DFA: Linoleic Acid (LA) and Intermediate Health Outcomes (2011)


Wang L, Folsom AR, Zheng ZJ, Pankow JS, Eckfeldt JH. Plasma fatty acid composition and incidence of diabetes in middle-ages adults: the Atherosclerosis Risk in Communities (ARIC) Study. Am J Clin Nutr. 2003. PMID: 12816776.

PubMed ID: 12816776
Study Design:
Prospective Cohort Study
B - Click here for explanation of classification scheme.
Quality Rating:
Positive POSITIVE: See Quality Criteria Checklist below.
Research Purpose:
  • Prospective investigation of the relation of plasma cholesterol ester (CE) and phospholipid (PL) fatty acid composition with the incidence of diabetes mellitus during nine years of follow-up
  • It was hypothesized that higher concentrations of SFAs and lower concentrations of PUFAs in plasma would be associated with increased risk of developing type 2 diabetes.
Inclusion Criteria:
  • Participant in the Atherosclerosis Risk in Communities (ARIC) study
  • 45 years to 64 years of age.
Exclusion Criteria:
  • Prevalent diabetes at baseline (N=301), defined as:
    1. Self-reported history of physician-diagnosed diabetes
    2. Current use of diabetes medications
    3. Eight-hour fasting serum glucose concentrations of at least 126mg per dL
    4. Non-fasting serum glucose concentration of at least 200mg per dL.
  • Unknown prevalent or incident diabetes status (N=104)
  • Missing fatty acid measurements (N=74)
  • Taking cholesterol-lowering medications (N=130)
  • Consuming a special diet (N=587)
  • Prevalent cardiovascular disease (N=222)
  • Non-whites, due to the small number enrolled (N=37).
Description of Study Protocol:
  • Recruitment: A probability sample was recruited
  • Design: Prospective cohort
  • Blinding used: Assumed for laboratory measurements.
  • Intervention: Grouped according to incident diabetes or no diabetes.

Statistical Analysis

  • Proportionate plasma fatty acid composition was compared between participants with and without incident diabetes by using a T-test
  • The association of plasma fatty acid quintiles with other risk factors was analyzed by using analysis of covariance
  • Age-adjusted mean values for baseline risk factors were compared across quintiles of plasma fatty acids after the assumptions of normal distribution and constant variance were confirmed
  • To reduce problems related to conducting multiple comparisons, P<0.01 was set as the significance level
  • Cox proportional hazards regression models were used to estimate the rate ratios (RRs) and 95% CIs for incident diabetes in relation to the quintiles of plasma fatty acid composition
  • Time at risk was calculated from baseline to the time of diabetes diagnosis or censoring
  • There was no multiplicative interaction between sex and plasma fatty acids in association with diabetes, so the data for men and women were combined
  • Baseline age (continuous) and sex (dichotomous) were adjusted for in Model One
  • In Model Two, additional adjustment was made for BMI (continuous), WHR (continuous), cigarette-years of smoking (continuous), alcohol intake (continuous), sports index (continuous), education (high school or less, college or vocational school, or graduate school) and parental family history of diabetes (yes, no or unknown)
  • The trend of RRs across quintiles of fatty acids was tested by using equal weight for each quintile
  • Analyses were conducted for grouped fatty acids and for the most common individual fatty acids, for CE and PL, separately
  • Supplemental analyses included the following. None of these analyses altered the conclusions; thus, for the most part, they were not presented:
    • Analyses were repeated with those participants having prevalent cardiovascular disease or consuming special diets at baseline included
    • Analyses were repeated with the time of incident diabetes assigned for everyone as the mid-point between visits without and with diabetes
    • Analyses were repeated with adjustment for baseline fasting serum glucose and insulin concentrations
    • Analyses were repeated, stratified on baseline fasting glucose concentrations (less than 110, compared with 110mg to 125mg per dL)
    • Analyses were repeated with adjustment for time-dependent body weight at all four ARIC examinations.
Data Collection Summary:

Timing of Measurements

  • Baseline measurements occurred 1987 to 1989
  • Participants were followed-up annually by telephone
  • Re-examinations were performed every three years, up to nine years.

