IMPAIRED
REVERSE CHOLESTEROL TRANSPORT IN INDIVIDUALS OF ASIAN INDIAN DESCENT
H.
Robert Superko, Enas A. Enas, Purushotham Kotha, Naras K Bhat
Berkeley
HeartLab, Berkeley, California
Abstract:
The purpose of the National Asian Indian Heart Disease Project is to collect information, blood samples, and archive plasma in order to determine the prevalence of inherited disorders linked to CAD in the Asian Indian population.
BACKGROUND:
Individuals of Asian Indian origin
(from the subcontinent of India, Pakistan, and Bangladesh) have a higher rate
of coronary artery disease (CAD) than other ethnic groups (1). Compared to men
in the Framingham study, men from New Delhi have four times the prevalence of CAD
(2). One million Asian Indians live in
the USA, and 27,000 are physicians and approximately 2,000 are
cardiologists. The increased incidence
of CAD is also present in physicians of Asian Indian origin who have moved to
the United States (3). This proclivity
to CAD is also apparent in other “Western” countries and the United Kingdom has
reported a 4-5 fold increase in CAD mortality in Asian Indian males compared to
a National average (4). Asian Indian
women also have a 3-4 fold higher CAD mortality rate compared to other
populations (4,5). Not only is the
incidence of CAD higher in the Asian Indian population, but the severity and
prematurity appears to be worse (6). The severity of CAD in this well-defined
ethnic group strongly suggests the presence of multiple inherited disorders
contributing to the unusually high incidence of CAD in Asian Indians.
In the Asian Indian population,
total cholesterol and LDL-C do not appear to be elevated, and in fact, TC is
approximately 20-40 mg/dl lower than Caucasian groups (7). Among Asian Indians with CAD, 45% had total
cholesterol < 200 mg/dl (8).
However, HDLC has been reported to be lower, along with higher apo B,
and characteristics of the “insulin resistance syndrome” which suggests a high
incidence of the Atherogenic Lipoprotein Profile (ALP) (7,9,10).
Lp(a) values have been found to be
higher in Asian Indians with mean values of approximately 20 mg/dl (11). It is reasonable to hypothesize that the
combination of inherited disorders, and the extent to which they are expressed,
interact with environmental issues that explains a considerable portion of the
excessive CAD seen in the Asian Indian population. Plasma homocysteine levels have also been reported to be higher
in Asian Indian populations compared to other ethnic groups (11a).
LDL, like HDL, is not a homogeneous category of
lipoproteins, but consists of a set of discrete subspecies with distinct
molecular properties (13,14). In normal
subjects, four to seven major LDL subspecies, distinguished by size and
density, can be identified. LDL-I is
the largest and least dense, and, the smallest, LDL-IV, is the most dense. Analysis of LDL subspecies is made possible
by a number of techniques, including gradient gel electrophoresis, which
separates LDL particles on the basis of their differing size, and
ultracentrifugation, which separates them on the basis of their differing
density (15). Often, but not always, associated with elevations in plasma
triglycerides is the dense LDL subclass pattern, (LDL pattern B), which is a
heritable trait determined by a single major dominant gene (the alp
locus) (16,17). The gene has
been designated ATHS (for atherosclerosis susceptibility) and located on the
short arm of chromosome 19, 0.5 CM from the LDL receptor (18). Based on Hardy-Weinberg equilibrium, 30-35%
of people are heterozygous for alp and another 5% are homozygous. The dense LDL subspecies increases CAD risk
3-fold.
This trait is associated with factors that may place the
Indian community at particularly high CAD risk. These include, insulin resistance and a predilection to diabetes,
slightly elevated triglycerides, slightly low HDL, low HDL2b implying impaired
reverse cholesterol transport, lipoprotein particles susceptible to oxidative
damage, and enhance postprandial lipemia.
The
clinical importance of clarifying true CAD risk can be seen from recent
arteriographic trials. Delay in the
rate of progression and some degree of regression of coronary atherosclerosis
has been documented in several well-conducted randomized trials with the use of
lipoprotein manipulation (19-21).
Specifically, change in LDL and HDL subclass distribution has been
linked to arteriographic change independent of LDLC change (22-25). In a male,
middle aged CAD population, the mean HDL2b is 8.9% and the mean in a healthy
middle aged male population with predominantly large LDL is 20.2% (26,27).
Subjects.
224 male individuals of Asian Indian descent, living in the USA were
recruited into Phase I of the NAIHDP. Subjects were recruited through recruitment lectures and and
community meetings. Informed consent
was obtained for inclusion in the NAIHDP.
