545

Clinica Chimica Acto, 69 (1976) 545-546 0 Elsevier Scientific Publishing Company,

Amsterdam

- Printed in The Netherlands

SHORT COMMUNICATION CGA 7855

DETERMINATION OF URINARY CHROMATOGRAPHY *

G. CASALE Euratom

(Received

PSEUDOURIDINE

BY GAS

** and G.B. GERBER

and Department

of Radiobiology,

C.E.N.S.C.K.,

B-2400

iVIol (Belgium)

February 4, 1976)

Pseudouridine, a specific constituent of transfer RNA, is a metabolic endproduct and its determination can indicate the turnover of transfer RNA. Increased levels of urinary pseudouridine have been observed after irradiation [l], as well as in leukemia and various metabolic disorders [5,6]. Determination of pseudouridine by column chromatography followed by paper chromatography is tedious and time consuming [ 2,4]. We have, therefore, investigated the possibility of determining pseudouridine by gas chromatography following ion exchange purification and trimethylsilytation. A column of 1 ml Biorex 50 WX 8H’, 200-400 mesh was prepared in a 2 ml plastic syringe (as described elsewhere), and 0.5 ml of urine was slowly passed through the column (< 0.5 ml/min), followed by 1 ml of water. In the case of the rat and particularly of the mouse, previous deproteinisation (e.g. by heating to 100°C) is required. If pseudouridine is determined together with the whole spectrum of indicators of radiation damage, 10 ml of urine can be passed through a 10 ml column washed with 15 ml water as described earlier [2] and a l-ml aliquot of this eluate can be assayed. An 0.3 ml aliquot of the eluate corresponding to 0.1 ml of urine is then dried under a stream of air at room temperature. As an internal standard, 20 ~1 of a 1% ethanolic solution of arachidic acid is added. Residual acid is removed by repeated evaporation with methanol and finally, water is azeotroped by heating with dichloromethane. Pseudouridine is silylated in screw-capped, teflon-lined tubes with 100 ~1 of BSTFA and 100 ~1 of pyridine, and 5 ~1 are injected into the gas chromatograph. A Hewlett Packard model 5700A equipped with a flame ionisation detector and a 1180 A Hewlett Packard In-

* Publication **

Euratom

No. fellow.

1246

of the

Euratom

Biology

Division,

Contract

0.95-72-18

IOC.

546 TABLE

I

EXCRETION

OF PSEUDOURIDINE

BY DIFFERENT

SPEClES

The percentage found after addition of pseudouridine is calculated on the basis of known pseudouridine added to urine and confirmed by experiments using radioactive pseudouridine.

pg/mgcreatinine (mean Man (10 samples) Rat (8 samples) Mouse (6 samples)

* S.D.)

57 i 16 108 * 35 198 + 45

amounts

of

Percent found after addition of 200 pg pseudouridine/ml urine (mean f S.E.) 85 + 7 80 +_9 91 ?- 8

tegrator were utilized. The 2 m X l/S inch internal diameter glass column was filled with 3% SP2100 on Supelcoport 100/120 mesh and was kept at 192°C. The temperature of the detector and of the injection port was 250°C the rate of the carrier gas was 22 ml/min. Pseudouridine was eluted after approx. 6.5 min and could be determined quantitatively by comparison with standards added to urine and carried through the procedure. Studies using radioactive pseudouridine confirmed that the recovery was in the order of 80% (Table I). The values observed for pseudouridine in urine from man, mouse and rat (Table I) correspond to the values found for the same sample using a column paper chromatographic procedure [ 2 1. The standard deviation between animals is also indicated in the table. The standard deviation of the method is in the order of 12%. The determination can be carried out in less than one day, the most timeconsuming procedure being the evaporation. References 1 2 3 4 5 6

Drabovsky, D., Winkler, A. and Skoda, J, (1965) Neoplasma 12,561 Gerber, G.B. and Decocq, J.P. (1975) Acta Radiol. 13. 556 Goldwasser, E. and Heinrickson, R.L. (1966) Progr. Nucl. AC. Res. Mol. Biol. 5.349 Rindorknecht, H. and Rinderknecht, J.F. (1965) J. Lab. Clin. Med. 65.1034 Weissman, S., Eisen, A.Z. and Karen, M. (1962) J. Lab. Clin. Med. 59,852 Weissman, S.. Lewis, M. and Karen, M. (1963) Blood 6.657

Determination of urinary pseudouridine by gas chromatography.

545 Clinica Chimica Acto, 69 (1976) 545-546 0 Elsevier Scientific Publishing Company, Amsterdam - Printed in The Netherlands SHORT COMMUNICATION C...
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