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Keywords:

  • biomarker;
  • brain-derived neurotrophic factor;
  • epilepsy;
  • Luminex;
  • serum

Abstract

  1. Top of page
  2. Abstract
  3. Introduction
  4. Methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. Disclosure of conflicts of interest
  9. References
  10. Supporting Information

Background and purpose

To clarify the utility of serum brain-derived neurotrophic factor (BDNF) as a biomarker for epilepsy diagnosis and severity.

Methods

Serum BDNF levels in 135 consecutive people with epilepsy meeting our criteria were assessed. Controls were 34 healthy individuals who over 12 months or longer had no complaints or signs of a neurological disorder. Serum BDNF concentrations were measured using Luminex technology.

Results

Gender, but not age, was found to be a significant factor related to serum BDNF levels in controls and people with epilepsy. Serum BDNF levels in people with epilepsy (mean 8798.5, SE 321.5 pg/ml) were not different from those of controls (mean 8919.5, SE 709.0 pg/ml). A multiple linear regression analysis, however, suggests that seizure frequency (P < 0.001) and epilepsy duration (P = 0.025) negatively correlate with serum BDNF levels independently of other factors. When BDNF cut-off values of 6260 pg/ml were used, the sensitivity for distinguishing people with daily or more frequent seizures from those with fewer seizures was 80% and specificity was 90%.

Conclusions

It seems that the concentration of BDNF in serum is associated with disease severity in people with epilepsy and may be a helpful marker for severity.


Introduction

  1. Top of page
  2. Abstract
  3. Introduction
  4. Methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. Disclosure of conflicts of interest
  9. References
  10. Supporting Information

There is currently no efficient method to evaluate epilepsy progression or to assess effectiveness of treatments other than limited clinical and electroencephalogram evaluation, which is neither complete nor definitive in confirming the diagnosis of epilepsy.

Brain-derived neurotrophic factor (BDNF) is found throughout the brain, particularly in the cerebral cortex and hippocampi [1, 2]. Recent evidence has implicated BDNF in the pathophysiology of epilepsy [3-5]. BDNF may be upregulated by limbic seizures in animal models [6, 7] and in people with epilepsy [8]. It has also been shown that acute application of BDNF in in vitro and in vivo models induces an increase in neuronal excitability, suggesting that BDNF facilitates epileptogenesis [9, 10]. A greater than twofold reduction in the rate of kindling development in BDNF heterozygous (±) mice in which one BDNF allele was inactivated by gene targeting has also been reported [11]. Thus, BDNF is an attractive candidate as a potential epileptic biomarker in human cerebrospinal fluid as well as in plasma or serum. Peripheral measures of BDNF may be obtained from blood samples.

Serum BDNF levels in adults with epilepsy have not been previously well described. A recent study found decreased plasma BDNF levels in people with epileptic seizures [12] (‘serum BDNF’ was corrected as ‘plasma BDNF’ in Neurology® 2011; 76: 935). This initial study, however, had some limitations including a small number of subjects (n = 12), a less than vigorous quantitative method and inadequate control for confounders.

To clarify the utility of serum BDNF as a biomarker for epilepsy diagnosis and severity a robust technology for BDNF quantification was used. This method is a microsphere-based liquid array, known as Luminex, which has a much higher sensitivity, throughput and efficiency [13-15] than enzyme-linked immunosorbent assays (ELISAs) or western blotting. Several factors (gender, age and clinical variables) were evaluated for their effects on serum BDNF levels. The diagnostic value of BDNF in a large cohort of people with epilepsy and in healthy controls was also determined.

Methods

  1. Top of page
  2. Abstract
  3. Introduction
  4. Methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. Disclosure of conflicts of interest
  9. References
  10. Supporting Information

Standard protocol approvals, registrations and patient consents

The study was approved by the Research Ethics Committee of the Medical School of Sichuan University. Each participant provided written informed consent prior to study enrollment.

Participants

Attendees (outpatients and inpatients) of a tertiary epilepsy center were recruited between November 2010 and June 2011. All were pre-screened for eligibility by the attending physicians. If diagnostic and inclusion criteria were met, a research assistant introduced the study and obtained informed consent.

