Understanding the Cholesterol Metabolism-Perturbing Effects of Docosahexaenoic Acid By

Understanding the cholesterol metabolism-perturbing effects of docosahexaenoic acid by gas chromatography-mass spectrometry targeted metabonomic profiling

Priti Bahety, Thi Hai Van Nguyen, Yanjun Hong, Luqi Zhang, Eric Chun Yong Chan* and Pui Lai Rachel Ee*

Department of Pharmacy, National University of Singapore, 18 Science Drive 4, Singapore 117543, Republic of Singapore

*Corresponding authors:

Pui Lai Rachel Ee, PhD

Mailing address: Department of Pharmacy, Faculty of Science, National University of Singapore, 18 Science Drive 4, Singapore 117543, Singapore.

Tel: +65 6516 2653; Fax: +65 6779 1554.

Email address:

Eric Chun Yong Chan, PhD

Mailing address: Department of Pharmacy, Faculty of Science, National University of Singapore, 18 Science Drive 4, Singapore 117543, Singapore.

Tel: + +65 65166137; Fax: + 65 67791554.

Email address:

Supplementary Information

Supplementary Table S1: Metabolites used in GC/MS -targeted metabolite profiling

Compound / Formula / Mass
Cholesterol / C27H46O / 458.00
Acetyl CoA sodium salt / C23H35O17N7P3S•Na3•H2O / 809.57
HMG CoA trisodium salt / C27H41N7Na3O20P3S / 977.62
Mevalonic acid lithium salt / C6H11O4 · xLi+ / 202.00
IPP trilithium salt / C5H9Li3O7P2 / 263.89
FPP ammonium salt / C15H37N3O7P2 / 433.42
Squalene / C30H50 / 410.00
Lanosterol / C30H50O / 498.00
Zymosterol / C27H44O / 456.00
Desmosterol / C27H44O / 456.00
7-dehydrocholesterol / C27H44O / 456.00
7β-hydroxycholesterol / C27H46O2 / 546.00
Lathosterol / C27H46O / 458.00
24S-hydroxycholesterol / C27H46O2 / 546.00
27-hydroxycholesterol / C27H46O2 / 546.00
5α-cholestane (IS) / C27H48 / 372.00

Supplementary Table S2: Retention times, quantifying m/z and qualifying m/z of metabolite standards and internal standard

Metabolite / Retention time (min) / Dwell time (ms) / Quantifying m/z / Qualifying m/z
Mevalonate / 5.50 / 30 / 115 / 145 / 143
Squalene / 10.37 / 30 / 137 / 136 / 121
5α-cholestane (IS) / 10.72 / 30 / 217 / 149 / 357
Cholesterol / 12.25 / 30 / 329 / 129 / 368
Desmosterol / 12.52 / 30 / 343 / 351 / 129
7-dehydrocholesterol / 12.57 / 30 / 325 / 351 / 143
Lathosterol / 12.70 / 30 / 458 / 255 / 459
Zymosterol / 12.75 / 30 / 441 / 129 / 351
7β-hydroxycholesterol / 12.83 / 30 / 457 / 456 / 73
Lanosterol / 13.83 / 30 / 393 / 109 / 483
24S-hydroxycholesterol / 14.32 / 30 / 145 / 413 / 159
27-hydroxycholesterol / 15.30 / 30 / 129 / 456 / 417

Supplementary Table S3: Concentration and fold changes of metabolites in DHA- and vehicle-treated CHO-wtcells

Metabolite / CHO-wt
Concentration (μM)a / Fold Δb
Vehicle-treated / DHA-treated
Mevalonate / 35.04 ± 7.74 / 41.45 ± 6.23 ns / 1.18
Squalene / 0.18 ± 0.03 / 0.28 ± 0.02* / 1.51
Cholesterol / 857.09 ± 45.51 / 931.80 ± 77.35ns / 1.09
Desmosterol / 62.16 ± 6.74 / 44.29 ± 3.90* / 0.71
7-dehydrocholesterol / 2.07 ± 0.20 / 1.69 ± 0.19* / 0.82
Lathosterol / 0.05 ± 0.01 / 0.06 ± 0.06 ns / 1.20
Zymosterol / 6.28 ± 0.55 / 4.87 ± 0.53* / 0.77
7β-hydroxycholesterol / 0.07 ± 0.01 / 0.08 ± 0.01 ns / 1.04
Lanosterol / 0.77 ± 0.24 / 0.98 ± 0.21ns / 1.20
27-hydroxycholesterol / 7.72 ± 1.10 / 5.95 ± 0.89* / 0.77

aConcentration values of the metabolites calculated from the respective calibration curves and expressed as mean ± S.E.M of six parallel replicates.

bFold change (Δ): concentration of metabolite in CHO-wt (DHA-treated) / concentration of metabolite in CHO-wt(vehicle-treated), and values >1 and <1 represent higher and lower metabolite levels induced by drug treatment, respectively.

* p<0.05 is significant while nsrefers to not significant when calculated using the independent t-test with Welch’s correction for concentration of metabolite in DHA-treated cells compared to vehicle-treated cells.

Supplementary Table S4: Concentration and fold changes of metabolites in DHA- and vehicle-treated CHO-AβPP695 cells

Metabolite / CHO-AβPP695
Concentration (μM)a / Fold Δb
Vehicle-treated / DHA-treated
Mevalonate / 35.84 ± 3.13 / 32.76 ± 4.10ns / 0.91
Squalene / 0.25 ± 0.04 / 0.25 ± 0.04 ns / 0.99
Cholesterol / 968.16 ± 83.94 / 892.42 ± 81.89ns / 0.92
Desmosterol / 76.43 ± 9.52 / 48.24 ± 5.69* / 0.63
7-dehydrocholesterol / 2.51 ± 0.32 / 1.86 ± 0.20* / 0.74
Lathosterol / 0.05 ± 0.01 / 0.06 ± 0.02 ns / 1.08
Zymosterol / 8.05 ± 0.90 / 5.69 ± 0.60* / 0.71
7β-hydroxycholesterol / 0.07 ± 0.01 / 0.07 ± 0.01 ns / 1.06
Lanosterol / 1.39 ± 0.21 / 1.56 ± 0.28ns / 1.12
27-hydroxycholesterol / 4.99 ± 0.76 / 3.27 ± 0.45* / 0.66

aConcentration values of the metabolites calculated from the respective calibration curves and expressed as mean ± S.E.M of six parallel replicates.

bFold change (Δ): concentration of metabolite in CHO-AβPP695 (DHA-treated) / concentration of metabolite in CHO-AβPP695 (vehicle-treated), and values >1 and <1 represent higher and lower metabolite levels induced by drug treatment, respectively.

* p<0.05 is significant while nsrefers to not significant when calculated using the independent t-test with Welch’s correction for concentration of metabolite in DHA-treated cells compared to vehicle-treated cells.

Supplementary Fig S1:

Supplementary Fig S1: DHA and simvastatin treatment inhibits HMG-CoA reductase and squalene epoxidase enzyme activity.Effect of DHA and simvastatin in inhibiting (a) HMG-CoA reductase and (b) squalene epoxidase enzyme activity was investigated by monitoring the slope of reduction in NADPH absorbance at 340 nm. Values are mean ± SEM from three independent experiments. . */$/#p< 0.05, **/$$p< 0.01 and ***/$$$p<0.001, *compared to vehicle-treated CHO-wt cells, $compared to vehicle-treated CHO-AβPP695 cells,# compared to 25 μM DHA-treated CHO-wt cells and compared to 1 μM simvastatin-treated CHO-wt cells.