215
Clinica Chimica Acta, 64 (1975) 215-216 @ Elsevier Scientific Publishing Company,
BRIEF TECHNICAL
Amsterdam
- Printed
NOTE
in The Netherlands
--
CCA 7272
A MODIFIED METHOD FOR PREPARATIVE OF PLASMA LIPOPROTEINS*
SAU W. CHEUNGa~**,
RALPH
A. JERSILD,
bDepartment aDepartment of Medical Genetics, of Medical Genetics, Indiana University School
(Received
ULTRACENTRIFUGATION
Jrb and JOE C. CHRISTIANC of Anatomy of Medicine,
and ‘Department Indianapolis, Ind.
(U.S.A.)
April 2, 1975)
The method of Have1 et al. [l] as previously modified by Hatch and Lees [2] was further modified to obtained very low density lipoproteins (VLDL), low density lipoproteins (LDL) and high density lipoproteins (HDL), by using high speed polycarbonate tubes (5/8 inch X 3 inch, Beckman Instrument Inc., Palo Alto, Calif., U.S.A.) which allowed centrifugation of partially full tubes as compared to thin plastic tubes [l] . The air space kept the lipoproteins from adhering to the cap and allowed flexibility of sample sizes. In addition the samples were spun at higher speeds which reduced the centrifugation time. Plasma was first centrifuged in a fixed rotor at 145 000 X g (12 hours at 10°C). The VLDL was pipetted off and density was adjusted to 1.063 [l],the sample was then spun at 151 000 X g (13 hours) for removal of LDL and finally adjusted to 1.21, spun at 158 000 X g (22 hours) for removal of HDL. These modifications save 29 hours of ultracentrifugation [2] and the method was reproducible with plasma samples from 4 to 8 ml. The lipoprotein fractions were tested for purity using cellulose acetate electrophoresis [3] and immunoelectrophoresis [4]. Only one band was found per fraction with each of these procedures. The fractions were examined by electron microscopy after negative staining with 2% aqueous phosphotungstate [5]. The VLDL fraction contained particles 300 to 750 ,& in diameter; LDL, 200-300 8, and the majority of the HDL particles 70-90 a with a few particles as large as 200 A. These findings are compatible with previously reported results [ 5,6] indicating that the present method gives adequate separation of plasma lipoproteins. * This is publication No. 75-01 from the Indiana University, Department of Medical Genetics and was supported in part by Contract number 71-2307 with the National Heart and Lung Institute of the National Institutes of Health, by PHS PO1 GM 21054, HSA 924, the John A. Hartford Foundation, Inc. and The Indiana Heart Association. ** Present address: Channing Laboratory, Boston City Hospital. Boston, Mass.. U.S.A.
216
References 1 2 3 4 6 6
Havel, R., Eder, H.A. and Bragdon, J.H. (1955) J. Clin. Invest. 34, 1345-1353 Hatch, F.T. and Lees, R.S. (1968) Adv. Lipid Res. 6, l-63 Christian, J.C. and Cheung, S.W. (1973) Clin. Chim. Acta 43, 23-26 Hirschfeld, J. (1960) Science Tools, The LKB instrument J. 7, 18-25 Hamilton. R.L., Havel, R.J., Kane. J.P., Blaurock, A.E. and Sata, T. (1970) Science Nichols, A.V. (1969) Proc. Natl. Acad. Sci. U.S.A. 64. 1128-1136
172. 475478
Clinica Chimica Acta, 64 (1975) 215-216 @ Elsevier Scientific Publishing Company,
BRIEF TECHNICAL
Amsterdam
- Printed
NOTE
in The Netherlands
--
CCA 7272
A MODIFIED METHOD FOR PREPARATIVE OF PLASMA LIPOPROTEINS*
SAU W. CHEUNGa~**,
RALPH
A. JERSILD,
bDepartment aDepartment of Medical Genetics, of Medical Genetics, Indiana University School
(Received
ULTRACENTRIFUGATION
Jrb and JOE C. CHRISTIANC of Anatomy of Medicine,
and ‘Department Indianapolis, Ind.
(U.S.A.)
April 2, 1975)
The method of Have1 et al. [l] as previously modified by Hatch and Lees [2] was further modified to obtained very low density lipoproteins (VLDL), low density lipoproteins (LDL) and high density lipoproteins (HDL), by using high speed polycarbonate tubes (5/8 inch X 3 inch, Beckman Instrument Inc., Palo Alto, Calif., U.S.A.) which allowed centrifugation of partially full tubes as compared to thin plastic tubes [l] . The air space kept the lipoproteins from adhering to the cap and allowed flexibility of sample sizes. In addition the samples were spun at higher speeds which reduced the centrifugation time. Plasma was first centrifuged in a fixed rotor at 145 000 X g (12 hours at 10°C). The VLDL was pipetted off and density was adjusted to 1.063 [l],the sample was then spun at 151 000 X g (13 hours) for removal of LDL and finally adjusted to 1.21, spun at 158 000 X g (22 hours) for removal of HDL. These modifications save 29 hours of ultracentrifugation [2] and the method was reproducible with plasma samples from 4 to 8 ml. The lipoprotein fractions were tested for purity using cellulose acetate electrophoresis [3] and immunoelectrophoresis [4]. Only one band was found per fraction with each of these procedures. The fractions were examined by electron microscopy after negative staining with 2% aqueous phosphotungstate [5]. The VLDL fraction contained particles 300 to 750 ,& in diameter; LDL, 200-300 8, and the majority of the HDL particles 70-90 a with a few particles as large as 200 A. These findings are compatible with previously reported results [ 5,6] indicating that the present method gives adequate separation of plasma lipoproteins. * This is publication No. 75-01 from the Indiana University, Department of Medical Genetics and was supported in part by Contract number 71-2307 with the National Heart and Lung Institute of the National Institutes of Health, by PHS PO1 GM 21054, HSA 924, the John A. Hartford Foundation, Inc. and The Indiana Heart Association. ** Present address: Channing Laboratory, Boston City Hospital. Boston, Mass.. U.S.A.
216
References 1 2 3 4 6 6
Havel, R., Eder, H.A. and Bragdon, J.H. (1955) J. Clin. Invest. 34, 1345-1353 Hatch, F.T. and Lees, R.S. (1968) Adv. Lipid Res. 6, l-63 Christian, J.C. and Cheung, S.W. (1973) Clin. Chim. Acta 43, 23-26 Hirschfeld, J. (1960) Science Tools, The LKB instrument J. 7, 18-25 Hamilton. R.L., Havel, R.J., Kane. J.P., Blaurock, A.E. and Sata, T. (1970) Science Nichols, A.V. (1969) Proc. Natl. Acad. Sci. U.S.A. 64. 1128-1136
172. 475478
