Lysine in protein is highly susceptible to damage through heat treatment as it possesses a reactive e-amino group, especially if reducing sugars are present. The follow-up products of lysine created during Maillard reactions for example (Eichner et al., 1994) are no longer available for animal or human protein synthesis. In 1960 Carpenter introduced an in vitro test for available, better reactive lysine, in which the protein was reacted with fluoro-2,4-dinitrobenzene (FDNB) and the resulting coloured dinitrophenyl (DNP)-lysine was determined photometrically following hydrolysis. Only converted lysine that was still reactive at the e-amino group is in theory available for the protein metabolism of animals. Other reagents and test conditions such as conversion with OPA or guanidination to homoargi-nine have also been described (see below). But such tests are beset by numerous problems, from the chemical aeeessiblity of the e-amino group for derivatization in the sample protein suspended in the solution to interferences and secondary reactions caused by the matrix. A more sophisticated FDNB method was therefore often used, where the total lysine from the underivatized sample and unconverted lysine were determined chromatographically after reaction with FDNB and hydrolysis, the difference being the available lysine. Carpenter et al. (1989) compared these and two further In vitro tests on lysine availability in fish, meat and plant-derived food products with the in vivo rat growth assay; all in vitro tests, except those in the pure protein samples casein and soy protein, failed. Faldet et al. (1991, 1992) applied the aforementioned differential method and the rat growth assay to soybeans subjected to heat treatment under different conditions in order to optimize protein utilization by ruminants and obtained a reasonable match for lysine utilization. More recent research went further in determining DNP-lysine by HPLC in cereal-based baby food, infant formulas and other food, but without drawing comparisons with biological tests (Castillo et al., 1997; Albala-Hurtado et al., 1997; Fernandez-Artigas et al., 1999; Hermandez et al., 2001).
Mauron and Bujard (1964) developed an assay of available lysine which is based on the guanidination of the e-amino group with o-methyl-isourea in an alkaline environment to homoarginine, which can then be chromatographically determined by EC following hydrolysis. Although this derivatization in protein is only possible at room temperature and requires a reaction time of several days, it is still in common use today. Mao et al. (1993) employed the method for the determination of available lysine in soy proteins treated with glucose or alkali and compared it with the total lysine assay. They showed that whereas total lysine declined by 17-40% after the treatments by reacting with glucose, available lysine fell by as much as 78-85%. Damage caused by heat treatment with alkali was also identified far more efficiently with guanidination, which was explained by a re-cleaving of some of the damaged lysine (e.g. fructose-lysine of the initial Maillard reaction) in acid hydrolysis. Imbeah et al. (1996) optimized the reaction conditions of guanidination in casein and soybean meal. They achieved 100% lysine conversion for the soluble milk protein and just under 80% for soy. These authors, like Ravindran et al. (1996), were interested in the preparative guanidination of feedstuff to determine endogenous amino acid secretions in digestibility trials. The latter studies focused mainly on optimal conversion in cottonseed protein, where up to 64% of the lysine was converted into homoarginine, and conversion rates of other feed raw materials are also given. Moughan and Rutherford (1996) also devised optimal derivatization conditions and compared the reactive lysine of guanidination with that determined in the FDNB test for heat-treated casein/lactose mixtures, with a good degree of concordance. This and subsequent studies (Rutherfurd and Moughan, 1997; Rutherfurd et al., 1997) focused primarily on the development of a new bioassay 'digestible reactive lysine', which would allow a more accurate determination of lysine digestibility in digestibility trials because the re-cleaving of previously damaged lysine which can occur during protein hydrolysis in lysine assays of raw material and digesta is eliminated. Research concerned with the determination of the available lysine content in proteins, dairy products or milk by reaction with OPA should also be mentioned (Vigo et al., 1992; Medina-Hernandez and Alvarez-Coque, 1992; Morales et al., 1996). A great advantage of this method is the rapidity and simplicity of fluorescence measurement as no prior protein hydrolysis is necessary.
In vitro methods are appropriate and helpful for comparing different treatments of a feed raw material or foodstuff. Relative effects are meaningful in such tests, but absolute results concerning lysine availability are heavily influenced by matrix effects on the test system and often diverge considerably from animal experiments.
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