355
Atherosclerosis, 29 (1978) 355-362 0 Elsevier/North-Holland Scientific Publishers,
LIPID AND LIPOPROTEIN SHUNT IN SWINE
A.C. NESTRUCK,
(Received (Revised, (Accepted
SECRETION
M. BERGSETH,
Znstitut de Recherches H2 W 1 R 7 (Canada)
Ltd.
M. BIDALLIER,
FOLLOWING
J. DAVIGNON
PORTACAVAL
and Y.L.
MARCEL
Cliniques de Mont&al, 110 avenue des Pins ouest, Montrkal
6 September, 1977) received 16 January, 25 January, 1978)
1978)
Lipid and lipoprotein secretion were studied over 24 h in normal and portacaval shunted (PCS) fasting miniature swine after an infusion of Triton WR1339 and [3H]amino acid mixture. The plasma triglyceride secretion rate and the secretion of triglyceride, total cholesterol and protein in the very low density lipoprotein fraction (VLDL) were linear for about 9 h after Triton. No net catabolism of the VLDL appeared to take place over the 24 h. Due to large variation, differences in the absolute secretion rates of plasma triglyceride and of the VLDL constituents could not be shown. However, the secretion of VLDL cholesterol in the PCS group appeared to be lower than that of the sham group when corrected for metabolic body size and plasma volume. Key words:
Hypercholesterolemia - Hypocholesterolemia - Portacaval shunt WR-1399 - Very low density lipoprotein secretion
7’riton
Introduction The cholesterol lowering action of portacaval shunt surgery, first used clinically by Starzl [l] and subsequently by others [2-41 for the treatment of homozygous familial hypercholesterolemia, has been investigated in numerous studies using animal models [5--111. A coherent concept for its mechanism This research was supported by grants from the Stewart Macdonald Foundation, The Quebec Heart Foundation and the Medical Research Council of Canada (MT-4011 and MA-5427). A.C.N. is the holder of a research scholarship from the Conseil de la Recherche en Sante du QuBbec.
356
however, has not been reached. The proposal that a decreased hepatic synthesis of cholesterol is induced by the shunt [ 5,6] has not been confirmed by others [S]. Further, it has been found that a reduced hepatic cholesterol synthesis does not necessarily result in a decreased circulating cholesterol level [ 51. The hypocholesterolemic effect of the shunt has been shown to result from decreased LDL levels in man [ 31 and swine [ 111. The metabolism of LDL has also been investigated and a decreased production of LDL rather than an increased removal was demonstrated in man [ 31 and swine [ 71. Suggestions of a reduced hepatic synthesis and/or secretion of lipoproteins [ 5,7] are also derived in part from the concept of diverted and diminished hepatotrophic factors [ 12,131, and from the indirect evidence of a hypoalbuminemia in portacaval shunted (PCS) pigs [ 11,141 and rats 191, and the histological changes of hepatocytes in one PCS patient [ 151 and shunted experimental animals [5,12] which indicate decreased lipoprotein secretion. The present study was designed to investigate the effect of PCS on VLDL secretion in swine by use of an infusion of the non-ionic detergent Triton WR1339. The accumulating VLDL were considered to be newly secreted hepatic lipoproteins [ 161. Although differences in the absolute secretion of the VLDL constituents were not significant between PCS and sham control swine, a trend to a slightly reduced cholesterol secretion in the PCS group was found when plasma volume and metabolic body size were considered. Methods Animals
Female miniature swine from Sinclair Research Farm, Univ. of Missouri, MO. were obtained at age 5-5; months. The animals were fed a growth hog chow (Maple Leaf Mills, Montreal) and allowed a stabilization period of 7-9 weeks prior to surgery. At operation a laterolateral anastomosis was performed; ligation and section of the portal vein just beneath the hepatic hilus transformed this into a functional end-to-side anastomosis. None of the venous tributaries of the portal vein were obstructed so that the portal flow was rerouted through the anastomosis. Triton study
Seventeen to 18 weeks following surgery, 4PCS and 3 sham-operated controls were subjected to the Triton study. Under anesthesia, a catheter was installed in an external jugular vein and exteriorized via a separate incision in the back of the neck. The fasting animal recovered with free access to water and all experiments were begun the following morning. The animal was weighed and housed in a metabolic cage and a blood sample was taken for hematocrit, plasma lipid and lipoprotein determinations. 2 mCi of an [3H] Lamino acid mixture (New England Nuclear) in 0.15 M NaCl, pH 7.4 was then injected via the catheter. Twenty-five minutes later a second blood sample was withdrawn and this was immediately followed by the infusion over 5-6 min of a 20% solution of Triton-WR 1339 (Ruger Chemical Company, N.Y.) (120 mg/kg) in 0.15 M NaCl, pH 7.4 previous filtered through a 0.45 pm Millipore filter. Blood samples of about 25 ml were taken 3-4 min and 0.5,1,1.5, 2, 3, 4, 6, 9, 15, 18, 21 and 24 h after infusion.
357
All blood samples were mixed with EDTA to kept on ice and the plasma and cells separated Plasma aliquots were centrifuged at 130,000 X g,, VLDL, and subsequently at density 1.02 for 16 h. lation of lipoproteins at density 1.08 (LDL) and carried out for 20 and 40 h respectively [ 171.
a concentration of 1 mg/ml, by low speed centrifugation. for 16 h for the isolation of In 3 experiments further isoat density 1.21 (HDL) were
Analyses The radioactivity of total plasma and lipoprotein was counted after precipitation, washing, and delipidation on filter paper discs [18]. Protein was determined by the method of Lowry [ 191. Lipids were extracted from plasma and lipoproteins with chloroform-methanol (2 : 1, v/v) [20]. The chloroform phase was evaporated to dryness under nitrogen and taken up in isopropanol for the determination of total cholesterol [ 211 and triglyceride by the method of Laurel1 [22], as modified by Kraml and Cosyns [23]. Triglyceride secretion rate was calculated from the formula: TG SR
C2 -C, = t2
-
=
pg/ml/min
t1
where T&R
C, (32
t, and t2
= triglyceride secretion rate, = concentration of TG pug/ml plasma at tl, = concentration of TG ,ug/ml plasma at t2. = sampling times with units of minutes.
