Summer/winter changes in serum S100B protein ...

Journal of Psychiatric Research 47 (2013) 791e795

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Summer/winter changes in serum S100B protein concentration as a source of research variance Armando L. Morera-Fumero a, *, Pedro Abreu-Gonzalez b, Manuel Henry-Benitez c, Silvia Yelmo-Cruz c, Estefania Diaz-Mesa c a b c

Departamento de Medicina Interna, Dermatologia y Psiquiatria, Facultad de Medicina, Universidad de La Laguna, 38071-La Laguna, Santa Cruz de Tenerife, Islas Canarias, Spain Departamento de Fisiologia, Facultad de Medicina, Universidad de La Laguna, Santa Cruz de Tenerife, Spain Servicio de Psiquiatria, Hospital Universitario de Canarias, La Laguna, Tenerife, Spain

a r t i c l e i n f o

a b s t r a c t

Article history: Received 7 March 2012 Received in revised form 21 May 2012 Accepted 5 March 2013

Background: S100B is a calcium binding protein that can be measured in cerebral and extra cerebral biological tissues and fluids. Circadian and seasonal variations have been described in several biological molecules such as melatonin, cortisol and testosterone. Healthy subjects do not have a circadian rhythm of S100B. There is no information on seasonal variations of S100B levels. The aim of this research is to study whether healthy subjects present summer/winter changes in serum S100B protein concentrations. Methods: Ninety-eight subjects were studied in summer, of those, 64 participated in the winter evaluation. Blood was drawn by venipuncture at 09:00 h, 12:00 h and 00:00 h in summer and winter. Serum was separated from blood by centrifugation and stored at 70 until analysis. Serum S100B concentrations were measured by ELISA. Results: Serum S100B concentrations were significantly higher in summer than winter (09:00 h: 43.4 24.6 ng/ml vs. 29.3 22.7 ng/ml, p < 0.001; 12:00 h: 42.8 25.0 ng/ml vs. 23.0 22.1 ng/ml, p < 0.001; 00:00 h: 44.5 23.2 ng/ml vs. 28.5 24.6 ng/ml, p < 0.001). Age, gender, body mass index and time points when blood was extracted did not affect serum S100B concentrations neither in summer nor in winter. Conclusions: Our results point to the fact that there is an important difference in serum S100B concentrations between summer and winter. It is strongly advisable to consider this summer/winter difference in serum S100B concentrations when researching into this area. Ó 2013 Elsevier Ltd. All rights reserved.

Keywords: S100B protein Seasonal rhythms Circadian rhythms Body mass index Age

1. Introduction The search for biological markers in psychiatry has been delayed for many years due to the fact that the brain is not directly accessible to observation in living human beings. At present time, important ethical considerations ought to be considered when researching into this area. Most of the available techniques to study brain dysfunctions are invasive and expensive, e.g. positron emission tomography, magnetic resonance imaging, CT-scan, lumbar puncture, etc. The problem could be partially solved if there were easily accessible peripheral brain markers. Finding brain peripheral parameters that could be measured and act as indicators of normal functioning, pathological changes or as markers of response to treatments would be extremely useful.

* Corresponding author. Tel.: þ34 922319281; fax: þ34 922319279. E-mail address: [email protected] (A.L. Morera-Fumero). 0022-3956/$ e see front matter Ó 2013 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.jpsychires.2013.03.001

Several molecules have been suggested as peripheral markers of brain dysfunction. Neuron specific enolase (NSE), glial fibrillary acidic protein (GFAP), myelin basic protein (MBP), neurofilament heavy chain (NF-H) and S100B protein are some of the proposed molecules (Vos et al., 2010; Gyorgy et al., 2011). In the last decade, S100B has attracted considerable interest because it is released into the cerebrospinal fluid and blood from brain tissue. S100B belongs to a multigenic family of calcium binding proteins and it is expressed in high abundance in the brain (Michetti et al., 2012). S100B interacts with target proteins within cells, altering their functions once secreted with the multiligand receptor for advanced glycation endproducts (RAGE) (Donato, 2007). As an intracellular regulator, S100B affects protein phosphorylation, energy metabolism, the dynamics of cytoskeleton constituents, calcium homeostasis, and cell proliferation and differentiation. At the extracellular level, S100B presents a dual role, it may act as a neuroprotective or neurotoxic and apoptotic molecule depending on its concentration. At low concentrations, pico or

