ANALYTICAL METHOD FOR THE SELECTIVE DETERMINATION OF
SIALIC ACID-CARRYING GIYCOLIPID COMPLEXES
The invention relates to an analytical method for the selective determination of sialic acid-carrying glycolipid complexes.
Sialic acid (N-acetyl neuraminic acid) appears in 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/) the se compounds can be classif ied into the groups of monosialogangliosides, disialogangliosides and trisialogangliosides. The hydrophobic part of gaoglioside 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-ecetyl 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 elaborated to determine the glycolipid-bound sialic acid content of blood. According to the method of
Svennerholm (Biochem. Biophys. Acta 24, 604-611 /1957/) blood plasm is extracted with a 2:1 mixture of chloroform end methanol to separate glyco sphingolipids from the other lipid fractions of blood, and the sialic acid content of glycosphingolipids is measured by a colorime tric reaction
produced with resorcine in the presence of a copper (II) salt . According to Katopodis (USP 4,342 ,567) the glycosphingolipid fraction is precipitated first from the me thanolic 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 be determined, however, by enzymatic reactions, too. According to the me thod disclosed in Clin. Chim. Acta 108, 493-498 (1980) the glycoprotein is split with neur¬aminidase 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 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 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 (value s below
18 mg % can be regarded as normal , whereas tho se exceeding 20 rag % are pathologic) , the results may refer to the existence of a malign disease but cannot be applied as an unambiguous proof for the existence of tumorous or tumour-free state o It is s further dissdvantage that certain benign
disease s , such as arthri tis, psoriasis , ulcer, inflamma
tions and gynaecological 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 elaborated 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.
Our aim was to elaborate an analytical method which may lead to a more reliable diagnosis, enables one to recognize the existence of tumor in a very early stage, gives reliable information on distinguishing tumorous and tumor- -free states from, one another and enables one to conclude on the probable localization of the tumor.
The invention is based on the recognition that, at an appropriate pH and ionic strength, different metal 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 determining the nature of the glycolipid to which sialic acid is bound conclusion can be drawn whether an increase in sialic acid level can be attributed to a tumorous process or to a benign disease (such as inflammation, arthritis, a gynaecological disorder, etc.), and in tumorous processes conclusion can be drawn on the Drobable localization of the
tumor or on the existence of metastasis . 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 precipitating agent.
Based on the above, the invention relates to a method for the selective determination 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-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 sa. According to the invention a water-soluble salt of a monovalant , 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/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 methanol is utilized as extracting agent .
The equeous methanolic supernatant (in the f ollowing: the methanolic supernatant) is processed further in the precipite
tion step, whereas the lower phase which is a chloroform solution of neutral lipids is discarded.
Thereaf ter the precipitating agent is added to the aqueous methanolic phase or to an aliquot thereof in an amount to obtain a mixture containing 0.005-0.1 mole/l of metal ion. The precipitating agent is utilized preferably as an aqueous solution. For some metal ions the preferred pH value s of precipitation are listed in the table below.
Metal ion pH
Fe2+ 3.0-3.5
Fe3+ 3.0-5.0
Co2+ 4.0-5.5
M2+ 4.5-5.5
Cd2+ 4.5-6.0
Zn2+ 1.5-5.0
Ag+ 6.0-8.0
Hg2+ 1.5-3.0
Cu2+ 3.0-4.5
Ca2+ 4.5-5.5
Mg2+ 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 as on the basis of the colour reaction produced with a mineral acid, a copper(II) salt and re sorcine as described in USP 4,342 , 567, or by the enzymatic me thod disclosed in Clin. Chim. Acta 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 chromatography as described in Clin. Chem. 22, 1516-1521 (1976) .
The differential diagnostic value of the me thod according 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 precipitating agent) are added up. According to our experiences the value of
decreases (for localized tumors) in the following order: mammary tumor - - colon tumor - ovarian tumor - head/neck tumor - adenocarcinoma - non-tumorous disease - healthy tumor-free state .
