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b-Casomorphins-7 in infants on different type of feeding and different levels of
;arina V. Gabaevaa, Yuriy A. Zolotarevc, !lexander K. Dadayanc, Sergei A. Grachevd,
+katerina V. Korneevab, Inna G. Mikheevab, !ndrey A. Zozulyaa
aNational Research Center for Mental Health RAMS, 113152 Moscow, Zagorodnoe shosse 2/2, Russia
bRussian State Medical University, 117997 Moscow, Ostrovityanova ul. 1, Russia
cInstitute of Molecular Genetics RAS, 123182 Moscow, Kurchatova pl. 2, Russia
dOvchinnikov-Shemyakin Institute of Bioorganical Chemistry R!S, 117997 Moscow, Miklukho-Maklay ul 16/10, Russia
Originally, more than 30 years ago, casomorphins (CMs) were
identified in enzymatic bovine casein digest as a substance with
defined opioid activity . Later, opioid peptides were shown to be
derived either from caseins or other proteins contained in
mammalian milk, including human and cow milk . Multiple
studies in animal models reported that these peptides and CMs
first of all decrease pain sensitivity [3,25,32], possess anxiolytic
[13,14], antihypertensive  and cardiotropic  effects,
improve learning and memory [12,35], regulate maternal motiva-
tion  and mother-oriented (‘‘child’s’’) behavior in rats ,
induce apnea and irregular breathing in adult rats and newborn
rabbits , increase prolactin release in rats , and expression
intestinal cells [44,45]. Most of these effects were opioid-
dependent, i.e. reversible by the opioid receptors antagonists
[3,10,16,25,27,32,35,44]. However, some effects of CMs were
opioid-independent [23,33], suggesting that there are additional
receptors and signaling pathways for CMs. In fact, b-CM-9 and
some other peptides derived from bovine milk casein have been
shown to inhibit angiotensine-converting enzyme , while
bovine and human b-CM-7 appeared to act as antagonists of 5-
HT2-serotonin receptors [38,39]. Non-opioid influence of CM-5 on
the b-adrenoceptor complex was detected in guinea pig heart
membranes. (D-Pro4)-b-CM-5 was shownto regulate synaptic
transmission of acetylcholine in hippocampus and dopamine in
striatum, presumably by interaction with the dopamine and
serotonin receptors .
The wide spectrum of the biological activity of CMs prede-
termines their role in human health and disease states. A number
of studies have data suggested that bovine b-CM-7 can act as a
Peptides 30 (2009) 1854–1860
A R T I C L E I N F O
Received 7 April 2009
Received in revised form 19 June 2009
Accepted 24 June 2009
Available online 1 July 2009
A B S T R A C T
Casomorphins are the most important during the first year of life, when postnatal formation is most
activeand milk isthemain source of bothnutritiveand biologicallyactivematerial for infants. This study
was conducted on a total of 90 infants, of which 37 were fed with breast milk and 53 were fed with
formula containing cow milk. The study has firstly indicated substances with immunoreactivity of
human (irHCM) and bovine (irBCM) b-casomorphins-7 in blood plasma of naturally and artificially fed
infants, respectively. irHCM and irBCM were detected both in the morning before feeding (basal level),
and 3 h after feeding. Elevation of irHCM and irBCM levels after feeding was detected mainly in infants in
the first 3 months of life. Chromatographic characterization of the material with irBCM has
demonstrated that it has the same molecular mass and polarity as synthetic bovine b-casomorphin-
7. The highest basal irHCM was observed in breast-fed infants with normal psychomotor development
and muscle tone. In contrast, elevated basal irBCM was found in formula-fed infants showing delay in
psychomotor development and heightened muscle tone. Among formula-fed infants with normal
development, the rate of this parameter directly correlated to basal irBCM. The data indicate that breast
feeding has an advantage over artificial feeding for infants’ development during the first year of life and
support the hypothesis for deterioration of bovine casomorphin elimination as a risk factor for delay in
psychomotor development and other diseases such as autism.
? 2009 Elsevier Inc. All rights reserved.
* Corresponding author at: National Research Center for Mental Health RAMS,
Laboratory of Pathophysiology, Zagorodnoe shosse 2/2, 113152 Moscow, Russia.
Tel.: +7 495 952 9090.
E-mail address: firstname.lastname@example.org (N.V. Kost).
