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The puzzle of high-density lipoprotein in cardiovascular prevention

El-Harchaoui, A.

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Citation for published version (APA):El-Harchaoui, A. (2009). The puzzle of high-density lipoprotein in cardiovascular prevention.

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part I | chapter 3high density lipoprotein size and particle concentration and

coronary risk

A Karim El Harchaoui, Benoit J Arsenault, Remco Franssen, Jean-Pierre Després,

G Kees Hovingh, Erik SG Stroes, James D Otvos, Nicholas J Wareham, John JP Kastelein,

Kay-Tee Khaw, S Matthijs Boekholdt.

Annals of Internal Medicine 2009;150:84-93

Chap

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32

absTracT

background: High-density lipoprotein (HDL) cholesterol levels are inversely related to risk of

coronary artery disease (CAD). Since HDL particles are heterogeneous in size and composition,

they may be differentially associated with other cardiovascular risk factors and cardiovascular

risk per se.

objective: To study the independent relationships of HDL size and HDL particle concentration

to risk of future CAD.

design: Nested case-control study within the EPIC-Norfolk cohort. Baseline survey between

1993 and 1997, follow-up until November 2003.

setting: Norfolk, United Kingdom.

participants: Cases were 822 apparently healthy men and women who developed CAD during

follow-up. Controls were 1401 participants who remained free of CAD, and were matched to

cases by sex, age and enrollment time.

measurements: First CAD events leading to either hospitalization or death.

results: Nuclear magnetic resonance spectroscopy-measured HDL particle concentration

(33.9±5 vs 32.9±6 µmol/l; p<0.001) and HDL size (8.9±0.5 vs 8.8±0.5 nm; p < 0.001) as well

as gradient gel electrophoresis-measured HDL size (88.6±4.2 vs 88.1±4.3 nm; p=0.005) were

lower in cases compared to controls. HDL size and HDL particle concentration were only weakly

correlated (for nuclear magnetic resonance spectroscopy-measured r= 0.08, for gradient gel

electrophoresis-measured r=0.10). HDL size was strongly associated with risk factors character-

istic of the metabolic syndrome, including waist-to-hip ratio, triglycerides, and apolipoprotein

B, whereas HDL particle concentration was not. HDL size and HDL particle concentration were

independently associated with CAD risk. The association between HDL size and CAD risk was

abolished upon adjustment for apolipoprotein B and triglycerides (adjusted odds ratio,1.00

[95% CI 0.71 to 1.39] for top versus bottom quartile), whereas HDL particle concentration

remained independently associated with CAD risk (adjusted odds ratio, 0.50 [95% CI 0.37 to

0.66]).

limitations: Measurements were performed in non-fasting blood samples and residual con-

founding cannot be excluded.

conclusions: HDL size and HDL particle concentration were independently associated with

other cardiovascular risk factors and with the risk of developing CAD. The relationship between

HDL size and CAD risk was completely explained by markers associated with the metabolic

syndrome, indicating that part of the relationship between HDL cholesterol and CAD risk is

merely a reflection of this constellation of metabolic risk.

HDL particle number and size and coronary risk 33

inTroducTion

The strong inverse relationship between high-density lipoprotein (HDL) cholesterol levels

and risk of coronary artery disease (CAD) is well established (1). However, at any given level

of HDL cholesterol, HDL particle concentration and HDL size distribution (see glossary table

for definitions) may differ substantially between individuals. The atheroprotective role of HDL

cholesterol is believed to be mediated mainly by its role in reverse cholesterol transport, the

biological pathway that facilitates removal of cholesterol from macrophages in the arterial wall

back to the liver (2). Substantial evidence suggests that HDL particles are heterogeneous in

their efficacy to facilitate ATP-binding cassette A1 (ABCA1)-mediated cholesterol efflux from

macrophages and scavenger receptor class BI (SR-BI)-mediated hepatic uptake of cholesterol

from HDL particles. In particular, this heterogeneity has been associated with HDL particle size

(3-5). In addition to its role in reverse cholesterol transport, accumulating evidence suggests

that HDL particles have anti-coagulant, anti-oxidative and anti-inflammatory properties which

contribute to the anti-atherogenic capacity of HDL. In fact, HDL lipoproteins were recently

shown to carry a wide array of proteins that mediate these properties (6). The binding affinity of

these proteins to the surface of HDL lipoproteins may depend on their size (7;8). Under certain

conditions, HDL lipoproteins may lose their anti-atherogenic capacity and become dysfunc-

tional (9;10). As a consequence, various HDL lipoprotein subpopulations may differ substan-

tially in their capacity to play an atheroprotective role. Despite this functional heterogeneity,

HDL has thus far been regarded as a single entity in epidemiological studies and the amount of

cholesterol transported by HDL particles is traditionally being used for this purpose. However,

we have recently shown that higher HDL cholesterol levels may not necessarily be associated

with lower cardiovascular risk (11) and the HDL cholesterol raising drug torcetrapib has recently

been shown to have detrimental effects (12). These findings emphasize that a broader perspec-

tive on HDL metabolism is warranted.

We hypothesized that HDL particle concentration and HDL size distribution are differen-

tially associated with cardiovascular risk factors and with cardiovascular risk. We tested these

hypotheses in a case-control study nested in the EPIC-Norfolk cohort.

meThods

We performed a nested case-control study among participants of the European Prospective

Investigation into Cancer and Nutrition (EPIC)-Norfolk study, a prospective population study of

25,663 men and women aged between 45 and 79 years, resident in Norfolk, United Kingdom,

who completed a baseline questionnaire survey and attended a clinic visit (Figure). Participants

were recruited from age-sex registers of general practices in Norfolk as part of the ten-country

collaborative EPIC study designed to investigate dietary and other determinants of cancer.

