JPS6078597A - Measurement of cyanuric acid having bonding property of lipid - Google Patents

Abstract

PURPOSE:To measure cyanuric acid having binding property of lipid simply and accurately, by measuring the concentration of cyanuric acid of a supernatant liquid having precipitate formed with phosphorus wolframate by enzymatic method, calculating the difference between the concentration of total cyanuric acid of specimen and that of cyanuric acid of the supernatant liquid. CONSTITUTION:The concentration of cyanuric acid of specimen is measured by enzymatic method, about 3-8%(w/v) calculated as final concentration of phosphorus wolframate is added to the specimen, and precipitate is formed. The precipitate is separated, and the concentration of cyanuric acid of the supernatant liquid is measured by using an enzyme to act on cyanuric acid. The concentration of cyanuric acid having bonding property of lipid is obtained by subtracting the concentration of cyanuric acid of the supernatant liquid from that of cyanuric acid.

Description

[Detailed description of the invention] It is about improvement.

Recently, it has become clear that the concentration of lipid-bound sialic acid in the blood increases significantly when suffering from cancer, and it is desired to develop a method for simply and accurately measuring this lipid-bound sialic acid.

Conventionally, lipid-bound sialic acid has been measured by a method in which a sample is treated with phosphotungstic acid to form a precipitate, and the precipitate is reacted with resorcinol. However, this method has the drawbacks of being complicated and requiring long steps, and requiring a great deal of time and effort for measurement.

The present inventors conducted extensive research to develop a simple and accurate method for measuring lipid-bound sialic acid, and as a result, they determined the concentration of sialic acid in the supernatant liquid obtained by generating a precipitate using phosphotungstic acid using an enzymatic method. devised a method of subtracting the sialic acid concentration of the supernatant from the total sialic acid concentration of the sample, and by this method, lipid-bound sialic acid could be measured easily and accurately, and a large amount of sample f-1 could be measured. The present invention was completed based on the discovery that it can be easily processed.

That is, the present invention measures the sialic acid concentration of a sialic acid-containing liquid containing lipid-bound alkalic acid, and separates the precipitate produced by adding phosphotungstic acid to this sialic acid-containing liquid. 7 Lipid binding property characterized by measuring alkyl acid concentration using an enzyme that acts on sialic acid/
The present invention relates to a method for measuring alkaline acid.

Sialic acid is a general term for various groups bonded to the skeleton of neuraminic acid, and various types are known, including lipid-bound sialic acid, glycoprotein-bound sialic acid, and free sialic acid. The sample to be measured by the method of the present invention contains lipid-bound sialic acid and other sialic acids, such as plasma and blood serum.

The measurement sample can be pretreated as necessary.

For example, dilution or extraction with an organic solvent such as chloroporum can be performed to remove free fatty acids from plasma or serum.

In the method of the present invention, the sialic acid concentration is measured before adding phosphotungstic acid. This sialic acid concentration may be omitted for the sample before pretreatment, or may be determined after pretreatment. The method for measuring the alkaline acid concentration may be, for example, a colorimetric method such as the or7nol method, the resorcinol method, the periodate-solnol method, or the thiobarbic acid method, but the enzymatic method described below is preferable.

The addition of phosphotungstic acid may be carried out in the same manner as in the known resorcinol trial method for measuring lipid-bound 7-alic acid.

That is, the final concentration of phosphotungstic acid is 3 to 8%.
(w/v) and left to stand for about 5 to 30 minutes at about 4 to 37°C, usually at room temperature, to form a precipitate.

Separation of the precipitate may be performed by centrifugation or p-filtration.

After separating the sediment, the sialic acid concentration of the supernatant is measured using an enzyme that acts on sialic acid. Enzymes that act on sialic acid can quantify changes in the system due to their action, such as neuraminidase.

When measuring using neuraminidase, for example, 7
Alkalic acid is converted to N-acetylneuramine with neuraminidase, and then N-acetylneuramine is converted with N-acetylneuramine aldolase.
Decomposes into acetylmannosamine and pyruvate. The generated pyruvate is oxidized with pyruvate oxidase/dase to H2
O2 is generated, and 4-aminoantipyrine and an oxidative condensing agent such as p-chlorophenol and N-ethyl-N-(β-hydroxyethyl)-m-)luidine are added to H20□ to produce a reddish-purple color in the presence of peroxidase. There is a method of generating a dye and measuring the absorbance at 550 nm. Reagent kits used in this method are commercially available.

