18-hydroxyoleic Acid Research Articles

Effect of 4 hydroxy fatty acids on lipid accumulation in the 3T3-L1 cells: a comparative study.

Notwithstanding the several investigations of the hydroxy fatty acids (hFAs)' physiological functions, studies focusing on their anti-obesity effects are limited. This study investigated the anti-obesity effects of 4 hFAs-10-hydroxy stearic acid (10-hSA), 12-hydroxy stearic acid (12-hSA), 9,12-hydroxy stearic acid (9,12-dhSA), and 12-hydroxy oleic acid (12-hOA)-on the 3T3-L1 cells. All hFAs suppressed lipid accumulation, with 10-hSA and 12-hOA exhibiting the strongest suppression, followed by 12-hSA and 9,12-hSA. This trend was similar to that observed for the glycerol-3-phosphate dehydrogenase (GPDH) activity level. Contrastingly, only 9,12-dhSA suppressed cell viability. The mRNA levels of HK1 and Aldoa were markedly suppressed by 10-hSA and 12-hSA compared to the control. Additionally, mRNA expression of Gyk was considerably suppressed by 12-hSA. Thus, all hFAs suppressed lipid accumulation by suppressing GPDH activity, although their molecular mechanisms were different. These findings will aid the application of hFAs in the food and medical industries.

Oleic acid and derivatives affect human endothelial cell mitochondrial function and vasoactive mediator production

BackgroundInhalation of common air pollutants such as diesel and biodiesel combustion products can induce vascular changes in humans which may contribute to increased mortality and morbidity associated with fine particulate matter exposures. Diesel, biodiesel, and other combustion byproducts contain fatty acid components capable of entering the body through particulate matter inhalation. Fatty acids can also be endogenously released into circulation following a systemic stress response to some inhaled pollutants such as ozone. When in the circulation, bioactive fatty acids may interact with cells lining the blood vessels, potentially inducing endothelial dysfunction. To examine whether fatty acids could potentially be involved in human vascular responses to air pollutants, we determined the effects of fatty acids and derivatives on important vascular cell functions.MethodsHuman umbilical vein endothelial cells (HUVEC) were exposed in vitro to oleic acid (OA) or OA metabolites for 4-48 h. Cytotoxicity, vasodilator production (by ELISA measurement), mitochondrial function (using Sea Horse assays), and iron metabolism (inferred by ICP-OES measurements) were examined, with standard statistical testing (ANOVA, t-tests) employed.ResultsDose-dependent cytotoxicity was noted at 24 h, with 12-hydroxy OA more potent than OA. Mitochondrial stress testing showed that 12-hydroxy OA and OA induce mitochondrial dysfunction. Analysis of soluble mediator release from HUVEC showed a dose-dependent increase in prostaglandin F2α, a lipid involved in control of vascular tone, at 24 h (85% above controls) after OA-BSA exposure. RT-PCR analysis revealed OA did not induce changes in gene expression at noncytotoxic concentrations in exposed HUVEC, but 12-OH OA did alter ICAM and COX2 gene expression.ConclusionsTogether, these data demonstrate that FA may be capable of inducing cytotoxic effects and altering expression of mediators of vascular function following inhalation exposure, and may be implicated in air pollutant-induced deaths and hospitalizations. (267 of max 350 words).

Discovery of Eicosapentaenoic Acid Esters of Hydroxy Fatty Acids as Potent Nrf2 Activators.

