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Journal of Diabetes and Its Complications 29 (2015) 923–927

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Journal of Diabetes and Its Complications


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Altered glutathione system is associated with the presence of distal


symmetric peripheral polyneuropathy in type 2 diabetic subjects
Mercedes Molina Mendez a, José Folgado a, Carmen Tormo e, Ana Artero a, Maria Ascaso a,
Sergio Martinez-Hervás a, b, c, F. Javier Chaves b, c, Juan F. Ascaso a, b, c, d, Jose T. Real a, b, c, d,⁎
a
Service of Endocrinology and Nutrition, Hospital Clínico Universitario de Valencia, Valencia, Spain
b
CIBER de Diabetes y Enfermedades Metabólicas asociadas (CIBERDEM), Barcelona, Spain
c
Laboratorio de Estudios Genéticos, Fundación para la Investigación. Instituto de Investigación INCLIVA, Valencia, Spain
d
Department of Medicine, University of Valencia, Valencia, Spain
e
Department of Biochemistry and Molecular Biology, University of Valencia and CIBER de Obesidad, Spain

a r t i c l e i n f o a b s t r a c t

Article history: Distal symmetric peripheral polyneuropathy (DSPN) is a highly prevalent complication of diabetes. However,
Received 21 March 2015 underlying pathophysiological mechanisms are multiple and not well understood. The aim of our study was to
Received in revised form 17 May 2015 analyze the oxidative stress levels in circulating mononuclear cells by measuring the glutathione system,
Accepted 31 May 2015
malondialdehyde and oxidized-LDL, in 60 type 2 diabetic patients from a well-characterized cohort of 196
Available online 10 June 2015
type 2 diabetic patients. Using a nested case–control design, we studied 30 type 2 diabetic patients with distal
Keywords:
symmetric polyneuropathy and 30 diabetic controls without this complication, according to the Neuropathy
Glutathione Disability Score. We have found that diabetic patients with distal symmetric polyneuropathy showed
Oxidative stress significantly lower values of reduced glutathione (GSH) and reduced glutathione/oxidized glutathione (GSH/
Neuropathy disability score GSSG) ratio. These data indicate an increased consumption of glutathione in mononuclear cells from patients
Peripheral polyneuropathy with distal symmetric polyneuropathy. No significant differences were found in malondialdehyde or in
Type 2 diabetes mellitus oxidized-LDL levels comparing both groups. These data show an altered glutathione response in circulating
monocytes from diabetic patients with distal symmetric polyneuropathy.
© 2015 Elsevier Inc. All rights reserved.

1. Introduction The etiology of diabetic DSPN seems to be heterogeneous, but


previous data strongly suggest that hyperglycemia-induced cellular
Distal symmetric peripheral polyneuropathy (DSPN) is a highly damage and an increased production of reactive oxygen species play
prevalent complication of diabetes mellitus. Signs and symptoms of major roles in the development of this disease (Brownlee, 2005).
peripheral nerve dysfunction in a patient with diabetes characterize Different evidences suggest that oxidative stress (OS) is implicated in
DSPN, once other causes of peripheral polyneuropathy have been the pathogenesis of diabetic neuropathy (Bandeira, da Fonseca,
excluded. Signs and symptoms should begin distally in the feet, on Guedes, et al., 2013; Brownlee, 2005).
both sides and be primarily sensory (including numbness, prickling Experimental studies have associated polyneuropathy with intra-
paresthesias, dysesthesias, burning or allodynia), motor (such as cellular hyperglycemia and succeeding altered polyols pathway and
weakness, atrophy or depressed tendon reflexes) or both. (England, increased intracellular advanced glycosylation end products formation,
Gronseth, Franklin, et al., 2005) which lead to reactive oxygen species-induced DNA damage (Aubert,
In addition, DSPN is the most important causal mechanism leading Michel, Gillery, et al., 2014; Gabbay, Merola, & Field, 1966; Sugimoto,
to foot ulceration, a significant cause of morbidity and mortality in Yasujima, & Yagihashi, 2008). In the presence of hyperglycemia
diabetic patients (Cheer, Shearman, & Jude, 2009). However, increased OS is detected in peripheral nerves (glial), dorsal nerves and
pathophysiological mechanisms underlying the development of vascular nerve endothelial cells (Nishikawa, Edelstein, Du, et al., 2000;
diabetic DSPN are multiple and not well understood (Pascuzzi, 2009). Pop-Busui, Sima, & Stevens, 2006; Schmeichel, Schmelzer, & Low, 2003).
Moreover, the presence of decreased reduced glutathione (GSH) levels
and increased oxidized lipoproteins is associated to nerve damage,
Conflict of interest: There is no conflict of interest to declare.
vacuolization of mitochondria and neuronal apoptosis (Vincent, Russell,
⁎ Corresponding author at: Service of Endocrinology and Nutrition, Hospital Clínico
Universitario Valencia and Department of Medicine University of Valencia, Avda Blasco Low, & Feldman, 2004). This pathological state induces a reduction in
Ibañez, 17, 46010, Valencia, Spain. Tel./fax: +34 963862665. nerve conduction, an altered neurotrophy and neuronal apoptosis; all
E-mail address: jtreal@uv.es (J.T. Real). typical findings of diabetic polyneuropathy (Obrosova, 2002).