Epilepsy was diagnosed clinically, requiring at least two unprovoked seizures [16], and confirmed by video-electroencephalogram monitoring. All had a blood screen including a full blood count, liver, renal and thyroid screen and these had to be within normal limits. All had a brain MRI scan, and completed the Beck Depression Inventory II (BDI-II). Exclusion criteria were (i) a history of epilepsy surgery; (ii) a history of psychogenic seizures or uncertain diagnosis; (iii) presence of an active neurological disorder; (iv) alcohol or substance abuse in the previous 12 months; (v) presence of other major neurological disabilities, including learning disability; (vi) taking part in a study of a new anti-epileptic drug (AED); (vii) active comorbid psychiatric illness; (viii) a history of taking psychotropic medication in the previous 6 months; or (ix) withdrawal of consent.

All participants were interviewed by neurologists who were study aware. Individuals were asked about socio-demographic details, history of epilepsy (duration, age of onset, recent seizure and history of AED taking) and clinical features of the condition in the year prior to the evaluation (seizure types and frequency). Each eligible subject was assigned to one of the following categories according to seizure frequency, treatment response and natural history: (i) newly diagnosed epilepsy (NDE), i.e. duration of less than 1 year and drug-naive; (ii) epilepsy in remission (SR), i.e. no seizures for more than 2 years; (iii) epilepsy in remission for 1–2 years or with occasional seizures (OS), i.e. seizures not requiring treatment changes in the opinion of the attending physician; (iv) active, non-drug-resistant (NDR) epilepsy (active seizures which, in the opinion of the attending physician, could be improved by treatment); and (v) active, drug-resistant (DR) epilepsy (seizures which, in the opinion of the attending physician, were unlikely to improve by further drug changes).

Controls were community volunteers in good health who underwent clinical assessment to exclude any lifetime history of a neurological, cognitive or psychiatric condition. All had a Mini-Mental State Examination score above 27; a Clinical Dementia Rating score of 0; and New York University paragraph recall scores (immediate and delayed) > 6. Controls were excluded if they smoked more than 100 cigarettes per year, used alcohol other than socially and used any psychoactive drugs. All had been followed for 12 months or longer (median of 1.5 years) without any symptoms or signs of neurological disorders, including mild cognitive impairment or depression.

Collection of serum and quality control

All samples were collected in the afternoon to limit any potential circadian fluctuation of blood proteins and metabolites. Ten milliliters of blood was drawn into a vacutainer tube(s) without anticoagulants from the antecubital vein and incubated in an upright position at room temperature for 30 min to clot. The sample was then centrifuged for 15 min at a speed of 1500 RCF. Serum was aspirated and removed. Samples were then flash-frozen in liquid nitrogen and stored at −80°C.

Measurement of serum BDNF

BDNF levels were assayed using the Luminex Human BDNF Antibody Bead Kit (Invitrogen, Camarillo, CA, USA) and Invitrogen's Growth Factor Buffer Reagent Kit, according to instructions. The detailed methodology is given in Appendix S1. This assay system has been independently validated as measuring BDNF serum levels accurately with high reproducibility. Using this kit, the minimum detectable concentration of human BDNF is < 10 pg/ml.

Statistical analysis

All analyses were performed using SPSS for Windows (version 12.0.1, SPSS Inc., Chicago, IL, USA). Chi-squared tests were used to compare gender between controls and people with epilepsy. To compare the demographic data, including age and BDI-II score, and serum BDNF levels between groups, Student's t-tests were used. Pearson's or the Spearman correlation coefficient was used for the correlation analyses. As levels of BDNF had a normal distribution (Kolmogorov–Smirnov test), multiple linear regression analysis was used to estimate the effects of different baseline risk factors (i.e. duration, prognostic category, seizure frequency, seizure type, age of onset, recent seizure or history of AEDs) in potentially changing the relative BDNF levels. In this analysis, epilepsy duration (years), seizure frequency (seizures/month), age of onset (years), seizure type (generalized epilepsy or localization-related epilepsy), recent seizure (days previously) or history of AED taking (in years) were used as continuous variables. Prognostic categories (NDE, SR, OS, NDR, DR) were used as a categorical variable in which NDE, SR, OS and NDR were compared with DR. In this analysis, to indicate possible situations where the explanatory variables are related by a linear function, making the inference of the regression coefficients impossible, multicollinearity statistics were used in the regression analysis. All tests were two-tailed and P values < 0.05 were considered statistically significant.

