1
Atherosclerosis, 28 (1977) l-14 0 Elsevier/North-Holland Scientific Publishers, Ltd.
CHANGES IN METABOLIC PROPERTIES OF RABBIT VERY LOW DENSITY LIPOPROTEINS BY DIETARY CHOLESTEROL, AND SATURATEDANDPOLYUNSATURATEDFAT
E. STANGE, M. ALAVI and J. PAPENBERG Medizinische
Universitiitsklinik,
Heidelberg
(W. Germany)
(Received 22 October, 1976) (Revised, received 11 May, 1977) (Accepted 25 May, 1977)
Summary
As was shown in a recent investigation, there is a marked change in the structural properties of rabbit lipoproteins by dietary cholesterol, saturated and polyunsaturated fats. Based on these electrophoretic, chemical and electron microscopic findings we examined the metabolic behaviour of ‘2SI-labeled VLDL (d < 1.006 g/ml) from normal rabbits (group I) as well as animals fed 1% cholesterol (group II), 1% cholesterol + 5% coconut oil (group III) or 1% cholesterol + 5% corn oil (group IV). The curve peeling technique resulted in a fractional catabolic rate per hour (FCR/h) of 0.082 in the normal VLDL. All hypercholesterolemic fractions II, III and IV were metabolised significantly faster (P< 0.05) with FCR/h values of 0.119,0.157 and 0.173 respectively. The difference in decay between VLDL II and both VLDL III or IV was statistically significant only during the late phase of metabolism, 2 h after injection. The apoprotein decay curves gave similar results. Ten min after the injection, most of the activity was recovered in the IDL class (d: 1.006-1.019 g/ml). The LDL fraction of the normal group had the lowest decay rate, whereas in all other groups HDL metabolism was delayed. It therefore may be concluded, that the metabolic behaviour of hypercholesterolemit VLDL differs from normal VLDL both quantitatively and qualitatively. Key words:
Atherosclerosis - Dietary cholesterol -Polyunsaturated rated fat - Very low density lipoprotein metabolism
This investigation was supported the 4th International Symposium
fat-Rabbits
-Satu-
in part by the Deutsche Forschungsgemeinschaft and was presented on Atherosclerosis in Tokyo, Japan. August, 1976.
at
2
Introduction In recent years knowledge of lipoprotein structure has expanded greatly [ 11. In different species it was possible to induce specific changes in the chemical, electrophoretic and electron microscopic properties of various lipoprotein density classes by feeding cholesterol. These alterations in guinea pigs [2], rabbits [3--81, swine [9] and dogs [lo] were suggested to play an important role in experimental atherosclerosis by their “inherent atherogenicity”. In a previous report we demonstrated the effect of saturated and polyunsaturated fat supplements to a cholesterol enriched chow on rabbit lipoprotein structure [ 111. Although we achieved a relative “normalization” of the agarose gel electrophoretic and electron microscopic properties by the unsaturated corn oil diet, accompanied ‘by a striking reduction in the extent of atherosclerosis in this group [12], the chemical and apoprotein composition as well as the behaviour in polyanionic precipitation was similar to the saturated coconut oil group. In the present study we examine the metabolism of ‘251-labeled VLDL from normal and dietary animals after injection into normal rabbits, Until now most of the effort focused on rat [13-161, monkey [ 17-191 as well as human [20-271 lipoprotein metabolism, whereas little information is available on rabbit [6,18,28]. However these investigations dealt with a heterogeneous lipoprotein fraction of d < 1.019 g/ml comprising VLDL and IDL [28] or used a biosynthetically labeled LDL of d: 1.006-1.063 g/ml and gave conflicting results [ 181. The findings reported in the present paper indicate that the diet induced structural alterations described previously in rabbit lipoproteins which significantly affect their metabolic behavior. Methods Animals and feeding
schedule
for experimental
diets
Male New Zealand White Rabbits weighing 2.5-3 kg were kept for the experimental period of 18 weeks as described previously [ 111. The animals were divided into 4 dietary groups: Group I received a control diet of normal rabbit chow (Georg Plange Co., Soest, W. Germany), group II was fed the control diet containing 1% cholesterol, groups III and IV had the control diet with 1% cholesterol and 5% coconut oil as a saturated fat or 5% corn oil as a polyunsaturated fat respectively. At the end of the dietary period the animals were bled from the carotid artery after an 18 h fast and the very low density lipoproteins were isolated as detailed below. The animals used in the metabolic experiments were normolipemics receiving their control diets ad libtum and were given additional 0.01% NaI in the drinking water prior to and after the injection of the 1251-labeled lipoproteins to block the thyroid gland. The serum lipid levels of both the recipient and the donor animals were in the same range as published previously [ll] for the different dietary groups.
3
Preparation and iodination of lipoproteins Lipoproteins were isolated by standard flotation technique and checked for purity and their typical mobility by immune electrophoresis in agarose gel as described elsewhere [ 111. Plasma was fractionated into 4 density classes: very low density lipoproteins (VLDL) of d < 1.006 g/ml, intermediate density lipog/ml, low density lipoproteins (LDL) of proteins (IDL) of d: 1.006-1.019 d: 1.019-1.063 g/ml, and high density lipoproteins (HDL) of d: 1.0631.210 g/ml. The VLDL samples were diluted to a final protein concentration of 10 mg/ 100 ml after dialysis in a 0.4 M glycine buffer at pH 10. For radioiodination 2 ml of the lipoprotein solution were mixed with 1 mCi of ‘*‘I carrier free (NEN Chemicals, Boston, U.S.A.) in 50 ~1 and various amounts of 0.0033 M ICl according to Fidge’s modification [ 291 of McFarlane’s iodine monochloride method [ 301. The concentration of ICl was adjusted to achieve a molar iodine/ protein ratio of approximately 1. Although this ratio is considered safe, the integrity of the radioiodinated particles was ascertained by immune electrophoretic and electron microscopic techniques. Determination of intramolecular activity distribution After exhaustive dialysis with at least 15 changes of 0.15 M NaCl the lipoprotein bound activity exceeded 95% in all preparations as determined by the extraction procedure according to Folch [ 311. The methanol/water phase was evaporated under nitrogen, the proteins were precipitated by 7% trichloroacetic acid and redissolved in 1 M NaOH to estimate the apoprotein bound activity. The chloroform phase containing the lipid moiety was counted in the dry state to avoid solvent quench, and the free iodide activity was assayed in the supernatant after TCA precipitation. The apoprotein bound activity plus the lipid bound activity was considered as lipoprotein bound activity. As a control an aliquot of labeled VLDL was lyophilised and delipidated with ethanol/ ether in 3 washings and 2 final ether extractions. This procedure gave similar results to the Folch technique, but of course did not yield the free iodide content of the preparation. The molar iodine/protein ratio was calculated according to the formula presented by Fidge [38], supposing a molecular weight of rabbit VLDL apoprotein of 250,000. The labeled lipid portion of [ 12’I]VLDL was separated by thin layer chromatography into mono-, di- and triglycerides, free fatty acids, phospholipids as well as free and esterified cholesterol. The silica gel layered plates were run with hexane/chloroform 1 : 3 (vol/vol) and methanol/chloroform 1 : 2 (vol/ vol) as the second solvent. Complete separation and recovery were checked by analysis of standard lipid mixture. All ‘*‘I activities were determined in a Searle Gamma Counter. Experimental design [‘*‘I]VLDL fractions of the 4 dietary groups were injected in a dose of lo7 cpm into the ear veins of normal animals on a control diet. The time lag between blood sampling and injection of the iodinated lipoproteins was always under 96 h. Blood samples were taken 10 min, 30 min, 1, 2, 4, 6, 12,24, 30,
