Earlier assessments of the PUFA biosynthesis pathway have centered on DHA and arachidonic acid synthesis. (Computer-100 microcentrifuge; Diamed, Mississauga, ON, Canada) and the plasma was gathered and kept at ?80C. Perseverance of plasma quantity Plasma quantity was determined utilizing the approach to Schreihofer, Locks. and Stepp (23) and altered by our laboratory (11). Briefly, a known quantity of Evans Blue dye was injected in to the jugular vein of another band of rats (n = 3). 15 minutes following injection, 1 ml of bloodstream was drawn from the carotid artery, two times. The plasma was gathered as Paclitaxel biological activity defined above and 100 l of plasma had been diluted into 1 ml of saline. Absorbance of plasma in saline was motivated at 604 nm with a Nanodrop 1000 and weighed against a typical curve, and the focus of the Evans Blue dye was motivated. Focus of the dye was after that used to find out plasma quantity. Lipid extractions from baseline serum Total lipid extracts (TLEs) for total fatty acid and for unesterified fatty acid measurements had been obtained from 10 and 40 l of serum, respectively, by way of a technique altered from Folch, Lees, and Sloane Stanley (24). Briefly, lipids had been extracted with 2:1:0.75 chloroform:methanol:0.88% potassium chloride (v:v:v) containing 1 and 4 g of heptadecanoic acid (17:0; Nu-Chek Prep, Inc.) as inner regular for unesterified and total essential fatty acids, respectively. The mixtures had been vortexed, centrifuged at 500 for 10 min, and the low chloroform lipid-containing level was pipetted right into a brand-new check tube. TLEs for total Paclitaxel biological activity fatty acid determinations had been kept for hydrolysis as defined afterwards. TLEs for unesterified fatty acid determinations had been evaporated under N2 gas and reconstituted in 50 l of chloroform, plated on a silica 60 G plate (Merck Millipore, Billerica, MA), and created using TLC with 60:40:2 hep-tane:diethyl ether:glacial acetic acid for separation of unesterified essential fatty acids, as previously defined (14). Unesterified essential fatty acids had been scraped from TLC plates and extracted from the silica by the technique described previous. Isolated unesterified fatty acids were stored until derivatization to pentafluorobenzyl (PFB) esters. Lipid extractions from stable isotope-infused plasma TLEs were obtained from 50 l of stable isotope-infused plasma by the Folch method, as described earlier, containing 5 g of heptadecanoic acid (17:0, Nu-Chek Prep, Inc.) Rabbit Polyclonal to PHLDA3 as internal standard. Four-fifths of the TLE was stored for hydrolysis and stable isotope enrichment of total fatty acids, and the remaining one-fifth of the TLE was used for isolation of isotopically labeled unesterified fatty acid by the TLC method described earlier. The isolated labeled unesterified fatty acids were stored for later on derivatization to PFB esters. Hydrolysis of total endogenous and isotopically labeled total fatty acid Paclitaxel biological activity pools The TLE from baseline serum and infused plasma used to determine endogenous and labeled fatty acid concentrations, respectively, was evaporated under nitrogen and the lipids were hydrolyzed in 2 ml of 10% potassium hydroxide in methanol (w:v), as previously explained (10, 14, 25). Hydrolyzed fatty acids from the total lipid pool were collected and stored at ?80C for later derivatization to PFB esters. PFB esterification The plasma unesterified fatty acids and hydrolyzed fatty acids from total lipid pools were dried under nitrogen and.