THE JOURNAL OF R~OLOQICAL CHEMISTRY Vol. 245, No. 20, Issue of October 25, PP. 5234-5238, 1970

Printed in U.S.A.

Epoxides as Obligatory Intermediates in the Metabolism of Olefins to Glycols*

(Received for publication, October 20, 1969)

E. W. n/IAYNERT, R. L. FOREMAN, AND T. WATABE$

From the Department of Pharmacology, College of Medicine, University of Illinois, Chicago, Illinois 60680

SUMMARY

In the presence of rat liver microsomes and NADPH n-l- octene, n-4-octene and 3-ethyl-2-pentene were converted to the glycols with no trace of epoxides. Increased substitution of ethylenic hydxogen atoms by alkyl groups was found to retard. the rate of biological oxid.ation but to enhance that of epoxidation by perbenzoic acid. in chloroform. Microsomes without cofactors hydrolyzed the monosubstituted ethylene oxide more rapidly than the di- or trisubstituted derivatives. The relative rates were in the opposite order of those pre- dicted for acid-catalyzed hydrolysis. The epoxides were found capable of inhibiting epoxide hydrolase. Incubation of microsomes and NADPH with 1 mM n-1-octene in the presence of 20 m&r 4,5-epoxy-n-octane yielded both 1,2- epoxy-n-octane and n-octane-l ,2-diol. However, in the presence of 20 mM 1,2-epoxy-n-octane, 1 mM n-4-octene yielded 4,5-epoxy-n-octane but no n-octane-4,5-diol. The complete replacement of n-octane-4,5-diol by 4,5-epoxy-n- octane in the presence of the inhibitor indicates that the epoxide is an obligatory intermediate in the conversion of n-4-octene to the glycol.

A few compounds containing a carbon-carbon double bond are metabolized to epoxides (l-3), but the usual products are glycols (4-6). At the beginning of the present investigation, the role of epoxides in the biological formation of glycols was un- certain. It seemed possible that the glycols might arise from either enzymatic or spontaneous hydrolysis of epoxides. How- ever, direct dihydroxylation of the ethylenic moiety could not be ruled out. An attractive approach to this problem was to focus attention on a few series of olefins, epoxides, and glycols in which the epoxides are quite stable in water. The compounds selected for this purpose were derived from n-1-octene, trans-n-

4-octene, and 3-ethyl-2-pentene.

METHODS

Chemical Syntheses-Commercial n-1-octene, trans-n-4-octene and 3-ethyl-2-pentene were purified by distillation. 1, ~-EPOXY-

* This work was supported by Grant NB-06288 from the Na- tional Institute of Neurological Diseases and Stroke. United States Public Health Servicer Preliminary communicatibns have appeared (15).

$ Present address, Department of Organic Chemistry, Tokyo College of Pharmacy, Tokyo, Japan.

n-octane (b.p.,, 6465”), 4,5-epoxy-n-octane (b.p.,,57-58’), and 2,3-epoxy-3-ethylpentane (b.p+g 53-54”) were prepared by the oxidation of the olefins with perbenzoic acid in chloroform (7). Their purity was confirmed by elemental analysis and by gas chromatography on 20% squalene and on 20% Apiezon L col- umns. Infrared spectroscopy of liquid films exhibited absorp- tion bands characteristic of the oxirane ring. n-Oct.ane-1,2-diol (b.p.,, 158”), n-octane-4,5-diol (m.p. 115-125”), and 3-ethyl- pentane-2,3-diol (b.p.r, 92”) were synthesized by treatment of the olefins with 30% HzOz in formic acid followed by alkaline hydrolysis (8). Their purity was confirmed by chromatography on 200/, Apiezon L. Like the two other glycols, n-octane-4,5- diol appeared as a single peak, although its melting point sug- gested a mixture of the meso and racemic forms. Infrared spec- troscopy indicated the absence of absorption bands from car- bony1 and olefinic groups in all three glycols. Heptachlor epoxide and dieldrin were supplied by Velsicol Corporation, Chicago, Illinois, through the courtesy of Dr. Milton Eisler.

Incubation Mi&ures-The livers from male Sprague-Dawley (Holtzman) rats, weighing 180 to 200 g, were homogenized at 4” in 2 volumes of isotonic KCI. The conditions for sedimenta- tion of the various subcellular fractions are specified elsewhere in the text. In all experiments the aliquots were equivalent to 2 g of liver. The composition of the incubation medium and other experimental details are presented in the legends of appro- priate figures and tables.

