Architect c4000 analyzer

Blood samples were collected from the heart, and the serum was separated by centrifugation (3000 rpm at 4°C for 15 min) and stored at −80°C for biochemical analyses. Serum total protein (T Prot) g/dL, total bilirubin (T Bil) mg/dL, direct bilirubin (D Bil) mg/dL, indirect bilirubin (I Bil) mg/dL, and albumin (Alb) g/dL levels were determined using an automated Architect C-4000 analyzer (Abbott Laboratories, Abbott Park, IL, USA). Aspartate aminotransferase (AST) IU/L, alanine aminotransferase (ALT) IU/L, and lactate dehydrogenase (LDH) IU/L levels were measured using an Architect C-8000 autoanalyzer (Abbott Laboratories) at Bozok University, School of Medicine, Central Laboratory, Yozgat, Turkey.

Brain concentrations of valproate, carbamazepine, phenytoin, and phenobarbital were measured to verify possible pharmacokinetic interactions between ranolazine and listed antiepileptic drugs. Control animals were applied with vehicle and a respective antiepileptic drug. Animals from investigated groups were administered with combinations of ranolazine with antiepileptics in the proportions of 1:3 and 1:1. In the two proportions, antagonistic interactions were found in the isobolographic analysis. At times used in behavioral tests, mice were decapitated, and their brains removed from the skulls. The brains were homogenized by Ultra Turax T8 homogenizer (IKA, Staufen, Germany) with Abbott buffer (2:1 vol/weight) and centrifuged at 10,000× g for 10 min. Supernatants obtained (75 µL) were used for drug concentration analysis by fluorescence polarization immunoassay (Architect c4000 analyzer, Abbott Laboratories, Warsaw, Poland). Results were expressed presented as means ± SD (µg/mL).

The content of PB and VPA in the brain tissue of experimental animals was measured by fluorescence polarization immunoassay, as reported earlier [44 (link),46 (link)]. Both PB and VPA were administered i.p. either alone or in combination with borneol and/or scoparone. The doses of PB and VPA reflected their ED₅₀ values from the tonic-clonic seizure model. Preparation of brain tissue and measurement of ASM content have been described elsewhere [44 (link),46 (link)]. Briefly, each mouse pretreated with the respective treatment (ASM alone, ASM with scoparone, ASM with borneol or ASM with scoparone and borneol) was decapitated at the time reflecting the peak of maximum anticonvulsant effect of the ASM in the MES test; subsequently, a whole mouse brain was removed from the skull, weighted, harvested and homogenized in the presence of Abbott buffer (1:2 weight/volume). The homogenates were centrifuged at 10.000 g for 10 min. and the supernatant was transferred to Abbott Architect c4000 analyzer (Abbott, Abbott Park, IL, USA) to perform fluorescence polarization immunoassay (FPIA) analysis with the manufacturer’s supplied kits for detection of PB and VPA. Total brain concentrations of ASMs were expressed in μg/g of wet brain tissue as means ± SD of 8 separate brain preparations.

Pharmacokinetic estimation of total brain ASM concentrations was performed only for combinations of VAR with CBZ for which the anticonvulsant effect in the MES seizure test was significantly greater than for control animals. In order to understand if pharmacokinetic interaction between CBZ and VAR was present, measurement of total brain concentrations of CBZ was conducted. The measurement was performed in the following way: each animal pretreated with CBZ alone or in combination with VAR was decapitated at time reflecting the peak of maximum anticonvulsant effect of CBZ in the MES test; subsequently a whole brain was removed from the skull, weighed, harvested, and finally homogenized with usage of Abbott buffer (1:2 weight/volume). The homogenates were centrifuged at 10,000× g for 10 min with usage of Centrifuge MPW 350 (MPW Med. Instruments, Warsaw, Poland). Supernatant obtained that way was analyzed with the fluorescence polarization immunoassay (FPIA) method and with Abbott Architect c4000 analyzer (Abbott Park, IL, USA). A manufacturer’s supplied kit for detection of CBZ was applied. Total brain concentration of CBZ (in µg/g of wet brain tissue) was measured as means ± S.E.M. of 8 samples with CBZ brain preparations as reference and 8 brain samples with CBZ+VAR preparations to exclude pharmacokinetic relationship.