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Characterization of elevated i-PGE2 in human female urine
CHARACTERIZATION HUMAN
OF
ELEVATED
FEMALE
i-PGE2 IN
URINE
D. W. SANDWlSCH,R. D. DYER, N. BRETZ, S. Y. TAN and P. J. MULROW Department of Medicine. Medical Colle,qe of Ohio, Toledo, Ohio 43699, U.S.A.
INTRODUCTION
Urinary PGE levels in male and females have been used to evaluate the production of prostaglandins (PG) by the kidney in various physiological and pathological states. 3"9"t3 In some cases, only female subjects were chosen. 4"s'a Several investigators have noted that i-PGE2 levels in male urines are higher and more variable than those of females T M and characterization of male PG composition has been conducted. 7 During the course of routine i-PGE2 measurement of female urines, several were found to contain unexplained abnormally high levels. The cause for their elevation was evaluated in this report. EQUIPMENT
Reagent grade hydrochloric, formic, and acetic acids were purchased from Mallincrodt, St. Louis, MO. All solvents for high pressure liquid chromatography (HPLC) and extraction were purchased from Burdick and Jackson, Muskegon, MI. The HPLC instrument was a solvent delivery system (model 6000), solvent programmer (model 660), injector (model U6K), and a 3.9 mm x 30 cm #-Porasil column from Waters Assoc., Milford, MA. A nonlinear program (Waters No. 4) of 2 solvents (Solvent C: chloroform, solvent D: chloroform/methanol/acetic acid, 500:50:11, v/v/v) at a flow rate of 1 ml/min was used. 2
Tritium labeled PGA2, PGE2, PGF2~, and 6-keto-PGFl, were obtained from New England Nuclear, Boston, MA. PCS scintillation cocktail was purchased from Amersham, Arlington Heights, IL. SAMPLE
COLLECTION
All 24 hr urine collections were refrigerated during collection and frozen after the completion of each collection: Seminal fluid was obtained from a normal volunteer on the same day it was added to and processed with a semen-free control urine. The 24 hr semen-free control urines were collected from an l 1-year-old prepubertal male and a healthy 30 year old female, both with assayed i-PGE2 levels less than 200 ng/24 hr. The normal mean for female i-PGE2 is 166 + 17 (SE) ng/24 hr (n = 38).
URINE
EXTRACTION
FOR
HPLC
AND
PGE2
RADIOIMMUNOASSAY
(RIA)
Fifty ml of each urine sample to which 105 cpm of [3H]PGE2 had been added, was adjusted to a pH of 3.5 with formic acid and extracted with 250 ml of chloroform. The aqueous phase was discarded and the organic phase was washed with 3 x 25 ml volumes of an acidic (pH = 3.5) saturated sodium chloride solution. The chloroform extract was evaporated under an air stream, and the residue dissolved in 1 ml of methanol. A 100 pl aliquot was counted for recovery. The remaining methanol extract was dried and reconstituted with a volume of chloroform that would give each extract a constant count rate per injection volume. Twenty #1 of chloroform extract was then injected into the HPLC and thirty 2 ml fractions were collected. Five-hundred #1 aliquots of each fraction were 791
792
D . W . Sandwisch et al.
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HPLC Elution Volume(ml) FIG. 1. lmmunochromatographic profiles of human urine extracts. No. I, female on furosemide i - P G E 2 = l . 6 1 + g / 2 4 h r : No. 2. female i - P G E 2 = 4 . 0 / J g / 2 4 h r : No. 3, female i - P G E 2 = 18.0 t+g/24 hr: No. 4. female (semen-free control) i-PGE2 = 168 ng/24 hr: No. 5. male (prepubertal semen-free control) i-PGE~ = 198 ng/24 hr: No. 6. prepubertal control + semen 2 p} semen/ml, of urine: No. 7. elution of [3HIPGA2. [3H]PGE2. [3H]-6-keto FI,. and [3H]PGF2,.
