Mol. Nutr. Food Res. 2014, 58, 1079–1086
1079
DOI 10.1002/mnfr.201300426
RESEARCH ARTICLE
Cardiometabolic risk factors are influenced by Stearoyl-CoA Desaturase (SCD) −1 gene polymorphisms and n-3 polyunsaturated fatty acid supplementation Iwona Rudkowska1,2 , Pierre Julien2 , Patrick Couture1 , Simone Lemieux1 , Andre´ Tchernof1,2 , Olivier Barbier2 and Marie-Claude Vohl1,2 1 2
Institute of Nutrition and Functional Foods (INAF), Laval University, Quebec, Canada ´ Department of Endocrinology and Nephrology, CHU de Quebec Research Center, Quebec, Canada
Scope: To determine if single nucleotide polymorphisms (SNPs) in stearoyl-CoA desaturase (SCD)-1 gene that encodes a key enzyme for fatty acid metabolism are associated with the response of cardiometabolic risk factors to n-3 PUFA supplementation. Methods and results: Two hundred and ten subjects completed a 2-week run-in period followed by 6-week supplementation with 5 g of fish oil (1.9–2.2 g eicosapentaenoic acid and 1.1 g docosahexaenoic acid). Risk factors were measured pre and post n-3 supplementation. Fatty acid composition of plasma phospholipids was analyzed by GC and the desaturase indices SCD16 (16:1n-7/16:0) and SCD18 (18:1n-9/18:0) were calculated. Genotyping of eight SNPs of the SCD1 gene was performed. N-3 PUFA supplementation decreased plasma triglycerides, as well as SCD16 and SCD18 indices, but increased fasting plasma glucose concentrations. SNPs in SCD1-modified cardiometabolic risk factors pre and post n-3 PUFA supplementation: triglyceride (rs508384, p = 0.0086), IL6 (rs3071, p = 0.0485), C-reactive protein (rs3829160, p = 0.0489), and SCD18 indices (rs2234970, p = 0.0337). A significant interaction effect between the SNP and n-3 PUFA supplementation was also observed for fasting plasma glucose levels (rs508384, p = 0.0262). Conclusion: These results suggest that cardiometabolic risk factors are modulated by genetic variations in the SCD1 gene alone or in combination with n-3 PUFA supplementation.
Received: June 12, 2013 Revised: October 11, 2013 Accepted: October 15, 2013
Keywords: Desaturase indices / Genetic variations / Inflammation / Nutrigenetics / Plasma lipids
1
Introduction
The fatty acid (FA) composition of plasma phospholipids (PL) reflects FA intake as well as concentrations of desaturating enzymes, such as stearoyl-CoA desaturase (SCD) [1–3]. SCD is the rate-limiting enzyme that catalyses the conversion of saturated to MUFAs [4]. Desaturation indices are calculated Correspondence: Dr. Marie-Claude Vohl, Institute of Nutrition and Functional Foods (INAF), Laval University, Pavillon des Services, ´ bureau 2729K, 2440, boulevard Hochelaga, Quebec, Canada G1V 0A6 E-mail: [email protected] Fax: +1-418-656-5877 Abbreviations: BP, blood pressure; CRP, C-reactive protein; EPA, eicosapentaenoic acid; FA, fatty acid; MetS, metabolic syndrome; PBMC, peripheral blood mononuclear cell; PL, phospholipids; SCD, stearoyl-CoA desaturase; SNPs, single nucleotide polymorphisms; TC, total cholesterol; TG, triglyceride; TNFA, tumor necrosis factor-alpha C 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
as the product-to-substrate ratio of the FAs metabolized by the enzyme SCD1 and may be used as a proxy for the enzyme’s activity [1–3]. Several studies have demonstrated that high SCD indices are associated with increased obesity and the metabolic syndrome (MetS) [5–8]. Reductions of SCD indices through genetic, pharmaceutical, or nutritional inhibition may elicit or reflect improvements in insulin resistance, glucose tolerance, and hypercholesterolemia as well as a reduction in adiposity [9]. In particular, intake of n-3 PUFAs, including eicosapentaenoic acid (EPA) and docosahexaenoic acid, are known to lower SCD indices [10, 11]. The SCD1 gene undergoes coordinate transcriptional downregulation in response to these unsaturated FAs [12]. An animal study suggested that downregulation of Scd1 is a mechanism by which n-3 PUFAs repress constitutive triglyceride (TG) biosynthesis [13, 14]. Overall, n-3 PUFAs intake may influence cardiometabolic risk factors via changes in SCD indices. Genetic variations in the SCD1 gene have also been associated with components of MetS: obesity, insulin sensitivity, www.mnf-journal.com
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blood pressure (BP), and plasma TG [15, 16]. A recent study identified the SCD1 gene single nucleotide polymorphism (SNP) rs1502593 and two commonly observed haplotypes comprising rs1502593, as being involved in the development of MetS in Costa Rican adults [16]. Taken together, n-3 PUFA supplementation and genetic variations in SCD1 may influence the development of MetS via potentially altered SCD1 expression, activity, and indices. Therefore, the aim of the present study was to determine if SCD1 gene SNPs are associated with cardiometabolic risk factors including: plasma lipid profiles, inflammation, and glycemic parameters as well as SCD indices, and if such association can interfere with the response to n-3 PUFA supplementation.
