Early development of essential fatty acid deficiency in rats: Fat-free vs. hydrogenated coconut oil diet

Pei-Ra Linga, Charlotte E. De Leona, Hau Leb, Mark Puderb, and Bruce R. Bistriana,*

aThe Laboratory of Nutrition/Infection, Beth Israel Deaconess Medical Center, Room 605, BakerBuilding, One Deaconess Road, 330 Brookline Ave., Boston, MA 02215, USAbThe Department of Surgery and The Vascular Biology Program, Children’s Hospital Boston,Harvard Medical School, Boston, MA, USA

AbstractThis study examined the effects of feeding an essential fatty acid deficient (EFAD) diet eitherwithout fat or with added hydrogenated coconut oil (HCO) on fatty acid profiles in rats. Both dietsinduced equivalent biochemical evidence of EFAD reflected by the triene/tetraene ratio in plasmaphospholipids within 2 weeks. However, the HCO diet led to larger increases of 16:1n7 and18:1n9 in muscle but smaller increases in fat tissue and plasma triglycerides than the fat-free diet,suggesting greater increases in hepatic de novo lipogenesis with the latter. In addition, the HCOdiet led to larger decreases of some 18:3n3 metabolites, particularly 22:6n3, in muscle, fat andbrain tissues than the fat-free diet, presumably related to lesser stimulation of elongation anddesaturation. Thus, these secondary effects of an EFAD diet on fatty acid metabolism can bemodified by the saturated fat in the diet while the primary impact of both diets on development ofEFAD is unaffected.

KeywordsEssential fatty acid deficiency; Fat-free diet; Hydrogenated coconut oil diet

1. IntroductionIn mammals, the nutritionally essential fatty acids (EFAs), linoleic acid (LA, 18:2n6) and α-linolenic acid (LNA, 18:3n3), must be present in the diet, since neither can be synthesizedde novo. Reduced consumption of both, but particularly LA, leads to marked biochemicaland functional consequences [1]. In man the development of EFA deficiency has beenobserved in many disease conditions when there is substantial intestinal malabsorption [2],or when induced by prolonged intravenous feeding without EFAs [3–5].

In order to better understand the nutritional needs for EFAs and their impact on lipidmetabolism as well as the development of deficiency disease, numerous studies have beenconducted in experimental animal models of EFA deficiency. Two diets, a fat-free (No fat)diet or a purified diet containing hydrogenated coconut oil (HCO), are commonly used toinduce EFA deficiency. It is well established that long-term feeding (8–14 weeks) of EFAD

© 2010 Elsevier Ltd. All rights reserved.*Corresponding author. Tel.: +1 617 632 8545; fax: +1 617 632 0204. bbistria@bidmc.harvard.edu (B.R. Bistrian).Conflict of interest statementThere are none.

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Published in final edited form as:Prostaglandins Leukot Essent Fatty Acids. 2010 ; 83(4-6): 229–237. doi:10.1016/j.plefa.2010.07.004.

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diets to rats leads to EFA deficiency including slower growth, and an increase in the n9eicosatrienoic acid (Mead acid, 20:3n9) associated with decreases in both 18:2n6 andarachidonic acid (20:4n6) in plasma phospholipids [6]. A triene–tetraene ratio(20:3n9/20:4n6) in plasma phospholipids >0.2 is considered pathognomonic for thediagnosis of EFA deficiency [6], although clinical evidence for EFA deficiency is notgenerally seen until the ratio is >0.4 [7–9]. Phospholipids are the major structural lipids ofmembranes, and changes in membrane phospholipid fatty acid composition stronglyinfluence membrane functions, such as fluidity, permeability, and anchoring of membraneproteins and associated enzyme systems [10]. The pathological severity of EFA deficiencyhas been shown to be influenced by the age at the time of diet initiation and the duration offeeding with the fat-free or HCO diets.

Evidence suggests there is paradoxical conservation of cardiac and renal arachidonatephospholipid content in EFA deficient rats after 8 weeks of feeding with EFA-deficient diet[11]. However, relatively little is known about the early effects of short-term feeding withEFAD diets on fatty acid profiles in phospholipids in various target tissues. It is also notknown whether the plasma phospholipids changes occur in various tissues at the same rateand to the same degree during a short-term feeding with EFAD diets. In the present study,we compared, in weanling rats, the effects of feeding two EFAD diets, one fat-free and theother containing HCO, to an adequate EFA diet containing soybean oil on phospholipidprofiles in plasma, liver, muscle, fat and brain tissues at 2, 3 and 4 weeks to see theprogressive changes in fatty acids in different organs and tissues. The total triglyceridecontents in plasma and liver were also compared.

