This study determined whether nitrogen balance was affected and whether dietary IAA were adequately obtained from the ad libitum consumption of diets at three levels of protein from different primary sources for 12 days. The successful implementation of the dietary protein intervention on each condition in both trials was confirmed by urinary nitrogen concentrations. There was a dose-dependent increase in nitrogen excretion with increasing dietary protein content, irrespective of the protein sources. Despite protein intake with the 5En%-protein diet being below the minimal protein requirement of 0.66 g.kgBW.d-1, it was sufficient to maintain nitrogen balance over 12 days. Nitrogen balance was positive on the 15En%- and 30En%-protein diets, irrespective of dietary protein composition. However, protein intake from the 5En%-protein diet did not reach the amount necessary to meet the calculated minimal IAA requirements, but IAA were sufficiently obtained from the 15En%- and 30En%-protein diets. In the 15En%- and 30En%-protein conditions, a higher protein intake from the soy-containing diets than from the whey with α-lactalbumin or beef containing diets was needed to meet the minimal IAA requirements.
Data of this study suggest that the lower concentration of protein provided with the 5En%-protein diet was not outside the range that is feasible to maintain nitrogen balance over 12 days. The Adaptive Demands model developed by Millward may provide an explanation for this observation by proposing that the metabolic demand for amino acids comprises a fixed component and a variable adaptive component . Short-term changes in protein intake are likely within the adaptive range . Adaptations in protein and amino acid metabolism to changes in protein intake largely occur via changes in whole body protein turnover and amino acid oxidation [2, 40]. Changes in amino acid oxidation were reflected as decreased and increased nitrogen excretion in response to the low- and high-protein diets respectively. The activity of the enzymes that regulates: 1) transamination, 2) the disposal of the carbon skeletons in intermediary metabolism, and 3) the disposal of nitrogen through the urea cycle was increased in response to high protein intake [41, 42]. Nevertheless, a positive nitrogen balance was observed in the present study following the high-protein diets despite increased enzyme activities. This is in line with earlier observations [2, 43–45], but does not automatically reflect an increase in protein anabolism . The capacity of the body to increase amino acid anabolism through an increase in lean body mass is limited . Only interventions using diets high in specific IAA, such as leucine, might be able to stimulate protein synthesis in specific target groups [46, 47]. Therefore, a transient retention or loss of body nitrogen because of a labile pool of body nitrogen may contribute to adaptations in amino acid metabolism in response to changes in protein intake . Transient adaptive mechanisms may be distinguished from mechanisms that maintain homeostasis in the body in the longer-term.
The calculated DIAAS for the 5En%-protein diet were well below 100%, which confirms that wheat is a low-quality protein. Especially at this low protein density, the intake of lysine was inadequate for satisfying the minimal IAA requirements. Protein intake should exceed 0.66 g.kgBW.d-1 to reach the minimal IAA intake. Consequently, measured intake from wheat protein being 0.39 g.kgBW.d-1 with the 5En%-protein diet means that minimal IAA requirements were not met. Protein intake was not spontaneously adjusted to reach the calculated optimal intake level of IAA. To meet the calculated minimal DIAAS level, subjects should have consumed ~63 MJ a day, from this diet. Interestingly, in animals, the detection of reduced concentrations of IAA in the anterior piriform cortex in the brain may result in deacetylation of the cognate transfer RNA . Subsequent activation of general amino acid nonderepressing kinase 2 may phosphorylate eukaryotic initation factor 2α, a factor involved in the control of the initiation of translation in protein synthesis. This may lead to behavioral responses including under-consumption of diets that lack a minimal amount of IAA [13, 16, 18]. The findings of this study do not show a behavioral response in humans [9, 10]. The lower concentration of protein provided in the present trials was not outside the range that is feasible to adjust protein intake. Nevertheless, a study  has documented acute food-choice compensation after low-protein meals in humans. After a low-protein meal, an increase in wanting and task-related signaling in the hypothalamus has been related to increased protein intake in a subsequent meal . Griffioen-Roose et al.  showed in a 4-day study that subjects increased their protein intake in a compensatory way during ad libitum feeding after a low-protein diet (5En% from protein). Since no shift towards a higher energy intake from the 5En% protein diet compared with the 15En% protein diet was observed (9, 10), the insufficient amount of IAA clearly does not trigger a possible compensatory protein intake over 12 days. Observations from studies in developing countries show health deterioration related to low protein intake, especially of IAA, at intakes < 0.66 g.kg BW.d-1. Thus, although we conclude that protein intake did not compensate for an insufficient IAA intake with a low-protein diet for 12 days, we do not propose that low-protein diets are sustainable.
Increasing the relative dietary protein content from whey with α-lactalbumin, soy or beef protein resulted in an improved protein quality of the diets. However, the higher DIAAS of the whey with α-lactalbumin and beef diets did not affect total daily protein intake differently compared with the lower DIAAS of the soy diets. Minimal requirements for IAA were reached with the ad libitum intake of each 15En% and 30En%-protein diet. However, a higher protein intake from the soy-containing diets than from the whey with α-lactalbumin or beef containing diets was needed to meet the minimal IAA requirements. This corresponds with the recommendations for vegetarians to consume more protein or to include a combination of different plant protein sources in the diet. Furthermore, a larger amount of protein from whey with α-lactalbumin or beef was available to fulfill roles beyond the obligatory role of maintenance. Since the subjects in this study were healthy, one of these roles may be the stimulation of muscle protein synthesis. The relative high abundance of leucine in whey with α-lactalbumin protein may be beneficial to stimulate muscle protein synthesis .
The DIAAS can be applied in practice to examine protein quality of food products or mixed diets. However, it should be emphasized that the calculations of the DIAAS rely on some assumptions. First, the use of digestibility coefficients based on animal data is unavoidable when human data is not available. Second, the calculated protein intake necessary to meet minimal IAA requirements is based on the assumptions of a daily minimal protein requirement. This value may vary depending on dietary protein composition and should be adjusted according to subject-specific protein requirements. Factors influencing protein and amino acid metabolism, subsequently affecting protein and IAA requirements, should be further elucidated. Longer-term intervention studies with measurements of blood concentrations of amino acids and other key factors in appetite regulation, whole body protein turnover and muscle protein synthesis would provide more insight in the changes in protein and amino acid metabolism in response to dietary protein intake. Furthermore, possible IAA sensing pathways involved in the regulation of energy and protein metabolism in humans remain to be investigated.