6.3 Protein and Amino Acid Metabolism
6.3.2. Dietary Effects on Amino Acid Utilization
6.3.2.1. A “Metabolically” Optimized Amino Acid Diet Formulation
Diet formulations for protein and indispensable amino acids require- ment are based entirely, or in large proportion, on free, synthetic amino acids. These diets seek to maximize the growth rates of fish to be compara- ble to those attained on diets based on proteins. It was realized early that protein hydrolysates (Aoeet al.,1974) and amino acid mixture diets (Aoe et al.,1970b; Muraiet al.,1981) were greatly inferior in supporting growth in common carp and tilapia (Mazidet al.,1978) in comparison to casein-based diets. Rainbow trout fed diets containing protein/amino acid mixtures grew more slowly than fish fed complete protein diets (Cowey and Luquet, 1983).
Coweyet al. (1992) used a 20% amino acid mixture in the experimental diets formulated for a methionine requirement study in rainbow trout and reported markedly inferior growth in comparison to that on an all-protein, control diet. This study was exemplary because of the duration of the experi- ment and more than 10-fold increase in fish weight to the estimate optimum requirements for methionine. Less convincing is a variable amount of food eaten by different groups, resulting in a confounding effect on fish growth of food intake in the lowest methionine treatment.
Quantitative requirements for the 10 indispensable amino acids for growth in fish should be reported as percentage of the dietary protein and be representative of the near-maximum growth rate. However, many data sets used for comparisons of individual amino acid requirements among fish were collected under rather unfavorable conditions of inferior growth rates due to inadequacy of amino acid test diets. For instance, the amino acid re- quirement for common carp is based on data where in many cases fish only doubled their body weights and/or growth rates were less than 2% per day in individuals 0.5–4 g in body weight (Nose 1979). Ravi and Devaraj (1991) determined amino acid requirements in Indian carp (Catla catla) using a
FIG. 6.13
Increase in body mass of juvenile Nile tilapia (40- to 50-mg individual weight) in response to varying dietary levels of threonine and lysine (Santiago and Lovell, 1988).
Threonine diet (6.5% protein, 23.04% free amino acids); lysine diet (18.15% protein, 14.05% free amino acids).
diet containing a 40% amino acid mixture and weight gains were two- to fourfold the initial body weight. More importantly, weight gains abruptly declined after a maximum weight gain, clearly indicating that the fish “tol- erance” for the particular amino acid was exceeded. The essential nutrient became toxic at increasing dietary concentrations.
Santiago and Lovell (1988) present an illustrious example of an amino acid requirement study in Nile tilapia where (1) the growth rate was nearly maximal, (2) the study was of sufficient duration, resulting in a body weight increase of 15- to 35-fold, and (3) no decline in weight gains was observed after a peak of response was reached (Fig. 6.13). However, the latter study was performed using diets containing 5.5–18.15% of a casein–gelatin base.
The growth response seemed to be related partially to the increase in protein base.
Nget al. (1996) added white sturgeon to a list of species almost-incapable of utilizing a diet based on 51% synthetic amino acids. Dietary pH adjust- ment to a neutral value was not helpful in sturgeon as demonstrated ear- lier in common cap (Noseet al.,1974) and channel catfish (Wilsonet al., 1977). Diet neutralization in the sturgeon study, however, significantly de- creased the urinary excretion of free amino acids. Although not directly
demonstrated in sturgeon, the authors concluded that amino acids absorbed were excreted via gills as shown earlier in common carp (Muraiet al.,1984).
Increased catabolism of amino acids ingested in diets based on synthetic amino acid mixtures was clearly not the case when ammonia excretion rates were measured in comparison to those on a casein-based diet (Kaushik and Dabrowski, 1983).
It therefore becomes intriguing that amino acids infused into the dor- sal aorta of rainbow trout (Kaczanowski and Beamish, 1996) or injected into the caudal aorta of channel catfish (Brown and Cameron, 1991a,b) in- duced increases in oxygen consumption mimicking an elevated metabolism.
