Comparison of different methods for LDL isolation and radioiodination on liver LDL receptor binding in vitro

Nucl. Med. Biol. Vol. 18, No. 5, pp. 513-517, 1991 Int. J. Radiat. Appl. Instrum. Part B Printed in Great Britain. All rights reserved

0883~2897/91 $3.00 + 0.00 Copyright Q 1991 Pergamon Press plc

Comparison of Different Methods for LDL Isolation and Radioiodination on Liver LDL Receptor Binding I. Vitro IRENE VIRGOLINI’*2~3*, PETER ANGELBERGER4, GRAZIANA LUPATTELLI’, SHUREN LI’, JOHANN PIDLICH’, EWALD MOLINARI’ and HELMUT SINZINGER’,2,3 ‘Atherosclerosis Research Group (ATK) of the Austrian Academy of Sciences, ‘Department of Nuclear Medicine, University of Vienna, sLudwig Boltzmann Institute for Nuclear Medicine, Vienna, Themistry Institute, Research Center Seibersdorf and 51mmuno AG, Austria (Received I March 1990; in revised form 10 September 1990)

Lipoproteins were isolated either by immunoaffinity chromatography (LDL and VLDL) or ultracentrifugation (LDL). Purified lipoproteins were labeled with rz31using either Iodogen or iodine-monochloride (ICl) each followed by purification with gel-chromatography or dialysis (total of 4 combinations). Lipoprotein-concentrations of 0.1-6 lg protein/mL were used for direct binding assays investigating the specific binding of labeled lipoproteins (in the presence of a 50-fold excess of unlabeled lipoproteins) to human liver apo-B,E-receptors. In separate experiments displacement of bound ‘231-lipoproteins (labeled by the methods mentioned) by unlabeled ones was studied. The binding capacities estimated by Scatchard analysis were similar to each other (141-163 ng protein bound/mg liver plasma membrane protein) independent from the method used for isolation and labeling. Also the affinity constants were very similar and ranged from 0.9 to 1.7 ng protein/L. It is concluded that immunoaffinity chromatography or ultracentrifugation for isolation of lipoproteins and the Iodogen or ICI-method for radiolabeling can be recommended to be equally good for in vitro receptor investigation.

Introduction Low-density lipoproteins (LDL) are internalized by endocytosis through high affinity apo-B,E-receptorbinding (Brown and Goldstein, 1976). These binding sites are located at many human cell types, predominantly in the liver (Goldstein and Brown, 1987), but also at adrenal gland cells, lymphocytes, monocytemacrophages and fibroblasts (for review see Mahley and Innerarity, 1983). Their primary function is to maintain cholesterol homeostasis. Elevated levels of cholesterol are associated with progression of atherosclerosis. Familial hypercholesterolemia (FH), due to a mutation in the LDL-receptor gene which leads to a reduced number or to a reduced activity of receptors, shows the highest degree of plasma cholesterol: patients affected by the homozygous form suffer from coronary heart disease at a rather young age (Goldstein and Brown, 1983). Goldstein’s group (Huettinger ef al., 1987) showed the possibility of detecting liver receptor activity in uivo through *All correspondence should be addressed to: Dr Irene Virgolini, Atherosclerosis Research Group (ASF) Vienna, Schwarzpanierstrasse 17, A-1090 Vienna, Austria.

scintiscanning using radiolabeled VLDL in rabbits and in man (Lupattelli et al., 1989). In fact, not only LDL but also other lipoproteins such as VLDL (Mahley and Innerarity, 1983) bind to the apo-B,E receptor. On the other hand, LDL isolated by ultracentrifugation and apo-B containing lipoproteins (LDL and VLDL) isolated by immunoaffinity chromatography and radiolabeled with ‘23I(Kaliman ef al., 1985; Sinzinger et al., 1985, 1989) ‘?‘I (Lees et al., 1983), “‘1 (Lupattelli et al., 1989) or “9mTc (Lees et al., 1988; Vallabhajosula et al., 1987) are clinically used for detecting human atherosclerotic lesions in carotid and femoral arteries indicating an increased LDL-entry into atherosclerotic vessels. Therefore, it is relevant to investigate if the different methods for lipoprotein labeling influence lipoprotein-receptor binding. This study investigated the specific binding of LDL isolated by ultracentrifugation and apo-B-containing lipoproteins isolated by immunoaffinity chromatography to the liver apoB,E-receptor. They were labeled with ‘23I using Iodogen or iodine-monochloride (ICI). Binding was tested to human liver tissue obtained at surgery. For each series of experiments lipoproteins were isolated and labeled from the same donor’s blood. 513

IRENE ViRooLiNret al.

