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pilepsy Research (2013) 107, 244—252

j ourna l h om epa ge: www.elsev ier .com/ locate /ep i lepsyres

nflammation and iron metabolism in adultatients with epilepsy: Does a link exist?

. Tombinia,∗, R. Squitti a,b, F. Cacciapagliac, M. Ventrigliaa,b,

. Assenzaa, A. Benvengaa, G. Pellegrinoa, C. Campanaa,. Assenzaa, M. Siottog, L. Pacificih, A. Afeltrad, P.M. Rossinib,e,f

Neurology, Campus Bio-Medico University, Rome, ItalyAFaR, Department of Neuroscience, Hosp. Fatebenefratelli, Isola Tiberina, Rome, ItalyInternal Medicine and Rheumatology, N. Melli Hospital, San Pietro Vernotico, Brindisi, ItalyDepartment of Clinical Medicine and Rheumatology, University ‘‘Campus Bio-Medico’’ of Rome, ItalyNeurology, Catholic University of Rome, ItalyCasa di Cura S. Raffaele and IRCCS S. Raffaele-Pisana, Rome, ItalyFondazione Don C. Gnocchi ONLUS, Milan, ItalyNeurology, Hosp. Fatebenefratelli, Isola Tiberina, Rome, Italy

eceived 20 January 2013 ; received in revised form 10 July 2013; accepted 17 September 2013vailable online 28 September 2013

KEYWORDSEpilepsy;Iron;Cytokines;Inflammation;IL-6;TNF-�

SummaryPurpose: Inflammation has been shown to play a key role in epilepsy, and may also affect boththe iron status and metabolism. Consequently, a relationship between iron metabolism andneuronal excitability and seizures could be expected.Methods: We aimed at characterizing in 37 adult patients affected by focal epilepsy duringthe interictal period serum inflammatory cytokines, such as interleukin 6 (IL-6), IL-6 solublereceptor (IL6-sR), interleukin 1 (IL-1), IL-1 receptor-antagonist (IL-1RA), tumor necrosis factor-� (TNF-�), and markers of iron status and metabolism: hemoglobin concentration (Hgb), meancorpuscular volume (MCV), hematocrit (Hct) red blood cell (RBC) count, serum iron and copperconcentrations, ceruloplasmin (iCp), the ceruloplasmin enzymatic activity (eCp), the specificceruloplasmin activity (eCp/iCp), total ferroxidase activity, transferrin (Tf), serum ferritin (SF),Tf saturation (Sat-Tf), and ratio of ceruloplasmin to transferrin (Cp/Tf). We investigated thecorrelations between these biological markers as well their relationship with patients’ clinical

features. A group of 43 healthy subjects had the same serologic measurements to serve ascontrols.Results: Our findings showed in the group of patients with epilepsy an increase of IL-6 (p = 0.026)and a decrease of TNF-� (p = 0.002) with respect to healthy subjects. For the first time, wealso detected significant changes in iron metabolism as an increase of Cp/Tf (p = 0.011) anda decrease of Tf (p = 0.031), possibly driven by cytokine modifications and consistent with

∗ Corresponding author at: Neurologia, Università Campus Bio-Medico, Via Alvaro del Portillo 200, Trigoria, 00128 Rome, Italy.el.: +39 06 225411286; fax: +39 06 225411028.

E-mail address: m.tombini@unicampus.it (M. Tombini).

920-1211/$ — see front matter © 2013 Elsevier B.V. All rights reserved.ttp://dx.doi.org/10.1016/j.eplepsyres.2013.09.010

Inflammation and iron metabolism in adult patients with epileps

inflammation as acute phase andcorrelated with Tf (p = 0.005). Fiquency and eCp (p = 0.046) and incorrelation between TNF-� and thConclusions: Our findings demoninflammation, with a consistent li

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and iron regulation and meta© 2013 Elsevier B.V. All right

