The principal finding of this study is that a combination of free EAA and whey protein is highly anabolic in healthy young volunteers. The anabolic response to the free EAA/protein composition was dose-dependent. Interestingly, the gain in NB following consumption of 12.6 g of free EAA plus whey protein was significantly greater than the response of NB to consumption of 6.3 g of the free EAA/protein product when normalized for the amount of product consumed (Fig. 4), due to a greater suppression of protein breakdown. The anabolic responses of either dose of free EAA/whey protein product was greater than the response to a whey protein-based commercial beverage when normalized to the amount consumed. When normalized for the amount of product consumed, the low dose free EAA/protein response of NB was approximately three- fold greater than the whey protein product, and the response of NB to the high dose of the free EAA/protein product was approximately six times greater than the response to the whey protein product.

The anabolic benefits of whey protein dietary supplements are well established, both in sedentary individuals as well as adjuncts to physical training (e.g., [21]. Similarly, the consumption of free EAA-based nutritional supplements are well-documented to stimulate muscle protein synthesis and net protein balance [5, 22], and sustained consumption improves physical function in older individuals [23]. The stimulation of muscle protein synthesis by consumption of less than 4 g of EAA has been reported to be as great as the response to consumption of 25 g dose of whey protein [24]. The enhanced anabolic effect of free EAA dietary supplements has been attributed by some to the activation of mTORC1 and related compounds involved in the initiation of protein synthesis [25]. Leucine in particular has been reported to play a key role in activating mTORC1, and thus the stimulation of muscle protein synthesis [26]. The notion that the addition of free leucine to a dose of dietary protein activates mTORC1, thereby amplifying the anabolic response to the amino acids in the protein, has been tested in previous studies [12, 13]. Results of studies in which free leucine has been added to dietary protein or to complete meals have been disappointing. In the circumstance of impaired anabolic responsiveness, such as occurs in cancer cachexia, the addition of leucine to a whey-based nutritional composition may enhance the anabolic response [27]. However, in healthy younger subjects, any beneficial effect of adding free leucine to intact protein is short-lived [25] or not detected [12, 28]. The problem with adding only leucine to dietary protein is that the availability of the other EAA becomes rate limiting. In particular, the plasma concentrations of the other branched chain amino acids (valine and isoleucine) fall below the fasting level when only extra leucine is added to intact protein [12].

The current study is the first of which we are aware in which a balanced formulation of free EAA has been combined with whey protein. The formulation differed from most EAA nutritional compositions in that leucine comprised only 20% of the free EAA. It has been postulated that the magnitude of anabolic response to dietary protein is determined by the increase in plasma leucine concentration, rather than to the amount of protein consumed [26]. In support of this perspective, EAA compositions designed for elderly individuals require a disproportionately high percentage of leucine to maximize the anabolic response than would be predicted from the composition of muscle protein [6]. However, disproportionately high leucine content in compositions designed to stimulate an anabolic response in younger heathy volunteers is not necessary [29]. Rather, in the current study the leucine content of the EAA/protein composition was based on the amount required to maintain a balance among all the protein synthetic precursors. By including only 20% of EAA as leucine, it was possible to increase the relative proportions of the other EAA, thereby providing all of the precursors necessary for synthesis of body proteins. Even with a low dose of free EAA comprised of only 20% leucine, the plasma leucine concentration rose almost 3-fold (Fig. 2), while the concentrations of the other EAA were increased in proportion to their requirements for muscle protein synthesis.

In addition to being able to produce a composition of exact proportions of EAA, free EAA have the advantage of being rapidly and completely absorbed [30]. The rapid peak response in plasma EAA is likely a key reason for their effectiveness [31]. On the other hand, the total duration of the response is limited, because just as the concentrations of EAA in the blood rise rapidly, they fall rapidly as well. For this reason, the composition tested in this study contains protein in addition to the EAA to prolong the anabolic response in the time after consumption.

Non-essential amino acids (NEAA) are not required for the acute anabolic response to EAA consumption [2,3,4]. This is because NEAA are normally produced in the body at fast enough rates to avoid deficiencies. On the other hand, studies performed in livestock suggest that maximal long-term animal growth and development is achieved with a balance of about 20–30% NEAA and 70–80% EAA [32]. The implication that NEAA availability can eventually become rate limiting for protein synthesis is supported by the fact that the NEAA, particularly alanine and glutamine, fall after consumption of a single dose of free-form EAA [33]. The addition of intact protein to a mixture of free-form EAA is the most efficient way to ensure an adequate amount of dietary NEAA to maximize long-term increases in lean body mass and physical function resulting from regular consumption. The action of peptides produced in the digestion of whey protein may have contributed to an interactive effect between free EAA and whey protein. Peptides of whey protein are reported to have a wide range of potential benefits (e.g., [8,9,10,11]), and amplifying the anabolic response to free EAA may be one such benefit. The current study design did not enable assessment of the role of peptides produced in the digestion of whey protein.

