Introduction. The Journal of Nutrition Biochemical, Molecular, and Genetic Mechanisms

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1 The Journal of Nutrition Biochemical, Molecular, and Genetic Mechanisms Effects of Dietary Palmitoleic Acid on Plasma Lipoprotein Profile and Aortic Cholesterol Accumulation Are Similar to Those of Other Unsaturated Fatty Acids in the F1B Golden Syrian Hamster 1 3 Nirupa R. Matthan,* Alice Dillard, Jaime L. Lecker, Blanche Ip, and Alice H. Lichtenstein Cardiovascular Nutrition Laboratory, Jean Mayer USDA Human Nutrition Research CenteronAgingatTuftsUniversity,Boston, MA Abstract The lower susceptibility of palmitoleic acid (16:1) to oxidation compared to PUFA may confer functional advantages with respect to finding acceptable alternatives to partially hydrogenated fats, but limited data are available on its effect on cardiovascular risk factors. This study investigated the effect of diets (10% fat, 0.1% cholesterol, wt:wt) enriched with macadamia [monounsaturated fatty acid (MUFA)16:1], palm (SFA,16:0), canola (MUFA,18:1), or safflower (PUFA,18:2) oils on lipoprotein profiles and aortic cholesterol accumulation in F1B Golden Syrian hamsters (n ¼ 16/group). After 12 wk, 8 hamsters in each group were killed (phase 1). The remaining hamsters fed palm oil were changed to a diet containing coconut oil, while hamsters in the other diet groups continued on their original diets for an additional 6 wk (phase 2). With minor exceptions, the time course and dietary SFA source did not alter the study outcomes. Macadamia oil-fed hamsters had lower non-hdl cholesterol and triglyceride concentrations compared with the palm and coconut oil-fed hamsters and higher HDL-cholesterol compared with the coconut, canola, and safflower oil-fed hamsters. The aortic cholesterol concentration was not affected by dietary fat type. The hepatic cholesterol concentration was higher in the unsaturated compared with the saturated oil-fed hamsters. RBC membrane and aortic cholesteryl ester, triglyceride, and phospholipid fatty acid profiles reflected that of the dietary oil. These data suggest that an oil relatively high in palmitoleic acid does not adversely affect plasma lipoprotein profiles or aortic cholesterol accumulation and was similar to other unsaturated fatty acid-rich oils. J. Nutr. 139: , Introduction Diet and lifestyle modification have long been advocated to decrease cardiovascular disease (CVD) 4 risk, the leading cause of death in the US (1). Dietary fatty acids of varying chain length and degree of saturation differentially alter plasma lipoprotein profiles and the subsequent risk of developing CVD (2). In 1 Supported by Dow AgroSciences (RPFA ) and USDA agreement Any opinions, findings, conclusions, or recommendations expressed in this publication are those of the author(s) and do not necessarily reflect the view of the USDA. 2 Supplemental Tables 1 3 and Supplemental Figure 1 are available as Online Supporting Material with the online posting of this paper at 3 Author disclosures: N. R. Matthan, A. Dillard, J. L. Lecker, B. Ip, and A. H. Lichtenstein, no conflicts of interest. 4 Abbreviations used: CVD, cardiovascular disease; CE, cholesteryl ester; EC, esterified cholesterol; FC, free cholesterol; MUFA, monounsaturated fatty acid; nhdl-c, non-hdl cholesterol; PL, phospholipid; TC, total cholesterol; TG, triglyceride. * To whom correspondence should be addressed. nirupa.matthan@ tufts.edu. general, relative to carbohydrate, SFA increase LDL and HDL cholesterol concentrations, monounsaturated fatty acids (MUFA) have a neutral effect, and PUFA decrease LDL and HDL cholesterol concentrations. Relative to SFA, both MUFA and PUFA decrease LDL and HDL cholesterol concentrations, PUFA more so than MUFA (2). The predominant MUFA in the diet is oleic acid (18:1). However, there are some plants that have high levels of palmitoleic acid (16:1). Although macadamia nut oil is the only source high in palmitoleic acid (;25 30%) that is readily available, there are other potential sources (e.g. sea buckthorn). Interest in the biological effects of palmitoleic acid has been spurred by the need to reduce the use of partially hydrogenated fat and the need for a stable alternative. However, there are limited and conflicting data in humans on the effect of high dietary palmitoleic acid on CVD risk factors. Palmitoleic acid has been reported to increase plasma total and LDL-cholesterol concentrations relative to oleic acid (MUFA) but have similar effects compared with palmitic acid (SFA) (3). In contrast, other studies have demonstrated that incorporating macadamia nuts into the diet favorably affects the /08 $8.00 ª 2009 American Society for Nutrition. Manuscript received September 18, Initial review completed October 1, Revision accepted November 20, First published online December 23, 2008; doi: /jn

