Radioimmunoassay of human plasma apolipoproteins

4therosclerosiq 21 (1975) 217-234

CC) Elsevier Scientific Publishing Company, Amsterdam - Printed in The Netherlands

RADIOIMMUNOASSAY

PART

OF

HUMAN

PLASMA

217

APOLIPOPROTEINS

1. ASSAY OF APOLIPOPROTEIN-B

G. J. BAUTOVICH, L. A. SIMONS*, P. F. WILLIAMS AND J. R. TURTLE Departtnmt of Medicine, UuiversitJj of Sydney, Sydney, N.S. W. 2006 (Australia) (Received August 19th, 1974) (Accepted October 23rd, 1974)

SUMMARY

human

A specific radioimmunoassay for apolipoprotein-B, plasma B-lipoprotein, is described. The antigen

density antibody

sub-class

of /3-lipoprotein

was produced

(density

by immunisation

the dominant apoprotein of for the assay was a narrow

1.020-1.050),

of the rabbit.

labelled

separated using a double antibody precipitation technique. sensitivity was approximately 5 ng protein, with a working Dilutions

of plasma and various lipoprotein

fractions

with 1251, and the

Bound and free fractions

were

The absolute lower limit of range of from 10 to 200 ng.

displaced

in a parallel fashion to the standard curve, demonstrating apo-B. There was limited cross-reactivity with monkey

labelled

B-lipoprotein

specificity of the assay for serum. Apoprotein-B was

undetectable in a-/I-lipoproteinaemia serum. Mean plasma apo-B concentration ( j: I S.D.) in healthy subjects was 90 & 24 mg/lOO ml, with a positively skewed distribution. Concentrations of apo-B were increased in all types of hyperlipoproteinaemia. There was a significant positive correlation between plasma apo-B and plasma cholesterol concentrations. In health, 94% of total plasma apo-B was confined to j%lipoprotein but there was a shift to pre-/3-lipoprotein in hypertriglyceridaemia.

Key words : A-P-lipoproteinaemia

-

Apolipoprotein-B

-

H,yperlipoproteinaemia

-

Radioimmunoassay

This work was supported by a grant from the National Heart Foundation of Australia. Requests for reprints should be addressed to Dr. L. Simons. * Present address: Medical Professorial Unit, St. Vincent’s Hospital, Darlinghurst, N.S.W. 2010 (Australia).

218

G. J. BAUTOVICH, L. A. SIMON& P. F. WILLIAMS, J. R. TURTLE

INTRODUCTION

The plasma

lipoproteins

have a unique

content of cholesterol, trigiycerides, fractions consist of a large number peptides

chemical

composition

based upon the

phospholipid and apoproteinl. The apoprotein of complex polypeptide molecules, these poly-

making a varying contribution

to the different

density classes2. In one classi-

fication the polypeptides are regarded as belonging to at least 3 broad apoprotein groupings or families-apo-A, apo-B and apo-C, based on their immunological reactivity3. APO-A is predominantly found in high density or a-lipoprotein, apo-B is low density or /I-lipoprotein and apo-C is very low density or pre-@-lipoprotein. However, this distribution is not absolute and all apoproteins contribute to each density class*. Recently, a fourth apoprotein, apo-D, was describedg. Apoprotein polypeptides have been subfractionated into constituent peptides, which have in turn been characterised immunologically

and

transport in plasma, formations8~g. Current

classifications

physicochemical flotationlo.

by amino

acid

analysis 6~7. The

as well as serving as essential

separations

AlaupoviC”

of plasma

lipoproteins

such as electrophoretic

has recently

proposed

apoproteins

cofactors

facilitate

lipid

in certain enzymic

trans-

and lipid disorders mobility

an alternative

and

are based on ultracentrifugal

apolipoprotein

family

concept, which is based on the theory that each of the apoproteins A, B and C exist free in the circulation with a characteristic complement of lipids. Evidence for this classification remains circumstantial. Whereas there has been much epidemiological evidence linking hypercholesterolaemia with premature atherosclerosis12J3, abnormalities of apoprotein metabolism have been relatively neglected. The technical difficulties of isolating apolipoproteins (delipidation, gel filtration, ion-exchange chromatography) have precluded any approach on an epidemiological scale. Recent metabolic studies involving the turnover of apolipoproteins have again focussed attention on their possible primary importance in hyperlipoproteinaemia1*,15. Since the principal apoprotein families are defined immunologically, they can be measured most easily by immunoassay, without preliminary fractionation techniques. Contemporary immunoassays of apo-B, an apoprotein of fundamental importance in cholesterol metabolism, are based on quantitative radial-immunodiffusion or electroimmunodiffusion in agar or agarose gel 16917,but do not lend themselves easily to automation, large sample capacity and precision of the radioimmunoassay procedure in a liquid system. The present communication describes the development and application of a specific radioimmunoassay procedure for apo-B in human plasma, based on a conventional double-antibody technique 18. Preliminary findings have been published previously 1g-23.

