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

Citation:

Krachler B, Norberg M, Eriksson, et al. Fatty acid profile of the erythrocyte membrane preceding development of Type 2 diabetes mellitus. Nutr Metab Cardiovasc Dis. 2008; 18: 503-510. PMID: 18042359.

PubMed ID: 18042359
 
Study Design:
Case Control Study
Class:
C - Click here for explanation of classification scheme.
Quality Rating:
Positive POSITIVE: See Quality Criteria Checklist below.
Research Purpose:

The aim of this study was to prospectively investigate the association of erythrocyte membrane fatty acid composition with development of Type 2 diabetes mellitus risk in a population based study.

Inclusion Criteria:
  • This research was a case-referent study nested within the larger Västerbotten Intervention Programme (VIP), a community program for the prevention of cardiovascular disease and type 2 diabetes mellitus
  • All inhabitants within a northern county of Sweden were invited to complete a health survey at the ages of 40, 50 and 60 years
  • Subjects chosen as part of this study were those that had:
    • Completed a oral glucose tolerance test at baseline
    • Provided a blood sample donation 
    • A verified diabetes diagnosis made via the local diabetes registry.
  • For each case, two referents were randomly assigned and matched for sex, age and year of VIP-survey.
Exclusion Criteria:
  • VIP subjects that were diagnosed with diabetes at baseline
  • Type 1 diabetes diagnosis
  • Incomplete oral glucose tolerance test
  • Unverified diabetes diagnosis
  • No blood sample donation
  • Individuals whose blood sample was used for other studies.
Description of Study Protocol:

Recruitment

  • This research was a case-referent study nested within the larger Västerbotten Intervention Programme (VIP), a community program for the prevention of cardiovascular disease and type 2 diabetes mellitus
  • All inhabitants within a northern county of Sweden were invited to complete a health survey at the ages of 40, 50 and 60 years
  • Cases were defined as free from diabetes at the time of the VIP-survey but diagnosed with type 2 diabetes mellitus during the follow-up period
  • For each case, two referents not diagnosed with diabetes during the follow-up period were randomly assigned from the original health survey cohort and individually matched for sex, age and year of VIP-survey.

Design

  • Subjects completed a health survey at their primary care center
    • Questionnaires on psycho-social conditions and lifestyle are completed
      • Dietary intake over the past year was assessed through a semi-quantitative self-administered food frequency questionnaire (FFQ)
      • FFQ varied between years and study centers
      • Only food items for which self-reported frequencies were available for more than 50% of the total population were used.
    • Biomedical measurements are performed
    • Blood donation was provided.
  • Registries of all diabetes diagnoses that were made in the area of participants and non-participants as of January 31, 2001, was linked to the subjects of the VIP study 
  • Cases were chosen as those who were not diagnosed with diabetes mellitus at baseline but who were then diagnosed with type 2 diabetes mellitus during the follow-up
  • Referents were randomly assigned to the cases and were sex- and age-matched 
  • Erythrocyte membrane fatty acid composition was obtained at baseline and compared between the two study groups
  • Correlations between food intake and erythrocyte membrane fatty acid composition were completed.

Intervention 

Erythrocyte membrane fatty acid composition: Expressed as the percentage of each fatty acid relative to the total sum of fatty acids.

Statistical Analysis

  • Subject characteristics are presented as means ± standard deviations
  • Statistical significance was tested for continuous and categorical variables by T-tests and chi-square tests, respectively
  • Association between increment of proportion of each erythrocyte membrane fatty acid composition by one standard deviation and risk of developing type 2 diabetes mellitus was estimated by conditional logistic regression
  • Adjustment for alcohol intake into quartiles was included in the basic model
  • BMI and HbA1c were adjusted for in an additional model
  • Level of self-reported physical activity and smoking were considered as explanatory variables
  • Correlations between single erythrocyte membrane fatty acid compositions and food items were estimated using Spearman correlation
  • Confidence intervals were estimated with Fisher's Z-transformation
  • SAS for Windows, version 9.1, was used.
Data Collection Summary:

Timing of Measurements

  • As part of the larger VIP study, participants were invited to their primary care center for a health survey at the ages of 40, 50 and 60 years: Baseline demographics and blood donations were made at this time
  • Diabetes diagnoses from the only local hospital and all primary care centers registry as of January 31, 2001
    • Mean duration of follow-up for both cases and referents was 8.8 years (range, 1.1 to 11.4 years)
    • Mean duration of follow-up until diabetes diagnosis among the cases was 5.4±2.6 years (range, 0.1 to 10.6 years).

