- Email: [email protected]
Specific elimination of albumin in serum lipoprotein preparations
ANALYTICAL
BIOCHEMISTRY
Specific
84,
598-603
Elimination Lipoprotein
(1978)
of Albumin Preparations
in Serum
In the preparation of pure serum lipoproteins by means of ultracentrifugation techniques, it is necessary to perform consecutive centrifugations in order to reduce the content of contaminating serum albumin (1) impairing apolipoprotein determination and purification and turnover studies with lz51-labeled lipoproteins. Extensive centrifugation, however, may cause in vitro changes in the composition of the lipoprotein particles (1,2). The present report describes the use of matrix-bound anti-albumin for specific elimination of albumin in very low-density lipoprotein (VLDL), low-density lipoprotein (LDL), and high-density lipoprotein (HDL) preparations from human serum. MATERIALS
AND METHODS
Materials. VLDL, LDL, and HDL were prepared from fasting subjects with different types of hyperlipoproteinemia, mainly type IV. Blood was allowed to clot at room temperature and serum was separated by low-speed centrifugation. EDTA was added to a final concentration of 0.05%. When not otherwise stated pooled VLDL, LDL, and HDL, respectively, from different subjects were used. Monovalent anti-human albumin (purified immunoglobulin fraction of the antiserum) produced in rabbits was purchased from DAKO-immunoglobulins, Copenhagen, Denmark. Cyanogen bromide-activated Sepharose 6MB from Pharmacia Fine Chemicals, Uppsala, Sweden, was used for imobilization of the antialbumin. Albumin-poor VLDL was isolated by density gradient centrifugation as described by Lindgren et al. (3) in an SW 40-Ti rotor using a Beckman Model L5-75 ultracentrifuge. A 40.3 rotor in a Beckman Model LSO ultracentrifuge was used for the isolation of albumin-poor LDL and HDL. LDL was isolated between densities of 1.006 and 1.063 g/cm3, and HDL, between 1.070 and 1.21 g/cm3 (4). Two centrifugations at each density were performed on these preparations of LDL and HDL. The centrifugation time was 16 hr at d = 1.006 g/cm3, 20 hr at d = 1.063 and 1.070 g/cm3, and 48 hr at d = 1.21 g/cm3. For the routine isolation of lipoprotein fractions a scheme with step0003-2697/78/0842-0598$02.00/O Copyright All rights
0 1978 by Academic Press, Inc. of reproduction in any form reserved.
598
SHORTCOMMUNICATIONS
599
wise increases in density was used with a single spin at each density. The procedure for VLDL and LDL isolation has been described in detail elsewhere (5). HDL was isolated from the bottom fraction after centrifugation at d = 1.063 g/cm3. The density was raised to d = 1.21 g/cm3 with a concentrated salt solution and HDL was isolated as a top fraction after centrifugation for 48 hr at 40,000 rpm in the 40.3 rotor. After centrifugation all samples isolated at a density greater than 1.006 g/cm3 were dialyzed against 0.9% sodium chloride. Agarose gel electrophoresis was performed as described before (5). Polyacrylamide disc gel electrophoresis of soluble apolipoproteins was carried out according to Ref. (6) in 11-cm-long glass tubes with an inner diameter of 5 mm. The separated proteins were stained with Coomassie brilliant blue R (Sigma) as described in Ref. (7). The technique of Lowry et al. was used for protein determination (8). The turbidity caused by lipids was eliminated by extraction with chloroform before photometry. Bovine serum albumin was used as standard and no correction was made for the difference in chromogenicity of the different proteins. Triglycerides and cholesterol were analyzed by means of a Technicon Auto Analyzer using standard methods (9,lO). Phospholipids were determined as described in Ref. (11). lz51-Labeled human serum albumin with a specific activity of 4 pCi/mg was prepared as described before (12) using purified albumin obtained from Kabi, Stockholm, Sweden. The labeled protein, after final dialysis against 0.9% sodium chloride, was bound to 99% by the antialbumin-Sepharose. lz51-Labeled LDL was prepared by a modified iodine monochloride method (13) yielding 100-300 pCi/mg of apolipoprotein. Column experitnents. The anti-albumin-Sepharose conjugate was prepared by mixing swollen CNBr-Sepharose 6MB (2 ml) with antialbumin (5 mg) in 0.1 M sodium bicarbonate buffer, 0.5 M with respect to sodium chloride (2 ml). After 2 hr at room temperature the gel was washed free of unbound protein and was treated with 1 M ethanolamine, pH 9, for 2 hr as previously described (14). The capacity of the column to bind albumin was in the range of 0.3-0.5 mg of albumin/ml of wet gel. To a column (5 x 25 mm) of anti-albumin-Sepharose was added lipoprotein preparation (0.5 ml) containing 1 mg of protein/ml. When the sample had drained into the gel, the column was washed three times with 0.5 ml of 0.195 m (1.006 g/ml) sodium chloride, and a total volume of 2 ml was collected in a volumetric flask. The flow rate was 0.1 mVmin. As gel support a polyamide filter was used. For recovery determinations 0.5 ml of the original lipoprotein preparation was diluted to 2 ml.
