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1 Composition and lipid spatial distribution of High Density Lipoprotein particles in subjects with Laxman Yetukuri, 1 Sanni Söderlund, 2 Artturi Koivuniemi, 3 Tuulikki Seppänen-Laakso, 1 Perttu S. Niemelä, 1 Marja Hyvönen, 4 Marja-Riitta Taskinen, 2 Ilpo Vattulainen, 3,5,6 Matti Jauhiainen, 7,8 and Matej Orešič 1,8, * 1 VTT Technical Research Centre of Finland, Espoo, Finland, 2 Division of Cardiology, Department of Medicine, University of Helsinki, Helsinki, Finland, 3 Department of Physics, Tampere University of Technology, Tampere, Finland, 4 Department of Physics, University of Oulu, Finland, 5 Department of Physics, Aalto University School of Science and Engineering, Espoo, Finland, 6 MEMPHYS Center for Biomembrane Physics, University of Southern Denmark, Odense, Denmark, 7 National Institute for Health and Welfare, Helsinki, Finland, 8 Institute for Molecular Medicine Finland FIMM, Helsinki, Finland *Correspondence to: Matej Orešič, VTT Technical Research Centre of Finland, P.O. Box 1000, Espoo, FI VTT, Finland. Tel.: ; Fax: ; matej.oresic@vtt.fi. Supporting Information 1
2 Contents Table S1. Identified lipids selected based on P-values (P<0.0001) from student t-test between high and low HDL-C subjects Table S2. All potential lipid peaks ordered based on P-values (t-test between high and low HDL-C subjects) Table S3. HDL subspecies 2b, 2a, 3a, 3b, and 3c in low- and high-hdl subjects divided by gender 13 Fig. S1. Gender specific clustering observed in HDL lipidomics data Fig. S2. Fold ratio (high vs. low HDL-C) of triacylglycerols as dependent on distribution of double bonds (degree of saturation) and total acyl carbon chain length Fig. S3. Heat map of Pearson correlations for top VIP variables vs. clinical variables Fig. S4. Correlation analysis between specific lipid variables (lysopc(18:0) and SM(18:1/16:0)) vs. selected clinical parameters (HDL-C and HDL particle size) Fig. S5. Correlation analysis between specific lipid variables (TG (16:0/18:1/20:1) and ChoE(18:1)) vs. selected clinical parameters (HDL-C and HDL particle size) Fig. S6. Radii of gyrations of HDL particles Fig. S7. Root mean square fluctuation (RMSF) profiles for apoa-is in each simulation Fig. S8. Radial distribution functions for other than TG molecules in high-hdl simulation Fig. S9. Radial distribution functions for other than TG molecules in normal-hdl simulation Fig. S10. Radial distribution functions for other than TG molecules in low-hdl simulation
