7 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 had been determined from ,50 mg of wet weight meat and organ tissue. The larger pieces of skeletal muscle and organ tissue samples (20000 g) had been reduce. Subsequently, smaller pieces of samples (,50 mg) were extracted in the different regions in the bigger tissue sample to provide 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 2 perchloric acid (PCA) were added. The tissue was then homogenized and centrifuged. The mixed tissue protein pellet was washed with 1.five mL of two PCA along with the pellet was lyophilized. Amino acids have been liberated by adding six M HCl immediately after which the hydrolyzed protein fraction was dried beneath a nitrogen stream even though being heated to 120C. The protein fraction was than dissolved inside a 25 acetic acid remedy and passed more than a Dowex exchange resin. The amino acids were eluted with 2 M NH4OH, dried, plus the purified amino acids wereStable Isotope InfusionAn outline with 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 (5 ) remedy (Braun Melsungen AG, Germany) with a final concentration of 278 mmol glucose/L and 60.five mmol L-[1-13C]phenylalanine/L. The amount of tracer and also the duration in the infusion was selected to attain high labeled milk protein and labeled meat of enough enrichment for use in human nutrition analysis [7].EIDD-1931 MedChemExpress The infusates have been stored at 4uC and permitted to warm to room temperature prior to use.THIQ supplier 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 below regional anaesthetics inside the suitable and left jugular vein for the intravenous tracer infusion and blood sampling, as previously described [6]. Straight following catheterization, a glucose infusion was initiated at a rate of 116 mmol/h. The continuous glucose infusion was maintained for 48 h prior to the experimental stable isotope infusion to maximize milk protein synthesis prices [12] andPLOS One | www.PMID:24818938 plosone.orgProtein Turnover in 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 establish the 13C enrichment of tissue protein phenylalanine employing gas chromatography-combustion-isotope ratio mass spectrometry (GC-C-IRMS; MAT 252, Finnigan, Breman, Germany). Standard regression curves were applied from a series of known common enrichment values against the measured values to assess the linearity in the mass spectrometer (r2 = 0.99; y = 0.9894x+0.0) and to account for any isotope fractionation which might have occurred through the analysis.period, the typical L-[1-13C]phenylalanine enrichments had been 36.061 MPE. Linear regression evaluation indicated that the slope on the plasma L-[1-13C]phenylalanine enrichments were not drastically various from zero for the duration of the continuous infusion (P = 0.11), indicating that an is.