**2. Identification of classic amino acid antagonisms in the chick**

#### **2.1 Lysine and Arginine**

In early work concerning the arginine requirement for poultry, Almquist and Merritt [20] found that the arginine requirement increased at a constant rate as crude protein was increased, citing requirement values of 0.9, 1.2, and 1.8% of the diet for arginine at crude protein levels of 15, 20, and 25%, respectively. Taking this into account, Anderson and Dobson [21] noticed that the arginine requirement fluctuated in diets containing similar crude protein levels [22], and postulated that amino acid balance was the more important variable than crude protein in general at the conclusion of their own experimentation. Furthermore, Anderson and Dobson [21] indicated that a relationship between arginine and lysine might be expected due to similarities in their chemical structure and potentially their metabolism. Likewise, Fisher et al. [23] indicated that the amino acid content of casein, used in purified diets to determine amino acid requirements, likely increased the arginine requirement compared to diets containing soybean meal, which contains approximately a third the lysine of casein. This disparity between purified and commercial-type diets had been previously discovered by Krautmann et al. in 1957 [24], but Krautmann et al. [24] had failed to make the connection of amino acid content and instead proposed an "unidentified factor of plant origin" was to blame for the disparity in arginine requirements among diet types.

Due to the extreme variation of requirement estimates that had been published at the time, Lewis et al. [25] attempted to establish to an arginine requirement based on commercial-type diets used in the United Kingdom. Lewis et al. [25] not only investigated the effects of varying crude protein levels, but also examined the influence of an amino acid imbalance induced by excess lysine based on the previous work of Anderson and Dobson [21]. The results of these studies indicated that under practical conditions using commercial-type diets it was unlikely that an arginine deficiency would occur unless excess lysine was introduced into the diet [25]. Despite these findings, work continued using purified diets in order to understand the mechanisms behind the lysine-arginine antagonism.

Jones [26] studied the antagonism between lysine and arginine using diets containing casein and gelatin as the protein contributing ingredients. In this study, Jones [26] indicated that excess lysine depressed the utilization of arginine in purified diets containing these protein sources. Boorman and Fisher [27] then reported that the antagonism was not reciprocal, indicating that excess levels of arginine did not result in further growth depressions when lysine was deficient. Boorman and Fisher [27] went further to indicate that a lysine-arginine antagonism did not exist, but that the results of their experiment showed a response of a general amino acid toxicity.

A major step in the identification of a mechanism behind the lysine-arginine antagonism was reported by Jones et al. in 1967 [28]. First, Jones et al. [28] showed that both control and excess lysine fed chicks were able to effectively digest and absorb arginine, dispelling the theory that lysine reduces the utilization (i.e., digestion and absorption) of arginine. Secondly, Jones et al. [28] proposed three potential mechanisms behind the antagonism, of which the primary effect of lysine was indicated to be increased catabolism of arginine or a reduction in renal tubular resorption of arginine. The increase in dietary lysine was associated with an increase in kidney arginase activity, but as this was a delayed response, Jones et al. [28] did not believe it

to be the primary cause of the increased arginine catabolism. Boorman et al. [29] later showed that intravenous infusions of lysine resulted in increased plasma lysine levels and inhibited renal reabsorption of arginine in cockerels.

Nesheim [30] studied the influence of lysine on chickens selected for high and low arginine requirements. In these studies, Nesheim [30] found that excess lysine had a greater growth depressing effect on chickens selected for high arginine requirements compared with those selected for low requirements. Despite the larger effect observed in the high arginine birds, Nesheim [30] observed growth depressing effects of lysine on the low arginine requirement birds, seemingly independent of kidney arginase levels. Neisheim [31] also observed an increase in urinary arginine loss when high levels of lysine were fed. Conversely, Austic and Nesheim [32] observed two to four-fold increases in arginase activity when excess lysine, histidine, tyrosine, and isoleucine were fed with and without arginine. These responses were determined to occur in concert with the depressions in body weight gain through the implementation of time-course studies. Therefore, Austic and Nesheim [32] concluded that arginase activity was a major factor in the variation of the arginine requirement, in stark contrast to previous research.

