Changes Of Postictal Serum Adenine Nucleotidases Activities In Patients With Epilepsy

BackgroudEpilepsy is one of emergency and severse cases of nervous system, there are ten millions of epilepsy patients in the world. They suffer a lot from epilepsy and become a big economic and mental burdens to the oneselt, family and society. Recent year, we have been made great advancements in etiopathogenisis, pathogenesy, pathology and treatment of epilepsy, but it is not illuminated of concrete pathogenesy of epilepsy, and it is not clear for the intermal mechanism of seisure stopping.Adenine nucleotides, such as adenosine triphosphate, adenosine diphosphate, adenosine monophosphate and derivate adenosine, are very significant signaling molecules in some physiological and pathological processes. It has been demonstrated that adenosine is an effective anticonvulsant, and has nerve profective property. These effects are manifested by activation of inhibitory adenosine Al and excitatory adenosine A2 receptors. Adenosine is produced from the hydrolysis of adenine nucleotides by adenine nucleotidases. So adenine anucleotidases control the availability of ligands for both adenine nucleotide and neucleotide receptors. Changes in adenine nucleotide hydrolysis occur after pentylenetetrazol-epileptic epileptic events.To our knowledge, Changes of postictal serum adenine nucleotidases activities in patients with epilepsy has not been studied and the modulation mechanism of adenine nucleotidases has not been determined. Now, in this study we try to explore the modulation mechanism of adenine nucleotidases during seizure by measureing serum ATP, ADP and AMP hydrolysis and soluble nucleotide phosphodiesterase activities after seizure in patients with epilepsy. ObjectiveTo investigate changes of serum ATP, ADP and AMP hydrolysis after seizure in patients with epilepsy and its clinical significane, and explore the modulation mechanism of serum ATP, ADP and AMP hydrolysis during seizure.Subject and Methods1. Subject(1) Epilepsy group There were 30 epilepsy patients hospitalized in our neurological ward from June 2008 to october 2009. It included 17 males and 13 femals; their age were at 12～63 years, average age of (30.6±11.2) years; the course were at 2～31 years, average course of (9±3.8)years. All patients underwent 24h videoelectroencephalogram and monitoring for the electroclinical characterization of seizures. All patients underwent head magnetic resonance imaging. All patients were classified according to the Tnternational League Against Epilepsy (ILAE,1981) diagnostic criteria. There were 12 patients with generalized clonic-clonus seizure and 18 patients with complex partial seizure. Patients with neurological progressive diseases and cardiovascular disorders and metabolic deseases were excluded. Eleven of the patients were on antiepileptic drug monotherapy, and nineteen were on polytherapy(median number of medications=2).(2) Control group It consisted of 20 healthy subjects from our hospital outpatient department. None of these subjects had suffered from epilepsy, neurological or cardiovascular disorders. There were 11 males and 9 femals, their age were at 20～50 years, average age of (28.3±7.86) years. Control group was matched for age and gender with epilepsy group.2. Blood samplingBlood were draw for two group subjects by ulnar vein after overnight fast in morning. All subjects needed to enter into an quiet room and had a rest for one hour before blood drawing. An indwelling gauge catheter was placed in an ulnar vein for epipesy patients. Epileptic patients were seizure free for at least 12 h before blood withdrawal. And blood samples were obtained postictally after 5,10,20,30 and 60 min, and at 10 h after seizure. All serum samples were placed for 30-60 min at room temperature, then were centrifugalizated for 15 min in 3000 r/min speed, then serum was obtained and placed in Eppendorf tube, stored at -70℃before analysis.3. Measurement of ATP, ADP and AMP hydrolysisHydrolysis rates of ATP, ADP and AMP were evaluated as Bruno et al reported. Briefly, the reaction mixture, which contained 5 mmol/L CaC12 and 44 mmol/L Tris-HCl pH 8.0 (pH 7.2 for the measurement of AMP hydrolysis) in a final volume of 175 ul, was preincubated with 5 ul of serum for 10 min at 37℃. The reaction was initiated by the addition of 10 ul ATP, or ADP or AMP, each to a final concentration of 1.0 mmol/L, incubated for 10 min at 37℃and stopped with 200 ul of 10% trichloroacetic acid (TCA). Light absorbance was measured at 630 nm. Controls to correct for nonenzymatic hydrolysis were performed by adding the serum after the reaction was stopped with TCA. All samples were evaluated in triplicate. Enzyme activities were expressed as nanomoles of Pi released per minute per milliliter of serum.Results1.24 cases of all patients were detected sharp wave or spike wave or sharp-slow resultant wave or spike-slow resultant wave or multispike-slow