Regioselectivity in enzymatic hydration of cis-1,2-disubstituted [18O]-epoxides

The hydration of $\text{cis}$-β-methylstyrene oxide, $\text{cis}$-2,3-octene oxide, and their ¹⁸O-enriched forms by epoxide hydrase of rat liver microsomes has been investigated. Both $\text{cis}$ epoxides underwent quantitative enzymatic hydration yielding exclusively the corresponding $\text{threo}$ diols, indicating that complete stereochemical inversion at a single oxirane carbon had occurred. Mass spectral analysis of diols formed enzymatically from the ¹⁸O enriched epoxides indicated they were formed with great regioselectivity, 89% and 85% of the ¹⁸O being located at the benzylic carbon of the styrene diol and at C-3 of the octane diol, respectively.     

Fate of naturally occurring epoxy acids: a soluble epoxide hydrase, which catalyzes cis hydration, from Fusarium solani pisi.

A cell-free extract prepared from Fusarium solani pisi grown on cutin, catalyzed the hydration of 18-hydroxy-9,10-epoxyoctadecanoic acid to 9,10,18-trihydroxyoctadecanoic acid while extracts from glucose-grown cells contained <6% of this activity. The product was identified by Chromatographic techniques and by radio gas-liquid chromatography of its periodate oxidation products. This epoxide hydrase activity had a pH optimum at 9.0 and it was located mainly in the 100,000g supernatant fraction. Rate of hydration of the epoxy acid was linear up to 15 min and up to a protein concentration of 30 μg/ml. This fungal epoxide hydrase has a molecular weight of 35,000, as determined by Sephadex G-100 gel filtration. It was partially purified by ammonium sulfate fractionation and gel filtration. The apparent K_m and V of the enzyme was 2 × 10^?4m and 222 nmoles/min/mg, respectively. Parachloromercuribenzoate strongly inhibited the enzyme, while N-ethylmaleimide was a less potent inhibitor. 1,1,1,-Trichloropropylene-2,3-oxide at 10^?3m gave 50% inhibition of the hydration of 18-hydroxy-9,10-epoxyoctadecanoic acid. Kinetic analysis showed that trichloropropylene oxide was a competitive inhibitor. 18-Acetoxy-9,10-epox-yoctadecanoic acid, methyl 18-acetoxy-9,10-epoxyoctadecanoate, 9,10-epoxyoctadecanoic acid, and styrene oxide were not readily hydrated by this fungal epoxide hydrase showing that it has a stringent substrate specificity. Analysis of the enzymatic hydration product on boric acid-impregnated silica gel plates showed that the product obtained from the cis epoxide was exclusively erythro while acid hydrolysis of this epoxide gave rise to the expected threo product. This enzyme is novel in that it catalyzes cis hydration of epoxide while the other epoxide hydrases heretofore isolated catalyzed trans hydration of epoxides.     

Mechanistic studies of epoxide hydrolase utilizing a continuous spectophotometric assay

A precise continuous photometric assay has been devised and utilized for mechanistic studies of chicken and rat liver microsomal epoxide hydrolase (EH). The assay is based on monitoring the hydration of p-nitrostyrene oxide (PNSO) at 310 nm. Rat liver EH hydrates S-(+)- and R-(?)-PNSO differentially, the K_m and V values for the former being ca. four times those for the latter; in contrast, enantiomeric differences are negligible with chicken liver EH. With rat EH V increases slightly from pH 7 to 8 and then falls rapidly from pH 8 to 9.5; K_m remains constant from pH 7 to 8 and then increases steadily from pH 8 to 9.5. In 86 mol% D₂O the solvent isotope effect on V ($\text{H}{}_2\text{O}\text{D}{}_2\text{O}$) is 1.103 ± 0.015. Both rat and chicken EH show a 3% inverse isotope effect for the hydration of [7-²H]PNSO and a 4% normal isotope effect for the hydration of [8-²H₂]PNSO. These observations are discussed in terms of the possible participation of acid as well as base catalysis in the enzymatic mechanism.     

