Endogenous Synthesis of Amino Acids Limits Growth, Lactation, and Reproduction in Animals^,

Abstract

Amino acids (AAs) are building blocks of protein. Eight AAs (Ala, Asn, Asp, Glu, Gln, Gly, Pro, and Ser) are formed by all animals, whereas de novo synthesis of Arg occurs in a species-specific manner in most mammals (e.g., humans, pigs, and rats). Synthesizable AAs were traditionally classified as nutritionally nonessential for animals, because they were thought to be formed in sufficient amounts. However, this assumption is not supported by evidence showing that 1) rats grow slowly when their diets do not contain Arg, Glu, or Gln despite adequate provision of all other proteinogenous AAs; 2) pigs cannot achieve maximum growth, lactation, or reproduction performance when fed corn- and soybean meal-based diets meeting National Research Council–recommended requirements of protein and AAs without supplemental Arg, Glu, Gln, Gly, or Pro; 3) chickens exhibit increases in lean tissue gain and feed efficiency when their diets are supplemented with Glu, Gln, Gly, and Pro; 4) lactating cows cannot obtain maximum milk protein production without a postruminal supply of Gln or Pro; 5) fish cannot achieve maximum growth when diets do not contain Gln or Pro; and 6) men fail to sustain spermatogenesis when fed an Arg-deficient diet. Quantitative analysis of nitrogen metabolism showed that AA synthesis in animals is constrained by both precursor availability and enzyme activity. Taken together, these findings support the conclusion that the endogenous synthesis of AAs limits growth, lactation, and reproduction in animals. This new knowledge can guide the optimization of human nutrition for improving health and well-being.

Introduction

Amino acids (AAs)⁶ are precursors for the synthesis of protein and low-molecular-weight substances (e.g., NO, polyamines, creatine, serotonin, dopamine, and glutathione) with enormous physiologic importance (1). Animals synthesize the carbon skeletons and intact molecules of Ala, Asn, Asp, Glu, Gln, Gly, Pro, and Ser in a cell- and tissue-specific manner but cannot form the carbon skeletons of Cys, His, Ile, Leu, Lys, Met, Phe, Thr, Trp, Tyr, or Val. De novo synthesis of Arg is species-specific, with most mammals (e.g., humans, pigs, cattle, sheep, mice, and rats) synthesizing this AA from Glu, Gln, and Pro via the intestinal-renal axis (2), whereas birds and some mammals (e.g., cats and ferrets) cannot synthesize Arg due to the lack or limited expression of ≥1 enzymes (e.g., pyrroline-5-carboxylate synthetase) in the intestinal pathway (2). Compared with mammals, the synthesis of Pro in chickens (3) and certain fish (4) is limited. This underscores the complexity of AA nutrition in the animal kingdom.

Historically, AAs whose carbon skeletons are not synthesized by animal cells were termed “nutritionally essential AAs” (EAAs) and must be included in diets, whereas AAs whose carbon skeletons are synthesized by animal cells were traditionally thought to be dispensable in diets and were classified as “nutritionally nonessential AAs” (NEAAs) (5, 6). Inconsistent with this long-standing nutrition concept, more-recent data indicate that animals have dietary requirements of NEAAs to fulfill their genetic potential for maximum growth, lactation, and reproduction (7–9). The main objective of this article is to quantitatively analyze the synthesis of AAs from dietary protein in young, lactating, and gestating animals, with the pig as the primary animal model.

Current Status of Knowledge

AA nutrition is a rapidly expanding field in both medicine and animal production (1). A major goal of researchers is to enhance protein synthesis and to reduce the production of ammonia, which is highly toxic to the organisms at elevated concentrations and which contributes to environmental pollution (10). Although much is known about the importance of EAAs in nutrition, there is a paucity of information with regard to the dietary need for synthesizable AAs by the organisms (9). Studies in pigs, rats, and humans have shown that dietary AAs undergo extensive first-pass catabolism in the small intestine and are metabolized in a cell- and tissue-specific manner (11–13). These metabolic transformations are expected to affect endogenous synthesis of AAs and the need for synthesizable AAs in diets under physiologic and pathological conditions.

Utilization of dietary AAs in the small intestine and its luminal microbes

Proteases and peptidases in the gastrointestinal tract, produced locally or by the pancreas, are responsible for the hydrolysis of dietary protein into free AAs, dipeptides, and tripeptides for absorption by enterocytes and luminal bacteria in the small intestine (1). Inside the cells, small peptides are rapidly hydrolyzed into free AAs. Studies in pigs have shown that mammalian enterocytes degrade nutritionally significant quantities of Ala, Asp, Glu, Gln, Pro, Ile, Leu, and Val in diets but have little or no ability to catabolize Asn, Gly, Ser, Cys, His, Lys, Met, Phe, Thr, Trp, and Tyr (14, 15). All of these AAs are utilized by bacteria in the small intestine for protein synthesis and catabolism at various rates (16–19).

Dietary AAs that escape metabolism in enterocytes and intestinal bacteria enter the lamina propria before transport into the microvasculature (1). During this passage, AAs are utilized by multiple cell types within the mucosa and submucosa, including fibroblasts, macrophages, lymphocytes, smooth muscle cells, and endothelial cells (20). Due to different activities of AA-catabolic pathways in the small intestine, the proportions of dietary AAs entering the portal vein vary greatly. For example, in 8-kg pigs fed a casein-based diet, 56% of dietary AAs enter the portal circulation, whereas 10% and 34% of dietary AAs are utilized in the small intestine via protein synthesis and catabolism, respectively (11). Likewise, results of our studies indicated that 95–97% of Glu and Asp, 67–70% of Gln, and 40% of Arg and Pro in the enteral diet are catabolized by the small intestine of weaned and gestating pigs (21–24). Of note, the mammalian small intestine takes up a large amount of Gln, but no other AAs, from the arterial blood (13). Products of intestinal AA metabolism include ammonia, citrulline, Arg, Ala, and Pro (1).

Knowledge of AA metabolism in the small intestine represents a new paradigm shift in our understanding of protein nutrition in animals. Traditionally, it was assumed that dietary AAs were not degraded by the small intestine and that the ratios of individual EAAs to lysine in the body were similar to those in the enteral diet, which constitutes the long-standing “ideal protein” concept in animal nutrition (25). This view is not consistent with the findings that substantial catabolism of dietary AAs in the small intestine selectively modifies their entry into the systemic circulation for utilization by extraintestinal tissues (21).

