Plasma levels of VLDL- + LDL-cholesterol, HDL-cholesterol, triglycerides and apoproteins B and A-I in a healthy population Influence of Several Risk Factors

559

31 (1980) 559-568 0 Elsevier/North-Holland Scientific Publishers, Ltd.

Atherosclerosis,

PLASMA LEVELS OF VLDL- + LDL-CHOLESTEROL, HDL-CHOLESTEROL, TRIGLYCERIDES AND APOPROTEINS IN A HEALTHY POPULATION Influence

of Several Risk Factors

E. DEDONDER-DECOOPMAN, C. FIEVET-DESREUMAUX P. DEWAILLY, G. SEZILLE and J. JAILLARD Laboratoire Laboratoire

B AND A-I

de Physiopathologie des Lipides, de la Clinique Me’dicale G&%-ale

*, E. CAMPOS, S. MOULIN,

Facultt de Pharmacie, 59045 Lille and Ouest, H6pital Rkgional, 59037 Lille (France)

(Received 21 March, 1980) (Revised, received 9 June, 1980) (Accepted 11 June, 1980)

Summary Plasma apo B and apo A-I were determined in 477 subjects (206 males and 271 females) by laser immuno-nephelometry. Measurements of VLDL- + LDLcholesterol, HDL-cholesterol and triglycerides were done simultaneously. VLDL- + LDL-cholesterol and apo B values were similar in males and females and increased with age. HDL-cholesterol and apo A-I values were higher in females but stable with age. Different regression curves (HDL-cholesterol vs apo A-I) were obtained in males and females and a negative correlation was found between HDL-cholesterol or apo A-I and triglycerides. Increased body weight was associated with higher values of VLDL- + LDL-cholesterol, apo B and triglycerides in both sexes but lower values of HDL-cholesterol and apo A-I essentially in males. Finally, the study provides evidence of a relationship between smoking and alcohol consumption on the one hand, and HDL-cholesterol and apo A-I on the other. Key words:

This

work

* Attach&

Alcohol Triglycerides

was supported de Recherche

Apoproteins - Body weight - VLDL- + LDL-cholesterol

by grant INSERM.

No.

070497

from

the CNRS.

-

HDL-cholesterol

-

Smoking -

Introduction Hypercholesterolaemia and hypertriglyceridaemia are associated with an increased risk of coronary heart disease. Because of their well established role in cholesterol transport and metabolism, the apoprotein B (apo B), major peptide of low density lipoproteins (LDL) and very low density lipoproteins (VLDL), and the apoprotein A-I (apo A-I), major peptide of high density lipoproteins (HDL) were considered to have better predictive value than lipids in atherosclerosis [ 11. In order to study the validity of this concept, we undertook simultaneously in a healthy control population, comparative determinations of apo B, apo A-I, cholesterol of VLDL and LDL (VLDL + LDL-chol.), cholesterol of HDL (HDL-chol.) and triglycerides (TG). At the same time, we examined the influence of increased body weight, alcohol consumption and smoking on the various parameters. Material and Methods Material

Fasting blood samples from 477 subjects (healthy control population of 206 males and 271 females) were drawn into EDTA (1 mg/ml). Plasma samples were stored at +4”C with sodium azide (0.05%) for no longer than 2 weeks. All subjects were submitted to medical examination in order to obtain information on age, height, smoking (number of cigarettes) and alcohol habits (amount of beer and/or wine consumed and calculation of alcohol intake (g) according to known concentrations). An alcohol consumption index (A.C.I.) corresponding to the ratio of alcohol consumption (g/day) over body weight (kg) was determined to allow statistical studies. In women, oral contraceptive users were excluded. Analytical

methods

Cholesterol and triglycerides were analyzed using a Technicon autoanalyzer II [ 21. HDL-cholesterol was evaluated by precipitation using heparin-manganese (Mn2’ = 92 mmol/l) [3]. VLDL- + LDL-chol. was calculated as follows: VLDL- + LDL-chol.

= Total chol. - HDL-chol.

Apo B and apo A-I were measured by laser immuno-nephelometry which is a precise and highly sensitive alternative method [4-6]. For apo B and apo A-I measurements, 200-fold and 250-fold dilutions of plasma were used respectively. Pure LPB (1.040-1.053 g/ml) and HDLJ (1.120-1.210 g/ml) assessed in apo A-I on the basis of moles of histidine [7] were used as standards. Both methods were very well correlated with corresponding electroimmunoassay ]4,51* Statistical

methods

Results were expressed as mean t SD. Statistical evaluation was performed by distribution laws, Student’s t-test, variance analysis and parametric regression analysis.