Dependent Variables

Type 2 diabetes mellitus diagnosis, according to the 1998 WHO definitions.

Independent Variables

Erythrocyte membrane fatty acid composition: Expressed as the percentage of each fatty acid relative to the total sum of fatty acids.

Control Variables

  • Dietary intake over the previous year assessed via a semi-quantitative, self-administered food frequency questionnaire
  • Body mass index
  • Blood pressure
  • Plasma lipids and HbA1c.
Description of Actual Data Sample:

Initial N

  • VIP survey: 33,336 (52% of eligible population) 
  • Diabetes registry: 6,739.

Attrition (Final N)

  • 159 cases
  • 291 referents.

Age

  • 51.7±7.7 years for cases
  • 51.5±7.8 years for referents.

Ethnicity

Not stated.

Anthropometrics

  • BMI: 29.6±4.2 for cases, 25.6±4.1 for referents
  • Weight (kg): 86.3±14.5 for cases, 75.4±12.8 for referents
  • Height (cm): 170.7±9.3 for cases, 171.8±9.1 for referents.

Location

Umeå in Northern Sweden.

Summary of Results:

Key Findings

Subjects who later on developed type 2 diabetes mellitus had higher baseline values for several components of the metabolic syndrome:

  • Higher BMI (P<0.0001)
  • Higher blood pressure: Systolic and diastolic (P<0.0001)
  • Higher HbA1c (P<0.0001)
  • Higher fasting blood glucose (P<0.0001)
  • Higher oral glucose tolerance test (P<0.001).

Erythrocyte fatty acid composition indicated that the case subjects that developed diabetes had:

  • Higher overall saturated fatty acid composition (P=0.004)
    • Higher myristic 14:0, palmitic 16:0 and stearic 18:0 saturated fatty acids
    • Lower pentadecanoic 15:0 and heptadecanoic 17:0 saturated fatty acids.
  • Higher overall monounsaturated fatty acid composition (P=0.016)
  • Lower overall polyunsaturated fatty acid composition (P<0.001): Lower linoleic 18:2 n-6 fatty acid composition (P<0.001)
  • Lower activity of Δ6desaturase (P<0.001).

Odds of developing type 2 diabetes for increments of the fractions of erythrocyte membrane fatty acids indicated:

  • Higher proportions of even numbered saturated fatty acids (P<0.05), palmitoleic 16:1 (P<0.001) and dihomo-γ-linolenic 20:3 n-6 (P<0.001) acids
  • Lower proportions of linoleic 18:2 n-6 (P<0.001) and clupanodonic 22:5 n-3 (P=0.007) acids.

After adjustment for BMI and HbA1c, the erythrocyte membrane fatty acids that remained significant for developing type 2 diabetes mellitus were:

  • Higher proportions of adrenic acid 22:4 n-6 (P=0.026)
  • Lower proportions of pentadecanoic 15:0 (P=0.033) and heptadecanoic 17:0 (P=0.005).

Other Findings
Baseline characteristics of participants

  Cases Referents P Valuea
Systolic Blood Pressure (mmHg)
140.2±18.2
128.7±18.9
<0.0001
Diastolic Blood Pressure (mmHg)
87.5±12.4
80.0±11.1
<0.0001
HbA1c
4.7±0.4
4.3±0.3
<0.0001
Fasting B-glucose (mmol/L)
5.9±0.8
5.2±0.7
<0.0001
OGTT: 2-Hour Blood Glucose (mmol/L)
8.1±2.2
6.6±1.6
<0.0001
Alcohol Intake (quartile 1/2/3/4; missing) Percentage of nb
19/23/24/27; 6%
18/28/28/21; 5%
0.348c
Physical Activity (never/
≥once a week; missing)
41/25/27; 7%
43/30/19; 9%
0.146c
Smoking (present/previous/never; missing) Percentage of nb
42/21/34, 3%
37/28/33; 1%
0.205c

aSignificance test for difference between means, two-tailed T-test
bQuartiles calculated separately for men and women, stratified for number of questions on food frequency questionnaire
cNon-parametrical test for difference: X2.

