Separation by high performance liquid chromatography of arachidonic acid metabolites from rabbit platelets

S E P A R A T I O N BY H I G H P E R F O R M A N C E LIQUID CHROMATOGRAPHY OF ARACHIDONIC ACID METABOLITES FROM RABBIT PLATELETS MIKE VANROLLINS,SAMUELH. K. Ho, JAMESE. GREENWALD,~"MICHAELALEXANDER,'~ NANCY J. DORMAN, LAN K. WONG~: and LLOYDA. HORROCKS Department of Physiolo~iical Chemistry and "fPharmacolo~ly, The Ohio State University, Columbus, Ohio 43210 and ~Pharmaceutics Department, University of Pittsburgh, Pittsbur~ih, Pennsylvania 15261, U.S.A.

INTRODUCTION

High performance liquid chromatography (HPLC) has recently been used to isolate underivatized prostaglandins from biological sources. One approach involves sequential use of two columns with different selectivities. T M Because multiple chromatographic steps were involved, maximal recoveries were only 70--80%.~ Partial resolution of prostaglandins has also been reported using various single-step HPLC separations, t'4'6'9'l°'t2 No single-column procedure has provided a complete separation of thromboxane B2 (TxB2), 6 keto-Ft~, the prostaglandins F2,, E2, and D2, and the hydroxylated derivatives 12-L-hydroxy-5,8,10-heptadecatrienoic acid (HHT) and 12-Lhydroxy-5,8,10,14-eicosatetraenoic acid (HETE). These metabolites are rapidly and completely resolved by the present method. This HPLC system may be used either quantitatively or qualitatively.

MATERIALS AND METHODS

The HPLC system consisted of a 25 x 0.46 (ID) era Ultrasphere-ODS (5/a particles, Altex) column attached to a 4 x 0.46 (ID) cm Spherisorb-ODS (5/z particles, Altex) guard column. Developing solvents were CH3CN (HPLC grade, Baker Chemical Co.) and H 2 0 (pH adjusted to 2.0 with H3PO4). Gradient chromatography was done using a Model 322 pump system (Altex) equipped with a variable wavelength detector (LC-75, Perkin Elmer) set at 192 nm. Prostaglandin standards and 5,8,11,14-eicosatetraynoic acid (ETYA) were generously provided by Drs. J. E. Pike (Upjohn Co.) and W. E. Scott (Hoffman-LaRoche), respectively. Radioactive HHT and 12-HETE were synthesized by incubation of [1-1'~C]arachi donic acid (AA) with platelets. After separation by thin layer chromatography (TLC), these standards were identified by gas chromatography-mass spectrometry (GC-MS). 2 Platelets were used to generate labeled cyclooxygenase and lipoxygenase products for quantitative comparisons between HPLC and TLC. Rabbit platelets (400/~g of protein) were washed according to Minkes et al., s then incubated at 37°C for 15-30 min in the presence of 200-1000 ng of [I-~4C]AA. After the pH was adjusted to 3.5 with 1.0 N HCI, products were extracted with ethyl acetate. The solution was dried under N2 and the products were redissolved in CHaOH. For HPLC, 2-9/~g of 6K-F~*, TxB2, F2,, E2, D2 and AA were added to platelet extracts to act as markers for retention times. For the first 28 min following injection of sample (20/zl), the CHaCN concentration was 31% (v/v). Linear increases in CHaCN concentrations were initiated at 28 rain to 75% over 12 min, and at 52 min to 95% over 4 min. The column was reequilibrated at 31~ CHaCN for 35 min prior to reuse. Collected fractions (0.7 ml) were counted in 4.3 ml of Ready-Solv EP (Beckman) using a Model 8100 counter (Beckman). 783

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~ . 1. ~P~C separation of arachidonic acid mctabolites ~rom rabbit platclets. Twent~ pl o~ ~0~ containin~ meta~olitcs [rom 3~ n~ of [l-~C]arach~donic acid and 2-~pg each o[ unlabeled prosta~landins an~ arachidonic acid, was injected onto an ~ltrasphere-ODS column (4.~ mm x ~5 cm). Elution was carried out at ~ ~ usin~ 3 1 - ~ % C ~ C ~ in aqueous phosphoric acid (p~ ~ ZO~ w~th a flow rate o~ I.~m]/min. Fractions of O.Tml were collected [or determinations of radioactivity.