Analytical Methods-The same analytical procedure was used for the quantification of the metabolites of the three olefins. The chilled reaction mixture plus 5 g of NaCl was extracted with 25 ml of ether. A 20-ml portion of the ether layer was removed after centrifugation and cautiously evaporated to minimize the loss of volatile metabolites. The chilled residue was dissolved in 1 ml of acetone, and aliquots of this solution were analyzed in a model 5000 Barber-Colman gas chromatograph equipped with an ionization detector. The composition of the column and other experimental conditions are specified in the legend of Fig. 1. When added to boiled subcellular fractions, the glycols (0.1 to 1 pmole) were recovered almost quantitatively (90%). The recovery of 1,2-epoxy-n-octane or 2,3-epoxy%ethylpentane averaged 90%, but that of 4,5-epoxy-n-octane was only 70%. All data were corrected for recovery losses.

With each olefin the identity of the epoxide and glycol metabo- lites was checked by both gas-liquid and thin layer cochromatog- raphy. After development with a 5: 1 benzene-acetone mixture and spraying with concentrated H&?Od on silica gel plates, 1,2-epoxy-n-octane, 4,5 -epoxy-n-octane, and 2,3 -epoxy-3- ethylpentane had Rp values of 0.75, 0.68, and 0.80, respectively.

5234

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Issue of October 25, 1970 E. W. Maynert, R. L. Foreman, and T. Watabe 5235

After development with a 2: 1 benzene-ethyl acetate mixture,

the RF values of n-octane-l ,2-diol, n-octane-4,5-diol, and 3-ethylpentane-2,3-diol were 0.40, 0.33, and 0.42, respectively.

RESULTS

Nonenzymatic Synthesis and Nydrolysis of Epoxides-Prior to the initiation of the biochemical work it seemed worthwhile to obtain some data on the influence of branching on the nonen- zymatic conversion of olefins to epoxides. Table I shows that the rate constants for perbenzoic acid oxidation increased strik- ingly with increased substitution of the ethylenic hydrogen atoms. Thus, in this reaction, the inductive effects of the alkyl groups outweigh steric hindrance. This finding is in agreement with the observations reviewed by Swern (9).

TABLE I

Rule constants for oxidation of ole$ns by perbenzoic acid at 10”

Chloroform solutions of the olefins and perbenzoic acid at 10” were mixed and agitated in stoppered vessels. The initial con-

centrations were 250 rnM for I-octene, 25 mM for 4-octene, and 3- ethyl-2-pentene, 50 rnx for perbenzoic acid when used with 1-octene and 25 rnM Then used with the other olefins. Aliquots taken at 10, 20, and 40 min were analyzed iodometrically for un- changed perbenzoic acid. The data are averages of closely agree- ing values of the second-order rate constant.

Ol&ll k

a-1 win-

n-1-Octene . . . . 0.07 trans-n-4-Octene. . . 3.30

3-Ethyl-2-pentene.. . 18.01

Nonenzymatic hydrolysis of epoxides was not investigated beyond demonstrating that at pH 3, 7, or 10 saturated aqueous solutions of 1,2-epoxy-n-octane, 4,5-epoxy-n-octane, or 2,3- epoxy-3-ethylpentane were stable at 37”. Incubation of 0.5 M

solutions of these epoxides in 40% acetone for 6 hours resulted in less than 0.01% conversion to the glycols. Information on the behavior of epoxides in neutral solutions has been scarce, but both acids and bases have been shown to catalyze cleavage of the ring. The study of Prichard and Long (10) indicates that electron-releasing substituents, including alkyl groups, facilitate acid-catalyzed hydrolysis of ethylene oxides. On the other hand, alkaline hydrolysis appears to be influenced by steric as well as inductive effects, and generalizations are not yet possible.

Emymatic Oxidation of Olejins-Incubation of n-1-octene, trans-n-4-octene, and 3-ethyl-2-pentene with the NADPH- enriched 9000 x g supernatant of a rat liver homogenate pro- duced the corresponding glycols, but the epoxides could not, be detected (Fig. 1). The limit for the gas chromatographic detec- tion of the epoxides was 0.3 nmoles or a 0.06% yield based on the initial amount of olefin. The identity of the glycols was

confirmed by thin layer and gas cochromatography. The rela- tive yields of the glycols (11.3%, 4.0’%, 0.12%) indicate that increasing substitution of the ethylenic moiety by alkyl groups decreases the rate of the reaction. Thus, here, steric hindrance from the alkyl groups appears to outweigh their inductive effects.

