A p C A J a b c a A R R 1 A A K E s F G s M T T 1 a d c [ s l a a T e t a h 0 International Journal of Biological Macromolecules 96 (2017) 817–832 Contents lists available at ScienceDirect International Journal of Biological Macromolecules j ourna l h o mepa ge: www.elsev ier .com/ locate / i jb iomac proteomic approach to identify metalloproteins and metal-binding roteins in liver from diabetic rats amila Pereira Bragaa,∗, José Cavalcante Souza Vieiraa, Ryan A. Groveb, Cory H.T. Booneb, line de Lima Leitec, Marília Afonso Rabelo Buzalaf c, Ana Angélica Henrique Fernandesa, iri Adamecb, Pedro de Magalhaes Padilhaa Department of Chemistry and Biochemistry, Institute of Bioscience of Botucatu, São Paulo State University, Botucatu, SP, Brazil Department of Biochemistry, University of Nebraska-Lincoln, Lincoln, NE, USA Bauru Dental School, University of São Paulo- USP, Bauru, SP, Brazil r t i c l e i n f o rticle history: eceived 13 September 2016 eceived in revised form 0 December 2016 ccepted 21 December 2016 vailable online 3 January 2017 eywords: lectrospray ionization-tandem mass pectrometry a b s t r a c t Proteins play crucial roles in biological systems, thus studies comparing the protein pattern present in a healthy sample with an affected sample have been widely used for disease biomarker discovery. Although proteins containing metal ions constitute only a small proportion of the proteome, they are essential in a multitude of structural and functional processes. The correct association between metal ions and proteins is essential because this binding can significantly interfere with normal protein function. Employment of a metalloproteomic study of liver samples from diabetic rats permitted determination of the differential abundance of copper-, selenium-, zinc- and magnesium-associated proteins between diabetic, diabetic treatment with insulin and non-diabetic rats. Proteins were detected by ESI–MS/MS. Seventy-five dif- ferent proteins were found with alterations in the metal ions of interest. The most prominent pathways lame atomic absorption spectrometry raphite furnace atomic absorption pectrometry etalloproteomic ype 1 diabetes wo-dimensional electrophoresis affected under the diabetic model included: amino-acid metabolism and its derivates, glycogen stor- age, metabolism of carbohydrates, redox systems and glucose metabolism. Overall, the current methods employed yielded a greater understanding of metal binding and how type 1 diabetes and insulin treat- ment can modify some metal bonds in proteins, and therefore affect their mechanism of action and function. © 2017 Elsevier B.V. All rights reserved. . Introduction Type 1 diabetes mellitus (DM1) is a severe metabolic disorder nd a public health concern. Clinical complications of DM1 include amage and dysfunction of multiple organs with secondary compli- ations, such as atherosclerosis, retinopathy and renal insufficiency 1]. Although metal ions comprise a small proportion of body tis- ues (4%), they are essential as structural components and in many ife processes. The main roles of these metal ions can be described s structural and functional [2]. Metal ions bound to proteins nd metalloproteins represent a large portion of the total protein. hese ions are responsible for many metabolic processes, such as nergy conversion in photosynthesis and respiration, gene regula- ion, substrate activation and other catalytic processes, transport nd storage [3]. ∗ Corresponding author. E-mail addresses: braga ca@ibb.unesp.br, braga ca@yahoo.com.br (C.P. Braga). ttp://dx.doi.org/10.1016/j.ijbiomac.2016.12.073 141-8130/© 2017 Elsevier B.V. All rights reserved. Copper (Cu) is an essential component of many metalloen- zymes, including cytochrome oxidase, lysyl oxidase, superoxide dismutase, dopamine-ß-hydroxylase and tyrosinase [4]. Superox- ide dismutase (SOD) is a metalloprotein that contains Cu and zinc (Zn) that catalyzes the decomposition of the superoxide anion. Therefore, it is a component of the cellular defense system protect- ing against oxidative damage. Cu is a vital component of electron transfer reactions of SOD, underlying its antioxidant function [5]. Experimental data shows that magnesium (Mg) is required as an important cofactor in many enzyme reactions. Importantly for DM1, Mg participates in phosphorylation reactions of glucose metabolism [6]. It is essential in almost all energy transduction sys- tems in the glycolytic pathway and in oxidative energy metabolism, which is required for the synthesis and oxidation of fatty acids, protein synthesis, muscle contraction and ATPase activity [7]. Additionally, Mg participates in the intracellular signaling system, phosphorylation and dephosphorylation reactions that activate or inhibit a multitude of enzymes [8]. Selenium (Se) is a nonmetal, essential micronutrient for the synthesis of selenoproteins, which play an important role in the dx.doi.org/10.1016/j.ijbiomac.2016.12.073 http://www.sciencedirect.com/science/journal/01418130 http://www.elsevier.com/locate/ijbiomac http://crossmark.crossref.org/dialog/?doi=10.1016/j.ijbiomac.2016.12.073&domain=pdf mailto:braga_ca@ibb.unesp.br mailto:braga_ca@yahoo.com.br dx.doi.org/10.1016/j.ijbiomac.2016.12.073 8 Biolog s m f d i i t z [ l p [ t e o a o Z t a t a C u f t i e 2 2 o u a C e t P 4 ( b t g a 2 i L o n m b c 2 t t t T 18 C.P. Braga et al. / International Journal of ynthesis, metabolism and action of thyroid hormones. Further- ore, Se modifies the expression of selenoproteins, including the amilies of peroxidases, selenoenzymes, glutathione and thiore- oxin [9]. Zn is a component of synthetases and transferases, ncluding DNA and RNA, digestive enzymes, and associates with nsulin. Zn also participates in metabolic pathways involving pro- ein synthesis, carbohydrates, lipids and nucleic acids. In addition, inc also plays a role in apoptotically driven programmed cell death 10]. Studies have reported alterations in metal ions by diabetes mel- itus and suggested that the imbalance of specific elements may lay an important role in normal glucose and insulin metabolism 11,12]. In the majority of studies, the focus is on analyzing concen- rations and on the status of a single element or combinations of lements in blood, plasma or tissues; in our study, the focus is the n the integration of traditional analytical studies with inorganic nd biochemical studies. For this study, we used a robust method- logy to assist in understanding the variability of Cu, Mg, Se and n in diabetes and how treatment with insulin can alter this condi- ion, providing valuable information about how Cu, Mg, Se and Zn re distributed with proteins, as well as the individual concentra- ion in specific proteins, which helps to elucidate the physiological nd functional aspects of biomolecules in the liver. In this context, the present study involves the investigation u, Mg, Se and Zn found in liver samples from diabetic rats sing flame atomic absorption spectrometry (FAAS) and graphite urnace atomic absorption spectrometry (GFAAS), by protein frac- ionation (two-dimensional gel electrophoresis, 2D-PAGE) and dentification by electrospray ionization-tandem mass spectrom- try (ESI–MS/MS). . Material and methods .1. Animals and experimental groups A total of 24 Wistar male rats (Rattus norvegicus), 45 days ld, were used in the experiment; these were kept in individ- al plastic cages with a controlled temperature and photoperiod, nd they received water and a commercial diet (Purina ® , Labina, ampinas-SP) ad libitum throughout the experimental period. The xperimental design was approved by the Ethics Committee on he Use of Animals (CEUA) at the Institute of Biosciences/São aulo State University (UNESP), Botucatu, Brazil (protocol: CEUA- 36/2012). The animals were divided into three groups (n = 8): C control group): normal rats, DM1: diabetic rats and DM1 + I: dia- etic rats that received insulin. Diabetes mellitus was induced with he administration of streptozotocin (STZ; 60 mg/body weight, sin- le dose, i.p.). Blood glucose was measured 48 h after the STZ dministration, and animals with glycemic levels greater than 20 mg dL−1 were considered diabetic. The animals received insulin n the form of Humulin N100UI neutral protamine Hagedorn (NPH; illy ® ) with an initial dose of 3 U/animal; this dose was adjusted r maintained so that serum glucose levels were kept within the ormal range. At the end of the 30 day experimental period, the ani- als were