4
36 and 48 h after injection. The 10 min value was taken as 100% of the injected dose. After slow centrifugation at 2500 rpm an aliquot of plasma was counted, lyophilised and delipidated as described above. Radioactivity was then determined both in the protein and lipid moieties. From another aliquot of plasma we isolated the VLDL, IDL, LDL and HDL fractions by ultracentrifugation as detailed before. These lipoproteins were counted and their activities corrected for the KBr quench of the high salt solutions. The correction factors were 1.275 3f IDL, 1.544 for LDL and 3.199 for HDL. Comparable values were published by Eisenberg [33] at the respective density cuts. The intravascular activity as a fraction of the injected dose was plotted on a logarithmic scale (ordinate) against time on linear scale (abscissa). These decay curves were analysed for the half life times and the fractional catabolic rates per hour according to the method of Matthews [34]. For statistical analysis we employed the chi square sum test, using the absolute values (cpm/ml) for all calculations. To check the metabolic steady state condition of the experimental animals we calculated the U/P ratio from the excreted urinary activity and the mean total plasma activity between 2 blood samples. Plasma volume was estimated from the Evans Blue method. Results Intramolecular
distribution
of activity
In the lipid moiety of the labeled VLDL fractions activity was bound predominantly to the phospholipids with 53% (mean of 3 determinations, range 49.5-58%). The rest of the lipid activity was rather evenly distributed between monoglycerides (8.3%), diglycerides (7.8%), triglycerides (12.1%), free fatty acids (3.0%), free (5.3%) and esterified cholesterol (6.4%) and a small unidentified fraction (4.1%). In the hypercholesterolemic VLDL the amount of lipid bound activity decreased from a mean of 50.7% in VLDL I to 18.8% in VLDL II, 15.3 and 22.7% in VLDL III and IV, respectively. To exclude interference of these varying proportions of lipid bound activity with the VLDL plasma decay of the 4 groups, total plasma samples were delipidated to obtain the apoprotein decay as such. The mean I/P (iodine/protein) ratio of all injected fractions was 1.06 (range 0.83-1.37). Immune electrophoresis of native and iodinated VLDL resulted in identical precipitin bands. In electron micrographs the labeled lipoprotein particles could not be distinguished from the native preparation [ 341. VLDL plasma activity decay
A mean of 45.9% (range: 31-75%) of the injected dose remained in the circulation 10 min after the injection of VLDL I. In groups II and IV a mean of 50.8% (17-63%) and 49.1% (25-77%) were found in total plasma after the same interval, whereas in group III only 36.8% (14-62%) were recovered. As can be seen from the plasma decay curves of the 4 groups of normal and dietary altered rabbit VLDL in normal animals, the catabolism of control
5
VLDL I was the slowest of all fractions (Fig. 1). This difference was also reflected in the longer half life times during both the initial rapid phase (t,,*,) and the late slow phase (tllzz) o f metabolism and the smaller fractional catabolic rate per hour (Table I). The statistical evaluation is presented in Table 2, demonstrating that all hypercholesterolemic fractions were metabolised significantly faster than normal VLDL I. Accordingly the FCR value for cholesterol VLDL was 32% higher than the control with 0.082, whereas it was nearly doubled in coconut and corn oil VLDL (Table 1). Concomitantly the half life times are reduced approximately by half. However, ‘he coconut and the corn oil VLDL plasma decay was significantly different from the cholesterol VLDL only during the later phase of decay 1 and 2 h after the injection. The decay rate of coconut and corn oil VLDL was similar. VLfiL
apoprotein
activity decay
The apoprotein activity curves are depicted in Fig. 2 and show the same distribution as described above for plasma decay. The catabolism of VLDL I was slowest, with a more rapid decay of apo VLDL II and the fastest apoprotein metabolism in VLDL III and IV (Table 1). The apo-VLDL differences also are statistically significant when control apo-VLDL is compared with the hypercholesterolemic groups, as well as apo VLDL II vs IV (Table 2). Coconut and
.
NORMAL
n:
.
CHOLESTEROL
nz 16
o
COCONUT CORN
26
2
6
OIL
n:
11
n= 12
0
12
4
OIL
14
12
24
30
36
48
TIME
Fig. 1. Plasma activity decay after injection of normal and dietary altered VLDL. Values represent median values of n experiments. The inserted plot depicts the urine/plasma activity ratio of a representative experiment.
6
TABLE
1
DECAY tl,z,
PARAMETERS is the half
life
OF time
NORMAL
during
AND
the early
DIETARY
phase.
ALTERED
tl ,z2
is the half
VLDL life
time
in days
during
the late phase
of
metabolism. _---
..-
_~
.-.-__
Normal
Cholesterol
Coconut
oil
-__
Corn .__~_~___
-___
oil
Plasma
*
1.111
0.590
0.479
t1/z2
0.118
0.056
0.042
0.069
FCR
0.082
0.119
0.157
0.173
tu2 *
1.146
0.951
0.674
0.604
tu2*
0.097
0.056
0.049
0.035
FCR
0.083
0.145
0.224
0.240
t1/2
0.660
Apoprotein
corn oil apo-VLDL decay was nearly identical. The fractional catabolic rate per hour of normal apo-VLDL of 0.083 was nearly the same as that found in total plasma decay, but in groups II, III and IV the apoprotein decay seems to be accelerated in comparison to plasma, thereby reflecting the slower metabolism of their cholesterol-rich lipid moiety (Table 1). The lipid bound activity in plasma was too low to give consistent results, due to the small amount of iodine bound to the lipid moiety of VLDL II, III and IV. Distribution of activity in lipoprotein classes The activity distribution in the different lipoprotein density classes VLDL, IDL, LDL and HDL indicates the rapidity of lipoprotein dynamics in the rabbit (Tables 3, 4, 5 and 6). After 10 min, most of the original VLDL bound activity was recovered in the intermediate density class irrespective of the lipoprotein group injected (Fig. 3 and 4). Only 27.5% of the injected activity was still
TABLE
2
STATISTICAL ns, Not horizontal protein
EVALUATION
significant. and decay
For
vertical in the
OF
comparison columns.
lower
left
VLDL
PLASMA
of two
groups
The
values
AND the
for plasma
PROTEIN
decay
Normal1
Cholesterol
P < 0.01
P < 0.05
-~____--~~-a Significant
b Significant
(P < 0.05) (P < 0.05)
are presented
in the upper
of the respective right
triangle. II
Coconut
I
IV
DECAY
P value is given at the cross-section
-~- .-.-
after
1 h.
after
2 h.
IIS ~_..__..
oil III
Corn
oil IV
triangle.
for
3
4
f
13.9
oil
9
Corn
k
18.5
6
coconut
oil
7
21.7
5.1
IN
PERCENT
10.8
9.9
’7.2
13.9
22.7
7.3
f
+ 4.8
6.4
? 10.0
f
OF
* 4.4
f 2.3
f 4.3
f: 1.6
30 min
PERCENT
9.1
11.0
9.7
20.0
30 min
? 2.4
+ 1.9
+ 4.0
4.2
8.3
11.2
15.7
lh
i 3.4
f 7.4
? 5.2
f 7.3
INJECTED
6.9
8.0
8.5
f 4.3
INJECTED
13.9
lh
THE
THE
OF
of n experiments.
