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Interrelationship between Apoproteins of Very Low Density Lipoprotein and Other Serum Lipoproteins in Lactating Goats
Interrelationship between Apoproteins of Very Low Density Lipoprotein and Other Serum Lipoproteins in Lactating Goats D. STEAD, M. TAMIR 1, and V. A. WELCH National Institute for Research in Dairying Shinfield, Reading, RG2 9AT, England ABSTRACT
intermediate density lipoprotein and with a transfer o f apoproteins between very low density and high density lipoproteins.
Lipoproteins of goat serum were labeled in vivo by intravenous injection of tritium labeled DL-lysine monohydrochloride. The specific radioactivity of the very low density plus intermediate density fractions reached a maximum at 1.2 h, declined sharply until about 9 h, and then more slowly. The specific radioactivities o f the low density and high density lipoproteins reached lower maxima at 6.5 h and 8.5 h, respectively. When iodine-125-labeled very low density lipoprotein o f goats was incubated in vitro with buffer there was a transfer of 11% of radioactivity, chiefly to the intermediate and low density lipoproteins, but on incubation in serum 29% of the radioactivity was distributed equally among the other lipoproteins. On gradient gel electrophoresis o f the apolipoproteins, the slowest migrating zones in the very low density, intermediate density, and low density lipoproteins were the most radioactive whereas over 90% of the radioactivity in the apoproteins o f the high density lipoprotein was in two zones, one of which was at the origin. The results are consistent with the formation o f low density lipoprotein from very low density lipoprotein via the
INTRODUCTION
In dairy cows certain lipoproteins in serum transport triglycerides to the m a m m a r y gland for synthesis o f milk fat (19), but it is n o t known whether the lipoproteins are interconvertible as are the lipoproteins of humans and rats (12) or, if so, which of their constituent apoproteins are transferred. The spectrum of lipoproteins is simpler in goat than in cow (5) and when, in connection with another study, a supply of goats' milk containing 3 H-labeled protein was required, the o p p o r t u n i t y was taken to study the turnover of lipoproteins of goat serum. The resultant specific radioactivity-time curves were complex and to reach a better understanding of them, 12s I-labeled VLDL 2 was incubated in buffer and in serum, and the distribution of radioactivity in the constituent apoproteins of the serum lipoproteins was studied. MATERIALS AND METHODS In Vivo Experiments
Received October 21, 1976. Dr. Tamir participated in the in vitro experiments while on sabbatical leave from Agricultural Research Organization, The Volcani Center, P. O. Box 6, Bet Dagan, Israel. 2Abbreviations: very low density lipoprotein, VLDL, d
A British Saanan goat 7.5 yr old in the 18th m o o f lactation giving 4 to 4.5 kg milk per day and fed on hay and concentrates (principally rolled barley and flaked maize plus mineral and vitamin supplements) to appetite was used. In the first experiment, 10 mCi of DL-[4,5 -3 H(n)]lysine monohydrochloride (Radiochemical Centre, Amersham, England) was administered. There was a little difficulty with the intravenous injection and 3 mCi were injected subcutaneously and 7 mCi via a cannula in the jugular vein. Seven days later 10 mCi of 3H-labeled mixed amino acids were administered via the jugular cannula, and milk samples only were
1529
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STEAD ET AL.
obtained. Thirty-six days after the second radioactive dose, 20 mCi of DL-[ 3 H] lysine HC1 were administered via the cannula (second experiment). Blood samples were obtained via a cannula in the jugular vein opposite to that in which the labeled lysine was introduced and from a cannula in the mammary vein. Dextran sulfate reagent (5) consisted of a mixture of 1 vol of 10% (wt/vol) clinical grade dextran sulfate (Mw ca. 5000) (Glaxo Laboratories Ltd., Greenford, Middx, U.K.) and 5 vol of 1M CaC12 solution. The optimum volume of reagent required to precipitate the serum lipoproteins of density less than 1.06 g/ml (.15 ml reagent/ml serum) was found by titration of small aliquots of serum with the reagent to maximum turbidity at 700 nm. The appropriate volume of reagent was added either to 10 ml serum (Experiment 1) or to 20 ml serum (Experiment 2). The precipitate was dissolved in sodium citrate solution (d = 1.019 g/ml) containing 1 mg/ml DL-lysine HC1 and reprecipitated with .1 M CaCI2 solution (5). Each sample was reprecipitated three times to reduce the free [3H]lysine content to negligible proportions. The precipitate then was dissolved in sodium citrate (6 ml, d = 1.019 g/ml), transferred to a polycarbonate centrifuge tube (17 mm x 63 mm, nominal capacity 10 ml), and the VLDL + IDL and LDL were separated by ultracentrifugation at 157,000 x g ( a v ) f o r 20 h at 12 C in the 10 × 10 ml angle rotor (tube angle 20 ° to axis) of an MSE Superspeed Ultracentrifuge (29). After removal of the precipitable lipoproreins, the HDL was obtained by ultracentrifugation of the supernatant, under the above condit!ons, after its density had been adjusted to 1.21 g/ml by the addition of solid KBr. The HDL was purified by further ultracentrifugation at the same density. To remove free [3H]lysine, the HDL samples were dialyzed overnight against running distilled water and then against three batches of distilled water on three successive nights. The purified lipoproteins were stored in sodium citrate solution (d -- 1.019 g/ml), and their purity was checked by electrophoresis (5). The protein contents were determined according to Lowry et al. (21) but with the addition of .1 ml of a 5% (vol/vol) Teepol solution to the VLDL + IDL assay mixtures to remove the turbidity which developed. J o u r n a l o f D a i r y S c i e n c e Vol. 6 1 , N o . 11, 1 9 7 8
Radioactivity was measured as in (2) in the scintillator solution containing methoxyethanol-anisole; 10 ml of scintillator solution were mixed with .6 ml (Experiment 1, 13% counting efficiency) or 1 ml (Experiment 2, 11% counting efficiency) of lipoprotein solution. The radioactivities of the lipoproteins/mg protein, i.e., the specific radioactivities of the apoproteins, were plotted semilogarithmically against time. In Experiment 2, the specific radioactivities were corrected for the low initial specific radioactivity which resulted from the earlier injections. For VLDL + IDL (Experiment 2), the points after the period of rapid decline in specific radioactivity which followed the maximum were fitted to a straight line by the method of least squares. In Vitro Experiments