Contents lists available at ScienceDirect
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Author's personal copy
causative agent of cardiovascular disease, type 1 diabetes, sudden
infant death syndrome, autism and schizophrenia [6,19]. However,
it appears highly unlikely that evolutional (natural) selection
retained a harmful product as the main nutritional component for
mammalian infants. Most experimental data indicate the positive
action of milk-derived peptides , and it can be inferred that
these exogenous peptides are most effective in early infancy, when
permeability of the intestinal mucosa is increased for small
peptides and large molecules of proteins . The only direct
identification of bovine CM-immunoreactive (irBCM) materials in
the blood was done in beagle puppies. The plasma level of irBCM
was shown to rise after feeding with cow milk based formula in
newborns, but did not in adult dogs . Immunoreactivity of
human CM (irHCM) has been detected in cerebrospinal fluid
[24,28], blood plasma [15,24,28], urine , and milk [17,28] of
pregnant, puerperal and lactating women. However, so far neither
The purpose of our study was to detect and characterize
materials with immunoreactivity of human and bovine b-CM-7 in
the blood plasma of naturally and artificially fed infants, and to
investigate the relationship between the level of b-CM-7-
immunoreactivity and the rate of the infants’ psychomotor
2. Materials and methods
Ninety infants younger than 1 year were enrolled in the current
parents of all participants. All infants were patients of the Moscow
Izmailovo Children’s Clinical Hospital, where they received
treatment for acute respiratory viral infection. All procedures
were performed right before the patients’ discharge, when they
had completely recovered from their infections. Pediatric exam-
ination of the infants included assessment of psychomotor
development using the 30-point scale method developed by Jurba
and Mastukova . This method differentially evaluates the
development of motor, language and mental functions in infants.
According to this test, 27–30 points on the scale corresponds to
normal psychomotor development, while 23–26 points are the
range for a risk of delayed development, and points below 26
correspond to a definite delay in development. Muscle tone was
referred as one of the dynamic functions of age development, and
measured in 4-point scale by deviation from physiological one
according to Jurba’s test. In addition, in general clinical investiga-
tion the patient’s muscle tone was evaluated as normal,
heightened, or lowered.
Blood samples were first obtained as a part of routine diagnostic
procedureinthe morningbeforefeeding.Agesforthe patients were
to 3-month old, 23 from 4- to 6-month old, and 17 from 7- to 12-
3-month old, 6 from 4- to 6-month old, and 5 older than 6 months.
The 4–10-month old naturally fed patients were combined into one
obtained from 39 infants 3 h after feeding with formula based on
cowmilkwithalbuminpredominance,andfrom20infants3 h after
breast feeding. Plasma samples from 5 healthy adult volunteers
were used as a negative control.
Blood samples were collected into tubes containing EDTA
(3 mg/ml) as an anticoagulant and peptidase inhibitor bacitracin
(150 mg/ml). Plasma was separated immediately by centrifugation
at 1000 ? g, at 4 8C for 10 min and stored at ?20 8C until analysis.
2.2.1. Extraction of b-casomorphins-7 from plasma
Peptides were extracted from plasma by boiling in a 10-fold
excessof 0.25 M acetic acid ina water bathfor 15 min. Theextracts
were frozen, lyophilized, and kept at ?70 8C. Just before the
analysis, all probes were dissolved in an appropriate buffer and
centrifuged at 1000 ? g, at 4 8C for 10 min, and the supernatant
was collected. The extraction recovery rate was about 70% as
assessed by the addition of [125I] b-CM-7 (about 50,000 cpm,
100 fmol) to 1 ml of human plasma. The recovery rate of
radioactivity after extraction was 82% for bovine and 76% for
human [125I] b-CM-7, while immunoreactivity of extracted
radioactive materials remained intact up to 90% of reference.
b-CM-7 antisera were prepared by immunization of rabbits
according to method described by Dmitriev et al. . Human (Tyr-
Pro-Phe-Val-Glu-Pro-Ile) and bovine (Tyr-Pro-Phe-Pro-Gly-Pro-
Ile) b-CM-7 (Sigma, USA) were coupled to a number of proteins:
bovine serum albumin (BSA), RNAase, chymotrypsin, thyroglobu-
lin, ovalbumin, cattle immunoglobulins, catalase, using glutaric
aldehyde as a cross-linking reagent. The coupling reaction was
performed overnight at 0 8C (ice bath) in the mixture contained
0.02% glutaric aldehyde, 0.4 mg/ml carrier protein and 0.2 mg/ml
either of casomorphins in 50 mM Na2HPO4(pH 8.0). Rabbits were
immunized with makeup conjugates subcutaneously in the back
(300 mg per rabbit in 0.5 ml of Friend’s adjuvant). The immuniza-
peptides were conjugated to different proteins to enhance the
specificity of the serum to CM. The serum was obtained by
centrifugation of the blood collected from the auricular vein and
stored at ?20 8C.