514

0.01 M NaOH, > 100 mCi/mL). The specific activity of [‘231]NaI used was co.08 nmol/mCi. To achieve (I) Isolation of pur#ied lipoproteins satisfactory reproducible labeling yields and a conFor isolation of human lipoproteins 36 mL blood stant molar ratio I/LDL with varying radioactivity, from overnight fasting normolipemic volunteers (6 1 nmol NaI carrier was added. At the resulting molar males, 8 females, 25-35 years) were drawn into 4 ratio I/LDL of about 0.5 no denaturation of the Monovette vials (Sarstadt, Germany) and anticoaguprotein is to be expected. lated 1: 10 with 3.8% sodium-citrate. The reaction mixture (500 p L) was stirred slowly at (a) ImmunoaJinity chromatography. Polyclonal 4°C for 10min and applied to a Sephadex G25Manti-apo-B-antibodies were obtained by immunizing column (bed size 9 x 100 mm) which had been sheep with LDL. Gamma-globulins were precipitated preeluted with identical unlabeled lipoproteins. The from sheep plasma with ammonium sulphate ‘231-LDL-peak was collected from the 2.5 to 4.5 mL (390 g/L, Sanabo, Vienna, Austria) to a final conceneluate, phosphate buffered saline (PBS), pH 7.5, using tration of 35% and further purified by immunoaffina radioactivity and a U.V. detector, stabilized by ity chromatography. For this purpose 3 g of pure addition of 20 mg human serum albumin/ml (HSA; LDL were coupled to 400mL of 0.5 M BrCN20 mg/mL product solution) and finally sterilized by activated Sepharose Cl 4B (Pharmacia, Uppsala, 0.2 pm membrane filtration. Alternatively the reacSweden) according to Axen et al. (1967). The imtion mixture was sterile filtered into a dialysis bag munopurified antibodies were themselves couped to which was kept in dialysis buffer (0.15 M NaCl CNBr-activated Sepharose Cl 4B and this support 0.01 M P04, pH 7.5, 0.2 mM EDTA) until appliwas used to isolate apo-B-containing lipoproteins cation for in vitro binding studies. (LDL, VLDL) from volunteers’ plasma: 10mL of Radiochemical purity was determined by (a) TCAanti-LDL-sepharose Cl 4B gel were filled into a glass precipitation, (b) electrophoresis on paper (Whatman column (22 x 2 cm). The gel was extensively washed No. 1; 0.1 M barbital buffer, pH 8.6, containing with 500 mL of isotonic NaCl-solution. 1OmL of 1 mM EDTA and 1% HSA, 300 V for 10 min) and (c) volunteer’s titrated plasma were recirculated for polyacrylamide gel electrophoresis [PAGE; gradient 30 min over the column at a flow of 10 mL/min. The gel (T = 8-18); gel buffer 0.12 M Tris, 0.12 M acetate column was then washed with isotonic saline solution and 0.1% SDS, pH 6.4,200 V/25 mA for 20 min, then until it was protein-free (E 260/280 nm < 0.002). 600 V/25 mA for 60 min]. Lipoproteins were desorbed from the column with 2 Iodine-monochloride (ICI)-method. An ICI stock bed volumes of 0.2 M glycine/HCl, pH 3.0, and solution (34 pmol/mL 6 M HCl) was purified before dialyzed overnight against 5 L of isotonic saline. The labeling by 3 extractions with CHCl, and diluted solution was then concentrated by ultrafiltration on 1: 100 with aqueous 2 M NaCl. To a microvial kept AMICON XM 100 filters (Vienna, Austria) until a at 4°C approx. 1 mg purified lipoproteins (100 pL), final concentration of 10 mg LDL/mL was achieved 200 FL of 1 M glycine buffer, pH 10, about 1 mCi (LDL-protein measured by the assay kit provided by [‘231]NaI/10pL (IRE, Belgium, in 0.01 M NaOH, BIORAD Laboratories, Commassie Blue Reagent > 100 mCi/mL, co.08 nmol I/mCi) and freshly G20, Richmond, Calif, U.S.A.). diluted ICl-solution were added to give a molar (6) Ultracentrifugation. Plasma was achieved by ratio ICl/apoprotein of 10/l. The reaction mixture centrifugation (3000 g, 15 min, 1SC) and overlayed (0.5-l mL) was slowly stirred for 10min at 4°C with 0.9% NaCl (4:2). Following an 18 h ultracenand sterile filtered into a dialysis bag which was trifugation (L5-75 ultracentrifuge, Beckman Instrukept in dialysis buffer (0.15 M NaCl, 0.01 M PO,, ments Inc., Palo Alto, Calif., U.S.A., Rotor 40.3 Ti, pH 7.5, 0.2 mM EDTA) until application for in vitro 40,000 rpm, 1O’C) the VLDL-fraction was withdrawn studies. Alternatively the reaction mixture was and the pellet suspended in KBr-solution (d = purified by Sephadex chromatography as described 1.063 g/mL) [g KBr = plasma volume (mL) x above. Analysis for radiochemical purity was per(1.063 - 1.006) x 0.94 (K)/l - (0.295 x 1.0631 and formed in a manner identical to that for the Iodogen centrifuged against the density gradient for 18 h method. (Rotor 40.3 Ti, 40,000 rpm, 10°C). The supernatant To summarize, four different combinations of (d = 1.019-l .063 g/mL) contained the LDL-fraction. radiolabeling and purification were compared in subsequent binding studies: Iodogen and Sephadex, (II) Radiolabeling of pur$ed LDL Iodogen and dialysis, ICI and Sephadex, ICI and dialysis. Typical radiochemical yields and final Iodogen-method. In a microvial 500 pL chloroform solution of 3Opg Iodogen was evaporated with a activity concentrations are summarized in Table 1. stream of nitrogen, redissolved and blown dry again (III) Liuer membrane prepration to produce a homogeneous surface coating. To Human liver samples were obtained from patients the Iodogen coated vial were added approx. 1 mg undergoing (gastro)intestinal surgery for different of purified lipoproteins in saline, 0.01 M phoscancers of the abdominal tract. Routine morphology phate buffer, pH 7.5, 0.15 pg NaI-carrier/S PL and was assessed by hematoxylin-eosin staining. Only about 1 mCi [‘231]NaI/10~L (IRE, Belgium, in

