ANALYTICAL METHOD FOR THE SELECTIVE DETERMINATION OF SIALIC ACID-CARRYING GLYCOLIPID COMPLEXES

Abstract: The invention relates to a method for the selective determination of sialic acid-carrying glycolipid complexes in blood, in which the serum is diluted with water, then extracted with a water-immiscible organic solvent or solvent mixture to remove neutral lipids, a lipoprotein fraction containing sialic acid-carrying glycolipids is precipitated from the aqueous phase, the precipitated fraction is redissolved, and the sialic acid content of the solution or the amount and quality of the sialic acid-carrying glycolipid is(are) determined by a method known per se. According to the invention a water-soluble salt of a monovalent, bivalent or trivalent metal other than alkali metal is applied as precipitating agent in an amount corresponding to a final metal ion concentration of 0.005-0.1 mole/l in the mixture, and precipitation is performed at a pH of 1.5 to 8.0.

Description: ANA YTICAL METHOD FOR THE SELECTIVE D3T3RLCQTA I0N OF SIALIC ACID-CABRYim GIYCOLIPID COMPISXBS The invention relates to an analytical method for the selective determination of sialic acid -carrying glycolipid complexes,, Sialic acid (N-εcetyl neuraminic acid) appears in living organism in free and bound states (Advances in Cancer Hesβ 28, 289-350 /1983/) • Sialic acid can be bound as s terminal group via an oligosaccharide chain to membrane -bound sphingolipids (J. of Supra molecular Structure °i, 157-177 /1978/) . The terminology of gangliosides, including glyco- sphingolipids, is not always unambiguous in the literature . According to Svennerholm (Comprehensive Biochemistry 18, 201 /Elsevier, Amsterdam, 1970/) the se compounds can be classi ie into the groups of αonosiεlogangliosides, disialogεngliosides and trisialogangliosidesβ The hydrophobic part of gaoglioside is cerε ide , which is bound to the membrane with a long un- saturated fatty acid chain β Cerεπiide is bound to oligosacchar ides, i.e. to glucose-, galactose , !T-ecetyl galεctosamine and sialic acid, through εn ε ino group. Owing to the presence of siεlic acid bound thereto, the glyco sphingolipid macromolecule has a negative charge , and this negative charge influences certain intercellular interactions . Due to the disturbances in biochemical equi¬ librium 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 elaborated to determine the glycolipid-bound sialic acid content of blood. According to the method of Svennerholm (Biochem. Biophys. Acta , 604-611 /1957/) blood plasm is extracted with a 2:τ_ mixture of chloroform end methanol to separate gl co sphingolipids from the other lipid fractions of blood, and the sialic acid content of glycosphingolipids is measured by a colorims ric reaction produced with resorcine in the presence of a coppe (II) salt According to Kβtopodis (USP 4,342 ,567) the glycosphingolipid fraction is precipitated first from the me thanolic supernata with phosphotungstic acid, and the redissolved precipitate is utilized in the colorimetric determination. In this way the specif ity of the Svennerholm method could be improved. Sialic acid can be determined, however, by enzymatic reac¬ tions, too. According to the me thod disclosed in Clin. Chim. Actε 108, 4 3-^ 8 (1980) the glycoprotein is split with neur aminidase enzyme and the liberated sialic acid is contacted with N-acetyl-ne raminic εcid-aldolase enzyme to obta in pyruvate and N-acetyl-D-mannosεmine . The resulting pyruvate is oxidised with pyruvate oxidasa into a peroxide which is converted into a coloured substance with p-chlorophenol and 4-amino-antipyrine in the presence of peroxidase, and the amount of the resulting coloured substance is determined a t 0 nm β All of the above methods have the common disadvantag that the error of determination exceeds 20 . Since εccord- ing to the authors there is a ra ther small difference betwee normal and pathologic sialic acid levels (vεlua s below 18 mg % can be regarded a s normal , whereas tho se exceeding 0 rag % are pa thologic) , the results may refer to the exist¬ ence of a ma lign disease but cannot be applied as an un- ambiguous proof for the existence of tumorous or tumour-free sta te o It is s further dissdvεntage that certain benign diεeε εe ε , such as arthri tis, psoriε sis , ulcer, inflεmma- tions and gynaecological disorders, may also result in an increase of the siεlic acid content of blood (J. Clin. Chem. Biochem. 22, 647-651 /1984/) . Consequently, the method elaborated so far for the determination of sialic acid content of blood may only give rise to ε suspicion of cancer but cannot form a basis for setting up a more accu¬ rate diagnosis. Our aim was to elaborate an analytical method which may lead to a more reliable diagnosis, enables one to re- cognize the existence of tumor in s very early stage, gives reliable information on distinguishing tumorous and tumor- -free states from, one εnother and enables one to conclude on the probεble localization of the tumor β The invention is based on the recognition that, at an appropriate pH and ionic strength, different metε-1 salts are able to separate well-defined subfractions from the complicated composite of glycolipid complexes present in blood. In cancerous processes of different origin or in non- tumorous diseases sialic acid binds preferentially to differ ent glycolipid fractions, thus, by measuring the sialic acid content of the separated glycolipid subfraction or by de¬ termining the nature of the glycolipid to which sialic acid is bound conclusion cεn be drawn whether an increase in sialic acid level can be attributed to a tumorous process or to ε benign disease (such as inflammation, arthritis, a gynaecological disorder, etc.)