Concentration and chemical composition of plasma lipoprotein subfractions in patients with peripheral vascular disease

Atherosclerosis, Elsevier

123

58 (1985) 123-137

ATH 03696

Concentration and Chemical Composition of Plasma Lipoprotein Subfractions in Patients with Peripheral Vascular Disease Evidence for Normal Apolipoprotein B but Low Cholesteryl Ester Content in Small VLDL Paolo

Pauciullo

*, Lars A. Carlson ‘, Brita Eklund and Anders G. Olsson ‘,**

2, Jan Johansson

V Research Institute Department ofClinical Physiology ‘, Internal Medicine I and King Gustaf Karolinska Institute and Karolinska Hospital, S - 104 01 Stockholm (Sweden)

’ ‘,

(Received 23 January, 1985) (Revised, received 7 June, 1985) (Accepted 8 June 1985)

Summary Subfractionation of the 3 major plasma lipoprotein classes was performed in 20 male patients with symptomatic peripheral vascular disease (PVD) and 18 male healthy controls of similar age and serum lipid levels as the patients in order to investigate if, at comparable levels of total serum lipids, any difference in the distribution or the chemical composition of the lipoprotein subfractions existed between patients and controls. Concentrations of free and esterified cholesterol, triglycerides, phospholipids, apolipoprotein B (apo B) and soluble apolipoproteins did not differ significantly in any lipoprotein subfraction of PVD patients compared to controls. Calculated

Supported by grants from the Swedish Medical Research Council (19X-204), the Swedish Association against Chest and Heart Diseases, Alfred and Gerda Svensson Foundation and King Gustaf Vth birthday Foundation. Address correspondence to: Professor Lars A. Carlson, King Gustaf V Research Institute, Box 60004, S-104 01 Stockholm, Sweden. * Visiting scientist from: II Clinica Medica, Nuovo Policlinico, Via Sergio Pansini, 5, 80131 Naples, Italy. ** Present address: Department of Internal Medicine, University Hospital, S-581 85 Linkoping, Sweden.

0021-9150/85/$03.30

0 1985 Elsevier Scientific

Publishers

Ireland,

Ltd.

124

molecular weights and numbers of lipoprotein particles/ml plasma were also similar in the 2 groups except that there were more heavy LDL particles in the patient group. Plotting concentrations of apo B against cholesteryl ester in the VLDL-D subfraction (Sr 20-100) yielded a linear regression in both groups. The PVD regression line was significantly steeper than that of controls. Calculation of the molecular mass of the various constituents of the VLDL-D fraction in the subjects with the highest content of esterified cholesterol in VLDL-D, where this difference was most pronounced, suggests that this difference was due entirely to a decreased number of cholesteryl ester molecules per lipoprotein particle in PVD. The findings suggest that a disordered metabolism of plasma cholesteryl esters may be present in certain PVD patients. Key words:

Apolipoprotein B - Cholesteryl ester - Lipoprotein protein subfractions - Peripheral vascular disease

composition

- Lipo-

Introduction Atherosclerotic peripheral vascular disease (PVD) is related to multiple risk factors such as elevated plasma lipids, smoking, diabetes and glucose intolerance [l-5]. Several studies testing the lipid hypothesis in PVD have shown a marked prevalence of type IV and IIB hyperlipoproteinemia as well as reduced levels of high density lipoprotein (HDL) [6-131. In some PVD patients, however, no increase of the lipid levels can be detected [14]. Recently the debate focused on the role of apoliprotein B (apo B) as an independent risk factor for the development of atherosclerotic disease [15-M]. Furthermore, other abnormalities in the serum lipoproteins have been considered in PVD such as abnormal chemical composition of the lipoproteins or altered partition of lipoprotein subclasses. Thus an Italian group found an increased ratio of apo B to cholesterol in very low density lipoproteins (VLDL) of PVD patients compared with plasma triglyceride-matched controls [19]. Concerning the altered distribution of lipoprotein subclasses in PVD, Rao et al. found a marked increase of the ‘light’ subfraction of low density lipoproteins (LDL) [20]. Teng et al. found that patients with hyperapobetalipoproteinemia differ from normal persons principally because of an increased amount of ‘heavy’ LDL [21]. The purpose of our study was to investigate if PVD patients, compared to plasma lipid-matched controls, showed any abnormality either in the partition of subfractions of the 3 major lipoprotein classes or in their chemical composition. Subfractionation of VLDL, LDL and HDL was undertaken and a complete chemical analysis was performed on each fraction to analyse the molecular composition of these particles.