Fifteen subjects were excluded from this analysis due to a history of
CAD (9 PTCA, 6 MI), 36 were excluded for current treatment with lipid altering
medications (19 statins, 2 Niacin, 1 fibrate, 3 beta blocker, 1 alpha blocker).
Asymptomatic male subjects (n=239) from the Berkeley HeartLab data base were
used for comparison. This age-matched
comparison group was reported to have no clinical evidence of CAD and were
determined to be non-Asian Indian based on name.
Laboratory: Fasting plasma was analyzed for triglycerides, total, LDL & HDL cholesterol by enzymatic methods, 7 LDL (I, IIa, IIb, IIIa, IIIb, IVa, IVb) and 5 HDL (2b, 2a, 3a, 3b, 3c) subclasses by S3 gradient gel electrophoresis using duplicate measures and internal standards and controls calibrated to analytic ultracentrifugation at the University of California, Berkeley (X). Lp(a), Apo B and homocysteine (tHcy) were determined by immunoassay (28-30). Triglyceride (TG), total cholesterol, low density lipoprotein cholesterol (LDL cholesterol), and high density lipoprotein cholesterol (HDL cholesterol) were determined by enzymatic methods and a modified heparin-2M MnCl2 procedure to precipitate very low density lipoproteins (VLDL) and LDL (31). This assay uses a monoclonal capture antibody immunospecific to apo(a) and a peroxidase-conjugated polyclonal detection antibody with recognition of the entire Lp(a) molecule. Internal quality assurance for apolipoproteins was monitored at two levels for each analyte on an ongoing basis using specifically prepared frozen pools.
Identification and densitometric measurements of LDL species were carried out using custom made, triple segmented polyacrylamide 2/16% gradient gels as described previously (32-34). Criteria described previously were used to classify the LDL subclass pattern as either pattern A which had the predominant peak larger than 262 angstrom with skewing to the right or pattern B which had the predominant peak less than 255 angstrom with skewing to the left (35). Peaks can be symmetric, broad, or multimodal and result in an intermediate LDL subclass pattern. For purposes of analysis, subjects classified as the intermediate pattern were included in the LDL pattern B group. Percent LDL distribution in 7 regions (I, IIa, IIb, IIIa, IIIb, IVa, IVb) was determined and assess the relative distribution of LDL particles in the large LDL I, Iia, and Iib regions, and the small IIIa, IIIb, IVa, and IVb regions. Region IIIa + IIIb correlates with the atherogenic region (Sf3-5) on analytic ultracentrifugation (r=0.77, p=0.0001).
HDL subclass distribution was determined by gradient gel electrophoresis of HDL is performed as previously described (35). Electrophoretic bands representing the HDL subspecies HDL-2b, HDL-2a, HDL-3a, HDL-3b, and HDL-3c are identified and densitometrically scanned using a computer assisted scanning procedure developed at the Donner Laboratory, University of California, Berkeley. The HDL bands are identified in the d<1.21 g/ml ultracentrifugal fraction by staining with Coomassie Blue, as described previously. In a typical male CAD population, the mean HDL2b is xx % (36).
Statistics. Chi-square analysis was used to test for
differences between groups. Analysis of variance (ANOVA), with a factorial
design, was used to test for significance between lipid value groups. The level
of significance was set at p < 0.05 (two-tailed). The STATVIEW (v.4.1)
statistical package was used for all analysis.
The relation of HDL2b to HDLC values above the male population mean (45
mg/dl) was based on the Lipid Research Clinics Population studies book (36a).
No significant differences were found
between groups in standard measures of age, plasma triglycerides, total
cholesterol, LDL-C or HDL-C (Table 1). However, despite no significant
difference in HDL-C the HDL2b was significantly lower (p = 0.0002) in the Asian
Indian group. Mean Apolipoprotein B
values were significantly lower in the Asian Indian group while mean Lp(a) and
tHcy values were significantly higher
(p<0.002). LDL subclass distribution
analysis revealed no significant difference in LDL peak particle diameter
between groups but a significant difference in LDL subclass distribution was
noted with a greater distribution of small LDLs in the non-Asian Indian
population (p<0.01). This was
significant in the LDL IIIb (p<0.003), IVa (p<0.003), and IVb (p<0.01)
regions.
