[41] ω- and (ω-1)-hydroxylation of eicosanoids and fatty acids by high-performance liquid chromatography

432

ENZYMEASSAYS

[41]

[41] to- a n d (to-1)-Hydroxylation of E i c o s a n o i d s a n d F a t t y Acids b y H i g h - P e r f o r m a n c e L i q u i d C h r o m a t o g r a p h y

By RICHARD T. OKITA, JOAN E. CLARK, JANICE RICE OKITA, and BETTIE SUE SILER MASTERS Introduction Cytochromes P450 of the IVA family catalyze the hydroxylation of a number of endogenous substrates including prostaglandins (PG), leukotriene B4 (LTB4), and fatty acids at their to and to-1 carbon atoms. 1 to-Oxidation is a major route in prostaglandin metabolism, with a large proportion of prostaglandins being excreted in the urine as to-oxidized products. 2 In certain tissues, hydroxylation of prostaglandins may form unique eicosanoid derivatives; for example, primate seminal fluid contains abundant quantities of 19-hydroxy-PGEl and 19-hydroxy-PGE2,3 and lung microsomes from pregnant rabbits contain a highly active prostaglandin to-hydroxylase.4 Hydroxylation of LTB4 to 20-hydroxy-LTB4 in human polymorphonuclear leukocytes represents a major route of inactivation for this eicosanoid. 5-7 Although a number of constitutive and inducible forms of cytochrome P450 which hydroxylate fatty acids have been identified,8'9 the function(s) of most of the to- and (to-l)-hydroxy fatty acids in mammalian organs is not yet known, to-Hydroxylated arachidonate has recently been reported to constrict rat aortic rings I° and dog small renal v e s s e l s . 11 Plant cells also contain a fatty acid to-hydroxylase which is

I B. S. S. Masters, A. S. Muerhoff, and R. T. Okita, in "Mammalian Cytochromes P-450" (F. P. Guengerich, ed.), p. 107. CRC Press, Boca Raton, Florida, 1987. 2 C. R. Pace-Asciak and N. S. Edwards, J. Biol. Chem. 255, 6106 (1980). 3 p. L. Taylor and R. W. Kelly, Nature (London) 250, 665 (1974). 4 W. S. PoweU, J. Biol. Chem. 253, 6711 (1978). 5 W. S. Pow¢ll, J. Biol. Chem. 259, 3082 (1984). 6 S. Shak and I. M. Goldstein, J. Biol. Chem. 259, 10181 (1984). 7 R. J. Soberman, R. T. Okita, B. Fitzsimmons, J. Rokach, B. Spur, and K. F. Austen, J. Biol. Chem. 2539 12421 (1987). 8 D. Kupfer, Pharmacol. Ther. 11, 469 (1980). 9 j. M. Hawkins, W. E. Jones, F. W. Bonner, and G. G. Gibson, Drug Metab. Rev. 18, 441 (1987). 10 B. Escalante, W. C. Sessa, J. R. Falck, P. Yadagiri, and M. L. Schwartzman, J. Pharmacol. Exp. Ther. 248, 229 (1989). II Y.-H. Ma, J. E. Clark, D. R. Harder, B. S. Masters, and R. J. Roman, Pharmacologist, 32, 32 (1990).

METHODS IN ENZYMOLOGY,VOL. 206

Copyright© 1991by AcademicPress, Inc. All rightsof reproductionin any formreserved.

[41]

