A TEST FOR THE DETERMINATION OF TAURINE IN URINE

BY E. IRENE PENTZ, CHARLENE H. DAVENPORT, W. GLOVER, AND DARREL D. SMITH

(From the Argonne Cancer Research Hospital and the Department of Biochemistry, University of Chicago, Chicago, Illinois)

(Received for publication, March 28, 1957)

No rapid, simple, chemically specific test for the quantitative determina- tion of taurine in biological fluids has been available. Previously, the pres- ence of taurine in urine was determined either by column chromatography (1) or by paper chromatography (2-5). While these methods are quanti- tatively satisfactory, they are inadequate for rapid routine examination of the large numbers of samples that are required in metabolic or clinical studies.

A description of a simple method for the determination of taurine in urine, which readily allows multiple analysis in a comparatively short period of time, is the subject of this paper. Crokaert et al. (6) made the observation that taurine does not adhere to the acid form of Dowex 50. The use of this property combined with Sanger’s reagent (7), which readily forms the colored dinitrophenol derivative of taurine, has made the test possible. It is reasonably specific and has the advantage that ammonia and the other nitrogenous constituents of urine do not interfere in the de- termination.

The method provides the opportunity for extension of the recent obser- vation that the excretion of taurine is increased in both the human (4) and the rat (8) following irradiation.

Methods and Materials

The sodium salt of dinitrophenol taurine (DNP-taurine) was synthesized and purified in the following manner: 0.5 gm. of taurine, 0.8 gm. of sodium bicarbonate in 10 ml. of water, 0.56 ml. of dinitrofluorobenzene,l and 20 ml. of absolute ethyl alcohol were shaken together in the dark for 16 hours at 160 oscillations per minute. The contents of the flask were then filtered through a Biichner filter and the filtrate was discarded. The solid material remaining on the filter paper was dissolved in approximately 40 ml. of warm 0.1 N HCl. This solution was extracted twice with 25 to 40 ml. of chloroform and then concentrated in a steam bath at reduced pressure to

r Dinitrofluorobenzene (DNFB) from Eastman Organic Chemicals or Eastern Chemical Corporation has been found to yield uniform results. The products of some other manufacturers have not been equally consistent.

433

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434 DETERMINATION OF TAURINE IN URINE

about 10 ml. It was then transferred to a beaker and allowed to stand in the cold overnight. The crystals that formed were separated by filtration and redissolved in approximately 17 ml. of 50 per cent ethyl alcohol. The crystals that formed subsequently were recrystallized from 50 per cent ethyl alcohol and then filtered from the mother liquor and dried in a desiccator; yield, 314 mg.; m.p., 230”.

Calculated. C 30.67, H 2.57, N 13.41, S 10.23, Na 7.34 Found. “ 30.75, “ 2.57, “ 13.05, “ 10.07, “ 7.22

This material was dissolved in 0.4 M HCl-KC1 buffer, pH 1.25, and the absorption spectrum was determined (Fig. 1).

b - Na DNP TAURINE IN KCL-HCL X BUFFER, 0.4 M, pH 1.20 I- 10.0

I5 - DNP TAURINE IN KCL-HCL BUFFER,

g 8.0 0.4 M, pH 1.25, FROM STANDARD CURVE DETERMINATION EQUIVALENT TO

IL : 8r TAURINE

u 6.0

t5 iz 4.0

is

g 2.0 w

0 iL t3 440 420 400 380 360 340 320 300 2

: WAVELENGTH (mp)

IO

FIG. 1. The absorption spectrum of sodium dinitrophenol taurine in KCl-HCl buffer, 0.4 M, pH 1.20 (light line) and of the solution obtained on analysis of an 8 y &urine standard (heavy line).

Appropriate dilutions of the above solution were made with HCl-KC1 buffer and read in the Beckman spectrophotometer, model B, at 355 rnp to establish the validity of the standard curve that was obtained by using the method outlined below.

