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