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Breast-milk contains a potent mixture of diverse components, such as the non-protein nitrogen fraction which includes nucleotides, whose variation in levels is evident throughout lactation. In addition, these substances play an important role in sleep homeostasis. In the present study, human milk samples were analyzed using a capillary electrophoresis system. The rhythmicity of each nucleotide was studied by cosinor analysis. It was found that the nucleotides 5'AMP, 5'GMP, 5'CMP, and 5'IMP have significant (P < 0.05) circadian rhythms, the acrophases of the first two being during the night, and of the latter two during the day. While 5'UMP did not show a clear circadian rhythm, there was an increase in its levels at night. In conclusion, the rise in nocturnal levels of 5'AMP, 5'GMP, and 5'UMP could be involved in inducing the 'hypnotic' action of breast-milk at night in the infant.
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2Nutritional Neuroscience 2009 Vol 12 No 1
The possible role of human milk nucleotides
as sleep inducers
Cristina L. Sánchez1, Javier Cubero1, Javier Sánchez2, Belén Chanclón1,
Montserrat Rivero3, Ana B. Rodríguez1, Carmen Barriga1
1Department of Physiology, Faculty of Science, University of Extremadura. Badajoz, Spain
2Laboratory of Metabolism, Hospital ‘Perpetuo Socorro’, Badajoz, Spain
3Ordesa Group, St Boi de Llobregat, Barcelona, Spain
Breast-milk contains a potent mixture of diverse components, such as the non-protein nitrogen
fraction which includes nucleotides, whose variation in levels is evident throughout lactation. In
addition, these substances play an important role in sleep homeostasis. In the present study,
human milk samples were analyzed using a capillary electrophoresis system. The rhythmicity of
each nucleotide was studied by cosinor analysis. It was found that the nucleotides 5AMP, 5GMP,
5CMP, and 5IMP have significant (P< 0.05) circadian rhythms, the acrophases of the first two
being during the night, and of the latter two during the day. While 5UMP did not show a clear
circadian rhythm, there was an increase in its levels at night. In conclusion, the rise in nocturnal
levels of 5AMP, 5GMP, and 5UMP could be involved in inducing the ‘hypnotic’ action of breast-
milk at night in the infant.
Keywords: nucleotides, circadian, sleep, human milk, capillary electrophoresis
Introduction
A joint declaration by the World Health Organization
(WHO) and the United Nations Childrens Fund
(UNICEF) stated that breast-milk is the optimal food
for infants and can never be equalled by artificial
substitutes. It covers all the child’s physiological and
nutritional needs during the first 4–6 months of life.1
For this reason, there is growing interest in attempting
to make infant formulas that more closely resemble
mother’s milk. Infant formulas are the only processed
food products that fully meet the nutritional needs of
infants during the first months of life until the
introduction of adequate supplementary feeding.2
The milk of every mammalian species has a
different composition, tailored to the digestive,
nutritional, and growth needs of its offspring. Human
milk is a living fluid that changes with time, with its
composition and volume being modified both during
the course of each day and throughout the breast-
feeding period. Its non-protein nitrogen fraction
includes nucleotides whose concentrations are known
to vary throughout lactation. In particular, there is an
increase in nucleotide concentration in the mature
milk (from day 15 postpartum) relative to the
colostrum (4–5 days’ postpartum).3
Nucleotides are the building blocks of nucleic acids
responsible for storing and transmitting genetic inform-
ation. They are precursors of energy-rich compounds that
control the metabolic processes (biosynthesis, funda-
mentally) in all cells. Their skeleton consists of a pentose
(carbohydrate), a nitrogen-containing base, and a
phosphate group. The commonest are nucleotides where
the nitrogen-containing base is a purine – adenosine
Research article
Correspondence to: Cristina L. Sánchez López, Department of
Physiology, Faculty of Science, University of Extremadura, Av. Elvas s/n,
06071 Badajoz, Spain. Tel: +34 (0)924 289388; Fax: +34 (0)924 289388;
E-mail: crissanchez@unex.es
Received 25 March 2008, revised manuscript accepted 19 August 2008
© W. S. Maney & Son Ltd 2009
DOI 10.1179/147683009X388922
Nutritional Neuroscience 2009 Vol 12 No 1 3
Sánchez et al. Human milk nucleotides and sleep
5monophosphate (5AMP), guanosine 5mono-
phosphate (5GMP), and their precursor, inosine
5monophosphate (5IMP) – or a pyrimidine – uridine
5monophosphate (5UMP), cytidine 5monophosphate
(5CMP), and thymidine 5monophosphate (5TMP).
The nucleotides act in cells as secondary messengers
through cAMP (cyclic 5AMP) and cGMP (cyclic
5GMP), and also supply the necessary chemical
energy. They can also act as components of many
enzyme co-factors such as flavin adenine dinucleotide
(FAD) and nicotinamide adenine dinucleotide (NAD),
in addition to having a strong influence on sleep – the
function which is the objective of the present study.
In reviewing the literature, we found that three
nucleotides are considered to be involved in the
physiological function of sleep – 5UMP, 5AMP, and
5GMP.