2. Materials and methods2.1. Animals

Weaning male Sprague-Dawley rats (45–60 g) were obtained from Taconic Farm(Germantown, NY) and placed in individual cages on a 12:12-h light–dark photoperiod at24–26 °C for 4 days before the experiments. Tap water and standard laboratory rat chow(Harlan Teklad, Madison, WI, USA) were provided ad libitum. All experimental protocolswere approved by the Institutional Animal Care and Use Committee of Beth IsraelDeaconess Medical Center.

At the end of 4 days of accommodation in the animal facility, a total of 45 animals werefasted overnight. The next morning, animals then were randomly assigned to three groups(15 rats/group) and fed ad libitum for 2–4 weeks, either a AIN 93M diet with 4% soybeanoil by weight (AIN group), or this diet without fat (No-fat group), or this diet with 4%hydrogenated coconut oil in place of soybean oil (HCO group). All the diets consisted of, byweight, 14% casein, 3.5% mineral mix and 1.0% vitamin mix and 0.25% choline bitartrate(Dyets, Inc., Bethlehem, PA, USA). The No-fat diet had a higher amount of sucrose thanothers for identical caloric density. Tables 1 and 2 list the dietary compositions and fattyacid compositions of all three diets.

During feeding, body weight and food intake were recorded. After 2, 3 or 4 weeks offeeding, five rats from each group were sacrificed, respectively. The experiment was carriedout in the fed state which is a more physiological condition limiting the influence ofendogenous lipid release from adipose tissue stores on fatty acid profiles. Blood and piecesof liver, muscle, brain and fat tissues were collected for fatty acid analysis.

2.2. Analysis of lipidThe lipid from samples was extracted with 6 volumes of chloroform–methanol 2:1 by themethod of Folch et al. [12]. Before the extraction, 30 μl of a 1 mg/ml solution of

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diheptadecanoyl phosphatidylcholine and 30 μl of 1 mg/ml solution of triheptadecanoylglycerol (Nu-Check Prep, Inc., Elysian, MN, USA) in chloroform–methanol (1:1, v/v) wereadded as an internal standard to all samples. Triglyceride and phospholipid fractions wereisolated by aminopropyl columns (Sigma, St. Louis, MO, USA) using chloroform/isopropanol (2:1, v/v) and methanol, respectively. The methyl esters were prepared usingsodium-methoxide and methanol base-boron trifluoride and washed with a saturated NaCIsolution. Fatty acid composition was determined by gas chromatography with a HewlettPackard 5890 II (Hewlett Packard, Palo Alto, CA, USA), using a supelcowax-10, 0.25 mmID column at a temperature of 260 °C. Fatty acid methyl ester peaks were identified bycomparison of retention times of a standard mixture and quantified using the internalstandard.

Plasma triglyceride was determined using a triglyceride determination kit (Sigma, St. Louis,MO, USA). Liver triglyceride content was calculated from the triglyceride profiles in livertissue determined by gas chromatography and presented as μmol/mg.

2.3. StatisticsResults are presented as mean±SD. To assess the statistical significance of differences inmean values among different diets and different feeding times, two-way analysis of variance(ANOVA) with Fisher least significant test was used. Significance for all analyses wasdefined as p≤0.05.

3. Results3.1. Food intakes and the changes in body and organ weight

Over the 4 weeks of feeding, food intakes were not different among the AIN, no-fat andHCO groups. Table 3 lists the average weekly food intake in different groups. Animals in allthe groups gained weight (p<0.001); the average body weight rose from 125±25 g (n=15) to341±23 g (n=5) in the AIN group, 115±9 g (n=15) to 323±14 g (n=5) in the No-fat groupand 117±10 g (n=15) to 329±18 g (n=5) in the HCO group. No significant differences inweight gain were found among the three groups. The livers were smaller but notsignificantly so in both the No-fat and the HCO groups compared to the AIN group. Therewere also no differences in brain weight among the three groups (data not shown).

3.2. Triene–tetraene ratio in plasma phospholipidsThe triene–tetraene ratio in plasma phospholipids is presented in Table 4. Rats fed the AINdiet for 2 weeks had a plasma phospholipid triene–tetraene ratio of 0.01 and this ratio wasmaintained for the entire experimental period. However, in rats fed the No-fat or HCO dietfor 2 weeks, this ratio increased to 0.36 which was significantly higher than for the AINgroup. From week 2 to week 4, this ratio further increased to 0.44 and 0.59 for the No-fatand HCO groups, respectively. There was no significant difference between the No-fat andHCO groups.

