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Redox Status Parameters and PBMC Membrane Fluidity in Diabetes Mellitus DENISA MARGINA1, MIHAELA ILIE2, DANIELA GRADINARU1, MARIA VLADICA3, CORNELIA PENCEA3, NICULINA MITREA1, EVA KATONA4 1
University of Medicine and Pharmacy, Faculty of Pharmacy, Biochemistry Department, 6, Traian Vuia Street, 020956, Bucharest, 2 University of Medicine and Pharmacy, Faculty of Pharmacy, Toxicology Department, 6, Traian Vuia Street, 020956, Bucharest, 3 N. Paulescu National Institute of Diabetes, Nutrition and Metabolic Diseases, Ion Movila Street, Bucharest, 4 University of Medicine and Pharmacy, Faculty of General Medicine, Biophysics Department, 8 Eroii Sanitari Street, 050474, Bucharest, Romania
The present study aims at finding correlations between certain biochemical and biophysical blood parameters for diabetes patients, focusing on the antioxidant status of the red blood cells and the membrane fluidity of peripheral blood mononuclear cells (PBMC), the endothelial function and the risk of stable interaction between the leucocytes and the endothelium. For that purpose we evaluated blood samples from 32 diabetes patients compared to a control group of 10 subjects for erythrocytes’ enzymatic activity of glucose-6phosphate dehydrogenase and of superoxide dismutase, their susceptibility to lipid peroxidation, the plasma nitric oxide stable end products level and the PBMC membrane fluidity. Our results showed that the erythrocytes’ antioxidant mechanisms and the PBMC membrane fluidity are impaired under chronic hyperglycemic conditions. Since microvascular complications of diabetes are mainly determined by redox mechanisms, the evaluation of these parameters might help in characterizing the risk of vascular complication for diabetes patients. Keywords: diabetes mellitus, blood, redox status, membrane fluidity
Abbreviations ATP/ADP = adenosine-triphosphate/adenosine-diphosphate; ESP = erythrocytes’ susceptibility to lipid peroxidation; FPG = fasting plasma glucose; G6PDH = glucose-6-phosphate dehydrogenase; HbA1c = glycated hemoglobin; IDDM = insulin dependent diabetes mellitus; LDL = low density lipoproteins; MDA = malondialdehyde; NADP+/NADPH = nicotinamide adenine dinucleotide phosphate oxidized/reduced; NIDDM = non-insulin dependent diabetes mellitus; NO = nitric oxide; NOS = nitric oxide synthase; PBMC = peripheral blood mononuclear cell; PBS = phosphate-buffered saline; PMS = phenazine methosulfate; RBC = red blood cell; ROS = reactive oxygen species; SOD = superoxide dismutase; TC = total cholesterol; TG = triglycerides; TMA-DPH = 1-(4-trimethylammoniumphenyl)-6-phenyl-1,3,5-hexatriene ptoluensulfonate Corresponding address: Denisa Margina MD. E-mail:
[email protected] DOI:10.1556/CEMED.3.2009.2.8
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The oxidation-reduction system plays such an essential role in living organisms, that life itself might be defined as a continuous redox reaction. Basic mechanisms underlying the transformation of organic constituents include metabolic sequences in which electron transfer occurs. As a result, oxygen reactive species (free radicals – ROS) are continuously generated in normal metabolic processes. The balance between the oxidative action of the free radicals and the level of antioxidants is essential for life and characterizes a living organism’s capacity of resistance to stress [1]. The redox stress is the factor which leads to lipid peroxidation, protein oxidation and DNA damage, thus generating the bio-degradation of the cell; the functional cell changes can result in the onset of auto-immune diseases, cardiovascular distress, neurological degeneration syndrome, mutations, tumorigenic processes and induce the ageing process as well [2]. Diabetes mellitus is a complex metabolic disorder characterized by pathological changes of glucose, lipid and protein metabolisms due to chronic hyperglycemia [3–5]. The etiopathogenesis of vascular complications in diabetes is complex and multifactorial [6–8[. The abnormal function of the vasculature precedes cardiovascular disease and is associated with impaired endothelium-dependent vasorelaxation and with pathological changes of the interactions between the blood cells and the vascular wall (9–11). Recently, endothelial dysfunction, such as nitric oxide (NO) impairment is regarded as an early step in the development of insulin resistance, atherosclerosis, and type II diabetes. Studies in the last years pointed out, among many other activities of NO, its ability to modulate peripheral and hepatic glucose metabolism and insulin secretion; it was suggested that NO alterations play an important role in the evolution of insulin resistance, type II diabetes and obesity associated pathologies [12, 13]. Membrane fluidity of blood cells was shown to have a decisive role in the direct cell to cell contact and the modulation of the activity of membrane enzymes and to be affected by the increased release of ROS [14]. These findings renewed the interest in the study of the redox systems involved in diabetes and related complications, showing the importance of complex studies involving biochemical and biophysical methods applied on diabetes patients. The aim of our study was to evaluate selected biochemical and biophysical parameters (glucose-6-phosphate dehydrogenase – G6PDH – and superoxide dismutase – SOD – activity in red blood cells, NO stable end products, peripheral blood mononuclear cell membrane fluidity) in patients with insulin-dependent diabetes mellitus (IDDM) and non insulin-dependent diabetes mellitus (NIDDM), in order to characterize both redox and blood cell-vascular wall risk interactions in the selected patients.
