ANALYTICAL SCIENCES JUNE 1998, VOL. 14 609 1998 © The Japan Society for Analytical Chemistry

Notes Spectrophotometric Determination of Urine by p-Dimethylaminobenzaldehyde

Yuji SUZUKI

Saitama College of Health, 519, Kamiokubo, Urawa, Saitama 338Ð0824, Japan

Keywords Urine bilirubin assay, spectrophotometry, bilirubinuria, p-dimethylaminobenzaldehyde

p-Dimethylaminobenzaldehyde (p-DMABA) in the solved with 1 dm3 of a strong hydrochloric acid solu- presence of strong hydrochloric acid has been widely tion. This reagent was stored in a refrigerator. used to determine in urine increasing due Bilirubin standard serum: Nescauto Bilirubin to certain pathologic status1,2, but has not yet been Standard (total bilirubin concentration 138 mg dm-3, applied to measuring bilirubin, a precursor of urobilino- direct bilirubin concentration 88 mg dm-3) was used as gen. However, the author reexamined the reaction of a standard material by redissolving it with distilled this reagent with bilirubin and the coexisting con- water. This standard solution was prepared just before stituents in serum by changing the preparation for the use. reagent, and showed that the serum total bilirubin can A solution of the coexisting constituents in urine: 1.0 also be determined by applying this reagent directly to g of glucose, 10 mg of tyrosine, 50 mg of tryptophan, serum.3 50 mg of phenylalanine, 0.5 g of hydroxyproline, 5.0 g Under physiological conditions bilirubin is present in of albumin, 1.0 g of creatinine, 1.0 g of ascorbic acid, small amounts in blood, and is excreted into the 1.0 g of urea, 20 mg of uric acid, 1.0 g of ammonia, 0.1 through the . However, under pathological condi- g of 5-, 0.2 g of hippuric acid, 1.0 g tions, different forms of liver damage, its concentration of acetone, 1.0 g of acetoacetic acid, 0.1 g of 3-hydroxy- in the blood increases, and its conjugated form is also butylic acid, 0.1 g of 5-hydroxyindole-3-acetic acid (5- excreted into the urine.4 Therefore, it is also very HIAA), 10 mg of indican, 1.0 g of oxalic acid, 1.0 g of important to measure the urine bilirubin concentration citric acid, 10 mg of , 20 mg of , and in order to grasp the accurate condition of the liver 20 mg of stercobilin were dissolved separately in 100 function. In general, it is difficult to accurately deter- cm3 of distilled water. mine urinary constituents by the method which is used A solution of urobilinogen was prepared by reducing to measure serum constituents, because the estimation urobilin as follows.5 To 50 cm3 of a 100 mg dm-3 uro- is often hampered by various colored substances and solution, 25 cm3 of 200 g dm-3 ferric sulfate solu- nonspecific-reactive coexisting interferences concen- tion and 25 cm3 of 2.5 mol dm-3 sodium hydroxide trated by the kidney. In this study the author attempted solution were added, and allowed to stand in the to design a spectrophotometric assay of the urine biliru- absence of sunlight or bright daylight at room tempera- bin concentration, using p-DMABA. ture for about 3 h. The reaction mixture was then fil- trated in order to eliminate a brown precipitate. The filtrate was used in the experiments. The concentration Materials and Methods of urobilinogen was estimated by a method of Henry et al. using Phenol Red as an artificial standard.6 Reagents Most of the reagents used were obtained from Wako Procedure of the urine bilirubin assay Pure Chemical Industries, Ltd., Osaka Japan, and were A reaction of the patients’ urine and the bilirubin of the best quality available. Biliverdin, ditaurobiliru- standard serum was performed as follows. If turbidity bin, urobilin and stercobilin were purchased from exists in urine, centrifugation of the sample is required. Funakoshi, Ltd., Tokyo Japan, bilirubin standard serum To 0.1 cm3 of the sample, 2.0 cm3 of the p-DMABA from Nippon Shoji Kaisha Ltd., Osaka Japan, and indi- reagent was added, well mixed and reacted for 30 min can from Sigma Chemical Co., USA. at 37¡C. The absorbance was then recorded at 640 nm A strong hydrochloric acid solution, 2.8 mol dm-3: with a Hitachi 7010 Clinical Spectrophotometer against 250 cm3 of concentrated hydrochloric acid was diluted a sample blank consisting of 0.1 cm3 of the urine and to 1000 cm3 with distilled water. 