Branched fatty acid esters of hydroxy fatty acids (FAHFAs) are a recently discovered class of biologically active lipids with anti-inflammatory and anti-diabetic properties. Despite the possible link between endogenous FAHFA levels and nuclear factor erythroid 2-related factor 2 (Nrf2), their possible function as antioxidants and the mechanisms involved in this are unknown. Here, we investigate FAHFAs’ plausible antioxidant potential with reference to their effect on the Nrf2 levels, oxidative stress, and lipid droplet oxidation in human hepatocytes (C3A). Six authentic FAHFAs were chemically synthesized and performed activity-based screening by reporter gene assay. Among them, eicosapentaenoic acid (EPA) esterified 12-hydroxy stearic acid (12-HSA) and 12-hydroxy oleic acid (12-HOA) FAHFAs showed less cytotoxicity compared to their free fatty acids and potent activators of Nrf2. To define their mode of action, relative levels of nuclear Nrf2 were determined, which found a higher amount of Nrf2 in nucleus of cells treated with 12-EPAHSA compared to the control. Furthermore, 12-EPAHSA increased the expression of Nrf2-dependent antioxidant enzyme genes (NQO1, GCLM, GCLC, SOD-1, and HO-1). Fluorescence imaging analysis of linoleic-acid-induced lipid droplets (LDs) in C3A cells treated with 12-EPAHSA revealed the strong inhibition of small-size LD oxidation. These results suggest that EPA-derived FAHFAs as a new class of lipids with less cytotoxicity, and strong Nrf2 activators with plausible antioxidant effects via the induction of cytoprotective proteins against oxidative stress, induced cellular damage.

12-hydroxy oleic acid impairs endothelium-dependent vasorelaxation

ABSTRACTDiesel and biodiesel emissions exposures reduce vascular responsiveness in vivo, but the components of PM responsible for this effect are poorly understood. Fatty acids (FAs) represent a significant fraction of the compounds that make up organic combustion by-products, and may be involved in vascular responses following inhalation. It was hypothesized that vascular tissue exposed to a model FA might impair responses to vasoactive agonists ex vivo. Rat aortic rings were exposed to oleic acid or 12-hydroxy oleic acid and responses determined by myography. 12-Hydroxy oleic acid was found to significantly reduce endothelium-dependent vasodilation at sub-cytotoxic concentrations. This approach demonstrates the potential for FAs, especially oxidized forms, to play a role in the vascular responses observed following air pollution exposure.

Chemo-enzymatic synthesis of 11-hydroxyundecanoic acid and 1,11-undecanedioic acid from ricinoleic acid

A practical chemoenzymatic synthetic method for 11-hydroxyundecanoic acid and 1,11-undecanedioic acid from ricinoleic acid (12-hydroxyoleic acid) was investigated.

Fruit Cuticle Composition of a Melting and a Nonmelting Peach Cultivar

Although postharvest quality of fruit is greatly affected by cuticle composition, structure, and properties, very few published studies have analyzed fruit cuticle composition from a postharvest perspective. In this work, the chemical composition of waxes and cutin, major cuticular components, was analyzed in fruit cuticle samples isolated from a melting ('October Sun') and a nonmelting ('Jesca') peach (Prunus persica L. Batsch.) cultivar at harvest and after a simulated shelf-life period of 5 days at 20 °C. Cutin composition was dominated by 18-hydroxyoleic acid, whereas the triterpenoid ursolic and oleanoic acids and the alkanes n-tricosane and n-pentacosane were quantitatively prominent among the wax compounds identified. Some quantitative differences were found between both cultivars for particular compound families and in their postharvest modifications. To the best of the authors' knowledge, this is the first study characterizing the composition of the cuticle of peach fruit and describing the changes therein after harvest.

The tomato SlSHINE3 transcription factor regulates fruit cuticle formation and epidermal patterning

Fleshy tomato fruit typically lacks stomata; therefore, a proper cuticle is particularly vital for fruit development and interaction with the surroundings. Here, we characterized the tomato SlSHINE3 (SlSHN3) transcription factor to extend our limited knowledge regarding the regulation of cuticle formation in fleshy fruits. We created SlSHN3 overexpressing and silenced plants, and used them for detailed analysis of cuticular lipid compositions, phenotypic characterization, and the study on the mode of SlSHN3 action. Heterologous expression of SlSHN3 in Arabidopsis phenocopied overexpression of the Arabidopsis SHNs. Silencing of SlSHN3 results in profound morphological alterations of the fruit epidermis and significant reduction in cuticular lipids. We demonstrated that SlSHN3 activity is mediated by control of genes associated with cutin metabolism and epidermal cell patterning. As with SlSHN3 RNAi lines, mutation in the SlSHN3 target gene, SlCYP86A69, resulted in severe cutin deficiency and altered fruit surface architecture. In vitro activity assays demonstrated that SlCYP86A69 possesses NADPH-dependent ω-hydroxylation activity, particularly of C18:1 fatty acid to the 18-hydroxyoleic acid cutin monomer. This study provided insights into transcriptional mechanisms mediating fleshy fruit cuticle formation and highlighted the link between cutin metabolism and the process of fruit epidermal cell patterning.