3 Table S1. Identified lipids selected based on P-values (P<0.0001) from student t-test between high and low HDL-C subjects. m/z, mass-to-charge ratio; RT, retention time; FDR, False discovery rate. m/z RT Lipid Name Mean fold change (High vs. low HDL-C) FDR q-value PC(16:0/20:3) 0.50 < PC(O-16:0/20:4) 0.62 < PC(34:0) 0.55 < PC(O-36:2) PC(O-36:5) TG(16:0/18:1/16:0) SM(d18:1/23:0) TG(16:0/18:1/20:1) PC(18:2/20:4) PC(18:0/22:5) TG(17:0/18:1/16:0) PC(18:2/18:2) SM(d18:1/23:1) PC(40:7) SM(d18:1/16:0)
4 Table S2. All potential lipid peaks ordered based on P-values (t-test between high and low HDL-C subjects). The reported lipids in Table 1 are the ones confirmed by MS/MS using ESI+ (and in some cases also ESI-). m/z, mass-to-charge ratio; RT, retention time in seconds; Lo, low HDL-C group; Hi, high HDL-C group; SEM, standard error of mean; MS/MS, not determined due to insufficient information at MS/MS, *two-sided t-test. Peak no M/Z RT LipidName Mode of identification Mean (Lo) Mean (Hi) SEM(Lo) SEM(Hi) P* PC(16:0/20:3) MS/MS < PC(O-16:0/20:4) MS/MS < PC(34:0) MS/MS < PC(O-36:2) MS/MS < PC(O-36:5) MS/MS < TG(16:0/18:1/16:0) MS/MS < SM(d18:1/23:0) MS/MS < TG(16:0/18:1/20:1) MS/MS < PC(18:2/20:4) MS/MS < PC(18:0/22:5) MS/MS < TG(17:0/18:1/16:0) MS/MS < PC(18:2/18:2) MS/MS < SM(d18:1/23:1) MS/MS < PC(40:7) MS/MS < SM(d18:1/16:0) MS/MS < PC(36:4)(sodiated) MS/MS < LysoPC(18:1) MS/MS < PC(33:4) MS/MS < PC(37:4)+PE(40:4) MS/MS < TG(18:1/16:0/18:1) MS/MS < PC(O-34:3) MS/MS < LysoPC(18:0) MS/MS < TG(17:0/18:1/18:2) MS/MS < PC(36:4)(sodiated) MS/MS < TG(18:2/16:0/18:1) MS/MS < TG(18:1/18:3/22:6) MS/MS < PC(38:6) MS/MS < TG(18:1/18:1/15:0) MS/MS < LysoPC(18:1)(sodiated) MS/MS < PC(18:2/18:2) MS/MS < PC(33:4) MS/MS <
5 SM(d18:1/24:1) MS/MS < SM(d18:1/22:0) MS/MS < PC(O-36:3) MS/MS < TG(18:1/16:0/16:1) MS/MS < PC(38:3) MS/MS PE(P-16:0/20:4) MS/MS TG(18:1/14:0/16:0) MS/MS SM(d18:1/21:0) MS/MS PC(O-34:2) MS/MS TG(18:0/18:1/18:1) MS/MS TG(16:0/18:0/16:0) MS/MS SM(d18:1/15:0) MS/MS PC(36:5) MS/MS TG(18:1/18:1/18:1) MS/MS SM(d18:1/23:0)(sodiated) MS/MS PC(sodiated) MS/MS PC(O-40:7) MS/MS SM(d18:1/14:0) MS/MS PC(16:0/20:5) MS/MS LysoPC(22:6) MS/MS PC(40:6) MS/MS SM(d18:1/16:0)(sodiated) MS/MS PC(40:4) MS/MS TG(18:2/18:1/18:1) MS/MS TG(16:0/18:2/18:2) MS/MS PC(36:5)(sodiated) MS/MS ChoE(18:1) MS/MS PC(O-32:1) MS/MS TG(18:2/16:1/17:0) MS/MS PC(34:3) MS/MS PC(O-40:3) MS/MS SM(d18:1/24:2) MS/MS PC(O-36:5) MS/MS PC(16:0/18:2) MS/MS PC(18:0/18:1) MS/MS TG(16:0/16:0/16:0) MS/MS PE(P-18:1/20:4) MS/MS LysoPC(18:0) MS/MS TG(18:1/18:2/18:2) MS/MS LysoPC(16:0) MS/MS ChoE(20:4) MS/MS TG(18:1/18:1/18:2) MS/MS