2 lipid and lipoprotein profile relative to an average American diet (4 7). Two additional studies in the area, one using sea buckthorn juice (high in 16:1) and another using a combination of 14:1 and 16:1 were inconclusive, primarily due to inadequate study designs and confounding due to the presence of other nonnutritive, lipidlowering components (8) or fatty acids (9). The aim of this study was to assess the effect of an oil high in palmitoleic acid (macadamia nut) relative to oils high in SFA (palm oil or coconut oil), MUFA (canola oil), and PUFA (safflower oil) on plasma lipoprotein profiles, RBC and aortic fatty acids profiles, hepatic lipid content, and aortic cholesterol accumulation in the F1B Golden Syrian hamster. The hamster was chosen as the experimental model due to the similarity with human cholesterol and bile acid metabolism (10,11) and responsiveness to alterations in dietary fatty acid profile and cholesterol (12 14). Materials and Methods Hamsters and diets. This investigation was a 2-phase study. During phase 1, 64 8-wk-old male F1B Golden Syrian hamsters (BioBreeders) were acclimatized for a 2-wk period and then assigned to 1 of 4 dietary groups (n ¼ 16 per group) by weight matching. They consumed the experimental diets ad libitum for 12 wk (Table 1). Experimental diets were based on a standard semipurified rodent diet with 10% (wt:wt) of either macadamia nut (16:1), palm (16:0), canola (18:1), or safflower (18:2) oil (Table 2) along with 0.1% cholesterol. At the end of 12 wk, 8 hamsters from each group were killed. During phase 2, the feeding duration for the remaining hamsters was continued for an additional 6 wk (n ¼ 8 per group). Hamsters in the macadamia nut, canola, and safflower oil groups continued receiving their original diets, while hamsters in the palm oil group were switched TABLE 1 Composition of diets enriched with either palm, coconut, macadamia, canola, or safflower oil fed to hamsters for wk 1 Ingredient Palm Coconut Macadamia Canola 2 Safflower Protein, % of energy Carbohydrate, % of energy Fat, % of energy Casein, g L-Methionine, g Maltodextrin, g Cornstarch, g Sucrose, g Cellulose, g Experimental oils, g Palm oil Coconut oil Macadamia oil Canola oil Safflower oil 3 Soybean oil, g Mineral mix (AIN93G), g Vitamin mix (AIN93), g Choline bitartrate, g Cholesterol, 4 g tbhq, g Semipurified diets (37) were formulated by Research Diets. 2 High oleic, low linolenic canola oil. 3 Supplied by Dow AgroSciences. 4 Supplied by Research Diets. TABLE 2 Fatty acid composition of the dietary oils incorporated into the hamster diets Fatty acid Palm Coconut Macadamia Canola Safflower % of total fatty acids SFA :0110: : : : : : : : MUFA :1(n-71n-9) :1(n-9) :1(n-9) :1(n-9) PUFA :2(n-6) :3(n-3)1(n-6) to another SFA diet enriched with coconut oil (rich in 12:0114:0), known to be atherogenic in hamsters (15). Hamsters were housed in groups of 4 in wire bottom cages to maximize atherogenic potential (16). Body weights were recorded every 2 wk and estimated food intake recorded bi-weekly. Retro-orbital bleeds under isoflurane anesthesia were performed after the acclimation period (0 wk) and after 6 and 12 wk (phase 1) and 18 wk (phase 2) to evaluate plasma lipid and lipoprotein profiles. Hamsters were food deprived for 14 h before blood draws. At the conclusion of phases 1 and 2, the hamsters were killed by CO 2 inhalation. The hearts were perfused in situ with PBS and harvested with the attached aorta. Aortas were cleaned of adventitia, weights recorded, flash frozen, and stored at 280 C. Livers were also removed, cleaned with PBS, weights recorded, flash frozen, and stored at 280 C. The animal protocol was approved by the Animal Care and Use Committee of the Jean Mayer Human Nutrition Research Center on Aging, Tufts University, and was in accordance with guidelines provided by the NIH Guide for the Care and Use of Laboratory Animals. Cholesterol concentration and fatty acid profile of experimental diets. Lipids were extracted overnight from desiccated aliquots of diet (17) and total cholesterol and fatty acid profiles were determined by GC as previously described (18,19). Analysis of the dietary fatty acid profile confirmed enrichment with the respective dietary oils (data not shown). The cholesterol concentration was also verified as being 0.1% (wt:wt) for each of the diets. Plasma lipid and lipoprotein profile. Retro-orbital blood was collected into EDTA-coated tubes and plasma was separated from RBC by centrifugation at g for 20 min at 4 C. Plasma total cholesterol, HDL cholesterol, and triglyceride concentrations were determined on a Cobas Mira automated analyzer using enzymatic reagents (Roche Diagnostics). Non-HDL cholesterol was calculated as the difference between total and HDL cholesterol. RBC and aortic fatty acid profile. Lipids were extracted from RBC membranes or aorta (17) followed by saponification and methylation by the method of Morrison and Smith (20). RBC FAME were quantified by GC as previously described (18). Aortic fatty acids were fractionated by solid phase extraction using aminopropyl columns (21) and then analyzed as previously described (18,19). Peaks of interest were identified by comparison with authentic fatty acid standards (Nu-Chek Prep) and expressed as mol% of total fatty acids. 216 Matthan et al.

