The cholesterol content of HDL2 and HDL3 subfractions of high density lipoproteins in different normocholesterolemic populations

Clrnrcu

Chimrco A&J,

319

142 (1984) 319-324

Elscvier

(‘(‘A

02967

The cholesterol content of HDL, and HDL, subfractions of high density lipoproteins in different normocholesterolemic populations D. Neel,

P. Beaudry,

I.ohorzrtorr~ de Btochrmre.

D. Erlich,

Il~pitol

Smut

E. Turpin,

Y. Goussault

Louis. .? place L/u Dr Fournrer

-

and C. Dreux

*

7547.5 Ports C&le.r IO (Fruttc~e)

(Received December 23, 19X3: revision June 12th. 1984)

Summary Two normocholesterolemic populations, selected for either high triglyceridemia or low HDL cholesterol content, both known to have increased artery disease risks, were studied

for their cholesterol

subfractions

were isolated

both populations,

content

total HDL

cholesterol

cholesterol

content

were decreased

percentage

of total

HDL

significantly

diminished

in HDL,

by a precipitation

and HDL,

method.

showed

values were similar and HDL,

when compared

cholesterol,

subfractions.

The results

the HDL,

only in female subjects

with a control

cholesterol

These that, in

and HDL,

population.

subfraction

for both populations

In

appeared

studied.

Introduction If the low density lipoprotein of coronary between

heart

diseases,

(LDL)

cholesterol

it has been

shown

these diseases and high density lipoprotein

[l]. HDL

are not homogeneous,

ultracentrifugation.

but comprise

The main ones are HDL,

importance

of the

emphasized

[2-31.

HDL, The

subfraction

existing

is directly related to the incidence that

an inverse

(HDL)

correlation

cholesterol

exists

concentration

several subfractions,

characterized

and HDL,.

evidence

in the protective

ultracentrifugal

methods

Recently role

of HDL

by

for the

has

for the quantitation

been of

HDL, and HDL, are often unsuitable for handling a large number of samples; so some workers have described a simpler method of separating HDL, and HDL, by a precipitation method [4]. * To whom correspondence should be addressed. 0009-8981/84/$03.00

0 1984 Elsevier Science Publishers B.V

Methods

Apoprotein addition

B containing

USP/ml)

and

heparin

100 ~1 of MnC‘t,t

0.046 mmol/ml, min at 4’c‘ and The

lipoprotein5

of 80 ~1 of sodium

precipitated

from

(sodium

salt,

2 ml ~)f herum

Sigma

to a final concentration

from

and

part

the heparin wax taken

manganese for

precipitation

immediate

an

assay

to stand

the

and

for 45

MJ;I\ \epar;ited ol‘

b>

I 5000

grade

of 184 IlSt),,~ml

respecti\,ely. After mixing. the sample wa\ allowed then centrifuged at 1500 X (y for 30 min at 4OC‘.

aupernatant

the precipitate

mot/t

were solution

from

cholesterol

tttll.

(11DLC‘). Scprrratron oj I-lDLa, und HDL; One

hundred

tubes

1600

x g

were

allowed

.subfruc~tion.s(41

of 1.21% dextran

’ supernatant,

heparin/Mn’ The

microlitres

giving to stand

for

for 30 min. The supernalants

The cholesterol (<‘HOD-PAP.

concentrations

sulphate

a final dextran 30 min

at room

added

to

I

temperalure.

ml

ol

the

of 0.1 19,

concentration then

spun

at

were separated.

were determined

Boehringer-Mannheim,

were

sulphate

FRG)

by a cholesterol

in the presence

enzymatic

of disodium

mmol/l. to eliminate positive interference by the precipitating The value for the HDL, cholesterol (HDL,C) was ohtaincd

reagents. by subtracting

method EDTA,

8

HILL 1

from total HDt> cholesterol. Total HDL and H IIL 3 cholesterol for the appropriate dilutions. enzymatic method values were obtained by using a gtycerolipase

cholesterol (HDL,C) values were corrected Triglycerides (Merck,

Darmstadt,

We sampled subjects

with

normal

hypertrigtyceridemic 21

mate

FRG).

42 female

and

HDL subjects

22 male

cholesterol with

normal

normochotesteroter~lic-normotrigtyccridcl~lic as controls: 12 female and

values,

values

normocholesterolemic-normotrigtyceridemic where the content (LHDLC)

of total

cholesterol:

subjects + VLDL)

(LDL cholesterol cholesterol ratio was higher than 4 [6]. All these subjects were between 1X and X0 years old with a mean groups exhibited a similar distribution. The hypertrigtyceridemic chosen from gtyceridemia.

among The

normocholesterolemic one5 whatever the origin normnchotesterolemic-normotrig1yceridemic-I,~iDL~~

30 malt

I9 female

and

with low’ HDL chotesterot/HDL ;II 45. The three SUbJech were of the hypertrisubjects

321

represent a population with increased artery disease risks and already described [6]. The frequent values observed in our clinical laboratory for normal healthy subjects were: triglycerides: 0.45-1.5 mmol/l. cholesterol: 4.1-6.2 mmol/l, HDL cholesterol: 1.1-2.05 mmol/l.

