Deuterated water ( <scp> ² H ₂ O </scp> ) can be used to quantify hemoglobin synthesis and red blood cell lifespan in humans

The stable isotope experiments by Shemin and Rittenberg in the 1940s employed ingested 15N-glycine to determine that red blood cell (RBC) lifespan was approximately 127 days.1 This estimate was much longer than that provided by agglutination methods2 and established the canonical 120-day lifespan for RBCs that appears in all textbooks today. While this finding is fundamental to our understanding of erythropoiesis in humans, the 15N-glycine method, like other cell labeling methods, models the decay in tracer abundance to estimate the average lifespan of RBCs, which requires an extended protocol (~5–6 months). While a fractional synthetic rate can be estimated from the lifespan (e.g., 120 days corresponds to a turnover rate of 0.8%/day), a more direct and rapid method to assess the rate at which RBCs are produced would be more practical and advantageous for research questions involving interventions. Characteristics of hemoglobin (Hb)—slow turnover and accessibility—make it feasible to assess its fractional synthetic rate with the deuterated water (2H2O) oral stable isotope method (Figure 1).3 Given that Hb comprises ~97% of all RBC protein,4 its synthesis rate is a surrogate of RBC production. While experiments with 2H2O by Ussing in 1941 indicated that this technique had potential in rodents, it was not developed further or applied to human research.5 After dosing with 2H2O, the Hb fractional synthetic rate can theoretically be measured when a linear increase in alanine-bound 2H isolated from Hb is observed in conjunction with stable 2H enrichment in the precursor pool. Although the precursor pool (i.e., bone marrow) is difficult to access in humans, measuring body water 2H enrichment is a noninvasive and valid surrogate.6 As almost all of the proteins in RBCs are synthesized during maturation in the bone marrow,7 there is a delay between the synthesis of Hb and its appearance in the blood. This process requires an extended protocol compared to the use of 2H2O in skeletal muscle (i.e., weeks vs. days), but it is possible to account for this delay in calculations of the fractional synthetic rate. Furthermore, because the total mass of Hb in circulation (i.e., hemoglobin mass or Hbmass) can be measured in humans,8 it is also theoretically possible to measure the absolute synthetic rate for Hb as well as fractional and absolute breakdown rates for Hb. Given the much shorter duration and relative simplicity of the protocol, this oral stable isotope method to measure Hb synthesis could provide insights into many areas of human physiology (e.g., exercise, environmental, clinical). Furthermore, this technique could improve diagnoses and treatments for conditions such as hemolytic anemia or chronic kidney disease through investigations of the underlying pathologies, measures of patient-specific RBC lifespan, or assessments of erythropoietic responses to treatments. As part of a larger study approved by the University of Calgary Conjoint Health Research Ethics Board (REB19-0215)—and conforming to the standards set by the Declaration of Helsinki, except for registration in a database—we assessed the fractional synthetic rate for Hb in humans. Representative data for one participant (female, 28 years) is reported. Using the 2H2O method, the Hb fractional synthetic rate for this individual was 0.78%/day, translating to a Hb lifespan of ~128 days, and an absolute synthetic rate of 5.3 g/day, given a Hbmass of 671 g. While the 2H2O method appears to be useful for quantifying Hb synthesis and could facilitate future studies in human physiology related to erythropoiesis, some assumptions used to calculate protein synthesis rates from metabolic tracer data must be clarified. First, we need to measure APE in the correct precursor pool. Since the measurement period in the present study was weeks rather than hours, it is almost certain that the 2H-labeled free alanine pool present in bone marrow was in equilibrium with 2H-labeled body water and therefore this assumption can be considered met. Second, we need to measure APE in the correct protein of interest. As 97% of RBC protein is Hb,4 we analyzed proteins liberated from the RBC fraction of blood samples without further Hb-isolating methods. Third, we must assume that no recycling of the precursor takes place. While ineffective erythropoiesis,11 the destruction of developing RBC in the marrow, can be significant in disorders of hemoglobin synthesis (e.g., folate or iron deficiency or thalassemia), our representative participant had no known conditions affecting erythropoiesis, meaning this process is unlikely to be a major factor contributing to precursor recycling. While significant shedding of Hb-containing vesicles from circulating RBCs is possible, it is unlikely to impact the 2H2O method, given this phenomenon seemingly occurs in the second half of the RBC lifecycle (i.e., after 60 days of age).12 Finally, because there is a delay in the appearance of labeled Hb relative to synthesis (Figure 1), the precursor and protein enrichments need to be aligned for calculation. Given that it took ~2 weeks for 2H label appearance in Hb to stabilize, we chose to use the body water APE from 2 weeks prior (i.e., first 14 days) when calculating the fractional synthetic rate for Hb using the final 14 days. As body water enrichment was relatively stable (Figure 1), this decision only had a minor effect on our calculations. In conclusion, the novel method to quantify the Hb synthetic rate by deuterated water labeling is feasible and provides results aligning with expected rates of erythropoiesis, previously only indirectly derived from RBC lifespan measurements. Future studies can use the technique to investigate the erythropoietic process in various clinical conditions or in response to exercise, nutritional, and environmental stressors, and for cross-validation of existing techniques in hematology. Hilkka Kontro: Conceptualization; investigation; writing – original draft; methodology; visualization; writing – review and editing; formal analysis; data curation. Chris McGlory: Conceptualization; investigation; methodology; visualization; writing – review and editing; formal analysis; resources; supervision; project administration. Martin J. MacInnis: Conceptualization; investigation; funding acquisition; writing – original draft; methodology; visualization; writing – review and editing; formal analysis; project administration; supervision; resources; data curation. The authors would like to thank the participants as well as Oluwatimilehin Ajayi for his help with data collection. This work was supported by an operating grant from the Natural Sciences and Engineering Research Council of Canada (NSERC; grant number RGPIN-2018-06424), start-up funding from the Faculty of Kinesiology (University of Calgary), and a University of Calgary VPR Catalyst Grant, all received by MJM. HK was supported by a Faculty of Kinesiology Dean’s Doctoral Scholarship. The data that support the findings of this study are available from the corresponding author upon reasonable request.