Unsaturated Metabolites Research Articles

Hypolipidemic effect of Sesame Seed Oil: A Review

Sesame seed (Sesamum indicum L) oil is an excellent source of unsaturated fatty acids and secondary metabolites. Most commonly used in Eastern countries, easily available and cheap source of essential nutrients. Objective: To identify scientific evidence regarding the hypolipidemic effect of sesame seed oil and its derivatives. Elevated levels of plasma lipids and cholesterol is known as hyperlipidemia, increased lipids levels are directly associated with vascular and metabolic disorders. Hyperlipidemia is the most common risk factor of cardiovascular diseases. Sesame seed oil is loaded in vitamin E, sesamin, sesamolin, sesamol, lignans, and phytoestrogen, these constituents have the potential to reduce hyperlipidemia, hypercholesterolemia, and elevated triglycerides levels. They also act as antioxidants to prevent lipid peroxidation and oxidative stress. The mechanism behind the hypolipidemic effect of sesame seed oil is its ability to increase superoxide dismutase SOD and glutathione peroxidase (antioxidants), reduction in triglycerides, and Malondialdehyde (MDA) concentration. MDA is the end product of lipid peroxidation and triggers lipid accumulation in blood. It has been studied that Sesaminol diglucoside helps to reduce white fat deposition by increasing the conversion of fat into brown fat. Conclusions: Results suggest that sesame seed oil is highly effective to manage hyperlipidemia and dyslipidemia due to its lipid ameliorating and anti-oxidative effect

The Artemiside-Artemisox-Artemisone-M1 Tetrad: Efficacies against Blood Stage P. falciparum Parasites, DMPK Properties, and the Case for Artemiside.

Because of the need to replace the current clinical artemisinins in artemisinin combination therapies, we are evaluating fitness of amino-artemisinins for this purpose. These include the thiomorpholine derivative artemiside obtained in one scalable synthetic step from dihydroartemisinin (DHA) and the derived sulfone artemisone. We have recently shown that artemiside undergoes facile metabolism via the sulfoxide artemisox into artemisone and thence into the unsaturated metabolite M1; DHA is not a metabolite. Artemisox and M1 are now found to be approximately equipotent with artemiside and artemisone in vitro against asexual P. falciparum (Pf) blood stage parasites (IC50 1.5–2.6 nM). Against Pf NF54 blood stage gametocytes, artemisox is potently active (IC50 18.9 nM early-stage, 2.7 nM late-stage), although against the late-stage gametocytes, activity is expressed, like other amino-artemisinins, at a prolonged incubation time of 72 h. Comparative drug metabolism and pharmacokinetic (DMPK) properties were assessed via po and iv administration of artemiside, artemisox, and artemisone in a murine model. Following oral administration, the composite Cmax value of artemiside plus its metabolites artemisox and artemisone formed in vivo is some 2.6-fold higher than that attained following administration of artemisone alone. Given that efficacy of short half-life rapidly-acting antimalarial drugs such as the artemisinins is associated with Cmax, it is apparent that artemiside will be more active than artemisone in vivo, due to additive effects of the metabolites. As is evident from earlier data, artemiside indeed possesses appreciably greater efficacy in vivo against murine malaria. Overall, the higher exposure levels of active drug following administration of artemiside coupled with its synthetic accessibility indicate it is much the preferred drug for incorporation into rational new artemisinin combination therapies.

Evaluation of the anti-oxidant property and cytotoxic potential of the metabolites extracted from the bacterial isolates from mangrove Forest and saltern regions of South India

The objective was to evaluate the anticancer and antioxidant activities of the methanolic extracts of halophilic bacteria, isolated from soil samples of Marakkanam saltern and Pichavaram mangrove forest, India. Radical Scavenging activity, reducing power, and metal ion chelation ability was used to evaluate the antioxidant potential of the metabolic extracts, whereas cell viability was determined by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. The methanolic extract of Bacillus VITPS7 exhibited significant antioxidant property. Bacillus VITPS14 and Bacillus VITPS16 extracts were cytotoxic against HeLa cell lines but not to A549 cell lines. Colorimetric assays for the presence of specific metabolites including, total flavonoid and β carotene content were performed. The presence of these specific classes of metabolites was confirmed by UV–Visible spectrophotometry, Nuclear Magnetic Resonance (NMR) spectroscopy and Gas Chromatography–Mass Spectrometry (GC–MS). Specific NMR signals revealed the presence of aromatic and unsaturated metabolites whereas GC–MS analysis indicated the presence of metabolites such as squalene and methyl hexadeconate. The present study thus reports for the first time the presence of squalene in Bacillus VITPS12 and Planococcus maritimus VITP21, in addition to other metabolites that contribute to the observed antioxidant or/and cytotoxicity, thus revealing the therapeutic potential of these selected halophilic bacterial isolates.