Serum was prepared from jugular blood withdrawn from lactating Saanen goats which were fed on hay and concentrates (principally barley, maize, and wheat bran plus vitamin and mineral supplements) to appetite. The blood samples were allowed to clot for 2 h at room temperature and centrifuged then at 2000 rpm for 20 min. Disodium ethylenediamine tetraacetare (EDTA) was added to serum to give a final concentration o f . 1%. Chylomicrons were removed from the serum by overlayering 6 ml of serum with 2 mi of a solution of density 1.006 g/ml (sodium citrate or saline) and were centrifuged for 30 min at 60,000 × g (av). Lipoprotein fractions were separated by sequential ultracentrifugation of 8 ml of chylomicron-free serum at increasing densities in the 50 Ti rotor of a Beckman L2-50 preparative ultracentrifuge at 14 C. Solid sodium citrate was used to obtain the required densities. Four lipoprotein fractions were isolated at densities of 1.006 g/ml (VLDL), 1.019 g/ml (IDL), 1.06 g/ml (LDL), and 1.21 g/ml (HDL). The VLDL and IDL were separated after 18 h and LDL and HDL after 24 h of centrifugation at 105,000 x g (av). Each fraction was brought to 8 ml with sodium citrate solution of its respective density and recentrifuged for 24 h. The purity of each fraction was checked by electrophoresis (5). The VLDL were iodinated by a modification
INTERRELATIONSHIPS BETWEEN SERUM LIPOPROTEINS of the iodine monochloride method of McFarlane (22) as described by Reif (26). The Na 12s I was obtained from the Radiochemical Centre, Amersham, England. Unreacted iodide was removed by dialysis against 8 to 10 changes of .15 M NaC1 and .01% EDTA, for 6 to 7 h at 4 C. The efficiency of labeling of the VLDL with 12sI was 25.85 -+ 3.1%, and protein recovery after iodination was 86.3 -+ 8.7% (means of three values -+ SD). The ratio of moles of iodide to 100,000 g of apoprotein was less than 1 in all preparations as recommended by Eisenberg and Rachmilewitz (13). Radioactivity was determined in the Autogamma Scintillation Spectrometer (Packard Tricarb). The labeled VLDL was incubated with serum for 1 h at 37 C. In the control, labeled VLDL was incubated with .15 M NaC1 in .05 M phosphate buffer, pH 7.6 under the same conditions. Each determination was in duplicate. Aliquot samples then were withdrawn for 12 s I monitoring. The lipoprotein fractions subsequently were isolated from the remaining samples by centrifuging at their respective densities. The 12s I content in each fraction was measured. The fractionated sampIes were lyophilized and delipidated as described by Brown, Levy and Fredrickson (3), and the 12 s I content of the lipid fraction was determined. The apoproteins were solubilized by incubation (37 C for 16 h) in .02 M tris/HC1 (pH 8.2) containing .1 M sodium dodecylsulfate and .001 M dithiothreitol, diluted 1:1 (vol/vol) with 40% (wt/vol) sucrose before gel application, and fractionated by electrophoresis on 5 to 11% polyacrylamide gradient gels containing 1% sodium dodecylsulfate. Bromophenol blue was used as a marker (7). The gels were stained with .05% Coomassie Blue R in 12.5% (wt/vol) trichloroacetic acid (6), destained in a solution of water to ethanol to acetic acid (65:25:8 by vol), (31), and sliced into zones whose radioactivities were determined. RESULTS In Vivo Experiments
Figure 1 shows that there was no cross contamination between the precipitable and nonprecipitable lipoproteins. In addition, it has been established (Stead and Welch unpublished) that when lipoproteins of goat serum are
1531
FIG. 1. Etectrophoresis of goat serum lipoproteins (prestained with Sudan Black B) on gradient polyacrylamide gel (5). The supernatant after removal of the dextran sulfate precipitate (DSS), the d>l.O19 g/ml fraction (LDL) of the dextran sulfate precipitate, and whole goat serum.
isolated by means of dextran sulfate as described here, the LDL has the same electrophoretic mobility as, and is immunologically identical to, LDL isolated by ultracentrifugation alone. Furthermore, the nonprecipitable lipoprotein is immunologieally identical to HDL isolated by uhracentrifugation, has the same flotation rate in the analytical ultracentrifuge, and does not contain LDL. The specific radioactivities o f the apoproteins of the lipoproteins from the second experiment are plotted semilogarithmically against time in Figur e 2a; the values for jugular and mammary venous serum appeared to be the same, and they were treated as equivalent when the curves were drawn. The maximum value for the specific radioactivity of the VLDL + IDL apoprotein was 22 dpm/ug, which was reached 1.2 h after injection, whereas that o f LDL was 7.1 dpm/ug at 6.5 h, and that of HDL 2.8 dpm/ug at 8.5 h. The decline in specific radioactivity did not follow a simple exponential curve for any of the lipoproteins. In particular, there was a marked change in the slope of the VLDL curve at about 9 h, which indicated that the curve represented apoproteins with different kinetic behavior. The specific radioactivity-time curves Journal of Dairy Science Vol. 61, No. 11, 1978
15 3 2
STEAD ET AL. b
In Vitro Experiment
4¢ VLDL ÷ IOL
10
•
•
i0 .I •
I
I
I
I
! "
HDL
HDL
Time, h
FIG. 2. Specific radioactivities of the protein moieties of goat very low density lipoprotein (VLDL), intermediate density lipoprotein (IDL), low density lipoprotein (LDL), and high density lipoprotein (I-IDL) after administration of DL-13Hllysine monohydrochloride. (a) Experiment 2; (b) Experiment 1. e, jugular venous serum; A, mammary venous serum.
which were obtained in the first experiment were similar to those of the second experim e n t in shape, and in time and relative size of the maximum (Fig. 2b).
The protein concentrations of the lipoprotein fractions of goat serum [expressed as mg! 100 ml + SD (means of six determinations)] were: VLDL, 1.28 + .12; IDL, 2.1 + .3; LDL, 8.1 + 1.8; HDL, 135.8 + 7.6. The distribution of radioactivity among the four lipoprotein classes after incubation of the 12s l-labeled VLDL with serum and phosphate/sodium chloride buffer is in Table 1. Approximately 80% of the total radioactivity in the lipoproteins recovered after incubation with either serum or buffer was bound to proteins. On incubation of the labeled VLDL with serum, 29% of the radioactivity originally in this fraction was transferred to other lipoproreins and was distributed approximately equally among the other three lipoprotein fractions. On incubation with buffer, however, only 11% of the radioactivity originally in the VLDL fraction was recovered in other lipoproteins, and there was approximately twice as much radioactivity in the IDL and LDL as in the HDL. After delipidation of the lipoproteins, the following proportions of radioactive protein were resolubilized: VLDL, 70%; IDL, 90%; LDL, 75%; HDL, 98%. The electrophoretic patterns of the apoproteins are shown diagrammatically in Figure 3. The distribution of radioactivity among the apoproteins after incubation of 12 s I-labeled VLDL in serum is in Table 2. The distribution of radioactivity was similar in the VLDL, IDL, LDL; the most radioactive bands were in zones 1 and 2. In the VLDL, zone 3 was also radioactive,
TABLE 1. Redistribution of 1251 from very low density lipoprotein to other lipoprotein fractions, a Incubation medium buffer + .15 M NaCI (control) (% recovered cpm)
PO 4
Lipoprotein fraction Very low density Intermediate density Low density High density
~ 71.0 10.5 10.1 8.6
Serum (% recovered cpm) Lipoprotein Protein SD ~ SD 3.6 2.5 3.4 1.5
aEach value is the mean of four determinations. Journal of Dairy Science Vol. 61, No. 11, 1978
56.4 7.5 8.7 7.2
4.6 1.4 3.7 1.7
Lipoprotein SD 88.7 4.6 3.4 1.9
3.6 2.0 .25 .19
~ 73.1 3.8 3.1 1.6
Protein SD 5.7 1.9 .44 0
INTERRELATIONSHIPS BETWEEN SERUM LIPOPROTEINS Zone
--~--
3
4
Bm 6 I B m
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i
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. 7
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i
9 .
