Heterogeneity of human plasma low density lipoprotein BERNARDRUBENSTEIN
Can. J. Biochem. Downloaded from www.nrcresearchpress.com by University of Regina on 11/18/14 For personal use only.
Department of Medicine, U~li\,ersitycPf Toronto, Toronto, O n f . ,Ccinuda MSS 1A8 Received June 9, 1978 Revised July 11, 1978
Rubenstein, B. (1978) Heterogeneity of human plasma low density lipoprotein. Cart. J . Biochern. 56,977-980 Low density lipoprotein (LDL) was fractionated into subspecies by the use of DEAE-agarose column chromatography and the peptide compositions of the LDL subspecies which eluted at different NaCl concentrations were determined. LDL which elutes at low NaCl concentration has relatively less non-B apoprotein than does LDL which elutes at high salt concentration. The LDL subspecies which elute at high NaCl concentration contain more apo A-1 than do those which elute at the lower NaCl molarity. These results indicate that LDL consists of subfractions which differ in their peptide compositions. Rubenstein, B. (1978) Heterogeneity of human plasma low density lipoprotein. Cun. 9. Biochern. 56,977-980 Nous awns fractionne les Bipoproteines de faible densit6 (LDL) en sous-especes par chromatographie sur colonne de DEAE agarose et nous avons determine la composition peptidique des sous-especes de LBL eluees i diffkrentes concentrations de NaCI. La LDL qui elue a faible concenti-ntion de NaCl contient reiativernent moins d9apoproteinenon-B que la LDL qui elue a forte concentration saline. Les sous-especes de LBL qui CIuent a forte concentration de NaCf contiennent plus d'apo A- 1que celles qui kluent 2 plus faible rnolarite de NaCI. Ces resuitats montrent que les LDL comportent des sous-fractions de composition peptidique diffkrente. [Traduit par le journal]
Introduction Most metabolic studies of LDL have been carried out using fraction d = 1.020- 1.050glml(1). This was primarily due to the fact that no reliable methods for sukfractionation of LDL were then available. Lee and Alaupsvlc (2) have divided %DL centrifugally, but this gives only a small number of subfractions in the d = 1.020- 1.050 g/ml range. However. recent work with LDL (3) and HDL (4) carried out in my laboratory as well as studies on HDL by Kostner and Holasek (5) have shown that these lipoproteins can be fractionated into subspecies by the use of ion-exchange column chromatography. The ability to subfractionate LDL will now permit more complete determination of the protein content of the L B L subspecies. The results show that LDL subspecies differ in their contents and compositions s f protein.
always carried out prior to obtaining LDL-2. The isolated LDL-2 was washed by centrifuging 12 mi of lipoprotein through 26ml of a solution of NaBr (d = B .050glml) for a further 22 h. The portion med for subsequent studies was always the top 6 ml from each tube.
--- NLDL aCl
e-*
Materials and Methods
LDL was isolated from 508-6Wrnl of pooled normolipemic human plasma by a modification of the method of Have1 et a]. (6). Each study, including those within a given series of experiments, represents analysis of LDL isolated from a different pool of human piasma. All centrifugation procedures were carried out at 16°C and 38000rpm in an L2-65B preparative ultracentrifuge using a type $0.2 Ti rotor. VLBL was isolated by centrifugation at a d = 1.006glml for 18h. Two fractions of 1.0%-1.019g/ml) and LDL-2 (d = LDL, LDL-8 (d 1.019- 1.050g/rnB). were separated by successive centrifugations of 22 h with appropriate adjustments of density by NaBr between spins. Although only LDL-2 was used in the results presented in this paper, isolation of VLDL and LBL-I was
-
ABBWEV~ATICPN~: LDL, low density Iipoprotein(s); HBL, high density Bipoprotein(s);VLDL. very Bow density lipoprotein(s).
FIG. 1. DEAE-agarose column chromatography pattern of LDL using a discontinuous NaCl gradient of 0.062, 0.079, and 0. ISM. The vertical lines enelosingarabic numerals indicate the aliquots used for further analysis.