http://dx.doi.org/10.1016/j.jdiacomp.2015.05.023
1056-8727/© 2015 Elsevier Inc. All rights reserved.
924 M.M. Mendez et al. / Journal of Diabetes and Its Complications 29 (2015) 923–927

Unfortunately human studies are scarce. In clinical studies, an plasma donors and investigators of our center. The inclusion criteria for
increase in perioxinitrate and oxidized lipoproteins levels in diabetic the control group were: BMI b 30 kg/m2, concentration of plasma total
patients with polyneuropathy compared to diabetic subjects with no cholesterol (TC) b 200 mg/dl and triglycerides (TG) b 150 mg/dl; fast-
polyneuropathy has been found; supporting the role of OS in DSPN ing plasma glucose b 110 mg/dl and absence of personal or family
(Kasznicki, Kosmalski, Sliwinska, et al., 2012). We have recently history of dyslipidemia, cardiovascular disease or diabetes.
shown that plasma homocysteine levels, an independent risk factor
for cardiovascular disease, are increased in diabetic patients with
DSPN and relate with the grade of peripheral neuropathy (Gónzalez, 2.2. Clinical and anthropometric parameters
Pedro, Martinez-Hervas, et al., 2012). Moreover, a decreased total
antioxidant status in type 2 diabetic patients (T2DM) with coexisting Complete medical history and physical examination were carried
DSPN has been reported (Kasznicki et al., 2012; Merzouk, Hichami, out in all the patients. Blood pressure was measured in the sitting
Madani, et al., 2003); although not all investigators support this position with a mercury sphygmomanometer after a 10-min rest, with
observation (El Boghdady & Badr, 2012). two separated measurements; body mass index (BMI) was calculated
Therefore, experimental data strongly implicate OS in the as the weight in kilograms divided by height in meters squared and
pathogenesis of diabetic polyneuropathy, but there is a need for abdominal circumference was measured in cm in the point between
stronger evidence from clinical studies. Besides, an early appropriate the low costal rim and the iliac crest. The same investigator performed
treatment of DSPN would be crucial for the patient’s prognosis and all measurements.
could prevent or delay this complication. However, no causative
treatment is known since the pathogenesis of DSPN is not fully
2.2.1. Assessment of distal symmetric polyneuropathy
elucidated (Lavery, Hunt, Lafontaine, et al., 2010).
The neuropathy disability score (NDS) was used to quantify the
Few clinical studies have evaluated OS levels in T2DM patients
presence and severity of DSPN obtained from physical examination
with DSPN; and little is known about the glutathione system,
(Boulton, 1998). An NDS score of 3 or greater was considered
malondialdehyde (MDA) or oxidized low-density lipoprotein (oxidi-
abnormal (presence of DSPN).
zed-LDL). Therefore, the aim of this study was to analyze the OS levels
We also evaluated the loss of protective sensation through
in circulating mononuclear cells by measuring MDA, oxidized-LDL and
application of the 10 g Semmes–Weinstein monofilament (SWM).
the glutathione system, in a well-characterized cohort of T2DM
Three plantar sites on each foot were explored: great toe and base of
patients. We assumed that the consumption of relevant intracellular
first and fifth metatarsals. The monofilament was not applied on areas
antioxidant factors (such as reduced glutathione [GSH]) or an increase
of calluses or other structural abnormalities. A score of 1 was given on
in OS markers (oxidized glutathione [GSSG], MDA and oxidized-LDL)
each site explored if the patient perceived the force applied in at least
would be associated with the presence of DSPN in T2DM patients.
2 of 3 attempts. A score of 0 was given if there was lack of perception.
A summation of all scores on both feet between 0 and 4 was
2. Subjects and methods
considered abnormal, while scores 5 or 6 were normal (Feng,
