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. Author manuscript; available in PMC: 2016 Jan 12.
Published in final edited form as: J Sci Technol Environ. 2015;5(1):3000251.

Increase in Blood Glutathione and Erythrocyte Proteins Related to Glutathione Generation, Reduction and Utilization in African-American Old Women with Diabetes

Guang Shan 1, Fang Yang 2, LiChun Zhou 3, Tian Tang 1, Emmanuel U Okoro 3, Hong Yang 3, ZhongMao Guo 3
PMCID: PMC4710176  NIHMSID: NIHMS711714  PMID: 26770888

Abstract

Data from this report demonstrate that the plasma and erythrocyte levels of total glutathione (TGSH) are significantly lower in nondiabetic old women than in their young counterparts, and significantly higher in diabetic patients than in age-matched nondiabetic controls. The ratio of reduced glutathione (GSH) to oxidized glutathione (GSSG) declines with age and diabetes, and shows an order as follows: nondiabetic young > nondiabetic old > diabetic old women. In addition, advanced glycation end-products (AGEs) accumulates in RBCs obtained from diabetic patients but not in those from young and old nondiabetic controls. The erythrocyte levels of glutamate cysteine ligase catalytic subunit (GCLC), glucose-6-phosphate dehydrogenase (G6PD), glutathione reductase (GR), glutathione peroxidase-1 (GPx1), glutathione S-transferase-ρ1 (GST-ρ1) and glyoxalase I (Glo1) are comparable in nondiabetic young and old women, but significantly higher in diabetic patients than in age-matched nondiabetic controls. Oxidative stress has been suggested to upregulate the expression of these proteins. It is possible that increase in oxidative stress in diabetes, reflected by reduced GSH/GSSG ratio and accumulation of AGEs, upregulates the expression of proteins involved in glutathione synthesis, reduction and utilization in erythrocyte precursor cells, and that overexpression of GCLC is, at least partially, responsible for the increased TGSH in diabetes.

Keywords: diabetes, aging, erythrocyte, glutathione, glutamate cysteine ligase

1. Introduction

The prevalence of diabetes mellitus in adults is ~1.6 fold higher in African-Americans (AA) than in European-Americans [1]. Diabetes is particularly common in AA women. It has been reported that ~28% of AA women have diabetes after 50 years-old [1, 2]. AAs with diabetes are more likely to develop complications and show higher mortality and disability from the complications than European-Americans with diabetes [1, 2]. Beyond possible socioeconomic contributions, the mechanism for the higher prevalence and poorer prognosis of diabetes in AAs remains undefined.

Glutathione has been implicated in many cellular activities, including scavenging oxidants and detoxifying toxic substances, such as polycyclic aromatic hydrocarbons and advanced glycation end-products (AGEs) [3]. Increase in generation of free radicals and AGEs has been reported to contribute to the complications of diabetes. Therefore, it is of a great interest to investigate the change of glutathione and the enzymes related to its generation, reduction and utilization in diabetic patients in different ethnic groups, including African-Americans [4, 5]. Data from previous studies indicated that glutathione levels in the plasma and red blood cells (RBCs) of diabetic patients compared to normal subjects were lower [4–6].

The cellular level of glutathione reflects a steady-state balance between the rates of its de novo synthesis and of transport in and out cells [7]. Plasma glutathione originates largely from liver cells. Glutathione molecules leave hepatocytes primarily in its reduced form. The kidney, lung, intestine and other organs that express γ-transpeptidase take up glutathione from the plasma and utilize liver-derived glutathione. It has been suggested that RBCs only release oxidized glutathione (GSSG) [8], and do not take up glutathione from the plasma due to absence of γ-transpeptidase.