Chap

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34

Additional data were obtained in EPIC-Norfolk to enable the assessment of determinants of

other diseases.

The design and methods of the study have been described in detail (13). In short, eligible

participants were recruited by mail. At the baseline survey between 1993 and 1997, partici-

pants completed a detailed health and lifestyle questionnaire. Non-fasting blood samples

were obtained by venipuncture into plain and citrate bottles. Blood samples were processed

for assay at the Department of Clinical Biochemistry, University of Cambridge, or stored at

–80˚C. All individuals have been flagged for death certification at the United Kingdom Office of

National Statistics, with vital status ascertained for the entire cohort. In addition, participants

admitted to hospital were identified using their unique National Health Service number by data

linkage with the East Norfolk Health Authority (ENCORE) database, which identifies all hospital

contacts throughout England and Wales for Norfolk residents. CAD was defined as codes 410-

414 according to the International Classification of Diseases 9th revision. Participants were

identified as having CAD during follow-up if they had a hospital admission and/or died with

CAD as underlying cause. Previous validation studies in our cohort indicate high specificity of

such case ascertainment (14). We report results with follow-up up to November 2003, an aver-

age of about 6 years. The study was approved by the Norwich District Health Authority Ethics

Committee and all participants gave signed informed consent.

Glossary

Adenosine triphospate-binding cassette A1 (ABCA1)

A cell membrane transporter that facilitates the delivery of cholesterol from cells to lipid-poor apolipoprotein A-I in the extracellular space

High density lipoproteins (HDL) The smallest (7.0 nm and 13 nm) and most dense (between 1.036 g/mL and 1.25 g/mL) of the plasma lipoproteins. They are heterogenous, comprising several subpopulations that vary in shape, size, composition and surface charge

HDL cholesterol The cholesterol content carried in all HDL particles

HDL particle concentration Number of HDL particles per plasma volume, expressed in µmol/L.

HDL size The mass-weighted average diameter of the HDL particles in a particular plasma sample

Large HDLs HDL particles with diameter between 7.3-8.2 nm

Medium HDLs HDL particles with diameter between 8.2-8.8 nm

Myeloperoxidase Leukocyte-derived enzyme with a role in the immune system.

Paraoxonase-1 Enzyme located on the surface of HDL is believed to protect against the oxidation of low-density lipoprotein (LDL) and therefore to affect the risk of coronary artery disease

Small HDL HDL particles with diameter between 8.8-13 nm

Scavenger receptor class B1 (SR-BI) HDL receptor, mainly present in the liver, that promotes the selectively uptake of HDL cholesterol


participants

We have previously described other analyses within this prospective nested case-control study

(14;15). Briefly, we excluded all individuals who reported a history of heart attack/stroke or

use of lipid lowering drugs at the baseline clinic visit. Cases were individuals who developed

fatal or non-fatal CAD during follow-up until November 2003. Controls were study participants

who remained free of any cardiovascular disease during follow-up. We matched two controls

Cases(n=1138)

Controls (n=2237)

EPIC-Norfolk Prospective Population Study (n=25.663)

Nested case-control study (n=3375)

Controls (n=1807)

Excluded (total n =270)* •no LDL cholesterol (n=75) •no HDL cholesterol (n=77) •no Triglycerides (n=15) •no blood pressure (n=2) •no ApoA1 level (n=161) •no ApoB level (n=109) •no CRP level (n=30) •no NMR data (n=10) •no Waist hip ratio (n=2)

Excluded (total n =430)* •no LDL cholesterol (n=112) •no HDL cholesterol (n=111) •no Triglycerides (n=31) •no blood pressure (n=5) •no ApoA1 level (n=289) •no ApoB level (n=171) •no CRP level (n=27) •no NMR data (n=7) •no Waist hip ratio (n=2)

Cases(n=868)

Excluded for lack of matching control participants (n=46)

Excluded for lack of matching case patients (n=406)

Analyzed Analyzed(n=822) (n=1401)

figure. Study flow diagramApoAI = apolipoprotein A-I; ApoB = apolipopotein B; CRP = C-reactive protein; EPIC = European Prospective Investigation into Cancer and Nutrition; HDL = high-density lipoprotein; LDL = low-density lipoprotein; NMR = nuclear magnetic resonance. *Individual case patients and control participants may have several missing variables.

Chap

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36

to each case by age (within 5 years), sex and time of enrolment (within 3 months). The current

analysis was performed on all participants who had a complete dataset available for baseline

characteristics, apolipoproteins A-I and B, gradient gel electrophoresis-measured HDL size and

lipoprotein nuclear magnetic resonance (NMR) spectroscopy.

We selected 1138 people who were apparently healthy at baseline but did develop CAD

during follow-up. We aimed to select 2 controls for every case who were healthy at baseline

and remained free of cardiovascular disease during follow-up. From the original dataset of 1138

cases and 2237 controls, 270 cases and 430 control patients were excluded because at least

one value was missing for any of the parameters mentioned above. A total of 46 cases were

excluded because they had no matching control patients, as were 406 control patients because

they had no matching cases. The analyses are therefore based on a dataset containing 822

cases and 1401 control patients (243 cases with 1 matching control patient, 579 cases were

matched with 2 control patients).

measurements

Data on smoking and alcohol consumption were obtained by health questionnaires at the

baseline clinic visit. Physical activity was obtained with a Physical Activity questionnaire (EPAQ2)

(13). In the original cohort smoking was classified into current cigarette smokers, former smok-

ers and never smokers. In the present study smoking was recoded into yes/no. Physical activity

was classified in four categories: inactive, moderately active, moderately inactive and active.