6 PC(36:2) MS/MS LysoPC(18:2) MS/MS PC(O-38:5) MS/MS PC(38:7) MS/MS PC(16:0/18:2) MS/MS PC(18:0/20:5) MS/MS PE(P-16:0/20:4) MS/MS PC(18:0/20:3) MS/MS PC(O-16:0/20:4) MS/MS TG(16:1/16:1/18:1) MS/MS PC(39:6) MS/MS TG(18:1/14:0/16:1) MS/MS PC(16:0/16:0) MS/MS PC(O-40:5) MS/MS PC(36:4) MS/MS TG(18:1/16:0/12:0) MS/MS PC(16:0/16:0) MS/MS TG(16:0/14:0/14:0) MS/MS PC(34:3) MS/MS PE(P-16:0/18:2) MS/MS PC(36:5) MS/MS PE(P-18:0/18:2)+PE(P :1/18:1) MS/MS TG(18:1/15:0/14:0) MS/MS PC(O-16:0/22:6) MS/MS TG(18:1/18:2/18:3) MS/MS PC(18:0/20:4) MS/MS TG(16:0/18:3/18:2) MS/MS SM(d18:1/18:0) MS/MS TG(18:1/18:2/18:3) MS/MS PC(16:1/16:0) MS/MS PC(40:4) MS/MS PC(18:0/18:2) MS/MS PC(34:3) MS/MS PC(36:2) MS/MS PC(38:4) MS/MS PE(O-38:6) MS/MS PC(40:6) MS/MS TG(18:1/18:1/18:2) MS/MS PC(16:0/22:6)(sodiated) MS/MS PC(O-38:7) MS/MS PE(O-38:7) MS/MS PC(34:1) MS/MS
7 TG(16:0/16:1/18:3) MS/MS ChoE(18:2) MS/MS PC(35:1) MS/MS TG(14:0/17:1/16:1) MS/MS PC(35:2) MS/MS PC(14:0/18:2) MS/MS SM(d18:1/18:1) MS/MS TG(16:0/18:2/14:1) MS/MS PC(O-16:0/22:6) MS/MS PC(33:0) MS/MS PC(16:0/18:2) MS/MS PC(30:0) MS/MS PC(37:1) MS/MS PC(40:7) MS/MS PC(30:1) MS/MS PC(40:8) MS/MS PC(18:2/20:4) MS/MS PC(36:6) MS/MS PE(O-36:3) MS/MS TG(18:3/18:2/18:2) MS/MS PC(O-40:4) MS/MS PC(40:6) MS/MS LysoPC(16:1) MS/MS PC(18:0/20:5) MS/MS DG(40:7) MS LysoPC(16:0)(sodiated) MS LysoPC(18:0)(sodiated) MS LysoPC(18:2) MS LysoPC(18:2)(sodiated) MS LysoPC(20:3) MS MAG(16:1) MS No match MS PC(26:1) MS PC(28:1) MS PC(32:1) MS PC(32:1)(sodiated) MS PC(32:2)(sodiated) MS PC(34:2e)(sodiated) MS PC(34:4) MS PC(34:5) MS < PC(34:5) MS PC(36:3) MS <
8 PC(36:5)(sodiated) MS PC(36:6) MS PC(36:7) MS PC(38:1) MS PC(38:6)(sodiated) MS PC(38:6)(sodiated) MS PC(38:9) MS PC(40:10) MS PC(40:5) MS PC(40:5)(sodiated) MS < PC(40:6)(sodiated) MS PC(40:7)(sodiated) MS PC(40:8) MS PC(40:9) MS PC(40:9) MS PC(42:5) MS PC(42:5) MS PC(42:6) MS PC(42:6) MS PC(42:6) MS PC(42:7) MS PC(42:8) MS PC(42:8) MS PC(O-32:1) MS PC(O-32:6) MS < PC(O-32:6) MS < PC(O-32:6) MS < PC(O-32:6) MS < PC(O-32:6) MS < PC(O-32:6) MS PC(O-36:2) MS PC(O-36:3) MS PC(O-36:5) MS PC(O-36:7) MS PC(O-38:5) MS PC(O-38:6) MS PC(O-38:6) MS PC(O-38:6) MS PC(O-38:7) MS < PC(O-38:8) MS PC(O-40:2) MS PC(O-40:5) MS
9 PC(O-40:6) MS PC(O-40:6) MS PC(O-40:7) MS PC(O-40:7) MS PC(O-40:7) MS PC(O-40:8) MS < PC(O-40:9) MS PE(32:0) MS PE(34:0) MS PE(34:3) MS PE(36:1) MS PE(36:2) MS < PE(38:0) MS PE(38:3) MS PE(38:3) MS PE(38:5) MS PE(40:2) MS PE(40:3) MS PE(40:3) MS PE(40:5) MS PE(42:1) MS PE(42:4) MS PE(42:9) MS PE(O-30:2) MS < PE(O-30:2) MS < PE(O-32:1) MS PE(O-34:2) MS PE(O-36:6) MS PE(O-38:3) MS PE(O-38:4) MS PE(O-40:6) MS < PE(O-40:6) MS PS(34:0) MS PS(38:2) MS PS(40:1) MS PS(40:2) MS PS(40:3) MS PS(40:4) MS PS(40:5) MS SM(d18:0/16:0) MS SM(d18:1/18:1)(sodiated) MS SM(d18:1/20:1) MS
10 SM(d18:1/22:5) MS SM(d18:1/22:5) MS SM(d18:1/23:5) MS SM(d18:1/24:2)(sodiated) MS SM(d18:1/24:4) MS SM(d18:1/24:6) MS SM(d18:1/25:1) MS TG(33:0) MS TG(36:0) MS TG(37:0) MS TG(46:2) MS TG(52:6) MS TG(54:3) MS TG(55:8) MS TG(55:8) MS TG(56:6) MS TG(56:6) MS TG(56:7) MS TG(56:8) MS PE(O-30:1) MS < PE(O-36:6) MS < SM(d18:1/22:0)(sodiated) MS < SM(d18:1/24:1)(sodiated) MS < PC(30:2) MS < PE(O-34:2) MS < PC(30:2) MS No match MS/MS No match MS/MS No match MS/MS < No match MS/MS < No match MS/MS < No match MS/MS < No match MS/MS < No match MS/MS < No match MS/MS No match MS/MS