Interorgan metabolism of dietary AAs

NO-dependent blood flow.

The utilization of dietary AAs depends on cooperation among many different organs. This requires adequate blood flow and AA transport by multiple cell types. Rates of blood flow, which partially affect the transport of AAs and other nutrients across tissues, are regulated by NO, a major vasodilator released by endothelial cells. An increase in utero-placental blood flow is particularly important for the growing fetus, which needs both oxygen and nutrients from its mother. Furthermore, the rate of blood flow across the lactating mammary gland critically influences the provision of substrates for milk production by the mother (26). Of note, NO is synthesized from Arg by tetrahydrobiopterin- and NAD(P)H-dependent NO synthase (1). In endothelial cells, Arg stimulates tetrahydrobiopterin synthesis by activating expression of GTP cyclohydrolase I. This shows an important regulatory role for AAs in the whole-body homeostasis and health of animals.

Intestinal-renal axis for Arg synthesis.

As noted previously, dietary Gln, Glu, and Pro, as well as arterial Gln, are converted into citrulline and Arg in the enterocytes of most mammals, with citrulline being taken up by the kidneys and extrahepatic tissues for Arg production (2). Arg is then available for utilization by tissues in the body. Rates of citrulline and Arg syntheses depend on developmental stages, substrate availabilities, and intestinal activities of key enzymes, such as N-acetylglutamate synthase, pyrroline-5-carboxylate synthase, and Pro oxidase (2).

Renal Gln utilization for regulation of acid-base balance.

Gln plays an important role in regulating acid-base balance through ammonia production in the kidneys, where NH₃ (ammonia) combines with H⁺ (proton) to form NH₄⁺ in the urine (27). Under acidotic conditions (e.g., intensive exercise, early lactation, late pregnancy, and diabetes), the renal uptake and catabolism of Gln are increased greatly to support ammoniagenesis and removal of H⁺. In normal growth and development, Gln is essential for the synthesis of aminosugars and glycoproteins. Both of these are of high relevance for embryos and fetuses in gestating dams (28). This is consistent with the fact that Gln is the most abundant AA in the plasma of humans and in most animals, including rats and chickens (1).

Gln synthesis from BCAAs.

Because most dietary Gln does not enter the portal circulation, Gln in the blood is derived primarily from de novo synthesis from BCAAs, glucose-derived α-ketoglutarate, and NH₄⁺ in extraintestinal tissues, including skeletal muscle, heart, adipose tissue, lungs, and placenta (1). Because of the limited activity of BCAA transaminase in the liver, dietary BCAAs are available for utilization by extrahepatic tissues, including skeletal muscle, the placenta in gestating dams, and the mammary gland in lactating mothers (29). Branched-chain α-ketoacids released by extrahepatic tissues are taken up primarily by the liver for either oxidation or gluconeogenesis (1).

Gly synthesis from hydroxyproline.

Milk is severely deficient in Gly (30, 31) but contains a high quantity of hydroxyproline in both peptide and free forms (32). For example, in piglets nursed by sows, milk provides, at most, only 23% of Gly for tissue protein synthesis and other metabolic pathways (33). Hydroxyproline is also formed from Pro residues in collagens as a post-translational modification. After these proteins are hydrolyzed, hydroxyproline (mainly 4-hydroxyproline) is released. Hydroxyproline is converted into Gly in kidneys, providing endogenous Gly to support neonatal growth, development, and health (33). Therefore, both carbons and nitrogen of the Pro molecule are effectively spared in milk-fed neonates. Similarly, a regular diet provides, at most, only 30% of Gly for metabolic utilization in adult humans (34).

Trp catabolism.

Trp is catabolized sequentially by Trp-2,3-dioxygenase or indoleamine-2,3-dioxygenase and kynurenine formamidase to generate kyrurenine in the liver and immune system cells (35). Through binding to its aryl hydrocarbon receptor, kynurenine increases mast cell activation, induces hypotension, and modulates apoptosis. Results of recent studies indicate that indoleamine-2,3-dioxygenase in lymphocytes and many cell types (e.g., peripheral blood mononuclear cells, dendritic cells, and mesenchymal stem cells) is induced by cytokines and that the kynurenine pathway plays an important role in responses to immune activation (35). Thus, Trp can modulate the catabolism of dietary AAs in the mucosa and luminal bacteria of the small intestine, thereby affecting their availability to extraintestinal tissues.

Insufficient synthesis of NEAAs in animals

The synthesis of AAs requires substrates (e.g., EAAs and α-ketoacids), cofactors, and enzymes (1). In animals and humans, dietary protein in excess to provide several limiting AAs is not advised due to the release of large amounts of ammonia from other AAs (9). Likewise, the consumption of protein below requirements is discouraged due to the degeneration of tissues (particularly skeletal muscle), impaired growth, low antioxidative capacity, and suboptimal immunity (36). The National Research Council has recognized that Arg and Gln are conditionally essential AAs for pigs but did not indicate the dietary needs for other synthesizable AAs (37), whereas the Institute of Medicine did not consider dietary needs for any NEAAs by humans (38).

Milk-fed neonates

Although the digestibilities of milk AAs are ≥96% (39), results of artificial feeding indicate that sow-fed piglets exhibit, at most, only 57.5% of their growth potential (40). In piglets, dietary EAAs greatly exceed protein accretion (Table 1). Among NEAAs, only Asn and Ser in diets exceed the needs for their accretion in the neonate and other dietary NEAAs cannot meet the needs for protein accretion in the neonate. These deficits are particularly severe for Asp, Glu, Gly, and Arg (Table 1). Accordingly, de novo synthesis of these AAs is highly active in milk-fed neonates (7). For example, in 14-d-old suckling pigs, net rates of synthesis of Arg, Gln, and Gly are at least 0.58, 1.15, and 1.20 g/kg body weight (BW) per day, respectively (24, 33, 40, 41).

TABLE 1.

