Fatty Acid Composition and Eicosanoid Levels (LTE 4 and PGE 2 ) of Human Milk from Normal Weight and Overweight Mothers

Abstract

Background:

Maternal obesity is known to affect human milk composition. Long-chain polyunsaturated fatty acids (LCPUFA) are vital nutrients to the nervous system development and precursors of eicosanoids related to obesity (prostaglandin E₂-PGE₂-and leukotriene E₄-LTE₄). The aim of the present research was to study the lipid profiles, with particular emphasis to LCPUFAs, and the concentrations of eicosanoids PGE₂ and LTE₄, involved in adipose tissue development, in human milk from overweight mothers compared with normal weight mothers.

Materials and Methods:

Study including 46 overweight and 86 normal weight breastfeeding volunteers was carried out. Fatty acids and eicosanoids (PGE₂ and LTE₄) were analyzed in mature human milk. Fatty acids quantification was determined by gas chromatography and mass spectrometry. PGE₂ and LTE₄ were measured by immununoassay.

Results:

Human milk of overweight mothers had lower contents of n-3 LCPUFA, including eicosapentaenoic acid (20:5n-3, EPA) and docosahexaenoic acid (22:6n-3, DHA) and higher levels of total n-6 LCPUFA, compared with normal weight mothers (0.45 ± 0.23 versus 0.58 ± 0.38, p = 0.016; 0.05 ± 0.04 versus 0.08 ± 0.08, p = 0.005; 0.26 ± 0.15 versus 0.34 ± 0.22, p = 0.015; 0.84 ± 0.25 versus 0.74 ± 0.20, p = 0.029; respectively). Multiple regression analyses showed that maternal overweight was associated with human milk fatty acid profile. The levels of PGE₂ and LTE₄ in human milk did not show significant differences between groups.

Conclusions:

Our findings support the hypothesis that mother weight status influences human milk n-3 LCPUFA lipid composition, but not its relationship with PGE₂ and LTE₄ levels.

Introduction

Obesity is associated with variations in lipid metabolism and an intensified inflammatory and oxidative status.¹ Biochemical and immunological parameters of human milk can be also modified by metabolic changes associated with obesity.^2,3 Increasing evidence suggests that milk composition differs between overweight and normal weight women in terms of metagenome and microbiome,⁴ intestinal regulation factors and immunological markers,^2,5 and lipid composition.² In this sense, few recent studies indicate that maternal obesity seems to be also associated with higher human milk n-6/n-3 fatty acid ratio, as well as with a higher proportion of longer-chain fatty acids, which present a lower digestibility by newborns' digestive system.^6–8

Among lipids, long-chain polyunsaturated fatty acids (LCPUFAs) are inseparably linked to human health, being part of a diverse range of physiological actions.^9,10 LCPUFAs such as eicosapentaenoic acid (EPA), docosahexaenoic acid (DHA), and arachidonic acid (20:4n-6, AA) play important roles during lactation as they are key constituents of cell membranes, the nervous system, and the retina of infants.^11,12

N-3 and n-6 LCPUFAs, and particularly EPA, DHA, and AA, are considered physiologically essential dietary components in most mammals, including humans, since they have a poor capacity to synthesize them from their 18C dietary fatty acid precursors, mainly 18:3n-3 (linolenic acid; LNA) and 18:2n-6 (linoleic acid; LA),¹³ respectively. Particularly limited is the human capacity to synthesize DHA, a fatty acid considered to be a dietary key ingredient of maternal nutrition during pregnancy and lactation, linked to its condition as an important nutrient for brain tissue development.¹¹ Infants have limited endogenous conversion to n-3 LCPUFAs, particularly DHA,^11,14 and therefore largely depend on the n-3 LCPUFAs provided by antenatal placental transfer or human milk.¹⁵

Moreover, EPA and DHA also have an important function in lipid homeostasis, acting as modulators of PPARs (peroxisome proliferator-activated receptors). These ligand-activated transcription factors can distinctly influence gene expression related to lipid homeostasis depending on its isoform. More specifically, activation of PPARα produces a decrease of lipid levels by stimulating fatty acid metabolism, PPARγ influences adipogenesis, energy balance, and lipid biosynthesis, while PPARβ/δ takes part in fatty acid oxidation. In this regard, n-3 essential fatty acids and specific eicosanoids, including leukotriene B4 and 8-(S)-HETE, act as natural ligands of PPARs and enhance lipid catabolism and homeostasis by stimulation of beta-oxidation of fatty acids.¹⁶

