JPH02242160A - Measurement of lipo-bobded sialic acid content in blood - Google Patents

Abstract

PURPOSE: To make it possible to take a measurement of sialic acid content by removing neutral lipid by diluting serum with water and performing extraction using an organic solvent, redisolving lipoprotein division containing sialic acid after sedimentation, and adding resorcin and causing colorimetic reaction. CONSTITUTION: A sediment is produced at a pH value of 8-7.5 by using 10,000 to 20,000 MW dextran of 15 to 35mg/100ml in concentration in a solution as a preciptant, at a pH value of 9-11 by using 4,000 to 10,000 MW polyetheylene glycol of 15 to 55mg/100ml in concentration in a solvent, or at a pH value of 5.5-7.5 by using water-soluble manganese (II) of 8.7 to 13g/100ml in manganese ion (Mg<2+> ) concentration in a solution. During the sedimentation, manganese oxide (II) is more preferable. This salt is used with 20 to 30g/100ml, preferably, 22 to 27g/100ml concentration and deposited preferably at a pH value of 6.0±0.1. The deposited lipoprotein fraction is preferably redissolved by using buffer liquid of a pH value of 7-9.

Description

DETAILED DESCRIPTION OF THE INVENTION [Field of Industrial Application] The present invention relates to a novel method for measuring the content of lipid-bound sialic acid in blood, mainly human blood.

[Conventional technology]

Early detection of malignant diseases (hereinafter referred to as cancer) is a decisive factor in curability and is therefore one of the major challenges in therapeutic management. In this sense, the development of biochemical methods is very promising.

One of the main aims of cancer research is the study of human body fluids (blood, urine,
Endogenous substances may be discovered whose concentrations in the brain, cerebrospinal fluid, etc. change significantly and characteristically during the progression of cancer. Such substances are called tumor markers (hereinafter referred to as markers).

Although more than 50 types of substances with marker properties have been discovered to date, there are still none that are widely recognized and used as so-called "cancer testing methods." In other words, there is no laboratory-grade diagnostic method that provides a clear and completely reliable yes-or-no answer for detecting cancer.

One of the reasons for this difficulty is that for a large number of marker substances, the biological basis of measurement, in particular the correlation between tumor status and marker quantity, is not understood or is difficult to obtain. One example is that the results are ambiguous. Furthermore, the complex physical structure of key markers makes their isolation and measurement extremely difficult, and in the overwhelming majority of cases, is beyond the reach of existing laboratory routine equipment and personnel. It is impossible. Another reason is that as a result, measurements are extremely expensive and results are not sufficiently reliable.

Sialic acid (N-acetylneuraminic acid) is a component of membrane-forming glycolipids and certain glycoproteins, and is also a blood group substance.

When examining changes in the composition of cell membrane glycoproteins and glycolipids, the amount of gangliosides increases in tumor cells and their plasma membranes. Therefore, it has been observed that measuring changes in ganglioside levels can be used as a marker testing method [Yogoswaren, G.
varen): cell surface glycoprotein in malignant transformation (
Cell 5 surface Glycoproteins
in Malignant Transform
ion) J, Advances in Cancer Research, Volume 38 (
1983), l5BN:0-12-006638-
6. Published by Academic Press].

However, currently known methods for the analytical determination of gangliosides are very complex and require a great deal of time and effort. Therefore, it is virtually impossible to use it as a routine method in clinical laboratories.

The amount of sialic acid in serum, i.e. lipid-bound sialic acid (
The concentration of LSA (a type of sialic acid fraction) has a strong correlation with the amount of gangliosides. This component can be detected using relatively simple analytical methods. Several researchers have already investigated and confirmed the applicability of LSA as a tumor marker in real clinical cases.

Katopodis et al.
Cancer Res, Volume 42 (198
2 years), pp. 5,270-5.275] of 850 subjects (670 with clinically confirmed cancer and 180 with confirmed non-neoplastic disease).
The researchers measured the amount of LSA in samples taken from cancer patients and found that there was a significant difference in the LSA measurements in the cancer patient group compared to the non-tumor group.

