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Urinary excretion of diols derived from eicosapentaenoic acid during N-3 fatty acid ingestion by man
PROSTAGLANDINS
URINARY EXCRETION OF DIOLS DERIVED FRC~ EICOSAPENTAENOIC ACID DURING N-3 F A T T Y A C I D INGESTION B Y M A N H.R. Knapp 1, A.J. Miller and J.A. Lawson Division of Cllnical Pharmacology, Vanderbilt Universlty, Nashville, TN 37232-6602. USA Abstraot Epoxides and fatty acid diols derived from arachidonate by the action of cytochrome P-450 appear in human urine and have biological activities. Dietary eicosapentaenoic acid gives rise to prostaglandins in vivo, but vascular effects of n-3 supplements do not all correlate with altered types or amounts of in vivo cyclooxygenase products. We investigated whether dietary eicosapentaenoic acid could also be metabolized by cytochrome P-450, by assessing the excretion of its vicinal diols. Utilizing gas chromatography/negative chemical ionization mass spectrometry, we have found that humans ingesting n-3 fatty acids excrete vicinal diols of eicosapentaenoic acid in substantial quantities. Introduction
Although originally described a decade ago (i,2), the cytochrome P-450 metabolites of arachidonic acid have only recently been confirmed to be endogenously synthesized by humans (3), and to have many in vitro activities to release hormones (4), affect the vasculature (5,6), and alter ion (7) and water (8) transport. The biochemistry and pharmacology of this class of mediators has been reviewed (9,10), and recent reports have suggested a role for this class of compounds in human pathophysiology (ii). With the surge of interest in the health benefits of marine oils containing n-3 polyunsaturated fatty acids (12,13), it has been shown that eicosapentaenoic acid (EPA) can also be metabolized by cytochrome P-450 i_nn vitro (14), and to be converted in vivo to a prostacyclin (15,16) and thromboxane (16) analogous to those formed from arachidonate. In some cases, these analogs possess similar biological activities to those derived from arachidonate, while in others there is a marked disparity between the two (17). Alterations in the endogenous formation of cyclooxygenase products induced by n-3 fatty acid ingestion are believed to be important in the resultant in vivo tendency to reduced platelet activation (16) and thrombosis (18). A number of other effects of dietary fish oil, such as the lowering of blood pressure, have not been readily explained by alterations in prostaglandin synthesis (19,20). In this IPresent address: Div. of Clinical Pharmacology, Dept. of Internal Medicine, Univ. of Iowa, Iowa City, IA 52240; to whom reprint requests should be addressed.
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PROSTAGLANDINS study, we have examined the urine of subjects before and during ingestion of fish oil supplements to determine whether cytochrome P-450 metabolites of eicosapentaenoic acid are formed in vivo. Materials
and Methods
Six healthy male subjects ingested 17 ml per day of an enriched n-3 ethyl ester concentrate derived from menhaden oil, providing approximately 9 gm eicosapentaenoic acid (EPA) daily, for one month. Urine collections were made with refrigeration for 24 hour periods prior to and at the end of the fourth w e e k of the n-3 supplementation. The study was approved by the Committee for the Protection of Human Subjects of Vanderbilt University. Standards of arachidonate-derived epoxides (Cayman Chemical, Ann Arbor, MI) were available to verify retention times and mass spectral characteristics of both epoxides and vicinal diols. Known amounts of the (8,9), (11,12) and (14,15) epoxides prepared from octadeuterated arachidonic acid (AA) using metachloroperoxybenzoic acid (7, a kind gift of Mr. Eric Petty, Vanderbilt Dept. of Pharmacology) were added to the urine samples. Since the epoxides and corresponding diols are present in urine in variable proportions (7), the urine samples were acidified overnight with 7% perchloric acid to convert all of the epoxides present to the diols. After neutralization and purification over C18 Sep-Paks (Supelco, Bellefonte, PA), the diols in the ethyl acetate eluates were purified by thin-layer chromatography (TLC), developing silica gel plates twice in heptane: ethyl acetate: acetic acid (25:15:0.3). This resolves the 8,9 diols of AA and EPA (Rf=0.26) from their 11,12 and 14,15 diols (Rf=0.34), and the 5,6 (AA and EPA) and 17,18 (EPA) diols (Rf=0.20). The diols were eluted from the TLC bands with ethyl acetate and derivatized with e-bromo-pentafluorotoluene (Aldrich, Milwaukee, WI) and diisoproplyamine to the pentafluorobenzyl esters as reported for other eicosanoids (16). Three different hydroxyl function derivatives were made to confirm the identity of the compounds on gas-chromatography/ negative chemical ionization mass spectrometry in the selected-ionmonitoring (SIM) mode, with methane as reagent gas on a Nermag 10-10C instrument with a i0 meter SPB-I column (Supelco) (16). The trimethylsilyl (TMS) ethers were formed with (bis) trimethylsilyl trifluoroacetamide (Supelco) and the derivatives of EPA, AA and octadeuterated AA were measured by SIM at m/z= 479,481, and 489, respectively. The nbutylboronates were formed with i% (w/w) n-butylboronic acid (Aldrich) in acetonitrile: dimethylformamide (4:1) for 20 min at room temperature, purified by TLC, and monitored at m/z= 401, 403, and 411, respectively. Finally, the t-butyldimethylsilyl (T-BuDMS) derivatives were formed by treatment with t-butyl-dimethylsilanol/imidazole (Supelco) in dimethylformamide at 40°C for 18 hours, purified from reagents by TLC, and monitored at m/z= 563, 565, and 573. ester
48
In addition to the above, samples of the 8,9 diol-PFB fraction were also chromatographed on TLC plates
JULY 1991 VOL. 42 NO. 1
PROSTAGLANDINS i m p r e g n a t e d with 10% AgNO 3 developed in ethyl a c e t a t e : m e t h a n o l (49:1) to separate the EPA (Rt=0.10) and AA (RI=0.23) derivatives. The resolved compounus were then d e r i v a t l z e d to their t r i m e t h y l s i l y l ethers and analyzed by SIM as above. RESULTS
Figure 1 shows the SIM tracings of the ii,12 and 14,15 diol f r a c t i o n as the (bis)TMS e t h e r - P F B esters. During i n g e s t i o n of the n-3 fatty acid supplements, peaks appear in the m/z=479 channel similar to those from the a r a c h i d o n i c acid d e r i v a t i v e being m o n i t o r e d in m/z=481, but d i f f e r i n g by two mass units, c o n s i s t e n t with an additional double bond.
