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Effects of bezafibrate on the serum lipoprotein lipid and apolipoprotein composition, lipoprotein triglyceride removal capacity and the fatty acid composition of the plasma lipid esters
257
Atherosclerosis, 37 (1980) 257-269 @ Elsevier/North-HollandScientific Publishers,Ltd.
EFFECTS OF BEZAFIBRATE ON THE SERUM LIPOPROTEIN LIPID AND APOLIPOPROTEIN COMPOSITION, LIPOPROTEIN TRIGLYCERIDE REMOVAL CAPACITY AND THE FATTY ACID COMPOSITION OF THE PLASMA LIPID ESTERS
BENGT VESSBY, HANS LITHELL, KRISTOFFER HELLSING, ANN-MARGRET ~~STLUND-LINDQVIST, INGA-BRITT GUSTAFSSON, JONASBoBERG and HELLMUTH LEDERMANN Departments
of Geriatrics and Clinical Chemistry,
University of Uppsala, Uppsala (Sweden)
(Received 8 February, 1980) (Revised, received 6 May, 1980) (Accepted 7 May, 1980)
Summary Bezafibrate has been shown to effectively reduce elevated levels of very low density lipoprotein (VLDL) in hypertriglyceridaemia. It has also been claimed to be particularly effective in increasing high density lipoprotein (HDL) concentrations. In the present study 15 hypertriglyceridaemic patients (9 type IIB, 1 type III and 5 type IV) were investigated before and after 2 months’ treatment with bezafibrate (600 mg daily) in an effort to ciarify further the effects of the drug on lipoprotein metabolism. The serum triglycerides decreased by 46% (P < 0.001) corresponding to a significant reduction of both the VLDL (-43%, P< 0.001) and the low density lipoprotein (LDL) (-27%, P< 0.05) triglycerides. The serum cholesterol decreased by 13% (P < O.OOl), mainly due to reduction of the VLDL cholesterol (-51%, P < 0.001). There was no significant reduction of the LDL cholesterol in the whole group (-lo%, n.s.). However, if only the patients with hypercholesterolaemia were considered, there was a significant reduction of LDL cholesterol by 19% (P < 0.01). HDL triglycerides and HDL cholesterol increased by 20% (P < 0.05) and 22% (P < 0.001) respectively. While the concentration of apolipoprotein (apo) B decreased by 15% (P < 0.01) there were significant increases of apo A-I (+14%, P< 0.001) and apo A-IX (+30%, P < 0.001) analogous to the increase of HDL lipid concentration. This study was financially supported by grants from the Swedish Medical Research Council (19P-5640 and 19X-6546-02). The bezafibrate tablets were generously provided by Boehringer Mannheim GmbH. Requests for reprints to: Dr. Ben& Vessby. Department of Geriatrics, Box 12042. S-750 12 UPPsala. Sweden.
258
However, the ratio between apo A-I and apo A-II decreased significantly during therapy (-12%, P < 0.001) indicating that the increase of HDL may have preferentially affected the HDL-3 fraction. There were highly significant reductions of both the ratio between LDL and HDL cholesterol (-24%, P < 0.001) and the ratio between apo B and apo A-I (-25%, P< O.OOl), changes which should be favourable with regard to atherogenicity. Significant increases of both the plasma post-heparin lipoprotein lipase activity (+20%, P < 0.001) and the fractional removal rate (K,) at the intravenous fat tolerance test (+30%, P < 0.001) indicate that one of the mochanisms behind the effects of the drug on lipoprotein metabolism may be an increased removal of circulating triglyceride-rich lipoproteins. Bezafibrate treatment caused significant changes in the plasma lipid fatty acid composition, mainly by increasing the mono-unsaturated fatty acids (palmitoleate and oleate) concomitant with a significant decrease in the content of linoleate. Very similar changes have been recorded during clofibrate treatment. The mechanisms behind these changes as well as their significance are at present obscure. Key words:
Apo-lipoproteins - Bezafibrate - Cholesterol proteins -Lipoprotein lipase activity
-Lipid-lowering
drug - Lipo-
Introduction In the search for new effective lipid-lowering drugs, bezafibrate (2-(4-[2-(4chlorobenzamido)ethyl] -phenoxy )-2-methylpropionic acid) has proved to be an efficient lipid-lowering drug in patients with hypertriglyceridaemia [ 141. It has been claimed that bezafibrate may be more effective than clofibrate in reducing increased concentrations of very low density lipoproteins (VLDL) [ 11. Also, bezafibrate has been suggested to be particularly effective in increasing the high density lipoprotein cholesterol (HDL-C) concentrations [ 21. However, there is presently sparse information regarding the effect of bezafibrate on serum apolipoprotein concentrations. The present study was performed to investigate further the effect of bezafibrate on the serum lipoprotein composition, especially with regard to the serum apo-lipoprotein concentrations. The effects of bezafibrate on the capacity to remove triglyceride-rich lipoproteins from the circulation, as judged from the post-heparin plasma lipoprotein lipase activity and the intravenous fat tolerance test, were also studied as well as the effects on the fatty acid composition of the plasma lipid esters. Materials and Methods Fifteen hypertriglyceridaemic patients were studied before and after 2 months of bezafibrate treatment. The clinical characteristics of the patients are shown in Table 1. All patients were on treatment with lipid-lowering diets for at least 2 months before the trial in order to obtain weight stabilisation and firm base line values for serum lipids. The hatients received 600 mg bezafibrate
259
TABLE
1
CLINICAL Patient
CHARACTERISTICS Initial
Sex
NO.
Age (Yr)
OF PATIENTS B.W.index a
PARTICIPATING
Lipoprotein
Clinical diagnosis
pattern
Before treatment
After treatment
1
BJ
M
43
1.25
IIB
IIB
2 3
LA RN
M M
61 44
1.11 1.21
IIB IIB
N IIB
4 5
SH MC
M F
63 60
0.86 1.07
III IIB
N(II1) N
6
SB
F
61
0.93
IIB
N
7 8 9 10 11
LS GL CGS NOH BK
M F M M M
61 53 46 49 49
1.16 1.42 1.31 1.28 1.16
IV IIB IV IV IIB
N IV IV N N
12 13 14 15
IS AS HS RT
F F M M
51 50 64 58
1.11 1.02 1.06 1.08
IV IIB IV IIB
N N N HA
a
BW_index
=
body weight height
(cm) -
IN THE TRIAL
Asymptomatic HLP. Poor response to gemfibrozil. Asymptomatic HLP. Asymptomatic HLP. Poor response to clofibrate Intermittent ckudication. Intermittent claudication. Angina pectoris? Stenosis of subclatian artery. Transitory global amnesia. Hypertension. Hypertension. Poor response to clofibrate. St. postmyocardial infarction. Hypertension and arthritis urica. Transitory ischaemic attacks and angina pectoris Hypertension. Hypertension; angina pectoris. Hypertension; angina pectoris. Asymptomatic HLP.
0%) 100
for 2 months. One tablet (200 mg) was given 3 times daily. The patients were seen at a clinical visit before the trial, after 1 month’s treatment and at the end of the 2 month treatment period. All patients declared that they had taken the full dose of bezafibrate throughout the trial. Subjects who had received other lipid-lowering drugs were only included in the trial after an interval of at least 2 months following discontinuation of the previous drug. All patients were on dietary treatment for at least 2 months before the trial and had demonstrated on this regimen firm and consistently elevated base lines for serum lipids. No patients with secondary hyperlipoproteinaemia due to hypothyroidism, liver disease, renal insufficiency or diabetes mellitus, or on medication known to affect the serum lipid levels, were included. The lipoprotein patterns (Table 1) were classified according to Fredrickson et al. [5] using the criteria suggested by Beaumont et al. 161. Cutoff points for hyperlipoproteinaemia (HLP) for men and women respectively, were for very low density lipoprotein triglycerides (VLDL-TG) 1.40 and 1.00 mmol/l, and for low density lipoprotein cholesterol (LDL-C) 5.20 and 5.70 mmol/l corresponding to the 85th percentile in healthy men and women in local control material [7]. Lipoprotein lipid concentrations were determined in serum after a 12-14 h overnight fast. EDTA was added to serum as a 5% solution to give a final concentration of 0.05%. VLDL were isolated as the top fraction after centrifu-
260
gation at d = 1.006 for 16 h at 15°C and 105,000 X g in a Beckman L2-65B preparative ultracentrifuge using a 40.3 rotor [ 81. LDL were precipitated from the bottom fraction at d = 1.006 by a heparin-manganese chloride solution. After low speed centrifugation, the HDL lipid levels were determined in the supernatant [9]. The concentrations of the lipids in LDL were obtained indirectly by subtracting the HDL lipid levels from lipid concentrations of the bottom fraction after centrifugation at d = 1.006. Cholesterol and triglyceride concentrations of serum and of the isolated lipoprotein classes were determined in isopropanol extracts by semi-automated methods in a Technicon AutoAnalyzer type II [lo]. The recoveries of triglyceride and cholesterol in the isolated density classes were within 100 f 10% in all samples. The top and bottom fractions at d = 1.006, as well as whole serum were analysed using agarose electrophoresis [ 111. The concentrations of serum apolipoprotein (apo) B, A-I and A-II were determined by electroimmunoassay according to Laurel1 [12] modified as suggested by Curry et al. [13]. Specific antibodies, anti-apo A-I, anti-apo A-II and anti-apo B were induced in rabbits against purified apo A-I, A-II and lipoprotein B. Apo A-I and lipoprotein B were prepared as described previously [14]. To prepare apo A-II, HDL was delipidated with chloroform-methanol (2 : 1) and apo HDL was fractionated by Sephadex G-200 chromatography followed by a further purification by chromatography on DEAE-cellulose in 6 M urea employing a linear gradient essentially as described previously [ 151. The electroimmunoassay was run overnight (16-20 h) at 10°C in a 0.05 M diemal buffer, pH 8.6, at 2.5 V/cm (apo B), 4.0 V/cm (apo A-I) and 3.0 V/cm (apo A-II) respectively using a 2% agarose gel containing 5% dextran. Samples and serial solutions of a reference serum were applied in duplicate and all samples from one patient were always run on the same plate to minimise intrapatient variation. The within-assay variations, S * were 2.6%, 2 8% and 3.6% for apo A-I, apo A-II and apo B respectively. The apolipoprotein concentrations in serum are expressed in qbitrsry units (A.U.) relative to the concentration in a reference serum obtained from a large pool of healthy blood donors (100 A.U.). The apolipoprotein concentrations in this reference serum, when standardised against a lipoprotein B preparation (d = 1.030-1.050) and a highly purified apolipoprotein A-I preparation, have been estimated to be 1.0 and 1.5 mg/ml, respectively. The lipoprotein lipase activity in post-heparin plasma was determined in an assay system using tritium trioleate labelled Intralipid@ as a substrate in a reaction medium containing glycine (2 mmol/l) and albumin (0.11 mmol/l). The triglyceride concentration was 3 mmol/l. The ionic strength was 0.08 and the pH value 8.5. The incubation was performed at 37°C for 40 min and the release of tritium oleic acid was used as a measure of post-heparin plasma lipoprotein lipase activity. In this system more than 90% of the total lipase is inhibited by protamine sulphate (1 mg/ml). This together with other inhibition criteria indicates that 90% or more of the total enzyme activity measured is lipoprotein-lipase activity. This is probably due to the fact that Intralipid is a poor substrate for measuring hepatic triglyceride activity under the conditions *&Cj=
d2
f- 2,
= standard deviation
of
the difference
(d) between
a number
of duplicate
measurements.
