A simplified protocol to obtain fatty acid methyl esters (FAME) directly from fresh tissue, oils, or feedstuffs, without prior organic solvent extraction, is presented. With this protocol, FAME synthesis is conducted in the presence of up to 33% water. Wet tissues, or other samples, are permeabilized and hydrolyzed for 1.5 h at 55 degrees C in 1 N KOH in MeOH containing C13:0 as the internal standard. The KOH is neutralized, and the FFA are methylated by H(2)SO(4) catalysis for 1.5 h at 55 degrees C. Hexane is then added to the reaction tube, which is vortex-mixed and centrifuged. The hexane is pipetted into a gas chromatography vial for subsequent gas chromatography. All reactions are conducted in a single screw-cap Pyrex tube for convenience. The method meets many criteria for fatty acid analysis, including not isomerizing CLA or introducing fatty acid artifacts. It is applicable to fresh, frozen, or lyophilized tissue samples, in addition to oils, waxes, and feedstuffs. The method saves time and effort and is economical when compared with other methods. Its unique performance, including easy sample preparation, is achieved because water is included rather than eliminated in the FAME reaction mixtures.
A quantitative calculation was made of the pentose phosphate pathway (PPP) activity in preimplantation mouse embryos from the 2-cell through the late blastocyst stage. This activity varied with development and showed repeated high and low values. Peak activities occurred at both the 2-cell (15.8%) and compacted morula (13.6%) stages, with lowest activity at the development of the late blastocyst (3.2%). The metabolic effectors dimitrophenol (DNP) and phenazine ethosulfate (PES) had opposite effects on PPP activity. Dinitrophenol, although stimulating total CO2 production, virtually eliminated PPP activity while PES stimulated the pentose cycle activity 6-fold. These results indicated that the PPP was under metabolic control and that the embryos had a potential for much larger PPP activities. There was no correlation between the C-1/C-6 ratio obtained from the metabolism of [1-14C] and [6-14C] glucose and calculated PPP activities. A metabolic incubation chamber was devised for these experiments that exhibited certain unique features, including continuous collection of 14CO2 and 3H2O. Single embryos were placed in the chamber and sampled momentarily for metabolic activity. Subsequently, such embryos were successfully transferred to pseudopregnant recipients.
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