A DIRECT BILIRUBIN ASSAY METHOD

Abstract: A method for assaying the direct bilirubin content of a sample comprising (a) mixing a sulfanilic acid solution comprising from about 0.02 to about 0.05 M sulfanilic acid and from about 0.02 to about 0.15 M hydrochloric acid with a sodium nitrite to thereby commence a diazotation process, the ratio of the sulfanilic acid solution to said sodium nitrate solution being preselected so as to yield a diazo solution comprising from about 0.0001 to about 0.001 M Diazo dye; (b) allowing the diazotation process to form the diazo solution for at least 15 seconds; (c) introducing an aliquot of the sample to be assayed into said diazo solution at a volume ratio of from about 1:100 to about 1:25 to thereby commence an azobilirubin process; and (d) measuring the instantaneous rate of azobilirubin formation at 545 nm in a time range of from about 12 to about 25 seconds after introduction of the aliquot of sample into the diazo solution.

Description: -1 - A DIRECT BILIRUBIN ASSAY METHOD Background of the Invention 1. Field of the Invention This invention relates to a method for assaying direct bilirubin. 2. Description of the Prior Art Direct bilirubin, also known as conjugated biliru¬ bin, is usually assayed by coupling it with a diazo dye in an acidic medium. In a typical Jendrassik-Grof pro¬ cedure, the dye is prepared by diazotizing sulfanilic acid with sodium nitrite. The azobilirubin formation reaction is carried out in•a dilute hydrochloric acid solution. The optical density (O.D.) change is then read at the end of a fixed reaction time, usually between 1 to 5 minutes, depending upon the specific procedure em¬ ployed, and compared with a bilirubin calibration curve for results. The testing procedure is time consuming and susceptible to error due to the degradation of the un¬ stable diazo reagent. Recently, Rizi et al. (1) have proposed a kinetic method for the determination of direct bilirubin. By design, their method is a modification of a one minute end-point procedure. The method of Rizi et al. elimi¬ nates the need of sample blank but does not improve the selectivity of the test. In an ingenious effort, Landis et al. (2) have de¬ veloped a rate method which can determine both total and direct bilirubin simultaneously in a single measure¬ ment. The drawback to Landis et al.'s method is that it does not allow direct bilirubin to be determined indepen- -2- dently. As a result any error in determination of uncon- jugated bilirubin becomes an error in the measurement of conjugated bilirubin. " Accordingly, it would be desirable to have a method capable of independently assaying direct bilirubin which also eliminates the reagent degradation problems and the time consuming aspects of the prior art procedure. Summary of the Invention In accordance with the present invention, there is provided an independent method for measuring direct bili¬ rubin which eliminates the problems associated with the degradation of the diazo reagent, shortens the time re¬ quired per test and eliminates the need for a sample blank. More particularly, the method of the instant invention for independently assaying the direct bilirubin content of a sample comprises: (a) mixing a sulfanilic acid solution comprising from about 0.02 to about 0.05 M sulfanilic acid and from about 0.02 to about 0.15 M hydrochloric acid with a so¬ dium nitrite solution comprising from about 0.001 to 0.015 M sodium nitrite to thereby commence a diazotiza- tion process, the ratio of the sulfanilic acid solution to the sodium nitrite solution being preselected so as to yield a diazo solution comprising from about 0.0001 to about 0.001 M diazo dye; (b) allowing the diazotization process to proceed to form the diazo solution for at least 15 seconds; (c) introducing an aliquot of the sample to be assayed into the dye solution at a volume ratio of from about 1:100 to about 1:25 to thereby commence, an azobili¬ rubin process; and OMPI A-. YΠPO . -3- (d) measuring the instantaneous rate of azobiliru¬ bin formation at 545 nm in a time range of from about 12 to about 25 seconds after introduction of the aliquot of the sample into the diazo solution. Still other features and attendant advantages of the present invention will become apparent to those skilled in the art from a reading of the following detailed de¬ scription of the preferred embodiment. Description of the Preferred Embodiment The method of the instant invention for assaying the direct bilirubin content