anesthetized (ketamine hydrochloride 10%, 0.1 mL/100 g ody weight, i.p.) and sacrificed by decapitation, and the liver was ollected. .2. Sample preparation Approximately 1.00 g of pooled sample (liver) was weighed in riplicate and ground with 2 mL of ultrapure water, macerated, and he protein extracts were separated from the solid portion by cen- rifugation at 10,000g at 4 ◦C for 10 min in a refrigerated centrifuge. he protein extracts were used to quantify protein resuspension in ical Macromolecules 96 (2017) 817–832 a protein pellet with 0.50 mol L−1 NaOH. The total protein concen- tration of the liver samples was determined by the Biuret method using bovine serum albumin as the standard. Analytical calibration curves were constructed with concentrations of 10–100 g L−1 from a stock solution of albumin (100 g L−1). The method used 50 mL of sample for the standard and 2.5 mL of Biuret reagent, which was mixed and placed in a water bath at 32 ◦C for 10 min. After 5 min at room temperature, absorbance readings were performed in a spectrophotometer at a wavelength of 545 nm. 2.3. Electrophoretic runs (2D-PAGE) With the standardization of the gels, the electrophoretic runs were performed using liver samples for the different experimen- tal groups. Six gels were made for each group (pooled). Aliquots of pooled liver were diluted in a urea solution containing 7 mol L−1; 2 mol L−1 thiourea, 2% (w/v) CHAPS (3-[(3-cholamidopropyl)- dimethylammonio]-1-propanesulfonate), 0.5% (v/v) ampholytes at a pH ranging from 3 to 10, 0.002% (w/v) bromophenol blue and 2.8 mg of dithiothreitol (DTT) were added to this buffer, and the mixture was used in the electrophoretic separations. A total of 3 �g �L−1 protein was added to 13 cm strips contain- ing polyacrylamide gel with ampholytes immobilized at pH 3–10. These strips were placed onto a first dimension focusing for 12 h at room temperature to be rehydrated with the protein extract. After this period, the strips were placed into an Ettan IPGphor iso- electric focusing (IEF) unit (GE Healthcare) for the first-dimension separation, after the strips were reduced for 10 min with a solu- tion containing 6 mol L−1 urea, 2% (w/v) SDS, 30% (v/v) glycerol, 50 mmol L−1 Tris-HCl, 0.002% (w/v) bromophenol blue and 2% (w/v) DTT and alkylated for 10 min with a similar solution, but with DTT replaced by 2.5% (w/v) iodoacetamide (IAA). For the second dimension, the strips were placed onto a 10% polyacrylamide gel; a piece of filter paper with 10 �L of a molec- ular weight standard (12–225 kDa range) was placed close to the strip in the gel, and they were sealed with a hot solution of 0.5% (m/v) agarose. The second dimension of the electrophoretic run was divided into two stages: 7.5 mA/gel for 30 min and 20 mA/gel for 6 h 40 min. After the first and second dimensions in the electrophoretic runs, the proteins in the gels were fixed in the gels using a staining solution containing 10% (v/v) acetic acid and 40% (v/v) ethanol for 1 h. In the sequence, the proteins were revealed with a colloidal Coomassie stain: 8% (m/v) ammonium sulfate, 1.6% (v/v) phospho- ric acid, 0.08% (m/v) Coomassie blue G-250 and 25% (v/v) methanol for 72 h. Afterwards, the Coomassie stain was removed and the gels were washed with ultrapure water. The gels were scanned using an ImageScanner III (GE Healthcare), and the images were analyzed by ImageMaster 2D Platinum 7.0 (GeneBio, Geneva, Switzerland). 2.4. Copper, magnesium, selenium and zinc mapping by FAAS or GFAAS The copper, selenium and zinc in the protein spots identified were determined by GFAAS, and magnesium was determined by FAAS after mineralizing the samples (spots and feed) as described by Moraes et al. [13]. The analyses used two different elec- trophoretic runs, and gels were obtained in duplicate for each run. The copper, magnesium, selenium and zinc determinations were performed with a Shimadzu AA-6800 atomic absorption spec- trometer using wavelengths of 324.7, 285.2, 190.0 and 213.9 nm, respectively. Copper, selenium and zinc operated with a cur- rent of 400 mA and magnesium with a current of 10 mA. The analytical curves were prepared using Merck Titrisol standard solutions. The curves for copper and zinc were constructed in Biolog c r 1 a c 0 2 c a s W a ( f d d t A t t e f f s t 6 A w o m M s s d fi a 1 t a t C F B D C.P. Braga et al. / International Journal of oncentration ranges of 5.00–20.00 �g L−1; for magnesium, the ange was 0.10–0.40 mg L−1 and for selenium the range was 0.00–60.00 �g L−1. The region of the gel where no protein spots ppeared was used for the analytical blank. The limits of quantifi- ation (LOQ) for copper, magnesium, selenium and zinc are 0.046, .94, 0.083 and 0.023 �g L−1, respectively. .5. Characterization of protein spots by ESI–MS/MS The protein spots were extracted from the gels using a scalpel, ut into segments of approximately 1 mm3 and prepared for MS ccording to Shevchenko et al. [14] with some modification. The ubsequent procedure with the segments was described by the aters’ Technical Bulletin, which can be summed up in four steps: ) dye removal (destained with 25 mM ammonium bicarbonate Ambic)/acetonitrile (ACN) (50:50 v/v), and after destaining, the ragments were dehydrated with two ACN baths for 10 min and ried at room temperature); b) reduction and alkylation (rehy- rated with 20 mM DTT in 50 mM Ambic for 40 min at 56 ◦C; after ime, the excess reagent was removed and 55 mM IAA in 50 mM mbic was added for 30 min at room temperature); c) tryptic diges- ion of proteins (incubated overnight at 37 ◦C with 10 ng �L−1 rypsin in 25 mM Ambic for 15 min; Trypsin Gold Mass Spectrom- try, Promega, Madison, USA) and d) elution of peptides (extracted rom gel by the addition of extraction buffer) A − 50% ACN with 1% ormic acid − to each tube and incubated for 15 min at 40 ◦C under onication; the supernatant was collected and transferred to a new ube, and this step was repeated with the extraction of buffer B − 0% methanol with 1% formic acid − and extraction buffer C − 100% CN; the extracts were dried in a vacuum centrifuge and peptides ere dissolved in 10 �L 3% ACN with 0.1% formic acid. Aliquots f solutions containing peptides were analyzed by obtaining the ass spectra using the nanoACQUITY UPLC-Xevo QT-MS (Waters, anchester, UK) system. Data was acquired over 20 min, and the can range was 50–2000 Da. ProteinLynx Global Server (PLGS) ver- ion 3.0 was used to process and search the continuum LC–MSE ata, setting carbamidomethylation of cysteines as the fixed modi- cation and oxidation of methionines as the variable modification, llowing one missing cleavage and a maximal error tolerance of 0 ppm [15]. The identification of proteins was performed using he UniProt database (UniProtKB/Swiss-Prot − www.uniprot.org), nd the search was conducted for the species Rattus norvegicus. The UniProt protein IDs were converted to gene symbols in order o analyze them using Reactome Functional Interaction (FI) [16], a ytoscape plugin [17]. Reactome uses a comparison with published ig. 1. A. Gel obtained by 2D-PAGE (pH 3–10) for liver tissue. The numbers indicate spots w . Qualitative analysis of Cu, Mg, Se and Zn in the C group. C. Qualitative analysis of Cu, M M1 + I group. ical Macromolecules 96 (2017) 817–832 819 knowledge about reactions, pathways and biological processes. Pathways with a false discovery rate (FDR) < 0.05 were considered to be significantly enriched. 3. Results and discussion 3.1. Protein separation by 2D-PAGE The total protein concentrations in the pool of pellets in the C, DM1 and DM1 + I groups were 131.12, 92.86 and 105.79 g L−1 in the total extract and 26.09, 20.05 and 21.79 g L−1 precipitated with acetone, respectively. The pellets obtained with the acetone pre- cipitation were used to calculate the volume of protein needed to obtain 3 �g �L−1 of protein to apply to the IEF strips. After stan- dardization of the protocol to be followed, the gels obtained from different experimental groups were scanned and analyzed using ImageMaster 2D Platinum 7.0. The gels were compared in pairs because the imaging is a laborious process. After the identifica- tion of equivalent spots through the matching process, we obtained results of the correlation between the pairs of gels. The correlation between the gel replicates, encompassing the three electrophoretic runs (six gels from each group studied), were, on average in the C, DM1 and DM1 + I groups, 86%, 79% and 91%, respectively. Correlation between the protein spots from gels was deter- mined, considering the percentage normalized volume (%V) during the matching process. The results obtained were as follows: G1 and G2 (R > 0.75), G1 and G3 (R > 0.90) and G2 and G3 (R > 0.76). Through the correlation analysis, we can see that the proteomic profile of the C group was closer to the proteomic profile of the DM1 + I group than to the profile of the DM1 group. Most spots were distributed in the molecular weight (Mw) range of 31–76 kDa with isoelec- tric points (pI) ∼= 6; in general, the Mw and pI distributions were homogeneous among the different experimental groups. 