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VLDL
IOk
.
.
.
Marker
i LDL
HDL
FIG. 3. Electrophoresis of the apolipoproteins of goat very low density lipoprotein (VLDL), intermediate density lipoprotein (IDL), low density lipoprotein (LDL), and high density lipoprotein (HDL), on gradient polyacrylamide gels containing sodium dodecyl sulfate. The gels were sliced into the zones indicated for counting of radioactivity.
but although the most intense band in the LDL was in this zone, it was not radioactive. In the HDL over 90% of the radioactivity was concentrated in two bands in zones 1 and 6. Zone 6 of the VLDL was the zone from which the greatest proportion of radioactivity was lost on incubation in serum as compared to incubation in buffer.
DISCUSSION
Human and rat VLDL contain apoproteins which are common to LDL (apoprotein B) and to HDL (apoprotein C). The apoprotein B of VLDL is converted rapidly in vivo to apoprotein B of the LDL via the formation of a lipoprotein of intermediate density (12, 17). This transfer does not take place to any great extent when labeled VLDL is incubated in serum for 20 to 30 min (1, 27), but when the incubation time is increased, and particularly when post-heparin plasma is used, there is an appreciable transfer (8). The apoprotein C of VLDL exchanges with the apoprotein C of the HDL in vitro and in vivo (11, 12), and there is also a net transfer of apoprotein C to HDL in rats in vivo and when VLDL is incubated in post-heparin plasma (15). The different phases of the complex disappearance curve of VLDL radioactivity after injection of 12 s I-labeled VLDL (13) can be attributed to differences in
1533
the rates of turnover of the VLDL apoproteins (11, 14). The specific radioactivity-time curves for the apoproteins of goat serum after an injection of DL-[3H] lysine were similar to those for human apoproteins after injection of [TSSe]seleno_ methionine (9, 28) and for five species of animals after injection of DL- or L-[3H] lysine (18). The early maximum and sharp fall in specific radioactivity of VLDL + IDL apoproteins of goat were consistent with the precursor-product relationship which has been suggested in humans between the apoprotein B of VLDL and that o f LDL (8, 9, 28). In support of this, most of the radioactivity in the IDL and LDL of goat serum, after incubation with 12s l-labeled VLDL, was in electrophoretic zones 1 and 2. In 10% polyacrylamide gels, apoprotein B remained at the origin (12), and although direct comparison is not possible because the electrophoretic system in our work was different, it seems likely that zones 1 and 2 could correspond to apoprotein B. Similarities between the apoproteins of VLDL and LDL2 (1.039< d< 1.060 g/ml) have been demonstrated also in cows (30). The complex decay curve o f the specific radioactivity of VLDL + IDL apoprotein of goats was also similar to that of VLDL radioactivity in rats after injection of x 2 s I-labeled VLDL (13). The slower turning over component of the decay curve of rat 12s I-labeled VLDL (13) was attributed to the turnover of apoprotein C which equilibrates between VLDL and HDL (12, 14). In our experiments more radioactivity transferred from 1 2 s I-labeled VLDL of goat to other lipoproteins when it was incubated w i t h serum than could be accounted for by control incubation in buffer (Table 1), and the relative increase in radioactivity was greatest in HDL (Table 1). On eleetrophoresis of apoproteins 42% of HDL radioactivity was recovered in zone 6 (Table 2). After incubation, zone 6 of the HDL contained more radioactivity than was recovered altogether from the corresponding zones of other lipoproreins, and this zone of the VLDL was the one which lost the greatest proportion of radioactivity after incubation in serum. It seems probable, therefore, that zone 6 corresponds to apoprotein C which exchanges between VLDL and HDL in humans (1, 10, 16) and in rats (14, 15, 27). Apart from transfer of apoprotein Journal o f Dairy Science Vol. 61, No. 11, 1 9 7 8
g~
t7
< o
o0
TABLE 2. Distribution of ~2 s 1 in apoproteins of lipoprotein fractions after in vitro incubation of labeled very low density lipoprotein with serum and buffer, a Lipoprotein fraction
1
2
3
Incubation with serum Very low density
15.5
37.3
18.9
Intermediate density
18.9
2.9
Low density High density
19.6 52.2
29.9 4.4 38.6 .4 .2 .3
32.9
10.9
Incubation with buffer Very low density
12.5
a) b) a) b) c)
avalues are percent of total radioactivity recovered/gel. bzones are as in Figure 3.
4
a) b) a) b)
.3 .3
4.0 1.1 3.4 1.7 2.5 .4
Z°neb 5
6
7
1.7
2.0
a) b)
3.1
2.0
3.3 3.6 2.6
3.2 .5
2.3 42.0
6.0 .3
a) b) a) b)
8
9
3.1 2.6 7.1 4.1 6.0 .6
1.9 4.2 2.1 1.4 .r"
a) b)
9.0 5.5
6.0
7.5
a) b)
3.5 2.4
a) b)
1.0 6.5
INTERRELATIONSHIPS BETWEEN SERUM LIPOPROTEINS f r o m V L D L to L D L a n d H D L in vivo, d i r e c t s y n t h e s i s o f L D L a n d H D L (18, 20), a n d recycling (9, 23), possibly m a y occur. In cows, w h o s e l i p o p r o t e i n s have b e e n studied more extensively than those of the goat, a p o p r o t e i n s o f V L D L ( d < 1.019 g / m l ) a n d H D L have similarities (30). A n a d d i t i o n a l l i p o p r o t e i n , LDL1 ( 1 . 0 1 9 < d < 1.03 9 g / m l ) w i t h a n e l e c t r o p h o r e t i c m o b i l i t y similar to t h a t o f HDL, is s o m e t i m e s p r e s e n t b u t has n o t b e e n o b s e r v e d in s h e e p a n d g o a t s (5). T h e LDL1 is i m m u n o l o g i c a l l y related to H D L (30) a n d like H D L can activate milk l i p o p r o t e i n lipase (4). Its c o n c e n t r a t i o n is variable, greater in l a c t a t i n g cows t h a n in dry cows (24, 25), a n d is increased in t h e b l o o d of cows fed p r o t e c t e d fat ( S t e a d a n d Welch u n p u b l i s h e d ) . A p o p r o t e i n C regulates t h e activity o f l i p o p r o t e i n lipase (12), a n d if it is in b o v i n e V L D L a n d H D L (as appears t o be t h e case in t h e g o a t ) and, t h e r e f o r e , possibly in L D L I , b u t n o t in LDL2 [ h u m a n L D L n o r m a l l y c o n t a i n s o n l y traces o f a p o p r o t e i n C ( 1 2 ) ] , this c o u l d explain w h y b o v i n e V L D L a n d LDL1 s u p p l y triglycerides for milk fat s y n t h e s i s w h e r e a s LDL2 m a y n o t (19). It does n o t e x p l a i n w h y b o v i n e H D L triglyceride is n o t a p r e c u r s o r o f m i l k fat a n d this t o g e t h e r w i t h a s t u d y o f t h e factors which affect the concentration of the b o v i n e l i p o p r o t e i n s a n d t h e i r i n t e r c o n v e r s i o n is under investigation. ACKNOWLEDGMENTS
T h e a u t h o r s wish to t h a n k H. L. B u t t l e f o r i n s e r t i o n o f cannulas, J. P o r t e r for t e c h n i c a l assistance, a n d R. F. G t a s c o c k f o r advice a n d encouragement.
