CN1044537A - The analytical approach of selective determination of sialic acid-carrying glycolipid complexes - Google Patents
The analytical approach of selective determination of sialic acid-carrying glycolipid complexes Download PDFInfo
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- CN1044537A CN1044537A CN89104207A CN89104207A CN1044537A CN 1044537 A CN1044537 A CN 1044537A CN 89104207 A CN89104207 A CN 89104207A CN 89104207 A CN89104207 A CN 89104207A CN 1044537 A CN1044537 A CN 1044537A
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/92—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving lipids, e.g. cholesterol, lipoproteins, or their receptors
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/52—Use of compounds or compositions for colorimetric, spectrophotometric or fluorometric investigation, e.g. use of reagent paper and including single- and multilayer analytical elements
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/66—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving blood sugars, e.g. galactose
Abstract
The present invention is relevant with the method for selectively determining of sialic acid-carrying glycolipid in the blood.In the method, with the serum dilute with water, then, with extracting with immiscible organic solvent of water or mixed solvent, so that remove neutral fats, the lipoprotein composition that contains sialic acid-carrying glycolipid precipitates from aqueous phase, dissolution precipitation thing again, and the sialic acid content in the solution or the character and the content that contain sialic glycolipid can be measured with known method itself.The used precipitation agent of the present invention is monovalence, divalence or trivalent water-soluble metal salt rather than alkalinous metal, and the ultimate density that precipitates metallic ion in the mixed liquor under pH1.5 to 8.0 condition is 0.005-0.1mole/l.
Description
The present invention is relevant with the square analysis method of selective determination of sialic acid-carrying glycolipid complexes.
There is (Advances in Cancer Res.38:289-350 in sialic acid (N-n acetylneuraminic acid n) with free and bonding state in vivo; 1983).Sialic acid can be used as end group and is attached to (J.of Supramolecular Strcture 9:157-177:1978) on the sphingolipid of getting in touch with biological membrane by an oligonucleotide chain.In the literature, the name that comprises the ganglion monoglyceride of sugared sphingolipid is not always clear and definite.According to Svennerholm, these compounds can be divided into a sialic acid ganglion monoglyceride, two sialic acid ganglion monoglycerides and three sialic acid ganglion monoglycerides (Comprehensive Biochemistry 18:201, Elsevier, Amstrdam, 1970).The hydrophobic part of ganglion monoglyceride is an acyl sphingosine, and the latter combines with the biological membrane with unsaturated long-chain fatty acid.Acyl sphingosine is by an amino group and oligosaccharides (such as glucose, galactose N-acetylgalactosamine and sialic acid) combination.
Because combined sialic acid exists, sugared sphingolipid giant molecule is electronegative, and this negative charge influences some intercellular interaction.In the tumour body, because biochemically balanced disorder, cell-cell interaction changes.Therefore, the sialic acid content that combines with glycolipid in the blood increases.
Based on understanding, set up the method for several mensuration blood sugar fat one sialic acid contents to above-mentioned theory.Method according to Svenerholm, blood plasma extracts through chloroform/methanol (mixing at 2: 1) liquid, other lipids are separated, sialic acid in the presence of cupric salt with resorcinol generation color reaction, sialic content can pass through colorimetric method for determining (Biochem.Biophys.Acta 24:604-611:1957) in the sugared sphingolipid.According to Katopodis(United States Patent (USP) 4,342,567) described, through the phosphotungstic acid effect, sugared sphingolipid composition at first is precipitated out in the methyl alcohol supernatant, and the dissolution precipitation thing carries out colorimetric estimation again.Like this, the Svennerholm method is improved.Certainly, sialic acid also the available enzyme reaction method measure.According to document (Clin.Chim.Acta 108:493-498:1980) method of reporting, glycoprotein is through the sweet enzymatic lysis of neuraminic acid, the sialic acid that discharges again with the effect of N-acetylneuraminate aldolase, form pyruvic acid and N-acetyl-D-mannosamine.The pyruvic acid that produces forms superoxide through the pyruvate oxidation oxydasis, and the latter is transformed into the colour substance that contains P-chlorophenol and 4-amino-antipyrine in the presence of peroxidase, and the amount of the colour substance of generation can be measured under the 505nm wavelength.
The common drawback of above-mentioned all methods is that error at measurment is all above 20%.Since these authors survey sialic acid level difference between normal person and patient very little (normally＜patient 18mg%＞20mg%), so may have malignant diseases though the result can point out, to whether existing tumour that clear and definite foundation can not be provided really.Another shortcoming is, some optimum disease, such as arthritis, psoriasis, ulcer, inflammation and gynecological disease, also can cause the blood sialic acid content increase (J.Clin.Chem.Biochem.22,647-651:1984).So the method for the mensuration blood sialic acid content of having delivered so far only can be suspected the existence of cancer, can not be as the basis of more accurate diagnosis.
Our purpose provides one can cause the more analytical approach of reliable diagnosis, is beneficial to the existence of early detection tumour, differentiates tumour and non-tumor disease and the possible position of inferring tumour.
The present invention is based on following understanding: under suitable pH and ionic strength, different metal salt can be isolated univocal Main Ingredients and Appearance from the complicated composition of blood sugar fat complexes.In the tumour and non-tumor disease of separate sources, sialic acid preferentially with the combination of different glycolipid composition.Therefore, measure the glycolipid character that sialic content in the inferior composition of separated glycolipid or mensuration combine saliva, can determine that increasing of sialic acid level is because examinee's tumour is caused, or because optimum disease is (as inflammation, arthritis, gynaecological disease or the like) caused, as also can determine the existence of its position or metastatic tumor for tumour.In addition, also observe, as precipitation agent, can earlier find tumour as precipitation agent than using phosphotungstic acid with some slaine (mainly being cadmium salt).