3.3. Fatty acid profiles in phospholipids (Figs. 1–7)After 2 weeks of feeding, the concentrations of linoleic acid (18:2n6) were significantlydecreased in plasma, liver, muscle and fat (p<0.001), but not in the brain in the No-fat andHCO groups as compared to AIN group, and was maintained at these low levels over thenext 2 weeks (Fig. 1a). In muscle, the concentration of 18:2n6 was further lowered in theHCO group after 4 weeks of feeding compared to the No-fat group (p<0.05). Theconcentrations of α-linolenic acid (18:3n3) (Fig. 1b) were also significantly decreased inplasma, liver, muscle and maintained at lower levels in the No-fat and HCO groupscompared to the AIN group. Although lower levels of 18:3n3 in the fat tissue were found in

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the NO-fat and HCO groups, no significant differences in this fatty acid were found amongthree groups due to the substantial variability. No differences of 18:3w3 in the brain werefound among the three groups.

The concentrations of palmitic acid (16:0) were significantly decreased in plasma (p<0.001)and liver (p<0.001) and maintained at the lower levels over the entire feeding period in theNo-fat and HCO groups compared to the AIN group (Fig. 2a). In muscle, after 2 weeks offeeding, this fatty acid was significantly lower in HCO group (p<0.05) but not in the No-fatgroup compared to the AIN group. At the end of 4 weeks of feeding, the concentrations ofthis fatty acid in the muscle in both the No-fat and HCO groups were significantly lowerthan those in the AIN group (p<0.02), although in the HCO group this fatty acid wasincreased to the level seen in the No-fat group after 3 weeks of feeding, In the fat and braintissues, no significant differences in 16:0 were found among the three groups.

Unlike the changes of 16:0, the concentrations of palmitoleic acid (16:1n7) weresignificantly increased and maintained at higher levels in plasma (p<0.001), liver (p<0.002),muscle (p<0.001) and fat tissues (p<0.01) in both the No-fat and HCO groups compared tothe AIN group (Fig. 2b). In addition, the concentration of 16:1n7 was significantly higher inthe HCO group as compared to the No-fat group in muscle (p<0.001) over the entire feedingperiod. No difference in 16:1n7 was found in the brain tissue among groups.

The concentrations of stearic acid (18:0) (Fig. 3a) were significantly increased in plasma(p<0.001) and liver (p<0.001) in the No-fat and HCO groups compared to the AIN groupafter 2 weeks of feeding and were maintained at higher levels over the entire experiment. Inmuscle tissue, the concentration of 18:0 was lower in the No-fat and HCO groups comparedwith the AIN group after 3 weeks of feeding (p<0.05), and lower in the HCO group than inthe No-fat and AIN groups after 4 weeks of feeding (p<0.05). In fat tissue, 2 weeks offeeding did not result in differences in 18:0 among the three groups. However, theconcentrations of 18:0 in fat tissue were significantly lower in the No-fat groups over thefeeding period compared to the AIN and HCO groups (p<0.005). No differences of this fattyacid were found between HCO and AIN groups in fat tissue. In the brain, no differenceswere found among the three groups over the feeding period.

The concentrations of oleic acid (18:1n9) in plasma, liver, muscle and fat were significantlyhigher (p<0.001) after 2 weeks of feeding and were maintained at high levels over the entirefeeding time in the No-fat and HCO groups compared to the AIN group (Fig. 3b). In muscle,the concentration of 18:1n9 was significantly increased in both the No-fat and HCO groupsover the feeding period, and more so in the HCO group compared to the No-fat group(p<0.005). In fat tissue, 18:1n9 was also significantly increased over time in both the No-fatand HCO groups compared to the AIN group (p<0.001), and was the highest in the No-fatgroup as compared to the HCO and AIN groups (p<0.005). In the brain, no differences werefound among the three groups.

The concentrations of Mead acid (20:3n9) (Fig. 4) were elevated in plasma, liver, muscle,fat and brain (p<0.001) after 2 weeks of feeding in both the No-fat and HCO groupscompared to the AIN group. No differences in 20:3n9 were found in liver, muscle and fattissues between the No-fat and the HCO group at any time point. In plasma, 20:3n9 wassignificantly increased in the No-fat and HCO groups over time (p<0.001), and more so inthe HCO group (p<0.001) compared to the No-fat group after 4 weeks of feeding. Incontrast, less 20:3n9 in the brain was found in the HCO group compared to the No-fat group(p<0.001).

Fig. 5 shows the changes in arachidonic acid (20:4n6) (Fig. 5a) and eicosapentaenoic acid(20:5n3) (Fig. 5b), the direct-downstream products from 18:2n6 and 18:3n3, respectively, in

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plasma and different tissues. The concentration of 20:4n6 in plasma, liver and muscle weresignificantly decreased in the No-fat and HCO groups as compared to the AIN group after 2weeks of feeding and was maintained at the lower levels over the feeding period (p<0.001).No differences of this fatty acid were found in plasma, liver and muscle between the No-fatand HCO groups. In fat tissue, 20:4n6 was not significantly different among the three groupsafter 2 weeks of feeding, but was slightly increased in the No-fat group after 3 weeks offeeding. After 4 weeks of feeding, the levels of 20:4n6 in fat tissue were increased in boththe HCO group and the No-fat group compared to the AIN group (p<0.002). In the brain, theconcentrations of 20:4n6 were not different among the three groups.

