Supplementarymaterial to: Plasma choline-containing phospholipids:
Potential biomarkers for colorectal cancer progression
Song Li•Bin Guo•Jianwen Song•Xiaoli Deng•Yusheng Cong•Pengfei Li•Ke Zhao• Lihong Liu•Gang Xiao•Feng Xu•Yingjiang Ye•Zhenwen Zhao•Menggang Yu•Yan Xu•Jianli Sang*•Junjie Zhang*
*Corresponding author:JL.Sang*•JJ.Zhang*
Key Laboratory of Cell Proliferation and Regulation Biology, Ministry of Education, Institute of Cell Biology, College of Life Sciences, Beijing Normal University, Beijing 100875, China; email: , . telephone: +86 10 58802137. Fax:+ 86 10 58807721
Supplementary material contains:
- Supplementarymethods
- SupplementaryTable 1
- SupplementaryTable 2
- SupplementaryTable 3
- SupplementaryFig. 1
- SupplementaryFig. 2
Supplementarymethods
Blood Processing, Lipid Extraction and Analysis of Phospholipids
Blood samples were collected in EDTA-containing tubes and centrifuged at 1,750g for 15 min at room temperature. Plasma samples were aliquoted into siliconized eppendorf tubes (Cat # 505-201; PGC Scientifics, Frederick, Maryland) and frozen at -80˚C until use without repetitive freezing and thawing. All extraction procedures were performed in 10 mL glass disposable centrifuge tubes (Kimble Glass Inc.). The same procedures of lipid extraction as described in the previous study were used (Zhao Z, Xiao Y, Elson P, et al. J Clin Oncol 25:2696-701, 2007). Plasma (10 μL for LPC, SPC and L-PAF analyses; 50 μL for LPA and S1P analyses) was diluted with phosphate-buffered saline (PBS) to 0.5 mL.Then10 μL 12:0 LPC (100 μmol/L) were added as internal standards for LPC, SPC and L-PAF analyses, or 50 μL 14:0 LPA (1 μmol/L) with 15 μL HCl (6N) were added as internal standardsfor LPA and S1P analyses.The sample was mixed with 3 mL MeOH/chloroform (2:1), vortexed for 30 seconds, and then incubated on ice for 10 min. Then, chloroform (1 mL) and H2O (1.3 mL) were added into the sample. The sample was vortexed for 30 seconds before centrifugation (1,750 gfor 6 minutes at room temperature). The lower phase was transferred to a new glass tube. The solvent of the lower phase extract was evaporated under nitrogen at room temperature. The dried lipids were resuspended in 1mL MeOH/H2O (9:1) for LPC, SPC, SM and Lyso-PAF MS analyses, while 150 μL MeOH/H2O (9:1) was added to the dried lipids for LPA and S1P analyses.
MS analyses of lipids
The extracted lipid samples (200 μL for LPC related lipids; 150 μL for LPA and S1P) were directly delivered into the electrospray ionization (ESI) -MS ion source through an Agilent 1100 series HPLC system (Agilent Technologies, Palo Alto, CA, USA) with an auto sampler. The mobile phase was MeOH/water/AmOH (90:10:0.1, v/v/v). The flow rate was 200 μL/min, and 20 μL/sample was injected. MS analyses were performed using an Applied Biosystems Sciex 3200QtrapTM mass spectrometer (Applied Biosystems Sciex, Ontario, Canada). Applied Biosystems/MDS SCIEX Analyst 1.4.1 software was used for data acquisition and processing.
Quantitative analysis was performed as described previously.[38] We established standard curves for each lipid form by mixing different concentrations of a particular form of lipids with the same concentration (100 μmol/L or 1 μmol/L) of an internal standard (12:0-LPC or 14:0-LPA). All standard lipid forms were purchased from Avanti Polar lipids), and then performing ESI-MS analyses. The peak intensity ratios (a lipid form/internal standard) versus the concentration ratios (a lipid form/internal standard) were plotted and fitted to a linear regression.
