CA1335071C - Diagnostic method of cirrhosis and hepatic cancer - Google Patents
Diagnostic method of cirrhosis and hepatic cancerInfo
- Publication number
- CA1335071C CA1335071C CA000597178A CA597178A CA1335071C CA 1335071 C CA1335071 C CA 1335071C CA 000597178 A CA000597178 A CA 000597178A CA 597178 A CA597178 A CA 597178A CA 1335071 C CA1335071 C CA 1335071C
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- CA
- Canada
- Prior art keywords
- iii
- hepatic cancer
- udp
- hepatic
- diagnosis
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
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- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
- Investigating Or Analysing Biological Materials (AREA)
Abstract
This invention relates to a method of diagnosing can-cerous diseases, which comprises measuring the amount of UDP-N-acetylglucosamine:glycoprotein N-acetylglucosaminyl-transferase in body fluid and evaluating the increase in its amount for the diagnosis of hepatic diseases.
AFP, CEA and ?-glutamyltranspeptidase have hitherto been used as tumor markers for the diagnosis of hepatic cancer. But these conventional tumor markers show a pos-itivity rate of about 60 %, making early diagnosis almost impossible.
The method of this invention employs UDP-N-acetyl-glucosamine:glycoprotein N-acetylglucosaminyltransferase as tumor marker, whereby early diagnosis of hepatic cancer can be made almost completely.
AFP, CEA and ?-glutamyltranspeptidase have hitherto been used as tumor markers for the diagnosis of hepatic cancer. But these conventional tumor markers show a pos-itivity rate of about 60 %, making early diagnosis almost impossible.
The method of this invention employs UDP-N-acetyl-glucosamine:glycoprotein N-acetylglucosaminyltransferase as tumor marker, whereby early diagnosis of hepatic cancer can be made almost completely.
Description
Diagnostic Method of Cirrhosis and Hepatic Cancer Field of the Invention This invention relates to a method of diagnosing cancerous diseases.
More particularly, it relates to a method of diagnosing cancerous diseases of the liver, etc. based on the increase in the amount of UDP-N-acetyl-glucos-amine:glycoprotein N-acetylglucosaminyltransferase (hereinafter abbreviated as Gn-T-III) in body fluid.
The method of this invention allows simple diagnosis of cancerous diseases such as hepatic cancer (hepato-cirrhosis) by measuring the increase in the amount of Gn-T-III in body fluid (e.g., serum, saliva and urine), and hence will be of much benefit to the medical and diagnostic fields.
Prior Art and Problems to be Solved by the Invention GOT, GPT, LDH, ChE and many other test items have been adopted for general diagnosis of hepatic functions.
These test items, however, are no more than to check the comparative degree of hepatic functions, and are far from direct diagnosis of hepatic diseases, particularly hepatic cancer.
Measurement of tumor markers, such as AFP and CEA, is - 2 - l 3350 7 1 ~ also known to be necessary for the diagnosis of hepatic cancer and has been put into practice.
But these conventional tumor markers show a positivity rate of 60~ at the highest, making early diagnosis almost impossible.
Recently, ~ -glutamyltranspeptidase is receiving attention as a new tumor market ( particularly for hepatic cancer ), because of the new fact that the blood of patients with hepatic cancer contains glycoproteins carrying different sugar-chain structure compared with normal subjects. However, this7~glutamyltrans-peptidase is not better than AFP, CEA and others as tumor marker.
Means to Solve the Problem Detailed studies on changes in the sugar-chain structure of IgG in patients with hepatic cancer revealed that N-acetylglucosamine is attached, through ~-1,4-linkage, to mannose (of ~-1,4-linkage) of the trimannosyl core of an asparagine-linked type sugar-chain. By "asparagine-linked type of sugar-chain~ is meant a sugar-chain having N-data-glycoside linkage at GlcNAc of Asn residue in animo acids sequence, Asn-X-Ser or Thr.
We continued our investigation on the assumption that this change might be accompanied by the increase in the amount of Gn-T-III --- an enzyme capable of trans-ferring this N-acetylgluco-samine. As a result, it was demonstrated that the sera of patients suffering hepatic diseases ( particularly hepatic cancer ) show a signifi-cantly higher Gn-T-III activity compared with normal sub-jects. We then succeeded in establishing a simple method for measuring the amount of this enzyme. The presentinvention was accomplished on the basis of these findinss.