EICOSANOID AND FATTY ACID HYDROXYLATION

433

involved in the synthesis of cutin, a major component of plant waxes. 12 Oxidation at the to end of the molecule produces compounds which are readily separable by reversed-phase high-performance liquid chromatography (RP-HPLC) on octadecasilyl (ODS or C~8) columns. This chapter describes the RP-HPLC procedures for separating to- and (to-1)-hydroxy derivatives of saturated and unsaturated fatty acids, prostaglandins, and 15-hydroxyeicosatetraenoic acid (15-HETE). Procedures Instrumentation. Chromatographic separations presented here were obtained with a Varian (Walnut Creek, CA) 5020 or 5060 HPLC system, and the radiolabeled substrates and products were detected by a Packard (Meriden, CT) Radiomatic Flo-One radioactivity detector and the data analyzed by Radiomatic software program FIB CU (Version 6a.001.0000). We have obtained similar results with two other HPLC systems, an LKB (Piscataway, N J) 2249 and the Beckman (Fullerton, CA) System Gold. Column. Separation procedures and retention times described in this chapter are for a 150 x 4 mm 5 ~m Bio-Sil ODS column (ODS-5S) and ODS-5 guard column (40 × 4.6 ram) which were purchased from Bio-Rad (Richmond, CA). The column flow rate is 0.7 ml/min. Comparable results have been obtained with the longer 250 x 4 mm Bio-Rad columns with a flow rate of 1 ml/min. Solvents. HPLC-grade acetonitrile and methanol as well as ethyl acetate were purchased from Burdick and Jackson (Muskegon, MI). Acetonitrile and methanol were passed through a Gelman (Ann Arbor, MI) DM Metricel 0.45 /zm pore size filter before use. Ethyl acetate and other organic solvents, if stored for extended periods and especially after being opened, should be examined for peroxides before use. The procedure described by Kates 13 was used for the detection of peroxides. Water was obtained from a Millipore (Bedford, MA) purification system consisting of one carbon cartridge, two ion-exchange cartridges, and an Organex-Q cartridge. Water was filtered through a 0.45/zm HV Millipore filter. Fatty Acid and Eicosanoid Substrates. Sodium laurate, sodium myristate, and palmitic acid were purchased from Sigma (St. Louis, MO). Stearic and arachidonic acids were from NuChek Prep (Elysian, MN). PGE 1 and 15-HETE were obtained from Cayman (Ann Arbor, MI). [1J4C]Lauric acid, -myristic acid, -stearic acid, and -arachidonic acid were

12p. E. Kolattukudy, R. Croteau, and J. S. Buckner, in "Chemistry and Biochemistry of Natural Waxes" (P. E. Kolattukudy, ed.), p. 289. Elsevier, Amsterdam and New York, 1976. t3 M. Kates, in "Techniquesof Lipidology:Isolation, Analysis,and Identificationof Lipids," 2nd Ed., p. 80. Elsevier, Amsterdam and New York, 1986.

434

ENZYME ASSAYS

[41]

purchased from Amersham (Arlington Heights, IL). [5,6-3H(N)]PGE1 and 15-[5,6,8,9,11,12,14,15-3H(N)]HETE were obtained from New England Nuclear (Boston, MA). The labeled arachidonic acid was purified by RPHPLC before use. A sample of approximately 8 ~Ci arachidonic acid is dried down and redissolved in 75/zl of 75% (v/v) acetonitrile and injected on a 250 x 4 mm Bio-Sil ODS-5S column which is equilibrated in 50% acetonitrile. A 40-min gradient from 50 to 100% acetonitrile is run and held at 100% acetonitrile for an additional 10 min. The eluant is collected between 28 and 33 rain in 0.5-ml fractions and aliquots taken for scintillation counting. Fractions with radioactivity are collected in a siliconized screw-top test tube and extracted immediately by adding 1 volume of deionized water and 4 volumes of ethyl acetate. The sample is vortexed and the ethyl acetate phase separated by centrifuging. The ethyl acetate layer is transferred to another tube and the aqueous layer reextracted with another 3 volumes of ethyl acetate. The ethyl acetate phases are combined, dried under a stream of nitrogen, stored under argon at - 8 0 °, and used within 1-2 weeks. Other labeled compounds are used without further purification. Enzymatic Reactions and Sample Preparations. Glassware used for enzymatic reactions and sample preparation should be siliconized to prevent adsorption of the fatty acids to the glass and maximize recovery of the substrates and metabolites. Otherwise, losses of substrates and/or metabolites result in erroneous estimations of rates of conversion. Tubes, vials, Pasteur pipettes, or other glassware are rinsed or immersed in 2.5% dimethyldichlorosilane (DMCS, Sigma) in chloroform (v/v), dried overnight at room temperature, then rinsed once with chloroform, then twice with methanol. The solution of DMCS may be reused for a number of tubes. If screw-top test tubes and vials are used, the caps should be Teflonlined. An alternative siliconizing procedure is to treat glassware with a 5% solution of DMCS in toluene, which is then followed by rinsing with methanol. 14 Hydroxylated products are formed enzymatically by incubating fatty acids or prostaglandins with lung microsomes from pregnant rabbits, liver microsomes from diethylhexyl phthalate (DEHP)-treated rats, or purified cytochromes P450 from these tissues in reconstituted enzyme systems. Reactions are normally performed in incubations of 0.5 or 1 ml as described by Williams et al.15 and Okita et al. 16The reactions are terminated by the 14D. D. Blumberg, in "Guide to Molecular Cloning Techniques" (S. L. Berger and A. R. Kimmel, eds.), p. 20. Academic Press, San Diego,California, 1987. is D. E. Williams, S. E. Hale, R. T. Okita, and B. S. S. Masters, J. Biol. Chem. 253, 14600 0984). 16 R. T. Okita, R. J. Soberman, J. M. Berghoite, B. S. S. Masters, R. Hayes, and R. C. Murphy, Mol. Pharmacol. 32, 706 (1987).

