US3511611A

US3511611A - Blood urea nitrogen test

Info

Publication number: US3511611A
Application number: US699770A
Authority: US
Inventors: Robert L Rush
Original assignee: Pfizer Inc
Current assignee: Pfizer Inc; Warner Lambert Co LLC
Priority date: 1968-01-23
Filing date: 1968-01-23
Publication date: 1970-05-12
Anticipated expiration: 1987-05-12
Also published as: GB1219555A

Description

United States Patent Int. Cl. G011! 21/06 U.S. Cl. 23230 Claims ABSTRACT OF THE DISCLOSURE A colorimetric procedure for the determination of urea nitrogen in human blood serum utilizing as the color developer an aqueous solution containing diacetyl monoxime, thiosemicarbazide, and ethylenediamine tetraacetic acid.

BACKGROUND OF THE INVENTION This invention relates to a novel diagnostic test. More particularly, it relates to an improved colorimetric method for determining the presence of urea nitrogen in blood. The principle of the test is the formation of a color complex between the urea in the serum with a thiosemicarbazide-diacetyl-monoxime combination (color developer reagent). The novel process modification consists of utilizing a novel, stabilized color developer reagent containing ethylenediamine tetraacetic acid (EDTA). This EDTA-containing color developer reagent exhibits a marked stability over previously known reagents. For instance, without the inclusion of EDTA, the color developer reagent discolors within a short time, becomes contaminated with a precipitate whose structure is not known at this time, when held at 30 C. for two days (immediate precipitation at 0 C.), and whose pH drops from about 6 to about 4 within a weeks time.

The determination of blood urea nitrogen is of particular importance when screening individuals who may be afilicted with kidney disfunction, for example, nephritis, and in the efiicacy of the therapeutic management of disorders mentioned above. Blood urea nitrogen determinations are also of importance in aiding in the diagnosis of liver disorder.

SUMMARY OF THE INVENTION This invention broadly comprises in a method for determining the presence of urea nitrogen in blood by:

(a) Commingling an aqueous acid catalyst solution containing about 0.0025% w./v. of FeC1 -6H 0, about 0.05% v./v. of 85% H PO and about 7.6% v./v. of 95% sulfuric acid and a sample of suspect serum, the volume ratio of said acid catalyst solution to said serum being approximately 150 to l; and

(b) Adding to the resulting mixture an aqueous color developer solution, heating the resulting mixture at approximately 100 C. for about minutes, cooling;

And determining the concentration of urea nitrogen spectrophotometrically by measuring the optical density at a wavelength of 515 to 525 mg;

The improvement which comprises using a color developer solution containing about 0.185% W./v. of diacetyl monoxime, about 0.03% w./v. of thiosemicanbazide and about 0.4% W./v. of ethylenediamine tetraacetic acid, said solution having a pH of about 6.5 to about 7.5 in an amount sufficient to provide a volume ratio of said color developer solution to said serum of about 100 to 1.

Experimentally, it is preferred to use the following amounts of materials when carrying out the aforedescribed diagnostic test:

Serum sample 0.02

Acid catalyst reagent (FeCl -6H OH P H SO 3 Color developer reagent (diacetyl monoxime-thiosemicarbazide-ethylenediamine tetraacetic acid) 2 Of course, it is to be understood that any equivalent volume proportions may be used in lieu of those above, however, the above amounts are preferred since the total resulting volume is appropriate for the subsequent optical density measurements.

With regard to the total wavelength at which the optical density measurements are carried out, it is preferred to use a wavelength of 520 millimicrons, however, a range from about 515 to 525 millimicrons is equally suitable.

DETAILED DESCRIPTION OF THE INVENTION The herein disclosed diagnostic method determines the amount of blood urea nitrogen in human serum wherein said amount is measured by a colorimetric procedure using a spectrophotometer or a colorimeter capable of measuring absorption at a wavelength of from 515 to 525 III/1.. The concentration of urea nitrogen is determined from an experimentally determined curve of optical density vs. mg./ 100 ml. of urea nitrogen. The normal range for urea nitrogen concentration predetermined experimentally by the herein subject method is from about 8 to about 25 mg./ 100 ml. A reading of less than 8 indicates one of the malfunctions described earlier.

Experimentally, the diagnostic procedure is carried out in the following manner: A 0.02 ml. sample of suspect serum is mixed with 3 ml. of an acid catalyst reagent solution which contains about 0.0025% w./v. of FeCl -6H O, about 0.05% v./v. of H PO and about 7.6% v./v. of sulfuric acid. To this mixture is added 2 ml. of an aqueous color developer reagent containing about 0.185% w./v. of diacetyl monoxime, about 0.03% w./v. of thiosemicarbazide and about 0.4% w./v. of ethylenediamine tetraacetic acid. Insofar as the said EDTA is concerned, it is preferred to use the disodium salt which is commercially available, however, the other sodium salts or EDTA itself can also be used. Furthermore, in preparing the EDTA solution prior to combining wtih the other two components, it should be noted that the pH is adjusted to a pH of about 6.5 to about 7.5, and preferably to 7.0 with l N aqueous sodium hydroxide. Mechanistically, the urea forms a color complex with the color developer reagent under the necessary acidic conditions provided for by the acid catalyst reagent. After the color developer reagent is added, the resulting mixture is agitated and then heated in rapidly boiling water for approximately 10 minutes. After this period, the container is cooled in either cold running water or an icebath until the solution reaches room temperature. It is transferred to a spectrophotometric cuvette, unless the conainer itself is a cuvette, and the optical density determined within 20 minutes. The aforedescribed method is well suited to any laboratory adapted for routine determinations.

