetic acid/methanol (75 : 20 : 5, v/v, at 21°C) as developing solvent system. After chromatography the plates were air-dried and lipids were visualized by iodine vapor. The intensities of lipid spots were measured by a Kipp and Zonen (Holland) DD2 microdensitometer with BCl digital integrator and BD5 micrograph. Apolipoprotein

analysis

After delipidation protein precipitates of VLDL were dissolved in a 0.05 M Tris-HCl buffer, pH 8.6 containing 8 M urea. The insoluble material was assumed to represent apo B. Apolipoproteins, soluble in 8 M urea, were analysed by disc electrophoresis on 7.5% polyacrylamide gel containing 8 M urea according to Reisfeld et al. [5]. The intensity of apolipoprotein fractions was measured by a Kipp and Zonen DD2 microdensitometer. The presence of particular VLDL apolipoproteins was also checled by immunodiffusion against monospecific antisera [6]. Monospecific goat antisera to human A I, A II, B, C I, C II and C III apoproteins were kindly given by Prof. Holasek, University of Graz, Austria. Electron

microscopy

Electron microscopy of VLDL particles was done by negative staining method [ 71. A drop of VLDL preparation was mixed with a drop of 2% sodium phosphotungstate on top of a copper grid and immediately examined with an Opton EM-9 Zeiss electron microscope.

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Lipid and protein de terminations Triglycerides were determined according to Eggstein [ 81, total cholesterol according to Watson [9], and proteins by the method of Lowry et al. [lo]. Results Lipoprotein and lipid concentrations in the pool of sera, with normal concentrations of triglycerides and total cholesterol, but with increased VLDL concentrations, are given in Table I. Fig. 1 shows the electrophoretic lipoprotein pattern of this pool. A high amount of VLDL can be clearly seen. Lipid and apolipoprotein compositions of VLDL isolated from this pool are shown in Table II. In the lipid composition a relatively small amount of triglyceride and a very high cholesterol content (especially free cholesterol) have been found (Fig. 2). Characterization of the apolipoprotein content in these VLDL by PAGE and immunodiffusion demonstrated a very small proportion of apo C (especially low was apo C II, apo C III, and apo C III2 content) and a high proportion of apo A (with prevailing apo A I) (Fig. 3). The intensities of

start

VLDL

LDL

IiDL

Fig. 1. Gel electrophoretogram and microdensitometric normal lipid concentrations and increased VLDL.

scan

of lipoproteins

of the pooled

serum

with

26 TABLE

I

LIPID

AND

LIPOPROTEIN

CONCENTRATIONS Serum

CONCENTRATIONS

AND

lipids

INCREASED mg/100

VLDL

ml

IN

THE

POOL

OF

SERA

WITH

Serum

lipoproteins

% Total

Total

lipids

850.0

HDL

28.6

Total

cholesterol

170.0

LDL

48.6

114.5

VLDL

22.8

Triglycerides

Chylomicrons

TABLE

NORMAL

LIPID

CONCENTRATIONS lipoproteins

0

II

LIPID

AND

APOLIPOPROTEIN

COMPOSITION

VLDL Phospholipids Nonesterified Esterified

cholesterol cholesterol

Triglycerides

TRIGLYCERIDE-POOR

VLDL % Total

Apolipoprotein

% Total

Lipids

OF

VLDL

proteins

lipids

20.5

ape

B

30.8

ape

A (AI

+ AII)

54.5 + CII + CIII)

15.0

ape

c (CI

33.7

ape

E

26.0 8.0 11.5

precipitation bands obtained by immunodiffusion against monospecific antibands of sera, apparently corresponded to the intensities of electrophoretic each particular apoprotein. Electron microscopy of negatively stained VLDL isolated from the pool

F

Fig. lipid HE.

2.

Ii

Chromatographic

concentrations cholesterol

and and

esters.

densitographic

increased

VLDL.

TC

presentation F,phospholipids;

HE

of the lipids

in VLDL

H,nonesterified

from

cholesterol;

the

sera with TG,

normal

triglycerides;

27

sta’rt

\ CI\A‘i:

A9 E

CII

CIII2

Fig. 3. Mel electrophoretogram and microdensitometric obtained by delipidation of triglyceride-poor VLDL.

scan of 200 /.lg of 8 M urea soluble apolipoproteins

Fig, 4. Electron micrograph of negatively stained triglyceride-poor VLDL.

