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US3792044A - Method for determining glucose with o-toluidine reagent containing an arsenic compound - Google Patents

Method for determining glucose with o-toluidine reagent containing an arsenic compound Download PDF

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US3792044A
US3792044A US00203840A US3792044DA US3792044A US 3792044 A US3792044 A US 3792044A US 00203840 A US00203840 A US 00203840A US 3792044D A US3792044D A US 3792044DA US 3792044 A US3792044 A US 3792044A
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glucose
toluidine
sodium
test
arsenic
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J Dooley
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Warner Lambert Co LLC
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Pfizer Corp Belgium
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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
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y10—TECHNICAL SUBJECTS COVERED BY FORMER USPC
    • Y10T—TECHNICAL SUBJECTS COVERED BY FORMER US CLASSIFICATION
    • Y10T436/00—Chemistry: analytical and immunological testing
    • Y10T436/14—Heterocyclic carbon compound [i.e., O, S, N, Se, Te, as only ring hetero atom]
    • Y10T436/142222—Hetero-O [e.g., ascorbic acid, etc.]
    • Y10T436/143333—Saccharide [e.g., DNA, etc.]
    • Y10T436/144444—Glucose

Definitions

  • This invention relates to a new and improved diagnostic composition which is useful for the quantitative determination of glucose in fluids, particularly body fluids such as urine, plasma, blood and the like.
  • glucose in blood and urine is of great importance for diabetic patients whose diets must be controlled so as to regulate sugar intake and who must frequently be guided in this regard by .regular checks on blood and urine glucose. Diabetes screening programs are dependent on the availability of rapid, inexpensive and accurate methods of glucose determination.
  • the Folin- Wu and Nelson-Somogyi test methods can be applied only to protein-free filtrates of blood serum or plasma. There is some loss of specificity and reliability because of saccharoids and other reducing compounds that cannot be completely removed in preparation of a protein-free filtrate with resulting values higher than true glucose levels. Other disadvantages include the limited stability of the test reagents, the instability of the final test color, and critical factors in the test procedure involving temperature, heating time and alkalinity of reaction mixture.
  • the glucose-oxidase test is a highly specific test for glucose when correctly done with pure materials. A protein-free filtrate is required. Other disadvantages include the sensitivity and limited stability of the test reagents, he necessary purity and standardization of the glucoseoxidase enzyme, the errors arising from interfering enzyme inhibitors, and the extreme care necessary during the three minute heating process where a matter of seconds can critically aifect the accuracy of results.
  • the o-toluidine test is a simple, rapid, reliable and economical method for glucose determination. Importantly, there is no need for protein-free filtrates although the test can be performed with such filtrates.
  • the test can be performed directly with blood serum or plasma.
  • Orthotoluidine is the only reagent required. Acetic acid solutions are stable for at least six months. This stability is enhanced by the addition of thiourea. The final test color has good stability.
  • An important disadvantage, however, of o-toluidine is its susceptibility to oxidation with its resultant effect on test sensitivity. The depressed sensitivity of the reagent results in a regression line with a lower slope when the optical densities of the final test colors are plotted against sugar concentrations, with serious loss of assay accuracy.
  • the object of this invention is to provide an improved diagnostic composition containing o-toluidine, thiourea and arsenic oxides for the simple, rapid, inexpensive and accurate quantitative determination of glucose in the biological fluids of humans.
  • This invention is concerned with a diagnostic composition containing o-toluidine for the quantitative determination of glucose in such biological fluids as blood and urine. It has been found that the incorporation of an oxide of arsenic in the o-toluidine reagent solution increases the sensitivity of the test reagent and increases the stability of the final test color in the assay procedure.
  • the o-toluidine test method for the quantitative determination of glucose in blood and urine involves the formation of a colored complex of glucose and o-toluidine in glacial acetic acid which follows Beers law. The green color, read at 630 nm., is stable 97%) after standing one hour in air at 25 C.
  • o-Toluidine is relatively sensitive to oxidation. Bulk supplies of uniform quality and purity are difficult to obtain, with Eastman Chemical Company the preferred source. Freshly distilled o-toluidine is, of course, eminently suitable. The stability of o-toluidine solutions in glacial acetic acid (6.25% w./v.) is increased by the addition of thiourea (1.50% w./v.). The addition of an arsenate salt or oxide of arsenic increases the sensitivity of the o-toluidine reagent as reflected in the slope of the optical density curve of the final test colors.
  • the arsenic compound may be selected from the group consisting of the following:
  • sodium orthoarsenate Na AsO 121-1 0 sodium mono-H-orthoa-rsenate: Na HAsO -7H O sodium mono-H-orthoarsenate: Na HAsO l2H O sodium di-H-orthoarsenate: NaH AsO -H O sodium metaarsenate: NaAsO sodium arsenite: NaAsO- arsenic trioxide: AS203 arsenolite: AS405 claudetite: As O arsenic pentoxide: AS205 sodium thioarsenate: Na AsS -8H O
  • Na arsenate (Na HAsO -7H O) increases the sensitivity to glucose up to An increase in sensitivity, over solutions containing no additive, is observed even with small 0.1% w./v.) concentrations of sodium arsenate additive.
  • the color formed by the o-toluidine, with and without arsenate additive, is quite stable for the first hour.
  • an increase in stability is noted for the sodium arsenate additive solution after 15 hours with a 32% decrease in color retained, compared to a 38% decrease for the control solution. This increased color stability is important when large numbers of test samples are run, and assay readings cannot be immediately made.
  • Test procedure (1) Using a suitable pipette add 0.05 ml. of blood serum or other sample to be tested to a 150 mm. x 15 mm., or similar, Pyrex test tube. If serum or plasma is used, deproteinization is not necessary.
  • EXAMPLE II The preparation of the o-toluidine test reagent of Example I is repeated in turn with sodium orthoarsenate (Na AsO -12H O), sodium mono-H-orthoarsenate (Na HAsO sodium di-R-orthoarsenate (NaH AsO -H O), sodium metaarsenate (NaAsO sodium arsenite (NaAsO arsenic trioxide (As o arsenolite (As O claudetite (ASaO arsenic pentoxide (AS305) and sodium thio- 4 arsenate (Na AsS -8H 0) in place of sodium mono-H- orthoarsenate (Na HAsO -7H O), with comparable test results.
  • sodium orthoarsenate Na AsO -12H O
  • Na HAsO sodium di-R-orthoarsenate NaH AsO -H O
  • an arsenic compound selected from the group consisting of sodium orthoarsenate (Na ASO 12H O) sodium mono-H-orthoarsenate (Na HAsO -7H O), sodium mono-H-orthoarsenate (NaHAsO -12H O), sodium di-H-orthoarsenate (NaH AsO -H O), sodium metaarsenate (NaAsO sodium arsenite (NaAsO arsenic trioxide (As O arsenolite (AS406), claudetite (As O arsenic pentoxide (As O and sodium thioarsenate (Na AsS 2.
  • arsenic compound is sodium mono-H-orthoarsenate (Na- HAsO (Na AsS 8H O 4.