REACTIVE OXYGEN AND NITROGEN SPECIES ROLE IN EXPERIMENTAL PERIODONTITIS DEVELOPMENT

Introduction. Activation of lipid peroxidation is one of the trigger mechanisms of periodontium injury, which is primary caused by cellular damage. Reactive oxygen and nitrogen species (RONS) are able to cause damage to a cell as well as final products of lipid peroxidation, including unsaturated aldehydes and other metabolites. Objective. The aim of the research was to determine the role of RONS and accumulation of lipid peroxidation derivatives in initial development and formation of chronical inflammatory process in periodontium. Methods. Experimental periodontitis was modeled in animals by injection of complex mixtures of microorganisms diluted in egg protein into periodontal tissues. The results of biochemical studies of free radical processes activity in blood serum were evaluated by content of diene, triene conjugates, TBA-active products and total quantity of metabolites of nitric oxide (NO2–+NO3–), which were determined on the 7th, 14th and 30th days of the experiment. Results. Generation of active forms of oxygen is more influential, providing longevity of inflammatory process. This pays attention to typical dynamics of changes in active processes of lipid peroxidation in the development and course of experimental periodontitis. The study of inflammatory process with a bacterial-immune component in the rats’ periodontal complex proved accumulation of lipid peroxidation and nitric oxide metabolites in blood serum.Conclusions. The preservation of increased lipid peroxidation and nitric oxide metabolites in blood serum of the experimental animals with acute periodontitis conduce enhance of alteration and delayed healing that result in its sequel into chronical periodontitis.

Clinical significance of CYP2C9-status guided valproic acid therapy in children.

Valproic acid (VPA)-induced adverse effects, which are sometimes serious in children, can be associated with alterations in VPA metabolism. VPA-evoked toxicity is attributed to both the parent compound and its unsaturated metabolites, primarily formed by the cytochrome P450 (CYP)2C9 enzyme. Thus, patients' CYP2C9-status may account for the predisposition to adverse reactions, and testing CYP2C9-status may contribute to the improvement and rationalization of VPA therapy in children. In the CYPtest group, children's CYP2C9-status was screened before initiating antiepileptic therapy. CYP2C9-status was estimated by the identification of defective CYP2C9 allelic variants (CYP2C9*2, CYP2C9*3) and current CYP2C9 expression in patients' leukocytes, which reflects hepatic CYP2C9 activities. When the results of CYP2C9 genotyping and CYP2C9 expression were combined, the patients' VPA-metabolizing capacity was predicted, and VPA dosing was adjusted to the patients' CYP2C9-status. Clinical and biochemical parameters, such as VPA serum levels, blood cell counts, liver function parameters, and adverse effects in patients of CYPtest group were compared with those of the control group treated with VPA according to conventional clinical practice. CYP2C9-guided treatment significantly reduced VPA misdosing and consequently decreased the ratio of patients out of the range of target VPA blood concentrations. In the CYPtest group of children who received CYP2C9-status adapted dose, serum alkaline phosphatase (ALP) level and the ratio of patients with abnormal ALP levels were substantially lower than in the control group. The incidence of serious side effects, notably hyperammonemia, was reduced in the CYPtest group; however, some other side effects, such as weight changes and somnolence, could not be avoided. The knowledge of pediatric patients' CYP2C9-status can contribute to the optimization of VPA dosing and to the avoidance of misdosing-induced side effects.

