Journal of the American College of Cardiology
© 1999 by the American College of Cardiology
Published by Elsevier Science Inc.
Vol. 33, No. 2, 1999
ISSN 0735-1097/99/$20.00
PII S0735-1097(98)00560-9
Risk Factors
Quantity and Function of High
Density Lipoprotein as an
Indicator of Coronary Atherosclerosis
Keijiro Saku, MD, PHD, FACP, Bo Zhang, MS, PHD, Takao Ohta, MD, PHD,*
Kikuo Arakawa, MD, PHD, FACC
Fukuoka and Okinawa, Japan
OBJECTIVES
To examine the association between the fractional esterification rate of cholesterol (C) in low
density lipoprotein- and very low density lipoprotein-depleted plasma (FERHDL) and
coronary artery disease (CAD) and the influence of serum HDL-C levels.
BACKGROUND The function of HDL in reverse cholesterol transport is involved in the antiatherogenic action
of HDL, and FERHDL is a newly established quantitative measure of HDL function in vivo.
METHODS
Cases (n 5 185, F/M: 43/142) and controls (n 5 74, F/M:27/47) were defined as subjects
with/without angiographically proven CAD, respectively.
RESULTS
The cases had significantly (p , 0.05) higher FERHDL values (13.2 6 0.3 %/h vs. 12.1 6 0.5
%/h) and lower HDL-C levels (39.0 6 1.0 mg/dL vs. 46.8 6 1.4 mg/dL) than the controls.
The associations of FERHDL and HDL-C with CAD were linear and significant (p , 0.05).
Multiple logistic regression analysis indicated that the association of FERHDL with CAD
varied with the HDL-C level: significant for the low HDL-C tertile (chi-square 5 6.20, p ,
0.05) but not significant for the middle and high HDL-C tertiles (chi-square 5 0.08 and
0.03, n.s.). The risk of CAD, relative to that in patients with low FERHDL and high
HDL-C, was higher in patients with low FERHDL and low HDL-C (odds ratio [95%
confidence interval]: 2.37 [1.12– 4.97], p , 0.05) and was highest in patients with high
FERHDL and low HDL-C (3.85 [1.84 – 8.06], p , 0.01).
CONCLUSIONS The functional assay of HDL (FERHDL) is an independent risk factor for CAD. The
combination of FERHDL and HDL-C could be a potent indicator for CAD, and may reflect
a potential mechanism of atherosclerosis. (J Am Coll Cardiol 1999;33:436 – 43) © 1999 by
the American College of Cardiology
An inverse relationship between plasma levels of high
density lipoprotein cholesterol (HDL-C) and the risk for
coronary artery disease (CAD) has been well established
(1,2). Direct evidence for the antiatherogenic effects of
HDL has recently been obtained in studies of the over- or
under-expression of apo A-I using genetic animal models of
reverse cholesterol transport (3–7).
HDL particles differ in size, structure and function (8).
Both HDL2 and HDL3 levels are reduced in patients with
CAD (9,10). However, although some studies have reported altered relative proportions of the HDL2 and HDL3
From the Department of Internal Medicine and Pathology, Fukuoka University
School of Medicine, Fukuoka 814-0180, Japan; and *Department of Pediatrics,
Ryukyus University School of Medicine, Okinawa 904-2300, Japan. Part of this work
was presented at the 46th Annual Scientific Session of the American College of
Cardiology (ACC), March 19th, 1997, Anaheim, California. This work was supported by grants-in-aid from the Ministry of Education, Science and Culture of Japan
(Nos. 04671503, 06670809, 07670827, 09670773, 09670773, and 10670693), by
research grants from the Ministry of Health and Welfare, and by research grants from
the Fukuoka University Research Fund.
Manuscript received April 15, 1998; revised manuscript received August 25, 1998,
accepted October 2, 1998.
subclasses, the functional aspects of HDL did not attract
much attention until Dobiasova and Frohlich (8) established a functional assessment of HDL: the fractional
esterification rate in low density lipoprotein (LDL)- and
very low density (VLDL)-depleted plasma (FERHDL).
They indicated that FERHDL is a functional test of HDL
particle interaction (8), and suggested that angiographically
proven CAD subjects had higher FERHDL values than
controls (11).
