Free radical production is increased during diabetes. Serum albumin is a major antioxidant agent, and structural modification of albumin induced by glucose or free radicals impairs its antioxidant properties. Therefore the aim of the present study was to compare the antioxidant capacities and structural changes in albumin in patients with T2DM (Type 2 diabetes mellitus) treated with MET (metformin) or SU (sulfonylureas) and in healthy control subjects. Structural changes in albumin were studied by fluorescence quenching in the presence of acrylamide. Albumin thiols and fructosamines, reflecting oxidized and glycation-induced changes in serum albumin respectively, were assessed. Structural changes in albumin were demonstrated by a significant decrease in fluorescence quenching in patients with T2DM, with patients treated with MET exhibiting a significant difference in the conformation of albumin compared with patients treated with SU. Oxidation, resulting in a significant decrease in thiol groups and plasma total antioxidant capacity, and glycation, associated with a significant increase in fructosamines, were both found when comparing healthy control subjects with patients with T2DM. When patients treated with MET were compared with those treated with SU, oxidative stress and glycation were found to be significantly lower in MET-treated patients. In conclusion, patients with T2DM have a decrease in the antioxidant properties of serum albumin which may aggravate oxidative stress and, thus, contribute to vascular and metabolic morbidities. Moreover, a significant protection of albumin was found in patients with T2DM treated with MET.

INTRODUCTION

MET (metformin) is an oral antihyperglycaemic agent universally used and recommended in the management of T2DM (Type 2 diabetes mellitus) [1]. MET also possesses antioxidant properties and decreases the production of ROS (reactive oxygen species) [2,3] implicated in the vascular complications of diabetes. A recent study [4] has investigated whether MET, at a pharmacological dose, was able to modulate the intracellular production of ROS in both quiescent BAECs (bovine aortic endothelial cells) and BAECs stimulated with a short (2 h) incubation of high levels of glucose (30 mmol/l for 2 h) or AngII (angiotensin II). The authors showed that MET was able to decrease the intracellular production of ROS in both non-stimulated and stimulated BAECs.

An in vivo antioxidant activity of MET has also been shown previously [5]. MET monotherapy ameliorates the imbalance between the free-radical-induced increase in lipid peroxidation [by decreasing the level of MDA (malondialdehyde) in both erythrocytes and plasma] and decrease in plasma and cellular antioxidant defences [by increasing the erythrocyte activities of Cu,ZnSOD (superoxide dismutase), catalase and glutathione] and decreased erythrocyte susceptibility to oxidative stress. Part of these effects were linked to its actions on lowering the production of AGEs (advanced glycated end-products), as these products contribute to vascular remodelling [6].

Modifications of albumin, through oxidative stress and glycation, have been shown to contribute to vascular complications in patients with diabetes [7]. Previous studies have focused on its antioxidant properties [8], and an inverse relationship between the albumin level and cardiovascular diseases has been established [8]. Patients with diabetes develop major vascular complications [9] and, in this disease, there are number of equally tenable hypotheses on the origins of vascular tissue damage, including protein glycation and the subsequent formation of AGEs. AGE proteins are chemically damaged and their biological properties are altered. Lysine, arginine and cysteine are the main residues affected.

Albumin represents the predominant circulating antioxidant agent in plasma exposed to continuous oxidative stress [10,11]. HSA (human serum albumin) contains one free cysteine-derived redox-reactive thiol (-SH) group (Cys34). Because of its reactive properties, the Cys34 thiol moiety confers a major role in serum antioxidant capacity to HSA, which accounts for 80% (500 μmol/l) of the thiols in plasma [12]. Moreover, a change in serum albumin structure accounts for its antioxidant properties, and these changes may be related to glycation of its lysine residues.

In the present study, we have investigated whether MET was able to protect albumin against free radical damage and protein glycation in patients with T2DM. To this end, we have compared the antioxidant capacity of albumin in patients with T2DM receiving either MET or SU (sulfonylureas) as their oral antidiabetic treatment. We show for the first time, using a fluorescence quenching method, in patients with T2DM having the same glycaemic control [i.e. the same HbA1c (glycated haemoglobin)], that MET significantly protects albumin and maintains its antioxidant properties through thiol protection.

