45

Biochimica et Biophysics Acta, 424 (1976) 45-56 @ Elsevier Scientific Publishing Company, Amsterdam - Printed in The Netherlands

BBA 56702

CHARACTERIZATION LIPOPROTEINS SATYA N. ~ATHUR

OF THE E~RLICH ASCITES TUDOR PLASMA

and ARTHUR A. SPECTOR

Departments of Biochemistry 52242 (U.S.A.)

and Internal Medicine. University of Iowa, Iowa City, Iowa

(Received July 28th, 1975)

Summary 1. The lipoproteins of the’ Ehrlich ascites tumor plasma were separated into 3 distinct fractions, very low density, low density and high density lipoproteins by preparative ultracentrifugation combined with agarose column chromatography. 2. High density lipoproteins contained 74% of the total protein in the lipoproteins. By contrast, most of the lipids were present in the very low density lipoprotein fraction. 3. The fatty acid compositions of the cholesteryl esters were appreciably different in the very low, low and high density lipoproteins, whereas phospholipid and triacylglycerol fatty acid compositions were quite similar in the 3 lipoprotein fractions. 4. Very low and high density apoprotein electrophoretic patterns on sodium dodecyl sulfate-acrylamide gels were similar to those observed in the corresponding lipoprotein fractions obtained from other mammalian species. The low density fraction, however, contained 7 apoprotein bands, and 32% of the low density apoprotein was soluble in ~tr~e~yl urea. 5. The average molecular weights as determined by analytical ultracentrifugation were 2 - 107 (very low density), 6 - 106 (low density) and 4.4 * lo5 (high density). Introduction Ehrlich cells grow in the peritoneal cavity of mice suspended in an ascites plasma [ 11. The ascitic fluid contains albumin-bound free fatty acids [ 21 and

Abbreviations: VLDL, very low density lipoprotein: density fipoprotein.

LDL,

low density lipoprotein;

HDL,

high

46

several classes of lipoproteins [3]. A number of studies indicate that the Ehrlich cells have a limited capacity to synthesize fatty acids and cholesterol de novo [ 3,4], and they derive much of the lipid that they require from the ascites plasma [ 3-51. In order to obtain some understanding of how lipid transfer occurs between the ascites plasma and the growing Ehrlich cells, it is essential to investigate the composition and structure of the ascites plasma lipoproteins. These data, together with a comparison between the tumor plasma lipoproteins and those present in the blood plasma of the tumor-bearing mice, are provided in the present communication. Materials and Methods The procedures for transplanting, harvesting and separating the tumor cells from the ascites plasma have been described [6] . The tumor was harvested 14 days after transplantation, and about 10-15 ml of tumor was obtained from each mouse. Chylomicrons were removed from the tumor ascites plasma by centrifugation at 20 000 X g for 30 min at 4” C [ 71. The remaining lipoproteins of the plasma were isolated by preparative ultracentrifugation into three fractions [8] ; very low density lipoproteins (VLDL, density < 1.006), low density lipoproteins (LDL, density 1.006-1.063), and high density lipoproteins (HDL, density .1.063-1.21). Each of the three lipoprotein fractions was washed twice by flotation through a salt solution of appropriate density and dialysed against 0.15 M NaCl containing 1 mM EDTA. The isolated lipoprotein fractions were purified further by chromatography on a 2% agarose column as described by Rude1 et al. [9]. When subjected to agarose gel electrophoresis [lo], each of the three lipoprotein fractions migrated as a single band (Fig. 1). Blood plasma from the tumor bearing mice (here after referred to as mouse blood plasma), also was analyzed. Because only very small quantities of blood could be obtained, the lipoprotein isolation procedure for blood plasma was modified as follows. First, the total lipoprotein fraction was isolated by adjusting the density of the blood plasma to 1.21 g/ml with solid KBr and then centrifuging it at 114 000 X g for 20 h. This fraction was washed twice by flotation at the same density. The total lipoprotein fraction was separated subsequently into three fractions viz., VLDL, LDL and HDL by agarose column chromatography. For comparative purposes, the total lipoprotein fraction from the tumor ascites plasma also was isolated by this procedure in several experiments. The SF, hydrated density, molecular diameter and molecular weight of the lipoproteins were estimated using the analytical ultracentrifugal (Beckman Model E) analysis described by Lindgren et al. [ 111. To determine the hydrated densities, solutions of density 1.063 and 1.21 g/ml were used for VLDL and LDL, and solutions of density 1.006 and 1.063 g/ml were used for HDL. For electron microscopy [ 121, the isolated VLDL were stained and fixed with 1% osmium tetroxide solution in 0.1 M phosphate buffer, pH 7.4 for 2 min. LDL and HDL were negatively stained with 1% potassium phosphotungstate, pH 7.0. A small drop of the suspension was placed on a 200 or 300 Formavarcoated grid (VLDL), or a 200 or 300 Formavar carbon-coated grid (LDL and HDL), and allowed to dry at room temperature. The grid was then examined

