Melatonin Concentrations and Antioxidative Capacity of Human Breast Milk According to Gestational Age and the Time of Day

Abstract

Background:

While changes in the composition of breast milk throughout the lactation period are well known, little is known about the antioxidative capacity of breast milk and its regulation as a function of time of day.

Objective:

The aim of this study was to evaluate the antioxidative capacity in breast milk and its regulation by time of day.

Methods:

Melatonin, superoxide dismutase (SOD), glutathione peroxidase 3 (Gpx3) concentrations, and the total antioxidative capacity (TAOC) were analyzed in 105 breast milk samples and 12 maternal serum samples from 21 healthy nursing mothers.

Results:

Comparison between daytime breast milk (collected from 1000-2200 h) and nighttime breast milk (collected from 2200-1000 h) revealed significantly higher concentrations of melatonin and Gpx3 in nighttime milk (melatonin: 1.5 pg/mL [1.0-2.1] day vs 7.3 pg/mL [3.8-13.6] night, median [quartiles], with an estimated mean night-to-day ratio of 5.2 [3.9, 7.1], P < .001; Gpx3: 1436 ng/mL [765-2060] day vs 1800 ng/mL [1242-2297] night, night-to-day difference 192.1 [0.6, 383.7], P = .049). Subgroup analysis showed that melatonin had a circadian rhythm in both preterm and term milk, with a significantly higher nighttime concentration (P < .001), while antioxidant enzymes had a circadian rhythm only in preterm milk, with a significantly higher nighttime concentration for Gpx3 and a significant higher daytime concentration for SOD and TAOC (P = .041 and P = .049, respectively). We found no significant correlation between the concentration of melatonin and the concentration of SOD, Gpx3, or TAOC. Moreover, there were no significant correlations observed between gestational age and the concentration of melatonin and antioxidant enzymes.

Conclusion:

Because of its higher melatonin and Gpx3 content, future research is needed to determine if preterm nighttime milk ought to be the first choice in the feeding of high-risk preterm infants.

Keywords

breast milk breastfeeding circadian rhythm glutathione peroxidase melatonin superoxide dismutase

Well Established

Human breast milk contains important enzymatic and nonenzymatic antioxidants. Antioxidant constituents of breast milk, in comparison to formula, grant the newborn significantly better protection against oxidative stress. Melatonin stimulates several antioxidant enzymes, including superoxide dismutase and glutathione peroxidase.

Newly Expressed

In human preterm breast milk, melatonin, glutathione peroxidase (Gpx), superoxide dismutase (SOD), and total antioxidative capacity (TAOC) exhibited diurnal rhythms, with a nighttime maximum level for melatonin and Gpx3 and a daytime maximum level for SOD and TAOC. The concentrations of SOD, Gpx, and TAOC were independent of the status of melatonin in breast milk.

Background

The diverse health benefits of breast milk for infants, including premature and sick newborns, have been increasingly recognized in recent years. The American Academy of Pediatrics recommends breast milk as the optimal source of enteral nutrition for preterm infants.¹ In accordance with this assessment of breast milk, previous studies have provided multiple lines of evidence suggesting that exclusive breast milk–based feeding is associated with a significant reduction in the prevalence of necrotizing enterocolitis (NEC) among premature newborns.² Although the exact pathogenesis of NEC remains poorly understood, injury of the intestine by oxidative stress is thought to play an important role.³

Breast milk contains not only major nutritive elements but also components that provide immunological protection, including biologically active substances such as hormones, cytokines, and growth factors.⁴ Moreover, human breast milk contains important enzymatic and nonenzymatic antioxidants including superoxide dismutase (SOD), glutathione peroxidase (Gpx), catalase, vitamin E, vitamin C, and melatonin.^5,6 Melatonin is a small lipophilic neurohormone that is mainly synthesized during the night and secreted into the blood by the pineal gland.⁷ In addition to its regulation of the circadian rhythm, melatonin and its metabolites (eg, 6-hydroxy-melatonin) are capable of detoxifying free radicals, including the superoxide anion, hydrogen peroxide; the hydroxyl radical, nitric oxide; and the peroxynitrite anion.⁸ In addition to its direct antioxidant potential, melatonin serves as an indirect antioxidant through the induction of several antioxidative enzymes, such as SOD and Gpx.⁹ It has been demonstrated in vitro and in animal model systems that all of these bioactive components of human milk not only regulate the maturation of the intestinal mucosal barrier but also have the ability to stimulate the healing and repair processes in injured intestinal epithelium.^10-13 Studies in animal models suggest that melatonin may attenuate NEC.^12-14 Moreover, it has been shown that antioxidant constituents of breast milk, in comparison to formula, grant the newborn a significantly better protection against oxidative stress.^15,16