3 Aortic lesion formation. The accumulation of free and esterified cholesterol in the aortic arch was used as a chemical endpoint to quantify the extent of atherosclerosis (22). Total and free cholesterol concentrations were measured using GC (19) as described for the diets and esterified cholesterol concentrations were computed by difference. Liver lipid concentration. Liver lipids were extracted (17) and total and free cholesterol, triglyceride, and phospholipid concentrations were determined using enzymatic reagents (Wako and Roche Diagnostics) (23). Esterified cholesterol was computed as the difference between total and free cholesterol. Statistical analysis. Data for phase 1 were collected at the 12-wk time point, (n ¼ 16 per group for plasma lipoprotein profile, n ¼ 8 per group for liver and aorta). Data for phase 2 were collected at the 18-wk time point for those hamsters for whom data were available throughout the entire study period (n ¼ 8 per group). Results are expressed as means 6 SD. Prior to statistical analysis, data were checked for normality and, if necessary, appropriate transformations were performed. An ANOVA (PROC ANOVA, SAS version 9.1, SAS Institute) followed by Tukey s post hoc test for multiple comparisons was used to identify significant differences between dietary treatment groups. The RBC and aortic cholesteryl ester, triglyceride, and phospholipid fatty acid data were analyzed by ANOVA of rank values. P-values, 0.05 were considered significant. Results RBC membrane fatty acid composition. The RBC fatty acid profile reflected the profile of the oils incorporated into the diets (Table 3). Data for the 8 hamsters per group killed at 18 wk (phase 2) and the 8 hamsters in the palm oil group killed at 12 wk (phase 1) are presented for comparison purposes. At 18 wk, hamsters fed the macadamia oil diet had proportionally more 16:1(n-7) compared with the other oil-fed groups. Hamsters fed the canola oil diet had a similar proportion of MUFA compared with hamsters fed the macadamia oil diet, although the overwhelming component was 18:1(n-9), with only trace amounts of 16:1(n-7). Hamsters fed the safflower oil diet had proportionally more PUFA compared with the other oil-fed groups, contributed to primarily by 18:2(n-6). At 12 wk, hamsters fed the palm oil diet had more 16:0 compared with the other oil-fed groups. At wk 18, the hamsters fed the palm/coconut oil diet sequence had proportionally more 14:0 than the other oil-fed groups. TABLE 3 RBC fatty acid profile after 12 or 18 wk of feeding hamsters diets enriched with either palm and/or coconut, macadamia, canola, or safflower oils 1 Fatty acid Palm 2 Palm/coconut 3,4 Macadamia 4 Canola 4 Safflower 4 mol% SFA a a b b ab 10: : ab a ab b ab 14: b a ab b b 16: a b b b b 18: b b b b a 20: : c b a bc bc 24: d a ab c bc MUFA c bc a ab c 14:1(n-9) :1(n-9) :1(n-7) b c b a c c 18:1(n-9) b b a a c 18:1(n-7) bc b a bc c 20:1(n-9) bc b a a c 22:1(n-9) b b a ab b 24:1(n-9) c a ab b c PUFA b b c b a 18:2(n-6) b b c b a 18:3(n-6) b a a a a 18:3(n-3) :2(n-6) b b c b a 20:3(n-6) :4(n-6) a b b b ab 20:5(n-3) :2(n-6) ab ab a ab b 22:4(n-6) b b c b a 22:5(n-6) b a b b a 22:5(n-3) a bc bc ab c 22:6(n-3) ab bc ab a c 1 Data are presented as mean 6 SD, n ¼ 6 8. Means in a row with superscripts without a common letter differ, P, Hamsters were fed the palm oil diet for 12 wk. 3 Hamsters were fed the palm oil diet for 12 wk and then switched to a coconut oil diet for the remaining 6 wk. 4 Hamsters were fed the respective diets for 18 wk. Palmitoleic acid and lipids in F1B hamsters 217