The high n-6/n-3 LCPUFA ratio, and particularly that of AA/EPA, could contribute to the prevalence of obesity by producing AA metabolites such as leukotriene E₄ (LTE₄) and prostaglandin E₂ (PGE₂), which have been shown to promote preadipocyte differentiation and obesity.^17,18 In addition, both are considered as biomarkers of inflammation. PGE₂ induces the production of interleukins IL-6, IL-1β, and tumor necrosis factor alpha (TNF-α), while LTE₄ promotes the recruitment and function of most subgroups of leukocytes at sites of inflammation.^19,20 Therefore, an adequate dietary proportion between n-6 and n-3 LCPUFAs is required to maintain a suitable inflammatory status in terms of prostaglandin and leukotriene metabolism.¹⁸

Although the bioactive lipid profile of human milk during the first month of lactation²¹ and in case of maternal inflammation²² has been previously reported, its relationship with maternal overweight has not yet been addressed. In addition, even though breastfeeding can protect against obesity,^23,24 little is known about the link between maternal obesity and human milk fatty acid profile and eicosanoid levels.

The present research aimed at studying the lipid profiles, with particular emphasis on LCPUFAs in human milk from overweight volunteers compared with normal weight volunteers. Since obesity shows certain features of a systemic chronic inflammation,¹ the concentrations of arachidonic acid metabolites LTE₄ and PGE₂, involved in adipose tissue development and inflammatory status, were also measured. As data available on both topics are still rather limited, the present research provides additional valuable information on human milk composition of overweight mothers.

Materials and Methods

Experimental design

The study corresponds to an observational and case–control approach. Breastfeeding volunteers were included in the investigation prospectively using nonprobability sampling. Mothers were regular donors of human milk and participants of support groups for breastfeeding. This study was approved by La Laguna University Ethics Committee and an informed written consent was provided by all the volunteers concerning their participation in the research.

Setting

The study was carried out from January 2012 to May 2013 on breastfeeding volunteers who were regular donors of human milk at the Breast Milk Bank of the “Hospital 12 de Octubre” in Madrid (Spain) as well as participants of breastfeeding support groups in Tenerife (Canary Islands, Spain).

Participation consisted in donating mature human milk samples (over 2 weeks of lactation) and completing a questionnaire that included the following information: mother's age, weight and height (recorded by a nurse in the last visit to the health center before pregnancy), weight gain during pregnancy, physical exercise, smoking status, parity (primiparous or multiparous), omega-3 supplementation, and weekly fish intake. With respect to infants, information about age, gestational age, and birth weight were also obtained.

The self-reported questionnaire was completed and delivered at the day of milk sample collection. This included information about country of birth, mother's health status, diagnosis of asthma among other diseases, and medications intake. Data were verified by means of a telephone call providing the new information requested. Gestational age and birth weight were obtained from the discharge report of the hospital's newborn. Maternal physical activity was measured according to the type of exercise practiced (gymnastics, walking, running, swimming, and so on) and their weekly hours of practice. Those who reported less than 1 hour per week of any physical activity were considered as not practicing exercise.

Sample description

A nonprobabilistic sampling was performed. Inclusion criteria: healthy adult breastfeeding volunteers, without any chronic disease, including thyroid affection or diabetes, with term infants over 2 weeks of lactation, were invited to participate. Therefore, mothers of premature infants, mothers in the early stages of lactation (≤2 weeks), and mothers with any chronic disease were all excluded. Use of aspirin/other nonsteroidal anti-inflammatory drugs was also part of the exclusion criteria.

Two milk samples (20 mL) were obtained from each participant. They were collected very early in the morning just before breastfeeding the baby, 2 hours since the last breastfeed and from the opposite breast to last offer. The milk samples were mainly extracted by means of a breast pump. Milk from the beginning (10 mL) and end of feeding (10 mL) were mixed to minimize the variability of its composition throughout breastfeeding.

Milk samples were stored in sterile plastic containers at 3–5°C for up to 2 hours, until divided into aliquots of 1.5–3 mL each and stored in closed rigid containers at −80°C until biochemical analysis.

Participants whose milk sample was not enough for the biochemical determinations and those who did not entirely complete the questionnaire were also excluded from the analysis. Table 1 displays a description of the clinical characteristics of the sample used in our study comprising a total of n = 132 volunteers.

Table 1.