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nanomolar levels, S100B protects neurons against apoptosis, stimulates neurite outgrowth and astrocyte proliferation, and negatively regulates astrocytic and microglia responses to neurotoxic agents. At high doses, micromolar levels, S100B produces neuronal death and exhibits properties of a damage-associated molecular pattern protein (Donato, 2009). The role of S100B as a marker of brain inflammation has led to some authors to consider S100B as the C-reactive protein of the brain (Sen and Belli, 2007). S100B can be measured in accessible peripheral biological fluids, such as blood or urine (Gazzolo et al., 2010). S100B has been used as a biological marker in a wide variety of medical specialties, such as pediatrics, dermatology, cardiology, neurology and psychiatry. S100B human research includes a broad variety of topics, ranging from the physiological ones, such as physical and psychological stress (Gazzolo et al., 2010) to the pathological, such as brain injuries (Beer et al., 2010) or schizophrenia psychosis (Zhang et al., 2010). Seasonal and circadian changes have been reported to affect several biological variables such as cortisol, melatonin and testosterone (Levine at al., 1994). The information about circadian rhythms of S100B is very scanty. In healthy subjects, an absence of a circadian rhythm of S100B levels has been reported (Ikeda and Umemura, 2005). There is no information about seasonal S100B variations. This investigation is aimed at studying whether healthy subjects present summer/winter changes in serum S100B protein concentrations. 2. Methods 2.1. Sample Subjects were recruited by an email announcement through the electronic mail of the University of La Laguna and by word of mouth among the acquaintances by the investigators. Exclusion criteria were: 1) pregnancy, 2) being physically or mentally ill, 3) taking drugs of abuse and 4) taking vitamin supplements. To ensure healthiness of the volunteers a general hematological, biochemical and urine analysis was carried out in July and December. Mental healthiness was assessed informally by asking the subjects if they had received psychiatric treatment in the past or were receiving treatment in the present or if any first-degree relative was in the past or at present receiving psychiatric treatment. Psychological treatment was considered as well as undergoing psychiatric treatment. The initial summer sample was comprised by 101 subjects, of whom 3 were excluded, a woman with a month of amenorrhea and a positive pregnancy test result and 2 men because the hepatic enzymes values were abnormally high. So the final summer sample was comprised of 98 subjects. Of those 98 subjects, in winter 3 women were pregnant, 1 man had died because of a car crash and another man was ill. Of the remaining volunteers, 29 subjects refused to participate in the second part of the study. The final winter sample was comprised of 64 subjects. 2.2. Methodology The study was carried out in July and December during two consecutive week-ends in order to minimize the interference with the subjects working life. The volunteers arrived at the School of Medicine at 07:30 and laid from 08:00 through 09:00 h when the first blood sample was drawn. This blood sample was drawn after fasting all night. Along the article the terms summer/winter and July/ December, and day/night and circadian will be used interchangeably. After the first blood extraction, the subjects had breakfast and they were free to go around the School of Medicine until 11:00 h.