3.2. Determination of Cu, Mg, Se and Zn in the spots The concentration of Cu, Mg, Se and Zn does not provide a lot of information without characterizing the proteins in these protein spots. Thus, we converted the estimate of the protein mass in the protein spots and the Cu, Mg, Se and Zn [18,19]. Fig. 1A shows the protein spots containing Cu, Mg, Se and/or Zn; Fig. 1B–D shows the qualitative analyses that were used to identify the percentage of spots with Cu, Se, Mg and Se. In the 75 spots analyzed, the C group showed the highest percentage of Cu, the DM1 group showed the here Cu, Mg, Se and/or Zn as detected and the proteins characterized by ESI–MS/MS. g, Se and Zn in the DM1 group. D. Qualitative analysis of Cu, Mg, Se and Zn in the http://www.uniprot.org http://www.uniprot.org http://www.uniprot.org 8 Biolog h p v i t o c s s f N c a r e a b T V 20 C.P. Braga et al. / International Journal of ighest percentage of Se and the DM1 + I group showed the highest ercentage of Mg. Se exerts a beneficial influence on health, including the pre- ention of cancer and neurodegenerative diseases, actuating the mmune system and mainly in providing antioxidant capacity hrough selenoproteins [20]. The literature reports that a number f metals (copper, zinc, vanadium and cadmium) have ions that are apable of eliciting insulin mimetic effects by activating the insulin ignaling cascade [21]. Recent epidemiological and intervention tudies related high Se levels to hyperglycemia and dyslipidemia; or example, a study conducted using the US National Health and utrition Examination Surveys associated a high serum selenium oncentration with an increased prevalence of diabetes [22,23], nd the French SUVIMAX trial population associated positive cor- elations between plasma Se and fasting plasma glucose [24]. The ffect of Se in carbohydrate metabolism is controversial, and the dverse effect on the insulin-regulated metabolic pathway could e a fake redox paradox that facilitates insulin action by an insulin- able 1 alues for copper, selenium and zinc concentration, determination by GFAAS and magne ical Macromolecules 96 (2017) 817–832 stimulated reactive oxygen species (ROS) [25], which could explain why most protein spots had Se presence, because most Se available can bind with proteins and interfere in their function and conse- quently in their pathways. The feed was analyzed for Cu, Mg, Se and Zn in order to exclude the role of feed in the results of this work. We found the following concentrations: Cu: 2.36 mg kg−1, Mg: 28.90 mg kg−1, Se: < LOQ and Zn: 51.40 mg kg−1. Thus, the feed was observed to be a major source of Mg and Zn. Table 1 shows the Cu, Mg, Se and Zn concentrations determined in each protein spot with their respective Mw and pI and the protein mass measured using ImageMaster 2D Platinum 7.0 software. 3.3. Protein spot analysis by ESI–MS/MS This study analyzed 75 spots of protein and found 114 different proteins that were associated with the presence of Cu, Mg, Se and/or Zn in the C, DM1 and DM1 + I groups, shown in Table 2. Reactome sium by FAAS in the protein spots for liver in different experimental groups. C.P. Braga et al. / International Journal of Biological Macromolecules 96 (2017) 817–832 821 Table 1 (Continued) F c I p a a m g 3 n t t p m r t e a g a t c a Z I was used to find the pathways of these 114 proteins that were onsidered significantly enriched (FDR < 0.05); the UniProt protein Ds were converted to gene symbols. Considering FDR < 0.05, 35 athways were considered to be significantly enriched (Fig. 2), nd the majority were related to the metabolism of amino acids nd derivates (19 genes), glycogen storage diseases (13 genes), etabolism of carbohydrates (13 genes), biological oxidations (12 enes) and glucose metabolism (12 genes). .3.1. Amino acid metabolism Carbamoyl phosphate synthase [ammonia] (CPS1) is ATP- and ucleotide-binding, and it is involved in the urea cycle (con- rols the entry of ammonia into the urea cycle). In the literature, here are no reports that this enzyme has a specific metal-binding roperty, but there is a report that it can be inactivated by a ixed-function oxidation binding a divalent metal and generally equiring a nucleotide [26]. However, in this work, the most impor- ant find was in the DM1 + I group, Cu and Zn were found; as these lements are divalent, it may be inferred that binding with Cu nd Zn can cause the inactivation of this enzyme in the DM1 + I roup associated with the control of hyperglycemia by insulin and decrease in the production of ammonia. Ornithine carbamoyl- ransferase (OTC) is another enzyme involved in the urea cycle (it atalyzes the formation of L-citrulline from carbamoyl phosphate nd L-ornithine). A study reported that this enzyme is regulated by n in two different ways: as an allosteric cofactor of the substrate- bound enzyme (site–site interactions) and by inducing inactivation by a slow, tight-binding inhibitor of the free enzyme [27]. The cys- teinyl residue at position 273 of the enzyme has been identified as a metal ligand [28]. In this study, the presence of Cu was found in the C group and Cu, Mg, Se and Zn were found in the DM1 + I group. Cu, Mg and Se have not yet been reported as binding with OTC; it can be suggested that in DM1 + I inactivation of this enzyme by hyperglycemia control is induced by insulin. Arginase-1 (AGR1) was found in spot ID 79, and sorbitol dehydrogenase (SORD) was also found in this spot. AGR1 is a binuclear manganese (Mn) metal- loenzyme that catalyzes the hydrolysis of arginine to ornithine and urea [29], and SORD is a Zn metalloenzyme (a tetramer containing one Zn atom/subunit) involved in the catalysis of sorbitol/fructose interconversion [30]. Some alterations in this conversion are associ- ated in diabetes with cataract formation, neuropathy, retinopathy and nephropathy [30]. The results showed in spot 79, the pres- ence of Zn in C, Se in DM1 and Cu, Mg and Zn in DM1 + I. No other previous reports have shown that ARG protein binds with Cu, Mg and Zn, and SORD with Cu and Mg. Beta-ureidopropionase (UPB1), 4-hydroxyphenylpyruvate dioxygenase (HPD) and fumary- lacetoacetase (FAH) were identified in spot 77, where the presence of Mg was found in C, Se in DM1 and Cu, Se, Mg and Zn in DM1 + I. UPB1 is a metalloenzyme that binds two Zn2+ ions per subunit [31], HPD is a metalloenzyme that binds one Fe cation per subunit [32] and FAH binds with Ca2+ and Mg2+ [33]. Other proteins associ- ated with this metabolism such as S-adenosylmethionine synthase 822 C.P. Braga et al. / International Journal of Biological M acrom olecules 96 (2017) 817–832 Table 2 Proteins identified by ESI–MS/MS in liver with the presence of copper, magnesium, selenium and/or zinc. Spot ID Protein entry Protein Score pI (theo- retical) Mw (Da, theoretical) Peptides Coverage (%) Biological process Metabolism associated Cu, Mg, Se and Zn presence 4 P07756 Carbamoyl-phosphate synthase [ammonia] mitochondrial OS = Rattus norvegicus GN Cps1 PE = 1 SV = 1 653 6.33 164,580 13 10.95 Urea cycle Amino-acid metabolism DM1 = Se; DM1 + I = Cu and Mg 8 P70498 Phospholipase D2 (GN = Pld2) 131 7.44 106,037 84 1.82 Response to hydrogen peroxide, hypoxia, organic cyclic compounds and peptide hormone Lipid metabolism DM1 = Se and Zn; DM1 + I = Cu and Mg 10 O88600 Heat shock 70 kDa protein 4 OS = Rattus norvegicus GN Hspa4 PE = 1 SV = 1 94 5.12 94,057 74 7.14 Stress response Chaperone DM1 + I = Cu and Se 11 P50475 Alanine–tRNA ligase cytoplasmic OS = Rattus norvegicus GN = Aars PE = 1 SV = 3 177 5.41 106,790 82 10.74 Protein biosynthesis Nucleotide DM1 + I = Cu and Se 14 Q63060 Glycerol kinase OS = Rattus norvegicus GN = Gk PE = 2 SV = 1 717 5.49 57,477 44 12.21 glycerol degradation via glycerol kinase pathway Glycerol metabolism DM1 = Se and Zn; DM1 + I = Cu and Mg 15 P28037 Cytosolic 10-formyltetrahydrofolate