Increased production of wax esters in transgenic tobacco plants by expression of a fatty acid reductase:wax synthase gene fusion.

Wax esters are hydrophobic lipids consisting of a fatty acid moiety linked to a fatty alcohol with an ester bond. Plant-derived wax esters are today of particular concern for their potential as cost-effective and sustainable sources of lubricants. However, this aspect is hampered by the fact that the level of wax esters in plants generally is too low to allow commercial exploitation. To investigate whether wax ester biosynthesis can be increased in plants using transgenic approaches, we have here exploited a fusion between two bacterial genes together encoding a single wax ester-forming enzyme, and targeted the resulting protein to chloroplasts in stably transformed tobacco (Nicotiana benthamiana) plants. Compared to wild-type controls, transgenic plants showed both in leaves and stems a significant increase in the total level of wax esters, being eight-fold at the whole plant level. The profiles of fatty acid methyl ester and fatty alcohol in wax esters were related, and C16 and C18 molecules constituted predominant forms. Strong transformants displayed certain developmental aberrations, such as stunted growth and chlorotic leaves and stems. These negative effects were associated with an accumulation of fatty alcohols, suggesting that an adequate balance between formation and esterification of fatty alcohols is crucial for a high wax ester production. The results show that wax ester engineering in transgenic plants is feasible, and suggest that higher yields may become achieved in the near future.

Wax esters of different compositions produced via engineering of leaf chloroplast metabolism in Nicotiana benthamiana.

In a future bio-based economy, renewable sources for lipid compounds at attractive cost are needed for applications where today petrochemical derivatives are dominating. Wax esters and fatty alcohols provide diverse industrial uses, such as in lubricant and surfactant production. In this study, chloroplast metabolism was engineered to divert intermediates from de novo fatty acid biosynthesis to wax ester synthesis. To accomplish this, chloroplast targeted fatty acyl reductases (FAR) and wax ester synthases (WS) were transiently expressed in Nicotiana benthamiana leaves. Wax esters of different qualities and quantities were produced providing insights to the properties and interaction of the individual enzymes used. In particular, a phytyl ester synthase was found to be a premium candidate for medium chain wax ester synthesis. Catalytic activities of FAR and WS were also expressed as a fusion protein and determined functionally equivalent to the expression of individual enzymes for wax ester synthesis in chloroplasts.

In vitro biodegradation of cyanotoxins in the rumen fluid of cattle.

BACKGROUND: In countries around the Baltic Sea grazing ruminants have access to and drink, surface water from lakes, rivers and in several coastal regions. The water quality of these naturally occurring reservoirs affects performance and health of livestock. In the Baltic Sea both microcystin (MC) and nodularin (NOD) occurs as cyclic peptides and have hepatotoxic effects. Although cattle obviously have died after consuming contaminated water very little information is available as to how susceptible ruminants are to the toxins produced by cyanobacteria. The critical question as to whether the rumen microflora might constitute a protective shield is unresolved. For this reason our aim is to investigate a possible degradation rate of these toxins in rumen. RESULTS: The ability of rumen microorganisms to degrade certain important cyanotoxins (MC-LR, YR, RR and NOD) was studied in vitro by incubating with rumen fluid at three different concentrations (0.05, 0.5 and 5 µg/mL) for 3 h. The degradation efficiencies were determined by LC-MS (ESI) positive mode. Degradation was observed in the following order MC-RR 36%, NOD 35%, MC-RR 25% and MC-LR 8.9% at lower concentrations within 3 h. However, average degradation was observed at concentration of 0.5 µg/mL. No degradation was observed in higher concentrations for entire 3 h. The present results reveal that the degradation was both dose and time dependent. CONCLUSIONS: In conclusion the present results suggest that the rumen microbial flora may protect ruminants from being intoxicated by Cyanotoxins.

Synthesis of hydroxylated sterols in transgenic Arabidopsis plants alters growth and steroid metabolism.

