A novel unsaturated fatty acid hydratase toward C16 to C22 fatty acids from Lactobacillus acidophilus

Hydroxy FAs, one of the gut microbial metabolites of PUFAs, have attracted much attention because of their various bioactivities. The purpose of this study was to identify lactic acid bacteria with the ability to convert linoleic acid (LA) to hydroxy FAs. A screening process revealed that a gut bacterium, Lactobacillus acidophilus NTV001, converts LA mainly into 13-hydroxy-cis-9-octadecenoic acid and resulted in the identification of the hydratase responsible, fatty acid hydratase 1 (FA-HY1). Recombinant FA-HY1 was purified, and its enzymatic characteristics were investigated. FA-HY1 could convert not only C18 PUFAs but also C20 and C22 PUFAs. C18 PUFAs with a cis carbon-carbon double bond at the Δ12 position were converted into the corresponding 13-hydroxy FAs. Arachidonic acid and DHA were converted into the corresponding 15-hydroxy FA and 14-hydroxy FA, respectively. To the best of our knowledge, this is the first report of a bacterial FA hydratase that can convert C20 and C22 PUFAs into the corresponding hydroxy FAs. These novel hydroxy FAs produced by using FA-HY1 should contribute to elucidating the bioactivities of hydroxy FAs.     

Epicuticular wax of Poa ampla leaves

Leaf wax of a glaucous variety of Poa ampla contains hydrocarbons (5%, C₂₃–C₃₅), esters (9%, C₃₆–C₅₆), free acids (3%, C₁₆–C₃₄), free alcohols (6%, mainly C₂₆); hentriacontane-14,16-dione (14%), 5-oxohentriacontane-14,16-dione (1%); hydroxy β-diketones (56%) and unidentified material (6%). The hydroxy β-diketones, which are more abundant in this wax than in others, were shown by ¹³C NMR to consist of 4-hydroxy (15%), 5-hydroxy (70%) and 6-hydroxy (15%) hentriacontane-14,16-diones.     

Essential structure of orexin 1 receptor antagonist YNT-707, Part II: Drastic effect of the 14-hydroxy group on the orexin 1 receptor antagonistic activity

The 14-dehydration- and 14-H derivatives of the orexin 1 receptor (OX₁R) antagonist YNT-707 (2) were synthesized. The obtained derivatives showed higher affinities for OX₁R than the corresponding 14-hydroxy derivatives. The conformational analysis suggested that the 17-sulfonamide groups in the derivatives without the 14-hydroxy group have a greater tendency to be oriented toward the upper side of the D-ring compared with the 14-hydroxy derivatives. Additionally, the 14-dehydration-derivative with 6α-amide side chain showed significantly higher affinity than the 14-hydroxy derivative, while the corresponding 14-H derivative showed only slightly higher affinity. Thus, the 14-hydroxy group strongly affects the affinity of the antagonist for the OX₁R.     

The mass spectra of the trimethylsilyl derivatives of the hydroxy and acid metabolites of delta 1- and delta 6-tetrahydrocannabinol

Deuterium labelling and high resolution mass measurements have been used to investigate the fragmentation mechanisms leading to diagnostic ions in the mass spectra of the trimethylsilyl derivatives of 58 hydroxy and acid metabolites of delta 1- and delta 6-tetrahydrocannabinol and of two related compounds, 2 alpha- and 2 beta-hydroxy-delta 6-tetrahydrocannabinol. The spectra of most of the hydroxy metabolites contained abundant ions which were characteristic of the position of hydroxy substitution. These could be used diagnostically to determine the structures of polysubstituted metabolites.     

Hydroxy fatty acids in Bacteroides species: D-(--)-3-hydroxy-15-methylhexadecanoate and its homologs.

Acid hydrolysates of 140 strains, representing 11 species of the genus Bacteroides, were analyzed by capillary gas-liquid chromatography for total cellular fatty acid. All samples contained components which appeared to be hydroxy fatty acid. The relative amount and chain length distribution of the hydroxy fatty acids, as well as the nonhydroxy fatty acids, varied according to species. To characterize the presumed hydroxy acids, a composite of some 40 of these samples was analyzed by thin-layer and capillary gas-liquid chromatography, mass spectrometry, infrared spectrophotometry, and polarimetry. The hydroxy acids were shown to be of the D-(--)-3-hydroxy acid family. The predominant component was the iso-branched D-(--)-3-hydroxy-15-methylhexadecanoic acid. Lesser amounts of the iso-branched 15-carbon, straight-chain 16-carbon, and anteiso-branched 17-carbon acids were also found.     