600
SHORT
COMMUNICATIONS
Elimination of albumin from lipoprotein preparations. To albumin-poor preparations of VLDL, LDL, and HDL containing 1 mg of protein/ml was added 40 pg of lz51-labeled human serum albumin/ml. The mixtures were left for 1 hr at room temperature. Aliquots of 0.5 ml of these preparations were applied to anti-albumin-Sepharose columns (5 x 25 mm) and the lipoproteins were eluted as described above. The antialbumin-Sepharose was then suspended in 1.5 ml of sodium chloride solution and the distribution of radioactivity between the effluent and the gel was measured. RESULTS
In order to examine if matrix-bound anti-albumin induced changes in the compositions or physical properties of VLDL, LDL, or HDL
FIG. 1. Electropherograms demonstrating the electrophoretic mobility of VLDL, LDL, and HDL in agarose gel. (1) Untreated lipoprotein preparation; (2) effluent from antialbumin-Sepharose column. The untreated preparations have been diluted to the same volume as the column effluents. See Materials and Methods.
SHORT COMMUNICATIONS
601
FIG. 2. Polyacrylamide disc gel electropherograms of soluble apohpoproteins of VLDL, LDL, and HDL. IF denotes the interface between the upper and lower gel, and R is the riboflavin marker. (1) Untreated lipoprotein preparations; (2) effluent from antialbumin-Sepharose columns. The untreated preparations have been diluted to the same volume as the column effluents.
from human serum, these lipoprotein fractions were isolated and purified by means of ultracentrifugation, and the different lipoproteins were passed through columns of anti-albumin-Sepharose. The content of triglyceride, total cholesterol, phospholipid, and protein in the different lipoprotein preparations, before and after passage through the columns was determined. Within experimental error the analyzed components of all lipoproteins were quantitatively recovered in the column effluents.
