ANALYTICAL BIOCHEMISTRY 93, 339--345 (1979)

Determination of Cyclooxygenase Products and Prostaglandin Metabolites Using High-Pressure Liquid Chromatography

and Radioimmunoassay I

IFTEKHAR ALAM, 2 K A Z U O OHUCHI, 3 AND LAWRENCE LEVINE 4

Department of Biochemistry, Brandeis University, Waltham, Massachusetts 02154

Received July 19, 1978

A method is described for measurement of the cyclooxygenase products, thromboxane, prostacyclin, and prostaglandins (PG), and several prostaglandin metabolites. The procedure involves separation of the compounds by high-pressure liquid chromatography combined with identification and estimation by serologic analysis. These combined procedures have been used to identify and estimate five such products, PGEz, PGEx, PGF2~, PGFI~, and 6-keto- PGFt~ in the culture fluids of dog kidney cells stimulated by a tumor-promoting phorbol diester. The prostaglandin metabolites, 13,14-dihydro-15-keto-PGEz, 13,14-dihydro-15-keto- PGF~, 13,14-dihydro-PGE2, and 13,14-dihydro-PGF~, were not found in these culture fluids.

Tumor-promoting phorbol diesters stimu- late deacylation of cellular lipids and pros- taglandin production in dog kidney (MDCK) 5 cells (1,2). Among the arachidonic acid metabolites produced by MDCK cells are PGF~ and PGE. These are identified by radioimmunoassay and by their rates of flow when subjected to thin-layer chromatog- raphy. The presence of thromboxane was not looked for since the tissue culture experiments were performed with media

1 This work was supported by Grant HD-07966 from the National Institute of Child Health and Human De- velopment and Grant CA-17309 from the National Can- cer Institute. This is Publication 1222 from the Depart- ment of Biochemistry, Brandeis University, Waltham, Mass. 02154.

Supported by Training Grant AI-07069 from the National Institute of Allergy and Infectious Diseases.

3 On leave from the Department of Biochemistry, Faculty of Pharmaceutical Sciences, Tohoku Univer- sity, Sendal 980, Japan.

4 Research Professor of Biochemistry of the Ameri- can Cancer Society (Award PRP-21). Author to whom reprint requests should be sent.

5 Abbreviations used: PG, prostaglandin; MDCK, canine kidney cells; hplc, high-pressure liquid chroma- tography; RIA, radioimmunoassay; TPA, 12-O-tetra- decanoyl-phorbol- 13-acetate.

containing 10% fetal bovine serum that most likely contained thromboxanes. Nor could we demonstrate the biosynthesis of prosta- cyclin since our radioimmunoassay for 6- keto-PGFl~ did not differentiate between 6- keto-PGFi~ and PGF~, and our thin-layer chromatographic system did not separate PGE2 from 6-keto-PGFa~.

Identification of arachidonic acid metabo- lites with antibodies depends on the sero- logic specificity of the antibodies. For ex- ample, our anti-PGF2~ and anti-PGE2 are specific for PGF~ and PGE, but they do not differentiate the dienoic from the monoenoic prostaglandins (3). If PGF2~ can be separated from PGFI,, or if PGE2 can be separated from PGEa, serologic analyses can be used to identify and quantify both the dienoic and monoenoic acids. Also, the anti-PGD2 used in this study is relatively nonspecific in that it reacts 8% with 13,14-dihydro-PGE2 and PGE2 and 4% with PGF~, it does not react with the 15-keto-prostaglandins. As will be shown in this paper, if PGD2 is separated from the cross-reacting 13,14-dihydro-PGE2, PGEz, and PGF2~, even this "nonspecific" antiPGDz can be used to quantify PGD2. We

339 0003-2697/79/040339-07502.00/0 Copyright © 1979 by Academic Press, Inc. All rights of reproduction in any form reserved.

340 ALAM, OHUCHI, AND LEVINE

have employed high-pressure liquid chro- matography to separate the prostaglandins (4,5) and have used the antibodies to quantify the resolved compounds in order to combat the uncertainty that results from the use of antibodies with undesirable specificity.