In sum, the present invention relates to the method for selectively determining of sialic acid-carrying glycolipid complexes in the blood (mainly being human blood).In the method, serum after water dilution, use can not be miscible with water organic solvent or mixed solvent remove neutral fats; The lipoprotein composition that contains sialic acid-carrying glycolipid is precipitated out from aqueous phase; Precipitation is dissolved again; Content and character with sialic content or mensuration sialic acid-carrying glycolipid in the known method mensuration solution own.The present invention uses water miscible monovalence, divalence or trivalent metal salt as precipitation agent, rather than with alkalinous metal as precipitation agent; In the mixed liquor, the ultimate density of metallic ion is 0.005～0.1mole/1, and pH is 1.5 to 8.0 during precipitation.
The method that the most handy United States Patent (USP) 4,342,567 is introduced is removed neutral fats from the serum of dilute with water, use chloroform/methanol (volume ratio 2: 1) mixed liquor as extract.Water methanol supernatant (being commonly referred to as the methyl alcohol supernatant later on) precipitates once more, and lower floor is the chloroformic solution of neutral fats, is dropped.
After, with precipitation agent be added in the water alcohol mutually in or be added to the part water methanol mutually in, the concentration that makes metallic ion in the potpourri is 0.005-0.1mole/1.Precipitation agent preferably uses with the form of aqueous solution.During the precipitation pH value that some metallic ions are preferably selected for use is listed in the table below:
Metallic ion pH
Ferrous (Fe
2+) 3.0-3.5
Iron (Fe
3+) 3.0-5.0
Cobalt (Co
2+) 4.0-5.5
Nickel (Ni
2+) 4.5-5.5
Cadmium (Cd
2+) 4.5-6.0
Zinc (Zn
2+) 1.5-5.0
Silver (Ag
+) 6.0-8.0
Mercury (Hg
2+) 1.5-3.0
Copper (Cu
2+) 3.0-4.5
Calcium (Ca
2+) 4.5-5.5
Magnesium (Mg
2+) 5.5-7.0
The precipitation that produces is separated, then,
A) detect sialic acid content in the sediment with a known method, as press United States Patent (USP) 4,342, the color reaction that a kind of mineral acid of 567 described usefulness, cupric salt and resorcinol produce is as foundation.Or the enzyme process of introducing with document (Clin.Chim.Acta 108:493-498:1980), or, b) measure the type and the amount of the glycolipid of bound sialic acid with known method, high pressure lipuid chromatography (HPLC) described in document (Clin.Chem.22:1516-1521:1976).
The differential diagnosis value of the inventive method clearly improves, after the sialic content sequence ground metallic ion different with at least 5 kinds measured in the duplicate blood specimen, the ratio addition of measured value and normal value (" normal value " refers to use the known metal ion as precipitation agent herein, records the statistical computation value of non-patient's sialic acid concentration).In our experience, measured value/normal value ratio sum is successively decreased in the following order for local tumor: tumor of breast-colon tumor-ovarian neoplasm-head/tumor colli-gland cancer-non-neoplastic disease-healthy disease-free state.
Equally, can obtain an antidiastole series.According to the present invention, survey the result that sialic result measured by known sialic acid (as United States Patent (USP) 4,342,567 results that deliver, the sialic acid measurement result of separate sources is different) with at least 5 kinds of different metal ions and remove, and will discuss addition.Different merchants also may have the value of antidiastole.
The present invention is by means of following non-limiting instance sets forth in detail in addition.In example, blood specimen is processed in advance, and sialic acid content is measured in order to following method in the sediment:
Venous blood is used for detecting, get blood after, sample is through 4000rpm, and is centrifugal 15 minutes, that the 0.6ml sample is frozen.The sialic acid concentration of fat combination in the sample-20 ℃ can keep 6 months constant.
Before beginning to analyze, test tube thoroughly soaks precooling in the dry rearmounted refrigerator earlier with methyl alcohol.Add 0.2ml serum specimen and 0.2ml distilled water in every test tube, mixed liquor stirred for 15 seconds through the Vortex stirrer, placed ice-water bath (0 ℃) then.Every part to be measured all prepares two parallel samples with control serum.
In each test tube, add chloroform/methanol (volume ratio 2: the 1) potpourri of 3ml precooling, stirred for 30 seconds with the Vortex stirrer.Test tube places ice-water bath again; Every pipe adds the distilled water that 0.5ml is chilled to 4 ℃ in advance again, in vitro tolerantly stirs for 15 seconds with the Vortex stirrer, and mixed liquor was placed under room temperature 5 minutes; Then, with sample centrifugal 10 minutes through 3000rpm; Every pipe is got the 1ml supernatant and is carefully added in the graduated centrifuge tube, adds the water-soluble metal salt solusion of prescribed concentration and amount in the example, adjusts pH of mixed value designated value to the example, and the content of each centrifuge tube left standstill 5 minutes after stirring with the Vortex stirrer.
With the content of centrifuge tube centrifugal 15 minutes, supernatant is separated (abandoning supernatant) fully with precipitation after leaving standstill with tilt-pour process with 4000rpm.
In each centrifuge tube, add 1ml Tris damping fluid, will precipitate dissolving again.The preparation of Tris damping fluid:, regulate the pH value to 8.6 that generates solution with 1 liter of distilled water dissolving 10mM three (methylol) aminomethane.
