subjects and data collection : the subjects were dogs examined at kakogawa 
 animal hospital , kakogawa , hyogo , japan between march 2005 and april 2010 .
cases were excluded , if 
 permission of the owner , interictal , hematological and biochemical data from physical and 
 neurologic examinations , csf findings or mri data were unavailable .
routine csf analysis included the total nucleated cell count ( tncc ) and measurement of 
 microprotein concentration using a micro total protein kit ( wako pure chemicals , osaka , 
 japan ) .
protein concentration was considered to be elevated if>25 mg / dl , 
 and pleocytosis was judged to be present if tncc in the csf was>5 /l . 

if csf analysis was abnormal , titers for infectious causes of encephalitis were evaluated , 
 including canine distemper virus , ehrlichia canis , neospora 
 canis , toxoplasma gondii , aspergillus sp . and 
 cryptococcus sp .
mri was performed with 0.2 t system ( signa , ge medical imaging systems , tokyo , japan ) , and 
 mri findings were evaluated independently of csf analysis .
routine imaging included t2 , 
 fluid attenuation inversion recovery and t1-weighted sequences performed before and after 
 administration of intravenous gadolinium ( gadoteridol , eizai , tokyo , japan ) .
images were 
 obtained in at least 3 orthogonal planes relative to the region of interest .
clinical diagnosis of ie was defined retrospectively by exclusion of cases that with a 
 positive csf titer or fungal or bacterial culture or showed morphological abnormality on mri 
 or based on the results of clinical and neurologic examinations .
cases that responded to 
 anticonvulsant therapy ( oral phenobarbital or bromide ) and required this therapy after a 
 presumptive diagnosis were included in the ie group .
se due to intracranial diseases was 
 diagnosed when abnormalities were found in brain mri , csf tap results or clinical course 
 after presentation .
normal csf was 
 sampled from a control group of 18 healthy domestic dogs with the owner s consent .
csf preparation : csf preparation for extraction and derivatization of 
 low - molecular weight metabolites was performed as described by kuhara et al 
 .
collected csf was immediately centrifuged at 
 5,000 g for 10 min at 4c , and the supernatant was transferred to a clean tube and stored at 
 80c until use . to extract low - molecular - weight metabolites ,
25 l of csf 
 was mixed with 900 l of 70% ethanol containing 10 ng of 
 2-isopropyl malic acid and centrifuged at 15,000 g for 10 min at 4c .
metabolites in the dried residue were converted to trimethylsilyl 
 derivatives with 100 l of n , o - bis ( trimethylsilyl ) 
 trifluoroacetamide and trimethylchlorosilane ( 10:1 ) ( thermo , tokyo , japan ) and analyzed by 
 gc - ms .
gc - ms analysis and data processing : gc - ms analysis was carried out using a 
 gc - ms - qp2010plus system ( shimadzu , kyoto , japan ) with inert cap 5 ms / np ( 0.25 mm i.d .
 30 
 m , 0.25 m ; gl science , tokyo , japan ) , as described in kuhara et 
 al .
chromatogram acquisition , 
 detection of mass spectral peaks and waveform processing were performed using shimadzu gc - ms 
 solution software ( ver .
identification of low - molecular - weight metabolites was 
 carried out using the library of the national institute of standards and technology . 

semiquantitative assessment was performed using the peak intensity of 2-isopropylmalic acid 
 as an internal standard .
multiple classification analysis and statics : a dataset for multiple 
 classification analysis was compiled from the metabolic profiling data .
a three - dimensional 
 matrix was constructed based on sample names ( observations ) , metabolite name ( variable 
 indices ) and normalized peak intensities ( variables ) .
partial least square discriminant 
 analysis ( pls - da ) was performed in metaboanalyst 2.0 .
subject characteristics and csf analysis : a total of 16 cases met the 
 inclusion criteria for canine ie .