Metabolism of AAs in 14-d-old piglets (3.9 kg BW) reared by sows¹

					Extraintestinal metabolism of AAs via non-protein-synthesis pathways5
AAs	AAs in sow milk,2 g/L	Dietary AAs entering the portal vein,3 g/d	AA accretion in extraintestinal tissues,4 g/d	Dietary AAs entering the portal vein/AA accretion in extraintestinal tissues, g/g	Total amount, g/d	Total nitrogen, mmol/d	Total carbon, mmol/d
Catabolism of AAs whose carbon skeletons are not synthesized by animal cells
Cys	0.72	0.50	0.40	1.25	0.10	0.83	2.48
His	0.92	0.76	0.63	1.21	0.13	2.51	5.03
Ile	2.28	1.41	1.07	1.32	0.34	2.59	15.6
Leu	4.46	2.78	2.06	1.35	0.72	5.49	32.9
Lys	4.08	3.09	1.82	1.70	1.27	17.4	52.1
Met	1.04	0.85	0.57	1.49	0.28	1.88	9.38
Phe	2.03	1.50	1.05	1.43	0.45	2.72	24.5
Thr	2.29	1.32	1.03	1.28	0.29	2.43	9.74
Trp	0.66	0.52	0.33	1.58	0.19	1.86	10.2
Tyr	1.94	1.43	0.80	1.79	0.63	3.48	31.3
Val	2.54	1.51	1.28	1.18	0.23	1.96	9.82
Subtotal	23.0	15.7	11.0	—	4.63	43.1	203
Net synthesis of AAs whose carbon skeletons can be formed by animal cells
Ala	1.97	1.38	1.98	0.70	−0.80	−8.98	−26.9
Arg	1.43	1.06	2.05	0.52	2.276	13.06	0
Asn	2.53	2.00	1.07	1.87	−0.93	−14.1	−28.2
Asp	2.59	0.11	1.29	0.085	1.18	8.87	35.5
Glu	4.57	0.21	2.36	0.089	2.15	14.6	73.1
Gln	4.87	1.42	1.52	0.93	0.10	1.37	3.40
Gly	1.12	0.82	3.41	0.24	2.59	34.5	69.0
Pro	5.59	3.12	3.667	0.85	0.37	3.21	16.1
Hyp	1.04	0.86	0.0	—	−0.86	−6.56	−32.8
Ser	2.35	1.72	1.34	1.28	−0.38	−3.62	−10.8
Subtotal	27.0	12.7	18.7	—	5.69	42.3	98.3

¹Values are means derived from previously published studies (21, 24, 47). The molecular weights of intact AAs were used for all of the calculations. AA, amino acid; BW, body weight; Hyp, hydroxyproline.

²Data are grams of AAs per liter of whole sow milk obtained on days 7–21 of lactation.

³Calculated on the basis of milk consumption (913 mL/d), the true ileal digestibility (%) of AAs in porcine milk protein (Arg, 90; His, 100; Ile, 90; Leu, 91; Lys, 92; Met, 99; Phe, 90; Pro, 94; Thr, 84; Trp, 96; Val, 87; Ala, 85; Asn, 96; Asp, 96; Cys, 84; Glu, 100; Gln, 100; Gly, 89; Ser, 89; and Tyr, 90), and the bioavailability (%) of orally administered AAs entering the portal vein (Arg, 90; His, 90; Ile, 75; Leu, 75; Lys, 90; Met, 90; Phe, 90; Pro, 65; Thr, 75; Trp, 90; Val, 75; Ala, 90; Asn, 90; Asp, 5; Cys, 90; Glu, 5; Gln, 32; Gly, 90; Ser, 90; and Tyr, 90). Products of intestinal AA metabolism that enter the portal vein are not included.

⁴Calculated on the basis of a BW gain of 235 g/d and the content of AAs in young pigs.

⁵Rates of extraintestinal net catabolism of AAs whose carbon skeletons are not synthesized by animal cells are calculated as dietary AAs entering the portal vein − (AA accretion in the body + AAs formed from intestinal catabolism of dietary AAs). Rates of extraintestinal net synthesis of AAs whose carbon skeletons can be synthesized by animal cells are calculated as AA accretion in the body − (dietary AAs entering the portal vein + AAs formed from intestinal catabolism of dietary AAs). Rates of AA formation from intestinal catabolism of dietary AAs (g/d) are estimated to be 1.40, 0.17, and 2.27 for Ala, Pro, and Arg, respectively, and to be negligible for other AAs. The minus sign (–) indicates the contribution of dietary AAs for extraintestinal AA synthesis in a cell- and tissue-specific manner.

⁶In pigs, citrulline (the precursor of Arg) is synthesized from Glu, Gln, and Pro exclusively in the small intestine, and citrulline is converted into Arg mainly in extraintestinal tissues. Asp provides an amino group for Arg synthesis from citrulline.

⁷Including Pro and Hyp for the calculation of extraintestinal carbon and nitrogen balance.

The total amount of nitrogen in EAAs available for extraintestinal utilization via non-protein-synthesis pathways exceeds that in synthesized AAs by only 1.9%, and the total amount of carbon in EAAs available for extraintestinal non-protein-synthesis pathways is 107% greater than that in synthesized AAs (Table 1). The amount of EAAs available for AA synthesis will not support even a 2% increase in the BW gain of 14-d-old suckling piglets. In absolute amounts, only 4.63 g EAAs (20% of dietary EAAs entering the portal vein) and 2.97 g Ala plus Asn plus hydroxyproline plus Ser (11% of dietary NEAAs entering the portal vein) are available for extraintestinal catabolism (Table 1). At present, it is unknown whether neonates can adequately synthesize all NEAAs even if substrate AA availability is not limiting (8, 42). Results from our piglet studies indicate that the extracellular concentration of Gln is not the major limiting factor for citrulline synthesis in enterocytes of 17- to 21-d-old piglets (43). Rather, low intramitochondrial concentrations of N-acetylglutamate (an allosteric activator of carbamoylphosphate synthase I) limit the formation of citrulline from Orn (41). Furthermore, piglets have low rates of Gln and Ser syntheses from supplemental Leu (44) and Gly (33), respectively. Thus, AA synthesis in neonates is constrained by both substrate availability and enzyme activity.

Thus, direct supplementation with NEAAs, their appropriate precursors, or high-quality protein (the source of all preformed AAs) to neonates may be needed to enhance their growth. In support of this view, supplementing 1% Gln (45), 1–2% Gly (46), 0.2–0.4% Arg (47), 1–2% Leu (a precursor for Glu, Asp, and Arg syntheses), or milk protein (doubling protein intake from the basal diet) to piglets with a normal birth weight (48) increased both lean tissue growth and BW gains. Much evidence shows that insufficient synthesis of Arg, Gln, and Gly limits the maximum growth of young pigs. Similar results have been reported for Arg in rats (49) and dairy calves (50) and for Glu and Gln in rats (9). In addition, the oral administration of Gln plus Gly can prevent and treat intestinal dysfunction in neonates (51), whereas the oral administration of Gln can improve intestinal health in preterm infants (52). Furthermore, dietary supplementation with Gly may ameliorate growth restriction of infants fed suboptimal amounts of milk protein (53).