Retention of n-3 polyunsaturated fatty acids in trimmed loin and belly is independent of timing of feeding ground flaxseed to growing-finishing female pigs1

A serial slaughter study, involving 45 individually housed Yorkshire female pigs, was conducted to evaluate the impact of feeding diets containing ground flaxseed (FS) during either the grower or late finisher phase on the incorporation of α-linolenic acid (18:3n-3) and n-3 highly unsaturated fatty acid metabolites (n-3 HUFA; all n-3 PUFA excluding 18:3n-3) in trimmed loin (longissimus dorsi) and belly (cross-section cut). Growth performance, carcass characteristics, and pork quality were also monitored. The three feeding regimens were 1) feeding a diet containing 10% FS between 25 and 50 kg BW, followed by FS-free diets (low n-3 PUFA content) until 110 kg BW (FS early, FSE; n = 16), 2) feeding no FS diets between 25 and 85 kg BW, followed by a diet containing 6% FS until 110 kg BW (FS late, FSL; n = 17), and 3) feeding FS-free diets between 25 and 110 kg BW (CON; n = 8). Pigs were assigned to 1 of 3 target slaughter weights for determining whole nutrient retention: 50 kg (n = 4 and 5 for FSE and FSL, respectively), 85 kg (n = 4 for FSE and FSL), and 110 kg (n = 8 pigs for FSE, FSL, and CON). An additional 4 pigs were slaughtered to determine initial body composition at 25 kg BW. Pigs on treatments FSE and FSL consumed equal cumulative amounts of FS (5.1 vs. 5.2 kg). Fatty acid content was expressed as milligrams per 100 g of fresh tissue. No treatment effect was observed for growth performance, body composition, carcass characteristics, loin meat quality, and fat content in the loin and belly samples. In the loin, the content of 18:3n-3 at 110 kg BW was greater for FSL than FSE and CON (P < 0.01; 143, 76.4, and 37 mg/100 g, respectively), whereas the contents of 18:3n-3 in the belly were greater for both FSL and FSE than CON (752, 667, and 207 mg/100 g for FSL, FSE, and CON, respectively). Within tissues, the content of n-3 HUFA was similar for FSL and FSE, and both were greater (P < 0.01) than CON for loin (41.7, 30.3, and 17.9 mg/100 g, respectively) and belly (168, 179, and 71.2 mg/100 g, respectively), except for docosahexaenoic acid (22:6n-3) in the loin. Feeding FS tended to reduce the content of arachidonic acid (20:4n-6) and adrenic acid (22:4n-6; P < 0.10) in the loin and belly. In the belly but not in the loin, feeding FS reduced the content of total SFA (P = 0.05) and tended to reduce the content of the sum of MUFA (P = 0.09). Within the loin and belly, the ratio of n-6 to n-3 PUFA and the content of n-3 HUFA were similar for FSE and FSL, with the exception of 18:3n-3 and 20:4n-6 in the loin, for which contents were greater for FSL than FSE (P < 0.05). These results indicate that providing modest amounts of FS leads to a substantial enrichment of 18:3n-3 and n-3 HUFA in pork products. The enrichment of n-3 HUFA appears to be independent of timing of feeding FS, providing flexibility as to when n-3 PUFA can be fed to generate n-3 PUFA-enriched pork products.

In vitro metabolism study of 2-isopropyl-9H-thioxanthen-9-one (2-ITX) in rat and human: evidence for the formation of an epoxide metabolite

2-Isopropyl-9H-thioxanthen-9-one (2-ITX) is one of the most extensively used photoinitiators in inks found in paper and/or packaging materials for foodstuffs. Recently, traces of 2-ITX as a contaminant were discovered in baby milk and other foodstuffs at a level sufficient to pose a risk to human health. However, little is known about the toxicological profile of 2-ITX.The high lipophilicity of this substance would suggest that it could be a good substrate for hepatic metabolizing enzymes and that these metabolites could have a role in the toxicological properties of 2-ITX.The metabolism of 2-ITX, using both rat and human subcellular preparations, was studied and has resulted in the formation of eight polar metabolites (M1–M8) as revealed in the liquid chromatograpy/ultraviolet (LC/UV) and liquid chromatograpy/mass spectrometry (LC/MS) analyses. Their structures highlight a marked regioselectivity in the metabolism of 2-ITX; it is directed mainly toward the isopropyl moiety and the sulfur atom.The unsaturated metabolite 6 causes the formation of a reactive epoxide metabolite 7. This finding was supported by identification in microsomal incubations of 1,2-diol metabolite 8 arising from the epoxide by hydrolysis and it was validated by incubating in the same conditions the synthetic epoxide 7: the formation of metabolite 8 was again observed.On the basis of these data, we propose that the metabolite 6 could be included in toxicological studies of 2-ITX.