A receiver operating characteristic (ROC) curve was used to calculate the relationship between sensitivity and specificity for the different seizure frequency groups and hence evaluate the serum BDNF diagnostic performance. The ‘optimum’ cut-off value from the ROC curve is the point at which the sum of sensitivity and specificity is maximal.

Results

  1. Top of page
  2. Abstract
  3. Introduction
  4. Methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. Disclosure of conflicts of interest
  9. References
  10. Supporting Information

Demographic data and characteristics of the participants

A total of 254 people with seizures were screened and 135 (79 males) were included. Thirty-four healthy controls (20 males) were recruited. Demographic details are provided in Table 1.

Table 1. Summary of the donor demographics and serum BDNF values
VariablePeople with epilepsy (n = 135)Controls (n = 34) P value
  1. AED, anti-epileptic drug; BDI, Beck Depression Inventory; BDNF, brain-derived neurotrophic factor; DR, drug-resistant epilepsy; NA, not available; NDE, newly diagnosed epilepsy; NDR, active non-drug-resistant epilepsy; OS, occasional seizures; SR, seizure remission. aBetween-group differences calculated using the chi-squared test; bbetween-group differences calculated using Student's t test.

Gender (n, %)
Female56 (41.5%)200.08a
Male79 (58.5%)14
Age (years)
Mean ± SD27.2 ± 11.431.8 ± 10.20.37b
Range11–6522–68
Age of onset (years)
Mean ± SD19.9 ± 11.5NANA
Range1–59
Duration of epilepsy (years)
Mean ± SD7.4 ± 7.6NANA
Range0.5–36
Seizure type
Generalized69NANA
Partial66
Frequency of seizure (seizures/month)
Mean ± SD2.9 ± 5.4NANA
Range0–30
Most recent seizure (days previously)
Mean ± SD176 ± 320NANA
Range0–1460
Prognostic category
NDE22NANA
SR16
OS21
NDR52
DR24
Type of epilepsy
Idiopathic epilepsy38NANA
Symptomatic epilepsy15
Cryptogenic epilepsy82
History of AEDs (years)
Mean ± SD3.7 ± 4.3NANA
Range0–20
Number of AEDs
026NANA
154
244
310
41
BDI-II score
Mean ± SD6.8 ± 4.54.8 ± 5.10.21b
Range0–130–13
Serum BDNF, pg/ml (all cases)
Mean ± SE8798.5 ± 321.58919.5 ± 709.00.83b
Range3.2–19 883.0163.5–21 126.5
Serum BDNF, pg/ml (cases excluding patients aged <21 years)
Mean ± SE8787.5 ± 384.08919.5 ± 709.00.53b
Range279.5–19 883.0163.5–21 126.5

The mean age of people with epilepsy was 27.2 years, SD 11.4 years (range 11–65 years) and of controls 31.8 years, SD 10.2 years (range 22–68 years). Onset of epilepsy ranged from 1 month to 59 years (mean 19.9, SD 11.5 years). The mean duration of epilepsy was 7.5 years (range 0.05–36 years). Seizures occurred at a rate of 0–35 per week. The most recent seizure occurred from 1 day to 17.9 years prior to the sample collection. The epilepsy syndrome was classified as generalized in 69 (51%) and localization-related in 66 (49%).

All but 26 of the people with epilepsy were taking AEDs and three were taking traditional Chinese medicines. AEDs had been taken for from 0 to 25 years (mean 3.7, SD 4.2 years) and nobody was on psychotropic drugs.

The mean depression scores (BDI-II) were 4.8, SD 5.1, in the control group and 6.8, SD 4.5, in the epilepsy group, and both groups were in the lowest range suggesting minimal depression (score 0–13) (P = 0.21 for the comparison between groups).

