Secondary Logo

Journal Logo

Short Communication: Nutrition

A Randomized Trial of Maternal Docosahexaenoic Acid Supplementation to Reduce Inflammation in Extremely Preterm Infants

Valentine, Christina J.∗,†,‡; Dingess, Kelly A.§; Kleiman, Jeanne||; Morrow, Ardythe L.; Rogers, Lynette K.

Author Information
Journal of Pediatric Gastroenterology and Nutrition: September 2019 - Volume 69 - Issue 3 - p 388-392
doi: 10.1097/MPG.0000000000002375

Abstract

What Is Known/What Is New

What Is Known

  • Preterm infants experience inflammatory responses to clinical interventions.
  • Unchecked inflammation can lead to further tissue injury.
  • Docosahexaenoic acid has anti-inflammatory properties.
  • Docosahexaenoic is essential to neurological development.
  • What Is New
  • Maternal docosahexaenoic supplementation provides anti-inflammatory effects for the mother.
  • Maternal docosahexaenoic supplementation provides anti-inflammatory effects to preterm infants through breast milk.

Low circulating concentrations of docosahexaenoic acid (DHA) have been associated with poor development in preterm infants (1). Unfortunately, intravenous sources of DHA are not widely used in the United States to meet third trimester targets of intrauterine accretion (55 mg · kg−1 · day−1). In addition, key nutrients such as DHA in the maternal diet can be limiting (2) and thus human milk DHA content is variable (3–5). Mother's milk can, however, reach intrauterine DHA accretion targets with maternal dietary supplementation of 1000 mg DHA per day (6,7). The effects of maternal DHA supplementation on reducing inflammation in the infant have been previously described (7). Proof of this concept has been demonstrated in our murine model that resulted in increased DHA concentrations, decreased inflammation, and improved lung development in the offspring (8); and in preterm infants correlating low DHA levels with increased risk of chronic lung disease (9). DHA is the fatty acid responsible for optimum neurological development, and supplementation with single source DHA is likely to provide additional benefits over fish oil, which is largely comprised of eicosapentaenoic acid (EPA) (10). For this study, our hypothesis was that 1000 mg (intervention) versus 200 mg (current clinical recommendations and control group) of DHA per day using an algal source, would result in improved maternal breast milk concentrations, and thus enhance infant DHA intake. Furthermore, we hypothesized that improved human milk DHA intake by the preterm infants would result in decreased inflammation.

METHODS

Study Design

The present study was designed as a prospective, randomized trial to give either 200 mg (low) or 1000 mg (high) DHA (Expecta) to mothers delivering and providing breast milk for their extremely preterm infants (ClinicalTrials.gov Identifier: NCT01732874, Institutional Review Board number 2012–0329, and the Food and Drug Administration number108241). All studies were conducted at University of Cincinnati Hospital (Cincinnati, OH). Mothers were recruited in the postpartum unit after delivery of an infant <28 weeks gestational age, who intended to provide breast milk for their infant, and whose infants did not have any congenital abnormalities that would interfere with the outcome parameters. Mother/infant dyads were randomized by the Investigational Pharmacy staff. The randomization scheme was generated by the Web site Randomization.com (http://www.randomization.com) using the method of randomly permuted blocks. The first randomization list was created using 6 blocks of 4 patients each, followed by a list containing 5 blocks of 2 patients each. Using computer-generated codes and corresponding labeled bottles, supplements were either mailed or picked up at the Pharmacy. The investigators and mothers were blinded to the groups. DHA supplementation was started on the day of birth. The study was powered to detect differences in inflammatory markers in infant samples at 4 weeks of age from mothers supplemented with 200 or 1000 mg/kg per day of DHA (alpha = 0.05 and power = 80%).

Fatty Acid Measurement

Maternal and infant blood or breast milk was collected to examine fatty acid levels at birth and at 2 and 4 weeks of age. Plasma and red blood cells (RBCs) were separated by centrifugation at the time of collection and stored at −80°C until analysis. Fatty acids were analyzed by gas chromatography with flame ionized detection as previously described (11).

