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

Citation:
 
Study Design:
Class:
- Click here for explanation of classification scheme.
Quality Rating:
Research Purpose:

To investigate the relationships of serum fatty acid (FA) composition and estimated desaturase activities with the liver fat marker alanine aminotransferase (ALT).

Inclusion Criteria:
  • Subjects were participants of the Uppsala Longitudinal Study of Adult Men (ULSAM)
  • 1,221 (7%) of 1,681 men participated in the third investigation cycle that was carried out between 1991 and 1995, when participants were 71±0.6 years of age
  • 1,181 men had a measure of ALT. Of these, 583 had data on serum FA composition.
Exclusion Criteria:
  • Participants with a previous diagnosis of liver disease were excluded
  • Subjects consuming at least 20g of alcohol per day and subjects with ALT levels below detection levels (less than 0.10µkat per L) were excluded.
Description of Study Protocol:

Recruitment

  • Subjects were participants of the Uppsala Longitudinal Study of Adult Men, a population-based study starting in 1970 in Uppsala, Sweden
  • 1,221 (7%) of 1,681 men participated in the third investigation cycle that was carried out between 1991 and 1995, when participants were 71±0.6 years of age
  • 1,181 men had measures of ALT: Of these, 583 had data on serum FA composition.

Design

546 Swedish elderly men of a population-based cohort particpatted in this cross-sectional study.

Statistical Analysis
  • The distribution of variables was examined by Shapiro-Wilk W-test. To promote a normal distibution the following variables were logarithmically transformed before analysis: ALT, 16:1, 18:0, 18:3n-6, 18:3n-3, 10:5n-3, Δ6-desaturase, SCD-1, fasting insulin, NEFA, triglycerides and adiponectin.
  • Alcohol intake was divided into quartiles. Linear regression analysis was used to associate FA composition (standardized to one SD) with ALT.
  • The relationship between FA composition and ALT was adjusted for abdominal obesity (measured by waist circumference), lifestyle factors (alcohol intake, physical activity) and insulin sensitivity in a multivariate analysis
  • To explore a potential independent link between FAs and liver fat, metabolic factors previously related to fatty liver, triglycerides, NEFA and adiponectin were added to the model
  • To avoid colinearity in Model Two, FA variables with a P-value of <0.001 in Model One (linoleic acid and SCD-1 index) were included
  • Potential selection bias was investigated by comparing participants with FA measurements (N=546) with those without FA measurements, in regard to ALT (N=634), WC, BMI, M and triglycerides.
Data Collection Summary:

Timing of Measurements

  • All measurements were performed under standardized conditions which were previously described
  • All blood samples were drawn after an overnight fast
  • Insulin sensitivity was determined by euglycemic insulin clamp with infusion rate of 56mU.min per body surface area (m2), instead of 40
  • Plasma glucose concentrations were maintained at 5.1mmol per L during the clamp by adjusting the rate of infusion of a 20% glucose solution
  • Glucose disposal; M (mg per kg body weight per minute) was calculated as the amount of glucose taken up during the last 60 minutes of the clamp
  • Serum adiponectin was analysed using a validated in-house time-resolved  immunofluorometric assay (TR-IFMA), based on commercial reagents
  • Physical activity was assessed by a self-administred questionnaire
  • Alcohol intake was assessed by a seven-day dietary record.

Dependent Variables

Serum fatty acid composition

  • Palmitic acid (16:0) percentage
  • Palmitoleic acid (16:1) percentage
  • Stearic acid (18:0) percentage
  • Oleic acid (18:1) percentage
  • Linoleic acid (18:2n-6) percentage
  • y-Linolenic acid (18:3n-6) percentage
  • Alpha ±-Linolenic acid (18:n-6) percentage
  • Dihomo-y-linolenic acid (20:3n-6) percentage
  • Arachidonic acid (20:4n-6) percentage
  • Eicosapentaenoic acid (20:5n-3) percentage
  • Docosahexaenoic acid (22:6m-3) percentage.

Desaturase activities

  • Δ5-desaturase index
  • Δ6-desaturase index
  • Stearoyl coA desaturase;1 index.

Independent Variables

Alanine aminotransferase (μkat per L).

Control Variables

  • Abdominal obesity
  • Lifestyle factors
  • Insulin sensitivity.
Description of Actual Data Sample:
  • Initial N: 1,221 of 1,681 men participated in the third investigation cycle of the Uppsala Longitudinal  Study of Adult Men (ULSAM). 1,181 men had ALT measures: Of these, 583 had serum FA composition data.
  • Attrition (final N): 583 elderly men
  • Age: 71.3 years (71.0 to 71.50 years)
  • Ethnicity: Swedish elderly men
  • Other relevant demographics: BMI, 26.3±3.4kg/m2
  • Anthropometrics: Participants with measurements of FA composition did not differ form the sub-sample without measurements of FA composition with regards to WC, BMI, ALT, M and triglycerides (P>0.28 for all)
  • Location: Sweden.
Summary of Results:

Key Findings

  • In linear regression analyses adjusting for lifestyle, abdominal obesity and insulin sensitivity, the dietary biomarker linoleic acid (n-6), but not n-3 FAs, was inversely related to ALT
  • Desaturation products including palmitoleic, oleic, y-linolenic and dihomo-y-linolenic acids and Δ6-desaturase and SCD-1 indices were directly related to ALT (all P<0.05)
  • After further adjustment for factors previously linked to fatty liver (serum lipids, adiponectin concentrations), SCD-1 index (P=0.004) and insulin resistance (P<0.0001) were independent determinants of ALT activity, whereas waist circumference, triglycerides, non-esterified FA and adiponectin were not.