The de novo synthesis of glutathione involves two steps [3]. The first step, i.e., synthesis of γ-glutamylcysteine from L-glutamate and cysteine, is catalyzed by glutamate cysteine ligase (GCL), previously known as γ-glutamylcysteine synthetase. GCL is a heterodimeric protein composed of a catalytic and a regulatory subunit. The second step adds a glycine to the C-terminal of γ-glutamylcysteine, which is catalyzed by glutathione synthetase. The reaction catalyzed by GCL is the rate-limiting step in glutathione synthesis. Glutathione synthesis occurs in virtually all cell types, including RBCs. Inhibition of GCL has been shown to significantly deplete glutathione in cultured cells and tissues within hours [9]. This underscores that regulation of GCL expression and activity is one of the major determinants of glutathione homeostasis.

The reduced glutathione (GSH) serves as a proton donor in several oxidation-reduction processes [3]. For example, glutathione peroxidases (GPxs) reduce hydrogen peroxide and oxidized macromolecule such as oxidized proteins using glutathione as a proton donor. In this process, GSH losses proton and becomes GSSG. To maintain the redox state of the cell, GSSG is recycled to GSH by glutathione reductase (GR) [10], using nicotinamide adenine dinucleotide phosphate (NADPH) as proton donor. Glucose-6-phosphate dehydrogenase (G6PD) is the rate-limiting enzyme for generation of NADPH [11]. Under normal conditions, cells have sufficient GR and NADPH for maintaining most cellular glutathione in its reduced form, i.e., GSH. However, under severe oxidative stress conditions or when GR and/or G6PD expression and activity are impaired, the ability of cells to reduce GSSG may be overwhelmed, resulting in its accumulation in cells.

GSH also functions as a cofactor of the glyoxalase system, which consists of glyoxalase I (Glo1) and Glo2 [12]. The glyoxalase system catalyzes the conversion of α-oxoaldehydes (e.g., methylglyoxal) to aldonate (e.g., D-lactate). The α-oxoaldehydes serve as precursors for AGEs. Thus, decrease in glutathione [13] and/or glyoxalases [14] might slow down the elimination of carbonyl intermediates and increase the generation of AGEs. In addition, GSH can interacts with a large variety of electrophilic substances to form glutathione conjugates. The conjugation reaction is catalyzed by glutathione S-transferases (GSTs). The glutathione-conjugated substances can be subsequently metabolized and excreted, and therefore reduce the toxicity induced by electrophilic substances [15].

In this study, we determined the concentrations of plasma and erythrocyte glutathione, and the protein levels of erythrocyte GCLC, GR, G6PD, GST-ρ1, GPx1 and Glo1 in diabetic old AA women, and nondiabetic young and old AA women. Our data showed that the TGSH level and GSH/GSSG ratio in the plasma and RBCs declined with age; however, the levels of proteins involved in glutathione synthesis and reduction, i.e., GCLC, G6PD and GR, were similar in nondiabetic young and old women. As compared to age-matched nondiabetic control subjects, the diabetic patients showed significantly higher levels of TGSH and lower levels of GSH/GSSG ratio in the plasma and RBCs, and higher levels of GCLC, GR, G6PD, GST-ρ1, GPx1 and Glo1 in RBCs. These results differ with previous reports that showed significantly lower levels of glutathione and proteins related to its generation, reduction and utilization in diabetic patients than in normal control subjects [4, 5]. Further studies are needed to reveal the different findings of our laboratory and others.

2. Materials and Methods

2.1. Materials

Assay kits for determination of glutathione concentration (703302), and the activities of glutathione peroxidase (GPx) (703102), glutathione S-transferase (GST) (703302) and glutathione reductase (GR) (703202) were purchased from Cayman Chemical (Ann Arbor, MI). Antibodies against human GPx1 (ab22604), GST-ρi (ab134934), GR (ab55075), glutamate cysteine ligase catalytic subunit (GCLC) (ab55435), glyoxalase I (Glo1) (ab96032), advanced glycation end products (AGEs) (ab176173) and catalase (ab16731) were purchased from Abcam (Cambridge, MA), while the antibodies against glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (sc305062), glucose-6-phosphate dehydrogenase (G6PD) (sc373887) and horseradish peroxidase-conjugated secondary antibodies were obtained from Santa Cruz Biotechnology (Santa Cruz, CA). Histopaque (10771) and assay kits for determination of glucose (GAGO-20), cholesterol (MAK043) and triglyceride (TR0100) concentrations were obtained from Sigma-Aldrich (St. Louis, MO).