Radioactively labeled peptides were obtained by radioiodina-
tion (125I: Amersham, U.K.) of b-CM-7 by a modified method
developed by Miller et al.  using chloramine T (Merck,
Germany) for the iodination and mercaptoethanol (Sigma, USA)
to terminate the reaction. The iodinated peptides were purified
from Na125I by ion exchange chromatography on QAE-Sephadex
A-25 column using 50 mM acetic acid with 0.5% BSA as an eluent.
b-CM-7-containing fractions were neutralized by ammonia to pH
7.5 and kept in 25% glycerin at ?10 8C for less than 1 month. The
purification efficiencyof both labeled peptides was more than 99%.
The specific radioactivity was 361 and 410 Ci/mmol for human and
bovine b-CM-7, respectively.
RIA was carried out in the buffer consisting of 0.15 M NaCl,
25 mM Na2HPO4(pH 7.5), 0.5% BSA, and 0.1% Tween 20, at 4 8C. For
the assay, the samples, chromatography fractions or b-CM-7
standards, were dissolved or further diluted by the buffer to the
volumeof600 mlandincubatedfor24 hwith100 mlofanti-b-CM-
7 antiserum (diluted 750-fold for bovine and 500-fold for human
b-CM-7 assay). One day later, 100 ml of [125I] b-CM-7 (about
20,000 cpm, 30 pg) was introduced into the mixture and incuba-
tion continued for 4 days. On day 4, 100 ml of goat anti-rabbit
serum was added for 2 h following by 100 ml of 50-fold diluted
neutral rabbit serum and 800 ml of 15% polyethyleneglycol 6000.
Afterthoroughmixing,thetubeswerecentrifugedat2000 ? g,4 8C
for 30 min. The supernatant was aspirated and the precipitate was
counted in a gamma counter (Gamma Trac, efficiency 70%).
Nonspecific binding (about 20%) was determined in the absence of
Cross-reactivity of human and bovine b-CM-7 to the obtained
antisera was negligible. Standard curves proved to be linear in a
logit/log plot between 10% and 90% inhibition of [125I] labeled
of 0.03–5.0 pmol/assay tube for human b-CM-7 and 0.01–
N.V. Kost et al./Peptides 30 (2009) 1854–1860
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2.0 pmol/assay tube for bovine b-CM-7. The detection limit of
human and bovine b-CM-7 was 50 and 15 fmol, respectively,
which is similar to the literature data [37,40].
2.2.3. Enzyme linked immunosorbent assay (ELISA)
ELISA was performed using purified antibodies from the same
antisera that were used for the RIA. Purification was carried out
by affine chromatography at BrCN-sepharose-4B conjugated
with bovine b-CM-7. Antisera (3 ml) were introduced to a
5 mm ? 15 mm column in PBS, the column was washed with
PBS to the disappearance of the optical density at 280 nm in the
eluate, followed by antibody elution with 0.1 M acetic acid (pH
2.2). The effluent was neutralized with ammonia to pH 7.0. Protein
output was about 0.3 mg.