there were subtle neurological deficits in some cases , 
 including delayed proprioceptive positioning on one side , in interictal neurological 
 examinations ( table 1table 1.characteristics of subjectsbreedsexage at csfcollectionage at first seizure onsetneurologicstatus at restdiagnosiscsf protein(mg / dl)beagles0.80.8nie15.7pomeranianc4.33.9nie46.8chihuahuaf3.03.0nie33.0bernese mountainc4.33.4nie9.9chihuahuam4.92.0nie26.4yorkshire terrierc1.13.4nie8.3toy poodlem2.00.5nie13.2beaglec1.61.4nie15.2miniature dachshundm5.63.5nie17.1shetland sheepdogc14.27.2nie3.6chihuahuam0.50.5nie27.2mongs13.57.3nie39.5welsh corgim8.26.4nie16.8welsh corgic7.27.5nie20.3miniature schnauzerc13.68.2aie16.4yorkshire terriers3.52.8nie16.4chihuahuam2.92.6nse ( mue)2.3pugm3.23.2ase ( mue)17.7labrador retrieverm8.58.2ase ( mue)43.0shih - tzum3.53.5ase ( mue)8.2mongf6.75.8ase ( mue)194.7mongm13.913.9ase ( mue)5.8mongc15.014.9ase ( mue)2.4golden retrieverf8.17.3nse ( brain tumor)10.0 labrador retrieverf7.87.8ase ( brain tumor)80.7welsh corgim8.78.6ase ( brain tumor)89.0golden retrieverm10.610.6nse ( brain tumor)2.6shibam11.111.0nse ( brain tumor)83.7welsh corgic11.811.8ase ( brain tumor)56.2shibam12.512.5ase ( brain tumor)30.5mongm15.915.8ase ( brain tumor)36.3pugm3.53.5ase ( brain tumor)64.5beaglef6.16.1nse ( meningoencephalitis)29.7french bulldogf6.36.3ase ( meningoencephalitis)202.9pugm13.313.2ase ( meningoencephalitis)82.9maltesec1.1-ncontrol40.3mongs1.4-ncontrol12.1shibam0.6-ncontrol32.0toy poodlem10.3-ncontrol27.8miniature dachshundc8.3-ncontrol9.2west highland white terriers7.3-ncontrol57.1papillonm0.8-ncontrol21.2shih - tzuc15.2-ncontrol8.6mongc12.0-ncontrol18.2corgif10.4-ncontrol23.2italian greyhoundm7.8-ncontrol20.1welsh corgim2.3-ncontrol17.7maltesef0.6-ncontrol8.9welsh corgim0.5-ncontrol46.3toy poodlem0.5-ncontrol8.1golden retrieverm4.9-ncontrol13.3shibam2.3-ncontrol13.1pomeranianf0.6-ncontrol18.6f , female ; m , male ; c , casted ; s , spayed ; a , abnormal ; n , normal . ) .
the mean age at csf collection was 5.5  4.5 years ( median 4.3 , range 0.5 to 
 14.2 ) , and that at first onset of seizure was 3.9  2.6 years ( median 3.4 , range 0.5 to 
 8.2 ) .
se was diagnosed in 19 cases , including 3 with meningoencephalitis , 7 with 
 meningoencephalomyelitis of undetermined etiology ( mue ) and 9 with brain tumors .
the mean 
 age at csf collection was 8.9  4.0 years ( median 8.5 , range 2.9 to 15.9 ) , and that at first 
 onset of seizure was 8.8  4.1 years ( median 8.2 , range 2.6 to 15.8 ) .
background data did 
 not differ significantly among the disease categories in the se group .
the mean age of the 
 healthy controls was 4.8  4.7 years ( median 2.3 , range 0.5 to 15.2 ) .
age at csf collection 
 in the ie group was slightly lower than that in the se group ( p=0.026 ) and 
 not significantly different from that in the control group .
age at csf collection in the se 
 group was significantly higher than that in the control group ( p=0.006 ) . 

