Serum adiponectin,leptin,and interleukin 6 levels as adipocytokines in children with febrile seizures

Introduction

Febrile seizures (FS) are the most common convulsive state in children less than 60 months. The pathophysiology of FS has not been clearly explained yet. ¹ The balance between proinflammatory and anti-inflammatory cytokines may play important role in the pathogenesis of FS. ^{2 –4} Adipocytokines like interleukin-6 (IL-6), leptin, and adiponectin released from adipose tissue play a role in inflammation. Leptin and IL-6 have roles similar to proinflammatory cytokine. Adiponectin is a double-acting cytokine, which primarily has anti-inflammatory effects, but it has also proinflammatory effects. ⁵

There are several studies investigating the relationship of cytokines and FS ^{2,3,6 –8} and serum leptin levels in children with FS ⁹ ; however, there has been no clinical study evaluating the role of adipose tissue in FS, as far as we know. Therefore, we proposed to evaluate the role of adipose tissue on the pathogenesis of FS by measuring serum IL-6, leptin, and adiponectin levels in children with FS and compared the results with the values of children of the same age with febrile illness without seizures and a healthy control group.

Materials and methods

This prospective study included patients with FS (n = 32) and acute febrile disease without seizures (febrile control, FC; n = 26) who had been hospitalized in the pediatric unit between March 2013 and December 2013. The healthy control group (HC) consisted of 29 subjects with no history of other potential health problems. The study protocol was approved by the Human Ethics Committee of the local committee, and informed consent was obtained from the parents of children (no: 2013-03/26).

Laboratory and clinical data including C-reactive protein (CRP), complete blood counts (CBCs), body temperature (in Celcius) at admission, duration of fever before obtaining the blood, and fever when hospitalized were obtained from the medical records. The inclusion criteria for FS were the age of the child should be from 6 to 60 months with an axillary temperature of at least 38°C or higher. Exclusion criteria for FS were any history of prior afebrile seizures, central nervous system infection, or metabolic imbalance. ^1,10 FS were divided into two groups, namely, simple FS (SFS) group (n = 21), defined as primary generalized seizures that lasted for less than 15 min and did not recur within 24 h, and complex FS (CFS) group (n = 11), defined as focal, prolonged (≥15 min), and/or recurrent within 24 h. ^1,10 Control groups were divided into two groups, namely, FC group (n = 26) hospitalized at our pediatric unit, children with fever due to infection except for central nervous system infection and absence of history of prior febrile and afebrile seizures and HC group (n = 29) with an absence of history of prior febrile and afebrile seizures and no fever or infection. Data regarding duration of the seizure and duration of fever before seizure were obtained from the parents by a questionnaire.

In this study, serum samples were collected as soon as venous lines were placed in study and control groups. Blood samples were centrifuged, and the serum was stored below −80°C in the refrigerator until analysis. All clinical chemical measurements were performed at the Clinical Laboratory of Cumhuriyet University Medical Research Hospital.

For all patients in the study and control groups, serum IL-6, leptin, and adiponectin levels were measured. The serum levels of IL-6 were assessed using an enzyme-linked immunosorbent assay (ELISA) according to the manufacturer’s instructions (CLB kit, Amsterdam, the Netherlands). Serum adiponectin levels were measured with the enzyme immunoassay kit on instant sandwich ELISA technology (eBioscience Campus Vienna Biocenter 2, Vienna, Austria). The intraassay coefficients of variation (CVs) value were 4.2% and the interassay CV was 3.1% for adiponectin. Serum leptin levels were determined by ELISA technology (BioVendor, Brno, Czech Republic). The intraassay CV value was 4.2% and the interassay CV was 6.7% for leptin.

Results

Table 1 shows the comparison of selected clinical and laboratory data among the FS group with control groups. The mean age was 23.7 ± 14.5 months; 18 (56%) FS were male. The FS, FC, and HC groups were found to be comparable with regard to the age, body weight, height, body mass index (BMI), and the ratios of gender (p > 0.05). Serum levels of white blood cells (WBCs), CRP, IL-6, leptin, and adiponectin were found to be significantly higher and serum hemoglobin (Hb) levels were found to be significantly lower in FS and FC groups than in the HC group (p < 0.001). When we compared the FS with the FC group, the serum Hb levels were significantly lower in the FS group than those in the FC group (p = 0.001). There was no significant difference among all the groups with regard to the thrombocyte count and serum levels of biochemical parameters such as glucose, sodium, potassium, calcium, blood urea nitrogen, creatinine, aspartate aminotransferase, and alanine aminotransferase (p > 0.05). Additionally, there was no significant difference between FS and FC group with regard to the body temperature on admission, serum levels of WBC, Hb, CRP, IL-6, leptin, and adiponectin and thrombocyte count (p > 0.05).

