A comparative study of the effects of acipimox and clofibrate in type III and type IV hyperlipoproteinemia

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Key words:

Acipimox - Apolipoproteins Lipolytic enzymes

-

Clofihrate

-

Hyperlipoproteinemia

-

Introduction Acipimox (5-methylpyrazinecarboxylic acid 4-oxide), an analogue of nicotinic acid, is a new lipid-lowering drug. It inhibits adipose tissue lipolysis, resulting in a decrease of free fatty acids and a decrease of synthesis of very low density lipoproteins (VLDL) [l]. The duration of activity is longer than that of nicotinic acid. Uncontrolled and placebo-controlled clinical trials showed its effect in type II and type IV hyperlipoproteinemia (HLP) [2]. In the type II patients a significant rise in high density lipoprotein (HDL) cholesterol was also observed [2]. At present clofibrate is the most commonly used and effective drug for the treatment of elevated levels of VLDL and VLDL-remnants [3]. Clofibrate is effective during long-term treatment, e.g. in type III HLP [4]. In order to compare the effect of acipimox with clofibrate we treated 10 patients with type III and 10 patients with type IV HLP with either drug in a cross-over study and determined serum lipids, lipoproteins,.apolipoproteins and lipoprotein lipases; routine laboratory and clinical measurements were performed to test its safety and tolerability. Patients and Methods Patients were selected from our lipid clinic on the basis of their willingness to cooperate. Two groups of patients, 10 with type III HLP and 10 with type IV HLP, included each 8 males and 2 females. All subjects gave informed consent and the study was approved by the University Hospital’s ethical committee. One type III patient dropped out of the study because of side-effects during treatment with acipimox (see below). The mean age of the type III patients was 53 years (range 33-68) and mean relative body weight (actual body weight X 100% divided by ideal body weight based on Metropolitan Life Insurance Life Time Tables, 1959) was 126% (range 101-169). Seven type III patients had palmar and tubero-eruptive xanthomas at the time of diagnosis. Cardiovascular disease was present in 4 and peripheral disease in 4 patients. Mean serum cholesterol at the entry of the study was 10.7 mmol/l (range 6.8-15.6), mean serum triglycerides (TG) 7.6 mmol/l (range 2.4-25.0). The diagnosis type III HLP was based on the presence of the following characteristics of the VLDL fraction: (a) P-mobility on agarose gel electrophoresis [5], (b) the relatively high cholesterol content: a ratio VLDL cholesterol/serum TG (in mmol/l) of more than 0.69 [6] and (c) the apoprotein E phenotype E2/2 [7]. The mean age of the type IV patients was 46 years (range 35-59). Their mean relative body weight was 120% (range 101-140). One patient had symptoms of cardiovascular disease. None had xanthomas or signs of peripheral vascular disease.