The product from n-1-octene contained a trace of an unknown metabolite with a retention time (ET) lower than that of the epoxide. Both of the other olefins yielded at least three or four

5 IO i5

RETENTION TIME (minutes)

0

FIG. 1. Gas chromatograms of t’he products from n-l-octene (A), trans-n-4-octene (B), and 3-ethyl-2-pentene (C) after incu- bation with the 9000 X g supernatant fluid of a rat liver homoge- nate. In each panel the left arrow indicates the expected position of the epoxide, and the right arrow, the glycol; the large peaks ore the left represent acetone and unchanged olefin. Mixtures of 10 Gmoles of an olefin dissolved in ethanol (0.2 ml), 100 pmoles of MgC&, 50 moles of nicotinamide, 3 pmoles of NADP, 50 kmoles of glucose g-phosphate, supernatant from a homogenate of 2 g of liver centrifuged at 9000 X g for 20 min, and sufficient 70 mM phosphate (pEI 7.4) to bring the volume to 9.5 ml were incubated for 60 min at 37’ in closed vessels containing an oxygen atmos- phere. Extracts were prepared as described under “Methods.” The chromatographic column (6 feet X 3 mm, inner diameter) packed with 20yo Apiezon 1, on 100 to 120 mesh Gas Chrom Q was eluted with argon (60 ml per min). The column temperature was held at 80” (60’ for the products from 3-ethyl-2-pentene) for 5 min and then increased to 180” at a rate of 10” per min. The yields of the glycols from 10 @molesof the olefins were 11.3% in A, 4.0% in B, and 0.12% in C.

compounds in addition to the glycols. The most abundant metabolite from n-4-octene (RT = 10.7 min), like the glycol (RT = 14.9 min), increased linearly with time of incubation. The following evidence suggests that this compound is an octenol. First, its RF is similar to that of 2-octanol (10.9 min). Second, treatment of the extract with an alcohol reagent (acetyl chloride) caused this substance to disappear and a new compound with a shorter RT to appear. Third, the addition of bromine to the extract converted the metabolite to a derivative with lower volatility. The formation of the other two unknown products of n-4-octene was not clearly related to incubation time. No attempt was made to identify these substances or the unidenti- fied metabolites of 3-ethyl-2-pentene.

The enzymatic conversion of the olefins to glycols was found to require microsomes and an NADPH-generating system (Ta- ble II). Twice-washed microsomes plus NADPH produced

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TABLE II When boiled 9,000 X g supernatant fluid was used, the epoxide

Formation of glycols from olefins by various fractions was recovered completely unchanged. About 85% of the hy- of a rat liver homogenate drolytic activity was localized in the microsomes; the remainder

The substrate was 10 rmoles of n-l-octene, trans-n4-octene, or could not be sedimented at 165,000 x g. The microsomal reac- 3-ethyl-2-pentene. The incubation time was 30 min. Methods tion did not require the presence of either NADPH or MgC12, and conditions were the same as those described in Fig. 1 except and the activity of the 165,000 x g supernatant was not dimin- for the subcellular fractions and cofactors employed. The micro- ished by dialysis. Inasmuch as the conversion of an epoxide to somes, sedimented at 105,000 X g for GO min, were washed twice a glycol must be formulated as hydrolysis, the enzyme responsi- with cold isotonic KCl. The 105,000 X g supernatant fluid was recentrifuged for 60 min at 165,000 X g. In the experiments

ble for this reaction may be called epoxide hydrolase.

involving microsomes and NADPH, 10 units of glucose 6-phos- The rate of hydrolysis of 4,5-epoxy-n-octane by microsomes

phate dehydrogenase was added to the medium. was slower than that of 1,2-epoxy-n-octane but faster than that of 2,3-epoxy-3-ethylpentane.’ Thus, this reaction diiers from

Fraction -O&tm&4,5- I-Ethylpen- t am-2,3-did

the acid-catalyzed conversion of epoxides to glycols in which in-

- creasing substitution by alkyl groups facilitates hydrolysis. It pnole jH+dt? p?wle may be presumed that alkyl groups hinder the approach of the

9,000 X g supernatant + substrate to the surface of the enzyme. In the experiments in NADPH . 0.50 0.11 0.01 which the NADPH-generating system and MgCIZ were omitted,