Similarly, a differential diagnostic series can be obtained when the results of the sialic acid determination according to the invention, performed ?άth at least five different metal ions , are divided with the result of a known sialic acid determination (e .g. with the result of the determination 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 elucidated in detail by the aid of the following non-limiting examples. In the examples the
blood samples were pre-treated 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 lipidbound 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 bidistilled 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 and methanol 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 bidistilled water pre-cooled to +4°C was added to the individual samples , the contents of the test tubes were stirred with a Vortex stirrer for 15 seconds, and 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 3000 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 adjusted 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 separated completely from the precipitate by decanting (the supernatant 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 dissolving 10 mmoles of tris (hydroxymethyl) 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 mg of analytically pure sialic acid were dissolved in 100 ml of bidistilled water, and the resulting solution was utilized as stock solution to prepare two further dilutions (25 mg/100 ml and 12.5 mg/100 ml in concentration) with bidistilled water. The standard solutions were stored at +4°C. Samples of 0.2 ml, each, of the standard solutions with three different concentrations were filled into
centrifuge tubes, and the volume of the solution was adjusted to 1 ml with bidistilled water in each of the centrifuge
tubes. 1 ml of bidistilled water was applied as blank.
1 ml, each, of resorcin reagent solution was added to all of the samples (standard, blank, control, test serum) . The resorcin reagent solution was 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 and 9.75 ml of bidistilled water, and 100 ml of anslytically pure concentrated aqueous hydrochloric acid were added to the mixture .
The test tubes were placed into a 100°C water bath for exactly 10 minutes . Thereafter all of the test tubes were immediately 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 and 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 at 2500 r.p .m. , and then the blue supernatants were filled from each centrifuge tube into a measuring cell. The. density of the blue colour formed doe s not change within 6 hours .
Photome tric measurement was performed at a wavelength of 580 nm. The zero point of the photometer was adjusted for bidistilled water . Thereafter, depending on the
type of the photome ter 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 :
E( corr) = E(measured) - S(blank)
Sialic acid content was calculated by one of the following two methods :
1) From the re sults obtained with s tandard sialic acid samples of known sialic acid concentrations, extinc tion vs . concentration curves were constructed either graphically or by calculation. Due to the dilution steps , the original concentrations should be recalculated according to the following table :
Original concentraMeasured concentration (mg/100 ml) tion ( μg/200 ml) Extinction 50 100 0.508
25 50 0.252
12.5 25 0.127
From the calibration curve construc ted as described above the sialic acid content (A) of the supernatant sample , 1 ml in volume , can be determined. The curve gives the sialic acid content in units of μg of sialic acid/l ml of supernatant. The sialic acid content of the blood serum can be calculated from the formula
Sialic acid content (mg/100 ml) = A x 0.72 ,
where A is the sialic acid content of the supernatant in units of μg/ml and 0.72 is a factor which comprises the degree of dilution and the c onversion of μg to mg .
2) According to the second method of calculation 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 sialic acid content of the sample to be tested can be calculated 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 a mixture of chloroform and me thanol as described above . The methanolic supernatant was admixed in a ratio of 10:1 with a 0.5 mole/litre solution of NiCl2 at a pH of 3<>5 to form a precipitate , and the sialic acid content of the precipitate was measured. A vslue of 4.65 mg % was obtained.
Example 2
Serum originating from a healthy parson 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 1.0 mole/litre solution of NiCl2 at a. pH of 5.3 to form a precipitate, and the sialic acid content of the precipitate 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 and methanol as described above. The methanolic supernatant was admixed in a ratio of 10:1 with a 0.1 mol/litre solution of CdCl2 at a pH of 5.9 to form a precipitate, and the sialic acid content of the precipitate 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 and me thanol as de scribed above . The me thanolic supernatant was admixed in a ratio of 10:1 with a 0.5 mole/li tre s olution of CdCl2 at a pH of 5.5 to form a precipitate , and the sialic acid content of the precipitate 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 me thanolic supernatant was admixed in a ratio of 10:1 with a 1.0 mole/litre solution of CdCl2 at a pH of 5.3 bo form a precipitate , and the sialic acid content of the precipitate 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 me thanol as described above . The me thanolic supernatant was admixed in a ratio of 10: 1 with a 0.1 mole/litre solution of FeSO4 at a pH of 3.3 to form a precipitate , and the sialic acid c ontent of the precipitate was measured. A vslue of 9.08 mg % was obtained.
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 FeSO4 at a pH of 3.0 to form a precipitate, and the sialic acid content of the precipitate was measured. A value of 9.54 mg % was obtained.