In 97% of samples collected from cancer patients, the LSA value was 20 mg/100 m.The average value per eye was 26.3 mg/1.
In contrast, the average LSA amount in the non-tumor group consisting of healthy people and patients with non-neoplastic benign diseases was 17.6*/100 m Q. The authors considered LSA values above 20 mg/100 mQ to be pathological. In addition, the authors believe that the measurement of LSA values is
The progress of the disease, i.e., is it getting better?
He acknowledges that it can also be applied to track whether things are getting worse.

Shamberger (R,S) [Journal of Clinical Chemistry and
Biochemistry (J, C11n, Chem, Bio
chem, ), vol. 22 (1984), 647-65
Page 1] for 134 non-neoplastic subjects (healthy subjects and patients with benign diseases) and 160 cancer patients.
The amount of sialic acid in serum was investigated.

In 94% of cancer patients, serum sialic acid concentrations were pathologically high. Among this group, sialic acid levels in 63 patients with metastases were significantly higher than those in patients without metastases, and there is a correlation between tumor stage and size and serum sialic acid concentrations. It was shown that a relationship exists. In 10 cancer patients, the authors compared the widely known carcinoembryonic antigen (CEA) test and sialic acid measurement to determine their effectiveness in diagnosis. Pathologically high CEA values were observed in only 4 patients, whereas all 10 patients had increased sialic acid levels. In addition, certain inflammatory diseases (arthritis, psoriasis, ulcers, etc.) were also accompanied by an increase in the amount of sialic acid.

Donistrian (A, M, Dnistrian) et al. [Clinical Chemistry (C1in, Chew,), 2
Volume 7, No. 10 (1981), pp. 1.737-1,739; Cancer, Volume 50 (198)
2), p. 1,815] investigated LSA as a tumor marker in breast cancer. The authors saw a significant increase in LSA values compared to a healthy control group. Furthermore, LSA in patients treated using various methods
We investigated changes in values and found that there was a correlation between the progress of the disease and changes in LSA values. The authors also. LSA and CEA values of serum samples obtained from 125 patients with clinically confirmed cancer were compared. patient 78
% had pathologically high LSA concentrations, whereas CEA concentrations were only high in 35% of the same patients.

According to the above-mentioned publications, measurement of LSA values is considered to be well applicable for indicating the presence of cancer, estimating the stage and size of tumor progression, and monitoring the progress of the disease. Ru.

Although many methods have been devised for measuring LSA content in human blood, there are relatively few methods that can be performed continuously and routinely under average clinical laboratory conditions. The only simple method is that disclosed in US Pat. No. 4,342,567.

According to this method, serum is diluted with distilled water, the diluted serum is extracted with a mixture of chloroform and methanol to remove the lipophilic fraction, and then phosphotungstic acid is used to remove the lipophilic fraction. Treat the phases, dissolve the separated precipitate in water, and add hydrochloric acid, copper(II) salt, and resorcinol to the aqueous phase.

Photometrically, based on the colorimetric reaction that occurs.

Measure the sialic acid content in serum.

[Problem to be solved by the invention]

The inventors attempted to repeat the method described above, strictly following the recipe of the instructions. Serum samples from three subjects were each analyzed in 30 parallel tests, and this analysis was repeated on four consecutive days. As a result, it was found that there was a very high deviation (20-30%) between the results of 30 measurements performed on the same serum sample. this is.

This is an unacceptable result for clinical chemistry purposes.

Reproducibility between different measurement days is similarly poor;
The deviation of the average value exceeded 20%. Furthermore, this method includes
The slope of the absorption vs. concentration curve is decisively gradual, resulting in its average and routine use in clinical laboratories. With a photometer with an accuracy of o, ooos angstrom units.