* Abbreviations used: VLDL, very low density lipoproteins (d 0.95-1.006); LDL, low density lipoproteins (d 1,006-l ,063); HDL, high density lipoproteins (d 1.063-I .21); Apo-VLDL, 55 % apo-B, 45 % apo-C and trace amounts of apo-A “; Apo-LDL, 95 ‘A apo-B and trace amounts of apo-A and apo-C2; Apo-DHL, SS-90% apo-A, 5% apo-B, and 10% apo-Cj.

RADIOIMMUNOASSAY

OF APOLIPOPROTEIN-B

219

METHODS

A narrow

sub-class

the radioimmunoassay class consists almost

of LDL (d 1.020-1.050)

for

procedure. It has been shown that the apoprotein of this subexclusively of apo-B, with oniy trace quantities of apo-A, and

apo-C2. These trace quantities under the existing

was used as an apo-B standard

are insufficient

experimental

to generate

significant

levels of antibody

conditionsa.

Preparation of LDL (density 1.020-1.050) Blood (500 ml) was collected in a plain plastic pack from a healthy donor and allowed to clot at room temperature over a period of 2 hr. All subsequent procedures were performed at 4°C. The cells were removed by low-speed centrifugation and EDTA added to the serum (1 mg/ml). Solid KBr was added to bring the sample to a solvent density of l.020z4. All 10 tubes of a Beckman-50 rotor were filled and the sample centrifuged The supernatant centrifugation

for 22 hr at 105,000 x g(Beckman

L-2 preparative

ultracentrifuge).

was removed by tube slicing and 2 ml infranatant was washed by re( x 2) at d 1.020 under the same conditions. Further solid KBr was

added to this washed infranatant

fraction

(d 1,020) to bring it to a final solvent density of 1.050. This sample was centrifuged for 22 hr at 105,000 x g. The 2 ml supernatant and washed by recentrifugation (1.020 < d < 1.050) was removed by tube-slicing ( x

2) under the same conditions. Purified LDL (d 1.020-I .050) was dialysed exhaustively against 0.01 “/, EDTA, pH 7.4 to remove all traces of NaCl and KBr. MerthiolateB (I :5000, w/v) was added to prevent bacterial growth. The sample was divided and solid NaCl added to one half only, to a final concentration of 0.15 M. Small aliquots were placed in glass ampoules,

sealed under nitrogen

and stored in the dark at 4°C. LDL was used subse-

quently for immunisation, iodination and as standard reference protein in the radioimmunoassay: it was observed to be stable under these storage conditions for at least 4 months. Antiserum

to LDL (anti-B)

0.5-I mg LDL protein was emulsified plete adjuvant and injected subcutaneously

with an equal volume of Freund’s into New Zealand white rabbits,

comusing

multiple injection sites. Three weeks later the injection was repeated with incomplete adjuvant. The animals were bled 2 weeks later by ear-vein and the serum separated by low-speed centrifugation. Merthiolate (1:5000) was added and the antiserum stored at -20°C. lodination of LDL Four hundred pg LDL protein (NaCl-free) was labelled with 2 mCi of lzjl the chloramine-T method at pH 7.5 and 4”C?s. 35 ,ug chloramine-T were used and reaction stopped 60 set later with 200 pg sodium metabisulphite. The efficiency iodination was approximately 70%, with an estimated molar iodide-protein ratio

by the of of

220

G. J. BAUTOVICH, L. A. SIMON& P. F. WILLIAMS, J. R. TURTLE

2:l (assuming

an LDL molecular

weight of 3 x 10s daltons).

Extraction

with chloro-

form-methanol (2: 1) showed that less than 5 “/, of label was bound to lipid. Inorganic radioiodide was removed by gel filtration through SephadexB G-50 (column 20 x 1 with 0.05 M sodium

cm, equilibrated

barbitone,

0.001 M EDTA,

pH 8.2, l-ml frac-

tions). Labelled LDL appeared in the void volume (6-9 ml). These fractions were pooled, diluted with an equal volume of 5 “/, bovine serum albumin in 0.05 M barbitone, 0.001 M EDTA,

pH 8.2 and stored at 4°C. The preparation

useful immunoreactivity for at least 1 month. On the morning of each assay a final definitive ed by gel filtration 0.5% bovine ml fractions).

