Zonal ultracentrifugation of plasma lipoproteins from normal and cholestatic pigs

187

Clinica

Chimica

0 Elsevier

Acta,

Scientific

65 (1975)

Publishing

187-196 Company,

Amsterdam

- Printed

in The Netherlands

CCA 7362

ZONAL ULTRACENTRIFUGATION OF PLASMA LIPOPROTEINS FROM NORMAL AND CHOLESTATIC PIGS B. DANIELSSON, Institute Surgery,

(Received

R. EKMAN,

B.G. JOHANSSON

of Biochemistry, University of Lund, University Hospital, Lund (Sweden)

and B.G. PETERSSON

and Departments

of Clinical

Chemistry

and

June 9,1975)

Summary

The plasma lipoproteins in normal and cholestatic pigs were isolated by zonal ultracentrifugation and studied with respect to apoprotein and lipid composition. In contrast with the distribution in human plasma, only one HDLpopulation but two LDL-populations were demonstrated. No cholestatic lipoprotein similar to the human lipoprotein X was observed. HDL and the lighter LDL component were largely unchanged in cholestasis. The immunochemical properties were changed to some extent. Thus HDL-reacting material was found in the heavier LDLcomponent and VLDL after cholestasis but not before.

Introduction

The human plasma lipoprotein pattern undergoes radical alterations in cholestasis affecting both the LDL- and the HDL-fractions. It has been shown that the commonly occurring changes in the LDL-fraction, which accompany obstructive jaundice in man, are due at least partly to the presence of an abnormal lipoprotein, called lipoprotein X [l-3]. Lipoprotein X is almost a regular finding in cholestasis and may be regarded as a diagnostic characteristic 14951. The LDL is increased in cholestasis while the HDL is often decreased, especially in long-standing icterus [6,7] , and the composition of the HDL is changed [ 81. In an endeavour to elucidate the mechanism of these lipoprotein changes, we induced obstructive jaundice in animals by ligation of the common bile duct. The present paper describes the alterations in porcine plasma lipoproteins by zonal ultracentrifugation and immunochemical methods.

Materials

and methods

Seven pigs weighing 20-25 kg, were fed carbohydrateand fat-rich food ad libitum for 14 days, after which they were anaesthetised (10 mg/kg Pentothal, Abbott) and the common bile duct sectioned and ligated. One of the pigs was subjected to a sham-operation with intra-abdominal incision and dissection around the common bile duct. The duration of the operation was the same as that of the bile-duct ligated pigs. Blood specimens for analysis were obtained from a catheterised jugular vein with EDTA as anticoagulant. Plasma samples were stored at +4”C and analysed within two days. All reagents used were of analytical grade. Antisera against porcine LDL and HDL were raised by immunisation of rabbits with lipoprotein fractions purified by preparative ultracentrifugation mainly as described for human lipoproteins by Have1 et al. [lo] . The antiserum against HDL was adsorbed with the infranatant fraction obtained by ultracentrifugation at 1.21 g/cm3 and with purified LDL. The antiLDL serum was absorbed with the infranatant obtained at a density of 1.07 g/cm3. On crossed immunoelectrophoresis both antisera were found to contain single immunoprecipitates when tested against porcine plasma. Zonal ultracentrifugation was performed in a Beckman model L2-65B ultracentrifuge equipped with a Ti-14 zonal rotor. Density gradients were made by mixing 0.01 M Tris buffer, pH 7.4, containing 1 mM EDTA, with sodium bromide (Mallinckrodt) dissolved in the same buffer (dZO = 1.200 or 1.300 g/cm3 ). Before use the solutions were filtered and degassed. The centrifugation technique was mainly as described by Wilcox et al. [ll]. Gradients, linear in density with respect to volume, were made by mixing buffer and sodium bromide solution with two peristaltic pumps programmed by a curve-follower. VLDL and LDL were isolated in a 600-ml gradient with a density range of 1.000-1.150 g/cm”. The plasma samples (15-25 ml) were made 1.160 g/cm” in density with solid sodium bromide before introduction and followed by a cushion of sodium bromide solution (d = 1.170) to fill the rotor completely. The centrifugation time was 3 h at 48 000 rpm (max. RCF 172 000 X g) and at +15”C. HDL was isolated in a 600 ml gradient with a density range of l.OOO1.290 g/cm”. The density of the sample was adjusted to 1.295 g/cm” with solid sodium bromide and sodium bromide solution (d = 1.300 g/cm3) was used as cushion. The centrifugation was carried out at 46 000 rpm (max. RCF 158 000 X g) at +15”C