602
SHORT
COMMUNICATIONS
Further, pure lipoproteins treated with anti-albumin-Sepharose and untreated lipoproteins yielded identical patterns upon electrophoresis in agarose gel (Fig. 1) and gave closely compatible electropherograms upon polyacrylamide gel electrophoresis of soluble apolipoproteins (Fig. 2). These results indicate that the lipoproteins pass through the anti-albumin-Sepharose columns without change in the content of lipids and soluble apolipoproteins and with no change in electrophoretic properties. Under the present experimental conditions, blue Sepharose CL-6B, which is reported to remove specifically albumin from serum (15), adsorbed VLDL, LDL, and HDL to 4, 39, and 33%, respectively, as examined by protein, cholesterol, and triglyceride determinations. In order to examine if apolipoprotein B, which is insolubIe in tetramethylurea extracts of VLDL and LDL (6), adsorbed to the matrixbound anti-albumin, LDL labeled with lz51 in the B protein was applied to a column of anti-albumin-Sepharose. By means of repeated ultracentrifugation the LDL preparation used for labeling was made apparently albumin free, as determined by polyacrylamide disc gel electrophoretic analyses (6). The recovery of lz51-labeled LDL after passage through a column of anti-albuminSepharose was better than 97%. Although unspecific binding of B protein may occur, the presence of minor amounts of albumin contaminating the LDL preparation more likely explain the small fraction of activity adsorbed on the column. Using 1251-labeled human serum albumin which was added to the serum before the isolation of the different lipoproteins by means of ultracentrifugation, the amount of albumin relative to the total amount of apolipoproteins and the volume of the lipoprotein preparation could be determined. Routine preparations of VLDL and LDL were found to contain 11 and 28 ,ug of albumin/mg of apolipoprotein respectively, whereas purified HDL preparation (4) contained about 10 pg/mg of apolipoprotein. Albumin may specifically bind to lipoproteins, and to examine if antialbumin-Sepharose could compete with the lipoproteins at the binding of albumin, lz51-labeled albumin was added to albumin-poor lipoprotein preparations. The amount of added albumin was in the range of the amount present in the VLDL and LDL obtained in the routine preparation procedure described above. Mixtures of 1251-labeled albumin and VLDL, LDL, and HDL prepared in this way were allowed to pass through the anti-albumin-Sepharose columns. In experiments with all preparations the added albumin was retained to more than 99% by the columns, which is identical to the results obtained when the lz51-labeled albumin preparation alone was applied to the column.
603
SHORT COMMUNICATIONS
The present results demonstrated that anti-albumin-Sepharose may be used for the highly specific elimination of albumin in VLDL, LDL, and HDL preparations without altering the compositions of lipoproteins or their electrophoretic mobility in agarose gel and without measurable losses of lipoproteins. ACKNOWLEDGMENT This work (19X-204).
was supported
by grants from the Swedish Medical
Research Council
REFERENCES 1. Herbert, P.N., Forte, T. M., Shulman, R. S., La Piana, M. J., Gong, E. L., Levy, R. L., Fredrickson, D. S., and Nichols, A. V. (1975) Prep. Biochem. 5, 93-129. 2. Scan, A., and Granda, J. L. (1966) Biochemistry 5, 446-455. 3. Lindgren, F. T., Jensen, L. C., and Hatch, F. T. (1972) in Blood Lipids and Lipoproteins: Quantitation, Composition and Metabolism (Nelson, G. I., ed.), pp. 181-174, Wiley-Interscience, New York. 4. Hatch, F. T., and Lees, R. S. (1968) Advan. Lipid Res. 6, l-68. 5. Carlson, K. (1973) J. C/in. Pathol. 26, Suppl. Ass. C/in. Patho!. 5, 32-37. 6. Kane, J. P. (1973) Anal. Biochem. 53, 350-364. 7. Fishbein, W. N. (1972) Anal. Biochem. 46, 388-401. 8. Lowry, 0. H., Rosenbrough, N. J., Farr, A. L., and Randall, R. J. (1951) J. Biol. Chem. 193, 265-275. 9. Kessler, G., and Lederer, H. (1965) in Automation in Analytical Chemistry (Skeggs, L. T., ed.), pp. 341-344, Medidad, Inc., New York. 10. Block, W. P., Jarrett, K. J., and Leoine, B. (1965) in Automation in Analytical Chemistry (Skeggs, L. T., ed.), pp. 345-347, Medidad, Inc., New York. 11. Svennerholm, L., and Vanier, M. T. (1972) Brain. Res. 47, 457-468. 12. MC Farlane, A. S. (1956) Biochem. J. 62, 135-143. 13. Shepherd, J., Bedford, D. K., Morgan, H. G., and Scott, E. (1976) Clin. Chim. Acfa 66, 97- 109. 14. Affmity Chromatography: Principles and Methods, Pharmacia Fine Chemicals AB, Uppsala, Sweden. 15. Travis, J., and Pannell, R. (1973) Clin. Chim. Acta 49, 49-52.
