129

Clinica Chimica Acta, 75 (1977) 129-135 0 ElsevierlNorth-Holland Biomedical Press

CCA 6334

THE ELECTROPHORETIC POSTHEPARIN PLASMA

MOBILITY

OF LIPOPROTEIN

X IN

STALE RITLAND a*, JOSTEIN SAUAR a, RAND1 HOLME b and JAN PETTER BLOMHOFF a a Medical Department A and b Laboratory for Electron Microscopy, Institute of Pathology, The National Hospital of Norway, Rikshospitalet, University of Oslo, Oslo (Norway) (Received September lOth, 1976)

Summary After incubation whole plasma and low density lipoproteins (LDL) taken before and 10 min after intravenous administration of heparin, from a patient with primary biliary cirrhosis and a patient with familial 1ecithin:cholesterol acyltransferase (LCAT) deficiency, have been tested for the presence of lipoprotein X (LP-X) by agar gel electrophoresis. LP-X was present in preheparin whole plasma and LDL. No precipitation lines on the cathodal side of the wells, indicating the absence of LP-X, were seen after electrophoresis of postheparin plasma and LDL. Immunodiffusion revealed the presence of apo-@-lipoproteins and LP-X in preheparin as well as postheparin LDL. After gel filtration three subfractions and similar patterns were observed in the preheparin and postheparin LDL. Electronmicroscopical examination of the intermediate subfractions showed LP-X-like particles in preheparin and postheparin samples. These observations indicate a changed electrophoretic mobility in agar gel of postheparin LP-X, giving a false negative LP-X test by the conventional agar gel electrophoresis.

Introduction Lipoprotein X (LP-X) is an abnormal lipoprotein occurring in the low density lipoprotein (LDL) fraction of plasma from patients with either cholestatic liver disorders [1,2] or familial 1ecithin:cholesterol acyltransferase (LCAT) deficiency [3,4]. The presence of LP-X is usually demonstrated by a test based in its cathodal migration on electrophoresis in agar gel [5]. Following incubation of plasma drawn some minutes after injection of heparin, LP-X may no longer be demonstrated or its concentration may be reduced [ 6,7].

* To

whom

correspondence

should

be addressed

130

The present investigation reports mobility of LP-X following incubation

on an alteration of the electrophoretic of postheparin plasma.

Materials and methods Patients Two patients were studied. One female patient had primary biliary cirrhosis diagnosed on the basis of histological changes, increased levels of immunoglobulin M and the presence of mitochondrial antibodies. The other female patient had familial LCAT deficiency. Blood sampling After an overnight fast, blood was drawn before and 10 min after intravenous injection of heparin (50 I.U./kg body weight). The blood was immediately cooled on ice and separated at 4°C. The plasma (ACD) was incubated at 37°C for 4 h before the isolation of the LDL. Other plasma aliquots (K,-EDTA, 2 mg/ml) were not incubated and kept at +15”C for subsequent lipid determination. Unfrozen aliquots of the unincubated plasma were used for analysis of LP-X. Isolation 0 f lipoproteins LDL (density 1.019-1.063 g/ml) were isolated by preparative gation as previously described [8]. Subsequently subfractionation performed by gel filtration on 2% agarose [ 81.

ultracentrifuof LDL was

Electron microscopy One drop of the lipoprotein solution was placed on a Formvar/carbon-coated copper grid and excess fluid was removed with a filter paper. Thereafter one drop of the staining solution, 1% sodium phosphotungstate (pH 7.4) was placed on the grid and excess fluid was removed with a filter paper. The negatively stained preparation was examined in a Jeol JEM 100 B electron microscope. Electrophoresis of lipoprotein-X LP-X was separated from the other lipoproteins by agar gel electrophoresis [ 51. It was visualized either by application of polyanions on the surface of the gel on the cathodal side of the well [9] or by immunodiffusion, applying the antiserum in a trough. Quantitative determination was performed as previously described [lo]. Antisera against human P-lipoproteins and LP-X (apolipoprotein-C) from Behringwerke AG, Hamburg, Lahn, G.F.R., were used.

Other methods Free cholesterol [ll], total and fractionated phospholipids sured by methods routinely used in our laboratory.

[12] were mea-

131

Results

Whole plasma In preheparin plasma LP-X was present before as well as after incubation. The levels were 509 mg/lOO ml in the patient with primary biliary cirrhosis and 251 mg/lOO ml in the patient with familial LCAT deficiency. In postheparin plasma LP-X was present before incubation, but could not be detected after incubation by the qualitative test, and quantitative determination revealed only traces (7 mg/lOO ml) in the patient with primary biliary cirrhosis and no LP-X in the other patient. Incubation studies revealed that with increasing incubation time the precipitation line became fainter and was situated closer to the well (Fig. 1). The changes occurred during a relatively short time. During incubation of preheparin plasma no changes in free cholesterol or phospholipids occurred. After incubation of postheparin plasma, the relative amount of lysolecithin increased, whereas that of lecithin decreased (Table I).