Inflammatory Markers

Aliquots of maternal and infant plasma were used for cytokine measurements at baseline (before 7 days after delivery) and at 2 or 4 weeks. The inflammatory markers were measured using the Meso Scale Discovery multiplex platform. Samples were analyzed for TNFα, IL-6, IL-8, IL-1β, IL-10, INFγ, and IL12p70 in a single analysis. sRAGE was also measured using the same technique using a single analysis platform.

Statistics

Data were analyzed by Univariate and Multivariate Linear Regression with treatment and time as variables and gestational age as a co-variate. Repeated measures analysis could not be performed because of the number of missing variables. Correlations were assessed by Spearman correlation.

RESULTS

Demographics

One hundred and thirty-nine mothers were assessed and 27 mothers met the criteria and were enrolled. Eligibility included <28 week's gestation and no genetic anomalies. Sixteen mother/infant dyads were enrolled in the 200 mg per day arm and 11 mothers and 14 infants in the 1000 mg/day arm. A total of 6 infants expired (2 after completing the study), 4 were withdrawn because of transfer, and 2 withdrew during the study. Eight infants completed the 200 mg/day arm (5 withdrawn or dropped, 3 deceased) and 10 completed the 1000 mg/day arm of the study (1 withdrawn, 3 deceased). Demographic data are indicated in Supplemental Table 1 (Supplemental Digital Content 1, http://links.lww.com/MPG/B640). There were no significant differences between 200 and 1000 mg/day DHA-supplemented groups in sex, race, gestational age at birth, maternal BMI, mode of delivery, or infant morbidities (Supplemental Table 1, Supplemental Digital Content 1, http://links.lww.com/MPG/B640).

Effects of Docosahexaenoic Acid Supplementation

DHA levels were measured in maternal breast milk and in both maternal and infant RBCs (Fig. 1A–C). Greater breast milk DHA concentrations were observed at 4 weeks of 1000 mg/day compared with 200 mg/day DHA supplementation. Maternal RBC DHA concentrations were higher in the 1000 mg/day DHA supplemented group than the 200 mg/day group at 4 weeks (Fig. 1B). Infant RBC DHA concentrations were not different as a result of enhanced maternal supplementation. However, there was an effect of treatment on arachidonic acid (ARA)/DHA ratio indicating either lower ARA or higher DHA levels (Fig. 1C and D).

FIGURE 1
FIGURE 1:
DHA measurements. DHA concentrations were measured in breast milk and maternal and infant RBCs as described in Methods section. White bars represent low DHA supplementation and gray bars represent high DHA supplementation. Data were analyzed by Univariate General Linear Regression with treatment and time as variables. Data from each time point were analyzed by multiple t-tests, significance was determined when P < 0.05.

Maternal Inflammatory Markers

Cytokine levels and sRAGE concentrations were measured in maternal plasma samples obtained at 0 and 4 weeks after birth (Table 1). Multivariate regression indicated no differences in the levels. Between subject analyses identified an effect of time on IL-6, an effect of treatment in IL-8, and an interaction between time and treatment in IFNγ levels.

TABLE 1
TABLE 1:
Inflammatory markers in infant and maternal plasma

Infant Inflammatory Markers

Cytokine levels and sRAGE concentrations were measured in infant plasma samples obtained at 0, 2, or 4 weeks after birth (Table 1). Multivariate regression indicated effects of time, treatment, and an interaction between time and treatment in the model. Between-subject analyses indicated an effect of time and treatment on TNFα levels and an effect of time on IL12p70. sRAGE levels in the 1000 mg/day DHA-supplemented group were disproportionately high at birth; however, they returned to levels matching the 200 mg/day DHA group at 4 weeks, indicating statistically, an effect of time, effect of treatment, and an interaction between time and treatment. Overall, our data suggest a decrease in inflammatory markers in the infants whose mothers received 1000 mg/day DHA supplement compared with infants whose mothers received 200 mg/day DHA supplement.

Correlations

Correlations were identified between maternal breast milk DHA levels and DHA levels detected in maternal RBCs (r2 = 0.55, P = 0.004, Supplemental Figure 1A, Supplemental Digital Content 2, http://links.lww.com/MPG/B641). Inverse correlations were observed between maternal plasma IL-6 (r2 = 0.523, P = 0.005) or IL-8 (r2 = 0.635, P = 0.006) levels and RBC DHA content (Supplemental Figure 1B and C, Supplemental Digital Content 2, http://links.lww.com/MPG/B641). Infant plasma levels of IL-12p70 were negatively correlated with infant RBC DHA concentrations (r2 = 0.635, P = 0.006, Supplemental Figure 1D, Supplemental Digital Content 2, http://links.lww.com/MPG/B641). These data suggest a correlation between higher circulating DHA levels and lower concentrations of specific inflammatory markers.