The concentrations of 20:5n3 (Fig. 5b) in plasma and liver were significantly lower in boththe No-fat and HCO groups compared to the AIN group at 2 weeks, with a further decreasesover the feeding period in the HCO group (p<0.05). In muscle tissue, the concentrations of20:5n3 were slightly but not significantly different among groups after 2 weeks of feeding.Over the feeding period, the 20:5n3 declined in all groups (p<0.05) with greater decreases inthe HCO group. As a result, 20:5n3 in muscle was significantly lower in the HCO comparedto the No-fat and AIN groups (p<0.01). In the fat and brain tissues, the changes in 20:5n3were not significantly different among groups.

The changes in 22:4n6, 22:5n6, 22:5n3 and docosahexaenoic acid (22:6n3), the furtherdownstream derivatives from 18:2n6 and 18:3n3, are shown in Figs. 6 and 7. In the w6pathway, the concentrations of 22:4n6 in plasma and brain were maintained over the feedingperiod, but was significantly lower only in the HCO group compared to the No-fat and AINgroups (p<0.05) at 4 weeks (Fig. 6a). At 2 weeks, 22:4n6 was significantly lower in liverand muscle tissues in the No-fat and HCO groups. The low levels of this fatty acid weremaintained over the feeding period. Only small amounts of 22:4n6 were found in fat tissueand there were no differences among the three groups.

The concentrations of 22:5n6 (Fig. 6b), a product derived from 22:4n6 by elongation anddesaturation, were significantly higher in plasma and liver in the No-fat and HCO groups(p<0.001) compared to the AIN group over the feeding period (Fig. 6). No differences werenoted in muscle. In fat tissue, the increased 22:5n6 was only found in the No-fat group after3 weeks of feeding as compared to the HCO and AIN groups. In the brain tissue, levels of22:5n6 were higher in both the No-fat and HCO groups compared to the AIN group but onlyreached significance after 4 weeks.

In the n3 pathway (Fig. 7), the concentrations of 22:5n3 (Fig. 7a) were significantly lower inplasma, liver, muscle, fat and brain in the No-fat and HCO groups compared to the AINgroup after 2 weeks of feeding and were maintained at these lower levels at all time points.In muscle, feeding with HCO diet further lowered 22:5n3 as compared to the No-fat group(p<0.05). In fat tissue, the No-fat group had further lowering of 22:5n3 at 4 weeks ascompared to the HCO group. The changes in 22:6n3, were different among groups over time(Fig. 7b). In plasma, lower levels were found only in the HCO group after 4 weeks offeeding as compared to the AIN group (p<0.05). In the liver, the lowest levels of 22:6n3 wasfound in the HCO group after 4 weeks of feeding compared to the AIN group (p<0.05),although this fatty acid was significantly decreased over time in all groups (p<0.001). Inmuscle, the lowest level of 22:6n3 were also in the HCO group which was significantlylower than those in the No-fat group (p<0.05) and the AIN group (p<0.001). In fat and braintissue, the concentrations of 22:6n3 was significantly lower in the HCO group at all timepoints compared to the AIN and No-fat groups (p<0.005).

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3.4. Triglyceride profiles and contents in plasma and liverThe triglyceride profiles were measured only in plasma and liver. As expected, both EFADdiets significantly reduced 18:2n6, 18:3n3 and 20:4n6 but significantly increased 20:3n9fatty acid in plasma and liver triglycerides after 2 weeks of feeding and maintained at thelower levels. The downstream products of 18:2n6 and18:3n3, including 20:5n3, 22:4n6,22:5w3 and 22:6n3, were also significantly lower in the No-fat and HCO groups comparedto the AIN group at 2 weeks of feeding and maintained at the lower levels over experimentalperiod. There were no differences in these fatty acids between the No-fat and HCO groups(data not shown). The changes of 16:0, 16:1n7, 18:0 and 18:1n9 in plasma and livertriglycerides are shown in Fig. 8. In plasma, the concentrations of 16:0, 16:1n7 and 18:1n9were significantly higher after 2 weeks of feeding and were maintained at the higher levelsover the next week in the No-fat and HCO groups compared to the AIN group. From weeks3–4, all these three fatty acids in the No-fat group were significantly higher compared to theAIN and HCO groups but there were no differences in these three fatty acids between theAIN and HCO groups. For 18:0, there were differences among the groups after 2 weeks offeeding. Over the feeding period, no differences were found between the No-fat and AINgroups. However, HCO feeding significantly increased 18:0 in plasma triglycerides from 2to 4 weeks of feeding.