Reagents for Taurine Determination- Dowex 50. 200 to 400 mesh, 8 per cent cross-linked. (Nalcite HCR-8,

H+ form) ;2 used without further treatment.

2 Two different lots of Nalcite HCR-8,200 to 400 mesh, H+ form, were used to carry out this work. Subsequently, small differences have been found among various lots of the product. These chiefly affect the optical density readings of the blanks, some lots yielding much lower blank readings than others. It is suggested that, on ob- taining a lot that yields a low blank, sufficient amounts of this material should be pro-

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PENTZ, DAVENPORT, GLOVER, AND SMITH 435

Potassium acid phthalate-sodium hydroxide buffer, pH 4.5. 50 ml. of 0.2 M potassium acid phthalate, 10 ml. of 0.2 M NaOH, 0.40 gm. of sodium benzoate; make to 200 ml. with water.

Sodium bicarbonate, 8 per cent. Dinitrofluorobenzene.l Hydrochloric acid, 6 N.

Potassium chloride-hydrochloric acid buffer, pH 1.25. 50 ml. of 0.4 M

potassium chloride, 41.5 ml. of 0.4 M hydrochloric acid; make to 200 ml. with water.

Chloroform, reagent grade. Taurine standard, 120.0 mg. of taurine in 100 ml. of phthalate buffer.

Dilute 1: 100 ml. with buffer. Use 10 ml. in Step 2 (see below). 1, 2, and 3 ml. aliquots of filtrate (see Step 5) provide final taurine values of 3.2, 6.4, and 9.6 y per 4 ml., respectively.

Specimens for analyses are as follows: (a) Human urine was usually di- luted 2: 10 ml. with phthalate buffer before treatment with Dowex and 1 ml. of filtrate from Step 4 used in Step 5. (b) Rat urine was diluted 1: 20 with phthalate buffer and 10 ml. were treated with Dowex. 1 ml. of fil- trate was used for analysis in Step 5.

Procedure

1. Introduce 5.0 gm. of Dowex 50 into a 50 ml. Erlenmeyer flask. 2. Add 10.0 ml. of the sample or the standard solution that is to be

analyzed. 3. Mix and let stand at room temperature for 5 minutes with occasional

agitation. 4. Filter through Whatman No. 40 filter paper. 5. Introduce 1.0 to 3.0 ml. of filtrate into a 25 ml. volumetric flask. 6. Add 5 ml. of 8 per cent sodium bicarbonate. 7. Add 0.28 ml. of DNFB. 8. Shake overnight (16 hours) at 160 oscillations per minute on a machine

that is protected from light. 9. Remove from shaking machine and add 1.0 ml. of 6 N HCI. 10. Add 2.0 ml. of KCl-HCl buffer, pH 1.25. 11. Add 10 ml. of chloroform. Stopper and extract by shaking vigor-

ously for 1 minute. 12. Add water to 25 ml. 13. Mix and transfer to a straight sided 25 ml. graduate.

cured to last for a considerable number of analyses. It has been observed, also, that the 12 per cent cross-linked material is equally satisfactory for the performance of this test.

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436 DETERMINATION OF TAURINE IN URINE

14. Transfer 10.0 ml. of the top layer to a 50 ml. straight sided, stop- pered graduate.

15. Add 15 ml. of chloroform. Extract by shaking for 1 minute. 16. Transfer 4.0 ml. of the top layer to matched calorimeter tubes and

read at 355 rnp. Standard Curve-The line of regression obtained from 50 analyses of

standard solutions is expressed by the equation

Y = 0.0073 + 0.0383X

where Y = optical density and X = micrograms of taurine per 4 ml. (Fig. 2). The calculated standard error was ~0.5 per cent. The calcu- lation is as follows:

Y oer 4 ml. found X 3.75 X original dilution3 X 0.903 X total urine volume

1000 = mg. per 24 hrs.