97 8
CAN. J. BIOCHEM. VOL. 56, 1938
Can. J. Biochem. Downloaded from www.nrcresearchpress.com by University of Regina on 11/18/14 For personal use only.
apo B
aps A-1
FIG.2. Eight percent polyacrylamide gel electrophoresis pattern of LDL subfractions. The arabic numerals indicate the aaiquots used as indicated in Fig. I . The initial A indicates LDL protein insoluble in 7 M urea while B indicates the urea-soluble apoprotein (9).
Ion-exchange chromatography of LDL was carried out using DEAE Biogel A agarose, lot No. 14627. Packing, sizing, and washing of the columns were carried out according to the method of Rubenstein and Steiner (3). Elution solutions always contained 0.OWM Tris pH 7.8 and 4.0mllC of a 5% EBTA solution pH 7.8. Protein was determined by the method of Lowry et (41. (7). &%ipidationof the LBL and separation of the apoprotein by polyacrylamide gel electrophoresis were carried out according to the methods of Rubenstein and Rubinstein (8). SolubiIization of the B and non-B LDL apoproteins was carried out in 7 M urea using the method of Rubenstein and Steiner (9). For gel filtration, Sephadex 6-2W was swollen in a solution containing 3 M urea and 0.01 M Tiis pH 7.8, packed into a column (1.6 x 100crn), and the flow rate adjusted to BO mllh. The column was washed with 200 mi of the above solution and then loaded with 5 mg of non-B LBL apoprotein. The elution fluid was collected in fractions of 6.0 ml. Between 90 and 98% sf the added protein was recovered from the column. Solubilization of the protein and elution from the Sephadex column was always carried out within 72 h to prevent prolonged exposure of the apoproteins to urea.
Results It has been shown that LBL can be fractionated into subspecies using DEAE-agarose column chromatography (3). The protein contents and carnpssitisns of the digerent LBL subfractions were investigated. LDL is composed of 95%B apoprotein and only 5% other peptides (9). A large quantity of LBL protein is therefore
required to obtain enough non-B peptide to do accurate analysis. In order to keep the elution volume within workable limits, a discontinuous WaCl gradient was employed. LBL was eluted using NaCl molarities of 8.862 and 8.879, since experiments carried out using a continuous NaCl gradient (3) showed a change in the shape of the elution pattern at these concentrations, and 0.15 to obtain all remaining LDL (Fig. 1). The elution pattern was divided into five parts and the total LBL protein in each determined (Table 1). It can be seen that the proportion of LDL eluted at a given NaCl concentration remains quite consistent, demonstrating the reproducibility of the method. Ninety-five percent of the added TABLE 1. Protein content of LBL subfraction
% tstaI LBL LBL subfraction
protein
1
9.32k2.1 23.65k2.1 4Q.53k 1 . 9 13.57L0.8 82.93+0.7
3
3 4 5
% urea soluble
-
insoluble
4.85+0.30 3.60k0.31 5.37i-0.47 8.35k0.56 8.55k8.56
95.85 96.40 94.63 98.65 91.45
Eash figure is the average of three experiments. I SEM. Ure?soluble protein was dissolved in 7 M urea. The remaining ~nsolubleprsteln was then dissolved in solution containing 7 1Lf urea and 0.4 M sodium dwyl sulphate. NOTE:
979
RURENSTBIN
Can. J. Biochem. Downloaded from www.nrcresearchpress.com by University of Regina on 11/18/14 For personal use only.