[41]

EICOSANOID AND FATTY ACID HYDROXYLATION

435

addition of HCI and the acidified solution extracted twice with 3 volumes of ethyl acetate. Incubations containing saturated fatty acids as substrates are terminated by the addition of 0.1-0.2 ml of 1 N HCI. It has been observed that if reactions which contain lauric acid as substrate are not acidified prior to the addition of ethyl acetate, acetoxy derivatives of the l 1-OH and 12-OH products are formed during the extraction process. ~7 For incubations which contain unsaturated fatty acids, 15-HETE, or prostaglandins, care must be taken when adding the HC1 to monitor the pH of the sample to prevent the pH from dropping below 3. Substantial degradation of the sample may occur, which is manifested by large amounts of radioactivity eluting at the solvent front. The ethyl acetate phases are combined in siliconized tubes and evaporated under a stream of nitrogen. If the sample is from a reconstituted cytochrome P450 system, the sample is redissolved in 50-100/zl of the appropriate solvent (i.e., 75% methanol for laurate or 75% acetonitrile for palmitate), a 10- to 50-/~1 aliquot is injected onto the RP-HPLC column, and a 5- to 10-tzl aliquot is taken for radioactivity counting to determine extraction efficiency. If the sample is from a microsomal incubation, it is redissolved in 3 ml ethyl acetate, 1-2 ml of deionized water added, and the mixture thoroughly vortexed. The ethyl acetate phase is separated by centrifugation and transferred to a 13 x 100 mm siliconized screw-top test tube. The aqueous phase is reextracted with 2 x 2 ml ethyl acetate. The organic phases are combined and evaporated by a stream of nitrogen. Samples are redissolved in 75% methanol or acetonitrile as described above. We normally do not filter the sample prior to HPLC injection. Incubations of decreased volume (200 tzl) are also performed to conserve enzymes, especially when only a small amount of microsomes can be obtained from sources such as COS cells and small renal vessels. In this case, aliquots of 50/~1 are removed at the desired times and mixed with 1-2 volumes of either methanol or acetonitrile. The samples are centrifuged in a microcentrifuge to pellet the precipitated protein, and the supernatant is injected directly onto the RP-HPLC column.

Separation of to- and (to-1)-Hydroxylated Metabolites A dual-pump HPLC system is used which delivers solvents/solutions from two reservoirs, A and B, at a combined flow rate of 0.7 ml/min. For most of the fatty acids and eicosanoids we study, solvent A is the organic solvent (either 100% methanol or 100% acetonitrile), and solution B is 17 A. S. Salhab, J. Applewhite, M. W. Couch, R. T. Okita, and K. T. Shiverick, Drug Metab. Dispos. 15, 233 (1987).