2
Materials and methods
2.1 Subjects and methods 2.1.1 Study population A total of 254 subjects from the greater Quebec City metropolitan area were recruited to participate in the study, and 210 subjects completed the protocol. Participants had a BMI between 25 and 40 kg/m2 , and were not currently taking any lipid-lowering medication. Subjects were excluded from the study if they had taken n-3 PUFA supplements for the preceding 6-month period, if they used oral hypolipidemic therapy, or had been diagnosed with diabetes, hypertension, hypothyroidism, or other known metabolic disorders such as severe dyslipidemia or coronary heart disease. The experimental protocol was approved by the ethics committees of Laval University Hospital Research Center and Laval University. This trial was registered at clinicaltrials.gov as NCT01343342.
Mol. Nutr. Food Res. 2014, 58, 1079–1086
to achieve the recommendations from Canada’s Food Guide. Subjects were asked to follow these recommendations and maintain their body weight stable throughout the protocol. Some specifications were given regarding the n-3 PUFA dietary intake: not exceed two fish or seafood servings per week (max 150 g), prefer white flesh fishes instead of fatty fishes (examples were given), and avoid enriched n-3 PUFA dietary products such as some milks, juices, breads, and eggs. Subjects were also asked to limit their alcohol consumption during the protocol, two regular drinks per week were allowed. In addition, subjects were not allowed to take n-3 PUFA supplements (such as flaxseed), vitamins, or natural health products during the protocol. After the 2-week run-in, each participant received a bottle containing needed fish oil capsules for the following 6 weeks. They were invited to take 5 (1 g of fish oil concentrate each) capsules per day (Ocean Nutrition, Nova Scotia, Canada), providing a total of 3 g of n-3 PUFA (including 1.9–2.2 g EPA and 1.1 g docosahexaenoic acid) per day. For a facilitated digestion, we recommended to take fish oil capsules while eating. Compliance was assessed from the return of bottles. Subjects were asked to report any deviation during the protocol, write down their alcohol and fish consumption as well as the side effects. Before each phase, subjects received detailed written and oral instructions on their diet. 2.2 Laboratory analyses 2.2.1 Biochemical parameters
Resting BP measurements were performed after a 5-min rest in a sitting position, phases I and V of Korotkoff sounds being respectively used for systolic BP and diastolic BP. Measurements were performed in duplicate and the mean was used for analyses.
Blood samples were collected from an antecubital vein into vacutainer tubes containing EDTA after 12-h overnight fast and 48-h alcohol abstinence. Blood samples were taken to identify and exclude individuals presenting exclusion criteria such as metabolic disorders, as mentioned. Afterward, selected participants had blood samples taken prior to and after the n-3 PUFA supplementation period. Plasma was separated by centrifugation (2500 × g for 10 min at 4⬚C) and samples were aliquoted and frozen at −80⬚C for subsequent analyses. Plasma total cholesterol (TC) and TG concentrations were measured using enzymatic assays [ [17, 18]. The repeatability determined by the variation in measurements within-run, was 0.7–1.1% for TC and TG. The precision determined using human samples and controls, was 1.4–1.6% for TC and 1.8–1.9% for TG. The HDL cholesterol fraction was obtained after precipitation of VLDL and LDL particles in the infranatant with heparin manganese chloride [19]. LDL cholesterol was calculated with the Friedewald formula [20]. Fasting insulinemia was measured by RIA with PEG separation [21]. Fasting glucose concentrations were measured enzymatically [22].
2.1.4 Study design and diets
2.2.2 Inflammatory markers
Subjects followed a run-in period of 2 weeks in which individual dietary instructions were given by a trained dietician
Plasma concentrations of IL6 and tumor necrosis factoralpha (TNFA) were measured with high-sensitivity ELISA kits
2.1.2 Anthropometric measurements Body weight, height, and waist girth were measured according to the procedures recommended by the Airlie Conference [26] and were taken by a nurse at each visit at the clinical investigation unit; during the run-in period as well as before and after the n-3 PUFA supplementation. BMI was calculated as weight per meter squared (kg/m2 ). 2.1.3 BP measurements
C 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
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Mol. Nutr. Food Res. 2014, 58, 1079–1086
including: Human IL6 Quantikine HS ELISA Kit Minneapolis, MN, USA (R&D Systems, Minneapolis, MN, USA (HS600B)) and Human TNF-alpha Quantikine HS ELISA Kit (R&D Systems (HSTA00D)). Plasma C-reactive protein (CRP) was measured by nephelometry (Prospec equipment Behring) using a sensitive assay, as described previously [23]. The repeatability was 6.9–7.4% for IL6, 3.1–8.5% for TNFA and 2.3–2.6% for CRP. The precision was 6.5–9.6% for IL6, 7.3–10.6% for TNFA and 1.3–4% for CRP. Subjects with CRP concentrations more than ± 3 SD from the mean were excluded from statistical analyses.
