METHODS Method to Produce 9(S)-Hydroperoxides of Linoleic and Linolenic Acids by Maize Lipoxygenase

Seed from maize (corn) Zea mays provides a ready source of 9-lipoxygenase that oxidizes linoleic acid and Iinolenic acid into 9(5)-hydroperoxy-1 0(8, 12(Zl-octadecadienoic acid and 9(SJ-hydroperoxy-1 0(£), 12(Zj, 15(Zl-octadecatrienoic acid, respectively. Corn seed has a very active hydroperoxide-decomposing enzyme, allene oxide synthase (AOS), which must be removed prior to oxidizing the fatty acid. A simple pH 4.5 treatment followed by centrifugation removes most of the AOS activity. Subsequent purification by ammonium sulfate fractional precipitation results in negligible improvement in 9-hyclroperoxide formation. This facile alternative method of preparing 9-hydroperoxides has advantages over other commonly used plant lipoxygenases. Paper no. L8749 in Lipids 36,529-533 (May 2001). Oxylipin research often requires the use of 9(S)-hydroperoxy1O(E),12(Z)-octadecadienoic acid [9S-HPODE: 9-HPODE is the abbreviation for stereochemically undefined 9-hydroperoxy-1O(E),12(Z)-octadecadienoic acid]. In our laboratory, 9SHPODE was routinely produced by oxidation of linoleic acid by 9-lipoxygenase (9-LOX) from a tomato fruit extract (1). Although tomato fruit is readily available, this method has a few disadvantages. Extraction of the product by chloroform as outlined by the method often gives intractable emulsions, and a variety of carotenoids co-elute by chromatographic clean-up of 9S-HPODE. Further. the use of certain varieties of tomato has given us amounts of 13(S)-hydroperoxy9(Z), 11 (E)-octadecadienoic acid as high as 15%. In addition, the I-mM solution of linoleic acid recommended (280 mglL) gives only partial conversion to 9S-HPODE. Probably because potato is a readily available commodity, potato 9-LOX has often been used to form 9S-HPODE (2). Our laboratory has limited experience with potato 9-LOX, but some purification (2) would be required in view of the biotransformations of 9S-HPODE known to occur in crude potato extracts (3,4). Although recombinant 9-LOX cloned into bacteria are known. these cultures must be maintained. "'To whom correspondence should be addressed at NCAUR. ARS. USDA. 1815 N. University St.. Peoria.IL 61604. E-mail: [email protected] Abbreviations: AOS. allene oxide synthase: CP-HPLC. chiral-phase highperformance liquid chromatography: EI. electron impact: FID-GC. llameionization detection-gas chromatography: GC-MS. gas chromatographymass spectrometry: 9S-HPODE. 9(S)-hydroperoxy-1 0(E).12(Z)-octadecadienoic acid: 9S-HPOTE. 9(S)-hydroperoxy-1 0(E).12(Z).15(Z)-octadecatrienoic acid: 9-LOX, 9-lipoxygenase: mes, 2-(N-morpholino)ethane sulfonic acid: OTMSi. trimethylsilyloxy: SP-HPLC. straight-phase high-perfomlance liquid chromatography. Zea mays, maize (corn), seed is another readily available commodity found worldwide. Although LOX from corn was the first 9-LOX identified (5), it has the disadvantage of possessing a highly efficient enzyme. allene oxide synthase (AOS, formerly known as hydroperoxide isomerase). AOS immediately transforms 9S-HPODE into 9,10and 1O,13-ketols. AOS can be mostly removed by selective ammonium sulfate precipitation to such an extent that 95-HPODE can be isolated (6), but further improvement in the ease of preparation is desirable. The germs of other cereals. such as rice (7), barley (8) and wheat (9), contain 9-LOX; however, multistep partial purification of these 9-LOX is required. In this report alternative methods are described to oxidize linoleic acid or linolenic acid into 95-HPODE or 9(5)hydroperoxy-l O(E), 12(Z), 15(Z)-octadecatrienoic acid (95HPOTE), respectively, by facile procedures using either corn germ (embryonic axis plus scutellum) or whole corn seed. MATERIALS AND METHODS Materials. The seed source was Z. mays, commonly known as maize (international terminology) or corn (U.S.A. terminology). Common field corn, otherwise known as "dent corn," was used. Corn seed preferably was from the most recent harvest. Ear corn, that is, "corn-on-the-cob," is recommended. The cob, upon which the kernels are attached, is a specialized receptacle with archaic origins from a stalk or tassel. In the past, corn was stored in bins as ear corn and permitted to airdry. Recently, most corn grain is harvested by a combined picker/sheller, and occasionally this grain is dried with heated air. Although not proven, it is believed that ear corn may be a more reliable product, not unduly subjected to variable heat conditions. Ear corn was purchased from a local animal feed store, and then the seed was shelled from the cob by hand. Almost all of the 9-LOX activity resides in the germ, whereas the endosperm has negligible activity (5). By weight. the germ makes up 10 to 14% of the total kernel and comprises several anatomically distinct tissues that are subcategorized by the general terminology of the embryonic axis plus the scutellum. The germ is identified as an oval depression on one side of the kernel that extends from the base of the kernel to about two-thirds upward. For a detailed explanation of germ tissue, consult the microscopic work of Wolf et al. (10). For those investigators who do not choose the tedium of hand-dissecting germ, an alternative method using whole corn kernels is outlined below (Procedure 3). Copyright lid 2001 by AGCS Press 529 Lipids, Vol. 36, no. 5 (2001)