Estrogenic action of estriol fatty acid esters

Recent studies suggest that, estriol, like estradiol, is biosynthetically esterified with fatty acids. We have synthesized the stearate estriol, at C-16 alpha, C-17 beta and the diester, C-16 alpha,17 beta and tested these D-ring esters for their estrogenic action both in vivo and in vitro, comparing them to estradiol, estriol and estradiol-17-stearate. None of the estriol esters bind to the estrogen receptor. They are only weakly estrogenic in a microtiter plate estrogen bioassay: stimulation of alkaline phosphatase in the Ishikawa endometrial cells. However, both estriol monoesters are extremely potent estrogens when injected subcutaneously (in aqueous alcohol) into ovariectomized mice. Compared to the free steroids, they produced a dramatically increased uterine weight with a greatly prolonged duration of stimulation. The 16 alpha,17 beta-diester also induced a protracted uterotrophic response, but the stimulation of uterine weight was comparatively low. Since the esters of estradiol and estriol do not bind to the estrogen receptor, their estrogenic signal must be generated through the action of esterase enzymes. These naturally occurring esters have the potential of being extremely useful pharmacological agents for long-lived estrogenic stimulation.     

Steroidal fatty acid esters

Several years ago we discovered an unexpected family of steroidal metabolites, steroidal fatty acid esters. We found that fatty acid esters of 5-ene-3β-hydroxysteroids, pregnenolone and dehydroisoandrosterone are present in the adrenal. Subsequently, others have shown the existence of these non-polar 5-ene-3β-hydroxysteroidal esters in blood, brain and ovaries. Currently, almost every family of steroid hormone is known to occur in esterified form. We have studied the esters of the estrogens and glucocorticoids in some detail, and have found that these two steroidal families are esterified by separate enzymes. In a biosynthetic experiment performed simultaneously with estrodiol and corticosterone, we established that the fatty acid composition of the steroidal esters is quite different. The corticoid is composed predominantly of one fatty acid, oleate, while the estradiol esters are extremely heterogeneous. Our studies have demonstrated that the estrogens are extremely long-lived hormones, that they are protected by the fatty acid from metabolism. They are extremely potent estrogens, with prolonged activity. Esterification appears to be the only form of metabolism that does not deactivate the biological effects of estradiol. We have demonstrated the biosynthesis of fatty acid esters of estriol, monoesters at both C-16 and C-17β. They too are very potent estrogens. These fatty acid esters of the estrogens are the endogenous analogs of estrogen esters, like benzoate, cypionate, etc., which have been used for decades, pharmacologically because of their prolonged therapeutic potency. We have found that the estradiol esters are located predominantly in hydrophobic tissues, such as fat. Sequestered in these tissues, they are an obvious reservoir of estrogenic reserve, requiring only an esterase for activation. To the contrary the biological activity of the fatty acid esters of the glucocorticoid, corticosterone, is not different from that of its free parent steroid. We have shown that the rapid kinetics of its induction of gluconeogenic responses is caused by its labile C-21 ester which is rapidly hydrolyzed by esterase enzymes. While it appears that the physiological role of the estrogen esters may be related to their long-lived hormonal activity, the role of the other families of steroidal esters is not yet apparent. They, and perhaps the estrogen esters as well, must serve other purposes. Indeed they may serve important biological functions beyond those which we ordinarily associate with steroid hormones.     

Neutral lipid biosynthesis in engineered Escherichia coli: jojoba oil-like wax esters and fatty acid butyl esters

Wax esters are esters of long-chain fatty acids and long-chain fatty alcohols which are of considerable commercial importance and are produced on a scale of 3 million tons per year. The oil from the jojoba plant (Simmondsia chinensis) is the main biological source of wax esters. Although it has a multitude of potential applications, the use of jojoba oil is restricted, due to its high price. In this study, we describe the establishment of heterologous wax ester biosynthesis in a recombinant Escherichia coli strain by coexpression of a fatty alcohol-producing bifunctional acyl-coenzyme A reductase from the jojoba plant and a bacterial wax ester synthase from Acinetobacter baylyi strain ADP1, catalyzing the esterification of fatty alcohols and coenzyme A thioesters of fatty acids. In the presence of oleate, jojoba oil-like wax esters such as palmityl oleate, palmityl palmitoleate, and oleyl oleate were produced, amounting to up to ca. 1% of the cellular dry weight. In addition to wax esters, fatty acid butyl esters were unexpectedly observed in the presence of oleate. The latter could be attributed to solvent residues of 1-butanol present in the medium component, Bacto tryptone. Neutral lipids produced in recombinant E. coli were accumulated as intracytoplasmic inclusions, demonstrating that the formation and structural integrity of bacterial lipid bodies do not require specific structural proteins. This is the first report on substantial biosynthesis and accumulation of neutral lipids in E. coli, which might open new perspectives for the biotechnological production of cheap jojoba oil equivalents from inexpensive resources employing recombinant microorganisms.     