2.0 cm3 of 2.8 mol dm-3 hydrochloric acid solution. A p-DMABA reagent: 200 g of p-DMABA was dis- When the concentration of urobilinogen in urine is 610 ANALYTICAL SCIENCES JUNE 1998, VOL. 14 more than 3+, which corresponds to about 80 mg dm-3 bilirubin, forming a stable green color with an absorp- measured by the semiquantitative method (UROPAPER tion maximum at about 640 nm. However, in many “Eiken” MUTI2 purchased from Eiken Chemical Co., urine samples having a negative test result for bilirubin, Ltd. Tokyo, Japan), it should first be removed by this reagent reacted with certain coexisting con- extraction, as follows. To 1.0 cm3 of urine one drop of stituents, giving a yellowish or red product, the absorp- a concentrated hydrochloric acid and 5.0 cm3 of petro- tion of which at 640 nm was very small. Thus, the leum ether7 were added, and well mixed. After a few reactivities of the urinary coexisting constituents minutes the lower layer was used for a bilirubin assay. described in the materials and methods were examined. Urea, albumin, tryptophan, 5-HIAA, indican, urobilino- gen, acetoacetic acid, hippuric acid, hydroxyproline Results and ascorbic acid formed a colored product. The reac- tion of urea, hippuric acid, hydroxyproline, ascorbic Effect of the hydrochloric acid concentration on the acid, indican and urobilinogen rapidly came to comple- color reaction of bilirubin tion, and that of tryptophan reached a maximum in The reaction of urinary bilirubin, ditaurobilirubin, 5- about 15 min. On the other hand, the reaction of albu- HIAA and albumin with p-DMABA was examined, min, 5-HIAA, and acetoacetic acid was relatively slow, changing the hydrochloric acid concentration from 2.8 and did not come to completion even after 30 min. The to 5.6 mol dm-3. In the reaction of these substances molar absorptivity of the colored product of urobilino- with p-DMABA urinary bilirubin and ditaurobilirubin gen was larger than that of the green product formed gave a green color, and 5-HIAA and albumin formed a from bilirubin. The spectral characteristics of the purple and red color, respectively. In the reaction of formed colored product and their reaction rates are urinary bilirubin and ditaurobilirubin, the changes in summarized in Table 1. the rate constant due to the hydrochloric acid concen- tration were comparatively small, while in the reaction Proportionality of the color intensity of 5-HIAA and albumin it markedly increased due to The relationship between the color intensity and the an increase in the acid concentration. Although the amount of bilirubin was examined. The absorbance at absorbance of the colored product of urinary bilirubin 640 nm was proportional for up to at least 200 mg dm-3 was almost constant over a wide range of hydrochloric of bilirubin. The apparent molar absorptivity of the acid concentrations, that of ditaurobilirubin decreased colored product at 640 nm was 2.8´107 mol-1 cm2. due to an increase in the acid concentration. The absorbances of 5-HIAA and albumin increased as the Precision of the present method hydrochloric acid concentration increased. Since the The precision was examined by analyzing several relative absorbances and rate constants of 5-HIAA and patients’ urines 10 times. The intra-assay RSD of the albumin to those of urinary bilirubin are small in the urine at mean bilirubin concentrations of 15.3, 25.3, range of a low concentration of hydrochloric acid, 2.8 30.4 and 46.7 mg dm-3 were less than 1.2%. The inter- mol dm-3 was selected for the hydrochloric acid con- assay RSD of the urine at mean bilirubin concentrations centration in order to reduce the effects of the coexist- of 24.6 and 85.8 mg dm-3 were less than 2.5%. ing constituents as much as possible. Analytical recoveries Effect of the p-DMABA concentration on the color The analytical recoveries were examined by adding reaction of bilirubin one amount of ditaurobilirubin to urine samples of fif- The reaction rates of the bilirubin standard serum, teen patients. Those ranged from 98.5 to 107.4% patients’ serum bilirubin and patients’ urine bilirubin (mean 102.0%). were examined, changing the p-DMABA concentration from 50 to 300 g per dm3 of 2.8 mol dm-3 hydrochloric Measurement value of bilirubin negative urine acid. In most of the tested specimens the reaction rate The measurement values of 80 spot urines with a neg- came to a maximum at a p-DMABA concentration of ative result for bilirubin and with a concentration of around 200 g per dm3 of the hydrochloric acid, while urobilinogen ranging from normal to about 10 mg dm-3, the absorbances gradually increased as the hydrochloric which were tested by a reagent strip (Pretest b and u for acid increased. From these results, although the color bilirubin and urobilinogen in urine purchased from intensity of the urinary bilirubin was not maximum, Wako Pure Chemical Industries, Ltd. Osaka, Japan), 200 g per dm3 of the hydrochloric acid was selected for were examined by the present method and the two the p-DMABA concentration in order to shorten the diazo methods using caffeineÐsodium benzoate10 or time required for the assay. methanol11 as a reaction accelerator. The measurement values (mean±SD) obtained by these methods were Reaction of bilirubin and the coexisting constituents in 4.7±3.7 and 3.1±1.6 mg dm-3 for the diazo methods urine with p-DMABA using methanol or caffeineÐsodium benzoate as a reac- The thus-prepared p-DMABA reagent gradually tion accelerator and 1.5±1.1 mg dm-3 for the present reacted with the patients’ urine bilirubin like serum method. Although these measurement values correlat- ANALYTICAL SCIENCES JUNE 1998, VOL. 14 611

