Development of a Food-Exchange Model to Replace Saturated Fat with MUFAs and n 6 PUFAs in Adults at Moderate Cardiovascular Risk 1 3

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1 The Journal of Nutrition Nutrition and Disease Development of a Food-Exchange Model to Replace Saturated Fat with MUFAs and n 6 PUFAs in Adults at Moderate Cardiovascular Risk 1 3 Michelle Weech, 4,5 Katerina Vafeiadou, 4,5,7 Marinela Hasaj, 4,5 Susan Todd, 6 Parveen Yaqoob, 4,5 Kim G. Jackson, 4,5 and Julie A. Lovegrove 4,5 * 4 Hugh Sinclair Unit of Human Nutrition, 5 Institute for Cardiovascular and Metabolic Research, and 6 Department of Mathematics and Statistics, University of Reading, Reading, United Kingdom Abstract The recommendation to reduce saturated fatty acid (SFA) consumption to #10% of total energy (%TE) is a key public health target aimed at lowering cardiovascular disease (CVD) risk. Replacement of SFA with unsaturated fats may provide greater benefit than replacement with carbohydrates, yet the optimal type of fat is unclear. The aim of the DIVAS (Dietary Intervention and Vascular Function) study was to develop a flexible food-exchange model to investigate the effects of substituting SFAs with monounsaturated fatty acids (MUFAs) or n 6 (v-6) polyunsaturated fatty acids (PUFAs) on CVD risk factors. In this parallel study, UK adults aged y with moderate CVD risk (50% greater than the population mean) were identified using a risk assessment tool (n = 195; 56% females). Three 16-wk isoenergetic diets of specific fatty acid (FA) composition (%TE SFA:%TE MUFA:%TE n 6 PUFA) were designed using spreads, oils, dairy products, and snacks as follows: 1) SFA-rich diet (17:11:4; n = 65); 2) MUFArich diet (9:19:4; n = 64); and 3) n 6 PUFA-rich diet (9:13:10; n = 66). Each diet provided 36%TE total fat. Dietary targets were broadly met for all intervention groups, reaching %TE SFA, %TE MUFA, and %TE n 6 PUFA in the respective diets, with significant overall diet effects for the changes in SFAs, MUFAs, and n 6 PUFAs between groups (P < 0.001). There were no differences in the changes of total fat, protein, carbohydrate, and alcohol intake or anthropometric measures between groups. Plasma phospholipid FA composition showed changes from baseline in the proportions of total SFAs, MUFAs, and n 6 PUFAs for each diet group, with the changes in SFAs and MUFAs differing between the groups (P < 0.001). In conclusion, successful implementation of the food-exchange model broadly achieved the dietary target intakes for the exchange of SFAs with MUFAs or n 6 PUFAs with minimal disruption to the overall diet in a free-living population. This trial was registered at clinicaltrials.gov as NCT J. Nutr. 144: , Introduction In the United Kingdom, cardiovascular disease (CVD) 8 is a major cause of mortality (1). Because diet is irrefutably associated with CVD risk, dietary recommendations are an 1 Supported by UK Food Standards Agency and Department of Health Policy Research Programme grant 024/0036. Unilever Research and Development produced and supplied in kind the study spreads and oils according to our specification. 2 Author disclosures: M. Weech, K. Vafeiadou, M. Hasaj, S. Todd, P. Yaqoob, K. G. Jackson, and J. A. Lovegrove, no conflicts of interest. 3 Supplemental Methods and Supplemental Tables 1 3 are available from the Online Supporting Material link in the online posting of the article and from the same link in the online table of contents at 7 Present address: School of Life and Medical Sciences, University of Hertfordshire, Hatfield, United Kingdom. 8 Abbreviations used: ALNA, a-linolenic acid; CVD, cardiovascular disease; DIVAS, Dietary Intervention and Vascular Function; LC, long-chain; NDNS, National Diet and Nutrition Survey; PAL, physical activity level; PC, principal component; PCA, principal component analysis; RISCK, Reading, Imperial, Surrey, Cambridge, and Kings; %TE, percentage of total energy. * To whom correspondence should be addressed. j.a.lovegrove@reading. ac.uk. essential strategy to reduce their incidence. Part of that strategy is the reduction of dietary SFAs to #10% of total energy (%TE) (2). Despite falling intakes of SFAs over the past 50 y, adults in the United Kingdom still exceed this recommendation, with a mean intake of 12.0%TE (3). A recent systematic review suggested that lowering dietary SFA intake, predominantly via the modification of dietary FAs rather than reduction in total fat, could reduce cardiovascular events by 14% (4). However, there is no clear guidance on which macronutrient should replace dietary SFA. Evidence suggests that replacement with unsaturated fats rather than carbohydrates may afford greater CVD risk reduction (5,6), in part by their ability to improve the lipid profile, yet there are no specific recommendations on which unsaturated fat is optimal because of insufficient and often conflicting evidence (4,7,8). Therefore, an important public health question remains unanswered: Does the reduction of SFA via the substitution of MUFA or n 6 PUFA have differential effects on CVD risk factors? Of paramount importance for any dietary study is the application of an effective dietary intervention and monitoring ã 2014 American Society for Nutrition. 846 Manuscript received January 13, Initial review completed February 5, Revision accepted March 11, First published online April 9, 2014; doi: /jn

2 of participant compliance. However, few studies describe the dietary intervention used, challenges faced, and strategies implemented to overcome these in sufficient detail (9 11). For the Dietary Intervention and Vascular Function (DIVAS) study, it was essential that the dietary FA intake of a free-living population was manipulated without changing other components of the diet. For this purpose, a food-exchange model was developed to produce 3 isoenergetic intervention diets rich in SFAs, MUFAs, or n 6 PUFAs, each having a specific target FA composition. Food-exchange models used previously in intervention studies successfully altered the amount and composition of dietary FAs in free-living individuals and sustained target intakes over many weeks (8 24 wk) while allowing a certain degree of flexibility as to food preferences and lifestyle (9 11). The DIVAS model enabled participants to make specific dietary substitutions (spreads, oils, snacks, and dairy products) while continuing to eat unrestricted with minimal disruption to their usual dietary habits for 16 wk. This study describes the DIVAS food-exchange model and the degree of success of its application (assessed using 4-d weighed dietary records and plasma phospholipid FA composition) in a free-living population of men and women aged y (n = 195) with moderate CVD risk (50% greater than the population mean, identified using a risk