Feeding high-protein formulas (50% greater than the protein intake from sow milk) resulted in reduced growth and 33% mortality in low-birth-weight piglets (54), likely because of their inability to detoxify excessive amounts of AA-derived ammonia via the hepatic urea cycle (2). Therefore, increased intake of dietary protein cannot be used to enhance the growth of preterm infants, and nutritionists should consider the use of specific AAs to achieve such a goal in medicine and animal production. This is important because low-birth-weight infants and piglets represent 5–10% and 15–25% of the total neonatal population, respectively.

Weanling animals

Weaning is associated with intestinal dysfunction, changes in AA metabolism, and reduced growth in mammals (24). A major difference in AA composition (expressed on the basis of dry matter) between plant-based diets and milk is that the former contains greater contents of Ala, Arg, His, and Gly but lower contents of BCAAs, Glu, Gln, Lys, Met, Pro, Ser, Thr, Trp, and Tyr (1). In weanling pigs fed corn- and soybean meal–based diets, dietary EAAs, as well as Asn, Ser, and Ala exceed the needs for protein synthesis, but dietary Asp, Glu, Gly, Pro, Arg, and Gln cannot meet the needs (Table 2).

TABLE 2.

Metabolism of AAs in 30-d-old piglets (7.8 kg BW) weaned at 21 d of age¹

					Extraintestinal metabolism of AAs via non-protein-synthesis pathways5
AAs	AAs in the diet,2g/kg	Dietary AAs entering the portal vein,3g/d	AA accretion in extraintestinal tissues,4g/d	Dietary AAs entering the portal vein/AA accretion in extraintestinal tissues, g/g	Total amount, g/d	Total nitrogen, mmol/d	Total carbon, mmol/d
Catabolism of AAs whose carbon skeletons are not synthesized by animal cells
Cys	3.74	0.91	0.52	1.75	0.39	3.22	9.66
His	5.73	1.43	0.81	1.77	0.62	12.0	24.0
Ile	8.91	1.79	1.38	1.30	0.41	3.13	18.8
Leu	17.8	3.63	2.66	1.36	0.97	7.39	44.4
Lys	14.2	3.14	2.35	1.34	0.79	10.8	32.4
Met	3.58	0.89	0.73	1.22	0.16	1.07	5.36
Phe	9.93	2.37	1.34	1.77	1.03	6.24	56.1
Thr	8.52	1.79	1.37	1.31	0.42	3.53	14.1
Trp	2.49	0.60	0.43	1.40	0.17	1.66	9.16
Tyr	7.62	1.89	1.06	1.78	0.83	4.58	41.2
Val	9.96	1.94	1.64	1.18	0.30	2.56	12.8
Subtotal	92.5	20.4	14.3	—	6.09	56.2	268
Net synthesis of AAs whose carbon skeletons can be formed by animal cells
Ala	13.0	3.50	2.56	1.37	−3.56	−40.0	−120
Arg	13.2	2.50	2.64	0.95	2.706	15.56	0
Asn	9.40	2.44	1.40	1.74	−1.04	−15.7	−31.5
Asp	13.2	0.23	1.67	0.14	1.44	10.8	43.3
Glu	17.2	0.21	3.29	0.064	3.08	20.9	105
Gln	18.4	1.79	2.00	0.90	0.21	2.87	7.18
Gly	8.81	2.21	4.57	0.48	2.36	31.4	62.9
Pro	15.8	2.91	4.837	0.60	1.07	9.29	46.5
Ser	7.86	2.04	1.73	1.18	−0.31	−2.95	−8.85
Subtotal	117	17.8	24.7	—	5.95	32.2	104

¹Values are means derived from previously published studies (21, 24). The molecular weights of intact AAs were used for all of the calculations. AA, amino acid; BW, body weight.

²Corn- and soybean meal–based diet containing 21.5% crude protein. The dry matter content in the diet was 89.5%.

³Calculated on the basis of feed intake (as-fed basis; 351 g/d), the true ileal digestibility (%) of AAs in the diet (Arg, 90; His, 89; Ile, 88; Leu, 88; Lys, 84; Met, 89; Phe, 87; Pro, 86; Thr, 83; Trp, 84; Val, 85; Ala, 88; Asn, 85; Asp, 86; Cys, 83; Glu, 87; Gln, 84.0; Gly, 87; Ser, 89; and Tyr, 88), and the bioavailability (%) of orally administered AAs entering the portal vein (Arg, 60; His, 80; Ile, 65; Leu, 66; Lys, 75; Met, 79; Phe, 78; Pro, 61; Thr, 72; Trp, 81; Val, 65; Ala, 87; Asn, 87; Asp, 5; Cys, 83; Glu, 4; Gln, 33; Gly, 82; Ser, 83; and Tyr, 80). Products of intestinal AA metabolism that enter the portal vein are not included.

⁴Calculated on the basis of a BW gain of 235 g/d and the content of AAs in young pigs.

⁵Rates of extraintestinal net catabolism of AAs whose carbon skeletons are not synthesized by animal cells are calculated as dietary AA entering the portal vein − (AA accretion in the body + AAs formed from intestinal catabolism of dietary AAs). Rates of extraintestinal net synthesis of AA whose carbon skeletons can be synthesized by animal cells are calculated as AA accretion in the body − (dietary AAs entering the portal vein + AAs formed from intestinal catabolism of dietary AAs). Rates of AA formation from intestinal catabolism of dietary AAs (g/d) are estimated to be 2.62, 0.85, and 2.70 for Ala, Pro, and Arg, respectively, and to be negligible for other AAs. The minus sign (–) indicates the contribution of dietary AAs for extraintestinal AA synthesis in a cell- and tissue-specific manner.

⁶In pigs, citrulline (the precursor of Arg) is synthesized from Glu, Gln, and Pro exclusively in the small intestine, and citrulline is converted into Arg mainly in extraintestinal tissues. Asp provides an amino group for Arg synthesis from citrulline.

⁷Including Pro and hydroxyproline for the calculation of extraintestinal carbon and nitrogen balance.

