BF: Dietary Factors, Breast Milk and Infant Outcomes (2008)


Smit EN, Koopmann M, Boersma ER, Musklet FAJ. Effect of supplementation of arachidonic acid (AA) or a combination of AA plus docosahexaenic acid on breastmilk fatty acid composition. Prostaglandins, Leukotrienes and Essential Fatty Acids 2000; 62 (6): 335-340.

PubMed ID: 10913225
Study Design:
Randomized controlled trial
A - Click here for explanation of classification scheme.
Quality Rating:
Neutral NEUTRAL: See Quality Criteria Checklist below.
Research Purpose:

The study investigated whether supplementation with a combination of arachidonic acid (AA) plus docosahexaenic acid (22:6-3, DHA) would affect human milk polyunsaturated fatty acid (PUFA) composition.

Inclusion Criteria:
  • Mothers who were breastfeeding for a period between three and 10 months were recruited to participate
  • All women gave their informed consent
  • The study protocol was in agreement with local ethical standards and the Helsinki declaration.


Exclusion Criteria:

 Exclusion criteria was not delineated.

Description of Study Protocol:


Strategies not delineated


Prospective Clinical Trial

Blinding used

Procedures were not described


Subjects were randomly allocated to receive daily supplements of 0.8ml AA oil (OPTIMAR, Gist-Brocades, The Netherlands), 2.5ml of a combination of AA and DHA oil (0.8ml AA oil and 1.7ml DHA oil (EPAX 0626 TG, Gist-Brocades. The Netherlands)), or to serve as unsupplemented controls.

The calculated daily intakes of the AA+DHA supplemented group corresponded with 321mg. AA, 399mg DHA mg ,113mg EPA, 387mg LCPUFA-6, 539mg LCPUFA-3, 873mg SAFA, 544mg MUFA and 1,083mg PUFA (further referred to as ’300mg AA,110mg EPA and 400mg DHA’).

Table 1

Fatty acid composition AA plus DHA Oil


3 series








22:6w 3






6 series




















9 series


18: 1-9












 Data represent a selection of fatty acids, expressed in mol percentage (mol/100mol).

Abbreviations: SAFA, saturated fatty acids; MUFA, monounsaturated fatty acid: PUFA, polyunsaturated fatty acid; LCPUFA, long chain PUFA (C≥20, double bonds≥3). Women were:

 Statistical Analysis  

  • Within-group differences in milk FA composition N days zero, one and seven were analyzed with the Wilcoxon signed-ranked test
  • Between group difference were analyzed with the Mann-Whitney U-test
  • P<0.017(adjusted for Bonferroni inequality rule type-1 errors) was considered significant
  • All statistical analysis were done with SPSS (SPSS 8.0  for Windows, SPSS Inc, Chicago, Illinois, USA).



Data Collection Summary:

Timing of Measurements 

  • The supplements were given during one week
  • Milk samples were collected on days zero (baseline), one and seven
  • The first milk samples were collected immediately prior to the intake of the first supplement day zero.
  • The next sample was taken on the following day in the morning before the intake of the supplement (day one)
  • The last sample was taken in the morning of day seven
  • Milk samples of controls at the same clock times
  • All milk samples *about 5ml were taken manually, mid-stream and from the same breast.

Dependent Variables

The fatty acid composition of breast milk: Milk was analyzed by capillary gas chromatograph with flame ionization detection

Independent Variables

2.5ml of a combination of AA and DHA oil (0.8ml AA oil and 1.7ml DHA oil (EPAX 0626 TG, Gist-Brocades. The Netherlands)) vs. unsupplemented controls

Control Variables 

The supplemented group received daily additional vitamin E (250 U; Ultra vit E forte, Kenpharm-Ultravit, Veghel, The Netherlands) together with the supplement.

Description of Actual Data Sample:

Initial N

19: (nine AA plus DHA) and 10 control

Attrition (final N)

16: (eight AA plus DHA) and eight control

Two women in the control group did not report for follow-up, and the first sample of a woman in the AA plus DHA group got lost during processing. These three women were excluded from the study.


The AA plus DHA and control groups did not differ in age (median [range]) (23 years [21-27], 24.5 [19-31], 23 [20-28].


The Netherlands 

Summary of Results:

The milk PUFA content and some ratios on days zero, one and seven are presented in Table 2.