assessment tool) in the United Kingdom. Anthropometric measurements are also described. The primary endpoint (vascular function) and secondary endpoints (blood pressure, plasma lipids and markers of inflammation, endothelial activation, and insulin resistance) will be published elsewhere. Participants and Methods Study participants and design. The DIVAS study was a single-blind randomized controlled parallel study registered at clinicaltrials.gov as NCT and conducted according to the guidelines of the Declaration of Helsinki. A favorable ethical opinion for conduct was given by the West Berkshire Local Research Ethics Committee (09/ H0505/56) and the University of Reading Research Ethics committee (09/40). All participants provided written informed consent before participating. To detect a 2% intergroup difference in the primary outcome (vascular function assessed by flow-mediated dilatation) using an SD of 2.3, power of 90%, and 5% significance level, n = 171 was required for power (equal to n = 57 per group) and increasing to n = 228 when including a 25% dropout rate. Nonsmoking males and females aged y with a moderate risk of CVD were recruited from the Reading area in the United Kingdom in 3 cohorts between November 2009 and July To identify individuals with moderate CVD risk, a scoring tool (12) based on the Framingham risk prediction algorithm (13) was used (Supplemental Table 1). A risk score of $2 points was required for participation, reflecting a 50% greater CVD risk than the population mean. This was achieved by the presence of single or multiple risk factors, including raised fasting total cholesterol, glucose, and blood pressure, low HDL cholesterol, overweight/obesity, and/or family history of premature myocardial infarction or type 2 diabetes. Participants at higher risk of total cholesterol ($8.0 mmol/l), glucose ($7.0 mmol/l), and blood pressure $160/100 mm Hg) were excluded and advised to visit their general practitioner. Additional inclusion criteria included the following: 1) normal blood biochemistry for liver and kidney function; 2) no dietary supplements; 3) no medication for hypertension, hypercholesterolemia, hyperlipidemia, or inflammatory disorders; 4) no diagnosis of a myocardial infarction, stroke, or diabetes; 5) not pregnant or lactating; 6) not consuming excessive amounts of alcohol (males: <21 units/wk; females: <14 units/wk); and 7) undertaking < min/wk of aerobic exercise. Dietary intervention. Participants were randomly assigned to 1 of 3 intervention diets using a minimization program matching for age, gender, BMI, and total risk score (14). The intervention diets (target compositions, %TE SFA:%TE MUFA:%TE n 6 PUFA) were rich in SFAs (17:11:4), MUFAs (9:19:4), and n 6 PUFAs (9:13:10). Because the n 6 PUFA-rich diet was restricted to 10%TE n 6 PUFA, in line with current recommendations (2), 8%TE SFA was replaced by 6%TE n 6 PUFA and 2%TE MUFA. All 3 isoenergetic diets provided 36%TE total fat. Protein, carbohydrates, and n 3 PUFAs were unchanged. The intervention diet groups are referred to as the SFA, MUFA, and n 6 PUFA diet groups going forward. Measures of vascular function (primary outcome) and other CVD risk markers (including blood pressure, plasma lipids, anthropometrics, NO and markers of endothelial activation, inflammation, and insulin resistance) were determined at baseline and after intervention. Presented below are the results from the 4-d dietary records and plasma phospholipid FA analysis to assess dietary compliance and the anthropometric measurements. All other clinical outcome measures will be published elsewhere. Food-exchange model. For the DIVAS dietary intervention, a foodexchange model was developed. This was adapted from the successful Reading, Imperial, Surrey, Cambridge, and Kings (RISCK) foodexchange model (10), which was based on the 2000/2001 National Diet and Nutrition Survey (NDNS) for adults aged y (15). For the DIVAS food-exchange model, the habitual energy, FA, and macronutrient intakes of men and women with increased risk of CVD were extracted from the RISCK study (n = 517) (10). It was preferred to use the RISCK population rather than the NDNS population because NDNS included all adults regardless of their CVD risk. Next, major sources of fat that were easily accessible within the diet ( exchangeable fat ) were identified as added oils, added fats (e.g., butter and spreads), milk, cheese, and snacks. The mean daily energy and FA intakes for these food groups were calculated using the most recent 2000/2001 NDNS percentage contribution of types of foods tables for energy and FAs (15) and combined to estimate daily energy and FA intakes for total exchangeable fat (Table 1). Because mean energy and FA intakes from NDNS and the RISCK study slightly differed (data not shown), the estimated intakes for total exchangeable fat were adjusted to reflect the mean RISCK habitual diet for the final food-exchange model. The proportion of exchangeable dietary fat in men and women was 47.6% and 48.5% of total fat, respectively. The difference between the RISCK total habitual intake and adjusted total exchangeable intake was identified as the non-exchangeable fat intake. This would not be affected by the dietary intervention and included intrinsic sources of fat (such as meat and fish) that, if substituted in the diet, would also affect other nutrient intakes. The sources of exchangeable fat in the replacement models and their corresponding daily quantities were designed so the dietary targets could be met while the participants continued to eat unrestricted for 16 wk (Table 2). This formed the dietary advice for the 3 intervention groups, which could be adjusted on an individual basis to achieve the target FA intakes. All diets were isoenergetic. Specially formulated spreads (80% total fat) and oils (Unilever Research and Development) were used for the MUFA diet (refined olive oil and olive oil/rapeseed oil blended spread) and n 6 PUFA diet (safflower oil and spread). Butter (Wyke Farm) was used as both a spread and oil replacement in the SFA diet. The nutritional compositions of the study foods (Supplemental Table 2) were obtained from food labels, food databases, or manufacturers. All snack products were commercially available. For the n 6 PUFA diet, the low MUFA content of the safflower oil and spread required the readdition of MUFAs into the diet via hazelnuts and MUFA-rich snacks. Implementation of the intervention diets. After completing the baseline clinical visit (week 0), a trained researcher provided 1:1 dietary advice specific to the randomly assigned 16-wk intervention diet. This was supplemented with a handbook containing clear guidelines. Participants were unaware of the dietary intervention. For each exchangeable food category (spreads, oils, snacks, and dairy), detailed instructions were provided on the types of foods to eat (e.g., study spread) and types to avoid (e.g., all other types of spread, margarine, and butter). Instructions were also given for the occasions when participants could not fully comply, such as holidays and when dining out. Deviation from the Exchange model for dietary fat manipulation 847

