1
Online Supplement to:
Developmental Changes of Polyunsaturated Fetal Plasma Phospholipids and Feto-Maternal Plasma Phospholipid Ratios and their Association with Bronchopulmonary Dysplasia
Wolfgang Bernhard1, Marco Raith1, Vera Koch1, Rebecca Kunze1, Christoph Maas1, Harald Abele3, Christian F. Poets1, Axel R. Franz1,2
Affiliations: Department of 1Neonatology, 2Center for Pediatric Clinical Studies, and 3Department of Gynecology, Faculty of Medicine, Eberhard-Karls-University, Tübingen, Germany.
Address of correspondance: Wolfgang Bernhard, MD PhD, Department of Neonatology, Faculty of Medicine, Eberhard-Karls-University, Calwer Straße 7, D-72076 Tuebingen, FRG; ; Phone: #-49 7071 29 86377
Short title: LC-PUFA-phospholipid changes during fetal development
Online supplemental Table S1: Characteristics of the study group.
Parturients / Newborns
Number of patients / 108 / 121
Singleton pregnancy / 94 (87%) / 94 (78%)
Twins/Triples / 13 (12%) / 1 (1%) / 24 (20%) / 3 (2%)
Sex of child (m / f) / - / 65/56
Maternal age (years) / 32.3 (28.3-36.4)* (17.9-43.8)† / -
PMA at Birth (weeks) / - / 34.9 (30.9-37.9)* (24.3-41.7) †
Birth weight (g) / 2395 (1390-3235) (450-4200)*
Postpartum time interval at blood sampling (days) / 0 (-1 to +1)† / 0.0
B / Characteristics of Offsprings
Numbers in PMA-subgroups‡ / Singleton / Twins/Triples
24-27 weeks / 11 / 5/3
28-31 weeks / 13 / 6/0
32-33 weeks / 11 / 2/0
34-36 weeks / 19 / 11/0
37-42 weeks / 40 / 0/0
Overall characteristics of the study group is indicated in A, whereas B indicates the numbers of cord blood samples from singletons and twins. Data in parentheses indicate 25th/75th interquartiles (*), range (†) and twins (‡) where appropriate.
Online supplemental Table S2: Breakpoint and linear regression analyses of phospholipid parameters in cord blood (fetal) versus maternal blood
(PMA, w) / Slope before breakpoint / r / p-value / Slope after breakpoint / r / p-value
Maternal PC Concentration / 32.6
(30.4;34.8) / -132.5
(-222.1;-42.9) / -0.4384 / 0.0066 / 119.6
(50.3;188.8) / 0.0010
Maternal PE Concentration / No breakpoint / 0.3887 / <0.0001 / -
Fetal PC Concentration / No breakpoint / -0.233 / 0.0101 / -
Fetal PE Concentration / 35.1
(33.5; 36.8) / -1.0834
(-1.7102; -0.4565) / 0.2942 / 0.053 / 2.1824
(1.1187; 3.2462) / 0.5124 / <0.0001
Feto-maternal C22:6-PC-ratio / 33.2
(30.5; 36.0) / 0.0212
(-0.0178; 0.0602) / 0.220 / 0.1514 / 0.1123
(0.0820; 0.1426) / 0.621 / <0.0001
Feto-maternal PC16:0/22:6-ratio / 33.0
(30.5; 35.9) / 0.0158
(-0.019; 0.0506) / 0.1701 / 0.2695 / 0.0912
(0.0642; 0.1183) / 0.5932 / <0.0001
Feto-maternal PC18:0/22:6-ratio / 33.4
(30.8; 36.1) / 0.0415
(-0.0246; 0.1076) / 0.3056 / 0.0367 / 0.2018
(0.1504; 0.2532) / 0.6024 / <0.0001
Feto-maternal C18:2-PC-ratio / 35.2
(33.0; 37.3) / 0.0030
(-0.0017; 0.0077) / 0.129 / 0.3222 / -0.0155
(-0.0234; -0.0077) / -0.5573 / <0.0001
Feto-maternal Sat. PC-ratio / 33.7
(31.3; 36.1) / 0.011
(-0.0309; 0.0529) / 0.0252 / 0.864 / -0.1125
(-0.156; -0.069) / -0.681 / <0.0001
Breakpoint analyses were performed for fetal and maternal total PC and PE concentrations, and for PC subgroups (expressed as feto-maternal ratios), when regression analyses indicated a non-linear function, as previously described [21]. Data indicate mean slope values and 95% confidence intervals. For individual PC species used for sub-grouping see: Online resource, supplemental Fig. S2+S3.
Online supplemental Fig. S1: Growth of Total Body (A), Brain (B) and Lungs (C) during fetal development. Body growth data are calculated body weights according to [[1]]. Head circumference data from [1] were used to calculate brain weight according to Jordaan et al [[2]], and lung growth according to Stocker& Dehner [[3]]. Data points in A & B indicate the 50th percentile, and bars the 25th and 75th percentile of male and female newborns, whereas C are mean values from authopsies of life born individuals.