Enzymatic formation of 13,26-Dihexyl-1,14-dioxacyclohexacosane-2,15-dione via Oligomerization of 12-Hydroxystearic acid

The enzymatic polymerization of 12-hydroxystearic acid (12-HSA) was carried out with Lipase CA® in benzene to produce poly(12-hydroxystearate) (PHS) with a low molecular weight. When this polymerization was continued for a long reaction time, the PHS once formed was depolymerized into a cyclic diester, 13,16-dihexyl-1, 14-dioxacyclohexacosane-2,15-dione (12-HSAD). Similar polymerization and depolymerization were observed when 12-hydroxyoleic acid (12-HOA) was treated with Lipase CA®, whereas only polymerization occurred when 12-hydroxydodecanoic acid (12-HDA) was treated in a similar manner. The preferential formation of cyclic diesters for 12-HSA was attributed to the structural requirements due to the bulkyn-hexyl side groups stemming from the ring systems.

Investigation of diacylglycerol acyltransferase (DGAT) activity of microsomes from the seeds of three euphorbs

Unusual fatty acids such as ricinoleate (12-hydroxyoleic acid) occurring in Ricinus communis L. or vernoleate (12,13-epoxyoleic acid) occurring in Euphorbia lagascae L. are suitable for industrial uses. Euphorbia lathyris L. is also a potential new oilseed crop on account of its high content of oleic acid in the seeds. The objective of this work was to test in vitro the preferences of E. lathyris microsomes for its native substrates (oleoyl-CoA and diolein) and to compare with R. communis and E. lagascae systems. The diacylglycerol acyltransferase (DGAT) catalyses the final step in transferring a fatty acid moiety to a diacylglycerol (DAG) into a triacylglycerol (TAG). To study the DGAT activity in microsomes of the three euphorbs: (1) plants of the three species were grown in a glasshouse at Instituto Murciano de Investigación y Desarrollo Agrario y Alimentario (Murcia, Spain), (2) endosperms were removed from developing seeds and the tissue was extracted, (3) in vitro DGAT assays using [ 14C]-oleoyl-CoA with or without 1,2-diolein were carried out and 4) labelled TAG were recorded using a molecular imager and a scintillation counter. Incorporation of [ 14C]-oleoyl into TAG was greater in R. communis and E. lathyris (72–89% of total TAG) than in E. lagascae. Adding exogenous 1,2-diolein (1 mM) to E. lagascae microsomes increased the amount of labelled TAG to 39%, suggesting that other acyl groups were being incorporated as well. R. communis and E. lagascae microsomes gave more-polar radiolabelled TAGs than E. lathyris possibly because endogenous DAGs (not 1,2-diolein) were being used in the reaction. Although E. lathyris microsomes showed specificity towards 1,2-diolein as a substrate, the preparations from R. communis, E. lagascae and E. lathyris were able to use the acyl donor and acyl receptor, possibly suggesting that DGAT enzymes would not be a limiting factor to engineer Euphorbiaceae crops with functionalized fatty acids.

Modified polycarbonate urethane: Synthesis, properties and biological investigation in vitro

A new polycarbonate urethane (PCU-I) was synthesized from aliphatic monomers, i.e. polyhexamethylene carbonate diol and 4,4'-methylene-bis cyclohexane diisocyanate, a mixture of low molecular diols, and castor oil (containing mainly the triglyceride of 12-hydroxyoleic acid). The second synthesized polymer (PCU-II) did not contain castor oil. Both PCUs had good tensile strength, i.e. 32.5 and 27.8 MPa for PCU-I and PCU-II, respectively. Modification by castor oil led to a decrease in glass transition temperature (T(g) = -14 degrees C for PCU-I and -6 degrees C for PCU-II) and an increase in the softening temperature (135 and 125 degrees C for PCU-I and PCU-II, respectively). Partial crosslinking of PCU-I increased the storage modulus of elasticity and provided better resistance to sterilization by ETO and gamma radiation. Both PCUs displayed good stability when subjected to sterilization by hydrogen peroxide plasma. Neither PCU caused cytotoxic effect in mouse fibroblasts (3T3 Balb C). They also had no toxic effects on the morphotic components and did not influence changes in the hematologic parameters or plasmatic coagulation system of human blood.

