Bioactive Lipids Are Generated to Micromolar Levels during RBC Storage, Even in Leukoreduced Units

Background: The transfusion of stored red blood cells (RBCs) has been linked to negative outcomes, such as Transfusion-Related Acute Lung Injury (TRALI) and Transfusion Related Immunomodulation (TRIM). Bioactive lipids have been implicated as playing an important role in TRALI pathogenesis, both as a "second hit" when anti-HLA antibodies are present and also in anti-HLA independent TRALI. Likewise, bioactive lipids have been suggested to play a role in TRIM.

Methods: To further study bioactive lipids in RBC storage, we developed a targeted metabolomics approach, with isotope dilution, to accurately and precisely quantify concentrations of multiple poly-unsaturated fatty acids (PUFAs) and their related oxidation products (oxylipins) in stored RBC units. Analytes in RBC samples were extracted using methanol with isotopically-labeled internal standards and analyzed by liquid chromatography-tandem mass spectrometry using multiple reaction monitoring (LC-MS/MS-MRM). Eight units of leukoreduced (LR) and non-leukoreduced (NLR) RBCs were analyzed longitudinally from 6 to 42 days of storage.

Results: LR RBCs had a significant increase in multiple PUFAs over storage, including: arachidonic acid (AA), linoleic acid (LA), docosahexaenoic acid (DHA), eicosapentaenoic acid (EPA), linolenic acid (ALA), dihomo-γ-linolenic acid (DGLA), adrenic acid (22:4), and docosapentaenoic acid (22:5). Only a modest increase was observed over the first 20 days of storage, followed by a rapid increase from day 20 to day 42. AA showed the biggest increase in concentration, from 1.0±0.4 µM at day 6 to 13.4±5.2 µM at day 42 in LR RBC units. Oxidative products of PUFA metabolism were also analyzed. The concentration of hydroxyeicosatetraenoic acids (HETEs), oxidation products of AA, including 5-, 8-, 9-, 11-, 12- and 15-HETEs increased 5 to 13 fold from day 6 to day 42 of storage in LR RBCs. The concentration of other oxylipins including hydroxyoctadecadienoic acids(9- and 13-HODEs)and dihydroxyoctadecenoic acids (, 9, 10- and 12,13-DiHOMEs), hydroxyl-DHA(14-, 16- and 17-HDoHEs) were also significantly increased during storage. If present, prostaglandins (PGE2 and PGD2) and Leukotriene (LTB4), were below the lower limits of detection. Juxta position of LR and NLR RBCs, showed that the concentrations of 12-HETE, 15-HETE, 14-HDoHE, and 17-HDoHE in NLR units were significantly higher at day 42 (p<0.01). Of particular note, the average concentration of 12-HETE reached 1.1±0.8 µM in NLR at day 42, which was 45 fold higher than that in LR units. No differences in LR and NLR RBCs were noted at day 6.

Conclusions: We report significant generation of PUFAs and products of lipid metabolism/oxidation over RBC storage, with a particular increase in generation after day 20 of storage. LR decreased the generation of some lipid species (12-HETE in particular); however, accumulation of many bioactive lipids occurs robustly despite LR. Because variation from unit to unit was small at day 6 but substantial at day 42, these findings also support significant donor-to-donor differences in the process of bioactive lipid generation during storage. Exactly how the generation of bioactive lipids effects medical outcomes of transfusion remains undetermined; however, the quantitation allowed by the current methods demonstrates the generation of bioactive lipids up to µM levels, which are known to be sufficient to have significant physiological and/or pathological impacts in other settings.