Direct Determination of Lipoprotein Particle Sizes and Concentrations By Ion Mobility Analysis

By Caulfield, Michael P Li, Shuguang; Lee, Gloria; Blanche, Patricia J; Salameh, Wael A; Benner, W Henry; Reitz, Richard E; Krauss, Ronald M

BACKGROUND: Current methods for measuring the concentrations of lipoprotein particles and their distributions in particle subpopulations are not standardized. We describe here and validate a new gas-phase differential electrophoretic macromolecular mobility- based method (ion mobility, or IM) for direct quantification of lipoprotein particles, from small, dense HDL to large, buoyant, very- low-density lipoprotein (VLDL). METHODS: After an ultracentrifugation step to remove albumin, we determined the size and concentrations of lipoprotein particles in serum samples using IM. Scan time is 2 min and covers a particle range of 17.2-540.0 [Angstrom]. After scanning, data are pooled by totaling the particle number across a predetermined size range that corresponds to particular lipoprotein subclasses. IM results were correlated with those of standard methods for cholesterol and apolipoprotein analysis.

RESULTS: Intra- and interassay coefficients of variation for LDL particle size were <1.0%. The intra- and interassay variation for LDL and HDL particle subfraction measurements was <20%. IM-measured non-HDL correlated well with apolipoprotein B (r = 0.92).

CONCLUSIONS: The IM method provides accurate, reproducible, direct determination of size and concentration for a broad range of lipoprotein particles. Use of this methodology in studies of patients with cardiovascular disease and other pathologic states will permit testing of its clinical utility for risk assessment and management of these conditions.

(c) 2008 American Association for Clinical Chemistry

Plasma concentrations of lipids and lipoproteins are strongly related to the development of atherosclerotic cardiovascular disease (CVD)4 and its complications (1). The Adult Treatment Panel III (ATP) guidelines include the use of standardized lipid and lipoprotein measurements in algorithms for assessing CVD risk and for reducing that risk by lifestyle and pharmacological interventions (2). A principal focus of these guidelines has been the designation of specific targets for LDL cholesterol concentrations that are based on overall risk status, with consideration of the impact on risk associated with other factors such as reduced plasma HDL cholesterol. However, global CVD risk scores that employ clinical markers leave a residual unexplained risk (3), particularly for individuals whose estimated risk according to the ATP 10-year scoring system (2) falls in the borderline range. Increased concentrations of atherogenic apolipoprotein B (apoB)-containing lipoprotein particles may contribute to risk that is disproportionate to the LDL cholesterol concentration (4). Thus, the use of LDL cholesterol targets alone may lead to undertreatment in some individuals with increased concentrations of smaller and more dense LDL particles or triglyceride-rich lipoprotein remnants and intermediate-density lipoprotein (IDL) (4). In this regard, several studies have reported that CVD risk status is more accurately reflected by measurement of plasma apoB-an index of the total number of atherogenic lipoprotein particles-than by LDL cholesterol (3-5); non-HDL cholesterol, calculated by subtracting HDL from total cholesterol, provides similar risk information. Nevertheless, evidence that measures of specific subfractions of apoB-containing lipoproteins provide clinical information beyond that assessed by total particle number (6-8) has led to growing implementation of methods designed to measure concentrations and distributions of these subfractions-in particular, small, dense LDL. In addition, measurements of larger HDL particle subspecies, and particularly the apoAI content of these particles (9), have been reported to be more strongly related to CVD risk than is total HDL cholesterol.

Fig. 1. Schematic of gas-phase differential electrical mobility analyzer showing the electrospray, differential mobility analyzer, and condensation particle counter. (Schematic kindly provided by Kathleen Ericksen, TSI Inc.)

Currently, there is no standardized method for measuring concentrations and distributions of lipoprotein particle subpopulations; each of the clinically available methods (vertical ultracentrifugation, nuclear magnetic resonance, and gradient gel electrophoresis) requires some form of algorithm to generate the lipoprotein profiles and/or particle concentrations.

Gas-phase differential electrical mobility, also known as ion mobility (IM), is well established in the field of aerosol science for particle analysis and measurement (10). The development of an electrospray interface (11) permitted the system to be used to analyze particles in solution, and its use was soon extended to analysis of DNA (12), proteins (13), and a variety of other biological materials (14-17). A patent describes its use for measuring the size distribution of lipoprotein particles (18). In this study, we describe and validate the use of IM to measure the distributions and concentrations of plasma lipoprotein particles, covering the spectrum of HDL (approximately 75 [Angstrom] diameter) to larger, very-low-density lipoprotein (VLDL) (approximately 520 [Angstrom] diameter), and establish summary statistics characterizing the result distributions commonly seen in healthy adults.

