Dynamic Simulation and Metabolome Analysis of Long-Term Erythrocyte Storage in Adenine–Guanosine Solution

<div><p>Although intraerythrocytic ATP and 2,3-bisphophoglycerate (2,3-BPG) are known as direct indicators of the viability of preserved red blood cells and the efficiency of post-transfusion oxygen delivery, no current blood storage method in practical use has succeeded in maintaining both these metabolites at high levels for long periods. In this study, we constructed a mathematical kinetic model of comprehensive metabolism in red blood cells stored in a recently developed blood storage solution containing adenine and guanosine, which can maintain both ATP and 2,3-BPG. The predicted dynamics of metabolic intermediates in glycolysis, the pentose phosphate pathway, and purine salvage pathway were consistent with time-series metabolome data measured with capillary electrophoresis time-of-flight mass spectrometry over 5 weeks of storage. From the analysis of the simulation model, the metabolic roles and fates of the 2 major additives were illustrated: (1) adenine could enlarge the adenylate pool, which maintains constant ATP levels throughout the storage period and leads to production of metabolic waste, including hypoxanthine; (2) adenine also induces the consumption of ribose phosphates, which results in 2,3-BPG reduction, while (3) guanosine is converted to ribose phosphates, which can boost the activity of upper glycolysis and result in the efficient production of ATP and 2,3-BPG. This is the first attempt to clarify the underlying metabolic mechanism for maintaining levels of both ATP and 2,3-BPG in stored red blood cells with <i>in silico</i> analysis, as well as to analyze the trade-off and the interlock phenomena between the benefits and possible side effects of the storage-solution additives.</p></div

Enzymatic reactions included in the model ^a.

a<p>From <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0071060#pone.0071060-Kinoshita1" target="_blank">[8]</a>.</p>b,c<p>The enzyme activities in the model were divided into 3 groups: activities of 8 enzymes in purine salvage pathway (b), the activity of the Na⁺/K⁺ pump (c), and the remaining other enzymatic or binding activities <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0071060#pone.0071060-Nishino1" target="_blank">[9]</a>.</p>d,e,f<p>Abbreviations used in this table are as follows: GLC, glucose; G6P, glucose 6-phosphate; F6P, fructose 6-phosphate; F1,6-BP, fructose 1,6-bisphosphate; DHAP, dihydroxyacetone phosphate; GA3P, glyceraldehyde 3-phosphate; 1,3-BPG, 1,3-bisphosphoglycerate; 2,3-BPG, 2,3-bisphosphoglycerate, 3PG, 3-phosphoglycerate; 2PG, 2-phosphoglycerate; PEP, phosphoenolpyruvate; PYR, pyruvate; LAC, lactate; GL6P, gluconolactone 6-phosphate; GO6P, gluconate 6-phosphate; RU5P, ribulose 5-phosphate; X5P, xylulose 5-phosphate; E4P, erythrose 4-phosphate; S7P, sedoheptulose 7-phosphate; R5P, ribose 5-phosphate; PRPP, 5-phosphoribosyl 1-phosphate; ADE, adenine; IMP, inosine monophosphate; R1P, ribose 1-phosphate; INO, inosine; ADO, adenosine; HX, hypoxanthine; AMP, adenosine monophosphate; ADP, adenosine diphosphate; ATP, adenosine triphosphate; NADP, nicotinamide adenine dinucleotide phosphate; NADPH, nicotinamide adenine dinucleotide phosphate (reduced); NAD, nicotinamide adenine dinucleotide; NADH, nicotinamide adenine dinucleotide (reduced); Ki, potassium ion; Nai, Sodium ion; Pi, inorganic phosphate; L_GC, l-glutamyl cysteine; GSH, glutathione (reduced); GSSG, glutathione (oxidized).</p

Pathway reactions in the mathematical model of PAGGGM-stored RBCs.

<p>Nodes indicate metabolites or ions, and edges indicate enzymatic reactions or transport processes, which were divided into 3 groups (<i>red, blue, gray boxes</i>) by the difference in sensitivity of enzymatic or reaction activity to temperature or pH as described in our previous study <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0071060#pone.0071060-Nishino1" target="_blank">[9]</a>. Abbreviations used in this figure are given in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0071060#pone-0071060-t001" target="_blank">Table 1</a>.</p

Prediction of intracellular metabolite levels depending on the combination of initial adenine and guanine concentrations.

<p>Predicted concentrations of ATP (<i>left column</i>), 2,3-BPG (<i>center column</i>), and total HX (<i>right column</i>) at 7, 14, 28, and 35 days after storage in various combinations of ADE and GUO concentrations. In each panel, the x and y axes represent initial concentration of ADE and GUO in the model, respectively. The initial setting of ADE and GUO varied between 0 to 3 mM.</p

Predicted transition of enzymatic activities and schematic representation of flux distributions in PAGGGM-stored RBCs.

