A comparison of microLC/electrospray ionization-MS and GC/MS for the measurement of stable isotope enrichment from a [2H2]-glucose metabolic probe in T-cell genomic DNA.
Measurement of the proliferation of lymphocytes and other high-turnover cell populations in vivo can be accomplished through the incorporation of an isotopically labeled DNA precursor into actively dividing cells and the subsequent determination of the isotope enrichment in the isolated genomic DNA from selected cell populations. Two published gas chromatography/mass spectrometry (GC/MS) methods were successfully modified by our laboratory whereby a postinjection methylation reaction, rather than silylation or acetylation, was used to form a volatile derivative of deoxyadenosine (dA). We also developed a second robust microcapillary liquid chromatography-electrospray ionization (microLC-ESI)/MS method that is faster and more sensitive than the GC/MS method and does not require sample derivatization. Following administration of [6,6-(2)H(2)]-glucose to human immunodeficiency virus-infected patients, peripheral blood was drawn; cells were obtained by lymphapheresis and fractionated. DNA was isolated from the desired cell subtypes and enzymatically hydrolyzed to the free deoxyribonucleosides. The digest was analyzed using both capillary GC/MS and microLC/ESI-MS to measure the levels of the dA and [(2)H(2)]-dA or their reaction products. Sample enrichments were calculated by comparison to standard curves prepared from dA and [(2)H(2)]-dA. The microLC/ESI-MS method required fewer cells, less sample preparation, shorter analysis times, and a single calibration curve. Overall, the microLC/ESI-MS method is superior to the GC/MS method in terms of precision and accuracy, while providing a 4-fold increase in sensitivity (from 20 pmol at 0.2% [(2)H(2)]-dA enrichment to 5 pmol at 0.1% [(2)H(2)]-dA enrichment).
Fox
S D
SD
Analytical Chemistry Laboratory, SAIC-Frederick, Inc., National Cancer Institute at Frederick, National Institutes of Health, P.O. Box B, Frederick, Maryland 21702-1201, USA.
Lempicki
R A
RA
Hosack
D A
DA
Baseler
M W
MW
Kovacs
J A
JA
Lane
H C
HC
Veenstra
T D
TD
Issaq
H J
HJ
D002853:Chromatography, Liquid / Q000379:methods*; D004247:DNA / Q000032:analysis*; D003903:Deuterium / Q000032:analysis; D005947:Glucose / Q000032:analysis*; D006801:Humans; D007553:Isotope Labeling / Q000379:methods*; D021241:Spectrometry, Mass, Electrospray Ionization / Q000379:methods*; D013601:T-Lymphocytes / Q000737:chemistry*
D004247:DNA; D003903:Deuterium; D005947:Glucose