Melatonin Stability in Human Milk

Abstract

Introduction:

Melatonin is an antioxidant, a circadian pacemaker, and an immune system stimulator. Studies have demonstrated beneficial effects of melatonin on various conditions in neonates. Melatonin is secreted in breast milk in circadian rhythm, but its half-life and stability in this medium and in real-life conditions of freezing and defrosting is unknown. The objective of this feasibility study was to evaluate stability of melatonin in breast milk after freezing and defrosting.

Methods and Results:

Breast milk samples of nocturnal milk and daytime milk were collected from 13 healthy breastfeeding mothers and were immediately frozen. Samples were defrosted in room temperature and were sampled for melatonin immediately and every hour for 4 hours and at 24 hours after defrosting. Melatonin levels were measured with Melatonin direct Saliva ELISA kit (IBL International).There was no statistically significant difference between levels at the different time points (p = 0.696). Melatonin levels in daytime milk were significantly lower than night-time levels (p = 0.028).

Conclusion:

Melatonin is stable in human milk for at least 4 hours after defrosting and even up to 24 hours. Further research of the therapeutic potential of night breast milk high in melatonin is needed.

Introduction

Melatonin (5-methoxy-N-acetyltryptamine) is an endogenously produced indoleamine, synthesized from the amino acid precursor tryptophan, primarily in the pineal gland. It is a highly effective antioxidant and a primary circadian pacemaker. Moreover, melatonin has an important stimulatory action in the immune system, protecting organisms against bacterial and viral infections.¹ Melatonin is secreted during the night in adults; however, in infants the circadian rhythm of melatonin biosynthesis develops only between the 6th and 8th weeks of life.² Moreover, according to Kennaway et al., in preterm infants born under certain circumstances such as premature rupture of membranes, intrauterine growth retardation, and fetal distress, melatonin biosynthesis develops even later.³

Several studies showed beneficial effects of melatonin (given by injections for a short period of time) on various conditions in neonates and young infants, such as improved outcome of neonates with respiratory distress syndrome, reduced inflammatory response in neonatal sepsis, and an analgesic effect in infants.^4–8

Interestingly, melatonin is secreted in breast milk in circadian rhythm. Engler et al. showed that melatonin levels were highest at 3:00 AM and that breastfed infants had a tendency toward longer nocturnal sleep and reduced irritability as compared with formula-fed infants.⁹ Katzer et al. observed the circadian rhythm of melatonin secretion in breast milk as well, and found no difference between the concentration of melatonin in breast milk between mothers of term and preterm infants.¹⁰

We believe that breast milk expressed during the night thus containing a high melatonin level may improve outcome of preterm infants, assuming that high levels are sustained until the milk is to be given.

Half-life of melatonin in serum varies between <30 and 60 minutes. It is metabolized primarily in the liver and is further excreted in urine.¹¹ To our knowledge, currently there is no sufficient data regarding the half-life of melatonin in breast milk. Furthermore, its stability in this medium and in real-life conditions of freezing and defrosting breast milk before it is given to the preterm infant is unknown.

The objective of this feasibility study was to examine the levels of melatonin in breast milk expressed during the night and evaluate its stability.

Materials and Methods

Participants

Breast milk samples were collected from 13 healthy breastfeeding mothers whose infants were delivered in Carmel Medical Center. The ethics committee of Carmel Medical Center approved this study in accordance with the declaration of Helsinki. Written informed consent was obtained from all participants.

Collection of samples

All women were asked to express milk at 3 AM, when melatonin levels are the highest. They were instructed to express in the dark upon arousal from sleep. A sample of 2 mL was collected in a sterile container and frozen immediately. As control, samples expressed at 10–12 AM, when melatonin levels are the lowest, were collected from the same women. The milk samples, protected from light, were brought by the participants to the Neonatal Intensive Care Unit (NICU) in a cooler and were placed in the freezer at −20°C. Afterward, the samples were delivered protected from light to the laboratory where they were placed in a freezer at −20°C until assayed. Sample collection took place anytime during the first 6 months of breastfeeding, at mother's convenience. The samples were stored for about 4 months and then assayed.

