Differential changes in the fatty acid composition of the main lipid classes of chick plasma induced by dietary coconut oil

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1 Comparative Biochemistry and Physiology Part B 133 (2002) Differential changes in the fatty acid composition of the main lipid classes of chick plasma induced by dietary coconut oil E. Garcıa-Fuentes, A. Gil-Villarino, M.F. Zafra, E. Garcıa-Peregrın* Department of Biochemistry and Molecular Biology, Faculty of Sciences, University of Granada, Granada, Spain Received 23 May 2002; received in revised form 20 August 2002; accepted 21 August 2002 Abstract For a better understanding of the hyperlipidemic function of saturated fat, we have studied the comparative effects of diet supplementation with 10 and 20% coconut oil on the main lipid classes of chick plasma. Changes in fatty acid composition of free fatty acid and triglyceride fractions were parallel to that of the experimental diet. Thus, the increase in the percentages of 12:0 and 14:0 acids may contribute to the hypercholesterolemic effects of coconut oil feeding. Plasma phospholipids incorporated low levels of 12:0 and 14:0 acids whereas 18:0, the main saturated fatty acid of this fraction, also increased after coconut oil feeding. The percentage of 20:4 n-6 was higher in plasma phospholipids than in the other fractions and was significantly decreased by our dietary manipulations. Likewise, minor increases were found in the percentages of 12:0 and 14:0 acids in plasma cholesterol esters. However, the percentage of 18:2 acid significantly increased after coconut oil feeding. Our results show a relationship between fatty acid composition of diets and those of plasma free fatty acid and triglyceride fractions, whereas phospholipids and cholesterol esters are less sensitive to dietary changes Elsevier Science Inc. All rights reserved. Keywords: Fatty acid composition; Free fatty acids; Triglycerides; Phospholipids; Cholesterol esters; Coconut oil; Gallus domesticus 1. Introduction It has been widely accepted that dietary manipulation, especially lipid modifications, can alter the lipid composition of different tissues and cells. Thus, fatty acid composition of dietary fat influences the membrane structural lipid composition and metabolic functions (Stubbs and Smith, 1984). Most research has focused on the effects of diet on plasma cholesterol levels, considered as the major cause of coronary heart disease. In this sense, the hypercholesterolemic effects of saturated *Corresponding author. Tel.: q ; fax: q address: garciap@ugr.es (E. Garcıa-Peregrın). fatty acids has been well established in humans and in several animal species (Grundy and Vega, 1988; Gil-Villarino et al., 1998). Monounsaturated fatty acids (MUFA) and polyunsaturated fatty acids (PUFA) of the n-3 and n-6 series reduce the plasma cholesterol concentration (Mensik and Katan, 1989; Castillo et al., 1999a). On the other hand, the association between lipid and protein in biological membranes modulates many membrane-associated enzymes by changes in the physical properties of the surrounding membrane lipids (Sanderman, 1978). The association between the different lipids is also involved in maintaining cell integrity and membrane fluidity (Yeagle, 1985). Thus, we have demonstrated that /02/$ - see front matter 2002 Elsevier Science Inc. All rights reserved. PII: S Ž

2 270 E. Garcıa-Fuentes et al. / Comparative Biochemistry and Physiology Part B 133 (2002) saturated fat induces rapid changes (24 h) in fatty acid composition of chick liver and hepatic mitochondria and microsomes (Gil-Villarino et al., 1997). Consequently, mitochondrial cytochrome oxidase and ATPase activities drastically changed after 24 h of dietary manipulation (Gil-Villarino et al., 1999) as did microsomal 3-hydroxy-3- methylglutaryl-coa reductase activity (Castillo et al., 1999b). The chick has been considered as a suitable model for studies on the comparative lipid metabolism because it is highly sensitive to dietary modifications (Chandler et al., 1979; Aguilera et al., 