Example 8
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 FeSO4 at a pH of 2.9 to form a precipitate, and the sialic scid content of the precipitate was measured. A value of 9.52 mg % was obtained.
Exsmple 9
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 0.1 mole/litre solution of ZnCl2 at a pH of 2.7 to form a precipitate, and the sialic acid content of the precipitate 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 and methsnol as described above. The methanolic supernatant was admixed in a ratio of 10:1 with a 0.5 mole/litre solution of ZnCl2 at a pH of 1.9 to form a precipitste, and the sialic acid content of the precipitate was measured. A value of 12.34 mg % was obtained.
Example 11
Serum originating from a healthy person was extracted with a mixture of chloroform end me thanol as described above . The me thanolic supernatant was admixed in a ratio of 10: 1 with a 0.1 mole/litre solution of Pb (NO3) 2 at a pH of 4.4 to form a precipitate , and the sialic acid content of the precipitate was measured. A vslue of 8.61 mg % was obtained.
Example 12
Serum originating from a healthy person was extracted with a mixture of chloroform and me thanol as de scribed above . The me thanolic supernatant was admixed in a ratio of 10:1 with a 0.5 mole/litre solution of Εb (NO3) 2 at a pH of 3.7 to form a pre cipitate , and the sialic acid content of the precipitate was measured. A value of 12.11 mg % was obtained.
Example 13
Serum origins ting from a healthy person was extracted with a mixture of chloroform and me thanol as described above . The me thanolic supernatant was admixed in a ratio of 10:1 with a 1.0 mole/litre solution of Pb (NO3) 2 at a pH of 3.5 to form a precipitate , and the sialic acid content of the precipitate was measured. A value of 12.35 mg % was obtained.
Example 14
Serum originating from a healthy person was extracted with a mixture of chlorof orm and me thanol as described above . The me thanolic supernatant was admixed in a ratio of 10: 1 with a 0.1 mole/litre solution of AgNO3 at a pH of 8.5 to form a precipitate and the sialic ac id content of the precipitate was measured. A value of 9.89 mg % was obtained.
Example 15
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 AgNO3 at a pH of 6.0 to form a precipitate , and the sialic acid content of the precipitate was measured. A value of 14.67 mg % was obtained.
Example 16
Serum originating from a healthy person was extracted with a mixture of chloroform and me thanol as described above . The methanolic supernatant was admixed in s ratio of 10: 1 with a 1.0 mole/litre solution of AgNO3 at a pH of 5.8 to form a precipitate , and the sialic acid content of the precipitate was measured. 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 methanolic supernstant was sdmixed in a ratio of 10: 1 with a 0.1 mole/litre solution of HgCl2 at a pH of 1.9 to form a precipitate , and the sialic acid content of the precipitate was measured. A value of 6.05 mg % was obtained.
Example 18
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 ,3 mole/litre solution of HgCl2 at a pH of 1.7 to form a precipitate, and the sialic acid content of the precipitate was measured. A value of 7.68 mg % was obtained.
Example 19
Serum originating from a healthy person was extracted with a mixture of chloroform and me thanol as described above . The me thanolic supernatant was admixed in a ratio of 10: 1 with a 1.0 mole/litre solution of HgCl2 at a pH of 1.5 to f orm a precipitate , and the sialic acid c ontent of the precipitate was measured. A value of 11.99 mg % was obtained.
Example 20
Serum originating from a healthy person was extracted . with a mixture of chloroform end methanol as described above. The me thanolic supernatant was admixed in a ratio of 10:1 with a 0.1 mole/litre solution of CuSO4 at a pH of 4.2 to form a precipitate , and the sialic acid content of the precipitate was measured . A value of 10.13 mg % was ob tained.
Example 21
Serum originating from a healthy person was extracted with a mixture of chloroform and me thanol as described above. The me thanolic supernstant was admixed in a ratio of 10: 1 with a 0.5 mole/litre solution of CuSO4 at a pH of 3.8 to form a precipitate , and the sialic acid content of the precipitate was measured. A value of 10.48 mg % was obtained.