53

Mean serum cholesterol was 6.1 mmol/l (range 5.4-7.4) and mean serum TG were 4.5 mmol/l (range 2.0-12.9). Type IV HLP was diagnosed when serum TG were above 2.2 mmol/l and LDL (low density lipoprotein) cholesterol below 5.2 mmol/l with exclusion of type III HLP. All patients continued their usual moderately fat-restricted diet (30-35s of total calories). Hypolipidemic drugs (mostly clofibrate) were discontinued at least 6 weeks prior to the start of the study. Causes of secondary hyperlipidemia were excluded. A cross-over open label study was performed during 4 consecutive 6-week periods on an outpatient basis. The first period was the base-line period. In the 2nd period the patients were randomly allocated to either clofibrate (1 g, twice a day) or acipimox (250 mg, 3 times a day). One half of the patients of each group started with acipimox, one half with clofibrate. The 3rd period was a wash-out period and in the 4th they took the other drug. Patients were seen at 3-week intervals at which time questions about adverse reactions were followed by a brief physical examination. At e&h visit blood samples were taken by venepuncture after an overnight fast for the determination of serum cholesterol, serum TG, HDL cholesterol and of several laboratory parameters (renal and hepatic function tests, blood cell counts, uric acid, blood glucose) and an urinanalysis was done. At the end of each 6-week period blood was taken for the determination of lipoproteins (VLDL, LDL, HDL and its subfractions, HDL, and HDL,), apoprotein A-I, A-II and B and the ratio between C-II and C-III. Lipoprotein lipase and hepatic lipase were determined in serum obtained 15 min after the injection of heparin (50 U/kg) at the end of the base-line period and of each treatment period. Prior to the study and at the end of each treatment period an electrocardiogram was obtained. Compliance with the medication was assessed by oral questioning and by pill counting. Cholesterol was determined by an enzymatic method [8] and triglycerides by a semi-automated calorimetric procedure [9]. VLDL (d < 1.006 g/ml) were isolated by ultracentrifugation [lo] and total HDL cholesterol was determined in whole serum using the polyethylene glycol 6000 precipitation method [ll]. LDL cholesterol was calculated by subtraction. HDL, cholesterol and HDL, cholesterol were determined after density gradient ultracentrifugation as described previously [12] using the density limit of 1.100 g/ml to separate HDL, and HDL,. The apoprotein E phenotypes and the apoproteins C of the VLDL were determined after isoelectric focusing and densitometric scanning [13,14]. Apoprotein A-I, A-II and B concentrations were determined in whole serum by rocket immuno-electrophoresis [15]. Apo A-I and A-II were isolated by Sephacryl S 200 column chromatography (Pharmacia, Uppsala, Sweden). Apo A-II was further purified by preparative flat bed isoelectric focusing [16]. Monospecific antisera against these pure apoproteins or against LDL (1.030 < d < 1.050 g/ml) were raised in rabbits. The 0.8% agarose gel contained besides the antisera 20 g polyethylene glycol 6000 per liter. The electrophoresis was performed overnight at 6.5 V/cm at room temperature. In the assay of apo A-I or A-II it was found that delipidation with n-butanol-diisoprophylether [17] or treatment with the detergent Tween 20 [18] did not improve the immunoreactivity. Furthermore, incubation of sera with TG concentration from l-7.5 mmol/l with purified bovine milk LPL (activity 750 pmol FFA . mll’ . h-‘) for 2 h at 37°C did

54

not increase apo B values [19]. Each serum was analysed in duplicate on the same plate; 2 plates with the same seca were always run on the same day. Thus, the final result obtained for a given apoprotein concentration is the mean of 4 measurements. Interassay coefficients of variation of 17 determinations were 5.8% for apo A-I, 5.4% for apo A-II and 7.9% for apo B. Apo A-I, A-II and B were determined in sera which had been stored frozen at -2O’C for 1-3 months, which does not lead to a loss in immunoreactivity [20,21]. Lipoprotein lipase and hepatic lipase in postheparin serum were measured with an immunochemical technique as previously described [ 131. All data are presented as mean f 1 standard deviation, unless otherwise indicated. For statistical analysis Wilcoxon’s test for paired observations was used. Results Nineteen of the 20 patients completed the study. Two patients (both type III) complained of flushing of the face during acipimox treatment. One of these had severe complaints, resulting in a drop-out after 1 week. The other patient continued the drug, resulting in a gradual decrease of the complaints of flushing. Flushing started 15 min after intake of the drug and remained during 2 h. Otherwise, both acipimox and clofibrate were tolerated well; most subjects were familiar with clofibrate from previous prescription. The compliance was good, because 93% (range SO-100%) of the prescribed medication was taken. During treatment with both drugs no consistent changes were noted during physical examination, including pulse rate and blood pressure, and routine laboratory analysis (blood cell count, creatinine, uric acid, alkaline phosphatase, ALAT, ASAT, creatine phosphokinase, blood sugar, urinanalysis) except an increase of creatine phosphokinase to slightly elevated levels in 4 patients during clofibrate treatment. Electrocardiography did not reveal abnormalities. Mean body weight changed less than 0.5 kg during each treatment period. Effect of clofibrate and acipimox in type III hyperlipoproteinemia In the base-line period there were no significant changes in mean concentrations of serum cholesterol, serum TG and HDL cholesterol in the 9 type III HLP patients (Fig. 1). There was no significant difference in the basal values of serum lipids before each treatment. During treatment with clofibrate and acipimox serum lipids dropped in all patients except one, who had no decline in serum TG during acipimox treatment. Serum cholesterol decreased 24% (P < 0.01) after 3 and 6 weeks of treatment with clofibrate in comparison with the last base-line value. Acipimox caused a decrease of 19% (P c 0.01) after 3 weeks and 30% (P < 0.01) after 6 weeks in serum cholesterol. Serum triglycerides decreased 34% (P < 0.01) after 3 and 6 weeks treatment with clofibrate. During acipimox treatment triglycerides dropped 44% (P < 0.01) after 3 weeks and 48% (P < 0.01) after 6 weeks (Fig. 1). There was an increase in HDL cholesterol of 15% (P < 0.05) and 10% (P < 0.05) after respectively 3 and 6 weeks treatment with clofibrate and of 14% (P < 0.01) and 7% (P < 0.01) with acipimox (Fig. 1). There was no significant difference in values of