12s

Material and Methods Patients Twenty males (aged 45-68 years) were selected from the patients attending the PVD outpatient Clinic of the Department of Internal Medicine, Karolinska Hospital, Stockholm, from February 1983 to March 1984. They were referred because of intermittent claudication of the lower limbs. Patients with rest pain or ulcers were excluded. PVD was assessed by evaluation of systolic ankle and toe pressure with a strain gauge plethysmograph [21-241. An oscillometric examination of the entire limb was carried out in order to localize the disease. Exercise test on treadmill with electrocardiogram recording was also performed. Angina was an exclusion criterium in our study while a symptomless S-T depression on the ECG was not. Thirteen patients were hyperlipidemic, 7 were normolipidemic. None of them was on diet or on drugs affecting lipid metabolism. Diabetes (fasting blood sugar greater than 6 mmol/l), hypertension (diastolic blood pressure exceeding 95 mm Hg), secondary causes of hyperlipidemia as well as any acute disease were other causes of exclusion. All patients were cigarette smokers. The control group included 18 apparently healthy, age-matched, smoking males showing on average approximately similar fasting levels of plasma cholesterol and triglycerides as the PVD patients. No disease was present in this group as evidenced by a routine clinical examination. In particular no leg pain was present on exercise and there were no abnormal pressure gradients in the legs (Table 1). None was on diet or chronic treatment with drugs. Twelve of the controls were normolipidemic subjects and 6 were hyperlipidemic. These controls were chosen in a large sample of healthy males randomly selected from the Stockholm population. Methods Blood was collected after overnight fasting into vacutainer EDTA for plasma and in normal vacutainer tubes for serum.

tubes

containing

Serum fractionation and routine lipoprotein analysis Blood was allowed to stand at room temperature for 2 h, serum and plasma were then recovered by low speed centrifugation (400 x g), and EDTA was added to serum for the routine analysis of serum lipoproteins [25]. In principle, VLDL was floated to the top by ultracentrifugation at d = 1.006 g/ml, whereafter LDL and other apo B-containing lipoproteins were precipitated by heparin/manganese, leaving HDL in solution. The fractions were extracted with chloroform-methanol and analyzed for total cholesterol and triglycerides [25]. VLDL subfractionation, recovery of IDL and LDL VLDL was subfractionated by cumulative rate centrifugation [26-281. In short, plasma was added with solid NaCl to increase the density to 1.10 g/ml and a 4-ml sample was transferred to a cellulose nitrate centrifuge tube. Three ml of d = 1.065, 3 ml of d = 1.020, and 3.4 ml of d = 1.006 g/ml NaCl solutions were layered above. Ultracentrifugation was carried out in a Beckman SW 40 Ti rotor at 20°C. Two consecutive runs were performed, calculated to float VLDL particles of the following

126 TABLE

1

AGE AND CLINICAL CHARACTERISTICS DISEASE AND OF CONTROLS Patient

Age (yr)

Body weight

Height (cm)

(kg) BK SR KB FB CA OB SK MR JH SH DB HH JK SKA KG BE FS AK HM LN Range

56 46 56 58 65 61 64 51 67 63 64 62 45 51 62 50 64 60 68 63 45-68

67 69 70 75 93 70 59 74 71 86 70 67 67 80 78 81 76 91 80 59-93

179 182 167 168 178 190 179 169 171 171 176 170 170 175 172 178 187 178 172 176 167-190