LDL protein was recovered. The five aliquots shown in Fig. H were lyophilized, delipidated, and the B and non-B peptides separated as previously described (9). The results (Table 1) show that the proportion of non-B geptide (urea soluble) increases in LDL which elutes at higher NaC1 concentration. The level in aliquot 3 is significantly higher than that in aliquot 2 (P< 8.05) and the levels in aliquots 4 and 5 are significantly higher than that in aliquot 3 (P< 0.05). Analysis was also carried out to determine if differences exist in the compositions of non-B apoproteins among the k D k subfractions. The LDL was fractionated by discontinuous NaCl gradient chromatography as shown in Fig. 1. Aliquots 1, 2, and 4 were IyophiIized, delipidated, and the B and non-B apoproteins dissolved and electrophoresed on polyacrylamide gels. A total of 250 pg protein was placed on each gel. It can be seen (Fig. 2) that in gels HA, 2A, and 4A, which contain the B apoprotein, all peptides are in the stacking gel or at the surface between the stacking and running gel. No protein has penetrated the running gel demonstrating an absence of non-B peptide in this fraction (9). With gels IB, 2B, and 4B, the gels which contain the mon-B apoprotein, the
pictures show that all three subspecies contain the fast moving or C peptides, but only the LBL subfraction isolated with high NaC1 concentration (4B) has large quantities of the slow moving peptide. The peptide was isolated by DEAE-cellulose column chromatography and the amino acid composition determined using a Beckman model 120C amino acid anallyser. The protein was found to be apo A-1. Lee and Alaupovic (2) also demonstrated the presence of this peptide in LBL. Subfraction 2B also contains apo A-H but in much lower quantity while 1B contains only trace quantities. The protein which appears at the top of gel 1B was present in all three gel electrophoreses experiments which were carried out. Since each experiment was carried out using different pool of LDL, its presence would not appear to be artifactual. The nature of this protein has as yet not been determined. Although the polyacrylamide gels show that a considerable difference in the quantity of apo A-1 exists among the LDL subspecies, this evidence is only qualitative. In order to provide quantitative evidence, the non-B apoproteins from LDL, subfractions 2 and 4 (Fig. 1). were chromatographed on Sephadex (3-200. It can be seen (Fig. 3) that the elution curve has three peaks. Peak A is the same size in both LDL subfractions containing 3% of the total apoprotein. Peak B contains 12% of the total protein of fraction 2 and 35% of the protein of fraction 4. Polyacrylamide gels of this fraction, shown as part of Fig. 3, demonstrate that this fraction contains primarily the apo A-1 protein. The gel pattern was identical when protein from fraction 2 and fraction 4 were used. Peak C which contains primarily the C peptide was 85% of the total fraction 2 protein and 62% of fraction 4. Discussion Although studies on LDL use the d = 1.0201.050glmB (10) or d = 1.020-1.060glm1 (1) fraction, the findings presented in this paper indicate that those results may be misleading, since they assume the peptide composition in this density range to be homogeneous. LDL within these density ranges contain subspecies separable by agarose column chromatography. These su bfractions differ in their protein contents and concentrations. The relative content of non-B apoprotein (A C) increases in subfractions which elute at higher NaCl molarity as does the amount of apo A (Fig. 1, Table 1). It might be postulated that subfraction 3 (Table 1, Fig. 1) is only a mixture of subfractions 2 and 4. This is unlikely, since for this to occur, subfraction 3 would have to be made up of 40% fraction 2 and 6Wo fraction 4 to bring its non-B protein up to 5.4% of total protein. Therefore, the amount of subfraction 2 present in subfraction 3 would almost be equal to the amount isolated as subfraction 2. The sharp peaks seen in Fig. 2 and the consistent results obtained for the percentage of protein in subfractions 2, 3, and 4 as seen in Table 1 would seem to rule out this possibility. Also, when subfraction 3 was rechromatographed with a stepwise elution, 95% of the protein was recovered in the 0.15 M NaCl elution frac-
+
ELUTION VOLUME/ML
FIG. 3. Sephadex G-280 column chromatography of ureasoluble LDL apoproteinsfrom subfractions2 and 4 as indicated in Fig. 2. The polyacrylamidegel patterns represent the apoproteins of the corresponding peak.
wnloaded from www.nrcresearchpress.com by University o For personal use only.