* and in tumorous processes conclusion cεn be drawn on the Drobεble localization of the tumor or on the existence of meta stasis . It has also been observed that when utilizing certain metal salts, primarily cadmium salts, as precipitating agent one can recognize tumorous state at a much earlier stage than it was possible when applying phosphotungstic acid as precipita ting εgent. Based on the above, the invention relates to a method for the selective determinεtion of sialic acid-carrying glycolipid complexes in blood, primarily in human blood, in which the serum is diluted with water, then extracted with a water-immiscible organic solvent or solvent mixture to remove neutral lipids, a lipoprotein fraction containing sialic acid-cεrrying glycolipids is precipita ted from the aqueous phase , the precipitated fraction is redissolved, and the sialic εcid content of the solution or the amount and quality of the sialic εcid-cεrrying glycolipid is (are) determined by a method known per se . According to the in¬ vention a water-soluble salt of a onovalεnt , bivalent or trivalent metal other than alkali metal is applied as pre¬ cipitating agent in an εmount corresponding to a final metal ion concentration of 0.005-0.1 mole/1 in the mixture , and precipitation is performed at a pH of 1.5 to 8.0. It is preferred to apply the me thod described in USP 4,342 ,567 to remove the neutral lipids from the serum diluted with water, according to which a 2:1 v/v mixture of chloroform and mβthanol is utilized as extracting agent . The equeous_methεnolic supernatant (in the f ollowing: the methε nolic superna tant) is processed further in the precipite tion step, wherea s the lower phase which is a chloroform solution of neutral lipids is discarded. Thereaf er the precipitating agent is added to the aqueous methanolic phase or to an aliquot thereof in an amount to obtain a mixture conta ining 0.005-0.1 mole/1 of metal ion. The precipitating agent is utilized preferably as an aqueous solution. For some metal ions the preferred pH value s of precipita ion are listed in the table below. Metal ion pH Fe 2 * 3.0-3.5 Fe 5+ 3.0-5.0 Co 2+ 4.0-5.5 2+ 4.5-5.5 Cd + 4.5-6.0 Zn + 1 * 5-5.0 Ag + 6.0-8.0 Hg 2 * 1.5-3.0 Cu 2+ 3.0-4.5 Ca 2+ 4.5-5.5 Mg 2+ 5.5-7.0 The resulting precipitate is separated and then a) the sialic acid content of the precipitate is determined by a known me thod, such εs on the basis of the colour reaction produced with a mineral εcid, a copper(II) salt εnd re sorcine as described in USP 4,342 , 567 » or by the enzymε tic me thod disclosed in Clin. Chim. Actε 108, 493-493 (1980) , or b) the type and amount of glycolipids bound to sialic acid are determined by a known me thod, such as by high pressure liquid chroma tography as described in Clin. Chem. 22, 1516-1521 (1976) . The differential diagnostic value of the me thod accord¬ ing to the invention can be increased considerably when the sialic acid content of a blood sample of one and the same origin is determined in serie s utilizing at. least f ive differ ent metal ions, ' and the ratios (where the term "normal value" refers to the statistically calculated non-pathologic sialic acid concentration utilizing the given me tal ion as precipita ting agent) are added up. According to our experiences the value of Σ. norma l decreases (for localized tumors) in the following order: mammary tumor - - colon tumor - ovεriεn tumor - head/neck tumor - adeno- carcino a - non- tumorous disease - healthy tumor-free state . Similarly, a differential diagnostic series can be obta ined when the results of the sialic acid determination according to the invention, performed ?άth a t least five different metal ions , are divided with the result of a known sialic acid dete mination (e .g. with the result of the de¬ termination disclosed in USP 4, 342,567 in which a sialic acid content independent of the origin of sialic acid is measured) , and these quotients are added up. The individual quotients may also have differential diagnostic value . The invention is elucida ted in detail by the aid of the following non-limiting examples. In the examples the blood samples were pre-trea ed and the sialic acid content of the precipitate was measured as follows : Venal blood was used for the determination. After blood sampling the sample was centrifuged for 15 minutes at 4000 r.p .m. , and 0.6 ml of the sample was frozen. The lipid- bound sialic acid concentration of the sample stored at -20°C remains unchanged for 6 months . Prior to starting the analysis the test tubes were thoroughly rinsed with methanol, dried, and pre-cooled in a refrigerator. 0.2 ml of the serum sample and 0.2 ml of bi- distilled water were filled into each of the test tubes, the mixture was stirred with a Vortex stirrer for 15 seconds and then placed into ice water bath (0°C) . Two parallel samples, each, were prepared from each of the sera to be examined and from the controls . 3 ml, each, of a pre-cooled 2:1 v/v mixture of chloroform εnd methεnol were filled into all of the test tubes, and the contents of the te st tubes were stirred with a Vortex, stirrer for 30 seconds. Then the te st tubes were pieced again into the ice water bath. 0.5 ml , each, of bi- distilled water pre-cooled to +4°C was added to the indi¬ vidual samples , the contents of the test tubes were stirred with a Vortex stirrer for 15 seconds, εnd then the mixture s were allowed to s tand at room temperature for 5 minutes . Thereafter the samples were centrifuged for 10 minute s at 3OOO r.p.m. 1 ml , each, of the supernatant was f illed cautiously into numbered centrifuge tubes, the acueous metal salt solution of the amount and concentration given in the examples was added, the pH of the mixture was adjust ed to the value given in the examples, the contents of the individual centrifuge tubes were stirred with a Vortex stirrer, and then allowed to stand for 5 minutes. At the end of the standing period the contents of the individual centrifuge tubes were centrifuged for 15 minutes at 4000 r.p.m., and then the supernatant was separa ed completely from the precipitate by decanting (the super- natεnt was discarded) . 1 ml, each, of a tris buffer solution was introduced into the individual centrifuge tubes, redissolving thereby the precipitate. Tris buffer solution was prepared by dis¬ solving 10 ππnoles of tris(hydroxymethy1) aminomethane in one litre of bidistilled water and adjusting the pH of the resulting solution to 8.6. Standard and blank samples were prepared for the photometric measurements as follows: 50 g of analytically pure sialic acid were dissolve in 100 ml of bidistilled water, εnd the resulting solution was utilized as stock solution to prepare two further dilu¬ tions (25 mg/100 ml and 12.5 mg/100 ml in concentra ion) with bidistilled water. The standard solutions were stored st +4°C. Samples of 0.2 ml, each, of the standard solutions with three different concentrations were filled into centrifuge tubes, εnd the volume of the solution was adjust ed to 1 ml with bidistilled water in each of the centrifuge tubes. 