JPH0611238B2 - Method for measuring argininosuccinate or argininosuccinate lyase - Google Patents

Description

The present invention relates to a method for measuring argininosuccinate or argininosuccinate lyase. Argininosuccinate and argininosuccinate lyase (EC4.3.2.1 Argininosucc
inate lyase (hereinafter referred to as “ASL”) is a member of the urea cycle that fixes free ammonia, which is a metabolite of nitrogen-containing compounds such as proteins, to harmless urea, and argininosuccinic acid in the liver is L- It is decomposed into arginine and fumaric acid, and then L-arginine is decomposed into L-ornithine and urea by the action of arginase, and urea is excreted from the kidney out of the body. Measurement of argininosuccinic acid or ASL is useful for diagnosis of inborn errors of metabolism such as argininosuccinic aciduria.

Conventionally, the measurement of argininosuccinic acid or ASL is derived from the double bond of fumaric acid, which is one of the reaction products of argininosuccinic acid and ASL. Method of measurement by absorption at 240 nm [Methods in enzymology (Meth
ods in Enzymology) 17A, 304, 1970], a method for measuring urea produced by reacting arginase with another reaction product, L-arginine [Journal of Biological Chemistry (J. Bio).
l.Chem.) 157, 507, 1945], a method using ¹⁴ C-labeled guanidino-L-argininosuccinic acid as a substrate [Methods in Enzymology (Meth
ods in Enzymology) Vol. 17A, p. 326, 1970]. However, since these methods require a large amount of sample and a centrifugation operation for deproteinization, the processing of a large number of samples requires a great deal of labor, time and reagents, and also has a high sensitivity. It was low.

The present inventors have examined a method for measuring argininosuccinate or ASL that can be used for mass screening for the diagnosis of metabolic disorders in children, and found that ornithine-keto acid aminotransferase [L-ornithine: 2- Oxo acid aminotransferase (EC 2.6.
1.13), hereinafter referred to as "OAT"], pyrroline-5-carboxylic acid reductase [L-proline: NAD (P) ⁺ 5-oxide reductase (EC1.5.1.2), hereinafter "P5CR".
], Arginase (L-arginine amidinohydrolase, EC3.5.3.1), α-ketoglutarate, reduced pyridine nucleotide coenzyme and either argininosuccinic acid or ASL are reacted to give reduced pyridine in the reaction solution. The inventors have completed the present invention, knowing that by measuring changes in nucleotide coenzymes, the drawbacks of the prior art in microquantitativeness, accuracy, speediness, simplicity, and multi-analyte measurement can be improved.

All enzymes used in the method of the present invention are known, for example, arginase is derived from Rad liver [Methods in Enzymology 17A, 313, 1970], OAT is Bacillus sphaericus (Ba).
cillus sphaericus) [FEBS letter (FEBS
Lett.) 105, 209, 1979], P5CR is derived from baker's yeast [Biochim. Biophys. Acta] 613, 318, 1980.
And ASL are exemplified by enzymes from Streptococcus faecalis.

The reaction system of the method of the present invention will be described in detail. First, argininosuccinic acid is decomposed into L-arginine and fumaric acid by the action of ASL, and then L-arginine is decomposed into L-ornithine and urea by the action of arginase.
-Ornithine produces glutamic acid γ-semialdehyde and glutamic acid by the action of OAT in the presence of α-ketoglutarate, and glutamic acid γ-semialdehyde is nonenzymatically changed to pyrroline-5-carboxylic acid. Pyroline-5-carboxylic acid produces proline under the action of P5CR in the presence of reduced pyridine nucleotide coenzyme,
At the same time, the reduced pyridine nucleotide coenzyme becomes oxidized. Argininosuccinic acid or ASL is quantified by measuring the change in reduced pyridine nucleotide coenzyme at this time, that is, the decrease thereof or the increase of oxidized coenzyme. Examples of the reduced pyridine nucleotide coenzyme include reduced nicotinamide adenine dinucleotide (hereinafter referred to as “NADH”) or reduced nicotinamide adenine dinucleotide phosphate (hereinafter referred to as “NADPH”). The measurement of these coenzymes is preferably performed by measuring the decrease of reduced pyridine nucleotide coenzyme from the change in absorbance or fluorescence intensity at around 340 nm, and in particular, it is preferable to measure the fluorescence intensity for microquantification. Good.

The reaction equation of the method of the present invention is as follows.

(1) Argininosuccinic acid → L-arginine + fumaric acid (2) L-arginine + H ₂ O → L-ornithine + urea (3) L-ornithine + α-ketoglutarate pyrroline-5-carboxylic acid + L-glutamic acid (4) pyrroline -5-carboxylic acid + NAD (P) H + 2H ⁺ → L-proline + NAD (P) ^{+ As} described above, the method of the present invention comprises OAT, P5CR, arginase, α-ketoglutarate, and reduced pyridine nucleotide coenzyme. And ASL are essential measurement methods for argininosuccinic acid and OSL, P5CR, arginase, α-ketoglutarate, reduced pyridine nucleotide coenzyme and argininosuccinic acid are essential measurement methods for ASL. The method of the present invention does not require pyridoxal phosphate, which is commonly used as a coenzyme for transposases such as OAT, but rather uses it for NAD (P) H
Adversely affect the fluorescence measurement of. The present inventors have found that the addition of α-ketoglutaric acid compensates for the action of pyridoxal phosphate, and as a result, it has become possible to perform microquantification by fluorescence measurement.

