ANALYTICAL
BIOCHEMISTRY
81,
??1-7,55
(1977)
Deuterium Isotope Dilution Method for the Specific Measurement of 6-Keto-prostaglandin F,, by Mass Fragmentography We recently reported the isolation of 6-keto-prostaglandin F,, (6KPGF,,) from incubations of rat stomach homogenates in the presence of exogenous arachidonic acid (1). This product was formed in far greater quantities (lo-20 times) than was PGE, in this preparation. It could also be derived from the intermediate prostaglandin endoperoxides PGH, and PGG, (2), indicating its formation by the “PG synthetase.” Its structure was unequivocally proven by comparison with an authentic sample derived through chemical synthesis (2). Because preliminary experiments indicated its presence in several other tissues (3,4), we set out to establish a simple, specific, and sensitive method for its quantitation by mass fragmentography, a method now used quite routinely in the assay of other prostaglandins (5-7) and many other biologically occurring products (8- 10). Pentadeuterated 6K-PGF1, was prepared from octadeuterated [5,6,8, 9,11,12,14, 15-2H,] arachidonic acid by a combined biochemical and chemical approach. Octadeuterated arachidonic acid was converted into heptadeuterated PGE, by conventional methods using microsomes of sheep seminal vesicles (ll), and the product was isolated and purified by thin-layer chromatography (tic). The purified compound was reduced with sodium borohydride in methanol, and the resulting heptadeuterated PGFza isomer was purified again by tic after prior conversion into the methyl ester derivative with diazomethane. The pure compound was chemically converted in good yield into methyl 6K-PGF,, (U. Axen, personal communication, method to be published). The final product which contained five deuterium atoms (positions 8,11,12,14, and 15) was purified by tic and was used in the assay. 6K-PGFi, displays good gc properties as the MeMOTMS derivative and is well resolved from PGE, (Fig. I). Its mass spectrum shows a multiplicity of ions of high intensity in the high end of the spectrum (Fig. 2), providing excellent potential for use in mass fragmentography. Fragment ions at m/e 508 and 513 [M-(90 + 31)] were selected for the protonated and deuterated forms because these ions were quite prominent in the mass spectrum (see Fig . 2 for the Do form). The loss of (90 + 31) from the molecular ion of the deuterated compound gives rise to two fragment ions, i.e., m/e 513 and 512, the latter being formed by the eliminaC opynghf 35 1977 by Academic t’res. Inc. All rights of reproductmn m any form reserved.
ISSN 0003-2697
252
SHORT
COMMUNICATIONS
FIG. I. Gas chromatographic separation of PGE, and 6K-PGF,, as the MeMOTMS derivatives. A 6ft x 0.25 in. column of 3%’ SE-30 on 100/120-mesh Gas Chrom Q was used at 24O”C, and 0.6 pg of each component was injected. 100
0 100
200
300
400
500
600
m/e FIG. 2. Mass spectrum of the authentic D,,-6K-PGF,, and the Dj product reported in this paper as the MeMOTMS derivative. A Varian MAT CH-5 coupled gas chromatograph-mass spectrometer-computer assembly was used. The operating conditions were: gas chromatograph oven at 265°C. separator at 280°C. source at 27O”C, electron multiplier at 2.5 kV. ionization voltage of 70 eV.
SHORT
253
COMMUNICATIONS
FIG. 3. Actual record of a mass fragmentogram of a mixture of 20 ng of protonated compound in 1 pg of deuterated 6K-PGF,,,. Fragments at rr~/e 508 and 513 were monitored using a two-channel peak-matching unit (Varian) and were displayed as shown on a Statos recorder. This concentration could be quantitated with an accuracy of i- 1.Y;.
I
tion of one deuterium atom with the trimethylsilyl group. The fragment at HZ/C 508 can also be used in conjunction with an assay for PGE, (M-31 fragment), since chromatographic resolution of both compounds can easily be accomplished on a nonpolar silicone phase column (Fig. 1). The amount of protonated background in the D, product was found to be 34 ng of D,,/l pg of D, product. This is not surprising since the initial part of the synthesis involved a biochemical approach. Figure 3 shows the relative intensities of the fragments at m/c 508 and 513 in a mixture containing 30 ng of protonated compound in 1 pg of deuterated carrier. A standard line of the ratios observed for peak heights for the protium and deuterium forms versus the composition of injected material is shown in Fig. 4.
