274 Analyst, April, 1971, Vol. 96, 99. 274-280 Quantitative Determination of Taurine by an o-Phthalaldehyde - Urea Reaction BY M. K. GAITONDE AND R. A. SHORT (Medical Research Council Neuropsychiatry Unit, Woodmansterne Road, Carshalton, Surrey) Taurine is made to react with o-phthalaldehyde in the presence of urea and phosphate ions, and on acidifying the mixture with acetic acid a purple product is formed with an extinction maximum at 560 nm. A method based on this reaction is described for the quantitative determination of taurine and it is applied to the determination of this amino-acid in rat brain after passage of the tissue extract through ion-exchange resins. The effect of other amino-acids on the accuracy of the method is discussed. AMINES react with o-phthalaldehydel s 2 to give coloured products and this reaction has also been used for their detection on paper chromatograms.Curzon and Giltrow3 used the reaction for identifying spots of taurine and other amino-acids and they also noted that different products resulted if the reaction was performed in the presence of urea. These observations have been made the basis of a specific quantitative method for the determination of taurine in tissue extracts. METHOD MATERIALS AND REAGENTS- Amino compounds were obtained from Koch-Light Laboratories Limited (Colnbrook, Buckinghamshire) or from California Corporation for Biochemical Research (Los Angeles, California). A sample of homotaurine (3-aminopropane-1-sulphonic acid) was kindly supplied by Dr. J. C. Watkins. o-Phthalaldehyde, urea, hydrochloric acid (sp.gr.1-18), acetic acid (99.6 per cent.), phosphoric acid (spgr. 1.75) and other reagents were products of B.D.H. (Chemicals) Limited, Poole, Dorset. Urea solution-A 50 per cent. urea solution was prepared by dissolving 50g of urea in water and diluting to 100 ml at room temperature (21 "C). o-Phthalaldehyde reagent-A saturated solution of o-phthalaldehyde (0-6 per cent.) was prepared by suspending 2 g of o-phthalaldehyde in 100 ml of water in a loosely stoppered conical flask and warming it in a boiling water bath for 2 to 3 minutes; the flask was then removed from the bath and stoppered tightly, and the contents were shaken vigorously. By repeated warming and mixing most of the residue in the flask dissolved in the water, although a small amount of yellow oily material remained adhered to the surface of the flask.The contents were then mixed on a mechanical mixer at room temperature for 1 to 2 hours and filtered through Whatman glass-fibre paper (GF/C). The clear filtrate was used directly or stored in a brown bottle for several weeks in the cold room a t about 8 to 10 "C until required. QUANTITATIVE DETERMINATION OF TAURINE WITH 0-PHTHALALDEHYDE- To 1-ml aqueous samples (containing 0.1 to 1.0pmole of taurine) in stoppered tubes, 1 ml of 0.02 M sodium phosphate buffer (pH 6.8) was added and the contents were mixed. The tubes were then placed in an ice-bath for 5 to 10 minutes, 1 ml of freshly prepared 50 per cent. urea solution was added consecutively to all tubes and the contents were mixed.The saturated (0.6 per cent. w/v) ice-cold solution of o-phthalaldehyde was then added in 1-ml amounts to all tubes. After mixing of the contents, the tubes were stoppered and placed in the ice-bath. After 5 minutes, 0.5 ml of 99.6 per cent. acetic acid was added, the contents were mixed immediately by repeatedly inverting the stoppered tubes, and the tubes were again placed in the ice-bath. The solution in the tubes was transferred to a 1-cm cell and the extinction at 560 nm measured against water until it reached a maximum value, which was then recorded. All optical measurements were made within 40 minutes of the addition of the acetic acid, 0 SAC and the authors.GAITONDE AND SHORT 275 and the time required for the extinction to reach the maximum value was between 2 and 3 minutes for those samples taken from the