Effects of interferon-beta on plasma lipid and lipoproteincomposition and post-heparin lipase activitiesin patients with chronic hepatitis C

R. J. ANDRADE, M. D. GARCIÂA-ESCANÄ O, P. VALDIVIELSO, R. ALCAÂ NTARA,

M. A. SAÂ NCHEZ-CHAPARRO & P. GONZAÂ LEZ-SANTOS

Liver Unit, Department of Medicine, School of Medicine, University of MaÂlaga, MaÂlaga, Spain

Accepted for publication 16 March 2000

INTRODUCTION

Interferons are naturally occurring cytokines involved

in host defenses against a variety of infectious agents

and in¯ammation.1 Interferons are clinically useful in

some malignant, viral, immmunological, angiogenic,

in¯ammatory and ®brotic diseases.2 Currently, inter-

ferons are also the ®rst line therapy for chronic hepatitis

C virus infection.3 Most studies of the effects of

interferons in patients with chronic hepatitis C have

used recombinant-derived (alpha 2a or 2b) or naturally

produced (lymphoblastoid) interferon-a,3, 4 and some

authors have found changes in lipid and lipoprotein

composition during interferon-a therapy. The most

notable changes have been an increase in total and

very low density lipoprotein (VLDL) triglycerides and a

decrease in high density lipoprotein (HDL) choles-

terol.5, 6 In addition, severe hypertriglyceridaemia and

chylomicronaemia have been reported in a few patients

treated with interferon-a.6±10 In such patients, pre-

treatment triglyceride levels were usually elevated.6, 7

Interferon-b, a naturally produced cytokine derived

from epithelial cells and ®broblasts is also marketed for

the treatment of chronic hepatitis C in several European

countries and Japan.3 Recently, interferon-b has also

shown ef®cacy in the therapy of multiple sclerosis.11

SUMMARY

Background: Interferon therapy has been shown to

induce lipid abnormalities.

Aim: We assessed the effects of interferon-b on the

lipoprotein pro®le and post-heparin lipase activities in

26 normolipaemic patients with chronic hepatitis C.

Methods: Interferon-b was administered subcutaneously

at doses of 6 ´ 106 U (units) three times a week, over

6 months, and lipoproteins and post-heparin lipases

were measured at baseline and at the end of therapy.

Results: Plasma triglycerides increased by 21% due to

preferential enrichment in those contained in the very

low density lipoprotein (VLDL) and low density lipo-

protein (LDL) fractions. The concentration of choles-

terol decreased slightly in the high density lipoprotein

(HDL) subfractions. Lipoprotein lipase, but not hepatic

lipase activity decreased by a 36%, and this change

showed a signi®cant negative correlation with changes

in plasma triglycerides. Five patients (19.5%) responded

to interferon-b therapy. The lipoprotein pro®le was no

different between responders and non-responders to

therapy.

Conclusions: Interferon-b treatment in normolipaemic

patients with chronic hepatitis C induced moderate

disturbances in plasma lipoproteins, associated with

inhibition of lipoprotein lipase activity.

Correspondence to: Dr R. J. Andrade, Unidad de HepatologõÂa, Departamento

de Medicina, Facultad de Medicina, Campus Universitario de Teatinos s/n,

29071, MaÂlaga, Spain.E-mail: andrade@uma.es

Aliment Pharmacol Ther 2000; 14: 929±935.

Ó 2000 Blackwell Science Ltd 929

Although lipid disturbances have been noted in associ-

ation with interferon-b therapy, neither the lipoprotein

pro®le nor the mechanism whereby interferon-b causes

such changes have been fully investigated.12, 13

In the present study we prospectively evaluated the

effects of interferon-b therapy on lipoprotein pattern and

post-heparin lipase activities in normolipaemic patients

with chronic hepatitis C, and examined the relationship

of these effects with the response to treatment.

MATERIALS AND METHODS

The study was designed and conducted at the Liver

Unit, University Hospital of MaÂlaga (Spain) and was

supported by Department of Medicine. It was not a part

of a sponsored study.

