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Changes in plasma lipoprotein constituents during constant infusions of heparin
Atherosclerosis,
22 (1975) 551-563
0 Elsevier Scientific Publishing Company, Amsterdam - Printed in The Netherlands
CHANGES
IN PLASMA
CONSTANT
Y. HOMMA Department
LIPOPROTEIN
INFUSIONS
AND
CONSTITUENTS
551
DURING
OF HEPARIN
P. J. NESTEL
of Clinical Science,
The Australian
National
University,
Canberra
(Australia)
(Received February 25th, 1975) (Accepted April 14th, 1975)
SUMMARY
Constant infusions of heparin of 4 to 6 hours’ duration were used to estimate the transfer of very low density lipoprotein constituents to other plasma lipoproteins. Eleven subjects were studied, 3 with Type III and the remainder with either Type IV or V hyperlipoproteinemia. Whereas only about 5 % of the triglyceride lost from VLDL was recovered in the other lipoproteins, 44% of the cholesterol was retained in the circulation, in equal amounts within low density (d 1.019-1.063) and intermediate density (d 1.006-l .019) lipoproteins. of protein, more than half of that originally lipoprotein.
By contrast, there was no apparent loss in VLDL being recovered in high density
In subjects with Type III hyperlipoproteinemia, lipid and protein was lost from the intermediate density lipoprotein as well as from VLDL. In subjects with marked hypertriglyceridemia, cholesterol became redistributed from larger to smaller VLDL. The esterification of plasma cholesterol as measured in vitro, was apparently suppressed during the early phase of the heparin infusions, but tended to recover later.
Key words:
Cholesterol ester$cation protein changes
- Heparin - Hyperlipidaemic subjects - Lipo-
INTRODUCTION
The plasma lipoproteins are metabolically interrelated. There is transfer and exchange of the individual lipid and protein moieties among the lipoproteins as well as conversion of one whole class of lipoprotein into another. It is probable that low
552
Y. HOMMA, P. J. NESTEL
density lipoproteins (LDL) are derived partly or entirely from the very low density lipoproteins (VLDL). This occurs during the breakdown of VLDL which is brought about by the hydrolysis and removal of the triglyceride; smaller species of VLDL emerge which in turn give rise to LDL. The evidence for this is based on the precursorproduct characteristics of the kinetics of the triglyceride fatty acid132 and proteins constituents of the respective lipoproteins. The esterified cholesterol of chylomicrons is not removed from the circulation as rapidly as the triglyceride4 but is at first retained within smaller lipoproteins or “remants” and subsequently removed in the liver4. The rate of transfer of lipid and protein from larger to smaller lipoproteins can be accelerated
with heparin
which stimulates
the catabolism
of VLDLs,6,7;
studies
of this sort have provided qualitative evidence about the metabolic fate of lipoprotein constituents. In this study we have used prolonged constant infusions of heparin which have allowed more accurate estimates of the net transfer of VLDL components to LDL and HDL. The results show that under these conditions
less than half of the
cholesterol lost from VLDL is recovered in the smaller lipoproteins, whereas most of the protein appears to be retained in the circulation. The reconstitution of intermediate classes of lipoproteins that takes place following
the removal
of triglyceride
from within the core of VLDL appears to require
the removal of excess surface membrane, a process that involves the enzyme lecithinof plasma cholesterol acyltransferase 8. We have measured the in vitro esterification cholesterol, the major function of the enzyme, since it has been reported that, perhaps paradoxically,
this is inhibited
by heparing.
METHODS
Eleven hyperlipidemic subjects were studied (Table 1). The lipoprotein phenotype was determined by plasma lipid electrophoresis on cellulose acetatetO, and, especially with the Type III patients, from the distribution of cholesterol and triglyceride among the lipoproteinstl. The subjects were not taking lipid-lowering drugs but several had been dieting for some time. The plasma lipids were therefore sometimes lower at the time of study than initially. All subjects fasted overnight, but most were given a small fat-free meal during the infusion. Sodium heparin, diluted in 0.15 M NaCl solution was infused intravenously at a constant rate to deliver 3000 international units/hr for 4-6 hr. Subjects 1 and 2 received only 1500 units of heparin for the first 165 min and then 3000 units for the last 2 hr. Two samples of blood were taken before the heparin infusion and a further 5 to 8 samples at approximately 60-min intervals during the infusion. Blood was placed into chilled tubes that contained 0.5 mg paraoxon for each ml blood to inhibit continued lipolysis in vitrolz. Plasma was separated at 4°C. Four major classes of
age (yr)
39 44 59 41 43 23 51 54 36 26 35
1 2 3 4 5 6 7 8 9 10 11
M M F M M M M M M M F
sex
AND THEIR
No.