Introduction

Epilepsy is a brain disorder that affects 50 million peopleworldwide and is characterized by an enduring predisposi-tion to generate seizures (Duncan et al., 2006). Nowadays,there is an increasing amount of clinical and experimentalevidence supporting the hypothesis that brain inflamma-tory processes might represent a key mechanism in thepathophysiology of seizures and epilepsy (Riazi et al.,2010; Vezzani and Granata, 2005; Vezzani et al., 2011).First, insights into this potential role come from clini-cal experiences, suggesting that immune and inflammatorymechanisms could be critical in febrile seizures (increaseof cytokines and proinflammatory agents) and some formsof epilepsies (i.e. Rasmussen encephalitis, limbic encephali-tis, etc.); furthermore, steroids and other anti-inflammatorytreatments were successfully used as anticonvulsant drugsin some drug-resistant epilepsies (Riikonen, 2004; Whelesset al., 2007; Wirrell et al., 2005). Second, over the pastdecade, several studies using in vivo and in vitro experi-mental models demonstrated a bidirectional link betweenepilepsy and inflammation, suggesting how seizures itselfcould generate an inflammatory response, which can inturn modulate epilepsy, inducing cortical hyperexcitabil-ity, cell loss and synaptic reorganization (for an extensivereview see Vezzani et al., 2011). In particular, many pre-clinical studies in experimental models of seizures haveextensively confirmed that proinflammatory cytokines suchas interleukin-1� (IL-1�), tumor necrosis factor-� (TNF-�)and interleukin-6 (IL-6) are over expressed in brain areasof seizure generation and propagation, prominently by glia(microglia and astrocytes) and to a lesser extent by neurons(Vezzani and Granata, 2005). Cytokine receptors seem to bealso upregulated in microglia, astrocytes and neurons, andthe related intracellular signaling is activated in these cellpopulations highlighting autocrine and paracrine actions ofcytokines in the brain (Vezzani et al., 2008). Furthermore,cytokines have been shown to promote neuronal hyperex-citability and affect seizures in rodents; with this regard,clear evidence exists in particular for IL-1�, exhibiting a sig-nificant proconvulsant activity in a large variety of seizuremodels (Vezzani and Granata, 2005). On the other hand, reg-ulatory mechanisms exist including production of proteinsthat compete with cytokines to bind their receptors, such asIL-1 receptor antagonist protein (IL-1RA) (Dinarello, 2009).

Furthermore, it has been also demonstrated thatinflammation and cytokine profile alterations may affect— at systemic level — both the iron status and red blood

cell (RBC) profile. Chronic immunostimulation is oftenassociated with a decrease in serum iron, thereby loweringthe bioavailability of iron in order to contrast uncontrolled

ewa

y 245

antioxidant activity markers. Accordingly, TNF-� positivelynally, a significant positive correlation between seizures fre-versely with Hgb (p = 0.038) and Hct (p = 0.041), and an inversee duration of epilepsy (p = 0.021) was detected.strate a relevant relationship between epilepsy and systemicnk between seizures, inflammatory cytokines (IL-6 and TNF-�)m, as acute phase and antioxidant markers.erved.

ellular proliferation, and reducing the excessive pro-uction of reactive oxygen radicals and/or to withhold ironrom pathogenic microorganisms (Koorts et al., 2011). Theffects of cytokines on iron bioavailability are largely medi-ted through a reduction in duodenal absorption of iron and

shift in the handling of iron by the macrophage in favor ofron storage, leading to hypoferremia (Alvarez-Hernandezt al., 1989; Fahmy and Young, 1993; Worwood, 1990)nd hemosiderosis of the macrophage (Finch et al., 1984;ipschitz et al., 1971; Worwood, 1990). This reduction inioavailable iron can contribute to the anemia of chronicisease (Weiss and Goodnough, 2005). In addition, theroinflammatory cytokines TNF-�, IL-1�, IL-6, and the anti-nflammatory cytokine IL-10 have all been shown to directlytimulate the transcription and translation of ferritin, theajor intracellular protein responsible for macrophage iron

torage (Ludwiczek et al., 2003; Miller et al., 1991; Rogers,996). Cytokines not only upregulate ferritin expression,ut also stimulate the macrophage to increase its uptakef iron by increasing the expression of the divalent metalransporter-1 (Ludwiczek et al., 2003). Moreover, cytokinesre responsible for the regulation of acute phase proteinynthesis during chronic immune stimulation (Castell et al.,989). Transferrin (Tf), the iron transport protein thatinds iron in the plasma and circulates it between theron-containing compartments (intercellular iron shuttling),s a negative acute phase protein, and as such serumerritin (SF) is reduced during immune stimulation (Weissnd Goodnough, 2005), resulting in less iron being availableor cellular processes. Ceruloplasmin (Cp), a multicoppernzyme carrying around 95% of circulating copper (Cu), isn acute phase protein, and cytokines (Il-6, IL-1 and TNF-�)ave been shown to increase its biosynthesis (Ramadorit al., 1988). Cp catalyzes the oxidation of ferrous ironFe2+) into ferric iron (Fe3+) that binds to Tf by which it isransported to cells. This enzymatic activity confers to Cp

