Medical Device Link.

Assay Development

Gradient-gel electrophoresis in the clinical evaluation of heart disease risk

A novel process for preparing high-resolution gradient gels for use in determining a new biomarker for risk is presented.

Anh Le

Gradient-gel electrophoresis (GGE) has long been used in research laboratories for isolating and identifying proteins. Application of the technology in clinical laboratories, however, has been limited owing to such obstacles as lack of standardization and the time-consuming postelectrophoresis processing required before the gel can be analyzed and the lack of standardization.

One clinical area in which GGE can be seen to have considerable potential is in the determination of risk for heart disease. Among numerous biochemical markers currently being investigated for their suitability in identifying individuals who may be at increased risk for experiencing a heart attack is low-density-lipoprotein (LDL) particle diameter, known as LDL size or LDL phenotype. This biomarker can be effectively assessed using GGE.

The following discussion provides some background on the apparent correlation between LDL size and heart disease and on GGE technology, and then focuses on a novel process for preparing ready-to-use high-resolution linear gradient gels instrumental in determining LDL particle diameter.

Low-Density Lipoproteins and Heart Disease

Figure 1. Schematic of a novel motorized device for the preparation of gradient gel. (click to enlarge)

Despite significant advances in cholesterol-lowering therapy, heart disease remains the leading cause of death in the United States and other industrialized countries. Cholesterol- and other lipid-bearing particles contribute to the development of cardiovascular disease in the following way.

Lipids are transported by the bloodstream as spherical particles ranging in diameter from 8 to 50 nm. The larger particles, those that are responsible for delivering triglycerides to peripheral tissues for storage, have a higher ratio of low-density lipid to high-density protein, and thus have a lower overall density. Smaller particles that remain in the circulation after the triglycerides have been unloaded are typically enriched in cholesterol and have a higher density.

Of the four major forms of cholesterol-carrying particles—chylomicrons, very-low-density lipoproteins (VLDLs), low-density lipoproteins (LDLs), and high-density lipoproteins (HDLs)—LDLs account for 60% of the cholesterol in the blood and are considered a major risk factor for heart disease. An abnormal form of LDL, lipoprotein(a) (Lp(a)) is also found in the plasma of some individuals. Lp(a) consists of an LDL molecule complexed to a molecule of a protein known as apo(a). The five types of lipid-carrying particles in plasma have widely different physical characteristics (see Table I).¹ In nonfasting plasma samples, a band containing larger, lipid-rich particles can be observed corresponding to remnant particles containing dietary fats. The presence of this band in fasting plasma of some individuals may be indicative of abnormal particle processing and convey an independent risk for heart disease.

A review study of clinical trials involving drugs found that twice as many patients receiving cholesterol-lowering medications experienced a slowdown in disease progression as did individuals in the placebo group.² However, 30% of the patients getting the drugs continued to develop more-severe atherosclerotic lesions in spite of their cholesterol being reduced to the desired goal level. Why might this be? The reason appears to be that some individuals may carry cholesterol predominantly in small-sized particles, which is more likely to contribute to the disease process. LDL size is a significant risk factor.

LDL Particle Size as a Disease Factor

Evidence Linking LDL Size and LDL Number. LDLs are spherical particles with a lipid core and a coat of hydrophilic components, including proteins. As mentioned, LDLs are responsible for the transport of 60% of the cholesterol in human blood. An LDL particle with a smaller than average core will contain a reduced amount of cholesterol. Therefore, to transport the same quantity of cholesterol within two individuals, one of which has generally smaller LDL particles, the cholesterol load in the person with small-diameter LDLs must be distributed among more particles. In view of the fact that the number of lipid molecules can determine the size of the particles, an individual with particles 10% smaller in diameter than those of another individual would, in order to transport the same concentration of LDL cholesterol, have to have 24% more particles in the bloodstream.¹

Figure 2. Digital camera images of GGE runs with segmental gradient gel 2.8/6.30 with a 16-hour run time (a) and, single gradient GGE 2.15 with 4-hour run time (b). C denotes the lanes containing the serum-based calibrator. The band corresponding to remnant is denoted as 1; Lp(a) as 2; LDL as 3; large HDL2 as 4; and small HDL3 as 5. (click to enlarge)