ice-bath 30 to 40 minutes after the addition of acetic acid.For rapid measurement of the highest extinction given by each sample, a limited number of tubes were removed from the ice-bath and left at room temperature, while the previous set of samples were investigated with the spectrophotometer to find their maximum extinctions. The amount of taurine in the sample was determined by reference to a calibration curve. The extinction at 560 nm was proportional to the amount of taurine in the range 0.2 to 1.0 pmole, and that at the isosbestic point at 490nm to the range of taurine from 0.05 to 0.7 pmole (Fig. 1). Taurine/pmole The relationship between the amount of taurine with o-phthalaldehyde when using 50 per cent.urea solution and 0.02 M phosphate buffer (pH 6.8), and the extinction a t 560 nm (-@-.-) and at the isosbestic point, 490 nm, (- 0- 0-) of the purple product formed after the addition of acetic acid Fig. 1. Concentration of o-p h t ha1 a1 de h yde, per cent. The effect of concentration of o-phthalaldehyde solution on the extinction a t 560 nm of the product formed on reaction with taurine (0.5 pmole) when using 0.02 M phosphate buffer (pH 6.8) and 50 per cent. urea solution Fig. 2. RESULTS FACTORS AFFECTING THE REACTION OF TAURINE WITH O-PHTHALALDEHYDE- Concentration of o-phthalaldehyde-The saturated 0.6 per cent. solution of o-phthalalde- hyde was diluted with water. The diluted reagent solutions gave lower extinction values than the saturated solution of the reagent when allowed to react with taurine in the presence of urea and phosphate (Fig.2). Concentration of urea-The typical purple product was formed from taurine after its reaction with o-phthalaldehyde only in the presence of urea. The extinction a t 560nm of the purple product formed on reaction of taurine (0.5 pmole) with o-phthalaldehyde in- creased with increasing concentration of urea, readings being 0.60, 0.63, 0-64, 0.65, 0-65 and 0.67 when using 10, 20, 25, 30, 40 and 50 per cent. urea solution, respectively. However, the stability of the purple product decreased with increasing concentration of urea solution (Fig. 3). The acid reagent for development of the coloztr-The intensity of the colour was greater on acidifying the reaction mixture with acetic acid than with hydrochloric, phosphoric or sulphuric acids.Temperature of incubation-The product formed on acidification of the reaction mixture was purple, provided that the temperature of the reaction mixture was between 0 and 39 "C. At higher temperatures the reaction product was yellow. The purple product was formed at pH values between 2.0 and 2.9.276 GAITONDE AND SHORT : QUANTITATIVE DETERMINATION OF [Analyst, Vol. 96 0.7 0.6 0.5 0.4 0.3 - 410 450 500 550 600 0 10 20 30 40 50 60 Wavelength/nrn Time after adding acetic acid/minutes Fig. 4. The optical spectra of the reac- tion products of taurine (1 pmole) with The effect of concentration of added o-phthalaldehyde when using 50 per cent. urea solution on the extinction a t 560 nm of the urea solution and 0.02 M phosphate buffer product formed on reaction of taurine (0.5 pmole) (pH 6.8) a t 0 "C.The spectra were recorded with o-phthalaldehyde in the presence of 0.06 M using a Unicam SPSOO spectrophotometer phosphate buffer (pH 6.6). A, 10 per cent. urea a t 0.5 minute (A) and a t 8-5 minutes (B) solution and B, 50 per cent. urea solution after the addition of acetic acid Fig. 3. Samples pre-incubated at 0 "C and subsequently acidified gave an orange product , with an extinction peak at 470 nm, which gradually changed into a purple product with maximum extinction at 560 nm (Fig. 4). The development of the purple product was almost complete in 40 to 50 minutes at 0 "C and could be accelerated on warming to between 16 and 21 "C (Fig. 5). The purple colour was stable for 2 to 3 minutes on reaching its maximum extinction at 560 nm (see Fig.3) and was stable for 10 minutes at the isosbestic point between 490 and 500 nm. Samples pre-incubated at higher temperatures (21 to 36°C) gave the purple product with maximum extinction at 560 nm immediately on acidification, its extinction increasing during the first 2 minutes and then showing a slow decrease. 