Then, they laid in bed until 12:00 h when the second blood sample was extracted. Subjects were free to go home until 23:00 h when they had to be back at the School of Medicine. The third blood sample was drawn at 00:00 h after laying in bed for 1 h. Subjects were instructed to have a light dinner not later than 20:30 h. The same routine was followed in December. All blood samples were extracted by venipuncture. The rationale to lay in bed 1 h before blood extraction was to allow the subjects to relax in order to minimize the psychological and physical stress that they may have had (Gazzolo et al., 2010). 2.3. Measurement of serum S100B concentrations After blood was extracted, samples were placed in vacutainer tubes without anticoagulant and allowed to clot. Samples were centrifuged at 3000 rpm during 5 min, then serum was aliquoted in Eppendorf tubes and stored frozen at 70 C until analysis. To minimize the assay variation all serum samples were analyzed the same day with the same laboratory batch and by the same analyst. The analyst was blind with respect to the samples pertaining to summer/winter or the time of the day when samples were extracted. S100B levels in serum samples were measured with an enzymelinked immunosorbent assay (ELISA) kit according to the manufacturer instructions (BioVendor, Candler, USA). The BioVendor Human S100B ELISA uses a polyclonal anti-cow S100B coated in microtitration wells. The absorbance of the resulting yellow color product was measured spectrophotometrically at 450 nm in a microplate spectrophotometer reader (BenchmarkPlus, Bio-Rad, Hercu1es, CA, USA). In this ELISA, the lowest detection limit was 10.8 pg/ml. Coefficients of variation were 3.92% and 5.03% for intraassay and inter-assay variability, respectively. 2.4. Statistical analysis Data were analyzed using the 15th version of the SPSS statistical package (SPSS, Chicago, Illinois, USA). Night/day (00:00 h, 09:00 h and 12:00 h) serum S100B differences were analyzed by means of an ANOVA for repeated measures. Summer/winter comparisons were analyzed by means of Paired t tests. Pearson correlations were applied to study the relationships between quantitative variables while the statistic chi-square was applied to study the association between qualitative variables. All statistical tests were two-tailed. Statistical significance level was set at 0.05. Quantitative data are presented as mean s.d. 3. Results Demographic and clinical characteristics of the samples are presented in Table 1. Summer-winter comparisons by gender and body mass index (BMI) did not elicit significant differences. Fig. 1 shows the comparison of serum S100B concentrations in summer and winter at the three time point studied (09:00 h, 12:00 h and 00:00 h). Time-point serum S100B concentrations were significantly higher in summer than winter. Table 2 shows the comparison of serum S100B levels by season and day/night times. Time-point measures of serum S100B

Table 1 Demographic and clinical characteristics of the sample by season. Variables

Summer

Age (mean sd, minemax) Sex (male/female) BMI (mean sd)

39.8 9.8, 18e59 48/50 30/34 24.8 3.7 24.9 3.9

Winter

P NA 0.793 0.693


Fig. 1. Comparison of serum S100B concentrations by season and time of the day.

concentrations did not elicited significant differences neither in summer nor in winter. Table 3 shows the correlation between age and S100B concentrations by season. There were no significant relationships between age and S100B concentrations. The comparison of serum S100B concentration by gender in both seasons is presented in Table 4. No differences between men and women were found in summer and winter. In Table 5 the correlations between BMI and S100B concentrations by season is presented. There was an absence of relationships between BMI and S100B in winter and summer. 4. Discussion As far as we know, this is the first time that a summer/winter difference in serum S100B protein levels is reported. The meaning of the serum summer S100B increase is uncertain. However, due to the fact that summer S100B concentrations almost double winter concentrations, such results should be taken into account to reduce the variability of research results in this area. It has been reported that long-term storage (for a period of eight years) increases serum S100B concentrations (Müller et al., 2006). Another research (Foerch et al., 2007) reported that long-term storage did not affect serum S100B concentrations. Müller et al. (2006) reported increases of serum S100B levels ranging between 0.02 and 0.08 ng/ml for an eight-year period of storage. Our summer samples were stored for seven months and the winter samples were stored one month. Assuming the worst of the scenarios, that our sample would have been stored eight years and they had the maximum increase in S100B concentrations (0.08 ng/ml in the summer samples), the summer/winter difference in S100B levels would remain. An alternative explanation of our results may stem from the change of temperature between seasons. In vitro studies have reported that the increase in temperature produces an increase in the permeability of the bloodebrain barrier (Wijsman and Shivers, 1993; Urakawa et al., 1995; Shivers and Wijsman, 1998), therefore it may allow the diffusion of S100B from brain to blood. The differences in mean environmental temperatures between summer and winter in our geographical area (Latitude: 28 270 N, Longitude: 16170 W) are approximately of eight degrees (summer 26 , winter 18 ), so, it remains a matter of speculation whether or not an environmental difference of eight degrees may explain the

Table 2 Comparison of time-point serum S100B levels by season. Season

S100B 9

S100B 12

S100B 00

P

Winter: mean (s.d.) Summer: mean (s.d.)