Because the Adult Treatment
Panel-III recommends HDLC < 40 mg/dl be considered a CAD risk factor,
subjects in both populations with HDLC > 40 mg/dl were examined to determine
if reduced HDL2b was still present despite what would be considered acceptable
HDLC vales for a middle aged male population (Table 2) (37). Despite HDLC values in excess of 40 mg/dl,
the Asian Indian population continued to exhibit significantly lower HDL2b
levels compared to non-Asian Indian subjects.
This is of additional interest since there were no significant
differences in standard lipid measurements, apolipoprotein B, insulin, or LDL
subclass distribution that would suggest the atherogenic lipoprotein profile as
the cause of the reduced HDL2b.
The percent of each population with
levels of triglycerides above 200 mg/dl, LDLC above 130 and 160 mg/dl, Lp(a)
greater than 20 mg/dl, total homocysteine greater than 14 umol/L, and HDL2b
< 10% and < 20% were examined to determine the prevalence of these
factors that may contribute to increased CAD risk (Table 3). There was no significant difference between
groups in the percent of subjects with elevated LDLC, small LDL IIIa+b, and
total homocysteine. However,
significantly more Asian Indian subjects had HDL2b distribution at levels
considered to be low (p<0.0001).
75.7% of non-Asian Indian subjects had HDL2b < 20% compared to 91.8%
of the Asian Indian subjects. The
percent of Asian Indian subjects with Lp(a) > 20 mg/dl (44.3%) was also
significantly higher (p<0.001) than the non-Asian Indian group (25.5%) as
were homocysteine (p=0.05) values > 14 umol/L, 7.7% and 3.1% respectively.
DISCUSSION:
In this investigation we report for the first time that Men of Asian Indian descent have significantly lower HDL2b compared to age matched men not of Asian Indian descent. This finding persists, and may be most clinically relevent, in Asian Indian men with HDLC in what is considered an acceptable range in regard to CAD risk stratification. The low HDL2b along with two other metabolic disorders that are at a significantly increased incidence in this Asian Indian population may contribute to the higher CAD risk noted in this ethnic group. Two of the disorders, elevated Lp(a), and elevated total homocysteine have been reported previously (11,11a).
The
low HDL2b in this ethnic population is particularly of interest since it was
not associated with a greater distribution of LDL particles in the small LDL
region. Previous studies, not conducted
in Asian Indians, have reported a significant inverse relationship of small LDL
and HDL2b (38). In fact, the Asian
Indian population had significantly less LDL distribution in the small LDL
IIIb, IVa, and IVb (p < 0.01), lower apoplipoprotein B (p < 0.005) and
significantly more large LDL I (p <
0.03) compared to the non-Asian Indian population. This suggests that low HDL2b in this population may be present
due to factors not associated with the Atherogenic Lipoprotein Profile. Low HDL2b is often associated with low
HDL-C. For this reason, the presence of
disorders in subjects with HDLC > 40 mg/dl was explored (Table 2). In this group of subjects who would be felt
to be a no increased CAD risk due to low HDLC values, significantly lower HDL2b
mean values (p=0.0001) persisted in the Asian Indian population. No difference in LDL subclass distribution
was seen in this patient subset while the Lp(a) remained significantly higher
in the Asian Indian group.
The
relative magnitude of the population at risk due to these disorders in the two
different groups is reflected by the percent with values generally associated
with increased CAD risk (Table 3).
Elevations in LDLC (> 130 or 160 mg/dl), and distribution of small
LDL in IIIa + IIIb > 20% were not found to be different between groups. The presence of elevated triglycerides (>
200 mg/dl) and low HDLC (< 40 mg/dl) were significantly more common in the
non-Asian Indian group compared to the Asian Indian group. Despite the lower incidence of low HDLC,
lower incidence of elevated triglycerides, and no difference in the distribution
of small LDL particles, there was a significantly higher incidence of low HDL2b
(p=0.0001) in the Asian Indian group (91.8%) compared to the non-Asian Indian
group (75.7%). Lp(a) values greater than 20 mg/dl were 44.3% in the Asian
Indian group and 25.5% in the non-Asian Indian group (p < 0.001). Elevations in plasma homocysteine :> 14
umol/L were significantly more common in the Asian Indian group (7.7% versus
3.1%) p=0.05.
Low
HDLC is established as a cardiovascular risk factor (37). Likewise, low HDL2b has been significantly
associated with arteriographically determined CAD severity and progression and
this relationship is most powerful in patients with “normal” triglyceride
values (25). This is relevant for this
study since while the mean fasting triglyceride value in the total male Asian
Indian population was 153+132 mg/dl, it was 115+54 mg/dl in the
men with HDLC > 40 mg/dl in whom low HDL2b was common in the Asian Indian
group. This may be clinically relevant
since an increase in HDLC, induced with a medication reported to increase
HDL2b, has been associated with a reduction in clinical CAD events
(39,40).