261
used. Theassay procedure has been described in detail elsewhere [ 161. Blood glucose concentrations were determined by a glucose oxidase method [ 171 and the serum insulin determinations by the Phadebas Insulin Test (Pharmacia AB, Uppsala) which is based upon a radioimmunosorbent technique [ 181. The serum samples were preincubated with the matrix-bound antibodies for 24 h before the isotope-labelled insulin was added. In this way, an increase of the sensitivity of the method was obtained. The coefficient of variation was 5% in the range 5-10 mu/l. Clinical laboratory analyses with regard to safety were performed according to the routines at the local clinical laboratory. The intravenous fat tolerance test (IVFTT) was performed according to Carlson and Rijssner [ 193. The fatty acid composition of plasma triglycerides, cholesterol esters, and phospholipids was determined in blood samples drawn into pre-cooled vacutainer tubes containing EDTA. After low-speed centrifugation of the blood at +4”C, the plasma lipids were extracted with chloroformlnethanol and separated by thin-layer chromatography according to a method previously described [20]. The fatty acids of the isolated lipid fractions (triglycerides, cholesterol esters and phospholipids) were transesterified to methylesters [ 211 and separated on a 4% EGSS-XH column in a Pye Unicam 104 gas-liquid chromatograph equipped with an automatic sample injector (Hewlett Packard Co.). Retention times of known fatty acid standards (obtained from Supelco Inc., Bellefonte, PA) were used for identifying the peaks of the chromatogram. The relative compositions of the different fatty acids (expressed in %) and their retention times were calculated by a Vidar 6300 digital integrator. Statistics The data presented are based on individual comparisons between results recorded before and after treatment with bezafibrate. The significance of the differences between mean values was estimated with paired t-test (two-tailed test). Results Body weight The average body weights before and after treatment did not differ cantly (mean body weight + SD, 79.7 4 13.4 kg before compared with 13.9 kg after treatment). The weight changes did in no case exceed during the 2 months of treatment and only in 3 patients did the body change exceed +2 kg.
signifi79.9 + 3.0 kg weight
Effects of bezafibrate on the serum lipoprotein lipid concentrations The serum triglyceride and cholesterol concentrations decreased highly significantly during treatment with bezafibrate (Table 2). This was mainly due to decreases of the VLDL-TG and VLDL-C concentrations. Also the LDL-TG concentration decreased significantly. The reduction of LDL-C did not reach statistical significance when calculated for the whole group of patients. Looking only at the patients with increased LDL-C concentrations before treatment (n = 9), a mean reduction in LDL cholesterol by 19% from 6.17 + 0.56 to
262
5.03 f 0.62 mmol/l (P < 0.01) was found. Eight of the hypercholesterolaemic patients showed reduced LDL-C concentrations during treatment, while 1 patient (patient No. 1) showed a slight increase. The HDL lipid concentrations increased significantly during treatment. Thirteen patients showed increasing HDL-C concentrations, in 1 patient there was no significant change and in 1 patient (patient No. 9) there was a slight reduction of the HDL concentration corresponding to a slight increase of the VLDL-TG concentration. The ratio LDL-C/HDL-C decreased on the average by 24% (P < 0.001) in the whole group of patients. Effects of bezafibrate on the serum apolipoprotein concentrations While there was a significant decrease in the serum apo B concentration (Table 3), the apo A-I and apo A-II concentrations increased significantly during treatment with bezafibrate. The relative increase of apo A-II was more pronounced than that of apo A-I which was shown by a significantly decreased ratio between apo A-I and apo A-II in serum. Compared with before treatment, there was a highly significant decrease in the Apo B/APO A-I ratio. Effect of bezafibrate on the relationships between apo-lipoprotein and lipoprotel’n-lipid concentrations While there was a slight but significant decrease of the ratio serum apo B/LDL-C by 8% (P = O.Ol), the composition of HDL, as judged from the ratio between A-I/HDL-C and A-II/HDL-C, did not change significantly during treatment with bezafibrate. There was a significant positive relationship between the changes of A-I (r = 0.69, P< 0.01) and A-II (r = 0.60, P< 0.05) respectively, and the changes in HDL-C concentration during treatment. Effects of bezafibrate on lipoprotein removal There was a significant increase by 20% of the post-heparin plasma lipoprotein lipase activity during treatment with bezafibrate. An increased capacity to remove triglyceride-rich lipoproteins was also demonstrated by an increased fractional removal rate (K,) at the intravenous fat tolerance test by 30% (Table
TABLE 2 SERUM LIPOPROTEIN
LIPID CONCENTRATIONS
(x * SD) BEFORE (BT) AND AFTER
Lipoprotein composition (mmol/l) LDL
VLDL
BT AT A%
ta Statistical significance
TG
CHOL
TG
CHOL
2.49 * 1.01 1.43 * 1.13 43 4.31 P < 0.001
1.43 * 0.80 0.70 I 0.60 -51 4.13 P < 0,001
0.92 f 0.49 0.61 k 0.13 -27 2.34 P < 0.05
5.25 f 1.29 4.71 f 0.71 -10 1.87 h.s.
* Tested with paired t-test (two-tailed test). TG. CHOL = triglyceride and cholesterol concentrations (mmoI/I).
(AT)
263 TABLE 3 SERUM APOLIPOPROTEIN MENT WITH BEZAFIBRATE
CONCENTRATIONS (n = 15)
Apolipoprotein
ET AT A% t Statistical significance
(ii
SD) BEFORE (BT) AND AFTER
(AT) TREAT-
concentrations (A.U.)
APO I3
APO A-I
APO A-II
APO A-II APO A-II
APO Bl APO A-I
186 i 40 158 * 31 -15 3.16 P < 0.01
81* 9 93 f 13 +14 -4.24 P < 0.001
105 * 14 136 * 28 c30 -5.35 P < 0.001
0.18 * 0.09 0.69 f 0.09 -12 5.36 P < 0.001
2.29 * 0.46 1.72 z? 0.42 -25 4.64 P < 0.001
4). There was a strong inverse relationship between the VLDL-TG concentrations and the KZ values both before (r = -0.74, P < 0.01) and after (r = -0.65, P < 0.01) treatment with bezafibrate. The relationship between the VLDL-TG concentrations and the post-heparin lipoprotein lipase activities was somewhat weaker before treatment (r = -0.58, P < 0.05) and did not quite reach statistical significance after treatment (r = -0.47, n.s.). A positive relationship existed between the HDL-C concentrations and the Kz values both before (r = 0.57, P < 0.05) and after (r = 0.56, P < 0.05) treatment with bezafibrate. Effects of bezafibrate on blood glucose and serum insulin concentrations No significant changes were recorded in fasting blood glucose or fasting
serum insulin concentrations
after treatment
with bezafibrate (Table 4).
Effects of bezafibrate on the fatty acid composition of the plasma lipid esters
Significant changes were recorded in all plasma lipid esters during treatment with bezafibrate (Table 5). In the cholesterol esters there was a higly significant increase of the mono-unsaturated fatty acids, while the linoleic acid (18 : 2) content decreased. There was also a slight, but significant, reduction of the amount of palmitate (16 : 0).
TREATMENT
WITH BEZAFIBRATE
HDL
(n = 15)
Serum
TG
CHOL
TG
CHOL
0.25 zt0.07 0.30 f 0.06 +20 -2.49 P < 0.05
0.94 * 0.15 1.15 f 0.23 +22 -4.40
3.65 f 1.09 2.34 f 1.10 46 5.11 P < 0.001
7.64 f 1.64 6.63 f 1.05 -13 3.22 P < 0.001
P < 0.001
A% = mean change during bezaffbrate treatment expressed as Percentage change of mean whe
before treatment.
264 TABLE 4 POST-HEPARLN PLASMA LIPOPROTEIN LIPASE ACTIVITY (PH-LPLA). FRACTIONAL REMOVAL RATE (Kz) AT THE I.V. FAT TOLERANCE TEST (IVFTT), FASTING BLOOD GLUCOSE AND SERUM nxwrm CONCENTRATIONS BEFORE (BT) AND AFTER (AT) TREATMENT WITH BEZAFIBRATE (x * SD. n = 15) K2 IVFTT (%/min)
f-B-glucose
f-S-insulin
(mUP)
(mmoW)
(mu/l)
195 * 31 233 i 41 +20 -4.40 P < 0.001
3.0 * 1.2 3.9 f 1.4 +30 4.94 P < 0.001
4.2 * 1.8 4.1 * 1.7 -1 0.35 n.s.
