View
5
Download
0
Category
Preview:
Citation preview
J. Nutr, Sci. Vitanzinol, 39, 63-71, 1993
Note
The Effect of Various Phospholipids on Plasma
Lipoproteins and Liver Lipids in
Hypercholesterolemic Rats
Toshio IWATA,1 Yoshiharu KIMURA,2 Kentarou TSUTSUMI,1 Yuji FURUKAWA,2 and Shuichi KIMURA2
1 Department of Research and Development, The Rinoru Oil Mills Co., Ltd., Minato-ku, Nagoya 455, Japan
2 Laboratory of Nutrition, Department of Applied Biological Chemistry, Faculty of Agriculture, Tohoku University,
Aoba-ku, Sendai 981, Japan
(Received May 19, 1992)
Summary Rats were fed a hypercholesterolemic diet (5% lard, 0.5% cholesterol, and 0.25% sodium cholate) containing 5% of dietary phospholipid as safflower phospholipid (SAP), soybean phospholipid (SOP), or egg yolk phospholipid (EGP), or 5% of soybean oil (SO) as a control for 4 weeks. The concentrations of plasma cholesterol were significantly higher in rats fed the EGP diet than those of the other diets. Similarly, the concentrations of chylomicron plus very low density lipoprotein (VLDL) cholesterol were higher in rats fed the EGP diet. The
phospholipid diets induced a significant increase of high density lipoprotein (HDL) cholesterol in comparision with the SO diet. The concentrations of liver cholesterol were significantly lower in rats fed the phospholipid diets than those of the SO diet. Among phospholipid-fed rats, the SAP and SOP diets decreased the concentrations of liver cholesterol compared with the EGP diet. The activity of plasma lecithin-cholesterol acyltransferase (LCAT) was significantly increased in rats fed the phos
pholipid diets. The phospholipid diets caused an enhanced excretion of neutral steroids into feces. Among phospholipid-fed rats, the SAP and SOP diets increased the excretion of fecal neutral steroids compared with the EGP diet. The fatty acid composition of HDL phospholipid was slightly reflected by the major dietary fat source. These results suggest that SAP and SOP inhibit markedly the absorption of dietary cholesterol in the small intestine of hypercholesterolemic rats and that the effect of SAP and SOP on plasma cholesterol metabolism may be different from that of EGP.Key Words safflower phospholipid, soybean phospholipid, egg yolk
phospholipid, plasma cholesterol, liver cholesterol, lipoprotein cholesterol, lecithin-cholesterol acyltransferase, fecal neutral steroids, hypercholesterolemia, rats
63
64 T. IWATA et al.
The effect of dietary phospholipids on serum lipids and lipoproteins has been
extensively studied in humans (1-3) and rats (4-9). However, the mechanism of the
cholesterol-lowering action of dietary phospholipids is still controversial.
We recently reported that, in addition to soybean phospholipid, safflower
phospholipid suppressed the elevation of plasma and liver cholesterol in rats fed a
hypercholesterolemic diet (10). Furthermore, we observed that safflower phospholip
id had various advantages in cholesterol metabolism over soybean phospholip
id (10). Egg yolk phospholipid containing phosphatidylcholine (PC) and
phosphatidylethanolamine (PE) caused a reduction in the serum cholesterol level
in rats (11, 24). In the present study, therefore, in order to investigate how dietary
phospholipids affect the lipid metabolism in plasma and liver of hypercholester
olemic rats, we have studied plasma and liver lipids and excretion of fecal steroids
in rats fed the hypercholesterolemic diets containing safflower phospholipid, soybean
phospholipid, or egg yolk phospholipid, compared with soybean oil.
Materials and methods. Animals and diets: Male Sprague-Dawley rats,
specific pathogen-free, were obtained from Funabashi Farm Co. (Funabashi). Each
rat was individually housed in an air-conditioned room (21-25•Ž) with lights on
from 08:00 to 20:00 h. Rats weighing approximately 140g were fed a commercial
nonpurified diet (Type F-2, Funabashi Farm Co.) for at least a week before initiation
of the experiments with purified diets. The composition of the basal diet is shown
in Table 1. For the experimental diet, 5% of dietary phospholipid as safflower
phospholipid (Rinoru Oil Mills Co., Tokyo), soybean phospholipid (Rinoru Oil
Mills Co.) or egg yolk phospholipid (Asahi Chemical Industry Co., Tokyo) or 5%
of soybean oil, as shown in Table 2, were added to the basal diets at the expense
of lard. A food-restricted pair-fed phospholipid groups of rats were given the
corresponding diets in an amount equal to that consumed on the previous day by
their respective paired-mate ad libitum-fed soybean oil groups. Food intake was
measured every day and weight gain was measured weekly.