45-64

67-91

168-195

OF PATIENTS

WITH

PERIPHERAL

VASCULAR

Hyperlipoproteinemia phenotype

Disease location

Ankle/arm systolic pressure ratio

N IIA IIB N N IV N N IIA IIB IV IIA N IV N IV IIA IIB IV IIA

A A C F A F F A F F Distal F A F A A F F F C

0.6 0.3 0.5 0.8 0.1 0.4 0.3 0.5 0.6 0.2 0.2 0.5 0.8 0.6 0.6 0.8 0.6 0.8 0.4 0.8 0.1-0.8

Controls (range)

N = normolipidemic;

A = aorto-iliac;

F = femoro-popliteal;

l-l.3 C = combined.

Svedberg flotation rates to the top of the tube: fraction ABC including previously defined [27,28] A particles, S,> 400, B particles, SF 175-400 and C particles, S/ loo-175 all together in the top 1 ml after the first run, and fraction D, Sr 20-100 after the second run. After the second centrifugation the top 1 ml (fraction D) was aspirated. Now the remaining top layer (3 ml) down to a level of 65 mm from the bottom of the tube contained intermediate density lipoproteins (IDL) which were also carefully aspirated. LDL was easily seen as a yellow band about 5-8 mm wide located 45-55 mm from the bottom [27]. This isolated LDL was also recovered by aspiration. Twelve ml of plasma in 3 centrifuge tubes, were required to provide sufficient material for all analyses, and the fractions from the 3 tubes were pooled. LDL subfractionation

Polyallomer Beckman tubes in a 50.3 Ti Beckman rotor were used. The density of plasma was raised to 1.019 g/ml by adding NaBr solution. At the end of the first

127

spin in the ultracentrifuge (24 h at 1.7 X lo8 X g X min at 4°C) tubes were sliced 27 mm from the top and the top fraction, containing VLDL and IDL, was discarded. After raising the density of the bottom fraction to 1.040 g/ml by adding NaBr solution, a second spin in the ultracentrifuge was performed (24 h at 1.7 x 10’ g x min at 4°C). At the end tubes were sliced 17 mm from the top and the top fraction is defined as ‘light’ LDL. The density of the bottom fraction was then raised to 1.070 [29], by adding NaBr solution. After a new ultracentrifugation for 24 h at 1.7 x 10’ x g X min at 4°C tubes were sliced again 17 mm from the top and the recovered fraction is called ‘heavy’ LDL. HDL subfractionation This was achieved by the method described by Kirstein and Carlson [30]. Bell-top Beckman tubes in a 50.3 Ti Beckman rotor were used. In short, serum density was raised to 1.125 g/ml by adding NaBr solution. After ultracentrifugation for 48 h, 3.3 X lo8 X g X min at average radius, tubes were cut 27 mm from the top. The bottom fraction was extracted for lipid analysis [31] and is called HDL,. The HDL, parameters were calculated as the difference between total HDL, obtained at the routine lipoprotein analysis, and HDL,. Analyses Before analysis the lipoprotein fractions were transferred to volumetric flasks. Lipids were extracted with chloroform-methanol [31]. Aliquots of the chloroform phase were used for analysis of triglycerides (in duplicate) [32] and phospholipids (in triplicate) as phosphorus [33] after wet combustion; triolein (Sigma, St. Louis, MO, U.S.A.) and KH,P04 were the respective standards. Free and esterified cholesterol were determined directly on 0.001-0.1 ml of the lipoprotein fractions, in triplicate, with an enzymatic method (Mercotest, E Merck, Darmstadt, F.R.G.) [25]. Dilutions of pooled human serum were used as standards, and standard curves of the second degree were fitted to the standards by the method of least squares. The content of free and esterified cholesterol in the standard was determined by Sperry and Webb [34]. Total protein was estimated in triplicate on aliquots of the lipoprotein fractions [35]. All samples, including the standards, were extracted with chloroform after colour development to remove any turbidity. Soluble proteins were estimated after extraction (and concomitant precipitation of apo B) with isopropanol [36,37] for VLDL and isobutanol for LDL by the same method. The content of apo B was calculated as the difference between total and soluble protein [37]. Bovine serum albumin was used as protein standard. Lipoprotein electrophoresis was performed in agarose gel [38]. Calculations Dimensions of lipoprotein particles such as diameters, molecular weight, S/-value, and particle numbers were calculated as described [27,28]. In essence these calculations were based on the assumption that lipoproteins are spherical particles with triglycerides and cholesteryl esters in the core and a constant thickness of the polar surface shell of 2.15 nm [39]. From calculations of total lipoprotein volume and core

