Evidence for Increased Red Cell Turnover in Unexplained Anemia in Patients Over 70 Years of Age
Authors: Friedman Jeff , Waalen Jill , Takeda Alisa , Beutler Ernest 
Published: October 2019

Abstract
Unexplained anemia in the elderly is a diagnosis of exclusion that becomes progressively more prevalent with each increasing decile of age. The frequency of anemia in individuals 85 and older is reported to be in excess of 20%, with as many as 1/3 of cases failing to be assigned to known causes such as renal disease, nutritional deficiency, chronic blood loss, malignancy or inflammatory disorder. We have collected samples from over 500 individuals 70 years and older who presented at a health appraisal clinic for routine evaluation, filled out a detailed questionnaire regarding medical history, diet and environmental exposures, and who donated blood for analysis. From this population, 11% were found to be anemic. Of these individuals, 68% were found to have no identifiable cause for their anemia (so called AUE or anemia of unknown etiology). Laboratory based exclusion criteria included low platelet count, elevated creatinine, evidence of nutritional deficiency, evidence of inflammatory disease or elevated ALT. AUE patient red cell samples and an equivalent number of age and sex matched non-anemic samples were analyzed for reticulocyte count, real time production of reactive oxygen species, protein carbonyl content of red cells, hexokinase activity, glutathione peroxidase activity and level of reduced glutathione. Granulocyte DNA was prepared from each sample for assessment of telomere length and telomere single stranded overhang. Interim analysis of these assays was performed when the number of control and AUE patient samples reached ~ 20 each. Interim analysis shows no correlation between anemia and telomere length, or anemia and the length of the telomeric single-strand overhang. Within our cohort of 70–90 year olds, we see no correlation between age and telomere length suggesting that our sample size in the interim analysis was insufficient to draw conclusions for these assays. Similarly, no significant differences were observed between AUE and control groups in the reactive oxygen species assay, glutathione peroxidase assay or measurement of reduced glutathione. Hexokinase activity was increased in the AUE group relative to control samples, and showed a strong negative correlation with hemoglobin levels. Reticulocyte count showed a weak negative correlation with hemoglobin levels and a weak positive correlation with hexokinase activity—although in 17/18 AUE samples reticulocyte counts were within the normal range.
Conclusions: An interim analysis of AUE samples versus age and sex matched controls demonstrates an increase in hexokinase activity in the anemic group. Compared to most other red cell enzymes, hexokinase activity shows a dramatic decline during red cell lifespan—and for this reason has been used as a surrogate marker of red cell age. Our data suggest that the average red cell age in AUE samples is younger than in control samples—providing evidence for increased red cell turnover in AUE. We are in the process of testing additional red cell enzymatic and metabolic markers that are reported to change with cell age to further investigate this finding.