55

serum cholesterol, TG and HDL cholesterol during treatment with acipimox and clofibrate, respectively. During treatment with both drugs lipids in the VLDL fraction decreased significantly (Table l), but the ratio between VLDL cholesterol and serum TG or VLDL-TG did not change. LDL cholesterol and HDL, cholesterol did not change significantly during treatment with clofibrate nor with acipimox (Table 1). HDL, cholesterol increased during treatment with clofibrate but not with acipimox. Clofibrate, but not acipimox, induced a significant increase of lipoprotein lipase activity (Table 2). For hepatic lipase a small but significant reduction during acipimox treatment and no change during clofibrate treatment was found. During clofibrate treatment an increase of apo A-I and A-II and a decrease of apo B were found (Table 2) resulting in a significant (P -C0.05) increase of the ratio between apo A-I and apo B by 31%. The ratio between apo A-I and A-II dropped significantly 14% (P < 0.01). During acipimox treatment there were no significant alterations in these apoprotein levels and ratios (Table 2). The ratio between apo C-II and C-III remained unaltered during treatment with both drugs. Effect of clofibrate and acipimox

in type IV hyperlipoproteinemia

In the base-line period before acipimox there was an increase of serum TG, which did not reach statistical significance (Fig. 2). Cholesterol did not change during treatment with both drugs. Serum TG fell in all patients during clofibrate treatment, but not in 2 patients during acipimox treatment. Serum TG decreased significantly TYPE

III

HLP

(mean?

1

SEM)

jACiPlMOXj

jCLOFlBRATE/

m mol / I 12

n=9

T

IO-

6-

s__~-;-T

-3

‘z

0

3

HDLc hoI

6

-3

0

3

6 weeks

rp<0.05

rrp
“5 week

0

Fig. 1. Effects of clofibrate (2 g) and acipimox (750 mg) on serum cholesterol (chol), serum triglycerides (TG) and high density lipoprotein cholesterol (HDL chol) in 9 patients with type III hyperiipoproteinemia. The percentages refer to changes after 6 weeks of treatment in comparison with the last value before treatment.

1

10.00* 3.19 7.05 * 1.04 -z 0.01 -30 -12 to -60

8.88 f 1.95 6.72+ 1.15 < 0.01 -24 -4 to -40

Chol.

7.99 f 6.30 4.15 f 1.51 < 0.01 -48 +4 to -73

5.57 f 2.39 3.68 f 1.61 < 0.01 -34 -15 to -61

TG

AND ACIPIMOX

LIPIDS

6.01 f 3.42 3.33 * 1.35 < 0.01 -45 -9 to -73

4.76 k 1.76 2.92 f 1.28 c 0.01 -40 -17 to -63

VLDL chol.

ON SERUM

a Mean f SD for last values before treatment. b Mean f SD for values obtained after 6 weeks of treatment.

Percent. change Range (I)

P

Base-line a Acipimox b

Percent. change Range (Sg)

P

Base-line a Clofibrate b

EFFECT OF CLOFIBRATE PROTEINEMIA

TABLE

6.34& 5.52 3.15*1.55 < 0.05 -50 +24 to -72

4.18 + 1.72 2.79 f 1.34 < 0.01 -33 -18 to -71

(mmol/l)

3.05 * 1.02 2.75 +0.85 NS -10 +30 to -43

3.14*1.01 2.79 f 0.82 NS -11 +38 to -32

LDL chol.

LIPOPROTEINS

VLDL-TG

AND

0.91 k 0.24 0.97 * 0.22 NS f7 -25 to +25

0.93 + 0.17 1.02 f 0.24 < 0.05 +10 -7 to +28

HDL chol.

IN 9 PATIENTS

chol.