1 ml of bidistilled water was applied a s blank. 1 ml, each, of resorcin reagent solution was added to all of the samples (standard, blank, control, test serum) . The resorcin reagent solution wa s prepared as follows : 2 g of analytically pure resorcin were dissolved in 100 ml of bidistilled water. 2.49 g of anhydrous copper(II sulfete were dissolved in 100 ml of bidistilled water. Thereafter 10 ml of the resorcin solution were admixed with 0.25 ml of the copper(II) sulfεte solution εnd 9.75 ml of bidistilled water, εnd 100 ml of εnslytic3lly pure con¬ centrated aqueous hydrochloric acid were added to the mix¬ ture . The test tubes were placed into ε 100°C water bath for exactly 10 minutes . Thereafter all of the test tubes were immediεtely immersed into a 0°C ice water bath and were kept there for 10 minutes . 2 ml , each, of a 85:15 v/v mixture of butyl acetate εnd n-butanol were introduced into the individual test tubes and the contents of the test tubes were stirred with a Vortex stirrer for 5 minutes. The mixtures were allowed to stand with occasional stirring , the sεmples were centrifuged f or 10 minutes εt 2500 r.p .m. , εnd then the blue supernatεnts were filled from each centri¬ fuge tube into a measuring cell. The. density of the blue colour formed doe s not chεnge within 6 hours . Photome tric measurement was performed a t a wave¬ length of 580 n . The zero point of the photometer wεs ad¬ justed or bidistilled water . Thereafter, depending on the type of the photometer used (single-beam or double-beam), the measurement was performed either against the blank, or the extinction of the blank was subtracted from the measured extinction of the sample to obtain a corrected value: S(corr) = E(measured) - S(blank) Sialic εcid content was calculated by one of the following two methods: 1) rom the results obtained with standard sialic acid samples of known sialic acid concentrations, extinction vs. concentration curves were constructed either graphically or by cεlculεtion. Due to the dilution steps, the original concentrations should be recalculated according to the following table: Original concentre- Measured concentra¬ tion (mg/100 ml) tion ( ,ug/200 ml) Extinction 50 100 0.508 25 50 0.252 12.5 25 0.127 From the calibration curve constructed as described εbove the siεlic acid content (A) of the supernatant εεmple, 1 ml in volume, cεn be determined. The curve gives the siεlic acid content in units of /Ug of sialic acid/1 ml of supe natεnt. The sialic acid content of the blood serum cεn be calculated from the formula Sialic εcid content (mg/100 ml) = A x 0.72, where A is the sis lie εcid content of the supernεtεnt in units of / g/ml εnd 0.72 is ε fεctor which comprises the degree of dilution εnd the conversion of U to πg. 2) According to the second method of calcula tion reference, serum samples with known sialic acid concentrations were applied. From several parallel measurements a factor F was calculated according to the formula F = C (ref)/E(ref) , where C (ref) is the sialic acid content of the reference serum in mg/100 ml and S(raf) is the extinction of the reference serum. The siεlic acid content of the sample to be tested can be calcula ted from the formula Sialic acid content (mg/100 ml) = S(m) x F, where 3(m) is the measured extinction of the sample to be tested. Example 1 Serum originating from a healthy person was extracted with s mixture of chloroform and me thanol a s described above . The iπethanolic superna tant was admixed in a ratio of 10:1 with a 0.5 mole/litre solution of C1 2 at a pH of 3<>5 to form a precipitate , and the sialic εcid content of the pre¬ cipitate was measured. A vslue of 4.65 mg % VJS S obtained. Example 2 Serum originating from a healthy parson was extracted with ε mixture of chloroform and methsnol as described εbove. The methanolic supernatant was εd ixed in a ratio of 10:1 wit a 1.0 mole/litre solution of ITiCl εt a. pH of 5.3 to form a precipitate, and the sialic acid content of the pre¬ cipitate was measured. A vslue of 6.43 mg % was obtained. Example 3 Serum originating from a healthy person was extracted with a mixture of chloroform εnd methanol as described above. The m thεnolic supernatant was admixed in a ratio of 10:1 with a 0.1 mol/litre solution of CdCl 2 at a pH of 5.9 to form a precipitate, and the sialic εcid content of the pre- # cipitate was measured. A value of 11.52 mg % was obtained. Example 4 Serum originating from a healthy person was extracted with a mixture of chloroform εnd methεnol εs described above. The methanolic supernatant was εd ixed in a ratio of 10:1 with a 0.5 mole/litre solution of CdC^ at a pH of 5.5 to form a precipitate, and the sialic acid content of the pre¬ cipitate was measured. A vslue of 10.48 mg % was obtained. Example 5 Serum originating from a healthy person was extracted with a mixture of chloroform and methanol as described above. The methanolic supernatant was admixed in a ratio of 10:1 with a 1.0 mole/litre solution of CdCl 2 at a pH of 5.3 bo form a precipitate, and the sialic acid content of the pre¬ cipitate was measured. A value of 14.81 mg % was obtained. Example 6 Serum originating from a healthy person was extracted with a mixture of chloroform and methanol as described above. The methanolic supernatant was εdmixed in a rεtio of 10:1 with a 0.1 mole/litre solution of FeSO^ at a pH of 3.3 to form a precipitate, εnd the siεlic εcid content of the pre¬ cipitate wss measured. A vslue of 9»03 mg % wεs obtεined. Example 7 Serum originating from a healthy person was extracted with a mixture of chloroform and methanol as described above. The methanolic supernatant was admixed in a ratio of 10:1 with a 0.5 mole/litre solution of FeSO^ at a pH of 3.0 to form a precipitate, and the sialic acid content of the pre¬ cipitate was measured. A value of . m % was obtained. Example 8 Serum originating from s healthy person was extracted with a mixture of chloroform and methanol as described above. The methanolic supernatant