resultant wave or multispike wave by 24 hour video electroencephalogram,6 cases were not detected seizure electrical discharge.13 cases head MRI showed abnormal signal. There were 3 cases with intracranial infection history,2 cases with cerebral vascular diseases history,2 cases with craniocerebral trauma history,1 case with cerebral atrophy history history,1 case with craniocerebral operation history.2. Compared with healthy control group, there were no differences in ATP, ADP and AMP hydrolysis rates in the interictal stage on epilepsy group, but significant increase (P<0.05) after seizure on epilepsy group.3. In comparison with healthy control group, ATP hydrolysis rates were significantly increased at 5min(all P<0.01), peaking at 20 min(all P<0.01), remained significantly increased at 60min (all P<0.01), returning to the basal level at 10 h after seizure on epilepsy group.4. In comparison with healthy control group, ADP and AMP hydrolysis rates were significantly increased at 5min (all P<0.01) and up to 30min, ADP hydrolysis was returning to the basal level atn 60 min, however at 60 min, AMP hydrolysis rate was significantly lower than in the control group, returning to basal level at 10h after the epileptic event. The pattern of changes in ADP hydrolysis partially overlapped that observed for AMP hydrolysis.Conclusions1. There are not significant increase in serum ATP, ADP and AMP hydrolysis rates in the interictal stage on epilepsy group.2. There are significant increase in serum ATP, ADP and AMP hydrolysis rates after seizure in patients with epilepsy.3. The pattern of changes in ADP hydrolysis partially overlapped that observed for AMP hydrolysis.4. It indicates adenine nucleotidases activities have a significant increased after seizure, adenine nucleotidases activities increased might play a role in the modulation of epileptic events. ObjectiveTo investigate changes of serum soluble nucleotide phosphodiesterase activities after seizure in patients with epilepsy and its clinical significane, and explore the modulation mechanism of adenine nucleotidases during seizure.Subject and Methods1. Thirty patients and twenty healthy controls were included in the study. There are 18 generalized tonic-clonic seizure patients and 12 complex partial seizure patients.2. Blood samples vere obtained after an overnight fast in two groups. Blood samples were obtained postictally at 5 min,10 min,20 min,30 min,60 min and 10 h after seizure in patients with epilepsy. All serum samples were stored at -70℃before measurment.3. Measurement of nucleotide phosphodiesterase activitiesSoluble nucleotide phosphodiesterase activities was determined as described by Bruno et al. The reaction mixture containing 0.5 mmol/L p-nitrophenil-5’-thymidinemonophosphate (p-Nph-5’-TMP) as a substrate in 40 mmol/L Tris-HCl (pH 8.9) was preincubated at 37℃for 10 min in a final volume of 230 ul. The reaction was initiated by the addition of 20ul serum, incubated for 10 min at 37℃and stopped with 750 ul of 0.2 mol/L NaOH. The amount of p-nitrophenol was measured by light absorbance at 400 nm. Controls to correct for nonenzymatic hydrolysis were performed by adding the serum after the reaction was stopped with NaOH. All samples were assayed in triplicate. Enzyme activities were expressed as nanomoles (nmol) ofp-nitrophenol released per minute per milliliter of serum.4. Measurement of Lactate dehydrogenase (LDH) activityLDH activity is a marker of tissue damage. LDH was determined in the serum of all subjects by OLYMPUS AU600 automatic biochemistry analyzer.Results1.Compared with healthy control group, there were no differences of soluble nucleotide phosphodiesterase activities in the interictal stage on epilepsy group, but significant increase (P<0.05) after seizure on epilepsy group.2.In comparison with healthy control group, soluble nucleotide phosphodiesterase activities were significantly increased at 5min(all P<0.01), peaking at 20 min(all P< 0.01), remained significantly increased at 60min (all P<0.01), returning to the basal level at 10 h after seizure on epilepsy group. The pattern of changes in postictal soluble nucleotide phosphodiesterase activities almost overlapped that observed for ATP hydrolysis rate.3. There were no significant differences in LDH activity in serum of patients with epilepsy compared with control subjects.Conclusions1. There are not significant increase in serum soluble nucleotide phosphodiesterase activities in the interictal stage on epilepsy group.2. There are significant increase in serum soluble nucleotide phosphodiesterase activities after seizure in patients with epilepsy. The pattern of changes in postictal soluble nucleotide phosphodiesterase activities almost overlapped that observed for ATP hydrolysis rate.3. Serum LDH activities have not marked increased after seizure in patients with epilepsy.4. Adenine nucleotidases activity increased might play a role in the modulation of epileptic events.