dehydrogenase OS = Rattus norvegicus GN = Aldh1l1 PE = 1 SV = 3 1633 6.15 101,440 79 21.51 Oxireductase Choline and glycine metabolism DM1 = Se and Zn; DM1 + I = Cu, Mg and Zn 16 P28037 Cytosolic 10-formyltetrahydrofolate dehydrogenase OS = Rattus norvegicus GN = Aldh1l1 PE = 1 SV = 3 1633 6.15 101,440 79 24.72 Oxireductase Choline and glycine metabolism DM1 = Se and Zn; DM1 + I = Cu and Zn 17 Q64380 Sarcosine dehydrogenase mitochondrial OS = Rattus norvegicus GN = Sardh PE = 1 SV = 2 1514 6.58 43,045 31 5.51 Kinase, transferase Creatine metabolism DM1 = Se; DM1 + I = Cu, Mg and Zn 20 P09034 Argininosuccinate synthase OS = Rattus norvegicus GN = Ass1 PE = 2 SV = 1 2849 7.63 46,496 33 36.65 Urea cycle Amino-acid metabolism DM1 = Se; DM1 + I = Cu and Mg 21 Q02253 Methylmalonate-semialdehyde dehydrogenase [acylating] mitochondrial OS = Rattus norvegicus GN = Aldh6a1 PE = 1 SV = 1 7429 8.47 57,808 44 35.14 Oxidoreductase Valine and pyrimidine metabolism C = Mg; DM1 = Se; DM1 + I = Cu and Se 22 O09171 Betaine–homocysteine S-methyltransferase 1 OS = Rattus norvegicus GN = Bhmt PE = 1 SV = 1 2238 8 39,929 29 6.06 Betaine-homocysteine S-methyltransferase activity, S-adenosylmethionine- homocysteine S-methyltransferase activity and S-methylmethionine- homocysteine S-methyltransferase activity Mehionine metabolism DM1 + I = Cu, Mg and Zn 24 P12346 Serotransferrin OS = Rattus norvegicus GN = Tf PE = 1 SV = 3 1104 7.14 76,395 65 14.76 ferric iron transmembrane transporter activit Transport DM1 = Se; DM1 + I = Cu Q5XHY5 Threonine–tRNA ligase cytoplasmic OS = Rattus norvegicus GN = Tars PE = 2 SV = 1 935 6.5 80,576 53 15.11 Protein biosynthesis Amino-acid metabolism 27 O35244 Peroxiredoxin-6 OS = Rattus norvegicus GN = Prdx6 PE = 1 SV = 3 6304 7.14 76,395 65 16.76 redox regulation Oxidative stress DM1 + I = Cu and Mg C.P. Braga et al. / International Journal of Biological M acrom olecules 96 (2017) 817–832 823 P12346 Serotransferrin OS = Rattus norvegicus GN = Tf PE = 1 SV = 3 709 7.14 76,395 55 14.49 ferric iron transmembrane transporter activit Transport 28 Q9ER34 Aconitate hydratase mitochondrial OS = Rattus norvegicus GN = Aco2 PE = 1 SV = 2 245 5.97 73,858 69 19 Response to toxic substance Chaperone DM1 = Se and Zn; DM1 + I = Cu 31 P34058 Heat shock protein HSP 90-beta OS = Rattus norvegicus GN Hsp90ab1 PE = 1 SV = 4 3813 4.96 83,281 67 30.66 chaperone, cellular response to interleukin-4, negative regulation of neuron apoptotic process Chaperone DM1 = Se; DM1 + I = Mg and Se P82995 Heat shock protein HSP 90-alpha OS = Rattus norvegicus GN Hsp90aa1 PE = 1 SV = 3 2081 4.93 84,815 91 18.14 Stress response Chaperone 32 P20059 Hemopexin OS = Rattus norvegicus GN Hpx PE = 1 SV = 3 182 7.58 51,351 36 15.65 cellular iron ion homeostasis Transport DM1 + I = Cu and Mg 33 P48721 Stress-70 protein mitochondrial OS = Rattus norvegicus GN Hspa9 PE = 1 SV = 3 3792 5.37 70,871 50 17.03 mRNA processing, mRNA splicing, stress response, transcription, transcription regulation Chaperone DM1 + I = Cu, Mg, Se and Zn P63018 Heat shock cognate 71 kDa protein OS = Rattus norvegicus GN Hspa8 PE = 1 SV = 1 93 5.50 69,642 57 17.68 Stress response Chaperone 35 P02793 Ferritin light chain 1 OS = Rattus norvegicus GN = Ftl1 PE = 1 SV = 3 220 5.98 20,749 16 27.87 Iron storage Other DM1 = Se and Zn; DM1 + I = Cu and Se 36 P02770 Serum albumin OS = Rattus norvegicus GN = Alb PE = 1 SV = 2 3023 6.09 68,731 50 20.07 Transport Other DM1 = Se; DM1 + I = Cu, Mg and Zn 37 P14882 Propionyl-CoA carboxylase alpha chain mitochondrial OS = Rattus norvegicus GN = Pcca PE = 1 SV = 3 531 7.6 81,623 60 16.01 cellular amino acid and fatty acid catabolic process Amino-acid metabolism C = Cu 38–39 P07379 Phosphoenolpyruvate carboxykinase cytosolic [GTP] OS = Rattus norvegicus GN = Pck1 PE = 1 SV = 1 2569 6.75 71,615 47 10.52 Tricarboxylic acid cycle Carbohydrate metabolism DM1 = Se and Zn; DM1 + I = Cu, Mg and Se Q920L2 Succinate dehydrogenase [ubiquinone] flavoprotein subunit mitochondrial OS = Rattus norvegicus GN = Sdha PE = 1 SV = 1 633 6.45 62,200 46 17.94 Glycolysis Carbohydrate metabolism P12928 Pyruvate kinase PKLR OS = Rattus norvegicus GN = Pklr PE = 2 SV = 2 107 6.23 60,647 49 9.54 Toxin transport Chaperone Q6P502 T-complex protein 1 subunit gamma OS = Rattus norvegicus GN Cct3 PE = 1 SV = 1 98 5.07 72,347 52 26.91 Stress response Chaperone 41 P06761 78 kDa glucose-regulated protein OS = Rattus norvegicus GN Hspa5 PE = 1 SV = 1 8726 5.91 70,549 51 16.54 Stress response Chaperone DM1 = Se; DM1 + I = Cu and Mg P55063 Heat shock 70 kDa protein 1-like OS = Rattus norvegicus GN Hspa1 L PE = 2 SV = 2 932 5.91 70,550 48 9.83 Stress response Chaperone Q07439 Heat shock 70 kDa protein 1A/1 B OS = Rattus norvegicus GN Hspa1a PE = 2 SV = 2 897 5.37 70,871 50 6.19 Stress response Chaperone P63018 Heat shock cognate 71 kDa protein OS = Rattus norvegicus GN Hspa8 PE = 1 SV = 1 894 5.50 69,642 50 6.32 Stress response Chaperone P14659 Heat shock-related 70 kDa protein 2 OS = Rattus norvegicus GN Hspa2 PE = 1 SV = 2 894 5.37 70,871 50 40.09 Stress response Chaperone 824 C.P. Braga et al. / International Journal of Biological M acrom olecules 96 (2017) 817–832 Table 2 (Continued) Spot ID Protein entry Protein Score pI (theo- retical) Mw (Da, theoretical) Peptides Coverage (%) Biological process Metabolism associated Cu, Mg, Se and Zn presence 42 P63018 Heat shock cognate 71 kDa protein OS = Rattus norvegicus GN Hspa8 PE = 1 SV = 1 7256 5.50 69,642 50 12.64 Stress response Chaperone DM1 = Se; DM1 + I = Cu, Se and Zn Q07439 Heat shock 70 kDa protein 1A/1 B OS = Rattus norvegicus GN Hspa1a PE = 2 SV = 2 3042 5.91 70,550 49 19.66 Stress response Chaperone P14659 Heat shock-related 70 kDa protein 2 OS = Rattus norvegicus GN Hspa2 PE = 1 SV = 2 2938 5.91 70,549 51 12.64 Stress response Chaperone P55063 Heat shock 70 kDa protein 1-like OS = Rattus norvegicus GN Hspa1 L PE = 2 SV = 2 2128 5.07 72,347 52 5.5 Stress response Chaperone 43 P38652 Phosphoglucomutase-1 OS = Rattus norvegicus GN = Pgm1 PE = 1 SV = 2 4463 6.3 61,403 44 37.01 Glycolysis/gluconeogenesis Carbohydrate metabolism DM1 + I = Cu, Mg and Se 44 P04762 Catalase OS = Rattus norvegicus GN Cat PE = 1 SV = 3 657 7.07 59,757 42 21.14 Response to reactive oxygen species Oxidative stress C = Mg P0C2 × 9 Delta-1-pyrroline-5-carboxylate dehydrogenase mitochondrial OS = Rattus norvegicus GN = Aldh4a1 PE = 1 SV = 1 627 7.13 61,869 33 30.10 Oxidoreductase Proline metabolism P38652 Phosphoglucomutase-1 OS = Rattus norvegicus GN = Pgm1 PE = 1 SV = 2 590 6.3 61,403 44 17.79 glycolysis/gluconeogenesis pathway Carbohydrate metabolism P32232 Cystathionine beta-synthase OS = Rattus norvegicus GN Cbs PE = 1 SV = 3 453 6.29 62,308 54 19.07 Carboxylic ester hydrolase activity Lipid metabolism Q64573 Liver carboxylesterase 4 OS = Rattus norvegicus PE = 2 SV = 2 426 6.25 62,495 57 11.23 Carboxylic ester hydrolase activity Lipid metabolism Q63010 Liver carboxylesterase B-1 OS = Rattus norvegicus PE = 1 SV = 1 372 6.10 62,147 36 11.68 Carboxylic ester hydrolase activity Lipid metabolism P16303 Carboxylesterase 1D OS = Rattus norvegicus GN Ces1d PE = 1 SV = 2 345 5.64 61,715 39 5.35 Carboxylic ester hydrolase activity Lipid metabolism Q63108 Carboxylesterase 1E OS = Rattus norvegicus GN Ces1e PE = 2 SV = 1 305 6.23 60,647 49 11.01 Toxin transport Chaperone Q6P502 T-complex protein 1 subunit gamma OS = Rattus norvegicus GN Cct3 PE = 1 SV = 1 199 6.45 62,200 46 14.98 Glycolysis Carbohydrate metabolism 45 P04762 Catalase OS = Rattus norvegicus GN Cat PE = 1 SV = 3 8595 7.07 59.757 42 21.49 Response to reactive oxygen species Oxidative stress DM1 = Se; DM1 + I = Cu, Mg and Zn P0C2 × 9 Delta-1-pyrroline-5-carboxylate dehydrogenase mitochondrial OS = Rattus norvegicus GN = Aldh4a1 PE = 1 SV = 1 2270 7.13 61.849 46 33.78 Oxidoreductase Proline metabolism 46 P04762 Catalase OS = Rattus norvegicus GN Cat PE = 1 SV = 3 4785 7 66,093 42 20.43 Glutamate and proline biosynthetic process Proline metabolism C = Se, DM1 = Se and Zn; DM1 + I = Cu, Mg and Se P0C2 × 9 Delta-1-pyrroline-5-carboxylate dehydrogenase mitochondrial OS = Rattus norvegicus GN = Aldh4a1 PE = 1 SV = 1 1043 7.13 61.849 20 24.57 Oxidoreductase Proline metabolism 47 P13255 Glycine N-methyltransferase OS = Rattus norvegicus GN = Gnmt PE = 1 SV = 2 12867 5.79 58,914 46 36.97 Lyase, Transferase Histidine metabolism DM1 = Se; DM1 + I = Cu, Mg, Se and Zn 49 P00564 Creatine kinase M-type OS = Rattus norvegicus GN Ckm PE = 1 SV = 2 63 6.58 43,045 