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Interaction of Fatty Acid Genotype and Diet on Changes in Colonic Fatty Acids in a 1 Mediterranean Diet Intervention Study 2

3 Shannon R. Porentaa, Yi-An Kob, Stephen B. Grubera*, 4

Bhramar Mukherjeeb, Ana Baylinc, Jianwei Rend, and Zora Djuricd 5 6

a Departments of Internal Medicine, b Biostatistics, c Epidemiology, and d Family Medicine 7 University of Michigan, Ann Arbor, MI 48109 8

*Present address: University of Southern California, Norris Comprehensive Cancer Center, 9 Keck School of Medicine, Los Angeles, CA 90089 10

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Corresponding author: 13 14 Zora Djuric, Ph.D. 15 1500 E. Medical Center Drive 16 Room 2150 Cancer Center 17 University of Michigan 18 Ann Arbor, MI 48109-5930 19 20 Phone: 734-615-6210 FAX: 734-647-9817 21 Email: [email protected] 22 23 Conflicts of Interest: 24 None of the authors have any conflicts of interest to declare with the reported research. 25 26 Acknowledgements 27 We thank all the individuals who volunteered for the Healthy Eating Study for Colon Cancer 28 Prevention. The parent study was designed and conducted in collaboration with Drs. Dean E. 29 Brenner, Mack T. Ruffin, D. Kim Turgeon and Ananda Sen. Mary Rapai was the coordinator for 30 the study and Maria Cornellier was the study dietitian. 31 This study was supported by a grant from the Ronald P. and Joan M. Nordgren Cancer Research 32 Fund, NIH grant RO1 CA120381, and Cancer Center Support Grant P30 CA046592. The study 33 used core resources supported by a Clinical Translational Science Award, NIH grant 34 UL1RR024986 (the Michigan Clinical Research Unit), the Michigan Diabetes Research Center 35 NIH grant 5P60 DK020572 (Chemistry Laboratory), and the Michigan Nutrition and Obesity 36 Research Center NIH grant P30 DK089503. 37

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Abstract 38 A Mediterranean diet increases intakes of n-3 and n-9 fatty acids and lowers intake of n-6 fatty 39 acids. This can impact colon cancer risk since n-6 fatty acids are metabolized to pro-40 inflammatory eicosanoids. The purpose of this study was to evaluate interactions of 41 polymorphisms in the fatty acid desaturase genes, FADS1 and FADS2, and changes in diet on 42 fatty acid concentrations in serum and colon. A total of 108 individuals at increased risk of colon 43 cancer were randomized to either a Mediterranean or a Healthy Eating diet. Fatty acids were 44 measured in both serum and colonic mucosa at baseline and after 6 months. Each individual was 45 genotyped for four single nucleotide polymorphisms in the FADS gene cluster. Linear regression 46 was used to evaluate the effects of diet, genotype and the diet by genotype interaction on fatty 47 acid concentrations in serum and colon. Genetic variation in the FADS genes was strongly 48 associated with baseline serum arachidonic acid (n-6, AA) but serum eicosapentaenoic acid (n-3) 49 and colonic fatty acid concentrations were not significantly associated with genotype. After 50 intervention, there was a significant diet by genotype interaction for AA concentrations in colon. 51 Subjects who had all major alleles for FADS1/2 and were following a Mediterranean diet had 52 16% lower AA concentrations in the colon after 6 months of intervention than subjects following 53 the Healthy Eating diet. These results indicate that FADS genotype could modify the effects of 54 changes in dietary fat intakes on AA concentrations in the colon. 55 56 57 58




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Abbreviations 59 FADS1: Fatty Acid Desaturase 1(Delta-5 desaturase), FADS2: Fatty Acid Desaturase 2 (Delta-6 60 desaturase); AA: Arachidonic Acid (20:4, n-6), EPA: Eicosapentaenoic Acid (20:5, n-3), DHA: 61 Docosahexaenoic Acid; MUFA: Monounsaturated Fatty Acids; PUFA: Polyunsaturated Fatty 62 Acids; SFA: Saturated Fatty Acids. 63 64 Key Words 65 Fatty acid desaturase, diet-genotype interaction, colon cancer prevention, nutrition 66 67 Running Title 68 Fatty Acid Genotype, Diet and Colonic Fatty Acids 69 70 71




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Introduction 72 Many studies have suggested that a Mediterranean diet, as compared to a typical Western 73

diet, may decrease the risk of various chronic diseases including colorectal cancer (1, 2). Rates of 74 colorectal cancer were very low in Greece and have increased as diet has drifted away from the 75 traditional eating pattern (3). The traditional Greek diet, relative to a Western diet, had lower 76 intakes of n-6 polyunsaturated fatty acids (PUFA) and red meat, but higher intakes of plant-77 based foods, fish and monounsaturated fatty acids (MUFA) chiefly from olive oil (2). The fat 78 content of the Mediterranean diet is of particular interest for colon cancer prevention since in 79 intervention studies increasing fiber alone does not appear to be preventive, and increased 80 intakes of fruit and vegetables have had modest preventive effects (4-6). In particular, we 81 hypothesized lower intakes of n-6 linoleic acid and higher intakes of n-3 fatty acids have 82 implications for preventing colon cancer since n-6 fatty acids are metabolized to eicosanoids 83 such as prostaglandin E2 (PGE2) that is pro-inflammatory in the colon (7). PGE2 is formed from 84 arachidonic acid (AA, 20:4 n-6) by cyclooxygenases in the colonic mucosa, and it plays an 85 important role in colonic crypt cellular expansion and subsequent adenoma formation (8). 86