Clinical Characteristics of Normal Weight and Overweight Women and of Their Infants Included in This Study (N = 132)

	Normal weight mothers^a (n = 86)	Overweight mothers^a (n = 46)
Parameter		M (SD)	p
Mother characteristics
Age (years)	34.95 (3.11)	34.06 (3.37)	0.132
BMI (kg m⁻²)	22.16 (1.75)	28.00 (2.88)^**	<0.0011
Pregnancy weight gain (kg)	12.19 (4.47)	13.54 (5.62)	0.067
Physical exercise (%yes)^b	41.20%	37.00%	0.710
Smoking (%yes)	3.50%	6.50%	0.252
Parity (%)
Primiparous	64.00%	64.40%	1.000
Multiparous	36.00%	35.60%
Omega-3 supplementation (%yes)^c	40.00%	26.10%	0.128
Fish intake (%)
<3 day week⁻¹	67.50%	79.50%	0.154
≥3 day week⁻¹	32.50%	20.50%
Infant characteristics
Age (months)	7.95 (8.23)	6.14 (5.65)	0.744
Gestational age (weeks)	39 (1.50)	39 (1.97)	0.528
Birthweight (g)	3,261 (611.86)	3,307 (609.50)	0.650

Data are presented as mean (SD) for quantitative variables and as percentage (%) for categorized variables. Differences between pairs of means were tested using Student's t-test in case of normal distribution and the nonparametric Mann–Whitney test was performed when normal distribution was not reached. For categorized variables a chi-square test or Fisher's exact test was used. ^**p < 0.01. The bold p-value denotes significant differences in BMI between normal weight and overweight mothers.

Maternal prepregnancy weight status was classified by the following definitions: normal weight = 18.5 ≥ BMI <25 kg m⁻² and overweight = 25 ≥ BMI <30 kg m⁻².

It was considered “yes” when the mothers reported more than 1 hour per week of any physical activity.

It was considered “yes” when the mother reported the intake of at least one daily dose of omega-3 supplements.

BMI, prepregnancy body mass index; SD, standard deviation.

Body mass index (BMI = kg m⁻²), an indirect measure of overweight and obesity,^2,6,7 was the index used to classify the breastfeeding volunteers into two groups: the overweight group, which included mothers with a BMI between 25 and 30 (25 ≥ BMI <30 kg m⁻²), and the normal weight group, mothers who had a BMI between 18.5 and 25 (18.5 ≥ BMI <25 kg m⁻²). The World Health Organization standard (World Health Organization, 1995) was used to establish groups, referring normal weight for BMI 18.5–24.9 and overweight for BMI 25–29.9.

Measurements

Human milk samples were analyzed to estimate their total contents of protein and lipids as well as the lipid class and fatty acid profiles, the lipid peroxidation and the levels of specific eicosanoids.

Total protein was determined by the colorimetric method described by Lowry et al.²⁵ using the Folin reagent, which is mixed with the sample to form a colored complex with the proteins. The absorbance was read at 750 nm in a spectrophotometer (DU 800; Beckman Coulter, Fullerton, CA).

Lipids were extracted with chloroform/methanol (2:1 by vol) containing 0.01% of butylated hydroxytoluene (BHT) as antioxidant, according to the method described by Christie.²⁶ The organic solvent was evaporated under a nitrogen stream and the lipid content was gravimetrically determined and stored at −20°C in chloroform/methanol (2:1), containing 0.01% of BHT.²⁶

Analysis of lipid classes was performed by one-dimensional double development high-performance thin layer chromatography. Lipid fractions were visualized by charring with 3% (w/v) aqueous cupric acetate containing 8% (v/v) phosphoric acid, and quantified by scanning densitometry²⁷ using a dual-wavelength flying spot Camag TLC visualizer (Muttenz, Switzerland).

Fatty acid methyl esters (FAMEs) were prepared by treating an aliquot of the lipid extract with 1% H₂SO₄ in methanol (by volume) at 50°C during 16 hours and the resultant FAMEs purified by thin layer chromatography (TLC). Before transmethylation, nonadecanoic acid (19:0) was added to the total lipid extract as an internal standard.

FAMEs separation and quantification was performed using a TRACE GC Ultra (Thermo Scientific, Milan, Italy) gas chromatograph (GC), following the chromatographic conditions described by Díaz-López et al.²⁸ The identity of uncertain fatty acids was further confirmed when necessary by mass spectrometry (DSQ II, Thermo Scientific) coupled to a GC of the same characteristics. Human milk fatty acid profiles were expressed as percentage of total fatty acids, also including the total content of fatty acids given as mg per mL of human milk (mg mL⁻¹).