128

volume, using the partial specific densities, the diameter is obtained [27,28,39]. The molecular weight and other characteristics were then calculated as described [27,28]. The molecular mass of the lipoprotein constituents in daltons was calculated as weight fraction times the molecular weight of the lipoprotein particle, and the number of molecules per particle was then obtained by dividing by the molecular weights of the respective constituents. Statistical calculations were as recommended by Snedecor and Cochran [40]. Differences between PVD and control subjects were tested with Student’s t-test. When distributions were not normal [40] the differences were checked with the Mann-Whitney test [40]. All calculations were performed on a computer Nord-100 (Norsk Data). The data were stored on magnetic disks. Programs were written in Fortran 77. Recovery Four runs, where the recovery of VLDL triglycerides (sum of VLDL subfractions) was less than 7055, were not accepted. The mean recovery for triglycerides in VLDL (sum of ABC + D) was 86%. The mean recovery for cholesterol in LDL (sum of ‘light’ and ‘heavy’ subfractions) was 80%. Results Table 1 shows age and clinical characteristics of PVD patients and controls. As seen in Table 2 no significant difference was present between the groups for the

TABLE 2 CHOLESTEROL CHOLESTEROL TROLS

AND TRIGLYCERIDE CONCENTRATION IN SERUM, VLDL, LDL, HDL AND CONCENTRATION IN HDL, AND HDL, IN PVD PATIENTS AND IN CON-

Values are presented as means + SEM.

Cholesterol (mmol/l) serum VLDL LDL HDL HDL, HDL, Triglycerides serum

Range Range Range Range

= = = =

Controls

1.29 f 0.33 a 0.74 f 0.11 5.20 + 0.30 1.3OkO.08 0.57*0.06 0.76 + 0.03

6.83 f 0.69 f 4.63 f 1.49 + 0.68 f 0.76 f

1.74kO.20’ 1.14*0.17 0.42 f 0.02 0.15 + 0.01

1.66 f 0.23 d 1.10+0.18 0.36 + 0.02 0.16 f 0.01

0.31 b 0.10 0.27 0.08 0.08 0.03

(mmoI/l)

VLDL LDL HDL a h ’ d

PVD Patients

4.15-9.81. 4.47-9.72. 0.61-3.80. 0.54-4.47.

129

u-

“a

LDL

8pre-fl-

a-

Fig. 1. Lipoprotein

electrophoretic

of whole serum and of the isolated lipoprotein fractions.

pattern

concentrations of cholesterol and triglycerides in either total serum or in the three major lipoprotein classes, or in the cholesterol concentration of HDL, and HDL,. The ranges of the values for both total serum cholesterol and total serum triglycerides were similar (Table 2). The electrophoretic behaviour of the isolated lipoprotein subfractions is shown in Fig. 1. VLDL-D always showed a slower mobility than VLDL-ABC. IDL had a mobility slightly faster than that of LDL but significantly slower than that of VLDL-D and was always free of visible LDL. All 3 LDL fractions, gradient mean

A = PVD 0::

PATIENTS

CONTROLS

PVD r = 0.96 bs0.31 CONTROLS , =a91 b=0.26 L1-

OJ& 0

14 VLDL

29 CHOLESTEROL

42

56

10

MG/DL

Fig. 2. Correlation between concentrations of apolipoprotein B and of cholesterol in total VLDL of PVD patients and of control subjects. The correlation is highly significant in both groups (P < 0.001) but the slopes of the two regression lines are not significantly different. In this and the following figures cholesterol is given in mg/dl for the comparability with ref. 19. A molecular weight of 650 was used for cholesteryl ester.