MATERIALS AND METHODS

The serologic specificities of many of the antisera used in this study have been de- scribed previously (3). However, where the cross-reactions are relevant to our analyses, they will be reemphasized.

The radiolabeled prostaglandins were purchased from New England Nuclear, Bos- ton, Massachusetts, and Amersham, Com- pany, Chicago, Illinois. The radiolabeled thromboxane B2 was a gift from Dr. Bernhard Peskar, University of Freiburg, Freiburg, West Germany. The unlabeled prostaglandins were gifts from Dr. Udo Axen, the Upjohn Company, Kalamazoo, Michigan. The 6-keto-PGF~ used for prep- aration of the anti-6-keto-PGFa~ and for standards was a gift from Dr. K. C. Nicolaou, University of Pennsylvania, Phil- adelphia.

For the high-pressure liquid chromatog- raphy, the Waters Associates, Milford, Massachusetts, instrument was used. The columns employed were 30 cm × 3.9 mm i.d. filled with silicic acid particles, 10/~m in diameter (/~-Porasil), and a 30-cm × 3.9- mm-i.d, reversed-phase column (Fatty Acid Analysis Column, Waters Associates, Mil- ford, Mass).

The cell culture procedures have been de- scribed in detail elsewhere (2). In brief, MDCK cells (2 × 105 cells/60-mm dish) were treated for 1 h with TPA (0.001/zg/ml) followed by three washes to remove residual TPA; the washed cells were incubated for 24 h in Eagle's minimal essential medium supplemented with 10% fetal bovine serum. For resolution by hplc, the culture fluids were extracted first for neutral lipids with petroleum ether followed by extraction with

ether at pH 3.2. The extract was washed with H~O (three times the volume of extract). After drying with anhydrous NazSO4, the ether layer was evaporated under N~ at room temperature and the residual extract was resolved on silicic acid and fatty acid analysis columns separately following the procedure of Whorton et al. (5). For resolution on the silicic acid column, the arachidonic acid metabolic products were eluted for 60 min with a linear gradient of CHCla to 6% methanol and 0.6% acetic acid in CHC13 (5). The flow rate was 1 ml/min/tube. For resolution on the fatty acid analysis column, the arachidonic acid metabolites were eluted isocratically with a solvent system containing 76.7% H~O, 23.0% CH3CH, 0.2% benzene, and 0.1% acetic acid (5). The flow rate was 2 milmin/tube. The fractions were analyzed for 6-keto- PGFI~ and PGF~ with anti-6-keto-PGFt~; PGE2 and PGEx with anti-PGE~; and PGF2~ with anti-PGF~.

RESULTS

Arachidonic acid, the primary prostaglan- dins, PGE, PGF~, and PGD2, and several of their metabolites are resolved by hplc on a silicic acid column (Fig. 1). The immuno- chromatogram (the unlabeled standards identified and quantified serologically) is shown in Fig. 1A, and the chromatogram of the radiolabeled standards is shown in Fig. lB.

Resolution by reversed-phase hplc of ra- diolabeled and unlabeled standards identified and quantified with antibodies is shown in Fig. 2. For these analyses, two previously unpublished radioimmunoassays, one for 6- keto-PGFl~ and the other for thromboxane B2, were used; the serologic specificities for these two immune reactions are shown in Tables 1 and 2, respectively. Whether measured by radioactivity or by radioim- munoassay, the separated standards cor- responded fairly well. The retention times after resolution by normal- or reversed-

HPLC AND RIA ANALYSIS FOR PROSTAGLANDINS 341

1.2

I.O

~ o.8 o

" 0.6

o.