Lipid peroxidation of milk samples was determined by using an iron-based spectrophotometric method with the absorbance read at 500 nm.²⁹ Results were expressed as hydroperoxide equivalents (meqO₂ kg⁻¹ fat) based upon the construction of a standard curve prepared with increasing concentrations of Fe III.

Eicosanoids PGE₂ and LTE₄ were measured by immununoassay (EIA) using commercial kits: “Prostaglandin E₂ EIA Kit-Monoclonal” of 96 wells for PGE₂ and “Leukotriene E₄ EIA Kit-Monoclonal” of 96 wells for LTE₄ (Cayman Chemical Company, Ann Arbor, MI), according to the manufacturer's instructions. Results were expressed in picograms per milliliter (pg mL⁻¹).

Statistical analysis

Data analyses were carried out using the IBM SPSS (version 21; IBM Corporation, Armonk, NY) statistical software. Results are presented as means (M) ± standard deviations.

Once confirmed that human milk lipid profiles did not vary with mother's origin, the samples were divided based on maternal BMI. As a result, the sample size for normal weight and overweight volunteers was 86 and 46, respectively. The homogeneity of variances was checked with the Levene test, and Student's t-test was used to identify differences between groups, in particular, the effect of maternal overweight on the fatty acid profile of human milk and eicosanoid levels. According to the central limit theorem, in large samples (>40), means of random samples from any distribution will themselves have normal distribution.³⁰ A study of normality has been also carried out, and differences between pairs of means were tested using Student's t-test in case of normal distribution and the nonparametric Mann–Whitney test when normal distribution was not reached.

For physical exercise, smoking status, parity, omega-3 supplementation, and mother's weekly fish intake, a chi-square test or Fisher's exact test, as appropriate, was used because of the categorized variables. To control for the effects of overweight group, breastfeeding duration, fish intake, and omega-3 supplement, multiple regression models were run with milk fatty acids as dependent variables. Linearity was assessed by partial regression plots and a plot of standardized residuals against the predicted values. Independence of residuals was assessed by a Durbin–Watson statistic. There was homoscedasticity as assessed by visual inspection of a plot of standardized residuals versus unstandardized predicted values. Outlier points were assessed by standardized deleted residual greater than ±3 standard deviations, leverage points by leverage values and influential points by Cook's distance. The unusual points had no effect on the results of the regression models proposed. The assumption of normality was met, as assessed by Q-Q Plots and the Kolmogorov–Smirnov tests. Pearson correlation coefficients were used to establish correlations between fatty acids and eicosanoid levels in breast milk. A p-value <0.05 was considered statistically significant.

Results

The clinical characteristics of the volunteers and their infants are summarized in Table 1. The time point after birth when milk samples were collected did not show significant differences between groups (7.95 and 6.14 months for normal weight and overweight volunteers, respectively). Studied population of subjects was comparable except for the prepregnancy BMI (p = 0.0001). Therefore, there were no significant differences in mean age, pregnancy weight gain, frequency of physical exercise, smoking status, parity, omega-3 supplementation and weekly fish intake among normal weight and overweight mothers, or infant characteristics (age, gestational age and birth weight) from both groups of study.

Total protein and total lipid contents, peroxidation index, and lipid class composition did not vary between human milk from normal or overweight volunteers (Table 2). Regarding analyzed eicosanoids, PGE₂ and LTE₄ concentrations were similar between both groups of women (Table 2).

Table 2.

Total Protein and Total Lipid Contents, Peroxidation Index, Lipid Class Composition, and Eicosanoid Levels of Human Milk from Normal Weight and Overweight Mothers (N = 132)

	Normal weight mothers ^a (n = 86)	Overweight mothers^a (n = 46)
Parameter		M (SD)	p
Total protein (mg mL⁻¹)	19.83 (6.14)	18.52 (6.24)	0.252
Total lipid (mg mL⁻¹)	33.66 (18.42)	32.57 (18.00)	0.750
PI (meqO₂ kg⁻¹ fat)	2.81 (2.64)	3.04 (2.84)	0.833
Lipid classes (%)
DAG	0.90 (0.75)	1.10 (0.94)	0.278
CHO	6.70 (3.04)	6.35 (2.46)	0.749
FFA	3.72 (3.26)	3.84 (2.85)	0.448
TG	85.10 (5.64)	84.60 (7.62)	0.943
SE	3.58 (1.58)	3.31 (1.64)	0.350
Eicosanoids (pg mL⁻¹)
PGE₂	189.64 (89.35)	219.74 (75.03)	0.342
LTE₄	6222.35 (3310.80)	4284.85 (2586.61)	0.135

Data are presented as mean (SD).