Visible light-induced oxidation of unsaturated components of cutins: a significant process during the senescence of higher plants

9-Hydroperoxy-18-hydroxyoctadec-10( trans)-enoic and 10-hydroperoxy-18-hydroxyoctadec-8( trans)-enoic acids deriving from type II (i.e. involving 1O 2) photooxidation of 18-hydroxyoleic acid were detected after visible light-induced senescence experiments carried out with Petroselinum sativum and subsequent cutin depolymerisation. These results showed that in senescent plants, where the 1O 2 formation rate exceeds the quenching capacity of the photoprotective system, 1O 2 can migrate outside the chloroplasts and affect the unsaturated components of cutins. Significant amounts of 9,18-dihydroxyoctadec-10( trans)-enoic and 10,18-dihydroxyoctadec-8( trans)-enoic acids resulting from the reduction of these photoproducts of 18-hydroxyoleic acid were also detected in different natural samples. These results well support the significance of the photooxidation of the unsaturated components of higher plant cutins in the natural environment.

Epoxidation catalysts

The major component of the cuticular hydroxyfatty acid polymer, cutin, of spinach leaf was identified as 18-hydroxy-cis-9,10-epoxystearic acid. A 3000g particulate fraction obtained from the homogenates of young spinach leaves catalyzed the epoxidation of 18-hydroxyoleic acid to 18-hydroxy-cis-9,10-epoxystearic acid. The epoxy acid was identified by thin-layer chromatography of the product of hydration of the oxirane function and subsequent reduction (octadecane-1,9,10,18-tetraol), on silica gel G and boric acid impregnated silica gel, and by radio gas-liquid chromatography of the periodate oxidation product of the tetraol derived from the epoxy acid. This enzymatic epoxidation required ATP, CoA, NADPH, and O2 for maximal activity. The pH optimum for epoxidation was 9.0, and the epoxidase activity was located mainly in the 3000g particulate fraction, which contained the cuticular membranes. The rate of epoxidation of the unsaturated acid was linear up to 2 h and up to a protein concentration of 1.5 mg/ml. The apparent Km and V of the enzyme was 7.5 × 10−5m and 180 nmol/1-h-mg protein, respectively. That the epoxidase has a stringent substrate specificity was indicated by the observation that 18-hydroxyelaidic acid, 18-acetoxyoleic acid, and oleic acid were not readily epoxidized by this enzyme preparation. Light reversible inhibition by CO suggested the involvement of cytochrome P-450 in the epoxidation. Chelators inhibited epoxidation, and this inhibition was reversed by FeSO4. The epoxidase was inhibited 30% by 0.5 mm NaCN, whereas the same concentration of NaN3 showed very little inhibition. The present epoxidase is unique in that it is associated with the 3000g particles which contain the cutin polymer and in that it requires activation of the carboxyl group distal to the epoxidation site. Enzyme preparations capable of epoxidizing 18-hydroxyoleic acid were also obtained from the skin of apple fruit and from the epidermis of Senecio odoris leaves. This is the first report on an enzyme involved in the biosynthesis of the C18 family of cutin monomer acids.

Differentiation of Malassezia species: selectivity of cremophor EL, castor oil and ricinoleic acid for M. furfur.

