Au400 autoanalyzer

Hp concentration was measured by an assay based on AlphaLisa technology previously used in pig saliva [51 (link)]. ITIH4 was measured using a porcine species-specific commercially available ELISA kit (Porcine ITIH4, ElabSciences). The kit showed values of intra and inter-imprecision lower than 15% and accurate results in linearity under dilution for quantifying the ITIH4 in the saliva of pigs. Total protein concentration was analyzed using a colorimetric kit to measure Low-Complexity Region (LCR) proteins (protein in urine and CSF, Spinreact). S100A8-A9 was analyzed with a commercially available immunoturbidimetric assay (BÜHLMANN fCal Turbo® assay, Laboratories AG) using an Olympus AU400 autoanalyzer. This assay is designed for humans, but it has been demonstrated to have cross-reactivity with CALPS100A8-A9 in pigs [38 (link)] and it has been validated in the saliva of this species [44 (link)]. SA100A12 was measured with the SEB080Po Cloud-Clone ELISA kit, which is a specific assay for the measurement of this protein in pigs. This assay provided an intra and inter-assay imprecision lower then 15% and was linear after serial sample dilutions.

Acute oral toxicity was assessed according to Organization for Economic Co-operation and Development (OECD) guidelines 423 (OECD, 2002 (link)). The animals were randomly distributed into two groups. The first group was treated with distilled water, and the second group was treated with P. heyneanus essential oil (PhEO) at 300 mg/kg. Treatments were carried out by gavage (10 mL/kg). After the treatments, the animals were observed for 2 h and then every 24 h for 14 consecutive days. During this period, behavioral and physiological changes were observed (alertness, spontaneous motor activity, locomotion, apathy, response to touch, stereotypy, aggression, ataxia, sweating, urination, diarrhea, convulsions, and death). On the 15th day, the animals were subjected to induced euthanasia (ketamine 300 mg/kg and xylazine 30 mg/kg) (Souzados et al., 2021 (link)).
Blood was obtained by cardiac puncture in order to analyze renal (creatine and urea) and liver [alanine aminotransferase (ALT-PGP), aspartate transaminase (AST-TGO) and alkaline phosphatase (ALP)] function. Analyses were carried out using an Olympus AU 400 Autoanalyzer (Hamburg, Germany) (Costa-Silva et al., 2008 (link)).

CK, CK-MB, AST, ALT, and lactate were measured using commercial kits from Beckman (Beckman Coulter Inc., Fullerton, CA, USA) while LDH was a commercial kit from Biosystem (Biosystem S.A., Barcelona, Spain) that was previously validated in the saliva of pigs [54 (link)]. These methods were performed using an Olympus AU400 autoanalyzer. Troponin I was quantified by a solid-phase enzyme-labeled chemiluminescent immunometric assay (IMMULITE Troponins I, Siemens Medical Solutions Diagnostics) in an IMMULITE 1000 immunoassay system.
CK, AST, ALT, LDH, lactate, and troponin I provided an intra- and inter-assay imprecision of < 15% and accurate results in linearity under dilution assays. In the case of troponin I determinations, values lower than the low limit of analytical detection of 0.2 ng/mL were established as 0.2 ng/mL.

Blood samples were taken from the tail vein after 8 hours of fasting. Sera were obtained by centrifugation and stored at −70°C. Serum glucose, total cholesterol, triglyceride and free fatty acid (FFA) levels were measured using an Olympus AU400 auto analyzer (Olympus GmbH, Germany). Insulin, IL-1β, IL-6 and TNFα were measured using a Milliplex Analyzer Luminex 200 System (Millipore, USA) with a Rat Cardiovascular Disease Panel kit.

Blood samples were collected from the tail vein after 8 hours of fasting, and serum was obtained by centrifugation and stored at −70°C. The levels of serum glucose, total cholesterol (TC), TG, aspartate aminotransferase (AST), alanine aminotransferase (ALT), and free-fatty acids (FFAs) were measured by using a model AU400 auto analyzer (Olympus GmbH, Hamburg, Germany) [16 (link)]. Insulin resistance (IR) was defined as a homeostasis model assessment of insulin resistance (HOMA-IR) as insulin (μU/mL) × fasting glucose (mmol/L)/22.5 [17 (link)].

FRAS was determined by the reduction of ferric-tripyridyltriazine (Fe3+-TPTZ, Sigma-Aldrich Co.) to the ferrous (Fe2+) form [52 (link)] and the AOPP based on a method previously described [53 (link)] and both were previously used in pig saliva [51 (link)]. These analyses were carried out using an Olympus AU400 autoanalyzer.