Example 22
Serum ori ginating from a healthy person was extracted with a mixture of chloroform and me thanol as de scribed above. The me thanolic supernatant was admixed in a ratio of 10: 1 with a 1,0 mole/litre solution of CuSO4 at a pH of 3.6 to form a pre cipitate , and the sialic acid content of the prec ipitate was measured. A value of 12.22 mg % was obtained.
Example 23
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 0.1 mole/litre solution of CaCl2 at a pH of 5.5 to form a precipitate, and the sialic acid content of the precipitate was measured. A value of 3.72 mg % was obtained.
Example 24
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 CaCl2 at a pH of 5.4 to form a precipitate, and the sialic acid content of the precipitate was measured. A value of 3.02 mg % was obtained.
Example 25
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 CaCl2 at a pH of 5.3 to form a precipitate, and the sialic acid content of the precipitate was measured. A value of 7.05 mg % was obtained.
Example 26
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 0.1 mole/litre solution of MgCl2 at a pH of 6.1 to form a precipitate, and the sialic acid content of the precipitate was measured. A value of 4.19 mg 55 was obtained.
Example 27
Serum originating from a healthy person was extracted with a mixture of chloroform and methanol as described above The methsnolic supernatant was admixed in a ratio of 10:1 with a 0.5 mole/litre solution of MgCl2 at a pH of 6.7 to form a precipitate, and the sialic acid content of the precipitate was measured. A value of 3.61 mg % was obtained.
Example 28
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 MgCl2 st a pH of 6.5 to form a precipitate, and the sialic scid content of the precipitate was measured. A value of 4.81 mg % was obtained.
Example 29
Serum originating from s patient with mammary cancer 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 NiCl2 at a pH of 5.3 to form a precipitate, and the sialic acid content of the precipitate was measured. A value of
28.87 mg % was obtained.
Example 50
Serum originating from a patient with mammary cancer was extracted with a mixture of chloroform and methanol as described above. The methanolic supernatant was admixed in a ratio of 10:1 with s 1.0 mole/litre solution of CdCl2
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 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 FeSO^ at a pH of 2.9 to form a precipitate , and the sialic acid content of the precipitate was measured. A value of
33 *39 mg % was obtained.
Example 32
Serum originating from a patient with mammary cancer 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 ZnCl2 at a pH of 1.7 to form a precipitate , and the sialic acid content of the precipitate was measured. A value of
39.41 mg % was obtained.
Example 35
Serum originating from a patient with mammary cancer 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 ZnSO4 at a pH of 1.7 to form a precipitate , and the sialic acid content of the precipitate was measured. A value of
38.90 mg % was obtained.
Example 54
Serum originating from a patient with mammary cancer was extracted with a mixture of chloroform and methanol as described above . The me thanolic supernatant was admixed in a ratio of 10:1 with a 1.0 mole/litre solution of Pb (NO3)2 at a pH of 3.5 to form a precipitate , and the sialic acid content of the precipitate was measured. A value of
45.02 mg % was obtained.
Example 35
Serum originating from a patient with mammary cancer 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 AgNO3 at a pH of 5.8 to form a precipitate , and the sialic acid content of the precipitate was measured. A value of
29.58 mg % was obtained.
Exsmple 36
Serum originating from a patient with mammary cancer was extracted with a mixture of chloroform and me thanol as described above . The methanolic supernatant was admixed in a rstio of 10: 1 with s 1.0 mole/litre solution of HgCl2 a t a pH of 1.5 to form a pre cipitate , and the sialic acid content of the precipitate was measured. A value of
33.89 mg % was obtained.
Example 57
Serum originating from a pa tient with mammary cancer was ex trac ted with a mix ture 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 CuSO4 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 cancer was extracted with a mixture of chloroform and metha nol as described above . The me thanolic supernatant was admixed in a ratio of 10: 1 with a 1.0 mole/litre solution of CaCl2 at a pH of 5.3 to form a precipitate , and the sialic acid content of the precipitate was measured. A value of
28.18 mg % was obtained.
Example 39
Serum originating from a patient with mammary cancer was extracted with a mixture of chloroform and me thanol as described above . The methanolic supernatant was admixed in a rstio of 10:1 with a 1.0 mole/litre solution of MgCl2 at a pH of 6.5 to form a precipita te , and the sialic acid content of the precipitate was measured. A value of
25.67 mg % was obtained.