Mean percentage of fatty acids in erythrocyte membrane

Fatty Acid (Percentage of Total Fatty Acids) and Fatty Acid Ratio Diabetes Cases (N=159)
(Mean ±SD)
Referentsa (N=291)
(Mean ±SD)
P-Value
(Case=ref)
14:0 (Myristic)
0.76±0.17
0.72±0.16
0.037
15:0 (Pentadecanoic)
0.24±0.07
0.25±0.05
0.002
16:0 (Palmitic)
30.00±2.40
29.46±1.63
0.005
17:0 (Heptadecanoic)
0.39±0.06
0.42±0.06
<0.001
18:0 (Stearic)
11.20±1.08
11.01±0.88
0.050
SFAs
42.58±3.05
41.86±2.12
0.004
 
16:1 (Palmitoleic)
1.08±0.32
0.92±0.29
<0.001
18:1 (Oleic)
18.89±1.42
18.69±1.30
0.136
MUFAs
19.97±1.59
19.61±1.43
0.016
 
18:2 n-6 (Linoleic)
14.44±1.81
15.18±1.70
<0.001
18:3 n-3 (Alpha-Linolenic)
0.35±0.10
0.36±0.13
0.191
20:3 n-6 (Dihomo-Gamma-Linolenic)
1.72±0.32
1.56±0.30
<0.001
20:4 n-6 (Arachidonic)
10.77±1.51
10.92±1.33
0.260
20:5 n-3 (Timnodonic)
1.31±0.45
1.37±0.46
0.188
22:4 n-6 (Adrenic)
2.12±0.47
2.06±0.41
0.162
22:5 n-3 (Clupanodonic)
2.14±0.35
2.24±0.35
0.005
22:6 n-3 (Cervonic)
4.61±1.01
4.83±1.02
0.031
PUFAs
37.45±3.49
38.53±2.78
<0.001
 
SCD-1 (16:1 n-7/16:0 Ratio)b
0.04±0.01
0.03±0.01
<0.001
SCD-1 (18:1 n-9/18:0 Ratio)b
1.70±0.21
1.71±0.18
0.763
D6D (20:3 n-6/18:2 n-6 Ratio)c
0.12±0.02
0.10±0.02
<0.001
D5D (20:4 n-6/20:3 n-6 Ratio)d
6.40±1.21
7.17±1.34
<0.001

aReferents were individually matched for age, sex and survey year
bRatios of 16:1 n-7/16:0 and 1:1 n-9/18:0 reflect activity of (microsomal) Stearoyl CoA desaturase =Δ9-desaturase
cRatio of 20:3 n-6/18:2 n-6 reflects activity of Δ6-desaturase (on the assumption that the step from 18:2 n-6 to 18:3 n-6 is rate-limiting)
dRatio of 20:4 n-6/20:3 n-6 reflects activity of Δ5-desaturase.

Odds of developing type 2 diabetes mellitus with one standard deviation increment

Fatty
Acid/Desaturase Activity
Basic (Crude Model Adjusted for Alcohol)a +Adjustment for BMI, HbA1c
ORb 95% CI P (OR=1) OR 95% CI P(OR=1)
14:0 (Myristic)
1.25
1.01-1.53
0.037
0.99
0.72-1.37
0.947
15:0 (Pentadecanoic)
0.65
0.50-0.85
0.002
0.71
0.52-0.97
0.033
16:0 (Palmitic)
1.39
1.07-1.79
0.013
0.96
0.69-1.34
0.822
17:0 (Heptadecanoic)
0.47
0.35-0.63
<0.001
0.54
0.35-0.83
0.005
18:0 (Atearic)
1.37
1.06-1.77
0.017
1.05
0.71-1.56
0.796
SFAs
1.45
1.11-1.89
0.006
0.96
0.68-1.36
0.818
 
16:1 n-7 (Palmitoleic)
1.82
1.42-2.34
<0.001
1.32
0.91-1.89
0.139
18:1 n-9 (Oleic)
1.19
0.96-1.49
0.113
0.86
0.62-1.19
0.357
MUFAs
1.31
1.05-1.62
0.015
0.91
0.66-1.26
0.583
 