A major hypothesis for explaining the antiatherogenic properties of HDL involves the role of HDL in reverse cholesterol
transport (RCT) (12). Reverse cholesterol transport is a multistep process that results in the net movement of cholesterol
from peripheral tissues back to the liver via the plasma
compartment (3). The efflux of cholesterol from the plasma
membrane of peripheral cells to HDL is the first step in the
RCT pathway (13). Promotion of this step may be antiatherogenic because it reduces the possibility of the overaccumulation
of cellular cholesterol, and this hypothesis has been supported
by studies using genetic animal models of RCT (4–6). Two
kinds of nascent HDL particles are believed to be secreted
JACC Vol. 33, No. 2, 1999
February 1999:436–43
Abbreviations and Acronyms
Apo
5 apolipoprotein
CAD
5 coronary artery disease
CETP 5 cholesterol ester transfer protein
CAG
5 diagnostic coronary angiography
FC
5 free cholesterol
FERHDL 5 fractional esterification rate in the HDL
fraction of plasma
HDL-C 5 high density lipoprotein cholesterol
LCAT 5 lecithin:cholesterol acyltransferase
LDL-C 5 low density lipoprotein cholesterol
RCT
5 reverse cholesterol transport
TC
5 total cholesterol
from the liver and (in humans) intestine: small spherical
HDL3-like particles (14) and small lipid-poor complexes (15)
that migrate with pre-b mobility on agarose gel electrophoresis
(16). These two subfractions of HDL remove cellular free
cholesterol by distinct mechanisms (17–19): diffusion-based or
receptor-dependent (15).
Cholesterol ester that has accumulated in HDL may then
transfer from HDL to apolipoprotein (apo)-B containing
lipoproteins (LDL and VLDL) in exchange for triglycerides
(TG) as a result of the activity of cholesterol ester transfer
protein (CETP) with subsequent uptake of TG-rich lipoprotein remnants by the liver. In humans, this second step
of RCT is illustrated by the dramatic accumulation of HDL
in subjects with CETP deficiency (20,21). TG-rich HDL2
particles are subjected to hydrolysis by hepatic lipase and
perhaps lipoprotein lipase (14) and are converted back to a
small HDL3-like particle. Apolipoprotein (Apo) A-I can
also be released from HDL2 to produce pre-b1 HDL (22).
Thus, the HDL3 3 HDL2 3 HDL3 cycle and pre-b1
HDL 3 HDL3 3 HDL2 3 pre-b1 HDL cycle are
completed. Promotion of this second step of RCT is
probably proatherogenic because CETP transfers cholesterol ester from “good” or “safe” lipoprotein HDL to
atherogenic apo-B containing LDL and VLDL, thereby
promoting cholesterol ester deposition (22). Therefore, we
propose that both the quantity of HDL, as measured by
HDL-C and the function of HDL in RCT, as quantitatively measured by FERHDL, play important roles in antiatherogenic properties of HDL.
In this case-control study, we tested the association of
FERHDL with CAD and its interaction with HDL-C, after
controlling for age, gender, conventional risk factors and
other lipid parameters.
METHODS
Patients. This study included 259 patients who underwent
diagnostic coronary angiography (CAG) for suspected or
known coronary atherosclerosis or for other reasons (mostly
atypical chest pain) at the Fukuoka University Hospital
from 1994 to 1996. This study was approved by the ethics
Saku et al.
Quantity and Function of HDL and CAD
437
committee of Fukuoka University Hospital, and informed
consent was obtained from each patient. Controls (CAD2
patients) were defined as those with less than 25% luminal
narrowing, and cases (CAD1 patients) were those who had
one, two or three stenosed (.50% luminal narrowing)
epicardial coronary arteries. Patients with luminal narrowing of between 25% and 50% were excluded. Patients with
spastic angina pectoris, i.e., acetylcholine-positive, were
excluded from the controls and none of the controls had
myocardial infarction (MI). Patients with acute MI (within
three weeks after onset), heart failure (Killip Class $2 after
myocardial infarction), vascular disease (aortitis treated by
prednisoline), hepatic dysfunction (virus and nonvirus,
transaminases more than three times the normal value) or
uncontrollable diabetes mellitus were excluded from the
study. Patients with systolic or diastolic blood pressure
.160 mm Hg or 95 mm Hg or who were under antihypertensive treatment were considered to have hypertension (HT). Patients under treatment for diabetes mellitus
(DM) and/or with symptoms of DM and a fasting glucose
concentration $126 mg/dL were considered to have DM.
Otherwise, the results of a 75 gm glucose tolerance test were
used to give a diagnosis of DM. About 98% of the women
were in menopause but none were receiving hormone
replacement therapy. None of the patients were being
treated with lipid-lowering agents at the time of sampling.
Coronary angiography. Coronary arteries were cannulated
by the Judkins technique (23) with 5F catheters, and
recorded on Kodak 35 mm cinefilm at a rate of 25 frames/s.
