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Correspondence to:
Benoît Lamarche, To whom correspondence should be addressed., benoit.lamarche{at}crchul.ulaval.ca (E-mail)
Low plasma levels of HDL cholesterol have been associated with an increased risk of coronary heart disease. HDL particles are heterogeneous with respect to size and apolipoprotein content. The objective of the present study was to develop a method to generate lipid-stainable calibrators that would allow the assessment of HDL particle size from whole plasma, using polyacrylamide gradient gel electrophoresis (PAGGE). Lipid-stainable HDL calibrators were obtained by subjecting isolated red blood cells to hemolysis either by freezing at - 20 or - 80°C overnight or by rapid exposure to liquid nitrogen and mixing of the hemolysis products with plasma aliquots. All three methods were highly reproducible in producing Sudan black lipid-stainable HDL calibrators ranging from 75 to 200 Å. The assessment of HDL particle size with these lipid-stainable HDL calibrators was also highly reproducible, with a coefficient of variation below 5.5%.
These lipid-stainable HDL calibrators simplify the assessment of HDL particle size by PAGGE using whole plasma, without the need for costly, time-consuming ultracentrifugation procedures. — Pérusse, M., A. Pascot, J-P. Després, C. Couillard, and B. Lamarche. A new method for HDL particle sizing by polyacrylamide gradient gel electrophoresis using whole plasma. J. Lipid Res. 2001. 42: 1331;–1334.
Supplementary key words:
HDL particle size, hemolysis, lipid staining
HDL cholesterol is recognized for its important role as an inverse risk predictor for the development of coronary heart disease (1). It must be stressed that the cholesterol content of HDL, that is, what is being measured in the routine clinical biochemistry procedure, is only one feature of the particles, which have been shown to be heterogeneous with respect to size, apolipoprotein content, and density (2).
Several methods such as sequential ultracentrifugation, precipitation, and immunoaffinity chromatography (3) (4) have been used in the past to isolate and characterize HDL particles. HDL particles can also be separated according to size, using nondenaturing polyacrylamide gradient gel electrophoresis (PAGGE) (5) (6). The only molecular weight (or size) standards currently available for the assessment of HDL particle size by PAGGE are the protein-stainable standards from Pharmacia (Piscataway, NJ) (6), which are revealed by Coomassie blue staining. Band sharpness and uniqueness of HDL isolated from whole plasma and revealed by Coomassie blue are not always optimal because of the presence of several proteins that comigrate in the HDL size range on PAGGE. For that reason, the assessment of HDL particle size by using protein-stainable standards requires that HDL first be separated by sequential ultracentrifugation, a costly and time-consuming procedure that greatly limits the use of this method, particularly in clinical settings. Lipid staining with Sudan black has been widely used in the past and has given reliable results on the characterization of LDL particle size by PAGGE (7). However, to the best of our knowledge, there are currently no lipid-stainable HDL standards or calibrators that allow for the determination of HDL particle size by PAGGE using whole plasma.
The objective of the present study was therefore to develop a rapid and simple method that produces lipid-stainable calibrators for the measurement of HDL particle diameter by PAGGE. We found that the hemolysis product of red blood cells generated reproducible lipid-stainable bands that comigrated in the HDL size range on the gels. We found that these bands can be used with reliability as lipid-stainable HDL diameter calibrators.
Blood samples were collected from an antecubital vein into Vacutainer tubes (Becton Dickinson, Franklin Lakes, NJ) containing Na2 EDTA (0.15%). After separation of plasma from blood cells by centrifugation (6,000 rpm, 4°C, 15 min), plasma and isolated erythrocytes were kept at 4°C separately for less than 1 day, until they were used as described below.
