nature publishing group Translational Investigation Articles Methylglyoxal concentrations differ in standard and washed neonatal packed red blood cells Autumn S. Kiefer1, Thomas Fleming2, George J. Eckert3, Brenda B. Poindexter1, Peter P. Nawroth2 and Mervin C. Yoder1 BACKGROUND: Preterm infants have a greater risk of nec- ≤28 wk of gestation, and this led to endothelial cell activa- rotizing enterocolitis following transfusion. It is hypothesized tion as evidenced by soluble intracellular adhesion molecule-1 that high glucose concentrations in red blood cell (RBC) pre- release (4). servatives lead to increased methylglyoxal (MG) metabolism, causing glycation-driven damage to transfused RBCs. Such Methylglyoxal and Advanced Glycation End Product Formation changes to the RBCs could promote a proinflammatory state Packed RBCs for transfusion are stored in preservatives that in transfusion recipients. allow the cells to remain metabolically active, utilizing the pre- METHODS: Standard and washed RBCs in Adsol-3, two servative’s glucose supply in glycolysis. Methylglyoxal (MG) is a common neonatal preparations, were studied. Consecutive natural by-product of glycolysis, formed from the spontaneous measurements were performed of glucose, MG, reduced glu- degradation of the triose phosphate intermediates (Figure 1). tathione, glyoxalase I activity (GLO-I), and D-lactate, the stable MG belongs to a class of small molecular weight compounds end product of MG detoxification by glyoxalase enzymes over known as the dicarbonyls, which are potent glycating agents. the 42-d storage period. During glycation, either intact glucose or dicarbonyls react RESULTS: RBC units consume glucose and produce D-lactate with proteins at lysine and arginine residues via the Maillard and MG during storage. In 28/30 units, the MG concentrations reaction to form advanced glycation end products (AGEs) showed only small variations during storage. Two units had (5,6). To minimize the extent of glycation, MG is metabolized elevated MG levels (>10 pmol/mg Hb) during the first half of by the glyoxalase I and II enzymes (GLO-I and GLO-II) using storage. Washing of the RBCs significantly reduced both MG reduced glutathione as a cofactor (7). The glyoxalase system and D-lactate. forms d-­lactate, which is distinct from the l-lactate stereo- CONCLUSION: This study shows two patterns of MG metabo- isomer formed by glycolysis under anaerobic conditions (8). lism in packed RBCs for neonatal transfusion and raises the pos- However, in the setting of hyperglycemia, higher levels of the sibility that RBC units with higher MG levels may have increased dicarbonyls are generated, and reduced glutathione may be glycation-driven damage in the transfused RBCs. Whether depleted (9,10). transfused MG could trigger an inflammatory response such The physiologic importance of MG and AGE formation as necrotizing enterocolitis in preterm neonates and whether remain under investigation; however, glycation can interfere washing could prevent this require further study. with the structure and function of proteins (11). Studies in patients with diabetes mellitus have shown a substantial link between MG and AGE accumulation and the development and ransfusion-related acute gut injury (TRAGI) is a condi- progression of nephropathy, neuropathy, and retinopathy (12– Ttion described in preterm infants who develop necrotiz- 14). AGEs also potentiate cellular dysfunction through inter- ing enterocolitis in the 48-h period following a packed red action with specific cell surface receptors, such as the receptor blood cell (RBC) transfusion (1). While blood product and for AGEs (RAGE) (15–18). AGE–RAGE interactions lead to transfusion recipient factors jointly contribute to the risk of a proinflammatory phenotype (17,18) through the expression transfusion-related acute lung injury in adult and pediatric of cytokines, growth factors, and adhesion molecules (tumor populations (2,3), only a few studies have investigated proin- necrosis factor-α, interleukin-1, platelet-derived growth fac- flammatory factors in blood products with respect to TRAGI. tor, insulin-like growth factor-1, interferon-γ´ intracellular Blau et al. (1) did not detect anti-HLA or antineutrophil anti- adhesion molecule-1, or vascular cell adhesion molecule-1) bodies in a single donor unit, whose recipient died follow- (19–21). RAGE activation also increases RAGE expression, ing TRAGI. However, plasma inflammatory cytokines, such suggesting that repeated exposures to AGEs can prime a cell as interleukin-8 and tumor necrosis factor-α, were found to for a larger magnitude inflammatory response on subsequent increase following routine transfusions of preterm infants exposures (22). 