relevant antioxidant power (Bielli and Calabrese, 2002).Iron plays a key role in the metabolism of several

nzymes and neurotransmitters, and in low iron statusldehyde oxidases and monoamine are reduced. In addi-ion, the expression of cytochrome C oxidase, a markerf neuronal metabolic activity, is decreased in iron defi-iency (DeUngria et al., 2000). Moreover, many studies havelearly demonstrated the effect of iron on development,ognition, behavior and neurophysiology, and especially onrain metabolism, neurotransmitter function and myelina-ion (Madan et al., 2011). Thus, a relationship between ironetabolism and neuronal excitability and seizures could be

xpected. This issue has been addressed in the past, butith results somewhat conflicting. Several studies reportedn association between iron-deficiency anemia and febrile

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onvulsions (Pisacane et al., 1996; Zareifar et al., 2012),nd found lower levels of serum ferritin (SF) in childrenith febrile seizure compared to controls (Daoud et al.,002; Naveed-Ur-Rehman and Billo, 2005), indicating thathildren with iron deficiency are more prone to febrileeizures. On the contrary, other authors failed in obtain-ng the same results (Momen and Hakimzadeh, 2003) orndeed showed opposite findings (Bidabadi and Mashouf,009), suggesting in some cases that iron-deficiency ane-ia could raise the threshold for febrile seizure (Kobrinsky

t al., 1995). In a recent meta-analysis (Idro et al., 2010),hat examined the association between iron deficiency andcute/febrile seizures, iron deficiency was associated with

significantly increased risk of seizures, weighted OR 1.7995% CI 1.03—3.09). Finally, Ozaydin et al. (2012) foundhat iron deficiency anemia was more frequently seenmong the patients with complex febrile seizures than theatients with simplex febrile seizures, and was associatedo lower levels of mean platelet volume (MPV) as an inflam-atory marker, supporting a consistent link between ironetabolism, inflammation and seizures.On the other hand, iron overload has been also reported

o be associated with several neurological disorders as wells epilepsy (Hughes and Oppenheimer, 1969; Rojas andessen, 1968). In fact, evidence from animal studies sug-ested that iron deposition might lead to epileptogenicesions (Campbell et al., 1984).

The aim of the present study is to characterize duringnterictal period in a sample of adult patients affected byocal epilepsy the inflammatory pattern as well as markersf iron status and metabolism, as evaluated in the bloodamples of patients. A correlation between cytokines andron metabolism markers, as well as between these param-ters and clinical features of epileptic patients (responseo antiepileptic therapy, duration of epilepsy, seizures fre-uency), will be also provided in order to understand theirelationship and their role in affecting epilepsy disorder.

ubjects and methods

atients and control subjects

hirty-seven patients affected by focal epilepsy (12 M/25; age 51.3 ± 18.8 years) were enrolled for the purposef the study. The study protocol was approved by localthics Committee and a written informed consent wasbtained from all patients. On the basis of neuroimagingata (CT scan and/or MRI), clinical and EEG features, 19ut of 37 patients were diagnosed as having cryptogenicocal epilepsy, and the remaining 18 as having symptomaticpilepsy; moreover, 13 patients resulted affected by tem-oral lobe epilepsy, and 24 by extratemporal one. Theeizure frequency was collected through clinical diaries,lled in by the patient himself or — when it was not pos-ible because of poor recognition of seizures — by thearents or caregivers. The mean seizure frequency duringhe previous year was less than 3 per month in all patients

mean 2.5 ± 2.6). Type, number, dosages and administra-ion schedule of drugs taken by patients were recorded.ighteen patients were in single therapy, and 14 were inolytherapy. The most common medications were valproate

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M. Tombini et al.

VPA; 8 patients) and carbamazepine (CBZ; 10 patients);n addition, some patients used other antiepileptic drugsAEDs) (phenobarbital, lamotrigine, levetiracetam, oxcar-azepine, topiramate). Twenty-nine percent of patients (11)ere newly diagnosed as having epilepsy and were not tak-

ng AEDs during the study recordings. The mean number ofrugs used was 1.2 (min 0/max 3).

The main features of patients were showed in Table 1.Forty-three healthy subjects (26 M/17 F) matched for age

47.3 ± 19.9 years) were enrolled as control group. The con-rol subjects were not taking any drug at the moment of thetudy.