2.2.3 FA composition of plasma PL Plasma lipids were extracted with chloroform:methanol (2:1, by volume) according to a modified Folch method [24]. Total PL were separated by TLC using a combination of isopropyl ether and acetic acid and FAs of isolated PL were then methylated. Capillary GC was then used to obtain FA profiles. The technique used for plasma analyses has been previously validated [25]. Values of FA concentrations are expressed as percent of total FA in plasma PL. Further, FA ratios were used as surrogate measures of desaturase activity. SCD16 was calculated with the ratio of palmitoleic acid (16:1n-7) on palmitic acid (16:0) as well as the SCD18 was calculated with the ratio of oleic acid (18:1n-9) on stearic acid (18:0).
2.3 Statistical analysis Data are shown as mean ± SD. Hardy-Weinberg equilibrium was tested with the Allele Procedure in SAS, version 9.2 (SAS Institute Inc., Cary, NC, USA). Distribution of alleles in the present study cohort was compared with the Caucasian population using the CEU data from the National Center for Biotechnology Information (www.ncbi.nlm.nih.gov/) and the Fisher Exact Test. Further, variables were checked for normality of distribution using the skewness and the kurtosis values. TG, CRP, IL6, and TNFA concentrations were log-transformed before analyses to normalize their distribution. First, the repeated ANOVA was used to test for the effects of the genotype, the time (pre- and post-n-3 PUFA supplementation) and the genotype by n-3 PUFA supplementation interaction effect on each variable when adjusted for the effects of age, sex and BMI. A second model also included energy intake. A statistical p-value was defined as p ≤ 0.05 and was used in order to avoid discounting true positive associations. When significant differences were found, a pairwise comparison was performed in a global analysis (Tukey tests; significance p ≤ 0.05). Statistical analyses were performed with SAS statistical software, version 9.2 (SAS Institute Inc.). A group of 152 subjects was calculated to provide an 80% probability at an alpha = 0.05 of to detect a difference of 0.25 mmol/L of plasma TG levels (primary endpoint) after 6 weeks of n-3 PUFA supplementation with a genetic variation that occurs at a relatively low frequency (5%) in the population [26].
2.2.4 DNA extraction and genotyping The SIGMA GenElute Gel Extraction Kit (Sigma-Aldrich Co. St.Louis, MO, USA) has been used to extract genomic DNA. Selected SNPs were genotyped using validated primers and TaqMan probes (Applied Biosystems, Foster City, CA, USA). DNA was mixed with TaqMan Universal PCR Master Mix (Applied Biosystems) and a gene-specific primer and probe mixture (predeveloped TaqMan SNP Genotyping Assays; Applied Biosystems) in a final volume of 10 L. Genotypes were determined using a 7500 FAST RT-PCR System and analyzed using ABI Prism SDS version 2.0.5 software (Applied Biosystems). SNPs in SCD were identified using the International HapMap Project SNP database, based on the National Center for Biotechnology Information B36 assembly Data Rel 28, phase II + III, build 126. Tagger procedure in Haploview V4.2 was used to determine tag SNPs using a minor allele frequency > 5% and pairwise tagging (r2 ≥ 0.8). Therefore, eight SNPs were selected from the SCD gene according to the specified parameters and genotyped: rs1502593, rs522951, rs11190480, rs3071, rs3829160, rs2234970, rs10883463, and rs508384. If a significant SNP that affects an amino acid substitution was found, the SIFT and PolyPhen Web-based softwares were used to predict the effect of amino acid substitution on protein structure and function. C 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
3
Results
3.1 Genotypic characteristics Genotype frequencies did not deviate from those predicted by the Hardy–Weinberg equilibrium except for the following two SNPs: rs1502593 and rs522951, which were excluded from statistical analyses (Table 1). In addition, allele frequencies in the current population were not different when compared to the CEU HapMap population (Table 1). 3.2 Effect of the n-3 PUFA supplementation Parameters pre and post n-3 PUFA supplementation for men (n = 97) and women (n = 113) are described in Table 2. Briefly, n-3 PUFA supplementation was associated with a similar decrease in fasting TG concentrations in men and women, 0.19 mmol/L (15%) and 0.20 mmol/L (17%), respectively. Plasma fasting glucose concentrations were increased significantly by 0.09 mmol/L (2%) in men and 0.12 mmol/L (2%) in women without changing fasting insulin concentrations. There were no other differences between the pre and post n-3 PUFA supplementation including BMI, systolic BP, diastolic BP, TC, LDL cholesterol, and HDL cholesterol www.mnf-journal.com
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Mol. Nutr. Food Res. 2014, 58, 1079–1086
Table 1. Genotypic and allelic distribution of SCD1 polymorphisms in study volunteers and comparison to HapMap population
SNP
Role
Genotype
Count (N)
Allele
Count
Frequency
HapMap (CEU) frequency
Fisher’s exact test (p value)
HWE (p value)
rs1502593
intron
166 254
0.40 0.60
0.41 0.59
0.82
0.03
intron
C G
220 200
0.52 0.48
0.49 0.51
0.67
0.03
rs11190480
intron
A G
378 42
0.90 0.10
0.92 0.08
0.45
0.44
rs3071
intron
A C
285 135
0.68 0.32
0.65 0.35
0.65
0.64
rs3829160
intron
A G
184 236
0.44 0.56
0.46 0.54
0.78
0.16
rs2234970
missense
A C
256 164
0.61 0.39
0.62 0.38
0.88
0.47
rs10883463
intron
C T
26 394
0.06 0.94
0.07 0.93
0.77
1
rs508384
3 UTR
25 116 69 49 122 39 171 36 3 95 95 20 35 114 61 75 106 29 0 26 184 7 59 144
A G
rs522951
AA AG GG CC CG GG AA AG GG AA AC CC AA AG GG AA AC CC CC CT TT AA AC CC
A C
73 347
0.17 0.83
0.15 0.85
0.70
0.81
HWE, Hardy–Weinberg equilibrium.
