title: Compound-specific stable isotope analysis of nitrogen-containing intact polar lipids.
abstract: BACKGROUND
  Compound-specific isotope analysis (CSIA) of nitrogen in amino acids has proven a valuable tool in many fields (e.g. ecology). Several intact polar lipids (IPLs) also contain nitrogen, and their nitrogen isotope ratios have the potential to elucidate food-web interactions or metabolic pathways. Here we have developed novel methodology for the determination of δ(15)N values of nitrogen-containing headgroups of IPLs using gas chromatography coupled with isotope-ratio mass spectrometry.
  
  
  METHODS
  Intact polar lipids with nitrogen-containing headgroups were hydrolyzed and the resulting compounds were derivatized by (1) acetylation with pivaloyl chloride for compounds with amine and hydroxyl groups or (2) esterification using acidified 2-propanol followed by acetylation with pivaloyl chloride for compounds with both carboxyl and amine groups. The δ(15)N values of the derivatives were subsequently determined using gas chromatography/combustion/isotope-ratio mass spectrometry.
  
  
  RESULTS
  Intact polar lipids with ethanolamine and amino acid headgroups, such as phosphatidylethanolamine and phosphatidylserine, were successfully released from the IPLs and derivatized. Using commercially available pure compounds it was established that δ(15)N values of ethanolamine and glycine were not statistically different from the offline-determined values. Application of the technique to microbial cultures and a microbial mat showed that the method works well for the release and derivatization of the headgroup of phosphatidylethanolamine, a common IPL in bacteria.
  
  
  CONCLUSIONS
  A method to enable CSIA of nitrogen of selected IPLs has been developed. The method is suitable for measuring natural stable nitrogen isotope ratios in microbial lipids, in particular phosphatidylethanolamine, and will be especially useful for tracing the fate of nitrogen in deliberate tracer experiments.
authors.affiliation: Department of Marine Organic Biogeochemistry, NIOZ Royal Netherlands Institute for Sea Research, P.O. Box 59, 1790 AB, Den Burg (Texel), The Netherlands.
authors.forename: Elisabeth
authors.identifier: http://orcid.org/0000-0002-5565-3266
authors.initials: E
authors.lastname: Svensson
authors.affiliation: Department of Marine Organic Biogeochemistry, NIOZ Royal Netherlands Institute for Sea Research, P.O. Box 59, 1790 AB, Den Burg (Texel), The Netherlands.|Department of Earth Sciences, Faculty of Geosciences, Utrecht University, P.O. Box 80021, 3508 TA, Utrecht, The Netherlands.
authors.forename: Stefan
authors.initials: S
authors.lastname: Schouten
authors.affiliation: Department of Marine Organic Biogeochemistry, NIOZ Royal Netherlands Institute for Sea Research, P.O. Box 59, 1790 AB, Den Burg (Texel), The Netherlands.
authors.forename: Axel
authors.initials: A
authors.lastname: Stam
authors.affiliation: Department of Earth Sciences, Faculty of Geosciences, Utrecht University, P.O. Box 80021, 3508 TA, Utrecht, The Netherlands.
authors.forename: Jack J
authors.initials: JJ
authors.lastname: Middelburg
authors.affiliation: Department of Marine Organic Biogeochemistry, NIOZ Royal Netherlands Institute for Sea Research, P.O. Box 59, 1790 AB, Den Burg (Texel), The Netherlands.|Department of Earth Sciences, Faculty of Geosciences, Utrecht University, P.O. Box 80021, 3508 TA, Utrecht, The Netherlands.
authors.forename: Jaap S
authors.initials: JS
authors.lastname: Sinninghe Damsté
mesh_terms: D000596:Amino Acids / Q000032:analysis*; D001419:Bacteria / Q000737:chemistry; D019856:Ethanolamine / Q000032:analysis*; D008401:Gas Chromatography-Mass Spectrometry / Q000379:methods*; D006868:Hydrolysis; D008055:Lipids / Q000737:chemistry*; D009584:Nitrogen / Q000032:analysis*; D009587:Nitrogen Isotopes / Q000032:analysis; D010714:Phosphatidylethanolamines / Q000737:chemistry; D010718:Phosphatidylserines / Q000737:chemistry
chemical_list: D000596:Amino Acids; D008055:Lipids; D009587:Nitrogen Isotopes; D010714:Phosphatidylethanolamines; D010718:Phosphatidylserines; C483858:phosphatidylethanolamine; D019856:Ethanolamine; D009584:Nitrogen