4 Phase 1 Food intake and body weight. All hamsters remained healthy throughout the 12-wk feeding period. Mean food intake [ g/(hamsterd)] and final body weights ( g/hamster) did not significantly differ among dietary fat groups. Plasma lipid and lipoprotein profile. At baseline, there was no significant difference in the plasma lipid and lipoprotein profiles among the 4 groups of hamsters (Table 4). Hamsters fed the macadamia nut and canola oil-enriched diets had significantly lower non-hdl cholesterol concentrations at the 6- and 12-wk time points than the hamsters fed the palm oil-enriched diet, with intermediate values for the safflower oil group. Plasma HDL cholesterol was higher in the palm and macadamia nut oil-fed hamsters at 6 wk but not 12 wk postintervention (Table 4). A similar pattern was observed for plasma triglyceride concentrations. The total:hdl cholesterol and non- HDL:HDL cholesterol ratios were more favorable in the hamsters fed the macadamia nut and canola oil diets than the palm oil diet at both the 6- and 12-wk time points. Aortic cholesterol concentration and fatty acid profile. Aortic total, free, and esterified cholesterol concentrations did not differ on the basis of dietary fat type (Supplemental Fig. 1A). Aortic cholesteryl ester, triglyceride, and phospholipid fatty acid profiles reflected that of the major fatty acids in the dietary oils (Supplemental Table 1). The cholesteryl ester and triglyceride fractions were most reflective of the dietary perturbations. This was not unexpected, given the specificity for membrane phospholipid fatty acids. In general, hamsters fed the palm oil diet had proportionally more 16:0 (;1.5- to 2-fold) than the other oil-fed groups. Hamsters fed the macadamia oil diet had more 16:1(n-7) (;2- to 4-fold) compared with the other oil-fed groups, as well as more 18:1(n-9) (consistent with the composition of the oil) compared with the palm or safflower oil-fed groups. The canola oil-fed hamsters had a similar proportion of total MUFA compared with the macadamia oil-fed hamsters, without the 16:1 component. Safflower oil-fed hamsters had proportionally more 18:2(n-6) (;3- to 5-fold) and a lower proportion of 18:1 compared with the other oil-fed hamsters. Liver lipid concentration. Liver weights were similar among the 4 diet groups ( g; mean of all 4 diets). Total cholesterol concentration was higher in the macadamia and canola oil groups than in the palm oil group (Fig. 1A). This was predominantly due to increases in the free cholesterol fraction (P, 0.05). Esterified cholesterol, triglyceride, and phospholipid concentrations did not differ among the diet groups. Phase 2 Food intake and body weight. Food intake did not significantly differ on the basis of dietary fat type [ g/(hamster d)], whereas body weight was significantly lower in the macadamia nut and canola oil-fed hamsters relative to those fed the palm/coconut oil diet (Supplemental Table 2). Plasma lipid and lipoprotein profile. After 18 wk of feeding (Table 5), hamsters fed the macadamia, canola, and safflower TABLE 4 Plasma lipid profile at baseline and 6 and 12 wk of feeding (phase 1) hamsters diets enriched with either palm, macadamia, canola, or safflower oil 1 Diet Lipid 2 Palm Macadamia Canola Safflower 0wk mmol/l TC nhdl-c HDL-C TG TC:HDL nhdl:hdl wk TC nhdl-c a c bc ab HDL-C a a ab b TG a b ab ab TC:HDL ab b b a nhdl:hdl ab b b a 12 wk TC nhdl-c a b b ab HDL-C TG TC:HDL a b b ab nhdl:hdl a b b ab 1 Data are presented as mean 6 SD, n ¼ 16. Means in a row with superscripts without a common letter differ, P, Blood samples were obtained from food-deprived hamsters. FIGURE 1 Liver lipid concentrations at the end of 12 wk (A)or18wk (B) in hamsters fed diets enriched in either palm/coconut, macadamia, canola, or safflower oils. Bars represent means 6 SD, n ¼ 16 (12 wk, phase 1) and n ¼ 8 (18 wk, phase 2). For each variable, labeled means without a common letter differ, P, Matthan et al.