2.2. Subjects

This study includes three groups, i.e., diabetic old (45–93 yrs) women, and nondiabetic young (19–35 yrs) and old (47–88 yrs) women. All study subjects self-identified as African-Americans. They were recruited from Metropolitan Nashville General Hospital at Meharry or from numerous health fairs affiliated with Meharry Medical College [16]. Immediately before the start of the study, a medical basal screening was performed by a physician. The 15 diabetic women participating in this study had suffered diabetes mellitus for 2 – 20 years and were receiving antidiabetic therapy. Patients whose fasting plasma glucose was higher than 130 mg/dl were excluded. The fasting plasma glucose level of the individuals in the nondiabetic young and old control groups was lower than 100 mg/dl. The general and clinical data are shown in Table 1. No participants were taking glutathione and N-acetylcysteine, which might affect cellular glutathione levels. Fully informed consent was obtained from all subjects. This study was approved by the Institutional Review Board of Meharry Medical College.

Table 1.

General Characteristics of the Study Population

Younger Controls Older Controls Older Patients
Age (years) (26±5) 24–35 56±13 (45 – 93) * 63±14 (47 – 88) *
Number of participants 15 15 15
Body mass index (kg/m2) 28.4±6.2 31.4±6.2 32.0±7.8
Baseline plasma glucose (mg/dl) 75±8 87±14 96±18
Total plasma cholesterol (mg/dl) 153±21 182±42 177±38
Total plasma triglycerides (mg/dl) 76±17 91±52 101±43
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Values are means ± SEM.

*

A statistically significant difference from younger or older nondiabetic controls (P<0.01).

2.3. Collection and preparation of blood samples

About 20 ml of venous blood was collected in tubes containing ethylene diamine tetra acetic acid as anticoagulant, and immediately centrifuged at 1000 rpm for 10 min at 4°C. Plasma was transferred to a new tube and mixed with butylated hydroxytoluene (final concentration 5 mM); platelets were separated from plasma by centrifugation at 2,400 rpm for 10 minutes. Blood cells were suspended in 10 ml of phosphate-buffered saline (PBS), and 10 ml of histopaque was gently overlaid on the cell suspension. The resulting cocktail was centrifuged at 4000 rpm for 30 min to separate white blood cells and red blood cells (RBCs). The RBCs were collected and suspended in 10 ml of PBS.

2.4. Analysis of plasma glucose, cholesterol, and triglycerides

Plasma glucose, cholesterol, and triglyceride concentrations were measured by spectrophotometric quantification using reagents obtained from Sigma-Aldrich. Briefly, 3 µl of plasma was incubated at 37 °C for 30 minutes with 297 µl of glucose-, cholesterol- or triglyceride- reaction reagents in a glass microplate. For measuring glucose and triglyceride, the absorbance was read at 540 nm with a Dynex microplate reader (Thermo Labsystems, Franklin, MA). For measuring cholesterol, the absorbance was read at 570 nm. Plasma glucose, cholesterol and triglyceride concentrations were calculated based on the absorbance obtained with the glucose, cholesterol and triglyceride standards provided by the manufacturer.

2.5. Measurement of plasma and erythrocyte glutathione

The level of glutathione in plasma and RBCs was determined using a Cayman glutathione assay kit. Briefly, 100 µl RBCs were lysed in 400 µl ice-cold HPLC grade water, and centrifuged at 10,000 × g for 15 min at 4°C. The RBC lysate or 100 µl plasma were mixed with equal volume of 10% metaphosphoric acid, and centrifuged at 5,000 × g for 5 min at room temperature. The supernatant was then mixed with 4 M triethanolamine (TEAM) at a ratio of 50 µl TEAM and 1 ml supernatant. For measurement of total glutathione (TGSH), 50 µl of RBC or plasma extract was mixed in a 96-well plate for 5 min at 25°C with 150 µl glutathione assay kit reagent, which contains glutathione reductase (GR), 5,5′-dithio-bis-(2-nitrobenzoic acid) (DTNB) and NADPH. The absorbance was read at 412 mm with a Dynex microplate reader (Thermo LabSystems). For measurement of oxidized glutathione (GSSG), 10 µl of 2-vinylpyridine (a GR inhibitor) solution was added into the glutathione assay reagent mixture, as the manufacture’s instruction advised. The absorbance was read as described above. The level of reduced glutathione (GSH) was calculated from the difference between TGSH and oxidized GSSG.

