Increased concentration of high density lipoprotein in plasma and decreased platelet aggregation in primary biliary cirrhosis

A /herosclerosis, 53

(1984)

Elsevier Scientific

Publishers

15 I- 162

151

Ireland.

Ltd.

ATH 03532

Increased Concentration of High Density Lipoprotein in Plasma and Decreased Platelet Aggregation in Primary Biliary Cirrhosis Y. Baruch, J.G. Brook, S. Eidelman Lipid Reseurch Unit, Depurtment of Internal Center, und Faculty of Medicine.

and

M. Aviram

Medicine B. and Gastroenterology Unit, Rambom Medical

Technion - Israel Institute of Technology. Haija

31090 (Israel)

(Received 31 January, 1984) (Revised, received 21 May, 1984) (Accepted 25 May, 1984)

Summary

Plasma lipoprotein concentration and composition were studied in 7 female patients with primary biliary cirrhosis and compared with 6 normal, age-matched controls. The effect of the lipoproteins derived from these patients on the function of normal platelets was also tested. High levels of plasma cholesterol and phospholipids and a raised free/esterified cholesterol ratio were found. In 4 of the patients, both HDL cholesterol and HDL protein were increased, and high levels of plasma apoprotein A-I and A-II were evident. This abnormal HDL did not contain excess apolipoprotein E. The VLDL and LDL fractions were also abnormal, as evidenced by a high cholesterol/protein ratio. Little correlation between lipoprotein disorders and clinical condition was found. Platelet function was reduced in all patients. LDL from the patients reduced aggregation of normal platelets, whereas HDL had a minima1 effect. The abnormal lipoproteins in these patients may contribute to their abnormal in vitro platelet aggregation. Key words:

Apolipoprotein - High density lipoprotein - Lipoprotein - Platelet aggregation - Primary biliary cirrhosis

Correspondence to: M. Aviram, Haifa 31096. Israel.

0021-9150/84/$03.00

Lipid Research

0 1984 Elsevier Scientific

Laboratory,

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Ireland,

Ltd.

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152

Introduction Liver disease is commonly associated with profound changes in lipoprotein concentration and composition [l]. Accelerated atherosclerosis is not a characteristic of chronic liver disease. Particularly in primary biliary cirrhosis, where both the presence of xanthoma and markedly raised plasma cholesterol levels are featured, the opposite would be expected. Study of lipoproteins in such patients might further our knowledge of factors underlying the degree of atherogenicity of the different lipoproteins. In human cholestasis., changes in both very low density (VLDL), low density (LDL) [2,3] and high density lipoproteins (HDL) have been described [4]. The protein and cholesterol content of the latter are low, and there is a disproportionately increased amount of the apoliprotein E (Apo E)-rich “nascent HDL” [5,6]. Many patients have high concentrations of an abnormal lipoprotein, LP-X [2,7]. These changes have been attributed to decreased levels of the enzyme lecithin: cholesterol acyltransferase (LCAT) in these patients [8-l 11. We have studied the lipoprotein concentrations and compositions of 7 patients with primary biliary cirrhosis (PBC) and have attempted to correlate clinical severity with lipoprotein derangement. We have also determined lipoprotein function by incubating these lipoproteins with normal platelets and studying the effect on in vitro platelet aggregation. Patients and Methods Seven female patients with long-standing cholestasis and diagnosed as suffering from PBC constituted the study group. The diagnosis was established on the basis of recognized clinical, immunological, and biochemical criteria, as well as liver biopsy [12]. All medications were terminated at least 2 weeks before commencing the study, with the exception of a patient (I.R.) who was diabetic and hypertensive, who continued on insulin and propranolol. None of the patients had been on steroids or immunosuppressive drugs. Six females of the same age-healthy and with normal liver function-comprised the control group. Blood samples were drawn into EDTA (1 mg/ml) after a 14-h fast. Plasma was separated after centrifugation for 20 min at 2000 X g. Lipoprotein separation Plasma lipoprotein was separated by sequential preparative ultracentrifugation [13] in a Beckman LS-65 ultracentrifuge. Cholesterol and protein were determined in each fraction, The lipoproteins were extensively dialyzed against concentrated saline (pH 8.6). Lipid and protein analysis Cholesterol and triglyceride were determined by enzymatic methods on a Gemsaec centrifugal fast analyzer (Electra-Nucleonics, Fairfield, NJ). Free cholesterol and cholesterol ester [14], plasma phospholipids [15] and lipoprotein protein concentration [16] were also determined.

