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BackgroundIncreases in the red blood cell (RBC) degree of fatty acid desaturation are reported in response to exercise, aging, or diseases associated with systemic oxidant stress. However, no studies have focused on the presence and activity of fatty acid desaturases (FADS) in the mature RBC.Study design and methodsSteady state metabolomics and isotope‐labeled tracing experiments, immunofluorescence approaches, and pharmacological interventions were used to determine the degree of fatty acid unsaturation, FADS activity as a function of storage, oxidant stress, and G6PD deficiency in human and mouse RBCs.ResultsIn 250 blood units from the REDS III RBC Omics recalled donor population, we report a storage‐dependent accumulation of free mono‐, poly‐(PUFAs), and highly unsaturated fatty acids (HUFAs), which occur at a faster rate than saturated fatty acid accumulation. Through a combination of immunofluorescence, pharmacological inhibition, tracing experiments with stable isotope‐labeled fatty acids, and oxidant challenge with hydrogen peroxide, we demonstrate the presence and redox‐sensitive activity of FADS2, FADS1, and FADS5 in the mature RBC. Increases in PUFAs and HUFAs in human and mouse RBCs correlate negatively with storage hemolysis and positively with posttransfusion recovery. Inhibition of these enzymes decreases accumulation of free PUFAs and HUFAs in stored RBCs, concomitant to increases in pyruvate/lactate ratios. Alterations of this ratio in G6PD deficient patients or units supplemented with pyruvate‐rich rejuvenation solutions corresponded to decreased PUFA and HUFA accumulation.ConclusionFatty acid desaturases are present and active in mature RBCs. Their activity is sensitive to oxidant stress, storage duration, and alterations of the pyruvate/lactate ratio.

Deficiency of Glucose 6 phosphate dehydrogenase (G6PD) is the single most common enzymopathy, present in approximately 400 million humans (e.g., 5% of humans). Its prevalence is hypothesized to be due to conferring resistance to malaria. However, G6PD deficiency also results in hemolytic sequelae from oxidant stress. Moreover, G6PD deficiency is associated with kidney disease, diabetes, pulmonary hypertension, immunological defects, and neurodegenerative diseases. To date, the only available mouse models have decreased levels of wild-type stable G6PD caused by promoter mutations. However, human G6PD mutations are missense mutations that result in decreased enzymatic stability. As such, this results in very low activity in red blood cells that cannot synthesize new protein. To generate a more accurate model, the human sequence for a severe form of G6PD deficiency (Med -) was knocked into the murine G6PD locus. As predicted, G6PD levels were extremely low in RBCs and deficient mice have increased hemolytic sequalae to oxidant stress. Non-erythroid organs had metabolic changes consistent with mild G6PD deficiency, consistent with what has been observed in humans. Juxtaposition of G6PD deficient and wild-type mice revealed altered lipid metabolism in multiple organ systems.Together, these findings both establish a new mouse model of G6PD deficiency that more accurately reflects human G6PD deficiency and also advance our basic understanding of altered metabolism in this setting. Introduction:Glucose-6-phosphate dehydrogenase (G6PD) is the first and rate determining enzyme in the pentose phosphate pathway (PPP), which utilizes glucose to generate NADPH; the latter is the major reducing equivalent that fuels multiple pathways by which cells handle oxidative stress.Deficiency in the activity of G6PD is the single most common enzymopathy in humans, estimated to be present in approximately 400 million people worldwide(1). The complete absence of G6PD is not compatible with life, and the vast majority of mutations leading to G6PD deficiency in humans are missense mutations leading to an unstable G6PD enzyme. Based upon the mutation and resulting G6PD levels, disease can range from mild to severe deficiency (2, 3). RBCs from humans with G6PD deficiency are particularly susceptible to oxidant stress for two prevailing reasons. First, because RBCs lack mitochondria, the PPP is their main source of NADPH. Second, mature RBCs are unable to synthesize new proteins. When G6PD deficient humans encounter an illness or consume a drug or food that increases oxidant stress (e.g., antimalarial quinone drugs or fava beans), they can manifest symptoms of acute hemolysis, ranging from mild to lethal(4, 5).Recent findings have also linked G6PD deficiency to many other diseases outside the RBC compartment, including immunological(1), cardiovascular(6), endocrine(7), renal(7), neurological(8), and pulmonary pathologies(9). Because G6PD deficiency is so prevalent in humans, and because its biology remains poorly understood, a translatable anim...

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