CA2337547A1 - A new method for hdl particle sizing by polyacrylamide gradient gel electrophoresis using whole plasma - Google Patents
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- 229940098773 bovine serum albumin Drugs 0.000 description 1
- UDSAIICHUKSCKT-UHFFFAOYSA-N bromophenol blue Chemical compound C1=C(Br)C(O)=C(Br)C=C1C1(C=2C=C(Br)C(O)=C(Br)C=2)C2=CC=CC=C2S(=O)(=O)O1 UDSAIICHUKSCKT-UHFFFAOYSA-N 0.000 description 1
- 230000003293 cardioprotective effect Effects 0.000 description 1
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- G01N27/00—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means
- G01N27/26—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means by investigating electrochemical variables; by using electrolysis or electrophoresis
- G01N27/416—Systems
- G01N27/447—Systems using electrophoresis
- G01N27/44704—Details; Accessories
- G01N27/44717—Arrangements for investigating the separated zones, e.g. localising zones
- G01N27/44721—Arrangements for investigating the separated zones, e.g. localising zones by optical means
- G01N27/44726—Arrangements for investigating the separated zones, e.g. localising zones by optical means using specific dyes, markers or binding molecules
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- G01N1/42—Low-temperature sample treatment, e.g. cryofixation
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- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
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- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/92—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving lipids, e.g. cholesterol, lipoproteins, or their receptors
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- G01N15/00—Investigating characteristics of particles; Investigating permeability, pore-volume or surface-area of porous materials
- G01N15/02—Investigating particle size or size distribution
- G01N15/0266—Investigating particle size or size distribution with electrical classification
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Abstract
Low plasma levels of high-density lipoprotein (HDL) cholesterol have been associated with an increased risk of coronary heart disease (CHD). HDL are very heterogeneous with respect to size and apolipoprotein content and the objective of the present study was to develop a method to generate lipid-stainable standards that would allow for the assessment of HDL particle size from whole plasma using polyacrylamide gradient gel electrophoresis (PAGGE). Lipid-stainable plasma standards were obtained by subjecting isolated red blood cells to hemolysis by either freezing at -20°C or -80°C
overnight or to rapid exposure to liquid nitrogen and mixing the hemolysis products with plasma aliquots. All three methods were highly reproducible in producing Sudan black lipid-stainable standards ranging from 75 .ANG. to 200 .ANG.. With the use of these standard bands, HDL particle size was assessed with intra and inter assay coefficients of variation below 0.8% and 1.2% respectively. This use of lipid-stainable HDL standards simplifies the assessment of HDL particle size by PAGGE using whole plasma, without having to go through cost and time consuming; ultracentrifugation procedures.
overnight or to rapid exposure to liquid nitrogen and mixing the hemolysis products with plasma aliquots. All three methods were highly reproducible in producing Sudan black lipid-stainable standards ranging from 75 .ANG. to 200 .ANG.. With the use of these standard bands, HDL particle size was assessed with intra and inter assay coefficients of variation below 0.8% and 1.2% respectively. This use of lipid-stainable HDL standards simplifies the assessment of HDL particle size by PAGGE using whole plasma, without having to go through cost and time consuming; ultracentrifugation procedures.
Description
A new method for HDL particle sizing by polyacrylamide gradient gel electrophoresis using whole plasma INTRODUCTION
High-density lipoprotein (HDI,) cholesterol is recognized for its important role as an inverse risk predictor for the development of coronary heart disease (1).
For instance, it has been suggested that for each 1 % increase in plasma HDL cholesterol concentration, the risk of CHD was. reduced by 1 to 1.5% (2). However, the cholesterol content of HDL, i.e. what is being measured in the routine clinical biochemistry procedure, represents only one aspect of the particles, which have been shown to be heterogeneous with respect to size, apolipoprotein content and density (3-5).
Several methods have been used in the past to isolate and characterize HDL
particles, such as sequential ultracentrifugation, precipitation and immunoaffinity chromatography (6-8). HDL particles can also be separated according to size using non-denaturing polyacrylamide gradient gel electrophoresis (PAGGE) (6,9,10).