Subjects
THE SUBJECTS
TABLE 1
76 82 75 85 85 73 73 90 101 89 65
(kg)
Weight
CHOLESTEROL
111 III III IV IV IV IV-V v v V V
Type of hyperlipoproteinemia
AND TRIGLYCERIDE
81 58 148 49 102 126 175 247 225 327 517
Chol
VLDL
120 72 189 200 465 498 1344 1213 1013 2555 2557
TG
CONCENTRATIONS
42 56 117 10 12 12 4 12 18 11 14
Chol
LDLl
25 29 30 13 18 16 9 17 27 19 38
TG
IN LIPOPROTEINS
112 140 125 87 124 43 46 48 45 28 91
Chol
LDLz
(IIlg/l(bo
Id)
20 23 27 19 32 10 35 20 41 40 56
TG
61 60 38 20 46 39 14 48 33 16 18
Chol
HDL
12 15 12 7 17 10 16 26 30 26 22
TG
554
Y. HOMMA, P. J. NESTEL
TABLE 2 CUMULATIVE
Subjects
4 5 6 7 8 9 10 11
CHANGES
IN LIPOPROTEIN
PROTEIN
CONCENTRATIONS
DURING
rime (hr)
time (hr)
0
3
1
2
3
4
VLDL 49 102 126 175 247 225 327 517
3 -14 - 6 - 6 + 1 -20 -35 +48
8 -15 -11 - 6 1 -22 -34 +23
-14 -14 -31 -12 -62 -31 -52 - 9
-18 -34 -35 -27 -22 -54 -30
- 22 34 - 48 - 50 57 58 - 69 -100
-
4
-
-28
-31
-
0
+2 +3 1-6
0
0 + 3 +1 +1 + 5
+3 +4 +9 +1 + 7 +1 +2 +13
+5 t-8 +7 +4 +16 f2 +4 $31
+ 4 +I2 + 7+13 + 6 + 18 + 23 + + 2+ 3f + 6+ 6+ + 45 + 52 +
24 5 7 54
0
+
+
f
+
22
LDLI 10 12 12 4 12 18 11 14
+1 +2 -
0 1 0
+1
LDLz 87 +2 124 +4 43 +7 46 -4 48 -5 45 -3 28 -1 56 1
Mean
Mean
AND
Triglyceride (mg/lOO ml)
Mean
4 5 6 7 8 9 10 11
TRIGLYCERIDE
Cholesterol
Mean
4 5 6 7 8 9 10 11
CHOLESTEROL,
HDL 29
9
2
5
9
5
-
12 +
0
3
1
- 87 - 72 60 -137
200 465 498 1344 1213 1013 2555 2557
26 53 - 49 -276 -384 87 -130 t-118
52 - 70 - 90 -417 -407 70 -285 -177
-110
-196
49
64 58 52 -125
54 -
6
70 -
19 +
89
+7 +2 +6 -8 +l -5 +3 +I3
+7 +9 +ll -1 +4 +2 +l +16
+4 -6 +9 +3 +2 f3 f38
+16 + 9 + 9+15 +12 + 8+ 7+17 4+ 4+ 6 + 4+ 5+ 6 + 27 + 43 + 57
0
+2
+6
+6
+10+15+21
0
+1
+1
+1
+
0
2
13-l 18 16 9 17 27 19 38
+ -
1+ 4+ 41+
0 4 9 5 4
+
0+ 2+
1 3
0+
9
0
19 32 10 35 20 41 40 91
19
-
+3
-
230-t 7 + 531+ 4 +
1 4 1 12 4 7 3 13
-
3+
1
+
1-k
3
555
HEPARIN INFUSION AND LIPOPROTEINS
HEPARININFUSIONSINEIGHTTYPEIVANDVHYPERLIPOPROTEINEMICSUBJECTS
Protein
time 2
-
3
66
4
5
6
0
-
74
-
81
47
-122
-
144
-
142
81
-145
-
151
-
151
-
-677
-
873
-
922
-1027
-
666
-
685
-
-193
-
199
-
272
-
319
-
-663
-
788
-
980
-
997
-740
-1110
-1349
-393
-
-
fl
+
2+
1
9
+2
+
5+
9
+9 -2
+ +
5+ 2+
2+ 3
8
9 10
+ +
23 l+
7 + 1+
9 l+
+ +
5+ 26 +
9+ 34 +
943
+
7 43
14
+
16
-538
+ -1
8
+3 +
13
477
+
570
(hr) 3
I
2
+14
-
-17
-22
-31
3
4
6
5
6
-5 -27
97
-21
725
175
-80
-
91
-
93
285
148
-25
-
20
-
27
-1135
261
-53
-
66
-
91
-1332
-1390
274
-67
-102
-161
-
-
-45
-
-
93
-3542
-1050
752
+
917
23 6
-3
63
j-2
-2
+2
-3
-2
+5
+5
+
5+9
8
+4
+
10
+
12
14
+2
+
5
+
6
13
-2
-
1
+
6
16
+7
+
24
+
35
$4
+
9
+
15
+
8+
8
-5
-
2
0
63
+8
+3
-
2+
3
+1
+
+3 -1
f7 -1-
+
2+ 6 -
115 38 15
46
-4
0
+
16
4
+10 -4
+ -4
20
+ -6
23
+15
+
46
+
48
+4
+
12
+
20
+12
+
37
+
46
+4
+1-5 +
+ 10
+
+
153
2+ 14
3
1+
4+
6
2-
5+
2+
2+ 32
8+ +
13
+1
+
4+
1+
+1
+
1+
1
+
7
30 56
30
+
42
114
9
+
17
+
3
5-t +
132
+5 -
3+
+
19
+
18 3
Y. HOMMA, I’. J. NESTEL
556 lipoproteins
were separated
in all studies 13: VLDL
(d <
1.006), LDLi
(d 1.006-
1.019), LDLa (d 1.019-1.063) and HDL (d 1.063-1.021). In subjects 8-l 1, who showed the highest plasma triglyceride values, the VLDL were further fractionated into S, > 400, Sf 100-400 and S, 20-100 subclassesi4. The mean
recoveries
of lipoprotein
cholesterol
and triglyceride
during
ultra-
centrifugal separation were 86 y0 and 83 % respectively. Most of this loss occurred with separation of VLDL. The values have not been corrected for losses. Lipids were extracted in Dole’s solutionlj and the cholesterol and triglyceride concentrations measured in a Technicon AutoanalyzerlG. The standard error in this laboratory for the assay of both lipids is less than 2 %. In general adequate volumes of plasma were separated to provide enough lipid for reliable assay; however differences of 2 mg/lOO ml or less, though tabulated in the paper, should be regarded with caution though they represent duplicate determinations. For the measurement of lipoprotein protein, each lipoprotein fraction was recentrifuged
at the appropriate
density to reduce contamination
by other proteins.
The
cholesterol concentration was measured for a second time to correct for losses during the second centrifugation. The fractions were dialyzed against 5 mM ammonium bicarbonate solution and the protein content measured17. Plasma cholesterol esterification was assayed by the method of Stokke and Norumi* before and on several occasions during the heparin infusions in subjects 3, 5, 7, 8 and 9. Plasma FFA were also measured in these experiments by the method of Dole and Meinertzlg.
RESULTS
The changes in the lipoprotein cholesterol and triglyceride concentrations in consecutive samples is shown in Tables 2 and 3. Table 2 shows all the data for the 8 type IV and V subjects and Table 3 shows only the mean values for the 3 type III subjects. In VLDL, the triglyceride and to a lesser extent the cholesterol levels tended TABLE
3
CUMULATIVE INFUSION
Lipoprotein
MEAN
CHANGES
IN THE THREE TYPE
IN CHOLESTEROL
AND
TRIGLYCERIDE
III HYPERLIPOPROTEINEMIC
Cholesterol
(mg/lOO ml)
Triglyceride _ time (hr)
time (hr) 0
+
I
2
VLDL
95
-6
-19
-28
LDLI LDLz
72 126
-4 +4
9 +16
-
HDL
53
+2
+2+2+3
IN THE LIPOPROTEINS
9
4
0
-36
127
-13 + 19
DURING
HEPARIN
SUBJECTS
(mg/lOO ml)
+
I
2
4
-27
-52
-62
-70
28 23
8 -1-l
-11
-14
-16 -2
13
+1
0
0
0
557
HEPARIN INFUSION AND LIPOPROTEINS
3000
i
U/hr
6-o
HEPARIN
ISO
li0
INFUSION
2io
360
360
Minutes
Fig. 1. Changes in lipoprotein
cholesterol concentration
during the heparin infusion in subject 11.