Being used for photometric standard and dummy is prepared as follows:
Dissolving 50mg analyzes pure sialic acid in the 100ml distilled water, and gained solution is as being used for preparing the stock solution of two kinds of distilled water dilutions (concentration is respectively 25mg/100ml and 12.5mg/100ml).Standard solution is+4 ℃ of preservations.The standard solution of three kinds of variable concentrations is respectively got 0.2ml, divides the centrifuge tube of packing into, with distilled water every pipe solution is added to 1ml.Other gets the 1ml distilled water as blank.
The all samples pipe respectively adds 1ml resorcinol reagent solution in (comprising standard, blank, contrast, experiment serum tube).The resorcinol reagent solution is prepared as follows: in the 100ml distilled water, dissolving 2g analyzes pure resorcinol; In the 100ml distilled water, dissolve the 2.49g anhydrous cupric sulfate in addition; Then, getting 10ml resorcinol solution mixes with 0.25ml copper-bath, 9.75ml distilled water and 100ml concentrated hydrochloric acid (analyzing pure).
Test tube was accurately placed 10 minutes in 100 ℃ of water-baths; Then, immediately all test tubes are immersed 0 ℃ of ice-water bath, kept 10 minutes.Every pipe adds
2Ml butyl acetate/n-butyric acie (volume ratio 85: 15) mixed liquor, in vitro tolerant with Vortex stirrer stirring 5 minutes.Mixed liquor is left standstill and between or stir.Sample centrifugal 10 minutes through 2500rpm.Blue supernatant in every pipe is respectively charged into measures pipe.In 6 hours, blue density remains unchanged.
Under the 580nm wavelength, carry out photometering.Adjust photometric zero point with distilled water; After, according to used photometric type (single beam or twin-beam), compare during mensuration or with blank, thereby or deduct blank extinction value with the sample extinction value that records and obtain corrected value.
E(proofreaies and correct)=the E(measured value)-the E(blank value)
Sialic acid content calculates by one of following two kinds of methods.
1) from the standard sialic acid sample measured result of known sialic acid concentration as calculated or mapping obtain the curve of extinction value to concentration.Because dilution step, concentration originally should recomputate according to following table.
Original concentration is measured the concentration extinction value
（mg/100ml） ·（μg/200ml）
50 100 0.508
25 50 0.252
12.5 25 0.127
Can record sialic content the 1ml supernatant sample from aforementioned gained calibration curve.The sialic acid content that curve marks is represented with μ g sialic acid in every ml supernatant.Sialic acid content can be calculated as follows in the serum:
Sialic acid content (mg/100ml)=A * 0.72
A refers to sialic acid content in the supernatant in the formula, and unit is μ g/ml; 0.72 be a coefficient, comprise the conversion between dilutability and μ g and the mg.
2) second kind of computing method are used the standard serum sample of known sialic acid concentration, get factor F from several replicate determination values by the following formula art.
F＝C（ref）/E（ref）
C(ref in the formula) is sialic acid content in the standard serum, the mg/100ml of unit; E(ref) be the extinction value of standard serum.Try to achieve the sialic acid content of test sample by following formula.
Sialic acid content (mg/100ml)=E(m) * F,
E(m in the formula) is the extinction value that test sample records.
Example 1
From a healthy people serum, extract with the chloroform-methanol mixed liquor as stated above, methyl alcohol supernatant and 0.5mole/1 nickel chloride (NiCl
2) solution was by mixing in 10: 1, formation precipitates when pH5.5, and sialic content is 4.65mg% in the mensuration sediment.
Example 2
From a healthy people serum, press last method and extract with the chloroform-methanol mixed liquor, the methyl alcohol supernatant is mixed by 10: 1 with the 1.0mole/1 nickel chloride solution, when pH5.3, form precipitation, sialic content is 6.43mg% in the mensuration sediment.
Example 3
From a healthy people serum, extract with the chloroform-methanol mixed liquor by last method, methyl alcohol supernatant and 0.1mole/1 caddy (CdCl
2) solution was by mixing in 10: 1, formation precipitates when pH5.9, and sialic content is 11.52mg% in the mensuration sediment.
Example 4
From a healthy people serum, press last method and extract with the chloroform-methanol potpourri, the methyl alcohol supernatant is mixed by 10: 1 with the 0.5mole/1 cadmium chloride solution, when pH5.5, form precipitation, sialic content is 10.48mg% in the mensuration sediment
Example 5
From a healthy people serum, press last method and extract with the chloroform-methanol mixed liquor, the methyl alcohol supernatant is mixed by 10: 1 with the 1.0mole/1 cadmium chloride solution, when pH5.3, form precipitation, sialic content is 14.81mg% in the mensuration sediment:
Example 6
From a healthy people serum, extract with the chloroform-methanol mixed liquor by last method, methyl alcohol supernatant and 0.1mole/1 ferrous sulphate (FeSO
4) solution was by mixing in 10: 1, formation precipitates when pH3.3, and sialic content is 9.08mg% in the mensuration sediment.
Example 7
From a healthy people serum, press last method and extract with the chloroform-methanol mixed liquor, the methyl alcohol supernatant is mixed by 10: 1 with the 0.5mole/1 copperas solution, when pH3.0, form precipitation, sialic content is 9.54mg% in the mensuration sediment.
Example 8
From a healthy people serum, press last method and extract with the chloroform-methanol mixed liquor, the methyl alcohol supernatant is connect 10: 1 with the 1.0mole/l copperas solution mix, when pH2.9, form precipitation, sialic content is 9.52mg% in the mensuration sediment.