3 TABLE 1 disease 1 Food-exchange model for adults in the United Kingdom at moderate risk of cardiovascular Total energy Total fat SFA MUFA n 6 PUFA M F M F M F M F M F MJ/d g/d g/d g/d g/d Total habitual intake Total habitual intake, %TE Exchangeable fat intake 3 Added oils Added fats Milk Cheese Snacks Total exchangeable fat intake Total exchangeable fat intake adjusted for habitual intake Non-exchangeable fat intake Total fat, SFAs, MUFAs, and n 6 PUFAs are given as grams per day unless noted otherwise. %TE, percentage of total energy. 2 Mean daily nutritional intakes for a population with increased risk of cardiovascular disease (10). 3 Proportion of exchangeable fat in the habitual diet calculated using the 2001/2002 National Diet and Nutrition Survey (15). 4 Included cakes, buns, chocolate, potato chips, pastries, and cookies. dietary intervention was minimized because the study foods could be taken on holiday. Participants were provided with all spreads, oils, nuts, and snacks, with spreads and oils being supplied in quantities for household usage to encourage consumption and provided in plain packaging identifiable only by the diet code (A, B, or C). Discussion about study foods and habitual diets enabled personalization of the dietary advice. The food-exchange model was flexible, allowing products to be interchanged, i.e., if oil was not consumed 1 d then a larger, specified quantity of spread would be consumed on the same day to compensate. Any food not listed in the dietary advice could be eaten as normal. Additional information regarding the implementation of the diets is provided in the Supplemental Methods. For support and dietary advice, participants were in close contact with the nutritionists throughout the study. To improve compliance, the study avoided the holiday season in December/January. Furthermore, tables listing the specific study foods and corresponding quantities were completed daily by the participants based on actual quantities consumed. These functioned as a reminder to consume the study foods, and, every 4-wk when participants collected their study foods, they were monitored and any issues with compliance were discussed. For the duration, body weight was to remain within 61 kg of baseline and was assessed every 4 wk. Weight change was addressed with advice to alter the intake of study snacks or carbohydrates and to avoid changes in activity levels. Assessment of dietary intake. Approximately 2 wk before the baseline clinical visit, trained researchers provided participants with digital food scales and full written and verbal instructions for recording 4-d weighed diet diaries. Emphasis was placed on not changing dietary habits. Participants completed diet diaries on 3 occasions (weeks 0, 8, and 16): the baseline diet diary (week 0) represented the habitual diet, and the combined week 8 and 16 diet diaries assessed participant compliance to the intervention diets. They were recorded on 1 weekend day and 3 weekdays, with the specific days of the week kept constant for all 3 diaries. During participant visits, diet diaries were assessed for completeness, and additional information was requested if necessary. Food prepared outside of the home was generally not weighed. Instead, portion sizes were estimated using household measures or portion-size images (16) and quantified using published food portion tables (17). Using the nutrient analysis software Dietplan 6.6 (Forestfield Software), diet diaries were analyzed using the NDNS nutrient databank, which included n 3 and n 6 PUFA values for all foods (15). Specific study foods were manually added to Dietplan. Mean daily intakes of energy, macronutrients, and alcohol were recorded, and the %TE was calculated to adjust for energy intakes. Mean daily intakes of dietary cholesterol, fiber (nonstarch polysaccharide), and sodium were also recorded but not energy adjusted. Assessment of underreporting. An estimate of dietary underreporting was determined. For all participants, basal metabolic rates were estimated using the Henry equation (18), as used preferentially by the Scientific Advisory Committee on Nutrition (United Kingdom) (19), and a physical activity level (PAL) of 1.2 was assigned to represent a sedentary lifestyle. The Goldberg lower 95% confidence limit was calculated as using the CV recommended by Black (20) (n = 192; 4 d). Under-reporters were identified by a ratio of reported energy intaketo-basal metabolic rate of < Anthropometric measurements. After an overnight fast, BMI and waist circumferences were recorded at weeks 0, 4, 8, 12, and 16. Height was measured to the nearest 0.5 cm using a wall-mounted stadiometer. While wearing light clothing, BMI was measured using the Tanita BC-418 digital scale (Tanita Europe) using standard settings (normal body type and 21 kg for clothing). With participants standing upright and at the end of expiration, waist circumference was measured at the minimum circumference halfway between the iliac crest and lowest rib margin to the nearest 0.5 cm by a trained researcher. Assessment of plasma phospholipid FA status. As a biomarker of short-term FA intake (days to weeks) (21), plasma phospholipid FA status was determined to assess compliance to the 16-wk dietary intervention. At weeks 0 and 16, fasting blood samples were collected into K 3 EDTA-containing vacutainers. After resting on ice for 30 min, samples were centrifuged at g for 15 min at 4 C, and 500 ml of plasma was stored at 280 C until required. Analysis of the plasma phospholipid FA fraction was performed by GC using the method by Burdge et al. (22) with a few modifications. FAME were synthesized by incubation with methanolic sulfuric acid (2% H 2 SO 4 v:v) at 70 C for 1 h and resolved using a CP-Sil 88 capillary column (60 m mm mm; Varian). Comparison of retention times against known standards (Supelco 37 component FAME mix and PUFA-3 Menhaden Oil standards; Sigma) identified the FAME. 1,2-Dipentadecanoyl-sn-glycero-3-phosphocholine (Sigma) was the internal standard. The area under the peak was analyzed using the Chemstation software (Agilent). Results were expressed as percentage of area of total plasma phospholipid FAs. Statistical analysis. For all variables, suitable checks for normality were implemented using SPSS (version 19; IBM SPSS) and, if necessary, variables were log transformed. Differences at baseline were determined 848 Weech et al.