through

SephadexO

serum albumin Three

in 0.05 M barbitone,

of LDL was performwith 40 x 2 cm, equilibrated

0.001 M EDTA,

yield of radioactivity

approximately 23 ml LDL eluted in the void volume. eluted was used in the assay (see RESULTS). Radioimmunoassay procedure The radioimmunoassay was performed using a buffer of 0.5 y0 bovine serum albumin

stable and

purification

G-200 (column

to 4 times the desired

retained

pH 8.2 at 4°C; 1 was applied

and

Only the first 2-3 ml of activity

in polystyrene tubes (6.2 x 0.8 cm), in 0.05 M sodium barbitone, 0.001 M

EDTA, pH 8.2 (BSA buffer). The principle of radioimmunoassay for apo-B is that of competition between labelled tracer and unlabelled antigen for a limited number of antibody-binding sites, as described for radioimmunoassay procedure is summarized in Table I. The assay employed

a conventional

second

of insulins6.

antibody-precipitating

The assay system,

to

TABLE 1 SUMMARY

OF THE RADIOIMMUNOASSAY

PROCEDURE

AND ITS OBJECTIVES

Reagents added

Requirements

0.014.3

standard diluted to I rn/lg//d of protein (Lowry); unknowns as specified. diluted to yield 50-70x binding of label in tube containing no unlabelled LDL (“zero”tube) to final volume 0.425 ml

ml LDL standard or unknown sample 0.125 ml specific antiserum (diluted) BSA buffer

Tubes vortexed and incubated for 24 hr at 4°C 0.1 ml labelled LDL

10,000 counts/min

Tubes vortexed and incubated further 24 hr at 4°C 0.1 ml normal rabbit serum (I :300) 0.02 ml donkey anti-rabbit precipitating serum (1) (2) (3) (4)

“carrier” rabbit protein “second antibody” system to yield “bound”

Tubes vortexed and incubated at least 12 hr at 4°C. 0.5 ml BSA buffer added and tubes centrifuged (20 min, 1100 x g, 4°C). Supernatant aspirated. 1.5 ml BSA buffer added and tubes recentrifuged. Supernatant aspirated and precipitate counted (“bound” fraction).

fraction

RADIOIMMUNOASSAY

separate “bound”

OF APOLIPOPROTEIN-B

from “free” label 18. Preliminary

221 experiments showed that the

usedweresufficient to produce maximum precipitation of the labelledLDL-

quantities

antibody complex. LDL standards were assayed in triplicate from 0 to 300 ng. Unknown samples were assayed in duplicate in appropriate dilutions (I:500 for whole serum and LDL; 1:50 for VLDL; 1:25 for HDL; 1:50 for chylomicrons). A standard curve was constructed with apo-B concentration (ngprotein) on a logarithmic abscissa versus percentage of total counts “bound” on the ordinate (see RESULTS). Subjects

and analytical

methods

Normal subjects (medical students and university employees, aged 20-35 yr, clinically healthy and free of metabolic disease) and a number of patients with hyperlipoproteinaemia (attending the Lipid Clinic, Royal Prince Alfred Hospital, Sydney, Australia) were bled in the absence of venostasis, after a 12-14 hr overnight fast. Blood was collected in EDTA containers and plasma was separated immediately at 4°C. Merthiolate (1:5000) was added, prior to storage at 4°C. Plasma lipoproteins were fractionated in the ultracentrifuge according to the method of Have1 et ~1.27. Plasma cholesterol was measured on the autoanalyser (Technicon N-24a) 28. Lipoprotein fractions were treated with 400 mg “Dowex” AG2-X8 (chloride form) to remove KBr where appropriate, prior to cholesterol estimation. Triglycerides were measured enzymatically after hydrolysis utilising the LKB-8600 Reaction Rate Analyser 29. Lipoprotein protein was measured by a modification of the Lowry procedureso, using crystalline bovine serum albumin as standard. Merthiolate was found to enhance colour development in the Lowry assay and was removed by preliminary treatment of all samples with resin (as for the cholesterol estimation). Gel double diffusion, immunoelectrophoresis, polyacrylamide gradient gel electrophoresis, and paper electrophoresis were all performed according to published proceduressl-34. MATERIALS

Carrier-free lzsI was obtained from the Radiochemical Centre, Amersham, U.K.; SephadexB from Pharmacia (South Seas) Pty. Ltd., Australia; “Dowex” resins from Bio-Rad Laboratories, California, U.S.A.; bovine serum albumin (fraction V) from Armour Pharmaceutical Co., U.K.; donkey anti-rabbit precipitating serum (RD 17) from Wellcome Diagnostics, U.K.; other commercial antisera from Behringwerke, Germany (rabbit antihuman @-lipoprotein, anti-a-lipoprotein, antiwhole human serum). Samples were counted on an LKB-Wallac auto-gamma counter. RESULTS

Specificity

of the antiserum

Rabbit anti-LDL serum produced a single precipitin line against whole human serum or LDL on immunoelectrophoresis in agarose gel. In agar gel double diffusion,

222

G. J. BAUTOVICH,L. A. SIMON&P. F. WILLIAMS, J. R. TURTLE

Fig. 1. Agar gel double-diffusion. A: anti-LDL LDL and anti-p-lipoprotein (Behringwerke).

