CA1146769A - Process for the determination of diabetes - Google Patents
Process for the determination of diabetesInfo
- Publication number
- CA1146769A CA1146769A CA000364791A CA364791A CA1146769A CA 1146769 A CA1146769 A CA 1146769A CA 000364791 A CA000364791 A CA 000364791A CA 364791 A CA364791 A CA 364791A CA 1146769 A CA1146769 A CA 1146769A
- Authority
- CA
- Canada
- Prior art keywords
- acid
- proteins
- trichloroacetic acid
- carried out
- colour reaction
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Expired
Links
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
- G01N33/72—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
Landscapes
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Hematology (AREA)
- Engineering & Computer Science (AREA)
- Molecular Biology (AREA)
- Biomedical Technology (AREA)
- Chemical & Material Sciences (AREA)
- Immunology (AREA)
- Urology & Nephrology (AREA)
- Biotechnology (AREA)
- Microbiology (AREA)
- Cell Biology (AREA)
- Food Science & Technology (AREA)
- Medicinal Chemistry (AREA)
- Physics & Mathematics (AREA)
- Analytical Chemistry (AREA)
- Biochemistry (AREA)
- General Health & Medical Sciences (AREA)
- General Physics & Mathematics (AREA)
- Pathology (AREA)
- Investigating Or Analysing Biological Materials (AREA)
Abstract
ABSTRACT
There is described a process for the determination of diabetes based on the hemoglobin species A1C. The hemo-globin species A1C is determined in principle according to the method of Gabbay, K.H. et al. in Diabetes Vol. 28, 1979, but the determination is effected in whole blood and not in hemolysates of red blood cells. In addition, the precipitation of the proteins and the color reaction are performed simultaneously.
There is described a process for the determination of diabetes based on the hemoglobin species A1C. The hemo-globin species A1C is determined in principle according to the method of Gabbay, K.H. et al. in Diabetes Vol. 28, 1979, but the determination is effected in whole blood and not in hemolysates of red blood cells. In addition, the precipitation of the proteins and the color reaction are performed simultaneously.
Description
- ~4~7t~9 It is known that the erythrocytes of human beings contain, besides the most representative haemoglobin S species A (ca 90~) and A2 (ca 2.5%) as well as small amounts of species F (ca 0.5%), further haemoglobin species.
These further haemoglobin species make up ca 5-8~ of the total haemoglobin. Of these further haemoglobin species, - species AlC is quantitatively the most significant, since it makes up 4-6~ of the entire haemoglobin. It is known that diabetics have high values of haemoglobin species AlC [Rahbars et al. Biochem. Biophys. Res. Commun. 36, 838--843 (1969)]. Furthermore, it is known that haemoglobin species AlC values do not reflect the actual blood sugar values, but are to be evaluated as the integral of the preceding four months values. Accordingly, the determination of haemoglobin species AlC is of particular diagnostic value.
Haemoglobin species AlC can be determined by cation--exchange chromatography of haemolysates of red blood cells [Trivelli L.A. et al. N.Engl. J. Med. 824, 353-357 (1971)], :
but this method is too expensive to be able to be used for carrying out a series of investigations. Furthermore, it is known that haemoglobin species AlC can be determined by Klt/4.11.1980 , . ~
These further haemoglobin species make up ca 5-8~ of the total haemoglobin. Of these further haemoglobin species, - species AlC is quantitatively the most significant, since it makes up 4-6~ of the entire haemoglobin. It is known that diabetics have high values of haemoglobin species AlC [Rahbars et al. Biochem. Biophys. Res. Commun. 36, 838--843 (1969)]. Furthermore, it is known that haemoglobin species AlC values do not reflect the actual blood sugar values, but are to be evaluated as the integral of the preceding four months values. Accordingly, the determination of haemoglobin species AlC is of particular diagnostic value.
Haemoglobin species AlC can be determined by cation--exchange chromatography of haemolysates of red blood cells [Trivelli L.A. et al. N.Engl. J. Med. 824, 353-357 (1971)], :
but this method is too expensive to be able to be used for carrying out a series of investigations. Furthermore, it is known that haemoglobin species AlC can be determined by Klt/4.11.1980 , . ~
- 2 -incubating a haemolysate of red blood cells in acidic solution, precipitating the proteins, carrying out a colour reaction after separation of the proteins and subsequently carrying out a photometric determination after incubation. The value obtained is thereafter compared with a standard in a manner known per se.
