In vivo D2O labeling to quantify static and dynamic changes in cholesterol and cholesterol esters by high resolution LC/MS.
High resolution LC/MS-MS and LC/APPI-MS methods have been established for the quantitation of flux in the turnover of cholesterol and cholesterol ester. Attention was directed toward quantifying the monoisotopic mass (M0) and that of the singly deuterated labeled (M+1) isotope. A good degree of isotopic dynamic range has been achieved by LC/MS-MS ranging from 3-4 orders of magnitude. Correlation between the linearity of GC/MS and LC atmospheric pressure photoionization (APPI)-MS are complimentary (r² = 0.9409). To prove the viability of this particular approach, male C57Bl/6 mice on either a high carbohydrate (HC) or a high fat (HF) diet were treated with ²H₂O for 96 h. Gene expression analysis showed an increase in the activity of stearoyl-CoA desaturase (Scd1) in the HC diet up to 69-fold (P < 0.0008) compared with the HF diet. This result was supported by the quantitative flux measurement of the isotopic incorporation of ²H into the respective cholesterol and cholesterol ester (CE) pools. We concluded that it is possible to readily obtain static and dynamic measurement of cholesterol and CEs in vivo by coupling novel LC/MS methods with stable isotope-based protocols.
Castro-Perez
Jose
J
Atherosclerosis Exploratory Biomarkers Group, Merck & Co., Inc., Rahway, NJ, USA. jose_castro-perez@merck.com
Previs
Stephen F
SF
McLaren
David G
DG
Shah
Vinit
V
Herath
Kithsiri
K
Bhat
Gowri
G
Johns
Douglas G
DG
Wang
Sheng-Ping
SP
Mitnaul
Lyndon
L
Jensen
Kristian
K
Vreeken
Robert
R
Hankemeier
Thomas
T
Roddy
Thomas P
TP
Hubbard
Brian K
BK
D000818:Animals; D002784:Cholesterol / Q000032:analysis* / Q000097:blood / Q000378:metabolism; D002788:Cholesterol Esters / Q000032:analysis* / Q000378:metabolism; D002853:Chromatography, Liquid / Q000379:methods*; D017666:Deuterium Oxide / Q000737:chemistry*; D007554:Isotopes; D008297:Male; D013058:Mass Spectrometry / Q000379:methods*; D051379:Mice; D008810:Mice, Inbred C57BL; D010168:Palmitates / Q000097:blood; D013230:Stearoyl-CoA Desaturase / Q000378:metabolism
D002788:Cholesterol Esters; D007554:Isotopes; D010168:Palmitates; D002784:Cholesterol; D013230:Stearoyl-CoA Desaturase; D017666:Deuterium Oxide