Paresh Chandra Dutta

Capillary column gas-liquid chromatographic separation of Δ5-unsaturated and saturated phytosterols

Abstract Diets consisting of food ingredients of both plant and animal origin contain a complex mixture of sterols in the lipid fraction. Analysis of these complex mixtures of sterols is generally accomplished by capillary column GC and GC–MS. A very low polarity liquid phase (methylsilicone) and medium polarity columns of various dimensions are used for the separation and quantitation of sterols. However, these columns may not be able to separate some of the sterols in the mixture. The objective of this study was to accomplish the complete separation of a complex mixture of sterols by GC. In order to achieve this objective, a fused-silica capillary column (14% cyanopropyl-phenyl-methylpolysiloxane) of low/medium polarity was used. Excellent separation of campesterol and campestanol, and sitosterol, sitostanol and Δ5-avenasterol was achieved. In addition to GC resolution, a few critical points for the analysis of sterols are presented.

Saturated Sterols (Stanols) in Unhydrogenated and Hydrogenated Edible Vegetable Oils and in Cereal Lipids

The content of saturated sterols (stanols) was investigated in a small number of samples of hydrogenated fats and oils, and in the free and bound lipids of various samples of cereals. The sterols, after saponification of the total lipids, were analysed as trimethylsilyl derivatives by GC and identified by GC-MS. Among the hydrogenated fats and oils, coconut oil contained the largest amounts of sitostanol followed by soybean oil (c 80 and 20 g kg -1 of total unsaponifiables, respectively). No sitostanol could be detected in hydrogenated palm oil under the present analytical conditions. Both free and bound lipids in various samples of wheat, except for wheat germ, contained c 70-120 g kg -1 campestanol and 100-150 g kg -1 sitostanol in total unsaponifiables. In lipids of oats and barley, no campestanol or sitostanol could be detected. Rye total lipids contained 60-90 g kg -1 of campestanol and 100-150 g kg -1 of sitostanol of total unsaponifiables in free and bound lipids, respectively.

Reduction of Cholesterol and Glycoalkaloid Levels in Transgenic Potato Plants by Overexpression of a Type 1 Sterol Methyltransferase cDNA

Transgenic potato (Solanum tuberosum cv Désirée) plants overexpressing a soybean (Glycine max) type 1 sterol methyltransferase (GmSMT1) cDNA were generated and used to study sterol biosynthesis in relation to the production of toxic glycoalkaloids. Transgenic plants displayed an increased total sterol level in both leaves and tubers, mainly due to increased levels of the 24-ethyl sterols isofucosterol and sitosterol. The higher total sterol level was due to increases in both free and esterified sterols. However, the level of free cholesterol, a nonalkylated sterol, was decreased. Associated with this was a decreased glycoalkaloid level in leaves and tubers, down to 41% and 63% of wild-type levels, respectively. The results show that glycoalkaloid biosynthesis can be down-regulated in transgenic potato plants by reducing the content of free nonalkylated sterols, and they support the view of cholesterol as a precursor in glycoalkaloid biosynthesis.

The effects of different cultural conditions on the accumulation of depot lipids notably petroselinic acid during somatic embryogenesis in Daucus carota L.

Total lipid content and the fatty acid composition of total lipids (TL), of triacylglycerols (TAG), and of total poplar lipids (PL) were investigated in tissue culture and somatic embryoids of Daucus carota L. Total lipids were approx. 0.2% of fresh weight in callus and suspension culture but approx. 0.5% in heart and torpedo shaped embryoids. Petroselinic acid (cis-6−8:1), which is approx. 70% of carrot seed oil, was not present in callus, suspension culture (0 time) and globular embryoids. The appearance of this fatty acid started in the heart shaped embryoids and reached a maximum level of 1.4% in the TAG of torpedo shaped embryoids. The major fatty acids invariably in all kinds of tissues where linoleic (18:2) and palmitic (16:0) acids, and the minor ones were stearic (18:0), oleic (cis-9−18:1), vaccenic (cis-11−18:1) and linolenic (18:3) acid. Different cultural conditions could not boost petroselinic acid accumulation but did increase TAG content in total lipids and total lipids in embryoids with a maximum level of approx. 17 mg TAG per g fresh weight. Abscisic acid, sorbitol and these in combination gave the maximum level of total lipids per g fresh weight; approx. 75% of the lipids were TAG. The results from this study, showing low levels of petroselinic acid in later stages embryoids but not in undifferentiated tissues point to weak but distinct embryo specific expression of gene(s) governing the biosynthesis of petroselinic acid.