Measurement of melatonin

Both night and morning milk samples were kept in −20°C until assayed. Melatonin was measured just after defrosting and not before freezing, because we wanted to mimic real-life conditions (as mentioned in the discussion section). Samples were defrosted in room temperature and mixed thoroughly. The samples were centrifuged in 16.2G for 10 minutes, and the middle phase was separated. Night milks' middle phase was sampled immediately and every hour for the duration of 4 hours and also at 24 hours after defrosting, and diluted 1:10 with the standard A from the ELISA kit, as described in Asher et al.¹² Morning milk middle phase was diluted with standard A immediately after defrosting. Melatonin levels were measured with commercial Melatonin direct Saliva ELISA kit (IBL International, Hamburg, Germany) according to manufacturer instructions. Optical densities were read with Infinite F50 absorbance microplate reader (Tecan, Switzerland). Melatonin concentrations were analyzed and calculated according to a standard curve using Logit–Log fit with Magellan software for Infinity F50.

Statistical analysis

Statistical analysis was performed using IBM statistics (SPSS) vs24. The continuous variables were presented by mean, standard deviation, and median with interquartile range. Differences in melatonin levels (after defrosting) over time were analyzed using Friedman test. Difference between day and night melatonin levels were analyzed using the Wilcoxon sign rank test. p value <0.05 (two tailed) was considered statistically significant.

The calculation of the sample size was based on the assumption that the standard deviation of melatonin degradation time is 2.5 hours and the standard error is 1.5. Based on confidence interval of 95% and power of 80%, the sample size needed is 11.

Results

Thirteen samples of night milk were collected. Three of them were used for calibration of the analysis system. Out of the 10 samples used for the analysis, four were of mothers of term infants and six were of mothers of preterm infants. Mean gestational age was 33 + 4 weeks. Seven samples of daytime milk were analyzed as control.

Melatonin levels were measured immediately after defrosting and then at 1, 2, 3, 4, and 24 hours after defrosting. There was no statistically significant difference between levels at the different time points (mean 45.6052 pg/mL, p = 0.696). Interestingly, an increase of the range of melatonin level across the samples was observed at 24 hours after defrosting (Fig. 1).

FIG. 1.

Melatonin levels (pg/mL) in nocturnal milk samples immediately after defrosting (time 0) and then at 1, 2, 3, 4, and 24 hours later (mean 45.6052 pg/mL, p = 0.696).

Melatonin levels in daytime milk were significantly lower than night-time levels (mean 34.3179 pg/mL, p = 0.028, Fig. 2).

FIG. 2.

A comparison between melatonin level (pg/mL) in daytime milk and nocturnal milk (mean 34.3179 pg/mL, p = 0.028).

Discussion

In the NICU, expressed breast milk is routinely used for extremely preterm or sick infants who cannot orally breastfeed. Usually, mothers express milk at the hospital or at home at their convenience, during both night and day. Preferably, fresh expressed breast milk is given immediately to the infant. However, due to practical limitations most breast milk is being frozen after expressing to be given later. The frozen milk is thawed and divided into portions that are then given for the next 24 hours. This feasibility study was designed to measure melatonin levels according to the realistic time frames of breast milk use in NICU.

In this preliminary study, we found that melatonin is stable in human milk for at least 4 hours after defrosting and even up to 24 hours. This important observation of the slow degradation rate of melatonin in human milk may allow for further research of the therapeutic potential of night breast milk high in melatonin.

At 2014, Karunanithi et al. published the validity of a new method of measuring melatonin in breast milk. They reported stable melatonin levels in human milk up to 60 hours; however, in this study melatonin was added to the milk and stored it in 10°C for up to 60 hours.¹³ Nonetheless, these findings are in keeping with our data about melatonin stability in human milk.

Our study has several limitations. As this study was designed as a feasibility study, the sample size was relatively small. In addition, we observed some variance in melatonin levels at 24 hours, as some of the milk samples had higher melatonin levels at 24 hours in comparison with the previous time points, and some had lower melatonin levels. This unexpected finding of a higher melatonin level at 24 hours may be due to some evaporation of breast milk water content while the samples were stored in the refrigerator. In addition, variance in melatonin measurements due to potentially interfering molecules was recently reported in melatonin assays with no extraction steps; however, this was reported in amniotic fluid and not in breast milk.¹⁴

The observation that nocturnal breast milk still contains high levels of melatonin after mimicking realistic conditions of storage and timeline opens the door for further studies designed to investigate the impact of melatonin in breast milk on the outcome of extremely preterm infants, aiming at improving the quality of care to this vulnerable population.

Conclusions

In this feasibility study, we showed that melatonin is stable in expressed frozen human milk up to 24 hours after defrosting. This data allow for further research on the role of night-time human milk, which contains a high melatonin level, in improving the outcome of preterm infants.

Footnotes

Acknowledgment

This study was supported by a grant from Materna Research Institute.

Disclosure Statement

No competing financial interests exist.

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