Role of Oxidative Metabolism in the Effect of Valproic Acid on Markers of Cell Viability, Necrosis, and Oxidative Stress in Sandwich-Cultured Rat Hepatocytes

Valproic acid (VPA) is a drug known for idiosyncratic hepatotoxicity and is associated with oxidative stress. It is metabolized extensively with at least one pathway leading to reactive metabolites. The primary aim of the present study was to determine whether oxidative metabolites of VPA generated in situ contribute to the toxicity of the parent drug in sandwich-cultured rat hepatocytes. Concentration-response experiments with VPA produced median effective concentration values (mean ± SEM) of 1.1 ± 0.4, 12.2 ± 1.4, and 12.3 ± 1.9mM in the 2-(4-iodophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfophenyl)-2H-tetrazolium (WST-1; cell viability), lactate dehydrogenase (LDH; necrosis), and 2',7'-dichlorofluorescein (DCF; oxidative stress) assays, respectively. At equimolar concentrations, only the unsaturated metabolites of VPA gave responses comparable to VPA, with 2,4-diene-VPA calculated to be 3-, 6-, and 10-fold more potent than VPA in the WST-1, LDH, and DCF assays, respectively. In support of a role for reactive metabolites, 2-fluoro-2-propylpentanoic acid, which is relatively resistant to biotransformation to form a 2,4-diene metabolite, yielded little or no toxicity when compared with the nonhepatotoxic octanoic acid or the vehicle-treated control. By comparison, attenuating the in situ formation of 2-propylpent-4-enoic acid (4-ene-VPA), 3-hydroxy-2-propylpentanoic acid, 4-hydroxy-2-propylpentanoic acid, and 5-hydroxy-2-propylpentanoic acid by an inhibitor of cytochrome P450 (1-aminobenzotriazole) did not alter the effects of VPA on the WST-1, LDH, or DCF assay. Overall, VPA toxicity in sandwich-cultured rat hepatocytes is independent of the in situ formation of cytochrome P450-dependent oxidative metabolites, including 4-ene-VPA. However, the data obtained from structural analogues of VPA suggest that biotransformation does appear to play a role in VPA toxicity in rat hepatocytes.

Metabolic oxidation regulates embryonic stem cell differentiation

Metabolites offer an important unexplored complement to understanding the pluripotency of stem cells. Using mass spectrometry-based metabolomics, we show that embryonic stem cells are characterized by abundant metabolites with highly unsaturated structures whose levels decrease upon differentiation. By monitoring the reduced and oxidized glutathione ratio as well as ascorbic acid levels, we demonstrate that the stem cell redox status is regulated during differentiation. Based on the oxidative biochemistry of the unsaturated metabolites, we experimentally manipulated specific pathways in embryonic stem cells while monitoring the effects on differentiation. Inhibition of the eicosanoid signaling pathway promoted pluripotency and maintained levels of unsaturated fatty acids. In contrast, downstream oxidized metabolites (e.g., neuroprotectin D1) and substrates of pro-oxidative reactions (e.g., acyl-carnitines), promoted neuronal and cardiac differentiation. We postulate that the highly unsaturated metabolome sustained by stem cells makes them particularly attuned to differentiate in response to in vivo oxidative processes such as inflammation.