47

Fig. 1. Agarose gel eiectrophoresis of ascites tumor plasma lipoproteins. (a) VLDL, 01) LDL and (e) HDL.

with a Hitachi HU-125-E electron microscope. The diameter (W) of the particles was calculated from photographs, in which the VLDL were magnified 9.9 - lo4 times and the LDL and HDL 1.27 * lo5 times. Urea-polyacrylamide gel electrophoresis of tetramethyl urea soluble apolipoproteins was performed according to the method of Kane [ 131. The sodium dodecyl sulfate-polyacrylamide gel electrophoresis was done aa described by Laemmli [ 141. Gels were stained with Brilliant Blue R (Sigma) according to the procedures described by Weber and Osborn [ 1511. The gels were destained by 4-5 changes of 7% acetic acid at 45°C. Bovine serum albumin, catalase, ovalbumin, lactate dehydrogenase, trypsin and cytochrome e were used as standard marker polypeptides, in sodium dodecyl sulfate gels, and molecular weights of the protein bands were determined from a semi-logarithmic plot of molecular weight versus relative mobility. Lipoprotein lipids were extracted by the procedure of Bligh and Dyer [IS]. Neutral lipids were separated from phospholipids on a silicic acid (Mallinckrodt, 100 mesh) column [ 171. Triacylglycerol, cholesterol esters and cholesterol were resolved on silica gel thin layer plates using a solvent system containing hexane/diethylether/methanol/glacial acetic acid (180 : 40 : 4 : 6 by vol.) [IS] . ‘I’riacylglycerol and cholesterol were measured by the Technicon Autoanalyzer II method ]19,20]. Phospholipid phosphorus was determined according to the method of Bartlett [ 211. Methyl esters of the fatty acids were prepared using the boron trifluoride/methanol reagent described by Metcalf et al. [ 22 1. The fatty acid methyl esters were separated by gas liquid chromatography on a SILAR-1OC (Applied Science Laboratories, Inc.) 100/120 mesh Gas Chrom Q, 4 mm internal diameter column using a 5710A Hewlett Packard Gas

48

Chromatograph with temperature programming from 180-210°C. Methyl ester standards were obtained from Supelco, Inc. Protein concentration was estimated by a modification of the Lowry et al. procedure [23] as described by Lees and Paxman [24]. Bovine serum albumin was used as the standard. Results

The elution patterns of ascites tumor plasma lipoproteins on a 2% agarose column (Bio-Gel A-50 m, 100-200 mesh, BioRad Laboratories) are shown in Fig. 2. The total lipoprotein fraction of the plasma was eluted with two major peaks which corresponded to the peaks produced by the isolated VLDL and HDL fractions. However, as the elution pattern in Fig. 2 indicates, substantial

Ascites

HDL

0.2

.&

0.0

_s

1.6

z

I.2 0.8

3 z g

0.4

- 0.2 .a.0 86-/

,*-\

Asc~tes

VLDL - 0.6

‘I5,

- 0.4 - 0.2 T&--K’

EFFLUENT

(ml

200

’ ‘0.0

1

Fig. 2. Separation of tumor plasma lipoproteins by agarose ly isolated lipoprotein fractions were applied to a Bio-Gel eluted with 0.15 M NaW0.0196 EDTA at 4OC.