In the liver, both the No-fat and HCO diets had significantly increased 16:0 after 2 weeks offeeding and were maintained at higher levels over the next 2 weeks compared to the AINdiet. There were no significant differences in 16:1n7 and 18:0 among the three groups. For18:1n9 in liver triglycerides, the No-fat diet significantly increased this fatty acid after 2weeks of feeding and the higher levels of 18:1n9 were maintained over feeding periodcompared to the AIN diet. However, the HCO diet significantly increased 18:1n9 in the liverafter 3 weeks of feeding and the higher levels of 18:1n9 were maintained over feedingperiod compared to the AIN diet.

The total content of triglycerides in plasma and liver are shown in Fig. 9. There were nodifferences in plasma triglycerides among the groups after 2 weeks of feeding with thedifferent diets. However, plasma triglycerides were significantly decreased in the No-fatgroup after 3 weeks of feeding and were maintained lower as compared to the AIN group,while no changes were seen in the HCO group over the feeding period. In the liver,triglycerides were significantly increased in the NO-fat and HCO groups compared to theAIN group after 2 and 3 weeks of feeding. No differences in liver triglyceride were observedamong groups at 4 weeks.

4. DiscussionThis study characterized the early development of EFA deficiency in weanling rats fedEFAD diets for up to 4 weeks. At a constant and equal energy intake for 2 weeks, providinganimals with the No-fat or HCO diets, devoid of 18:2n6 and 18:3n3, significantly increasedthe 20:3n9 (Mead acid) in plasma associated with reductions of 18:2n6, 18:3n3 and 20:4n6compared to the AIN diet which contains ample amounts of 18:2n6 and 18:3n3. As aconsequence, the ratio of Mead acid/20:4n6 in plasma phospholipids rose above 0.2, whichwas significantly increased from 0.01 in the AIN group to 0.36 in both No-fat and HCOgroups, providing definitive biochemical evidence of EFA deficiency. The increases in thisratio were mainly due to 51% increases in 20:3n9 with only 18% decreases in 20:4n6 inplasma phospholipids. After an additional 2 weeks of feeding these EFAD diets, the ratio ofMead acid/20:4n6 in plasma phospholipids further increased to 0.44 in the No-fat and to0.59 in the HCO group, mainly attributable to the significant increases of 20:3n9 in bothgroups over time, reflecting enhancement of elongation and desaturation in the omega 9pathway that would be increasingly limited by substrate availability in the omega 3 and 6

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pathways. In addition, the concentration of 20:3n9 was markedly increased by 63.5% in theliver, 82.1% in the muscle, 93.7% in the fat and 96% in the brain but remained about 4% oftotal phospholipids in the liver, 2% in the muscle and less than 1% in the fat and brain fromweek 2 to week 4. Over the feeding period, however, the concentration of 20:3n9 in plasmawas increased at least 100-fold with both EFAD diets compared to the AIN diet.

Interestingly, moreover, the increases of 20:3n9 and the decreases of 18:2n6, 18:3n3 and20:4n6 in plasma and all the measured tissues were almost identical between the No-fat andHCO groups indicating these changes to principally reflect EFA deficiency.

It has been proposed that the addition of saturated fat, especially hydrogenated coconut oil,to an EFAD diet accentuates the development of EFA deficiency in comparison with a fat-free diet [13,14]. The discrepancy between the results of our study and others may be relatedto the relative contents of HCO in the experimental diets used. The previous conclusion wasbased on experiments which were done primarily to obtain information on the relationshipbetween the onset of external symptoms of EFA deficiency and tissue levels of EFAs in ratfed EFAD diets containing 25–30% HCO or in those fed a fat-free diet [15]. In the presentstudy, the HCO diet contained only 4% HCO. Thus, the present results would suggest thatfeeding with a 4% HCO diet without EFA or a fat-free diet (No-fat diet) for 4 weeks equallyincreased the concentrations of 20:3n9 and decreased 20:4n6 and EFAs in most organ andtissues, but did not retard body growth or cause other clinical symptoms of EFA deficiencyin rats. Beyond this feeding period, clinical signs presumably would have been observedbecause a value of triene/tetraene ratio of 0.4 is generally the level at which clinicalevidence of EFA deficiency is detected [7–9] The two principal factors in the production of20:3n9 are the degree of enhancement of elongation and desaturation in the n9 pathway as aconsequence of EFA deficiency and the availability of 18:1n9 as substrate which wouldresult from de novo lipogenesis and from the diet providing 16:0 and 18:0.