4 TABLE 2 Replacement model for diets rich in SFAs, MUFAs, and n 6 PUFAs for use in adults in the United Kingdom at moderate risk of cardiovascular disease 1 Quantity Total energy Total fat SFAs MUFAs n 6 PUFAs M F M F M F M F M F M F g/d MJ/d g/d g/d g/d g/d Non-exchangeable fat intake SFA-rich diet Exchangeable fat intakes Spread (butter) Oil replacement (butter) Cheese (full fat) Milk (semi-skimmed) SFA-rich snacks (e.g., crackers, cookies, chocolate) Total intake Total intake, %TE Target intake, %TE MUFA-rich diet Exchangeable fat intakes Spread (olive and rapeseed oil blend) Oil (refined olive) Cheese (very low fat, e.g., cottage cheese) Milk (skimmed) MUFA-rich snacks (e.g., potato chips, cereal bars, olives, cookies) 2 Hazelnuts Total intake Total intake, %TE Target intake, %TE n 6 PUFA-rich diet Exchangeable fat intakes Spread (safflower oil blend) Oil (safflower) Cheese (very low fat, e.g., cottage cheese) Milk (skimmed) n 6 PUFA-rich and MUFA-rich snacks (e.g., popcorn, cake, potato chips, olives) 2 Hazelnuts Total intake Total intake, %TE Target intake, %TE Total fat, SFAs, MUFAs, and n 6 PUFAs are given as grams per day unless noted otherwise. %TE, percentage of total energy. 2 Quantity varied depending on the snack food. The replacement model was based on 2 snacks per day (32). 3 Total intake is the sum of exchangeable and non-exchangeable intakes based on megajoules per day for energy and grams per day for FAs. using ANOVA or, if non-normally distributed, using the Kruskal-Wallis test. For the analysis of dietary assessments, plasma phospholipid FA composition, and anthropometric measurements, the overall effect of diet was derived by univariate analysis (general linear model) using the change from baseline (week 16 2 week 0), with TukeyÕs correction for post hoc analysis. Baseline values of the corresponding variable, BMI, age, gender, and intervention diet were used as prognostic variables. A probability level of P # 0.05 was considered significant. Data presented represent means 6 SEMs. To investigate the unexpected plasma phospholipid 18:2n 6 status of the n 6 PUFA group, univariate analysis was performed on subgroups of participants whose proportions of plasma phospholipid 18:2n 6 increased (subgroup 1) or decreased (subgroup 2) by >1% relative to baseline for dietary n 6 PUFAs and plasma phospholipid 18:2n 6 and total n 6 PUFAs. The same prognostic variables were used with subgroup replacing diet. Correlations between dietary intake and plasma phospholipid for SFAs, MUFAs, and n 6 PUFAs were determined using Pearson correlation coefficients (2-tailed). To display the FA intake profiles of participants in each intervention group at baseline and after intervention and their proximity to the target intake profiles, recorded dietary FA intakes (%TE of SFAs, MUFAs, and n 6 PUFAs) were subjected to principal component analysis (PCA) using Minitab (version 16; Minitab). PCA was run on the correlation matrix. No rotation was applied because the resulting score plots from unrotated and varimax-rotated components yielded an identical conclusion. With eigenvalues $1 and explaining >80% variance, 2 principal components (PCs) were retained that produced score plots representing the component score for each participant at baseline and after intervention. Target FA profiles were added to the plots to represent the theoretical target compositions (%TE of SFAs, MUFAs, and n 6 PUFAs) of the 3 intervention diets. Factor loadings of the 2 PCs were used to interpret the FA profiles of the clusters on the score plots. Results Participant flow through the study is shown in Figure 1. Of 202 participants who were randomly assigned and commenced the study, 195 (85 males and 110 females) completed it. At baseline, Exchange model for dietary fat manipulation 849

5 Dietary analysis. For the dietary assessment (n = 192), there were no significant differences between the diet groups for any dietary variable at baseline. The mean recorded intakes of each group during the intervention broadly met the target intakes and were maintained for the 16-wk intervention period (Table 3). There was no overall diet effect (based on changes from baseline between the intervention groups) for total dietary fat intake. In line with target intakes, there were significant changes in dietary SFAs, MUFAs, and n 6 PUFAs between the 3 dietary groups. The mean increase in SFA intake from baseline in the SFA group was significantly different from the reductions in the MUFA and n 6 PUFA groups relative to baseline, which did not differ significantly from one another. For the changes in both dietary MUFAs and n 