MATERIALS AND METHODS

Patients

Patients with T2DM between 51 and 65 years of age were enrolled. Patients had systematic blood sampling for routine analysis (plasma glucose, HbA1c and kidney function) in the Diabetes Department, University Hospital, Grenoble, France. Plasma was collected to determine oxidative stress parameters and the chemico–physical modifications of albumin. A total of 23 patients were treated with MET (16 men and seven women) and 21 were treated with SU (16 men and five women). Healthy control subjects (n=12) were volunteers from the laboratory. The main metabolic characteristics of the patients with T2DM and control subjects are shown in Table 1.

Table 1
Metabolic characteristics of patients with T2DM and healthy control subjects

Values are means±S.D. *P<0.05 compared with MET- and SU-treated groups; †P<0.05 compared with the SU-treated group.

Patients with T2DM treated with
MET (n=23)SU (n=21)Control subjects (n=12)
Age (years) 53±7 49±8 40±4* 
Fasting plasma glucose (mmol/l) 8.1±3.1 7.5±2.9 4.9±1.2* 
Fasting plasma insulin (mmol/l) 8.1±3.1 7.9±2.5 5.9±1.4* 
HbA1c (%) 7.2±2.1 7.4±2.3 4.6±0.9* 
Fructosamines (μmol/l) 271±11† 320±12 241±9* 
Patients with T2DM treated with
MET (n=23)SU (n=21)Control subjects (n=12)
Age (years) 53±7 49±8 40±4* 
Fasting plasma glucose (mmol/l) 8.1±3.1 7.5±2.9 4.9±1.2* 
Fasting plasma insulin (mmol/l) 8.1±3.1 7.9±2.5 5.9±1.4* 
HbA1c (%) 7.2±2.1 7.4±2.3 4.6±0.9* 
Fructosamines (μmol/l) 271±11† 320±12 241±9* 

The study protocol conformed to the Ethical Guidelines of the Declaration of Helsinki (2000) and was approved by our Institutional Review Board. All the subjects gave their written informed consent.

Biological analysis

After separation of plasma from blood cells by centrifugation, aliquots of plasma were frozen at −80 °C for subsequent analysis of insulin, albumin and glucose, and oxidative stress parameters. Plasma glucose, lipid and lipoprotein cholesterol concentrations in all of the samples were determined using reagents from Boehringer Mannheim, and an automated clinical chemistry analyser (Boehringer Mannheim/Hitachi 917) was used to assess plasma glucose, using a hexokinase method, and albumin, using an immunoanalysis method.

Albumin study

Isolation of albumin from human plasma

Blue Sepharose®6 Fast Flow (Pharmacia Fine Chemicals) was used to purify plasma albumin, as described previously [10]. The gel was packed into a column (1.5 cm×4 cm) and was washed with starting buffer [50 mmol/l Tris/HCl (pH 7.4) and 0.5 mol/l NaCl].

All of the procedures were performed at 4 °C. The column was equilibrated with five bed vols. of starting buffer. The gel was then transferred into tubes (5 ml), and batch separation was used to obtain solutions of purified and concentrated albumin from plasma. Briefly, plasma (1.350 ml) was applied on to the gel (2.5 ml) and, after the first centrifugation (4000 g for 10 min at 4 °C), the supernatant was discarded. The gel was washed three times with starting buffer under the same conditions. Gel-bound albumin was eluted (650 μl) with 50 mmol/l Tris/HCl (pH 7.4) containing 1.5 mol/l NaCl. The tightly bound proteins were removed with 0.5 mol/l sodium thiocyanate, and the gel was re-equilibrated with three bed vols. of starting buffer. Aliquots of the albumin fraction recovered from the gel were separated by SDS/PAGE. Gel staining with Coomassie Blue and densitometry showed that the purity of albumin was >92%.

Fluorescence quenching

The intrinsic fluorescence of albumin and fluorescence quenching in the presence of acrylamide is able to be used [13], as albumin contains two tryptophan residues (Trp218 and Trp134) that generate autofluorescence. Moreover, it has been shown previously that acrylamide does not alter the conformation of the protein [13]. Fluorescence measurements were performed using a PerkinElmer LS 50 fluorimeter at excitation and emission wavelengths of 278 and 343 nm respectively. As shown previously [14], acrylamide was used as a quenching agent and was added to the albumin solution at a final concentration of 20 g/l in 0.5 mol/l phosphate buffer (pH 7.4) to a final concentration up to 60 mmol/l acrylamide. The results are expressed as mean values of I0/I (Stern–Volmer constant, Kq), where I0 is albumin fluorescence before adding the quencher and I is albumin.