Quantitative analysis shows that, in weanling pigs, the total amounts of nitrogen and carbon in EAAs available for extraintestinal utilization via non-protein-synthesis pathways greatly exceed those in synthesized AAs (Table 2). On the basis of the half-lives of Arg, Asp, Glu, Gln, Leu, Lys, and Pro in the plasma (21), rates of their degradation per kilogram of BW are similar between 14- and 35-d-old piglets. Assuming that rates of extraintestinal AA synthesis per kilogram of BW do not differ between 30-d-old weaned piglets and 14-d-old sow-fed piglets, the great disparity in the amounts of nitrogen and carbon between EAAs available for extraintestinal catabolism and AAs synthesized in extraintestinal tissues suggests that endogenous synthesis of AAs in weaned pigs is also limited by activities of key enzymes. This would necessitate direct supplementation with synthesizable AAs to diets for weaned piglets, because a high intake of protein results in intestinal dysfunction and diarrhea, partly due to excessive intestinal production of histamine from His (8).

Several lines of evidence showed that synthesis of NEAAs is insufficient for maximum growth in weanling pigs (45, 55, 56). First, the oral administration of Gln (1 g/kg BW per day) to low-birth-weight piglets improved their survival and growth, particularly in response to endotoxin treatment (57). Second, dietary supplementation with 1% Gln to early-weaned piglets prevented jejunal atrophy during the first week postweaning and increased feed efficiency (gain:feed ratio) by 25% during the second week postweaning (58). Third, dietary supplementation with 0–4% monosodium Glu to weanling pigs dose-dependently increased jejunal growth, intestinal antioxidative capacity, BW gain, and feed efficiency, while reducing the incidence of diarrhea (56). Fourth, supplementing 0–2.1% Pro to a Pro-free diet increased BW gain by 69 g/d and feed efficiency by 20% in young pigs, while reducing concentrations of urea in plasma by 50% (59). Similar results were obtained for weanling pigs fed a corn- and soybean meal–based diet supplemented with 1% Pro (60). Fifth, dietary supplementation with 0.4% Arg to 7- to 21-d-old milk-fed piglets reduced plasma ammonia concentrations by 35% and increased BW gain by 66% (47). Sixth, supplementing Arg to diets of weanling pigs improved intestinal vascular and mucosal growth (61) and immunity (62). Seventh, dietary supplementation with 1% Arg to finishing-stage pigs increased skeletal muscle gain by 5.5% while decreasing carcass fat content by 11% (63). Thus, weanling piglets cannot synthesize sufficient Arg, Glu, Gln, or Pro to support their maximum growth or optimal intestinal health. Similar findings have also been reported for rats (9, 49).

Gestating mammals

The rapid growth of the placenta and fetus during pregnancy requires the provision of large amounts of AAs from maternal diets. To prevent the development of overweight or obesity during gestation, maternal food intake must be controlled (64). Differences between dietary AAs entering the portal vein and uterine uptake of AAs can provide useful information on AA metabolism in gestating dams (65). For example, in gilts fed 2 kg of a corn- and soybean meal–based diet/d containing 12.2% crude protein and dietary Lys and Trp cannot meet the needs of their uterine uptake during late gestation, dietary Met just matches its uterine uptake, and all other EAAs exceed their uterine uptake (Table 3). Ala, Asn, and Ser are the only synthesizable AAs whose supplies in the diet exceed uterine uptake. Other NEAAs (including Arg, Glu, Gln, and Gly) must be synthesized by the gestating dam to support fetal growth and development.

TABLE 3.

Metabolism of AAs in gestating gilts¹

						Extraintestinal metabolism of AAs via non-protein-synthesis pathways in maternal tissues5
AAs	AAs in the diet,2g/kg	Dietary AAs entering the portal vein,3g/d	Uterine uptake of AAs, g/d	AA accretion in fetuses,4g/d	Uterine uptake of AAs/AA accretion in fetuses, g/g	Total amount, g/d	Total nitrogen, mmol/d	Total carbon, mmol/d
Catabolism of AAs whose carbon skeletons are not synthesized by animal cells
Cys	2.30	3.28	2.02	1.36	1.49	1.92	15.9	47.5
His	3.32	4.54	2.81	2.40	1.17	2.14	41.4	82.8
Ile	5.08	5.70	4.16	3.28	1.27	2.42	18.5	111
Leu	11.7	13.3	10.3	7.56	1.36	5.74	43.8	263
Lys	5.81	7.48	7.62	6.61	1.15	0.87	11.9	35.7
Met	1.79	2.45	2.45	2.02	1.21	0.43	2.88	14.4
Phe	6.22	8.32	4.52	3.81	1.19	4.51	27.3	246
Thr	4.94	6.07	4.82	3.61	1.34	2.46	20.7	82.6
Trp	1.30	1.81	2.04	1.37	1.49	0.44	4.31	23.7
Tyr	4.49	6.19	3.40	2.54	1.34	4.70	25.9	234
Val	6.52	7.27	5.85	4.55	1.29	2.72	23.2	116
Subtotal	53.5	66.4	50.0	39.1	—	27.3	230	1203
Net synthesis, for fetal growth, of AAs whose carbon skeletons can be formed by animal cells
Ala	7.76	11.7	8.91	7.23	1.23	13.6	152	457
Arg	7.01	7.22	7.49	7.32	1.02	7.666	44.06	0
Asn	5.80	8.68	2.64	3.82	0.69	4.86	73.6	147
Asp	7.58	0.65	0.76	4.61	0.16	−3.96	−29.8	−119
Glu	10.7	0.74	4.20	9.22	0.46	−8.48	−57.6	−288
Gln	12.2	6.92	25.7	5.95	4.32	0.97	13.3	33.2
Gly	5.50	7.76	16.2	13.8	1.18	−5.99	−79.8	−160
Pro	10.3	10.8	11.8	13.27	0.89	0.16	1.39	6.90
Ser	4.52	7.14	5.75	4.71	1.22	2.43	23.1	69.4
Subtotal	71.4	61.6	83.5	69.8	—	11.2	141	147

¹Values are means derived from previously published studies (65). The molecular weights of intact AAs were used for all the calculations. AA, amino acid.

²Corn- and soybean meal–based diet containing 12.2% crude protein. The dry matter content in the diet was 89.8%.

