Human milk contains S100B protein

doi:10.1016/S0304-4165(02)00499-3

Biochimica et Biophysica Acta (BBA) - General Subjects

Volume 1619, Issue 2, 20 January 2003, Pages 209-212

https://doi.org/10.1016/S0304-4165(02)00499-3 Get rights and content

Abstract

The present study constitutes the first finding of the calcium-binding protein S100B and of its mRNA in human milk, as revealed by a quantitative immunoluminometric assay, by Western blot analysis and by reverse transcription-polymerase chain reaction (RT-PCR) assay followed by restriction enzyme digestion. The concentration of S100B in milk is markedly higher than that observed in other biological fluids such as cord blood, peripheral blood, urine, cerebrospinal fluid and amniotic fluid. This finding could be related to a possible trophic role, which has been hypothesized for the protein.

Introduction

S100B is an acidic calcium-binding protein originally isolated from the nervous system, where it is highly concentrated in glial cells, but later detected also in definite extra-nervous cell types [1]. Although many hypotheses have been formulated, the biological role of S100B is still a matter of debate. Among the different functions attributed to this protein, the possibility that it acts as a cytokine with a neurotrophic effect at physiological concentrations, essentially supported in experimental models on laboratory animals and cell cultures, appears interesting [1]. It is relevant, in this respect, that the caudo-rostral pattern of accumulation of the protein has been related to the biochemical, morphological and electrophysiological maturation of the human nervous system [2]. Recently, this hypothesis has also been supported in clinical studies demonstrating a correlation between S100B cord blood levels and physiological events related to brain maturation [3], [4].

The protein is known to be a normal constituent of human biological fluids, including cerebrospinal fluid, blood, urine and amniotic fluid [5], [6], [7]. Human milk is believed to contain biological factors involved in the regulation of newborn growth, including brain development [8], [9]. The present study investigates the presence of S100B in human milk to add to our knowledge regarding its distribution in biological fluids, which could also be useful to clarify the function of the protein.

Section snippets

Materials and methods

Breast milk samples for S100B protein assessment were drawn at day 5 from delivery from 16 women with consecutive singleton physiological pregnancies, whose deliveries were between 37 and 42 weeks' gestation. Exclusion criteria were: multiple pregnancies, gestational hypertension, diabetes and infections, fever, chromosomal abnormalities, metabolic diseases, diseases of the breast or central nervous system, and malnutrition.

The study protocol was approved by the Ethics Committee of the Giannina

Results

All mothers showed normal clinical conditions and no overt neurological injury and/or infections were observed at the sampling time-point or on discharge from the hospital.

All the samples investigated exhibited measurable levels of S100B, ranging from 62 to 212 μg/l (median 116.2 μg/l). Western blot analysis of milk samples (Fig. 1) using an anti-S100B rabbit antiserum revealed a major band that migrated with an apparent molecular mass comparable to that of purified ox brain S100B (lower than

Discussion

The present data constitute the first observation of the presence of S100B protein in breast milk. Western blot analysis confirms that the immunoreactivity observed using immunoluminometric assay reasonably refers to S100B protein and RT-PCR analysis also detects human S100B mRNA in the human milk.

These findings add milk to the list of biological fluids that contain this calcium-binding protein. The presence of a calcium-binding protein in a biological fluid such as milk, in which calcium is

Acknowledgements

This work was partially supported by Grants from Università Cattolica del S. Cuore to Fabrizio Michetti and from “Let's improve neonatal life” Foundation to Diego Gazzolo. We also thank Sangtec Medical, Bromma, Sweden, and Byk Gulden Italia for supplying analysis kits.