Patients

A total of 26 consecutive patients with chronic hepatitis

C were prospectively included in the study between

April and June 1996. To be included, a patient had to

have hepatitis C virus antibodies detectable by ELISA

(Ortho Diagnostics, NeckargemuÈ n, Germany),1 RNA

positivity by the polymerase chain reaction (Amplicor,

Hoffmann-La Roche, Basel, Switzerland), and biopsy-

proven chronic hepatitis within 6 months prior to the

study. Exclusion criteria were: positive serology for

hepatitis B virus infection or human immunode®ciency

virus; cirrhosis; marked obesity (body mass index > 30);

diabetes; hyperlipemia (cholesterol > 240 mg/dL; tri-

glycerides > 150 mg/dL), or any other disease known

to alter the lipid pro®le; history of alcohol use; or

therapy with drugs known to in¯uence plasma lipid or

lipoproteins. Patients were instructed not to alter their

dietary habits during the course of the study.

Patients received interferon-b (Frone, Serono, Madrid,

Spain) subcutaneously at a dose of 6 ´ 106 U, three

times a week over a 6-month period. The objective of this

study, speci®cally designed to evaluate lipid changes

during interferon-b therapy, was thoroughly explained

to all participants, who signed an informed consent.

Assessment of ef®cacy

Parameters monitored in all patients at baseline and

at the end of therapy were: body mass index, serum

glucose, aspartate aminotransferase, alanine amino-

transferase, bilirubin, albumin and prothrombin activity.

The response to treatment was de®ned according to the

criteria of the National Institutes of Health Consensus

Development Conference on Hepatitis C as normal

serum alanine aminotransferase concentrations and

undetectable serum hepatitis C virus RNA levels at the

end of therapy.14

Liver biopsy specimens obtained before treatment and

at the end of the follow-up period were interpreted by a

single pathologist in a blinded fashion. The degree of

hepatic in¯ammation and ®brosis was scored with the

Knodell histological activity index.15 In¯ammation and

®brosis scores were evaluated separately.

Lipoprotein analysis

Lipid and lipoproteins were evaluated at baseline and at

the end of therapy. Fasting blood specimens were

collected by venipuncture into tubes containing sodium

EDTA, and were centrifuged at 1500 g for 30 min at

4 °C. Total cholesterol, triglycerides and phospholipids

in plasma, and lipoprotein fractions were assayed

enzimatically in a Cobas MIRA autoanalyser (Hoff-

mann-La Roche). Very low density lipoprotein (VLDL),

low density lipoprotein (LDL), and high ensity lipopro-

tein subfractions (HDL2 and HDL3) were isolated by

sequential ¯otation in a preparative ultracentrifuge

(Beckman L8±55 M, Palo Alto, California, USA) accord-

ing to the method of Havel et al.,2 using a 50.3 Ti

Beckman rotor.16 With the addition of a KBr + NaCl

mixture, each lipoprotein was fractionated at the follow-

ing densities: VLDL, d < 1.006 g/mL; LDL, d � 1.006±

1.063 g/mL; HDL2, d � 1.063±1.125 g/mL; and HDL3,

d � 1.125±1.210 g/mL.

Activities of lipoprotein lipase and hepatic lipase

in postheparin plasma

Lipolytic enzymes are anchored to the vessel wall, but

can be removed by infusing molecules with high af®nity

to lipases3 , such as heparin. Lipolytic activities should

therefore be assayed in post-heparin plasma. Plasma

samples were obtained 10 min after the intravenous

injection of heparin at a dose of 50 UI/kg body weight,

and stored at )70 °C until assay. The activities of

lipoprotein lipase and hepatic lipase were measured by a

radioisotopic method using H3-Triolein as a substrate,

emulsi®ed by sonication with Triton X-100 (Branson

250, Danbury, Connecticut, USA). Post-heparin lipase

activity was measured as the difference in salt sensitiv-

930 R. J. ANDRADE et al.

Ó 2000 Blackwell Science Ltd, Aliment Pharmacol Ther 14, 929±935

ity: the enzymatic activity of lipoprotein lipase is

inhibited by high salt concentrations, whereas hepatic

lipase is active in 1 M NaCl.17 In addition, apolipopro-

tein (Apo) C-II is required as a co-factor for maximal

lipoprotein lipase activity, whereas hepatic lipase has no

co-factor requirements.

