GB2101740A - Method and kit for determining glycosylated proteins in biological fluids - Google Patents
Method and kit for determining glycosylated proteins in biological fluids Download PDFInfo
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- 238000000034 method Methods 0.000 title claims abstract description 35
- 102000035122 glycosylated proteins Human genes 0.000 title claims abstract description 11
- 108091005608 glycosylated proteins Proteins 0.000 title claims abstract description 11
- 239000013060 biological fluid Substances 0.000 title claims abstract description 6
- MUBZPKHOEPUJKR-UHFFFAOYSA-N Oxalic acid Chemical compound OC(=O)C(O)=O MUBZPKHOEPUJKR-UHFFFAOYSA-N 0.000 claims abstract description 30
- 150000001720 carbohydrates Chemical class 0.000 claims abstract description 19
- 235000014633 carbohydrates Nutrition 0.000 claims abstract description 19
- 239000003153 chemical reaction reagent Substances 0.000 claims abstract description 16
- RVBUGGBMJDPOST-UHFFFAOYSA-N 2-thiobarbituric acid Chemical compound O=C1CC(=O)NC(=S)N1 RVBUGGBMJDPOST-UHFFFAOYSA-N 0.000 claims abstract description 13
- 230000002452 interceptive effect Effects 0.000 claims abstract description 11
- 239000007791 liquid phase Substances 0.000 claims abstract description 10
- 235000006408 oxalic acid Nutrition 0.000 claims abstract description 10
- 230000002378 acidificating effect Effects 0.000 claims abstract description 8
- YNJBWRMUSHSURL-UHFFFAOYSA-N trichloroacetic acid Chemical compound OC(=O)C(Cl)(Cl)Cl YNJBWRMUSHSURL-UHFFFAOYSA-N 0.000 claims abstract description 5
- 102000004169 proteins and genes Human genes 0.000 claims description 17
- 108090000623 proteins and genes Proteins 0.000 claims description 17
- QTBSBXVTEAMEQO-UHFFFAOYSA-N Acetic acid Chemical compound CC(O)=O QTBSBXVTEAMEQO-UHFFFAOYSA-N 0.000 claims description 12
- 230000003287 optical effect Effects 0.000 claims description 8
- 239000000126 substance Substances 0.000 claims description 7
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 6
- 239000002253 acid Substances 0.000 claims description 5
- 239000003795 chemical substances by application Substances 0.000 claims description 5
- 238000011534 incubation Methods 0.000 claims description 5
- 150000007524 organic acids Chemical class 0.000 claims description 5
- 235000005985 organic acids Nutrition 0.000 claims description 5
- 230000001376 precipitating effect Effects 0.000 claims description 5
- 150000003839 salts Chemical class 0.000 claims description 5
- 239000012279 sodium borohydride Substances 0.000 claims description 5
- 229910000033 sodium borohydride Inorganic materials 0.000 claims description 5
- 108090000790 Enzymes Proteins 0.000 claims description 4
- 102000004190 Enzymes Human genes 0.000 claims description 4
- VEXZGXHMUGYJMC-UHFFFAOYSA-N Hydrochloric acid Chemical compound Cl VEXZGXHMUGYJMC-UHFFFAOYSA-N 0.000 claims description 4
- 239000003638 chemical reducing agent Substances 0.000 claims description 4
- 238000001556 precipitation Methods 0.000 claims description 4
- YCPXWRQRBFJBPZ-UHFFFAOYSA-N 5-sulfosalicylic acid Chemical compound OC(=O)C1=CC(S(O)(=O)=O)=CC=C1O YCPXWRQRBFJBPZ-UHFFFAOYSA-N 0.000 claims description 3
- 239000002535 acidifier Substances 0.000 claims description 3
- 150000001298 alcohols Chemical class 0.000 claims description 3
- 150000001728 carbonyl compounds Chemical class 0.000 claims description 3
- 150000002894 organic compounds Chemical class 0.000 claims description 3
- 150000005846 sugar alcohols Polymers 0.000 claims description 3