== 0.4 1

02.

f

IO

v

20 30 Fraction Number

4 0 50 6 0

IO

0 -~ 8

P, ~- 6 ?,

4

E 2

B < [ t ¢ l :=~

=2- £ -

J , L , ~ J ,

I0 20 50 40 50 60 Fraction Number

FIG. 1. Separation of some arachidonic acid metabolites by normal-phase hplc. Experimental pro- cedures for the hplc are given under Materials and Methods. With both unlabeled (A) and radiolabeled (B) standards, individual metabolites, or in some experimental runs mixtures of metabolites, were added to the appropriate solvent prior to injection. Several recovery experiments from the normal-phase hplc were performed with the [3H]PGF2~, [3H]PGE2, PGFz~, and PGE2. These recoveries varied from 70 to 85%. All of the data were corrected for the average loss. Only the major peaks of radioactive standards and serologic activities are shown. For RIA of the unlabeled standards, aliquots containing subnanogram quantities were used, because with the levels injected (microgram quantities) cross- reactions with many of the metabolites would have been found. Several antisera were used for assay of all fractions, and in most cases only one major peak was seen after dilution to subnanogram quantities. Abbreviations used in the figures: TXBz, thromboxane Bz; H2Fz~, 13,14-dihydroprostaglandin F2~; HzE2, 13,14-dihydroprostaglandin E2; Hzl5KF2~, 13,14-dihydro-15-keto-prostaglandin F~; H215KEz, 13,14-dihydro-15-keto-prostaglandin E2; AA, arachidonic acid.

phase hplc were reproducible usually within 2 but always within 3 min.

Polycyclic and heterocyclic aromatic hy- drocarbon carcinogens, aflatoxin B1 (6), epi- dermal growth factor (7), and tumor-pro- moting phorbol esters (1,2) stimulate canine kidney cells to deacylate cellular lipids and to produce prostaglandins. One of the most effective stimulants ofprostaglandin biosyn-

thesis is the tumor promoter, 12-O-tetra- decanoyl-phorbol- 13-acetate.

Shown in Fig. 3 are the immunochromato- grams of arachidonic acid metabolites pro- duced by control MDCK cells and TPA-stim- ulated cells determined after high-pressure normal and reversed-phase chromatography. The cyclooxygenase products, PGE2, PGE1, PGF2~, PGFI~, and prostacyclin (in the form

342 ALAM, OHUCHI, AND LEVINE

1.2

1.0

"~ 0.8

" 0.6 n

~ 0.4 IL

0.2

I0 60

=

I-- "I" :Z - -

2 0 3 0 4 0 5 0

Fraction Number

t~

u. 4 ID a.

E 2

IO 20 :50 40 50 60 Fraction N u m b e r

FIG. 2. Separation of some arachidonic acid metabofites by reversed-phase hplc. Experimental procedures for the bplc are given under Materials and Methods. Individual, or in some experiments mixtures of unlabeled (A) and labeled (B) metabolites, were added to the bplc solvent just prior to injection. Several recovery experiments from the reversed-phase hplc were run with [3H]PGF2~, [3H]- PGE2, PGF2~, PGE2, and 6-keto-PGFl~. These recoveries varied from 75 to 90%. All of the data were corrected for the average loss. Only the major peaks of radioactive materials and serologic activities are shown, although all of the fractions were assayed with some antisera, and after appropriate dilutions of the fractions only one major peak of serologic activity was seen. With anti-6-keto-PGFl~, two peaks corresponding to 6-keto-PGFl~ and PGFI~ were found when both were chromatographed. The sensitivity of detection varied with the immune systems used, but in general, at least 50 pg of ligand could be detected. For the 6-keto-PGFl~ standard shown in (A), anti-6-keto-PGFl~ (the serologic speci- ficity of which is shown in Table 1) was used. The average recovery in two experiments was 85%, most of the unlabeled 6-keto-PGF~ chromatographed as a single peak.

o f i ts n o n e n z y m a t i c p r o d u c t , 6 - k e t o - P G F ~ ) we re ident i f ied a f te r r e s o l u t i o n b y hp lc o f the e x t r a c t s o f cu l tu re f luids o f the T P A - s t imu la t ed M D C K cel ls . In t hese exper i - men t s , PGE2, PGF2~, and 6 -ke to -PGFl~ were s t imu la t ed 1 5 - 2 5 t imes b y T P A t r ea tmen t . Such s t imu la t ions b y T P A t rea t - m e n t have b e e n o b s e r v e d a f te r a n a l y s e s o f cu l tu re fluids for PGE2 and PGFz~ b y R I A