In 1970, D'Mello and Lewis [33] published the first of their series of papers on amino acid interactions in chick nutrition, focusing on the lysine-arginine antagonism. As previous researchers had challenged the existence of a lysine-arginine antagonism [27], D'Mello and Lewis [33] utilized a basal diet limiting in methionine and only marginally adequate in arginine. When excess lysine was added to the diet, depressions in chick performance could not be corrected with additional methionine, but only when arginine was added. These responses suggested a restructuring of the order of limitation in the basal diet and confirmed a direct relationship between lysine and arginine. The third paper in the D'Mello and Lewis [34] series defined the arginine requirement when diets contained excess lysine. When arginine was titrated at four lysine levels, D'Mello and Lewis [34] reported a linear increase in the arginine requirement (**Figure 1**).

Allen et al. [35] titrated arginine in diets containing dietary lysine levels of 0.55, 0.95, 1.35, 1.95, and 2.55%. These titrations allowed for the comparison of growth curves to show the declining efficiency of arginine to promote weight gain. Arginine efficacy decreased linearly to 58.8% of control levels as lysine was increased to 1.84%.

**Figure 1.** *Influence of dietary lysine level on the determined arginine requirement. Adapted from D'Mello and Lewis [34].*

#### *Broiler Amino Acid Research: Then and Now DOI: http://dx.doi.org/10.5772/intechopen.101896*

Further increases of dietary lysine had no effect on arginine efficiency. Based on these observations, Allen et al. [35] concluded that the lysine-arginine antagonism was based on lysine magnifying the effects of an arginine deficiency. Allen and Baker [36] then determined the arginine requirement when dietary lysine levels were 0.95 and 1.95%. The required arginine level increased by 52 and 37% for body weight gain and feed conversion, respectively (**Figure 2**).

Wang et al. [37] investigated the influence of excess dietary lysine and arginine on the enzyme activity of lysine-ketoglutarate reductase and arginase. Increased supplementation of L-lysine HCl, ranging from 0 to 1.0%, resulted in a in an approximate two and five-and-a-half-fold increase in lysine-ketoglutarate reductase and arginase, respectively. Conversely, supplementing L-arginine from 0 to 2.0% resulted in an approximate two-fold increase in kidney arginase activity, but arginine supplementation had no effect on lysine-ketoglutarate reductase activity.

Kadirvel and Kratzer [38] examined the intestinal uptake of L-arginine and L-lysine when excesses of lysine, leucine, and glycine in vitro. Focusing on arginine and lysine, it was discovered that arginine absorption was reduced when lysine was added to the solution, but progressive amounts of lysine had no further influence on arginine absorption, indicating that limited competition between lysine and arginine exists during absorption. Kadirvel and Kratzer [38] then displayed the effects of feeding the aforementioned amino acids to broilers and evaluated their effects on bird performance. During the in vivo study, only excess lysine resulted in the appearance of arginine deficiency symptoms, which Kadirvel and Kratzer [38] interpreted to indicate that lysine-arginine antagonism is mediated through a metabolic effect as opposed to competitive absorption. Robbins and Baker [39] revisited the influence of amino acid excess on kidney arginase activity. They found that not only did lysine, arginine, and histidine influenced arginase activity, in agreement with Austic and Nesheim [32], but also an effect of total nitrogen that exceeded that of individual amino acids. Robbins and Baker [39] concluded that total nitrogen level was equally important in the activity of arginase as dietary arginine and lysine.

Based on research evaluating lysine-arginine antagonism from its discovery until the early 1980s several conclusions can be drawn characterizing the antagonism. First, a specific antagonism exists among arginine and lysine that appears to be

#### **Figure 2.**

*Influence of dietary lysine level on the arginine requirement for body weight gain (solid line) and feed conversion (dashed line). Adapted from Allen and Baker [35].*

non-reciprocal, displaying only effects of lysine on arginine metabolism. Secondly, the reason for the discovery of said antagonism lies in the amino acid contents of specific protein sources that were used in the diets of the period used to characterize amino acid requirements, namely casein due to its low arginine content relative to lysine. Lastly, the mechanism behind the lysine-arginine antagonism has not been cleanly defined but it does appear to be linked to the reduced capacity for the renal tubes to reabsorb arginine. While the role of arginase, and lysine's effect on it, is still debated, the findings of Robbins and Baker [39] combined with the findings of Keene and Austic [40] twenty years later may potentially explain the conflicting reports on arginase activity. Keene and Austic [40] found that catabolic enzymes are stimulated more by dietary protein than by the single amino acid targeted by the enzyme. The response of arginase by multiple amino acids is likely the response of increased dietary nitrogen, or in the case of Robbins and Baker [39] a balanced amino acid mixture, as opposed to the individual amino acids.
