BF: Dietary Factors, Breast Milk and Infant Outcomes (2008)
Makrides M, Neumann MA, Gibson RA. Effect of maternal docosahexaenoic acid (DHA) supplementation on breast milk composition. European Journal of Clinical Nutr. 1996; 50: 352-357.
PubMed ID: 8793415
NEUTRAL:To assess the effect of varying maternal intake of docosahexaenoic acid (DHA, 22:6-3), in the absence of other dietary polyunsaturates, on breast milk fatty acids.
- Mothers of normal infants (37 weeks to 42 weeks gestation) who intended to breast feed for at least 12 weeks were recruited to participate in the study
- Written consent was obtained from all mothers according to the protocol approved by the Committee on Clinical Investigations (Ethics) at Flinders Medical Center.
Women with diabetes, epilepsy, lipid metabolism disorders or a history of drug or alcohol abuse were excluded.
Recruitment
The recruitment procedures were not described.
Design
Prospective randomized clinical trial.
Blinding Used
- All research and clinical staff associated with the trial were unaware of each mother's dietary grouping
- Each mother was unaware of her treatment group.
Intervention
- Mothers were randomly allocated to one of five daily DHA doses: 0.0g (placebo group), 0.2g, 0.4g, 0.9g and 1.3g
- These doses were calculated to achieve breast milk DHA levels that reflected the range of values reported in cross-sectional studies
- Each mother was asked to consume a 0.5g capsule six times per day
- Active treatment capsules contained 0.2g DHA in the form of triglycerides derived from unclear algae (DHASCO, Martek Biosciences)
- Placebo capsules were identical in size and shape but contained soy oil
- All capsules were provided by Martek Biosciences Inc., Columbia, Maryland.
Fatty Acid Composition of DHA-Oil and Placebo Capsules (Fatty Acids Expressed as a Percentage of Total Fatty Acids)
| Fatty Acid | DHA-Oil | Placebo Oil | |
| Total Saturated |
37.7
|
14.9
|
|
| Total Monounsaturated |
17.4
|
25.9
|
|
| 18:2 n-6 |
0.9
|
54.7
|
|
| 18:3 n-6 |
0.1
|
0.1
|
|
| 20:4 n-6 |
0.4
|
ND
|
|
| Total N-6 |
1.4
|
54.9
|
|
| 18:3 n-3 |
0.1
|
4.2
|
|
| 20:5 n-3 |
ND
|
ND | |
| 22:5 n-3 |
0.4
|
ND
|
|
| 22:6 n-3 |
42.9
|
ND
|
|
| Total N-3 |
43.4
|
4.2
|
|
ND: Not detected less than 0.05% total fatty acids.
Statistical Analysis
- Differences in plasma, erythrocyte and breast milk fatty acids between diet groups were examined by one-way analysis of variance (ANOVA)
- Associations between DHA dose (dietary intake and body mass index) and maternal and breast milk fatty acids were assessed by Pearson correlation co-efficients
- All analysis were by SPSS for Windows 6.0 (SPSS Inc., Chicago, Illinois)
- The participants who ceased exclusive breastfeeding (defined as less than one 250ml bottle of formula per week during the 12-week study period were excluded from the statistical analysis.
Timing of Measurements
- Supplementation of maternal diet was between day five and week 12 postpartum
- Regular contact was maintained with all mothers during the study period to encourage and support breastfeeding
- Fasting maternal blood samples (ml) were taken by venipuncture at 12 weeks postpartum and placed into tubes containing lithium heparin
- Samples of expressed breast milk (two ml), taken at the same feed each day for seven consecutive days, were collected by each mother
- Aliquots (0.5ml) from each daily sample were pooled and the combined milk extracted to obtain a representative breast milk total lipid sample for each mother in the study.
Dependent Variables
- Fatty acids
- Fatty acid methyl esters were identified based on the retention time to authentic lipid standards obtained from the Nu Chek Prep Inc., (Elysian, Minnesota). See Table One.