However, the only molecular weight (or size) standards currently available for the assessment of HDL
particle size by PACJGE are the protein-stainable standards from Pharmacia ( 10), which are revealed by Coomassie blue staining. As band sharpness and uniqueness of HDL
isolated from whole plasma and revealed by Coomassie blue is not always optimal, the assessment of HDL particle size using these protein-stainable standards first requires that HDL 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 low density lipoprotein (LDL) particle size by PAGGE ( 11 ).
However, to the bfat of our knowledge, there are currently no lipid-stainable HDL
standards 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 standards 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 standards.
METHODS
Blood samples were; collected from an antecubital vein into vacutainer tubes containing Na2 EDTA (0.15%). After separation of plasma from blood cells by centrifugation (6000 rpm, 4°C, 15 min), plasma and hemolysed erythrocytes were kept at 4°C separately.
Hemolysis of blood red cells Three hemolysis conditions were compared. Isolated red blood cells were first subjected to overnight freezing at -20 °C or -f30 °C. Hemolysis was also carried out by subjecting cryovials of isolated red blood cells to liquid nitrogen for 3 cycles of freezing (5 seconds) while being thawed with warm water between each cycle. Then, 2 ~1 of the isolated hemolyzed erythrocytes were mixed with 200 y1 of plasma from the same individual ( 1 :100) (v/v) (herea:fter refered to a~~ hemolyzed plasma).
Electrophoresis of hemolyzed plasma Briefly, aliquot of 10 ~.1 of the henuolyzed plasma sample were mixed 1:1 (v/v) with a sampling buffer containing 20% sucrose and 0.25% bromophenol blue and loaded onto a nondenaturing 4-30% polyacrylamide gradient gel. A 1 S min pre-run at 75 V
preceeded the electrophoresis of hemolyzed plasma samples at 150V for 15 hours. Gels were stained with Sudan black 0.07% overnight and destained for a period of three hours with of ethanol 45%. Gels were then stored in a solution containing acetic acid 9% and methanol (20%) until they recovered their original size.
Size determination of lipid-stainable bands in hemolyzed plasma 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 standards from Pharmacia, were then cut and subjected to Coomassie blue staining for 1 hour; 3) the Coomassie blue stained extremities were finally allowed to retrieve their original sizes in a solution of acetic acid (9%) and methanol (20%) for 3 hours (Figure 1 ).
Image and statistical analyses Gels were analyzed using the Imagemaster I D computer software (Pharmacia LKB, Uppsala, Sweden). 'The diameter of each lipid-stainable band of the hemolyzed plasma was computed using; a standard curve derived from the relative migration distance (Rf) of the protein-stainable Pharmacia standards. Statistical analyses were performed with the SAS statistical package (SAS Institute, Carry, North Caarolina).
RESULTS
Data showed that size determination of each lipid-stainable HDL standard was highly reproducible with a coefficient of variation based on 20 consecutives measurements of less than 3%. Figure 2 shows tha~.t the three methods used to hemolyze erythrocytes, namely freezing at -20°C, -80°C or with liquid nitrogen, yielded nearly identical lipid-stainable bands. These lipid-stainalble standards remained stable for at least 6 months when kept at -80°C (data not shown). Red blood cells had to be mixed with plasma to generate lipid-stainable bands. Indeed, no band appeared on the gel when saline 0.9%
was mixed with red blood cells or when isolated red blood cells alone were subjected to PAGGE. Erythrocytes and plasma from both normolipidemic or hyperlipidemic individuals could both yield distinct and measurable lipid-stainable HDL
standards following the hemolysis procedure.
Measurement of HDL particle size Two distinct approaches for the measurement of HDL particle size have been used: 1- an integrated HDL diameter that took into account the relative contribution of each subclass of HDL for a given individual (Figure 3), 2- HDL "peak" particle size, which corresponded to the diameter of the most prominent HDL subclass (12). The integrate HDL particle diameter measurement was found to be accurate and reliable with intra and inter assay coefficients of variation of 0.7% and 0.9% respectively for a subject with small HDL particles and of 1.0% and 1.4% for an individual with large HDL
particles (Table 1). Relatively similar results were obtained when computing HDL peak particle size (not shown).