[Gabbay K. H. et al. Diabetes Vol. 28 (1979)].
In accordance with the present invention it has now been found that the aforementioned photometric determination method for haemoglobin species AlC can be substantially simplified by on the one hand carrying out the determination not with a haemolysate of red blood cells but in whole blood and by on the other hand simultaneously carrying out the precipitation of the proteins and the colour reaction.
, The present invention is accordingly concerned with a method for the determination of the haemoglobin species AlC according to the photometric method,~which comprises incubating a whole blood sample at an elevated temperature in acidic solution, simultaneously precipitating the proteins and carrying out the colour reaction and after incubation and separation of the denatured proteins, carrying out a photometric determination and comparing the value obtained with a standard in a manner known per se.
: ' 6'~
The first incubation of the whole blood sample is preferably carried out at a temperature of 95-105C.
This incubation is preferably carried out for 1 to 5 hours, particularly for 5 hours.
The acidic solution is conveniently a 0.2-lN oxalic acid, sulphuric acid or hydrochloric acid solution. A 0.4N
oxalic acid solution is preferred.
.
The separation of the proteins is carried out using a suitable protein-precipitation agent such as, for example, trichloroacetic acid.
The colour reaction is carried out using an agent which forms a colour complex. Thiobarbituxic acid is especially suitable for thls purpose. ~ -The agent which forms a colour complex should be :
soluble in sufficient amounts in the protein-precipitation agent. In accordance with an especlally preferred embodiment there is used trichloroacetic acid which is almost saturated with thiobarbituric acid.
'7~
Before the separation of the denatured proteins the mixture is preferably incubated for about 30 minutes at abou~ 37C.
The separation of the denatured proteins is carried out according to customary methods; for example, by centrifugation or filtration.
The photometric measurement is carried out at 443 nm.
The wavelength can vary when filters are used.
4~7~
~ 5 --The following Examples illustrate the present invention:
Example 1 0.1 ml blood samples are placed in test tubes.
1.4 ml of 0.2M (0.4N) oxalic acid are added thereto and the tubes are closed. The tubes are incubated on a heating block at 100C for 5 hours. Thereupon, the tubes are cooled down to room temperature.
` l ml of a 0.025M solution of thiobarbituric acid in 20~ aqueous trichloroacetic acid is added to each tube.
Since the thiobarbituric acid does not dissolve very well, the tubes are warmed to 60C. The solution obtained can ; ~ be used immediately or can be stored for 7 days. The tubes are incubated for 30 minutes at 37C and then left to stand for 30 minutes at room temperature. The tubes are centrifuged at 3000 rpm;for 5 minutesl whereupon the supernatants are measured photometrically at 430 nm and a layer thickness of l cm. At this wavelength the colour ~ complex formed by the thiobarbituric acid exhibits a - 20 molar extinction coefficient of 26.
Example 2 Blood samples from diabetics and healthy test persons ~ ' ' , :
.
were investigated according to the method described in Example 1. In this case the following haemoglobin species Al~ values were ascertained:
Diabetic Healthy test person Patient 1 13.5~ Test person 1 4.2 Patient 2 12.9% Test person 2 4.
Patient 3 12.3% Test person 3 4.6~
Patient 4 10.6~ Test person 4 4.9%
Patient 5 10.2~ Test person 5 5.2%
Patient 6 10.0~ Test person 6 5~3 From these values it is clearly evident that the diabetics have high values of haemoglobin species AlC.
Exam~e 3 For the preparation of the standard, samples which contain known, increasing amounts of haemoglobin species ; AlC are prepared in a manner known per se. The haemoglobin species AlC content of these samples is determined according to the method described in Example 1, whereupon a standard curve is prepared in the usual manner.
[Gabbay K. H. et al. Diabetes Vol. 28 (1979)].
In accordance with the present invention it has now been found that the aforementioned photometric determination method for haemoglobin species AlC can be substantially simplified by on the one hand carrying out the determination not with a haemolysate of red blood cells but in whole blood and by on the other hand simultaneously carrying out the precipitation of the proteins and the colour reaction.
, The present invention is accordingly concerned with a method for the determination of the haemoglobin species AlC according to the photometric method,~which comprises incubating a whole blood sample at an elevated temperature in acidic solution, simultaneously precipitating the proteins and carrying out the colour reaction and after incubation and separation of the denatured proteins, carrying out a photometric determination and comparing the value obtained with a standard in a manner known per se.