for 18 h. In some experiments the two-step technique described by Wilcox et al. [ll] was tried. It involves partial emptying of the rotor (for recovery of VLDL and LDL) after a short centrifugation step, replenishing the gradient, and finally a longer centrifugation step for the separation of HDL. The rotor was center-unloaded at 3000 rpm and the rotor content was continuously monitored for UV-absorbance at 280 nm with a Uvicord II (LKBProdukter, Stockholm, Sweden) via a transmission-absorbance converter. 20 ml fractions were collected and the density of the fractions were checked by weighing 10 ml aliquots. Fractions for further investigations were concentrated, when necessary, by membrane ultrafiltration (UM-20, Amicon, 1J.S.A.).

189

Gel filtration was carried out with a 5 cm X 90 cm column filled with Sephadex G-ZOO with a particle size of 20-30 pm obtained by dry elutriation of Sephadex G-200 superfine (Pharmacia Fine Chemicals, Uppsala, Sweden) (Ekman et al., unpublished technique). Ascending chromatography of plasma samples was performed in 0.02 M Tris buffer, pH 7.4, containing 0.2 M NaCl and 5 mM EDTA at a flow rate of 15 ml per hour in the cold-room. 10 ml fractions were collected and the absorbance at 280 nm was determined with a Zeiss PMQ II spectrophotometer. Lipoprotein electrophoresis in agarose [12], electroimmunoassay [13], and crossed immunoelectrophoresis [ 141 were performed in the way described previously. Agar gel electrophoresis in Difco Bacto Agar with subsequent polyanion precipitation as described by Seidel et al. [ 151 was used for detecting abnormal lipoproteins similar to human lipoprotein X occurring in porcine cholestatic plasma. Cholestatic human serum was used as a control in all experiments. Chemical analyses. The amount of triglycerides in plasma was determined according to Laurel1 and Tibbling [16]. Total cholesterol in plasma was measured with a Technicon AutoAnalyzer, and the proportions of free and esterified cholesterol were estimated after thin-layer chromatography according to Abel1 et al. [17]. The following analyses were performed on serum samples with methods used in the clinical routine laboratory: bilirubin, alkaline phosphatases, glulamic-pyruvic transaminase (GPT), and y -glutamyltransferase (GT). Apoproteins were separated by SDS-gel electrophoresis as described by Garrard and Bonner [18], after the samples had been heated at 90°C for three min. The following proteins were used as molecular weight standards: human serum albumin, pepsin, myoglobin, and cytochrome C. The proteins were stained with Coomassie Brilliant Blue R 250 in 10% TCA and densitometric recordings were made with a Zeiss PMQ II spectrophotometer equipped with a gel scanner (type ZK 4). Determination of lipids in lipoproteins. Unesterified cholesterol and cholesterol esters were determined according to Abel1 et al. [ 171 after separation on thin layer chromatography: phospholipids and triglycerides as described by Belfrage et al. [ 191 and Laurel1 and Tibbling [ 161. Results General observations Ligation of the common bile duct invariably resulted in a few days in liver function values typical of extrahepatic jaundice (Fig. la). No changes in the liver function were found in the sham-operated animal. Also histological examination of the livers revealed damage characteristic of obstructive jaundice: “bile lakes” in widened canaliculi, degenerative changes of liver cells, and bile duct proliferations. The cholesterol and triglyceride levels in the serum increased during the first week after the operation and then returned to preoperative levels (Fig. lb). In the control pig, the levels were essentially constant. Agarose gel electrophoresis revealed no visible changes in the lipoprotein patterns and crossed immunoelectrophoresis showed no changes in the electro-

