An improved method for rapid analysis of the fatty acids of glycerolipids

Ichihara, Kazuo; Shibahara, Akira; Yamamoto, Kohei; Nakayama, Takao

doi:10.1007/bf02522648

Cited by 377 publications

(163 citation statements)

References 8 publications

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“…After preincubation of embryos for 20 min under light of 35 mmoles/cm 2 /s, temperature at 24°C, and relative humidity of 35% with constant shaking, the labeling of 26 embryos per sample was started by replacing the 1 mL preincubation medium with 1 mL fresh culture medium containing [1-14 C]-acetate (0.5 mM) or [1,[3][4][5][6][7][8][9][10][11][12][13][14] C] glycerol (0.2 mM) in a 2-mL Eppendorf tube (Eppendorf North America). For each time point, the labeling reaction was stopped by removing the 1.0 mL culture medium containing radioactive substrate and freezing the embryos in the tube immediately in liquid N. For [ 14 C]glycerol-labeled lipid classes including PC, DAG, and TAG, the proportion of label in the acyl chains versus the backbone was determined by base-catalyzed transmethylation of TLC-separated and purified lipids and scintillation counting of the separated organic and aqueous phases (Ichihara et al, 1996).…”

Section: [ 14 C]acetate and [ 14 C]glycerol Labelingmentioning

confidence: 99%

Phospholipase Dζ Enhances Diacylglycerol Flux into Triacylglycerol

Yang

Wang

et al. 2017

Plant Physiol.

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Plant seeds are the primary source of triacylglycerols (TAG) for food, feed, fuel, and industrial applications. As TAG is produced from diacylglycerol (DAG), successful engineering strategies to enhance TAG levels have focused on the conversion of DAG to TAG. However, the production of TAG can be limited by flux through the enzymatic reactions that supply DAG. In this study, two Arabidopsis phospholipase D z genes (AtPLD z1 and AtPLD z2 ) were coexpressed in Camelina sativa to test whether the conversion of phosphatidylcholine to DAG impacts TAG levels in seeds. The resulting transgenic plants produced 2% to 3% more TAG as a component of total seed biomass and had increased 18:3 and 20:1 fatty acid levels relative to wild type. Increased DAG and decreased PC levels were examined through the kinetics of lipid assembly by [ 14 C]acetate and [ 14 C]glycerol incorporation into glycerolipids. [ 14 C]acetate was rapidly incorporated into TAG in both wild-type and overexpression lines, indicating a significant flux of nascent and elongated acyl-CoAs into the sn-3 position of TAG. Stereochemical analysis revealed that newly synthesized fatty acids were preferentially incorporated into the sn-2 position of PC, but the sn-1 position of de novo DAG and indicated similar rates of nascent acyl groups into the Kennedy pathway and acyl editing. [ 14 C]glycerol studies demonstrated PC-derived DAG is the major source of DAG for TAG synthesis in both tissues. The results emphasize that the interconversions of DAG and PC pools can impact oil production and composition.

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Section: [ 14 C]acetate and [ 14 C]glycerol Labelingmentioning

confidence: 99%

Phospholipase Dζ Enhances Diacylglycerol Flux into Triacylglycerol

Yang

Wang

et al. 2017

Plant Physiol.

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“…Before the fatty acid extraction, C23:0 (Nu-Chek Prep Inc. Elysian, MN, USA) was added to the samples as an internal standard. The meat samples were methylated according to Ichihara et al (1996) and the pasture samples according to Hartman and Lago (1973). The samples were analyzed with a gas chromatograph (GC-2010 Plus; Shimadzu®, Kyoto, Japan) equipped with an FID detector.…”

Section: Fatty Acid Compositionmentioning

confidence: 99%

Finishing strategies for steers based on pasture or silage plus grain and time on feed and their effects on beef quality

et al. 2015

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The physicochemical and sensory attributes and the fatty acid composition of the longissimus lumborum muscle were determined. The beef from pasture-fed animals tended to be tenderer, darker, less red, and with yellower fat than the beef from grain-fed steers. The beef from steers fed wheat plus silage for 75 d had lower tenderness scores than beef from steers fed for 35 d on grain plus silage. The beef from pasture-fed steers had higher levels of conjugated linoleic acid c-9 t-11 and n-3 fatty acids and a lower n-6:n-3 fatty acid ratio than the beef from grain-fed steers. The time fed on grain plus silage had a significant effect on the fatty acid composition of the beef from steers fed wheat, but no similar effect was observed in beef from steers fed oats. However, the n-6:n-3 ratio of beef was more favorable when steers were fed grain (wheat or oats) plus silage for 35 d than for 75 d.