Maternal prepregnancy weight status was classified by the following definitions: normal weight = 18.5 ≥ BMI <25 kg m⁻² and overweight = 25 ≥ BMI <30 kg m⁻². Differences between pairs of means were tested using Student's t-test in case of normal distribution, and the nonparametric Mann–Whitney test was performed when normal distribution was not reached. No difference between groups.

CHO, cholesterol; DAG, diacylglycerols; FFA, free fatty acids; LT₄, leukotriene E₄; PGE₂, prostaglandin E₂; PI, peroxidation index; SE, sterol esters; TG, triacylglycerols.

Table 3 shows the fatty acid profiles of milk from the two groups of breastfeeding volunteers. 18C and 20C saturated fatty acids, 20:5n-3, 22:6n-3, total n-3 LCPUFA, and %n-3 LCPUFA/total LCPUFA were significantly lower in the milk of overweight volunteers, whereas 16:1 isomers, 20:2n-6, 20:3n-6, 22:4n-6, total n-6 LCPUFA, and %n-6 LCPUFA/total LCPUFA presented the opposite trend. As a consequence, human milk from overweight volunteers contained lower EPA/AA, DHA/AA, and (DHA+EPA)/AA ratios and higher n-6/n-3 and LA/LNA ratios than those of normal weight women. Other ratios commonly used to characterize fatty acid composition of human milk such as unsaturated/saturated, monounsaturated/saturated, polyunsaturated/saturated, and polyunsaturated/monounsaturated did not vary between groups.

Table 3.

Main Fatty Acid Composition of Human Milk from Normal Weight and Overweight Mothers (N = 132)

	Normal weight mothers^a (n = 86)	Overweight mothers^a (n = 46)
Fatty acid		M (SD)	P
∑FA (mg mL⁻¹)	26.93 (14.94)	26.33 (14.93)	0.692
Saturated (%)	36.21 (5.37)	35.93 (4.54)	0.764
14:0	5.78 (2.49)	5.59 (2.00)	0.890
16:0	20.14 (2.67)	20.21 (2.56)	0.886
18:0	5.84 (1.04)	5.45 (0.93)^*	0.034
20:0	0.16 (0.02)	0.15 (0.03)^*	0.028
Monounsaturated (%)	46.46 (5.21)	45.51 (5.12)	0.318
16:1n-9	0.47 (0.09)	0.50 (0.08)^*	0.044
16:1n-7	1.83 (0.53)	2.03 (0.62)^*	0.032
18:1n-9	40.06 (5.13)	38.97 (4.84)	0.236
18:1n-7	2.86 (0.40)	2.83 (0.41)	0.652
20:1n-9	0.40 (0.12)	0.37 (0.08)	0.318
Polyunsaturated n-6 (%)	15.12 (4.07)	16.52 (4.37)	0.083
18:2n-6	13.99 (3.98)	15.25 (4.20)	0.119
18:3n-6	0.12 (0.07)	0.14 (0.06)	0.069
20:2n-6	0.25 (0.06)	0.27 (0.08)^*	0.033
20:3n-6	0.27 (0.09)	0.32 (0.10)^**	0.003
20:4n-6	0.38 (0.10)	0.41 (0.12)	0.363
22:4n-6	0.06 (0.02)	0.08 (0.03)^**	0.004
Polyunsaturated n-3 (%)	1.31 (0.53)	1.14 (0.36)	0.051
18:3n-3	0.65 (0.28)	0.62 (0.22)	0.738
20:5n-3	0.08 (0.08)	0.05 (0.04)^**	0.005
22:6n-3	0.34 (0.22)	0.26 (0.15)^*	0.015
LCPUFA	1.32 (0.49)	1.29 (0.38)	0.947
n-6 LCPUFA	0.74 (0.20)	0.84 (0.25)^*	0.029
n-3 LCPUFA	0.58 (0.38)	0.45 (0.23)^*	0.016
Ratios
n-6/n-3	12.90 (4.92)	15.68 (6.11)^*	0.011
LA/LNA	23.11 (7.94)	26.39 (9.59)^*	0.038
DHA/EPA	4.80 (2.06)	5.38 (2.33)	0.168
EPA/AA	0.20 (0.18)	0.13 (0.09)^**	0.001
DHA/AA	0.90 (0.53)	0.67 (0.38)^**	0.001
(DHA+EPA)/AA	1.11 (0.68)	0.79 (0.46)^**	0.001
LCPUFA balance (%)
%n-6 LCPUFA	58.59 (11.08)	65.79 (9.54)^***	<0.001
%n-3 LCPUFA	41.41 (11.08)	34.21 (9.54)^***	<0.001

Data are presented as mean (SD).