11 No match MS/MS No match MS/MS < No match MS/MS No match MS/MS No match MS/MS No match MS/MS No match MS/MS No match MS/MS No match MS/MS No match MS/MS No match MS/MS No match MS/MS No match MS/MS No match MS/MS No match MS/MS No match MS/MS No match MS/MS No match MS/MS No match MS/MS No match MS/MS No match MS/MS < No match MS/MS < No match MS/MS < No match MS/MS < No match MS/MS < No match <
12 No match No match No match No match MS/MS MS/MS <0.001 MS/MS <0.001 MS/MS <0.001 MS/MS <
13 Table S3. HDL subspecies 2b, 2a, 3a, 3b, and 3c in low- and high-hdl subjects divided by gender. LH,low HDL-C group; HH, high HDL-C group; IQR, interquartile range; *Mann-Whitney U test. HDL 2b (%) LH men Median (IQR) 12.9 ( ) LH women Median (IQR) 18.2 ( ) P-value* HH men Median (IQR) 46.8 ( ) HH women Median (IQR) 50.8 ( ) P-value* HDL 2a (%) 24.1 ( ) 28.5 ( ) ( ) 22.5 ( ) HDL 3a (%) 35.6 ( ) 33.3 ( ) ( ) 17.8 ( ) HDL 3b (%) 21.8 ( ) 14.5 ( ) ( ) 6.0 ( ) HDL 3c (%) 5.0 ( ) 3.3 ( ) ( ) 1.7 ( )
14 Scores on LV 2 (18.53%) L.Yetukuri et al., Composition and lipid spatial distribution of High Density Lipoprotein particles in subjects with Fig. S1. Gender specific clustering observed in HDL lipidomics data. Multivariate analysis of lipidomics data shows that subjects are clustered based on the HDL-cholesterol status, not on the gender. Supervised PLS-DA model was constructed using random subsets corss validation method based on four classes (two genders and HDL-C status). No gender specific clusters are observed as shown PLS-DA scores plot below. 40 LoHDLF 30 PLS-DA Scores plot LoHDLM LoHDLM HiHDLM HiHDLM HiHDLF HiHDLF HiHDLF HiHDLF HiHDLF LoHDLF HiHDLM HiHDLF HiHDLMHiHDLM HiHDLF HiHDLM LoHDLMLoHDLF HiHDLM LoHDLF HiHDLF LoHDLM LoHDLF LoHDLF LoHDLM LoHDLM LoHDLM Scores on LV 1 (32.37%) 14
15 Fold change Fold change L.Yetukuri et al., Composition and lipid spatial distribution of High Density Lipoprotein particles in subjects with Fig. S2. Fold ratio (high vs. low HDL-C) of triacylglycerols as dependent on distribution of double bonds (degree of saturation) and total acyl carbon chain length. Spearman rank correlations were applied to correlate fold ratio TG chain length or degree of saturation, respectively r = -0.17, p = Triglyceride carbon chain length r = 0.28, p = Total number of TG fatty acyl chain double bonds 15