In addition to the possible effects of dietary intakes, genetic variation in fatty acid desaturase 87 genes has been shown to influence serum and tissue AA concentrations (9-15). Delta-5 88 desaturase (FADS1) and delta-6 desaturase (FADS2) are key desaturase enzymes involved in the 89 synthesis of AA and eicosapentaenoic acid (EPA, 20:5, n-3) from 18 carbon precursor fatty 90 acids. Dietary intake of AA is low in humans; however, AA comprises between 5-10% of the 91 phospholipids in cells due to elongation and desaturation of linoleic acid (18:2 n-6) to AA (16). 92

Polymorphisms in the FADS1 and FADS2 genes have been identified, and these significantly 93 affect PUFA concentrations in serum. The minor alleles are associated with lower desaturase 94




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activity and lower concentrations of AA in blood (9-15). Analogous associations for EPA and 95 docosahexaenoic acid (DHA) have not been consistent across studies, perhaps since certain types 96 of fish can supply high amounts of pre-formed EPA and DHA. Dietary intakes are important to 97 consider since conversion of dietary linolenic acid to longer chain n-3 fatty acids competes with 98 the analogous process for n-6 fatty acids (17). (In addition to diet, desaturase activity appears to 99 be important in cardiovascular health, and presence of the minor allele in FADS1/2 has been 100 associated with improved measures of blood lipids, C-reactive protein, insulin and fasting 101 glucose (18-21). This indicates that lower AA levels are associated with lower pro-inflammatory 102 states. The prevalence of minor alleles appears to have evolved in response to Western diets that 103 are plentiful in n-6 fatty acids, and they are more prevalent in persons of European descent than 104 of African descent (11, 22). 105

Much less research is available on how FADS polymorphisms might affect changes in fatty 106 acids in response to changes in diet, and the available studies have generally focused on n-3 fatty 107 acid supplementation. Flaxseed supplementation, which provides linolenic acid (18:3, n-3), was 108 less effective in increasing EPA concentrations in minor allele carriers of either FADS1 or 109 FADS2, resulting in significant diet by genotype interactions on plasma concentrations of EPA 110 and AA (23). Dietary n-3 fatty acids also may interact with FADS genotype in affecting 111 concentrations of blood cholesterol and triglycerides, with significant beneficial effects for 112 carriers of all minor alleles being found in some but not all studies (20, 24-26). 113

The goal of this present study was to assess potential interactions of polymorphisms in 114 FADS1 and FADS2 with changes in diet on levels of arachidonic acid (AA) and 115 eicosapentaenoic acid (EPA) in the serum and in the colonic mucosa of persons at increased risk 116 for colon cancer. This was a secondary analysis of a randomized clinical trial that evaluated 117




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changes in fatty acids and carotenoids elicited by six months of intervention with either a 118 Mediterranean or a standard Healthy Eating diet. In that study we observed that dietary changes 119 had little effect on colon fatty acids, which led to the hypothesis that metabolic factors may be 120 limiting for changes in fatty acids (27). The randomized study obtained both blood and colon 121 biopsies. Here, the relationships of FADS polymorphisms with serum and colonic fatty acid 122 concentrations were evaluated at baseline and after six months of dietary intervention. 123 124 125 126 Methods 127 128 Study Design and Eligibility 129

Details of recruitment and conduct of the Healthy Eating for Colon Cancer Prevention Study 130 have been published previously (27, 28). The study was approved by the University of Michigan 131 Medical Internal Review Board and was registered at the ClinicalTrials.org (NCT00475722). 132 Briefly, 120 individuals at increased risk of colon cancer gave informed consent and were 133 randomized to follow a modified Mediterranean diet or to Healthy People 2010 diet for 6 134 months. Blood and colonic mucosal tissue samples were collected at baseline and at 6 months by 135 flexible sigmoidoscopy without prior preparation of the bowels. Blood was drawn after an 136 overnight fast. At baseline, a Health Status Questionnaire was filled out by participants that 137 included health and demographic data. Health information was asked again at 6 months. Dietary 138 data was collected at 0 and 6 months using two days of food records and two 24-hour recalls. 139

The decision to genotype subjects with regard to fatty acid desaturases was made after the 140 study began, and consent for genotyping could not be obtained from nine individuals, two of 141 whom completed 6 months of study and seven of whom had dropped out after enrolling. Three 142




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samples were not genotyped successfully. The present analysis therefore included 108 of 120 143 subjects enrolled in the study and randomized to 6 months of counseling for either a 144 Mediterranean or a Healthy Eating diet. 145