Table 2. Parameters pre and post n-3 PUFA supplementation for men (n = 97) and women (n = 113)
Men (n = 97) Pre n-3 PUFA Age (years) BMIb) (kg/m2 ) BP Systolic BPb) (mmHg) Diastolic BPb) (mmHg) Glycemic parameters Glucoseb) (mmol/L) Insulin (pmol/L) Lipids parameters Total Cholesterol (mmol/L) LDL-Cholesterolb) (mmol/L) HDL-Cholesterolb) (mmol/L) Triglycerides1b) (mmol/L) Ratio TC/HDLb) Inflammation parameters CRP1b) (mg/L) IL61b) (pg/mL) TNFA1 (pg/mL)
Women (n = 113) Post n-3 PUFA
31.1 ± 8.12 27.4 ± 3.59 27.5 ± 3.66
Pre n-3 PUFA
p-value for timea) Post n-3 PUFA
30.5 ± 9.11 28.2 ± 3.92 28.3 ± 4.06
118.0 ± 11.6 68.5 ± 8.23
118.2 ± 12.0 69.9 ± 8.89
107.2 ± 8.56 67.5 ± 8.53
106.8 ± 9.80 67.1 ± 8.70
NS NS
5.04 ± 0.45 90.0 ± 101
5.13 ± 0.43 80.1 ± 37.8
4.87 ± 0.45 84.7 ± 42.1
4.99 ± 0.53 86.6 ± 42.9
0.02 NS
4.75 2.90 1.28 1.26 3.89
± ± ± ± ±
0.95 0.89 0.30 0.66 1.11
1.21 ± 1.47 1.31 ± 1.38 1.62 ± 1.08
4.70 2.92 1.29 1.07 3.86
± ± ± ± ±
0.97 0.91 0.32 0.54 1.18
1.36 ± 1.86 1.16 ± 0.94 1.66 ± 1.07
4.75 2.64 1.57 1.17 3.15
± ± ± ± ±
0.85 0.73 0.35 0.60 0.82
3.75 ± 4.85 1.45 ± 0.85 1.74 ± 1.65
4.73 2.65 1.63 0.97 3.03
± ± ± ± ±
0.92 0.78 0.40 0.50 0.82
3.74 ± 5.38 1.49 ± 1.00 1.70 ± 1.40
NS NS NS 0.0001 NS NS NS NS
Data are shown as mean ± SD, significance p ≤ 0.05, 1 p value derived from transformed data. Abbreviations: blood pressure (BP); high density lipoprotein-cholesterol (HDL-C); low density lipoprotein-cholesterol (LDL-C); C-reactive protein (CRP); tumor necrosis factor-alpha (TNFA). a) p value derived from model which included the effects of the time (pre and post n-3 PUFA supplementation) on variables when age, sex, and BMI were taken into account in the entire population (n = 210). b) Significant difference between sexes in the model. C 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
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Mol. Nutr. Food Res. 2014, 58, 1079–1086 Table 3. Phospholipid fatty acid profiles pre and post n-3 PUFA supplementation for men (n = 97) and women (n = 113)
Percentage of total FA
Men (n = 97) Pre n-3 PUFA
Palmitic acidb) Palmitoleic acidb) SCD 16 indiceb) Stearic acidb) Oleic acid SCD 18 indiceb)
27.07 0.59 0.022 14.1 8.59 0.615
± ± ± ± ± ±
1.22 0.18 0.006 0.97 1.06 0.092
Women (n = 113) Post n-3 PUFA 27.1 0.49 0.018 14.5 7.96 0.551
± ± ± ± ± ±
1.14 0.16 0.006 1.23 1.28 0.080
Pre n-3 PUFA 28.3 0.76 0.027 13.1 8.56 0.662
± ± ± ± ± ±
1.50 0.24 0.008 1.37 0.95 0.108
p-value for timea) Post n-3 PUFA 28.2 0.65 0.023 13.5 8.01 0.599
± ± ± ± ± ±
1.44 0.22 0.007 1.30 1.08 0.104
NS
1079
DOI 10.1002/mnfr.201300426
RESEARCH ARTICLE
Cardiometabolic risk factors are influenced by Stearoyl-CoA Desaturase (SCD) −1 gene polymorphisms and n-3 polyunsaturated fatty acid supplementation Iwona Rudkowska1,2 , Pierre Julien2 , Patrick Couture1 , Simone Lemieux1 , Andre´ Tchernof1,2 , Olivier Barbier2 and Marie-Claude Vohl1,2 1 2
Institute of Nutrition and Functional Foods (INAF), Laval University, Quebec, Canada ´ Department of Endocrinology and Nephrology, CHU de Quebec Research Center, Quebec, Canada
Scope: To determine if single nucleotide polymorphisms (SNPs) in stearoyl-CoA desaturase (SCD)-1 gene that encodes a key enzyme for fatty acid metabolism are associated with the response of cardiometabolic risk factors to n-3 PUFA supplementation. Methods and results: Two hundred and ten subjects completed a 2-week run-in period followed by 6-week supplementation with 5 g of fish oil (1.9–2.2 g eicosapentaenoic acid and 1.1 g docosahexaenoic acid). Risk factors were measured pre and post n-3 supplementation. Fatty acid composition of plasma phospholipids was analyzed by GC and the desaturase indices SCD16 (16:1n-7/16:0) and SCD18 (18:1n-9/18:0) were calculated. Genotyping of eight SNPs of the SCD1 gene was