METHODE DIAGNOSTIQUE DE LA CIRRHOSE ET DU CANCER DU FOIE

DIAGNOSTIC METHOD OF CIRRHOSIS AND HEPATIC CANCER

Abrégé anglais

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.

Revendications

- 8 -
THE EMBODIMENTS OF THE INVENTION IN WHICH AN EXCLUSIVE
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:
<IMG>
(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.

Description

-1- 1 335071
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

1 335071
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 ~
1 2
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):

- 5 - 1 3 3 5 0 7 1
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.

6 - 1 335071
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

- 7 - 1335071
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