The instrument was equipped with an electrospray ion (ESI) source, and was operated in the positive and negative ion modes. The LC-MS/MS detector was operated at unit resolution in the MRM mode using the mass transition ion-pairs m/z 440 (the parent ion) -184 (the product ion) for 12:0LPC (as an internal standard), 465-184 for SPC, 468-184 for 14:0LPC, 482-104 for L-PAF, 496-184 for 16:0LPC, 522-184 for 18:1LPC, 524-184 for 18:0LPC, 552-184 for 20:0LPC, 580-184 for 22:0LPC, 703.9-184 for 16:0 SM, 731.9-184 for 18:0 SM, 520-184 for 18:2LPC, 540-184 for 20:4LPC, 568-184 for 22:6 LPC. In order to optimize MS parameters, a standard solution of analyte and IS was infused into the mass spectrometer using a syringe pump. Optimized parameters were as follows: Curtain gas, gas 1 and gas 2 (nitrogen) were 15, 30 and 40 units, respectively; dwell time 100 ms; source temperature 500C; IonSpray voltage 5500 V. Declustering Potential (DP) and Collision Energy (CE) were, respectively, 53 V and 36 eV for palonosetron and 25 V and 40 eV for tramadol. The collision gas was set to high mode and the interface heater to on mode.
Supplementary Table1Comparison of plasma SPC, Lyso-PAF, LPC and SM levels betweenmales and females
Lipids / Male / Female / paMale vs Female
Mean sd / Mean sdb
SPC / 0.064 0.043 / 0.0610.045 / 0.2637
Lyso-PAF / 1.2010.667 / 1.055 0.531 / 0.0130
14:0LPC / 1.4200.876 / 1.3510.826 / 0.2285
16:0LPC / 175.34491.976 / 156.26272.478 / 0.0210
18:2LPC / 64.78930.805 / 57.07723.750 / 0.0069
18:1LPC / 33.95615.523 / 30.25011.880 / 0.0093
18:0LPC / 77.55042.274 / 71.22836.624 / 0.0757
20:4LPC / 0.264 0.128 / 0.266 0.120 / 0.4426
20:0LPC / 1.003 0.886 / 0.865 0.436 / 0.0480
22:6LPC / 5.2182.406 / 4.6621.980 / 0.0126
22:0LPC / 0.8451.238 / 0.6920.583 / 0.0922
16:0SM / 152.506 51.852 / 157.097 43.384 / 0.1962
18:0SM / 29.72910.708 / 31.5399.357 / 0.0537
total LPC / 360.390173.993 / 322.651 135.989 / 0.0167
total SM / 182.23560.948 / 188.63650.680 / 0.1549
Sat. LPC / 256.162134.118 / 230.397108.490 / 0.0309
Unsat. LPC / 104.22746.198 / 92.25435.560 / 0.0054
All lipid concentrations are inM. Sat: saturated; Unsat: unsaturated
a p-value from the Wilcoxon rank sum test
b sd, standard deviation
Supplementary Table2Comparison of plasma choline-containing phospholipid levels betweenthe healthy control and the diseased(AP and CRC)group
Lipids / ControlMean±sd b / AP and CRC
Mean±sd b / P a
Control vs. AP and CRC
SPC / 0.078±0.051 / 0.055 ±0.037 / <0.0001
Lyso-PAF / 1.613±0.719 / 0.920 ±0.408 / <0.0001
14:0 LPC / 1.915±0.993 / 1.136 ±0.640 / <0.0001
16:0 LPC / 240.326±97.203 / 132.437 ±49.536 / <0.0001
18:2 LPC / 78.828±30.140 / 53.560 ±23.807 / <0.0001
18:1 LPC / 43.401±15.575 / 27.209 ±10.142 / <0.0001
18:0 LPC / 109.458±46.779 / 58.146 ±21.852 / <0.0001
Total 18 LPC / 231.690±83.879 / 138.915 ±50.294 / <0.0001
20:4 LPC / 0.290±0.124 / 0.252 ±0.124 / 0.0030
20:0 LPC / 1.087±0.973 / 0.885 ±0.612 / 0.0084
22:6 LPC / 5.956±2.349 / 4.546 ±2.086 / <0.0001