29.82 (22.91) 42.10 (22.97)

26.34 (22.34) 41.93 (24.10)

28.86 (24.93) 43.13 (22,94)

0.346 0.499

793

differences in serum S100B levels between summer and winter in healthy subjects. With respect to day/night S100B differences, our results show that there is no circadian rhythm of S100B levels in healthy subjects. This finding is in accordance with a previous report that found no circadian variation in S100B serum levels in healthy subjects (Ikeda and Umemura, 2005). Previously we have reported the presence of a circadian rhythm of serum S100B protein in schizophrenic inpatients when hospitalized but when patients were clinically stable that circadian rhythm was not present (Diaz-Mesa et al., 2008). Those data point to the fact that the presence of a circadian rhythm of S100B could be an indicator of clinical abnormality and when “normality” (clinical improvement) is reached the circadian rhythm disappears. Age is maybe another variable that could have biased our results. In adult subjects, a negative correlation (Wiesmann et al., 1998), a positive correlation (Steiner et al., 2010) and an absence of correlation (Portela et al., 2002; Ikeda and Umemura, 2005) between age and S100B have been reported. Our results do not find a correlation between age and serum S100B concentrations in any of the studied seasons, therefore confirming the results of Portela et al. (2002) and Ikeda and Umemura (2005) but not those of Wiesmann et al. (1998) and Steiner et al. (2010). In the case of Wiesmann et al. (1998), they found, what they consider a weak negative correlation between age and S100B (r ¼ 0.144, p ¼ 0.04). The authors found nothing at all in the data that would explain their results, although they suggested an age bias of the sample because eight of the 10 subjects with higher concentrations of S100B were younger than 30. Steiner et al. (2010) reported a positive correlation between age and serum S100B (r ¼ 0.303, p ¼ 0.018). The authors do not give an explanation for their results. From our point of view, differences in the subject samples and analytical techniques may explain the differences. First, our sample was comprised of healthy subjects while the sample of Steiner et al. (2010) was comprised by healthy subjects and patients from an outpatient lipid clinic. Second, we used an ELISA to measure serum S100B while Steiner et al. (2010) used an immunoluminometric technique. According to Pham et al. (2010) S100B ELISA measurements are less contaminated by extracranial sources of S100B while Western-blot techniques, like inmunoluminometry, are more contaminated by extracranial sources of S100B. The recent research of Steiner et al. (2010) considered that BMI was an important caveat in S100B neuropsychiatric research because they found a positive correlation between BMI and serum S100B concentrations. Steiner et al. (2010) suggested that adiposerelated factors such as leptin and A-FABP (Adipocyte-type Fatty Acid Binding Protein) could influence serum S100B concentrations. In a stepwise regression analysis to predict S100B from age, leptin and A-FABP, only A-FABP significantly predicted S100B and this prediction was age independent. The authors strongly advise taking into account adipose-related factors when examining peripheral blood S100B levels. Our results are in accordance with the results of a recent research that has found no correlation between BMI and serum S100B concentrations (Pham et al., 2010). Our results and the results from Pham et al. (2010) are very similar with respect to the two variables that do not affect S100B levels, BMI and age. By the contrary, the results of Steiner et al. (2010) showed that age and BMI affect S100B levels. From our point of view those differences may stem from the analytical technique used by the different authors in measuring S100B. While Pham et al. (2010) and ourselves measured S100B with an ELISA, a quantitative technique, Steiner et al. (2010) measured S100B with a Western-blot analysis, a semi quantitative technique, less precise than the ELISA. It has been reported that there are extracranial sources of S100B production, mainly by fat tissue and the muscle (Steiner et al.,

794


Table 3 Correlations between age and serum S100B levels by season. S100B winter

S100B summer

Age

9h

12 h

00 h

9h

12 h

00 h

Pearson Correlation (P)

0.152 (0.244)

0.170 (0.194)

0.194 (0.137)

0.060 (0.576)

0.018 (0.863)

0.031 (0.777)

Table 4 Comparison of serum S100B levels by gender. S100B

Gender

Mean

Standard deviation

p

S100B 9 Winter S100B 12 Winter S100B 00 Winter S100B 9 Summer S100B 12 Summer S100B 00 Summer