980
CAN. J. BIOCHEM. VOL. 56. 1978
tion. Lee and Aiaupovic ( I I) have demonstrated that LBL peptides can exist in the free as well as associated form. However, they have also shown that the d 1.020-1 -850fraction, the range which is covered in these experiments, gives only the associated forin ( 6 ) . The compositional heterogeneity of LBL raises the
-
possibility that LDE is also metabolically heterogeneous. Streja et al. (12) have pointed out that VLDL is metabolically heterogeneous and that all parts of the VLDL spectrum may be catabolized to EDL. It is also possible that the LDL subspecies represent products sf partial EDL catabolism. This aspect of the study is currently under investigation. Acknowkdgements This work was supported by a grant from the Medical Research Cuuncii of Canada. 1. Granger, T., Sta-oker, W. & Levy, R. I. (1972) J. C6irt. bnilesf.51, 1528- 1536
2. Lee, D. M. & Alaupcwic, P. (1970) Biochenzisfvg, 9 , 2244-2252 3. Rubenstein, B. & Steiner, G. (1976) Can. J . Biociaerzt. 34, 1023- 1028 4. Rubenstein, B., Evans, S. & Steiner, G. (1977). Can. J. Bioc+frcm.53,766-768 5. Kostnes. G . ha. & Holasek, A. (1977) Bicpckem. Biophys. ,4cta 488,417-431 6. Havel. 8. J . , Eder, H. A. & Bragdon, J. H . (1955). J . Clin. drzvrst. 34, 1345-1353 7. Lowry, 0. H . , Rosebrough, N. J., Fan., A. L. & Randall, R. J. (1951) J. B i d . G e m . 193.265-275 8. Rubenstein, B. & Rubinstein, D. (1972) J. Lipid Res. 13, 3 17-324 9. Rubenstein. B . 6% SSteiner, G . (1975) Carl. J. Biochene. 53, 128- 134 lo. Slack, J. $c Mills, A. J . (1970) Clin. Chem. Actu 29, 15-25 l 1. Lee. D. M.& Alaupovic, C. (1972) Circislation 46-1 1,268 12. Streja, D., Mallai, hf. A. & Steiner, G. (1977) Metabolism 26,1333-1343
Can. J. Biochem. Downloaded from www.nrcresearchpress.com by University of Regina on 11/18/14 For personal use only.
Department of Medicine, U~li\,ersitycPf Toronto, Toronto, O n f . ,Ccinuda MSS 1A8 Received June 9, 1978 Revised July 11, 1978
Rubenstein, B. (1978) Heterogeneity of human plasma low density lipoprotein. Cart. J . Biochern. 56,977-980 Low density lipoprotein (LDL) was fractionated into subspecies by the use of DEAE-agarose column chromatography and the peptide compositions of the LDL subspecies which eluted at different NaCl concentrations were determined. LDL which elutes at low NaCl concentration has relatively less non-B apoprotein than does LDL which elutes at high salt concentration. The LDL subspecies which elute at high NaCl concentration contain more apo A-1 than do those which elute at the lower NaCl molarity. These results indicate that LDL consists of subfractions which differ in their peptide compositions. Rubenstein, B. (1978) Heterogeneity of human plasma low density lipoprotein. Cun. 9. Biochern. 56,977-980 Nous awns fractionne les Bipoproteines de faible densit6 (LDL) en sous-especes par chromatographie sur colonne de DEAE agarose et nous avons determine la composition peptidique des sous-especes de LBL eluees i diffkrentes concentrations de NaCI. La LDL qui elue a faible concenti-ntion de NaCl contient reiativernent moins d9apoproteinenon-B que la LDL qui elue a forte concentration saline. Les sous-especes de LBL qui CIuent a forte concentration de NaCf contiennent plus d'apo A- 1que celles qui kluent 2 plus faible rnolarite de NaCI. Ces resuitats montrent que les LDL comportent des sous-fractions de composition peptidique diffkrente. [Traduit par le journal]
Introduction Most metabolic studies of LDL have been carried out using fraction d = 1.020- 1.050glml(1). This was primarily due to the fact that no reliable methods for sukfractionation of LDL were then available. Lee and Alaupsvlc (2) have divided %DL centrifugally, but this gives only a small number of subfractions in the d = 1.020- 1.050 g/ml range. However. recent work with LDL (3) and HDL (4) carried out in my laboratory as well as studies on HDL by Kostner and Holasek (5) have shown that these lipoproteins can be fractionated into subspecies by the use of ion-exchange column chromatography. The ability to subfractionate LDL will now permit more complete determination of the protein content of the L B L subspecies. The results show that LDL subspecies differ in their contents and compositions s f protein.
