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Urinary excretion of o-tyramine
105
Clinica Chimico Acta 51 (1974) 105-107 @ Elsevier Scientific Publishing Company, Amsterdam - Printed in The Netherlands
SHORT COMMUNICATION CCA 6183
URINARY
EXCRETION
OF o-TY RAMINE
G.S. KING, B.L. GOODWIN, C.R.J. RUTHVEN and M. SANDLER Bernhard Baron Memorial Research Laboratories and Institute of Obstetrics and Gynaecology, Queen Charlotte’s Maternity Hospital, Goldhawk Road, London W6 OXG (7%K.) (Received October 10, 1973)
The presence of p-tyramine in urine is well known [1,2] and m-tyramine excretion has similarly been demonstrated [l-4]. o-Tyramine, however, has not, to our knowledge, been detected as a normal urinary metabolite although Jepson et al. [l] identified it in human urine after monoamine oxidase blockade. Large amounts of o-hydroxyphenylacetic acid are excreted in phenylketonuria [5] ; whilst this compound is thought to derive largely from the action of p-hydroxyphenylpyruvate oxidase on phenylpyruvic acid [6] it seemed important to establish whether it might also derive from an alternative metabolic route, the oxidative deamination of o-tyramine. Appropriate samples were therefore subjected to gas chromatography-mass spectrometry (GC-MS) using a multiple ion monitoring technique (mass fragmentography [ 7]), which offers a very high degree of sensitivity and selectivity for the identification of compounds of this type. Six urine samples from untreated white Wistar rats, about 200 g weight (2-ml portions; about 10% of the 24 h collection) and 2 normal human urine samples (lo-ml portions; about 1% of the 24 h collection) were examined qualitatively and semi-quantitatively for tyramines. Specimens were hydrolysed overnight with O.l-ml portions of a sulphatase-glucuronidase preparation (Sue d’Helix pomatia, Industrie Biologique Francaise, Gennevilliers-en-Seine, France), at pH 5.5 and 37”. Amines were extracted by passage through a 1 cm column of 1 g dry weight of Dowex 50W X 8 [2] followed by elution with 3 M HCl in 50% ethanol. The eluate was evaporated to dryness, borate buffer, pH 11, added and the amines extracted with ethyl acetate. After evaporation they were derivatised with pentafluoropropionic anhydride [ 81, and the derivatives, after thorough removal of anhydride [ 91, examined for o-, m- and p-tyramine using an LKB 9000s GC-MS on a 1% OVl column at 145”. This was done with the multiple ion monitor unit [lo] by continuously monitoring at 24 eV the ions at m/e 266 and 253, which are present as strong peaks in the mass spectra of authentic samples of the three compounds (Fig. 1). A compound in rat urine extracts gave a response at both m/e values and had the same retention time as authentic o-tyramine. The peak (Fig. 2) corre-
Fig. 1. Mass spectra of: (A) o-Tyramine pentafluoropropionate; (C) p-Tyramine pentafluoropropionate.
(B) m-Tyramine pentafluoropropionate;
sponded to a daily excretion of about 1 ,ug of o-tyramine. We were unable to detect it in normal human urine samples even at a sensitivity corresponding to 0.5 pg/24 h. All the urine extracts contained m- and p-tyramine (about 5 pg/24 h and 200 pg/24 h, respectively, in rats and 20 pg/24 h and 1 mg/24 h, respectively, in man.
107
Human
urine
Standards
A
266 t 253[401 t 0
2
L
6
8
10
Retentiontime[minsl Fig. 2. Detection
of o-, m-
and p-tyramine
(A, B and C. respectively)
in urine by
fragmentograpby.
References P. Zaltzman, J.A. Oates, A. Sjoerdsma and S. Udenfriend. Biochem. 1 J.B. Jepson, W. Lovenberg, (1960) 5P. 2 Y. Kakimoto and M.D. Armstrong, J. Biol. Chem., 237 (1962) 208. 3 R.F. Coward, P. Smith and OS. Wilson, Clin. Chim. Acta. 9 (1964) 381. 12 (1963) 358. 4 T.L. Perry and W.A. Schroeder, J. Chromatog.. J. Biol. Chem., 213 (1955) 797. 5 M.D. Armstrong, K.N.F. Shaw and K.S. Robinson, 6 K. Taniguchi and M.D. Armstrong, J. Biol. Chem.. 238 (1963) 4091. 73 (1972) 401. I A.E. Gordon and A. Frigerio, J. Chromatog., 8 K.P. Won& C.R.J. Ruthven and M. Sandier. Clin. Chim. Acta, 47 (1973) 215. 9 G.S. King and M. Sandier. Clin. Chim. Acta, 49 (1973) 295. Isle of Elba, Italy, 1972. 10 C.G. Hammar, in A. Frigerio (Ed.), “Proc. Int. Symp. on GC-MS”, bwini Editore, Milano. p. 5.