The frequency of counseling sessions was the same in both study arms. The Healthy Eating 146 diet had dietary goals based on the Healthy People 2010 diet. The goals were to include 2 147 servings/day of fruit, 3 servings/day of vegetables with at least one of those servings being dark 148 green or orange, 6 servings/day of grains with at least 3 from whole grains, less than 10% of 149 calories from saturated fat and less than 30% of calories from total fat. The Mediterranean diet 150 had goals for consumption of high n-3 foods such as fish or flax at least 2 times a week, 151 consumption of foods in a manner to increase MUFA and decrease n-6 PUFA intakes, 6 152 servings/day of grains with at least 3 from whole grains, and 7-9 fruits and vegetable 153 servings/day in specified variety. 154

155 Serum and Colonic Fatty Acids 156

Fatty acid analysis was performed by gas chromatography with mass spectral detection (GC-157 MS) of fatty acid methyl esters. Total lipids were extracted from serum using a 1:1 mixture of 158 chloroform and methanol, and 17:0 (1,2-diheptadecanoyl-sn-glycero-3-phosphocholine) was 159 used as the internal standard. For colon tissue, one biopsy of about 5 mg was sufficient for 160 analysis of fatty acids. The biopsy was pulverized in liquid nitrogen, sonicated in 150 μl of ice-161 cold phosphate buffered saline containing 0.1% BHT and 1mM EDTA with an Ultrasonic 162 processor (30 seconds twice), and then total lipids were extracted with 1 ml of chloroform and 163 methanol (1:1). The organic layer in either case was used to prepare fatty acid methyl esters with 164 METH-PREP II derivatization reagent (Alltech, Deerfield, IL). The GC-MS analysis was carried 165




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out with A SupelcoSP2330 column, 30m X 0.32mm X 0.2µm film thickness (Sigma-Aldrich, St. 166 Louis, MO), a HP 7673/5971 GC-MS and helium as the carrier gas with a validated assay (29). 167 The following fatty acids in serum and colon tissue were measured in 12 analytical different 168 batches: 12:0, 14:0, 16:0, 16:1, 18:0, 18:1, 18:2 (n6), 18:3 (n3), 20:0, 20:1, 20:3 (n6), 20:4 (n6), 169 20:5 (n3) and 22:6 (n3). 170

171 DNA Extraction and Genotyping 172

Many polymorphisms have been identified in the FADS1/2 gene cluster. Haplotypes have 173 been constructed using 3 to 18 single nucleotide polymorphisms, and AA concentrations were 174 typically about 30% higher in carriers of all major alleles (9, 12, 16, 30). This literature has 175 indicated that there was little additional benefit from genotyping more than three SNPs, we 176 therefore chose to genotype the three SNPs used in the study of the Rzehak et al. (30). A 177 subsequent genome-wide association study indicated that another polymorphism in the FADS1/2 178 region explained 18% of the inter-individual variation in AA concentrations, we therefore added 179 rs174537 to the present analysis (21). 180

DNA was extracted from the buffy coat of heparinized blood samples. The buffy coat had 181 been collected for each blood sample and mixed with 1% sodium dodecyl sulfate/1 mM EDTA 182 prior to freezing at -80˚C. After all the samples had been collected, they were treated with 183 RNases A and heat-treated RNase T1 followed by digestion with protease K, solvent extraction 184 and precipitation of DNA. The DNA was purified using MinElute Reaction Cleanup Kit 185 (QIAGEN) to ensure high quality DNA for genotyping. Four SNPs were genotyped; one in the 186 FADS2 gene (rs3834458), two SNPs located in FADS1 (rs174556 and rs174561), and one SNP 187 located in the intragenic region between FADS1 and FADS2 (rs174537). 188




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Three of the SNPs (rs174556, rs174561, and rs174537) were genotyped using TaqMan SNP 189 Genotyping assays (Applied Biosystems). All assays were done both in real time and post read 190 mode for allelic discrimination on an AB7900 system. The rs3834458 polymorphisms was 191 detected by sequencing. For quality control 10% of all samples were re-genotyped. All plates 192 incorporated positive and negative controls. 193

PCR reactions for rs3834458 included 5 μl (20 pmol/μl) of both forward and reverse primers, 194 12 μl AmpliTaq Gold master mix, 10 μg/μl genomic DNA, and Millipore water for a total 195 volume of 25 μl. Primers used for rs3834458 were 5’-TCCACGATTCCCAAAGAGAC-3’ and 196 5’-TCTGCAACCTCCCTAGAGACA-3’. Samples were covered in mineral oil, denatured for 10 197 minutes at 95°C, were passed through 40 cycles of amplification consisting of 1 minute of 198 denaturation at 95°C, 1 minute of primer annealing at 55°C, and 1 minute of elongation at 72°C. 199 The PCR products were checked by running on a 2% agarose gel stained with ethidium bromide 200 prior to sequencing. Sequencing was done on an ABI 3730 sequencer in the University of 201 Michigan Sequencing Core Facility. 202

203 Statistical Analyses 204

The distributions of fatty acid variables were first checked for normality and transformed to 205 approximate normality as needed prior to analyses. The transformations applied prior to analysis 206 are given in the table footnotes, and variables were back transformed to calculate percent 207 increases or differences. Untransformed means are shown in the Tables for ease of interpretation. 208 Deviations from Hardy-Weinberg equilibrium for the genotypes of each SNP were tested using 209 chi-square tests. Differences in baseline parameters between diet arms were assessed using 210 independent t-tests or chi-square tests, as appropriate (Tables 1 and 2). Genotype data for the 211