Maternal prepregnancy weight status was classified by the following definitions: normal weight = 18.5 ≥ BMI <25 kg m⁻², overweight = 25 ≥ BMI <30 kg m⁻².

Asterisks and the bold p-values denote significant differences (^*p < 0.05; ^**p < 0.01; ^***p < 0.001) between pairs of means corresponding to human milk of normal weight and overweight mothers (Student's t-test or Mann–Whitney test).

AA, arachidonic acid (20:4n-6); BMI, body mass index; DHA, docosahexaenoic acid (22:6n-3); EPA, eicosapentaenoic acid (20:5n-3); FA, fatty acids; LA, linoleic acid (18:2n-6); LCPUFA, long-chain polyunsaturated fatty acids (C ≥ 20 and ≥3 double bounds); LNA, linolenic acid (18:3n-3); %n-6 LCPUFA, %n-6 LCPUFA/total LCPUFA); %n-3 LCPUFA, %n-3 LCPUFA/total LCPUFA; SD, standard deviation.

For all fatty acids, multiple regression models were carried out. In all cases, the design was identical: fatty acids were defined as dependent variables and normal versus overweight women, breastfeeding duration (in months), fish intake (less than three times per week versus more than three times per week), and omega-3 supplementation (yes or not) were assigned as independent variables (Table 4). Results showed a very consistent pattern: overweight group was associated with significant effects on fatty acids (Table 3), but neither fish intake nor omega-3 supplements were (Table 4).

Table 4.

Summary of Multiple Regression Analysis

	B		SE_B		β
Variable	20:3n-6	n-6 LCPUFA	20:3n-6	n-6 LCPUFA	20:3n-6	n-6 LCPUFA
Intercept	0.307	0.797	0.017	0.038
Normal versus overweight women	0.046	0.093	0.017	0.039	0.218^**	0.201^*
Omega-3 supplementation	0.017	0.043	0.018	0.040	0.081	0.092
Breastfeeding duration	−0.073	−0.124	0.016	0.037	−0.372^***	−0.288^**
Fish intake	0.001	−0.015	0.018	0.042	0.007	−0.031

p < 0.05; ^**p < 0.01; ^***p < 0.001.

B, unstandardized regression coefficient; β, standardized coefficient; LCPUFA, long-chain polyunsaturated fatty acids (C ≥ 20 and ≥3 double bounds); SEB, standard error of the coefficient.

Regarding breastfeeding duration, which also corresponds to infant age, we found that this variable only proved to have a significant effect on the levels of 20:3n-6 (p < 0.001) and total n-6 LCPUFA (p < 0.001) (Table 4). Once the effect of breastfeeding duration was controlled, maternal overweight continued to have a significant effect on the levels of these n-6 polyunsaturated fatty acids in breast milk: p < 0.01 for 20:3n-6; and p < 0.05 for total n-6 LCPUFA (Table 4). For the rest of the fatty acids, the regression models are equivalent to comparison between pairs of means corresponding to human milk fatty acids of normal and overweight volunteers (Table 3).

Finally, no significant correlation existed between LTE₄ levels and AA, EPA, and DHA contents in human milk, neither for normal weight nor for overweight groups. Similarly, PGE₂ and AA, EPA, and DHA levels were not significantly correlated (Table 5).

Table 5.

Analysis of the Associations Between Fatty Acids and Eicosanoids Levels in Human Milk

	Normal weight mothers (n = 86)		Overweight mothers (n = 46)
	r	p	r	p
LTE₄
AA	0.084	0.648	0.066	0.802
EPA	0.089	0.628	−0.124	0.637
DHA	0.129	0.480	−0.103	0.693
PGE₂
AA	−0.077	0.584	−0.358	0.062
EPA	−0.134	0.339	−0.195	0.320
DHA	−0.238	0.087	−0.202	0.302

Pearson correlation between AA, EPA, and DHA and LTE₄ and PGE₂ levels in human milk from normal weight and overweight mothers (n = 132).

r = Pearson correlation coefficient, p = two-tailed significance level (p-value) of the correlation coefficient.

AA, arachidonic acid (20:4n-6); DHA, docosahexaenoic acid (22:6n-3); EPA, eicosapentaenoic acid (20:5n-3); LT₄, leukotriene E₄; PGE₂, prostaglandin E₂.

