title: Reconciling Estimates of Cell Proliferation from Stable Isotope Labeling Experiments.
abstract: Stable isotope labeling is the state of the art technique for in vivo quantification of lymphocyte kinetics in humans. It has been central to a number of seminal studies, particularly in the context of HIV-1 and leukemia. However, there is a significant discrepancy between lymphocyte proliferation rates estimated in different studies. Notably, deuterated (2)H2-glucose (D2-glucose) labeling studies consistently yield higher estimates of proliferation than deuterated water (D2O) labeling studies. This hampers our understanding of immune function and undermines our confidence in this important technique. Whether these differences are caused by fundamental biochemical differences between the two compounds and/or by methodological differences in the studies is unknown. D2-glucose and D2O labeling experiments have never been performed by the same group under the same experimental conditions; consequently a direct comparison of these two techniques has not been possible. We sought to address this problem. We performed both in vitro and murine in vivo labeling experiments using identical protocols with both D2-glucose and D2O. This showed that intrinsic differences between the two compounds do not cause differences in the proliferation rate estimates, but that estimates made using D2-glucose in vivo were susceptible to difficulties in normalization due to highly variable blood glucose enrichment. Analysis of three published human studies made using D2-glucose and D2O confirmed this problem, particularly in the case of short term D2-glucose labeling. Correcting for these inaccuracies in normalization decreased proliferation rate estimates made using D2-glucose and slightly increased estimates made using D2O; thus bringing the estimates from the two methods significantly closer and highlighting the importance of reliable normalization when using this technique.
authors.affiliation: Institute for Infection and Immunity, St. George's, University of London, London, United Kingdom.
authors.forename: Raya
authors.initials: R
authors.lastname: Ahmed
authors.affiliation: Laboratory of Translational Immunology, Department of Immunology, University Medical Center Utrecht, Utrecht, The Netherlands.
authors.forename: Liset
authors.initials: L
authors.lastname: Westera
authors.affiliation: Laboratory of Translational Immunology, Department of Immunology, University Medical Center Utrecht, Utrecht, The Netherlands; Theoretical Biology & Bioinformatics, Utrecht University, The Netherlands.
authors.forename: Julia
authors.initials: J
authors.lastname: Drylewicz
authors.affiliation: Department of Medicine, Imperial College, London, United Kingdom.
authors.forename: Marjet
authors.initials: M
authors.lastname: Elemans
authors.affiliation: Institute for Infection and Immunity, St. George's, University of London, London, United Kingdom.
authors.forename: Yan
authors.initials: Y
authors.lastname: Zhang
authors.affiliation: Institute for Infection and Immunity, St. George's, University of London, London, United Kingdom.
authors.forename: Elizabeth
authors.initials: E
authors.lastname: Kelly
authors.affiliation: Institute for Infection and Immunity, St. George's, University of London, London, United Kingdom.
authors.forename: Rajko
authors.initials: R
authors.lastname: Reljic
authors.affiliation: Laboratory of Translational Immunology, Department of Immunology, University Medical Center Utrecht, Utrecht, The Netherlands.
authors.forename: Kiki
authors.initials: K
authors.lastname: Tesselaar
authors.affiliation: Theoretical Biology & Bioinformatics, Utrecht University, The Netherlands.
authors.forename: Rob J
authors.initials: RJ
authors.lastname: de Boer
authors.affiliation: Institute for Infection and Immunity, St. George's, University of London, London, United Kingdom.
authors.forename: Derek C
authors.initials: DC
authors.lastname: Macallan
authors.affiliation: Laboratory of Translational Immunology, Department of Immunology, University Medical Center Utrecht, Utrecht, The Netherlands.
authors.forename: José A M
authors.initials: JA
authors.lastname: Borghans
authors.affiliation: Department of Medicine, Imperial College, London, United Kingdom.
authors.forename: Becca
authors.initials: B
authors.lastname: Asquith
mesh_terms: D000465:Algorithms; D049109:Cell Proliferation / Q000502:physiology*; D003903:Deuterium / Q000032:analysis / Q000737:chemistry*; D017666:Deuterium Oxide / Q000032:analysis / Q000737:chemistry; D005947:Glucose / Q000737:chemistry / Q000378:metabolism*; D006801:Humans; D007553:Isotope Labeling / Q000379:methods; D018655:Lymphocyte Count / Q000379:methods*; D008214:Lymphocytes / Q000166:cytology* / Q000378:metabolism*; D011865:Radioisotope Dilution Technique; D019275:Radiopharmaceuticals / Q000032:analysis / Q000737:chemistry; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity
chemical_list: D019275:Radiopharmaceuticals; D003903:Deuterium; D005947:Glucose; D017666:Deuterium Oxide