Trichothecene Biosynthesis in Fusarium sporotrichioides: Origin of the Oxygen Atoms of T-2 Toxin

Mass spectral analysis of T-2 toxin formed during the growth of Fusarium sporotrichioides (ATCC 24043) in the presence of H₂¹⁸O showed incorporation of up to three ¹⁸O atoms per toxin molecule. The carbonyl oxygens of the acetates at C-4 and C-15 and of the isovalerate at C-8 were derived from H₂O. Toxin formed in the presence of ¹⁸O molecular oxygen incorporated up to six ¹⁸O atoms per toxin molecule. The overall incorporation was 78 and 92% of toxin molecules labeled for H₂¹⁸O and ¹⁸O₂ labeled samples, respectively. The oxygens of position 1, the 12,13-epoxide, and the hydroxyl groups at C-3, C-4, C-8, and C-15 were all derived from molecular oxygen.     

18O Labeling of Chlorophyll d in Acaryochloris marina Reveals That Chlorophyll a and Molecular Oxygen Are Precursors

The cyanobacterium Acaryochloris marina was cultured in the presence of either H₂¹⁸O or ¹⁸O₂, and the newly synthesized chlorophylls (Chl a and Chl d) were isolated using high performance liquid chromatography and analyzed by mass spectroscopy. In the presence of H₂¹⁸O, newly synthesized Chl a and d, both incorporated up to four isotopic ¹⁸O atoms. Time course H₂¹⁸O labeling experiments showed incorporation of isotopic ¹⁸O atoms originating from H₂¹⁸O into Chl a, with over 90% of Chl a ¹⁸O-labeled at 48 h. The incorporation of isotopic ¹⁸O atoms into Chl d upon incubation in H₂¹⁸O was slower compared with Chl a with ∼50% ¹⁸O-labeled Chl d at 115 h. The rapid turnover of newly synthesized Chl a suggested that Chl a is the direct biosynthetic precursor of Chl d. In the presence of ¹⁸O₂ gas, one isotopic ¹⁸O atom was incorporated into Chl a with approximately the same kinetic incorporation rate observed in the H₂¹⁸O labeling experiment, reaching over 90% labeling intensity at 48 h. The incorporation of two isotopic ¹⁸O atoms derived from molecular oxygen (¹⁸O₂) was observed in the extracted Chl d, and the percentage of double isotopic ¹⁸O-labeled Chl d increased in parallel with the decrease of non-isotopic-labeled Chl d. This clearly indicated that the oxygen atom in the C3¹-formyl group of Chl d is derived from dioxygen via an oxygenase-type reaction mechanism.     

The Human Enzyme That Converts Dietary Provitamin A Carotenoids to Vitamin A Is a Dioxygenase

β-Carotene 15–15′-oxygenase (BCO1) catalyzes the oxidative cleavage of dietary provitamin A carotenoids to retinal (vitamin A aldehyde). Aldehydes readily exchange their carbonyl oxygen with water, making oxygen labeling experiments challenging. BCO1 has been thought to be a monooxygenase, incorporating oxygen from O₂ and H₂O into its cleavage products. This was based on a study that used conditions that favored oxygen exchange with water. We incubated purified recombinant human BCO1 and β-carotene in either ¹⁶O₂-H₂¹⁸O or ¹⁸O₂-H₂¹⁶O medium for 15 min at 37 °C, and the relative amounts of ¹⁸O-retinal and ¹⁶O-retinal were measured by liquid chromatography-tandem mass spectrometry. At least 79% of the retinal produced by the reaction has the same oxygen isotope as the O₂ gas used. Together with the data from ¹⁸O-retinal-H₂¹⁶O and ¹⁶O-retinal-H₂¹⁸O incubations to account for nonenzymatic oxygen exchange, our results show that BCO1 incorporates only oxygen from O₂ into retinal. Thus, BCO1 is a dioxygenase.     