Removal of these LDL particles from the blood by the liver, however, is dependent on a limited number of dedicated gates known as receptors. When 24% more particles contend for access to these receptors, the rate of removal of LDLs is reduced, the number of particles in the same vicinity rises, and the increased traffic flow of LDL particles can result in a higher incidence of damaged particles in the blood. These damaged forms of LDL contribute to the development of lesions. Experiments exposing cells to high concentrations of normal LDLs failed to result in the formation of foam cells similar to those found in atherosclerotic lesions, whereas the addition of minimal amounts of damaged LDL particles could cause foam cell formation.³

The first evidence directly linking LDL size and LDL particle number in heart disease was published in 1980.⁴ While other lipid-carrying particles in human blood contain a mixture of proteins, LDL has one single-protein molecule. Thus, the protein content of LDL in plasma is related directly to particle number. The 1980 investigators demonstrated that patients with extremely elevated LDL cholesterol due to a genetic deficiency in hepatic receptors were unable to remove LDL from the bloodstream. As a result, these patients had a concomitant increase in LDL protein. The observed relationship between LDL cholesterol and LDL protein was actually no different from that seen in healthy controls with normal cholesterol levels. By contrast, patients with a comparable level of LDL cholesterol who had premature coronary artery disease (CAD) had 20% higher LDL protein. With fewer lipids per particle, these LDL particles are smaller and also more dense than normal LDL particles.

Evidence Linking LDL Size and LDL Damage. In vitro studies have indicated clearly that exposure to concentrations of LDL cholesterol two to three times higher than normal cannot alone cause cholesterol accumulation in cells with the subsequent formation of foam cells that is seen in human lesions.³ Several forms of chemical modification, including oxidative modification by myeloperoxidase, an enzyme that is found in abundance in human lesions, have been demonstrated to be sufficient to cause lipid accumulation.⁵

Of interest are data that have demonstrated different levels of susceptibility to oxidative modification among subpopulations of LDLs isolated from the plasma. Regardless of the clinical characteristics of the donor, a subpopulation of small, higher-density LDL particles was much more susceptible to oxidation than a subpopulation of larger LDL particles isolated from the same plasma sample. One possible hypothesis was that smaller particles have less antioxidant protection.⁶

Evidence Linking LDL Size and Risk for Heart Attack. While most evidence linking LDL size and risk for heart disease comes from case-control studies, several prospective studies have been published in support of this hypothesis as well. The Boston Area Health Study conducted more than a dozen years ago found that individuals with small, dense LDL particles had a threefold greater risk of myocardial infarction than those who did not, independent of age, sex, and obesity.⁷ Two six-year-old studies discovered that participants who eventually developed CAD had had significantly smaller LDL size at the baseline of the studies. The relationship was independent of triglyceride level, HDL, smoking status, blood pressure, and obesity, according to one of the studies.⁸ In the other, after the risk associated with triglyceride level had been taken into account, LDL particle diameter was not found more useful in predicting who would develop a heart attack during the follow-up period.⁹ In 1999, a study reported quantitative results: individuals with small, dense LDL particles at the time of entry into the study were 2.6 times more likely to have a heart attack over the following four years.¹⁰

Research into therapeutic outcomes, meanwhile, has concluded that individuals with small, dense LDL benefited the most from aggressive CAD risk-reduction therapy and also that therapeutic interventions that increase LDL size can result in improvement in coronary stenosis with an actual increase in the lumen diameter of coronary arteries.^11,12

Moreover, other work has demonstrated an association between small, dense LDL and insulin resistance syndrome, a metabolic abnormality closely related to increased risk of CAD.¹³

Gel Electrophoresis and GGE Technologies

Figure 3. Reproducibility of the gel scan for the biological calibrator and actual plasma samples. The relative distance of migration from the top of the gel is presented on the x-axis and the absorbance of 405 nm is presented on the y-axis. (click to enlarge)

