The mushroom Lentinus edodes has a potent hypocholesterolemic effect when fed to rats (Kaneda and Tokuda 1966), and eritadenine [2(R),3(R)-dihydroxy-4-(9-adenyl)-butyric acid] was isolated from the mushroom as a hypocholesterolemic factor (Chibata et al. 1969, Rokujo et al. 1970). Previous studies have suggested that the hypocholesterolemic action of eritadenine might be elicited through depressed secretion of lipoprotein cholesterol from the liver or increased uptake of plasma cholesterol by tissues (Takashima et al. 1973 and 1974). However, the detailed mechanism is not yet fully elucidated.

Recently we found that dietary supplementation with eritadenine markedly decreased the ratio of phosphatidylcholine (PC)⁴ to phosphatidylethanolamine (PE) in liver microsomes of rats; there was a significant correlation between the PC:PE ratio and the plasma cholesterol concentration (Sugiyama et al. 1995a and b). Furthermore, eritadenine could modify the molecular species composition of plasma PC, a major phospholipid class of plasma lipoproteins, in rats when added to a diet containing corn oil as a fat source (Sugiyama and Yamakawa 1996). With regard to the latter finding, it is interesting that the uptake rate of cholesteryl ester of reconstituted HDL by perfused rat livers was largely influenced by the difference in PC molecular species used for the reconstitution of HDL (Kadowaki et al. 1993). These findings suggest that eritadenine may alter the molecular species composition of plasma lipoprotein phospholipids and may thereby accelerate the uptake of lipoprotein cholesterol by the liver. However, little information is available concerning the relationship between plasma PC molecular species composition and the regulation of plasma cholesterol concentration. The fatty acid composition and probably molecular species composition of plasma lipoprotein phospholipids can be readily modified by the type of dietary fats. It is therefore interesting to compare the effect of dietary eritadenine on plasma PC molecular species profile with that of dietary fat sources, in terms of clarifying the mechanism of hypocholesterolemic action of eritadenine.

This study was conducted to determine whether dietary eritadenine and fat sources have distinct, additive or interacting effects on the plasma cholesterol concentration and on the molecular species composition of plasma PC in rats fed different types of fats (olive oil, corn oil and linseed oil).

MATERIALS AND METHODS

Six different diets were utilized in this study: diets with or without eritadenine contained olive oil, corn oil or linseed oil. The diets without eritadenine consisted of the following (g/kg): casein (Nacalai Tesque, Kyoto, Japan), 250; corn starch (Fuji-seifun, Shimizu, Japan), 398; sucrose (Fuji-seito, Shimizu, Japan), 200; olive oil (Wako Pure Chemical, Osaka, Japan), corn oil (Honen, Tokyo, Japan) or linseed oil (Wako), 100; AIN-76 mineral mixture (Oriental Yeast, Tokyo, Japan), 35; AIN-76 vitamin mixture (Oriental Yeast), 10; choline chloride (Wako), 6; lactose (Wako), 1. The fatty acid composition of fat sources used is shown in Table . Eritadenine, which was isolated from dried L. edodes mushroom according to the method of Tokita et al. (1971), was mixed with lactose and added to the diet at a level of 50 mg/kg at the expense of lactose. Forty-eight rats were divided into six group of eight rats, each with similar initial mean weights, and given free access for 2 wk to the six experimental diets and water. The body weight and food consumption of rats were measured daily. The experimental design was approved by the Laboratory Animal Care Committee of the Faculty of Agriculture, Shizuoka University.

Table 1. Fatty acid composition of dietary fats [View Table]

RESULTS

Table 2. Effects of eritadenine supplementation on body weight gain, food intake, liver weight, liver and plasma lipid concentrations and liver microsomal phospholipids in rats fed different types of fat1 [View Table]

Table 3. Effects of eritadenine supplementation on the fatty acid composition of plasma phosphatidylcholine in rats fed different types of fat1 [View Table]

Eritadenine supplementation significantly increased the proportion of linoleic acid and decreased the proportion of arachidonic acid in plasma cholesteryl esters (Table ). In contrast, the proportions of saturated fatty acids, such as palmitic acid and stearic acid, were slightly increased by eritadenine. In rats fed linseed oil, eritadenine supplementation significantly increased the proportion of -linolenic acid and inversely decreased the proportion of 20:5(n-3).