1Department of Pediatrics, Indiana University School of Medicine, Indianapolis, Indiana; 2Department of Internal Medicine and Clinical Chemistry, University of Heidelberg, Heidelberg, Germany; 3Department of Biostatistics, Indiana University School of Medicine, Indianapolis, Indiana. Correspondence: Autumn S. Kiefer ([email protected]) Received 30 May 2013; accepted 5 September 2013; advance online publication 22 January 2014. doi:10.1038/pr.2013.243 Copyright © 2014 International Pediatric Research Foundation, Inc. Volume 75 | Number 3 | March 2014 Pediatric REsEarch 409 Kiefer et al. Articles Glucose ATP Hexokinase D-Lactate ADP Glucose-6-phosphate H2O G-6-P Glyoxalase II Isomerase Fructose-6-phosphate ATP Phospho- fructokinase ADP Glutathione S-D-lactoylglutathione Fructose-6-diphosphate Aldolase TPI 1.3- Glyoxalase I Glyceraldehyde-3 diphospho- phosphate glycerate Cellular dysfunction & AGEs proinflammatory phenotype Pyruvate Methyglyoxal Hypoxia L-Lactate dehydrogenase L-Lactate Figure 1. Intracellular methylglyoxal (MG) formation and removal. The breakdown of glucose to pyruvate via the Embden–Meyerhof pathway of glycoly- sis is shown on the left. The early steps of glycolysis and the formation of triose phosphate intermediates (TPI) are detailed, as the spontaneous dephos- phorylation of TPI leads to the formation of MG and other dicarbonyls. MG either forms AGEs by binding to free amino groups or it is removed by the activity of the glyoxalase enzymes with conversion to D-lactate (dashed arrow on right). The gray arrows show the effects of increased glucose supplies, with increased MG and AGE formation, leading to a proinflammatory state. The shadowed arrow shows the distinct pathway by whichL -lactate may form. ADP, adenosine diphosphate; AGE, advanced glycation end product; ATP, adenosine triphosphate. Few studies have evaluated the effects of high glucose storage antibodies, hepatitis B and C viruses, human immunodefi- conditions on transfused RBCs. Lysenko et al. (23) found that ciency virus, human T-lymphotropic virus, West Nile Virus, AGE levels increased in packed RBCs over a 42-d storage period. and syphilis antibody. None of the packed RBC units had Mangalmurti et al. (24) showed that stored RBCs had significantly bacterial infection on day 20 or day 42. One unit did have a greater levels of a lysine-derived AGE, carboxymethyllysine, and contaminated culture at day 20, but subsequent cultures on stored RBCs caused significantly more in vitro RAGE activation days 25 and 42 were negative for any bacterial growth without than fresh RBCs. It can therefore be hypothesized that this AGE intervention. accumulation occurs due to MG generation by the stored RBCs during glycolysis. Under physiologic conditions, MG rapidly Glucose Metabolism forms AGEs or is removed by the glyoxalase enzymes. However, Glucose concentrations were determined in packed RBC sam- we hypothesize that the low temperature and pH conditions used ples, which were hemolyzed for analysis. The mean initial glu- for blood storage will slow MG binding to free amino groups cose concentration was 907 ± 10 mg/dl, and this decreased to and lead to a larger pool of unbound MG. Thus, the previously 578 ± 14 mg/dl by the end of the storage life (Figure 2). With described elevations in AGE levels may not represent the full washing, the mean glucose concentration decreased signifi- extent of glycation damage within stored RBCs. This hypothesis cantly to 27 ± 1 mg/dl (P < 0.0001). Washing did not reduce is supported by the observation that another intermediate in the glucose concentration as much if it was performed at the glycation reactions, labile hemoglobin A1c (HbA1c), is a minor end of the storage period (P < 0.0001), with a mean decline in variant of HbA1c in vivo but can represent a larger proportion of glucose on day 0 of 886 ± 51 mg/dl, day 20 of 628 ± 100 mg/dl, Hb in stored blood (25). Determining the amount of unbound and day 42 of 550 ± 32 mg/dl. MG in the transfused blood is a key first step to understanding the potential impact of glycation-damaged RBCs on transfusion Glucose-Mediated Changes in Packed RBCs recipients and ultimately determining if these changes are linked Donor whole blood samples had a mean labile HbA1c of to the development of posttransfusion inflammatory responses 2.1 ± 0.05% and mean stable HbA1c of 5.6 ± 0.17%. In the like TRAGI in preterm infants. packed RBC units from these donors, the stable form slowly increased over the 42 d of storage (5.8 ± 0.17% on day 42; P < RESULTS 0.0001). Stable HbA1c was higher for AB+ than for A+ units Donor Unit Profiles regardless of storage age (P = 0.0499). Labile HbA1c had more Of the 30 packed RBC units, 7 were blood type AB positive abrupt changes with the greatest percent present on day 0 of and 23 were A positive. All units tested negative for red cell storage (4.7 ± 0.05%; P < 0.0001), declining to 2.9 ± 0.07% on 410 Pediatric REsEarch Volume 75 | Number 3 | March 2014 Copyright © 2014 International Pediatric Research Foundation, Inc. Methylglyoxal in transfused blood Articles 1,200 2,500 1,000 mol/l) 2,000 µ ( 800 1,500 1,000 600 500 Glucose (mg/dl) 400 -Lactate concentration D 0 200 0510 15 20 25 30 35 40 45 Days of storage 0 0510 15 20 25 30 35 40 45 Figure 3. D-Lactate concentrations significantly increase until the limits Days of storage of packed RBC storage.