JPS60227171A - Measuring method of glucose conjugated to hemoglobin - Google Patents

Abstract

PURPOSE:To measure a blood sugar value without the need for removing the disturbing material in specimen blood by mixing nitroblue tetrazolium (NBT) and hemolytic blood in a liquid compd. in which hemoglobin of tetramer can be made monomeric and measuring the amt. at which the NBT is reduced by measuring the absorbancy. CONSTITUTION:The hemolysis sample is mixed together with the NBT into a carbonic acid buffer soln. (0.03mol NaHCO3+0.07mol Na2CO3) and the absorbancy at 540nm is measured while the specific absorption at 500-600nm by hemoglobin is decreased or eliminated by making use of the monomeric state of the hemoglobin, which is usually tetramer, in the NBT liquid adjusted to 10.8 or 12.5pH by said buffer soln. The measurement of the conjugated glucose with good accuracy is made possible from the absorbancy occuring in the change of the NBT to a blue color as a result of the reduction caused by the glucose conjugated to the hemoglobin without the need for the removal of the other reducing component, i.e., vitamin C or flutose in the blood or anticlotting agent, etc. and without the need for such a high temp. heating treatment as 100 deg.C. The measuring method is used for diagnosis of the diabetes, etc.

Description

Detailed Description of the Invention (Field of Industrial Application) The present invention relates to a method for determining the amount of glucose bound to hemoglobin produced by red blood cells in blood using absorbance measurement. This is an effective method for managing related blood sugar levels.

(Prior art) Glucose is not bound to hemoglobin produced by red blood cells at the beginning, but during the approximately four-month lifespan of red blood cells, it reacts with glucose in the blood and forms bonds similar to syn-2 bases. It turns into aldimine. This reaction is reversible, but when it is converted into a ketoamine, which is a type of ketone, it does not return to the original aldimine and becomes a stable compound. Because this reaction is nonenzymatic, slow, and proceeds slowly through the circulation of red blood cells, the amount produced reflects the past blood sugar concentration, and the higher the blood sugar level and the longer the period. It gets bigger. That is, hemoglobin to which glucose is bound (this is also called glycosylated hemoglobin or HbAl, and is a minute component distinguished from the main component HbA) HbAla, HbAl
If you know the concentration of HbAlc), you can cumulatively know the blood sugar level for the past few months dating back to the time of blood collection, and it is more useful in diagnosing diabetes than the measurement of blood sugar itself, which tends to fluctuate. This can be an effective guideline.

In addition, when used in conjunction with the measured value of blood sugar itself, it is extremely useful in the treatment of diabetes, and has attracted attention in recent years.

The most common method for measuring H'bA+ is a simple column method using a small cation exchange column (hereinafter referred to as the blade ram method). In this column method, hemolyzed blood is dropped into a cation exchange column, and an eluent adjusted to a predetermined salt concentration and pH is added.
Since HbAI can be eluted and separated more quickly than HbA, the absorbance of this HbAl-containing solution and the absorbance of the HbA-containing solution that will be eluted later are calculated to determine the amount of HbAI and the total amount of hemoglobin. This method calculates the concentration of HbAI by taking the ratio of . )lbAl a
+) HbAI b and 1(bA+c) may be eluted separately to calculate their respective absorbances.

However, this column method does not allow HbA using a column.
Since this method requires an elution operation for I and HbA and absorbance measurement, there are problems in that it is time-consuming and the measurement cannot be automated. Also, this method has low accuracy;
In particular, there is a problem in that the measured values vary greatly depending on the temperature.

Further, a conventional method includes a TBA colorimetric method. This method removes glucose bound to hemoglobin by heating hemolyzed blood at 100°C for several hours under mildly acidic conditions.
- separated as hydroxymethylfurfural, TB
A is a method in which the absorbance is decreased by adding color (2-thiiz<rubituric acid).

However, this method using TBA, as mentioned above,
'C1 Since several hours of heat treatment are required, it is inefficient and cannot be automated like the above method.

A conventional method that can be automated is a method using phytic acid. In this method, phytic acid does not bind to the N-terminus of hemoglobin when glucose is already bound to hemoglobin, and phytic acid binds to hemoglobin only when glucose is not bound, and the absorbance changes as the wavelength changes. In this method, the concentration of bound glucose is measured from the amount of change in absorbance at a certain wavelength.

However, this method has the disadvantage that, in most cases, sodium fluoride, which is added to the blood for the purpose of preventing coagulation and suppressing the activity of glycolytic enzymes, acts as an interfering substance when measuring blood sugar levels. be.