³Calculated on the basis of feed intake (2 kg/d), the true ileal digestibility of 86% for each AA in the diet, and the bioavailability (%) of orally administered AAs entering the portal vein (Arg, 60; His, 80; Ile, 65; Leu, 66; Lys, 75; Met, 79; Phe, 78; Pro, 61; Thr, 72; Trp, 81; Val, 65; Ala, 87; Asn, 87; Asp, 5; Cys, 83; Glu, 4; Gln, 33; Gly, 82; Ser, 83; and Tyr, 80). Products of intestinal AA metabolism that enter the portal vein are not included.

⁴Calculated on the basis of 10 fetuses per gilt. The weight of the gilt at 110–140 d of gestation is 170 kg (130 kg maternal body weight + 40 kg conceptus).

⁵Rates of extraintestinal net catabolism of AAs whose carbon skeletons are not synthesized by animal cells are calculated as dietary AAs entering the portal vein − (AA accretion in the body + AAs formed from intestinal catabolism of dietary AAs). Rates of extraintestinal net synthesis of AAwhose carbon skeletons can be synthesized by animal cells are calculated as AA accretion in the body − (dietary AAs entering the portal vein + AAs formed from intestinal catabolism of dietary AAs). Rates of AA formation from intestinal catabolism of dietary AAs (g/d) are estimated to be 9.10, 2.57, and 7.66 for Ala, Pro, and Arg, respectively, and to be negligible for other AAs. The minus sign (–) indicates the contribution of dietary AAs for extraintestinal AA synthesis in a cell- and tissue-specific manner.

⁶In pigs, citrulline (the precursor of Arg) is synthesized from Glu, Gln, and Pro exclusively in the small intestine, and citrulline is converted into Arg mainly in extraintestinal tissues. Asp provides an amino group for Arg synthesis from citrulline.

⁷Including Pro and hydroxyproline for the calculation of extraintestinal carbon and nitrogen balance.

Excessive intake of protein and AAs reduces the survival and growth of embryos and fetuses due to the toxicity of ammonia and possibly adverse effects of other metabolites (e.g., histamine, hydrogen sulfide, and homocysteine) (8). When gestating gilts and sows are fed 2- or 2.2-kg diets daily during late gestation (65), the endogenous synthesis of some AAs may be inadequate for optimal pregnancy outcome. In support of this view, dietary supplementation with 0.83% Arg between days 30 and 114 of gestation increased the number of live-born piglets by 2 per litter and litter birth weight by 24% (66). Similarly, supplementing 0.4% or 0.8% Arg to the diet of gilts between days 14 and 28 of gestation enhanced placental weight, as well as embryonic survival, growth, and development (67). Beneficial effects of Arg on improving pregnancy outcome in swine were also shown under practical production conditions (68). Likewise, the intravenous administration of Arg enhanced fetal growth in underfed ewes (69) and in prolific ewes gestating multiple fetuses (70). Furthermore, dietary supplementation with 1.3% Arg-HCl either throughout pregnancy or during the first 7 d of gestation increased the number of live-born rats by 3 per litter (71). Finally, daily intravenous infusion of Arg (20 g/d) for 7 d during late (week 33) gestation in women with intrauterine growth restriction increased birth weight at term (week 39) by 6.4% (72), whereas Arg administration to women with intrauterine growth restriction fetuses improved fetal growth and development (73).

Experimental data indicate that the synthesis of Gly and Gln in gestating dams with either inadequate protein intake or infection is also insufficient for optimal fetal growth and offspring health (74, 75). First, maternal dietary supplementation with Gly prevented hypertension and vascular dysfunction in the offspring of rats fed a low-protein diet (74). Second, maternal Gln supplementation enhanced fetal growth in gilts fed a 12% crude-protein diet (24) and in ewes receiving an intravenous infusion of alcohol (76), as well as and fetal survival in mice infected with porcine circovirus type II (75). The underlying mechanisms may involve improvements in antioxidative function, immune response, acid-base balance, and protein synthesis (1).

Lactating mammals

Work on AA metabolism for milk production in lactating dams, to our knowledge, is very limited in the literature. Studies in sows, cows, and rats have shown that the lactating mammary gland increases milk production as dietary protein intake is augmented (77). Lactating sows fed a corn- and soybean meal–based diet containing 18% crude protein loses ∼0.5 kg BW per day between days 1 and 21 of lactation (78) or even 1.4 kg BW per day between days 1 and 18 of lactation (79) (Table 4). This indicates that dietary protein intake in the current feeding program is substantially insufficient for milk protein production by prolific sows. It is unknown whether EAAs, synthesizable AAs, or both in diets limit lactogenesis.

TABLE 4.

Metabolism of AAs in lactating sows¹

					Extraintestinal metabolism of AAs via non-protein-synthesis pathways5
AAs	AAs in the diet,2g/kg	Dietary AAs entering the portal vein,3g/d	AA output in milk,4g/d	Dietary AAs entering the portal vein/AA output in milk, g/g	Total amount, g/d	Total nitrogen, mmol/d	Total carbon, mmol/d
Catabolism of AAs whose carbon skeletons are not synthesized by animal cells
Cys	3.04	13.0	6.13	2.12	6.87	56.7	170
His	4.37	18.5	7.84	2.36	10.7	206	412
Ile	7.44	25.5	19.4	1.31	6.07	46.3	278
Leu	15.8	54.8	38.0	1.44	16.8	128	769
Lys	9.00	35.4	34.8	1.02	0.64	8.70	26.2
Met	2.83	11.9	8.86	1.34	3.04	20.4	102
Phe	8.61	35.6	17.3	2.06	18.3	111	997
Thr	6.53	24.0	19.5	1.23	4.49	37.7	151
Trp	2.08	8.70	5.62	1.55	3.08	30.1	166
Tyr	6.95	29.6	16.5	1.79	17.1	94.5	850
Val	8.17	27.7	21.6	1.28	6.06	51.7	259
Subtotal	74.8	285	196	—	89.1	769	3978
Net synthesis, for milk protein production, of AAs whose carbon skeletons can be formed by animal cells
Ala	9.35	42.4	16.8	2.53	−58.3	−655	−1964
Arg	10.8	34.5	12.2	2.83	34.86	2006	0
Asn	7.68	34.0	21.6	1.58	−12.4	−188	−377
Asp	11.0	2.80	22.1	0.13	19.3	145	579
Glu	14.9	3.10	38.9	0.08	35.8	244	1218
Gln	16.1	26.7	41.5	0.64	14.8	202	506
Gly	8.51	36.1	9.54	3.78	−26.6	−354	−708
Pro	14.3	45.0	56.57	0.80	−0.21	−1.84	−9.20
Ser	8.31	36.5	20.0	1.82	−16.5	−157	−470
Subtotal	101	261	239	—	–9.30	–565	–1225

¹Values are means derived from previously published studies (24, 78). The molecular weights of intact AAs were used for all the calculations. AA, amino acid.

