Colorimetric method for determining gamma-glutamyl transpeptidase and compositions useful therein

Claim 2

In a colorimetric method for the determination of gamma-glutamyl transpeptidase enzyme activity in a biological fluid, wherein said enzyme in said fluid is reacted catalytically with a substrate of a synthetic peptide of L-glutamic acid in aqueous medium to yield a diazotizable cleavage product of the peptide and the cleavage product is diazotized and reacted with a coupling compound to produce a dye having a color intensity proportional to the enzyme activity, the improvement which comprises employing as said substrate a water-soluble salt of a compound selected from the group consisting of L-gamma-glutamyl-3-sulfoanilide and L-gamma-glutamyl-4-sulfoanilide.

Claim 3

The method defined in claim 1 wherein said substrate is a water-soluble salt of L-gamma-glutamyl-3-sulfoanilide.

Claim 4

The method defined in claim 1 wherein said substrate is a water-soluble salt of L-gamma-glutamyl-4-sulfoanilide.

Claim 5

The method defined in claim 1 wherein said coupling compound is N-(1-naphthyl)ethylenediamine.

Claim 6

A colorimetric method for the determination of gamma-glutamyl transpeptidase enzyme activity in a biological fluid which comprises catalytically reacting said enzyme in said fluid with a substrate of a water-soluble salt of a compound selected from the group consisting of L-gamma-glutamyl-3-sulfoanilide and L-gamma-glutamyl-4-sulfoanilide in aqueous solution to produce the corresponding aniline sulfonic acid salt as a cleavage product, diazotizing said cleavage product, and coupling the diazotized product with N-(1-naphthyl)ethylenediamine in water-soluble salt form.

Claim 7

A colorimetric method for the determination of gamma-glutamyl transpeptidase enzyme activity in a biological fluid which comprises catalytically reacting said enzyme in said fluid with a substrate of a water-soluble salt of a compound selected from the group consisting of L-gamma-glutamyl-3-sulfoanilide and L-gamma-glutamyl-4-sulfoanilide in aqueous solution to produce the corresponding aniline sulfonic acid salt as a cleavage product, diazotizing said cleavage product, and coupling the diazotized product with N-(1-naphthyl)ethylenediamine in water-soluble salt form.

Claim 8

The method defined in claim 5 wherein said fluid comprises human blood serum.

Claim 9

A colorimetric method for the determination of gamma-glutamyl transpeptidase enzyme activity in human blood serum which comprises incubating the serum with an aqueous substrate solution having a pH of about 7.2, containing an excess amount of the sodium sulfonate salt of a compound selected from the group consisting of L-gamma-glutamyl-3-sulfoanilide and L-gamma-glutamyl-4-sulfoanilide, and containing glycyl glycine as a receptor of the glutamyl radical at about 37° C and for a period of time of about 10 minutes to produce the corresponding aniline sodium sulfonate as a cleavage product, inactivating said enzyme, diazotizing said cleavage product in the resulting solution with nitrous acid, adding to the resulting solution N-(1-naphthyl)ethylenediamine in water-soluble salt form for coupling with the diazotized product to form a dye, and measuring the intensity of light absorption by the resulting solution at 535 nanometers.

Claim 10

A colorimetric method for the determination of gamma-glutamyl transpeptidase enzyme activity in human blood serum which comprises incubating the serum with an aqueous substrate solution having a pH of about 7.2, containing an excess amount of the sodium sulfonate salt of a compound selected from the group consisting of L-gamma-glutamyl-3-sulfoanilide and L-gamma-glutamyl-4-sulfoanilide, and containing glycyl glycine as a receptor of the glutamyl radical at about 37° C and for a period of time of about 10 minutes to produce the corresponding aniline sodium sulfonate as a cleavage product, inactivating said enzyme, diazotizing said cleavage product in the resulting solution with nitrous acid, adding to the resulting solution N-(1-naphthyl)ethylenediamine in water-soluble salt form for coupling with the diazotized product to form a dye, and measuring the intensity of light absorption by the resulting solution at 535 nanometers.

Claim 11

The method defined in claim 7 wherein said enzyme inactivation and diazotization are accomplished by lowering the solution pH to a maximum of about 1.6.

Claim 12

A substrate solution for reaction with gamma-glutamyl transpeptidase enzyme in a colorimetric method for the determination of the activity of the enzyme in a biological fluid, which comprises an aqueous solution having a pH of about 7.2 and containing the following materials in the proportions indicated: ______________________________________ Material Amount Per Liter ______________________________________ Sodium sulfonate salt of L-gamma-glutamyl-3-sulfoanilide or L-gamma-glutamyl-4-sulfoanilide 2.5-5 millimoles Glycyl glycine 130-210 millimoles Sodium nitrite 0.5-2 millimoles Tris(hydroxymethyl)aminomethane hydrochloride 10-100 millimoles Sodium chloride 3-5 millimoles Polyoxyethylene(23) lauryl ether 1-2 grams. ______________________________________

Claim 13

A substrate solution for reaction with gamma-glutamyl transpeptidase enzyme in a colorimetric method for the determination of the activity of the enzyme in a biological fluid, which comprises an aqueous solution having a pH of about 7.2 and containing the following materials in the proportions indicated: ______________________________________ Material Amount Per Liter ______________________________________ Sodium sulfonate salt of L-gamma-glutamyl-3-sulfoanilide or L-gamma-glutamyl-4-sulfoanilide 2.5-5 millimoles Glycyl glycine 130-210 millimoles Sodium nitrite 0.5-2 millimoles Tris(hydroxymethyl)aminomethane hydrochloride 10-100 millimoles Sodium chloride 3-5 millimoles Polyoxyethylene(23) lauryl ether 1-2 grams. ______________________________________