WYSUSLDL and whole serum; B: LDL VPYSUS anti-

anti-LDL serum produced a single precipitin arc against whole human serum and LDL, demonstrating a line of identity (Fig. 1A). Anti-LDL serum and a commercial antihuman /?-lipoprotein serum produced further line of identity (Fig. IB).

a single arc against

LDL, demonstrating

a

Immunochemical and radiochemical purity qfLDL Purified LDL (d 1.020-1.050) produced only a single precipitin line against rabbit anti-whole human serum and specific anti-LDL serum on immuno-electrophoresis. Double diffusion of LDL against anti-LDL serum produced a single precipitin arc (Fig. IS) and a single arc for LDL against anti-whole human serum. Radioimmunoelectrophoresis of labelled LDL against anti-LDL serum demonstrated a single

radioactive

precipitin

line.

In polyacrylamide

gradient

gel electrophoresis,

LDL migrated as a single band. Paper electrophoresis of labelled LDL eluted from Sephadex G-50 and stored for a few days showed 3 radioactive peaks, A, B, and C, indicating the need for further purification

(Fig. 2A). Gel filtration

of this impure

product

through

Sephadex

G-200

also resolved the sample into 3 radioactive peaks, I, II, and III (Fig. 2B). Eluate from 23-30 ml (peak I corresponding to peak A) reacted with specific antiserum in the immunoassay, whereas eluate from 40-55 ml (peak II corresponding to peak B) showed no such immunoreactivity. Peak III (corresponding to peak C) showed no immunoreactivity and coincided with the elution of inorganic iodide. Only the first 2-3 ml of peak 1 eluted from G-200 were used as labelled antigen in the radioimmunoassay, to utilise full immunoreactivity and achieve maximum specific radioactivity. Antiserum dilution curve A radioimmunoassay

was performed

with 2 variations

from the usual protocol.

RADIOIMMUNOASSAY OF APOLIPOPROTEIN-B

APER

ELECTROPHCflESI.5

223

(G-50)

C

I

JI

-

MIGRATION --,

ELUATE

VOLUME

ML

Fig. 2. A : radioscan of paper electrophoretic strip from labelled LDL after Sephadex G-50 chromatography and 3 days storage; B: radioactivity elution profile of the above material following filtration through Sephadex G-200.

3

6

9

12

RECIPROCAL ,poAi’&lS~~l

6

18

21

DILUTION

Fig. 3. Antiserum dilution curve relating percentage of label bound dilution (see text for details).

versus

reciprocal of antiserum

224

G. J. BAUTOVICH,

L. A. SIMON& P. F. WILLIAMS, J. R. TURTLE

NANOGRAMS LD;Ap;OIEIN (LOWRY)

Fig. 4. Standard curve for radioimmunoassay of apolipoprotein-B. Percentage binding of labelled LDL standard is plotted against ng protein (assayed by the method of Lowry). Antiserum dilution 1:6250 (see text for details).

Unlabelled antigen was omitted and the volume replaced with BSA buffer. Secondly, 0.125 ml specific antiserum was used in a series of doubling dilutions. Reciprocal of anti-serum dilution was plotted on the abscissa against percentage “bound” on the ordinate (Fig. 3): 93 % binding of label in the region of antibody excess was evidence of full immunoreactivity of the tracer; 3 % binding of label in the absence of specific antibody

was evidence

of an acceptable

degree of “non-specific

binding”.

The steep

fall of the curve was indicative of a potentially useful assay system. In the radioimmunoassay, optimal sensitivity was achieved by using an antiserum dilution yielding 50-70% binding in a tube containing no unlabelled antigen. This dilution varied from one batch of antiserum to another (I : I ,000-l : 65,000). The radioimmunoassay and its speciJicity A representative standard curve for the radioimmunoassay of apo-B is shown in Fig. 4, plotting percentage of label bound versus ng protein (Lowry assay). 64% labelled LDL was precipitated in the absence of unlabelled LDL, this figure falling to 5% in the presence of 300 ng LDL standard. Variation between replicates was less than 3%. The working range of the assay was IO-200 ng protein, with maximal sensitivity between 20-100 ng; samples were appropriately diluted with BSA buffer to

RADIOIMMUNOASSAY

OF APOLIPOPROTEIN-B

10000

RECIPROCAL

IO

1000

OF

225

DILUTIOf?