Effect of diet, sex and age on fatty acid metabolism in broiler chickens: SFA and MUFA

The present study was conducted to evaluate the effect of different dietary lipid sources, age and sex on the SFA and MUFA metabolism in broiler chickens using a whole body fatty acid balance method. Four dietary lipid sources (palm fat, Palm; soyabean oil, Soya; linseed oil, Lin; and fish oil, Fish) were added at 3 % to a basal diet containing 5 % Palm. Diets were fed to female and male chickens from day 1 to either day 21 or day 42 of age. The accumulation (percentage of net intake and ex novo production) of SFA and MUFA was significantly lower in broilers fed on Palm than in broilers fed on the other diets (85.7 v. 97.4 %). Conversely, beta-oxidation was significantly higher in Palm-fed birds than the average of the other dietary treatments (14.3 v. 2.6 %). On average, 33.1 % of total SFA and MUFA accumulated in the body were elongated, and 13.8 % were Delta-9 desaturated to longer chain or more unsaturated metabolites, with lower proportions being elongated and desaturated for the Palm and Fish diets than for the Soya and Lin diets. Total in vivo apparent elongase activity decreased exponentially in relation to the net intake of SFA and MUFA, while it increased with age. Total in vivo apparent Delta-9 desaturase activity was not significantly affected by dietary treatment or age. Total ex novo production and beta-oxidation of SFA and MUFA showed a negative and positive curvilinear relationship with net intake of SFA and MUFA, respectively. Sex had no effect on SFA and MUFA metabolism.

Flavouring Group Evaluation 15, Revision 1 (FGE.15Rev1) - Aryl-substituted saturated and unsaturated primary alcohol/aldehyde/acid/ester derivatives from chemical group 22 - Opinion of the Scientific Panel on Food Additives, Flavourings, Processing Aids and Materials in contact with Food (AFC).

Flavouring Group Evaluation 15, Revision 1 (FGE.15Rev1) - Aryl-substituted saturated and unsaturated primary alcohol/aldehyde/acid/ester derivatives from chemical group 22 - Opinion of the Scientific Panel on Food Additives, Flavourings, Processing Aids and Materials in contact with Food (AFC).

Highly oxygenated and unsaturated metabolites providing a diversity of hispidin class antioxidants in the medicinal mushrooms Inonotus and Phellinus

Three new highly oxygenated and unsaturated metabolites named interfungins A ( 1), B ( 2), and C ( 3), which provide a diversity of hispidin class compounds in the fungi Inonotus and Phellinus, were isolated from the methanolic extract of the fruiting body of the fungus Inonotus xeranticus (Hymenochaetaceae). Their structures were established by spectroscopic methods. The existence of these functionalized metabolites implies that inoscavin A, davallialactone, and phelligridin F, which were previously isolated from the fungi Inonotus and Phellinus spp., are derived from 1. Compound 1 is derived from the condensation of hispidin and hispolon. Inoscavins B and C previously isolated from the fungus I. xeranticus are most probably derived from 2 which stemmed from the oxidative coupling of 3,4-dihydroxybenzalacetone and hispidin. This class of compounds exhibited significant free radical scavenging activity against the superoxide radical cation, ABTS radical anion, and DPPH radical.

Metabolic products and pathways of fluorotelomer alcohols in isolated rat hepatocytes