130 TABLE

3A

CONCENTRATION OF CONSTITUENTS IN THE TWO VLDL IDL FRACTION IN PATIENTS (P) AND CONTROLS (C) Values are means f SEM. Differences difference.

between

Groups

Constituents

Total protein (mg/l) Soluble protein (mg/l) Apo B (mg/l) Total cholesterol ( mmol/l) Free cholesterol (mmol/l) Esterified cholesterol (mmol/l) Phospholipids (mmol/l) Triglycerides (mmol/l) Ratio cholesterol to triglycerides

groups

SUBFRACTIONS

were tested by Student’s

t-test;

VLDL

P C P C P C P C P C P C P C P C P C

AND

IN THE

P-value

for the

IDL

ABC

D

82.2 f14 81.7 +16 59.0 *10 58.1 f12 23.3 f 4 23.6 f 4 0.19* 0.04 0.20* 0.04 0.09f 0.02 0.10* 0.02 0.10* 0.02 0.10* 0.02 0.18k 0.03 0.18* 0.04 0.65* 0.13 0.65 f 0.06 0.29* 0.01 0.30* 0.02

109 *14.0 109 *12.0 48.9 f 6.44 50.6 f 6.10 60.7 f 8.10 58.8 * 7.0 0.38* 0.06 0.39* 0.06 0.16+ 0.02 0.16+ 0.02 0.21 f 0.03 0.23k 0.04 0.23k 0.03 0.22+ 0.03 0.44* 0.06 0.45 f 0.06 0.81 f 0.03 0.87* 0.05

99.0 *9.80 87.5 f9.70 9.30*0.97 8.20f1.20 89.7 +9.0 79.3 *8.60 0.53 f 0.06 0.48 f 0.06 0.18kO.02 0.15 f 0.02 0.35 f 0.04 0.32kO.04 0.18 f 0.02 0.15 f 0.02 0.12 f 0.01 0.09 f 0.01 4.47 f 0.36 5.30 +0.29

= ‘P < 0.05.

recovered total LDL, light and heavy LDL, had the same apparent mobility. No differences in electrophoretic mobility were observed patients and controls.

A= PVD

electrophoretic between PVD

PATIENTS

0 = CONTROLS $

16 PVD

% m

: 0

r =O.OS (p~O.001)

12

b=0.35* CONTROLS

6

r = 0.97 (p
: 5 >

b=0.2#* 4 *t 12.34;

VLDL-D

CHOLESTEROL

peo.05

MG/DL

Fig. 3. Correlation between the concentrations fraction of PVD patients and of control subjects. is statistically significant (P < 0.05).

of apolipoprptein B and of cholesterol in VLDL-D The difference in slope between the two regression lines

131 TABLE

3B

CONCENTRATION OF CONSTITUENTS IN THE TOTAL LDL FRACTION AND IN THE TWO LDL SUBFRACTIONS (‘LIGHT’ AND ‘HEAVY’) IN PATIENTS (P) AND CONTROLS (C) Constituents

Groups

LDL

‘Light’ LDL

‘Heavy’ LDL

Total protein

P C P C P C P C P C P C P C P C P C

578 *35 498 +28 l3.30* 3.50 5.0 f 0.60 570 *33 493 &28 2.825 0.18 2.44* 0.15 0.81 f 0.06 0.67f 0.04 2.01* 0.12 1.78f 0.12 0.805 0.06 0.67* 0.05 0.14* 0.01 a 0.11* 0.01 20.8 f 1.10 22.8 f 1.70