Comparison of fatty acid composition and astaxanthin content in healthy and by M74 affected salmon eggs from three Swedish river stocks

M74 is an extremely lethal syndrome appearing during the yolk sac-swim up stage. The syndrome is known in the Baltic Sea salmon but is still unknown in Atlantic salmon. In this study, Baltic salmon eggs from females showing M74 syndrome were compared with those from healthy females. Further, the study also included a comparison between eggs from a western (Atlantic) and two eastern (Baltic) Swedish stocks. We found differences between the Atlantic and Baltic stocks in the content of 22:6 (n-3) both in the phospholipid and triacylglycerol lipid fractions (P<0.013, P<0.0001, respectively). In addition, the eggs from the Baltic stocks had a much lower astaxanthin content than the west coast stock (P<0.0001). In the phospholipid fraction, an inverse relation was found between river temperature and 22:6 (n-3) content, when rivers were arranged according to day degrees during egg development. M74 affected eggs had a higher content of 22:6 (n-3) compared to non-affected M74 ones. We found differences in the (n-3)/(n-6) ratio between healthy and M74 eggs (P<0.0046). Also, the astaxanthin content was significantly lower in M74 eggs (P<0.0018). We suggest that egg lipid composition is under strong genetic influence. Further, we suggest that in healthy eggs of Baltic salmon, there is a balance between oxidative potential and antioxidant capacity and that a disturbance of this balance at the cellular level could possibly be a cause of the M74 syndrome.

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 fatty acid patterns in developing seed, leaf, root, and in tissue culture initiated from embryos of Daucus carota L

Abstract Accumulation of lipids in different development stages of carrot seed and other tissues were studied. The fatty acids were determined in two fractions, the triacylglycerols (TAGs) and total polar lipids using a polar capillary column gas chromatography system. In the early stage, the triacylglycerols had high percentage, approx. 43%, of linoleic acid and almost equal proportions of myristic, palmitic, petroselinic, oleic and linolenic acids. Petroselinic acid content increased rapidly up to approx. 3 weeks after pollination and then reached a steady state of approx. 75%. Linoleic acid was the major component in total polar lipids at both early and mature stage, approx. 39% and 32%. Linolenic acid decreased from approx. 27% at early to approx. 3% at mature stage with concomitant increase in oleic and petroselinic acids. In roots, both triacylglycerols and total polar lipids had linoleic acid as major fatty acid, 75% and 69%, respectively. No petroselinic acid was found in leaves before flowering. However, a very small amount of petroselinic acid was found besides the predominant linoleic and linolenic acids in the triacylglycerol fraction in leaf lipids of plants carrying maturing seed. Seed coats had 45% petroselinic acid in triacylglycerol but only 1.8% in total polar lipids. Somatic embryos initiated from developing seeds of domestic carrot failed to accumulate any detectable amount of petroselinic acid in the lipids.

Lipid bodies in tissue culture, somatic and zygotic embryo of Daucus carota L.: a qualitative and quantitative study

Abstract Qualitative and quantitative electron microscopic examinations were made to study lipid body accumulation in zygotic embryos and in cells and tissues of Daucus carota L. cultured in vitro. Lipid bodies were present in all types of tissue analysed. The somatic embryos, callus and cells in suspension culture had lipid bodies rather similar in appearance and no protein bodies were detected. A few lipid bodies which were indistinct and diffuse in structure without any bordering membrane, perhaps were the so-called nascent lipid bodies. Quantitative analysis using an image analysis system showed that the torpedo-shaped somatic embryos had a significantly higher proportion of lipid body area/section area than callus cells, cells in suspension culture, and other early stages of somatic embryos. Somatic embryos grown in media with different supplements had higher numbers of lipid bodies as well as a higher proportion of lipid body area/section area compared to torpedo-shaped embryos grown in basal medium containing 2% sucrose. Zygotic embryos 17 days after pollination had an ultrastructure similar to that of somatic embryos. Late stage embryos 45 days after pollination were very rich in lipid bodies and ultrastructurally similar to other common oil-bearing seeds.

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 β 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.

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-β-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-β-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.