Measurement of oxidative stress parameters

The measurement of thiol groups was performed on isolated albumin using Ellman's reagent {DNTB [5,5′-dithiobis-(2-nitrobenzoic acid)]} [15]. Briefly, 2.5 mmol/l DNTB in 0.2 mol/l phosphate buffer (pH 8.0) was mixed with 500 μl of sample and 750 μl of 50 mmol/l phosphate buffer (pH 8.0), and baseline absorbance was recorded at 412 nm. Subsequently, 250 μl of freshly prepared DNTB was added, the reaction allowed to proceed for 15 min at room temperature (20 °C) in the dark and the final absorbance was measured. Thiol values are expressed in μmol/g of albumin using a molar absorption coefficient of 13600 litre·mol−1·cm−1 for the thiol–DNTB complex. A calibration curve was performed by sequential dilution of a 1 mmol/l N-acetylcysteine stock solution. Total antioxidant capacity was measured using a kit from Randox. Total blood glutathione was measured using the method described by Gunzler et al. [16]. MDA was measured using HPLC as described previously [17]. Briefly, plasma was subjected to alkaline hydrolysis, acid deproteinization, derivatization with thiobarbituric acid and n-butanol extraction. After this, MDA was determined by HPLC at 532 nm. The assay was linear from 0.35–7.4 μmol/l. The intra-run reproducibility was obtained with a <5% coefficient of variation, and the inter-run reproducibility was obtained with a <9% coefficient of variation. The accuracy (bias) ranged from 2.5–4.8%, and the recovery was >96%.

The 8-isoPGF (8-isoprostaglandin F) isoprostane was analysed using an enzyme immunoassay kit from Cayman Chemicals, according to the manufacturer's specifications. This assay is based on the competition between 8-isoprostane and an 8-isoprostane acetylcholinesterase conjugate for 8-isoprostane-specific rabbit antiserum-binding sites, as described previously [18].

Statistical analysis

Statistical analyses were performed using SPSS software. The normality of the distribution of the data was assessed. Continuous data are expressed as means±S.D. Relationships between the continuous variables were evaluated by Pearson's correlation analysis, or by Spearman's correlation analysis when data were not normally distributed. Differences in the mean Stern–Volmer constants between the two groups and according to the different acrylamide concentrations were determined by two-factor repeated measures ANOVA. Group comparisons of continuous variables were performed with a Student's t test (or Mann–Whitney test for groups with data not normally distributed). Both univariate and multivariate analyses were performed in order to explain the contribution of the variance in albumin thiols and fructosamines. P values <0.05 were considered significant.

RESULTS

Characteristics of the patients

No metabolic differences were observed between the two groups of patients with T2DM, except for fructosamines which were lower in the MET-treated group compared with the SU-treated group. With regard to lipid metabolism, renal function and albumin levels, no differences were observed between the MET- and SU-treated groups (results not shown). HbA1c was not significantly different between the MET- and SU-treated groups on the day of sampling for the albumin study. With regard to this parameter, it was also measured 2 months before the experiment and no significant difference was observed compared with the present findings in the MET- and SU-treated groups respectively (7.4±2.3 and 7.5±2.2% at 2 months compared with 7.2±2.1 and 7.4±2.3% at the time of the study), indicating a good metabolic control of the patients.

Oxidative stress parameters

Albumin thiols were significantly elevated in MET-treated patients compared with the SU-treated group (Table 2). Control subjects had significantly higher plasma thiols compared with patients with T2DM. Plasma TAS (total antioxidant status) was lower in SU-treated patients, but was higher in control subjects compared with both groups of patients with T2DM.

Table 2
Oxidative stress parameters of the patients with T2DM and the control subjects

Values are means±S.E.M. *P<0.05 compared with the remaining group(s); †P<0.05 compared with the SU-treated group.