- JOXIMZWYDAKGHI-UHFFFAOYSA-N toluene-4-sulfonic acid Chemical class CC1=CC=C(S(O)(=O)=O)C=C1 JOXIMZWYDAKGHI-UHFFFAOYSA-N 0.000 claims description 3
- QAOWNCQODCNURD-UHFFFAOYSA-N Sulfuric acid Chemical compound OS(O)(=O)=O QAOWNCQODCNURD-UHFFFAOYSA-N 0.000 claims description 2
- 235000011167 hydrochloric acid Nutrition 0.000 claims description 2
- 239000000203 mixture Substances 0.000 claims description 2
- 239000001117 sulphuric acid Substances 0.000 claims description 2
- 235000011149 sulphuric acid Nutrition 0.000 claims description 2
- 239000012530 fluid Substances 0.000 claims 5
- 239000012490 blank solution Substances 0.000 claims 4
- 229940077731 carbohydrate nutrients Drugs 0.000 claims 4
- 239000012488 sample solution Substances 0.000 claims 3
- 239000000243 solution Substances 0.000 claims 3
- 108010093096 Immobilized Enzymes Proteins 0.000 claims 1
- 235000011054 acetic acid Nutrition 0.000 claims 1
- 229910017053 inorganic salt Inorganic materials 0.000 claims 1
- 238000011533 pre-incubation Methods 0.000 claims 1
- 239000002244 precipitate Substances 0.000 claims 1
- 239000007790 solid phase Substances 0.000 abstract description 2
- 229960004319 trichloroacetic acid Drugs 0.000 abstract 1
- 239000000523 sample Substances 0.000 description 16
- 210000002966 serum Anatomy 0.000 description 10
- 238000012360 testing method Methods 0.000 description 10
- RJGBSYZFOCAGQY-UHFFFAOYSA-N hydroxymethylfurfural Natural products COC1=CC=C(C=O)O1 RJGBSYZFOCAGQY-UHFFFAOYSA-N 0.000 description 7
- 239000006228 supernatant Substances 0.000 description 7
- NOEGNKMFWQHSLB-UHFFFAOYSA-N 5-hydroxymethylfurfural Chemical compound OCC1=CC=C(C=O)O1 NOEGNKMFWQHSLB-UHFFFAOYSA-N 0.000 description 6
- 102000009027 Albumins Human genes 0.000 description 5
- 108010088751 Albumins Proteins 0.000 description 5
- 206010012601 diabetes mellitus Diseases 0.000 description 5
- 230000007062 hydrolysis Effects 0.000 description 5
- 238000006460 hydrolysis reaction Methods 0.000 description 5
- 150000007513 acids Chemical class 0.000 description 4
- 238000004458 analytical method Methods 0.000 description 4
- 238000006243 chemical reaction Methods 0.000 description 4
- 238000000502 dialysis Methods 0.000 description 4
- 230000005484 gravity Effects 0.000 description 3
- PMZURENOXWZQFD-UHFFFAOYSA-L Sodium Sulfate Chemical compound [Na+].[Na+].[O-]S([O-])(=O)=O PMZURENOXWZQFD-UHFFFAOYSA-L 0.000 description 2
- 210000004369 blood Anatomy 0.000 description 2
- 239000008280 blood Substances 0.000 description 2
- 238000009835 boiling Methods 0.000 description 2
- 238000005119 centrifugation Methods 0.000 description 2
- 238000001816 cooling Methods 0.000 description 2
- 230000013595 glycosylation Effects 0.000 description 2
- 238000006206 glycosylation reaction Methods 0.000 description 2
- 238000004255 ion exchange chromatography Methods 0.000 description 2
- 230000002503 metabolic effect Effects 0.000 description 2
- QXLPXWSKPNOQLE-UHFFFAOYSA-N methylpentynol Chemical class CCC(C)(O)C#C QXLPXWSKPNOQLE-UHFFFAOYSA-N 0.000 description 2
- VLTRZXGMWDSKGL-UHFFFAOYSA-N perchloric acid Chemical compound OCl(=O)(=O)=O VLTRZXGMWDSKGL-UHFFFAOYSA-N 0.000 description 2
- 239000008363 phosphate buffer Substances 0.000 description 2
- KBPLFHHGFOOTCA-UHFFFAOYSA-N 1-Octanol Chemical compound CCCCCCCCO KBPLFHHGFOOTCA-UHFFFAOYSA-N 0.000 description 1
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 1
- COQLPRJCUIATTQ-UHFFFAOYSA-N Uranyl acetate Chemical compound O.O.O=[U]=O.CC(O)=O.CC(O)=O COQLPRJCUIATTQ-UHFFFAOYSA-N 0.000 description 1