The first, 5UMP, is distributed throughout the
body (including the brain), and has a depressive effect
on the CNS. The nightly administration of low doses
of this nucleotide produces a moderate increase in the
number of REM and non-REM sleep episodes,4but
has little or no influence on their duration.5,6 The
plasma concentration of uridine in mice has a marked
circadian rhythm,6with the time of the maximum
concentration (acrophase) coinciding with the time of
least activity.
The second, 5AMP, is the nucleotide which is most
referred to in the literature as a sleep inducer. Indeed,
its hypnotic properties have been recognized now for
over 30 years.7More recent evidence confirming its
role in sleep induction is based on several facts:
extracellular concentrations (through the secondary
messenger cAMP) present circadian variations, its
administration induces an hypnotic effect, and its
levels decline during the period of wakefulness.8–11
The third, 5GMP, is also a second messenger in its
cyclic form (cGMP), which mediates most of the
neuronal effects of nitric oxide (NO). Many studies
have pointed to the role of NO in sedation. For
instance, the injection of a cGMP inhibitor into rats
was found to increase wakefulness at the same time as
suppressing REM and non-REM sleep.12 Human
studies have shown that cGMP plasma concentrations
rise when the subject goes to bed and remain high
throughout the night, reflecting its role in stimulating
the secretion of the pineal hormone melatonin.13,14
Recently there has been growing interest in
studying nucleotides in the diet, since they seem to
play an important role in human nutrition at different
stages of life. This is especially so in infancy, as they
influence neonatal development by the synthesis of
phospholipids, by modifying the microflora and
repairing any damage in the gut, and also by
participating in the T-lymphocyte mediated immune
response.15–18 It has been suggested that both the
nucleotides and the nucleosides found in human milk
may be important for tissue development in infants.19
The Co-ordinated International Expert Group of
the European Society for Paediatric Gastroenterology,
Hepatology, and Nutrition (ESPGHAN) recommends
the following maximum concentrations for nucleotides
added to infant formulas: 1.75 mg/100 kcal of 5CMP,
1.5 mg/100 kcal of 5UMP, 1.5 mg/100 kcal of 5AMP,
0.5 mg/100 kcal of 5GMP, and 1 mg/100 kcal of
5IMP. Also, the total of all nucleotides must not
exceed 5 mg/100 kcal.20
Although the most extensively validated method
for nucleotide assay in human milk is high
performance liquid chromatography (HPLC), our
research group has demonstrated that capillary
electrophoresis (CE) is another perfectly viable
technique.21Nearly all nucleotide determinations have
studied the variations in their concentrations over the
months of lactation. The novelty of the present work
is the study of the possible circadian rhythms in the
nucleotide content of breast-milk by determining the
changes that occur during each 24-h period. This is
essentially the reason for using CE as against HPLC,
since measurements with CE are significantly faster
(approximately 30 min compared with 2 h in HPLC).
Also, the efficiency of the method is much greater
(more than 200,000 plates theoretically, compared
with 5000 for HPLC), and the expenditure in terms of
the volumes of reagents and samples is much lower.
Subjects and methods
Subjects
The study population consisted of 30 healthy mothers
from the region of Extremadura (Spain) who had been
breast-feeding for 3 months. Their median age was 33
years (minimum-to-maximum range, 26-39 years), and
the mean ± SD values for weight, height, and body
mass index (BMI) were 62.3 ± 7.3 kg, 164 ± 6 cm, and
23.1 ± 2.4 kg/m2, respectively. The subjects were
considered healthy on the basis of their breast-feeding
success, a physical examination, and a follow-up. All
subjects were informed about the investigation, and
gave their written consent.
During the study, the subjects took no drugs that
would disturb the levels of nucleotides. The Ethical
Investigation Committee of University of Extremadura
approved the study.
4Nutritional Neuroscience 2009 Vol 12 No 1
Sánchez et al. Human milk nucleotides and sleep
Samples
Samples of breast-milk were collected in polystyrene
tubes before each feed over a 24-h period, during
March to July, and stored frozen at –30ºC until assay
in duplicate. In general, between 6 and 8 samples of
breast-milk were obtained from each mother.
Equipment and components
The CE system used was a P/ACE MDQ System 5510
equipped with a diode array detector (Beckman
Coulter, Inc., USA). The system can be rapidly
reconfigured from a flexible research platform to a
tightly regulated routine-use platform. Automated
fractionation of a detected peak allows isolation of
newly resolved compounds for external identification.
Capillary cartridges
The capillaries are housed in user-assembled cartridges
which are compatible with all current CE capillaries. For
the present study of nucleotides, the CE separations
were carried out in an uncoated silica capillary (75 µm
i.d. × 375 µm o.d.; Polymicro Technologies®, LLC,
USA) with an effective length of 20 cm.
Detector modules
To allow for flexible method development and rugged
routine use, the design of the P/ACE MDQ makes it
easy to interchange high-sensitivity diode array
(DAD), UV/Vis, and laser-induced fluorescence (LIF)
detection modules. An external detector adapter
allows the capillary to be extended to additional
detection systems.
Software for the CE analysis
The 32Karat™ software package specific to capillary
electrophoresis includes mobility plot generation,
advanced reports, and new 2-D algorithms to couple
mobility and spectral signatures for peak identification.
All of this results in a fully integrated CE control and
data analysis workstation.
The methods are defined and edited in table format.
All functions for the system are handled in a single
window, including programming of the buffer array
for the automation of strategies for the development
of methods, using filters such as scan range,
wavelength maximum, and mobility.