Studies on platelet lipoxygenase specificity towards icosapolyenoic and docosapolyenoic acids

Two docosapolyenoic acids (22:5(n-3) and 22:5(n-6)) were isolated from the liver of normal and 18:3(n-3)-deficient trout, respectively. They were prepared by combined thin-layer chromatography (TLC) and reversed-phase high performance liquid chromatography (HPLC). Their purity, checked by capillary gas liquid chromatography, was greater than 95%. Each fatty acid was oxygenated into monohydroxy derivatives by human platelets. The hydroxy compounds were purified by TLC and HPLC and then derivatized for gas chromatography-mass spectrometry analysis. Whereas 22:5(n-6) was only converted into 14-OH-22:5, three hydroxy derivatives (11, 13 and 14) were obtained from 22:5(n-3). However, 13-hydroxy was not formed in the presence of aspirin, indicating that platelet lipoxygenase catalyses the formation of both 11- and 14-hydroxy derivatives from 22:5(n-3), as described previously, from 22:6(n-3). Further studies showed that 22:4(n-6) and 20:5(n-3) were only converted into 14- and 12-hydroxy derivatives. We conclude then that, besides the well-known n-9 oxygenation, lipoxygenase of human platelets is able to catalyse an n-12 oxygenation on docosapolyenoic acids of the n-3 family.     

The numerous effector functions of Nef.

P450BM-3, a catalytically self-sufficient, soluble bacterial P450, contains on the same polypeptide a heme domain and a reductase domain. P450BM-3 catalyzes the oxidation of short- and long-chain, saturated and unsaturated fatty acids. The three-dimensional structure of the heme domain both in the absence and in the presence of fatty acid substrates has been determined; however, the fatty acid in the substrate-bound form is not adequately close to the heme iron to permit a prediction regarding the stereoselectivity of oxidation. In the case of long-chain fatty acids, the products can also serve as substrate and be metabolized several times. In the current study, we have determined the absolute configuration of the three primary products of palmitic acid hydroxylation (15-, 14-, and 13-OH palmitic acid). While the 15- and 14-hydroxy compounds are produced in a highly stereoselective manner (98% R, 2% S), the 13-hydroxy is a mixture of 72% R and 28% S. We have also examined the binding of these three hydroxy acids to P450BM-3 and shown that only two of them (14-OH and 13-OH palmitic acid) can bind to and be further metabolized by P450BM-3. The results indicate that in contrast to the flexibility of palmitoleic acid bound to the oxidized enzyme, palmitic acid is rigidly bound in the active site during catalytic turnover.     

Effect of the position of the phenolic group in morphinans on their affinity for opiate receptor binding

Homologous series of N-methyl, N-allyl and N-cyclopropylmethylmorphinans, differing only in the position of the plenolic hydroxy group, were examined with respect to their binding affinities for the opiate receptor. IC₅₀'s were determined for competition with ³H-naltrexone in the presence and absence of 100 mM NaCl. While the compounds with the hydroxy in the 3-position had, as expected, by far the highest affinity, the corresponding molecules with the hydroxy in the 2- or 4- position had significant binding affinity ranging from 30 nM in the cyclopropyl- methyl series to 400 nM for the 2-hydroxy N-methyl morphinan. The sodium indices were also very similar to those of the corresponding 3-hydroxy compounds. The only 1-hydroxy derivative available was about 5-fold weaker than the corresponding 2- and 4-hydroxy compounds. Covering or removing the hydroxy group greatly weakened the binding but did not totally destroy it. There was good correlation between binding affinity and pharmacological potency for all except the methoxy compounds. Their high potency is consonant with $\text{in vivo}$ hydrolysis of the methyl ether.     

Quantification of ergosterol and 3-hydroxy fatty acids in settled house dust by gas chromatography-mass spectrometry: comparison with fungal culture and determination of endotoxin by a Limulus amebocyte lysate assay.