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four SNPs were summarized to yield the count of minor alleles (the minimum and maximum 212 counts were 0 and 8, respectively). Linear regression was used to evaluate the effect of number 213 of minor alleles on fatty acid concentrations. Subsequently, a binary variable for genotype group 214 was created by the presence/absence of minor alleles, i.e., all major alleles versus one or more 215 minor alleles. 216

A linear mixed model was used to evaluate whether the presence of any FADS variant affects 217 baseline fatty acid concentrations (AA, EPA). Each of the baseline fatty acids in both serum and 218 colonic mucosa was regressed on genotype group (Table 2). Batch number was a random effect 219 to account for heterogeneity since fatty acids were measured in different batches. The covariates 220 in the model included age, gender, body mass index (BMI; in kg/m2), and dietary intake 221 measures of n-6 PUFA, n-3 PUFA and long chain n-3 PUFA (sum of the n-3 fatty acids 20:5, 222 22:5 and 22:6) as a percentage of energy using 9 kcal/gram. 223

Next, we used linear mixed models to evaluate the changes in fatty acid concentrations after 224 6 months of diet intervention: dietary intake, serum, and colon fatty acid concentrations were 225 regressed on time (baseline, 6 month) with a random intercept for each individual. For serum and 226 colon fatty acids, batch number was included in the random effects. Separate analyses were 227 performed for the two diet groups (Table 3). Finally, analyses were done to compare the changes 228 in fatty acid composition over 6 months between the two diet arms and to assess if the changes 229 were modified by the presence of minor alleles in FADS. For these analyses, each of the 230 outcome variables (AA, EPA for both serum and colonic mucosa) at 6-month follow-up was 231 regressed on genotype group, diet arm, and genotype group*diet assignment interaction by a 232 linear mixed model (Table 4). The model was adjusted for age, BMI, and the concentration of 233 each corresponding fatty acid at baseline. In all the models, batch number was incorporated as a 234




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random effect when appropriate. All reported P values were two-tailed. The statistical 235 significance was set α = 0.05 level. Analyses were performed using SAS version 9.1 (SAS 236 Institute, Cary, NC). 237 238 239 240 241 Results 242

243 Baseline Characteristics and Genotyping 244

The overall study consisted of 108 study participants after exclusions for lack of genotyping 245 consent (n=9) and incomplete genotype data (n=3). Genotyping success rate of the 4 SNPs 246 chosen to define the FADS1/2 haplotype as described in Methods, was between 96.7% and 247 98.3%. Minor allele frequencies were in the range of 25.0% to 32.9%.The genotype distribution 248 for each SNP did not deviate from Hardy-Weinberg equilibrium (p >0.05). 249

Baseline characteristics for the Healthy Eating diet group (n = 54) and the Mediterranean diet 250 group (n = 54) were summarized in Table 1. No significant differences were found in minor 251 allele frequency of any SNP, gender, race, age, or BMI between the two diet groups at baseline. 252 Likewise, baseline measurements of AA, EPA, and long chain n-3 fatty acids (the sum of EPA 253 and DHA) did not differ significantly in the serum or the colonic mucosa between the two diet 254 groups (Table 1). 255

256 Baseline measures 257

Linear regression analysis indicated that the number of minor alleles was a significant 258 predictor of baseline serum AA concentration (p < 0.001) and almost significant for colonic AA 259 concentration (p = 0.058). A greater number of minor alleles was significantly associated with 260




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lower AA concentration in serum. Dietary AA intakes were not a significant predictor of either 261 serum of colon concentrations. For long chain n-3 fatty acids, however, the situation was the 262 reverse. Dietary intake of long chain n-3 fatty acids was a significant predictor of baseline serum 263 long chain n-3 concentration (p < 0.001) and colonic long chain n-3 concentration (p = 0.044) 264 while the number of minor alleles was not a significant predictor of either. 265

Subsequent analyses were done categorizing subjects into two groups by presence or absence 266 of any minor alleles in the FADS gene cluster. The only dietary or demographic factor to differ 267 by genotype at baseline was BMI, which was lower in carriers of any minor alleles (mean of 268 27.8, SD 3.7, in all major allele carriers and mean of 26.1, SD 3.6, in carriers of any minor 269 alleles, p=0.02 by the 2-sided t-test). Age was not significantly different (p=0.11) but was 270 retained as a covariate. No significant difference was found for other demographic characteristics 271 (race, gender, smoking, common medication use) between minor allele and all major allele 272 carriers. Results were similar when using any one SNP individually versus all minor SNPS (not 273 shown). 274

Serum and colon fatty acid concentrations at baseline by genotype group are shown in Table 275 2. Linear mixed models were used to evaluate differences between genotype groups. The 276 presence of any minor alleles was highly significantly associated with baseline serum 20:4, n-6 277 concentrations (p<0.0001) and 18:3, n-3 concentration (p=0.01), and marginally significant for 278 colonic 20:4, n-6 concentration (p = 0.07), with adjustment for age, BMI, and dietary intakes of 279 n-6 PUFA, n-3 PUFA and/or long chain n-3 PUFA as a percentage of energy. Specifically, mean 280 serum 20:4, n-6 concentration (% of total fatty acids) for minor allele carriers were estimated to 281 be 2% (95% CI = [1%, 3%]) lower, whereas mean serum 18:3, n-3 concentration for minor allele 282 carriers were estimated to be 21% (95% CI = [4%, 41%]) higher, compared to those individuals 283