16 PC(O-36:4) PC(34:0) PC(O-36:5) TG(16:0/18:1/18:1) TG(16:0/18:1/20:1) TG(16:0/18:1/16:0) TG(16:0/18:2/18:1) TG(16:0/18:1/16:1) P(O-.32:1) PC(16:0/16:0) PC(O-40:3) SM(d18:1/21:0) lysopc(22:6) PC(O-36:3) PC(O-34:3) PC(O-36:2) SM(d18.1:/16.0) SM(d18:1/23:1) PC(O-34:.2) SM(d18:1/15:0) PC(18:2/18:2) SM(d18:1/23:0) PC(18:2/:20:4) lysopc(18:1) lysopc(18:0) L.Yetukuri et al., Composition and lipid spatial distribution of High Density Lipoprotein particles in subjects with Fig. S3. Heat map of Pearson correlations for top VIP variables vs. clinical variables. Pearson correlation HDL_SIZE ApoA-I HDL-C Paraoxonase PLTP activity CETP activity PLTP mass Total Cholesterol TG Insulin BMI TNF_α CRP il_6 LDL-C 16
17 Conc n, mol/g Conc n, mol/g Conc n, mol/g L.Yetukuri et al., Composition and lipid spatial distribution of High Density Lipoprotein particles in subjects with Fig. S4. Correlation analysis between specific lipid variables (lysopc(18:0) and SM(18:1/16:0)) vs. selected clinical parameters (HDL-C and HDL particle size). A lysopc(18:0) SM(d18:1/16:0) x x 10 Low HDL-C Low HDL-C r=0.54, p= r=0.72, p= x 10-4 x 10-4 High HDL-C High HDL-C r=-0.06, p= r=0.71, p= B C HDL-C (mmol/l) lysopc(18:0) x 10-4 x r = 0.50, p = p = r = 0.580, p < p < D HDL-C (mmol/l) SM(d18:1/16:0) HDL size (nm) HDL size (nm) 17
18 Conc n, mol/g Conc n, mol/g Conc n, mol/g L.Yetukuri et al., Composition and lipid spatial distribution of High Density Lipoprotein particles in subjects with Fig. S5. Correlation analysis between specific lipid variables (TG (16:0/18:1/20:1) and ChoE(18:1)) vs. selected clinical parameters (HDL-C and HDL particle size). A TG(16:0/18:1/20:1) x10-4 x Low HDL-C Low HDL-C r=-0.06, p=0.75 r=0.33, p= x10-4 x High HDL-C HighHDL-C r=-0.43, p= r=0.02, p= ChoE(18:1) B HDL-C (mmol/l) HDL-C (mmol/l) C x r =-0.51, p = TG(16:0/18:1/20:1) HDL size (nm) 18
19 Fig. S6. Radii of gyrations of HDL particles. Results indicate that when moving from high HDL-C to low HDL-C the particle becomes smaller. This finding agree with size data acquired by electrophoresis. 19
20 Fig. S7. Root mean square fluctuation (RMSF) profiles for apoa-is in each simulation. The residual RMSF fluctuations differ markedly between the simulations. The reason for this could be that either the lipid composition or the conformation of apoa-i around the particles is different in each simulation. 20
21 Fig. S8. Radial distribution functions for other than TG molecules in high-hdl simulation. Note that one SM molecule was trapped under the ApoA-I in this particular simulation, which produced the noticeable region in SM plot near 1 nm. 21
22 Fig. S9. Radial distribution functions for other than TG molecules in normal-hdl simulation. 22
23 Fig. S10. Radial distribution functions for other than TG molecules in low-hdl simulation. 23
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