Role of oxygenases in pisatin biosynthesis and in the fungal degradation of maackiain

Some isolates of the plant pathogen Nectria haematococca detoxify the isoflavonoid phytoalexin (−)maackiain by hydroxylation at carbon 6a. Precursor feeding studies strongly suggest that the penultimate step in (+)pisatin biosynthesis by Pisum sativum is 6a-hydroxylation of (+)maackiain. We have used ¹⁸O labeling to test the involvement of oxygenases in these two reactions. When fungal metabolism of maackiain took place under ¹⁸O₂, the product was labeled with 99% efficiency; no label was incorporated by metabolism in H₂¹⁸O. Pisatin synthesized by pea pods in the presence of ¹⁸O₂ or H₂¹⁸O was a mixture of molecules containing up to three labeled oxygen atoms. Primary mass spectra of such mixtures were complex but were greatly simplified by tandem MS. This analysis indicated that the 6a oxygen of pisatin was derived from H₂O and not from O₂. Labeling patterns for the other five oxygen atoms were consistent with the proposed pathway for biosynthesis of pisatin and related isoflavonoids. We conclude that the fungal hydroxylation of maackiain is catalyzed by an oxygenase, but the biosynthetic route to the 6a hydroxyl of pisatin is unknown.     

Heme-Dependent Rubber Oxygenase RoxA of Xanthomonas sp. Cleaves the Carbon Backbone of Poly(cis-1,4-Isoprene) by a Dioxygenase Mechanism

Oxidative cleavage of poly(cis-1,4-isoprene) by rubber oxygenase RoxA purified from Xanthomonas sp. was investigated in the presence of different combinations of ¹⁶O₂, ¹⁸O₂, H₂¹⁶O, and H₂¹⁸O. 12-Oxo-4,8-dimethyl-trideca-4,8-diene-1-al (ODTD; m/z 236) was the main cleavage product in the absence of ¹⁸O-compounds. Incorporation of one ¹⁸O atom in ODTD was found if the cleavage reaction was performed in the presence of ¹⁸O₂ and H₂¹⁶O. Incubation of poly(cis-1,4-isoprene) (with RoxA) or of isolated unlabeled ODTD (without RoxA) with H₂¹⁸O in the presence of ¹⁶O₂ indicated that the carbonyl oxygen atoms of ODTD significantly exchanged with oxygen atoms derived from water. The isotope exchange was avoided by simultaneous enzymatic reduction of both carbonyl functions of ODTD to the corresponding dialcohol (12-hydroxy-4,8-dimethyl-trideca-4,8-diene-1-ol (HDTD; m/z 240) during RoxA-mediated in vitro cleavage of poly(cis-1,4-isoprene). In the presence of ¹⁸O₂, H₂¹⁶O, and alcohol dehydrogenase/NADH, incorporation of two atoms of ¹⁸O into the reduced metabolite HDTD was found (m/z 244), revealing that RoxA cleaves rubber by a dioxygenase mechanism. Based on the labeling results and the presence of two hemes in RoxA, a model of the enzymatic cleavage mechanism of poly(cis-1,4-isoprene) is proposed.     

Oxygen-18 and deuterium labeling studies of choline oxidation by spinach and sugar beet

Chenopods synthesize betaine by a two-step oxidation of choline: choline → betaine aldehyde → betaine. The pathway is chloroplastic; the first step has been shown in isolated spinach (Spinacia oleracea L.) chloroplasts to be O₂- and light-dependent, the role of light being to provide reducing power (P Weigel, EA Weretilnyk, AD Hanson 1988 Plant Physiol 86: 54-60). Here, we report use of in vivo¹⁸O- and ²H-labeling in conjunction with fast atom bombardment mass spectrometry to test for two hypothetical choline-oxidizing reactions that would explain the observed requirements for O₂ and reductant: a desaturase or an oxygenase. Simple syntheses for ²H₃-choline, ²H₃, ¹⁸O-choline, and ²H₃, ¹⁸O-betaine are given. A desaturase mechanism was sought by giving choline deuterated at the 2-carbon, or choline unlabeled at this position together with ²H₂O and by analyzing newly synthesized betaine. About 15% of the ²H at C-2 was lost during oxidation of choline to betaine, and about 10% of the betaine made in the presence of 50% ²H₂O was monodeuterated. These small effects are more consistent with chemical exchange than with a desaturase, because 10 to 15% losses of ²H from the C-2 position also occurred if choline was converted to betaine by a purified bacterial choline oxidase. To test for an oxygenase, the incorporation of ¹⁸O from ¹⁸O₂ into newly synthesized betaine was compared with that from ¹⁸O-labeled choline, in light and darkness. Incorporation of ¹⁸O from ¹⁸O-choline was readily detectable and varied from about 15 to 50% of the theoretical maximum value; the ¹⁸O losses were attributable to exchange of the intermediate betaine aldehyde with water. In darkness, incorporation of ¹⁸O from ¹⁸O₂ approached that from ¹⁸O-choline, but in the light was severalfold lower, presumably due to isotopic dilution by photosynthetic ¹⁶O₂. These data indicate that the chloroplast choline-oxidizing enzyme is an oxygenase.     