153

Plusmu upolipoprotein ana/+ Apo A-I, A-II and B in plasma munoelectrophoresis [17]. Plasma lipoprotein electrophoresis Electrophoresis was performed

were measured

on cellulose

acetate

Red-Gel etectrophoresis Redi-Gel electrophoresis of plasma lipoprotein kits (Ames, Miles Laboratories, Elkhart, IN).

quantitatively

by rocket

im-

paper strips [18].

was performed

using ready-made

Isoelectric focusing gel electrophoresis Isoelectric focusing gel electrophoresis was performed [19]. Gels comprised 7.5% acrylamide (30 : 8 of acrylamide : bis-acrylamide), 6 M urea, 5% ampholin pH 4-6 (Bio-Lyte 4/6, Bio-Rad Laboratories, Richmond, CA), 0.07% ammonium persulfate solution and 2 ~1 tetramethylenediamine. The solution was introduced immediately into tubes (0.6 cm x 12.5 cm) up to a height of 8 cm. The lower (anode) chamber was filled with 10 mM phosphoric acid, and the upper (cathode) chamber with 20 mM NaOH. Lipoproteins were delipidated [20] with 1,1,3,3_tetramethylurea (TMU). Seventy-five micrograms of lipoprotein protein, 50 mM dithiotreitol, 0.05% Triton X-100 and 10% sucrose were carefully overlaid on the gels, which were focused at 250 V for 16 h at 4°C. The gels were removed from the tubes into 0.125% Coomassie brilliant blue G-250 (Sigma Chemical Co., St. Louis, MO) in 50% methanol and 10% acetic acid for 1 h at room temperature and then transferred into destaining solution (7.5% acetic acid, 10% methanol). After 72 h the gels were photographed, and the relative proportions of the proteins were determined by densitometry at 520 nm with a scanning densitometer (GD-4, Pharmacia Fine Chemicals, Uppsala, Sweden). The area under each peak was cut and weighed and the relative weight calculated. Platelet preparation Blood was drawn into 3.8% sodium citrate (v :v = 9 : 1). and platelet-rich plasma (PRP) was obtained after centrifugation at 23’C for 20 min at 200 X g. Gel-filtered platelets (GFP) were prepared by gel-filtration chromatography of PRP on 27 mm x 90 mm Sepharose 2B (Pharmacia Fine Chemicals) column using Hepes buffer (10 mM Hepes, 120 mM NaCl, 2.4 mM KCI, 1.2 mM MgSO, and 0.1% glucose) [21]. Plutelet incubation Normal GFP were diluted with Hepes buffer to 100,000 PI/ml prior to incubation with normal and patient lipoproteins. Incubation was performed, using physiological lipoprotein concentrations (pg of protein/ml: VLDL, 250; LDL, 750; HDL, 1500), for 30 min at 37°C in polyethylene tubes. Platelet aggregation Platelet aggregation was performed in a dual-chronalog from 5 of the patients was tested using ADP (5 PM),

aggregometer [22]. PRP epinephrine (5 PM) and

154

thrombin (0.2 U/ml) as aggregating agents. The results were compared to those of 10 age- and sex-matched, normolipidemic controls. GFP were also prepared and tested from each of the patients and controls. For aggregation of GFP, fibrinogen (mg/ml) and CaCl, (2 mM) were added prior to the addition of the aggregating agent. A control tube containing all the added constituents except GFP, which were replaced by Hepes buffer, was also included. Thrombin (0.2 U/ml) was used as the aggregating agent. Results were expressed as the percentage maximal aggregation after 4 min. Statistics The Wilcoxon

rank test was used

Results The clinical and laboratory data of the PBC patients are summarized in Tables 1A and 1B. The group was heterogeneous, the patients being at different clinical and had raised plasma pathological stages of their disease. Six of the 7 patients cholesterol levels and increased ratio of free/esterified cholesterol (Table 2).

TABLE

1A

CLINICAL Patient

AND

Disease duration

LABORATORY

DATA OF PATIENTS

WITH

PRIMARY

BlLlARY

Age (yr)

Weight (kg)

Pruritus

Xanthomas

Enlarged liver

Enlarged spleen

Esophageal varices

CIRRHOSIS Histological stage A

(yr) S.H. F.R. K.M. C.L.

1 5 5 1

38 38 70 50

70 50 60 50

+ _ + _

+ + _

+ + + _

+ + + _

_ + _

1.R. L.M. R.B.

5 5 15

53 35 61

50 50 65

+ + +

+ + +

+ + +

+ + +

_ + +

a Histological TABLE

stage at time of diagnosis

S.H. F.R. K.M. C.L. I.R. L.M. R.B.

to Scheuer

[12].