m/z=479
m/z=481
~' ' ~ « 2;õ e'''' 2 ~ '~'-'-2;-? PRE FISH O I L
~=;o"' ';= ;2"" ~' :, ó x ' " ~=; x POST
FISH O I L
Figure i. S e l e c t e d - i o n - m o n i t o r i n g of the 11,12 and 14,15 diols derived from EPA, AA, and o c t a d e u t e r a t e d AA (m/z= 479, 481 and 489, respectively) as their (bis) TMS e t h e r - P F B esters. Shows tracings from urine collected prior to (PRE) and after 4 weeks of (POST) i n g e s t i o n of the supplement c o n t a i n i n g EPA. Peaks clearly a n a l o g o u s to those in the m/z=481 (AA-derivative) channel are not seen in before EPA ingestion, w h e r e a s the peaks r e p r e s e n t i n g the EPA d e r i v a t i v e are obvious afterwards. Figure 2 d e m o n s t r a t e s that a putative EPA d e r i v a t i v e also appears in the 8,9 diol fraction during fish oil ingestion. Panel A shows an SIM trace of the n - b u t y l b o r o n a t e - P F B ester derivatives, with the prominent (M-181-) ions (loss of PFB) at a p p r o p r i a t e m/z values for the vicinal diols d e r i v e d from
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49
PROSTAGLANDINS epoxides. Scans of these Deaks also showed the ratio of doublet signals (1:4..2 --.. ~°B:IIB) at adjacent m/z values expected for boron-containing compounds; for straight-chain dihydroxy fatty acids, boronates form under these conditions when hydroxyls are within two carbons of each other (21). In panel B a r e SIM traces for t-Bu-DMS derivatives of EPA and AA diols at m/z 84 units higher than for the TMS derivatives, consistent with two hydroxyl groups in the molecules. m/z=563
B
~ ~ ~
m/z=565
,~
_~-,-,~
_~e-~.~.~~
34
m/z=573
m/z=411
,
3'3O
3,30
PRE
FISH
OIL
POST
4'OO
FISH
OIL
,
. . . . .
i
3,30
PRE FISH OIL
--T
,
,
~
*
5~30
«-
"r~,
~«r
~
,
4:00
,
'
i
,
4,30
POST FISH OIL
Figure 2. Selected-ion-monitoring traces of the 8,9 diols derived from EPA, AA and octadeuterated AA. Panel A shows the three compounds (m/z= 401, 403 and 411, respectively) as their n-butylboronate-PFB esters, before (PRE) and after (POST) ingestion of the EPA-containing supplement. Panel B shows SIM tracings of the same compounds as their (bis)t-butyl-DMS ether-PFB esters (m/z = 563, 565, 573, respectively). Both derivatives show appearance of the EPA-derived species in the urine during EPA ingestion. After resolution of the EPA and AA diols on argentation TLC, the TLC bands were extracted, derivatized to their TMS ether, PFB esters, and yielded characteristic (M-181") ions at m/z=479 for the EPA-diol and m/z=481 and 489 for AA and octadeuterated-AA diols, respectively (not shown). Although deuterated internal standards for the EPAderived diols are not currently available, an attempt was made to quantify both the EPA- and AA-derived analogs of several diols by SIM using the deuterated AA-diol internal standards. The excretion of both types of compounds before and at the end of ingesting the fish oil concentrate is presented in Table I. It appears that regioisomers of EPA are present in
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PROSTAGLANDINS
A
B
m/z=479
m/z=481
m/z=489
EPA DIOL BAND
AA DIOL BAND
Figure 3. Shows SIM t r a c i n g s from individual i n j e c t i o n of E P A - d i o l f r a c t i o n and AA- + d e u t e r a t e d A A - d i o l f r a c t i o n from a single urine sample, r e s o l v e d on a r g e n t a t i o n TLC. Panel A i n d i c a t e s lower b a n d (EPA-diol, m/z=479) w h i l e i n j e c t i o n for Panel B c o n t a i n s the latter two c o m p o u n d s (m/z=481 and 489). The a r g e n t a t i o n TLC p r o v i d e d e s s e n t i a l l y c o m p l e t e r e s o l u t i o n of the two fractions.
The e x c r e t i o n of both types of c o m p o u n d s by the s u b j e c t s b e f o r e and at the end of ingesting the fish oil c o n c e n t r a t e is p r e s e n t e d in Table i. It appears that r e g i o i s o m e r s of EPA are p r e s e n t in d i f f e r e n t p r o p o r t i o n s than those of AA. Also, there a p p e a r s to be a n o n - s i g n i f i c a n t d e c l i n e in the amounts of the A A - d e r i v e d compounds when those from EPA are b e i n g released, r e s u l t i n g in a higher overall e x c r e t i o n of this class of compounds.
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PROSTAGLANDINS T a b l e i. Intake
Excretion
of
EPA-
and
AA-Derived
Before
Diols
with
N-3
After
Diols
AA
EPA
AA
EPA
8,9
48.3-+19.6
n.d.
