Conversion of Essential Fatty Acids by Delta 6-Desaturase in Dog Liver Microsomes

Abstract

EXPANDED ABSTRACT [18:3(n-3), ALA] are converted to long-chain polyunsaturated fatty acids (LCPUFAs) by desaturase and chain-elongation enzyme systems (1). The LCPUFAs are important because they serve as eicosanoid precursors. In addition, several LCPUFAs have specific structural and functional roles in development or maintenance of neural tissues such as brain, retina and other tissues (2).The rate-limiting step for desaturation and elongation is controlled by ⌬6-desaturase, which adds a double bond at the sixth carbonyl carbon. Hence, LA is converted to 18:3(n-6) and ALA is converted to 18:4(n-3) and competition between these substrates for this enzymatic step exists among the fatty acid families. Some reports indicate a higher specificity for (n-3) fatty acid desaturation compared to that for (n-6) fatty acids (3).Dogs are important to humans not only as companion animals but also serve as a model for human metabolism (4,5). This study addresses EFA metabolism in dogs using a classical enzyme kinetic approach. MATERIALS AND METHODS AnimalsFresh liver tissues were removed from 11 normal, healthy Coonhound dogs and three Sprague-Dawley rats at the termination of animal use protocols approved by Texas A&M University Laboratory Animal Use Committee. The animals had been fed once daily and were not denied food before euthanasia. Liver microsomes were prepared with rat serving as a positive control group for enzyme activity determinations. Microsomal preparationLiver tissue was immediately transferred to ice-cold saline after collection, blotted dry, minced and rinsed twice with saline. A phosphate buffer (40 mM, Buffer A) containing 0.1 M sucrose (pH 7.4) with tissue-to-buffer ratio of 1:6 was used for homogenization. Homogenates were centrifuged for 20 min at 4°C and 10,000 ϫ g with fixed-angle rotor. Supernates were then centrifuged for 1 h at 4°C and 105,000 ϫ g to pellet the microsomes. Microsomes were resuspended in fresh Buffer A and protein concentrations determined before freezing at Ϫ80°C. Microsomal lipid composition determinationMicrosomes were extracted by the method of Folch et al. (6) with internal standards for phospholipids (PL), nonesterified fatty acids (FFA) and triacylglyerol (TG) containing 22:1(n-9). Lipid subclasses were separated by thin-layer chromatography (TLC) on silica gel plates with 80:20:1 hexane:ether:glacial acetic acid (v/v/v). The PL, FFA and TG were scraped and fatty acid methyl esters (FAME) prepared. Samples were quantified by capillary gas chromatography (GC) on a Restek Stabilwax column (0.32 mm ID ϫ 30 m ϫ 0.25 mm film) with He gas carrier. A temperature program was begun at 170°C, held for 10 min, ramped to 228°C at 2°C/min then held for 20 min. IncubationsIncubation conditions for ALA as substrate in the absence of malonyl-CoA were determined by independently varying C 14 -ALA or C 14 -LA substrate content, protein concentrations and incubation times. Protein (4 mg), 15-min incubation and 50 M ALA substrate concentration were found to be suitable conditions for ⌬6-desatu...

Rice varietal differences in bioactive bran components for inhibition of colorectal cancer cell growth

“…Total lipids were extracted using chloroform:methanol (2:1, v/v) via a modified Folch procedure (Dunbar & Bauer, 2002; Folch, Lees & Stanley, 1957) and the triacylglycerol fraction was subfractionated via thin layer chromatography. Fatty acid methyl esters were prepared after recovery of this fraction and fatty acid profiles were determined via capillary gas chromatography and flame ionization detection (GC-FID) as previously reported (Dunbar et al , 2002).…”

“…Fatty acid methyl esters were prepared after recovery of this fraction and fatty acid profiles were determined via capillary gas chromatography and flame ionization detection (GC-FID) as previously reported (Dunbar et al , 2002). …”

Early and Sustained Enrichment of Serum n‐3 Long Chain Polyunsaturated Fatty Acids in Dogs Fed a Flaxseed Supplemented Diet

“…Because dogs, like humans, can convert either ALA or stearidonic acid to longer chain n-3 derivatives [19,20], the ability of dietary ALA to provide enough EPA to be similarly cardioprotective is unknown. Knowledge of dietary ALA metabolism in dogs will therefore provide a basis for future investigations of the canine arrhythmic model using dietary interventions with n-3 fatty acids.…”

Fat Requirements

“…6 In addition, adult dogs are capable of converting alpha-linolenic to both EPA and to a precursor of DHA, docosapentaenoic acid (DPA), although at relatively low rates of conversion. In dogs, as in humans, the rate of conversion of the parent fatty acid to other LCPUFAs is regulated by delta-6desaturase.…”