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Table 1.

Comparison of clinical and laboratory findings among the patient group with control groups.^a

Characteristics	FS group (n = 32)	FC group (n = 26)	HC (n = 29)	p Value (within groups)	p Value (FS and HC)	p Value (FC and HC)	p Value (FS and FC)
Age (months)	23.7 ± 14.5	34.2 ± 20.3	25.5 ± 16.2	0.054	0.911	0.145	0.055
Gender (male/female)	18/14	14/12	17/12	0.939	0.853	0.724	0.856
BT (°C) on admission	38.6 ± 0.4	38.5 ± 0.4	–	0.530	–	–	0.715
Body weight (kg)	12.5 ± 3.6	14.4 ± 4.0	13.5 ± 4.4	0.147	0.358	0.357	0.069
Height (cm)	85.3 ± 12.7	92.7 ± 14.9	88.9 ± 13.5	0.172	0.308	0.340	0.072
BMI	14.1 ± 2.4	15.7 ± 2.8	16.7 ± 2.1	0.104	0.506	0.152	0.053
Laboratory findings
Glucose (mg/dL)	98.3 ± 12.0	95.4 ± 14.2	91.8 ± 12.9	0.216	0.069	0.371	0.415
Sodium (mmol/L)	136.5 ± 2.9	137.4 ± 2.3	139.0 ± 1.8	0.114	0.209	0.053	0.336
Potassium (mmol/L)	4.3 ± 0.5	4.1 ± 0.5	4.5 ± 0.4	0.314	0.534	0.149	0.297
Calcium (mg/dL)	9.5 ± 0.5	9.7 ± 0.6	9.9 ± 0.4	0.347	0.133	0.495	0.481
BUN (mg/dL)	11.6 ± 2.8	11.2 ± 3.3	11.7 ± 3.0	0.804	0.857	0.550	0.618
Creatinine (mg/dL)	0.4 ± 0.1	0.4 ± 0.1	0.5 ± 0.1	0.085	0.119	0.167	0.894
AST (IU/L)	38.0 ± 9.1	33.4 ± 12.5	33.2 ± 6.3	0.700	0.086	0.365	0.643
ALT (IU/L)	21.7 ± 7.3	22.6 ± 9.3	18.8 ± 4.8	0.063	0.092	0.07	0.662
WBC (×109 cells/L)	14.2 ± 5.4	13.0 ± 5.3	8.2 ± 2.4	<0.001	<0.001	<0.001	0.416
Hb (g/dL)	11.5 ± 0.8	12.3 ± 1.0	13.5 ± 0.9	<0.001	<0.001	<0.001	0.001
Plt (×109 cells/µL)	286.3 ± 70.0	332.6 ± 108.6	326.8 ± 97.7	0.246	0.114	0.846	0.217
CRP (mg/L)	14.8 (1.2−96.3)	26.9 (1.1−423.0)	2.1 (0.5−39.2)	0.001	<0.001	<0.001	0.080
IL-6 (ng/mL)	22.8 (2.2−397.1)	17.7 (2.3−456.9)	2.5 (0.1−7.0)	0.009	<0.001	<0.001	0.669
Leptin (ng/mL)	1.2 (0.5−3.5)	1.0 (0.7−4.8)	0.6 (0.1−13.9)	0.002	0.002	0.003	0.962
Adiponectin (ng/mL)	612.5 (113.0−1042.0)	684.0 (353.0−888.0)	291.0 (15.0−728.0)	<0.001	<0.001	<0.001	0.291

FS: febrile seizures; FC: febrile control; HC: healthy control; BT: body temperature; BMI: body mass index; BUN: blood urea nitrogen; AST: aspartate aminotransferase; ALT: alanine aminotransferase; WBC: white blood cell; Hb: hemoglobin; Plt: platelet count; IL-6: interleukin 6; CRP: C-reactive protein.

^aData shown mean ± standard deviation and median (min−max).