31 5.51 phosphocreatine biosynthetic process, response to heat Other DM1 = Se; DM1 + I = Cu, Mg and Se C.P. Braga et al. / International Journal of Biological M acrom olecules 96 (2017) 817–832 825 50 Q6P9T8 Tubulin beta–4 B chain OS = Rattus norvegicus GN = Tubb4b PE = 1 SV = 1 5873 4.79 49,801 30 43.37 microtubule-based process Other DM1 = Mg and Se; DM1 + I = Cu and Mg P69897 Tubulin beta-5 chain OS = Rattus norvegicus GN = Tubb5 PE = 1 SV = 1 4701 4.78 49,671 30 24.32 microtubule-based process Other 56 O88618 Formimidoyltransferase- cyclodeaminase OS = Rattus norvegicus GN = Ftcd PE = 1 SV = 4 5832 5.88 56,623 53 26.53 Cell redox homeostasis Oxidative stress DM1 = Se; DM1 + I = Cu and Se P11598 Protein disulfide-isomerase A3 OS = Rattus norvegicus GN = Pdia3 PE = 1 SV = 2 5409 6.41 60,806 37 41.39 sulfur metabolism Energy metabolism Q07116 Sulfite oxidase mitochondrial OS = Rattus norvegicus GN = Suox PE = 1 SV = 2 1751 6.41 60,806 39 18.88 energy metabolism; sulfur metabolism Energy metabolism 60 Q63150 Dihydropyrimidinase OS = Rattus norvegicus GN = Dpys PE = 1 SV = 2 2776 6.77 56,815 44 12.9 beta-alanine metabolic process, thymine catabolic process, uracil metabolic and catabolic process Other C = Zn; DM1 = Se and Zn; DM1 + I = Cu, Mg, Se and Zn P70619 Glutathione reductase (Fragment) OS = Rattus norvegicus GN = Gsr PE = 2 SV = 2 120 8.06 46,301 40 33.47 cell redox homeostasis Oxidative stress 62 P10719 ATP synthase subunit beta mitochondrial OS = Rattus norvegicus GN = Atp5b PE = 1 SV = 2 10854 5.18 56,354 36 68.62 ATP synthesis, Hydrogen ion transport, Ion transport, Transport Energy metabolism C = Zn; DM1 + I = Se Q63081 Protein disulfide-isomerase A6 OS = Rattus norvegicus GN = Pdia6 PE = 1 SV = 2 188 5 48,173 34 14.09 cell redox homeostasis Chaperone 65 Q8VIF7 Selenium-binding protein 1 OS = Rattus norvegicus GN = Selenbp1 PE = 1 SV = 1 1690 5.1 52,532 31 5.77 protein transporter Transport C = Zn; DM1 = Se; DM1 + I = Cu, Mg and Se 67 P04764 Alpha-enolase OS = Rattus norvegicus GN = Eno1 PE = 1 SV = 4 5530 6.16 47,128 31 38.71 gglycolysis/gluconeogenesis Carbohydrate metabolism DM1 = Cu and Se; DM1 + I = Cu, Mg, Se and ZnP15429 Beta-enolase OS = Rattus norvegicus GN = Eno3 PE = 1 SV = 3 2236 7.8 47,014 29 19.59 gglycolysis/gluconeogenesis Carbohydrate metabolism P07323 Gamma-enolase OS = Rattus norvegicus GN = Eno2 PE = 1 SV = 2 2108 5.03 47,141 29 26.96 gglycolysis/gluconeogenesis Carbohydrate metabolism Q9JLJ3 4-trimethylaminobutyraldehyde dehydrogenase OS = Rattus norvegicus GN = Aldh9a1 PE = 1 SV = 1 417 6.57 53,653 40 18.42 amine and polyamine biosynthesis; carnitine biosynthesis Lipid metabolism P25409 Alanine aminotransferase 1 OS = Rattus norvegicus GN = Gpt PE = 1 SV = 2 305 6.08 55,110 36 17.74 amino-acid degradation, biosynthetic process, L-alanine catabolic process Amino-acid metabolism 68 P13444 S-adenosylmethionine synthase isoform type-1 OS = Rattus norvegicus GN = Mat1a PE = 1 SV = 2 4457 5.61 43,698 31 21.16 amino-acid biosynthesis; S-adenosyl-l-methionine biosynthesis; S-adenosyl-l-methionine from L-methionine Amino-acid metabolism DM1 = Se; DM1 + I = Cu, Mg, Se and Zn P18298 S-adenosylmethionine synthase isoform type-2 OS = Rattus norvegicus GN = Mat2a PE = 1 SV = 1 1377 5.93 43,716 32 6.58 amino-acid biosynthesis; S-adenosyl-l-methionine biosynthesis Amino-acid metabolism 70 P10860 Glutamate dehydrogenase 1 mitochondrial OS = Rattus norvegicus GN = Glud1 PE = 1 SV = 2 3974 8.05 61,416 44 19.81 oxidoreductase, allosteric regulation Other DM1 = Se and Zn; DM1 + I = Cu, Mg and Zn 826 C.P. Braga et al. / International Journal of Biological M acrom olecules 96 (2017) 817–832 Table 2 (Continued) Spot ID Protein entry Protein Score pI (theo- retical) Mw (Da, theoretical) Peptides Coverage (%) Biological process Metabolism associated Cu, Mg, Se and Zn presence 73 P85968 6-phosphogluconate dehydrogenase decarboxylating OS = Rattus norvegicus GN = Pgd PE = 1 SV = 1 110 6.57 53,236 36 3.52 Gluconate utilization, Pentose shunt Carbohydrate metabolism DM1 + I = Cu, Mg, Se and Zn 75 Q64640 Adenosine kinase OS = Rattus norvegicus GN = Adk PE = 1 SV = 3 2104 5.72 40,134 36 26.59 purine salvage Lipid metabolism DM1 = Se; DM1 + I = Cu, Mg, Se and Zn 77 Q03248 Beta-ureidopropionase OS = Rattus norvegicus GN = Upb1 PE = 1 SV = 1 2812 6.74 44,042 33 25.45 amino-acid biosynthesis; beta-alanine biosynthesis Amino-acid metabolism C = Mg, DM1 = Se and DM1 + I = Cu, Mg, Se and ZnP32755 4-hydroxyphenylpyruvate dioxygenase OS = Rattus norvegicus GN Hpd PE = 1 SV = 3 2625 6.29 45,112 29 15.04 L-phenylalanine catabolic proces, tyrosine catabolic process Amino-acid metabolism P25093 Fumarylacetoacetase OS = Rattus norvegicus GN = Fah PE = 1 SV = 1 1804 6.67 45,976 31 20.47 Phenylalanine catabolism, Tyrosine catabolism Amino-acid metabolism 78 Q6P756 Adaptin ear-binding coat-associated protein 2 OS = Rattus norvegicus GN Necap2 PE = 1 SV = 2 139 7.72 28,405 18 10.27 endocytosis Transport C = Mg and Zn; DM1 = Mg and Se; DM1 + I = Cu and Mg 79 P07824 Arginase-1 OS = Rattus norvegicus GN = Arg1 PE = 1 SV = 2 8906 6.76 34,973 31 41.8 Urea cycle Amino-acid metabolism C = Zn; DM1 = Se; DM1 + I = Cu, Mg and Zn P27867 Sorbitol dehydrogenase OS = Rattus norvegicus GN = Sord PE = 2 SV = 4 176 7.14 38,235 30 7.84 Fructose biosynthesis Carbohydrate metabolism 86 P46844 Biliverdin reductase A OS = Rattus norvegicus GN = Blvra PE = 1 SV = 1 807 5.82 33,566 28 13.9 biliverdin reductase activity Other C = Cu; DM1 = Se, DM1 + I = Mg 87 D3ZHP7 Serine/threonine-protein kinase ULK3 OS = Rattus norvegicus GN = Ulk3 PE = 3 SV = 1 181 6.34 53,419 20 51.85 Autophagy Nucleotide C = Cu; DM1 + I = Mg, Se and Zn 88 Q5I0J9 Putative L-aspartate dehydrogenase OS = Rattus norvegicus GN = Aspdh PE = 2 SV = 1 5716 5.52 31,260 23 29.21 aspartate dehydrogenase activity Other C = Cu; DM1 = Se, DM1 + I = Mg and Zn P17988 Sulfotransferase 1A1 OS = Rattus norvegicus GN = Sult1a1 PE = 1 SV = 1 2500 6.37 33,906 20 26.62 Transferase Lipid metabolism 91 P13255 Glycine N-methyltransferase OS = Rattus norvegicus GN = Gnmt PE = 1 SV = 2 12867 7.1 32,549 20 24.57 methyltransferase, transferase Other DM1 = Se; DM1 + I = Cu, Mg and Zn 92 P00481 Ornithine carbamoyltransferase mitochondrial OS = Rattus norvegicus GN Otc PE = 1 SV = 1 2516 9.12 39,886 27 44.63 Urea cycle Amino-acid metabolism C = Cu, DM1 + I = Cu, Mg, Se and Zn 94 P04797 Glyceraldehyde-3-phosphate dehydrogenase OS = Rattus norvegicus GN = Gapdh PE = 1 SV = 3 3136 8.14 35,828 29 19.88 glycolysis/gluconeogenesis Carbohydrate metabolism C = Cu; DM1 = Se; DM1 + I = Mg, Se and Zn P04642 L-lactate dehydrogenase A chain OS = Rattus norvegicus GN = Ldha PE = 1 SV = 1 812 8.45 36,451 24 2.71 lactate Carbohydrate metabolism P19629 L-lactate dehydrogenase C chain OS = Rattus norvegicus GN = Ldhc PE = 1 SV = 3 180 7.56 35,687 30 24.26 lactate Carbohydrate metabolism P04636 Malate dehydrogenase mitochondrial OS = Rattus norvegicus GN = Mdh2 PE = 1 SV = 2 170 8.93 35,684 19 4.26 tricarboxylic acid cycle Carbohydrate metabolism 95 P00884 Fructose-bisphosphate aldolase B OS = Rattus norvegicus GN = Aldob PE = 1 SV = 2 4190 8.66 39,618 19 25.34 Glycolysis Carbohydrate metabolism C = Cu; DM1 = Se; DM1 + I = Mg, Se and Zn P00507 Aspartate aminotransferase mitochondrial OS = Rattus norvegicus GN = Got2 PE = 1 SV = 2 1130 6.73 46,429 21 9.01 Amino-acid biosynthesis Amino-acid metabolism C.P. Braga et al. / International Journal of Biological M acrom olecules 96 (2017) 817–832 827 P04903 Glutathione S-transferase alpha-2 OS = Rattus norvegicus GN = Gsta2 PE = 2 SV = 2 1111 8.89 25,559 20 21.62 glutathione transferase activity Oxidative stress P00502 Glutathione S-transferase alpha-1 OS = Rattus norvegicus GN = Gsta1 PE = 1 SV = 3 1111 8.87 25,607 21 10.11 glutathione transferase activity Oxidative stress 98 P08010 Glutathione S-transferase Mu 2 OS = Rattus norvegicus GN = Gstm2 PE = 1 SV = 2 8573 6.91 25,703 21 16.51 glutathione transferase activity Oxidative stress C = Cu; DM1 = Se; DM1 + I = Cu, Mg and Zn P08009 Glutathione S-transferase Yb-3 OS = Rattus norvegicus GN = Gstm3 PE = 1 SV = 2 2575 6.84 25,681 22 30.28 glutathione transferase activity Oxidative stress P04905 Glutathione S-transferase Mu 1 OS = Rattus norvegicus GN = Gstm1 PE = 1 SV = 2 1602 8.27 25,914 15 7.11 glutathione transferase activity Oxidative stress 103 P14141 Carbonic anhydrase 3 OS = Rattus norvegicus GN Ca3 PE = 1 SV = 3 1436 6.89 29,431 23 9.23 Response to oxidative stress Oxidative stress