the mean protein 
 concentrations in the csf were 22.0 mg / dl ( median 18.4 , range 8.1 to 57.1 
 mg / dl ) in controls , 20.4 mg / dl ( median 16.6 , range 3.6 
 to 46.8 mg / dl ) in the ie group and 54.9 mg / dl ( median 
 36.3 , range 2.3 to 202.9 mg / dl ) in the se group with no significant 
 differences among the groups .
f , female ; m , male ; c , casted ; s , spayed ; a , abnormal ; n , normal .
evaluation of differences in csf profiles using multiple classification 
 analysis : a total of 60 metabolites identified in the csf were analyzed by 
 pls - da .
a. gc - ms data for the control 
 ( squares ) , ie ( triangles ) and se ( circles ) groups were analyzed using pls - da , and the 
 results are represented as a score plot . b. loading plot of canine csf 
 metabolites . ) .
the metabolites largely responsible for this separation included ascorbic acid , 
 glyceric acid , threonic acid , glutaric acid , isoleucine , leucine , phosphoric acid , 
 methionine , phenylalanine and pyruvic acid ( fig . 

the levels of 20 metabolites differed significantly among the groups , and 16 
 metabolites showed significant changes in ie cases compared to controls ( fig .
2.levels of metabolites in csf in the control ( black ) , ie ( stripes ) and se ( white ) 
 groups .
levels are expressed in 
 arbitrary units ( see materials and methods ) . ) .
the levels of glutamic acid , isoleucine , phosphoric acid , malonate , stearic acid , 
 glutaric acid , glutamine , acetic acid , oxanilic acid , glyceric acid , threonic acid and 
 succinic acid increased , and those of ascorbic acid , pyruvic acid and 3-hydroxy-2-butanone 
 decreased in ie .
the levels of 13 metabolites differed significantly between ie and se , 
 including higher levels of glutamic acid , isoleucine , methionine and serine in ie . the 
 levels of 9 metabolites
were significantly different in se cases compared to controls with 
 glutamic acid , methionine , phenylalanine , ascorbic acid , malic acid , acetone and pyruvic 
 acid all decreasing in se .
a. gc - ms data for the control 
 ( squares ) , ie ( triangles ) and se ( circles ) groups were analyzed using pls - da , and the 
 results are represented as a score plot .
levels of metabolites in csf in the control ( black ) , ie ( stripes ) and se ( white ) 
 groups .
epileptic seizure is a common neurological disease , but is difficult to diagnose by 
 observation . in most patients ,
the present study was performed in a population of epileptic dogs encountered 
 commonly in veterinary practice .
classification of canine epilepsy as ie or se is currently 
 performed by exclusion of identifiable underlying causes for epileptic seizures , and there 
 is a need to establish a method for rapid diagnosis of these types of epilepsy .
recent developments in metabolomics permit mining of metabolic information in disease 
 states and during treatment .
the approach is 
 founded on the assumption that a disease signature exists within a given biological sample , 
 but requires no prior knowledge of sample content .
suitable body fluids for metabolomics 
 include urine , plasma , serum , saliva and tissue homogenates , all of which carry the 
 signature of biochemical and metabolic responses to a myriad of genetic and environmental 
 influences .
metabolomics also holds promise for 
 early diagnosis of disease and for identification of metabolic pathways as targets for 
 disease amelioration .
serum metabolomics of cns 
 disorders has been vindicated by identification of blood biomarkers of parkinson s disease 
 .
serum is the basic sample for metabolomics , 
 being readily collectable by minimally invasive techniques , but serum metabolic profiles of 
 responders and non - responders to anti - epilepsy drugs may not be clearly distinguishable 
 .
this suggests that serum may not always be 
 optimal for evaluation of heterogeneous disorders , such as epilepsy , because of significant 
 blood - brain barrier interference with metabolite exchange between blood and csf .
for this reason , we used 
 gc - ms - based canine csf metabolic profiling to detect potential biomarkers applicable for 
 diagnosis of epilepsy . in canine csf ,
16 of 60 identified metabolites had significantly different levels in ie 
 cases compared to controls .
pls - da separated ie from se and controls , and metabolites with 
 higher loading values in this analysis were identified ( fig .
the levels of 20 metabolites , including amino acids , fatty acids and 
 organic acids that could be potential diagnostic biomarkers for epilepsy , showed significant 
 differences among the groups ( fig .