Patients and controls differed for sex (Chi square = 6.189, = 0.013); so that all the statistical analyses were correctedor sex.

ethods

iochemical and molecular investigations

era from fasting blood samples were collected in the morn-ng and rapidly stored at −80 ◦C. Samples were taken in thenterictal period.

The following inflammatory cytokines were evaluated inerum sample IL-6, IL-6 soluble receptor (IL6-sR), TNF-�,L-1 and IL-1RA and measured by ELISA method. Humanytokines kits from Invitrogen (Paisley, UK) were usedccording to the manufacturer’s instructions. A microplateeader (SEAC Sirio S), set at 450 nm, was used to determineerum concentrations from a standard curve.

We also measured, in the interictal period, the fol-owing iron-status and metabolism markers: hemoglobinoncentration (Hgb), mean corpuscular volume (MCV),ematocrit (Hct) red blood cell (RBC) count, serum iron andopper (Cu), ceruloplasmin concentration (iCp), ceruloplas-in activity (eCp) and the specific ceruloplasmin activity

eCp/iCp), total ferroxidase activity, transferrin (Tf), serumerritin (SF), Tf saturation (Sat-Tf), and we calculated theeruloplasmin-transferrin ratio (Cp/Tf).

Briefly, measurements of serum copper were obtainedither by atomic absorption spectroscopy (A Analyst00 Perkin Elmer atomic absorption spectrophotometerquipped with a graphite furnace platform HGA 800) orolorimetrically, following the method of Abe et al. (1989)RandoxLaboratories Ltd., Crumlin, UK) automated on Cobasira Plus (Horiba ABX, Montpellier, France). Data producedy the latter were used in the statistical analysis. Iron waseasured by photometric test using Ferene (Higgins, 1981).f (Skikne et al., 1990) and iCp (Wolf, 1982) levels wereeasured by immunoturbidimetric assays. More details on

hese methods can be found in Hussain et al. (2002). Ferritinas measured by latex-enhanced turbidimetric immunoas-

ay (Simo et al., 1994) (all reagents were from Horiba ABX,ontpellier, France). eCp measurements were assayed byn automation of the o-dianisidine method (Boyett et al.,976; Lehmann et al., 1974; Martinez-Subiela et al., 2007;chosinsky et al., 1974), and data were expressed in IU/L.

otal serum Ferroxidase activity measurements were per-ormed by applying an automation of the kinetic ferroxidaseethod (Somani and Ambade, 2007) and were expressed

n Abs/min. Reagents employed in enzymatic assays were

Inflammation and iron metabolism in adult patients with epileps

Tabl

e

1

Clin

ical

feat

ures

of

pati

ents

.

No.

ofsu

bjec

tsSe

xM

/FAg

e

(yea

rs)

Mea

n

(SD

)D

urat

ion

ofep

ileps

y(y

ears

)M

ean

(SD

)

Etio

logy

(sym

p-to

mat

ic/c

rypt

ogen

ic)

Type

of

epile

psy

(tem

po-

ral/

extr

atem

pora

l)

Ther

apy

(tre

ated

/unt

reat

ed)

Mea

nnu

mbe

r

ofdr

ugs

(min

/max

)

VPA

(num

ber

ofpa

tien

ts)

CBZ

(num

ber

ofpa

tien

ts)

37

12/2

5

51.3

(18.

8)

22.1

(19.

4)

18/1

9

13/2

4

26/1

1

1/3

8

10

VPA,

valp

roat

e;

CBZ,

carb

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tcs

S

AS

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y 247

ll from Sigma (St. Louis, MO, USA). We also computedhe ratio between Cp and Tf serum concentrations (Cp/Tf),hich is a statistical index conceived on the basis of exper-

mental analysis of electron paramagnetic signals of theeruloplasmin — binding a copper in the oxidized state (Cu+2)

and of the apotransferrin. Sat-Tf (%) was calculated byividing serum iron (�g/dL) by the total iron-binding capac-ty (TIBC = Tf in mg/dL × 1.25) and multiplying by 100. Thepecific enzymatic activity, eCp/iCp, which represents thenzymatic activity per mg of ceruloplasmin concentration,as calculated as the ratio between eCp and iCp.

Finally, data about RBC count, erythrocyte sedimenta-ion rate (ESR), liver enzymes (SGOT, SGPT and �-GT) wereollected from medical records up to 3 months before thetudy.

tatistical analysis

ll the statistical analysis was performed by using IBM SPSStatistics version 20.