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Fig. 1. a. Serum concentrations of GT (y-glutamyltransferase), bilirubin. and GPT (glutamic-pyruvic transaminax) during the first two weeks after ligation. b. Serum concentrations of cholesterol (total) and triglycerides during the first two weeks after ligation. Each point represents the mean value (k S.D.) of the observations made in 6 pigs.

phoretic distribution of lipoproteins reacting with anti-oc-lipoprotein or anti-plipoprotein immune sera. No lipoprotein with the peculiar, cathodic migration in agar gel electrophoresis characteristic of human lipoprotein X was detected in cholestatic porcine serum during the observation period of 14 days after the operation. The molecular size distribution of HDL was studied by gel filtration in Sephadex G-200. Although no drastic changes were revealed, material with immunoreactivity to anti-HDL was somewhat more heterogeneously distributed, with an increased amount of larger particles after ligation of the common bile duct.

Studies of lipoproteins

by zonal ultracentrifugation

Complete separation of VLDL and LDL from normal or cholestatic porcine plasma could be achieved within three hours (Fig. 2). LDL was generally partially resolved into two subclasses (provisionally called LDL, and LDLl). The ratio between these subclasses varied widely and was probably not influenced by the induction of cholestasis. On some occasions only one broad peak was obtained. With a longer centrifugation time and a steeper gradient it was also possible to isolate HDL fairly well separated from the rest of the plasma proteins. The porcine HDL appeared to be confined to only one symmetrical peak with no separation into HDLz and HDL3 (Fig. 3). The two-step technique of Wilcox

191

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Effluent volumeImU Fig. 2. Preparations of VLDL and LDL by zonal ultiacentrifugation in a Ti-14 rotor from (a) 17 ml porcine plasma before, (b) 25 ml 8 days after ligation of ductus choledochus, and (c) 15 ml human cholestatic plasma. Run time three hours at 48 000 rpm. -. A280nm continuously recorded; A- - - - - -A, density gradient after centrifugation determined at 2O’C; O-----cI, and l electroimmuno assay against anti-LDL and anti-HDL, respectively. In (a) and (b) a normal porcine plasma and in (c) pooled normal human plasma was used as standard for the immunochemical determinations. Fractions were pooled as indicated by the bars.

et al. [ 111 employed in some experiments gave less clear resolution, especially in the LDL region, and was therefore not generally used. No distinct changes in the zonal ultracentrifugal pattern were found after

192

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Fig. 3. Preparation of HDL by zonal ultracentrifugation in a Ti-14 rotor from (a) 15 ml porcine plasma before and (b) 25 ml plasma 8 days after ligation of the common bile duct. Run time 18 h at 46 000 rpm. The symbols used are the same as in Fig. 2.

the operation, with the exception of an increase in VLDL. No analogue to the human lipoprotein X was found in the VLDL-LDL region (Fig. 2). There were no significant changes in the densities at which the different lipoproteins were situated after the runs, or in the shape or width of the peak. Although no change was found in the distribution of HDL as judged from the absorption at 280 nm (Fig. 3), immunochemical analyses revealed the presence of anti*-lipoprotein reacting material in the LDL region (LDL*). This reaction occurred within three days of the operation and became more intense with time. Fractions from zonal ultracentrifugation were pooled (Figs. 2 and 3) and concentrated. Agar gel electrophoresis showed no analogue to human lipoprotein X in any of these fractions. The apoprotein composition of the isolated lipoproteins was determined

193

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Day

0

8

0

8

0

0

LDL,

LDL,

VLDL

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Fig. 4. SDS-gel electrophoresis of apoproteins from lipoproteins isolated by zonal ultracentrifugation before and 8 days after ligation. The bands of main interest are indicated by numbers and have the same localization as the following human apoproteins: 1, B; 2, albumin; 3, “arginine-rich protein”; 4, A-I. and 5. C-proteins.

by SDS-polyacrylamide gel electrophoresis (Fig. 4). Normal porcine VLDL showed several components with a slight preponderance of low molecular weight material (molecular weight around 10 000). Most of the protein in LDL did not penetrate into the separation gel, but like the other lipoprotein classes, LDL also contained material with a molecular weight around 70 000. In addition, LDLz contained a distinct component with a molecular weight of about 25 000. The HDL was dominated by a large component with the same localization (molecular weight 25 000).

