Quantitative 2 H NMR analysis of deuterium distribution in petroselinic acid isolated from parsley seed Se´bastien Guiet a , Richard J. Robins a , Michele Lees b , Isabelle Billault a, * a Isotopic Fractionation in Metabolism Group, LAIEM, CNRS UMR6006, University of Nantes, BP 99208, F-44322 Nantes, France b Eurofins Scientific, rue Pierre Adolphe Bobierre, BP 42301, F-44323 Nantes, France Received 14 January 2003; received in revised form 18 March 2003 Dedicated to the memory of Professor Jeffrey B. Harborne Abstract We have previously demonstrated that 2 H distribution in fatty acids is non-statistical and can be related to isotopic discrimina- tion during chain extension and desaturation. Petroselinic acid (C18:1 6 ), a fatty acid characteristic of the seeds of the Apiaceae, has been shown to be biosynthesised from palmitoyl-ACP (C16:0) by two steps, catalysed by a dedicated  4 -desaturase and an elongase. We have now demonstrated that the isotopic profile resulting from this pathway is similar to that of the classical plant fatty acid pathway but that the isotopic fingerprint from both the desaturase and elongase steps show important differences relative to oleic and linoleic acid biosynthesis. # 2003 Elsevier Ltd. All rights reserved. Keywords: Petroselinum crispum; A.W. Hill; Apiaceae; Dehydrogenation; 2 H NMR; 2 H Distribution; Fatty acids; Isotope effects; Petroselinic acid 1. Introduction Petroselinic acid (C18:1 6 ) is a fatty acid character- istic of the seeds of the Apiaceae, where it typically accumulates as the major (up to 80%) storage FA in the seed (Kleinman and Spencer, 1982). While there is no reason not to assume the  6 -desaturation to be intro- duced by a  6 -desaturase equivalent to the  9 -desatur- ase responsible for the formation of oleic acid (C18:1 9 ), this has recently been proved not to be the case. Rather, petroselinic acid is formed by a unique pathway, in which five specific enzymes have been iden- tified. Critically, the biosynthesis differs from that of oleic acid in that the desaturation is introduced prior to elongation. Thus, a specific  4 -desaturase acts on palmitoyl-ACP to produce  4 -hexadecenoyl-ACP (C16:1 4 )(Cahoon et al., 1994), itself the substrate for a dedicated elongase that produces petroselinoyl-ACP. In the elongation, specificity appears related to the properties of the 3-ketoacyl-ACP synthase (KAS) (Mekhedovetal.,2001).Thisstepisfollowedbytwosteps of reduction, comparable to the fatty acid synthase (FAS) responsiblefortheC2toC16chainelongation(Harwood, 1997; Ohlrogge and Browse, 1995). In either case, elonga- tioninvolvestheincorporationofhydrogenatomsintothe chain,eitherfrommalonyl-CoAorfromNAD(P)H+H + . It has previously been shown that the hydrogen atoms in different positions of plant long-chain fatty acids contain different levels of 2 Handthatthedistributionof 2 H is non-statistical (Royer et al., 1999; Que´merais et al., 1995; Billault et al., 2001; Duan et al., 2002). Fur- thermore, this distribution can largely be explained in terms of the origin of individual hydrogen atoms and the kinetic isotope effects (KIEs) intrinsic to the perti- nent enzymes (Billault et al., 2001; Duan et al., 2002; Robins et al., 2003). Notably, during the 3-ketoacyl- reductase and enoyl reductase steps, two hydrogen atoms are incorporated from NAD(P)H+H + at each uneven position of the chain and one at each odd posi- tion. The other odd-position hydrogen is derived from malonyl-CoA. Thus, positional variation in the (D/H) i 0031-9422/03/$ - see front matter # 2003 Elsevier Ltd. All rights reserved. doi:10.1016/S0031-9422(03)00278-4 Phytochemistry 64 (2003) 227–233 www.elsevier.com/locate/phytochem Abbreviations: ACP, acyl carrier protein; FA, fatty acid; FAME, fatty acid methyl ester; FAS, fatty acid synthase complex; KAS, 3- ketoacyl-ACP synthase; KIE, kinetic isotope effect; LAME, linoleic acid methyl ester; OAME, oleic acid methyl ester; PAME, petroselinic acid methyl ester. * Corresponding author. Tel.: +332-5112-5709; fax: +332-5112- 5712. E-mail address: isabelle.billault@chimbio.univ-nantes.fr (I. Billault).