Gel electrophoresis is commonly used to separate macromolecules according to their net electrostatic charge. In a conventional system, the concentration of the gel is uniform, and separation is based entirely on the rates of migration of the charged particles in the electrical field. The position of a band of molecules once the electrical current is stopped depends on the size and charge of the particles. A disadvantage of this technique is that the macromolecules can continue to migrate off the gel if the electrical field exists for too long. Thus, with a uniform gel, the distance of migration is a function of the size of the particle, its charge, and the duration of the electrophoresis.¹⁴

Gradient Gels. If gels are prepared with different concentrations of acrylamide, macromolecules can be separated with higher resolution and can be prevented from running off the gel. Acrylamide is usually caused to form a gel that is cross-linked with N,N'-methylene bisacrylamide. The basic principle is that, with a higher gel-solution concentration, the matrix that is formed upon polymerization will be more tightly meshed, and larger macromolecules will not be able to migrate past layers of predetermined pore size. In these gradient gels, the distance of migration is thus a function of the size of the macromolecule and the concentration gradient of the gel.¹⁵

In a typical GGE setup, the rate of mixing of two gel solutions with different acrylamide concentrations will determine the gradient of concentrations. The two different concentrations of gel solution are allowed to mix in equal proportion in a linear gradient, with the highest concentration being layered at the bottom and the lowest concentration constituting the top layer.¹⁵ Theoretically, a discontinuous gradient gel could be prepared that would consist of several layers with distinct concentrations. The highest-concentration solution would be the foundation layer, topped by a series of solutions with progressively lower concentrations.

Systems Used in LDL Size Studies. Most studies that have demonstrated an association between LDL size and heart disease risk have used gradient polyacrylamide gel to determine the diameter of LDL particles.

Conventional gradient gels are typically thin (1–1.5 mm) and narrow (10–15 cm). Thin gel can accept only small volumes of sample. It has to be stained with a protein-specific stain and then destained to reduce background before the position of the LDL bands can be determined. During these steps the gel has to be removed from its casing, a step that can distort the gel and affect the true position of the band. Narrow gels can accommodate only six to eight samples per gel, and thus have a relatively low throughput. These performance limitations of conventional gradient gel are due to one thing: because the gel solutions are layered in the gel chamber through a stationary dispensing tube, variability in the diffusion rate between areas near the dispensing tip and those at the sides of the gel raise the possibility of gel concentration from left to right being inconsistent.

The system just described is basically the one used by the Donner Laboratories at the University of California, Berkeley, for the past 20 years.^6–9 It is available through Berkeley HeartLab Inc. (Burlingame, CA). Two separate gradient gels have to be used to analyze LDL and HDL from the sample plasma. Furthermore, Lp(a) cannot be visualized as a distinct band.

Another process based on gel electrophoresis uses a nongradient gel and determines the areas under the seven bands of LDL to assess lipoprotein distribution.¹⁶ This system, LipoPrint, is available from Quantimetrix (Redondo Beach, CA).

Figure 4. Resolution of a segmental gradient-gel system displaying results for a donor plasma sample in which LDL particles became smaller after incubation. The relative distance of migration from the top of the gel is presented on the x-axis and the absorbance of 405 nm is presented on the y-axis. (click to enlarge)

The separation of lipoprotein classes of different densities in a gravitational field is the basis of a novel process. Called VAP-II, it is available exclusively through Atherotech Inc. (Birmingham, AL). At the end of this procedure, a fraction of heavier density is at the bottom of the tube and a fraction of lower density is at the top. The contents of the tube can be fractionated by means of a fraction recovery system that allows a heavy (i.e., high-density) liquid to be introduced at the bottom of the tube so that the contents are displaced upward through an on-line derivatization process that generates a cholesterol profile. This profile is mathematically fitted to a series of Gaussian distribution to determine the position of the peak and the area under each peak. The position of the peak is related to density and thus indirectly to particle size, while the areas provide an estimate of the distribution of cholesterol among these subclasses. Lp(a) appears as a shoulder on the left of the LDL peak. Lp(a) concentration is assumed to account for the portion of the profile that cannot be explained by the major lipoprotein subclasses (VLDL, LDL, and HDL); a distinct peak for Lp(a) typically cannot be demonstrated.¹⁷