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with all major alleles in the four SNPs in FADS. There was no significant association of 284 genotype with EPA nor with long chain n-3 fatty acids (the sum of EPA and DHA). Genotype 285 group also had no significant effects on total cholesterol, LDL, HDL, triglycerides, insulin, 286 glucose, and CRP with p>0.11 in each case (not shown). 287

288 Effects of Dietary Intervention on Fatty Acid Intakes and Fatty Acid Concentrations in Serum 289 and Colon 290

We first evaluated changes in fatty acids by diet group assignment alone without considering 291 the genotype groups. Table 3 displays dietary intakes, serum, and colon fatty acid concentrations 292 for the two diet arms at baseline and after 6 months of intervention. Based on data from food 293 records and 24-hour recalls, dietary intakes of saturated fats (SFA) and monounsaturated fats 294 (MUFA) were significantly reduced (p<0.0001) and long chain n-3 PUFA was significantly 295 increased (p=0.004) in the Healthy Eating group after 6 months. The decrease in mean SFA 296 resulted in an increased polyunsaturated fat: saturated fat ratio from 0.60 to 0.92 in the Healthy 297 Eating group (p=0.008 from mixed linear regression models controlling for age). In the 298 Mediterranean group, dietary intakes of SFA and n-6 PUFA both significantly decreased 299 (p<0.0001), while MUFA and long chain n-3 PUFA significantly increased (p<0.0001), in 300 accord with the counseling goals. The mean polyunsaturated fat: saturated fat ratio increased 301 non-significantly from 0.72 to 0.77 in the Mediterranean group. 302

Serum 18:2 n-6 significantly decreased (p=0.02), and both MUFA and n-3 PUFA 303 significantly increased (p=0.0005 and p=0.01, respectively) in the Mediterranean arm only 304 (Table 3). There was little change in colon fatty acid concentrations. The only significant change 305




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was for long chain n-3 PUFA that significantly increased in both Healthy Eating (p=0.01) and 306 Mediterranean groups (p=0.01). 307 308 Interactions of Genotype and Diet Intervention 309

Figures 1 and 2 show the raw means in each group over time. Table 4 shows the linear 310 mixed model results for the analysis of the genotype by diet interaction. There was a significant 311 interaction of genotype by diet for 20:4, n-6 (AA) concentrations in the colon (p=0.004). No 312 significant genotype-by-diet interactions were found for AA in serum nor for EPA. Among 313 subjects with no minor alleles, mean colon AA concentrations were estimated to be 16% (95% 314 CI = [5%, 26%]) lower for the Mediterranean arm than the Healthy Eating arm at 6 months. 315 These results indicate that after adjusting for baseline AA concentrations, mean colon AA 316 concentrations at 6 months were significantly different between diet arms only in persons with 317 no minor alleles in the FADS1/2 gene cluster. This was mainly due to an increase in colon AA in 318 the Healthy Eating diet arm while colon AA concentrations remained fairly constant in the 319 Mediterranean group. 320

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Discussion 324 325 This randomized, dietary intervention study afforded the opportunity to evaluate the impact 326

of FADS genotype and diet on fatty acid concentrations in both serum and colonic mucosa of 327 individuals at increased risk for colon cancer. The number of minor alleles in the FADS gene 328 cluster, but not diet, predicted serum AA concentrations. This agrees well with results of 329 previous studies, namely that carriers of minor alleles have lower AA concentrations (9-15). For 330 EPA concentrations in serum, genotype had no effect while diet did have a significant effect, 331 likely because n3 fatty acid intakes were fairly low and limiting in this study population. It 332 should, however, be noted that diet in this study was assessed using self-report on four separate 333 days. In addition to the possibility of mis-reporting of intakes, those four days might not 334 represent usual intakes over the last month of study and therefore will weaken any apparent 335 associations with diet. 336

In epidemiological studies, relatively higher dietary intakes of both n-3 and n-9 fatty acids 337 are thought to be protective while high intakes of n-6 fatty acids increase risk of several cancers 338 including that of the colon (31). This has been confirmed in experimental models of colon 339 cancer, and low versus high n6 fatty acid diets are associated with decreased tumors and lower 340 production of certain eicosanoids such as prostaglandin E2 (PGE2) (32, 33). In the colon, 341 prostaglandin E2 (PGE2) has been tightly linked with colon cancer risk (34). Increased n-3 fatty 342 acid intakes also reduce PGE2 production (35-39). Interestingly, a reduction in n-6 fatty acid 343 intakes can augment increases in EPA after n-3 fatty acid supplementation (40). Bartoli et al. 344 observed inhibition of aberrant crypt foci, adenocarcinomas, decreased mucosal arachidonate 345 (20:4) and decreased PGE2 in rats fed either n-9 or n-3 diets relative to rats fed diets high in n-6 346 fatty acids (41). The levels of colon mucosal PGE2 were directly proportional to arachidonate 347




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levels in the colon in that study (41). This data makes it important to better understand factors 348 that could affect AA and EPA levels in the human colon. 349

