Plasma Fatty Acids and Markers of Insulin Resistance
Plasma Fatty Acids and Markers of Insulin Resistance
Participants recruited from the University of Toronto campus (2004–2009) for the Toronto Nutrigenomics and Health (TNH) study were used for the current investigation. Participants were between the ages of 20–29 yrs and classified as either Caucasian (n = 461; 132 men/329 women), East Asian (n = 362; 95 men/267 women), or South Asian (n = 104; 41 men/63 women) by self-reported ethnicity. Participants were excluded from the investigation if they had a body mass index (BMI) greater than 30 kg/m, did not complete a food frequency questionnaire (FFQ), or were diagnosed with diabetes or cancer. The TNH study was approved by the Research Ethics Boards at the University of Toronto and the University of Guelph. All subjects provided informed written consent.
Plasma samples were collected following a 12 hour overnight fast, and were used for analysis of glucose and insulin at LifeLabs Laboratories (Toronto, Canada). The homeostatic model assessment of insulin resistance (HOMA-IR) was calculated using the HOMA Calculator v2.2.2 (http://www.dtu.ox.ac.uk/homacalculator/index.php).
Subjects were asked to complete a one-month 196-item food frequency questionnaire (FFQ), adapted from the Willet questionnaire. This FFQ data was used to estimate total dietary intake of fat (in grams) per day. Subjects also completed lifestyle and general health questionnaires, which were used to calculate a physical activity (PA) score.
Fasting plasma samples were assessed using gas chromatography (GC) as previously described. Briefly, 5 μg of a C17:0 internal standard was added to plasma samples and mixed with a 2:1 chloroform:methanol solution containing KCl. Following an overnight incubation at 4°C, samples were centrifuged and the organic layer was dried under a gentle stream of nitrogen gas. Extracted lipids were saponified with 0.5 M KOH in methanol for 1 hour at 100°C, and cooled for 10 minutes at room temperature. 14% BF3-MeOH and hexane were added to samples, prior to methylation, for 1 hour at 100°C. After samples had cooled, double-distilled water was added, and samples were centrifuged at 1000 rpm for 10 minutes. The upper-layer was extracted and dried under nitrogen gas, prior to reconstitution in hexane. Samples were analyzed using an Agilent Technologies 7890A GC system (Agilent Technologies, Mississauga, CA). All FA peaks were identified by comparison to retention times of FA methyl ester standards. Only those FA that were consistently detected across all plasma samples were considered for the current study (i.e. 24 of the 62 FA that can be measured by GC). Absolute FA values were calculated by comparing individual FA peaks to the internal standard and are reported as μg FA/mL plasma ± SE.
All statistical analyses were conducted with JMP Genomics software Version 5.1 (SAS Institute, Cary, NC). The Shapiro-Wilk test was used to assess the distribution of all variables. Fasting insulin was subsequently log transformed for all analyses. Fourteen outliers were identified by performing a Jackknife test and removed prior to the analyses. Baseline anthropometric, clinical, and FA data were examined using a Kruskal-Wallis analysis of variation (ANOVA) test followed by a post-hoc Mann–Whitney-Wilcoxon test. Individual associations between FA and markers of IR (i.e. fasting insulin and glucose, and HOMA-IR) were examined using mixed-effects linear regression models, which accounted for both fixed effects (age, sex, BMI, PA, and average daily fat intake (in grams) per day) and one random effect (date of GC processing). For analyses of sex-specific associations, sex was removed from the aforementioned list of variables. We used a two-step process to determine statistical significance: (1) ethnic-specific associations between FA and markers of IR were considered significant only if they satisfied a Bonferroni correction for multiple testing; followed by (2) an investigation of sex-specific differences for those FA that were statistically significant in the previous step. A P-value < 0.05 was considered statistically significant.
Methods
Study Participants
Participants recruited from the University of Toronto campus (2004–2009) for the Toronto Nutrigenomics and Health (TNH) study were used for the current investigation. Participants were between the ages of 20–29 yrs and classified as either Caucasian (n = 461; 132 men/329 women), East Asian (n = 362; 95 men/267 women), or South Asian (n = 104; 41 men/63 women) by self-reported ethnicity. Participants were excluded from the investigation if they had a body mass index (BMI) greater than 30 kg/m, did not complete a food frequency questionnaire (FFQ), or were diagnosed with diabetes or cancer. The TNH study was approved by the Research Ethics Boards at the University of Toronto and the University of Guelph. All subjects provided informed written consent.
Clinical Measurements
Plasma samples were collected following a 12 hour overnight fast, and were used for analysis of glucose and insulin at LifeLabs Laboratories (Toronto, Canada). The homeostatic model assessment of insulin resistance (HOMA-IR) was calculated using the HOMA Calculator v2.2.2 (http://www.dtu.ox.ac.uk/homacalculator/index.php).
Food Frequency and Health Questionnaires
Subjects were asked to complete a one-month 196-item food frequency questionnaire (FFQ), adapted from the Willet questionnaire. This FFQ data was used to estimate total dietary intake of fat (in grams) per day. Subjects also completed lifestyle and general health questionnaires, which were used to calculate a physical activity (PA) score.
Plasma Fatty Acid Analysis
Fasting plasma samples were assessed using gas chromatography (GC) as previously described. Briefly, 5 μg of a C17:0 internal standard was added to plasma samples and mixed with a 2:1 chloroform:methanol solution containing KCl. Following an overnight incubation at 4°C, samples were centrifuged and the organic layer was dried under a gentle stream of nitrogen gas. Extracted lipids were saponified with 0.5 M KOH in methanol for 1 hour at 100°C, and cooled for 10 minutes at room temperature. 14% BF3-MeOH and hexane were added to samples, prior to methylation, for 1 hour at 100°C. After samples had cooled, double-distilled water was added, and samples were centrifuged at 1000 rpm for 10 minutes. The upper-layer was extracted and dried under nitrogen gas, prior to reconstitution in hexane. Samples were analyzed using an Agilent Technologies 7890A GC system (Agilent Technologies, Mississauga, CA). All FA peaks were identified by comparison to retention times of FA methyl ester standards. Only those FA that were consistently detected across all plasma samples were considered for the current study (i.e. 24 of the 62 FA that can be measured by GC). Absolute FA values were calculated by comparing individual FA peaks to the internal standard and are reported as μg FA/mL plasma ± SE.
Statistical Analysis
All statistical analyses were conducted with JMP Genomics software Version 5.1 (SAS Institute, Cary, NC). The Shapiro-Wilk test was used to assess the distribution of all variables. Fasting insulin was subsequently log transformed for all analyses. Fourteen outliers were identified by performing a Jackknife test and removed prior to the analyses. Baseline anthropometric, clinical, and FA data were examined using a Kruskal-Wallis analysis of variation (ANOVA) test followed by a post-hoc Mann–Whitney-Wilcoxon test. Individual associations between FA and markers of IR (i.e. fasting insulin and glucose, and HOMA-IR) were examined using mixed-effects linear regression models, which accounted for both fixed effects (age, sex, BMI, PA, and average daily fat intake (in grams) per day) and one random effect (date of GC processing). For analyses of sex-specific associations, sex was removed from the aforementioned list of variables. We used a two-step process to determine statistical significance: (1) ethnic-specific associations between FA and markers of IR were considered significant only if they satisfied a Bonferroni correction for multiple testing; followed by (2) an investigation of sex-specific differences for those FA that were statistically significant in the previous step. A P-value < 0.05 was considered statistically significant.
