Ultima gold xr

Uniformly ¹⁴C ring‐labelled isoproturon [3‐(4‐isopropylphenyl)‐1,1‐dimethylurea] (¹⁴C‐IPU) with a specific radioactivity of 9.96 kBq μg⁻¹ and radiochemical purity of 98% according to the producer was purchased from GE Healthcare (Little Chalfont, UK) and used as a representative pesticide for phenylurea herbicides. The ¹⁴C‐IPU was mixed with unlabelled IPU to provide a final concentration of 1.75 mg ml⁻¹ and a specific radioactivity of 154 Bq μg⁻¹. The new mix was denominated ‘¹⁴C‐IPU standard mix’. Non‐labelled IPU, MDIPU [3‐(4‐isopropylphenyl)‐1‐methylurea], 2‐OH‐MIPU 3‐(4‐(2‐hydroxyisopropylphenyl))1‐methylurea, DD‐IPU [3‐(4‐isopropylphenyl)‐urea] and 4‐isopropyl‐aniline (4‐IPA) were purchased from Dr. Ehrenstorfer (Augsburg, Germany; purity 99.5%). Scintillation cocktails (Ultima Gold XR and Ultima Flo AF) were obtained from Packard (Dreieich, Germany). All other chemicals and solvents were of analytical grade and were purchased from Merck (Darmstadt, Germany).

At the end of the second phase, pieces of liver, muscle and adipose tissue (about 0.2 g) collected from each group were cut finely in ice-cold 0.25 M-sucrose medium containing 2 mM-EGTA and 10 mM-2-amino-2-hydroxymethyl-propane-1,3-diol-HCl, pH 7.4, rinsed five times in the same medium, blotted with absorbent paper and weighed. The tissues were respectively diluted (1:40, 1:20 and 1:10, w/v) in the chilled sucrose medium and homogenized by using a drill-driven Teflon glass homogenizer (Ningbo Scientz Biotechnology co., China) with 4-6 strokes. The 1 ml samples of homogenate were used for the immediate measurement of [1-¹⁴C] palmitate oxidation20 (link)47 (link). Palmitate oxidation rate was measured at 28 °C using a media allowing both mitochondrial and peroxisomal FA oxidation to occur as already described53 (link). After 30 min, the reaction was stopped by addition of 10% (w/v) perchloric acid, which precipitated proteins. The media were filtered using Millipore filters (0.45 μm pore size) under very low pressure and the filtrate containing the acid-soluble products (ASP, the short metabolites from FA oxidation) was mixed with Ultima Gold XR (Packard) for radioactivity measurements.

After the eight-week trial, Nile tilapia from control and high cholesterol (1.6%) treatments were fasted overnight and then used for metabolic tracking of ¹⁴C-labelled nutrient. Six fish from each treatment were subjected to ¹⁴C-labelled nutrient tracking, including D-[1-¹⁴C]-glucose (Glu^∗), [1-¹⁴C]-palmitic acid (PA^∗) and L-[¹⁴C(U)]-amino acid mixture (AA^∗) (PerkinElmer). The doses for intraperitoneal injection of ¹⁴C-labelled were based on our previous studies (Li et al. 2020c (link)). To measure the ¹⁴CO₂ released from the oxidation of different nutrients, the injected fish were immediately transferred to oxygen-saturated water in sealed bottles, which were connected to another glass bottle containing 1 mol/L potassium hydroxide (KOH) solution. The fish were digested with a strong lysate (HClO₄ /30% H₂O₂, 2:1, v/v; 1:5, w/v) at 60 °C in a water bath for 6 h to ensure total retention of ¹⁴C in the whole body. The radioactivities of strong lysate and KOH solution (500 μl) were measured using the Tri-Carb 4910TR Liquid Scintillation Analyzer (PerkinElmer) with 2 ml of scintillation fluid (Ultima Gold XR; Packard, Conroe, TX, USA).