DISCUSSION

Maternal dietary supplementation has been shown to influence breast milk composition of select nutrients, including DHA (6). Supplementation with preformed DHA is important as studies with the precursor, linolenic acid, do not correlate with increased breastmilk DHA levels (12). Furthermore, preterm infants likely have limited lipid absorption and synthetic capacity. DHA has been shown to have anti-inflammatory properties (13). Consequently, supplementing preterm infants with milk that contains higher concentrations of DHA could attenuate inflammation, and thus improve development.

Although the mechanisms are not completely understood, the anti-inflammatory effect of DHA likely includes changes in receptor-mediated signaling, changes in membrane fluidity, and/or enhancement of the production of anti-inflammatory lipid mediators because of the availability of DHA as substrate (14,15). More specifically, modulation of immune cell activity by long chain fatty acids, such as DHA can affect macrophage adhesion and decrease phagocytosis (16). Premature infants, who miss DHA accretion during the third trimester of development have lower overall intake of DHA, and this deficit is likely to contribute to their vulnerability to inflammation and inflammation-mediated tissue injury after birth.

This study tested the hypothesis that maternal dietary DHA supplementation, while providing breast milk for their infant, would provide anti-inflammatory effects to the infant. We developed a Consort Framework for a randomized control trial of 1000 versus 200 mg/day DHA (control group-current standard of care). No statistical differences were found in demographics or postnatal morbidities between the 2 groups (Supplemental Table 1, Supplemental Digital Content 1, http://links.lww.com/MPG/B640).

As we have previously reported, supplementation with 1000 mg/day peaked maternal breast milk DHA levels within 4 weeks (Fig. 1A) (6). Furthermore, increased breast milk DHA concentrations correlated with increases in maternal RBC DHA levels (Fig. 1B). No differences were observed in infant RBC DHA levels (Fig. 1C). However, as an alternative measurement, the relative ratio of ARA to DHA were calculated and this ratio was significantly lower in the infants receiving milk from high DHA-supplemented mothers indicating that relative amounts of DHA were greater in the infant plasma (Fig. 1D). Collectively, our data support several other studies that maternal DHA supplementation provides additional DHA to the mother, and by means of breast milk to the infant.

Our findings suggest that maternal DHA supplementation of 1000 mg/day suppresses the expression of inflammatory cytokines both in the mother and in the infant (Table 1). Furthermore, the expressions of specific cytokines are inversely related to RBC DHA content (Supplemental Figure 1, Supplemental Digital Content 2, http://links.lww.com/MPG/B641). We propose that the attenuated inflammation in the 1000 mg/day DHA-supplemented infants provides a more suitable environment to promote growth. Poor growth has been associated with other chronic inflammatory diseases in children (17) and elevation in inflammatory cytokines, such as IL-6 (18). DHA supplementation in animal models has been shown to increase bone growth (19,20) and may offer a mechanism by which linear growth in infants may be improved.

The primary limitations of our findings are the small sample size and the complex morbidities found in such immature infants. Our sample size prevented the use of Repeated Measures Analyses, which would have given the study more power to detect differences. In addition, the small size also prevented the ability to statistically control for any of the infant morbidities defined in Supplemental Table 1 (Supplemental Digital Content 1, http://links.lww.com/MPG/B640), however, the low and high supplementation groups had similar diagnoses. In conclusion, maternal supplementation with a higher dose of DHA can impact maternal milk DHA concentrations and may influence infant cytokine levels. Although not conclusive, our data provides information that the clinical team may use to discuss maternal diet strategies.

