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1 LDL Particle Size Distribution Is Associated With Carotid Intima-Media Thickness in Healthy 50-Year-Old Men Camilla Skoglund-Andersson, Rong Tang, M. Gene Bond, Ulf de Faire, Anders Hamsten and Fredrik Karpe Arterioscler Thromb Vasc Biol. 1999;19: doi: /01.ATV Arteriosclerosis, Thrombosis, and Vascular Biology is published by the American Heart Association, 7272 Greenville Avenue, Dallas, TX Copyright 1999 American Heart Association, Inc. All rights reserved. Print ISSN: Online ISSN: The online version of this article, along with updated information and services, is located on the World Wide Web at: Permissions: Requests for permissions to reproduce figures, tables, or portions of articles originally published in Arteriosclerosis, Thrombosis, and Vascular Biology can be obtained via RightsLink, a service of the Copyright Clearance Center, not the Editorial Office. Once the online version of the published article for which permission is being requested is located, click Request Permissions in the middle column of the Web page under Services. Further information about this process is available in the Permissions and Rights Question and Answer document. Reprints: Information about reprints can be found online at: Subscriptions: Information about subscribing to Arteriosclerosis, Thrombosis, and Vascular Biology is online at: Downloaded from by guest on February 21, 2013

2 LDL Particle Size Distribution Is Associated With Carotid Intima-Media Thickness in Healthy 50-Year-Old Men Camilla Skoglund-Andersson, Rong Tang, M. Gene Bond, Ulf de Faire, Anders Hamsten, Fredrik Karpe Abstract Results of cross-sectional and prospective studies have suggested that small, dense low-density lipoprotein (LDL) particles predispose to coronary heart disease. We investigated the relationships between plasma concentrations of LDL subfractions and intima-media thickness (IMT) of the common carotid artery (CCA), quantified by B-mode ultrasound, in 94 healthy, 50-year-old men, all of whom were homozygous for the apolipoprotein E3 allele. A novel 3% to 7.5% polyacrylamide gradient gel was developed to provide separation of LDL subfractions with high resolution, as was a procedure to quantify plasma concentrations of these LDL subspecies. The LDL particle size distribution pattern obtained by the gradient gel electrophoresis procedure was in good agreement with the one obtained by a well-established, single-spin density gradient ultracentrifugation technique. LDL-II (particle size, 23.5 to 25.0 nm) was the most abundant subfraction, and its plasma concentration correlated closely with the total LDL cholesterol concentration (r 0.61, P 0.001) but not with CCA IMT (r 0.13, NS). In contrast, the plasma concentration of the predominant small, dense LDL particle subfraction (LDL-III; particle size, 22.5 to 23.5 nm) correlated strongly with CCA IMT (r 0.42, P 0.001). In multivariate analysis, the plasma concentration of the LDL-III subfraction contributed significantly to the variation in CCA IMT (R ). When plasma triglycerides and LDL cholesterol were forced into the multivariate model, 10% of the variation in CCA IMT was still accounted for by the LDL-III subfraction. In summary, use of a novel and sensitive gradient gel electrophoresis method for evaluation of LDL heterogeneity provided the basis for demonstrating an independent relation between the plasma concentration of small LDL and IMT of the CCA in healthy, middle-aged men. (Arterioscler Thromb Vasc Biol. 1999;19: ) Key Words: intima-media thickness atherosclerosis low-density lipoprotein subfractions gradient gel electrophoresis apolipoprotein B The LDL fractions have been shown to be heterogeneous with regard to several structural and functional properties, such as size, 1 3 density, 3,4 electric charge, 5 7 and lipid and apolipoprotein composition. 5,8,9 Regarding potentially atherogenic properties, small, dense LDL species have been shown to be less resistant to oxidative modification and to bind more tightly to arterial wall proteoglycans 13,14 than larger and lighter LDL species. Both of these properties suggest an increased atherogenicity of small, dense LDL particles. Furthermore, small, dense LDL particles bind less efficiently to the LDL receptor 15,16 and hence have a prolonged residence time in plasma, giving them an extended opportunity to infiltrate the arterial wall, undergo oxidative modification, and exert atherogenic effects. Cross-sectional studies have consistently demonstrated the importance of LDL heterogeneity in coronary heart disease (CHD) In addition, results of recent prospective studies indicate that a small LDL particle size predicts CHD However, the association between LDL particle size and CHD risk has been independent of the plasma triglyceride level only when a quantitative variable has been used to describe LDL particle size distribution. 