A Comparison of μLC/Electrospray Ionization-MS and GC/MS for the Measurement of Stable Isotope Enrichment from a [<sup>2</sup>H<sub>2</sub>]-Glucose Metabolic Probe in T-Cell Genomic DNA

Fox, Stephen D.; Lempicki, Richard A.; Hosack, Douglas A; Baseler, M; Kovacs, Joseph A.; Lane, H. Clifford; Veenstra, Timothy D.; Issaq, Haleem J.

doi:10.1021/ac030186v

Cited by 9 publications

(10 citation statements)

References 15 publications

(23 reference statements)

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“…The highest increases were observed for the [1- 13 C 1 ]glucose tracer (i.e., to 93.4% at a TTR level of 0.1%) and the lowest for the [6,6- 2 H 2 ]- and [U- 13 C 6 ]glucose tracers (i.e., to 25.4% at a TTR level of 0.1%). Our observation of higher uncertainties in the enrichment determination of the [1- 13 C 1 ]glucose tracer, which required correction for the high ion intensity contribution due to the naturally occurring 13 C 1 12 C 5 1 H 12 16 O 6 isotopologue, is consistent with previous findings showing that uncertainties in the determination of tracer enrichments become larger as the extent of correction applied to the raw ion intensity data increases. , Furthermore, CV values obtained for the [6,6- 2 H 2 ]-, [U- 13 C 6 ]-, or [1- 2 H 2 ]glucose tracer in the various triple combinations are similar to or better than those obtained when the enrichment of each of these tracers was determined in the presence or absence of a second glucose tracer (serving as internal standard) using conventional GC/Q-MS or LC/Q-MS methods. ,− …”

Section: Resultssupporting

confidence: 91%

Determination of the Isotopic Enrichment of ¹³C- and ²H-Labeled Tracers of Glucose Using High-Resolution Mass Spectrometry: Application to Dual- and Triple-Tracer Studies

Trötzmüller

Triebl²,

Ajsic³

et al. 2017

Anal. Chem.

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Multiple-tracer approaches for investigating glucose metabolism in humans usually involve the administration of stable and radioactive glucose tracers and the subsequent determination of tracer enrichments in sampled blood. When using conventional, low-resolution mass spectrometry (LRMS), the number of spectral interferences rises rapidly with the number of stable tracers employed. Thus, in LRMS, both computational effort and statistical uncertainties associated with the correction for spectral interferences limit the number of stable tracers that can be simultaneously employed (usually two). Here we show that these limitations can be overcome by applying high-resolution mass spectrometry (HRMS). The HRMS method presented is based on the use of an Orbitrap mass spectrometer operated at a mass resolution of 100 000 to allow electrospray-generated ions of the deprotonated glucose molecules to be monitored at their exact masses. The tracer enrichment determination in blood plasma is demonstrated for several triple combinations of C- andH-labeled glucose tracers (e.g., [1-H]-, [6,6-H]-, [1,6-C]glucose). For each combination it is shown that ions arising from H-labeled tracers are completely differentiated from those arising fromC-labeled tracers, thereby allowing the enrichment of a tracer to be simply calculated from the observed ion intensities using a standard curve with curve parameters unaffected by the presence of other tracers. For each tracer, the HRMS method exhibits low limits of detection and good repeatability in the tested 0.1-15.0% enrichment range. Additionally, due to short sample preparation and analysis times, the method is well-suited for high-throughput determination of multiple glucose tracer enrichments in plasma samples.

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Section: Resultssupporting

confidence: 91%

Determination of the Isotopic Enrichment of ¹³C- and ²H-Labeled Tracers of Glucose Using High-Resolution Mass Spectrometry: Application to Dual- and Triple-Tracer Studies

Trötzmüller

Triebl²,

Ajsic³

et al. 2017

Anal. Chem.

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“…Other researchers in the field have utilized deuterated water with quantitation of the dA M+1 moiety 9,13 and aqueous deuterated glucose with quantitation of the dA M+2 moiety. 10–12 Therefore, a single methodology that is developed and optimized to concurrently measure dA and its stable isotopologues (e.g. dA M+1, dA M+2) would be advantageous.…”

Section: Resultsmentioning

confidence: 99%

“…The MethElute™ derivatization approach is similar to Fox et al 10 ; however our method does not require the use of an organic modifier (e.g. dimethylformamide), or processing a large number of cells for analysis.…”

Section: Introductionmentioning

confidence: 99%

Sensitive GC-MS/MS Method to Measure Deuterium Labeled Deoxyadenosine in DNA from Limited Mouse Cell Populations

