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Blood, Vol. 93 No. 1 (January 1), 1999:
pp. 376-384
Evaluation of Biochemical Changes During In Vivo Erythrocyte
Senescence in the Dog
Michael P. Rettig,
Philip S. Low,
J. Aura Gimm,
Narla Mohandas,
Jiazhen Wang, and
John A. Christian
From the Departments of Chemistry and Veterinary Pathobiology, Purdue
University, West Lafayette, IN; and Lawrence Berkeley National
Laboratory and UCSF/UCB Bioengineering Graduate Group, Berkeley, CA.
One hypothesis to explain the age-dependent clearance of red blood
cells (RBCs) from circulation proposes that denatured/oxidized hemoglobin (hemichromes) arising late during an RBC's life span induces clustering of the integral membrane protein, band 3. In turn,
band 3 clustering generates an epitope on the senescent cell surface
leading to autologous IgG binding and consequent phagocytosis. Because
dog RBCs have survival characteristics that closely resemble those of
human RBCs (ie, low random RBC loss,  115-day life span), we decided
to test several aspects of the above hypothesis in the canine model,
where in vivo aged cells of defined age could be evaluated for
biochemical changes. For this purpose, dog RBCs were biotinylated in
vivo and retrieved for biochemical analysis at various later dates
using avidin-coated magnetic beads. Consistent with the above
hypothesis, senescent dog RBCs were found to contain measurably
elevated membrane-bound (denatured) globin and a sevenfold enhancement
of surface-associated autologous IgG. Interestingly, dog RBCs that were
allowed to senesce for 115 days in vivo also suffered from compromised
intracellular reducing power, containing only 30% of the reduced
glutathione found in unfractionated cells. Although the small quantity
of cells of age  110 days did not allow direct quantitation of band 3 clustering, it was nevertheless possible to exploit single-cell microdeformation methods to evaluate the fraction of band 3 molecules that had lost their normal skeletal linkages and were free to cluster
in response to hemichrome binding. Importantly, band 3 in RBCs 112
days old was found to be 25% less restrained by skeletal interactions
than band 3 in control cells, indicating that the normal linkages
between band 3 and the membrane skeleton had been substantially
disrupted. Interestingly, the protein 4.1a/protein 4.1b ratio, commonly
assumed to reflect RBC age, was found to be maximal in RBCs isolated
only 58 days after labeling, implying that while this marker is useful
for identifying very young populations of RBCs, it is not a very
sensitive marker for canine senescent RBCs. Taken together, these data
argue that several of the readily testable elements of the above
hypothesis implicating band 3 in human RBC senescence can be validated
in an appropriate canine model.
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