WV)

Fig. 5. Displacement of labelled LDL by dilutions of plasma, VLDL, LDL, HDL, chylomicrons, a-fi-lipoproteinaemic plasma and a plasma fraction of density :, 1.21. (Chylomicrons were from a patient with Type V hyperlipoproteinaemia.)

fall within this range. This assay has been in routine production for 2 years; many batches of LDL standard have been prepared, and more than 100 standard curves obtained-all

standard

curves

have been virtually

superimposable.

Four

samples

of

plasma were assayed repeatedly over a 4-month period using varying dilutions and different standard LDL preparations. There was no tendency for the values to change over this period, and the samples had respective coefficients of variation of 6 %,5 X),7 ‘4 and 7”/,. To assess the quantitative recovery of the assay, known amounts of LDL protein (30-150 ng) were added to normal and hypo-@-lipoproteinaemic plasma and the samples re-assayed. protein

There was a high correlation

(Y = 0.994, P < 0.001). Absolute specificity of the radioimmunoassay

between added and assayed LDL for apo-B was demonstrated

by

assaying dilutions of serum from a patient with a-/I-lipoproteinaemia. This sample in dilutions down to 1 :lOOO showed no displacement of label whatsoever (Fig. 5). Assay of a plasma fraction of density > 1.21 produced a similar result, indicating the complete absence of apo-B (Fig. 5). Further specificity was established by demonstration of clear parallelism between displacement curves produced by serial dilutions of whole plasma, chylomicrons, VLDL, LDL and HDL (Fig. 5). Species specificity was established by a similar approach comparing displacement curves produced by dilutions of animal sera. Of the species examined (monkey, pig, rat, cow, chicken, guinea pig, rabbit, horse, cat, mouse and sheep), only monkey serum showed significant displacement of labelled human LDL; and that curve was not parallel to one from human plasma.

G. J.

226

50-69

m-49

PLASMA

Apoprotein-B Plasma

90-109

70-69

APO-B

Fig. 6. Frequency distribution subjects.

110-129

L. A. SIMON&

130-149

P. F. WILLIAMS,

J. R. TURTLE

149+

CONCENTRATION mg/looml

of plasma apolipoprotein-B

concentrations was obtained

BAUTOVICH,

concentrations

in normal subjects from 82 healthy subjects,

in a population of healthy

aged 20-35 yr, with sampling

from both sexes (33/49). The mean cholesterol of the population + 1 S.D. was 190 & 33 mg/lOO ml. Mean triglycerides were 77 f 28 mg/lOO ml. There was no significant sex difference with respect to plasma lipids. The frequency distribution of apo-B levels in normal human plasma is demonstrated in Fig. 6. This distribution was skewed to the right but could be “normalised” by logarithmic transformation. The mean apo-B level was 90 & 24 mg/lOO ml with a range of 49-152 mg/lOO ml. Mean levels in females

TABLE 2 PLASMA

LIPIDS

AND

APOLIPOPROTEIN-B

CONCENTRATIONS

IN

NORMAL

SUBJECTS

AND

IN

HYPERLIPO-

PROTEINAEMIAa

Class

No.

APO-B (mg/IOO ml)

Cholesterol (mgj100 ml)

Triglycerides (tngllO0 tnl)

Normals Type IIab homozygotes Type Ilab heterozygotes Type IIb Type IV Type V

82 4 8 10 11 10

90 i 383 & 237 & 257 & 132 * 126 +

190 &

765 385 438 247 482

77 I 28 90& 21 90 + 48 402 + l17r 492 -1 186’ 3006 & 2301c

24 43? 47” 6V 21c 30c

5 Means i 1 S.D. b Familial disease according to published criteriaa”. Groups compared with normal subjects by Student’s r-test. c Significantly different P < 0.001.

I I + & rt

33 62C 6gC 106’ 38’ 226r

RADIOIMMUNOASSAY

: 8

227

OF APOLIPOPROTEIN-B

A

6 NORMAL SUBJECTS

II 100 PLASMA

HYPERLIPIDAEMIC SUBJECTS ’

I 150

200

CHOLESTEROL

250

CONCENTRATION

mg/lOOml

’

I

200 PLASMA

400 CHOLESTEROL

II

600

I

600 ‘im

CONCENTRATION

mg@3,,1

Fig. 7. The relationship between plasma apolipoprotein-B and plasma cholesterol concentrations. A : normal subjects; B: hyperlipidaemic subjects. (For the sake of clarity, only half of the population represented in Fig. 6 is presented in Fig. 7A).

were about significant.