Table 1. Composition of the basal diet.
1 Composition according to Harper.
J. Nutr. Sci. Vitaminol.
VARIOUS PHOSPHOLIPIDS AND HYPERCHOLESTEROLEMIA 65
Table 2. Phospholipid class and fatty acid composition of dietary fats used in the
experiments.
SO, soybean oil; SAP, safflower phospholipid; SOP, soybean phospholipid; EGP, egg
yolk phospholipid.
Analytical procedures: After 28-day feeding all rats were fasted overnight,
and blood was collected from the abdominal aorta under diethyl ether anesthesia.
Plasma lipoprotein was isolated at 4•Ž from each plasma sample by sequential
ultracentrifugation in an RPL42T fixed-angle rotor of Hitachi ultracentrifuge
(Model 70P-72, Hitachi, Tokyo) according to the method of Havel et al. (13). The
density ranges of chylomicron plus VLDL, low density lipoproteins (LDL) and
HDL were d<1.006 (g/ml), 1.006<d<1.063, and 1.063<d< 1.21, respectively,
using KBr solution.
Total cholesterol levels in plasma and lipoprotein fraction and free cholesterol,
triacylglycerol and phospholipid levels in plasma were determined as described
previously (9). Liver lipids were extracted in chloroform/methanol=2:1(14). Total
cholesterol and triacylglycerol in liver were determined as described previously (9).
For the determination of fatty acid composition of phospholipid of HDL, thin-layer
chromatography (TLC) was carried out by using the solvent system of hexane/diethyl
ether/acetic acid (70:30:1, v/v/v). With this system, phospholipids run near the
origin. The spots of phospholipids visualized with iodine vapor were scraped and
eluted, subjected to methanolysis with BF3/CH3OH (15) for gas-liquid chromato
Vol. 39, No. 1, 1993
66 T. IWATA et al.
Table 3. Effect of dietary phospholipids on weight gain and liver weight in rats fed
the experimental diets
SO, soybean oil, SAP, safflower phospholipid; SOP, soybean phospholipid; EGP, egg
yolk phospholipid. * Mean•}SE (n=8). Means in the same column not sharing a
common superscript letter are significantly different (p<0.05).
graphic analyses.
Activity of lecithin-cholesterol acyltransferase [LCAT, EC 2.3.1.43] was
determined in plasma using [3H]cholesterol as a substrate according to the method
of Stokke and Norum (16).
Feces were collected for 2 days prior to sacrifice and immediately frozen at
-20•Ž . Analyses of the fecal neutral steroids were performed as described
previously (10).
Statistical analysis: Statistical evaluation of data was carried out by analysis
of variance (ANOVA) coupled with a Duncan's New Multiple Range Test for the
classification of the means. The acceptable level of probability was set at 95%.
When necessary to achieve homogeneity of variance, the data were subjected to
logarithmic transformation.
Results. Weight gain and liver weight: As shown in Table 3, there were no
statistically significant differences in the weight gain among the various groups .
Liver weight (g/100g body weight) of rats fed the SAP and SOP diets was significantly
lower than that of the other diets.
Plasma lipids: Plasma lipid concentrations of rats fed the various experimental
diets are shown in Table 4. The concentrations of total cholesterol in plasma of
rats fed the EGP diet were significantly increased in comparison with those of the
other diets. Plasma free cholesterol concentrations in rats fed the SO diet were
lower than those of the SOP and EGP diets. Plasma triacylglycerol concentrations
were not different among the various groups . Plasma phospholipid concentrations
in rats fed the EGP diet were significantly higher than those of the other diets .
Lipoprotein cholesterol: The concentrations of total cholesterol in the different
lipoprotein fractions are shown in Table 4. The phospholipid diets induced a
significant increase of HDL cholesterol concentrations compared with the SO diet .
The concentrations of LDL cholesterol did not differ among the various groups .