DISCUSSION
Many epidemiological studies have shown a negative relationship between HDL
cholesterol concentrations and the risk of CHD (2,13-15). Furthermore, there is growing evidence suggesting that most of the cardioprotective properties of HDL are associated with the HDLz fraction (larger particles) rather than the HDL3 fraction (smaller particles) (4,16-19). The study of HDL particle size by PAGGE has been traditionally quite tedious. Indeed, HDL first had to bf: isolated from other plasma lipoproteins using costly and time-consuming ultracentrifugation procedures. This was necessary because 1) the only adequate high molecular weight standards co-migrating in the HDL region on PAGGE were Coomassie blue stainable protein standards and 2) the large number of plasma proteins co-migrating with HDL on PAGGE did not allow for an accurate and reliable determination of particle size by Coomassie blue staining. For these reasons, HDL size determination has been quite difficult particularly in clinical settings where a large number of samples are processed simultaneously.
The present study describes a new reliable and reproducible method to measure HDL
particle size by PAGGE using whole plasma and lipid-stainable standards.
Indeed, we have developed a rapid procedure that generates standards of high molecular weights, which comigrate in the HDL region on PAGGE and are stainable with Sudan black.
This approach greatly facilitates the analysis of HDL particle size by PAGGE since it can be carried out using whole plasma, without having to go through time-consuming ultracentrifugation procedures necessary to isolate HDL. Lipid-stainable standards obtained as a result of blood hemolysis have been generated using blood samples from several individuals displaying various metabolic characteristics (normolipidemic vs hyperlipidemic, as vvell as different blood types). Analyses indicated that blood from a majority of subjects will provide distinct and identifiable lipid-stainable bands on PAGGE following the freezing-thawing cycles (20,21 ). It was interesting to note that the lipid-stainable bands obtained by ,subjecting red blood cells to hemolysis were consistant in terms of number and molecular weight (diameter) irrespective of the subjects'characteristi.cs. This suggests that the process leading to the formation of lipid-stainable macromolecules does not appear to be dependant upon specific aspects of the blood donor. However, it is recommended to use blood from individuals with low plasma HDL cholesterol concentration, in order to maximize the definition and the visibility of the lipid-stainable bands on the gel. Indeed, the lipid-stainable HDL
standards are less likely to be obscured by the HDL itsf:lf when the latter is present in a small quantity.
The effects of freezing on red blood cells has been investigated previously.
Chanutin and Curnish (21 ) 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"C or -79°C. T he 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 and found with PAGGE has yet to be documented.
In conclusion, we have developped a new method to generate lipid-stainable standards that are very helpful for HDL particle size determination from whole plasma using PAGGE. We found this method to be rapid and reproducible while providing several identifiable .and distinct bands. Our data showed that HDL particle size can be determined with great accuracy using these lipid-stainable standards. From a clinical standpoint, this technique will greatly facilitate the study of the relationship between HDL pauticle size and CHD. Indeed, HDL particle diameter can be assessed by PAGGE
from whole plasma instead of having to first isolate the lipoprotein fraction.
Considering the importance of HDL in the etiology of CHD, substantive new information could therefore be obtained using this new simple and accessible technique.
REFERENCE LIST
1. Castelli, W.P., J.T. Doyles, and T. Cordon. 1977. HDL-cholesterol and other lipids in coronam heart disease: The cooperative lipoprotein phenotyping study.
Circulation 55:767-772.
High-density lipoprotein (HDI,) cholesterol is recognized for its important role as an inverse risk predictor for the development of coronary heart disease (1).
For instance, it has been suggested that for each 1 % increase in plasma HDL cholesterol concentration, the risk of CHD was. reduced by 1 to 1.5% (2). However, the cholesterol content of HDL, i.e. what is being measured in the routine clinical biochemistry procedure, represents only one aspect of the particles, which have been shown to be heterogeneous with respect to size, apolipoprotein content and density (3-5).
Several methods have been used in the past to isolate and characterize HDL
particles, such as sequential ultracentrifugation, precipitation and immunoaffinity chromatography (6-8). HDL particles can also be separated according to size using non-denaturing polyacrylamide gradient gel electrophoresis (PAGGE) (6,9,10).