231 +19 217 +21 55.6 *12 55.2 k17 176 +17 161 +ll 0.97* 0.09 0.95 f 0.07 0.29k 0.03 0.28 + 0.02 0.68+ 0.06 0.67+ 0.05 0.32* 0.04 0.30* 0.04 0.09f 0.01 0.08+ 0.01 10.5 + 0.68 = 13.2 + 1.12

670 *39 605 +34 57.0 + 5.0 54.9 f 6.20 613 +39 550 +33 3.09* 0.21 2.79+ 0.19 0.89k 0.06 0.77+ 0.05 2.19* 0.15 2.03+ 0.14 0.90+ 0.06 0.73* 0.05 0.15+ 0.01 a 0.11 f 0.01 21.3 & 1.80 26.3 * 2.14

(mg/l) Soluble protein (mg/l) Apo B (mg/l) Total cholesterol (mmol/l) Free cholesterol (mmol/l) Cholesteryl esters (mmol/l) Phospholipids (mmol/l) Triglycerides (mmol/l) Ratio cholesterol to triglycerides See legend to Table 3A.

The analysis of constituents in the 2 VLDL subfractions and in the 2 LDL subfractions as well as in IDL and total LDL gave similar results (Tables 3A and B) in patients and in controls. Only triglycerides seem to be significantly higher in LDL and ‘heavy LDL’ of patients although a mere chance may be responsible for this significance at the 5% level due to the great number of comparisons made in these tables.

A= PVD

PATIENTS

O=CONTROLS

r =0.99

(p
CONTROLS r = 0.94 (p
0 0

9 VLDL-D

19 CHOLESTERYL

27

36 ESTER

45

MG/DL

Fig. 4. Correlation between the concentration of apolipoprotein fraction of PVD patients and of control subjects. The difference is statistically significant (P i 0.05).

B and of cholesteryl ester in VLDL-D in slope between the two regression lines

weight ( x 106)

MOLECULAR

See legend to Table 3A.

Number of particles ( X lO’*/mI

Molecular

Parameters

CALCULATED

TABLE 4

of plasma)

P C

P C 19.0 f 3.03 18.3 + 3.86

26.3 f 1.98 31.5 f 3.24

ABC

VLDL

OF LIPOPROTEIN

Groups

PARAMETERS

54.0 50.3

*7.35 k5.67

9.59 + 0.47 10.3 +0.55

D

FRACTIONS

85.3 71.7

kg.59 k7.40

4.50k0.22 4.59 f 0.21

IDL

FOR PATIENTS

487 383

0.20 0.13 f45.1 k24.4

3.83+ 4.01*

LDL

(C)

192 162

0.22 0.18 f 19.6 k13.6

3.62* 3.84+

‘Liaht’ LDL

(P) AND CONTROLS

577 448

0.15 0.19

LDL

f46.8 = *31.0

3.51* 3.95+

‘Heaw’

E

133

TABLE

5

MOLECULAR

MASS OF LIPOPROTEIN

Triglyceride VLDL-ABC VLDL-D IDL LDL ‘Light’ LDL ‘Heavy’ LDL

CONSTITUENTS

(DALTONS

x 106)