Blanks-A small amount of material that absorbs at 355 rnF is introduced into the filtrate at Step 4 because of the use of unwashed Dowex. For this reason, it is necessary to prepare blanks from a filtrate of phthalate buffer and Dowex for each different volume of filtrate used in Step 5. The day to day reading of these blanks against water has been extremely con- sistent and thus this practice has introduced no error into the method. It was found quite possible to wash the Dowex free of this colored material, but the difficulty of subsequently drying the Dowex to constant water content introduced more errors than did the use of Dowex without pre- treatment.

HydrolysisSpecimens of urine were hydrolyzed by adding an equal volume of HCl (concentrated) and autoclaving for 5 hours at 15 pounds pressure and 250”. This is conveniently carried out in 25 X 200 mm. tubes that have a screw cap with a Vinylite liner in the cap. Total volume before autoclaving never exceeded 10 ml. After autoclaving, the inside of the cap was washed with a small amount of water and the contents of

3 A correction for dilution caused by the water content of the Dowex 50 must be applied. It is necessary to determine the value of this factor for each lot of Dowex used. This is done by developing a standard curve without using the resin, that is, by starting at Step 5 of the procedure and using appropriate dilutions of the stand- ard solution directly. The difference between the slope of this curve and that of the standard curve obtained by using Dowex is the correction factor. It may either be applied directly in the equation, or, as has been done here, as a percentage of the final figure. Inspection of Fig. 2 shows that the standard curve obtained with Dowex cuts the origin and there is, therefore, no question of taurine loss by virtue of its adherence to the resin. For the determinations reported here this correction factor has been found to be 0.90.

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PENTZ, DAVENPORT, GLOVER, AND SMITH 437

the tubes were taken to dryness by placing them in a water bath at 95” and passing a continuous stream of air over the surface. Water, to the amount of the original volume of specimen, was added again to the tube. A volume of phthalate buffer, such that the appropriate dilution was achieved, was then added directly to the tubes. The residue on the sides and bottom of the tubes was stirred into solution and, after mixing, the total volume was filtered. 10 ml. of this filtrate were then used in Step 2 of the pro- cedure.

Recovery Experiments-Solutions of taurine were added quantitatively to urine that had been analyzed previously for its taurine content and

1.0 -

0 4 8 12 16 20 24 28

f TAURINE/4ml.

FIG. 2. The relationship of optical density of the dinitrophenol derivative to the concentration of taurine as determined by the steps outlined under “Procedure.”

analyses were made for recovery of the added taurine. The results ob- tained are shown in Table I.

XpeciJcity-Substances normally present in urine or that might be pres- ent in the urine from diseased subjects or those undergoing therapy were tested to rule out the possibility of their interference in the test. The sub- stances tested and the results obtained are shown in Table II. Also in- cluded at the bottom of Table II are some analyses on hydrolyzed samples of sodium taurocholate, pantoyltaurine, and ox bile.

In$uence of Hydrogen Ion and Xalt Concentrations on Adsorption by Dowex &-An amino acid mixture having the following composition per 1500 ml. was made: aspartic acid 246 mg., threonine 96 mg., serine 150 mg., glutamic acid 600 mg., proline 93 mg., glycine 999 mg., alanine 84 mg., cystine 60 mg., valine 36 mg., isoleucine 24 mg., leucine 30 mg., tyrosine 195 mg., phenylalanine 48 mg., histidine 450 mg., methylhistidine 138 mg.,

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438 DETERMINATION OF TAURINE IN URINE

lysine 75 mg., arginine 12 mg. The above mixture was divided into three parts and taurine was added to two of them at levels of 30 mg. per 200 ml. and 60 mg. per 200 ml. These three solutions were each diluted 1:5 with the following buffer solutions: potassium phthalate buffer, 0.2 M, pH 2.7 and 4.5; potassium-acid phosphate buffer, 0.2 M, pH 7.0; boric acid-KCl- NaOH buffer, 0.2 M, pH 10.0; and water. Analyses were then made for recovery of taurine. The results are shown in Table III. In addition, triplicate analyses were made on an amino acid solution containing no tau- rine but higher total concentrations of amino acids. These were diluted with 0.2 M potassium phthalate buffer, pH 4.5, and with water to deter-

TABLE I

Recovery of Added Taurine from Urine That Had Been Previously Analyzed for Taurine Content

Taurine content of urine Taurine added

y per 4 nal. y per 4 ml.

