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Lipoprotein (a) is not a metabolic product of other lipoproteins containing apolipoprotein B
63
Biochimica et Biophysics Acta, 575 (1979) 0 Elsevier/North-Holland Biomedical Press
63-70
BBA 57440
LIPOPROTEIN LIPOPROTEINS
F. KREMPLER,
(a) IS NOT A METABOLIC PRODUCT OF OTHER CONTAINING APOLIPOPROTEIN B
G. KOSTNER,
K. BOLZANO
and F. SANDHOFER
First Department of Medicine, Landeskrankenanstalten, A-5020 Salzburg and Institute of Medical Biochemistry, University of Graz, A-8010 Graz (Austria) (Received
March 19th,
Key words: Lipoprotein
1979)
(a); Apolipoprotein
B; VLDL
Summary 12’1-Labeled autologous very low density lipoprotein (VLDL) was injected intravenously into three lipoprotein (a) positive individuals. One other lipoprotein (a) positive subject received ‘251-labeled VLDL from a lipoprotein (a) negative donor. Specific activity of apolipoprotein B in VLDL, low density lipoprotein (LDL) and lipoprotein (a) was measured for 5 days. In the lipoprotein (a) fraction only traces of radioactivity could be detected, which were caused by contamination with labeled LDL. No precursor-product relationship existed between apolipoprotein B in VLDL or LDL and apolipoprotein B in lipoprotein (a). One lipoprotein (a)-positive individual was kept on a fat-free diet for 4 days to prevent chylomicron formation; no change in the serum level of lipoprotein (a) could be detected under these conditions. The data of this study indicate that lipoprotein (a) is not a metabolic product of VLDL or LDL. Also chylomicrons are not likely to play a role as a precursor for lipoprotein (a). It is concluded that lipoprotein (a) is synthesized as a separate lipoprotein.
Introduction Since the first description of lipoprotein (a) [l] numerous studies on the physicochemical properties of this lipoprotein, its incidence and concentration in the serum, and its relation to coronary artery disease have been published (for a review see Ref. 2). A possible role of lipoprotein (a) as a risk factor for
Abbreviations: VLDL, very low density lipoproteins (d < 1.006 g/ml); LDL. low density lipoproteins Cd = 1.006-1.063 g/ml); HDL, high density lipoproteins (d = 1.063-1.21 g/ml); SDS. sodium dodecyl sulfate.
64
atherosclerotic vascular disease has already been postulated in earlier studies [ 3-51. Albers et al. [6] developed a very sensitive radioimmunoassay for the qu~titative estimation of lipoprotein (a). With this method, lipopro~in (a) could be detected in practically all individuals with apolipoprotein B. Albers et al. [6] also found a positive association between high lipoprotein (a) levels and coronary heart disease, which was even stronger for subjects younger than 50 years. In many respects, lipoprotein (a) is very similar to low density lipoproteins (LDL). There is no si~ific~t difference in the composition of neutral lipids and phospholipids f 7-91, and in both lipoproteins the main protein is apolipoprotein B [lo]. The carbohydrate content of lipoprotein (a), however, is several times higher than that of LDL [lo] and, in contrast to LDL, lipoprotein (a) contains a specific lipoprotein (a) apolipoprotein [ lO,ll]. The density of lipoprotein (a) is between 1.055 and 1.110 g/ml [2]. Because of its higher c~bohydra~ content lipoprotein (a) has a higher electrophoretie mobility than LDL and, therefore, was also described as ‘sinking pre-P’ or ‘pre-p,’ lipoprotein
[4,5,121. The site and mechanism of synthesis of this lipoprotein are not yet known. Because of the chemical similarities between lipoprotein (a) and LDL, it is possible that lipopro~in (a) is formed during the metabolic catabolism of other apoprotein B containing lipoproteins, that is chylomicrons, VLDL or LDL. This study was performed in order to assess the possibility that one of these lipoproteins could be a precursor for lipoprotein (a). Methods Subjects. The study was performed on four male subjects after obtaining informed consent (Table I). All were strongly positive for lipoprotein (a), as checked by immunodiffusion with specific antisera to lipoprotein (a). They were hospitalized during the investigation; none received any treatment known to influence lipid metabolism and all were kept on a ‘normal’ hospital diet. No change in body weight or serum cholesterol and triglyceride levels was observed throughout the study. KI (3 X 60 mg daily) was administered to each individual, beginning 3 days prior to the study and extending throughout the whole study. Preparation of labeled lipoproteins. VLDL (d < 1.006 g/ml) was isolated from serum after fasting by ultracen~ifugation at serum density at 42 000 rev./
TABLE CLINICAL
I DATA
OF
SW
THE
SUBJECTS Body
Serum
Serum
weight
cholesterol
triacylglyceral
(kg)
tmgidl)
(mgidl)
G.F.