44.5-+23.3
16.7-+ 8.0
11,12
25.6+ 6.5
n.d.
21.8+ 5.2
22.3_+ 5.5
14,15
22.5+ 7.4
n.d
19.0+ 4.8
7.7_+ 4.5
V a l u e s are means±sem, in ng excreted/24 h o u r s p r i o r to (Before) and at the end of (After) 4 w e e k s of d i e t a r y n-3 supplements, n . d . = n o n e d e t e c t e d above limits of approx. 0.05 ng/24 hours. Discussion T h e s e studies d e m o n s t r a t e for the first t i m e t h a t d i o l s of EPA, a n a l o g o u s to those b e l i e v e d to be f o r m e d by c y t o c h r o m e P-450 from AA, are excreted by humans i n g e s t i n g fish oil supplements. The a p p r o p r i a t e r e t e n t i o n times and p e a k shapes of three different derivatives on capillary gas chromatography, m i g r a t i o n on a r g e n t a t i o n TLC, and i n f o r m a t i o n on functional groups, their r e l a t i v e positions, and m o l e c u l a r w e i g h t s p r o v i d e d by mass spectrometry, appear to e s t a b l i s h the p r e s e n c e of E P A - d e r i v e d vicinal diols in h u m a n u r i n e w i t h a h i g h degree of confidence. Similar v a l u e s for t h e s e diols were o b t a i n e d from urines c o l l e c t e d in the p r e s e n c e of an antioxidant, so it is u n l i k e l y that the c o m p o u n d s we have m e a s u r e d w e r e formed ex vivo. Such c o m p o u n d s in u r i n e could p o s s i b l y o r i g i n a t e in the kidney, but f u r t h e r w o r k will be needed to define the m e c h a n i s m and locus of t h e i r formation. We h a v e r e p o r t e d that similar p h a r m a c o l o g i c d o s i n g w i t h n-3 p o l y u n s a t u r a t e s lowers the b l o o d p r e s s u r e of p a t i e n t s w i t h m i l d essential hypertension, but that this e f f e c t was not e x p l a i n e d by the a l t e r a t i o n s found in the e n d o g e n o u s s y n t h e s i s of thromboxanes, p r o s t a c y c l i n s or p r o s t a g l a n d i n E (20). The r e d u c t i o n in p l a t e l e t activation and t h r o m b o x a n e s y n t h e s i s by fish oils appears to account for the a m e l i o r a t i o n of p l a t e l e t v a s c u l a r i n t e r a c t i o n s (15) and a n t i - t h r o m b o t i c e f f e c t s (16) p r e v i o u s l y described. Other b e n e f i c i a l effects, such as r e d u c t i o n in r e s t e n o s i s rates after c o r o n a r y a n g i o p l a s t y (21), have been noted in patients taking aspirin, w h i c h by itself o b l i t e r a t e s t h r o m b o x a n e p r o d u c t i o n by platelets. Therefore, v a s c u l a r b e n e f i t s found in such p a t i e n t s w o u l d not d e p e n d u p o n c y c l o o x y g e n a s e p r o d u c t s of n-3 fatty acids. C y t o c h r o m e P-450 m e t a b o l i t e s of a r a c h i d o n i c a c i d were d e c r e a s e d to a n o n - s i g n i f i c a n t degree w h i l e t h o s e from e i c o s a p e n t a e n o i c acid were increasing, but t h e r e was m u c h v a r i a b i l i t y in this and standards of all of the r e g i o i s o m e r s of AA and EPA d i o l s were not available. T h e r e f o r e , further studies will be n e c e s s a r y to define both the f a c t o r s g o v e r n i n g p r o p o r t i o n s of the AA- and E P A - d e r i v e d r e g i o i s o m e r s formed,
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PROSTAGLANDINS as well as the d i f f e r e n c e s in b i o l o g i c a l a c t i v i t i e s of the c y t o c h r o m e P-450 m e t a b o l i t e s of e i c o s a p e n t a e n o i c acid.
Acknowledgments This w o r k was supported in part by a g r a n t from the N a t i o n a l I n s t i t u t e s of Health (HL-35380). Dr. K n a p p is an E s t a b l i s h e d I n v e s t i g a t o r of the A m e r i c a n H e a r t A s s o c i a t i o n .
Referen¢es i. Oliw, E.H., J.A. Lawson, A.R. A r a c h i d o n i c A c i d M e t a b o l i s m in Biol. Chem. 256:9924. 1981.
Brash, Rabbit
and J.A. Oates. R e n a l Cortex. J.