Effects of confounding factors such as sample preparation, age and gender on the serum BDNF level

To estimate any potential variations arising from sample preparation, fractions of control samples were assessed. For BDNF, no apparent alterations were seen when the control samples were frozen and thawed twice or stored at −80°C for up to 6 months.

Amongst controls, males had higher levels of BDNF (mean 11 026.5, SE 1219.0 pg/ml) than females (mean 7397.5, SE 667.0 pg/ml), P = 0.009 (Fig. 1). Similarly, in people with epilepsy, males had higher levels of BDNF (mean 9503.0, SE 417.5 pg/ml) than females (mean 8213.5, SE 470.5 pg/ml), P = 0.045 (see Fig. 1). The age dependence of serum BDNF levels was also assessed (see Fig. 2). Linear regression and association analyses showed no significant association between BDNF levels and age in controls (Pearson correlation, P = 0.618) or in people with epilepsy (P = 0.989) within the range of ages in our cohort. Similarly, no age dependence of BDNF levels was found in either gender group (Fig. 2).

image

Figure 1. Gender dependence on the BDNF levels in serum. Serum BDNF levels were measured in individual control (CTL) and disease (EP, epilepsy) samples using the Luminex assay. The BDNF levels of female, male or both male and female samples in the control, disease or all cases were assessed. A significant difference was observed between female and male subjects when the average BDNF levels were compared, in all cases, including the control and disease groups. No significant difference amongst the groups was observed in either of the gender samples. The data shown are the mean ± SE. *P < 0.05.

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image

Figure 2. The relationship of age and BDNF levels in serum. Serum BDNF levels were measured in individual control (CTL) and disease (EP, epilepsy) samples using the Luminex assay. The data shown are female (a) or male (b) cases. The correlation coefficient (r) and P value of the linear regression for each group were as follows: BDNF in CTL,= −0.01 (= 0.93) and = 0.10 (= 0.73), for female and male cases, respectively; BDNF in EP,= 0.14 (= 0.32) and = −0.12 (= 0.29), for female and male cases, respectively.

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Serum BDNF in controls and people with epilepsy

As shown in Fig. 1 and Table 1, our analyses suggest that BDNF levels in people with epilepsy were not significantly different from those of controls (t test, P = 0.83). As there was no control in the < 21-year-old age group, to control for potential age-dependent changes, only cases in the same range of age as the control group were used to compare BDNF levels amongst the different groups, i.e. subjects younger than 21 were excluded (leaving 34 controls and 88 cases) After these exclusions, our overall conclusions did not change (cases versus control: P = 0.533).

The t tests showed no differences in BDNF levels between controls and cases in either males (P = 0.875) or females (P = 0.530) (Fig. 1).

Serum BDNF levels related to epilepsy factors

To determine if serum BDNF levels were altered during the course of epilepsy or other epilepsy factors, BDNF levels were assessed according to clinical characteristics such as disease duration, prognostic category, seizure frequency, seizure type, age of onset, recent seizure and history of AEDs. Multiple linear regression analysis showed a significant negative correlation between seizure frequency and BDNF levels (P < 0.001) and disease duration and BDNF levels (P = 0.025) which was independent of other factors (Table 2 and Appendix S2). When gender data were analyzed separately, findings were similar. In both males and females, seizure frequency was a significant predictor of changes in BDNF levels (P = 0.008 in males and P = 0.009 in females) (Table 2 and Appendix S2).

Table 2. Stepwise multiple linear regression analysis of the potential predictors of changes in serum BDNF levelsa
Model Non-standardized coefficientStandardized coefficient
B SE β P
  1. AED, anti-epileptic drug; BDNF, brain-derived neurotrophic factor.Model 1: All people with epilepsy included. Excluded variables: prognostic category, seizure type, age of onset, recent seizure, history of AEDs. Adjusted R2 = 0.204. Model 2: Only males with epilepsy included. Excluded variables: prognostic category, seizure type, age of onset, recent seizure, history of AEDs, disease duration. Adjusted R2 = 0.139. Model 3: Only females with epilepsy included. Excluded variables: prognostic category, seizure type, age of onset, recent seizure, history of AEDs, disease duration. Adjusted R2 = 0.172. aDependent variable, BDNF.