0 20 40 60 80 100 120 140 Time/minutes Fig. 5. Rate of development of purple colour after reaction of taurine (1 pmole) with o-phthalaldehyde when using 0-06 M sodium phosphate (pH 6.4) and 50 per cent. urea solution. Curve A gives the extinction a t 560 nm recorded directly after withdrawal of the reaction mixture from the ice-bath a t different times after the addition of acetic acid.Curve B gives the extinction at 560 nm of the same samples on allowing thc development of the purple colour up to its maximum value in the spectrophotometer .- t; 0.2 *G 0.1 U O 0.02 0.04 0.06 0.08 0.10 0.20 0-30 Molarity of sodium phosphate (pH 6.6) Fig. 6. The effect of phosphate ions (pH 6-6) on the extinction a t 560 nm of the product formed on reaction of taurine (0.5 pmole) with o-phthalaldehyde when using 50 per cent. urea solution (A), and 25 per cent. urea solution (R)April, 19711 TAURINE BY AN O-PHTHALALDEHYDE - UREA REACTION TABLE I EXTINCTION OF THE PRODUCTS OF AMINO COMPOUNDS ON TREATMENT WITH O-PHTHALALDEHYDE Amino compound Taurine . . . . . . Homotaurine .. .. H ypotaurine .. .. Glycine . . . . . . Lysine . . . . ..6-Hydroxylysine . . . . y-Aminobutyric acid . . /3-Alanine . . . . . . Ethanolamine . . .. Ornithine . . . . . . Cysteamine . . . . . . Cystamine . . .. .. Tyramine . . . . . . 3-Hydroxytyramine (dopamine) Phenvlethvlamine . . Tryptamiie . . . . 5-Hydroxytryptamine 5-H ydroxytryptophan Histamine . . . . N-E-Methyllysine . . a-Alanine . . .. Arginine . . .. Aspartic acid . . Asparagine . . . . Citrulline . . .. Cysteine hydrochloride S-Methylcysteine , . Penicillamine .. Cysteic acid .. Cysteine sulphinic acid Cystine . . . . Cystine disulphoxide Homocysteic acid . . Glutathione, GSH . . ,, GSSG . . L-Cystathionine . . Methionine . . . . Methionine sulphoxide Methionine sulphone Glutamic acid . . Glutamine . . . . Histidine . . . . l-Methylhistidine . . 3-Methylhistidine .. Proline . . . . Serine .. .. Homoserine . . . . Threonine . . . . Leucine . . . . Isoleucine . . . . Valine . . .. Diaminopimelic acid Phenylalanine . . Tyrosine . . . . Ammonium chloride Ergothioneine . . Glucosamine .. Tryptophan . . .. Homocystine .. .. .. .. .. . . .. .. .. .. .. .. .. .. .. .. .. .. . . .. . . .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. . . .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. . . . . .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. . . .. .. .. .. .. .. .. .. . . . . .. .. .. Amax 560 560 500 560 560 560 560 560 560 560 560 560 520 to 570 530 to 630 510 to 560 480 to 495 480 450 475 560 - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - Extinction a t 560 nm - a t o oc 1.24 0.75 0.32 0-97 1-05 1.00 0.32 0-58 0.40 0.26 0.27 0.46 0.27 0.57 0.41 0.17 0.33 0.07 0.15 0.12 0.08 0.05 0.06 0-04 0.05 0.05 0.04 0.09 0.05 0.04 0.02 0.04 0.11 0.04 0.04 0.10 0.11 0-04 0-05 0.04 0.04 0-04 0.02 0.02 0.02 0.05 0.06 0.04 0.04 0.03 0.06 0.06 0.04 0.04 0.03 0.02 0.02 - - a t 36 "C 1-13 0.66 0.23 0.22 0.72 0.28 0.12 0-24 0-14 0.30 0.65 0.69 0.25 0.34 0.16 0.11 0-26 0-16 0-28 0.2 1 0-33 0.17 0.1 1 0-27 0.14 0.09 0-30 0.34 0.27 0.27 0-29 0.20 0.36 0-17 0-20 0.2 1 0.3 1 0.12 - - - - - - - - - 0.12 1.19 0.13 - - - - 0-26 0.34 0-05 0.02 - Each compound (1 pmole) was treated with 0-6 per cent.of o-phthalaldehyde solution while using 50 per cent. urea solution and 0.06 M sodium phosphate solution (pf-I.6.4) a t 0 "C for 5 minutes or a t 36 "C for 30 minutes.The reaction mixture was acidified with acetic acid and the maximum extinction value was recorded. 