In recent years, the genus Malassezia has been reclassified based on molecular data. In addition to M. furfur, M. pachydermatis and M. sympodialis, four new species, M. globosa, M. obtusa, M. restricta and M. slooffiiae, have been described. However, apart from their lipid dependence, little is known about the metabolism and nutritional requirements of all the seven species. Further to recent studies, 10 hydrophilic emulsifiers (HLB > 10) were examined in an agar diffusion test to determine their growth-promoting effect on reference strains of the different Malassezia species. Polyethylene glycol (PEG) 7 glyceryl monoalcanoate (Cetiol HE). PEG-glyceryl stearate (Tagat S2) and macrogol-50 stearate (Myrj 53) were metabolized by all strains, while PEG-35 castor oil (Cremophor EL) was metabolized only by M. furfur. The latter observation is due to a different metabolism of castor oil and its main component, ricinoleic acid (12-hydroxy oleic acid), which may also give an insight into the pathogenesis of diseases that are associated with Malassezia spp. As hydroxy fatty acids are important in maintaining the epidermal structure and function, their metabolism specifically by M. furfur might clarify some clinical aspects of pityriasis versicolor. Apart from this speculation, use of Cremophor EL, with splitting of esculin as an additional key character, improves the distinction of the species M. furfur, M. slooffiae and M. sympodialis.

Composition of Australian Honey Extractives. 1. Norisoprenoids, Monoterpenes, and Other Natural Volatiles from Blue Gum (Eucalyptusleucoxylon)and Yellow Box (Eucalyptus melliodora)Honeys

Chemical fingerprinting of Australian honey requires information on the composition of natural honey volatiles if it is to be useful as a honey-sourcing method. The naturally occurring volatiles of Australian blue gum (Eucalyptus leucoxylon) and yellow box (Eucalyptus melliodora) honeys were isolated by solvent (ethyl acetate) extraction. Compounds in the extracts were analyzed by gas chromatography (GC) and gas chromatography−mass spectrometry (GC−MS). These procedures have permitted the identification of 55 compounds that include norisoprenoids, monoterpenes, benzene derivatives, aliphatic compounds, and Maillard reaction products. The following 13 compounds were quantitatively identified for the first time in honey: four isomeric 3,4-dihydro-3-oxoactinidols; 8,9-dehydrotheaspirone; two isomeric 3-oxoretro-α-ionols; megastigm-4-ene-3,9-dione; 1-phenylbutane-2,3-diol; 1-phenylbutane-2,3-dione; 18-hydroxyoleic acid lactone; 3,5-dihydroxy-2-methyl-4H-pyran-4-one; and 2,5-dimethyl-2,4-dihydroxy-3(2H)-furan...

An oleate 12-hydroxylase from Ricinus communis L. is a fatty acyl desaturase homolog.

Recent spectroscopic evidence implicating a binuclear iron site at the reaction center of fatty acyl desaturases suggested to us that certain fatty acyl hydroxylases may share significant amino acid sequence similarity with desaturases. To test this theory, we prepared a cDNA library from developing endosperm of the castor-oil plant (Ricinus communis L.) and obtained partial nucleotide sequences for 468 anonymous clones that were not expressed at high levels in leaves, a tissue deficient in 12-hydroxyoleic acid. This resulted in the identification of several cDNA clones encoding a polypeptide of 387 amino acids with a predicted molecular weight of 44,407 and with approximately 67% sequence homology to microsomal oleate desaturase from Arabidopsis. Expression of a full-length clone under control of the cauliflower mosaic virus 35S promoter in transgenic tobacco resulted in the accumulation of low levels of 12-hydroxyoleic acid in seeds, indicating that the clone encodes the castor oleate hydroxylase. These results suggest that fatty acyl desaturases and hydroxylases share similar reaction mechanisms and provide an example of enzyme evolution.

ω-Hydroxylation of Z9-octadecenoic, Z9,10-epoxystearic and 9,10-dihydroxystearic acids by microsomal cytochrome P450 systems from Vicia sativa

A microsomal fraction from etiolated Vicia sativa seedlings incubated aerobically with [1-14C]oleic acid (Z9-octadecenoic acid) or [1-14C]9,10-epoxystearic acid or [1-14C]9,10-dihydroxystearic acid catalyzed the NADPH-dependent formation of hydroxylated metabolites. The chemical structure of compounds formed from oleic, 9,10-epoxystearic or 9,10-dihydroxystearic acids was established by gas chromatography/mass spectra analysis to be 18-hydroxyoleic acid, 18-hydroxy-9,10-epoxystearic acid and 9,10,18-trihydroxystearic acid, respectively. The reactions required O2 and NADPH and were inhibited by carbon monoxide. As expected for monooxygenase reactions involving cytochrome P450, inhibition could be partially reversed by light and all three reactions were inhibited by antibodies raised against NADPH-cytochrome P450 reductase from Jerusalem artichoke. The ω-hydroxylation of the three substrates was enhanced in microsomes from clofibrate induced seedlings.