The following examples are provided by way of illustration and should not be interpreted as limiting the invention, many variations of which are possible without departing from the spirit or scope thereof.

3 PREPARATION OF REAGENTS (A) Acid catalyst reagent.--To a one liter flask is added 500 ml. of distilled water, 2.5 ml. of a 1% w./v. aqueous solution of ferric chloride (FeCl .6H O) and 0.5 ml. of phosphoric acid (85% v./v.). This solution is cooled to 4 C. whereupon 76.6 ml. of sulfuric acid (95%) is added and mixed. Sufiicient distilled water is then added to provide a one liter solution.

(B) Color developer reagent.To a one liter flask is added 125 ml. of a 1.4% w./v. aqueous solution of diacetyl monoxime, 125 ml. of 0.297 w./v. aqueous solution of thiosemicarbazide and 500 ml. of 0.744% w./v. aqueous solution of ethylenediamine tetraacetic acid (2H O disodium salt). In preparing the EDTA solution, the final pH of the resulting solution is adjusted to 7.0 by the addition of 1 N aqueous sodium hydroxide. After the above said three components are combined, the pH is adjusted to 7.0 by adding additional 1 N aqueous sodium hydroxide. Once this desired pH reading is obtained, sufficient distilled water is added to provide a one liter final solution.

EXAMPLE I A sample of non-hemolized suspect serum (0.02 ml.) is combined with 3 ml. of reagent (A) (acid catalyst reagent) as prepared by the procedure outlined above (in a pyrex tube 150 mm. x 15 mm.). Reagent (B) (2 ml.) is then added and the reaction mixture is agitated and then heated in rapidly boiling water for ten minutes. After this time period, the tube or container is cooled in cold running water or an ice-bath until the solution reaches room temperature. The solution is then transferred to a 12 mm. spectrophotometer cuvette. The wavelength of the spectrophotometer is set at 520 m adjusted to zero optical density with the blank, and the optical density of the sample is determined within 20 minutes.

EXAMPLE II The procedure of Example I is repeated wherein the following amounts of sample and reagents are used:

Sample 0.04 Reagent (A) Reagent (B) liter Mix liter Mix M1. Sample 0.08 Reagent (A) l2 Reagent (B) 8 Equivalent results are obtained.

What is claimed is:

1. In a method for determining the presence of urea nitrogen in blood serum by: nitrogen in blood serum by:

(a) commingling an aqueous acid catalyst solution containing about 0.0025% w./v. of FeCl .6H O, about 0.05% v./v. of H PO and about 7.6% v./v. of sulfuric acid and a sample of suspect serum, the volume ratio of said acid catalyst solution to said serum being approximately 150 to 1; and

(b) adding to the resulting mixture an aqueous color developer solution, heating the resulting mixture at approximately C. for about 10 minutes, cooling;

and determining the concentration of urea nitrogen spectrophotometrically by measuring the optical density at a wavelength of 515 to 525 my; the improvement which comprises using as the color developer an aqueous solution containing about 0.185% W./v. of diacetyl monoxime, about 0.03% w./v. of thiosemicarbazide and about 0.4% w./v. of ethylenediamine tetraacetic acid, said solution having a pH of about 6.5 to about 7.5 and being in an amount sufficient to provide a volume ratio of said color developer solution to said serum of about 100 to 1.

2. The method of claim 1 wherein the pH of the aqueous color developer solution is maintained at a pH of 7.0 with 1 N aqueous sodium hydroxide.

3. The method of claim 1 wherein said heating period is 10 minutes at the temperature of boiling water.

4. The method of claim 1 wherein the amounts of said serum sample, said acid catalyst solution and said color developer solution are 0.02 ml., 3 ml. and 2 ml., respectively.

5. The method of claim 1 wherein said ethylenediamine tetraacetic acid is in the form of its disodium salt.

References Cited UNITED STATES PATENTS 3,068,071 12/1962 Velluz et al 23--230 3,119,751 l/1964 Chaney 23-230 X 3,145,086 8/1964 Free 23230 OTHER REFERENCES C. A. 54:19812g (1960). R. C. Hoseney et al., Anal. Chem, 36, 2145 ;48 (1964).

5 MORRIS O. WOLK, Primary Examiner S. MARANTZ, Assistant Examiner U.S. Cl. X.R. 252-408