JPH0543359B2 - - Google Patents

Description

[Detailed description of the invention]

[Industrial Field of Application] The present invention relates to a reagent for quantifying lipase, and particularly to a reagent for quantifying lipase by turbidity measurement. [Prior Art] In clinical diagnosis, quantifying lipase activity in serum or urine is useful for diagnosing pancreatitis and the like. Lipase is an enzyme that cleaves the ester bonds of triglycerides containing fatty acid residues and decomposes them into diglycerides, monoglycerides, and fatty acids.
Various methods are known and actually used to measure lipase, including a titration method, a so-called turbidimetric method for measuring turbidity, and a colorimetric method using a synthetic substrate. however,
The titrimetric method is partially difficult to handle,
Because of the long reaction time and the need for a large amount of sample, it is not widely used in routine clinical chemistry tests. Colorimetric methods using synthetic substrates have the drawback of lacking specificity, quantitative performance, and the like. on the other hand,
A nephelometric method, in which lipase activity is followed by photometric clarification of the turbidity of triglyceride-water emulsions, largely avoids the above-mentioned drawbacks. However, in the turbidimetric method, colipase and urea must coexist as essential components.
The component removes the enzyme inhibition caused by the high content of bile hydrochloric acid and improves the linearity of the reaction progress, as well as influences the water-oil surface of the emulsion and stabilizes the emulsion. According to
See Publication No. 28275]. That is, if the component is not present, the reaction will not proceed linearly, and the enzymatic reaction will be inhibited. However, when using colipase, measurements can only be carried out at a relatively low temperature of 25 to 30°C, and colipase also has the drawbacks of poor stability and high cost. [Object of the Invention] An object of the present invention is to provide a lipase quantitative reagent that does not contain colipase and urea and can be used in the measurement of lipase by turbidimetry. [Structure of the Invention] The gist of the present invention is (a) liquid triglycerides, (b) at least one anionic surfactant selected from the group consisting of alkyl sulfates and alkyl sulfonates, (c) bile acids or The present invention provides a reagent for quantifying lipase by turbidimetry, which comprises an alkali salt thereof, (d) calcium ions, and (v) a buffer that maintains a pH of 7 to 10. A feature of the present invention is the presence of an anionic surfactant in the reaction system in a turbidimetric method in which lipase activity is monitored by photometrically measuring the clarification of turbidity in a triglyceride-water emulsion. By the way, in the method of quantifying lipase activity by colorimetry using S-acyl ester as a substrate,
Albumin in serum inhibits lipase activity when present in excess, but this inhibitory effect is known to be restored by the addition of anionic surfactants (mainly SDS) [Japanese Patent Publication No. 4559/1983] . However, according to the publication, the SDS concentration is 0 to 2.0.
In the mM range, bovine serum albumin (BSA) is 0.
When present at a concentration of ~2 mg/ml, lipase activity is
It has been shown to vary greatly depending on BSA concentration. Therefore, in the method of colorimetrically quantifying lipase activity using S-acyl ester as a substrate, the effect of albumin on lipase activity is not eliminated by the addition of SDS. In other words, when SDS is not added, lipase activity is
It is activated in the presence of BSA 0.25 mg/ml compared to when BSA concentration is added, but when BSA concentration is 0.5 to 2
mg/ml, lipase activity is in turn inhibited by BSA. However, with the addition of SDS, the reversal of the BSA concentration dependence of lipase activity is no longer observed, indicating that lipase activity increases as the BSA concentration increases. Therefore, it is considered that the effect of adding an anionic surfactant in the present invention is clearly different from the effect of adding an anionic surfactant in the method described in this publication using S-acyl ester as a substrate. To quantify lipase using the quantitative reagent of the present invention, first add bile acid or its alkali salt, an anionic surfactant, calcium ions, and a preservative if necessary to a known buffer such as Tris buffer. A solution of triglyceride (water-soluble organic solvent solution) is added to the prepared solution under stirring to form an emulsion to prepare a substrate. After keeping the substrate solution at a temperature slightly higher than room temperature (for example, 30 to 40 degrees Celsius) for a certain period of time (usually 5 to 10 minutes), a sample such as serum or urine is added, and changes in absorbance are measured using a photometer. This can be done by As a triglyceride, the number of carbon atoms is about 4 to 22