2.6. Activity assays of Glutathione Peroxidase (GPx), Glutathione S-Transferase, and GR

Three Cayman assay kits were used to determine the activities of GPx, GST and GR in RBCs. Briefly, 0.5 ml RBCs were lysed in 2 ml ice-cold HPLC grade water, and centrifuged at 10,000 × g for 15 min at 4°C. RBC protein extracts in the supernatant were collected for enzyme activity assays. For measurement of GPx activity, 20 µl RBC extract was mixed with 180 µl GPx activity assay reagent, which contains assay buffer, glutathione, NADPH, GR and cumene hydroxide. For measurement of GST activity, 20 µl of plasma mixed with 180 µl GST activity assay reagent, which contains assay buffer, 1-chloro-2,4-dinitrobenzene (CDNB) and GSH. The activity of GST was measurement as the amount of CDNB conjugation with reduced glutathione per minute. For measurement of GR activity, 20 µl of RBC extract mixed with 180 µl GR activity assay reagent, which contains assay buffer, GSSG and NADPH. For all three assays, absorbance at 340 nm was monitored using a Dynex microplate reader (Thermo LabSystems).

2.7. Western blot analysis

The protein levels of GCLC, G6PD, GR, GST-ρ1, GPx1, Glo1, catalase, AGEs and GAPDH in RBCs were measured by western blot analysis. Protein sample were prepared as described previously [16]. Briefly, 100 µl of RBCs was washed thrice with ice-cold 0.9 % Nacl, and then vigorously vortexed with 400 µl of 0.5% Triton X-100 and sonicated for 30 sec to break the plasma membranes. The hemolysate was mixed with 500 µl of chloroform/ethanol solution (15/25 v/v), and the mixture was centrifuged at 13,000 rpm at 4 °C for 5 min. The resultant supernatant was lyophilized with a freeze dry system (Labconco, Kansas city, MO). The pellet was dissolved in 100 µl of 0.9 % Nacl. Final concentrations of protein were determined by the BCA protein assay kit (Thermo Scientific). The RBC proteins were resuspended in PBS and separated by a 10 % SDS-polyacrylamide gel. Proteins were transferred to a polyvinylidene difluoride (PVDF) membrane (Millipore, Billerica, MA). After blocked with 3% fat-free milk, the membranes were incubated with antibodies against human GCLC, G6PD, GR, GST-ρ1, GPx1, Glo1, catalase, AGEs and GAPDH, and then incubated with horseradish peroxidase-conjugated secondary antibodies. Immunoreactive bands were visualized using ECL-plus chemiluminescence reagent (GE Healthcare–Amersham) and analyzed with imaging densitometer (Bio-Rad, Hercules, CA) [17]. The uneven sample loading was normalized using the intensity ratio of the immunoreactive bands of the tested proteins relative to GAPDH.

2.8. Statistical analysis

For each variable, results are expressed as mean ± standard error of the mean (SE). Data distribution was examined by Shapiro-Wilk normality test. Comparisons between control and hypertensive groups were carried out with Student’s t test. Differences were considered significant at a P value less than 0.05. All statistical analyses were performed using Stastix software (Analytical Software, Tallahassee, FL).