Low density lipoproteins (density 1 .019-l .063 g/ml) After electrophoresis side of the well yielded

in agar gel, application of polyanion on the cathodal a precipitation line, indicating the presence of LP-X, in

Fig. 1. Agar gel electrophoresis of plasma taken 10 min. after the injection of heparin (50 I.U./kg body weight). The postheparin samples have been incubated at 37°C from 0 to 20 min. Cathode to the left.

132 TABLE

I

PLASMA

LIPIDS

PLASMA

AND

FROM

LP-X

BEFORE

A PATIENT

WITH

incubation incubation

Postheparin

OF

PRE-

AND

POSTHEPARIN

CIRRHOSIS

Phospho-

LY so-

Lecithin

LP-x

cholesterol

lipids

lecithin

(%)

(mg/100

ml)

(mg/lOO

ml)

ml)

(%)

282

522

1.6

83

298

498

2.2

84

298

508

1.6

85

299

488

5.5

80

509

plasma

Before After

INCUBATION

BILIARY

plasma

Before After

AFTER

Freee (m&!/100 Preheparin

AND

PRIMARY

incubation incubation

I

the preheparin sample but not in the postheparin sample (Fig. 2). Immunodiffusion with anti-apolipoprotein-C after electrophoresis demonstrated a faint precipitation line on the cathodal side of the well in preheparin sample but not in the postheparin sample (Fig. 3). Double immunodiffusion, however, revealed the presence of apolipoprotein-B and apoliprotein-C in the preheparin as well as in the postheparin sample (Fig. 4). After gel filtration three fractions were identified. The large molecular weight LDL eluted with the void volume, the normal LDL fraction eluted at volumes about 300 ml and the intermediate fraction as a shoulder in the patient with primary bilk-y cirrhosis (Fig. 5A) and as a plateau in the patient with familiar LCAT deficiency (Fig. 5B). The gel filtration pattern of the postheparin LDL was similar to that of the preheparin LDL. Electron microscopy Electron microscopic

Fig.

2.

Electrophoresis

cirrhosis. Fig. well.

3.

Upper The

After

same

well

in 1%

of the intermediate

examination

agar

preheparin

of LDL

LDL

samples

as in Fig.

electrophoresis

anti-LP-X

and 2.

(density lower

Preheparin was

1.019-a.063

well

applied

postheparin

sample in the

g/ml)

trough

from

LDL.

in upper

subfraction

a patient

Cathode

well between

and

with

primary

biliaw

to the left.

postheparin

the

of LDL

wells.

sample

Cathode

to

in lower the

left.

133

Fig. 4. Immunodiffusion of LDL (density 1.019-10.63 g/ml) from a patient with familial LCAT deficiency. Upper well preheparln LDL, lower well postheparin LDL. In the well to the left (1). anti&lipoprotein, and the well to the right (2), anti-LP-X were applied.

la

-

E’

,.

O.l_

!i2 N

“,i”,

8 z *

0.05,

0”

- -

Prcheparin

-

Postheparin

3 4 - -

$00

-._.

4

500~Volume

ml.

Fig. 5. Gel filtration (2% agarose) of preheparin and postheparin LDL (density 1.019-1.063 from a patient

with primary

biliary cirrhosis.

B, from a patient

with familial LCAT deficiency.

g/ml).

A.

134

from preheparin and postheparin plasma from the patient with primary biliary cirrhosis showed particles with diameter 300-650 ,& (Fig. 6). Some of the particles had a disc-like appearance with thickness about 100 8. This is the characteristic feature of the LP-X particles [13,14]. A few particles with diameter

Fig. 6. Electron micrograph cirrhosis. X 300 000. Upper,

of the intermediate LDL subfraction from preheparin LDL; lower, postheparin LDL.

the patient

with

primary

biliary

135

ZOO-250 8, similar to the particles in normal LDL, were also seen. In the intermediate subfractions of LDL from the patient with familial LCAT deficiency similar observations were made. LP-X-like particles were found in preheparin as well as in postheparin LDL. Discussion Injection of heparin is known to release lipases from the liver and also from other tissues [ 15,16,17 J. LP-X has been found to decrease or disappear from postheparin plasma [6,7]. These changes occur during incubation of the postheparin plasma and are not caused by heparin itself as in vitro addition of heparin to preheparin plasma does not induce any change in the level of LP-X [ 71. Changes in the composition of phospholipids with increase in lysolecithin and decrease in lecithin favoured the hypothesis that the changes were due to a phospholipolytic activity [7], i.e. a metabolic effect. In the present study we have confirmed previous observations that metabolic changes of the phospholipids do occur during incubation of postheparin plasma [ 71. This investigation has demonstrated that despite the disappearance of LP-X on agar gel electrophoresis after incubation of postheparin whole plasma or LDL, C-peptides are present in the LDL fraction, and the in~rmediate subfraction of LDL on gel filtration is unchanged. Finally LP-X-like particles are still found by electron microscopy. These findings indicate an altered electrophoretic mobility of LPX in agar gel after incubation of postheparin plasma. Acknowledgement Excellent technical assistance has been given us by Mrs. Hilde Letnes, Mrs. Ingrid Wiencke and Mr. E. Jenssen. This investigation was financially supported by the Norwegian Research Council for Science and Humanities and Tom Wilhelmsen’s Foundation, and the Norwegian Cancer Society. References 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17

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