Coronary arteries were divided into 15 segments, according
to the classification of the American Heart Association
Grading Committee. In this study, reliable and reproducible
measurements were obtained. Coronary artery segments
were carefully selected by two expert cardiologists on the
basis of smooth luminal borders and the absence of stenotic
changes. The presence of stenosis was determined using a
computer-assisted coronary angiography analysis system
(Mipron 1; Kontron Co., Tokyo, Japan) after the direct
intracoronary injection of isosorbide dinitrate (ISDN) (2.5
mg/5 mL solution), as described previously (24). Arterial
stenosis, that produced more than 50% luminal narrowing,
was considered significant.
Determination of serum lipids, lipoproteins, and apolipoproteins. Blood was drawn in the morning after an
overnight fast. Serum total cholesterol (TC) and triglyceride
(TG) concentrations were determined enzymatically.
HDL-C was determined by the heparin Ca21 precipitation
method (25). HDL subfraction (HDL2 and HDL3) were
separated by standard sequential preparative ultracentrifugation techniques (26). Apo A-I, apo A-II, apo B, apo C-II,
apo C-III and apo E were determined by the turbidity
immunoassay method (27). Serum lipoprotein (a) (Lp[a])
levels were measured by an enzyme-linked immunosorbent
assay using Tint Eliza Lp(a) (Biopool Co., Stockholm,
Sweden) (28). For all measurements in our laboratory, the
438
Saku et al.
Quantity and Function of HDL and CAD
coefficients of interassay and intraassay variation were less
than 5.0%, and blinded quality-control specimens were
included in each assay.
Assay for FERHDL in plasma. VLDL-LDL-depleted
plasma was prepared by precipitating apolipoprotein
B-containing lipoproteins with phosphotungstate-MgCl2
(11). FERHDL was determined according to the method of
Ohta et al. (29) with minor modifications. [3H] Free
cholesterol (FC) was incorporated onto polystyrene tissueculture wells (Corning, New York, New York) as follows:
absolute ethanol (100 ml) containing 1 mCi of [3H] FC was
placed in wells and dried off by flushing with nitrogen. Next,
100 ml of the VLDL- and LDL-depleted plasma samples,
in 400 ml of PBS was added to each well and [3H] FC was
equilibrated with the FC in each sample by incubation at
4° C for 16 h. [3H] FC-labeled VLDL- and LDL-depleted
plasma samples were incubated in a shaking water bath for
3 h at 37° C. The enzyme reaction was stopped by immersing the sample tubes in an ice bath. The lipids in incubation
samples were extracted with methanol/chloroform (2:1,
v/v). The extract was dried by flushing it with nitrogen and
was then dissolved in 60 ml of isopropanol. Aliquots (20 ml)
of lipid extracts were spotted in duplicate on a thin-layer
chromatography plate (Merck, West Point, Pennsylvania)
and developed in n-hexane/diethyl ether/acetic acid/
methanol (85:20:1:1, v/v). Spots corresponding to FC and
cholesteryl ester (CE) were cut from the plate and the
radioactivities were determined. The increase in [3H] CE
was linear within 3 h of incubation. The fractional esterification rate was expressed as the difference between the
percentage of radioactive cholesterol esterified before and
after incubation at 37° C. The samples were measured in
triplicate and the coefficient of variation of the assay was
0.75%. The coefficient of variation for the interassay variability was 6.2%.
Statistical analysis. Statistical analysis was performed using the SAS Software Package (Version 6, Statistical Analysis System, SAS Institute Inc., Cary, North Carolina).
Categorical variables (such as gender) were compared between cases and controls by a chi-square analysis. Differences between cases and controls or among patients with 1-,
2-, and 3-vessel diseases were examined by an analysis of
variance (ANOVA). Comparisons of 1-vessel and 2- or
3-vessel disease patients were performed with the multiple
comparison test of Dunnett (30). Age and gender were
adjusted for by an analysis of covariance (ANCOVA) (30).
The logistic model was used to evaluate linear associations
between CAD and lipid variables (continuous). In addition,
odds ratios were simultaneously adjusted for age, gender and
potentially confounding variables by a multiple logistic
regression analysis (30). For all of the odds ratios, we
calculated 95% confidence intervals (CI). For logistic regression coefficients, we show either 95% CI or the standard
error. A multiple regression analysis was used to test the
correlation between HDL-C or FERHDL and lipid variables
JACC Vol. 33, No. 2, 1999
February 1999:436–43
Table 1. Patient Characteristics
Age (yr.)