Materials and Methods Study Design We selected 42 patients: – 14 with non-insulin dependent diabetes mellitus (NIDDM group); – 18 with insulin dependent diabetes mellitus (IDDM group); – 10 normoglycemic, normolipidemic patients that constituted the control group. The diagnosis of diabetes (either IDDM or NIDDM) was based on current World Health Organization criteria [15]. Patients with severe renal, hepatic or hematological disease, overt car2009 n Volume 3, Number 2
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diovascular disease or malignancy were excluded from the study. None of the subjects had taken known antioxidants-containing supplements (vitamin C, vitamin E, probucol, etc). The protocol was approved by the local ethics committee and the informed consent of the patients was obtained. The body mass index (BMI) for every patient was calculated according to the equation (1): kg weight BMI 2 = m height 2
(1)
For all the subjects the following parameters were evaluated on á jeun venous blood samples: fasting plasma glucose (FPG), total cholesterol (TC), triglycerides (TG), low density lipoproteins (LDL) and glycated hemoglobin (HbA1c), red blood cell G6PDH and SOD activity, erythrocytes susceptibility to lipid peroxidation (ESP), NO stable end products in plasma, membrane anisotropy and fluidity of peripheral blood mononuclear cells (PBMC).
Materials Biologic material: 5 ml peripheral venous blood sampled on Na2EDTA as anticoagulant. Reagents: sodium citrate, sodium chloride, triethanolamine chloride, digitonin, nicotinamide adenine dinucleotide phosphate (NADP+), nicotinamide adenine dinucleotide reduced (NADH), glucose-6-phosphate (G6PDH), nitro-blue tetrazolium, phenazine methosulfate (PMS), phosphate-buffered saline (PBS), thiobarbituric acid, sodium azide, trichloracetic acid, Hystopaque 1077® were purchased from Merck and Sigma, RPMI 1640 medium with sodium bicarbonate and L-glutamine were purchased from Biochrom AG, 1-(4-trimethylammoniumphenyl)-6-phenyl-1,3,5-hexatriene p-toluensulfonate (TMA-DPH) was purchased from Molecular Probes (Invitrogen Inc.). Devices: Cary 100 BIO UV-VIS absorption spectrophotometer (Varian Inc.), equipped with Peltier thermostated cell holder; LS50 B spectrofluorimeter (Perkin Elmer), equipped with thermostated cell holder, magnetic stirring and fluorescence polarisation accessory.
Sample Preparation Fasting venous blood samples were drawn from the patients. The separation of the cells was performed using the gradient density method: the samples were centrifuged at 600xg for 20 minutes at 25°C on Hystopaque 1077®; platelet rich plasma was separated and used for the evaluation of the NO stable end products. The PBMC ring was collected and processed as following. The red blood cells were harvested, washed twice with 5 ml of 0.9% NaCl solution and standardized at 1 g hemoglobin / 100 mL erythrocyte suspension. The erythrocyte enzyme activity was evaluated on freshly prepared hemolysate: the standardized suspension of erythrocytes was mixed with distilled water (1:3 v/v), and 0.3 mL of digitonin (1%) was added. After 15 min standing at room temperature, the mixture was centrifuged at 300xg for 15 minutes to remove the insoluble material and the hemolysate was used for the enzymatic assay. The PBMC cell ring obtained, containing mainly lymphocytes and monocytes, was suspended in 7 mL of RPMI 1640 medium, and the suspension was centrifuged at 450xg for 10 minutes at 4°C. The PBMC pellet was collected, resuspended in RPMI medium, and once again washed CEMED
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with RPMI medium (210xg, 10 min, 4°C). The supernatant was discarded, the pellet was resuspended in 3 mL of RPMI medium and the cells were counted in a Burker–Türk chamber. To be used in fluorescence anisotropy measurements, the PBMC suspension was standardized at 105 cells/mL in RPMI 1640 medium.