A comment about the relation between the whole-body protein and muscle protein FSR response is appropriate. Qualitatively the responses of muscle protein FSR were similar to the responses of whole-body protein synthesis with the three treatments. Further, the muscle FSR responses in the current study were generally in line with the results from comparable studies. For example, Churchward-Venne, et al., [7] reported that consumption of 1.5 g or 6 g of an EAA composition increased muscle FSR by 40 and 36%, respectively, as compared to a 50% increase following consumption 40 g of whey protein. The corresponding values in our study were 39 and 76% increases in FSR in response to 6.3 g and 12.6 g, respectively, of the free EAA/protein composition, and a 28% increase in response to the 12.6 g of whey protein in Gatorade Recover. However, in the current study the magnitude of the differences in whole-body net balance response between treatments was much greater than the differences in FSR, owing to a suppression of whole-body protein breakdown in addition to a greater stimulation of protein synthesis in the high-dose EAA/protein treatment. The two doses of the EAA/protein compositions resulted in increases in net protein balance of 3.6 ± 1.9 and 11.8 ± 1.8 g protein /4 h for the low- and high-dose free EAA/protein compositions, respectively, as compared to an increase of 3.0 ± 0.9 g for the Gatorade Recover. These results underscore the importance of quantifying both the rates of protein synthesis and breakdown when assessing the net anabolic response to a nutritional intervention.

The quantification of the response of whole-body net balance to nutrient consumption enabled the comparison of the amount of amino acids ± protein consumed with the net gain in body protein. The increase in body protein was approximately 24% of the amount of whey protein consumed with Gatorade Recover (Fig. 3). This percentage of net protein gain is consistent with the long-established relationship between N intake and N retention at levels of N intake above minimal requirements [34], and provides support for the quantitative validity of the whole-body protein model. In contrast to the response to whey protein, the gain in body protein was approximately 64 and 105% of the low-and high-doses of the free-form EAA/protein composition, respectively. The extraordinary increase of body protein in relation to the amount of amino acids in the free-form in the EAA/protein composition reflects the activation of the synthetic capacity by the rapid increase in EAA (including leucine) concentrations, the suppressive effects of a high-dose of EAA on protein breakdown [35,36,37], and the increased reutilization of endogenous NEAA to produce complete proteins.

It is appropriate to consider some of the advantages and limitations of quantifying the anabolic response by measurement of whole body protein synthesis and breakdown. Consideration of the response to nutrient ingestion at the whole body level is reasonable, since nutrients are consumed at the whole-body level. Importantly, whole-body protein turnover methodology enables the simultaneous determination of rates of protein synthesis and breakdown, and recent studies have highlighted the previously underappreciated role of protein breakdown in the anabolic response to protein intake [38]. Direct measurement of muscle protein FSR, on the other hand, provides information only on the protein synthetic response. Accurate measurement of net balance of muscle protein requires the invasive procedure of arterial and deep venous catheterization. Balanced against the advantages of whole-body protein kinetics, there are limitations. Calculated results reflect a pooling of the responses of all proteins in the body, and muscle protein may constitute as little as 25% of the total rate of whole body protein synthesis in some circumstances. Because the majority of whole body protein synthesis occurs elsewhere than the muscle, the rate of whole-body protein synthesis may not directly correspond to muscle protein FSR in some circumstances. However, with regard to the current study, the response of muscle FSR generally corresponded to the changes in whole-body protein synthesis, suggesting that at least some of the gain in net protein balance occurred in the muscle.

There are different methodological approaches to quantifying whole body protein synthetic and breakdown rates, all of which have advantages and limitations. We have recently discussed in detail the methodology used in the current study [39]. Importantly, we concluded that the necessary assumptions, while potentially contributing variability to the results, do not cause systematic over- or under-estimations of the calculated values. The validity of the whole-body methodology used in the current study is supported by comparison of the results to the results of other studies using different methodologies. As discussed above, there is a close relation between the net gain of body N following consumption of whey protein calculated by the tracer method and the value expected on the basis of previous N-balance studies. In addition, a key finding in the current study was that whole body protein breakdown was significantly suppressed with the highest dose of the EAA/protein composition. The suppressive effect of high concentrations of plasma amino acids on muscle protein breakdown in human subjects has been well-established for more than 20 years by arterial-venous balance studies [35,36,37].