Direct evidence for the involvement of epoxide intermediates in the biosynthesis of the C18 family of cutin acids

Cutin, the structural component of plant cuticle, is a polymer of C₁₆ and C₁₈ hydroxy fatty acids. Previous results have suggested that oleic acid undergoes ω-hydroxylation, epoxidation of the double bond, and, finally, hydration of the epoxide to give rise to the three major components of the C₁₈ family of cutin acids. 18-Hydroxy [18-³H]oleic acid and 18-hydroxy-9,10-epoxy[18-³H]stfaric acid have been synthesized and, with these synthetic substrates, the conversion of 18-hydroxyoleic acid to 18-hydroxy-9,10-epoxystearic acid and the hydrolysis of 18-hydroxy-9,10-epoxystearic acid to 9,10,18-trihydroxystearic acid were directly demonstrated in apple fruit skin and in the leaves of apple and Senecio odoris. Trichloropropene oxide, an inhibitor of microsomal epoxide hydrases of animals, specifically inhibited the conversion of [1-¹⁴C]oleic acid into 18-hydroxy-9,10-epoxystearic acid and 9,10,18-trihydroxystearic acid, while it had no effect on the conversion of [1-¹⁴C]palmitic acid into hydroxylated palmitic acid, a process which does not involve epoxy acid intermediates. Therefore, it appears that this inhibitor affects epoxidation and or epoxide hydration steps involved in cutin biosynthesis.     

Interaction of epoxide hydrolase with itself and other microsomal proteins

The interactions of rat liver epoxide hydrolase (EC 3.3.2.3) with itself and with cytochromes P-450 and NADPH-cytochrome P-450 reductase were investigated in microsomal preparations and in reconstituted systems in which all of the enzymes are functionally active. Hydrodynamic measurements indicated that purified epoxide hydrolase behaves as a single aggregate of approximately 16 monomeric units and that further aggregation of the protein only occurs in the presence of high concentrations of phospholipid. Neither guanidine-HCl nor the nonionic detergent Lubrol PX was able to completely dissociate the aggregate into monomers. The interactions of epoxide hydrolase with NADPH-cytochrome P-450 reductase and the major forms of cytochrome P-450 isolated from phenobarbital- and 5,6-benzoflavone-treated rats were studied by Soret difference spectroscopy, by perturbation of the fluorescence of NADPH-cytochrome P-450 reductase and fluorescein-labeled epoxide hydrolase, and by CD spectroscopy. The spectra provided evidence that binding of the proteins to each other occurs and some of the results suggest that affinity constants are on the order of 10⁷, m^?1. The spectral perturbations were not observed with other intrinsic membrane proteins. When microsomes were treated with the crosslinking reagent dimethylsuberimidate and solubilized with detergents, epoxide hydrolase could be precipitated with antibodies raised to cytochromes P-450 or NADPH-cytochrome P-450 reductase. Transient times were determined for the conversion of 1-octene to octene-1,2-dihydrodiol in a reconstituted enzyme system and for the conversion of naphthalene to naphthalene-1,2-dihydrodiol in rat liver microscomes and compared to the transient times predicted from the enzymatic rates of hydrolysis of the intermediate epoxides. In all cases the observed transient times were shorter than expected, in support of the view that coupling of epoxide hydrolase with cytochromes P-450 occurs. These results support the view that epoxide hydrolase couples with cytochrome P-450-containing mixed-function oxidase systems and may have relevance to the metabolism of potentially harmful xenobiotics by these enzymes.     

Enzymatic hydration of leukotriene A4. Purification and characterization of a novel epoxide hydrolase from human erythrocytes