Table 1 Spectral characteristics of the colored product produced from the constituents in urine and their reaction rate

Apparent molar An amount of Absorption A /A Rate constant/ Constituent absorptivity/ lmax 640 excretion/ peak/nm ratio minÐ1 molÐ1 cm2 mmol dÐ1

Albumin 550 3.62´106 12.9 0.2 1.36´10Ð2 580 3.65´106 13.2 Tryptophan 545 2.60´104 4 150 4.89´10Ð1 570 2.30´104 3.5 Hydroxyproline 435 5.70´104 300 < 330 react rapidly 5-HIAA 560 8.29´106 4.9 30 1.49´10Ð1 590 8.16´106 4.8 Indican 585 8.81´106 1500 < 100 5.72´10Ð1 Acetoacetic acid 440 1.10´104 25 0.6 2.44´10Ð2 Hippuric acid 430 4.30´104 130 < 3400 8.48´10Ð1 Ascorbic acid 430 4.00´103 64 < 60 react rapidly Urea 440 2.13´106 2000 < 500000 react rapidly Urobilinogen 565 5.00´107 97 4 react rapidly Bilirubina 640 2.80´107 1 6 1.27 The apparent molar absorptivities were calculated from the absorbances measured when the color reaction completed at 37ûC.

Almax and A640 indicate the absorbances of each substance at the absorption peak and 640 nm, respectively. An amount of excretion of each substance is the approximate upper limit of the reference interval in normal adults.8,9 a. The bilirubin standard serum.

ed with urine specific gravities (r=0.805) measured by a refractometer, which varied from 1.002 to 1.025 Discussion (mean±SD=1.012±0.006), each urine diluted with 2.8 mol dm-3 hydrochloric acid (the mixture ratio, 1:20) The author previously indicated that the p-DMABA indicated little absorbance (0 Ð 0.001) at 640 nm reagent can also be utilized to determine the total serum whether or not the specific gravity of the urines was bilirubin concentration by changing the concentrations high or low. This result suggests that the effect of a of hydrochloric acid and p-DMABA. In general, urine shade of a color tone of urine itself on the measurement contains a large number of soluble waste products of value is either very small or can be neglected. By the metabolism and of the excesses of ingestants, which are way, in the assay of these urine samples by the oxida- excreted in a concentrated state. Therefore, it involves tion method12 and the enzymatic method13, it was difficulties to establish a method for measuring urinary impossible to calculate their measurement values in compounds, because there are many coexisting sub- most urine samples, because the absorbance of the stances which are inclined to interfere with the detec- reagent blank exceeded that of the reaction mixture. tion reaction of urinary substances.5 As indicated in the experiments, the p-DMABA Comparison of the urine bilirubin concentration reagent, even after changing its recipe, reacted with measured by five methods various substances (urea, 5-HIAA, indican and so on), The urine bilirubin concentration in 17 bilirubinurias giving a yellowish or red color. However, favorably was determined by the two diazo methods with diazo- unlike these interfering substances, bilirubin formed a tized sulfanilic acid10 and 3-nitroaniline11, the oxidation green color with an absorption maximum at about 640 method with vanadic acid12 and the enzymatic method nm, where the absorption of the colored products with bilirubin oxidase13, using Nescauto Bilirubin formed from the coexisting constituens was very small. Standard Serum as a standard material in all of the In the present method the measurement value methods. The present method correlated well with the (mean±SD) of the urine samples with a negative test four conventional methods (r=0.985 Ð 0.992), and the result for bilirubin was 1.5±1.1 mg dm-3, which was mean value of the present method (25.6 mg dm-3) was lower than those obtained by the two diazo methods. close to those of the enzymatic method (25.5 mg dm-3), Since most of the urine samples diluted with 2.8 mol the oxidation method (25.0 mg dm-3), and the diazo dm-3 hydrochloric acid solution without containing p- method (23.8 mg dm-3) using the diazotized sulfanilic DMABA absorbed little at 640 nm, irrespective of the acid. Also, the measurement values obtained by the size of the urine specific gravity, the measurement val- present method were parallel to those by the reagent ues for the urine samples with a negative test result for strip test (UROPAPER “Eiken” MULTI2), as indicated bilirubin is thought to result from the formation of col- in Table 2. ored products from the coexisting constituents in the 612 ANALYTICAL SCIENCES JUNE 1998, VOL. 14