: ' 6'~
The first incubation of the whole blood sample is preferably carried out at a temperature of 95-105C.
This incubation is preferably carried out for 1 to 5 hours, particularly for 5 hours.
The acidic solution is conveniently a 0.2-lN oxalic acid, sulphuric acid or hydrochloric acid solution. A 0.4N
oxalic acid solution is preferred.
.
The separation of the proteins is carried out using a suitable protein-precipitation agent such as, for example, trichloroacetic acid.
The colour reaction is carried out using an agent which forms a colour complex. Thiobarbituxic acid is especially suitable for thls purpose. ~ -The agent which forms a colour complex should be :
soluble in sufficient amounts in the protein-precipitation agent. In accordance with an especlally preferred embodiment there is used trichloroacetic acid which is almost saturated with thiobarbituric acid.
'7~
Before the separation of the denatured proteins the mixture is preferably incubated for about 30 minutes at abou~ 37C.
The separation of the denatured proteins is carried out according to customary methods; for example, by centrifugation or filtration.
The photometric measurement is carried out at 443 nm.
The wavelength can vary when filters are used.
4~7~
~ 5 --The following Examples illustrate the present invention:
Example 1 0.1 ml blood samples are placed in test tubes.
1.4 ml of 0.2M (0.4N) oxalic acid are added thereto and the tubes are closed. The tubes are incubated on a heating block at 100C for 5 hours. Thereupon, the tubes are cooled down to room temperature.
` l ml of a 0.025M solution of thiobarbituric acid in 20~ aqueous trichloroacetic acid is added to each tube.
Since the thiobarbituric acid does not dissolve very well, the tubes are warmed to 60C. The solution obtained can ; ~ be used immediately or can be stored for 7 days. The tubes are incubated for 30 minutes at 37C and then left to stand for 30 minutes at room temperature. The tubes are centrifuged at 3000 rpm;for 5 minutesl whereupon the supernatants are measured photometrically at 430 nm and a layer thickness of l cm. At this wavelength the colour ~ complex formed by the thiobarbituric acid exhibits a - 20 molar extinction coefficient of 26.
Example 2 Blood samples from diabetics and healthy test persons ~ ' ' , :
.
were investigated according to the method described in Example 1. In this case the following haemoglobin species Al~ values were ascertained:
Diabetic Healthy test person Patient 1 13.5~ Test person 1 4.2 Patient 2 12.9% Test person 2 4.
Patient 3 12.3% Test person 3 4.6~
Patient 4 10.6~ Test person 4 4.9%
Patient 5 10.2~ Test person 5 5.2%
Patient 6 10.0~ Test person 6 5~3 From these values it is clearly evident that the diabetics have high values of haemoglobin species AlC.
Exam~e 3 For the preparation of the standard, samples which contain known, increasing amounts of haemoglobin species ; AlC are prepared in a manner known per se. The haemoglobin species AlC content of these samples is determined according to the method described in Example 1, whereupon a standard curve is prepared in the usual manner.
Claims (11)
1. A method for the determination of the haemoglobin species A1C according to the photometric method, which com-prises incubating a whole blood sample at an elevated tempera-ture in an acidic solution, simultaneously precipitating pro-teins and carrying out a colour reaction and, after incubation and separation of denatured proteins, carrying out a photo-metric determination and comparing the value obtained with a standard in a manner known per se.
2. A method according to claim 1, wherein the whole blood sample is incubated at 95° - 105°C.
3. A method according to claim 1, wherein the acidic sol-ution is oxalic acid solution.
4. A method according to claim 2, wherein the acidic sol-ution is oxalic acid solution.
5. A method according to claim 3, wherein the acidic sol-ution is 0.4N oxalic acid solution.
6. A method according to any one of claims 1 to 3, wherein the proteins are precipitated using trichloroacetic acid and simultaneously the colour reaction is carried out using thiobar-bituric acid.
7. A method according to any one of claims 4 or 5, wherein the proteins are precipitated using trichloroacetic acid and simultaneously the colour reaction is carried out using thiobar-bituric acid.