Unlike serum fatty acids, genotype had no significant effects on fatty acid concentrations in 350 the colon at baseline (Table 2). It may be the case that serum concentrations of fatty acids are 351 affected by first pass liver metabolism more so than tissues. After absorption of fatty acids, 352 mainly in the small intestine, the liver is the initial site of fatty acid metabolism. The subsequent 353 distribution of fatty acids from the circulation to tissues will be dependent on lipoprotein lipase 354 activity in each tissue site and on tissue-specific metabolic conversions. In a well-controlled 355 study in pigs, increased dietary intakes of linolenic acid and/or linoleic acid significantly affected 356 metabolism of each other to longer chain fatty acids in the liver, but the effect was minimal in 357 brain cortex (42). In a human lipodomic study, fatty acid desaturase activity of blood reflected 358 activity in the liver but not in adipose tissue (43). Serum and colon fatty acid concentrations 359 therefore not only diet and genotype, but any tissue-specific regulation of fatty acid metabolism. 360

Since the present study was a randomized clinical trial, we then evaluated the effects of the 361 two dietary interventions on changes in fatty acid intakes and levels over time. Both dietary 362 interventions decreased SFA intakes and increased n-3 PUFA intakes. Only the Mediterranean 363 intervention resulted in increased MUFA and decreased n-6 PUFA intakes. Serum fatty acids in 364 the Mediterranean arm reflected these changes in diet (Table 3). In the colon, however, the only 365 significant change was an increase in n-3 PUFA. This indicates that tissue-specific processes 366 may limit the impact of dietary changes in colon fatty acids. The increase in colon n-3 PUFA is 367 interesting, however, since the increases in dietary n-3 PUFA were modest in each diet arm. 368

The effect of FADS genotype on fatty acid concentrations in colon was only evident after 369 intervention (Table 4). Study subjects who were carriers of all major alleles and randomized to 370




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the Healthy Eating intervention had higher colon AA concentrations after 6 months than subjects 371 with all major alleles in the Mediterranean group. It is not entirely clear why this should be the 372 case, but the Healthy Eating intervention did result in a higher relative amount of n-6 PUFA to 373 other dietary fats. This could have helped increase the percentage of AA in the colon fatty acids 374 after the Healthy Eating intervention. In addition to polymorphisms in FADS, other factors could 375 be operative to affect fatty acid desaturation such as diet-induced changes in the expression and 376 the activity of FADS, and to changes in substrate competition (44). In carriers of all major alleles 377 randomized to the Mediterranean intervention, AA levels stayed relatively low at both time 378 points and were estimated to be 16% lower than in the Healthy arm after 6 months of 379 intervention. 380

Limitations of this study include the small sample size, the relatively short intervention 381 length, and the self-report of diet which is known to be subject to biases. It may take longer for a 382 change in diet to be fully manifest, especially in tissues. In addition, the measurement of fatty 383 acids was done as a percentage of total fatty acids such that increases in one fatty acid on a 384 volume basis would result in decreases in other fatty acids. An additional consideration is that 385 AA concentrations are not easily modifiable by changes in n6 fatty acids in the diet, especially if 386 AA is not elevated at the outset (45). Strengths of the study include that it was a randomized 387 study, and measures were available before and after diet change in both serum and colonic 388 mucosa of individuals at increased risk for colon cancer. 389

In conclusion, this study showed that those subjects with no minor alleles in the FADS1/2 390 cluster had higher concentrations of AA in serum. Polymorphism in FADS1/2 had no effect on 391 concentrations of EPA, perhaps because concentrations of this fatty acid are more highly driven 392 by dietary intakes. The trends were similar in colon tissue fatty acids but not significant. After 393




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randomization to Mediterranean or Healthy Eating intervention for 6 months, there was a 394 significant gene*diet interaction for colon AA concentrations. Subjects who had all major alleles 395 for FADS1/2 had significantly lower AA concentrations in the colon after 6 months if they were 396 in the Mediterranean diet arm. Since AA is the substrate for prostaglandin E2 production, these 397 results indicate that a Mediterranean diet could be especially favorable for reducing colon cancer 398 risk in the subset of subjects with all major alleles in FADS1/2. Future work should evaluate the 399 effects of these FADS polymorphisms on colonic pro-inflammatory states. 400 401




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Table 1. Characteristics of study subjects at baseline by diet arm. The data shown are mean (SD) or number (%). _________________________________________________________________________________ Characteristic Healthy Eating arm (n=54) Mediterranean arm (n=54) P-value a ________________________________________________________________________________ Gender, female 38 (70%) 40 (74%) 0.67 Age, years 51.1 (13.3) 54.9 (9.9) 0.10 Caucasian 49 (91%) 45 (83%) 0.26 BMI, kg/m2 26.9 (3.5) 26.8 (3.9) 0.88 Completed 6 months of study 43 (80%) 47 (87%) 0.31 Minor Allele Frequency FADS2 rs3834458 29 (54%) 26 (48%) 0.87 FADS1 rs174556 28 (52%) 24 (44%) 0.68 FADS1 rs174561 27 (50%) 23 (43%) 0.74 FADS1 rs174537 30 (55%) 27 (50%) 0.73 Number of minor alleles 2.6 (2.2) 2.2 (2.5) 0.47