With progressive development of EFAD, there were substantial increases in triglycerides of16:0 in plasma and liver in both EFAD groups (Fig. 8). At various times there were alsoincreases in 16:1n7, 18:0, and 18:1n9 in plasma and 18:1n9 in liver triglycerides withEFAD. In addition, EFAD also significantly increased the concentrations of 16:1n7 (Fig. 2b)and 18:0 phospholipids in plasma and liver (Fig. 3a), and 18:1n9 phospholipids in plasma,liver and muscle (Fig. 3b). These four fatty acids can be endogenously synthesized and two,16:0 and 18:0, are found in the HCO diet. In this study, the higher 16:1n7 and 18:1n9 weremost likely a consequence of endogenous synthesis of 16:0 and 18:0 and increased Δ9desaturation, both reflecting de novo lipogenesis, because there were only small amounts(0.09% and 1.14%, respectively) of these two latter fatty acids in the HCO diet and none inthe No-fat diet. The increased 16:1n7 and 18:1n9 reflect increases in stearyl coenzyme Areductase, or Δ9 desaturase activity [16]. Feeding small amounts of PUFA for 3 or 4 dayscan significantly reduce the activities of these enzymes in EFAD rats fed a fat-free diet[17,18]. Thus, the present results suggest that adding modest amounts of saturated fattyacids (4% by weight) into an EFAD diet which is fat-free has substantially less or limitedeffect on inhibition of lipogenic enzyme activities. It should also be noted that feeding withthe HCO diet significantly increased 16:1n7 and 18:1n9 phospholipids in muscle tissuescompared to the No-fat diet, implying that dietary saturated fat might have a local effect tostimulate de novo lipogenesis and/or lead to the accumulation of these fatty acids withinmuscle tissue. Dietary saturated fatty acids have also been shown to play a major role in thedevelopment of insulin resistance, which has a major impact on the fatty acid metabolism inmuscle tissue in diabetes and obesity [19]. The determination of lipogenic enzyme proteincontent and activity in muscle tissue would help to clarify this point.

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In the 18:2n6 and 18:3n3 pathways, their respective distal metabolites in plasma, liver,muscle, fat and brain were substantially altered given the dietary absence of precursor. In thew6 pathway, the concentration of 20:4n6 declined 20% in plasma, liver and muscle tissues,and 22:4n6, its elongation product, declined in plasma (15%), liver (25%) and muscle (35%)in rats fed the No-fat or HCO diet as compared to the AIN diet. These reductions would be aconsequence of limited substrates while Δ6 and Δ5 desaturation would likely be acceleratedin an attempt to maintain 20:4n6 levels as near normal as possible for as long as possible.No changes in these fatty acids were noted in fat and brain tissues among AIN, No-fat andHCO groups presumably reflecting their slower turnover [20]. The concentration of 22:5n6,formed by elongation, desaturation, and beta oxidation from 22:4n6, was significantlyincreased in plasma (2.9 times), liver (2.8 times), fat (1.2 times) and brain (1.5 times) in theNo-fat and HCO group compared to the AIN group, presumably reflecting increased enzymeactivity at each point in this pathway. In the w3 pathway, 20:5n3 in plasma, liver and muscledeclined more in the HCO group compared to the No-fat group. Moreover, 22:5n3 in muscleand 22:6n3 in liver, muscle, fat and brain tissues also declined more in the HCO groupcompared to the No-fat group. These changes suggest that the presence of fat as HCO in theEFA deficient diet reduces desaturase activity throughout w3 pathway when compared to adiet without fat. As a consequence in these two models of EFA deficiency, although 20:4n6in the brain was equally maintained, 22:6n3 levels were significantly lower with the HCOdiet than with the No-fat diet.

It is appreciated that a fat-free diet as well as an EFAD diet enhance Δ6 and Δ5 desaturaseactivity and saturated fats inhibit Δ6 and Δ5 desaturases in general, which would explain thelower levels of the important w3 metabolites, 20:5n3 and 22:6n3 with the HCO diet.However, this does not explain the differential effects of HCO on n3 and w6 PUFAs. Thereasons are not clear but may be related to the primacy of the n6 pathway in EFAmetabolism making the defense of adequate 20:4 n6 levels as a higher priority than relativedesaturase enzyme activity in the w6 compared to the n3 pathway. On the other hand dietary18:3n3 deficiency promotes accumulation of brain docosapentaenoic acid (22:5n6) andupregulates expression of arachidonic acid (20:4n6)-metabolizing enzymes, thereby furtherincreasing 20:4n6 levels in the brain [20]. Excess arachidonic metabolism can contribute toneuronal damage in experimental models [21,22]. It is clear that further studies are needed,particularly kinetic ones, to explore these possibilities.