to stabilize in the last 2 to 3 samples in about half the studies; in the remainder the rate of fall was diminishing towards the end of the infusions. In LDLi, both cholesterol and triglyceride concentrations fell in the three Type III subjects but rose in the other 8. The cholesterol levels rose in LDLz in all 11 subjects; in the eight Type IV and V subjects, by about as much as in LDLi. The triglyceride levels in LDLz rose substantially in 2 subjects with initially high plasma triglyceride concentrations, especially after 6 hr of heparin. The changes in HDL lipids were small, cholesterol
levels tending
to rise and only the mean values for the
group are shown. The progressive changes in lipoprotein cholesterol are shown for one subject (No. 11) in Fig. 1. Considering the mean falls in VLDL lipids and rises in LDL and HDL lipids in the eight Type IV and V subjects, only 2 ‘A of the triglyceride that was lost from VLDL after 4 hr was recovered in the other lipoproteins; the corresponding value at 6 hr was 4 %. The net balance for cholesterol after 4 hr of heparin was 44 ‘A for the Type IV and V and 49 % for the Type III subjects (for the latter, cholesterol loss was computed from VLDL + LDLi). By contrast there was no apparent loss of protein for the group of Type IV and V subjects, (though there was considerable individual variation): at 6 hr, mean loss of protein from VLDL was 93 mg/lOO ml and gain in LDLi + LDLs + HDL was 82 mg/lOO ml. The changes within the VLDL, studied in 4 subjects are shown in Table 4. Both cholesterol and triglyceride levels declined in the Sf > 400 fraction from the earliest sampling time. In the Sf lOWlO and Sf 20-100 fractions the cholesterol concentration tended to rise or remain unchanged initially and declined clearly only
558
Y.
HOMMA,P. J. NESTEL
TABLE 4 CHOLESTEROL
Subject
AND
TRIGLYCERIDE
CONCENTRATIONS
WITHIN
DURING
HEPARIN
Cholesterol (mgjlO0 ml)
Triglyceride (mg/lOO ml)
time (hr)
Time (hr)
0
1
2
4
6
6
277 200 274
267 201 868
222 206 -
1138
703
-
415
890
712
598
-
-
544 641 643 1169
393 579 853 1138
256 489 701 703
209 430 592 -
196 414 415
749
740
537
-
-
25 30
100 135 121
87 140
75 124
70 108
69 106
86
397
110 244
115 268
113 -
175
188
145
145
-
-
9 10 11
-
155
Mean
140
-
-
108
96
89 39 94
73 39
8 9 10 11
100400 lipoproteins 104 119 73 139 125 134 135 151 145 205 260 223
71 95 125 -
137
Mean
145
-
-
0
I
2
567 233 1781
325 222 1308
1169
INFUSIONS
4
Sf > 400 lipoproteins 116 96 85 51 43 42 167 116 98 229 177 162
8
VLDL
Sf
163
138
60 82
-
Sf 20-100 lipoproteins
8 9 10 11
25 35 23
30 33 25
25 36 30
91
102
124
-
Mean
43
47
53
-
29 32 37
-
-
during the latter part of the infusion in the Sf 100-400 fraction. The findings in subject 11 are shown in Fig. 2. VLDL subfractionation was carried out in one Type III subject (No. l), except that the isolated classes were Sf 12-20, 20-100 and > 100. The fall in the cholesterol concentration in the Sf > 100 fraction was associated with a rise in the Sf 12-20 fraction (Fig. 2). The in vitro esterification of plasma cholesterol, measured during 5 heparin infusions, is shown in Fig. 3. There was an early suppression of in vitro esterification in every subject, followed by a gradual recovery, which was partial or complete in 4 studies. The changes in plasma FFA concentration (also shown in Fig. 4) appeared not to be related to the inhibition of cholesterol esterification”, as suggested by Rutenberg, Lacko and Soloff.
* In further studies, the addition of bovine albumin to the assay medium did not reverse the suppression. The addition of heparin to the assay medium of the preinfusion samples, in concentrations of 0.2, 2 and 20 U/ml, did not inhibit cholesterol esterification.
559
HEPARIN INFUSION AND LIPOPROTEINS Subject
II
(Type 5 1 U hr
3000
Subject
HEPARIN
I (Type
3
)
HEPARIN 1500
0
INFUSION
INFUSION
U hr
I
I
I
I
30
60
120
3000
U hr
I
180
I 240
Minutes
Fig. 2. Changes in cholesterol heparin infusions.
concentration
in subfractions
of very low density lipoprotein
during
DISCUSSION
The main purpose of these studies was to obtain semi-quantitative estimates of the intravascular redistribution of VLDL constituents. Others have shown conclusively that a single injection of heparin which accelerates the catabolism of VLDL, will increase the cholesterol and protein6 content of LDL and HDL. These findings, together with the precursor-product characteristics of triglyceride fatty acidslJ and
Y. HOMMA, P. J. NESTEL
MINUTES
MINUTES
Fig. 3. Plasma cholesterol esterification measured in vitro at intervals during the heparin infusions. The changes in plasma free fatty levels are shown in the upper panel.
of protein3 in VLDL and LDL have shown that at least part of the catabolized VLDL particle remains in the circulation as LDL. The extent of this conversion is unclear; the protein moiety or at least some of the peptides appear to interchange between VLDL and HDL during triglyceride transport without being lost from the circulation2a. By contrast triglyceride is largely removed as VLDL are broken down, although some is recovered in smaller lipoproteins. The fate of VLDL cholesterol is unclear. It is known that the cholesterol in alimentary particles, such as chylomicrons, is cleared more slowly from the circulation of animals than the triglyceride 4. A proportion of the cholesterol circulates within reconstituted remnants of chylomicrons and is removed in the liver495. It is probable that at least some VLDL cholesterol transfers to LDL in view of the reciprocal relationship between the amounts of cholesterol in these two classes of lipoproteins that occurs whenever VLDL levels are lowered 21. The present studies suggest that about half of VLDL cholesterol may transfer to other lipoproteins during heparin infusions (mostly to LDLr and LDLe) (Table 2). This contrasts with the simultaneous loss from the circulation of more than 95% of VLDL triglyceride and the apparent retention of most of the protein. These estimates are clearly only approximations: in the first place a new steady state was not always achieved for VLDL lipid
HEPARIN INFUSION
concentrations
561
AND LIPOPROTEINS
even after 6 hr and secondly,
little is known about the effect of heparin
on the metabolism of other lipoproteins. However, it is not unreasonable to assume that the loss of cholesterol from LDL during the infusion would be small relative to the gain from VLDL, since the turnover of plasma cholesterol pool is measured in terms of daysz2. It is also unlikely from elsewhere,
since other
that LDL cholesterol
studies
with labeled
had been substantially
VLDL
suggested that newly synthesized cholesterol enters The importance of the class of lipoprotein
derived
and LDL cholesterol
have
plasma mainly within VLDL23. with density 1.006-1.019 (also
known as LDLr or Sf 12-20 or intermediate density lipoprotein) has also been demonstrated in these studies (Table 2). Heparin accelerated the loss of lipid from this fraction in the three Type III subjects in whom there was an excess of lipoprotein of this class. In each of the other subjects, this lipoprotein was enriched in both lipid and protein difference injection
during
heparin
infusion
as has been shown
by othersss~sJ5.