Dietary fatty acids, % of energy b

n-6 PUFA 6.55 (1.63) 7.17 (2.14) 0.09 n-3 PUFA 0.77 (0.26) 0.88 (0.46) 0.14 Long chain n-3 PUFA c 0.06 (0.11) 0.07 (0.12) 0.52

Serum fatty acids, as % of total

20:4, n-6 8.7 (1.9) 9.2 (2.1) 0.17 Long chain n-3 PUFA c 2.98 (1.20) 3.04 (1.37) 0.82

Colon fatty acids, as % of total

20:4, n-6 10.5 (3.1) 10.1 (3.0) 0.53 Long chain n-3 PUFA c 3.52 (1.53) 3.18 (1.62) 0.26

______________________________________________________________________________ a P-values for differences at baseline between diet arms are from two-sided t-tests for continuous variables or

Chi-square tests for proportions. Natural log transformations were used to normalize the data before analysis

except for dietary n-3 PUFA and serum 20:4, n-6, which did not require transformation. Data is shown

untransformed for ease of interpretation. b For dietary intakes, n-6 polyunsaturated fatty acids (n-6 PUFA) was the sum of 18:2, and 20:4, n-3 PUFA

was the sum of 18:3, 20:5, 22:5, and 22:6, and ‘long chain n-3’ was the sum of 20:5, 22:5 and 22:6. c Long chain n-3 PUFA in serum and colon were the sum of 20:5, n-3 and 22:6, n-3.




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Table 2. Dietary intakes, serum fatty acid concentrations and colon fatty acid concentrations at

baseline by genotype. The data shown are mean and SD.

_____________________________________________________________________________ Fatty Acid Number of Minor Alleles P-Value a none 1-8 n=46 n=62 ______________________________________________________________________________ Dietary intakes (% of calories) N-6 PUFA 7.0 (2.2) 6.8 (1.7) 0.54 N-3 PUFA 0.8 (0.3) 0.8 (0.4) 0.98 Long chain n-3 PUFA b 0.08 (0.14) 0.04 (0.06) 0.22 Serum fatty acids (% of fatty acids) 20:4, n-6 10.2 (1.9) 8.0 (1.7) <0.001 20:5, n-3 0.8 (0.4) 0.8 (0.6) 0.19 22:6, n-3 2.18 (0.81) 2.20 (1.00) 0.78 Long chain n-3 PUFA 3.0 (1.1) 3.0 (1.4) 0.55 18:2 n-6 27.5 (6.1) 26.7 (6.8) 0.63 18:3 n-3 0.64 (0.23) 0.87 (0.53) 0.01 N-3/N-6 ratio 0.10 (0.03) 0.12 (0.07) 0.04 Colon fatty acids (% of fatty acids) 20:4, n-6 10.7 (3.1) 10.0 (3.0) 0.07 20:5, n-3 1.2 (0.9) 1.2 (1.0) 0.66 22:6, n-3 2.12 (0.77) 2.20 (0.88) 0.95 Long chain n-3 PUFA 3.3 (1.4) 3.4 (1.7) 0.94 18:2 n-6 19.3 (4.3) 20.0 (7.6) 0.97 18:3 n-6 1.14 (0.90) 1.58 (1.41) 0.19 N-3/N-6 ratio 0.15 (0.08) 0.18 (0.11) 0.15 _____________________________________________________________________________ a P-values were obtained from linear mixed models with each of the baseline fatty acids

regressed on genotype group with batch number as a random effect. The covariates of the model

included age, baseline BMI, and baseline dietary intake measures of n-6 PUFA, n-3 PUFA

and/or long chain n-3 PUFA as a percentage of energy. An inverse squared root transformation

was applied to colon 18:2, n-6 and colon 22:6, n-3 to approximate normality, respectively.

Natural logarithm transformations were used to normalize the rest fatty acid data except for

serum 20:4, n-6 and 18:2, n-6, which did not require transformation. Means and SDs are shown

untransformed for ease of interpretation.




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b Long chain n-3 PUFA in serum and colon were the sum of 20:5, n-3 and 22:6, n-3. In the diet,

grams/day of the n-3 fatty acids 20:5, 22:5 and 22:6 were summed and expressed as a percentage

of energy using 9 kcal/gram.




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Table 3. Effects of dietary intervention on changes in dietary intakes and fatty acid

concentrations.

______________________________________________________________________________ Fatty Acid Healthy Eating Mediterranean Baseline 6 Months Baseline 6 Months (n=54) (n=43) (n=54) (n=47) ______________________________________________________________________________

Dietary Fatty Acids (% of energy)

SFA a 12.3 (2.7) 7.9 (2.3) b 11.2 (2.6) 8.3 (2.3) b MUFA 13.0 (2.2) 10.6 (3.3) b 13.0 (3.4) 16.4 (3.7) b

n-6 PUFA 6.5 (1.6) 6.3 (2.2) 7.2 (2.1) 6.2 (1.9) b

18:3, n-3 0.72 (0.22) 0.78 (0.32) 0.81 (0.43) 0.90 (0.67) Long-chain n-3 PUFA 0.06 (0.11) 0.12 (0.16) b 0.07 (0.12) 0.17 (0.22) b

