In Vivo Analysis of Human Phosphatidylcholine Synthesis and Turnover: Implications For

1 1 1Online Resource 2 to:

2Plasma phospholipids indicate impaired fatty acid homeostasis in preterm infants

4Wolfgang Bernhard1, Marco Raith1, Vera Koch1, Rebecca Kunze1, Christoph Maas1, Harald Abele2,

5Christian F. Poets1, Axel R. Franz1,3

7Affiliations: Departments of 1Neonatology and 2Gynecology, and 3Center for Pediatric Clinical

8Studies, Faculty of Medicine, Eberhard-Karls-University, Tübingen, Germany.

10Address of correspondance: Wolfgang Bernhard, MD PhD, Department of Neonatology, Faculty of

11Medicine, Eberhard-Karls-University, Calwer Straße 7, D-72076 Tuebingen, FRG;

[email protected]; Phone: #-49 7071 29 86377

13Short title: Plasma phospholipid alterations in preterm infants

14 2 2

15ONLINE Resource 2: Molecular species of choline containing phospholipids (phosphatidylcholine

16[PC], lyso-PC and palmitoyl-sphingomyelin [16:0-SPH]), and of phosphatidylethanolamine (PE).

LysoPC12:0 (m/z = +440) PE14:0/14:0 (m/z = +636, internal standard) LysoPC14:0 (m/z = +468) PE16:0/14:0 (m/z = +648) PE14:0/14:0 (m/z = +658, sodiated internal LysoPC16:0 (m/z = +496) standard) LysoPC18:0 (m/z = +524) PE16:0/14:0 (m/z = +664) LysoPC18:1 (m/z = +522) aaPE16:0/16:1 (m/z = +674) LysoPC18:2 (m/z = +520) aaPE16:0/16:0 (m/z = +676) LysoPC20:0 (m/z = +550) PE16:0/16:1 (m/z = +690) LysoPC22:5 (m/z = +570) PE16:0/16:0 (m/z = +692) PC14:0/14:0 (m/z = +678) aaPE16:0/18:3 (m/z = +698) 16:0-SPH (m/z = +703) aaPE16:0/18:2 (m/z = +700) PC14:0/16:0 (m/z = +706) aaPE16:0/18:1 (m/z = +702) PC16:0/16:1 (m/z = +732) aaPE16:0/18:0 (m/z = +704) PC16:0/16:0 (m/z = +734) PE16:0/18:3 (m/z = +714) PC16:0/18:3 (m/z = +756) PE16:0/18:2 (m/z = +716) PC16:0/18:2 (m/z = +758) PE16:0/18:1 (m/z = +718) PC16:0/18:1 (m/z = +760) PE16:0/18:0 (m/z = +720) PC16:0/18:0 (m/z = +762) aaPE16:0/20:5 (m/z = +722) PC14:0/22:6 (m/z = +778) aaPE16:0/20:4 (m/z = +724) PC16:0/20:5 (m/z = +780) aaPE18:1/18:2 (m/z = +726) PC16:0/20:4 (m/z = +782) aaPE18:0/18:2 (m/z = +728) PC18:1/18:2 (m/z = +784) aaPE18:0/18:1 (m/z = +730) PC18:0/18:2 (m/z = +786) aaPE18:0/18:0 (m/z = +732) PC18:0/18:1 (m/z = +788) PE14:0/22:6 (m/z = +736) PC18:0/18:0 (m/z = +790) PE16:0/20:5 (m/z = +738) aaPC16:0/22:6 (m/z = +792) PE16:0/20:4 (m/z = +740) aaPC18:0/20:5 (m/z = +794) PE18:1/18:2 (m/z = +742) PC16:0/22:6 (m/z = +806) * PE18:0/18:2 (m/z = +744) PC18:1/20:4 (m/z = +808) * PE18:0/18:1 (m/z = +746) PC18:0/20:4 (m/z = +810) aaPE16:0/22:6 (m/z = +748) PC18:0/20:5 (m/z = +808) * aaPE18:1/20:4 (m/z = +750) PC20:5/20:5 (m/z = +826) aaPE18:0/20:4 (m/z = +752) 3 3