male
52
67
261
97
F.J.
male
36
71
218
195
Z.E.
male
68
79
171
75
G.L.
male
47
82
209
169
Diagnosis
emphysema normal coronary normal
heart
disease
65
min for 18 h using a Beckman Ti 50.2 rotor. The top fraction was removed by slicing the tube and washed once. LDL (d = 1.006-1.063 g/ml) were obtained after adjusting the infranatant to d = 1.063 g/ml by addition of solid NaCl and ultracentrifugation at 42 000 rev./min for 20 h. The densities were measured at 15°C with a calculating digital density meter DMA 46 (Anton Paar K.G., Graz, Austria). All ultracentrifugation procedures were carried out at 15°C. The top fraction which contained LDL and some lipoprotein (a) was chromatographed on an agarose column to separate LDL from contaminating lipoprotein (a). VLDL and LDL were radioiodinated with ‘*‘I (The Radiochemical Centre, Amersham) according to McFarlane [ 131 as modified by Bilheimer et al. [ 141. Free iodine was removed by filtration on Sephadex G-25 and subsequent dialysis against 0.15 M NaCl with several bath changes. 7-15s of the radioactivity in the labeled VLDL was found in the lipid moiety after extraction by the method of Folch et al. [ 151. Labeled and unlabeled VLDL showed the same electrophoretic mobility on agarose and identical immunoreactive behavior. Prior to the injection, the labeled lipoproteins were sterilized by passage through a Millipore filter (0.45 pm). The injected radioactivity was between 20 and 60 &!i. Determination of specific activity of apolipoprotein B in VLDL, LDL and lipoprotein (a). After the injection of labeled VLDL, venous blood was collected
at several time intervals (Fig. 3), allowed to clot at room temperature and centrifuged for 20 min at 3000 rev./mm 1 mg/ml NaN3 and 1 mg/ml disodium EDTA were added to the serum. VLDL were isolated as described above. After removal of VLDL, the infranatant was brought to a density of 1.055 g/ml by addition of solid NaCl and centrifuged for 20 h at 42 000 rev./min. The top fraction which contained most of the LDL was removed by slicing the tube. The infranatant was adjusted to d = 1.110 g/ml and centrifuged for 22 h at 42 000 rev./min. From the top fraction (d = 1.055-1.110 g/ml) which contained lipoprotein (a) and some LDL and high density lipoproteins (HDL), lipoprotein (a) was purified by filtration on an agarose column (0.9 X 100 cm) using BioGel A-5m (Bio-Rad Laboratories, Richmond, CA). Elution of the lipoproteins was carried out at room temperature with 0.15 M NaCl containing 1 mg/ml disodium EDTA and 1 mg/ml NaN,, pH adjusted to 8.5 by addition of 25% NH40H (E. Merck, Darmstadt). The elution pattern of the lipoproteins of density 1.055-l .llO g/ml is shown in Figs. 1 and 2. The protein content of the peaks was identified by immunodiffusion using specific antisera [16,17], and the lipoprotein (a) peak was further characterized by its electrophoretic mobility on agarose gel, immunoelectrophoresis, immunodiffusion and chemical composition. It was identical with lipoprotein (a) preparations described in earlier reports [ 2,161. Immunodiffusion and immunoelectrophoresis as well as agarose electrophoresis were carried out in 1% agarose gels using 0.05 M barbital buffer (pH 8.2). Electrophoretic separations were performed at 5 V/cm for 65 min. Sudan Black and Amido Black 10 B were used for lipid staining and protein staining, respectively. For the determination of the specific activity of apolipoprotein B, the fractions containing the lipoproteins were first dialysed against distilled water and then lyophilized. The lipoproteins were delipidated with diethyl ether/ethanol (1 : 3, v/v) [ 181. The delipidated apolipoproteins were washed several times
66
with 6 M urea to remove the ‘soluble” apolipoproteins. Removal of ‘soluble’ apo~~opro~ins was considered complete when no radioactivity could be detected in the urea solution for the last three washes. The rem~nin~ apolipoproteins which were insoluble in 6 M urea were solubilized by addition of SDS. This fraction contained less than 1% of apolipoproteins other than apolipoprotein B as judged from SDS-polyacrylamide gel electrophoresis. The protein content was estimated by the method of Lowry et al. [ 191; radioactivity was measured in a Packard Autogamma Sc~t~lati~n Spectrometer 5160. The co~c~n~at~on of lipoprotein (a) in the serum was estimated by Laurel13 one-dimensional rocket immunoelectrophoresis method [ 201. Results Fig. 1 shows the elution pattern