Diagnostic method of cirrhosis and hepatic cancer

Details Diagnostic method of cirrhosis and hepatic cancer Diagnostic method of cirrhosis and hepatic cancer

1989-05-01

1991-02-19

Cashion, Jr., Merrell C.

Chemistry: molecular biology and microbiology

Measuring or testing process involving enzymes or...

Involving transferase

435 14, 424 9, 424945, 424531, 424553, C12Q 148, A61K 3752, A61K 3516

Patent

active

049943740

DESCRIPTION:

BRIEF SUMMARY
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-glucosamine:glycoprotein N-acetylglucosaminyltransferase III (hereinafter abbreviated a Gn-T-III ) in body fluid.
The method of this invention allows simple diagnosis of cancerous diseases such as hepatic cancer (hepatocirrhosis) 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 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, .gamma.-glutamyltranspeptidase is receiving attention as a new tumor marker (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, this .gamma.-glutamyltranspeptidase is not better than AFP, CEA and others as a tumor marker.


MEANS TO SOLVE THE PROBLEMS

Detailed studies on the change in sugar-chain structure in patients with hepatic cancer revealed that N-acetylglucosamine is attached, through .beta.1,4-linkage, to the mannose (of .beta.-1,4-linkage) bound to the trimannosyl core of sugar chain of asparagine linked type. 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 transferring this N-acetylglucosamine. As a result, it was demonstrated that the sera of patients suffering hepatic diseases (particularly hepatic cancer) show a significantly higher Gn-T-III activity compared with normal subjects. We then succeeded in establishing a simple method for measuring the amount of this enzyme. The present invention was accomplished on the basis of these findings.
It was first found by the present inventors that the sera of normal subJects 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 have 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 Meeting of Japanese Biochemical Society, is described a method of measuring Gn-T-III activity, in which N-acetylglucosamine is transferred to GnGn sugar chain and the product thus formed is measured by high-performance liquid chromatography. However, it is not known at all to apply this method to the diagnosis of cancerous diseases.
In the method of this invention, the amount of Gn-T-III is preferably measured by allowing it to act upon uridine 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 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). ##STR1##
.beta.-Galactosidase is then allowed to act upon this sugar chain, giving pyridylaminated GnGn sugar chain of formula (II). #

REFERENCES:
The Levels of Nucleotide-Sugar: Glycoprotein Sialyl-and N-Acetyl Glucosaminyl-Transferases in Normal and Pathological Human Sera., Mookerjea et al, Can. J. Biochem. 50, 738-740 (1972).
Glycosyl Transferase and Glycosidase Activities in Ovarian Cancer Patients, Chatterjee et al., Cancer Res. 39, 1943 (1979).
Studies on VDP-N-Acetyl Glucosamine: .alpha. Mannoside .beta.-N-Acetyl-Glucosaminyl Transferase of Rat Liver Hepatomas, Miyagi et al., BBA 661, 148 (1981).
Serum Levels of Glycosyltransferases . . . Weiser et al., CRC Critical Reviews in Clinical Lab Sciences.

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