(Magnification 100 000X.)

28

revealed spherical particles being rather diameter) and in shape (Fig. 4).

homogeneous

in size (300-450

A in

Discussion VLDL isolated from sera with normal lipid concentrations and the increased pre-/3 should be classified according to their density and size as VLDL (d < 1.006, diameter 300-450 A). However, their lipid and apolipoprotein composition differs appreciably from that of the “normal”, i.e. triglyceride-rich VLDL. Although different values have been reported it is well accepted that the “normal” VLDL consist of 8-12% proteins and 88-92s lipids. Their lipid part is composed of approximately 59-70% triglycerides, 16-20% phospholipids, 5-10s free cholesterol and 814% cholesterol esters. The protein part of normal VLDL consists of about 58% apo B, 13% apo A and more than 29% apo C. The VLDL we isolated have twice as few triglycerides than the normal VLDL (we therefore designated them as triglyceride-poor VLDL) and 3 to 6 times more free cholesterol. The cholesterol/triglycerides ratio in these VLDL is 1 : 0.74 and the same ratio in the normal VLDL is about 1 : 3.5. The outstanding feature of the VLDL lipid composition is also the predomination of free cholesterol over cholesterol esters, Cholesterol esters/free cholesterol ratio was 1 : 2, while in normal VLDL this ratio is approximately 1 : 1. The apolipoprotein B content of triglyceride-poor VLDL was about the same as in normal VLDL but the apo C content was more than three times lower than in normal VLDL. However, there was twice as much apo A as in the normal VLDL. The presence of apo E is logical in view of its affinity towards cholesterol [ 111. Electron microscopy showed that triglyceride-poor VLDL particles tended to be smaller than the average VLDL particles (300-800 A in diameter). Their homogeneity in size must be stressed because normal VLDL show greater size polydispersion. Considering all the above characteristics, the differences between triglyceridepoor VLDL and any other serum lipoprotein are also significant and quite obvious. Cholesterol enriched VLDL have been found in hypothyroid humans, type II hyperlipoproteinaemia, and in cholesterol-fed rabbits [ll] but until now nothing could be concluded about the clinical significance and the origin of the VLDL described in this paper. However, clinical studies now in progress indicate that an increased concentration of pre-fl with normal serum triglycerides and cholesterol values were found in 60 among 3132 patients, i.e. with frequency of 1.92%, that it is more often in males and in old age and that it is accompanied by lower concentrations of cu-lipoproteins. Acknowledgement This work was financially supported Scientific Research of SR Croatia.

by the Self-managing

Authority

for

29

References 1 Frederickson. D.S., Levy, R.J. and Lees. R.S. (1967) New Engl. J. Med. 276,32-44; 94-102; 148156; 215-226; 273-281 2 Dahlen. G.. Ericson. C. and Furberg, C. (1972) Acta Med. Stand. Suppl. 521, 25 3 Karen, E.. Mihatov. S.. Bonid, I., Gjurovib. J. and MiIkovie. S. (1973) Angiology 24.708 4 Scanu, A.M. and Granda, J.L. (1968) Progr. Biochem. Pharmacol. 4.153 5 Reisfeld. R.A.. Lewis, V.J. and Williams, D.E. (1962) Nature 195, 281 6 Ouchterlony, 0. (1953) Acta Pathol. Microbial. Stand. 32. 231 7 Forte, T. and Nichols, A.V. (1972) Adv. Lipid Res. 10.1 8 Eggstein. M. (1966) Km. Wochenschr. 44.267 9 Watson, D. (1960) Clin. Chim. Acta 5. 637 10 Lowry. O.H., Rosebrough. N.J., Farr. A.L. and Randall, R.J. (1951) J. Biol. Chem. 193. 265-275 11 Shore. B., Shore, V., SaIel, A., Mason, D. and Zelis, R. (1974) Biochem. Biophys. Res. Commun. 58.1