1984; Gonzalez-Pacanowska et al., 1986). Our previous results also show that young chicks respond to saturated fatty acids more sharply and rapidly than other animal species (Gil-Villarino et al., 1998). The rapid response of very low density lipoprotein to saturated fatty acids (Castillo et al., 1996) and PUFA (Castillo et al., 1999a,b, 2000) in the chick corroborates its suitability as a model for studying the development of atherogenesis. In addition, avian models have been used in both surgical and medical therapeutic trials of atherosclerosis. Thus, we have cultured smooth muscle cells from hypercholesterolemic chicks for molecular study of changes in these arterial cells induced by a cholesterol-enriched diet (Carazo et al., 1998). The intimal thickening in the aortas from these hypercholesterolemic chicks can be detected after only 20 days. This short period contrasts with data for other experimental animal models, such as non-human primates, which require 1 18 months of cholesterol diet to produce atherosclerotic lesions (Faggiotto et al., 1984). Therefore, we have studied the effects of diet supplementation with 10 20% coconut oil on the fatty acid composition of the main lipid classes in chick plasma: free fatty acids, triglycerides, phospholipids and cholesterol esters. 2. Materials and methods 2.1. Animals and feeding procedures White Leghorn male chicks (Gallus domesticus) from a commercial hatchery were maintained in a chamber at 28 8C under a light cycle from 09:00 to 21:00 h. A minimal of 6 chicks, housed together, were treated in each experiment. Control animals were fed ad libitum for the first 3 weeks of life on a cholesterol-free commercial diet (Sanders A- 00, Granada, Spain) containing (wyw) 42.0% carbohydrate (mainly starch), 6.6% vegetable fat, 20.6% vegetable protein (cereal seeds), 8.6% mineral mix, and 7.8% humidity, as well as supplements of Vitamin A ( IUykg), Vitamin D 3 (2000 IUykg), Vitamin E (10 mgykg), and cupric sulphate (10 mgykg). The energy content of this standard diet, normally used for growing chicks, was approximately 340 kcaly100 g. Experimental diets were prepared by supplementation of 10 20% commercial cooking coconut oil to the standard diet and were used from day 14 of chick life. These experimental diets were enriched in saturated fatty acids, especially lauric and myristic acids, which were nearly absent in the standard diet (Table 1). The energy content of these experimental diets was approximately 390 kcaly100 g and 450 kcaly100 g, respectively. Experimental treatment was prolonged for 1 week (from 14-day-old to 21-day-old) because the rapid hypercholesterolemic response (24 h) of young chicks to coconut oil feeding (Gil-Villarino et al., 1998). To ascertain whether the coconut oil contained toxic products as a result of its preparation systems, we analyzed the content in hexane, xylene, toluene, and other possible impurities. No toxic element reached 0.01 ppm, well below limits permitted for human consumption Plasma lipid analysis Chicks were decapitated 6 h after free access to water and food (postprandial conditions).the use of mixed blood did not influence our results. Blood was taken from each chick and maintained at 4 8C for 2 h. Plasma was separated by centrifugation at 2500 rpm for 20 min at 4 8C. Plasma lipids were extracted with chloroformymethanol 2:1 (vyv) as described by Folch et al. (1957). Different lipid classes were separated by thin layer chromatography according to the method of Skipski et al. (1968). Fatty acid methyl esters were prepared according to Lepage and Roy (1986) and analyzed by gas chromatography as previously described in detail (Gil-Villarino et al., 1997) Statistical analysis Results are expressed as mean values"s.e.m. of three experiments. Equal amounts of plasma from six chicks were pooled to one sample for each experiment. Each experimental value was