Materials and Methods

GENERAL DESCRIPTION

The number of particles detected in 0.1 -s bins is recorded and used to determine distributions. The data in a time bin is converted into particle number concentration (number/mL) and also into particle mass concentration (mass/mL). When these values are plotted against particle diameter, particle number or particle mass distributions are generated. An example of the raw particle count data is shown in Fig. 2A (particle number corrected for dilution) for a control sample, prepared by an overnight ultracentrifugation. Fig. 2B shows the data converted to arbitrary mass units using the formula: mass = density x volume (a density of 1.0 was used for all lipoprotein particles).

SAMPLE PREPARATION

We briefly mix serum samples or controls by vortexmixing, then mix 5 [mu]L of sample or control with 20 [mu]L of an albumin removal reagent [7.5 g/L Reactive green 19 dextran (RGD), Sigma-Aldrich; 2.5 g/L dextran sulfate, Sigma-Aldrich; and 0.5 g/L EDTA, Spectrum Chemicals] and incubate them on ice for 15 min. After incubation, we overlay the sample mixture on 200 [mu]L deuterium oxide (Medical Isotopes) in a 42.2 ultracentrifuge tube (Beckman Coulter). The samples are ultracentrifuged at 10 [degrees]C for 135 min at 223 000g (42 000 rpm), and then we remove the top 85 [mu]L (i.e., the lipid fraction) of the sample. We dilute the samples 1:800 for HDL analysis using 25 mmol/L ammonium acetate, 0.5 mmol/L ammonium hydroxide, pH 7.4. For LDL analysis, samples are diluted 1:200 with the same diluent containing 5 [mu]g/mL dextran sulfate to help prevent LDL particles from sticking to the capillary surfaces. Final dilutions are made in deep-well 96-well plates and placed in a Leap HTC PAL autosampler (Eksigent) with the cooled stack maintained at 6 [degrees]C.

LIPOPROTEIN ANALYSIS

The autosampler is connected to the electrospray generator (Model 3480; TSl) via methyl-deactivated silica capillary (50 [mu]m i.d.; SGE). Flow is introduced by nano-LC pumps (Eksigent) running a mobile phase of 25 mmol/L ammonium acetate, 0.5 mmol/L ammonium hydroxide, pH 7.4. By means of a capillary metal union (Upchurch Scientific), an autosampler injects 10 [mu]L sample at 6 [mu]L/min into a transfer capillary (methyl-deactivated, SGE, 50 [mu]m, 33 cm long). High voltage (2.1 kV) is applied to the metal capillary union located 33 cm upstream of the electrospray unit. The electrospray Taylor cone is monitored visually and amperometrically to ensure stability. After the sample has filled the capillary and reached the electrospray chamber, the flow is decreased to 200 nL/min and the data recording process is started. The air (containing approximately 5% CO2) flowing into the electrospray chamber is regulated at 1.6 L/ min.

The electrosprayed particles pass through a particle-charge neutralizing chamber and then enter the DMA. Scan time is 2 min and covers a particle range of 17.2 to 542.0 [Angstrom]. After a scan is completed, data for specific ranges of particles corresponding to lipoprotein subclasses are pooled by totaling the particles across a predetermined set of 0.1-s bins that corresponds to particular subclasses, and the predominant LDL particle size (modal diameter) is determined.

VOLUNTEERS FOR DETERMINATION OF REFERENCE DISTRIBUTIONS FOR LIPOPROTEIN PARTICLE SIZE AND SUBFRACTION CONCENTRATIONS

Nonmedicated (including hormone replacement therapy and birth control), apparently healthy Quest Diagnostics employee volunteers were recruited to establish reference distributions for the lipoprotein fractions detected by IM. The study was approved by the institutional review board, and all volunteers gave informed consent. We analyzed samples from 650 individuals (469 women, 181 men) by IM before applying specific exclusion criteria. All subjects had normal complete blood counts and chemistry panels. For inclusion in the reference population, individuals had to meet NCEP ATP guidelines (2) for optimal plasma lipid and lipoprotein concentrations: total cholesterol <200 mg/ dL, triglycerides <150 mg/ dL, LDL cholesterol <130 mg/dL, and HDL cholesterol >40 mg/dL (men) or >50 mg/dL (women). After applying the exclusion criteria, 68 men and 191 women remained. Total cholesterol, triglycerides, and HDL cholesterol to determine eligibility for inclusion in the reference distributions and for method comparison were run on a Hitachi 917 (Roche Diagnostics) using Roche Diagnostics reagents. Fig. 2. Ion mobility data.