Relationships of Serum Fatty Acid Composition and Desaturase Indices with Serum Alanine Aminotransferase

Fatty Acid Standardized to 1 SD Univariable Model Multivariable Model One
β P-Value β P-Value
Palmitic Acid (16:0)
0.06
0.0009
0.03
0.08
Palmitoleic Acid (16:1)
0.11
<0.0001
0.09
0.08
Stearic Acid (18:0)
0.02
0.25
0.02
0.37
Oleic Acid (18:1)
0.05
0.01
0.04
0.03
Linoleic Acid (18:2n-6)
-0.09
<0.0001
-0.07
0.0002
y-Linolenic Acid (18:3n-6)
0.08
<0.0001
0.05
0.02
α-Linolenic Acid (18:n-6)
-0.03
0.15
-0.02
0.25
Dihomo-y-Linolenic Acid (20:3n-6)
0.11
<0.0001
0.06
0.002
Arachidonic Acid (20:4n-6)
0.04
0.01
0.02
0.25

Eicosapentaenoic Acid (20:5n-3)
0.05

0.004

0.04
0.03
Docosahexaenoic Acid (22:6m-3)
0.02
0.25
0.01
0.53
Δ5-Desaturase Index
-0.05
0.01
-0.02
0.34
Δ6-Desaturase Index
0.09
<0.0001
0.06
0.004
Stearoyl coA Desaturase; 1 Index
0.10
<0.0001
0.09
<0.0001

β, regression coefficient
Δ5-desaturase=(20:4m-6/20:3n-6)
Δ6-desaturase=(18:3n-6/18:2n-6)
Stearyl coA desaturase-1=(16:1/16:0).

Model 1
N=460
Mutivariable model adjusting for waist circumference, alcohol intake, physical activity and insulin sensitivity.

Associations Between Serum Alanine Aminotransferase Levels and Metabolic Variables (N=460)

Variables Standarized to 1 SD Univariable Regression Multivariable Model Two
β P-Value β P-Value
Waist Circumference
0.10
<0.0001
0.02
0.44
Insulin Sensitivity (M)
-0.15
<0.0001
-0.13
<0.0001
Serum Triglycerides
0.08
<0.0001
0.0001
0.996
Serum Non-Esterified Fatty Acids
0.08
<0.0001
0.02
0.34
Linoleic Acid (18:2n-6)
-0.11
<0.0001
-0.03
0.21
Stearoyl coA Desaturase-1 Index
0.12
<0.0001
0.07
0.004
Serum Adiponectin
-0.05
0.007
0.02
0.35

β, regression coefficient
M, glucose disposal measured by euglycemic insulin clamp
Stearoyl coA desaturase-1=(16:1/16:0)

Model 2 also included alcohol intake and physical activity. None of these variables were significantly related to ALT in the univariable or multivariable models and are therefore not shown.

Author Conclusion:
  • Low dietary intake of linoleic acid and elevated SCD-1 index may contribute to higher ALT activity in elderly men, even independent of obesity and insulin resistance
  • Increased proportions of palmitoleic, oleic, y-linoleic and dihomo-y-linolenic acids and high Δ6-desaturase, SCD-1 and lipogenic indices were realted to ALT, independent of obesity, lifestyle factors and insulin resistance
  • SCD-1 may play a role in the development of fatty liver.
Funding Source:
Other: Nordforsk (Nordic Centre of Excellent in Food, Nutrition and health (SYSDIET), Swedish Nutrition Foundation (SNF) and Swedish council for working life and social research (FAS)/
Reviewer Comments:
  • As an observational cross-sectional study no conclusion regarding causality can be drawn
  • ALT activity was used as a liver fat marker instead of histology or imaging techniques
  • Serum ALT correlates with liver fat, but only partially explains the variation. This would weaken the observed associations rather than create false associations.
  • Desaturase activities were estimated and not directly measured, but desaturation indices are established in epidemiological studies and are related to desaturase activities in vitro and in animals
  • FA composition was measured in a sub-group, but the sub-group did not differ from those without FA composition measurement with regard to WC, BMI, ALT, M and triglycerides. Therefore, the generalizability was probably not affected.
  • Only men of the same age participated without data on women, other ethnic groups or other ages. Therefore, the study is not generalizable.
  • Alcohol intake was self-reported and thus possibly under-reported.
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) Yes
  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) Yes
 
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? 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? N/A
  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? 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.) 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? N/A
  4.1. Were follow-up methods described and the same for all groups? N/A
  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%.) N/A
  4.3. Were all enrolled subjects/patients (in the original sample) accounted for? N/A
  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? Yes
  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? Yes
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? N/A
  6.8. In diagnostic study, were details of test administration and replication sufficient? Yes
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? N/A
  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? N/A
  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