- 238000005903 acid hydrolysis reaction Methods 0.000 description 1
- 238000001042 affinity chromatography Methods 0.000 description 1
- BFNBIHQBYMNNAN-UHFFFAOYSA-N ammonium sulfate Chemical compound N.N.OS(O)(=O)=O BFNBIHQBYMNNAN-UHFFFAOYSA-N 0.000 description 1
- 229910052921 ammonium sulfate Inorganic materials 0.000 description 1
- 239000001166 ammonium sulphate Substances 0.000 description 1
- 235000011130 ammonium sulphate Nutrition 0.000 description 1
- 230000015572 biosynthetic process Effects 0.000 description 1
- 239000012496 blank sample Substances 0.000 description 1
- 238000012937 correction Methods 0.000 description 1
- ISNBJLXHBBZKSL-UHFFFAOYSA-N ethyl n-[2-(1,3-benzothiazole-2-carbonylamino)thiophene-3-carbonyl]carbamate Chemical compound C1=CSC(NC(=O)C=2SC3=CC=CC=C3N=2)=C1C(=O)NC(=O)OCC ISNBJLXHBBZKSL-UHFFFAOYSA-N 0.000 description 1
- 230000007774 longterm Effects 0.000 description 1
- 238000005259 measurement Methods 0.000 description 1
- 150000007522 mineralic acids Chemical class 0.000 description 1
- 238000012544 monitoring process Methods 0.000 description 1
- 239000000047 product Substances 0.000 description 1
- 230000000750 progressive effect Effects 0.000 description 1
- 238000009877 rendering Methods 0.000 description 1
- 239000011347 resin Substances 0.000 description 1
- 229920005989 resin Polymers 0.000 description 1
- 238000000926 separation method Methods 0.000 description 1
- 239000002002 slurry Substances 0.000 description 1
- 229910052938 sodium sulfate Inorganic materials 0.000 description 1
- 235000011152 sodium sulphate Nutrition 0.000 description 1
- 238000003756 stirring Methods 0.000 description 1
- 239000000758 substrate Substances 0.000 description 1
- 208000024891 symptom Diseases 0.000 description 1
- 238000010998 test method Methods 0.000 description 1
- 230000001225 therapeutic effect Effects 0.000 description 1
Classifications
-
- 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
- G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
- G01N33/6803—General methods of protein analysis not limited to specific proteins or families of proteins
- G01N33/6842—Proteomic analysis of subsets of protein mixtures with reduced complexity, e.g. membrane proteins, phosphoproteins, organelle proteins
-
- 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
- G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
- G01N33/6803—General methods of protein analysis not limited to specific proteins or families of proteins
-
- 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
- G01N33/72—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving blood pigments, e.g. haemoglobin, bilirubin or other porphyrins; involving occult blood
- G01N33/721—Haemoglobin
- G01N33/723—Glycosylated haemoglobin
Abstract
The presence and the quantity of glycosylated proteins in biological fluids are determined by a method comprising removing from the sample interfering factors such as carbohydrates, incubating the sample in a hot acidic environment, separating the liquid and solid phases so found and determining carbohydrate in the liquid phase. A reagent kit comprises oxalic acid, trichloracetic acid and thiobarbituric acid. <IMAGE>
Description
SPECIFICATION
Method and kit for determining glycosylated proteins in biological fluids
It is extremely important to keep under close control the level of glicose in the blood of diabetic patients, who, as is well known, have the symptoms ofglycemia to a lesser or greater extent.