counted for peak location and the remaining 1.5 ml was dried and reconstituted with 500/d of the assay buffer for the PGE2 RIA. All i-PGE2 levels for 24 hr urine collections and H PLC fractions were determined as previously described tl with several modifications. 1° RESULTS
The elution profile No. 7 in Fig. 1 gives the elution volumes for the 4 tritium labeled PG's in the H PLC system. Inspection of the 2 semen-free control profiles Nos. 4 and 5 in Fig. 1 indicates that only the 23-24ml fraction of the HPLC elution contains i-PGE2 activity, where F3H]PGE2 elutes. The elution profiles of female urines Nos. 2 and 3 contain increases in i-PGE2 activity in the [-3H]PGE2 area as well as several other elution areas as compared to the controls (Nos. 4 and 5). The greatest increase occurs in the 20-26 ml fractions, where I-3H]PGE2 and [3H]6-keto-PGFl~ elute. The second largest increase was noted in the 47-50 ml fraction indicating a more polar component than [3H]PGF2~. Two smaller increases are found in the 15-16ml fraction, slightly more polar than [3H]PGA2, and in the 37-42 ml region where [3H]PGF2~ elutes. Urine profile No. 1 shows an increase in i-PGE2 activity only in the PGE2 area, and 23-24 ml fraction. When the extract of 50 ml of prepubertal male urine containing 100 pl of semen is chromatographed in profile No. 6, large increases in i-PGE2 are found in the same areas as the increases in profiles Nos. 2 and 3.
Elevated i-PGE2 in human female urine DISCUSSION
AND
793
CONCLUSION
Increased i-PGE2 values in male urines have been indicated by Patrono e t al. 7 to be the result of extra-renal seminal contamination. Profile No. 6 (Fig. l) illustrates that concentrations of semen as low as 2 pl/ml of urine can lead to extremely large increases in i-PGE2 activity in the PGE2 elution area as well as in several other locations. Since seminal PGE2 and renal PGE2 can not be differentiated, an alternative method for identifying seminal contamination is required. Elevations in urinary PGE2 can occur without evidence of a seminal type elution profile, as in profile No. 1. Perhaps the presence of PGE2 of seminal origin can be indicated by the appearance of the i-PGE2 area that is more polar than PGF2, and common to profiles Nos. 2, 3 and 6. This area in the same HPLC system has been reported by Tusell and Gelpi 12 to contain 19-OH-PGEI&2 which, when quantitated in semen by Jonsson e t al., 6 was found to be 3-5 times the concentration of PGEI&2. The mobility of the 19-OH-PGE in the thin layer chromatography system of Patrono e t al. 7 is unknown. Because the 19-OH-PGE compounds are not available, their cross-reactivity in this RIA and those of other groups remains to be determined. Female urines with abnormally elevated i-PGE2 levels can give immunoreactive elution patterns similar to a seminally contaminated urine, but increases in i-PGE2 in the PGE2 fractions without semen was demonstrated in the urine with increased PGE2 content produced by furosemide. To identify those urines in which semen is responsible for increased i-PGE2, the presence or absence of the area more polar than PGF'2, should be established. SUMMARY
In routine measurements of urinary immunoreactive prostaglandin E 2 (i-PGE2), it was noted that some female urines contained greater quantities of i-PGE2 (1.6-18/~g/24 hr) than the normal female (166 + 17ng/24hr, n = 38). Elevated and variable urinary i-PGE2 values frequently encountered in male urines are probably due to seminal contamination. This could account for elevated i-PGE2 levels in some female urines. Lipid extracts of the urines with elevated i-PGE2 were fractionated by HPLC and the i-PGE2 elution profiles were determined with a PGE2 radioimmunoassay (RIA). Two semen-free control urines exhibited only one i-PGE2 peak that coeluted with [3H]PGE2. The female urines with elevated i-PGE2 values of 4 and 18/1g/24 hr had elution profiles with increased i-PGE2 in 4 regions. The elution profile for control urine with 100/A of human semen/50 ml of urine contained increases in i-PGE2 activity in the same regions as the female urines with 4 and 18 pg/24 hr of i-PGE2. A urine from a female on furosemide treatment with a high i-PGE2 of 1.6/~g/24 hr had an elution pattern with an i-PGE2 peak only where [3H]PGE2 eluted. These results suggest that some but not all elevated female urinary i-PGE2 values may occur as a result of seminal contamination.
