Extracranial sources of S100B do not affect serum levels

Abstract

S100B, established as prevalent protein of the central nervous system, is a peripheral biomarker for blood-brain barrier disruption and often also a marker of brain injury. However, reports of extracranial sources of S100B, especially from adipose tissue, may confound its interpretation in the clinical setting. The objective of this study was to characterize the tissue specificity of S100B and assess how extracranial sources of S100B affect serum levels. The extracranial sources of S100B were determined by analyzing nine different types of human tissues by ELISA and Western blot. In addition, brain and adipose tissue were further analyzed by mass spectrometry. A study of 200 subjects was undertaken to determine the relationship between body mass index (BMI) and S100B serum levels. We also measured the levels of S100B homo- and heterodimers in serum quantitatively after blood-brain barrier disruption. Analysis of human tissues by ELISA and Western blot revealed variable levels of S100B expression. By ELISA, brain tissue expressed the highest S100B levels. Similarly, Western blot measurements revealed that brain tissue expressed high levels of S100B but comparable levels were found in skeletal muscle. Mass spectrometry of brain and adipose tissue confirmed the presence of S100B but also revealed the presence of S100A1. The analysis of 200 subjects revealed no statistically significant relationship between BMI and S100B levels. The main species of S100B released from the brain was the B-B homodimer. Our results show that extracranial sources of S100B do not affect serum levels. Thus, the diagnostic value of S100B and its negative predictive value in neurological diseases in intact subjects (without traumatic brain or bodily injury from accident or surgery) are not compromised in the clinical setting.

Figure 1. S100B Antibody Comparison.

Twelve different types of human tissues were assessed for S100B expression by two different antibodies by Western blot. The OriGene monoclonal antibody was made by immunizing against a synthetic peptide corresponding to residues on the C-terminus of human S100B. The polyclonal Sangtec antibody was raised against the whole human protein. (A) shows the tissue specific expression level of S100B using the Sangtec-Diasorin antibody and (B) OriGene antibody after Western blot analysis. Regardless of the antibody used, S100B was found in tissues other than brain. (C) We quantified and compared the results of the two Western blots obtained by the two different antibodies as well as (D) this data normalized to brain tissue. The rank order of S100B expression is different depending on the antibody used.

Figure 2. Extracranial Detection of S100B.

Extracranial sources of S100B revealed on Western blots do not affect the clinical detection of S100B using the Sangtec-Diasorin immunoassay. (A) The results of two clinically relevant Sangtec Diasorin immunoassay systems; the fully automated Liaison (x-axis) was compared with the manual LIA-mat assay kit. There was good correlation between the two systems. (B) A good correlation existed between two automated (Elecsys by Roche Diagnostics and Liaison by DiaSorin) immunoassays for S100B. (C) The same human tissue protein extracts used for Western blotting previously were analyzed with the Sangtec-Diasorin immunoassay. In contrast to what we found in Western blots, the brain is the main chief expressor detected by this method.

Figure 3. BBB Opening: Comparison of Various Forms of S100B.

The main species of S100B released from BBB disruption is the B-B dimer as measured using the CanAg/Fujirebio ELISA system. The rise in total S100B after BBB opening was 0.011 ng/ml, which was found to be accounted for by the concomitant rise of the B-B dimer. There was virtually no change in the concentration of the A1-B dimer with BBB opening. We determined the concentration of the A1-B dimer by using the CanAg S100A1B EIA solid-phase, two-step, non-competitive immunoassay based on two mouse monoclonal antibodies specific for two different epitopes specifically expressed in S100A1B. The assay thus determines S100A1B with very low cross-reactivity with S100BB or other forms of S100. Similarly, to measure the B-B dimer we used the CanAg S100BB EIA solid-phase, one-step, non-competitive immunoassay based on two mouse monoclonal antibodies specific for two different epitopes specifically expressed in S100BB. This assay thus determines S100BB with very low cross-reactivity with S100A1B or other forms of S100.

Figure 4. Correlation Between BMI and S100B.

Given the expression of S100B in adipocytes, we investigated the relationship between fat content and S100B levels using 200 subjects. No correlation was found between BMI and S100B levels.