Packard tri carb 2900tr

After diet treatments, rats were starved for 12 h during the day. Three hours after light offset they received an intragastric application (1 ml per 100 g) of an amino acid mixture of all proteinogenic amino acids with a final concentration 10-fold higher than the plasma concentration (gavage-amino acid mixture, Table 2). The mixture was supplemented with 0.5 μCi/ml ³H-mannitol and 0.05 μCi/ml ¹⁴C-radiolabeled L-isoleucine. After 1 h, rats were anesthetized with Attane^TM Isolfurane ad us. vet (Piramal Healthcare, India) and euthanized by heart cut. The blood was collected in heparin-coated tubes (Braun Medical AG, Sempach, Switzerland), plasma was purified and 20 μl were used for ultra performance liquid chromatography amino acid measurements. The small intestine was harvested and each 10 cm of the proximal, middle and distal part were washed with 1 ml PBS. The content was collected and sections were inverted followed by mucosa scraping. The intestinal content, scraped mucosa and plasma samples were lysed overnight on a shaker in 1 ml Solvable^TM (Perkin Elmer, Waltham, MA, USA), followed by addition of Ultimate Gold^TM scintillation fluid (Perkin Elmer, Waltham, MA, USA) and determination of radioactivity using the liquid scintillation analyzer (Packard Tri-Carb 2900TR, PerkinElmer, Waltham, MA, USA).

RPF measurements were performed as terminal experiments in most animals in which GFR had been previously tested. An osmotic pump (2ML1 Charles River, Germany) containing a solution of [³H] PAH (Perkin Elmer, USA) and 10 μM unlabeled PAH (with HEPES as a buffer) in saline was implanted into the rat that was put into the metabolic cage for 24 h. The following day, food was taken away for 1 h before the animal was anesthetized (3% isoflurane), and blood was collected from both the renal vein and the aorta for RPF calculations. The urine collected in the metabolic cage provided the information for urinary flow rate and urinary tracer measurements. Tubes were prepared containing either 100 μl of plasma or 100 μl of urine. A volume of 3 ml of ultimate GoldTM scintillation fluid (Perkin Elmer, Waltham, MA, USA) was added and the tubes were shaken for 2 h following which the level of radioactivity was measured using the liquid scintillation analyzer (Packard Tri-Carb 2900TR, PerkinElmer, USA). The RPF was then calculated by using the formula RPF (ml/min) = (U*V)/(Pa − Pv) where U is the urinary concentration of [³H] PAH, V is the urinary flow rate in ml/min, Pa is the arterial plasma concentration of [³H] PAH, and Pv is the venous plasma concentration of [³H] PAH.