Control and analysis
Peak identification using either time or mobility,
coupled with spectral signature confirmation, creates
powerful 2-D peak identification schemes.
Velocity-calibrated peak area and CAESAR©
integration ensure reproducible quantification at low
limits of detection.
Reagents
Adenosine 5monophosphate, uridine 5monophos-
phate, guanosine 5monophosphate, thymidine 5mono-
phosphate, cytidine 5monophosphate, inosine
5monophosphate, boric acid, and sodium dodecyl-
sulphate (SDS) were purchased from Sigma-Aldrich
(USA). All other chemicals were of analytical purity
grade. Perchloric acid 60%, sodium hydroxide and
potassium hydroxide 85% pellets were purchased from
Panreac, Spain. All solutions were prepared using de-
ionized water (Milli-Q System).
Procedures
Preparation of stock solutions
The values reported in the literature indicated that the
nucleotide concentrations in human milk would be in
the range 0–9 µg/ml. Stock nucleotide solutions were,
therefore, prepared in the following concentrations:
10, 5, 1, and 0.5 µg/ml of 5AMP, 5CMP, 5GMP,
5TMP, 5UMP, and 5IMP.
Extraction of nucleotides from breast-milk
We followed the technique of Perrin et al.22 with
certain modifications. We started from milk samples
of healthy women of at least 12 weeks’ lactation.
Aliquots of 0.75 ml of each sample were hydrolysed
with 0.75 ml of 13% perchloric acid, mixing for 45 min
on a roller mixer. After centrifuging at 5000 gfor 20
min at room temperature, the supernatant was
collected, discarding the fatty halo.
The solution was then adjusted to neutral pH with
5 M KOH, and left in an ice bath for 1 h for all the
potassium perchlorate to precipitate. It was then
filtered through a 0.45 µm membrane filter (Millex;
Millipore, USA) before assay.
CE analysis
All experiments were performed on a P/ACE System
5510 (Beckman Coulter). The CE separations were
carried out in an uncoated silica capillary (75 µm i.d.
× 375 µm o.d.; Polymicro Technologies) with an
effective length of 20 cm. Detection was by UV light
over the range 190–300 nm (cartridge detection
window 100 × 800 µm) and the limit of detection
(LOD) was 60 ng/ml.
Samples were loaded by low-pressure injection (3.45
kPa) for 6 s (14.3 nl, 2.7% of the total capillary volume
injected). Borate buffers were prepared from boric acid,
then SDS was added, and the solution was adjusted with
500 g/l NaOH to the appropriate pH. The capillary was
washed at the beginning of each working day with de-
ionized water, 0.1 M sodium hydroxide, water, and
finally with a separation buffer for 5 min.
Nutritional Neuroscience 2009 Vol 12 No 1 5
Sánchez et al. Human milk nucleotides and sleep
Between runs, it was rinsed with water for 1 min
and with a separation buffer for 2 min. The assays
were run at constant voltage using a ramp of 1 min.
The alkaline (borate) separation system as described
by Adam et al.23 was used as follows. The capillary was
operated at 30ºC. The separation buffer was prepared
from boric acid (60 mmol/l), SDS (80 mmol/l), and
adjusted with 2-amino-2-methyl-1-propanol to neutral
pH. Assays were run at +10 kV (positive outlet). The
detector’s data rate was set at 4 Hz.
Chronobiological analysis
The chronobiological analysis of the data was
performed using Ritme®for Windows software
package. The rhythmicity of each nucleotide was
studied by cosinor analysis.24The sinusoidal function
used for the fit is the following:
y(t) = M + A × cos [(2 × π/τ) × t – Φ] Eq. 1
where y(t) is the value of the cosine function at time t,
M is the mean level of oscillation or the MESOR
(acronym of midline-estimating statistic of rhythm,
the mean value about which the oscillation occurs,
equal to the arithmetic mean of equidistant data
covering a whole number of cycles), A is the amplitude
(measure of the extent of a rhythmic change in a cycle as
estimated by the sinusoidal function that best fits the
data), the frequency (ω=2×π/τ) where πis the number
pi and τis the period (24 h in our case), and Φis the
acrophase (a phase angle measuring the timing of the
peak activity, expressed as the lag from a reference
time to the crest time of the best fit sinusoidal
function). Therefore, cosinor analysis determines the
best-fitting sinusoidal wave by estimating three
parameters – mesor, amplitude, and acrophase.
Sample distribution
Given that the times at which milk samples were
extracted did not exactly coincide from one mother to
another, we selected those hours of the 24-h period for
which there were the greatest numbers of samples
under the constraint of requiring reasonably uniform
distribution of those hours.
By cosinor analysis, we determined the confidence
limits of the MESOR, amplitude, and acrophase at
95% probability level. When the range determined by
the confidence limits of the amplitude contains the
value 0, it cannot be excluded that the amplitude is 0
and, therefore, the existence of a rhythm is not
statistically significant. In other words, to test the
statistical significance of the rhythm, we determined
whether the null hypothesis of zero amplitude is or is
not rejected at 0.05 of alpha level. The P-value
indicates the significance of the fit of the cosine curve
to the data.