277278 GAITONDE AND SHORT : QUANTITATIVE DETERMINATION OF [Arcalyst, Vol. 96 Molarity and $H of the phosphate bu$er--The presence of small amounts of phosphate ions was necessary for the formation of the purple product but phosphate buffer solutions of higher molarity (greater than 0-1 M phosphate) gave a considerable decrease in the extinction of the purple solution (Fig. 6). The extinction was highest if the reaction mixture was incubated at a pH between 6.5 and 7.2 (Fig. 7 ) . A reagent of 0.02 M phosphate buffer (pH 698) was chosen for use in the quantitative determination of taurine. 0.7 0.6 PH Fig. 7. The effect of 0.02 M and 0.06 M sodium phosphate buffers the extinction a t 560 nm of the product formed on reaction of taurine (Oi5 .pmole) with o-phthalaldehyae when using 50 per cent.urea solution. A, 0 . 0 2 ~ sodium phosphate buffer and B, 0.06~ sodium phosphate buffer The presertce of chloride iouts-Samples of taurine solutions up to 0.05 M in sodium chloride However, higher concentrations of the did not show any appreciable interference effect. chloride ions gave a decrease in the extinction of the purple product. REACTION OF AMINO COMPOUNDS WITH 0-PHTHALALDEHYDE IN THE PRESENCE OF UREA- Several amino compounds were treated with o-phthalaldehyde and 50 per cent. urea solution at 0 or 36 "C. After 5 minutes, the reaction mixture was acidified with acetic acid, and the extinction maximum of the product and its extinction at 560nm were recorded (Table I).The first twelve amino compounds, except hypotaurine, listed in Table I gave products with extinction maxima a t 560 nm. Hypotaurine gave an extinction maximum at 500 nm, with an extinction of 1.26 under the conditions described in Table I. The products of other amino compounds had extinction maxima below 490 nm or had a wide absorption band between 510 and 630nm. For the quantitative determination of taurine, the reaction with o-phthalaldehyde a t 0 "C was preferred to that at 36 "C because (i) under the conditions used several a-aminocar- boxylic acids gave a yellow product ( E ~ ~ ~ = 2 250) at 36 "C but no coloured product at 0" C. The extinction of the products at 560 nm was less when these a-aminocarboxylic acids were reacted at 0 "C than at 36 "C; (ii) the calibration curve was a straight line; and (iii) the procedure is applicable under certain conditions to the determination of lysine, hydroxylysine and glycine (e.g., in protein hydrolysates). Lysine gave a brownish-black product (A,,, 450) and glycine a yellowish green product (A,,, 450) on treatment with o-phthalaldehyde.EXTRACTION OF THE REACTION PRODUCTS WITH CHLOROFORM- After optical measurement the reaction mixture was equilibrated with 4 ml of ch1oroform.l The purple products formed from glycine, cysteamine and cystamine were found to be extractable with chloroform, in which the products of the two sulphur-containing amino-acids were stable for 16 to 20 hours. This property can be utilised in the detection of these acids in the presence of other sulphur-containing amino-acids.The reaction products of tryptamine and phenylethylamine were extractable into the chloroform phase but those given by 5-hydroxytryptamine, tyramine and 3-hydroxytyramine remained predominantly in the aqueous phase.April, 19711 TAURINE BY AN 0-PHTHALALDEHYDE - UREA REACTION 279 MECHANISM OF REACTION OF TAURINE WITH 0-PHTHALALDEHYDE IN THE PRESENCE OF UREA- Taurine also gave rise to a purple product if urea solution in the reaction mixture was replaced by thiourea solution (10 per cent. w/v), acetamide solution (50 per cent. w/v) or glutamine solution (5 mM), but not if urea solution was replaced by acetone or ethyl methyl ketone. It was found that under conditions in which a mixture of urea and phosphate was pre- incubated for 5 minutes with taurine and o-phthalaldehyde, or with taurine and subsequently o-phthalaldehyde, or with o-phthalaldehyde and subsequently taurine, the reaction product formed on the addition of acetic acid was yellow - orange, changing with time into a purple product.The purple product was not formed if phosphate was omitted from the incubation mixture, or if taurine was omitted from the incubation mixture but added to the acidified reaction mixture. The following mechanism, which is consistent with the experimental findings, is proposed : (i) urea + phosphate + o-phthalaldehyde -+ [A] (ii) [A] + taurine--+ [B] (iii) [B] + acid -+ [C] -+ [D] -+ [El orange purple yellow It may be assumed that urea, thiourea and glutamine form addition products [A] similar to those reported for a~etamide.