The drawback is that measurements cannot be made without large errors.

Studies using blinded samples (i.e. samples consisting of distilled water instead of serum) have shown that, in particular, LSA concentrations of 20 mg/
For samples below 100 m Q, the effective signal/noise ratio is 3:1, which is accepted as a critical limit of measurability.
was shown to be below. In such a situation,
It is likely that the method of US Pat. No. 4,342,567 has never been put into practice.

When considering the individual steps of the method described in US Pat. No. 4,342,567, it was found that the precipitation of the sialic acid-containing fraction has a decisive role for the measurement accuracy. . According to US Pat. No. 4,342,567, the phosphotungstic acid used as a precipitant is not able to selectively precipitate the sialic acid-containing fraction. In addition to the target fraction, other interfering protein fractions are similarly precipitated and denatured.

This can be seen from the fact that even in the cited literature it is not possible to completely redissolve the precipitated protein fraction. Control of the quality and quantity of the interferingly acting protein fraction is not possible.

Therefore, the measurement results will be distorted under uncontrollable circumstances. As the biggest defect, U.S. Patent Nos. 4 and 3
No. 42,567, it is not clear which fractions should be separated from the multicomponent proteins present in human blood, whose structure and composition vary widely; therefore, the precipitation results are independent. It cannot be confirmed using other methods.

[Means to solve the problem]

The inventors first devised the centrifugation method as an independent method for determining the properties of the protein fractions to be examined. The ultracentrifugation applied to this is performed under the following conditions.

Temperature: 20°C Volume of sample centrifuge tube: 11.5 mfi Density gradient: 8.5 mfi of 0.9% by weight sodium chloride aqueous solution as the upper layer, serum (specific gravity = 1.3 g/
ml) 3m12 Rotation speed: 50,0OOr, p, m.

Rotation time: 120 min Kinematic viscosity (K'): 68.7 The theoretical basis of ultracentrifugation is that individual protein and lipoprotein fractions are separated at different speeds depending on the density of their hydrated forms. The result of the floating is that they separate from each other to form individual, distinct bands.

The fraction targeted for LSA quantification is separated as the fourth band from the meniscus. This zone is quantitatively removed from the centrifuge tube using a syringe.

It can also be used for various subsequent measurements.

Thus, according to ultracentrifugation, the fraction targeted for LSA quantification can be measured very accurately.The physicochemical properties of the separated fraction are as follows.

Density: 1.125-1.210g/mQ Molecular weight: (
3,86-1.86) It has high accuracy and is used as a reference method to confirm the reliability of chemical precipitation methods.

Furthermore, the inventors intend to devise a method for selective precipitation of LSA-containing fractions, which can be selectively and quantitatively precipitated by any of the following methods. I discovered something.

(b) pi (5.8 to 7.5, 15 to 35 g
/100m Q of dextran sulfate (molecular weight io, oo
o ~ 20,000) solution is used.

(b) At pH 9-11, 15-55 g/100+
* Polyethylene glycol of Q (molecular weight 4,000~
10,000) using a solution.

(c) p) 8.7 to 13 g at 15.5 to 7.5
A water-soluble manganese (II) salt solution corresponding to a manganese ion (Hn2*) concentration of /100 mQ is used.

In addition, the sialic acid content of the precipitated fraction was determined by U.S. Pat.
Using the colorimetric reaction described in No. 342,567,
It has also been found that measurements are reproducible and can be made with very high precision. When the precipitate is redissolved using a buffer solution with a pH of 7 to 9, measurement accuracy can be improved.

Based on the above, one aspect of the present invention is to dilute serum with water and then
removing neutral lipids by extraction with a water-immiscible organic solvent or solvent mixture; precipitating a polyprotein fraction containing lipid-bound sialic acids from the aqueous phase; and precipitating the precipitated fraction. Calculating the content of lipid-bound sialic acid in the serum based on the steps of redissolving, adding an inorganic acid, a copper (II) salt, and resorcin to the solution, and the colorimetric reaction caused by the resorcin. The present invention relates to a method for measuring the content of lipid-bound sialic acid in blood, mainly human blood, including steps.