Human erythrocytes contained a soluble cytosolic epoxide hydrolase for stereospecific enzymatic hydration of leukotriene A4 into leukotriene B4. The enzyme was purified 1100-fold, to apparent electrophoretic homogeneity, by conventional DEAE-Sephacel fractionation followed by high performance anion exchange and chromatofocusing procedures. Its characteristics include a molecular weight of 54,000 +/- 1,000, an isoelectric point 4.9 +/- 0.2, a Km apparent from 7 to 36 microM for enzymatic hydration of leukotriene A4, and a pH optimum ranging from 7 to 8. The enzyme was partially inactivated by its initial exposure to leukotriene A4. There was slow but detectable enzymatic hydration (pmol/min/mg) of certain arachidonic acid epoxides including (+/-)-14,15-oxido-5,8-11-eicosatrienoic acid and (+/-)-11,12-oxido-5,8,14-eicosatrienoic acid, but not others, including 5,6-oxido-8,11,14-eicosatrienoic acid. Human erythrocyte epoxide hydrolase did not hydrate either styrene oxide or trans-stilbene oxide. In terms of its physical properties and substrate preference for leukotriene A4, the erythrocyte enzyme differs from previously described versions of epoxide hydrolase. Human erythrocytes represent a novel source for an extrahepatic, cytosolic epoxide hydrolase with a potential physiological role.     

Assay and partial purification of epoxide hydrase from rat liver microsomes

Solubilized cytochrome P-450 monooxygenase and epoxide hydrase activities from rat liver microsomes have been separated by column chromatography. The highly active epoxide hydrase fraction is still contaminated with cytochrome P-450, which has very low monooxygenase activity. The highly purified cytochrome P-450 fraction possesses high monooxygenase activity and is essentially devoid of epoxide hydrase activity. Purification factors for the epoxide hydrase through four purification steps are similar with [³H]styrene oxide, [³H]naphthalene oxide, [³H]cyclohexene oxide, and benzene oxide as substrates. Failure of benzene oxide to inhibit hydration of styrene or naphthalene oxide in the most purified preparations in indicative of the presence of at least two hydrases. These purified cytochrome monooxygenase and hydrase preparations represent valuable tools for the study of the intermediacy of arene oxides in drug metabolism. Thus, with naphthalene, only naphthol is formed with the monooxygenase, while both naphthol and the dihydrodiol are formed in the presence of monooxygenase and hydrase. A convenient radiochemical synthesis of [³H]naphthalene 1,2-oxide and assays for the measurement of the hydration of [³H]naphthalene oxide and benzene oxide, based on differential extractions and high-pressure liquid chromatography, respectively, are described.     

Oxygen Isotope Exchange between Metabolites and Water during Biochemical Reactions Leading to Cellulose Synthesis

Cellulose was produced heterotrophically from different carbon substrates by carrot tissue cultures and Acetobacter xylinum (a cellulose-producing bacterium) and by castor bean seeds germinated in the dark, in each case in the presence of water having known concentration of oxygen-18 (¹⁸O). We used the relationship between the amount of ¹⁸O in the water and in the cellulose that was synthesized to determine the number and ¹⁸O content of the substrate oxygens that exchanged with water during the reactions leading to cellulose synthesis. Our observations support the hypothesis that oxygen isotope ratios of plant cellulose are determined by isotopic exchange occurring during hydration of carbonyl groups of the intermediates of cellulose synthesis.     

Inhibition of insect juvenile hormone epoxide hydrolase: asymmetric synthesis and assay of glycidol-ester and epoxy-ester inhibitors of trichoplusia ni epoxide hydrolase

Juvenile hormone (JH) undergoes metabolic degradation by two major pathways involving JH esterase and JH epoxide hydrolase (EH). While considerable effort has been focussed on the study of JH esterase and the development of inhibitors for this enzyme, much less has been reported on the study of JH-EH. In this work, the asymmetric synthesis of two classes of inhibitors of recombinant JH-EH from Trichoplusia ni, a glycidol-ester series and an epoxy-ester series is reported. The most effective glycidol-ester inhibitor, compound 1, exhibited an I(50) of 1.2x10(-8) M, and the most effective epoxy-ester inhibitor, compound 11, exhibited an I(50) of 9.4x10(-8) M. The potency of the inhibitors was found to be dependent on the absolute configuration of the epoxide. In both series of inhibitors, the C-10 R-configuration was found to be significantly more potent that the corresponding C-10 S-configuration. A mechanism for epoxide hydration catalyzed by insect EH is also presented.     

