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Conversion of essential fatty acids by Delta 6 desaturase in dog liver microsomes
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Long Chain Polyunsaturated Fatty Acids (LCPUFA) are physiologically important precursors for eicosanoids, leukotrienes, and prostanoids. Desaturation of essential fatty acids (EFAs) by 6-desaturase is considered the rate-limiting step in conversion of EFAs to LCPUFA. This study was designed to study the conversion of EFAs by 6-desaturase in dog liver microsomes. Microsomes were prepared using fresh liver from healthy dogs. Microsomes were incubated with ¹⁴C labeled 18 carbon EFA substrates. Following incubation, lipids were extracted, saponified and phenacylated. The resulting fatty acid phenacyl esters (FAPES) were separated by HPLC. Radioactivity was measured with a liquid scintillation counter, converted to enzymatic activity, and expressed as pmol/min/mg protein. Using 18:3n-3 as substrate, the apparent 6-desaturase maximal velocity was 50.9 pmol/min/mg protein and using 18:2n-6 substrate, the maximal velocity was 5.4 pmol/min/mg protein. Apparent K values were 20.8 M for 18:3n-3 and 41.8 M for 18:2n-6. Maximal velocities were lower than previously reported in dogs and other species. Possible explanations include the presence of high endogenous fatty acid concentration (especially 18:2n-6) inherent in the dog liver microsome preparations providing high amounts of competitive non-radioactively labeled substrate, or methodological differences used in other studies. After accounting for endogenous 18:2n-6 fatty acids, the corrected V and K for the α-LA substrate was 62.4 pmol/min/mg protein and 12.4 M, respectively. Corrected values of V and K for LA substrate were not calculated due to interference from high endogenous LA ubstrate concentrations in the liver microsomes. Dog liver microsomes have the ability to desaturate EFAs. Also, the maximal velocity of 6-desaturase of 18:3n-3 is considerably higher than that of 18:2n-6 in vitro, while K values are similar. Physiologically, 18:3n-3 concentration in liver (2.4 M) may never exceed the K for desaturation in the absence of high dietary amounts while 18:2n-6 amounts are readily converted because its concentration (64.4 M) easily exceeds the K. These phenomena may explain the low in vivo conversion rate of 18:3n-3 in dogs and other species. These findings suggest that high levels of 18:3n-3 supplementation may be necessary to exceed the 6-desaturase K and significantly affect physiological levels of n-3 LCPUFA in the dog.
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Includes bibliographical references (leaves 72-81).
Issued also on microfiche from Lange Micrographics.
Dunbar, Brent Lee (2001). Conversion of essential fatty acids by Delta 6 desaturase in dog liver microsomes. Master's thesis, Texas A&M University. Available electronically from
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