of a sample comprises: (a) mixing a sulfanilic acid solution comprising from about 0.02 to about 0.05 M sulfanilic acid and from about 0.03 to about 0.15 M hydrochloric acid with a so¬ dium nitrite solution comprising from about 0.001 to about 0.015 M sodium nitrite to thereby commence a diazo- tization process, the ratio of the sulfanilic acid solu¬ tion to the sodium nitrite solution being preselected so as to yield a diazo solution comprising from about 0.0001 to about 0.001 M diazo dye; (b) allowing the diazotization process to proceed to form the diazo solution for at least 15 seconds; (c) introducing an aliquot of sample to be assayed into the diazo solution at a volume ratio of from about 1:100 to about 1:25 to thereby commence an azobilirubin process; and (d) measuring the instantaneous rate of the azo¬ bilirubin formation at 545 nm in a time range of from about 12 to about 25 seconds after introduction of the aliquot of the sample into the diazo solution. -4- The ratio of the sulfanilic acid solution to the sodium nitrite solution is preferably preselected so as to yield a diazo solution comprising about 0.0004 M diazo dye. The sulfanilic acid solution preferably comprises about 0.03 iM sulfanilic acid and about 0.05 M hydrochlor¬ ic acid. The sodium nitrite solution preferably comprises about 0.004 M sodium nitrite. The diazotization process is preferably allowed to proceed for about 30 seconds, and more preferably, is allowed to proceed for about 30 seconds at 37°C. In addition, it is preferred that the aliquot of sample be introduced into the diazo solution at a volume ratio of about 1:55. Furthermore, it is preferred that the instantaneous rate measurement be made about 18 seconds after introduc¬ tion of the aliquot of the sample into the diazo solu¬ tion * A preservative may optionally be incorporated into the sodium nitrite solution. Typical preservatives in¬ clude, but are not limited to, (ethylenedinitrilo)tetra- acetic acid, tetrasodium salt. The sulfanilic acid solution and sodium nitrite solution employed in the method involved in the instant invention can be prepared by any method well known to those skilled in the art. __ OMPI -5- The following examples are provided for purposes of further illustration only and are not intended to be limitations on the disclosed invention. Example 1 Solutions of sulfanilic acid (0.03 M sulfanic acid and 0.05M hydrochloric acid) and sodium nitrite (0.004 M sodium nitrite) were mixed in a 10:1 ratio to yield a diazo solution of 1.1 ml comprising about 0.004 M diazo dye. After 30 seconds of diazotization, a 20 μl aliquot of a serum sample was added to the diazo solution. The instantaneous rate of azobilirubin formation was read 18 seconds after sample addition. This procedure was re¬ peated for a total of 20 serum samples and the data ob¬ tained therefrom is set forth in Table I. Example 2 The same 20 serum samples in Example 1 were also assayed via American Monitor Corporation's modified Jendrassik and Grof procedure. The data obtained there¬ from is also set forth in Table I. -6- Table I Direct Bilirubin Serum Correl,ation, g/dl Serum Example 1 Example 2 Sample (Instant Invention) (Prior Art) 1 0.11 0.03 2 1.16 - 1.62 3 0.42 0.44 4 2.00 1.67 5 4.40 4.45 6 0.14 0.24 7 0.32 0.33 8 0.07 0.14 9 0.11 0.11 10 0.95 0.90 11 0.60 0.68 12 6.93 6.78 13 0.11 0.06 14 0.32 0.34 15 0.11 0.13 16 0.11 0.02 17 2.11 2.38 18 5.90 6.14 19 2.95 2.63 20 0.32 0.53 Y Ex1 = 0.9983 EX 2 - 0.215 r = 0.9959 The data set forth in Table I show that the method of the instant invention correlates well (r = 0.9959) with a commercially available modified Jendrassik and Grof pro¬ cedure. However, since the procedure of the instant inven¬ tion forms the diazo reagent in situ and because the sulfanilic acid and sodium nitrite solutions are much more stable in comparison to the diazo reagent, the meth¬ od of this invention enables one to employ solutions which have a shelf-life considerably longer than that of OMPI the reagents employed in the prior art (i.e., the solu¬ tions employed in this inventin have a shelf-life of at least 30 days at room temperature vs. the 4 hour room temperature stability of the diazo reagent employed in the prior art) . Accordingly, in practicing the method of the instant invention one gains a substantial amount of time by not having to frequently prepare the reagent employed therein. In addition, the increased stability of the solutions employed in this invention reduces the frequency at which one must calibrate the instrument and thereby improves the accuracy of the results obtained via the assays performed. Based on this disclosure, many other modifications and ramifications will naturally suggest themselves to those skilled in the art. These are intended to be com¬ prehended as within the scope of this invention. Bibliography 1. Rizi et al., Clin. Che .- 23:1128 (1977). 2. Landis et al., Clin. Che ., 24:1700 (1978). OMPI ^ Zie