The Production of γ-Decalactone by Fermentation of Castor Oil

A microbial process for the production of optically-active γ-decalactone from the ricinoleic acid present as triglycerides in castor oil has been developed, γ-decalactone (γDL) is a component of some fruit flavours, being an important organoleptic component of peach flavours. Screening showed two red yeast microorganisms, Rhodotorula glutinis and Sporobolomyces odortts to be especially suitable for this biotransformation. The process involves lipase-mediated hydrolysis of the castor oil to give free ricinoleic acid, uptake of the acid by the cells and aerobic fermentation to achieve abbreviated β-oxidation of the ricinoleic acid (12-hydroxyoleic acid) into 4-hydroxydecanoic acid (4HDA), lactonisation of the acid into γ-DL, followed by solvent extraction and distillation. γ-DL broth concentrations of 0.5-1.2g · 1-t were obtained after 3-5 days from fermentation media containing 10 g · 1-1 castor oil, representing an 8.3-20.0% theoretical yield. Intermediates detected were consistent with the operation of the β-oxidation pathway. Appreciable amounts of novel metabolites identified as cis and trans isomers of a tetrahydrofuran (C10) were also produced. Their formation from 4HDA appeared to be non-enzymic and was favoured by anaerobic conditions. Yields of γ-DL were inversely proportional to the concentration of castor oil present in the medium, indicating that substrate inhibition takes place. The highest yields of γ-DL were obtained when castor oil was present from the beginning of the fermentation, rather than when added once the fermentation had become established, demonstrating that the β-oxidation pathway and/or transport system require continual induction. Significant amounts of γ-DL were not produced from other fatty acids, including ricinelaidic acid, the trans isomer of ricinoleic acid. γ-DL formation was dramatically inhibited by antibiotic inhibitors of oxidative phosphorylation, indicating the importance of intact β-oxidation pathways, whereas inhibitors of protein synthesis and cell-wall synthesis had much less marked effects. Selective extraction of 4HDA from the fermentation broths, and of γDL from broth lactonised by heating at low pH, could be achieved by adsorption to Amberlite XAD-1 and XAD-7 resins respectively. Some product could be recovered from the exit gases of the fermenter by passing through propylene glycol traps. This pathway is unusual in that it is a rare example of the truncated β-oxidation of a fatty acid by microorganisms. This effect probably occurs because of partial inhibition of one or more enzymes of the β-oxidation pathway by the C10 hydroxylated fatty acid intermediate(s) allowing intracellular accumulation of the 4HDA, followed by leakage out of the cell; although further metabolism of this C10 intermediate does take place slowly.

