Quantitation of blood and plasma amino acids using isotope dilution electron impact gas chromatography/mass spectrometry with U-13C amino acids as internal standards

Improved representation of postabsorptive N metabolism in lactating dairy cows requires a better understanding of protein synthesis regulation in the mammary glands. This study aimed to determine the quantitative effects of Ile, Leu, Met, and Thr on the phosphorylation state of signaling proteins that regulate protein synthesis. The experiment used a composite design with a central point, 2 axial points per AA, and a complete 2(4) factorial. All of the other AA were provided at the concentrations in Dulbecco's modified Eagle's medium. The experiment was replicated with tissues from 5 lactating cows. Mammary tissue slices (0.12 ± 0.02 g) were incubated for 4h. Total and site-specific phosphorylated mammalian target of rapamycin (mTOR; Ser2448), eukaryotic elongation factor (eEF) 2 (Thr56), ribosomal protein S6 (Ser235/236), and eukaryotic initiation factor 2α (Ser51) were determined by western immunoblotting. Tissue concentrations of the 4 AA studied responded linearly to media supply. Addition of Ile, Leu, Met, or Thr had no effect on eukaryotic initiation factor 2α phosphorylation. Isoleucine and Thr positively affected mTOR phosphorylation. However, the 2 AA had an antagonistic relationship. Similarly, Ile linearly increased ribosomal protein S6 phosphorylation, and Thr inhibited the Ile effect. In addition, eEF2 phosphorylation was linearly decreased by Ile and Leu. Threonine curvilinearly decreased eEF2 phosphorylation, Ile and Leu negatively interacted on eEF2, and Thr tended to inhibit Leu effects on eEF2. This work demonstrated saturable responses and interactions between AA on activation of the mTOR pathway. Incorporation of these concepts into milk protein response models will help to improve milk and milk protein yield predictions and increase postabsorptive N efficiency and reduce N excretion by dairy cows.

Section: Tissue Free Aa Concentrationsmentioning

confidence: 99%

Isoleucine, leucine, methionine, and threonine effects on mammalian target of rapamycin signaling in mammary tissue

Apelo

Singer

Lin³

et al. 2014

“…Plasma concentrations of pAH were determined after deacetylation of pAH by heating the samples at 90°C for 2 h in acidic conditions (Lobley et al, 1995). Heparinized plasma samples were analyzed for AA by GC-MS using the isotope dilution method (Calder et al, 1999) with (heptafluorobutyric) AA derivative for Arg, and the N-(tert-butyldimethyl) AA derivative for all other AA (Doepel and Lapierre, 2010). Arterial plasma concentrations of urea-N were determined in fresh plasma samples at the sampling day using a Technicon AutoAnalyzer system (Seal Analytical Inc., Mequon, WI; Huntington, 1984).…”

Section: Analytical Proceduresmentioning

confidence: 99%

Abomasal amino acid infusion in postpartum dairy cows: Effect on whole-body, splanchnic, and mammary amino acid metabolism