Schlösser, & Sumpio, 2009). The same experienced physician
2.1. Subjects
performed all procedures in each participant.
We studied 196 non-related T2DM patients, selected by consec-
utive sampling at the Diabetic Unit of our center and at two primary 2.2.2. Assessment of peripheral vascular disease
health care centers (45% of patients); all subjects were from the Peripheral vascular disease was assessed through the ankle
metropolitan area of Valencia, Spain. brachial index (ABI). ABI is the ratio of systolic blood pressure in the
A nested case–control study was conducted. The cases and controls ankle to that in the brachial artery. Measurement of ABI is made in
proceeded from a well-characterized cohort of 196 T2DM. The study supine position, using a hand-held continuous-wave Doppler ultra-
included 30 T2DM patients with DSPN and 30 diabetic controls sound (Bi-directional Smartdrop TM 20). PVD is defined by an ABI b 0.9
without DSPN who were matched to cases on gender, age, body mass in either leg (American Diabetes Association, 2003). Scores above 1.2
index (BMI), HbA1c and renal function. This was done in a stepwise were excluded from the statistical analysis due to possible arterial
way to obtain similar age, gender distribution, BMI, HbA1c and stiffness and the inability to accurately assess the grade of peripheral
glomerular filtration rate (GFR). It is well known that all these vascular disease.
parameters can influence redox status, and we use this strategy in
order to minimize the effect of these confounding factors (Aranda,
Doménech, Rus, et al., 2007; Jones, 2006; Lodovici, Giovannelli, 2.2.3. Biochemical parameters
Pitozzi, et al., 2008; Miller, Michael De Silva, Jackman, & Sobey, 2007). Blood samples were drawn following an overnight 12 h fasting
Inclusion criteria were as follows: adult patients (aged ≥ 40 years) period. HbA1c was measured using a high-performance liquid
with type 2 diabetes (identified based on fasting glucose and/or HbA1c chromatography assay. Total cholesterol (TC), triglycerides (TG),
or a prescription for oral or subcutaneous hypoglycemic drugs or HDL cholesterol (HDL-C) and apolipoprotein B were determined using
insulin). Exclusion criteria were type 1 diabetes, heart failure NYHA class standard methods previously described (Ascaso, Merchante, Lorente,
III or IV, renal disease (GFR b 30 ml/min/1.72 m 2), cirrhosis, vitamin et al., 1998). Urinary albumin concentration was determined by a
supplementation, vitamin B12 or folate deficiency, hypothyroidism, standard radioimmunoassay. Glomerular filtration rate was calculated
positive HIV, systemic illness, cancer, smoking habit, consumption of using the Modification of Diet in Renal Disease equation (MDRD).
N 30 g alcohol/day or any other disease, condition or drugs known to Markers of OS were determined in circulating mononuclear cells
cause neuropathy. Patients with Charcot´s arthropathy and/or bilateral isolated by Ficoll-Hypaque methods, as previously reported (Oltra,
amputation were also excluded. Carbonell, Tormos, Iradi, & Sáez, 2001). Oxidized glutathione and
All patients continued on their usual hypoglycemic treatment, as reduced glutathione (GSSG and GSH) were determined using high
well as drug treatment for other cardiovascular risk factors. The performance liquid columns (HPLC) and UV detection (Navarro,
ethical committee of our hospital approved the study and all patients Obrador, Pellicer, et al., 1997). MDA was analyzed by HPLC (Wong,
gave their written informed consent. Knight, Hopfer, et al., 1987). Oxidized-LDL was measured by
We also have studied a control group of 55 healthy volunteers commercial enzyme-linked immunosorbent assay (Biomedica,
ranging age from 18 to 65 years that were randomly recruited among Wien, Austria).
M.M. Mendez et al. / Journal of Diabetes and Its Complications 29 (2015) 923–927 925