6 mg/lOO ml higher than in males but this difference

Apo-B concentrations

was not statistically

in hyperlipoproteinaemia

Plasma was obtained from patients with different types of hyperlipoproteinaemia. Diagnoses were confirmed by clinical features, lipoprotein electrophoresis and preparative centrations

ultracentrifugation. are fully tabulated

The mean cholesterol. triglycerides and apo-B conin Table 2. Plasma apo-B levels were significantly ele-

vated in Type II, and also to a lesser extent in Types IV and V. The correlations

between

plasma

apo-B and plasma

cholesterol

in normal subjects and hyperlipoproteinaemics are presented respectively. In normal subjects there was a significant positive plasma

apo-B

and cholesterol

concentrations

concentrations

in Figs.

correlation

7A and B between

(r = 0.74, P CC 0.001). In hyperlipo-

proteinaemia the precise functional relationship between plasma apo-B and cholesterol differed between the types. Plasma apo-B and cholesterol were not significantly correlated in Type IIa heterozygotes (r = 0.69, 0.05 < P < 0.1). The results were scattered and the numbers small. Plasma apo-B and cholesterol were significantly correlated in Types IIb, IV and V (r = 0.86, P < 0.01 ; r = 0.82, P < 0.05; r = 0.74, P c.. ’ 0.01, respectively). In the hyperlipidaemic group all the data were grouped close to the regression line for normal subjects, with the exception of Type V patients. In Types IIa, IIb and IV, plasma apo-B and plasma cholesterol appeared to have the same functional relationship as in healthy subjects. A different relationship clearly applied to Type V (Fig. 7B). There was no significant correlation between plasma

_

c

II

a

27c 20

19Y 48

196~ 69 99 27 67” 26

14.1c 9.4 16.6C 6.4 28~ 12

6.2

1.1

2.8 2

85 18 369~

2.3 1.5 1.6

8.3C 4.9 5.1” 3.2 6.8C 4.2

5.4” 2.6

0.5

3 1.6 4.9

219c 178 179c 108

83c 51 78c 37

26 13

17 12 25 17

VLDL

Data for each group are presented in two rows: (1) Mean in mg/lOO ml; (2) 1 S.D. Significantly different P < 0.01. Significantly different P < 0.001. Means compared with corresponding value in normal subjects by Student’s t-test.

(10)

HDL Chylos

LDL

Chylos

VLDL

Cholesterol

APO-B

UISTRIBUTIONSa

Normals (431 Type IIa homozygotes (41 Type IIa heterozygotes (8) Type IIb (10) Type IV (11) Type V

Group

LIPOPROTEIN

TABLE 3

280c 86 106 26 50c 20

294c 81

112 24 626C 59

LDL

65 16 44c 8 35’ 9

53 17

62 12 43” 9

HDL

1816C 1953

Chylos

117 357’ 216 1106r 505

262~

35 32

28 17

37 20

VLDL

Triglycerides

1lOC 28 76c 24 58 40

45c 14

11 61’ 2

25

LDL

31h 17 58c 4.7 26? 9

8

10

-

9 4c

15

HDL

RADIOIMMUNOASSAY

OF APOLIPOPROTEIN-B

apo-B and triglyceride Apo-B concentration Plasma

concentrations

from about

into the respective

The respective

in normal

subjects

or hyperlipoproteinaemics.

in lipoproteins

samples

fractionated

229

concentrations

half the normal

lipoprotein

subjects

and all the patients

classes by preparative

of apo-B, cholesterol

ultracentrifugation.

and triglycerides

Table 3. In Type 11 there were significantly increased (P < O.OOl), and in Type V a significantly reduced

were

concentrations concentration

are presented

in

of LDL apo-B (P < 0.01). In

Types Ilb, IV and V there were significantly increased concentrations of VLDL apo-B (P < 0.001). There were significant increases in HDL apo-B levels in all types; this was of little quantitative importance, but was suggestive of a qualitative change in composition. Plasma apo-B and LDL-cholesterol were not significantly correlated in normals or in Types IIa, IV or V. However, they were significantly correlated in Type IIb (r = 0.68, P < 0.05). Ninety-four percent of plasma apo-B of healthy subjects was confined to LDL. This was little changed in Type II, but fell to 83 “/, in Type IV and to 51 “/, in Type V. VLDL apo-B was 2.5 % of the plasma total in normal subjects, increasing to about 15 T/Qin Types IIB and IV and to 28 % in Type V. Chylomicrons accounted for 27 “,‘iof the plasma apo-B total in Type V, but these samples were not exhaustively washed and may have been contaminated. Lipid-apo-B ratios in lipoproteins have revealed some important changes in composition. For example, cholesterol/ape-B of VLDL in Type IIa homozygotes was 15.6, in Type IIa heterozygotes 9.3, and in Type IV, 4.7, compared to 7.4 in normals. Cholesterol/ape-B of LDL in Type V was 0.7 compared to I .3 in normals. Similarly, triglyceride/ape-B of VLDL in Type V was 39.5 compared to 16. I in normals; comparison

of LDL it was 0.9 in Type V compared

and statistical

analysis

of lipoprotein

composition

to 0.3 in normals. will be published

A full later.