561

Results VLDL- + LDL-chol. and 2)

and apo B modifications

according

to sex and age (Figs. 1

VLDL- + LDL-chol. distribution appears to be very similar in males and females, mean values being, respectively 1.38 f 0.33 g/l and 1.31 * 0.35 g/l (P < 0.05), but apo B distribution shows higher values in males (1.29 k 0.31 g/l) than in females (1.20 + 0.34 g/l) (P < 0.01). Moreover, VLDL- + LDL-chol. and apo B tend to increase in females with age, reaching male values at menopause. VLDL- + LDL-chol. and apo B are positively correlated in males and females (r = +0.74 and r = +0.84, respectively). Regression studies do not show statistical differences between males and females, giving a common sample regression equation: VLDL- + LDL-chol.

= 0.85 apo B + 0.28.

_____

Females

30.

r-x.__r--1 I I L

20.

I I L I #--

J :I +--

IO-

I k I L-..

J

I c, 1.2

0.8

0.4

1.6

_--L

2.0

VLDL-

+LDL-Chol.

O/f

2.4

Frequ.nry 70

30

I

I r-

20.

,oI----’

PI I

+-

’

I I I I

I I I I

I , I

L_. I___( I I -

0.4

Fig.

0.8

1. Distribution

1.2

of VLDL-

1.6

2.0

+ LDL-chol.

2.4

and

ape

Ape

I

WI B in relatuion

to sex.

562

VL,DL-+LDL-Chol.

(.j

,

0

19

, 29

, 39

. 1_‘_ A9

59

A9

59

Ago

0.5 -

0

19 .

29

p .z 0.001

39

( I number

69

of subjects

Fig. 2. VLDL- + LDL-chol. and ape B variations in relation to sex and age.

HDL-chol.

and apo A-I variations with sex and age (Figs. 3 and 4)

HDL-chol. appears very different in relation to sex, the female population showing higher values: mean concentrations are 0.58 + 0.14 g/l in males and 0.70 + 0.15 g/l in females (P < 0.001). Apo A-I concentration also differs with sex, mean values being 1.32 f 0.23 g/l in males and 1.44 -+ 0.25 g/l in females (P< 0.001). No real variation with age is found for HDL-chol. or apo A-I in either sex. HDL-chol. is positively correlated to apo A-I (r = +0.68) in males as in females, but the regression studies differ significantly with sex, the sample regression equations being: in males, HDL-chol. = 0.43 apo A-I + 0.027 in females, HDL-chol. = 0.40 apo A-I + 0.11 The statistical study exhibits the intercept is significantly zero.

no difference between regression coefficients, but higher in females and statistically different from

563

40.

----

f,maIar

r-1

I

L_

1

1 r’

30-

i-

1

I-C

-Chol

J :I I

IO_

L_, I

I

I I

----I’

J

:I 08

Fig. 3. Distribution

I 1.2

Ape

I.6

of HDL-chol.

2.4

2.0

and ape A-I in relation

VLDL- + LDL-chol./HDL-chol. according to sex and age (Table

and I)

A-I

WI

apo

to sex.

B/ape

A-I

ratios : modifications

Both ratios are lower in females than in males; they do not change significantly with age in males, but increase significantly in females. Finally the VLDL- + LDL-chol./HDL-chol. ratio appears to be more sensitive than the apo B/ape A-I ratio, giving a higher significant difference between males and females (t = 6.52 vs 4.64). TG modifications apo A-I

according

to sex and age. Correlations

with HDL-chol.

and

TG distribution is asymmetrical in both sexes and has to be assimilated to a log normal distribution. Mean TG values do not differ significantly (P < 0.05) with regard to sex (1.1’7 +_0.40 g/l in males, 1.08 +_0.44 g/l in females), but a slight increase is observed with age (Fig. 5). Log TG vs HDL-chol. or apo A-I are

564 HDL-Chol. _

Moles

--

1 -

T

0

19

29

39

Females S

D

A9

59

69

A9

59

69

APO A-I

g/l 2.0

J

0.5-

5

0

19 1, P <

29

39

0.01

Fig. 4. Variations of HDL-chol.