Ergosterol and 3-hydroxy fatty acids, chemical markers for fungal biomass and the endotoxin of gram-negative bacteria, respectively, may be useful in studies of health effects of organic dusts, including domestic house dust. This paper reports a method for the combined determination of ergosterol and 3-hydroxy fatty acids in a single dust sample and a comparison of these chemical biomarkers determined by gas chromatography-mass spectrometry with results from fungal culture and Limulus assay. Analyses of replicate house dust samples resulted in correlations of 0.91 (ergosterol in six replicates; P < 0.01) and 0.94 (3-hydroxy fatty acids in nine replicates; P < 0.001). The amounts of ergosterol (range, 2 to 16.5 ng/mg of dust) correlated with those of total culturable fungi (range, 6 to 1,400 CFU/mg of dust) in 17 samples, (r = 0.65; P < 0.005). The amounts of endotoxin (range, 11 to 243 endotoxin units/mg of dust) measured with a modified chromogenic Limulus assay correlated with those of lipopolysaccharide (LPS) determined from 3-hydroxy fatty acid analysis of 15 samples. The correlation coefficient depended on the chain lengths of 3-hydroxy acids used to compute the LPS content. The correlation was high (r = 0.88 +/- 0.01; P < 0.001) when fatty acid chains of 10 to 14 carbon atoms were included; the correlation was much lower when hydroxy acids of 16- or 18-carbon chains were included. In conclusion, the results of the described extraction and analysis procedure for ergosterol and 3-hydroxy fatty acids are reproducible, and the results can be correlated with fungal culture and endotoxin activity of organic dust samples.     

Further Oxygenated Compounds Produced from Methyl Linoleate Monohydroperoxides at the Process of Autoxidation

The primary stable products, methyl linoleate monohydroperoxides (MLHPO), formed by the autoxidation of methyl linoleate were characterized by gas chromatography-mass spectrometry. MLHPO was converted into methyl hydroxy stearates which consisted of two isomers, methyl 9-hydroxy and methyl 13-hydroxy stearate. Trimethylsilyl ether derivatives of these hydroxy isomers were separated directly by gas chromatography and mass fragmentgraphy. MLHPO was degradated by incubating under aerobic condition at 37°C for a week, and the quantity of MLHPO was determined as hydroxy derivatives. Decrease of MLHPO was almost similar to that of conjugated diene structure, but the peroxide value was not appreciably decreased during the incubation. This fact based on the formation of further oxygenated compounds. After chemical reduction, these compounds were identified as methyl 9,13-hydroxy octadecenoate and methyl 9,12,13- or 9,10,13-trihydroxy octadecenoate, in which oxygen attached to the conjugated diene. The formation mechanisms of these oxygenated compounds are proposed.     

Plant growth retardant activity of 4-homoisotwistane derivatives

A variety of simple derivatives of 3-substituted 4-homoisotwistane derivatives were prepared, and their effect on the growth of cucumber seedlings in complete darkness was investigated. The 3-hydroxy derivative was found to show a strong inhibitory activity at 50 μg/ml, so a series of other hydroxy derivatives of 4-homoisotwistane, endo-2-, exo-2-, and 5-hydroxy- and exo-2,3-dihydroxy-4-homoisotwistane were prepared in order to obtain information on structure-activity relationships. The endo-2-hydroxy derivative inhibited the growth of cucumber and the germination of lettuce seed at 12.5 μg/ml. All the hydroxy derivatives tested increased the number of adventitious roots in hypocotyls of kidney bean at 100 μg/ml, but they inhibited root formation at the lowest part of the cuttings, and the effect was again exhibited most strongly by the endo-2-hydroxy compound. It is suggested that the 2- and 3-hydroxy derivatives possess a potent activity as plant growth retardants.     

Optical Configuration Analysis of Hydroxy Fatty Acids in Bacterial Lipids by Chiral Column High-Performance Liquid Chromatography

Through the adoption of a chiral stationary phase in high-performance liquid chromatography and a simple derivatization method for hydroxy fatty acids, it became easy to separate and identify the optical isomers of 2- and 3-hydroxy fatty acids composing several kinds of microbial lipids. The 2- and 3-hydroxy fatty acids were converted with dinitrophenyl isocyanate to their 3, 5-dinitrophenyl urethane derivatives (DU-derivatives), which were analyzable by HPLC using a chiral column. By varying the composition of an eluent, separation of the DU-derivatives of hydroxy fatty acids differing in optical configuration, chain length and position of hydroxyl group was achieved. The general elution orders of these DU-derivatives were determined with authentic 2- and 3-hydroxy fatty acids. Small amounts (~300 μg) of ornithine-containing lipids isolated from the Serratia marcescens strains were examined by this method to identify 3-hydroxy fatty acids of the lipids as D isomers.     