The anticlastogenic potential of fatty acid methyl esters

To test for possible anticlastogenic effects of fatty acids, the methyl esters of fatty acids--short-chain to long-chain--were examined on busulfan in Chinese hamster bone-marrow cells using the chromosome aberration test. When the experimental animals were treated with fatty acid esters and the mutagen, the chromosome-breaking actions of busulfan were not modulated by the short-chain fatty acids, but the fatty acids from lauric acid (C12) up to nonadecanoic acid (C19) reduced the rate of aberrant metaphases from 9.4 to about 3% at doses of 100 mg/kg and less. Other chemical properties of the fatty acids (saturated or not, number of double bonds, even- or odd-numbered) had no influence on the anticlastogenic effects. The only exceptions to this rule were arachidonic acid, which had no effect, and gamma-linolenic acid, which had no consistent effect on the action of busulfan.     

The biosynthesis of benzoic acid glucosinolate esters in Arabidopsis thaliana

The siliques and seeds of Arabidopsis thaliana accumulate a series of glucosinolates containing an alkyl side chain of varying length with a terminal benzoate ester function. The biosynthesis of these unusual nitrogen- and sulfur-containing natural products was investigated by feeding isotopically-labeled precursors to detached flowering stems. Glucosinolates were extracted, purified and analyzed by tandem mass spectrometry. Phenylalanine and benzoic acid were incorporated into the benzoate ester function, and methionine and acetate were incorporated into the aliphatic portion of the side chain in a position-specific manner. The labeling patterns observed were consistent with the chain extension of methionine by a three-step elongation cycle which begins with the condensation of acetyl-CoA with a 2-oxo acid derived from methionine and ends with an oxidative decarboxylation forming a new 2-oxo acid with an additional methylene group. Incorporation of desulfo-4-methylthiobutyl glucosinolate into 4-benzoyloxybutyl olucosinolate suggested chain-extended methionine derivatives are first converted to their corresponding methylthioalkyl glucosinolates with further side chain modification occurring later. Transformation of the methylthiol function to a hydroxyl group is followed by esterification with benzoic acid. The siliques appear to possess the complete machinery for carrying out all of the reactions in the biosyntheis of these complex glucosinolates.     

Antibacterial targets in fatty acid biosynthesis

The fatty acid biosynthesis pathway is an attractive but still largely unexploited target for the development of new antibacterial agents. The extended use of the antituberculosis drug isoniazid and the antiseptic triclosan, which are inhibitors of fatty acid biosynthesis, validates this pathway as a target for antibacterial development. Differences in subcellular organization of the bacterial and eukaryotic multienzyme fatty acid synthase systems offer the prospect of inhibitors with host versus target specificity. Platensimycin, platencin, and phomallenic acids, newly discovered natural product inhibitors of the condensation steps in fatty acid biosynthesis, represent new classes of compounds with antibiotic potential. An almost complete catalog of crystal structures for the enzymes of the type II fatty acid biosynthesis pathway can now be exploited in the rational design of new inhibitors, as well as the recently published crystal structures of type I FAS complexes.     

Red blood cell fatty acid ethyl esters: a significant component of fatty acid ethyl esters in the blood

Although alcohol abuse is known to cause an array of ethanol-induced red blood cell (RBC) abnormalities, the underlying molecular mechanisms remain poorly understood. Fatty acid ethyl esters (FAEEs) are toxic, nonoxidative ethanol metabolites that have been found in blood, plasma, and tissues. Because FAEEs have been shown to be incorporated into phospholipid bilayers, we conducted a controlled ethanol intake study to test the hypothesis that FAEEs accumulate and persist within RBCs following ethanol ingestion. We demonstrated that RBC FAEEs account for approximately 5% to 20% of total whole-blood FAEEs, and that the fatty acid composition of FAEEs in RBCs and plasma are different and vary differently over time. These data indicate that a significant percentage of FAEEs in the blood is associated with RBCs and that the metabolism of RBC FAEEs and that of plasma FAEEs (bound to albumin or lipoproteins) are largely independent.     