Methods

in vitro

Comparative

study on

human

515

liver 1231-LDL-receptors

Table 1. Radiochemical yields and tinal activity Radiochemical yield (%)

Specific activity

Activity concentration

Labeling method

I/LDL molar ratio

(mCi/mg)

(pCi/mL)

Iodogen-Sephadex lodogen-dialysis El-Sephadex ICI-dialvsis

0.5 0.5 IO IO

88 92 69 72

0.88 0.92 0.69 0.72

50 50 50 50

Table 2. Radiochemical nuritv of ‘2’I-liooproteins at 5 min and 2 h after purification TCA-precipitate (%) Labelina method

5 min

2h

Iodogen-Sephadex Iodogen-dialysis ICI-Sephadex ICI-dialvsis

90+6 91+4 95 + 2 96 f 3

79 + 80 f 86 f 85 +

2h

5 min 5 5 3 5

)

Paperzlectrophoresis (% I

3.4 2.5 I.1 0.7

F 2.3 f 1.3 kO.2 * 0.02

9.6 6.0 4.9 3.2

+ + * f

4.2 2.3 2.2 2.2

f*SD,n=6.

tissue samples proven to show normal morphology were used for binding assays. Liver plasma membranes were prepared according to Neville (1968) as modified by us (Virgolini er al., 1989) on the day of tissue removal. The membranes were taken up at a protein concentration of 50 p g/ 100 p L assayed by dye-binding using

(A)

l

0

20

40 Lipoprotein

120

60

Iodogan - Saphodax Iodogen- Dialysis ICI-saphodex ICI-Dialysis

80

100

120

(V) Statistics

(pg protein/ml)

Binding data were analyzed by the Scatchard plot (Scatchard, 1949). Values are given as mean f SD. Significance was calculated by Student’s t-test.

r IB) l

t x 0

20

(IV) Binding studies In order to compare ligand binding to the lipoprotein receptor of human liver plasma membranes, direct binding experiments were carried out. The liver membranes (50 yg/lOO pL) were incubated with increasing concentrations of ‘231-lipoproteins (0.1-6 pg/mL) for 45 min at room temperature. After incubation the tubes were rapidly centrifuged (SOOOg, 10 min, 4°C) to separate membrane-bound from free radioligand. The pellet was counted in a gamma counter for 1 min. In typical experiments, nonspecific binding (determined in presence of a 50-fold excess of unlabeled LDL) amounted to less than 10% of total binding [SB = B - NSB = 100 - (< 10) = > 901.