C = Cu B0BNN3 Carbonic anhydrase 1 OS = Rattus norvegicus GN Ca1 PE = 1 SV = 1 322 6.86 28,300 18 8.81 Response to oxidative stress Oxidative stress 106 Q497B0 Omega-amidase NIT2 OS = Rattus norvegicus GN Nit2 PE = 1 SV = 1 4320 6.9 30,701 21 16.55 omega-amidase activity Other C = Cu, Mg and Se; DM1 = Se; DM1 + I = Mg and ZnQ6AXX6 Redox-regulatory protein FAM213A OS = Rattus norvegicus GN = Fam213a PE = 1 SV = 1 117 9.19 25,763 24 31.83 Antioxidant Oxidative stress 107 P13803 Electron transfer flavoprotein subunit alpha mitochondrial OS = Rattus norvegicus GN = Etfa PE = 1 SV = 4 1862 8.62 34,951 12 6.94 electron transport Energy metabolism C = Cu and Se; DM1 + I = Cu, Mg and Zn 108 P52847 Sulfotransferase family cytosolic 1 B member 1 OS = Rattus norvegicus GN = Sult1b1 PE = 1 SV = 2 935 8.16 34,835 23 8.46 lipid metabolism, steroid metabolism Lipid metabolism C = Cu, Mg and Se; DM1 = Se; DM1 + I = Cu, Mg and Zn 113 P19112 Fructose-1 6-bisphosphatase 1 OS = Rattus norvegicus GN = Fbp1 PE = 1 SV = 2 5045 5.54 39,609 33 34.16 gluconeogenesis Carbohydrate metabolism C = Cu; DM1 = Se; DM1 + I = Cu, Mg and Zn Q9Z1N1 Fructose-1 6-bisphosphatase isozyme 2 OS = Rattus norvegicus GN = Fbp2 PE = 2 SV = 1 424 6.76 36,887 26 5.31 gluconeogenesis Carbohydrate metabolism 120 O35244 Peroxiredoxin-6 OS = Rattus norvegicus GN = Prdx6 PE = 1 SV = 3 8422 7.14 76,395 17 6.84 redox regulation Oxidative stress C = Cu and Zn; DM1 = Se; DM1 + I = Cu, Mg, Se and ZnP34067 Proteasome subunit beta type-4 OS = Rattus norvegicus GN = Psmb4 PE = 1 SV = 2 117 6.44 29,197 20 9.73 Hydrolase, Protease, Threonine protease Other Q9Z0V6 Thioredoxin-dependent peroxide reductase mitochondrial OS = Rattus norvegicus GN = Prdx3 PE = 1 SV = 2 107 7.14 28,295 18 18.59 negative regulation of neuron apoptotic process Oxidative stress 122 P04041 Glutathione peroxidase 1 OS = Rattus norvegicus GN = Gpx1 PE = 1 SV = 4 2858 7.7 22,305 15 38.81 Oxidoreductase, Peroxidase Oxidative stress C = Cu; DM1 = Cu and Se; DM1 + I = Mg and Zn 124 Q63716 Peroxiredoxin-1 OS = Rattus norvegicus GN = Prdx1 PE = 1 SV = 1 2729 8.27 22,109 19 33.33 redox regulation Oxidative stress C = Cu P40307 Proteasome subunit beta type-2 OS = Rattus norvegicus GN = Psmb2 PE = 1 SV = 1 2650 6.96 22,912 17 6.31 Hydrolase, Protease, Threonine protease Other 828 C.P. Braga et al. / International Journal of Biological M acrom olecules 96 (2017) 817–832 Table 2 (Continued) Spot ID Protein entry Protein Score pI (theo- retical) Mw (Da, theoretical) Peptides Coverage (%) Biological process Metabolism associated Cu, Mg, Se and Zn presence 127 P07895 Superoxide dismutase [Mn] mitochondrial OS = Rattus norvegicus GN = Sod2 PE = 1 SV = 2 3761 8.96 24,674 18 13.07 response to oxidative stress Oxidative stress C = Cu; DM1 = Mg and Se 128 Q63716 Peroxiredoxin-1 OS = Rattus norvegicus GN = Prdx1 PE = 1 SV = 1 2872 8.27 22,109 19 31.56 redox regulation Oxidative stress C = Cu 129 P30713 Glutathione S-transferase theta-2 OS = Rattus norvegicus GN = Gstt2 PE = 1 SV = 3 4035 7.75 27,439 22 33.03 glutathione metabolic process Oxidative stress C = Cu and Se P04905 Glutathione S-transferase Mu 1 OS = Rattus norvegicus GN = Gstm1 PE = 1 SV = 2 3785 8.27 25,914 18 18.59 olfaction, Sensory transduction Other P08010 Glutathione S-transferase Mu 2 OS = Rattus norvegicus GN = Gstm2 PE = 1 SV = 2 119 6.91 25,703 19 16.29 olfaction, Sensory transduction Other P04904 Glutathione S-transferase alpha-3 OS = Rattus norvegicus GN = Gsta3 PE = 1 SV = 3 110 8.78 25,319 20 13.96 Transferase Oxidative stress Q6AXY0 Glutathione S-transferase A6 OS = Rattus norvegicus GN = Gsta6 PE = 1 SV = 1 87 5.9 25,808 19 3.62 glutathione metabolic process Oxidative stress P46418 Glutathione S-transferase alpha-5 OS = Rattus norvegicus GN = Gsta5 PE = 1 SV = 2 79 8.42 25,347 20 3.6 xenobiotic catabolic process, response to drug, response to nutrient levels Oxidative stress P14942 Glutathione S-transferase alpha-4 OS = Rattus norvegicus GN = Gsta4 PE = 1 SV = 2 79 6.77 25,510 21 3.6 glutathione metabolic process Oxidative stress P04903 Glutathione S-transferase alpha-2 OS = Rattus norvegicus GN = Gsta2 PE = 2 SV = 2 79 8.89 25,559 20 3.6 glutathione metabolic process Oxidative stress P00502 Glutathione S-transferase alpha-1 OS = Rattus norvegicus GN = Gsta1 PE = 1 SV = 3 79 8.87 25,607 46 31.46 glutathione metabolic process Oxidative stress 132 P15999 ATP synthase subunit alpha mitochondrial OS = Rattus norvegicus GN = Atp5a1 PE = 1 SV = 2 1435 9.22 59,754 22 7.34 ATP synthesis, Hydrogen ion transport, Ion transport, Transport Energy metabolism C = Cu; DM1 + I = Cu, Mg, Se and Zn 133 P52844 Estrogen sulfotransferase isoform 1 OS = Rattus norvegicus GN = Sult1e1 PE = 2 SV = 1 1811 5.78 35,509 29 21.36 Transferase Other C = Cu; DM1 + I = Cu, Mg and Zn P49889 Estrogen sulfotransferase isoform 3 OS = Rattus norvegicus GN = Ste PE = 1 SV = 1 1500 5.57 35,416 29 16.61 Transferase Other P52845 Estrogen sulfotransferase isoform 2 OS = Rattus norvegicus GN = Ste2 PE = 2 SV = 1 1500 5.57 35,365 28 16.61 Transferase Other 140 P08010 Glutathione S-transferase Mu 2 OS = Rattus norvegicus GN = Gstm2 PE = 1 SV = 2 550 6.91 25,703 30 25 olfaction, Sensory transduction Other C = Cu, Mg and Zn; DM1 = Se 142 P09634 Homeobox protein Hox-A7 (Fragment) OS = Rattus norvegicus GN Hoxa7 PE = 3 SV = 1 62 4.86 12,552 9 25.71 Developmental protein Other C = Cu and Zn; DM1 = Se; DM1 + I = Mg and Zn C.P. Braga et al. / International Journal of Biological M acrom olecules 96 (2017) 817–832 829 143 O35077 Glycerol-3-phosphate dehydrogenase [NAD(+)] cytoplasmic OS = Rattus norvegicus GN = Gpd1 PE = 1 SV = 4 782 6.16 37,453 28 18.62 glycolysis/gluconeogenesis Carbohydrate metabolism C = Cu and Zn; DM1 = Mg and Se; DM1 + I = Mg and Zn Q8CG45 Aflatoxin B1 aldehyde reductase member 2 OS = Rattus norvegicus GN = Akr7a2 PE = 1 SV = 2 245 8.35 40,675 21 28.34 Oxidoreductase Other 145 P31210 3-oxo-5-beta-steroid 4-dehydrogenase OS = Rattus norvegicus GN = Akr1d1 PE = 1 SV = 1 2741 6.18 37,378 101 26.38 bile acid catabolic process Lipid metabolism C = Cu; DM1 = Se; DM1 + I = Mg and Se 146 P24329 Thiosulfate sulfurtransferase OS = Rattus norvegicus GN = Tst PE = 1 SV = 3 1131 7.77 33,407 20 10.44 epithelial cell differentiation synthesis Other C = Cu 147 Q63797 Proteasome activator complex subunit 1 OS = Rattus norvegicus GN = Psme1 PE = 2 SV = 1 1544 5.77 28,577 25 20.08 innate immune response Other C = Cu; DM1 = Se; DM1 + I = Mg, Se and Zn P13803 Electron transfer flavoprotein subunit alpha mitochondrial OS = Rattus norvegicus GN = Etfa PE = 1 SV = 4 182 8.62 34,951 24 5.11 electron transport Energy metabolism P23680 Serum amyloid P-component OS = Rattus norvegicus GN = Apcs PE = 2 SV = 2 106 5.5 26,176 17 5.26 innate immune response Other 148 Q5XI73 Rho GDP-dissociation inhibitor 1 OS = Rattus norvegicus GN = Arhgdia PE = 1 SV = 1 3089 5.1 23,407 44 22.06 cellular response to redox state Oxidative stress C = Cu; DM1 + I = Mg P46953 3-hydroxyanthranilate 3 4-dioxygenase OS = Rattus norvegicus GN Haao PE = 1 SV = 2 2132 5.57 32,582 100 27.62 cofactor biosynthesis; NAD(+) biosynthesis Other 149 Q63797 Proteasome activator complex subunit 1 OS = Rattus norvegicus GN = Psme1 PE = 2 SV = 1 733 5.77 28,577 25 21.69 innate immune response Other C = Cu, Mg and Zn; DM1 + I = Mg 150 P85973 Purine nucleoside phosphorylase OS = Rattus norvegicus GN = Pnp PE = 1 SV = 1 5621 6.46 32,302 21 38.41 immune response Nucleotide C = Cu; DM1 = Se; DM1 + I = Mg, Se and Zn 156 Q63716 Peroxiredoxin-1 OS = Rattus norvegicus GN = Prdx1 PE = 1 SV = 1 2026 8.27 22,109 18 21.11 redox regulation Oxidative stress DM1 = Se 160 Q68FU3 Electron transfer flavoprotein subunit beta OS = Rattus norvegicus GN = Etfb PE = 2 SV = 3 7067 7.61 27,687 17 26.27 electron transport, transport Energy metabolism DM1 = Mg P08010 Glutathione S-transferase Mu 2 OS = Rattus norvegicus GN = Gstm2 PE = 1 SV = 2 2130 6.91 25,703 22 33.49 glutathione transferase activity Oxidative stress 161 P04905 Glutathione S-transferase Mu 1 OS = Rattus norvegicus GN = Gstm1 PE = 1 SV = 2 10826 8.27 25,914 22 36.24 glutathione transferase activity Oxidative stress C = Cu; DM1 + I = Mg and Zn P29411 GTP:AMP phosphotransferase AK3 mitochondrial OS = Rattus norvegicus GN = Ak3 PE = 2 SV = 2 278 8.89 25,438 20 14.98 homeostasis of cellular nucleotides Nucleotide 830 C.P. Braga et al. / International Journal of Biological Macromolecules 96 (2017) 817–832 Fig. 2. Pathways with FDR < 0.05 using the Reactome