(

1 number

Age

of subjects

and ape A-I in relation to sex and age.

negatively correlated: r = -0.273 (P < 0.001) and -0.139 (P < O.Ol), respectively, the correlation being more significant with HDL-chol:than apo A-I. Increased body weight influence The ideal body weight is calculated according to Lorentz-formula and the body weight index (B.W.I.) corresponds to the ratio, real body weight/ideal body weight. The study was carried out on 453 subjects (199 males and 254 females) divided into 3 groups corresponding to different B.W.I. (Table 2): B.W.I. < 0.99, 1 < B.W.I. < 1.19 and B.W.I. > 1.20. In males, a very significant decrease of HDL-chol. but less significant decrease of apo A-I are observed when B.W.I. is increasing. Simultaneously, a very significant increase of TG, VLDL- + LDL-chol. and apo B is registered. In females, the HDL-chol. decrease is less significant with a higher B.W.I. and apo A-I does not change significantly. Nevertheless, TG, VLDL- + LDL-chol. and apo B increase significantly with B.W.I. Finally, a significant negative correlation between B.W.I. and HDL-chol. is found in both males (r = -0.18; P < 0.01)

565

TABLE VLDL(mean

1 + LDL-CHOLJHDL-CHOL. values

AND

APO

B/APO

A-I

RATIOS

IN

RELATION

TO

SEX

AND

AGE

f SD) Females

Males n

VLDL-

+

LDL-chol.

APO

B

Apo

A-I

n

VLDL-

HDL-chol. <19

+

LDL-chol.

APO

B

APO

A-I

HDL-chol.

13

2.47

k 0.88

0.91

+ 0.29

16

1.64

f 0.42

0.66

F 0.13

20-29

37

2.64

f 0.94

1.03

? 0.29

53

1.95

t 0.85

0.85

+ 0.36

3w-39

67

2.39

+ 0.94

0.98

+ 0.34

64

1.86

t 0.88

0.83

? 0.30

40-49

58

2.52

+ 0.69

1.06

?: 0.25

12

1.97

f 0.72

0.86

+ 0.25

50-59

26

2.57

f 1.00

1.03

5 0.30

57

2.26

f 0.79

1.00

? 0.25

60-69

5

2.54

? 0.78

0.96

+ 0.30

9

2.43

? 0.77

1.04

f 0.20

2.50

+ 0.81

1 .oo

+ 0.30

2.00

-L 0.80

0.88

f 0.40

Total

206

a

a Significantly

different

from

females,

Significantly

different

from

females,

b

b

271

P < 0.001. P < 0.001.

--1 I

20.

I

r--’

-Lb_

I

I I

I

I I

L

L---

I r--J

IO-

L--l

I I

0.2

0.6

1.0

1.4

1.8

2.2

2.6

s/l

TO

TG

w

/As-

0

Fig.

19

29

A P~o.05

I

5. Distribution

39 ) number

and variations

49 of

59

subjects

of triglycerides

69 T 5 (TG)

D

in relation

to sex and

age.

TABLE 2 INFLUENCE B.W.J.

OF BODY WEIGHT INDEX (B.W.I.) IN RELATION n

VLDL- + LDL (g/I)

TO SEX (mean values t SD) HDL (g/l)

Chol

APO B a

TGa

Chol

APO A-I a

46 110 43

1.20 f 0.32 1.38 d * 0.30 1.55 d + 0.32

1.11 f 0.30 1.31 d * 0.28 1.44 d f 0.23

1.00 f 0.31 1.17 c C 0.36 1.34 d i 0.46

0.63 k 0.14 0.56 d f 0.11 0.54 d * 0.09

1.36 * 0.23 1.28 b f 0.21 1.30 * 0.21

81 110 63

1.16 + 0.27 1.34 d f 0.40 1.43 d * 0.32

1.07 ?r 0.24 1.22 d f 0.37 1.36 d f 0.31

0.92 f 0.33 1.08 d f 0.43 1.29 d + 0.55

0.71 i 0.14 0.69 k 0.13 0.66 b + 0.14

1.41 1.44 1.37

Males (199) GO.99 l-1.19 21.20 Females (254) GO.99 l-1.19 al.20 a b c d

Triglycerides Significantly Significantly Significantly

(TG), APO B and APO A-I different from controls, P different from controls, P different from controls, P

f 0.22 + 0.25 + 0.21

measured in total plasma. < 0.05. < 0.01. < 0.001.