Larsen

Galindo

Ouellet

et al. 2015

Nine Holstein cows with rumen cannulas and indwelling catheters in splanchnic blood vessels were used in a generalized randomized incomplete block design with repeated measures to study the effect of increased early postpartum AA supply on splanchnic and mammary AA metabolism. At calving, cows were blocked according to parity (second and third or greater) and allocated to 2 treatments: abomasal infusion of water (CTRL; n=4) or free AA with casein profile (AA-CN; n=5) in addition to a basal diet. The AA-CN infusion started with half of the maximal dose at the calving day (1 d in milk; DIM) and then steadily decreased from 791 to 226 g/d until 29 DIM. On 5, 15, and 29 DIM, 6 sample sets of arterial, portal, hepatic, and mammary blood were taken at 45-min intervals. Over the whole period, increasing AA supply increased milk (+7.8 ± 1.3 kg/d) and milk protein yields (+220 ± 65 g/d) substantially. The increased milk yield was not supported by greater dry matter intake (DMI) as, overall, DMI decreased with AA-CN (-1.6 ± 0.6 kg/d). Arterial concentrations of essential AA were greater for AA-CN compared with CTRL. The net portal-drained viscera (PDV) release of His, Met, and Phe was greater for AA-CN compared with CTRL, and the net PDV recovery of these infused AA ranged from 72 to 102% once changes in DMI were accounted for. The hepatic removal of these AA was increased equivalently to the increased net PDV release, resulting in an unaltered net splanchnic release. The net PDV release of Ile, Leu, Val, and Lys tended to be greater for AA-CN, and the net PDV recovery of these infused AA ranged from 69 to 73%, indicating increased PDV metabolism with AA-CN. The fractional hepatic removal of these AA did not differ from zero and was unaffected by the increased supply. Consequently, the splanchnic release of these AA was approximately equivalent to their net PDV release for both CTRL and AA-CN. Overall, greater early postpartum AA supply increased milk and milk protein yields substantially based on increased mammary AA uptake. The PDV metabolism of branched-chain AA and Lys were increased, whereas it seemed to be unaffected for other essential AA when the intestinal AA supply was increased. On a net basis, the liver removed more group 1 AA (His, Met, Phe, and Trp) for anabolism and catabolism when the early postpartum AA supply was increased. Thus, increasing the postpartum AA supply increased splanchnic and mammary consumption of AA; hence, the protein deficiency persisted.

“…The plasma concentration of NO was measured calorimetrically (Green et al, 1982) using the Griess reaction with a commercial NO test kit (Nanjing Jiancheng Bioengineering Research Institute, Nanjing, China). The concentrations of total AA in the plasma were analyzed according to the procedure described by Calder et al (1999) using kits (DiaSys Diagnostic Systems; Shanghai Co. Ltd., Shanghai, China). The plasma ammonia N concentration was determined by the method developed by Seligson and Hirahara (1957).…”

Section: Sampling Measurement and Analysesmentioning

confidence: 99%

Effect of dietary N-carbamoylglutamate on milk production and nitrogen utilization in high-yielding dairy cows

Chacher

Zhu

et al. 2014

The objective of the current study was to investigate the effect of N-carbamoylglutamate (NCG) supplementation on milk production and nitrogen (N) utilization in Chinese Holstein dairy cows. Sixty multiparous cows (78±17.3 d in milk, 635±61.00kg of body weight, and 41.9±7.9kg/d milk yield; mean ± SD) were blocked by parity, days in milk, and milk yield and randomly allocated to 1 of 4 groups, each of which was fed a dietary treatment containing 0 (control), 10, 20, or 30g of NCG/d. Milk yield was recorded weekly. Dry matter intake, milk composition, plasma variables, and urea N contents in plasma, urine, and milk were determined every other week. Blood samples were collected from the coccygeal vein. Rumen microbial protein synthesis was estimated based on the purine derivatives in the urine. Dry matter intake was found to be similar between the treatments. Addition of 20g of NCG/d tended to increase milk yield (40.2 vs. 38.1kg/d) and increased the content (2.83 vs. 2.74%) and yield (1.12 vs. 1.02kg/d) of milk protein compared with the control. The yield and content of milk fat were similar between the treatments, whereas the contents of lactose and total solids increased linearly with an increase in NCG. Dietary supplementation of NCG linearly increased the plasma nitric oxide level and decreased the plasma ammonia N level. Compared with the control, the plasma Arg concentration in cows fed 10, 20, and 30g of NCG/d was increased by 1.1, 10.4, and 16.0%, respectively. The urea N concentrations in the milk, plasma, and urine decreased with the addition of NCG, although the lowest urea N concentrations were observed with the addition of 20g of NCG/d. The conversion of dietary crude protein to milk protein exhibited quadratic trends of improvement by NCG supplementation, with a peak at 20g of NCG/d. The rumen microbial protein synthesis was not altered by NCG supplementation, but the metabolizable protein tended to show a quadratic increase in cows fed 20g of NCG/d. In conclusion, supplementation of 20g of NVG/d may alter the plasma metabolites, optimize the AA profile, increase the metabolizable protein utilization, and thereby improve the lactation performance and N utilization of high-yielding dairy cows.