REFERENCES
1 Bilheimer, D. W., S. Eisenberg, and R. I. Levy. 1972. The metabolism of very low density lipoprotein proteins. I. Preliminary in vitro and in vivo observations. Biochim. Biophys. Acta 260:212. 2 Bishop, C., T. Davies, R. F. Glascock, and V. A. Welch. 1969. Studies on the origin of milk fat. A further study of bovine serum lipoproteins and an estimation of their contribution to milk fat. Biochem. J. 113:629. 3 Brown, W. V., R. I. Levy, and D. S. Fredrickson. 1969. Studies of the proteins in human plasma very low density lipoproteins. J. Biol. Chem. 244: 5687. 4 Brumby, P. E., J. E. Storry, and J. D. Sutton. 1972. Metabolism of cod-liver oil in relation to
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milk fat secretion. J. Dairy Res. 39:167. 5 Brumby, P. E., and V. A. Welch. 1970. Fractionation of bovine serum lipoproteins and their characterization by gradient gel electrophoresis. J. Dairy Res. 37:121. 6 Chrambach, A., R. A. Reisfeld, M. Wyckoff, and J. Zaccari. 1967. A procedure for rapid and sensitive staining of protein fractionated by polyacrylamide gel electrophoresis. Anal. Biochem. 20:150. 7 Davis, B. J. 1964. Disc electrophoresis. II. Method and application to human serum proteins. Ann. N. ¥. Acad. Sci. 121:404. 8 Eaton, R. P., S. Crespin, and D. M. Kipnis. 1976. Incorporation of 7 s Se-selenomethionine into human apoproteins. II. Characterization of metabolism of very low-density and low-density lipoproteins in vivo and in vitro. Diabetes 25:44. 9 Eaton, R. P., and D. M. Kipnis. 1972. Incorporation of S e 7 s selenomethionine into a protein component of plasma very-low-density lipoprotein in man. Diabetes 21:744. 10 Eisenberg, S., D. W. Bilheimer, and R. I. Levy. 1972. The metabolism of very low density lipoprorein proteins. II. Studies on the transfer of apoproteins between plasma lipoproteins. Biochim. Biophys. Acta 280:94. 11 Eisenberg, S., D. W. Bilheimer, R. I. Levy, and F. T. Lindgren. 1973. On the metabolic conversion of human plasma very low density lipoprotein to low density lipoprotein. Biochim. Biophys. Acta 326:361. 12 Eisenberg, S., and R. I. Levy. 1975. Lipoprotein metabolism. Adv. Lipid Res. 13:1. 13 Eisenberg, S., and D. Rachmilewitz. 1973. Metabolism of rat plasma very low density lipoprotein. I. Fate in circulation of the whole lipoprotein. Biochim. Biophys. Acta 326:378. 14 Eisenberg, S., and D. Rachmilewitz. 1973. Metabolism of rat plasma very low density lipoprotein. II. Fate in circulation of apoprotein subunits. Biochim. Biophys. Acta 326:391. 15 Eisenberg, S., and D. Rachmilewitz. 1975. Interaction of rat plasma very low density lipoprotein with lipoprotein lipase-rich (post heparin) plasma. J. Lipid Res. 16:341. 16 Eisenberg, S., H. G. Windmueller, and R. I. Levy. 1973. Metabolic fate of rat and human lipoprotein apoproteins in the rat. J. Lipid Res. 14:446. 17 Faergeman, O., S. Tsunako, J. P. Kane, and R. J. Havel. 1975. Metabolism of apoprotein B of plasma very low density lipoproteins in the rat. J. Clin. Invest. 56:1396. 18 Fried, M., H. G. Wilcox, G. R. Faloona, S. P. Eoff, M. S. Hoffman, and D. Zimmerman. 1968. The biosynthesis of plasma lipoproteins in higher animals. Comp. Biochem. Physiol. 25:651. 19 Glascock, R. F., and V. A. Welch. 1974. Contribution of the fatty acids of three low density serum lipoproteins to bovine milk fat. J. Dairy Sci. 57:1364. 20 lllingworth, D. R. 1975. Metabolism of lipoproreins in nonhuman primates. Studies on the origin of low density lipoprotein apoprotein in the plasma of the squirrel monkey. Biochim. Biophys. Acta 388:38. Journal o f Dairy Science Vol. 61, No. l 1, 1978
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21 Lowry, O. H., N. J. Rosebrough, A. L. Farr, and R. J. Randall. 1951. Protein measurement with the Folin phenol reagent. J. Biol. Chem. 193:265. 22 McFarlane, A. S. 1958. Efficient trace-labelling of proteins with iodine. Nature 182:53. 23 Phair, R. D., M. G. Hammond, J. A. Bowden, M. Fried, W. R. Fisher, and M. Berman. 1975. A preliminary model for human lipoprotein metabolism in hyperlipoproteinemia. Fed. Proc. 34:2263. 24 Puppione, D. L., B. Raphael, R. D. McCarthy, and P. S. Dimick. 1972. Variations in the electrophoretic distribution of low density lipoproteins of Holstein cows. J. Dairy Sci. 55:265. 25 Raphael, B. C., P. S. Dimick, and D. L. Puppione. 1973. Electrophoretic characterization of bovine serum lipoproteins throughout gestation and lactation. J. Dairy Sci. 56:1411. 26 Reif, A. E. 1968. A simple procedure for high-
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efficiency radioiodination of proteins. J. Nuc. Med. 9:148. Rubenstein, B., and D. Rubinstein. 1972. Interrelationship between rat serum very low density and high density lipoproteins. J. Lipid Res. 13:317. St~ihelin, H. B. 1975. 7 SSe_selenomethionine_ labeled lipoproteins in hypedipidemic and normolipidemic humans. Metabolism 24:505. Stead, D., and V. A. Welch. 1975. Lipid composition of bovine serum lipoproteins. J. Dairy Sci. 58:122. Stead, D., and V. A. Welch. 1976. Immunological properties of bovine serum lipoproteins and chemical analysis of their protein moieties. J. Dairy Sci. 59:1. Vesterberg, O. 1971. Staining of protein zones after isoelectric focusing in polyacrylamide gets. Biochim. Biophys. Acta 243: 345.
intermediate density lipoprotein and with a transfer o f apoproteins between very low density and high density lipoproteins.