IB

CLINICAL AND LABORATORY BILIARY CIRRHOSIS Patient

according

I I 11 I 111 11 III

Bilirubin (mg/lOO 2.5 1.5 1.6 1.0 5.2 15.0 3.8

ml)

DATA OF PATIENTS

WITH

PRIMARY

Alkaline phosphatase (IU)

SGOT

Prothrombin

(lU)

(%)

Albumin (g/l00 ml)

Cuprum (mg/lOO

1700 960 1290 340 4700 990 600

56 55 31 17 80 80 60

94 55 45 45 45 50 38

4.4 4.0 3.4 4.0 4.0 3.8 4.0

262 193 180 180 357 257 276

SGOT = transminase; AMA = antimitochondrial HBsAg = hepatitis B surface antigen.

antibodies;

Ig = immunoglobulin;

ml)

AMA (l/10)

+ + + + + + +

155 TABLE

2

PLASMA

LIPID

LEVELS

Each value represents Patient

S.H. F.R. K.M. C.L. I.R. L.M. R.B. Normal

’

IN PBC PATIENTS

the mean of 4 determinations.

Total cholesterol

Free cholesterol

(mM)

(mM)

11.9 6.7 1.9 6.6 23.2 6.6 4.1

7.8 1.8 4.2 3.9 23.0 6.1 2.3

3.9kl.l

0.9 f 0.2

5.2 f 0.9

1.3kO.4

a Values are the mean f SD of 6 normal

Fig. 1. Plasma lipoprotein L = LDL: H = HDL.

12.5 19.5 30.0 14.0 14.0 27.8 19.0

ml)

(mUlO > > > > > > >

3.8 3.8 3.8 3.8 3.8 3.8 3.8

Phospholipids

(mM)

(mM)

(mM)

4.1 4.9 3.5 2.1 0.2 0.5 2.2

0.5 1.1 1.0 1.3 2.2 1.4 1.0

9.0 3.2 3.5 4.0 16.2 3.3 1.9 k1.1

controls.

on Redi-Gel.

1 = control;

2 = S.H.;. 3 = R.B.;

HBsAg

IN

W (mg/lOO

age-matched

electrophoresis

Triglyceride

Cholesterol ester

ml) _ -

V = VLDL

156

TABLE

3

LIPOPROTEIN Patient

COMPOSITION

IN PBC PATIENTS

VLDL (d < 1.006 S/ml)

LDL (d=1.006-1.063

HDL (d =1.063-1.210

S/ml)

g/ml)

Cholesterol

Protein

Chol/prot.

Cholesterol

Protein

Chol/prot.

Cholesterol

Protein

Chol/prot.

@M)

(w/W

(x 10’)

@M)

(w/W

( x 103)

@M)

(w/W

( x 10’)

S.H.

0.16

20

0.30

6.90

98

2.72

3.60

317

0.44

F.R.

0.10

26

0.15

2.72

90

1.17

2.30

224

0.39

K.M.

0.25

26

0.37

3.40

66

1.99

3.16

256

0.47

C.L.

0.49

13

1.45

3.00

88

1.32

1.20

171

0.27

LR.

1.16

19

2.35

14.60

163

3.46

2.00

145

0.53

L.M.

0.14

10

0.54

2.60

94

1.07

0.95

137

0.27

R.B.

0.34

9

1.46

2.92

76

1.48

1.10

151

0.28

Normal a

0.18

17

0.41

2.40

77

0.96

1.25

176

* 0.03

f0.12

*4

+ 0.53

a Values are the mean f SD of 6 age-matched

k13

50.20

f0.19

+39

0.27 + 0.08

controls.

Only one patient (I.R.) had a raised plasma level of triglyceride, whereas 4 of 7 patients had elevated phospholipids. The composition of the lipoproteins in each patient is illustrated in Table 3. All lipoprotein classes were abnormal in composition. In 4 of 7 patients, raised levels of both HDL cholesterol and protein were found. An increased cholesterol/protein ratio was apparent in the LDL of all patients and the HDL of 5 out of 7 patients. The cholesterol and protein content of VLDL varied markedly on plasma lipoprotein electrophoresis. Three patients (S.H., F.R. and K.M.) had a markedly pronounced alpha lipoprotein band. On electrophoresis of the isolated lipoproteins, both the LDL and the HDL of patients S.H. and F.R. demonstrated different mobility in comparison to the controls. The increased HDL concentration in patient S.H. was confirmed on Redi-Gel electrophoresis (Fig. 1). TABLE

4

PLASMA

APOLIPOPROTEIN

Each value represents Patient

S.H. F.R. K.M. C.L. I.R. L.M. R.B. Normal

’

CONCENTRATION

IN PBC PATIENTS

the mean of 3 determinations. Apo A-I

Apo A-II

Apo B

(mg/dU

(mg/dU

(mg/dU

213 260 168 135 114 95 136

12 66 77 67 31 48 33

98 103 79 95 139 98 86

150+31

40*7

a Values are the mean k SD of 6 age-matched

female controls.