between Type III and Type IV subjects has also been observed of heparin26.
lipoproteinemia
Catabolism
can therefore
of the accumulated
be stimulated,
though
lipoprotein
This
after a single
in Type III hyper-
the heparin-induced
increase
in
intravascular lipolytic activity cannot be regarded as entirely physiological. It is furthermore not certain which of the heparin-released lipases is responsible for the accelerated interconversion of lipoproteins. The present studies also confirm the interconversion of one species of VLDL into another (Table 4). This has been previously demonstrated by isotope kinetic studies for triglyceride fatty acids7, esterified cholestero12* and protein7. The frequent sampling
during
the constant
infusion
of heparin
clearly shows the increase in choles-
terol concentration in the smaller species as the larger species are catabolized (Fig. 2). It is interesting to compare the findings in Type III with the Type IV and V subjects. The characteristic accumulation of lipoprotein in Type III hyperlipoproteinemia is within the Sf 20-100 range, which, as shown in Fig. 2, was susceptible to rapid catabolism with heparin in the Type III subject. In the four Type IV and V subjects, the cholesterol concentration in the Sf 20-100 fraction either rose or changed little (Table 4, Fig. 2), a finding that has also been previously observed for the triglyceride24 and protein3 moieties. Eisenberg et al.3 have therefore suggested that heparininduced lipolysis may not operate effectively on lipoprotein species smaller than Sf 60. This is not entirely borne out by the present studies and in any case, the faillure for the concentration of lipid or protein to change may simply indicate a balance between the influx from the larger VLDL and efflux into the LDL class. We have confirmed the findings of Rutenberg et al.9 that the intravenous injection of heparin suppresses the esterification of plasma cholesterol measured in vitro. However, our studies showed a partial or complete recovery in 4 of 5 studies despite the contirming infusion of heparin (Fig. 3). The very rapid suppression makes it unlikely that heparin prevented the release of enzyme from the liver. The possibility of an artefactual effect, other than through the direct action of heparin which has been ruled out, is supported by two findings. Firstly, the ratios of free:esterified cholesterol were unchanged in the plasma lipoproteins throughout the infusions.
562
Y. HOMMA, P. J. NESTEL
Secondly, unpublished studies in 3 other subjects showed that the in vivo conversion of free into esterified cholesterol, following the injection of radiomevalonatezg, was not reduced during a 3-day infusion of heparin (1000 units/hr). It is possible that heparin altered the availability of lecithin substrate. ACKNOWLEDGEMENTS
Two subjects with Type III hyperlipoproteinemia were kindly referred for study by Professor R. B. Blacket of the University of New South Wales. Technical help was provided
by Mrs Geraldine
Power.
REFERENCES 1 HAVEL, R. J., Conversion of plasma free fatty acids into triglycerides of plasma lipoprotein fractions in man, Merabolism, 10 (1961) 1031. 2 QUARFORDT, S. H., FRANK, A., SHAMES, D. M., BERMAN, M. AND STEINBERG, D., Very low density lipoprotein triglyceride transport in Type IV hyperlipoproteinemia and the effect of carbohydraterich diets, J. Clin. Invest., 49 (1970) 2281. 3 EISENBERG, S., BILHEIMER, D. W., LEVY, R. I. AND LINDGREN, F. T., On the metabolic conversion of human plasma very low density lipoprotein to low density lipoprotein, Biochim. Bioghys. Acta, 326 (1973) 361. of constituent lipids of dog cylomicrons, 4 NESTEL, P. J., HAVEL, R. J. AND BEZMAN, A., Metabolism J. Clin. Invest., 42 (1963) 1313. 5 REDGRAVE, T. G., Formation of cholesterol ester-rich particulate lipid during metabolism of cylomicrons, J. Clin. Invest., 49 (1970) 465. 6 LAROSA, J. C., LEVY, R. I., BROWN, W. V., AND FREDRICKSON, D. S., Changes in high-density lipoprotein composition after heparin-induced lipolysis, Am. J. Physiol., 220 (1971) 785. 7 NICHOLS, A. V., STRISOWER, E. H., LINDGREN, F. T., ADAMSON, G. L. AND COGGIOLA, E. L., Analysis of change in ultracentrifugal lipoprotein profiles following heparin and ethyl-p-phenC/in. Chim. Acta, 20 (1968) 277. oxyisobutyrate administration, 8 GLOMSET, J. A. AND NORUM, K. R., The metabolic role of lecithin-cholesterol acyltransferase Perspectives from pathology, Adv. Lipid Res., 11 (1973) 1. of lecithin-cholesterol acyltransferase 9 RUTENBERG, H. L., LACKO, A. G. AND SOLOFF, A., Inhibition following intravenous administration of heparin in man, Eiochim. Bioghys. Actu, 326 (1973) 419. 10 KOHN, J., A lipoprotein staining method for zone electrophoresis, Nature, 189 (1961) 312. 11 HAZZARD, W. R., PORTE, JR., D. AND BIERMAN, E. L., Abnormal lipid composition of very low density lipoproteins in diagnosis of broad-beta disease (type III hyperlipoproteinemia), Metabolism, 21 (1972) 1009. 12 PORTE JR. D. AND BIERMAN, E. L., The effect of heparin infusion on plasma triglyceride in vivo and in vitro with a method for calculating triglyceride turnover, J. Lab. Clin. Med., 73 (1969) 631. 13 HAVEL, R. J., EDER, H. A. AND BRAGDON, J. H., The distribution and chemical composition of ultracentrifugally separated lipoproteins in human serum, J. C&z. Invest., 34 (1955) 1345. 14 GUSTAFSSON, A., ALAUPOVIC, P. AND TIERMAN, R. H., Studies of the composition of serum lipoproteins - Isolation, purification and characterization of VLDL of human serum, Biochem., 4 (1965) 596. 15 DOLE, V. P., A relation between nonesterified fatty acids in plasma and the metabolism of glucose, J. Clin. Invest., 35 (1956) 150. 16 Autoanalyzer Manual, Technicon Instruments Corporation, New York, 1971. 17 LOWRY, 0. H., ROSENBROUGH, N. J., FAN, A. L. AND RANDALL, R. J., Protein measurement with the Folin-phenol reagent, J. Biol. Chem., 193 (1951) 265. 18 STOKKE, K. T. AND NORUM, K. R., Determination of lecithin-cholesterol acyltransferase in human blood plasma, Scund. J. Clin. Lab. Invest., 27 (1971) 21.