REFERENCES

1. Martin CR. Fatty acid requirements in preterm infants and their role in health and disease. Clin Perinatol 2014; 41:363–382.
2. Copp K, DeFranco EA, Kleiman J, et al. Nutrition Support Team Guide to maternal diet for the human-milk-fed infant. Nutr Clin Pract 2018; 33:687–693.
3. Elsevier, Valentine CJ, Morrow AL. Human Milk Feeding of the High Risk Neonate. 2012.
4. Dingess KA, Valentine CJ, Ollberding NJ, et al. Branched-chain fatty acid composition of human milk and the impact of maternal diet: the Global Exploration of Human Milk (GEHM) Study. Am J Clin Nutr 2017; 105:177–184.
5. Koletzko B, Agostoni C, Carlson SE, et al. Long chain polyunsaturated fatty acids (LC-PUFA) and perinatal development. Acta Paediatr 2001; 90:460–464.
6. Valentine CJ, Morrow G, Pennell M, et al. Randomized controlled trial of docosahexaenoic acid supplementation in midwestern U.S. human milk donors. Breastfeed Med 2013; 8:86–91.
7. Makrides M, Neumann MA, Gibson RA. Effect of maternal docosahexaenoic acid (DHA) supplementation on breast milk composition. Eur J Clin Nutr 1996; 50:352–357.
8. Velten M, Britt RD Jr, Heyob KM, et al. Maternal dietary docosahexaenoic acid supplementation attenuates fetal growth restriction and enhances pulmonary function in a newborn mouse model of perinatal inflammation. J Nutr 2014; 144:258–266.
9. Martin CR, Dasilva DA, Cluette-Brown JE, et al. Decreased postnatal docosahexaenoic and arachidonic acid blood levels in premature infants are associated with neonatal morbidities. J Pediatr 2011; 159:743.e1–749.e2.
10. Collins CT, Gibson RA, Anderson PJ, et al. Neurodevelopmental outcomes at 7 years’ corrected age in preterm infants who were fed high-dose docosahexaenoic acid to term equivalent: a follow-up of a randomised controlled trial. BMJ Open 2015; 5:e007314.
11. Eder K. Gas chromatographic analysis of fatty acid methyl esters. J Chromatogr B Biomed Appl 1995; 671:113–131.
12. Lapillonne A, Moltu SJ. Long-chain polyunsaturated fatty acids and clinical outcomes of preterm infants. Ann Nutr Metab 2016; 69 (Suppl 1):35–44.
13. Jasani B, Simmer K, Patole SK, et al. Long chain polyunsaturated fatty acid supplementation in infants born at term. Cochrane Database Syst Rev 2017; 3:CD000376.
14. Calder PC. The relationship between the fatty acid composition of immune cells and their function. Prostaglandins Leukot Essent Fatty Acids 2008; 79:101–108.
15. Martin CR, Zaman MM, Gilkey C, et al. Resolvin D1 and lipoxin A4 improve alveolarization and normalize septal wall thickness in a neonatal murine model of hyperoxia-induced lung injury. PLoS One 2014; 9:e98773.
16. Calder PC, Bond JA, Harvey DJ, et al. Uptake and incorporation of saturated and unsaturated fatty acids into macrophage lipids and their effect upon macrophage adhesion and phagocytosis. Biochem J 1990; 269:807–814.
17. Cirillo F, Lazzeroni P, Sartori C, et al. Inflammatory diseases and growth: effects on the GH-IGF axis and on growth plate. Int J Mol Sci 2017; 18: pii: E1878.
18. Denson LA, McDonald SA, Das A, et al. Cytokines Study Subcommittee of the NICHD Neonatal Research Network. Early elevation in interleukin-6 is associated with reduced growth in extremely low birth weight infants. Am J Perinatol 2017; 34:240–247.
19. Currie LM, Tolley EA, Thodosoff JM, et al. Long chain polyunsaturated fatty acid supplementation in infancy increases length- and weight-for-age but not BMI to 6 years when controlling for effects of maternal smoking. Prostaglandins Leukot Essent Fatty Acids 2015; 98:1–6.
20. Watkins BA, Lippman HE, Le Bouteiller L, et al. Bioactive fatty acids: role in bone biology and bone cell function. Prog Lipid Res 2001; 40:125–148.
Keywords:

breast milk; docosahexaenoic acid; inflammation; maternal supplementation; prematurity

Supplemental Digital Content

Copyright © 2019 by European Society for Pediatric Gastroenterology, Hepatology, and Nutrition and North American Society for Pediatric Gastroenterology, Hepatology, and Nutrition