Urinary and plasma metabolome of farm mink (Neovison vison) after an intervention with raw or cooked poultry offal: a ¹H NMR investigation

Abstract

Deuterium oxide (D2O, 99.9 atom % D), monobasic potassium phosphate (KH2PO4, ≥99.0%), dibasic potassium phosphate (K2HPO4, ≥98.0%), monosodium hydrogen phosphate (NaH2PO4 99% anhydrous), disodium hydrogen phosphate (NaH 2 PO 4 99% anhydrous) 3-(Trimethylsilyl) propionic-2,2,3,3-d4 acid sodium salt (TSP, 98 atom % D, ≥98.0% (NMR)), and sodium azide (NaN3, ≥99.5%) were purchased from Sigma-Aldrich (Søborg, Danmark). Water used throughout the study was purified using a Millipore lab water system (Merck KGaA, Darmstadt, Germany) equipped with a 0.22 μm filter membrane. NMR Sample preparation -BiofluidsA total of 80 μl phosphate buffer was mixed with 720 μl of urine sample in 2.0 ml plastic Eppendorf tubes. Samples were vigorously vortexed for 5 s and a 600 μl aliquot was transferred into NMR SampleJet tubes (Bruker Biospin, Ettlingen, Germany) of L = 103.5 mm and O.D. = 5.0 mm. For plasma samples, a total of 350 μl buffer was mixed with 350 μl of plasma sample.The phosphate buffer was prepared as described by Khakimov et al. (2020). Plasma buffer was prepared as described by Dona et al. [45]. NMR Sample preparation -FeedThree replicates of feed samples for each group and each time-point available were prepared for NMR analysis in two ways: one to obtain the hydrophilic extract and one to obtain the hydrophobic extract. Samples were homogenised with an ultraturrax, then 500 mg were weighted. Twelve millilitre of a methanol/chloroform solution (1:2) were then added and the solution was homogenised, then stored in darkness for 30 min. Four millilitre of deuterated water (D2O) were then added and samples were shaken manually, then stored in darkness in the same manner. Samples were then filtered through Whatman #4 filter paper and centrifuged for 1 h at 1300 rpm and 4°C to separate the two phases. One millilitre of hydrophilic phase was then