Bovine b-CM-7 was biotinylated with NHS-LC-LC-biotin
(succinimidyl-6-(biotinamido)-6-hexanamido hexanoate, Pierce,
USA) in equimolar concentrations by overnight incubation of the
substances in 0.1 M NaHCO3(pH 8.0) at 4 8C with subsequent
purification by HPLC at ProntoSIL-120-5-C18 column using
gradient elution. Acetonitrile with 0.1% TFA was used as a mobile
phase. The concentration of the product was evaluated by optical
density at 280 nm. For the assay, purified antibodies were diluted
in 0.05 M Na-carbonate buffer (pH 9.5) to 4 mg/ml, added to 96-
well plates (Nunc, Denmark) (0.25 ml/well), and the plates were
incubated for 3 days at 4 8C in a humidified atmosphere. The plates
were then washed 5 times with distilled water. The samples,
buffer consisting of 0.15 M NaCl, 25 mM Na2HPO4(pH 7.5), 0.2%
BSA, and 0.05% Tween 20 (ELISA buffer), added (200 ml/well) to the
washed antibody-coated wells together with 50 ml of biotin-b-
CM-7 (10 fmol/well) in the same buffer, and incubated for 1.5 h at
37 8C. After subsequent rinsing with water, 250 ml of conjugate of
streptavidin with peroxidase (100 ng/ml, Imtek Ltd., Russia)
diluted in ELISA buffer were added into each well for 30 min
(37 8C). Plates were rinsed again with distilled water 8 times and
250 ml of 2.5% tetramethylbenzidine (Bioservice, Russia) in 0.2 M
Na2HPO4–citrate buffer (pH 4.0) with 0.01% H2O2 and 0.05%
ProClin were added. The reaction was stopped by the addition of
50 ml of 1 M H2SO4 after 15 min at room temperature and
absorbance was immediately measured at 450 nm using plate
reader (Uniplan, Russia). Nonspecific binding (about 10%) was
determined in wells not coated with antibodies.
Standard curves were linear in a logit/log plot between 10% and
90% inhibition of biotin-labeled bovine b-CM-7 binding to the
antibody over the range of concentrations of 0.015–1.2 pmol/well
for bovine b-CM-7, IC50was about 0.1 pmol/well. The detection
limit of bovineb-CM-7 was 25 fmol. Humanb-CM-7 andb-casein
from bovine milk (Sigma, USA) in 100-fold excessive concentra-
tions did not displace labeled peptide in this assay.
The molecular weight of b-CM-7 immunoreactive material in
infants’ plasma was evaluated by gel filtration at HPLC system
(Gilson, USA). Lyophilized extracts of plasma samples prepared as
above were reconstituted in 25 mM Na2HPO4 buffer pH 6.0,
centrifuged, and the dissolved material was separated on
7.8 mm ? 300 mm Protein Pak 60 column. The elution was carried
out with the same buffer at a flow rate of 0.7 ml/min with
absorbance detection at 226 nm. Seven fractions (about 2 ml each)
were collected, lyophilized and reconstituted in ELISA buffer.
Control trialswere performedtoevaluatethe rateof elutionofBSA,
b-casein from bovine milk, bovine b-CM-7, and synthetic
hexapeptide dalargin (Tyr-D-Ala-Gly-Phe-Leu-Arg).
Chromatographic characterization of b-CM-7 immunoreactive
material was also done by reverse phase chromatography. Extracts
were reconstituted in 1.5 ml of 50% acetonitrile with 0.1% TFA,
centrifuged, evaporated, dissolved in 100 ml of 0.1% TFA and
separated on 2.0 mm ? 75 mm ProntoSIL-120-5-C18 column (flow
rate of 0.2 ml/min, 35 8C) using gradient elution in acetonitrile (0–
60%) with 0.1% TFA as a mobile phase. Four fractions (about 1.5 ml
each) were collected in the range of b-CM-7 elution (identified by
control runs of reference substances), evaporated and applied to
The data analyses were performed using Statistica program
(version 6.0). The results were expressed as mean values ? SE. The
mean values in different groups were compared using Student’s t-
test. Non-parametric correlations were calculated by Spearman’s
rank correlation coefficient.
3.1. Immunoreactivity of bovine and human b-CM-7 in
the blood of infants
The consequent immunization of the rabbits with bovine and
human b-CM-7 conjugated with different proteins resulted in
antisera with a high titer to the peptides. As measured by half-
maximum binding with iodinated peptides (about 20,000 cpm/ml)
the titer was 1:7500 and 1:5000 for bovine and human b-CM-7,
respectively.Using the antisera,ELISA and RIA of the peptideswere
created. The immunoreactivity of both bovine and human b-CM-7
in all the infants’ blood plasma was measured by RIA.
RIA detected immunoreactivity of bovine b-CM-7 (irBCM) in
plasma specimens of infants fed with formula containing the
proteins of cow milk, with no difference in this parameter before
feeding (basal level) in the 1–3-month-old infants (67 ? 19 fmol/
ml, n = 13) and 4–6-month-old patients (59 ? 16 fmol/ml, n = 23)
(Fig. 1). However, after the age of sixth month, the basal level of
plasmairBCMincreasedupto90 ? 14(n = 17)(Fig.1). Themeanlevel
7–12-month-old infants, but not for the group of 4–6-month-old
patients (p < 0.05, Fig. 1). There was no detectable irBCM in the
plasma of mostof the adult volunteers,whilein the breast-fed infants
it was in the range of the cross-reactivity between human and bovine
b-CM-7 (about 3% from the same parameter tested for human CM).