Claims: . -8- The embodiments of the invention in which an exclu¬ sive property or privilege is claimed are defined as follows: 1. A method for assaying the direct bilirubin con¬ tent of a sample comprising: (a) mixing a sulfanilic acid solution comprising from about 0.02 to about 0.05 M sulfanilic acid and from about 0.02 to about 0.15 M hydrochloric acid with a so¬ dium nitrite solution comprising from about 0.001 to about 0.015 M. sodium nitrite to thereby commence a diazo- tization process, the ratio of said sulfanilic acid solu¬ tion to said sodium nitrite solution being preselected so as to yield a diazo solution comprising from about 0.0001 to about 0.001 M diazo dye; (b) allowing said diazotization process to proceed to form said diazo solution for at least 15 seconds; (c) introducing an aliquot of said sample to be assayed into said diazo solution at a volume ratio of from about 1:100 to about 1:25 to thereby commence an azobilirubin process; and (d) measuring the instantaneous rate of azobiliru¬ bin formation at 545 nm in a time range of from about 12 to about 25 seconds after introduction of said aliquot of said sample into said diazo solution. 2. The method of claim 1 wherein the ratio of said sulfanilic acid solution to said sodium nitrite solution is preselected so as to yield said diazo solution com¬ prising about 0.004 M diazo dye. 3. The method of claim 1 wherein said sulfanilic acid solution comprises about 0.03 M sulfanilic acid and about 0.05 M hydrochloric acid. 4. The method of claim 1 wherein said sodium ni¬ trite solution comprises about 0.004 M sodium nitrite. 5. The method of claim 1 wherein said diazotization process is allowed to proceed for about 30 seconds. 6. The method of claim 1 wherein said diazotization process is allowed to proceed for about 30 seconds at about 37 β C. 7. The method of claim 1 wherein said aliquot of said sample is introduced into said diazo solution at a volume ratio of about 1:55. 8. The method of claim 1 wherein said instantaneous rate measurement is made at about 18 seconds after intro¬ duction of said aliquot of said sample into said diazo solution. 9. The method of claim 1 „wherein: (a) said sulfanilic acid solution comprises about 0.03 M sulfanilic acid and about 0.05 M hydrochloric acid; (b) said sodium nitrite solution comprises about 0.004 M sodium nitrite; (c) the ratio of said sulfanilic acid solution to said sodium nitrite solution is preselected so as to yield said diazo solution comprising about 0.004 M diazo dye; OMPI -1 0- (d) said diazotization process is allowed to pro¬ ceed for about 30 seconds; (e) said aliquot of said sample is introduced into said diazo solution at a volume rate of about 1:55; and (f) said instantaneous rate measurement is made at about 18 seconds after introduction of said aliquot of said sample into said diazo solution. 10. The method of claim 9 wherein said diazotiza¬ tion process is allowed to proceed at about 37 β C. 11. The method of claim 9 wherein said sodium ni¬ trite solution further comprises a preservative. 12. The method of claim 1 wherein: (a) said sulfanilic acid solution comprises about 0.03 M sulfanilic acid and about 0.05 M hydrochloric acid.; (b) said sodium nitrite solution comprises about 0.004 M sodium nitrite and further comprises a preserva¬ tive; (c) the ratio of said sulfanilic acid solution to said sodium" nitrite solution is preselected so as to yield said diazo solution comprising about 0.004 M diazo dye; (d) said diazotization process is allowed to pro¬ ceed for about 30 seconds at about 37°C; (e) said aliquot of said sample is introduced into said diazo solution at a volume ratio of about 1:55; and OMPI -1 1 - (f) said instantaneous rate measurement is made at about 18 seconds after introduction of said aliquot of said sample into said diazo solution. OMPI ^ < W1PO ,

Inventors: OLANDER GLENN J, SHU FRANK R, OLANDER, GLENN, J., SHU, FRANK, R.
Applicants: BECKMAN INSTRUMENTS INC, BECKMAN INSTRUMENTS, INC.
Application year: 1983
Main classification: G01N33
IPC codes: G01N33