It is known that the composition of breast milk changes continuously throughout the lactation period. However, little is known about the circadian rhythm of the antioxidative enzymes in human breast milk. First, we analyzed the concentration of SOD, Gpx3, and total antioxidative capacity (TAOC) in breast milk depending on the time of day. Second, we analyzed the relationship between melatonin concentration and concentration of SOD, Gpx3, and TAOC. Analyses were carried out in breast milk samples of mothers with term and preterm delivery. We further compared the antioxidative capacities between term and preterm milk.

Methods

Participants

The Ethics Committee of the University of Bonn (244/09) approved this study in accordance with the Declaration of Helsinki. We obtained written informed consent from all women. We enrolled 21 healthy mothers whose child was delivered at the University of Bonn. The mean maternal age was 30 years (17-41 years). Mothers with chronic diseases (eg, hypertension, diabetes mellitus, thyroid disorders, hepatitis, bronchial asthma, chronic renal failure, and heart failure), as well as acute infections, were excluded.

Group 1 (preterm) comprised 14 mothers who gave birth between 27.6 and 36.1 weeks of gestation (mean, 32 weeks). Group 2 (term) included 7 mothers who gave birth between 37.0 and 39.4 weeks of gestation (mean, 38.4 weeks). Both term and preterm infants born to these mothers were sick and unable to be breastfed; therefore, all of these mothers used breast pumps to obtain breast milk for nursing.

Collection of Breast Milk Samples

The mothers were enrolled in the study between the 5th and 10th day postpartum. All women were asked to fill 2 mL breast milk in tubes after each expressing at 4- to 6-hour intervals and to freeze them at −20°C. A total of 5 milk samples were collected from each mother in one 24-hour period. The milk samples were divided into 2 groups based on the time of day of collection: daytime milk (collected from 1000-2200 h) and nighttime milk (2200-1000 h). The women carried the tubes to us, and they were then frozen at −80°C until analysis. Prior to analysis, the samples were thawed and centrifuged at 3000 g for 5 min. The supernatant was pipetted and used for analysis.

To analyze the concentration of melatonin, SOD, Gpx3, and TAOC in maternal blood, we collected 12 blood samples immediately before breast milk was obtained from 12 mothers via routine blood withdrawal. Blood samples were centrifuged at 3000 g for 5 min. The plasma was pipetted and frozen at −80°C until analysis.

Measurement of Melatonin

The concentration of melatonin in plasma was measured using a commercial radioimmunoassay kit (Melatonin ELISA; IBL-International, Hamburg, Germany). According to the manufacturer, the intra- and interassay coefficients of variation were 3.9% to 6.9% at a range of 28.8 to 266 pg/mL and 6.2% to 15.9% at a range of 3.5 to 281 pg/mL, respectively. The mean recovery of melatonin was 102%, and the sensitivity of the assay was 0.9 pg/mL.

Measurement of SOD

Cu/Zn SOD concentration was measured using a commercially available enzyme-linked immunosorbent assay (ELISA) kit (Cu/ZnSOD ELISA; IBL-International, Hamburg, Germany). The intra- and interassay coefficients of variation were 5.1% and 5.8%, respectively. The Cu/Zn SOD Human ELISA kit used in our study can detect both SOD1 and SOD3 in body fluids.

Measurement of GPx3

Gpx3 concentration was assayed using a commercially available ELISA kit (ALPCO Diagnostics, Salem, New Hampshire, USA). The intra- and interassay coefficients of variation for Gpx3 were 4.21% to 9.64% and 1.12% to 5.04%, respectively.