Fluorotelomer alcohols (FTOHs; CF 3(CF 2) x C 2H 4OH; where x = 3, 5, 7, 9) are a novel class of polyfluorinated contaminants, recently detected in the North American atmosphere, that are possible precursors to the series of perfluoroalkyl carboxylates (PFCAs) in human blood. An in vivo rat study validated earlier independent work that poly- and per-fluoroalkyl carboxylates were metabolites of FTOHs, but our detection of several novel metabolites prompted us to examine their pathways in greater detail using isolated rat hepatocytes. Using 8:2 FTOH (i.e. where x = 7) as a model compound, the metabolic products formed by isolated rat hepatocytes were identified, and three synthesized intermediates were incubated separately to elucidate the metabolic pathways. For 8:2 FTOH, a major fate was direct conjugation to form the O-glucuronide and O-sulfate. Using 2,4-dinitrophenylhydrazine (DNPH) trapping, the immediate oxidation product of 8:2 FTOH was identified as 8:2 fluorotelomer aldehyde (8:2 FTAL; CF 3(CF 2) 7CH 2C(H)O). 8:2 FTAL was transient and eliminated HF non-enzymatically to yield 8:2 fluorotelomer α,β-unsaturated aldehyde (8:2 FTUAL; CF 3(CF 2) 6CF CHC(H)O) which was also short-lived and reacted GSH and perhaps other endogenous nucleophiles. Four polyfluorinated acid intermediates were also detected, including 8:2 fluorotelomer carboxylate (8:2 FTCA; CF 3(CF 2) 7CH 2C(O)O −), 8:2 fluorotelomer α,β-unsaturated carboxylate (8:2 FTUCA; CF 3(CF 2) 6CFCHC(O)O −), tetrahydroperfluorodecanoate (CF 3(CF 2) 6(CH 2) 2CO 2 −), and dihydroperfluorodecenoate (CF 3(CF 2) 6CH CHCO 2 −). The pathways leading to 8:2 FTCA and FTUCA involve oxidation of 8:2 FTAL, however, the pathways leading to the latter two polyfluorinated acids remain inconclusive. The fate of the unsaturated metabolites, 8:2 FTUAL and FTUCA, included conjugation with GSH and dehydrofluorination to yield α,β-unsaturated GSH conjugates, and GS-8:2 FTUAL which was subsequently reduced to the corresponding alcohol. Perfluorooctanoate (PFOA) and minor amounts of perfluorononanoate (PFNA) were confirmed as metabolites of 8:2 FTOH, and the respective roles of β- and α-oxidation mechanisms are discussed. The analogous acids, aldehydes, and conjugated metabolites of 4:2, 6:2, and 10:2 FTOH (i.e. where x = 3, 5, and 9, respectively) were also detected, and metabolite profiles among FTOHs generally differed only in the length of their perfluoroalkyl chains. Preincubation with aminobenzotriazole, but not pyrazole, inhibited the formation of metabolites from all FTOHs, suggesting that their oxidation was catalyzed by P450, not alcohol dehydrogenase.

Proteomics of Collagen Peptides: A Method to Reveal Minor Changes in Post‐Translationally Modified Collagen by HPLC and Capillary Electrophoresis

Depository effects in slowly metabolized proteins, typically glycation or the estimation of products arising from the reaction of unsaturated long chain fatty acid metabolites (possessing aldehydic groups) are very difficult to assess owing to their extremely low concentration in the protein matrix. Posttranslational nonenzymatic modifications of collagen with sugars and oxidation products of lipid metabolism in tail tendons of two groups of rats (controls and hypertriglyceridemics with a high fructose diet) were studied. Collagen (a mixture of type I and III) was digested by bacterial collagenase and separated by reversed‐phase HPLC; the profile obtained was divided into five fractions, which were further characterizated by capillary electrophoresis (CE) in a bare fused silica capillary (37/30 cm×75 µm I.D.). As the chromatographic and electromigration behaviour of individual peptides was considerably different, the combination of HPLC and CE appeared as a suitable approach capable of acceptable separation of complex peptide mixtures (over 150 peptide peaks were obtained on the electropherograms). This two‐stepped peptide mapping with subsequent statistical evaluation (linear regression analysis) was shown to represent a reliable approach for revealing posttranslational modifications in slowly metabolized model proteins in vivo.

Ethanol toxicity in pancreatic acinar cells: mediation by nonoxidative fatty acid metabolites.

Ethanol causes pancreatic damage by an unknown mechanism. Previously, we demonstrated that a sustained rise of the cytosolic Ca(2+) concentration ([Ca(2+)](i)) causes pancreatic acinar cell injury. Here we have investigated the effects of ethanol and its metabolites on Ca(2+) signaling in pancreatic acinar cells. Most cells exposed to ethanol (up to 850 mM) showed little or no increase in [Ca(2+)](i) (and never at concentrations <50 mM). During sustained exposure to 850 mM ethanol, acetylcholine (ACh) evoked a normal [Ca(2+)](i) elevation and following ACh removal there was a normal and rapid recovery to a low resting level. The oxidative metabolite acetaldehyde (up to 5 mM) had no effect, whereas the nonoxidative unsaturated metabolite palmitoleic acid ethyl ester (10-100 microM, added on top of 850 mM ethanol) induced sustained, concentration-dependent increases in [Ca(2+)](i) that were acutely dependent on external Ca(2+) and caused cell death. These actions were shared by the unsaturated metabolite arachidonic acid ethyl ester, the saturated equivalents palmitic and arachidic acid ethyl esters, and the fatty acid palmitoleic acid. In the absence of external Ca(2+), releasing all Ca(2+) from the endoplasmic reticulum by ACh (10 microM) or the specific Ca(2+) pump inhibitor thapsigargin (2 microM) prevented such Ca(2+) signal generation. We conclude that nonoxidative fatty acid metabolites, rather than ethanol itself, are responsible for the marked elevations of [Ca(2+)](i) that mediate toxicity in the pancreatic acinar cell and that these compounds act primarily by releasing Ca(2+) from the endoplasmic reticulum.