Table 2: PUFA contents of AA plus DHA supplemented women at baseline, day one and day seven

 Fatty Acid   Controls  AA plus DHA 
 18:3-3  Day zero  1.01±0.28  0.94±0.30
   Day one  1.18±0.56  0.91±0.26&
   Day seven  1.25±0.27  1.21±0.38&
 20:5-3  Day zero  0.05±0.05  0.05±0.04&
   Day one  0.05±0.06  0.07±0.04&a
   Day seven  0.04±0.02a,b  0.06±0.03b,c
 22:6-3  Day zero  0.13±0.06  0.20±0.13
   Day one  0.22±0.16  0.25±0.09a
   Day seven  0.15±0.05  0.21±0.08a
 sum- 3  Day zero  1.28±0.37  1.30±0.39
   Day one  1.57±0.64  1.36±0.31
   Day seven  1.54±0.31  1.60±0.41a
 LCPUFA -3  Day zero  0.28±0.13  0.36±0.19&
   Day one  0.39±0.22  0.44±0.17&,a
   Day seven   0.29±0.09  0.38±0.12a
 18:2-6  Day zero  17.07±4.20  18.55±5.82
   Day one  18.67±4.39  17.13±6.29
   Day seven  19.61±3.12  18.16±3.90
 20:4-6  Day zero  0.46±0.06  0.55±0.15
   Day one  0.47±0.09  0.61±0.12
   Day seven  0.49±0.07  0.69±0.23
 22:5-6  Day zero  0.05±0.03  0.05±0.02
   Day one  0.04±0.01  0.05±0.02
   Day seven  0.05±0.01  0.05±0.01
 sum-6  Day zero  18.56±4.27  20.19±5.73
   Day one  20.22±4.64  18.77±6.24
   Day seven  21.13±3.29  19.98±3.82
 LCPUFA-6  Day zero  1.31±0.17  1.44±0.34
   Day one  1.37±0.22  1.46±0.28
   Day seven  1.37±0.19  1.62±0.39
 18:2-6/18:3-3  Day zero  17.82±5.43  21.99±10.97
   Day one  16.89±3.49  19.96±9.23
   Day seven  16.13±2.97 16.04±5.15& 
 20:4-6/22:6-3  Day zero  3.85±1.15  3.34±1.44
   Day one  2.92±1.49  2.63±0.72&,a
   Day seven  3.50±0.81  3.55±0.93&
 6/3  Day zero  15.22±4.85  17.58±9.49
   Day one  13.75±3.30  14.83±7.54
   Day seven  13.94±1.94  13.19±4.06
 LCPUFA-6/LCPUFA-3  Day zero  5.33±1.80  4.72±2.06&
   Day one  4.32±1.73  3.61±1.17&,$,a
   Day seven    



Author Conclusion:
  • Administration of a combination of AA and DHA and tended to increase both milk AA and long chain PUFA (LCPUFA)-3 content
  • Between Group differences: On day seven, the control group had a lower 20:5-3 compared with the AA plus DHA group (P=0.004). Within group differences
  • The control group did not exhibit changes in milk PUFA during the one week study period
  • In the AA plus DHA group 20:5-3 (P=0.012) and LCPUFA-3 (P=0.012) increased from day zero to one
  • From day one to day seven 18:3-3 (P=0.012), the 20:4-6/22:6-3 (P=0.012) and the LCPUFA-6/LCPUFA-3 ratio (P=0.012) and the LCPUFA-6/LCPUFA-3 ratio (P=0.012) also increased


The investigation might have been hampered the uniformity of the breastmilk AA content, despite the differences in dietary AA intakes and the possible adverse effects of extremely high AA intake (6g daily) on prostaglandin synthesis.


Funding Source:
University/Hospital: UniversityHospital Gronongen, The Netherlands
Reviewer Comments:

The review extracted information and data for AA plus DHA and control group only. AA oil group was not of our interest.

Poor reporting of mother's demographic, anthropmetric, and health data.



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? No
  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? ???
  2.3. Were health, demographics, and other characteristics of subjects described? No
  2.4. Were the subjects/patients a representative sample of the relevant population? ???
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.) 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? 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? No
  5.1. In intervention study, were subjects, clinicians/practitioners, and investigators blinded to treatment group, as appropriate? No
  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.) N/A
  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? 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? Yes
  6.2. In observational study, were interventions, study settings, and clinicians/provider described? N/A
  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? Yes
  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? 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)? Yes
  8.5. Were adequate adjustments made for effects of confounding factors that might have affected the outcomes (e.g., multivariate analyses)? N/A
  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