22,24,25 A number of methods have been developed to characterize LDL heterogeneity. Density gradient ultracentrifugation (DGUC) of plasma 26,27 or isolated LDL 1,8,28 has been commonly used to separate the LDL particle spectrum according to density. An advantage of the ultracentrifugation techniques is the possibility for compositional studies of LDL subfractions. Nondenaturing polyacrylamide gradient gel electrophoresis (GGE), on the other hand, separates LDL according to particle size, is fairly easy to perform, and has been extensively used in clinical studies. GGE has been reported to enable separation of as many as 7 discrete subclasses within the LDL population. 29 However, the qualitative nature of the GGE method is a disadvantage in its current application, because a protocol for quantification of LDL subspecies has Received Received October 23, 1998; revision accepted February 17, From the Atherosclerosis Research Unit, King Gustaf V Research Institute (C.S.-A., A.H., F.K.), and the Department of Emergency and Cardiovascular Medicine (U.d.F., A.H., F.K.), Karolinska Hospital, and Institute of Environmental Medicine, Division of Cardiovascular Epidemiology, Karolinska Institute (U.d.F.), Stockholm, Sweden; and Center for Medical Ultrasound, Division of Vascular Ultrasound Research, Wake Forest University School of Medicine, Winston-Salem, NC (R.T., M.G.B.). Correspondence to Camilla Skoglund-Andersson, King Gustaf V Research Institute, Karolinska Hospital, S Stockholm, Sweden. camilla@instmed.ks.se 1999 American Heart Association, Inc. Arterioscler Thromb Vasc Biol. is available at Downloaded from by guest on February 21, 2013

3 Skoglund-Andersson et al LDL Particle Size and Carotid IMT 2423 not been established. Typically, LDL particle size distribution has been categorized into 2 patterns, A and B, 30 and the peak particle diameter has been the sole quantitative estimate. B-mode ultrasound examination of the common carotid artery (CCA) enables measurement of its size and the width of the vascular wall components. An increased CCA intimamedia thickness (IMT) is considered a good surrogate marker of early atherosclerosis. Furthermore, it has been shown to correlate significantly with the presence of coronary artery disease (CAD) and to predict coronary events Factors suggested to influence CCA IMT are age, smoking, hypertension, LDL cholesterol concentration, and postprandial lipemia In accordance with its clinical benefit, lipidlowering treatment also retards progression of CCA IMT Recently, the common apoe polymorphism was shown to have an effect on carotid atherosclerosis. 42,43 The purpose of this study was to investigate the relation between LDL particle size distribution and IMT of the CCA in healthy, 50-year-old men. A 3% to 7.5% polyacrylamide gradient gel in which separation of LDL particles is achieved with high resolution was developed, as was a procedure to quantify the apob mass of defined LDL subfractions. Methods Subjects A total of 95 healthy, 50-year-old men living in the county of Stockholm were recruited after a random, population-based screening. Inclusion criteria were Northern European descent, homozygosity for the apoe3 allele, and a technically satisfactory carotid ultrasound examination. Exclusion criteria were chronic disease of any kind, history of CHD or arterial thromboembolic disease, continuous treatment with antihypertensive or lipid-lowering agents, body mass index (BMI) 32 kg/m 2, alcohol abuse or psychiatric disorders that would interfere with compliance, and participation in other ongoing studies. Of 411 men originally recruited for the study, 324 (79%) accepted, of whom 80 were found during the interview to meet 1 or more exclusion criteria. A total of 244 men came to the screening visit. Of these, 110 (45%) were found not to carry the apoe3/e3 genotype. In addition, 30 were excluded by other criteria, and in 2 subjects no LDL was obtained. Furthermore, a satisfactory ultrasound examination was not performed in 7 subjects. One of the 95 subjects included in the study was considered to have a plaque (CCA IMT 1.41 mm) and was therefore not included in the analyses. The study was approved by the Ethics Committee of Karolinska Hospital, and all subjects gave informed consent to participate. Blood Sampling Venous blood samples were drawn into precooled sterile tubes (Vacutainer, Becton Dickinson) containing Na 2 -EDTA (final concentration, 4 mmol/l), and plasma was immediately recovered by low-speed centrifugation (1.750g, 20 minutes, 1 C). PMSF (10 mmol/l, dissolved in isopropanol) and aprotinin (1.4 mg/ml; Trasylol, Bayer) were immediately added to the isolated plasma to final concentrations of 10 mol/l and 28 mg/ml, respectively. Major Plasma Lipoproteins Fasting plasma concentrations of cholesterol and triglycerides in VLDL, LDL, and HDL were determined by a combination of preparative ultracentrifugation, precipitation of apob-containing lipoproteins, and lipid determinations. 44 ApoE genotype was determined as described elsewhere. 45,46 Subfractionation of ApoB-Containing Lipoproteins and Determination of ApoB Content Large and small VLDL (Svedberg flotation rate, 60 to 400 and 20 to 60, respectively), IDL, and LDL fractions were isolated by DGUC. 47 Downloaded from The content of apob-100 in the VLDL subfractions and IDL was determined by analytical SDS polyacrylamide gel electrophoresis. 