Immunoreactivity of human b-CM-7 (irHCM) was detected in
the plasma specimens of breast-fed infants. Prior to the feeding, the
average value was also the same in the 1–3-month old (208 ?
23 fmol/ml, n = 26) and 4–10-month-old patients (197 ? 30 fmol/ml,
n = 11).SimilarlytoirBCM,irHCMsignificantlyroseafterfeedinginthe
case of 1–3-month-old infants, but not in the older ones (Fig. 2). As a
old infants was significantly higher than in the oldest ones (p < 0.05,
Fig. 1. Immunoreactivity of bovine b-casomorphin-7 in the blood plasma of
different age infants. Bars are the mean ? SE.#p < 0.05 refers to comparison of base-
line values with values 3 h after feeding.
N.V. Kost et al./Peptides 30 (2009) 1854–1860
Author's personal copy
Fig. 2). The plasma irHCM of adult volunteers was below the detection
limit, but a significant amount of human CM-immunoreactive
materials was shown in the plasma of several infants who were fed
Thus the immunoreactivity of bovine and human b-CM-7 was
detected in the blood of first year life infants. It increased after
feeding mainly in 1–3-month-old patients.
3.2. Immunoreactivity of b-CM-7 in the blood and psychomotor
development of infants
We found no difference in the mean values of psychomotor
development of infants who were on breast feeding versus those
on artificial feeding. However, there was only one case of
developmental delay in the group of breast-fed infants, while 16
The rate of psychomotor development tested on a 30-point scale
positively correlated with the basal level of irHCM in the total
group of breastfed infants (R = 0.35, p < 0.05). This correlation was
morepronouncedandwasrevealedbothbefore(R = 0.45,p < 0.05)
and after (R = 0.66, p < 0.05) feeding in the group of 1–3-month-
old breast-fed infants. The basal level of irHCM in the blood of
infants with normal development was twice as high as that in
infants with a risk of developmental delay (Table 1).
One of the parameters tested using Jurba’s method was
muscle tone. A statistical analysis revealed a direct correlation
between the values of this parameter and the basal irHCM in
breast-fed infants (R = 0.51, p < 0.01). This means that the higher
the level of irHCM is in the blood, the closer muscle tone is to a
normal (physiological) level. The same correlation was detected
in both 1–3 (R = 0.51, p < 0.05) and 4–10 (R = 0.61, p < 0.05)
month old breast-fed infants. There are two polar deviations of
muscle tone from normal values. Our study has shown that the
level of irHCM in the infants with heightened muscle tone was
significantly lower than in those with normal muscle tone
The opposite results were obtained in infants fed with formula.
The basal level of irBCM increased 2-fold in the bloodplasma of the
infants who displayed delays in development (estimated by the
Jurba’s test to be less than 23 points) (Table 1). At the same time,
the rate of psychomotor development positively correlated with
post-feeding irBCM (R = 0.49, p < 0.05) in the common group of
infants with normal development and those with a risk of
developmental delay, and this correlation was more pronounced
at 4–12 months of age (R = 0.57, p < 0.05). The increase in irBCM
3 h after feeding was detected only in infants with normal rate of
psychomotor development (Table 1). There was no correlation
between the points of muscle tone and irBCM levels in artificially
fed infants. However, the average irBCM level in plasma of the
infants with heightened muscle tone was twice as high as in
patients with normal muscle tone (Table 2).
3.3. Chromatographic analysis of extracted material with irBCM
Chromatographic study of the material with irBCM extracted
from plasma was performed by HPLC, using specially elaborated
ELISA to detect the immunoreactivity in fractions. The assay was
found to be as sensitive to bovine b-CM-7 as RIA, but ELISA is a
more efficient and environmentally appropriate method than RIA.
The molecular weight of bovine b-CM-7 immunoreactive
material from infants’ plasma was evaluated by HPLC gel filtration.