5.2 3.8 4.2 3.8 5.5 7.8 5.5 6.2

12.4 5.3 4.7 16.0 5.6 13.2 4.0 5.1 8.0 5.1 4.9 5.0

ReCOV.Ty

per cent 100

98 102 102 102

99 98

103 102 104

mine the efficiency of the conditions of the test for removal of amino acids other than taurine. These results are also included in Table III.

Inspection of Table III reveals the importance of both the ionic strength and the pH of the diluent buffer solution for both the completeness of re- covery of taurine and the exclusion of other amino acids. Two of the amino acid concentrations tested, 251 y per 4 ml. and 313 y per 4 ml., represent, respectively, about 1.0 gm. and 1.3 gm. of free amino nitrogen (calculated on total urine volume of 1500 ml.). Therefore, in urine having very high amino nitrogen values, a small increase in the apparent taurine values might be expected. However, the margin of safety appears to be reasonably wide.

It was originally believed that water as a diluent followed by a pH ad- justment might prove satisfactory in the method, but subsequent irregu- larities in standard curve determinations proved this to be in error.

Influence of Length of Shaking Time-Two standard solutions containing,

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PENTZ, DAVENPORT, GLOVER, AND SMITH 439

TABLE II Color Production by Substances Other Than Taurine As Well As Taurine

Content of Some Conjugates of Taurine before and after Hydrolyses

Compound

Urea. ..................................... Creatinine. ............................... Ammonia .................................

Uric acid. ................................ Allantoin ................................. Guanine .................................. Adenine ................................... Xanthine ................................. Kynurenic acid. .......................... Creatine .................................. p-Aminobenzoic acid. ..................... Xanthopterin ............................. Thiamine hydrochloride. .................. Riboflavin. ............................... Hemoglobin ............. :. ................ Phenylserine .............................. Glycylglycine ............................. 2,4-Dimethylbutyric acid .................. p-Hydroxyphenylglycine .................. 7-Methylxanthine ......................... 1,7-Dimethylxanthine. .................... Sulfapyridine. ............................ Sulfanilamide. ............................ Sulfathiazole .............................. Sulfadiazine ............................... Sulfaguanidine ............................ Sulfamerazine ............................. Phenylethylamine. ........................ Benzidine ................................. Tyramine hydrochloride ................... Guanidine acetate ......................... 3-Indoleacetic acid ........................ Acetamide ................................ Bilirubin ..................................

Vitamin A palmitate. ..................... “ a acetate ........................

Adrenaline ................................ Histamine ................................

Final concen- tration tested

per 4 ml.

Equivalent concentration in urine, mg.

per 24 hrs. (1500 ml.)

w. m. 10.6 298 .o 0.53 14.8 2.1 59.1 7

25 16 16 16 16 5.3

26.6 5.3 5.3 5.3 9.6 5.3 5.3 5.3 5.3 5.3 5.3

26.6 26.6 26.6 26.6 26.6 26.6 26.6 10.7 26.6 26.6 26.6 26.6 26.6 26.6

u. s. P. units

400 400

Y 26.6 26.6

0.7 0.0022 0.45 0.0000 0.45 0.0022 0.45 0.0000 0.45 0.0362 0.15 0.0177 0.75 0.0000 0.15 0.0177 0.15 0 .oooo 0.15 0.0000 0.27 0.0200 0.15 0.0223 0.15 0.0223 0.15 0.0223 0.15 0.0223 0.15 0.0223 0.15 0.0088 0.75 0.0044 0.75 0 .OllO 0.75 0.0110 0.75 0.0110 0.75 0.0000 0.75 o.oooo 0.75 o.oooo 0.30 0.0223 0.75 0.0066 0.75 0.0066 0.75 0.0066 0.75 0.0088 0.75 0.0088 0.75 0.0339

0.75 0.75

Optical density at

35.5 nip

0.0044 0.0088 0.0088

0.0022 0.0315

0.0555 0.0000

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440 DETERMINATION OF TAURINE IN URINE

TABLE II-Concluded

Compound

Threonine.. . . . . . . . . . . Serine.................................... Sodium taurocholate (Pfanstiehl). . . . Pantoyltaurine. .