47 Total VLDL apob-100 concentration is the sum of apob-100 content in the small and large VLDL subfractions. LDL for GGE was isolated from plasma by a single-spin DGUC procedure according to the method of Redgrave and Carlson 48 with modifications. After a 16-hour spin at rpm and 15 C (Beckman SW40), the top 0.5-mL layer was aspirated (VLDL). The tube was then sliced 57 mm from the top to harvest the fraction (density, to kg/l) containing both IDL and LDL. The protein concentration of the isolated fraction was determined according to the method of Lowry et al 49 after addition of SDS to the reagent mixture to clear turbidity. Aliquots of isolated LDL were then stored at 80 C after addition of one-fifth of the volume of sucrose (50% wt/vol), NaCl (0.15 mol/l), and EDTA (0.24 mmol/l, ph 7.4) 50 until later GGE analysis. Nondenaturing Polyacrylamide GGE of LDL Gel Casting Polyacrylamide gradient gels were cast using a 2-chamber gradient mixer (GM-1, Pharmacia-LKB). The gels consisted of a short 3.0% stacking gel (Acrylamide, BioRad Laboratories), followed by a linear 3.0% to 7.5% acrylamide gradient. Ammonium persulfate (10% wt/vol) was added to the acrylamide solutions to attain a polymerization time of 90 minutes. The gel casting cassette (Hoefer Scientific) prepared for 10 gels (1.5-mm spacers, 10-well combs) was filled from the bottom with 20 ml of a 3% acrylamide solution (acrylamide, g/l; bisacrylamide, 0.75 g/l; Tris, mol/l, ph 8.35; Temed, 0.9 ml/ml; and ammonium persulfate, 0.4 g/l; prepared on ice). Then, 44 ml of the 3% acrylamide solution and 44 ml of a 7.5% acrylamide solution (acrylamide, g/l; bisacrylamide, g/l; Tris, mol/l, ph 8.35; Temed, 0.6 ml/ml; and ammonium persulfate, 0.2 g/l; prepared on ice) were poured into the 2 chambers of the gradient mixer. The acrylamide gradient was formed by allowing the gradient mixture to fill the gel casting cassette from the bottom by hydrostatic pressure during 10 to 15 minutes. When the gradient had been formed, 27 ml of 50% (vol/vol) glycerol was added manually, displacing the gradient upward into the cassette. The glycerol was added slowly with a syringe, taking extreme care to keep a constant flow and to avoid any stirring of the gradient. A grain of methyl green was added to the glycerol solution to observe any perturbation in the interface. Gels were then left to polymerize at room temperature for at least 2 hours, after which they could be stored under moist conditions at 4 C for no longer than 2 weeks. Electrophoresis The vertical slab gels were run in the Hoefer Mighty Small II apparatus equipped with an EPS 500/400 Pharmacia-LKB power supply. Preelectrophoresis (60 minutes at 50 V) and electrophoresis were performed by using Tris (180 mmol/l), boric acid (160 mmol/l), and Na 2 -EDTA (6 mmol/l ph 8.35) as running buffer with cooling from a thermostatic circulator (Multitemp II, Pharmacia- LKB) set at 10 C. To avoid smearing, low-melting-point agarose (final concentration, 0.4% wt/vol) was added to the sample immediately before application on the gel. A total volume of 25 L of sample, containing 3 g of LDL protein, was applied to each well. Reference proteins were run in 2 lanes on each gel. Wells closest to the edges of the gel were not used. Electrophoresis was conducted at 50 V for 60 minutes, followed by 100 V for 20 hours. Gels were stained for protein (Coomassie) in glass petri dishes using a newly filtered solution of 0.04% Coomassie Brilliant Blue G-250 (Serva) in 3.5% perchloric acid for a minimum of 3 hours. Gels were then destained in 7% acetic acid for 20 hours with 1 change of destaining solution. Reference Proteins Four calibration standard size reference proteins or lipoproteins were used. The largest was a Lp(a) species, which was isolated by sequential overnight ultracentrifugation at densities of and kg/l and rpm in a 50.3 Ti rotor (Beckman Instruments) 51 and characterized as follows. The purity of the fraction was verified by guest by itson pre- February mobility 21, during 2013agarose gel electrophoresis and

4 2424 Arterioscler Thromb Vasc Biol. October 1999 subsequent lipid staining. 52 Scanning of the gel showed that 80% of the stainable material was confined to the pre- region, whereas the remaining stainable material was in the region. An isolated LDL sample was also used as a size reference and as a distribution pattern reference for rapid estimation of running quality and reproducibility. The Lp(a)-containing fraction and the isolated LDL sample were dialyzed against 1% ammonium bicarbonate and thereafter subjected to electron microscopy (Philips EM 400) using a negative stain (1% phosphotungstic acid, ph 8.5) at magnification. The particle diameter of the respective fraction was measured in a dehydrated state. The micrograph was photographically enlarged 5 times. The diameters of 100 particles were then determined on each micrograph. The calculated mean diameter of the Lp(a) particles was nm (n 200). For the LDL sample, 115 particles were measured, and mean particle size was nm. The LDL sample was further characterized by DGUC 27 to establish the LDL density profile. Aliquots of the Lp(a) fraction and LDL sample were mixed with the sucrose-containing buffer previously described, kept frozen at 80 C, and thawed immediately before application on the gel. The smaller reference proteins were thyroglobulin (Pharmacia- LKB) and its dimer. The hydrated sizes of thyroglobulin and its dimer were calculated to be 17 and 21.4 nm, respectively. 