We compared the results using the Kolmogorov-Snlirnov test which is one of the most convenient for such a population in which the Gaussian distributiol~ was not proved. Therefore the means and SD were given only for information. Results

As shown in Table I, the hypertriglyceridemic subjects were chosen to have normal values of total blood serum cholesterol and the subjects with low HDL cholesterol (LHDLC) to have normal triglyceride values and normal total blood serum cholesterol values; the subjects with low or high total blood serum cholesterol were eliminated. So the two populations were identical considering whole cholesterol content. The hypertrigly~er~demic subjects exhibited a decrease of the total HDLC and HLD,C and HDL,C. This decrease was significant in both subpopulations of male and female subjects. The same results were found in the LHDL C subjects (Table II). The comparison of the hypertriglyceridemic population with the LHDL C population showed that these two populations appeared identical for total HDLC’, HDL,C and HDL,C for both male and female subjects.

C‘ontrol

0.76 + 0.28 2.01 + 0.49 ks 57 (S) ,’ -=c0.01

Hypertriglyceridcmic normocholeaterolemic

5.11 * 0.69 5.14iO.64 ks 0.64 (NS)

0.81 + 0.32 2.07 i 0.66 k.\ 53.33 (Sj ,’ < 0.01

5.21 IO.54 5.21 t O.hO k\ 0.72 (NS)

Normcltriglvce~idcmic nortnclcholcsterolemi~ with low HDL cholestcrol content (LHDLC) ks = vai~e obtained

0.X6 _c0.26 k> 1.46 tNS)

4.90 i 0.58 ks 2.83 (f-3

rrom the K.S. test: S = significant;

1.11 to.26 ks 14.02 (S) p < 0.05

NS = n~n-~ig~ifi~~~nt

5.20 + 0.55 k.\O7 INSi

Table 111 shows the variations of HDL?C’ expressed in percentage of total I iDI_, : it appeared significantly decressed in female aubpopulatinns of the two popul:~t~on~ studied.

Control H!pcrtriglycer1dcrnic LHDLC

(low HDL cholesterol)

Total HDL

HDL,

1.52 i 0.23 0 97 i 0.23 * ks42.G (S) I’ -C 0.01 0.95 + 0.15 * kx 52.3X (S) ,’ i 0.01

0 22 i 0. I1 0 1 I I 0.0’) * ks 14.31 (S) I’ C 0.05 0. lo t 0.w * k\ 14.5X (S) ,’ ( 0.05

** ks = 0.77 (NS)

1.53 + 0.30 0.96 r 0.23 * k> 32.4Y (S) ,I < 0.01 0.96 t 0.13

** ks = 2.Yl

* ks 3X.XX (S) I’ < 0.01

(NS)

0.22 i_O.ih 0.0’) -f 0.0x * ka 9.53 (S) ,’ i 0.05 0.10 t 0.08 * ks 6.74 (S) 1’ C 0.05

171 to77 0.X6 / 0 73 * k,\ 2303 (S) ,I . Ol)l 0 X6 + 0 13 * kx7S.71 (S) /> c ti.oi

** ki = 1.37 (NS)

** lx\ 1 45 (NS)

ks = value obtained from the K.S. tot: S = Ggniflwnt: NS = non >lgnificant: * = comparison with normal bubyx’t value; ** = wtnpar~xm of h\ipertrigl~c~ridctnic and LDHC’ zuhyxt.

TABLE

III

HDL, cholesterol subjects

expressed

in percent

of total

HDL

cholesterol;

statistical

comparison

with normal __I_

Females (% HDL,) Control Hypertriglycetidemic

LHDLC

ks = value obtained

14.42 rt 6.79 10.33 + 7.86 ks 6.58 (S) p c. 0.05 10.02 f. 9.48 ks 10.07 (S) p < 0.05 from the KS. test; S = significant;

Males (5%HDL,) 13.X2&9.19 IO.53 * 9.79 ks 5.23 (NS) 10.28 f 8.02 ks 3.50 (NS) NS = non significant.

--

323 TABLE

IV

Statistical

comparisons

Subjects

between

males and females for HDL. Total HDL

HDL,

and HDL,

cholesterol

HDL,

HDL,

Control

ks 2.24

ks 5.41

ks 1.36

Hypertriglyceridemic

(NS) ks 0.86

(N’S) ks 1.16

(NS) ks 0.501

(NS) ks 4.10

WS) ks 0.30

(NS) ks 1.34

(NS)

(NS)

(NS)

Hypocholesterolemic HDL ks = value obtained

values.

from the KS. test; NS = non significant.