However, the only molecular weight (or size) standards currently available for the assessment of HDL
particle size by PACJGE are the protein-stainable standards from Pharmacia ( 10), which are revealed by Coomassie blue staining. As band sharpness and uniqueness of HDL
isolated from whole plasma and revealed by Coomassie blue is not always optimal, the assessment of HDL particle size using these protein-stainable standards first requires that HDL 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 low density lipoprotein (LDL) particle size by PAGGE ( 11 ).
However, to the bfat of our knowledge, there are currently no lipid-stainable HDL
standards 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 standards 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 standards.
METHODS
Blood samples were; collected from an antecubital vein into vacutainer tubes containing Na2 EDTA (0.15%). After separation of plasma from blood cells by centrifugation (6000 rpm, 4°C, 15 min), plasma and hemolysed erythrocytes were kept at 4°C separately.
Hemolysis of blood red cells Three hemolysis conditions were compared. Isolated red blood cells were first subjected to overnight freezing at -20 °C or -f30 °C. Hemolysis was also carried out by subjecting cryovials of isolated red blood cells to liquid nitrogen for 3 cycles of freezing (5 seconds) while being thawed with warm water between each cycle. Then, 2 ~1 of the isolated hemolyzed erythrocytes were mixed with 200 y1 of plasma from the same individual ( 1 :100) (v/v) (herea:fter refered to a~~ hemolyzed plasma).
Electrophoresis of hemolyzed plasma Briefly, aliquot of 10 ~.1 of the henuolyzed plasma sample were mixed 1:1 (v/v) with a sampling buffer containing 20% sucrose and 0.25% bromophenol blue and loaded onto a nondenaturing 4-30% polyacrylamide gradient gel. A 1 S min pre-run at 75 V
preceeded the electrophoresis of hemolyzed plasma samples at 150V for 15 hours. Gels were stained with Sudan black 0.07% overnight and destained for a period of three hours with of ethanol 45%. Gels were then stored in a solution containing acetic acid 9% and methanol (20%) until they recovered their original size.
Size determination of lipid-stainable bands in hemolyzed plasma 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 standards from Pharmacia, were then cut and subjected to Coomassie blue staining for 1 hour; 3) the Coomassie blue stained extremities were finally allowed to retrieve their original sizes in a solution of acetic acid (9%) and methanol (20%) for 3 hours (Figure 1 ).
Image and statistical analyses Gels were analyzed using the Imagemaster I D computer software (Pharmacia LKB, Uppsala, Sweden). 'The diameter of each lipid-stainable band of the hemolyzed plasma was computed using; a standard curve derived from the relative migration distance (Rf) of the protein-stainable Pharmacia standards. Statistical analyses were performed with the SAS statistical package (SAS Institute, Carry, North Caarolina).
RESULTS
Data showed that size determination of each lipid-stainable HDL standard was highly reproducible with a coefficient of variation based on 20 consecutives measurements of less than 3%. Figure 2 shows tha~.t the three methods used to hemolyze erythrocytes, namely freezing at -20°C, -80°C or with liquid nitrogen, yielded nearly identical lipid-stainable bands. These lipid-stainalble standards remained stable for at least 6 months when kept at -80°C (data not shown). Red blood cells had to be mixed with plasma to generate lipid-stainable bands. Indeed, no band appeared on the gel when saline 0.9%
was mixed with red blood cells or when isolated red blood cells alone were subjected to PAGGE. Erythrocytes and plasma from both normolipidemic or hyperlipidemic individuals could both yield distinct and measurable lipid-stainable HDL
standards following the hemolysis procedure.
Measurement of HDL particle size Two distinct approaches for the measurement of HDL particle size have been used: 1- an integrated HDL diameter that took into account the relative contribution of each subclass of HDL for a given individual (Figure 3), 2- HDL "peak" particle size, which corresponded to the diameter of the most prominent HDL subclass (12). The integrate HDL particle diameter measurement was found to be accurate and reliable with intra and inter assay coefficients of variation of 0.7% and 0.9% respectively for a subject with small HDL particles and of 1.0% and 1.4% for an individual with large HDL
particles (Table 1). Relatively similar results were obtained when computing HDL peak particle size (not shown).