Example 40
Serum originating from a patient with ovarian cancer 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 NiCl2 at a pH of 5.3 to f orm a precipitate , and the sialic acid
content of the precipita te was measured. A value of
22.09 mg % was obta ined.
Example 41
Serum originating from a patient with ovarian cancer 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 CdCl2 at a pH of 5.3 to form a precipitate, and the sialic acid content of the precipitate was measured. A value of
25.80 mg % was obtained.
Example 42
Serum originating from a patient with ovarian cancer was extracted with a mixture of chloroform and me thanol. as described above . The methanolic supernatant was admixed in a ratio of 10:1 with a 1.0 mole/litre solution of FeSO4 at a pH of 2.9 to form a precipitate , and the sialic a cid content of the precipitate was measured. A value of
18.75 mg % was obtained.
Example 43
Serum originating from a patient with ovarian cancer was extracted with a mixture of chloroform and methanol as described above . The methanolic supernatant wa s admixed in a ratio of 10:1 with a 1.0 mole/litre solution of ZnCl2 at a pH of 1.7 to form a precipitate , and the sialic acid content of the precipitate was measured. A value of
23.77 mg % was obtained.
Example 44
Serum originating from a patient with ovarian ca ncer was extracted with a mixture of chloroform and me thanol as described above . The me thanolic supernatant was admixed in a ratio of 10:1 with a 1.0 mole/litre solution of ZnSO4 at a pH of 1.7 to form a precipitate , and the sialic acid content of the precipitate was measured. A value of
26.87 mg % was obtained.
Example 45
Serum originating from a pa tient with ovarian cancer was extracted with a mixture of chloroform and me thanol as described above . The methanolic supernatant was admixed in a ratio of 10:1 with a 1.0 mole/litre solution of Pb ( NO3)2 at a pH of 3.5 to form a precipitate , end the sialic acid content of the precipitate was measured. A value of
26.63 mg % was obtained.
Example 46
Serum originating from a patient with ovarian cancer 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 Ag NO3 at a pH of 5.8 to form a precipita te , and the sialic acid content of the precipitate was measured. A value of
28.66 mg % was obtained.
Example 47
Serum originating from a patient with ovarian cancer wa s 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 HgCl2 at a pH of 1.5 to form a precipitate , and the sialic acid content of the precipitate was measured. A value of
33.08 mg % was obtained.
Example 48
Serum originating from a patient with ovarian cancer was extracted with a mixture of chloroform and methanol as described above . The me thanolic superna tant was admixed in a ratio of 10 :1 with a 1.0 mole/litre solution of CuSO4 at a pH of 3.6 to form a precipitate , and the sialic acid content of the precipitate was measured. A value of
26.63 mg % was obtained.
Example 49
Serum originating from a patient with ovarian cancer was extracted with s mixture of chloroform and me thanol as described above . The methanolic supernatant was admixed in a ratio of 10: 1 with a 1.0 mole/litre solution of CaCl2 at a pH of 5.3 to form a precipitate , and the sialic acid content of the precipitate was measured. A value of
13.26 mg % was obtained.
Example 50
Serum originating from a patient with ovarian cancer was extracted with a mixture of chloroform and methanol as described above . The me tha nolic superna tant was admixed in a ratio of 10 : 1 with a 1.0 mole/litre so lution of MgCl2 at a pH of 6.5 to f orm a precipitate , and the sialic a cid
content of the precipita te was measured. A value of
11.82 mg % was obtained.
Example 51
Serum originating from a pa tient with head/neck cancer was extracted with a mixture of chloroform and me thanol as described above . The me thanolic supernatant was admixed in a ratio of 10:1 with a 1.0 mole/litre solution of MC12 at a pH of 5.3 to form a precipitate , and the sialic acid content of the precipitate was measured. A value of
12.62 mg % wa s obtained.
Example 52
Serum originating from a patient with head/neck cancer was extracted with a mixture of chloroform and me thanol as described above . The methanolic suparnatant was admixed in a ra tio of 10 :1 with a 1.0 mole/litre solution of CdCl2 at a pH of 5.3 to form a precipitate , and the sialic acid content of the pre cipitate was measured. A value of
21.99 mg % was obtained.