The mean age was 26.2 ± 15.3 months in the SFS group and 18.8 ± 12.2 months in the CFS group. The SFS and CFS groups were found to be comparable regarding the age, body weight, height, BMI, and the ratios of gender (p > 0.05). When we compared the SFS with the CFS group, body temperature on admission was significantly higher in the SFS group (38.8 ± 0.3°C) than those in the CFS group (38.4 ± 0.4°C; p = 0.02). There were no significant differences between the SFS and CFS groups with regard to the serum levels of WBC, Hb, CRP, IL-6, leptin, adiponectin and thrombocyte count, and biochemical parameters (p > 0.05). Results of the clinical and laboratory characteristics of the SFS and CFS group are listed in Table 2.

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Table 2.

Comparison of clinical and laboratory findings between the patient groups.^a

Characteristics	SFS group (n = 21)	CFS group (n = 11)	p Value
Age (months)	26.2 ± 15.3	18.8 ± 12.2	0.174
Gender (male/female)	12/9	6/5	0.890
Body weight (kg)	13.2 ± 3.8	11.3 ± 2.8	0.208
Height (cm)	87.1 ± 13.7	81.9 ± 10.3	0.374
BMI	17.4 ± 2.8	16.6 ± 1.6	0.606
BT (°C) on admission	38.8 ± 0.3	38.41 ± 0.35	0.020
Duration of fever before FS (<24 h/>24 h)	14/7	7/4	0.907
Laboratory findings
Glucose (mg/dL)	99.0 ± 12.3	96.8 ± 11.9	0.450
Sodium (mmol/L)	138.7 ± 2.3	137.8 ± 1.8	0.213
Potassium (mmol/L)	4.2 ± 0.5	4.4 ± 0.4	0.647
Calcium (mg/dL)	9.4 ± 0.5	9.8 ± 0.6	0.091
BUN (mg/dL)	11.5 ± 2.9	11.7 ± 2.9	0.905
Creatinine (mg/dL)	0.4 ± 0.1	0.4 ± 0.2	0.368
AST (IU/L)	35.6 ± 8.8	38.0 ± 4.2	0.223
ALT (IU/L)	21.2 ± 7.6	22.6 ± 7.0	0.282
WBC (×109 cells/L)	14.3 ± 5.5	14.1 ± 5.4	0.984
Hb (g/dL)	11.6 ± 0.7	11.4 ± 0.8	0.487
Plt (×109 cells/µL)	294.8 ± 62.0	270.1 ± 84.1	0.351
CRP (mg/L)	16.6 (2.5−96.3)	9.8 (1.2−23.9)	0.060
IL-6 (ng/mL)	22.5 (4.3−397.1)	28.8 (2.2−201.4)	0.751
Leptin (ng/mL)	1.1 (0.50−3.50)	1.2 (0.6−1.9)	0.968
Adiponectin (ng/mL)	631.0 (113.0−1042.0)	571.0 (129.0−869.0)	0.439

FS: febrile seizures; SFS: simple febrile seizures; CFS: complex febrile seizures; BT: body temperature; BMI: body mass index; BUN: blood urea nitrogen; AST: aspartate aminotransferase; ALT: alanine aminotransferase; WBC: white blood cell; Hb: hemoglobin; Plt: platelet count; IL-6: interleukin 6; CRP: C-reactive protein.

^aData shown mean ± standard deviation and median (min−max).

Discussion

FS are the most common form of convulsions in children. The pathophysiology of this age-specific condition still remains uncertain. ¹ The balance between proinflammatory and anti-inflammatory cytokines may play a key role in the pathogenesis of FS. ^{2 –4} The children who are prone to seizures secrete high amount of proinflammatory cytokine, so it may be said that a high amount of anti-inflammatory cytokine secretion may protect children from seizures. This situation may occur as a direct effect of the ionic current. It may also be formed indirectly by increasing extracellular glutamate concentrations or decreasing function of γ-aminobutyric acid (GABA)-A receptor. ⁴ Adiponectin, IL-6, leptin, ometin, tumor necrosis factor α (TNF-α), vaspin, and visfatin are adipocytokines and are excreted from adipose tissue. These adipocytokines are thought to play an important role in the etiopathogenesis of variable physiologic and metabolic functions. ¹¹

A few nonclinical studies have noted the double role of IL-6 in seizures. De Sarro et al. ¹² reported that seizure sensitivity to glutamate receptor agonists had increased IL-6 in knockout mice. Increased seizure sensitivity to glutamate receptor agonists was also reported in transgenic mice overexpressing IL-6 in astrocytes. This may be due to reduced GABA-mediated inhibition. ¹³ In the study of Furukawa and Mattson, intranasal administration of IL-6 to developing rats prolonged the latent period and decreased the duration of seizure induced by hyperthermia. ¹⁴ They suggested that IL-6 had an anticonvulsant effect on febrile seizures. Although the studies showed that serum levels of IL-6 increased in patients with febrile seizure, ^3,8,15 in our study, we found no significant difference between FS and FC groups with regard to the serum IL-6 levels and between SFS and CFS as well. In addition, we found higher levels of IL-6 in FS and FC groups than HC groups. So it may be said that IL-6 was increased as an acute-phase reactant.