Patients with T2DM treated with
MET (n=23)SU (n=21)Control subjects (n=12)
TAS (μmol/l) 1.31±0.21† 1.02±0.17* 1.45±0.31* 
Albumin thiol groups (μmol/g of protein) 2.6±0.51† 2.1±0.60* 3.2±0.6 
Blood total glutathione(μmol/l) 870±41 590±31* 980±44 
MDA (μmol/l) 2.82±0.05† 3.12±0.04 2.51±0.07* 
8-isoPGF isoprostane (pg/ml) 25.2±4.1† 31.3±6.2 17.2±3.9* 
Patients with T2DM treated with
MET (n=23)SU (n=21)Control subjects (n=12)
TAS (μmol/l) 1.31±0.21† 1.02±0.17* 1.45±0.31* 
Albumin thiol groups (μmol/g of protein) 2.6±0.51† 2.1±0.60* 3.2±0.6 
Blood total glutathione(μmol/l) 870±41 590±31* 980±44 
MDA (μmol/l) 2.82±0.05† 3.12±0.04 2.51±0.07* 
8-isoPGF isoprostane (pg/ml) 25.2±4.1† 31.3±6.2 17.2±3.9* 

Blood total glutathione was higher in MET-treated patients compared with the SU-treated group; however, this parameter was also higher in the control group. Plasma MDA and 8-isoPGF isoprostane were significantly lower in the MET-treated patients compared with SU-treated patients. These parameters were significantly lower in the control group compared with both groups of patients with T2DM.

Fluorescence quenching of albumin

Fluorescence of HSA was quenched by increasing concentrations of acrylamide (Figure 1). The quasi-linearity of the plots indicated that the quenching process followed a collision-type process. The Stern–Volmer curves, representing tryptophan accessibility to the quencher, were significantly different for the various albumin preparations tested when comparing control subjects and patients with T2DM (P<0.001, as determined by ANOVA). The extent of the increase in the Stern–Volmer constant with higher acrylamide concentrations was lower in MET-treated patients compared with SU-treated patients (P<0.001, as determined by ANOVA).

Structural changes in albumin determined by fluorescence quenching in the presence of acrylamide

Figure 1
Structural changes in albumin determined by fluorescence quenching in the presence of acrylamide

The Stern–Volmer curves, representing tryptophan accessibility to the quencher, were significantly different in the albumin preparations tested (control subjects compared with MET- and SU-treated groups; P<0.001, as determined by ANOVA). Moreover the increase in Stern–Volmer constant with acrylamide concentration was different in control subjects compared with the patients with T2DM (P<0.001, as determined by ANOVA). This shows that both global accessibility to tryptophan and the magnitude of the decrease in SU-treated patients is due to structural changes in albumin.

Figure 1
Structural changes in albumin determined by fluorescence quenching in the presence of acrylamide

The Stern–Volmer curves, representing tryptophan accessibility to the quencher, were significantly different in the albumin preparations tested (control subjects compared with MET- and SU-treated groups; P<0.001, as determined by ANOVA). Moreover the increase in Stern–Volmer constant with acrylamide concentration was different in control subjects compared with the patients with T2DM (P<0.001, as determined by ANOVA). This shows that both global accessibility to tryptophan and the magnitude of the decrease in SU-treated patients is due to structural changes in albumin.

DISCUSSION

Albumin is the most important extracellular antioxidant due to the presence of thiol groups and its high plasma concentration [13], and a number of potential associations between its plasma concentration or modification and mortality have been observed [19]. In the present study, we investigated how MET could interfere with the antioxidant capacity of albumin.

We investigated the antioxidant property of serum albumin by determining albumin oxidation (albumin thiol groups) and the structural changes in albumin assessed by tryptophan accessibility (fluorescence quenching by acrylamide). The decreased intrinsic fluorescence of albumin reflected the more difficult access to the two tryptophan residues; this is explained by changes in the conformational properties of the protein. Albumin thiol groups provide protection from lipid peroxidation propagated by ROS, with the Cys34 moiety the source of the antioxidant protection. In previous studies, H2O2 and peroxynitrite have been shown to oxidize Cys34 to a sulfenic acid derivative [16], leading to a dramatic loss in extracellular free radical protection and the subsequent consequences for cardiovascular disease.