Results Effect of the surgery The 4 PCS swine had patent portacaval anastomoses at sacrifice. At the time of this study, 18 weeks after surgery (about 1 week before sacrifice), these PCS animals showed a 3-9% body weight gain in contrast to the sham, whose weight gain was 28-40%. Body weight, metabolic body size [24] and plasma volume are summarized in Table 1. Plasma volume was measured at sacrifice by the indocyanine green dye extraction method, at almost the same body weight and was corrected for the hematocrit on the Triton day. The PCS swine showed a trend to a lower plasma volume/body size. The estimated hepatic blood flow of the animals however, was not different from the sham controls [ 111. Hepatic function as judged by serum levels of total proteins, serum glutamate-oxalacetate transaminase, alkaline phosphatase and lactate dehydrogenase, appeared normal after surgery, although the PCS swine showed a decreased serum albumin level [ 1 l] . The changes in plasma lipid and lipoprotein levels for these swine within a larger group of PCS and sham-operated animals after surgery are reported separately [ 111. In addition to significantly lowered plasma lipoprotein levels, the VLDL were shown to be lipid depleted, the LDL to have a reduced complement of cholesterol with an increased triglyceride content and the HDL were compositionally depleted in protein and phospholipid ([ 111 and Table 2).
358 TABLE 1 BODY WEIGHT, PLASMA VOLUME
AND METABOLIC
BODY SIZE
Sham Body weight (kg) Metabolic body size (kg314) Plasma volume (ml)
PCS
54.0 19.92 2590
60.5 21.69 2252
49.5 18.66 2066
130.0
103.8
110.7
42.5 16.64 1412 84.8
64.0 22.62 1963
42.3 16.59 1357
86.8
51.2 19.14 2200
81.8
114.9
Effect of Triton All animals showed a large accumulation of lipid, protein and associated radioactivity in the d < 1.006 density fraction (VLDL) over the 24 h after the injection of Triton. Essentially no radioactivity and no lipoprotein was recovered in the density range d 1.006-1.02. In the experiments were higher densities were surveyed, the LDL (d 1.02-1.08) had a constant level of protein and lipid and no radioactivity indicating no conversion of the VLDL protein to LDL. The HDL (d 1.08-1.21) showed a marked decrease in protein content immediately after the Triton infusion. This was probably due to a Triton-induced lipid : protein dissociation of the HDL as described by Scanu [ 251. No further analyses were carried out on the HDL. A linear increase in the specific activity (cpm/pg) of the total plasma proteins was found in all animals in the first 3 h after Triton. Between 4 and 6 h, the specific activity curve reached a maximum and remained at a high plateau to 24 h. The specific activity curve of the VLDL protein also increased sharply from O-l h, peaked at l-l.5 h and declined gradually such that the specific activity of the 24-h sample was similar to the pre-Triton low level. The pre-Triton fasting VLDL protein level of the sham group was 10.3 f 1.0 mg/dl plasma and of the PCS group 6.7 f 1.3 mg/dl. At 21-24 h after infusion all swine had
TABLE 2 EFFECT OF PORTACAVAL SHUNT ON COMPOSITIONAL DENSITY LIPOPROTEINS 19 WEEKS AFTER SURGERY Lipid d < 1.006 (VLDL) Sham b PCS
5.6 f 1.0 c 3.6 f 1.0 * Lipid
d < 1.02-1.07 (LDL) Sham PCS
:Pa
:P
1.9 ?: 0.1 2.0 f 0.3
TG
:P
4.3 t 0.9 2.7 ? 0.8 *
TG
: TC
RATIOS
TC
:P
10.8 ? 1.1 1.5 f 2.0 **
0.52 + 0.23 0.39 + 0.11
P : TG
Ch:TG
TC
10.2 + 2.9 5.3 t 0.8 **
12.2 f 3.2 6.4 + 0.9 **
1.2 1.2
:P f 0.1 ? 0.2
a P = protein, TG = triglyceride. TC = total cholesterol, PL = phospholipids, b No. of animaIs = 4 sham, 5 PCS. c Values are means f SD. Significantly different, sham vs. PCS. * P < 0.05,
** P < 0.025.
OF VERY
LOW AND
LOW
PL:TG
UC : TC
0.18 f 0.06 0.19 * 0.02
0.53 + 0.18 0.43 f 0.13
PL:TG
UC
6.7 2.9
0.22 + 0.03 0.26 f 0.05
f 2.3 f 1.2 *
: TC
UC = unesterified cholesterol.
359 TABLE
3
THE EFFECT OF SECRETION RATE
PORTACAVAL SHUNT AFTER TRITON
Hours post-Triton
ON
PLASMA
TRIGLYCERIDE
AND
TRIGLYCERIDE
Plasma triglyceride (mg/ml)
0.5 2.0 4.0 9.0
Sham a
PCS
0.75 1.83 3.58 7.92
1.03 2.20 3.80 8.19
r ? * t
0.15 b 0.37 1.40 1.18
f * f f
0.39 0.76 0.92 1.99
13.5 r
3.9
Triglyceride secretion rate t~g/mI/min) O-12
12.8 _+ 2.8 (mg/h/kg314)
O-12
77.1 f 16.2
69.0 ? 16.8
a 3 shamaperated controls and 4 PCS swine were infused with Triton WR 1339 (120 mg/kg) over 6 min immediately prior to time 0. b The values are means + SEM.