436

[41]

ENZYME ASSAYS TABLE I SEPARATION OF HYDROXYLATED METABOLITES BY ELUTION a Conditions

Substrate and solvents

Initial

Gradient

Retention (min) Final

ca-1

ca

Substrate

Laurate A: 100% Methanol B: 0.2% Acetic acid in water Laurate (alternative procedure) A: 100% Acetonitrile B: 0.2% Acetic acid in water

62% A, 38% B, 18 min

Isocratic

100%A

13

15

26

35% A, 65% B, 5 min

100% A

14.9

16.3

31

Myristate A: 100% Methanol B: 0.2% Acetic acid in water Palmitate A: 100% Acetonitrile B: 0.2% Acetic acid in water Dicarboxylic acid elutes at 8 rain

66% A, 34% B, 28 min

35-55% A over 10 rain, hold 2.5 rain Isocratic

100% A

23

26

40

100% A

12

13

29

Arachidonate A: 100% Acetonitrile B: 0.2% Acetic acid in water Prostaglandin Et A: 0.4% Benzene in acetonitrile B: 0.4% Acetic acid in water 15-HETE A: 0.4% Benzene in acetonitrile B: 0.4% Acetic acid in water

48% A, 52% B, 30 rain

100% A

24

26.5

43

--

3.5

7.5

--

4.5

14

60% A, 40% B, 3 rain

60-80% A over 12.5 rain, to 100% A over 3.4 min Isocratic

40% A, 60% B

Isocratic

60% A, 40% B

Isocratic

Same as initial conditions Same as initial conditions

Column: 150 × 4 mm 5 pm ODS column with 40 x 4.6 mm ODS guard column (Bio-Rad), flow rate 0.7 ml/min.

0.2-0.4% acetic acid in Millipore-filtered water (v/v). For some eicosanoids, 0.4% benzene is added to the organic solvent in reservoir A, resulting in sharper elution peaks. An isocratic mixture of the two solvents is used while the sample is loaded on the column. At this step, the proportion of organic solvent and aqueous solution is varied according to the substrate used and other conditions such as column usage. Appropriate resolution of the hydroxylated products is achieved in many cases with an isocratic elution; however, linear gradient elution is used in some cases for expediency. In most cases, after elution of the hydroxylated products, the solvent is changed to 100% solvent A to elute the unmetabolized substrate peak quickly. The column is then reequilibrated with the initial solvent mixture before injection of the next sample. See Table I for complete descriptions of elution conditions used for each substrate. Separation o f l l - and 12-OH-Laurate. For laurate, the elution is isocratic at 62% methanol and 38% Solution B for the initial 18 min. The

[41]

EICOSANOID AND FATTY ACID HYDROXYLATION I

I

1

150x4mm Bio-Sil ODS

I

I

Lauric

12-•H•

1000-

[

Acid-

I"

L

.........,.....

:z o. O

['

437

-~.,J

-100 -80

o C

-60

500 -

F 40

•

20

0

I

I

4

8

III

12

I,

16

20

I 24

Ll 28

0

Time (mln)

FIG. 1. Separation of 1 l-OH-laurate, 12-OH-laurate, and laurate by RP-HPLC as described in Table I.

11- and 12-OH-laurates elute with retention times of 13 and 15 min, respectively (Fig. 1). At 18 min the solvent system is changed to 100% methanol to elute the unmetabolized lauric acid which has a retention time of 26 min. An alternative elution procedure utilizes acetonitrile (see Table I). Aoyama and Sato Is have reported that p-bromophenacyl may be used to derivatize the OH-laurate products which may then be separated on normal-phase or RP-HPLC columns and products monitored with a UV detector. A small amount of 10-OH laurate has also been detected in the l l-OH-laurate peak in reactions catalyzed by rat liver microsomes. If separation of the 10-OH-laurate is needed, the HPLC procedure of Romano et al.19 is suggested. Separation of to- and (to-1)-OH Fatty Acids. The to- and (to-D-OH derivatives of other fatty acids (myristate, palmitate) are also separated by RP-HPLC using conditions similar for the 11- and 12-OH-laurates (see Table I). In some cases, a small amount of the dicarboxylic acid (DCA) product is formed which elutes just prior to the (to-1)-OH product. With 18 T. Aoyama and R. Sato, Anal. Biochem. 170, 73 (1988). 19 M. C. Romano, K. M. Straub, L. A. P. Yodis, R. D. Eckardt, and J. F. Newton, Anal. Biochem. 170, 83 (1988).