3. Results

3.1 Increase in blood glutathione in diabetic patients

High variations of glutathione concentrations in human plasma (2–20 µM) and RBCs (1–10 µmol/g Hb) have been reported from different laboratories [18–20]. Data in Fig. 1 show that the plasma and erythrocyte levels of total glutathione (TGSH) are ~3.2 µM and 4.1 µmol/g protein, respectively; and the ratios of GSH to GSSG in the plasma and RBCs are ~4.5 and 11.5, respectively, in nondiabetic young women. As compared to these young subjects, the nondiabetic old women show ~35 and 27% lower plasma and erythrocyte TGSH, and ~60 and 41% lower GSH/GSSG ratio in the plasma and RBCs, respectively. Glutathione is an important antioxidant. The GSH/GSSG ratio is an indicator of the cellular redox state. Decrease in TGSH level and GSH/GSSG ratio in old nondiabetic women suggests an age-related increase in oxidative stress in the plasma and RBCs.

FIGURE 1.

FIGURE 1

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The effect of diabetes on erythrocyte glutathione levels. Plasma and red blood cells were obtained from old women with diabetes mellitus (O DM), and nondiabetic young and old control women (Y Ctrl and O Ctrl). The level of plasma and erythrocyte total and oxidized glutathione (TGSH and GSSG) was determined using a glutathione assay kit, and the level of reduced glutathione (GSH) was calculated by subtraction of GSSG from TGSH. Data represent the mean ± SE (n=15/group). *P<0.05 vs. Y Ctrl, and † P <0.05 vs. O Ctrl.

The data in Fig. 1 also show that the plasma TGSH and GSSG levels are ~1.62 and 2.54 fold higher, and the erythrocyte TGSH and GSSG levels are ~1.46 and 4.85 fold higher in diabetic patients than in age-matched nondiabetic subjects. No significant difference is observed between these two groups of subjects with regard to the plasma and erythrocyte GSH levels. A remarkable increase in GSSG without a significant change in GSH results in a significant reduction in plasma and erythrocyte GSH/GSSG ratios in diabetic patients compared to the age-matched controls (Fig. 1B and 1D). The plasma and erythrocyte TGSH levels in diabetic old women are comparable to those in the nondiabetic young women (Fig. 1A and 1C). However, the GSH and GSSG levels between these two groups are significantly different. The plasma and erythrocyte GSH/GSSG ratios are ~5.3 and 6.8 fold lower in diabetic old women than in nondiabetic young women. These results suggest an increased oxidative stress in the plasma and RBCs obtained from diabetic old women compared to those obtained from either young or age-matched nondiabetic women.

3.2 Increase in proteins for synthesis, reduction and utilization of glutathione in the RBCs of diabetic patients

To explore the mechanism underlying the age- and diabetes-related changes in erythrocyte glutathione, we determined the level of proteins involved in glutathione synthesis, reduction and utilization, including GCLC, G6PD, GR, GST-ρ1, Glo1 and GPx1, as well as the activity of some of these proteins in RBCs. As the data in Fig. 2 show, the protein level of erythrocyte GST-ρ1 is ~33% lower in nondiabetic old women than their young counterparts, while the GCLC, G6PD, GR, Glo1 and GPx1 protein levels are comparable in these two age groups of nondiabetic women. The data in Fig. 2 also show that the levels of all the aforementioned proteins are significantly higher in the diabetic old women than in both nondiabetic young and old women. For example, the levels of GCLC, G6PD, GR, Glo1, GST-ρ1 and GPx1 in the old diabetic patients increased by ~3, 1.7, 2.6, 1.9, 4.8 and 1.8 fold, respectively, when compared to those in age-matched nondiabetic controls.

FIGURE 2.

FIGURE 2

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The effects of diabetes on proteins involved in glutathione synthesis, reduction and utilization, as well as catalase (Cat) and advanced glycation end products (AGEs). Red blood cells were obtained from old women with diabetes mellitus (O DM), and nondiabetic young and old control women (Y Ctrl and O Ctrl). The protein level of glutamate cysteine ligase catalytic subunit (GCLC), glucose-6-phosphate dehydrogenase (G6PD), glutathione reductase (GR), glyoxalase I (Glo1) glutathione S-transferase-ρ1 (GST- ρ1), glutathione peroxidase-1 (GPx1), Cat and AGEs was determined with western blot analysis, and expressed relative to glyceraldehyde-3-phosphate dehydrogenase (GAPDH). Data represents the mean ± SE (n=15/group). *P<0.05 vs. Y Ctrl, and † P <0.05 vs. O Ctrl.