FI. Genes related in each pathway. Sulfide oxidation to sulfate: Tst,Suox; Phenylalanine and tyrosine catabolism: Fah,Hpd; Uptake and function of diphtheria toxin: Hsp90ab1,Hsp90aa1; Purine catabolism: Pnp,Gpx1,Cat; Attenuation phase: Hsp90ab1,Hsp90aa1,Hspa8; Citric acid cycle (TCA cycle): Mdh2,Aco2,Sdha; Urea cycle: Ass1,Arg1,Cps1,Otc; HSF1-dependent transactivation: Hspa1l,Hsp90ab1,Hsp90aa1,Hspa8; Sulfur amino acid metabolism: Mat1a,Tst,Suox,Cbs; Purine metabolism: PNP,GPX1,CAT,ADK; Cellular response to heat stress: Pnp,Gpx1,Cat,Adk; Pyruvate metabolism and Citric Acid (TCA) cycle: Ldha,Mdh2,Aco2,Sdha; Glycolysis: Aldob,Pklr,Eno2,Eno3,Gapdh,Eno1; Metabolism of nucleotides: Pnp,Gpx1,Gsr,Cat,Adk,Dpys; Detoxification of Reactive Oxygen Species: Prdx3,Prdx1,Gpx1,Gsr,Cat,Prdx6,Sod2; Glu- tathione conjugation: Gstm1,Gstm2,Gstm3,Gsta1, Gsta2,Gsta3,Gsta4; The citric acid (TCA) cycle and respiratory electron transport: Ldha,Mdh2,Atp5b,Aco2,Etfb,Etfa,Sdha,Atp5a1; Gluconeogenesis: Got2, Fbp1, Fbp2, Mdh2, Aldob, Eno2, Eno3, Gapdh, Eno1, Pck1; Defective TPMT causes Thiopurine S-methyltransferase deficiency (TPMT defi- ciency), Defective SLC35D1 causes Schneckenbecken dysplasia (SCHBCKD), Defective GSS causes Glutathione synthetase deficiency (GSS deficiency), Defective MAT1A causes Methionine adenosyltransferase deficiency (MATD), Defective AHCY causes Hypermethioninemia with S-adenosylhomocysteine hydrolase deficiency (HMAHCHD), Defective GGT1 causes Glutathionuria (GLUTH), Defective OPLAH causes 5-oxoprolinase deficiency (OPLAHD), Defective UGT1A4 causes hyperbiliru- binemia, Phase II conjugation, Defective GCLC causes Hemolytic anemia due to gamma-glutamylcysteine synthetase deficiency (HAGGSD) and Defective UGT1A1 causes hyperbilirubinemia: Gstm1,Gstm2,Gstm3,Mat1a,Sult1a1,Sult1e1,Gsta1,Gsta2,Gsta3,Gsta4,Mat2a; Cellular responses to stress: Hspa1l, Hsp90ab1, Prdx3, Prdx1, Gpx1, Gsr, Cat, Hsp90aa1, Prdx6, Hspa8, Sod2; Glucose metabolism: Got2, Fbp1, Fbp2, Pgm1, Mdh2, Aldob, Pklr, Eno2, Eno3, Gapdh, Eno1, Pck1; Biologicaloxidations: G ycoge M rivativ O i e m m M Z s a b l c a ( 3 p h a s S h H stm1,Gstm2,Gstm3,Mat1a,Sult1a1,Akr7a2,Sult1e1,Gsta1,Gsta2,Gsta3,Gsta4,Mat2a; Gl dh2, Aldob, Pklr, Eno2, Eno3, Gapdh, Eno1, Pck1; Metabolism of amino acids and de tc, Ckm, Psme1, Haao, Ftcd, Cbs. soform type 1 and 2 (MAT1A and MAT2A) are reported in the lit- rature as metal ion-binding proteins; this protein has Mg-finger etal-binding domains [34]. MAT1A and MAT2A catalyze the for- ation of S-adenosylmethionine from methionine and ATP [34]. ost significant was the protein bound in DM1 and DM1 + I with n, as Zn and Mg have the same properties that can interfere with ome modifications in these proteins. Propionyl-CoA carboxylase lpha chain (PCCA) was detected with Cu in C. Cu is expected to ind by amino acids containing side chains with soft or border- ine ligands such as histidine, cysteine and methionine [35], which ould explain this presence because PCCA is composed of 737 amino cids where 2.4% are histidine, 1.5% cysteine and 2.8% methionine UniProtKB/Swiss-Prot − www.uniprot.org). .3.2. Chaperone and oxidative stress metabolism Heat shock proteins protect other proteins from stress, promote rotein folding and prevent protein aggregation [36]. In spot 10, eat shock 70 kDa protein 4 (HSPA4) showed the presence of Cu nd Se in DM1 + I. In spot 33, stress-70 protein (HSPA9) and heat hock cognate 71 kDa (HSPA8) showed the presence of Cu, Mg, e and Zn. In spot 41, 78 kDa glucose-regulated protein (HSPA5), eat shock 70 kDa protein 1-like and 1A/1 B (HSPA1L and HSPA1A), SPA8 and heat shock-related 70 kDa protein 2 (HSPA2) showed n storage diseases and Metabolism of carbohydrates: Pgd, Got2, Fbp1, Fbp2, Pgm1, es: Fah, Got2, Mat1a, Ass1, Glud1, Arg1, Psmb4, Psmb2, Aldh4a1, Cps1, Tst, Hpd, Suox, the presence of Se in DM1 and Cu and Mg in DM1 + I. In spot 42, HSPA8, HSPA1A, HSPA2 and HSPA1L showed the presence of Se in DM1 and Cu, Mg and Se in DM1 + I. The literature reported metal binding just in the small heat shock protein �-crystallin that binds with Cu in the core domain, inducing increased dynamics at the dimer interface and modulating anti-aggregation [37]. The literature does not have any other report of metal binding or asso- ciation with metals in heat shock proteins; our study could suggest some alterations to treatment with insulin in that the ions can bind with these proteins to protect them from oxidative stress gener- ated in the diabetic condictions. Peroxiredoxins are responsible for the degradation of peroxides and thiol-dependent enzymes; peroxiredoxin-1 (PRX1) was found in three spots (124, 128 and 156) with the presence of Cu detected in C and Se in DM1. Another isoform of PRX (peroxiredoxin-6, PRX6) was found in two spots: spot 27 with Cu and Mg in DM1, and spot 120 with Cu and Zn in C, Se in DM1 and Cu, Mg, Se and Zn in DM1 + I. The literature does not report any binding or association of these enzymes with Cu, Mg, Se and Zn; in DM1 the PRX-1 and 6 had a presence of Se that could change the function of these proteins in the degradation of perox- ides. Different isoforms of glutathione S-transferase were found in this study. This enzyme plays a key role in enzymatic detoxification [38]. The results showed association with Cu in C, Se in DM1 and http://www.uniprot.org http://www.uniprot.org http://www.uniprot.org Biolog M o a s Z a 3 d g p P s T p a g i a I C i d m t i b a s t n L p n t a 4 b c f t i c a R [ [ [ [ [ [ [ [ [ [ [ [ [ [ [ [ [ [ [ [ [ [ [ [ C.P. Braga et al. / International Journal of g and Zn in DM1 + I; can be associated with some modification f GST thiol groups that can interfere in their function. Carbonic nhydrase 1 and 3 (CA1 and CA3) are Zn metalloenzymes. In this tudy, these enzymes were found with the presence of Cu in C. As n has many characteristics similar to Cu, Cu can bind to the CA1 nd CA3 domains. .3.3. Carbohydrate and energy metabolism Phosphoglucomutase-1 (PGM1) plays a key role in carbohy- rate metabolism by reversibly catalyzing the interconversion of lucose-1-phosphate to glucose-6-phosphate by the transfer of a hosphate between the C6 and C1 hydroxyl groups of glucose [39]. GM1 is a ubiquitous metalloenzyme that binds one Mg2+ ion per ubunit. In this study, Cu, Mg and Se presence was found in PGM1. he presence of cysteine (weak base) in the peptide sequence may romote the association of Cu and Se in this enzyme. Alpha, beta nd gamma enolase (ENO1, ENO3 and ENO2) that are involved in luconeogenesis and glycolysis were found in spot 67 [40]. ENO1 s a Mg metalloenzyme that binds two Mg2+ per subunit, and ENO2 nd 3 require Mg2+ for catalysis and for stabilizing the dimer [41]. n our study, the presence of Cu and Se was found in DM1 and u, Mg, Se and Zn in DM1 + I. Thus, it may imply some alteration n this enzyme in DM1 and DM1 + I that can interfere in carbohy- rate metabolism and glycemia control. Spot 113 showed two Mg etalloenzymes: fructose 1,6-bisphosphatase 1 (FBP1) and fruc- ose 1,6-bisphosphatase isozyme 2 (FBP2) which bind three Mg2+ ons per subunit and are involved in the production of fructose 1,6- isphosphate [42]. They showed the presence of Cu in C, Se in DM1 nd Cu, Mg and Se in DM1 + I. The cysteine present in the peptide equence may promote the association of Cu and Se observed in his enzyme. In spot 34, glyceraldehyde-3-phosphate dehydroge- ase (GAPDH), L-lactate dehydrogenase A and C chain (LDHA and DHC) and malate dehydrogenase (MDH2) were found with the resence of Se in DM1 and Se and Zn in DM1 + I. The literature does ot report any metal-binding association with these enzymes. Since hese enzymes have cysteine in their sequences, we can infer this ssociation in groups DM1 and DM1 + I. . Conclusion The determination of copper, magnesium, selenium and zinc ound to different proteins related to different metabolic pro- esses can provide important information about the activity and unction of the entire proteome. The literature has poor informa- ion about these associations, particularly related to diabetes and nsulin treatment. However, the results shown in this manuscript ould represent a mechanism for understanding the interactions nd changes in DM1 pathology. eferences [1] P. Manna, J. Das, J. Ghosh, P.C. Sil, Contribution of type 1 diabetes to rat liver dysfunction and cellular damage via activation of NOS, PARP, IkappaBalpha/NF-kappaB, MAPKs, and mitochondria-dependent pathways: prophylactic role of arjunolic acid, Free Radic. Biol. Med. 48 (2010) 1465–1484. [2] I.T.L. Bresolin, E.A. Miranda, S.M.A. Bueno, Cromatografia de afinidade poríons Metálicos Imobilizados (IMAC) de biomoléculas: aspectos fundamentais e aplicaç ões tecnológicas, Quim. Nova. 32 (2009) 1288–1296. [3] J.S. Garcia, C.S. De Magalhães, M.A.Z. Arruda, Trends in metal-binding and metalloprotein analysis, Talanta 69 (2006) 1–15. [4] M. Baierle, J. Valentini, C. Paniz, A. Moro, F.B. Junior, S. Garcia, Possible effects of blood copper on hematological parameters in elderly, J. Bras. Patol. Med. Lab. 46 (2010) 463–470. [5] C.B. Netto, I.R. Siqueira, C. Fochesatto, L.V. Portela, M.D.P. Tavares, D.O. Souza, R. Giugliani, C.A. Gonç alves, S100 B content and SOD activity in amniotic fluid of pregnancies with Down syndrome, Clin. Biochem. 