Example 9
From a healthy people serum, extract with the chloroform-methanol mixed liquor by last method, methyl alcohol supernatant and 0.1mole/1 zinc chloride (ZnCl
2) solution was by mixing in 10: 1, formation precipitates when pH2.7, and sialic content is 6.52mg% in the mensuration sediment.
Example 10
From a healthy people serum, press last method and extract with the chloroform-methanol mixed liquor, the methyl alcohol supernatant is mixed by 10: 1 with the 0.5mole/1 liquor zinci chloridi, when pH1.9, form precipitation, sialic content is 12.34mg% in the mensuration sediment.
Example 11
From a healthy people serum, extract with the chloroform-methanol mixed liquor by last method, methyl alcohol supernatant and 0.1mole/1 plumbi nitras (Pb(NO
3)
2) solution was by mixing in 10: 1, formation precipitates when pH4.4, and sialic content is 8.61mg% in the mensuration sediment.
Example 12
From a healthy people serum, press last method and extract with the chloroform-methanol potpourri, the methyl alcohol supernatant is mixed by 10: 1 with the 0.5mole/1 lead nitrate solution, when pH3.7, form precipitation, sialic acid content is 12.11mg% in the mensuration sediment.
Example 13
From a healthy people serum, press last method and extract with the chloroform-methanol mixed liquor, the methyl alcohol supernatant is mixed by 10: 1 with the 1.0mole/1 lead nitrate solution, when pH3.5, form precipitation, sialic content is 12.35mg% in the mensuration sediment.
Example 14
From a healthy people serum, extract with the chloroform-methanol mixed liquor by last method, methyl alcohol supernatant and 0.1mole/1 silver nitrate (AgNO
3) solution was by mixing in 10: 1, formation precipitates when pH8.5, and sialic content is 9.89mg% in the mensuration sediment.
Example 15
From a healthy people serum, press last method and extract with the chloroform-methanol potpourri, the methyl alcohol supernatant is mixed by 10: 1 with the 0.5mole/1 liquor argenti nitratis ophthalmicus, when pH6.0, form precipitation, sialic content is 14.67mg% in the mensuration sediment
Example 16
From a healthy people serum, press last method and extract with the chloroform-methanol mixed liquor, the methyl alcohol supernatant is mixed by 10: 1 with the 1.0mole/1 liquor argenti nitratis ophthalmicus, when pH5.8, form precipitation, sialic content is 14.81mg% in the mensuration sediment
Example 17
From a healthy people serum, extract with the chloroform-methanol mixed liquor by last method, methyl alcohol supernatant and 0.1mole/1 mercuric chloride (HgCl
2) solution was by mixing in 10: 1, formation precipitates when pH1.9, and sialic content is 6.05mg% in the mensuration sediment.
Example 18
From a healthy people serum,, that methyl alcohol supernatant and 0.5mole/1 mercuric chloride solution is mixed by 10: 1 by last method with the extraction of chloroform-methanol potpourri, when pH1.7, form precipitation, sialic content is 7.68mg% in the mensuration sediment.
Example 19
From a healthy people serum, press last method and extract with the chloroform-methanol potpourri, the methyl alcohol supernatant is mixed by 10: 1 with the 1.0mole/1 mercuric chloride solution, when pH1.5, form precipitation, sialic content is 11.99mg% in the mensuration sediment.
Example 20
From a healthy people serum, extract with the chloroform-methanol mixed liquor by last method, methyl alcohol supernatant and 0.1mole/1 copper sulphate (CuSO
4) solution was by mixing in 10: 1, formation precipitates when pH4.2, and sialic content is 10.13mg% in the mensuration sediment.
Example 21
From a healthy people serum, press last method and extract with the chloroform-methanol mixed liquor, the methyl alcohol supernatant is mixed by 10: 1 with the 0.5mole/1 copper-bath, when pH3.8, form precipitation, sialic content is 10.48mg% in the mensuration sediment
Example 22
From a healthy people serum, press last method and extract with the chloroform-methanol mixed liquor, the methyl alcohol supernatant is mixed by 10: 1 with the 1.0mole/1 copper-bath, when PH3.6, form precipitation, sialic content is 12.22mg% in the mensuration sediment.
Example 23
From a healthy people serum, extract with the chloroform-methanol mixed liquor by last method, methyl alcohol supernatant and 0.1mole/1 lime chloride (CaCl
2) solution was by mixing in 10: 1, formation precipitates when pH5.5, and sialic content is 3.72mg% in the mensuration sediment.
Example 24
From a healthy people serum, press last method and extract with the chloroform-methanol mixed liquor, the methyl alcohol supernatant is mixed by 10: 1 with the 0.5mole/1 calcium oxide solution, when pH5.4, form precipitation, sialic content is 3.02mg% in the mensuration sediment.
Example 25
From a healthy people serum, press last method and extract with the chloroform-methanol mixed liquor, the methyl alcohol supernatant is mixed by 10: 1 with the 1.0mole/1 calcium chloride solution, when pH5.3, form precipitation, sialic content is 7.05mg% in the mensuration sediment.
Example 26
From a healthy people serum, extract with the chloroform-methanol mixed liquor by last method, methyl alcohol supernatant and 0.1mole/1 magnesium chloride (MgCl
2) solution was by mixing in 10: 1, formation precipitates when pH6.1, and sialic content is 4.19mg% in the mensuration sediment.
Example 27
From a healthy people serum, press last method and extract with the chloroform-methanol mixed liquor, the methyl alcohol supernatant is mixed by 10: 1 with the 0.5mole/1 magnesium chloride solution, when pH6.7, form precipitation, sialic content is 3.61mg% in the mensuration sediment.