The data in Fig. 3 show that the activities of erythrocyte GSTs and GR is reduced by ~53 and 37% in nondiabetic old women compared to young women, but no significant difference in GPx activity is observed between these two age groups (Fig. 3). The activities of GST-ρ1, GR and GPx1 in unit of cellular proteins are comparable in RBCs obtained from diabetic patients and young nondiabetic subjects (Fig. 3), though the expression level of these proteins is much higher in RBCs obtained from the former than those obtained from the latter (Fig. 2).

FIGURE 3.

FIGURE 3

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The effect of diabetes on the activities of glutathione reductase (GR), glutathione S-transferase (GSTs) and glutathione peroxidases (GPxs). Red blood cells were obtained from old women with diabetes mellitus (O DM), and nondiabetic young and old control women (Y Ctrl and O Ctrl). The activity of GR, GSTs and GPxs was determined using commercial assay kits. Data represent the mean ± SE (n=15/group). *P<0.05 vs. Y Ctrl, and † P <0.05 vs. O Ctrl.

3.3 Increase in erythrocyte AGEs and decrease in catalase activity in diabetic patients

AGEs are formed from nonenzymatic reaction of the carbonyl groups of reducing sugars with the free amino groups on proteins. AGE formation is accelerated under conditions of hyperglycemia and increased oxidative stress [21]. Hemoglobin accounts for over 95% of the soluble proteins in RBCs. The data in Fig. 2 show that RBCs obtained from the diabetic patients, including those whose fasting plasma glucose concentrations were controlled under 100 mg/dl, show high immunereactivity to an anti-AGE antibody. However, AGEs are barely detectable in the RBCs obtained from both nondiabetic young and old women.

Catalase is a protein that coverts hydrogen to oxygen and water. The data in Fig. 2 show that the catalase protein level is comparable among these 3 groups. The activity of catalase is comparable between nondiabetic young and old women, but is reduced by 32 and 27% in diabetic patients when compared to young and old normal women, respectively.

4. Discussions

Data from the present report showed significant lower concentrations of TGSH and GSH, and lower GSH/GSSG ratios in the plasma and RBCs obtained from old nondiabetic women than in those obtained from young nondiabetic women. This is in consistent with previous reports showing age-related decrease in glutathione and increase in oxidative stress in human plasma [6], RBCs [22] and whole blood [23]. It has been reported that RBCs release GSSG to the plasma, while other cells, such as the liver cells, transport GSH out of cells [6, 24]. The age-related decrease in plasma TGSH and GSH thus might be a result of glutathione deficiency in the liver and other tissues in old subjects.

This report also demonstrated that the protein level of glutamate cysteine ligase catalytic subunit (GCLC), G6PD and GR was comparable between young and old nondiabetic women. Thus, the age-related decrease in erythrocyte glutathione level and GSH/GSSG ratio is unlikely due to a reduced expression of GCLC, G6PD and GR. Data from a previous study suggests that decrease in glutathione precursors could be a mechanism for age-related glutathione deficiency [23]. Specifically, the concentrations of glutathione, cysteine and glycine in RBCs obtained from old subjects were significantly lower than in those obtained from young controls, and dietary supplementation with cysteine and glycine fully restored age-related decrease in erythrocyte glutathione synthesis and glutathione concentration [23]. It has been reported that the activity of G6PD [25] and GR [26] in RBCs declined with age. Data from this report also showed a reduced erythrocyte GR activity in old nondiabetic women than in young controls, though the protein level of GR was comparable in these two age groups. Decrease in GR activity might be, at least partially, responsible for the age-related reduction in erythrocyte GSH level and GSH/GSSG ratio.