Preference is given to natural as well as synthetic triglycerides having fatty acid residues of . Among these triglycerides, triglycerides with long-chain unsaturated fatty acid residues that are not hydrolyzed by carboxylesterases and other esterases and are specifically hydrolyzed by lipases, especially those whose fatty acid residues are Triglycerides having 10 to 20 carbon atoms and 1 to 8, particularly preferably 1 to 3 carbon-carbon double bonds are preferred. In particular, triolein and olive oil are very suitable. Further, the triglyceride solution used as a substrate in the present invention is prepared as a solution of a water-soluble organic solvent, and the concentration of triglyceride is preferably 5 to 20 mM. As the water-soluble organic solvent, for example, ethanol is preferably used. Preparation of the substrate liquid emulsion may be carried out by adding the triglyceride solution under stirring to a buffer containing bile acids or bile salts, anionic surfactants, calcium ions, and optionally preservatives. The concentration of triglyceride in the substrate solution is usually 0.1 to 0.5 mM. Bile acids include known deoxycholic acid, taurodeoxycholic acid or its alkali salts,
For example, sodium salts are applicable. Its concentration is 0.1
~0.7% (weight/volume) is preferred. Anionic surfactants are chosen among alkyl sulfates and alkyl sulfonates, especially sodium and lithium salts. Among these, those in which the alkyl residue has 10 to 20 carbon atoms are preferred. In particular, it is most preferable to use sodium laurate (SDS), which has a large effect on improving the reaction linearity of lipase and is easily available. The amount of anionic surfactant added is preferably 0.01 to 0.5% (weight/volume). The concentration of calcium ions is 0.01-0.1
mM is preferred. As the buffer, all known buffers that can adjust the pH value to 7-10 can be used. A particularly good buffer is Tris-buffer, which
The M concentration is a particularly preferred buffer concentration. As a preservative, alkali azide, which does not inhibit the enzymatic activity of lipase, is suitable within the scope of the present invention. The preferred amount of preservative is 0.005-0.01% by weight
It is. The reagents for quantifying lipase by turbidimetry described above are preferably stored in a form depending on their properties and intended use. For example, triglycerides are best stored as solutions in water-soluble organic solvents;
Bile acids, anionic surfactants, calcium ions, and preservatives are preferably dissolved in a buffer solution and stored. [Effects of the Invention] According to the present invention, an anionic surfactant can be used without using colipase and urea, which are necessary components for improving reaction linearity in the conventional turbidimetric lipase determination method. The addition of bile acids or their alkaline salts improves the linearity of the reaction and makes it possible to quantify lipase activity in serum or urine specifically and with high sensitivity. Hereinafter, the present invention will be explained in more detail with reference to Examples. Example 1 Preparation of reagent (a) Substrate stock solution (10mM triolein) A solution of 10mM triolein in absolute ethanol is prepared and used as a substrate stock solution. (b) Deoxycholate (0.1-0.7%), SDS
(0.01~0.5%), calcium chloride (0.01~0.1m
M) and 25m containing 0.01% sodium azide
Prepare M Tris-HCl buffer (PH9.2) and use it as a buffer. In order to use these for quantifying lipase, a substrate solution is prepared as follows. i.e. buffer
Prepare the substrate solution by adding 5 ml of (a) substrate stock solution to 150 ml while stirring. This substrate solution is used for quantifying lipase. In addition, when quantifying a small number of samples, take out the required amount of buffer solution and substrate stock solution, prepare the substrate solution according to the above, and use it for lipase quantification. ~
Preferably, it should be stored at 8°C. Example 2 General quantitative method for lipase [Reagents] (a) Substrate stock solution (10mM triolein) (b) Buffer solution [Quantitative method] Add reagents (a) and (b) to a cuvette (3 ml volume) with an optical path length of 1 cm. Take 2.5ml of the prepared substrate solution and heat at 37℃ for 5 minutes.
Incubate for 1 minute, then remove an appropriate amount of sample (10μ for human serum).
) and measured photometrically against water at 37° C. at 340 nm. Measure the change in absorbance with a photometer, calculate the change in absorbance per unit time, and measure the lipase activity. Assuming that the absorbance of the substrate solution before mixing the sample is A sub, the absorbance after mixing the sample is A ₀ min, and the absorbance after 5 minutes is A ₅ min, the lipase activity per liter of sample is calculated using the following formula. Lipase activity (unit/L) = A ₀ min - A ₅ min/Asub × 1500 The unit of lipase activity described here is 1 at 37°C.
1 μmol of triolein per minute
It is taken as a unit. Comparative example A certain amount of lipase extracted and purified from human pancreas was added to human serum, and 100μ of the lipase was added to other surfactant (Triton