2.3. Statistical analyses Table 2


Reduced glutathione (GSH), oxidized glutathione (GSSG), GSH/GSSG ratio, MDA and
oxidized-LDL in diabetic subjects with and without distal peripheral polyneuropathy.
All analyses were conducted using the Statistical Package for Social
Sciences (SPSS) software (version 22, SPSS, Chicago, IL, USA). Results T2DM without DSPN (n = 30) T2DM with DSPN (n = 30)
are expressed as the mean ± standard deviation. The p-values were GSH (nmol/ml) 22.40 ± 2.54 ⁎ 18.34 ± 4.18
two-tailed and a p-value of less than 0.05 was considered significant. GSSG (nmol/ml) 3.45 ± 0.29 3.35 ± 0.37
For the comparison of means of continuous variables we used GSH/GSSG ratio 6.52 ± 0.95 ⁎ 5.54 ± 1.41
Oxidized-LDL (U/I) 185.9 ± 19.80 205.20 ± 21.30
T-student test. Percentages were compared using the chi-squared test.
MDA (nmol/ml) 1.84 ± 0.15 1.78 ± 0.19
Pearson’s correlation or Spearman, as appropriate, was used to assess
the degree of association between two quantitative variables, such as All values are indicated as mean ± standard deviation. Abbreviations: T2DM: type 2
diabetic subjects, DSPN: distal symmetric polyneuropathy, MDA: malondialdehyde,
that found between GSH or GSSG and clinical or biological parameters. GSH: reduced glutathione, GSSG: oxidized glutathione, oxidized-LDL: oxidized
low-density lipoprotein.
* indicates p b 0.01.
3. Results

