The effect of l -carnitine on the apolipoprotein pattern of rats fed a cholesterol-rich diet

Comp. Biochem. Physiol. Vol. 89B, No. 1, pp. 69-73, 1988 Printed in Great Britain

0305-0491/88 $3.00 + 0.00 © 1987 Pergamon Journals Ltd

THE EFFECT OF L-CARNITINE ON THE APOLIPOPROTEIN PATTERN OF RATS FED A CHOLESTEROL-RICH DIET P. MONDOLA,A. BELFIORE,F. SANTANGELOand M. SANTILLO Institute of Physiology, Medical Faculty, Naples University, 80131 Naples, Italy (Tel: 081-25-3022) (Received 2 March 1987)

Abstract--1. L-Carnitine was administered to hypercholesterolemic rats for eight days, after which the blood was collected by intracardiac puncture and lipoproteins were collected by preparative ultracentrifuge. 2. The chemical composition of lipoproteins of normal rats and hypercholesterolemic rats before and after L-carnitine treatment was determined. 3. The total serum B and E apoproteins were assayed by rocket electrophoresis, while apoproteins in VLDL, IDL, LDL and HDL were determined by densitometric scanning of SDS and TMU polyacrylamide disc gel electrophoresis. 4. The results obtained showed that the L-carnitine treatment tends to restore to normal values both the chemical composition of lipoproteins and the apoproteins pattern of rats fed on a diet enriched with cholesterol.

diet (Nath et al., 1959) enriched with 1.5% of cholesterol for a period of two months; all the rats had free access to water and food. After 60 days of diet the animals were fasted for 16 hr and 4 ml of blood were collected by intracardiac puncture under diethylether anesthesia. The rats of the second group were given an intraperitoneal injection of 0.5 ml of saline solution of NaCI 0.15 M for eight days. The third group received 50 mg of L-carnitine intraperitoneally also for eight days. Throughout the treatment period all the rats were fed on the Nath diet. The serum of each rat was separated after light centrifugation and stored in the presence of 0.1% of EDTA disodium salt and 0.1% of sodium azide to prevent the bacterial degradation of apoproteins.

INTRODUCTION L-Carnitine plays an important role in the transport o f activated long chain fatty acids into the mitochondrial matrix (Fritz and Marquis, 1965). It is also known that serum triglycerides are significantly reduced after carnitine administration in patients with type IV hyperlipoproteinemia (Maebashi et ai., 1978). Oral carnitine has been reported to have a lipid lowering effect with a concomitant increase in high density lipoprotein cholesterol ( H D L cholesterol) (Rossi and Siliprandi, 1982). Similar results have been obtained (Vadra et al., 1983) in hemodialyzed patients with low H D L cholesterol values and hypertriglyceridemia. It has been demonstrated that apolipoproteins regulate lipoprotein metabolism and are involved in the transport and redistribution of lipids a m o n g many tissues (Jonas et al., 1984; Steinmetz and Utermann, 1983). The delivery of lipids to specific ceils involves the recognition of specific apolipoproteins (B-100 and E) by cell surface lipoprotein receptors of liver and extrahepatic tissues (Mahley and Innerarity, 1983; Brown and Goldstein, 1983). Given the lack o f information on the role of carnitine in apolipoproteins, we have studied the effects o f L-carnitine on lipids and on the apolipoprotein pattern o f rats fed a cholesterol-rich diet.

Analytical methods Cholesterol, triglycerides and phospholipid were determined by the method of Siedel et al. (1981), Wahlefeld and Bergmeyer (1974) and Takayama et al. (1977) respectively. The protein content in the lipoprotein fractions was determined by the method of Lowry et al. (1951). The serum of rats of each group was pooled and lipoproteins were separated according to the method described by Havel et al. (1955) after ultracentrifugation in a Beckman L3-50 ultracentrifuge using a 40.3 rotor. Very Low Density Lipoproteins (VLDL), density
MATERIALS AND M E T H O D S Animals Thirty male Wistar strain rats, weighing 300-330 g, were used in the experiment. The rats, maintained at a constant temperature (25°C) throughout the experiment, were divided in three groups of 10 animals each. The first group was used to determine the normal values of serum cholesterol, trigiycerides, phospholipids, total apo B and apo E. The second and third groups were fed the Nath

Tetramethylurea polyacrylamide gel electrophoresis ( T M U PAGE) To delipidate VLDL, IDL and HDL, the denaturating solvent, TMU, was used; in fact it will delipidate and

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P. MONDOLA et al.

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solubilize all apolipoproteins except B apoproteins that are precipitated. Electrophoresis was according to the method described by K a n e (1973). The gels were loaded with 15/tg o f VLDL, 15 # g of I D L and 2 0 # g o f HDL.