BMI (Kg/m2)
Gender
Female
Age # 63†
Age . 63
Male
Age # 63
Age . 63
Smoking
Hypertension
Diabetes mellitus
CAD1
Patients
(n 5 185)
CAD2
Patients
(n 5 74)
62.6 6 7.9
23.3 6 3.5
62.1 6 7.2
22.8 6 3.3
43 (23.2%)
13 (13.5%)
30 (33.7%)
142 (76.8%)
83 (86.5%)
59 (66.3%)
100 (54.3%)
74 (40.2%)
58 (31.4%)
27 (36.5%)
15 (39.5%)
12 (33.3%)
47 (63.5%)*
23 (60.5%)*
24 (66.7%)
30 (40.5%)*
30 (40.5%)
17 (23.0%)
*p , 0.05, by chi-square. †, median age of all patients.
CAD 5 coronary artery disease; BMI 5 body mass index.
while controlling for age, gender and other lipid parameters
(30). Because some variables were not normally distributed,
we used rank scores in the regression analysis to simplify the
calculation (31). All p values are two-tailed. The significance level was considered to be 5% unless otherwise
indicated.
RESULTS
Table 1 shows the patient characteristics. There were
significantly more men (especially in patients younger than
63 years) and smokers among CAD1 patients than among
CAD2 patients. On the other hand, the prevalence of
diabetes mellitus and hypertension was not significantly
different between the two groups. The prevalences of
smokers and diabetes in both CAD1 and CAD2 patients
were both higher than those seen in the United States (32).
In Table 2 serum lipids, lipoproteins, apolipoproteins,
FERHDL and HDL-FC were compared between CAD1
and CAD2 patients after controlling for age and gender.
Serum levels of HDL-C, HDL2-C, HDL3-C, apo A-I, apo
A-II, apo E and HDL-FC were significantly lower, and
serum Lp(a) levels and FERHDL values were significantly
higher in CAD1 patients than in CAD2 patients. However, these variables (except for HDL3-C levels) were not
associated with the extent of stenosis as judged by the
number of vessels affected (data not tabulated). As shown in
Table 2, serum levels of total cholesterol in CAD1 patients
were much lower than those in equivalent American patients with proven atherosclerotic disease but similar to
those reported by another Japanese study group (n 5 133,
193 6 36 mg/dl) (33). The levels of LDL and HDL-C in
CAD1 patients were almost the same as those in the
Cholesterol and Recurrent Events (CARE) Study (34).
Table 3 shows the results of the test for the linearity of
the associations of CAD-related variables (continuous) after
controlling for age and gender, and with (indicated by †)
Saku et al.
Quantity and Function of HDL and CAD
JACC Vol. 33, No. 2, 1999
February 1999:436–43
439
Table 2. Age- and Gender-Adjusted Mean Values of Lipids, Lipoproteins Lipid,
Apolipoproteins, and FERHDL in Patients With (CAD1) and Without (CAD2) Significant
Coronary Stenosis
CAD1
(1V/2V/3V 5 93/68/24)
CAD2
p value
39.0 6 1.0
26.1 6 0.9
17.1 6 0.3
104.5 6 1.8
27.5 6 0.5
114.2 6 2.4
3.6 6 0.1
9.3 6 0.3
4.9 6 0.1
199 6 3
125 6 5
135 6 3
50.8 6 0.81
3.0 6 0.1
13.2 6 0.3
7.6 6 0.2
46.8 6 1.4
31.3 6 1.3
19.3 6 0.5
119.9 6 2.6
29.9 6 0.7
107.2 6 3.4
3.8 6 0.2
10.0 6 0.4
5.4 6 0.2
198 6 5
113 6 7
129 6 4
50.0 6 1.3
2.6 6 0.1
12.1 6 0.5
9.0 6 0.3
, 0.05
, 0.05
, 0.05
, 0.05
, 0.05
n.s.
n.s.
n.s.
, 0.05
n.s.
n.s.
n.s.
n.s.
, 0.05
, 0.05
, 0.05
HDL-C (mg/dL)
HDL2-C (mg/dL)
HDL3-C (mg/dL)
Apo A-I (mg/dL)
Apo A-II (mg/dL)
Apo B (mg/dL)
Apo C-II (mg/dL)
Apo C-III (mg/dL)
Apo E (mg/dL)
TC (mg/dL)
TG (mg/dL)
LDL-C (mg/dL)
FC (mg/dL)
loge[Lp(a)] (mg/dL)
FERHDL (%/h)
HDL-FC (mg/dL)
Values are least-square means 6 SEM.