HEPARIN INFUSION AND LIPOPROTEINS
563
of long chain fatty acids in plasma and 19 DOLE, V. P. AND MEINERTZ,H., Microdetermination tissues, J. Biol. C&m., 235 (1960) 2595. 20 HAVEL, R. J., KANE, J. P. AND KASHYAP,M. L., Interchange of apolipoproteins between cyiomicrons and high density lipoproteins during alimentary lipemia in man, J. Clin. Invest., 52 (1973) 32. 21 WILSON, D. E. AND LEES, R. S., Metabolic relationships among the plasma lipoproteins - Reciprocal changes in the concentrations of very low and low density lipoproteins in man, J. C/in. Invest., 51 (1972) 1051. 22 NESTEL,P. J., Cholesterol turnover in man, Adv. Lipid Rex, 8 (1970) 1. 23 CLIFTON-BLIGH,P., MILLER, N. E. AND NESTEL,P. J., Changes in plasma lipoprotein-lipids in hypercholesterolemic patients treated with the bile acid-sequestering resin, colestipol, Clin. Sci. Molec. Med., 47 (1974) 547. 24 BARTER,P. J. AND NESTEL, P. J., The distribution of triglyceride in subclasses of very low density plasma lipoproteins, J. Lab. Clin. Med., 76 (1970) 925. 25 SHORE,B. AND SHORE,V., Some physical and chemical properties of the lipoproteins produced by lipolysis of human serum .Sf 20-400 lipoproteins by post-heparin plasma, J. Atheroscler. Res., 2 (1962) 104. 26 QUARFORDT,S., LEVY, R. I., AND FREDRICKSON,D. S., On the lipoprotein abnormality in Type III hyperlipoproteinemia, J. Clin. Invest., 50 (1971) 754. 21 BARTER, P. J. AND NESTEL, P. J., Precursor-product relationship between pools of very low density lipoprotein triglyceride, J. Clin. Znvest., 5 (1972) 174. 28 BARTER,P. J., Origin of esterified cholesterol transported in the very low density lipoproteins of human plasma, J. Lipid Res., 15 (1974) 1 I. 29 NESTEL,P. J. ANDMONGER,E. A., Turnover of plasma esterified cholesterol in normocholesterolemic and hypercholesterolemic subjects and its relation to body build, J. Clin. Invest.. 46 (1967) 967.
22 (1975) 551-563
0 Elsevier Scientific Publishing Company, Amsterdam - Printed in The Netherlands
CHANGES
IN PLASMA
CONSTANT
Y. HOMMA Department
LIPOPROTEIN
INFUSIONS
AND
CONSTITUENTS
551
DURING
OF HEPARIN
P. J. NESTEL
of Clinical Science,
The Australian
National
University,
Canberra
(Australia)
(Received February 25th, 1975) (Accepted April 14th, 1975)
SUMMARY
Constant infusions of heparin of 4 to 6 hours’ duration were used to estimate the transfer of very low density lipoprotein constituents to other plasma lipoproteins. Eleven subjects were studied, 3 with Type III and the remainder with either Type IV or V hyperlipoproteinemia. Whereas only about 5 % of the triglyceride lost from VLDL was recovered in the other lipoproteins, 44% of the cholesterol was retained in the circulation, in equal amounts within low density (d 1.019-1.063) and intermediate density (d 1.006-l .019) lipoproteins. of protein, more than half of that originally lipoprotein.
By contrast, there was no apparent loss in VLDL being recovered in high density
In subjects with Type III hyperlipoproteinemia, lipid and protein was lost from the intermediate density lipoprotein as well as from VLDL. In subjects with marked hypertriglyceridemia, cholesterol became redistributed from larger to smaller VLDL. The esterification of plasma cholesterol as measured in vitro, was apparently suppressed during the early phase of the heparin infusions, but tended to recover later.
Key words:
Cholesterol ester$cation protein changes
- Heparin - Hyperlipidaemic subjects - Lipo-
INTRODUCTION
The plasma lipoproteins are metabolically interrelated. There is transfer and exchange of the individual lipid and protein moieties among the lipoproteins as well as conversion of one whole class of lipoprotein into another. It is probable that low
552
Y. HOMMA, P. J. NESTEL
density lipoproteins (LDL) are derived partly or entirely from the very low density lipoproteins (VLDL). This occurs during the breakdown of VLDL which is brought about by the hydrolysis and removal of the triglyceride; smaller species of VLDL emerge which in turn give rise to LDL. The evidence for this is based on the precursorproduct characteristics of the kinetics of the triglyceride fatty acid132 and proteins constituents of the respective lipoproteins. The esterified cholesterol of chylomicrons is not removed from the circulation as rapidly as the triglyceride4 but is at first retained within smaller lipoproteins or “remants” and subsequently removed in the liver4. The rate of transfer of lipid and protein from larger to smaller lipoproteins can be accelerated
with heparin
which stimulates
the catabolism
of VLDLs,6,7;
studies
of this sort have provided qualitative evidence about the metabolic fate of lipoprotein constituents. In this study we have used prolonged constant infusions of heparin which have allowed more accurate estimates of the net transfer of VLDL components to LDL and HDL. The results show that under these conditions
less than half of the
cholesterol lost from VLDL is recovered in the smaller lipoproteins, whereas most of the protein appears to be retained in the circulation. The reconstitution of intermediate classes of lipoproteins that takes place following
the removal
of triglyceride
from within the core of VLDL appears to require
the removal of excess surface membrane, a process that involves the enzyme lecithinof plasma cholesterol acyltransferase 8. We have measured the in vitro esterification cholesterol, the major function of the enzyme, since it has been reported that, perhaps paradoxically,
this is inhibited
by heparing.