~~ 0
~-' 50
100
ng Do in lpg D5 GK-PGF,, FIG. 4. Standard line constructed from injection of a mixture of D,, in I pg of D, 6K-PGF,,. Response (peak height) versus composition of mixture injected is shown. Since the fragment selectedfortheD,product i.e., M-(90 + 31). is split int0twopeaks.i.e.. ~nic 512(37’7,)and513 (63%). the composition of the protium component trrric 508) had to be compensated accordingly. The reproducibility of this fragmentation was -+ 0.05? (five determinations). Data in the figure have been corrected for a D,, background of 3.47;
254
SHORT
Incubation 20 4
FIG. fundus Tissue directly samples
10’ 37”
COMMUNICATIONS
-
-
+ -
+ +
5. Quantitation of the 6K-PGF,, present in fresh homogenates of the rat stomach after incubation in the presence and absence of exogenous arachidonic acid (100 kg/g). was homogenized in 0.05 M KH,PO,-NaOH (20 vol. pH 7.4) and was extracted (or after incubation) with ethanol (5 vol). Carrier 6K-PGF,, ( 1 wg. Dj) was added, and were worked up as described in the text.
The assay consists of the addition of 1 pg of Dj-6K-PGF,, as carrier as the methyl ester (or the free acid) to an ethanol extract of a tissue sample or homogenate. The total extract is methylated with diazomethane, and the sample is purified on tic. Using chloroform/methanol/acetic acid/water (90:9:1:0.6.5, v/v) as developing solvent, the Rf is 0.45 (2). The sample is eluted from the silica gel with methanol, and the product is converted into the methoxime trimethylsilyl derivative as previously reported (2). The method was applied to an investigation of the capacity of rat stomach fundus homogenates to biosynthesize 6K-PGF,,. The amounts of 6KPGF,, detected are representative of the combined capacity of the prostaglandin endoperoxide synthetase and the prostagiandin 6(9)oxy cyclase (12). As shown in Fig. 5, the amounts of 6K-PGF, in unincubated homogenates are quite small, as compared to the amounts formed after incubation, especially when exogenous arachidonic acid is added prior to the incubation. The large amounts formed under the latter conditions are indicative of the high capacity of this tissue to be directed along the 6(9) oxy pathway of endoperoxide catabolism. No other tissue tested forms equal or larger amounts of 6K-PGF,, than the fundus of the rat stomach, which is principally directed to this product, PGE, and PGF.,, being almost undetected (13). The assay can be greatly improved if a deuterated derivative with a lower protium content is prepared chemically as for other prostaglandins (background about 0.5%). Also. if the product contained only four deuterium atoms, it would be possible to quantitate PGE, (available as the
SHORT
COMMUNICATIONS
25.5
D, derivative) and 6K-PGF,, in the same sample through a single injection, since prior resolution of both compounds can be obtained by gas chromatography. Further work along these lines is under way. ACKNOWLEDGMENTS This research was supported by a grant from the Medical Research Council of Canada (No MA-4181). I thank Mr. Lajos Marai for operating the Varian Mat CH-5 mass spectrometer assembly, an MRC regional facility at the Best Institute, University of Toronto. Authentic unlabeled 6K-PGF,, was generously provided by Dr. U. Axen. the Upjohn Co., Kalamazoo, Mich.
REFERENCES 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13.
Pace-Asciak. C. ( 1976) &perirnlia 32. 291-392. Pace-Asciak. C. (1976) J. Amer. Churn. Sot. 98, 1348-2349. Pace-Asciak. C., and Nashat, M. (1976) Proc. Canard. Fed. Biol. SW. 19, 268. Dawson. W.. Boot. J. R., Cockerill. A. F.. Mallen. D. N. B., and Osborne. D. J. (1976) Nature (London) 262, 699-702. Samuelsson. B.. Hamberg. M.. and Sweeley, C. C. (1970) Anal. Biochem. 38, 3Ol304. Green. K., Granstrom. E., Samuelsson. B., and Axen, U. (1973) Anal. Biochrm. 54, 434-453. Nicosia. S.. and Galli. G. (1974) Am/. Biochem. 61, 192-199. Brandenberger. H., and Schnyder, D. (1972) Z. Anul. Chem. 261, 297-305. Jenden. D. J. (1973) in Advances in Biochemical Psychopharmacology (Costa. E., and Holmstedt. B., eds.). Vol. 7. pp. 69-81, Raven Press, New York. Cattabeni, F.. Koslow. S. J., and Costa. E. (1972) Science 178, 166- 168. Wlodawer, P., and Samuelsson, B. (1973) J. Eiol. Chem. 248, 5673-5678. Pace-Asciak, C. R., and Nashat. M. (1977) Biochim. Biophys. Actu, in press. Pace-Asciak, C. R., and Rangaraj. G. (1977) Biochim. Biophys. Acta. 486. 579-582.