of the lipoproteins of the density 1.0551.110 g/ml on agarose gel. From the immunolo~c~ behavior against specific antisera, peak I is identified as lipoprotein (a), peak II as lipoprotein B (part of serum LDL), and peak III as HDL. Fig. 1 also shows the radioactivity of the eluted lipoprotein fractions 24 h after the injection of the labeled VLDL (subject Z.E.). It can be seen that the radioactivity peaks coincide with the extinction peaks II and III, The radioactivity in the HDL fraction (peak III) can be explained by exchange of apolipoprotein C between VLDL and HDL [21, 221. There are traces of radioactivity in the lipoprotein (a) peak, but these small amounts of radioactivity do not follow the concentration of this lipoprotein. From the curve of the radioactivity it may be assumed that these traces of radioactivity are caused by conflation with labeled LDL, To prove this assumption, labeled LDL was mixed in vitro with lipoprotein
Fig. 1. Elution profile of the lipoproteins of density 1.055-1.110 g/ml on BioGel A-5m. The samPle was taken from subject Z.E. 24 h after the injection of labeled VLDL. -, extinction at 280 nm; . --. - *, radioactivity (cpm/ml). The immunological reactivities of the eluted fractions against antibodies to lipoprotein (af apohpoprotein, apoBpopr&eio A, B and C are shown in the figure, Peak f rwresents hPwrotein (a), peak II lipoprotein B
67
i
ld05&
.?4
ri
?i
hours
C
9k
Z.E.
G.L.
1
5x104
5x10
4
,
A
6 hours
24
18
72
96
125
hours
Fig. 3. Specific activity-time curves of apolipoprotein B in VLDL, LDL and lipoprotein (a) after intravenous injection of labeled VLDL in subject G.F. (A), subject F.J. (B), subject Z.E. (C), and subject G.L. (D). o----o, VLDL; +-----., LDL; X-X. lipolprotein (a).
(a) positive serum. Specific activity of apolipopro~~ B in LDL and lipoprotein (a) was then determined in the same way as for in vivo studies. Fig. 2 shows the elution pattern of the lipoproteins of the density 1.055-l .110 g/ml of this in vitro assay. The same elution pattern and the same distribution of radioactivity was found except for HDL (no labeled apolipoprotein C). Traces of radioactivity were found in the lipoprotein (a) fraction to a similar extent as in the in vivo studies.
68
The specific activity-time curves of apolipoprotein B in VLDL, LDL and lipoprotein (a) after the injection of labeled VLDL are given in Fig. 3. In the subjects G.F., F.J., and Z.E., autologous VLDL were labeled and reinjected. In the case of subject G.L., VLDL was obtained from a lipoprotein (a)-negative donor, labeled and injected to subject G.L., who was lipoprotein (a)-positive as the other recipients. In all cases, the specific activity-time curves of apolipoprotein B in VLDL and LDL showed the well known precursor-product relationship as already described by others [14,23,24]. Only traces of radioactivity could be detected in apolipopro~~ B of the lipopro~in (a) fraction. The specific activity of apolipoprotein B in the lipoprotein {a) fractions was extremely low when compared with that of VLDL until 4 days after the injection of labeled VLDL. At that time, radioactivity in both fractions had practically disappeared. Specific activity of apolipoprotein B in the lipoprotein (a) fraction was between 4 and 9% (R = 6.5%) of that in the LDL throughout the whole study. The same percentage was found in the lipoprotein (a) fraction after the in vitro addition of labeled LDL to lipoprotein (a) positive serum. Therefore, the specific activity of apolipoprotein I3 in the lipoprotein (a) fraction found in vivo after the injection of labeled VLDL can be accounted for by contamination of lipoprotein (a) with labeled LDL. Obviously, no precursor-product relationship exists between apolipopro~in B in VLDL or LDL and apolipoprotein B in lipoprotein (a). It is possible that chylomicrons are a precursor for lipoprotein (a). To study this possibility, a lipoprotein (a)-positive individual was kept on an extremely low-fat diet (less than 2 g fat/day) for 4 days and serum samples were obtained daily for rocket immunoelectrophoresis of lipoprotein (a). No change in the lipopro~~ (a) level could be observed as judged from the peak height in immunoelectrophoresis. Discussion In recent years, many experiment have been performed to study the metabolism of apolipoproteins in various lipoprotein classes. A unidirection~ catabolic pathway from VLDL to ‘intermediate lipoproteins’ and then to LDL has been established [14,21,23-251. In the course of this catabolic process, apolipoprotein B is not removed from the lipoprotein particle. From analysis of the specific activity-time curves of apolipoprotein B in different lipoprotein fractions after the injection of labeled VLDL, a precu~or-product relationship between VLDL and LDL has clearly been demonstrated by other investigators [ 14,23,24]. Since apolipoprotein B represents approx. 