Microsomes . 0.00 the gas chromatograms of the products contained only two peaks Microsomes + NADPH 0.56 0.13 0.03 Microsomes + NADP. 0.00

representing the glycol and unchanged epoxide. However,

Microsomes + NADH.. . <O.Ol when the cofactors were added, the yields of n-octane-4,5-diol

Boiled microsomes + NADPH 0.00 and 3-ethylpentane-2,3-diol were lower, and the chromatograms

165,000 X g supernatant i- revealed additional peaks. The retention time on 20% Apiezon

NADPH . 0.00 L of the new product from 4,5-epoxy-n-octane was 13.1 min, - and from 2,3-epoxy-3-ethylpentane, 14.0 min (cj. Fig. 1). In

the former case, the peak height was only 30% as large as that TABLE III of the glycol, but in the latter it was 80% as large. These new

Hydrolysis of epoxides by various fractions of rat compounds did not appear to be isomers of the pure glycols, liver homogenate because they could not be detected in crude synthetic products

The substrate was 10 pmoles of 1,2-epoxy-n-octane, 4,5-epoxy- expected to contain all possible isomers. They may represent n-octane, or 2,3-epoxy-3-ethylpentane added to the incubation alcoholic derivatives of the epoxides. In respect to its relative mixture dissolved in 0.2 ml of ethanol. Methods and conditions activity in hydrolyzing 1,2- and 4,5-epoxy-n-octane, the enzyme were t,he same as those described in Table II and Fig. 1 except in the 165,000 X g supernatant behaved much the same as that that. the incubation time was 15 min. The experiment without in the microsomes. This observation indicates that the two cofactors involved the omission of nicotinamide and MgClz as

well as the NADPH-generating system. enzymes may be the same.

Inhibition of Epoxide Hydrolase-Recognition that microsomes

Fraction n-Oc&e-1,2- n-octfi-4,s 3-Ethylpen- 0 tane-Z+Iiol

contain an epoxide hydrolase suggested that inhibition of this enzyme would provide useful information on the role of epoxides

p?wles umoles J&moles in the conversion of olefins to glycols. Among a number of

9,000 X g supernatant + epoxides and glycols examined for such inhibitory action, only NADPH . . 9.95 4, B-epoxy-n-octane was found capable of revealing 1,2-epoxy-n-

Boiled 9,000 X g supernatant + octane as a metabolite of n-1-octene in a system containing NADPH 0.00 0.00 0.00 microsomes and NADPH (Table IV). In this particular experi-

Microsomes + NADPH. 10.00 2.11 0.85 Microsomes (without cofactors) 10.00

ment, the epoxide appeared in somewhat greater quantity than 4.99 2.08

Dialyzed 165,000 X g superna- the glycol (0.40 versus 0.23 pmole). The sum of the two com-

tant........................ 1.42 0.42 pounds was practically the same as the amount of glycol formed in the absence of the inhibitor (0.63 versus 0.64 pmole). Con- trol experiments established the requirement of NADPH and

the glycols in about the same amounts as did the 9000 x g super- gaseous oxygen for the appearance of the epoxide. Although

natant. Moreover, the products from the individual olefins apparently ineffective as inhibitors of epoxide hydrolase, hepta-

did not differ appreciably from those shown in Fig. 1. Thus, chlor epoxide and dieldrin reduced the formation of n-octane-

it would appear that soluble enzymes are not responsible for the 1 , 2-diol. It seems possible that these epoxides may have an

formation of any of the unidentified metabolites. inhibitory effect on the epoxidase involved in the conversion of

Enzymatic Hydrolysis of Epoxides-The absence of epoxides n-1-octene to 1,2-epoxy-n-octane. However, this question was

and the presence of glycols in the products from the biological not investigated.

oxidation of the olefins invited an examination of the action of The indication that 4,5-epoxy-n-octane inhibited microsomal

liver homogenates on 1,2-epoxy-n-octane, 4,5-epoxy-n-octane, epoxide hydrolase, whereas 2,3-epoxy-3-ethylpentane did not,

and 2,3-epoxy-3-ethylpentane, for these compounds had been 1 In separate experiments the rates of hydrolysis by washed shown to be quite stable in aqueous solution. The data in Table microsomes of 1,2-epoxy-n-octane and 4,5-epoxy-n-octane were III show that incubation of 10 pmoles of 1,2-epoxy-n-octane for found to be 695 and 78 nmoles per min per g of liver, respectively.