It was first found by the present inventors that the sera of normalsubjects generally show a Gn-T-III activity as low as about 2.0+0.5 nmol/ml/h, while the sera of patients with hepatic cancer about 2 to 3 times the activity, the sera of patients with hepatocirrhosis about 1.5 times and the sera of patients with chronic hepatitis 1.2 times.
On page 634 of Preliminary Notes for the 60th Mee_ing of Japanese Biochemical Society, is described a method of me~suring Gn-T-III activity, in which N-acetylglucosamine is trans-ferred to GnGn sugar chain an the product thus former is measured by high-performance liquid chromatogra?hy.
However, it is not known at all to apply this method to the diagnosis of cancerous diseases.
Tn the method of this invention, the amount of Gn-T-III
is preferably measured by allowing it to act upon uri~ine diphospho N-acetylglucosamine ( hereinafter abbreviated as UDP-GlcNAc ) and to transfer N-acetylglucosamine to GnGn sugar chain. Thus the product formed is detected by high-performance liquid chromatography. In this case, if the GnGn sugar chain is previously fluorescence-labelled, the - product can be easily detected by monitoring the fluorescence intensity. The GnGn sugar chain used in this invention is isolated from human transferrin, and then _ 4 _ 1 3 3 5 0 7 1 pyridylaminated ( fluorescence labelling ) by the method of Hase et al. ( S. Hase et al, Journal of - Biochemistry, 197-203 (1984) ), as shown by formula (I).
Gal~1-4GlcNAc~1-2Man~1 Man~1 - (I) Gal~1-4GlcNAc~1-2Man~1/
4GlcNAc~1-4GlcNAc-2-aminopyridine ~-Galactosidase is then allowed to act upon this sugar chain, giving pyridylaminated GnGn sugar chain of for~ula (II).
5' 4' GlcNAC~1-2Man~1~ 6 3 Man~1 - (II) GlcNAc~1 -2Man~1 ~
4GlcNAc~1-4GlcNAc-2-aminopyridine The GnGn sugar chain herein means the part of com~ound (II) from which 2-amino?vridine ( fluorescent substance ) is removed, and it also includes a derivative thereof in which fucose is attached to the 1-position ( GlcNAc ).
The reaction of Gn-T-III in the method of this invention is shown by the following equation (III):
Fluorescence-labelled GnGn Sugar Chain UDP-GlcNAc ~
- ~ Gn-T-III
UDP J (III) GlcNAc~l-2Man~ -6 GlcNAc~1 4 Man~1 GlcNAc~1-2Mand1~~~~
L
- 4GlcNAc~1-4GlcNAc-2-aminopyridine The reaction mixture was subjected to high-perfor~ance liquid chromatography, and the amount of reaction prcduct was determined from the fluorescence-intensity, thus measuring the enzyme activity of Gn-T-III.
The amount of Gn-T-III may also be measured by other methods, such as by the antigen-antibody reaction.
Effects Achieved by the Invention It was demonstrated that hepatic disease increases the Gn-T-III activity in the serum, and that this enzyme activity can be easily measured by allowing it to act upon UDP-GlcNAc to transfer N-acetylglucosamine to GnGn sug-~
chain and determining the amount of reaction product by high-performance liquid chromatography. This invention provides a simple method for diagnosing cancerous diseases such as hepatic cancer based on these findings.
Presented below is an Example of this invention.
Example Reagent 250mM MES ( 2-(N-morpholino)ethanesulfonic acid monohydrate ) ( pH: 6.25 ) 400mM GlcNAc ( N-Acetylglucosamine ) 20mM MnC12 40mM UDP-GlcNAc 1~0% Triton X 100*
l50~M GnGn sugar chain ( flurorescence-labelled ) Into fifty containers each containing 50 ~1 of the ~bove reagent, were added 50 ~ of sera taken from patients with primary hepatic cancer, patients with hepatocirrhosis, patients with chronic hepatitis, patients with fatty liver and normal persons ( 10 cases each ), the mixtures were incubated at 37C for one hour, and the reaction was terminated by adding 20 ~l each of a solution containing 0.2M EDTA and 0.1M sodium borate.
Each of the reaction mixtures ( 1 ~l ) was subjected to high-performance liquid chromatography, fluorescene-inten-sity chromatograms were prepared, and the Gn-T-III relative activity was determined for each case.
The result is shown in Table 1 below.
