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Technical Briefs |
1 Departamento de Bioquímica, ICBS, UFRGS, Porto Alegre, RS, 90035-003 Brazil
2 Laboratório Municipal de Novo Hamburgo, RS, 93310-003 Brazil
3 Departamento de Psiquiatria do Hospital de Clínicas de Porto Alegre, Porto Alegre, RS, 90035-003 Brazil
4 Centro de Cirurgia de Epilepsia, CIREP, Hospital de Clínicas, FMRP-USP, Campus Universitário, Ribeirão Preto, SP, 14048-900 Brazil
5 Laboratório de Patologia Clínica, HSL-PUCRS, Porto Alegre, RS, 91530-001 Brazil
6 Serviço de Hemoterapia, Hemosul, Cachoeira do Sul, RS, 96508-970 Brazil
aaddress correspondence to this author at: Departamento de Bioquímica, ICBS, UFRGS, Rua Ramiro Barcelos 2600-Anexo, Porto Alegre, RS, 90035-003 Brazil; e-mail diogo{at}ufrgs.br
S100B is a 21-kDa calcium-binding protein produced and released primarily by astrocytes in the central nervous system (CNS), where it exerts neurotropic and gliotropic actions (1). Several studies have investigated the potential role of S100B as a peripheral biochemical marker of neural injury, including reactive gliosis, astrocytic death, and/or blood-brain barrier dysfunction (2). Increased cerebrospinal fluid (CSF) and/or serum S100B has been reported in several acute and chronic injuries, including traumatic brain injury (3), stroke (4), Alzheimer disease (5), schizophrenia (6), HTLV-I-associated myelopathy (7), and systemic lupus erythematosus (8).
Previous neurodevelopmental studies have demonstrated age-related changes in S100B tissue expression and distribution in the CNS in mammals, which may be related to the different roles of this protein in distinct brain regions during the fetal period, adulthood, and aging (1)(9). Furthermore, S100B concentrations in CSF have been noted to increase with age in healthy individuals (10). Because these increases are more evident in men than women (11), S100B concentrations in CSF may be sex and age dependent, suggesting that age- and sex-matched controls may be necessary for studies of S100B in CSF. To determine the usual concentrations for plasma S100B in adults, Wiesmann et al. (12) measured the protein in healthy blood donors and found no differences with sex and age in individuals 18–65 years of age, in agreement with our findings in previous studies in adults (6)(7)(8).
To our knowledge, however, S100B concentrations in blood in childhood and adolescence were not established until recently. The present study was performed to verify S100B serum concentrations in healthy individuals of different ages, ranging from neonates to adults.
We collected blood samples from 19 healthy term neonates (<48 h of age) and from 25 healthy children 4–16 years of age undergoing routine clinical and laboratory evaluation. These children had no previous history of neurologic deficits or any other serious disorder, and their blood samples revealed no abnormal findings. Samples were also obtained from 85 healthy adults 18–70 years of age. This study was approved by local Ethics Committee of the Hospital de Clínicas de Porto Alegre (RS, Brazil). Informed consent was obtained from the adults and parents.
All blood samples were collected without anticoagulants by venipuncture. Serum was obtained by centrifugation at 3000g for 5 min and, soon thereafter, was frozen at -70 °C until analysis.
Serum S100B was measured using a monoclonal immunoluminometric assay (LIA-mat® Sangtec®100; Sangtec Medical) in a Lumat LB9507 luminometer (EG&G Berthold). Serum samples and calibrators (100 µL of each) were diluted with 100 µL of bovine serum albumin in test tubes provided with the assay. These tubes were already coated with anti-S100B antibody (three monoclonal antibodies: SMST 12, SMSK 25, and SMSK 28). After incubating for 1 h, the tubes were washed three times with wash buffer, and 200 µL of an antibody labeled with isoluminol as a tracer was added to each tube. After an additional 2-h incubation, during which this antibody bound to the immobilized S100B, the unbound tracer was washed out and the residual antibody was measured in a luminometer. S100B concentrations were derived by comparison with the calibration curve based on the total luminescence for each given calibrator provided with the assay. All determinations were carried out within the same experiment. The S100B calibration curve was linear up to 20 µg/L, and the CVs for duplicates across the entire concentration range for the calibrators and samples were <5%. The detection limit of the assay is 0.02 µg/L, as provided by the supplier of the LIA-mat Sangtec100 assay.
Statistical analyses were performed using the Mann–Whitney test for comparisons between sexes and the Spearman correlation coefficient to evaluate correlation between serum S100B and age. Comparisons of serum S100B concentrations among different age groups were performed by Kruskal–Wallis ANOVA, with Mann–Whitney post hoc testing. P <0.05 was considered statistically significant.
No statistically significant difference was noted between serum S100B concentrations in male and female term neonates (age <48 h; P = 0.60), children (4–16 years; P = 0.59), adults (18–70 years; P = 0.18), or in all age groups combined (P = 0.67). We found a negative correlation between S100B and age (Fig. 1
; r = -0.70; P <0.001), with the higher concentrations in neonates, as shown in the inset of Fig. 1
. Moreover, a correlation between age and S100B was not evident in individuals >20 years of age (21–70 years; n = 76; r = -0.05; P = 0.76), but was present in individuals <20 years of age (0–20 years; n = 53; r = -0.87; P <0.001). Table 1
shows the medians and interquartile ranges obtained for the various age groups.
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In agreement with the results obtained by Wiesmann et al. (12), we also found no sex dependence in serum S100B concentrations in adults. In addition, our data revealed no sex-related difference in S100B concentrations in the blood of neonates and children. Importantly, when we compared serum S100B in all individuals with their corresponding age, we found a statistically significant negative correlation (r = -0.70; P <0.001).