Thus, an elevated metabolism is not always associated with the amount or the composition of nutrients ingested. Mixtures which were deficient in essential amino acids or unbalanced with respect to optimum requirements, resulted in a higher oxygen consumption than infusions of amino acids of profiles equivalent to those of fish body proteins. Brown and Cameron (1991b) suggested, based on the use of the protein synthesis inhibitor, cyclo- hexamide, that an increase in oxygen uptake was correlated with the protein synthesis rate. In catfish, ammonia excreted following amino acid infusion constituted 21% of the amino acid nitrogen (Brown and Cameron, 1991a), whereas in common carp fed amino acid mixture diets, metabolic loss con- stituted only 6% of the dietary nitrogen source (Kaushik and Dabrowski, 1983). This comparison, with all interspecies limitations, demonstrates that amino acid catabolism is elevated in fish fed “parenterally.”
Both studies with catfish and trout infused with amino acids suffered from several physiological artifacts which make the findings uncertain. First, in- fusion of only amino acids, without other nutrients, minerals, and vitamins, may profoundly affect transport and metabolism. Second, infusion of amino acid into the dorsal aorta (trout) or caudal artery (catfish) by bypassing re- ceptors on the absorption site of digestive tract and liver (Holstein and Haux, 1982) must have sent some awkward neurosignals to the central ner- vous system, affecting the compartmentalization of blood flow (Axelsson and Fritsche, 1991; Thorarensenet al.,1993), motoricity, and secretory func- tions of the digestive tract. Despite these shortcomings, experimentation on the whole fish, with further refinement of the method, should be used to address the metabolism of amino acids.
The strong statement by Aoe and Saito (1970) that “salmonids show no difference in growth between the casein-gelatin diet and corresponding amino acid test diet” requires further investigation. For instance, Barroso et al. (1999) observed that rainbow trout fed diets with a 15% amino acid mixture replacing fish meal on a weight basis showed a significant decline in body weights. An increase in the frequency of feeding from twice to four times daily improved fish growth on these experimental diets, whereas it did
not have any effect on the growth of fish fed controls diets. Rodehutscord et al. (1995a) declared that “free amino acids can replace protein-bound amino acids” in diets for rainbow trout. However, their conclusions do not seem to be warranted, as supplementation of essential amino acids at 2.6 and 4.0% did not restore weight gain to their respective controls (experiment A).
The results of experiment A are also questionable because glutamic acid was withdrawn from diets with a lower protein content. Schuhmacheret al.
(1995) demonstrated that the source of nonessential amino acids has a highly significant effect on rainbow trout growth. In experiment C of Rodehutscord et al. (1995a), where fish meal was completely replaced by wheat gluten and supplemented with essential amino acids, the level of protein was decreased to 30–31%. Only under these conditions of dietary protein levels below the optimum for growth did the fish final weight be- tween treatments not differ significantly. Experiments with 12% wheat gluten replacement as a protein source for indispensable amino acids (4%) and dispensable amino acids (11.4%) resulted in body weight gains that did not differ among treatments. However, daily growth rates in this study, 0.8%
per day, were inferior to those normally occurring in 40- to 50-g rainbow trout [4.1% (Bassompierreet al.,1998)]. These findings are not sufficient to generalize that dietary free amino acids do not negatively affect the efficiency of utilization of amino acids for growth in comparison to protein-bound amino acids. In retrospect, the results obtained by Rodehutscord et al.
(1995a) may be considered as disappointing and of limited use in practical or experimental diet formulations. Ogataet al. (1983) demonstrated that re- placement of casein with 4.47% essential amino acids on a mass basis resulted in growth enhancement by 18–21% in two juvenile salmonids. Supplemen- tation of a casein-based diet with six essential amino acids (total of 2.38%) highly reduced mortality and more than doubled the final body mass of juvenile Atlantic salmon (Rumsey and Ketola, 1975). In conclusion, it seems that more studies are required to optimize the ratio of synthetic amino acids to protein in high-nutrient diets aimed at examining requirements for indivi- dual amino acids.