15 min with a 9,000 x g supernatant equivalent to 2 g of rat The substrate concentrations were 1 mM, and the enzyme con- centration, 100 mg of liver per ml. Both rate curves were linear

liver resulted in a quantitative conversion to n-octane-l ,2-diol. for at least 10 min.

5236 Epoxide Intermediates in OleJin Metabolism Vol. 245, No. 20

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Issue of October 25, 1970 E. W. Maylaert, R. L. Foreman, awl T. TPatabe 5237

TABLE Iv OH OH

E$P( 1 of potential inhibztors oj’ epoxitle /yd~olase on metabolism of n-l -0ctene by rat liver mcro.somes

The sltbstrate was 10 pmoles of the olefin added to the incuba-

tion mixtllre in 0.2 ml of ethanol in Tvhich the inhibitor u’as also dissolved or, in the case of heptachlor eposide and dieldrin, SK-

pended. The medium colitnined the XADPH-generating sys- tem. The incubation time was 30 min. Other methods and con- ditions mere the same as those described in Fig. 1 and Table II.

In several other experiments employing the 4,5-epoxide, the yields of the 1,2-epoxide and the 1,2-din1 equalled that of the glycol in the absence of the inhibitor.

El \c=c: ~-> / 1; ‘/ -C / \

\ E2

0 ///1 E3

Lid\,/ F - c\

FIG. 3. Alternative pathurays for the biological conversion of an olefin to a glycol. Experiments described in the text appear to obviate the possibility of a direct dihydroxylating enzyme (El) in the transformation of n-4-octene to n-octane-4,Sdiol.

Inhibitor (2 X 10-Z M)

Kane

4,5-Epoxy-n-octane 2,3-~;posy-3-ethylpentane

Heptachlor epoxide Dieldrin . Ethylene glycol Propylene glycol

1 .I filnole 0.00

/mole

0.64

0.40 0.23 0.00 O.G3 0.00 0.50

0.00 0.54 0.00 0.64 0.00 0.64

:

6 2 $. 4 IP RETENTION TIME (minutes)

-1 nhibition of glycol

f ormation

%

(i4 2

22

16 0 0

FIG. 2. Gas chromatogram showing the inhibitory effect of 20 mnr 1,2-epoxy-n-octane on the metabolism of n-4-octene to n-octane-4,5-diol by rat liver microsomes. The left arrow indi- cates the peak from 4,5-epoxy-n-octane, and t.he right arrow, the expect’ed position of n-oct.ane-4,5-diol. Reading from left to right, the other large peaks represent acet,one, n-4-octene, 1,2- epoxy-n-octane, and n-octane-1,2-diol. In separate experiments all the peaks specified were identified by cochromatography of authentic compounds and the extract. Methods and conditions were the same as those in Table IV. The slight differences in retention times from those in Fig. 1 can be att,ributed to aging of t’he chromatographic column.

suggested that a monosubstituted ethylene oxide might provide maximal blocking activity. Accordingly, Dhe effect of 20 mM 1,2-epoxy-n-octane on the metabolism of n-4-octene was in- vestigated. Fig. 2 reveals that this monosubstituted epoxide completely inhibited the appearance of n-octane-4,5-diol. The quantity of 4,5-epoxy-n-octane in the product was approxi- mately equivalent to the amount of glycol formed in the absence of the inhibitor. An exact numerical comparison was precluded by the magnification of a trace (0.2yC) of the 4,5-epoxide in the 1,2-epoxide to substantial proportions as a result of the high concentraOion of inhibitor used in this experiment.

Direct evidence that these results should be attributed to in- hibition of epoxide hydrolase was obtained by employing an epoxide as the substrate. ,4 low concentration (0.3 m$ of 1,2-epoxy-n-octane inhibited completely for 10 min the hydroly- sis of 5 rn>I 4,5-epoxy-n-octane by washed microsomes at a concentration of 25 mg of liver per ml.

DISCUSSIOK

The ability of hepatic microsomes, an KADPH-generating system, and oxygen to convert n-1-octene, trans-n&octene, and 3-ethyl-2-pentene to the corresponding glycols was antici- pated, for earlier work2 had demonstrated the formation of 5-(2,3-dihg-drox~propyl)-5-(l-methylbutyl)barbituric acid from 5-&lyl-5-(l-methylbutyl)barbituric acid (secobarbital) under the same conditions. Recently, Leibman and Ortiz (11) reported the dihydroxylation of indene and styrene by a similar micro- somal preparation. i2ls0, Jerina et al. (12) have mentioned similar requirements for the transformation of naphthalene to trans-1 ,2-dihydro-l , 2-dihydroxynaphthalene. Thus, it would appear that hepatic microsomes contain the necessary enzymes for the conversion of both aliphatic and aromatic double bonds to diols. The capacity of microsomes and NADPH to form stable epoxides from cyclodiene insecticides such as aldrin and heptachlor has been recognized for several years (13, 14).