Lipoproteins of goat serum were labeled in vivo by intravenous injection of tritium labeled DL-lysine monohydrochloride. The specific radioactivity of the very low density plus intermediate density fractions reached a maximum at 1.2 h, declined sharply until about 9 h, and then more slowly. The specific radioactivities o f the low density and high density lipoproteins reached lower maxima at 6.5 h and 8.5 h, respectively. When iodine-125-labeled very low density lipoprotein o f goats was incubated in vitro with buffer there was a transfer of 11% of radioactivity, chiefly to the intermediate and low density lipoproteins, but on incubation in serum 29% of the radioactivity was distributed equally among the other lipoproteins. On gradient gel electrophoresis o f the apolipoproteins, the slowest migrating zones in the very low density, intermediate density, and low density lipoproteins were the most radioactive whereas over 90% of the radioactivity in the apoproteins o f the high density lipoprotein was in two zones, one of which was at the origin. The results are consistent with the formation o f low density lipoprotein from very low density lipoprotein via the
INTRODUCTION
In dairy cows certain lipoproteins in serum transport triglycerides to the m a m m a r y gland for synthesis o f milk fat (19), but it is n o t known whether the lipoproteins are interconvertible as are the lipoproteins of humans and rats (12) or, if so, which of their constituent apoproteins are transferred. The spectrum of lipoproteins is simpler in goat than in cow (5) and when, in connection with another study, a supply of goats' milk containing 3 H-labeled protein was required, the o p p o r t u n i t y was taken to study the turnover of lipoproteins of goat serum. The resultant specific radioactivity-time curves were complex and to reach a better understanding of them, 12s I-labeled VLDL 2 was incubated in buffer and in serum, and the distribution of radioactivity in the constituent apoproteins of the serum lipoproteins was studied. MATERIALS AND METHODS In Vivo Experiments
Received October 21, 1976. Dr. Tamir participated in the in vitro experiments while on sabbatical leave from Agricultural Research Organization, The Volcani Center, P. O. Box 6, Bet Dagan, Israel. 2Abbreviations: very low density lipoprotein, VLDL, d
A British Saanan goat 7.5 yr old in the 18th m o o f lactation giving 4 to 4.5 kg milk per day and fed on hay and concentrates (principally rolled barley and flaked maize plus mineral and vitamin supplements) to appetite was used. In the first experiment, 10 mCi of DL-[4,5 -3 H(n)]lysine monohydrochloride (Radiochemical Centre, Amersham, England) was administered. There was a little difficulty with the intravenous injection and 3 mCi were injected subcutaneously and 7 mCi via a cannula in the jugular vein. Seven days later 10 mCi of 3H-labeled mixed amino acids were administered via the jugular cannula, and milk samples only were
1529
1530
STEAD ET AL.
obtained. Thirty-six days after the second radioactive dose, 20 mCi of DL-[ 3 H] lysine HC1 were administered via the cannula (second experiment). Blood samples were obtained via a cannula in the jugular vein opposite to that in which the labeled lysine was introduced and from a cannula in the mammary vein. Dextran sulfate reagent (5) consisted of a mixture of 1 vol of 10% (wt/vol) clinical grade dextran sulfate (Mw ca. 5000) (Glaxo Laboratories Ltd., Greenford, Middx, U.K.) and 5 vol of 1M CaC12 solution. The optimum volume of reagent required to precipitate the serum lipoproteins of density less than 1.06 g/ml (.15 ml reagent/ml serum) was found by titration of small aliquots of serum with the reagent to maximum turbidity at 700 nm. The appropriate volume of reagent was added either to 10 ml serum (Experiment 1) or to 20 ml serum (Experiment 2). The precipitate was dissolved in sodium citrate solution (d = 1.019 g/ml) containing 1 mg/ml DL-lysine HC1 and reprecipitated with .1 M CaCI2 solution (5). Each sample was reprecipitated three times to reduce the free [3H]lysine content to negligible proportions. The precipitate then was dissolved in sodium citrate (6 ml, d = 1.019 g/ml), transferred to a polycarbonate centrifuge tube (17 mm x 63 mm, nominal capacity 10 ml), and the VLDL + IDL and LDL were separated by ultracentrifugation at 157,000 x g ( a v ) f o r 20 h at 12 C in the 10 × 10 ml angle rotor (tube angle 20 ° to axis) of an MSE Superspeed Ultracentrifuge (29). After removal of the precipitable lipoproreins, the HDL was obtained by ultracentrifugation of the supernatant, under the above condit!ons, after its density had been adjusted to 1.21 g/ml by the addition of solid KBr. The HDL was purified by further ultracentrifugation at the same density. To remove free [3H]lysine, the HDL samples were dialyzed overnight against running distilled water and then against three batches of distilled water on three successive nights. The purified lipoproteins were stored in sodium citrate solution (d -- 1.019 g/ml), and their purity was checked by electrophoresis (5). The protein contents were determined according to Lowry et al. (21) but with the addition of .1 ml of a 5% (vol/vol) Teepol solution to the VLDL + IDL assay mixtures to remove the turbidity which developed. J o u r n a l o f D a i r y S c i e n c e Vol. 6 1 , N o . 11, 1 9 7 8
Radioactivity was measured as in (2) in the scintillator solution containing methoxyethanol-anisole; 10 ml of scintillator solution were mixed with .6 ml (Experiment 1, 13% counting efficiency) or 1 ml (Experiment 2, 11% counting efficiency) of lipoprotein solution. The radioactivities of the lipoproteins/mg protein, i.e., the specific radioactivities of the apoproteins, were plotted semilogarithmically against time. In Experiment 2, the specific radioactivities were corrected for the low initial specific radioactivity which resulted from the earlier injections. For VLDL + IDL (Experiment 2), the points after the period of rapid decline in specific radioactivity which followed the maximum were fitted to a straight line by the method of least squares. In Vitro Experiments
Serum was prepared from jugular blood withdrawn from lactating Saanen goats which were fed on hay and concentrates (principally barley, maize, and wheat bran plus vitamin and mineral supplements) to appetite. The blood samples were allowed to clot for 2 h at room temperature and centrifuged then at 2000 rpm for 20 min. Disodium ethylenediamine tetraacetare (EDTA) was added to serum to give a final concentration o f . 1%. Chylomicrons were removed from the serum by overlayering 6 ml of serum with 2 mi of a solution of density 1.006 g/ml (sodium citrate or saline) and were centrifuged for 30 min at 60,000 × g (av). Lipoprotein fractions were separated by sequential ultracentrifugation of 8 ml of chylomicron-free serum at increasing densities in the 50 Ti rotor of a Beckman L2-50 preparative ultracentrifuge at 14 C. Solid sodium citrate was used to obtain the required densities. Four lipoprotein fractions were isolated at densities of 1.006 g/ml (VLDL), 1.019 g/ml (IDL), 1.06 g/ml (LDL), and 1.21 g/ml (HDL). The VLDL and IDL were separated after 18 h and LDL and HDL after 24 h of centrifugation at 105,000 x g (av). Each fraction was brought to 8 ml with sodium citrate solution of its respective density and recentrifuged for 24 h. The purity of each fraction was checked by electrophoresis (5). The VLDL were iodinated by a modification
