Measurement of mitochondrial DNA synthesis in vivo using a stable isotope-mass spectrometric technique

Collins, Michelle; Eng, Shannon; Hoh, R.; Hellerstein, Marc K.

doi:10.1152/japplphysiol.00691.2002

Cited by 63 publications

(57 citation statements)

References 27 publications

(63 reference statements)

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“…To observe multiphasic kinetics and thereby tease out cells with different turnover rates in any system by use of a continuous labeling strategy, it is necessary to administer label for a considerably longer period than the turnover time of the short-lived pool (i.e., 2-3 weeks for T cells) (23,24). This had not been possible before the development of the 2 H 2 O-labeling technique (10,(21)(22)(23). Second, these are the first studies of long-term label retention in the m/e-surface phenotype compartment of human T cells.…”

Section: Discussionmentioning

confidence: 99%

“…The long-term 2 H 2 O labeling technique has recently been developed for measuring the proliferation of slowturnover cells (14,21,22). 2 H 2 O enters into each of the C-H bonds of deoxyribose in replicating DNA through the de novo nucleotide synthesis pathway (19).…”

Section: Methodsmentioning

confidence: 99%

“…Cell-cycle markers such as Ki67 do not provide information concerning the fate of a cell, whereas labeling with BrdU or 3 H-thymidine is not readily interpretable with regard to cell survival (13) (see below), in addition to having toxicities that have generally precluded use in humans. We recently developed stable isotope-labeling methods for measuring the replication of DNA in dividing cells (9,10,14,19,20), including a deuterated water-labeling ( 2 H 2 O-labeling) technique that allows very long-term studies of cell proliferation (10,15,(20)(21)(22)(23). Here, we describe for the first time the kinetics of long-term label incorporation into T cell DNA in humans.…”

Section: Subpopulations Of Long-lived and Short-lived T Cells In Advamentioning

confidence: 99%

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Subpopulations of long-lived and short-lived T cells in advanced HIV-1 infection

Hellerstein

Hoh²,

Hanley³

et al. 2003

J. Clin. Invest.

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156

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Section: Discussionmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Section: Subpopulations Of Long-lived and Short-lived T Cells In Advamentioning

confidence: 99%

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Subpopulations of long-lived and short-lived T cells in advanced HIV-1 infection

Hellerstein

Hoh²,

Hanley³

et al. 2003

J. Clin. Invest.

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122

156

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“…The acetylene gas was then removed with a syringe and injected into a GC vial containing 10% bromine in carbon tetrachloride and incubated at room temperature for 2 h to produce tetrabromoethane. Excess bromine was neutralized with 25 l of 10% cyclohexene, and the sample was suspended in ethyl acetate (16). Alternatively, the acetylene gas was directly measured by a new mass spectrometric method (17).…”

Section: Measurements Of 2 H 2 O Enrichment In Body Water-mentioning

confidence: 99%

Physiologic and Pharmacologic Factors Influencing GlyceroneogenicContribution to Triacylglyceride Glycerol Measured by Mass IsotopomerDistribution Analysis

Chen

Peacock

Samady

et al. 2005

Journal of Biological Chemistry

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An imbalance between triacylglycerol synthesis and breakdown is necessary for the development of obesity. The direct precursor for triacylglycerol biosynthesis is ␣-glycerol phosphate, which can have glycolytic and glyceroneogenic origins. We present a technique for determining the relative glyceroneogenic contribution to triacylglyceride glycerol by labeling the glycerol moiety with 2 H 2 O. The number of hydrogen atoms (n) incorporated from H 2 O into C-H bonds reflects the metabolic source of ␣-glycerol phosphate and can be calculated by combinatorial analysis of the distribution of mass isotopomers in triacylglyceride glycerol. Three physiological settings with potential effects on glyceroneogenesis and glycolysis were studied in rodents. Adipose tissue acylglyceride glycerol in mice fed a low carbohydrate diet had significantly higher values of n than in mice fed a high carbohydrate diet, suggesting an increased contribution from glyceroneogenesis of from 17 to 50% on the low carbohydrate diet. Similarly, mice administered rosiglitazone had a significant relative increase in glyceroneogenesis (from 17 to 53%), indicated by an increase in adipose acylglyceride glycerol n. Fructose infusion in overnight fasted rats rapidly lowered plasma triacylglyceride glycerol n, reflecting a decreased contribution from glyceroneogenesis (from 66 to 34%) presumably because of increased glycolytic input. In conclusion, we demonstrate that the number of C-H atoms derived from cellular H 2 O in triacylglyceride glycerol is an informative indicator of ␣-glycerol phosphate origin and, ultimately, triacylglycerol metabolism. Under certain physiological conditions, glyceroneogenesis can be upregulated in adipose (e.g. low carbohydrate diet) or down-regulated in liver (e.g. fructose infusion). Additionally, stimulation of glyceroneogenesis by rosiglitazone in adipose tissue may be an important factor in the antilipolytic actions of thiazolidinediones.