IN
9.4
t 18.5
f
Cholesterol
33.4
5
IDL
10 min
Time
OF
k 5.4
* 4.4
+ 7.1
k 5.3
NOllnal
KINETICS
13.2
13.6
12.4
27.5
10 min
n
RABBITS
9
VLDL
deviation
Time
+ standard
OF
VLDL
NORMAL
RADIOACTIVITY
TABLE
oil
6
coconut
Corn
7
Cholesterol
oil
5
Normal
mean
KINETICS
n
represent
RABBITS
VLDL
Values
NORMAL
RADIOACTlVITY
TABLE
DOSE
+ 1.2
+ 1.0
? 1.3
f 1.8
AFTER
2.1
2.6
4.6
f 0.4
C 0.5
* 2.7
+ 4.3
AFTER
11.2
6h
3.3
2.5
4.0
8.2
6h
DOSE
1.8
-___
1.4
1.4
1.8
3.6
OF
f 0.6
+ 0.8
k 0.76
? 2.2
.___
OF
* 1.0
r 1.0
? 0.7
+ 0.9
12 h
INJECTION
_
1.7
2.2
5.0
12 h
INJECTION
0.7
0.6
0.8
1.5
AND
k 0.6
f 0.4
* 0.4
f. 0.5
AND
* 0.7
+ 0.4
f 0.5
k 0.28
24 h
.~_
NORMAL
0.9
0.6
1.1
2.3
24 h
NORMAL
___-
0.3
0.3
0.39
0.9
36
DIETARY
0.4
0.3
0.57
1.3
h
36 h
DIETARY
* 0.2
+ 0.2
f 0.3
+ 0.4
ALTERED
* 0.3
* 0.3
f 0.4
‘_ 0.24
-_
ALTERED
h
0.11
0.2
0.25
0.5
48
h
VLDL
0.2
0.16
0.4
0.7
48
VLDL
* 0.07
+_ 0.1
+ 0.2
+ 0.1
INTO
+ 0.1
+ 0.1
k 0.26
+ 0.24
-
INTO
-1
5
6
oil
9
6
coconut
corn
7
Cholesterol
oil
5
Normal
KINETICS
n
RABBITS
9
VLDL
NORMAL
RADIOACTIVITY
TABLE
oil
6
coconut
Corn
7
Cholesterol
oil
5
Normal
KINETICS
n
RABBITS
VLDL
NORMAL
RADIOACTIVITY
TABLE
LDL
HDL
f 1.9
* 9.0
f 4.1
k 2.0
9.0
8.0
17.1
10.9
? 2.6
+ 2.6
+ 5.9
k 5.9
10 min
Time
OF
7.1
15.4
9.8
14.8
10 min
Time
OF
IN
IN
f 2.6 f 0.7
8.8
4.4
7.6
6.0
15.4
f 4.0
k 2.1
? 7.2
? 2.8
min
8.8
30
PERCENT
* 1.4
5.5
t 1.0
* 1.2
f 3.1
f 1.1
+ 1.2
6.0
6.0
15.0
6.4
lh
r 2.8
f 2.5
* 7.8
* 3.1
INJECTED
3.3
6.7
4.8
7.7
INJECTED
9.0
THE
THE
lh
OF
OF
30 min
PERCENT
3.5
3.3
7.8
4.1
6h
DOSE
1.9
2.4
2.8
6.3
6h
DOSE
t 1.8
+ 0.8
r 5.6
r 1.0
AFTER
t 0.3
f; 1.0
+ 1.2
* 1.9
~____
AFTER
+ 0.7
+ 0.8
f 0.5
+ 1.7
~_
2.3
2.5
3.8
3.5
+ 1.6
? 0.8
+ 0.9
? 1.0
12 h
INJECTION
1.4
1.4
1.6
5.7
12h
~___
INJECTION
OF
1.2
1.2
1.8
1.9
24
f 0.6
+ 0.4
* 0.9
+ 0.8
AND
+ 0.4
0.6
NORMAL
? 0.4
0.73
f 0.7 f 0.5
h
h
AND
0.7
2.6
24
NORMAL
~. ___~_.
OF
0.8
1.0
1.5
1.3
ALTERED
+ 0.5
+ 0.3
* 0.7
t 0.5
ALTERED
f 0.4
k 0.2
* 0.3
* 1.0
36 h
DIETARY
0.4
0.3
0.5
2.1
36 h
DIETARY
h
0.6
0.7
0.9
0.9
INTO
i 0.3
+ 0.3
+ 0.4
INTO
f 0.1
+ 0.1
+ 0.26
i 0.3
f 0.5
48 h
VLDL
0.14
0.2
0.3
0.6
48
VLDL
co
9
.
NORMAL
n=
6
I
CHOLESTEROL
n=
15
0
COCONUT
n=
9
n=
7
OIL
0CORNOIL
0.0 1
,h 2
4
6
12
24
30
36
s
48
TIME
Fig. 2. Apoprotein activity decay median values of n experiments.
after injection
of normal
and dietary
altered
VLDL.
Values
represent
1.0
0.3
9 1
. VLDL
(d:Wo6)
x
(d 1.006
IDL
A LDL
0.1
S
i o HDL
(d
-1.019)
1.019-1.063)
(d.1.063-1.210)
s i
:
2
z FoxI1 'e' 5 f ._ t e v
O.COl
. I. 246
I
.h 12
Fig. 3. Distribution of activity in lipoprotein represent mean values of 5 experiments.
24
lime
density
30 classes
36 after
injection
48 of normal
VLDL.
Values
10
1.a
7 1
0.3
9 B Y 5
??
0.1
: p” z ‘0 .% C
VLDL
x
ID1
A
LDL
0
HDL
T .E t z Y
0.01
0.001
h
246
.
-
12
Fig. 4. Distribution of activity in lipoprotein represent mean values of 7 experiments.
24 Time density
30
36
classes after injection
c
46 of cholesterol
h VLDL.
Values
bound to the control VLDL, compared with 12.4, 13.6 and 13.2% in the hypercholesterolemic VLDL fractions (Table 3). In the control group the LDL fraction had the lowest decay rate, whereas in all other groups HDL decay was delayed relative to the other density classes (Figs. 3 and 4). It is obvious from Fig. 4 that the more rapid plasma decay after injection of cholesterol VLDL is due to an accelerated metabolism of all lipoprotein classes of d < 1.063 g/ml. There was no significant difference between the lipoprotein decay curves of the 4 density classes after injection of cholesterol VLDL when compared with coconut or corn oil VLDL, so these two groups are not depicted separately. Urinary iodine excretion and metabolic steady state The ratio of excreted urinary activity to total plasma. activity (U/P ratio) was fairly constant from 9-48 h after the injection of labeled VLDL. A representative experiment is inserted in Fig. 1. A total of 6 such studies were performed, allowing the estimation of the fractional catabolic rates from the urine activity. This method gave comparable results under steady state conditions to the curve peeling technique. However, it is evident from Fig. 1 that there was a time lag in urinary iodine excretion at the beginning of the experiment which was compensated for rather rapidly.
11
Discussion The data presented in this study provide evidence for a more rapid metabolism of the abnormal cholesterol rich VLDL as compared to the control VLDL. This effect was even more pronounced in the lipoprotein fractions isolated from rabbits fed additional coconut or corn oil to their cholesterol enriched diets. Since the radio-labeled VLDL fractions were injected into normolipemic animals, differences in the catabolic rate have to be due to structural alterations in the particle itself as described previously [ 111. In contrast to the striking dissimilarity in agarose gel electrophoresis and electron micrographs between both the cholesterol and the coconut oil group as compared to the corn oil VLDL, all hypercholesterolemic fractions resembled each other in chemical and apoprotein composition as well as in their high heparin affinity. It therefore seems possible that the differing apoprotein patterns are responsible for an activation or an inhibition of lipoprotein lipase in the respective group [ 361. Our findings exclude the possibility that the increase in catabolic rate is only due to the lipid portion of the lipoproteins, because the activity decay in delipidated plasma, reflecting the apoprotein catabolism, was even more enhanced than total plasma decay. This difference on the contrary indicates a slightly delayed lipid metabolism in the cholesterol rich fractions. Recent investigations by Rodriguez et al., suggested a slower rate of catabolism in hypercholesterolemic rabbit “VLDL” of the density d < 1.019 g/ml. However this density range comprises two structurally and metabolically heterogeneous lipoprotein classes of VLDL and IDL. As was shown conclusively by Eisenberg [24] for human VLDL and IDL these two fractions have a precursor-product relationship and should therefore be studied separately in metabolic experiments. Furthermore there were no data given on the metabolic steady state of the animals, and the thyroid was not blocked. Experiments by Camejo [6] as well as the data reported in the present paper also emphasize that the IDL fraction in the rabbit may be the immediate product of VLDL decay, since most of the original VLDL bound activity was found in this density class 10 min after the injection. In a later phase of metabolism the activity found in the LDL density range increased above the IDL. However, specific activity data proving the precursor-product relationship in rabbits are still lacking and are now being investigated. The results reported for biosynthetically labeled rabbit LDL indicated an accelerated catabolism in the lipoproteins from hypercholesterolemic animals and are in good agreement with our findings for VLDL. By contrast, Bilheimer’s findings of a decreased rate of VLDL catabolism in patients with type III hyperlipoproteinemia, which is considered to be similar to the metabolic situation in cholesterol-fed rabbits, is not in accordance with these findings [ 261. To conclude, the structurally altered lipoproteins of the low, intermediate and very low density range obtained from hypercholesterolemic rabbits are characterised by their rapid metabolism. This may have significant implications on the inherent atherogenicity of these particles, emphasizing their role in experimental atherosclerosis [ 371.
