CUN. CHEM.40/8, 1554-1558(1994)
Lipids and Lipoproteins
#{149}
Structural and Functional Assessment of High-Density Lipoprotein Heterogeneity
Milada
DobiIovI’
and Jiri .1. Frohlich2
We studied the heterogeneity of high-density lipoproteins
(HDL) in plasma of 110 subjects, using three different
methods: (a) gradient gel electrophoresis (GGE); (b) electroimmunoassay, to measure the concentration of lipoprotein particles containing apoprotein (apo) Al but no apoAll (LP Al); and (c cholesterol esterification rate (FERHDJ
in very-low- and low-density lipoprotein-depleted plasma.
There were two study groups: patients with hypertension,
whose plasma lipid profile was similar to their respective
controls, and patients with hypoalphalipoproteinemia (hy-
poalpha), whose family members served as controls. Values for FERHOL were significantly higher in both risk
groups than in their respective controls. LP Al was significantly decreased only in the hypoalpha subjects. Generally, LP Al and FERHDL were inversely related. LP Al
correlated strongly with plasma HDL-cholesterol, apo Al,
and LP Al/All; FERHDL correlated with those values inversely. LP Al, but not FERHDL,correlated with HDL free
cholesterol. On the other hand, FERHDL correlated
strongly with plasma concentrations of triglycerides and
with the plasma ratio of totat/HDL-cholesterol while LP Al
did not. GGE determination of the composition of HDL
subspecies showed that both FERHDL and LP Al were
significantly related to the content of HDL2b particles:
FERHDI_inversely, LP Al directly; the relative amount of
HDLC particles correlated only with FERHDL.We conclude that GGE and FERHDLcan be used to quantify both
the apparently protective (HDL) and risk-associated
(HDL.C) particles, whereas the concentration of LP Al in
plasma mainly reflects the concentration of the HDL2
subpopulation.
Indexing
Terms: apvtens/cho1stero1-este’ficaflon
rate/eiectrophoresis. gradient gei/iecithin:cholesteroi
acyltransferase/radioaay/eecoimmunoay/hyptensn/hypoaiphaJipopmteinemia
Over the last decade increasing attention has been paid
to the heteroneity
of high-density
lipoprotem
(HDL)
particles (1 )#{149}3
The antiatherogenic
potential of HDL has
long been known; analysis of the Framinghaxn data mdi1 Institute
of Physiology, Academy of Sciences of the Czech Republic, VldeflskI 1083, 14220 Praha 4 -Kr#{235},
Czech Republic. Fax
hit + 422-471-3220; E-mail <[email protected]>
(author for correspondence).
2University
Hospital Lipoprotein Research Group, Department
of Pathology, University of British Columbia, Vancouver, BC,
Canada.
3Nonstandard
abbreviations:
and very-low-density
lipoprotein,
HDL, LDL, VLDL, high-, low-,
respectively; CAD, coronary artery disease; GGE, gradient gel electrophoresis; apo, apoproteun;
LP A!, lipoproteun containing ape A! but not ape All; LP Al/All,
lipoprotein containing both ape Al and All; LCAT; lecithin:cholesterol acyltransferase;
FEE, fractional esterification rate (of cholesterol); and hypoalpha, hypoalphalipoproteinemia.
Received November 17, 1993; accepted April 12, 1994.
1554
CUNICAL CHEMISTRY, Vol. 40, No. 8, 1994
cated that the predictive value of serum concentrations
of
HDL-cholesterol
was about twice that of low-density 11poprotein (LDL) cholesterol
(2). However, members of the
HDL family of particles differ in size, structure, and function. The relative proportions
of various HDL particles
might affect the direction of lipid transport between extraand intravascular
pools. Subfractionation
of HDL by ultracentrifugation
indicated that the antiatherogemc
properties were mostly associated with the HDL2 subclass (1).
The predictive
power of HDL2 for risk of coronary artery
disease (CAD) was recently confirmed by Drexel et al. (3),
who measured HDL2 cholesterol after precipitation
with
dextran sulfate.
Gradient gel electrophoresis
(GGE) resolves the HI)L
population
into five distinct
subspecies
of particles
of
different sizes: HDLb
and
HDL2b, like total
H1)L2, as determined
by either ultracentrifugation
or
precipitation,
is “antiatherogenic.”
HDL3, however, includes the smallest HDL particles (HDLsb,), which have
been associated
with increased
CAD risk in several
studies (4-7).