VLDL protein levels of 65-73 mg/dl. When the VLDL protein specific activity curves were corrected for the greater dilution of the incorporated amino acids into the larger circulating VLDL pool of the sham animals, no difference between groups in the initial rate or the peak time was found. In all animals the radioactivity of the VLDL protein was a constant percentage of the total plasma protein radioactivity over the 24 h: sham 12.0 f 0.5%; PCS 10.9 f 1.1%. Triglyceride and cholesterol in VLDL and plasma The triglyceride secretion rate for all animals was linear over at least the first TABLE
4
SECRETION
RATES
OF VLDL
CONSTITUENTS
OVER
Sham &minim1 plasma protein triglyceride cholesterol b
0.28 5.71 0.79
6 HOURS
AFTER
TRITON
IN SWINE a
PCS
0.58 14.87 1.63
0.84 16.06 1.86
1.15 12.96 1.47
0.63 14.92 1.11
0.29 7.84 0.68
0.27 7.18 0.70
mg/h/kg314 c protein triglyceride cholesterol
2.2 44.6 6.2
3.6 92.6 9.6
6.6 106.6 12.4
5.9 121.9 7.5
3.3 77.7 5.8
1.4 38.6 3.3
1.9 49.5 4.8
Ratio TG TC TG
20.1 2.4 7.2
25.6 2.6 9.7
19.2 2.2 8.6
20.1 1.3 16.3
23.7 1.8 13.4
26.1 2.3 11.5
26.0 2.5 10.3
of constituents d :P :P : TC
a 3-shamaperated controls and 4 PCS swine were infused with T&on WR 1339 (120 mg/kg) over 6 min Immediately preceeding time 0. Values are derived from multiple samples over 6 h. b Total cholesterol. c Secretion rates corrected for plasma volume and metabolic body size. d TG : triglyceride, P : protein. TC = total cholesterol.
360
12 h following Triton, Table 3 presents the actual plasma triglyceride levels and the secretion rates for both groups. The VLDL triglyceride accounted for 97% of the total plasma triglyceride after the 1 h post-Triton sample. The secretion rates of the VLDL constituents are shown in Table 4. After correction of the absolute rates for metabolic body size and plasma volume, the variation between animals and the overlap between groups persisted, although the trend to a lower cholesterol secretion in the PCS group was apparent. When the ratio of the VLDL constituents was calculated, a significantly higher triglyceride : total cholesterol ratio in the PCS group was found: sham 8.53 + 1.22, PCS 12.88 f 2.02 (mean + SD) (P < 0.05). Discussion It has been suggested that the hypolipidemia of portacaval shunt surgery is due to reduced lipoprotein synthesis [ 51 or production [ 71, and demonstrated that LDL synthesis was decreased in a familial hypercholesterolemic homozygote [3] and in normal miniature swine after surgery [7]. We have attempted to corroborate these findings by the direct study of VLDL production in PCS and sham-operated swine. Studies in man [ 261, rats [ 271 and squirrel monkeys [28] have indicated that the intravascular catabolism of VLDL leads to the sequential production of LDL via the formation of intermediates or remnants. The model chosen to study the production of VLDL was the injection of Triton WR-1339. Intravenous injection of Triton has frequently been used to determine rates of hepatic triglyceride secretion in various animals [ 16,25,2932]. The effect of Triton is believed to be the formation of a surface layer around the lipoprotein which prevents hydrolysis by lipoprotein lipase [25]. Triton has also been shown to inhibit the enzyme lecithin : cholesterol acyltransferase [ 321. Thus, the accumulated plasma lipid which follows the injection of Triton is believed to be newly secreted VLDL lipoprotein, refractory to its normal catabolism [ 161. In this study, all animals were fasting for 20-24 h before the infusion of Triton. We therefore assume that the intestinal contribution to the accumulated VLDL was minor [32]. In the present study, essentially no radioactivity or lipoprotein was recovered in the plasma density fraction d 1.006-1.02, nor was there any radioactivity recovered in the d l.O21.08 (LDL) fraction. This indicates that no net catabolism of the VLDL took place over the 24 h studied, and we have taken the increments in VLDL concentration as being representative of newly secreted lipoprotein [ 161. The rate of appearance of the incorporated label in the proteins of VLDL and in total plasma proteins was unchanged in the PCS swine, compared to the controls. Further the radioactivity of the VLDL remained a constant percentage of total plasma protein radioactivity over the 24 h and was equivalent in all animals. Additionally, the levels of VLDL protein accumulating in the plasma after Triton could not be shown to be different in the PCS group. Thus the fasting state hepatic synthesis and secretion of apo-VLDL appeared to be unchanged by the shunt. Recently., the incorporation of radioactivity in total apolipoproteins of PCS rats has been measured and found decreased in the VLDL [lo]. However, this cannot be taken as an index of synthesis since secretion
361
rates were not specifically measured and the known exchange of VLDL apoproteins in the plasma was not considered [ 331. We have previously reported ([ 111 and Table 2) that portacaval shunted fasting miniature swine have significantly lowered levels of VLDL which are lipiddepleted with significantly decreased triglyceride and slightly decreased cholesratios. Further, the fasting PCS LDL showed an terol : protein compositional increased content of triglyceride. In the present study, the absolute secretion rates of VLDL protein, triglyceride and cholesterol were variable between animals and no significant difference between groups could be shown. The trend to a lower cholesterol secretion in the PCS VLDL was evident however, when plasma volume and metabolic body size were considered. The compositional ratios of the newly synthesized VLDL of PCS swine showed a small but significant reduction in cholesterol content and a normal complement of triglyceride. In the normal fasting state these VLDL, after intravascular exchange and modification [33] have a significantly reduced triglyceride and slightly reduced cholesterol complement [ll]. Although the apoprotein composition of nascent VLDL in PCS swine has not been investigated, it is possible that a changed proportion of apoproteins accompanied their modified lipid composition. Finally this study has been conducted in fasting animals, and it is interesting to speculate that, in the normal fed state, the diversion of hepatotrophic factors [5] would further alter the VLDL production capacity of the liver with consequent decreased circulating lipoprotein levels, hypolipidemia and LDL synthesis. References 1 Starzl. 2 3
4 5 6 7 8 9 10
11
12
T.E..
Chase,
H.P.. Putnam.
C.W. and Porter,
K.A..
