JPS6057840B2 - Cholesterol quantitative agent and method - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to a quantitative agent and method for accurately quantifying cholesterol in a sample.

In general, even healthy people contain a small amount of cholesterol in their blood, but if the amount is too large, it can lead to deposition of cholesterol in the blood vessels, causing blood circulation disorders. Therefore, quantifying the amount of cholesterol in the blood is extremely important for the diagnosis and follow-up of these diseases. Conventionally, a precipitation measurement method using digitonin has been used as a method for quantifying cholesterol in blood, but this method cannot be considered a general method because it is time-consuming and requires a large amount of blood.

The present inventors conducted research in search of a method for quantifying cholesterol that was completely unaffected by metabolites in the blood, and found that they added methanol to a quantitative reagent in the presence of cholesterol oxidase and catalase to generate formaldehyde. NAD is converted to NADH by the action of formaldehyde dehydrogenase in the presence of reduced glutathione.
By reducing the amount to NADH and measuring the increase in NADH, we were able to invent a simple quantitative measurement method for cholesterol.

The cholesterol quantitative agent of the present invention contains the following composition. Methanol 0.5~Solid reduced glutathione 0.5~1.5n1MNA
D0.5-5.0rnM cholesterol oxidase 0.2-5.0U/TfLl catalase
25-1000U/ml formaldehyde dehydrogenase 0.5-5.0
U/T! LtPH7. O~9.0 where the enzyme unit is
It means an international unit, and one unit is the amount of enzyme that transfers 1 micromole of substrate per minute under standard conditions.

The present invention also involves treating a cholesterol-containing sample with cholesterol oxidase and catalase in the presence of methanol and oxygen, and treating the resulting formaldehyde with formaldehyde dehydrogenase in the presence of reduced glutathione to convert NAD to NADH. This is a method of quantifying cholesterol by measuring the amount of increased NADH.

The special feature of the present invention is that cholesterol is extracted in the presence of methanol and oxygen.
Formaldehyde, which is not found in living organisms at all, is produced by the action of oxidase and catalase, and by the action of formaldehyde dehydrogenase, NAD is converted to NADH.Cholesterol in the blood is subjected to a series of reactions, and the increase in NADH is measured, and the amount of increase in NADH is measured. It is extremely unique in that it can be carried out reliably in a short period of time.

The reaction for quantifying cholesterol in the present invention is schematically shown as follows.

One condition for the quantitative method of the present invention is that the reaction pH is 7.0 to 9.0.
Any buffer solution may be used as long as it has a buffering effect at a temperature of 9.0 to 9.0, but phosphate buffer is particularly preferred.

Next, test examples and examples of the present invention will be shown.

Test example (ml) 0
．． 1M Calibathar Phosphate (PH=7.5) (Contains 10% methanol) ・・・1.90100
mM reduced glutathione...0.0210
0mMNAD...0.02400U/ml formaldehyde dehydrogenase...0.0
11 (1'u/Mt catalase ・
・0.02400m9/dl cholesterol
...0.02 (Boehringer standard material) 200U
/ml cholesterol oxidase...0.001~
For compositions of 20 (variable) or more, the amount of cholesterol oxidase was varied and each mixture was reacted at 37°C,
The absorbance at 340r1m was measured.

The results are shown in FIG. 1, and it can be seen that the absorbance at 340 nm reaches its maximum when the amount of cholesterol oxidase added is 1 U/ml.

Example 1 (ml) 0
．． 1M potassium phosphate buffer (PH=7.5) (10
% methanol content)...1.9001
00mM reduced glutathione...20
100mMNAD...20400U/ml formaldehyde dehydrogenase...101 (
PU/ml catalase...2
0200U/ml cholesterol oxidase
・・10400m9/Dt cholesterol ・・1～20μm (manufactured by Boehringer)
(Variable) Pretice Cholesterol) A mixed solution was obtained by removing cholesterol from the above.

The cholesterol quantitative agent of the present invention consists of this mixed solution. To this cholesterol quantification agent, 400 m9/dl cholesterol solution was added in varying amounts of 1 to 20 p1, and reacted at 3 rC with 340 m9/dl solution.
The absorbance was measured at n.

The results are shown in FIG. 2, and it was found that ΔE increased according to the amount of cholesterol, and there was a proportional relationship between the two.

This Figure 2 can be used as a standard graph. Example 2 Serum was separated from blood collected from a patient who appeared to have a high cholesterol disease.

Add 0.02 ml of this serum to 1.98 ml of the mixture of Example 1.
When added to the water, reacted for 1 minute at 3TC, and measured by increasing 340r1m, the value immediately after addition was 0.147.
The value after 5 minutes was 0.574. Therefore 340nm
The ΔE value was 0.417. When this was applied to the standard graph prepared in Example 1, it became clear that the serum contained 310 mg/Dt of cholesterol.

[Brief explanation of the drawing]

FIG. 1 is a diagram showing changes in absorbance due to changes in cholesterol oxidase concentration. FIG. 2 is a graph showing a calibration curve for cholesterol determined using the quantitative agent of the present invention.

Claims (1)

[Scope of Claims] 1. Cholesterol quantitative agent containing the following composition: methanol 0.5-5M, reduced glutathione 0.5-1.5mM, NAD 0.5-5.0mM, cholesterol oxidase 0.2-5. 0u/ml
Catalase 25-1000u/ml Formaldehyde dehydrogenase 0.5-5.0u/ml pH 7.0-
9.0 2 Formaldehyde generated when quantitatively measuring cholesterol by acting cholesterol oxidase and catalase in the presence of methanol and oxygen on a cholesterol-containing sample is subjected to formaldehyde dehydrogenase in the presence of reduced glutathione. N.A.D.
A method for quantifying cholesterol, which comprises using NADH as NADH, and measuring the amount of NADH that increases.