53 Densitometry and Determination of LDL Particle Size Gels were scanned by a laser densitometer (Ultroscan XL, Pharmacia-LKB) linked to a personal computer, and the area under the absorbance curve (AUC) was calculated automatically with use of Gelscan XL software (Pharmacia-LKB). Before scanning, an OH film with a straight black line was placed on top of the gel to align the line to the bottom of the wells. The migration distance of each sample was calculated from the position of the straight line. Every absorbance value along the distance from the line to a point slightly beyond the peak of the thyroglobulin monomer corresponded to a migration distance in millimeters. The AUC was calculated across the LDL particle size range. On the basis of the linear gradient profile of the gel, a linear regression analysis was conducted on the relative migration distance of the reference proteins on the gel and their respective sizes. The regression line was calculated for each gel and was used to convert migration distance of the LDL samples into LDL particle diameter size, to determine major peak size, and to calculate borders between subfractions. The mean correlation coefficient for the relationship between the migration distance and size of the standard references from 10 consecutive runs was Downloaded from Figure 1. Subfractions of LDL were isolated by a DGUC procedure and subjected to electrophoresis on the 3% to 7.5% polyacrylamide gel. Top, Photograph of electrophoretic band pattern of density subfractions. LDL subfractions are shown in lanes 2 through 9 (from left). Bottom, Major peak particle size of each subfraction plotted against fraction density. Definition of LDL Subfractions After LDL was separated from pooled plasmas by use of an equilibrium DGUC procedure described by Chapman et al, 8 densitydefined subfractions were collected and subjected to GGE along with the standard size reference proteins. The density profile of the ultracentrifugation gradient was controlled by replacing LDL with a salt solution (1.040 kg/l). The density-defined subfractions were then recovered, and the density of each fraction was determined with use of a precision densitometer (Paar, DMA 60). After the isolated LDL subfractions were evaluated, the LDL particle sizes of the respective density subfractions were measured by using the GGE method. Major peak sizes of the DGUC fractions were plotted against fraction densities (Figure 1) and were thereafter used to convert density cutoffs into LDL particle sizes on the gel. Boundaries of the total LDL particle size interval were defined as 21.0 nm LDL particle diameter 27.0 nm. Size cutoffs were set at 25.0 nm (corresponding to kg/l), 23.5 nm ( kg/l), and 22.5 nm ( kg/l). Subsequently, 4 LDL subfractions were defined: LDL-I (27.0 to 25.0 nm, to kg/l), LDL-II (25.0 to 23.5 nm, to kg/l), LDL-III (23.5 to 22.5 nm, to kg/l), and LDL-IV (22.5 to 21.0 nm, to kg/l). The density cutoff for small, dense LDL (1.040 kg/l) was originally defined by Krauss and Burke 2 and has been used to relate LDL heterogeneity to severity of CAD. 18 LDL particle size distribution was expressed with use of the defined size cutoffs, and the resulting subfractions were denoted LDL-I to LDL-IV, from the largest to the smallest particle size. The relative AUC for stainable material within each LDL subfraction was calculated by the software. Three different parameters reflecting LDL particle size distribution were derived from evaluation of the gel: (1) The major peak size (nm) denotes the particle size of the predominant peak (corresponds to PPD); (2) the relative distribution fraction shows the fraction of the total AUC accounted for by each LDL subfraction; and (3) the plasma LDL subfraction concentration was quantified by multiplying the total LDL protein concentration by the relative distribution fraction. To examine the stoichiometry for Coomassie binding between different subfractions of LDL, purified subfractions were isolated by DGUC 8 and subjected to electrophoresis on the 3% to 7.5% gradient gel. Total AUC (21.0 to 27.0 nm) of the density subfractions, along with cholesterol, triglyceride, and protein content, was measured. The reproducibility of the GGE procedure was estimated by calculating within-subject variation. LDL samples obtained from 61 subjects on 2 occasions were evaluated, and the difference between measurements was plotted against the subject mean. Absence of correlation between the 2 indicated that the standard deviation was independent of the measurement and therefore that the coefficient of variation (CV) was not an appropriate representation of measurement error. The within-subject standard deviation was for the relative distribution of LDL-I, for LDL-II, for LDL-III, and for LDL-IV. The within-subject standard deviation for major peak was 0.14 nm (CV, 0.006). CCA Ultrasound Examinations IMT was measured essentially according to the ultrasound