“ . . . . . . . . . . . . . . . . . . . Sulfanilic acid. . Cysteic acid. Tyrosine-O-sulfate* .

“ . . . . . . . . . . . . . . . . .

<‘

Sodium taurocholate (Sample 1, hydrolyzed) Pantoyltaurine (hydrolyzed). Sodium taurocholate (Sample 1, hydrolyzed

Y w. 18.1 0.51 0.0000 18.1 0.51 0.0000 34.4 0.98 0.0022 17.8 0.50 0.0482 17.8 0.50 0.0555 26.6 0.75 0.392 26.6 0.75 0.745

637.3 2.39 0.6240 318.6 1.19 0.3170

12.7 0.047 0.0000 34.4 0.1643 17.85 0.305

34.4 0.1707 34.4 0.2716 25.6 0.1107

* Kindly supplied by Dr. Harris T. Tallan of The Rockefeller Institute for Medi- cal Research.

7 hrs.). Sodium taurocholate (Sample 2, hydrolyzed) Ox bile extract? (Sample A, hydrolyzed)

Final concen- tration tested

per 4 ml.

Equivalent concentration in urine, mg.

per 24 hrs. (1500 ml.)

t Donated by Dr. Claire E. Graham of the Wilson Laboratories, Chicago.

-

respectively, 15.3 and 9.7 y per 4 ml. of taurine and an unhydrolyzed urine containing 7.6 y per 4 ml. of taurine were analyzed for their taurine content after shaking for 3, 4, or 5 hours, respectively, with DNFB at Step 8 of the procedure. The per cent recovery in each case is shown in Fig. 3.

Other points in the procedure are not critical. Amounts larger than 3 ml. aliquots of the solutions to be analyzed have not been tried, but there is no apparent reason why they should not be satisfactory. The only ex- ception to this is in the case of very concentrated urine having a high salt content. Occasionally, the aqueous phase of such fluids is cloudy at Step 11 of the procedure. This clears on the addition of the water at Step 12 and therefore previous allowance for dilution at this step is expeditious. The amount, 0.28 ml., of DNFB was chosen because it had been used suc- cessfully by Sanger (9) in preparing amino acid derivatives under similar conditions. In this procedure 0.3 ml. has also been used with satisfactory results. Equivalent values are obtained as long as there isan excesspresent and two chloroform extractions are used subsequently. The pH at Step 10 may vary from 0.91 to 1.49 without altering the optical density values obtained. The color obtained is permanent, at least over a 3 day period,

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‘aurine Test solution Diluent PH pz14

Re- COWy’

found) -.

Y ger cent

Amino acids, 125 y per 4 ml. 0.2 M potassium phthal- 2.6 0.93 “ “ + 8 y taurine ate-HCl buffer 2.6 8.62 96.2 ‘I “ +16” “ 2.6 16.39 96.6

Amino acids, 125 y per 4 ml. 0.2 M potassium-acid 6.9 0.38 I‘ “ + 8 y taurine phosphate buffer 6.9 7.62 95.2 “

“ + 16 “ “ 6.9 15.56 95.9

Amino acids, 125 y per 4 ml. 0.2 M boric acid-potassium 10.0 1.22 I‘ “ + 8 y taurine chloride-sodium hy- 10.0 9.27 100.5 “ “ + 16 “ I‘ droxide buffer 10.0 16.84 97.6

Amino acids, 125 y per 4 ml. 0.2 M potassium phthal- 4.5 0.0 “ “ + 8 y taurine ate-HCl buffer 4.5 8.06100.75 “

“ +16“ “ 4.5 15.86 99.0

Amino acids, 125 y per 4 ml. Water 2.0! 0.0 “ “ + 8 y taurine 2.0! 8.12 101.4 “ “

+ 16 “ “ 2.0! 16.39101.8

Amino acids 0.2 M potassium phthal- 4.5 1.84 Total content, 251 y per 4 ml. ate-HCl buffer

Amino acids Water 4.5 2.03 Total content, 251 y per 4 ml.