HDL-C 5 high density lipoprotein cholesterol; Apo 5 apolipoprotein; TC 5 total cholesterol; TG 5 triglyceride;
LDL-C 5 low density lipoprotein cholesterol; FC 5 free cholesterol; Lp(a) 5 lipoprotein (a); FERHDL 5 fractional
esterification rate in the HDL fraction of plasma; HDL-FC 5 free cholesterol content in HDL.
and without also adjusting for smoking, hypertension,
diabetes and body mass index. For each variable shown in
Table 3, the association with CAD was significantly linear:
The relative risk of CAD (odds ratio) decreased with
increasing serum levels of HDL-C, HDL2-C, HDL3-C,
apo A-I, apo A-II, apo E, and HDL-FC and with decreasing serum Lp(a) levels and FERHDL values. After adjusting
for conventional risk factors (Table 3, right columns), the
association of HDL-C, apo A-I, apo E, Lp(a), and
FERHDL with CAD remained significant, suggesting that
these variables are independently associated with CAD.
Table 4 shows the influence of HDL-C on the
association between FERHDL and CAD and the effect of
interaction between FERHDL and HDL-C. The association of FERHDL with CAD varied with the serum level
of HDL-C: FERHDL was significantly associated with
CAD in the low tertile of HDL-C (Wald x2 5 6.20, p 5
0.01) but not in the middle and high tertiles (Wald x2 5
0.08 and 0.03, n.s.). Figure 1 shows a plot of the
prevalence of CAD, FERHDL (in tertiles), and HDL-C
(in tertiles). These results show that the association of
FERHDL with CAD was modified by HDL levels. It is
Table 3. Age- and Gender-Adjusted Linear Trends Across Fractional Ranks of Coronary Atherosclerosis-related Variables, as Tested
by a Multiple Logistic Regression Analysis
Adjusted for Age and Gender
Variable*
Regression
Coefficient
(Standard
Error)
Wald
chi-square
HDL-C
HDL2-C
HDL3-C
Apo A-I
Apo A-II
Apo E
Lp(a)
FERHDL
HDL-FC
22.5 (0.57)
22.19 (0.63)
22.15 (0.62)
22.52 (0.57)
21.24 (0.52)
21.4 (0.53)
1.3 (0.51)
1.38 (0.64)
22.99 (0.74)
19.3
12.1
12.2
19.4
5.66
7.08
6.44
4.5
16.2
Adjusted for All†
(p value)
Regression
Coefficient
(Standard
Error)
Wald
chi-square
(p value)
(, 0.001)
(, 0.001)
(, 0.001)
(, 0.001)
(0.017)
(0.008)
(0.011)
(0.033)
(, 0.001)
20.45 (0.12)
20.11 (0.09)
20.14 (0.09)
20.44 (0.11)
20.2 (0.10)
20.31 (0.11)
0.28 (0.10)
0.3 (0.11)
20.13 (0.09)
13.7
1.18
2.03
14.4
3.45
7.23
7.06
7.14
1.77
(, 0.001)
(0.28)
(0.15)
(, 0.001)
(0.06)
(0.007)
(0.008)
(0.008)
(0.18)
*Fractional ranks rather than original values are used for the regression analysis and were calculated by dividing the rank by the number of observations (31). †Adjusted for age,
gender, smoking, hypertension, diabetes and body mass index.
HDL-C 5 high density lipoprotein cholesterol; Apo 5 apolipoprotein; Lp(a) 5 lipoprotein (a); FERHDL 5 fractional esterification rate in the HDL fraction of plasma;
HDL-FC 5 free cholesterol content in HDL.
440
Saku et al.
Quantity and Function of HDL and CAD
JACC Vol. 33, No. 2, 1999
February 1999:436–43
Table 4. Age- and Gender-Adjusted Associations between FERHDL and Coronary
Atherosclerosis by HDL-C (in Tertiles) and HDL-C-by-FERHDL Interaction as Assessed by a
Multiple Logistic Regression Analysis
HDL-C tertile
I (low)
II (middle)
III (high)
Independent
Variable
Regression
Coefficient
(standard error)
Wald x2
(p value)
FERHDL
FERHDL
FERHDL
0.90 (0.36)
0.10 (0.34)
0.07 (0.39)
6.20
0.08
0.03
(0.01)
(0.77)
(0.86)
x2, chi-square.
also possible that some other factor(s) mediate the
relationship of both HDL-C and FERHDL with coronary
atherosclerosis.
We tested the relationships between FERHDL and lipid
parameters in patients with and without CAD by a multiple
regression analysis after controlling for age and gender. As
shown in Table 5, the age- and gender-adjusted parameter
estimates were significant between FERHDL and HDL-C,
HDL2-C, apo A-II, apo B, apo C-III, TG, and HDL-FC
in both groups, and between FERHDL and apo A-I, apo
C-II, apo E, TC, and LDL-C in the CAD1 patients.