²Corn- and soybean meal–based diet containing 18% crude protein. The dry matter content in the diet was 89.8%.

³Calculated on the basis of feed intake (as-fed basis; 5.94 kg/d), the true ileal digestibility (%) of AAs in the diet (Arg, 90; His, 89; Ile, 89; Leu, 89; Lys, 88; Met, 90; Phe, 89; Pro, 87; Thr, 86; Trp, 87; Val, 88; Ala, 88; Asn, 86; Asp, 86; Cys, 87; Glu, 86; Gln, 84; Gly, 87; Ser, 89; and Tyr, 90), and the bioavailability (%) of orally administered AAs entering the portal vein (Arg, 60; His, 80; Ile, 65; Leu, 66; Lys, 75; Met, 79; Phe, 78; Pro, 61; Thr, 72; Trp, 81; Val, 65; Ala, 87; Asn, 87; Asp, 5; Cys, 83; Glu, 4; Gln, 33; Gly, 82; Ser, 83; and Tyr, 80). Products of intestinal AA metabolism that enter the portal vein are not included.

⁴Calculated on the basis of 9 piglets nursed by the lactating sow (174 kg body weight) and the average consumption of milk by the piglet at day 14 of lactation (246 mL milk/kg body weight per day). This amounts to total production of 8.52 L milk/d per sow.

⁵Rates of extraintestinal net catabolism of AAs whose carbon skeletons are not synthesized by animal cells are calculated as dietary AAs entering the portal vein − (AA accretion in the body + AAs formed from intestinal catabolism of dietary AAs). Rates of extraintestinal net synthesis of AAs whose carbon skeletons can be synthesized by animal cells are calculated as AA accretion in the body − (dietary AAs entering the portal vein + AAs formed from intestinal catabolism of dietary AAs). Rates of AA formation from intestinal catabolism of dietary AAs (g/d) are estimated to be 32.7, 11.7, and 34.8 for Ala, Pro, and Arg, respectively, and to be negligible for other AAs. The minus sign (–) indicates the contribution of dietary AAs for extraintestinal AA synthesis in a cell- and tissue-specific manner.

⁶In pigs, citrulline (the precursor of Arg) is synthesized from Glu, Gln, and Pro exclusively in the small intestine, and citrulline is converted into Arg mainly in extraintestinal tissues. Aspartate provides an amino group for Arg synthesis from citrulline.

⁷Including Pro and hydroxyproline for the calculation of extraintestinal carbon and nitrogen balance.

Except for Lys, dietary EAAs entering the portal vein exceed their output in sow milk (Table 4). Dietary Lys just matches its output in milk, whereas dietary Ala, Arg, Asn, Gly, and Ser exceed their output in milk. As noted previously, these AAs are catabolized in the maternal body to support cell-specific needs, and they are also synthesized via interorgan coordination for utilization by the mammary gland, reflecting the dynamic metabolism of AAs during lactation. Dietary Asp, Glu, Gln, and Pro cannot meet their output in milk (Table 4), and these AAs must be synthesized de novo from dietary EAAs. Thus, when EAAs were supplemented to a low-protein diet for lactating sows, their BW loss decreased and piglet growth increased (79).

At present, little information is available for the uptake of all AAs by the lactating mammary gland. The uptake of BCAAs and Arg by the sow mammary gland greatly exceeds their output in milk (79). In mammary epithelial cells, BCAAs are metabolized to form Glu, Asp, and Gln (80), whereas Arg is catabolized by arginase to produce Pro and polyamines (81). Because polyamines are essential to DNA and protein synthesis, their production at the expense of Arg by mammary epithelial cells is of physiologic importance for the development of the neonatal small intestine (8). Because the gut converts Pro into Arg by using Glu and Asp, which are abundant in milk (2), there is no net loss of Arg carbons or nitrogen in the so-called Arg-Pro cycle between mother and neonate (81). Another salient aspect of our studies is that the amount of dietary Gly is much greater than the output of Gly in milk (Table 4). It is unknown how Gly is utilized beyond protein synthesis in lactating dams, but Gly metabolism likely plays an important role in regulating milk production. Experimental data are needed to support this hypothesis.

As noted previously, lactating sows mobilize their protein stores to provide AAs for milk production. However, this event cannot be without a limit because an excessive loss of body protein is not compatible with survival (8). Thus, lactating dams may not be able to synthesize sufficient AAs to support maximal milk production. This view is substantiated by the findings that 1) supplementing 1% Arg-HCl to corn- and soybean meal–based diets stimulates milk production by lactating sows and piglet BW gains (78); 2) dietary supplementation with 1% Gln enhances milk production by sows, as well as piglet growth and survival (24, 82); and 3) supplementing 1% and 2% monosodium Glu to a corn- and soybean meal–based diet for lactating sows increased milk production, piglet growth and survival, and efficiency of feed use for lactation (83). Similar findings have been reported for cows. For example, abomasal infusion of Glu into lactating cows increased milk protein production (84). In addition, the infusion of 80 g Pro/d into the duodenum of cows in midlactation promoted milk protein synthesis (85). Thus, lactating mothers need sufficient amounts of synthesizable AAs in their diets to maximize milk production.