- Vitamin A and vitamin E
- Plasma vitamin A (retinol) and vitamin E (α-tocopherol) were quantified by high-pressure liquid chromatography
- A Waters 470 data module analyzer was used to monitor peak height and the ratio external: Internal standards
- Concentration was calculated by linear regression analysis of standard dilutions to give correlations co-efficients which were calculated for each individual run
- Retinol was detected to 0.1umol per liter and α-tocopherol to 3.0umol per liter.
Independent Variables
- Age
- Weight
- Body-mass index.
Control Variables
All women were omnivorous and so were asked to limit their fish and seafood intake to no more than one meal per week. Dietary compliance was checked at the end of the study by questionnaire.
- Initial N: 89
- Attrition (final N): 52
- Age: See table three
- Ethnicity: Not available
- Location: Adelaide, Australia.
Of 89 subjects enrolled, 37 subjects were excluded or withdrew their consent and the reasons are documented in the following table:
|
Group |
Placebo |
0.2g DHA |
0.4g DHA |
0.9g DHA |
1.3g DHA |
|
Ceased Breastfeeding |
2 |
4 |
5 |
4 |
5 |
|
Social and Family Reasons |
2 |
1 |
3 |
0 |
5 |
|
Disliked Taking Capsules |
1 |
0 |
0 |
0 |
0 |
|
Baby Unsettled |
1 |
0 |
1 |
1 |
0 |
|
Aggravated Irritable Bowel A |
0 |
0 |
0 |
0 |
1 |
|
Maternal Upset Stomach |
0 |
0 |
0 |
0 |
1 |
|
Total |
6 |
5 |
9 |
5 |
12 |
- A Irritable bowel was a pre-existing condition.
Table Three: Description of Subjects Completing the Study
|
Fatty Acid |
Placebo |
0.2g DHA |
0.4g DHA |
0.9g DHA |
1.3g DHA |
|
Age (years, ±SD) |
30±4 |
32±3 |
29±3 |
30±6 |
29±3 |
|
Weight (kg ±SD) |
62±9 |
60±7 |
69±14 |
66±15 |
64±10 |
|
Body Mass Index (kg/m2 ±SD) |
23.2±2.5 |
23.0±2.2 |
25.2±6.2 |
24.0±4.7 |
23.0±3.4 |
Antioxidant status as assessed by plasma vitamin A and E was similar in all mothers. Dietary treatments had no effects on plasma or erythrocyte levels of total saturates, total monounsaturates, linoleic acid (LA, 18:2n-6) or the n-3 PUFA, α linolenic acid (ALA, 18:3n-3 and EPA).
Table Four: Maternal Plasma Phospholipids and Levels of Plasma Vitamin A and E at 12 Weeks Postpartum
(Fatty acids expressed as a percentage of total fatty acids ±SD)
|
Fatty Acid |
Placebo (N=12) |
0.2g DHA (N=10) |
0.4g DHA (N=12) |
0.9g DHA (N=10) |
1.3g DHA (N=8) |
|
Total Sats |
43.6±0.8 |
43.4±0.9 |
43.9±1.0 |
44.5±1.3 |
43.8±0.5 |
|
Total Mono |
12.8±1.0 |
13.0±1.5 |
13.0±1.2 |
13.5±1.5 |
12.6±1.4 |
|
18:2n-6 |
23.7±2.6 |
24.1±1.4 |
22.5±3.4 |
21.7±3.3 |
22.4±2.4 |
|
18:3n-6 |
0.1±0.1 |
0.1±0.0 |
0.1±0.0 |
0.1±0.1 |
0.1±0.0 |
|
20:3n-6 |
3.2±0.6 |
2.8±0.4 |
2.9±0.7 |
2.8±0.5 |
2.5±0.7 |
|
20:4n-6 |
9.6±1.0a |
8.5±1.1a,b |
8.9±1.9a,b |
7.2±0.9a |
7.7±1.0a |
|
22:4n-6 |
0.4±0.1a |
0.3±0.1a,b |
0.3±0.1b |
0.2±0.0a |
0.2±0.0a |
|
22:5n-6 |
0.3±0.1a |
0.2±0.0b |
0.2±0.1a,b |
0.1±0.0a |
0.1±0.0a |
|
Total n-6 |
37.6±1.3a |
|
35.2±2.2b,c |
32.4±2.9 d |
33.3±2.5d |
|
18:3n-3 |
0.2±0.1 |
0.2±0.1 |
0.2±0.1 |
0.3±0.1 |
0.2±0.1 |
|
20:5n-3 |
0.9±0.4 |
0.8±0.3 |
0.8±0.3 |
1.0±0.3 |
1.1±0.4 |
|
22:5n-3 |
1.1±0.1a |
0.9±0.2b |
0.9±0.2b |
0.7±0.1b,c |
0.6±0.1c |
|
22:6n-3 |
2.8±0.8a |
4.1±0.7b |
4.8±1.9b |
6.4±1.9a |
7.5±1.8c |
|
Total n-3 |
5.0±1.2a |
6.1±0.7a,b |
6.7±2.0b |