2. Capdevila, J., N. Chacos, J. Werringler, R. Prough, and K.W. Estabrook. Liver M i c r o s o m a l C y t o c h r o m e P450 and the O x i d a t i v e M e t a b o l i s m of A r a c h i d o n i c Acid. Proc. Natl. Acad. Sci. U S A 7_88:5362. 1981. 3. Toto, R., A. Siddhanta, S. Manna, B. Pramanik, J.R. Falck, and J. Capdevila. A r a c h i d o n i c Acid Epoxygenase: Detection of E p o x y e i c o s a t r i e n o i c Acids in H u m a n Urine. Biochim. Biophys. Acta, 919:132-139. 1987. 4. Junier, M.-P., F. Dray, I. Blair, J. Capdevila, E. Dishman, J.R. Falck, and S.R. Ojeda. E p o x y g e n a s e P r o d u c t s of Arachidonic Acid Are Endogenous Constituents of the Hypothalamus. Endocrinology, 126:1534. 1989. 5. Proctor, K.G., S. Shatkin, P.M. Kaminski, J.R. Falck, and J.H. Capdevila. M o d u l a t i o n of A r t e r i o l a r B l o o d F l o w by I n h i b i t o r s of A r a c h i d o n i c Acid O x i d a t i o n A f t e r T h e r m a l Injury: P o s s i b l e Role for a Novel Class of V a s o d i l a t o r Metabolites. C i r c u l a t i o n 7/7(5):1185. 1988. 6. Roslowsky, M., J.R. Falck, J.T. Willerson, and W.B. Campbell. Synthesis of L i p o x y g e n a s e and Epoxygenase P r o d u c t s of A r a c h i d o n i c Acid by Normal and S t e n o s e d C a n i n e C o r o n a r y Arteries. Circ. Res. 66:608. 1990. 7. Schlondorff, D., E. Petty, J.A. Oates, M. Jacoby, and S.D. Levine. Epoxygenase Metabolites of A r a c h i d o n i c Acid Inhibit V a s o p r e s s i n Response in Toad Bladder. Am. J. Physiol. 253:F464. 1987. 8. Hirt, D.L., J. Capdevila, J.R. Falck, M.D. Bryer, and H.R. Jacobson. C y t o c h r o m e P450 M e t a b o l i t e s of A r a c h i d o n i c A c i d Are P o t e n t I n h i b i t o r s of V a s o p r e s s i n A c t i o n on R a b b i t Cortical C o l l e c t i n g Duct. J. Clin. Invest. 8__4:1805. 1989. 9. Fitzpatrick, F.A. and R.C. Murphy. C y t o c h r o m e P-450 M e t a b o l i s m of A r a c h i d o n i c Acid: F o r m a t i o n and B i o l o g i c a l A c t i o n s of " E p o x y g e n a s e " - D e r i v e d Eicosanoids. Pharm. Rev. 40~229. 1989.
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PROSTAGLANDINS i0.
Schwartzman, M.L., P. Martasek, A.R. Rios, R.D. Levere, K. Solangi, A.I. Goodman, and N.G. Abraham. Cytochrome P 4 5 0 - D e p e n d e n t A r a c h i d o n i c A c i d M e t a b o l i s m in H u m a n Kidney. K i d n e y Int. 3_/7:94. 1990.
ii.
Catella, F., J.A. Lawson, D.J. Fitzgerald, and G.A. FitzGerald. E n d o g e n o u s B i o s y n t h e s i s of A r a c h i d o n i c A c i d E p o x i d e s in Humans: Increased F o r m a t i o n in P r e g n a n c y I n d u c e d H y p e r t e n s i o n . Proc. Natl. Acad. Sci. U S A 8_/7:5893. 1990. 12. C. Von Shacky. Prophylaxis of A t h e r o s c l e r o s i s W i t h M a r i n e Omega-3 F a t t y Acids: A C o m p r e h e n s i v e Strategy. Ann. Intern. Med. 107:890. 1987. 13. Leaf, A., and P.C. Weber. Cardiovascular Effects n-3 Fatty Acids. N. Engl. J. Med. 318:549. 1988.
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14. VanRollins, M., P.D. Frade, and O.A. Carretero. O x i d a t i o n of 5 , 8 , 1 1 , 1 4 , 1 7 - E i c o s a p e n t a e n o i c A c i d by H e p a t i c and Renal Microsomes. Biochim. Biophy. Acta, 966:133. 1988. 15. Fischer, S., and P.C. Weber. P r o s t a g l a n d i n 13 is Formed In V i v o in M a n A f t e r Dietary E i c o s a p e n t a e n o i c Acid. Nature, 707:165. 1984. 16. Knapp, H.R., I.A.G. Reilly, P. Alessandrini, and G.A. FitzGerald. In Vivo Indexes of P l a t e l e t and V a s c u l a r F u n c t i o n D u r i n g Fish-Oil A d m i n i s t r a t i o n in P a t i e n t s with Atherosclerosis. N. Engl. J. Med. 314:937. 1986. 17. Needleman, P., A. Raz, M.S. Minkes, J.A. Ferrendeli, and H. Sprecher. Triene Prostaglandins: P r o s t a c y c l i n and T h r o m b o x a n e B i o s y n t h e s i s and U n i q u e B i o l o g i c a l Properties. Proc. Natl. Acad. Sci. USA. 76:944. 1979. 18. Braden, G.A., H.R. Knapp, D.J. Fitzgerald, and G.A. FitzGerald. D i e t a r y Fish Oil A c c e l e r a t e s the R e s p o n s e to Coronary Thrombolysis With Tissue-Type Plasminogen Activator. C i r c u l a t i o n 82:178. 1990. 19.
Knapp, H.R., and G.A. FitzGerald. The A n t i h y p e r t e n s i v e Effects of Fish Oil: A C o n t r o l l e d Study of P o l y u n s a t u r a t e d Fatty Acid S u p p l e m e n t s in E s s e n t i a l Hypertension. N. Engl. J. Med. 320:1037. 1989.
