JPS5832599B2 - Differential determination method for lactate dehydrogenase isozyme - Google Patents

Description

[Detailed Description of the Invention] In recent years, in the field of clinical testing, progress has been made in elucidating various diseases and enzyme activities in body fluids such as phlegmon, and it has been found that certain enzyme activities in oil serum become abnormal in certain diseases. It has become known that it increases in height and then disappears again.

For example, human lactate dehydrogenase (rLD)
The activity (abbreviated as Hj) is very low in normal people, but it increases markedly in patients with malignant tumors, myocardial infarction, hepatitis, etc.

In addition, since it changes characteristically as various diseases worsen or improve, it is extremely useful for monitoring the progress of diseases and determining the effectiveness of treatments.

Furthermore, it is known that the isozyme composition of enzymes of the same type exhibits characteristic variations depending on the type of disease.

The aforementioned human LDH consists of two types of enzymes (subunits), H type (cardiac type) and M type (skeletal muscle type), and it is known that under normal conditions it exists as a tetramer of these subunits. ing.

Since there are two types of subunits, there are five types of combinations that can form a tetramer.

The polymerization is random bonding, and the composition of the polymer is H
It is said that it is determined by the abundance of M type and M type.

LDH in oil serum increases due to LD from diseased organs.
This is because H is eluted, and in the case of hepatitis, the LDH eluted into the oil serum is mostly M-type isozyme originating from the liver, and in the case of myocardial infarction, the H-type isozyme originating from the heart accounts for the majority. .

Therefore, by differentially quantifying the LDH isozyme eluted in the oil serum, it is possible to estimate the organ affected by the disease.

Conventional methods for separately quantifying LDH isozymes include methods of separating them by electrophoresis and then quantifying them, methods of separating them using an ion exchanger and then quantifying them, and methods that utilize the difference in thermal stability to select isozymes that are more unstable to heat. There are methods such as inactivation and quantitative determination.

However, these methods have drawbacks such as the need for a separation process, which takes time and effort, and the setting of processing conditions is delicate, resulting in poor accuracy.

The present invention relates to a method for directly, quickly and easily fractionating and quantifying ume pickles without separating LDH isozymes while they coexist.

That is, the present invention provides reduced nicotinamide adenine dinucleotide (r
A method for differentially quantifying LDH isozymes is provided, which is characterized in that an LDH activity inhibitor produced from NADH is contained in a reaction solution for measuring LDH activity using NADHJ (abbreviated as NADHJ) as a coenzyme.

The LDH activity inhibitor referred to herein is a substance having a structure similar to nicotinamide coenzyme, which is produced from NADH when NADH powder or aqueous solution is stored for a long period of time.

(46th Annual Meeting of the Japanese Biochemical Society: Yamauchi et al.
Regarding DH reaction inhibitors") It is widely known that these inhibitors exhibit a very strong inhibitory effect on LDH activity.

(Biochimica et Biophysica
Acta.

Vol, 54. p, 210 (1961); 5
candinavian Journal of C1
1nical Laboratory Investig
ation, Vol. 33. Supplem
(1974)) It is known that the degree of inhibitory action of the inhibitor differs for LDH of different origins; however, (Quality
Control of C1inicC11nica
lChe, Transactions ofInte
National Symposium, 6th
;Edited by Anido, Van Kam
pen.

Rosalki; de Gruyter, Be
rlin, pa 291° (1975)) The relationship between the degree of inhibition of LDH isozyme activity and the pattern of LDH isozyme has not been elucidated. They discovered that there is such a regularity and completed the present invention based on this new knowledge.

Here, various active factors for samples containing a mixture of LDH isozymes are shown in the following table.

That is, AH, AM, and A represent the activity when using a normal reaction solution that does not contain the above inhibitor when measuring the activity,
A'H2XM and A' each represent the LDH activity when a certain amount of the above inhibitor is present in the reaction solution.

Also RH=AH/A RM=AM/A P = A7A PH=A'H/AH PM=A’M/AM Then, The following relationship holds between P and RH.

P = A7A = (A'H + AM) / A = (PHAH + PMAM) / A = PHRH + PMRM = PHRH + PM - PMRH = (pH - PM ) RH + RM ......
(1) Set This relationship shows that, as shown in the nomograph in Figure 1, if P is plotted on the vertical axis and RH is plotted on the horizontal axis, the P ring intercept is PM.
The gradient is represented by a straight line of (PHPM).

P is the activity ratio when a certain amount of the reaction inhibitor is added, and RH is the activity of H-type LDH that accounts for the total LDH activity.

As a result, a reaction solution containing no activity inhibitor (reaction solution 1), a reaction solution containing a certain concentration of an LDH activity inhibitor (reaction solution 2), and two types of LDH samples whose isozyme compositions are known in advance. While creating a standard line using
By applying the obtained activity ratio P (A7A) to the standard line, we get
Its isozyme composition can be determined separately.

P changes depending on the concentration of the LDH activity inhibitor, but is also greatly influenced by the concentration of pyruvic acid, which is a substrate for the reaction, the concentration of NADH, and pH.

Therefore, in order to obtain highly accurate RH, it is desirable to set conditions such that the difference between PM and PH is as large as possible.

In clinical tests, it is desirable that the difference between PM and PH be 50% or more.

The measurement method may be a normal method for measuring LDH activity, and no special equipment or operation is required.

As described above, according to the method of the present invention, LDH isozyme can be easily differentially quantified using the same operations as those for measuring normal LDH activity.

Example 1 Purified porcine LDH as a sample (subunit:
HHHH) (manufactured by Boehringer), porcine LDH5 (subunit: MMMM) (manufactured by Boehringer), and a mixture of these in the ratios shown in Table 1 were used.

The reaction solution was 0.05M phosphate buffer (pH 7.5),
1mM EDTA, 0.16mM NADH and 0.
Reaction solution containing 3 mM sodium pyruvate (reaction solution 1
) and NADH as an LDH activity inhibitor in reaction solution 1.
-Inhibitor-■ (manufactured by Oriental Yeast Co., Ltd.) was added at 1.1 units/- (reaction solution 2).

However, here, 1 unit of inhibitor activity is the concentration of inhibitor per rnl that inhibits 50 LDH reactions.

To 3rnl of each of the above reaction solutions, add 5μ of the enzyme preparation (protein] -00μ/-) shown in Table 1,
The reaction was started at °C, and the decrease in absorption at 340 nm was measured by spectrophotometry.

The results are shown by black circles in Figure 2.

This is pig LDH. (H type 100+) and pig LDH5 (M
It matched well with the nomograph (straight line in the figure) obtained from equation (1) from the 100 types.

[Brief explanation of drawings]

FIG. 1 is a nomograph for calculating the fraction of H-type isozyme from the degree of inhibition of LDH activity. Figure 2 shows that various samples with known isozyme compositions are
This is a graph in which the activity ratio P when a DH activity inhibitor is applied and when it is not applied is plotted against the fraction of H-type isozyme.

Claims (1)

[Claims] 1. A lactate dehydrogenase activity inhibitor produced from reduced nicotinamide adenine dinucleotide is contained in the reaction solution when measuring lactate dehydrogenase activity using reduced nicotinamide adenine dinucleotide as a coenzyme. A method for differentially quantifying lactate dehydrogenase isozymes.