Although red blood cell (RBC) life span is a known determinant of percentage hemoglobin A1c (HbA1c), its variation has been considered insufficient to affect clinical decisions in hematologically normal persons. However, an unexplained discordance between HbA1c and other measures of glycemic control can be observed that could be, in part, the result of differences in RBC life span. To explore the hypothesis that variation in RBC life span could alter measured HbA1c sufficiently to explain some of this discordance, we determined RBC life span using a biotin label in 6 people with diabetes and 6 nondiabetic controls.

OBJECTIVE-To determine whether interindividual heterogeneity in the erythrocyte (red blood cell [RBC]) transmembrane glucose gradient might explain discordances between A1C and glycemic control based on measured fructosamine. RESEARCH DESIGN AND METHODS-We modeled the relationship between plasma glucose and RBC glucose as the concentration distribution (C i -to-C o ratio) of a nonmetabolizable glucose analog 14 C-3-O-methyl glucose ( 14 C-3OMG) inside (C i ) and outside (C o ) RBCs in vitro. We examined the relationship between that distribution and the degree of glycation of hemoglobin in comparison with glycation of serum proteins (fructosamine), the glycation gap. A1C, fructosamine, and in vitro determination of the 14 C-3OMG distribution in glucose-depleted RBCs were measured in 26 fasted subjects.RESULTS-The C i -to-C o ratio 0.89 Ϯ 0.07 for 3-O-methyl-Dglucopyranose (3OMG) ranged widely (0.72-1.04, n ϭ 26). In contrast, urea C i -to-C o (1.015 Ϯ 0.022 [range 0.98 -1.07], P Ͻ 0.0001) did not. Concerning mechanism, in a representative subset of subjects, the C i -to-C o ratio was retained in RBC ghosts, was not dependent on ATP or external cations, and was reestablished after reversal of the glucose gradient. The 3OMG C i -to-C o ratio was not correlated with serum fructosamine, suggesting that it was independent of mean plasma glucose. However, C i -to-C o did correlate with A1C (R 2 ϭ 0.19) and with the glycation gap (R 2 ϭ 0.20), consistent with a model in which differences in internal glucose concentration at a given mean plasma glucose contribute to differences in A1C for given level of glycemic control.CONCLUSIONS-The data demonstrate interindividual heterogeneity in glucose gradients across RBC membranes that may affect hemoglobin glycation and have implications for diabetes complications risk and risk assessment. Diabetes 57: [2445][2446][2447][2448][2449][2450][2451][2452] 2008 A 1C is the current gold standard for determination of chronic glycemic control in people with diabetes. Yet it is common to find hematologically normal people with diabetes in whom A1C appears discordant from other measures of glycemic control. Some have suggested the notion of a "hemoglobin glycation index" to assess A1C discordance from mean blood glucose (1-3). We have quantitated this discordance with the use of the glycation gap (GG), a measure of the disparity between two integrated measures of glycemic control, one intracellular in red blood cells (RBCs), A1C, and the other extracellular, glycated serum protein (GSP) measured as fructosamine (4). A non-zero GG could result from differences between the ambient glucose concentrations or rates of glycation in the intracellular and extracellular compartments, and/or interindividual differences in the turnover/metabolism of underlying proteins. We have demonstrated that the GG is reproducible within subjects over time and is associated with important clinical end points. In longstanding type 1 diabetes, there is a over a twofold rise in prevalence of nephropathy in pati...

HbA1c is commonly used to monitor glycemic control. However, there is growing evidence that the relationship between HbA1c and mean blood glucose (MBG) is influenced by variation in red blood cell (RBC) lifespan in hematologically normal individuals. Correction of HbA1c for mean RBC age (MRBC) requires a noninvasive, accurate, and affordable method to measure RBC survival. In this study, we evaluated whether a stable isotope approach would satisfy these requirements. RBC lifespan and MRBC were determined in a group of nine hematologically normal diabetic and nondiabetic subjects using oral 15N-glycine to label heme in an age cohort of RBC. The MRBC was 58.7 ± 9.1 (2SD) days and RBC lifespan was 106 ± 21 (2SD) days. This degree of variation (±15 - 20%) is consistent with previous studies using other techniques. In a subset of seven subjects, MRBC determined with the biotin label technique were available from approximately five years prior, and strongly correlated with the stable isotope values (R2 = 0.79). This study suggests that the MRBC is stable over time but varies substantially among individuals, and supports the importance of its variation in HbA1c interpretation. The characteristics of the stable isotope method support its suitability for studies to directly evaluate the impact of variation in MRBC on the interpretation of HbA1c.

Hemolysis is a key feature of sickle cell anemia (HbSS). Direct quantitation of hemolysis could be used as an objective outcome in clinical trials of new therapeutics for HbSS and would also enable better human studies of the pathogenesis of complications of HbSS that are ostensibly hemolysis-related, such as pulmonary hypertension. However, contemporary human studies in HbSS have used only surrogate markers of hemolysis rather than direct measurements of RBC survival. We directly quantified hemolysis in HbSS by measuring survival of an age cohort of RBCs labeled with a stable isotope, administered orally as 15N-glycine, a metabolic precursor of heme. The atomic excess of 15N in heme extracted from blood was monitored by mass spectrometry over time. We performed 13 labeling experiments in 11 individuals with HbSS. Mean RBC survival was 31.9 days (range 14.1 – 53.6). Both HbF level, a known determinant of hemolysis, and absolute reticulocyte count (ARC), an index of the marrow’s response to hemolysis, correlated with directly measured RBC survival (r=0.61, P<0.002; r=−0.84, P<0.001). However, commonly used biochemical surrogates of hemolysis (LDH, AST, bilirubin and plasma free hemoglobin) did not correlate with directly measured RBC survival. These biochemical surrogates should be interpreted cautiously, at best, in clinical trials and human physiologic studies in HbSS. ARC was the best correlate of total hemolysis, but only 70% of the variation in RBC survival was reflected in this marker. If greater accuracy is required in human studies, 15N-glycine RBC labeling can directly and accurately quantify hemolysis.