- Email: [email protected]
Human milk contains S100B protein
Biochimica et Biophysica Acta 1619 (2003) 209 – 212 www.bba-direct.com
Human milk contains S100B protein Diego Gazzolo a, Giovanni Monego b, Valentina Corvino b, Matteo Bruschettini a, Pierluigi Bruschettini a, Giovanni Zelano b, Fabrizio Michetti b,* a
Department of Pediatrics, Giannina Gaslini Children’s University Hospital, I-16147, Genoa, Italy b Institute of Anatomy, Catholic University, Largo Francesco Vito, 1, I-00168, Rome, Italy Received 15 July 2002; received in revised form 1 November 2002; accepted 6 November 2002
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. D 2002 Elsevier Science B.V. All rights reserved. Keywords: S100B protein; Newborn; Human milk
1. 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 extranervous 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 – 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.
2. 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 Gaslini Children’s Hospital, Genoa University, and the parents of the subjects examined gave informed consent. 2.1. S100B measurements
* Corresponding author. Tel.: +39-630-155-848; fax: +39-630-154813. E-mail address: [email protected] (F. Michetti).
Milk was collected and samples were immediately centrifuged at 900 g for 10 min, and the supernatants stored
0304-4165/02/$ - see front matter D 2002 Elsevier Science B.V. All rights reserved. doi:10.1016/S0304-4165(02)00499-3
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D. Gazzolo et al. / Biochimica et Biophysica Acta 1619 (2003) 209–212
ity-purified antirabbit IgG made in goat (Vector Laboratories, Burlingame, CA) diluted in 1:300 for 60 min at RT and Vectastein Elite ABC reagent (Vector Laboratories) diluted 1:300 for 30 min at RT. The paper was then incubated at RT with diaminobenzidine (Peroxidase substrate kit DAB; Vector Laboratories) to visualize antibody reactivity. 2.3. RT-PCR analysis
Fig. 1. Western blot analysis of human milk. Human milk blotted onto nitrocellulose paper and reacted with anti-S100B antiserum.
at 70jC before measurement. The S100B protein concentration was measured in all samples, using a commercially available immunoluminometric assay (Lia-mat Sangtec 100, AB Sangtec Medical, Bromma, Sweden). This assay is specific for the h subunit of the protein. Each measurement was performed in duplicate according to the manufacturer’s recommendations and the averages were reported. The limit of sensitivity of the assay was 0.02 Ag/l. The S100B levels in the milk are expressed as mean F S.D.
RNA was extracted from 10 ml of human milk using the ‘‘micro-to-midi total RNA purification system’’ (InvitrogenLife Technologies). After initial centrifugation of the sample at 900 g for 10 min, the cell-pellet was processed according to the manufacturer’s RNA extraction protocol. Reverse transcription (RT) was performed on 2 Ag of human milk RNA and on 2 Ag of RNA from human brain as positive control. The final volume reaction was 40 Al and the enzyme employed was the SUPERSCRIPT II (Invitrogen-Life Technologies) reverse transcriptase according to the manufacturer’s protocol and RT conditions (50 min at 42jC and 15 min at 72jC). The PCR assay was performed on 2 Al of cDNA of each sample in 25 Al of final volume. The reaction conditions for S100B were 10 pmol of each primer, 200 AM of each dNTP, 2.5 units of Taq polymerase (AmpliTaq Gold DNA Polymerase, Applied Biosystems), 2.2 mM MgCl2 and 1 PCR buffer I (Applied Biosystems). The primers used were 5V-CATTTCTTAGAGGAAATC3V(sense) and 5V-ATGTTCAAAGAACTCGTG-3V(anti(antisense). These primers are specific for human S100B mRNA as reported by Riol et al. [11]. Amplification conditions were 95jC for 9 min followed by 43 cycles at 95jC for 15 s, cycles at 46jC for 15 s, and cycles at 72jC for 30 s, followed by final extension at 72jC for 5 min.
