JP2628340B2 - Method for stabilizing insulin component in blood - Google Patents

Description

Description: TECHNICAL FIELD The present invention relates to a method for preventing a change in the amount of components after blood collection and obtaining an accurate measurement value when measuring the amount of insulin in blood. .

"Prior art" Blood glucose is usually measured as a test for diabetes, but insulin measurement has recently been performed as an indispensable test. This test (measurement) was conducted by Berson and
Radioimmunoassa using a specific antibody developed by Yallow
It is common to use the y method (hereinafter referred to as the RIA method).

To measure the insulin concentration, serum is separated from the collected blood by centrifugation or the like, and the insulin concentration in the serum is measured.

[Problems to be Solved by the Invention] By the way, it is actually impossible to separate serum immediately after blood collection and directly measure insulin concentration, and it takes some time after blood collection and before measurement. Is common. Then, for example, a paper by Masashi Chiba and Wataru Ueki, "Analysis of the decrease in the insulin level of hemolyzed serum" (Hygiene Inspection, No. 3
As shown in 5, pp. 715 to 719, 5.1986), when hemolysis occurs in blood and the components of red blood cells are contained in serum, an abnormal pattern of measured values of insulin is observed. There is. This hemolysis phenomenon cannot be safely prevented. Therefore, conventionally, there is a problem that the measurement of the insulin concentration has instability due to hemolysis.

"Means to solve the problem".

The present invention invents a method for inhibiting the degradation of sugars by adjusting the pH of a blood sample, and disclosed on May 10, 1985, JP-A-60-100161.
No. "Method for preventing glycolysis in blood".

Further, as a result of intensive studies, the inventor of the present invention has found that using a method similar to the above-mentioned invention in which acid is added to the collected blood to adjust the acidity of the sample so that the pH of the blood becomes 5.0 to 6.5, even if there is hemolysis, It has been found that the present invention has been found to be able to prevent a change in the concentration of glycerol (a chemical change in insulin).

This chemical change in insulin is caused by the formation of the S--S bond of insulin having an S--S bond as a kind of amino acid by a component having an S--H bond in a chemical structure such as reduced glutathione present in erythrocytes effluxed by hemolysis. Probably due to being destroyed. Therefore, when blood is made acidic as in the present invention, reduced glutathione or the like is transformed into acidic glutathione or the like, so that it is presumed that a chemical change of insulin is prevented.

An object of the present invention is to prevent a change in insulin concentration. However, as described in the above-mentioned application, the method of the present invention can simultaneously prevent a change in sugar content, and a single sample can be used. This has the advantage that both components can be measured.

The pH of blood is usually 7.4, but in the present invention, p is added by adding an acid.
Adjust so that it becomes H5.0-6.5. The acid may be either an inorganic acid or an organic acid, but becomes powdery with an organic acid, puts a fixed amount in a sampling tube, and when blood is collected in the collection tube, it is automatically mixed with blood and easily adjusted to an appropriate pH. What can be made to become preferable is preferable.

In particular, mixing 3.0 to 5.0 mg / ml of citric acid in the blood is preferable because the pH of the blood becomes 6.5 to 5.8, and not only insulin but also the effect of inhibiting glycolysis is maintained.

Insulin is stabilized by adjusting the pH with an acid, and the addition of a fluorine compound such as NaF further improves the glycolytic inhibition effect. In this case, it is not necessary to use the conventional 1.25 mg / ml of NaF, and a very small amount of about 0.1 to 0.5 mg / ml is sufficient.

In order to carry out the present invention, for example, an aqueous solution mainly composed of citric acid and NaF is added to a collection tube for collecting 1.5 to 2.0 ml of blood so that the above-mentioned pH concentration is obtained when mixed with blood. Heat to evaporate the water, leave the components in powder form and adhere to the bottom of the collection tube.
It is advisable to collect 2.0 ml, store and transfer as is, and measure the insulin concentration. At that time, if the budosugar (blood sugar level) or lactic acid and pyruvic acid are simultaneously quantified, the glycolytic inhibition effect can also be used.

"Example" Blood was collected in a sampling tube to produce serum without hemolysis by centrifugation, while human blood cells were artificially hemolyzed to produce hemolyzed blood in which hemoglobin concentration was adjusted.

Serum citrate 5mg / ml (per blood) and EDTA ・ 2Na
Citric acid and EDTA ・ 2N to be 2mg / ml (per blood)
a was added and mixed. Serum is at pH 7.4, pH after addition
It was 5.8.

The rate of decrease in insulin concentration with time was measured using the serum without pH adjustment and the serum without pH adjustment, with and without hemolysis, and with hemolyzed hemoglobin at 500 ml / dl and hemoglobin at 1500 ml / dl. As a result of the examination, the results are as shown in FIGS.

As can be seen from the graph, in the serum without pH adjustment, the measured concentration of insulin sharply decreases when hemolyzed is added. However, when the pH is adjusted according to the present invention, the insulin concentration is maintained even when hemolyzed is added and 24 hours have passed. It turns out that it is very stable.

[Effects of the Invention] As described in detail above, the present invention requires only adding acid, especially citric acid to a collected blood sample to adjust the blood pH to 5.0 to 7.0 when measuring insulin in blood. Insulin in the sample blood is stable, and accurate concentration values can be obtained even after storing for several tens of hours before sampling and measuring, which is very useful for measuring insulin values, especially when measuring large amounts of samples. This is an effective method. This method also stabilizes the blood glucose level, which is particularly effective when measuring both at the same time. As described in claims 2 and 3, citric acid is easy to use as an acid, and is very effective in inhibiting glycolysis. It is more effective to add a trace amount of a fluorine compound and adjust the pH during pH adjustment, as the inhibitory action of the glycolytic reaction is maintained.

[Brief description of the drawings]

Fig. 1 is a graph showing the change over time of the insulin concentration when hemolysin was added to serum without pH adjustment and the hemoglobin concentration was adjusted to 500 mg / dl and 1500 ml / dl. FIG. 3 is a graph showing the time-dependent change in the concentration of insulin when the pH-adjusted serum of the present invention is measured under the same conditions.

Claims (5)

(57) [Claims] 1. A method for stabilizing an insulin component in blood, comprising measuring the concentration of insulin in blood by adding an acid to the collected blood to adjust the pH of the blood to 5.0 to 6.5. 2. The method for stabilizing an insulin component in blood according to claim 1, wherein citric acid is used as the acid. 3. A citric acid of 3.0 to 5.0 mg / ml (per blood)
The method for stabilizing an insulin component in blood according to claim 1 or 2, characterized by mixing. 4. The method for stabilizing an insulin component in blood according to claim 1, wherein a fluorine compound is used in combination. 5. The method for stabilizing an insulin component in blood according to claim 1, wherein NaF is mixed at 0.1 to 0.5 mg / ml (per blood).