Immunoaflinity
chromatography
(8-12) and immunoelectrophoresis
techniques
(13-15) offer an alternative
approach
to the resolution
of lipoprotein
subpopulations. Two major types of particles can be separated:
those containing
apoprotein
(apo) Al but no ape Al! (LP
Al) and particles containing
both ape Al and ape Al!
(LP Al/All). All these particles
occur within the whole
range of HDL subpopulations,
whether characterized
by
ultracentrifugation
(8) or GGE (9, 11). LP Al particles
have apparently the greatest
antiatherogenic
potential
(10, 13), and 30-50% of these particles
(depending
on
sex) are located in the HDL2b population;
most of the LP
Al/Al! particles
are found in HDL3 (12, 16). All of the
above-mentioned
techniques assess the heterogeneity
of
HDL by measuring
the concentration
of particles according to differences in charge, size, and (or) apoprotein
composition.
We have developed
a functional
assessment
of HDL
heterogeneity
(4, 5, 17) based on esterification
of cholesterol by lecithin:cholesterol
acyltransferase
(LCAT)
within HDL particles (18). In this reaction, neither substrate (free cholesterol and lecithin) nor the amount of
enzyme (LCAT) is rate limiting.
The most important
factor in this reaction apparently
is the nature of the
HDL particle substrate,
i.e., the physicochemical
characteristics
of the particle surface where the interaction
of LCAT with substrate takes place (19). Small HDL
particles
provide
the most active surface
for LCAT
reaction
(20, 21), whereas
large HDL particles
may
inhibit the reaction rate (22, 23). Removal of very-lowdensity lipoprotein (VLDL) and LDL from an individual’s plasma
allows measurement
of the (fractional)
esterification
rate (FER) as determined
by interactions
among HDL subspecies only (FERunL). Plasma depleted
of LDL and VLDL contains all the components
needed
for esterification
to proceed without interference
from
large masses of cholesterol
in less suitable
substrates.
FERunL
is strongly
and positively
correlated
with
plasma concentrations
of HDLSb,C particles and strongly
but inversely
correlated
with the concentration
of
HDL2b.
This functional
assessment
seems to be a good indicator of CAD risk (4,5). Subjects with other risk factors
such as hypertension
or those with angiographically
proven CAD have significantly
higher FERunL values
than controls do. In subjects with hypertension,
changes
in HDL subpopulations
appear to be their most distinct
lipoprotein abnormality.
As shown (4, 5), these changes
determine
FERL.
We compared the concentration
of LP Al (as assayed
by immunoelectrophoresis)
with the measurement
of
cholesterol esterification
rate (FER)
to assess their
ability to reflect the subfraction
composition
of plasma
HDL as determined
by GGE.
Materials and Methods
Subjects
Two case-control
groups were studied. In Prague, 22
were recruited
from apparently healthy laboratory workers and 27 hypertensive
patients were recruited from among the outpatients
of the Nephrolor
Clinic of Institute for Clinical
and Experimental
Medicine. The age of the subjects ranged from 27 to 70 years.
In Vancouver, we examined
28 patients with primary
hypoalpha.
The 31 controls used in this study were relatives of these patients.
All these subjects had HDL
cholesterol
values
>0.9 mmol/L and a wide range of
other lipoprotein values, as described earlier (24).
volunteers
Procedures
Lipid analysis.
Blood was collected into EDTA-contaming tubes after a 12-h overnight
fast, placed on ice,
and centrifuged
within 2 h at 1750g for 10 mm to separate plasma
Plasma was analyzed within 48 h if kept
on ice, or within 3 months if stored at -20#{176}C,
or within
2 years at -70#{176}C.
We have verified that those storage
conditions
do not affect the analysis
of FERunL described below. Concentrations
of total and free cholesterol and triglycerides
were estimated
enzymatically
(Boehringer
Mannheim,
Mannheim,
Germany).
VLDII
LDL-depleted
plasma was prepared by precipitating
ape
B-containing
lipoproteins
with
phosphotungstateMgCl2 (25).
Determination
of esterification
rates and LPAI. Determination
of FERL
is based on a method described in
detail elsewhere
(5, 17). The essential
step of the
method consists in transferring
a trace amount of [!lJcholesterol
(spec. acty. 5 kCi/mol; Amersham,
Bucks,
UK) from a paper disc to HDL in VLDL/LDL-depleted
plasma. Labeled samples are placed in a shaking water
bath and incubated
there for 30 min at 37#{176}C.
The lipid
content is extracted
with ethanol (980 mL/L) and the
extract
components
are separated
by thin-layer chromaGermany).