always carried out prior to obtaining LDL-2. The isolated LDL-2 was washed by centrifuging 12 mi of lipoprotein through 26ml of a solution of NaBr (d = B .050glml) for a further 22 h. The portion med for subsequent studies was always the top 6 ml from each tube.
--- NLDL aCl
e-*
Materials and Methods
LDL was isolated from 508-6Wrnl of pooled normolipemic human plasma by a modification of the method of Have1 et a]. (6). Each study, including those within a given series of experiments, represents analysis of LDL isolated from a different pool of human piasma. All centrifugation procedures were carried out at 16°C and 38000rpm in an L2-65B preparative ultracentrifuge using a type $0.2 Ti rotor. VLBL was isolated by centrifugation at a d = 1.006glml for 18h. Two fractions of 1.0%-1.019g/ml) and LDL-2 (d = LDL, LDL-8 (d 1.019- 1.050g/rnB). were separated by successive centrifugations of 22 h with appropriate adjustments of density by NaBr between spins. Although only LDL-2 was used in the results presented in this paper, isolation of VLDL and LBL-I was
-
ABBWEV~ATICPN~: LDL, low density Iipoprotein(s); HBL, high density Bipoprotein(s);VLDL. very Bow density lipoprotein(s).
FIG. 1. DEAE-agarose column chromatography pattern of LDL using a discontinuous NaCl gradient of 0.062, 0.079, and 0. ISM. The vertical lines enelosingarabic numerals indicate the aliquots used for further analysis.
97 8
CAN. J. BIOCHEM. VOL. 56, 1938
Can. J. Biochem. Downloaded from www.nrcresearchpress.com by University of Regina on 11/18/14 For personal use only.
apo B
aps A-1
FIG.2. Eight percent polyacrylamide gel electrophoresis pattern of LDL subfractions. The arabic numerals indicate the aaiquots used as indicated in Fig. I . The initial A indicates LDL protein insoluble in 7 M urea while B indicates the urea-soluble apoprotein (9).
Ion-exchange chromatography of LDL was carried out using DEAE Biogel A agarose, lot No. 14627. Packing, sizing, and washing of the columns were carried out according to the method of Rubenstein and Steiner (3). Elution solutions always contained 0.OWM Tris pH 7.8 and 4.0mllC of a 5% EBTA solution pH 7.8. Protein was determined by the method of Lowry et (41. (7). &%ipidationof the LBL and separation of the apoprotein by polyacrylamide gel electrophoresis were carried out according to the methods of Rubenstein and Rubinstein (8). SolubiIization of the B and non-B LDL apoproteins was carried out in 7 M urea using the method of Rubenstein and Steiner (9). For gel filtration, Sephadex 6-2W was swollen in a solution containing 3 M urea and 0.01 M Tiis pH 7.8, packed into a column (1.6 x 100crn), and the flow rate adjusted to BO mllh. The column was washed with 200 mi of the above solution and then loaded with 5 mg of non-B LBL apoprotein. The elution fluid was collected in fractions of 6.0 ml. Between 90 and 98% sf the added protein was recovered from the column. Solubilization of the protein and elution from the Sephadex column was always carried out within 72 h to prevent prolonged exposure of the apoproteins to urea.