Oxidation of 15-hydroxyeicosatetraenoic acid and other hydroxy fatty acids by lung prostaglandin dehydrogenase

The oxidation of the 15-hydroxy group of prostaglandins of the A, E, and F series by the NAD+-dependent prostaglandin dehydrogenase (PGDH) has been well documented. In addition to prostaglandins, we have observed that the purified lung PGDH also will oxidize 15-HETE to a novel metabolite that was isolated by reverse-phase HPLC and identified by gas chromatography-mass spectrometry as the 15-keto-5,8,11-cis-13-trans-eicosatetraenoic acid (15-KETE). The Km for 15-HETE was 16 microM, which was 2.5 times lower than the value obtained for PGE1. In addition to 15-HETE, 5,15-diHETE and 8,15-diHETE also were substrates for the lung PGDH with Km values of 138 and 178 microM, respectively. Other hydroxy derivatives of eicosatetraenoic acid that did not have a hydroxy group at carbon atom 15 did not support the PGDH-mediated reduction of NAD+. In addition to the 15-hydroxy derivatives of eicosatetraenoic acid, 12-HHT also was a substrate for the lung enzyme with a Km of 12 microM. These data indicate that omega 6-hydroxy fatty acids, in addition to prostaglandins, are also substrates of the lung NAD+-dependent PGDH and that the enzyme does not require the cyclopentane ring of prostaglandins.     

Porpoisamides A and B, two novel epimeric cyclic depsipeptides from a Florida Keys collection of Lyngbya sp

NMR-guided fractionation of a non-polar extract of a Florida Keys collection of Lyngbya sp. resulted in the isolation of two novel epimeric cyclic depsipeptides, porpoisamides A (1) and B (2). The planar structures of these compounds were determined using NMR spectroscopic techniques. The absolute configurations of amino and hydroxy acid subunits were assigned by enantioselective HPLC analysis. These compounds showed weak cytotoxicity towards HCT-116 colorectal carcinoma and U2OS osteosarcoma cells. The porpoisamides are a unique pair of cyclic depsipeptides that are epimeric at C-2 of the β-amino acid, 3-amino-2-methyloctanoic acid.     

Neo-clerodane diterpenoids from ajuga chamaepitys

From the whole plant of Ajuga chamaepitys, 15-ethoxy-14-hydroajugapitin and a C-15 epimeric mixture of 14-hydro-15-hydroxyajugapitin have been isolated.     

Metabolism of 18:2(n - 6), 18:3(n - 3), 20:4(n - 6) and 20:5(n - 3) by the fungus Gaeumannomyces graminis: identification of metabolites formed by 8-hydroxylation and by w2 and w3 oxygenation.

The present study was aimed at developing a cell-free preparation of Gaeumannomyces graminis to biosynthesize w2-hydroxy, w3-hydroxy and related metabolites of essential fatty acids. 14C-labelled linoleic acid (18:2(n - 6)), linolenic acid (18:3(n - 3)), arachidonic acid (20:4(n - 6)) and eicosapentaenoic acid (20:5(n - 3)) were incubated with the cytosolic and microsomal fractions and NADPH. Significant metabolism was only found in the cytosol. The main products were purified by high-performance liquid chromatography and identified by gas chromatography-mass spectrometry (GC-MS). 18:2(n - 6) was metabolized mainly to 8-hydroxy-9,12-octadecadienoic acid (8-HODE), while the w2 and the w3 alcohols were formed in relatively small amounts. The absolute configuration of the 8-hydroxyl was found to be R by ozonolysis of the diastereoisomeric (-)-menthoxycarbonyl derivative of 8-HODE and GC-MS analysis. In analogy, 18:3(n - 3) was converted to 8-hydroxy-9,12,15-octadecatrienoic acid and to smaller amounts of the 15,16-diol (15,16-DiHODE). In contrast, 8-hydroxy metabolites of 20:4(n - 6) or 20:5(n - 3) could not be detected. 20:4(n - 6) was efficiently converted to 18(R)-hydroxyeicosatetraenoic acid (18(R)-HETE) and 19(R)-HETE and to traces of 17-HETE, while 20:5(n - 3) was mainly metabolized to the 17,18-diol (17,18-DiHETE) and to smaller amounts of the w2 alcohol. In conclusion, the cytosol of G. graminis can be used for stereoselective biosynthesis of some hydroxy metabolites of essential fatty acids.     

Alkylation and carbamoylation intermediates from the carcinostatic 1-(2-chloroethyl)-3-cyclohexyl -1-nitrosourea (CCNU)

Evidence is presented which shows that 1-(2-chloroethyl) -3-cyclohexyl-1-nitrosourea (CCNU) upon degradation provides a 2-chloroethyl alkylating intermediate, possibly 2-chloroethyl carbonium ion, and 2-chloroethanol. Thiol alkylation occurs $\text{in vivo}$ and a major urinary metabolite of CCNU is thiodiacetic acid. A rapid microsomal hydroxylation of the cyclohexyl ring occurs which yields varying ratios of at least five metabolites: cis or trans 2-hydroxy, trans- 3-hydroxy, cis-3-hydroxy, cis-4-hydroxy and trans-4- hydroxy-CCNU. $\text{In vivo}$ carbamoylation appears to not be due to cyclohexylisocyanate but to the various hydroxy-cyclohexylisocyanates which are formed from hydroxy CCNU metabolites.     