The spots corresponding
to free cholesterol and cholesteryl
esters are
then cut out and their radioactivity
determined
in a
liquid scintillation
counter. FERunL is calculated as the
difference between the percentage
of labeled esterifled
cholesterol before and after incubation.
We estimated
the concentration
of LP Al in samples of
plasma as described by Parra et al. (13), using a Hydragel LP Al Particles
Kit (Sebia, Issy-les-Moulineaux,
France) for electroimmunoassay.
The concentration
of
particles
containing
both apoproteins,
LP Al/Al!, was
calculated
as a difference between the total concentration of apo A! and the concentration
of LP Al. Apo Al
and ape B were estimated by using Beckman (Fullerton,
CA) Apo A and Ape B kits.
Gradient gel electrophoresis
of HDL. Plasma lipeproteins were removed by ultracentrifugation
and electrophoresed in a 4-30% polyacrylamide
gradient gel (Midget electrophoresis;
LKB, Bromma, Sweden) as described
(26). Three of the HDL subclasses
were distinctly
resolved in the gel: HDL2b (particle
size 9.5-12.9
tim),
HDL
(possibly with HDL,
8.2-9.5 nm), and HDL3b
(with HDL,
7.0-8.2 tim). These particle
sizes were
similar to those previously
reported
(27). The relative
content of the HDL subpopulations
was estimated
with
a laser densitometer
(LKB Ultroscan
XL).
Statistical analysis. Student’s t-test was used to establish significant
differences between the mean values of
each group. The relations between FERunL, LP Al, and
other variables
measured
during this study were determined by multiple linear regression analysis.
tography (Merck, Darmstadt,
Results
FERHDL, LP Al, Plasma Lipids, and HDL Subclasses
The data obtained from hypertensive
and normal subjects of comparable age and body mass index are shown
in Table 1. The most significant
differences
between
these groups were in FERL,
plasma total cholesterol,
and triglycerides.
The concentration
of LP Al was not
significantly
different between
controls and hypertensive patients; however, the control women had significantly higher content of LP Al than did the control men
(P <0.01). Values for FERL
were also significantly
different
between
the control men and the women (P
<0.01). Hypertensive
subjects had a tendency toward
higher values of HDL3bC and reduced HDL2b, particularly if the ratio of HDLSb,C to HDL2b was used. A similar tendency can be seen in the differences
between
men and women, namely,
lower HDL2b and higher
HDL3bC in control men than in control women.
Table 2 shows significant
differences between
patients
with hypoalpha
and their unaffected
family members
for several analytes.
The most statistically
significant
difference was an increase in FERImL and a decrease in
HDL-cholesterol
and total apo Al in the hypoalpha
subjects. The concentration
of LP Al/Al! particles was significantly
reduced
and the ratio of total cholesterol
to
HDL-cholesterol
increased.
As expected, LP Al was significantly lower in hypoalpha
subjects than in their conCUNICAL CHEMISTRY, Vol. 40, No. 8, 1994
1555
Table 1. Mean (SD) data for subjects
with hypertension.
Men
Controls (n = 9)
329
(75)
19 Al, mg/L
FERHOL,
%/h
TC, mmol/L
Women
(4.74)
17.66
24.90
6.55
5.06(0.62)
1.2 (0.5)
1.06(0.30)
217
(48)
3.53 (0.59)
TG, mmol/L
HDL-TC, mmol/L
HDL-FC, moVL
LDL-TC, mmoi/L
HDL,, %
(O.83)t)
5.27 (1.15)
3.4 (2.8)a
249
3.2 (1.7)
4.65 (1)
TC, total cholesterol; FC,free cholesterol; TG, triglycerides.
(110)
4.53 (0.6)
9.4 (5.90)
32.1 (13.7)
5.3 (3.7)
7.08 (2.67)
HDL.,JHDL,
TC/HDL-TC
Hypertensives (n =
382
(70)
22.13 (5.73)C
6.21 (1.40)
2.0 (0.6)C
0.96 (0.25)
228
(80)
4.44 (0.91)
13.5 (8.20)
24.7 (13.1)
3
(2.5)
6.54 (5.62)
0.9 (0.4)
1.16 (0.28)
275
(78)
3.73 (0.71)
19.7 (9.4)
16.2 (9.0)
1.2 (1)
0.93 (0.31)
18.8 (6.10)
23.9 (13.1)
HDLJ,C, %
(6.91)
Controls (n = 10)
461
(115)
12.55 (4.67)
Hypsrtsnslv.. (n = 13)
409
(138)
4.54 (4.1)
SignifIcance of differences from controls:
a
P <0.05,
b
p <0.01,
C
14)
P <0.001.
trols, but the differences were not as significant
as those
for FERImL.