The
combination of low HDLC and elevated Lp(a) identifies a particularly high risk
group in the REGRESS study (41). In
this investigation, while Lp(a) predicted only 2.6% of arteriographic change,
the combination of low HDLC and elevated Lp(a) predited 37% of the
arteriographic change. This argues for
the potential role of multiple metabolic disorders in the Asian Indian
population contributing to the significant elevation in CAD risk in this ethnic
group. Low HDL2b may be an important
contributor since we found 91.8% of
Asian Indian middle aged men to have HDL2b less than the mean in a healthy
non-Asian Indian population. 42.1% of
the Asian Indian men had both HDL2b < 20% and Lp(a) > 20 mg/dl compared
to 18.8% of the non-Asian Indian men (p<0.0001). This combination of both elevated Lp(a) and low HDL2b may
identify a particularly high risk group in this ethnic population. Since many of these disorders have a genetic
component, cultural aspects of marriage may impact CAD risk in the Asian Indian
community.
N 173 239
Age (years) 49.0+11.6 49.2+10.1 0.89
TG (mg/dl) 153+132 167+121 0.21
TC (mg/dl) 197+37 204+53 0.13
LDLC (mg/dl) 123+35 127+40 0.30
HDLC (mg/dl) 44.0+9.9 42.5+12.6 0.19
HDL2b 11.6+5.0 14.3+8.3 0.0002
Apo B 88.6+19.9 96.4+25.2 0.005
Lp(a) (mg/dl) 22.2+17.2 15.3+14.0 0.001
THcy (umol/L) 9.9+2.9 8.9+3.1 0.002
Insulin (uU/l) 11.1+10.6 9.3+4.7 0.41
LDL size pk#1 256.8+9.9 257.4+8.9 0.51
LDL I % 21.9+20.0 19.0+5.3 0.03
LDL IIa % 19.2+6.5 17.2+6.2 0.09
LDL IIb % 23.9+7.0 22.7+6.2 0.09
LDL IIIa % 19.7+8.2 20.9+7.9 0.56
LDL IIIb % 5.9+4.2 7.3+4.8 0.003
LDL IVa % 5.6+3.1 6.6+3.7 0.003
LDL IVb % 5.2+4.0 6.2+3.8 0.01
Table
1. Mean (+SD) demographics,
standard lipid values, Lp(a) and tHcy in the two groups.
N 65 83
Age (years) 51.1+11.9 50.3+9.2 0.67
TG (mg/dl) 115+54 110+72 0.66
TC (mg/dl) 208+36 205+47 0.68
LDLC (mg/dl) 130+35 126+41 0.58
HDLC (mg/dl) 54.6+6.3 56.2+9.5 0.26
HDL2b 15.1+5.7 19.9+7.5 0.0001
Apo B 87+20 88+27 0.91
Insulin 8.6+5.8 7.5+2.2 0.64
Lp(a) (mg/dl) 27.8+20.8 15.6+13.6 0.0001
THcy (umol/L) 9.5+2.4 9.5+4.5 0.98
LDL size pk#1 261.1+10.9 263.8+7.0 0.08
LDL I % 23.6+8.1 22.3+5.8 0.25
LDL IIa % 21.6+6.3 21.7+5.6 0.92
LDL IIb % 23.5+6.4 24.5+5.5 0.28
LDL IIIa % 16.4+7.1 15.7+6.1 0.48
LDL IIIb % 4.8+1.9 4.9+2.4 0.75
LDL IVa % 5.4+2.2 5.7+2.5 0.46
LDL IVb % 4.6+2.5 5.1+2.6 0.20
Table
2. HDLC > 40 mg/dl
Triglycerides > 200 mg/dl 16.2% 29.7% 0.002
LDLC > 160 (mg/dl) 15.0% 17.7% 0.46
LDLC > 130 (mg/dl) 39.5% 42.6% 0.53
LDL IIIa+b > 20% 61.8% 67.1% 0.27
HDLC < 40 mg/dl 36.9% 49.1% 0.02
HDL2b<20% 91.8% 75.7% 0.0001
Lp(a) > 20 (mg/dl) 44.3% 25.5% 0.0001
THcy > 14 (umol/L) 7.7% 3.1% 0.05
Table 3.
Percent of subjects in each
group with values below or above cut points that reflect increased CAD risk
(Chi-square).
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