1Constant10 258.571559.842 <0.001
Seizure frequency (seizures/month)−288.95168.012−0.437<0.001
Disease duration (years)−107.12346.832−0.2360.025
2Constant9583.112601.672 <0.001
Seizure frequency (seizures/month)−243.50787.204−0.4000.008
3Constant9319.021674.739 <0.001
Seizure frequency (seizures/month)−331.473118.357−0.4440.009

Correlation analyses and t test further indicated that there was a significant negative association between serum BDNF levels and frequency of seizures (Appendix S3).

Evaluation of serum BDNF as a marker for epileptic severity

To evaluate the utility of serum BDNF levels in discriminating cases with different seizure frequency, ROC curve analysis was performed. As shown in Table 3 and Fig. 3, when a cut-off value of 6260 pg/ml was chosen for BDNF, the sensitivity and specificity for distinguishing cases with daily or more frequent seizures from the ones with low seizure frequency were 80% and 90%.

Table 3. Summary of receiver operating characteristic curve results for serum BDNFa
 All people with epilepsyMales with epilepsyFemales with epilepsy
AUCP valueCut-off value (pg/ml)Sens (%)Spec (%)AUCP valueCut-off value (ng/ml)Sens (%)Spec (%)AUCP valueCut-off value (ng/ml)Sens (%)Spec (%)
  1. AUC, area under the curve; BDNF, brain-derived neurotrophic factor; Sens, sensitivity; Spec, specificity; monthly, patients with monthly or more frequent seizures; weekly, patients with weekly or more frequent seizures; daily, patients with daily or more frequent seizures. aReceiver operating characteristic curve analysis with AUC using Statistical Package of the Social Sciences (SPSS). Cut-off is given for sensitivity + specificity maximized for each comparison.

Monthly versus less frequent0.600.037651580400.630.039705080360.540.61548359027
Weekly versus less frequent0.660.014633080530.710.035633585600.580.34548358524
Daily versus less frequent0.86<0.001626080900.880.012604088750.850.00948358760
image

Figure 3. Receiver operating characteristic (ROC) curves to evaluate the serum BDNF as a biomarker for epileptic severity. Combined ROC curves for the BDNF of patients with monthly or more (dotted line), weekly or more (dot-dash line) or daily or more (solid line) seizures versus patients with a lower frequency of seizures. The data shown are for all the people with epilepsy (a), males with epilepsy (b) and females with epilepsy (c).

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Discussion

  1. Top of page
  2. Abstract
  3. Introduction
  4. Methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. Disclosure of conflicts of interest
  9. References
  10. Supporting Information

Factors that seem critical to BDNF levels in human serum were identified, i.e. influence of gender and age, as well as the effects of freezing and thawing cycles and storage time of the samples. Our results suggest that BDNF in human serum may be useful as a biomarker of epilepsy severity. The effects of different risk factors (i.e. epilepsy duration, prognostic category, seizure frequency, seizure types, age of onset, recent seizures or use of AEDs) on the potential changes in the relative levels of serum BDNF were estimated.

Data on the age dependence of serum BDNF have been inconsistent, or provide evidence of a decrease [17, 18] or exhibit no change in BDNF with age [19, 20]. Our results were largely consistent with those reported in a meta-analysis of 11 studies examining the differences in serum BDNF content between depressed and non-depressed subjects (total n = 748) [20], which also found no age dependence. Studies reporting a decline in serum BDNF levels with advancing age only recruited people over the age of 59 [17, 18]. Thus, our results may not be conflicting with these reports, as people in a different age range were being investigated.

