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PIMT and RBC metabolism
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The potential role of PIMT in RBC blood storage remains unclear. As with any negative finding, the lack of change in PTR for PCMT1 KO RBC does not formally rule out that PIMT may be involved in the biology of stored RBC, if one evokes the possibility of redundant pathways. However, given the metabolic changes seen in PCMT1 KO RBC, we do not believe redundant pathways are likely. More impor- tantly, a number of the metabolites that are increased in PCMT1 KO RBC have shown a strong inverse correlation to PTR in both human and murine RBC, in particular increased hypoxanthine30 and oxidized lipid species.16 This correlation has held under multiple experimental conditions and contexts; as such, oxidized lipids have been posited to play a causal role in post-transfusion clearance of stored RBC. However, the current report instantiates a condition in which there is an increase in hypoxanthine and certain oxidized lipids, but normal PTR. Thus, this counts as importance evidence against the lipid oxidation hypothesis of RBC clearance after storage. On the other hand, it is worth noting that PCMT1 KO RBC have a compensatory activation of the PPP, which is relevant in the light of the recently reported decreases in PTR in blood donors with G6PD deficiency,29,32 the most common enzymopathy in humans and thus clinically relevant for ~400 million indi-
Figure 4. Metabolic effect of diamide treatment on PCMT1-/- knockout mouse red blood cells. (A) Metabolic effect of diamide treatment on PCMT1-/- knockout (PCMT1 KO) mouse red blood cells (RBC); (B and C) an overview of the Partial Least Square-Discriminant Analysis and hierarchical clustering analysis of sig- nificant metabolites by two-way ANOVA in wild-type (WT) and PCMT1 KO RBC in the presence and absence of diamide for 0, 3 and 6 hours; (D and E) highlight- ed metabolites from the heat map in (C) showing metabolites that increase or decrease in PCMT1 KO mice, respectively.
viduals worldwide. It remains to be assessed whether ablat- ed PIMT activity may result in poor post-transfusion per- formances of the stored RBC in the context of G6PD defi- ciency or other genetic factors that have been shown to negatively impact post-transfusion recoveries in mice (e.g., STEAP316). In addition, the murine recipients here were all healthy, which is not consistent with the pro-oxidant envi- ronment the stored RBC faces upon transfusion in the crit- ically ill or chronically hypoxic recipient (e.g., trauma or sickle cell patient, respectively).33,34
Several unanticipated alterations in metabolism were observed in PCMT1 KO mice, which serve as grounds for reasonable hypothesis driven speculation in the context of broader pathological consequences. Increased kynurenine pathway metabolites have been suggested to be responsi- ble for neurotoxicity, as reported in Down syndrome.28 Prior reports had also suggested an increase in isoaspartyl dam- age and PIMT activation in Down syndrome.7 Dysregulation of methionine metabolism has also been reported in RBC from individuals with Down syndrome;35 this has been partially explained by the localization on chromosome 21 of genes coding for enzymes involved in homocysteine metabolism. Of note, alterations of RBC kynurenines and indoles (breakdown product of trypto-
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