performed. N-3 PUFA supplementation decreased plasma triglycerides, as well as SCD16 and SCD18 indices, but increased fasting plasma glucose concentrations. SNPs in SCD1-modified cardiometabolic risk factors pre and post n-3 PUFA supplementation: triglyceride (rs508384, p = 0.0086), IL6 (rs3071, p = 0.0485), C-reactive protein (rs3829160, p = 0.0489), and SCD18 indices (rs2234970, p = 0.0337). A significant interaction effect between the SNP and n-3 PUFA supplementation was also observed for fasting plasma glucose levels (rs508384, p = 0.0262). Conclusion: These results suggest that cardiometabolic risk factors are modulated by genetic variations in the SCD1 gene alone or in combination with n-3 PUFA supplementation.
Received: June 12, 2013 Revised: October 11, 2013 Accepted: October 15, 2013
Keywords: Desaturase indices / Genetic variations / Inflammation / Nutrigenetics / Plasma lipids
1
Introduction
The fatty acid (FA) composition of plasma phospholipids (PL) reflects FA intake as well as concentrations of desaturating enzymes, such as stearoyl-CoA desaturase (SCD) [1–3]. SCD is the rate-limiting enzyme that catalyses the conversion of saturated to MUFAs [4]. Desaturation indices are calculated Correspondence: Dr. Marie-Claude Vohl, Institute of Nutrition and Functional Foods (INAF), Laval University, Pavillon des Services, ´ bureau 2729K, 2440, boulevard Hochelaga, Quebec, Canada G1V 0A6 E-mail: [email protected] Fax: +1-418-656-5877 Abbreviations: BP, blood pressure; CRP, C-reactive protein; EPA, eicosapentaenoic acid; FA, fatty acid; MetS, metabolic syndrome; PBMC, peripheral blood mononuclear cell; PL, phospholipids; SCD, stearoyl-CoA desaturase; SNPs, single nucleotide polymorphisms; TC, total cholesterol; TG, triglyceride; TNFA, tumor necrosis factor-alpha C 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
as the product-to-substrate ratio of the FAs metabolized by the enzyme SCD1 and may be used as a proxy for the enzyme’s activity [1–3]. Several studies have demonstrated that high SCD indices are associated with increased obesity and the metabolic syndrome (MetS) [5–8]. Reductions of SCD indices through genetic, pharmaceutical, or nutritional inhibition may elicit or reflect improvements in insulin resistance, glucose tolerance, and hypercholesterolemia as well as a reduction in adiposity [9]. In particular, intake of n-3 PUFAs, including eicosapentaenoic acid (EPA) and docosahexaenoic acid, are known to lower SCD indices [10, 11]. The SCD1 gene undergoes coordinate transcriptional downregulation in response to these unsaturated FAs [12]. An animal study suggested that downregulation of Scd1 is a mechanism by which n-3 PUFAs repress constitutive triglyceride (TG) biosynthesis [13, 14]. Overall, n-3 PUFAs intake may influence cardiometabolic risk factors via changes in SCD indices. Genetic variations in the SCD1 gene have also been associated with components of MetS: obesity, insulin sensitivity, www.mnf-journal.com
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I. Rudkowska et al.
blood pressure (BP), and plasma TG [15, 16]. A recent study identified the SCD1 gene single nucleotide polymorphism (SNP) rs1502593 and two commonly observed haplotypes comprising rs1502593, as being involved in the development of MetS in Costa Rican adults [16]. Taken together, n-3 PUFA supplementation and genetic variations in SCD1 may influence the development of MetS via potentially altered SCD1 expression, activity, and indices. Therefore, the aim of the present study was to determine if SCD1 gene SNPs are associated with cardiometabolic risk factors including: plasma lipid profiles, inflammation, and glycemic parameters as well as SCD indices, and if such association can interfere with the response to n-3 PUFA supplementation.
