Trace elements status in diabetes mellitus type 2 ...

diabetes research and clinical practice 91 (2011) 333–341

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Diabetes Research and Clinical Practice journ al h omepage: www .elsevier.co m/lo cate/diabres

Trace elements status in diabetes mellitus type 2: Possible role of the interaction between molybdenum and copper in the progress of typical complications Crescencio Rodrı´guez Flores a, Mo´nica Preciado Puga b, Katarzyna Wrobel a, Ma. Eugenia Garay Sevilla b, Kazimierz Wrobel a,* a b

Department of Chemistry, University of Guanajuato, L de Retana N85, 36000 Guanajuato, Mexico Department of Medical Research, University of Guanajuato, L de Retana N85, 36000 Guanajuato, Mexico

article info

abstract

Article history:

It is well established that both, the deficiency and possible overload of mineral micronu-

Received 7 October 2010

trients have adverse health effects. It is also generally accepted that non-essential xeno-

Received in revised form

biotics contribute to oxidative damage, which is considered one of the principal factors in

30 November 2010

diabetes and its complications. The purpose of this work was to gain an insight on the global

Accepted 6 December 2010

role of metal/metalloids in the progress of diabetes mellitus type 2. In such approach,

Published on line 5 January 2011

aluminum, vanadium, chromium, manganese, cobalt, nickel, copper, zinc, arsenic, selenium, molybdenum, mercury, cadmium and lead were determined by inductively coupled

Keywords:

plasma-mass spectrometry (ICP-MS) in serum and urine of 76 diabetic patients (age 52 8

Diabetes mellitus type 2

years, 5–16 years of DM2, 52 subjects with slight-to-moderate complications and 24 with

Complications

severe complications). A series of anthropometric and clinical parameters usually evaluated

Trace elements

in the follow-up of patients were assessed by standard methods. Statistical analysis

Inductively coupled plasma-mass

(unpaired t-test, analysis of correlation and principal component analysis) was then carried

spectrometry

out in search of possible relationships existing among metals/metalloids and these parameters. The results obtained suggest that antagonistic interaction between molybdenum and copper might be involved in the progress of diabetes complications. # 2010 Elsevier Ireland Ltd. All rights reserved.

1.

Introduction

The main feature of diabetes is chronic hyperglycemia, which leads to the disturbance of carbohydrate, fat and protein metabolism. Even though diabetes is classified as a single disease, various secondary complications may occur such as, renal dysfunction and failure, cardiac abnormalities, diabetic retinopathy, neuropathy, atherosclerosis, among others [1]. The etiology of diabetes and its complications still is not clear, however several factors as aging, obesity and oxidative damage have been implicated [2,3]. Owing to the increasing

prevalence of diabetes, multidisciplinary study aimed at preventing and treating diabetes is one of the world-wide research priorities. Several micronutrients have beneficial effects in healthy subjects and also in diabetes. In particular, copper, zinc, selenium, iron or manganese are essential components of metalloenzymes such as Se–cys containing glutathione peroxidase, Cu/Fe cytochrome C oxidase or different types of superoxide dismutases, all of them important in intra- and extra-cellular antioxidant defense [4,5]. On the other hand, some metallic species are considered important in glucose

* Corresponding author. Tel.: +52 473 7327555; fax: +52 473 7326252. E-mail addresses: [email protected] (C.R. Flores), [email protected] (M.P. Puga), [email protected], [email protected] (K. Wrobel), [email protected] (M.E. Garay Sevilla), [email protected] (K. Wrobel). 0168-8227/$ – see front matter # 2010 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.diabres.2010.12.014