The dependence of the association of FERHDL with
CAD on HDL-C was tested by a multiple logistic regression analysis (Table 6, upper panel). As shown in Models 1
and 2, HDL-C (in tertiles) and FERHDL (in tertiles) were
significantly and linearly associated with CAD after controlling for age and gender. When FERHDL was added to
Model 1 (Model 3), the fit was improved, as judged by the
model fitting criterion, 22 Log Likelihood (Table 6).
Addition of the HDL-C-by-FERHDL interaction term to
Model 3 (Model 4) further improved the fit, with the
association of FERHDL. These results suggest that the
association of FERHDL with CAD is independent of
HDL-C levels. Adding smoking, HT and DM to Model 4
(Model 5) slightly improved the model fit, and adding
Lp(a), TG, apo E and apo C-II to Model 5 (Model 6)
further improved the fit.
Table 6 (lower panel) also shows the odds ratio for each
combination of FERHDL (two levels) and HDL-C (two
levels) after controlling for age and gender. As shown,
patients with low FERHDL-low HDL-C had a significantly
higher relative risk than patients with low FERHDL-high
HDL-C, and patients with high FERHDL-low HDL-C
had the highest relative risk. Patients with high-FERHDLlow HDL-C also had the highest prevalence of CAD (Fig.
1). These results suggest that FERHDL modified the risk
associated with HDL levels, and when HDL-C was low,
high FERHDL values further increased the risk of CAD.
Figure 1. Relationship between the percentage of patients with
angiographically defined coronary atherosclerosis (prevalence of
CAD) and FERHDL (in tertiles) according to HDL-C (in tertiles).
Open, striped and solid bars represent patients with low, middle
and high FERHDL values.
*p , 0.05; †p , 0.01, parameter estimates significantly different from zero.
HDL-C 5 high density lipoprotein cholesterol; Apo 5 apolipoprotein; TC 5
total cholesterol; TG 5 triglyceride; LDL-C 5 low density lipoprotein cholesterol;
FC 5 free cholesterol; Lp(a) 5 lipoprotein (a); FERHDL 5 fractional esterification
rate in the HDL fraction of plasma. Data are presented as age- and gender-adjusted
parameter estimates (95% confidence interval).
DISCUSSION
In the present study, we investigated the relationship
between the function of HDL as quantitatively measured by
Table 5. Age- and Gender-Adjusted Relationships Between
FERHDL Values and Other Lipid Variables in Patients With
and Without Coronary Artery Disease as Assessed by a
Multiple Regression Analysis
HDL-C
HDL2-C
HDL3-C
Apo A-I
Apo A-II
Apo B
Apo C-II
Apo C-III
Apo E
TC
TG
LDL-C
FC
Lp(a)
HDL-FC
Patients without CAD
Patients with CAD
20.59 (20.75, 20.38)†
20.66 (20.79, 20.47)†
20.02 (20.30, 0.26)
20.24 (20.48, 0.04)
0.28 (0.00, 0.52)*
0.53 (0.29, 0.70)†
0.27 (20.01, 0.51)
0.31 (0.03, 0.54)*
0.09 (20.20, 0.36)
0.09 (20.19, 0.36)
0.61 (0.39, 0.76)†
0.19 (20.10, 0.44)
0.11 (20.18, 0.38)
0.05 (20.23, 0.32)
0.69 (0.52, 0.82)†
20.68 (20.75, 20.58)†
20.73 (20.80, 20.64)†
20.01 (20.16, 0.15)
20.32 (20.46, 20.18)†
0.19 (0.04, 0.35)*
0.57 (0.45, 0.67)†
0.41 (0.26, 0.53)†
0.29 (0.14, 0.43)†
0.32 (0.18, 0.46)†
0.21 (0.05, 0.36)*
0.51 (0.38, 0.62)†
20.29 (20.43, 20.14)†
0.17 (0.01, 0.32)
0.02 (20.14, 0.18)
0.76 (0.62, 0.86)†
Saku et al.
