Multi-Omics Evidence for Inheritance of Energy Pathways in Red Blood Cells
Authors: Weisenhorn Erin M , Raife Thomas , van't Erve Thomas J , Riley Nicholas M , Hess John , Coon Joshua J 
Published: October 2019

Abstract
Introduction
Each year over 90 million units of blood are transfused worldwide. Our dependence on this blood supply requires optimized blood collection and storage. During storage, red blood cells (RBCs) undergo degenerative processes resulting in altered metabolic characteristics. In the past decade numerous studies have implicated longer storage of RBCs in adverse patient outcomes. The post-storage ATP level in blood is the single best predictor of transfused RBC in vivo recovery. Although the rate of ATP decline is highly variable between individuals, post-storage ATP levels are primarily determined by inheritance. Understanding the effect of storage on energy metabolism pathways is thus of vital importance to maintaining a safe and effective blood supply.
Methods
We performed comprehensive metabolomics and proteomics studies of mono- and di-zygotic twin pairs to measure heritability of molecules and identify correlations with ATP and other markers in energy metabolism. Metabolite levels were measured at six time points from 0-56 days to elucidate changes that occur during storage. An obstacle for RBC proteomics is the massive quantity of hemoglobin, constituting 97% of protein material. This was avoided by preparing RBC membrane fractions, which mitigated the need for hemoglobin depletion. All proteomics data was collected on an Orbitrap Elite hybrid ion trap-orbitrap mass spectrometer (Thermo Fisher Scientific). Metabolomics data was collected by Metabolon Inc. and was collated with proteomics results to give a complete view of RBC metabolism.
Preliminary Data
Our optimized method for collecting proteomics data in RBCs has yielded the greatest depth of coverage observed without the use of commercial hemoglobin depletion. Purified RBCs were lysed and centrifuged to collect membrane fractions allowing us to identify 1280 proteins and 330 metabolites from mono and di-zygotic twins. Of these, 146 proteins and 148 metabolites were found to be over 30% heritable. We observe a high degree of heritability in metabolites involved in energy metabolism, especially glycolysis. This is supported by the heritability in key regulatory enzymes including phosphofructokinase (PFK) (57%) and bisphosphoglycerate mutase (BPGM) (50%). Additionally we observe high correlations between both glycolytic proteins and metabolites suggesting that this crucial energy metabolism pathway is inherited en blocat various levels.
A number of the correlations we observed can be combined to produce a model to predict post-storage ATP levels. Five key parameters in this model include PFK, carbonic anhydrase 1 (CA1), band 3, BPGM, and pH. Strikingly, concentrations of all protein components of this model were at least 45% heritable. Band 3, BPGM, and CA1 correlate negatively with post storage ATP levels and together shuttle flux away from glycolysis and ATP production. We also observe a positive correlation between pH and post-storage ATP.
A negative correlation observed between CA1, which is 84% heritable, and post-storage ATP, is especially significant in that it provides a hypothetical model for the heritability of ATP decline during storage. Our model proposes that RBC units, which are stored in gas permeable bags allowing CO2 to diffuse into the bag, are subject to genetically determined, CA1-mediated production of carbonic acid, resulting in inhibition of PFK. This model is further supported by negative correlations between CA1 and pH during storage. We propose that heritable concentrations of CA1 negatively influence pH, which allosterically inhibits PFK and impedes energy metabolism and subsequently ATP production.
We conclude that individuals inherit a phenotype composed of higher or lower concentrations of key energy metabolism proteins that regulate flux through glycolysis during RBC storage. Heritability of energy metabolism can result in markedly different RBC storage profiles and knowledge of heritable RBC energy metabolism can be used to improve and individualize RBC storage methods.

Disclosures
Hess: ASH: Patents & Royalties: 4 US patents related to RBC storage solution AS-7.