12
Acknowledgements The author wish to thank Prof. Dr. Immich (Institut fiir Dokumentation und Statistik, Heidelberg) for his help in the statistical calculations. The assistance of Mrs. E. Bauer is gratefully acknowledged. References 1 Morrissett, J.D.. Jackson, R.L. and Gotto, Jr.. A.M., Lipoproteins: Structure and function, Ann. Rev. Biochem., 44 (1975) 183. 2 Sardet. C., Hansma. H. and Ostwald, R., Characterization of guinea pig lipoproteins - The appearance of new lipoproteins in response to dietary cholesterol, J. Lip. Res., 13 (1972) 624. 3 Gofman. J.W., Lindgren, F., Elliot. H., Mantz. W., Hewitt, J.. Strisower, B. and Herring, V., The role of lipids and lipoproteins in atherosclerosis, Science, 111 (1950) 166. 4 Schumaker. V.N.. Cholesterolemic rabbit lipoproteins - Serum lipoproteins of cholesterolemic rabbits. Amer. J. Physlol.. 184 (1956) 35. 5 Camejo. G.. Bosch, V.. Arreaza, C. and De Mantez, H., The changes in the plasma lipoprotein structure and biosynthesis in cholesterol-fed rabbits. J. Lip. Res.. 14 (1973) 61. 6 Camejo, G.. Bosch, V. and Lopez, A., The very low density lipoproteins of cholesterol-fed rabbits, Atherosclerosis, 19 (1974) 139. 7 Shore, V.G., Shore. B. and Hart, R.C., Changes in apolipoproteins and properties of rabbit lipoproteins on induction of cholesterolemia, Biochem.. 13 (1974) 1597. 8 Stange, E., Agostini. B. and Papenberg, J., Changes induced in rabbit plasma lipoproteins by polyunsaturated fat. Naturwiss.. 61 (1974) 408. 9 Mahley, R.W., Weisgraber. K.H., Innerarity. T.. Brewer, H.B. and Assmann, G., Swine lipoproteins and atherosclerosis - Changes in the lipoproteins and apoproteins induced by cholesterol feeding, Biothem.. 14 (1975) 2817. 10 Mahley. R.W., Welagraber. K.H. and Innerarity, T.. Canine lipoproteins and atherosclerosis -Characterization of the plasma lipoproteins associated with atherogenic and nonatherogenic hyperlipidemia, Circ. Res.. 36 (1974) 722. 11 Stange, E.. Agostini, B. and Papenberg, J., Changes in rabbit lipoprotein properties by dietary cholesterol, and saturated and polyunsaturated fats, Atherosclerosis, 22 (1975) 125. 12 Stange, E. and Papenberg, J., Die Wirkung van Maisol, Kokosfett und/oder Cholesterin auf die Serumund Aortenwandhpide sowie den Aortenwandstoffwechsel helm Kaninchen, Verh. Deutsch. Ges. Inn. Med., SO (1974) 1251. 13 Eisenberg. S. and Rachmilewitz, D., Metabolism of rat very low density lipoproteins. I. Fate in circulation of the whole lipoproteins, Biochim. Biophys. Acta. 326 (1973) 378. of rat very low density lipoproteins. II. Fate in 14 Eisenberg. S. and Rachmllewitz, D., Metabolism circulation of apoprotein subunits, Biochlm. Biophys. Acta. 326 (1973) 392. 15 Faergeman. 0.. Tsunako, S., Kane. J.P. and Havel, R.J., Metabolism of apoprotein B of plasma very low density lipoproteins in the rat, J. Clin. Invest., 56 (1975) 1396. 16 Fidge, N. and Poulis. P.. Studies on the metabolism of rat serum very low density apolipoproteln, J. Lip. Res., 16 (1975) 367. 17 Illingworth, D.R., Metabolism of lipoproteins in nonhuman primates. Studies on the origin of low
18 19
20
21 22 23
density lipoprotein apoprotein in the plasma of the squirrel monkey, Biochlm. Biophys. Acta, 388 (1976) 38. Portman, O.W.. Illingworth. D.R. and Alexander, M.. The effect of hyperlipidemia on lipoprotein metabolism ln squirrel monkeys and rabbits, Biochim. Biophys. Acta. 396 (1975) 55. 0.. Metabolism of lipoproteins in nonhuman primates, Illlngworth, R., Whipple, E. and Portman, reduced secretion of very low density lipoproteins in squirrel monkeys with diet induced hypercholesterolemia, Atherosclerosis, 22 (1976) 325. Gitlln, D., Cornwell, G., Nakasato, D.. Oncley. J., Hughes, J.R. and Janeway. C.. Studies on the metabolism of plasma proteins ln the nephrotic syndrome. II. The lipoproteins, J. Clin. Invest., 37 (1968) 172. Scott, P. and Hurley. P., The distribution of radioiodinated serum albumin and low density lipoprotein in tissue and the arterial wall, Atherosclerosis, 11 (1970) 77. Bllhelmer, D., Eisenberg, S. and Levy, R., The metabolism of very low density lipoprotein Proteins. I. Preliminary in vitro and in viva observations, Biochim. Biophys. Acta, 260 (1972) 212. Eisenberg, S., Bilheimer. D. and Levy. R.. The metabolism of very low density lipoprotein Proteins. II. Studies on the transfer of apoproteins between plasma lipoproteins, Biochim. Biophys. Acta. 280 (1972) 94.
13 24
Eisenberg, S.. Bilheimer. D.. Levy, R. and Lindgren. F., On the metabolic conversion of very low density lipoproteins to low density lipoproteins, Biochim. Biophys. Acta. 326 (1973) 361. 25 Langer, T., Strober, W. and Levy, R., The metabolism of low density lipoproteins in familial type II hyperlipoproteinemia. J. Clin. Invest., 51 (1972) 1528. 26 BiIheimer, D.W.. Eisenberg, S. and Levy, R.I., Abnormal metabolism of very low density lipoproteins (VLDL) in type III hyperlipoproteinemia, Circulation, 44 (1971) Suppl. II. abstr. 186. 27 Sigurdsson, G.. Nicoll. A. and Lewis, B., The metabolism of low density lipoprotein in endogenous hypertriglyceridemia, Europ. J. Clin. Invest., 6 (1976) 151. 28 Rodriguez. J.L.. Catapano. A., Ghiselli. G.C. and Sirtori, C.R., Very low density lipoproteins in normal and cholesterol fed rabbits: Lipid and protein composition and metabolism. Part 2: Metabolism of very low density lipoproteins in rabbits. Atherosclerosis, 23 (1976) 85. 29 Fidge, N.. A review of methods and metabolic studies associated with the radioiodination of lipoproteins. Clin. Chim. Acta. 52 (1974) 5. 30 McFarlane, AS.. Efficient trace-labeling of proteins with iodine, Nature, 182 (1958) 53. 31 Folch, J., Lees, M. and Stanley, S.G.H.. A simple method for the isolation and purification of total lipids from animal tissues, J. Biol. Chem., 224 (1957) 497. 32 Kane, J.P.. Sata. T., Hamilton, R.L., Havel, R.J., Apoprotein composition of very low density lipoproteins of human serum. J. Clin. Invest., 56 (1975) 1622. 33 Eisenberg, S., Stein, 0. and Stein, Y.. Radioiodinated lipoproteins: absorption of 1125 radioactivity by high density solutions. J. Lip. Res., 16 (1975) notes on methodology. 34 Matthews. C.M.E., The theory of tracer experiments with * 3 ’I-labeled plasma proteins. Physiol. in Med. Biol.. 2 (1957) 36. in electron microscopy of the integrity of 35 Stange, E., Agostini, B. and Papenberg. J.. Evaluation iodinated lipoproteins. Histochem., 52: 2 (1977) 145. 36 La Rosa, J.C., Levy, R.I.. Herbert, P.. Lux, S.E. and Fredrickson, D.S., A specific apoprotein activator for lipoprotein lipase, Biochem. Biophys. Res. Comm.. 51 (1974) 57. 37 Zilversmit, D.B., A proposal linking atherogenesis to the interaction of endothelial lipoprotein lipase with triglyceride-rich lipoproteins, Circ. Res.. 33 (1973) 633. 38 Fidge, N. and Poulls. P., Studies on the radioiodination of very low density lipoprotein obtained from different mammalian species, Clin. Chim. Acta. 52 (1974) 15.