Leptin and adiponectin effects are mediated directly and indirectly. Direct effect occurs through actions on specific tissues, and the indirect effect is through central nervous system, endocrine and neural mechanisms. ⁵ As the condition of blood leptin’s possibility of accessing its receptors on these neurons, leptin modulation of neuronal excitability outside the hypothalamus becomes potentially important. Leptin transporters in the brain provide easy access of leptin to its receptors on the hippocampus and other cortical neurons. ¹⁶ Leptin has controversial effects on seizures. According to some animal and experimental studies, ^{17 –19} it has an anticonvulsant effect, while in some studies, it has opposite effects such as proconvulsant and convulsant. ^{20 –22} For studies on animals, exercising the arcuate nucleus of leptin on rat hypothalamic slices decreases the impulse of glutamatergic excitatory postsynaptic. ¹⁷ Leptin has also decreased stimulated evoked epileptiform-like activity in hippocampal brain cell cultures in rats. ^18,19 The effects of leptin on convulsant-related activity are produced by various selective glutamate receptor (N-methyl-d-aspartate receptor, α-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid receptor, and kainate receptor) agonists as well as by the glutamate itself. ^{20 –22} According to a study on children with febrile convulsion by Khoshdel et al., there was no important change in serum leptin levels between children with simple febrile seizures (n = 25) and febrile children without seizures (n = 25). ⁹ Our results were compatible with the results of Khosdel et al.

Adiponectin regulates hypothalamic and brainstem neuronal activity as well as centrally controlling the peripheral metabolism. ²³ Adiponectin has a protective role against ischemic brain injury due to modulating inflammatory pathways and endothelial function. ^24,25 According to the animal study by Jeon et al., intracerebral injection of adiponection lightened hippocampal neuronal damage induced by kainic acid. ²⁶ Adiponectin induces the secretion of various anti-inflammatory cytokines such as IL-10 and IL-1 receptor antagonist (RA) by human monocytes, macrophages, and dendritic cells. In addition, it decreases the production of interferon-γ ²⁷ as well as proinflammatory mediators such as TNF-α and IL-6 and inhibit adiponectin gene expression. ²⁸ Straussberg et al. studied the production of IL-1β, IL-6, and IL-10 and TNF-α in children with FS. ² They reported that higher IL-1β levels detected after 5 h of incubation with lipopolysaccharide were suppressed by IL-10 after 24 h of incubation. In one study, it was reported that FS were not related to the IL-1β promoter, IL-1β exon 5, IL-6 promoter, IL-8, IL-10, and TNF-α promoter gene polymorphisms. But IL-1 RA had been found important in predicting FS in Taiwanese children (104 subjects with FS and 143 HC subjects). ⁴ There were two different results reported from Turkey related to IL-1 RA polymorphism. ^29,30 In the study by Haspolat et al., there was no significant effects of IL-1 RA intron 2 variable tandem repeat polymorphisms on FS. ²⁹ According to these results, adiponectin might play an important role in the pathogenesis of FS. In literature, we couldn’t see any study related to adiponectin levels in children with FS. In our study, serum adiponectin was found higher in FS and FC groups than the HC group. But we couldn’t find any difference between FS and HC groups and between SFS and CFS in terms of serum adiponectin. This study showed that elevated levels of adipocytokines as acute-phase reactants in FS and FC groups did not contribute to the development of febrile seizures.

Conclusion

Although many studies associated the role of cytokines related with FS have existed in the literature, any etiopathogenetic relationship between cytokines originating from adipose tissue and FS has not been shown in this study. Therefore, for a more accurate assessment of the role of adipose tissue in FS, further studies involving more patients and investigating the serum and CSF levels of those and other cytokines (i.e. TNF α, vaspin, and visfatin) released from adipose tissue are needed.