22:0 LPC / 0.836±0.935 / 0.766 ±1.104 / 0.2766
Saturated LPC / 353.621±143.841 / 193.369 ±69.969 / <0.0001
Unsaturated LPC / 128.478±45.869 / 85.567 ±33.315 / <0.0001
Sat. LPC/unsat. LPC / 2.792±0.785 / 2.360 ±0.600 / <0.0001
Total LPC / 482.099±179.935 / 278.936 ±97.464 / <0.0001
16:0 SM / 176.776±51.363 / 142.877 ±43.564 / <0.0001
18:0 SM / 34.423±9.941 / 28.368 ±9.831 / <0.0001
Total SM / 211.205±59.450 / 171.244 ±51.582 / <0.0001
All lipid concentrations are inM. Sat: saturated; Unsat: unsaturated
a p-value from the Wilcoxon rank sum test
b sd, standard deviation
Supplementary Table 3Comparison of the sensitivity and specificity for males versus females in theestablishedmodels to distinguishbetween healthy control, adenomatous polyps (AP)and CRC subjects
Health vs CRC a / Health vs CRC / Health vs AP / AP vs CRCEquation / 11.406 ×18:2 LPC% + 7.43 ×18:1 LPC% + 0.024 × Saturated LPC = 14.58 / 0.023 × Saturated LPC + 5.96 ×18:2 LPC% + 17.06 × SPC = 9.66 / 0.0093 × saturated LPC – 1225 ×20:4 LPC% + 0.02 × sum of 16:0 and 18:0 SM = 3.3 / 0.044 × sum of 16:0 and 18:0 SM – 60.1 × SPC – 0.027 total saturated LPC = -1.06
Sensitivity
（%） / Specificity
（%） / Sensitivity
（%） / Specificity
（%） / Sensitivity
（%） / Specificity
（%） / Sensitivity
（%） / Specificity
（%）
Male / 84.5 / 85.7 / 87.3 / 83.8 / 88.2 / 79.8 / 90.1 / 95.5
Female / 81.6 / 85.1 / 89.8 / 78.6 / 95.5 / 79.2 / 91.8 / 86.5
Total / 83 / 86 / 88.3 / 80 / 89 / 80 / 90 / 92.5
aThe adjusted formula with the same LPC markers as published previously in the USA study (Zhao, Z., et al, 2007)was used to separate CRC from the healthy controls in the Chinese population
Supplementary Fig.1.The plasma levels of SM and SPC in healthy, AP and CRC subjects. The plasma levels of SPC (A)，16:0 SM (B)，18:0 SM (C) andtotal SM (D) in healthy control, AP and CRC groups were detected as described in Materials and Methods. The collected data were analyzed by origin 7.5 software (OriginLab).Statistical analysis was performed using Student’s t tests. Significance was assumed at a p value of less than 0.0001.
Supplementary Fig.2. Evaluate the LPC markers identified in the previous USA study for distinguishing CRC from healthy controls in Chinese population. (A) The ROC curve for healthy vs. CRC cases. (B)Plasma levels of (18:2 LPC% – 18:1 LPC%) from CRC cases (c) and healthy controls (h) are plotted against total saturated LPC levels in each sample. The equation [18:2LPC% –18:1LPC% + 0.053 × total saturated LPC = 16.8] is also plotted (the line in the figure). Subjects whose values fall to the left of this line are classified as diseased, while subjects whose data are to the right of this line would be classified as unaffected (healthy) subjects. A total of 120 CRC and 120 healthycontrols are involved. The 18:2 LPC% and 18:1 LPC% in the formula are calculated as the percentages (with range from 0-100%) of 18:2 LPC and 18:1 LPC with respect to unsaturated LPC.
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