Example 28
From a healthy people serum, press last method and extract with the chloroform-methanol mixed liquor, the methyl alcohol supernatant is mixed by 10: 1 with the 1.0mole/1 magnesium chloride solution, when pH6.5, form precipitation, sialic content is 4.81mg% in the mensuration sediment.
Example 29
From a breast cancer patient serum, press last method and extract with the chloroform-methanol mixed liquor, the methyl alcohol supernatant is mixed by 10: 1 with the 1.0mole/1 nickel chloride solution, when pH5.3, form precipitation, sialic content is 28.87mg% in the mensuration sediment.
Example 30
From a breast cancer patient serum, press last method and extract with the chloroform-methanol mixed liquor, the methyl alcohol supernatant is mixed by 10: 1 with the 1.0mole/1 cadmium chloride solution, when pH5.3, form precipitation, sialic content is 41.31mg% in the mensuration sediment.
Example 31
From a breast cancer patient serum, press last method and extract with the chloroform-methanol mixed liquor, the methyl alcohol supernatant is mixed by 10: 1 with the 1.0mole/1 copperas solution, when pH2.9, form precipitation, sialic content is 33.39mg% in the mensuration sediment.
Example 32
From a breast cancer patient serum, press last method and extract with the chloroform-methanol mixed liquor, the methyl alcohol supernatant is mixed by 10: 1 with the 1.0mole/1 liquor zinci chloridi, when pH1.7, form precipitation, sialic content is 39.41mg% in the mensuration sediment.
Example 33
From a breast cancer patient serum, press last method and extract with the chloroform-methanol mixed liquor, the methyl alcohol supernatant is mixed by 10: 1 with the 1.0mole/1 solution of zinc sulfate, when pH1.7, form precipitation, sialic content is 38.90mg% in the mensuration sediment.
Example 34
From a breast cancer patient serum, press last method and extract with the chloroform-methanol mixed liquor, the methyl alcohol supernatant is mixed by 10: 1 with the 1.0mole/1 lead nitrate solution, when pH3.5, form precipitation, sialic content is 45.02mg% in the mensuration sediment.
Example 35
Serum from a breast cancer patient, extract with the chloroform-methanol mixed liquor by last method, the methyl alcohol supernatant is mixed by 10: 1 with the 1.0mole/1 liquor argenti nitratis ophthalmicus, form precipitation when pH5.8, sialic content is 29.58 mg% in the mensuration sediment.
Example 36
From a breast cancer patient serum, press last method and extract with the chloroform-methanol mixed liquor, the methyl alcohol supernatant is mixed by 10: 1 with the 1.0mole/1 mercuric chloride solution, when pH1.5, form precipitation, sialic content is 33.89mg% in the mensuration sediment.
Example 37
From a breast cancer patient serum, press last method and extract with the chloroform-methanol mixed liquor, the methyl alcohol supernatant is mixed by 10: 1 with the 1.0mole/1 copper-bath, when pH3.6, form precipitation, sialic content is 37.20mg% in the mensuration sediment.
Example 38
From a breast cancer patient serum, press last method and extract with the chloroform-methanol mixed liquor, the methyl alcohol supernatant is mixed by 10: 1 with the 1.0mole/1 calcium chloride solution, when pH5.3, form precipitation, sialic content is 28.18mg% in the mensuration sediment.
Example 39
From a breast cancer patient serum, press last method and extract with the chloroform-methanol mixed liquor, the methyl alcohol supernatant is mixed by 10: 1 with the 1.0mole/1 magnesium chloride solution, when pH6.5, form precipitation, sialic content is 25.67mg% in the mensuration sediment.
Example 40
From an oophoroma patient serum, extract with the chloroform-methanol mixed liquor by last method, the methyl alcohol supernatant is mixed by 10: 1 with the 1.0mole/1 nickel chloride solution, when PH5.3, form precipitation, sialic content is 22.09mg% in the sediment.
Example 41
From an oophoroma patient serum, press last method and extract with the chloroform-methanol mixed liquor, the methyl alcohol supernatant is mixed by 10: 1 with the 1.0mole/1 cadmium chloride solution, when pH5.3, form precipitation, sialic content is 25.80mg% in the mensuration sediment.
Example 42
From an oophoroma patient serum, press last method and extract with the chloroform-methanol mixed liquor, the methyl alcohol supernatant is mixed by 10: 1 with the 1.0mole/1 copperas solution, when pH2.9, form precipitation, sialic content is 18.75mg% in the mensuration sediment.
Example 43
From an oophoroma patient serum, press last method and extract with the chloroform-methanol mixed liquor, the methyl alcohol supernatant is mixed by 10: 1 with the 1.0mole/1 liquor zinci chloridi, when pH1.7, form precipitation, sialic content is 23.77mg% in the mensuration sediment.
Example 44
From an oophoroma patient serum, press last method and extract with the chloroform-methanol mixed liquor, the methyl alcohol supernatant is mixed by 10: 1 with the 1.0mole/1 solution of zinc sulfate, when pH1.7, form precipitation, sialic content is 26.87mg% in the mensuration sediment.
Example 45
Serum from an oophoroma patient, extract with the chloroform-methanol mixed liquor by last method, the methyl alcohol supernatant is mixed by 10: 1 with the 1.0mole/1 lead nitrate solution, form precipitation when pH3.5, sialic content is 26.63mg% in the mensuration sediment.
Example 46
From an oophoroma patient serum, press last method and extract with the chloroform-methanol mixed liquor, the methyl alcohol supernatant is mixed by 10: 1 with the 1.0mole/1 liquor argenti nitratis ophthalmicus, when pH5.8, form precipitation, sialic content is 28.66mg% in the mensuration sediment.
