Method for quantitative and semi-quantitative detection of l-phenylalanine, l-tyrosine, l-3,4-dihydroxyphenylalanine and their corresponding keto-acids, phenylpiruvic acid, 3-hydroxyphenylpyruvic acid and 3,4-dihydroxyphenylpyruvic acid in biological fluids, related kits for diagnosis and monitoring of altered metabolisms of these aminoacids
OwnerGiorgio Ricci, Francesco Ricci +1inventors
InventorsGiorgio Ricci, Francesco Ricci, Giorgio Federici
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Abstract
Method for quantitative and semi-quantitative determination of endogenous amino acids L-phenyalanine, L-tyrosine, L-3,4-dihydroxyphenylalanine and their corresponding keto-acids, phenylpiruvic acid, 3-hydroxyphenylpyruvic acid and 3,4-dihydroxyphenylpyruvic in biological fluids useful for diagnosis and monitoring of metabolic disorders of said amino acids or diseases involving said amino acids
Claims20
Method for quantitative and semi-quantitative determination of endogenous amino acids L-phenyalanine, L-tyrosine, L-3,4-dihydroxyphenylalanine and their corresponding keto-acids, phenylpiruvic acid, 3-hydroxyphenylpyruvic acid and 3,4-dihydroxyphenylpyruvic in biological fluids useful for diagnosis and monitoring of metabolic disorders of said amino acids or diseases involving said amino acids, comprising the following steps:
- a) reaction of phenylpiruvic acid, 3-hydroxyphenylpyruvic acid and 3,4-dihydroxyphenylpyruvic, present as such in biological fluids or coming from the parent endogenous amino acids L-phenyalanine, L-tyrosine, L-3,4-dihydroxyphenylalanine by deamination, with an organic salt of phenazine derivatives in the presence of at least one alkaline buffer to give colored charge transfer complexes;
- b) measurement of the absorbance values due to said charge transfer complexes in the wavelength range from 650 to 690 nm and quantification of the keto acids or amino acids concentrations in biological fluids.
Kit for diagnosis and monitoring of metabolic disorders of L-phenyalanine, L-tyrosine, L-3,4-dihydroxyphenylalanine or diseases involving said amino acids, based on quantitative detection of the parent keto-acids phenylpiruvic acid, 3-hydroxyphenylpyruvic acid and 3,4-dihydroxyphenylpyruvic comprising:
- a) an organic salt of phenazine derivative in solution or adsorbed on solid support;
- b) an alkaline buffer such as the extinction coefficients at 663 nm of complexes of phenylpiruvic acid, 3-hydroxyphenylpyruvic acid and 3,4-dihydroxyphenylpyruvic with the organic salt of phenazine derivatives are higher than 13,000 M−1 cm−1.
Kit according to anyone of the claims from 11 to 16, which also comprises:
- d) a deaminating enzyme for L-phenyalanine, L-tyrosine, L-3,4-dihydroxyphenylalanine.
Dependencies are inferred from the claim text.