In the present invention, (a) as a precipitant, the concentration in the solution is 15 to 35 mg/
100mj) molecular weight of 10,000 to 20,0
00 dextran and generate a precipitate at pH 5.8 to 7.5.

(b) As a precipitant, the concentration in the solution is 15 to 55 mg/
Molecular weight of 1001 4,000 to 10,000
(c) using manganese ion (Mg") in the solution as a precipitant, or
”) using a water-soluble manganese(II) salt in an amount such that the concentration is 8.7 to 13 g/100ml, and at a pH of 5.5.
A precipitate is formed at ~7.5.

According to the present invention, the precipitated lipoprotein fraction is purified from pH 7 to
Preferably, the precipitate is redissolved using a buffer solution of No. 9.

Particularly suitable is manganese sulfate (■
), manganese(II) nitrate, manganese(II) chloride.

Using water-soluble manganese(II) salts such as manganese(II) bromide and manganese(II) iodide, inter alia,
Manganese chloride (■) is suitable for the -layer. This salt has a concentration of 20 to 30 g/100m Q, preferably 22 to
27g/100m Used as Q, pH 6.0±0.1
Preferably, the precipitate is generated at .

To redissolve the precipitate, add 10 mmol/Ω tris(hydroxymethyl)aminomethane buffer solution (pH=8.6
) has been found to be particularly suitable.

It is known that when serum is stored at -20°C, the LSA content remains unchanged for 6 months. It is also known that the color intensity (proportional to LSA concentration) of colored complexes formed using resorcin does not change within 6 months at room temperature. This is an essential element for continuous analysis.

The method of the present invention is mainly applied to diagnosis for the purpose of rapid preliminary examination of cancer in humans, and is performed continuously and monitors the progress of healing of cancerous disease and evaluates the treatment method used. can be lowered.

However, the method of the present invention can also be applied to pharmacological tests aimed at cancer research, such as evaluation of the effects of drugs expected to be antitumor agents.

〔Example〕

Hereinafter, the present invention will be described in detail with reference to examples that do not limit the present invention.

Venous blood is used to measure the LSA content in human blood. 4.00 for blood sample after collection
Or, p, s, centrifugation for 15 minutes, sample 0.6
■Freeze U. The concentration of lipid-bound sialic acid in samples stored at -20°C remains unchanged for 6 months.

Before starting the analysis, wash the test tubes thoroughly with methanol, dry them, and keep them cool in the freezer. Each test tube contains 0.2 mm of serum sample.

and inject 0.2Ω of double-distilled water. The mixture is stirred for 15 minutes using a Vortex stirrer and then placed in an ice-cold water bath (0° C.). Two parallel samples are used from each of the test serum and control groups.

3 mQ of a pre-cooled mixture of chloroform and methanol in a volume ratio of 2:1 is injected into all test tubes and the contents of the test tubes are stirred for 30 seconds using a Portex stirrer. The test tube is then placed in the ice-cold water bath again.

Add 0.0 ml of double-distilled water to each sample, pre-cooled to +4°C.
Add 5+Q and transfer the contents of the test tube to a Portex stirrer.
Stir for 15 seconds. The mixture is then allowed to stand at room temperature for 5 minutes.

The sample was then centrifuged at 3,0 OOr, p, m for 1
Carefully inject 1 mg of each supernatant into numbered centrifuge tubes, add 0.1 ml of manganese(II) chloride solution to each, stir the mixture using a Portex stirrer, and then Let stand for 5 minutes. Manganese chloride (
II) The method for preparing the solution is as follows.

25g of anhydrous manganese(II) chloride in 80ml of double distilled water
The pH of the solution was adjusted to 6.0 ± 0.1 using a 0.1N aqueous sodium hydroxide solution or a 0.1N aqueous hydrochloric acid solution, and the volume of the solution was distilled twice. Make Loom Q using water.