~ 0:;: + NHzCONH;, - Q - F H N-CO-NH2 0”H ‘H 0-p ht ha lalde h yd e urea N-arnido-1,3 -dihydroxyisoindoline The compound [A] is then converted into the purple product according to the mechanism postulated in reactions (ii) and (iii) above.Phosphate probably acts as a catalyst in the formation and stabilisation of the reaction product [A]. In view of the fact that glutamine can replace urea in the reaction it appears that only one amid0 group of urea is reacting with o-phthalaldehyde. In general, several amino compounds of the type NH2-CH2-R, where R is -COOH or an aliphatic carbon chain (e.g., taurine, homotaurine, lysine, glycine, cystamine, cysteamine, y-aminobutyric acid, p-alanine, ornithine, ethanolamine or butylamine) , gave purple products on treatment with o-phthalaldehyde at 0 “C; hyptotaurine gave a pink product.An intense purple product was also given by isopropylamine. a-Aminocarboxylic acids, except those mentioned above, gave no purple products on acidification of their reaction products with o-phthalaldehyde. In experiments in which urea was not present during the incubation but was added at the end of 5 minutes of incubation and then allowed to react for 1 to 5 minutes, no purple product resulted on subsequent acidification. This observation, which showed that taurine also reacts with o-phthalaldehyde in the absence of urea, was also found to be dependent on the presence of phosphate ions in the incubation mixture.The reaction mechanism may be similar to the one postulated above for urea. The addition compound so formed is probably responsible for fluorescent products reported for several amines (and a-aminocarboxylic acids) by earlier workers5 to lo APPLICATION OF THE METHOD TO TISSUE EXTRACTS The method can be applied directly to the determination of taurine (and homotaurine) in the in vitro assay of enzymes involved in metabolism with methionine and other sulphur- containing amino-acids. Taurine is present in large amounts in rat brain and certain other tissues. Its concentration in tissue extracts was determined by the present method after removal of y-aminobutyric acid, glycine and lysine, which interfere to some extent, depending on their concentration in the tissue extract.Rat brain was homogenised in ice-cold 5 per cent. w/v perchloric acid or 10 per cent. w/v trichloroacetic acid. The suspension was centrifuged and the clear extract decanted, after which the tissue residue was washed once with the same acid. The washing was combined with the main extract, which was then neutralised to precipitate perchlorate ions or washed three times with ether to remove tri-280 GAITONDE AND SHORT chloroacetic acid. The aqueous extract was filtered into a calibrated cylinder and made up to a suitable volume (e.g., 25ml). A fraction containing taurine that was free from inter- fering amino-acids was obtained from the extract by one of the following two procedures. In the first, a portion of the aqueous extract (10 to 20 m1) was passed through a two- column assembly consisting of Zeo-Karb 225 (Hf form, 10 cm) fitted on top of Dowex 1 X-10 (carbonate form, 12 cm).After the passage of the sample, the column assembly was washed with 50ml of water. The Dowex 1 column was disconnected from the assembly; taurine adsorbed on this resin was eluted with 0.1 N acetic acid under pressure of nitrogen gas until all the carbonate was