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  • Diabetes (AREA)
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  • General Health & Medical Sciences (AREA)
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Abstract

A DIAGNOSTIC C~MPOSITION CONTAINING O-TOLUIDINE, THIOUREA AND AN OXIDE OF ARSENIC IS USED FOR THE QUANTITATIVE DETERMINATION OF GLUCOSE IN THE BODY FLUIDS OF HUMANS.

Description

United States Patent US. Cl. 23-230 B 4 Claims ABSTRACT OF THE DISCLOSURE A diagnostic composition containing o-toluidine, thiourea and an oxide of arsenic is used for the quantitative determination of glucose in the body fluids of humans.
BACKGROUND OF THE INVENTION This invention relates to a new and improved diagnostic composition which is useful for the quantitative determination of glucose in fluids, particularly body fluids such as urine, plasma, blood and the like.
The detection and quantitative determination of glucose in blood and urine is of great importance for diabetic patients whose diets must be controlled so as to regulate sugar intake and who must frequently be guided in this regard by .regular checks on blood and urine glucose. Diabetes screening programs are dependent on the availability of rapid, inexpensive and accurate methods of glucose determination.
Procedures for the detection. of sugar in blood and urine are well known in clinical chemistry. The Folin-Wu and Nelson-Somogyi procedures utilize copper reduction for the detection and determination of sugar. US. Pat. 3,008,879 describes a means for testing biological fluids for their glucose content which involves a combination comprising an enzyme system having glucose oxidase activity, a primary indicator material which is capable of being preferentially oxidized to a colored form in the presence of hydrogen peroxide and the material having peroxidative activity and, as a secondary indicator, a material which is capable of reducing the preferentially oxidized primary indicator material while simultaneously becoming colored.
The use of o-toluidine solutions in glacial acetic acid for the determination of aldosaccharides in serum or plasma is described by Hultman, E., Nature, 183, 108 (1959) and Dubowski, K. M. Clin. Chem. 8, 215 (1962). Ordinarily, glucose is the only aldohexose present in cli-nically significant amounts in 'blood serum; other aldohexoses that may be present in minute quantities are mannose and galactose. This test method is based on the formation of a colored complex of glucose and o-toluidine in glacial acetic acid. The concentration of this complex, and thus the intensity of the green color developed, is directly proportional to the concentration of glucose present in the reaction mixture, and can be measured photometrically.
For the determination of glucose in blood, the Folin- Wu and Nelson-Somogyi test methods can be applied only to protein-free filtrates of blood serum or plasma. There is some loss of specificity and reliability because of saccharoids and other reducing compounds that cannot be completely removed in preparation of a protein-free filtrate with resulting values higher than true glucose levels. Other disadvantages include the limited stability of the test reagents, the instability of the final test color, and critical factors in the test procedure involving temperature, heating time and alkalinity of reaction mixture.
The glucose-oxidase test is a highly specific test for glucose when correctly done with pure materials. A protein-free filtrate is required. Other disadvantages include the sensitivity and limited stability of the test reagents, he necessary purity and standardization of the glucoseoxidase enzyme, the errors arising from interfering enzyme inhibitors, and the extreme care necessary during the three minute heating process where a matter of seconds can critically aifect the accuracy of results.
The o-toluidine test is a simple, rapid, reliable and economical method for glucose determination. Importantly, there is no need for protein-free filtrates although the test can be performed with such filtrates. The test can be performed directly with blood serum or plasma. Orthotoluidine is the only reagent required. Acetic acid solutions are stable for at least six months. This stability is enhanced by the addition of thiourea. The final test color has good stability. An important disadvantage, however, of o-toluidine is its susceptibility to oxidation with its resultant effect on test sensitivity. The depressed sensitivity of the reagent results in a regression line with a lower slope when the optical densities of the final test colors are plotted against sugar concentrations, with serious loss of assay accuracy.
The object of this invention is to provide an improved diagnostic composition containing o-toluidine, thiourea and arsenic oxides for the simple, rapid, inexpensive and accurate quantitative determination of glucose in the biological fluids of humans.
SUMMARY OF THE INVENTION This invention is concerned with a diagnostic composition containing o-toluidine for the quantitative determination of glucose in such biological fluids as blood and urine. It has been found that the incorporation of an oxide of arsenic in the o-toluidine reagent solution increases the sensitivity of the test reagent and increases the stability of the final test color in the assay procedure.