Three samples of blood plasma extracts from the artificially fed
infants eluted on Protein Pak 60 column as multiple peaks as
detected by UV absorption. A typical elution profile (Fig. 3) shows
that the major protein fraction eluted from the column with a void
volume within the first 10 min of elution just as high molecular
weight BSA and b-casein did. The retention time of short peptides
such as b-CM-7 and dalargin was about 20 min, and their UV
absorption at 226 nm was relatively low. irBCM was detected in
the fractions eluted very close to the position of synthetic bovine
b-CM-7, but not in high molecular weight fractions (Fig. 3).
Reverse phase chromatography performed using ProntoSIL-
120-5-C18 column also showed that bovine b-CM-7 immunor-
eactive material from infants’ plasma had the same retention time
as the synthetic peptide (Fig. 4). Thus chromatographic analysis of
the samples demonstrated that the material with irBCM extracted
and polarity similar to synthetic bovine b-CM-7.
Fig. 2. Immunoreactivity of human b-casomorphin-7 in the blood plasma of
different age infants. Bars are mean ? SE.#p < 0.05 refers to comparison of base-line
values with values 3 h after feeding, *p < 0.05 refers to comparison of post-feding
values in the group of different age infants.
Immunoreactivity of human and bovine b-casomorphins-7 in the blood plasma of infants with different level of psychomotor development.
Psychomotor developmentirHCM (fmol/ml) irBCM (fmol/ml)
Before feeding After feeding Before feedingAfter feeding
Normal (>26 points)
Risk of delay (26–23 points)
Delay in development (<23 points)
236 ? 23 (n = 28)
122 ? 29#(n = 8)
154 (n = 1)
208 ? 29 (n = 15)
116 ? 42 (n = 4)
189 (n = 1)
52 ? 10 (n = 21)
123 ? 38#(n = 16)
108 ? 22#(n = 16)
122 ? 32*(n = 12)
103 ? 29 (n = 15)
135 ? 30 (n = 12)
Values are mean ? SE.
*p < 0.05 refers to comparison of base-line values with values 3 h after feeding.
#p < 0.05 refers to comparison of base-line values in the group of infants showing normal development with values in other groups.
Basal immunoreactivity of human and bovine b-casomorphins-7 in the blood
plasma of infants with different state of muscle tone.
Muscle tone irHCM (fmol/ml)irBCM (fmol/ml)
225 ? 25 (n = 17)
207 ? 27 (n = 9)
127 ? 30 (n = 12)#
60 ? 12 (n = 18)
99 ? 34 (n = 17)
132 ? 33 (n = 18)#
Values are mean ? SE.
#p < 0.05 refers to comparison of base-line values in the group of infants
showing physiological muscle tone with values in other groups.
N.V. Kost et al./Peptides 30 (2009) 1854–1860
Author's personal copy
The biological activity of materials entering the human
organism with food is the main issue in dietology. It is most
important during the first year of life when postnatal formation of
all organism systems is most active. At this time, milk is the main
source of both nutritive and biologically active materials for
infants. Thus the aim of our study was to estimate the level of CM-
immunoreactive material in blood plasma of infants on different
types of feeding and investigate the possible relation between this
immunoreactivity and the level of their psychomotor develop-
The substances with the immunoreactivity of human and
bovine b-CM-7 were detected in the blood plasma of most of the
infants fed with breast milk and cow milk based formula,
respectively. Chromatographic analysis of blood plasma extract
from formula-fed infants demonstrated that the material with
irBCM has the same molecular mass and polarity as synthetic
bovine b-CM-7. The level of irBCM in infants was similar to that
measured earlier in newborn dogs fed with cow milk .
Both irBCM and irHCM were detected in infants’ plasma
samples taken in the morning after 6 h intermission between
feeding (basal level) and 3 h after morning nursing. The average
basal level of irBCM and irHCM did not significantly vary according
to age. While the average value of both bovine and human CM
immunoreactivity increased 3 h after feeding mainly in infants of
1–3-month age (Figs. 1 and 2). This is in agreement with the well
known fact that the intestinal mucosa of newborns is permeable
for peptides and even for relatively large proteins . It is worth
noting that irBCM also increased after bottle-feeding in infants
6-month of age and older. This might be due to an increase in
protein abundance in formula used for feeding this age group. In
fact, we have observed that the greater the protein content was of
the formula, the higher was the level of irBCM detected in blood
obtained after feeding. However, this relationship was traced only
as a tendency.