To measure post-heparin lipase activities, each assay

was performed in triplicate tubes, including a no-plasma

blank, with plasma, and with plasma plus 1 M NaCl. All

tubes (refrigerated on ice) contained 200 lL of substrate

and 10 lL of post-heparin plasma. For the tubes with

salt (hepatic lipase activity), 50 lL 5 M NaCl was added.

For tubes without a high salt concentration, 50 lL of

human plasma with Apo-CII (0.1 M NaCl; total activity

of post-heparin plasma) was added. The tubes were

incubated at 37 °C for 1 h and the reaction was then

stopped by adding 3 mL of methanol/chloroform/hep-

tane (1.4:1.25:1.0; v/v/vol) with 1 mL carbonate/

borate buffer, pH 10.5. Fatty acids were extracted by

the method of Belfrage and Vaughan.18 After vortexing,

the samples were centrifuged at 1500 g4 for 15 min at

4 °C. One millilitre of the top fraction (aqueous phase)

was then removed and radioactivity was determined.

Lipoprotein lipase activity was determined by subtract-

ing hepatic lipase activity (samples plus 1 M NaCl) from

total Phepatic lipase activity. Paired samples (i.e. before

and after treatment) were assayed in the same batch.

Whitin-batch coef®cient of variation for total post-

heparin lipase activity and hepatic lipase activity were

2.3% and 9.4%, respectively. All enzyme activities are

expressed as micromoles of free fatty acids per hour per

millilitre of plasma.

Statistical analysis

All values are expressed as the mean � s.d. Statistical

analysis was performed with statistical software (SPSS/

PC+, SPSS Inc, Chicago, IL) on a personal computer.

Student's paired t-test was used for comparisons

between values before and after treatment with inter-

feron-b, when the means of variables were distributed

normally. For lipoproteins and post-heparin lipase

activities, pairwise comparisons were tried. However,

the Wilcoxon signed ranks test was used because the

values did not show a normal distribution. For other

comparisons we used the non-parametric Mann±Whit-

ney test. Data were analysed by calculating Pearson's

correlation coef®cient.

RESULTS

The characteristics of the patients at the start of

interferon-b therapy are shown in Table 1. No patient

had any serious or potentially life-threatening complica-

tions, and all patients completed the trial, receiving the

same total dose of interferon-b (432 ´ 106 U). No

signi®cant changes were observed, after interferon-btreatment, in body mass index, leucocyte and platelet

count or liver function as measured by bilirubin, albumin

and prothrombin time. A slight increase in haemoglobin

levels and a decrease in serum glucose (both within the

normal range) at the end of therapy was noted (median

values: basal 15 � 1.1 g/dL; 6 months 15.5 � 1.2 g/dL,

P < 0.01; basal 106.3 � 19.1 mg/dL; 6 months

102.6 � 19.5 mg/dL, P < 0.05, respectively).

Table 2 shows the effect of interferon-b therapy on the

concentration of cholesterol, triglycerides and phospho-

lipids in plasma and on the lipoprotein fractions. After

6 months of treatment, mean plasma triglyceride level

had increased by 21%, whereas no changes were noted

in the plasma concentration of total cholesterol and

phospholipids. All lipoprotein fractions were enriched in

triglycerides, but the increases were statistically signif-

icant only in the VLDL and LDL fractions. In contrast

there was a slight (but not statistically signi®cant)

decrease in HDL cholesterol due to that contained in

both subfractions HDL2 and HDL3.

Table 1. General characteristics and demographic variables

before treatment with interferon-b in patients with chronic

hepatitis C*

Age (years) 39 � 12

Male sex (%) 15 (58%)

Body mass index (kg/m2) 27.1 � 3.5

Serum glucose (mg/dL) 106 � 19

Leucocyte ´109/L 6.9 � 1.8

Platelet ´109/L 190 � 43

Haemoglobin (g/dL) 15 � 1.1

Bilirubin (mg/dL) 0.75 � 0.2

AST (U/L) 61 � 28

ALT (U/L) 97 � 48

c-GT (U/L) 51 � 32

Alkaline phosphatase (U/L) 182 � 62

Albumin (g/dL) 4.6 � 0.3

Prothrombin activity (%) 115 � 15

Knodell HAI 9 � 4

* Values are means � s.d. or number of cases (%).

