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Direct determination of urinary pseudouridine by high-performance liquid chromatography
ANALYTICAL
BIOCHEMISTRY
170,387-389
(1988)
Direct Determination of Urinary Pseudouridine Performance Liquid Chromatography TETSUYAYAMAMOTO,KAZUYAHIGASHINO, SHINSUKE YOSHIKIAMURO,ANDTOSHIWUHADA
by High-
TAMURA,HIROSHIFLJJIOKA,
The Third Department of Internal Medicine, Hyogo College of Medicine, Mukogawa-cho Nishinomiya Hyogo 663, Japan
I-1,
Received January 13, I987 A reversed-phase high-performance liquid chromatographic method was developed for the determination of pseudouridine in urine. This method does not need pretreatment by boronate affinity gel. Therefore, it can be used in screening patients with malignant disease and for monitoring clinical response to chemotherapy with other tumor markers. Q 1988 Academic Press, Inc.
The urinary excretion of modified nucleosides derived from transfer ribonucleic acid has been reported to increase in patients with different types of cancer (1). Of the modified nucleosides, the excretion of pseudouridine was known to increase most frequently and significantly (2,3). Furthermore, the determination of urinary pseudouridine suggested that it could be used as a tumor marker in the follow-up of neoplastic diseases (4,5). In these studies, the determination of urinary pseudouridine needed pretreatment with boronate affinity gel before high-performance liquid chromatography was used (6). The method is sensitive and accurate for the determination of urinary pseudouridine but tedious because of the need for two-step analysis. We therefore developed the direct determination of urinary pseudouridine by HPLC without using boronate affinity gel. MATERIALS
AND METHODS
Samples. Urine from healthy subjects and patients with radiologically and/or histologically proven primary hepatocellular carcinoma was collected for 24 h and was stored at -20°C until used. The urine was thawed and centrifuged at 800s. A 0.5-ml aliquot of
the supernatant was then diluted fivefold with distilled water, and a 20-~1 aliquot of the diluted urine was loaded onto the column. Apparatus and technique. The chromatographic system consisted of an LC-6A highperformance liquid chromatograph, an SPDdAV uv-vis spectrophotometric detector, and a C-R3A Chromatopac recorder with computer function (all obtained from Shimadzu Co., Kyoto, Japan). The two columns of PBondasphere 5 Cl8 (3.0 X 150 mm, particle size 5 pm) (Waters Assoc., Milford, MA) were combined in series. The mobile phase was 0.02 M KH2P04 adjusted to pH 3.3 with phosphoric acid and the flow rate was 0.5 ml/min. The absorbance intensity was monitored at 254 nm. The calibration curve was obtained between 12.5 and 200 nmol/ml of pseudouridine. In order to compare the results of the determination of urinary pseudouridine by the present method with those by the method of Gehrke et al. (6), purification and determination of urinary pseudouridine was performed according to the method of Gehrke et al. In brief, 1 ml of urine was buffered with 0.3 ml of 2.5 M CH$OONH4 at pH 9.5 and then applied to a 5 X 40-mm column of phenyl boronate affinity gel packed into a glass column, 387
0003-2697188 $3.00 Copyright Q 1988 by Academic Press, Inc. All rights of reproduction in any form reserved.
388
YAMAMOTO
bmol/ml)
(
r=o.ss
ET AL.
dard deviation was below 2.0% at all concentrations between 20 and 100 nmol/ml. These results were comparable with those of Gehrke et al. (6).
Identification of Pseudouridine in Urine
0 1Xld
ZXld
FIG. 1. Relationship between concentration douridine and peak area.
(area )
of pseu-
which had been equilibrated with 0.25 M CHsCOONH4 at pH 8.8. Pseudouridine was eluted with 5 ml of 0.01 M HCOOH, lyophilized to dryness, and redissolved in 1 ml of distilled water. Twenty microliters of this solution was loaded onto the Crs+Bondapak column (Waters Assoc.) of an LC-5A liquid chromatograph, equipped with an SPD-2A uv spectrophotometer and a C-R1 B Chromatopac Recorder (all obtained from Shimadzu Co.). Recovery of pseudouridine was 95% when we measured its concentration using this method. Reagents. Pseudouridine was purchased from Sigma (St. Louis, MO). RESULTS AND DISCUSSION
Linearity (Peak Area as a Function of Pseudouridine Concentration)
As recommended by Krstulovic et al. (7), we investigated the retention time and absorbance ratio in order to identify pseudouridine in a urine sample. Any deviation from the retention time and absorbance ratio expected for pseudouridine may indicate other compounds eluting near to or simultaneously with pseudouridine. The A&AzS4 and A270,254 of the peak in urine having the same retention time as had pseudouridine in standard aqueous solution were 0.49 and 1.07, respectively. These values were identical to those for pseudouridine, indicating that the peak in urine having the same retention time as had pseudouridine was indeed that of pseudouridine.