Hemolysis of blood red cells
Electrophoresis of hemolyzed plasma
Size determination of lipid-stainable bands in hemolyzed plasma
Image and statistical analyses
Data presented in Table 1 show the reproducibility of calibrating nine lipid-stainable HDL bands obtained from a representative hemolyzed plasma mixture, using the Pharmacia HMW standards. Intra- and interassay coefficients of variation were all <5.5%. Of note is the fact that bands with smaller diameter tended to be measured with more accuracy than larger bands. The three methods used to hemolyze erythrocytes, namely freezing at -20 or -80°C or with liquid nitrogen, yielded nearly identical lipid-stainable bands (data not shown). Lipid-stainable calibrators remained stable for at least 6 months when kept at -80°C.
Hemolyzed red blood cells had to be mixed with plasma to generate lipid-stainable bands. Indeed, no band appeared on the gel when 0.9% saline was mixed with red blood cells or when isolated red blood cells alone were subjected to PAGGE. On the other hand, lipoprotein-depleted plasma (d > 1.21 g/ml), when mixed with hemolyzed erythrocytes, generated distinct and identifiable bands on PAGGE when stained with Sudan black (data not shown). Finally, erythrocytes and plasma from both normolipidemic or hyperlipidemic individuals yielded distinct and measurable lipid-stainable HDL standards after the hemolysis procedure. Fig 2 depicts the HDL calibration bands obtained from two individual donors having distinct lipid profiles. The diameters of each of their calibration bands were similar but not identical. Thus, each preparation of lipid-stainable HDL standards must be independently calibrated with the Pharmacia HMW standards.
HDL particle size was computed as two distinct measures that have been described previously (6) (8): 1) an integrated HDL diameter that took into account the relative contribution of each subclass of HDL for a given individual, and 2) HDL "peak" particle size, which corresponded to the diameter of the most prominent HDL subclass ( Fig 3). As shown in Table 2, both the peak and integrated HDL particle diameter measurements were found to be accurate and reliable in individuals characterized by either large or small HDL particles.
Finally, analyses of a sample of 55 men (mean age, 35.4 ± 11.7 years) indicated that the integrated HDL particle size correlated significantly with plasma HDL cholesterol (r = 0.65, P < 0.001), HDL2 cholesterol (r = 0.63, P < 0.001), HDL3 cholesterol (r = 0.37, P = 0.005), HDL phospholipids (r = 0.58, P < 0.001), and apolipoprotein A-I (apoA-I) only-containing particles (r = 0.34, P = 0.01) but not with apoA-I- and apoA-II-containing lipoproteins (r = -0.18, P = 0.19). Similar correlations, but of lower magnitude, were observed between the HDL peak particle size and other HDL characteristics (data not shown).
Many epidemiological studies have shown a negative relationship between HDL cholesterol concentrations and the risk of CHD (9) (10) (11) (12). Furthermore, there is growing evidence suggesting that most of the cardioprotective properties of HDL are associated with the HDL2 fraction (larger particles) rather than the HDL3 fraction (smaller particles) (13) (14). The study of HDL particle size by PAGGE has been traditionally quite tedious. Indeed, HDL first had to be isolated from other plasma lipoproteins by costly and time-consuming ultracentrifugation procedures. This was necessary because 1) the only adequate HMW standards comigrating in the HDL region on PAGGE were Coomassie blue-stainable protein standards; and 2) the large number of plasma proteins comigrating with HDL on PAGGE did not allow for an accurate and reliable determination of particle size by Coomassie blue (protein) staining. This has made HDL size determination particularly difficult in clinical settings, where a large number of samples are processed concurrently.
The present study describes a new reliable and reproducible method to measure HDL particle size by PAGGE using whole plasma and lipid-stainable calibrators. We have developed a rapid procedure that generates standards of HMW, which comigrate in the HDL region on PAGGE and that are identifiable with Sudan black staining. This approach greatly facilitates the analysis of HDL particle size by PAGGE because it can be carried out with whole plasma, without having to go through time-consuming ultracentrifugation procedures necessary to isolate HDL. Lipid-stainable calibrators obtained as a result of blood hemolysis have been generated with blood samples from several individuals displaying various metabolic characteristics (normolipidemic vs. hyperlipidemic, as well as different blood types). Previous analyses indicated that blood from a majority of subjects may give rise to distinct and identifiable lipid-stainable bands on PAGGE after the freezing-thawing cycles (15) (16). However, it is recommended that each preparation of lipid-stainable HDL standards be independently calibrated because small variations between different donor preparations could lead to a systematic bias in HDL particle size analysis. It is also recommended that blood from individuals with low plasma HDL cholesterol concentration or lipoprotein-depleted plasma be used in order to maximize the definition and visibility of the lipid-stainable bands on the gel. Indeed, the lipid-stainable HDL calibrators are less likely to be obscured by the HDL itself when the latter is present in a small quantity.