Biochemical Evaluation Fasting plasma glucose (FPG), total cholesterol (TC), triglycerides (TG), low density lipoproteins (LDL) and glycated hemoglobin (HbA1c) were assessed using standard automatic devices and commercial kits (Merck and Biorad). The activity of G6PDH (EC 1.1.1.49) was assayed by the method of Lohr and Waller (1974). The rate of NADPH formation, which is a measure of the enzyme activity, was evaluated spectrophotometrically, at 340 nm [16]. The activity of SOD was assayed using a non-enzymatic system for the generation of superoxide anions (PMS-NADH) and nitro-blue tetrazolium for the generation of a formazan dye. The measurements were performed spectrophotometrically at 560 nm, and the activity of the enzyme was expressed in international units/g haemoglobin. The red blood cells susceptibility to lipid peroxidation was evaluated on the standardized erythrocyte suspension as follows: the erythrocytes were washed twice with buffer-phosphate-saline solution containing 4 mM natrium azide for the inhibition of the endogenous catalase; the erythrocyte suspension was further treated for 1 hour with 10 mM H2O2 (1:1, v/v); the malondialdehyde (MDA) resulted from the red blood cell membrane lipid peroxidation was evaluated spectrophotometrically at 535 nm using thiobarbituric acid (1%, 3:0.75 v/v). The NO stable end products (nitrites and nitrates) in plasma were evaluated using the Griess method [17, 18]. After the reduction of plasma nitrates to nitrites with cadmium (1:1w/v), the samples were mixed 5 minutes on a vortex mixer with the Griess reagent (1:1 v/v), then centrifuged at 100 x g for 20 minutes, at room temperature. Results were expressed as µmoles (nitrites and nitrates)/L of plasma.
Fluorescence Anisotropy Measurements The fluorescence anisotropy of PBMC was assessed by the determination of TMA-DPH steady state fluorescence polarization after the cell membrane exterior phospholipid layer permeation of the probe [19, 20]. For the measurement of the changes in the TMA-DPH fluorescent properties following the membrane permeation we added to a 2 mL of normalized PBMC suspension an aliquot of TMA-DPH stock solution in DMSO to get 2.5 µM TMA-DPH in the measurement cuvette. The cell suspension with the fluorescent probe was incubated for 2 minutes at 37°C under continuous magnetical stirring. Steady state fluorescent polarization of TMA-DPH was measured using a Perkin Elmer LS50 spectrofluorimeter; the TMA-DPH was excited with polarized light at 340 nm and the emission intensities were detected at 425 nm, through a polarizer system. The measurements were carried out for unstained cells (as background) and TMA-DPHlabeled cells, in the four possible positions of the polarizers in the excitation and emission beam; each intensity value was computed as a mean of 200 points (measurements in a total time of 4 s 2009 n Volume 3, Number 2
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in steps of 0.02 s). The calculation of the fluorescence anisotropy (r) was performed according to equation (2): I − GI vh (2) r = vv I vv + 2GI vh where Ivv, Ivh, Ihv and Ihh represent the emission intensity when the polarisors in the excitation end emission beams are oriented vertical-vertical, vertical-horizontal, horizontal-vertical and horizontal-horizontal, respectively and G = Ihv/Ihh is a correction factor [21]. The membrane lipid order (fluidity) in the polar head-group region of the plasma membrane bilayer, f, was computed following equation (3), as a function of limiting initial r0 and long-time r∞ values of the fluorescence anisotropy [20, 22].
f =
r0 (3) r∞ where r∞ = 1.270·r – 0.076 for 0 < r < 0.28 and r∞ = 1.100·r – 0.032 for 0.28 0.05), but significantly higher compared to the control group (p = 0.008). The diabetes groups (both IDDM and NIDDM) are characterized by a significantly higher value of the FPG (p < 0.001) and of the HbA1c (p < 0.001) compared to the control group. From the lipid metabolism point of view, the patients with diabetes had similar levels of total cholesterol and LDL; both IDDM (p = 0.01) and NIDDM (p = 0.034) had significantly higher levCEMED
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Table 1 The general biochemical parameters evaluated for the study groups Parameter
NIDDM group
Control 2
BMI (Kg/m )
21.47 !