For TLC, products containing 17,000 dpm in 50/al of CH3OH were spotted on LK-6D plates (5 x 20 cm, 250/~, Whatman). The plates were developed according to the methods of Ho et al. 5 and of Hamberg et al., ~ scanned (Model 7720, Packard Instruments), marked, scraped and counted in 10 ml of ACS-II (Amersham, Arlington Heights). RESULTS

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DISCUSSION

Baseline separations of underivatized cyclooxygenase and lipoxygenase products were achieved within 70min (Fig. 1). Major metabolites were identified by comparing: (1) retention times to those of standards, (2) distribution of radioactivity after HPLC to that obtained after TLC, and (3) the effects of indomethacin and ETYA on the distribution of TABLE !. Comparison of H P L C and T L C for Separation of Radioactive Metabolites from Arachidonic Acid Incubated with Rabbit Platelets HPLC ~ Solvent Front TxB., F2, E2 D2 ab HHT b 12-HETE c AA

6.1 21.3 0.7 1.4 1.8 5.9 14.0 4.6 37.7 4.5 2.1

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~ Each value represents the mean percentage + SD I n - - 4 ) . An average of 99.6 + 7.5% of the radioactivity applied to the column was recovered in three other H P L C runs. Each peak was collected in one fraction. b Letters correspond to fractions in Fig. 1.

Separation by high performance liquid chromatography

785

radioactivity. Indomethacin prevented the labeling of all components except 12-HETE, AA and those in the intervening Fraction c (Fig. 1). ETYA prevented the labeling of all components except the AA and phospholipid (solvent front) fractions. With boiled platelets, only AA was labeled suggesting an enzymic origin for all other components. Thus, both major and minor enzymic products of arachidonic acid could be detected by HPLC. The distribution of label in metabolites was also determined (Table 1). HPLC values for TxB2, E2 and AA agree well with TLC determinations. In contrast, TLC values for 12-HETE are 21% higher and for HHT are 9% lower than the corresponding HPLC values. Such differences are probably due to incomplete resolution of underivatized 12-HETE and HHT by conventional TLC methods) The accuracy of HPLC determinations was reaffirmed when HPLC results were compared with values obtained by argentation TLC. 2 HHT was completely resolved from 12-HETE in the latter TLC system. Thus, this new HPLC system provides rapid, accurate and precise determinations of all major AA metabolites found in platelets.

REFERENCES 1. CARR, K., SWEETMAN,B. J. and FROLICH, J. C. Prostatclandins II, 3-14 (1976). 2. GREENWALD,J. E., ALEXANDER,M., VANROLLINS, M., WONG, L. K. and BIANCHINE, J, R. Prosta,qlandin.s 21, 3. 4. 5. 6. 7. 8. 9. 10. 11. 12,

33-39 (1981). HAMBERG,M., SVENSSON,J. and SAMUELSSON,B. Proc. Natl. Acad. Sci. U.S.A. 71, 3824-3828 (1974). HILL, G. T. J. Chromatogr. 176, 407-412 11979). Ho, P. P. K.. WALTERS, P. and SULLIVAN, H. R. Prosta~tlandins 12, 951-970 11976), HUBBARD,W. C. and WATSON, J. T. Prostaglandins 12, 21-35 (1976). HUBBARD,W. C., WATSON, J. T. and SWEETMAN, B. J. In Biological Applications of Liquid Chromato,qraphy, Vol. 10, pp. 31-55. (G. HAWK, ed.) Marcel Dekker, New York, 1979. MINKES, M., STANFORD, N., CHI, M. M-Y., ROTH, G. J., RAZ, A., NEEDLEMAN, P. and MAJERUS, R. W. J. Clin. Ire'est. 59, 449-454 (1977), NAGAYO, K. and Mlzuuo, N. J. Chromato,qr. 178, 347-349 (1979). RUSSELL, A. F. and DE'~KIN, D. Prosta~llandins 18, 11-18 0979). WHORTON, A. R., CARR, K., SMIGEL, M., WALKER, L., ELLIS, K. and DATES, J. A. J. Chromato.qr. 163, 64-71 (1979). WYNALDA, M. A., LINCOLN. F. H. and FITZPATRICK, F. A. J. Chromato.qr. 176, 413-417 (1979).

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