Joseph Wilfred Fraser, Jr., Osceola, and Charles T. W. Lam and Raymond L. Mast, Elkhart, Ind., assignors to Miles Laboratories, Inc., Elkhart, Ind. 

U.S. CI. 23-230 B 

4,4’-biphenyldiazonium salts in test compositions and devices capable of differential determination of both bili› rubin and urobilinogen in an aqueous solution or body fluid such as urine. In addition to a 4,4’-biphenyldiazo› nium salt, the test compositions preferably include an acid constitutent capable of producing an acid pH upon con› tacting the aqueous solution to be tested. The test de› vice comprises a bibulous carrier member such as paper incorporated with the test composition in dry form. The method includes contacting the test composition with the aqueous solution to be tested and observing the response either visually or spectrophotometrically. 

Bilirubin is a degradation product of the heme moiety of hemoglobin molecules. In plasma bilirubin is normally unconjugated and is bound to various proteins. Conjuga› tion results at the liver and possibly at other sites and the conjugated bilirubin is excreted into the duodenum as a constituent of bile. The action of bacteria normally pres› ent in the intestinal tract causes the reduction of bilirubin to other compounds, one of which, urobilinogen, is nor› mally found in urine. Normal urine does not contain an appreciable amount (usually le.ss than 0.05 mg./100 ml.) of either conjugated or unconjugated bilirubin. 

The diagnostic value of bilirubin and urobilinogen de› terminations in body fluids is well known. Various dis› eased conditions cause abnormal levels of both com› pounds in urine. Such diseased conditions include hemo› lytic and hepatic diseases, biliary obstruction and other liver and bile duct dysfunctions. However, it is not pos› sible to diagnose differentially a hemolytic condition, a hepatic condition or a biliary obstruction by the perform› ance of a test for only one of these two compounds in urine. Isolated determinations do not indicate particular diseased conditions. For instance, a negative bilirubin determination, i.e. less than 0.05 mg./100 ml. indicates either a normal or a hemolytic condition. On the other hand in urine urobilinogen determinations, abnormally high ’1evels are indicative of either a hemolytic or hepatic condition while abnormally low levels indicate biliary obstruction. 

The performance of both a bilirubin and a urobilinogen determination provides much more specific results due to the interrelationship between the levels of the two com› pounds in urine and certain diseased conditions. In nor› mal urine the bilirubin level may be considered as nega› tive (less ’than 0.05 mg./100 ml.). In a hemolytic con~li› tion, more bilirubin is formed than normal due to m› creased degradation of the heme moiety. This results in an increase in the amount of bilirubin reduction products, in› cluding urobilinogen. Therefore, in a hemolytic con_di› tion urine urobilinogen levels become abnormally high whiie bilirubin remains negative. Levels of bilirubin in the blood stream increase in hepatic conditions and biliary obstruction due to the decrease in the ability of the liver to excrete bile into the intestinal tract. Thus, the amount of bilirubin excreted by the kidneys as a urine constituent 

# COMPOSITION, METHOD AND DEVICE FOR DETERMINING BILffiUBIN AND UROBILINOGEN 

Filed Jan. 2, 1973, Ser. No. 320,117 

Int. Cl. G01n 21/20, 33/16 

## ABSTRACT OF THE DISCLOSURE 

## BACKGROUND OF THE INVENTION 

3,814,586 

23 Claims 

increases resulting in positive urine bilirubin determina› tions. Urobilinogen levels in urine are found to _increase in hepatic conditions but decrease in biliary obstruction. based on a classification of bilirubin levels in urine as positive or negative as hereinbefore defined, and urobi› linogen levels as low, normal or high, diagnosis proceeds as follows for each of the following possible bilirubin› urobilinogen levels: negative-normal, normal; negative- high, hemolytic disease; positive-high, hepatic disease; and positive-low, biliary obstruction. 

Bilirubin determinations which are known in the art are based on the reaction of bilirubin with diazotized ani- line test reagents. These single benzene ring compounds are generally substituted with such constituents as nitro, sulfonic, chloride and methoxy groups. The more com› monly employed test reagents are diazotized 2,4-dichloro› aniline and diazotized sulfanilic acid. The reaction of bili- rubin molecules with the diazo group of the test reagent results in conjugation which shifts the absorption spectrum of the azo reaction complex into the visible spectrum. This colorimetric response provides quantitation in aque› ous solutions based on Beer’s Law of light energy absorp- tion. The molar extinction coefficient of 2,4-dichloroani› line is such that determinations of bilirubin using this preferred test reagent impregnated in paper for use in a dry test device are sensitive to a minimum concentration of about 1 mg./100 ml. Therefore, the range of bilirubin levels in urine between the normal level of about 0.05 mg./100 ml. and this minimum detectable amount are incapable of being determined using the traditional test reagents incorporated in a dry test device. 

The prior art methods for detecting urobilinogen em- ploy benzaldehyde test reagents. The most commonly used benzaldehyde test reagent by far is para-dimethyl› aminobenzaldehyde which is commonly referred to as Ehrlich’s reagent. The reaction of urobilinogen and Ehr› lich’s reagent yields a conjugated complex with absorption in the visible spectrum. The colorimetric responses ex› hibited by this reaction are interpreted on the basis of a measurement unit known as an Ehrlich unit, rather than on mg. urobilinogen/100 ml. This is due to the fact that Ehrlich’s reagent is known to react with various inter- ferring substances sometimes present in urine such as para-aminosalicyclic acid, porphobilinogen and indole. An Ehrlich unit is defined in the art as the colorimetric re› sponse yielded by the reaction of Ehrlich’s reagent with 1 mg./100 ml. of urobilinogen. In certain instances the lack of specificity in the detection of urobilinogen, partic› ularly in urine determinations, detracts from the use of such determinations in diagnosis. The use of diazonium salts in the colorimetric detection of urobilinogen is un› reported. Urobilinogen determinations therefore are pres- ently based almost exclusively on the use of the non› specific Ehrlich’s reagent. 

As a result of limitations in the prior art, bilirubin and urobilinogen determinations must be performed sepa› rately. Also, known methods employing reagent impreg- nated paper for the detection of bilirubin are capable of detecting bilirubin only in concentrations greater than 1 mg./100 ml. 

It is therefore an object of the present invention to provide a test composition which is capable of detecting both bilirubin and urobilinogen. Such a test composition would exhibit differential colorimetric responses with re› spect to the two compounds. Such a composition would also allow differential diagnoses to ,be made between normal, hemolytic and hepatic conditions and biliary obstructions using a single test. 

Another object of the present invention is to provide a test composition which is more specific with respect to 

## DESCRIPTION OF THE PRIOR ART 