More recently, nuclear magnetic resonance (NMR) has been introduced to achieve the high-resolution isolation of lipoproteins.¹⁸ A 400-MHz NMR analyzer is used to detect the spectroscopic signals generated by the methyl group on lipid molecules. Deconvolution techniques have been employed to extract from the composite waveform 15 different spectra corresponding to 6 subpopulations of VLDL, 4 subpopulations of LDL, and 5 subpopulations of HDL.¹⁸ The spectrum reflects the clustering of methyl groups within the particles and thus corresponds to size distribution.

The NMR method is by far the fastest of all approaches now available. However, attempts to correlate results derived from this type of spectral analysis with physical separation of the lipoprotein fractions have been unsuccessful. In this system, the final data result from a three-step process: measurement of the plasma NMR spectrum from the sample, deconvolution of the spectral data by computer, and conversion of the signal amplitudes for each subclass to the desired units of concentration. The NMR technique represents a novel approach to dealing with the heterogeneity of cholesterol-carrying particles, but it requires an entirely different database to be built to demonstrate the clinical significance of the parameters.

A High-Resolution GGE System

In order to increase gel resolution in gradient-gel electrophoresis and to reduce postelectrophoresis processing time, thicker gels that can accommodate more samples must be developed. A patented process for gradient-gel preparation developed by Clinical Laboratory Development Group Inc. (Winter Park, FL) introduces a motorized assembly that allows the gel-solution dispensing tip to travel at a uniform speed from left to right (see Figure 1). The rate of movement of the dispensing arm in this system is synchronized with the solution flow rate and the width of the gel so as to deliver a uniform gradient solution from one edge of the gel to the other.

This gel-making station can prepare 3-mm-thick gels that can accommodate up to 25 samples for LDL size determination. The greater thickness of the gel allows the use of plasma or serum samples that have been stained for lipids. Migration of the LDL bands can be visualized in real time. At the end of the electrophoretic run, the gel can be captured for analysis by a high-resolution digital camera within four hours of the time the sample application is completed. The gel need not be removed from its casing.

The reproducibility of this process can be optimized by applying the concept of gel constant (Cgel) rather than using the conventional approach based on the relative mobility (Rf), common in sodium decyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE), which relates the migration distance to molecular weight in a semilogarithmic scale. In a linear gradient, by contrast, the gel constant relates particle diameter to a linear relationship between the distance of migration and the gel concentration thus:

where S_calibrator is the diameter of the known calibrator, L is the concentration of the lower gel solution, d_calibrator is the position of the band corresponding to the calibrator as measured from the top of the gradient, and Slope_gradient is the slope of the gradient.

While LDL is the major lipid-carrying particle of interest, the use of plasma samples that have been stained for lipids allows other cholesterol-carrying particles such as HDL and Lp(a) to be visualized as well. Sudan black generally is used as the lipid stain, though other stains may be available for this application.

Lp(a) levels vary widely among individuals; some people have undetectable levels. Only Lp(a) levels greater than 35 mg/dl can be visualized. Elevated Lp(a) has been suggested as an independent risk factor for CAD.²⁰

High levels of HDL are believed to be protective against heart disease. Individuals with elevated HDL typically exhibit two subpopulations of HDL designated as HDL₂ and HDL₃. The distribution of lipids between HDL₂ and HDL₃, as well as the size of these HDL fractions, has been shown to relate to CAD risk.²¹

Because the diameter of Lp(a) ranges between 28 and 32 nm and the diameter of HDL is approximately 9.5 nm for HDL₃ and 11.5 nm for HDL₂, for a test of the new process two linear gradients were prepared stacked one on top of the other: a 6–30% gel for the separation of HDL subclasses and a 2–8% gradient gel for the separation of Lp(a) and LDL. This is called a segmental gradient gel.