DETAILED DESCRIPTION OF THE INVENTION The o-toluidine test method for the quantitative determination of glucose in blood and urine involves the formation of a colored complex of glucose and o-toluidine in glacial acetic acid which follows Beers law. The green color, read at 630 nm., is stable 97%) after standing one hour in air at 25 C.
o-Toluidine is relatively sensitive to oxidation. Bulk supplies of uniform quality and purity are difficult to obtain, with Eastman Chemical Company the preferred source. Freshly distilled o-toluidine is, of course, eminently suitable. The stability of o-toluidine solutions in glacial acetic acid (6.25% w./v.) is increased by the addition of thiourea (1.50% w./v.). The addition of an arsenate salt or oxide of arsenic increases the sensitivity of the o-toluidine reagent as reflected in the slope of the optical density curve of the final test colors.
The arsenic compound may be selected from the group consisting of the following:
sodium orthoarsenate: Na AsO 121-1 0 sodium mono-H-orthoa-rsenate: Na HAsO -7H O sodium mono-H-orthoarsenate: Na HAsO l2H O sodium di-H-orthoarsenate: NaH AsO -H O sodium metaarsenate: NaAsO sodium arsenite: NaAsO- arsenic trioxide: AS203 arsenolite: AS405 claudetite: As O arsenic pentoxide: AS205 sodium thioarsenate: Na AsS -8H O Theaddition of sodium arsenate (Na HAsO -7H O) to the o-toluidine reagent increases the sensitivity to glucose up to An increase in sensitivity, over solutions containing no additive, is observed even with small 0.1% w./v.) concentrations of sodium arsenate additive. The
sensitivity increase levels off in the region of 0.5% w./v. additive, and finally attains a value of 120% increase at saturated levels of sodium arsenate (35% w./v.).
The color formed by the o-toluidine, with and without arsenate additive, is quite stable for the first hour. However, an increase in stability is noted for the sodium arsenate additive solution after 15 hours with a 32% decrease in color retained, compared to a 38% decrease for the control solution. This increased color stability is important when large numbers of test samples are run, and assay readings cannot be immediately made.
The following examples are provided to illustrate the present invention, but not to limit its scope.
EXAMPLE I Preparation of reagent Thiourea (0.3 g., 1.5% w./v.) is placed in a 500 ml. Erlenmeyer flask and dissolved in 150 ml. of fresh glacial acetic acid. Fresh opened o-toluidine from Eastman Chemical Company (12.5 ml., 6.25% w./v.) is added dropwise to the stirred acetic acid solution. Sodium mono- H-orthoarsenate, Na HAsO -7H O, (100 mg., 0.5% w./v.) is added all at once and dissolved by gentle stirring under nitrogen. The solution is stored at room temperaure in a dark glass bottle under nitrogen.
Test procedure (1) Using a suitable pipette add 0.05 ml. of blood serum or other sample to be tested to a 150 mm. x 15 mm., or similar, Pyrex test tube. If serum or plasma is used, deproteinization is not necessary.
(2) Add 4.0 ml. of o-toluidine reagent to the tube. Mix sample and reagent thoroughly, preferably with a vortex mixer.
(3) In a similar manner, prepare a reagent blank using 0.05 ml. of distilled water instead of sample, and prepare a glucose standard using 0.05 ml. of standard glucose solution instead of serum or other sample.
(4) Place the test tubes in a boiling water bath for exactly 10 minutes.
(5) Cool the solutions to room temperature, and determine optical densities at 630 nm.
(6) Determine the concentration of glucose in the sample from a previously prepared curve of optical density vs. glucose concentration, using a series of at least three dilferent glucose concentrations in the range of expected results (50-400 mcgs. percent).
EXAMPLE II The preparation of the o-toluidine test reagent of Example I is repeated in turn with sodium orthoarsenate (Na AsO -12H O), sodium mono-H-orthoarsenate (Na HAsO sodium di-R-orthoarsenate (NaH AsO -H O), sodium metaarsenate (NaAsO sodium arsenite (NaAsO arsenic trioxide (As o arsenolite (As O claudetite (ASaO arsenic pentoxide (AS305) and sodium thio- 4 arsenate (Na AsS -8H 0) in place of sodium mono-H- orthoarsenate (Na HAsO -7H O), with comparable test results.
What is claimed is:
1. In the method for quantitatively determining glucose in the biological fluids of humans using a diagnostic composition containing o-toluidine, the improvement which comprises incorporating an arsenic compound selected from the group consisting of sodium orthoarsenate (Na ASO 12H O) sodium mono-H-orthoarsenate (Na HAsO -7H O), sodium mono-H-orthoarsenate (NaHAsO -12H O), sodium di-H-orthoarsenate (NaH AsO -H O), sodium metaarsenate (NaAsO sodium arsenite (NaAsO arsenic trioxide (As O arsenolite (AS406), claudetite (As O arsenic pentoxide (As O and sodium thioarsenate (Na AsS 2. The method of claim 1 wherein said arsenic compound is sodium mono-H-orthoarsenate (Na- HAsO (Na AsS 8H O 4. The diagnostic composition of claim 3 wherein said arsenic compound is sodium mono-H-orthoarsenate (Na HAsO References Cited UNITED STATES PATENTS 3,001,915 9/1961 Fonner 23-253 TP 3,453,180 7/1969 Fraser 23-253 TP 3,607,077 9/1971 Hartel 23-230 B 3,615,228 10/1971 Thiegs 23230 B 3,653,836 4/ 1972 Gruher 23230 B 3,653,841 4/1972 Klein 23230 B 3,660,559 5/1972 Buckert 23230 B MORRIS O. WOLK, Primary Examiner S. MARANTZ, Assistant Examiner US. Cl. X.R. 252-408
US00203840A 1971-12-01 1971-12-01 Method for determining glucose with o-toluidine reagent containing an arsenic compound Expired - Lifetime US3792044A (en)