Another observation in our study was the significant association between serum BDNF levels and gender in controls and people with epilepsy (Fig. 1). Previous studies, however, have reported conflicting results regarding the gender dependence of serum BDNF as determined by ELISA. It has been reported that women have lower BDNF serum levels [21] or BDNF platelet content [22] than men, although several other studies have reported that BDNF levels did not show any significant gender differences [17-19]. The discrepancy between this study and those using ELISA may be attributed to some experimental differences. First, compared with ELISA, the Luminex assays are more robustly quantitative and exhibit a greater dynamic range. The Luminex assays have a standard curve that can assess BDNF levels more precisely at both the low and high end and exhibit less variability. All controls were evaluated for 12 months or longer (median 1.5 years) and showed no symptoms or signs of neurological disorders, including mild cognitive impairment or depression (the variables were not controlled in other reports). Thus, some confounding factors were avoided as it has been well documented that both mild cognitive impairment [23, 24] and depression [20] significantly influence BDNF levels. Finally, different antibodies used in the two methods may also have contributed, at least in part, to the disparity in BDNF levels.

A previous study showed that BDNF levels in the peripheral blood (plasma) measured using ELISA were lower in people with epilepsy than in controls (17 controls versus 12 cases) [25]. In our study, although BDNF levels in people with epilepsy were not different from those of controls, levels were found to be negatively correlated with severity (determined by seizure frequency and epilepsy duration). Issues such as low sensitivity of the earlier ELISA assays and different antibodies may account for the differences. A larger cohort (34 healthy controls and 135 people with epilepsy) providing adequate statistical power to resolve several factors, such as gender dependence and seizure frequency, which may influence BDNF levels was assessed. Our study population was also recruited consecutively, reducing the risk of ascertainment and participation bias.

Our results provide evidence that decreases in serum BDNF are correlated with seizure frequency. In humans, determining whether BDNF is simply a biomarker for epileptic severity or alternatively is causally associated with epileptic severity is methodologically challenging, if not impossible. Despite the strong evidence linking changes in BDNF with the physiopathological features of epilepsy, there are several issues that remain unclear. It remains to be seen whether circulating levels of BDNF precisely reflect its expression in the brain as different cell types in the periphery, such as endothelial and mononuclear cells, can synthesize it [26]. BDNF may also be stored in platelets, which function as a ‘buffer system’ to regulate the peripheral levels of BDNF [27, 28]. This may be a confounding factor when assessing the relationship between serum BDNF and central BDNF and its relevance to the pathophysiology of epilepsy.

The association between epilepsy progression and serum BDNF levels was investigated. Several selected variables were controlled for but there might still be factors that confound this association. These results are nevertheless relevant to both clinicians who work with people with epilepsy and researchers who design clinical trials of drugs that target epilepsy with potential disease-arresting effects.

Acknowledgements

  1. Top of page
  2. Abstract
  3. Introduction
  4. Methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. Disclosure of conflicts of interest
  9. References
  10. Supporting Information

This work was funded by the National Natural Science Fund of China (no. 30900471), the China Postdoctoral Foundation (no. 20090451411) and the Sichuan Science and Technology Support Program (no. 2013SZ0003). We are grateful to Dr Gail S. Bell for critically reviewing the manuscript and to Dr LaFrance for his kind suggestions. JWS is supported by the Marvin Weil Epilepsy Research Fund.

Disclosure of conflicts of interest

  1. Top of page
  2. Abstract
  3. Introduction
  4. Methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. Disclosure of conflicts of interest
  9. References
  10. Supporting Information

The authors declare no financial or other conflicts of interest.

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  2. Abstract
  3. Introduction
  4. Methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. Disclosure of conflicts of interest
  9. References
  10. Supporting Information
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Supporting Information

  1. Top of page
  2. Abstract
  3. Introduction
  4. Methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. Disclosure of conflicts of interest
  9. References
  10. Supporting Information
FilenameFormatSizeDescription
ene12232-sup-0001-AppendixS1.docWord document29KAppendix S1. Measurement of serum BDNF.
ene12232-sup-0002-AppendixS2.docWord document77KAppendix S2. Excluded variables in the stepwise multiple linear regression analysis of BDNF.
ene12232-sup-0003-AppendixS3.docWord document819KAppendix S3. Relationship between seizure frequency and serum BDNF.

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