5 TABLE 5 Plasma lipid profile at baseline and 6, 12, and 18 wk of feeding (phase 2) hamsters diets enriched with either palm/coconut, macadamia, canola, or safflower oils 1 Diet Lipid 2 Palm/coconut Macadamia Canola Safflower 0wk mmol/l TC nhdl-c HDL-C TG TC:HDL nhdl:hdl wk TC a b b b nhdl-c a c bc ab HDL-C ab a b b TG a c bc b TC:HDL a b ab a nhdl:hdl a b ab a 12 wk TC nhdl-c a b ab ab HDL-C b a b b TG a b ab b TC:HDL a c b ab nhdl:hdl a c b ab 18 wk TC a a ab b nhdl-c a b b b HDL-C b a b b TG a b b b TC:HDL a b b b nhdl:hdl a b b b 1 Data are presented as mean 6 SD, n ¼ 8. Means in a row with superscripts without a common letter differ, P, Blood samples were obtained from food-deprived hamsters. oil-enriched diets had similar non-hdl cholesterol concentrations, which were significantly lower than in hamsters fed the palm/coconut oil-enriched diet. This pattern was also reflected in triglyceride concentrations. Plasma HDL cholesterol concentrations were higher in the macadamia nut oil-fed hamsters compared with the other 3 diet groups. The total:hdl cholesterol and non-hdl:hdl cholesterol ratios were similar among the macadamia, canola, and safflower oil groups and more favorable than the palm/coconut oil group. Aortic cholesterol concentration and fatty acid profile. As observed for the phase 1 data, aortic total, free, and esterified cholesterol concentrations did not differ on the basis of dietary fat type (Supplemental Fig. 1B) at the end of phase 2. Likewise, the fatty acid profiles of each aortic lipid fraction reflected that of the major fatty acids in the dietary oils (Supplemental Table 3). Hamsters fed the palm/coconut oil diet had proportionally more 12:0 (;1- to 10-fold), 14:0 (;2- to 8-fold), and 16:0 (;0.2- to 0.5-fold) compared with the other oil-fed groups. Hamsters fed the macadamia oil diet had more 16:1 (;2- to 4-fold) compared with the other oil-fed groups, as well as more 18:1(n-9) than the palm/coconut or safflower oil-fed groups. Hamsters fed the canola oil diet had a similar proportion of MUFA compared with hamsters fed the macadamia oil diet, without the 16:1 component. Hamsters fed the safflower oil diet had proportionally more 18:2(n-6) (;1.5- to 5-fold) than the other oil-fed groups. Liver lipid concentration. Liver weights were higher at the end of phase 2 than phase 1, but the 4 dietary groups did not differ ( g; overall mean). The triglyceride, phospholipid, free, and esterified cholesterol concentrations did not differ among diet groups. However, the total cholesterol concentration was significantly higher in the MUFA-fed hamsters (macadamia and canola) compared with the palm/coconut group (Fig. 1B). Discussion Palmitoleic acid is a minor dietary MUFA in Western diets but is found in significant quantities in certain plants. Interest in palmitoleic acid has been spurred by the potential of using oils rich in this fatty acid as an alternative to partially hydrogenated fat. An advantage compared with oils containing a high proportion of PUFA is its lower susceptibility to oxidation. However, little is known about the effects of dietary palmitoleic acid on CVD risk factors. The aim of the present study was to determine whether increasing intake of an oil high in palmitoleic acid (macadamia nut oil) resulted in incorporation into selected tissues and altered plasma lipoprotein profiles, relative to oils high in SFA (palm and/or coconut oil, 16:0/12:0114:0), MUFA (canola oil, 18:1), and PUFA (safflower oil, 18:2). The results demonstrate that macadamia nut oil was similar to other unsaturated fatty acid-rich oils and did not have adverse effects on plasma lipoprotein profiles, aortic cholesterol accumulation, or hepatic lipid content. Our data are consistent with the results of 2 clinical studies in which macadamia nut-enriched diets (40 90 g/d) resulted in significantly lower plasma LDL cholesterol concentrations ( %) relative to an average American diet (4,6). Effects observed on triglyceride concentrations were inconsistent. Two other human studies have also reported improvements in lipoprotein profile with macadamia nut-containing diets compared with SFA-enriched diets (5,7). However, in these studies, there was a concomitant decrease in carbohydrate and/or SFA intake caused by the large increase in the nut component consumption, thus making it difficult to attribute the effects on plasma lipoprotein profile to the fatty acid profile of the nuts themselves. In contrast to these studies, a