6 PUFAs, all 3 intervention groups were significantly different (P < 0.001), with the MUFA and n 6 PUFA groups reaching their target intake of 19%TE MUFAs and 10%TE n 6 PUFAs, respectively. PCA of the dietary records showed a lack of distinction between FA intakes at baseline (Fig. 2A). Factor loadings (Table 4) of PC1 and PC2 at baseline identified that participants with factor scores located in the right quadrants of Figure 2A had high intakes of SFAs and MUFAs, as denoted by high positive loadings for PC1 (0.72 and 0.70, respectively), whereas those in the bottom quadrants had higher intakes of n 6 PUFAs identified by a high negative loading (20.95) for PC2. In contrast to baseline, the post-intervention score plot showed complete separation of the FA profiles recorded from participants following the 3 intervention diets (Fig. 2B). Furthermore, these factor scores clustered around the respective target intakes, suggestive of compliance. Loading weights identified the cluster in the bottom right quadrant, generally belonging to the MUFA group (Fig. 2B), as being rich in MUFAs but low in SFAs and n 6 PUFAs (Table 4). In contrast, the cluster on the far left side, belonging primarily to the SFA group (Fig. 2B), displayed high intakes of SFAs but low intakes of MUFAs and n 6 PUFAs, whereas FA intakes for the cluster of participants in the n 6 PUFA group (top right quadrant) were rich in n 6 PUFAs but low in SFAs. Despite considerable effort to maintain isoenergetic intervention diets, there was a significant overall diet effect for the changes in reported total energy intake (P = 0.023), with significant differences between the SFA and MUFA diet groups. Total energy intake remained constant after the SFA diet but reduced significantly from baseline after both the MUFA (change: MJ; P = 0.001) and n 6 PUFA diets (change: MJ; P = 0.015). Additionally, the SFA group had lower intakes of n 3 PUFAs and higher intakes of trans fat and dietary cholesterol relative to baseline. These were significantly different from the changes in the MUFA and n 6 PUFA groups that showed small increases in n 3 PUFAs (significant for MUFA) and significant reductions in trans fat and dietary cholesterol. There were no significant effects of diet for changes in protein, carbohydrates, alcohol, and sodium, although there was for dietary fiber (P = 0.022) whereby the reduction in the SFA group relative to baseline was significantly different from the increase in the n 6 PUFA group. FIGURE 1 Flow of participants through the different stages of the Dietary Intervention and Vascular Function study. there were no significant differences between the intervention groups (Supplemental Table 3). The ethnicity of the study group was as follows: 1) whites, 84%; 2) Asians, 8%; 3) blacks, 7%; and 4) Chinese/Far Easterners, 2%. Underestimation. It was estimated that, if the participants were in energy balance, then the reported energy intakes were insufficient for 38.0% of participants at baseline and 48.4% after intervention. However, underreporting was not considered further in the statistical analysis. Anthropometric measures. The changes in BMI were not significantly different between the SFA ( vs kg/m 2 for baseline vs. after intervention; n = 65), MUFA ( vs kg/m 2 ; n = 64), and n 6 PUFA ( vs kg/m 2 ; n = 66) groups. Furthermore, there was no significant overall diet effect for changes in waist circumference between the intervention groups (n = 56 63). Relative to baseline, post-intervention waist circumference did not differ significantly in the SFA ( vs cm for baseline vs. after intervention) and n 6 PUFA ( vs cm) groups, although there was a small yet significant decrease in the MUFA group ( vs cm, P = 0.008). Plasma phospholipid FA analysis. After the 16-wk intervention, there were significant changes in plasma phospholipid FA composition between the intervention groups (n = 190) (Table 5). For the changes in phospholipid total SFA and MUFA, there were significant overall effects of diet (P < 0.001), with differences between the SFA group and the MUFA and n 6 PUFA groups for both classes of plasma phospholipid FAs (P < for all). However, the changes were not significantly different between the latter 2 diet groups. Compared with baseline, phospholipid total SFA, 14:0, and 16:0 increased in the SFA group but decreased in the MUFA and n 6 PUFA groups. For phospholipid total MUFAs, there were significant post-intervention increases in the MUFA and n 6 PUFA groups vs. baseline (P < 0.001), as opposed to a near significant decrease in the SFA group (P = 0.057). Similar effects were apparent for 18:1n Weech et al.