The confidence limits of the acrophase allow one to
determine whether there were significant differences
between the acrophases of different variables. When
the range determined by the confidence limits of the
acrophase of one variable overlaps that of another, the
possibility that both acrophases are equal cannot be
discarded.25
Results
Figure 1 shows the levels of 5AMP in human milk
over a 24-h period. The levels increase as night falls
(after 20:00), and the levels are higher at the first hours
of the night relative to the interval before dawn.
Figure 2 shows the equivalent results for 5UMP. In
this case, there was an increase in the middle of the
night with respect to the previous hours and with
respect to the light hours.
Figures 3–6 present the results for the other four
nucleotides (5GMP, 5CMP, 5IMP, and 5TMP) in
which variations between the different time hours
showed no difference. In Figure 3, however, there was
an apparent increasing trend of the levels of 5GMP
for the nocturnal period (20:00–08:00). A similar
Figure 1 Sinusoidal function obtained by cosinor analysis
of the nucleotide 5AMP for a 24-h period (n= 30)
Figure 2 Sinusoidal function obtained by cosinor analysis
of the nucleotide 5UMP for a 24-h period (n= 30)
6Nutritional Neuroscience 2009 Vol 12 No 1
Sánchez et al. Human milk nucleotides and sleep
trend, but during daylight hours (08:00–20:00), is
observed for 5CMP and 5IMP (Figs 4 and 5,
respectively). This contrasts with the apparent down -
ward trend in the daylight intervals for 5AMP (Fig. 1).
The results of the chronobiological study (Table 1)
of particular interest were the significant circadian
rhythms of 5AMP (Fig. 1) and 5GMP (Fig. 3) with
acrophases during the period of darkness (at 20:19
and 05:08, respectively). The other two nucleotides
having significant circadian rhythms were 5CMP
(Fig. 4) and 5IMP (Fig. 5) but with acrophases during
the daytime period (at 18:40 and 19:14, respectively).
Discussion
Breast-milk is not static in its composition, but
changes with time,26,27 in parallel with the infant’s
energy demands and tissue growth. For the newborn,
there is an accentuated protein demand because of the
anabolic requirement involved in the first weeks of
growth.
Nonetheless, there has until now been no
consideration of the possibility that, through her milk,
the mother is preparing her baby’s adaptation to the
changing environmentday and night, for example. It is
now known that high levels of melatonin in breast-milk
appear during the night and low levels during the day.28
Since melatonin is the hormone that regulates the
sleep/wake cycle, these changes in breast-milk will
doubtless be the signal to help the baby adapt as quickly
Figure 3 Sinusoidal function obtained by cosinor analysis
of the nucleotide 5GMP for a 24-h period (n= 30)
Figure 4 Sinusoidal function obtained by cosinor analysis
of the nucleotide 5CMP for a 24-h period (n= 30)
Figure 5 Sinusoidal function obtained by cosinor analysis
of the nucleotide 5IMP for a 24-h period (n= 30)
Figure 6 Sinusoidal function obtained by cosinor analysis
of the nucleotide 5TMP for a 24-h period (n= 30)
Table 1 Chronobiological parameters of each nucleotide for a 24-h period
Nucleotide MESOR (µg/ml) Amplitude (µg/ml) Acrophase (h:min) Cosinor significance P-value
5AMP 5.17 (4.61–5.73) 1.03 (0.02–2.03) 20:19 (15:08–25:30) 0.04457*
5UMP 6.14 (4.91–7.37) 1.20 (–) 02:00 (–) 0.36425
5GMP 3.63 (3.42–3.85) 0.46 (0.07–0.84) 05:08 (01:18–08:58) 0.01955*
5CMP 2.44 (2.25–2.64) 0.42 (0.16–0.68) 18:40 (14:59–22:20) 0.01645*
5IMP 3.06 (2.91–3.21) 0.44 (0.18–0.70) 19:14 (16:48–21:41) 0.00149*
5TMP 4.27 (3.94–4.60) 0.36 (–) 04:45 (–) 0.28860
MESOR values and amplitudes are in the corresponding parameter units. Acrophases are given as times of day (08:00–20:00
light/dark cycle). Confidence limits are in parentheses. The P-value indicates significance of the fit of the cosine curve to the data.
*P< 0.05 was considered statistically significant (n= 30).
