Nai Wen Chang a, *, Po Chao Huang b

Transcription

1 Atherosclerosis 142 (1999) Comparative effects of polyunsaturated- to saturated fatty acid ratio versus polyunsaturated- and monounsaturated fatty acids to saturated fatty acid ratio on lipid metabolism in rats Nai Wen Chang a, *, Po Chao Huang b a Department of Biochemistry, China Medical College, 91 Hsueh-Shih Road, Taichung 404, Taiwan, ROC b Department of Biochemistry, College of Medicine, National Taiwan Uni ersity, Taipei, Taiwan, ROC Received 30 March 1998; accepted 5 August 1998 Abstract This study examined the effects of various polyunsaturated fatty acid (PUFA, P)/saturated fatty acid (SFA, S) ratio versus PUFA+monounsaturated fatty acid (MUFA, M)/SFA ratio on lipid metabolism. The P/S ratio of dietary fat was fixed at a certain level (0.5, 1, 2, or 4, respectively) for each of four pairs of rat groups respectively, and then the P+M/S ratio was changed for the four pairs of rat groups. When the P/S ratio was fixed at 0.5, 1, 2, or 4, the plasma total cholesterol, LDL-cholesterol (C), and HDL-C levels did not show any significant difference in each pair of groups with different P+M/S ratios. However, when the P/S ratio was fixed at 1.0, the higher P+M/S ratio of 5.7 (M/S=4.7, P/M=0.2) resulted in significantly higher plasma total triacylglycerol (TAG), VLDL-TAG, VLDL-C, and VLDL-phospholipid (PL) than the lower P+M/S ratio of 1.4 (M/S=0.4, P/M=2.4). Whereas when SFA was fixed at a similar level, it therefore had approximately the same P+M/S ratio(5.3, 5.6, 5.7), and by increasing the P/S ratio from 1, 2 to 4 (70.4, 52.7 and 23.2% of the total fatty acids as MUFA respectively), the plasma VLDL-C, VLDL-TAG, and VLDL-PL decreased progressively. When PUFA or MUFA was kept on a similar level (14.9 or 53% respectively), the higher P+M/S ratio (5.7 or 5.3, respectively) resulted in significantly greater accumulation of liver cholesterol than the lower P+M/S ratio of 2.2. When the P/S ratio was fixed at 1 or 4, the diet of higher P+M/S ratio in a pair of the groups also resulted in greater accumulation of liver cholesterol. The results of the study suggests that if the P+M/S ratio was below 3, the change in the P/S ratios (0.5, 1 or 2) did not affect the levels of plasma total and lipoprotein cholesterol and TAG. Increases in the plasma VLDL-C and VLDL-TAG were related to increased MUFA content in the diet. And high MUFA content resulted in greater accumulation of liver cholesterol. It seems that the prerequisites for keeping low plasma and liver cholesterol are (a) low M/S ratio and (b) high P/M ratio and (c) P+M/S ratio not to exceed Elsevier Science Ireland Ltd. All rights reserved. Keywords: Monounsaturated fatty acid; PUFA/SFA ratio; PUFA+MUFA/SFA ratio; Cholesterol; Triacylglycerol; Phospholipid 1. Introduction A reduction in the atherogenic LDL-C is favorable, but a reduction in high density lipoprotein (HDL) cholesterol has been reported to have a negative effect on coronary heart disease (CHD) in epidemiological * Corresponding author. Tel.: , ext. 8608; fax: ; nwchang@mail.cmc.edu.tw. studies [1 3]. Dietary fatty acid composition is one of the most important factors determining plasma lipid and consequently affecting CHD risk [4]. A high content of saturated fatty acids (SFA) in the diet leads to an increase in the of plasma total cholesterol and LDL-C, whereas a decrease can be achieved by replacing SFAs with polyunsaturated fatty acids (PUFA) [4 6]. In the past, monounsaturated fatty acids (MUFA) were considered to be neutral with /99/$ - see front matter 1999 Elsevier Science Ireland Ltd. All rights reserved. PII: S (98)