3 E. Garcıa-Fuentes et al. / Comparative Biochemistry and Physiology Part B 133 (2002) Table 1 Fatty acid composition of control and experimental diets (% of total fatty acids) Fatty acid Diet Control CO10 CO20 8:0 N.D. 1.71"0.04 a 2.18"0.05 a 10:0 N.D. 3.02"0.05 a 3.72"0.08 a 12:0 N.D 29.03"0.21 a 37.29"0.28 a 14:0 0.77" "0.18 a 15.11"0.22 a 16: " "0.11 a 13.02"0.12 a 18:0 8.56" "0.07 a 4.78"0.08 a S Saturated 31.69" "0.30 a 76.10"0.40 a 16:1 n " "0.02 a 0.82"0.01 a 18:1 n " "0.05 a 13.63"0.06 a S MUFA 35.70" "0.06 a 14.45"0.06 a 18:3 n " "0.01 a 0.21"0.01 a 22:5 n " "0.02 a 0.42"0.02 a S n " "0.02 a 0.63"0.02 a 18:2 n " "0.06 a 7.42"0.04 a 20:2 n " "0.01 a 0.63"0.01 a 20:3 n " "0.01 a 0.27"0.01 a 20:4 n " "0.02 a 0.39"0.01 a S n " "0.06 a 8.71"0.04 a S PUFA 32.33" "0.05 a 9.34"0.02 a S Unsaturated 68.03" "0.08 a 23.81"0.08 a SaturatedyUnsaturated 0.47" "0.02 a 3.20"0.02 a a a Results are expressed as means"s.e.m. of three determinations. Significantly different from the corresponding control value: P N.D., not detected. CO10, standard diet supplemented with 10% (wyw) coconut oil. CO20, standard diet supplemented with 20% (wyw) coconut oil. mean of duplicate determinations. Data were analyzed by two-way analysis of variance (ANOVA). When overall F-statistic was significant (P-0.05), analyses of significance were determined by the Student s t-test. 3. Results Despite the different fat contents of control and experimental diets, no interference in the growth rate of the chicks was found. No significant differences were observed in body and liver weight gains among groups fed different diets: 99.2% in control chicks, 95.7% in those fed a 10% coconut oil-enriched diet and 100.7% in those fed a 20% coconut oil-enriched diet for body weight gains and 40.8, 41.1 and 37.6%, respectively, for liver weight gains. No differences were observed in the amount of diet consumed by chicks fed different diets. Previous results showed that no influence derived from protein, vitamin or mineral deficiency has been observed after the addition up to 20% coconut oil to the diet (Gil-Villarino et al., 1997). No cellular death was observed in the histological examination of hepatic 1 mm cuts from chicks fed 20% commercial cooking coconut oil in the diet up to 14 days. Some glycogen and lipid droplets were observed in these hepatocytes (Gil-Villarino et al., 1997; Gil-Villarino, 1998). Table 2 shows that both dietary manipulations drastically increased the percentages of 12:0 and 14:0 acids in the fraction of free fatty acids of plasma with respect to the control values. These increases were more patent with 20% than with 10% coconut oil supplementation to the diet. Results in Table 2 also show that percentages of 18:1 n-9 and total MUFA in plasma free fatty acids from treated animals were significantly lower than in control. Similar results were found in 18:2 n-6, total n-6 fatty acids and total PUFA. Minor but significant decreases also resulted in the levels of other fatty acids as 16:1 n-7 and 20:4 n-6. The changes induced by coconut oil feeding in the fatty acid composition of plasma triglycerides (Table 2) were similar to those observed in the free fatty acid fraction. Both 12:0 and 14:0 acids increased in a similar proportion, on the contrary to that found in the free fatty acid fraction, in which the increase in the percentage of 12:0 was more pronounced than that of 14:0. Likewise, the

4 272 E. Garcıa-Fuentes et al. / Comparative Biochemistry and Physiology Part B 133 (2002) Table 2 Effects of 10% (CO10) or 20% (CO20) coconut oil supplementation to the diet on fatty acid composition of free fatty acid and triglyceride fractions of chick plasma Fatty acid Free fatty acid fraction Triglyceride fraction Control CO10 CO20 Control CO10 CO20 8:0 1.15" " "0.18 N.D. N.D. N.D. 10:0 0.30" "0.18 c 1.79"0.08 c,d 0.08" "0.05 c 0.68"0.12 c 12:0 1.52" "1.39 c 19.93"0.36 c,d 1.90" "0.57 c 12.39"0.53 c,e 14:0 2.12" "0.48 c 11.60"0.55 c,d 1.58" "0.36 c 10.16"0.24 c,e 16: " "1.30 a 21.72"0.77 c 33.63" "0.78 a 27.45"0.78 a 18: " " "0.61 a,d 18.13" " "0.87 S Sat " "0.90 c 65.64"1.66 c 55.40" "0.77 a 65.38"0.54 c,e 16:1 n " "0.59 a 4.11"0.29 a 3.60" " " :1 n " "0.98 c 19.33"0.72 a 25.54" " "1.43 S MUFA 31.58" "0.61 c 23.44"0.63 c 29.14" " " :2 n " "0.56 b 9.84"0.51 c,d 11.99" "0.29 b 4.51"0.63 c,f 20:2 n " " " " " " :4 n " "0.05 a 0.67"0.17 b,d 1.57" "0.10 a 0.33"0.10 a S n " "0.27 c 10.91"0.55 c,f 15.44" "0.18 b 7.14"0.32 c,f S PUFA 18.37" "0.27 c 10.91"0.55 c,f 15.44" "0.18 b 7.14"0.32 c,f S Unsat " "1.19 c 34.35"1.58 c 44.58" "1.20 a 34.61"1.60 a SatyUnsat. 