Effect of the methodology on peptide amino acid concentrations in blood and plasma of sheep.

Different methodologies for the measurement of peptide amino acid (PAA) in blood and plasma were compared in sheep. Preparation of blood and plasma samples consisted of a deproteinization, either chemical with sulfosalicylic acid (0.04 g for 1 ml of sample) or physical by ultrafiltration (10,000-MW cut-off filters), with or without a subsequent ultrafiltration through a 3,000-MW cut-off filter. Peptide concentrations were determined by quantification of amino acid concentrations before and after acid hydrolysis of samples. Free amino acid concentrations were similar by all the method used (about 2.5 and 2.7 mM, for blood and plasma respectively). Peptide concentrations were higher with chemical deproteinization (10.6 and 4.2 mM, for blood and plasma respectively) than with physical deproteinization (5.7 and 3.3 mM, for blood and plasma respectively). When the deproteinized samples were further treated to remove material of molecular weight above than 3 kDa, peptide concentrations were significantly reduced, which indicates inefficiencies in the ability of the deproteinizing procedures in removing all the proteinaceous materials. Concentration of small PAA (< 3 kDa) in blood was about 1.5-fold that in plasma, mainly due to peptide Gly and Glu derived from the hydrolysis of the erythrocyte glutathione. The choice of a methodology for quantifying circulating peptides is discussed.