was εdmixed in a ratio of 10:1 with a 1.0 mole/litre solution of FeSO^ at a pH of 2.9 to form a precipitate, and the sialic scid content of the pre¬ cipitate was measured. A value of 9 * 52 mg % was obtained. Exsmple 9 Serum originating from a healthy person was extrεcted with a mixture of chloroform and methanol εs described εbove. The methεnolic supernatant was admixed in a ratio of 10:1 with a 0.1 mole/litre solution of ZnCl 2 at a pH of 2.7 to form a precipitate, and the sialic acid content of the pre¬ cipitate was measured. A value of 6.52 mg % was obtained. Example 10 Serum originating from a heslthy person was extracted with a mixture of chloroform εnd methsnol as described above. The methεnolic supernatant was admixed in a ratio of 10:1 with a 0.5 mole/litre solution of ZnCl 2 at a pH of 1.9 to form s precipitste, εnd the siεlic εcid content of the pre- cipitεte wεε meεsured. A vεlue of 12.34 g % wεs obtained. Example 11 Serum originating from ε healthy person was extracted with a mixture of chloroform end methsnol as described above. The methanolic supernatant v;as admixed in a ratio of 10:1 with a 0.1 mole/litre solution of Pb(ΪTO 5 ) 2 at a pH of 4.4 to form a precipitate, and the sialic acid content of the pre¬ cipitate was measured. A vslue of 8.61 mg % was obtained. Example 12 Serum originεting from a healthy person was extracted with s mixture of chloroform snd methsnol as described above. The methanolic supernatant was admixed in a ratio of 10:1 with a 0.5 mole/litre solution of Εb{H0--) 2 εt s pH of 3.7 to form a precipitate, and the siεlic acid content of the pre¬ cipitate wεs messured. A value of 12.11 mg % was obtained. Exsmple 13 Serum origins ting from s healthy person was extracted with a mixture of chloroform and methsnol as described above. The methanolic supernstsnt was admixed in ε ratio of 10:1 with a 1.0 mole/litre solution of Pb(NO j ) 2 at - pH of 3 ' .5 to form a precipitate, and the siεlic acid content of the pre¬ cipitate was measured. A value of 12.35 m % was obtained. Example 14 Serum originating from a healthy person wεs extracted with a mixture of chloroform and methanol as described above. The methanolic supernstsnt wss εdmixed in a rstio of 10:1 with s 0.1 mole/litre solution of AgNO, at s pH of 8.5 to form ε precipitate εnd the sialic εcid content of the pre¬ cipitate wss eεεured. A vslue of 9.89 mg % wss obtεined. Example 15 Serum originating from a healthy person was extracted with a mixture of chloroform and methsnol as described above. The methanolic supernatant was admixed in a ratio of 10:1 with a 0.5 mole/litre solution of g£?0 5 a t a pH of 6.0 to form a precipitate , and the sialic acid content of the pre¬ cipita te was measured. A value of 14.67 mg % was obtained. Example 16 Serum origina ting from a healthy person was extracted with a mixture of chloroform and me thanol as described above . The methεnolic supernatant was admixed in s ratio of 10: 1 with a 1.0 mole/litre solution of AgϊK at a pH of 5.8 to form a . precipitate , and the siεlic acid content of the pre¬ cipitate ws s mea sured. A value of 14.81 mg % was obtained. Example 17 Serum originating from a healthy person was extracted with a mixture of chloroform end methanol as described above . The methsnolic supernstεnt wss sdmixed in s rs tio of 10: 1 with a 0.1 mole/litre solution of HgCl 2 st a pH of 1.9 to form a precipita te , and the sialic acid content of the pre- cipita te was measured. A value of 6.05 mg % was obtained. Example 18 Serum originating from a healthy person was extracted with s mixture of chloroform εnd methεnol as described above. The methanolic supernatant was admixed in a rεtio of 10:1 with a , mole/litre solution of EgCl 2 εt a pH of 1.7 to form a precipitate, and the siεlic εcid content of the pre¬ cipitate wεε measured. .. vεlue of .68 mg % v.-εs obtεined. Example 19 Serum originating from a healthy person was extrscted with a mixture of chloroform and methsnol as described above,, The methεnolic supernatεnt was admixed in a ratio of 10:1 with s 1.0 mole/litre solution of HgCl 2 at a pH of 1.5 to form a precipάiate, and the sialic acid content of the pre- cipitate was measured. A vslue of 11.99 mg % wεs obtsined. Sxs ple 20 Serum originating from a healthy person was extracted. with s mixture of chloroform end methsnol ss described above. The methεnolic supernatant wεs sdmixed in a rεtio of 10:1 with a 0.1 mole/litre solution of CuSO^ st a pH of 4.2 to form ε precipitate, and the sialic acid content of the pre¬ cipitate wss measured. A vslue of 10.13 mg % was obtained. Example 21 Serum originating from s healthy person wss extrscted with a mixture of chloroform εnd methanol as described above. The methεnolic supernstεnt wεs εdmixed in s rstio of 10:1 with a 0.5 mole/litre solution of CuSO^ at a pH of 3.8 to form ε precipitate, εnd the siεlic εcid content of the pre¬ cipitate wεs measured. A value of 10.48 mg % wεs obtained. ■Example 22 Serum originating from s heεlthy person wss extracted with a mixture of chloroform εnd methsnol εs described εbove. The methεnolic supernεtεnt wss sdmixed in s ratio of 10:1 with s 1,0 mole/litre solution of C SO^ εt ε pH of 3. to form s precipitεte, and the siεlic εcid content of the pre¬ cipitate wεs measured. .4 vslue of 12.22 zp fi was obtained. Exs ple 23 Serum originsting from a heslthy person wεs extrscted with a mixture of chloroform snd methεnol ss described above. The methεnolic supernatant wεs admixed in s ratio of 10:1 with a 0.1 mole/litre solution of CεCl 2 at a pH of 5.5 to form a precipitate, and the siεlic εcid content of the pre- cipitεte was measured. A value of 3.72 mg % was obtained. Example 24 Serum originsting from a healthy person wεs extracted with a mixture of chloroform and methεnol as described above. The methεnolic supernatant wss admixed in ε rεtio of 10:1 with ε 0.5 mole/litre solution of CεCl 2 at a pH of 5.4 to form s precipitate, and the sialic εcid content of the pre¬ cipitate was measured. A vslue of- 3-02 mg % was obtained. Exsmple 25 Serum originating from s heslthy person wss extracted with s mixture of chloroform εnd methsnol εs described above. The methεnolic supernatant was εdmixed in a ratio of 10:1 with a 1.0 mole/litre solution of CsCl 2 at a pH of 5 * 3 to