Atherosclerosis, 28 (1977) l-14 0 Elsevier/North-Holland Scientific Publishers, Ltd.
CHANGES IN METABOLIC PROPERTIES OF RABBIT VERY LOW DENSITY LIPOPROTEINS BY DIETARY CHOLESTEROL, AND SATURATEDANDPOLYUNSATURATEDFAT
E. STANGE, M. ALAVI and J. PAPENBERG Medizinische
Universitiitsklinik,
Heidelberg
(W. Germany)
(Received 22 October, 1976) (Revised, received 11 May, 1977) (Accepted 25 May, 1977)
Summary
As was shown in a recent investigation, there is a marked change in the structural properties of rabbit lipoproteins by dietary cholesterol, saturated and polyunsaturated fats. Based on these electrophoretic, chemical and electron microscopic findings we examined the metabolic behaviour of ‘2SI-labeled VLDL (d < 1.006 g/ml) from normal rabbits (group I) as well as animals fed 1% cholesterol (group II), 1% cholesterol + 5% coconut oil (group III) or 1% cholesterol + 5% corn oil (group IV). The curve peeling technique resulted in a fractional catabolic rate per hour (FCR/h) of 0.082 in the normal VLDL. All hypercholesterolemic fractions II, III and IV were metabolised significantly faster (P< 0.05) with FCR/h values of 0.119,0.157 and 0.173 respectively. The difference in decay between VLDL II and both VLDL III or IV was statistically significant only during the late phase of metabolism, 2 h after injection. The apoprotein decay curves gave similar results. Ten min after the injection, most of the activity was recovered in the IDL class (d: 1.006-1.019 g/ml). The LDL fraction of the normal group had the lowest decay rate, whereas in all other groups HDL metabolism was delayed. It therefore may be concluded, that the metabolic behaviour of hypercholesterolemit VLDL differs from normal VLDL both quantitatively and qualitatively. Key words:
Atherosclerosis - Dietary cholesterol -Polyunsaturated rated fat - Very low density lipoprotein metabolism
This investigation was supported the 4th International Symposium
fat-Rabbits
-Satu-
in part by the Deutsche Forschungsgemeinschaft and was presented on Atherosclerosis in Tokyo, Japan. August, 1976.
at
2
Introduction In recent years knowledge of lipoprotein structure has expanded greatly [ 11. In different species it was possible to induce specific changes in the chemical, electrophoretic and electron microscopic properties of various lipoprotein density classes by feeding cholesterol. These alterations in guinea pigs [2], rabbits [3--81, swine [9] and dogs [lo] were suggested to play an important role in experimental atherosclerosis by their “inherent atherogenicity”. In a previous report we demonstrated the effect of saturated and polyunsaturated fat supplements to a cholesterol enriched chow on rabbit lipoprotein structure [ 111. Although we achieved a relative “normalization” of the agarose gel electrophoretic and electron microscopic properties by the unsaturated corn oil diet, accompanied ‘by a striking reduction in the extent of atherosclerosis in this group [12], the chemical and apoprotein composition as well as the behaviour in polyanionic precipitation was similar to the saturated coconut oil group. In the present study we examine the metabolism of ‘251-labeled VLDL from normal and dietary animals after injection into normal rabbits, Until now most of the effort focused on rat [13-161, monkey [ 17-191 as well as human [20-271 lipoprotein metabolism, whereas little information is available on rabbit [6,18,28]. However these investigations dealt with a heterogeneous lipoprotein fraction of d < 1.019 g/ml comprising VLDL and IDL [28] or used a biosynthetically labeled LDL of d: 1.006-1.063 g/ml and gave conflicting results [ 181. The findings reported in the present paper indicate that the diet induced structural alterations described previously in rabbit lipoproteins which significantly affect their metabolic behavior. Methods Animals and feeding
schedule
for experimental
diets
Male New Zealand White Rabbits weighing 2.5-3 kg were kept for the experimental period of 18 weeks as described previously [ 111. The animals were divided into 4 dietary groups: Group I received a control diet of normal rabbit chow (Georg Plange Co., Soest, W. Germany), group II was fed the control diet containing 1% cholesterol, groups III and IV had the control diet with 1% cholesterol and 5% coconut oil as a saturated fat or 5% corn oil as a polyunsaturated fat respectively. At the end of the dietary period the animals were bled from the carotid artery after an 18 h fast and the very low density lipoproteins were isolated as detailed below. The animals used in the metabolic experiments were normolipemics receiving their control diets ad libtum and were given additional 0.01% NaI in the drinking water prior to and after the injection of the 1251-labeled lipoproteins to block the thyroid gland. The serum lipid levels of both the recipient and the donor animals were in the same range as published previously [ll] for the different dietary groups.
3
Preparation and iodination of lipoproteins Lipoproteins were isolated by standard flotation technique and checked for purity and their typical mobility by immune electrophoresis in agarose gel as described elsewhere [ 111. Plasma was fractionated into 4 density classes: very low density lipoproteins (VLDL) of d < 1.006 g/ml, intermediate density lipog/ml, low density lipoproteins (LDL) of proteins (IDL) of d: 1.006-1.019 d: 1.019-1.063 g/ml, and high density lipoproteins (HDL) of d: 1.0631.210 g/ml. The VLDL samples were diluted to a final protein concentration of 10 mg/ 100 ml after dialysis in a 0.4 M glycine buffer at pH 10. For radioiodination 2 ml of the lipoprotein solution were mixed with 1 mCi of ‘*‘I carrier free (NEN Chemicals, Boston, U.S.A.) in 50 ~1 and various amounts of 0.0033 M ICl according to Fidge’s modification [ 291 of McFarlane’s iodine monochloride method [ 301. The concentration of ICl was adjusted to achieve a molar iodine/ protein ratio of approximately 1. Although this ratio is considered safe, the integrity of the radioiodinated particles was ascertained by immune electrophoretic and electron microscopic techniques. Determination of intramolecular activity distribution After exhaustive dialysis with at least 15 changes of 0.15 M NaCl the lipoprotein bound activity exceeded 95% in all preparations as determined by the extraction procedure according to Folch [ 311. The methanol/water phase was evaporated under nitrogen, the proteins were precipitated by 7% trichloroacetic acid and redissolved in 1 M NaOH to estimate the apoprotein bound activity. The chloroform phase containing the lipid moiety was counted in the dry state to avoid solvent quench, and the free iodide activity was assayed in the supernatant after TCA precipitation. The apoprotein bound activity plus the lipid bound activity was considered as lipoprotein bound activity. As a control an aliquot of labeled VLDL was lyophilised and delipidated with ethanol/ ether in 3 washings and 2 final ether extractions. This procedure gave similar results to the Folch technique, but of course did not yield the free iodide content of the preparation. The molar iodine/protein ratio was calculated according to the formula presented by Fidge [38], supposing a molecular weight of rabbit VLDL apoprotein of 250,000. The labeled lipid portion of [ 12’I]VLDL was separated by thin layer chromatography into mono-, di- and triglycerides, free fatty acids, phospholipids as well as free and esterified cholesterol. The silica gel layered plates were run with hexane/chloroform 1 : 3 (vol/vol) and methanol/chloroform 1 : 2 (vol/ vol) as the second solvent. Complete separation and recovery were checked by analysis of standard lipid mixture. All ‘*‘I activities were determined in a Searle Gamma Counter. Experimental design [‘*‘I]VLDL fractions of the 4 dietary groups were injected in a dose of lo7 cpm into the ear veins of normal animals on a control diet. The time lag between blood sampling and injection of the iodinated lipoproteins was always under 96 h. Blood samples were taken 10 min, 30 min, 1, 2, 4, 6, 12,24, 30,