it has been proposed that the balance of excitatory and inhibitory neurotransmitters is a 
 key regulator of seizure .
glutamic acid and 
 -aminobutyric acid ( gaba ) are major excitatory and inhibitory compounds in brain , 
 respectively . in this study ,
the glutamic acid content in ie increased significantly 
 compared to those in se and in controls .
levels of related compounds ( glutamine , which is 
 synthesized from glutamic acid and serine ) in ie were also high compared to those in 
 controls , whereas gaba levels in ie and se did not differ from that in controls ( data not 
 shown ) . the glutamic acid level in csf
is regulated by glutamate transporters located on the 
 surface of epithelial cells of veins , glia and neuronal cells and depends on the 
 concentration in serum .
it was also reported that 
 some metabolites in brain are often excluded into extracellular fluid   and that the glutamic acid level in csf has been related to the 
 severity of neurological disorder in dog and cat models , although the level in csf has also been found to decrease in canine epilepsy 
 .
these findings indicate that a disorder of 
 glutamic acid regulation and metabolism might be related to the pathogenesis of canine ie 
 and that further evaluation of glutamic acid is required as a potential diagnostic biomarker 
 for ie .
it was shown that levels of ascorbic acid , pyruvic acid , glyceric acid , threonic acid and 
 glutamine were significantly changed in epilepsy ( fig . 

reactive oxygen species ( ros ) formation increases during seizures and removal of ros 
 depends on the antioxidant system .
the protective effect of antioxidants may partly be due 
 to antioxidant nutrients , such as ascorbic acid and carotenoids .
ascorbic acid is a powerful water - soluble antioxidant that has a 
 protective role against oxidative damage in tissues , including in central nervous system . 

ascorbic acid contents in csf of ie and se cases were significantly lower than that in 
 controls , but there was no significant difference between ie and se .
threonic acid , a 
 metabolite of ascorbic acid , was also elevated in both epilepsy groups .
these findings 
 indicate that ros - related oxidative stress might be present in the brain in ie and se and 
 that ascorbic acid may be a potent biomarker for epilepsy .
further investigation is needed 
 to evaluate these findings in chronic intracranial disease , but it is possible that ascorbic 
 acid and its metabolite in csf could be a marker of epileptic symptoms .
the decrease of pyruvic acid and increase of glyceric acid in epilepsy were significant in 
 se ( fig .
high glycolytic activity is common in 
 tumors known as the warburg effect , and serum levels of metabolites related to it are also 
 known to be changed in tumors .
thus , se cases 
 with tumors might have contributed to the differences in the metabolites among the groups in 
 this study .
thus , in cases with epileptic symptoms , investigation of metabolic changes in 
 csf in those with brain tumors is needed to distinguish these cases from those with 
 inflammatory disease . in conclusion , gc - ms - based canine csf metabolic profiles in ie differed from those in se 
 and in healthy controls .
glutamic acid may be a potent biomarker for ie , and ascorbic acid 
 and threonic acid may be useful for general detection of epilepsy .
 abstractepilepsy is a common neurological disorder with seizures , but diagnostic 
 approaches in veterinary clinics remain limited . 
 cerebrospinal fluid ( csf ) is a body fluid 
 used for diagnosis in veterinary medicine . in this study , we explored canine epilepsy 
 diagnostic biomarkers using gas chromatography - mass spectrometry ( gc - ms)-based metabolic 
 profiling of csf and multivariate data analysis . 
 profiles for subjects with idiopathic 
 epilepsy differed significantly from those of healthy controls and subjects with 
 symptomatic epilepsy . among 60 identified metabolites , the levels of 20 differed 
 significantly among the three groups . 
 glutamic acid was significantly increased in 
 idiopathic epilepsy , and some metabolites including ascorbic acid were changed in both 
 forms of epilepsy . 
 these findings show that metabolic profiles of csf differ between 
 idiopathic and symptomatic epilepsy and that metabolites including glutamic acid and 
 ascorbic acid in csf may be useful for diagnosis of canine epilepsy . 
MATERIAL AND METHODS
RESULTS
DISCUSSION