- Original Articles
- Open Access
Characterization of a Novel Hemoglobin-Glutathione Adduct That Is Elevated in Diabetic Patients
© Picower Institute Press 2001
- Accepted: 15 June 2001
- Published: 1 September 2001
Typically, a diagnosis of diabetes mellitus is based on elevated circulating blood glucose levels. In an attempt to discover additional markers for the disease and predictors of prognosis, we undertook the characterization of HbA1d3 in diabetic and normal patients.
Material and Methods
PolyCAT A cation exchange chromatography and liquid chromatography-mass spectroscopy was utilized to separate the α- and β-globin chains of HbA1d3 and characterize their presence in normal and diabetic patients.
We report the characterization of HbA1d3 as a glutathionylated, minor hemoglobin subfraction that occurs in higher levels in diabetic patients (2.26 ± 0.29 %) than in normal individuals (1.21 ± 0.14%, p < 0.001). The α-chain spectrum displayed a molecular ion of m/z 15126 Da, which is consistent with the predicted native mass of the HbA0 α-globin chain. By contrast, the mass spectrum of the β-chain showed a mass excess of 307 Da (m/z = 16173 Da) versus that of the native HbA0 β-globin chain (m/z = 15866 Da). The native molecular weight of the modified β-globin chain HbA0 was regenerated by treatment of HbA1d3 with dithiothreitol, consistent with a glutathionylated adduct.
We propose that HbA1d3 (HbSSG) forms normally in vivo, and may provide a useful marker of oxidative stress in diabetes mellitus and potentially other pathologic situations.
Once a mammalian red cell is released into the blood stream, it loses its capacity to synthesize protein. During a red cell’s 120-day life span, its proteins are susceptible to several posttranslational modifications, including nonenzymatic glycation and oxidation. The accumulation of such protein modifications is considered to be typical of the cellular and molecular changes associated with the aging process (1–4). Accordingly, an important clinical marker of diabetes mellitus is the hemoglobin species known as HbA1c. HbA1c occurs when the amino terminal valine residue of the β-chain of globin becomes covalently derivatized with an Amadori product via the Maillard reaction (5,6). Because the formation of the slowly reversible Amadori product depends on circulating glucose levels, this marker accumulates to a higher degree in the red cells of those diabetics with higher hyperglycemia. Similarly, the accumulation of advanced glycation endproducts (AGEs) on hemoglobin and other proteins has been used as a long-term marker of glucose control in diabetes. Using AGEs as a marker, rather than HbA1c, has the advantage of presenting an indication of circulating glucose levels over an extended period of time (1–4,7–10).
Although no de novo protein synthesis takes place in erythrocytes, the cells exhibit very active enzymatic synthesis of reduced glutathione (GSH), which is present at an intracellular concentration of 2.3 mM. In the red cell, GSH is an essential component for the maintenance of HbA0 in a physiologically active form. Glutathione disulfide (GSSG), the oxidized form of GSH (present at a concentration of 4.0 µM) is either continuously reduced by the glutathione reductase system or actively transported out of the erythrocyte so as to maintain a high intracellular GSH/GSSG ratio. It has been widely reported that diabetics have lower intracellular levels of GSH, and this is considered to be indicative of increased oxidative stress in these patients (11–14). Reduced GSH levels are believed to be a result of at least three circumstances within the cell: (1) a decrease in the activity of γ-glutamyl-cysteine synthetase (the enzyme that is responsible for the first step in glutathione synthesis), possibly due to nonenzymatic glycation of the enzyme (15); (2) a decrease in the activity of glutathione reductase, the enzyme that catalyzes the reduction of GSSG to GSH (also a result of enzyme glycation) (16); and (3) a decrease in the activity of the Mg+2 ATPase transporter responsible for GSSG export resulting in an increase of the intraerythrocytic level of GSSG (13). These factors result in decreased synthesis of new glutathione, as well as decreased regeneration of GSH from GSSG, and decreased transport of GSSG to the outside of the red cell. This creates an ideal environment for the formation of hemoglobin (and other protein)-glutathione adducts (HbSSG).
HbSSG can be synthetically prepared by reacting GSSG (17–19) with human hemoglobin in vitro. Successful experimental formation of HbSSG in vitro led to the suggestion that HbSSG might also form under in vivo conditions, but this has been shown only to occur under extraordinary circumstances of long-term therapy with red-ox active drugs. In a population of patients treated with the anti-epileptic agents phenobarbital and caramazepin, Niketic et al. (20) detected by isoelectric focusing a minor hemoglobin, designated HbA1x, which they propose to be a glutathione-modified form.