120 r

+ x 0

the assay kit provided by Biorad Laboratories (Commassie Blue Reagent, G20, Richmond, Calif, U.S.A.). These membranes were directly used for binding studies if available on the same day, or were stored at -80°C for not longer than 14 days.

40 Lipoprotein

Iodogen-Sq2had8x Iodogan-Dialysis XC1- Sapbadax ICI -0ialyslr

60

80

(pg

protein/ml)

loo

Results Molar ratios of I/LDL used for labeling, typical radiochemical yields, specific activities and activity concentrations of final products are summarized in Table 1. Radiochemical purity of the four

120

Fig. 1. Ability of unlabeled human lipoproteins from normolipemic subjects to compete with ‘%LDL for binding to human liver plasma membranes. Each assay tube contained human 1231-LDL(0.75 ~g protein/ml; 625 cpm/ng protein) and the indicated concentrations of unlabeled lipoproteins. (A) Competition study after lipoprotein isolation by immunoaffinity chromatography and (B) by ultracentrifugation. For corresponding IC, values see Table 3.

Table 3. Concentration of unlabeled lipoproteins causing half-maximal inhibition (IC,) of ‘2’I-lipoorotein-bindinn to human liver olasma membranes IC, value (pg protein/ml) Labelinn method Iodogen-Sephadex Iodogen-dialysis ICI-Sephadex ICI-dialysis

IAC 7.9 8.1 9.3 8.0

* k f k

0.9 I.0 1.1 0.9

UC 7.7 f 8.0 f 8.8 * 7.22

I.1 0.9 1.0 I.1

f +_SD, n = 6; IAC: immunoaffinity chromatography; UC: uhracentrifugation

IRENEkk3OLlNI

516

2

(A)

?!

g

et al.

.E

s2

16Or

16Or

(AI

; 140 .s 120

7

v . +

F

x

e 100

IOdogen-sepdladex

Iodogen-Olalyrir ICI - S&laLX

f

; J 9 A ro 1231-LDL (PQ protein/ml)

n

+ x 0

60

o 60 40 20 0

1

2

c!

4

3

6

6

7

i231-LDL (pg protein/ml)

Iodcqen-Ssphadex t Iodogen-Diolyris X ICI -Sephodex 0 ICI-Dialysis l

Iodogen-Sghadrx Iodogen-Dialyrir ICI -saphadDx ICI -Dialyses

0.04 0.02 0 So ‘23*-LDZng Bound

160 protein/ml)

J x)0

Fig. 2. Saturation curve (A) and Scatchard analysis (B) of specific ‘231-lipoprotein-binding to human liver apo-B,E-receptors after lipoprotein isolation by immunoaffinity chromatography. Each assay tube contained the indicated concentrations of ‘231-lipoproteins (625 cpm/ng of protein). Specific binding was calculated by subtracting the amount of ‘*Wipoproteins bound in the presence of excess of unlabeled lipoproteins (50 pg protein/ml) from that bound in its absence. For the corresponding binding data see Table 4. measured by TCA-precipitation and electrophoresis at 5 min and 2 h after purification showed a slightly better result for the ICI-method of radiolabeling (Table 2). Specific binding reached equilibration within the 45 min incubation time at 4°C. In the presence of an excess of unlabeled lipoprotein (100 pg protein/ml) binding specificity amounted to approx. 95% [i.e. unlabeled LDL (1OOpg protein/ml) caused 95% inhibition of ‘231-LDL (0.1-6 pg/mL)-binding] independent of the use of ultracentrifugation (LDL) or immunoaffinity chromatography (LDL and VLDL) for lipoprotein-isolation or the use of the Iodogen- or ICI-method for radiolabeling (Fig. 1). The concentrations of unlabeled lipoproteins causing half maximal inhibition were very similar, and ranged from 7.7 to 9.3 pg protein/ml (Table 3). ‘231-lipoproteins (0.1-6 pg/mL) bound to liver plasma membrane receptors in a high affinity manner, independent of the method used for isolation or labeling. Bound/free ratios ranged from 0.08 to 0.17 indicating a single class of high affinity binding sites, Typical experiments for comparison of the Iodogen preparations