In a further series of tests, dextran sulfate is used as a precipitating agent. 20 μg of dextran sulfate with a molecular weight of 15,000 was added to 100 μg of a 0.9% by weight aqueous sodium chloride solution.
Dissolve 1 mu of the prepared solution in mQ, supernatant 1
mj2 and the mixture is stirred for 15 seconds using a portex stirrer and then left to stand at room temperature for 5 minutes.

Furthermore, a polyethylene glycol solution is used as a precipitant in a series of tests. 20 μm of polyethylene glycol with a molecular weight of 6,000 is dissolved in 0.2 mol/a glycine buffer 80a+Q, and a solution is prepared using a 0.1 N aqueous hydrochloric acid solution or a 0.1 N aqueous sodium hydroxide solution. The pH of the solution is adjusted to 10.0±0.1 and the volume of the solution is brought to 100 mA using double-distilled water.

Add 1 mA of the prepared solution to 1 mQ of supernatant, stir the mixture using a Portex stirrer, and then leave it at room temperature for 5 minutes.

At the end of the standing period, add 4.0 OOr to the contents of each centrifuge tube.
, p, m, then centrifuge for 15 min.

Gently drain off the supernatant to completely separate it from the precipitate (discard the supernatant).

Inject 1 mff of Tris buffer into each centrifuge tube to remove the precipitate.
Redissolve in this. Tris buffer is prepared by dissolving 10 mmol of tris(hydroxymethyl)aminomethane in double distilled water IIIQ and adjusting the po of the solution to 8.6.

The method for preparing standard samples and blind samples for photometric measurements is as follows.

50 μg of analytically pure sialic acid in double distilled water 100 μl
Dissolve it in mQ, use this solution as a stock solution, and dilute it two more times with double-distilled water (concentration is 25 x 7100 mQ).
, and 12.5 mg/room Q tonal) -@
The quasi-solution is stored at +4°C.

0.2 IIQ of each of these three concentration standard solutions is injected into a centrifuge tube as a sample, and the volume of the solution in each centrifuge tube is adjusted to 1+aΩ using double distilled water. 1 ml of double distilled water is used as a blind sample.

Add 1 ml of each reagent grade resorcinol solution to all samples (standard sample, blind sample, control sample, test serum).

The iIJ manufacturing method of the resorcinol solution for reagents is as follows.

2 g of analytically pure resorcinol was added to double distilled water Lo
Dissolve in om Q. Anhydrous copper sulfate (It) 2.49
g is dissolved in 100 w n of double-distilled water. Then, add 10+s Q of resorcinol solution and 0.0s of cupric sulfate solution.
25 mQ, and 9.15 rsQ of double-distilled water.
100+w Q of analytically pure concentrated aqueous hydrochloric acid solution is added to this mixture.

Place the test tube in a 100°C water bath for exactly 10 minutes. All test tubes are then immediately immersed in an ice-cold water bath at 0° C. and left there for 10 minutes. 2 .OMEGA. of a mixture of butyl acetate and n-butanol in a volume ratio of 85:15 are injected into each test tube, and the contents of the test tubes are stirred for 5 minutes using a Portex stirrer.

The mixed solution was left to stand while stirring occasionally, and the sample was heated at 2.500 r.
, p. 11. centrifugation for 10 minutes, then.

The supernatant, which has a blue color, is transferred from each centrifuge tube to a measuring cuvette; the intensity of this blue color does not change within 6 months.

Photometric measurements are performed using a wavelength of 580 rt+s. Adjust the zero point of the photometer to match the double-distilled water. after that,
Depending on the type of photometer (single beam or dual beam), measurements can be made on a blind sample, or the absorbance of the blind sample can be calculated from the measured absorbance of the test sample using the formula below. Find the correction value by subtracting, etc.