Although a simple diagnosis of “diabetes mellitus” can be made on the basis of abnormally elevated blood sugar levels, additional markers may be valuable in the diagnosis and management of the disease and in the formation of a prognosis of the development of multi-organ complications (3). Herein, we report the isolation and molecular characterization of a variant of hemoglobin, termed HbSSG (HbA1d3), which occurs at higher levels in Type I diabetics than in normal subjects. Its formation from GSSG suggests that this novel hemoglobin species may represent a specific marker of oxidative stress.
Human diabetic whole blood, preserved in EDTA, was generously provided by Dr. Helen Vlassara (Mount Sinai School of Medicine, NY, NY, USA). Control blood was obtained from age-matched, nondiabetic volunteers. Blood samples were centrifuged at 1800 ×g for 10 min at 4°C to obtain packed red blood cells (RBCs). To 1 ml of packed RBCs, 3 ml dH2O was added to lyse cells and 2 ml of toulene was added for delipidation. Vigorous vortexing for 3 min ensured complete extraction of lipids. Hemolysates then were centrifuged at 1800 ×g for 10 min at 4°C to separate the aqueous from the nonaqueous phases. The nonaqueous phase was removed by aspiration and a glass pipette was used to remove the hemolysate. Hemolysates were stored at −70°C until use.
Fractionation of Hemoglobin Species
To achieve separation of the various glycated hemoglobin species, PolyCAT A (PolyLC, Columbia, MD, USA) cation exchange chromatography (200 × 4.6 mm) was employed. The chromatographic system consisted of a Waters 600E controller, a 60F pump, a 996 photodiode array detector, and a 717 plus auto sampler. Buffer A was 35 mM Bis-Tris (all chemicals in buffers purchased from Sigma, St. Louis, MO, USA), 16.85 mM ammonium acetate, 90 mM sodium acetate, and 1.5 mM potassium cyanide, and was buffered to a pH of 6.8 with acetic acid. Buffer B was 35 mM Bis-Tris, 3 mM ammonium acetate, and 1.5 mM potassium cyanide, and was buffered to pH 6.5 with acetic acid. The gradient started isocratically at 22% A, 78% B for the first 3 min and was maintained at a flow rate of 3.0 ml/min throughout the run. From 3–30 min, buffer A increased linearly to 50%, and then linearly to 100% from 3040 min. Buffer A was maintained at 100% until 40.5 min and then decreased to 22% over the next 5 min. The column then was equilibrated over the next 20 min with the initial solvent ratio. Fractions were collected upon monitoring absorbance at 415 nm.
A C4-reversed phase column (1 × 50 mm) (Vyadec, 5 µM) at a flow rate of 50 µλ/min was used for α- and β-chain separation. Solvent C contained 0.05% triflouroacetic acid (TFA) in ddH2O, and solvent D contained 0.05% TFA in acetonitrile. The column was eluted with a binary C:D solvent gradient beginning at 40% D and linearly increasing to 60% D in 20 min. Column eluate was monitored at 214 nm, and peaks were analyzed by electrospray ionization mass spectrometry (combined LC-ESI) wherein ESI spectra were scanned from 10,00–35,000 mass units at a scan cycle of 5 sec/scan.
Isolation of Human Hemoglobin
Separation and Characterization of the α- and β-Globin Chains of HbA1d3
Susceptibility of Glycated Hb to Glutathione Modification
HbA1d3 and HbA1d3 Levels in Non-diabetic and Diabetic Individuals
We previously identified a glutathionylated hemoglobin in diabetic rats using ion exchange chromatography and LC-MS (21). Niwa et al. (22,23) recently reported the identification of a glutathionylated HbA0 that is present at higher levels in diabetic (Type I) and hyperlipidemic patients. It was concluded in that study that there is no correlation between the formation of HbA1c and HbA1d3 in diabetic and hyperlipidemic patients. Their finding was based however on detecting a mass unit of 16,173 Da for a modified β-globin subfraction that corresponds to glutathionylated β-globin and runs parallel with the native β-globin chain in reverse phase HPLC. The elevated level of this glutathionylated Hb in diabetic (Type I) and hyperlipidemic patients was based on a nonquantitative mass spectra analysis.
In summary, we have isolated and characterized HbA1d3, a normally occurring minor hemoglobin subfraction that results from a glutathione adduct at the β-93 cysteine residue (HbSSG). This adduct accounts for 1.21 ± 0.14% versus 2.26 ± 0.29% of the total hemoglobin in normal individuals and diabetic patients, respectively. We propose that the minor hemoglobin species HbA1d3, or HbSSG, forms normally in vivo, and may provide a useful marker of oxidative stress in a variety of pathologic situations.
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