INTERRELATIONSHIPS BETWEEN SERUM LIPOPROTEINS of the iodine monochloride method of McFarlane (22) as described by Reif (26). The Na 12s I was obtained from the Radiochemical Centre, Amersham, England. Unreacted iodide was removed by dialysis against 8 to 10 changes of .15 M NaC1 and .01% EDTA, for 6 to 7 h at 4 C. The efficiency of labeling of the VLDL with 12sI was 25.85 -+ 3.1%, and protein recovery after iodination was 86.3 -+ 8.7% (means of three values -+ SD). The ratio of moles of iodide to 100,000 g of apoprotein was less than 1 in all preparations as recommended by Eisenberg and Rachmilewitz (13). Radioactivity was determined in the Autogamma Scintillation Spectrometer (Packard Tricarb). The labeled VLDL was incubated with serum for 1 h at 37 C. In the control, labeled VLDL was incubated with .15 M NaC1 in .05 M phosphate buffer, pH 7.6 under the same conditions. Each determination was in duplicate. Aliquot samples then were withdrawn for 12 s I monitoring. The lipoprotein fractions subsequently were isolated from the remaining samples by centrifuging at their respective densities. The 12s I content in each fraction was measured. The fractionated sampIes were lyophilized and delipidated as described by Brown, Levy and Fredrickson (3), and the 12 s I content of the lipid fraction was determined. The apoproteins were solubilized by incubation (37 C for 16 h) in .02 M tris/HC1 (pH 8.2) containing .1 M sodium dodecylsulfate and .001 M dithiothreitol, diluted 1:1 (vol/vol) with 40% (wt/vol) sucrose before gel application, and fractionated by electrophoresis on 5 to 11% polyacrylamide gradient gels containing 1% sodium dodecylsulfate. Bromophenol blue was used as a marker (7). The gels were stained with .05% Coomassie Blue R in 12.5% (wt/vol) trichloroacetic acid (6), destained in a solution of water to ethanol to acetic acid (65:25:8 by vol), (31), and sliced into zones whose radioactivities were determined. RESULTS In Vivo Experiments
Figure 1 shows that there was no cross contamination between the precipitable and nonprecipitable lipoproteins. In addition, it has been established (Stead and Welch unpublished) that when lipoproteins of goat serum are
1531
FIG. 1. Etectrophoresis of goat serum lipoproteins (prestained with Sudan Black B) on gradient polyacrylamide gel (5). The supernatant after removal of the dextran sulfate precipitate (DSS), the d>l.O19 g/ml fraction (LDL) of the dextran sulfate precipitate, and whole goat serum.
isolated by means of dextran sulfate as described here, the LDL has the same electrophoretic mobility as, and is immunologically identical to, LDL isolated by ultracentrifugation alone. Furthermore, the nonprecipitable lipoprotein is immunologieally identical to HDL isolated by uhracentrifugation, has the same flotation rate in the analytical ultracentrifuge, and does not contain LDL. The specific radioactivities o f the apoproteins of the lipoproteins from the second experiment are plotted semilogarithmically against time in Figur e 2a; the values for jugular and mammary venous serum appeared to be the same, and they were treated as equivalent when the curves were drawn. The maximum value for the specific radioactivity of the VLDL + IDL apoprotein was 22 dpm/ug, which was reached 1.2 h after injection, whereas that o f LDL was 7.1 dpm/ug at 6.5 h, and that of HDL 2.8 dpm/ug at 8.5 h. The decline in specific radioactivity did not follow a simple exponential curve for any of the lipoproteins. In particular, there was a marked change in the slope of the VLDL curve at about 9 h, which indicated that the curve represented apoproteins with different kinetic behavior. The specific radioactivity-time curves Journal of Dairy Science Vol. 61, No. 11, 1978
15 3 2
STEAD ET AL. b
In Vitro Experiment
4¢ VLDL ÷ IOL
10
•
•
i0 .I •
I
I
I
I
! "
HDL
HDL
Time, h
FIG. 2. Specific radioactivities of the protein moieties of goat very low density lipoprotein (VLDL), intermediate density lipoprotein (IDL), low density lipoprotein (LDL), and high density lipoprotein (I-IDL) after administration of DL-13Hllysine monohydrochloride. (a) Experiment 2; (b) Experiment 1. e, jugular venous serum; A, mammary venous serum.
which were obtained in the first experiment were similar to those of the second experim e n t in shape, and in time and relative size of the maximum (Fig. 2b).
The protein concentrations of the lipoprotein fractions of goat serum [expressed as mg! 100 ml + SD (means of six determinations)] were: VLDL, 1.28 + .12; IDL, 2.1 + .3; LDL, 8.1 + 1.8; HDL, 135.8 + 7.6. The distribution of radioactivity among the four lipoprotein classes after incubation of the 12s l-labeled VLDL with serum and phosphate/sodium chloride buffer is in Table 1. Approximately 80% of the total radioactivity in the lipoproteins recovered after incubation with either serum or buffer was bound to proteins. On incubation of the labeled VLDL with serum, 29% of the radioactivity originally in this fraction was transferred to other lipoproreins and was distributed approximately equally among the other three lipoprotein fractions. On incubation with buffer, however, only 11% of the radioactivity originally in the VLDL fraction was recovered in other lipoproteins, and there was approximately twice as much radioactivity in the IDL and LDL as in the HDL. After delipidation of the lipoproteins, the following proportions of radioactive protein were resolubilized: VLDL, 70%; IDL, 90%; LDL, 75%; HDL, 98%. The electrophoretic patterns of the apoproteins are shown diagrammatically in Figure 3. The distribution of radioactivity among the apoproteins after incubation of 12 s I-labeled VLDL in serum is in Table 2. The distribution of radioactivity was similar in the VLDL, IDL, LDL; the most radioactive bands were in zones 1 and 2. In the VLDL, zone 3 was also radioactive,
TABLE 1. Redistribution of 1251 from very low density lipoprotein to other lipoprotein fractions, a Incubation medium buffer + .15 M NaCI (control) (% recovered cpm)
PO 4
Lipoprotein fraction Very low density Intermediate density Low density High density
~ 71.0 10.5 10.1 8.6
Serum (% recovered cpm) Lipoprotein Protein SD ~ SD 3.6 2.5 3.4 1.5
aEach value is the mean of four determinations. Journal of Dairy Science Vol. 61, No. 11, 1978
56.4 7.5 8.7 7.2
4.6 1.4 3.7 1.7
Lipoprotein SD 88.7 4.6 3.4 1.9
3.6 2.0 .25 .19
~ 73.1 3.8 3.1 1.6
Protein SD 5.7 1.9 .44 0
INTERRELATIONSHIPS BETWEEN SERUM LIPOPROTEINS Zone
--~--
3
4
Bm 6 I B m
u
.