To explore mechanisms in plant sterol homeostasis, we have here increased the turnover of sterols in Arabidopsis (Arabidopsis thaliana) and potato (Solanum tuberosum) plants by overexpressing four mouse cDNA encoding cholesterol hydroxylases (CHs), hydroxylating cholesterol at the C-7, C-24, C-25, or C-27 positions. Compared to the wild type, the four types of Arabidopsis transformant showed varying degrees of phenotypic alteration, the strongest one being in CH25 lines, which were dark-green dwarfs resembling brassinosteroid-related mutants. Gas chromatography-mass spectrometry analysis of extracts from wild-type Arabidopsis plants revealed trace levels of α and ß forms of 7-hydroxycholesterol, 7-hydroxycampesterol, and 7-hydroxysitosterol. The expected hydroxycholesterol metabolites in CH7-, CH24-, and CH25 transformants were identified and quantified using gas chromatography-mass spectrometry. Additional hydroxysterol forms were also observed, particularly in CH25 plants. In CH24 and CH25 lines, but not in CH7 ones, the presence of hydroxysterols was correlated with a considerable alteration of the sterol profile and an increased sterol methyltransferase activity in microsomes. Moreover, CH25 lines contained clearly reduced levels of brassinosteroids, and displayed an enhanced drought tolerance. Equivalent transformations of potato plants with the CH25 construct increased hydroxysterol levels, but without the concomitant alteration of growth and sterol profiles observed in Arabidopsis. The results suggest that an increased hydroxylation of cholesterol and/or other sterols in Arabidopsis triggers compensatory processes, acting to maintain sterols at adequate levels.

Cholesterol and lipid oxidation in raw and pan-fried minced beef stored under aerobic packaging.

BACKGROUND: The type of packaging atmosphere has been reported as a technological factor that consistently affects the quality of lipid fraction in meat. Oxidation of cholesterol and lipids was evaluated before and after pan frying in commercial refrigerated minced beef stored under aerobic atmosphere for 1 and 8 days. RESULTS: In raw beef, cholesterol and lipid oxidation developed at a slow rate. Cholesterol oxidation products (COPs) did not significantly vary (approximately 8 microg COPs g(-1) of fat) over 8 days, while in the same period thiobarbituric acid reactive substances (TBARS) less than doubled (from 0.7 to 1.2 malondialdehyde equivalents kg(-1) of muscle). Pan frying did not influence the oxidative degree in the fresh product but consistently catalyzed cholesterol oxidation in stored beef. A significant increase was assessed in beef at the end of storage: from 8.6 to 30.0 microg COPs g(-1) of fat in raw and cooked beef, respectively. CONCLUSION: Aerobic packaging did not appear as a pro-oxidant factor in fresh minced beef with a good oxidative quality during a short period of refrigerated storage.

A single step solid-phase extraction method for complete separation of sterol oxidation products in food lipids.

One of the crucial steps in determination of sterol oxidation products (SOPs) in foods is their enrichment and purifications by various preparative methods for further analysis by GC and GC-MS. Among the preparative methods, SPE of various adsorbents and solvent systems, are being used most widely. At present, no single step SPE method is suitable to completely separate the SOPs. In this study, a SPE (1g silica) method, suitable for both transesterified and cold saponified oil samples, was developed to separate completely SOPs from other lipid components. This method resulted in high recovery from rapeseed oil of added 5beta,6beta-epoxycholestan-3beta-ol (94-96%), cholest-5-en-3beta-ol-7-one(94%), cholestane-3beta,5alpha,6beta-triol (88-91%), cholest-5-en-3beta,7alpha-diol and 5alpha,6alpha-epoxycholestan-3beta-ol (88-90%). The method has a high sample capacity of up to 1g transesterified or cold-saponified oil sample. The method was tested and applied to different vegetable oils and to monitor the effects of refining processes on POPs in hazelnut oil.

Evaluation of cholesterol and lipid oxidation in raw and cooked minced beef stored under oxygen-enriched atmosphere.