2.2. Western blot analysis Western blotting for detection of S100B protein was performed as described by Van Eldik and Wolchok [10]. Twenty microliters of human milk (900 g supernatant) was added to the gels and the proteins were separated by SDS-polyacrylamide gel electrophoresis (15% acrylamide w/v) and electrophoretically transferred to nitrocellulose paper (100 W for 60 min at 4jC). After transfer, the nitrocellulose was rinsed briefly in PBS and incubated in 0.2% (v/v) glutaraldehyde in PBS for 45 min at room temperature (RT) to enhance the retention of proteins on the nitrocellulose. The unreacted sites were blocked in PBS containing BSA 2% (w/v), gelatine 0.1% (w/v) and 0.1% (v/ v) Tween 20 for 60 min at RT. The nitrocellulose was then incubated sequentially in solutions containing primary rabbit polyclonal anti-S100B antiserum 1:2000 (Dako, Glostrup, Denmark) for 60 min at RT, biotinylated affin-
Fig. 2. RT-PCR amplified fragments of S100B mRNA from human brain and milk. M = pUC Mix Marker 8.
D. Gazzolo et al. / Biochimica et Biophysica Acta 1619 (2003) 209–212
As mentioned above, the positive control for RT-PCR was human RNA obtained from a human brain obtained at autopsy. A negative control (cDNA replaced with H2O) was also used to detect possible contamination. The amplification products were examined by electrophoresis on 1.7% agarose gel stained with ethidium bromide. All the samples were analyzed in triplicate. The amplicon expected was a 147-bp band and was matched with pUC Mix Marker 8 (MBI Fermentas). To confirm the specificity of amplification products, the amplicons were digested with HaeIII enzyme (MBI Fermentas), which has a unique restriction site within the S100B cDNA fragment. The digested fragments were analyzed on 2.5% agarose gel stained with ethidium bromide.
3. 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 Ag/l (median 116.2 Ag/l). Western blot analysis of milk samples (Fig. 1) using an antiS100B rabbit antiserum revealed a major band that migrated with an apparent molecular mass comparable to that of purified ox brain S100B (lower than 14,400 kDa). The RT-PCR analysis produced the 147-bp S100B amplicon starting from milk RNA and from brain RNA (Fig. 2). Positive PCR products were subjected to restriction
Fig. 3. Restriction enzyme digestion of RT-PCR amplicons of S100B mRNAs from human brain and milk with HaeIII (u = undigested, d = digested). The whole fragment is 147 bp long, whereas the digested fragment is 100 bp long. M = pUC Mix Marker 8.
211
enzyme digestion and subsequent gel electrophoresis, which showed the expected bands of 100 and 47 bp, respectively, confirming the presence of a HaeIII restriction site in amplified fragments (Fig. 3).
4. 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 abundant, is not surprising, in the light of the consideration that other calcium-binding proteins (e.g. alpha-lactalbumin, calmodulin, osteocalcin) [12 –14] have already been detected in milk. It is interesting that 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 [3– 7]. It is tempting to correlate the presence of a high concentration of S100B in breast milk with its putative neurotrophic role, because breast-feeding is believed to exert a stimulating effect on brain maturation [8,9]. More detailed information on the fate of the S100B molecule in the gastrointestinal tract is also needed to corroborate the possibility that S100B may participate in the nutritional effects of milk, including a role in brain development. Similarly, no information is currently available to support the hypothesis of a trophic role for the protein in intestinal development. However, a trophic role for S100B in milk would not be surprising, given that human breast milk is known to contain a variety of substances that may actively influence the growth and development of the infant, including hormones, growth factors and cytokines [8,15] through which biochemical communication between mother and child is established [16,17]. Human milk is known to contain cell types expressing S100B protein, including mammary epithelial cells and lymphocytes [18], which may reasonably be supposed to be the sources of S100B mRNA, detected by RT-PCR and of S100B protein detected by immunoluminometric and immunoblotting assays. It is also reasonable to suppose that a significant part of the S100B protein present in milk is secreted by mammary epithelial cells, which are known to express the protein [18], taking into consideration the ascertained extracellular role of the protein and its high concentration in human milk. However, whereas fat cells are a site of concentration for S100B [19], lipids are known to be present in milk only as membrane-surrounded globules secreted by mammary epithelial cells [20]. In conclusion, the presence of S100B in human milk adds to our knowledge of the possible biological roles of
212
D. Gazzolo et al. / Biochimica et Biophysica Acta 1619 (2003) 209–212
this protein, also suggesting the possibility, which for the present is only a matter of speculation, that S100B may contribute to biochemical communications between mother and child.
[8] [9]
Acknowledgements This work was partially supported by Grants from Universita` 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.