438

ENZYME ASSAYS ! 60:40

t

t

80:20

100:0

[41]

! 6-OH

15 -OH

:i O.

o

DCA

t 0

10

2"0

30

MINUTES (ELUTION OFF OOS COLUMN) FIG. 2. Separation of 15-OH-palmitate, 16-OH-palmitate, and palmitate by RP-HPLC as described in Table I. DCA represents the dicarboxylic acid derivative of palmitate, hexadecadioic acid. Full scale is 500 cpm.

palmitic acid as the substrate, the DCA (hexadecadioic acid) is formed as shown in Fig. 2. Separation of 19- and 20-OH-Eicosatetraenoic Acids (HETE). The 19and 20-OH products of eicosatetraenoic acid (arachidonic acid) may also be separated by RP-HPLC using conditions described in Table I. Briefly, the solvent is 48% acetonitrile and 52% solution B (v/v) for 30 min to elute 19- and 20-HETE at 24 and 26.5 min, respectively. The solvent is then changed to 100% acetonitrile to elute the unmetabolized arachidonic acid at 43 rain (Fig. 3). Capdevila et al. 2° have also described the separation of 19- and 20-HETE by first isolating the HETEs by RP-HPLC and then separating the 19-HETE from the 20-HETE by normal-phase HPLC. Separation of 19- and 20-Hydroxyprostaglandins. To study cytochrome P450-mediated hydroxylation of PGE 1, we have used two HPLC systems, depending on the source of the cytochrome P450 and the products formed. When lung microsomes from pregnant rabbits are used, prostaglandins are converted predominantly to the 20-OH derivative with only negligible amounts of 19-OH products formed. Reservoir A contains 0.4% benzene in acetonitrile (v/v), and reservoir B contains 0.4% acetic acid in water (v/v). The HPLC is programmed to run an isocratic mixture of 40% 20 j. Capdevila, Y. R. Kim, C. Martin-Wixtrom, J. R. Falck, S. Manna, and R. W. Estabrook, Arch. Biochem. Biophys. 243, 8 (1985).

[41]

EICOSANOID AND FATTY ACID HYDROXYLATION

439

20-OH

X a. O

19-OH

A o

1'o

'

2'0

' ;o

'

io

MINUTES (ELUTION OFF ODS COLUMN) FIG. 3. Separation of 19-HETE, 20-HETE, and arachidonic acid by RP-HPLC as described in Table I. Full scale is 500 cpm.

solvent A and 60% solution B. The 20-OH-PGE1 elutes at 3.5 min, and the unmetabolized PGEI elutes at 7.5 min from the 150 × 4 mm Bio-Rad ODS-5 column. This HPLC system will allow for product separation by an isocratic solvent system without switching to 100% organic solvent and reequilibration of the column, saving time and solvents. When studying cytochromes P450 which catalyze the hydroxylation of PGE~ at both the 19- and 20-carbon atoms such as those from liver and kidney microsomes, we have used the methanol-water-acetic acid HPLC procedure described by Powell. 2~ Reservoir A contains 100% methanol, and Reservoir B contains 0.2% acetic acid in water; the HPLC is programmed to deliver 50% solvent A and 50% solution B to elute the 19- and 20-OH-PGE~ products. The unmetabolized PGE~ is eluted after switching the solvent to 100% methanol.

Separation of 15,20-DihydroxyeicosatetraenoicAcid (15,20-diHETE). Separation of 15,20-diHETE from the substrate, 15-HETE, can be obtained with an isocratic solvent system composed of 60% solvent A (0.4% benzene in acetonitrile) and 40% solution B (0.4% acetic acid in water) (Table I). 12-Hydroxyeicosatetraenoic acid (12-HETE) may also undergo to-hydroxylation, and a HPLC procedure for the separation of 12,20-di21 W. S. Powell, this series, Vol. 86, p. 168.

