Biochrom version 30

Concentrations of serum amino acids, except tryptophan, were determined as previously described (Espe et al., 2014 (link)). In brief, serum samples were oxidized using a hydrogen peroxide solution (containing phenolic formic acid) at 0 °C for 16 h, and sodium pyrosulfite was added after the reaction to decompose the excess peroxyformic acid. Subsequently, 6 mol/L HCl solution was added to the oxidized samples and hydrolyzed at 110 °C for 23 h to release free amino acids. The concentrations of free amino acids were then determined on an amino acid analyzer (Biochrom, Version 30, Biochrom Ltd., Cambridge, UK) equipped with an ion-exchange column. Tryptophan was determined by a high-performance liquid chromatography (HPLC; Shimadzu, Tokyo, Japan). In brief, samples were saponified under alkaline conditions with barium hydroxide solution in the absence of air at 110 °C for 20 h in an autoclave. After adjusting the hydrolysate pH to 3.0, the tryptophan was separated by reversed-phase chromatography RP-18 on a HPLC column, and the chromatograms were integrated using Labsolutions software with a fluorescence detector (Shimadzu, Tokyo, Japan).

Amino acids were determined using an amino acid analyser (Biochrom, Version 30, Biochrom Ltd., Cambridge, UK). In brief, samples were oxidized in a mixture of hydrogen peroxide and phenolic formic acid solution and then left to react for 16 h at 0 °C for the oxidation of cystine and methionine. Samples were subsequently neutralised with a spatula tip of sodium disulfide and then hydrolysed with 6 mol/L HCl (containing phenol) for 23 h at 110 °C. The amino acids were then detected on an ion-exchange column, and the chromatograms were integrated using the OPENLAB software with amino acids simultaneously detected at 570 nm and 440 nm. $$\mathrm{AID}\left(\%\right)=\left(1,-,\frac{{\mathrm{TiO}}_2\mathrm{in}\mathrm{diet}\left(\%\right)}{{\mathrm{TiO}}_2\mathrm{in}\mathrm{digesta}\left(\%\right)},\times ,\frac{\mathrm{Digesta}\mathrm{nutrient}\left(\%\right)}{\mathrm{Diet}\mathrm{nutrient}\left(\%\right)}\right)\times 100$$