AST, aspartate aminotransferase; ALT, alanine aminotransferase;

HAI, histological activity index.

INTERFERON-BETA AND POST-HEPARIN LIPASES IN CHRONIC HEPATITIS C 931

Ó 2000 Blackwell Science Ltd, Aliment Pharmacol Ther 14, 929±935

Post-heparin lipase activities measured in the 26

patients before and after interferon-b treatment are

shown in Figure 1. The activity of post-heparin lipase

showed a tendency to decrease as a result of interferon-btherapy (median values: basal 5.7 � 4.1 lmol;

6 months 5.1�3.9 lmol free fatty acids/mL per hour)

due to a signi®cant decrease in lipoprotein lipase activity

(median values: basal 1.9 � 1.3 lmol; 6 months

1.2 � 1.2 lmol free fatty acids/mL per hour,

P < 0.001). This decrease was noted in 24 of the 26

patients (88.4%). The mean percentage decrease in

lipoprotein lipase activity was 36%. In contrast, hepatic

lipase activity did not change with interferon-b treat-

ment (median values: basal 3.8 � 3.2 lmol; 6 months

3.9 � 3.4 lmol free fatty acids/mL per hour).

Five patients (19.5%) responded to interferon-b ther-

apy. Lipid and lipoprotein changes were no different

between responders and non-responders. However, in

the whole group, alanine aminotransferase and aspar-

tate aminotransferase correlated with lipoprotein lipase

activity at the end of treatment (r � 0.569, P < 0.01

for both).

Thirteen patients underwent a second liver biopsy

12 months after the end of the trial. Although there

was only one responder in this group, the histological

activity index improved signi®cantly after interferon-b

Table 2. Plasma lipid and lipoprotein concentrations before and

after 6 months of treatment with interferon-b in patients with

chronic hepatitis Cã

Before After

Plasma

Cholesterol 163.5 � 31.7 169.5 � 35.2

Triglyceride 93.03 � 29.2 112.1 � 47.5*

Phospholipid 192.5 � 26.5 189.5 � 27.4

VLDL

Cholesterol 8.7 � 6.9 10.1 � 7.7

Triglyceride 32.3 � 23.5 40.6 � 33.2**

Phospholipid 15.3 � 12.4 15 � 10.5

LDL

Cholesterol 91 � 37.1 102.8 � 34.2

Triglyceride 33.2 � 14.4 42.6 � 17.4**

Phospholipid 71.6 � 30.7 80 � 23.8

HDL2

Cholesterol 28.5 � 13.2 25.6 � 10.2

Triglyceride 12.7 � 7.2 14.4 � 7.9

Phospholipid 35.9 � 16.2 33.7 � 13.6

HDL3

Cholesterol 35 � 10.7 30.7 � 9.8

Triglyceride 14.5 � 5 17.2 � 8.9

Phospholipid 69.8 � 15.2 60.5 � 17

HDL-CH 63.5 � 17.9 56.4 � 15.3

HDL2/HDL3 0.87 � 0.49 0.94 � 0.55

T-CH/HDL-CH 2.8 � 0.9 3.1 � 0.9

ã14 The values are presented by mean � s.d., and the unit is mg/dL.

* P < 0.01, **P < 0.05.HDL-CH = HDL cholesterol, T-CH = total cholesterol.

Figure 1. Changes in post-heparin lipase activities before and

after a course of interferon-b in 26 patients with chronic hepatitis

C. (A) Total post-heparin lipase (PHL) activity; (B) hepatic lipase

(HL) activity and (C) lipoprotein lipase (LPL) activity. Individual

values are expressed with thin lines, and mean values with thick

lines. *P < 0.05. FFA: free fatty acids.

932 R. J. ANDRADE et al.

Ó 2000 Blackwell Science Ltd, Aliment Pharmacol Ther 14, 929±935

treatment (median values: ®rst biopsy 9.8 � 3.75 ; second

biopsy 7.2 � 3.36 , P < 0.05). Histological improvement

was exclusively due to a decrease in the in¯ammatory

scores from 7.6 � 2.77,8 to 5.0 � 2.67,8 (P < 0.01); there

was no changes in the ®brosis score (median values:

®rst biopsy 2.0 � 1.19;10 ; second biopsy 2.1 � 1.19;10 ).