Influence of buffer system and pH on the amount of oxygen exchanged between solvent H2O and the CO2 produced in the aerobic oxidation of Cypridina luciferin catalyzed by Cypridina luciferase.

Incorporation of ¹⁸O into CO₂ was measured under various buffer conditions when the bioluminescent oxidation of Cypridina luciferin, catalyzed by luciferase, was carried out either in H₂¹⁶O medium with ¹⁸O₂ gas, or in H₂¹⁸O medium with ¹⁶O₂ gas. The results indicate that (1) the exchange of oxygen between CO₂ and solvent H₂O is significantly influenced by the kind of buffer as well as by pH, (2) the exchange of oxygen between solvent H₂O and CO₂ produced from luciferin in a neutral buffer can be reasonably well estimated from the exchange that takes place when the same amount of CO₂ gas is introduced into the same buffer by the presently employed method, and (3) in the Cypridina bioluminescent reaction, one of two oxygens of O₂ is quantitatively incorporated into the product CO₂ prior to the exchange of oxygen between CO₂ and solvent H₂O.     

Mechanism of oxidation of carbon monoxide by bacteria

Double label experiments were performed employing ¹³CO and either H₂¹⁸O or ¹⁸O₂ in the presence of a CO utilizing bacterium. CO₂ generated was trapped and $\text{m}\text{e}$ ratios $\text{47}\text{45}$ showed that the second oxygen atom in the oxidation of CO to CO₂ by this bacterium comes neither from O₂ nor H₂O.     

Inactivation of Bacteriophages by ε-Poly-l-lysine Produced by Streptomyces

Degradation of a β-O-4lignin substructure model dimer by a white rot fungus, Phanerochaete chrysosporium, was investigated using a culture containing H₂¹⁸O, and the following conclusions were made. a) The direct hydrolysis at C_β of the β-O-4 dimer was not involved in formation of arylglycerol. b) About half of the oxygen at the benzyl (C_α) position of the glycerol was derived from H₂O (H₂¹⁸O) and the other half was from the oxygen at the benzyl (C_α) position of the substrate β-O-4 dimer. c) But, the oxygen at the C_α position of the substrate β-O-4 dimer did not migrate to the C_α position of the aryglycerol.     

O2 and H2O are each the source of one O in NO−2 produced from NH3 by Nitrosomonas: 15N-NMR evidence

The exchange of ¹⁸O between H₂¹⁸O and exogeneously added ¹⁵N¹⁶O^?₂ which occurs during oxidation of ammonia by Nitrosomonas is shown to occur one oxygen at a time. Conditions in which the exchange is diminished (notably the presence of ¹⁴NO^–₂ and CCCP) allowed demonstration that water and dioxygen are each the source of one oxygen in nitrite produced from ¹⁵NH₃. The nitrate produced in the presence of ¹⁸O₂ consisted of 67 and 0% ¹⁵N¹⁸O¹⁶O^? and ¹⁵N¹⁸O¹⁸O^?, respectively. Analysis was made using the ¹⁸O-isotope shift in ¹⁵N-NMR.     

Study on the properties of the S3-state by mass spectrometry in the filamentous cyanobacterium Oscillatoria chalybea

In the present paper we analyzed the properties of the S₃-state in the filamentous cyanobacterium Oscillatoria chalybea by mass spectrometry. In this organism a substantial O₂-signal due to a single flash is observed even after extensive dark adaptation (20 min). This signal can be measured by mass spectrometry as well as amperometrically on an oxygen electrode and is not due to an interference of respiratory and photosynthetic electron transport in the prokaryotic membrane. The mass spectrometric analysis shows that, if S₃ is generated by two flashes in a medium containing only H₂¹⁶O, addition of H₂¹⁸O and subsequent firing of a third flash yields O₂ evolution labelled with ¹⁸O. It appears that the isotopic composition of the O₂ evolved corresponds to the isotopic composition of the water in the suspension. This experiment shows that water oxidation does not proceed via an oxygen precursor or water derivatives bound to the S₃-state. This conclusion has been reached shortly before ours by Radmer and Ollinger [15] in the reverse marker experiment. From our study with O. chalybea it appears that freshly generated S₃ can be distinguished from metastable S₃ by the mass spectrometric method. It looks as if in contrast to freshly generated S₃ metastable S₃ contained bound unexchangeable water or an oxidized water derivative.     