Mechanism of catalysis for the hydration of substituted styrene oxides by hepatic epoxide hydrase

The effect of aryl substituents on the rate at which epoxide hydrase catalyzes the addition of water to styrene and cis-stilbene oxides has been examined. Plots of log V_m for each substrate versus the Hammett σ constants for the substituent suggest that a nucleophilic attack occurs and that a free carbonium ion form of the substrate is not involved at the rate-determining step in the mechanism. For the stilbene oxides, high selectivity for attack by water at the carbon atom with [S] absolute stereochemistry was observed.     

Determination of the absolute stereochemistry of the epoxide group in alpinia epoxide by NMR

The absolute stereochemistry of the epoxide group in alpinia epoxide [14,15-epoxylabda-8(17),12-dien-16-al (E)] has been determined by simultaneous reduction of the aldehyde and epoxide functional groups in this molecule to primary and secondary alcohols, followed by selective protection of the primary alcohol and derivitization of the secondary alcohol with S(+) and R(−) MTPCl as Mosher esters. Changes in ¹H NMR chemical shifts for all positions in these two esters were determined by 2D-NMR and used to infer the absolute stereochemistry of the epoxide group in the natural product alpinia epoxide.     

The epoxide hydrolase catalyzed hydrolysis of trans-3-bromo-1,2-epoxycyclohexane. A direct proof for a general base catalyzed mechanism of the enzymatic hydration

The hydrolysis of (±)-$\text{trans}$-3-bromo-1,2-epoxycyclohexane in the presence of rabbit liver microsomes was investigated, and found to yield, beside $\text{c}$-3-bromocyclohexane-$\text{r}$-1,$\text{t}$-2-diol, 2,3-epoxycyclohexanol. It was demonstrated that the latter compound was the only product of the enzymatic reaction, whereas the diol resulted from a non enzymatic hydration in the reaction medium. These data provide the first direct proof for a general base catalysis in the enzymatic epoxide hydration, previously hypothesized on the basis of several lines of indirect evidence, and disprove alternative mechanisms involving protonation of the oxirane oxygen.     

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.     

The reactions of OH radicals with D-ribose in deoxygenated and oxygenated aqueous solution

Aqueous solution ofD-ribose (10^?2M) saturated with N₂O and N₂O/O₂ (4/1) were γ-irradiated (dose rate: 3.85 x 10¹⁸ eV.g^?1.h^?1) at room temperature. The following products were identified:D-ribonic acid (1). D-erythro-pentos-2-ulose (2). D-erythro-pentos-4-ulose (3),D-erythro-pentos-3-ulose (4), D-ribo-pentodialdose (5), 2-deoxy-D-erythro-pentonic acid (6), 2-deoxypentos-3-ulose (7)(7), 4-deoxylpentos-3-ulose (8), 3-deoxypentos-4-ulose (9), 3-deoxypentos-2-ulose (10), 5-deoxypentos-4-ulose (11), erythrose (12), erythro-tetrodialdose (13), erythronic acid (14), threose/erythrulose (15). threonic acid (16), 2-deoxytetrose (17), and glyceraldehyde (18). In deoxygenated solutions, 13, 14, and 16 were absent. In the presence of oxygen, the formation of 6–11 and 17 was suppressed. From quantitative measurements, G-values were calculated for both deoxygenated and oxygenated conditions. Five different, primary, ribosyl radicals are formed which, in deoxygenated solution, undergo disproportionation reactions (to give 1-5), and transformations such as elimination of water and carbon monoxide followed by disproportionation reactions (to give6-12.17). Material-balance considerations indicate the formation of dimers (not measured). In oxygenated solutions, oxygen rapidly adds to the primary ribosyl radicals, thus preventing the transformation reactions, and the main products are 1–5 and 13. Possible mechanistic routes are discussed. The attack of HO radicals on D-ribose involves C-1, ～20%; C-2 and C-4, ～35%: C-3, ～ 20%; and C-5, ～25%     

Effect of changes in water content on photosynthesis,transpiration and discrimination against 13CO2 and C18O16O in Pleurozium and Sphagnum

