<p> 1 1 1Online Resource 2 to:</p><p>2Plasma phospholipids indicate impaired fatty acid homeostasis in preterm infants</p><p>3</p><p>4Wolfgang Bernhard1, Marco Raith1, Vera Koch1, Rebecca Kunze1, Christoph Maas1, Harald Abele2,</p><p>5Christian F. Poets1, Axel R. Franz1,3</p><p>6</p><p>7Affiliations: Departments of 1Neonatology and 2Gynecology, and 3Center for Pediatric Clinical</p><p>8Studies, Faculty of Medicine, Eberhard-Karls-University, Tübingen, Germany. </p><p>9</p><p>10Address of correspondance: Wolfgang Bernhard, MD PhD, Department of Neonatology, Faculty of</p><p>11Medicine, Eberhard-Karls-University, Calwer Straße 7, D-72076 Tuebingen, FRG;</p><p>[email protected]; Phone: #-49 7071 29 86377</p><p>13Short title: Plasma phospholipid alterations in preterm infants</p><p>14 2 2 </p><p>15ONLINE Resource 2: Molecular species of choline containing phospholipids (phosphatidylcholine</p><p>16[PC], lyso-PC and palmitoyl-sphingomyelin [16:0-SPH]), and of phosphatidylethanolamine (PE).</p><p>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 </p><p>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</p><p>18Abbreviations: The numbers in the PC and PE species, separated by a slash, define the two fatty acids</p><p>19in the sn-1 and sn-2 position of the glycerol backbone. The figure before the colon defines the number</p><p>20of carbon units, while the figure behind defines the number of double bonds of the respective fatty</p><p>21acid. aa in front of the PC or PE represents a component comprising one acyl and one alk(en)yl group</p><p>22rather than 2 acyl groups. Consequently, PC16:0/18:2 is a PC species comprising a hexadecanoic</p><p>23(palmitic) acid and a octadecadienoic (linoleic) acid residue. m/z, mass by charge.</p><p>24</p><p>25Routine analysis of PC and PE molecular species</p><p>26The choline containing phospholipids phosphatidylcholine (PC), lyso-PC and 16:0-sphingomyeline</p><p>27(16:0-SPH) were analyzed via specific reaction monitoring (SRM) of individual masses (mass by</p><p>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-</p><p>30eicosanoyl-PC (PC20:0/20:0) and di-myristoyl-PE (PE14:0/14:0). As sodiation is common among PE</p><p>31components, samples were checked for sodium adduct formation by measuring the mass by charge</p><p>32ratio (m/z) = +658 for Na-PE14:0/14:0 relative to m/z = +636 for PE14:0/14:0, which was below 10%</p><p>33throughout. Species with identical mass by charge values (indicated by *) were assessed further as</p><p>34described in the following paragraph. </p><p>35</p><p>36Identification of PC and PE components of identical mass by charge values, but theoretically</p><p>37comprising different molecular species</p><p>38Molecular identity of individual components was analyzed in negative ionization mode, with fatty acid</p><p>39anions as daughter ions. Mobile phase for PE was the same as for analysis in positive ionization</p><p>40(chloroform:methanol:water:25%NH4OH; 20:78:1.7:0.3,v/v). For PC fatty acid analysis methanol:</p><p>41dichloromethane: 200mM ammoniumacetate (66:30:4, v/v) was used, resulting in a mass shift of</p><p>42m/z=44 (acetate anion: m/z=59; minus methyl group: m/z=15) in the 1st quadrupole, followed by the</p><p>43measurement of fatty acid daughter ions in the 3rd quadrupole clarifying the molecular identity of</p><p>44individual m/z values. For example, m/z=808 was clearly identified as PC18:1/20:4, with an m/z =</p><p>45-303 fragment (arachidonic acid anion, C20:4) and an m/z = -281 fragment (oleic acid anion, C18:1)</p><p>46predominating, and m/z = -301 (eicosapentaenoic acid anion, C20:5) and m/z= -283 (stearic acid</p><p>47anion, C18:0) being virtually absent. Similarly, m/z = 786 comprised PC18:0/18:2, with PC18:1/18:1.</p><p>48</p><p>49Accurateness of analysis:</p><p>50Repetitive analyses of samples showed that accurateness depended on the fraction and concentration of</p><p>51individual components. For lyso-PC and SPH, coefficients of variation were 4.7-7.3%, for individual</p><p>52PC species coefficients of variation ranged from 2.7-10.6% depending on their concentrations. For PC</p><p>53subgroups OA-PC, LA-PC, ARA-PC, EPA-PC and DHA-PC coefficients of variation were 3.1%,</p><p>541.6%, 3.0%, 4.4% and 2.3%, respectively. For OA-, LA-, ARA-, EPA- and DHA-PE coefficients of</p><p>55variation were 11.7%, 12.3%, 4.2%, 69.6% and 7,3%, respectively.</p>