0

50 Bound

100

-J 200

150

1231-LDL (ng protein/ml)

Fig. 3. Saturation curve (A) and Scatchard analysis (B) of specific 1231-lipoprotein-binding to human liver apo-B,E-receptors after lipoprotein isolation by ultracentrifugation. For conditions see Fig. 2 and for the corresponding binding data see Table 4. Table 4. Binding capacity (B,,,, ng protein/mg liver plasma membrane protein) and binding affinity constant (Kd, pg protein/ml) for “‘I-lipoprotein-binding to human liver plasma membranes UC

IAC Labelina method

E_..

Iodogen-Sephadex Iodogen-dialysis ICI-Sephadex ICI-dialvsis

162 + 151 + 141 f 156+_

z? 5 SD, n = 6; IAC:

17 13 12 15

K,

B.“..

KA

I .3 f 0.8

163 + 21 154* 17 155 f 13 160 + _ 12

1.2kO.9 1.3 f 0.9 1.2_+ 1.1 0.9 + - 0.9

1.4+ 1.0

I .o+ 0.9 0.9+ I.1

immunoaffinity

chromatography;

UC:

and ICI-method of radiolabeling (and lipoproteins isolated by either immunoaffinity chromatography or ultracentrifugation) are presented in Figs 2 and 3. The &-value was somewhat lower for the IClmethods indicating an insignificantly higher binding affinity (Table 4). The B,,,,,-values as given in pg protein bound/mg liver protein, however, were in the same range (141-163 pg protein/mg liver protein).

Discussion 12’I-LDL, labeled by the method of MacFarlane (1958) (ICI-method) or its modification (Langer et al., 1972), has been used for many years to study the kinetic behavior of LDL in experimental animals and humans as well. In addition, almost all in oitro receptor studies have utilized the same labeling

Comparative in oitro study on human liver ‘2’I-LDL-receptors methodology (Kovanen et al., 1981). It was subsequently of interest to investigate whether lipo-

isolated through different methods proteins (ultracentrifugation, immunoaffinity chromatography) exhibit a similar binding behavior towards their receptors after different methods of radiolabeling, either with ICI (McFarlane, 1958) or Iodogen (Fraker and Speck, 1978). It is known that in various species more than half of the total LDL receptors are located in the liver (for review see Steinberg, 1983). Therefore, the liver seemed to be the optimal organ for comparative ligand studies after LDL isolation and radiolabeling. The results of this study show that lipoprotein preparations consisting of LDL (isolated by ultracentrifugation) and those consisting of LDL and VLDL (isolated by immunoaffinity chromatography) derived from the same donor maintain the same binding characteristics (specificity, saturation, competition behavior) for liver LDL receptor binding. Therefore, the presence of VLDL is not likely to affect the ability of LDL receptor binding to the liver. This observation is in accordance with the findings of Mahley and Innerarity (1983) who have previously shown that VLDL from normolipidemic subjects were nearly as active as LDL in binding to the LDL receptor of fibroblasts. In addition, the differences in radiochemical purity and stability between the four labeling purification methods were rather small (Tables I and 2), and we also observed a similar binding affinity between the two differently labeled preparations. This could be due to the fact that with both lableling methods, native LDL presenting a physiological receptor binding character was labeled. Apart from the observation of Naruzsewicz et al. (1984) that the fractional catabolic rate of LDL is similar after labeling with the iodogen method (Fraker and Speck, 1978) or iodine monochloride method (McFarlane, 1958), the results of this comparative study indicate that isolation of lipoprotein via immunoaffinity chromatography or ultracentrifugation and radiolabeling via the Iodogen- or ICI-method can be recommended as equivalent techniques for production of tracers for in vitro receptor evaluation. Acknowledgements-The

authors would like to thank Drs W. Klepetko (2nd Department of Surgery, University of Vienna, Austria) and M. Hermann (Department of Surgery, Kaiserin Elisabeth Hospital Vienna) and L. Lonsky, M.D. (Immuno AG, Vienna, Austria) for their continuous cooperation.

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