80% of the apolipoproteins in lipoprotein (a) [lo], it is possible that lipoprotein (a) is formed during the catabolism of either chylomicrons, VLDL or LDL. Apolipoprotein B does not exchange between. the various apolipoprote~ B-con~n~g lipoproteins [21], Therefore, from the specific activity-time curves of apolipoprotein B in VLDL, LDL and lipoprotein (a) after the injection of labeled VLDL a possible metabolic conversion of VLDL or LDL to lipoprotein (a) can be demonstrated or excluded. If a precursor-product relationship existed between VLDL or LDL and lipo-
69
protein (a), a cross-over of the specific activity-time curves of apolipoprotein B in VLDL or LDL and apolipoprotein B in lipoprotein (a) must occur within the first 2 days, as estimated from the known half-lives of apolipoprotein B in these lipoproteins [ 16,261. In our experiments, only traces of radioactivity could be detected in the lipoprotein (a) fraction that were due to contamination with labeled LDL. Our results show that neither VLDL nor LDL can be considered as a precursor for lipoprotein (a). To exclude chylomicrons as a possible precursor for lipoprotein (a), a lipoprotein (a)-positive subject was kept on a fat-free diet for 4 days. One can assume that under such a diet the formation of chylomicrons is drastically reduced as compared with a normal, exogenous fat-containing diet. If lipoprotein (a) were a catabolic product of chylomicrons, a decrease in the serum concentration of lipoprotein (a) is to be expected during this time when the known half-lives of chylomicrons [ 271, triacylglycerol-rich ‘intermediates’ [ 281 and lipoprotein (a) [ 161 are taken into consideration. Since under the conditions described above the concentration of lipoprotein (a) in the serum remained constant we believe that chylomicrons do not play a significant role in the formation of lipoprotein (a). From our studies, it is evident that lipoprotein (a) is not formed from VLDL, LDL or chylomicrons through the catabolic pathway of these lipoproteins. Therefore it can be concluded that lipoprotein (a) is secreted into the blood as a separate lipoprotein. This could explain the fact that the plasma concentration of lipoprotein (a) is not influenced by various dietary manipulations which are known to drastically influence the level of the other apolipoprotein B-containing lipoproteins [6]. Until now no information concerning the site of production of this lipoprotein is available. Acknowledgments This work was supported by Fonds zur Fijrderung Forschung (Project Nr. 3631). The skillful technical TaIman and Miss E. Meisl is greatfully acknowledged.
der Wissenschaftlichen assistance of Miss H.
References 1
Berg,
2
Kostner,
K.
Plenum 3
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Acta
G.M.
(1976)
Press.
Dahlen.
New
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Microbial.
in Low
Stand.
Density
59.
369-382
Lipoproteins
(Day,
C.E.
and
Levy,
R.S..
eds.),
PP.
229-269,
York
G..
Ericson,
C.,
Furberg.
G.,
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Ramberg.
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L. and
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K.
(1972)
Acta
Med.
Stand.
SUPPI.
531.1-29 4
Dahlen,
5
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6
Alhers,
7
Simons.
K.,
Dahlen,
J.J.,
K..
G. and
Adolphson,
K.,
J.L.
Ehnholm,
U. and
Frick.
C.,
M.H.
and
Tamm.
A.
(1974)
Hazard.
Renkonen,
0.
(1974)
Acta
Genet.
6. 23+235
Clin. W.J. and
(1977)
Med.
J. Lipid
Bloth.
B. (1970)
(1973)
2.
Res.
Stand. 18.
Acta
196,
327-331
331-338
Pathol.
Microbic&
Stand.
78.459-466 8
Vogelberg,
9
J&gem.
K.H., G. and
Uterman. Kostner.
G. and G.M.
10
Ehnholm.
C.,
Garoff,
11
Uterman,
G.,
LiPP,
K. and
Cabana,
V.G..
12
Albers.
13
McFarlane,
J.J.,
A.S.
14
BiIheimer,
D.W..
H..
(1958)
Renkonen, Wiegandt. Warnik,
Nature
Eisenberg,
Gries,
(1975)
182.
S. and
F.A.
Immunogenetics 0.
and
Simons,
H. (1972) G.R.,
KIin.
K.
(1972)
Humangenetik
and
Hazzard,
R.I.
(1972)
Chem.
KIin.
Biochem.
11,
W.R.
Biochemistry
11.