76+10

157 TABLE

5

APOLIPOPROTEIN COMPOSITION OF PLASMA CONTROL (PERCENTAGE OF TOTAL PROTEIN) VLDL

Apo C-III2 Apo C-III, Apo C-II Apo C-III,l/Apo Unidentified

A-II ’

(1) (2) Apo A-l Apo E

LIPOPROTEINS

LDL

IN PATIENT

S.H. AND

HDL

Control

Patient

Control

Patient

Control

Patient

(8)

(%)

(%)

(S)

(B)

(%)

48.0 30.0 17.0 5.0

40.0 35.0 17.5 7.5

23.0 47.0 6.0 3.5

26.0 46.0 10.0 12.5

2.0 1.0 1.0 20.0

4.0 4.0 2.5 25.0

6.0

13.5 51.0 1 .o

1.0

13.0

IN

2.5 12.5 40.0 2.5

’ Apo A-II only in HDL.

Fig. 2. Apolipoprotein analysis by isoelectric focusing on polyacrylamide gels. A = VLDL; B = LDL; C = HDL; c = control; p = patient (S.H.); l= Apo C-III,; 2 = Apo C-III,; 3 = Apo C-II; 4 = Apo C-III,/Apo A-II; 5, 6 = unidentified; 7 = Apo A-I; 8 = Apo E.

158 TABLE

6

PLATELET TROLS

AGGREGATION

Values expressed

as percentage

of amplitude

Maximal

Patient

a Ten normal, healthy N.S. = not statistically

FROM

PBC PATIENTS

aggregation

Epinephrine

(5 PM)

(5 PM)

Thrombin (0.2 U/ml)

76 65 15 82 65

94 93 93 100 98

73+7 95*7 0.05

96k5 98+12 N.S.

subjects constituted significant.

AND CON-

(%)

ADP

65 +6 93 +16 < 0.01

a

PLASMA

at 4 min. as mean f SD.

53 54 14 78 65

S.H. F.R. 1.R. L.M. R.B. Mean Controls P

IN PLATELET-RICH

the control

group.

In the patients with increased levels of HDL, plasma concentrations of Apo A-I and A-II were increased, whereas in the other patients these levels were decreased. Apo B was increased in all 7 patients (Table 4). The apolipoprotein composition of the lipoproteins of patient S.H., who demonstrated the highest levels of HDL, was studied by isoelectric focusing analysis on TABLE

7

THE EFFECT LETS

OF PATIENT

LIPOPROTEINS

ON THE AGGREGATION

OF NORMAL

PLATE-

Normal GFP were incubated with purified lipoproteins from either PBC patients or normal subjects for 30 min at 37 o C as described under Patients and Methods. Platelet aggregation was studied in response to thrombin (0.2 U/ml). In GFP alone (without the addition of plasma lipoproteins) platelet aggregation was 73 + 9%. Patient

Amplitude

(W)

VLDL

LDL

HDL

S.H. F.R. I.R. L.M. R.B. K.M. C.L.

80 90 70 15 100 100 70

34 53 58 51 22 24 51

86 86 12 66 61 15 71

Mean Normal subjects (n = 10) P vs. patients a

85&13 89k6 N.S.

42&-15 84*5 0.01

75+8 65k6 0.02

-

a P-value refers to the difference on normal platelets. N.S. = not significant.