Fig. 3. Lipoprotein fractionation profiles derived from ion mobility.

DETERMINATION OF PARTICLE DIAMETER BOUNDARY INTERVALS FOR LIPOPROTEIN FRACTIONS QUANTIFIED BY THE IM PROCEDURE

Fig. 3 displays quantitative profiles of lipoprotein particles as a function of particle diameter for 3 representative subjects. Shown in this figure are the boundaries used to define major lipoprotein subfraction groupings. We ascertained 2 HDL subfraction categories, HDL large (equivalent to HDL2b, 105-145 ?) and HDL small (equivalent to HDL3 + 2a, 76.5-105 A), using visual inspection of individual profiles and based on conformity of these size intervals with those previously determined by other methods (20). HDL large was discerned as a distinct peak in most samples (Fig. 3). Particle size boundaries within the IDL to LDL range were determined by IM analysis of subir/actions isolated by density gradient ultracentrifugation from 8 subjects using published procedures (21, 22). The major subfractions were defined according to previous nomenclature (6) as IDL large (IDLl, 268.2-296 [Angstrom]), IDL small (IDL2, 238-268.2 [Angstrom]), LDL large (LDL I, 219.9-238 [Angstrom]), LDL medium (LDL II, 211-219.9 [Angstrom]), LDL small (LDL III, 201.7-21 1 [Angstrom]), and LDL very small (LDL IV, 180- 201.7 [Angstrom]). In the case of VLDL-sized particles, IM particle distributions were inspected visually to select boundaries that were used to define 3 regions: small (296-335 [Angstrom]), medium (335- 424 [Angstrom]), and large (424-520[Angstrom]). VLDL particles >520 [Angstrom] were not measured in this scan but will be the focus of future studies.

INTERFERENCE

Matrix specificity was minimized in this procedure through a) initial enrichment of the lipoproteins from the serum/plasma proteins and b) separation of the lipoprotein particles by size in the IM. Compounds such as hemoglobin are removed during the ultracentrifiigation; small compounds such as bilirubin, while not completely sedimenting in the ultracentrifuge, appear before the lipoproteins in the IM analysis because of their small size.

STATISTICS

We used regression analysis to assess the relationships between biochemical and IM lipoprotein measurements.

Results

SENSITIVITY

Based on these data, the LOD and LOQ for particle concentrations ranged from 0.01 nmol/L and 0.02 nmol/L, respectively, for the VLDL large category to 10.6 nmol/L and 38.6 nmol/L for the HDL small category (see Supplemental Table 1 that accompanies the online version of this article at http://www. clinchem.org/content/vol54/ issue8).

REPRODUCIBILITY AND PRECISION

The intraassay precision for all lipoprotein fractions was <20% (see Supplemental Table 2, A-C, in the online Data Supplement). The highest CVs were associated with IDL and VLDL, fewer particles of which were present. The variation seen in these fractions is a combination of the sample preparation as well as the IM analysis. The intraassay variation of a control sample run 22 times was <9.0% for all HDL and LDL fractions and < 13.1% for all lipoprotein fractions (see Supplemental Table 2D in the online Data Supplement).

The reproducibility (interassay mean, SD, and CV) for each sample is shown in Supplemental Table 3 in the online Data Supplement. As was the case for intraassay variation, higher CVs were observed for fractions with lower particle concentrations. CVs were <20% for LDL and HDL and <26% for IDL and VLDL. Both intra- and interassay CVs for LDL particle size were < 1.0%.

SPECIMEN STABILITY

We used EDTA plasma samples from 5 individuals to evaluate the stability of the lipoproteins at room temperature (20-26 [degrees]C), under refrigeration (2-8 [degrees]C), after freezing (< - 20 [degrees]C), and after multiple freeze/thaw cycles at <- 70 [degrees]C. Samples were divided into sufficient numbers of aliquots for the appropriate time points at each temperature. Values were expressed as a percentage of the day 0 time-point value. Samples were stable for 5 days at 20-26 [degrees]C, 7 days at 2-8 [degrees]C, and 28 days at -20 [degrees]C. Five freeze-thaw cycles had no significant effects on the results (see Supplemental Table 4 in the online Data Supplement).