Discussion

Our present results provide valuable information about the relationship among the condition of maternal overweight and human milk fatty acid composition. To our knowledge, there are only very few published studies comparing the human milk fatty acid profile of obese and normal weight mothers, which in turn show some contradictory results.^2,6,7,31 Therefore, the research area is novel and this is a valuable contribution to the literature regarding human milk composition of overweight volunteers. More specifically, as far as we are concerned, our investigation is the first one to analyze PGE₂ and LTE₄ contents, and their relationship with LCPUFA levels in human milk of overweight and normal weight mothers.

Human milk fatty acids are either synthesized in the mammary gland or provided by maternal plasma. Some studies suggest that the availability of circulating fatty acids in the mammary gland and their endogenous synthesis are affected by the dietary habits of the mother.^32–36 Thus, human milk DHA levels are related to maternal consumption of this essential fatty acid, being reported that the concentration of this nutrient increases linearly with maternal intake.³²It is also known that DHA content in human milk of vegetarian and vegan mothers is around 0.1%, compared with its levels in mothers who eat fish and other essential lipid sources more frequently, such as those from China, Malaysia, Japan, and northern Canada who have up to 0.8% DHA in their milk.³²

It is generally accepted that fatty acid desaturases control the production of essential LCPUFAs from their 18C dietary precursors through genetic processes.³⁷ Thus, it is also postulated that, in addition to diet, other factors such as genetic differences in desaturases may influence the fatty acid profile of human milk. In our study, we focused on determining if excess maternal weight has an impact on the fatty acid profile of their milk by comparing overweight breastfeeding volunteers with normal weight volunteers, both with a similar pattern of fish intake.

In the present research and in agreement with Fujimori et al.² and Panagos et al.,⁷ no differences in total lipid content, lipid class composition, and total protein levels of human milk between normal and overweight mothers were observed. Also, in concordance with Panagos et al.,⁷ we found significant lower levels of n-3 LCPUFAs and higher contents of n-6 LCPUFAs in human milk of overweight volunteers.

In accordance with our results, Storck Lindholm et al.⁶ in a study performed in Sweden, found that milk from obese mothers presented lower levels of n-3 LCPUFAs and a higher n-6/n-3 fatty acid ratio compared with that of normal weight mothers. Also, these authors found values of DHA and EPA similar to those obtained in our present work. In addition, Mäkelä et al.³⁸ and Panagos et al.⁷ reported a decrease in n-3 LCPUFAs, especially DHA and EPA, and higher n-6/n-3 fatty acid relationship in overweight volunteers' breast milk compared with normal weight women even after adjusting for maternal diet. Only a study developed in Argentina did not find differences in the levels of n-3 PUFA in human milk from obese and normal weight mothers.³¹

Human milk is considered as the most relevant nutrient in metabolic and immunological programming of the child's health.³⁹ Therefore, the lower levels of n-3 LCPUFAs, the higher LA/LNA ratio, and consequently, the higher n-6/n-3 ratio in human milk from overweight volunteers obtained in the present study, could have a negative impact on the optimal offspring nutrition and development.⁴⁰ Although it is suggested that milk composition from overweight mothers is partly proinflammatory with respect to that of normal weight ones,⁷ the benefits of breastfeeding is still evident due to its protective function providing all the macronutrients and bioactive factors required for the optimal infant development.⁴¹

It is known that n-3 LCPUFAs have anti-inflammatory properties, whereas most n-6 LCPUFAs and eicosanoids derived from them present proinflammatory effects. As a result, an imbalance in the n-6/n-3 LCPUFA ratio in favor of n-6 LCPUFAs is described as highly proinflammatory.¹⁸ For this reason, specific n-6 LCPUFA-derived eicosanoids such as PGE₂ and LTE₄ were considered as potential biomarkers of human milk proinflammatory properties. Moreover, in a recent study, it is highlighted that apart from this ratio, the relevance of the proportions between both C18 precursors may have a significant impact on DHA levels. Considering that LA and LNA compete for the same desaturases enzymes, even in the case of a n-3 LCPUFA healthy status, an increase of LA could negatively affect DHA conversion from its shorter chain fatty acid.⁴²

Regarding eicosanoids, only two previous studies have been found in the literature focusing on the analysis of eicosanoid levels in adipose tissue and urine of obese and normal weight subjects.^43,44 García-Alonso et al.⁴⁴ found higher levels of PGE₂ in adipose tissue and Bäck et al.⁴³ a higher concentration of LTE₄ in urine of the obese group.

