Neurology

A new biomarker in the differential diagnosis of epileptic seizure: Neurogranin

a b s t r a c t

Aim: The aim of this study was to determine the level of serum NGRN in epilepsy patients presenting at the Emer- gency Department with complaints of an epileptic seizure, and to thus evaluate the utility of this biomarker in the differentiation of epilepsy and PNES patients from each other.

Material methods: The study included patients aged >18 years who had experienced an epileptic seizure or were experiencing an epileptic seizure proven with EEG. All patients with brain disease of structural or infectious cause were excluded from the study (dementia, stroke, intracranial mass, meningitis, encephalitis, Creutzfeldt-Jacobs disease, abscess, etc). Patients were also excluded if they had traumatic brain injury or a severe systemic disease such as sepsis, which was thought to impair brain blood flow. The control group was formed of completely healthy volunteers.

Results: Evaluation was made of a total of 49 patients, comprising 19 (38.78%) males and 30 (61.22%) females, and a control group of 53 healthy volunteers comprising 28 (52.83%) males and 25 (47.17%) females. The serum neurogranin value was median 184.16 ng/dl (range: 110.1-1172.98) in the patient group and 97.90 ng/dl (range: 73.71-282. 11) in the control group. The serum neurogranin value was determined to be statistically sig- nificantly higher in the patient group than in the control group (p < 0.005).

Conclusion: The differential diagnosis of ES from PNES remains a challenging situation for emergency service phy-

sicians. Based on the findings of this study, it can be said that the serum NRGN level is high in patients who have experienced an epileptic seizure. Therefore, this new biomarker can be considered for use in the differential diagnosis of epileptic seizure and PNES.

(C) 2022

  1. Introduction

An epileptic seizure is a transient occurrence of signs and/or symp- toms due to abnormal excessive or synchronous neuronal activity in the brain [1]. epileptic seizures have been reported to account for 5% of 911 calls in the USA and for 1% of presentations at the Emergency De- partment (ED) [2]. One of the most important tasks of ED doctors is to differentiate an epileptic seizure (ES) from a psychogenic non- epileptic seizure (PNES). Although PNES is seen in 70% of patients in the second decade of life, it can also be seen in childhood and old age. In the differential diagnosis of ES and PNES, the patient history, and physical examination findings are important. Many of the clinical

* Corresponding author at: University of Health Sciences, Prof. Dr. Cemil Tascioglu City Hospital, Department of Emergency Medicine, Istanbul 34100, Turkey.

E-mail address: [email protected] (A. Kalkan).

features of these diseases are similar, and 10% of PNES patients even have a history of an actual ES. Therefore, the gold standard diagnosis method is video-EEG, which is able to differentiate epileptiform activi- ties of ES and PNES [3].

Neurogranin (NRGN) is a small neuronal protein responsible for postsynaptic signal transmission, which is found in the apical and basal dendritic extensions of pyramidal neurons and the cell bodies of cerebral cortex neurons. Previous experimental rat model studies have shown a relationship between NRGN and the management of Cognitive functions (orientation, learning) and the development of stress and anxiety [4]. NRGN can be determined in the serum or cerebral spinal fluid (CSF) in acute or chronic conditions where there is brain tissue damage (traumatic brain injury, Alzheimer’s disease, etc) [5]. Therefore, it is thought that this biomarker, which shows early brain damage and impaired signal transmission, may be able to differentiate an actual ES from PNES.

https://doi.org/10.1016/j.ajem.2022.02.010

0735-6757/(C) 2022

The aim of this study was to determine the level of serum NGRN in epilepsy patients presenting at the Emergency Department with com- plaints of an epileptic seizure, and to thus evaluate the utility of this bio- marker in the differentiation of epilepsy and PNES patients from each other.

  1. Material methods
    1. Study design

This prospective study included patients who had experienced an epileptic seizure and presented at the Emergency Department of Health Sciences University Prof. Dr. Cemil Tascioglu City Hospital between 01.02.2020 and 01.02.2021, and a healthy control group. Written in- formed consent of voluntary participation was obtained from all pa- tients and Control subjects. All procedures were applied in strict compliance with the Good Clinical Practices guidelines and the Helsinki Declaration. Approval for the study was granted by the Local Ethics Committee (decision no: 9, dated: 07/01/2020).