form s precipitate, and the sialic acid content of the pre¬ cipitate was measured. A value of 7.05 mg % was obtained. Example 26 Serum originating from a healthy person was extracted with ε mixture of chloroform εnd methεnol εs described above. The methanolic supernεtεnt wss εdmixed in ε rεtio of 10:1 with s 0.1 mole/litre solution of IgCl 2 at ε pH of 6.1 to form ε precipitεte, εnd the sialic εcid content of the pre¬ cipitate was measured. A vεlue of 4.19 mg 55 wε s obtsined. Exεmple 27 Serum originating from ε heslthy person wεs extracted with s mixture of chloroform εnd methεnol as described above The methsnolic supernatant was admixed in a ratio of 10:1 with a 0.5 mole/litre solution of MgCl 2 at a pH of 6.7 to form a precipitate, εnd the sislic εcid content of the pre¬ cipitate wss measured. A value of 3.61 g % wss obtained. Example 28 Serum originsting from s heslthy person wεs extracted with s mixture of chloroform and methεnol as described above The methεnolic supernstsnt wεs admixed in a ratio of 10:1 with a 1.0 mole/litre solution of MgCl 2 st a pH of 6.5 to form s precipitate, and the siεlic scid content of the pre¬ cipitate wss messured. A vslue of 4.81 mg % wss obtsined. Exsmple 29 Serum originsting from s patient with msmmsry csncer was extracted with a mixture of chloroform and methsnol ss described above. The methεnolic supernstsnt was εdmixed in a rεtio of 10:1 with a 1.0 mole/litre solution of NiCl 2 εt ε pH of 5.3 to form a precipitate, and the sialic acid content of the precipitate was measured. value of 28.87 mg % was obtained. Example 50 Serum originating from a patient with mammary cancer was extrscted with s mixture of chloroform and methεnol εs described sbove. The methεnolic supernstsnt wεs sdmixed in a rεtio of 10:1 with s 1.0 mole/litre solution of CdCl 2 at a pH of 5 «3 to form a precipitate, and the sialic acid Content of the prec ipitate was measured. A value of 41.31 mg % was obtained. Example 31 Serum originating from a patient with mammary cancer was extrscted with a mixture of chloroform and methsnol as described above . The methanolic supernatant was admixed in a rεtio of 10:1 with a 1.0 mole/litre solution of eSO^ a t a pH of 2.9 to form a precipitate , and the sialic acid content of the precipita te was measured. A value of 33 * 39 mg % was obtained. Example 32 Serum originsting from a patient with msmmsry csncer was extracted with a mixture of chloroform and methsnol as described above . The methanolic supernstsnt was admixed in a rεtio of 10:1 with a 1.0 mole/litre solution of ZnCl 2 a t a pH of 1.7 to form a precipitate , and the sislic εcid content of the precipitate was measured. A value of 39.41 mg % was obtsined. Exsmple 35 Serum originsting from ε patient with msmmεry csncer was extracted with a mixture of chloroform and methanol as described above . The methanolic superna tant wε s sdmixed in a ratio of 10:1 with a 1.0 mole/litre solution of ZnSO^ 8 t a pH of 1.7 to form a precipita te , and the sislic εcid content of the precipitste ws s measured. A value of 38.90 eg % was obtsined. Example 54 Serum originating from a patient with mammsry cancer was extrscted with a mixture of chloroform and methanol as described above. The methanolic supernatant was admixed in a rstio of 10:1 with a 1.0 mole/litre solution of Pb(N0z) 2 at a pH of 3.5 to form a precipitste, and the sislic acid content of the precipitste was measured. A value of 45.02 g % was obtained. Example 35 Serum originating from a patient with mammary cεncer was extracted with a mixture of chloroform and methsnol as described above. ' The methanolic supernstsnt was sdmixed in a rεtio of 10:1 with a 1.0 mole/litre solution of AgKO, at a pH of 5 * 8 to form a precipitate, and the siεlic scid content of the precipitste was measured. A vslue of 29.58 g % wss obtεined. Exsmple 36 Serum originsting from a patient with mammary cancer wss extracted with s mixture of chloroform and methsnol as described above. The methanolic supernstsnt was sdmixed in a rstio of 10:1 with s 1.0 mole/litre solution of HgCl 2 st a pH of 1.5 to form a precipitate, snd the siεlic scid content of the precipitate was measured. A value of 33.89 mg % was obtained. Example 57 Serum originsting from a pstient with msm sry csncer wεs extrscted with s mixture of chloroform εnd methεnol ss described above . The methsnolic supernstsnt wss admixed in a ratio of 10:1 with a 1.0 mole/litre solution of CuSO^ at a pH of 3.6 to form a precipitate , and the sialic acid content of the precipitate was measured. A value of 37.20 mg % was obtained. Example 58 Serum originating from a patient with mammary csncer was extracted with a mixture of chloroform εnd meths nol as described sbove . The me thsnolic supernatant was admixed in a rεtiό of 10: 1 with a 1.0 mole/litre solution of CaCl 2 a t a pH of 5.3 to form a precipitate , and the sialic acid content of the precipita te was measured. A value of 28.18 mg % was obtained. Example 39 Serum origina ting from a patient with mεmmεry cεncer was extracted with a mixture of chloroform and me thanol as described above . The methεnolic supernatant was admixed in a rstio of 10:1 with a 1.0 mole/litre solution of MgCl 2 a t s pH of 6.5 to form ε precipits te , and the sislic scid content of the precipitate was measured. A value of 25.67 mg % was obtsined. Example 40 Serum originating from a patient with ovεrian cancer was extracted with a mixture of chloroform and methsnol ss described sbove . The methsnolic supernatant was sdmixed in s rstio of 1C:1 with ε 1.0 mole/litre solution of EiCl 2 st s pH of 5.3 to f orm a precipitste , snd the siεlic scid content of the precipits te wss measured. A vslue of 22.09 mg % wss obts ined. Example 41 Serum originsting from a patient with ovarian cancer was extrscted with a mixture of chloroform and methanol as described above. The methanolic supernstsnt was admixed in a ratio of 10:1 with a 1.0 mole/litre solution of CdCl at a pH of .3 to form a precipitate, and the sialic acid content of the precipitate wss measured. A vslue of 25.80 mg % was obtained. Example 42 Serum origina ting from a patient with ovarian cancer wss extrscted with a mixture of chloroform and me thsnol. as described above . The methanolic superna tant was sdmixed in a rεtio