C. R.
PACE-ASCIAK
BIOCHEMISTRY
81,
??1-7,55
(1977)
Deuterium Isotope Dilution Method for the Specific Measurement of 6-Keto-prostaglandin F,, by Mass Fragmentography We recently reported the isolation of 6-keto-prostaglandin F,, (6KPGF,,) from incubations of rat stomach homogenates in the presence of exogenous arachidonic acid (1). This product was formed in far greater quantities (lo-20 times) than was PGE, in this preparation. It could also be derived from the intermediate prostaglandin endoperoxides PGH, and PGG, (2), indicating its formation by the “PG synthetase.” Its structure was unequivocally proven by comparison with an authentic sample derived through chemical synthesis (2). Because preliminary experiments indicated its presence in several other tissues (3,4), we set out to establish a simple, specific, and sensitive method for its quantitation by mass fragmentography, a method now used quite routinely in the assay of other prostaglandins (5-7) and many other biologically occurring products (8- 10). Pentadeuterated 6K-PGF1, was prepared from octadeuterated [5,6,8, 9,11,12,14, 15-2H,] arachidonic acid by a combined biochemical and chemical approach. Octadeuterated arachidonic acid was converted into heptadeuterated PGE, by conventional methods using microsomes of sheep seminal vesicles (ll), and the product was isolated and purified by thin-layer chromatography (tic). The purified compound was reduced with sodium borohydride in methanol, and the resulting heptadeuterated PGFza isomer was purified again by tic after prior conversion into the methyl ester derivative with diazomethane. The pure compound was chemically converted in good yield into methyl 6K-PGF,, (U. Axen, personal communication, method to be published). The final product which contained five deuterium atoms (positions 8,11,12,14, and 15) was purified by tic and was used in the assay. 6K-PGFi, displays good gc properties as the MeMOTMS derivative and is well resolved from PGE, (Fig. I). Its mass spectrum shows a multiplicity of ions of high intensity in the high end of the spectrum (Fig. 2), providing excellent potential for use in mass fragmentography. Fragment ions at m/e 508 and 513 [M-(90 + 31)] were selected for the protonated and deuterated forms because these ions were quite prominent in the mass spectrum (see Fig . 2 for the Do form). The loss of (90 + 31) from the molecular ion of the deuterated compound gives rise to two fragment ions, i.e., m/e 513 and 512, the latter being formed by the eliminaC opynghf 35 1977 by Academic t’res. Inc. All rights of reproductmn m any form reserved.
ISSN 0003-2697
252
SHORT
COMMUNICATIONS
FIG. I. Gas chromatographic separation of PGE, and 6K-PGF,, as the MeMOTMS derivatives. A 6ft x 0.25 in. column of 3%’ SE-30 on 100/120-mesh Gas Chrom Q was used at 24O”C, and 0.6 pg of each component was injected. 100
0 100
200
300
400
500
600
m/e FIG. 2. Mass spectrum of the authentic D,,-6K-PGF,, and the Dj product reported in this paper as the MeMOTMS derivative. A Varian MAT CH-5 coupled gas chromatograph-mass spectrometer-computer assembly was used. The operating conditions were: gas chromatograph oven at 265°C. separator at 280°C. source at 27O”C, electron multiplier at 2.5 kV. ionization voltage of 70 eV.
SHORT
253
COMMUNICATIONS
FIG. 3. Actual record of a mass fragmentogram of a mixture of 20 ng of protonated compound in 1 pg of deuterated 6K-PGF,,,. Fragments at rr~/e 508 and 513 were monitored using a two-channel peak-matching unit (Varian) and were displayed as shown on a Statos recorder. This concentration could be quantitated with an accuracy of i- 1.Y;.
I
tion of one deuterium atom with the trimethylsilyl group. The fragment at HZ/C 508 can also be used in conjunction with an assay for PGE, (M-31 fragment), since chromatographic resolution of both compounds can easily be accomplished on a nonpolar silicone phase column (Fig. 1). The amount of protonated background in the D, product was found to be 34 ng of D,,/l pg of D, product. This is not surprising since the initial part of the synthesis involved a biochemical approach. Figure 3 shows the relative intensities of the fragments at m/c 508 and 513 in a mixture containing 30 ng of protonated compound in 1 pg of deuterated carrier. A standard line of the ratios observed for peak heights for the protium and deuterium forms versus the composition of injected material is shown in Fig. 4.