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metabolism. For example, trivalent chromium was proposed as a structural part of glucose tolerance factor and even though such compound has not been confirmed, it is believed that Cr participates in stimulation of insulin signaling [6]. Other element to be mentioned is zinc, which is necessary for the synthesis of insulin hexamer [7]. Vanadium salts have been used to lower glucose levels in diabetes patients even before the discovery of insulin, however the molecular mechanisms underlying the observed effects are not clear [8]. Finally, different chemical forms of zinc, vanadium, molybdenum and selenium have been examined as insulin mimetics and used in diabetes treatment [8–10]. It should be stressed however that, the above beneficial effects of specific elements can be observed only within relatively sharp range of their concentrations, while the deficiency or possible overload usually cause adverse health effects [11]. It is well established that trace element status in diabetic patients is altered as compared to healthy subjects [12]. The disturbed metabolism of some micronutrients has been reported and it was postulated that certain metals/metalloids might have specific roles in the pathogenesis and progress of diabetes mellitus [5,7,13–15]. Logically, the deficiency of essential elements may lead to the failure of antioxidant defense and also to glucose intolerance, both important in the progress of diabetes. Furthermore, the overload of some transition metals may be responsible for oxidative damage [13]. Redox-cycling metals as copper, iron or chromium are involved in the formation of reactive oxygen species; in particular the catalytic function of Fe(II)/Fe(III) and Cu(I)/Cu(II) in Fenton reaction–mediated generation of hydroxyl radical should be mentioned [5]. Otherwise, non-essential xenobiotics like cadmium, mercury or arsenic deplete glutathione levels due to their high affinity to sulfhydryl group [7]. Even though molybdenum has not been directly associated with diabetes in humans, different thiomolybdate species have been used in the treatment of Wilson disease to eliminate the excess of Cu accumulated in liver and brain [16]. On the other hand, several studies have been carried out on ruminant animals [17,18]. In particular, since the mid1980s, a so called ‘‘moose sickness’’ had been afflicting the moose population of south-western Sweden [19]. Systematic studies revealed that the principal cause of this disease was molybdenum overload and related secondary deficiency of copper [20]. The increased oxidative stress and the disturbed carbohydrate metabolism have been reported [21,22]. The evaluation of trace element status in diabetic patients has often been undertaken [23–25]. The determinations have been made preferentially in biological fluids, hair and nails [26]. Some studies have focused a single element in order to understand its specific biological role [6,27,28], yet multielemental determinations have also been carried out [12,23– 25,29,30]. The results reported in the above mentioned studies indicate higher serum concentration levels of copper, lead, arsenic, cadmium, nickel, aluminum and lower levels of selenium, chromium, manganese and zinc in diabetic patients with respect to healthy individuals. The purpose of the present study was to gain a further insight on the global role of metal/metalloids in diabetes and particularly, in its complications. In such approach, serum and urine concentration levels of several elements were

determined by inductively coupled plasma mass spectrometry and their possible relationship with anthropometric and clinical parameters typically evaluated in the follow-up of diabetic patients were statistically evaluated in a group of subjects at different stages of diabetes mellitus type 2 (DM2). The results obtained suggest that the antagonistic interaction between molybdenum and copper could be related to the progress of diabetes complications.

2.

Material and methods

2.1.

Design of study (subject, groups and samples)

The cross-sectional study was carried out in 76 DM2 patients recruited in the Clinical Services of Health Department and referred to the facilities at the Department of Medical Research, University of Guanajuato. The Institutional Review Board approved the protocol, and all subjects signed the informed consent. The DM2 patients were of either sex, 52 8 years old, 5–16 years from diagnostics, with slight-to-severe diabetes complications, no smoking and with no other chronic or infectious diseases. The subjects were grouped according to the stage of diabetes complications: nephropathy, neuropathy and retinopathy. Among 76 patients, 52 were classified in a group of slight-to-moderate complications and 24 in a group of severe complications. Additionally, a small group of twelve healthy volunteers was recruited exclusively for the determination of trace elements. Blood serum was obtained from a 3– 5 ml venous fasting blood and 24 h urine was collected for each subject. Precautions to avoid metal/metalloid contamination were carefully observed.

2.2.

ICP-MS determination of trace elements

The wet digestion of samples was carried out as follows: to an aliquot of serum (400 ml), 100 ml of IS (In + Bi), 500 ml concentrated nitric acid and 200 ml 30% hydride peroxide were added, the mixture was incubated 24 h at 90 8C; after cooling down the volume was brought to 6 ml with deionized water, the samples were centrifuged and introduced to ICP-MS system. For urine, the sample aliquot was 500 ml, 200 ml of nitric acid and 100 ml of IS solution were added and the samples were incubated during 12 h at 90 8C. The final volume was 5 ml. Each analysis was carried out in triplicate and the blank was run in parallel. A model 7500ce inductively coupled plasma mass spectrometer (Agilent Technologies, Tokyo, Japan) with a Meinhard nebulizer and Peltier-cooled spray chamber (2 8C) was used. The instrumental operating conditions were as follows: forward power 1500 W, plasma gas flow rate 15 l min 1, carrier gas flow rate 0.89 l min 1, make-up gas flow rate 0.15 l min 1, sampling depth 10 mm, platinum sampling and skimmer cones, dwell time 300 ms per isotope, collision/reaction cell gas He, 4 ml min 1. The isotopes 27Al, 51V, 52Cr, 55Mn, 59Co, 60Ni, 63Cu, 66 Zn, 75As, 82Se, 95Mo, 111Cd, 202Hg, 208Pb, were monitored (115In and 209Bi as internal standards). Calibration was performed using the Agilent multi-element standard solution (0.1% nitric acid (v/v)) at element concentration levels: 0; 0.2; 0.4; 1.0; 2.0; 5.0 and 10 mg l 1 with addition of the internal standards (5.0 mg l 1 In, 5.0 mg l 1 Bi). The analytical accuracy was demonstrated by