LEIF HOLMQUIST KERSTIN CARLSON King Gustav V Research Institute Karolinska Hospital 104 01 Stockholm, Sweden Received February 22, 1977; accepted
September
19. 1977
BIOCHEMISTRY
Specific
84,
598-603
Elimination Lipoprotein
(1978)
of Albumin Preparations
in Serum
In the preparation of pure serum lipoproteins by means of ultracentrifugation techniques, it is necessary to perform consecutive centrifugations in order to reduce the content of contaminating serum albumin (1) impairing apolipoprotein determination and purification and turnover studies with lz51-labeled lipoproteins. Extensive centrifugation, however, may cause in vitro changes in the composition of the lipoprotein particles (1,2). The present report describes the use of matrix-bound anti-albumin for specific elimination of albumin in very low-density lipoprotein (VLDL), low-density lipoprotein (LDL), and high-density lipoprotein (HDL) preparations from human serum. MATERIALS
AND METHODS
Materials. VLDL, LDL, and HDL were prepared from fasting subjects with different types of hyperlipoproteinemia, mainly type IV. Blood was allowed to clot at room temperature and serum was separated by low-speed centrifugation. EDTA was added to a final concentration of 0.05%. When not otherwise stated pooled VLDL, LDL, and HDL, respectively, from different subjects were used. Monovalent anti-human albumin (purified immunoglobulin fraction of the antiserum) produced in rabbits was purchased from DAKO-immunoglobulins, Copenhagen, Denmark. Cyanogen bromide-activated Sepharose 6MB from Pharmacia Fine Chemicals, Uppsala, Sweden, was used for imobilization of the antialbumin. Albumin-poor VLDL was isolated by density gradient centrifugation as described by Lindgren et al. (3) in an SW 40-Ti rotor using a Beckman Model L5-75 ultracentrifuge. A 40.3 rotor in a Beckman Model LSO ultracentrifuge was used for the isolation of albumin-poor LDL and HDL. LDL was isolated between densities of 1.006 and 1.063 g/cm3, and HDL, between 1.070 and 1.21 g/cm3 (4). Two centrifugations at each density were performed on these preparations of LDL and HDL. The centrifugation time was 16 hr at d = 1.006 g/cm3, 20 hr at d = 1.063 and 1.070 g/cm3, and 48 hr at d = 1.21 g/cm3. For the routine isolation of lipoprotein fractions a scheme with step0003-2697/78/0842-0598$02.00/O Copyright All rights
0 1978 by Academic Press, Inc. of reproduction in any form reserved.
598
SHORTCOMMUNICATIONS
599
wise increases in density was used with a single spin at each density. The procedure for VLDL and LDL isolation has been described in detail elsewhere (5). HDL was isolated from the bottom fraction after centrifugation at d = 1.063 g/cm3. The density was raised to d = 1.21 g/cm3 with a concentrated salt solution and HDL was isolated as a top fraction after centrifugation for 48 hr at 40,000 rpm in the 40.3 rotor. After centrifugation all samples isolated at a density greater than 1.006 g/cm3 were dialyzed against 0.9% sodium chloride. Agarose gel electrophoresis was performed as described before (5). Polyacrylamide disc gel electrophoresis of soluble apolipoproteins was carried out according to Ref. (6) in 11-cm-long glass tubes with an inner diameter of 5 mm. The separated proteins were stained with Coomassie brilliant blue R (Sigma) as described in Ref. (7). The technique of Lowry et al. was used for protein determination (8). The turbidity caused by lipids was eliminated by extraction with chloroform before photometry. Bovine serum albumin was used as standard and no correction was made for the difference in chromogenicity of the different proteins. Triglycerides and cholesterol were analyzed by means of a Technicon Auto Analyzer using standard methods (9,lO). Phospholipids were determined as described in Ref. (11). lz51-Labeled human serum albumin with a specific activity of 4 pCi/mg was prepared as described before (12) using purified albumin obtained from Kabi, Stockholm, Sweden. The labeled protein, after final dialysis against 0.9% sodium chloride, was bound to 99% by the antialbumin-Sepharose. lz51-Labeled LDL was prepared by a modified iodine monochloride method (13) yielding 100-300 pCi/mg of apolipoprotein. Column experitnents. The anti-albumin-Sepharose conjugate was prepared by mixing swollen CNBr-Sepharose 6MB (2 ml) with antialbumin (5 mg) in 0.1 M sodium bicarbonate buffer, 0.5 M with respect to sodium chloride (2 ml). After 2 hr at room temperature the gel was washed free of unbound protein and was treated with 1 M ethanolamine, pH 9, for 2 hr as previously described (14). The capacity of the column to bind albumin was in the range of 0.3-0.5 mg of albumin/ml of wet gel. To a column (5 x 25 mm) of anti-albumin-Sepharose was added lipoprotein preparation (0.5 ml) containing 1 mg of protein/ml. When the sample had drained into the gel, the column was washed three times with 0.5 ml of 0.195 m (1.006 g/ml) sodium chloride, and a total volume of 2 ml was collected in a volumetric flask. The flow rate was 0.1 mVmin. As gel support a polyamide filter was used. For recovery determinations 0.5 ml of the original lipoprotein preparation was diluted to 2 ml.