Inasmuch as the evolution of the progressive secondary complications of diabetes is closely related to the intensity of the controls of the metabolic behaviour of the patient, attempts have been made to discover parameters which might be capable of supplying data independent of the rapid variations of glycemia, which variations, as is known from the literature, are caused by a number of factors.
One way of ascertaining the extent of glycemia in such patients is to measure the level of nonenzymically glycosylated proteins (Yue, K. et al., Diabetes, 29: 296 (1980)). The formation of such glycosylated proteins is a reaction which is common to a number of proteins, and is a glicose-blocking process (Day, J. F. et al, J. Biol. Chem. 254: 595 (1979)). The monitoring ofthis reaction gives a significant indication of the degree of metabolic behavious of diabetic patients, since it has been shown that a rise in the extent of glycemia causes a rise in the amount of glycosylated proteins.
The determination of glycosylated haemoglobin (Hb At) may serve as a long-term measurement of the extent of glycemia since the lifetime of this protein is very long (about 120 days). On the other hand, this determination may prove to be of poor utility if it is desired to follow the progress of a therapeutical treatment. To the latter purpose, it might be more valuable to evaluate the degree of glycosylation of albumin since its lifetime is shorter than that of haemoglobin.
Glycosylated haemoglobin is isolated from total haemoglobin by ion-exchange chromatography on a weakly acidic resin, and is then determined colorimetrically. It can also be determined by evaluating, by reaction with thiobarbituric acid (TBA), the quantity of S - hydroxymethylfurfural (5-HMF) which is produced by acidic treatment.
In connection with the glycosylation of albumin, this parameter has been evaluated by methods in which the glycosylated albumin is isolated by affinity chromatography on certain particular dyestuffs and is subjected to a subsequent colorimetric determination after reaction with TBA, or, as an alternative, in which it is subjected to ion-exchange chromatography and is subjected to a subsequent determination by measuring the optical density of the fractions thus obtained.
After it had been discovered that a very close correlation exists between the quantity of glycosylated albumin and the quantity of glycosylated seral proteins, both being colorimetrically assessed, there was proposed a procedure for determining the latter, thus avoiding the need to isolate the albumin frcm the serum, this procedure having considerable advantages both from the point of view of the time necessary for the test and the simplicity of the test procedures. Unfortunately however, as was shown later, in such a determination, the free glicose (and also other carbohydrates) interferes with the determination, and it is necessary to render these carbohydrates incapable of interfering with the determination.
To overcome this difficulty, Kennedy et al (Diabetes,29: 413 (1980)) suggested a 15 hour hydrolysis at 40C against an appropriate saline solution. According to this procedure, the time required for the analysis is considerably extended and the analytical method becomes more intricate. Thus, taking into account the requirement of rendering the seral glicose incapable of interfering prior to the analysis, the procedure suggested by Kennedy et al is summarized in the following Table 1.
Table 1
pgz31Ppm"ee qi = Reagent Sample Blank Operation details Serum 1.0 ml - hour dialysis Dialyzed serum 0.1 ml 0.1 ml Physiological solution (pH 7.4) 0.9 ml Physiological solution +NaBH4 - 0.9 ml 1 molar oxalic acid - 0.5 ml 0.5 ml Boiling steam bath for 5 hours Trichloroacetic acid 1.0 ml 1.0 ml Centrifugation Supernatant 1.5 ml 1.5 ml TBA 0.5 ml 0.5 ml Keep for 30 minutes at 40"C and read optical density at 443 nm (microns) The overall time required for analysis is 21-22
hours (15 of which are necessaryforthe dialysis) and
the various operational steps, even though they are
not actually difficult, differ, at least in certain cases,
from routine laboratory procedures.