Results It has been shown that LBL can be fractionated into subspecies using DEAE-agarose column chromatography (3). The protein contents and carnpssitisns of the digerent LBL subfractions were investigated. LDL is composed of 95%B apoprotein and only 5% other peptides (9). A large quantity of LBL protein is therefore
required to obtain enough non-B peptide to do accurate analysis. In order to keep the elution volume within workable limits, a discontinuous WaCl gradient was employed. LBL was eluted using NaCl molarities of 8.862 and 8.879, since experiments carried out using a continuous NaCl gradient (3) showed a change in the shape of the elution pattern at these concentrations, and 0.15 to obtain all remaining LDL (Fig. 1). The elution pattern was divided into five parts and the total LBL protein in each determined (Table 1). It can be seen that the proportion of LDL eluted at a given NaCl concentration remains quite consistent, demonstrating the reproducibility of the method. Ninety-five percent of the added TABLE 1. Protein content of LBL subfraction
% tstaI LBL LBL subfraction
protein
1
9.32k2.1 23.65k2.1 4Q.53k 1 . 9 13.57L0.8 82.93+0.7
3
3 4 5
% urea soluble
-
insoluble
4.85+0.30 3.60k0.31 5.37i-0.47 8.35k0.56 8.55k8.56
95.85 96.40 94.63 98.65 91.45
Eash figure is the average of three experiments. I SEM. Ure?soluble protein was dissolved in 7 M urea. The remaining ~nsolubleprsteln was then dissolved in solution containing 7 1Lf urea and 0.4 M sodium dwyl sulphate. NOTE:
979
RURENSTBIN
Can. J. Biochem. Downloaded from www.nrcresearchpress.com by University of Regina on 11/18/14 For personal use only.
LDL protein was recovered. The five aliquots shown in Fig. H were lyophilized, delipidated, and the B and non-B peptides separated as previously described (9). The results (Table 1) show that the proportion of non-B geptide (urea soluble) increases in LDL which elutes at higher NaC1 concentration. The level in aliquot 3 is significantly higher than that in aliquot 2 (P< 8.05) and the levels in aliquots 4 and 5 are significantly higher than that in aliquot 3 (P< 0.05). Analysis was also carried out to determine if differences exist in the compositions of non-B apoproteins among the k D k subfractions. The LDL was fractionated by discontinuous NaCl gradient chromatography as shown in Fig. 1. Aliquots 1, 2, and 4 were IyophiIized, delipidated, and the B and non-B apoproteins dissolved and electrophoresed on polyacrylamide gels. A total of 250 pg protein was placed on each gel. It can be seen (Fig. 2) that in gels HA, 2A, and 4A, which contain the B apoprotein, all peptides are in the stacking gel or at the surface between the stacking and running gel. No protein has penetrated the running gel demonstrating an absence of non-B peptide in this fraction (9). With gels IB, 2B, and 4B, the gels which contain the mon-B apoprotein, the
pictures show that all three subspecies contain the fast moving or C peptides, but only the LBL subfraction isolated with high NaC1 concentration (4B) has large quantities of the slow moving peptide. The peptide was isolated by DEAE-cellulose column chromatography and the amino acid composition determined using a Beckman model 120C amino acid anallyser. The protein was found to be apo A-1. Lee and Alaupovic (2) also demonstrated the presence of this peptide in LBL. Subfraction 2B also contains apo A-H but in much lower quantity while 1B contains only trace quantities. The protein which appears at the top of gel 1B was present in all three gel electrophoreses experiments which were carried out. Since each experiment was carried out using different pool of LDL, its presence would not appear to be artifactual. The nature of this protein has as yet not been determined. Although the polyacrylamide gels show that a considerable difference in the quantity of apo A-1 exists among the LDL subspecies, this evidence is only qualitative. In order to provide quantitative evidence, the non-B apoproteins from