13 C Nuclear Magnetic Resonance Spectra of Natural Undiluted Lipids: Docosahexaenoic-Rich Phospholipid and Triacylglycerol from Fish C. LEIGH BROADHURST, ² WALTER F. SCHMIDT,* ,² MICHAEL A. CRAWFORD, ‡ YIQUN WANG, ‡ AND RONG LI § Nuclear Magnetic Resonance Facility, Environmental Quality Laboratory, Agricultural Research Service, U.S. Department of Agriculture, Beltsville, Maryland 20705; Institute of Brain Chemistry and Human Nutrition, London Metropolitan University, London N7 8DB, United Kingdom; and Department of Nutrition and Food Science, University of Maryland, College Park, Maryland 20742 In the 13 C NMR spectra of natural, unenriched docosahexaenoic acid-rich sardine oil and cod muscle glycerophosphocholine significant signal intensity differences across the 1D spectrum between undiluted and diluted samples were observed. In undiluted samples 13 C- 13 C 2D nuclear Overhauser enhancement spectroscopy (NOESY) interchain cross-peaks for CH, CH 2 , CH 3 , and CdC structures were observed. Results indicate that in undiluted natural lipids, NMR signal intensity is influenced by polarization transfer from the extended lipid structure. The NOE enhancement of specific molecular sites especially in unsaturated lipids is evidence that some natural lipids remain oriented relative to each other and in an orderly arrangement at the molecular level long enough for the effect to be detected by the NMR experiment. The presence of polyunsaturated fatty acids in mixtures of natural lipids could stabilize specific local molecular conformations within the remaining less saturated lipids. KEYWORDS: 13 C- 13 C 2D NOESY; 13 C NMR; polarization transfer; polyunsaturated lipids; fish oil; fish phospholipid; glycerophosphocholine; triacylglycerol INTRODUCTION Natural triacylglycerols and phospholipids are high molecular weight viscous liquids. Closeness among the constituent lipid chains within triacylglycerols and phospholipids and between adjacent lipid molecules can result in two distinct distinguishable hydrophobic phases detectable by 13 C nuclear magnetic reso- nance (NMR) (1). In the absence of dilution with deuterated solvents, the 1 H frequencies in these lipids are poorly resolved overlapping peaks. Line broadening limits the ability to iden- tify and assign sites close in space using two dimensional (2D) 1 H- 1 H chemical exchange nuclear Overhauser enhancement spectroscopy (NOESY) experiments. Diluting lipid samples with solvent sharpens the proton spectrum but innately alters the relative distances between sites within and between lipid structures. In binary mixtures of phospholipids with cholesterol, proton- proton broadening was dramatically improved and intramolecu- lar cross-peaks were identified with 2D 1 H magic angle spinning NOESY techniques (2). Other NMR techniques including deuterium labeling of lipids can provide useful information (3). However, because the proton and deuterium sites are structurally and/or conformationally redundant, it cannot be determined which of multiple sites are close in space within a given sample (4). Two-dimensional heteronuclear 1 H- 31 P techniques enable quantitative analysis of constituent phospholipids in membranes, but conformational information using heteronuclear NOESY is innately limited to short distances close to the phosphate headgroup (5). The intensity of 13 C peaks in a one-dimensional (1D) ex- periment depends on polarization transfer from 1 H to 13 C atoms that are close in spacesthe NOE effect (6). For quantitative analysis, long recycle delays in highly diluted lipids are often used to minimize NOE-related intensity differences (7). There- fore, maximizing the NOE effect is a means whereby conforma- tion and closeness in space within lipid structures can in principle be detected. The standard 2D NOE pulse sequence (6, 9) using undiluted samples could enable detection of NOE- related changes in peak intensity because slowly relaxing sites are potential reservoirs of polarization that could be transferred to more rapidly relaxing sites. Thus, proton-proton-carbon NOE transfers occurring during the same time interval as relaxation would be detectable in appropriate 2D experiments; the product operators that define the 2D experiments are identical whether the spin 1 / 2 nuclei is 1 H or 13 C(8). High- resolution 13 C spectra can be obtained in only 32 scans in undiluted lipid, which makes this approach both feasible and practical. Assignment of the sites at which proton-proton- * Author to whom correspondence should be addressed [fax (301) 504- 5992; e-mail schmidtw@ba.ars.usda.gov]. ² U.S. Department of Agriculture. ‡ London Metropolitan University. § University of Maryland. 4250 J. Agric. Food Chem. 2004, 52, 4250-4255 10.1021/jf0353178 CCC: $27.50 © 2004 American Chemical Society Published on Web 05/25/2004