3229-3232
14.142-150 (1975)
Metabolism
24.1047-1054
53 Levy,
291-296
1, 56Ce574
Biochim.
Biophys.
Acta
260.
212-221
Sect.
B
70 15 16 17
Folch, J.M., bees, M. and Sloane-Stanley. G.H. (1957) J. Biol. Chem. 226, 497-509 Krempier, F., Kostner, G.. Bolzano, K. and Sandhofer, F. (1978) Atherosclerosis 30, 57-65 Kostner, G.&f., Patsch. J.R., Sailer, S.. Braunsteiner. H. and Holasek, A. (1974) Eur. 6. Biochem.
611-621 18 Brown. W.V.. Levy, R.I. and Fredrickson, D.S. (1969) J. Biol. Chem. 244, 5687-5694 19 Lowry, O.H., Rosebrough. N.J., Farr, A.L. and Randall, R.J. (1951) J. Biol. Chem. 193,265-275 20 LaurelI. C.B. (1972) Stand. J. Clin. Lab. Invest. 29 Suppl. 124, 21-37 21 Eisenberg, S. and Rachmilewitz. D. (1975) J. Lipid Res. 16, 341-351 22 Eisenberg, S. (1978) J. Lipid Res. 19, 229-236 23 Eisenberg, S., Bilheimer. D.W. and Levy, R.I. (1972) Biochim. Biophys. Acta 280.94-104 24 Eisenberg, S., Bilheimer, D.W. and Levy, R.I. (1973) Biochim. Biophys. Acta 326, 361-377 25 Langer, T., Strobrr. W. and Levy, R.I. (1972) J. Clin. Invest. 51. 1528-1536 26 Berman, M.. Hall, M.. III. Levy. R.I., Eisenberg, S., Bitheimer. D.W., Phair, R.D. and Goebel, 27 28
(1978) J. Lipid Res. 19. 38-56 Nest& P.J. (1964) J. Clin. Invest. 43, 943-949 Schaefer, E.J.. Eisenberg, S., Levy, R.I. (1978)
J. Lipid Res. 19, 667-87
45.
R.H.
Biochimica et Biophysics Acta, 575 (1979) 0 Elsevier/North-Holland Biomedical Press
63-70
BBA 57440
LIPOPROTEIN LIPOPROTEINS
F. KREMPLER,
(a) IS NOT A METABOLIC PRODUCT OF OTHER CONTAINING APOLIPOPROTEIN B
G. KOSTNER,
K. BOLZANO
and F. SANDHOFER
First Department of Medicine, Landeskrankenanstalten, A-5020 Salzburg and Institute of Medical Biochemistry, University of Graz, A-8010 Graz (Austria) (Received
March 19th,
Key words: Lipoprotein
1979)
(a); Apolipoprotein
B; VLDL
Summary 12’1-Labeled autologous very low density lipoprotein (VLDL) was injected intravenously into three lipoprotein (a) positive individuals. One other lipoprotein (a) positive subject received ‘251-labeled VLDL from a lipoprotein (a) negative donor. Specific activity of apolipoprotein B in VLDL, low density lipoprotein (LDL) and lipoprotein (a) was measured for 5 days. In the lipoprotein (a) fraction only traces of radioactivity could be detected, which were caused by contamination with labeled LDL. No precursor-product relationship existed between apolipoprotein B in VLDL or LDL and apolipoprotein B in lipoprotein (a). One lipoprotein (a)-positive individual was kept on a fat-free diet for 4 days to prevent chylomicron formation; no change in the serum level of lipoprotein (a) could be detected under these conditions. The data of this study indicate that lipoprotein (a) is not a metabolic product of VLDL or LDL. Also chylomicrons are not likely to play a role as a precursor for lipoprotein (a). It is concluded that lipoprotein (a) is synthesized as a separate lipoprotein.
Introduction Since the first description of lipoprotein (a) [l] numerous studies on the physicochemical properties of this lipoprotein, its incidence and concentration in the serum, and its relation to coronary artery disease have been published (for a review see Ref. 2). A possible role of lipoprotein (a) as a risk factor for
Abbreviations: VLDL, very low density lipoproteins (d < 1.006 g/ml); LDL. low density lipoproteins Cd = 1.006-1.063 g/ml); HDL, high density lipoproteins (d = 1.063-1.21 g/ml); SDS. sodium dodecyl sulfate.