REFERENCES 1. ABE, K., YASUYING, M.. CHIBA, S., IROKAMA, N., ITO, T. and YOSHINAGA, K. Prostaolandins 14, 513-521 (1977). 2. CARR, K.. SWEETMAN, B. J. and FROLICH, J. C. Prostaglandins I I, 3-14 (1976). 3. DRAY, F. Clin. Sci. Mol. Med. 54, i15-118 (1978). 4. FROLICH. J. C., WILSON, T, W,, SWEETMAN, B. J., SMIGEL, M., N1ES, A. S., CARR, K., WATSON, J. T. and OATES, J. A. J. Clin. Invest. 55, 763-770 (1975). 5. GILL, J. R., JR., FROLICH. J. C., BOWDEN, R. E., TAYLOR, A. A., KEISER. H. R., SEYBERTH, H. W., OATES, J. A. and BAR'r~R, F. C. Am. J. Med. 610 43-51 (1976). 6. JONSSON, H. T., JR., MIDDLEDITCH, B. S., SCHEXMAYDON, M. A. and DESIDERIO, D. M. J. Lipid Res. 17, 1-6 (1976). 7. PATRONO, C., WENNMALM. A., CIABATTOUI, G., NOWAK, J., PUGLIESE, F. and Cxr4oTn, G. A. Prostaolandins 18, 623-629 (1979). 8. SCHERER, B., SCHNERMAN, J., SOFRONIEU, M. and WEBER, P. Prostaglandins 15, 255-266 (1978). 9. SCHERER,B. and WEBER, P. C. Clin. Sci. 56, 77-81 [1979). 10. TAN. S. Y., SANDWISCH, D. W. and MULROW. P. J. Prostacllandins Med. 4, 53-63 (1980t.
794
D.W. Sandwisch et al.
11. Tan, S. Y.. Sweet, P. and Muurow. P. J. Prostaqlamlins 15, 139-150 (1978). 12. TUSELL,J. M. and GELPI, E. J. Chromato.qr. 181,295-310 (1980). 13. YASUJIMA.M., ABE. K.. OTSUKA. Y.. CHIn^. S.. RITSU, K., IrOKAWA. N,. SEINO, M., SAKUrAI, Y., Salto, K, 1TO. T. and YOSmNAGA, K. Tohokll J. Exp. Med. 123, 271--278 (1977}.
OF
ELEVATED
FEMALE
i-PGE2 IN
URINE
D. W. SANDWlSCH,R. D. DYER, N. BRETZ, S. Y. TAN and P. J. MULROW Department of Medicine. Medical Colle,qe of Ohio, Toledo, Ohio 43699, U.S.A.
INTRODUCTION
Urinary PGE levels in male and females have been used to evaluate the production of prostaglandins (PG) by the kidney in various physiological and pathological states. 3"9"t3 In some cases, only female subjects were chosen. 4"s'a Several investigators have noted that i-PGE2 levels in male urines are higher and more variable than those of females T M and characterization of male PG composition has been conducted. 7 During the course of routine i-PGE2 measurement of female urines, several were found to contain unexplained abnormally high levels. The cause for their elevation was evaluated in this report. EQUIPMENT
Reagent grade hydrochloric, formic, and acetic acids were purchased from Mallincrodt, St. Louis, MO. All solvents for high pressure liquid chromatography (HPLC) and extraction were purchased from Burdick and Jackson, Muskegon, MI. The HPLC instrument was a solvent delivery system (model 6000), solvent programmer (model 660), injector (model U6K), and a 3.9 mm x 30 cm #-Porasil column from Waters Assoc., Milford, MA. A nonlinear program (Waters No. 4) of 2 solvents (Solvent C: chloroform, solvent D: chloroform/methanol/acetic acid, 500:50:11, v/v/v) at a flow rate of 1 ml/min was used. 2
Tritium labeled PGA2, PGE2, PGF2~, and 6-keto-PGFl, were obtained from New England Nuclear, Boston, MA. PCS scintillation cocktail was purchased from Amersham, Arlington Heights, IL. SAMPLE
COLLECTION
All 24 hr urine collections were refrigerated during collection and frozen after the completion of each collection: Seminal fluid was obtained from a normal volunteer on the same day it was added to and processed with a semen-free control urine. The 24 hr semen-free control urines were collected from an l 1-year-old prepubertal male and a healthy 30 year old female, both with assayed i-PGE2 levels less than 200 ng/24 hr. The normal mean for female i-PGE2 is 166 + 17 (SE) ng/24 hr (n = 38).