METHODS
Eleven hyperlipidemic subjects were studied (Table 1). The lipoprotein phenotype was determined by plasma lipid electrophoresis on cellulose acetatetO, and, especially with the Type III patients, from the distribution of cholesterol and triglyceride among the lipoproteinstl. The subjects were not taking lipid-lowering drugs but several had been dieting for some time. The plasma lipids were therefore sometimes lower at the time of study than initially. All subjects fasted overnight, but most were given a small fat-free meal during the infusion. Sodium heparin, diluted in 0.15 M NaCl solution was infused intravenously at a constant rate to deliver 3000 international units/hr for 4-6 hr. Subjects 1 and 2 received only 1500 units of heparin for the first 165 min and then 3000 units for the last 2 hr. Two samples of blood were taken before the heparin infusion and a further 5 to 8 samples at approximately 60-min intervals during the infusion. Blood was placed into chilled tubes that contained 0.5 mg paraoxon for each ml blood to inhibit continued lipolysis in vitrolz. Plasma was separated at 4°C. Four major classes of
age (yr)
39 44 59 41 43 23 51 54 36 26 35
1 2 3 4 5 6 7 8 9 10 11
M M F M M M M M M M F
sex
AND THEIR
No.
Subjects
THE SUBJECTS
TABLE 1
76 82 75 85 85 73 73 90 101 89 65
(kg)
Weight
CHOLESTEROL
111 III III IV IV IV IV-V v v V V
Type of hyperlipoproteinemia
AND TRIGLYCERIDE
81 58 148 49 102 126 175 247 225 327 517
Chol
VLDL
120 72 189 200 465 498 1344 1213 1013 2555 2557
TG
CONCENTRATIONS
42 56 117 10 12 12 4 12 18 11 14
Chol
LDLl
25 29 30 13 18 16 9 17 27 19 38
TG
IN LIPOPROTEINS
112 140 125 87 124 43 46 48 45 28 91
Chol
LDLz
(IIlg/l(bo
Id)
20 23 27 19 32 10 35 20 41 40 56
TG
61 60 38 20 46 39 14 48 33 16 18
Chol
HDL
12 15 12 7 17 10 16 26 30 26 22
TG
554
Y. HOMMA, P. J. NESTEL
TABLE 2 CUMULATIVE
Subjects
4 5 6 7 8 9 10 11
CHANGES
IN LIPOPROTEIN
PROTEIN
CONCENTRATIONS
DURING
rime (hr)
time (hr)
0
3
1
2
3
4
VLDL 49 102 126 175 247 225 327 517
3 -14 - 6 - 6 + 1 -20 -35 +48
8 -15 -11 - 6 1 -22 -34 +23
-14 -14 -31 -12 -62 -31 -52 - 9
-18 -34 -35 -27 -22 -54 -30
- 22 34 - 48 - 50 57 58 - 69 -100
-
4
-
-28
-31
-
0
+2 +3 1-6
0
0 + 3 +1 +1 + 5
+3 +4 +9 +1 + 7 +1 +2 +13
+5 t-8 +7 +4 +16 f2 +4 $31
+ 4 +I2 + 7+13 + 6 + 18 + 23 + + 2+ 3f + 6+ 6+ + 45 + 52 +
24 5 7 54
0
+
+
f
+
22
LDLI 10 12 12 4 12 18 11 14
+1 +2 -
0 1 0
+1
LDLz 87 +2 124 +4 43 +7 46 -4 48 -5 45 -3 28 -1 56 1
Mean
Mean
AND
Triglyceride (mg/lOO ml)
Mean
4 5 6 7 8 9 10 11
TRIGLYCERIDE
Cholesterol
Mean
4 5 6 7 8 9 10 11
CHOLESTEROL,
HDL 29
9
2
5
9
5
-
12 +
0
3
1
- 87 - 72 60 -137
200 465 498 1344 1213 1013 2555 2557
26 53 - 49 -276 -384 87 -130 t-118
52 - 70 - 90 -417 -407 70 -285 -177
-110
-196
49
64 58 52 -125
54 -
6
70 -
19 +
89
+7 +2 +6 -8 +l -5 +3 +I3
+7 +9 +ll -1 +4 +2 +l +16
+4 -6 +9 +3 +2 f3 f38
+16 + 9 + 9+15 +12 + 8+ 7+17 4+ 4+ 6 + 4+ 5+ 6 + 27 + 43 + 57
0
+2
+6
+6
+10+15+21
0
+1
+1
+1
+
0
2
13-l 18 16 9 17 27 19 38
+ -
1+ 4+ 41+
0 4 9 5 4
+
0+ 2+
1 3
0+
9
0
19 32 10 35 20 41 40 91
19
-
+3
-
230-t 7 + 531+ 4 +
1 4 1 12 4 7 3 13
-
3+
1
+
1-k
3
555
HEPARIN INFUSION AND LIPOPROTEINS
HEPARININFUSIONSINEIGHTTYPEIVANDVHYPERLIPOPROTEINEMICSUBJECTS
Protein
time 2
-
3
66
4
5
6
0
-
74
-
81
47
-122
-
144
-
142
81
-145
-
151
-
151
-
-677
-
873
-
922
-1027
-
666
-
685
-
-193
-
199
-
272
-
319
-
-663
-
788
-
980
-
997
-740
-1110
-1349
-393
-
-
fl
+
2+
1
9
+2
+
5+
9
+9 -2
+ +
5+ 2+
2+ 3
8
9 10
+ +
23 l+
7 + 1+
9 l+
+ +
5+ 26 +
9+ 34 +
943
+
7 43
14
+
16
-538
+ -1
8
+3 +
13
477
+
570
(hr) 3
I
2
+14
-
-17
-22
-31
3
4
6
5
6
-5 -27
97
-21
725
175
-80
-
91
-
93
285
148
-25
-
20
-
27
-1135
261
-53
-
66
-
91
-1332
-1390
274
-67
-102
-161
-
-
-45
-
-
93
-3542
-1050
752
+
917
23 6
-3
63
j-2
-2
+2
-3
-2
+5
+5
+
5+9
8
+4
+
10
+
12
14
+2
+
5
+
6
13
-2
-
1
+
6
16
+7
+
24
+
35
$4
+
9
+
15
+
8+
8
-5
-
2
0
63
+8
+3
-
2+
3
+1
+
+3 -1
f7 -1-
+
2+ 6 -
115 38 15
46
-4
0
+
16
4
+10 -4
+ -4
20
+ -6
23
+15
+
46
+
48
+4
+
12
+
20
+12
+
37
+
46
+4
+1-5 +
+ 10
+
+
153
2+ 14
3
1+
4+
6
2-
5+
2+
2+ 32
8+ +
13
+1
+
4+
1+
+1
+
1+
1
+
7
30 56
30
+
42
114
9
+
17
+
3
5-t +
132
+5 -
3+
+
19
+
18 3
Y. HOMMA, I’. J. NESTEL
556 lipoproteins
were separated
in all studies 13: VLDL
(d <
1.006), LDLi
(d 1.006-
1.019), LDLa (d 1.019-1.063) and HDL (d 1.063-1.021). In subjects 8-l 1, who showed the highest plasma triglyceride values, the VLDL were further fractionated into S, > 400, Sf 100-400 and S, 20-100 subclassesi4. The mean
recoveries
of lipoprotein
cholesterol
and triglyceride
during
ultra-
centrifugal separation were 86 y0 and 83 % respectively. Most of this loss occurred with separation of VLDL. The values have not been corrected for losses. Lipids were extracted in Dole’s solutionlj and the cholesterol and triglyceride concentrations measured in a Technicon AutoanalyzerlG. The standard error in this laboratory for the assay of both lipids is less than 2 %. In general adequate volumes of plasma were separated to provide enough lipid for reliable assay; however differences of 2 mg/lOO ml or less, though tabulated in the paper, should be regarded with caution though they represent duplicate determinations. For the measurement of lipoprotein protein, each lipoprotein fraction was recentrifuged
at the appropriate
density to reduce contamination
by other proteins.