It has been outlined in the foregoing to a certain
extent that the determination of non-enzymically glycosylated proteins is subject to analytical errors which are due to interference factors and that the only correction measure which has been suggested by the prior art is a preliminary dialysis of the sample.
The present invention provides two alternative to the method mentioned in the foregoing, namely, on the one hand, the dialysis stage is replaced either by an enzymic step which suppresses the carbohydrates which are not bound to the proteins, and, on the other hand, the determination is carried out in the presence of free carbohydrates but under such analytical conditions (such as controlled hydrolysis) that such carbohydrates cannot interfere. In the latter case, the acidic hydrolysis is carried out in a time of 21 hours or less. If the fi rst of these two alterna- tives is used, a preliminary incubation of the sample being tested is suggested.
According to one aspect, the present invention provides an improved method for determining nonenzymically glycosylated proteins present in biological fluids (blood serum among others), the method comprising hydrolysis of the sample to be tested in an acidic environment and at a temperature above 80"C (the hydrolysis being optionally preceded by a preliminary incubation and the removal of carbohydrate and/or interfering substances), effecting subsequent precipitation of the proteins, separating the liquid phase and adding thereto a reagent capable of detecting carbohydrates and/ortheir derivatives, and comparing the optical densities ofthe sample and of a test blank.
The removal of carbohydrates and of possibly interfering substances of different nature is carried out, more particularly, in two ways.
According to the first way, all of the proteins in question are precipitated by the addition of a particu larprecipitation agent selected from a number of reagents, these reagents being organic compounds such as alcohols, polyhydric alcohols, carbonyl compounds, organic acids or salts thereof. In addition, salts of inorganic acids can be used. In particular, p-toluenesulphonic acid, trichloroacetic acid, perchloric acid, uranyl acetate, sulphosalicylic acid, ammonium sulphate or sodium sulphate, are used.
The temperature is maintained to a value which is that of the environment or lower.
The removal of the carbohydrates and/or other interfering substances can also be carried out enzymically, possibly after having adjusted the pH of the blood medium to an appropriate value. In this case, the enzymes (added either separately at differend times or admixed with each other) are appropriately selected so as to convert such interfering substances into products which do not interfer.
The enzymes can be introduced as such or in properly immobilized form, for example occluded in fibres or bonded (covalently or otherwise) to natural or synthetic polymeric substrates.
As outlined above, the operations for the removal of the interfering substances can be preceded by an incubation of the sample, with a view to overcoming potential interfering factors which could disturb the subsequent determination. Typical examples are preliminary treatment ofthe sample with appropri ate enzymes (such as syalidase, galactoxidase and others) or with acids at a temperature in the range from 80"C and 100"C and for a time not longer than 30 minutes. The preferred acids are oxalic acid, sulphuric acid, hydrochloric acid, sulphosalicylic acid and p-toluenesulphonic acids, and mixtures of such acids with each other and with other acids.
The method according to the present invention then proceeds with a hydrolysis stage in an acidic environment for a time over 22 hours and at a temperature above 80"C. Subsequently, the proteins are precipitated. The same precipitating agents as mentioned above can be used for this purpose. This step is followed by separation of the solid phase from the supernatant liquor, the latter being divided into two portions. To a first portion of the liquid phase a carbohydrate detecting reagent (and/or a reagent for detecting their derivatives such as thiobarbituric acid) is added, whereas water is added to the second portion of the liquid phase to obtain a test blank if no
NaBH4 or reducing agent is used. Finally, the optical densities are measured (at an appropriate wavelength) both for the sample and for the blank.
The method can be applied to the determination of glycosylated proteins which are contained in biological media or liquors of any nature and origin.
The method has proven to be particularly advantageous for determining glycosylated proteins which are contained in blood serum samples, and we have deemed it useful to limit the Examples given below to that specific case on account of the importance of that application. However, the use ofthe method on samples of different origin does not produce any difficulty whatsoever and any extension of the method exemplified below will be an obvious routine operation lying within the scope of the present invention.