Influence of the position of the side chain hydroxy group on the gastric antisecretory and antiulcer actions of E1 prostaglandin analogs

The influence of transposing the C-15 hydroxy group of prostaglandin E₁ methyl ester (PGE₁ME) on gastric antisecretory and antiulcer actions was investigated. The compound (±)15-deoxy- 16,β-hydroxy PGE₁ME (SC-28904) was equipotent to the reference standard PGE₁ME in suppressing histamine-stimulated gastric secretion in the Heidenhain pouch (HP) dog. In contrast to PGE₁ME, SC-28904 was longer acting when administered intravenously and also showed significant oral activity in the histamine-stimulated gastric fistula dog. SC-28904 was also equipotent to PGE₁ME (range of active doses of 0.5 to 5.0 mg/kg, s.c.) in inhibiting forced-exertion gastric ulceration in rats.

The compound (±)15-deoxy- 17,β-hydroxy PGE₁ME (SC-30693) was an inactive antisecretory agent in the dog at the 1.0 mg/kg i.v. bolus dose. This dose was 100 times greater than the active antisecretory dose of PGE₁ME. Likewise, SC-30693, when administered subcutaneously at a 5.0 mg/kg dose, was also totally inactive in preventing gastric ulcers induced by forced exertion in rats.

The important implications of this work are that some of the receptor sites for the PGE₁ molecule could easily accommodate the side chain hydroxy group either in the C-15 or C-16 position. Moreover, the hydroxy group in the latter position significantly improved the biological activity of PGE₁ME.     

Microbial hydroxylation of chiral bicyclic enones by Chaetomium sp.1 and Didymosphaeria igniaria cultures

The biotransformations of (R)-(?)-methyloctalone and (S)-(+)-methyloctalone were investigated using Chaetomium sp.1 KCH 6651 and Didymosphaeria igniaria KCH 6670 as biocatalysts, yielding mostly 6β- and 7β-hydroxy derivatives. During the incubation of (R)-methyloctalone with the Chaetomium sp.1 culture, three products were obtained: the trans-7β-hydroxy derivative in 50% yield, trans-6β-hydroxy derivative in 30% yield and cis-8α-hydroxy derivative in 6% yield. The (S)-enone was hydroxylated mainly in the 6α-position (with 60% yield). 6β- and 7β-hydroxy derivatives are new compounds, not previously described in the literature. The biotransformation of the two enantiomers of methyloctalone with D. igniaria also afforded monohydroxy derivatives. In both cases the main product of transformation was the allylic hydroxy derivative (27–35% yield). D. igniaria transformed the (R)-methyloctalone more rapidly whereas Chaetomium sp. 1 preferred the (S)-methyloctalone.     

LC-MS/MS analysis of epoxyalcohols and epoxides of arachidonic acid and their oxygenation by recombinant CYP4F8 and CYP4F22

CYP4F22 and CYP4F8 are expressed in epidermis, and mutations of CYP4F22 are associated with lamellar ichthyosis. Epoxyalcohols (HEETs) and epoxides (EETs) of 20:4n−6 appear to be important for the water permeability barrier of skin. Our aim was to study the MS/MS spectra and fragmentation of these compounds and to determine whether they were oxidized by CYP4F22 or CYP4F8 expressed in yeast. HEETs were prepared from 15-hydroperoxyeicosatetraenoic acid (15-HPETE), 12-HPETE, and their [²H₈]labeled isotopomers, and separated by normal phase-HPLC with MS/MS analysis. CYP4F22 oxygenated 20:4n−6 at C-18, whereas metabolites of HEETs could not be identified. CYP4F8 formed ω3 hydroxy metabolites of HEETs derived from 12R-HPETE with 11,12-epoxy-10-hydroxy configuration, but not HEETs derived from 15S-HPETE. 8,9-EET and 11,12-EET were also subject to ω3 hydroxylation by CYP4F8. We conclude that CYP4F8 and CYP4F22 oxidize 20:4n−6 and that CYP4F8 selectively oxidizes 8,9-EET, 11,12-EET, and 10,11R,12R-HEET at the ω3 position.