[10]
[11]
[12] [13]
References [1] C.W. Heizmann, Ca2 +-binding S100 proteins in the central nervous system, Neurochem. Res. 24 (1999) 1097 – 1100. [2] J.E. Zuckerman, H.R. Herschman, L. Levine, Appearance of a brain specific antigen (th S-100 protein) during human foetal development, J. Neurochem. 17 (1970) 247 – 251. [3] D. Gazzolo, P. Vinesi, E. Marinoni, R. Di Iorio, M. Marras, M. Lituania, P. Bruschettini, F. Michetti, S100B protein concentrations in cord blood: correlations with gestational age in term and preterm deliveries, Clin. Chem. 46 (2000) 998 – 1000. [4] D. Gazzolo, E. Marinoni, R. Di Iorio, M. Lituania, P.L. Bruschettini, F. Michetti, Circulating S100h is increased in intrauterine growthretarded fetuses, Pediatr. Res. 51 (2002) 215 – 219. [5] F. Nygaard, B. Langbakk, B. Romner, 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. 43 (1997) 541 – 543. [6] D. Gazzolo, M. Bruschettini, V. Corvino, R. Oliva, R. Sarli, M. Lituania, P. Bruschettini, F. Michetti, S100B protein concentrations in amniotic fluid correlate with gestational age and cerebral ultrasound scanning results in healthy fetuses, Clin. Chem. 47 (2001) 954 – 956. [7] D. Gazzolo, M. Bruschettini, M. Lituania, G. Serra, E. Gandullia, F.
[14] [15]
[16] [17]
[18]
[19]
[20]
Michetti, S100B protein concentrations in urine are correlated with gestational age in healthy preterm and term newborns, Clin. Chem. 47 (2001) 1132 – 1133. N. Gordon, Nutrition and cognitive function, Brain Dev. 19 (1997) 165 – 170. S.B. Amin, K.S. Merle, M.S. Orlando, L.E. Dalzell, R. Guillet, Brainstem maturation in preterm infants as a function of enteral feeding type, Pediatrics 106 (2000) 318 – 322. L.J. Van Eldik, S.R. Wolchok, Conditions for reproducible detection of calmodulin and S100h in immunoblots, Biochem. Biophys. Res. Commun. 124 (1984) 752 – 759. H. Riol, M. Tardy, B. Rolland, G. Le`vesque, M.R. Ven Murthy, Detection of the peripheral nervous system (PNS)-type glial fibrillary acidic protein (GFAP) and its mRNA in human lymphocytes, J. Neurosci. Res. 48 (1997) 53 – 62. B. Lonnerdal, C. Glazier, Calcium binding by alpha-lactalbumin in human milk and bovine milk, J. Nutr. 115 (1985) 1209 – 1216. S. MacNeil, R.A. Dawson, G. Crocker, C.H. Barton, L. Hanford, R. Metcalfe, M. McGurk, D.S. Munro, Extracellular calmodulin and its association with epidermal growth factor in normal human body fluids, J. Endocrinol. 118 (1988) 501 – 509. W.B. Pittard III, K.M. Goddis, B.W. Hollis, Osteocalcin and human milk, Biol. Neonate 63 (1993) 61 – 63. A.S. Goldman, S. Chheda, R. Garofalo, F.C. Schmalstieg, Cytokines in human milk: properties and potential effects upon the mammary gland and the neonate, J. Mammary Gland Biol. Neoplasia 1 (1996) 251 – 258. K.M. Bernt, W.A. Walker, Human milk as a carrier of biochemical messages, Acta Paediatr. Suppl. 88 (1999) 27 – 41. A. Donnet-Hughes, N. Duc, P. Serrant, K. Vidal, E.J. Schffrin, Bioactive molecules in milk and their role in health and disease: the role of transforming growth factor-beta, Immunol. Cell Biol. 78 (2000) 74 – 79. H. Haimoto, S. Hosoda, K. Kato, Differential distribution of immunoreactive S100-a and S100-h proteins in normal nonnervous human tissues, Lab. Invest. 57 (1987) 489 – 498. F. Michetti, E. Dell’Anna, G. Tiberio, D. Cocchia, Immunochemical and immunocytochemical study of S100 protein in rat adipocytes, Brain Res. 262 (1983) 352 – 356. I.H. Mather, T.W. Keenan, Origin and secretion of milk lipids, J. Mammary Gland Biol. Neoplasia 3 (1998) 259 – 273.