600
SHORT
COMMUNICATIONS
Elimination of albumin from lipoprotein preparations. To albumin-poor preparations of VLDL, LDL, and HDL containing 1 mg of protein/ml was added 40 pg of lz51-labeled human serum albumin/ml. The mixtures were left for 1 hr at room temperature. Aliquots of 0.5 ml of these preparations were applied to anti-albumin-Sepharose columns (5 x 25 mm) and the lipoproteins were eluted as described above. The antialbumin-Sepharose was then suspended in 1.5 ml of sodium chloride solution and the distribution of radioactivity between the effluent and the gel was measured. RESULTS
In order to examine if matrix-bound anti-albumin induced changes in the compositions or physical properties of VLDL, LDL, or HDL
FIG. 1. Electropherograms demonstrating the electrophoretic mobility of VLDL, LDL, and HDL in agarose gel. (1) Untreated lipoprotein preparation; (2) effluent from antialbumin-Sepharose column. The untreated preparations have been diluted to the same volume as the column effluents. See Materials and Methods.
SHORT COMMUNICATIONS
601
FIG. 2. Polyacrylamide disc gel electropherograms of soluble apohpoproteins of VLDL, LDL, and HDL. IF denotes the interface between the upper and lower gel, and R is the riboflavin marker. (1) Untreated lipoprotein preparations; (2) effluent from antialbumin-Sepharose columns. The untreated preparations have been diluted to the same volume as the column effluents.
from human serum, these lipoprotein fractions were isolated and purified by means of ultracentrifugation, and the different lipoproteins were passed through columns of anti-albumin-Sepharose. The content of triglyceride, total cholesterol, phospholipid, and protein in the different lipoprotein preparations, before and after passage through the columns was determined. Within experimental error the analyzed components of all lipoproteins were quantitatively recovered in the column effluents.