PC20:4/20:5 (m/z = +828) aaPE18:0/20:3 (m/z = +754) PC18:0/22:6 (m/z = +834) PE16:0/22:6 (m/z = +764) PC18:0/22:5 (m/z = +836) PE18:1/20:4 (m/z = +766) PC18:0/22:4 (m/z = +838) PE18:0/20:4 (m/z = +768) PC20:0/20:0 (m/z = +846, internal standard) PE18:0/20:3 (m/z = +770) PC20:5/22:6 (m/z = +852) aaPE18:0/22:6 (m/z = +776) PC20:0/22:6 (m/z = +862) aaPE18:0/22:5 (m/z = +778) PC22:0/20:5 (m/z = +864) aaPE18:0/22:4 (m/z = +780) PC22:6/22:6 (m/z = +878) PE20:5/20:5 (m/z = +784) PE20:4/20:5 (m/z = +786) PE20:4/20:4 (m/z = +788) PE18:0/22:6 (m/z = +792) PE18:0/22:5 (m/z = +794) PE18:0/22:4 (m/z = +796) PE20:0/20:0 (m/z = +804) aaPE22:0/20:5 (m/z = +806) PE20:5/22:6 (m/z = +810) PE20:0/22:6 (m/z = +820) PE22:0/20:5 (m/z = +822) PE22:6/22:6 (m/z = +836) 17

18Abbreviations: The numbers in the PC and PE species, separated by a slash, define the two fatty acids

19in the sn-1 and sn-2 position of the glycerol backbone. The figure before the colon defines the number

20of carbon units, while the figure behind defines the number of double bonds of the respective fatty

21acid. aa in front of the PC or PE represents a component comprising one acyl and one alk(en)yl group

22rather than 2 acyl groups. Consequently, PC16:0/18:2 is a PC species comprising a hexadecanoic

23(palmitic) acid and a octadecadienoic (linoleic) acid residue. m/z, mass by charge.

25Routine analysis of PC and PE molecular species

26The choline containing phospholipids phosphatidylcholine (PC), lyso-PC and 16:0-sphingomyeline

27(16:0-SPH) were analyzed via specific reaction monitoring (SRM) of individual masses (mass by

28charge = m/z values) in the positive ionization mode. PE species were analyzed by neutral loss scan. 4 4 29The table indicates the m/z values of the analytes identified, together with the internal standards di-

30eicosanoyl-PC (PC20:0/20:0) and di-myristoyl-PE (PE14:0/14:0). As sodiation is common among PE

31components, samples were checked for sodium adduct formation by measuring the mass by charge

32ratio (m/z) = +658 for Na-PE14:0/14:0 relative to m/z = +636 for PE14:0/14:0, which was below 10%

33throughout. Species with identical mass by charge values (indicated by *) were assessed further as

34described in the following paragraph.

36Identification of PC and PE components of identical mass by charge values, but theoretically

37comprising different molecular species

38Molecular identity of individual components was analyzed in negative ionization mode, with fatty acid

39anions as daughter ions. Mobile phase for PE was the same as for analysis in positive ionization

40(chloroform:methanol:water:25%NH4OH; 20:78:1.7:0.3,v/v). For PC fatty acid analysis methanol:

41dichloromethane: 200mM ammoniumacetate (66:30:4, v/v) was used, resulting in a mass shift of

42m/z=44 (acetate anion: m/z=59; minus methyl group: m/z=15) in the 1st quadrupole, followed by the

43measurement of fatty acid daughter ions in the 3rd quadrupole clarifying the molecular identity of

44individual m/z values. For example, m/z=808 was clearly identified as PC18:1/20:4, with an m/z =

45-303 fragment (arachidonic acid anion, C20:4) and an m/z = -281 fragment (oleic acid anion, C18:1)

46predominating, and m/z = -301 (eicosapentaenoic acid anion, C20:5) and m/z= -283 (stearic acid

47anion, C18:0) being virtually absent. Similarly, m/z = 786 comprised PC18:0/18:2, with PC18:1/18:1.

49Accurateness of analysis:

50Repetitive analyses of samples showed that accurateness depended on the fraction and concentration of

51individual components. For lyso-PC and SPH, coefficients of variation were 4.7-7.3%, for individual

52PC species coefficients of variation ranged from 2.7-10.6% depending on their concentrations. For PC

53subgroups OA-PC, LA-PC, ARA-PC, EPA-PC and DHA-PC coefficients of variation were 3.1%,

541.6%, 3.0%, 4.4% and 2.3%, respectively. For OA-, LA-, ARA-, EPA- and DHA-PE coefficients of

55variation were 11.7%, 12.3%, 4.2%, 69.6% and 7,3%, respectively.