LDL, subfractions 2 and 4 (Fig. 1). were chromatographed on Sephadex (3-200. It can be seen (Fig. 3) that the elution curve has three peaks. Peak A is the same size in both LDL subfractions containing 3% of the total apoprotein. Peak B contains 12% of the total protein of fraction 2 and 35% of the protein of fraction 4. Polyacrylamide gels of this fraction, shown as part of Fig. 3, demonstrate that this fraction contains primarily the apo A-1 protein. The gel pattern was identical when protein from fraction 2 and fraction 4 were used. Peak C which contains primarily the C peptide was 85% of the total fraction 2 protein and 62% of fraction 4. Discussion Although studies on LDL use the d = 1.0201.050glmB (10) or d = 1.020-1.060glm1 (1) fraction, the findings presented in this paper indicate that those results may be misleading, since they assume the peptide composition in this density range to be homogeneous. LDL within these density ranges contain subspecies separable by agarose column chromatography. These su bfractions differ in their protein contents and concentrations. The relative content of non-B apoprotein (A C) increases in subfractions which elute at higher NaCl molarity as does the amount of apo A (Fig. 1, Table 1). It might be postulated that subfraction 3 (Table 1, Fig. 1) is only a mixture of subfractions 2 and 4. This is unlikely, since for this to occur, subfraction 3 would have to be made up of 40% fraction 2 and 6Wo fraction 4 to bring its non-B protein up to 5.4% of total protein. Therefore, the amount of subfraction 2 present in subfraction 3 would almost be equal to the amount isolated as subfraction 2. The sharp peaks seen in Fig. 2 and the consistent results obtained for the percentage of protein in subfractions 2, 3, and 4 as seen in Table 1 would seem to rule out this possibility. Also, when subfraction 3 was rechromatographed with a stepwise elution, 95% of the protein was recovered in the 0.15 M NaCl elution frac-
+
ELUTION VOLUME/ML
FIG. 3. Sephadex G-280 column chromatography of ureasoluble LDL apoproteinsfrom subfractions2 and 4 as indicated in Fig. 2. The polyacrylamidegel patterns represent the apoproteins of the corresponding peak.
wnloaded from www.nrcresearchpress.com by University o For personal use only.
980
CAN. J. BIOCHEM. VOL. 56. 1978
tion. Lee and Aiaupovic ( I I) have demonstrated that LBL peptides can exist in the free as well as associated form. However, they have also shown that the d 1.020-1 -850fraction, the range which is covered in these experiments, gives only the associated forin ( 6 ) . The compositional heterogeneity of LBL raises the
-
possibility that LDE is also metabolically heterogeneous. Streja et al. (12) have pointed out that VLDL is metabolically heterogeneous and that all parts of the VLDL spectrum may be catabolized to EDL. It is also possible that the LDL subspecies represent products sf partial EDL catabolism. This aspect of the study is currently under investigation. Acknowkdgements This work was supported by a grant from the Medical Research Cuuncii of Canada. 1. Granger, T., Sta-oker, W. & Levy, R. I. (1972) J. C6irt. bnilesf.51, 1528- 1536
2. Lee, D. M. & Alaupcwic, P. (1970) Biochenzisfvg, 9 , 2244-2252 3. Rubenstein, B. & Steiner, G. (1976) Can. J . Biociaerzt. 34, 1023- 1028 4. Rubenstein, B., Evans, S. & Steiner, G. (1977). Can. J. Bioc+frcm.53,766-768 5. Kostnes. G . ha. & Holasek, A. (1977) Bicpckem. Biophys. ,4cta 488,417-431 6. Havel. 8. J . , Eder, H. A. & Bragdon, J. H . (1955). J . Clin. drzvrst. 34, 1345-1353 7. Lowry, 0. H . , Rosebrough, N. J., Fan., A. L. & Randall, R. J. (1951) J. B i d . G e m . 193.265-275 8. Rubenstein, B. & Rubinstein, D. (1972) J. Lipid Res. 13, 3 17-324 9. Rubenstein. B . 6% SSteiner, G . (1975) Carl. J. Biochene. 53, 128- 134 lo. Slack, J. $c Mills, A. J . (1970) Clin. Chem. Actu 29, 15-25 l 1. Lee. D. M.& Alaupovic, C. (1972) Circislation 46-1 1,268 12. Streja, D., Mallai, hf. A. & Steiner, G. (1977) Metabolism 26,1333-1343