Human milk contains S100B protein Diego Gazzolo a, Giovanni Monego b, Valentina Corvino b, Matteo Bruschettini a, Pierluigi Bruschettini a, Giovanni Zelano b, Fabrizio Michetti b,* a
Department of Pediatrics, Giannina Gaslini Children’s University Hospital, I-16147, Genoa, Italy b Institute of Anatomy, Catholic University, Largo Francesco Vito, 1, I-00168, Rome, Italy Received 15 July 2002; received in revised form 1 November 2002; accepted 6 November 2002
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. D 2002 Elsevier Science B.V. All rights reserved. Keywords: S100B protein; Newborn; Human milk
1. 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 extranervous 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 – 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.
2. 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 Gaslini Children’s Hospital, Genoa University, and the parents of the subjects examined gave informed consent. 2.1. S100B measurements
* Corresponding author. Tel.: +39-630-155-848; fax: +39-630-154813. E-mail address: [email protected] (F. Michetti).
Milk was collected and samples were immediately centrifuged at 900 g for 10 min, and the supernatants stored
0304-4165/02/$ - see front matter D 2002 Elsevier Science B.V. All rights reserved. doi:10.1016/S0304-4165(02)00499-3
210
D. Gazzolo et al. / Biochimica et Biophysica Acta 1619 (2003) 209–212
ity-purified antirabbit IgG made in goat (Vector Laboratories, Burlingame, CA) diluted in 1:300 for 60 min at RT and Vectastein Elite ABC reagent (Vector Laboratories) diluted 1:300 for 30 min at RT. The paper was then incubated at RT with diaminobenzidine (Peroxidase substrate kit DAB; Vector Laboratories) to visualize antibody reactivity. 2.3. RT-PCR analysis
Fig. 1. Western blot analysis of human milk. Human milk blotted onto nitrocellulose paper and reacted with anti-S100B antiserum.
at 70jC before measurement. The S100B protein concentration was measured in all samples, using a commercially available immunoluminometric assay (Lia-mat Sangtec 100, AB Sangtec Medical, Bromma, Sweden). This assay is specific for the h subunit of the protein. Each measurement was performed in duplicate according to the manufacturer’s recommendations and the averages were reported. The limit of sensitivity of the assay was 0.02 Ag/l. The S100B levels in the milk are expressed as mean F S.D.
RNA was extracted from 10 ml of human milk using the ‘‘micro-to-midi total RNA purification system’’ (InvitrogenLife Technologies). After initial centrifugation of the sample at 900 g for 10 min, the cell-pellet was processed according to the manufacturer’s RNA extraction protocol. Reverse transcription (RT) was performed on 2 Ag of human milk RNA and on 2 Ag of RNA from human brain as positive control. The final volume reaction was 40 Al and the enzyme employed was the SUPERSCRIPT II (Invitrogen-Life Technologies) reverse transcriptase according to the manufacturer’s protocol and RT conditions (50 min at 42jC and 15 min at 72jC). The PCR assay was performed on 2 Al of cDNA of each sample in 25 Al of final volume. The reaction conditions for S100B were 10 pmol of each primer, 200 AM of each dNTP, 2.5 units of Taq polymerase (AmpliTaq Gold DNA Polymerase, Applied Biosystems), 2.2 mM MgCl2 and 1 PCR buffer I (Applied Biosystems). The primers used were 5V-CATTTCTTAGAGGAAATC3V(sense) and 5V-ATGTTCAAAGAACTCGTG-3V(anti(antisense). These primers are specific for human S100B mRNA as reported by Riol et al. [11]. Amplification conditions were 95jC for 9 min followed by 43 cycles at 95jC for 15 s, cycles at 46jC for 15 s, and cycles at 72jC for 30 s, followed by final extension at 72jC for 5 min.