8.5 f 4.1 8.5 f 5.6 *O 0.05
PH-LPLA
BT AT A% t Statistical significance
Il.S.
In the triglycerides there was a significant decrease of linoleate (18 : 2), and also of linoleate (18 : 3), while the arachidonate (20 : 4) content increased highly significantly. Both myristate (14 : 0) and stearate (18 : 0) decreased, while palmitate (16 : 0) and plamitoleic acid (16 : 1) increased. In the phospholipids there was a pattern which was very similar to that seen in the cholesterol esters with increasing relative content of the mono-unsaturated fatty acid8 and reduction of the content of linoleate (18 : 2). Adverse effects of bezafibrate treatment
All patients completed the trial taking the prescribed dose8 of 600 mg bezafibrate a day. However, 1 patient (patient No. 2) complained of vertigo, dizziness and malaise and another patient (patient No. 5) experienced gastrointestinal discomfort, which disappeared on stopping the medication. No other adverse reactions attributable to the trial drug were reported by the patients. Discussion The present study confirmed that bezafibrate is an efficient lipid-lowering drug in hypertriglyceridaemic patient8 in accordance with the findings in several
TABLE 5 FATTY
ACID
Fatty acid (shorthand)
COMPOSITION
LIPID ESTERS
1.00 11.90 4.20 1.20 18.55 57.73 0.62 4.82
A%
BT
11.43 * 0.70 5.86 f 1.92 1.29 * 0.40
+15 n.s. * 4 +40 *** +3 n.s.
22.11 52.96 0.59 4.73
+19 -8 -5 -2
2.34 26.16 5.92 4.16 39.12 19.99 1.40 0.87
AT f * * f f * * *
0.27 0.90 1.92 0.25 2.77 5.37 0.23 1.30
(W) BEFORE
(BT) AND AFTER
(AT)
Triglycerides
Cholesterol esters BT
14:o 16:0 16:l 18:0 18:l 18:2 18:3 20~4
IN PLASMA
1.15 * 1.00
f f * *
3.28 5.60 0.32 1.30
*** *** n.s. n.s.
AT f * * f f f * f
0.73 2.66 0.99 0.77 2.23 4.96 0.35 0.25
A%
1.83 f 0.50 28.11 f 2.39 6.48 3.72 39.94 17.37 1.07
+ f * f f
1.45 0.78 1.59 4.27 0.23
1.43 f 0.52
-21*** +7 ** +9 -11** +2 -13 -24
n.s. ** ***
+64 ***
A% = Mean change during bezafibrate treatment expressed as percentage change of mean value before *** = Significant change during treatment on the 5%. 1% and 0.1% level when tested
treatment. * , * *, with paired t-test.
*
265
previous studies [ 141. The main objective of the present study, however, was to investigate further the effect of bezafibrate on the serum lipoprotein composition, especially on the serum apolipoprotein concentrations. Significant average reductions of the VLDL lipid levels were associated with an inverse increase of the HDL-TG and HDL-C concentrations. However, there was no significant relationship between the individual changes in VLDL-TG and HDL-C concentrations, calculated either as absolute or relative changes. There was in this material a highly significant, inverse relationship (r = -0.81, P < 0.001) between the percentage decrease of the VLDL-TG and the pretreatment body weight index (weight in kg/height in cm - 100) of the patients (Fig. 1). This means that the relative reduction of the VLDL-TG was much more pronounced in the lean than in the obese patients. Separating the patients into a group with a pretreatment body weight index above 1.20 (n = 5) and a group with a body weight index less than 1.20 (n = lo), the average VLDL-TG reduction in the overweight group was -8%, and in the group with a normal body weight -62%. The difference in the VLDL-TG reduction between these 2 groups was statistically highly significant. The pretreatment VLDL-TG concentration was not different in the 2 groups, nor did the pretreatment activity of post-heparin lipoprotein lipase or the fractional removal at the intravenous fat tolerance test differ significantly. It thus seems as if bezafibrate would be a more efficient lipid-lowering drug in normal weight subjects than in obese patients. However, these results have to be interpreted with care as three of the overweight patients had been poor responders also to other lipid-lowering drugs, which might mean that the present results are due rather to the patient selection than to effects specifically caused by this drug. There was no decrease of the mean LDL-C concentration in the whole group of patients. As shown earlier by Olsson and coworkers [ 11, the effect of bezafibrate on LDL-C depended on the pre-treatment LDL-C concentration. Thus, the 2 patients with the lowest LDL-C concentrations before treatment both showed increased LDL-C concentrations during treatment by 19 and 64%, respectively. This was accompanied by a slight increase of the ratio between
TREATMENT
WITH BEZAFIBRATE
(z f SD; n = 15)
Phospholipids
0.43 34.17 1.35 16.18 12.64 26.43 0.38 8.59
AT
BT * f f f f f * *
0.14 1.65 0.24 1.98 1.06 3.07 0.08 1.81
0.39 f 0.16 34.95 * 2.21 1.59 f 0.36 15.84 * 2.03
A% -9 -2
n.s. n.s.
+18 *** -2 as.
14.93 f 1.82
+18 ***
23.50 f 4.10 0.41 * 0.14 8.50 * 1.85
-11 ** +a as. _l n.s. ~-
-
266 VLDL TG
A% +20 -I B.W. index
r = 0.81
Fig. 1. Relationships between body weight (B.W.) index and change of VLDL-T treatment with bezafibrate (n = 15).
concentration
during
LDL- and HDL-C in the first patient while in the second patient this ratio actually decreased in spite of the rise of the LDL-C, because of a concomitant pronounced increase of the HDL-C concentration. However, both these patients also had normal LDL-C concentrations after treatment. These 2 patients also showed a slightly increasing apo B concentration during treatment with bezafibrate in contrast to the majority of the patients who showed reductions of the serum apo B levels. Also, the decrease of the serum apo B concentration was directly related to the pretreatment concentrations of apo B (r = 0.65, P < 0.01). During treatment of hypertriglyceridaemic patients with bezafibrate, Weisweiler and Schwandt [22] found increasing HDL-C but unchanged apo A-I concentrations in serum. In this study the concentrations of serum apo A-I and A-II showed parallel changes with the HDL-C concentrations during treatment. Highly significant increases were seen with regard to both HDL-C, serum apo A-I and A-II concentrations. However, although the ratio between A-I and HDL-C and A-II and HDL-C did not change significantly, there was a tendency to a decrease of A-I and an increase of A-II in relation to HDL-C and the ratio between A-I and A-II decreased highly significantly during treatment as the relative increase of A-II was more pronounced than that of A-I. A preferential increase of A-II in relation to A-I has also been seen during lipid-lowering treatment with clofibrate [23] and gemfibrozil (B. Vessby, to be published), drugs which are both chemically related to bezafibrate. This is in contrast to what is seen during treatment with nicotinic acid where the concentration of serum apo A-I increases to a greater extent than apo A-II [24]. The significance of the changing ratio between A-I and A-II during bezafibrate treatment is at present unclear. However, as the apo A-I/ape A-II ratio is inversely related to the density of the HDL fractions [25] it may be that bezafibrate and related drugs cause an increase of HDL-3 (d = 1.121-1.210) rather than HDL-2 (d = 1.0631.121).