Incorporation of 18 O into homovanillic acid of rat brain during exposure to oxygen 18 containing atmospheres

Rats were exposed to air containing ¹⁸O₂ at atmospheric pressure. $\text{In vivo}$ incorporation of ¹⁸O in brain homovanillic acid (HVA) was determined by gas chromatography-mass spectrometry. One ¹⁸O atom was incorporated into each molecule of HVA indicating that tyrosine is the predominant precursor of brain dopamine and that the oxygen in the 3-position is of atmospheric origin. Intraperitoneal administration of ¹⁸O-enriched water did not alter the ¹⁸O content of brain HVA Mass fragmentography (2) was used to measure the increase in ¹⁸O and the decrease in ¹⁶O in HVA from rat brain over several hours of exposure to an ¹⁸O enriched atmosphere. These experiments demonstrate the possibility to pulse label brain dopamine and its metabolites by $\text{in vivo}$ inhalation of stable oxygen isotopes. The procedure should be useful for quantitative determinations of the turnover of brain dopamine in animals and man.     

New Type of Oxygenase Involved in the Metabolism of Propane and Isobutane

Nocardia paraffinicum (Rhodococcus rhodochrous), a hydrocarbon-degrading microorganism, was used in a study of propane and isobutane metabolism. The bacterium was able to utilize propane or isobutane as a sole source of carbon, and oxygen was found to be essential for its metabolism. Gas chromatographic analysis showed that n-propanol was the major compound recovered from the metabolism of propane by resting cells, although trace amounts of isopropanol and acetone were detected. When a mixture of propane and isobutane was used, drastic inhibition (72 to 88%) of hydrocarbon utilization by resting cells occurred. The ratio of hydrocarbon to oxygen consumed was found to be approximately 2:1 during the metabolism of propane or isobutane by resting cells when these substrates were provided individually to the organism. Gas chromatographic-mass spectrometric analysis of products formed from ¹⁸O₂ confirmed that the initial oxidative step in the metabolism of these substrates involved molecular oxygen. The proportion of the alcohol containing ¹⁸O was the same as that of ¹⁸O₂ in the gas mixture. Only a negligible amount of ¹⁸O was detected in the alcohol when H₂¹⁸O was incorporated into the system. The observed 2:1 ratio of hydrocarbon to oxygen consumption suggests that the oxygenase in N. paraffinicum, unlike the conventional mono- or dioxygenases, requires two hydrocarbon-binding sites for each of the oxygen-binding sites and is therefore an intermolecular dioxygenase. The newly described oxygenase, which catalyzes the reaction of two molecules of propane with one molecule of oxygen to yield two molecules of a C₃ alcohol, is proposed as the initial oxidation step of the hydrocarbon substrate.     

Abscisic Acid Biosynthesis in Leaves and Roots of Xanthium strumarium

Research on the biosynthesis of abscisic acid (ABA) has focused primarily on two pathways: (a) the direct pathway from farnesyl pyrophosphate, and (b) the indirect pathway involving a carotenoid precursor. We have investigated which biosynthetic pathway is operating in turgid and stressed Xanthium leaves, and in stressed Xanthium roots using long-term incubations in ¹⁸O₂. It was found that in stressed leaves three atoms of ¹⁸O from ¹⁸O₂ are incorporated into the ABA molecule, and that the amount of ¹⁸O incorporated increases with time. One ¹⁸O atom is incorporated rapidly into the carboxyl group of ABA, whereas the other two atoms are very slowly incorporated into the ring oxygens. The fourth oxygen atom in the carboxyl group of ABA is derived from water. ABA from stressed roots of Xanthium incubated in ¹⁸O₂ shows a labeling pattern similar to that of ABA in stressed leaves, but with incorporation of more ¹⁸O into the tertiary hydroxyl group at C-1′ after 6 and 12 hours than found in ABA from stressed leaves. It is proposed that the precursors to stress-induced ABA are xanthophylls, and that a xanthophyll lacking an oxygen function at C-6 (carotenoid numbering scheme) plays a crucial role in ABA biosynthesis in Xanthium roots. In turgid Xanthium leaves, ¹⁸O is incorporated into ABA to a much lesser extent than it is in stressed leaves, whereas exogenously applied ¹⁴C-ABA is completely catabolized within 48 hours. This suggests that ABA in turgid leaves is either (a) made via a biosynthetic pathway which is different from the one in stressed leaves, or (b) has a half-life on the order of days as compared with a half-life of 15.5 hours in water-stressed Xanthium leaves. Phaseic acid showed a labeling pattern similar to that of ABA, but with an additional ¹⁸O incorporated during 8′-hydroxylation of ABA to phaseic acid.     