2.2. Western blot analysis Western blotting for detection of S100B protein was performed as described by Van Eldik and Wolchok [10]. Twenty microliters of human milk (900 g supernatant) was added to the gels and the proteins were separated by SDS-polyacrylamide gel electrophoresis (15% acrylamide w/v) and electrophoretically transferred to nitrocellulose paper (100 W for 60 min at 4jC). After transfer, the nitrocellulose was rinsed briefly in PBS and incubated in 0.2% (v/v) glutaraldehyde in PBS for 45 min at room temperature (RT) to enhance the retention of proteins on the nitrocellulose. The unreacted sites were blocked in PBS containing BSA 2% (w/v), gelatine 0.1% (w/v) and 0.1% (v/ v) Tween 20 for 60 min at RT. The nitrocellulose was then incubated sequentially in solutions containing primary rabbit polyclonal anti-S100B antiserum 1:2000 (Dako, Glostrup, Denmark) for 60 min at RT, biotinylated affin-
Fig. 2. RT-PCR amplified fragments of S100B mRNA from human brain and milk. M = pUC Mix Marker 8.
D. Gazzolo et al. / Biochimica et Biophysica Acta 1619 (2003) 209–212
As mentioned above, the positive control for RT-PCR was human RNA obtained from a human brain obtained at autopsy. A negative control (cDNA replaced with H2O) was also used to detect possible contamination. The amplification products were examined by electrophoresis on 1.7% agarose gel stained with ethidium bromide. All the samples were analyzed in triplicate. The amplicon expected was a 147-bp band and was matched with pUC Mix Marker 8 (MBI Fermentas). To confirm the specificity of amplification products, the amplicons were digested with HaeIII enzyme (MBI Fermentas), which has a unique restriction site within the S100B cDNA fragment. The digested fragments were analyzed on 2.5% agarose gel stained with ethidium bromide.
3. 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 Ag/l (median 116.2 Ag/l). Western blot analysis of milk samples (Fig. 1) using an antiS100B rabbit antiserum revealed a major band that migrated with an apparent molecular mass comparable to that of purified ox brain S100B (lower than 14,400 kDa). The RT-PCR analysis produced the 147-bp S100B amplicon starting from milk RNA and from brain RNA (Fig. 2). Positive PCR products were subjected to restriction
Fig. 3. Restriction enzyme digestion of RT-PCR amplicons of S100B mRNAs from human brain and milk with HaeIII (u = undigested, d = digested). The whole fragment is 147 bp long, whereas the digested fragment is 100 bp long. M = pUC Mix Marker 8.
211
enzyme digestion and subsequent gel electrophoresis, which showed the expected bands of 100 and 47 bp, respectively, confirming the presence of a HaeIII restriction site in amplified fragments (Fig. 3).
4. 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 abundant, is not surprising, in the light of the consideration that other calcium-binding proteins (e.g. alpha-lactalbumin, calmodulin, osteocalcin) [12 –14] have already been detected in milk. It is interesting that 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 [3– 7]. It is tempting to correlate the presence of a high concentration of S100B in breast milk with its putative neurotrophic role, because breast-feeding is believed to exert a stimulating effect on brain maturation [8,9]. More detailed information on the fate of the S100B molecule in the gastrointestinal tract is also needed to corroborate the possibility that S100B may participate in the nutritional effects of milk, including a role in brain development. Similarly, no information is currently available to support the hypothesis of a trophic role for the protein in intestinal development. However, a trophic role for S100B in milk would not be surprising, given that human breast milk is known to contain a variety of substances that may actively influence the growth and development of the infant, including hormones, growth factors and cytokines [8,15] through which biochemical communication between mother and child is established [16,17]. Human milk is known to contain cell types expressing S100B protein, including mammary epithelial cells and lymphocytes [18], which may reasonably be supposed to be the sources of S100B mRNA, detected by RT-PCR and of S100B protein detected by immunoluminometric and immunoblotting assays. It is also reasonable to suppose that a significant part of the S100B protein present in milk is secreted by mammary epithelial cells, which are known to express the protein [18], taking into consideration the ascertained extracellular role of the protein and its high concentration in human milk. However, whereas fat cells are a site of concentration for S100B [19], lipids are known to be present in milk only as membrane-surrounded globules secreted by mammary epithelial cells [20]. In conclusion, the presence of S100B in human milk adds to our knowledge of the possible biological roles of
212
D. Gazzolo et al. / Biochimica et Biophysica Acta 1619 (2003) 209–212
this protein, also suggesting the possibility, which for the present is only a matter of speculation, that S100B may contribute to biochemical communications between mother and child.
[8] [9]
Acknowledgements This work was partially supported by Grants from Universita` 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.