Corrected absorbance=measured absorbance−absorbance of blind sample LSA content is calculated using one of two methods:

(1) From the results obtained using a standard sialic acid sample with a known sialic acid concentration, the absorbance versus concentration curve is shown on the graph as
Or it can be obtained by calculation. Since there are stages of dilution, the original concentration must be recalculated according to the table below.

100 0.50825
50 0.25212.5
25 0.127
From the calibration curve prepared as described above, the sialic acid content (A) of the supernatant sample in a volume of 1 m 12 can be determined. This curve shows the sialic acid content in μg of sialic acid per mfi of supernatant.

The sialic acid content in serum is determined by the formula % (where A means the sialic acid content in the supernatant in μg/mQ, and 0.72 is the dilution and the change from μg to It is a coefficient consisting of the conversion rate.).

(2) In the second calculation method, a reference serum sample with a known LSA concentration is used. From several parallel measurements, the formula % (%) (where C (ref) is the LSA content of the reference serum expressed in mg/100 ml, and E (ref) is the absorbance of the reference serum ) Calculate the coefficient F by

The LSA content of the test sample can be calculated from the formula % (where E(m) means the absorbance measured in the test sample).

The reliability of the measurements was confirmed using the ultracentrifugation method described above. LSA of serum samples collected from 15 different subjects
content by ultracentrifugation (IIC), manganese(II) chloride
11!2 (MnC1□), dextran sulfate precipitation (DS), polyethylene glycol precipitation (PEG), and U.S. Patent No. 4,342,5.
It was measured by the method described in No. 67 (known). The results are shown in Table 1.

(Margin below)! -yo one number 2 19.0 18.8 19,1 19.5
2183 169 16.6 16.5
1? , 1 1914 21.0 22.0 2
3.1 20.1 2655 195 19.
0 1g, 4 21.0 1516 30.8
31,3 32.1 29.1 2427
256 25.0 24.2 27.1 1
828 26.9 27.1 25.6 2g
, 0 2029 14°9 L4.3 16
．． 0 15.5 17910 23.5 21
．． 6 22.4 23.7 18.711 2
0.3 19.6 1g, 7 21.6 24
．． 412 16.1 16.4 18.0 1
7.3 12.813 18.3 18.3
16.7 1? , 1 23.514 28.5
29.3 31.2 26.9 21.8 1st
According to the data shown in the table, 1 school was selected as the standard. The data obtained in the ultracentrifugation method, which provides extremely accurate data, has a very strong correlation with the results obtained by the method of the present invention.

The best results are obtained when manganese(II) chloride is used as the precipitating agent, and the correlation coefficient for this method is 0.995. In contrast, the results obtained using the known method differ significantly from those obtained in the reference measurements.

In order to test the reproducibility of the method of the present invention within the same laboratory, the LSA content of Hyland's normal reference serum was measured again every day for 30 days. Statistical evaluation of the results showed that the reproducibility in different measurement series was better than ±10% and was acceptable.

Several hundred serum samples were analyzed in two different laboratories to test reproducibility between different laboratories. The basic characteristics of the analyzes (personnel, type of equipment, etc.) are all different. Comparison of the results showed that when applied to the same reference serum, the reproducibility between different laboratories does not exceed a limiting precision of ±10%.

Based on experience with hundreds of LSA measurements, it has been found that a well-trained inspector is able to perform at least 80-100 measurements per working day without any reliance on machines.

The applicability of the method of the invention was tested in a real clinical case. The results were classified according to the 5th order of isoastometry.

Normal value: 18m (/100m m or less Boundary between normal value and abnormal value = 18-20mg/100mQ Abnormal value: 20mg
/100aΩ or more (1)-2I! However, L was conducted on 115 healthy people or patients with benign diseases.
From SA measurements, in 95% of cases the test value is 20m
g/100+++ Q or less. In five patients actually receiving treatment for pneumonia, LS
Test value of A is 20-25mg/100I! /l, fully corresponding to the findings described in the literature.