We checked whether the variables required to be normal-zed by applying the Kolmogorov—Smirnov test. Four out of9 variables (IL6, TNF-�, IL1, IL1ra) had a distribution signif-cantly different from a normal one, at least for one of thewo groups (healthy subjects and epileptic patients). In thatase we applied a Ln transformation obtaining a distributionhich did not differ from a normal one. The comparisons of

he variable under study across groups were performed withNOVA ‘between groups’ tests, the relations between vari-bles by means of Spearman’s rho correlation coefficients.or the purpose of the study we analyzed the correlationsetween some clinical features (i.e. seizures frequency,uration of epilepsy) and different biological variables. Tohis aim we decided to apply Spearman’s rho correlation,ecause some variables within the epileptic patients groupere not normally distributed, and in order to address aore general monotonic relationship instead of a linear rela-

ion. In other words, Spearman’s correlation may be used onhatever type of data pattern is seen and it has the advan-

age of not specifically assessing linear association but moreeneral association.

We checked for possible effects of AEDs on the biologicalariables under study. To do so, we first split the subjectample defining three ‘classes of therapy’, i.e. VPA, CBZnd all the other drugs different from VPA and CBZ. Then,e separately compared with the ANOVA ‘between groups’

est all the biological variables under study considering theroups of subjects (corresponding to the three classes) gen-rated by the classification described above. Subsequently,e compared patients who were drug free (29%) with those

aking at least one drug. No effect of therapies on the con-idered biological variables was found (all p values >0.2)part from �-GT, which was higher in patients taking ateast one drug than in those who were drug free, being4.6 ± 32.9 UI in treated patients and 14.70 ± 6.15 UI in non-reated (F = 8.015, p = 0.008). Specifically, we did not findny significant effect of VPA on Hgb and other biologicalarameter, differently from previous reports (Taher et al.,

009; Ounjaijean et al., 2011).

Given the small sample size, we decided to avoid cor-ections for multiple comparisons in order to increase theensitivity in identifying any association potentially useful

248 M. Tombini et al.

Table 2 Serum levels of the investigated variables in epileptic patients and controls [means (SD)].

Epileptic patients Controls Test

IL-6 (pg/mL) 1.40 (1.15) 0.75 (0.58) p = 0.026*

Cp/Tf × (10−2) 11.14 (2.11) 9.51 (2.34) p = 0.011*

Tf (g/L) 2.3 (0.38) 2.6075 (0.47) p = 0.031*

TNF-�(pg/mL) 0.74 (0.47) 1.09 (0.32) p = 0.002*

IL6-sR (pg/mL) 774.98 (288.03) 843.41 (355.51) NSIL-1 (pg/mL) 7.05 (1.70) 7.41 (1.78) NSIL-1RA (pg/mL) 523.75 (233.48) 912.11 (248.74) NSSF (ng/mL) 72.85 (69.28) 111.58 (100.82) NSiCp (mg/dL) 26.26 (5.13) 24.26 (4.64) NSeCp (IU/L) 97.29 (21.97) 86.29 (14.49) NSCu (�mol/L) 14.37 (3.64) 12.75 (2.57) NSIron (�g/dL) 87.26 (43.34) 81.03 (30.01) NSSat-Tf (%) 27.15 (17.53) 24.71 (10.49) NSeCp/iCp, IU/mg × (10−1) 3.77 (0.84) 3.64 (0.58) NSTotal ferroxidase activity (Abs/min) 78.19 (19.85) 73.01 (11.17) NSHgb (g/dL) 13.34 (1.71) 14.02 (1.35) NSHct (%) 40.36 (4.26) 42.34 (3.22) NSRBC (no. × 103/�L) 4.63 (0.56) 4.79 (0.36) NSMCV (fL) 88.43 (6.73) 88.55 (3.83) NS

IL-6, interleukin 6; IL6-sR, IL-6 soluble receptor; IL-1, interleukin 1; IL-1RA, IL-1 receptor-antagonist; TNF-�, tumor necrosis factor-�;Hgb, hemoglobin concentration; MCV, mean corpuscular volume; Hct, hematocrit; RBC, red blood cell count, Cu, copper concentration;iCp, ceruloplasmin concentration; eCp, ceruloplasmin activity; eCp/iCp, specific ceruloplasmin activity, Tf, transferrin; SF, serum ferritin;

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Sat-Tf, transferrin saturation; Cp/Tf, ceruloplasmin-transferrin ra* p-value < 0.05.

o unveil relevant aspects of epilepsy pathophysiology. How-ver, this issue could be a limitation of the study.

