Measurement in vivo of proliferation rates of slow turnover cells by ² H ₂ O labeling of the deoxyribose moiety of DNA

Abstract

We describe here a method for measuring DNA replication and, thus, cell proliferation in slow turnover cells that is suitable for use in humans. The technique is based on the incorporation of 2 H2O into the deoxyribose (dR) moiety of purine deoxyribonucleotides in dividing cells. For initial validation, rodents were administered 4% 2 H2O in drinking water. The proliferation rate of mammary epithelial cells in mice was 2.9% per day and increased 5-fold during pregnancy. Administration of estradiol pellets (0 -200 g) to ovariectomized rats increased mammary epithelial cell proliferation, according to a doseresponse relationship up to the 100 g dose. Similarly, proliferation of colon epithelial cells was stimulated in a dose-response manner by dietary cholic acid in rats. Bromodeoxyuridine labeling correlated with the 2 H2O results. Proliferation of slow turnover cells was then measured. Vascular smooth muscle cells isolated from mouse aorta divided with a half-life in the range of 270 -400 days and die-away values after 2 H2O wash-out confirmed these slow turnover rates. The proliferation rate of an adipocyte-enriched fraction from mouse adipose tissue depots was 1-1.5% new cells per day, whereas obese ad libitum-fed ob͞ob mice exhibited markedly higher fractional and absolute proliferation rates. In humans, stable long-term 2 H2O enrichments in body water were achieved by daily 2 H2O intake, without toxicities. Labeled dR from fully turned-over blood cells (monocytes or granulocytes) exhibited a consistent amplification factor relative to body 2 H2O enrichment (Ϸ3.5-fold). The fraction of newly divided naive-phenotype T cells after 9 weeks of labeling with 2 H2O was 0.056 (CD4 ؉ ) and 0.043 (CD8 ؉ ) (replacement rate <0.1% per day). In summary, 2 H2O labeling of dR in DNA allows safe, convenient, reproducible, and inexpensive measurement of cell proliferation in humans and experimental animals and is well suited for slow turnover cells. deuterated water ͉ cell proliferation ͉ DNA synthesis ͉ adipogenesis ͉ vascular smooth muscle cell proliferation

Assessment of protein synthesis in highly aerobic canine species at the onset and during exercise training

“…Determination of 2 H incorporation into purine deoxyribose (dR) of DNA was performed as previously described (4,5,14,15). Briefly, DNA isolated from whole tissue was hydrolyzed overnight at 37°C with nuclease S1 and potato acid phosphatase.…”

“…Hydrolysates were reacted with pentafluorobenzyl hydroxylamine and acetic acid and then acetylated with acetic anhydride and 1-methylimidazole. Dichloromethane extracts were dried, resuspended in ethyl acetate, and analyzed by GC/MS as previously described (4,5,14,15).…”

Aging and T-cell diversity☆

“…In the absence of exogenous antigen, memory and even naïve T cells exhibit a constant turnover. The daily replacement rate of T cells has been estimated to be on the order of 1%, which means that with about 3×10 11 T cells in a human body, approximately 3×10 9 T cells have to be generated each day Hellerstein et al, 1999;Neese et al, 2002). Memory T cells have higher homeostatic proliferation than naïve T cells , raising the question of whether they can out compete naïve T cells and, over time, significantly compromise the integrity of the naïve T-cell compartment.…”

DNA digestion to deoxyribonucleoside: A simplified one-step procedure

“…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.…”