Nutritional Neuroscience 2009 Vol 12 No 1 7
Sánchez et al. Human milk nucleotides and sleep
as possible to the day/night versus sleep/wakefulness
environment.29–31
The present study continues this line of inquiry into
the change and temporal evolution of the macro- and
micro-nutrients in breast-milk. Our purpose was to
study some of the possible variations, but on a much
shorter time scale, in particular the 24-h period
variation of the nucleotides belonging to the non-
protein nitrogen fraction. As was first described some
30 years ago and has been confirmed in recent years,
these nucleotides have a great genetic importance32via
their action on the flora in the gut,15 and neuro -
chemically via their intracellular action as secondary
messengers, particularly the physiological action of
the purine nucleotides on sleep.8Also, in the last few
years, their hypnotic action in infants has been
demonstrated by the results of applied research with
starter milks for infants with sleep problems.21,30,31
The higher nocturnal levels of the purine
nucleotide 5AMP were consistent with its nature as a
sleep inducer as found in earlier studies.33–35 In
addition, as a novel result compared to those reported
by other workers,36 we demonstrated the existence of a
circadian rhythm for this nucleotide. The increase was
confined to the beginning of the night (with acrophase
at 20:19, and a MESOR of 5.17 µg/ml), and could
mean that the cAMP which is used in the release of
GABA, an inhibitory and ‘sleep-promoting’ neuro -
transmitter,37 originates from this nucleotide in the
milk. It is notable that the increase of this nucleotide
coincides with the onset of darkness at 20:00, and that
the raised levels are maintained over a long time to
conserve the cAMP-mediated intracellular response,
especially in brain tissue in order to maintain
homeostasis during sleep.34
The other purine nucleotide, 5GMP, showed a
tendency to increase during the night, unlike the
periods of daylight during which its levels were more
irregular. This nucleotide is a precursor of another
intracellular messenger (cGMP) which, during the
night, is involved in the secretion of the hormone
melatonin, thereby inducing and entraining nocturnal
rest.13,38,39 Our chronobiological study showed this
nucleotide to have a clear circadian rhythm, with the
acrophase in the final hours of darkness, at 05:08 (an
acrophase that is very similar to that reported by Skala
et al.36) and a MESOR of 3.63 µg/ml.
With respect to 5UMP, this nucleotide did not
describe a clear circadian rhythm, but its concent -
rations gradually decreased during the hours of
daylight, followed by a clear increasing trend during
the period of darkness, indicating a possible ultradian
rhythm, which is understood as being part of the
stimulation and functioning of the hypnotic
mechanism.4–6,40
Of the other nucleotides, 5CMP had a significant
circadian rhythm with acrophase at 18:40 (during
daylight hours), and a MESOR of 2.44 µg/ml.
Because 5IMP is the precursor of the other two
purine nucleotides, it was not surprising that it showed
a significant circadian rhythm that was in synchrony
with the other two purine nucleotides, 5AMP and
5GMP. Indeed, its acrophase was at 19:14 (just before
the onset of darkness when the sleep inducers, 5AMP
and 5GMP, reach their acrophases) and its MESOR
was 3.06 µg/ml.
Conclusions
The assay of nucleotides in the breast-milk of the
study population showed that their levels were not
constant over a 24-h period. This was particularly so
for 5AMP, 5UMP, and 5GMP, which showed
increased concentrations at night and may, therefore,
be involved in inducing hypnotic action in the infant.
Acknowledgements
Laboratorios Ordesa S.L. financed this work through
project 167/06. Thanks are also due to the University
of Extremadura for the research grant ‘II Plan de
Iniciación a la Investigación, Desarrollo Tecnológico e
Innovación awarded to Cristina L. Sánchez López,
and to Elena Circujano for her technical assistance.
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... In mature milk, clear rhythms were detected also in methionine, aspartic acid, histidine, phenylalanine, and tyrosine, with an acrophase at different times of the day (7). In mature milk, there are also rhythms of nucleotides, adenosine 5'monophosphate (5'AMP), guanosine 5'monophosphate (5'GMP), uridine 5'monophosphate (5'UMP),cytidine5'monophosphate (5'CMP) and inosine 5'mono'hosphate (5'IMP), the first three with higher levels during the night and the latter two during the day (44). In mature milk, 2-AG showed significantly higher concentration during the day than during the night, which mirrored plasma levels in the mother [(33); Figure 2A]. ...
... In agreement, there is an increase of 5'GMP in adult human plasma during sleep in the night in comparison to the period of wakefulness (59). In human milk, the concentration of 5'AMP, 5'GMP, and 5'UMP is higher during the night than during the day (44). The effect of the differential concentration of tryptophan and the nucleotides was tested in children. ...
Article
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During pregnancy the human fetus receives timed cues from the circadian rhythms of temperature, metabolites, and hormones from the mother. This influence is interrupted after parturition, the infant does not secrete melatonin and their circadian rhythms are still immature. However, evolution provided the solution to this problem. The newborn can continue receiving the mother's timed cues through breastmilk. Colostrum, transitional, and mature human milk are extraordinary complex biofluids that besides nutrients, contain an array of other non-nutritive components. Upon birth the first milk, colostrum, is rich in bioactive, immunological factors, and in complex oligosaccharides which help the proper establishment of the microbiome in the gut, which is crucial for the infants' health. Hormones, such as glucocorticoids and melatonin, transfer from the mother's plasma to milk, and then the infant is exposed to circadian cues from their mother. Also, milk components of fat, proteins, amino acids, and endogenous cannabinoids, among others, have a markedly different concentration between day and night. In the present review, we give an overview of nutritive and non-nutritive components and their daily rhythms in human milk and explore their physiological importance for the infant. Finally, we highlight some interventions with a circadian approach that emphasize the importance of circadian rhythms in the newborn for their survival, proper growth, and development. It is estimated that ~600,000 deaths/year are due to suboptimal breastfeeding. It is advisable to increase the rate of exclusive breastfeeding, during the day and night, as was established by the evolution of our species.
... Numerous substances in breast milk fluctuate on a circadian schedule (207), and several of these components may be involved in promoting sleep onset. Specifically, concentrations of melatonin (99,233,374), tryptophan (109), and the nucleotides adenosine 5 ′ monophosphate (5 ′ AMP) and guanosine 5 ′ monophosphate (5 ′ GMP) (411) all peak at night in human breast milk and influence sleep onset. The rhythmicity of these breast milk components is likely important in the development of the infant circadian system (20). ...