267
With regard to atherogenicity, the reduction of the ratio between LDL cholesterol and HDL-C, as well as the reduction of the ratio between apo B and apo A-I in serum, should be considered as probably favourable changes. Little is known regarding the mode of action of bezafibrate. An increase of the lipoprotein-lipase activity in post-heparin plasma, as well as an increased fractional removal rate at the intravenous fat tolerance test, indicated that treatment causes an improved capacity to remove triglyceride-rich lipoproteins. This might be the explanation, or one of the explanations, for the reductions of the VLDL concentrations during the treatment. Klose et al. [26] reported significantly increased activities of heparin releasable lipoprotein-lipase and hepatic triglyceride lipase in plasma when bezafibrate was given to healthy volunteers. Increased post-heparin lipoprotein-lipase activity [ 27,281 and improved intravenous fat tolerance [30] have also been demonstrated for clofibrate. Apparently, the main reason for the increased removal capacity during clofibrate treatment is an increase of the lipoprotein-lipase activity in skeletal muscle [ 301. Whether the same is true for bezafibrate remains to be shown. No significant changes were recorded in fasting blood glucose or fasting serum insulin concentrations after treatment with bezafibrate in this study. This seems to be somewhat in contrast to the results obtained during treatment with clofibrate as it has been shown [31] that clofibrate treatment coincides with decreasing serum insulin concentrations, the reduction being most pronounced in subjects with initially high fasting serum insulin concentrations. Rather, a significant increase of the fasting insulin concentration has been reported in hypertriglyceridaemic patients during treatment with bezafibrate [ 321. In the present study, although there was a significant pretreatment correlation between the serum insulin concentration and the body weight index (r = 0.64, P = O.Ol), there was no correlation between the changes in the serum insulin concentrations and in the VLDL-TG. Treatment with bezafibrate was associated with most significant changes in the fatty acid composition of the plasma lipid esters. In the cholesterol esters, as well as in the phospholipids, the major changes recorded were due to significant increases of the mono-unsaturated fatty acids, palmitoleate and oleate, concomitant with significant decreases of the content of linoleic acid. In the triglycerides there were also other significant changes, including reductions of some of the saturated fatty acids, reduction of linolenate and a highly significant increase, although numerically small, of arachidonate. Closely similar changes have been recorded during clofibrate-treatment [33,34]. The mechanism behind these changes, as well as the significance of the changes, are still obscure. Based on the present data it may be suggested that bezafibrate should never be instituted without a dietary regimen including an increased dietary content of polyunsaturated fats, as it has been shown that this regimen may counteract the tendency to reduction of the content of linoleic acid in the plasma lipid esters during treatment with clofibrate [ 341. All patients in the present study were already on a lipid-lowering diet and no changes were made in their dietary regimen during this treatment. The changes demonstrated in the fatty acid pattern during bezafibrate treatment may be a reason for taking those patients off the drug, who responded poorly with regard to decreasing
268
VLDL or LDL concentrations. Otherwise a net effect in these patients may be a potentially negative change in the fatty acid composition of their circulating serum lipoproteins with a reduction of the relative content of linoleate. On comparing the fatty acid composition of the cholesterol esters of the obese with that of the lean patients, there was no significant difference regarding the group mean values of the two groups for either the content of oleate or linoleate before treatment or for the changes of these fatty acids during treatment. This means that the obese patients, in spite of virtually unaffected VLDL lipid levels, showed qualitative changes of the composition of the lipoproteins indicating that they had taken the drug as prescribed. Bezafibrate was well tolerated by the patients, as shown previously in other studies. Two patients complained of subjective side-effects, possibly attributable to the trial drug. With regard to the laboratory analyses performed in the interest of safety there was a moderate, but highly significant, increase of the serum creatinine concentration during bezafibrate treatment as seen in previous studies [ 21. However, all patients showed post-treatment values well within the normal range for serum creatinine concentrations and the significance of these changes is as yet unclear. References 1 OIsson, A.G., Rossner, S.. WalIdius. G., Carlson, L.A. and Lang, P.D.. Effect of BM 15.076 on lipoprotein concentrations in different types of hyperhpoproteinaemia. Atherosclerosis, 27 (1977) 279. 2 OIsson, A.G. and Lang, P.D.. Dose.+esponse study of besafibrate on serum Iipoprotein concentrations in hyperlipoproteinaemia, Atherosclerosis, 31 (1978) 421. 3 Ohson, A.G. and Lang, P.E., One-year study of the effect of bezafibrate on serum lipoprotein concentrations in hyperhpoproteinaemia. Atherosclerosis, 31 (1978) 429. 4 Kaffarnik, H.. Schneider, J., Schubots, R.. MtihIfeBner, 0.. MtihlfeBner. G., Hausman. L. and Ziifel. P.. Long term results with bezafibrate, a new derivative of clofibrate. In: L.A. C&son et al. (Eds.). International Conference on Atherosclerosis, Raven Press, New York, 1978. p. 129. 5 Fredrickson, D.S.. Levy, R.I. and Lees. R.S.. Fat transport in lipoproteins -An integrated approach to mechanisms and disorders, New EngI. J. Med., 276 (1967) 34.94. 149.215.273. 6 Beaumont, J.L., Carison. L.A.. Cooper, G.R.. Fejfar. Z., Fredrickson, D.S. and Strasser. T., Classifications of hyperlipidaemias. BuB. WId Hlth. Org., 43 (1970) 891. 7 CarIson, L.A. and Ericsson, M., Quantitative and qualitative serum lipoprotein analysis. Part 1 (Studies in healthy men and women), Atherosclerosis, 21 (1975) 417. 8 Havel. Rd., Eder. H.A. and Bragdon, J.H.. The determination and chemical composition of ultracentrifugsBy separated lipoproteins in human serum, J. Clin. Invest., 34 (1955) 1345. 9 Burstein, M. and SamaiBe, J.. Sur un dosage rapide du cholesterol he aux o- et fl-Iipoprotbines du serum, Chn. Chim. Acta. 5 (1960) 609. 10 Rush, R.L., Leon, L. and TurreII. J., Automated simuRaneous cholesterol and triglyceride determination on the auto-AnaIyserR II Instrument. In: Advances in Automated Analysis, Vol. 1. Thurman, New York, 1971, p. 603. 11 Noble, R.P.. Electrophoretic separation of plasma lipoproteins in agarose gel. J. Lipid Res.. 9 (1969) 693. 12 LaureII, C.G., Electroimmuno assay. Scand. J. Clin. Lab. Invest., 29, Suppl. 124 (1972) 21. 13 Curry, M.D., Alapovic. P. and Suenram, C.A.. Determination of apo-lipoprotein A and its constitutive A-I and A-II polypeptides by separate electroimmunoaasays. Chn. Chem.. 22 (1976) 315. 14 Vessby, B., Boberg, J., Gustafsson, I.-B., KarIsti6m. B.. Lithe& H. and Gsthmd-Lindqvist. A.-M., Reduction of high density Iipoprotein cholesterol and apohpoprotein A-I concentrations by a Iipidlowering diet, Atherosclerosis, 35 (1980) 21. 15 Scanu, A., Toth. J.. EdeIstein. C.. Koga, S. and StiBer, E., Fractionation of human serum high density lipoprotein in urea solution. Evidence for polypeptide heterogenicity. Biochemistry, 8 (1969) 3309. 16 Lithe& H.. Boberg, J.. HeBsing. K.. LjungdaB, S.. Lundqvist. G.. Vessby. B. and Wide. L., Serum Iipoprotein and apolipoprotein concentrations and tissue BPoProtain-lipase activity in overt and subchnicaI hypothyroidism -The effect of substitution therapy. Europ. J. CBn. Invest. (1980) In press.
269 17 Hjelm. M. and de Verdier. C.H.. A methodological study of the enzymatic determination of glucose in blood. Stand. J. Clin. Lab. Invest., 15 (1963) 415. 18 Wide, L., Ax&. R. and Porath. J., Radlohnmunosorbent assay of proteins. Chemical couplings of antibodies to insoluble dextran. Immunochemistry, 4 (1967) 381. 19 Carlson. L.A. and Riissner, S., A methodological study of an intravenous fat tolerance test with Intralipid@emulsion. Stand. J. Clin. Lab. Invest., 29 (1972) 271. 20 Boberg. J., Separation of labelled plasma and tissue lipids by thin layer chromatography - A quantitative methodological study, Clin. Chlm. Acta, 14 (1966) 325. 21 Mason, M.F. and Wailer, G.E., Dimethoxypropane induced transesterification of fats and oils in the preparation of methyl esters for gas-chromatographlc analysis, Anal. Chem., 36 (1964) 359. 22 Weisweiler, P. and Schwandt, P., Einfluss von Bezafibrat auf Lipoproteinhpide und Apolipoproteine A und B bei Patienten mit Hyperlipoprotciniimie Typ IV und V. In: H. Greten, P.D. Lang and G. Schettler (Eds.) Lipoproteine und Herzlnfarkt. Verlag Gerhard Witzroch Baden-Baden. Cologne, New York, 1979, p. 151. 23 Miettinen. T.A., Huttunen. J.K., Kumlln, T., Naukkarinen, V., Mattila. S. and E@iholm, C.. High density lipoprotein cholesterol and apollpoproteins A-I and A-II during long-term treatment with clofibrate and probucol. In: 13th Annual Meeting, Europ. Sot. Clin. Inv. Med., 1979, Abstr. No. 141. 24 Blum, C.G., Levy, R.I.. Eisenberg. S., HsIl III. M.. Goebel, R.H. and Berman, M.. High density lipoprotein metabolism in man, J. Clin. Invest., 60 (1977) 795. 25 Cheung, M.C. and Albers. J.J.. The measurement of apolipoprotein A-I and A-II levels in men and women by immunoassay, J. Clin. Invest., 60 (1977) 43. 26 Klose. G., Behrendt, J., Vollmar. J. and Greten. H., Wirkung von Bezafibrat auf die Lipoproteinlipasenaktlvitat und die Lebertriglyceridhydrolase bei gesunden Versuchspersonen. In: H. Greten, P.D. Lang, G. Schettler (Eds.) Lipoproteine und Herzinfsrkt, Verlag Gerhard Witzstroch Baden-Baden, Cologne, New York, 1979, p. 185. 27 Boberg. J., Boberg. M.. Gross, R.. Grundy, S., Augustin. J. and Brown, V., The effect of treatment with clofibrate on hepatic triglyceride and lipoprotein lipase activities of post-heparin plasma in male patients with hyperlipoproteinemia. Atherosclerosis, 27 (1977) 499. 28 Nikkilii, E.A., Huttunen, J.K. and Ehnbolm. C.. Effect of clofibrate on postheparln plasma triglyceride lipsse activities in patients with hypertriglycerldemla. Metabolism, 26 (1977) 179. 29 Greten, H., Labile, V., Zipperle, W. and Augustin. J.. Comparison of assay methods for selective measurement of plasma llpase, Atherosclerosis, 26 (1977) 563. 30 Lithell, H., Boberg. J.. Hellsing. K.. Lundqvist. G. and Vessby. B., Increase of the lipoprotein-lipase activity in human skeletal muscle during clofibrate administration, Europ. J. Clin. Invest., 8 (1978) 67. 31 Lithell, H., Boberg, J.. Hedstrand, H., Hell&g. K.. Ljungdall, S. and Vessby, B.. Effects of clofibrate on glucose tolerance. serum insulin, serum lipoproteins and plasma fibrinogen. Europ. J. Clin. Pharmacol.. 12 (1977) 51. 32 Weisweiler. P., Neureuther. G.. Schwandt. P., Einfluss van Besafibrate auf Glucosetolersnz und Insulin Sekretion van Patienten mit Hyperllpoprotainiimie Typ IV und V. In: H. Greten, P.D. Lang, G. Schettler (Eds.), Llpoproteine und Herzinfarkt, Verlag Gerhard Witzstroch, Baden-Baden, Cologne, New York, 1979. p. 185. 33 Jurand, J. and Oliver, M.J., The effects of ethyl chlorophenoxyisobutyrate on serum cholesterol, triglyceride and phosphohpid fatty acids, J. Atheroscler. Res., 3 (1968) 457. 34 Vessby, B., LithelI, H.. Gustafsson, I.-B. and Boberg, J., Changes in the fatty acid composition of the plasma lipid esters during lipid lowering treatment with diet, clofibrate and niceritrol. Reduction of the proportion of hnoleate by clofibrate but not by nlceritrol, Atherosclerosis, 35 (1980) 51. 35 Vessby, B.. Lithell. H.. Gustafsson, I.-B. and Boberg. J., Pronounced lipoprotein lipid reduction obtained by combined lipid-lowering treatment in patients with atherosclerotic disease, Atherosclerosis, 33 (1978) 457.