Table 4. Effects of eritadenine supplementation on the fatty acid composition of plasma cholesteryl esters in rats fed different types of fat1 [View Table]

Table 5. Effects of eritadenine supplementation on the molecular species composition of plasma phosphatidylcholine in rats fed different types of fat1 [View Table]

In the sn-1 position, the proportion of palmitic acid was significantly increased and the proportion of stearic acid was significantly decreased by eritadenine supplementation, irrespective of dietary fat sources (Table ). In the sn-2 position, the proportion of linoleic acid was significantly increased by eritadenine, irrespective of dietary fat sources. In rats fed the olive oil diet, the proportion of 18:1 in both the sn-1 and sn-2 positions was significantly increased by eritadenine supplementation.

Table 6. Composition of major fatty acids in the sn-1 and sn-2 positions of plasma phosphatidylcholine in rats fed experimental diets1 [View Table]

DISCUSSION

The fatty acid composition, and probably molecular species composition, of plasma phospholipids can be affected by the type of dietary fat. Therefore, one of the objectives of this study was to compare the effect of eritadenine on the fatty acid and molecular species composition of plasma PC in rats fed various dietary fats. Of interest is that rats fed linseed oil had the highest level of linoleic acid in plasma PC, despite the fact that the linoleic acid concentration of linseed oil was much lower than that of corn oil. This could be explained by the fact that -linolenic acid, a major fatty acid of linseed oil, competes with linoleic acid at the step of 6-desaturation (Mohrhauer and Holman 1963). Higher levels of 18:1 or linoleic acid in plasma PC in rats fed olive oil or corn oil, respectively, are considered to be the result of direct effects of these dietary fatty acids. In addition to direct or indirect effects of dietary fatty acids, eritadenine increased the proportion of linoleic acid in plasma PC, irrespective of dietary fat type. In our previous study (Sugiyama and Yamakawa 1996) we showed that the increased proportion of linoleic acid in plasma PC caused by eritadenine was reflected mainly in the increase in 16:0-18:2 molecular species and the decreased proportion of arachidonic acid was reflected mainly in the decrease in 18:0-20:4 in rats fed a diet containing corn oil at a lower level (50 g/kg). The present study confirmed that these eritadenine-induced selective alterations of plasma PC molecular species could occur in rats fed all the dietary fat sources tested. In contrast, the alteration of plasma PC molecular species composition due to different dietary fat sources appears to be less specific, since the proportion of 18:0-18:2 and 16:0-20:4 molecular species was also largely influenced by the type of dietary fat. This study demonstrates that eritadenine could affect the fatty acid profile of plasma PC not only in the sn-2 position, but also in the sn-1 position. Both the increase in 16:0-18:2 molecular species and the decrease in 18:0-20:4 molecular species might be associated with the eritadenine-induced alteration of the fatty acid profile in the sn-1 position of plasma PC. In this study, the fatty acid composition of plasma cholesteryl esters changed in a similar manner to that of plasma PC in response to both dietary fat sources and eritadenine supplementation. The primary explanation may be that the fatty acid composition of plasma PC was reflected in that of plasma cholesteryl esters through the action of LCAT, which transfers fatty acid of phospholipids, especially in the sn-2 position, to free cholesterol on the surface of HDL particles.

Although our previous studies have suggested that alterations of both hepatic phospholipid class profile (Sugiyama et al. 1995a and b) and plasma phospholipid molecular species profile (Sugiyama and Yamakawa 1996) might participate in the hypocholesterolemic action of eritadenine, the exact mechanism is unclear. In principle, plasma cholesterol can be reduced either by decreasing the secretion rate of lipoprotein cholesterol from tissues (e.g., liver) into the blood circulation or by increasing the uptake rate of plasma lipoprotein cholesterol by tissues. We have demonstrated that eritadenine may decrease the plasma cholesterol concentration, but not triacylglycerol concentration, without development of fatty liver when an adequate amount of choline was included in the diet (Sugiyama et al. 1995b). This was also the case for the present study. These observations suggest that the essential hypocholesterolemic action of eritadenine might be independent of depressed secretion of triacylglycerol-rich lipoproteins (VLDL) from the liver. In supporting this assumption, the secretion of triacylglycerol and cholesterol in a form of VLDL, as measured after Triton WR-1339 injection, was not impaired in eritadenine-fed rats compared with control rats (unpublished data). In contrast, an earlier report has shown that the clearance rate of plasma cholesterol was faster in eritadenine-treated rats than in control rats (Takashima et al. 1974), suggesting that eritadenine would increase the uptake of plasma lipoprotein cholesterol by tissues.

