Two-dimensional analysis of glycated hemoglobin heterogeneity in pediatric type 1 diabetes patients James M. Hempe a,b,⇑ , Amanda M. McGehee a , Stuart A. Chalew a,b a Research Institute for Children, Children’s Hospital, New Orleans, LA 70118, USA b Department of Pediatrics, Louisiana State University Health Sciences Center, New Orleans, LA 70118, USA article info Article history: Received 5 June 2013 Received in revised form 7 July 2013 Accepted 9 July 2013 Available online 20 July 2013 Keywords: Glycated hemoglobin Hemoglobin A1c Glucose Diabetes Multidimensional analysis abstract Interindividual and ethnic variation in glycated hemoglobin levels, unrelated to blood glucose variation, complicates the clinical use of glycated hemoglobin assays for the diagnosis and management of diabetes. Assessing the types and amounts of glycated hemoglobins present in erythrocytes could provide insight into the mechanism. Blood samples and self-monitored mean blood glucose (MBG) levels were obtained from 85 pediatric type 1 diabetes patients. Glycated hemoglobin levels were measured using three pri- mary assays (boronate-affinity chromatography, capillary isoelectric focusing (CIEF), and standardized DCA2000+ immunoassay) and a two-dimensional (2D) analytical system consisting of boronate-affinity chromatography followed by CIEF. The 2D system separated hemoglobin into five subfractions, four of which contained glycated hemoglobins. Glycated hemoglobin measurements were compared in patients with low, moderate, or high hemoglobin glycation index (HGI), a measure of glycated hemoglobin con- trolled for blood glucose variation. MBG was not significantly different between HGI groups. Glycated hemoglobin levels measured by all three primary assays and in all four glycated 2D subfractions were significantly different between HGI groups and highest in high HGI patients. These results show that interindividual variation in glycated hemoglobin levels was evident in diabetes patients with similar blood glucose levels regardless of which glycated hemoglobins were measured. Ó 2013 Published by Elsevier Inc. Hemoglobin is a tetrameric red blood cell (RBC) 1 protein con- sisting of two pairs of a,b globin heterodimers [1,2]. High blood glu- cose concentrations promote the nonenzymatic posttranslational chemical modification of specific globin amino groups via the Mail- lard reaction. These spontaneous reactions occur at rates determined by glucose concentration and amino group pK a . Glycation proceeds through a series of intramolecular rearrangements and fragmenta- tions that produce an assortment of early, intermediate, and late gly- cation products. Glycated hemoglobin is a general term used to describe the heterogeneous set of hemoglobin complexes derived by the Maillard reaction. Glycated hemoglobin assays can be divided into three general types based on their mode of separation [3]. Boronate affinity chro- matography divides hemoglobin molecules into boronate-reactive and boronate-unreactive fractions based on the presence or ab- sence, respectively, of chemical modifiers that contain accessible coplanar vicinal diol groups (two hydroxyl groups on adjacent car- bon atoms) [4,5]. In contrast, modern immunoassays use monoclo- nal antibodies developed against synthetic peptides that mimic the glycated N-terminus of b globin [3,6]. Charge-based assays, such as cation-exchange chromatography and isoelectric focusing, can sep- arate hemoglobin molecules into multiple fractions based on dif- ferences in surface charge and isoelectric point (pI) [7]. In this report, the abbreviation A1c is used as originally intended [8], to identify a specific low pI hemoglobin fraction isolated by charge- based separation techniques. Glycated hemoglobin assays are used clinically to indirectly monitor blood glucose levels in diabetes patients [9]. Because dif- ferent glycated hemoglobin assays measure different subsets of chemically modified hemoglobins, they also give different results for the same blood sample. Widespread interassay variation prompted the development of national and international standard- ization programs to ‘‘harmonize’’ assays used by hospital laborato- ries and clinics [10,11]. In this report, the abbreviation HbA1c is used to specifically refer to results obtained using standardized glycated hemoglobin assays. Glycated hemoglobin assay standardization has unquestionably improved the care and outcomes of diabetes patients. New con- cerns have emerged, however, about the way HbA1c assay results are used to diagnose and manage diabetes [12–15]. At issue is the entrenched practice of using HbA1c as an unbiased estimate of blood glucose concentration. The clinical use of HbA1c first arose 0003-2697/$ - see front matter Ó 2013 Published by Elsevier Inc. http://dx.doi.org/10.1016/j.ab.2013.07.016 ⇑ Corresponding author at: Research Institute for Children, Children’s Hospital, New Orleans, LA 70118, USA. Fax: +1 504 896 9413. E-mail address: jhempe@chnola-research.org (J.M. Hempe). 1 Abbreviations used: RBC, red blood cell; HGI, hemoglobin glycation index; PBS, phosphate-buffered saline; CIEF, capillary isoelectric focusing; MBG, mean blood glucose; CBG, capillary blood glucose; GHb, glycated hemoglobin; pI isoelectric point. Analytical Biochemistry 442 (2013) 205–212 Contents lists available at ScienceDirect Analytical Biochemistry journal homepage: www.elsevier.com/locate/yabio