Use of large amounts of alcohol was defined as more than 21 units of alcohol/week. Diabetes

mellitus and use of hormone replacement therapy was self-reported. Participants were asked

about medical history with the question “Has a doctor ever told you that you have any of the

following?”, followed by a number of choices including diabetes. Blood pressure was recorded

by taking two measurements of diastolic and systolic blood pressure using the Accutor Sphyg-

momanometer (Datascope, UK), after 3 min of resting.

Serum levels of total cholesterol, HDL cholesterol and triglycerides were measured on fresh

samples with the RA 1000 (Bayer Diagnostics, Basingstoke, United Kingdom). Low-density lipo-

protein (LDL) cholesterol levels were calculated with the Friedewald formula to closely approach

current clinical procedures. Plasma concentrations of C-reactive protein were measured with a

sandwich-type enzyme linked immuno sorbent assay as previously described (16). Serum levels

of apolipoprotein A-I and apolipoprotein B were measured by rate immunonephelometry

(Behring Nephelometer BNII, Marburg, Germany) with calibration traceable to the International

Federation of Clinical Chemistry primary standards (17). Serum concentration of myeloperoxi-

dase was measured by use of a commercially available enzyme-linked immuno sorbent assay

(CardioMPO Test, Prognostix, Cleveland, Ohio, USA) (15). Paraoxonase-1 activity was measured

as previously described (18). HDL size was measured by 4-30% nondenaturing polyacrylamide

gradient gel electrophoresis (GGE) as previously described (19). Lipoprotein subclass particle

concentrations and average size of particles were also measured by proton NMR spectroscopy


(LipoScience, Inc., NC, USA) as previously described (20). In brief, particle concentrations of

lipoprotein subclasses of different size were obtained directly from the measured amplitudes

of their spectroscopically distinct lipid methyl group NMR signals. HDL particle concentrations

are expressed in µmoles of particles per liter (µmol/L). Summation of the HDL subclass levels

provides total HDL particle concentration. The following 5 HDL subclasses were defined: H5

(10-13 nm, mean 11.5 nm), H4 (8.8-10 nm, mean 9.4 nm), H3 (8.2-8.8 nm, mean 8.5 nm), H2

(7.8-8.2 nm, mean 8.0 nm), H1 (7.3-7.7 nm, mean 7.5 nm). These diameter range estimates were

based on size measurements of the isolated HDL subclass reference standards by GGE. The HDL

subclasses H5, H4, H3, H2 and H1 are closely related to the GGE subclass designations 2b, 2a,

3a, 3b and 3c, respectively (21). To simplify data analysis and interpretation, we grouped these

HDL subclasses as follows: Small HDL (H1 + H2), Medium HDL (H3), Large HDL (H4 + H5).(22)

NMR-measured HDL size was calculated as the mass-weighted average diameter of the HDL

particles in a particular plasma sample. The average HDL particle size is computed as the sum

of the diameter of each subclass multiplied by its relative mass percentage as estimated from

the amplitude of its measured NMR signal. It has been shown that the reproducibility of NMR

lipoprotein profile assessments is very good, storing at -70°C and thawing has no significant

influence on the quality of the HDL associated measurements (20). Samples were analyzed in

random order to avoid systemic bias. Researchers and laboratory personnel were blinded to

identifiable information, and could identify samples by number only.

statistical analysis

The hypotheses of the present study were generated prior to data analysis. Baseline character-

istics were compared between cases and controls taking into account the matching. A mixed

effect model was used for continuous variables and conditional logistic regression was used for

categorical variables. Pearson’s correlation coefficients and corresponding p-values were cal-

culated to assess associations between HDL-related parameters, metabolic and inflammatory

covariates. Mean values of risk factors were calculated per quartile of HDL particle concentration

and HDL size. Differences between categories were calculated by analysis of variance (ANOVA).

In order to assess the strength of the associations between HDL particle concentration or HDL

size and the risk of future CAD, we calculated odds ratios and corresponding 95% confidence

intervals (95% CI) using conditional logistic regression, taking into account matching for sex,

age and enrollment time and additionally adjusting for smoking. Regression analyses were also

performed with additional adjustment for the inflammatory covariates myeloperoxidase, para-

oxonase and C-reactive protein levels and for the metabolic covariates apolipoprotein B, and

log-transformed triglycerides. The first quartile was used as reference group. P-values represent

significance for linearity across the quartiles. Statistical analyses were performed using SPSS

software (version 12.0.1, Chicago, Illinois). A P-value < 0.05 was considered to indicate statistical

significance.

Chap

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38

role of the funding source

EPIC-Norfolk is supported by program grants from the Medical Research Council United King-

dom and Cancer Research United Kingdom and with additional support from the European

Union, Stroke Association, British Heart Foundation, Department of Health, Food Standards

Agency and the Wellcome Trust. Part of the lipid and apolipoprotein measurements described

in this article were funded by an educational grant from the Future Forum. LipoScience Inc.

performed all nuclear magnetic resonance spectroscopy measurements. The funding sources

had no role in study design, conduct, collection, management, analysis, and interpretation of

the data, and preparation, review, or approval of the manuscript.

resulTs

A full dataset was available for 822 cases and 1401 controls; 579 cases were matched to two

controls and 243 cases were matched to one control only. A total of 680 cases had a hospital

admission during which 104 cases subsequently died. A total of 246 cases had a fatal event

without a hospital admission. As expected, cases were more likely than controls to be smokers

and have diabetes mellitus (Table 1). Cases had higher levels of LDL cholesterol, higher systolic

blood pressure, higher body mass index and waist hip ratio, and lower HDL cholesterol levels.

