Au400 analyser

At the beginning and at 5-years follow-up laboratory assessments were completed for all participants. Venous blood samples were obtained after an 8-h fasting period. HbA1c levels were determined by high-performance liquid chromatography with the use of a method certified by the National Glycohemoglobin Standardization Program. All HbA1c values were converted to Diabetes Control and Complications Trial-aligned units (26 (link)). Glucose levels were measured in the fasting serum samples using the glucose oxidase peroxidase method. Routine laboratory determinations were performed with an Olympus AU-400 analyser (Olympus, Tokyo, Japan).

Blood samples obtained from jugular venipuncture were collected in 10 ml Vacutainer tubes without anticoagulant. Sera were obtained by centrifugation at 1,500 g for 10 min and transferred to new tubes to be stored in aliquots at −80°C until further analysis. At the day of analysis, aliquots were thawed. Measurement of serum clinical biochemistry analytes was performed on the Olympus AU400 analyser except cortisol and TNF-alpha. The protocols for the use of Olympus System Reagents (OSRs) and other commercial reagents were in reference of previous studies (16 (link), 17 (link)). Quality control protocols were based on the daily quantification of two control sera of low and high concentration (Control serum I and Control serum II, Beckman Coulter). For cortisol and TNF-alpha in piglets, two commercial ELISA kits were used: Cortisol ELISA Kit (DRG, Marburg, Germany) and Porcine TNF-alpha Quantikine ELISA Kit (R&D Systems, Abingdon, UK). The analytes, methods, reagents, and coefficients of variation (CV%) are shown in Table S3.

PBS or 2 × 10¹⁰ vp of Adwt or AdCPE were injected intravenously into the tail vein of 6- to 8-week-old male immunocompetent C57BL/6 mice. Animals were weighed and examined daily for any clinical signs of toxicity. Three days later, mice were killed, organs were isolated and blood samples were collected by intracardiac puncture. Serum aspartate aminotransferase and alanine aminotransferase were determined on an Olympus AU400 Analyser (Olympus, Tokyo, Japan) at the Clinical Biochemistry Service, School of Veterinary Medicine, Autonomous University of Barcelona. All animal procedures met the guidelines of European Community Directive 86/609/EEC and were approved by the ethical committee (CEEA-University of Barcelona) and by the local authorities of the Generalitat de Catalunya.

Serum biochemistry analytes were analysed with the Olympus AU400 analyser with following techniques and OSR reagents (Olympus System Reagent)^{35 (link),36 (link)}: glucose (HK method), urea (GLDH method), creatinine (Jaffé method), cholesterol (CHOP-PAP-method), triglyceride (GPO-PAP method), total protein (Biuret method), ALT (International Federation of Clinical Chemistry (IFCC) method), AST (IFCC method) and GGT (IFCC method). NEFAs were determined with NEFA-C reagent (Wako Chemicals GmbH, Neuss, Germany). Quality control protocols were based on the daily quantification of two control sera of low and high concentration (Control serum I and Control serum II, Beckman Coulter) Haptoglobin was determined by a colorimetric method (Tridelta, Ireland). Insulin and IGF-I were determined by ELISA (Bovine Insulin ELISA from Mercodia, Uppsala, Sweden and Human IGF-I ELISA E20 from Mediagnost, Reutlingen, Germany). This reagent is suitable for bovine samples, as declared by the manufacturer, but the cross-reactivity was not assessed by the authors.
Intra-assay Coefficients of Variation (CV) were: glucose (1.7%), urea (2.1%), creatinine (1.4%), cholesterol (1.1%), triglycerides (3.0%), total protein (0.9), ALT (1.7%), AST (1.0%), GGT (0.6%), NEFAs (2.7%), Haptoglobin (4.1%), insulin (4.1%) and IGF-1 (4.4%).