Location and biosynthesis of monoterpenyl fatty acyl esters in rose petals

The upper epidermal layer of cells and the epicuticular wax surface of Lady Seton rose petals are sites of biosynthesis and accumulation, respectively, of a family of terpenyl fatty acyl esters. These esters are based mainly on the acyclic monoterpene alcohol geraniol coupled primarily to fatty acids of chain lengths 16-20 and in mass terms represent from 14% to 64% of the total monoterpenes present in the petals. The lipophilic nature of these non-volatile esters of the monoterpene alcohols contrasts with that of the lipophilic volatile parent alcohols themselves and with the hydrophilic, non-volatile, glucoside derivative of the other principal petal fragrant compounds, the phenylpropanoids, beta-phenyl ethanol and benzyl alcohol. These latter compounds are also synthesised and are resident in the petal. Biosynthetic studies confirmed that the petal upper epidermal cell layer has the capacity to incorporate mevalonic acid into the monoterpene component of the fatty acyl ester. The biosynthesis of the monoterpene component of the fatty acyl ester occurs via the mevalonic acid pathway in Lady Seton as well as in the hybrid tea rose Fragrant Cloud. In the latter flower the biosynthesis of geraniol was biosynthetically trans as was the formation of nerol and citronellol. Both geraniol and nerol were shown to be precursors of citronellol via an NADPH dependent reductase reaction. Oleic acid is assimilated into the acyl moiety of the terpenyl ester in Lady Seton isolated petal discs. It is probable that the lipophilic non-volatile terpenyl fatty acyl esters represent a stable storage form of the corresponding alcohols from their residency within the epicuticular wax layer. These acyl esters may realise, on hydrolysis, additional aroma notes from the living flower and potentially commercially significant quantities of the fragrant terpenols during oil of rose essence production.     

Plasma estriol levels after intramuscular injection of estriol and two of its esters

Twelve female volunteers from Berlin and 9 from Stockholm, all using a contraceptive pill (30 micrograms ethinyl estradiol and 150 micrograms levonorgestrel), received an intramuscular injection of estriol (E3; 1 mg in oil) on day 5 of withdrawal bleeding. Blood samples were collected at increasing time intervals during 4 weeks. Three months later, on day 5 of their withdrawal bleeding, 6 women were given intramuscularly (in oil) estriol 3,17-dipropionate (E3-prop) and 15 women estriol 3,17-dihexanoate (E3-hex). The doses were equivalent to 5 mg of estriol, i.e. 6.94 and 8.90 mg, respectively. Blood samples were collected during a period of 9 weeks. Estriol was analyzed by radioimmunoassay in all plasma samples. The average half-life of E3 ranged from 1.5 to 5.3 h after the administration of E3. It was 12.7 h and between 187 and 221 h after the administration of E3-prop and E3-hex, respectively. The average areas under the curve (in nmol.l-1.h) of E3 were between 82.5 and 161 after the administration of E3-prop or E3-hex, and between 27.1 and 37.9 when E3 had been given. As E3 was administered in a 5-fold lower dose than the esters, the areas under curve appeared to be comparable. Thus, the total exposure to E3 seemed to be almost independent of the type of E3 derivatization, while the time and intensity of exposure were very different.     

The biosynthesis of ethyl esters of lithocholic acid and isolithocholic acid by rat intestinal microflora

Recent reports concerning the tumor-promoting action of lithocholic acid in the colon and liver suggest that the metabolism of this major fecal bile acid may be important in carcinogenesis at various target sites. The metabolism of [¹⁴COOH]-lithocholic acid by rat intestinal microflora derived from standard laboratory chow-fed animals produced slightly more non-polar metabolites than those incubations which utilized flora from animals on a high lean-beef regimen. Purification of the crude bacterial extracts by Sephadex LH-20 chromatography and analysis of the radioactive peaks by glass fiber paper chromatography resulted in the identification of two neutral metabolites. Confirmation of their identity as ethyl lithocholate and ethyl isolithocholate was achieved by gas-liquid chromatography and combined gas-liquid chromatography-chemical ionization mass spectrometry. The formation of ethyl esters of lithocholic acid and isolithocholic acid by the intestinal microflora requires the presence of ethanol and anaerobic incubation conditions. These data support results obtained previously with single human fecal microorganisms. Since the formation of these derivatives in vitro occurs under anaerobic conditions only, it is possible that such derivatives may form physiologically in the colon. The carcinogenic potential of these derivatives is under investigation.     