Photosynthetic gas exchange characteristics of two common boreal forest mosses, Sphagnum (section acutifolia) and Pleurozium schreberi, were measured continuously during the time required for the moss to dry out from full hydration. Similar patterns of change in CO₂ assimilation with variation in water content occurred for both species. The maximum rates of CO₂ assimilation for Sphagnum (approx. 7 mol m^–2 s^–1) occurred at a water content of approximately 7 (fresh weight/dry weight) while for Pleurozium the maximum rate (approx. 2 mol m^–2 s^–1) occurred at a water content of approximately 6 (fresh weight/dry weight). Above and below these water contents CO₂ assimilation declined. In both species total conductance to water vapour (expressed as a percentage of the maximum rates) remained nearly constant at a water content above 9 (fresh weight/dry weight), but below this level declined in a strong linear manner. Short-term, on-line ¹³CO₂ and C¹⁸O¹⁶O discrimination varied substantially with changes in moss water content and associated changes in the ratio of chloroplast CO₂ to ambient CO₂ partial pressure. At full hydration (maximum water content) both Sphagnum and Pleurozium had similar values of ¹³CO₂ discrimination (approx. 15). Discrimination against ¹³CO₂ increased continuously with reductions in water content to a maximum of 27 in Sphagnum and 22 in Pleurozium. In a similar manner C¹⁸C¹⁶O discrimination increased from approximately 30 at full hydration in both species to a maximum of 150 in Sphagnum and 90 in Pleurozium, at low water content. The observed changes in C¹⁸O¹⁶O were strongly correlated to predictions of a mechanistic model of discrimination processes. Field measurements of moss water content suggested that photosynthetic gas exchange by moss in the understory of a black spruce forest was regularly limited by low water content.     

Novel Types of N6,2′-Cyclonucleosides

Abstract

Upon oxidation followed by treatment with hydroxylamine, the 3′,5′-diblocked uridine 1 gave the expected oxime 2 together with the N⁶,2′.cyclonucleoside 3 formed by nucleophilic attack of hydroxylamine at both C-6 and C-2′ positions. Reduction of 2 took place predominantly from the α face and the major D-arabino compound obtained gave the cyclonucleosides, 7 via Michael type addition. The structures of the novel cyclonucleosides, particularly their configuration at C-6 were established by X-ray diffraction.     

Purine nucleoside phosphorylase cleaves the C--O bond of ribose 1-phosphate. Evidence from the 18O shift in 31P NMR.

An equilibrium mixture of highly enriched [18(O)]Pi (represents the mixture of [[18(O)4]Pi, [[18(O)3]Pi, [18(O)2]Pi as represented in the figures, unless otherwise specified), alpha-D-ribose 1-[16(O)]phosphate, and hypoxanthine plus inosine was equilibrated with calf spleen purine-nucleoside phosphorylase (EC 2.4.2.1). The 31P NMR spectrum clearly indicated the formation of alpha-D-ribose 1-[18(O)4]-phosphate and of [16(O)]Pi. Incubation for the same time span in the absence of alpha-D-ribose 1-phosphate left the [18(O)4]Pi isotopic distribution unchanged. The results clearly demonstrated that the C--O bond of alpha-D-ribose 1-phosphate is cleaved in the enzymatic reaction. It is unlikely that the enzyme catalyzes the exchange of oxygen between Pi and H2O. Several possible mechanistic pathways are ruled out by the results, which demand attack by a phosphate oxygen at the anomeric C-1' atom.     