Table 3 shows simple correlation coef®cients for the

changes in lipoprotein lipase, hepatic lipase and post-

heparin lipase activities and those of serum lipids and

lipoproteins during 6 months of treatment with inter-

feron-b. The change (D) in post-heparin lipase and

lipoprotein lipase activities before and after 6 months of

treatment with interferon-b showed a signi®cant neg-

ative correlation with D plasma triglycerides. In addi-

tion, the change in HL11 activity after treatment with

interferon-b showed a signi®cant positive correlation

with D histological activity index in all 13 patients

evaluated (r � 753, P < 0.01).

DISCUSSION

Among their multiple systemic effects, interferons have

been found to in¯uence lipid and lipoprotein metabo-

lism.19, 20 Taking into account that lipoprotein lipase

and hepatic lipase are two key enzymes in the

metabolism and interconversion of lipoproteinsÐas

demonstrated by the major derangements of lipopro-

teins in individuals with genetic defectsÐlipase

activities should be measured in studies of the effects

of diet, drugs or other factors on lipoprotein metabo-

lism.21

In this prospective study a 6-month course of inter-

feron-b in normolipemic patients with chronic hepatitis

C was associated with disturbances in plasma lipopro-

tein metabolism as de®ned by a signi®cant increase in

total, VLDL and LDL triglycerides as well as a tendency

for HDL cholesterol levels to decrease. These changes

paralleled a marked inhibition in lipoprotein lipase (but

not in hepatic lipase) activity. Because no other

confounding factors such as diabetes, liver function

deterioration, changes in body mass index or concur-

rent therapy with other drugs were present during the

trial, the lipid alterations were probably related to

interferon-b. We did not include hyperlipaemic patients

because of the potential risk of severe hypertrygliceri-

daemia and to avoid interferences with lipases assay.

We used 6 ´ 106 U of interferon-b three times a week.

However, the optimal dose regimen of interferon-b for

chronic hepatitis C has not yet been established.3, 22

Doses of 9±12 ´ 106 U three times a week have been

reported to be as effective as 6 ´ 106 U of interferon-a in

terms of response to treatment.23

The effects of interferon-b on lipid metabolism are little

known. When administered for 10 days, to patients

with stable renal cancer, daily interferon-b serine (a

recombinant DNA-derived interferon-b) injections dur-

ing 9 days produced rapid and dose-dependent increases

in plasma triglyceride concentrations of 74% at 4.5 MU,

and 136% at 9.0 MU.24 Furthermore, in this study the

reductions in LDL cholesterol of 28% and 50% occurred

later than the reductions in triglycerides. In a small

study involving patients with chronic hepatitis C, 6 MU

of interferon-b daily caused a time-dependent increase

in triglycerides during 14 days of therapy.12 Therefore,

the more modest increase in triglyceride concentrations

and the absence of reduction in LDL cholesterol

observed in our study could be due to the different dose

regimen and time-point.

Similar changes have recently been reported in

association with interferon-a therapy.5, 6 Shinohara

et al. also tested the activity of post-heparin lipases

before and after a short-term course (2 weeks) of high

daily doses (9 MU) of interferon-a 2a, and found a

Table 3. Simple correlation coef®cients between changes in lipo-

protein lipase, hepatic lipase and post-heparin lipase activities and

serum lipids and lipoproteins during 6 months of interferon-btreatment in patients with chronic hepatitis C

DLPL

Activity

D HL

activity

D PHL

activity

D Total cholesterol 0.024 0.383 0.353

D Triglycerides )0.397* )0.321 )0.468*

D HDL-cholesterol )0.016 0.312 0.264

VLDL

D Cholesterol )0.295 0.012 )0.123

D Triglycerides )0.306 )0.103 0.037

LDL

D Cholesterol 0.075 0.050 0.084

D Triglycerides )0.220 )0.252 0.192

HDL2

D Cholesterol 0.050 0.319 0.306

D Triglycerides )0.051 )0.304 )0.309

HDL3

D Cholesterol )0.073 0.203 0.137

D Triglycerides 0.028 )0.001 0.083

* P < 0.05.

D, change in value after treatment with interferon-b.

