COMPARISON OF TESTS FOR BILIRUBIN IN URINE*

Transcription

1 COMPARISON OF TESTS FOR BILIRUBIN IN URINE* ALVIN G. FOORD, AND CECIL F. BAISINGER From the Laboratories of the Collis P. and Howard Huntington Memorial Hospital, Pasadena, California, and the Department of Pathology of the University of Southern California Medical School, Los Angeles, California Experience with the time honored nitric acid contact test of Gmelin or the Rosenbach filter paper modification and the iodine ring method has proved these methods to be relatively insensitive and particularly hard to read when pigments other than bilirubin are present in the urine. A preliminary experiment with 200 urines to which fresh gallbladder bile was added and icteric urines from 20 jaundiced patients showed that even the simple Kapsinow 1 technique, consisting of adding 0.5 cc. Obermeyer's reagent to 5 cc. urine producing a green color if bilirubin is present, is somewhat more sensitive and easier to read than the above methods. Extensive routine use of the Huppert-Nakayama 2 test for 15 years, controlled on many specimens with the above tests, and numerous tests in which bile-containing urines were diluted serially with bile-free urine, containing albumin or not, has shown that the Huppert-Nakayama test is still more sensitive. In order to compare these tests with others as to sensitivity and ease of reading, bilirubin-containing urine from cases of obstructive jaundice were diluted serially with a mixture of hospital specimens of urine, and the various bilirubin tests were done. The readings are charted in charts 1, 2 and 3. The technic of the methods used is as follows: Rosenbach's? Urine is filtered through filter paper, the paper nearly dried, and 1 to 2 drops yellow HNO3 is dropped on. A succession of rings colored red, reddish yellow, violet, and blue results if positive. * Received for publication, May 27, Read before the American Society of Clinical Pathologists at St. Louis, Missouri, May 12-14,

2 COMPARISON OF TESTS FOR BILIRUBIN IN URINE 239 CHART I SENSITIVITY OF TESTS FOR BILIRUBIN IN URINE METHOD ROSENBACH </ 5 H- DILUTION '',o ^ '/ S o 1/ '160 i/ 3ZO IODINE HULLER MULL Eft CONC. HUPPERT NAKAYAMA DIA2.Q NAUMANN CONOL HUNTER -H -H H- H H HT HH r f H -H THT HT -rff -H H r n f Hr TV -H 1/ 2.0 H- - -H- - -H- -r '/zee 2S60 URINE FROM PATIENT WITH INTENSE OBSUCTIVE. JAUNDICE. DILUTED WITH M/X D ROUTINE HOSPITAL URINES CHART H SENSITIVITY OF TESTS FOR BILIRUBIN IN URINE METHOD ROSEN BACH l /s DILUTION 1/ >/ 4o 'so '40 '/so 'i&o 3ZO IODINE riuller OAZO NAUMANN ZINS HARRISON CONOL SPOT '// ft H h T -h h r T T/? HULLER CONC- HUPPERT NAKAYAMA OIAZO HUNTER H HH- H m H H H -H f -H- H -m H H H -Hf 1 -HH- H -H H H H Hr H- -HH- -HH- H H H- H H H H r -f- Tn f 1/ 6A0 '/ izao Tie Iodine method} Stratify on surface of urine a layer of alcoholic iodine solution made by diluting tincture of iodine 1 to 10 with 95 per Cent alcohol. Positive green ring at contact.