1.00" "0.07 c 1.91"0.09 c 1.24" "0.06 a 1.89"0.09 c,d Results (%) are expressed as means"s.e.m. of 3 experiments carried out with pools of six animals. Each experimental value was a,b,c a b c the mean of duplicate determinations. Significantly different from the corresponding control value: P-0.05; P-0.005; P d,e,f d e f Significantly different from the corresponding CO10 value: P-0.05; P-0.005; P N.D., not detected. CO10, standard diet supplemented with 10% (wyw) coconut oil. CO20, standard diet supplemented with 20% (wyw) coconut oil. percentages of MUFA did not change significantly by coconut oil supplementation to the diet. Changes in the fatty acid composition of plasma phospholipids were lower than those found in the previously studied fractions (Table 3). It is important to note the low levels of 12:0 and 14:0 acids incorporated from coconut oil-enriched diet in plasma phospholipids, as well as the high content of 18:0 in this lipid fraction from control animals and the increase induced by dietary coconut oil. The percentage of arachidonic acid (20:4 n-6) in plasma phospholipids was higher than in the other fractions. This percentage was significantly decreased by coconut oil feeding, especially when the diet was supplemented with 20% fat. Table 3 also shows that n-3 PUFA were present in plasma phospholipid fraction although only in low percentages. Finally, we have studied the changes in fatty acid composition of plasma cholesterol esters induced by coconut oil feeding. Only minor variations were detected in the percentages of 12:0 and 14:0 as well as in those of other fatty acids (Table 3). However, linoleic acid (18:2 n-6), which was the most abundant fatty acid in this plasma fraction, significantly increased after coconut oil feeding. 4. Discussion The effects of dietary saturated fat raising plasma cholesterol in fasting conditions are well documented (Grundy and Vega, 1988). There is evidence that different saturated fatty acids can exert specific and in some cases opposite effects on plasma cholesterol level (Fernandez et al., 1992). Myristic (14:0) and lauric (12:0) acids, mainly present in coconut and palm kernel oil, appear to be the principal saturated fatty acids that raise plasma cholesterol (Hayes et al., 1991). Our results in young chick showed a response to coconut oil that is more rapid than that reported in humans and other animal species: a significant hypercholesterolemic effect was observed after 1 day of this dietary manipulation (Gil-Villarino et al., 1998). However, little is known about the effects of dietary saturated fat on postprandial lipid metabolism (Lay and Ney, 1995) and, more concretely, on fatty acid composition of the different plasma lipid classes. Free fatty acids are considered to be the most abundant lipids in young chick plasma (approx. 35%) under postprandial conditions (Gil-Villarino, 1998), followed by phospholipids (approx. 28%). Each cholesterol ester and triglyceride fractions

5 E. Garcıa-Fuentes et al. / Comparative Biochemistry and Physiology Part B 133 (2002) Table 3 Effects of 10% (CO10) or 20% (CO20) coconut oil supplementation to the diet on fatty acid composition of phospholipid and cholesterol ester fractions of chick plasma Fatty acid Phospholipid fraction Cholesterol ester fraction Control CO10 CO20 Control CO10 CO20 12:0 1.07" " " " "0.51 a 3.95"0.15 c 14:0 0.97" "0.05 a 1.97"0.08 a,f 0.82" "0.68 a 3.01"0.51 b 16: " " " " "0.36 c 12.39"0.32 c 18: " "0.27 a 31.00"0.64 b,a 8.56" "0.26 c 5.14"0.41 b S Sat " " "0.30 c,f 27.85" "0.29 a 24.49"0.30 b 16:1 n " " " " " "0.13 a 18:1 n " " " " "0.71 a 16.32"0.84 a 20:1 n " " " " " "0.10 S MUFA 13.97" " " " "0.32 c 20.07"0.35 c 18:2 n " " " " "0.86 b 51.01"0.72 b 20:2 n " " " " " " :4 n " "0.22 a 5.25"0.64 b,f 3.26" "0.11 a 2.49"0.17 a 22:5 n " "0.05 a 0.44"0.05 b,d N.D. N.D. N.D. S n " " "0.34 c,f 46.59" "0.37 c 53.93"0.30 c 18:3 n " "0.02 c 0.63"0.04 c,f 0.62" " " :6 n " " "0.15 b,a 1.09" " "0.12 S n " " "0.07 c,f 1.71" "0.11 a 1.02"0.11 a S PUFA 34.02" " "0.35 c,f 48.30" "0.38 c 54.95"0.30 c S Unsat " " "1.05 a,f 72.96" " "1.13 Sat.yUnsat. 