Online supplemental Fig. S2: Molecular species composition of maternal and fetal plasma PC (A) and PE (B) of the study group (24-42w postmenstral age). PC and PE molecular species were analyzed with tandem mass spectrometry as described in Materials and Methods. PE masses (B) below m/z = 704 (palmitoyl-linoleoyl-PE and beyond 806 (di-eicosanoyl PE), were below 0.1% of total PE and are not indicated. Abbreviations: PC, phosphatidylcholine; PE, phosphatidylethanolamine; aa in front of the PC or PE represents a component comprising an alk(en)yl-residue in position sn-1 and an acyl-residue in sn-2 position of the glycerol backbone, rather than 2 acyl groups. The numbers, separated by a slash, define the two fatty acids of PC and PE, respectively. Here, the figure before the colon defines the number of carbon units, while the figure behind defines the number of double bonds of the respective fatty acid. Consequently, PC16:0/18:2 is a PC species comprising a hexadecanoic (palmitic) acid and an octadecadienoic (linoleic) acid residue, connected in ester bonds to the glycerol backbone.
Online supplemental Fig. S3: Concentrations of PC subgroups in maternal and fetal plasma PC (A) and PE (B) from 24-42w postmenstral age. Maternal and cord blood samples were analyzed as indicated in Fig. S2. Individual molecular species were then grouped according to their content in oleic acid (C18:1), linoleic acid (C18:2), arachidonic acid (C20:4), eicosapentaenoic acid (C20:5) or two saturated fatty acid residues (Sat.). Other defines the sum of those components containing other minor fatty acids. LC-PUFA (long chain poly-unsaturated fatty acid) defines the sum of those PC/PE components comprising C20:4, C20:5 or C22:6. For details see Online resource to [[4]]. Data are depicted as medians and 25th/75th percentiles. Abbreviations: PC, phosphatidylcholine; PE, phosphatidylethanolamine; *, p<0.05, **, p<0.01, ***, p<0.001 relative to maternal blood; †, p<0.05, ††, p<0.01, †††, p<0.001 relative to cord blood.
Online supplemental Fig. S4: PC sub-group composition in cord plasma of male versus female offsprings. PC data from cord blood samples of the respective post-menstrual age (PMA) groups were separated into female and male sex, and compared. Data indicate medians and 25th/75th percentiles. Abbreviations: see legend to Fig. S3.
Online supplemental Fig. S5: PC sub-group composition in maternal serum with male compared to female offsprings. PC data from maternal blood samples of the respective post-menstrual age (PMA) groups were separated into those with a femal and male offspring, and compared. Data indicate medians and 25th/75th percentiles. Abbreviations: see legend to Fig. S3.
Online supplemental Fig. S6: Fractions of PE subgroups in fetal compared to maternal blood in postmenstrual age (PMA) groups. PE species were sub-grouped according to their fatty acid content, and PMA was sub-grouped into comprehensive intervals of 24-27w, 28-31w, 32-33w, 34-36w and 37-42w, as clinically relevant hallmarks of fetal development. Data are medians and 25th/75th percentiles. Abbreviations: C18:1, oleic acid; C18:2, linoleic acid; C20:4, arachidonic acid; C20:5, eicosapentaenoic acid; C22:6, docosahexaenoic acid; PE, phosphatidylethanolaminee; *, p<0.05; **, p<0.01; ***, p<0.001;.****, p<0.0001;.n.s., not significant.
Online supplemental Fig. S7: Changes in feto-maternal ratios of PE-subgroups from 24-42w PMA. PE data of individual samples were sub-grouped into saturated PE (Sat.-PE, A) and those containing an oleic (C18:1-PE, B), linoleic (C18:2-PE, C), C20:4chidonic (C20:4-PE, D), eicosapentaenoic (C20:5-PE, E) or docosahexaenoic acid (C22:6-PE, F) residue. Then the ratios between cord blood and their corresponding maternal values were formed. For comprehension, a value of 2 means that the fraction of a component is twice as high in fetal as in maternal blood, whereas a value of 0.5 means that it is 50% lower in fetal compared to maternal blood. Abbreviations: w, week; PMA, postmenstrual age; r, correlation coefficient; p, significance value.
References
[1] Voigt M, Fusch C, Olbertz D, Hartmann K, Rochow N, Renken C, Schneider KTM (2006) Analysis of the neonatal collective in the federal republic of germany 12th report: presentation of detailed percentiles for the body measurement of newborns. Geburtsh Frauenheilk 66: 956-970.
[2] Jordaan HV, Sattar FA, Shah P, Makarachi A (1980) Simplified method of determining fetal brain weight in utero. Obstet Gynecol. 55:673-677.
[3] Stocker JT, Dehner LP, Husain AN (2002) Means and standard deviations of weights and measurements of lifeborn infants by body weight (Appendix 28-29). 2nd ed. In: JT Stocker & LP Dehner, ed. Stocker & Dehner’s Pediatric Pathology, Lippinkott Williams & Wilkins, Philadelphia, PA 2002:1302-1304.
[4] Bernhard W, Raith M, Koch V, Kunze R, Maas C, Abele H, Poets CF, Franz AR (2014) Plasma phospholipids indicate impaired fatty acid homeostasis in preterm infants. Eur J Nutr.53:1533-1547.