DISCUSSION
Many epidemiological studies have shown a negative relationship between HDL
cholesterol concentrations and the risk of CHD (2,13-15). Furthermore, there is growing evidence suggesting that most of the cardioprotective properties of HDL are associated with the HDLz fraction (larger particles) rather than the HDL3 fraction (smaller particles) (4,16-19). The study of HDL particle size by PAGGE has been traditionally quite tedious. Indeed, HDL first had to bf: isolated from other plasma lipoproteins using costly and time-consuming ultracentrifugation procedures. This was necessary because 1) the only adequate high molecular weight standards co-migrating in the HDL region on PAGGE were Coomassie blue stainable protein standards and 2) the large number of plasma proteins co-migrating with HDL on PAGGE did not allow for an accurate and reliable determination of particle size by Coomassie blue staining. For these reasons, HDL size determination has been quite difficult particularly in clinical settings where a large number of samples are processed simultaneously.
The present study describes a new reliable and reproducible method to measure HDL
particle size by PAGGE using whole plasma and lipid-stainable standards.
Indeed, we have developed a rapid procedure that generates standards of high molecular weights, which comigrate in the HDL region on PAGGE and are stainable with Sudan black.
This approach greatly facilitates the analysis of HDL particle size by PAGGE since it can be carried out using whole plasma, without having to go through time-consuming ultracentrifugation procedures necessary to isolate HDL. Lipid-stainable standards obtained as a result of blood hemolysis have been generated using blood samples from several individuals displaying various metabolic characteristics (normolipidemic vs hyperlipidemic, as vvell as different blood types). Analyses indicated that blood from a majority of subjects will provide distinct and identifiable lipid-stainable bands on PAGGE following the freezing-thawing cycles (20,21 ). It was interesting to note that the lipid-stainable bands obtained by ,subjecting red blood cells to hemolysis were consistant in terms of number and molecular weight (diameter) irrespective of the subjects'characteristi.cs. This suggests that the process leading to the formation of lipid-stainable macromolecules does not appear to be dependant upon specific aspects of the blood donor. However, it is recommended to use blood from individuals with low plasma HDL cholesterol concentration, in order to maximize the definition and the visibility of the lipid-stainable bands on the gel. Indeed, the lipid-stainable HDL
standards are less likely to be obscured by the HDL itsf:lf when the latter is present in a small quantity.
The effects of freezing on red blood cells has been investigated previously.
Chanutin and Curnish (21 ) 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"C or -79°C. T he 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 and found with PAGGE has yet to be documented.
In conclusion, we have developped a new method to generate lipid-stainable standards that are very helpful for HDL particle size determination from whole plasma using PAGGE. We found this method to be rapid and reproducible while providing several identifiable .and distinct bands. Our data showed that HDL particle size can be determined with great accuracy using these lipid-stainable standards. From a clinical standpoint, this technique will greatly facilitate the study of the relationship between HDL pauticle size and CHD. Indeed, HDL particle diameter can be assessed by PAGGE
from whole plasma instead of having to first isolate the lipoprotein fraction.
Considering the importance of HDL in the etiology of CHD, substantive new information could therefore be obtained using this new simple and accessible technique.
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2. Cordon, D.J. and B.M. Rifkind. 1989. High density lipoprotein: the clinical implications of recent studies. NEngl Jlhled 321:1311-1316.
3. Fruchart, J.C., G. Ailhaud, and J.M. Bard. 1993. Heterogeneity of high density lipoprotein particles. Circulation 87:III22-III27 4. Silverman, D.L, G.S. Ginsburg, and R.C. Pasternak. 1993. High density lipoproprotein subfractions. Am JMed '94:636-645.
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6. Atger, V., D. Malon, M.C. Bert:iere, F. N'Diaye, and A. Girard-Globa. 1991.
Cholesterol distribution between high-density-lipoprotein subfractions HDL2 and HDL3 determined in serum by discontinuous gradient gel electrophoresis. Clip Chenz 37:1149-1152.
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7. Cheung, M.C., J.P. Segrest, J.J. Albers, J.T. Cone, C.G. Brouillette, B.H.