2 186 N. Wen Chang, P. Chao Huang / Atherosclerosis 142 (1999) Table 1 Fatty acid composition of the test diets a Fatty acids Diet 1 (%) Diet 2 (%) Diet 3 (%) Diet 4 (%) Diet 5 (%) Diet 6 (%) Diet 7 (%) Diet 8 (%) 6: : : : : : : : : : : : : : SFA MUFA PUFA P/S ratio P+M/S ratio M/S ratio P/M ratio a Diet 1: 55.4% coconut oil+44.6% soybean oil; Diet 2: 100% modified lard; Diet 3: 68.5% soybean oil+31.5% coconut oil; Diet 4: 12.3% soybean oil+87.7% olive oil; Diet 5: 87.6% soybean oil+12.4% coconut oil; Diet 6: 55.5% olive oil+44.5% soybean oil; Diet 7: 100% soybean oil; Diet 8: 44.6% olive oil+55.4% safflower oil. regard to their influence on plasma lipids and lipoproteins [5,6]. Since 1985, however, studies using MUFA-rich diets have reported either no changes [7 9], decrease [10], or increase [11 15] in the plasma HDL-C when compared with PUFA- or carbohydrate-rich diets. Moreover, MUFA-rich diets are also found to decrease [10,13,15], increase [14,16], or leave unchanged [9] plasma LDL-C when compared with PUFA- or carbohydrate-rich diets. Thus, the effect of diets enriched in monounsaturated fatty acids on plasma lipids has been controversial. These conflicting results are probably due to the variety of experimental designs used by different investigators. We have found in the cross-over feeding trials (total of 6 weeks) with healthy humans that when the dietary P/S ratio is fixed at 1.0, those subjects given a diet with P+M/S ratio of 4.5 showed significantly higher plasma triacylaglycerol (TAG), VLDL-TAG, LDL-TAG, and LDL-C levels than those subjects given a diet with P+M/S ratio of 1.5 [16]. On the other hand, MUFA was found in animal experiments to increase the plasma and liver total cholesterol levels in rats [17 19], rabbits [18], and hamsters [20]. Oleate may also raise the heart cholesterol level in rats in comparison with linoleate [21]. It appears that the advantageous effect of MUFA on the hypocholesterolemic responses is uncertain. In the present study, the relationship between P/S ratio and P+M/S ratio on lipid metabolism was determined. The P/S ratio of dietary fat was fixed at a certain level (0.5, 1, 2 or 4, respectively) for each of four pairs of rat groups, and then the P+M/S ratio of each group was varied as described under materials and methods. The effect of different P+M/S, M/S, and P/M ratios on plasma and liver lipid was studied. 2. Materials and methods 2.1. Animals Used for this study were 48 male Wistar rats (Laboratory Animal Center, College of Medicine, National Taiwan University), each weighing about 130 g. These rats were divided into eight groups of six rats in each group on the basis of their weight. They were housed individually in stainless steel, wire bottomed cages, and were given a diet and water ad libitum for 21 days. The food consumption of each rat and their individual body weight were measured every 2 days Test diets The composition of the eight semisynthetic diets contained (wt./wt.): 4% salt mixture (AIN-76, ICN), 1%

3 N. Wen Chang, P. Chao Huang / Atherosclerosis 142 (1999) vitamin mixture (AIN76, ICN), 3% methyl cellulose, 20% casein, 52% corn starch, and 20% fat. As shown in Table 1, the different fat mixtures were used in different test diets and prepared with various ratios of soybean oil, coconut oil, modified lard, safflower oil and olive oil. The fatty acid composition of each diet was analyzed by high-performance liquid chromatography (HPLC) [22] for verification of the correct P/S and P+M/S ratios. Each diet supplied 15% of total energy as protein, 45% as carbohydrate and 40% as fat. The first two diets (Diet l and Diet 2) had a same P/S ratio of 0.5, but their P+M/S ratio was 0.7 and 2.2 respectively. The second two diets (Diet 3 and Diet 4) had a same P/S ratio of 1.0, but Diet 3 had a P+M/S ratio of 1.4 and Diet 4 had a P+M/S ratio of 5.7. The third two diets (Diet 5 and Diet 6) had a same P/S ratio of 2.0, but their P+M/S ratio was 2.8 and 5.3 respectively. The last two diets (Diet 7 and Diet 8) had a same P/S ratio of 4.0, but their P+M/S ratio was 5.6 and 7.4 respectively (Table 1). In this feeding protocol, Diet 4, Diet 6, and Diet 7 had a similar SFA level, and it therefore had approximately the same P+M/S ratios of 5.7, 5.3, and 5.6 respectively, but they differed in their P/S ratios of 1, 2, and 4 respectively. While Diet 2 and Diet 6 had a similar MUFA level of 53.5 and 52.7%, but differed in P/S ratios of 0.5 and 2.0 and P+M/S ratios of 2.2 and 5.3 respectively. Whereas Diet 2 and Diet 4 had approximately the same amount of PUFA (14.9%), but differed in P/S ratios of 0.5 and 1.0 and P+M/S ratios of 2.2 and 5.7 respectively Blood sample collection and analyses Blood samples were taken by decapitating rats after an overnight fast of 12 h and centrifuged (365 g) for 