6 TABLE 3 Reported daily composition of diets at baseline (week 0) and after diets rich in SFAs, MUFAs, and n 6 PUFAs (week 16) in adults with moderate risk of cardiovascular disease and target FA intakes 1 SFA diet (n = 65) MUFA diet (n = 64) n 6 PUFA diet (n = 63) Baseline Post T 2 D Baseline Post T D Baseline Post T D P 3 Energy, MJ/d a *** b * b Total fat, %TE *** *** ** SFA, %TE *** a *** b *** b,0.001 MUFA, %TE *** a *** b ** c,0.001 n 6 PUFA, %TE *** a ** b *** c,0.001 n 3 PUFA, %TE *** a * b b,0.001 Total PUFA, %TE *** a * b *** c,0.001 trans fat, %TE *** a *** b *** b,0.001 Protein, %TE * Carbohydrates, %TE *** * Alcohol, %TE * Dietary fiber (NSP), g/d a ab * b Cholesterol, mg/d a *** b *** b,0.001 Sodium, g/d * Values are given as means 6 SEMs. Dietary intakes estimated from 4-d weighed diet diaries at baseline (week 0) and after intervention (mean of weeks 8 and 16). Post-intervention intakes significantly different from baseline identified as follows: *P # 0.05; **P # 0.01; ***P # Post hoc analyses used TukeyÕs correction. Different superscript letters within a row identify comparisons significantly different from one another (P # 0.05). NSP, nonstarch polysaccharide; Post, after the intervention; T, target; %TE, percentage of total energy. 2 Target FA intakes for the 3 intervention diets. 3 Overall effect of diet based on change from baseline was derived by univariate analysis (general linear model) for between-group comparisons using baseline values of variable of interest, BMI, age, intervention diet, and gender as prognostic factors. Exchange model for dietary fat manipulation 851

7 FIGURE 2 PCA score plots at baseline (A) and after intervention (B) for the dietary FA intake profiles of adults with moderate risk of cardiovascular disease (n = 192) on the SFA, MUFA, and n 6 PUFA intervention diets. Mean FA intakes (%TE) for each participant at baseline and after intervention were determined using 4-d weighed diet diaries. PCA was run on the correlation matrix. No rotation was applied. Score plots of PC1 vs. PC2 represent the component score for each participant at baseline (explaining 84.9% variation) and after intervention (89.6%). Target FA profiles added to the post-intervention plot (B) represent the theoretical target compositions of the intervention diets (%TE SFA:%TE MUFA:%TE n 6 PUFA): 1) SFA diet (17:11:4); 2) MUFA diet (9:19:4); and 3) n 6 PUFA diet (9:13:10). PC, principal component; PCA, principal component analysis; %TE, percentage of total energy. Regarding changes in phospholipid total n 6 PUFAs, 18:2n 6, 18:3n 6, 20:3n 6, and 20:4n 6, there were no significant overall effects of diet, although 18:2n 6 was reduced in the MUFA group relative to baseline (P = 0.021). For the n 6 PUFA group, the mean post-intervention increase in phospholipid total n 6 PUFAs was smaller than anticipated for a %TE increase in dietary n 6 PUFAs. Subgroup analysis of participants whose plasma phospholipid 18:2n 6 either increased (subgroup 1; n = 14) or decreased (subgroup 2; n = 17) by >1% relative to baseline showed differences within the n 6 PUFA intervention group. For phospholipid 18:2n 6, the mean change from baseline was % in subgroup 1 compared with % in subgroup 2 (P < 0.001), similar to phospholipid total n 6 PUFAs (change: vs %, respectively; P < 0.001). However, at baseline, there were significantly higher proportions of plasma phospholipid 18:2n 6 ( vs %; P = 0.035) and phospholipid total n 6 PUFAs ( vs ; P = 0.015) in subgroup 2 compared with subgroup 1. Baseline intakes of dietary n 6 PUFAs did not differ significantly ( vs % TE for subgroup 1 and subgroup 2, respectively). Although an increase from baseline for total dietary n 6 PUFAs was reported for subgroup 2, this was significantly smaller than the increase for subgroup 1 (change: vs %TE; P < 0.001). There were no significant overall diet effects for the changes in the phospholipid total n 3 PUFA, total long-chain (LC) n 3 PUFA, 20:5n 3, and 22:6n 3. The n 6 PUFA group was significantly different from the MUFA group for 18:3n 3 (P = for the overall diet effect), with post-intervention decreases in the n 6 PUFA group vs. baseline (P = 0.002). However, the SFA group was significantly different from the MUFA and n 6 PUFA groups for 22:5n 3 (P < for the overall diet effect), with reductions observed in the latter 2 groups relative to baseline (P < 0.001). Correlation analysis between dietary intake and plasma phospholipid. There were moderate correlations between dietary intake and plasma phospholipid for SFAs (r = 0.36, P < 0.001) and MUFAs (r = 0.22, P = 0.003) based on changes from baseline (n = 187). No significant correlation was shown for n 6 PUFAs. Discussion The DIVAS food-exchange model allowed the successful implementation of 3 16-wk dietary interventions of specific FA composition, with no significant change in macronutrient intake (total fat, protein, and carbohydrates), in a free-living population with moderate CVD risk. This was confirmed by the analysis of the 4-d weighed diet diaries. Furthermore, the postintervention PCA plots showed very clear differences between the diet groups. The dietary FA intakes recorded from participants on each of the intervention diets clustered discretely around the corresponding target FA profile, providing additional evidence for the successful implementation of the food-exchange model. Used in this capacity, PCA is a novel method for analyzing dietary intakes and their deviation from target intakes, which could prove a useful technique for visualizing dietary compliance in future studies. Compliance was further confirmed by the FA composition in the plasma phospholipid fraction. This broadly reflected the dietary analysis, with significant moderate associations observed between the dietary records and plasma phospholipid FA composition for SFAs and MUFAs. The small difference of note was the change in n 6 and n 3 PUFA proportions in the plasma phospholipid fraction. Compared with baseline, the change in phospholipid total n 6 PUFAs in the n 6 PUFA group was smaller than might be expected from the large increase in %TE n 6 PUFAs. Epidemiologic studies show that dietary intakes of FAs associate well with plasma FAs that are not synthesized endogenously (e.g., n 6 and n 3 PUFAs), although the associations are TABLE 4 Factor loadings of PC1 and PC2 identified by principal component analysis of FA intakes at baseline (week 0) and after intervention (week 16) by adults at moderate cardiovascular risk 1 Baseline After intervention Dietary FA intake PC1 PC2 PC1 PC2 SFA MUFA n 6 PUFA Principal component analysis was run on the correlation matrix (n = 192). No rotation was applied. PC, principal component. 852 Weech et al.