protocol of the European Lacidipine Study on Atherosclerosis 54 and included 2 components, the scanning and reading procedures. Two certified ultrasonographers performed the scanning, with subjects in the supine position with the head slightly turned from the sonographer. The ultrasound device used was a Biosound 2000 II S.A. (Biosound Inc) with an 8-MHz high-resolution annular array scanner. The far and near walls of the right and left CCA were scanned in the anterior, lateral, and posterior angles. Scanning was recorded on S-VHS videotapes, which were sent to the Center for Medical Ultrasound, Division of Vascular Ultrasound Research, Wake Forest University, Winston-Salem, NC, for reading. IMT was estimated by measuring the linear distance, perpendicular to the luminal axis, between 2 points defined by the ultrasonic interfaces, which indicate the boundary between the lumen and intimal surface and the boundary between the medial-adventitial interface. CCA IMT was calculated as the mean of the right and left CCA far-wall IMT. Measurements of IMT in the bifurcation and internal carotid artery were not used in this report. All subjects were routinely scanned twice on the same occasion by guest by the on February same sonographer 21, 2013 to evaluate intrasonographer

5 Skoglund-Andersson et al LDL Particle Size and Carotid IMT 2425 Figure 2. Digital photograph of LDL samples on the 3% to 7.5% polyacrylamide gradient gel. Lanes 1 and 2 (from left), Size standard references (LDL/thyroglobulin and Lp(a), respectively). Lanes 3 through 8, Isolated LDL samples from study participants. reproducibility. To evaluate intersonographer reproducibility, the 2 sonographers each scanned, on the same occasion, all subjects appearing during 1 month at 6-month intervals. Intrasonographer CVs were 3.8% and 5.1%. The intersonographer CV was 4.7%. Statistical Analysis Statistical calculations were performed by using JMP software (SAS) for Macintosh. Deviation from normality was tested by using the Shapiro-Wilk W test. If logarithmic transformation did not normalize the variable, nonparametric tests were used. Not once did nonparametric test results deviate from those obtained with log 10 -transformed variables. The distribution of continuous variables was represented by the median and interquartile range. Associations between parameters were determined by linear regression analysis, and results are shown as correlation coefficients (r). Variables showing a statistically significant univariate association with CCA IMT were included in the multiple stepwise linear regression analysis. An F value of 4.0 was entered in the forward stepwise regression model. Results LDL Particle Size Distribution and Study Group Characteristics The electrophoretic procedure separated the LDL particles over a distance of 25 to 30 mm of the 3% to 7.5% polyacrylamide gel. Resolution of the LDL particle size spectrum was thus improved compared with previous GGE procedures, in which the LDL fraction was distributed over a 5- to 10-mm portion of the gel. The distance between the largest and the smallest reference proteins was 45 mm, which covered the whole spectrum of LDL particle sizes. A photograph of a gel with LDL samples from 6 subjects is shown in Figure 2. All samples displayed a continuous particle size distribution without signs of discrete subclasses. A qualitative comparison between the 3% to 7.5% GGE method and an established ultracentrifugation procedure for separation of LDL subfractions 27 is shown in Figure 3. LDL particle size distribution patterns obtained with the 2 methods were in good agreement. The relative chromogenicity of LDL subfractions separated by DGUC 8 is shown in Table 1, along with the cholesterol, triglyceride, and protein contents of the respective subfraction. The total AUC (21.0 to 27.0 nm) for Figure 3. Qualitative comparison of the 3% to 7.5% GGE method (bottom), using an isolated sample of LDL (density, to kg/l) and an established ultracentrifugation procedure 27 for separation of LDL subfractions (top) from plasma, showing LDL size/density distribution of 3 subjects. The 2 methods are in good agreement with regard to the LDL particle distribution pattern. Subject C is the LDL sample used as size reference standard in the GGE protocol, and it is applied on the gel together with thyroglobulin (the isolated peak to the right). each subfraction is compared with the mean AUC for all 10 LDL subfractions and is shown as relative AUC. The standard deviation from mean total AUC was 12%. LDL heterogeneity characteristics of the 94 subjects are described in Table 2. The LDL-II subfraction (23.5 to 25.0 nm) comprised 50% of total LDL, and the major peak was most often found within this subfraction (Table 2). The greatest heterogeneity was seen within LDL-III (22.5 to 23.5 nm), which constituted 22% (16% to 33%) of total LDL. Only a small portion (5%) of the LDL particles were contained in the LDL-IV subfraction (21.0 to 22.5 nm). TABLE 1. Fraction LDL Subfraction Chromogenicity Density, kg/l rauc Cholesterol, mmol/l Triglycerides, mmol/l Protein, mg/l Downloaded from rauc by indicates guest on relative February area under 21, 2013 the curve.

