Old RBCs deplete carnitine pools and have impaired Lands cycle. (A) Double biotinylation studies in mice afford labeling and sorting of in vivo RBCs based on their age in circulation. Numbers indicate the main steps for this experiment, from double biotinylation of the first mouse (old RBCs), to the biotinylation of the second mouse (young RBCs), to leukodepletion and streptavidin and thioflavin-T labeling, to separation of young (9-15 days) and old RBCs (41-47 days) via sorting. (B) Metabolomics analyses of young vs old RBCs shows age-dependent depletion in carnitine pools in the latter group. Asterisks next to each row of the heat map denote significance (unpaired t test: ∗∗P < .01 and ∗∗∗P < .0001). (C) Percoll-density gradient-based separation of human RBCs of different ages shows a decline in RBC carnitine content in older, smaller erythrocytes, with progressively smaller mean cell volumes (MCV) and higher band 4.1a:4.1b ratios. (D) Incubation of the old, young, and average age populations of RBCs with radioactive palmitate shows a carnitine/age-dependency of labeled palmitate incorporation in phosphatidylethanoloamines.