Clinica Cfiinaico Acta, 80 (1977) 181-185 @ EI~vier/North-Holland Biomedical Press
THE URINARY EXCRETION OF THE NUCLEOSIDES PSEUDOURIDINE AND l-METHYLINOSIN~ DURING NORMAL MENSTRUAL CYCLE
and PEKKA H. MAENPb;;d;
Lfepartment of Biochemistry, (Received
University of Kuopio, Kuopio (Finland)
April 29th, 1977)
Summary The excretion levels of the nucleosides pseudouridine and l-methylinosine were determined by gas-liquid chromatography in 24-h urine specimens from young women during normal menstrual cycle. These nucleosides are derived primarily from transfer RNA and their excretion reflects the turnover of tRNA. The excretion levels were found to be essentially unaltered by the cycle and the average excretion values with standard deviations were 0.70 * 0.078 and 0.051 * 0.011 mg/kg/24 h for pseudou~dine and 1-methylinosine, respectively.
Introduction ~od~ied nucleosides, ~cluding pseudou~d~e and methyla~d derivatives, are found predominantly in transfer RNA and, to a lesser extent, as components of ribosomal and messenger RNA, Pseudouridine is a normal urinary constituent and elevated excretion levels have been reported in patients with different types of malignant diseases [ 11. Improvements in analytical techniques, which include gas-liquid chromatography and high-pressure ion-exchange liquid chromato~aphy, allow identification and accurate qu~ti~tion of other modified nudeosides which are present in urine in much smaller quantities. With these techniques, elevated levels of 1-methylinosine and N2,N2dimethylguanosine have been reported in the urine of patients with advanced cancer and of some patients with diseases other than cancer [ 11. Increased urinary excretion of modified nucleosides reflects changes in activities of tRNA methyltr~sferases, which are greatly elevated in almost every type of tumor examined . tRNA methyltransferases are also under hormonal control . For instance, the activity is one half of that in the normal organ in the uterus of ovsriectomized animals and the administration of physiological doses of estrogen restores the enzyme activity to normal . The aim of the present ~vestigation was to study whether the physiological variation in
steroid secretion during normal menstrual cycle is accompanied by alterations in the urinary excretion of pseudouridine and methylated nucleosides, from which previous data are not available. This would be of interest in evaluating patients with suspected malignancies. Materials and methods Sample
Women volunteers between 17 and 24 years in apparent good health and in no hormonal medication with cycle lengths ranging from 26 to 30 days were studied. All the volunteers carried on with their usual activities during the period of the study. The volume of the 24-h urine specimens was measured, the sample was mixed thoroughly, and ahquot samples were frozen and stored at -20°C until analyzed. The volunteers were on a regular diet eliminating meat during the 24-h collection period. Reagents
Pseudouridine and 1-methylinosine were purchased from Sigma Chemical Co. Internal standards, n-octacosane (CZ8Hs8) and n-tetratriacontane (C&HYO), were from Applied Science Laboratories, Inc. Bis(trimethylsilyl)trifluoroacetamide (BSTFA) was from Fluka A.G. Other reagents were of highest purity available. Preparation
0 f samples
Determination of pseudouridine and 1-methylinosine in urine was performed Commercially by the method of Chang et al.  with some modifications. obtained charcoal was powdered and an SO/l00 mesh fraction was used for the adsorption cleanup procedure. Frozen urine samples were thawed, thoroughly mixed and filtered through a 0.45 pm nitrocellulose filter. For pseudouridine (method A), exactly 1.0 ml of filtered urine was applied to a l-ml charcoal column. After adsorption and washes with distilled water and 1% pyridine, the nucleosides were eluted with pyridine/96% ethanol (3 : 1, v/v). The eluate was concentrated with rotary evaporation at 45°C under reduced pressure and evaporated to dryness at 70°C using nitrogen gas. 1 ml of dichloromethane was added and the sample was evaporated to remove any remaining traces of water. For determination of 1-methylinosine (method B), exactly 1.0 ml of filtered urine was applied to a 0.5-ml charcoal column. After adsorption and washes with distilled water, 0.5 N NaOH, distilled water, 2% pyridine and 1% pyridine, the nucleosides were eluted as in method A. Internal standards were added in heptane to dried samples, 60 and 6 pg/sample of n-octacosane (IS,) and 70 and 7 ,ug/sample of n-tetratriacontane (IS,) in methods A and B, respectively. After evaporation, 0.5 ml of the silylating reagent (BSTFA/CH&N, 1 : 1, in method A and BSTFA/CH2C12/CH3CN, 2 : 1 : 1, in method B) were added and the samples were heated in closed tubes for 15 min at 150°C. Chromatographic analysis of the trimethylsilyl derivative was performed in triplicate within 48 h of the derivatization. Instrumental