.
.
.
.
.
.
i
.
.
.
.
.
. 7
.
.
.
.
i.
.
.
.
.
.
i.
.
.
.
.
.
I .
.
.
.
i
9 .
.
.
.
i
.
.
VLDL
IOk
.
.
.
Marker
i LDL
HDL
FIG. 3. Electrophoresis of the apolipoproteins of goat very low density lipoprotein (VLDL), intermediate density lipoprotein (IDL), low density lipoprotein (LDL), and high density lipoprotein (HDL), on gradient polyacrylamide gels containing sodium dodecyl sulfate. The gels were sliced into the zones indicated for counting of radioactivity.
but although the most intense band in the LDL was in this zone, it was not radioactive. In the HDL over 90% of the radioactivity was concentrated in two bands in zones 1 and 6. Zone 6 of the VLDL was the zone from which the greatest proportion of radioactivity was lost on incubation in serum as compared to incubation in buffer.
DISCUSSION
Human and rat VLDL contain apoproteins which are common to LDL (apoprotein B) and to HDL (apoprotein C). The apoprotein B of VLDL is converted rapidly in vivo to apoprotein B of the LDL via the formation of a lipoprotein of intermediate density (12, 17). This transfer does not take place to any great extent when labeled VLDL is incubated in serum for 20 to 30 min (1, 27), but when the incubation time is increased, and particularly when post-heparin plasma is used, there is an appreciable transfer (8). The apoprotein C of VLDL exchanges with the apoprotein C of the HDL in vitro and in vivo (11, 12), and there is also a net transfer of apoprotein C to HDL in rats in vivo and when VLDL is incubated in post-heparin plasma (15). The different phases of the complex disappearance curve of VLDL radioactivity after injection of 12 s I-labeled VLDL (13) can be attributed to differences in
1533
the rates of turnover of the VLDL apoproteins (11, 14). The specific radioactivity-time curves for the apoproteins of goat serum after an injection of DL-[3H] lysine were similar to those for human apoproteins after injection of [TSSe]seleno_ methionine (9, 28) and for five species of animals after injection of DL- or L-[3H] lysine (18). The early maximum and sharp fall in specific radioactivity of VLDL + IDL apoproteins of goat were consistent with the precursor-product relationship which has been suggested in humans between the apoprotein B of VLDL and that o f LDL (8, 9, 28). In support of this, most of the radioactivity in the IDL and LDL of goat serum, after incubation with 12s l-labeled VLDL, was in electrophoretic zones 1 and 2. In 10% polyacrylamide gels, apoprotein B remained at the origin (12), and although direct comparison is not possible because the electrophoretic system in our work was different, it seems likely that zones 1 and 2 could correspond to apoprotein B. Similarities between the apoproteins of VLDL and LDL2 (1.039< d< 1.060 g/ml) have been demonstrated also in cows (30). The complex decay curve o f the specific radioactivity of VLDL + IDL apoprotein of goats was also similar to that of VLDL radioactivity in rats after injection of x 2 s I-labeled VLDL (13). The slower turning over component of the decay curve of rat 12s I-labeled VLDL (13) was attributed to the turnover of apoprotein C which equilibrates between VLDL and HDL (12, 14). In our experiments more radioactivity transferred from 1 2 s I-labeled VLDL of goat to other lipoproteins when it was incubated w i t h serum than could be accounted for by control incubation in buffer (Table 1), and the relative increase in radioactivity was greatest in HDL (Table 1). On eleetrophoresis of apoproteins 42% of HDL radioactivity was recovered in zone 6 (Table 2). After incubation, zone 6 of the HDL contained more radioactivity than was recovered altogether from the corresponding zones of other lipoproreins, and this zone of the VLDL was the one which lost the greatest proportion of radioactivity after incubation in serum. It seems probable, therefore, that zone 6 corresponds to apoprotein C which exchanges between VLDL and HDL in humans (1, 10, 16) and in rats (14, 15, 27). Apart from transfer of apoprotein Journal o f Dairy Science Vol. 61, No. 11, 1 9 7 8
g~
t7
< o
o0
TABLE 2. Distribution of ~2 s 1 in apoproteins of lipoprotein fractions after in vitro incubation of labeled very low density lipoprotein with serum and buffer, a Lipoprotein fraction
1
2
3
Incubation with serum Very low density
15.5
37.3
18.9
Intermediate density
18.9
2.9
Low density High density
19.6 52.2
29.9 4.4 38.6 .4 .2 .3
32.9
10.9
Incubation with buffer Very low density
12.5
a) b) a) b) c)
avalues are percent of total radioactivity recovered/gel. bzones are as in Figure 3.
4
a) b) a) b)
.3 .3
4.0 1.1 3.4 1.7 2.5 .4
Z°neb 5
6
7
1.7
2.0
a) b)
3.1
2.0
3.3 3.6 2.6
3.2 .5
2.3 42.0
6.0 .3
a) b) a) b)
8
9
3.1 2.6 7.1 4.1 6.0 .6
1.9 4.2 2.1 1.4 .r"
a) b)
9.0 5.5
6.0
7.5
a) b)
3.5 2.4
a) b)
1.0 6.5
INTERRELATIONSHIPS BETWEEN SERUM LIPOPROTEINS f r o m V L D L to L D L a n d H D L in vivo, d i r e c t s y n t h e s i s o f L D L a n d H D L (18, 20), a n d recycling (9, 23), possibly m a y occur. In cows, w h o s e l i p o p r o t e i n s have b e e n studied more extensively than those of the goat, a p o p r o t e i n s o f V L D L ( d < 1.019 g / m l ) a n d H D L have similarities (30). A n a d d i t i o n a l l i p o p r o t e i n , LDL1 ( 1 . 0 1 9 < d < 1.03 9 g / m l ) w i t h a n e l e c t r o p h o r e t i c m o b i l i t y similar to t h a t o f HDL, is s o m e t i m e s p r e s e n t b u t has n o t b e e n o b s e r v e d in s h e e p a n d g o a t s (5). T h e LDL1 is i m m u n o l o g i c a l l y related to H D L (30) a n d like H D L can activate milk l i p o p r o t e i n lipase (4). Its c o n c e n t r a t i o n is variable, greater in l a c t a t i n g cows t h a n in dry cows (24, 25), a n d is increased in t h e b l o o d of cows fed p r o t e c t e d fat ( S t e a d a n d Welch u n p u b l i s h e d ) . A p o p r o t e i n C regulates t h e activity o f l i p o p r o t e i n lipase (12), a n d if it is in b o v i n e V L D L a n d H D L (as appears t o be t h e case in t h e g o a t ) and, t h e r e f o r e , possibly in L D L I , b u t n o t in LDL2 [ h u m a n L D L n o r m a l l y c o n t a i n s o n l y traces o f a p o p r o t e i n C ( 1 2 ) ] , this c o u l d explain w h y b o v i n e V L D L a n d LDL1 s u p p l y triglycerides for milk fat s y n t h e s i s w h e r e a s LDL2 m a y n o t (19). It does n o t e x p l a i n w h y b o v i n e H D L triglyceride is n o t a p r e c u r s o r o f m i l k fat a n d this t o g e t h e r w i t h a s t u d y o f t h e factors which affect the concentration of the b o v i n e l i p o p r o t e i n s a n d t h e i r i n t e r c o n v e r s i o n is under investigation. ACKNOWLEDGMENTS
T h e a u t h o r s wish to t h a n k H. L. B u t t l e f o r i n s e r t i o n o f cannulas, J. P o r t e r for t e c h n i c a l assistance, a n d R. F. G t a s c o c k f o r advice a n d encouragement.