2.02
FPG (mg/dL)
85.80 !
7.25
4.82 !
0.53
HbA1c (%)
30.18 !
IDDM group
5.60
33.27 ! 13.40
149.78 ! 23.65
259.54 ! 74.61
7.94 !
1.03
13.69 !
1.06
TC (mg/dL)
176.60 ! 34.46
209.91 ! 23.65
232.87 ! 79.98
LDL (mg/dL)
100.18 ! 39.10
130.45 ! 36.48
150.80 ! 72.38
HDL (mg/dL)
60.20 ! 13.17
43.74 ! 10.87
TG (mg/dL)
94.1 ! 22.06
178.60 ! 46.46
34.44 !
9.21
238.14 ! 112.02
els of LDL compared to the control. All of the diabetes subjects had significantly lower values of the protective anti-atherosclerotic HDL parameter (p < 0.001) and significantly higher values of the TG level (p < 0.001), compared to the control. Still, the cardioprotective factor was higher in the NIDDM group, where 42.85% of the patients had a normal HDL level (HDL > 45 mg/dL) compared to the IDDM group, where only 5.55% of the patients reached this cardioprotective value. The diabetes patients, both IDDM and NIDDM, had a significantly lower activity of SOD (0.82 ! 0.31 IU/g Hb for NIDDM, and 0.45 ! 0.17 IU/g Hb, for IDDM patients compared to 1.41 ! 0.14 IU/g Hb for the control, p < 0.001) and a significantly higher ESP value (712.67 ! 202.80 mM MDA/g Hb for NIDDM and 1019.07 ! 301.73 mM MDA/g Hb for IDDM vs. 166.86 ! 35.56 mM MDA/g Hb for the control, p < 0.001) compared to the control group.
G6PDH (IU/ml) ESP (mM MDA/g Hb) 200
1200
180 100
160 140
800
120 100
600
80 400
60 40
200
20 0
0 Control group NIDDM group IDDM group
Fig. 1 Correlation between the activity of G6PDH and the erythrocyte susceptibility to lipid peroxidation (ESP) in NIDDM and IDDM patients compared to the control group
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2.5 NO stable end products [mmoles (nitrites + nitrates)/l plasma]
2
SOD activity (IU/g Hb) 1.5
1
0.5
0 Control group
NIDDM group
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Fig. 2 Evaluation of the SOD activity and NO stable end products level in NIDDM and IDDM patients compared to control subjects
The activity of G6PDH was also higher in the NIDDM group (141.35 ! 33.84 IU/mL, p < 0.001) compared to the IDDM (47.67 ! 24.62 IU/mL), and significantly lower in both study groups compared to the control (190.44 ! 27.89IU/mL) (Fig. 1). The evaluation of NO stable end products illustrates the synthesis of the endothelial derived relaxing factor. The results revealed the fact that the level of nitrites and nitrates was significantly increased in IDDM patients [2.17 ! 0.90 µmoles (nitrites + nitrates)/L of plasma) and in NIDDM patients 2.09 ! 0.50 µmoles (nitrites + nitrates)/L of plasma], compared to the control group [1.19 ! 0.50 µmoles (nitrites + nitrates)/l of plasma]. The increase of the NO stable end products was correlated with the decrease of the SOD activity (Fig. 2). The comparative analysis of the fluorescence polarization emission intensity reveals that the increase of the fluorescence upon the addition of the probe was similar for the control and NIDDM groups; this increase was much higher in control and NIDDM groups compared to the IDDM group (Fig. 3). The mean membrane binding parameter f was different for the PBMCs belonging to the three groups (515.2 ! 217.4 for control vs. 166.8 ! 63.5 for NIDDM group, p = 0.0003 and 48.12 ! 11.8 for IDDM group, p = 0.00004) (Fig. 4). In the IDDM group, the fluorescence anisotropy could be computed only for a few patients (for most of the subjects, the computed anisotropy was greater than the limiting initial anisotropy of the molecular probe, r0 = 0.36). However, the computed membrane fluidity was not significantly different for the control and IDDM groups (1.14 ! 0.8 for control, and 1.14 ! 0.11 for IDDM groups, respectively) but was slightly higher for NIDDM patients (1.30 ! 0.17) (Fig. 5). The Spearman multiple regression analysis showed a positive correlation (u = 0.561, p < 0.0001) between the fluorescence anisotropy (r) and the TC level for the control group. For the IDDM group, the fluorescence anisotropy significantly correlated (p < 0.0001) with the NO plasma level; we also obtained a significant positive correlation (p < 0.0001) of the G6PDH level and the binding parameter f. For the NIDDM group our data showed a positive significant corCEMED
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a)
Control Control + TMA-DPH IDDM IDDM + TMA-DPH
Fluorescence emission intensity (r. u.)