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analyzing NIST 1643d certified reference material and by the method of standard addition. For three independent analyses of certified reference material, the results obtained were 34.7 1.2 mg l 1 for V, 18.2 0.3 mg l 1 for Cr, 38.0 0.9 mg l 1 for Mn, 22.2 0.1 mg l 1 for Co, 20.2 1.6 mg l 1 for Cu, 72.9 0.6 mg l 1 for Zn, 53.9 1.3 mg l 1 for As, 9.9 2.2 mg l 1 for Se, 115 3 mg l 1 for Mo, 6.37 0.2 mg l 1 for Cd, 19.3 0.8 mg l 1 for Pb (certified values respectively: 35.1 1.4 mg l 1 for V, 18.5 0.2 mg l 1 for Cr, 37.7 0.8 mg l 1 for Mn, 25.0 0.6 mg l 1 for Co, 20.5 3.8 mg l 1 for Cu, 72.5 0.7 mg l 1 for Zn, 56.0 0.7 mg l 1 for As, 11.4 0.2 mg l 1 for Se, 113 2 mg l 1 for Mo, 6.47 0.4 mg l 1 for Cd, 18.2 0.6 mg l 1 for Pb).

2.3.

Clinical and anthropometric measurements

The questionnaire was answered by each patient by oral inquiry of an investigator at the facilities of our institution. The following data were collected: age in years, years since diagnosis of diabetes, data for the evaluation of body mass index (BMI, kg/m2), systolic and diastolic blood pressure (sBP, dBP). Metabolic control was assessed by measurement of serum fasting glucose (Glc, enzymatic method GODPAP, Lakeside, Me´xico City) and HbA1c by ion-exchange chromatography (Sigma, St. Louis, MO); total cholesterol (Chol), HDL-cholesterol, LDL/VLDL-cholesterol and triglycerides (TG) by conventional enzymatic methods; urine creatinine (Cre_U) and total proteins (Prot_U); serum creatinine (Cre_S), urea (U) and uric acid (UA) by conventional methods [6,15,31]. For serum malondialdehyde (MDA), spectrophotometric assay was used [32].

2.4.

Assessment of diabetic complications

Actual stage of diabetes nephropathy, neuropathy and retinopathy was evaluated in an arbitrary scale ranging from

0 to 3: absent (0), slight (1), moderate (2) and severe (3). Renal function was evaluated based on the albumin excretion in 24 h urine, retinopathy was assessed by direct and indirect ophthalmoscopy with pupil dilatation and neuropathy was ´ s questionnaire [31]. classified with the scrutiny Michigan

2.5.

Statistical analysis

Descriptive statistics was performed to obtain means and standard deviations. An unpaired t-test was used to compare mean values of each parameter among the groups. To observe possible relationships between parameters, principal component analysis (PCA) and analysis of correlation were performed. Significance level was established at p < 0.05. The software used were: Statistica for Window (StatSoft Inc., Tulsa, OK) and Unscrambler 7.5 (Camo, Norway).

3.

Results

The concentration levels of trace elements in serum and urine of diabetic patients and healthy subjects are presented in Table 1. Significantly higher serum concentrations of Al, Cd, Cu, Mn, Hg, Ni and lower Cr, Co, V were found in diabetic patients as compared to healthy subjects (unpaired t-test, p < 0.05). A tendency toward higher urine levels of Cr, As, Cu, Zn and lower levels of Cd, Co, Pb, Mn, Mo, Ni, Se in diabetic patients can be perceived, as compared to healthy subjects, however statistically significant differences were observed only for Cd and Zn. For DM2 patients (n = 76), statistically important correlations were observed between pairs of essential micronutrients in serum: Mn–Cu (b = 0.3104, p = 0.070), Se–Cu (b = 0.6764, p = 0.000), Zn–Cu (b = 0.5571, p = 0.001), Se–Mn (b = 0.3782, p = 0.025), Mn–Zn (b = 0.3944, p = 0.019) and Se–Zn (b = 0.7938,

Table 1 – Serum and urine concentration levels found in the group of diabetic patients (n = 76) and in healthy individuals (n = 12). For each element, the range of reference values reported for healthy populations in different geographical regions is also presented (serum [33–35]–urine [35,37,38]. Element

Serum (mg l 1) Reference range

Ala As Cda,c Cra Coa Cua Pb Mna Hga Mo Nia Se Va Znc a b c

1.8–6.1 0.5–1.8 0.2–1.0 0.1–0.5 0.1–0.4 700–1300