The concentrations of chylomicron plus VLDL cholesterol were significantly higher
in rats fed the EGP diet than those of the other diets .
J. Nutr. Sci. Vitaminol.
VARIOUS PHOSPHOLIPIDS AND HYPERCHOLESTEROLEMIA 67
Table 4. Effect of dietary phospholipids on various lipids and activity of lecithin cholesterol acyltransferase (LCAT) in plasma of rats fed the experimental diets.
SO, soybean oil; SAP, safflower phospholipid; SOP, soybean phospholipid; EGP, egg
yolk phospholipid. * Mean•}SE (n=8). Means in the same horizontal column not
sharing a common superscript letter are significantly different (p<0.05). ** Data sub
jected to logarithmic transformation.
Table 5. Effect of dietary phospholipids on various lipids in liver of rats fed the
experimental diets.
Abbreviations and mean values are the same as Table 3.
LCAT activity: As shown in Table 4, the LCAT activity was significantly
higher when rats were fed the phospholipid diets than when rats were fed the SO
diet. But this activity did not differ among the phospholipid diets.
Liver lipids: Liver lipid concentrations of rats fed the various experimental
diets are shown in Table 5. The concentrations of liver cholesterol in rats fed the
phospholipid diets decreased markedly in comparison with those of the SO diet. Among phospholipid-fed rats, the liver cholesterol in rats fed the SAP and SOP
diets were significantly lower than that of the EGP diet. The phospholipid diets
induced a reduction in liver total lipid compared with the SO diet; the SAP diet
indicated the lowest value. The concentrations of liver triacylglycerol in rats fed
the SO diet were significantly higher than those of the other diets.
Vol. 39, No. 1, 1993
68 T . IWATA et al.
Table 6. Effect of dietary phospholipids on excretion of neutral steroids into feces in
rats fed the experimental diets.
Abbreviations and other notations are the same as Table 4.
Table 7. Effect of dietary phospholipids on fatty acid composition of phospholipid in
plasma high density lipoproteins of rats fed the experimental diets.
Abbreviations and other notations are the same as Table 4.
Fecal lipids: Feces dry weight and excretion of neutral steroids into feces in rats fed the various experimental diets are shown in Table 6. Feces weight (g/2 days) was similar within the various diets. The phospholipid diets caused an enhanced excretion of neutral steroids into feces compared with the SO diet. Among
phospholipid-fed rats, the SAP and SOP diets induced a higher excretion of neutral steroids compared with the EGP diet. Although the increase due to the SAP and EGP diets of fecal neutral steroids was more prominent in cholesterol than in coprostanol, the increase due to the SOP diet was more prominent in coprostanol than in cholesterol.
Fatty acid composition of HDL phospholipid: The fatty acid composition of HDL phospholipid was slightly, but significantly, modified by the constituent fatty acid of the dietary phospholipids, as shown in Table 7. Oleic acid increased under the SOP and EGP diets in comparison with the SO diet; the EGP diet indicated the highest value. Linoleic acid decreased by the EGP diet, compared with the other diets. Arachidonic acid increased by the SAP and EGP diets; the EGP diet indicated the highest value. The EGP diet induced an increase of docosahexaenoic acid in
J. Nutr. Sci. Vitaminol.
VARIOUS PHOSPHOLIPIDS AND HVPERCHOLESTEROLEMIA 69
comparison with the other diets.
Discussion. In addition to SOP, hypocholesterolemic action of SAP has been
demonstrated in rats given a cholesterol-enriched diet (9,10). Furthermore, we
showed that SAP caused a favorable alteration in plasma and liver lipids and
excretion of neutral steroids compared with SOP (10). In the present experiment ,
we have studied plasma and liver lipids and the excretion of neutral steroids into
feces in rats fed the SAP, SOP, or EGP diets compared with the SO diet. The EGP
diet significantly increased the concentrations of plasma cholesterol compared with
the other diets. Similarly, the concentrations of chylomicron plus VLDL cholesterol
also were higher in the EGP diet. However, the EGP diet induced a reduction of
the liver cholesterol as well as a rise of HDL cholesterol in comparision with the
SO diet. Although the SAP and SOP diets suppressed the elevation of both plasma
and liver cholesterol, the EGP diet could not suppress the elevation of plasma
cholesterol. These results suggest that the effect of SAP and SOP on plasma
cholesterol metabolism may be different from that of EGP .