It has been suggested that hepatic lipase participates in the uptake by the liver of constituents of lipoproteins, such as HDL (Kadowaki et al. 1992) and remnants of chylomicron (Shafi et al. 1994) or VLDL (Murase and Itakura 1981). Hepatic lipase has phospoholipase A₁ activity in addition to triacylglycerol lipase activity, and the hydrolysis of HDL phospholipids by hepatic lipase is thought to be necessary for the subsequent uptake of HDL constituents by the liver (Kadowaki et al. 1992). With regard to this, Kadowaki et al. (1993) have shown that the uptake of cholesteryl oleate of reconstituted HDL by perfused rat livers was most stimulated by 16:0-18:2 molecular species of the five molecular species tested (16:0-18:2, 16:1-16:1, 18:0-18:2, 18:1-16:0 and 20:1-20:1), and that 16:0-18:2 PC was hydrolyzed the most in vitro by hepatic lipase. These findings suggest that the phospholipid molecular species composition of certain plasma lipoproteins has an important role in the regulation of plasma cholesterol concentration. At present, it is unknown whether some hypo- or hypercholesterolemic substances exert their action through such a mechanism. However, the fact that one of the marked changes in plasma PC molecular species caused by eritadenine was an increase in 16:0-18:2 molecular species suggests that eritadenine may increase the uptake of plasma lipoprotein cholesterol by the liver through altered plasma phospholipid molecular species profile, thereby leading to the reduction of plasma cholesterol. Another possibility is that the altered plasma PC molecular species may enhance plasma LCAT activity, thereby accelerating the metabolism of plasma cholesterol. The LCAT activity is thought to be influenced by both the substrate specificity of LCAT and the molecular species composition of PC, in addition to various activators such as apoproteins. Unlike human LCAT, rat LCAT prefers PC molecular species containing arachidonic acid in the sn-2 position (Subbaiah and Liu 1996). It is therefore conceivable that plasma LCAT activity may not be stimulated by eritadenine-induced alteration of plasma PC molecular species composition, although the effect of eritadenine on plasma LCAT activity appears to deserve further investigation.

The present study demonstrated that dietary eritadenine and fat source had additive effects on plasma cholesterol concentration, since the order of plasma total cholesterol concentration among the fat groups was not changed by eritadenine supplementation. A number of studies have shown that fish oil rich in eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) decreases plasma cholesterol concentration in rats, but not in humans, whereas fish oil decreases plasma triacylglycerol concentration in both rats and humans (Harris 1989 and 1996). In rats, linseed oil and perilla oil, which are rich in -linolenic acid, also possess hypolipidemic action (Garg et al. 1988, Lee et al. 1989). Depressed secretion of triacylglycerol-rich lipoproteins (VLDL) resulting from both a decrease in triacylglycerol synthesis and an increase in -oxidation of fatty acids in the liver is associated with the hypotriacylglycerolemic action of fish oil (Herzberg et al. 1996, Surette et al. 1992, Wong et al. 1984) and perilla oil (Ide et al. 1996). Consistent with this, the plasma triacylglycerol concentration apparently changed in parallel with hepatic triacylglycerol concentration in the fat groups in this study. In perfused rat livers, EPA and DHA reduced the secretion of VLDL cholesteryl esters (Zhang et al. 1991). The decrease in the activity of 3-hydroxy-3-methylglutaryl-CoA reductase due to fish oil or DHA, but not EPA, has also been shown in rats (Frøyland et al. 1996, Roach et al. 1987). These findings suggest that depressed secretion of lipoprotein cholesterol from the liver contributes to the hypocholesterolemic action of (n-3) fatty acids in rats. In addition, it was shown that fatty acid composition of plasma lipoproteins could affect their uptake by the liver in rats fed different types of fats. For instance, the removal rate of chylomicron remnant cholesterol derived from fish oil-fed rats was faster than that of chylomicron remnant cholesterol derived from rats fed palm oil, olive oil or corn oil (Lambert et al. 1995). These results, together with results presented here, suggest that alteration of fatty acid or molecular species composition of plasma lipoprotein phospholipids may also contribute, at least in part, to the hypocholesterolemic action of (n-3) fatty acids in rats.