Atherosclerosis, 37 (1980) 257-269 @ Elsevier/North-HollandScientific Publishers,Ltd.
EFFECTS OF BEZAFIBRATE ON THE SERUM LIPOPROTEIN LIPID AND APOLIPOPROTEIN COMPOSITION, LIPOPROTEIN TRIGLYCERIDE REMOVAL CAPACITY AND THE FATTY ACID COMPOSITION OF THE PLASMA LIPID ESTERS
BENGT VESSBY, HANS LITHELL, KRISTOFFER HELLSING, ANN-MARGRET ~~STLUND-LINDQVIST, INGA-BRITT GUSTAFSSON, JONASBoBERG and HELLMUTH LEDERMANN Departments
of Geriatrics and Clinical Chemistry,
University of Uppsala, Uppsala (Sweden)
(Received 8 February, 1980) (Revised, received 6 May, 1980) (Accepted 7 May, 1980)
Summary Bezafibrate has been shown to effectively reduce elevated levels of very low density lipoprotein (VLDL) in hypertriglyceridaemia. It has also been claimed to be particularly effective in increasing high density lipoprotein (HDL) concentrations. In the present study 15 hypertriglyceridaemic patients (9 type IIB, 1 type III and 5 type IV) were investigated before and after 2 months’ treatment with bezafibrate (600 mg daily) in an effort to ciarify further the effects of the drug on lipoprotein metabolism. The serum triglycerides decreased by 46% (P < 0.001) corresponding to a significant reduction of both the VLDL (-43%, P< 0.001) and the low density lipoprotein (LDL) (-27%, P< 0.05) triglycerides. The serum cholesterol decreased by 13% (P < O.OOl), mainly due to reduction of the VLDL cholesterol (-51%, P < 0.001). There was no significant reduction of the LDL cholesterol in the whole group (-lo%, n.s.). However, if only the patients with hypercholesterolaemia were considered, there was a significant reduction of LDL cholesterol by 19% (P < 0.01). HDL triglycerides and HDL cholesterol increased by 20% (P < 0.05) and 22% (P < 0.001) respectively. While the concentration of apolipoprotein (apo) B decreased by 15% (P < 0.01) there were significant increases of apo A-I (+14%, P< 0.001) and apo A-IX (+30%, P < 0.001) analogous to the increase of HDL lipid concentration. This study was financially supported by grants from the Swedish Medical Research Council (19P-5640 and 19X-6546-02). The bezafibrate tablets were generously provided by Boehringer Mannheim GmbH. Requests for reprints to: Dr. Ben& Vessby. Department of Geriatrics, Box 12042. S-750 12 UPPsala. Sweden.
258
However, the ratio between apo A-I and apo A-II decreased significantly during therapy (-12%, P < 0.001) indicating that the increase of HDL may have preferentially affected the HDL-3 fraction. There were highly significant reductions of both the ratio between LDL and HDL cholesterol (-24%, P < 0.001) and the ratio between apo B and apo A-I (-25%, P< O.OOl), changes which should be favourable with regard to atherogenicity. Significant increases of both the plasma post-heparin lipoprotein lipase activity (+20%, P < 0.001) and the fractional removal rate (K,) at the intravenous fat tolerance test (+30%, P < 0.001) indicate that one of the mochanisms behind the effects of the drug on lipoprotein metabolism may be an increased removal of circulating triglyceride-rich lipoproteins. Bezafibrate treatment caused significant changes in the plasma lipid fatty acid composition, mainly by increasing the mono-unsaturated fatty acids (palmitoleate and oleate) concomitant with a significant decrease in the content of linoleate. Very similar changes have been recorded during clofibrate treatment. The mechanisms behind these changes as well as their significance are at present obscure. Key words:
Apo-lipoproteins - Bezafibrate - Cholesterol proteins -Lipoprotein lipase activity
-Lipid-lowering
drug - Lipo-
Introduction In the search for new effective lipid-lowering drugs, bezafibrate (2-(4-[2-(4chlorobenzamido)ethyl] -phenoxy )-2-methylpropionic acid) has proved to be an efficient lipid-lowering drug in patients with hypertriglyceridaemia [ 141. It has been claimed that bezafibrate may be more effective than clofibrate in reducing increased concentrations of very low density lipoproteins (VLDL) [ 11. Also, bezafibrate has been suggested to be particularly effective in increasing the high density lipoprotein cholesterol (HDL-C) concentrations [ 21. However, there is presently sparse information regarding the effect of bezafibrate on serum apolipoprotein concentrations. The present study was performed to investigate further the effect of bezafibrate on the serum lipoprotein composition, especially with regard to the serum apo-lipoprotein concentrations. The effects of bezafibrate on the capacity to remove triglyceride-rich lipoproteins from the circulation, as judged from the post-heparin plasma lipoprotein lipase activity and the intravenous fat tolerance test, were also studied as well as the effects on the fatty acid composition of the plasma lipid esters. Materials and Methods Fifteen hypertriglyceridaemic patients were studied before and after 2 months of bezafibrate treatment. The clinical characteristics of the patients are shown in Table 1. All patients were on treatment with lipid-lowering diets for at least 2 months before the trial in order to obtain weight stabilisation and firm base line values for serum lipids. The hatients received 600 mg bezafibrate
259
TABLE
1
CLINICAL Patient
CHARACTERISTICS Initial
Sex
NO.
Age (Yr)
OF PATIENTS B.W.index a
PARTICIPATING
Lipoprotein
Clinical diagnosis
pattern
Before treatment
After treatment
1
BJ
M
43
1.25
IIB
IIB
2 3
LA RN
M M
61 44
1.11 1.21
IIB IIB
N IIB
4 5
SH MC
M F
63 60
0.86 1.07
III IIB
N(II1) N
6
SB
F
61
0.93
IIB
N
7 8 9 10 11
LS GL CGS NOH BK
M F M M M
61 53 46 49 49
1.16 1.42 1.31 1.28 1.16
IV IIB IV IV IIB
N IV IV N N
12 13 14 15
IS AS HS RT
F F M M
51 50 64 58
1.11 1.02 1.06 1.08
IV IIB IV IIB
N N N HA
a
BW_index
=
body weight height
(cm) -
IN THE TRIAL
Asymptomatic HLP. Poor response to gemfibrozil. Asymptomatic HLP. Asymptomatic HLP. Poor response to clofibrate Intermittent ckudication. Intermittent claudication. Angina pectoris? Stenosis of subclatian artery. Transitory global amnesia. Hypertension. Hypertension. Poor response to clofibrate. St. postmyocardial infarction. Hypertension and arthritis urica. Transitory ischaemic attacks and angina pectoris Hypertension. Hypertension; angina pectoris. Hypertension; angina pectoris. Asymptomatic HLP.
0%) 100
for 2 months. One tablet (200 mg) was given 3 times daily. The patients were seen at a clinical visit before the trial, after 1 month’s treatment and at the end of the 2 month treatment period. All patients declared that they had taken the full dose of bezafibrate throughout the trial. Subjects who had received other lipid-lowering drugs were only included in the trial after an interval of at least 2 months following discontinuation of the previous drug. All patients were on dietary treatment for at least 2 months before the trial and had demonstrated on this regimen firm and consistently elevated base lines for serum lipids. No patients with secondary hyperlipoproteinaemia due to hypothyroidism, liver disease, renal insufficiency or diabetes mellitus, or on medication known to affect the serum lipid levels, were included. The lipoprotein patterns (Table 1) were classified according to Fredrickson et al. [5] using the criteria suggested by Beaumont et al. 161. Cutoff points for hyperlipoproteinaemia (HLP) for men and women respectively, were for very low density lipoprotein triglycerides (VLDL-TG) 1.40 and 1.00 mmol/l, and for low density lipoprotein cholesterol (LDL-C) 5.20 and 5.70 mmol/l corresponding to the 85th percentile in healthy men and women in local control material [7]. Lipoprotein lipid concentrations were determined in serum after a 12-14 h overnight fast. EDTA was added to serum as a 5% solution to give a final concentration of 0.05%. VLDL were isolated as the top fraction after centrifu-
260
gation at d = 1.006 for 16 h at 15°C and 105,000 X g in a Beckman L2-65B preparative ultracentrifuge using a 40.3 rotor [ 81. LDL were precipitated from the bottom fraction at d = 1.006 by a heparin-manganese chloride solution. After low speed centrifugation, the HDL lipid levels were determined in the supernatant [9]. The concentrations of the lipids in LDL were obtained indirectly by subtracting the HDL lipid levels from lipid concentrations of the bottom fraction after centrifugation at d = 1.006. Cholesterol and triglyceride concentrations of serum and of the isolated lipoprotein classes were determined in isopropanol extracts by semi-automated methods in a Technicon AutoAnalyzer type II [lo]. The recoveries of triglyceride and cholesterol in the isolated density classes were within 100 f 10% in all samples. The top and bottom fractions at d = 1.006, as well as whole serum were analysed using agarose electrophoresis [ 111. The concentrations of serum apolipoprotein (apo) B, A-I and A-II were determined by electroimmunoassay according to Laurel1 [12] modified as suggested by Curry et al. [13]. Specific antibodies, anti-apo A-I, anti-apo A-II and anti-apo B were induced in rabbits against purified apo A-I, A-II and lipoprotein B. Apo A-I and lipoprotein B were prepared as described previously [14]. To prepare apo A-II, HDL was delipidated with chloroform-methanol (2 : 1) and apo HDL was fractionated by Sephadex G-200 chromatography followed by a further purification by chromatography on DEAE-cellulose in 6 M urea employing a linear gradient essentially as described previously [ 151. The electroimmunoassay was run overnight (16-20 h) at 10°C in a 0.05 M diemal buffer, pH 8.6, at 2.5 V/cm (apo B), 4.0 V/cm (apo A-I) and 3.0 V/cm (apo A-II) respectively using a 2% agarose gel containing 5% dextran. Samples and serial solutions of a reference serum were applied in duplicate and all samples from one patient were always run on the same plate to minimise intrapatient variation. The within-assay variations, S * were 2.6%, 2 8% and 3.6% for apo A-I, apo A-II and apo B respectively. The apolipoprotein concentrations in serum are expressed in qbitrsry units (A.U.) relative to the concentration in a reference serum obtained from a large pool of healthy blood donors (100 A.U.). The apolipoprotein concentrations in this reference serum, when standardised against a lipoprotein B preparation (d = 1.030-1.050) and a highly purified apolipoprotein A-I preparation, have been estimated to be 1.0 and 1.5 mg/ml, respectively. The lipoprotein lipase activity in post-heparin plasma was determined in an assay system using tritium trioleate labelled Intralipid@ as a substrate in a reaction medium containing glycine (2 mmol/l) and albumin (0.11 mmol/l). The triglyceride concentration was 3 mmol/l. The ionic strength was 0.08 and the pH value 8.5. The incubation was performed at 37°C for 40 min and the release of tritium oleic acid was used as a measure of post-heparin plasma lipoprotein lipase activity. In this system more than 90% of the total lipase is inhibited by protamine sulphate (1 mg/ml). This together with other inhibition criteria indicates that 90% or more of the total enzyme activity measured is lipoprotein-lipase activity. This is probably due to the fact that Intralipid is a poor substrate for measuring hepatic triglyceride activity under the conditions *&Cj=
d2
f- 2,
= standard deviation
of
the difference
(d) between
a number
of duplicate
measurements.