wi thou t hplc (1,2). The leve ls o f PGE2, PGF2~, PGE1, PGFI~, and 6-keto-PGFx~ [after t r e a t m e n t o f the cel ls wi th T P A (1.0 ng/ml) for 1 h and a f te r r e m o v a l o f the r e s idua l TPA] that had accumulated in the media during the 24-h i nc uba t i on in s e r u m - s u p p l e m e n t e d m e d i u m are s h o w n in T a b l e 3. S e v e r a l m e t a b o l i c p r o d u c t s o f p r o s t a g l a n d i n s we re no t f o u n d in this T P A - s t i m u l a t e d m e d i u m .

HPLC AND RIA ANALYSIS FOR PROSTAGLANDINS 343

DISCUSSION

There are several examples that illustrate the advantages of the combined hplc-RIA procedure. Some of these are: (i) The anti- PGE2 does not distinguish the dienoic from the monoenoic acids. PGE2 and PGE1 chro- matograph with identical retention times on normal-phase hplc, but they are separated on reversed-phase hplc. Thus, both PGE2 and PGE1 can be quantified with the anti- PGE2. (ii) Anti-15-keto-PGF2~ (which reacts <1% with PGD2 and 13,14-dihydro-PGE2), anti-PGD2 (which reacts < 1% with 15-keto- PGF2~ and 8% with 13,14-dihydro-PGE2), and anti-PGE2 (which reacts 100% with 13,14-dihydro-PGE~, but <0.01% with 15- keto-PGF2~ and PGD~) were used to quantify PGD2, 13,14-dihydro-PGE~, and 15-keto- PGF2~ even in the chromatographic peak unresolved by normal-phase hplc. It should be noted here that PGD2, 13,14-dihydro- PGE2, and 15-keto-PGF~ were present at similar concentrations. For positive identifi- cation and quantification in a biological sample, however, PGD2, 13,14-dihydro- PGE2, and 15-keto-PGF2~ would have to be resolved by reversed-phase hplc before serologic analyses. (iii) PGE2 and 6-keto- PGF~ are not separated on normal-phase hplc, at least with the solvent that was used.

TABLE 1

SEROLOGIC SPECIFICITY OF THE [zH]PGFI~ ANTI-6-KETo-PGFx~ BINDING

REACTION

Ligand

Amount required for 50%

inhibition (ng)

6-keto-PGFl~ PGFI~ PGF2~ PGD2 PGE1 PGE2

0.14 0.25

11.0 12.0

110.0 > 100.0 a

Zero inhibition with 100 ng.

TABLE 2

SEROLOGIC SPECIFICITY OF THE [ZH]THROMBOXANE B2-ANTI-THROMBOXANE B 2 REACTION

Ligand

Amount required for 50%

inhibition (ng)

Thromboxane B2 0.075 PGDz 50.0 PGF2~ > 100.0 PGEz > 100.0

The anti-PGE2 and anti-6-keto-PGFl~ are sufficiently specific to quantitate them even in the unresolved fractions if they are not present at grossly unequal levels (anti-PGE2 reacts 0.02% with 6-keto-PGFl~ and anti-6- keto-PGFl~ reacts <0.1% with PGE2). But PGE~ and 6-keto-PGFl~ are separated by reversed-phase hplc (Figs. 2A, 3) and thus their levels can be estimated. The results of the quantitative analyses of PGE2, 6-keto- PGFI~, and PGF~ were similar regardless of the chromatographic separation procedure used.

Some antisera may be sufficiently specific to identify and quantify products without separation procedures. For example, the anti-13,14-dihydro-15-keto-PGE2 cross-re- acts 7% with 15-keto-PGE2, 5% with 13,14- dihydro-15-keto-PGF~, 2% with 13,14-di- hydro-15-keto-PGA~ (8), and <1% with all the other known prostaglandins or prosta- glandin metabolites. Thus, it is not surprising that only fractions with retention times around 20 min (normal-phase hplc, Fig. 1A) or around 46 min (reversed-phase hplc, Fig. 2A) inhibited this immune system. Of course, even with this relatively specific antiserum, if about 10 times more 13,14-dihydro-15-keto- PGF2~ were present, fractions with re- tention times around 25 min (Fig. 1A) and around 40 min (Fig. 2A) would react; or if, for example, 200 times more PGE2 were pres- ent (PGE2 cross-reacts 0.1%), fractions con- taining PGEz would react. Anti-13,14-dihy-