Another important finding from this report is that old women with diabetes have increased plasma and erythrocyte TGSH when compared to age-matched nondiabetic subjects. In addition, the protein level of GCLC was significantly higher in the RBCs obtained from diabetic patients than in those obtained from age-matched controls. Changes in GCLC expression and activity are mechanisms to upregulate glutathione synthesis and cellular glutathione level [27, 28]. The increased erythrocyte TGSH content in diabetic patients might be a result of overexpression of GCLC protein. These results are conflicting with previous reports showing that diabetic patients have reduced erythrocyte TGSH level and glutathione synthesis [4–6]. The mechanism underlying the difference of the results obtained from our laboratory and others remains unknown.

The expression of GCLC is regulated by a number of factors at the transcriptional, posttranscriptional, translational, and posttranslational levels [29, 30]. For instance, oxidative stress and electrophilic substances that can deplete cellular GSH or induce its oxidation to GSSG have been suggested to induce GCLC expression [29, 30]. Diabetes increases oxidative stress through various mechanisms, such as hyperglycemia, accumulation of AGEs and impairment of antioxidant enzymes [31]. Data from this report showed accumulation of AGEs and decrease in catalase activity in RBCs obtained from diabetic patients. In addition, insulin has been reported to stimulate cysteine uptake by cells and upregulate GCL expression, and therefore accelerate glutathione synthesis [30, 32]. The diabetic patients participating in this study had received long-term insulin therapy. Thus, increase in oxidative stress and long-term use of insulin might be, at least partially, responsible for the increased GCLC in the diabetic patients studied in this report. This report also demonstrated that the protein levels of erythrocyte G6PD, GR, GST-ρ1, GPx1 and Glo1 were much higher in diabetic patients than in age-matched controls. This is consistent with the view that oxidative stress upregulates the expression of G6PD, GR, GSTs, GPxs and glyoxalases [33–36]. Mature RBCs cannot synthesize RNAs and proteins. The increased level of the GCLC, G6PD, GR, GST-ρ1, GPx1 and Glo1 in RBCs obtained from diabetic patients highly likely results from an increased synthesis of these proteins during early phases of erythropoiesis or reduced degradation rate in mature RBCs.

Data from this report implies that the activity of the proteins involved in glutathione generation, reduction and utilization was depressed in RBCs obtained from diabetic patients. Specifically, although the protein levels of erythrocyte GCLC, G6PD and GR were much higher in diabetic old women than in nondiabetic young women, the erythrocyte TGSH level was similar and the GSH level was significantly lower in diabetic old women compared to those in nondiabetic young women. In addition, the erythrocyte GR, GST-ρ1 and GPx1 protein levels were significantly higher in diabetic old women than in nondiabetic young women, while the GR, GST and GPx activities in unit of cellular protein were comparable in these two groups. Damage due to glycation has been suggested to disturb the functions of proteins. Increase in protein glycation, as indicated by accumulated AGEs, might be a mechanism for the reduced activities of these glutathione generation and utilization proteins in RBCs obtained from diabetic patients.

5. Conclusions

In summary, the plasma erythrocyte TGSH and GSH levels declined with age in nondiabetic AA women. This age-related change unlikely results from a reduced expression of proteins related glutathione synthesis and reduction, as the protein level of GCLC, G6PD and GR is comparable in nondiabetic young and old women. In contrast to previous reports that showed reduced glutathione levels in diabetic patients, this report demonstrated that the plasma and erythrocyte TGSH levels, as well as the erythrocyte GCLC protein level were significantly higher in diabetic old than in nondiabetic old women. Further studies are needed to define the mechanism(s) underlying these conflicting findings. The plasma and erythrocyte GSH level, GSH/GSSG ratio and catalase activity were significantly lower in RBCs obtained from diabetic patients than those obtained from age-matched controls. In addition, substantial AGEs accumulated in RBCs of diabetic patients. These findings suggest an increased oxidative stress in RBCs of diabetic patients. Herein we proposed that the increased level of the proteins related to glutathione metabolism in the RBCs of diabetic patients results from an oxidative stress-induced expression of these proteins in erythrocyte precursor cells, because matured RBCs cannot synthesize proteins and because oxidative stress is able to induce the expression of these proteins [29, 30, 33–36].

Acknowledgement

This study was supported by NIH grant SC1HL101431 (HY), U54MD0007593 (ZMG).

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