In the case of this method using phytic acid, it is possible to use EDTA, which does not act as an interfering substance, as an anticoagulant, but in this case, it may be insufficient as a sample for blood glucose measurement in terms of inhibiting glycolytic enzyme activity. Therefore, it is not suitable for the purpose of simultaneously measuring blood sugar and HbAI from the same specimen. Similarly, anti-caking agents such as pepperin cannot be used because they interfere with the phytic acid method.
At present, this situation significantly hinders the clinical application of the phytic acid method. Furthermore, this method has the disadvantage of poor accuracy because it measures normal hemoglobin and back calculates the amount of glycosylated hemoglobin.

On the other hand, proteins during hemorrhage are also glycosylated (glycosylated proteins) by the same mechanism as glycosylated hemoglobin, and this is also known to be useful as a clinical indicator similar to glycosylated hemoglobin. ing. As a method for measuring this glycosylated protein, NBT (nitroblue tet
orazolium) is reduced by fructosamine bound to protein y4, and NBT changes color from colorless or yellow to blue.
．． After serum was added to the carbonate buffer in step 8 and mixed, the change in absorbance at 530 nm after 10 to 15 minutes was measured, and the same procedure was used to prepare DMF (1-deoxy, l-
It has been reported that a new method for controlling blood sugar levels has been obtained by comparing the change in absorbance when morphalinofructose (morphalinofructose) is added to albumin (Ro
ger N, Johnson et al, F-ruc.
tosamine: a new approach
o the ego+5tion of serum
Glycasyl protein, An 1ndex
of diabe-tic control, EIs
evier Biomedical Press, 19
83°87-95).

However, when this method using NET is applied to the measurement of glycosylated hemoglobin, aggregation of hemoglobin occurs during one reaction, resulting in turbidity. At a wavelength of 5°00 to 600 nm, there is a specific absorption of hemoglobin, and the absorbance of this portion changes during the reaction and becomes unstable, so it is practically impossible.

(Object of the Invention) An object of the present invention is to provide a measuring method that can accurately measure the amount of glucose bound to hemoglobin using the NET, and can also automate the measurement.

(Structure of the Invention) The method for measuring glucose bound to hemoglobin according to the present invention involves mixing NBT and hemolysate in a liquid composition that can monomerize hemoglobin, which consists of tetramers. The NBT is reduced by the glucose bound to hemoglobin, and the absorbance is measured with the specific absorption at 500 to 600 nm by hemoglobin reduced or eliminated, and an index value indicating gluco-19 degrees is calculated from the absorbance. It is characterized by the following.

That is, hemoglobin is normally a tetramer consisting of two α chains and two β chains, but when a certain buffer is used, the PH
If α is 12 or more, each monomer is separated, that is, each α
, is known to separate into β chains. In addition, it is generally known that adjusting the pH and salt concentration of the solution, or using surfactants and thiol compounds are effective means to suppress protein association (Eibu Ando et al. ;
"Protein Chemistry, 3. Higher Order Structure, Published by Kyoritsu Publishing Co., Ltd., September 20, 1976). The present inventors have discovered that hemoglobin, in the presence of a dissociation promoter such as a surfactant,
Even if the pH is below 12 (for example, 11,0), due to the dissociation of the tetramer, the specific absorption of the hemoglobin at 500 to 600 na+ disappears, and the aggregation property of hemoglobin also disappears. We found that the amount of NBT reduced by glucose bound to hemoglobin can be calculated by monomerizing hemoglobin and eliminating the specific absorption. By measuring absorbance, it provides a new guideline for blood sugar management.

(Example) Figure 1 is an absorbance curve that serves as the background of the present invention, and 1 is a carbonate buffer solution of 0.1M (0.03M NaHG).
O3+ 0,07 MNa2 GO3), the PH
This is an absorbance curve of the NBT (manufactured by Gyudon Chemical Co., Ltd., NC1, 24720) reagent 51 solution adjusted to 10.8 or 12.5. There is an absorption range in the area, and the color is mostly transparent or pale yellow.

Curve 2A shows red blood cells obtained by washing the entire surface containing the anticoagulant EDTA three times with twice the amount of physiological saline, and dissolving the red blood cells by adding water and stirring (hereinafter referred to as a hemolyzed sample). This is the absorbance curve of a sample obtained by taking 1100 pL of the sample and diluting it with 1 mJ1 of water. When adjusted to PHt-11.5 or less with carbonate buffer, P)l
It becomes like curve 2A regardless of the height of 540n
There is a specific absorption with peaks near m and 580 nm. Curve 2B shows that in the same concentration of hemolyzed sample, only the pH is 12
．． This is the case when it is raised to 0, and as you can see from the figure,
The height of the peak in the specific absorption region has been halved.