Example 55
Serum origina ting from a patient with head/neck cancer was extracted with a mixture of chloroform and methanol as described above . The methanolic supernatant wa s admixed in a ratio of 10:1 with a 1.0 mole/litre solution of FeSO4 at a pH of 2.9 to f orm a precipitate , and the sialic acid content of the precipita te was measured. A value of
16.09 mg % was obtained.
Example 54
Serum originating from a patient with head/neck cancer 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 ZnCl2 at a pH of 1.7 to form a precipitate , and the sialic acid content of the precipitate was measured . A value of
19.56 mg % was obtained.
Example 55
Serum originating from a patient with head/neck cancer 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 ZnSO4 at a pH of 1.7 to form a precipitate , and the sialic acid content of the precipitate was mes sured. A value of
21.41 mg % was obtained.
Example 56
Serum originating from a patie nt with head/neck cancer was extracted with a mixture of chloroform and methanol as de scribed above . The methanolic supernatant was admixed in a ratio of 10:1 with a 1.0 mole/litre solution of Pb(NO3)2 at a pH of 3.5 to form a precipitate , and the sialic acid content of the precipitate was measured. A value of
29.98 mg % was obtained.
Example 57
Serum originating from a patient with head/neck cancerwas extracted with a mix ture 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 Ag NO3 at a pH of 5.8 to form a precipitate , and the sialic acid content of the precipitate was measured. A value of
14.93 mg % was obtained.
Example 58
Serum originating from a patient with head/neck cancer 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 HgCl2 at a pH of 1.5 to form a precipitate , and the sialic acid content of the precipitate was measured. A value of
18.87 mg % was obtained.
Example 59
Serum originating from a patient with head/neck cancer 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 CuSO4 at a pH of 3.6 to form a precipitate , and the sialic acid content of the precipitate 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 thanol as described above . The methanolic supernatant was admixed in a ratio of 10:1 with a 1.0 mole/litre solution of CaCl2 at a pE of 5.3 to form a precipitate , and the sialic acid
content of the pre cipitate was measured. 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 methanol as described above . The methanolic supernatant was admixed in a ratio of 10: 1 with a 1.0 mole/litre solution of MgCl2 at a pH of 6.5 to form a precipitate , and the sialic acid content of the precipitate was measured. A value of
12.27 mg % was obtained.
Example 62
Serum originating from a patient with a denocarcinoma 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 NiCl2 at a pH of 5.3 to form a precipitate , and the sialic acid content of the precipitate was measured. A value of
11.77 mg % was ob tained .
Example 63
Serum originating from a patient with adenocarcinoma was extracted with a mixture of chloroform and me thanol as described above . The methanolic supernatant was admixed in a ratio of 10:1 with a 1.0 mole/litre solution of CdCl2 at a pH of 5.3 to form a precipitate , and the sialic acid content of the precipitate was measured. A value of
20.09 mg % was obtained.
Example 64
Serum originating from a patient with a denocarcinoma 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 FeSO4 at a pH of 2.9 to form a precipitate , and the sialic acid content of the precipitate was measured. A value of
16.13 mg % was obtained.
Example 65
Serum originating from a patient with adenocarcinoma 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 ZnCl2 at a pH of 1.7 to f orm a precipitate , and the sialic acid content of the precipitate 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 and methanol as described above . The methanolic supernatant was admixed in a ratio of 10 :1 with a 1.0 mole/litre solution of ZnSO4 at a pH of 1.7 to form a precipitate , and the sialic acid content of the precipitate was measured. A value of
17.15 mg % was obtained.
Example 67
Serum originating from a patient with adenocarcinoms 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 soluti on of Pb (NO3) 2 at a pH of 3.5 to form a precipitate , and the sialic acid content of the precipitate was measured. A value of
22.66 mg % was obtained.
Example 68
Serum originating from a patient with adenocarcinoma 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 AgNO3 at a pH of 5.8 to f orm a pre cipitate , and the sialic acid content of the precipitate was measured. A value of
16.51 mg % was obtained.
Example 69
Serum originating from a patient with adenocarcinoma 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 HgCl2 at a pH of 1.5 to form a precipitate , and the sialic acid content of the precipitate was measured. A value of
18.94 mg % was obtained.