2
Materials and methods
2.1 Subjects and methods 2.1.1 Study population A total of 254 subjects from the greater Quebec City metropolitan area were recruited to participate in the study, and 210 subjects completed the protocol. Participants had a BMI between 25 and 40 kg/m2 , and were not currently taking any lipid-lowering medication. Subjects were excluded from the study if they had taken n-3 PUFA supplements for the preceding 6-month period, if they used oral hypolipidemic therapy, or had been diagnosed with diabetes, hypertension, hypothyroidism, or other known metabolic disorders such as severe dyslipidemia or coronary heart disease. The experimental protocol was approved by the ethics committees of Laval University Hospital Research Center and Laval University. This trial was registered at clinicaltrials.gov as NCT01343342.
Mol. Nutr. Food Res. 2014, 58, 1079–1086
to achieve the recommendations from Canada’s Food Guide. Subjects were asked to follow these recommendations and maintain their body weight stable throughout the protocol. Some specifications were given regarding the n-3 PUFA dietary intake: not exceed two fish or seafood servings per week (max 150 g), prefer white flesh fishes instead of fatty fishes (examples were given), and avoid enriched n-3 PUFA dietary products such as some milks, juices, breads, and eggs. Subjects were also asked to limit their alcohol consumption during the protocol, two regular drinks per week were allowed. In addition, subjects were not allowed to take n-3 PUFA supplements (such as flaxseed), vitamins, or natural health products during the protocol. After the 2-week run-in, each participant received a bottle containing needed fish oil capsules for the following 6 weeks. They were invited to take 5 (1 g of fish oil concentrate each) capsules per day (Ocean Nutrition, Nova Scotia, Canada), providing a total of 3 g of n-3 PUFA (including 1.9–2.2 g EPA and 1.1 g docosahexaenoic acid) per day. For a facilitated digestion, we recommended to take fish oil capsules while eating. Compliance was assessed from the return of bottles. Subjects were asked to report any deviation during the protocol, write down their alcohol and fish consumption as well as the side effects. Before each phase, subjects received detailed written and oral instructions on their diet. 2.2 Laboratory analyses 2.2.1 Biochemical parameters
Resting BP measurements were performed after a 5-min rest in a sitting position, phases I and V of Korotkoff sounds being respectively used for systolic BP and diastolic BP. Measurements were performed in duplicate and the mean was used for analyses.
Blood samples were collected from an antecubital vein into vacutainer tubes containing EDTA after 12-h overnight fast and 48-h alcohol abstinence. Blood samples were taken to identify and exclude individuals presenting exclusion criteria such as metabolic disorders, as mentioned. Afterward, selected participants had blood samples taken prior to and after the n-3 PUFA supplementation period. Plasma was separated by centrifugation (2500 × g for 10 min at 4⬚C) and samples were aliquoted and frozen at −80⬚C for subsequent analyses. Plasma total cholesterol (TC) and TG concentrations were measured using enzymatic assays [ [17, 18]. The repeatability determined by the variation in measurements within-run, was 0.7–1.1% for TC and TG. The precision determined using human samples and controls, was 1.4–1.6% for TC and 1.8–1.9% for TG. The HDL cholesterol fraction was obtained after precipitation of VLDL and LDL particles in the infranatant with heparin manganese chloride [19]. LDL cholesterol was calculated with the Friedewald formula [20]. Fasting insulinemia was measured by RIA with PEG separation [21]. Fasting glucose concentrations were measured enzymatically [22].
2.1.4 Study design and diets
2.2.2 Inflammatory markers
Subjects followed a run-in period of 2 weeks in which individual dietary instructions were given by a trained dietician
Plasma concentrations of IL6 and tumor necrosis factoralpha (TNFA) were measured with high-sensitivity ELISA kits
2.1.2 Anthropometric measurements Body weight, height, and waist girth were measured according to the procedures recommended by the Airlie Conference [26] and were taken by a nurse at each visit at the clinical investigation unit; during the run-in period as well as before and after the n-3 PUFA supplementation. BMI was calculated as weight per meter squared (kg/m2 ). 2.1.3 BP measurements
C 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
www.mnf-journal.com
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Mol. Nutr. Food Res. 2014, 58, 1079–1086
including: Human IL6 Quantikine HS ELISA Kit Minneapolis, MN, USA (R&D Systems, Minneapolis, MN, USA (HS600B)) and Human TNF-alpha Quantikine HS ELISA Kit (R&D Systems (HSTA00D)). Plasma C-reactive protein (CRP) was measured by nephelometry (Prospec equipment Behring) using a sensitive assay, as described previously [23]. The repeatability was 6.9–7.4% for IL6, 3.1–8.5% for TNFA and 2.3–2.6% for CRP. The precision was 6.5–9.6% for IL6, 7.3–10.6% for TNFA and 1.3–4% for CRP. Subjects with CRP concentrations more than ± 3 SD from the mean were excluded from statistical analyses.