Biochemistry of Suberization

Biosynthesis of the aliphatic components of suberin was studied in suberizing potato (Solanum tuberosum) slices with [1-(14)C]oleic acid and [1-(14)C]acetate as precursors. In 4-day aged tissue, [1-(14)C]oleic acid was incorporated into an insoluble residue, which, upon hydrogenolysis (LiA1H(4)), released the label into chloroform-soluble products. Radio thin layer and gas chromatographic analyses of these products showed that (14)C was contained exclusively in octadecenol and octadecene-1, 18-diol. OsO(4) treatment and periodate cleavage of the resulting tetraol showed that the labeled diol was octadec-9-ene-1, 18-diol, the product expected from the two major components of suberin, namely 18-hydroxyoleic acid and the corresponding dicarboxylic acid. Aged potato slices also incorporated [1-(14)C]acetate into an insoluble material. Hydrogenolysis followed by radio chromatographic analyses of the products showed that (14)C was contained in alkanols and alkane-alpha,omega-diols. In the former fraction, a substantial proportion of the label was contained in aliphatic chains longer than C(20), which are known to be common constituents of suberin. In the labeled diol fraction, the major component was octadec-9-ene-1,18-diol, with smaller quantities of saturated C(16), C(18), C(20), C(22), and C(24)-alpha,omega-diols. Soluble lipids derived from [1-(14)C]acetate in the aged tissue also contained labeled very long acids from C(20) to C(28), as well as C(22) and C(24) alcohols, but no labeled omega-hydroxy acids or dicarboxylic acids were detected. Label was also found in n-alkanes isolated from the soluble lipids, and the distribution of label among them was consistent with the composition of n-alkanes found in the wound periderm of this tissue; C(21) and C(23) were the major components with lesser amounts of C(19) and C(25). The amount of (14)C incorporated into these bifunctional monomers in 0-, 2-, 4-, 6-, and 8-day aged tissue were 0, 1.5, 2.5, 0.8, and 0.3% of the applied [1-(14)C]oleic acid, respectively. Incorporation of [1-(14)C]acetate into the insoluble residue was low up to the 3rd day of aging, rapid during the next 4 days of aging, and subsequently the rate decreased. These changes in the rates of incorporation of exogenous oleic acid and acetate reflected the development of diffusion resistance of the tissue surface to water vapor. As the tissue aged, increasing amounts of the [1-(14)C]acetate were incorporated into longer aliphatic chains of the residue and the soluble lipids, but no changes in the distribution of radioactivity among the alpha-omega-diols were obvious. The above results demonstrated that aging potato slices constitute a convenient system with which to study the biochemistry of suberization.

Biosynthesis of hydroxyfatty acid polymers: Enzymatic epoxidation of 18-hydroxyoleic acid to 18-hydroxy- cis-9,10-epoxystearic acid by a particulate preparation from spinach ( Spinacia oleracea)

The major component of the cuticular hydroxyfatty acid polymer, cutin, of spinach leaf was identified as 18-hydroxy- cis-9,10-epoxystearic acid. A 3000 g particulate fraction obtained from the homogenates of young spinach leaves catalyzed the epoxidation of 18-hydroxyoleic acid to 18-hydroxy- cis-9,10-epoxystearic acid. The epoxy acid was identified by thin-layer chromatography of the product of hydration of the oxirane function and subsequent reduction (octadecane-1,9,10,18-tetraol), on silica gel G and boric acid impregnated silica gel, and by radio gas-liquid chromatography of the periodate oxidation product of the tetraol derived from the epoxy acid. This enzymatic epoxidation required ATP, CoA, NADPH, and O 2 for maximal activity. The pH optimum for epoxidation was 9.0, and the epoxidase activity was located mainly in the 3000 g particulate fraction, which contained the cuticular membranes. The rate of epoxidation of the unsaturated acid was linear up to 2 h and up to a protein concentration of 1.5 mg/ml. The apparent K m and V of the enzyme was 7.5 × 10 −5 m and 180 nmol/1-h-mg protein, respectively. That the epoxidase has a stringent substrate specificity was indicated by the observation that 18-hydroxyelaidic acid, 18-acetoxyoleic acid, and oleic acid were not readily epoxidized by this enzyme preparation. Light reversible inhibition by CO suggested the involvement of cytochrome P-450 in the epoxidation. Chelators inhibited epoxidation, and this inhibition was reversed by FeSO 4. The epoxidase was inhibited 30% by 0.5 m m NaCN, whereas the same concentration of NaN 3 showed very little inhibition. The present epoxidase is unique in that it is associated with the 3000 g particles which contain the cutin polymer and in that it requires activation of the carboxyl group distal to the epoxidation site. Enzyme preparations capable of epoxidizing 18-hydroxyoleic acid were also obtained from the skin of apple fruit and from the epidermis of Senecio odoris leaves. This is the first report on an enzyme involved in the biosynthesis of the C 18 family of cutin monomer acids.

Direct evidence for the involvement of epoxide intermediates in the biosynthesis of the C 18 family of cutin acids

Cutin, the structural component of plant cuticle, is a polymer of C 16 and C 18 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 C 18 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.