220 200 180 160 140 120 100 80 60 40 20 0 0
1
2 Time (seconds)
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4
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b)
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120 100 80 60 0
1
2 Time (seconds)
3
4
Fig. 3 The measured fluorescence emission intensity Ivv for the cell suspensions with and without TMA-DPH (a) control and IDDM group, b) control and NIDDM group)
relation of the SOD activity with the membrane fluidity (u = 0.691, p = 0.019) illustrating the influence of the redox homeostasis on the membrane function. For all 32 diabetes patients, membrane fluidity correlated significantly with SOD activity (u = 0.691, p = 0.019), with the glucose level (u = –0.556, p = 0.039) and the erythrocyte susceptibility to peroxidation (u = –0.640, p = 0.014).
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f (%)
600
400
200
0
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IDDM
Fig. 4 The membrane-binding parameter f for the three groups
Discussion According to literature, for studies of oxidative damage physiopathology, erythrocytes are often used (as a model system) because of their simplicity and availability. The human red blood cell (RBC) is known to be susceptible to oxidant damage when the endogenous antioxidant protection (SOD, catalase, etc) is overcome; oxygen radicals formed during oxidative stress may promote the oxidation of polyunsaturated fatty acids that are present in high concentrations within cell membranes. Lipid peroxidation causes polymerization of the membrane components, their crosslinking and/or fragmentation, both structural and functional properties being altered. Such oxidant-related alterations may lead to changes of RBC rheologic behavior (i.e., deformability, aggregability, fluidity) [23–28]. The results of this study show that in diabetes mellitus patients, erythrocyte superoxide dismutase activity is decreased. Thus, the diabetes patients, having significantly lower activity of the protective SOD activity, were susceptible to oxidative changes of the RBC membrane, leading to the increase of the ESP. Possible explanations for our results include reduced antioxidant protection in both IDDM and NIDDM and greatly increased amounts of free radicals that are generated in hyperglycemic conditions, and overwhelm the defense system.
Membrane fluidity
1.5
1
0.5
0 Control
NIDDM Subject type
IDDM
Fig. 5 Membrane fluidity values for the three groups
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The oxidized/reduced forms of nicotinamide adenine dinucleotide (NAD+/NADH) and nicotinamide adenine dinucleotide phosphate (NADP+/NADPH) are common electron acceptor/donor molecules within the cell [3, 29]. A major source of NADPH in the cytosol is derived from the pentose phosphate pathway. The first and rate-limiting step in this pathway is carried out by the enzyme glucose-6-phosphate dehydrogenase (G6PDH) that catalyzes the conversion of glucose-6-phosphate (G6P) to 6-phosphogluconolactone, generating also NADPH from NADP+. Studies have emphasized that hyperglycemia, by inhibiting glucose-6-phosphate dehydrogenase (G6PDH) through a cAMP mechanism, plays a crucial role in NADPH/NADP+ ratio and thus in the pro-oxidant/anti-oxidant cellular status [3–5]. Genetic studies pointed out that insulin acts as an inductor of G6PDH expression [30]. Our data showed that the activity of the G6PDH was higher in the NIDDM group compared to the IDDM. Both diabetes groups had significantly lower levels of G6PDH compared to the control, thus confirming the fact that the G6PDH activity is inhibited under hyperglycemic conditions (Fig. 1). Glucose-6-phosphate dehydrogenase (G6PDH) is essential to control the intracellular reductive potential by increasing the glutathione intracellular level, which, in turn, decreases the amount of reactive oxygen species [3, 4, 14]. All the NIDDM subjects received individualized doses of metformin during the study. It has been reported that metformin has many different cellular effects according to the experimental models and/or conditions. Metformin is a mild inhibitor of respiratory chain complex 1, it activates G6PDH in a model of high-fat related insulin resistance, and it has antioxidant properties by a mechanism, which is not completely elucidated as yet [5]. This effect of the hypoglycemic drug might explain the results obtained for the activity of the antioxidant systems (G6PDH and SOD) at NIDDM patients. As genetic studies pointed out that insulin acts as an inductor of G6PDH expression, the lack of insulin action in the IDDM subjects might explain the extremely low activity of G6PDH observed at these subjects [24]. The antioxidant systems that protect the erythrocyte are highly impaired under hyperglycemic conditions, but the changes seem to be more profound in the case of IDDM subjects. Our results are in agreement with literature data, which point out a significantly higher concentration of erythrocyte MDA in metabolically decompensated diabetes patients compared to patients with proper metabolically compensated diabetes [6]. The evaluation of NO stable end products illustrates the synthesis of the endothelial derived relaxing factor; our results revealed the fact that the level of nitrites and