2.2.3 FA composition of plasma PL Plasma lipids were extracted with chloroform:methanol (2:1, by volume) according to a modified Folch method [24]. Total PL were separated by TLC using a combination of isopropyl ether and acetic acid and FAs of isolated PL were then methylated. Capillary GC was then used to obtain FA profiles. The technique used for plasma analyses has been previously validated [25]. Values of FA concentrations are expressed as percent of total FA in plasma PL. Further, FA ratios were used as surrogate measures of desaturase activity. SCD16 was calculated with the ratio of palmitoleic acid (16:1n-7) on palmitic acid (16:0) as well as the SCD18 was calculated with the ratio of oleic acid (18:1n-9) on stearic acid (18:0).
2.3 Statistical analysis Data are shown as mean ± SD. Hardy-Weinberg equilibrium was tested with the Allele Procedure in SAS, version 9.2 (SAS Institute Inc., Cary, NC, USA). Distribution of alleles in the present study cohort was compared with the Caucasian population using the CEU data from the National Center for Biotechnology Information (www.ncbi.nlm.nih.gov/) and the Fisher Exact Test. Further, variables were checked for normality of distribution using the skewness and the kurtosis values. TG, CRP, IL6, and TNFA concentrations were log-transformed before analyses to normalize their distribution. First, the repeated ANOVA was used to test for the effects of the genotype, the time (pre- and post-n-3 PUFA supplementation) and the genotype by n-3 PUFA supplementation interaction effect on each variable when adjusted for the effects of age, sex and BMI. A second model also included energy intake. A statistical p-value was defined as p ≤ 0.05 and was used in order to avoid discounting true positive associations. When significant differences were found, a pairwise comparison was performed in a global analysis (Tukey tests; significance p ≤ 0.05). Statistical analyses were performed with SAS statistical software, version 9.2 (SAS Institute Inc.). A group of 152 subjects was calculated to provide an 80% probability at an alpha = 0.05 of to detect a difference of 0.25 mmol/L of plasma TG levels (primary endpoint) after 6 weeks of n-3 PUFA supplementation with a genetic variation that occurs at a relatively low frequency (5%) in the population [26].
2.2.4 DNA extraction and genotyping The SIGMA GenElute Gel Extraction Kit (Sigma-Aldrich Co. St.Louis, MO, USA) has been used to extract genomic DNA. Selected SNPs were genotyped using validated primers and TaqMan probes (Applied Biosystems, Foster City, CA, USA). DNA was mixed with TaqMan Universal PCR Master Mix (Applied Biosystems) and a gene-specific primer and probe mixture (predeveloped TaqMan SNP Genotyping Assays; Applied Biosystems) in a final volume of 10 L. Genotypes were determined using a 7500 FAST RT-PCR System and analyzed using ABI Prism SDS version 2.0.5 software (Applied Biosystems). SNPs in SCD were identified using the International HapMap Project SNP database, based on the National Center for Biotechnology Information B36 assembly Data Rel 28, phase II + III, build 126. Tagger procedure in Haploview V4.2 was used to determine tag SNPs using a minor allele frequency > 5% and pairwise tagging (r2 ≥ 0.8). Therefore, eight SNPs were selected from the SCD gene according to the specified parameters and genotyped: rs1502593, rs522951, rs11190480, rs3071, rs3829160, rs2234970, rs10883463, and rs508384. If a significant SNP that affects an amino acid substitution was found, the SIFT and PolyPhen Web-based softwares were used to predict the effect of amino acid substitution on protein structure and function. C 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
3
Results
3.1 Genotypic characteristics Genotype frequencies did not deviate from those predicted by the Hardy–Weinberg equilibrium except for the following two SNPs: rs1502593 and rs522951, which were excluded from statistical analyses (Table 1). In addition, allele frequencies in the current population were not different when compared to the CEU HapMap population (Table 1). 3.2 Effect of the n-3 PUFA supplementation Parameters pre and post n-3 PUFA supplementation for men (n = 97) and women (n = 113) are described in Table 2. Briefly, n-3 PUFA supplementation was associated with a similar decrease in fasting TG concentrations in men and women, 0.19 mmol/L (15%) and 0.20 mmol/L (17%), respectively. Plasma fasting glucose concentrations were increased significantly by 0.09 mmol/L (2%) in men and 0.12 mmol/L (2%) in women without changing fasting insulin concentrations. There were no other differences between the pre and post n-3 PUFA supplementation including BMI, systolic BP, diastolic BP, TC, LDL cholesterol, and HDL cholesterol www.mnf-journal.com
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Mol. Nutr. Food Res. 2014, 58, 1079–1086
Table 1. Genotypic and allelic distribution of SCD1 polymorphisms in study volunteers and comparison to HapMap population
SNP
Role
Genotype
Count (N)
Allele
Count
Frequency
HapMap (CEU) frequency
Fisher’s exact test (p value)
HWE (p value)
rs1502593
intron
166 254
0.40 0.60
0.41 0.59
0.82
0.03
intron
C G
220 200
0.52 0.48
0.49 0.51
0.67
0.03
rs11190480
intron
A G
378 42
0.90 0.10
0.92 0.08
0.45
0.44
rs3071
intron
A C
285 135
0.68 0.32
0.65 0.35
0.65
0.64
rs3829160
intron
A G
184 236
0.44 0.56
0.46 0.54
0.78
0.16
rs2234970
missense
A C
256 164
0.61 0.39
0.62 0.38
0.88
0.47
rs10883463
intron
C T
26 394
0.06 0.94
0.07 0.93
0.77
1
rs508384
3 UTR
25 116 69 49 122 39 171 36 3 95 95 20 35 114 61 75 106 29 0 26 184 7 59 144
A G
rs522951
AA AG GG CC CG GG AA AG GG AA AC CC AA AG GG AA AC CC CC CT TT AA AC CC
A C
73 347
0.17 0.83
0.15 0.85
0.70
0.81
HWE, Hardy–Weinberg equilibrium.
