Chemistry: analytical and immunological testing – Cancer
Patent
1991-06-03
1994-01-04
Housel, James C.
Chemistry: analytical and immunological testing
Cancer
436 71, 436813, 436 95, 436 63, G01N 3392
Patent
active
052759520
DESCRIPTION:
BRIEF SUMMARY
CROSS REFERENCE TO RELATED APPLICATIONS
This application is a national phase of PCT/HU89/00021 filed May 12, 1989 and based, in turn, on Hungarian national application 249/89 of Jan. 23, 1989 and 2190/89 of May 5, 1989 under the International Convention.
FIELD OF THE INVENTION
The invention relates to an analytical method for the selective determination of sialic acid-carrying glycolipid complexes.
BACKGROUND OF THE INVENTION
Sialic acid (N-acetyl neuraminic acid) appears in the living organism in free and bound states (Advances in Cancer Res. 38, 289-350; 1983). Sialic acid can be bound as a terminal group via an oligosaccharide chain to membrane-bound sphingolipids (J. of Supramolecular Structure 9, 157-177; 1978). The terminology of gangliosides, including glycosphingolipids, is not always unambiguous in the literature. According to Svennerholm (Comprehensive Biochemistry 18, 201; Elsevier, Amsterdam, 1970) these compounds can be classified into the groups of monosialogangliosides, disialogangliosides and trisialogangliosides. The hydrophobic part of the ganglioside is ceramide, which is bound to the membrane with a long unsaturated fatty acid chain. Ceramide is bound to oligosaccharides, i.e. to glucose, galactose, N-acetyl galactosamine and sialic acid, through an amino group.
Owing to the presence of sialic acid bound thereto, the glycosphingolipid macromolecule has a negative charge, and this negative charge influences certain intercellular interactions. Due to the disturbances in biochemical equilibrium in tumorous organisms the intercellular interactions change whereupon the amount of glycolipid-bound sialic acid increases in the blood.
Based on this theoretical recognition several methods have been developed to determine the glycolipid-bound sialic acid content of blood. According to the method of Svennerholm (Biochem. Biophys. Acta 24, 604-611 1957) blood plasma is extracted with a 2:1 mixture of chloroform and methanol to separate glycosphingolipids from the other lipid fractions of blood, and the sialic acid content of glycosphingolipids is measured by a colorimetric reaction produced with resorcinol in the presence of a copper(II) salt. According to Katopodis (U.S. Pat. No. 4,342,567) the glycosphingolipid fraction is precipitated first from the methanolic supernatant with phosphotungstic acid, and the redissolved precipitate is utilized in the colorimetric determination. In this way the specifity of the Svennerholm method could be improved. Sialic acid can also be determined, however, by enzymatic reactions. According to the method disclosed in Clin. Chim. Acta 108, 493-498 (1980) the glycoprotein is split with neuraminidase enzyme and the liberated sialic acid is contacted with N-acetyl-neuraminic acid-aldolase enzyme to obtain pyruvate and N-acetyl-D-mannosamine. The resulting pyruvate is oxidized with pyruvate oxidase into a peroxide which is converted into a colored substance with p-chlorophenol and 4-amino-antipyrine in the presence of peroxidase, and the amount of the resulting coloured substance is determined at 505 nm.
All of the above methods have the common disadvantage that the error of determination exceeds 20%. Since according to the authors there is a rather small difference between normal and pathologic sialic acid levels (values below 18 mg % can be regarded as normal, whereas those exceeding 20 mg % are pathologic), the results may refer to the existence of a malignant disease but cannot be applied as an unambiguous proof of the existence of tumorous or tumour-free state. It is a further disadvantage that certain benign diseases, such as arthritis, psoriasis, ulcer, inflammations and gynecological disorders, may also result in an increase of the sialic acid content of blood (J. Clin. Chem. Biochem. 22, 647-651 1984). Consequently, the method developed so far for the determination of sialic acid content of blood may only give rise to a suspicion of cancer but cannot form a basis for setting up a more accurate diagnosis.
OBJECT OF THE INVENTION
It is the object of our
REFERENCES:
patent: 4115062 (1978-09-01), Morre et al.
patent: 4486531 (1984-12-01), Ziegenhorn et al.
patent: 4520111 (1985-05-01), Miller
patent: 4746605 (1988-05-01), Kerscher et al.
patent: 4748128 (1988-05-01), Katopodis
patent: 4923439 (1990-05-01), Seidel et al.
Babarczi Jolan
Gesztesi nee Koszegi Anna
Ivony nee Kladiva Katalin
Kinczel Edit
Milovan Iren
Dubno Herbert
Housel James C.
Myers Jonathan
Reanal Finomveygszergyar
Tran Lien
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