6 2426 Arterioscler Thromb Vasc Biol. October 1999 TABLE 2. LDL Particle Size Distribution (n 94) Median (IQR) Major peak size, nm 23.6 ( ) Relative distribution of LDL subfractions LDL-I 0.20 ( ) LDL-II 0.51 ( ) LDL-III 0.22 ( ) LDL-IV 0.05 ( ) Plasma concentration, mg/l LDL-I 186 ( ) LDL-II 413 ( ) LDL-III 179 ( ) LDL-IV 46 (39 59) IQR indicates interquartile range. Descriptive statistics of the CCA IMT along with plasma lipids and major lipoproteins are reported in Table 3. Relations Between Major Plasma Lipoproteins and LDL Particle Size Distribution Relations between the major plasma lipoproteins and LDL particle size distribution are shown in Table 4. As expected, the major peak size was inversely related to VLDL triglycerides (r 0.63, P 0.001) and positively related to HDL cholesterol (r 0.50, P 0.001). An inverse correlation was also seen between VLDL triglycerides and the fraction of total LDL contained in LDL-II, with a reciprocal positive relation to HDL cholesterol. These relations were reversed for LDL-III. The LDL-IV subfraction was not significantly correlated with any major plasma lipoprotein fraction. When actual plasma concentrations of LDL subfractions were related to plasma concentrations of major lipoproteins, the pattern changed slightly. As expected, all LDL subfractions TABLE 4. LDL Subclass Distribution in Relation to Major Plasma Lipoproteins VLDL-TG LDL-C HDL-C Major peak size Relative distribution of LDL subfractions LDL-I 0.22* LDL-II LDL-III LDL-IV Plasma concentration LDL-I LDL-II LDL-III LDL-IV * 0.21* Values are correlation coefficients (r ). VLDL-TG indicates VLDL triglycerides; LDL-C, LDL cholesterol; and HDL-C, HDL cholesterol. *P 0.05, P 0.01, P were now associated with LDL cholesterol. Plasma concentrations of the LDL subfractions containing smaller-particle species (LDL-III and -IV) were also strongly and positively correlated with VLDL triglycerides and inversely related to HDL cholesterol. In contrast, plasma levels of LDL subfractions containing larger-particle species (LDL-I and -II) correlated only with the plasma concentration of LDL cholesterol. Major Plasma Lipoprotein and LDL Subfraction Correlations With CCA IMT Linear regression analysis was used to determine the relations of plasma lipids and lipoprotein lipids to CCA IMT (Table 5). Of the lipid variables, plasma cholesterol showed the strongest correlation with CCA IMT (r 0.29, P 0.01). This TABLE 3. Characteristics of Study Group (n 94) Median (IQR) TABLE 5. Univariate Correlations of Plasma Lipids and Major Lipoproteins with CCA IMT CCA IMT CCA IMT, mm 0.85 ( ) Cholesterol Cholesterol, mmol/l Plasma 5.34 ( ) Plasma VLDL VLDL 0.30 ( ) LDL 0.24* LDL 3.60 ( ) HDL 0.06 HDL 1.18 ( ) Triglycerides Triglycerides, mmol/l Plasma 1.20 ( ) Plasma VLDL 0.25* 0.24* VLDL 0.75 ( ) LDL 0.19 LDL 0.27 ( ) HDL 0.02 HDL 0.11 ( ) ApoB-100 ApoB-100, mg/l VLDL 43.0 ( ) VLDL IDL * IDL 44.9 ( ) LDL 0.12 LDL 870 ( ) BMI 0.02 BMI (kg/m 2 ) 25.1 ( ) SBP 0.02 SBP (mm Hg) 122 ( ) DBP 0.07 DBP (mm Hg) 80 (75 85) Values are correlation coefficients, (r ). IQR indicates interquartile range. Downloaded from *P 0.05, by guest P on February 21, 2013

7 Skoglund-Andersson et al LDL Particle Size and Carotid IMT 2427 TABLE 6. Univariate Correlations Between LDL Particle Size Distribution and CCA IMT CCA IMT Major peak size 0.40 Relative distribution of LDL subfractions LDL-I 0.50 LDL-II 0.28 LDL-III 0.44 LDL-IV 0.18 Plasma concentration LDL-I 0.32 LDL-II 0.13 LDL-III 0.42 LDL-IV 0.23* Values are correlation coefficients (r ). *P 0.05, P 0.01, P Figure 4. Subjects were divided into quartiles with respect to plasma concentration of LDL-III (22.5 to 23.5 nm, corresponding to to kg/l), and the quartiles were related to CCA IMT. Subjects in the highest quartile had significantly increased CCA IMT values compared with subjects in the lowest quartile ( 50%, P 0.001). correlation was accounted for by the association between LDL cholesterol and CCA IMT (r 0.24, P 0.05). Plasma triglycerides (r 0.25, P 0.05) as well as VLDL triglycerides (r 0.24, P 0.05) were also significantly related to the CCA IMT. In contrast, plasma concentrations of apob-100 in the VLDL and LDL fractions were not significantly associated with CCA IMT, whereas IDL apob-100 was. Neither BMI, systolic blood pressure (SBP), nor diastolic blood pressure (DBP) correlated significantly with CCA IMT. Major LDL peak size was strongly inversely related to the IMT of the CCA, suggesting that a predominance of small LDL particles is associated with an increased IMT (Table 6). The relative distribution of larger-particle LDL species (LDL-I and -II) was negatively related to IMT, but when the actual plasma concentrations of these LDL subfractions were considered, only that of LDL-I remained significantly associated (r 0.32, P 0.01) with IMT. Of the smaller-particle LDL species, the major fraction (LDL-III) was strongly and positively associated with CCA IMT, and this relation persisted when the plasma concentration of this subfraction was considered (r 0.42, P 0.001). Relations between quartiles of LDL-III plasma concentration and CCA IMT are depicted in Figure 4. The opposite relations of large- and small-particle LDL to CCA IMT likely explain the relative weakness of the LDL cholesterol association with IMT. Multivariate Analysis Multiple stepwise linear regression analysis was performed to identify independent