20. Knapp, H.R. Omega-3 Fatty Acids, E n d o g e n o u s P r o s t a g l a n d i n s , and Blood Pressure R e g u l a t i o n in Humans. Nutr. Rev. 4_~7:301, 1989. 21. Oliw, E~H. A n a l y s i s of 1,2-Diols of Linoleic, «L i n o l e n i c and A r a c h i d o n i c Acid by Gas C h r o m a t o g r a p h y - M a s s S p e c t r o m e t r y U s i n g Cyclic Alkyl Boronic Esters. J. Chromatogr. 275:245. 1983. 22. Dehmer, G.J., J.J. Popma, E.K. van den Berg, et al. R e d u c t i o n in the Rate of Early R e s t e n o s i s A f t e r C o r o n a r y A n g i o p l a s t y by a Diet S u p p l e m e n t e d with n-3 F a t t y Acids. N. Engl. J. Med. 319:733. 1988. Editor:
54
E. G r a n s t r o m
Received:
12-3-90
Accepted:
3-26-91
JULY 1991 VOL. 42 NO. 1
URINARY EXCRETION OF DIOLS DERIVED FRC~ EICOSAPENTAENOIC ACID DURING N-3 F A T T Y A C I D INGESTION B Y M A N H.R. Knapp 1, A.J. Miller and J.A. Lawson Division of Cllnical Pharmacology, Vanderbilt Universlty, Nashville, TN 37232-6602. USA Abstraot Epoxides and fatty acid diols derived from arachidonate by the action of cytochrome P-450 appear in human urine and have biological activities. Dietary eicosapentaenoic acid gives rise to prostaglandins in vivo, but vascular effects of n-3 supplements do not all correlate with altered types or amounts of in vivo cyclooxygenase products. We investigated whether dietary eicosapentaenoic acid could also be metabolized by cytochrome P-450, by assessing the excretion of its vicinal diols. Utilizing gas chromatography/negative chemical ionization mass spectrometry, we have found that humans ingesting n-3 fatty acids excrete vicinal diols of eicosapentaenoic acid in substantial quantities. Introduction
Although originally described a decade ago (i,2), the cytochrome P-450 metabolites of arachidonic acid have only recently been confirmed to be endogenously synthesized by humans (3), and to have many in vitro activities to release hormones (4), affect the vasculature (5,6), and alter ion (7) and water (8) transport. The biochemistry and pharmacology of this class of mediators has been reviewed (9,10), and recent reports have suggested a role for this class of compounds in human pathophysiology (ii). With the surge of interest in the health benefits of marine oils containing n-3 polyunsaturated fatty acids (12,13), it has been shown that eicosapentaenoic acid (EPA) can also be metabolized by cytochrome P-450 i_nn vitro (14), and to be converted in vivo to a prostacyclin (15,16) and thromboxane (16) analogous to those formed from arachidonate. In some cases, these analogs possess similar biological activities to those derived from arachidonate, while in others there is a marked disparity between the two (17). Alterations in the endogenous formation of cyclooxygenase products induced by n-3 fatty acid ingestion are believed to be important in the resultant in vivo tendency to reduced platelet activation (16) and thrombosis (18). A number of other effects of dietary fish oil, such as the lowering of blood pressure, have not been readily explained by alterations in prostaglandin synthesis (19,20). In this IPresent address: Div. of Clinical Pharmacology, Dept. of Internal Medicine, Univ. of Iowa, Iowa City, IA 52240; to whom reprint requests should be addressed.
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47
PROSTAGLANDINS study, we have examined the urine of subjects before and during ingestion of fish oil supplements to determine whether cytochrome P-450 metabolites of eicosapentaenoic acid are formed in vivo. Materials
and Methods
Six healthy male subjects ingested 17 ml per day of an enriched n-3 ethyl ester concentrate derived from menhaden oil, providing approximately 9 gm eicosapentaenoic acid (EPA) daily, for one month. Urine collections were made with refrigeration for 24 hour periods prior to and at the end of the fourth w e e k of the n-3 supplementation. The study was approved by the Committee for the Protection of Human Subjects of Vanderbilt University. Standards of arachidonate-derived epoxides (Cayman Chemical, Ann Arbor, MI) were available to verify retention times and mass spectral characteristics of both epoxides and vicinal diols. Known amounts of the (8,9), (11,12) and (14,15) epoxides prepared from octadeuterated arachidonic acid (AA) using metachloroperoxybenzoic acid (7, a kind gift of Mr. Eric Petty, Vanderbilt Dept. of Pharmacology) were added to the urine samples. Since the epoxides and corresponding diols are present in urine in variable proportions (7), the urine samples were acidified overnight with 7% perchloric acid to convert all of the epoxides present to the diols. After neutralization and purification over C18 Sep-Paks (Supelco, Bellefonte, PA), the diols in the ethyl acetate eluates were purified by thin-layer chromatography (TLC), developing silica gel plates twice in heptane: ethyl acetate: acetic acid (25:15:0.3). This resolves the 8,9 diols of AA and EPA (Rf=0.26) from their 11,12 and 14,15 diols (Rf=0.34), and the 5,6 (AA and EPA) and 17,18 (EPA) diols (Rf=0.20). The diols were eluted from the TLC bands with ethyl acetate and derivatized with e-bromo-pentafluorotoluene (Aldrich, Milwaukee, WI) and diisoproplyamine to the pentafluorobenzyl esters as reported for other eicosanoids (16). Three different hydroxyl function derivatives were made to confirm the identity of the compounds on gas-chromatography/ negative chemical ionization mass spectrometry in the selected-ionmonitoring (SIM) mode, with methane as reagent gas on a Nermag 10-10C instrument with a i0 meter SPB-I column (Supelco) (16). The trimethylsilyl (TMS) ethers were formed with (bis) trimethylsilyl trifluoroacetamide (Supelco) and the derivatives of EPA, AA and octadeuterated AA were measured by SIM at m/z= 479,481, and 489, respectively. The nbutylboronates were formed with i% (w/w) n-butylboronic acid (Aldrich) in acetonitrile: dimethylformamide (4:1) for 20 min at room temperature, purified by TLC, and monitored at m/z= 401, 403, and 411, respectively. Finally, the t-butyldimethylsilyl (T-BuDMS) derivatives were formed by treatment with t-butyl-dimethylsilanol/imidazole (Supelco) in dimethylformamide at 40°C for 18 hours, purified from reagents by TLC, and monitored at m/z= 563, 565, and 573. ester
48
In addition to the above, samples of the 8,9 diol-PFB fraction were also chromatographed on TLC plates
JULY 1991 VOL. 42 NO. 1
PROSTAGLANDINS i m p r e g n a t e d with 10% AgNO 3 developed in ethyl a c e t a t e : m e t h a n o l (49:1) to separate the EPA (Rt=0.10) and AA (RI=0.23) derivatives. The resolved compounus were then d e r i v a t l z e d to their t r i m e t h y l s i l y l ethers and analyzed by SIM as above. RESULTS
Figure 1 shows the SIM tracings of the ii,12 and 14,15 diol f r a c t i o n as the (bis)TMS e t h e r - P F B esters. During i n g e s t i o n of the n-3 fatty acid supplements, peaks appear in the m/z=479 channel similar to those from the a r a c h i d o n i c acid d e r i v a t i v e being m o n i t o r e d in m/z=481, but d i f f e r i n g by two mass units, c o n s i s t e n t with an additional double bond.