Portacavai
shunt in hyperhpidemia,
Lance&
2 (1973) 940. Stein, E.A.. Pettifor, J., Mieny. C.. Heimann, K.W., Spitz, L., Bersohn. I.. Saaron. I. and Dinner, N.. Portacaval shunt in four patients with homozygous hypercholesterolemia. Lancet, 1 (1974) 832. BiIheimer, D.W., Goldstein, J.L., Grundy. S.M. and Brown, M.S., Reduction in cholesterol and low density lipoprotein synthesis after portacaval shunt surgery in a patient with homozygous familial hypercholesterolemia, J. Chn. Invest.. 56 (1975) 1420. Cywes, S.. Davies, M.R., Louw, J.H.. Berger. G.M.B.. Bonnici, F. and Joffre. H.S.. Portacaval shunt in two patients with homozygous type II hyperhpoproteinaemia, S.A. Med. J.. 50 (1976) 239. Starr& T.E., Lee. Y.J., Porter, K.A. and Putnam, C.W.. The influence of portal blood upon lipid metabolism in normal and diabetic dogs and baboons, Surg. Gynecol. Obstet., 140 (1975) 381. Chase, H.P. and Morris, T.. Cholesterol metabolism following portacaval shunt surgery in the pig. Atherosclerosis, 24 (1976) 141. Carew. T.E.. Saik, R.P.. Johansen, K.H., Dennis, C.A. and Steinberg, D.. Low density and high density lipoprotein turnover following portacaval shunt in swine, J. Lipid Res., 17 (1976) 441. Coyle, J.J., Schwartz. N.Z., Marubbio. A.T., Varco, R.L. and BuchwaId, H.. The effect of portacaval shunt on plasma lipids and tissue cholesterol synthesis in the dog, Surgery, 80 (1976) 54. Edwards, K.D.G., Bradley. S.E. and Herr, R., Blood pressure cholesterol and triglyceride lowering effects of portacaval anastomosis, Med. J. Aust.. 1 (1976) 450. Magide, A.A., Press, C.M., Myant, C.B.. Mitropoulos, K.A.. and Balasubramanian, S., The effect of portacavai anastomosis on plasma lipoprotein metabolism in rats, Biochim. Biophys. Acta, 441 (1976) 302. Nestruck, A.C.. Lussier-Cacan. S., Bergseth, M., BidaBier, M., Davignon, J. and Marcel, Y.L.. The effect of portacavai shunt on plasma lipids and lipoproteins in swine. Biochim. Biophys. Acta, 488 (1977) 43. Starzl. T.E.. Porter, K.A., Kashinwagi, N., Lee. I.Y., Russell, W.J.I. and Putnam, C.W., The effect of diabetes meIIitus on portal blood hepatotrophic factors in dogs. Surg. Gynecol. Obstet., 140 (1975) 549.
362 13 Star& T.E.. Porter, K.A.. Kashiwagi. N. and Putnam, C.W., Portal hepatotrophic factors diabetes mellitus and acute liver atrophy. hypertrophy and regeneration. Surg. Gynecol. Obstet.. 141 (1975) 843. 14 Cushieri, A.. Baker, P.R., Halley, M.P. and Hanson, C.. Portacaval shunt in the pig. Part 1 (Effect on survival behavior, nutrition and liver function). J. Surg. Res.. 17 (1974) 387. 15 Star& T.E.. Chase, H.P., Putnam, C.W. and Nora. J.J.. Follow-up of patient with portacaval shunt for treatment of hyperlipidemia, Lancet, 2 (1974) 714. 16 Zilversmit. D.B.. Hughes, L.B. and Remington, M., Hypolipidemic effect of pregnancy in the rabbit, J. Lipid Res., 13 (1972) 750. 17 Havel, R.J., Eder, H.A. and Bragdon, J.H.. The distribution and chemical composition of ultracentrifugally separated lipoproteins in human serum, J. Clin. Invest., 34 (1955) 1345. 18 Mans, R.J. and Novelli. G.D.. Measurement of the incorporation of radioactive amino acids into protein by a filter-paper disc method, Arch. Biochem. Biophys., 94 (1961) 48. 19 Lowry, O.H.. Rosebrough. N.J.. Farr, A.L. and Randall, R.J., Protein measurement with the Folin phenol reagent, J. Biol. Chem. 193 (1951) 265. 20 Folch, J.. Lees, M. and SloaneStanley. G.N., A simple method for the isolation and purification of total lipids from animal tissues, J. Biol. Chem., 226 (1957) 497. 21 Block, W.D.. Jarret. K.J. and Levine, L.B., An improved automated determination of serum total cholesterol with a single colour reagent, Clin. Chem., 12 (19661 681. 22 Laurell, S.. A method for routine determination of plasma triglycerides, Scand. J. Clin. Lab. Invest., 18 (1966) 688. 23 Kraml. N. and Cosyns, L.. A semi-automated determination of serum triglycerides, Clin. Biochem., 2 (1969) 373. 24 Kleiber, M.. The Fire of Life, Wiley, New York, 1960. P. 200. 25 Scsnu. A. and Oriente. P.. Triton hyperlipidemia in dogs. J. EXP. Med., 113 (1961) 735. 26 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. Biophys. Acta. 326 (1973) 361. 27 Eisenberg. S. and Rachmilewitz, D., Metabolism of rat plasma very low density lipoprotein. Part 1 (Fate in circulation of the whole lipoprotein). Biochim. Biophys. Acta. 326 (1973) 378. 28 Illingworth, R.D.. Metabolism of lipoproteins in non human primates - Studies on the origin of low density lipoprotein apoprotein the plasma of the squirrel monkey, Biochim. Biophys. Acta., 388 29 30 31 32
33
(19751 28. Robertson, R.P., Gavareski, D.J.. Henderson, J.D.. Porte, Jr., D. and Bierman, E.L.. Accelerated triglyceride secretion, a metabolic consequence of obesity, J. Clln. Invest., 52 (1973) 1620. Kremmer, T. and Holczinger. L.. Effect of vlncristine on Triton WR-1339 induced hyperlipidemia in mice. Biochem. Pharmacol., 23 (1974) 3317. Fiser, R.H.. Denniston. J.C.. Rinds& R.B. and Be&l. W.R.. Triglyceride secretion rates -Use of Triton WR-1339 in the Rhesus monkey, J. Nutr.. 104 (1974) 223. Illingworth. D.R., Portman, O.W. and Whipple. L.E., Metabolic interrelationships between the lipids of very low. low and high density lipoproteins in the squirrel monkey, Biochim. Biophys. Acta. 369 (1974) 304. Rubenstein, B. and Rubinstein. D.. Interrelationship between rat serum very low and high density lipoproteins. J. Lipid Res.. 13 (1972) 317.