Two-column gas-liquid chromatography of fatty acid methyl esters

Fatty acid methyl esters were separated into fractions according to chain length on a nonpolar gas-liquid chromatographic column. These fractions were collected and rechromatographed on a polar column. Temperature programming was used in both cases. Data are given for the accuracy of the double procedure applied to a synthetic mixture.     

Growth inhibitory properties of lactose fatty acid esters

Sugar esters are biodegradable, nonionic surfactants which have microbial inhibitory properties. The influence of the fatty acid chain length on the microbial inhibitory properties of lactose esters was investigated in this study. Specifically, lactose monooctanoate (LMO), lactose monodecanoate (LMD), lactose monolaurate (LML) and lactose monomyristate (LMM) were synthesized and dissolved in both dimethyl sulfoxide (DMSO) and ethanol. Minimum inhibitory concentrations (MIC) and minimum bactericidal concentrations (MBC) were determined in growth media. LML was the most effective ester, exhibiting MIC values of <0.05 to <5 mg/ml for each Gram-positive bacteria tested (Bacillus cereus, Mycobacterium KMS, Streptococcus suis, Listeria monocytogenes, Enterococcus faecalis, and Streptococcus mutans) and MBC values of <3 to <5 mg/ml for B. cereus, M. KMS, S. suis, and L. monocytogenes. LMD showed MIC and MBC values of <1 to <5 mg/ml for B. cereus, M. KMS, S. suis, L. monocytogenes, and E. faecalis, with greater inhibition when dissolved in ethanol. LMM showed MIC and MBC values of <1 to <5 mg/ml for B. cereus, M. KMS, and S. suis. LMO was the least effective showing a MBC value of <5 mg/ml for only B. cereus, though MIC values for S. suis and L. monocytogenes were observed when dissolved in DMSO. B. cereus and S. suis were the most susceptible to the lactose esters tested, while S. mutans and E. faecalis were the most resilient and no esters were effective on Escherichia coli O157:H7. This research showed that lactose esters esterified with decanoic and lauric acids exhibited greater microbial inhibitory properties than lactose esters of octanoate and myristate against Gram-positive bacteria.     

Long-acting estrogenic responses of estradiol fatty acid esters

Estradiol esters at C-17 and C-3 with palmitic, stearic and oleic acids were chemically synthesized and then evaluated for their long-acting estrogenic responses in ovariectomized rats. The duration of the biological effects was measured after a single subcutaneous dose of 0.1 mumol of each ester and compared with those observed with 17 beta-estradiol, estradiol 3-benzoate and estradiol 17-enanthate. Vaginal citology, uterophyc action, serum gonadotropins inhibition and 17 beta-estradiol levels were measured 0, 5, 10, 20, 30 and 60 days after injection. The results disclosed that most of the estradiol derivatives evaluated exhibited a long-acting estrogenic action. However, the monoesters at C-17 showed longer effects that monoesters at C-3, while the estradiol diesters exhibited the shortest effects. In addition as shown by its low serum levels, all estradiol esters with unsaturated fatty acids show a decreased E2 absorption. The overall results indicated that esterification of E2 with long chain fatty acids provided long-acting properties to it, being higher with C-17 esters. Whether some of these compounds could be employed in substitutive endocrine therapy remains to be established.     

Synthesis and properties of fatty acid starch esters

Being completely bio-based, fatty acid starch esters (FASEs) are attractive materials that represent an alternative to crude oil-based plastics. In this study, two synthesis methods were compared in terms of their efficiency, toxicity and, especially, product solubility with starch laurate (C₁₂) as model compound. Laurates (DS > 2) were obtained through transesterification of fatty acid vinylesters in DMSO or reaction with fatty acid chlorides in pyridine. The latter lead to higher DS-values in a shorter reaction time. But due to the much better solubility of the products compared to lauroyl chloride esterified ones, vinylester-transesterification was preferred to optimize reaction parameters, where reaction time could be shortened to 2 h. FASEs C₆–C₁₈ were also successfully prepared via transesterification. To determine the DS of the resulting starch laurates, the efficient ATR-IR method was compared with common methods (elementary analysis, ¹H NMR). Molar masses (M_w) of the highly soluble starch laurates were analyzed using SEC-MALLS (THF). High recovery rates (>80%) attest to the outstanding solubility of products obtained through transesterification, caused by a slight disintegration during synthesis. Particle size distributions (DLS) demonstrated stable dissolutions in CHCl₃ of vinyl laurate esterified – contrary to lauroyl chloride esterified starch. For all highly soluble FASEs (C₆–C₁₈), formation of concentrated solutions (10 wt%) is feasible.