m/z=479
m/z=481
~' ' ~ « 2;õ e'''' 2 ~ '~'-'-2;-? PRE FISH O I L
~=;o"' ';= ;2"" ~' :, ó x ' " ~=; x POST
FISH O I L
Figure i. S e l e c t e d - i o n - m o n i t o r i n g of the 11,12 and 14,15 diols derived from EPA, AA, and o c t a d e u t e r a t e d AA (m/z= 479, 481 and 489, respectively) as their (bis) TMS e t h e r - P F B esters. Shows tracings from urine collected prior to (PRE) and after 4 weeks of (POST) i n g e s t i o n of the supplement c o n t a i n i n g EPA. Peaks clearly a n a l o g o u s to those in the m/z=481 (AA-derivative) channel are not seen in before EPA ingestion, w h e r e a s the peaks r e p r e s e n t i n g the EPA d e r i v a t i v e are obvious afterwards. Figure 2 d e m o n s t r a t e s that a putative EPA d e r i v a t i v e also appears in the 8,9 diol fraction during fish oil ingestion. Panel A shows an SIM trace of the n - b u t y l b o r o n a t e - P F B ester derivatives, with the prominent (M-181-) ions (loss of PFB) at a p p r o p r i a t e m/z values for the vicinal diols d e r i v e d from
JULY 1991 VOL. 42 NO. 1
49
PROSTAGLANDINS epoxides. Scans of these Deaks also showed the ratio of doublet signals (1:4..2 --.. ~°B:IIB) at adjacent m/z values expected for boron-containing compounds; for straight-chain dihydroxy fatty acids, boronates form under these conditions when hydroxyls are within two carbons of each other (21). In panel B a r e SIM traces for t-Bu-DMS derivatives of EPA and AA diols at m/z 84 units higher than for the TMS derivatives, consistent with two hydroxyl groups in the molecules. m/z=563
B
~ ~ ~
m/z=565
,~
_~-,-,~
_~e-~.~.~~
34
m/z=573
m/z=411
,
3'3O
3,30
PRE
FISH
OIL
POST
4'OO
FISH
OIL
,
. . . . .
i
3,30
PRE FISH OIL
--T
,
,
~
*
5~30
«-
"r~,
~«r
~
,
4:00
,
'
i
,
4,30
POST FISH OIL
Figure 2. Selected-ion-monitoring traces of the 8,9 diols derived from EPA, AA and octadeuterated AA. Panel A shows the three compounds (m/z= 401, 403 and 411, respectively) as their n-butylboronate-PFB esters, before (PRE) and after (POST) ingestion of the EPA-containing supplement. Panel B shows SIM tracings of the same compounds as their (bis)t-butyl-DMS ether-PFB esters (m/z = 563, 565, 573, respectively). Both derivatives show appearance of the EPA-derived species in the urine during EPA ingestion. After resolution of the EPA and AA diols on argentation TLC, the TLC bands were extracted, derivatized to their TMS ether, PFB esters, and yielded characteristic (M-181") ions at m/z=479 for the EPA-diol and m/z=481 and 489 for AA and octadeuterated-AA diols, respectively (not shown). Although deuterated internal standards for the EPAderived diols are not currently available, an attempt was made to quantify both the EPA- and AA-derived analogs of several diols by SIM using the deuterated AA-diol internal standards. The excretion of both types of compounds before and at the end of ingesting the fish oil concentrate is presented in Table I. It appears that regioisomers of EPA are present in
50
JULY 1991 VOL. 42 NO. 1
PROSTAGLANDINS
A
B
m/z=479
m/z=481
m/z=489
EPA DIOL BAND
AA DIOL BAND
Figure 3. Shows SIM t r a c i n g s from individual i n j e c t i o n of E P A - d i o l f r a c t i o n and AA- + d e u t e r a t e d A A - d i o l f r a c t i o n from a single urine sample, r e s o l v e d on a r g e n t a t i o n TLC. Panel A i n d i c a t e s lower b a n d (EPA-diol, m/z=479) w h i l e i n j e c t i o n for Panel B c o n t a i n s the latter two c o m p o u n d s (m/z=481 and 489). The a r g e n t a t i o n TLC p r o v i d e d e s s e n t i a l l y c o m p l e t e r e s o l u t i o n of the two fractions.
The e x c r e t i o n of both types of c o m p o u n d s by the s u b j e c t s b e f o r e and at the end of ingesting the fish oil c o n c e n t r a t e is p r e s e n t e d in Table i. It appears that r e g i o i s o m e r s of EPA are p r e s e n t in d i f f e r e n t p r o p o r t i o n s than those of AA. Also, there a p p e a r s to be a n o n - s i g n i f i c a n t d e c l i n e in the amounts of the A A - d e r i v e d compounds when those from EPA are b e i n g released, r e s u l t i n g in a higher overall e x c r e t i o n of this class of compounds.
JULY 1991 VOL. 42 NO. 1
51
PROSTAGLANDINS T a b l e i. Intake
Excretion
of
EPA-
and
AA-Derived
Before
Diols
with
N-3
After
Diols
AA
EPA
AA
EPA
8,9
48.3-+19.6
n.d.