    1. Study groups

The study included patients aged >18 years who had experienced an epileptic seizure or were experiencing an epileptic seizure proven with EEG. All patients with brain disease of structural or infectious cause were excluded from the study (dementia, stroke, intracranial mass, meningitis, encephalitis, Creutzfeldt-Jacobs disease, abscess, etc). Pa- tients were also excluded if they had traumatic brain injury or a severe systemic disease such as sepsis, which was thought to impair brain blood flow. The control group was formed of completely healthy volun- teers. Patient data were recorded of age, gender, body temperature, pulse, blood pressure, peripheral oxygen saturation, previous diseases, consciousness status, type of seizure, time of presenting at ED after the seizure, and serum Creatinine kinase, lactate, and neurogranin levels. The data were compared between the two groups.

    1. Neurogranin analysis

A 10 cc blood sample was taken from the patients and the control group subjects for measurement of the serum NRGN level. After waiting for 30 mins, the blood samples were centrifuged at 1000 rpm for 20 mins. The sera obtained were placed in Eppendorf tubes and stored at

-80 ?C until assay. All the samples were removed from the deepfreeze 3 h before the biochemical analysis, left to thaw, then all were examined at the same time. The NRGN levels in the serum samples were measured using a commercially available SEA404Mi Neurogranin ELISA Kit (Wuhan USCN Business Co., Ltd./ USCN life science KIT INC. PRC- Lot: L 190110633) in an automatic microELISA device (ChemWell Awareness Technology, INC.). The results were stated as ng/ml.

    1. Statistical analysis
  1. Results

Evaluation was made of a total of 49 patients, comprising 19 (38.78%) males and 30 (61.22%) females, and a control group of 53

healthy volunteers comprising 28 (52.83%) males and 25 (47.17%) fe- males. In the histories of the patient group, there was hypertension in 4 (8.16%) patients, diabetes mellitus in 2 (4.08%), ischemic heart disease in 5 (10.2%), and asthma in 2 (4.08%). It was recorded that 45 (91.84%) patients had experienced a generalized tonic clonic seizure, and 4 (8.16%) patients had experienced a tonic seizure. The patients were sep- arated into 3 groups according to the time of presentation after the sei- zure, as 0-1 h, 1-6 h, and > 6 h. In the blood tests of the patients, WBC, hemoglobin, platelet, glucose, urea, creatinine, serum electrolyte, and liver function test mean values were within the normal range.

The mean CRP value was determined to be 2.33 mg/L (range: 0.20-27.02), mean lactate was 3.30 mmol/L (range: 0.60-19 mmol/L) and mean creatine kinase (CK) was 95 U/L (range: 0.60-19). The mean age of 29 years (range:18-60) in the patient group and 33 years (range:35-760) in the control group were determined to be higher than the normal values (Table 1). No statistically significant difference was determined between the groups in respect of mean age (p > 0.005). In the patient group, median systolic blood pressure was deter- mined as 120 mmHg (range:90-165), diastolic blood pressure as mean 70 + 9.98 mmHg, body temperature mean 36 ?C (range:36-37.5), pulse median 84 bpm (range:60-133), and the saturation median value was 97% (range:92-100). In the control group, median systolic blood pres- sure was determined as 116 mmHg (range: 101-132), diastolic blood pressure as mean 74 + 6.97 mmHg, body temperature mean 36.5 ?C (range:36.2-36.9), pulse median 84 bpm (range:66-95), and the satu- ration median value was 97% (range:95-100). No statistically signifi- cant differences were determined between the groups in respect of these vital signs (p > 0.05).

The serum neurogranin value was median 184.16 ng/dl (range: 110.1-1172.98) in the patient group and 97.90 ng/dl (range: 73.71-282. 11) in the control group. The serum neurogranin value

Table 1

Demographic characteristics of patients in the study.