Oxygen-enriched modified atmosphere packaging (MAP) represents an important means to stabilize meat colour but may lead to an increase in lipid oxidation, influencing the acceptability and safety of the product. In this work, the effect on cholesterol and lipid susceptibility to oxidation was investigated in commercial minced beef held under MAP (80% O(2)/20% CO(2)). Cholesterol oxidation products (COPs), peroxide value (PV) and thiobarbituric acid reactive substances (TBARS) were determined, before and after pan frying, at 1, 8 and 15 days since packaging under refrigerated storage (3-4°C). 7α-Hydroxycholesterol, 7ß-hydroxycholesterol and 7-ketocholesterol were the more abundant COPs identified. COPs significantly increased in raw beef during storage: after 1, 8 and 15 days since packaging COPs were at the levels of 10.4, 30.7 and 60.5µg/g of fat, respectively. Cooking did not affect cholesterol oxidation in freshly packaged minced beef but led to a rise in COPs amount with respect to raw muscle after 8 and 15 days of storage. The trend in cholesterol oxidation reflected the progressive increase in lipid peroxidation rate brought by MAP conditions.

Transcript profiling of two potato cultivars during glycoalkaloid-inducing treatments shows differential expression of genes in sterol and glycoalkaloid metabolism.

Steroidal glycoalkaloids (SGA) are sterol-derived neurotoxic defence substances present in several members of the Solanaceae. In the potato (Solanum tuberosum), high SGA levels may render tubers harmful for consumption. Tuber SGA levels depend on genetic factors, and can increase as a response to certain stresses and environmental conditions. To identify genes underlying the cultivar variation in tuber SGA levels, we investigated two potato cultivars differing in their SGA accumulation during wounding or light exposure; two known SGA-inducing treatments. Using microarray analysis coupled to sterol and SGA quantifications, we identified a small number of differentially expressed genes that were associated with increased SGA levels. Two of these genes, encoding distinct types of sterol Δ24-reductases, were by sense/antisense expression in transgenic potato plants shown to have differing roles in sterol and SGA metabolism. The results show that an increased SGA level in potato tubers during both wounding and light exposure is mediated by coordinated expression of a set of key genes in isoprenoid and steroid metabolism, and suggest that differences in this expression underlie cultivar variations in SGA levels. These results may find use within potato breeding and quality assessment.

Novel solid-phase extraction method to separate 4-desmethyl-, 4-monomethyl-, and 4,4'-dimethylsterols in vegetable oils.

Conventional methods for sterol fractions separation by TLC have some drawbacks such as low recovery and time consuming. A new solid-phase extraction (SPE) method was developed with stepwise elution by increasing the polarity of solvents mixture: n-hexane and diethyl ether. This method was applied to separate sterol fractions of hazelnut and virgin olive oils, and our results were compared with those of TLC method. The recovery of spiked authentic sample of 4-desmethylsterols in oil was higher with the SPE method (94%) compared with the TLC method (62%). The amount of 4,4'-dimethylsterols and 4-desmethylsterols separated with SPE in both hazelnut and virgin olive oil samples were at least 75% and 35%, respectively, higher than that of TLC. Generally, both methods obtained similar results for 4-monomethylsterols of the two oils. This new SPE method to separate phytosterol fractions was less time consuming, simpler and can be used instead of preparative TLC to detect adulteration of virgin olive oil with hazelnut oil.

Separation of phytosterol oxidation products by combination of different polarity gas chromatography capillary columns.

The number of characterized phytosterol oxidation products (POPs) from both ring- and side-chain structures has increased during recent decades, resulting in difficulties in the separation of POPs on different gas chromatography (GC) capillary columns. The main objective of this study was to separate a mixture of 29 purified and characterized oxidation products from sito-, campe- and stigmasterol using GC capillary columns with different polarity. For the first time in the area of POPs analysis, the separation efficiency of the combination of two capillary GC columns with different polarities was investigated. A non-polar 5% phenyl coated (DB5-MS) and a mid-polar 35% phenyl coated (DB35-MS) column was combined with a pressfit connector. The main improvement was enhanced base line separation for many of the analyzed POPs, compared with the separations achieved using the individual columns. However, three pairs of POPs co-eluted: 24-hydroxysitosterol/campesterol-5beta,6beta-epoxide, stigmasterol-5beta,6beta-epoxide/campesterol-5alpha,6alpha-epoxide and stigmasterol-5alpha,6alpha-epoxide/campestanetriol.

Transient silencing of the KASII genes is feasible in Nicotiana benthamiana for metabolic engineering of wax ester composition.