Female Male Female Male Female Male Female Male Female Male Female Male

31.40 26.71 25.98 25.83 29.84 27.06 45.98 38.52 47.05 39.68 47.09 39.32

25.06 19.54 21.56 22.80 28.84 19.00 23.58 21.27 26.27 20.33 24.25 20.92

0.389 0.977 0.648 0.102 0.094 0.094

endothermic mammals such us the human being has to be clarified in the future. An alternative explanation also mediated by the summer/winter change in temperature would be that the expression of the S100B SNPs rs9722 and the S100B haplotype TeGeGeA were increased in summer, therefore it would be associated with elevated serum S100B concentrations. In conclusion, our results point to the fact that there is an important difference in serum S100B concentrations between summer and winter. When researching into this area, it is strongly advisable to consider this summer/winter difference. It remains a matter of speculation and future studies should clarify whether or not the temperature differences between summer and winter could be a feasible explanation for such results. Conflict of interest

2007). A recent paper has reported that the contribution of the extracranial sources of S100B may or may not affect serum S100B concentrations (Pham et al., 2010). While Western-blot techniques show an important contribution of the extracranial sources of S100B to the total S100B serum concentration, ELISA techniques do not have such disadvantage. In our case we have measured S100B concentration by means of an ELISA, so according to Pham et al. (2010), the presence of extracranial sources of S100B in our serum samples should be minimum. Another source of variation that could have contaminated our results may stem from racial and genetic differences. Higher levels of serum S100B concentrations have been reported in Asians and Blacks compared to Caucasians (Ben et al., 2003). The racial effect on serum S100B concentrations has not been studied in our sample because all subjects were Caucasians. Recently, it has been reported that in particular the S100B single nucleotide polymorphism (SNP) rs9722 and the S100B haplotype TeGeGeA are associated with elevated serum S100B concentrations (Hohoff et al., 2010). The genetic base of the S100B regulation is not within the scope of our research, but we cannot exclude that part of the variance in the research results of S100B could be explained by genetic differences. We think that the main known sources of S100B variation measurement have been controlled in our study and did not affect our results. It remains a matter of speculation and future studies whether or not the differences between summer and winter temperature per se could be a possible explanation of our results. Animal and in vitro studies have shown that changes in the environmental conditions of temperature are one source of harmful stress affecting the central nervous system (Shivers and Wijsman, 1998). The extent to which these changes affect homoeothermic/

Table 5 Serum S100B values and BMI correlations by season. S100B

Season

BMI

p

S100B 9

Summer Winter Summer Winter Summer Winter

0.057 0.180 0.073 0.189 0.021 0.201

0.589 0.173 0.489 0.165 0.842 0.127

S100B 12 S100B 00

None. Contributors -

Conceived and designed the experiments: ALMF, PAG, MHB. Performed the experiments: ALMF, PAG, MHB, EDM, SYC. Biochemical analysis: PAG. Statistical analysis: ALMF, MHB. Wrote the paper: ALMF, PAG, MHB, EDM, SYC.

Funding This study was partly supported by a grant (PI: 08/115) of the Fundacion Canaria de Investigacion y Salud (FUNCIS). The funders had no role in the study design, data collection, analysis, preparation of the manuscript or decision to publish. Acknowledgments The authors want to thank to all volunteers that kindly participated in the study. References Beer C, Blacker D, Bynevelt M, Hankey GJ, Puddey IB. Systemic markers of inflammation are independently associated with S100B concentration: results of an observational study in subjects with acute ischemic stroke. Journal of Neuroinflammation 2010;7:71. Ben AO, Vally J, Adem C, Foglietti MJ, Beaudeux JL. Reference values for serum S100B protein depend on the race of individuals. Clinical Chemistry 2003;49: 836e7. Diaz-Mesa E, Morera AL, Abreu-Gonzalez P, Henry M, Intxausti A, Garcia-Valdecasas-Campelo J. Circadian protein S100B rhythm in acute schizophrenic patients. International Journal of Neuropsychopharmacology 2008;11:144. Donato R. RAGE: a single receptor for several ligands and different cellular responses: the case of certain S100 proteins. Current Molecular Medicine 2007;7: 711e24. Donato R, Sorci G, Riuzzi F, Arcuri C, Bianchi R, Brozzi F, et al. S100B’s double life: intracellular regulator and extracellular signal. Biochimica et Biophysica Acta 2009;1793:1008e22. Foerch C, Wunderlich MT, Dvorak F, Humpich M, Kahles T, Goertler M, et al. Elevated serum S100B levels indicate a higher risk of hemorrhagic transformation after thrombolytic therapy in acute stroke. Stroke 2007;38:2491e5.

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