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Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US3947377A (en) * 1972-02-08 1976-03-30 Boehringer Mannheim Gmbh Stabilized indicator compositions containing 9-γ-aminopropyl)-3-aminocarbazole
US4058366A (en) * 1976-02-06 1977-11-15 Continental Oil Company Determination of flow characteristics of petroliferous formations
US4705756A (en) * 1983-01-26 1987-11-10 University Of Medicine And Dentistry Of New Jersey Method of determining the existence and/or the monitoring of a pathological condition in a mammal
US4814247A (en) * 1983-01-26 1989-03-21 University Of Medicine And Dentistry Of New Jersey Method for determining the existance and/or the monitoring of a pathological condition in a mammal

Families Citing this family (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4330299A (en) * 1981-03-09 1982-05-18 Evreka, Inc. Article and method for measuring glucose level in body fluids

Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US3947377A (en) * 1972-02-08 1976-03-30 Boehringer Mannheim Gmbh Stabilized indicator compositions containing 9-γ-aminopropyl)-3-aminocarbazole
US4058366A (en) * 1976-02-06 1977-11-15 Continental Oil Company Determination of flow characteristics of petroliferous formations
US4705756A (en) * 1983-01-26 1987-11-10 University Of Medicine And Dentistry Of New Jersey Method of determining the existence and/or the monitoring of a pathological condition in a mammal
US4814247A (en) * 1983-01-26 1989-03-21 University Of Medicine And Dentistry Of New Jersey Method for determining the existance and/or the monitoring of a pathological condition in a mammal

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