Impaired Synthesis of Acetylcholine by Mild Hypoxic Hypoxia or Nitrous Oxide

The effect of mild hypoxic hypoxia on brain metabolism and acetylcholine synthesis was studied in awake, restrained rats. Since many studies of hypoxia are done with animals anesthetized with nitrous oxide (N₂O), the effects of N²O were evaluated. N²O (70%) increased the cerebral cortical blood flow by 33% and the cortical metabolic rate of oxygen by 26%. In addition, the synthesis of acetylcholine in N²O-anesthetized animals, measured with [U-¹⁴C]glucose and [1-²H₂,2-²H₂]choline, decreased by 45 and 53%, respectively. Consequently, mild hypoxia was studied in unanesthetized rats. Control rats breathing 30% O₂ (partial pressure of oxygen, Pao₂= 120 mm Hg) were compared with rats exposed to 15% O₂ (Pao₂= 57 mm Hg) or 10% O₂ (Pao₂= 42 mm Hg). The synthesis of acetylcholine, measured with [U-¹⁴C]glucose, was decreased by 35 and 54% with 15% O₂ and 10% O₂ respectively; acetylcholine synthesis, measured with [1-²H₂,2-²H₂]choline, was decreased by 50 and 68% with 15% O₂ and 10% O₂ respectively. Animals breathing either 15% or 10% O₂ had normal cerebral metabolic rates of oxygen but had increased brain lactates and increased cortical blood flows compared with animals breathing 30% O₂. These results show that even mild hypoxic hypoxia impairs acetylcholine synthesis, which in turn may account for the early symptoms of brain dysfunction associated with hypoxia.     

Kinetics of Energetic O<Superscript>−</Superscript> Ions in the Discharge Plasmas of Water Vapor and H<Subscript>2</Subscript>O-Containing Mixtures

The process of relaxation of energetic O^– ions formed via dissociative attachment of electrons to molecules in the discharge plasmas of water vapor and H₂O: O₂ mixtures in a strong electric field is studied by the Monte Carlo method. The probability of energetic ions being involved in threshold ion–molecular processes is calculated. It is shown that several percent of energetic O^– ions formed via electron attachment to H₂O molecules in the course of plasma thermalization transform into OH^– ions via charge exchange or are destroyed with the formation of free electrons. The probabilities of charge exchange of O^– ions and electron detachment from them increase significantly (up to 90%) when O^– ions are formed via electron attachment to O₂ molecules in water vapor with an oxygen additive. This effect decreases with increasing oxygen fraction in the mixture but remains appreciable even when the fraction of H₂O molecules in the H₂O: O₂ mixture does not exceed several percent.     

Incorporation of Oxygen into Glycolate, Glycine, and Serine during Photorespiration in Maize Leaves

Glycolate, glycine, and serine extracted from excised Zea mays L. leaves which had been allowed to photosynthesize in the presence of ¹⁸O₂ were analyzed by gas chromatography-mass spectrometry. In each case, only one of the oxygen atoms of the carboxyl group had become labeled. The maximum enrichment observed in glycine and serine was attained after 5 minutes and 15 minutes of exposure to ¹⁸O₂ at the CO₂ compensation point; the labeling was very high, reaching 70 to 73% of that in the applied O₂. Thus, it appears that all or nearly all of the glycine and serine are synthesized in maize leaves via fixation of O₂. In the presence of CO₂ (380 or 800 microliters per liter), ¹⁸O-labeling was markedly slower.

Glycolate enrichment was variable and much lower than that in glycine and serine. It is possible that there are additional pathways of glycolate synthesis which do not result in the incorporation of ¹⁸O from molecular oxygen. An estimation of the metabolic flow of O₂ through the photorespiratory cycle was made. It appeared that less than 75% of the O₂ taken up by maize leaves is involved in this pathway. Therefore, other processes of O₂ metabolism must occur in the light.