261
used. Theassay procedure has been described in detail elsewhere [ 161. Blood glucose concentrations were determined by a glucose oxidase method [ 171 and the serum insulin determinations by the Phadebas Insulin Test (Pharmacia AB, Uppsala) which is based upon a radioimmunosorbent technique [ 181. The serum samples were preincubated with the matrix-bound antibodies for 24 h before the isotope-labelled insulin was added. In this way, an increase of the sensitivity of the method was obtained. The coefficient of variation was 5% in the range 5-10 mu/l. Clinical laboratory analyses with regard to safety were performed according to the routines at the local clinical laboratory. The intravenous fat tolerance test (IVFTT) was performed according to Carlson and Rijssner [ 193. The fatty acid composition of plasma triglycerides, cholesterol esters, and phospholipids was determined in blood samples drawn into pre-cooled vacutainer tubes containing EDTA. After low-speed centrifugation of the blood at +4”C, the plasma lipids were extracted with chloroformlnethanol and separated by thin-layer chromatography according to a method previously described [20]. The fatty acids of the isolated lipid fractions (triglycerides, cholesterol esters and phospholipids) were transesterified to methylesters [ 211 and separated on a 4% EGSS-XH column in a Pye Unicam 104 gas-liquid chromatograph equipped with an automatic sample injector (Hewlett Packard Co.). Retention times of known fatty acid standards (obtained from Supelco Inc., Bellefonte, PA) were used for identifying the peaks of the chromatogram. The relative compositions of the different fatty acids (expressed in %) and their retention times were calculated by a Vidar 6300 digital integrator. Statistics The data presented are based on individual comparisons between results recorded before and after treatment with bezafibrate. The significance of the differences between mean values was estimated with paired t-test (two-tailed test). Results Body weight The average body weights before and after treatment did not differ cantly (mean body weight + SD, 79.7 4 13.4 kg before compared with 13.9 kg after treatment). The weight changes did in no case exceed during the 2 months of treatment and only in 3 patients did the body change exceed +2 kg.
signifi79.9 + 3.0 kg weight
Effects of bezafibrate on the serum lipoprotein lipid concentrations The serum triglyceride and cholesterol concentrations decreased highly significantly during treatment with bezafibrate (Table 2). This was mainly due to decreases of the VLDL-TG and VLDL-C concentrations. Also the LDL-TG concentration decreased significantly. The reduction of LDL-C did not reach statistical significance when calculated for the whole group of patients. Looking only at the patients with increased LDL-C concentrations before treatment (n = 9), a mean reduction in LDL cholesterol by 19% from 6.17 + 0.56 to
262
5.03 f 0.62 mmol/l (P < 0.01) was found. Eight of the hypercholesterolaemic patients showed reduced LDL-C concentrations during treatment, while 1 patient (patient No. 1) showed a slight increase. The HDL lipid concentrations increased significantly during treatment. Thirteen patients showed increasing HDL-C concentrations, in 1 patient there was no significant change and in 1 patient (patient No. 9) there was a slight reduction of the HDL concentration corresponding to a slight increase of the VLDL-TG concentration. The ratio LDL-C/HDL-C decreased on the average by 24% (P < 0.001) in the whole group of patients. Effects of bezafibrate on the serum apolipoprotein concentrations While there was a significant decrease in the serum apo B concentration (Table 3), the apo A-I and apo A-II concentrations increased significantly during treatment with bezafibrate. The relative increase of apo A-II was more pronounced than that of apo A-I which was shown by a significantly decreased ratio between apo A-I and apo A-II in serum. Compared with before treatment, there was a highly significant decrease in the Apo B/APO A-I ratio. Effect of bezafibrate on the relationships between apo-lipoprotein and lipoprotel’n-lipid concentrations While there was a slight but significant decrease of the ratio serum apo B/LDL-C by 8% (P = O.Ol), the composition of HDL, as judged from the ratio between A-I/HDL-C and A-II/HDL-C, did not change significantly during treatment with bezafibrate. There was a significant positive relationship between the changes of A-I (r = 0.69, P< 0.01) and A-II (r = 0.60, P< 0.05) respectively, and the changes in HDL-C concentration during treatment. Effects of bezafibrate on lipoprotein removal There was a significant increase by 20% of the post-heparin plasma lipoprotein lipase activity during treatment with bezafibrate. An increased capacity to remove triglyceride-rich lipoproteins was also demonstrated by an increased fractional removal rate (K,) at the intravenous fat tolerance test by 30% (Table
TABLE 2 SERUM LIPOPROTEIN
LIPID CONCENTRATIONS
(x * SD) BEFORE (BT) AND AFTER
Lipoprotein composition (mmol/l) LDL
VLDL
BT AT A%
ta Statistical significance
TG
CHOL
TG
CHOL
2.49 * 1.01 1.43 * 1.13 43 4.31 P < 0.001
1.43 * 0.80 0.70 I 0.60 -51 4.13 P < 0,001
0.92 f 0.49 0.61 k 0.13 -27 2.34 P < 0.05
5.25 f 1.29 4.71 f 0.71 -10 1.87 h.s.
* Tested with paired t-test (two-tailed test). TG. CHOL = triglyceride and cholesterol concentrations (mmoI/I).
(AT)
263 TABLE 3 SERUM APOLIPOPROTEIN MENT WITH BEZAFIBRATE
CONCENTRATIONS (n = 15)
Apolipoprotein
ET AT A% t Statistical significance
(ii
SD) BEFORE (BT) AND AFTER
(AT) TREAT-
concentrations (A.U.)
APO I3
APO A-I
APO A-II
APO A-II APO A-II
APO Bl APO A-I
186 i 40 158 * 31 -15 3.16 P < 0.01
81* 9 93 f 13 +14 -4.24 P < 0.001
105 * 14 136 * 28 c30 -5.35 P < 0.001
0.18 * 0.09 0.69 f 0.09 -12 5.36 P < 0.001
2.29 * 0.46 1.72 z? 0.42 -25 4.64 P < 0.001
4). There was a strong inverse relationship between the VLDL-TG concentrations and the KZ values both before (r = -0.74, P < 0.01) and after (r = -0.65, P < 0.01) treatment with bezafibrate. The relationship between the VLDL-TG concentrations and the post-heparin lipoprotein lipase activities was somewhat weaker before treatment (r = -0.58, P < 0.05) and did not quite reach statistical significance after treatment (r = -0.47, n.s.). A positive relationship existed between the HDL-C concentrations and the Kz values both before (r = 0.57, P < 0.05) and after (r = 0.56, P < 0.05) treatment with bezafibrate. Effects of bezafibrate on blood glucose and serum insulin concentrations No significant changes were recorded in fasting blood glucose or fasting
serum insulin concentrations
after treatment
with bezafibrate (Table 4).
Effects of bezafibrate on the fatty acid composition of the plasma lipid esters
Significant changes were recorded in all plasma lipid esters during treatment with bezafibrate (Table 5). In the cholesterol esters there was a higly significant increase of the mono-unsaturated fatty acids, while the linoleic acid (18 : 2) content decreased. There was also a slight, but significant, reduction of the amount of palmitate (16 : 0).
TREATMENT
WITH BEZAFIBRATE
HDL
(n = 15)
Serum
TG
CHOL
TG
CHOL
0.25 zt0.07 0.30 f 0.06 +20 -2.49 P < 0.05
0.94 * 0.15 1.15 f 0.23 +22 -4.40
3.65 f 1.09 2.34 f 1.10 46 5.11 P < 0.001
7.64 f 1.64 6.63 f 1.05 -13 3.22 P < 0.001
P < 0.001
A% = mean change during bezaffbrate treatment expressed as Percentage change of mean whe
before treatment.
264 TABLE 4 POST-HEPARLN PLASMA LIPOPROTEIN LIPASE ACTIVITY (PH-LPLA). FRACTIONAL REMOVAL RATE (Kz) AT THE I.V. FAT TOLERANCE TEST (IVFTT), FASTING BLOOD GLUCOSE AND SERUM nxwrm CONCENTRATIONS BEFORE (BT) AND AFTER (AT) TREATMENT WITH BEZAFIBRATE (x * SD. n = 15) K2 IVFTT (%/min)
f-B-glucose
f-S-insulin
(mUP)
(mmoW)
(mu/l)
195 * 31 233 i 41 +20 -4.40 P < 0.001
3.0 * 1.2 3.9 f 1.4 +30 4.94 P < 0.001
4.2 * 1.8 4.1 * 1.7 -1 0.35 n.s.