Patient group (n = 49)

Age (years) 29 (18:60)

Gender

Male 19 (38.78%)

Female 30 (61.22%)

Comorbidities

HT

4 (8.16%)

DM

2 (4.08%)

Ischaemic heart disease

5 (10.20%)

Asthma/COPD

2 (4.08%)

Time of presentation after seizure

0-1 h 24 (48.98%)

1-6 h 21 (42.86%)

Data obtained in the study were analyzed statistically using SPSS for

>6 h

3

4 (8.16%)

Windows, Version 21.0 software (IBM Corpn., released 2012. IBM SPSS Statistics, Armonk, NY, USA). Conformity of the data to normal distribu- tion was assessed with the Shapiro Wilk test. Continuous variables were presented as median (minimum: maximum) and mean +- standard de- viation values. Categorical variables were reported as number (n) and percentage (%). According to the normality test results, the Mann Whit- ney U test or the Independent Samples t-test was used in comparisons between two groups. For the comparisons of categorical variables, the Pearson Chi-square test or Fisher’s Exact Chi-square test was used. Cor- relations between continuous variables were analyzed using correlation analysis. According to the normality test results, the Spearman correla- tion coefficient was calculated. A value of p < 0.05 was considered sta- tistically significant.

WBC (10 /uL) 8.44 (4.30:23.46)

HGB (g/L) 13.50 (7.80:15.90)

PLT (103/uL) 236.73 +- 52.04

Glucose (mg/dl) 101 (84:252)

Urea (mg/dl) 26 (0.90:63)

Creatinin (mg/dl) 0.72 (0.32:27)

Sodium (mmol/L) 139 (125:145)

Potassium (mmol/L) 4.13 +- 0.35

ALT (U/L) 16 (6:74)

AST (U/L) 21 (11:62)

CRP (mg/L) 2.30 (0.20:27.02)

Lactate (mmol/L) 3.30 (0.60:19)

CK (U/L) 95 (35:760)

GCS 15 (3:15)

Data are expressed as n(%), median(minimum:maximum) and mean

+- standard deviation.

Table 2

Comparisons between the groups

Total

(n = 102)

Control Group (n = 53)

Patient Group (n = 49)

p value

higher levels compared to a control group. This elevation in NRGN levels was associated with neuronal damage [5]. The determination of higher serum NRGN levels in the patients in the current study compared to the control group could be due to the neuronal damage caused in an epi-

Age (years) 31.50 (18:67) 33 (18:67) 29 (18:60) 0.925a

Gender

Male 47 (46.08%) 28 (52.83%) 19 (38.78%) 0.155b

Female 55 (53.92%) 25(47.17%) 30 (61.22%)

lepsy attack.

The blood-brain barrier (BBB), a unique anatomic and physiological interface between the central nervous system (CNS) and the peripheral circulation, is essential for the function of neural circuits. Interactions be-

Neurogranin

(ng/ml)

Blood pressure (mm/hg)

126.98

(73.71:1172.9)

97.90

(73.71:282.1)

184.16

(110.10:1172.)

<0.001a

tween the BBB, cerebral blood vessels, neurons, astrocytes, microglia, and pericytes form a dynamic functional unit known as the neurovascular unit (NVU). NVU-BBB crosstalk plays a key role in the regulation of blood flow,

Systolic 120 (90:165) 116 (101:132) 120 (90:165) 0.024a

Diastolic 72.39 +- 8.68 74.06 +- 6.97 70.59 +- 9.98 0.047d

response to injury, neuronal firing, and synaptic plasticity. Dysfunction of

the BBB is a hallmark of brain injury, and a prominent finding in status

Body temperature (?C)

36.60

(36:37.50)

36.50

(36.20:36.90)

36.60

(36:37.50)

0.066a

epilepticus [17].

Pulse 80 (60:133) 78 (66:95) 84 (60:133) 0.015a

Saturation (%) 97 (92:100) 97 (95:100) 97 (92:100) 0.851a

Data are expressed as n (%), median (minimum:maximum) and mean +- standard deviation.

a: Mann-Whitney U test, b: Pearson Chi-square test, c: Fisher’s Exact test, d: Independent Samples t-test.

Table 3

Neurogranin (n = 49)

rs p-value

The BBB has been shown to be disrupted in rat models of

experimentally-induced epilepsy. These studies have shown that some neuromediators from the BBB, which is disrupted in status epilepticus or temporal lobe epilepsy, participate in blood circulation [18,19].

