title: Isolation of α-linolenic acid biohydrogenation products by combined silver ion solid phase extraction and semi-preparative high performance liquid chromatography.
abstract: Polyunsaturated fatty acids typically found in cattle feed include linoleic (LA) and α-linolenic acid (ALA). In the rumen, microbes metabolize these resulting in the formation of biohydrogenation products (BHP), which can be incorporated into meat and milk. Bioactivities of LA-BHP, including conjugated linoleic acid (cis (c) 9,trans (t) 11-18:2 and t10,c12-18:2) and trans fatty acid isomers (t9-, t10- and t11-18:1) have been investigated, but effects of several BHP unique to ALA have not been extensively studied, and most ALA-BHP are not commercially available. The objective of the present research was to develop methods to purify and collect ALA-BHP using silver ion (Ag(+)) chromatography in sufficient quantities to allow for convenient bioactivity testing in cell culture. Fatty acid methyl esters (FAME) were prepared from perirenal adipose tissue from a cow enriched with ALA-BHP by feeding flaxseed. These were applied to Ag(+)-solid phase extraction, and eluted with hexane with increasing quantities of acetone (1, 2, 10, 20%) or acetonitrile (2%) to pre-fractionate FAME based on degree of unsaturation and double bond configuration. Fractions were collected, concentrated and applied to semi-preparative Ag(+)-high performance liquid chromatography (HPLC) for the isolation and collection of purified isomers, which was accomplished using isocratic elutions with hexane containing differing amounts of acetonitrile (from 0.015 to 0.075%). Purified trans-18:1 isomers collected ranged in purity from 88 to 99%. Purity of the ALA-BHP dienes collected, including c9,t13-18:2, t11,c15-18:2 and t10,c15-18:2, exceeded 90%, while purification of other dienes may require the use of other complementary procedures (e.g. reverse phase HPLC).
authors.affiliation: Agriculture and Agri-Food Canada, Lacombe Research Centre, 6000 C & E Trail, Lacombe, AB, Canada T4L 1W1; Thompson Rivers University, 900 McGill Road, Kamloops, BC, Canada V2C 0C8.
authors.forename: T D
authors.initials: TD
authors.lastname: Turner
authors.affiliation: Agriculture and Agri-Food Canada, Lacombe Research Centre, 6000 C & E Trail, Lacombe, AB, Canada T4L 1W1.
authors.forename: W J
authors.initials: WJ
authors.lastname: Meadus
authors.affiliation: Department of Animal Sciences, Faculty of AgriSciences, Stellenbosch University, P. Bag X1, Matieland 7602, South Africa.
authors.forename: C
authors.initials: C
authors.lastname: Mapiye
authors.affiliation: Agriculture and Agri-Food Canada, Lacombe Research Centre, 6000 C & E Trail, Lacombe, AB, Canada T4L 1W1.
authors.forename: P
authors.initials: P
authors.lastname: Vahmani
authors.affiliation: Livestock Gentec, 1400 College Plaza 8215 112 Street, Edmonton Alberta, Canada T6G 2C8.
authors.forename: Ó
authors.initials: Ó
authors.lastname: López-Campos
authors.affiliation: Agriculture and Agri-Food Canada, Lacombe Research Centre, 6000 C & E Trail, Lacombe, AB, Canada T4L 1W1.
authors.forename: P
authors.initials: P
authors.lastname: Duff
authors.affiliation: Agriculture and Agri-Food Canada, Lacombe Research Centre, 6000 C & E Trail, Lacombe, AB, Canada T4L 1W1.
authors.forename: D C
authors.initials: DC
authors.lastname: Rolland
authors.affiliation: Thompson Rivers University, 900 McGill Road, Kamloops, BC, Canada V2C 0C8.
authors.forename: J S
authors.initials: JS
authors.lastname: Church
authors.affiliation: Agriculture and Agri-Food Canada, Lacombe Research Centre, 6000 C & E Trail, Lacombe, AB, Canada T4L 1W1. Electronic address: duganm@agr.gc.ca.
authors.forename: M E R
authors.initials: ME
authors.lastname: Dugan
mesh_terms: D002851:Chromatography, High Pressure Liquid / Q000379:methods*; D044243:Linoleic Acids, Conjugated / Q000302:isolation & purification; D052616:Solid Phase Extraction / Q000379:methods*; D017962:alpha-Linolenic Acid / Q000302:isolation & purification*
chemical_list: D044243:Linoleic Acids, Conjugated; D017962:alpha-Linolenic Acid
keywords: Ag(+)-HPLC; Ag(+)-SPE; Biohydrogenation; α-Linolenic acid