The two groups described in Tables 1 and 2 differ in
their respective
controls. In hypertension,
the patients
and their controls had comparable plasma lipid concentrations. However, the hypoalpha
subjects, selected because of a serum HDL <0.9 mmolIL, often had other
family members
(controls) with some abnormal
values
for serum lipids. Nevertheless,
regardless
of the type of
cardiovascular
risk factor-hypertension
or hypoalPha-FERL
values were markedly
higher in patients
than in controls. In contrast, the concentration
of LP Al,
as expected, was significantly
lower only in hypoalpha
patients.
nificant relation between U’ Al and hltsb,c,
FERL
showed a strong positive correlation
with the concentration of this subclass. The plasma concentration
of LP Al is
apparently
independent of serum triglycerides
and has no
correlation with the ratio of total cholesterol to HDL cholesterol; FERunL,
however,
correlates
positively
with
these factors. Interestingly,
the free cholesterol content in
HDL has a strong positive correlation with plasma LP Al
but not with FERL,
again confirming our previous find-
Correlations
Data analysis of both groups of patients dearly shows
an inverse correlation
between the plasma concentration
of LP Al and FERWL. Despite the weakness
of the correlation (Table 3), the relation is more apparent if we consider other factors. Both LP AT (positively) and FERunL
(negatively) correlated strongly with plasma HDL-cholesterol and with the concentrations
of apo Al and LP Al/All.
Both variables were also strongly correlated with HDL,
as determined
by gel electrophoresis
(LP Al positively,
FERHDL negatively).
However, although there was no sig-
Discussion
ings(5).
Other correlations
of interest
are those of triglycerides to HDL3bC (r = 0.513, P <0.001) and of total HDLcholesterol
to HDL2b (r = 0.485, P <0.001)
and to
HDL3bC (r = -0.461, P <0.01).
Our data show that the composition
of HDL subspecies (as estimated
by GGE) is strongly correlated
with
the concentration
of LP Al particles and the cholesterol
esterification
rate in the HDL pool.
The amount
of LP Al correlated
positively
with the
amount of HDL2b particles.
LP Al correlated
also with
concentrations
of HDL total and free cholesterol,
with
apo Al, and with LP Al/AlT particles.
We have confirmed the findings of James and Pometta that women
have higher concentrations
of LP Al than do men (16)
Table 2. Mean (SD) data for subjects with
Women
Men
Controls (n = 12)
400
(100)
LPAI, mg/L.
FERHOL,
%/h
18.49 (7.28)
TC, mmoUL
Hypoalpha (n
300
=
25)
36.98 (13.50)#{176}
1G. mmol/L
4.64(1.04)
1.06 (0.57)
5.89(1.56)”
3.23 (4.10)”
HDL-TC, mmol/L
1.26(0.29)
0.71 (O.13)c
4.23 (1.35)C
1000
(15)C
LDL-TC,mmol/L
2.88 (0.95)
1310
ApoAI,mg/L
Apo B, mg/L
LPM/A1l,mgfL
TC/HDL-TC
CLINICAL
(160)
(190)
910
(170)
3.85 (0.96)
IC. total cholesterol;
1556
750
1G. triglycerIdes. Significance of differences
CHEMISTRY,
Vol. 40, No. 8, 1994
Controls (n
(80)8
480
=
19)
(180)
19.57 (9.00)
5.89 (1.00)
1.30 (1.00)
1.32 (0.40)
3.97 (1.08)
1470 (390)
900
(29)C
840
(260)
680
(190)”
990
(230)
(1.70)
4.80
8.53 (2.88)#{176}
from controls: 8P <0.05, “P <0.01,
C
P
<0.001.
Hypoalpha (n
=
8)
250
(110)”
31.48 (5.58)c
5.57 (1.39)
1.54 (0.40)
0.78 (0.04)c
4.10 (1.31)
1030
(90)#{176}
890
(250)
780
7.17
(80)8
(l,81)b
probably because
esterification
of cholesterol
occurs
preferentially
on these small particles,
which contain
only a small proportion of the LP Al in plasma.
The
Hypoalpha (n = 64)
HypertensIon (n = 46)
relative plasma content of small HDL particles
correLP Al
FERH
LP Al
FERH
lates well with the concentration
of plasma triglycerides
1
1
N.S.