Serum Fatty Acids (% of total fatty acids)

SFA 34.0 (5.3) 33.9 (5.0) 34.0 (5.4) 33.5 (5.3) MUFA 24.7 (6.0) 24.2 (5.2) 24.1 (4.6) 26.6 (4.1) b 18:2, n-6 27.1 (6.0) 27.6 (5.9) 27.1 (6.9) 25.1 (5.3) b 20:4, n-6 8.67 (1.92) 8.54 (2.30) 9.2 (2.1) 8.9 (2.2) 18:3, n-3 0.79 (0.28) 0.81 (0.32) 0.75 (0.55) 0.74 (0.43) Long-chain n-3 PUFA 2.97 (1.20) 3.26 (1.45) 3.04 (1.38) 3.42 (1.39) b Colon Fatty Acids (% of total fatty acids) SFA 32.4 (4.9) 32.4 (3.6) 32.4 (3.3) 31.9 (3.4) MUFA 30.5 (4.0) 31.4 (3.8) 32.3 (5.0) 32.8 (4.7) 18:2, n-6 20.2 (7.9) 19.1 (4.8) 19.3 (4.4) 18.9 (5.3)




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20:4, n-6 10.5 (3.1) 10.5 (2.6) 10.1 (3.0) 10.3 (2.7) 18:3, n-3 1.39 (1.24) 1.26 (1.03) 1.39 (1.24) 1.16 (1.07) Long-chain n-3 PUFA 3.52 (1.53) 3.86 (1.47) b 3.18 (1.62) 3.47 (1.60) b ____________________________________________________________________________

a The transformations used for dietary variables were natural log both for 18:3, n-3 and for long

chain n-3 fatty acids (sum of 20:5 and 22:6). The transformations used for serum concentrations

were squared for saturated fat (SFA), and natural log both for 18:3, n-3 and long chain n-3 fatty

acids. The transformations used for colon fatty acids were: squared for SFA, inverse squared

root for 18:2, n-6 and natural log for MUFA, 20:4, n-6, 18:3, n-3, and long chain n-3 fatty acids.

Means and standard deviations are shown un-transformed for ease of interpretation. b Significant different from baseline for that diet arm (p<0.05).




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Table 4. Interactions of diet group assignment with genotype on fatty acid concentrations in serum and colonic mucosa at 6 months a.

Sample Fatty Acid (at 6 months)

Effect Estimate SE p-Value

Serum 20:4, n-6 Diet 0.368 0.458 0.42

Genotype -0.286 0.483 0.56

Diet*Genotype -0.630 0.602 0.30

Baseline 20:4, n-6 0.804 0.085 <0.001

Age -0.016 0.014 0.25

BMI 0.011 0.039 0.78

20:5, n-3 b Diet 0.092 0.149 0.54

Genotype -0.019 0.145 0.90

Diet*Genotype -0.015 0.199 0.94

Baseline 20:4, n-6 0.442 0.097 <0.001

Age 0.009 0.005 0.04

BMI -0.013 0.013 0.31

Colon 20:4, n-6 b Diet -0.178 0.066 0.01

Genotype -0.226 0.066 0.001

Diet*Genotype 0.266 0.089 0.004

Baseline 20:4, n-6 0.293 0.071 <0.001

Age <0.001 0.002 0.85

BMI 0.001 0.006 0.82

20:5, n-3 b Diet -0.014 0.120 0.91

Genotype -0.027 0.116 0.82

Diet*Genotype 0.063 0.158 0.69

Baseline 20:4, n-6 0.721 0.057 <0.001

Age 0.004 0.004 0.28

BMI -0.003 0.011 0.79 a Analyses were performed by linear mixed models by including each fatty acid concentration at

6 months as the response variable and covariates: diet arm (Mediterranean versus Healthy

Eating), genotype (presence of any minor alleles versus all major alleles at the four SNPs in the

FADS gene), interaction of diet and genotype, baseline age, baseline BMI and baseline

concentration of each fatty acid. Batch of sample analysis was treated as a random effect. b Natural log transformation was applied to fatty acid variables approximate normality in

analysis.




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Figure Legends

Figure 1. Serum concentrations of arachidonic acid (AA) and eicosapentaenoic acid (EPA) at baseline and after 6 months of intervention. Subjects were grouped by presence/absence of any minor alleles in the FADS1/FADS2 gene cluster (rs3834458, rs174556, rs174561, and rs174537). The data shown are untransformed mean and SE. Figure 2. Colon mucosa concentrations of arachidonic acid (AA) and eicosapentaenoic acid (EPA) at baseline and after 6 months of intervention. Subjects were grouped by presence/absence of any minor alleles in the FADS1/FADS2 gene cluster (rs3834458, rs174556, rs174561, and rs174537). There was a significant gene*diet interaction for AA in persons with no minor alleles in any of the single nucleotide polymorphisms in linear mixed models. The data shown are untransformed mean and SE.




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Published OnlineFirst September 10, 2013.Cancer Prev Res Shannon Porenta, Yi-An Ko, Stephen B. Gruber, et al. Colonic Fatty Acids in a Mediterranean Diet Intervention StudyInteraction of Fatty Acid Genotype and Diet on Changes in

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