Quantity and Function of HDL and CAD
JACC Vol. 33, No. 2, 1999
February 1999:436–43
441
Table 6. Multiple Logistic Function Analysis of Coronary Risk Factors and Accompanying Odds Ratios for Each Combination of
FERHDL and HDL-C After Adjusting for Age and Gender
Model No. and Description
Model 1: HDL-C, age, gender
Model 2: FERHDL, age, gender
Model 3: Model 1 1 FERHDL
Model 4: Model 3 1 HDL-C* FERHDL
Model 5: Model 4 1 HT, DM, smoking
Model 6: Model 5 1 Lp(a), TG, apo CII
Regression
Coefficients
Wald x2
(p value)
HDL-C: 20.73 6 0.19
FERHDL: 0.59 6 0.19
HDL-C: 20.58 6 0.21
FERHDL: 0.42 6 0.21
HDL-C: 20.08 6 0.44
FERHDL: 1.16 6 0.54
HDL-C: 20.13 6 0.45
FERHDL: 1.17 6 0.54
HDL-C: 0.17 6 0.48
FERHDL: 1.26 6 0.56
14.3
9.74
7.98
3.98
0.03
4.71
0.08
4.73
0.13
5.12
(,0.001)
(0.002)
(0.005)
(0.048)
(0.864)
(0.030)
(0.772)
(0.030)
(0.718)
(0.024)
22 Log
Likelihood
(p Value)
21.31
16.35
25.68
(,0.001)
(0.003)
(,0.001)
27.37
(,0.001)
28.88
(,0.001)
44.01
(,0.001)
Odds ratio for each combination of FERHDL and HDL-C
High FERHDLHigh HDL-C*
High FERHDLLow HDL-C*
1.84 (0.64–5.29)
3.85 (1.84–8.06)†
Low FERHDLHigh HDL-C
1.00
Low FERHDLLow HDL-C*
2.37 (1.12–4.97)†
*Odds ratio and 95 percent confidence interval are given; †p , 0.05.
HDL-C 5 high density lipoprotein cholesterol; FERHDL 5 fractional esterification rate in the HDL fraction of the plasma; HT 5 hypertension; DM 5 diabetes mellitus;
apo 5 apolipoprotein.
FERHDL and CAD and its interaction with the quantity of
HDL as measured by serum levels of HDL-C. Our findings
seem to support the most recent hypothesis of reverse
cholesterol transport and the atherogenic remnant hypothesis which are beginning to be seen as a single concept (12).
Figure 2 shows a schematic view of this model for HDL
metabolism. We propose that the process by which cholesterol moves from the periphery to apo B-containing particles consists of two steps: the first step is antiatherogenic, as
measured by HDL-C levels, and the second is proatherogenic, as measured by FERHDL. The antiatherogenic role
that HDL plays in the first step of RCT has been wellestablished, and direct evidence has been obtained in studies
of the over- or under-expression of apo A-I using genetic
animal models of reverse cholesterol transport (3–7). Our
finding that HDL-C levels are linearly and inversely related
with CAD (Table 3) supports this hypothesis.
Figure 2. Model for HDL metabolism in the two-steps reverse
cholesterol transport process. The first step is the efflux of cellular
free cholesterol to HDL, which is an antiatherogenic step. The
second step is the transfer of cholesterol ester from HDL to apo
B-containing lipoproteins (LDL and VLDL) via CETP, which
may be proatherogenic and is what FERHDL measures.
Our results show that patients with angiographically documented CAD have an increased value of FERHDL. This agrees
with the results of Dobiasova and Frohlich (8,35) but is
contrary to the generally accepted belief that an increased rate
of cholesterol esterification may be beneficial because it contributes to the HDL cholesterol content of plasma (35). Our
finding is supported by several facts that suggest that increased
CETP activity may be atherogenic and reduced activity may
protect against the development of atherosclerosis. Mice that
lack CETP are resistant to the deleterious effects of highcholesterol diets, and humans and rabbits with CETP are
susceptible to hypercholesterolemia and atherosclerosis when
fed diets high in fat and cholesterol. Elevated levels of CETP
in transgenic mice containing simian CETP result in the
formation of atherosclerotic lesions (36). Increased CETP
activity is associated with acceleration of the rate of atherosclerotic plaque formation in human dyslipidemias such as dysbetalipoproteinemia (37), familial hypercholesterolemia (38)
and hypercholesterolemia (39).
HDL is remodeled during the process of RCT, with
changes in structure and size (40), which are the most important factors in determining the rate of LCAT-catalyzed cholesterol esterification (8) and CETP-mediated transport of
cholesterol ester (40). This is reflected in our findings that
FERHDL is inversely related to HDL-C and that the association of FERHDL with CAD is modified by HDL levels. The
net flux of cellular cholesterol to HDL particles is generated by
a gradient of the cholesterol content between cells and the
lipoprotein surface and provided by an LCAT reaction (41).
Because the size of circulating HDL particles as reflected by
FERHDL (8) depends on the balance between the opposing
processes of cholesterol acceptance (which increases particle
size) and lipolytic digestion (which reduces it), we can speculate
442
Saku et al.
