Sistema de Monitoreo, Seguimiento, Control y Evaluación

The enzyme hydrolysed protein method, also referred to as the ultrafiltration method or

peptide alimentation method, was first proposed by Moughan et al. (1990a) and allows

the simultaneous determination of both endogenous nitrogen and amino acid flows when the test animals are fed a diet containing peptides and amino acids as the only source of nitrogen. The dietary mixture contains peptides (none larger than 5kDa), and amino acids from hydrolysed casein that simulate the natural products of protein digestion. Enzyme hydrolysed casein is almost completely digested by the growing pig (Kies et al., 1986; Moughan and Smith, 1984, 1985). Digesta collected from the test animal are centrifuged before being ultrafiltered to remove any compounds smaller than a filtration cut-off of 10kDa; thus removing any unabsorbed dietary amino acids and peptides. The retentate (>10kDa fraction of digesta) is added to the precipitate from the centrifugation step and this material is used for the determination of endogenous nitrogen and amino acids. The enzyme hydrolysed protein method has been applied to determine gut endogenous nitrogen losses in several species of simple- stomached animals including: the rat (Butts et al., 1991; Darragh et al., 1990); the pig

(Butts et al., 1993; Hodgkinson and Moughan, 2001; Moughan et al., 1992); the cat

(Hendriks et al., 1996); the dog (Hendriks et al., 2002); the chicken (Cowieson and

Ravindran, 2007; Ravindran and Hendriks, 2004); and humans (Deglaire et al., 2007;

Moughan et al., 2005b).

One of the main advantages of this method over other methods is that it allows the determination of total nitrogen, and all amino acids, directly, in experimental animals with a positive body nitrogen balance (Moughan et al., 1998).

However, in addition to unabsorbed dietary peptides and amino acids the filtrate (< 10kDa) may also contain small peptides and free amino acids of endogenous origin, and not accounting for this may lead to an underestimation of endogenous nitrogen and amino acid flows. The amount of endogenous molecules < 10kDa has been estimated

by ultrafiltering digesta from animals fed a protein-free diet, where all protein, peptides and amino acids are endogenous. In the pig Moughan and Schuttert (1991) found that only 14% of the total nitrogen was from molecules that were < 10kDa.

However, in the rat, Butts et al. (1992) found the percentage nitrogen from molecules

less than 10kDa to be 33%. In another study Leterme et al. (1996a) found the total

nitrogen present in the < 10kDa fraction was between 19% – 27% for pigs, fed pea

fibre isolates; however, this percentage may have been overestimated as the pH of the digesta was not lowered sufficiently to prevent autolysis.

To improve the accuracy of the determination of endogenous flows calculated using this method and to reduce any underestimation, high quality ultrafiltration devices with a molecular weight cut-off of 3kDa are now available commercially and have been used by Hodgkinson and Moughan (2003) to estimate the difference in endogenous nitrogen and amino acid flows between the two ultrafiltration cut offs (i.e. 3kDa and 10kDa). They found that the endogenous ileal nitrogen was 17% greater when using < 3kDa ultrafiltration than for the < 10kDa ultrafiltration. The difference in endogenous ileal amino acid flows ranged from 1.7% for arginine and phenylalanine to 24.5% for glutamic acid and 26% for serine, with the mean amino acid increase being 12%. Interestingly serine and glutamic acid are two of the six amino acids present in the highest proportions in the polypeptide core of mucins. Hodgkinson and Moughan (2003) recommended that the molecular weight cut off for this assay be reduced to < 3kDa.

Leterme et al. (1996a) also used the 3kDa ultrafiltration cut off for discarding

undigested dietary peptides/amino acids and suggested (although not quantified), that even at this low level a significant proportion of endogenous amino acids are

discarded. They claimed that the observed increase in amino acid flows, when enzyme hydrolysed casein was fed to their experimental animals, was largely due to the

presence of undigested dietary molecules < 1kDa. A flawed argument as these small peptides should have been discarded, if they had indeed used the ultrafiltration

technique. They stated that the three main amino acid flow increases were observed for glutamic acid (accounting for one fifth of casein), followed by aspartic acid, and proline, these amino acids are three of the six most prolific amino acids in gastrointestinal mucins; the secretion of which is known to increase when

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protein/peptides are present in the diet (Claustre et al., 2002; Hodgkinson et al., 2000b;

Lien et al., 1996; Montagne et al., 2004).