Chung, M.
Kashyap, M.A. Glasscock:, and G.M. Anantharamaiah. 1987. Characterization of high density lipoprotein subspecies: structural studies by single vertical spin ultracentrifugation and imununoaffinity chromatography. JLipidRes 28:913-929.
Chung, M.
Kashyap, M.A. Glasscock:, and G.M. Anantharamaiah. 1987. Characterization of high density lipoprotein subspecies: structural studies by single vertical spin ultracentrifugation and imununoaffinity chromatography. JLipidRes 28:913-929.
8. Warnick, G.R., J. Benderson, and J.J. Albers. 1982. Dextran sulfate-Mg2+
precipitation procedi.cre for quantitation of high- density-lipoprotein cholesterol. Clin ChenZ
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28:137!x-1388.
9. Blanche, P.J., E.L. Gong, T.M. Forte, and A.V. Nichols. 1981.
Characterization of human high-density lipoproteins by gradient gel electrophoresis. Biochim Biophys Acta 665:408-419.
Characterization of human high-density lipoproteins by gradient gel electrophoresis. Biochim Biophys Acta 665:408-419.
10. Li, Z., J.R. McNamara, J.M. Grdovas, and E.J. Schaefer. 1994. Analysis of high density lipoproteins by a modified gradient gel eletrophoresis method. JLipid Res 35:1698-1711.
11. Krauss, R.M. and P.J. Blanche. 1992. Detection and quantitation of LDL
subfractions.
Curr Opin Lipidul 3: 37 7-383.
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12. Lamarche, B., K.D. Uffelman, H.R. Barrett, G. Steiner, and G.F. Lewis.
1998. Analysis of particle size and lipid composition as determinants of the metabolic clearance of human high density lipoproteins in a rabbit model. JLipid Res 39:1162-1172.
1998. Analysis of particle size and lipid composition as determinants of the metabolic clearance of human high density lipoproteins in a rabbit model. JLipid Res 39:1162-1172.
13. Jacobs, D.R.J., :1.L. Mebane, S.I. Bangdiwala, M.H. Criqui, and H.A.
Tyroler. 1990. High density lipoprotein cholesterol as a predictor of cardiovascular disease mortality in men and women: the follow-up study of the Lipid Research Clinics Prevalence Study. Ana JEpiderniol 131:32-47.
Tyroler. 1990. High density lipoprotein cholesterol as a predictor of cardiovascular disease mortality in men and women: the follow-up study of the Lipid Research Clinics Prevalence Study. Ana JEpiderniol 131:32-47.
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Jacobs, Jr., S. Bangdiwala, and H.A. Tyroler. 1989. High-density lipoprotein cholesterol and cardiovascular disease. Four prospective American Studies.
Circulation 79:8-15.
Jacobs, Jr., S. Bangdiwala, and H.A. Tyroler. 1989. High-density lipoprotein cholesterol and cardiovascular disease. Four prospective American Studies.
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Despres. 1997.
Associations of HDLG and HDL3 subfractions with ischemic heart disease in men.
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Despres. 1997.
Associations of HDLG and HDL3 subfractions with ischemic heart disease in men.
Prospective results from 'the Quebec Cardiovascular Study. Arterioscler Thromb vast Biol 17:1098-1105.
17. Calabresi, L., G. Franceschini, M. Sirtori, G. Gianfranceschi, P. Werba, and C.R. Sirtori.
1990. Influence of serunn triglycerides on the HDL pattern in normal subjects and patients with coronary arl:ery disease. Atherosclerosis 84:41-48.
1990. Influence of serunn triglycerides on the HDL pattern in normal subjects and patients with coronary arl:ery disease. Atherosclerosis 84:41-48.
18. Johansson, J., L.A. Carlson, C. Landou, and A. Hamster. 1991. High density lipoproteins and coronary atherosclerosis. A strong inverse relation with the largest particles is confined to normotriglycl:ridemic patients. Arterioscler Thromb 11:174-182.
19. Fruchart, J.C., G. Castro, and P. L)uriez. 1996. Apolipoprotein-AI-containing particles and atherosc:lerosis. Isr J Abed S'ci 32:498-502.