exchanged with acetate and free acid emerged from the column. The neutral eluate was evaporated to dryness and the residue dissolved in 5 ml of water. In the alternative procedure a portion of the aqueous tissue extract (10 to 20ml) was passed through a two-column assembly consisting of Zeo-Karb 225 (H+ form, 10 cm) fitted on top of Dowex 1 X-10 (acetate form, 12 cm).The effluent emerging from the column assembly contained taurine and neutral compounds, which were collected quantitatively by washing the column assembly with water (50 ml) . The effluent (@us washings) was evaporated to dryness and the residue dissolved in 5 ml of water. Because chloride ions inhibit the o-phthalaldehyde - urea reaction, the substitution of Dowex 1 in the acetate form with a Dowex 1 column in the chloride form should be avoided. If it nevertheless occurs, the chloride ions (washed out as hydrochloric acid) should be removed by repeated evaporation of the effluent fraction. Homotaurine, when present in the tissue extracts, was found in the taurine fraction on ion-exchange chromatography. The taurine fraction obtained by these two procedures con- tained phosphoethanolamine and, probably, glycerylphosphorylethanolamine.They were resolved on paper chromatograms developed with acetone - ethyl methyl ketone - water (2 + 2 + l), which gave relative RF values, with respect to that of taurine, of 0.32 for phospho- ethanolamine and 0-55 for glycerylphosphorylethanolamine. Moreover, the fraction obtained by the second procedure also contained free sugars. None of these compounds interfered in the determination of taurine in the tissue extracts. A mean value of 6.76 +_ 0.09 (s.e.m.) prnole of taurine per gram of brain was obtained for Wistar albino rats of 100-g body weight. From the same tissue extracts taurine was isolated by combined ion-exchange and paper Chromatography. Taurine was eluted from paper chromatograms and the eluate, after removal of interfering volatile materialll and reaction with ninhydrin,12 gave a mean value of 6.88 0.16 (s.e.m.) pmole of taurine per gram of brain.The present value may be compared with a value of 6.94 pmole per gram of brain for rats of 73-g body weight.13 An inspection of the values reported for taurine content of brains of rats of different body weights shows a wide scatter (this has been reviewedl4). This suggests that, besides age, other factors such as strain and nutritional state of the animal may also affect the taurine content of the brain. As all the interfering amino compounds are adsorbed on the cation-exchange resin (Zeo-Karb 225, H+ form), the use of a second column of anion-exchange resin is not necessary in enzyme assay systems. The effluent emerging from the cation-exchange resin is either assayed for taurine directly, or after concentration of the material by evaporation. 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. REFERENCES Klein, G., and Linser, H., Hoppe-Seyler’s 2. physiol. Chem., 1932, 205, 251. Patton, A. R., J . Biol. Chem., 1935, 108, 267. Curzon, G., and Giltrow, J., Nature, 1954, 173, 314. Reynolds, R. D., and Conboy, R. J., J . Org. Chem., 1965, 30, 2251. Shore, P. A., Burkhalter, A., and Cohn, V. H., jun., J . Pharmac. Exp. Ther., 1959, 127, 182. Juhlin, L., and Shelley, W. B., J . Histochem. Cytochem., 1966, 14, 525. Cohn, V. H., jun., and Shore, P. A., Analyt. Biochem., 1961, 2, 237. Kremzner, L. T., Ibid., 1966, 15, 270. Rogers, C. J., Chambers, C. W., and Clarke, N. A., Ibid., 1967, 20, 321. Sen, N. P., Somers, E., and O’Brien, R. C., Ibid., 1968, 26, 457. Gaitonde, M. K., Dahl, D. R., and Elliott, K. A. C., Biochem. J., 1965, 94, 345. Sorbo, B., Clinica Chim. Acta, 1961, 6, 87. Garvin, J. E., Archs. Biochem. Biophys., 1960, 91, 219. Gaitonde, M. K., in Lajtha, A., Editor, “Handbook of Neurochemistry,” Volume 111, Plenum Received August 3rd, 1970 Accepted September 30th, 1970 Press, New York, 1970, p. 225.