64
atherosclerotic vascular disease has already been postulated in earlier studies [ 3-51. Albers et al. [6] developed a very sensitive radioimmunoassay for the qu~titative estimation of lipoprotein (a). With this method, lipopro~in (a) could be detected in practically all individuals with apolipoprotein B. Albers et al. [6] also found a positive association between high lipoprotein (a) levels and coronary heart disease, which was even stronger for subjects younger than 50 years. In many respects, lipoprotein (a) is very similar to low density lipoproteins (LDL). There is no si~ific~t difference in the composition of neutral lipids and phospholipids f 7-91, and in both lipoproteins the main protein is apolipoprotein B [lo]. The carbohydrate content of lipoprotein (a), however, is several times higher than that of LDL [lo] and, in contrast to LDL, lipoprotein (a) contains a specific lipoprotein (a) apolipoprotein [ lO,ll]. The density of lipoprotein (a) is between 1.055 and 1.110 g/ml [2]. Because of its higher c~bohydra~ content lipoprotein (a) has a higher electrophoretie mobility than LDL and, therefore, was also described as ‘sinking pre-P’ or ‘pre-p,’ lipoprotein
[4,5,121. The site and mechanism of synthesis of this lipoprotein are not yet known. Because of the chemical similarities between lipoprotein (a) and LDL, it is possible that lipopro~in (a) is formed during the metabolic catabolism of other apoprotein B containing lipoproteins, that is chylomicrons, VLDL or LDL. This study was performed in order to assess the possibility that one of these lipoproteins could be a precursor for lipoprotein (a). Methods Subjects. The study was performed on four male subjects after obtaining informed consent (Table I). All were strongly positive for lipoprotein (a), as checked by immunodiffusion with specific antisera to lipoprotein (a). They were hospitalized during the investigation; none received any treatment known to influence lipid metabolism and all were kept on a ‘normal’ hospital diet. No change in body weight or serum cholesterol and triglyceride levels was observed throughout the study. KI (3 X 60 mg daily) was administered to each individual, beginning 3 days prior to the study and extending throughout the whole study. Preparation of labeled lipoproteins. VLDL (d < 1.006 g/ml) was isolated from serum after fasting by ultracen~ifugation at serum density at 42 000 rev./
TABLE CLINICAL
I DATA
OF
SW
THE
SUBJECTS Body
Serum
Serum
weight
cholesterol
triacylglyceral
(kg)
tmgidl)
(mgidl)
G.F.
male
52
67
261
97
F.J.
male
36
71
218
195
Z.E.
male
68
79
171
75
G.L.
male
47
82
209
169
Diagnosis
emphysema normal coronary normal
heart
disease
65
min for 18 h using a Beckman Ti 50.2 rotor. The top fraction was removed by slicing the tube and washed once. LDL (d = 1.006-1.063 g/ml) were obtained after adjusting the infranatant to d = 1.063 g/ml by addition of solid NaCl and ultracentrifugation at 42 000 rev./min for 20 h. The densities were measured at 15°C with a calculating digital density meter DMA 46 (Anton Paar K.G., Graz, Austria). All ultracentrifugation procedures were carried out at 15°C. The top fraction which contained LDL and some lipoprotein (a) was chromatographed on an agarose column to separate LDL from contaminating lipoprotein (a). VLDL and LDL were radioiodinated with ‘*‘I (The Radiochemical Centre, Amersham) according to McFarlane [ 131 as modified by Bilheimer et al. [ 141. Free iodine was removed by filtration on Sephadex G-25 and subsequent dialysis against 0.15 M NaCl with several bath changes. 7-15s of the radioactivity in the labeled VLDL was found in the lipid moiety after extraction by the method of Folch et al. [ 151. Labeled and unlabeled VLDL showed the same electrophoretic mobility on agarose and identical immunoreactive behavior. Prior to the injection, the labeled lipoproteins were sterilized by passage through a Millipore filter (0.45 pm). The injected radioactivity was between 20 and 60 &!i. Determination of specific activity of apolipoprotein B in VLDL, LDL and lipoprotein (a). After the injection of labeled VLDL, venous blood was collected