Atherosclerosis, 29 (1978) 355-362 0 Elsevier/North-Holland Scientific Publishers,
LIPID AND LIPOPROTEIN SHUNT IN SWINE
A.C. NESTRUCK,
(Received (Revised, (Accepted
SECRETION
M. BERGSETH,
Znstitut de Recherches H2 W 1 R 7 (Canada)
Ltd.
M. BIDALLIER,
FOLLOWING
J. DAVIGNON
PORTACAVAL
and Y.L.
MARCEL
Cliniques de Mont&al, 110 avenue des Pins ouest, Montrkal
6 September, 1977) received 16 January, 25 January, 1978)
1978)
Lipid and lipoprotein secretion were studied over 24 h in normal and portacaval shunted (PCS) fasting miniature swine after an infusion of Triton WR1339 and [3H]amino acid mixture. The plasma triglyceride secretion rate and the secretion of triglyceride, total cholesterol and protein in the very low density lipoprotein fraction (VLDL) were linear for about 9 h after Triton. No net catabolism of the VLDL appeared to take place over the 24 h. Due to large variation, differences in the absolute secretion rates of plasma triglyceride and of the VLDL constituents could not be shown. However, the secretion of VLDL cholesterol in the PCS group appeared to be lower than that of the sham group when corrected for metabolic body size and plasma volume. Key words:
Hypercholesterolemia - Hypocholesterolemia - Portacaval shunt WR-1399 - Very low density lipoprotein secretion
7’riton
Introduction The cholesterol lowering action of portacaval shunt surgery, first used clinically by Starzl [l] and subsequently by others [2-41 for the treatment of homozygous familial hypercholesterolemia, has been investigated in numerous studies using animal models [5--111. A coherent concept for its mechanism This research was supported by grants from the Stewart Macdonald Foundation, The Quebec Heart Foundation and the Medical Research Council of Canada (MT-4011 and MA-5427). A.C.N. is the holder of a research scholarship from the Conseil de la Recherche en Sante du QuBbec.
356
however, has not been reached. The proposal that a decreased hepatic synthesis of cholesterol is induced by the shunt [ 5,6] has not been confirmed by others [S]. Further, it has been found that a reduced hepatic cholesterol synthesis does not necessarily result in a decreased circulating cholesterol level [ 51. The hypocholesterolemic effect of the shunt has been shown to result from decreased LDL levels in man [ 31 and swine [ 111. The metabolism of LDL has also been investigated and a decreased production of LDL rather than an increased removal was demonstrated in man [ 31 and swine [ 71. Suggestions of a reduced hepatic synthesis and/or secretion of lipoproteins [ 5,7] are also derived in part from the concept of diverted and diminished hepatotrophic factors [ 12,131, and from the indirect evidence of a hypoalbuminemia in portacaval shunted (PCS) pigs [ 11,141 and rats 191, and the histological changes of hepatocytes in one PCS patient [ 151 and shunted experimental animals [5,12] which indicate decreased lipoprotein secretion. The present study was designed to investigate the effect of PCS on VLDL secretion in swine by use of an infusion of the non-ionic detergent Triton WR1339. The accumulating VLDL were considered to be newly secreted hepatic lipoproteins [ 161. Although differences in the absolute secretion of the VLDL constituents were not significant between PCS and sham control swine, a trend to a slightly reduced cholesterol secretion in the PCS group was found when plasma volume and metabolic body size were considered. Methods Animals
Female miniature swine from Sinclair Research Farm, Univ. of Missouri, MO. were obtained at age 5-5; months. The animals were fed a growth hog chow (Maple Leaf Mills, Montreal) and allowed a stabilization period of 7-9 weeks prior to surgery. At operation a laterolateral anastomosis was performed; ligation and section of the portal vein just beneath the hepatic hilus transformed this into a functional end-to-side anastomosis. None of the venous tributaries of the portal vein were obstructed so that the portal flow was rerouted through the anastomosis. Triton study
Seventeen to 18 weeks following surgery, 4PCS and 3 sham-operated controls were subjected to the Triton study. Under anesthesia, a catheter was installed in an external jugular vein and exteriorized via a separate incision in the back of the neck. The fasting animal recovered with free access to water and all experiments were begun the following morning. The animal was weighed and housed in a metabolic cage and a blood sample was taken for hematocrit, plasma lipid and lipoprotein determinations. 2 mCi of an [3H] Lamino acid mixture (New England Nuclear) in 0.15 M NaCl, pH 7.4 was then injected via the catheter. Twenty-five minutes later a second blood sample was withdrawn and this was immediately followed by the infusion over 5-6 min of a 20% solution of Triton-WR 1339 (Ruger Chemical Company, N.Y.) (120 mg/kg) in 0.15 M NaCl, pH 7.4 previous filtered through a 0.45 pm Millipore filter. Blood samples of about 25 ml were taken 3-4 min and 0.5,1,1.5, 2, 3, 4, 6, 9, 15, 18, 21 and 24 h after infusion.
357
All blood samples were mixed with EDTA to kept on ice and the plasma and cells separated Plasma aliquots were centrifuged at 130,000 X g,, VLDL, and subsequently at density 1.02 for 16 h. lation of lipoproteins at density 1.08 (LDL) and carried out for 20 and 40 h respectively [ 171.
a concentration of 1 mg/ml, by low speed centrifugation. for 16 h for the isolation of In 3 experiments further isoat density 1.21 (HDL) were
Analyses The radioactivity of total plasma and lipoprotein was counted after precipitation, washing, and delipidation on filter paper discs [18]. Protein was determined by the method of Lowry [ 191. Lipids were extracted from plasma and lipoproteins with chloroform-methanol (2 : 1, v/v) [20]. The chloroform phase was evaporated to dryness under nitrogen and taken up in isopropanol for the determination of total cholesterol [ 211 and triglyceride by the method of Laurel1 [22], as modified by Kraml and Cosyns [23]. Triglyceride secretion rate was calculated from the formula: TG SR
C2 -C, = t2
-
=
pg/ml/min
t1
where T&R
C, (32
t, and t2
= triglyceride secretion rate, = concentration of TG pug/ml plasma at tl, = concentration of TG ,ug/ml plasma at t2. = sampling times with units of minutes.