Comparison between the Concentration of Pseudouridine Measured by the Present Method and That of Gehrke et al. Figure 2 is the chromatogram of a urine sample by the present method. Figure 3 shows a relationship between the concentration of urinary pseudouridine measured by the present method and that measured by the method of Gehrke et al. (6). The correlation
Linearity was tested on standards in aqueous solution at various concentrations, and the linearity was excellent up to 200 nmol/ml for pseudouridine (Fig. 1). The regression line was Y (concentration of pseudouridine) = 0.000201742 X peak area + 0.52 and the correlation coefficient was 0.999.
Detection Limits, Recovery, and Relative Standard Deviation The detection limit of pseudouridine was 7 nmol, recovery was lOO%, and relative stan-
FIG. 2. Chromatogram Pseudouridine.
of urinary pseudouridine.
*,
CHROMATOGRAPHIC
coefficient is 0.95 but urinary pseudouridine method are somewhat the method of Gehrke that recovery by the complete.
DETERMINATION
the concentrations of measured by our higher than those by et al. (6), indicating latter method is not
The Ratio of Pseudouridine/Creatinine in Urine of Normal Subjects and Patients with Hepatocellular Carcinoma We have previously described that the urinary concentration of pseudouridine in patients with primary hepatocellular carcinoma was significantly higher than that found in both normal subjects as well as in patients with liver cirrhosis (8). We therefore reinvestigated using the present method to determine whether or not the same results could be obtained in patients with primary hepatocellular carcinoma. Figure 4 shows the results. Although the samples examined were small, the ratio of pseudouridine/creatinine in urine is significantly higher in patients with primary hepatocellular carcinoma than in normal subjects. The method which we have developed is simple and rapid. In addition, it was comparable with the method of Gehrke et al. (6) in
OF URINARY
g Y
loo-
20. 8f6. (Mean+S
2 D.)
44.7*b?. (Mean+S.D
Fi c B, .5 .c ;;; ;
389
PSEUDOURIDINE
3 )
. . . 50-
8 :
3 5 4 2 h
+
:
:. 3
!
0 (1)
(2)
FIG. 4. Comparison of pseudouridine/creatinine in urine from patients with primary hepatocellular carcinoma and control subjects (1) Control, (2) hepatoma.
precision and accuracy. Tamura et al. (8,5) suggested that serial measurement of urinary pseudouridine concentration in patients with cirrhosis of the liver might serve as an indicator for the early detection of malignant transformation. Therefore, this method can be used in screening patients with malignant diseases and in monitoring therapeutic effects. REFERENCES
.
500 Urinary measured
Pseudouridine by the method
I& (nmol/ml) of Gehrke
et al
Rci. 3. Comparison of concentration of urinary pseudouridine between our method and the method of Gehrkes’ et al. (6).
1. Waalkes, T. P., Gehrke, C. W., Zumvalt, R. W., et al. (1976) Cancer 36,390-398. 2. Salvatore, F., Colonna, A., Constanzo, F., Russo, T., Esposito, F., and Cimiso, F. (1983) Recent Results Cancer Rex 84, 360-377. 3. Salvatore, F., Russo, T., Colonna, A., Cimiso, L., Mazzacca, G., and Cimiso, F. (1983) Cancer Detect. Prev. 6, 531-536. 4. Russo, T., Colonna, A., Salvatore, F., Cimiso, F., Brides, S., and Gurge, C. (1984) Cancer Res. 44, 2567-2570. 5. Tamura, S., Fujii, J., Nakano, T., Hada, T., and Higashino, K. (1986) Clin. Chim. Actn 154, 125-132. 6. Gehrke, C. W., Kuo, K. C., Davis, G. I., and Suits, R. D. (1978) J. Chromafogr. 150,455-476. 7. Krstulovic, A. M., Brown, P. R., and Rosie, D. M. (1977) Anal. Chem. 49,2237-224 I, 8. Tamura, S., Amuro, Y., Nakano, T., Fujii, J., Moriwaki, Y., Yamamoto, T., Hada, T., and Higashino, K. (1986) Cancer57, 1571-1575.