A p-value < 0.05 was considered significant for all statis-ical analysis.

esults

aking into account the inflammatory variables we foundhat IL-6 concentrations (F = 5.345, p = 0.026) were highern epileptic patients with respect to controls while TNF-

values were lower (F = 10.159, p = 0.002) (Table 2). Noifferences between groups (healthy subjects vs epilepticatients) were observed for IL6-sR, IL-1 and IL-1RA.

Considering markers of iron metabolism we observedn patients group a significant increase of Cp/Tf systemF = 6.897, p = 0.011) while Tf values were lower (F = 4.872,

= 0.031) than in controls (Table 2). No significant differ-nces between groups were detected for all other variablesiCp, eCp, serum copper and iron concentrations, totalerroxidase activity, eCp/iCp, Sat-Tf and SF, Hgb, MCV, Hctnd RBC count). Moreover, no significant differences of alliological variables were found when splitting the patientsroup according to the epileptic focus site (temporal vsxtratemporal) and epilepsy etiology (symptomatic vsryptogenetic).

The evaluation of relationship between iron metabolismariables and cytokines revealed that TNF-� positivelyorrelated with Tf (Spearman’s rho = 0.373, p = 0.027). Con-

idering the relationship of the markers of hepatocytesecrosis with the biological variables of iron metabolism, weound no significant correlations as well as between indicesf liver necrosis and cytokines.

D

Ot

Taking into account the correlation between clinical fea-ures of epilepsy, specifically seizures frequency, and alliological variables under study, we observed that this clin-cal parameter positively correlated with eCp (Spearman’sho = 0.38, p = 0.046) and negatively with Hgb (Spearman’sho = −0.32, p = 0.038) and Hct (Spearman’s rho = −0.32,

= 0.041) (Table 3). Finally, the duration of epilepsynversely correlated with TNF-� (Spearman’s rho = −0.414,

= 0.021), while had not any effect on the other biologicalarameters.

As expected, Hgb correlated positively with SF (Spear-an’s rho = 0.63, p < 0.001), iron concentration (Spearman’s

ho = 0.42, p = 0.015), and negatively with Cp/Tf ratioSpearman’s rho = −0.33, p = 0.048). RBC count showed aositive correlation with iron concentration (Spearman’sho = 0.46, p = 0.006) and Sat-Tf (Spearman’s rho = 0.42,

= 0.010). In addition, Hct correlated positively withF (Spearman’s rho = 0.69, p < 0.001), iron concentrationSpearman’s rho = 0.41, p = 0.016) and eCp/iCp (Spearman’sho = 0.34, p = 0.043), and negatively with IL-1 (Spear-an’s rho = −0.32, p = 0.035), iCp (Spearman’s rho = −0.33,

= 0.048). ESR correlated positively with iCp (Spear-an’s rho = 0.41, p = 0.015) and eCp (Spearman’s rho = 0.34,

= 0.044) and negatively with iron concentration (Spear-an’s rho = −0.49, p = 0.004) and Sat-Tf (Spearman’s

ho = −0.49, p = 0.003).

iscussion

ur findings confirmed an imbalance of inflammatory pat-ern in adult patients with focal epilepsy compared to

Inflammation and iron metabolism in adult patients with epileps

Table 3 Correlations between seizures frequency and bio-logical variables under study.

Biological variables Spearman’s rho correlations

IL-6rho = −0.331p = 0.133

IL6-sRrho = 0.263p = 0.326

IL-1rho = 0.061p = 0.724

IL-1RArho = 0.073p = 0.674

iCp (mg/dL)rho = 0.014p = 0.944

SF (ng/mL)rho = −0.116p = 0.557

Cu (�mol/L)rho = 0.267p = 0.219

Iron (�g/dL)rho = 0.198p = 0.342

eCp (IU/L)rho = 0.38p = 0.046*

Sat-Tf (%)rho = 0.105p = 0.594

eCp/iCp × (10−1)rho = 0.296p = 0.134

Total ferroxidaseActivity (Abs/min)