REFERENCES
1 Bilheimer, D. W., S. Eisenberg, and R. I. Levy. 1972. The metabolism of very low density lipoprotein proteins. I. Preliminary in vitro and in vivo observations. Biochim. Biophys. Acta 260:212. 2 Bishop, C., T. Davies, R. F. Glascock, and V. A. Welch. 1969. Studies on the origin of milk fat. A further study of bovine serum lipoproteins and an estimation of their contribution to milk fat. Biochem. J. 113:629. 3 Brown, W. V., R. I. Levy, and D. S. Fredrickson. 1969. Studies of the proteins in human plasma very low density lipoproteins. J. Biol. Chem. 244: 5687. 4 Brumby, P. E., J. E. Storry, and J. D. Sutton. 1972. Metabolism of cod-liver oil in relation to
153 5
milk fat secretion. J. Dairy Res. 39:167. 5 Brumby, P. E., and V. A. Welch. 1970. Fractionation of bovine serum lipoproteins and their characterization by gradient gel electrophoresis. J. Dairy Res. 37:121. 6 Chrambach, A., R. A. Reisfeld, M. Wyckoff, and J. Zaccari. 1967. A procedure for rapid and sensitive staining of protein fractionated by polyacrylamide gel electrophoresis. Anal. Biochem. 20:150. 7 Davis, B. J. 1964. Disc electrophoresis. II. Method and application to human serum proteins. Ann. N. ¥. Acad. Sci. 121:404. 8 Eaton, R. P., S. Crespin, and D. M. Kipnis. 1976. Incorporation of 7 s Se-selenomethionine into human apoproteins. II. Characterization of metabolism of very low-density and low-density lipoproteins in vivo and in vitro. Diabetes 25:44. 9 Eaton, R. P., and D. M. Kipnis. 1972. Incorporation of S e 7 s selenomethionine into a protein component of plasma very-low-density lipoprotein in man. Diabetes 21:744. 10 Eisenberg, S., D. W. Bilheimer, and R. I. Levy. 1972. The metabolism of very low density lipoprorein proteins. II. Studies on the transfer of apoproteins between plasma lipoproteins. Biochim. Biophys. Acta 280:94. 11 Eisenberg, S., D. W. Bilheimer, R. I. Levy, and F. T. Lindgren. 1973. On the metabolic conversion of human plasma very low density lipoprotein to low density lipoprotein. Biochim. Biophys. Acta 326:361. 12 Eisenberg, S., and R. I. Levy. 1975. Lipoprotein metabolism. Adv. Lipid Res. 13:1. 13 Eisenberg, S., and D. Rachmilewitz. 1973. Metabolism of rat plasma very low density lipoprotein. I. Fate in circulation of the whole lipoprotein. Biochim. Biophys. Acta 326:378. 14 Eisenberg, S., and D. Rachmilewitz. 1973. Metabolism of rat plasma very low density lipoprotein. II. Fate in circulation of apoprotein subunits. Biochim. Biophys. Acta 326:391. 15 Eisenberg, S., and D. Rachmilewitz. 1975. Interaction of rat plasma very low density lipoprotein with lipoprotein lipase-rich (post heparin) plasma. J. Lipid Res. 16:341. 16 Eisenberg, S., H. G. Windmueller, and R. I. Levy. 1973. Metabolic fate of rat and human lipoprotein apoproteins in the rat. J. Lipid Res. 14:446. 17 Faergeman, O., S. Tsunako, J. P. Kane, and R. J. Havel. 1975. Metabolism of apoprotein B of plasma very low density lipoproteins in the rat. J. Clin. Invest. 56:1396. 18 Fried, M., H. G. Wilcox, G. R. Faloona, S. P. Eoff, M. S. Hoffman, and D. Zimmerman. 1968. The biosynthesis of plasma lipoproteins in higher animals. Comp. Biochem. Physiol. 25:651. 19 Glascock, R. F., and V. A. Welch. 1974. Contribution of the fatty acids of three low density serum lipoproteins to bovine milk fat. J. Dairy Sci. 57:1364. 20 lllingworth, D. R. 1975. Metabolism of lipoproreins in nonhuman primates. Studies on the origin of low density lipoprotein apoprotein in the plasma of the squirrel monkey. Biochim. Biophys. Acta 388:38. Journal o f Dairy Science Vol. 61, No. l 1, 1978
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21 Lowry, O. H., N. J. Rosebrough, A. L. Farr, and R. J. Randall. 1951. Protein measurement with the Folin phenol reagent. J. Biol. Chem. 193:265. 22 McFarlane, A. S. 1958. Efficient trace-labelling of proteins with iodine. Nature 182:53. 23 Phair, R. D., M. G. Hammond, J. A. Bowden, M. Fried, W. R. Fisher, and M. Berman. 1975. A preliminary model for human lipoprotein metabolism in hyperlipoproteinemia. Fed. Proc. 34:2263. 24 Puppione, D. L., B. Raphael, R. D. McCarthy, and P. S. Dimick. 1972. Variations in the electrophoretic distribution of low density lipoproteins of Holstein cows. J. Dairy Sci. 55:265. 25 Raphael, B. C., P. S. Dimick, and D. L. Puppione. 1973. Electrophoretic characterization of bovine serum lipoproteins throughout gestation and lactation. J. Dairy Sci. 56:1411. 26 Reif, A. E. 1968. A simple procedure for high-
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28 29 30
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efficiency radioiodination of proteins. J. Nuc. Med. 9:148. Rubenstein, B., and D. Rubinstein. 1972. Interrelationship between rat serum very low density and high density lipoproteins. J. Lipid Res. 13:317. St~ihelin, H. B. 1975. 7 SSe_selenomethionine_ labeled lipoproteins in hypedipidemic and normolipidemic humans. Metabolism 24:505. Stead, D., and V. A. Welch. 1975. Lipid composition of bovine serum lipoproteins. J. Dairy Sci. 58:122. Stead, D., and V. A. Welch. 1976. Immunological properties of bovine serum lipoproteins and chemical analysis of their protein moieties. J. Dairy Sci. 59:1. Vesterberg, O. 1971. Staining of protein zones after isoelectric focusing in polyacrylamide gets. Biochim. Biophys. Acta 243: 345.