*Trade Mark Table 1 Gn-T-III Relative Activity Serum of patients with primary 3.7+2.3 hepatic cancer Serum of patients with hepato- 3.3+1.8 ~irrhnsis Serum of patients with chronic 2.0+0.5 hepatitis Se~um of patients with fatty 2.0+0.5 liver Serum of normal persons 2.0+0.5
More particularly, it relates to a method of diagnosing cancerous diseases of the liver, etc. based on the increase in the amount of UDP-N-acetyl-glucos-amine:glycoprotein N-acetylglucosaminyltransferase (hereinafter abbreviated as Gn-T-III) in body fluid.
The method of this invention allows simple diagnosis of cancerous diseases such as hepatic cancer (hepato-cirrhosis) by measuring the increase in the amount of Gn-T-III in body fluid (e.g., serum, saliva and urine), and hence will be of much benefit to the medical and diagnostic fields.
Prior Art and Problems to be Solved by the Invention GOT, GPT, LDH, ChE and many other test items have been adopted for general diagnosis of hepatic functions.
These test items, however, are no more than to check the comparative degree of hepatic functions, and are far from direct diagnosis of hepatic diseases, particularly hepatic cancer.
Measurement of tumor markers, such as AFP and CEA, is - 2 - l 3350 7 1 ~ also known to be necessary for the diagnosis of hepatic cancer and has been put into practice.
But these conventional tumor markers show a positivity rate of 60~ at the highest, making early diagnosis almost impossible.
Recently, ~ -glutamyltranspeptidase is receiving attention as a new tumor market ( particularly for hepatic cancer ), because of the new fact that the blood of patients with hepatic cancer contains glycoproteins carrying different sugar-chain structure compared with normal subjects. However, this7~glutamyltrans-peptidase is not better than AFP, CEA and others as tumor marker.
Means to Solve the Problem Detailed studies on changes in the sugar-chain structure of IgG in patients with hepatic cancer revealed that N-acetylglucosamine is attached, through ~-1,4-linkage, to mannose (of ~-1,4-linkage) of the trimannosyl core of an asparagine-linked type sugar-chain. By "asparagine-linked type of sugar-chain~ is meant a sugar-chain having N-data-glycoside linkage at GlcNAc of Asn residue in animo acids sequence, Asn-X-Ser or Thr.
We continued our investigation on the assumption that this change might be accompanied by the increase in the amount of Gn-T-III --- an enzyme capable of trans-ferring this N-acetylgluco-samine. As a result, it was demonstrated that the sera of patients suffering hepatic diseases ( particularly hepatic cancer ) show a signifi-cantly higher Gn-T-III activity compared with normal sub-jects. We then succeeded in establishing a simple method for measuring the amount of this enzyme. The presentinvention was accomplished on the basis of these findinss.
It was first found by the present inventors that the sera of normalsubjects generally show a Gn-T-III activity as low as about 2.0+0.5 nmol/ml/h, while the sera of patients with hepatic cancer about 2 to 3 times the activity, the sera of patients with hepatocirrhosis about 1.5 times and the sera of patients with chronic hepatitis 1.2 times.
On page 634 of Preliminary Notes for the 60th Mee_ing of Japanese Biochemical Society, is described a method of me~suring Gn-T-III activity, in which N-acetylglucosamine is trans-ferred to GnGn sugar chain an the product thus former is measured by high-performance liquid chromatogra?hy.
However, it is not known at all to apply this method to the diagnosis of cancerous diseases.
Tn the method of this invention, the amount of Gn-T-III
is preferably measured by allowing it to act upon uri~ine diphospho N-acetylglucosamine ( hereinafter abbreviated as UDP-GlcNAc ) and to transfer N-acetylglucosamine to GnGn sugar chain. Thus the product formed is detected by high-performance liquid chromatography. In this case, if the GnGn sugar chain is previously fluorescence-labelled, the - product can be easily detected by monitoring the fluorescence intensity. The GnGn sugar chain used in this invention is isolated from human transferrin, and then _ 4 _ 1 3 3 5 0 7 1 pyridylaminated ( fluorescence labelling ) by the method of Hase et al. ( S. Hase et al, Journal of - Biochemistry, 197-203 (1984) ), as shown by formula (I).
Gal~1-4GlcNAc~1-2Man~1 Man~1 - (I) Gal~1-4GlcNAc~1-2Man~1/
4GlcNAc~1-4GlcNAc-2-aminopyridine ~-Galactosidase is then allowed to act upon this sugar chain, giving pyridylaminated GnGn sugar chain of for~ula (II).
5' 4' GlcNAC~1-2Man~1~ 6 3 Man~1 - (II) GlcNAc~1 -2Man~1 ~
4GlcNAc~1-4GlcNAc-2-aminopyridine The GnGn sugar chain herein means the part of com~ound (II) from which 2-amino?vridine ( fluorescent substance ) is removed, and it also includes a derivative thereof in which fucose is attached to the 1-position ( GlcNAc ).