344 ALAM, OHUCHI, AND LEVINE

"L-I )19

I ,n

uo!l.::)o.~.:l Jocl Ou

~3+s3 e ~ - - - - ~ ' ~

I ~o

I I

uo!.l.:)o.L-I Sad Ou

0

o~ .~_

B:, 4

PL.-I)I 9

El I

uoH.=o.L.-I ,~td ()u

"L-I ~ 9 , , ~ 13+ z3

rn

uo!,~ooJ..-I JOd Ou

~ . = ~ b ~

L 0 I~ g~

o ~ : ~ ~ "~ 0 ,,,= "E~ 0

~ .~ " ~1 ~ I=

o ~ ~

HPLC AND RIA ANALYSIS FOR PROSTAGLANDINS

TABLE 3

IMMUNOCHROMATOGRAPHIC ANALYSES OF CYCLOOXYGENASE PRODUCTS OF MDCK CELLS a

345

TPA stimulated Control

Cyclooxygenase Reversed Normal Reversed Normal product phase phase phase phase

PGE2 + PGE1 - - 17 - - 0.3 PGE2 11 - - 0.7 - - PGE1 2 - - - - - - PGF2~ 17 15 1.0 0.7

PGFI~ 3 3 - - - - 6-keto-PGFx~ 7 6 0.3 0.2 Thromboxane B2 . . . . 13,14-H2-15-keto-PGE2 b b _ __ 13,14-H2-15-keto-PGF2~ b b __ __ 13,14-H2-PGF2~ b b __ __ 13,14-H2-PGE2 b b __ __

a The data were calculated from the experiments shown fluid. - - , not analyzed.

b <0.1.

in Fig. 3 and are expressed in ng/mt culture

dro-15-keto-PGF2~ is another example of a relatively specific antiserum that can be used without fractionation of most biological flu- ids. This antiserum cross-reacts 6% with 15- keto-PGF2~ and 1% with 13,14-dihydro-15- keto-PGE2 (3). It reacts only with fractions with retention times around 25 min (Fig. 1A) and 39 rain (Fig. 2A), unless 10 to 50 times more of the cross-reacting derivatives are present in the unfractionated sample.

Positive identification and estimation of some cyclooxygenase products and prosta- glandin metabolites require knowledge of the retention times of the compounds. For example, the anti-PGE2 reacts equally with PGE2, PGA2, and PGB2 (3). If PGE2, PGB2, and PGA2 were present in a biological sample resolved by normal phase hplc and all 60 fractions were assayed with anti- PGE2, fractions 15 to 17, 18 to 20, and 32 to 36 would be positive. Since PGA2, PGB2,

and PGE2 are separable, PGE2 is identified in fractions 32-36. With an antiserum that reacts specifically with PGBz, not with PGA2 or PGEz, only fractions having retention times around 15-18 min would inhibit.

REFERENCES

1. Levine, L., and Hassid, A. (1977) Biochem. Bio- phys. Res. Commun. 79, 477-484.

2, Ohuchi, K., and Levine, L. (1978) J. Biol. Chem. 253, 4783-4790.

3. Pong, S. S., and Levine, L. (1977) in The Prosta- glandins (Ramwell, P. W., ed.), Vol. 3, pp. 41-76, Plenum, New York.

4. Hubbard, W. C., and Watson, J. T. (1976) Pros- taglandins 12, 21-35.

5. Whorton, A. R., Smigel, M., Oates, J. A., and Frrlich, J. C. (1978)Biochim. Biophys. Acta 529, 176-180.

6. Levine, L. (1977) Nature (London) 268, 447-448. 7. Levine, L., and Hassid, A. (1977) Biochem. Bio-

phys. Res. Commun. 76, 1181-1187. 8. Levine, L. (1977) Prostaglandins 14, 1125-1139.