Results Effect of the surgery The 4 PCS swine had patent portacaval anastomoses at sacrifice. At the time of this study, 18 weeks after surgery (about 1 week before sacrifice), these PCS animals showed a 3-9% body weight gain in contrast to the sham, whose weight gain was 28-40%. Body weight, metabolic body size [24] and plasma volume are summarized in Table 1. Plasma volume was measured at sacrifice by the indocyanine green dye extraction method, at almost the same body weight and was corrected for the hematocrit on the Triton day. The PCS swine showed a trend to a lower plasma volume/body size. The estimated hepatic blood flow of the animals however, was not different from the sham controls [ 111. Hepatic function as judged by serum levels of total proteins, serum glutamate-oxalacetate transaminase, alkaline phosphatase and lactate dehydrogenase, appeared normal after surgery, although the PCS swine showed a decreased serum albumin level [ 1 l] . The changes in plasma lipid and lipoprotein levels for these swine within a larger group of PCS and sham-operated animals after surgery are reported separately [ 111. In addition to significantly lowered plasma lipoprotein levels, the VLDL were shown to be lipid depleted, the LDL to have a reduced complement of cholesterol with an increased triglyceride content and the HDL were compositionally depleted in protein and phospholipid ([ 111 and Table 2).
358 TABLE 1 BODY WEIGHT, PLASMA VOLUME
AND METABOLIC
BODY SIZE
Sham Body weight (kg) Metabolic body size (kg314) Plasma volume (ml)
PCS
54.0 19.92 2590
60.5 21.69 2252
49.5 18.66 2066
130.0
103.8
110.7
42.5 16.64 1412 84.8
64.0 22.62 1963
42.3 16.59 1357
86.8
51.2 19.14 2200
81.8
114.9
Effect of Triton All animals showed a large accumulation of lipid, protein and associated radioactivity in the d < 1.006 density fraction (VLDL) over the 24 h after the injection of Triton. Essentially no radioactivity and no lipoprotein was recovered in the density range d 1.006-1.02. In the experiments were higher densities were surveyed, the LDL (d 1.02-1.08) had a constant level of protein and lipid and no radioactivity indicating no conversion of the VLDL protein to LDL. The HDL (d 1.08-1.21) showed a marked decrease in protein content immediately after the Triton infusion. This was probably due to a Triton-induced lipid : protein dissociation of the HDL as described by Scanu [ 251. No further analyses were carried out on the HDL. A linear increase in the specific activity (cpm/pg) of the total plasma proteins was found in all animals in the first 3 h after Triton. Between 4 and 6 h, the specific activity curve reached a maximum and remained at a high plateau to 24 h. The specific activity curve of the VLDL protein also increased sharply from O-l h, peaked at l-l.5 h and declined gradually such that the specific activity of the 24-h sample was similar to the pre-Triton low level. The pre-Triton fasting VLDL protein level of the sham group was 10.3 f 1.0 mg/dl plasma and of the PCS group 6.7 f 1.3 mg/dl. At 21-24 h after infusion all swine had
TABLE 2 EFFECT OF PORTACAVAL SHUNT ON COMPOSITIONAL DENSITY LIPOPROTEINS 19 WEEKS AFTER SURGERY Lipid d < 1.006 (VLDL) Sham b PCS
5.6 f 1.0 c 3.6 f 1.0 * Lipid
d < 1.02-1.07 (LDL) Sham PCS
:Pa
:P
1.9 ?: 0.1 2.0 f 0.3
TG
:P
4.3 t 0.9 2.7 ? 0.8 *
TG
: TC
RATIOS
TC
:P
10.8 ? 1.1 1.5 f 2.0 **
0.52 + 0.23 0.39 + 0.11
P : TG
Ch:TG
TC
10.2 + 2.9 5.3 t 0.8 **
12.2 f 3.2 6.4 + 0.9 **
1.2 1.2
:P f 0.1 ? 0.2
a P = protein, TG = triglyceride. TC = total cholesterol, PL = phospholipids, b No. of animaIs = 4 sham, 5 PCS. c Values are means f SD. Significantly different, sham vs. PCS. * P < 0.05,
** P < 0.025.
OF VERY
LOW AND
LOW
PL:TG
UC : TC
0.18 f 0.06 0.19 * 0.02
0.53 + 0.18 0.43 f 0.13
PL:TG
UC
6.7 2.9
0.22 + 0.03 0.26 f 0.05
f 2.3 f 1.2 *
: TC
UC = unesterified cholesterol.
359 TABLE
3
THE EFFECT OF SECRETION RATE
PORTACAVAL SHUNT AFTER TRITON
Hours post-Triton
ON
PLASMA
TRIGLYCERIDE
AND
TRIGLYCERIDE
Plasma triglyceride (mg/ml)
0.5 2.0 4.0 9.0
Sham a
PCS
0.75 1.83 3.58 7.92
1.03 2.20 3.80 8.19
r ? * t
0.15 b 0.37 1.40 1.18
f * f f
0.39 0.76 0.92 1.99
13.5 r
3.9
Triglyceride secretion rate t~g/mI/min) O-12
12.8 _+ 2.8 (mg/h/kg314)
O-12
77.1 f 16.2
69.0 ? 16.8
a 3 shamaperated controls and 4 PCS swine were infused with Triton WR 1339 (120 mg/kg) over 6 min immediately prior to time 0. b The values are means + SEM.