Clinical, anthropometric and biochemical characteristics of the two subjects and T2DM patients divided according to the presence or not
studied groups (T2DM patients with or without DSPN) are shown in of DSPN.
Table 1. As expected there were no differences in age, gender The levels of oxidized-LDL, a marker of lipoprotein-associated
distribution, BMI, HbA1c or GFR between groups. In addition, the two oxidative stress, were non-significantly higher in diabetic patients
studied groups showed similar waist circumference, ABI, systolic and with DSPN (oxidized-LDL: 205.20 ± 21.30 vs 185.90 ± 19.80 U/l). In
diastolic blood pressure values, hypoglycemic treatment and lipid addition, no significant differences between groups were found when
profile. There were significant differences in disease duration (p = comparing MDA levels (MDA: 1.78 ± 0.19 vs 1.84 ± 0.15 nmol/ml).
0.05), prevalence of dyslipidemia (p = 0.01) and altered SWM test In the whole group, we found a significant negative association of
(p = 0.001). GSH with fasting triglycerides (r = − 0.251, p = 0.038), and GSSG
As shown in Table 2, a significant decrease in GSH values was with fasting plasma triglycerides (r = 0.222, p = 0.06). No signifi-
found in T2DM patients with DSPN compared to those without DSPN cant associations were found between OS (GSH or GSSG) and clinical,
(GSH: 18.34 ± 4.18 vs 22.4 ± 2.56 mmol/ml, p b 0.001). The GSH/ anthropometric or metabolic parameters (age, gender, evolution time
GSSG ratio was significantly lower in the group with DSPN (GSH/GSSG of diabetes, fasting glucose or HBA1c, ABI or renal function).
ratio: 5.54 ± 1.41 vs 6.52 ± 0.95, p = 0.003), despite higher GSSG
values in patients with DSPN. Moreover, when we compared GSH 4. Discussion
values, GSH/GSSG ratio and GSSG values obtained in a control group
and the T2DM group, we found significant differences: GSH value Our results show that diabetic patients with DSPN present
22.53 ± 3.82 in controls vs 20.37 ± 4.01 mmol/ml in T2DM patients, significantly decreased levels of GSH and GSH/GSSG ratio compared
p = 0,004; GSH/GSSG ratio 10.11 ± 5.29 vs 6.03 ± 1.29 p = 0,001 with diabetic patients without DSPN, suggesting that subjects with
and GSSG 2.78 ± 1.28 vs 3.41 ± 0.33 mmol/ml p = 0,001, respec- neuropathy have an increased consumption of glutathione. In
tively, Table 3). In Fig. 1 we show GSH and GSSG values in healthy addition, compared to healthy non obese, normolipidemic, non
diabetic subjects, GSH values were decreased and GSSG values
increased in T2DM patients independently of the presence of DSPN,
Table 1 indicating, as expected, a higher fasting OS status in T2DM patients.
General, anthropometric and biochemical characteristics of the studied groups. Data from previous studies underline the importance of a
T2DM without T2DM with
significant decrease in the effectiveness of antioxidant defenses in
DSPN (n = 30) DSPN (n = 30) diabetic patients, leading to an increase in OS (Brownlee, 2005;
Ceriello, 2003). Insufficient cellular antioxidative defense mecha-
Age (years) 65.93 ± 8.43 68.10 ± 6.90
Gender (M/F) 20/10 20/10 nisms may also be involved in nerve damage. Kasznicki J et al.
BMI (kg/m2) 30.70 ± 5.30 31.48 ± 4.91 demonstrated that the activity of cellular antioxidant enzymes, such
Waist circumference (cm) 107.03 ± 9.98 109.65 ± 10.73 as superoxide dismutase (SOD), catalase (CAT), and glutathione
Systolic blood pressure (mmHg) 148.97 ± 15.63 151.03 ± 20.33
peroxidase (GPx), was decreased in peripheral blood cells of diabetic
Diastolic blood pressure (mmHg) 84.93 ± 10.64 79.48 ± 12.8
TC (mg/dl) 184.46 ± 40.31 185.79 ± 38.63 patients with coexisting DSPN. Moreover, the evaluation of total
TG (mg/dl) 146.11 ± 78.76 147.41 ± 69.95
HDLc (mg/dl) 48.82 ± 14.03 47.31 ± 8.75
Fasting glucose (mg/dl) 152.04 ± 49.60 177.86 ± 53.50 Table 3
HbA1c (%) 7.5 ± 1.69 7.6 ± 1.59 General and anthropometric characteristics, lipids and glutathione values in control
Creatinine (mg/dl) 0.99 ± 0.35 1.01 ± 0.42 and type 2 diabetic subjects.
GFR (ml/min/1.72 m2) 93.09 ± 36.67 90.19 ± 37.51
Controls (n = 55) T2DM (n = 60)
Duration of diabetes (years) 9.68 ± 8.01 ⁎⁎ 14.97 ± 11.77
Prevalence of hypertension, n (%) 23 (76.67) 22 (73.33) Age (years) 57.93 ± 4.56 67.07 ± 7.71⁎
Prevalence of dyslipidemia, n (%) 17 (56.67) ⁎ 25 (83.33) Gender (M/F) 31/24 40/20
ABI 0.88 ± 0.16 0.84 ± 0.17 BMI (kg/m2) 27.08 ± 4.74 31.09 ± 5.08 ⁎⁎
Altered SWM test, n (%) 8 (26.67) ⁎⁎ 22 (73.33) Waist circumference (cm) 86.02 ± 10.59 108.34 ± 10.36⁎⁎
Diabetic treatment TC (mg/dl) 187.21 ± 32.51 185.14 ± 38.62
Oral agents, n (%) 18 (60.00) 16 (53.33) TG (mg/dl) 97.3 ± 43.2 146.77 ± 73.74⁎
Insulin, n (%) 5 (16.67) 7 (23.33) GSH (nmol/ml) 22.53 ± 3.82 20.37 ± 4.01⁎
Both, n (%) 7 (23.33) 7 (23.33) GSSG (nmol/ml) 2.78 ± 1.28 3.41 ± 0.33⁎⁎
GSH/GSSG ratio 10.11 ± 5.29 6.03 ± 1.29⁎⁎
All values are indicated as mean ± standard deviation (SD). Abbreviations: T2DM:
type 2 diabetic subjects, DSPN: distal symmetric polyneuropathy, TC: total cholesterol, All values are indicated as mean ± standard deviation. Abbreviations: T2DM: type 2
TG: triglycerides, HDLc: high density lipoprotein cholesterol, GFR: glomerular filtration diabetic subjects, TC: total cholesterol, TG: triglycerides, GSH: reduced glutathione,
rate, ABI: ankle brachial index, SWM: Semmes–Weinstein monofilament test. GSSG: oxidized glutathione.
* indicates p b 0.05. * indicates p b 0.01.
⁎⁎ indicates p b 0.01. ⁎⁎ indicates p ≤ 0.001.
926 M.M. Mendez et al. / Journal of Diabetes and Its Complications 29 (2015) 923–927