1.08" " "0.04 b,f 0.38" "0.01 a 0.33"0.01 a Results (%) are expressed as means"s.e.m. of 3 experiments carried out with pools of six animals. Each experimental value was a,b,c a b c the mean of duplicate determinations. Significantly different from the corresponding control value: P-0.05; P-0.005; P d,e,f d e f Significantly different from the corresponding CO10 value: P-0.05; P-0.005; P N.D., not detected. CO10, standard diet supplemented with 10% (wyw) coconut oil. CO20, standard diet supplemented with 20% (wyw) coconut oil. represent approximately 11 12% of total lipids, whereas free cholesterol represents less than 10% (Garcıa-Fuentes et al., 2000, 2002). Our results showed that in the free fatty acid fraction the relative percentage gain of 12:0 was higher than that of 14:0, suggesting that these changes may be related to the fatty acid composition of the experimental diets. However, our previous results indicated that percentages of both saturated fatty acids in total plasma lipids were similar, despite that the percentage of 12:0 in both diets was approximately 2.5-fold that of 14:0. Thus, the response of plasma free fatty acids appears to parallel that of dietary fatty acids. Changes in the percentages of the other fatty acids may result from increased percentages of 12:0 and 14:0, although these alterations would be of special interest because functional and pathological consequences may be correlated (Mc- Murchie, 1988). However, the increases induced by 10 and 20% coconut oil in the diet regarding S-saturated and S-unsaturated fatty acids were similar, suggesting a maximal response to 10% saturated fat in this free fatty acid fraction. The decrease detected in the arachidonic acid levels in phospholipid fraction may be of interest for a lower production of its derived eicosanoids. Arachidonic acid was normally synthesized from 6 dietary linoleic acid (18:2 n-6) by D -desaturation, 5 elongation and D -desaturation reactions (Rosenthal, 1987) occurring in liver microsomes. Thus, the lower content of 18:2 n-6 acid in the coconut oil enriched diets with respect to the standard diet may explain the lower levels of 20:4 n-6 acid found in plasma phospholipids when chicks were fed the experimental diets. The n-3 PUFA are mainly metabolized in the hepatic cells by desaturation and elongation of essential fatty acids. It is accepted that n-3 PUFA levels in plasma phospholipids may be related to those in diets (Bjerve et al., 1993), so that the decreases found after coconut oil feeding may be accounted for the low percentages of these fatty acids in our experimental diets. In any case, changes in the phospholipid fraction may be of considerable significance, given that these components largely determine the chemical and phys-

6 274 E. Garcıa-Fuentes et al. / Comparative Biochemistry and Physiology Part B 133 (2002) ical properties of biological membranes andyor membrane enzyme proteins. Finally, fatty acid composition of plasma cholesterol esters can be related to the lecithin: cholesterol acyltransferase activity (Glomset, 1978). Our results agree with the greater chick plasma esterified cholesterol previously noted when animals were fed a 10% (Castillo et al., 2000) or 20% (Gil-Villarino et al., 1998) coconut oil enriched diet. In summary, our results showed a relationship between fatty acid composition of saturated fatenriched diet and those of plasma free fatty acid and triglyceride fractions, corroborating the suitability of our animal model for studies in human nutrition and health, especially for the development of experimental hypercholesterolemic conditions under to study the hypolipidemic effects of different diets (Castillo et al., 2000) and drugs (Garcıa- Fuentes et al., 2002). 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