nitrates was significantly increased in IDDM and NIDDM patients compared to the control group. The increase of the NO stable end products was correlated with the decrease of the SOD activity (Fig. 2). These results suggest that the hyperglycemic oxidative stress might induce a compensatory reaction at the endothelial level; the lower the activity of the antioxidant systems, the higher the level of NO stable end products, suggesting that NO alteration plays an important role in the evolution of diabetes and associated pathologies [12, 13]. Our results showed that the increase of the fluorescence upon the addition of the probe was similar for the control and NIDDM groups; this increase was much lower in the IDDM group compared to control and the NIDDM group (Fig. 3). These results suggest that, in the case of IDDM patients, the membranes of the PBMC had an impaired structure/function, that did not allow the fluorescent probe to interact with the membrane constituents. The mean membrane binding parameter f obtained for the three groups can also be interpreted in terms of an impaired membrane functionality of the PBMC belonging to the IDDM group 2009 n Volume 3, Number 2
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compared to the control and NIDDM groups: for these subjects the PBMC membrane did not allow the fluorescent dye to penetrate between the two phospholipid layers. The fact that the computed membrane fluidity was higher for NIDDM patients compared to IDDM supports the hypothesis of Wiernsperger that metformin corrects membrane fluidity in the diabetes state and restores the function regulating glucose transport and metabolism [23, 24]. The Spearman multiple regression analysis showed an interesting positive correlation between the fluorescence anisotropy (r) and the TC level for the control group. This illustrates the fact that, under normal metabolic conditions, the increase of the cholesterol level leads to a decrease of the PBMC membrane fluidity. In order to point out the influence of the oxidative stress exerted under hyperglycemic conditions on the membrane status, we performed a multivariate analysis for the whole group of diabetes patients. For all 32 diabetes patients, membrane fluidity correlated significantly with SOD activity and the glucose level as well as with the erythrocyte susceptibility to peroxidation, thus pointing out the fact that increased generation of reactive oxygen species greatly influences the membrane fluidity.
Conclusions Our results showed an impairment of the erythrocyte antioxidant systems (G6PDH and SOD) in both NIDDM and IDDM patients. Diabetes mellitus is associated with an increased risk of RBC membrane peroxidation and also with an impairment of the NO synthesis. The increase of the ESP in diabetes patients, and especially in IDDM subjects, can be explained by the fact that the oxidative stress determines the fragmentation of the membrane polyunsaturated fatty acids, that can form adducts. This might lead to a decrease of the RBC membrane permeability for nutrients; our results are in agreement with literature data showing that glucose transport across the membranes may be strongly influenced by a variety of membrane functions, such as membrane fluidity [27, 28]. The PBMC membranes are rather impermeable for the TMA-DPH probe in IDDM subjects (as seen from the membrane binding parameter value). As a result, the membrane fluidity could be computed only for a few IDDM patients; the mean obtained for the membrane fluidity for IDDM patients is the same as in the control group, and the membrane fluidity in NIDDM patients is slightly increased compared to control. These findings suggest the impairment of the cell membrane function and/or structure in diabetes patients. For IDDM subjects, the membrane fluidity correlated with the NO end products level. The impairment of the membrane function might lead to an increase of the endothelium-leukocyte interaction probability, explaining the risk of vascular complication in diabetes subjects. These results should be completed in the future by correlation studies of the PBMC membranes and adhesion molecules (ICAM, VCAM, selectines), in order to determine if the impairment of the cell membrane behavior might activate inflammatory phenomena at the endothelial level. The correlations of the PBMC membrane fluidity obtained for all diabetes subjects illustrate the influence of the antioxidant defense system on the membrane function. The increase of the PBMC membrane fluidity is correlated with an increase of the SOD activity and with the decrease of the ESP. At glucose values higher that 110 mg/dL, the increase of the glucose level leads to a decrease of the PBMC membrane fluidity and an increase of the vascular risk complications. CEMED
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Our results show that a complex evaluation, both biochemical and biophysical, of the blood cells from diabetes patients might help for a better metabolic characterization and also for the assessment of the vascular risk complication at the studied patients. Our method links biochemical and biophysical blood parameters to find new pathways in the study of diabetes mellitus mechanisms in order to improve the treatment of the patients.