<p><i>Panel A</i>: Transition of enzymatic activities. Each enzymatic activity was represented as a normalized value between −1.0 and 1.0 Each value was normalized by an absolute maximum value during 0–35 days of storage simulation. <i>Panel B</i>: Schematic representation of metabolic pools and flux distributions in PAGGGM-stored RBCs during 0–7 days of storage (<i>top panel</i>) and 8–35 days of storage (<i>bottom panel</i>). Total HX represents the sum of intracellular and extracellular HX. Circles and arrows indicate metabolic pools and fluxes, respectively. The width of arrows is proportional to fluxes of metabolic reactions predicted by the model. Abbreviations used in this figure are given in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0071060#pone-0071060-t001" target="_blank">Table 1</a>.</p

Measured and predicted time courses of pH and metabolic intermediates in PAGGGM-stored RBCs.

<p><i>Panel A</i>: Time courses of intracellular pH at 37°C (measured values; ▪) and at 4°C (estimated values; •); fitted value used in the model (–). <i>Panel B</i>: ATP and 2,3-BPG were measured by CE-TOFMS (<i>left column</i>) and predicted by the mathematical model (<i>right column</i>) in which free-form (<i>solid black</i>) and total amount (<i>broken black</i>) of metabolites are shown separately. Since intracellular ATP and 2,3-bisphophoglycerate (2,3-BPG) are known to bind to hemoglobin and band 3 membrane protein <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0071060#pone.0071060-Kinoshita1" target="_blank">[8]</a>, in our model, free-form metabolite represents its amount in plasma, and total amount of metabolite represents the sum of metabolites (free-form) in plasma and their binding to the proteins. <i>Panel C</i>: Measured or predicted time courses of intermediates in glycolysis (G6P, F1,6-BP, DHAP, PYR, LAC), non-oxidative pentose phosphate pathway (R5P, RU5P), and purine salvage pathway (PRPP, intracellular HX, and ADE), respectively. pH and capillary electrophoresis time-of-flight mass spectrometry (CE-TOFMS) data are expressed as means ± SD of 6 separate experiments. An asterisk indicates that a two-sided <i>p</i>-value is <0.01 versus the Day 0 values.</p

Prediction of adenine- and guanosine-dependent metabolic alterations during cold storage.

<p><i>Panel A</i>: Time-related changes of metabolic intermediates with or without adenine (ADE) and guanine (GUO). Simulation with both ADE and GUO (<i>solid black</i>, PAGGGM solution), without GUO (<i>broken black</i>), without ADE (<i>solid gray</i>), and without neither ADE nor GUO (<i>dotted black</i>), respectively. Upper glycolysis represents the total concentrations of G6P and F6P; middle glycolysis represents the total of F1,6-BP, DHAP, and GA3P; and non-oxidative pentose phosphate pathway (non-ox PPP) represents the total of R5P, RU5P, X5P, and R1P. The adenylate pool represents the sum concentration of AMP, ADP, and ATP. Total HX represents the sum of intracellular and extracellular HX. <i>Panel B</i>: Time-related change of ADE flux distribution through AMP to HX or to the adenylate pool. The ratio of ADE flux distribution into HX (<i>gray filled curve)</i> is calculated as <i>J_HX</i>/(<i>J_HX</i> + <i>J_ANPpool</i>) ×100, where <i>J_HX</i> is the sum of enzymatic activities of purine salvage pathway, including AMP deaminase, AMPase, and adenosine kinase, and <i>J_ANPpool</i> is the activity of APK. <i>1^st</i> and <i>2^nd panels</i> show the ratio of ADE flux distribution into HX in the ADE(+)GUO(+) model and ADE(+)GUO(−) model, respectively. <i>3^rd panel</i> shows the time-related changes of total ADE flux (<i>J_HX</i> + <i>J_ANPpool</i>) in the ADE(+)GUO(+) (<i>solid black</i>) and ADE(+)GUO(−) <i>(broken black</i>) model. <i>Panel C</i>: Time-related change of GUO flux distribution through R5P to glycolysis or purine salvage pathway. The ratio of GUO flux distribution into glycolysis (<i>gray filled curves</i>) is calculated as <i>J_glycolysis</i>/(<i>J_glycolysis</i> + <i>J_purine</i>) ×100, where <i>J_glycolysis</i> is the sum of enzymatic activities of R5PI and TK1 and <i>J_purine</i> is the activity of PRPPsyn. <i>1^st</i> and <i>2^nd panels</i> show the ratio of GUO flux distribution into glycolysis in the ADE(+)GUO(+) model and ADE(−)GUO(+) model, respectively. <i>3^rd panel</i> shows the total GUO flux (<i>J_glycolysis</i> + <i>J_purine</i>) in the ADE(+)GUO(+) (<i>solid black</i>) and ADE(−)GUO(+) (<i>broken black</i>) models.</p