A Varian Aerograph
Series 1400 gas chromatograph
with a flame
ionization detector and an automatic sequential temperature programmer was used. The column was an 1.8 m X 2 mm coiled glass tube which was silanized and packed with 3% OV-7 on 80-100 mesh Gas Chrom Q. The instrument settings were as follows: column ~mperatu~ was linearly programmed from 190°C to 310°C at a rate of &J3 C/min. The temperature was subsequently kept at 310°C for 6 min. The injector was at 280°C and the detector at 350°C. The flow rates of nitrogen, hydrogen and air were 40, 25 and 300 ml/mm respectively. The silylated samples were warmed up to 70°C and exactly 3 ~1 were injected. The amounts of nucleosides (N) present in each sample of a 24-h urine collection were calculated as follows [ 53. mg,/sample
RWR = -X-
The relative weight response (RWR) values for p~udou~dine and l-methylinosine were determined by repeated analyses of calibration standards. Measurement of peak areas was used as a method for calculation of the amount of nucleosides. The relative standard deviations for pseudouridine and l-methylinosine in urine samples were about 6%. The statistical significances were determined ‘by Student’s t-test. Results and discussion Figs. 1 and 2 present representative chromatograms obtained for the analysis of pseudouridine and 1-methylinosine in the urine of normal young women using methods A and B. The daily excretion rate of p~udou~dine (Fig. 3) and 1-methylinosine (Fig. 4) during menstrual cycle was found to vary very little. The average amount of urinary pseudouridine and 1-methylinosine was 38.9 and 2.8 mg/24 h with standard deviation values of 6.2 and 0.6. On a body weight basis, the co~esponding values were 0.70 + 0.078 and 0.051 rt 0.011 mg/kg/24 h. Only few data are available on urine levels of modified nucleosides for normal subjects. However, the present values are in good agreement with those obtained in a recent study by gas-liquid chromatography for normal male and female adult control subjects, age 18 to 60, with average excretion values of 0.78 mg/kg/24 h for pseudouridine and 0.04 mg/kg/24 h for l-methylinosine [ 61. N2,N2-Dimethylguanosine is determined simultaneously with l-methylinosine by method B, but due to a large variation of the values in repeated analyses the results are not presented here. This variation may be due to the presence of other methylated guanosine nucleosides in the same chromatographic peak (unpublished observations). The results indicate that although tRNA methyltransferases have been shown to be under hormonal control, especially by steroids, this is not reflected in significant alterations of the urinary excretion levels of pseudouridine and l-methylinosine during normal menstrual cycle. Changes may occur, however,
222 234 210 310 _e-p+ -. _c~_ e4 8 12 16
f?O _ 0
310 “c 20 min
310 s: 20 min
Fig. 1. Representative chromatogram pseudouridine.
for urine analysis derived by method A used in the determination of
Fig. 2. Representative chromatogram l-methylinosine.
for urine analysis derived by method B used in the determination of
60 I 50
Fig. 3. Urinary 24-h pseudouridine excretion values during normal menstrual cycle.
f -- ---~---
Fig. 4. Urinary 24-h I-methylinosine excretion values during normal menstrual cycle.
as a result of more pronounced physiological alterations such as during gravidity or during rapid growth and in patients with diseases of endocrine origin or under hormonal medication. Acknowledgements This work was supported in part by a grant from the Sigrid Jus&us Foundation, Finland. References 1 Waalkes, T.P. and Borek, E. (1976) in Advanced in Tumour Prevention, Detection and Characterization (C. Maltoni. ed), Vol. 3. pp. 15-31. Excerpta Medics, Amsterdam 2 Borek, E. and Kerr, S.J. (1972) Adv. Cancer Res. 15.163-190 3 Sharma, O.K. and Borek, E. (1974) Adv. Ent. Regul. 12,85-102 4 Sham+ O.K., Kerr, S.J., Wiesner, R.L. and Borek, E. (1971) Fed. Proc. 30,167 5 Charm. S.Y.. Lakins, D.B.. Zumwalt. R.W.. Gehrke, C.W. and Waalkes. T.P. (1974) J. Lab. Clin. Med. 83,816-830 6 Waalkes, T.P.. Gehrke, C.W., ZumwaIt, R.W., Charm, S.Y., L&ins. D.B., Tormey. D.C., Ahmann, D.L. and Moertel, C.G. (1975) Cancer 36.390-398