The invention also provides a means to be used for performing the method. This means is a kit which contains, alone in discrete containers, or separate but assembled in a single container, reagents selected from (a) medium-acidifying agents, (b) protein precipitants, (c) ketone-reducing agents, and (d) detecting agents for carbohydrates and/or their derivatives. More particularly, the kit comprises oxalic acid or acetic acid, trichloroacetic acid (TCB) sodium borohydride, and thiobarbituric acid (TBA).
The foregoing and other operative details will become clearer from the following illustrative
Examples which however do not limit the invention.
EXAMPLE 1
A serum portion equivalent to 3.3 mg of proteins was diluted, after addition of traces of octyl alcohol, with 1 ml of phosphate buffer (0.01 molar, pH 7.4) containing 0.15 molar NaC (PBS) and 0.85 molar sodium borohydride. The sample was allowed to stand at room temperature for 1 hour and, then, for 18 hours at 4"C. This procedure is for preparing a test blank.
Another serum portion containing the same amount of proteins was diluted with 1 ml of the usual phosphate buffer (pH 7) but without the reducing agent Both this sample and the test blank were treated with 1 ml of 40% TCA and, after a 1 5-minute stay at room temperature, the samples and the test blank were centrifuged for 15 minutes at 2500 x gravity. The pricipitates, reslurried in 1.5 ml of2 normal acetic acid, were kept for 24 hours at 1 OO"C.
Upon cooling, the slurry was treated with 0.5 ml of 400% TCA and centrifuged to recover a clear supernatant. Portions of 1.5 ml each of the clear solution were reacted with 0.5 ml of TBA for 30 minutes at 40"C. The difference between the optical densities (O.D.) at443 nm of the blanks and the samples was reported on an appropriate plot (a calibration plot with 5-HMF) so that the millimols of 5-HMF contained in the sample can be determined.This method is summarized in the following Table 2.
Table2
Reagent Blank Sample Operation details Serum 0.05 ml 0.05 ml PBS - 1 ml PBS + NaHBH4 1 ml - 1 hour at room temperature plus 18 hours at4 C 40%TCA 1 ml 1 ml Centrifuge 2 normal acetic acid 1.5 ml 1.5 ml Keep for 24 hours at 1 00 C TCA 0.5 ml 0.5 ml Centrifuge Supernatant 1.5 ml 1.5 ml TBA 0.5 ml 0.5 ml Read O.D. at 443 nm after 30 minutes at40"C EXAMPLE2
The method of this Example includes a preiimi
nary treatment for 15 minutes at 1000C and the pre capitation of the proteins with 40% TCA.
A portion of serum (0.4 ml) was diluted in a ratio of from 1:2 to 1:20 (1:5 is preferred) with 0.6 normal oxalic acid, directly in a test tube with a screwed plug and, upon stirring, it was kept for 15 minutes at 100"C. After cooling to room temperature, 2 ml of 40% TCA were added. The sample was stirred, allowed to stand for 15 minutes at room temperature and centrifuged at 2500 x gravity for 10 minutes.
The supernatant was discarded by allowing the upturned tube test to stay for a few minutes on blotting paper pads. The residue was then mixed with 2.8 ml of 0.6 normal oxalic acid and, once the test tubes had been sealed, they were held in a boiling steam bath for 5 hours, so that the glicose bonded to the proteins was converted to 5-HMF. The several test tubes were cooled to room temperature in a water bath for 10 minutes, and 1 ml of 40% TCA was added to each tube. After 10 additional minutes at room temperature, centrifugation was carried out at 2500 x gravity. The clear supernatant was collected and divided into two portions of 1.5 ml each. One portion, mixed with 0.05 molar thiobarbituric acid, is the sample, whereas the other portion, mixed with water only, is the blank.
The determination of the quantity of 5-HMF which is contained in the sample was carried out by determining the difference between the respective optical densities at 443 nm of the sample and of the blank and by comparing the difference so found with a calibration plot obtained with standard 5-HMF.
