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.