of 10:1 with a 1.0 mole/litre solution of FeSO^ at a pH of 2.9 to form a precipita te , snd the sislic s cid content of the precipitεte wss messured. A value of 18.75 mg % was obta ined. Example 43 Serum originating from a patient with ovεrisn csncer wss extrscted with a mixture of chloroform and methsnol as described above . The methεnolic supernatsnt ws s admixed in a rεtio of 10:1 with a 1.0 mole/litre solution of ZnCl 2 at a pH of 1.7 to form a precipitste , and the siεlic acid content of the precipita te wa s measured. A vslue of 23.77 mg % was obtsined. Example 44 Serum originating from a pa tient with ovsrisn cε ncer wss extracted with s mixture of chloroform and me thsnol as described above . The me thanolic supernatant was admixed in a rstio of 10:1 with a 1.0 mole/litre solution of ZnSO^ a t a pH of 1.7 to form a precipita te , and the sialic acid content of the precipitate was measured. A value of 26.87 mg % was obtained. Example 45 Serum origina ting from s ps tient with ovsrisn cancer wss extrscted with a mixture of chloroform and me thanol as described above . The methsnolic superna tant was admixed in a ratio of 10:1 with a 1.0 mole/litre solution of ?b (I 0,) 2 at a pH of 3.5 to form s precipitste , end the sialic acid content of the precipita te was measured. A value of 26.63 mg % was obtained. Example 46 Serum originating from a patient with ovarisn cεncer wss extrscted with a mixture of chloroform and methεnol as described above . The methsnolic supernstsnt wss sdmixed in a ratio of 10:1 with s 1.0 mole/litre solution of gl O^ at a pH of 5.8 to form a precipits te , and the sialic εcid content of the precipitate was measured. A vslue of 28.66 mg % was obtsined. Example 47 Serum originsting from a patient with ovarian cancer wε s extrεcted with a mixture of chloroform εnd methεnol εs described sbove. The methεnolic supernatant was admixed in a rεtio of 10:1 with a 1.0 mole/litre solution of HgCl 2 at a pH of 1.5 to form a precipitste, snd the sialic acid content of the precipitate was measured. A value of 33.08 mg % was obtsined. Exsmple 48 Serum originating from a patient with ovarian csncer was extracted with a mixture of chloroform snd methsnol as described sbove. The methεnolic supe nstεnt was sdmixed in a ratio of 10:1 with a 1.0 mole/litre solution of CuSO^ at a pH of 3»6 to form s precipitste, and the sialic εcid content of the precipitate was measured. A value of 26.63 mg % was obtained. Exsmple 49 Serum originsting from s patient with ovεriεn cεncer was extrεcted with s mixture of chloroform εnd methεnol ss described εbove. The methεnolic supernstsnt wss sdmixed in s rεtio of 10:1 with s 1.0 mole/litre solution of CaCl 2 st ε pH of 5.3 to form a precipitste, and the siεlic εcid content of the precipitate wss meεsured. A vεlue of 13.26 mg % wss obtsined. Example 50 Serum originating from a pεtient with ovsrian cεncer wεs extrεcted with a mixture of chloroform εnd methεnol as described above. The methεnolic supernεtεnt wεs εdmixed in ε rεtio of 10:1 with s 1.0 mole/litre solution of IύgCl 2 st ε pH of 6.5 to form ε precipitate, end the εiεlic εcid content of the precipits te wss messured. A vslue of 11.82 mg % wss obtsined. Exsmple 51 Serum originsting from s ps tient ?;ith head/neck cεncer wεs extrεcted with s mixture of chloroform snd me thsnol ss described εbove . The me thεnolic supernstsnt wεs sdmixed in a ratio of 10:1 with a 1.0 mole/litre solution of C1 2 at ε pH of 5.3 to form a precipitate , and the sialic εcid content of the precipitεte wεs measured. A value of 12.62 mg % wε s obtained. Example 52 Serum originating from a pstient with hesd/neck csncer was extrscted with a mixture of chloroform and me thanol as described εbove . The methanolic suparnatsnt wss sdmixed in a rs tio of 10 :1 with a 1.0 mole/litre solution of CdCl 2 at s pH of 5.3 to form a precipitate , snd the siεlic acid content of the pre cipitate was measured. A vεlue of 21.99 mg % was obtained. Example 55 Serum origins ting from a patient with heεd/neck cεncer wss extrscted with a mixture of chloroform and methanol as described above . The methsnolic εupernεtεrit wε s sdmixed in s rεtio of 10:1 with a 1.0 mole/litre solution of FeSO^ at a pH of 2.9 to f orm ε precipitste , εnd the sislic scid content of the precipits te wss measured. A value of 16.09 mg % wεs obtsined. Exsmple 54 Serum origins ting from a ps tient with head/neck cance was extrεcted with ε mixture of chloroform εnd methsnol as described above . The methεnolic supernstsnt wss sdmixed in s rεtio of 10:1 with s 1.0 mole/litre solution of ZnCl 2 st ε pH of 1.7 to form a precipita te , and the sialic scid content of the precipitate wss eεsured . A vεlue of 19.56 mg % wε s obtεined. Exε ple 55 Serum originsting from ε pεtient with head/neck cεnce wss extracted with a mixture of chloroform snd methanol εs described above . The methsnolic supernstsnt wεs sdmixed in s rεtio of 10:1 with a 1.0 mole/litre solution of ZnSO^ s t a pH of 1.7 to form a precipitate , εnd the sislic εcid content of the precipitεte wεε mes sured. A vεlue of 21.41 mg % ws s obtained. Example 56 Serum originsting from a patie nt with heεd/neck cance wss extra cted with a mixture of chloroform snd methsnol ss de scribed sbove . The methsnolic supernstsnt ws s sdmixed in ε rstio of 10:1 with a 1.0 mole/litre solution of Pb(lTO 5 ) 2 at a pH of 3.5 to form a precipitate , snd the sislic scid content of the precipita te was eεεured. A vεlue of 29.98 mg % wss obtsined. Example 57 Serum originsting from a pstient with hesd/neck csnce s s extrεcted with s mix ture of chloroform and methsnol ss described above . The methanolic supernatant wss sdmixed in a rεtio of 10: 1 with a 1.0 mole/litre solution of Agl O, st a pH of 5.8 to form a precipitate , and the sislic εcid content of the precipitste wss measured. A value of 14.93 mg % was obtained. Example 58 Serum origina ting from a patient with head/neck cancer was extrs cted with a mixture of chloroform εnd methεnol as described above . The methεnolic superna tant was admixed in a rεtio of 10:1 with a 1.0 mole/litre solution of HgCl 2 at a pH of 1.5 to form a precipitate , snd the sislic scid content of the precipitate was measured. A value of 18.87 g % was obtained. Example 59 Serum origina ing from