(nmol/ml) 25 peripheral polyneuropathy in diabetic patients. However additional


studies are needed to confirm these findings.
20 * Nevertheless, our study has several limitations that must be kept
in mind. First, as a case–control study a causal relationship cannot
15 be established. The number of studied subjects is low, although
Controls (n=55) nested case–control design can increase the power of the study. Other
T2DM - DSPN (n=30) kinds of diabetic neuropathies were not studied. Neuropathy was
10 defined clinically, through signs scoring systems: Neuropathy Dis-
T2DM + DSPN (n=30)
ability Score (NDS) without electrodiagnostic testing. Finally, the
5 ** * activities of the main antioxidant enzymes or the total antioxidant
capacity were not measured.
0 In conclusion, in diabetic patients with distal symmetric peripheral
GSH GSSG polyneuropathy there is an alteration in the glutathione system in
circulating monocytes cells. This finding could be important for
Fig. 1. Reduced and oxidized glutathione values in controls and type 2 diabetic subjects understanding how OS impacts on the development of neuropathic
divided according to the presence (+) or absence (−) of distal peripheral polyneuropathy.
complications in diabetic patients. However, further prospective and
* p b 0.01 controls vs T2DM + DSPN. ** p b 0.01 controls vs T2DM − DSPN.
intervention studies are necessary to evaluate the impact of OS in the
pathogenesis of peripheral neuropathy in diabetic patients.

Acknowledgements

plasma antioxidant capacity revealed a decrease in the group with We thank the patients for their cooperation. This study has been
DSPN (Kasznicki et al., 2012). supported by grants from Conselleria de Sanitat to JT Real (AP-04/06
Reduced glutathione (GSH) is an important part of reactive and AP 068/08) and CIBERDEM (ISCIII). CIBER de Diabetes and
oxygen species defense system (Little & O’Brien, 1968). In addition, Enfermedades Metabólicas Asociadas (CIBERDEM) is an Instituto de
GSH is involved in a large number of intracellular detoxifications. Salud Carlos III initiative.
Interestingly, El Boghdady and Badr (2012) found a significant María Ascaso has a scholarship grant from Ministerio de Educación
decrease of erythrocyte GSH in diabetic patients with and without y Ciencia (Universitat de Valencia Estudi General curso 2013/2014).
DSPN compared to healthy subjects. Similar results have been
found in our study, revealing a decrease of GSH and GSH/GSSG ratio
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