HDL particle concentration was higher in controls compared to cases (p<0.001). Concentra-

tions of medium and small HDL particles did not differ between cases and controls. However,

controls had an increased number of large HDL particles, suggesting that the reduction in HDL

cholesterol levels observed in cases is attributable to a lower number of large HDL particles.

Consistently, both NMR-measured and GGE-measured HDL size were lower in cases than con-

trols. Controls were more physically active than cases. Use of hormone replacement therapy

and intake of large amounts of alcohol (defined as more than 21 units of alcohol/week) did not

differ between cases and controls.

relationships between hdl size, hdl particle concentration and cardiovascular risk factors

NMR-measured HDL size and GGE-measured HDL size were strongly correlated (r= 0.78,

p<0.001) (Appendix Table 1). In contrast, NMR-measured HDL size and GGE-measured HDL size

were only weakly correlated with HDL particle concentration (r=0.08 and r=0.10, respectively).

Both NMR-measured HDL size and GGE-measured HDL size were much more strongly correlated

with large HDL particles than with medium or small HDL particles. As expected, triglycerides

were inversely correlated with HDL cholesterol (r= -0.38, p<0.001). Apolipoprotein A-I was also

strongly correlated with HDL particle concentration but this was entirely explained by a strong

correlation with large HDL, whereas the correlation with medium and small HDL particles was

weak.


Consistent with the low correlation between HDL particle concentration and HDL size, rela-

tionships with other cardiovascular risk factors differed substantially between NMR-measured

HDL size and HDL particle concentration (Table 2). As expected, HDL cholesterol levels differed

significantly between HDL particle concentration quartiles and between HDL size quartiles

(p<0.001 for each). Consistently, apolipoprotein A-I differed significantly between HDL particle

Table 1. Baseline characteristics

Controls Cases

(n = 1401) (n = 822) P Value

Men, % (n) 64 (523) 63 (878) Matched

Mean age (SD), y 65 ± 8 65 ± 8 Matched

Diabetes, % (n) 1.6 (23) 6.1 (49) < 0.001

Smoking, % (n) 8.1 (114) 16.4 (135) < 0.001

Mean waist-to-hip ratio (SD) 0.88 ± 0.08 0.90 ± 0.08 < 0.001

Hormone replacement therapy, % (n) 14.0 (73) 12.0 (36) 0.7

Alcohol intake > 21 units/week, % (n) 8.4 (117) 8.3 (68) 0.9

Systolic blood pressure (SD) mmHg 139 ± 18 144 ± 19 < 0.001

Diastolic blood pressure (SD) mmHg 84 ± 11 86 ± 12 < 0.001

Physical activity, % (n)

- inactive 31.4 (440) 43.1 (354) < 0.001

- moderately inactive 27.4 (384) 25.2 (207)

- moderately active 23.6 (331) 17.9 (147)

- active 17.6 (246) 13.9 (114)

Total cholesterol (SD) mmolL 6.2 ± 1.1 6.4 ± 1.2 < 0.001

(mg/dL) (239.4±42.5) (247.1 ±46.3)

HDL cholesterol (SD) mmol/L 1.4 ± 0.4 1.3 ± 0.4 < 0.001

(mg/dL) (54.1±15.4) (50.1±15.4)

LDL cholesterol (SD) mmol/L 4.1 ± 1.0 4.3 ± 1.1 < 0.001

(mg/dL) (158.3±38.7) (166±42.5)

Triglycerides (IQR) mmol/L 1.6 [1.1-2.2] 1.8 [1.3-2.6] < 0.001

(mg/dL) (141.6 [97.3-194.6]) (159.3 [115.0-230.1])

Apolipoprotein A-I (SD) g/L 1.62 ± 0.29 1.55 ± 0.30 < 0.001

Apolipoprotein B (SD) g/L 1.28 ± 0.29 1.38 ± 0.32 < 0.001

C-reactive protein (SD) mg/L 3.3 ± 5.5 4.6 ± 7.4 < 0.001

Myeloperoxidase (SD) pmol/L 744 ± 560 822 ± 676 < 0.001

Paraoxonase (SD) U/L 62.9 ± 46 59.9 ± 44 0.1

HDL size (SD) nm (GGE-measured) 88.6 ± 4.2 88.1 ± 4.3 0.005

HDL size (SD) nm (NMR-measured) 8.9 ± 0.5 8.8 ± 0.5 < 0.001

HDL particle concentration (SD) µmol/L 33.9 ± 5 32.9 ±6 < 0.001

Large HDL (SD) µmol/L 6.2 ± 3.6 5.2 ± 3.4 < 0.001

Medium HDL (SD) µmol/L 3.2 ± 3.0 3.2 ± 3.0 0.9

Small HDL (SD) µmol/L 24.5 ± 4.7 24.5 ± 4.9 0.8

Data are presented as mean ± SD, percentage (n), or median [interquartile range]. Values may be based on fewer observations than the indicated number of subjects. HDL indicates high-density lipoprotein; LDL indicates low-density lipoprotein; Triglyceride levels were log-transformed before analysis. Ddifferences between cases and controls were calculated taking into account the matching procedure. A mixed effect model was used for continuous variables and conditional logistic regression was used for categorical variables

Chap

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concentration quartiles and between HDL size quartiles (p<0.001 for each). Triglycerides were

inversely associated with HDL size (p<0.001), whereas they were positively associated with HDL

particle concentration. Apolipoprotein B was inversely associated with HDL-size (p<0.001) but

levels did not differ between HDL particle concentration quartiles. Waist-to-hip ratio had an

inverse relation with HDL-size (p <0.001) but was not associated with HDL particle concentra-

tion. Paraoxonase levels were associated with HDL particle concentration such that levels were

higher in higher HDL particle concentration quartiles. Paraoxonase levels were not associated

with HDL size quartiles. For analyses according to HDL size quartiles, adjustment for sex, age,

smoking and BMI resulted in loss of statistical significance for waist-to-hip ratio and apolipo-

protein B whereas for paraoxonase it resulted in statistically significant differences between

quartiles. For HDL particle concentration, adjustment resulted in the opposite effects such

that statistical significance was lost for paraoxonase whereas differences became statistically

significant for apolipoprotein B and waist-to-hip ratio. In summary, participants with low HDL

size had higher levels of metabolic syndrome features (triglycerides, apolipoprotein B, LDL size

and waist-to-hip ratio) than those with higher HDL size. Similar analyses were performed after

using GGE-measured HDL size or NMR-measured HDL size instead of NMR-measured HDL size.