et al. 2013

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A rapid and sensitive GC-MS/MS method was developed to quantitatively measure low levels of DNA base deoxyadenosine (dA) and its isotopologues (e.g. dA M+1) from limited mouse cell populations. Mice undergoing allogeneic hematopoietic transplantation (AHSCT) received deuterated water at biologically relevant time intervals post AHSCT, allowing labeling of DNA upon cell division, which was detected as the dA M+1 isotopologue. Targeted mouse cell populations were isolated from lymphoid organs and purified by multi-parameter fluorescence activated cell sorting. Cell lysis, DNA extraction and hydrolysis were accomplished using available commercial procedures. The novel analytical method utilized a hydrophilic-lipophilic balanced sample preparation, rapid on-line hot GC inlet gas phase sample derivatization, fast GC low thermal mass technology, and a recently marketed GC-MS/MS system. Calibration standards containing dA and fortified with relevant levels of dA M+1 (0.25–20%) and dA M+5 (internal standard) were used for sample quantitation. The method employed a quadratic fit for calibration of dA M+1 (0.25–20%) and dA, demonstrated excellent accuracy and precision, and had limits of detection of 100 fg on-column for the dA isotopologues. The method was validated and required only 20,000 cells to characterize population dynamics of cells involved in the biology of chronic graft-versus-host disease, the main cause of late morbidity and non-relapse-mortality following AHSCT. The high sensitivity and specificity of the method makes it useful for investigating in vivo kinetics on limited and important cell populations (e.g. T regulatory cells) from disease conditions or in disease models that are immune-mediated, such as diabetes, HIV/AIDS, arthritis, inflammatory bowel disease, and multiple sclerosis.

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“…Genomic DNA was hydrolyzed to mono-deoxyribonucleosides with deoxyribonuclease I, phosphodiesterase I, and bacterial alkaline phosphatase, desalted using Oasis HLB 96-well extraction plate (Waters Corp.), and either converted to the permethylated derivatives of deoxyadenosine (292 m/z) and 2 H-deoxyadenosine (294 m/z) for gas chromatography/mass spectrometry analysis or analyzed directly by liquid chromatography/mass spectrometry as described in ref. 24. Sample enrichments were calculated by comparison with standard curves prepared from 2 H-deoxyadenosine.…”

Section: Patients Hiv-infected Patients With No Major Clinical or Lamentioning

confidence: 99%

Induction of prolonged survival of CD4+ T lymphocytes by intermittent IL-2 therapy in HIV-infected patients

Kovacs¹,

Lempicki²,

Sidorov³

et al. 2005

J. Clin. Invest.

114

101

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HIV infection leads to decreases in the number of CD4 + T lymphocytes and an increased risk for opportunistic infections and neoplasms. The administration of intermittent cycles of IL-2 to HIV-infected patients can lead to profound increases (often greater than 100%) in CD4 cell number and percentage. Using in vivo labeling with 2 H-glucose and BrdU, we have been able to demonstrate that, although therapy with IL-2 leads to high levels of proliferation of CD4 as well as CD8 lymphocytes, it is a remarkable preferential increase in survival of CD4 cells (with half-lives that can exceed 3 years) that is critical to the sustained expansion of these cells. This increased survival was time-dependent: the median half-life, as determined by semiempirical modeling, of labeled CD4 cells in 6 patients increased from 1.7 weeks following an early IL-2 cycle to 28.7 weeks following a later cycle, while CD8 cells showed no change in the median half-life. Examination of lymphocyte subsets demonstrated that phenotypically naive (CD27 + CD45RO -) as well as central memory (CD27 + CD45RO + ) CD4 cells were preferentially expanded, suggesting that IL-2 can help maintain cells important for host defense against new antigens as well as for long-term memory to opportunistic pathogens.

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A Comparison of μLC/Electrospray Ionization-MS and GC/MS for the Measurement of Stable Isotope Enrichment from a [²H₂]-Glucose Metabolic Probe in T-Cell Genomic DNA

Cited by 9 publications

References 15 publications

Determination of the Isotopic Enrichment of ¹³C- and ²H-Labeled Tracers of Glucose Using High-Resolution Mass Spectrometry: Application to Dual- and Triple-Tracer Studies

Determination of the Isotopic Enrichment of ¹³C- and ²H-Labeled Tracers of Glucose Using High-Resolution Mass Spectrometry: Application to Dual- and Triple-Tracer Studies

Sensitive GC-MS/MS Method to Measure Deuterium Labeled Deoxyadenosine in DNA from Limited Mouse Cell Populations

Induction of prolonged survival of CD4+ T lymphocytes by intermittent IL-2 therapy in HIV-infected patients

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A Comparison of μLC/Electrospray Ionization-MS and GC/MS for the Measurement of Stable Isotope Enrichment from a [2H2]-Glucose Metabolic Probe in T-Cell Genomic DNA

Cited by 9 publications

References 15 publications

Determination of the Isotopic Enrichment of 13C- and 2H-Labeled Tracers of Glucose Using High-Resolution Mass Spectrometry: Application to Dual- and Triple-Tracer Studies

Determination of the Isotopic Enrichment of 13C- and 2H-Labeled Tracers of Glucose Using High-Resolution Mass Spectrometry: Application to Dual- and Triple-Tracer Studies

Sensitive GC-MS/MS Method to Measure Deuterium Labeled Deoxyadenosine in DNA from Limited Mouse Cell Populations

Induction of prolonged survival of CD4+ T lymphocytes by intermittent IL-2 therapy in HIV-infected patients

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Product

Resources

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A Comparison of μLC/Electrospray Ionization-MS and GC/MS for the Measurement of Stable Isotope Enrichment from a [²H₂]-Glucose Metabolic Probe in T-Cell Genomic DNA

Determination of the Isotopic Enrichment of ¹³C- and ²H-Labeled Tracers of Glucose Using High-Resolution Mass Spectrometry: Application to Dual- and Triple-Tracer Studies

Determination of the Isotopic Enrichment of ¹³C- and ²H-Labeled Tracers of Glucose Using High-Resolution Mass Spectrometry: Application to Dual- and Triple-Tracer Studies