The beta-ketoacyl-ACP synthase II (KASII) is an enzyme in fatty acid biosynthesis, catalyzing the elongation of 16:0-acyl carrier protein (ACP) to 18:0-ACP in plastids. Mutations in KASII genes in higher plants can lead to lethality, which makes it difficult to utilize the gene for lipid metabolic engineering. We demonstrated previously that transient expression of plastid-directed fatty acyl reductases and wax ester synthases could result in different compositions of wax esters. We hypothesized that changing the ratio between C16 (palmitoyl-compounds) and C18 (stearoyl-compounds) in the plastidic acyl-ACP pool by inhibition of KASII expression would change the yield and composition of wax esters via substrate preference of the introduced enzymes. Here, we report that transient inhibition of KASII expression by three different RNAi constructs in leaves of N. benthamiana results in almost complete inhibition of KASII expression. The transient RNAi approach led to a shift of carbon flux from a pool of C18 fatty acids to C16, which significantly increased wax ester production in AtFAR6-containing combinations. The results demonstrate that transient inhibition of KASII in vegetative tissues of higher plants enables metabolic studies towards industrial production of lipids such as wax esters with specific quality and composition.

Fatty acid alterations in liver and milk of cadmium exposed rats and in brain of their suckling offspring.

Fatty acid composition was studied in milk at day 14 and in liver at day 24 after parturition of lactating rats exposed to 0 ppm, 5 ppm or 25 ppm cadmium (Cd) via drinking water for 17 days during lactation, and in the brain of their offspring at day 19 after birth. In the liver phospholipid fraction, 22:5(n-3) was significantly higher, while in the triacylglycerol fraction 22:6(n-3)/20:5(n-3) ratio was significantly lower in the 25 ppm group compared to the controls. Significantly higher proportions of 16:0 and lower proportions of medium-chain fatty acids, 8:0-14:0, were observed in milk of dams in the 25 ppm group, indicating decreased enzymatic activity of thiotransferase II in the mammary gland. Slightly increased levels of 20:3(n-6) were observed in brains of pups in the 25 ppm group compared to control. The results indicate that Cd exposure influences fatty acid metabolism in lactating rats.

Inhibition of stigmasterol oxidation by antioxidants in purified sunflower oil.

A study was conducted to analyze the effect of the antioxidants butylated hydroxytoluene, alpha-tocopherol, ethanolic extracts of rosemary, and green tea on stigmasterol resistance against degradation and formation of its oxidation products in purified triacylglycerols (TAG) from sunflower oil. The content of stigmasterol and its oxidation products 7alpha- and 7beta-hydroxy, alpha- and beta-epoxy, triol, and 7-ketostigmasterol were determined during incubation at 60 degrees C for 3, 6, and 9 days. In addition, peroxide value and fatty acid composition were also determined in the samples. Correlation between the levels of the accumulated stigmasterol oxides and peroxide value of the TAG with antioxidants during incubation was significant only for rosemary extract (R = 0.6799, p < 0.05). The lack of correlation precludes the use of peroxide values to determine the level of sterol oxidation products in the used model system. Correlation between stigmasterol content and the level of stigmasterol oxides was significant for all samples (R = 0.8874, p < 0.05). The total increase of the stigmasterol oxidation products was the lowest in samples with alpha-tocopherol, but the content of stigmasterol-triol increased the most in this sample. In all the analyzed samples, alpha-epoxy-stigmasterol was formed in the highest amounts among the analyzed stigmasterol oxidation products.

Harmonization of methods for analysis of cholesterol oxides in foods--the first portion of a long road toward standardization: interlaboratory study.