While getting to the circulatory system, b-casomorphins and
been confirmed by post-mortal study that detected b-CM-
immunoreactivity in a number of brain regions of human infants
. As CMs reach the brain, they might affect the development of
the infants’ central nervous system. Our study has circumstantially
confirmed this. In fact, irHCM in breast-fed infants with normal
psychomotor development and normal muscle tone was twice as
high as in infants with a risk of developmental delay and a
heightened tone, respectively (Tables 1 and 2). The rate of
psychomotor development directly correlated with irHCM in
breast-fed infants, and this correlation was most pronounced in
the infants in the first 3 months of life—that very age when
permeabilityof the intestinal mucosa for peptides is most probable.
A similar correlation was detected for the formula-fed group,
but only for those whose psychomotor development was close to
their age norm. Moreover, a difference between basal and post-
feeding irBCM was detected only in normally developing infants
(Table 1). So under regular uptake and removal, bovine CM such as
human CM is supposed to have a positive action upon infants’
development. However, in formula-fed infants with risk and delay
in development as well as with heightened muscle tone, basal
irBCM was significantly greater than in normal ones (Tables 1 and
2). High basal irBCM in those infants might be because of any
disturbances in degradation and elimination of food-born pep-
tides, especially CM. Prolonged circulation of bovine CM in high
Fig. 3. Gel filtration analysis of bovine b-casomorphin-7 immunoreactive material
from blood plasma of formula-fed infants. Elution profile was obtained by HPLC on
Protein Pak-60 column and shown as 226 nm absorbance and immunoreactivity of
corresponding fractions. Arrows indicate peak elution position for BSA, b-casein,
synthetic b-casomorphin-7 and synthetic hexapeptide dalargin.
Fig. 4. Reverse phase chromatography of bovineb-casomorphin-7 immunoreactive
material from blood plasma of formula-fed infants. Elution profile was obtained by
HPLC on ProntoSIL-120-5-C18 column and shown as 226 nm absorbance and
immunoreactivityof correspondingfractions. Arrowindicatespeakelutionposition
for synthetic b-casomorphin-7.
N.V. Kost et al./Peptides 30 (2009) 1854–1860
Author's personal copy
concentration could induce pathological reactions that entail a
delay in development. For comparison, a study in animal has
shown that b-CM-5 at a low dose could ameliorate impaired
memory function in mice, while at high dose it induces amnesia
It should be noted that hyperpeptidemia is considered to be
involved in pathogenesis of autistic spectrum disorders in children
[6,30]. Moreover, of other dietary protein-derived peptides bovine
CM and gliadorphins are supposed to be the most pathophysio-
logically important in autism [30,36]. However, the data on direct
identification of these peptides in any samples from children with
this disease are very inconsistent. Chromatographic analysis of
urine samples from the patients with autism spectrum disorders
showed apparent peptiduria and presence of material with the
same retention time as bovine CM, and irBCM [1,31], while mass
spectrometry failed to detect CM in the urine of both healthy and
autistic children [7,8].
The explanation of opposite effects of human and bovine CM on
infants’ psychomotor development and muscle tone probably lay in
their species-specificity. Indeed, the structure of bovine and human
b-casein is matched only at 47% and the sequence of bovine and
human CMs differs in 2 amino acids. This likely defines a difference
in their biological properties. Both human and bovine CMs are
shown to interact with opioid  and serotonin [38,39] receptors
which are known to be of great importance for CNS maturation
[22,43], but bovine CM has higher affinity to m- and d-opioid
receptors  and also to 5-HT2-serotonin receptors  versus
humanCM. Therefore under durable circulation inorganism bovine
CM more likely provokes desensitization of those receptors than
human CM does. The data on desensitization of guinea pig heartb-
adrenoceptors in the presence of b-CM-5 in relatively low
concentrations  circumstantially support the hypothesis.
This is the first study that identified substances with immunor-
infants under 1 year of age who were fed with breast milk and
formula containing cow milk, respectively. Chromatographic
analysis of the material with irBCM has demonstrated that it has
the same molecular mass and polarity as synthetic bovine b-CM-7.
levels 3 h after feeding was detected mainly in infants of 3 and less
months old. The greatest basal irHCM was revealed in breastfed
infants with normal psychomotor development and muscle tone. In
contrast, elevated basal irBCM was found in formula-fed infants
tone, while among formula-fed infants with normal development
support known opinion that the breast feeding has an advantage
bovine CM elimination as a risk factor for delay in psychomotor
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