-033O’
and with the ratio for total/HDL-cholesterol.
As recently
N.S.
N.S.
0.407c
N.S.
reported, increased
proportions
of HDL3bC appear to be
NS
0.789#{176} atherogemc
NS
0 7090
(5, 7). We found that hypertensive
subjects
-0.683#{176} had high values of FERL
-0.646#{176} 0.288a
0.6580
(with low HDL2b and high
N.D.
0.576#{176} N.S.
N.D.
HDL3b,C concentration)
compared
with the concentraN.S.
N.S.
N.S.
0.358”
tions in their age-, weight-, and lipid profile-matched
0.8080
N.D.
N.D.
controls (5). Williams
et al. (7) pointed
out that, in
0.356”
N.D.
N.D.
N.S.
healthy subjects, other CAD risk factors-e.g.,
male sex,
0.425”
N.D.
N.D.
triglycerides,
increased
LDL-are
associated
with
0.633#{176}
N.S.
0.810#{176} N.S.
higher concentrations
of HDLSb.
Table 3. CorrelatIon between LP Al, FERHOL,and
other variables.
FERHOL
IC
TG
HDL-TC
HDL-FC
LDL-TC
Apo Al
Apo B
LI’ Al/All
TCIHDL-TC
HDL,,,
HDL,,
HDL,I,,C
HDL,JHDL2b
N.D.
N.D.
N.D.
ND.
N.D.
N.D.
N.D.
ND.
0436”
-0.583#{176}
-0.482#{176}
N.S.
N.S.
We conclude,
therefore, that FERHJ,L, which reflects
of HDL subspecies
(specifically,
the relative content of HDL2b and HDLSb,C or their interactions), might be a better test than LP Al assay to evaluate the risk of CAD and (or) the effectiveness
of
treatment
for CAD. Assessment
of FERunL is a functional test of HDL particle interactions,
whereas the LP
Al assay mostly reflects only the concentrations
of the
HDL2 or HDL2b subclasses.
Although,
in our opinion,
FERL
yields a useful information
about the composition of patients
plasma
HDL, it is indeed not the ultimate risk indicator; other risk factors, both lipid-related
and others, must be assessed in individual patients.
0.734#{176}
the composition
0.6340
N.S., not significant; N.D., not done. TC, total cholesterol; FC, free cholesterol; TG, triglycerides. Significance of differences from controls: 8 P <0.05,
bp<)
#{176}P<O.001.
but found no significant
correlations
between plasma LP
Al and triglycerides
or between LP Al and the ratio of
total cholesterol
to HDL-cholesterol.
The plasma
concentrations
of LP Al in controls were not significantly
different from those in hypertensive
subjects with similar lipid profiles. As expected,
we saw a significant
decrease of LP Al in hypoalpha
patients. Our finding
that LP Al correlated
significantly
with the free cholesterol in HDL is consistent
with the data of Ohta et al.
(11) and James and Pometta (16), who found a higher
free cholesterol
content in LP Al than in LP Al/All.
FERL
correlated
also with the concentrations
of ape
Al, LP Al/All particles,
and HDL total (but not free)
cholesterol,
but this correlation was in the opposite direction from that of LP Al. Both hypertensive
patients
and persons with hypoalpha
had markedly
higher values of FERL.
The data on FERunL in this study are
consistent with our previously reported findings (5, 19);
we confirmed
the inverse correlation between FERunL
and the relative concentration
of Hl)L2b particles,
and
the positive correlation
of FERL
with the relative
concentration
of HDL3b,C particles.
FERL
was also
strongly
correlated
with the concentration
of plasma
triglycerides,
perhaps because the HDL3bC subfraction
also correlated
significantly
with the plasma triglycerides. The correlation between HDL2b and triglycerides
was not significant.
As we showed previously
(4), individuals with identical concentrations
of HDL free (or total) cholesterol may
differ dramatically
in their respective
FERunL. This
confirms
the effect of specific HDL particles/particle
sizes distribution
on the rate of cholesterol esterification
in Hl)L.
The data indicate
that LP Al and FERL
are comparable
indicators
of relative content of HDL2b particles
(Table 3). However, only FERL,
but not LP Al, correlates strongly with relative concentration
of HDLSb,C-
We thank Marie Schutzova and Lida Adler for their valuable
assistance with this project and J. F. Bowden in preparation of this
manuscript.
The study was supported by Grant Agency of Czech
Republic, Czech Health Care Foundation MZ 121 and by the British Columbia Heart Foundation.
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