We also assessed the stability of prepared samples to determine the length of time they could be stored before analysis. Analysis of samples stored in the cooled autosampler tray for a period of 3 days showed no change in lipoprotein profile (see Supplemental Fig. 1 in the online Data Supplement).

SAMPLE CARRYOVER

The capillaries in the system are flushed between samples to minimize the potential for carryover. To confirm that this washing is sufficient, we analyzed sample diluent (ammonium acetate with dextran sulfate) used for LDL sample dilutions after a 1:200 dilution sample. There was a small amount of carryover in the LDL region (<1.3%). The overall lipoprotein carryover was <1.5% (see Supplemental Fig. 2 in the online Data Supplement).

EFFECT OF SPECIMEN TYPE

Matched samples were drawn in red top (serum), serum separator (SST), EDTA, and heparin collection tubes. Each collection type from the same individual was prepared and analyzed in the same assay. We evaluated the data using the serum as the target concentration for each lipoprotein region and determining the percent recovery of the other sample types relative to it. Recoveries for the 3 collection tubes were as follows: SST, 82%-101%; EDTA plasma, 91%-114%; and heparin plasma, 88%-95%.

Fig. 4. Relationships between IM and biochemical measurements in 79 subjects.

METHOD COMPARISON

We correlated IM-determined concentrations of total HDL, HDL large, total LDL, total IDL, total VLDL, and non-HDL cholesterol (sum of LDL, IDL, and VLDL fractions) with biochemically measured triglycerides, HDL cholesterol, LDL cholesterol (calculated), and plasma apoAl and apoB. Fig. 4 shows the individual data and correlation (r) for the comparisons among the most closely related measurements. As expected, given that IM measures particle concentrations and not the lipid or protein content of lipoproteins, there were varying degrees of correlation among the measure- ments. Notably, total LDL particle concentrations as measured by IM correlated more strongly with apoB (r = 0.90) than with LDL cholesterol (r = 0.79), and the correlation with apoB increased slightly for the non-HDL (r = 0.92) (Fig. 4, A-C). Although linear regression analysis fit these data well, the results using a polynomial fit were slightly improved and went through the origin (non-HDL, r = 0.94). Significant correlations were also observed between VLDL and triglycerides, and between HDL large and total HDL and HDL cholesterol (Fig. 4D-F). Similar correlations were seen for apoAl (see Supplemental Fig. 3 in the online Data Supplement).

Table 1. Summary statistics of individual lipoprotein fractions in healthy adults.8

Table 1 gives summary statistics characterizing the result distributions observed in our healthy adult population for the respective lipoprotein fractions as described in Materials and Methods and shown for representative subjects in Fig. 3.

Discussion

We herein describe and validate a new method for fractionation, sizing, and quantification of lipoprotein particles using the principles of aerosol science. IM has been used routinely in analyzing particles in aerosols (10). We have adapted this instrument to analyze large biological macromolecules in a highthroughput environment. Lipoproteins are ideal candidates, as they can be isolated from other serum proteins on the basis of their densities. An important step in this quantification is the separation of albumin from HDL. This was achieved by incorporating an albumin-binding dye (reactive green 19 dextran), thereby creating an albumin particle of increased density. Binding of multiple albumin molecules to the RGD would presumably increase the difference in density between albumin and HDL, permitting separation of the two with a lower-density solution. Incorporation of the RGD together with deuterium oxide as the dense underlayer obviated the need for dialysis before sample analysis by IM. In addition, this procedure eliminates the effects of added salts on lipoprotein particle structure and composition that may occur with ultracentrifugation. The method permits lipoproteins to be isolated from 72 samples in <2.5 h and provides optimal removal of albumin with equivalent recovery of HDL and LDL, IDL, and VLDL. Albumin removal was estimated to be >99% by a process of standard addition (data not shown).

The method described here is accurate and reproducible, with intra- and interassay CVs of <1.0% for LDL particle size and <18% and 16% for HDL and LDL fractions, respectively. There was more variation in the IDL and VLDL regions (intra- and interassay CVs of <26%) because of the absolute lower number of particles. This variation could be reduced by incorporating a lower dilution and an additional scan on the IM. We did not investigate this in the present study.