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Fig. 5. Densitometric tracings at 565 nm of LDL2 apoproteins separated by SDS-gel electrophoresis before and (b) 8 days after ligation. The bands are numbered in the same way as in Fig. 4.

(a)

19‘4

No changes were found in LDL, and HDL in fractions obtained eight days after induction of cholestasis. VLDL on the other hand showed a distinct band with a molecular weight of approximately 25 000. LDL, showed the most pronounced changes which are also visualized in the densitogram given in Fig. 5. The increase in components 3 and 4 in the diagram is striking. The lipid composition of the fractions was not systematically studied. No profound changes were found apart from an increase in phospholipids from that induction of 12% to 25% (of total lipoprotein) in LDL2. It is noteworthy cholestasis was not followed by any changes in total cholesterol or in the cholesterol/cholesterol ester ratio. Discussion The zonal ultracentrifugation technique has been successfully used in investigations of lipoprotein patterns in human cholestasis. The discovery and isolation of a new cholestatic HDL (HDL, ) [8] can fully be ascribed to the availability of this technique. The analytical quality of zonal ultracentrifugation is valuable in studies of the present type not only because it gives more information, but also because the density distribution of the lipoproteins may change during the study, as it does in man. As will be discussed below, uncritical application of differential flotation on species less well studied than man may prove misleading. Rather large sample volumes can, if necessary, be used in zonal ultracentrifugation. The resolution between porcine LDLi and LDL, is almost as good with samples of 75 ml as of 15 ml. When only small amounts of material are available, the two-step technique developed by Wilcox et al. [ll] may be advantageous, since it permits isolation of all density classes from one sample, but the loss of resolution is considerable compared with that of separate LDL and HDL runs. The normal lipoprotein pattern obtained by zonal centrifugation of porcine plasma differed from the human pattern in that no subclasses were found in porcine HDL, whereas LDL appeared more heterogeneous and could generally be separated into two subclasses. This is consistent with the observations made by Janado et al. in 1966 [20] on analytical ultracentrifugation of porcine plasma lipoproteins. Their results have, however, generally not been considered in later studies of porcine plasma lipoproteins [ 21,221. Moreover, porcine LDL should be isolated at a higher density interval at differential flotation than human LDL (Fig. 2). Despite the unequivocal signs of cholestasis demonstrated by the laboratory analyses and histological examination of the livers, the lipoprotein patterns in the pigs were only slightly changed during the observation period of two weeks after the operation. The zonal ultracentrifugation pattern suggested no analogue to the human lipoprotein X, which is clearly separated from human normal LDL. Studies on the protein and lipid composition of the isolated lipoproteins in plasma from cholestatic pigs confirmed the absence of any abnormal lipoprotein similar to human lipoprotein X, although the lipid composition of one of the LDL-subclasses (LDL2) resembled to some degree that of human lipo-