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“…To measure the turnover of DNA [1,2] or cells [3][4][5] in various tissues, DNA can be labeled with isotope-labeled precursor. In such studies, the DNA is extracted, hydrolyzed, and analyzed to determine label incorporation by mass spectrometry.…”

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confidence: 99%

DNA digestion to deoxyribonucleoside: A simplified one-step procedure

Quinlivan

Gregory

2008

Analytical Biochemistry

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We present a simple and inexpensive 'one-step' protocol for the hydrolysis of DNA to deoxyribonucleosides. Unlike the older DNA hydrolysis protocol which is cumbersome and labor intensive, thes new protocol is ideal for high-throughput assays and is suitable automation. Using this protocol we were able to hydrolyze several hundred samples within an 8-hour period. The new protocol is fully compatible with LC-MS/MS and gives similar recoveries for all five major deoxyribonucleosides when compared to the older protocol.The requirement to hydrolyze DNA to deoxyribonucleosides is a common component of several assays. To measure the turnover of DNA [1,2] or cells [3][4][5] in various tissues, DNA can be labeled with isotope-labeled precursor. In such studies, the DNA is extracted, hydrolyzed, and analyzed to determine label incorporation by mass spectrometry. Likewise, in vivo experiments in humans, such as examination of the effect of gene polymorphisms [6] and nutritional status [6,7] on DNA metabolism, have used labeled one-carbon donors to label monocyte DNA. Nucleoside hydrolysates of DNA also have been used in determining the base composition of DNA, including investigation of epigenetic modification such as deoxycytosine methylation [8,9], oxidative damage [10,11], or unique DNA composition [12].One of the most commonly used protocols for digesting DNA is the tri-enzyme protocol devised by Crain [13]. However, a major weakness of the Crain protocol is that the first enzyme in the reaction, nuclease P1 (E.C. 3.1.30.1), only can digest single stranded DNA and has a pH optimum (pH ~5) significantly lower and a reaction temperature significantly higher (~50°C) than the other two enzymes (pH >8 and ~37°C) in the reaction. This leads to an overly complex protocol [Fig 1], whereby the DNA must be boiled to denature (and rapidly cooled to prevent reversion), the pH of the sample is adjusted twice, and the sample undergoes three separate enzyme incubations. These complexities limit the number of samples that can be prepared. For logistical reasons (repetitive manipulations of tubes), we were typically [6] restricted to preparing ~60 samples per day when we used the Crain protocol. Furthermore, the Crain protocol uses high buffer concentrations (e.g., 50-100 mmol/L ammonium bicarbonate, pH 7.9) which can interfere with downstream reactions and assays. Therefore, we devised a simple *Corresponding Author: Email: epq@ufl.edu (E.P. Quinlivan). 1 Abbreviations Used: LC-MS/MS: liquid chromatography -tandem mass spectrometry; H-ESI: heated electrospray ionization; dCyt: deoxycytidine; MdCyt: 5-methyldeoxycytidine; dThy: thymidine; dAdn: deoxyadenosine; dGua: deoxyguanosine Publisher's Disclaimer: This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final citable form. Please note that dur...

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Measurement of mitochondrial DNA synthesis in vivo using a stable isotope-mass spectrometric technique

Cited by 63 publications

References 27 publications

Subpopulations of long-lived and short-lived T cells in advanced HIV-1 infection

Subpopulations of long-lived and short-lived T cells in advanced HIV-1 infection

Physiologic and Pharmacologic Factors Influencing GlyceroneogenicContribution to Triacylglyceride Glycerol Measured by Mass IsotopomerDistribution Analysis

DNA digestion to deoxyribonucleoside: A simplified one-step procedure

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