Table 2 Comparison of the present method with the semiquantitative method (UROPAPER)

Sample number 1 2 3 4 5 6 7 8 9 10 11 12 13 Present method/mg dmÐ3 85.8 84.7 77.8 35.3 24.6 12.6 8.0 8.3 7.1 6.0 1.9 0.9 0.6 UROPAPER bil 3+ 3+ 3+ 3+ 2+ 2+ 2+ + + + Ð Ð Ð uron +nn+nnnnnnnn Abbreviation used: bil, bilirubin; uro, urobilinogen; n, normal level. The semiquantitative methods for bilirubin and urobilinogen by the UROPAPER are both based on the diazo method.

urine and urinary bilirubin excreted in a small amount, “Clinical Chemistry; Principles and Technics”, 2nd ed., even in normal adults.14 From this result, it assumes p.1079, Harper & Row, Publishers, Inc., New York, 1974. that it is reasonable to think that the abnormal excretion 6. R. J. Henry, A. A. Fernandez and S. Berkman, Clin. Chem., of bilirubin into urine does not occur when the mea- 10, 440 (1964). surement value of urine sample is approximately less 7. N. Imai and K. Arisue, Medical Technology, 13, 1095 -3 (1978). than 4 mg dm (corresponding to mean+2SD=1.5+2´ 8. I. Kanai and M. Kanai, “Rinshokensahoteiyo (A Handbook -3 1.1=3.7 mg dm ). Since the measurement values of of Clinical Examinations, in Japanese)”, 30th ed., p. 48, the present method were parallel to the semiquantita- Kinbara Shuppan, Tokyo, 1993. tive values by the UROPAPER “Eiken” MULTI2, and 9. “Seikagaku data book (Data book of biochemistry, in were correlated with those obtained by the four conven- Japanese)“, ed. Japanese Biochemical Society, p.1598, tional methods, it seems that the p-DMABA reagent Tokyokagakudojin, Tokyo, 1979. can also be applied to the urinary bilirubin assay. 10. L. Jendrassik and P. Gróf, Biochem. Ztschr., 297,81 (1938). 11. Y. Suzuki and Y. Sakagishi, Jpn. J. Clin. Chem., 23, 158 References (1994). 12. K. Tokuda and K. Tanimoto, Jpn. J. Clin. Chem., 22, 116 (1993). 1. P. Ehrlich, Med. Wochschr., 1, 151 (1901). 13. A. Kosaka, in “Methods of enzymatic analysis”, ed. H. U. 2. S. Schwartz, V. Sborov and C. J. Watson, Am. J. Clin. Bergmyer, 3rd ed., p.591, VCH, Weinheim, 1985. Pathol., 14, 598 (1944). 14. M. Michaëlsson, Scand. J. Clin. Lab. Invest., 13 (suppl.), 3. Y. Suzuki, Anal. Sci., 13, 291 (1997). 64 (1961). 4. R. L. Wolf, M.Pizette, A. Richman, D. A. Dreiling, W. Jacobs, O. Fernandez and H. Popper, Am. J. Med., 28,32 (Received October 16, 1997) (1960). (Accepted March 19, 1998) 5. R. J. Henry, D. C. Cannon and J. W. Winkelman, in