20. Skrabut, E.M., J.P. Crowley, N. C'atsirnpoolas, and C.R. Valeri. 1976. The effect of cryogenic storage on human erythrocyte membrane proteins as' determined by polyacrylamide-gel electrophoresis. Cryobiology 13:395-403.
21. Chanutin, A. and R.R. CL11'111S11. 1966. Effect of freezing red blood cells and hemolyzates at different temperatures. Electrophoretic and methemoglobin studies. Arch Biochena Biophys 113:114-121.
Table 1: Coefficients of variation for the determination of an integrated HDL
particle size by PAGGE using whole plasma and lipid-stainable standards.
Coefficient of variation (%) HDL particles diameter " Intra assay Inter assay Small 0.70 0.89 Large 0.95 1.40 " These numbers are based on 24 consecutive measures performed in one individual with small HDL particles (mean HDL size 83.0 ~ 0.74 (SD) ~) and one individual with large HDL particles (mean HDL size 89.4 ~ 1.25 ~).
FIGURES HEADINGS
Figure 1 : Size determination of lipid-stainable bands by 4-30% PAGGE.
A: Pharmacia high molecular weight protein standards stained with Coomassie blue (Thyroglobulin 170~~, ferritin 122, catalase 104, lactate dehydrogenase 81.61, bovine serum albumin 71A). B : Hemolyzed plasma stained with Sudan black (lipid staining).
The "standard" bands were obtained by subjecting isolated red blood cells to either freezing at -20°C or -80°C overnight or to rapid exposure to liquid nitrogen and by mixing the hemolysis products with plasma aliquots.
Figure 2 : Comparison of three diifferent hemolysis approaches that generate lipid-stainable HDL standards for PA.GGE. A : The standards (hemolyzed plasma). B
Hemolysis product obtained at -:?0"C. C : Hemolysis at -80°C. and D :
The hemolysis with liquid nitrogen. The arrows indicate the presence of lipid stainable bands.
Figure 3 : Determination of HDL particle size with lipid-stainable standard.
HDL
particle size was determined by den~sitometric scanning of stained 4-30%
polyacrylamide gradient gel lipid-st;~inable standards based on relative distance (Rfj of migration. 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 third subclass (80.7t~) while the integrated HDL diameter = (25% x 99f~) + (13% x 85.60 + (50% x 80.70 + (12% x 75.70 = 85.3.
Table 1: Coefficients of variation for the determination of an integrated HDL
particle size by PAGGE using whole plasma and lipid-stainable standards.
Coefficient of variation (%) HDL particles diameter " Intra assay Inter assay Small 0.70 0.89 Large 0.95 1.40 " These numbers are based on 24 consecutive measures performed in one individual with small HDL particles (mean HDL size 83.0 ~ 0.74 (SD) ~) and one individual with large HDL particles (mean HDL size 89.4 ~ 1.25 ~).
FIGURES HEADINGS
Figure 1 : Size determination of lipid-stainable bands by 4-30% PAGGE.
A: Pharmacia high molecular weight protein standards stained with Coomassie blue (Thyroglobulin 170~~, ferritin 122, catalase 104, lactate dehydrogenase 81.61, bovine serum albumin 71A). B : Hemolyzed plasma stained with Sudan black (lipid staining).
The "standard" bands were obtained by subjecting isolated red blood cells to either freezing at -20°C or -80°C overnight or to rapid exposure to liquid nitrogen and by mixing the hemolysis products with plasma aliquots.
Figure 2 : Comparison of three diifferent hemolysis approaches that generate lipid-stainable HDL standards for PA.GGE. A : The standards (hemolyzed plasma). B
Hemolysis product obtained at -:?0"C. C : Hemolysis at -80°C. and D :
The hemolysis with liquid nitrogen. The arrows indicate the presence of lipid stainable bands.
Figure 3 : Determination of HDL particle size with lipid-stainable standard.
HDL
particle size was determined by den~sitometric scanning of stained 4-30%
polyacrylamide gradient gel lipid-st;~inable standards based on relative distance (Rfj of migration. 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 third subclass (80.7t~) while the integrated HDL diameter = (25% x 99f~) + (13% x 85.60 + (50% x 80.70 + (12% x 75.70 = 85.3.