     

Mechanisms of hydroxylation by cytochrome P-450: Exchange of iron-oxygen intermediates with water

Highly-purified rat liver microsomal cytochrome P-450 converted cyclohexane to cyclohexanol in the presence of iodosobenzene. Oxygen from ¹⁸O-iodosobenzene was not incorporated into cyclohexanol but oxygen from H₂¹⁸O was readily incorporated. Cytochrome P-450 catalyzed the facile exchange of oxygen between iodosobenzene and water but neither cytochrome P-420 nor the apoenzyme did. Under these conditions cytochrome P-450 readily incorporated oxygen from ¹⁸O₂ into cyclohexanol in the presence of NADPH-cytochrome P-450 reductase and NADPH. The results are interpreted in a mechanism in which cytochrome P-450 forms a common hydroxylating species in the presence of iodosobenzene or O₂ plus NADPH.     

克隆植物结缕草的水分生理整合格局特征及其生态效应分析

以克隆植物结缕草为研究对象,采用18 O作为示踪元素,从克隆植株不同生长发育阶段的复合节根系引入H218 O,在"异质高水"、"均质低水"两种环境条件下,探测和分析结缕草克隆植株复合节根、匍匐茎、A和B分株叶各构件组分系列内的水分生理整合格局特征及其生态效应。结果表明:(1)在两种水分环境条件下,H218 O在克隆植株主匍匐茎内各构件组分系列中均表现出双向传输的趋势,但更倾向于向顶传输。(2)H218 O向顶传输时,在"异质高水"生境内,基部复合节根系吸收的H218 O呈先增加后降低的趋势,而中部复合节根系吸收的H218 O呈先降低后增加的趋势;在"均质低水"生境内,中部复合节根系吸收的H218 O呈持续增加趋势。(3)在两种生境的3种引入情况下,H218 O均向顶传输到尖端生长点。其中在"异质高水"和"均质低水"生境内H218 O在克隆植株中向基传输过程中,传输强度整体上呈下降趋势;H218 O在主匍匐茎中传输时18 O分配于分株叶片中的量较多;H218 O在二级匍匐茎中的传输都呈现出明显的向顶趋势,传输距离都到达了二级匍匐茎的顶端生长点。(4)在绝大多数情况下,A分株叶系列的18 O丰度均明显高于B分株叶系列,这与A、B分株系列的生长发育特征相一致;但在"异质高水"生境内,中部分株吸收的H218 O在二级匍匐茎中传输时,分配于B分株叶系列的18 O明显高于A分株叶系列,即A分株系列相对于B分株系列的比较优势并不是一成不变的,在某些情况下还可以发生逆转。     

Hydroperoxide-dependent oxygenation of trans-7,8-dihydroxy-7,8-dihydrobenzo[a]pyrene by ram seminal vesicle microsomes. Source of the oxygen

Incubation of 15-hydroperoxy-5,8,11,13-eicosatetraenoic acid with ram seminal vesicle microsomes (RSVM) triggers the oxygenation of $\text{trans}$-7,8-dihydroxy-7,8-dihydrobenzo[a]pyrene (BP-7,8-diol). The principal oxidation products are 7,8,9,10-tetrahydroxy-7,8,9,10-tetrahydrobenzo[a]pyrenes which are non-enzymatic hydrolysis products of r-7,t-8-dihydroxy-t-9,10-oxy-7,8,9,10-tetrahydrobenzo[a]pyrene. At short incubation times, an additional product is isolated which is identified as r-7,t-8,t-9-trihydroxy-c-10-methoxy-7,8,9,10-tetrahydrobenzo[a]pyrene. This product appears to arise by solvolysis of the extracted diolepoxide during high performance liquid chromatography using methanol-water solvent systems. The incubation of ¹⁸O-labeled 15-hydroperoxy-5,8,11,13-eicosatetraenoic acid with BP-7,8-diol and RSVM leads to very little incorporation of ¹⁸O into the stable solvolysis products (analyzed by gc-ms of their peracetates). Parallel incubations conducted with ¹⁶O-labeled hydroperoxide under an ¹⁸O₂ atmosphere indicate that the principle source of the epoxide oxygen is molecular oxygen.