The method according to this Example is summarized in the following Table 3.
Table3
Reagent . Operation Details Serum 0.4 ml 0.6 normal oxalic acid 1.6 ml 15 minutes at 100"C 400cTCA 2 ml Centrifuge 0.6 normal oxalic acid 2.8 ml Keep for 5 hours at 100"C 40%TCA 1.0 ml Centrifuge Sample Blank Supernatant 1.5 ml 1.5 ml TBA 0.5 ml Water - 0.5 ml Read O.D. at 433 nm after 30 minutes at40"C
Claims (15)
1. A method for determining glycosylate protein contained in a biological fluid, which comprises treating the fluid in an acidic environment at a temperature above 80"C (with or without preliminaryincubation and the removal of carbohydrates and/or other interfering substances), effecting precipitation of the proteins, separating the liquid phase, adding thereto a reagent for detecting the presence of carbohy drates and/or thei r derivatives to obtai n asample solution, adding water or an appropriate solution to a portion of the liquid phase to form a blank solution (if or since no NaBH4 or other reducing agent is employed), and comparing the optical densities of the sample solution and the blank solution.
2. A method according to claim 1, wherein the acidic treatment of the fluid is the first operation and is carried out for a time which does not exceed 22 hours.
3. A method according to claim 1, which comprises (a) effecting preincubation of the fluid, (b) removing the carbohydrates and/orthe interfering substances, (c) subjecting to acidic treatment the residue of the previous step for a time longer than 22 hours, (d) precipitating the proteins, (e) separating the liquid phase from the precipitate, (f) adding to a portion ofthe liquid phase a reagent for detecting carbohydrates and/or their derivatives to obtain a sample solution, (g) adding water or an appropriate solution to a portion of the liquid phase in the same ratio as in the previous step to obtain a blank solution, and (h) comparing the optical densities ofthe sample solution and the blank solution.
4. A method according to claim 3, wherein step (b) is carried out by precipitating the proteins con tainedinthefluid.
5. A method according to claim 4, wherein the precipitation is carried out by adding to the fluid a precipitating agent selected from monohydric alcohols, polyhydric alcohols, organic acids, salts of organic acids, carbonyl compounds, and other organic compounds.
6. A method according to claim 4, wherein the precipitation is carried out by adding an inorganic salt to the fluid.
7. A method according to claim 3, wherein step (b) is carried out by adding a specific enzyme of the carbohydrate(s) to be removed, either each alone individually or in admixture and/or at different times.
8. A method according to claim 7, wherein step (b) is carried out by using immobilized enzyme(s).
9. A method according to any of claims 3 to 8, wherein the preliminary incubation step is carried out at a temperature of from 80 to 1 OO"C.
10. A method according to claim 9, wherein the incubation is carried out in the presence of an acid selected from oxalic acid, acetic acid, sulphuric acid, hydrochloric acid, sulphosalicylic acid, p-toluene sulphonic acids, and mixtures thereof.
11. A method according to claim 3, wherein step (d) is carried out by adding a precipitating agent selected from monohydric alcohols, polyhydric alcohols, organic acids, salts of organic acids, carbonyl compounds, other organic compounds, and inorganic salts.
12. A method according to claim 1, substantially as hereinbefore described.
13. A kit of diagnostic reagents for the determination of glycosylated protein in a biological fluid, comprising, alone in separate containers, or separate but assembled in a single container, reagents selected from (a) an acidifying agent such as a medium-acidifying agent, (b) a protein precipitant, and (c) a reagent for detecting carbohydrate and/or derivative thereof.