0.21* 0.09 0.23 kO.10 NS +10 -36 to +200

chol.

0.65 + 0.17 0.71 *0.14 NS +9 -28 to +55

0.66 + 0.07 0.81 f 0.21 < 0.05 +23 -23 to +57

HDL,

TYPE III HYPERLIPO-

0.23 f 0.09 0.18 +0.05 NS -22 -44 to +33

HDL,

WITH

57

TABLE

2

EFFECT OF CLOFIBRATE AND ACIPIMOX ON LIPOLYTIC ENZYME ACTIVITIES (LIPOPROTEIN LIPASE (LPL) AND HEPATIC LIPASE (HL) IN POSTHEPARIN SERUM (50 U/kg)) AND APOPROTEINS IN 9 PATIENTS WITH TYPE III HYPERLIPOPROTEINEMIA LPL

HL

A-I

prnol FFA.ml-‘.h-’

A-II

B

mg/ml

Base-line A Clofibrate h

6.6+1.6

16.6 k 6.4

1.6+0.2

0.6 f 0.2

1.29kO.29

9.7 f 2.0

15.6k5.0

1x+0.3

0.8 *0.3

1.14kO.36

P

< 0.01

NS

< 0.05

< 0.01

i 0.05

Percent. change

+47

-6

+13

+ 33

-12

+ 19 to +103

-26

0 to +80

+8 to -33

Base-line a

6.6+ 1.6

16.6k6.4

1.6kO.2

0.6kO.2

1.28k0.32

Acipimox

7.1 k2.1

14.7 + 4.9

1.7kO.2

0.6kO.2

1.28kO.26

P

NS

< 0.05

NS

NS

NS

Percent. change

+ 14

-11

+6

0

0

Range (‘%)

-25

0 to 1-33

-25

(%)

Range

h

-26

to i36

-12

to +29

to +7

-7

to +21

to t27

to +25

“ Mean _+SD for values before treatment. h Mean? SD for values obtained after 6 weeks of treatment

(P < 0.01) 31% and 35% after 3 and 6 weeks administration of clofibrate, respectively (Fig. 2). Acipimox caused a drop of 28% and 34% (P -C 0.05) respectively. Because of the variability in serum TG in the period before treatment with acipimox, we also compared the effect of acipimox after 6 weeks on serum TG with the mean of the 4 basal values before treatment with acipimox and clofibrate. The difference in values obtained this way, was also significant (5.02 f 3.36 vs 3.95 _t 2.30 mmol/l, TYPE

n =lO

IIZ HLP

(mean

+ SEM)

mmol/l 8-l

6

I

J-T-T-

r

TCHOL’A\ /

II:\,;

4

1

I

35 % I----~‘TG

2

.T-_J

\

-34% I’j

1

-3 +p
0

3

?? p
6

-3

0

3

6 weeks

vs

week

0

Fig. 2. Effects of clofibrate (2 g) and acipimox (750 mg) on serum cholesterol (chol), serum triglycerides (TG) and high density lipoprotein cholesterol (HDL chol) in 10 patients with type IV hyperlipoproteinemia. The percentages refer to changes after 6 weeks of treatment in comparison with the last value before treatment.

3

OF CLOFIBRATE

-c 0.01

-35

-9

5.95 + 5.77

3.95 + 2.30

< 0.05

-34

+83

NS

- 2

+21

6.55 + 1.40

6.07kO.88

NS

- 7

+3 to -30

P

Percent. change

Range (W)

Base-line a

Acipimox

P

Percent. change

Range (X)

treatment.

after 6 weeks of treatment.

to +35

-1

-16

+s9

+63

to +27

+3

-36

-34 to -55

+11

NS

NS

< 0.01 to -50

< 0.05

3.80 f 0.96

2.99k1.95

1.37kO.77

b Mean + SD for values obtained

to -60

0.81 kO.18 0.90 * 0.21

3.70 +0.72

4.65 + 4.58

2.08 f 1.50

to +29

-16

to +85

-8

to -62

-19

-21

to -59

( 0.05 +11

< 0.05 +24

i 0.01 -50

< 0.01 -46

0.18 + 0.09

-44

to +35

-5 -47

to +100

< 0.05 +10

0

0.66 f 0.14 0.20 + 0.04 NS

0.60 + 0.09 0.20+0.10

to +37

-8

+6 to i44

i 0.05 +11

NS

0.62 f0.13 0.69 + 0.15

0.17 + 0.06

0.88 f 0.20

chol.