Acknowledgements The research was performed within the frame of grant 41-067/2007 of the Romanian Ministry of Education and Research, the National Program Management Center.
References [1] Bonnefont-Rousselot, D., Bastard, J. P. et al.: Consequences of the diabetic status on the oxidant/ antioxidant balance. Diab. Metab., 2000, 26, 163–176. [2] Hayden, M. R., Tyagi, S. C.: Intimal redox stress: accelerated atherosclerosis in metabolic syndrome and type 2 diabetes mellitus. Atheroscleropathy. Cardiovasc. Diabetol., 2002, 1, 1–27. [3] Bugdayci, G., Altan, N. et al.: The effect of the sulfonylurea glyburide on glutathione-S-transferase and glucose-6-phosphate dehydrogenase in streptozotocin-induced diabetic rat liver. Acta Diabetol., 2006, 43, 131–134. [4] Costagliola, C.: Oxidative state of glutathione in red blood cells and plasma of diabetic patients: in vivo and in vitro study. Clin. Physiol. Biochem., 1990, 8, 204–210. [5] Leverve, X. M., Guigas, B. et al.: Mitochondrial metabolism and type-2 diabetes: a specific target of metformin. Diabetes Metab., 2003, 29, 6S88–6S94. [6] Rysz, J., Blaszczak, R. et al.: Evaluation of selected parameters of the antioxidative system in patients with type 2 diabetes in different periods of metabolic compensation. Arch. Immunol. Ther. Exp., 2007, 55, 335–340. [7] Nishikawa, T., Kukidome, D. et al.: Impact of mitochondrial ROS production on diabetic vascular complications. Diabetes Res. Clin. Pract., 2007, 77, S41–45. [8] Toth, E., Racz, A. et al.: Contribution of polyol pathway to arteriolar dysfunction in hyperglycemia. Role of oxidative stress, diminished NO and enhanced PGH2/TXA2 mediation. Am. J. Physiol. Heart Circ. Physiol., 2007, 293, H3096–H30104. [9] Sena, C. M., Nunes, E. et al.: Endothelial dysfunction in type 2 diabetes: effect of antioxidants. Rev. Port. Cardiol., 2007, 26, 609–619. [10] Rabini, R. A., Cester, N. et al.: Modifications induced by LDL from type 1 diabetic patients on endothelial cells obtained from human umbilical vein. Diabetes, 1999, 48, 2221–2228. [11] Wever, R. M. F., Lûscher, T. F. et al.: Atherosclerosis and the two faces of endothelial nitric oxide synthase. Circulation, 1998, 97, 108–112. [12] Monti, L. D., Barlassina, C. et al.: Endothelial nitric oxide synthase polymorphisms are associated with type 2 diabetes and the insulin resistance syndrome. Diabetes, 2003, 52, 1270–1275. [13] Pieper, G. M.: Enhanced, unaltered and impaired nitric oxide-mediated endothelium dependent relaxation in experimental diabetes mellitus: importance of disease duration. Diabetologia, 1999, 42, 204 –213. [14] Hollan, S.: Membrane fluidity of blood cells. Haematologia (Budapest), 1996, 27, 109–127. [15] Expert Committee: Report of the expert committee on the diagnosis and classification of diabetes mellitus. Diabet. Care, 1997, 201, 183–97 [16] Shibib, B. A., Khan, L., Rahman, R.: Hypoglycaemic activity of Coccinia indica and Momordica charantia in diabetic rats: depression of the hepatic gluconeogenic enzymes glucose-6-phosphatase and fructose-1,6-bisphosphatase and elevation of both liver and red-cell shunt enzyme glucose-6-phosphate dehydrogenase. Biochem. J., 1993, 292, 267–270. 2009 n Volume 3, Number 2
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