trial in hypercholesterolemic men concluded that a diet rich in 16:1 (macadamia nut oil) resulted in similar total and LDL cholesterol concentrations compared with 16:0 (palm oil rich in SFA) and both diets were hypercholesterolemic relative to a diet rich in 18:1 (high oleic acid sunflower seed oil) (3). In this study, the palmitoleic rich dietary food/beverage contributed 4% of dietary energy, which is lower than the 15% of energy used in other human interventions and could account for the discrepancy in results. The mechanism responsible for the hypocholesterolemic effect of dietary MUFA or PUFA has been attributed to the upregulation of LDL receptor activity, whereas dietary SFA attenuates this effect by partially downregulating or suppressing hepatic LDL receptor activity (24 26). MUFA, specifically 18:1, are preferred substrates for acyl CoA:cholesterol acyltransferase, which catalyzes the esterification of hepatic free cholesterol to an inert cholesteryl ester pool (27). This in turn reduces the putative regulatory pool of intracellular free cholesterol, increasing LDL receptor activity and subsequently decreasing circulating cholesterol concentrations (28). Consistent with this observation, hepatic total cholesterol content was highest in both the MUFA groups (macadamia and canola oils), intermediate in the PUFA Palmitoleic acid and lipids in F1B hamsters 219

6 group (safflower oil), and lowest in the SFA groups (palm and/or coconut oils), with esterified cholesterol contributing 75 86% of the total cholesterol content of the liver. These changes could account for the significantly lower non-hdl cholesterol concentrations in hamsters fed the MUFA-enriched diets (macadamia and canola) compared with the SFA-enriched diets (palm and/or coconut). Additionally, differences in body weight could have also contributed to the observed differences. In phase 2, the SFA-fed hamsters weighed more than the hamsters in the macadamia- and canola-fed diet groups. Although this difference in weight was consistent with the food intake data, we cannot fully exclude the impact of this variable on the lipoprotein differences. Feeding African green monkeys a high MUFA (oleic acid-rich safflower oil) or (n-6) PUFA (linoleic acid-rich safflower oil) diet resulted in lower plasma LDL and higher plasma HDL-cholesterol concentrations than monkeys fed a high SFA (palm oil) diet (29,30). Nonetheless, when atherosclerosis was quantified, monkeys fed the high MUFA diet developed more atherosclerosis than those fed the high PUFA diet and similar amounts of lesion as those fed the SFA diet. The adverse effect of the MUFA-rich diet was attributed to higher secretion of cholesteryl oleate-enriched lipoproteins by the liver (30). Similar findings were reported when the same experimental question was assessed in the LDL receptor null and apolipoprotein B-100 overexpressing transgenic mouse (22). The effect of palmitoleic acid was not evaluated in this model. Present results do not support an atherogenic role for either 18:1 or 16:1 MUFA in the hamster model. Unexpectedly, at the end of phase 1, hamsters fed the PUFA (safflower oil) enriched diet had similar non-hdl cholesterol concentrations as the SFA (palm oil) diet. In phase 2, switching the SFA source from palm to coconut oil resulted in a marked increase in plasma non-hdl cholesterol and triglyceride concentrations. This is consistent with previous reports where feeding 12:0114:0 (coconut oil) has been shown to elicit a greater hyperlipidemic response than 16:0 (palm oil) in the hamster model (31). The response has been attributed to the higher content of oleic and linoleic acids in palm than in coconut oil. Thus, it appears that palm oil was less potent in suppressing LDL receptor activity and subsequently increasing circulating cholesterol concentrations than coconut oil. An unanticipated observation was the similar and higher plasma HDL cholesterol concentrations between the palm oiland macadamia nut oil-fed hamsters in phase 1 and the sustained higher HDL cholesterol concentrations in the macadamia nut oil relative to the coconut, canola, and safflower oil fed hamsters at the end of phase 2. This higher HDL cholesterol concentration has been observed in some (5) but not all macadamia nut intervention studies (3,4,6). This may be a consequence of the metabolic sequelae associated with the lower triglyceride concentrations (32). Isocaloric substitution of SFA by MUFA has been reported to