PVD patients

Controls

16.6 *1.43 4.30 f 0.25 0.76 + 0.06 0.16 * 0.01 0.28 i 0.03 0.13*0.01

20.3 4.63 0.66 0.16 0.26 0.14

*2.41 kO.34 f 0.04 f 0.01 f 0.03 + 0.01

Esterified cholesterol VLDL-ABC VLDL-D IDL LDL ‘Light’ LDL ‘Heavy’ LDL

1.83&0.17 1.48+0.09 1.57+0.12 1.74 f 0.12 1.49kO.12 1.57*0.09

2.26 + 0.24 1.68 * 0.09 1.73*0.11 1.85+0.08 1.65 fO.10 1.83+0.11

Free cholesterol VLDL-ABC VLDL-D IDL LDL ‘Light’ LDL ‘Heavy’ LDL

1.03*0.10 0.66 f 0.03 0.49 + 0.03 0.41 f 0.03 0.37 f 0.03 0.37 + 0.01

1.20+0.07 0.71 f0.04 0.50 f 0.02 0.41 f 0.02 0.41 f 0.02 0.41 f 0.02

Phospholipids VLDL-ABC VLDL-D IDL LDL ‘Light’ LDL ‘Heavy’ LDL

4.24 1.88 0.96 0.76 0.74 0.71

0.22 0.06 0.02 0.02 0.04 0.02

4.66 1.97 0.99 0.79 0.76 0.73

f 0.34 f 0.07 f 0.03 f 0.02 f 0.03 *0.02

Soluble apoprotein VLDL-ABC VLDL-D IDL LDL ‘Light’ LDL ‘Heavy’ LDL

1.86 f 0.10 0.57 kO.03 0.07 f 0.01 0.01+ 0 0.16 kO.02 0.07 f 0.01

2.22 0.62 0.07 0.01 0.15 0.08

f 0.26 f 0.05 f 0.01 f0 kO.01 f 0.01

Apoprotein B VLDL-ABC VLDL-D IDL LDL ‘Light’ LDL ‘Heavy’ LDL

0.72 zt 0.06 0.70 * 0.03 0.66*0&l 0.76 f 0.05 0.58 f 0.03 0.66 f 0.03 a

0.81 0.72 0.68 0.79 0.62 0.76

f f It f k f

See legend to Table 3A.

k f f f + f

0.05 0.04 0.03 0.03 0.03 0.04

134 TABLE

6

x lo* OF ESTERIFIED CHOLESTEROL AND APO CALCULATED NUMBER OF MOLECULES B PER VLDL-D PARTICLE FROM CONTROLS AND PVD PATIENTS IN THE UPPER RANGE OF CONCENTRATION OF ESTERIFIED CHOLESTEROL IN VLDL-D ( > 0.29 MMOL/L) Values are presented

as means f SEM.

Constituent Esterified ApoBC a b c

PVD patients cholesterol

Number of patients. P < 0.02. Expressed as number

26f2b 68+4

(4) a

Controls

(5) a

33*1 69*3

of amino acids using an average residue weight of 100.

Calculated molecular weight, number of particles and molecular mass were approximately similar in the 2 groups (Tables 4 and 5). ‘Heavy’ LDL of patients were more numerous than in controls. Because of the earlier reported changed relationship between apo B and cholesterol of VLDL in PVD [19] we have studied this relationship. In total VLDL (sum of ABC + D subfractions), the amount of apo B correlated well to that of cholesterol in both groups but no significant difference in the slopes of these two regression lines was present (Fig. 2). Fig. 3 shows the same relationship for the VLDL-D fraction. Now the slope of the PVD line was significantly steeper than that of the controls The esterified moiety of VLDL-D cholesterol was the (b = 0.35 vs 0.29, P -G0.05). main cause (Fig. 4) of this difference in composition of VLDL-D between patients and controls (b = 0.62 vs 0.49, P < 0.05). When apo B was plotted against free cholesterol of VLDL-D no difference in slopes of the regression lines for patients and controls was found. The regression lines in Fig. 4 indicate that the difference between patients and controls is only present at a cholestexyl ester level above 18 mg/dl and that the difference increased with rising VLDL-D cholesteryl ester levels. To investigate the molecular basis for this difference in relationship to apo B and cholesteryl ester, we have calculated the number of cholesteryl ester molecules as well as the number of amino acid residues of apo B in VLDL-D for subjects having the highest cholesteryl ester concentration in VLDL-D, i.e. > 0.29 mmol/l. Table 6 shows that the number of apo B amino acid residues is almost identical in the two groups, while the number of cholesteryl ester molecules per VLDL-particle is significantly lower in patients than in controls. The raised ratio apo B to cholesteql esters in VLDL-D of this subgroup of the PVD patients was thus due to a decreased number of cholesteryl ester molecules per lipoprotein particle. Discussion The purpose of our study was to study possible abnormalities in the distribution and chemical composition of lipoprotein subclasses. Therefore our control material