4
36 and 48 h after injection. The 10 min value was taken as 100% of the injected dose. After slow centrifugation at 2500 rpm an aliquot of plasma was counted, lyophilised and delipidated as described above. Radioactivity was then determined both in the protein and lipid moieties. From another aliquot of plasma we isolated the VLDL, IDL, LDL and HDL fractions by ultracentrifugation as detailed before. These lipoproteins were counted and their activities corrected for the KBr quench of the high salt solutions. The correction factors were 1.275 3f IDL, 1.544 for LDL and 3.199 for HDL. Comparable values were published by Eisenberg [33] at the respective density cuts. The intravascular activity as a fraction of the injected dose was plotted on a logarithmic scale (ordinate) against time on linear scale (abscissa). These decay curves were analysed for the half life times and the fractional catabolic rates per hour according to the method of Matthews [34]. For statistical analysis we employed the chi square sum test, using the absolute values (cpm/ml) for all calculations. To check the metabolic steady state condition of the experimental animals we calculated the U/P ratio from the excreted urinary activity and the mean total plasma activity between 2 blood samples. Plasma volume was estimated from the Evans Blue method. Results Intramolecular
distribution
of activity
In the lipid moiety of the labeled VLDL fractions activity was bound predominantly to the phospholipids with 53% (mean of 3 determinations, range 49.5-58%). The rest of the lipid activity was rather evenly distributed between monoglycerides (8.3%), diglycerides (7.8%), triglycerides (12.1%), free fatty acids (3.0%), free (5.3%) and esterified cholesterol (6.4%) and a small unidentified fraction (4.1%). In the hypercholesterolemic VLDL the amount of lipid bound activity decreased from a mean of 50.7% in VLDL I to 18.8% in VLDL II, 15.3 and 22.7% in VLDL III and IV, respectively. To exclude interference of these varying proportions of lipid bound activity with the VLDL plasma decay of the 4 groups, total plasma samples were delipidated to obtain the apoprotein decay as such. The mean I/P (iodine/protein) ratio of all injected fractions was 1.06 (range 0.83-1.37). Immune electrophoresis of native and iodinated VLDL resulted in identical precipitin bands. In electron micrographs the labeled lipoprotein particles could not be distinguished from the native preparation [ 341. VLDL plasma activity decay
A mean of 45.9% (range: 31-75%) of the injected dose remained in the circulation 10 min after the injection of VLDL I. In groups II and IV a mean of 50.8% (17-63%) and 49.1% (25-77%) were found in total plasma after the same interval, whereas in group III only 36.8% (14-62%) were recovered. As can be seen from the plasma decay curves of the 4 groups of normal and dietary altered rabbit VLDL in normal animals, the catabolism of control
5
VLDL I was the slowest of all fractions (Fig. 1). This difference was also reflected in the longer half life times during both the initial rapid phase (t,,*,) and the late slow phase (tllzz) o f metabolism and the smaller fractional catabolic rate per hour (Table I). The statistical evaluation is presented in Table 2, demonstrating that all hypercholesterolemic fractions were metabolised significantly faster than normal VLDL I. Accordingly the FCR value for cholesterol VLDL was 32% higher than the control with 0.082, whereas it was nearly doubled in coconut and corn oil VLDL (Table 1). Concomitantly the half life times are reduced approximately by half. However, ‘he coconut and the corn oil VLDL plasma decay was significantly different from the cholesterol VLDL only during the later phase of decay 1 and 2 h after the injection. The decay rate of coconut and corn oil VLDL was similar. VLfiL
apoprotein
activity decay
The apoprotein activity curves are depicted in Fig. 2 and show the same distribution as described above for plasma decay. The catabolism of VLDL I was slowest, with a more rapid decay of apo VLDL II and the fastest apoprotein metabolism in VLDL III and IV (Table 1). The apo-VLDL differences also are statistically significant when control apo-VLDL is compared with the hypercholesterolemic groups, as well as apo VLDL II vs IV (Table 2). Coconut and
.
NORMAL
n:
.
CHOLESTEROL
nz 16
o
COCONUT CORN
26
2
6
OIL
n:
11
n= 12
0
12
4
OIL
14
12
24
30
36
48
TIME
Fig. 1. Plasma activity decay after injection of normal and dietary altered VLDL. Values represent median values of n experiments. The inserted plot depicts the urine/plasma activity ratio of a representative experiment.
6
TABLE
1
DECAY tl,z,
PARAMETERS is the half
life
OF time
NORMAL
during
AND
the early
DIETARY
phase.
ALTERED
tl ,z2
is the half
VLDL life
time
in days
during
the late phase
of
metabolism. _---
..-
_~
.-.-__
Normal
Cholesterol
Coconut
oil
-__
Corn .__~_~___
-___
oil
Plasma
*
1.111
0.590
0.479
t1/z2
0.118
0.056
0.042
0.069
FCR
0.082
0.119
0.157
0.173
tu2 *
1.146
0.951
0.674
0.604
tu2*
0.097
0.056
0.049
0.035
FCR
0.083
0.145
0.224
0.240
t1/2
0.660
Apoprotein
corn oil apo-VLDL decay was nearly identical. The fractional catabolic rate per hour of normal apo-VLDL of 0.083 was nearly the same as that found in total plasma decay, but in groups II, III and IV the apoprotein decay seems to be accelerated in comparison to plasma, thereby reflecting the slower metabolism of their cholesterol-rich lipid moiety (Table 1). The lipid bound activity in plasma was too low to give consistent results, due to the small amount of iodine bound to the lipid moiety of VLDL II, III and IV. Distribution of activity in lipoprotein classes The activity distribution in the different lipoprotein density classes VLDL, IDL, LDL and HDL indicates the rapidity of lipoprotein dynamics in the rabbit (Tables 3, 4, 5 and 6). After 10 min, most of the original VLDL bound activity was recovered in the intermediate density class irrespective of the lipoprotein group injected (Fig. 3 and 4). Only 27.5% of the injected activity was still
TABLE
2
STATISTICAL ns, Not horizontal protein
EVALUATION
significant. and decay
For
vertical in the
OF
comparison columns.
lower
left
VLDL
PLASMA
of two
groups
The
values
AND the
for plasma
PROTEIN
decay
Normal1
Cholesterol
P < 0.01
P < 0.05
-~____--~~-a Significant
b Significant
(P < 0.05) (P < 0.05)
are presented
in the upper
of the respective right
triangle. II
Coconut
I
IV
DECAY
P value is given at the cross-section
-~- .-.-
after
1 h.
after
2 h.
IIS ~_..__..
oil III
Corn
oil IV
triangle.
for
3
4
f
13.9
oil
9
Corn
k
18.5
6
coconut
oil
7
21.7
5.1
IN
PERCENT
10.8
9.9
’7.2
13.9
22.7
7.3
f
+ 4.8
6.4
? 10.0
f
OF
* 4.4
f 2.3
f 4.3
f: 1.6
30 min
PERCENT
9.1
11.0
9.7
20.0
30 min
? 2.4
+ 1.9
+ 4.0
4.2
8.3
11.2
15.7
lh
i 3.4
f 7.4
? 5.2
f 7.3
INJECTED
6.9
8.0
8.5
f 4.3
INJECTED
13.9
lh
THE
THE
OF
of n experiments.