In suggesting that the enzyme hydrolysed casein is not absorbed completely they cite

Rouanet et al. (1990), stating that the activity of amino peptidases in the brush border

is lower when a protein is given in a hydrolysed form. However, what Leterme et al.

(1996a) fail to say is that Rouanet et al., concluded that the hydrolysate, containing di-

and tri-peptides, is utilized efficiently in healthy growing rats, although the efficacy is

not higher than with intact dietary bovine plasma proteins. But Leterme et al. (1996a)

miss the point as the dietary peptides do not need to be fully absorbed if they are being discarded in the residue left from ultrafiltration. When using different dietary

concentrations of enzyme hydrolysed casein, to study its effect on the endogenous flow

of amino acids in broiler chickens Ravindran et al. (2009) observed that increasing

dietary peptide concentrations increased the flow of endogenous amino acid flow at the terminal ileum in a dose-dependent manner that also caused changes in the

composition of endogenous protein. They concluded that such changes in the amino acid profile of endogenous protein may be reflective of changes in the output of one or

more of the components of endogenous protein (Ravindran et al., 2009). A conclusion

which would seem to corroborate the notion that increasing the quantity of dietary proteins/peptides increases the secretion of gastrointestinal mucins.

Leterme et al. (1996a) also dispute the accuracy of ultrafiltration, stating that it is

designed for the preparative purification of large proteins and not for the quantitative separations of proteins and peptides. Citing Amicon´s Guide to Users, they state that the loss of proteins with a molecular mass of 12.4 kDa typically reaches 15% with the Centriprep-10 (cut-off: 10 kDa). However, the retention and recovery performance of the Centriprep-10 quoted by Millipore (Millipore, 2009) when used to filter the

12.4kDa cytochrome C is 96%; and for the Amicon® Ultra (Millipore, 2001) recovery

ranges between 93% and 95% (for the Amicon Ultra-15 and Amicon Ultra-4

respectively), suggesting losses of just 5% – 7%, which is somewhat better than the

performance cited by Leterme et al. (1996a).

Leterme et al. (1996a) conclude with the suggestion that nitrogen free diets should be

“40 to 50 g·kg-1 dry matter intake of a highly digestible protein such as casein

or egg yolk protein instead of 100 g kg-1 dry matter intake of a hydrolysed

form. The digestive secretions would be stimulated, the presence of dietary

nitrogen (sic) molecules in the ileal digesta would be very limited, the total

endogenous amino acid losses would be collected, and this would enable the flow of the endogenous amino acids under normal feeding conditions to be

estimated easily and accurately.”

The notion that a whole protein such as casein is more efficiently utilized than its

hydrolysate is untenable and contrary to the conclusion of Rouanet et al. (1990)

discussed above.

When Jansman et al. (2002) reviewed different methods of determining the amino acid

composition of basal endogenous crude protein in the pig, they reported that in two studies the enzyme hydrolysed casein method produced a higher mean value for the flow of basal endogenous crude protein and amino acids (17.1 ± 0.4 g crude protein per kg dry matter intake) when compared to other methods, including that of eleven studies using casein/wheat gluten. Such uncontrolled cross-study comparisons are

fraught with difficulties. Deglaire et al. (2008) found no evidence of a heightened ileal

endogenous protein loss due to the dietary peptides along the entire digestive tract from the stomach to the colon. Caution however, is still needed, as more research is required to compare directly the effect of feeding intact protein with its hydrolysed oligopeptides products in the same species.

In summary the enzyme hydrolysed protein method, employing ultrafiltration with a molecular mass cut off of 3kDa, may underestimate endogenous ileal nitrogen and amino acid flows, due to the presence of some endogenous amino acids and peptides in the discarded ultrafiltrate, but such an underestimation is suggested likely to be

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