These analyses showed similar results (data not shown).

Table 2. Relationships between quartiles of HDL size and HDL particle concentration and cardiovascular risk factors

HDL size quartile

HDL particle concentration quartile, by cardiovascular risk factor

1 2 3 4 P*

1 0.89 ± 0.14 1.00 ± 0.15 1.12 ± 0.17 1.55 ± 0.31 < 0.001

HDL cholesterol 2 0.97 ± 0.15 1.10 ± 0.18 1.28 ± 0.20 1.64 ± 0.34 < 0.001

(mmol/L) 3 1.10 ± 0.17 1.16 ± 0.17 1.42 ± 0.23 1.78 ± 0.31 < 0.001

4 1.22 ± 0.21 1.32 ± 0.25 1.56 ± 0.26 2.02 ± 0.38 < 0.001

P† < 0.001 < 0.001 < 0.001 < 0.001

Triglycerides 1 1.93 ± 0.71 1.78 ± 0.68 1.42 ± 0.60 1.05 ± 0.42 < 0.001

(mmol/L) 2 2.23 ± 0.74 1.95 ± 0.76 1.62 ± 0.64 1.26 ± 0.51 < 0.001

3 2.36 ± 0.85 2.11 ± 0.76 1.58 ± 0.62 1.26 ± 0.54 < 0.001

4 2.51 ± 0.89 2.39 ± 0.99 1.82 ± 0.71 1.42 ± 0.60 < 0.001

P† < 0.001 < 0.001 0.001 < 0.001

Apolipoprotein A-I 1 1.29 ± 0.19 1.34 ± 0.23 1.42 ± 0.16 1.67 ± 0.27 < 0.001

(g/L) 2 1.36 ± 0.13 1.44 ± 0.17 1.56± 0.19 1.78 ± 0.22 < 0.001

3 1.49 ± 0.17 1.54 ± 0.14 1.66 ± 0.18 1.88 ± 0.20 < 0.001

4 1.61 ± 0.17 1.69 ± 0.18 1.82 ± 0.20 2.09 ± 0.26 < 0.001

P† < 0.001 < 0.001 < 0.001 < 0.001


HDL size quartile

HDL particle concentration quartile, by cardiovascular risk factor

1 2 3 4 P*

Apolipoprotein B 1 1.47 ± 0.30 1.22 ± 0.23 1.21 ± 0.24 1.15 ± 0.29 < 0.001

(g/L) 2 1.46 ± 0.23 1.27 ± 0.30 1.29 ± 0.27 1.13 ± 0.26 < 0.001

3 1.46 ± 0.34 1.34 ± 0.27 1.26 ± 0.27 1.16 ± 0.25 < 0.001

4 1.47 ± 0.33 1.38 ± 0.26 1.25 ± 0.24 1.16 ± 0.25 < 0.001

P† 0.9 0.001 0.4 0.7

Small dense LDL 1 1362 ± 431 1020 ± 374 816 ± 360 581 ± 239 < 0.001

(nmol/L) 2 1417 ± 430 1022 ± 354 824 ± 295 599 ± 265 < 0.001

3 1377 ± 454 1132 ± 376 817 ± 287 617 ± 233 < 0.001

4 1350 ± 423 1165 ± 377 864 ± 285 698 ± 264 < 0.001

P† 0.8 0.02 0.6 0.007

C-reactive protein 1 4.60 ± 7.44 4.48 ± 5.57 3.89 ± 5.40 3.47 ± 6.45 0.006

(mg/L) 2 4.18 ± 5.53 3.51 ± 6.55 3.27 ± 4.40 2.36 ± 4.03 0.006

3 3.87 ± 6.80 3.05 ± 3.95 2.69 ± 3.99 2.73 ± 4.71 0.06

4 3.13 ± 5.50 2.98 ± 4.19 2.74 ± 3.45 2.52 ± 4.40 0.05

P† 0.4 0.4 0.5 0.9

Myeloperoxidase 1 874 ± 647 795 ± 546 898 ± 921 746 ± 530 0.5

(pmol/L) 2 805 ± 573 687 ± 630 684 ± 384 644 ± 415 0.2

3 743 ± 654 719 ± 586 702 ± 432 698 ± 597 0.9

4 564 ± 437 666 ± 444 686 ± 634 893 ± 396 0.3

P† 0.03 0.5 0.1 0.1

Paraoxonase 1 53 ± 40 48 ± 32 48 ± 32 50 ± 37 0.9

(u/L) 2 53 ± 39 58 ± 38 57 ± 38 62 ± 44 0.3

3 60 ± 44 66 ± 43 65 ± 45 76 ± 55 0.1

4 81 ± 64 64 ± 48 80 ± 55 76 ± 58 0.2

P† 0.007 0.06 0.001 0.001

Waist-to-hip ratio 1 0.93 ± 0.06 0.93 ± 0.07 0.89 ± 0.09 0.83 ± 0.07 < 0.001

2 0.93 ± 0.06 0.92 ± 0.07 0.89 ± 0.08 0.84 ± 0.08 < 0.001

3 0.92 ± 0.06 0.92 ± 0.07 0.87 ± 0.08 0.82 ± 0.08 < 0.001

4 0.91 ± 0.07 0.91 ± 0.08 0.87 ± 0.08 0.81 ± 0.08 < 0.001

P† 0.1 0.3 0.2 0.1

Data are given as mean ± SD per quartile. *P value for analysis of variance across HDL size quartiles; †P value for analysis of variance across HDL particle concentration quartiles. The riskfactors are distributed on the basis of HDL size quartiles (horizontal) and HDL particle concentration quartiles (vertical). To convert HDL cholesterol values to mg/dL, divide by 0.0259. To convert triglycerides values to mg/dL divide by 0.0113.