In the previous experiment, the activity of plasma LCAT was greatly high
when phospholipid diets were fed (9, 10). The present experiment also showed the
similar results. Jimenez ct al. (8) reported that the LCAT activity increased when
lecithin was fed to rats. This enzyme is intimately involved in the cholesterol
metabolism (17). The phospholipid diets induced a rise of HDL cholesterol. We
suppose that dietary phospholipids may increase the formation of mature HDL via
activation of LCAT. Fatty acid composition of HDL phospholipid was measured,
since the principal molecular reaction catalyzed by LCAT is the transfer of an acyl
chain from the sn-2 position of PC to cholesterol (18). As shown in Table 7 , the
ratio of arachidonate to linoleate was not altered by the SAP and SOP diets except
for the EGP diet. Such a result may be due to the presence of cholesterol in the
diet, since cholesterol supplementation itself markedly suppresses the ‡™6-desaturase
activity (19). It is not clear at present whether or not the fatty acid composition of
HDL phospholipid relates to LCAT activity.
As shown in Table 6, the phospholipid diets caused an enhanced excretion of
fecal neutral steroids compared with the SO diet. In the previous experiment , we
observed the enhanced excretion of fecal neutral steroids by SAP and SOP (10). A
similar effect of dietary lecithin on the excretion of fecal neutral steroids has been
reported in rats fed a cholesterol-enriched diet (20). We suppose that the
phospholipid diets inhibit the absorption of dietary cholesterol in the small intestine.
Among phospholipid4ed rats, both the SAP and SOP diets increased the excretion
of fecal neutral steroids compared with the EGP diet. Furthermore, the SAP and
SOP diets significantly decreased the liver cholesterol compared with the EGP diet.
These results suggest that a factor of liver cholesteroliowering action may be the
inhibition of dietary cholesterol absorption in the small intestine of rats fed a
hypercholesterolemic diet.
The SAP and SOP diets caused a favorable alteration in plasma and liver
cholesterol and excretion of fecal neutral steroids, compared with the EGP diet.
Vol. 39, No. 1, 1993
70 T. IWATA et al .
The factor of these different results among phospholipid-fed rats cannot be explained in the present experiment. The fatty acid composition of EGP is different from that of SAP and SOP. Although EGP is comprised of 64% of PC and 23% of PE, SAP and SOP contain several classes of phospholipid. It has been reported that PE contained in EGP was responsible for the hypocholesterolemic action (11, 21). More
precisely, the constituent base, ethanolamine, was responsible for lowering of the plasma cholesterol. The major constituent in dietary phospholipids, PC, appeared to be less effective (11, 21). Other investigators indicated that the inositol moiety of
phosphatidylinositol (PI) may have a significant role in the regulation of lipid metabolism (22). However, it is not clear in the present experiment whether or not the respective phospholipids influence the hypocholesterolemic action in hypercholesterolemic rats. This is worthy of further investigation.
REFERENCES
1) Childs, M. T., Bowlin, J. A., Ogilvie, J. T., Hazzard, W. R., and Albers , J. J. (1981): The contrasting effects of a dietary soya lecithin product and corn oil on lipoprotein lipids in normolipidemic and familial hypercholesterolemic subjects. Atherosclerosis , 38, 217-228.
2) Cobb, M., Tukki, P., Linscheer, W., and Raheja, K. (1980): Lecithin supplementation in healthy volunteers. Effect on cholesterol esterification and plasma , and bile lipids.
Nutr. Metab., 24, 228-237.3) Greten, H., Raetzer, H., Stichl, A.,and Schettler, G. (1980): The effect of polyunsaturated
phosphatidlycholine on plasma lipids and fecal sterol excretion. Atherosclerosis, 36, 81-88.
4) Clark, S. B., Clark, V. E., and Small, D. M. (1981): Effects of lecithin ingestion on
plasma and lymph lipoproteins of normo and hyperlipemic rats, Am. J. Physiol., 241, G422-G430.
5) Samochowiec, L., Kadlubowska, D., and Rozewicka, L. (1976): Investigations in experimental atherosclerosis. Part 1. The effects of phosphatidylcholine (EPL) on experimental atherosclerosis in white rats. Atherosclerosis, 23, 305-317.
