Ion-exchange column chromatographic method for simultaneous separation and determination of 5-hydroxyindoles

Ion-exchange column chromatographic method for simultaneous separation and determination of 5-hydroxyindoles

314 SHORT COMMUNICATIONS 10. KLAUS, R., Pharm. Ztg. 112, 480 (1967). 11. MALINS, D. C., AND MANGOLD, H. K., J. Amer. Oil Chem. Sot. 37, 576 (1960). ...

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314

SHORT COMMUNICATIONS

10. KLAUS, R., Pharm. Ztg. 112, 480 (1967). 11. MALINS, D. C., AND MANGOLD, H. K., J. Amer. Oil Chem. Sot. 37, 576 (1960). 12. STAHL, E., Anger. Chem. Int. Ed. Engl. 3, 784 (1964). 13. STAHL, E., L‘Diinnschicht-Chro,matographic,” 2nd ed. Springer-Verlag, Berlin/ New York, 1967. 14. TURINA, S., SOIJIC, Z., AND MARJANOVIC, V., J. Chromatogr. 39, 81 (1969). NICOL~~S G. BAZLN, JR.~ STEVE CELLIK Department University

of Biochemistry

of

Toronto,

and

Toronto,

Clarke Canada

Institute

of

Psychiatry

and Znstituto de Investigaciones l&q&micas Universidad National de1 Sur Bahia Blanca, Argentina Received

December

9, 1970

1 Address all correspondence to : Institute de Investigaciones versidad National de1 Sur, Avenida Alem 1253, Bahia Blanca,

Ion-Exchange Simultaneous

Column

Chromatographic

Separation

and

Bioquimicas, Argentina.

Method

Uni-

for

Determination

of 5Hydroxyindoles

It has been shown that 5-hydroxy-L-tryptophan (5HTP), 5-hydroxytryptamine (5HTPA), and 5-hydroxyindole-3-acetic acid (5HIA) are biosynthesized from L-tryptophan in the brain and small intestine of some mammals (1-12). Although some procedures for the determination of each 5-hydroxyindole from biological materials have been published (X3-15)) there are few or no reports on the quantitative analysis of all these physiologically important tryptophan metabolites together. In this communication, a convenient and reproducible ion-exchange chromatographic method is described for the separation and determination of the three 5-hydroxyindoles. Macroscale Determination. The mixture (0.1 mg, each) of 5HTP, 5HTPA, and 5HIA in 5 ml water (pH 6.8) was applied to a column (1 x 7 cm) of Dowex 1 (acetate form). The column was washed with 100 ml water, 100 ml 0.01 N acetic acid, and 100 ml 6 N acetic acid. The effluent and all three eluates were fractionated in 5 ml fractions and the optical density of each fraction at 275 rnp was measured. As shown in @ 1972 by Academic Press, Inc.

SHORT

b

315

COMMUNICATIONS

0.01 -+-ACETIC+-ACID

WTER

N .&:TI[:r(

.4CIr! SHIA

0.3SHTPA

0

511TP

50

100

150

ELIfATE FIG. 1. Elution behavior of 5-hydroxyindoles on column of Dowex 1 (acetate form).

20ll

250

3nn

\‘C)LlNE

(ml)

during

ion-exchange

chromatography

Fig. 1, 5HTPA appeared in the mixture (F-I) of the effluent and water eluate, 5HTP in 0.01 N acetic acid eluate (F-II), and 5HIA in 6 N acetic acid eluate (F-III). For the calculation of the recovery rate of the 5-hydroxyindoles, the molar absorption coefficients at 275 mp were taken as 5200 for 5HTP in 0.01 N acetic acid, 5800 for 5HTPA in water, and 6400 for 5HIA in 6 N acetic acid, respectively. The recovery rate of each 5-hydroxyindole was nearly complete. Microscale Determination. The mixture of small amounts (30-600 nmoles) of the three 5-hydroxyindoles in 4 ml water (pH 6.8) was passed through a column (1 X 7 cm) of Dowex 1 (acetate form) and

Recovery

TABLE 1 of 5-Hydroxyindoles

5HTP

Added (nmoles) 30 90 150 210 300 450 600

5HTPA Recovery rate (7%)

Added (nmoles)

95.0 97.7 99.7 100.1 100.3 97.6 95.4

30 90 150 210 300 450 600

5HTP, 5-hydroxy-ttryptophan. indole-a-acetic acid.

