Evidence for interindividual heterogeneity in the glucose gradient across the human red blood cell membrane and its relationship to hemoglobin glycation

Abstract

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 +/- 0.07 for 3-O-methyl-d-glucopyranose (3OMG) ranged widely (0.72-1.04, n = 26). In contrast, urea C(i)-to-C(o) (1.015 +/- 0.022 [range 0.98-1.07], P < 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) = 0.19) and with the glycation gap (R(2) = 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.

FIG. 1.

A: Time course of ¹⁴C-3OMG uptake into glucose-depleted human erythrocytes at 37°C at a series of initial external sugar concentrations. Values shown are mean of triplicate incubations. B: Dependence of the 3OMG gradient on external 3OMG. The concentration gradient decreases (i.e., C_i-to-C_o approaches 1) with increasing 3OMG across a concentration range broader than the physiologic (n = 7; different symbols and lines denote individual subjects). C: Distribution of C_i-to-C_o determined at steady state in human subjects (Table 1).

FIG. 2.

Paired values of the C_i-to-C_o ratio determined for 3OMG vs. urea within subject are different (P < 0.0001). The C_i-to-C_o ratio approximates unity and has a narrow distribution for urea in all subjects but is below unity and more heterogeneous in the same population for 3OMG.

FIG. 3.

3OMG (3-O-methyl-glucose) C_i-to-C_o dependence on external 3OMG in pink (PG) (A) and white (WG) (B) erythrocyte ghosts compared with intact RBCs (S1–S5 denote different human subjects). The 3OMG concentration gradient was not reduced, and in fact was more pronounced as hemoglobin decreased. A is representative of 17 experiments in eight subjects, and B is representative of one experiment in each of three subjects.

FIG. 4.

A: Effect of transmembrane cation gradient on the 3OMG C_i-to-C_o dependence on external 3OMG. The dependence of 3OMG C_i-to-C_o on 3OMG was compared in extracellular medium containing 137 mmol/l KCl vs. the standard PBS containing 139 mmol/l NaCl. The use of KCl buffer failed to overcome the gradient. The data are representative of one experiment from each of three subjects. B: ATP effects. Preparations of pink ghosts (PG) sealed with the addition of the enzyme apyrase to deplete intracellular ATP failed to shift C_i-to-C_o toward unity. However, the preparation of ghosts reduced the 3OMG dependence of the C_i-to-C_o ratio and was further reduced by apyrase. The data are representative of one experiment in each of four subjects.

FIG. 5.

Membrane gradient reversal. In this experiment, RBCs were first incubated with 3OMG until an apparent steady-state outside-to-inside gradient was reached. Next, the samples (arrow) were diluted twofold to expand the extracellular volume. If the observed gradient was due to a failure to reach true equilibrium, then the gradient would be expected to reverse after dilution. However, after dilution, there was a prompt reestablishment of essentially the same steady-state outside-to-inside gradient, indicating that the sugar moved up a concentration gradient. The data are representative of one experiment in each of 15 subjects.

FIG. 6.

A1C (A) and GG (B) rise as the ¹⁴C-3OMG C_i-to-C_o ratio increases. The GG is a measure of variance in A1C relative to glycated serum proteins. Across the population, intracellular sugar rises relative to extracellular, as does hemoglobin glycation relative to the glycation of extracellular proteins. Values of r² are shown to demonstrate the fraction of the variance in A1C and GG accounted for by C_i-to-C_o ratio. In contrast (Table 2), fructosamine has no significant slope relative to C_i-to-C_o ratio.