Chapter
Circadian rhythms are endogenously generated, daily patterns of behavior and physiology that are essential for optimal health and disease prevention. Disruptions to circadian timing are associated with a host of maladies, including metabolic disease and obesity, diabetes, heart disease, cancer, and mental health disturbances. The circadian timing system is hierarchically organized, with a master circadian clock located in the suprachiasmatic nucleus (SCN) of the anterior hypothalamus and subordinate clocks throughout the CNS and periphery. The SCN receives light information via a direct retinal pathway, synchronizing the master clock to environmental time. At the cellular level, circadian rhythms are ubiquitous, with rhythms generated by interlocking, autoregulatory transcription-translation feedback loops. At the level of the SCN, tight cellular coupling maintains rhythms even in the absence of environmental input. The SCN, in turn, communicates timing information via the autonomic nervous system and hormonal signaling. This signaling couples individual cellular oscillators at the tissue level in extra-SCN brain loci and the periphery and synchronizes subordinate clocks to external time. In the modern world, circadian disruption is widespread due to limited exposure to sunlight during the day, exposure to artificial light at night, and widespread use of light-emitting electronic devices, likely contributing to an increase in the prevalence, and the progression, of a host of disease states. The present overview focuses on the circadian control of endocrine secretions, the significance of rhythms within key endocrine axes for typical, homeostatic functioning, and implications for health and disease when dysregulated. © 2022 American Physiological Society. Compr Physiol 12: 1-30, 2022.
... Montgomery Downs et al. stated that even though mothers who exclusively breastfed their babies woke up frequently at night, exclusive breastfeeding had a sleep-compensating effect, and that they might be exposed to the conditions of physical environment (light, formula preparation, etc.) less than did the mothers who prepared the formula (Montgomery-Downs, Clawges, & Santy, 2010). This situation can be explained by the fact that more stimulation of the prolactin hormone causes a relaxation effect, or that breastfeeding success makes the mother feel more confident (Sánchez et al., 2009). ...
... ПРИРОДНОЕ СОДЕРЖАНИЕ И ФУНКЦИОНАЛЬНЫЕ СВОЙСТВА НУКЛЕОТИДОВ В КОЗЬЕМ МОЛОКЕ Нуклеотиды являются строительным материалом для ДНК и играют важную роль в энергетическом обмене каждой живой клетки. Кроме того, они участвуют в процессах регуляции сна [50]. Присутствующие в составе детских молочных смесей нуклеотиды принимают участие в созревании иммунной системы и способствуют комфортному пищеварению [51]. ...
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The early stages of child development are characterized by various processes of maturation such as brain growth and development. The digestive and immune systems are developing as well at the same time. The intestinal microbiota plays significant role in the development of all organs and systems. Various disorders of microbial colonization of digestive system can negatively affect food programming processes. Nutrition type (breastfeeding or artificial) also has its own noticeable effect on early development. Breastfeeding is the “gold standard” in children’s nutrition. Modern approaches on creation of adapted formulas based on goat’s milk allow to develop its optimal composition (protein fraction for easy digestion, -palmitate, oligosaccharides, and natural nucleotides) that has positive effect on the child’s development.
... Cubero et al. [98], demonstrated that breastfed infants had greater sleep times overall and longer sleep bouts compared to formula-fed infants. Furthermore, nucleotides such as purine adenosine 5′ monophosphate (5′GMP) and pyrimidineuridine 5′ monophosphate (5′UMP) in breastmilk are controlled by circadian rhythms and peak at night [99]. Formula enriched with 5′GMP, 5′UMP, and tryptophan improved sleep parameters in infants including sleep duration, assumed sleep, and wake periods [100,101], indicating that not only the type of nutrition is important during this time, but the timing also plays an important role in developing the sleep/wake cycle. ...
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The microbes that colonize the small and large intestines, known as the gut microbiome, play an integral role in optimal brain development and function. The gut microbiome is a vital component of the bi-directional communication pathway between the brain, immune system, and gut, also known as the brain-gut-immune axis. To date there has been minimal investigation into the implications of improper development of the gut microbiome and the brain-gut-immune axis on the sleep-wake cycle, particularly during sensitive periods of physical and neurological development, such as childhood, adolescence, and senescence. Therefore, this review will explore the current literature surrounding the overlapping developmental periods of the gut microbiome, brain, and immune system from birth through to senescence, while highlighting how the brain-gut-immune axis affects maturation and organisation of the sleep-wake cycle. We also examine how dysfunction to either the microbiome or the sleep-wake cycle negatively affects the bidirectional relationship between the brain and gut, and subsequently the overall health and functionality of this complex system. Additionally, this review integrates therapeutic studies to demonstrate when dietary manipulations, such as supplementation with probiotics and prebiotics, can modulate the gut microbiome to enhance health of the brain-gut-immune axis and optimize our sleep-wake cycle.