Identification of 7(8) and 8(9) unsaturated adrenal steroid metabolites produced by patients with 7-dehydrosterol-Δ 7-reductase deficiency (Smith–Lemli–Opitz syndrome)

Patients with Smith–Lemli–Opitz syndrome have impaired ability to synthesize cholesterol due to attenuated activity of 7-dehydrosterol-Δ 7-reductase which catalyses the final step in cholesterol synthesis. Accumulation of 7- and 8-dehydrocholesterol is a result of the disorder and potentially these sterols could be used as precursors of a novel class of Δ 7 and Δ 8 unsaturated adrenal steroids and their metabolites. In this study, we have analyzed urine from SLOS patients in the anticipation of characterizing such metabolites. Gas chromatography/mass spectrometry (GC/MS) was used in the identification of two major metabolites as 7- and 8-dehydroversions of the well-known steroid pregnanetriol. Other steroids, such as 8-dehydro dehydroepiandrosterone (8-dehydro DHEA) and 7- or 8-dehydroandrostenediol were also identified, and several more steroids are present in urine but remain uncharacterized. As yet, the study provides no evidence for the production of ring-B unsaturated metabolites of complex steroids, such as cortisol. We believe that the following transformations can utilize ring-B dehydroprecursors: StAR transport of cholesterol, p450 side chain cleavage, 17-hydroxylase/17,20-lyase, 3β-hydroxysteroid dehydrogenase, 3α-hydroxysteroid dehydrogenase, 17β-hydroxysteroid dehydrogenase, 20α-hydroxysteroid dehydrogenase and 5β-reductase. We have yet to prove the activity of adrenal 21-hydroxylase, 11β-hydroxylase or 5α-reductase towards 7- or 8-dehydroprecursors.

Production of new unsaturated lipids during wood decay by ligninolytic basidiomycetes.

Lipids were analyzed by gas chromatography-mass spectrometry for a 7-week in vitro decay of eucalypt wood by four ligninolytic basidiomycetes. The sound wood contained up to 75 mg of lipophilic compounds per 100 g of wood. Hydrolysis of sterol esters, which represented 38% of total wood lipids, occurred during the fungal decay. The initial increase of linoleic and other free unsaturated fatty acids paralleled the decrease of sterol esters. Moreover, new lipid compounds were found at advanced stages of wood decay that were identified from their mass spectra as unsaturated dicarboxylic acids consisting of a long aliphatic chain attached to the C-3 position of itaconic acid. These dicarboxylic acids were especially abundant in the wood treated with Ceriporiopsis subvermispora (up to 24 mg per 100 g of wood) but also were produced by Phlebia radiata, Pleurotus pulmonarius, and Bjerkandera adusta. We hypothesize that three main alkylitaconic acids (tetradecylitaconic, cis-7-hexadecenylitaconic, and hexadecylitaconic acids) are synthesized by fungi in condensation reactions involving palmitic, oleic, and stearic acids. We suggest that both wood unsaturated fatty acids (present in free form or released from esters during natural decay) and unsaturated metabolites synthesized by fungi could serve as a source for peroxidizable lipids in lignin degradation by white rot basidiomycetes.