at several time intervals (Fig. 3), allowed to clot at room temperature and centrifuged for 20 min at 3000 rev./mm 1 mg/ml NaN3 and 1 mg/ml disodium EDTA were added to the serum. VLDL were isolated as described above. After removal of VLDL, the infranatant was brought to a density of 1.055 g/ml by addition of solid NaCl and centrifuged for 20 h at 42 000 rev./min. The top fraction which contained most of the LDL was removed by slicing the tube. The infranatant was adjusted to d = 1.110 g/ml and centrifuged for 22 h at 42 000 rev./min. From the top fraction (d = 1.055-1.110 g/ml) which contained lipoprotein (a) and some LDL and high density lipoproteins (HDL), lipoprotein (a) was purified by filtration on an agarose column (0.9 X 100 cm) using BioGel A-5m (Bio-Rad Laboratories, Richmond, CA). Elution of the lipoproteins was carried out at room temperature with 0.15 M NaCl containing 1 mg/ml disodium EDTA and 1 mg/ml NaN,, pH adjusted to 8.5 by addition of 25% NH40H (E. Merck, Darmstadt). The elution pattern of the lipoproteins of density 1.055-l .llO g/ml is shown in Figs. 1 and 2. The protein content of the peaks was identified by immunodiffusion using specific antisera [16,17], and the lipoprotein (a) peak was further characterized by its electrophoretic mobility on agarose gel, immunoelectrophoresis, immunodiffusion and chemical composition. It was identical with lipoprotein (a) preparations described in earlier reports [ 2,161. Immunodiffusion and immunoelectrophoresis as well as agarose electrophoresis were carried out in 1% agarose gels using 0.05 M barbital buffer (pH 8.2). Electrophoretic separations were performed at 5 V/cm for 65 min. Sudan Black and Amido Black 10 B were used for lipid staining and protein staining, respectively. For the determination of the specific activity of apolipoprotein B, the fractions containing the lipoproteins were first dialysed against distilled water and then lyophilized. The lipoproteins were delipidated with diethyl ether/ethanol (1 : 3, v/v) [ 181. The delipidated apolipoproteins were washed several times
66
with 6 M urea to remove the ‘soluble” apolipoproteins. Removal of ‘soluble’ apo~~opro~ins was considered complete when no radioactivity could be detected in the urea solution for the last three washes. The rem~nin~ apolipoproteins which were insoluble in 6 M urea were solubilized by addition of SDS. This fraction contained less than 1% of apolipoproteins other than apolipoprotein B as judged from SDS-polyacrylamide gel electrophoresis. The protein content was estimated by the method of Lowry et al. [ 191; radioactivity was measured in a Packard Autogamma Sc~t~lati~n Spectrometer 5160. The co~c~n~at~on of lipoprotein (a) in the serum was estimated by Laurel13 one-dimensional rocket immunoelectrophoresis method [ 201. Results Fig. 1 shows the elution pattern of the lipoproteins of the density 1.0551.110 g/ml on agarose gel. From the immunolo~c~ behavior against specific antisera, peak I is identified as lipoprotein (a), peak II as lipoprotein B (part of serum LDL), and peak III as HDL. Fig. 1 also shows the radioactivity of the eluted lipoprotein fractions 24 h after the injection of the labeled VLDL (subject Z.E.). It can be seen that the radioactivity peaks coincide with the extinction peaks II and III, The radioactivity in the HDL fraction (peak III) can be explained by exchange of apolipoprotein C between VLDL and HDL [21, 221. There are traces of radioactivity in the lipoprotein (a) peak, but these small amounts of radioactivity do not follow the concentration of this lipoprotein. From the curve of the radioactivity it may be assumed that these traces of radioactivity are caused by conflation with labeled LDL, To prove this assumption, labeled LDL was mixed in vitro with lipoprotein
Fig. 1. Elution profile of the lipoproteins of density 1.055-1.110 g/ml on BioGel A-5m. The samPle was taken from subject Z.E. 24 h after the injection of labeled VLDL. -, extinction at 280 nm; . --. - *, radioactivity (cpm/ml). The immunological reactivities of the eluted fractions against antibodies to lipoprotein (af apohpoprotein, apoBpopr&eio A, B and C are shown in the figure, Peak f rwresents hPwrotein (a), peak II lipoprotein B
67
i
ld05&
.?4
ri
?i
hours
C
9k
Z.E.
G.L.
1
5x104
5x10
4
,
A
6 hours
24
18
72
96
125
hours
Fig. 3. Specific activity-time curves of apolipoprotein B in VLDL, LDL and lipoprotein (a) after intravenous injection of labeled VLDL in subject G.F. (A), subject F.J. (B), subject Z.E. (C), and subject G.L. (D). o----o, VLDL; +-----., LDL; X-X. lipolprotein (a).
(a) positive serum. Specific activity of apolipopro~~ B in LDL and lipoprotein (a) was then determined in the same way as for in vivo studies. Fig. 2 shows the elution pattern of the lipoproteins of the density 1.055-l .110 g/ml of this in vitro assay. The same elution pattern and the same distribution of radioactivity was found except for HDL (no labeled apolipoprotein C). Traces of radioactivity were found in the lipoprotein (a) fraction to a similar extent as in the in vivo studies.
