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 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.
Keywords: nucleotides, circadian, sleep, human milk, capillary electrophoresis
A joint declaration by the World Health Organization
(WHO) and the United Nations Children’s 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
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;
Received 25 March 2008, revised manuscript accepted 19 August 2008
© W. S. Maney & Son Ltd 2009
Nutritional Neuroscience 2009 Vol 12 No 1 3
Sánchez et al. Human milk nucleotides and sleep
5′monophosphate (5′AMP), guanosine 5′mono-
phosphate (5′GMP), and their precursor, inosine
5′monophosphate (5′IMP) – or a pyrimidine – uridine
5′monophosphate (5′UMP), cytidine 5′monophosphate
(5′CMP), and thymidine 5′monophosphate (5′TMP).
The nucleotides act in cells as secondary messengers
through cAMP (cyclic 5′AMP) and cGMP (cyclic
5′GMP), 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 – 5′UMP, 5′AMP, and
The first, 5′UMP, 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
The second, 5′AMP, 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, 5′GMP, 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 5′CMP,
1.5 mg/100 kcal of 5′UMP, 1.5 mg/100 kcal of 5′AMP,
0.5 mg/100 kcal of 5′GMP, and 1 mg/100 kcal of
5′IMP. 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
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 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.
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.
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
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.
Adenosine 5′monophosphate, uridine 5′monophos-
phate, guanosine 5′monophosphate, thymidine 5′mono-
phosphate, cytidine 5′monophosphate, inosine
5′monophosphate, 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).
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 5′AMP, 5′CMP, 5′GMP,
5′TMP, 5′UMP, and 5′IMP.
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.
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.
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.
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
Figure 1 shows the levels of 5′AMP 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 5′UMP. 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 (5′GMP, 5′CMP, 5′IMP, and 5′TMP) 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 5′GMP
for the nocturnal period (20:00–08:00). A similar
Figure 1 Sinusoidal function obtained by cosinor analysis
of the nucleotide 5′′AMP for a 24-h period (n= 30)
Figure 2 Sinusoidal function obtained by cosinor analysis
of the nucleotide 5′′UMP 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 5′CMP and 5′IMP (Figs 4 and 5,
respectively). This contrasts with the apparent down -
ward trend in the daylight intervals for 5′AMP (Fig. 1).
The results of the chronobiological study (Table 1)
of particular interest were the significant circadian
rhythms of 5′AMP (Fig. 1) and 5′GMP (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 5′CMP
(Fig. 4) and 5′IMP (Fig. 5) but with acrophases during
the daytime period (at 18:40 and 19:14, respectively).
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
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 environment – day 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 5′′GMP for a 24-h period (n= 30)
Figure 4 Sinusoidal function obtained by cosinor analysis
of the nucleotide 5′′CMP for a 24-h period (n= 30)
Figure 5 Sinusoidal function obtained by cosinor analysis
of the nucleotide 5′′IMP for a 24-h period (n= 30)
Figure 6 Sinusoidal function obtained by cosinor analysis
of the nucleotide 5′′TMP 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
5′AMP 5.17 (4.61–5.73) 1.03 (0.02–2.03) 20:19 (15:08–25:30) 0.04457*
5′UMP 6.14 (4.91–7.37) 1.20 (–) 02:00 (–) 0.36425
5′GMP 3.63 (3.42–3.85) 0.46 (0.07–0.84) 05:08 (01:18–08:58) 0.01955*
5′CMP 2.44 (2.25–2.64) 0.42 (0.16–0.68) 18:40 (14:59–22:20) 0.01645*
5′IMP 3.06 (2.91–3.21) 0.44 (0.18–0.70) 19:14 (16:48–21:41) 0.00149*
5′TMP 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
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 5′AMP 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, 5′GMP, 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 5′UMP, 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
Of the other nucleotides, 5′CMP had a significant
circadian rhythm with acrophase at 18:40 (during
daylight hours), and a MESOR of 2.44 µg/ml.
Because 5′IMP 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, 5′AMP and
5′GMP. Indeed, its acrophase was at 19:14 (just before
the onset of darkness when the sleep inducers, 5′AMP
and 5′GMP, reach their acrophases) and its MESOR
was 3.06 µg/ml.
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 5′AMP, 5′UMP, and 5′GMP, which showed
increased concentrations at night and may, therefore,
be involved in inducing hypnotic action in the infant.
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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