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“…One fraction was used to measure total radioactivity by scintillation counting. The second fraction was subjected to base transmethylation (Ichihara et al, 1996). Lipids were vortexed with 2 M KOH in methanol (0.6 mL) plus hexane (4 mL) for 3 min at room temperature (Ichihara et al, 1996).…”

Section: Individual Glycerolipid Analysismentioning

confidence: 99%

“…The second fraction was subjected to base transmethylation (Ichihara et al, 1996). Lipids were vortexed with 2 M KOH in methanol (0.6 mL) plus hexane (4 mL) for 3 min at room temperature (Ichihara et al, 1996). The reaction was quenched by adding 4 mL of 1 M aqueous acetic acid.…”

Section: Individual Glycerolipid Analysismentioning

confidence: 99%

Metabolic Responses to the Reduction in Palmitate Caused by Disruption of the FATB Gene in Arabidopsis

et al. 2004

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Disruption of the FATB gene in Arabidopsis results in a two-thirds reduction in saturated fatty acids, largely palmitate, in the leaf extra-plastidic phospholipids and a reduction in the growth rate of the mutant compared to wild type (Bonaventure G, Salas JJ, Pollard MR, Ohlrogge JB [2003] Plant Cell 15: 1020-1033. In this study, we report that although fatb-ko seedlings grow more slowly than wild type, the rate of fatty acid synthesis in leaves of the mutant increases by 40%. This results in approximately the same amount of palmitate exported from the plastid as in wild type but an increase in oleate export of about 55%. To maintain constant amounts of fatty acids in leaves, thereby counterbalancing their higher rate of production, the mutant also increases its rate of fatty acid degradation. Although fatb-ko leaves have higher rates of fatty acid synthesis and turnover, the relative proportions of membrane lipids are similar to wild type. Thus, homeostatic mechanisms to preserve membrane compositions compensate for substantial changes in rates of fatty acid and glycerolipid metabolism in the mutant. Pulse-chase labeling studies show that in fatb-ko leaves there is a net increase in the synthesis of both prokaryotic and eukaryotic lipids and consequently of their turnover. The net loss of palmitate from phosphatidylcholine plus phosphatidylethanolamine is similar for wild type and mutant, suggesting that mechanisms are not present that can preferentially preserve the saturated fatty acids. In summary, the leaf cell responds to the loss of saturated fatty acid production in the fatb-ko mutant by increasing both fatty acid synthesis and degradation, but in doing so the mechanisms for increased fatty acid turnover contribute to the lowering of the percentage of saturated fatty acids found in eukaryotic lipids.In plants, the major site for de novo fatty acid synthesis (FAS) occurs in the plastid (Ohlrogge et al., 1979). Fatty acids are either utilized in this organelle or exported to supply diverse cytoplasmic biosynthetic pathways and cellular processes. Production of fatty acids for export depends on the activity of acyl-ACP thioesterases (FATs) that hydrolyze acyl-acyl carrier protein (acyl-ACP) to release free fatty acids and ACP (for review, see Voelker et al., 1997). After export, the free fatty acids are re-esterified to CoA to form the cytosolic acyl-CoA pool (Pollard and Ohlrogge, 1999). In mesophyll cells, acyl-CoAs are primarily used for the biosynthesis of membrane glycerolipids in the endoplasmic reticulum (Browse and Somerville, 1991). However, in other cell types exported fatty acids have different fates. For example, in embryo cells of oilseeds, the major fraction is incorporated into triacylglycerols, while in epidermal cells a large fraction is utilized for the synthesis of waxes and cutin (Post-Beittenmiller, 1996;Kolattukudy, 2003). Furthermore, all cells synthesize sphingolipids (Lynch, 1993), and we recently estimated that as much as 30% to 40% of exported palmitate is needed for sphingoid ba...

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An improved method for rapid analysis of the fatty acids of glycerolipids

Cited by 377 publications

References 8 publications

Phospholipase Dζ Enhances Diacylglycerol Flux into Triacylglycerol

Phospholipase Dζ Enhances Diacylglycerol Flux into Triacylglycerol

Finishing strategies for steers based on pasture or silage plus grain and time on feed and their effects on beef quality

Metabolic Responses to the Reduction in Palmitate Caused by Disruption of the FATB Gene in Arabidopsis

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