8.4±2.0a |
9.4±2.0c |
- The study's results demonstrated that DHA in the diet has a strong, specific and dose-dependent effect on breast milk DHA
- Antioxidant status as assessed by plasma vitamin A and E was similar in all mothers. Dietary treatments had no effects on plasma or erythrocyte levels of total saturates, total monounsaturates, linoleic acid (LA, 18:2n-6) or the n-3 PUFA, alpha-linolenic acid (ALA, 18:3n-3 and EPA).
- Dietary treatments had no effects on plasma or erythrocyte levels of total saturates, total monounsaturates, linoleic acid (LA, 18:2n-6) or the n-3 PUFA, alpha-linolenic acid (ALA, 18:3n-3 and EPA)
- Mean breast milk DHA levels ranged from 0.2 to 1, 1% of total fatty acids. There was a minimal effect of dietary docosahexaenoic acid (DHA) on breast milk arachidonic acid (AA) levels.
- There was a linear association between dietary dose of DHA when expressed per body mass index and DHA in maternal plasma and erythrocyte phospholipids
- There was a direct correlation between maternal dose and BMI of DHA and breast milk DHA (R2=0.12, P<0.05)
- Breast milk was AA was not affected by increasing doses of DHA (R2=0.7, NS). The levels of other n-6 PUFA in breast milk, such as LA (R2=0.12, P<0.05), dihomo-γ-linolenic acid (DGLA, 20:3n-6; R2=0.11, P<0.05) and 22:4n-6 (R2=0.17, P<0.01) showed weak, negative associations with maternal dose of DHA. Levels of the n-3 PUFA were unaffected by increased maternal dietary DHA.
Limitations
Despite the sensitive nature of the inter-relationship between maternal DHA and breast milk DHA, it was difficult to quantify the proportion of dietary DHA that is transferred by breast milk.
Analysis were done for 52 (58%) of the 89 women completed the study only. Significantly more women in the DHA groups withdrew from the study.
Randomized Clinical Trials (RCTs) are considered the gold standard of research practice and are accepted as producing the most reliable scientific evidence. The use of the RCT methodology contributes to improving the overall quality and rigor of the research. Within an RCT, participants are allocated at random to either a control group or experimental group, thereby eliminating all forms of spurious or additional causality. This ensures that both groups are statistically equivalent enabling valid conclusions to be drawn regarding the effect of a treatment or intervention on the experimental group.
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Quality Criteria Checklist: Primary Research
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| 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? | No | |
| 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? | No | |
| 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? | No | |
| 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%.) | No | |
| 4.3. | Were all enrolled subjects/patients (in the original sample) accounted for? | No | |
| 4.4. | Were reasons for withdrawals similar across groups? | No | |
| 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? | Yes | |
| 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? | 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)? | N/A | |
| 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 | |