decrease cholesteryl ester transfer protein activity. Cholesteryl ester transfer protein mediates the transfer of cholesteryl esters and triglycerides between HDL and lower activity has been associated with higher plasma HDL cholesterol concentrations (33). Consistent with these observations, hamsters fed the macadamia nut oil had higher HDL cholesterol concentrations than those fed the coconut, canola, and safflower oil-enriched diets. Additionally, the contribution of the 16:0 component as well as a synergistic effect of other nonfatty acid components of macadamia nut oil (phytosterols, tocopherols, polyphenols) on plasma lipoprotein concentrations cannot be excluded. Despite the differences in plasma lipoprotein profile induced by dietary oil type, total aortic cholesterol concentrations were low and did not significantly differ among diet groups. Free cholesterol, rather than esterified cholesterol, was the predominant form of cholesterol in the aortas, indicative of early atherosclerotic lesion formation (11). This observation is contrary to prior reports that have shown significant atherosclerotic lesion development in hamsters after wk of consuming a high SFA and cholesterol diet (15). It has been documented that in the F1B strain of hamster, the majority of cholesterol is carried in VLDL and HDL rather than in LDL (34,35). In the present study, plasma HDL cholesterol concentrations were comparable and, in some diets, higher than plasma non-hdl cholesterol concentrations. Given the inverse relationship between plasma HDL cholesterol concentrations and atherosclerotic lesion development, this lipoprotein profile would favor less, rather than more, atherosclerotic lesion development, which could account for the lower degree of atherogenesis observed in our study (35,36). Nonetheless, the response to dietary fatty acids appears qualitatively, if not quantitatively, similar to those shown by others (11,35). In conclusion, the effect of an oil (macadamia nut) high in palmitoleic acid on plasma lipoprotein profiles was similar to that of another oil high in MUFA (canola oil) as well as an oil high in PUFA (safflower oil). All these oils resulted in a less atherogenic plasma lipoprotein profile compared with hamsters fed a diet enriched in palm oil for 12 wk or palm and then coconut oil for an additional 6 wk. Acknowledgments We gratefully acknowledge the technical assistance of Dr. Donald Smith, Comparative Biology Unit (Tufts University, Boston) for assistance with the hamster feeding and maintenance protocol. We also thank Dr. Daniel Gachotte and Virgina Stoltz at Dow AgroSciences (Indianapolis, IN) for the fatty acid analyses of the oils used in this study. Literature Cited 1. AHA. Heart disease and stroke statistics: 2008 update [cited October 2008]. Available from: identifier¼ Lichtenstein AH. Thematic review series: patient-oriented research. Dietary fat, carbohydrate, and protein: effects on plasma lipoprotein patterns. J Lipid Res. 2006;47: Nestel P, Clifton P, Noakes M. Effects of increasing dietary palmitoleic acid compared with palmitic and oleic acids on plasma lipids of hypercholesterolemic men. J Lipid Res. 1994;35: Curb JD, Wergowske G, Dobbs JC, Abbott RD, Huang B. Serum lipid effects of a high-monounsaturated fat diet based on macadamia nuts. Arch Intern Med. 2000;160: Garg ML, Blake RJ, Wills RB. Macadamia nut consumption lowers plasma total and LDL cholesterol levels in hypercholesterolemic men. J Nutr. 2003;133: Griel AE, Cao Y, Bagshaw DD, Cifelli AM, Holub B, Kris-Etherton PM. A macadamia nut-rich diet reduces total and LDL-cholesterol in mildly hypercholesterolemic men and women. J Nutr. 2008;138: Hiraoka-Yamamoto J, Ikeda K, Negishi H, Mori M, Hirose A, Sawada S, Kitamori K, Onobayashi Y, Kitano S, et al. Serum lipid effects of a monounsaturated (palmitoleic) fatty acid -rich diet based on macadamia nuts in healthy, young Japanese women. Clin Exp Pharmacol Physiol. 2004;31:S Eccleston C, Baoru Y, Tahvonen R, Kallio H, Rimbach GH, Minihane AM. Effects of an antioxidant-rich juice (sea buckthorn) on risk factors for coronary heart disease in humans. J Nutr Biochem. 2002;13: Smith DR, Knabe DA, Cross HR, Smith SB. A diet containing myristoleic plus palmitoleic acids elevates plasma cholesterol in young growing swine. Lipids. 1996;31: Chen J, Song W, Redinger RN. Effects of dietary cholesterol on hepatic production of lipids and lipoproteins in isolated hamster liver. Hepatology. 1996;24: Matthan et al.