20 min. The plasma (1 mg EDTA/ml) was stored at 4 C in a refrigerator before analysis. Plasma VLDL, LDL, and HDL were obtained by ultracentrifugation at densities of 1.006, 1.063, and 1.21 g/ml, respectively [16]. Plasma total and lipoprotein cholesterol and TAG were determined by commercial kit enzymatic methods (Boehringer Mannheim) Plasma total and lipoprotein phospholipid was determined by using an enzyme kit (BioMerieux) Assay of li er lipids Liver was excised, blotted, weighed and frozen until analysis. The liver lipids were extracted with chloroform-methanol 2:1 mixture according to Folch et al. [23]. Liver total cholesterol was determined by the method of Abell [24]. Liver TAG and phospholipid were determined according to the method of Soloni [25] and Stewart [26]. The fatty acid pattern of liver lipid was analyzed by using HPLC [22] Statistical analysis The data were compared between groups by analysis of variance (ANOVA). Student s t-test was used whenever a statistically significant difference between the two groups was shown by ANOVA. All data are presented in tables and figures as mean S.D. 3. Results 3.1. Body weight gain and feed efficiency There was no significant difference in body weight gain and feed efficiency (weight gain/100 g diet) among groups of rats (data are not shown) Plasma lipid When the P/S ratio was held constant at 0.5, 1, 2, or 4 respectively, the plasma total cholesterol, LDL-C, and HDL-C levels did not show any significant difference in each pair of groups with different P+M/S ratios (Table 2). When the P/S ratio was fixed at 4.0, a high P+M/S diet (Diet 8) resulted in a significant increase in VLDL-C (Table 2), total TAG (Table 3), and VLDL-PL (Table 4) (P 0.05). Whereas when the P/S ratio was fixed at 1.0, the higher P+M/S ratio (5.7, Diet 4) resulted in significantly higher plasma VLDL-C (Table 2), total TAG, VLDL-TAG (Table 3), and VLDL-PL (Table 4) than the lower P+M/S ratio (1.4, Diet 3), while no significant effect was seen in LDL and HDL fractions. In addition, as shown in Tables 2, 3 and 4, when P+M/S ratio was kept on a similar level (Diet 4=5.7, Diet 6=5.3, Diet 7=5.6), and the P/S ratio raised from 1, 2 to 4 (MUFA content reduced from 70.4, 52.7 to 23.2), there was a progressive decrease in plasma VLDL-C, total TAG, VLDL-TAG, and VLDL-PL. On the other hand, when PUFA was fixed at a certain level (Diet 2 and Diet 4), the plasma total and lipoprotein cholesterol, TAG, and PL were not significantly different between the different P/S ratio groups of 0.5 and 1. While similar results were also found when MUFA was on a same level (Diet 2 and Diet 6). However, if the P+M/S ratio was below 3 (Diets 1, 2, 3, and 5), the change in the P/S ratios (0.5, 1, or 2) did not affect the levels of plasma total and lipoprotein cholesterol and TAG Li er lipid When the P/S ratio was fixed at 1 or 4, the diet of higher MUFA content in a pair of the groups resulted

4 188 N. Wen Chang, P. Chao Huang / Atherosclerosis 142 (1999) Table 2 Cholesterol levels of various plasma fractions a Groups P/S P+M/S M/S P/M Total cholesterol VLDL-C (mg/dl) LDL-C (mg/dl) HDL-C (mg/dl) Diet Diet Diet l.l b Diet be Diet Diet d,e Diet c,d,f Diet c a Results are shown as Mean S.D. C: Cholesterol. b Matched superscripts in the same column indicate that there is a significant difference between the mean values (P 0.05) by the Student s t-test. cf, same as b. in significantly greater accumulation of liver cholesterol, but not TAG and PL (shown in Fig. 1). When the P/S ratio was fixed at 0.5, higher P +M/S ratio (2.2) significantly decreased the liver TAG and PL than lower P+M/S ratio (0.7) (P 0.05). When SFA content was on the same level, as in Diets 4, 6, and 7 (with similar P+M/S ratios), it did not show any significant difference in liver cholesterol, TAG and PL. However when MUFA was on the same level (Diets 2 and 6), the higher PUFA content (Diet 6, 31.5%) resulted in greater accumulation of liver cholesterol and TAG than the lower PUFA group (Diet 2, 14.9%). When PUFA content was similar (Diets 2 and 4), the higher MUFA Diet 4 also resulted in greater accumulation of liver cholesterol (Fig. 1). 