A compilation of literature data on the content of cholesterol oxidation products (COP) in various food products and in blood demonstrates a large variation in content in products or tissues of very similar nature when analyzed in different laboratories according to a large number of methods. The lack of validated, internationally recognized methodology with published accuracy and precision has so far hindered such assessments. Hence an interlaboratory comparision of methodologies of COP analysis was undertaken on egg yolk powders (EYP), whole milk powders (WMP), skim milk powders (SMP), and lard (L). Each product type had one fresh sample (low) and one aged (high) in COP contents. A total of 17 sets of results on WMP, 15 on SMP and EYP, and 13 on L were compared. Overall results (mg/kg sample) varied extensively: Fresh EYP 0.72-265, aged EYP 2.51-361; fresh WMP 0.02-18.1, aged WMP 0.02-26.9; fresh SMP 0.02-6.51, aged SMP <0.01-6.51; fresh L 0.18-97, aged L 4.15-452. Some results were questioned, viz., those from laboratories not indicating substantial differences between samples "low" and "high" in total COP. Others were excluded because of lack of verification of identity of gas chromatographic peaks by mass spectrometry. Then a more narrow range of core results (mg/kg sample) was observed: Fresh EYP 5.69-29.5 sample, aged EYP 11.8-79.0; fresh WMP 0.12-1.76, aged WMP 1.17-13.7; fresh SMP <0.30-<1.21, aged SMP 0.30-2.26; fresh L 0.18-5.07, aged L 94.4-231. At a workshop discussing the results, numerous recommendations were made toward more reliable methodology for determination of COP in foods.

Protective effect of Actiniopteris radiata (Sw.) Link. against CCl4 induced oxidative stress in albino rats.

ETHNOPHARMACOLOGICAL RELEVANCE: Actiniopteris radiata is a herb with great medicinal value and is evaluated for hepatoprotective activity. To investigate the protective effect of ethanolic extract of Actiniopteris radiata (EEAR) on CCl4 induced oxidative stress in male Wistar albino rats. MATERIALS AND METHODS: EEAR were administered for 8 consecutive weeks to rats. Group I - control; Group II - toxin control (30% CCl4); Group III and Group IV received EEAR (250 and 500 mg/kg respectively). Antioxidant status in liver were estimated by determining the activities of the antioxidant enzymes, superoxide dismutase (SOD), catalase (CAT) and glutathione peroxidase (GPx); as well as by determining the levels of lipid peroxidation (LPO) and reduced glutathione (GSH). In addition, isoenzyme pattern and mRNA expression of the antioxidants were studied. Partial characterization of EEAR was performed by Liquid chromatography-mass spectrometry (LC-MS). RESULTS: CCl4 induced oxidative stress as evidenced from increase in LPO along with reduction of SOD, CAT, GPx and GSH. Treatment with EEAR (250 and 500 mg/kg) mitigated the CCl4 induced oxidative stress. An analysis of the isozyme pattern of these antioxidant enzymes revealed variations in SOD2, CAT, GPx2 and GPx3 in CCl4 treated rats, which were normalized after EEAR treatment. Furthermore, expression of genes for the antioxidant enzymes, were down-regulated by CCl4 treatment, which were reversed by EEAR. The results of partial characterization of EEAR by LC-MS revealed the presence of rutin and other 7 unknown phenolic derivatives. CONCLUSIONS: These findings suggest the protective effect of EEAR against CCl4 induced oxidative stress might be attributed to the presence of flavonoids and phenolic compounds.

Conversion of exogenous cholesterol into glycoalkaloids in potato shoots, using two methods for sterol solubilisation.

Steroidal glycoalkaloids (SGA) are toxic secondary metabolites naturally occurring in the potato, as well as in certain other Solanaceous plant species, such as tomato, eggplant and pepper. To investigate the steroidal origin of SGA biosynthesis, cut potato shoots were fed cholesterol labelled with deuterium (D) in the sterol ring structure (D5- or D6-labelled), or side chain (D7-labelled), and analysed after three or five weeks. The labelled cholesterol and presence of D-labelled SGA were analysed by GC-MS and LC-MS/MS, respectively. When feeding D-labelled cholesterol solubilised in Tween-80, labelled cholesterol in free form became present in both leaves and stems, although the major part was recovered as steryl esters. Minor amounts of D-labelled SGA (α-solanine and α-chaconine) were identified in cholesterol-treated shoots, but not in blank controls, or in shoots fed D6-27-hydroxycholesterol. Solubilising the labelled cholesterol in methyl-ß-cyclodextrin instead of Tween-80 increased the levels of labelled SGA up to 100-fold, and about 1 mole% of the labelled cholesterol was recovered as labelled SGA in potato leaves. Both side chain and ring structure D labels were retained in SGA, showing that the entire cholesterol molecule is converted to SGA. However, feeding side chain D7-labelled cholesterol resulted in D5-labelled SGA, indicating that two hydrogen atoms were released during formation of the SGA nitrogen-containing ring system. Feeding with D7-sitosterol did not produce any labelled SGA, indicating that cholesterol is a specific SGA precursor. In conclusion, we have demonstrated a superior performance of methyl-ß-cyclodextrin for delivery of cholesterol in plant tissue feeding experiments, and given firm evidence for cholesterol as a specific sterol precursor of SGA in potato.