Studies that address the mechanism of amino acid absorption and trans- port at the digestive tract levelin vivoadd substantially to the understanding of amino acid utilization and the way they must be presented in formulated diets. A unique feature of double capillarization of the venous system in teleost fish allowed Muraiet al. (1987) to examine concentrations of amino acids in the hepatic portal vein, reflecting the level derived from intestinal absorption, and then in the hepatic vein when a decline due to absorp- tion in liver can be demonstrated based on comparisons to the level in the previous location. They reported that concentration differences resulting from liver absorption (or protein synthesis) of essential amino acids were
consistently higher in fish fed a casein-based diet rather than a diet com- posed of 38% synthetic amino acids between 3 and 12 hr after force-feeding.
This would actually mean that “flooding” of the major protein synthesis site in rainbow trout is much more severe in fish fed a casein-based diet.
This would contradict a major argument harbored in the literature of im- paired utilization for protein synthesis of dietary synthetic amino acids in comparison to dietary protein-bound amino acids. This difference between dietary treatments in liver absorption of circulating amino acids extends for many passages of blood through a portal liver system (assuming that 2 hr is needed for complete equilibration of the blood level). In effect, there was no evidence of extensive hepatic catabolism of amino acids leading to excretion of ammonia into the hepatic blood vessel in rainbow trout fed an amino acid mixture diet. In the arteriovenous blood collected in the caudal vessels, ammonia concentrations were also not different between the two diets (Muraiet al.,1987). Muraiet al. (1984) reported that 36% of the total N excretion was in the form of free amino acids. Alternatively, Murai and Ogata (1990) found that the injection of exogenous insulin reduced free amino acid concentrations in tissues of common carp and suggested accelerated synthesis and deposition of protein. However, they did not provide evidence in terms of fish growth response to support this hypothesis.
These data demonstrate the high efficiency of amino acid uptake in the liver. It would not be unreasonable to assume that in fish, as in alligators (Coulson et al., 1990), essential amino acid removal from body fluids is accomplished severalfold more rapidly by incorporation into synthesized proteins than by catabolism.
Ash et al. (1989) and McLean and Ash (1989) described another ap- proach to analyze net absorption of amino acids in the digestive tract of fish, where the difference in concentrations between the dorsal aorta and the hepatic portal vein multiplied by the blood flow rate would provide a direct estimate of absorption. The decrease in nonessential amino acids (glutamic acid and glycine) was 10-fold between arterial blood and hepatic venous concentrations (see also Table 6.1), whereas the decreases in lysine, arginine and methionine concentrations were much more moderate, 2- to 4-fold. This method may provide additional information on postabsorptive handling of amino acids if strengthened by data related to changes in blood flow.
Espe and Njaa (1991) experimented with 0.88-g Atlantic salmon offered a diet based exclusively on free amino acids (50.2%) along with a diet based on fish meal and found that the body weight gain was fivefold lower on the former diet. They concluded that free amino acid leakage could be partly responsible for impaired growth. Yamada and Yone (1986) gave some more
Table 6.1
Concentrations of Amino Acids in Blood Plasma of Rainbow Trouta (6)
Amino acid (1) (2) (3) (4) (5) G AA
Lysine 747 121 824 457 318 (4) 331.26 (6) 361.3 (3)
Arginine 262 65 236 241 65 (4) 413.8 (24) 481.0 (9)
Methionine 252 10 121.1 202 328 (12) 234.5 (18) 264.8 (15) Leucine 672 126 622.6 571 814 (12) 1,615.1 (18) 1,360 (15)
Hydroxyproline — 69 42.4 — — — —
Proline 587 34 810.3 — 760 (4) — —
Glycine 894 397 523.9 2.338 929 (12) 1,800 (12) 2,120 (9)
aUnder different conditions after feeding and different diets: (1) Yamadaet al.