A comparison of digital gel images displays 12 samples and (in lanes 1, 8, and 15) three biological calibrators captured during runs with the segmental gradient gel (designated 2.8/6.30; see Figure 2a) and the single-gradient 2–15% gel (designated 2.15; see Figure 2b). As illustrated in Figure 2, the segmental gradient gel can provide information on the subclasses of HDL while the single gradient gel, which can be completed in 4–6 hours, is more specific for the larger particles. The calibrator in this experiment was a serum-based single-donor sample for which LDL, Lp(a), HDL₂, and HDL₃ particle sizes (molecular weight) had been determined via a combination of protein standards. Run times were 16 hours for the segmental GGE and 4 hours for the single-gradient GGE.

The reproducibility of the gel scan for the serum-based calibrator on four separate lanes for two different gels is evident (see Figure 3). Serum-based calibrator can be stored at –80°C for up to five years.

The reproducibility of the distance of migration for all four major lipoproteins present in the biological-serum-based calibrator used in the experiment has been charted (see Table II).¹⁹ With the known diameters for the four lipoprotein bands in the calibrator being 30.4 nm for Lp(a), 25.70 nm for LDL, 11.80 nm for HDL₂, and 9.60 nm for HDL₃, gel constants for these lipoproteins for a typical segmental gradient gel (2.8/6.30), calculated using the equation above, are 111.863, 112.748, 118.255, and 115.044, respectively.

The high resolution of segmental GGE makes possible the detection of small changes in particle size (see Figure 4). For the experiment, portions of the same whole-plasma sample were incubated for 4 hours at 4°C and 4 hours at 37°C. At body temperature, 37°C, a number of enzymes in the plasma were active, resulting in the redistribution of plasma lipids among lipid-carrying particles in time-dependent fashion. The net result for the sample donor was changes in size to smaller LDL particles and to larger HDL particles. Preliminary experimental data indicated that this process does not result in the formation of smaller LDLs in all individuals: only 30% of the plasma samples demonstrated the formation of smaller LDLs following incubation. (Incubations of whole plasma from some donors under identical conditions have resulted in either no change in LDL size or the generation of larger LDL particles.) Whether this propensity to form small LDL particles in vitro reflects some metabolic characteristics of the plasma is currently under investigation, as is its link to CAD risk.

Conclusion

Although determination of LDL size is recognized by Medicare as a reimbursable test procedure for the management of patients with lipid abnormalities, no FDA-approved kit is available for clinical use. The gradient-gel preparation technique described here holds promise in this regard. The next step in the development of an FDA-approvable kit will be validation of a size calibrator that can be standardized by the National Institute of Standards and Technology.

Thicker gels of 5 mm are now being investigated to allow the application of larger sample volumes in preparative isolation of charged macromolecules 3–35 nm in size. Gels can be prepared for the isolation of large amounts of macromolecules as an alternative to column chromatography, which tends to be slower. While current application of the segmental GGE process described here is focused on a diagnostic test that is increasingly gaining the interest of physicians and researchers, the availability of thick, highly reproducible gradient gels that can accept large sample volumes, as well as a greater number of samples, opens the door to other potential uses.

In addition, different lipid stains are being examined to determine their utility in directly quantitating lipid content in remnant, Lp(a), LDL, and HDL subclasses. Protein stains and affinity-specific stains can be used to detect proteins of interest. And the use of more-specific bioactive materials in the preparation of the gels is also under study.

References

1. AM Scanu, "Plasma Lipoproteins: An Overview," in Biochemistry and Biology of Plasma Lipoproteins, ed. AM Scanu and AA Spector (New York: Marcel Decker, 1986), 1–10.

2. GR Thompson, "Angiographic Trials of Lipid-Lowering Therapy: End of an Era," British Heart Journal 74 (1995): 343–347.

3. MS Brown and JL Goldstein, "Lipoprotein Metabolism in the Macrophage: Implications for Cholesterol Deposition in Atherosclerosis," Annual Review of Biochemistry 52 (1983): 223–261.