Example 47
From an oophoroma patient serum, press last method and extract with the chloroform-methanol mixed liquor, the methyl alcohol supernatant is mixed by 10: 1 with the 1.0mole/1 mercuric chloride solution, when pH1.5, form precipitation, sialic content is 33.08mg% in the mensuration sediment.
Example 48
From an oophoroma patient serum, press last method and extract with the chloroform-methanol mixed liquor, the methyl alcohol supernatant is mixed by 10: 1 with the 1.0mole/1 copper-bath, when pH3.6, form precipitation, sialic content is 26.63mg% in the mensuration sediment.
Example 49
From an oophoroma patient serum, use the chloroform-methanol mixed liquor to extract by last method, methyl alcohol supernatant and 1.0mole/1 chlorine examination calcium solution were mixed by 10: 1, when pH5.3, form precipitation, sialic content is 13.26mg% in the mensuration sediment.
Example 50
Serum from an oophoroma patient, extract with the chloroform-methanol mixed liquor by last method, the methyl alcohol supernatant is mixed by 10: 1 with the 1.0mole/1 magnesium chloride solution, form precipitation when pH6.5, sialic content is 11.82mg% in the mensuration sediment.
Example 51
From one/neck carninomatosis people's serum, press last method and extract with the chloroform-methanol mixed liquor, the methyl alcohol supernatant is mixed by 10: 1 with the 1.0mole/1 nickel chloride solution, when pH5.3, form precipitation, sialic content is 12.62mg% in the mensuration sediment.
Example 52
From one/neck carninomatosis people's serum, press last method and extract with the chloroform-methanol mixed liquor, the methyl alcohol supernatant is mixed by 10: 1 with the 1.0mole/1 cadmium chloride solution, when pH5.3, form precipitation, sialic content is 21.99mg% in the mensuration precipitated liquid.
Example 53
From one/neck carninomatosis people's serum, press last method and extract with the chloroform-methanol mixed liquor, the methyl alcohol supernatant is mixed by 10: 1 with the 1.0mole/1 copperas solution, when pH2.9, form precipitation, sialic content is 16.09mg% in the mensuration sediment.
Example 54
From one/neck carninomatosis people's serum, press last method and extract with the chloroform-methanol mixed liquor, the methyl alcohol supernatant is mixed by 10: 1 with the 1.0mole/1 liquor zinci chloridi, when pH1.7, form precipitation, sialic content is 19.56mg% in the mensuration sediment.
Example 55
From one/neck carninomatosis people's serum, press last method and extract with the chloroform-methanol mixed liquor, the methyl alcohol supernatant is mixed by 10: 1 with the 1.0mole/1 solution of zinc sulfate, when pH1.7, form precipitation, sialic content is 21.41mg% in the mensuration sediment.
Example 56
From one/neck carninomatosis human serum, press last method and extract with the chloroform-methanol mixed liquor, the methyl alcohol supernatant is mixed by 10: 1 with the 1.0mole/1 lead nitrate solution, when pH3.5, form precipitation, sialic content is 29.98mg% in the mensuration sediment.
Example 57
From one/neck carninomatosis people's serum, press last method and extract with the chloroform-methanol mixed liquor, the methyl alcohol supernatant is mixed by 10: 1 with the 1.0mole/1 liquor argenti nitratis ophthalmicus, when pH5.8, form precipitation, sialic content is 14.93mg% in the mensuration sediment.
Example 58
From one/neck carninomatosis people's serum, press last method and extract with the chloroform-methanol mixed liquor, the methyl alcohol supernatant is mixed by 10: 1 with the 1.0mole/1 mercuric chloride solution, when pH1.5, form precipitation, sialic content is 18.87mg% in the mensuration sediment.
Example 59
Serum from one/neck carninomatosis people, extract with the chloroform-methanol mixed liquor by last method, the methyl alcohol supernatant is mixed by 10: 1 with the 1.0mole/1 copper-bath, form precipitation when pH3.6, sialic content is 14.70 mg% in the mensuration sediment.
Example 60
From one/neck carninomatosis people's serum, press last method and extract with the chloroform-methanol mixed liquor, the methyl alcohol supernatant is mixed by 10: 1 with the 1.0mole/1 calcium chloride solution, when pH5.3, form precipitation, sialic content is 9.72mg% in the mensuration sediment.
Example 61
From one/neck carninomatosis people's serum, press last method and extract with the chloroform-methanol mixed liquor, the methyl alcohol supernatant is mixed by 10: 1 with the 1.0mole/1 magnesium chloride solution, when pH6.5, form precipitation, sialic content is 12.27mg% in the mensuration sediment.
Example 62
From a gland cancer patient serum, press last method and extract with the chloroform-methanol mixed liquor, the methyl alcohol supernatant is mixed by 10: 1 with the 1.0mole/1 nickel chloride solution, when pH5.3, form precipitation, sialic content is 11.77mg% in the mensuration sediment.
Example 63
From a gland cancer patient serum, press last method and extract with the chloroform-methanol mixed liquor, the methyl alcohol supernatant is mixed by 10: 1 with the 1.0mole/1 cadmium chloride solution, when pH5.3, form precipitation, sialic content is 20.09mg% in the mensuration sediment.
Example 64
Serum from a gland cancer patient, extract with the chloroform-methanol mixed liquor by last method, the methyl alcohol supernatant is mixed by 10: 1 with the 1.0mole/1 copperas solution, form precipitation when pH 2.9, sialic content is 16.13mg% in the mensuration sediment.