602
SHORT
COMMUNICATIONS
Further, pure lipoproteins treated with anti-albumin-Sepharose and untreated lipoproteins yielded identical patterns upon electrophoresis in agarose gel (Fig. 1) and gave closely compatible electropherograms upon polyacrylamide gel electrophoresis of soluble apolipoproteins (Fig. 2). These results indicate that the lipoproteins pass through the anti-albumin-Sepharose columns without change in the content of lipids and soluble apolipoproteins and with no change in electrophoretic properties. Under the present experimental conditions, blue Sepharose CL-6B, which is reported to remove specifically albumin from serum (15), adsorbed VLDL, LDL, and HDL to 4, 39, and 33%, respectively, as examined by protein, cholesterol, and triglyceride determinations. In order to examine if apolipoprotein B, which is insolubIe in tetramethylurea extracts of VLDL and LDL (6), adsorbed to the matrixbound anti-albumin, LDL labeled with lz51 in the B protein was applied to a column of anti-albumin-Sepharose. By means of repeated ultracentrifugation the LDL preparation used for labeling was made apparently albumin free, as determined by polyacrylamide disc gel electrophoretic analyses (6). The recovery of lz51-labeled LDL after passage through a column of anti-albuminSepharose was better than 97%. Although unspecific binding of B protein may occur, the presence of minor amounts of albumin contaminating the LDL preparation more likely explain the small fraction of activity adsorbed on the column. Using 1251-labeled human serum albumin which was added to the serum before the isolation of the different lipoproteins by means of ultracentrifugation, the amount of albumin relative to the total amount of apolipoproteins and the volume of the lipoprotein preparation could be determined. Routine preparations of VLDL and LDL were found to contain 11 and 28 ,ug of albumin/mg of apolipoprotein respectively, whereas purified HDL preparation (4) contained about 10 pg/mg of apolipoprotein. Albumin may specifically bind to lipoproteins, and to examine if antialbumin-Sepharose could compete with the lipoproteins at the binding of albumin, lz51-labeled albumin was added to albumin-poor lipoprotein preparations. The amount of added albumin was in the range of the amount present in the VLDL and LDL obtained in the routine preparation procedure described above. Mixtures of 1251-labeled albumin and VLDL, LDL, and HDL prepared in this way were allowed to pass through the anti-albumin-Sepharose columns. In experiments with all preparations the added albumin was retained to more than 99% by the columns, which is identical to the results obtained when the lz51-labeled albumin preparation alone was applied to the column.
603
SHORT COMMUNICATIONS
The present results demonstrated that anti-albumin-Sepharose may be used for the highly specific elimination of albumin in VLDL, LDL, and HDL preparations without altering the compositions of lipoproteins or their electrophoretic mobility in agarose gel and without measurable losses of lipoproteins. ACKNOWLEDGMENT This work (19X-204).
was supported
by grants from the Swedish Medical
Research Council
REFERENCES 1. Herbert, P.N., Forte, T. M., Shulman, R. S., La Piana, M. J., Gong, E. L., Levy, R. L., Fredrickson, D. S., and Nichols, A. V. (1975) Prep. Biochem. 5, 93-129. 2. Scan, A., and Granda, J. L. (1966) Biochemistry 5, 446-455. 3. Lindgren, F. T., Jensen, L. C., and Hatch, F. T. (1972) in Blood Lipids and Lipoproteins: Quantitation, Composition and Metabolism (Nelson, G. I., ed.), pp. 181-174, Wiley-Interscience, New York. 4. Hatch, F. T., and Lees, R. S. (1968) Advan. Lipid Res. 6, l-68. 5. Carlson, K. (1973) J. C/in. Pathol. 26, Suppl. Ass. C/in. Patho!. 5, 32-37. 6. Kane, J. P. (1973) Anal. Biochem. 53, 350-364. 7. Fishbein, W. N. (1972) Anal. Biochem. 46, 388-401. 8. Lowry, 0. H., Rosenbrough, N. J., Farr, A. L., and Randall, R. J. (1951) J. Biol. Chem. 193, 265-275. 9. Kessler, G., and Lederer, H. (1965) in Automation in Analytical Chemistry (Skeggs, L. T., ed.), pp. 341-344, Medidad, Inc., New York. 10. Block, W. P., Jarrett, K. J., and Leoine, B. (1965) in Automation in Analytical Chemistry (Skeggs, L. T., ed.), pp. 345-347, Medidad, Inc., New York. 11. Svennerholm, L., and Vanier, M. T. (1972) Brain. Res. 47, 457-468. 12. MC Farlane, A. S. (1956) Biochem. J. 62, 135-143. 13. Shepherd, J., Bedford, D. K., Morgan, H. G., and Scott, E. (1976) Clin. Chim. Acfa 66, 97- 109. 14. Affmity Chromatography: Principles and Methods, Pharmacia Fine Chemicals AB, Uppsala, Sweden. 15. Travis, J., and Pannell, R. (1973) Clin. Chim. Acta 49, 49-52.
LEIF HOLMQUIST KERSTIN CARLSON King Gustav V Research Institute Karolinska Hospital 104 01 Stockholm, Sweden Received February 22, 1977; accepted
September
19. 1977