Claims (9)
1. Method for the preparation of Sudan black-stainable standards for use within a non-denaturing polyacrylamide gradient gel electrophoresis, the method comprising the steps of:
a) subjecting isolated red blood cells to hemolysis and therefore obtaining a solution of hemolysed red blood cells;
b) standardising the Sudan black-stainable molecules of the solution of hemolysed red blood cells in order to obtained Sudan black-stainable standards;
1) migrating separately in a non-denaturing polyacrylamide gradient gel electrophoresis i) an aliquot of the solution obtained in step (b); and ii) a solution of protein-stainable standards including proteins of known particle sizes;
a) subjecting isolated red blood cells to hemolysis and therefore obtaining a solution of hemolysed red blood cells;
b) standardising the Sudan black-stainable molecules of the solution of hemolysed red blood cells in order to obtained Sudan black-stainable standards;
1) migrating separately in a non-denaturing polyacrylamide gradient gel electrophoresis i) an aliquot of the solution obtained in step (b); and ii) a solution of protein-stainable standards including proteins of known particle sizes;
2) staining with Sudan black a portion of the gel electrophoresis where the aliquot of the solution of hemolysed red blood cells have migrated and therefore obtaining Sudan black-stained bands;
3) staining with Coomassie blue a portion of the gel electrophoresis where the solution of protein-stainable standards have migrated and therefore obtaining Coomassie blue-stained bands;
4) calibrating the particle size of each Sudan black-stained band by using the relative migration distance of each Sudan black-stained band in comparison with the relative migration distances of the Coomassie blue-stained bands which correspond to known particle sizes respectively.
2. Method of claim 1, wherein the red blood cells are isolated from a normolipidemic or hyperlipidemic individual.
3. Method of claim 2, wherein the individual is normolipidemic.
4. Sudan black-stainable standards for use within a non-denaturing polyacrylamide gradient gel electrophoresis prepared by the method of claim 1, or 3.
2. Method of claim 1, wherein the red blood cells are isolated from a normolipidemic or hyperlipidemic individual.
3. Method of claim 2, wherein the individual is normolipidemic.
4. Sudan black-stainable standards for use within a non-denaturing polyacrylamide gradient gel electrophoresis prepared by the method of claim 1, or 3.
5. Sudan black-stainable standards of claim 4, in a range of particles sizes of about 220 to 70.ANG..
6. Sudan black-stainable standards of claim 4, in a range of particles sizes of human HDL.
7. Method for identifying the particle size of one or more Sudan black-stainable molecules in a sample, comprising the steps of:
a) migrating separately in a non-denaturing polyacrylamide gradient gel electrophoresis:
i) an aliquot of them Sudan black-stainable standards prepared by the method of claim 1, 2 or ;3; and ii) an aliquot of the sample;
d) staining with Sudan black the gel electrophoresis and therefore obtaining stained bands; and e) determining the particle size of each Sudan black-stained band in the migration area of the sample by using their relative migration distance in comparison with the relative migration distances of the bands in the migration area of the Sudan black-stainable standards which correspond to known particle sizes respectively.
a) migrating separately in a non-denaturing polyacrylamide gradient gel electrophoresis:
i) an aliquot of them Sudan black-stainable standards prepared by the method of claim 1, 2 or ;3; and ii) an aliquot of the sample;
d) staining with Sudan black the gel electrophoresis and therefore obtaining stained bands; and e) determining the particle size of each Sudan black-stained band in the migration area of the sample by using their relative migration distance in comparison with the relative migration distances of the bands in the migration area of the Sudan black-stainable standards which correspond to known particle sizes respectively.
8. Method of claim 7, wherein the sample is a plasma sample.
9. Method of claim 8, wherein the plasma sample is a human plasma sample and the Sudan black-stainable molecules of the sample are HDL; and wherein the Sudan black-stainable standards are prepared by the method of claim 3.
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| CN102297842B (en) * | 2010-06-22 | 2014-12-24 | 国家海洋局第三海洋研究所 | Method for rapidly testing lipid content of microalgae with sudan black B |
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