Example 65
From a gland cancer patient serum, extract through the chloroform-methanol potpourri by last method; The methyl alcohol supernatant mixes by 10: 1 with the 1.0mole/1 zinc chloride liquid, forms precipitation at pH1.7, and sialic acid content is 18.69mg% in the mensuration sediment.
Example 66
From a gland cancer patient serum, press last method and extract with the chloroform-methanol mixed liquor, the methyl alcohol supernatant is mixed by 10: 1 with the 1.0mole/1 solution of zinc sulfate, when pH1.7, form precipitation, sialic content is 17.15mg% in the mensuration sediment.
Example 67
From a gland cancer patient serum, press last method and extract with the chloroform-methanol mixed liquor, the methyl alcohol supernatant is mixed by 10: 1 with the 1.0mole/1 lead nitrate solution, when pH3.5, form precipitation, sialic content is 22.66mg% in the mensuration sediment.
Example 68
From a gland cancer patient serum, press last method and extract with the chloroform-methanol potpourri, the methyl alcohol supernatant is mixed by 10: 1 with the 1.0mole/1 liquor argenti nitratis ophthalmicus, when pH5.8, form precipitation, sialic content is 16.51mg% in the mensuration sediment.
Example 69
Serum from a gland cancer patient, extract with the chloroform-methanol potpourri by last method, the methyl alcohol supernatant is mixed by 10: 1 with the 1.0mole/1 mercuric chloride solution, form precipitation when pH 1.5, sialic content is 18.94mg% in the mensuration sediment.
Example 70
From a gland cancer patient serum, extract with the chloroform-methanol mixed liquor by last method; The methyl alcohol supernatant is mixed by 10: 1 with the 1.0mole/1 copper-bath, form precipitation when pH3.6, sialic content is 17.41mg% in the mensuration sediment.
Example 71
From a gland cancer patient serum, press last method and extract with the chloroform-methanol mixed liquor, the methyl alcohol supernatant is mixed by 10: 1 with the 1.0mole/1 calcium chloride solution, when pH5.3, form precipitation, sialic content is 9.73mg% in the mensuration sediment.
Example 72
From a gland cancer patient serum, extract with the chloroform-methanol mixed liquor by last method; The methyl alcohol supernatant is mixed by 10: 1 with the 1.0mole/1 magnesium chloride solution, form precipitation when pH6.5, sialic content is 7.94mg% in the mensuration sediment.
Example 73
From the serum of a colon cancer patient, press last method and extract with the chloroform-methanol mixed liquor, the methyl alcohol supernatant is mixed by 10: 1 with the 1.0mole/1 nickel chloride solution, when pH5.3, form precipitation, sialic content is 20.70mg% in the mensuration sediment.
Example 74
Serum from a colon cancer patient, extract with the chloroform-methanol mixed liquor by last method, the methyl alcohol supernatant is mixed by 10: 1 with the 1.0mole/1 cadmium chloride solution, form precipitation when pH5.3, sialic content is 32.14mg% in the mensuration sediment.
Example 75
From the serum of a colon cancer patient, press last method and extract with the chloroform-methanol mixed liquor, the methyl alcohol supernatant is mixed by 10: 1 with the 1.0mole/1 copperas solution, when pH2.9, form precipitation, sialic content is 24.23mg% in the mensuration sediment.
Example 76
From the serum of a colon cancer patient, press last method and extract with the chloroform-methanol mixed liquor, the methyl alcohol supernatant is mixed by 10: 1 with the 1.0mole/1 liquor zinci chloridi, when pH1.7, form precipitation, sialic content is 27.52mg% in the mensuration sediment.
Example 77
From the serum of a colon cancer patient, press last method and extract with the chloroform-methanol mixed liquor, the methyl alcohol supernatant is mixed by 10: 1 with the 1.0mole/1 solution of zinc sulfate, when pH1.7, form precipitation, sialic content is 3.63mg% in the mensuration sediment.
Example 78
From the serum of a colon cancer patient, press last method and extract with the chloroform-methanol mixed liquor, the methyl alcohol supernatant is mixed by 10: 1 with the 1.0mole/1 lead nitrate solution, when pH3.5, form precipitation, sialic content is 35.55mg% in the mensuration sediment.
Example 79
Serum from a colon cancer patient, extract with the chloroform-methanol mixed liquor by last method, the methyl alcohol supernatant is mixed by 10: 1 with the 1.0mole/1 liquor argenti nitratis ophthalmicus, form precipitation when pH5.8, sialic content is 29.46mg% in the mensuration sediment.
Example 80
From the serum of a colon cancer patient, press last method and extract with the chloroform-methanol mixed liquor, the methyl alcohol supernatant is mixed by 10: 1 with the 1.0mole/1 mercuric chloride solution, when pH1.5, form precipitation, sialic content is 31.66mg% in the mensuration sediment.
Example 81
From the serum of a colon cancer patient, press last method and extract with the chloroform-methanol mixed liquor, the methyl alcohol supernatant is mixed by 10: 1 with the 1.0mole/1 copper-bath, when pH3.6, form precipitation, sialic content is 32.51mg% in the mensuration sediment.
Example 82
From the serum of a colon cancer patient, press last method and extract with the chloroform-methanol mixed liquor, the methyl alcohol supernatant is mixed by 10: 1 with the 1.0mole/1 calcium chloride solution, when pH5.3, form precipitation, sialic content is 18.51mg% in the mensuration sediment.
