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A. D'Alessandro et al.
PCMT1 KO RBC was observed compared to WT RBC fol- lowing two consecutive (6 h apart) injections of PHZ in vivo (Figure 6B), resulting in double the clearance of the PCMT1 KO RBC within the first 30 h from PHZ injection. Of note, the metabolic phenotypes of PCMT1 KO→WT RBC upon PHZ-induced stress showed significant decreases in the lev- els of glutathione pools (both reduced and oxidized) and accumulation of several short chain fatty acids (e.g., 5:0, 6:0, 8:0, 9:0-OH, 12:0) markers of fatty acid breakdown and oxi- dation in the mitochondria-devoid mature RBC (Figure 6C).
Increased oxidant stress but normal circulation in red blood cells from PCMT1 mice following blood storage
We hypothesized that RBC from PCMT1 KO mice would be more susceptible to storage-induced damage (Figure 7A). RBC were collected from WT and PCMT1 KO mice (at 5 weeks of age prior to seizures) and stored at 2-6 oC for 12 days. Stored RBC were mixed with fresh tracer cells (HOD RBC that express a trackable transgene) in order to control for differences in injection or blood volume. The mixture was transfused into Ubi-GFP+ recipient mice. This approach allows us to determine the percentage of test RBC (GFP- HOD-) as a function of control RBC (GFP-HOD+) that are circulating at 24 h after transfusion, a parameter referred to as post-transfusion recovery (PTR) and one of the Food and Drug Administration-mandated gold stan- dards for RBC storage quality in humans.29
Contrary to our prediction, both fresh and stored WT and PCMT1 KO RBC showed comparable PTR (Figure 7B). Despite the lack of a phenotype with respect to PTR, PCMT1 KO RBC were characterized by (i) a decrease in methionine consumption and accumulation of SAM, but not SAH (consistent with the lack of PCMT1 activity); (ii) significantly lower glutathione pools (especially GSSG) and
AC
B
increased oxidation of methionine thiols to sulfoxide; (iii) increased levels of purine and lipid oxidation markers (hypoxanthine; 15-HETE, 12,13-diHOME, 9-oxononanoic acid) (Figure 7C), previously identified as predictors of poor post-transfusion recoveries in mice and humans.16,30,31 On the other hand, stored PCMT1 KO mouse RBC showed sig- nificantly higher levels of PPP metabolites (gluconolactone phosphate), the CoA precursor pantotheine (indirectly involved in the Lands cycle for the recycling of oxidized lipids) and sphingosine 1-phosphate, at least at baseline (Figure 7C and D).
Discussion
Herein, we report that while PCMT1 is not required for normal erythropoiesis or RBC circulatory lifespan, upon exposure to oxidative stress, RBC lacking PCMT1 are more rapidly cleared from circulation. Metabolically, the absence of PIMT results in a severe depletion of the glutathione pools and a compensatory activation of the PPP to generate the reducing equivalents required for the recycling of oxi- dized thiols. This occurs in both RBC and also peripheral organs. Additional metabolic perturbations are also observed, including increased levels of tryptophan oxidation products (especially metabolites in the kynurenine pathway) and dopamine in both RBC and various organs, including the brain. Since metabolites in the kynurenine pathway can be neurotoxicant,28 it is interesting to note how our metabolomics analysis could provide a potential clue as to the mechanisms that drive early onset of seizures in mice lacking both copies of PCMT1. To our knowledge, this is the first report showing an essential role of PIMT in mainte- nance of RBC circulatory capability during oxidative stress.
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haematologica | 2021; 106(10)