Selective determination of cholesterol in high density lipoprotein subfractions (HDL2 and HDL3) in patients with cerebral and peripheral arteriosclerosis

233

Clinica Chimica Acta, 147 (1985) 233-240 Elsevier

CCA 03147

Selective determination of cholesterol in high density lipoprotein subfractions (HDL, and HDL,) in patients with cerebral and peripheral arteriosclerosis R. Fellin a, L. Baroni a, M.R. Baiocchi a, G. Baldo Enzi a, F. Grego b and G. Valerio a ’ Department ofInternal Medicine (Received Key words: HDL,

and HDL,

and ’ Department of Vascular Surgery, University of Padua, Polictinico, Via Gwtiniani 2, 35100 Padova (Italy) August

20th. 1984; revision January

3rd. 1985)

cholesterol; Polyanion precipitation; Arteriosclerosis; Cerebral; Peripheral

Summary

Cholesterol levels in high density lipoprotein subfractions (HDL, and HDL,) were evaluated in 69 patients (55 males, average age k SD 58.3 k 8.8, and 14 females, average age k SD 63.1 k 10.3) with extra-coronary arteriosclerosis (lower limbs, supraaortic trunks and both sites), and in 79 healthy age-matched control subjects. HDL cholesterol was significantly reduced in male and female patients. The HDL cholesterol decrease was due to a fall in both HDL, and HDL, cholesterols; nonetheless, an analysis of the HDL,-cholesterol/HDL,-cholesterol ratio disclosed that HDL, cholesterol was the most reduced. Slightly higher plasma cholesterol and triglyceride levels were found in the patients as well as a higher plasma cholesterol/ HDL-cholesterol ratio. On the contrary, the HDL,-cholesterol/HDL,-cholesterol ratio was significantly reduced in the patients. These preliminary findings suggest that, as in ischemic heart disease, the HDL cholesterol reduction in cerebral and peripheral arteriosclerosis is also mainly due to a reduction in the HDL, subfraction. These results also lend further support to the proposal that determination of the HDL subfractions is useful for a better assessment of the risk profile for arteriosclerosis.

Introduction

Low plasma high density lipoprotein cholesterol (HDL-C) levels are one of the main risk factors for coronary [1,2], cerebral [3,4] and peripheral [5-71 arteriosclerosis. 0009-8981/85/$03.30

0 1985 Elsevier Science Publishers

B.V. (Biomedical

Division)

234

HDL, (d= 1.063-1.125 g/ml) and HDL, (d= 1.125 - 1.21 g/ml) are the two most important subfractions constituting the high density lipoproteins (HDL), and seem to have different predictive values for ischemic heart disease (IHD). Following the first report by Gofman et al [8], the major role of the HDL, subfraction in the evolution of IHD was confirmed by other workers [9-111. Unlike HDL,, HDL, plasma levels cover a wide range, mainly contributing to influence the concentrations of total HDL [lo]. Low HDL, levels are associated with several conditions, such as a sedentary life style [12], obesity [13], hypertriglyceridemia [14], carbohydrate-rich diet [15] and the male sex [ll]. High levels, instead, are found in females [ll] and physically active individuals [12]. The traditional methods of separating the HDL subclasses by means of analytic and preparative ultracentrifugation, and the high costs and long operating times involved have constantly hindered a wide-scale practical application of these assays. However, Gidez et al [lo] recently proposed a simple and accurate method for the separation of the HDL, and HDL, subfractions using a double precipitation with heparin-MnCl, and dextran sulphate. The results of this technique correlate well with those obtained by analytic and preparative ultracentrifugation. The present study was designed to determine which HDL subfraction is primarily responsible for variation in the plasma levels of HDL cholesterol in patients with cerebral and peripheral arteriosclerosis. Materials and methods Patients Sixty-nine patients (55 males, average age + SD = 58.3 t_ 8.8; 14 females, average age _+ SD = 63.1 _C 10.3) with clinical evidence of cerebral and peripheral arteriosclerosis form the basis of this study. Diagnosis was based on clinical history and had been confirmed by angiography. Arteriosclerotic lesions were localized in the lower limbs in 39 patients (31 males and 8 females), in the supraaortic trunks in 17 patients (11 males and 6 females) and in both areas in the remaining 13 (males) (Table I). Information about non-lipid related risk factors, such as smoking, hypertension and diabetes was also collected. Seventy-nine healthy age-matched, randomly selected subjects from various social groups (47 males, average age + SD = 54.7 _t 13; 32 females, average age + SD = 62.9 * 10.2) were taken as controls. No control subject was receiving drug therapy; grounds for exclusion were diabetes or other endocrine disorders, a history or electrocardiographic evidence of IHD, absence of peripheral pulses or symptoms of peripheral arterial disease (PAD), recent weight change and gross obesity. Methods Venous blood samples (10 ml) were drawn in the morning and placed in graduated test tubes containing 3.8 mmol/l separated by centrifugation at 1,500 X g for 10 min and determinations were carried out on the same day.