4. A Sniderman et al., "Association of Coronary Atherosclerosis with Hyperapobetalipoproteinemia [Increased Protein but Normal Cholesterol Levels in Human Plasma Low Density (b) Lipoproteins]," in Proceedings of the National Academy of Sciences USA 77 (1980): 604–608.

5. AM Fogelman et al., "Malondialdehyde Alteration of LDL Leads to Cholesterol Accumulation in Human Monocyte-Macrophages," in Proceedings of the National Academy of Sciences USA 77 (1980): 2214–2218.

6. DL Tribble et al., "Variations in Oxidative Susceptibility among Six LDL Subfractions of Differing Density and Particle Size," Atherosclerosis 93 (1992): 189–199.

7. MA Austin et al., "LDL Subclass Patterns and Risk of Myocardial Infarction," Journal of the American Medical Association 260 (1988): 1917–1921.

8. CD Gardner, SP Fortman, and RM Krauss, "Association of Small LDL Particles with the Incidence of CAD in Men and Women," Journal of the American Medical Association 276 (1996): 875–881.

9. MJ Stampfer et al., "A Prospective Study of Triglyceride, LDL Particle Diameter, and Risk of Myocardial Infarction," Journal of the American Medical Association 276 (1996): 882–888.

10. B Lamarche et al., "Small, Dense LDL Particles as a Predictor of the Risk of Ischemic Heart Disease in Men: Prospective Results from the Quebec Cardiovascular Study," Circulation 95 (1997): 69–75.

11. BD Miller et al., "Predominance of Dense LDL Particles Predicts Angiographic Benefit of Therapy in the Stanford Coronary Risk Intervention Project," Circulation 94 (1996): 2146–2153.

12. A Zambon et al., "Evidence for a New Pathophysiological Mechanism for CAD Regression: Hepatic Lipase-Mediated Changes in LDL Density," Circulation 99 (1999): 1959–1964.

13. SM Haffner et al., "LDL Size in African Americans, Hispanics, and Non-Hispanic Whites: The Insulin Resistance Atherosclerosis Study," Arteriosclerosis, Thrombosis, and Vascular Biology 19 (1999): 2234–2240.

14. I Smith, Chromatographic and Electrophoretic Techniques, vol. 2 (London: Heineman Medical Books, 1976).

15. JJ Pratt and WG Dangerfield, "Polyacrylamide Gels of Increasing Concentration Gradient for the Electrophoresis of Lipoproteins," Clinica Chimica Acta 23 (1969): 189–201.

16. I Rajman et al., "Investigation of LDL Subfractions as a Coronary Risk Factor in Normotriglyceridemic Men," Atherosclerosis 125 (1996): 231–242.

17. KR Kulkarni et al., "Identification and Cholesterol Quantification of Low-Density Lipoprotein Subclasses in Young Adults by VAP-II Methodology," Journal of Lipid Research 36 (1995): 2291–2302.

18. JD Otvos, EJ Jeyarajah, and DW Bennet, "Quantification of Plasma Lipoproteins by Proton Nuclear Magnetic Resonance Spectroscopy," Clinical Chemistry 37 (1991): 377–386.

19. X Li et al., "Protocol for the Preparation of a Segmental Linear Polyacrylamide Gradient Gel: Simultaneous Determination of Lp(a), LDL and HDL Particle Sizes," Journal of Lipid Research 38 (1997): 2603–2614.

20. EJ Schaefer et al., "Lp(a) Levels and Risk of Coronary Heart Disease in Men: The Lipid Research Clinics Coronary Primary Prevention Trial," Journal of the American Medical Association 271 (1994): 999–1003.

21. MC Cheung et al., "Altered Particle Size Distribution of apoA-I Containing Lipoproteins in Subjects with CAD," Journal of Lipid Research 32 (1991): 383–397.

Anh Le, PhD, is founder and chief scientific officer of Clinical Laboratory Development Group Inc. (Winter Park, FL). He can be reached via e-mail at ale@edgeconnect.com.

Photos and Illustrations Courtesy Clinical Laboratory Development Group Inc.

Copyright ©2002 IVD Technology