BIOCHEMICAL
MEDICINE
Age Dependence
22, 387-390 (1979)
of Human
Urinary
Pseudouridine
Excretion
Urinary pseudouridine ($U) may be used as an index of the rate of turnover of tRNA and rRNA. By use of gas-liquid chromatography, urinary IJJUwas quantitated in healthy individuals with a broad range of ages. +U excretion/24 hr increased from adolescence to advancing age at a rate of 8 mg/24 hr per decade from an average of 28 mg at age 25 to 83 mg at age 90, and at a rate of 11 mg/g creatinine per decade from an average of 22 mg/g at age 25 to 82 mg/g at age 79. This change reflects an age-related metabolic control mechanism which may be related to diminution in RNA reutilization efficiency or an age-related difference in the JIU content of the various RNA molecules.
The excretion rates of pseudouridine (+U, 5-P-ribofuranosyluracil), and other modified nucleosides have been proposed as indicators of neoplastic disease activity (1). During our continuing study of this, we examined $IJ excretion in healthy individuals over a broad range of ages to match our cancer patient population, and became aware of alterations in urinary $U as a function of age (2). $U, a modified nucleoside constituent of all mammalian tRNA’s and ribosomal RNA, is excreted into the urine during RNA turnover without being further metabolized (3,4). This unusual metabolic stability is due to the fact that the ribose moiety is linked to C-5 of the pyrimidine ring rather than to the conventional N- 1. Like all other modified nucleosides, it is not incorporated into RNA, but is formed by modification of specific uridine residues within the polynucleotide. Its excretion may thus be used as an index of the rate of RNA turnover. Individuals maintained on a reasonably steady protein diet exhibit little day-to-day variation in urinary $IJ (9). Increased urinary +U levels have been reported in leukemia (5,6), adenovirus- 12 induced tumors in rodents (7), mammary carcinoma (8), in other diseases such as gout, Paget’s disease of bone, psoriasis (3), and surgical stress (9). Urinary JlU was quantitated by modification of the method of Chang et al. (10). Urine specimens were obtained from the donors and stored not more than 2 days at 4°C. Preliminary urine purification was on a column (1.5 cm i.d.) of OS-cm Celite-545 (Johns Manville Co.) on top, 2.5 cm of 1: 1 Celite:charcoal (Norite-N, Fisher Scientific Co.), and l-cm Celite on 387 0006-2944/79/060387-04$02.00/O Copyright @ 1979 by Academic Press. Inc. All rights of reproduction in any form reserved.
388
I .ETTERS TO THE EDITOR
L IO
L--I 20
30
I40
.L 50
IU 60
70
60
w
loo
AGE (years)
100 901
FIG. 1. (a) Pseudouridine excretion of ostensibly normal individuals in terms of mg/24 hr as a function of age. (b) Pseudouridine excretion of ostensibly normal individuals in terms of mg/g creatinine as a function of age. The straight lines were calculated by the method of least squares.
the bottom. Both the charcoal and the Celite had been previously refluxed with water for 1 hr, refluxed with cone HCO,H for 1 hr, washed with water to neutrality, and dried. One milliliter of urine was pipetted onto the column, which was then washed with 50 ml water followed by 75 ml 1% pyridine in water. t,tLJ was then eluted with 30 ml of 3:l pyridine-95% ethanol. This eluate was evaporated to dryness in a rotary evaporator, transferred quantitatively to a l-ml Reactivial (Pierce Chemical Co.), and dried under N,. The residue was treated with 200 ~1 Tri-Sil 2 (Pierce
LETTERS
TO THE EDITOR
389
Chemical Co.) at room temp. for 10 min to prepare the trinethylsilyl derivative. Four microliters of this was injected onto a 6-ft x 2-mm i.d. U-shaped glass column packed with 3% OV-7 on Chromosorb W-HP, 80/100 mesh (Regis Chemical Co.) in a Varian 2100 gas chromatograph. Injector port and detector temperature was 300°C. The flow rates for Nz, Hz, and air were 40,30, and 400 mYmin, respectively. Column temperature was 190°C for 4 min, followed by an increase of 6’Cimin to 290°C. The silated derivative of $JU eluted 9 min into the program at 220°C and was detected with a flame ionization detector and quantitated with an electronic digital integrator. The recovery from the charcoal column was 95% and the relative standard error for the gas chromatography was 7.5% (n =4). Creatinine was estimated as the complex with alkaline picrate with a Creatinine II Rapid Stat Kit (Pierce Chemical Co.) with a relative standard error of 0.4% (n=8). Our findings are illustrated in Fig. 