The LSA test values of 94 patients with various malignant diseases (lung cancer, colon cancer, gastric cancer, kamaru cancer, breast cancer, laryngeal cancer, etc.) were measured in parallel with the measurement of their tumor marker substances (neopritene, polyamines, etc.).

In the - group of patients (30 patients), measurements were taken during the period of clinically confirmed disease activity.

Pathologically high LSA values were measured in each patient, apart from one patient who had advanced liver cancer.

The remaining patient group (64 patients) had their tumors surgically removed several years ago. In this group, the goal of testing was to determine whether the patient remained tumor-free or had any indicators of recurrence.

In 20% of patients (13 patients), both the LSA values and the values obtained for other markers invariably showed signs of malignant disease or an active phase. These patients were diagnosed as ``likely to have active tumor progression.''

In 25% of patients (16) both LSA values and values obtained for other markers showed equivocal results. In other words, while some values were normal, others were pathologically high.

These patients are recognized as “suspicious cases” and
The doctor recommended more frequent tests and the use of other diagnostic tools, while alerting the doctor to the possibility of inflammation or other non-malignant conditions.

In 55% of patients (35 people), LSA values and other marker values were within the normal range;

It was assumed that there was a high probability that the tumor did not exist.

(2) Examination of β I Among malignant gynecological tumors, we particularly examined ovarian cancer. Patients with a complete and detailed case record (including histological findings, surgical procedure, type and time of therapeutic intervention) were included, which would allow a realistic determination of LSA's behavior as a tumor marker. This is because it can be lowered.

L in 142 patients with histologically confirmed ovarian cancer.
SA values were measured preoperatively and then an average of 5 to 7 tests were performed in conjunction with various therapeutic interventions. L.S.
Among the laboratory parameters tested along with the measurement of the A value, the test value for monoclonal antigen CA-125 was selected and compared with the LSA value. The reason is that CA-125
This is because it is now internationally recognized as the most sensitive tumor marker used as an indicator of epithelial ovarian cancer and as a means of tracking it.

The results are summarized as follows.

(b) In 95% of patients, pathologically high LSA values were measured before surgery, but at the same time,
95% of patients had pathological CA-125 levels
It was below.

(b) After successful and complete removal of the tumor, LSA values returned to normal levels within an average of 45 days. This number of days is effectively 10 times the half-life of CA-125 (48-50
day).

(c) LSA could be used to monitor disease progression with virtually the same sensitivity as CA-125.

LSA was an indicator of metastasis with 100% certainty.

LSA also served as an indicator of the improvement and recovery process occurring during treatment, although the rate and degree of decrease in LSA values was lower than that of CA-125 values.

(d) LSA was an indicator of recurrence before the appearance of clinical signs in 80% of cases.

(e) In all cases of clinically confirmed recurrence, LSA values were pathologically high.

(f) Non-epithelial tumors accounted for approximately 20% of ovarian cancer cases (
CA-125 could not be used as a reliable indicator for embryonic tumors (embryonic tumors, etc.). It is very important that LSA was found to be a tumor marker whose sensitivity does not change in such cancer tests.

(G) 25 patients with fibroids for the purpose of evaluating their value as a factor for distinguishing between benign and malignant tumors.

and 12 patients with benign ovarian cysts. Pathologically high LSA values were not observed in any of these cases.

The main advantages resulting from implementation of the invention are as follows.

(1) Viewpoint from the laboratory side: (a) Measurements do not require special training and can be performed by any person with ordinary skill in laboratory work.

(b) Measurements can be carried out in a laboratory equipped with average standard equipment and equipment and do not require any special additional equipment.

(c) If the analytical prescription is strictly followed, reliable measurements can be performed even in different laboratories.

(d) Due to the low labor and time consumption required, the method can be used as a continuous analysis tool for large-scale measurements.