0.79+0.16

HDL,

IV HYPERLIPO-

3.36 + 0.72 4.16 f 0.66

chol.

TYPE

2.17 f 2.25

HDL,

WITH

4.17k2.17

IN 10 PATIENTS

1.11 +oso

(mmol/l)

2.06 f 0.98

LIPOPROTEINS

HDL chol.

AND

LDL chol.

chol.

LIPIDS

VLDL-TG

VLDL

ON SERUM

a Mean f SD for last value before

b

to -53

3.36 rt 1.77

to -15

5.14*2.45

6.28 f 0.94

6.15_+0.81

b

Base-line a

Clofibrate

ACIPIMOX

TG

AND

Chol.

PROTEINEMIA

EFFECT

TABLE

59 TABLE

4

EFFECT OF CLOFIBRATE AND ACIPIMOX ON LIPOLYTIC ENZYME ACTIVITIES (LIPOPROTEIN LIPASE (LPL) AND HEPATIC LIPASE (HL) IN POSTHEPARIN SERUM (50 U/kg)) AND APOPROTEINS IN 10 PATIENTS WITH TYPE IV HYPERLIPOPROTEINEMIA LPL

A-l

HL

pmol FFA.ml-‘.h-’ Base-line a Clofibrate h

P Percent. change Range (‘%) Base-line ’ Acipimox h

P Percent. change Range (‘%)

A-II

B

mg/ml

6.Ok2.6 8.3k3.2 < 0.05 +38 -22 to +220

22.1 f 1.4 22.1 + 8.4

1.5 * 0.2 1.6? 0.3

0.4+0.1 0.5 + 0.1

NS 0 -26

NS -+I -31

< 0.05 +25 0 to +50

1.8kO.3 1.7kO.4 NS -6 -25 to +11

6.0 & 2.6 6.7 + 2.8 NS +12 -39 to +93

22.1 * 7.4 19.4 + 8.9 c 0.01 -12 -90 to +2

0.5 * 0.1 0.5 & 0.1 NS 0 010 +33

1.7*0.4 1.7kO.4 NS 0 -14to +8

to +35

to +21

1.4kO.2 1.6kO.3 < 0.01 + 14 0 to +20

’ Mean k SD for values before treatment. ’ Mean + SD for values obtained after 6 weeks of treatment.

P -=z0.05). The increase of HDL cholesterol during treatment with clofibrate and acipimox after 3 weeks was 9% and 6% respectively, and after 6 weeks 11% and 11% (p < 0.05) (Fig. 2). There was no significant difference in the values of serum cholesterol, TG and HDL cholesterol during treatment with acipimox and clofibrate, respectively. During treatment with both drugs VLDL cholesterol and VLDL-TG decreased significantly with the exception of VLDL-TG during acipimox treatment (Table 3). LDL cholesterol increased significantly during clofibrate treatment, but remained constant during acipimox treatment (Table 3). HDL, cholesterol was unaltered during treatment with both drugs, while HDL, cholesterol increased significantly (Table 3). Lipoprotein lipase activity increased significantly during clofibrate treatment and did not change during acipimox treatment (Table 4). Hepatic lipase activity remained unchanged during clofibrate treatment, but decreased significantly during acipimox treatment (Table 4). Clofibrate did not induce changes of apoprotein A-I and B, but caused an increase in apo A-II (Table 4). The ratio between apo A-I and A-II decreased 12% (P < 0.05); the ratio between apo A-I and B did not change. During acipimox treatment apo A-I increased significantly, but apo A-II and apo B remained unchanged (Table 4). The ratios between these apoproteins were stable. The ratio between apo C-II and C-III was unaltered during treatment with both drugs. Discussion Nicotinic acid has a strong hypolipidemic effect in type II, III and IV HLP, usually in doses of 3-6 g/day [3]. The use of this drug is limited, however, because