Our preliminary report (15) that 1,2-epoxy-n-octane was readily hydrolyzed to n-octane-l ,2-diol by microsomes coincided with an announcement by Leibman and Ortiz (16) that the same behavior had been observed with indene, cyclohexene, and styrene epoxides. More recently (17), the enzymatic formation of 1,2-dihydro-l , 2-dihydroxynaphthalene from 1,2-naphthalene oxide was recorded. Inasmuch as the conversion of an epoxide to a glycol ha:: classically been formulated as a hydrolytic reac- tion, the appropriate name for the enzyme would appear to be epoxide hydrolase. However, other workers (17, 18) have called it epoxide hgdrase. Like other hydrolytic enzymes such as esterases and amidases, it does not require NADPH or mag- nesium ion as a cofactor.

The systematic studp of 1,2-epoxy-n-octane, 4,5-eposy-n- octane, and 2,3-epoxy-3-ethylpentane revealed that increasing the number of alkyl groups in the oxirane ring retards the rate of cleavage by microsomes, whereas acid-catalyzed hydrolysis is enhanced. Presumably, the bulk of the alkyl groups hinders the approach of the epoxide to the surface of the enzyme. The facile cleavage of the oxirane ring by the hydrolase could account for the fact that epoxides have only rarely been detected as end products of metabolism. Heptachlor epoxide and dieldrin, which are stable enough to be excreted in substantial amounts,

2 S. Toki and E. W. htaynert, unpublished data.

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5238 Epoxide Intermediates in Olefin Metabolism Vol. 245, No. 20

may be presumed to be poor substrates for the enzyme, although rats can convert dieldrin to the trans form of the corresponding glycol (19). In the present investigation, the metabolism of heptachlor epoxide and dieldrin was not examined beyond dem- onstrating that they did not inhibit the action of microsomal epoxide hydrolase on 1, Z-epoxy-n-octane.

The discovery that high concentrations of some epoxides could inhibit the microsomal hydrolase provided the means for examin- ing the role of epoxides in the conversion of olefins to glycols. In the presence of 20 mM 4,5-epoxy-n-octane, the product from n-1-octene contained both 1,2-epoxy-n-octane and n-octane- 1 ,2-diol, whereas in the absence of the inhibitor only the glycol could be detected. This result could be explained in two ways. (a) The only route from the olefin to the glycol involves the epoxide, but inhibition of the hydrolase was incomplete. (b) Glycol may be formed by a mechanism other than epoxidation, for example, by direct dihydroxylation (Fig. 3). A similar experiment in which the substrate was n-4-octene and the in- hibitor was 20 mM 1 ,2-epoxy-n-octane gave unequivocal results. In the presence of the inhibitor, the product contained the epoxide but not the glycol, whereas, in the absence of the in- hibitor, the glycol was present, but the epoxide was not. This observation appears to prove that the epoxide is an obligatory intermediate in the conversion of n&octene to the corresponding glycol.

It now seems likely that the biological conversion of both aliphatic and aromatic carbon-carbon double bonds proceeds through epoxides. In connection with aromatic compounds, the evidence of Holtzman, Gillette, and Milne (20) is strong, although indirect. They found that trans-1 ,2-dihydro-1 , Z-di- hydroxynaphthalene formed from naphthalene in the presence of microsomes, NADPH, and gaseous l8O2 contained only 1 atom of heavy oxygen. Recently, Jerina et al. (17) reported the detection of 1 ,2-naphthalene oxide as a metabolite of naph- thalene and suggested its role as an obligatory intermediate in

the formation of the dihydrodiol as well as 1-naphthol and a premercapturic acid. Whether a single microsomal hydrolase acts on both aliphatic and aromatic epoxides is not yet clear. Studies involving inhibitors such as 1, Z-epoxy-n-octane should answer this question.

1.

2.

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5. 6. 7. 8.

9. 10.

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18. 19. 20.

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E. W. Maynert, R. L. Foreman and T. WatabeEpoxides as Obligatory Intermediates in the Metabolism of Olefins to Glycols

1970, 245:5234-5238.J. Biol. Chem. 

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