IN
9.4
t 18.5
f
Cholesterol
33.4
5
IDL
10 min
Time
OF
k 5.4
* 4.4
+ 7.1
k 5.3
NOllnal
KINETICS
13.2
13.6
12.4
27.5
10 min
n
RABBITS
9
VLDL
deviation
Time
+ standard
OF
VLDL
NORMAL
RADIOACTIVITY
TABLE
oil
6
coconut
Corn
7
Cholesterol
oil
5
Normal
mean
KINETICS
n
represent
RABBITS
VLDL
Values
NORMAL
RADIOACTlVITY
TABLE
DOSE
+ 1.2
+ 1.0
? 1.3
f 1.8
AFTER
2.1
2.6
4.6
f 0.4
C 0.5
* 2.7
+ 4.3
AFTER
11.2
6h
3.3
2.5
4.0
8.2
6h
DOSE
1.8
-___
1.4
1.4
1.8
3.6
OF
f 0.6
+ 0.8
k 0.76
? 2.2
.___
OF
* 1.0
r 1.0
? 0.7
+ 0.9
12 h
INJECTION
_
1.7
2.2
5.0
12 h
INJECTION
0.7
0.6
0.8
1.5
AND
k 0.6
f 0.4
* 0.4
f. 0.5
AND
* 0.7
+ 0.4
f 0.5
k 0.28
24 h
.~_
NORMAL
0.9
0.6
1.1
2.3
24 h
NORMAL
___-
0.3
0.3
0.39
0.9
36
DIETARY
0.4
0.3
0.57
1.3
h
36 h
DIETARY
* 0.2
+ 0.2
f 0.3
+ 0.4
ALTERED
* 0.3
* 0.3
f 0.4
‘_ 0.24
-_
ALTERED
h
0.11
0.2
0.25
0.5
48
h
VLDL
0.2
0.16
0.4
0.7
48
VLDL
* 0.07
+_ 0.1
+ 0.2
+ 0.1
INTO
+ 0.1
+ 0.1
k 0.26
+ 0.24
-
INTO
-1
5
6
oil
9
6
coconut
corn
7
Cholesterol
oil
5
Normal
KINETICS
n
RABBITS
9
VLDL
NORMAL
RADIOACTIVITY
TABLE
oil
6
coconut
Corn
7
Cholesterol
oil
5
Normal
KINETICS
n
RABBITS
VLDL
NORMAL
RADIOACTIVITY
TABLE
LDL
HDL
f 1.9
* 9.0
f 4.1
k 2.0
9.0
8.0
17.1
10.9
? 2.6
+ 2.6
+ 5.9
k 5.9
10 min
Time
OF
7.1
15.4
9.8
14.8
10 min
Time
OF
IN
IN
f 2.6 f 0.7
8.8
4.4
7.6
6.0
15.4
f 4.0
k 2.1
? 7.2
? 2.8
min
8.8
30
PERCENT
* 1.4
5.5
t 1.0
* 1.2
f 3.1
f 1.1
+ 1.2
6.0
6.0
15.0
6.4
lh
r 2.8
f 2.5
* 7.8
* 3.1
INJECTED
3.3
6.7
4.8
7.7
INJECTED
9.0
THE
THE
lh
OF
OF
30 min
PERCENT
3.5
3.3
7.8
4.1
6h
DOSE
1.9
2.4
2.8
6.3
6h
DOSE
t 1.8
+ 0.8
r 5.6
r 1.0
AFTER
t 0.3
f; 1.0
+ 1.2
* 1.9
~____
AFTER
+ 0.7
+ 0.8
f 0.5
+ 1.7
~_
2.3
2.5
3.8
3.5
+ 1.6
? 0.8
+ 0.9
? 1.0
12 h
INJECTION
1.4
1.4
1.6
5.7
12h
~___
INJECTION
OF
1.2
1.2
1.8
1.9
24
f 0.6
+ 0.4
* 0.9
+ 0.8
AND
+ 0.4
0.6
NORMAL
? 0.4
0.73
f 0.7 f 0.5
h
h
AND
0.7
2.6
24
NORMAL
~. ___~_.
OF
0.8
1.0
1.5
1.3
ALTERED
+ 0.5
+ 0.3
* 0.7
t 0.5
ALTERED
f 0.4
k 0.2
* 0.3
* 1.0
36 h
DIETARY
0.4
0.3
0.5
2.1
36 h
DIETARY
h
0.6
0.7
0.9
0.9
INTO
i 0.3
+ 0.3
+ 0.4
INTO
f 0.1
+ 0.1
+ 0.26
i 0.3
f 0.5
48 h
VLDL
0.14
0.2
0.3
0.6
48
VLDL
co
9
.
NORMAL
n=
6
I
CHOLESTEROL
n=
15
0
COCONUT
n=
9
n=
7
OIL
0CORNOIL
0.0 1
,h 2
4
6
12
24
30
36
s
48
TIME
Fig. 2. Apoprotein activity decay median values of n experiments.
after injection
of normal
and dietary
altered
VLDL.
Values
represent
1.0
0.3
9 1
. VLDL
(d:Wo6)
x
(d 1.006
IDL
A LDL
0.1
S
i o HDL
(d
-1.019)
1.019-1.063)
(d.1.063-1.210)
s i
:
2
z FoxI1 'e' 5 f ._ t e v
O.COl
. I. 246
I
.h 12
Fig. 3. Distribution of activity in lipoprotein represent mean values of 5 experiments.
24
lime
density
30 classes
36 after
injection
48 of normal
VLDL.
Values
10
1.a
7 1
0.3
9 B Y 5
??
0.1
: p” z ‘0 .% C
VLDL
x
ID1
A
LDL
0
HDL
T .E t z Y
0.01
0.001
h
246
.
-
12
Fig. 4. Distribution of activity in lipoprotein represent mean values of 7 experiments.
24 Time density
30
36
classes after injection
c
46 of cholesterol
h VLDL.
Values
bound to the control VLDL, compared with 12.4, 13.6 and 13.2% in the hypercholesterolemic VLDL fractions (Table 3). In the control group the LDL fraction had the lowest decay rate, whereas in all other groups HDL decay was delayed relative to the other density classes (Figs. 3 and 4). It is obvious from Fig. 4 that the more rapid plasma decay after injection of cholesterol VLDL is due to an accelerated metabolism of all lipoprotein classes of d < 1.063 g/ml. There was no significant difference between the lipoprotein decay curves of the 4 density classes after injection of cholesterol VLDL when compared with coconut or corn oil VLDL, so these two groups are not depicted separately. Urinary iodine excretion and metabolic steady state The ratio of excreted urinary activity to total plasma. activity (U/P ratio) was fairly constant from 9-48 h after the injection of labeled VLDL. A representative experiment is inserted in Fig. 1. A total of 6 such studies were performed, allowing the estimation of the fractional catabolic rates from the urine activity. This method gave comparable results under steady state conditions to the curve peeling technique. However, it is evident from Fig. 1 that there was a time lag in urinary iodine excretion at the beginning of the experiment which was compensated for rather rapidly.
11
Discussion The data presented in this study provide evidence for a more rapid metabolism of the abnormal cholesterol rich VLDL as compared to the control VLDL. This effect was even more pronounced in the lipoprotein fractions isolated from rabbits fed additional coconut or corn oil to their cholesterol enriched diets. Since the radio-labeled VLDL fractions were injected into normolipemic animals, differences in the catabolic rate have to be due to structural alterations in the particle itself as described previously [ 111. In contrast to the striking dissimilarity in agarose gel electrophoresis and electron micrographs between both the cholesterol and the coconut oil group as compared to the corn oil VLDL, all hypercholesterolemic fractions resembled each other in chemical and apoprotein composition as well as in their high heparin affinity. It therefore seems possible that the differing apoprotein patterns are responsible for an activation or an inhibition of lipoprotein lipase in the respective group [ 361. Our findings exclude the possibility that the increase in catabolic rate is only due to the lipid portion of the lipoproteins, because the activity decay in delipidated plasma, reflecting the apoprotein catabolism, was even more enhanced than total plasma decay. This difference on the contrary indicates a slightly delayed lipid metabolism in the cholesterol rich fractions. Recent investigations by Rodriguez et al., suggested a slower rate of catabolism in hypercholesterolemic rabbit “VLDL” of the density d < 1.019 g/ml. However this density range comprises two structurally and metabolically heterogeneous lipoprotein classes of VLDL and IDL. As was shown conclusively by Eisenberg [24] for human VLDL and IDL these two fractions have a precursor-product relationship and should therefore be studied separately in metabolic experiments. Furthermore there were no data given on the metabolic steady state of the animals, and the thyroid was not blocked. Experiments by Camejo [6] as well as the data reported in the present paper also emphasize that the IDL fraction in the rabbit may be the immediate product of VLDL decay, since most of the original VLDL bound activity was found in this density class 10 min after the injection. In a later phase of metabolism the activity found in the LDL density range increased above the IDL. However, specific activity data proving the precursor-product relationship in rabbits are still lacking and are now being investigated. The results reported for biosynthetically labeled rabbit LDL indicated an accelerated catabolism in the lipoproteins from hypercholesterolemic animals and are in good agreement with our findings for VLDL. By contrast, Bilheimer’s findings of a decreased rate of VLDL catabolism in patients with type III hyperlipoproteinemia, which is considered to be similar to the metabolic situation in cholesterol-fed rabbits, is not in accordance with these findings [ 261. To conclude, the structurally altered lipoproteins of the low, intermediate and very low density range obtained from hypercholesterolemic rabbits are characterised by their rapid metabolism. This may have significant implications on the inherent atherogenicity of these particles, emphasizing their role in experimental atherosclerosis [ 371.