a patient with head/neck csncer wss extrscted with a mixture of chloroform εnd methsnol as described above . The methanolic supernatant wss εdmixed in a ratio of 10 :1 with a 1.0 mole/litre solution of CuSO^ at a pH of 3.6 to form s precipits e , snd the sislic acid content of the precipitste was measured. A value of 14.70 mg % was obtained. Example 60 Serum originating from a patient with head/neck cancer was extracted with a mixture of chloroform and me thsnol as described sbove . The methεnolic supernεtεnt wεs εdmixed in s rstio of 10:1 with a 1.0 mole/litre solution of CaCl 2 at s pE of 5 * 3 to form ε precipitste , snd the sislic εcid content of the precipitate was messured. A value of 9.72 mg % was obtained. Example 61 Serum originating from a patient with head/neck cancer was extracted with a mixture of chloroform and methsnol as described above. The methεnolic supernstsnt was admixed in a ratio of 10:1 with a 1.0 mole/litre solution of MgClg at a pH of 6.5 to form a precipitate, and the sialic acid content of the precipitate wss messured. A vslue of 12.27 mg % was obtained. Example 62 Serum originating from a patient with de no carcinoma wss extracted with a mixture of chloroform εnd methsnol as described above. The methanolic supernstsnt was εdmixed in a rεtio of 10:1 with a 1.0 mole/litre solution of KiCl 2 at a pH of 5.3 to form a precipitate, and the sislic scid content of the precipitste wεs messured. A value of 11.77 mg % wss obtained. Example 63 Serum originating from a patient with εdenocarcinoma was extracted with a mixture of chloroform and methanol as described above. The methεnolic supernatant was admixed in a rstio of 10:1 with a 1.0 mole/litre solution of CdCl 2 at a pH of 5.3 to form a precipitate, and the sislic acid content of the precipitste wss messured. A vεlue of 20.09 mg % was obtained. Example 64 Serum originsting from s patient with a deno carcino a was extracted with a mixture of chloroform and methanol as described above . The methεnolic supernatant wss sdmixed in a rstio of 10: 1 with a 1.0 mole/litre solution of FeSO^ at a pH of 2.9 to form a precipitste , and the siεlic scid content of the precipitate was measured. A vslue of 16.13 mg % was obtained. Example 65 Serum originating from a patient with sdenocsrcinoma wss extracted with a mixture of chloroform and methanol as described above. The methsnolic supernstsnt was admixed in s rεtio of 10:1 with a 1.0 mole/litre solution of ZnCl 2 at a pH of 1.7 to f orm s precipitate , and the sialic acid content of the precipita te was measured. A value of 18.69 mg % was obtained. Example 66 Serum originating from a patient with adenocarcinoma was extracted with a mixture of chloroform εnd methsnol ss described sbove . The methsnolic supernstsnt was εdmixed in a rstio of 10 :1 with a 1.0 mole/litre solution of ZnSO^ at s pH of 1.7 to form a precipitate , snd the siεlic scid content of the precipits te was messured. A vslue of 17.15 mg % was obtsined. Exsmple 67 Serum origins ting from s patient with adenocsrcinoms wεs extrε cted with a mixture of chloroform εnd methεnol ss described above. The methsnolic supernstsnt wss sdmixed in s rstio of 10:1 with a 1.0 mole/litre solution of Pb(ΪJOz) 2 at a pH of 3«5 to form a precipitate, and the siεlic acid content of the precipitate was easured.A value of 22.66 mg % was obtained. Example 68 Serum originating from a patient with sdenocsrcinoma wss extracted with a mixture of chloroform and methsnol as described above. The methanolic supernatant was admixed in a rεtio of 10:1 with a 1.0 mole/litre solution of gNO at ε pH of 5.8 to form a precipitate, and the sialic acid content of the precipitate was measured. A vslue of 16.51 mg % wss obtained. Example 69 Serum originating from a patient with sdenocsrcinoma was extrεcted with s mixture of chloroform and methanol as described above. The methanolic supernatant wss sdmixed in s rεtio of 10:1 with a 1.0 mole/litre solution of HgCl 2 εt ε pH of 1.5 to form s precipitste, and the sislic acid content of the precipitste wss messured. A vslue of 18.94 g % was obtained. Example 70 Serum originating from a patient with a deno carcinoma wss extrscted with a mixture of chloroform and methsnol as described εbove. The methsnolic supernstsnt was sdmixed in s rstio of 10:1 with a 1.0 mole/litre solution of CuSO^ εt e pH of 3.6 to form a precipitate, and the sialic acid - 3? - content of the precipitste wss messured. A vslue of 17.41 mg % was obtained. Example 71 Serum originsting from a ps tient with sdenocsrcinoma ws s extracted with a mixture of chloroform and methanol as described above . The methsnolic supernstsnt was admixed in a ra tio of 10: 1 with a 1.0 mole/litre solution of CsCl 2 at a pH of 5.3 to form s precipits te , and the sislic scid content of the precipitste wss measured. A vεlue of 9.73 mg % wss obtained. Example 72 Serum originεting from a patient with sdenocsrcinoma wss extrs cted with s mixture of chloroform εnd methsnol as described above . The methsnolic superns tsnt was sdmixed in s rεtio of 10 :1 with a 1.0 mole/litre solution of gCl 2 at a pΞ of 6.5 to form a precipitste , snd the sislic scid content of the precipits te wss mes sured. A vεlue of 7.94- mg % was obtained. Example 73 Serum originating from a patient with colon cancer was extracted with a mixture of chloroform and methεnol as described above . The me thanolic supernatant was εdmixed in a ratio of 10:1 with a 1.0 mole/litre solution of NiCl 2 at a pH of 5.3 to f orm ε pre cipitε te , and the siεlic εcid content of the precipitate wεs measured. A vεlue of 20.70 mg % was obtained. Example 74 Serum originating from s pstient with colon cεncer wss extrscted with s mixture of chloroform and methsnol as described above. The methεnolic supernstsnt wεs εdmixed in ε rstio of 10:1 with a 1.0 mole/litre solution of 0dCl 2 st a pH of 5.3 to form a precipitate, εnd the siεlic εcid content of the precipitεte wss messured. A vslue of 32.14 mg % wεs obtained. Exsmple 75 Serum originsting from a pεtient with colon cancer was- extracted with a mixture of chloroform and methsnol εs described sbove. The methsnolic supernstsnt wεs admixed in a rεtio of 10:1 with a 1.0 mole/litre solution, of FeSO^ at ε pH of 2.9 to form a precipitate, and the sialic scid