BIOCHEMISTRY
170,387-389
(1988)
Direct Determination of Urinary Pseudouridine Performance Liquid Chromatography TETSUYAYAMAMOTO,KAZUYAHIGASHINO, SHINSUKE YOSHIKIAMURO,ANDTOSHIWUHADA
by High-
TAMURA,HIROSHIFLJJIOKA,
The Third Department of Internal Medicine, Hyogo College of Medicine, Mukogawa-cho Nishinomiya Hyogo 663, Japan
I-1,
Received January 13, I987 A reversed-phase high-performance liquid chromatographic method was developed for the determination of pseudouridine in urine. This method does not need pretreatment by boronate affinity gel. Therefore, it can be used in screening patients with malignant disease and for monitoring clinical response to chemotherapy with other tumor markers. Q 1988 Academic Press, Inc.
The urinary excretion of modified nucleosides derived from transfer ribonucleic acid has been reported to increase in patients with different types of cancer (1). Of the modified nucleosides, the excretion of pseudouridine was known to increase most frequently and significantly (2,3). Furthermore, the determination of urinary pseudouridine suggested that it could be used as a tumor marker in the follow-up of neoplastic diseases (4,5). In these studies, the determination of urinary pseudouridine needed pretreatment with boronate affinity gel before high-performance liquid chromatography was used (6). The method is sensitive and accurate for the determination of urinary pseudouridine but tedious because of the need for two-step analysis. We therefore developed the direct determination of urinary pseudouridine by HPLC without using boronate affinity gel. MATERIALS
AND METHODS
Samples. Urine from healthy subjects and patients with radiologically and/or histologically proven primary hepatocellular carcinoma was collected for 24 h and was stored at -20°C until used. The urine was thawed and centrifuged at 800s. A 0.5-ml aliquot of
the supernatant was then diluted fivefold with distilled water, and a 20-~1 aliquot of the diluted urine was loaded onto the column. Apparatus and technique. The chromatographic system consisted of an LC-6A highperformance liquid chromatograph, an SPDdAV uv-vis spectrophotometric detector, and a C-R3A Chromatopac recorder with computer function (all obtained from Shimadzu Co., Kyoto, Japan). The two columns of PBondasphere 5 Cl8 (3.0 X 150 mm, particle size 5 pm) (Waters Assoc., Milford, MA) were combined in series. The mobile phase was 0.02 M KH2P04 adjusted to pH 3.3 with phosphoric acid and the flow rate was 0.5 ml/min. The absorbance intensity was monitored at 254 nm. The calibration curve was obtained between 12.5 and 200 nmol/ml of pseudouridine. In order to compare the results of the determination of urinary pseudouridine by the present method with those by the method of Gehrke et al. (6), purification and determination of urinary pseudouridine was performed according to the method of Gehrke et al. In brief, 1 ml of urine was buffered with 0.3 ml of 2.5 M CH$OONH4 at pH 9.5 and then applied to a 5 X 40-mm column of phenyl boronate affinity gel packed into a glass column, 387
0003-2697188 $3.00 Copyright Q 1988 by Academic Press, Inc. All rights of reproduction in any form reserved.
388
YAMAMOTO
bmol/ml)
(
r=o.ss
ET AL.
dard deviation was below 2.0% at all concentrations between 20 and 100 nmol/ml. These results were comparable with those of Gehrke et al. (6).
Identification of Pseudouridine in Urine
0 1Xld
ZXld
FIG. 1. Relationship between concentration douridine and peak area.
(area )
of pseu-
which had been equilibrated with 0.25 M CHsCOONH4 at pH 8.8. Pseudouridine was eluted with 5 ml of 0.01 M HCOOH, lyophilized to dryness, and redissolved in 1 ml of distilled water. Twenty microliters of this solution was loaded onto the Crs+Bondapak column (Waters Assoc.) of an LC-5A liquid chromatograph, equipped with an SPD-2A uv spectrophotometer and a C-R1 B Chromatopac Recorder (all obtained from Shimadzu Co.). Recovery of pseudouridine was 95% when we measured its concentration using this method. Reagents. Pseudouridine was purchased from Sigma (St. Louis, MO). RESULTS AND DISCUSSION
Linearity (Peak Area as a Function of Pseudouridine Concentration)
As recommended by Krstulovic et al. (7), we investigated the retention time and absorbance ratio in order to identify pseudouridine in a urine sample. Any deviation from the retention time and absorbance ratio expected for pseudouridine may indicate other compounds eluting near to or simultaneously with pseudouridine. The A&AzS4 and A270,254 of the peak in urine having the same retention time as had pseudouridine in standard aqueous solution were 0.49 and 1.07, respectively. These values were identical to those for pseudouridine, indicating that the peak in urine having the same retention time as had pseudouridine was indeed that of pseudouridine.