3 240 ALVIN G. FOORD AND CECIL F. BAtSlNGER HuppertrNakayama. 2 Mix equal quantities of urine and 5 per cent BaCU solution, centrifuge, and pour off liquid. Add to sediment 2 cc. of reagent (4 grams FeCU in 990 cc. of 95 per cent alcohol plus 10 cc. concentrated HC1). Stir and heat to boiling. Positive testdeep green color. Muller. h Direct method: Add to 10 cc. urine 1 cc. of 0.1 per cent NaNC>2 solution and 1 cc. Ehrlich's reagent (2 grams of paradimethylaminobenzaldehyde (Eastman) in 100 cc. of 20 per cent HC1). If bilirubin content is high, green color develops. Mutter. Concentration method: Mix 10 cc. urine with 5 cc. of 10 per cent BaCU. Filter, wash precipitate with water. Add to precipitate 1 drop of 0.1 per cent NaNOa solution and 1 drop Ehrlich's reagent. Blue-green to green color develops if positive. Diazo-Hunter. 6 Acidify urine with acetic acid if alkaline. Add 2 cc. 10 per cent BaClj solution to 5 cc. urine. Centrifuge, wash precipitate with water add 0.5 cc. diazo reagent (same as for VandenBergh test on blood serum) CHART Iff SENSITIVITY OF TESTS FOR BILIRUBIN IN URINE. DIUUTION URINE A VRINE B METHOD 1/ '40 *'«> l 4eo ^20 '/zo '<40 DIAZO HUNTER TK DIATO SPOT- -H -H- - TP HARRISON /SML/MANN -ff -h r/e - %o?. 2 cc. of 95 per cent alcohol and 0.3 cc. of 6 per cent Na2HPO-4 12H 2 0. Positivered color similar to VandenBergh test in serum. Dilute urine 1 to 4 or more if large amount of bilirubin is expected. Use 10 cc. urine if trace is present. For special technique with urines highly pigmented with urobilin, etc., see original article. Diazo spot (Godfried). 7 Precipitate bilirubin with BaCl2 as above, filter, spread filter paper on dry filter paper, and add 1 drop diazo reagent, 4 drops of 95 per cent alcohol, and 1 drop phosphate buffer (as above). Positivered to pink color. Test works best with small amounts of bilirubin. If precipitate is deep yellow in color, add more of reagents, or better, dilute urine and repeat. A quantitative technique by diazo method is given by this author. Harrison spot (Godfried). 7 Add to 10 cc. urine 5 cc. of 10 percent BaCl 2 solution, mix and filter. Spread filter paper on dry filter paper. Add 1 to 2 drops of Fouchet's reagent (Trichloracetic acid25 grams; H s O100 cc, 10 per cent FeCl 3 10 cc). Positiveblue to green color. Zins. s Add to 5 cc. urine 3-5 drops 1 per cent NaNC>2 plus few crystals

4 COMPARISON OF TESTS FOR BILIRUBIN IN URINE 241 of BaClj (or J volume of 10 per cent BaCU). Mix and filter. Take precipitate off paper, put on watch glass or fresh paper. Add a few drops of 20 per cent trichloracetic acid. Positiveblue to green color. Naumann* Filter through paper in a Buchner funnel of 3i cm. diameter 5 cc. of a 10 per cent emulsion of talc in water. Suck nearly dry. Add 5 cc. urine and suck nearly dry. Add 1 drop of Fouchet's reagent, or 10 per cent HNOs in middle of disc. Blue is positive. Fellinger and Menkes. 10 Add 0.2 per cent aqueous methylene blue solution drop by drop to 5 cc. urine. Positiveintense green. Blue color of reagent is imparted to negative urines after 1 to 2 drops. The above results and numerous single examinations show that all of the methods in which the bilirubin is concentrated by BaCU precipitation are much more sensitive than those in which no concentration was done. Miiller's method on direct urine is much more plainly read than any of the other non-concentration methods. A few preliminary tests with the methylene blue method of Fellinger and Menkes were satisfactory and as sensitive as the other non-concentration methods. The most easily read and at the same time the most sensitive of all the tests used is the diazo spot test developed by Godfried. The original diazo method of Hunter gives a clear cut result, but is not as sensitive as the filter paper modification. Possible objection to the diazo tests may be raised because of the necessity of making fresh diazo mixture for the occasional urine to be tested for bile. In the other barium chloride concentration tests (Huppert-Nakayama, Muller concentration test, Harrison, and Zins methods) the reagents are stable. The tests where the reagent is added to a precipitate on filter paper are much more easily read and detect bilirubin in smaller amounts than those in which the reagent is added to the centrifuged precipitate in the tube. Purposely the Muller method was changed in tests recorded in chart 2, and the Ehrlich reagent added to the precipitate in the tube, rather than to the filtered precipitate. The observed readings were one or two tubes less sensitive than those of repeat tests on filter paper. The oxidizing reagents chosen for the various oxidation methods appear satisfactory. Other reagents are used in some technics not investigated by us at this time, such as 3 cc. alcohol, 1 drop