rho = 0.118p = 0.549

TNF-�rho = −0.192p = 0.476

Tf (g/L)rho = −0.172p = 0.523

Cp/Tf × (10−2)rho = 0.311p = 0.241

Hgbrho = −0.32p = 0.038*

Hctrho = −0.32p = 0.041*

RBCrho = −0.30p = 0.050

IL-6, interleukin 6; IL6-sR, IL-6 soluble receptor; IL-1, inter-leukin 1; IL-1RA, IL-1 receptor-antagonist; TNF-�, tumor necrosisfactor-�; Hgb, hemoglobin concentration; MCV, mean corpus-cular volume; Hct, hematocrit; RBC, red blood cell count, Cu,copper concentration; iCp, ceruloplasmin concentration; eCp,ceruloplasmin activity; eCp/iCp, specific ceruloplasmin activity,Tf, transferrin; SF, serum ferritin; Sat-Tf, transferrin saturation;

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Cp/Tf, ceruloplasmin-transferrin ratio.* p-value < 0.05.

healthy subjects; in particular, a clear increase of IL-6 anda decrease of TNF-� were detected in the serum of patientsduring the interictal phase. Therefore, as in human studiescytokine levels in blood have been shown to reflect the cen-tral nervous system’s production (Peltola et al., 2000), ourdata are consistent with the increasing evidence support-ing the role of brain inflammation in seizures and epilepsy

disorders.

With regard to IL-6, it has been shown in animalstudies that IL-6 knockout mice are more susceptible toseizures induced by different convulsive stimuli compared

1Tco

y 249

o wild type (De Sarro et al., 2004; Penkowa et al., 2001),nd exogenously applied IL-6 increases the severity ofentylenetetrazole-induced seizures in rats (Kalueff et al.,004). In humans, IL-6 concentration increases rapidly inerebrospinal fluid (CSF) after tonic-clonic seizures (Peltolat al., 1998). Moreover, in patients with refractory epilepsy,levated levels of IL-1 and IL-6, and a decreased level ofL-1RA have been observed (Hulkkonen et al., 2004). Virtat al. (2002) reported that the plasma IL-1 RA/IL-1� rationd plasma IL-6 levels were significantly higher in 55 patientsith febrile seizures, compared to 20 age-matched, febrileealthy subjects. Finally, other authors (Liimatainen et al.,009) recently described a strong association between tem-oral lobe epilepsy and the chronic overproduction of IL-6.ur data did not reveal this association, but our group ofatients is more heterogeneous than that one described byiimatainen et al. (2009), who analyzed mostly patients withrug-resistant epilepsy.

Taking into account the TNF-� experimental evidence, itsffects on seizures seem to depend on the receptor subtypesredominantly stimulated (p55 and p75 receptors). Balossot al. showed that intra-hippocampal injection of murineecombinant TNF-� potently inhibits seizures in mice andhis action is mediated by neuronal p75. Transgenic miceith an astrocytic overexpression of TNF-� showed reduced

usceptibility to seizures, whereas mice lacking TNF-� p75eceptors showed prolonged seizures (Balosso et al., 2005).hile the neuroprotective effect is mediated by TNF-�

eceptor p75 (Bernardino et al., 2005), p55 receptors con-ain an intracellular ‘‘death domain,’’ and they appear toontribute predominantly to cell damage (MacEwan, 2002).ven if there is more evidence that p55 receptor mediates aeurotoxic effect of TNF-� (MacEwan, 2002; Shinoda et al.,003), Lu et al. demonstrated that it may play a protectiveole on seizures; in fact, mice with a genetic deletion of the55 receptor showed significantly more severe seizures thanhe wild-type mice after a nasal application of kainic acidLu et al., 2008). In humans several observations reportedn increase of TNF-� in the post-ictal phase (Vezzani et al.,011) as well as other pro-inflammatory cytokines (IL-1, IL-), but less data exist about the TNF-� levels in the interictaleriod. Interestingly, in our patients sample the durationf epilepsy inversely correlated with TNF-�, suggesting theypothesis that epilepsy and seizures could provoke inflam-atory response and cytokines alterations. However, on

he basis of the experimental evidence above elucidatede cannot definitively rule out that decreased levels of

his cytokine in the serum of patients could play a pro-pileptogenic role.