Only a few studies have been published on the determination of S100B in the blood of neonates and children (13)(14)(15)(16). Although these previous studies did not attempt to correlate S100B concentrations in the blood with age, their results suggested that the baseline blood concentrations in neonates and young infants could be higher than in adults. Our results suggest that S100B concentrations in term neonates are much higher than in older individuals. It is worth pointing out that bilirubin up to 150 mg/L did not interfere with the S100B assay (data not shown). On the basis of our data, blood S100B concentrations appear to decrease considerably in the first two decades of life (r = -0.87; P <0.001) and remain relatively constant during adulthood (r = -0.05; P = 0.76), which is in accordance with the results obtained by Wiesmann et al. (12).
Several theories can be offered to explain the results obtained in our study. The higher concentrations of serum S100B protein found in neonates and children compared with adults could reflect the ongoing central neurodevelopmental processes occurring during these different stages of life (9). The proliferation and maturation of glial cells, extension of neurites, and formation of synapses have been documented to be the most important functions of S100B in morphogenesis (1)(17)(18), possibly involving mitogen-activated protein kinase (19) and activation of receptors for advanced glycation end products in neurons (20). It is also interesting to note that some nerve fibers are positive for S100B during development, implicating a possible role for S100B in regulating the activity of fiber sprouting (9). Therefore, S100B, like other glial-derived cytokines, may be involved in coordinating the development and maintenance of the CNS by synchronously stimulating the differentiation of neurons and the proliferation of astroglia.
Functional maturation of the human brain has been studied with positron emission tomography, and the regional or local cerebral metabolic rates of glucose utilization (LCMRglc) have been used as indirect measurement of synaptogenesis in the human brain (21)(22). These studies showed that the LCMRglc in the cerebral cortex of children between the ages of 
4 and 9–10 years exceed adult rates by more than twofold. After 9–10 years, LCMRglc for cerebral cortex begin to decline and gradually reach adult values by 16–18 years. In addition, magnetic resonance imaging has shown an increase of gray matter during childhood, which decreases before adulthood (23). From the electrophysiologic point of view, electroencephalographic findings become similar to those in adults early in the second decade of life (24).
The time window of maximal plateau and decline of LCMRglc in cerebral cortex and the consolidation of adult electroencephalographic patterns observed in older children are closely associated with the variations in serum concentrations of S100B in healthy individuals. Considering that the S100B protein exerts tropic effects on neural cells, we believe that it could play an important role during the early phase of human brain development, which is reflected in its serum concentrations.
In conclusion, we have shown a negative correlation between blood S100B and age in the first 20 years of life; after age 20, S100B does not appear to vary with age. Therefore, in studies involving measurement of blood S100B in pediatric and adolescent patients, it is important to establish age-matched reference values. Further work will be required to clarify the role of S100B during neural development.
Acknowledgments
This work was supported by Consello Nacional de Pesquisa (CNPq) and Programa de Apoio a Núcleos de Excelência (PRONEX) No. 41960904.
References
The following articles in journals at HighWire Press have cited this article:
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  J Ambrozic, M Bunc, J Osredkar, and M Brvar S100B protein in benzodiazepine overdose Emerg. Med. J., February 1, 2008; 25(2): 90 - 92. [Abstract] [Full Text] [PDF]
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 C. Foerch, M. T. Wunderlich, F. Dvorak, M. Humpich, T. Kahles, M. Goertler, J. Alvarez-Sabin, C. W. Wallesch, C. A. Molina, H. Steinmetz, et al. Elevated Serum S100B Levels Indicate a Higher Risk of Hemorrhagic Transformation After Thrombolytic Therapy in Acute Stroke Stroke, September 1, 2007; 38(9): 2491 - 2495. [Abstract] [Full Text] [PDF]
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P. M. Bokesch, G. A. Izykenova, J. B. Justice, K. A. Easley, and S. A. Dambinova NMDA Receptor Antibodies Predict Adverse Neurological Outcome After Cardiac Surgery in High-Risk Patients Stroke, June 1, 2006; 37(6): 1432 - 1436. [Abstract] [Full Text] [PDF]
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 A. P. Schmidt, A. B.L. Tort, O. B. Amaral, A. P. Schmidt, R. Walz, J. Vettorazzi-Stuckzynski, S. H. Martins-Costa, J. G. L. Ramos, D. O. Souza, and L. V.C. Portela Serum S100B in Pregnancy-Related Hypertensive Disorders: A Case-Control Study Clin. Chem., February 1, 2004; 50(2): 435 - 438. [Full Text] [PDF]
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D. Gazzolo, F. Michetti, M. Bruschettini, N. Marchese, M. Lituania, S. Mangraviti, E. Pedrazzi, and P. Bruschettini Pediatric Concentrations of S100B Protein in Blood: Age- and Sex-related Changes Clin. Chem., June 1, 2003; 49(6): 967 - 970. [Full Text] [PDF]
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O. Ben Abdesselam, J. Vally, C. Adem, M.-J. Foglietti, and J.-L. Beaudeux Reference Values for Serum S-100B Protein Depend on the Race of Individuals Clin. Chem., May 1, 2003; 49(5): 836 - 837. [Full Text] [PDF]
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, males; 
, females. Inset shows serum S100B in term neonates. A statistically significant negative correlation was observed (r = -0.70; P <0.001). The fitted curve (solid line) was obtained using a third-degree polynomial regression. The dashed line represents the detection limit of the assay (0.02 µg/L).