Example 83
From the serum of a colon cancer patient, press last method and extract with the chloroform-methanol mixed liquor, the methyl alcohol supernatant is mixed by 10: 1 with the 1.0mole/1 magnesium chloride solution, when pH6.5, form precipitation, sialic content is 16.68mg% in the mensuration sediment.
Claims (12)
1, the method of selectively determining of sialic acid-carrying glycolipid in the blood, this method comprises the serum dilute with water, then, with extracting with immiscible organic solvent of water or mixed solvent, so that remove neutral fats, make the lipoprotein composition that contains sialic acid-carrying glycolipid from the phase water sedimentation, again dissolution precipitation thing, sialic acid content in the solution or contain the character of sialic glycolipid and content can be measured with known method itself, it is characterized in that monovalence, divalence or trivalent water-soluble metal salt as precipitation agent rather than with alkalinous metal as precipitation agent, it is 0.005-0.1mole/1 that the amount that precipitation agent is used is equivalent in the potpourri final concentration of metal ions, and is 1.5 to 8.0 to precipitate at PH.
2, the method for claim 1, its characteristics are that precipitation agent is water miscible divalent iron salt and is to precipitate for 3.0 to 3.5 times at pH.
3, the method for claim 1, its characteristics are that precipitation agent is water miscible trivalent iron salt, and are to precipitate for 3.0 to 5.0 times at pH.
4, the method for claim 1, its characteristics are that precipitation agent is water miscible divalent cobalt and is to precipitate for 4.0 to 5.5 times at pH.
5, the method for claim 1, its characteristics are that precipitation agent is water miscible divalent nickel salt and is to precipitate for 4.5 to 5.5 times at pH.
6, the method for claim 1, its characteristics are that precipitation agent is water miscible divalence cadmium salt and is to precipitate for 4.5 to 6.0 times at pH.
7, the method for claim 1, its characteristics are that precipitation agent is water miscible divalent zinc salt and is to precipitate for 1.5 to 5.0 times at PH.
8, the method for claim 1, its characteristics are that precipitation agent is water-soluble monovalence silver salt, and are to precipitate for 6.0 to 8.0 times at pH.
9, the method for claim 1, its characteristics are that precipitation agent is water-soluble mercuric salt, and are to precipitate for 1.5 to 3.0 times at pH.
10, the method for claim 1, its characteristics are that precipitation agent is a water soluble bivalent nantokite, and precipitate under pH3.0 to 4.5.
11, the method for claim 1, its characteristics are that precipitation agent is water-soluble divalence calcium salt and is to precipitate for 4.5 to 5.5 times at pH.
12, the method for claim 1, its characteristics are that precipitation agent is water-soluble divalence magnesium salts and is to precipitate for 5.5 to 7.0 times at pH.
Applications Claiming Priority (4)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| HU249-89 | 1989-01-23 | ||
| HU24989 | 1989-01-23 | ||
| HU219089A HU208377B (en) | 1989-05-08 | 1989-05-08 | Analytic method for selective detecting glycolipid complexes carrying sial acid |
| HU2190-89 | 1989-05-08 |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN1044537A true CN1044537A (en) | 1990-08-08 |
Family
ID=26317184
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN89104207A Pending CN1044537A (en) | 1989-01-23 | 1989-06-20 | The analytical approach of selective determination of sialic acid-carrying glycolipid complexes |
Country Status (10)
| Country | Link |
|---|---|
| EP (1) | EP0515355A1 (en) |
| JP (1) | JPH02206763A (en) |
| KR (1) | KR910700457A (en) |
| CN (1) | CN1044537A (en) |
| AU (1) | AU3735689A (en) |
| DK (1) | DK226590D0 (en) |
| ES (1) | ES2012728A6 (en) |
| FI (1) | FI913518A0 (en) |
| NO (1) | NO903877D0 (en) |
| WO (1) | WO1990008317A1 (en) |
Family Cites Families (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US4342567A (en) * | 1981-07-06 | 1982-08-03 | Sloan Kettering Institute For Cancer Research | Sialic acid determination method |
-
1989
- 1989-05-12 WO PCT/HU1989/000021 patent/WO1990008317A1/en not_active Application Discontinuation
- 1989-05-12 EP EP89905744A patent/EP0515355A1/en not_active Ceased
- 1989-05-12 AU AU37356/89A patent/AU3735689A/en not_active Abandoned
- 1989-05-12 KR KR1019900701970A patent/KR910700457A/en not_active Application Discontinuation
- 1989-05-26 JP JP1131718A patent/JPH02206763A/en active Pending
- 1989-06-06 ES ES8901972A patent/ES2012728A6/en not_active Expired - Fee Related
- 1989-06-20 CN CN89104207A patent/CN1044537A/en active Pending
-
1990
- 1990-09-05 NO NO1990903877A patent/NO903877D0/en unknown
- 1990-09-20 DK DK226590A patent/DK226590D0/en not_active Application Discontinuation
-
1991
- 1991-07-23 FI FI913518A patent/FI913518A0/en not_active Application Discontinuation
Also Published As
| Publication number | Publication date |
|---|---|
| KR910700457A (en) | 1991-03-15 |
| NO903877L (en) | 1990-09-05 |
| DK226590A (en) | 1990-09-20 |
| AU3735689A (en) | 1990-08-13 |
| FI913518A0 (en) | 1991-07-23 |
| JPH02206763A (en) | 1990-08-16 |
| DK226590D0 (en) | 1990-09-20 |
| EP0515355A1 (en) | 1992-12-02 |
| NO903877D0 (en) | 1990-09-05 |
| ES2012728A6 (en) | 1990-04-01 |
| WO1990008317A1 (en) | 1990-07-26 |
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