between

the effect of the patient-

and the normal-derived

lipoproteins

159

polyacrylamide gel electrophoresis (Fig. 2). The results of the densitometric estimation are shown in Table 5. In the patient VLDL, the percentages of Apo C-III,, and C-III, were slightly elevated, whereas Apo C-III, levels were decreased. In the LDL fraction, both Apo C-II and C-III, were elevated, whereas Apo E concentration appeared less than that of the controls. Similar results were found for patient F.R., who also had high HDL levels. The HDL contained an increased concentration of all Apo Cs. The percentage of Apo A-I was decreased, despite the absolute increase of Apo A-I in plasma. The percentage concentration of Apo E was only slightly increased. Similar changes were obtained in the other patients with high HDL levels. Aggregation of platelets derived from the patients was reduced in response to epinephrine and ADP and unchanged in response to thrombin in comparison to the controls (Table 6). The effect of lipoproteins derived from the PBC patients on normal platelet aggregation was studied by incubating the lipoproteins with normal platelets (GFP preparation) (Table 7). The maximal aggregation of these patients without the addition of lipoproteins in response to thrombin was 73 f 9%. On adding patient LDL, a reduced response of 42 + 15% was noted, whereas LDL derived from the controls increased the response to 84 f 5%. The addition of patient HDL had no significant effect (75 k 8%) but control HDL diminished the response (65 + 6%). Both patientand control-derived VLDL increased the aggregation response. Discussion Abnormal lipoprotein metabolism is a consequence of chronic liver disease, and in both intrahepatic and extrahepatic cholestasis abnormal lipids and lipoproteins have been described [23]. Furthermore, the severity of the liver disease can often be correlated with features of the plasma lipid abnormality [24,25]. Our PBC patients represent different degrees of liver damage and biliary obstruction and a consistent change in lipoprotein concentration and composition could not have been expected. The pattern emerging from our patients was not uniform, and there appeared to be little correlation between clinical status and degree of lipoprotein changes. Abnormalities in the lipoprotein concentration and composition of our patients’ lipoproteins were obtained. A high total cholesterol/protein ratio in most of the lipoproteins was observed, and this confirms what has already been described in patients with liver disease [3,26,27]. Of interest were the findings regarding HDL. Changes in HDL in liver disease have been attributed to altered LCAT activity [ll]. Increased activity is associated with either normal or high HDL levels, whereas in low LCAT activity both HDL cholesterol and protein levels are depressed [28]. Since most of our patients established a low cholesterol ester/free cholesterol ratio, decreased LCAT activity can be assumed [28]. However, 4 of our patients had raised levels of HDL, and plasma Apo A-I and A-II were increased. The raised levels were, furthermore, confirmed on both cellulose acetate and Red&Gel electrophoresis. With one exception [25], these findings contrast with what has previously been reported in

160

patients with cholestatic liver disease [29,30], who usually have no demonstrable alpha band on agarose electrophoresis [31]. Our patients’ HDL was also abnormal in composition, containing relatively more Apo A-II and C-III and relatively less Apo A-I. The raised HDL levels could be a result of either defective catabolism or decreased production. There is no evidence in support of increased production in the damaged liver, especially in the presence of low LCAT activity. It is thus likely that the accumulation of HDL is a result of decreased hepatic catabolism in which production of HDL has not yet been impaired. The liver is responsible for the sialization of Apo C-III [32]. The increase in the levels of Apo C-III, observed in our patients as well as the partial fall in Apo C-III, and C-III, indicate an impairment in the sialization process in these patients. This process would be impaired in severe liver disease. The expected result would be an increase in C-III, levels, which was indeed observed in our patients, and a drop in C-III, and C-III,, which was only partially noted in our patients. Platelets derived from 4 patients with familial hypercholesterolemia exhibited enhanced in vitro aggregation and release [33]. Conversely, platelets obtained from 4 patients with chronic liver disease had an impaired response [34-361. The platelets of our patients also demonstrated reduced in vitro aggregation in response to epinephrine, ADP and thrombin. These changes have been variously attributed to changes in the platelet content of cholesterol, of phospholipids, of fatty acids and of arachidonic acid [36]. Alternatively, the presence of some abnormal plasmatic factor may be responsible for the impairment [34]. Since platelets possess specific binding sites for plasma lipoproteins [21] and since lipoproteins, when incubated with GFP, have a profound influence on their aggregation response as well as on their release of [‘4C]serotonin [37], lipoprotein-platelet interactions may well be instrumental in determining platelet function. Normal VLDL and LDL enhance platelet activation, whereas normal HDL has the opposite effect [38-401. The lipoproteins of the PBC patients differed in their concentration and composition from normal lipoproteins. We employed our model of platelet-lipoprotein interaction to study the effect of these lipoproteins on GFP derived from normal donors. LDL was found to reduce platelet aggregation, whereas HDL had no significant effect. The increased Apo C content of the LDL noted in these patients may be of relevance in this respect. These abnormal responses must reflect the abnormal lipoprotein composition, and it is likely that the abnormal platelet membrane composition noted in these patients also plays a significant role [36]. It thus appears that in some patients with PBC and possible low LCAT activity, abnormal HDL accumulates in the plasma. This may reflect a decreased hepatic catabolism of HDL. The abnormal lipoprotein composition may contribute to the decreased in vitro platelet aggregation described in our patients. Acknowledgements We wish to thank Mrs. Gertrude Dankner and Mrs. Carmela Lindenbaum their technical assistance and Miss Ruth Singer for the secretarial work.

for

161

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