Lipase activity was measured in the same manner as in Example 2, except that 100, Breezy-35 and Tween-20) were added. As a result, these surfactants rather inhibited lipase activity, and the improvement in reaction linearity obtained by the addition of SDS was not observed. Example 3 A certain amount of human pancreatic lipase was added to human pool serum and 10 mM Tris buffer (PH
8) The lipase activity was measured in the same manner as in Example 2, except that the SDS concentration in the substrate solution was changed to 0 or 0.05% using each sample added to the substrate solution, and the results shown in Figure 1 were obtained. . As is clear from Figure 1, in the case of a 10mM Tris buffer sample, the rate of decrease in absorbance, that is, the lipase activity, was almost the same regardless of whether SDS was added or not, and the reaction proceeded linearly in terms of reaction kinetics. There is. However, in the case of serum samples, when SDS is not added, the reaction does not progress linearly, and the rate of decrease in absorbance decreases rapidly during the reaction, inhibiting lipase activity. On the other hand, when 0.05% SDS was added, such inhibition during the reaction was removed, and the reaction proceeded linearly, showing lipase activity equivalent to that of the buffer sample. It is understood that lipase activity in serum can be accurately quantified by adding Further, the sample in which lipase serum was measured was extracted with n-hexane and concentrated, and then the product was analyzed by thin layer chromatography using silica gel G.
As a result, diolein, monoolein, and fatty acids, which are degradation products of triolein by lipase, were confirmed in the sample with an SDS concentration of 0.05%, and the SDS concentration of 0% obtained in the serum-free buffer sample was confirmed.
It was the same as the decomposition product at the time of . Therefore, SDS
It was found that the decrease in absorbance of the substrate solution observed upon addition reflected lipase activity. Example 4 The samples were a fixed amount of human pancreatic lipase added to human pool serum and 10mM Tris buffer sample (PH8), except that the SDS concentration was varied and 3 μg of colipase was added or not. The lipase activity was measured in the same manner as in Example 2, and the results shown in FIG. 2 were obtained. As is clear from Figure 2, (1) SDS concentration 0%
The lipase activity of serum samples was suppressed to about 50% of that of 10mM Tris buffer samples, but SDS
(2) The suppression of lipase activity in the serum sample is released by the addition of , and lipase activity equivalent to or greater than that of the 10mM Tris buffer sample is expressed.
(3) The addition of SDS releases the suppression of lipase activity in the same way that the addition of colipase releases the suppression of lipase activity in serum samples at an SDS concentration of 0%. The lipase activity recovered by the addition of colipase cannot be further activated; (4) the SDS concentration is
It is found that the inhibition of lipase activity can be most effectively recovered within the range of 0.01 to 0.1%. Example 5 Human pancreatic lipase with various lipase activities was added to human pool serum or 10mM Tris buffer sample (PH8) as the sample, and Example 2
Lipase activity was measured in the same manner as above, and the results shown in FIG. 3 were obtained. As is clear from Figure 3, the measured lipase activity is proportional to the amount of lipase added;
Also, considering that there is no difference in lipase activity values between serum samples and 10mM Tris buffer samples, this measurement system specifically measures the lipase activity in serum samples and the added lipase activity. It can be seen that measurements can be made accurately with the so-called ideal addition recovery rate. Example 6 A fixed amount of human pancreatic lipase was added to human pool serum or 10mM Tris buffer as a sample, and the SDS concentration in the buffer was adjusted to 0% or 0.05%.
Lipase activity was measured in the same manner as in Example 2, except that a buffer solution containing bovine serum albumin (BSA) at a final concentration of 0 to 2 mg/ml was used in each case. The results were obtained. As is clear from Figure 4, when serum samples were measured at an SDS concentration of 0%, lipase activity was suppressed as the BSA concentration increased;
In Tris buffer samples, lipase activity is constant regardless of BSA concentration, as well as SDS concentration of 0.05.
% shows constant lipase activity for both samples, independent of BSA concentration. Therefore, the special public official 1983
Lipase activity as shown in Publication No. 4559 is SDS
It can be seen that a phenomenon that is largely dependent on the BSA concentration even when added is not observed in this measurement system. Example 7 Lipase activity in five human serum samples was measured using the reagent of the present invention (Example 2) and a commercially available kit (Boehringer Mannheim) (kit described in Japanese Patent Publication No. 57-28275). I got the following results.