4. Discussion 4.1. The effects of different P/S ratio and P+M/S ratio on plasma total and lipoprotein lipid When the P/S ratio was fixed at 0.5, 1, 2, or 4, the plasma total cholesterol, LDL-C, and HDL-C did not show any significant difference in each pair of groups with different P+M/S ratios. But, when the P/S ratio was fixed at 1.0, a high MUFA content (P+M/S= 5.7) increased the plasma total TAG, VLDL-TAG, VLDL-C, and VLDL-PL. When the P/S ratio was fixed at 1.0, a high MUFA diet seems to increase the VLDL secretion in this experimental design which also occurs in human study [16]. We found that the diets highest in MUFA (Diet 4, 70.4%) had the greatest plasma TAG increment (Table 3) and similar results were also observed in most of the recent studies [7,27 29]. As shown in previous reports [7 9], plasma HDL-C did not differ significantly within each pair of test groups. Mattson and Grundy [27] have observed a greater decline in HDL-C in a PUFA group as compared with a MUFA group at 30% level of total energy. Thus, the decline in HDL appears to be a result of the removal of SFA from the diet and occurs regardless of which unsaturated fatty acid is included Saturated fatty acid and plasma lipid In order to eliminate the effects of different contents of PUFA, MUFA, and SFA, we fixed the amounts of Table 3 Triacylglycerol levels of various plasma fractions a Groups P/S P+M/S M/S P/M Total TAG VLDL-TAG (mg/dl) LDL-TAG (mg/dl) HDL-TAG (mg/dl) Diet Diet Diet b e Diet bg,h e,i,j Diet Diet c,g f,i Diet c,d,h f,j Diet d a Results are shown as Mean+S.D. TAG: Triacylglycerol. b Matched superscripts in the same column indicate that there is a significant difference between the mean values (P 0.05) by the Student s t-test. cj, same as b.

5 N. Wen Chang, P. Chao Huang / Atherosclerosis 142 (1999) Table 4 Phospholipid levels of various plasma fractions a Groups P/S P+M/S M/S P/M Total PL VLDL-PL (mg/dl) LDL-PL (mg/dl) HDL-PL (mg/dl) Diet b c Diet b c Diet d Diet d,g Diet Diet e Diet e,f,g Diet f a Results are shown as Mean S.D. PL: Phospholipid. b Matched superscripts in the same column indicate that there is a significant difference between the mean values (P 0.05) by the Student s t-test. cg, same as b. PUFA, MUFA, and SFA, respectively. When SFA was fixed at a certain level, the P/S ratio was increased from 1, 2 to 4 (70.4, 52.7 and 23.2% of MUFA, respectively), the plasma VLDL-C, VLDL-TG, and VLDL-PL decreased progressively. Howard et al. also found a substitution of PUFA for MUFA from 11.9 to 48.1% of the total fatty acids which resulted in a progressive decline in total cholesterol and less TAG elevations, without any effect on the HDL-C in human study [30] Monounsaturated fatty acid and plasma lipid When MUFA was fixed at a certain level (52% of the total fatty acids), there was no significant difference in plasma total and lipoprotein lipid between the P/S ratio of 0.5 and 2.0 groups. Similar results were also found in our previous study, in which the MUFA content was fixed at 41% of the total fatty acids and the P/S ratio was 0.2 or 4.1 respectively (unpublished observations) Polyunsaturated fatty acid and plasma lipid In this study we found that when PUFA was fixed at a certain level (14.9% of the total fatty acids), the plasma total and lipoprotein lipid did not show any significant difference with different P+ M/S ratio of 2.2 (Diet 2) or 5.7 (Diet 4) when cholesterol was not supplemented. However, with 1% cholesterol supplement, high MUFA diet (Diet 4, P+ M/S=5.7) increased the plasma total cholesterol, VLDL-C and liver cholesterol (data are not shown). These finding are not consistent with the observation by Brousseau et al. [31] in cynomolgus monkeys. Brousseau et al. reported that when PUFA was fixed at a certain level (19.8% of the total fatty acids), the high MUFA diet (MUFA 58.5%, SFA Fig. 1. Liver lipid contents of the rats. Diet 1: P/S=0.5, P+M/S= 0.7; Diet 2: P/S=0.5, P+M/S=2.2; Diet 3: P/S=1.0, P+M/S= 1.4; Diet 4: P/S=1.0, P+M/S=5.7; Diet 5: P/S=2.0, P+M/S=2.8; Diet 6: P/S=2.0, P+M/S=5.3; Diet 7: P/S=4.0, P+M/S=5.6; Diet 8: P/S=3.9, P+M/S=7.4. Each bar represents the mean+s.d. (ah): Matched superscripts indicate that there is a significant difference between the mean values (P 0.05) by the Student s t-test.