440

ENZYME ASSAYS

HETE from 12-HETE has been described by Wong

[41] e t al. 22 and

Marcus

et

al. 23

Comments

The enzymatic rates of formation of products are calculated based on the percentage of radioactivity for the given metabolite relative to the total radioactivity eluting from the HPLC column. This method requires that the substrates and products are recovered with equal efficiency. It is important to determine extraction efficiencies to be certain substrates and products have been extracted by the ethyl acetate from the aqueous phase. It is also critical to ascertain that the radioisotope detector has the same radioisotope efficiency with both the products and substrate. We have never observed a change in efficiency with a change in solvent composition. However, we have observed a change in efficiency depending on the amount of radioactivity. Efficiencies are lower and more variable with 100-500 counts/rain (cpm) per peak, depending on radioisotope detector, solvent composition, and scintillation cocktail used, and should be verified for each set of conditions. We have sometimes found that following the injection of several samples, the OH fatty acid peaks lose their sharpness. To improve resolution, chloroform can be injected with the column equilibrated in 100% methanol. For the laurate studies, for instance, we routinely inject 25-50/~l chloroform every fifth sample when the column is equilibrated in 100% methanol. Resolution of peaks will also decrease because of column usage, but separation between peaks may still be obtained by decreasing the solvent strength slightly. The elution conditions listed in Table I may also require modification due to column-to-column variations. Greater variations of elution conditions may be required when other substrates are used. To develop a method for separation of OH products from a different fatty acid or eicosanoid use the following procedure. (1) Start with the HPLC conditions for the substrate closest to the new substrate, for example, for stearate choose the conditions for palmitate. (2) If the OH products are eluted quickly but are not separated, decrease the solvent strength by decreasing the proportion of organic solvent in the initial solvent mixture. Large changes in solvent composition (5-10% steps) can be undertaken until a point is reached where the OH products are retained on the column 22 p. Y.-K. Wong, P. Westlund, M. Hamberg, E. Granstrom, and P. H.-W. Chao, J. Biol. Chem. 259, 2683 (1984). 23 A. J. Marcus, L. B. Sailer, H. L. Ullman, M. J. Broekman, N. Islan, T. D. Oglesby, and R. R. Gorman, Proc. Natl. Acad. Sci. U.S.A. 81, 903 (198,*).

[42]

P450 ARACHIDONICACID EPOXYGENASE

441

for a relatively long time (45 min). (3) At this point, increase the solvent strength in small steps (1-2%) until the separation and elution times are optimized. By this procedure, variations on the basic elution conditions summarized in Table I can be applied for the determination of a wide variety of fatty acid and eicosanoid to- and (to-1)-hydroxylations catalyzed by c y t o c h r o m e s P450. Acknowledgments This work was supported by National Institutes of Health Grants ES 03771 (RTO) and GM 31296(BSSM). J.E.C. was a Parker B. Francis Fellow in pulmonary research during the time in which these studies were performed. B.S.S.M. is The Robert A. Welch Foundation Professor in Chemistry at the University of Texas Health Science Center at San Antonio.

[42] C y t o c h r o m e P 4 5 0 A r a c h i d o n i c A c i d E p o x y g e n a s e : Stereochemical Characterization of Epoxyeicosatrienoic Acids By J O R G E H. C A P D E V I L A , E L I Z A B E T H D I S H M A N , A R M A N D O KARARA,

and J. R. FALCK Introduction The c y t o c h r o m e P450 epoxygenase catalyzes the N A D P H - d e p e n d e n t epoxidation o f arachidonic acid (AA) to 5,6-, 8,9-, 11,12-, and 14,15-cisepoxyeicosatrienoic acids (EETs) according to the following stoichiomettic relationship: AA + NADPH + 02 H ÷ --, EET + NADP÷ + H20 The epoxygenase regio- and stereoselectivity o f oxygenation is under the control o f the c y t o c h r o m e P450 protein catalyst.l'2 The potential functional significance of this metabolic pathway has been highlighted by (a) the potent biological activities of the E E T s 3 and (b) the demonstration of a role for the hemoprotein in the in vivo metabolism of endogenous arachidonic acid. 1 The enantioselective nature o f the endogenous E E T pools 1 A. Karara, E. Dishman, I. Blair, J. R. Falck, and J. H. Capdevila, J. Biol. Chem. 264, 19822 (1989).

2j. H. Capdevila, A. Karara, D. Waxman, M. V. Martin, J. R. Falck, and F. P. Guengerich, J. Biol. Chem. 265, 10865 (1990). 3 F. A. Fitzpatrick and R. C. Murphy, Pharmacol. Rev. 40, 229 (1989), and references therein. METHODS IN ENZYMOLOGY,VOL. 206

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