SHTPA,

5HIA Recovery rat,e (%) 96.2 96.7 96.6 98.7 99.4 96.6 95.5

5-hgdroxytryptamine.

Added (nmoles)

Recovery rate (%I

30 90 150 210 300 450 600

97.5 96.6 94.8 98.2 99.6 100.2 96.4 5HIA,

5-hydroxy-

316

SHORT

COMMUNICATIONS

eluted as described above. Each fraction was evaporated to dryness in vacua at 40’ t 2°C under nitrogen gas and 5-hydroxyindole in each residue were determined by the nitrosonaphthol reaction of Udenfriend, Weissbach, and Clark (13,14), Table 1 shows the recovery rate for 5HTP, 5HTPA, and 5HIA over the range of 30-600 nmoles. The mean per cent recovery was: for 5HTP, 98.0%, with a range of 95-100.3%; for 5HTPA, 97.1%, with a range of 96.2-99.4s; and for 5HIA, 97.6%, with a range of 97.5-100.2oJo. This method is reproducible and useful for the determination of 5HTP, 5HTPA, and 5HIA present together in biological fluids. REFERENCES 1. GRAHAME-SMITH, D. G., B&hem. Biophys. Res. Commun. 16, 586 (1964). 2. GRAHAME-SMITH, D. G., Biochem. J. 92, 529 (1964). 3. GRAHAME-SMITH, D. G., AND MOLONEY, L., Biochem. J. 96, 66 (1965). 4. GREEN, H., AND SAWER, J. L., Anal. Biochem. 15,539 (1966). 5. GAL, M., ARMSTRONG, J. C., AND GINSBERG, B., J. Neurochem. 13, 643 (1966). 6. LOVENBERG, W., JEQTJIER, E., AND SJOERDSMA, A., Science 155,217 (1967). 7. JEQUIER, E., LOVENBERG, W., AND SJOERDSMA, A., Mol. Pharmacol. 3, 274 (1967). 8. GRAHAME-SMITH, D. G., Biochem. J. 605,35 (1967). 9. HAKANSON, R., AND HOFFMAN, G. J., Biochem. Pharmacol. 16,1677 (1967). 10. ICHIYAMA, A., NAKAMURA, S., NISHIZUKA, Y., AND HAYAISHI, O., J. Biol. Chem.

245, 1699 (1970). 11. COOPER, J. R., AND MELCER, I., J. Phamzacol. Exp. Ther. 132, 265 (1961). 12. N~CXJCHI, T., NISHINO, M., AND KIDO, R., Life Sci. 10,583 (1971). 13. CLARK, C. T., WEBSBACH, H., AND UDENFRIEND, S., J. Bid. Chem. 210, 139 (1954). 14. UDENFRIEND, S., WEISSBACH, H., AND CLARK, C. T., J. Biol. Chem. 215, 337 (1955). 15. WELCH, A. S., AND WELCH, B. L., Anal. Biochem. 30, 161 (1969). MIHO NISHINO TOMOO NOGUCHI RYO KIDO Department of Biochemistry Wakayama Medical College Wakaycsma 640, Japan Received

Simple

March

94,1971

Gel

Electrophoresis

of Achromobacter

Procedure fkcheri

Nitrite

for

Purification

Reductase

The purification and properties of the nitrite reductase of Achromobatter fischeri has been described in an earlier communication (1). The @ 1972 by Academic

Press, Inc.