6) Kobatake, Y., Kuroda, K., Saito, M., Nishide, E., and Yamaguchi, M. (1988): Differential effect of a dietary soybean phosphatidylcholine and phospholipid mixture on hematologic parameters, and serum lipids in rats. Nippon Eiyo Shokuryo Gakkaishi
(J. Jpn. Soc. Nutr. Food Sci.), 41, 457-463.7) Imaizumi, K., Murata, M., and Sugano, M. (1982): Effect of dietary polyunsaturated
phospholipid on the chemical composition of serum lipoproteins in rat. J. Nutr. Sci. Vitaminol., 28, 281-294.
8) Jimenez, M. A., Scarino, M. L., Vignolini, F., and Mengheri, E. (1990): Evidence that polyunsaturated lecithin induces a reduction in plasma cholesterol level and favorable changes in lipoprotein composition in hypercholesterolemic rats. J. Nutr.,120, 659-667.
9) Iwata, T., Hoshi, S., Tsutsumi, K., Furukawa, Y., and Kimura, S. (1991): Effect of dietary safflower phospholipid on plasma and liver lipids in rats fed a hypercholesterolemic diet. J. Nutr. Sci. Vitaminol., 37, 591-600.
10) Iwata, T., Hoshi, S., Takehisa, F., Tsutsumi, K., Furukawa, Y., and Kimura, S. (1992): The effect of dietary safflower phospholipid and soybean phospholipid on plasma and
J. Nutr. Sci. Vitaminol,
VARIOUS PHOSPHOLIPIDS AND HYPERCHOLESTEROLEMIA 71
liver lipids in rats fed a hypercholesterolemic diet. J. Nutr. Sci. Vitaminol., 38, 471-479.11) Murata, M., Imaizumi, K., and Sugano, M. (1982): Effect of dietary phospholipids
and their constituent bases on serum lipids and apoliporoteins in rats. J. Nutr.,112, 1805-1808.
12) Imaizumi, K., Sakono, M., Sugano, M., Shigematsu, Y., and Hasegawa, M. (1989): Influence of saturated and polyunsaturated egg yolk phospholipids on hyperlipidemia in rats. Agric. Biol. Chem., 53, 2469-2474.
13) Havel, R. J. Eder, H. A., and Bragdon, J. H. (1955): The distribution and chemical composition of ultracentrifugally separated lipoproteins in human serum. J. Gun. Invest., 34, 1345-1353.
14) Folch, J., Lees, M., and Stanley, G. H. S. (1957): A simple method for the isolation and purification of total lipids from animal tissues. J. Biol. Chem., 226, 497-509.
15) Metcalfe, L. D., and Schmitz, A. A. (1961): The rapid preparation of fatty acid esters for gas chromatographic analysis. Anal. Chem., 33, 363-364.
16) Stokke, K. T., and Norum, K. R. (1971): Determination of lecithin: cholesterol acyltransfer in human blood plasma. Scand. J. Clin. Lab. Invest., 27, 21-27.
17) Glomset, J. A. (1968): The plasma lecithin: cholesterol acyltrasferase reaction. J. Lipid Res., 9, 155-167.
18) Glomset, J. A. (1972): Plasma lecithin: cholesterol acyltransferase in Blood Lipids and Lipoproteins, ed by Nelson, G., Wiley, New York, pp. 745-787.
19) Leikin, A. I., and Brenner, R. R. (1987): Cholesterol-induced microsomal changes modulate desaturase activities. Biochim. Biophys. Acta, 922, 294-303.
20) O'Mullane, Y. E., and Hawthorne, J. N. (1982): A comparison of the effects of feeding linoleic acid-rich lecithin or corn oil on cholesterol absorption and metabolism in the rat. Atherosclerosis, 45, 81-90.
21) Imaizumi, K., Mawatari, K., Murata, M., Ikeda, I., and Sugano, M. (1983): The contrasting effect of dietary phosphatidylethanolamine and phosphatidylcholine on serum lipoproteins and liver lipids in rats. J. Nutr., 113, 2403-2411.
22) Ishida, T., Koba, K., Sugano, M., Imaizumi, K., Watanabe, S., and Minoshima, R.
(1988): Cholesterol levels and eicosanoid production in rats fed phosphatidylinositol or soybean lecithin. J. Nutr. Sci. Vitaminol., 34, 237-244.
Vol. 39, No. 1, 1993
Recommended