8 TABLE 5 Proportion of plasma phospholipid FAs at baseline (week 0) and after diets rich in SFAs, MUFAs, and n 6 PUFAs (week 16) in adults with moderate risk of cardiovascular disease 1 SFA diet (n = 65) MUFA diet (n = 62) n 6 PUFA diet (n = 63) Baseline Post D Baseline Post D Baseline Post D P 2 % of area % of area % of area SFAs 12: : ** a ** b b, : ** a *** b *** b, : Total SFAs *** a *** b ** b,0.001 MUFAs 18:1n * a *** b *** b,0.001 Total MUFAs a *** b *** b,0.001 n 6 PUFAs 18:2n * :3n :3n :4n Total n 6 PUFAs n 3 PUFAs 18:3n ab a ** b :5n * :5n a ** b *** b, :6n Total n 3 PUFAs Total LC n 3 PUFAs Values given as means 6 SEMs. Post-intervention values differing from baseline identified as follows: *P # 0.05; **P # 0.01; ***P # Post hoc analyses used TukeyÕs correction. Different superscript letters within a row identify comparisons significantly different from one another (P # 0.05). LC, long-chain; Post, after the intervention. 2 Overall effect of diet for change from baseline was derived by univariate analysis (general linear model) for between-group comparisons with baseline values for the variable of interest, BMI, age, diet, and gender as prognostic factors. 3 Total LC n 3 PUFAs included 20:5n 3, 22:5n 3, and 22:6n 3, and change was log transformed before statistical analysis. weaker with FAs synthesized endogenously from carbohydrates (e.g., SFAs and MUFAs) (21). After a 5-wk n 6 PUFA-rich diet (10%TE SFAs and 13%TE n 6 PUFAs), 18:2n 6 in the plasma phospholipid fraction increased by 5.2 mol% relative to an SFArich diet (17%TE SFAs and 4%TE n 6 PUFAs). However, interestingly, it was observed that the increase in 18:2n 6 was much greater in the cholesteryl ester fraction (10 mol%) (23). Therefore, the lack of significant increase in n 6 PUFAs in the plasma phospholipid was a little unexpected. Subgroup analysis of the n 6 PUFA intervention group identified differences between the participants whose proportions of phospholipid 18:2n 6 either increased or decreased by $1% at week 16 relative to baseline (data not shown). It was observed that the participants who consumed greater amounts of total n 6 PUFAs during the intervention period and who had lower proportions of phospholipid 18:2n 6 at baseline showed increases of $1% in plasma phospholipid 18:2n 6 in response to the n 6 PUFA dietary intervention. This suggests that both dietary intake and baseline phospholipid FA status may have influenced the changes in plasma phospholipid total n 6 PUFAs observed. Plasma FA composition may not directly reflect dietary intake as a result of the competitive hyperbolic relation between intakes of n 3 and n 6 PUFAs and proportions of highly unsaturated n 3 PUFAs and n 6 PUFAs in plasma phospholipid. For example, dietary n 3 PUFA intake was significantly reduced in response to the SFA diet, the majority of which would have been provided as 18:3n 3 [or a-linolenic acid (ALNA)]. However, there was a significant diet effect for plasma phospholipid 22:5n 3, which did not change in the SFA group but decreased significantly in the n 6 PUFA and MUFA groups. This finding was not unexpected. A reduction in LC n 3 PUFAs, specifically 20:5n 3, was reported previously in healthy men in response to diets rich in n 6 PUFAs (10.5%TE vs. 3.8%TE), which is assumed to result from increased competition for the desaturase and elongase enzymes with high intakes of n 6 PUFAs (24). This hyperbolic relation could be explored further using the Lands models (25). The diets were designed to be isoenergetic, which was confirmed by the maintenance of mean BMI and waist circumference over the 16-wk intervention period. Because weight change could act as a confounding factor [for example, a study in overweight/obese adults reported significant improvements in arterial stiffness after 6 mo of weight loss (26)], weight maintenance was imperative. This was achieved by frequent monitoring and close contact between the participants and study investigators, who provided specific advice when they observed weight changes. According to the dietary analysis, there were statistically significant reductions in reported energy intake in the MUFA group and, to a lesser extent, in the n 6 PUFA group compared with the habitual diet. However, all 3 spreads provided ;3000 kj/100 g, and the mean energy content of the snacks per portion were very similar (SFA diet: 470 kj; MUFA diet: 478 kj; n 6 PUFA diet: 476 kj) (data not shown). Reducing the SFA content in the MUFA and n 6 PUFA diets by substituting whole-fat dairy products for low-fat dairy products may have inadvertently contributed to the reduced energy intake in these groups. However, regardless of energy intake, BMI and Exchange model for dietary fat manipulation 853

9 waist circumference did not significantly differ between diet groups. According to the dietary analysis, there were small but significant differences in dietary cholesterol and trans fat intakes between the intervention groups, which mirrored the changes in dietary SFAs. It is often difficult to separate intakes of these nutrients, because foods rich in SFAs (such as butter and cheese) also contain cholesterol and ruminant-derived trans fats, although this was kept to a minimum within the food-exchange model. The changes in trans fats and dietary cholesterol could be considered as possible confounding factors. Trans fats affect CVD risk independently from SFAs (27), although, at current intakes, dietary cholesterol has little impact (28,29). Changes in trans fats and dietary cholesterol were significantly different between intervention groups, but the changes were quantitatively small compared with the large differences in postintervention SFA intakes between the SFA group and the MUFA (9.5%TE) and n 6 PUFA (9.6%TE) groups, accounting for ;27% of total dietary fat. Importantly, the trans fat and cholesterol intakes for all intervention groups were lower than the recommended maximum dietary intake of 2% of food energy (27) and 300 mg/d (30), respectively. Detrimental effects of these nutrients on CVD risk and lipid concentrations are reported for