Table 2. Parameters pre and post n-3 PUFA supplementation for men (n = 97) and women (n = 113)
Men (n = 97) Pre n-3 PUFA Age (years) BMIb) (kg/m2 ) BP Systolic BPb) (mmHg) Diastolic BPb) (mmHg) Glycemic parameters Glucoseb) (mmol/L) Insulin (pmol/L) Lipids parameters Total Cholesterol (mmol/L) LDL-Cholesterolb) (mmol/L) HDL-Cholesterolb) (mmol/L) Triglycerides1b) (mmol/L) Ratio TC/HDLb) Inflammation parameters CRP1b) (mg/L) IL61b) (pg/mL) TNFA1 (pg/mL)
Women (n = 113) Post n-3 PUFA
31.1 ± 8.12 27.4 ± 3.59 27.5 ± 3.66
Pre n-3 PUFA
p-value for timea) Post n-3 PUFA
30.5 ± 9.11 28.2 ± 3.92 28.3 ± 4.06
118.0 ± 11.6 68.5 ± 8.23
118.2 ± 12.0 69.9 ± 8.89
107.2 ± 8.56 67.5 ± 8.53
106.8 ± 9.80 67.1 ± 8.70
NS NS
5.04 ± 0.45 90.0 ± 101
5.13 ± 0.43 80.1 ± 37.8
4.87 ± 0.45 84.7 ± 42.1
4.99 ± 0.53 86.6 ± 42.9
0.02 NS
4.75 2.90 1.28 1.26 3.89
± ± ± ± ±
0.95 0.89 0.30 0.66 1.11
1.21 ± 1.47 1.31 ± 1.38 1.62 ± 1.08
4.70 2.92 1.29 1.07 3.86
± ± ± ± ±
0.97 0.91 0.32 0.54 1.18
1.36 ± 1.86 1.16 ± 0.94 1.66 ± 1.07
4.75 2.64 1.57 1.17 3.15
± ± ± ± ±
0.85 0.73 0.35 0.60 0.82
3.75 ± 4.85 1.45 ± 0.85 1.74 ± 1.65
4.73 2.65 1.63 0.97 3.03
± ± ± ± ±
0.92 0.78 0.40 0.50 0.82
3.74 ± 5.38 1.49 ± 1.00 1.70 ± 1.40
NS NS NS 0.0001 NS NS NS NS
Data are shown as mean ± SD, significance p ≤ 0.05, 1 p value derived from transformed data. Abbreviations: blood pressure (BP); high density lipoprotein-cholesterol (HDL-C); low density lipoprotein-cholesterol (LDL-C); C-reactive protein (CRP); tumor necrosis factor-alpha (TNFA). a) p value derived from model which included the effects of the time (pre and post n-3 PUFA supplementation) on variables when age, sex, and BMI were taken into account in the entire population (n = 210). b) Significant difference between sexes in the model. C 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
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Mol. Nutr. Food Res. 2014, 58, 1079–1086 Table 3. Phospholipid fatty acid profiles pre and post n-3 PUFA supplementation for men (n = 97) and women (n = 113)
Percentage of total FA
Men (n = 97) Pre n-3 PUFA
Palmitic acidb) Palmitoleic acidb) SCD 16 indiceb) Stearic acidb) Oleic acid SCD 18 indiceb)
27.07 0.59 0.022 14.1 8.59 0.615
± ± ± ± ± ±
1.22 0.18 0.006 0.97 1.06 0.092
Women (n = 113) Post n-3 PUFA 27.1 0.49 0.018 14.5 7.96 0.551
± ± ± ± ± ±
1.14 0.16 0.006 1.23 1.28 0.080
Pre n-3 PUFA 28.3 0.76 0.027 13.1 8.56 0.662
± ± ± ± ± ±
1.50 0.24 0.008 1.37 0.95 0.108
p-value for timea) Post n-3 PUFA 28.2 0.65 0.023 13.5 8.01 0.599
± ± ± ± ± ±
1.44 0.22 0.007 1.30 1.08 0.104
NS
