Limits...
Reconciling Estimates of Cell Proliferation from Stable Isotope Labeling Experiments.

Ahmed R, Westera L, Drylewicz J, Elemans M, Zhang Y, Kelly E, Reljic R, Tesselaar K, de Boer RJ, Macallan DC, Borghans JA, Asquith B - PLoS Comput. Biol. (2015)

Bottom Line: 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.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.

View Article: PubMed Central - PubMed

Affiliation: Institute for Infection and Immunity, St. George's, University of London, London, United Kingdom.

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.

No MeSH data available.


Related in: MedlinePlus

Enrichment curves of D2-glucose labeled mice.Best fits to the fraction of deuterium enrichment in plasma, and the percentage of label enrichment in the DNA of PBMC, splenocytes and thymocytes after seven-days of D2-glucose labeling. Dots represent individual mice. The end of label administration at day 7 is marked by a dashed vertical line. (A) Conventional normalization: measurements are normalized to the mean plasma enrichment x bg. (B) Normalized with respect to thymocytes: thymocytes were used to determine the maximum percentage of labeled DNA that cells could possibly attain (Methods), and all measured enrichments were scaled to this maximum. The raw data of the % DNA labeled in splenocytes and PBMC is the same in panel A and B but has been normalized using two different approaches.
© Copyright Policy
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC4593553&req=5

pcbi.1004355.g003: Enrichment curves of D2-glucose labeled mice.Best fits to the fraction of deuterium enrichment in plasma, and the percentage of label enrichment in the DNA of PBMC, splenocytes and thymocytes after seven-days of D2-glucose labeling. Dots represent individual mice. The end of label administration at day 7 is marked by a dashed vertical line. (A) Conventional normalization: measurements are normalized to the mean plasma enrichment x bg. (B) Normalized with respect to thymocytes: thymocytes were used to determine the maximum percentage of labeled DNA that cells could possibly attain (Methods), and all measured enrichments were scaled to this maximum. The raw data of the % DNA labeled in splenocytes and PBMC is the same in panel A and B but has been normalized using two different approaches.

Mentions: All cell types showed a progressive increase in DNA enrichment. Strikingly, the maximum enrichment in thymocyte DNA in glucose-labeled mice (about 4.5%) exceeded the estimated precursor (D2-glucose) enrichment, which averaged about 3.3% during the labeling period. Initially, the raw data were normalized following the conventional approach. That is, both D2-glucose and D2O data were first adjusted for plasma deuterium enrichment. The data were then scaled to the maximal level of enrichment; for D2O labeling this was determined using the plateau enrichment of a rapidly turning over cell population (thymocytes in this case), and for D2-glucose by using the in vitro derived constant factor bg = 0.65. The kinetic heterogeneity model was then fitted to the normalized data. This analysis (Figs 3A and 4) yielded substantially different proliferation rates for the D2O and D2-glucose labeling experiments (Fig 5). Similar (differences in) estimates were found using a multi-exponential model.


Reconciling Estimates of Cell Proliferation from Stable Isotope Labeling Experiments.

Ahmed R, Westera L, Drylewicz J, Elemans M, Zhang Y, Kelly E, Reljic R, Tesselaar K, de Boer RJ, Macallan DC, Borghans JA, Asquith B - PLoS Comput. Biol. (2015)

Enrichment curves of D2-glucose labeled mice.Best fits to the fraction of deuterium enrichment in plasma, and the percentage of label enrichment in the DNA of PBMC, splenocytes and thymocytes after seven-days of D2-glucose labeling. Dots represent individual mice. The end of label administration at day 7 is marked by a dashed vertical line. (A) Conventional normalization: measurements are normalized to the mean plasma enrichment x bg. (B) Normalized with respect to thymocytes: thymocytes were used to determine the maximum percentage of labeled DNA that cells could possibly attain (Methods), and all measured enrichments were scaled to this maximum. The raw data of the % DNA labeled in splenocytes and PBMC is the same in panel A and B but has been normalized using two different approaches.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4593553&req=5

pcbi.1004355.g003: Enrichment curves of D2-glucose labeled mice.Best fits to the fraction of deuterium enrichment in plasma, and the percentage of label enrichment in the DNA of PBMC, splenocytes and thymocytes after seven-days of D2-glucose labeling. Dots represent individual mice. The end of label administration at day 7 is marked by a dashed vertical line. (A) Conventional normalization: measurements are normalized to the mean plasma enrichment x bg. (B) Normalized with respect to thymocytes: thymocytes were used to determine the maximum percentage of labeled DNA that cells could possibly attain (Methods), and all measured enrichments were scaled to this maximum. The raw data of the % DNA labeled in splenocytes and PBMC is the same in panel A and B but has been normalized using two different approaches.
Mentions: All cell types showed a progressive increase in DNA enrichment. Strikingly, the maximum enrichment in thymocyte DNA in glucose-labeled mice (about 4.5%) exceeded the estimated precursor (D2-glucose) enrichment, which averaged about 3.3% during the labeling period. Initially, the raw data were normalized following the conventional approach. That is, both D2-glucose and D2O data were first adjusted for plasma deuterium enrichment. The data were then scaled to the maximal level of enrichment; for D2O labeling this was determined using the plateau enrichment of a rapidly turning over cell population (thymocytes in this case), and for D2-glucose by using the in vitro derived constant factor bg = 0.65. The kinetic heterogeneity model was then fitted to the normalized data. This analysis (Figs 3A and 4) yielded substantially different proliferation rates for the D2O and D2-glucose labeling experiments (Fig 5). Similar (differences in) estimates were found using a multi-exponential model.

Bottom Line: 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.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.

View Article: PubMed Central - PubMed

Affiliation: Institute for Infection and Immunity, St. George's, University of London, London, United Kingdom.

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.

No MeSH data available.


Related in: MedlinePlus