The reaction of Gn-T-III in the method of this invention is shown by the following equation (III):
Fluorescence-labelled GnGn Sugar Chain UDP-GlcNAc ~
- ~ Gn-T-III
UDP J (III) GlcNAc~l-2Man~ -6 GlcNAc~1 4 Man~1 GlcNAc~1-2Mand1~~~~
L
- 4GlcNAc~1-4GlcNAc-2-aminopyridine The reaction mixture was subjected to high-perfor~ance liquid chromatography, and the amount of reaction prcduct was determined from the fluorescence-intensity, thus measuring the enzyme activity of Gn-T-III.
The amount of Gn-T-III may also be measured by other methods, such as by the antigen-antibody reaction.
Effects Achieved by the Invention It was demonstrated that hepatic disease increases the Gn-T-III activity in the serum, and that this enzyme activity can be easily measured by allowing it to act upon UDP-GlcNAc to transfer N-acetylglucosamine to GnGn sug-~
chain and determining the amount of reaction product by high-performance liquid chromatography. This invention provides a simple method for diagnosing cancerous diseases such as hepatic cancer based on these findings.
Presented below is an Example of this invention.
Example Reagent 250mM MES ( 2-(N-morpholino)ethanesulfonic acid monohydrate ) ( pH: 6.25 ) 400mM GlcNAc ( N-Acetylglucosamine ) 20mM MnC12 40mM UDP-GlcNAc 1~0% Triton X 100*
l50~M GnGn sugar chain ( flurorescence-labelled ) Into fifty containers each containing 50 ~1 of the ~bove reagent, were added 50 ~ of sera taken from patients with primary hepatic cancer, patients with hepatocirrhosis, patients with chronic hepatitis, patients with fatty liver and normal persons ( 10 cases each ), the mixtures were incubated at 37C for one hour, and the reaction was terminated by adding 20 ~l each of a solution containing 0.2M EDTA and 0.1M sodium borate.
Each of the reaction mixtures ( 1 ~l ) was subjected to high-performance liquid chromatography, fluorescene-inten-sity chromatograms were prepared, and the Gn-T-III relative activity was determined for each case.
The result is shown in Table 1 below.
*Trade Mark Table 1 Gn-T-III Relative Activity Serum of patients with primary 3.7+2.3 hepatic cancer Serum of patients with hepato- 3.3+1.8 ~irrhnsis Serum of patients with chronic 2.0+0.5 hepatitis Se~um of patients with fatty 2.0+0.5 liver Serum of normal persons 2.0+0.5
Claims
PROPERTY OR PRIVILEGE IS CLAIMED ARE DEFINED AS FOLLOWS:
1. A method for diagnosing hepatocirrhosis or hepatic cancer which comprises the following steps:
(a) adding fluorescence-labelled GnGn sugar chain and UDP-G1cNAc to a serum sample to react with UDP-N-acetyl-glucosamine:glycoprotein N-acetylclucosaminyltrans-ferase III (Gn-T-III) in said serum sample to produce the following compound:
(b) subjecting the resulting reaction solution containing said compound to high-performance liquid chromato-graphy; and (c) examining the increase in degree of Gn-T-III activity.
(a) adding fluorescence-labelled GnGn sugar chain and UDP-G1cNAc to a serum sample to react with UDP-N-acetyl-glucosamine:glycoprotein N-acetylclucosaminyltrans-ferase III (Gn-T-III) in said serum sample to produce the following compound:
(b) subjecting the resulting reaction solution containing said compound to high-performance liquid chromato-graphy; and (c) examining the increase in degree of Gn-T-III activity.
Applications Claiming Priority (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JPPCT/JP88/00898 | 1988-09-06 | ||
| PCT/JP1988/000898 WO1989002474A1 (en) | 1987-09-07 | 1988-09-06 | Method for diagnosis of cancer diseases |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CA1335071C true CA1335071C (en) | 1995-04-04 |
Family
ID=13930802
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CA000597178A Expired - Fee Related CA1335071C (en) | 1988-09-06 | 1989-04-19 | Diagnostic method of cirrhosis and hepatic cancer |
Country Status (1)
| Country | Link |
|---|---|
| CA (1) | CA1335071C (en) |
-
1989
- 1989-04-19 CA CA000597178A patent/CA1335071C/en not_active Expired - Fee Related
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| Date | Code | Title | Description |
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