intakes far higher than those consumed by all 3 intervention groups. In addition, the change in dietary n 3 PUFAs was significantly different between the intervention groups, with a significant reduction after the SFA diet compared with the unsaturated diets. However, these changes were due to differences in ALNA intake rather than LC n 3 PUFA intake. At these intake amounts, ALNA has been associated with minimal effects on vascular function or fasting lipid profiles (7,31). There was also a significant diet effect for dietary fiber, with a difference of 1.9 g/d between the SFA and n 6 PUFA groups. However, mean baseline and post-intervention dietary fiber intakes for all groups were #17.7 g/d, which were lower than the suggested $25 g/d required to significantly improve cardiovascular risk factors in the middle-aged (32). A major challenge was to motivate a large group of participants with differing lifestyles and dietary habits to comply with the 16-wk intervention diets. This was achieved through frequent contact with the participants and careful monitoring of their dietary intake and weight. The dropout rate of participants who attended the first clinical visit but failed to complete was very low at 3.5% (n = 7), suggesting that the dietary advice was relatively easy to follow and not too disruptive to their overall diet or lifestyle. However, a major challenge faced by many participants on the 2 unsaturated fat diets was consuming sufficient oil to reach their dietary targets, which resulted in additional spread being consumed to compensate. In contrast, it was observed that participants from an Asian background consumed more oil (data not shown). Therefore, flexibility between spread and oil intake was essential. Participants who did not habitually snack struggled to incorporate these items into their diets, especially without gaining weight. When designing the food-exchange model, identifying snacks with suitable FA compositions proved challenging. There was a limited choice of savory snacks for the SFA diet because of the increased use of sunflower and high-oleic sunflower oils in the manufacture of many potato-based snacks and of sweet snacks for the MUFA diet. There is always a degree of underreporting in any dietary assessment. In the DIVAS study, 65% of participants were categorized as overweight or obese, a population known for selective bias in dietary assessments, whereby unhealthy foods are omitted, leading to underreporting of dietary intakes (33). For example, a significantly greater degree of underreporting was identified in obese females vs. normal-weight females as assessed by 24-h diet recalls and 3-d food records (34). Using a PAL of 1.2 to represent a sedentary lifestyle, 38% of participants were identified as underreporters at baseline in the DIVAS study, which was similar to 2 of the 4 studies that used weighed food records (37.5% and 38.5%) identified in a review of dietary misreporting (35). However, using the Goldberg equation on 7-d diet records of 1551 adults from the 2000/2001 NDNS, 75% of men and 77% of women were classified as underreporters, although the PAL were based on individual activity levels (36). To adjust for the variation of energy intakes in the DIVAS study, macronutrients were expressed as %TE rather than using absolute intakes of macronutrients. Furthermore, the participants who underreported did so consistently at baseline and throughout the intervention. Therefore, the change from baseline, on which the statistical analysis was based, may not be greatly affected. In conclusion, a flexible food-exchange model for the longterm substitution of dietary SFAs with MUFAs or n 6 PUFAs was successfully developed. In a free-living population with a moderate CVD risk (50% greater than the population mean), the 3 intervention diets with distinct FA compositions were successfully implemented with minimal disruption to the overall diet while effectively influencing the intake of specific dietary FAs. Acknowledgments The authors thank R. Mihaylova, J. Luff, and the research students (A. Curran, E. Hobby, M. Kellermann-Thornton, and B. Doyle) for their help with recruitment and study visits and P. Zock and M. Cosgrove at Unilever, who produced and supplied in kind the study spreads and oils according to the authorsõ specification. J.A.L., K.G.J., P.Y., and S.T. designed the study; M.W. and J.A.L. designed the food-exchange model; M.W., K.V., and M.H. implemented the research; M.W., K.V., and M.H. analyzed the data; S.T. provided statistical advice; M.W. wrote the manuscript, which was modified by all coauthors; and J.A.L. had primary responsibility for final content and study management. All authors read and approved the final manuscript. Literature Cited 1. Scarborough P, Bhatnagar P, Wickramasinghe K, Smolina K, Mitchell C, Rayner M. Coronary heart disease statistics. London: British Heart Foundation; Department of Health Committee on Medical Aspects of Food Policy. Dietary reference values for food energy and nutrients in the United Kingdom: report on the Panel of Dietary Reference Values of the Committee on Medical Aspects of Food Policy. Report of Health and Social Subjects, No. 41. London: The Stationary Office; Bates B, Lennox A, Prentice A, Bates C, Swan G. National diet and nutrition survey: headline results from years 1, 2 and 3 (combined) of the Rolling Programme (2008/ /11) [cited 2013 May 24]. Available from: 4. Hooper L, Summerbell CD, Thompson R, Sills D, Roberts FG, Moore HJ, Davey Smith G. Reduced or modified dietary fat for preventing cardiovascular disease. Cochrane Database Syst Rev. 2012;5:CD Jakobsen MU, OÕReilly EJ, Heitmann BL, Pereira MA, Balter K, Fraser GE, Goldbourt U, Hallmans G, Knekt P, Liu S, et al. Major types of dietary fat and risk of coronary heart disease: a pooled analysis of 11 cohort studies. Am J Clin Nutr. 2009;89: Micha R, Mozaffarian D. Saturated fat and cardiometabolic risk factors, coronary heart disease, stroke, and diabetes: a fresh look at the evidence. Lipids. 2010;45: Weech et al.

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