Measurement of TAOC

The total antioxidative capacity in breast milk was measured using a commercial antioxidant assay kit according to the manufacturer’s protocol (Sigma-Aldrich, St Louis, MO, USA).

Statistical Analyses

Statistical analyses were performed using SPSS 22.0 (SPSS, Inc, an IBM Company, Chicago, Illinois, USA) and SAS version 9.2 (SAS Institute, Cary, North Carolina, USA). Data are expressed as medians (interquartile range [IQR]). To account for the repeated measurements on all participants, the analyses and estimations were based on a mixed-linear model approach taking the participants as random factors. Because the variation of the melatonin values was quite different between day and night, we applied a logarithmic transformation to the melatonin values prior to analysis. The mean values given for melatonin are therefore retransformed means of the logarithmic values (ie, geometric means). For the other variables and for melatonin after transformation, a visual check of the distributions revealed that the application of parametric methods was feasible. The estimated night-to-day differences (ratio for melatonin) are given together with 95% confidence limits. Because melatonin, SOD, Gpx3, and TAOC were not normally distributed, the relationships among these variables were estimated using the Spearman correlation coefficient.

Results

Concentrations of Melatonin, SOD, Gpx3, and TAOC in Breast Milk by Time of Day

Compared with daytime milk, the concentrations of melatonin and Gpx3 were significantly higher at nighttime. In contrast, no significant differences were observed between daytime and nighttime concentrations for SOD and TAOC (Table 1). The subgroup analysis showed that melatonin had a circadian rhythm in both preterm and term milk, with a significantly higher nighttime concentration (P < .001). Antioxidant enzymes, on the other hand, had a circadian rhythm only in preterm milk, with a significantly higher nighttime concentration of Gpx3 (preterm daytime milk vs preterm nighttime milk: 1480 ng/mL [765-1857] per day vs 1860 ng/mL [1273-2275] per night, night-to-day difference 264 [13, 515], P = .041), with a significant higher daytime concentration for SOD and TAOC (283 ng/mL [193-364] per day vs 233 ng/mL [167-291] per night, day-to-night difference 55.7 [15.7, 95.8], P = .010, and 0.44 mM Trolox [0.35-0.5] per day vs 0.42 mM Trolox [0.33-0.46] per night, day-to-night difference 0.025 [0.000, 0.05], P = .049, for SOD and TAOC, respectively).

Table 1.

Concentration of Melatonin, SOD, Gpx3, and TAOC in Breast Milk According to the Time of Day.^a

	Daytime Milk (n = 54)	Nighttime Milk (n = 51)	P Value
Melatonin, pg/mL	1.5 (1.0-2.1)	7.3 (3.8-13.6)	<.001
SOD, ng/mL	227 (158-338)	229 (168-276)	.056
Gpx3, ng/mL	1436 (765-2060)	1800 (1242-2297)	.049
TAOC, mM Trolox	0.43 (0.37-0.49)	0.42 (0.33-0.46)	.152

Abbreviations: Gpx3, glutathione peroxidase 3; SOD, superoxide dismutase; TAOC, total antioxidative capacity.

Data are expressed as median (interquartile range).

Concentration of Antioxidants in Breast Milk According to Gestational Age

The concentrations of melatonin, SOD, Gpx3, and TAOC were compared between breast milk from 14 mothers with preterm delivery (n = 70) and 7 mothers with term delivery (n = 35). Although the preterm breast milk tended to have a higher SOD concentration than the term breast milk, this difference was not statistically significant (Table 2). The concentration of melatonin, Gpx3, and TAOC did not show significant differences between preterm and term breast milk (Table 2). No significant correlations were found between gestational age and the concentration of melatonin and antioxidant enzymes (P > .05).

Table 2.

Melatonin, SOD, Gpx3, and TAOC Concentrations in Breast Milk According to Gestational Age.

	Preterm Milk (n = 70)	Term Milk (n = 35)	P Value
Melatonin,^a pg/mL	2.7 (0.1-30.5)	3.9 (0.8-36.2)	.124
SOD,^b ng/mL	245 (179-353)	202 (117-243)	.052
Gpx3,^b ng/mL	1591 (1174-2208)	1505 (799-2269)	.793
TAOC,^b mM Trolox	0.43 (0.33-0.47)	0.42 (0.37-0.49)	.584

Abbreviations: Gpx3, glutathione peroxidase 3; SOD, superoxide dismutase; TAOC, total antioxidative capacity.