The measurement sample may be whole blood, serum, plasma, urine, tissue extract, etc., but for mass screening, a so-called blood filter paper on which hemopigment is immobilized after soaking blood into filter paper and drying it is used. Is good. The method of immobilizing hemoglobin is sure to use a composition consisting of ether (or methylene chloride): water: acetone: alcohol,
It is preferable because it is quick. The blood filter paper thus prepared is punched out into a disk having a diameter of about 3 mm, and this is added as a sample to the reaction mixture, whereby the measurement can be easily carried out.

The present invention will be specifically described below with reference to examples.

Example 1 50 μl of a reaction mixture (0.1 M Tris-HCl buffer, pH 8.0) was added with the following reagents, 5 mu ASL (manufactured by Sigma Chemicals), 2
0 mu arginase [Used by dissolving in 1.0 M glycine buffer solution (pH 7.5) containing 10 mM manganese sulfate], 0.25 μmol
α-ketoglutaric acid, 0.5 nmol dithiothreitol, 12 m
Each reaction mixture containing uOAT, 12 mu P5CR, 5 nmol NADH and 0-10 nmol of argininosuccinic acid as a sample was reacted at 37 ° C. for 1 hour. After adding 3.0 m of water to the reaction solution, 0.29 μM quinine sulfate standard solution was used as the excitation wavelength.
The fluorescence intensity was measured at 340 nm and a fluorescence wavelength of 450 nm. A value obtained by operating in the same manner except that ASL was removed from the above reaction mixture was used as a blank value, and the value (percentage) obtained by dividing the difference between this value and the above measured value by the blank value was obtained. When the calibration curve was created by plotting the relationship with the acid concentration on the graph, it was as shown in the figure.

Next, when plasma was used as a sample and operated in the same manner as above, and the measured fluorescence intensity was compared with the calibration curve, the argininosuccinic acid concentration was 0.62 mM. NA instead of NADH
Similar results were obtained with DPH.

Example 2 A few drops of blood were soaked in Guthrie's filter paper (manufactured by Daiichi Pure Chemicals Co., Ltd.) used for mass screening for neonatal metabolic disorders and air dried. This was punched into a disc with a diameter of 3 mm and put into a test tube, and then 20 μm of a composition having a volume ratio of ether: water: acetone: methanol of 70: 5: 5: 20 was added, and the mixture was allowed to stand at room temperature for 1 hour to remove blood pigment A denaturing immobilization treatment was performed.
When this filter paper disk was used as a sample and operated in the same manner as in Example 1, the concentration of argininosuccinic acid was 0.61 mM.

Example 3 50 μl of a reaction mixture (0.1 M Tris-HCl buffer, pH 8.0) was dissolved in the following reagents: 1.0 mmol argininosuccinate, 20 mu arginase [1.0 M glycine buffer containing 10 mM manganese sulfate (pH 7.5). Used)], 0.25 μmol α-ketoglutarate, 0.5 nmol dithiothreitol, 12 muOA
A reaction mixture containing T, 12 mu P5CR, 10 nmol NADH and a serum sample was reacted at 37 ° C. for 1 hour. Water in the reaction solution 3.
After adding 0 m, the fluorescence intensity was measured at an excitation wavelength of 340 nm and a fluorescence wavelength of 450 nm using 0.29 μM quinine sulfate as a standard solution, and the ASL activity was determined from the decrease amount of NADH.

[Brief description of drawings]

The figure shows a calibration curve of argininosuccinic acid according to the method of the present invention.

 ─────────────────────────────────────────────────── ─── Continuation of the front page Judge Judge Toshiko Otaka (56) Reference JP-A-53-89493 (JP, A) J Biol chen, Volume 157, pp. 507-518, 1945

Claims (2)

[Claims] 1. A test sample is reacted with one of ornithine-keto acid aminotransferase, pyroline-5-carboxylic acid reductase, arginase, α-ketoglutarate, reduced pyridine nucleotide coenzyme and argininosuccinate lyase or argininosuccinate. A method for measuring argininosuccinic acid or argininosuccinic acid lyase, which comprises measuring a change in a pseudo-reducing pyridine nucleotide coenzyme. 2. The method for measuring argininosuccinic acid or argininosuccinate lyase according to claim 1, wherein the reduced pyridine nucleotide coenzyme is reduced nicotinamide adenine dinucleotide or reduced nicotinamide adenine dinucleotide phosphate.