135

has been matched for serum lipid levels in order to be sure that possible differences should reflect a true difference between PVD and patients and should not merely be due to a significantly different lipid content in total serum. Our results are not consistent with the hypothesis of gross abnormalities either in the distribution or in the composition of lipoprotein subclasses in PVD. Neither for VLDL, nor for LDL or HDL was there any difference in the amount of lipids or protein recovered in the various subfractions between controls and PVD patients. Furthermore there were no differences concerning IDL between the two groups. These are new observations concerning VLDL, IDL and HDL subfractions in a comparison between such individuals selected at similar lipid levels. There was, however, an increased number of heavy LDL particles in PVD which is in contrast to the finding of Rao et al. reporting that in a group of 4 PVD patients there was a shift from light to heavy LDL [20]. The density criteria to subdivide LDL were the same as used here but the patients and the controls had dissimilar total LDL levels in contrast to our material. There was also a decrease of the mass of apo B in the heavy LDL fraction of the PVD patients. The PVD patients thus had more numerous and possibly smaller heavy LDL particles than ‘controls, a finding analogous to that of Teng et al. [21] in hyperapobetalipoproteinemia. Our patient group did not, however, have hyperapobetalipoproteinemia. Franceschini et al. [18] plotted apo B content in VLDL against VLDL cholesterol in PVD patients and in controls and found a steeper slope (b = 0.36) of the regression line of PVD than of controls (b = 0.22). When we made the same plot, the slope for PVD (b = 0.31) was slightly but insignificantly steeper than that of controls (b = 0.26). No obvious explanation for this difference in results is apparent. The materials from the two cities appear to be comparable although the Milan sample of PVD had included patients with more advanced disease. Information on smoking is not available in the Milan study. The Italian and Swedish populations studied had similar lipid and lipoprotein levels although the Milan sample had a few with higher VLDL levels. We could not with statistical significance confirm the Milan finding of an increased slope for the regression line of apo B on cholesterol in total VLDL of PVD patients. However, in our material similar results to those in Milan were obtained in this context in the subfraction D of VLDL, these particles containing most of the apo B and cholesterol of VLDL. A further analysis of the steeper slope for the regression of apo B on cholesterol in VLDL-D from PVD showed that this was present only for esterified and not for free cholesterol suggesting that the problem is related to the core but not to the surface of the VLDL particle. There are two possible changes in the molecular make up that may explain the increased ratio of apo B to cholesteryl esters. Either the VLDL-D particle may be enriched by apo B, as suggested by the Milan group [19], or it may contain less cholesteryl ester. To test this we calculated the number of molecules of these two constituents in the VLDL-D particle. These calculations clearly established that there was no difference in the apo B content of VLDL-D particles between controls and patients. However, VLDL-D from PVD cases had about 20% fewer cholesteryl ester molecules per particle than the controls (Table 6). The difference in the slopes of the two lines for

136

the regression of apo B on cholesteryl ester can thus be explained by a lack of cholesteryl esters in VLDL-D from PVD patients. Since cholesteryl esters are formed in plasma by the action of lecithin : cholesterol acyl transferase (LCAT) on HDL particles and then progressively transferred to VLDL [41], changes in both LCAT activity and in cholesteryl ester transfer protein may be involved in the presently demonstrated abnormality with low number of cholesteryl ester molecules in VLDL-D subfraction of PVD patients. References 1 Kannel, W.B. and Shurtleff, 2 3 4 5 6 7 8 9 10 11 12 13 14

15

16

17

18 19

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