Amino acids Total content, 313 y per 4 ml.

Amino acids Total content, 313 y per 4 ml.

0.2 M potassium phthal- 4.5 3.20 ate-HCI buffer

Water 4.5 3.17

PENTZ, DAVENPORT, GLOVER, AND SMITH 441

and the individual tubes may therefore be read when it is convenient to do so.

Taurine Values Found in Normal Male Urine-24 hour urine specimens were collected (under toluene) from nineteen normal healthy males be-

TABLE III

Recovery of Taurine from Amino Acid Mixtures of Various Hydrogen Ion Concentration with and without Added Buffer Solutions

* When color was obtained from the solution of amino acids containing no taurine, this amount was subtracted from the color obtained from the solutions containing taurine to calculate the per cent recovery.

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442 DETERMINATION OF TAURINE IN URINE

100 - & 5 s

80-

it 60 -

5 g 40- - 15.31/4 ml. Std.

E ---- -x- 9.77 / 4 20 - 7.61/4

ml. ml. Urine Std.

07 I 2 3 4 5 6 7 8 9 3

HOURS

FIG. 3. The per cent recovery of taurine from a standard solution containing 9.7 7 per 4 ml. and 15.3 y per 4 ml. and from a urine sample containing 7.6 y per 4 ml. after 3, 4, and 5 hours of shaking.

TABLE IV

Taurine values Found for Unhydrolyzed and Hydrolyzed Urine Obtained from Nineteen Male Subjects

J. K. J. S. W. G. L. s. J. N. E. S. A. L. C. H. G. J. R. H. s. P. R. H. P. H. F. S. R. Z. J. S. v. w. G. B. F. S.

Average. . . . . . . . . . .

Unhydrolyzed Hydrolyzed

mg. per 24 hrs. mg. per 24 hrs.

194.76 241.47 287.28 310.95 131.22 131.13 253.35 287.19 178.38 173.52 119 88 136.17 211.14 239.58 191.25 229.41 165.15 216.99 513.90 512.10 230.40 216.90 269.73 343.35 210.42 226.62 139.77 165.60

43.83 52.92 268.20 256.05 260.37 319.68 213.57 232.29 140.04 145.35

211.6 233.5

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PENTZ, DAVENPORT, GLOVER, AND SMITH 443

tween the ages of 20 and 40 years. They were analyzed for taurine both before and after hydrolysis. The results obtained are shown in Table IV.

DISCUSSION

The presence of taurine was first reported in normal urine by Dent (10) less than 10 years ago. Stein (1) subsequently reported excretion values of 86 to 294 mg. per 24 hours obtained on unhydrolyzed specimens from eight normal males. After hydrolyses, three specimens gave 24 hour values of 147, 180, and 302 mg., respectively. In the current series the range reported, 44 to 514 mg. per 24 hours, is the same both before and after hy- drolysis. The average value is, however, slightly higher in the hydrolyzed specimens. These increased values are probably due to the presence of cysteic acid formed during the hydrolytic treatment or taurine liberated from traces of taurocholic acid originally present in urine.

Tyrosine-O-sulfate, a constituent of normal urine, has been reported not to adhere to the acid form of Dowex 50 (11). It was therefore expected that the presence of this substance in urine would introduce an error since its DNFB derivative would be expected to have an absorption maximum very close to 355 rnp. However, inspection of Table II shows that, at values double the reported average daily excretion value (28 mg.), no in- crease in optical density resulted due to the presence of this substance. If exceedingly large amounts of tyrosine-O-sulfate are present, some color does appear, but these are so high that they may be considered to be well be- yond physiological limits.