68
The specific activity-time curves of apolipoprotein B in VLDL, LDL and lipoprotein (a) after the injection of labeled VLDL are given in Fig. 3. In the subjects G.F., F.J., and Z.E., autologous VLDL were labeled and reinjected. In the case of subject G.L., VLDL was obtained from a lipoprotein (a)-negative donor, labeled and injected to subject G.L., who was lipoprotein (a)-positive as the other recipients. In all cases, the specific activity-time curves of apolipoprotein B in VLDL and LDL showed the well known precursor-product relationship as already described by others [14,23,24]. Only traces of radioactivity could be detected in apolipopro~~ B of the lipopro~in (a) fraction. The specific activity of apolipoprotein B in the lipoprotein {a) fractions was extremely low when compared with that of VLDL until 4 days after the injection of labeled VLDL. At that time, radioactivity in both fractions had practically disappeared. Specific activity of apolipoprotein B in the lipoprotein (a) fraction was between 4 and 9% (R = 6.5%) of that in the LDL throughout the whole study. The same percentage was found in the lipoprotein (a) fraction after the in vitro addition of labeled LDL to lipoprotein (a) positive serum. Therefore, the specific activity of apolipoprotein I3 in the lipoprotein (a) fraction found in vivo after the injection of labeled VLDL can be accounted for by contamination of lipoprotein (a) with labeled LDL. Obviously, no precursor-product relationship exists between apolipopro~in B in VLDL or LDL and apolipoprotein B in lipoprotein (a). It is possible that chylomicrons are a precursor for lipoprotein (a). To study this possibility, a lipoprotein (a)-positive individual was kept on an extremely low-fat diet (less than 2 g fat/day) for 4 days and serum samples were obtained daily for rocket immunoelectrophoresis of lipoprotein (a). No change in the lipopro~~ (a) level could be observed as judged from the peak height in immunoelectrophoresis. Discussion In recent years, many experiment have been performed to study the metabolism of apolipoproteins in various lipoprotein classes. A unidirection~ catabolic pathway from VLDL to ‘intermediate lipoproteins’ and then to LDL has been established [14,21,23-251. In the course of this catabolic process, apolipoprotein B is not removed from the lipoprotein particle. From analysis of the specific activity-time curves of apolipoprotein B in different lipoprotein fractions after the injection of labeled VLDL, a precu~or-product relationship between VLDL and LDL has clearly been demonstrated by other investigators [ 14,23,24]. Since apolipoprotein B represents approx. 80% of the apolipoproteins in lipoprotein (a) [lo], it is possible that lipoprotein (a) is formed during the catabolism of either chylomicrons, VLDL or LDL. Apolipoprotein B does not exchange between. the various apolipoprote~ B-con~n~g lipoproteins [21], Therefore, from the specific activity-time curves of apolipoprotein B in VLDL, LDL and lipoprotein (a) after the injection of labeled VLDL a possible metabolic conversion of VLDL or LDL to lipoprotein (a) can be demonstrated or excluded. If a precursor-product relationship existed between VLDL or LDL and lipo-
69
protein (a), a cross-over of the specific activity-time curves of apolipoprotein B in VLDL or LDL and apolipoprotein B in lipoprotein (a) must occur within the first 2 days, as estimated from the known half-lives of apolipoprotein B in these lipoproteins [ 16,261. In our experiments, only traces of radioactivity could be detected in the lipoprotein (a) fraction that were due to contamination with labeled LDL. Our results show that neither VLDL nor LDL can be considered as a precursor for lipoprotein (a). To exclude chylomicrons as a possible precursor for lipoprotein (a), a lipoprotein (a)-positive subject was kept on a fat-free diet for 4 days. One can assume that under such a diet the formation of chylomicrons is drastically reduced as compared with a normal, exogenous fat-containing diet. If lipoprotein (a) were a catabolic product of chylomicrons, a decrease in the serum concentration of lipoprotein (a) is to be expected during this time when the known half-lives of chylomicrons [ 271, triacylglycerol-rich ‘intermediates’ [ 281 and lipoprotein (a) [ 161 are taken into consideration. Since under the conditions described above the concentration of lipoprotein (a) in the serum remained constant we believe that chylomicrons do not play a significant role in the formation of lipoprotein (a). From our studies, it is evident that lipoprotein (a) is not formed from VLDL, LDL or chylomicrons through the catabolic pathway of these lipoproteins. Therefore it can be concluded that lipoprotein (a) is secreted into the blood as a separate lipoprotein. This could explain the fact that the plasma concentration of lipoprotein (a) is not influenced by various dietary manipulations which are known to drastically influence the level of the other apolipoprotein B-containing lipoproteins [6]. Until now no information concerning the site of production of this lipoprotein is available. Acknowledgments This work was supported by Fonds zur Fijrderung Forschung (Project Nr. 3631). The skillful technical TaIman and Miss E. Meisl is greatfully acknowledged.
der Wissenschaftlichen assistance of Miss H.
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