7 11. Nistor A, Bulla A, Filip DA, Radu A. The hyperlipidemic hamster as a model of experimental atherosclerosis. Atherosclerosis. 1987;68: Dorfman SE, Smith DE, Osgood DP, Lichtenstein AH. Study of dietinduced changes in lipoprotein metabolism in two strains of Golden- Syrian hamsters. J Nutr. 2003;133: Dorfman SE, Wang S, Vega-Lopez S, Jauhiainen M, Lichtenstein AH. Dietary fatty acids and cholesterol differentially modulate HDL cholesterol metabolism in Golden-Syrian hamsters. J Nutr. 2005;135: Spady DK, Dietschy JM. Interaction of dietary cholesterol and triglcyerides in the regulation of hepatic low density lipoprotein transport in the hamster. J Clin Invest. 1988;81: Nicolosi RJ. Dietary fat saturation effects on low-density-lipoprotein concentrations and metabolism in various animal models. Am J Clin Nutr. 1997;65:S Smith D, Pedro-Botet J, Cantuti-Castelvetri I. Influence of photoperiod, laboratory caging and aging on plasma lipid response to an atherogenic diet among F1B hamsters. Int J Neurosci. 2001;106: Folch J, Lees M, Sloane-Stanley GH. A simple method for the isolation and purification of total lipid from animal tissues. J Biol Chem. 1957; 226: Lichtenstein AH, Matthan NR, Jalbert SM, Resteghini NA, Schaefer EJ, Ausman LM. Novel soybean oils with different fatty acid profiles alter cardiovascular disease risk factors in moderately hyperlipidemic subjects. Am J Clin Nutr. 2006;84: Matthan NR, Ausman LM, Lichtenstein AH, Jones PJ. Hydrogenated fat consumption affects cholesterol synthesis in moderately hypercholesterolemic women. J Lipid Res. 2000;41: Morrison WR, Smith LM. Preparation of fatty acid methyl esters and dimethylacetals from lipids with boron fluoride-methanol. J Lipid Res. 1964;5: Agren JJ, Julkunen A, Penttila I. Rapid separation of serum lipids for fatty acid analysis bya single aminopropylcolumn. J Lipid Res. 1992;33: Rudel LL, Kelley K, Sawyer JK, Shah R, Wilson MD. Dietary monounsaturated fatty acids promote aortic atherosclerosis in LDL receptornull, human ApoB100-overexpressing transgenic mice. Arterioscler Thromb Vasc Biol. 1998;18: Carr TP, Andresen CJ, Rudel LL. Enzymatic determination of triglyceride, free cholesterol, and total cholesterol in tissue lipid extracts. Clin Biochem. 1993;26: Fernandez ML, West KL. Mechanisms by which dietary fatty acids modulate plasma lipids. J Nutr. 2005;135: Woollett LA, Spady DK, Dietschy JM. Saturated and unsaturated fatty acids independently regulate low density lipoprotein receptor activity and production rate. J Lipid Res. 1992;33: Woollett LA, Spady DK, Dietschy JM. Regulatory effects of the saturated fatty acids 6:0 through 18:0 on hepatic low density lipoprotein receptor activity in the hamster. J Clin Invest. 1992;89: Rumsey SC, Galeano NF, Lipschitz B, Deckelbaum RJ. Oleate and other long chain fatty acids stimulate low density lipoprotein receptor activity by enhancing low density lipoprotein receptor activity by enhancing acyl coenzyme A:cholesterol acyltransferase activity and altering intracellular regulatory cholesterol pools in cultured cells. J Biol Chem. 1995;270: Daumerie CM, Woollett LA, Dietschy JM. Fatty acids regulate hepatic low density lipoprotein receptor activity through redistribution of intracellular cholesterol pools. Proc Natl Acad Sci USA. 1992;89: Rudel LL, Haines JL, Sawyer JK. Effects on plasma lipoproteins of monounsaturated, saturated, and polyunsaturated fatty acids in the diet of African green monkeys. J Lipid Res. 1990;31: Rudel LL, Haines JL, Sawyer JK, Shah R, Wilson MS, Carr TP. Hepatic origin of cholesteryl oleate in coronary artery atherosclerosis in African green monkeys. J Clin Invest. 1997;100: Wilson TA, Nicolosi R, Kotyla T, Sundram K, Kritchevsky D. Different palm oil preparations reduce plasma cholesterol concentrations and aortic cholesterol accumulation compared to coconut oil in hypercholesterolemic hamsters. J Nutr Biochem. 2005;16: Tokgözoglu SL. The interaction between HDL and triglycerides. Atherosclerosis. 1999;146:S Jansen S, Lopez-Miranda J, Castro P, et al. Low-fat and highmonounsaturated fatty acid diets decrease plasma cholesterol ester transfer protein concentrations in young, healthy, normolipidemic men. Am J Clin Nutr. 2000;72: Cheema SK, Cornish ML. Bio F1B hamster: a unique animal model with reduced lipoprotein lipase activity to investigate nutrient mediated regulation of lipoprotein metabolism. Nutr Metab (Lond). 2007;4: Mangiapane EH, McAteer MA, Benson GM, White DA, Salter AM. Modulation of the regression of atherosclerosis in the hamster by dietary lipids: comparison of coconut oil and olive oil. Br J Med. 1999; 82: Stein O, Dabach Y, Hollander G, Halperin G, Thiery J, Stein Y. Relative resistance of the hamster to aortic atherosclerosis in spite of prolonged vitamin E deficiency and dietary hypercholesterolemia. Putative effect of increased HDL? Biochim Biophys Acta. 1996;1299: Reeves PG, Nielsen FH, Fahey GCJ. AIN-93 purified diets for laboratory rodents: final report of the American Institute of Nutrition ad hoc writing committee on the reformulation of the AIN-76A diet. J Nutr. 1993;123: Palmitoleic acid and lipids in F1B hamsters 221