Comparison between the Concentration of Pseudouridine Measured by the Present Method and That of Gehrke et al. Figure 2 is the chromatogram of a urine sample by the present method. Figure 3 shows a relationship between the concentration of urinary pseudouridine measured by the present method and that measured by the method of Gehrke et al. (6). The correlation
Linearity was tested on standards in aqueous solution at various concentrations, and the linearity was excellent up to 200 nmol/ml for pseudouridine (Fig. 1). The regression line was Y (concentration of pseudouridine) = 0.000201742 X peak area + 0.52 and the correlation coefficient was 0.999.
Detection Limits, Recovery, and Relative Standard Deviation The detection limit of pseudouridine was 7 nmol, recovery was lOO%, and relative stan-
FIG. 2. Chromatogram Pseudouridine.
of urinary pseudouridine.
*,
CHROMATOGRAPHIC
coefficient is 0.95 but urinary pseudouridine method are somewhat the method of Gehrke that recovery by the complete.
DETERMINATION
the concentrations of measured by our higher than those by et al. (6), indicating latter method is not
The Ratio of Pseudouridine/Creatinine in Urine of Normal Subjects and Patients with Hepatocellular Carcinoma We have previously described that the urinary concentration of pseudouridine in patients with primary hepatocellular carcinoma was significantly higher than that found in both normal subjects as well as in patients with liver cirrhosis (8). We therefore reinvestigated using the present method to determine whether or not the same results could be obtained in patients with primary hepatocellular carcinoma. Figure 4 shows the results. Although the samples examined were small, the ratio of pseudouridine/creatinine in urine is significantly higher in patients with primary hepatocellular carcinoma than in normal subjects. The method which we have developed is simple and rapid. In addition, it was comparable with the method of Gehrke et al. (6) in
OF URINARY
g Y
loo-
20. 8f6. (Mean+S
2 D.)
44.7*b?. (Mean+S.D
Fi c B, .5 .c ;;; ;
389
PSEUDOURIDINE
3 )
. . . 50-
8 :
3 5 4 2 h
+
:
:. 3
!
0 (1)
(2)
FIG. 4. Comparison of pseudouridine/creatinine in urine from patients with primary hepatocellular carcinoma and control subjects (1) Control, (2) hepatoma.
precision and accuracy. Tamura et al. (8,5) suggested that serial measurement of urinary pseudouridine concentration in patients with cirrhosis of the liver might serve as an indicator for the early detection of malignant transformation. Therefore, this method can be used in screening patients with malignant diseases and in monitoring therapeutic effects. REFERENCES
.
500 Urinary measured
Pseudouridine by the method
I& (nmol/ml) of Gehrke
et al
Rci. 3. Comparison of concentration of urinary pseudouridine between our method and the method of Gehrkes’ et al. (6).
1. Waalkes, T. P., Gehrke, C. W., Zumvalt, R. W., et al. (1976) Cancer 36,390-398. 2. Salvatore, F., Colonna, A., Constanzo, F., Russo, T., Esposito, F., and Cimiso, F. (1983) Recent Results Cancer Rex 84, 360-377. 3. Salvatore, F., Russo, T., Colonna, A., Cimiso, L., Mazzacca, G., and Cimiso, F. (1983) Cancer Detect. Prev. 6, 531-536. 4. Russo, T., Colonna, A., Salvatore, F., Cimiso, F., Brides, S., and Gurge, C. (1984) Cancer Res. 44, 2567-2570. 5. Tamura, S., Fujii, J., Nakano, T., Hada, T., and Higashino, K. (1986) Clin. Chim. Actn 154, 125-132. 6. Gehrke, C. W., Kuo, K. C., Davis, G. I., and Suits, R. D. (1978) J. Chromafogr. 150,455-476. 7. Krstulovic, A. M., Brown, P. R., and Rosie, D. M. (1977) Anal. Chem. 49,2237-224 I, 8. Tamura, S., Amuro, Y., Nakano, T., Fujii, J., Moriwaki, Y., Yamamoto, T., Hada, T., and Higashino, K. (1986) Cancer57, 1571-1575.