Plasma S-100b Protein Concentration in Healthy Adults Is Age- and Sex-Independent

¹ Neuroradiology, Klinikum Grosshadern, Ludwig Maximilian University, 81377 Munich, Germany, and
² Immunology and Transfusion Medicine, University of Luebeck, School of Medicine, 2400 Luebeck, Germany;
^a address for correspondence: Abteilung fuer Neuroradiologie, Klinikum Grosshadern, Ludwig-Maximilians-Universitaet, Marchioninistr. 15, 81377 Muenchen, Germany

S-100 protein (S-100) is a calcium-binding protein found predominantly in the cytosol of glial cells in all parts of the central nervous system (CNS). Three different subtypes, designated S-100a, S-100b, and S-100a0, are known. S-100b predominates in the brain.

The concentration of S-100 can be measured in cerebrospinal fluid (CSF) and in blood. For methodological reasons, most studies reported in the literature measured S-100 concentrations in CSF, and an increased concentration of S-100 in the CSF has been found to be a sensitive although nonspecific indicator of nervous system damage in patients with various neurological disorders (1).

Increased concentrations of S-100 have also been found in the blood of patients suffering from CNS tumors or cerebrovascular insults, and maximal concentrations of S-100 in the blood are correlated with the infarct volume after acute ischemic stroke (1)(2)(3)(4)(5)(6). It has recently been reported that concentrations of S-100 in blood may be of prognostic value in patients with minor head injury (7) and may indicate acute exacerbation of multiple sclerosis (8).

Van Engelen et al. (9) reported that S-100 concentrations in CSF increase with age. Nygaard et al. (10) confirmed this and also found a difference in the mean concentration of S-100 in the CSF of male and female subjects. For routine clinical use, these findings would necessitate age- and sex-corrected reference intervals.

We have described a method for determining the concentration of S-100 in blood that is sensitive enough to detect the concentration of S-100 in the plasma of healthy subjects (1). We believe that this method could be useful clinically, but the dependency on age or sex of S-100 concentrations in blood has not been studied yet. We therefore conducted a study to determine whether S-100 concentrations in blood change with age or depend on the individual's sex.

Blood samples were obtained from 200 healthy blood donors between 18 and 65 years of age who had no history of previous neurological deficit or any other serious disorder. Subjects were receiving no medications, and the results of physical examination and routine laboratory tests were normal. The study was in accordance with the current revision of the Helsinki Declaration of 1975, and all subjects gave informed consent to the procedure.

Subjects were grouped according to age: 18–25 years, 26–35 years, 36–45 years, 46–55 years, and 56–65 years. Each group was composed of 40 individuals, 20 men and 20 women. Blood was collected in heparin-containing tubes, centrifuged within 12 h of collection, and stored at -70 °C until analysis.

S-100 concentrations were determined with an immunofluorometric sandwich assay as described earlier (1). In brief, all measurements were set up in duplicate. Microtiter plates coated with 10 µL of anti-S-100 ß-chain (Sigma Chemical) in 20 mL of phosphate buffer (0.05 mol/L, pH 8.6) were incubated with 200 µL per well of S-100 calibrators, controls, or samples for 120 min. Biotin-labeled rabbit anti-S-100 antibody (DAKO) in a Tris (0.05 mol/L), NaCl (0.15 mol/L), CaCl₂ (10 mmol/L), NaN₃ (0.15 mmol/L) buffer was added, and the plates were incubated for another hour. After the plates had been washed, 200 µL of streptavidin-europium in assay buffer (0.05 mol/L Tris; 0.15 mol/L NaCl; 1 g/L bovine serum albumin; 0.5 g/L bovine -globulin, both from Sigma; and 0.15 mmol/L NaN₃) was added to each well, and the plates were incubated for 30 min. As a last step, 200 µL of enhancement solution (0.01 mol/L acetic acid, 38 mg/L tri-n-octylphosphine oxide, 1.3 g/L potassium phthalate, 222 mg/L thenoyltrifluoroacetone, and 2 mL/L Triton X-100) was added to each well, and the plates were incubated for 15 min. The resulting fluorescence was measured with a DELFIA 1234 fluorometer (Wallac). The threshold for detection of S-100 with this assay was 0.015 µg/L.

For each age group, the median concentration of S-100 and the 25th, 75th, 10th, and 90th percentiles of S-100 concentration were calculated. The significance of differences in median concentration between groups was examined using the Mann–Whitney U-test, and the correlation between age and S-100 concentration was calculated using the Spearman rank order correlation coefficient.

For all 200 healthy volunteers, the median plasma concentration of S-100b was 0.052 µg/L (10th percentile, 0.023 µg/L; 90th percentile, 0.097 µg/L; Fig. 1 ). The difference in median concentrations for men (median, 0.055 µg/L) and women (median, 0.048 µg/L) was not statistically significant (P >0.05). With increasing age, plasma concentrations of S-100b decreased slightly. The correlation between decreasing concentration and increasing age was weak, however, (r = -0.144; P = 0.04), and differences between age groups were not significant (P >0.05; Fig. 1 ).

View larger version (32K): [in this window] [in a new window]   Figure 1. Plasma concentrations of S-100 protein in healthy individuals (n = 200) according to age (n = 40 in each group). Box-whisker plots depict the median concentration for each group (horizontal line in the box), the 25th and 75th percentiles (upper and lower edges of box), and the 10th and 90th percentiles (lines extending above and below box). Median S-100 protein concentrations decreased slightly with increasing age in the older age groups but the differences between groups were not statistically significant (P >0.05).

The possibility that the concentration of S-100 in CSF might be age-dependent is based on two published reports. Van Engelen et al. (9) studied S-100 concentrations in the CSF of patients who were undergoing neurological examination but who had no evidence of an organic neurological disease. That study found that the concentration of S-100 increased slightly with increasing age but did not differ substantially by sex. Nygaard et al. (10) examined patients undergoing various surgical procedures with spinal anesthesia. That study found substantially higher concentrations of S-100 in the CSF with increasing age. They also found that S-100 concentrations in the CSF were substantially higher in men than in women. Both of these groups used analytical methods that detected predominantly S-100b but were not sensitive enough to detect S-100 concentrations in the blood of healthy subjects.

Several explanations have been offered as to why the concentration of S-100 in CSF might increase with increasing age: (a) the concentration of S-100 in CSF might parallel the rate of myelin loss, which increases with age; (b) the turnover of CNS cells remains constant, but S-100 concentrations in the cells increase with age; and (c) a reduced CSF bulk flow with older age leads to an increased half-life of S-100 in CSF (10).

In contrast to these findings regarding S-100 concentrations in CSF, our study found a slight decrease in the S-100b concentration in blood with increasing age, although the differences between age groups were not significant. The trend toward decreasing concentrations of S-100b with increasing age was mainly because of the finding of relatively high concentrations of S-100b in a few young individuals. Eight of the 10 subjects with the highest concentrations of S-100b were younger than 30 years. We found nothing in the data that would explain this finding.

The concentration of S-100 in blood may be useful clinically to screen for and monitor progression of damage to the CNS. Our study found no evidence that age- and sex-corrected reference values need to be established for measurements of S-100 concentrations in the blood of adults. Such may not be the case, however, for determinations of S-100 concentrations in children because the results of studies in animals indicate that S-100 concentrations in the brain change substantially during postnatal development of the brain (11). Therefore, reference values for different age groups of children should be established before this method is used routinely to evaluate acute brain damage in pediatric patients.

Footnotes

fax 0049 (89) 7095 3270, e-mail wiesmann{at}ikra.med.uni-muenchen.de

References

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