8. A method according to any one of claims 1 to 3, wherein the proteins are precipitated using trichloroacetic acid and simultaneously the colour reaction is carried out using thiobarbit-turic acid, and wherein the trichloroacetic acid concentration of the sample is adjusted to ca 6-10% with aqueous trichloro-acetic acid which is almost saturated with thiobarbituric acid.
9. A method according to any one of claims 1 to 3, wherein the proteins are precipitated using trichloroacetic acid and simultaneously the colour reaction is carried out using thiobarbit-turic acid, and wherein the trichloroacetic acid concentration of the sample is adjusted to ca 8% with aqueous trichloroacetic acid which is almost saturated with thiobarbituric acid.
10. A method according to any one of claims 1 to 3, wherein the sample is incubated for about 30 minutes at 37°C before the separation of the denatured proteins.
11. A method according to any one of claims 1 to 3, wherein the denatured proteins are separated by centrifugation or fil-tration.
Applications Claiming Priority (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CH86880 | 1980-02-04 | ||
| CH868/80 | 1980-02-04 |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CA1146769A true CA1146769A (en) | 1983-05-24 |
Family
ID=4197050
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CA000364791A Expired CA1146769A (en) | 1980-02-04 | 1980-11-17 | Process for the determination of diabetes |
Country Status (4)
| Country | Link |
|---|---|
| EP (1) | EP0033543B1 (en) |
| JP (1) | JPS56122959A (en) |
| CA (1) | CA1146769A (en) |
| DE (1) | DE3167992D1 (en) |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US4675185A (en) * | 1985-12-06 | 1987-06-23 | Baxter Travenol Laboratories, Inc. | Solution for stabilizing red blood cells during storage |
Families Citing this family (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| IT1167460B (en) * | 1981-06-26 | 1987-05-13 | Sclavo Inst Sieroterapeut | DOSAGE OF GLYCOSYLATE PROTEINS IN ORGANIC FLUIDS AND MEDIA SUITABLE FOR THE PURPOSE |
| HU186976B (en) * | 1982-10-01 | 1985-10-28 | Reanal Finomvegyszergyar | Process for determation of glucose containt of glucolized in non-ensimatic way proteins and reagent lutes for this process |
| EP0729031A1 (en) * | 1995-02-24 | 1996-08-28 | F. Hoffmann-La Roche Ag | Set of reagents determining the content of total haemoglobin |
| GB9913560D0 (en) * | 1999-06-10 | 1999-08-11 | Cortecs Diagnostics Limited | Mixing apparatus and method of mixing |
| CN105203528A (en) * | 2015-09-22 | 2015-12-30 | 深圳市希莱恒医用电子有限公司 | Glycosylated hemoglobin detecting device |
Family Cites Families (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US4108603A (en) * | 1977-05-18 | 1978-08-22 | Purdue Research Foundation | Method for rapid analysis of hemoglobin variants by high speed liquid chromatography |
| DE2820310A1 (en) * | 1978-05-10 | 1979-11-15 | Univ Rockefeller | Specific antibody against human haemoglobin A-1c - used for quantitative determination of haemoglobin A-1c levels in diabetics by radioimmunological assay |
| GB2024829B (en) * | 1978-06-28 | 1982-08-04 | Amicon Corp | Method and product for separation of glycoproteins |
| US4200435A (en) * | 1978-12-26 | 1980-04-29 | Abbott Laboratories | Determination of glycosylated hemoglobin in blood |
| US4268270A (en) * | 1979-04-30 | 1981-05-19 | Children's Hospital Medical Center | Glycosylated hemoglobin measurement |
-
1980
- 1980-11-17 CA CA000364791A patent/CA1146769A/en not_active Expired
-
1981
- 1981-02-02 JP JP1421081A patent/JPS56122959A/en active Pending
- 1981-02-03 DE DE8181100762T patent/DE3167992D1/en not_active Expired
- 1981-02-03 EP EP81100762A patent/EP0033543B1/en not_active Expired
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US4675185A (en) * | 1985-12-06 | 1987-06-23 | Baxter Travenol Laboratories, Inc. | Solution for stabilizing red blood cells during storage |
Also Published As
| Publication number | Publication date |
|---|---|
| EP0033543B1 (en) | 1985-01-02 |
| JPS56122959A (en) | 1981-09-26 |
| EP0033543A1 (en) | 1981-08-12 |
| DE3167992D1 (en) | 1985-02-14 |
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Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| MKEX | Expiry |