The
cholesterol concentration was measured for a second time to correct for losses during the second centrifugation. The fractions were dialyzed against 5 mM ammonium bicarbonate solution and the protein content measured17. Plasma cholesterol esterification was assayed by the method of Stokke and Norumi* before and on several occasions during the heparin infusions in subjects 3, 5, 7, 8 and 9. Plasma FFA were also measured in these experiments by the method of Dole and Meinertzlg.
RESULTS
The changes in the lipoprotein cholesterol and triglyceride concentrations in consecutive samples is shown in Tables 2 and 3. Table 2 shows all the data for the 8 type IV and V subjects and Table 3 shows only the mean values for the 3 type III subjects. In VLDL, the triglyceride and to a lesser extent the cholesterol levels tended TABLE
3
CUMULATIVE INFUSION
Lipoprotein
MEAN
CHANGES
IN THE THREE TYPE
IN CHOLESTEROL
AND
TRIGLYCERIDE
III HYPERLIPOPROTEINEMIC
Cholesterol
(mg/lOO ml)
Triglyceride _ time (hr)
time (hr) 0
+
I
2
VLDL
95
-6
-19
-28
LDLI LDLz
72 126
-4 +4
9 +16
-
HDL
53
+2
+2+2+3
IN THE LIPOPROTEINS
9
4
0
-36
127
-13 + 19
DURING
HEPARIN
SUBJECTS
(mg/lOO ml)
+
I
2
4
-27
-52
-62
-70
28 23
8 -1-l
-11
-14
-16 -2
13
+1
0
0
0
557
HEPARIN INFUSION AND LIPOPROTEINS
3000
i
U/hr
6-o
HEPARIN
ISO
li0
INFUSION
2io
360
360
Minutes
Fig. 1. Changes in lipoprotein
cholesterol concentration
during the heparin infusion in subject 11.
to stabilize in the last 2 to 3 samples in about half the studies; in the remainder the rate of fall was diminishing towards the end of the infusions. In LDLi, both cholesterol and triglyceride concentrations fell in the three Type III subjects but rose in the other 8. The cholesterol levels rose in LDLz in all 11 subjects; in the eight Type IV and V subjects, by about as much as in LDLi. The triglyceride levels in LDLz rose substantially in 2 subjects with initially high plasma triglyceride concentrations, especially after 6 hr of heparin. The changes in HDL lipids were small, cholesterol
levels tending
to rise and only the mean values for the
group are shown. The progressive changes in lipoprotein cholesterol are shown for one subject (No. 11) in Fig. 1. Considering the mean falls in VLDL lipids and rises in LDL and HDL lipids in the eight Type IV and V subjects, only 2 ‘A of the triglyceride that was lost from VLDL after 4 hr was recovered in the other lipoproteins; the corresponding value at 6 hr was 4 %. The net balance for cholesterol after 4 hr of heparin was 44 ‘A for the Type IV and V and 49 % for the Type III subjects (for the latter, cholesterol loss was computed from VLDL + LDLi). By contrast there was no apparent loss of protein for the group of Type IV and V subjects, (though there was considerable individual variation): at 6 hr, mean loss of protein from VLDL was 93 mg/lOO ml and gain in LDLi + LDLs + HDL was 82 mg/lOO ml. The changes within the VLDL, studied in 4 subjects are shown in Table 4. Both cholesterol and triglyceride levels declined in the Sf > 400 fraction from the earliest sampling time. In the Sf lOWlO and Sf 20-100 fractions the cholesterol concentration tended to rise or remain unchanged initially and declined clearly only
558
Y.
HOMMA,P. J. NESTEL
TABLE 4 CHOLESTEROL
Subject
AND
TRIGLYCERIDE
CONCENTRATIONS
WITHIN
DURING
HEPARIN
Cholesterol (mgjlO0 ml)
Triglyceride (mg/lOO ml)
time (hr)
Time (hr)
0
1
2
4
6
6
277 200 274
267 201 868
222 206 -
1138
703
-
415
890
712
598
-
-
544 641 643 1169
393 579 853 1138
256 489 701 703
209 430 592 -
196 414 415
749
740
537
-
-
25 30
100 135 121
87 140
75 124
70 108
69 106
86
397
110 244
115 268
113 -
175
188
145
145
-
-
9 10 11
-
155
Mean
140
-
-
108
96
89 39 94
73 39
8 9 10 11
100400 lipoproteins 104 119 73 139 125 134 135 151 145 205 260 223
71 95 125 -
137
Mean
145
-
-
0
I
2
567 233 1781
325 222 1308
1169
INFUSIONS
4
Sf > 400 lipoproteins 116 96 85 51 43 42 167 116 98 229 177 162
8
VLDL
Sf
163
138
60 82
-
Sf 20-100 lipoproteins
8 9 10 11
25 35 23
30 33 25
25 36 30
91
102
124
-
Mean
43
47
53
-
29 32 37
-
-
during the latter part of the infusion in the Sf 100-400 fraction. The findings in subject 11 are shown in Fig. 2. VLDL subfractionation was carried out in one Type III subject (No. l), except that the isolated classes were Sf 12-20, 20-100 and > 100. The fall in the cholesterol concentration in the Sf > 100 fraction was associated with a rise in the Sf 12-20 fraction (Fig. 2). The in vitro esterification of plasma cholesterol, measured during 5 heparin infusions, is shown in Fig. 3. There was an early suppression of in vitro esterification in every subject, followed by a gradual recovery, which was partial or complete in 4 studies. The changes in plasma FFA concentration (also shown in Fig. 4) appeared not to be related to the inhibition of cholesterol esterification”, as suggested by Rutenberg, Lacko and Soloff.