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A robust method using HPLC-UV was developed to improve the accuracy and repeatability of a quantitative method to detect 5 nucleotides (cytidine-monophosphate, uridine monophosphate, adenosine monophosphate, guanine monophosphate, and inosine monophosphate) in infant formulas. The results showed that efficient separation could not be achieved without strict conditions. The proposed method displayed a strong linear relationship (R2 > 0.9999) of single nucleotide in infant formula milk powder in the range of 10 to 1,000 mg/kg, a steady recovery (80.0% ∼110.0%) with relative standard deviation from 0.5% to 3.5%, under strict conditions of hydrophilic C18 column with di-isopropyl at 62.5 ± 2.5°C (± standard deviation), 0.65 ± 0.1 mg/mL tetrabutylammonium bisulfate, and mobile phase of pH of 2.75 ± 0.02. By applying this method on a series of milk products in the Chinese market, we found a few of them exceeded the legal limits of nucleotides.
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Background Breastfed infants have a reduced risk of infections and allergies. The study of chrononutrition in human milk seeks to understand the circadian variation of various human milk immune factors. Methods Empirical studies on human milk, chrononutrition, and immune factors were searched through PUBMED, Google Scholar, and SCOPUS. Keywords included “chrononutrition,” “breastmilk composition,” “human milk,” “day-night cycles,” “sleep-wake cycles” and the names of various immune factors. After excluding duplicate articles, animal studies, studies looking at other human milk components, studies that did not collect human milk samples over a 24 hour period, and studies that were not in English, eleven studies on the topic remained and ten studies were included in the review. The excluded study had a sample size of two. Results This review identified the circadian variation of certain immune factors found in human milk such as antibodies, complement proteins, cytokines, by-products of phagocyte activity, nucleotides, microRNAs, and antioxidants. Conclusion The circadian variation observed in some human milk components highlights the unique ability of human milk to vary in composition based on the circadian rhythms of mothers and infants. The limited number of studies makes it difficult to make conclusive recommendations and creates an opportunity for further research in this growing field.
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Our knowledge about the complexity of human milk, in particular fatty acid, protein and oligosaccharide profiles, has increased considerably in recent years. However, little attention has been paid to nucleotides, which account for ∼2-5% of the non-protein nitrogen fraction of breast milk and provide important cellular and metabolism functions for the infant. We examined literature published in the last 25 years to provide an updated review of concentrations of nucleotides in breast milk across lactational stages in mothers around the world. The free mononucleotides found in highest concentrations in breast milk are, from highest to lowest in the order of cytidine 5’-monophosphate (CMP), uridine 5’-monophosphate (UMP) and adenosine 5’-monophosphate (AMP), guanosine 5’-monophosphate (GMP), and inosine 5’-monophosphate (IMP). Levels of nucleotides varied considerably amongst individual mothers and with stage of lactation. They could be further influenced by time of day and season and the mother's diet. Levels of free nucleotides varied between studies undertaken in different regions, however, in studies that measured total potentially available nucleotides (TPAN) levels regional differences were not apparent. Some studies report higher amounts in colostrum and transition milk compared with mature milk, whilst other studies report the converse. the Recently clinical studies showed that there are benefits to supplement nucleotides in infant formula. Although comparing data in literature remain a challenge due to different milk collection methodologies and measurement protocols used by different studies, the information may provide insights for designing of formula products for infant at different stages of development.
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Background: Although most studies have reported unfavorable short-term effects of breastfeeding on early-childhood sleep-wake behaviors that potentially attenuate over time, findings have remained inconsistent. Objectives: We assessed associations of breastfeeding with longitudinal day-, night-, and total-sleep trajectories and with sleep-wake behaviors in healthy infants and preschoolers. Methods: Caregivers of naturally conceived, term, singleton infants (n = 654) completed the Brief Infant Sleep Questionnaire (3, 6, 9, 12, 18, and 24 mo) and/or Children's Sleep Habits Questionnaire (54 mo), and provided information on their infants' breastfeeding status at 3 mo. Trajectory analyses derived 4 day- (n = 243), 3 night- (n = 248), and/or 4 total- (n = 241) sleep trajectories, each differing in length of sleep duration (short/moderate/long) and variability (variable/consistent). Sleep-wake behaviors from 3 to 24 mo (day/night/total-sleep durations and duration/number of night awakenings) were also assessed for associations with breastfeeding. Results: After adjusting for potential covariates, formula-fed infants, relative to fully breastfed (predominant or exclusive) infants, were significantly less likely to exhibit moderate (OR: 0.28; 95% CI: 0.11, 0.70) and long consistent (OR: 0.18; 95% CI: 0.07, 0.50) night-sleep trajectories and less likely to exhibit moderate (OR: 0.21; 95% CI: 0.07, 0.61) and long consistent (OR: 0.12; 95% CI: 0.04, 0.38) and long variable (OR: 0.16; 95% CI: 0.05, 0.56) total-sleep trajectories, instead of short variable night- and total-sleep trajectories. Partially breastfed infants did not differ from fully breastfed infants for both night- and total-sleep trajectories. No significant differences were found between all groups for day-sleep trajectories. Fully breastfed infants had longer night- (6, 9, 12, and 24 mo) and total- (3 and 12 mo) sleep durations than formula-fed infants, albeit a greater number of night awakenings (from 6 to 12 mo). Conclusions: Despite more night awakenings, fully breastfed infants have overall longer night- and total-sleep durations (sleep trajectories) than formula-fed infants.
