Ual portions of concentrate given at 6:00, 14:00, and 22:00 h (provided in the milk collection instances). The animal was housed in an individual tie stall and water was accessible ad libitum. The experiment and animal handling procedures were approved by the Institutional Animal Care and Use Committee of Wageningen University and carried out under the Dutch Law on Animal Experimentation.Cow Plasma and Protein AnalysisPlasma phenylalanine and tyrosine were derivatized to their tbutyldimethylsilyl (TBDMS) derivatives and their 13C enrichments have been determined by electron ionization gas chromatography-mass spectrometry (GC-MS, Agilent 6890N GC/5973N MSD Little Falls, USA) applying selected ion monitoring of masses 336 and 337 for unlabeled and labeled L-[1-13C]phenylalanine, respectively; and masses 466, 467, for unlabeled and labeled L-[1-13C]tyrosine, respectively. The tissue protein-bound L-[1-13C]phenylalanine enrichments were determined from ,50 mg of wet weight meat and organ tissue. The bigger pieces of skeletal muscle and organ tissue samples (200?00 g) were reduce. Subsequently, smaller sized pieces of samples (,50 mg) have been extracted in the different regions of the larger tissue sample to supply a general enrichment value. The ,50 mg wet wt. tissue was freeze-dried, collagen, blood, and also other non-relevant material had been removed in the muscle or organ tissue below a light microscope. The isolated tissue mass (eight mg dry weight) was weighed and 8 volumes (86dry weight of isolated tissue6wet/dry ratio) ice-cold two perchloric acid (PCA) have been added. The tissue was then homogenized and centrifuged. The mixed tissue protein pellet was washed with 1.5 mL of two PCA as well as the pellet was lyophilized. Amino acids had been liberated by adding six M HCl right after which the hydrolyzed protein fraction was ?dried below a nitrogen stream although getting heated to 120C. The protein fraction was than dissolved inside a 25 acetic acid option and passed over a Dowex exchange resin. The amino acids have been eluted with two M NH4OH, dried, along with the purified amino acids wereStable Isotope InfusionAn outline from the experimental tracer infusion protocol is shown in Figure 1. A total of 400 g L-[1-13C]phenylalanine (Cambridge Isotopes Laboratories, Andover, MA, USA) was dissolved in 40 L of an isotonic glucose (five ) answer (Braun Melsungen AG, Germany) with a final concentration of 278 mmol glucose/L and 60.77545-45-0 Order five mmol L-[1-13C]phenylalanine/L.681004-50-2 Purity The volume of tracer and the duration of the infusion was selected to attain higher labeled milk protein and labeled meat of sufficient enrichment for use in human nutrition study [7].PMID:34337881 The infusates have been stored at 4uC and allowed to warm to space temperature prior to use. Two days just before the tracer infusion (248 h), catheters (Careflow 16 gauge6300 mm with 18 gauge670 mm needle introducer; Becton Dickinson, BD, Netherlands) had been inserted percutaneously under neighborhood anaesthetics within the ideal and left jugular vein for the intravenous tracer infusion and blood sampling, as previously described [6]. Directly following catheterization, a glucose infusion was initiated at a rate of 116 mmol/h. The continuous glucose infusion was maintained for 48 h before the experimental steady isotope infusion to maximize milk protein synthesis prices [12] andPLOS One | plosone.orgProtein Turnover within a Dairy CowFigure 1. Study protocol from the cow infusion protocol. doi:ten.1371/journal.pone.0068109.gderivatized into their N(O,S)-ethoxycarbonyl ethyl esters to identify the 13C enri.