Chap

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hdl size, hdl particle concentration and risk of future coronary heart disease

The unadjusted odds ratio for future CAD for people in the top quartile compared to those in

the bottom quartile was 0.40 (95%CI 0.31 to 0.54, p<0.001) for HDL cholesterol, 0.60 (95%CI

0.47 to 0.79, p<0.001) for HDL particle concentration, 0.51 (95%CI 0.39 to 0.67, p<0.001) for

NMR-measured HDL size, and 0.65 (95%CI 0.50 to 0.84, p<0.001) for GGE-measured HDL size.

Table 3 shows the odds ratios for future CAD associated with increasing quartiles of HDL

particle concentration and HDL size or large HDL entered in the same regression model. HDL

particle concentration and NMR-measured HDL size were independently associated with CAD

risk, with an odds ratio for individuals in the highest quartile compared with those in the lowest

quartile of 0.60 (95%CI 0.46 to 0.79) and 0.52 (95%CI 0.40 to 0.69), respectively (p for linearity

<0.001 for both) (model 1b). Analyses with HDL particle concentration and GGE-measured HDL

size showed similar results, with respective odds ratios of 0.63 (95%CI 0.48 to 0.82) and 0.67

(95%CI 0.52 to 0.87) ( model 1). HDL particle concentration and large HDL particle concentra-

tion were also independently associated with CAD risk, with odds ratios of 0.72 (95%CI 0.55

to 0.94, p=0.002) and 0.45 (95%CI 0.33 to 0.59, p<0.001) for individuals in the highest quartile

compared with those in the lowest quartile (model 1). Adjustment for myeloperoxidase, para-

oxonase and C-reactive protein levels (model 2) did not affect the risk estimates for HDL size

or large HDL particle concentration. However, this adjustment did attenuate the risk estimate

in the top HDL particle concentration quartile substantially (for the models with HDL size) or

abolished the association completely (for the model with large HDL). By contrast, adjustment

for the “metabolic” parameters triglycerides and apolipoprotein B had limited effect on the risk

estimate for the HDL particle concentration top quartile but abolished the association for the

top quartiles of HDL size or large HDL completely (model 3). Analyses with additional adjust-

ment for alcohol intake, hormone replacement therapy and physical activity showed similar

results (Appendix Table 2).

Table 3. Odds ratios for risk for future coronary heart disease

Model Parameter Odds Ratio (95% CI), by Quartile of HDL Parameter P Value

1 2 3 4

Model 1HDL particle concentration 1.00 0.59 (0.46-0.76) 0.61 (0.47-0.79) 0.72 (0.55-0.94) 0.002

Large HDL 1.00 0.78 (0.61-1.01) 0.65 (0.50-0.85) 0.45 (0.33-0.59) < 0.001


Large HDL 1.00 0.78 (0.61-1.01) 0.66 (0.50-0.86) 0.46 (0.35-0.62) < 0.001

Model 3HDL particle concentration 1.00 0.52 (0.41-0.69) 0.50 (0.38-0.66) 0.53 (0.40-0.72) < 0.001

Large HDL 1.00 0.99 (0.76-1.30) 0.99 (0.74-1.33) 0.85 (0.60-1.20) 0.38


discussion

Traditionally, the HDL fraction is quantified by measuring its cholesterol content. However,

individuals with similar HDL cholesterol levels can differ substantially in terms of HDL size

distribution and particle concentration. In this study, we measured HDL size and HDL par-

ticle concentration among apparently healthy men and women and observed that these

parameters were differentially associated with other cardiovascular risk factors, and were

independently associated with the risk of future CAD. HDL size was strongly associated with

well-known components of the metabolic syndrome, whereas HDL particle concentration

was not. As a consequence, the relationship between HDL size and risk of future CAD risk was

virtually abolished upon adjustment for metabolic parameters. The relationship between HDL

particle concentration and CAD risk was independent of metabolic parameters. These findings

indicate that HDL is a heterogeneous lipid fraction and that various HDL subpopulations are

differentially associated with other cardiovascular risk factors and with cardiovascular risk.

Model Parameter Odds Ratio (95% CI), by Quartile of HDL Parameter P Value


NMR spectrometry-measured HDL size 1.00 0.89 (0.68-1.15) 0.76 (0.58-0.98) 0.52 (0.40-0.69) < 0.001




NMR spectrometry-measured HDL size 1.00 1.09 (0.83-1.43) 1.15 (0.86-1.53) 1.00 (0.71-1.39) 0.92


GGE-measured HDL size 1.00 0.70 (0.54-0.90) 0.68 (0.52-0.88) 0.67 (0.52-0.87) 0.003





GGE= gradient gel electrophoresis; HDL = high-density lipoprotein; NMR = nuclear magnetic resonance.Odds ratios were calculated by conditional logistic regression, taking into account matching for sex and age, and adjusting for smoking (model 1). Model 2: as model 1 with additional adjustment for myeloperoxidase, paraoxonase and C-reactive protein levels. Model 3: as model 1 with additional adjustment for apolipoprotein B, and log-transformed triglycerides. †P for linear trend.