Poultry

Chickens grow very rapidly and respond sensitively to the dietary intake of AAs. De novo synthesis of Gly and Pro is insufficient for the maximal growth of poultry (3). Thus, supplementing 0.3% Gly to a 17% crude-protein diet increased fat absorption, mucin production, weight gain, and feed efficiency in 21- to 35-d-old broilers (86). Similar results were obtained by supplementing 0.2% Gly to an 18% crude-protein diet in 5- to 21-d-old broilers (87). In addition, there is evidence that dietary Glu and Gln are needed for maximal growth of young chicks (88, 89), suggesting that they do not synthesize sufficient Glu or Gln. In support of this notion, dietary supplementation with 0.5–1% Glu augmented small-intestinal villus height, BW gain, and feed efficiency, while reducing posthatching mortality in 1- to 42-d-old broilers (90). Dietary supplementation with either 1% or 0.7% Gln was also reported to enhance BW gain and feed efficiency in 1- to 21-d-old broilers (91) or 21- to 42-d-old turkey poults (92), respectively. Furthermore, supplementing a mixture of synthesizable AAs (0.8% Gly, 0.43% Pro, 0.33% Ala, 1.21% Glu, and 0.99% Asp) to a 16.2% crude-protein diet also improved BW gain and feed efficiency in 1- to 42-d-old broilers (93). Collectively, these findings indicate that chickens cannot sufficiently synthesize Gly, Glu, and Gln when fed a normal-protein diet or Gly, Pro, Ala, Glu, and Asp when fed a low-crude-protein diet.

Fish

In contrast to livestock and poultry, research on AA metabolism in aquatic animals is limited. Nonetheless, there is evidence that fish cannot sufficiently synthesize many NEAAs. For example, the endogenous synthesis of Pro cannot meet requirements for Pro in rainbow trout (94). In addition, dietary supplementation with Gln increased intestinal villus height and whole-body growth in red drum (Sciaenops ocellatus), Jian carp, and hybrid striped bass while enhancing the proliferation of T and B lymphocytes, as well as immunity, in fish (95). Likewise, dietary supplementation with Glu improved fillet quality in Atlantic salmon and reduced fat accumulation in the liver and whole body of Atlantic salmon while increasing the growth, antioxidant capacity, and intestinal expression of antioxidative genes (96). Similarly, dietary supplementation with 4% Glu or Gln promoted protein retention and feed efficiency in gilthead seabream juveniles (97). Furthermore, dietary Gln supplementation improved survival and growth of postlarval tongue sole, Cynoglossus semilaevis through enhancing activities of digestive enzymes, antioxidative abilities, and resistance to hypoxic stress (98). Finally, dietary Gly was required for optimal feed intake, fat digestion, nutrient absorption, adaptation to a change in the environment (e.g., transfer from seawater to freshwater), and growth in fish (4).

Conclusions and Perspectives

Over the past century, EAAs have received much attention in the nutrition of livestock, poultry, fish, and humans because these nutrients are not synthesized in animal cells. In contrast, dietary requirements of synthesizable AAs had long been largely ignored because they were assumed to be sufficiently synthesized de novo in animals. However, recent studies in healthy rats, pigs, poultry, and fish do not support this assumption (9). In animals, AAs are synthesized in part from EAAs and most of the synthesizable AAs are interconverted (7). Importantly, the provision of synthesizable AAs through either diet or endogenous synthesis can spare EAAs for protein synthesis (1, 3, 95, 99). It should be kept in mind that dietary AAs are almost the exclusive source of nitrogen for animals and that the nitrogen atom cannot be made in the body. Thus, nitrogen balances between entry into the portal vein and availability for extraintestinal catabolism, between uterine uptake and fetal growth, or between mammary gland uptake and milk output can provide useful information about factors affecting AA synthesis in mammals (8). Some of these data are now available for swine but are limited for other species.

Quantitative analysis of swine nitrogen metabolism showed that AA synthesis in animals is constrained by both precursor availability and enzyme activity under current feeding programs. Much evidence shows that synthesizable AAs in diets are required for maximum growth, reproduction, and lactation (100–102). It can be surmised that the well-recognized protein-sparing effect of glucose in animals may result from the provision of pyruvate, oxaloacetate, and α-ketoglutarate for synthesis of Ala, Asp, and Glu plus Gln, respectively. These AAs may be particularly important for maintaining whole-body homeostasis under stressful conditions (e.g., pregnancy, lactation, weaning, high environmental temperature, long-distance transportation, injury, infection, and disease). Because genetic strains of animals likely affect their requirements of dietary AAs, it would be important to determine rates of whole-body AA synthesis and its contribution to muscle growth in fast- compared with slow-growing pigs.

Growth is a sensitive criterion for identifying quantitative and qualitative needs for dietary AAs in young animals, as are sperm number and motility for males, embryonic survival for gestating dams, and milk protein yield for lactating mothers (8). On the other hand, nitrogen balance is not a highly sensitive indicator for dietary essentiality of AAs in adults (1). For example, the near absence of Arg from diets for 9 d did not influence nitrogen balance in men but reduced their sperm quantity and motility by 90% (103). Similarly, a deficiency of dietary Arg in young male rats over a period of 2 mo did not affect nitrogen balance but resulted in progressive damage to testes, no production of spermatozoa, and the abnormal filling of the lumina of the tubules with cellular debris, leukocytes, and macrophages (103).

Work on dietary requirements of synthesizable AAs in laboratory and farm animals can help to guide research in human beings. To date, except for taurine, data on the dietary requirements of synthesizable AAs in humans are very limited. Dietary taurine is essential for normal development and function of vision, cardiovascular, and muscular systems in human infants and cats (37). One must wonder whether so-called NEAAs are indeed required for the maintenance of normal BW (particularly skeletal muscle mass) in adults and for normal growth in the young, particularly under conditions of stress and disease. Human infants cannot sufficiently synthesize the following: 1) Pro and Arg under burn and injury conditions (104), 2) Gly when fed low-protein diets (53), and 3) Gln and Arg in response to intestinal inflammation (105). It is unknown whether elderly subjects who consume 0.8 g protein/kg BW per day synthesize sufficient NEAAs to maintain skeletal muscle mass and prevent oxidative stress.

Beyond protein synthesis, the functional needs for AAs for vascular, reproductive, immune, oxidative defense, and gastrointestinal functions, as well as whole-body homeostasis, well-being, and neurological development should be considered in recommendations for dietary requirements of AAs (including synthesizable AAs) in animals and humans (7). For example, certain metabolites of AAs and some AAs (e.g., Glu, Asp, and Gly) themselves are neurotransmitters (1). To our knowledge, no studies have been published that evaluate optimal intakes of dietary AAs to support cerebral-neuronal development in any species. Recent advances in the understanding of AA biochemistry, physiology, and nutrition are transforming the practice of livestock, poultry, and fish feeding worldwide. The availability of feed-grade Arg, Asp, Glu, Gln, Gly, and Pro is expected to improve the efficiency and economic returns of global animal production. Knowledge gained from studies in laboratory and agriculturally important animals has important implications for improving growth and health in humans.