Data are expressed as geometric mean (range).

Data are expressed as median (interquartile range).

Correlation of Melatonin, Gpx3, SOD, and TAOC Levels between Maternal Blood and Breast Milk

The median (IQR) concentration of SOD in breast milk was significantly higher compared with the corresponding maternal blood samples (224 ng/mL [149-297] in breast milk vs 27 ng/mL [24-43] in maternal blood, P = .005).

In contrast, the median (IQR) concentrations of melatonin and Gpx3 were significantly higher in maternal blood than in breast milk (melatonin and Gpx3 in the breast milk vs maternal blood: 2.3 pg/mL [1.5-6.2] vs 19.3 pg/mL [14.4-24.3] and 1193 ng/mL [602-2081] vs 8644 ng/mL [8239-10 362], P = .008 and P = .012, respectively). Concentration of TAOC did not differ between breast milk and maternal blood (0.47 mM Trolox [0.37-0.56] vs 0.38 mM Trolox [0.24-0.48], P = .507).

We found no significant correlation between the maternal blood and breast milk concentrations of melatonin, SOD, Gpx3, and TAOC (P > .05).

Correlations between Melatonin and SOD, Gpx3, and TAOC

We found no significant correlation between the concentration of melatonin and the concentration of SOD, Gpx3, or TAOC (P > .05).

Discussion

In human preterm breast milk, not only melatonin but also Gpx3, SOD, and TAOC showed a diurnal rhythm, with a nighttime maximum level for melatonin and Gpx3 and a daytime maximum level for SOD and TAOC. Moreover, melatonin also had a diurnal rhythm in term breast milk, exhibiting a nighttime maximum level. The concentrations of SOD, Gpx3, and TAOC were independent of the status of melatonin in breast milk.

The SOD family consists of 3 antioxidative isoenzymes (SOD1, SOD2, and SOD3) that catalyze the dismutation of superoxide into oxygen and hydrogen peroxide. SOD1 (Cu/ZnSOD) is located in the cytosol, and SOD2 (MnSOD) is found in the mitochondrial matrix.¹⁷ SOD3 (Cu/Zn SOD; EC-SOD, extracellular SOD) is the major SOD isoenzyme in extracellular fluids such as plasma, lymph, and synovial fluid.¹⁸ The Gpx family consists of 7 antioxidant isoenzymes that have the ability to reduce organic hydroperoxides to their corresponding alcohols as well as free hydrogen peroxide to water in the presence of reduced glutathione.¹⁹ Gpx3, also known as plasma or extracellular Gpx, is the extracellular isoform that is secreted into plasma, lungs, and milk.²⁰

Previously, Illernova et al⁶ and Rowe et al²¹ reported on the circadian rhythm of melatonin in rat and human milk. SOD, Gpx, and TAOC are also known to exhibit a circadian rhythm in several tissues of different species.^22-25 In human plasma, Luo et al²³ reported a circadian rhythm of SOD, with highest concentrations during the evening. A circadian rhythm of Gpx was also described in the brain of chick and the midgut of Procambarus.^24,25 The serum TAOC has shown a synchronous circadian rhythm with nocturnal peaks and daytime troughs correlating with the levels of melatonin in serum.²⁶ Nevertheless, Cubero et al²⁷ demonstrated that afternoon breast milk has a significantly higher TAOC relative to the 3 other intervals of the day. In the present study, we confirmed the diurnal fluctuation of melatonin levels not only in term but also in preterm milk. However, SOD, Gpx3, and TAOC had a diurnal rhythm only in preterm milk. It has been previously reported that the concentrations of biological components of human breast milk may change according to time of day and the milk maturation.²⁸ Therefore, it is possible that this diurnal variation of antioxidant enzymes in human preterm breast milk may represent an additional biological mechanism to improve adaptation of preterm infants to environmental changes.