14. A kit according to claim 13, wherein the reagents are oxalic acid, trichloroacetic acid and thiobarbituric acid.
15. A kit according to claim 13, substantially as hereinbefore described.
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| IT8122582A IT1167460B (en) | 1981-06-26 | 1981-06-26 | DOSAGE OF GLYCOSYLATE PROTEINS IN ORGANIC FLUIDS AND MEDIA SUITABLE FOR THE PURPOSE |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| GB2101740A true GB2101740A (en) | 1983-01-19 |
Family
ID=11198083
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| GB08217960A Withdrawn GB2101740A (en) | 1981-06-26 | 1982-06-21 | Method and kit for determining glycosylated proteins in biological fluids |
Country Status (6)
| Country | Link |
|---|---|
| JP (1) | JPS5821167A (en) |
| DE (1) | DE3223837A1 (en) |
| ES (1) | ES514387A0 (en) |
| FR (1) | FR2508646A1 (en) |
| GB (1) | GB2101740A (en) |
| IT (1) | IT1167460B (en) |
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| FR2559267A1 (en) * | 1982-10-01 | 1985-08-09 | Reanal Finomvegyszergyar | Determn. of glucose content of glycosylated proteins |
| EP0215170A1 (en) * | 1985-09-19 | 1987-03-25 | Isolab, Inc. | Single color reading method for determining fructosamine |
| EP0271996A1 (en) * | 1986-11-18 | 1988-06-22 | Scripps Clinic And Research Foundation | Immunochemical method for assaying stable glycosylated hemoglobin |
| US5571723A (en) * | 1991-02-07 | 1996-11-05 | Evans; Cody A. | Method of testing for diabetes that reduces the effect of interfering substances |
Families Citing this family (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JPS62215398A (en) * | 1986-01-06 | 1987-09-22 | アイソラブ，インク． | Method and kit for measuring quantity of phosphatidyl glycerol in amniotic fluid as indication element for medicaldiagnosis |
| GB9024771D0 (en) * | 1990-11-14 | 1991-01-02 | Axis Research | Assay |
Family Cites Families (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US4268270A (en) * | 1979-04-30 | 1981-05-19 | Children's Hospital Medical Center | Glycosylated hemoglobin measurement |
| CA1146769A (en) * | 1980-02-04 | 1983-05-24 | Beat E. Glatthaar | Process for the determination of diabetes |
-
1981
- 1981-06-26 IT IT8122582A patent/IT1167460B/en active
-
1982
- 1982-06-21 GB GB08217960A patent/GB2101740A/en not_active Withdrawn
- 1982-06-23 ES ES514387A patent/ES514387A0/en active Granted
- 1982-06-25 JP JP57109652A patent/JPS5821167A/en active Pending
- 1982-06-25 DE DE19823223837 patent/DE3223837A1/en not_active Ceased
- 1982-06-25 FR FR8211192A patent/FR2508646A1/en not_active Withdrawn
Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| FR2559267A1 (en) * | 1982-10-01 | 1985-08-09 | Reanal Finomvegyszergyar | Determn. of glucose content of glycosylated proteins |
| EP0215170A1 (en) * | 1985-09-19 | 1987-03-25 | Isolab, Inc. | Single color reading method for determining fructosamine |
| EP0271996A1 (en) * | 1986-11-18 | 1988-06-22 | Scripps Clinic And Research Foundation | Immunochemical method for assaying stable glycosylated hemoglobin |
| US4876188A (en) * | 1986-11-18 | 1989-10-24 | Scripps Clinic And Research Foundation | Novel immunochemical method for assaying stable glycosylated hemoglobin |
| US5571723A (en) * | 1991-02-07 | 1996-11-05 | Evans; Cody A. | Method of testing for diabetes that reduces the effect of interfering substances |
Also Published As
| Publication number | Publication date |
|---|---|
| IT1167460B (en) | 1987-05-13 |
| FR2508646A1 (en) | 1982-12-31 |
| DE3223837A1 (en) | 1983-01-13 |
| JPS5821167A (en) | 1983-02-07 |
| IT8122582A0 (en) | 1981-06-26 |
| ES8305934A1 (en) | 1983-05-01 |
| ES514387A0 (en) | 1983-05-01 |
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Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| WAP | Application withdrawn, taken to be withdrawn or refused ** after publication under section 16(1) |