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Sleep is not the mere absence of wakefulness, but an active state which is finely regulated. The homeostatic facet of sleep-wake regulation is keeping track of changes in 'sleep propensity' (or 'sleep need'), which increases during wakefulness and decreases during sleep. Increased sleep propensity following extended prior wakefulness (sleep deprivation) is counteracted by prolonged sleep duration, but also by enhanced non-rapid-eye-movement (nonREM) sleep intensity as measured by electroencephalographic (EEG) slow-wave activity (SWA, power within approximately 1-4 Hz). This highly reliable regulatory feature of nonREM sleep may be the most important aspect of sleep in relation to its function. The neurochemical mechanisms underlying nonREM sleep homeostasis are poorly understood. Here we provide compelling and convergent evidence that adenosinergic neurotransmission plays a role in nonREM sleep homeostasis in humans. Specifically, a functional polymorphism in the adenosine metabolizing enzyme, adenosine deaminase, contributes to the high inter-individual variability in deep slow-wave sleep duration and intensity. Moreover, the adenosine receptor antagonist, caffeine, potently attenuates the EEG markers of nonREM sleep homeostasis during sleep, as well as during wakefulness. Finally, adenosinergic mechanisms modulate individual vulnerability to the detrimental effects of sleep deprivation on neurobehavioral performance, and EEG indices of disturbed sleep after caffeine consumption. While these convergent findings strongly support an important contribution of adenosine and adenosine receptors to nonREM sleep homeostasis, further research is needed to elucidate the underlying mechanisms that mediate the actions of adenosine on sleep and the sleep EEG.
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The effect of nucleotides in the newborn is a determinant in this first stage of life, and their correctlevel in breastmilk is vital. We have designed a new method for the assay of nucleotides in milk bycapillary electrophoresis (CE) after acid hydrolysis. Breastmilk samples were collected from healthymothers (ages, 25–35 years) of one month lactation, and stored at -20°C. The duplicated sampleswere dissociated by acidic hydrolysis (HClO4) and the CE assay was performed in an uncoatedfused-silica capillary using an alkaline (borate) electrophoretic separation system.The method gave good recoveries of 5´-mononucleotides. Under the conditions used, the actualCE analysis time was less than 20 minutes. The physiologically and nutritionally importantnucleotides were detected at concentrations of 387 μg/100ml for UMP-5P, 385.3 μg/100ml forAMP-5P, 67 μg/100ml for CMP-5P, 172 μg/100ml for TMP-5P and 315 μg/100ml for GMP-5P.Nucleotides are a significant nutrient in infant growth, and capillary electrophoresis is a sensitiveand efficient tool for the assay of nucleotides with a purine or pyrimidine base in breastmilk.
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A method is described for the determination of four 5′-mononucleotides (cytidine 5′-monophosphate, uridine 5′-monophosphate, adenosine 5′-monophosphate and guanosine 5′-monophosphate) in infant formulae. Nucleosides which may be formed during processing can also be analysed simultaneously. This method is based on deproteinisation of samples and direct analysis by ion-pair HPLC using two Nucleosil 120-C18 columns in series, followed by diode-array detection. This method gives good recoveries of 5′-mononucleotides from spiked infant formula products. However, some chromatographic interferences were observed when analysing hypoallergenic infant formulae containing hydrolysed proteins which made peak quantification difficult. To overcome this problem a strong anion-exchange solid-phase extraction (SPE) column was used. Four SPE columns from different suppliers were evaluated, but the best recoveries of all four 5′-mononucleotides and highest reproducibility of results were obtained with Bakerbond® quaternary amine columns. Nucleosides, which may occur in very low concentrations in hypoallergenic products, are not retained on the SPE columns and so cannot be analysed by this technique.
The effects on sleep of N6-L-(phenylisopropyl) adenosine, cyclohexyladenosine and adenosine-5'-ethylcarboxamide were studied in rats. Also, the effects on sleep of deoxycoformycin, a potent inhibitor of adenosine deaminase, and adenosine were examined. In addition, we determined the effects of 48 h of REM sleep deprivation on adenosine (A1) receptors in specific brain structures. N6-L-(phenylisopropyl) adenosine and cyclohexyl-adenosine increased deep slow wave sleep and REM sleep whereas adenosine-5'-ethylcarboxamide increased only deep slow-wave sleep. At the dose of 0.9 mumol/kg all three adenosine analogs suppressed REM sleep and except for adenosine-5-ethylcarboxamide, were without an effect on deep slow-wave sleep. In accordance, administration of deoxycoformycin increased REM and deep slow-wave sleep. Intracerebroventricular administration of 1, 10 and 100 nmoles of adenosine to rats decreased waking, increased deep slow-wave sleep and increased total sleep. In addition, REM sleep deprivation significantly increased the number of A1 receptors (Bmax) in cerebral cortex and corpus striatum which correlates with the increased pressure for REM sleep and the onset of REM sleep rebound. When these data are taken together, they indicate a role for adenosine in the regulation of sleep and, in contrast to barbiturate and benzodiazepine hypnotics, increase in behaviorally deep and REM sleep.
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During 48 and 72 hrs paradoxical sleep deprivation (PSD) in rats, the adenosine values determined in brain homogenates by a chromatographic method on Dowex I X4 columns were proportionally increased. Intraventricular injections of 0.2 ml adenosine solution 2.5 mg/ml in dogs induced behavioral and electroencephalographical alterations of the hypnogenic type.