Measurement of Ribosomal RNA Turnover In Vivo by Use of Deuterium-Labeled Glucose

Abstract

AbstractBackground: Most methods for estimation of rates of RNA production are not applicable in human in vivo clinical studies. We describe here an approach for measuring ribosomal RNA turnover in vivo using [6,6-2H2]-glucose as a precursor for de novo RNA synthesis. Because this method involves neither radioactivity nor toxic metabolites, it is suitable for human studies.Methods: For method development in vitro, a lymphocyte cell line (PM1) was cultured in the presence of [6,6-2H2]-glucose. RNA was extracted, hydrolyzed enzymatically to ribonucleosides, and derivatized to either the aldonitrile tetra-acetate or the pentafluoro triacetate derivative of the pentose before GC-MS. We identified optimum derivatization and analysis conditions and demonstrated quantitative incorporation of deuterium from glucose into RNA of dividing cells.Results: Pilot clinical studies demonstrated the applicability of this approach to blood leukocytes and solid tissues. A patient with chronic lymphocytic leukemia received [6,6-2H2]-glucose (1 g/kg) orally in aliquots administered every 30 min for a period of 10 h. When we analyzed CD3– B cells that had been purified by gradient centrifugation and magnetic-bead adhesion, we observed deuterium enrichment, a finding consistent with a ribosomal RNA production rate of about 7%/day, despite the slow division rates observed in concurrent DNA-labeling analysis. Similarly, in 2 patients with malignant infiltration of lymph nodes, administration of [6,6-2H2]-glucose (by intravenous infusion for 24 h) before excision biopsy allowed estimation of DNA and RNA turnover in lymph node samples.Conclusions: Our study results demonstrate the proof-of-principle that deuterium-labeled glucose may be used to analyze RNA turnover, in addition to DNA production/cell proliferation, in clinical samples.