~~ 0
~-' 50
100
ng Do in lpg D5 GK-PGF,, FIG. 4. Standard line constructed from injection of a mixture of D,, in I pg of D, 6K-PGF,,. Response (peak height) versus composition of mixture injected is shown. Since the fragment selectedfortheD,product i.e., M-(90 + 31). is split int0twopeaks.i.e.. ~nic 512(37’7,)and513 (63%). the composition of the protium component trrric 508) had to be compensated accordingly. The reproducibility of this fragmentation was -+ 0.05? (five determinations). Data in the figure have been corrected for a D,, background of 3.47;
254
SHORT
Incubation 20 4
FIG. fundus Tissue directly samples
10’ 37”
COMMUNICATIONS
-
-
+ -
+ +
5. Quantitation of the 6K-PGF,, present in fresh homogenates of the rat stomach after incubation in the presence and absence of exogenous arachidonic acid (100 kg/g). was homogenized in 0.05 M KH,PO,-NaOH (20 vol. pH 7.4) and was extracted (or after incubation) with ethanol (5 vol). Carrier 6K-PGF,, ( 1 wg. Dj) was added, and were worked up as described in the text.
The assay consists of the addition of 1 pg of Dj-6K-PGF,, as carrier as the methyl ester (or the free acid) to an ethanol extract of a tissue sample or homogenate. The total extract is methylated with diazomethane, and the sample is purified on tic. Using chloroform/methanol/acetic acid/water (90:9:1:0.6.5, v/v) as developing solvent, the Rf is 0.45 (2). The sample is eluted from the silica gel with methanol, and the product is converted into the methoxime trimethylsilyl derivative as previously reported (2). The method was applied to an investigation of the capacity of rat stomach fundus homogenates to biosynthesize 6K-PGF,,. The amounts of 6KPGF,, detected are representative of the combined capacity of the prostaglandin endoperoxide synthetase and the prostagiandin 6(9)oxy cyclase (12). As shown in Fig. 5, the amounts of 6K-PGF, in unincubated homogenates are quite small, as compared to the amounts formed after incubation, especially when exogenous arachidonic acid is added prior to the incubation. The large amounts formed under the latter conditions are indicative of the high capacity of this tissue to be directed along the 6(9) oxy pathway of endoperoxide catabolism. No other tissue tested forms equal or larger amounts of 6K-PGF,, than the fundus of the rat stomach, which is principally directed to this product, PGE, and PGF.,, being almost undetected (13). The assay can be greatly improved if a deuterated derivative with a lower protium content is prepared chemically as for other prostaglandins (background about 0.5%). Also. if the product contained only four deuterium atoms, it would be possible to quantitate PGE, (available as the
SHORT
COMMUNICATIONS
25.5
D, derivative) and 6K-PGF,, in the same sample through a single injection, since prior resolution of both compounds can be obtained by gas chromatography. Further work along these lines is under way. ACKNOWLEDGMENTS This research was supported by a grant from the Medical Research Council of Canada (No MA-4181). I thank Mr. Lajos Marai for operating the Varian Mat CH-5 mass spectrometer assembly, an MRC regional facility at the Best Institute, University of Toronto. Authentic unlabeled 6K-PGF,, was generously provided by Dr. U. Axen. the Upjohn Co., Kalamazoo, Mich.
REFERENCES 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13.
Pace-Asciak. C. ( 1976) &perirnlia 32. 291-392. Pace-Asciak. C. (1976) J. Amer. Churn. Sot. 98, 1348-2349. Pace-Asciak. C., and Nashat, M. (1976) Proc. Canard. Fed. Biol. SW. 19, 268. Dawson. W.. Boot. J. R., Cockerill. A. F.. Mallen. D. N. B., and Osborne. D. J. (1976) Nature (London) 262, 699-702. Samuelsson. B.. Hamberg. M.. and Sweeley, C. C. (1970) Anal. Biochem. 38, 3Ol304. Green. K., Granstrom. E., Samuelsson. B., and Axen, U. (1973) Anal. Biochrm. 54, 434-453. Nicosia. S.. and Galli. G. (1974) Am/. Biochem. 61, 192-199. Brandenberger. H., and Schnyder, D. (1972) Z. Anul. Chem. 261, 297-305. Jenden. D. J. (1973) in Advances in Biochemical Psychopharmacology (Costa. E., and Holmstedt. B., eds.). Vol. 7. pp. 69-81, Raven Press, New York. Cattabeni, F.. Koslow. S. J., and Costa. E. (1972) Science 178, 166- 168. Wlodawer, P., and Samuelsson, B. (1973) J. Eiol. Chem. 248, 5673-5678. Pace-Asciak, C. R., and Nashat. M. (1977) Biochim. Biophys. Actu, in press. Pace-Asciak, C. R., and Rangaraj. G. (1977) Biochim. Biophys. Acta. 486. 579-582.
C. R.
PACE-ASCIAK