* In further studies, the addition of bovine albumin to the assay medium did not reverse the suppression. The addition of heparin to the assay medium of the preinfusion samples, in concentrations of 0.2, 2 and 20 U/ml, did not inhibit cholesterol esterification.
559
HEPARIN INFUSION AND LIPOPROTEINS Subject
II
(Type 5 1 U hr
3000
Subject
HEPARIN
I (Type
3
)
HEPARIN 1500
0
INFUSION
INFUSION
U hr
I
I
I
I
30
60
120
3000
U hr
I
180
I 240
Minutes
Fig. 2. Changes in cholesterol heparin infusions.
concentration
in subfractions
of very low density lipoprotein
during
DISCUSSION
The main purpose of these studies was to obtain semi-quantitative estimates of the intravascular redistribution of VLDL constituents. Others have shown conclusively that a single injection of heparin which accelerates the catabolism of VLDL, will increase the cholesterol and protein6 content of LDL and HDL. These findings, together with the precursor-product characteristics of triglyceride fatty acidslJ and
Y. HOMMA, P. J. NESTEL
MINUTES
MINUTES
Fig. 3. Plasma cholesterol esterification measured in vitro at intervals during the heparin infusions. The changes in plasma free fatty levels are shown in the upper panel.
of protein3 in VLDL and LDL have shown that at least part of the catabolized VLDL particle remains in the circulation as LDL. The extent of this conversion is unclear; the protein moiety or at least some of the peptides appear to interchange between VLDL and HDL during triglyceride transport without being lost from the circulation2a. By contrast triglyceride is largely removed as VLDL are broken down, although some is recovered in smaller lipoproteins. The fate of VLDL cholesterol is unclear. It is known that the cholesterol in alimentary particles, such as chylomicrons, is cleared more slowly from the circulation of animals than the triglyceride 4. A proportion of the cholesterol circulates within reconstituted remnants of chylomicrons and is removed in the liver495. It is probable that at least some VLDL cholesterol transfers to LDL in view of the reciprocal relationship between the amounts of cholesterol in these two classes of lipoproteins that occurs whenever VLDL levels are lowered 21. The present studies suggest that about half of VLDL cholesterol may transfer to other lipoproteins during heparin infusions (mostly to LDLr and LDLe) (Table 2). This contrasts with the simultaneous loss from the circulation of more than 95% of VLDL triglyceride and the apparent retention of most of the protein. These estimates are clearly only approximations: in the first place a new steady state was not always achieved for VLDL lipid
HEPARIN INFUSION
concentrations
561
AND LIPOPROTEINS
even after 6 hr and secondly,
little is known about the effect of heparin
on the metabolism of other lipoproteins. However, it is not unreasonable to assume that the loss of cholesterol from LDL during the infusion would be small relative to the gain from VLDL, since the turnover of plasma cholesterol pool is measured in terms of daysz2. It is also unlikely from elsewhere,
since other
that LDL cholesterol
studies
with labeled
had been substantially
VLDL
suggested that newly synthesized cholesterol enters The importance of the class of lipoprotein
derived
and LDL cholesterol
have
plasma mainly within VLDL23. with density 1.006-1.019 (also
known as LDLr or Sf 12-20 or intermediate density lipoprotein) has also been demonstrated in these studies (Table 2). Heparin accelerated the loss of lipid from this fraction in the three Type III subjects in whom there was an excess of lipoprotein of this class. In each of the other subjects, this lipoprotein was enriched in both lipid and protein difference injection
during
heparin
infusion
as has been shown
by othersss~sJ5.
between Type III and Type IV subjects has also been observed of heparin26.
lipoproteinemia
Catabolism
can therefore
of the accumulated
be stimulated,
though
lipoprotein
This
after a single
in Type III hyper-
the heparin-induced
increase
in
intravascular lipolytic activity cannot be regarded as entirely physiological. It is furthermore not certain which of the heparin-released lipases is responsible for the accelerated interconversion of lipoproteins. The present studies also confirm the interconversion of one species of VLDL into another (Table 4). This has been previously demonstrated by isotope kinetic studies for triglyceride fatty acids7, esterified cholestero12* and protein7. The frequent sampling
during
the constant
infusion
of heparin
clearly shows the increase in choles-
terol concentration in the smaller species as the larger species are catabolized (Fig. 2). It is interesting to compare the findings in Type III with the Type IV and V subjects. The characteristic accumulation of lipoprotein in Type III hyperlipoproteinemia is within the Sf 20-100 range, which, as shown in Fig. 2, was susceptible to rapid catabolism with heparin in the Type III subject. In the four Type IV and V subjects, the cholesterol concentration in the Sf 20-100 fraction either rose or changed little (Table 4, Fig. 2), a finding that has also been previously observed for the triglyceride24 and protein3 moieties. Eisenberg et al.3 have therefore suggested that heparininduced lipolysis may not operate effectively on lipoprotein species smaller than Sf 60. This is not entirely borne out by the present studies and in any case, the faillure for the concentration of lipid or protein to change may simply indicate a balance between the influx from the larger VLDL and efflux into the LDL class. We have confirmed the findings of Rutenberg et al.9 that the intravenous injection of heparin suppresses the esterification of plasma cholesterol measured in vitro. However, our studies showed a partial or complete recovery in 4 of 5 studies despite the contirming infusion of heparin (Fig. 3). The very rapid suppression makes it unlikely that heparin prevented the release of enzyme from the liver. The possibility of an artefactual effect, other than through the direct action of heparin which has been ruled out, is supported by two findings. Firstly, the ratios of free:esterified cholesterol were unchanged in the plasma lipoproteins throughout the infusions.
562
Y. HOMMA, P. J. NESTEL
Secondly, unpublished studies in 3 other subjects showed that the in vivo conversion of free into esterified cholesterol, following the injection of radiomevalonatezg, was not reduced during a 3-day infusion of heparin (1000 units/hr). It is possible that heparin altered the availability of lecithin substrate. ACKNOWLEDGEMENTS
Two subjects with Type III hyperlipoproteinemia were kindly referred for study by Professor R. B. Blacket of the University of New South Wales. Technical help was provided
by Mrs Geraldine
Power.
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