Quantity and Function of HDL and CAD
that an increased FERHDL value reflects a detrimental condition that facilitates accumulation of intracellular cholesterol via
LDL. This is confirmed by our finding that increased
FERHDL is associated with an increased prevalence of CAD.
However, a sufficient number of HDL particles may overcome
this condition by retaining cholesterol esters in the HDL
fraction. This is reflected in our finding that when HDL-C
levels were high, increased FERHDL did not significantly
increase the risk of CAD (Table 4, Fig. 1). Because low
HDL-C levels also tended to be linked to a reduction in the
expression of lipoprotein lipase and a rise in hepatic lipase, both
of which are enzymatic changes that would lead to a decrease
in triglyceride-rich particle lipolysis (12), increased FERHDL
may cause an increased accumulation of cholesteryl ester-rich
remnants of VLDL and chylomicra under conditions of low
HDL-C and then confer an increased risk of CAD. This is
reflected in our findings that patients with both low HDL-C
and high FERHDL had the highest risk of CAD (Table 6, Fig.
1) and that the combination of both FERHDL and HDL is
more strongly related to CAD than is either measure alone
(Table 6).
Our results show that low HDL is a better indicator of
CAD than high FERHDL, as is indicated by a better model
fit for HDL than FERHDL (22 log likelihood: 21.31 vs.
16.35, Table 6) and as is apparent in Figure 1. This may be
attributed to other functions of HDL particles, e.g., the
antioxidant properties of HDL (which inhibits the oxidation of LDL particles) (42) and the ability of HDL particles
to inhibit the expression of adhesion molecules on the
surface of endothelial cells (43).
Limitations. In this angiographic case-control study, we
demonstrated that FERHDL is independently associated
with CAD and this association was modified by HDL-C
levels. However, whether or not FERHDL plays a causal role
is unclear and cannot be determined from a case-control
study. In this case-control study, cases were not matched
with controls with regard to the number of patients or
gender (Table 1). Although when the cost of sampling for
cases and controls is equal and the relation between disease
and exposure is not known beforehand, matching 1 case
with 1 control can minimize the variance of the estimated
odds ratio (44). We did not obtain a suitable number of
controls due to limited funds. Differences in the gender ratio
between cases and controls may have caused biased estimates of the odds ratio, since HDL-C levels were different
(p , 0.05) between males and females among the controls
(43.2 6 2.0 mg/dL vs. 51.1 6 2.6 mg/dL) and in all of the
patients together (38.4 6 0.90 mg/dL vs. 44.5 6
1.5 mg/dL) after controlling for age. Although we tried to
avoid this possible bias by adjusting for gender in the logistic
regression analysis and found no gender interaction (group*
gender: F value 5 1.86, p 5 0.17; HDL-C* gender: x2 5
0.01, p 5 0.99), the variance of the estimated odds ratio
may not be as low as that in a matched study.
We selected angiographically defined normal subjects as
JACC Vol. 33, No. 2, 1999
February 1999:436–43
controls. However, a selection bias is known to exist: angiographically defined normal subjects generally have more risk
factors for coronary disease than patients with clinical symptoms but who have not been selected for angiography, because
a person with both a chest pain and a known risk factor, such
as smoking, may be more likely to be referred for angiography
than a person with just a clinical symptom (45). The lack of
significance for the fairly substantial difference in the prevalence of diabetes between cases and controls and the lack of an
association between FERHDL and the severity of coronary
disease as judged by the number of involved vessels (Table 2)
in the present study may be due to this bias. Previous
angiographic studies such as the Coronary Artery Surgery
Study have also failed to show associations between classic lipid
risk factors and the severity of disease, presumably due to the
biases involved in selecting patients for angiography. The
controls were defined as having less than 25% luminal narrowing by conventional coronary angiography. However, since
even mild coronary atherosclerotic lesions in the 25% range can
result in significant acute events when based on unstable plaque
(46), our controls are not absolute controls in the sense of
having no significant coronary atherosclerosis or its eventual
sequela. Thus, limitations regarding the controls may have
limited the power of this study.
Conclusions. FERHDL, a quantitative measure of the
HDL function, when combined with serum HDL-C levels,
is a new epidemiological marker for the risk of CAD that is
superior to HDL-C levels alone. Since FERHDL values are
fairly constant, this test may be of great value in clinical
screening. The clinical significance of this finding needs to
be demonstrated in a prospective trial.
Reprint requests and correspondence: Keijiro Saku, Department of Internal Medicine, Fukuoka University School of Medicine, 7-45-1 Nanakuma Jonan-ku, Fukuoka 814-80, Japan. Email: [email protected].
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