URINE
EXTRACTION
FOR
HPLC
AND
PGE2
RADIOIMMUNOASSAY
(RIA)
Fifty ml of each urine sample to which 105 cpm of [3H]PGE2 had been added, was adjusted to a pH of 3.5 with formic acid and extracted with 250 ml of chloroform. The aqueous phase was discarded and the organic phase was washed with 3 x 25 ml volumes of an acidic (pH = 3.5) saturated sodium chloride solution. The chloroform extract was evaporated under an air stream, and the residue dissolved in 1 ml of methanol. A 100 pl aliquot was counted for recovery. The remaining methanol extract was dried and reconstituted with a volume of chloroform that would give each extract a constant count rate per injection volume. Twenty #1 of chloroform extract was then injected into the HPLC and thirty 2 ml fractions were collected. Five-hundred #1 aliquots of each fraction were 791
792
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HPLC Elution Volume(ml) FIG. 1. lmmunochromatographic profiles of human urine extracts. No. I, female on furosemide i - P G E 2 = l . 6 1 + g / 2 4 h r : No. 2. female i - P G E 2 = 4 . 0 / J g / 2 4 h r : No. 3, female i - P G E 2 = 18.0 t+g/24 hr: No. 4. female (semen-free control) i-PGE2 = 168 ng/24 hr: No. 5. male (prepubertal semen-free control) i-PGE~ = 198 ng/24 hr: No. 6. prepubertal control + semen 2 p} semen/ml, of urine: No. 7. elution of [3HIPGA2. [3H]PGE2. [3H]-6-keto FI,. and [3H]PGF2,.
counted for peak location and the remaining 1.5 ml was dried and reconstituted with 500/d of the assay buffer for the PGE2 RIA. All i-PGE2 levels for 24 hr urine collections and H PLC fractions were determined as previously described tl with several modifications. 1° RESULTS
The elution profile No. 7 in Fig. 1 gives the elution volumes for the 4 tritium labeled PG's in the H PLC system. Inspection of the 2 semen-free control profiles Nos. 4 and 5 in Fig. 1 indicates that only the 23-24ml fraction of the HPLC elution contains i-PGE2 activity, where F3H]PGE2 elutes. The elution profiles of female urines Nos. 2 and 3 contain increases in i-PGE2 activity in the [-3H]PGE2 area as well as several other elution areas as compared to the controls (Nos. 4 and 5). The greatest increase occurs in the 20-26 ml fractions, where I-3H]PGE2 and [3H]6-keto-PGFl~ elute. The second largest increase was noted in the 47-50 ml fraction indicating a more polar component than [3H]PGF2~. Two smaller increases are found in the 15-16ml fraction, slightly more polar than [3H]PGA2, and in the 37-42 ml region where [3H]PGF2~ elutes. Urine profile No. 1 shows an increase in i-PGE2 activity only in the PGE2 area, and 23-24 ml fraction. When the extract of 50 ml of prepubertal male urine containing 100 pl of semen is chromatographed in profile No. 6, large increases in i-PGE2 are found in the same areas as the increases in profiles Nos. 2 and 3.