Sodium dodecylsulphate (SDS) polyacrylamide gel electrophoresis

Trygticerides [ ] 200

I00

SDS P A G E was performed according to the method o f K a n e (1980) in 3.4% o f acrylamide; the disc _gels were loaded with 20 # g o f V L D L , II)L and L D L delipidated with ethanol-diethyl ether in a ratio 3: i (v/v). The densitometric scanning o f both SDS a n d T M U polyacrylamide gel electrophoresis was in an L K B 2202 ultra-scan laser densitomer.

Electroimmunodiffusion B and E apoproteins in total serum were determined by rocket electrophoresis as previously described (Mondola and Reichl, 1982) with 2% of anti apo B and anti apo E rat antibodies. The anti rat apo B antibodies were prepared by four subcutaneous injections o f 160 # g of rat L D L (density range 1.019-1.050g/ml) and complete F r e u n d ' s adjuvant (Dico Laboratories Michigan, USA) at intervals o f 10 days. The antibodies against rat apo E were kindly provided by Prof. P. S. Roheim, Louisiana State University, USA. The intra-assay coet~cient of variation between samples assayed three times was 3.4%.

Chol.estero[ r-]

3 0 0 -

--

--

A

B

C

Fig. 1. Serum levels of total cholesterol and tryglicerides of normal rats (A), before (B) and after L-carnitine treatment (C); P < 0.005.

Serum cholesterol and triglycerides were relatively unchanged in the rats of the group treated intraperitoneally with NaCI 0.15 M (group 2). In fact, the cholesterol level increased from 358 mg%ml _+ 38.6 (SE) to 375 mg%ml + 37.1 (SE) after saline administration, and the triglycerides level 800

600

q-

RESULTS

After carnitine treatment, the rats of the third group showed a very strong decrease of both total serum cholesterol and triglyccrides (P < 0.005) (Fig. 1), associated with a significant (P < 0.005) drop in total serum apo B (Figs 2 and 3). A similar finding was obtained for the total serum apo E, (P < 0.05), however since the standard apo E is not available, we have expressed the change in apo E as the percent decrease of single samples compared to the control (rat before treatment) (Fig. 4).

x. 40(3

A B C Fig. 2. Total serum rat apo B: normal values (A), before L-camitine treatment (B), and after L-carnitine treatment (C); P < 0.005.

Fig. 3. Rocket electrophoresis o f total rats apo B before (wells 2, 4, 6, 8, 10, 12, 14, 16) and after L-carnitine treatment (wells 3, 5, 7, 9, 11, 13, 15, 17). In wells 1 and 18 standard apo B two times assayed. P < 0.005. Table 1. Chemical composition of rat lipoprotcins before and after L-carnitine administration mg/dl

VLDL

Cholesterol Triglycerides Phospholipids Proteins pg/ml VLDL d < 1.006; IDL

Normals IDL LDL

9 0.11 8.7 60 8.1 8.2 22 3.3 6.5 186 75 129 d 1.006-1.019 g/ml; LDL d

HDL

VLDL

Before treatment IDL LDL

HDL

VLDL

31.5 110 20 7 9.6 3.3 73 11 5.5 2.8 31.4 69 12.4 3.3 10.6 720 849 119 66 310 1.019-1.063 g/ml; HDL d 1.063-1.210 g/ml.

67.6 61.2 42.2 510

After treatment IDL LDL 12 8.4 7.2 47

9 6.2 6.3 86

HDL 20.4 2.9 18.3 660

Carnitine affects hypercholesterolemia

71

Fig. 4. Rocket electrophoresis of total rats ape E before (wells 1, 3, 5, 7, 9, I1, 13, 15) and after L-carnitine treatment (wells 2, 4, 6, 8, 10, 12, 14, 16). P <0.05.

rose from 95 mg%ml + 8.5 (SE) to 100 mg%ml + 10.6 (SE). Table 1 shows the chemical composition of rat lipoproteins before and after carnitine administration. As can be observed cholesterol, triglycerides,

Z

phospholipids and proteins of rat lipoproteins tend to return to normal values after carnitine administration. Figure 5 illustrates the SDS polyacrylamide disc gel electrophoresis of rat VLDL, IDL and LDL and Fig.

:

Large Apo B

SmaLl, Apo B

iil

Z;

i ~2

I 23

(a)

I 2

(b)

3

I

23

(c)

Fig. 5. SDS PAGE ofVLDL (A), IDL (B) and LDL (C). Line 1 represents normal samples; line 2: samples before treatment and line 3: samples after L-carnitine treatment.

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P. MONDOLAet al.