In Table IV, the total HDLC and the cholesterol content of HDL, and HDL, subfractions in male and female subjects are compared. In control subjects, the results indicated no difference between male and female subjects; however, some authors have shown that total HDL cholesterol was higher in female subjects, particularly before the menopause [7]. This discrepancy could be explained in our study by the presence of post-menopausal women and by the absence of subjects with low HDL cholesterol often found among male subjects. As regards total HDL C, HDL,C and HDL,C, no difference between male and female subjects was observed either in hypertriglyceridemic population or in LHDLC samples. Discussion

The precipitation procedure used here for HDL,, HDL, fractionation was described by Gidez et al in 1979 [4]. For SR Kahn et al [8], this procedure is valid in that the subclasses of HDL obtained are analogous to those obtained by ultracentrifugation. For Simpson et al [9] there is a higher value of HDL,C as measured by the precipitation procedure than by zonal ultracentrifugation. Nevertheless, this precipitation method is a simpler method for handling a large number of samples despite the inherent variability of precipitation methods and the probably low precision of HDL,C values, which had a low cholesterol content and were determined by difference between total HDLC and HDL,C. The two populations studied are known to have some increased ischaemic artery disease risks, though they are both normocholesterolemic [5,6]. In 1978, Schaefer showed that HDL cholesterol levels are inversely correlated with serum or VLDL triglyceride levels [lo]. In our study, we confirmed, for the hypertriglyceridemic patients, a HDL cholesterol decrease observed for both HDL, and HDL, subfractions, possibly explaining the higher incidence of ischaemic artery diseases in this population. The decrease of HDL,C, in the populations studied appeared more important than that of HDL,C when the results are expressed in percentage of total HDLC, but it was only significant according to the Kolmogorov-Smirnov test, in female subjects (Table III). However the HDL,C was about 50% decreased in male or female subjects, whereas it was only 35% decreased in HDL,C fractions, Alau-

povic

]I I] suggested

that

provided

the biochemical

particles,

and

that.

or in VLDL content both

on

cfficiencv

the

Here

such

subpopulations

where

particles

cholesterol,

the same

should of

the

and

results increase

an

be considered

serum

found.

in VLDL

the triglyceride

could

population.

L>w

f l1)l.C

of

w;15 oh4cr\ ccl in which is hncm 11 c<)n(cnt

the lack of ape

01

C’-containing

non r-call\ he \uggcsted.

like. for instance.

in this

of tripl\c~cride

decrease

;I decrease subfraction

As regards

were

t 1111

in t 11)1 ) )

a decrease the

to

in the \\hc)le

decrca\c

of

tIDl.:

the apo A, or A ,, were probablv also implicated. It would hc intrrcstinp now the apolipoprotein content of HDL, and HDL, subfractions in thcae

to study

two normocholesterolemic valuea.

However.

the

and

(mainI\

mechanism\

explain

p,~rt~c~lc~

Vl.DL

E in HDL

removal

could

subjects.

between

and not only in the HDL,

in HDL

mechanisms C content

of the

part of apo C lipoproteins.

C samples.

lipoprotein Other

of HDL

the main

relationship

C and ape

;I hypothesis

~~I‘ Ili)l.

lipoproteina

E contatning

of ape

in the hyprrtriglyceridemic

LHDL

apo

apt)

for the inverse

the distribution

[12].

contain

to

C‘ and

basis

depends

metabolism

apcj

populations,

but are phvsiologicallv

which

different

had the same

with

respect

1cholealcrol

HDL,-HDI.

to their

lipid

mctahnlism.

References

3 Miller

NE.

Hammett

F. Saltlsst.

Ilpoprotelns

to plasma

4 Ci~dez LI. Miller

GJ.

Burst&n

procedure-correlation HDL

with

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5 Carlsaon

MA.

Bottiger

J. Chanu

chez da 7 Kraus

5uJets KM,

on high X Kahn

Undgren

SR,

Actn

Ahfrlat Stand

HS. 1982;

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Risks

factors

206:

351~360.

Med

J. Bradley

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14:

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h\

suhclasw\

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Y, Dreux

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ct LDL

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ronal

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high

Alaupvvic

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by differential

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Invat

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by a precipitation

ah measured

C‘hem

1

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Anelys~s

32X, no. 7’). Usdew:

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t1A.

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wbclasse~

M. bder

Med

B. Nerl

of :cnglographicall\

and apollpopr~,teins.

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suhfractwns

EA.

between Metabolic

197X: 8: 111-113.

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drnslty VLDL regulation

Jr

HB.

lipoproteins memoral and

HDL.

Lrvj

RI.

Danner

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The

Can

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of plasma

high

hiochernud

density

Blackweldcr

KN.

Lancet

197X: 2: 3Yl and

clInical

W.C‘.

Met;itx,lic

7Y2. ugniflcance

of the

19X1: 59: 565.-579. lipoproteins

conccntr;~tIon\.

Eur

.I (‘lln