Chap

ter 3

44

app

endi

x Ta

ble

1. P

ears

on c

orre

latio

n co

effici

ents

Risk

Fac

tor

HD

L-C

TGA

poA

-IA

poB

CRP

MPO

PON

HD

L si

ze(G

GE)

HD

L si

ze(N

MR)

HD

L-P

Larg

eH

DL

Med

ium

HD

LSm

all

HD

L

HD

L-C

1.00

TG-0

.38*

1.00

Apo

A-I

0.82

*-0

.22*

1.00

Apo

B-0

.22*

0.43

*-0

.09

1.00

CRP

-0.0

7§0.

07§

-0.0

7§0.

031.

00

MPO

-0.1

1*0.

02-0

.12*

0.00

0.26

*1.

00

PON

-10.

16*

-0.0

30.

12*

0.00

8-0

.03

-0.0

51.

00

HD

L si

ze (G

GE)

0.70

*-0

.34*

0.58

*-0

.29*

-0.0

6§-0

.05

0.07

§1.

00

HD

L si

ze (N

MR)

0.76

*-0

.47*

0.63

*-0

.38*

-0.0

9¶-0

.07§

0.07

§0.

78*

1.00

HD

L-P

0.44

*0.

17*

0.54

*0.

01-0

.07§

-0.1

2*0.

23*

0.10

*0.

08§

1.00

Larg

e H

DL

0.80

*-0

.43*

0.69

*-0

.38*

-0.1

1*-0

.07§

0.12

*0.

71*

0.88

*0.

33*

1.00

Med

ium

HD

L0.

09*

0.25

*0.

11*

-0.0

9*0.

13*

-0.0

40.

11*

-0.0

6¶-0

.11*

0.39

*-0

.12*

1.00

Smal

l HD

L-0

.17*

0.36

*0.

008

0.36

*-0

.08§

-0.0

50.

09*

-0.3

9*-0

.52*

0.62

*-0

.32*

-0.1

0*1.

00

WH

R-0

.45*

0.27

*-0

.43*

0.08

§0.

07§

0.10

*-0

.07§

-0.4

0*-0

.47*

-0.1

4*-0

.45*

-0.0

20.

20*

HD

L-C

= hi

gh d

ensi

ty li

popr

otei

n ch

oles

tero

l; TG

= tr

igly

cerid

es; A

poA

-I =

apol

ipop

rote

in A

-I; A

poB

= ap

olip

opro

tein

B; C

RP =

C-r

eact

ive

prot

ein;

PO

N =

par

aoxo

nase

; M

PO =

mye

lope

roxi

dase

; HD

L-P

= H

DL

part

icle

con

cent

ratio

n; W

HR

= w

aist

hip

ratio

. Trig

lyce

ride

leve

ls w

ere

log-

tran

sfor

med

bef

ore

anal

ysis

. * in

dica

tes

p<0.

001,

¶ in

dica

tes

p=0.

001;

§ indi

cate

s p<

0.05

; † indi

cate

s p=

0.05

.


hdl size, hdl particle concentration and cardiovascular risk

The published literature about the relationship between HDL subclasses and cardiovascular

risk is limited to cross-sectional studies that have used different methods to quantify HDL

subclasses and used different measures of cardiovascular risk (23). Several studies using GGE

to measure HDL size have reported that CAD patients tend to have more smaller HDL particles

and that large HDL particles may protect against the development of atherosclerosis (4;24;25)

whereas others have reported that only small HDL3 particle were atheroprotective (26).

Studies that used NMR to measure HDL subclasses have usually analyzed HDL subclasses

in concert, which makes the results difficult to interpret because HDL subclasses are highly

appendix Table 2. Odds ratios for risk for Coronary artery disease in patients who did not smoke, use hormone replacement therapy, or drink large amounts of alcohol

Model Parameter Odds Ratio (95% CI), by Quartile P Value

1 2 3 4

Model 1 HDL particle concentration 1.00 0.58 (0.42-0.74) 0.62 (0.45-0.85) 0.65 (0.46-0.91) 0.03

Large HDL 1.00 0.87 (0.63-1.19) 0.64 (0.46-0.90) 0.49 (0.35-0.70) < 0.001


Large HDL 1.00 0.86 (0.63-1.18) 0.64 (0.45-0.90) 0.49 (0.34-0.70) < 0.001

Model 3 HDL particle concentration 1.00 0.52 (0.38-0.72) 0.50 (0.36-0.70) 0.46 (0.31-0.66) < 0.001

Large HDL 1.00 1.12 (0.80-1.56) 1.03 (0.71-1.51) 0.97 (0.64-1.48) 0.8






NMR spectrometry-measured HDL size 1.00 1.10 (0.78-1.57) 1.06 (0.73-1.54) 1.03 (0.69-1.55) 0.9







GGE= gradient gel electrophoresis; HDL = high-density lipoprotein; NMR = nuclear magnetic resonance.Odds ratios were calculated by conditional logistic regression, taking into account matching for sex and age, and adjusting for smoking (model 1). Model 2: as model 1 with additional adjustment for myeloperoxidase, paraoxonase and C-reactive protein levels. Model 3: as model 1 with additional adjustment for apolipoprotein B, and log-transformed triglycerides. †P for linear trend.