and in females (r = -0.15; P< 0.05), whereas B.W.I. and apo A-I are not significantly negatively correlated. Alcohol consumption and smoking Using the previously described alcohol consumption index (A.C.I.) we have studied the effect of alcohol in 197 males and 244 females excluding subjects giving no precise information on alcohol intake. A very significant positive correlation is registered between A.C.I. and HDL-chol. in men (r = +0.316; P< 0.001) as in women (r = +0.215; P < 0.001). The correlation is similar when calculated for apo A-I in males (r = +0.315; P< 0.001) or in females (r = +0.293; P < 0.001). No correlation was found between A.C.I. and the VLDL- + LDL-chol. in either sex. To study the effect of smoking, subjects with A.C.I. > 1 were excluded, and 3 groups including males and females were established as follows (Table 3): non-smokers, moderate smokers (15 cigarettes/day) and heavy smokers (>15 cigarettes/day). A significant decrease of HDL-chol. and apo A-I values is shown in both sexes between heavy smokers and non-smokers. Discussion Our study confirms recent data, obtained in a Health Survey Program, on VLDL- + LDL-chol., HDL-chol., apo B and apo A-I variations with sex and age [8]. In males as in females, VLDL- + LDL-chol. and apo B increase with age, whereas HDL-chol. and apo A-I are fairly constant in both sexes. Such stability of HDL-chol. and apo A-I has already been shown by several studies [9,10]. Moreover, apo A-I values are similar to Alaupovic’s data in normal subjects, being higher in females than in males [ 111. Premenopausal women seem to be better protected against atherosclerosis than men since they have lower levels of atherogenic lipoproteins (VLDL and LDL) but higher levels of anti-atherogenie lipoproteins (HDL). In addition, the regression line of HDL-chol. vs apo A-I varies according to

TABLE

3

INFLUENCE

OF

SMOKING

ON

HDL-CHOL.

AND

APO

A-I

IN

RELATION

TO

SEX

(mean

values

f SD)

Females

Males

n

HDL-chol.

APO

(g/I)

(g/I)

n

A-I

A

69

0.59-f

0.11

1.39

+ 0.26

B

45

0.56

t 0.13

1.30

f 0.24

C

50

0.54

+ 0.12

1.26

+ 0.26

A = nonsmokers,

B = moderate

a smokers

a Significantly

different

from

controls,

b Significantly

different

from

controls,

(<15

226

b

cig/d)

and

HDL-chol.

APO

(g/I)

(g/I)

A-I

0.71

? 0.14

1.46

? 0.24

29

0.66

? 0.18

1.39

-L 0.28

11

0.60

+ 0.16

1.32

? 0.28

C = heavy

smokers

(>15

b cig/d).

P < 0.05. P < 0.02.

sex, the intercept being statistically different from zero in females. Such differences in regression studies according to sex indicate that in females, HDL-chol. is not only related to apo A-I but also includes some cholesterol from non-containing apo A-I lipoproteins. It seems to corroborate the Alaupovic’s hypothesis [ 121 considering HDL as a heterogeneous mixture of lipoproteins. The highly significant HDL-chol. differences between sexes also explain the apparently better predictive value of the VLDL- + LDL-chol./HDL-chol. ratio compared to the apo B/ape A-I ratio [ 11. Our study also demonstrates the influence of B.W.I. on the different parameters measured in relation to sex. In both sexes, we found a parallel increase of B.W.I. and VLDL or LDL concentrations indirectly appreciated by TG or apo B measurements. The increased plasma levels of such lipoproteins have already been related to a VLDL overproduction by the liver [13] and a slower intravascular lipolytic process [ 141. On the contrary, HDL-chol. decreases significantly when B.W.I. increases [ 151, but the relationship is still more significant in males than in females, the lipolytic system remaining more efficient in females. The origin of such differences between sexes is not yet known but may be related to the lipolytic enzyme activity or the lipoprotein composition. Finally, alcohol consumption, as has already been published [16], increases HDL-chol. and apo A-I in a similar manner, probably affecting HDL synthesis by the liver. Smoking, on the other hand, is associated with a slight decrease of both values, the mechanisms still being unclear [ 171. In summary, our study emphasizes the numerous factors affecting apo B and apo A-I plasma values such as sex, age, increased body weight, alcohol consumption or smoking. Acknowledgements We wish to thank Mrs. M.C. Mullet and Miss E. Carpentier technical assistance.

for their excellent

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i (1979)

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15