DISCUSSION

There is abundant

evidence

associating

premature

coronary

heart disease with

elevations of plasma cholesterol and triglyceride concentrations12>1”,36,37. The specific apo-proteins which accompany cholesterol and triglycerides in plasma, apo-A, B and C, have been technically very difficult to assay in the past. Although the apoproteins have been demonstrated to exist in atheromatous lesions 38, their unique role in pathogenesis, if any, has remained somewhat obscure. It is clear that apoproteins do not play merely a passive metabolic role as “solubilising” agents. They are essential cofactors in the regulation of lipoprotein lipases and lecithin-cholesterol acyl transferContemaseg, as well as being absolutely essential for the synthesis of chylomicrons. porary research has not as yet been able to provide any simple assay of apoprotein for widespread application in the clinical laboratory or as an epidemiological tool. The aim of the present work has been to develop simple, rapid methods for assay of the major apoprotein families of plasma. We have chosen apo-B (also known as apo-LDL or apo-LP-Ser), because of its fundamental role in cholesterol metabolism. Using

G.

230 methods

J. BAUTOVICH,

which are totally analogous

L. A. SIMON&

P. F. WILLIAMS,

to the assay of plasma insulin,

J. R. TURTLE

we have succeeded

in producing a radioimmunoassay for human apo-B which is simple, absolutely specific, highly sensitive and capable of automation for clinical and epidemiological research. Validation

In designing

a radioimmunoassay

one requires

either a pure antigen

or a mono-

specific antibody. Apo-LDL consists predominantly of apo-B, but also contains trace quantities of apo-A and apo-C2. Alaupovic et a1.2 have stated that these trace quantities are insufficient to immunise rabbits under the conditions we have chosen. We have also shown that immunisation with LDL (d 1.020-l .050) only produces a single antibody species which gives a reaction of identity with commercial anti-P-lipoprotein. Radioimmunoelectrophoresis of labelled LDL demonstrated only one labelled precipitin arc. We have immunoassayed the LDL standard for apo-A by a new assay, currently approaching completion (Simons, L. A., unpublished observations). APO-A content of LDL standard was 0.012 % of total protein. These findings support the concept that we are measuring indeed only a single protein species. Other material which might be measured simultaneously cannot be of quantitative significance. We have chosen to label LDL with the chloramine-T methodz5. This method is in general use in many radioimmunoassay laboratories and has proven very satisfactory. We have employed precisely the same techniques to study the in Go metabolism of apo-LDLts. Turnovers of apo-LDL labelled by this method have given comparable results in normal subjects to those in whom LDL was labelled by the iodine monochloride methodi4J9. After labelling, we have noted the presence of damaged nonimmunoreactive protein. This material may have arisen before or after labelling. Filtration

through

Sephadex

G-200 was successful

in yielding

a completely

immuno-

reactive labelled tracer. The product was stable for at least 7 days and was well suited for use in the assay. Assay of serum from a patient with a-b-lipoproteinaemia failed to demonstrate any immunoreactivity

whatever.

This rare disorder

is characterised

by the inability

to

synthesise apo-B, and by zero plasma levels of chylomicrons, VLDL and normal LDL40. Our finding confirms the absolute specificity of the radioimmunoassay for apo-B. Further specificity was confirmed by parallelism of displacement curves for LDL, VLDL, HDL and whole serum. Seidel et al.41 have described antigenic masking of albumin in lipoprotein-X, the obstructive jaundice lipoprotein. Parallelism of our displacement curves would suggest that this is an unlikely event for apo-B, which is presumably disposed on the molecular surface of the respective lipoproteins. Addition of 30-I 50 ng LDL standard to whole plasma produced complete recovery in the assay. The absolute sensitivity of the assay, defined as the minimum quantity of protein detectable from zero, is approx. 5 ng. In the range lo-80 ng, we are able to distinguish differences of 1 ng. However, the objective of the assay is specificity as well as sensitivity. Plasma levels are high by immunoassay standards and all samples require high dilution. This is inconvenient and a possible source of error. The standard curve may

RADIOIMMUNOASSAY OF APOLIPOPROTEIN-B

231

be shifted to the right with reduced absolute tion

interval

gents

after addition

of label.

at the commencement

of

sensitivity

by prolongation

This may also be achieved

the assay

(label,

standard,

of the incuba-

by addition

of all rea-

The method of separation of “bound” from “free” label was based on a conventional second antibody technique. A modification of this step was attempted by coupling of the specific antibody body exhibited

to a solid phase (CNBr-Sephadex,

a dramatic

felt that the conventional against

combination

loss of binding

capacity

assay was preferable.

specific antibody).

ultrafine).

However,

under these conditions

This may represent

the antiand it was

a steric hindrance

with large moleculesaa.