1. $U excretion increased from adolescence to advancing age at a rate of 8 mg/24 hr per decade, from an average of 28 mg at age 25 to 83 mg at age 90. In earlier experiments (2) we measured $U excretion per gram creatinine and found the increase to be 11 mg/g creatinine per decade from an average of 22 mg/g at age 25 to 82 mglg at age 79. Expression of gU excretion per 24 hr or per gram creatinine gives comparable results, and implies that creatinine excretion remains relatively constant between ages 25 and 90. This change in $U excretion herein observed clearly reflects a fundamental metabolic control mechanism which is age related. We propose as most plausible a diminution in the efficiency of RNA reutilization in protein biosynthesis with higher rates of turnover with increasing age. Less likely appears to be an alteration with age in the $U content of the various RNA molecules or the amount of $IJ containing RNA. Agedependent changes in the efficiency of gene transcription have been noted in experimental systems (I 1) and it is likely that similar regulatory mechanisms would be involved in man. It is clearly evident that age-matched controls are essential to the interpretation of t,f~Uexcretion data in disease states. REFERENCES I. Waalkes, T. P., Gehrke, C. W., Zumwalt, R. W., Chang, S. Y., Lakings, D. B., Tormey, D. C., Ahmann, D. L.. and Moertel, C. G., Cancer 36, 390 (1975). 2. Luch, J., Mittelman, A., and Tritsch, G. L., Fed. Proc. 36, 122 (1977) (Abstmct). 3. weissman, S., Eisen, A. Z., and Karen, M., J. Lab. Clin. Med. 59, 852 (1962). 4. Chheda, G. B., L$e Sci. 8, 979 (1%9). 5. Adams, W. S., Davis, F., and Nakatani, M., Amer. J. Med. 28, 726 (MO). 6. Park, R. W., Holland, J. F., and Jenkins, A., Cancer Res. 22, 4% (1%2).
390
I,ETTERS
7.
McFarlane,
8.
Mandel.
E. S.. and I,.
R..
Shaw.
Srinivasan.
9. Mittelman. A., Chheda. IO. Chang. S. Y.. Lakingx.
6. P. K.,
TO
THE
J.. (‘o~IoL/. and
G.. and Grace, D. B., Zumwalt.
Borek.
EDIT-OK
J. Mic.,
MEDICINE
Age Dependence
22, 387-390 (1979)
of Human
Urinary
Pseudouridine
Excretion
Urinary pseudouridine ($U) may be used as an index of the rate of turnover of tRNA and rRNA. By use of gas-liquid chromatography, urinary IJJUwas quantitated in healthy individuals with a broad range of ages. +U excretion/24 hr increased from adolescence to advancing age at a rate of 8 mg/24 hr per decade from an average of 28 mg at age 25 to 83 mg at age 90, and at a rate of 11 mg/g creatinine per decade from an average of 22 mg/g at age 25 to 82 mg/g at age 79. This change reflects an age-related metabolic control mechanism which may be related to diminution in RNA reutilization efficiency or an age-related difference in the JIU content of the various RNA molecules.
The excretion rates of pseudouridine (+U, 5-P-ribofuranosyluracil), and other modified nucleosides have been proposed as indicators of neoplastic disease activity (1). During our continuing study of this, we examined $IJ excretion in healthy individuals over a broad range of ages to match our cancer patient population, and became aware of alterations in urinary $U as a function of age (2). $U, a modified nucleoside constituent of all mammalian tRNA’s and ribosomal RNA, is excreted into the urine during RNA turnover without being further metabolized (3,4). This unusual metabolic stability is due to the fact that the ribose moiety is linked to C-5 of the pyrimidine ring rather than to the conventional N- 1. Like all other modified nucleosides, it is not incorporated into RNA, but is formed by modification of specific uridine residues within the polynucleotide. Its excretion may thus be used as an index of the rate of RNA turnover. Individuals maintained on a reasonably steady protein diet exhibit little day-to-day variation in urinary $IJ (9). Increased urinary +U levels have been reported in leukemia (5,6), adenovirus- 12 induced tumors in rodents (7), mammary carcinoma (8), in other diseases such as gout, Paget’s disease of bone, psoriasis (3), and surgical stress (9). Urinary JlU was quantitated by modification of the method of Chang et al. (10). Urine specimens were obtained from the donors and stored not more than 2 days at 4°C. Preliminary urine purification was on a column (1.5 cm i.d.) of OS-cm Celite-545 (Johns Manville Co.) on top, 2.5 cm of 1: 1 Celite:charcoal (Norite-N, Fisher Scientific Co.), and l-cm Celite on 387 0006-2944/79/060387-04$02.00/O Copyright @ 1979 by Academic Press. Inc. All rights of reproduction in any form reserved.