Example 70
Serum originating from a patient with adenocarcinoma was extracted with a mixture of chloroform and me thanol as described above . The me thanolic supernatant was admixed in a ratio of 10:1 with a 1.0 mole/litre solution of CuSO4 at a pH of 3.6 to form a precipitate , and the sialic acid
content of the precipitate was measured. A value of
17.41 mg % was obtained.
Example 71
Serum originating from a patient with adenocarcinoma 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 CaCl2 at a pH of 5.3 to form a precipitate , and the sialic acid content of the precipitate was measured. A value of
9.73 mg % was obtained.
Example 72
Serum originating from a patient with adenocarcinoma 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 MgCl2 at a pH of 6.5 to form a precipitate , and the sialic acid content of the precipitate was measured. A value of
7.94 mg % was obtained.
Example 73
Serum originating from a patient with colon cancer was extracted with a mixture of chloroform and methanol as described above . The me thanolic supernatant was admixed in a ratio of 10:1 with a 1.0 mole/litre solution of NiCl2 at a pH of 5.3 to f orm a pre cipitate , and the sialic acid content of the precipitate was measured. A value of
20.70 mg % was obtained.
Example 74
Serum originating from a patient with colon cancer was extracted with a mixture of chloroform and me thanol as de scribed above . The me thanolic supernatant was admixed in a ratio of 10 : 1 with a 1.0 mole/litre solution of CdCl2 st a pH of 5.3 to form a precipitate , and the sialic acid content of the precipitate was measured. A value of
32.14 mg % was obtained.
Example 75
Serum originating from a patient with colon cancer was extracted with a mixture of chloroform and methanol as described above . The me thanolic supernatant was admixed in a ratio of 10: 1 with a 1.0 mole/litre s olution of FeSO4 at a pH of 2.9 to form a precipitate , and the sialic acid content of the precipitate was measured. A value of
24.23 mg % was obtained.
Example 76
Serum originating from a patient with colon cancer was extracted with a mixture of chloroform and me thanol as described above. The methanolic supernatant was admixed in a ratio of 10:1 with a 1.0 mole/litre solution of ZnCl2 at a pH of 1.7 to form a precipitate , and the sialic acid conte nt of the precipitate was measured . A value of
27.52 mg % was obtained.
Example 77
Serum originating from a patient with colon cancer was extrac ted with a mixture of chlorof orm and methanol as
described above . The methanolic supernatant was admixed in a ratio of 10: 1 with a 1.0 mole/litre solution of ZnSO4 at a pH of 1.7 to form a precipitate , and the sialic acid content of the precipitate was measured. A value of
3.63 mg % was obtained.
Example 78
Serum originating from a patient with colon cancer 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 Pb ( NO3) 2 at a pH of 3.5 to form a pre cipitate , and the sialic acid content of the precipitate was measured. A value of
35.55 mg % was obtained.
Example 79
Serum originating from a patient with colon cancer 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 AgNO3 at a pH of 5.8 to form a precipitate , and the sialic acid content of the precipitate was measured. A value of
29.46 mg % was obtained.
Example 80
Serum origins ting from a patient with colon cancer 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 HgCl2 at s pH of 1.5 to form a precipitate , and the siolic acid
content of the precipitate was measured. A value of
31.66 mg % was obtained.
Example 81
Serum originating from s patient with colon cancer was extracted with a mixture of chloroform and methanol as described above . The me thanolic supernatant was admixed in a ratio of 10: 1 with a 1.0 mole/litre solution of CuSO4 at a pH of 3.6 to form a precipitate , and the sialic acid content of the precipitate was measured. A value of
32.51 mg % was obtained.
Example 82
Serum originating from a patient with colon cancer was extracted wi th a mix ture of chloroform and me thanol as de scribed above . The me thanolic supernatant was admixed in a ratio of 10: 1 with a 1.0 mole/litre solution of CaCl2 at a pH of 5.3 to form a precipitate , and the sialic acid content of the precipitate was measured. A value of
18.51 mg % was obtsined.
Example 83
Serum originating from a patient with colon cancer was extrac ted with a mixture of chloroform and me thanol as described above . The me thanolic supernatant was admixed in a ratio of 10:1 with a 1.0 mole/litre solution of MgCl2 at a pH of 6.5 to form a precipitate , and the sialic acid content of the precipitate was measured. A value of
16.68 mg % was obtained.