VLDL protein levels of 65-73 mg/dl. When the VLDL protein specific activity curves were corrected for the greater dilution of the incorporated amino acids into the larger circulating VLDL pool of the sham animals, no difference between groups in the initial rate or the peak time was found. In all animals the radioactivity of the VLDL protein was a constant percentage of the total plasma protein radioactivity over the 24 h: sham 12.0 f 0.5%; PCS 10.9 f 1.1%. Triglyceride and cholesterol in VLDL and plasma The triglyceride secretion rate for all animals was linear over at least the first TABLE
4
SECRETION
RATES
OF VLDL
CONSTITUENTS
OVER
Sham &minim1 plasma protein triglyceride cholesterol b
0.28 5.71 0.79
6 HOURS
AFTER
TRITON
IN SWINE a
PCS
0.58 14.87 1.63
0.84 16.06 1.86
1.15 12.96 1.47
0.63 14.92 1.11
0.29 7.84 0.68
0.27 7.18 0.70
mg/h/kg314 c protein triglyceride cholesterol
2.2 44.6 6.2
3.6 92.6 9.6
6.6 106.6 12.4
5.9 121.9 7.5
3.3 77.7 5.8
1.4 38.6 3.3
1.9 49.5 4.8
Ratio TG TC TG
20.1 2.4 7.2
25.6 2.6 9.7
19.2 2.2 8.6
20.1 1.3 16.3
23.7 1.8 13.4
26.1 2.3 11.5
26.0 2.5 10.3
of constituents d :P :P : TC
a 3-shamaperated controls and 4 PCS swine were infused with T&on WR 1339 (120 mg/kg) over 6 min Immediately preceeding time 0. Values are derived from multiple samples over 6 h. b Total cholesterol. c Secretion rates corrected for plasma volume and metabolic body size. d TG : triglyceride, P : protein. TC = total cholesterol.
360
12 h following Triton, Table 3 presents the actual plasma triglyceride levels and the secretion rates for both groups. The VLDL triglyceride accounted for 97% of the total plasma triglyceride after the 1 h post-Triton sample. The secretion rates of the VLDL constituents are shown in Table 4. After correction of the absolute rates for metabolic body size and plasma volume, the variation between animals and the overlap between groups persisted, although the trend to a lower cholesterol secretion in the PCS group was apparent. When the ratio of the VLDL constituents was calculated, a significantly higher triglyceride : total cholesterol ratio in the PCS group was found: sham 8.53 + 1.22, PCS 12.88 f 2.02 (mean + SD) (P < 0.05). Discussion It has been suggested that the hypolipidemia of portacaval shunt surgery is due to reduced lipoprotein synthesis [ 51 or production [ 71, and demonstrated that LDL synthesis was decreased in a familial hypercholesterolemic homozygote [3] and in normal miniature swine after surgery [7]. We have attempted to corroborate these findings by the direct study of VLDL production in PCS and sham-operated swine. Studies in man [ 261, rats [ 271 and squirrel monkeys [28] have indicated that the intravascular catabolism of VLDL leads to the sequential production of LDL via the formation of intermediates or remnants. The model chosen to study the production of VLDL was the injection of Triton WR-1339. Intravenous injection of Triton has frequently been used to determine rates of hepatic triglyceride secretion in various animals [ 16,25,2932]. The effect of Triton is believed to be the formation of a surface layer around the lipoprotein which prevents hydrolysis by lipoprotein lipase [25]. Triton has also been shown to inhibit the enzyme lecithin : cholesterol acyltransferase [ 321. Thus, the accumulated plasma lipid which follows the injection of Triton is believed to be newly secreted VLDL lipoprotein, refractory to its normal catabolism [ 161. In this study, all animals were fasting for 20-24 h before the infusion of Triton. We therefore assume that the intestinal contribution to the accumulated VLDL was minor [32]. In the present study, essentially no radioactivity or lipoprotein was recovered in the plasma density fraction d 1.006-1.02, nor was there any radioactivity recovered in the d l.O21.08 (LDL) fraction. This indicates that no net catabolism of the VLDL took place over the 24 h studied, and we have taken the increments in VLDL concentration as being representative of newly secreted lipoprotein [ 161. The rate of appearance of the incorporated label in the proteins of VLDL and in total plasma proteins was unchanged in the PCS swine, compared to the controls. Further the radioactivity of the VLDL remained a constant percentage of total plasma protein radioactivity over the 24 h and was equivalent in all animals. Additionally, the levels of VLDL protein accumulating in the plasma after Triton could not be shown to be different in the PCS group. Thus the fasting state hepatic synthesis and secretion of apo-VLDL appeared to be unchanged by the shunt. Recently., the incorporation of radioactivity in total apolipoproteins of PCS rats has been measured and found decreased in the VLDL [lo]. However, this cannot be taken as an index of synthesis since secretion
361
rates were not specifically measured and the known exchange of VLDL apoproteins in the plasma was not considered [ 331. We have previously reported ([ 111 and Table 2) that portacaval shunted fasting miniature swine have significantly lowered levels of VLDL which are lipiddepleted with significantly decreased triglyceride and slightly decreased cholesratios. Further, the fasting PCS LDL showed an terol : protein compositional increased content of triglyceride. In the present study, the absolute secretion rates of VLDL protein, triglyceride and cholesterol were variable between animals and no significant difference between groups could be shown. The trend to a lower cholesterol secretion in the PCS VLDL was evident however, when plasma volume and metabolic body size were considered. The compositional ratios of the newly synthesized VLDL of PCS swine showed a small but significant reduction in cholesterol content and a normal complement of triglyceride. In the normal fasting state these VLDL, after intravascular exchange and modification [33] have a significantly reduced triglyceride and slightly reduced cholesterol complement [ll]. Although the apoprotein composition of nascent VLDL in PCS swine has not been investigated, it is possible that a changed proportion of apoproteins accompanied their modified lipid composition. Finally this study has been conducted in fasting animals, and it is interesting to speculate that, in the normal fed state, the diversion of hepatotrophic factors [5] would further alter the VLDL production capacity of the liver with consequent decreased circulating lipoprotein levels, hypolipidemia and LDL synthesis. References 1 Starzl. 2 3
4 5 6 7 8 9 10
11
12
T.E..
Chase,
H.P.. Putnam.
C.W. and Porter,
K.A..