8.5 f 4.1 8.5 f 5.6 *O 0.05
PH-LPLA
BT AT A% t Statistical significance
Il.S.
In the triglycerides there was a significant decrease of linoleate (18 : 2), and also of linoleate (18 : 3), while the arachidonate (20 : 4) content increased highly significantly. Both myristate (14 : 0) and stearate (18 : 0) decreased, while palmitate (16 : 0) and plamitoleic acid (16 : 1) increased. In the phospholipids there was a pattern which was very similar to that seen in the cholesterol esters with increasing relative content of the mono-unsaturated fatty acid8 and reduction of the content of linoleate (18 : 2). Adverse effects of bezafibrate treatment
All patients completed the trial taking the prescribed dose8 of 600 mg bezafibrate a day. However, 1 patient (patient No. 2) complained of vertigo, dizziness and malaise and another patient (patient No. 5) experienced gastrointestinal discomfort, which disappeared on stopping the medication. No other adverse reactions attributable to the trial drug were reported by the patients. Discussion The present study confirmed that bezafibrate is an efficient lipid-lowering drug in hypertriglyceridaemic patient8 in accordance with the findings in several
TABLE 5 FATTY
ACID
Fatty acid (shorthand)
COMPOSITION
LIPID ESTERS
1.00 11.90 4.20 1.20 18.55 57.73 0.62 4.82
A%
BT
11.43 * 0.70 5.86 f 1.92 1.29 * 0.40
+15 n.s. * 4 +40 *** +3 n.s.
22.11 52.96 0.59 4.73
+19 -8 -5 -2
2.34 26.16 5.92 4.16 39.12 19.99 1.40 0.87
AT f * * f f * * *
0.27 0.90 1.92 0.25 2.77 5.37 0.23 1.30
(W) BEFORE
(BT) AND AFTER
(AT)
Triglycerides
Cholesterol esters BT
14:o 16:0 16:l 18:0 18:l 18:2 18:3 20~4
IN PLASMA
1.15 * 1.00
f f * *
3.28 5.60 0.32 1.30
*** *** n.s. n.s.
AT f * * f f f * f
0.73 2.66 0.99 0.77 2.23 4.96 0.35 0.25
A%
1.83 f 0.50 28.11 f 2.39 6.48 3.72 39.94 17.37 1.07
+ f * f f
1.45 0.78 1.59 4.27 0.23
1.43 f 0.52
-21*** +7 ** +9 -11** +2 -13 -24
n.s. ** ***
+64 ***
A% = Mean change during bezafibrate treatment expressed as percentage change of mean value before *** = Significant change during treatment on the 5%. 1% and 0.1% level when tested
treatment. * , * *, with paired t-test.
*
265
previous studies [ 141. The main objective of the present study, however, was to investigate further the effect of bezafibrate on the serum lipoprotein composition, especially on the serum apolipoprotein concentrations. Significant average reductions of the VLDL lipid levels were associated with an inverse increase of the HDL-TG and HDL-C concentrations. However, there was no significant relationship between the individual changes in VLDL-TG and HDL-C concentrations, calculated either as absolute or relative changes. There was in this material a highly significant, inverse relationship (r = -0.81, P < 0.001) between the percentage decrease of the VLDL-TG and the pretreatment body weight index (weight in kg/height in cm - 100) of the patients (Fig. 1). This means that the relative reduction of the VLDL-TG was much more pronounced in the lean than in the obese patients. Separating the patients into a group with a pretreatment body weight index above 1.20 (n = 5) and a group with a body weight index less than 1.20 (n = lo), the average VLDL-TG reduction in the overweight group was -8%, and in the group with a normal body weight -62%. The difference in the VLDL-TG reduction between these 2 groups was statistically highly significant. The pretreatment VLDL-TG concentration was not different in the 2 groups, nor did the pretreatment activity of post-heparin lipoprotein lipase or the fractional removal at the intravenous fat tolerance test differ significantly. It thus seems as if bezafibrate would be a more efficient lipid-lowering drug in normal weight subjects than in obese patients. However, these results have to be interpreted with care as three of the overweight patients had been poor responders also to other lipid-lowering drugs, which might mean that the present results are due rather to the patient selection than to effects specifically caused by this drug. There was no decrease of the mean LDL-C concentration in the whole group of patients. As shown earlier by Olsson and coworkers [ 11, the effect of bezafibrate on LDL-C depended on the pre-treatment LDL-C concentration. Thus, the 2 patients with the lowest LDL-C concentrations before treatment both showed increased LDL-C concentrations during treatment by 19 and 64%, respectively. This was accompanied by a slight increase of the ratio between
TREATMENT
WITH BEZAFIBRATE
(z f SD; n = 15)
Phospholipids
0.43 34.17 1.35 16.18 12.64 26.43 0.38 8.59
AT
BT * f f f f f * *
0.14 1.65 0.24 1.98 1.06 3.07 0.08 1.81
0.39 f 0.16 34.95 * 2.21 1.59 f 0.36 15.84 * 2.03
A% -9 -2
n.s. n.s.
+18 *** -2 as.
14.93 f 1.82
+18 ***
23.50 f 4.10 0.41 * 0.14 8.50 * 1.85
-11 ** +a as. _l n.s. ~-
-
266 VLDL TG
A% +20 -I B.W. index
r = 0.81
Fig. 1. Relationships between body weight (B.W.) index and change of VLDL-T treatment with bezafibrate (n = 15).
concentration
during
LDL- and HDL-C in the first patient while in the second patient this ratio actually decreased in spite of the rise of the LDL-C, because of a concomitant pronounced increase of the HDL-C concentration. However, both these patients also had normal LDL-C concentrations after treatment. These 2 patients also showed a slightly increasing apo B concentration during treatment with bezafibrate in contrast to the majority of the patients who showed reductions of the serum apo B levels. Also, the decrease of the serum apo B concentration was directly related to the pretreatment concentrations of apo B (r = 0.65, P < 0.01). During treatment of hypertriglyceridaemic patients with bezafibrate, Weisweiler and Schwandt [22] found increasing HDL-C but unchanged apo A-I concentrations in serum. In this study the concentrations of serum apo A-I and A-II showed parallel changes with the HDL-C concentrations during treatment. Highly significant increases were seen with regard to both HDL-C, serum apo A-I and A-II concentrations. However, although the ratio between A-I and HDL-C and A-II and HDL-C did not change significantly, there was a tendency to a decrease of A-I and an increase of A-II in relation to HDL-C and the ratio between A-I and A-II decreased highly significantly during treatment as the relative increase of A-II was more pronounced than that of A-I. A preferential increase of A-II in relation to A-I has also been seen during lipid-lowering treatment with clofibrate [23] and gemfibrozil (B. Vessby, to be published), drugs which are both chemically related to bezafibrate. This is in contrast to what is seen during treatment with nicotinic acid where the concentration of serum apo A-I increases to a greater extent than apo A-II [24]. The significance of the changing ratio between A-I and A-II during bezafibrate treatment is at present unclear. However, as the apo A-I/ape A-II ratio is inversely related to the density of the HDL fractions [25] it may be that bezafibrate and related drugs cause an increase of HDL-3 (d = 1.121-1.210) rather than HDL-2 (d = 1.0631.121).