The effects of freezing on red blood cells has been investigated previously. Chanutin and Curnish (16) have shown that new, fast-moving components appeared on electrophoresis after subjecting washed, intact human erythrocytes to freezing for 1, 2, 3, and 7 days at ;–20 or –79°C. The authors described in great details these new electrophoretic patterns identified by Coomassie blue (protein) staining but did not discuss their lipid content. Thus, the exact composition of the lipid-stainable bands of hemolyzed red blood cells observed on PAGGE will have to be documented through future investigations.
In conclusion, we have developed a new method to generate lipid-stainable HDL calibrators that may be helpful for HDL particle size determination from whole plasma by PAGGE. This method provides several identifiable and distinct bands and is rapid and reproducible. Our data showed that HDL particle size can be determined with great accuracy, using these lipid-stainable standards. From a clinical standpoint, this technique may greatly facilitate the study of the relationship between HDL particle size and coronary heart disease (CHD). Indeed, HDL particle diameter can be assessed by PAGGE from whole plasma instead of having first to isolate the lipoprotein fraction. Considering the importance of HDL in the etiology of CHD, substantive new information could therefore be obtained by this new simple and accessible technique.
Abbreviations: HMW, high molecular weight; PAGGE, polyacrylamide gradient gel electrophoresis.
This study was supported in part by an operating grant from the Canadian Institute for Health Research (MOP-35970). Benoît Lamarche is the recipient of a Canada Research Chair in Nutrition, Functional Foods, and Cardiovascular Health.
Manuscript received October 16, 2000; and in revised form March 22, 2001
Copyright © 2001 by Lipid Research, Inc.
methods
A new method for HDL particle sizing by polyacrylamide gradient gel electrophoresis using whole plasma
Mélanie Pérussea,b,
Agnès Pascotb,
Jean-Pierre Desprésa,b,c,
Charles Couillardb, and
Benoît Lamarchea,b
a Department of Food Sciences and Nutrition, Laval University, Québec, Canada G1K 7P4
b Lipid Research Center, CHUL Research Center, Québec, Canada G1V 4G2
c Québec Heart Institute, Québec, Canada G1V 465
ABSTRACT
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
INTRODUCTION
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
MATERIALS AND METHODS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
Three hemolysis conditions were compared. Isolated red blood cells were first subjected to overnight freezing at -20 or -80°C. Hemolysis was also carried out by subjecting cryovials of isolated red blood cells to liquid nitrogen for three cycles of freezing (5 s) while being thawed with warm water between each cycle. Aliquots of 2 µl of the isolated hemolyzed erythrocytes were mixed with 200 µl of plasma from the same individual (1:100) (v/v) (hereafter referred to as hemolyzed plasma).
Briefly, aliquots of 8;–10 µl of the hemolyzed plasma mixture were mixed 1:1 (v/v) with a sampling buffer containing 20% sucrose and 0.25% bromophenol blue. The entire preparation (16;–20 µl) was loaded onto a nondenaturing 4;–30% polyacrylamide gradient gel. All gels were prepared in batches in our laboratory, using a multicasting chamber (Bio-Rad Laboratories Canada, Mississauga, ON, Canada). A 3% stacking gel was cast on top of the gradient gel to allow for the loading of the samples onto the gels. Gels without samples were first subjected to a 15-min prerun at 125 V. Hemolyzed plasma samples were then loaded onto the gels and subjected to electrophoresis at 70 V for 20 min and at 150 V thereafter for 15 h. Gels were stained with a 0.07% (w/v) Sudan black-ethanol solution overnight and destained for a period of 3 h with 45% ethanol. Gels were then incubated for a minimum of 6 h in a 0.8% acetic acid and 4% methanol solution. This allowed the gels to resume their original rectangular shape.