12
Acknowledgements The author wish to thank Prof. Dr. Immich (Institut fiir Dokumentation und Statistik, Heidelberg) for his help in the statistical calculations. The assistance of Mrs. E. Bauer is gratefully acknowledged. References 1 Morrissett, J.D.. Jackson, R.L. and Gotto, Jr.. A.M., Lipoproteins: Structure and function, Ann. Rev. Biochem., 44 (1975) 183. 2 Sardet. C., Hansma. H. and Ostwald, R., Characterization of guinea pig lipoproteins - The appearance of new lipoproteins in response to dietary cholesterol, J. Lip. Res., 13 (1972) 624. 3 Gofman. J.W., Lindgren, F., Elliot. H., Mantz. W., Hewitt, J.. Strisower, B. and Herring, V., The role of lipids and lipoproteins in atherosclerosis, Science, 111 (1950) 166. 4 Schumaker. V.N.. Cholesterolemic rabbit lipoproteins - Serum lipoproteins of cholesterolemic rabbits. Amer. J. Physlol.. 184 (1956) 35. 5 Camejo. G.. Bosch, V.. Arreaza, C. and De Mantez, H., The changes in the plasma lipoprotein structure and biosynthesis in cholesterol-fed rabbits. J. Lip. Res.. 14 (1973) 61. 6 Camejo, G.. Bosch, V. and Lopez, A., The very low density lipoproteins of cholesterol-fed rabbits, Atherosclerosis, 19 (1974) 139. 7 Shore, V.G., Shore. B. and Hart, R.C., Changes in apolipoproteins and properties of rabbit lipoproteins on induction of cholesterolemia, Biochem.. 13 (1974) 1597. 8 Stange, E., Agostini. B. and Papenberg, J., Changes induced in rabbit plasma lipoproteins by polyunsaturated fat. Naturwiss.. 61 (1974) 408. 9 Mahley, R.W., Weisgraber. K.H., Innerarity. T.. Brewer, H.B. and Assmann, G., Swine lipoproteins and atherosclerosis - Changes in the lipoproteins and apoproteins induced by cholesterol feeding, Biothem.. 14 (1975) 2817. 10 Mahley. R.W., Welagraber. K.H. and Innerarity, T.. Canine lipoproteins and atherosclerosis -Characterization of the plasma lipoproteins associated with atherogenic and nonatherogenic hyperlipidemia, Circ. Res.. 36 (1974) 722. 11 Stange, E.. Agostini, B. and Papenberg, J., Changes in rabbit lipoprotein properties by dietary cholesterol, and saturated and polyunsaturated fats, Atherosclerosis, 22 (1975) 125. 12 Stange, E. and Papenberg, J., Die Wirkung van Maisol, Kokosfett und/oder Cholesterin auf die Serumund Aortenwandhpide sowie den Aortenwandstoffwechsel helm Kaninchen, Verh. Deutsch. Ges. Inn. Med., SO (1974) 1251. 13 Eisenberg. S. and Rachmilewitz, D., Metabolism of rat very low density lipoproteins. I. Fate in circulation of the whole lipoproteins, Biochim. Biophys. Acta. 326 (1973) 378. of rat very low density lipoproteins. II. Fate in 14 Eisenberg. S. and Rachmllewitz, D., Metabolism circulation of apoprotein subunits, Biochlm. Biophys. Acta. 326 (1973) 392. 15 Faergeman. 0.. Tsunako, S., Kane. J.P. and Havel, R.J., Metabolism of apoprotein B of plasma very low density lipoproteins in the rat, J. Clin. Invest., 56 (1975) 1396. 16 Fidge, N. and Poulis. P.. Studies on the metabolism of rat serum very low density apolipoproteln, J. Lip. Res., 16 (1975) 367. 17 Illingworth, D.R., Metabolism of lipoproteins in nonhuman primates. Studies on the origin of low
18 19
20
21 22 23
density lipoprotein apoprotein in the plasma of the squirrel monkey, Biochlm. Biophys. Acta, 388 (1976) 38. Portman, O.W.. Illingworth. D.R. and Alexander, M.. The effect of hyperlipidemia on lipoprotein metabolism ln squirrel monkeys and rabbits, Biochim. Biophys. Acta. 396 (1975) 55. 0.. Metabolism of lipoproteins in nonhuman primates, Illlngworth, R., Whipple, E. and Portman, reduced secretion of very low density lipoproteins in squirrel monkeys with diet induced hypercholesterolemia, Atherosclerosis, 22 (1976) 325. Gitlln, D., Cornwell, G., Nakasato, D.. Oncley. J., Hughes, J.R. and Janeway. C.. Studies on the metabolism of plasma proteins ln the nephrotic syndrome. II. The lipoproteins, J. Clin. Invest., 37 (1968) 172. Scott, P. and Hurley. P., The distribution of radioiodinated serum albumin and low density lipoprotein in tissue and the arterial wall, Atherosclerosis, 11 (1970) 77. Bllhelmer, D., Eisenberg, S. and Levy, R., The metabolism of very low density lipoprotein Proteins. I. Preliminary in vitro and in viva observations, Biochim. Biophys. Acta, 260 (1972) 212. Eisenberg, S., Bilheimer. D. and Levy. R.. The metabolism of very low density lipoprotein Proteins. II. Studies on the transfer of apoproteins between plasma lipoproteins, Biochim. Biophys. Acta. 280 (1972) 94.
13 24
Eisenberg, S.. Bilheimer. D.. Levy, R. and Lindgren. F., On the metabolic conversion of very low density lipoproteins to low density lipoproteins, Biochim. Biophys. Acta. 326 (1973) 361. 25 Langer, T., Strober, W. and Levy, R., The metabolism of low density lipoproteins in familial type II hyperlipoproteinemia. J. Clin. Invest., 51 (1972) 1528. 26 BiIheimer, D.W.. Eisenberg, S. and Levy, R.I., Abnormal metabolism of very low density lipoproteins (VLDL) in type III hyperlipoproteinemia, Circulation, 44 (1971) Suppl. II. abstr. 186. 27 Sigurdsson, G.. Nicoll. A. and Lewis, B., The metabolism of low density lipoprotein in endogenous hypertriglyceridemia, Europ. J. Clin. Invest., 6 (1976) 151. 28 Rodriguez. J.L.. Catapano. A., Ghiselli. G.C. and Sirtori, C.R., Very low density lipoproteins in normal and cholesterol fed rabbits: Lipid and protein composition and metabolism. Part 2: Metabolism of very low density lipoproteins in rabbits. Atherosclerosis, 23 (1976) 85. 29 Fidge, N.. A review of methods and metabolic studies associated with the radioiodination of lipoproteins. Clin. Chim. Acta. 52 (1974) 5. 30 McFarlane, AS.. Efficient trace-labeling of proteins with iodine, Nature, 182 (1958) 53. 31 Folch, J., Lees, M. and Stanley, S.G.H.. A simple method for the isolation and purification of total lipids from animal tissues, J. Biol. Chem., 224 (1957) 497. 32 Kane, J.P.. Sata. T., Hamilton, R.L., Havel, R.J., Apoprotein composition of very low density lipoproteins of human serum. J. Clin. Invest., 56 (1975) 1622. 33 Eisenberg, S., Stein, 0. and Stein, Y.. Radioiodinated lipoproteins: absorption of 1125 radioactivity by high density solutions. J. Lip. Res., 16 (1975) notes on methodology. 34 Matthews. C.M.E., The theory of tracer experiments with * 3 ’I-labeled plasma proteins. Physiol. in Med. Biol.. 2 (1957) 36. in electron microscopy of the integrity of 35 Stange, E., Agostini, B. and Papenberg. J.. Evaluation iodinated lipoproteins. Histochem., 52: 2 (1977) 145. 36 La Rosa, J.C., Levy, R.I.. Herbert, P.. Lux, S.E. and Fredrickson, D.S., A specific apoprotein activator for lipoprotein lipase, Biochem. Biophys. Res. Comm.. 51 (1974) 57. 37 Zilversmit, D.B., A proposal linking atherogenesis to the interaction of endothelial lipoprotein lipase with triglyceride-rich lipoproteins, Circ. Res.. 33 (1973) 633. 38 Fidge, N. and Poulls. P., Studies on the radioiodination of very low density lipoprotein obtained from different mammalian species, Clin. Chim. Acta. 52 (1974) 15.