Portacavai
shunt in hyperhpidemia,
Lance&
2 (1973) 940. Stein, E.A.. Pettifor, J., Mieny. C.. Heimann, K.W., Spitz, L., Bersohn. I.. Saaron. I. and Dinner, N.. Portacaval shunt in four patients with homozygous hypercholesterolemia. Lancet, 1 (1974) 832. BiIheimer, D.W., Goldstein, J.L., Grundy. S.M. and Brown, M.S., Reduction in cholesterol and low density lipoprotein synthesis after portacaval shunt surgery in a patient with homozygous familial hypercholesterolemia, J. Chn. Invest.. 56 (1975) 1420. Cywes, S.. Davies, M.R., Louw, J.H.. Berger. G.M.B.. Bonnici, F. and Joffre. H.S.. Portacaval shunt in two patients with homozygous type II hyperhpoproteinaemia, S.A. Med. J.. 50 (1976) 239. Starr& T.E., Lee. Y.J., Porter, K.A. and Putnam, C.W.. The influence of portal blood upon lipid metabolism in normal and diabetic dogs and baboons, Surg. Gynecol. Obstet., 140 (1975) 381. Chase, H.P. and Morris, T.. Cholesterol metabolism following portacaval shunt surgery in the pig. Atherosclerosis, 24 (1976) 141. Carew. T.E.. Saik, R.P.. Johansen, K.H., Dennis, C.A. and Steinberg, D.. Low density and high density lipoprotein turnover following portacaval shunt in swine, J. Lipid Res., 17 (1976) 441. Coyle, J.J., Schwartz. N.Z., Marubbio. A.T., Varco, R.L. and BuchwaId, H.. The effect of portacaval shunt on plasma lipids and tissue cholesterol synthesis in the dog, Surgery, 80 (1976) 54. Edwards, K.D.G., Bradley. S.E. and Herr, R., Blood pressure cholesterol and triglyceride lowering effects of portacaval anastomosis, Med. J. Aust.. 1 (1976) 450. Magide, A.A., Press, C.M., Myant, C.B.. Mitropoulos, K.A.. and Balasubramanian, S., The effect of portacavai anastomosis on plasma lipoprotein metabolism in rats, Biochim. Biophys. Acta, 441 (1976) 302. Nestruck, A.C.. Lussier-Cacan. S., Bergseth, M., BidaBier, M., Davignon, J. and Marcel, Y.L.. The effect of portacavai shunt on plasma lipids and lipoproteins in swine. Biochim. Biophys. Acta, 488 (1977) 43. Starzl. T.E.. Porter, K.A., Kashinwagi, N., Lee. I.Y., Russell, W.J.I. and Putnam, C.W., The effect of diabetes meIIitus on portal blood hepatotrophic factors in dogs. Surg. Gynecol. Obstet., 140 (1975) 549.
362 13 Star& T.E.. Porter, K.A.. Kashiwagi. N. and Putnam, C.W., Portal hepatotrophic factors diabetes mellitus and acute liver atrophy. hypertrophy and regeneration. Surg. Gynecol. Obstet.. 141 (1975) 843. 14 Cushieri, A.. Baker, P.R., Halley, M.P. and Hanson, C.. Portacaval shunt in the pig. Part 1 (Effect on survival behavior, nutrition and liver function). J. Surg. Res.. 17 (1974) 387. 15 Star& T.E.. Chase, H.P., Putnam, C.W. and Nora. J.J.. Follow-up of patient with portacaval shunt for treatment of hyperlipidemia, Lancet, 2 (1974) 714. 16 Zilversmit. D.B.. Hughes, L.B. and Remington, M., Hypolipidemic effect of pregnancy in the rabbit, J. Lipid Res., 13 (1972) 750. 17 Havel, R.J., Eder, H.A. and Bragdon, J.H.. The distribution and chemical composition of ultracentrifugally separated lipoproteins in human serum, J. Clin. Invest., 34 (1955) 1345. 18 Mans, R.J. and Novelli. G.D.. Measurement of the incorporation of radioactive amino acids into protein by a filter-paper disc method, Arch. Biochem. Biophys., 94 (1961) 48. 19 Lowry, O.H.. Rosebrough. N.J.. Farr, A.L. and Randall, R.J., Protein measurement with the Folin phenol reagent, J. Biol. Chem. 193 (1951) 265. 20 Folch, J.. Lees, M. and SloaneStanley. G.N., A simple method for the isolation and purification of total lipids from animal tissues, J. Biol. Chem., 226 (1957) 497. 21 Block, W.D.. Jarret. K.J. and Levine, L.B., An improved automated determination of serum total cholesterol with a single colour reagent, Clin. Chem., 12 (19661 681. 22 Laurell, S.. A method for routine determination of plasma triglycerides, Scand. J. Clin. Lab. Invest., 18 (1966) 688. 23 Kraml. N. and Cosyns, L.. A semi-automated determination of serum triglycerides, Clin. Biochem., 2 (1969) 373. 24 Kleiber, M.. The Fire of Life, Wiley, New York, 1960. P. 200. 25 Scsnu. A. and Oriente. P.. Triton hyperlipidemia in dogs. J. EXP. Med., 113 (1961) 735. 26 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. Biophys. Acta. 326 (1973) 361. 27 Eisenberg. S. and Rachmilewitz, D., Metabolism of rat plasma very low density lipoprotein. Part 1 (Fate in circulation of the whole lipoprotein). Biochim. Biophys. Acta. 326 (1973) 378. 28 Illingworth, R.D.. Metabolism of lipoproteins in non human primates - Studies on the origin of low density lipoprotein apoprotein the plasma of the squirrel monkey, Biochim. Biophys. Acta., 388 29 30 31 32
33
(19751 28. Robertson, R.P., Gavareski, D.J.. Henderson, J.D.. Porte, Jr., D. and Bierman, E.L.. Accelerated triglyceride secretion, a metabolic consequence of obesity, J. Clln. Invest., 52 (1973) 1620. Kremmer, T. and Holczinger. L.. Effect of vlncristine on Triton WR-1339 induced hyperlipidemia in mice. Biochem. Pharmacol., 23 (1974) 3317. Fiser, R.H.. Denniston. J.C.. Rinds& R.B. and Be&l. W.R.. Triglyceride secretion rates -Use of Triton WR-1339 in the Rhesus monkey, J. Nutr.. 104 (1974) 223. Illingworth. D.R., Portman, O.W. and Whipple. L.E., Metabolic interrelationships between the lipids of very low. low and high density lipoproteins in the squirrel monkey, Biochim. Biophys. Acta. 369 (1974) 304. Rubenstein, B. and Rubinstein. D.. Interrelationship between rat serum very low and high density lipoproteins. J. Lipid Res.. 13 (1972) 317.