6 190 N. Wen Chang, P. Chao Huang / Atherosclerosis 142 (1999) %; P+M/S=3.6) resulted in a significantly lower plasma total cholesterol and HDL-C levels than the high SFA diet (MUFA 19.2%, SFA 60.0%; P+M/S=0.6). But the low content of SFA rather than the high content of MUFA in the high MUFA diet might be responsible for decreasing plasma cholesterol level Li er lipid High MUFA diets in each pair of groups resulted in greater accumulation of liver cholesterol. Similar results were also found in rats [17 19], rabbits[18], and hamsters [20]. When SFA was fixed at a certain level (Diets 4, 6 and 7), high MUFA content (Diet 4) increased the VLDL secretion, but did not change the liver lipid. When both MUFA (Diets 2 and 6) and PUFA (Diets 2 and 4) were fixed at a certain level, high P+M/S ratio (Diet 6 or Diet 4) also resulted in greater accumulation of liver cholesterol. It is worthwhile to notice that when the amount of MUFA was fixed at a certain level (53%), the high PUFA diet (Diet 6, P+M/S=5.3, P/M=0.6, M/S=3.3) resulted in the same plasma cholesterol level but higher liver cholesterol and TAG levels than did the high SFA diet (Diet 2, P+M/S=2.2, P/M=0.3, M/S=1.7). The reason that Diet 6 resulted in increased liver cholesterol might be due to its high M/S ratio and P+M/S ratio unlike Diet 2. Similar results were also observed in the high PUFA diets (Diet 6: P+M/S=5.3, P/M=0.6, M/S=3.3; Diet 7: P+M/S=5.6, P/M=2.7, M/S=1.5; Diet 8: P +M/S=7.4, P/M=1.1, M/S=3.5) resulted in increased liver cholesterol level than did the high SFA diets (Diet 1: P+M/S=0.7, P/M =2.0, M/S=0.2; Diet 2: P+M/S=2.2, P/M=0.3, M/S=1.7; Diet 3: P +M/ S=1.4, P/M=2.4, M/S=0.4). The reason might be due to the P+M/S ratio in Diet 6, 7 or 8 were somewhat higher than those in Diet 1, 2 or 3. It seems that the prerequisites for keeping low plasma and liver cholesterol are (a) low M/S ratio and (b) high P/M ratio and (c) P+M/S ratio not to exceed 3. In summary, the results of the present study show that if the P+M/S ratio was below 3, the change in the P/S ratio (0.5, 1 or 2) did not affect the levels of plasma total and lipoprotein cholesterol and TAG. An increase in the plasma VLDL-C and VLDL-TAG was related to increased MUFA content in the diet. High dietary MUFA content also resulted in greater accumulation of liver cholesterol. In the present study we cannot predict a suitable P+M/S ratio or P/S ratio in the food supply, however, the P+M/S ratio of dietary fats under 3 may be considered. Acknowledgements This work was supported by a grant (NSC B ) from the National Science Council, Taiwan, ROC. References [1] Keys A. Coronary heart disease in seven countries. Circulation 1970;41(Suppl 1):1211. [2] Kannel WB, Castelli WP, Cordon T. 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