The IM method offers advantages over other procedures for lipoprotein particle analysis in that it not only measures particle size accurately on the basis of physical principles, but also directly counts the particles present at each size. This approach thereby provides the only direct measurement of lipoprotein particle size and concentration for each lipoprotein subclass, from small HDL to large VLDL. The differences between IM and standard measurements of plasma lipid and lipoprotein cholesterol concentrations are due in part to interindividual variations in the compositions of lipoprotein particles. However, there were strong correlations between plasma concentrations of apoB and IM measurements of both LDL and non-HDL particle concentrations (r = 0.90 and 0.92, respectively). The correlations reflect the fact that 1 molecule of apoB is present in each of these particles, and hence that apoB provides a measure of total particle concentration. The data for the large, apoB-containing particles were fitted slighdy better by a polynomial regression passing through zero than by a linear regression. This indicates that directly measured particle concentrations are overestimated by apoB, particularly at lower plasma concentrations. This may be due to a limitation of the immunoassay to accurately measure apoB in all lipoprotein particles, and/or the immunoassay measures both apoB100 and apoB48. Further investigation will be required to determine the basis for this finding, but it may indicate a limitation in the use of plasma apoB concentration as a surrogate measure of lipoprotein particle number. The poorer correlation (r = 0.79) between total LDL and LDL cholesterol is likely due to the varying concentrations of cholesterol in these particles. VLDL correlated with triglycerides (r = 0.84) and demonstrated the expected increased content of triglycerides with larger VLDL particles. HDL cholesterol was strongly correlated with HDL large (r = 0.86), but the correlation was weaker for total HDL (r = 0.59). This may reflect a greater variation in either the recovery or composition of HDL small, the densest lipoprotein particle. Similar results were obtained for correlations of HDL large and total HDL with plasma apoAl.

We used the IM procedure to determine reference distributions for particle concentrations for a series of fractions throughout the lipoprotein particle spectrum, as well as for peak LDL diameter, based on a population of individuals who met the NCEP ATP criteria for optimal plasma lipid and lipoprotein cholesterol measurements (2). Use of this methodology in studies of patients with cardiovascular disease and other pathologic states will permit testing of its clinical utility in the assessment of risk and management of these conditions.

Grant/Funding Support: This study was funded by Quest Diagnostics Incorporated.

Financial Disclosures: M. P. Caulfield, S. Li, G. Lee, W. A. Salameh, and R. E. Reitz are employees of Quest Diagnostics Inc. W. H. Benner was a consultant for Quest Diagnostics during this study. R. M. Krauss received financial support from Quest Diagnostics for his laboratory during this study.

Acknowledgments: We thank J. C. Geaney and Katie Wojnoonski for technical assistance, Colette Scheele and Esther Carlton for their help in collecting and analyzing the reference distribution data, Earl Cornell for help with the programming of the ion mobility instrument interface, and Jeff Radcliff for help with the manuscript preparation.

4 Nonstandard abbreviations; CVD, cardiovascular disease; ATP, Adult Treatment Panel III; apo, apolipoprotein; IDL, intermediate- density lipoprotein; IM, ion mobility; VLDL, very-low-density lipoprotein; DMA differential mobility analyzer; RGD, reactive green 19 dextran; LOD, limit of detection; LOQ, limit of quantification.

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Michael P. Caulfield,1* Shuguang Li,1 Gloria Lee,1 Patricia J. Blanche,2 Wael A. Satameh,1 W. Henry Benner,3 Richard E. Reitz,1 and Ronald M. Krauss2

1 Quest Diagnostics Nichols Institute, San Juan Capistrano, CA; 2 Children’s Hospital Oakland Research Institute (CHORI), Oakland, CA; 3 Lawrence Livermore National Laboratory, Livermore, CA.

* Address correspondence to this author at: Quest Diagnostics Nichols Institute, 33608 Ortega Highway, San Juan Capistrano, CA 92675. E-mail michael.p.caulfield@questdiagnostics.com.

Received November 19, 2007; accepted April 16, 2008.

Previously published online at DOI: 10.1373/dinchem.2007.100586

Copyright American Association for Clinical Chemistry Aug 2008

(c) 2008 Clinical Chemistry. Provided by ProQuest LLC. All rights Reserved.