Differential effect of valproate and its Δ 2- and Δ 4-unsaturated metabolites, on the β-oxidation rate of long-chain and medium-chain fatty acids

Overall fatty acid oxidation rates were investigated in rat hepatocytes using [9,10- 3H]-palmitic, [9,10- 3H]-oleic, [9,10- 3H]-myristic and [2,3- 3H]-phenylpropionic acids. The effect of both valproate (VPA) (0–10 mM) and two of its unsaturated metabolites, Δ 2(E)-VPA and Δ 4-VPA (0–10 mM), on the overall 3H 2O production rate was studied. The results give evidence of a general inhibitory effect of VPA on the β-oxidation rate of all the tested substrates. Similar effects were observed with both VPA metabolites but these effects appeared to be dependent on the chain length of the substrate. When the effect on the oxidation of the medium-chain fatty acid 3-phenylpropionate (PPA) was studied, Δ 2(E)-VPA at 0.5 mM caused a 94% inhibition of the overall β-oxidation rate. However, with long-chain substrates, 0.5 mM Δ 4-VPA was a more potent inhibitor (20–30% of control activity) than 0.5 mM Δ 2(E)-VPA (60–80% of control activity). Our results suggest that VPA and/or its metabolites inhibit fatty acyl-CoA metabolism within the mitochondrion by two different mechanisms. The first mechanism involves CoASH sequestration, which affects the oxidation rate of all fatty acids with different chain length. The second mechanism is more specific in nature and involves selective inhibition of particular enzymes implicated in fatty acid β-oxidation.

Function of human mitochondrial 2,4-dienoyl-CoA reductase and rat monofunctional Δ3-Δ2-enoyl-CoA isomerase in β-oxidation of unsaturated fatty acids

Human 2,4-dienoyl-CoA reductase (2,4-reductase; DECR) and rat monofunctional Delta(3)-Delta(2)-enoyl-CoA isomerase (rat 3, 2-isomerase; ECI) are thought to be mitochondrial auxiliary enzymes involved in the beta-oxidation of unsaturated fatty acids. However, their function during this process has not been demonstrated. Although they lack obvious peroxisomal targeting signals (PTSs), both proteins have been suggested previously to also occur in the mammalian peroxisomal compartment. The putative function and peroxisomal location of the two mammalian proteins can be examined in yeast, since beta-oxidation of unsaturated fatty acids is a compartmentalized process in Saccharomyces cerevisiae requiring peroxisomal 2,4-dienoyl-CoA reductase (Sps19p) and peroxisomal 3, 2-isomerase (Eci1p). A yeast sps19Delta mutant expressing human 2, 4-reductase ending with the native C-terminus could not grow on petroselinic acid [cis-C(18:1(6))] medium but could grow when the protein was extended with a PTS tripeptide, SKL (Ser-Lys-Leu). We therefore reason that the human protein is a physiological 2, 4-reductase but that it is probably not peroxisomal. Rat 3, 2-isomerase expressed in a yeast eci1Delta strain was able to re-establish growth on oleic acid [cis-C(18:1(9))] medium irrespective of an SKL extension. Since we had shown that Delta(2,4) double bonds could not be metabolized extra-peroxisomally to restore growth of the sps19Delta strain, we postulate that rat 3,2-isomerase acted on the Delta(3) unsaturated metabolite of oleic acid by replacing the mutant's missing activity from within the peroxisomes. Immunoblotting of fractionated yeast cells expressing rat 3, 2-isomerase in combination with electron microscopy supported our proposal that the protein functioned in peroxisomes. The results presented here shed new light on the function and location of human mitochondrial 2,4-reductase and rat monofunctional 3,2-isomerase.

Valproate metabolites in the rat brain--regional distribution in various brain areas.

To investigate the regional distribution of valproic acid (VPA) and 10 of its metabolites in the rat brain, the animals were treated with 300 mg/kg/day VPA i.p. on a 3 times daily dose regimen for 5 days, and the concentrations of the compounds in serum and 15 brain regions were measured. 2-(n-propyl)-(Z)-2-pentenoic acid [(Z)-2-en], a VPA metabolite expected to possess neurotoxic potency in humans, was determined in brain tissue for the first time. The brain/serum concentration ratio of (Z)-2-en was found to be about 14-fold higher than the ratio for its (E)-isomer, thereby demonstrating the influence of the double-bond configuration in the unsaturated metabolites on their ability to penetrate into the central nervous system. The concentrations of VPA and its metabolites in the brain regions were compared to c(hom), the calculated concentration for an assumed homogeneous distribution. The parent drug and its metabolites exhibited individual distribution patterns with varying degrees of inhomogeneity. Elevated metabolite concentrations were found especially in the motorium and the medulla oblongata. Decreased concentrations of VPA and several metabolites were found in the visual cortex.