INTERFERON-BETA AND POST-HEPARIN LIPASES IN CHRONIC HEPATITIS C 933

Ó 2000 Blackwell Science Ltd, Aliment Pharmacol Ther 14, 929±935

decrease in both lipoprotein lipase and hepatic lipase

activities.5

The mechanism(s) for such interferon-b-induced dis-

turbances in lipoprotein metabolism are unclear. A

variety of cytokines, including TNF12 -a, interleukin-1,

interferon-a and interferon-c, inhibit lipoprotein lipase

activity in post-heparin plasma, adipose tissue, or both,

and these cytokines and interferon-b inhibit adipose

tissue lipogenesis.25±28 The reduced lipoprotein lipase

activity found in our study might be associated with a

lesser availability rather than an inhibition of the

released enzyme into plasma. Indeed, some studies

indicate that cytokines down-regulate mRNA of lipo-

protein lipase.29 Measurement of lipoprotein lipase mass

in addition to lipoprotein lipase activity may con®rm

this view. The inhibition of lipoprotein lipase activity is

consistent with the major changes in lipoproteins

observed in our study, since lipoprotein lipase is thought

to be necessary for the clearance of VLDL, which

transfers surface constituents to HDL particles, enhanc-

ing HDL cholesterol.30 In fact, the negative correlation

between D lipoprotein lipase activity and D plasma

tryglycerides supports an interferon-b-induced impair-

ment in the lipolytic pathway. In addition, the enrich-

ment in LDL triglycerides could be ascribed to impaired

cholesterol ester transfer protein or lecithin cholesterol

acyltransferase activities.31 Reduced lecithin cholesterol

acyltransferase activity is seen in cholestatic syndromes

and/or in hepatic failure.32 Because no patient in our

study had evidence of worsening liver function or

cholestasis, this mechanism is unlikely. Cholesterol ester

transfer protein de®ciency in humans may also result in

triglyceride-rich LDL.33 A decrease in cholesterol ester

transfer protein activity and mass was recently demon-

strated in patients with chronic hepatitis C during

treatment with interferon-a.5 Nevertheless, interferon-

b, by inhibiting the activity of lipoprotein lipase, could

also have an effect on cholesterol ester transfer protein

activity, since adequate activity of lipoprotein lipase

enhances the ability of cholesterol ester transfer protein

to bind triglyceride-rich substrates such as VLDL.34 On

the other hand, an interferon-b-induced increase in

lipogenesis and VLDL secretion in the liver is unlikely in

our patients, because interferon-b does not stimulate

hepatic fatty acid synthesis as do other cytokines,

including interferon-a, TNF-a and interleukin-1.35

We found no differences in lipoprotein pro®le between

patients strati®ed into responders and non-responders to

interferon-b, according to accepted criteria of response to

treatment, and this has also been observed with inter-

feron-a.6 However, liver enzyme behaviour correlated

with that of lipoprotein lipase, suggesting that the effects

of interferon-b on liver in¯ammation paralleled, to some

degree, the effects on lipoprotein metabolism. In addition,

although in our study hepatic lipase activity was not

modi®ed after interferon-b therapyÐin contrast to the

effect of interferon-aÐthe signi®cant positive correlation

between D HL activity and the improvement in histolog-

ical lesions in the subset of patients who had a second

biopsy also suggests this possibility.5, 10 In fact, whereas

the short-term effects of high daily doses of interferon-awere found to be associated with an inhibition of hepatic

lipase activity, the stable hepatic lipase activity after long-

term therapy with interferon-b observed in our study

might re¯ect less in¯ammatory activity in hepatocytes.5

No patient in our study had marked increases of serum

triglycerides. This is probably due to the fact that we

included only patients with normal triglycerides at

baseline, since severe hypertriglyceridemia after inter-

feron-a therapy has been reported in relationship to

high pre-treatment triglyceride levels and recommen-

dations for monitoring triglyceride levels have been

given for this subset of patients.6±10 We believe that the

same policy is reasonable for hypertriglyceridemic

patients who are to receive treatment with interferon-b.

ACKNOWLEDGEMENTS

We thank to Javier Rubio for reviewing the liver biopsy

specimens and Karen Shashok for improving the use of

English in the manuscript.

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