Dependent Variables

  • Diabetes: Incident diabetes was identified in the three ARIC follow-up visits among those who were free of diabetes at baseline
  • Time of diabetes diagnosis was unknown and therefore imputed. For participants who reported diabetes diagnosed by a physician or who were taking diabetes medication, the date was assigned to the mid-point between the last visit without diabetes and the next visit with diabetes. For diabetes defined by serum glucose concentration, the diagnosis date was estimated as the point when the serum glucose concentration crossed the diabetes cut-offs (126mg per dL fasting or 200mg per dL non-fasting), on a regression line of glucose concentrations by visit date. For participants who did not develop diabetes, the censoring date for analysis was the last completed follow-up visit. 

Independent Variables

  • Plasma fatty acids: The CE and PL fractions were separated by thin-layer chromatography with a silica gel plate and two-stage mobile phase development. Fatty acids were measured by gas chromatography. Plasma SFAs, MUFAs and PUFAs were calculated by summing the respective fatty acids with 12 to 24 carbon atoms. Test-retest reliability coefficients (individuals were sampled three times, two weeks apart) for various plasma fatty acids ranged from 0.50 to 0.93 for CE and from 0.31 to 0.89 for PL.
  • Serum glucose: Hexokinase-glucose-6-phosphate dehydrogenase method.

 Control Variables

  • Education level
  • Sports activity: Sports index, derived from the survey of Baecke et al (ref 10), ranged from one (low) to five (high) for physical activity from sports during leisure time
  • Smoking status: Cigarette-years of smoking was defined as the average number of cigarettes smoked per day multiplied by the number of years of smoking
  • Alcohol intake
  • Family history 
  • Height, weight, waist circumference, BMI, waist-to-hip ratio.
Description of Actual Data Sample:

Initial N

  • 15,792 enrolled in ARIC study
  • 4,009 at the Minneapolis site only had plasma saved at baseline and was analyzed for fatty acids.

Attrition (Final N)

  • 2,909 after exclusion criteria were applied (2,657 with no diabetes; 252 with incident diabetes)
  • Men: 45.8% with no diabetes; 58.3% with incident diabetes (P<0.01)


  • No diabetes: 53.5±5.5
  • Incident diabetes: 54.0±5.5.


  • No information given
  • Non-whites were excluded due to low number on recruitment. Therefore, it is assumed participants were white.

Other Relevant Demographics

  • Less than or equal to high school education: No diabetes, 39.4%; diabetes, 46.4%
  • Current smoking: No diabetes, 22.5%; diabetes, 25.4%
  • Parental history of diabetes: No diabetes, 12.9%; diabetes, 22.6% (P<0.01)
  • Alcohol intake (g per week): No diabetes, 60.0±101; diabetes, 71.9±137
  • Cigarette-years of smoking: No diabetes, 305±384; diabetes, 395±449 (P<0.01)
  • Sports index: No diabetes, 2.60±0.81; diabetes, 2.50±0.80.


  • BMI (kg/m2): No diabetes, 26.3±4.2; diabetes, 30.6±4.8
  • Waist-to-hip ratio: No diabetes, 0.902±0.084; diabetes, 0.966±0.071 (P<0.01)
  • Fasting serum glucose (mmol per L): No diabetes, 5.46±0.45; diabetes, 6.05±0.51 (P<0.01)
  • Fasting serum insulin (pmol per L): No diabetes, 62±43; diabetes, 106±65 (P<0.01).


Participants from selected suburbs of Minneapolis, MN.

Summary of Results:

Key Findings

  • 252 developed incident diabetes over a mean of 8.1 years of follow-up
  • Compared with those who remained free of diabetes, persons who developed incident diabetes had significantly (P<0.01) higher proportions of total SFAs in both the CE and PL fractions
  • In CE only, total PUFAs were significantly lower among persons with incident diabetes than among those who did not develop diabetes. In the PL fraction, total PUFAs did not differ significantly by incident diabetes status.
  • Proportions of linoleic acid (as a percentage of total fatty acids) in CE and PL were significantly lower in participants with incident diabetes than those without (no diabetes, 54.3±4.8; diabetes, 52.9±4.4)
  • Persons in the highest quintile of CE PUFAs were more likely to be female and to be more physically active
  • After adjustment for age and sex (Model One), the incidence of diabetes was significantly (P<0.05 for the RR of the highest quintile, compared with the lowest quintile; P<0.05 for trend) and positively associated with total SFAs and MUFAs and significantly and inversely associated with total PUFAs in CE (as shown in Figure 1 of the article)
  • After further adjustment for confounding variables (Model Two), the associations were attenuated and the trend across quintiles remained significant only for total SFAs
  • Compared with the lowest quintile of individual fatty acids in CE, the RR of incident diabetes was 0.46 (95% CI, 0.28, 0.74) for the highest quintile of linoleic acid
  • A tendency for decreased incidence of diabetes across quintiles was observed for PL linoleic acid.

Other Findings

  • For the CE fraction, Pearson correlations between baseline fatty acid concentrations were R=0.51 for SFAs with MUFAs, R=-0.68 for SFAs with PUFAs and R=-0.96 for MUFAs with PUFAs
  • For the PL fraction, these respective correlations were R=0.00, R=-0.36 and R=-0.84.
Author Conclusion:

Limitations as Stated by Authors

  • The measurement of tissue fatty acid composition does not perfectly represent the proportion of fatty acids in the diet because of variability between individuals in the cellular utilization and endogenous synthesis of fatty acids. Although non-dietary determinants of fatty acids were controlled for in the analyses, the associations observed may still, to some degree, reflect the effect of varying metabolic responses to dietary fat.
  • Individual fatty acids were expressed as proportions of total fatty acids, thus making these proportional fatty acid measurements inherently interdependent. This makes it difficult to interpret the effect of single fatty acid constituents, independent of other fatty acids.
  • In the ARIC study, the short-term and long-term reliability of plasma fatty acid composition was better for the major fatty acids (reliability coefficients over 0.65) and lower for fatty acids that compose less than 1% of total fatty acids
  • Adjustments for BMI and waist-to-hip ratio could have been an over-adjustment
  • Did not take into account other dietary factors that have been hypothesized to be related to diabetes development, such as the glycemic index, vitamin E or cereal fiber
  • The proportional saturated fatty acid composition of plasma was positively associated with the development of diabetes. The findings with the use of this biomarker suggest indirectly that the dietary fat profile, particularly that of saturated fat, may contribute to the etiology of diabetes.
Funding Source:
Government: multiple NIH grants
Reviewer Comments:
Quality Criteria Checklist: Primary Research
Relevance Questions
  1. Would implementing the studied intervention or procedure (if found successful) result in improved outcomes for the patients/clients/population group? (Not Applicable for some epidemiological studies) N/A
  2. Did the authors study an outcome (dependent variable) or topic that the patients/clients/population group would care about? Yes
  3. Is the focus of the intervention or procedure (independent variable) or topic of study a common issue of concern to dieteticspractice? Yes
  4. Is the intervention or procedure feasible? (NA for some epidemiological studies) N/A
Validity Questions
1. Was the research question clearly stated? Yes
  1.1. Was (were) the specific intervention(s) or procedure(s) [independent variable(s)] identified? Yes
  1.2. Was (were) the outcome(s) [dependent variable(s)] clearly indicated? Yes
  1.3. Were the target population and setting specified? Yes
2. Was the selection of study subjects/patients free from bias? Yes
  2.1. Were inclusion/exclusion criteria specified (e.g., risk, point in disease progression, diagnostic or prognosis criteria), and with sufficient detail and without omitting criteria critical to the study? Yes
  2.2. Were criteria applied equally to all study groups? N/A
  2.3. Were health, demographics, and other characteristics of subjects described? Yes
  2.4. Were the subjects/patients a representative sample of the relevant population? No
3. Were study groups comparable? Yes
  3.1. Was the method of assigning subjects/patients to groups described and unbiased? (Method of randomization identified if RCT) N/A
  3.2. Were distribution of disease status, prognostic factors, and other factors (e.g., demographics) similar across study groups at baseline? N/A
  3.3. Were concurrent controls or comparisons used? (Concurrent preferred over historical control or comparison groups.) N/A
  3.4. If cohort study or cross-sectional study, were groups comparable on important confounding factors and/or were preexisting differences accounted for by using appropriate adjustments in statistical analysis? Yes
  3.5. If case control study, were potential confounding factors comparable for cases and controls? (If case series or trial with subjects serving as own control, this criterion is not applicable.) N/A
  3.6. If diagnostic test, was there an independent blind comparison with an appropriate reference standard (e.g., "gold standard")? N/A
4. Was method of handling withdrawals described? Yes
  4.1. Were follow-up methods described and the same for all groups? Yes
  4.2. Was the number, characteristics of withdrawals (i.e., dropouts, lost to follow up, attrition rate) and/or response rate (cross-sectional studies) described for each group? (Follow up goal for a strong study is 80%.) Yes
  4.3. Were all enrolled subjects/patients (in the original sample) accounted for? Yes
  4.4. Were reasons for withdrawals similar across groups? N/A
  4.5. If diagnostic test, was decision to perform reference test not dependent on results of test under study? N/A
5. Was blinding used to prevent introduction of bias? Yes
  5.1. In intervention study, were subjects, clinicians/practitioners, and investigators blinded to treatment group, as appropriate? N/A
  5.2. Were data collectors blinded for outcomes assessment? (If outcome is measured using an objective test, such as a lab value, this criterion is assumed to be met.) Yes
  5.3. In cohort study or cross-sectional study, were measurements of outcomes and risk factors blinded? No
  5.4. In case control study, was case definition explicit and case ascertainment not influenced by exposure status? N/A
  5.5. In diagnostic study, were test results blinded to patient history and other test results? N/A
6. Were intervention/therapeutic regimens/exposure factor or procedure and any comparison(s) described in detail? Were interveningfactors described? Yes
  6.1. In RCT or other intervention trial, were protocols described for all regimens studied? N/A
  6.2. In observational study, were interventions, study settings, and clinicians/provider described? Yes
  6.3. Was the intensity and duration of the intervention or exposure factor sufficient to produce a meaningful effect? Yes
  6.4. Was the amount of exposure and, if relevant, subject/patient compliance measured? Yes
  6.5. Were co-interventions (e.g., ancillary treatments, other therapies) described? N/A
  6.6. Were extra or unplanned treatments described? N/A
  6.7. Was the information for 6.4, 6.5, and 6.6 assessed the same way for all groups? Yes
  6.8. In diagnostic study, were details of test administration and replication sufficient? N/A
7. Were outcomes clearly defined and the measurements valid and reliable? Yes
  7.1. Were primary and secondary endpoints described and relevant to the question? Yes
  7.2. Were nutrition measures appropriate to question and outcomes of concern? Yes
  7.3. Was the period of follow-up long enough for important outcome(s) to occur? Yes
  7.4. Were the observations and measurements based on standard, valid, and reliable data collection instruments/tests/procedures? Yes
  7.5. Was the measurement of effect at an appropriate level of precision? Yes
  7.6. Were other factors accounted for (measured) that could affect outcomes? Yes
  7.7. Were the measurements conducted consistently across groups? N/A
8. Was the statistical analysis appropriate for the study design and type of outcome indicators? Yes
  8.1. Were statistical analyses adequately described and the results reported appropriately? Yes
  8.2. Were correct statistical tests used and assumptions of test not violated? Yes
  8.3. Were statistics reported with levels of significance and/or confidence intervals? Yes
  8.4. Was "intent to treat" analysis of outcomes done (and as appropriate, was there an analysis of outcomes for those maximally exposed or a dose-response analysis)? N/A
  8.5. Were adequate adjustments made for effects of confounding factors that might have affected the outcomes (e.g., multivariate analyses)? Yes
  8.6. Was clinical significance as well as statistical significance reported? Yes
  8.7. If negative findings, was a power calculation reported to address type 2 error? N/A
9. Are conclusions supported by results with biases and limitations taken into consideration? Yes
  9.1. Is there a discussion of findings? Yes
  9.2. Are biases and study limitations identified and discussed? Yes
10. Is bias due to study's funding or sponsorship unlikely? Yes
  10.1. Were sources of funding and investigators' affiliations described? Yes
  10.2. Was the study free from apparent conflict of interest? Yes