Apo A Apo E Apo A

Apo A IV Apo E Apo A T- - [ - - "

Apo E

C's [

C's t c'sl

(a)

(b)

(c]

Fig. 6. TMU disc gel electrophoresis of VLDL (A), IDL (B) and HDL (C). Line I represents normal samples; line 2: samples before treatment and line 3: samples after L-carnitine treatment. 6 shows the 4.2 M tetrametylurea polyacrylamide disc gel electrophoresis of rat VLDL, IDL and HDL. The values of densitometric scanning of TMU delipidated VLDL, IDL and H D L before and after L-carnitine administration are reported in Table 2. In Table 3 the per cent values of B-100 and B-48 apoproteins of VLDL, IDL and LDL, delipidated by ethanol-diethylether 3:1 (v/v), are reported. DISCUSSION

It is Well known that L-carnitine lowers the serum lipid level (Maebashi et al., 1978; Rossi and Siliprandi, 1982) and increases high density lipoprotein

cholesterol and apoprotein A (Vadra et al., 1983) in various conditions. The present study confirms the finding that L-carnitine lowers serum lipids. Moreover, we have shown that L-carnitine restores the apolipoproteins pattern of rat fed a cholesterol-rich diet to normal values. Moreover the change of both total serum B and E apoproteins, that fall by 30 and 8% respectively, after carnitine treatment (Figs 3 and 4), indicates that the marked decrease in total serum lipids is associated with a significant decrease in the level of these apoproteins. In addition, Fig. 5 and Table 3 show that the

Table 2. Apolipoprotein per cent composition in TMU delipidated rat VLDL, IDL and HDL normal, before and after L-earnitine administration VLDL Normal (% of values)

VLDL Before treatment (% of values)

VLDL After treatment (% of values)

Apo E Apo C

9.7 40.8 13.4 36.1 IDL Normal (% of values) undetectable undeteetable

4.3 39.2 13.9 42.6 IDL Before treatment (% of values) 77.6 22.4

2.9 39.1 12.4 45.6 IDL After treatment (% of values) 54.5 45.5

Apo Apo Apo Apo

HDL Normal (% of values) undetectable 34.5 57.1 8.4

HDL Before treatment (% of values) undetectable 9 77.9 13.1

HDL After treatment (% of values) undetectable 22.1 68.9 9

Apo Apo Apo Apo

A IV E A I C

A IV E A I C

Carnitine affects hypercholesterolemia

73

Table 3. The B apoproteins per cent composition in ethanol-diethylether delipidated rat VLDL, IDL and LDL normal, before and after L-carnitine administration VLDL Normal VLDL Before treatment VLDL After treatment (% of values) (% of values) (% of values) Apo B~ 45 62 53 Apo Bi00 55 38 47 Apo B4a/Apo Bl0o 0.8 1.6 1.1 IDL Normal IDL Before treatment IDL After treatment (% of values) (% of values) (% of values) Apo B~ undetectable 40 39 Apo B~00 undetectable 60 61 Apo B~/Apo Bt00 undetectable 0.7 0.6 LDL Normal LDL Before treatment LDL After treatment (% of values) (% of values) (% of values) Apo B48 38 50 43 Apo Bi00 62 50 57 Apo B,~/APO Bi00 0.6 1.0 0.7 greater decrease of B apoproteins involves the small apo B or apo B-48, which in rats is synthesized by the intestinal mucosa and the liver (Elovson et al., 1981). The densitometric scanning of H D L shows that its apolipoprotein composition, following L-carnitine administration, tends to return to the normal per cent values. The post L-carnitine increase in H D L proteins (Table 1) is due mainly to A p o A-I, because this apoprotein represents 68.9% of proteins in lipoproteins of a density greater than 1.063 g/ml. We were unable to scan the apo A-IV because its concentration in H D L was very low. The percentage change of B apoproteins in V L D L , I D L and L D L reported in Table 3, shows that a cholesterol diet strongly increases the a m o u n t o f the small apo B in V L D L and L D L , so that the apo B-48/apo B-100 ratio is increased; the carnitine treatment tends to normalize this ratio. Taken together our results indicate that L-carnitine treatment restores the apolipoprotein pattern of rat lipoproteins, remarkably modified by cholesterol diet. Since it is well known that apolipoproteins play an important role in lipoprotein metabolism (Jonas et al., 1984; Steinmetz and Utermann, 1983; Brown and Goldstein, 1983), our data support the hypothesis that the effect of L-carnitine administration on lipid metabolism can be ascribed also to the capacity of this substance to affect the serum apolipoproteins. SUMMARY We have investigated the effect of L-carnitine administration on apolipoproteins of rats fed a cholesterol-rich diet. L-carnitine restored the rat apolipoprotein pattern, that was greatly altered by the cholesterol diet. These data support the hypothesis that the effect o f L-carnitine on lipid metabolism could be ascribed also to the capacity of this substance to affect serum apolipoproteins. Acknowledgements--We thank Mr Lucio Cammarota for his excellent technical assistance. L-Carnitine was kindly supplied by Sigma-Tau, Industrie Farmaceutiche Riunite S.p.A. Rome, Italy. REFERENCES

Brown M. S. and Goldstein J. L. (1983) Lipoprotein receptors in the liver. Control signals for plasma cholesterol traffic. J. clin. Invest. 72, 743-747. Elovson J., Huang Y. O., Baker N. and Kannan R. (1981)

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