content of the precipitate wss messured. A vεlue of 24.23 mg % wεs obtained. Exεmple 76 Serum originsting from s pstient with colon csncer wss extrscted with s mixture of chloroform snd methsnol as described sbove. The methsnolic supernstsnt wss sdmixed in s rstio of 10:1 with a 1.0 mole/litre solution of ZnCl 2 at a pH of 1.7 to form ε precipitate, and the sialic scid content of the precipitste wss messured. A vslue of 27.52 mg % wss obtεined. Sxεαple 77 Serum originating from ε pεtient with colon cεncer wεs extrεcted with s mixture of chloroform εnd methεnol as - 5 - described sbove . The methεnolic supernstsnt wεs εdmixed in s rεtio of 10: 1 with ε 1.0 mole/litre solution of ZnSO^ at a pH of 1.7 to form a precipits te , snd the sislic scid content of the precipitste wss messured. A vεlue of 3.63 mg % wss obtained. Example 78 Serum originating from s patient with colon cεncer was extrεcted with s mixture of chloroform end methεnol ε s described εbove . The methεnolic supernstεnt wss admixed in a rstio of 10 :1 with a 1.0 mole/litre solution of Pb (l 0,) 2 st a pH of 3 -5 to form a pre cipitste , εnd the sislic εcid content of the precipitste wss measured. A vslue of 35 * 55 mg % wss obtained. Example 79 Serum originsting from s pstient with colon cεncer wss extrεcted with ε mixture of chloroform and methanol ss described sbove . The .methsnolic supernεtεnt was εdmixed in s rεtio of 10:1 with a 1.0 mole/litre solution of gNO^ at a pH of 5.8 to form a precipits te , and the siεlic εcid content of the precipitste was measured. A value of 29.46 g % wss obtained. Example 80 Serum origins ting from a pstient with colon cancer was extrεcted with a mixture of chloroform snd methsnol as described sbove. The methεnolic supernatant wss sdmixed in a rstio of 10: 1 with s 1.0 mole/litre solution of HgCl ε t s pH of 1.5 to form a precipitate , εnd the εiolic εcid content of the precipitste was measured. A vεlue of 31.66 mg % was obtsined. Example 81 Serum originating from s pεtient with colon cεncer wεs extrscted with a mixture of chloroform and methanol as described above. The methsnolic supernεtεnt wεs εdmixed in s rεtio of 10:1 with a 1.0 mole/litre solution of CuSO^ st s pH of 3.6 to form ε precipitste, εnd the siεlic εcid content of the precipitate was measured. A value of 32.51 g % was obtained. Example 82 Serum originsting from a patient with colon csncer wεs extrscted with a mixture of chloroform and methsnol ss described εbove. The methsnolic supernstsnt wss εdmixed in s rstio of 10:1 with a 1.0 mole/litre solution of CsCl 2 st s pH of 5.3 to form a precipitste, snd the sislic scid content of the precipitste wss messured. A vslue of 18.51 mg % wss obtsined. Exsmple 83 Serum originating from a pstient with colon csncer was extracted with a mixture of chloroform and methsnol as described above. The methanolic supernatant wss sdmixed in ε rεtio of 10:1 with a 1.0 mole/litre solution of MgCl 2 εt a pH of 6.5 to form ε precipitate, εnd the sislic acid content of the precipitate wss messured. A vεlue of 16.68 mg f wss obtained.

Claims: VTha we claim is : 1. A me thod for the selective de termination of sielic acid-cεrrying glycolipid complexes in blood, in which the serum is diluted with water, then extracted with a water-im- miεciblθ organic solvent or solvent mixture to remove neutral lipids, a lipoprotein fraction contεining sialic scid-cεrry- ing glycolipids is precipitated from the aqueous phase , the precipita ted fraction is redissolved, snd the sislic scid content of the solution or the εmount snd quality of the sislic scid-csrrying glycolipid is (sre) determined by s metho .known per se , chεrscteriεed in thεt s wster-soluble εslt of s monovslent, bivalent or trivεlent metal other than alkali me tal is applied as precipitating agent in sn amount corresponding to s final etεl ion concentrεtion of 0.005-0.1 mole/1 in the mixture , εnd precipitation is per¬ formed st ε pH of 1.5 to 8.0. •i 2. A method ss clsi ed in clsim 1, characterised in that a water-soluble Fe 2+ sslt is spplied ss precipitating sgent, snd precipitation is performed εt a pH of 3«0 to 3 * 5 * 3o A method as claimed in claim 1, characterised in that a water-soluble Fe^ salt is applied as precipitating sgent, snd precipitstion is performed st a pH of 3 * 0 to 5.0. 4. ϋ method ss clsimed in clsim 1, chsrεcterised in thεt ε wster-soluble Co 2+ sslt is spplied as precipitating sgent, snd precipitstion is performed st s pH of 4.0 to 5.5. 5. A method ss claimed in clsim 1, chεrecteriεed in that a wster-soluble Hi 2+ salt is spplied ss precipitating sgent, εnd precipitstion is performed at a pH of 4.5 to 5 * 5. 60 A method εs clsimed in clsim 1, characterised in thεt a water-soluble Cd 2-t* sslt is spplied ss precipitating sgent, εnd precipitation is performed at a pH of 4.5 to 6.0. 7. method ss clsimed in claim 1, characterised in thεt a water-soluble Zn 2+ salt is applied as precipitating sgsntj.εnd precipitation is performed at a pΞ of 1.5 to 5 * 0. 8. A method ss claimed in clsim 1, characterised in that ε wster-soluble Ag + salt is spplied ss precipitεting εgent, εnd precipitεtion is performed st s pH of 6.0 to 8 0 0. 9. A method as claimed in clsim 1, chsrεcterised in that ε wster-soluble Eg 2+ sslt is applied ss precipitεting agent, and precipitation is performed at a pB of 1.5 to 3 * 0. 10. A method as claimed in claim 1, characterised in thst a wster-soluble Cu 2+ εslt is spplied as precipitating sgent, snd precipitstion is performed εt s pH of 3 * 0 to 4.5. 11. A method ss clsimed in clsim 1, chsrsctariεed in thst s water-soluble Cs 2+ εεlt is εpplied ss precipitεting sgent, snd precipitstion is performed st ε pE of 4.5 to 5 * 5. 12. A method as claimed in clεim 1, characterised in thεt a water-soluble Li sslt is εpplied εs precipitating sgent, and precipitstion is performed εt a pH of 5 * 5 to 7 * 0.

Inventors: BABARCZI JOLAN, GESZTESI GYULANE, IVONY JOZSEFNE, KINCZEL EDIT, MILOVAN IREN, PRIBEK FERENC, SCHUMANN BELA, SIKLOSINE PATAY EDIT, SZAJANI BELA, BABARCZI, JOLAN, GESZTESI, GYULANE, IVONY, JOZSEFNE, KINCZEL, EDIT, MILOVAN, IREN, PRIBEK, FERENC, SCHUMANN, BELA, SIKLOSINE, PATAY, EDIT, SZAJANI, BELA
Applicants: REANAL FINOMVEGYSZERGYAR
Application year: 1989
Main classification: G01N33
IPC codes: G01N33