5 242 ALVIN G. FOORD AND CECIL F. BAISINGER of C.P. sulphuric acid and 1 drop 5 per cent KC10 3 in the Huppert-Cole 11 reaction. Three cc. alcohol containing 3 per cent HC1 and 1 drop of 0.5 per cent NaN0 2 is used in the Schiffers and Steensma 12 modification of the Huppert-Salkowski test. In this test, CaCl 2 is used instead of BaCl 2 as the precipitant. Naumann calls attention to the fact that the first step in oxidation of bilirubin is to the green biliverdin, next to the blue bilicyanin and finally to pink choletelin. Others believe the green color obtained is merely a mixture of blue oxidized and yellow non-oxidized bilirubin. A table of various colors obtained with various oxidizing agents is given by Naumann, concentrated nitric acid giving the strongest reaction (pink). Naumann's test depends on the fact that bilirubin is adsorbed to the surface of talc granules and is free to react with oxidizing reagents. It is quite striking how yellow the talc becomes even when only small amounts of bilirubin are present in the urine. Also, there is a stronger reaction if the urine is sucked through a layer of talc than if it is mixed with the talc and then filtered in the Buchner filter. Oxidation with Fouchet's reagent yields a highly satisfactory color in fairly low dilutions comparable with other tests, and in one test (chart 1) a reaction was obtained in more dilute specimens than was obtained by the Huppert- Nakayama or the Miiller concentration test. " However, the color is rather hard to see in the puffy talc disc when only minute amounts are present, and one is apt to imagine he sees color in urine containing faint traces of bilirubin when he really might be mistaken. The end point in a series of diluted urines is very hard to make out. Naumann states that he obtains a color with normal urines, but in our hands this is so far questionable. He stated tests done by dissolving pure bilirubin in urine freed from pre-formed bilirubin with nitric acid and subsequent neutralization, concentration of parts of bilirubin per million could be detected. Godfried claims that Harrison's and the diazo spot methods are as sensitive, stating that Harrison's method detects to mgm. of bilirubin per 100 cc. of urine, and the diazo spot test to mgm. In endeavoring to add pure bilirubin (Eastman's) to urine, we found that the substance would

6 V* COMPARISON OF TESTS FOB BILIRUBIN IN URINE 243 not dissolve in urine but did in strongly alkaline solution, and on adding the latter solution to various urines, it precipitated out. Too much alkali was necessary to dissolve the bilirubin in urine to allow us to do all the various tests we wished to use. Consequently, we cannot report at this time as to numerical sensitivity figures with pure bilirubin. Not all of the above tests have been used in testing urines pigmented with medicinal dyes which might confuse laboratory workers, such as pyridium, prontosil, etc. The Huppert-Nakayama reaction, however, has given negative reactions in all cases tested over a period of years, except in the presence of bilirubin. Most probably the other oxidation reactions and the Vanden- Bergh would react likewise. Excess urobilin also has given a negative test. Of interest is Bensley's 13 paper in which he showed that the Ehrlich test for urobilinogen is negative if nitrite is present in the urine. Miiller used this finding to avoid the red color of urobilinogen, obtaining a green color when bilirubin containing urines are treated with sodium nitrite and Ehrlich's reagent. CONCLUSION In doing tests for bilirubin in the urine, particularly to avoid missing small quantities, some method of concentration of the pigment should be used, either precipitation and adsorption by barium chloride or adsorption by talc. The most commonly used tests on unconcentrated specimens are not sensitive and often are difficult to read. The diazo spot method of Godfried is a highly satisfactory method, as are also the Harrison spot and the Naumann methods. The latter is perhaps more time consuming. The red color developed in the diazo spot method is more easily read than the colors produced by the other methods. REFERENCES (1) KAPSINOW, R.: A new test for bile pigments in urine, bile and blood serum. J. A. M. A., 82: 687 (Mar. 1) (2) MATHEWS, A. P.: Physiologic Chemistry. 4th edition, Wm. Wood & Co., New York, 1925, 1155.

7 244 ALVIN G. FOORD AND CECIL F. BAISINGER (3) MATTICE, M. R.: Chemical Procedures for Clinical Laboratories. Lea & Febiger, Philadelphia, 1936, 237. (4) TODD, J. C, AND SANFORD, A. H.: Clinical Diagnosis by Laboratory Methods. 8th edition, W. B. Saunders Co., Philadelphia, 1935,134. (5) MULLER, A.: Zur Natur der Griinen Farbereaktion des Harnes mit paradimethyl-amino-benzaldehyde und die Bestimmung des Bilirubins und der Nitrite. Klin. Wchnschr., 17: 564 (Apr. 16) (6) HUNTER, GEORGE: A diazo method for detecting bilirubin in urine. Canad. Med. Assn. Jr., 23: 823 (Dec.) (7) GODFRIED, E. G.: Clinical tests for bilirubin in urine. Biochem. J., 28: pt. 2, 2056, (8) ZINS, B.: Zur Methodik des Nachweises von Bilirubin in Harn. Klin. Woch., 2: 978 (May 21) (9) NAUMANN, H. N.: Studies on bile pigments. II. A new test for bilirubin in the urine and its use for detection of bilirubin in normal urine. Biochem. J., 30: 762,1936. (10) FELLINGER, K., AND MENKES, K.: Ueber quantitative Bilirubinbestimmungen im Harn mit der Methylenblaumethode. Wien. Klin. Woch., 46: 133,1933. (11) OSGOOD, E. E.: Laboratory Diagnosis. 2nd edition, P. Blakiston's Son & Co. Inc., Philadelphia, 1935, 296. (12) Quoted by GODFRIED (7). (13) BENSLEY, E. H.: The interference of nitrites in the detection and estimation of urobilinogen in urine. Jour. Lab. & Clin. Med., 21: 1195 (Aug.) 1936.