Novel Conjugates of 1,3-Diacylglycerol and Lipoic Acid: Synthesis, DPPH Assay, and RP-LC-MS-APCI Analysis.

1,3-Diacylglycerol is known to reduce body weight and fat deposits in humans. α-Lipoic acid is a potent antioxidant and effective against many pathological conditions, including obesity and related metabolic syndromes. The present work is based on the hypothesis that the hybrid molecules of 1,3-diacylglycerol and lipoic acid possess synergistic and/or additive effects compared with the parent compounds against obesity, overweight, and related metabolic syndromes. Laboratory scale synthesis of 1,3-dioleoyl-2-lipoyl-sn-glycerol (yield 80%) and 1,3-dioleoyl-2-dihydrolipoyl-sn-glycerol (yield 70%) was performed for the first time and supported by NMR and MS data. Free radical scavenging capacity of the conjugates was assayed using DPPH test. A remarkably high in vitro free radical scavenging capacity was demonstrated for the 1,3-dioleoyl-2-dihydrolipoyl-sn-glycerol (EC(50) value 0.21). RP-HPLC-MS-APCI analysis showed satisfactory separation between the conjugates (R~1). Protonated molecular ion of the conjugates at m/z 809 and m/z at 811, respectively, and their characteristic fragment ions were abundant.

Overexpression of CYP710A1 and CYP710A4 in transgenic Arabidopsis plants increases the level of stigmasterol at the expense of sitosterol.

Sitosterol and stigmasterol are major sterols in vascular plants. An altered stigmasterol:sitosterol ratio has been proposed to influence the properties of cell membranes, particularly in relation to various stresses, but biosynthesis of stigmasterol is poorly understood. Recently, however, Morikawa et al. (Plant Cell 18:1008-1022, 2006) showed in Arabidopsis thaliana that synthesis of stigmasterol and brassicasterol is catalyzed by two separate sterol C-22 desaturases, encoded by the genes CYP710A1 and CYP710A2, respectively. The proteins belong to a small cytochrome P450 subfamily having four members, denoted by CYP710A1-A4, and are related to the yeast sterol C-22 desaturase Erg5p acting in ergosterol synthesis. Here, we report on our parallel investigation of the Arabidopsis CYP710A family. To elucidate the function of CYP710A proteins, transgenic Arabidopsis plants were generated overexpressing CYP710A1 and CYP710A4. Compared to wild-type plants, both types of transformant displayed a normal phenotype, but contained increased levels of free stigmasterol and a concomitant decrease in the level of free sitosterol. CYP710A1 transformants also displayed higher levels of esterified forms of stigmasterol, cholesterol, 24-methylcholesterol and isofucosterol. The results confirm the findings of Morikawa et al. (Plant Cell 18:1008-1022, 2006) regarding the function of CYP710A1 in stigmasterol synthesis, and show that CYP710A4 also has this capacity. Furthermore, our results suggest that an increased stigmasterol level alone is sufficient to stimulate esterification of other major sterols.

Lipids and lipid oxidation with emphasis on cholesterol oxides in some Indian sweets available in London.

Ghee (clarified butter oil), a major ingredient in Indian sweets, is an important source of saturated fatty acids, cholesterol and cholesterol oxidation products (COP) that are considered risk factors for atherosclerosis. The high frequency of atherosclerotic complications reported among the Indian immigrants in England prompted determination of lipids and lipid oxidation status of a ghee sample and 15 Indian sweets available in London supermarkets. The fatty acid profile of the samples shows saturated fats (about 73%), mainly composed of myristic, palmitic and stearic acids, except in two samples. There were large variations in thio-barbituric acid reacting substance values (19-260 microg/100 g) and total COP (1.4-51.2 microg/g lipids) among the sweet samples. Regular consumption of some of these sweets can be a source of considerable amounts of saturated fatty acids, cholesterol and COP in the diet and may contribute to atherosclerosis.