18:2 n-6 (Linoleic)
0.51
0.39-0.68
<0.001
0.93
0.62-1.39
0.721
18:3 n-3 (Alpha-Linolenic)
0.85
0.67-1.09
0.206
0.85
0.61-1.20
0.358
20:3 n-6 (Dihomo-Gamma-Linolenic)
1.64
1.31-2.06
<0.001
1.21
0.88-1.65
0.236
20:4 n-6 (Arachidonic)
0.94
0.75-1.17
0.569
1.19
0.86-1.64
0.302

20:5 n-3 (Timnodonic)

0.87
0.69-1.10
0.247
0.75
0.53-1.05
0.095
22:4 n-6 (Adrenic)
1.17
0.95-1.45
0.142
1.49
1.05-2.13
0.026
22:5 n-3 (Clupanodonic)
0.74
0.59-0.92
0.007
0.90
0.66-1.24
0.520
22:6 n-3 (Cervonic)
0.79
0.63-1.00
0.052
1.02
0.74-1.40
0.908
PUFAs
0.67
0.53-0.85
0.001
1.08
0.77-1.52
0.645
 
SCD-1 (16:1 n-7/16:0 Ratio)c
1.74
1.36-2.22
<0.001
1.33
0.94-1.89
0.108
SCD-1 (18:1 n-9/18:0 Ratio)c
0.95
0.75-1.22
0.711
0.86
0.60-1.24
0.422
D6D (20:3 n-6/18:2 n-6 Ratio)d
2.14
1.66-2.76
<0.001
1.31
0.92-1.88
0.132
D5D (20:4 n-6/20:3 n-6 Ratio)e
0.57
0.45-0.72
<0.001
0.83
0.60-1.14
0.251

aAdjusted for alcohol intake (potential confounder of relation dietary fat-intake EMFA)
bReferents were individually matched for sex, age and survey year
cRatio of 16;1 n-7/16:0 and 18:1 n-9/18:0 reflect activity of (microsomal) Stearoyl CoA desaturase =Δ9-desaturase
dRatio of 20:3 n-6/18:2 n-6 reflects activity of Δ6-desaturase (on the assumption that the step from 18:2 n-6 to 18:3 n-6 is rate-limiting)
eRatio of 20:4 n-6/20:3 n-6 reflects activity of Δ5-desaturase.

Association of food intake and two saturated fatty acids (pentadecanoic 15:0 and heptadecanoic 17:0) indicated:
  • Positive correlation with intakes of butter, 3% fat milk and bran flakes
  • Negative correlation with hamburgers and liquor.
Author Conclusion:
  • Erythrocyte membrane fatty acid composition patterns predicted the development of Type 2 diabetes
  • After adjustment for BMI, HbA1c, alcohol intake, smoking and physical activity, the significant predictors were pentadecanoic acid (15:0) and heptadecanoic (17:0) as protective factors and adrenic (22:4 n-6) as a risk factor. 
Funding Source:
Government: Joint Committee of the local county councils of Jamtland, Norrbotten, Vasternorrland and Vasterbotten, Joint Committee of Northern Sweden Health Care Region, Swedish Public Health, Swedish Medical Research Council, the Heart and Chest Foundation, the Stroke Fund
Other: Grant by Norrbottensakademin
Reviewer Comments:
  • The food frequency questionnaires varied between survey years and only food items that were available for more than 50% of the population were used
  • Information on the 44 participants that were excluded as cases due to participation in other studies was not provided and may have added a selection bias.
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? No
  2.2. Were criteria applied equally to all study groups? Yes
  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? Yes
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) Yes
  3.2. Were distribution of disease status, prognostic factors, and other factors (e.g., demographics) similar across study groups at baseline? Yes
  3.3. Were concurrent controls or comparisons used? (Concurrent preferred over historical control or comparison groups.) Yes
  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? N/A
  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.) Yes
  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? Yes
  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? N/A
  5.4. In case control study, was case definition explicit and case ascertainment not influenced by exposure status? Yes
  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? N/A
  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? N/A
  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? Yes
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? No
  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