[10]
[11]
[12] [13]
References [1] C.W. Heizmann, Ca2 +-binding S100 proteins in the central nervous system, Neurochem. Res. 24 (1999) 1097 – 1100. [2] J.E. Zuckerman, H.R. Herschman, L. Levine, Appearance of a brain specific antigen (th S-100 protein) during human foetal development, J. Neurochem. 17 (1970) 247 – 251. [3] D. Gazzolo, P. Vinesi, E. Marinoni, R. Di Iorio, M. Marras, M. Lituania, P. Bruschettini, F. Michetti, S100B protein concentrations in cord blood: correlations with gestational age in term and preterm deliveries, Clin. Chem. 46 (2000) 998 – 1000. [4] D. Gazzolo, E. Marinoni, R. Di Iorio, M. Lituania, P.L. Bruschettini, F. Michetti, Circulating S100h is increased in intrauterine growthretarded fetuses, Pediatr. Res. 51 (2002) 215 – 219. [5] F. Nygaard, B. Langbakk, B. Romner, 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. 43 (1997) 541 – 543. [6] D. Gazzolo, M. Bruschettini, V. Corvino, R. Oliva, R. Sarli, M. Lituania, P. Bruschettini, F. Michetti, S100B protein concentrations in amniotic fluid correlate with gestational age and cerebral ultrasound scanning results in healthy fetuses, Clin. Chem. 47 (2001) 954 – 956. [7] D. Gazzolo, M. Bruschettini, M. Lituania, G. Serra, E. Gandullia, F.
[14] [15]
[16] [17]
[18]
[19]
[20]
Michetti, S100B protein concentrations in urine are correlated with gestational age in healthy preterm and term newborns, Clin. Chem. 47 (2001) 1132 – 1133. N. Gordon, Nutrition and cognitive function, Brain Dev. 19 (1997) 165 – 170. S.B. Amin, K.S. Merle, M.S. Orlando, L.E. Dalzell, R. Guillet, Brainstem maturation in preterm infants as a function of enteral feeding type, Pediatrics 106 (2000) 318 – 322. L.J. Van Eldik, S.R. Wolchok, Conditions for reproducible detection of calmodulin and S100h in immunoblots, Biochem. Biophys. Res. Commun. 124 (1984) 752 – 759. H. Riol, M. Tardy, B. Rolland, G. Le`vesque, M.R. Ven Murthy, Detection of the peripheral nervous system (PNS)-type glial fibrillary acidic protein (GFAP) and its mRNA in human lymphocytes, J. Neurosci. Res. 48 (1997) 53 – 62. B. Lonnerdal, C. Glazier, Calcium binding by alpha-lactalbumin in human milk and bovine milk, J. Nutr. 115 (1985) 1209 – 1216. S. MacNeil, R.A. Dawson, G. Crocker, C.H. Barton, L. Hanford, R. Metcalfe, M. McGurk, D.S. Munro, Extracellular calmodulin and its association with epidermal growth factor in normal human body fluids, J. Endocrinol. 118 (1988) 501 – 509. W.B. Pittard III, K.M. Goddis, B.W. Hollis, Osteocalcin and human milk, Biol. Neonate 63 (1993) 61 – 63. A.S. Goldman, S. Chheda, R. Garofalo, F.C. Schmalstieg, Cytokines in human milk: properties and potential effects upon the mammary gland and the neonate, J. Mammary Gland Biol. Neoplasia 1 (1996) 251 – 258. K.M. Bernt, W.A. Walker, Human milk as a carrier of biochemical messages, Acta Paediatr. Suppl. 88 (1999) 27 – 41. A. Donnet-Hughes, N. Duc, P. Serrant, K. Vidal, E.J. Schffrin, Bioactive molecules in milk and their role in health and disease: the role of transforming growth factor-beta, Immunol. Cell Biol. 78 (2000) 74 – 79. H. Haimoto, S. Hosoda, K. Kato, Differential distribution of immunoreactive S100-a and S100-h proteins in normal nonnervous human tissues, Lab. Invest. 57 (1987) 489 – 498. F. Michetti, E. Dell’Anna, G. Tiberio, D. Cocchia, Immunochemical and immunocytochemical study of S100 protein in rat adipocytes, Brain Res. 262 (1983) 352 – 356. I.H. Mather, T.W. Keenan, Origin and secretion of milk lipids, J. Mammary Gland Biol. Neoplasia 3 (1998) 259 – 273.