following a 12-h fast, EDTA. Plasma was than stored at 4°C;

235 TABLE I Mean age (yr) (R f SD) and number of patients (subdivided according to lesion distribution), males (M), females (F) and controls

M F M+F

M F

Patients

Controls

No. Age No. Age No. Age

55 58.3& 8.8 14 63.1 f 10.3 69 59.3* 9.3

41 54.7 f 13 32 62.9 + 10.2 79 58.0 f 12.5

No. Age No. Age

Lower limbs 31 58+9 8 64k9.5

Supraaortic trunks 11 58 +lO 6 61.8 * 12.1

Both sites 13 59.1 f 8

Patients and control subjects were allowed to follow their usual diet before this study, but all drugs interfering with lipid metabolism had been suspended in the patients for at least 2 wk before. HDL, as well as HDL, and HDL, subfractions, were isolated by the precipitation procedure of Gidez et al [lo], which is accomplished in two steps: (a) to precipitate Apo B containing lipoproteins (VLDL, LDL), 0.3 ml of a 1.06 mol/l MnCl, solution and Na-heparin (40 U:l) (Lipo-Hepin, Riker Pharma, Borken, Westfalen, FRG) were added to 3 ml plasma. This mixture was held at 4°C for 20 min, and then centrifuged at 1,300 X g for 1 h at 4°C. The supernatant containing total HDL was drawn off; (b) 0.2 ml of a 14.3 g/l Dextran sulphate (15000 M,; Sochibo, Boulogne, France) solution in 0.15 mol/l NaCl were added to 2 ml of the above supematant. After 20 min at room temperature, the mixture was centrifuged at 1,300 x g for 30 min, thus obtaining HDL, in the supematant. Total cholesterol (TC), HDL-C and HDL, cholesterol (HDL,-C) were determined by Abel1 and Kendall’s extraction method [16]. HDL, cholesterol was calculated from the difference, HDL-C minus HDL,-C. Plasma triglycerides (TG) were assayed by Wahlefeld’s enzymatic method [17]. Statistical analysis Student’s double-tailed t test was employed to compare the means, and correlations between the various parameters were evaluated by simple linear regression analysis. Results Cholesterol and triglyceride mean values in patients and controls are reported in Table II. TC levels were significantly higher (p < 0.05) in patients (both males and females); TG levels were also higher in patients, but the difference was significant

236 TABLE

Ii

Mean values (mmol/l,

M rt SD) of TC, TG, HDL-C,

HDL,-C

and HDL,-C

in patients

and controls

Sex

No.

TC

TG

HDL-C

HDL,-C

HDL,-C

Patients

M

55

Controls

M

47

Patients

F

14

Controls

F

32

5.77 a il.22 5.31 kO.91 6.17 a i 1.37 5.49 i 0.81

1.41 +0.61 1.32 _c0.49 1.47 h i 0.63 0.98 + 0.36

0.93 c f 0.25 1.19 ~0.27 1.12h + 0.27 1.41 f 0.34

0.27 ’ +0.14 0.40 &O.lR 0.33 il *0.1.5 0.50 +0.23

0.66 ’ t 0.16 0.79 iO.16 0.78 ,’ kO.17 0.92 10.17

a p < 0.05. b p -=0.01. c p
only in females (p < 0.01). HDL-C levels instead were significantly lower in patients, ( p -c 0.001 in males and p < 0.01 in females). This decrease was due to a reduction in both HDL,-C and HDL,-C. HDL-C levels were significantly higher in females in both patient and control groups (p < 0.05 and p -=z0.01, respectively).

TABLE

III

Mean values (mmol/l, R *SD) according to lesion distribution

of TC, TG,

HDL-C,

HDL,-C

and

HDL,-C

in patients

subdivided

Site

Sex

No.

TC

TG

HDL-C

HDL,-C

HDLI-C

Lower limbs only

M

31

F

8

Supraaortic trunks only

M

11

F

6

Both sites

M

13

5.82 = f 1.23 6.96 b f 1.24 5.83 *1.20 5.13 * 0.64 5.59 i 1.29

1.41 F 0.55 1.77 b * 0.59 1.66 f 0.61 1.08 rfr0.48 1.47 f 0.77

0.97 b kO.23 1.20 +0.25 0.80 b f 0.27 1.02a rt 0.27 0.95 d 50.27

0.28 c ,0.14 0.38 rtO.17 0.21 c f0.15 0.27 = +0.11 0.29 * rto.12

0.68 d 50.15 0.85 *0.12 0.58 b kO.16 0.70 a +0.20 0.66 d io.19

Controls

M

47

F

32

5.31 kO.91 5.49 + 0.81

1.32 f 0.49 0.98 f 0.36

1.19 * 0.27 1.41 + 0.34

0.40 kO.18 0.50 i 0.23

0.79 i0.16 0.92 +0.17

a p -z 0.05. b p < 0.001. c pi0.005.

d p < 0.01.

237

TABLE IV Mean values (I%%f

SD)

of

TC/HDL-C and HDL,-C/HDL,-C

Sex

ratios in patients

and controls

No.