column chromatography. A-50 m agarose column,

The ultracentrifugal2.6 cm X 41 cm, and

49

amounts of intermediate size lipoproteins also were present. The ultracentrifugally isolated VLDL fraction was eluted as a single peak (elution volume 70 ml) just after the void volume (68 ml), The LDL fraction resolved into two distinct peaks, with a main peak at 129 ml and a minor peak corresponding to that of VLDL. More than 94% of the LDL protein, however, was present in the main peak. The HDL fraction was eluted as a single peak (elution volume 165 ml) which contained more than 99.5% of the protein. Although a very small amount of turbidity appeared near the void volume, this accounted for less than 0.5% of the protein in the HDL fraction. ~though there was some overlap between the LDL and HDL peaks on the 2% agarose column, these 2 lipoprotein fractions were resolved completely on a 4% agarose column. When the total lipoprotein fraction isolated at density 1.21 g/ml from the blood plasma of the tumor-bearing mice was applied on the 2% agarose column, the elution pattern (Fig. 2) was very similar to that observed with the total lipoprotein fraction of the ascites plasma. The fractions corresponding to main VLDL, LDL and HDL peaks of blood or ascites plasma were collected as follows: VLDL, elution volume 66-88 ml; LDL, elution volume 111-143 ml; HDL. elution volume 158-180 ml.

The protein distribution and lipid composition of the ascites lipoproteins are shown in Table I. HDL contained 74% of the total lipoprotein protein, whereas VLDL and LDL each accounted for 13%. By contrast, most of the lipids were contained in the VLDL, with more than 86% of the triacylglycerol being present in this fraction. Most of the cholesterol present in the VLDL was unes~rifi~, whereas in LDL and HDL, most of the cholesterol was in the form of cholesteryl esters. As shown in Table II, there were significant differences in the fatty acid composition of the cholesteryl esters contained in the three lipoprotein fractions. The major fatty acids of the cholesteryl esters of VLDL were 18 : 1 (n-9) and 13: 2 (n-6), w h ereasl8:2~~-6)~d20:4(~-6~werethe predominant fatty acids of the cholesteryl esters in HDL. LDL cholesteryl esters were rich in 18 : 1 (n - 9) and 18 : 2 (n - 6) as well as 20 : 4 (n - 6). TABLE I LIPID COMPOSITION Mean values (*S.E.) Lipoprotein Class

VLDL LDL HDL

OF ASCITES TUMOR PLASMA LIPOPROTEINS

are given and the number of samples anaiyzed is shown in parentheses,

Protein (mg/lOO ml) plasma

Phospholipids Wmol P/mg) protein

Triacylglycerol

Cholesterol

(mg/mg protein)

Free

9.7 f 1.5 (6)

2.60 f 0.30 (11)

11.0 (11)

10.2 i: 1.8 (6)

1.15 f 0.08 (11)

56.4 ?: 4.2 (6)

0.41 f 0.02 (12)

0.06 (12)

Esterified -._.___

fmgimg protein) 0.98 * 0.02

0.69 f 0.02

(5)

(5)

1.35 + 0.10 (11)

0.34 + 0.02 (5)

0.89 f 0.06

+ 0.01

0.05 f 0.01 (6)

0.39 * 0.03

+ 0.9

Free/ester

1.42 0.38

(5) (6)

0.12

16:0 16:l (n - 9) 18:0 18:l (n - 9) 18:2 (n - 6) 20:3 (n - 6) + 22:l 20:4(n-6) 22:6 (n - 3) Others

T

T

3 f 0.8 9

1 * 0.1 6f 1.0 2 t 0.4 5

1.6 0.6 0.9 1.0 0.9

T 12 k 0.8 T 1

f + + f t

19 4 I 34 23

zk0.8 + 0.4 f 0.5 k 3.0 + 0.6

18 3 4 41 20

2 0.4 0.1 2 1

23 3 4 35 23

f + i f f

HDL

LDL

OF ASCITES TUMOR

VLDL

TriaeylgIycerol

% cornposItion

of 6 samples are given.

Fatty acid

ACID COMPOSITION

PERCENT FATTY

Mean vr,Iues (*S.E.)

TABLE II LIPOPROTEINS

2 f 0.4 14 ? 1.0 T 8 I f 1.3 22 c 1.9 T 2

f 0.9 _+0.4 f 0.3 f 1.8 f 1.2

6 3 2 21 31

1.0 0.3 0.5 2.8 0.9

12 4 4 34 22

* f + f i

LDL

VLDL

Cholesterol ester

PLASMA

5 + 1.0 38 + 3.4 3 + 0.8 3

T 10 f 0.5 37 f 3.2

3 f 0.6 1 _+0.2

HDL

f ? + f k

0.7 0.6 1.0 0.9 1.0 4 f 0.2 20 f 1.6 5 + 0.6