Chap

ter 3

46

correlated. In a study among patients undergoing coronary angiography, higher levels of small

HDL particles were associated with more severe atherosclerosis(27), whereas a study among

patients with type 1 diabetes observed no relationship between HDL subclasses and carotid

intima-media thickness (28). The only study investigating a relationship with clinical events was

a substudy of the Veterans Affairs High-Density Lipoprotein Intervention Trial, which investi-

gated the effect of gemfibrozil on CAD risk (29). In this trial HDL particle concentrations were

associated with cardiovascular risk independent of traditional lipid levels. In the current study

we decided not to analyze these highly related HDL subclasses, but focused our analyses on

HDL particle concentration and either HDL size or large HDL. We observed that HDL cholesterol,

HDL size and HDL particle concentration were each individually associated with decreased risk

of CAD. When HDL size and HDL particle concentration were analyzed conjointly, both were

independently and inversely associated with cardiovascular risk. For HDL size, this relationship

was confounded by characteristics of the metabolic syndrome because adjustment for these

variables resulted in abolition of the relationship between HDL size and CAD risk.

hdl size and hdl particle concentration versus metabolic dysregulation

We observed that HDL size was significantly associated with several features of the metabolic

syndrome including high plasma levels of triglycerides, apolipoprotein B, small LDL particles

and higher waist-to-hip ratio, whereas HDL particle concentration was completely independent

of these variables. Our results are consistent with previous studies which have shown reduced

HDL size in people with diabetes mellitus (30), insulin resistance (31;32), hypertriglyceridemia

(33), and parameters associated with the metabolic syndrome (34). In other words, the low HDL

cholesterol levels observed among people with metabolic features was explained by reduced

HDL size, not by a low HDL particle concentration. In addition, we observed that both reduced

HDL size and low HDL particle concentration were associated with cardiovascular risk. How-

ever, upon correction for metabolic parameters (apolipoprotein B and triglyceride levels), HDL

size was no longer associated with risk of future CAD. In contrast, HDL particle concentration,

which is numerically unaffected in the metabolic syndrome, does retain its predictive value

after correction for metabolic parameters. These data suggest that the association between

HDL size and cardiovascular risk is confounded by metabolic dysregulation.

hdl size and hdl particle concentration versus inflammation

Whereas HDL particle concentration was only weakly correlated with metabolic parameters, it

did show an association with inflammatory and oxidative markers, including myeloperoxidase

and paraoxonase. These surface-bound enzymes may explain part of the atheroprotective

effect of HDL particles. The association between paraoxonase levels and HDL particle con-

centration is interesting and suggests that higher particle concentration may facilitate more

surface-bound molecules. This observation is not consistent with in vitro studies showing that

the difference in shape and size of several HDL subpopulations is critical for binding of these


anti-oxidative enzymes (7;8). In contrast we did not observe a strong association between HDL

size and paraoxonase levels. In fact, several lines of evidence have supported a direct anti-

inflammatory effect of apolipoprotein A-I. Infusion of minute dosages of apolipoprotein A-I had

potent anti-atherogenic effects in rabbit models,(35;36) apolipoprotein A-I or its Milano variant

had potent anti-inflammatory effects in monocytic cell lines(37;38), and the anti-inflammatory

effect of HDL was shown to be due to its apolipoproteins, not to protein-free phospholipid (39).

limitations

A number of issues have to be taken into account when interpreting the results of our study.

Measurements were performed in non-fasting blood samples which could have affected the

relation of triglycerides with the various HDL subclasses. However, HDL subclass concentra-

tions are not significantly altered by freezing or in the postprandial state (20). Diurnal variation,

variation over time, and differences in the time since the last meal could have affected our

results. However, the relation of non-fasting triglycerides with other lipids and CAD risk in the

EPIC-Norfolk cohort is not different than expected for fasting triglycerides. Moreover, in the

Western world, people live under constant postprandial conditions, and studies on the associa-

tions between lipids, lipoproteins, and CAD risk may be more physiologically relevant under

non-fasting conditions, as has recently been demonstrated (40-42). Second, CAD events were

scored through death certification and hospital admission data, which may have resulted in

under-ascertainment or misclassification. Previous validation studies in this cohort, however,

indicate high specificity of such case ascertainment (14). In addition, any misclassification

leads to underestimation of true associations and therefore does not negate our results. Third,

because only non-fasting samples were obtained in the EPIC-Norfolk cohort, we were not able

to define the metabolic syndrome, which requires the measurements of parameters in the

fasting state. As a consequence, we were not able to analyze our results in the context of the

metabolic syndrome, but only in relation to separate metabolic parameters. Finally, we adjusted

for confounding factors, but residual confounding by imperfectly measured or unmeasured

confounders cannot be excluded. However this is a common limitation of a non-randomized

study.

conclusion

In summary, we observed that HDL size and HDL particle concentration were independently

associated with other cardiovascular risk factors and with cardiovascular risk. For HDL size,

these associations appeared to be explained by features of the metabolic syndrome. By

contrast, the relationships for HDL particle concentration were not affected by adjustment

for metabolic parameters. These results suggest that part of the relationship between HDL

cholesterol levels and CAD risk is explained by metabolic parameters, and that this part can be

Chap

ter 3

48

quantified by measuring HDL size or the concentration of large HDL particles. The other part

of the association between HDL cholesterol levels and CAD risk is independent of metabolic

parameters, is weakly associated with inflammatory parameters, and can be quantified by the

concentration of HDL particles. Future research into the relationship between HDL cholesterol

and cardiovascular risk should take these aspects into account. In addition, these findings

may have implications for the development of therapeutic strategies aimed at modifying HDL

metabolism. Our results suggest that HDL-raising strategies that primarily affect HDL-size may

have different effects on cardiovascular risk than those affecting HDL particle concentration.


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UvA-DARE (Digital Academic Repository) The puzzle of high-density lipoprotein … · Chapter 3 32 absTracT background: High-density lipoprotein (HDL) cholesterol levels are inversely