In addition, there is a method in which lactose dehydrogenase is allowed to act on the generated pyruvate in the presence of NADH as a coenzyme, and the consumption of NADH is determined by measuring the decrease in absorbance at 340 to 370 nm.

The enzymatic method can be used to measure only the supernatant, and precipitation is not suitable because it requires a long reaction time.

The method of the present invention utilizes the fact that lipid-bound sialic acid is precipitated by phosphotungstic acid. It is obtained by subtracting the sialic acid concentration of the supernatant liquid from which the precipitate produced by the addition was separated.

The method of the present invention allows lipid-bound sialic acid to be measured simply and accurately, and a large amount of samples can be measured in a short period of time.
It is particularly effective in cancer screening.

Example: Put 50 μt of test serum into a centrifuge tube and add 20 m
After adding 150 μt of M'J sodium phosphate buffer and mixing, the mixture was cooled in a water bath. Two parts of chloroform and methanol:
Add 3 ml of cold mixed solvent mixed at a ratio of 1 (v/v), mix well, then add 0.5 ml of cold 20 mM
Sodium phosphate buffer pH 6.5 was added. After shaking, this was centrifuged at 2500 jpm at room temperature for 5 minutes, and the resulting upper layer was divided into 100 μt and 500 μt portions and placed into two test vessels.

1 g of 25 μt in a test tube containing 500 μt of upper layer liquid,
4ni l) Add tungstic acid aqueous solution and mix 5
Leave it for a minute. Centrifuge at 2500 rpm for 5 minutes at room temperature to obtain a supernatant.

100μ of this supernatant liquid and 100 ltt of the above upper layer liquid
into separate test tubes, and in each tube add Q, 5 ml of reagent 1 (0.2 U/ml neuraminidase, J rule NA).
NA-aldolase, 50mM phosphate buffer PI (6
, s) was added, mixed, and then reacted at 45°C for 30 minutes.

After reaction 1 me reagent 2 (48U/Inl pyruvate oxidase, 0,67 μmoleAll thiamine pyrophosphate, 002μm o l e7ft+l flavin adenine dinucleotide, 27mM p-chloropheno-/
L/, 3.2 UAnl peroxidase, 0.5
Jtmole//fn14-7mi/77 Tibilin, 7.
2mM MgCt2.20mM phosphate buffer pH 7,4)
was added and reacted at 37°C for 30 minutes. 1゜5 to this
ml.

Reagent 3 (0, IMEDTA -39/l) Rydon X-40!5-0.1 M Disodium Phosphate -0,1
M sodium citrate buffer P) 19.0) e added, 1
After being left at room temperature for 0 minutes, the absorbance at 505 nm was measured within 60 minutes. The sialic acid concentration of the combined solution was determined from the standard curve obtained by measuring an appropriately diluted aqueous solution of 1 mfiAni's N-acetylneuraminic acid (NANA) standard.

After correcting the liquid volume of the obtained value, the amount of sialic acid binding to silipid was calculated using the following formula.

Lipid-binding sialic acid (nvdl) = sialic acid in the upper layer liquid - sialic acid in the supernatant liquid The amount of lipid-binding sialic acid was determined using the resorcinol reagent method for the phosphotungsten precipitated residue of the same test serum.

The results of these measurements are shown in the table below.

A 17.5 17.9 B 16, 8 18. O C24,325,4 D 29.8 27.4 E32.1 30.8 F' 20.5 19.4

Claims (1)

[Claims] The sialic acid concentration of the sialic acid-containing liquid containing lipid-bound sialic acid is measured, and the precipitate generated by adding phosphotungstic acid to the sialic acid-containing liquid is separated, and the sialic acid concentration of the supernatant liquid is determined by measuring the sialic acid concentration of the sialic acid-containing liquid. A method for measuring lipid-bound sialic acid, characterized by measuring using an enzyme that acts on