The diameter of each lipid-stainable band of hemolyzed plasma samples was determined as follows: 1) gels were stained with Sudan black as described above; 2) the extremities of the gel, which contained the protein-stainable high molecular weight (HMW) standards from Pharmacia, were then cut and subjected to Coomassie blue staining for 1 h; and 3) the Coomassie blue-stained extremities were finally allowed to resume their original sizes and shapes in a solution of acetic acid (9%) and methanol (20%) for 3 h ( Fig 1).
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Figure 1.
Size determination of lipid-stainable bands by 4;–30% PAGGE. A: Pharmacia HMW protein standards stained with Coomassie blue (thyroglobulin, 170 Å; ferritin, 122 Å; catalase, 104 Å; lactate dehydrogenase, 81.6 Å; bovine serum albumin, 71 Å). B: Hemolyzed plasma stained with Sudan black (lipid staining). The HDL "calibrator" bands were obtained by subjecting isolated red blood cells to freezing at - 20 or - 80°C overnight, or rapid exposure to liquid nitrogen, and mixing the hemolysis products with plasma aliquots.
Gels were analyzed with Imagemaster 1D Prime computer software (Pharmacia LKB, Uppsala, Sweden). The diameter of each lipid-stainable band of hemolyzed plasma was calibrated by computing a log-linear standard curve of the protein-stainable Pharmacia HMW standards as a function of their relative migration distance (Rf). A similar approach was used to assess HDL particle size, using the calibrated lipid-stainable bands. Statistical analyses were performed with the SAS statistical package (SAS Institute, Cary, NC).
RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
View this table:
[in a new window]
Table 1.
Coefficients of variation associated with the calibration of lipid-stainable HDL bands, using Pharmacia high molecular weight standards
View larger version (130K):
[in a new window]
Figure 2.
Example of lipid-stainable HDL calibrators generated with red blood cells and plasma from donors with different plasma lipid levels. A: A 44-year-old man (body mass index, 36.5 kg/m2) with LDL cholesterol (LDL-C) = 3.2 mM, HDL-C = 0.72 mM, and triglycerides = 3.6 mM. B: A 40-year-old man (body mass index, 32.8 kg/m2) with LDL-C = 3.6 mM, HDL-C = 1.08 mM, and triglycerides = 1.4 mM. STD: Lipid-stainable HDL calibrators.
View larger version (33K):
[in a new window]
Figure 3.
Determination of HDL particle size, using lipid-stainable HDL calibrators. HDL particle size (A) was determined by densitometric scanning of the 4;–30% polyacrylamide gradient gel stained with Sudan black. The standard curve was computed as a log-linear curve of the lipid-stainable calibrators (STD) as a function of their relative distance of migration (Rf). The calibration band that migrated the farthest into the gel was attributed an Rf of 1.0. HDL peak particle size corresponded to the size of the most prominent HDL subclass for a given individual. The integrated HDL particle size was calculated by multiplying the size of each HDL subclass by its relative contribution (in percent). In this example, the HDL peak particle size corresponded to the size of the fourth subclass (78.0 Å) while the integrated HDL diameter = (16% x 100.1 Å) + (15% x 91.4 Å) + (10% x 85.8 Å) + (52% x 78.0 Å) + (7% x 75.7 Å) = 84.2 Å.
View this table:
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Table 2.
Mean coefficient of variationa for the determination of HDL particle size by PAGGE using lipid-stainable HDL calibrators in a sample of 55 men
DISCUSSION
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
FOOTNOTES 
ACKNOWLEDGMENTS
REFERENCES
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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