On the other hand, our results contrast with those from previous studies that have shown Gpx and SOD to follow the diurnal dynamics of melatonin.^9,29 Mayo et al⁹ demonstrated that a physiological (1 nM or 0.232 ng/mL [= 232 pg/mL]) or a low pharmacological dose of melatonin (100 nM or 23.2 ng/mL) increases the messenger RNA of both superoxide dismutases and GPx in neuronal cell lines. However, it should be noted that the melatonin concentrations (1 nM = 232 pg/mL) in the study by Mayo et al were very high compared with the concentration of melatonin in the breast milk and serum of mothers reported here. It is therefore possible that melatonin may stimulate the synthesis of antioxidant enzymes in breast milk if its concentration is sufficiently high.

It remains unclear whether melatonin and antioxidative enzymes in the breast milk originate from blood or intracellular compartments. The concentration of SOD in our study was approximately 6 times higher in breast milk than in maternal serum. Maternal serum may be one of the potential sources of SOD in breast milk. The much higher levels of SOD in breast milk may be due to an accumulation of SOD between 2 intervals of expressing breast milk. Another source may be the mammary gland itself. However, no information exists on the ability of the mammary gland to express SOD3, although it is known that SOD3, unlike SOD1 and SOD2, shows tissue- and cell-specific expression.^18,30 Hence, because regional macrophages in other tissues are known for their SOD3 expression, it might be that not the mammary gland itself but regional macrophages constitute the SOD3 source for the SOD3 concentration measured in human breast milk.³¹

In contrast to SOD, we found significantly lower concentrations of Gpx3 (7 times lower) and melatonin (5 times lower) in breast milk compared with those measured in maternal serum. Furthermore, we found no correlation between maternal blood and the milk concentrations of these components. It is known that not only the pineal gland but also many other tissues possess the necessary enzymes required for melatonin synthesis. Moreover, the concentration of melatonin in many tissues is found to be higher than in plasma, independent of the time of day.³² Based on the assumption that Gpx3 and melatonin in breast milk may originate from blood, it could be expected that Gpx3 and melatonin would show higher levels in breast milk compared with the corresponding serum such as SOD. Although it cannot be ruled out that the Gpx3 found in breast milk partially originates from maternal blood, it seems more likely that the mammary gland itself represents the main source for the Gpx3 found in breast milk.³³ Thus, the main source of melatonin and antioxidative enzymes in the human breast milk remains uncertain, and further studies are necessary to determine the synthesis and transport pathways of these enzymes and bioactive factors in human breast milk.

The importance of antioxidant enzymes in breast milk has been well established. Friel et al,³⁴ for instance, added SOD, catalase, and Gpx to formula and were able to document a significant increase in its antioxidant capacity. As in other tissues, melatonin exhibits endocrine, paracrine, autocrine, and luminal actions in the gastrointestinal tract.^35,36 Exogenous administration of melatonin or its precursor, L-tryptophan, increases the melatonin content in the gastrointestinal tract.^35,37 Guven et al¹³ demonstrated melatonin’s ability to ameliorate NEC-like injuries in an NEC model of newborn rats receiving intraperitoneal injections. Ustundag et al¹² demonstrated melatonin’s efficacy in the protection of injury reperfusion–induced intestinal injury. The intraperitoneal administration of melatonin led to a detectable increase in Gpx and SOD concentration in the gastrointestinal system tissue that may have helped to protect the intestine. It is also possible that melatonin and antioxidant enzymes, ingested by the preterm infants via breast milk, can contribute to protecting the gastrointestinal mucosa against oxidative and inflammatory damage, such as NEC.

Conclusion

Our results add to the knowledge base on the antioxidative capacity/status of human breast milk. Because of the higher melatonin and Gpx3 content in nighttime breast milk, a prospective clinical study on the outcomes of preterm infants is needed to determine whether preterm nighttime milk ought to be the first choice in the feeding of these high-risk infants.

Footnotes

Acknowledgements

We thank Gabriele Strackbein and the nursing staff of the Department of Neonatology for their contributions.

Declaration of Conflicting Interests

The authors declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

Funding

The authors disclosed receipt of the following financial support for the research, authorship, and/or publication of this article: Research Fund of the Department of Neonatology, Children’s Hospital, University of Bonn.

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