60

of side-effects such as almost obligatory flushing and abnormalities in hepatic function. We compared acipimox, a new nicotinic acid derivative, and clofibrate, for treatment of patients with type III and type IV HLP. Both drugs were well tolerated. Flushing occurred in only 2 patients during acipimox treatment. One patient dropped out of the study because of this complaint; in the other subject flushing diminished during the course of the study. Other side-effects have not been noted. Thus, acipimox used in a dose of 750 mg/day seems to be better tolerated than nicotinic acid. Clofibrate (2 g/day) and acipimox (750 mg/day) had a similar hypolipidemic effect during 6 weeks of treatment in type III and type IV HLP. A decrease of serum TG of 3540% in both types was observed. Serum cholesterol did not change in type IV patients, and decreased 24-30s in type III patients. Clofibrate seemed to have reached its maximal effect after 3 weeks of treatment, whereas during acipimox treatment the serum lipids were still falling (Figs. 1 and 2). The effect of acipimox in type IV patients is in agreement with a previous report [2]. There are no earlier studies of acipimox in patients with type III HLP. The decrease of serum cholesterol and TG was due to a decrease of these lipids in the VLDL-fraction. These reductions were rather similar during treatment with both drugs in both types of patients. The VLDL-fraction of the type III patients remained cholesterol-rich during treatment with both drugs, indicating the presence of remnant-particles. In type IV patients there was no change in LDL cholesterol during acipimox treatment, in contrast to the known increase during clofibrate treatment caused by enhanced catabolism of VLDL [22]. There was a striking difference in the effect of both drugs on lipolytic enzymes. Clofibrate induced a considerable rise in lipoprotein lipase but no change in hepatic lipase in the 2 groups, whereas acipimox gave a slight decrease of hepatic lipase and no change in lipoprotein lipase. These results are in agreement with the hypothesis that clofibrate acts by stimulating the conversion of VLDL [22,23], while acipimox, as nicotinic acid, decreases VLDLsynthesis [24]. During treatment with both drugs HDL cholesterol increased in both groups of patients. The effect of clofibrate and nicotinic acid on HDL cholesterol is known [25-301. Acipimox was studied so far only in type II patients for its effect on HDL cholesterol, showing an increase [2]. The increase of HDL cholesterol in our study was mainly due to a rise of HDL, cholesterol. HDL, cholesterol did not change. These findings are at variance with a previous study, indicating a rise in HDL, cholesterol in type IV HLP patients during clofibrate treatment [25]. These contrasting results can be ascribed to differences in methodology (sequential preparative ultracentrifugation versus density gradient ultracentrifugation), and more important, to the different densities used to separate HDL, and HDL, (1.125 vs 1.100 g/ml), because a major variable component of HDL is found between these densities [12]. There are some reports of the effect of nicotinic acid and clofibrate treatment on HDL, and HDL,, isolated by rate zonal ultracentrifugation [26-281. During nicotinic acid treatment an increase of HDL, cholesterol has been described in normolipidemic subjects [26] and in type IV patients [27]; in the latter group a slight increase of HDL, cholesterol was also found. We only observed an increase of

61

HDL, cholesterol during acipimox treatment in the type IV patients. In typ.e III HLP clofibrate and nicotinic acid increase HDLz and HDL, [28]. We only observed an increase of HDL, cholesterol in the type III patients during clofibrate treatment. These results are less likely explained by differences in methodology, because rate zonal ultracentrifugation and density gradient ultracentrifugation may expect to give similar results [12]. It can be due to selection of subjects or difference in drugs or dose used. Previous studies reported an increase of apo A-I during nicotinic acid treatment [26,27]. We observed the same effect in type IV patients during acipimox treatment. During clofibrate treatment apo A-I and A-II increased in type III patients as reported earlier [29]. Decreases of apoprotein A have not been noted. In summary, clofibrate and acipimox in a relatively low dose have a similar hypolipidemic effect in type III and type IV hyperlipoproteinemia. Both drugs increase HDL cholesterol. Because of the absence of an increase of LDL cholesterol during acipimox treatment and because of the doubts around clofibrate since the outcome of the WHO-trial [29], acipimox could be an alternative for clofibrate in these conditions. A higher dose of acipimox may even have a more pronounced effect. However, its long-term safety and efficacy still have to be established. Acknowledgements The excellent technical assistance of Mrs. D. van Sommeren-Zondag and Mrs. A. Hijmans, the support of Dr. J. Verjans and the preparation of the manuscript by Mrs. I. ten Have are gratefully acknowledged. Acipimox was provided by Farmitalia, Brussels, Belgium. References 1 Fucella

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