(1981)—dorsal aorta, 6 hr postprandially, amino acid (AA) mixture, 10◦C; (2) Ogata and Arai (1985)—caudal vein–artery, commercial feed; (3) Muraiet al. (1987)—hepatic portal vein, 3 hr postprandially, AA diet; (4) Ashet al. (1989)—hepatic portal vein, 3 hr postprandially; (5) Walton and Wilson (1986)—caudal vein, maximum postfeeding in parentheses (hr), casein; and (6) Schuhmaeheret al. (1997)—heart, gluten (G)- or AA-based diet (AA), maximum in parentheses (hr).
evidence to the “leakage hypothesis” with respect to the use of an amino acid mixture diet. In common carp, fish that masticate food by pharyngeal teeth, loss of water soluble amino acids may reach 60% and contribute to the loss of other dietary nutrients.
The research group of Bergeet al. (1994) compared diets containing a 13.4% amino acid mixture with free lysine versus protein-bound lysine in Atlantic cod. Incorporation into muscle protein was threefold higher in the case of the latter diet, indicating that free amino acid supplements are ca- tabolized extensively, in contrast to amino acids released after hydrolysis.
This is most likely the same mechanism that governs incorporation of hy- drolyzed algal protein14C-labeled amino acids into the systemic blood of rainbow trout (Cowey and Walton, 1988). These authors reported a higher activity and an earlier peak of amino acids in the acid-soluble fraction when delivered as hydrolyzed algal protein. Interestingly, the “dietary protein history” did not influence the rate of amino acid incorporation into protein from either protein- or free amino acid-containing diets. Therefore, it appears that (1) the proportion of free amino acid mixture to protein in a diet formulation and (2) the protein source are critical for utilization of amino acid supplements. Espe and Lied (1994) argued that the inclusion of 20–30% free amino acid in the total protein(or a 14% absolute amount)
was the optimal ratio for salmonid fish growth. However, this conclusion may be quite speculative. First, the protein source was fish muscle meal devoid of the peptide and free amino acid fraction. In this context, the original composition of fish meal was merely reconstituted in a diet sup- plemented with “amino acids mixtures of identical composition” (Espe and Lied, 1994). Second, the growth studies lasted only 2 weeks, with weight gains of 10–20% of the initial value. This reservation is reinforced by the results of Ogataet al. (1983), who demonstrated that salmon juveniles fed a casein diet supplemented with a mixture of 3.37–4.37% of essential amino acids performed significantly better than fish fed a fish meal-based diet of equal protein contents.
Schuhmacheret al. (1995a) addressed the question of possible advantages of supplying the dispensable amino acids in the “free” portion of the dietary protein over providing a single amino acid (e.g., glycine) which would satisfy all amino nitrogen needs for synthesis of other nonessential amino acids. In rainbow trout of 48-g individual weight, glutamine was proven to be superior to glycine and glutamic acid as a source of nitrogen. However, fish less than doubled the body weight during the 84-day experiment. Assessment of these results with less than adequate growth rates (0.13–0.3% per day) is somewhat difficult and most likely a consequence of using a pure synthetic amino acid mixture diet, in some cases supplemented with 30% glutamic acid. Glutamate incorporation into the protein fraction in rainbow trout is severalfold less than that of acetate (Fauconneauet al.,1989), and in fact, glutamate dietary toxicity was suggested in these fish (Hughes, 1985). In tilapia diets, supplements of 9.5% glutamate or an equivalent mixture of six amino acids did not result in significant improvements in the growth rate (Mambrini and Kaushik, 1994). Therefore, other nonessential amino acids which are at high concentrations in the postprandial state and low during fasting, such as alanine, glycine; and proline, need to be examined in diet formulations.