determinants of CCA IMT. Variables that were significant in univariate analyses were used as independent variables in multivariate analyses. These variables included LDL cholesterol, VLDL triglycerides, IDL apob-100, and the plasma concentration of the major LDL subfraction containing smaller-particle LDL species (LDL- III), which was included as the single measure of LDL particle size distribution in the multivariate model. In the multivariate forward stepwise model (model A, Table 7), LDL-III was the first variable to enter, contributing 19% to the variation in CCA IMT. Once LDL-III had entered, none of the other lipoprotein or apolipoprotein variables remained significantly related to CCA IMT. In model B, VLDL triglycerides was first forced to enter (r ), and stepwise Downloaded from forward progression subsequently allowed LDL-III to enter the model (r ), followed by LDL cholesterol. Finally, in model C, VLDL triglycerides, LDL cholesterol, and IDL apob-100 were forced to enter the model. Together they accounted for 10% of the variation in CCA IMT, which indicates a substantial degree of interaction between these variables. Nevertheless, LDL-III entered the model and increased the value of r 2 by This indicates that the plasma concentration of LDL-III correlates significantly and independently with CCA IMT in healthy, middle-aged men. Using total cholesterol and total triglyceride concentrations instead of LDL cholesterol and VLDL triglycerides did not change the outcome of the multivariate analysis. Discussion To the best of our knowledge, this is the first study of LDL particle size distribution in relation to measurements of vascular change, which is believed to precede atherosclerosis, in healthy and essentially normolipidemic individuals. A novel polyacrylamide GGE procedure was developed to quantify plasma concentrations of LDL subfractions according to particle size. As anticipated, the plasma level of small-particle LDL was strongly associated with plasma triglyceride concentration and inversely related to plasma HDL cholesterol content. In contrast, plasma levels of the larger-particle LDL species (LDL-I and -II) were determined by total LDL concentration. Although the metabolic origin of small, dense LDL is not fully understood, the distribution of LDL particle size in human plasma is believed to be the result of the coordinated actions of hepatic and endothelial lipases as well as mediators of neutral lipid exchange. One current hypothesis 25,55 is that the large, light LDL particles are direct products of the action of lipoprotein lipase on small VLDL species (VLDL 2 ). In contrast, small, dense LDL particles are believed to arise through the exchange of cholesteryl esters for triglycerides, between LDL and large VLDL (VLDL 1 ), mediated by the cholesteryl ester transfer protein, producing triglycerideenriched LDL particles, which are then lipolyzed by hepatic lipase. by This guest metabolic on February relation 21, 2013 can, in part, explain the strong

8 2428 Arterioscler Thromb Vasc Biol. October 1999 TABLE 7. Determinants of CCA IMT According to Multivariate Analysis correlation between triglyceride levels and the plasma concentration of small, dense LDL particles. In our study, the strongest correlation with CCA IMT was found for LDL-III. In the multivariate analysis, the plasma concentration of LDL-III accounted for 19% of the variation in CCA IMT, and after LDL cholesterol, VLDL triglycerides, and IDL apob-100 were forced into the model, this specific LDL subfraction still accounted for 10% of the variation in CCA IMT. The present method separates LDL particles with high resolution and enables quantification of subtle differences in the LDL particle size distribution pattern between individuals. In the majority of previous clinical studies, evaluation of LDL heterogeneity was based on qualitative classification of LDL particle distribution. 21 It is our opinion that the dichotomization of the LDL particle size distribution into subclass patterns A and B limits the information to be gained from studies of the LDL heterogeneity trait. The most common procedure for separating LDL particles by GGE uses the Pharmacia PAA 2/16 gel. This gel has been useful for obtaining a crude estimate of the LDL particle size distribution pattern. However, because of the steep progression of the polyacrylamide gradient, the resolution of the LDL particle size spectrum is relatively poor in comparison with ultracentrifugation techniques. In this regard, our protocol provides an improved resolution due to the very slow progression of the polyacrylamide gradient. The cutoffs for defining individual LDL particle size subfractions were derived from densitometric analyses of LDL particle distribution. 2 Although conversion of the LDL subfraction density to LDL particle size is approximate, the LDL subfractions obtained by this procedure proved to be appropriate. A couple of points pertaining to determination of LDL particle size require consideration. The commonly used latex beads 56 could not be used as a reference marker in our procedure because they either did not enter the gel or failed to focus on the gel. The variation in size of latex beads seems to be too wide for a slowly inclining polyacrylamide gradient. Instead, the particle size of an isolated Lp(a) fraction was determined by electron microscopy, and the Lp(a) sample was then used as the largest reference protein. The advantage of using Lp(a) as a reference marker is its resemblance to LDL in structure and particle size (corresponds to the size of the largest IDL or LDL particles). The disadvantages are difficulties with stability and standardization, which were avoided by freezing large batches of well-defined Lp(a) at a high sucrose concentration. 