388
I .ETTERS TO THE EDITOR
L IO
L--I 20
30
I40
.L 50
IU 60
70
60
w
loo
AGE (years)
100 901
FIG. 1. (a) Pseudouridine excretion of ostensibly normal individuals in terms of mg/24 hr as a function of age. (b) Pseudouridine excretion of ostensibly normal individuals in terms of mg/g creatinine as a function of age. The straight lines were calculated by the method of least squares.
the bottom. Both the charcoal and the Celite had been previously refluxed with water for 1 hr, refluxed with cone HCO,H for 1 hr, washed with water to neutrality, and dried. One milliliter of urine was pipetted onto the column, which was then washed with 50 ml water followed by 75 ml 1% pyridine in water. t,tLJ was then eluted with 30 ml of 3:l pyridine-95% ethanol. This eluate was evaporated to dryness in a rotary evaporator, transferred quantitatively to a l-ml Reactivial (Pierce Chemical Co.), and dried under N,. The residue was treated with 200 ~1 Tri-Sil 2 (Pierce
LETTERS
TO THE EDITOR
389
Chemical Co.) at room temp. for 10 min to prepare the trinethylsilyl derivative. Four microliters of this was injected onto a 6-ft x 2-mm i.d. U-shaped glass column packed with 3% OV-7 on Chromosorb W-HP, 80/100 mesh (Regis Chemical Co.) in a Varian 2100 gas chromatograph. Injector port and detector temperature was 300°C. The flow rates for Nz, Hz, and air were 40,30, and 400 mYmin, respectively. Column temperature was 190°C for 4 min, followed by an increase of 6’Cimin to 290°C. The silated derivative of $JU eluted 9 min into the program at 220°C and was detected with a flame ionization detector and quantitated with an electronic digital integrator. The recovery from the charcoal column was 95% and the relative standard error for the gas chromatography was 7.5% (n =4). Creatinine was estimated as the complex with alkaline picrate with a Creatinine II Rapid Stat Kit (Pierce Chemical Co.) with a relative standard error of 0.4% (n=8). Our findings are illustrated in Fig. 1. $U excretion increased from adolescence to advancing age at a rate of 8 mg/24 hr per decade, from an average of 28 mg at age 25 to 83 mg at age 90. In earlier experiments (2) we measured $U excretion per gram creatinine and found the increase to be 11 mg/g creatinine per decade from an average of 22 mg/g at age 25 to 82 mglg at age 79. Expression of gU excretion per 24 hr or per gram creatinine gives comparable results, and implies that creatinine excretion remains relatively constant between ages 25 and 90. This change in $U excretion herein observed clearly reflects a fundamental metabolic control mechanism which is age related. We propose as most plausible a diminution in the efficiency of RNA reutilization in protein biosynthesis with higher rates of turnover with increasing age. Less likely appears to be an alteration with age in the $U content of the various RNA molecules or the amount of $IJ containing RNA. Agedependent changes in the efficiency of gene transcription have been noted in experimental systems (I 1) and it is likely that similar regulatory mechanisms would be involved in man. It is clearly evident that age-matched controls are essential to the interpretation of t,f~Uexcretion data in disease states. REFERENCES I. Waalkes, T. P., Gehrke, C. W., Zumwalt, R. W., Chang, S. Y., Lakings, D. B., Tormey, D. C., Ahmann, D. L.. and Moertel, C. G., Cancer 36, 390 (1975). 2. Luch, J., Mittelman, A., and Tritsch, G. L., Fed. Proc. 36, 122 (1977) (Abstmct). 3. weissman, S., Eisen, A. Z., and Karen, M., J. Lab. Clin. Med. 59, 852 (1962). 4. Chheda, G. B., L$e Sci. 8, 979 (1%9). 5. Adams, W. S., Davis, F., and Nakatani, M., Amer. J. Med. 28, 726 (MO). 6. Park, R. W., Holland, J. F., and Jenkins, A., Cancer Res. 22, 4% (1%2).
390
I,ETTERS
7.
McFarlane,
8.
Mandel.
E. S.. and I,.
R..
Shaw.
Srinivasan.
9. Mittelman. A., Chheda. IO. Chang. S. Y.. Lakingx.
6. P. K.,
TO
THE
J.. (‘o~IoL/. and
G.. and Grace, D. B., Zumwalt.
Borek.
EDIT-OK
J. Mic.,