TC/HDL-C

HDL,-C/HDL,-C 0.43 f 0.23 ’ 0.53 + 0.24

Patients Controls

M

55 47

6.54+ 1.96 a 4.68 f 1.37

Patients Controls

F

14 32

5.76+1.82= 4.06 + 1.03

0.44 f 0.20 0.55 k 0.23

Patients Controls

M+F

69 79

6.38 k 1.94 a 4.43 + 1.27

0.43 + 0.23 ’ 0.54 + 0.23

a p < 0.001. h p < 0.05. c p < 0.01.

TABLE

V

Correlations controls

(r)

between

the concentration

of TG and

HDL-C,

HDL,-C,

HDL,-C

TG/HDL-C

TG/HDL,-C

TG/HDL,-C

Patients

-0.21

- 0.24

- 0.08

(No. = 69) Controls

-0.38

- 0.43 a

-0.18

a

in patients

and

(No. = 79)

The lowest values of HDL-C, HDL,-C and HDL,-C were observed in patients with arteriosclerosis in the supraaortic trunks (Table III). Mean values of the TC/HDL-C and HDL,-C/HDL,-C ratios are reported in Table IV. The TC/HDL-C ratio was significantly higher ( p -=z 0.001)in both male and female patients; the HDL2-C/HDL3-C ratio was significantly lower in male patients (p -e0.05)and in patients as a whole ( p < 0.01). The same tendency was also observed in the female patients, but was not significant. The correlation coefficients between TG and HDL-C, HDL,-C and HDL,-C are reported in Table V. A statistically significant, negative correlation between TG and HDL-C and between TG and HDL,-C was present only in controls. No significant correlation between TG and HDL,-C was observed. Discussion In agreement with other workers [l&19], we found a significant reduction in HDL cholesterol associated with a slight increase in plasma cholesterol and triglyceride levels, and a significant increase in the TC/HDL-C ratio in patients with cerebral

23R

and peripheral arteriosclerosis. The HDL reduction was due to a fall in both HDL, and HDL, cholesterols; nonetheless, an analysis of the HDL,-C/HDL,-C ratio disclosed that HDL, cholesterol was the most reduced. To our knowledge, this is the first description of HDL, and HDL, cholesterol behaviour in patients with extra-coronary arteriosclerosis. Similar changes in these two HDL subfractions had been observed by Gofman et al [S] in IHD patients (HDL,-C = - 32%, HDL,-C = - 8.4%) and according to Anderson et al [20], the HDL, cholesterol level defines the ‘coronary risk profile’ with 50% more accuracy than the HDL cholesterol level. Referring specifically to extra-coronary arteriosclerosis, Bradby et al [19] reported a significant reduction in Apo A-I (main HDL, apoprotein) in individuals with peripheral arteriosclerosis. Taken together, these studies tend to suggest that HDL, may be the major antiatherogenic component of the two HDL subfractions. This view is supported by the observation that premenopausal women (who are generally resistant to arteriosclerosis) have higher HDL, levels than age-matched males, while HDL, levels in both sexes do not differ significantly [21-231. However, case-specific longitudinal data on HDL, and HDL, cholesterol concentrations in patients with arteriosclerosis and controls are lacking. In our study HDL-C, HDL,-C and HDL,-C levels were significantly higher in female than in male controls; in patients, HDL-C and HDL,-C levels were also significantly higher in females, while no significant difference in HDL,-C levels was present. Studies on the formation of HDL, and HDL, suggest that their plasma concentrations are reciprocally regulated by the activity of lipoprotein lipase. The data are compatible with a concept proposing conversion of HDL, to HDL, through assimilation of cholesterol, phospholipids and apoproteins from triglyceride-rich lipoproteins during their degradation by lipoprotein lipase [24,25]. In particular, the concentration of HDL, seems closely related to the rate of intravascular lipolysis [14,26]. The statistically significant negative correlation between triglycerides and HDL-C and HDL,-C, but not between triglycerides and HDL,-C, demonstrated in controls indirectly fits in with this hypothesis. The absence of significant correlation between these parameters in patients suggests a possible alteration of this metabolic process. In conclusion, these preliminary results in patients with cerebral and peripheral arteriosclerosis show that the reduction in HDL cholesterol is due to a reduction in both HDL, and HDL, cholesterols but the decrease is greater in the former. Since HDL cholesterol is a parameter in the definition of the arteriosclerosis-risk profile, the determination of cholesterol in the HDL subfractions should be carried out, in our opinion. References 1 Miller GL, Miller NE. Plasma high density lipoprotein concentration and development of ischaemic heart disease. Lancet 1975; 1: 16-19. 2 Castelli WP, Doyle JT, Gordon T, Hames CG, Hjortland MC, Hulley SB, Kagan A, Zukel WJ. HDL

239

cholesterol Circulation

and other lipids in coronary 1977; 55: 767-112.

heart disease. The cooperative

lipoprotein

phenotyping

study.

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