Of the many substances tested for interference in the taurine values de- termined by this method, only cysteic acid appears likely to be present in sufficient amounts (1) to produce artificially high values. Since the nor- mal cysteic acid content of urine is reported to be low (I), the error intro- duced by its presence is one of low magnitude.

It has been demonstrated by Portman and Mann (12) that prefeeding animals a diet low in organic sulfur results in tissue retention of taurine and a decrease in its urinary excretion. Since no effort was made in this study to control the diet of the ninet’een males chosen for urine analysis, this factor no doubt influenced the results obtained.

It has previously been demonstrated (13-16) that methionine, cysteine, sulfate, and glutathione are precursors of taurine. Kay et al. (17) have been able to show an increased excretion of taurine following total body x-irradiation of rats. This suggests increased oxidation of the sulfur-con- taining compounds of the body. In view of the well established effect of irradiation on sulfhydryl groups (18), this is undoubtedly the case. Kay and coworkers (17) also report an increase in the excretion of cysteic acid following irradiation. However, the increase in taurine is 10 times greater

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444 DETERMINATION OF TAURINE IN URINE

than that in any of the other amino acids. Use of the current test to de- termine the amount of taurine present following irradiation will therefore yield values that are increased by the value of any cysteic acid which is also present in the urine samples. It has been suggested (17) that meas- urement of the increased excretion of taurine following irradiation may have some diagnostic value. If so, simultaneous measurement of the in- creased excretion of cysteic acid would be of no particular disadvantage.

Some clinical applications for a test of this kind may well develop in view of the recent studies of Mann et al. (12, 19-21) on taurine metabolism and on the relationship of the sulfur-containing amino acids to hypercholes- terolemia.

SUMMARY

The sodium salt of dinitrophenol taurine has been synthesized and its absorption spectrum from 450 to 300 rnp described.

A method for the determination of taurine in urine is outlined. The method depends upon the fact that taurine does not adhere to the acid form of Dowex 50 and that its colored derivative with I-fluoro-2,4-dinitroben- zene is formed readily and measured at 355 mp.

One compound normally present in urine, cysteic acid, has been found to yield color under the conditions described. Since this compound is nor- mally present in urine in only small amounts, the test is reasonably specific for taurine.

The 24 hour taurine excretion values of nineteen normal males are re- ported for both hydrolyzed and unhydrolyzed specimens. The average value for the unhydrolyzed samples was 211.6 mg. per 24 hours and for the hydrolyzed, 233.5 mg. per 24 hours.

The authors take pleasure in acknowledging the interest and encourage- ment of Dr. Robert J. Hasterlik and the helpful suggestions of Dr. Leif Sorensen. Mr. Rodney Sung also contributed to the early phases of the work.

BIBLIOGRAPHY

1. Stein, W. H., J. Biol. Chem., 201, 45 (1953). 2. Block, R. J., LeStrange, R., and Zweig, G., Paper chromatography, New York

(1952). 3. Williams, R. J., Univ. Texas Pub. No. 6109 (1951). 4. Katz, E. J., and Hasterlik, R. J., J. Nut. Cancer Inst., 16, 1085 (1955). 5. Kay, R. E., Harris, D. C., and Entenman, C., Research and development tech-

nological report USNRDL-TR40, Mar. (1955). 6. Crokaert, R., Moore, S., and Bigwood, E. J., Bull. Sot. Aim. biol., 33, 1209 (1951). 7. Sanger, F., Biochem. J., 39, 507 (1945).

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PENTZ, DAVENPORT, GLOVER, AND SMITIE 445

8. Kay, R. E., and Entenman, C., Abstracts, Radiation Research Society, May (1956).

9. Sanger, F., Biochem. J., 39, 33 (1945). 10. Dent, C. E., Biochem. J., 43, 169 (1948). 11. Tallan, H. H., Bella, S. T., Stein, W. H., and Moore, S., J. Biol. Chem., 217, 703

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