267
With regard to atherogenicity, the reduction of the ratio between LDL cholesterol and HDL-C, as well as the reduction of the ratio between apo B and apo A-I in serum, should be considered as probably favourable changes. Little is known regarding the mode of action of bezafibrate. An increase of the lipoprotein-lipase activity in post-heparin plasma, as well as an increased fractional removal rate at the intravenous fat tolerance test, indicated that treatment causes an improved capacity to remove triglyceride-rich lipoproteins. This might be the explanation, or one of the explanations, for the reductions of the VLDL concentrations during the treatment. Klose et al. [26] reported significantly increased activities of heparin releasable lipoprotein-lipase and hepatic triglyceride lipase in plasma when bezafibrate was given to healthy volunteers. Increased post-heparin lipoprotein-lipase activity [ 27,281 and improved intravenous fat tolerance [30] have also been demonstrated for clofibrate. Apparently, the main reason for the increased removal capacity during clofibrate treatment is an increase of the lipoprotein-lipase activity in skeletal muscle [ 301. Whether the same is true for bezafibrate remains to be shown. No significant changes were recorded in fasting blood glucose or fasting serum insulin concentrations after treatment with bezafibrate in this study. This seems to be somewhat in contrast to the results obtained during treatment with clofibrate as it has been shown [31] that clofibrate treatment coincides with decreasing serum insulin concentrations, the reduction being most pronounced in subjects with initially high fasting serum insulin concentrations. Rather, a significant increase of the fasting insulin concentration has been reported in hypertriglyceridaemic patients during treatment with bezafibrate [ 321. In the present study, although there was a significant pretreatment correlation between the serum insulin concentration and the body weight index (r = 0.64, P = O.Ol), there was no correlation between the changes in the serum insulin concentrations and in the VLDL-TG. Treatment with bezafibrate was associated with most significant changes in the fatty acid composition of the plasma lipid esters. In the cholesterol esters, as well as in the phospholipids, the major changes recorded were due to significant increases of the mono-unsaturated fatty acids, palmitoleate and oleate, concomitant with significant decreases of the content of linoleic acid. In the triglycerides there were also other significant changes, including reductions of some of the saturated fatty acids, reduction of linolenate and a highly significant increase, although numerically small, of arachidonate. Closely similar changes have been recorded during clofibrate-treatment [33,34]. The mechanism behind these changes, as well as the significance of the changes, are still obscure. Based on the present data it may be suggested that bezafibrate should never be instituted without a dietary regimen including an increased dietary content of polyunsaturated fats, as it has been shown that this regimen may counteract the tendency to reduction of the content of linoleic acid in the plasma lipid esters during treatment with clofibrate [ 341. All patients in the present study were already on a lipid-lowering diet and no changes were made in their dietary regimen during this treatment. The changes demonstrated in the fatty acid pattern during bezafibrate treatment may be a reason for taking those patients off the drug, who responded poorly with regard to decreasing
268
VLDL or LDL concentrations. Otherwise a net effect in these patients may be a potentially negative change in the fatty acid composition of their circulating serum lipoproteins with a reduction of the relative content of linoleate. On comparing the fatty acid composition of the cholesterol esters of the obese with that of the lean patients, there was no significant difference regarding the group mean values of the two groups for either the content of oleate or linoleate before treatment or for the changes of these fatty acids during treatment. This means that the obese patients, in spite of virtually unaffected VLDL lipid levels, showed qualitative changes of the composition of the lipoproteins indicating that they had taken the drug as prescribed. Bezafibrate was well tolerated by the patients, as shown previously in other studies. Two patients complained of subjective side-effects, possibly attributable to the trial drug. With regard to the laboratory analyses performed in the interest of safety there was a moderate, but highly significant, increase of the serum creatinine concentration during bezafibrate treatment as seen in previous studies [ 21. However, all patients showed post-treatment values well within the normal range for serum creatinine concentrations and the significance of these changes is as yet unclear. References 1 OIsson, A.G., Rossner, S.. WalIdius. G., Carlson, L.A. and Lang, P.D.. Effect of BM 15.076 on lipoprotein concentrations in different types of hyperhpoproteinaemia. Atherosclerosis, 27 (1977) 279. 2 OIsson, A.G. and Lang, P.D.. Dose.+esponse study of besafibrate on serum Iipoprotein concentrations in hyperlipoproteinaemia, Atherosclerosis, 31 (1978) 421. 3 Ohson, A.G. and Lang, P.E., One-year study of the effect of bezafibrate on serum lipoprotein concentrations in hyperhpoproteinaemia. Atherosclerosis, 31 (1978) 429. 4 Kaffarnik, H.. Schneider, J., Schubots, R.. MtihIfeBner, 0.. MtihlfeBner. G., Hausman. L. and Ziifel. P.. Long term results with bezafibrate, a new derivative of clofibrate. In: L.A. C&son et al. (Eds.). International Conference on Atherosclerosis, Raven Press, New York, 1978. p. 129. 5 Fredrickson, D.S.. Levy, R.I. and Lees. R.S.. Fat transport in lipoproteins -An integrated approach to mechanisms and disorders, New EngI. J. Med., 276 (1967) 34.94. 149.215.273. 6 Beaumont, J.L., Carison. L.A.. Cooper, G.R.. Fejfar. Z., Fredrickson, D.S. and Strasser. T., Classifications of hyperlipidaemias. BuB. WId Hlth. Org., 43 (1970) 891. 7 CarIson, L.A. and Ericsson, M., Quantitative and qualitative serum lipoprotein analysis. Part 1 (Studies in healthy men and women), Atherosclerosis, 21 (1975) 417. 8 Havel. Rd., Eder. H.A. and Bragdon, J.H.. The determination and chemical composition of ultracentrifugsBy separated lipoproteins in human serum, J. Clin. Invest., 34 (1955) 1345. 9 Burstein, M. and SamaiBe, J.. Sur un dosage rapide du cholesterol he aux o- et fl-Iipoprotbines du serum, Chn. Chim. Acta. 5 (1960) 609. 10 Rush, R.L., Leon, L. and TurreII. J., Automated simuRaneous cholesterol and triglyceride determination on the auto-AnaIyserR II Instrument. In: Advances in Automated Analysis, Vol. 1. Thurman, New York, 1971, p. 603. 11 Noble, R.P.. Electrophoretic separation of plasma lipoproteins in agarose gel. J. Lipid Res.. 9 (1969) 693. 12 LaureII, C.G., Electroimmuno assay. Scand. J. Clin. Lab. Invest., 29, Suppl. 124 (1972) 21. 13 Curry, M.D., Alapovic. P. and Suenram, C.A.. Determination of apo-lipoprotein A and its constitutive A-I and A-II polypeptides by separate electroimmunoaasays. Chn. Chem.. 22 (1976) 315. 14 Vessby, B., Boberg, J., Gustafsson, I.-B., KarIsti6m. B.. Lithe& H. and Gsthmd-Lindqvist. A.-M., Reduction of high density Iipoprotein cholesterol and apohpoprotein A-I concentrations by a Iipidlowering diet, Atherosclerosis, 35 (1980) 21. 15 Scanu, A., Toth. J.. EdeIstein. C.. Koga, S. and StiBer, E., Fractionation of human serum high density lipoprotein in urea solution. Evidence for polypeptide heterogenicity. Biochemistry, 8 (1969) 3309. 16 Lithe& H.. Boberg, J.. HeBsing. K.. LjungdaB, S.. Lundqvist. G.. Vessby. B. and Wide. L., Serum Iipoprotein and apolipoprotein concentrations and tissue BPoProtain-lipase activity in overt and subchnicaI hypothyroidism -The effect of substitution therapy. Europ. J. CBn. Invest. (1980) In press.
269 17 Hjelm. M. and de Verdier. C.H.. A methodological study of the enzymatic determination of glucose in blood. Stand. J. Clin. Lab. Invest., 15 (1963) 415. 18 Wide, L., Ax&. R. and Porath. J., Radlohnmunosorbent assay of proteins. Chemical couplings of antibodies to insoluble dextran. Immunochemistry, 4 (1967) 381. 19 Carlson. L.A. and Riissner, S., A methodological study of an intravenous fat tolerance test with Intralipid@emulsion. Stand. J. Clin. Lab. Invest., 29 (1972) 271. 20 Boberg. J., Separation of labelled plasma and tissue lipids by thin layer chromatography - A quantitative methodological study, Clin. Chlm. Acta, 14 (1966) 325. 21 Mason, M.F. and Wailer, G.E., Dimethoxypropane induced transesterification of fats and oils in the preparation of methyl esters for gas-chromatographlc analysis, Anal. Chem., 36 (1964) 359. 22 Weisweiler, P. and Schwandt, P., Einfluss von Bezafibrat auf Lipoproteinhpide und Apolipoproteine A und B bei Patienten mit Hyperlipoprotciniimie Typ IV und V. In: H. Greten, P.D. Lang and G. Schettler (Eds.) Lipoproteine und Herzlnfarkt. Verlag Gerhard Witzroch Baden-Baden. Cologne, New York, 1979, p. 151. 23 Miettinen. T.A., Huttunen. J.K., Kumlln, T., Naukkarinen, V., Mattila. S. and E@iholm, C.. High density lipoprotein cholesterol and apollpoproteins A-I and A-II during long-term treatment with clofibrate and probucol. In: 13th Annual Meeting, Europ. Sot. Clin. Inv. Med., 1979, Abstr. No. 141. 24 Blum, C.G., Levy, R.I.. Eisenberg. S., HsIl III. M.. Goebel, R.H. and Berman, M.. High density lipoprotein metabolism in man, J. Clin. Invest., 60 (1977) 795. 25 Cheung, M.C. and Albers. J.J.. The measurement of apolipoprotein A-I and A-II levels in men and women by immunoassay, J. Clin. Invest., 60 (1977) 43. 26 Klose. G., Behrendt, J., Vollmar. J. and Greten. H., Wirkung von Bezafibrat auf die Lipoproteinlipasenaktlvitat und die Lebertriglyceridhydrolase bei gesunden Versuchspersonen. In: H. Greten, P.D. Lang, G. Schettler (Eds.) Lipoproteine und Herzinfsrkt, Verlag Gerhard Witzstroch Baden-Baden, Cologne, New York, 1979, p. 185. 27 Boberg. J., Boberg. M.. Gross, R.. Grundy, S., Augustin. J. and Brown, V., The effect of treatment with clofibrate on hepatic triglyceride and lipoprotein lipase activities of post-heparin plasma in male patients with hyperlipoproteinemia. Atherosclerosis, 27 (1977) 499. 28 Nikkilii, E.A., Huttunen, J.K. and Ehnbolm. C.. Effect of clofibrate on postheparln plasma triglyceride lipsse activities in patients with hypertriglycerldemla. Metabolism, 26 (1977) 179. 29 Greten, H., Labile, V., Zipperle, W. and Augustin. J.. Comparison of assay methods for selective measurement of plasma llpase, Atherosclerosis, 26 (1977) 563. 30 Lithell, H., Boberg. J.. Hellsing. K.. Lundqvist. G. and Vessby. B., Increase of the lipoprotein-lipase activity in human skeletal muscle during clofibrate administration, Europ. J. Clin. Invest., 8 (1978) 67. 31 Lithell, H., Boberg, J.. Hedstrand, H., Hell&g. K.. Ljungdall, S. and Vessby, B.. Effects of clofibrate on glucose tolerance. serum insulin, serum lipoproteins and plasma fibrinogen. Europ. J. Clin. Pharmacol.. 12 (1977) 51. 32 Weisweiler. P., Neureuther. G.. Schwandt. P., Einfluss van Besafibrate auf Glucosetolersnz und Insulin Sekretion van Patienten mit Hyperllpoprotainiimie Typ IV und V. In: H. Greten, P.D. Lang, G. Schettler (Eds.), Llpoproteine und Herzinfarkt, Verlag Gerhard Witzstroch, Baden-Baden, Cologne, New York, 1979. p. 185. 33 Jurand, J. and Oliver, M.J., The effects of ethyl chlorophenoxyisobutyrate on serum cholesterol, triglyceride and phosphohpid fatty acids, J. Atheroscler. Res., 3 (1968) 457. 34 Vessby, B., LithelI, H.. Gustafsson, I.-B. and Boberg, J., Changes in the fatty acid composition of the plasma lipid esters during lipid lowering treatment with diet, clofibrate and niceritrol. Reduction of the proportion of hnoleate by clofibrate but not by nlceritrol, Atherosclerosis, 35 (1980) 51. 35 Vessby, B.. Lithell. H.. Gustafsson, I.-B. and Boberg. J., Pronounced lipoprotein lipid reduction obtained by combined lipid-lowering treatment in patients with atherosclerotic disease, Atherosclerosis, 33 (1978) 457.