44.5-+23.3
16.7-+ 8.0
11,12
25.6+ 6.5
n.d.
21.8+ 5.2
22.3_+ 5.5
14,15
22.5+ 7.4
n.d
19.0+ 4.8
7.7_+ 4.5
V a l u e s are means±sem, in ng excreted/24 h o u r s p r i o r to (Before) and at the end of (After) 4 w e e k s of d i e t a r y n-3 supplements, n . d . = n o n e d e t e c t e d above limits of approx. 0.05 ng/24 hours. Discussion T h e s e studies d e m o n s t r a t e for the first t i m e t h a t d i o l s of EPA, a n a l o g o u s to those b e l i e v e d to be f o r m e d by c y t o c h r o m e P-450 from AA, are excreted by humans i n g e s t i n g fish oil supplements. The a p p r o p r i a t e r e t e n t i o n times and p e a k shapes of three different derivatives on capillary gas chromatography, m i g r a t i o n on a r g e n t a t i o n TLC, and i n f o r m a t i o n on functional groups, their r e l a t i v e positions, and m o l e c u l a r w e i g h t s p r o v i d e d by mass spectrometry, appear to e s t a b l i s h the p r e s e n c e of E P A - d e r i v e d vicinal diols in h u m a n u r i n e w i t h a h i g h degree of confidence. Similar v a l u e s for t h e s e diols were o b t a i n e d from urines c o l l e c t e d in the p r e s e n c e of an antioxidant, so it is u n l i k e l y that the c o m p o u n d s we have m e a s u r e d w e r e formed ex vivo. Such c o m p o u n d s in u r i n e could p o s s i b l y o r i g i n a t e in the kidney, but f u r t h e r w o r k will be needed to define the m e c h a n i s m and locus of t h e i r formation. We h a v e r e p o r t e d that similar p h a r m a c o l o g i c d o s i n g w i t h n-3 p o l y u n s a t u r a t e s lowers the b l o o d p r e s s u r e of p a t i e n t s w i t h m i l d essential hypertension, but that this e f f e c t was not e x p l a i n e d by the a l t e r a t i o n s found in the e n d o g e n o u s s y n t h e s i s of thromboxanes, p r o s t a c y c l i n s or p r o s t a g l a n d i n E (20). The r e d u c t i o n in p l a t e l e t activation and t h r o m b o x a n e s y n t h e s i s by fish oils appears to account for the a m e l i o r a t i o n of p l a t e l e t v a s c u l a r i n t e r a c t i o n s (15) and a n t i - t h r o m b o t i c e f f e c t s (16) p r e v i o u s l y described. Other b e n e f i c i a l effects, such as r e d u c t i o n in r e s t e n o s i s rates after c o r o n a r y a n g i o p l a s t y (21), have been noted in patients taking aspirin, w h i c h by itself o b l i t e r a t e s t h r o m b o x a n e p r o d u c t i o n by platelets. Therefore, v a s c u l a r b e n e f i t s found in such p a t i e n t s w o u l d not d e p e n d u p o n c y c l o o x y g e n a s e p r o d u c t s of n-3 fatty acids. C y t o c h r o m e P-450 m e t a b o l i t e s of a r a c h i d o n i c a c i d were d e c r e a s e d to a n o n - s i g n i f i c a n t degree w h i l e t h o s e from e i c o s a p e n t a e n o i c acid were increasing, but t h e r e was m u c h v a r i a b i l i t y in this and standards of all of the r e g i o i s o m e r s of AA and EPA d i o l s were not available. T h e r e f o r e , further studies will be n e c e s s a r y to define both the f a c t o r s g o v e r n i n g p r o p o r t i o n s of the AA- and E P A - d e r i v e d r e g i o i s o m e r s formed,
52
JULY 1991 VOL. 42 NO. 1
PROSTAGLANDINS as well as the d i f f e r e n c e s in b i o l o g i c a l a c t i v i t i e s of the c y t o c h r o m e P-450 m e t a b o l i t e s of e i c o s a p e n t a e n o i c acid.
Acknowledgments This w o r k was supported in part by a g r a n t from the N a t i o n a l I n s t i t u t e s of Health (HL-35380). Dr. K n a p p is an E s t a b l i s h e d I n v e s t i g a t o r of the A m e r i c a n H e a r t A s s o c i a t i o n .
Referen¢es i. Oliw, E.H., J.A. Lawson, A.R. A r a c h i d o n i c A c i d M e t a b o l i s m in Biol. Chem. 256:9924. 1981.