195

protein X with a high phospholipid content and a decreased content of triglycerides. It should be noted that no change in the cholesterol/cholesterol ester ratio was found in any of the studied fractions. The considerable amount of material reacting with antiserum against normal porcine LDL, and probably consisting of apoprotein B, argues against this fraction containing significant amounts of a component similar to human lipoprotein X. The absence of positive lipoprotein X tests in agar gel electrophoresis according to Seidel et al. [6] with this fraction corroborates the absence of lipoprotein X in pig’s plasma. This is in contrast with the findings obtained in other animals, viz. dog [ 231. The marked fall in the plasma HDL levels common in human obstructive jaundice, especially when prolonged, was, not demonstrable in the pigs, which were observed for two weeks after operation. The only noticeable change in the HDL composition was a decrease in the triglycerides and an increase in the phospholipid content. The changes in apoprotein distribution of different lipoprotein classes obviously accompanying the biliary obstruction of the pigs are difficult to interpret with the present scarce knowledge of the porcine apolipoproteins. It is reasonable to assume from the SDS-gel electrophoretic patterns that apoproteins similar to human C-proteins and A-proteins are present in porcine plasma lipoproteins. According to Cox and Tanford [24] , the main component of porcine HDL is analogous to human A-I, whereas no A-II could be demonstrated. These results are consistent with the present data, i.e. preponderance of a component with an apparent molecular weight of around 25 000. The subtle changes of apoproteins occurring in porcine cholestasis bore no resemblance to those found in human cholestasis which is associated with profound apoprotein changes both in LDL [ 251 and HDL [ 81. It can be concluded that the lipoprotein changes in cholestatic pigs are of only limited value for studying the mechanism behind the lipoprotein alterations in human cholestasis. This is in contrast with the situation in other animals, e.g. dogs, where conditions more analogous to human conditions can be found [23,26]. Studies are in progress in our laboratory to isolate abnormal lipoproteins in cholestatic dogs by means of zonal ultracentrifugation. Acknowledgements This work was supported by grants from the Swedish Medical Research Council (03X-41473, the Royal Physiographic Society, and the Medical Faculty, University of Lund. References 1 2 3 4 5 6 7 8 9

Seidel. D., Agostini. B. and Miiller, P. (1972) Biochim. Biophys. Acta 260,146 Quarfordt, S.H., Oelschlaeger, H. and Krigbaum. W. (1972) J. Clin. Invest. 51, 1979 Danielsson, B., Johansson, B.G. and Petersson. B.G. (1973) Clin. Chim. Acta 47.365 Seidel. D., Gretz, H. and Ruppert, C. (1973) Clin. Chem. 19. 86 Petek. W.. Kostner, G. and Holasek. A. (1973) Z. Klin. Chem. 11,415 Seidel. D.. Greten, H.. G&en, H.P., Wengeler, H. and Wieland, H. (1972) Eur. J. Clin. Invest. 2. 359 Ekman, R., Johansson, B.G. and Petersson, B.G. (1975) Stand. J. Immunol. 4, Suppl. 2, 13 Danielsson. B., Ekman, R. and Petersson, B.G. (1975) FEBS Lett. 50,180 Wengeler, H., Greten, H. and Seidel, D. (1972) Eur. J. Clin. Invest. 2. 372

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Havel, R.J., Eder. H.A. and Bragdon. H.J. (1955) J. Clin. Invest. .X4, 1MR Wilcox, H.J., Davis, D.C. and Heimberg, M. (1971) J. Lipid Res. 12, 160 Johansson, B.G. (1972) Sand. J. Clin. Lab. Invest., Suppl. 124, 7 Laurell, C.-B. (1972) &and. J. Clin. Lab. Invest., Suppl. 124, 21 Ganrot, P.O. (1972) Sand. J. Clin. Lab. Invest.. Suppl. 124. 39 Seidel, D., Wieland, H. and Ruppert, C. (1973) Clin. Chem. 19, 737 Laurell, S. and Tibbling. G. (19fi6) Clin. Chin Acta 13, 317 Abell. L.L., Levy. B.B., Brodie. B.B. and Kendall, F.A. (1952) J. Biol. Chem. 195, 357 Garrard. W.T. and Banner, J. (1974) J. Biol. Chem. 249, 5570 Belfrage, P., Wiebe, T. and Lundquist, A. (1970) Sand. J. Clin. Lab. Invest. 26, 53 Janado, M.. Martin, W.G. and Cook, W.H. (1966) Can. J. Biochem. 44,120l Fidge, N. (1973) Biochim. Biophys. Acta 295, 258 Davis. M.A.F., Henry, R. and Leslie, R.B. (1974) C amp. Biochem. Physiol. 47B. 831 Miiller, P., Faser, U., Fellin. R., Wieland, H. and Seidel, D. (1973) FEBS Lett. 38. 53 Cox, A.C. and Tanford, C. (1968) J. Biol. Cbem. 243, 3083 Seidel, D., Alaupovic. P., Furman, R.H. and McConathy. W.J. (1970) J. Clin. Invest. 49. 2396 Ritland, S. and Bergan, A. (1975) Stand. J. Gastroenterol. 10. 17