In summary, feeding with a No-fat or HCO EFAD diet induces biochemical evidence ofEFA deficiency in as early as 2 weeks in rats. During short term feeding, the absence ofdietary EFA is the predominant factor in the development of EFA deficiency. In response toan EFAD diet, however, metabolic changes related to hepatic de novo lipogensis and PUFAsinterconversions are sensitive to the presence of dietary saturated fat, which may haverelevance to metabolic, functional and behavioral effects in the study of EFA deficiency inanimal models.

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4. Riella MC, Broviac JW, Wells M, Scribner BH. Essential fatty acid deficiency in human adultduring total parenteral nutrition. Ann Intern Med. 1975; 83:788–789.

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16. Melin T, Nilsson A. Delta-6-desaturase and delta-5-desaturase in human Hep G2 cells are bothfatty acid interconversion rate limiting and are upregulated under essential fatty acid deficientconditions. Prostaglandins Leukot Essent Fatty Acids. 1997; 56:437–442. [PubMed: 9223654]

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Fig. 1.Phospholipid fatty acid content of linoleic acid (C18:2n6) and alpha-linolenic acid(C18:3n3) in plasma and selected tissues after 2, 3 and 4 weeks of feeding with AIN (●),No-fat (○) or HCO (▼) diets. AIN: AIN-93M diet; No-fat: AIN-93M diet without fat; HCO:AIN diet with 4% of hydrogenated coconut oil in place of soybean oil. Values are means±SD. *p<0.001, AIN vs. all others; ^p<0.02, No-fat vs. HCO.

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Fig. 2.Phospholipid fatty acid content of palmitic acid (C16:0) and palmitoleic acid (C16:1n7) inplasma and selected tissues after 2, 3 and 4 weeks of feeding with AIN (●), No-fat (○) orHCO (▼) diets. AIN: AIN-93M diet; No-fat: AIN-93M diet without fat; HCO: AIN dietwith 4% of hydrogenated coconut oil in place of soybean oil. Values are means±SD.*p<0.001, AIN vs. all others; ^p<0.05, HCO vs. all others; ^^p<0.05, AIN vs. all others;**p<0.002, AIN vs. all others; ^^^p<0.001, No-fat vs. HCO; #p<0.01, AIN vs. all others.

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Fig. 3.Phospholipid fatty acid content of stearic acid (C18:0) and oleic acid (C18:1n9) in plasmaand selected tissues after 2, 3 and 4 weeks of feeding with AIN (●), No-fat (○) or HCO (▼)diets. AIN: AIN-93M diet; No-fat: AIN-93M diet without fat; HCO: AIN diet with 4% ofhydrogenated coconut oil in place of soybean oil. Values are means±SD. *p<0.001, AIN vs.all others; **p<0.05, AIN vs. all others; ^p<0.05, HCO vs. all others; #p<0.005, No-fat vs.HCO.

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Fig. 4.Phospholipid fatty acid content of Mead acid (C20:3n9) in plasma and selected tissues after2, 3 and 4 weeks of feeding with AIN (●), No-fat (○) or HCO (▼) diets. AIN: AIN-93Mdiet; No-fat: AIN-93M diet without fat; HCO: AIN diet with 4% of hydrogenated coconutoil in place of soybean oil. Values are means±SD. *p<0.001, AIN vs. all others; ^p<0.001,No-fat vs. HCO.

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Fig. 5.Phospholipid fatty acid content of arachidonic acid (C20:4n6) and eicosapentaenoic acid(C20:5n3) in plasma and selected tissues after 2, 3 and 4 weeks of feeding with AIN (●),No-fat (○) or HCO (▼) diets. AIN: AIN-93M diet; No-fat: AIN-93M diet without fat; HCO:AIN diet with 4% of hydrogenated coconut oil in place of soybean oil. Values are means±SD. *p<0.001, AIN vs. No-fat and HCO; ^p<0.05, week 3 vs. week 2 and 4; **p<0.002,AIN vs. No-fat and HCO; ^^p<0.05, week4 vs. week 2 and 3; #p<0.05, HCO vs. No-fat andHCO.

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Fig. 6.Phospholipid fatty acid content of docosatetraenoic acid (C22:4n6) and docosapentaenoicacid (w6) (C22:5n6) in plasma and selected tissues after 2, 3 and 4 weeks of feeding withAIN (●), No-fat (○) or HCO (▼) diets. AIN: AIN-93M diet; No-fat: AIN-93M diet withoutfat; HCO: AIN diet with 4% of hydrogenated coconut oil in place of soybean oil. Values aremeans±SD. *p<0.001, AIN vs. all others; ^p<0.05 HCO vs. all others; ^^p<0.05 No-fat vs.all others.