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2. Capdevila, J., N. Chacos, J. Werringler, R. Prough, and K.W. Estabrook. Liver M i c r o s o m a l C y t o c h r o m e P450 and the O x i d a t i v e M e t a b o l i s m of A r a c h i d o n i c Acid. Proc. Natl. Acad. Sci. U S A 7_88:5362. 1981. 3. Toto, R., A. Siddhanta, S. Manna, B. Pramanik, J.R. Falck, and J. Capdevila. A r a c h i d o n i c Acid Epoxygenase: Detection of E p o x y e i c o s a t r i e n o i c Acids in H u m a n Urine. Biochim. Biophys. Acta, 919:132-139. 1987. 4. Junier, M.-P., F. Dray, I. Blair, J. Capdevila, E. Dishman, J.R. Falck, and S.R. Ojeda. E p o x y g e n a s e P r o d u c t s of Arachidonic Acid Are Endogenous Constituents of the Hypothalamus. Endocrinology, 126:1534. 1989. 5. Proctor, K.G., S. Shatkin, P.M. Kaminski, J.R. Falck, and J.H. Capdevila. M o d u l a t i o n of A r t e r i o l a r B l o o d F l o w by I n h i b i t o r s of A r a c h i d o n i c Acid O x i d a t i o n A f t e r T h e r m a l Injury: P o s s i b l e Role for a Novel Class of V a s o d i l a t o r Metabolites. C i r c u l a t i o n 7/7(5):1185. 1988. 6. Roslowsky, M., J.R. Falck, J.T. Willerson, and W.B. Campbell. Synthesis of L i p o x y g e n a s e and Epoxygenase P r o d u c t s of A r a c h i d o n i c Acid by Normal and S t e n o s e d C a n i n e C o r o n a r y Arteries. Circ. Res. 66:608. 1990. 7. Schlondorff, D., E. Petty, J.A. Oates, M. Jacoby, and S.D. Levine. Epoxygenase Metabolites of A r a c h i d o n i c Acid Inhibit V a s o p r e s s i n Response in Toad Bladder. Am. J. Physiol. 253:F464. 1987. 8. Hirt, D.L., J. Capdevila, J.R. Falck, M.D. Bryer, and H.R. Jacobson. C y t o c h r o m e P450 M e t a b o l i t e s of A r a c h i d o n i c A c i d Are P o t e n t I n h i b i t o r s of V a s o p r e s s i n A c t i o n on R a b b i t Cortical C o l l e c t i n g Duct. J. Clin. Invest. 8__4:1805. 1989. 9. Fitzpatrick, F.A. and R.C. Murphy. C y t o c h r o m e P-450 M e t a b o l i s m of A r a c h i d o n i c Acid: F o r m a t i o n and B i o l o g i c a l A c t i o n s of " E p o x y g e n a s e " - D e r i v e d Eicosanoids. Pharm. Rev. 40~229. 1989.
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PROSTAGLANDINS i0.
Schwartzman, M.L., P. Martasek, A.R. Rios, R.D. Levere, K. Solangi, A.I. Goodman, and N.G. Abraham. Cytochrome P 4 5 0 - D e p e n d e n t A r a c h i d o n i c A c i d M e t a b o l i s m in H u m a n Kidney. K i d n e y Int. 3_/7:94. 1990.
ii.
Catella, F., J.A. Lawson, D.J. Fitzgerald, and G.A. FitzGerald. E n d o g e n o u s B i o s y n t h e s i s of A r a c h i d o n i c A c i d E p o x i d e s in Humans: Increased F o r m a t i o n in P r e g n a n c y I n d u c e d H y p e r t e n s i o n . Proc. Natl. Acad. Sci. U S A 8_/7:5893. 1990. 12. C. Von Shacky. Prophylaxis of A t h e r o s c l e r o s i s W i t h M a r i n e Omega-3 F a t t y Acids: A C o m p r e h e n s i v e Strategy. Ann. Intern. Med. 107:890. 1987. 13. Leaf, A., and P.C. Weber. Cardiovascular Effects n-3 Fatty Acids. N. Engl. J. Med. 318:549. 1988.
of
14. VanRollins, M., P.D. Frade, and O.A. Carretero. O x i d a t i o n of 5 , 8 , 1 1 , 1 4 , 1 7 - E i c o s a p e n t a e n o i c A c i d by H e p a t i c and Renal Microsomes. Biochim. Biophy. Acta, 966:133. 1988. 15. Fischer, S., and P.C. Weber. P r o s t a g l a n d i n 13 is Formed In V i v o in M a n A f t e r Dietary E i c o s a p e n t a e n o i c Acid. Nature, 707:165. 1984. 16. Knapp, H.R., I.A.G. Reilly, P. Alessandrini, and G.A. FitzGerald. In Vivo Indexes of P l a t e l e t and V a s c u l a r F u n c t i o n D u r i n g Fish-Oil A d m i n i s t r a t i o n in P a t i e n t s with Atherosclerosis. N. Engl. J. Med. 314:937. 1986. 17. Needleman, P., A. Raz, M.S. Minkes, J.A. Ferrendeli, and H. Sprecher. Triene Prostaglandins: P r o s t a c y c l i n and T h r o m b o x a n e B i o s y n t h e s i s and U n i q u e B i o l o g i c a l Properties. Proc. Natl. Acad. Sci. USA. 76:944. 1979. 18. Braden, G.A., H.R. Knapp, D.J. Fitzgerald, and G.A. FitzGerald. D i e t a r y Fish Oil A c c e l e r a t e s the R e s p o n s e to Coronary Thrombolysis With Tissue-Type Plasminogen Activator. C i r c u l a t i o n 82:178. 1990. 19.
Knapp, H.R., and G.A. FitzGerald. The A n t i h y p e r t e n s i v e Effects of Fish Oil: A C o n t r o l l e d Study of P o l y u n s a t u r a t e d Fatty Acid S u p p l e m e n t s in E s s e n t i a l Hypertension. N. Engl. J. Med. 320:1037. 1989.
20. Knapp, H.R. Omega-3 Fatty Acids, E n d o g e n o u s P r o s t a g l a n d i n s , and Blood Pressure R e g u l a t i o n in Humans. Nutr. Rev. 4_~7:301, 1989. 21. Oliw, E~H. A n a l y s i s of 1,2-Diols of Linoleic, «L i n o l e n i c and A r a c h i d o n i c Acid by Gas C h r o m a t o g r a p h y - M a s s S p e c t r o m e t r y U s i n g Cyclic Alkyl Boronic Esters. J. Chromatogr. 275:245. 1983. 22. Dehmer, G.J., J.J. Popma, E.K. van den Berg, et al. R e d u c t i o n in the Rate of Early R e s t e n o s i s A f t e r C o r o n a r y A n g i o p l a s t y by a Diet S u p p l e m e n t e d with n-3 F a t t y Acids. N. Engl. J. Med. 319:733. 1988. Editor:
54
E. G r a n s t r o m
Received:
12-3-90
Accepted:
3-26-91
JULY 1991 VOL. 42 NO. 1