We observed significant differences regarding markers ofron metabolism between patients and healthy subjects. Inarticular, for the first time, we found a decrease of Tf andn increase of Cp/Tf system in sera of patients comparedo controls. As already mentioned, Tf is a negative acutehase protein and is reduced during immune stimulationWeiss and Goodnough, 2005). Cytokines, and in particularL-6, significantly modulate the acute phase protein syn-hesis during chronic immune stimulation (Castell et al.,

989). Our analysis showed a positive correlation betweenNF-� and Tf, suggesting the hypothesis that changes ofytokines in the plasma — the increase of IL-6 and decreasef TNF-� — could drive the reduction of Tf, probably

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eflecting a chronic immune stimulation. Accordingly, as itas been recognized that IL-6 has both pro-inflammatorynd anti-inflammatory activities mediated by differentignaling (Scheller et al., 2011) and TNF-� is a pro-nflammatory cytokine, the observed changes of cytokineseem to reflect an anti-inflammatory response.

No significant differences in SF and Tf saturationetween patients and controls were recognized as previ-usly reported in patients with febrile seizures and epilepsyDaoud et al., 2002; Ikeda, 2001; Naveed-Ur-Rehman andillo, 2005).

In the same direction we could interpret the increase ofp/Tf ratio observed in the serum of patients. In the Cp-Tfystem, Cp has a relevant antioxidant power and a signif-cant role in iron homeostasis. The ferroxidase activity ofp is fundamental because it eliminates from circulatione2+, which takes part in Fenton reaction in the oxidativetress cascade (Bielli and Calabrese, 2002). In fact, Fe2+ canlso convert hydrogen peroxide into the highly reactive rad-cals. In other words, if it were not oxidized and removedy the coordinated ceruloplasmin and Tf actions, iron wouldyclically produce highly reactive radicals, which the circu-ating blood would then distribute all over the organism. Cpncrease and Tf decrease were identified in acute phase oftroke (Altamura et al., 2009) and in different pathologicalonditions as a sign of inflammation (Squitti et al., 2010)nd as defense mechanism reflecting the body’s resistanceo an oxidant insult (Kozlov et al., 1984). In this regard,n our patients sample the frequency of seizures corre-ates with eCp. This result is consistent with the findingsbout the antioxidant role of ceruloplasmin and corrobo-ates the strong bidirectional relationship between seizuresnd inflammation.

Finally, we remarkably observed a strong negative cor-elation between seizures frequency and hematologicalarameters, such as Hgb and Hct: we hypothesize that thiselationship could be mediated by inflammation. In fact, asreviously elucidated (Ludwiczek et al., 2003; Miller et al.,991; Rogers, 1996; Weiss and Goodnough, 2005), inflamma-ion and cytokines could directly influence iron metabolismnd red blood cell status, leading to anemia in some cases. Inur study, patients were not affected by anemia, nor showedgb values significantly different from control subjects. Aigher number of seizures was strongly paired with reducedevels of Hgb and Hct. Even if there is not a significant directorrelation between cytokines and these biological parame-ers except for IL-1, Hgb and Hct were negatively correlatedespectively with Cp/Tf ratio and, iCp, well-known mark-rs of inflammation as previously discussed by Altamurat al. (2009), and, as expected, positively with iron con-entration and SF. Our hypothesis is that seizures with highrequency could boost both brain and systemic inflamma-ion and alter cytokines profile, able in turn — at centralervous system (CNS) level — to increase cortical excitabil-ty also in the interictal period, and — at systemic level

to decrease Tf and modulate iron metabolism and redlood cells parameters; thus, the increase of Cp/Tf and iCp,ith their antioxidant activity is a reactive phenomenon,

iming to contrast inflammation and the oxidative stressascade. We believe that the findings of our study couldelp to explain the epidemiological observations linkingron-deficiency anemia and febrile seizures (Pisacane et al.,

D

M. Tombini et al.

996; Zareifar et al., 2012): inflammation and cytokinesrofile alterations, already documented in this conditionVezzani et al., 2008; Virta et al., 2002), could probably behe link between these two disorders.

onclusions

ur findings showed in a sample of patients with focalpilepsy during the interictal period clear alterations inlasma pattern of cytokines with respect to healthy controlsonsisting of an increase in IL-6 and a decrease in TNF-� plas-atic levels, in part related to duration of epilepsy. For therst time we also detected in these patients statistically sig-ificant changes in iron metabolism, as an increase of Cp/Tfatio and a decrease of Tf, possibly driven by cytokine mod-fications and consistent with inflammation as acute phasend antioxidant activity markers. Finally, we may speculatehat the strong correlation between seizures frequency andCp and inversely with Hgb and Hct count be interpretedy the bidirectional link between seizures and inflammationoth at CNS as well as at systemic level.

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