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Thus, determination of extracellular S100B level may be useful in diagnosing and monitoring of these diseases. Studies on S100B during pregnancy have shown that the protein is present in placenta, amnion, umbilical cord, fetal membranes, cord blood, amniotic fluid, maternal serum and milk [55,114–117]. In some of these tissues and fluids, such as amnion, amniotic fluid and serum, the level of S100B in preeclamptic patients is significantly higher [118–122].
S100 proteins bind Ca²⁺ and regulate various signaling pathways inside and outside the cell. They are expressed in vertebrates and exhibit tissue and cell specific distribution. Of note, increased level of S100 proteins is observed in different pathologies such as cancers, nervous system diseases/neurodegeneration, inflammation or cardiovascular diseases. Certain S100 proteins can be found in serum and/or other body fluids, at an especially high level in pathological states. Thus, S100 proteins might serve as diagnostic markers in the clinic. Interestingly, expression of many S100 proteins is found to be associated with pregnancy, which suggests that alterations in their expression during pregnancy may regulate processes involved in embryo/fetus formation. In this review we summarize available literature data concerning the expression and possible function of S100 proteins in a disease of pregnant women known as preeclampsia or EPH-gestosis.
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As a rather small protein with a molecular weight of 21 kDa in its biologically active homodimeric form, S100B passes the BBB, and is thus detectable as a brain-derived protein in peripheral blood [29]. S100B has been widely studied in different biological fluids such as CSF, amniotic fluid, blood, urine, saliva and milk [30–39]. More recently, in high-risk pregnancies the protein has been detected in fetal-maternal bloodstreams [40,41].
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The importance of certain neurotrophic proteins found in maternal blood and milk for breastfed infants has remained ambiguous. This study was conducted to present evidence of the impact of an induced deficit of active S100B protein on neonate development. Newborn mice from two groups of mothers, immunized or sham-immunized against S100B, were subjected to various behavioral tests, and the development of their morphological characteristics was recorded from birth until weaning. Morphological problems, including weight gain and fur coating, a delay in the maturation of neurobehavioral systems and a deficit in neuromotor functions, including visual abilities, somato-sensory and posture reactions, muscular strength, locomotion, and fear/orienting processes, were observed in pups of immunized mothers. The S100B protein of external or internal origin in infants may be considered to be a specific factor that determines neuro- and morphological development and a risk-avoidance (‘homeward-bent’ or fearful) phenotype. The suppression of activity of the S100B protein results in a slower neonatal development and the formation of a risk-tolerant (fearless) phenotype of the offspring. This study thus considers the mechanism of neuroplastic regulation on the extent of sensation-seeking or risk-taking (homeless-like or fearless) and sensation- or risk-avoidance (home-bound or fearful) features in individual phenotypes.
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However, there is growing evidence that fatty acid content (PUFA, AA; EPA, DHE, etc.), per se, cannot be considered the only responsible of these strategical properties and the hypothesis that other molecules of extreme interest namely “trophic factors” could be involved in these development processes is to date consistent. These latter include neuro-oxidative stress biomarkers, neurotrophic proteins and calcium binding proteins [8,13–16] that are known to be involved in a cascade of events leading to brain, cardiac and vascular development/damage [8,13–19]. The present findings are also emphasized by the presence in human milk, at different periods of maturation, of highest concentrations of these trophic factors than on other biological fluids [i.e. cerebrospinal (CSF), blood, amniotic, urine] up to 20 times more [8,15,17–19].
Mother milk is widely accepted to be a unique product believed to contain biological factors involved in the regulation of newborn optimal growth including brain when compared to milk-formula milks. In this setting, there is growing evidence that in milk-formula neuro-oxidative stress biomarkers, neurotrophic proteins and calcium binding proteins, known to be involved in a cascade of events leading to brain, cardiac and vascular development/damage, are to date lacking or at a lower concentration than breast milk.
Therefore, this review is aimed at offering additional insights to the role in human milk of some selected biomarkers such as: i) neurotrophic factors such as Activin A; ii) Calcium binding protein such as S100B and, iii) heat shock protein known to be involved in oxidative stress response (namely hemeoxygenase-1, HO-1 or Heat shock Protein 32, HSP32).
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The S100B astroglial protein is widely used as a parameter of glial activation and/or death in several conditions of brain injury. Cerebrospinal fluid and serum S100B variations have been proposed to evaluate clinical outcomes in these situations. Here, we briefly broach some aspects, commonly not sufficiently valorized, concerning the biology and measurements of this protein. S100B has molecular targets and activities in and outside of astrocytes, and variations of intra and extracellular content are not necessarily coupled. We discuss the extracellular origin of this protein in brain tissue, as well as extracerebral sources of this protein in serum, comparing it with other available protein markers of brain damage. The superestimation of the heterodimer S100A1-B in the current clinical literature is also analyzed. We affirm that poor dualistic views that consider S100B elevation as “bad” or “good” simplify clinical practice and delay our comprehension of the role of this protein, both in physiological conditions and in brain disorders.
A simple, sensitive and widely applicable ELISA for S100B: Methodological features of the measurement of this glial protein
2008, Journal of Neuroscience Methods
S100B expression, particularly extracellular S100B, is used as a parameter of glial activation and/or death in several situations of brain injury. Several immunoassays for S100B measurement are available, which differ with regard to specificity, sensitivity, sample application, and, of course, economic costs. We standardized two protocols for S100B measurement (range between 1.9 pg and 10 ng/mL) in human and rat samples from brain and adipose tissues, blood serum, cerebrospinal fluid, urine and cell culture. Abundance and secretion of this protein in adipose tissue reinforces the caution about its origin in blood serum. Interestingly, S100B recognition was affected by the redox status of the protein. This aspect should be considered in S100B measurement, assuming that oxidized and reduced forms possibly coexist in vivo and the equilibrium can be modified by oxidative stress of physiological or pathological conditions or even by obtaining sample conditions.

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View full text

Human milk contains S100B protein

Abstract

Introduction

Section snippets

Materials and methods

Results

Discussion

Acknowledgements

Brain Dev.

Biochem. Biophys. Res. Commun.

J. Nutr.

Brain Res.

Ca2+-binding S100 proteins in the central nervous system

Neurochem. Res.

Appearance of a brain specific antigen (th S-100 protein) during human foetal development

J. Neurochem.

S100B protein concentrations in cord blood: correlations with gestational age in term and preterm deliveries

Clin. Chem.

Circulating S100β is increased in intrauterine growth-retarded fetuses

Pediatr. Res.

Age- and sex-related changes of S100 protein concentrations in cerebrospinal fluid and serum in patients with no previous history of neurological disorders

Clin. Chem.

S100B protein concentrations in amniotic fluid correlate with gestational age and cerebral ultrasound scanning results in healthy fetuses

Clin. Chem.

Ca²⁺-binding S100 proteins in the central nervous system