Curve 2C shows that in the hemolyzed sample at the same concentration, only the pH is 1.
2.5, and as can be seen from the figure, the peaks in the specific absorption region have disappeared. This is consistent with the report that when P)I becomes 12 or more, hemoglobin changes from a tetramer to a monomer.

Curve 4 in Figure 2 is an absorbance curve obtained by simply diluting the hemolysed sample with water, and curves 5 and 6 are the absorbance curves obtained by diluting the hemolysed sample with alkyl nonionic surfactants added for the purpose of promoting the dissociation of hemoglobin. -Phenoxypolyethoxy-
Ethanol (Rohmand)! Tri made by aas company
tons)! -100)' P in carbonate buffer containing 3%
This is an absorbance curve obtained by adjusting H11.0 and PH11.5 or higher, respectively, without adding the surfactant,
When the pH is simply adjusted to 11.0, curve 2A in Figure 1
As shown in Table 1, although the dissociation of hemoglobin was insufficient, it can be seen from curves 5 and 6 that monomerization proceeded sufficiently when the surfactant was added.

Curve 3A shows the hemolyzed sample taken at 100 gM and 5
This is an absorbance curve of a sample obtained by adding 0.00 μl of water and 50 μl of NBT solution having a concentration of 0.5 mM and adjusting the pH to 10.8 with carbonate buffer, and reacting at 37°C for 30 minutes. Yes, in this PH10.8,
The liquid is cloudy and the absorbance is high even in the long wavelength range. Curve 3B is obtained by changing only the pH of the solution of curve 3A to 12.0, and in this case, although the solution has some turbidity, the turbidity has decreased and the absorbance has decreased. Curve 3C shows that the surfactant Triton is added to the solution of curve 3A.
This is the case when X-100 was added to a concentration of 3% and P)I was changed to ii, o. This solution was not cloudy, and the absorbance also changed to curve 3B.
It decreases slightly compared to the solution of

In Figure 1, the absorbance for the length of the line a or b is the change in absorbance due to the reduction of NBT, and 3% Tr
If the pH is 11 or higher in the presence of iton Note that there is no particular problem with Triton X-100 interfering with the reaction system, and the concentration does not necessarily need to be fixed at 3%; however, if the concentration is low, no effect will be obtained.

Next, a method for actually determining a value that is an index of the amount of glucose bound to hemoglobin in the present invention will be explained using a specific example.

[Example 1] Take the hemolyzed sample with looILM and apply 1 mJJ of T to it.
Add 3% aqueous solution of riton
The absorbance at m was measured (this absorbance corresponds to the concentration of hemoglobin, and is referred to as A.

). On the other hand, take 1 oopLJL of the hemolyzed sample,
5001L of carbonate buffer with pH 11.5 containing 3% Triton X-100! L and NE at a concentration of 0.5mM
500% T solution (containing 3% Triton X-100)
After adding p and reacting at 37℃ for 30 minutes, 540na
The absorbance at m was measured (this absorbance is referred to as A). Then, from these values, the index value x1 is calculated using equation (1).
I met.

X+ = (A+ Ao)/Ao (1) where AIAO (=ΔA) corresponds to the concentration of NBT reduced to ketonized glucose. In this method,
In Figure 1, ΔA corresponds to the length of line C (that is, the absorbance d of the solution containing NET minus the absorbance e corresponding to the hemoglobin concentration), so when the concentration of ketonized glucose is small, Although the error will be large, a tentative indicator can be provided. When the measured data obtained by this method was compared with the measured data obtained by the conventional column method, a relationship expressed by the following equation was established, as shown in FIG. 3, and it was found that this method can be put to practical use.

[Example 2] Take ioogi of the hemolyzed sample and add 3% of 1 mJJ to it.
Add Triton X-100 aqueous solution and 540 nm
(This absorbance corresponds to the concentration of hemoglobin, and its value is defined as A2.)
. On the other hand, take 100p of the hemolyzed sample separately from the above,
This includes PH1 containing 3% Triton X-100.
Add 500 JL of carbonate buffer 1 and 5, react at room temperature for 15 minutes, and measure the absorbance at 540 nm (
This absorbance is designated as A3). Next, to the solution to which this carbonate buffer was added was an NBT solution (3% Trit) with a concentration of 0.5mM.
Add 500 p-1 of on
After reacting for 30 minutes, the absorbance at 540nI11 was measured (this absorbance is designated as A4). Then, the index value x2 was determined from these values using equation (2).