50 We also used a carefully characterized LDL preparation (electron microscopy to establish Model A Model B Model C Partial Correlation r 2 Partial Correlation r 2 Partial Correlation r 2 LDL-C f VLDL-TG 0.25 f 0.06 f IDL apob f } 0.10 LDL-III Multiple r LDL-C indicates LDL cholesterol; f, forced variable; VLDL-TG, VLDL triglycerides. Downloaded from the size of the most abundant particle and DGUC to establish the LDL density profile of the sample) as a reference marker. This had several advantages. First, the LDL sample constituted a fourth point on the standard curve, which therefore became more stable and reliable. Second, after the lane containing the reference LDL sample was evaluated, the quality and applicability of the run could quickly be determined. Together, these factors, along with standardized procedures for staining, destaining, and evaluation, contributed to the creation of a highly reproducible GGE procedure. Use of a different set of reference markers, isolated samples of Lp(a) and LDL (measured by electron microscopy in a dehydrated state) and the absence of the formerly used latex bead (which is very large, artificial, and incompressible) explain why our estimated lipoprotein diameter sizes are somewhat smaller than those reported from earlier studies based on GGE techniques. 2 The results of this study suggest that the plasma concentration of LDL-III (particle size 22.5 to 23.5 nm) is independently related to CCA IMT in a homogenous group of healthy, 50-year-old men with a fairly narrow IMT distribution (0.59 to 1.19 mm). Of the major lipoproteins, only VLDL triglycerides, LDL cholesterol, and IDL apob-100 related significantly to the CCA IMT and together explained 10% of its variation, which is in agreement with results of an earlier study. 57 In our GGE procedure, an LDL sample of density to kg/l is applied to the gel. Hence, the LDL subfraction containing the largest particles (LDL-I; particle size, 25.0 to 27.0 nm) is likely to contain IDL particles. Results of a recent study 58 indicated that IDL is associated with progression of atherosclerosis, measured as an increase in CCA IMT. In our study, the IDL apob-100 concentration was associated with CCA IMT in univariate analysis but failed to contribute to the variation in IMT independently of LDL cholesterol and VLDL triglycerides. It is likely that the linear relationship between plasma levels of small LDL particles and CCA IMT would not have been detected if a method with less precision had been used to determine the LDL particle size distribution. GGE methods have been used in several studies to investigate LDL peak particle size in relation to the presence of CAD, 17,20 but a graded relationship between LDL particle size and the extent of atherosclerosis assessed by coronary angiography has not been detected. This could be due to the fact that once CAD is present, several mechanisms contribute to the development of the atherosclerotic lesions and that the relative contribution of the small LDL species is difficult to disentangle. An alternative by explanation guest February could 21, be 2013 that the relation between LDL

9 Skoglund-Andersson et al LDL Particle Size and Carotid IMT 2429 particle size and CAD depends on the quantity of a particularly atherogenic LDL species and that a more sensitive method of measuring LDL particle size distribution is needed to demonstrate such a relation. However, coronary angiography and ultrasonographic measurement of carotid IMT are 2 principally different methods of measuring vascular lesions and are likely to reflect different stages of the atherosclerotic process. Our results suggest that the small, dense LDL particle species could play an important role in causing vascular change, leading to atherosclerosis, and indicate that the evaluation of LDL particle size distribution with a highresolution GGE method may be a valuable approach to estimating the plasma levels of particularly atherogenic LDL species in asymptomatic individuals. Acknowledgments This study was supported by grants from the Swedish Medical Research Council (8691, 9533, and 12659), the Swedish Heart-Lung Foundation, the Marianne and Marcus Wallenberg Foundation, the Petrus and Augusta Hedlund Foundation, and the Swedish Society of Medicine. The authors are grateful to Ulla Hellmark-Augustsson and Linda Nilsson for assisting with B-mode ultrasound image acquisition and to Dr Susanna Boquist for recruitment of study participants. References 1. Shen MM, Krauss RM, Lindgren FT, Forte TM. Heterogeneity of serum low density lipoproteins in normal human subjects. J Lipid Res. 1981; 22: Krauss RM, Burke DJ. Identification of multiple subclasses of plasma low density lipoproteins in normal humans. J Lipid Res. 1982;23: Musliner TA, Giotas C, Krauss RM. Presence of multiple subpopulations of lipoproteins of intermediate density in normal subjects. Arteriosclerosis. 1986;6: Lindgren FT, Jensen LC, Wills RD, Freeman NK. 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