(e) The cost of chemicals and equipment is much cheaper (about one-tenth) than that required for the measurement of currently used tumor markers (CEA, CA-125, AFP, etc.).

(f) According to the method of the present invention, a standardized test instrument-formula can be constructed very easily, which can be used to save time and effort and to provide reliable measurements between different laboratories. You can increase the degree.

(2) Clinical viewpoint: The content of lipid-bound sialic acid in human blood can be used as information particularly in the treatment of malignant diseases in the following fields.

(b) For preliminary examination, early detection and support of alternative diagnostic methods: Depending on the form and stage of the progression of malignant disease, LSA values indicate the possibility of the presence of the disease with a probability of 70-93%. The method is relatively easy and inexpensive to perform, so it can be used for high volume preliminary testing. Introducing this as a boat fishing pre-inspection method does not require special additional financial resources and investments.

Needless to say, it should be emphasized that measuring LSA levels alone does not solve the problem of cancer detection, yet it is a powerful element of a well-organized diagnostic system.

In preliminary testing programmes, LSA can be used to detect the presence of a high probability of various diseases and is therefore a more complex and consequently more expensive option that can be performed in a very narrow range of subjects. Other specific diagnostic tests can be applied intensively to suspected cases.

By introducing such a multi-stage preliminary inspection system,
It should be possible to discover diseases much more efficiently than current implementation systems.

(b) Monitoring the condition of patients with malignant tumors and checking the effectiveness of therapeutic interventions: From the point of view of chances of recovery, continuous monitoring of the condition of patients with malignant tumors is at least as important as early detection. I can say that. When used in conjunction with other laboratory and physician diagnostic methods, measurement of LSA levels can be used as a powerful component of an objective cancer surveillance system.

In such applications, changes in LSA values over time provide important information for distinguishing between a tumor-free stage and a tumor-progressing stage, and for confirming the effectiveness of various therapeutic interventions.

Claims (5)

[Claims] (1) Serum is diluted with water, neutral lipids are removed by extraction with a water-immiscible organic solvent or solvent mixture, and the lipoprotein fraction containing lipid-bound sialic acids is extracted from the aqueous phase. precipitate, redissolve the precipitated fraction,
Lipid-bound sialic acid in blood consists of adding an inorganic acid, copper(II) salt, and resorcin to this solution, and calculating the content of lipid-bound sialic acid in serum based on the colorimetric reaction caused by resorcin. A method for measuring acid content, comprising: (a) a precipitant having a concentration in the solution of 15 to 35 mg;
/100ml molecular weight 10,000-20,0
00 dextran and at pH 5.8 to 7.5, or (b) As a precipitant, the concentration in the solution is 15 to 55 mg/
Molecular weight 4,000 to 10,000 for 100ml
or (c) using manganese (II) ions (
Using water-soluble manganese (II) salt in an amount such that the Mg^2^+) concentration is 8.7 to 13 g/100 ml, and the pH is 5.5.
7. A method for measuring lipid-bound sialic acid content in blood, which comprises producing a precipitate in step 7.5. (2) As a precipitant, the concentration is 20-30g/100ml
2. The method for measuring lipid-bound sialic acid content according to claim 1, characterized in that a manganese (II) chloride solution is used. (3) Claim (1) characterized in that the precipitated lipoprotein fraction is redissolved using a buffer solution of pH 7 to 9.
Or the method for measuring lipid-bound sialic acid content according to (2). (4) As a precipitant, the concentration is 22-27g/100ml
using a manganese(II) chloride solution with a pH of 6.0±0.
．． Claim 1 characterized in that a precipitate is produced in step 1.
) or the method for measuring lipid-bound sialic acid content described in (3). (5) Any one of claims (1) to (4), characterized in that the precipitated lipoprotein content is redissolved using a 10 mmol/l tris(hydroxymethyl)aminomethane aqueous solution as the buffer solution. The method for measuring lipid-bound sialic acid content described above.