X2 = (A4-(8/+1)A3)/At-
(2) In equation (2), as the coefficient of A3, (
8/11) because the hemoglobin concentration in this solution is 11/6 times higher than that in the final solution. In this method, A4-(8/11)A3
will show an absorbance corresponding to the line b (=df) in FIG. 1, so a more accurate value will be obtained compared to the method of Example 1.

In addition, when carrying out this method, the ratio of the amount of carbonate buffer and the amount of NBT solution can also be changed variously. In contrast to the method of Example 1, which allows two types of absorbance measurements to be carried out simultaneously and can be called a one-step method, this method can be called a two-step method.

[Example 3] Next, we will show an example of a 3-step method in which water, carbonate buffer, and NBT solution are sequentially added to the same container, the absorbance of each is measured, and an index value is calculated from the results. state Add 100p of the hemolyzed sample to 500p.
gJj (7) Add 3% Triton
. Next, add 3% cy) Triton X-1 to this solution.
Added 250jLl of carbonate buffer solution containing 0.00 at pH 11.5 (double concentration of the solution in Example 1.2 above), reacted for 15 minutes at room temperature, and measured the absorbance at 540nm (
Let this absorbance be 86). Next, a NET solution (containing 3% Triton XJOO) with a concentration of 1mM was added to this solution.
After adding 2501 Ll of and reacting at 37°C for 30 minutes, the absorbance at 540 nm was measured (this absorbance is designated as A7). Then, the index value x3 was determined from these values using equation (3).

X3 = (A7 (8,5/11) As l / (
(6/11) As )...(3) The method of Example 3 is a method of sequentially adding each reagent to one cell, and is therefore the easiest method to automate. In the case of Example 3 as well, the concentration and amount of the liquid can be variously selected.

In addition, when determining the concentration of hemoglobin in each of the above, it is also possible to determine it by measuring the absorbance in the main absorption region around 415 nm.

[About fluctuations in measurement results due to hemoglobin concentration]

Next, the results of testing the influence of hemoglobin concentration on the index value X+ etc. will be described. FIG. 4 shows that in Examples 1 and 2, the degree of dilution of the sample was changed (that is, the concentration of hemoglobin was changed) in the same sample,
The results are shown in which the index value X1 or x2 was obtained by keeping the concentrations of other carbonate buffers and NBT solutions the same. In the figure, line 7.8
are the measurement results for the same sample taken from a diabetic patient, line 7 is the measurement result according to Example 2, and line 8 is the measurement result according to Example 1.
These are the measurement results. 9 in equation (2) of Example 2,
Instead of the absorbance A2, which corresponds to the hemoglobin concentration (6
/11)・Using A3, X2 = fAa (8/11
) A3 ) / ((8/11)・A3 ) is the result.

As can be seen from Figure 4, the index values x,, x2 tend to decrease as the hemoglobin concentration increases, but the actual range of variation in human hemoglobin concentration is generally much larger than this measurement range. Because the area is narrow, it can be considered that the effect of hemoglobin concentration on the measurement results is small.

[About interference]

When performing measurements according to the present invention, excess amounts of substances thought to be contained in a specimen were added to each sample and measured using the method of Example 2, and the influence on the measurement results was discussed.

[The l-anticoagulant]

Regarding the anticoagulants used in the pre-liquid, such as sodium fluoride, pepperin, and EDTA, measurements were taken with each concentration added in higher amounts than usual, as shown in Figure 5.
It was found that these did not substantially affect the measurement results.

[Part 2 - Reducing Substances in Blood] Figure 6 shows the results for fructose, vitamin C, and glutathione. It can be seen that there is virtually no effect in this case as well.

On the other hand, free glucose that is not bound to hemoglobin is ubiquitous in the blood as Jf11 sugar, and is expected to be at an extremely high concentration in severe diabetic patients (several times higher than in normal people). Therefore, regarding the interference effect of free glucose, P)l concentration, Triton X-10
FIG. 7 shows the results obtained by changing the combination conditions of 0 concentration. As shown in FIG. 7, at pH 12.5, where monomerization is possible without using Tri-ton X-100, some interference with M# glucose was observed. On the other hand, Tri
When 3% of ton X-100 was added and P)l was lowered to 11.5, there was no interference from free glucose;
NET reaction was also good.

[Part 3 - Substances related to sugar metabolism in red blood cells] Among the glycolytic intermediates contained in red blood cells, lactic acid, pyruvic acid, 2,3
-About glycerophosphoric acid, 3-glycerophosphoric acid Chapter 8
Figure 8 (B) shows the results for 2-glycerophosphate, glucose-6-phosphate, fructose-6-phosphate, and fructose-1,6-diphosphate.
It can be seen that there is virtually no effect in these cases as well.