To our knowledge, our study is the first one to determine the amounts of LTE₄ and PGE₂ in human milk from overweight mothers and its relationship with the LCPUFA contents. Nonetheless, we found no differences in LTE₄ and PGE₂ levels between both groups of study. The disparity between our results and those previously reported on adipose tissue and urine could be explained because the levels of these components in human milk do not fully reflect their levels in other fluids.² On the contrary, there are diverse factors influencing leukotriene production, including the amount of arachidonic acid that is released from membrane phospholipids by the action of phospholipase A₂ as well as the level of each protein involved in the 5-lipoxygenase enzymatic pathway.¹⁹ This is supported through the finding of similar amount of AA in the human milk from both groups of mothers included in our investigation.

Despite the fact that previous reports indicate that a high n-6/n-3 LCPUFA relationship is linked to a higher production of PGE₂ and LTE₄ in obese individuals,^17,18,43,45 no significant correlations have been found between the levels of these fatty acids and the concentration of PGE₂ and LTE₄ in the human milk of overweight volunteers.

Wada et al.⁴⁶ reported that the relative intensity of eicosanoid's formation and action depends on many factors, including the balance between n-3 and n-6 LCPUFAs, giving rise to a high variability of results in available literature. Thus, these authors pointed out that prostaglandins and cysteinyl leukotrienes production is less strong when the enzymes involved in their synthesis pathway use n-3 LCPUFA rather than n-6 LCPUFA substrates.⁴⁶ Moreover, Fischer et al.⁴⁷ indicate that an increase of n-3 LCPUFA eicosanoids in human urine is associated with a high proportion of n-3 fatty acid precursors. In contrast, and in accordance with our results, Bibus and Lands⁴⁸ show no significant association between the human blood levels of some fatty acids related to eicosanoid biosynthesis. Nonetheless, as far as we know, no previous study has analyzed the possible correlation between PGE₂ and LTE₄ levels and the fatty acid profile of human milk of overweight mothers.

According to existing studies, the sampling method may limit to a certain extent the results generalization, since human milk, as with other corporal fluids, does not have a constant composition. It is known that its components vary throughout the day and between the beginning and ending of breastfeeding.⁴⁹ To minimize the variability of these factors, in the present investigation all the samples were collected from each participant early in the morning and mixing milk from the beginning and end of feeding. The consistency of our fatty acid profiles with previous studies^6,7 also makes the authors think that the collection method did not greatly affect the assay parameters.

Although the aim of our research was to compare the milk from overweight mothers with that of normal weight, in complex metabolic diseases, including obesity, genetic factors, and others, in particular, diet, may influence LCPUFA and eicosanoid profile. In this sense, although fish is the main source of omega-3, the authors consider a limitation of the present study, the lack of a more detailed dietary questionnaire, which in addition to the frequency of weekly fish intake, may include information about quantity and fish species consumed by the volunteers, their origin, and other sources of omega-3 intake. In addition, a higher and unregistered amount of LA+AA in the diets, which may compete with omega-3 for absorption and deposition, might modify the relative proportions of other fatty acids, including the omega-3 in breast milk.

Therefore, authors believe that it could be desirable for future approaches to also record the omega-6 fatty acid intake, to give a robust measure of their potential influence in the fatty acid profiles of human milk. Moreover, considering that leptin and eicosanoids derived from n-6 LCPUFAs play an important role in adiposity,⁵⁰ it would be also of great interest to evaluate the relationship between their levels in human milk of overweight mothers.

Conclusions

In summary, our findings support the hypothesis that mother's weight status influences human milk fatty acid composition, but it is not related to its levels of PGE₂ and LTE₄. The higher contents of n-3 LCPUFAs, specifically EPA and DHA, found in human milk from normal weight mothers, and the elevated levels of n-6 LCPUFAs in that of overweight mothers, might suggest some dissimilarity in terms of metabolic functions among both groups of women.

Footnotes

Acknowledgments

The authors thank Flora Barroso (Faculty of Health Science), Nieves Guadalupe Acosta, and José Antonio Pérez (Faculty of Science) from the University of La Laguna for laboratory support, Jose Miguel Díaz for his help in statistic analysis of the results, and Paul Mrocek for reviewing the English language version of the article. This research was supported by “Instituto de La Mujer”-Ministerio de Sanidad, Servicios Sociales e Igualdad. Gobierno de España (Project 2011-0004-INV-00196), and La Caixa and CajaCanarias Banks through a predoctoral contract to Sara García Ravelo.

Disclosure Statement

No competing financial interests exist.

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