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Fig. 7.Phospholipid fatty acid content of docosapentaenoic acid (C22:5n3) and docosahexaenoicacid (22:6n3) in plasma and selected tissues after 2, 3 and 4 weeks of feeding with AIN (●),No-fat (○) or HCO (▼) diets. AIN: AIN-93M diet; No-fat: AIN-93M diet without fat; HCO:AIN diet with 4% of hydrogenated coconut oil in place of soybean oil. Values are means±SD. *p<0.001, AIN vs. all others; ^^p<0.05, HCO vs. No-fat; **p<0.05, HCO vs. AIN;#p<0.05, HCO vs. all others.

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Fig. 8.Triglyceride of palmitic acid (16:0), palmitoleic acid (16:1n7), stearic acid (18:0) and oleicacid (18:1n9) in plasma and liver after 2, 3 and 4 weeks of feeding with AIN (●), No-fat (○)or HCO (▼) diets. AIN: AIN-93M diet; No-fat: AIN-93M diet without fat; HCO: AIN dietwith 4% of hydrogenated coconut oil in place of soybean oil. Values are means±SD.*p<0.001, AIN vs. all others; ^p<0.001, AIN vs. No-fat; #p<0.001, HCO vs. AIN and No-fat, and week 2 vs. weeks 3 and 4.

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Fig. 9.The triglyceride content in plasma (mg/ml) and liver (μmol/mg) after 2, 3 and 4 weeks offeeding with AIN (empty bar), No-fat (gray bar) or HCO (darker bar) diets. AIN: AIN-93Mdiet; No-fat: AIN-93M diet without fat; HCO: AIN diet with 4% of hydrogenated coconutoil in place of soybean oil. Values are means±SD. *p<0.05 No-fat vs. AIN and HCO;^p<0.001, AIN vs. No-fat and HCO.

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Table 1

Dietary compositions (g/kg).

Ingredient AIN-93M No-fat HCO

Casein 140 140 140

L-Cystine 1.8 1.8 1.8

Sucrose 100 100 100

Cornstarch 465.692 495.525 465.692

Dextrose 155 165.175 155

Soybean oil 40 0 0

Hydrogenated coconut oil 0 0 40a

Cellulose 50 50 50

Mineral Mix#210050 35 35 35

Vitamin Mix #310025 10 10 10

Choline bitartrate 2.5 2.5 2.5

AIN-93M: AIN-93M purified rodent diet; No-fat: AIN-93M purified rodent diet without fat; HCO: Modified AIN-93M purified rodent diet with4% hydrogenated coconut oil replacing 4% soybean oil.

aContains 98% saturated fat, <2% mono-unsaturated fat and <0.1% trans fat provided by manufacturer (Welch, Holme & Clark Co., Inc., Newark,

NJ, USA).

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Table 2

Fatty acids (% nmol in total fat) in diets.

Fatty acids AIN 93M diet No-fat diet HCO diet

C8:0 – – 6.22

C10:0 – – 6.32

C12:0 2.41 – 49.30

C14:0 1.02 – 18.14

C16:0 13.01 – 9.10

C16:1 0.39 – 0.09

C18:0 4.77 – 9.06

C18:1w9 21.57 – 1.14

C18:2 49.14 – 0.64

C18:3w6 – – –

C18:3w3 6.70 – –

C20:3w9 – – –

C20:3w6 – – –

C20:4w6 0.12 – –

C20:5w3 0.36 – –

C22:4w6 – – –

C22:5w6 0.14 – –

C22:5w3 0.12 – –

C22:6w3 – – –

The results were obtained from three individual samples. –, not detected. AIN93M diet contains 4% soybean oil by weight; No-fat diet: AIN93Mdiet without fat; HCO diet: AIN93M diet with 4% of hydrogenated coconut oil in place of soybean oil.

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Table 3

Average weekly food intakes (g).

Week 2 Week 3 Week 4

AIN 21.22±1.31 22.14±0.47 22.13±0.47

No-fat 20.92±2.21 22.56±0.61 23.52±0.42

HCO 21.28±1.61 22.14±0.44 23.40±0.32

Mean±SD.

AIN: a group fed by AIN-93M diet; No-fat: a group fed by AIN-93M diet without fat; HCO: a group fed by AIN diet with 4% of hydrogenatedcoconut oil in place of soybean oil.

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Table 4

Triene–tetraene ratio in plasma phospholipids (N=5).

Week 2 Week 3 Week 4

AIN* 0.01±0.01 0.00±0.00 0.00±0.00

No-fat 0.36±0.01 0.52±0.10 0.44±0.23

HCO 0.36±0.10 0.52±0.21 0.59±0.20

Mean±SD.

AIN: a group fed by AIN-93M diet; No-fat: a group fed by AIN-93M diet without fat; HCO: a group fed by AIN diet with 4% of hydrogenatedcoconut oil in place of soybean oil.

*p≤0.001 AIN vs. No-fat and HCO at week 2, 3 and 4 of feeding with AIN, No-fat and HCO diets.

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