Elevated i-PGE2 in human female urine DISCUSSION
AND
793
CONCLUSION
Increased i-PGE2 values in male urines have been indicated by Patrono e t al. 7 to be the result of extra-renal seminal contamination. Profile No. 6 (Fig. l) illustrates that concentrations of semen as low as 2 pl/ml of urine can lead to extremely large increases in i-PGE2 activity in the PGE2 elution area as well as in several other locations. Since seminal PGE2 and renal PGE2 can not be differentiated, an alternative method for identifying seminal contamination is required. Elevations in urinary PGE2 can occur without evidence of a seminal type elution profile, as in profile No. 1. Perhaps the presence of PGE2 of seminal origin can be indicated by the appearance of the i-PGE2 area that is more polar than PGF2, and common to profiles Nos. 2, 3 and 6. This area in the same HPLC system has been reported by Tusell and Gelpi 12 to contain 19-OH-PGEI&2 which, when quantitated in semen by Jonsson e t al., 6 was found to be 3-5 times the concentration of PGEI&2. The mobility of the 19-OH-PGE in the thin layer chromatography system of Patrono e t al. 7 is unknown. Because the 19-OH-PGE compounds are not available, their cross-reactivity in this RIA and those of other groups remains to be determined. Female urines with abnormally elevated i-PGE2 levels can give immunoreactive elution patterns similar to a seminally contaminated urine, but increases in i-PGE2 in the PGE2 fractions without semen was demonstrated in the urine with increased PGE2 content produced by furosemide. To identify those urines in which semen is responsible for increased i-PGE2, the presence or absence of the area more polar than PGF'2, should be established. SUMMARY
In routine measurements of urinary immunoreactive prostaglandin E 2 (i-PGE2), it was noted that some female urines contained greater quantities of i-PGE2 (1.6-18/~g/24 hr) than the normal female (166 + 17ng/24hr, n = 38). Elevated and variable urinary i-PGE2 values frequently encountered in male urines are probably due to seminal contamination. This could account for elevated i-PGE2 levels in some female urines. Lipid extracts of the urines with elevated i-PGE2 were fractionated by HPLC and the i-PGE2 elution profiles were determined with a PGE2 radioimmunoassay (RIA). Two semen-free control urines exhibited only one i-PGE2 peak that coeluted with [3H]PGE2. The female urines with elevated i-PGE2 values of 4 and 18/1g/24 hr had elution profiles with increased i-PGE2 in 4 regions. The elution profile for control urine with 100/A of human semen/50 ml of urine contained increases in i-PGE2 activity in the same regions as the female urines with 4 and 18 pg/24 hr of i-PGE2. A urine from a female on furosemide treatment with a high i-PGE2 of 1.6/~g/24 hr had an elution pattern with an i-PGE2 peak only where [3H]PGE2 eluted. These results suggest that some but not all elevated female urinary i-PGE2 values may occur as a result of seminal contamination.
REFERENCES 1. ABE, K., YASUYING, M.. CHIBA, S., IROKAMA, N., ITO, T. and YOSHINAGA, K. Prostaolandins 14, 513-521 (1977). 2. CARR, K.. SWEETMAN, B. J. and FROLICH, J. C. Prostaglandins I I, 3-14 (1976). 3. DRAY, F. Clin. Sci. Mol. Med. 54, i15-118 (1978). 4. FROLICH. J. C., WILSON, T, W,, SWEETMAN, B. J., SMIGEL, M., N1ES, A. S., CARR, K., WATSON, J. T. and OATES, J. A. J. Clin. Invest. 55, 763-770 (1975). 5. GILL, J. R., JR., FROLICH. J. C., BOWDEN, R. E., TAYLOR, A. A., KEISER. H. R., SEYBERTH, H. W., OATES, J. A. and BAR'r~R, F. C. Am. J. Med. 610 43-51 (1976). 6. JONSSON, H. T., JR., MIDDLEDITCH, B. S., SCHEXMAYDON, M. A. and DESIDERIO, D. M. J. Lipid Res. 17, 1-6 (1976). 7. PATRONO, C., WENNMALM. A., CIABATTOUI, G., NOWAK, J., PUGLIESE, F. and Cxr4oTn, G. A. Prostaolandins 18, 623-629 (1979). 8. SCHERER, B., SCHNERMAN, J., SOFRONIEU, M. and WEBER, P. Prostaglandins 15, 255-266 (1978). 9. SCHERER,B. and WEBER, P. C. Clin. Sci. 56, 77-81 [1979). 10. TAN. S. Y., SANDWISCH, D. W. and MULROW. P. J. Prostacllandins Med. 4, 53-63 (1980t.
794
D.W. Sandwisch et al.
11. Tan, S. Y.. Sweet, P. and Muurow. P. J. Prostaqlamlins 15, 139-150 (1978). 12. TUSELL,J. M. and GELPI, E. J. Chromato.qr. 181,295-310 (1980). 13. YASUJIMA.M., ABE. K.. OTSUKA. Y.. CHIn^. S.. RITSU, K., IrOKAWA. N,. SEINO, M., SAKUrAI, Y., Salto, K, 1TO. T. and YOSmNAGA, K. Tohokll J. Exp. Med. 123, 271--278 (1977}.