[Table] From the above table, the correlation between this method and Boehringer was determined as follows: Y=0.283X-2.01 γ=0.991 (X: Boehringer, Y: present invention). Therefore, a very good correlation was obtained with a correlation coefficient of 0.991 between the lipase activity measured according to the present invention and the lipase activity measured by the Boehringer method. In addition, since the display unit of lipase activity is different between the present invention and the Boehringer method, the regression equation is
Considering that = 0.283X - 2.01, it is also possible to convert to international units (V) by multiplying the value obtained by this method by 3.5. Furthermore, the day-to-day reproducibility (n=10) of lipase activity measured according to the present invention is 6.0-13.7%,
The simultaneous reproducibility (n=10) was 2.1 to 9.2%, indicating that lipase activity in serum can be accurately measured by the present invention. Example 8 In normal serum samples, it is possible to quantify lipase activity by adding SDS as described above, but serum often contains substances that inhibit or interfere with lipase activity. It may be mixed. Such substances include hemoglobin (Clinical Examination 20, 75-77, 1976), high triglyceride specimens (Treatment 64, 179-183, 1982), emulsion specimens,
In addition, there are bilirubin samples with high values,
This measurement method was able to quantify lipase activity without being interfered with or inhibited by any of these substances.

[Brief explanation of the drawing]

Figure 1 is a chart of lipase activity measurement of serum and buffer samples with and without SDS in Example 3. Figure 2 is a diagram showing the effect of SDS concentration on lipase activity in Example 4. Figure 3 4 is a diagram showing the relationship between lipase amount and lipase activity in Example 5, and FIG. 4 is a diagram showing the influence of BSA on lipase activity in Example 6.

Claims (1)

[Scope of Claims] 1. (a) liquid triglyceride, (b) at least one anionic surfactant selected from the group consisting of alkyl sulfates and alkyl sulfonates, (c) bile acid or its alkali salt, (d) Calcium ion; (e) A reagent for quantifying lipase by nephelometry, which comprises a buffer that maintains a pH of 7 to 10. 2 The fatty acid residue of triglyceride has 10 carbon atoms
20. The reagent for quantifying lipase according to claim 1, having 1 to 8 carbon-carbon double bonds. 3. The lipase quantitative reagent according to claim 1 or 2, wherein the triglyceride concentration is 0.2 to 3.0% by weight. 4. The lipase quantitative reagent according to any one of claims 1 to 3, which contains triglyceride as a solution in a water-soluble organic solvent. 5. The lipase quantitative reagent according to any one of claims 1 to 4, wherein the alkyl residue of the alkyl sulfate and the alkyl sulfonate has 10 to 20 carbon atoms. 6. The lipase quantitative reagent according to claim 5, wherein the alkyl sulfate and the alkyl sulfonate are sodium salts or lithium salts. 7. The lipase quantitative reagent according to any one of claims 1 to 5, wherein the concentration of the anionic surfactant is 0.01 to 0.5 (weight/volume)%. 8. The lipase quantitative reagent according to any one of claims 1 to 7, wherein the bile acid or its alkali salt is deoxycholic acid or deoxycholate. 9 The concentration of bile acids or alkali salts is 0.1~
Claims 1 to 1.0 (weight/volume)%
The reagent for quantifying lipase according to any one of Item 7.