Neurogranin is a small neuronal protein responsible for postsynaptic signal transmission, which is found in the apical and basal dendritic ex- tensions of pyramidal neurons and the cell bodies of cerebral cortex neurons. The elevated levels of NGRN in epileptic seizures are thought to be associated with disruption of the blood brain barrier. The use of this type of biomarker in ED to differentiate real epileptic seizures from PNES in particular would be useful. The results of this study found higher levels of serum NGRN in the patient group than in the healthy control group. Strong aspects of the study were that the epilep-

CK

0.13

0.384

tic seizures were confirmed with EEG and that the CK levels were also

LACTATE

0.02

0.888

high in the patient group.

rs: spearman correlation coefficient.

was determined to be statistically significantly higher in the patient group than in the control group (p < 0.005) (Table 2) (See Table 3).

No statistically significant correlation was determined between neurogranin and CK and lactate levels (p > 0.05). In all the patients who had experienced an epileptic seizure, as neurogranin increased, so the CK and lactate levels increased, but this positive increase showed only a mild correlation.

  1. Discussion

The results of this study demonstrated that the serum neurogranin levels of patients who had experienced an epileptic seizure were higher than those of healthy individuals. The level of NRGN protein has previ- ously been determined to be elevated both in the serum and in CSF in acute and chronic diseases of the brain [6]. This protein first started to be a subject of research in chronic brain damage. High levels of NGRN have been determined in both serum and CSF in patients with chronic diseases such as Alzheimer’s disease [7-11]. There are also recent stud- ies reporting that serum NGRN levels are significantly elevated in acute brain damage.

One such study by Yang et al. found higher serum NGRN levels in pa- tients with traumatic brain injury compared to a control group [12]. In another study of patients with Mild head trauma with a Glasgow coma score of 13-15, the relationship was examined between brain im- aging findings and the serum NRGN levels. It was determined that the serum NRGN levels were higher in patients with pathological imaging findings, and the elevated NRGN levels were attributed to disruption of the blood brain barrier [13]. Studies of both ischaemic and hemor- rhagic stroke patients have also reported high NRGN levels [14,15].

De Vos et al. determined that with an increase in ischaemic area, serum NRGN levels were much more increased in ischemic stroke pa- tients, and thus it was stated that NRGN could be a good biomarker to determine ischemic stroke [16]. Yesilyurt et al. evaluated serum NRGN levels in patients with carbon monoxide poisoning, and determined

  1. Limitations

The most important limitations of this study were the low number of patients in the sample and that the serum NRGN levels were not followed up at later times. Impairment of the blood brain barrier can last for a long time after an epileptic seizure, and therefore NRGN levels may have been higher at later times. In all but 4 patients in this study, the NRGN levels of the patients were only examined at up to 6 h after the seizure. If there had been follow-up of serum NRGN during the hos- pitalization period, this could have provided further support for the study findings. Nevertheless, the findings obtained support the study hypothesis that NRGN could be used in ED for early stage differential di- agnosis from PNES. Another limitation of the study could be said to be that the epileptic seizures of the patients were only confirmed with EEG and serum CK levels. There are some studies in literature that have compared multiple biomarkers [20-22] and reported that new bio- markers have supported biomarkers that have been previously proven to be elevated in epileptic seizures. That the findings were supported by serum CK levels, which is increased in the early period and readily available in ED, was thought to strengthen the value of this study.

  1. Conclusion

The differential diagnosis of ES from PNES remains a challenging sit- uation for emergency service physicians. Based on the findings of this study, it can be said that the serum NRGN level is high in patients who have experienced an epileptic seizure. Therefore, this new biomarker can be considered for use in the differential diagnosis of epileptic seizure and PNES.

Credit authorship contribution statement

Asim Kalkan: Conceptualization, Formal analysis, Methodology, Project administration, Supervision, Writing – original draft, Writing – review & editing. Ahmet Demirel: Supervision, Methodology. Seref Emre Atis: Software, Formal analysis, Conceptualization. Edip Burak

Karaaslan: Writing – review & editing, Methodology, Investigation, For- mal analysis, Data curation. Mehmet Esat Ferhatlar: Writing – original draft, Supervision, Project administration, Formal analysis, Data curation. Mucahit Senturk: Methodology, Investigation, Conceptualization.

Declaration of Competing Interest

The authors have no conflict of interests to disclose.

Acknowledgements

No financial support was received for this manuscript.

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