[When using whole blood]

Although the use of separated and washed red blood cells has the advantage of removing blood leaf components and measuring only glucose bound to hemoglobin, it has a major disadvantage in terms of simplicity as a clinical test. When whole blood samples are used, in addition to glucose bound to hemoglobin, glucose bound to proteins as shown by R.H. It is understood to reflect harmony. Conventionally, such a measurement method has not been put into practical use, and the present invention provides a new index.

FIG. 9 shows the results of measuring the loOgM of 50 pL of whole blood containing EDTA with 1 mM of water added thereto in the same manner as in Example 2, and comparing it with the HbAt value determined by the column method. Thus, the absorbance change amount x2 in this example correlated well with the HbA+ value, and was recognized to be highly useful as a clinical index.

(Effects of the Invention) As described above, the present invention does not require a hemoglobin elution operation as in the conventional column method, and requires heating for several hours at a high temperature of 100°C as in the TBA method. Since the J index value can be calculated by mixing reagents and measuring absorbance without any work, automation of the measurement is easy and the measurement work can be carried out efficiently. Further, when carrying out the present invention, there is no interfering effect due to sodium fluoride or other substances that may be present in the sample, and it is easy to put it into practical use. In addition, the present invention measures the concentration of glucose itself, which has become a stable ketone form by binding multiple units to hemoglobin, rather than so-called glycosylated hemoglobin HbA, so the amount of the substance to be detected is large. Therefore, measurement accuracy is improved.

[Brief explanation of drawings]

FIG. 1 shows the absorbance curves of hemoglobin, NBT, and NBT after reduction, which are the premise of the present invention. FIG. 2 shows the absorbance curves of the present invention when the combination conditions of surfactant and PH are changed. FIG. 3 is a diagram showing the measurement results when implementing the present invention and the results by the conventional column method, FIG. 4 is a diagram showing the influence of hemoglobin concentration in the present invention, FIGS. 5, 6, Figures 7 and 8 respectively show blood anticoagulants, reducing substances in blood, free glucose in blood,
A diagram showing the results of investigating the interfering effects of sugar metabolism-related substances in red blood cells. FIG. 9 is a diagram showing a comparison of the measurement results when the present invention was carried out on whole blood samples and the results by the conventional column method. . l...Absorbance curve of NET, 2A...Absorbance curve of hemolyzed sample (PH11, 5 or less), 2B...Absorbance curve of hemolyzed sample (PH-12, 0), 2C...Absorbance curve of hemolyzed sample - Light curve (PH12,5), 3A... Absorbance curve of hemolyzed sample + NBT (PH10,8), 3B...
・Absorbance curve of hemolyzed sample + NBT (PH12, O), 3
C... Absorbance curve of hemolyzed sample + NBT (PH12,5
), 4...absorbance curve when the hemolyzed sample is diluted with water,
5゜6...Absorbance curves at PH11,0 and PH11,5 when a surfactant is added to a hemolyzed sample Patent applicant Masaru Wakata, Patent attorney, Sogo Biomedical Research Institute Co., Ltd. Figure 2) 1bA1 (z) (cuff d+=, r; C: (I) Huppict-A (n) Big'n C (In) Evening end + on Fig. 7 (1) Triotna x -tpoiJ'Jt, -(P,
l, 25) (Dori Ri (P rain 2, O) <1[') f (1'l-1+0.f) (W) Trr
tc++c ×-+oo4e issue/, /i<, (1 in 1'
．． Figure 8 (A) (II) Pill-bottle can, 0n) 2,3-Grisero = Gin- ( (■) 3-Goo) Loli Mimasaki (B) (1) 2-da 2 and chestnut L bite (2) gu IV cor 6-li L
71--S1,6-Nira Sakaki Ryo Figure 9 HbAl (Phantom Kakumu Hiro Continuation of page 10 Inventor 50 Hiroshi Arashi On-site in Nakano-ku, Tokyo Inventor Supervisor Seishin On-site in Nakano-ku, Tokyo 0 Inventor Toshisada Ishido Inside Nakano-ku, Tokyo

Claims (1)

[Claims] By mixing NET and the dust solution in a liquid composition that can monomerize hemoglobin, which is composed of tetramers, the NBT is reduced by glucose bound to hemoglobin as fructosamine, and the
A method for measuring glucose bound to tosulhemocrohin, characterized in that absorbance is measured with specific absorption at 00 to 600 nm reduced or eliminated, and an index value indicating glucose concentration is calculated from the absorbance.