Induction, activation and inhibition of epoxide hydrase: an anomalous prevention of chlorobenzene-induced hepatotoxicity by an inhibitor of epoxide hydrase

Epoxide hydrase activity, measured with [³H]styrene oxide as substrate, is present in mammalian liver, kidney, lung, intestine and skin. The hepatic level of the enzyme, measured in vitro with [³H]styrene oxide, benzene oxide or naphthalene-1,2-oxide, is elevated substantially by pretreatment of rats with phenobarbital and to a lesser extent by pretreatment with 3-methylcholanthrene. Metyrapone and 1-(2-isopropylphenyl)-imidazole, two monooxygenase inhibitors, activate epoxide hydrase in vitro, but have no demonstrable effect on the enzyme in vivo. 3,3,3-Trichloropropene oxide, a potent in vitro inhibitor of epoxide hydrase, has no effect on monooxygenase activity measured in vitro with [³H]benzenesulfonanilide. Trichloropropene oxide is extremely toxic. In sub-lethal dosages, it does not significantly inhibit epoxide hydrase activity in vivo, although it and several other epoxides do react with and thereby reduce hepatic levels of glutathione. Cyclohexane oxide, another potent in vitro inhibitor of epoxide hydrase, reduces hepatic glutathione levels to 10% of control values. This relatively non-toxic substance should potentiate the hepatotoxicity of chlorobenzene by inhibiting further metabolism of the toxic chlorobenzene oxide intermediate through either hydration or conjugation with glutathione. Instead, co-administration of cyclohexene oxide and chlorobenzene significantly reduces the rate of metabolism of [¹⁴C]chlorobenzene and prevents the hepatic centrilobular necrosis caused by chlorobenzene in rats. Arene oxide-mediated hepatotoxicity apparently is dependent upon a variety of factors including both rates of formation and degradation of arene oxides in tissue. The presently known hydrase inhibitors are not sufficiently selective in their effects on liver cells to permit a quantitative assessment of the relative importance of these factors.     

Comparison of nuclear and microsomal epoxide hydrase from rat liver

The specific activities of hydration of nine arene and alkene oxides by purified nuclei prepared from the livers of 3-methylcholanthrene-pretreated rats were found to fall within the range of 2.2 to 9.1% of the corresponding microsomal values. Pretreatment with phenobarbital enhanced both the nuclear and microsomal hydration of phenanthrene-9,10-oxide, benzo(a)pyrene-11,12-oxide, and octene-1,2-oxide. 3-Methylcholanthrene pretreatment enhanced the nuclear hydration of these three substrates by 30–60% but had no significant effect on microsomal hydration. An epoxide hydrase modifier, metyrapone, stimulated the hydration of octene-1,2-oxide by the two organelles to quantitatively similar extents, but affected the nuclear and microsomal hydration of benzo(a)pyrene-4,5-oxide differentially. Cyclohexene oxide also exerted differential effects on nuclear and microsomal epoxide hydrase which were dependent both on the substrate and on the organelle. The inhibition by this agent of nuclear and microsomal epoxide hydrase was quantitatively similar only for a single substrate, benzo(a)anthracene-5,6-oxide. When purified by immunoaffinity chromatography, nuclear and microsomal epoxide hydrases from 3-methylcholanthrene-pretreated rats were shown to have identical minimum molecular weights (? 49,000) on polyacrylamide gels in the presence of sodium dodecyl sulfate. These findings support the assertion that microsomal metabolism can no longer be considered an exclusive index of the cellular activation of polycyclic aromatic hydrocarbons.     

Enzymatic formation of (20R, 22R)-20,22-dihydroxycholesterol from cholesterol and a mixture of 16O2 and 18O2: random incorporation of oxygen atoms

[4-¹⁴C]Cholesterol was incubated with an adrenocortical preparation in the presence of ¹⁶O₂ and ¹⁸O₂ devoid of significant ¹⁶O¹⁸O. Isolated (20R,22R)-20,22-dihydroxycholesterol was converted to a trimethylsilyl derivative and analyzed by gas chromatography - mass spectrometry to determine the isotope distribution of the oxygen atoms at C-20 and C-22. The ions of $\text{m}\text{e}$ 289, 291, and 293 (comprising the C₈ C-20 to C-27 side-chain and containing, respectively, ¹⁶O₂, ¹⁶O¹⁸O, and ¹⁸O₂) exhibited a binomial distribution indicating that the oxygen atoms of the vicinal glycol were drawn at random from the atomic pool of the oxygen molecules. If both side-chain hydroxyl groups had originated from the atoms of the same oxygen molecule, the ion of $\text{m}\text{e}$ 291 would have been absent.