Using different systems, we observed that the antioxidant capacity and physico–chemical properties of albumin were dramatically modified in patients with T2DM, but were less so in MET-treated patients compared with SU-treated patients. Induced modification of albumin is suggested by the alteration in the Stern–Volmer constant in patients with T2DM. These can be attributed to changes in cysteine and methionine residues [20] and the subsequent conformational modifications of albumin, leading to decreased accessibility following methylglyoxal binding [21]. Bourdon et al. [13], using fluorescence quenching, demonstrated that glycation of BSA leads to conformational changes, as glycated BSA and native BSA have different Stern–Volmer constants. Any autofluorescence study of albumin must take into account that it can be affected by factors other than conformational changes. Hence albumin modification by glycation results from the formation of Schiff bases at the beginning of the reaction, which then form further modification products via rearrangements [22]. These rearrangements, leading to minor conformation modifications, may change the environment of the tryptophan residues, as measured by fluorescence. In these conditions, it is interesting to observe that patients treated with MET had a Stern–Volmer constant significantly different from that in SU-treated patients. Hence fructosamine, which is correlated with albumin glycation, was significantly lower in MET-treated patients. Knowing the kinetics of fructosamine formation, it can be a consequence of an improvement in glycaemic equilibrium from approx. 20 days. It could also result from a decrease in albumin glycation in the MET-treated group, as described previously [6]. HbA1c was not significantly different between the groups on either the day of the experiment or 2 months before, suggesting that differences in glycation may be due to lower albumin glycation in MET-treated patients through a mechanism other than its metabolic effects. Correlation between albumin thiols (reflecting albumin antioxidant capacity) and fructosamines (reflecting glycaemic control and subsequent protein glycation) was calculated. A significant negative correlation was found between these two parameters in MET-treated patients (r=−0.78, P<0.002), but not in SU-treated patients (r=−0.58, P=0.71). Therefore the additional antioxidant capacity of MET could explain the lower TAS in MET-treated patients. Glycated albumin has been demonstrated to be associated with cardiovascular changes in both animal models [23] and human diabetes [7,24]. It has also been shown in mice that endothelial cell damage caused by glycated albumin can be reversed by monoclonal antibodies against glycated albumin [19]. In patients with diabetes, the increase in glycated albumin levels has been reported to be associated with an increase in pulse pressure, a well-recognized marker of cardiovascular risk [7]. Glycated albumin has also been shown to be correlated with plasma markers of endothelial dysfunction and cell adhesion molecules [24], and serum albumin is also able to increase the proliferation, as well as migration, of vascular smooth muscle cells [25]. Finally, these intermediate glycated products may lead to AGE formation. An increase in antioxidant protection, as shown by TAS, total blood glutathione and plasma thiols, was also observed. TAS is a global measure of serum antioxidant properties, including different pathways, and it can thus be hypothesized that changes in the antioxidant capacity of albumin are part of the explanation for the decrease in TAS. In our present study, an antioxidant effect of albumin was found in patients with T2DM receiving MET compared with those receiving SU. This is not completely explained by glycaemic control, as shown by similar HbA1c, and thus other mechanisms need to be determined. It is known that MET has antiglycation properties, which could explain these results. Laboratory and clinical findings support the hypothesis that one important explanation of the effect of MET on diabetic complications is its ability to decrease toxic dicarbonyls and AGEs [6]. This effect could be related either to the binding of the α-dicarbonyls methylglyoxal or 3-deoxyglucosone or to an increase in enzymatic detoxification. It is also interesting to observe that a series of oxidative stress markers, including MDA, glutathione and plasma 8-iso PGF2α isoprostane, were significantly improved in MET-treated patients, which is another means of highlighting the better control of oxidative stress in these patients, through a decreased lipid peroxidation pathway and lower arachidonic acid oxidation.

Other potential effects of MET on in vitro albumin protection will help to clarify this potentially important effect of MET and provide a further rationale for using this drug to prevent long-term complications.

In conclusion, the present study confirms that patients with T2DM have a dramatic decrease in the antioxidant capacity of albumin, potentially due to the physico–chemical modifications of albumin by diabetes. This underlines that the quality of albumin is important in maintaining its biological properties. We also show that MET can protect albumin against oxidation and leads to an increased antioxidant protection in addition to its glycaemic effect. These results could partly explain the better vascular protection in patients with T2DM, as shown in the UKPDS (UK Prospective Diabetes Study) [26].

Abbreviations

     
  • AGE

    advanced glycated end-product

  •  
  • BAEC

    bovine aortic endothelial cell

  •  
  • DNTB

    5,5′-dithiobis-(2-nitrobenzoic acid)

  •  
  • HbA1c

    glycated haemoglobin

  •  
  • HSA

    human serum albumin

  •  
  • 8-isoPGF

    8-isoprostaglandin F

  •  
  • MDA

    malondialdehyde

  •  
  • MET

    metformin

  •  
  • ROS

    reactive oxygen species

  •  
  • T2DM

    Type 2 diabetes mellitus

  •  
  • SU

    sulfonylureas

  •  
  • TAS

    total antioxidant status

We thank Fabienne Rochette for her technical support.

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