Casein synthesis is independently and additively related to individual essential amino acid supply.

Specific AA affect rates of milk protein synthesis in the mammary glands of lactating cows. The objective of this study was to quantify the rate of αS1-casein synthesis in response to Ile, Leu, Met, and Thr supplementation, and to test the single-limiting AA theory for milk protein synthesis by exploring interactions among these AA. Effects of Ile, Leu, Met, and Thr were studied in vitro with a composite design containing a central point repeated 4 times, with 2 axial points per AA and a complete 2(4) factorial. Other AA were at the concentration in Dulbecco's modified Eagle medium/F12 medium (DMEM). The experiment was replicated with mammary tissue from 5 lactating cows. Mammary tissue slices (0.12 ± 0.02 g) were incubated for 4h at 37°C in 5 mL of treatment medium containing (2)H5-Phe. Caseins were precipitated from cell homogenate supernatants. Enrichment with (2)H5-Phe of the N[34]LLRFFVAPFPE αS1 peptide was determined by matrix-assisted laser desorption/ionization-tandem time-of-flight (MALDI-TOF-TOF), which was used to determine enrichment of Phe in the transfer (t)RNA pool and αS1-casein fractional synthesis rates (CFSR). Data were analyzed with a polynomial mixed model containing linear, quadratic, and 2-factor interactions for Ile, Leu, Met, and Thr, and cow and residual as random factors. Interactions were not significant at P<0.1 and were removed from the model. Increasing concentrations of Ile, Leu, Met, and Thr simultaneously increased CFSR curvilinearly with a predicted maximum response of 4.32 ± 0.84%/h at 63% of DMEM concentrations. The maximum response to each of the 4 AA was at 71, 49, 60, and 32% of the concentration in DMEM, for Ile, Leu, Met, and Thr, respectively. These values correspond to 270, 120, 440, and 140% the plasma concentrations of Ile, Leu, Met, and Thr observed in lactating cows fed to meet National Research Council requirements, respectively. The CFSR estimated at those maxima were similar among AA (3.6 ± 0.6%/h). Individual AA effects on CFSR did not correlate with mammalian target of rapamycin (mTOR) signaling. Independent responses of CFSR to individual essential AA observed in this study contradict the single-limiting AA theory assumed in current requirement systems. The saturable responses in CFSR to these 4 AA also highlight the inadequacy of using a fixed postabsorptive AA efficiency approach for determining AA requirements for milk protein synthesis.