J., 74
Tam-
Clinica Chimico Acta 51 (1974) 105-107 @ Elsevier Scientific Publishing Company, Amsterdam - Printed in The Netherlands
SHORT COMMUNICATION CCA 6183
URINARY
EXCRETION
OF o-TY RAMINE
G.S. KING, B.L. GOODWIN, C.R.J. RUTHVEN and M. SANDLER Bernhard Baron Memorial Research Laboratories and Institute of Obstetrics and Gynaecology, Queen Charlotte’s Maternity Hospital, Goldhawk Road, London W6 OXG (7%K.) (Received October 10, 1973)
The presence of p-tyramine in urine is well known [1,2] and m-tyramine excretion has similarly been demonstrated [l-4]. o-Tyramine, however, has not, to our knowledge, been detected as a normal urinary metabolite although Jepson et al. [l] identified it in human urine after monoamine oxidase blockade. Large amounts of o-hydroxyphenylacetic acid are excreted in phenylketonuria [5] ; whilst this compound is thought to derive largely from the action of p-hydroxyphenylpyruvate oxidase on phenylpyruvic acid [6] it seemed important to establish whether it might also derive from an alternative metabolic route, the oxidative deamination of o-tyramine. Appropriate samples were therefore subjected to gas chromatography-mass spectrometry (GC-MS) using a multiple ion monitoring technique (mass fragmentography [ 7]), which offers a very high degree of sensitivity and selectivity for the identification of compounds of this type. Six urine samples from untreated white Wistar rats, about 200 g weight (2-ml portions; about 10% of the 24 h collection) and 2 normal human urine samples (lo-ml portions; about 1% of the 24 h collection) were examined qualitatively and semi-quantitatively for tyramines. Specimens were hydrolysed overnight with O.l-ml portions of a sulphatase-glucuronidase preparation (Sue d’Helix pomatia, Industrie Biologique Francaise, Gennevilliers-en-Seine, France), at pH 5.5 and 37”. Amines were extracted by passage through a 1 cm column of 1 g dry weight of Dowex 50W X 8 [2] followed by elution with 3 M HCl in 50% ethanol. The eluate was evaporated to dryness, borate buffer, pH 11, added and the amines extracted with ethyl acetate. After evaporation they were derivatised with pentafluoropropionic anhydride [ 81, and the derivatives, after thorough removal of anhydride [ 91, examined for o-, m- and p-tyramine using an LKB 9000s GC-MS on a 1% OVl column at 145”. This was done with the multiple ion monitor unit [lo] by continuously monitoring at 24 eV the ions at m/e 266 and 253, which are present as strong peaks in the mass spectra of authentic samples of the three compounds (Fig. 1). A compound in rat urine extracts gave a response at both m/e values and had the same retention time as authentic o-tyramine. The peak (Fig. 2) corre-
Fig. 1. Mass spectra of: (A) o-Tyramine pentafluoropropionate; (C) p-Tyramine pentafluoropropionate.
(B) m-Tyramine pentafluoropropionate;
sponded to a daily excretion of about 1 ,ug of o-tyramine. We were unable to detect it in normal human urine samples even at a sensitivity corresponding to 0.5 pg/24 h. All the urine extracts contained m- and p-tyramine (about 5 pg/24 h and 200 pg/24 h, respectively, in rats and 20 pg/24 h and 1 mg/24 h, respectively, in man.
107
Human
urine
Standards
A
266 t 253[401 t 0
2
L
6
8
10
Retentiontime[minsl Fig. 2. Detection
of o-, m-
and p-tyramine
(A, B and C. respectively)
in urine by
fragmentograpby.
References P. Zaltzman, J.A. Oates, A. Sjoerdsma and S. Udenfriend. Biochem. 1 J.B. Jepson, W. Lovenberg, (1960) 5P. 2 Y. Kakimoto and M.D. Armstrong, J. Biol. Chem., 237 (1962) 208. 3 R.F. Coward, P. Smith and OS. Wilson, Clin. Chim. Acta. 9 (1964) 381. 12 (1963) 358. 4 T.L. Perry and W.A. Schroeder, J. Chromatog.. J. Biol. Chem., 213 (1955) 797. 5 M.D. Armstrong, K.N.F. Shaw and K.S. Robinson, 6 K. Taniguchi and M.D. Armstrong, J. Biol. Chem.. 238 (1963) 4091. 73 (1972) 401. I A.E. Gordon and A. Frigerio, J. Chromatog., 8 K.P. Won& C.R.J. Ruthven and M. Sandier. Clin. Chim. Acta, 47 (1973) 215. 9 G.S. King and M. Sandier. Clin. Chim. Acta, 49 (1973) 295. Isle of Elba, Italy, 1972. 10 C.G. Hammar, in A. Frigerio (Ed.), “Proc. Int. Symp. on GC-MS”, bwini Editore, Milano. p. 5.
J., 74
Tam-