Our findings suggest that we are assaying a homogenous protein species, c.g., monospecific antibodies, one labelled antigen, parallel displacement curves, absent protein in a-P-lipoproteinaemia, and, in addition, the steep slope of the antiserum dilution

curve (Fig. 5). It is important

to know whether we are able to measure

any of

the antigenic variants of LDL, e.g., Lp(a) variant 42. Lp(a) positive lipoprotein is capable of precipitation by anti-LDL antisera, but it has been found to occur only at d :> l.05042. On this basis it has probably been excluded from the labelled antigen (d 1.020-I .050) and from the assay. Applications

of the assay

In the absence of delipidation, apo-B has previously been assayed by a number 16,43~47and electroimmunoof methods, including quantitative radial immunodiffusion diffusion17. Plasma levels of apo-B in the present assay were in excellent agreement with earlier published data, both for normal subjects and hyperlipoproteinaenncslti~r~. The earlier methods suffer from the disadvantage that they are not readily automated and that larger lipoprotein molecules, such as chylomicrons and VLDL, are poorly diffusible

in solid media and may not be assayed. The frequency-distribution

of plasma

apo-B in normal subjects appeared to parallel previously published data for cholesterol and triglycerides with a positive skew12 (Fig. 6). If there is a significant sex difference in plasma levels we will require a larger population sample to define it. APO-B levels in plasma

were significantly

elevated

in hyperlipoproteinaemia,

particularly in the presence of increased LDL (Type IIa and Ilb). There was no functional relationship between plasma apo-B and plasma triglycerides, but there was a correlation between plasma apo-B and plasma cholesterol. This correlation was statistically significant for all groups except Type IIa. In Type Ila the data were scattered above the regression line for normal subjects (Fig. 7B) and a significant correlation might have resulted from a larger group with less variability. An overall lack of correlation between plasma apo-B and LDL-cholesterol emphasised the contribution of apo-B from other density classes (particularly chylomicrons and VLDL in Type V) and the changes in lipoprotein composition between individuals and between lipoprotein typeslc. Whereas there was a similar functional relationship between plasma apo-B and plasma cholesterol in normals and in hyperlipoproteinaemia Types 11 and IV, the findings in Type V were unusual. We have previously shown that the plasma pool of LDL in Type V may be reduced to less than half and that its size

G. J. BAUTOVICH,

232 varies inversely

L. A. SIMON& P. F. WILLIAMS,

with the size of the lipoprotein

pool of density

J. R. TURTLE

< 1.0064*. In health

about 94 % of plasma apo-B is in LDL, whereas in Type V some 44 % is in chylomicrons and VLDL. This finding emphasises the importance of apo-protein B beyond the plasma

pool of LDL.

In preliminary studies”2 we attempted to express apo-B as a percentage of total apo-VLDL (according to the method of Lowry). These studies were performed using washed VLDL,

which contained

no albumin

as estimated

by immunodiffusion

and

immunoelectrophoresis. However, polyacrylamide gradient gel electrophoresis was successful in demonstrating the presence of significant quantities of albumin, sufficient to invalidate

the ratio

of apo-B

to apo-VLDL

(L.A.

Simons

and P. F. Williams,

unpublished observations). Clearly, immunodiffusion methods are not of sufficient sensitivity for the investigation of this problem. Our present analysis of apolipoprotein-lipid interactions has been more or less guided

by the W.H.O.

rewarding

partially

levels in relation series of normal

classification

to ignore

to plasma

of hyperlipoproteinaemiata.

this classification

and to simply

lipids and lipoprotein

subjects and patients

classesll.

It may, however be consider

apo-protein

The results in the present

with hyperlipoproteinaemia

can only be regard-

ed as a preliminary application of this new assay technique. We are now able to measure with ease plasma and lipoprotein levels of one specific apoprotein. A long period of evaluation must necessarily follow in order to relate the apoprotein to vascular disease, to existing classifications of hyperlipoproteinaemialo, classifications11 and to lipid metabolism in general.

to proposed

ACKNOWLEDGEMENTS

We are grateful to Mrs. K. Cooper for technical assistance, and to Miss M. Bakker and Miss H. Blake for the manuscript. We wish to thank Drs. N. B. Myant (London) and M. Mancini (Naples) for supplying plasma from patients with familial Type II hyperlipoproteinaemia in the homozygous form, and to Associate Professor P. J. Scott (Auckland)

for supplying

a-P-lipoproteinaemic

plasma.

ADDENDUM

Following completion of this manuscript we noted the publication of a similar method for the radioimmunoassay of apo-B [Schonfeld, G., Lees, R. S., George, P. K. and Pfleger, B., Assay of total plasma apolipoprotein-B concentration in human subjects, J. C/in. Invest., 53 (1974) 14581. The methods were developed quite independently of one another, but show general agreement.

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