37 (2004) 134–137. [6] N.E.L. Saris, E. Mervaala, H. Karppanen, J.A. Khawaja, A. Lewenstam, Magnesium, Clin. Chim. Acta. 294 (2000) 1–26. [7] M.J. Dacey, Hypomagnesemic disorders, Crit. Care Clin. 17 (2001) 155–173. ical Macromolecules 96 (2017) 817–832 831 [8] S. Bo, E. Pisu, Role of dietary magnesium in cardiovascular disease prevention, insulin sensitivity and diabetes, Curr. Opin. Lipidol. 19 (2008) 50–56. [9] M.P. Rayman, Selenium and human health, Lancet 379 (2012) 1256–1268 d. 10] K.J.C. Cruz, A.R.S. Oliveira, D.N. Marreiro, Antioxidant role of zinc in diabetes mellitus, World J. Diabetes 6 (2015) 333–337. 11] A.H. Zargar, M.I. Bashir, S.R. Masoodi, B.A. Laway, A.I. Wani, A.R. Khan, F.A. Dar, Copper, zinc and magnesium levels in type-1 diabetes mellitus, Saudi Med. J. 23 (2002) 539–542. 12] T.G. Kazi, H.I. Afridi, N. Kazi, M.K. Jamali, M.B. Arain, N. Jalbani, G.A. Kandhro, Copper chromium, manganese, iron, nickel, and zinc levels in biological samples of diabetes mellitus patients, Biol. Trace Elem. Res. 122 (2008) 1–18. 13] P.M. Moraes, F.A. Santos, B. Cavecci, C.C.F. Padilha, J.C.S. Vieira, P.S. Roldan, P.D.M. Padilha, GFAAS determination of mercury in muscle samples of fish from Amazon, Brazil, Food Chem. 141 (2013) 2614–2617. 14] A. Shevchenko, H. Tomas, J. Havlis, J.V. Olsen, M. Mann, In-gel digestion for mass spectrometric characterization of proteins and proteomes, Nat. Protoc. 1 (2006) 2856–2860. 15] G.Z. Li, J.P.C. Vissers, J.C. Silva, D. Golick, M.V. Gorenstein, S.J. Geromanos, Database searching and accounting of multiplexed precursor and product ion spectra from the data independent analysis of simple and complex peptide mixtures, Proteomics 9 (2009) 1696–1719. 16] M.S. Cline, M. Smoot, E. Cerami, A. Kuchinsky, N. Landys, C. Workman, R. Christmas, I. Avila-Campilo, M. Creech, B. Gross, K. Hanspers, R. Isserlin, R. Kelley, S. Killcoyne, S. Lotia, S. Maere, J. Morris, K. Ono, V. Pavlovic, A.R. Pico, A. Vailaya, P.L. Wang, A. Adler, B.R. Conklin, L. Hood, M. Kuiper, C. Sander, I. Schmulevich, B. Schwikowski, G.J. Warner, T. Ideker, G.D. Bader, Integration of biological networks and gene expression data using Cytoscape, Nat. Protoc. 2 (2007) 2366–2382. 17] G. Wu, X. Feng, L. Stein, A human functional protein interaction network and its application to cancer data analysis, Genome Biol. 11 (2010) R53. 18] F.A. Silva, B. Cavecci, W.A. Baldassini, P.M. Lima, P.M. Moraes, P.S. Roldan, C.C.F. Padilha, P.M. Padilha, Selenium fractionation from plasma, muscle and liver of Nile tilapia (Oreochromis niloticus), J. Food Meas. Charact. 7 (2013) 158–165. 19] P.M. Lima, R.D.C.F. Neves, F.A. Dos Santos, C.A. Pérez, M.O.A. Da Silva, M.A.Z. Arruda, G.R. Castro, P.M. Padilha, Analytical approach to the metallomic of Nile tilapia (Oreochromis niloticus) liver tissue by SRXRF and FAAS after 2D-PAGE separation: preliminary results, Talanta 82 (2010) 1052–1056. 20] H. Steinbrenner, H. Sies, Protection against reactive oxygen species by selenoproteins, Biochim. Biophys. Acta − Gen. Subj. 1790 (2009) 1478–1485. 21] H. Steinbrenner, B. Speckmann, A. Pinto, H. Sies, High selenium intake and increased diabetes risk: experimental evidence for interplay between selenium and carbohydrate metabolism, J. Clin. Biochem. Nutr. 48 (2011) 40–45. 22] M. Laclaustra, A. Navas-Acien, S. Stranges, J.M. Ordovas, E. Guallar, Serum selenium concentrations and diabetes in U.S. adults: national health and nutrition examination survey (NHANES) 2003–2004, Environ. Health Perspect. 117 (2009) 1409–1413. 23] J. Bleys, A. Navas-Acien, E. Guallar, Serum selenium and diabetes in U.S. adults, Diabetes Care 30 (2007) 829–834. 24] S. Czernichow, A. Couthouis, S. Bertrais, A.C. Vergnaud, L. Dauchet, P. Galan, S. Hercberg, Antioxidant supplementation does not affect fasting plasma glucose in the Supplementation with Antioxidant Vitamins and Minerals (SU.VI.MAX) study in France: association with dietary intake and plasma concentrations, Am. J. Clin. Nutr. 84 (2006) 395–399. 25] B.J. Goldstein, M. Kalyankar, X. Wu, Redox paradox: insulin action is facilitated by insulin-stimulated reactive oxygen species with multiple potential signaling targets, Diabetes 54 (2005) 311–321. 26] E. Alonso, J. Cervera, A. García-España, E. Bendala, V. Rubio, Oxidative inactivation of carbamoyl phosphate synthetase (ammonia). Mechanism and sites of oxidation, degradation of the oxidized enzyme, and inactivation by glycerol, EDTA, and thiol protecting agents, J. Biol Chem. 267 (1992) 4524–4532. 27] S. Lee, W.H. Shen, A.W. Miller, L.C. Kuo, Zn2+ regulation of ornithine transcarbamoylase. I. Mechanism of action, J. Mol. Biol. 211 (1990) 255–269. 28] L.C. Kuo, C. Caron, S. Lee, W. Herzberg, Zn2+ regulation of ornithine transcarbamoylase. II. Metal binding site, J. Mol. Biol. 211 (1990) 271–280. 29] B.C. Yan, C. Gong, J. Song, T. Krausz, M. Tretiakova, E. Hyjek, H. Al-Ahmadie, V. Alves, S.Y. Xiao, R.A. Anders, J.A. Hart, Arginase-1: a new immunohistochemical marker of hepatocytes and hepatocellular neoplasms, Am. J. Surg. Pathol. 34 (2010) 1147–1154. 30] C. Karlsson, H. Jornvall, J.O. Hoog, Sorbitol dehydrogenase: cDNA coding for the rat enzyme. Variations within the alcohol dehydrogenase family independent of quaternary structure and metal content, Eur. J. Biochem. 198 (1991) 761–765. 31] K.L. Kvalnes-Krick, T.W. Traut, Cloning sequencing, and expression of a cDNA encoding beta-alanine synthase from rat liver, J. Biol. Chem. 268 (1993) 5686–5693. 32] M.H. Lee, Z.H. Zhang, C.H. MacKinnon, J.E. Baldwin, N.P. Crouch, The C-terminal of rat 4-hydroxyphenylpyruvate dioxygenase is indispensable for enzyme activity, FEBS Lett. 393 (1996) 269–272. 33] D. Phaneuf, Y. Labelle, D. Berube, K. Arden, W. Cavenee, R. Gagne, R.M. Tanguay, Cloning and expression of the cDNA encoding human fumarylacetoacetate hydrolase, the enzyme deficient in hereditary tyrosinemia: assignment of the gene to chromosome 15, Am. J. Hum. Genet. 48 (1991) 525–535. http://refhub.elsevier.com/S0141-8130(16)31614-2/sbref0005 http://refhub.elsevier.com/S0141-8130(16)31614-2/sbref0005 http://refhub.elsevier.com/S0141-8130(16)31614-2/sbref0005 http://refhub.elsevier.com/S0141-8130(16)31614-2/sbref0005 http://refhub.elsevier.com/S0141-8130(16)31614-2/sbref0005 http://refhub.elsevier.com/S0141-8130(16)31614-2/sbref0005 http://refhub.elsevier.com/S0141-8130(16)31614-2/sbref0005 http://refhub.elsevier.com/S0141-8130(16)31614-2/sbref0005 http://refhub.elsevier.com/S0141-8130(16)31614-2/sbref0005 http://refhub.elsevier.com/S0141-8130(16)31614-2/sbref0005 http://refhub.elsevier.com/S0141-8130(16)31614-2/sbref0005 http://refhub.elsevier.com/S0141-8130(16)31614-2/sbref0005 http://refhub.elsevier.com/S0141-8130(16)31614-2/sbref0005 http://refhub.elsevier.com/S0141-8130(16)31614-2/sbref0005 http://refhub.elsevier.com/S0141-8130(16)31614-2/sbref0005 http://refhub.elsevier.com/S0141-8130(16)31614-2/sbref0005 http://refhub.elsevier.com/S0141-8130(16)31614-2/sbref0005 http://refhub.elsevier.com/S0141-8130(16)31614-2/sbref0005 http://refhub.elsevier.com/S0141-8130(16)31614-2/sbref0005 http://refhub.elsevier.com/S0141-8130(16)31614-2/sbref0005 http://refhub.elsevier.com/S0141-8130(16)31614-2/sbref0005 http://refhub.elsevier.com/S0141-8130(16)31614-2/sbref0005 http://refhub.elsevier.com/S0141-8130(16)31614-2/sbref0005 http://refhub.elsevier.com/S0141-8130(16)31614-2/sbref0005 http://refhub.elsevier.com/S0141-8130(16)31614-2/sbref0005 http://refhub.elsevier.com/S0141-8130(16)31614-2/sbref0005 http://refhub.elsevier.com/S0141-8130(16)31614-2/sbref0005 http://refhub.elsevier.com/S0141-8130(16)31614-2/sbref0005 http://refhub.elsevier.com/S0141-8130(16)31614-2/sbref0005 http://refhub.elsevier.com/S0141-8130(16)31614-2/sbref0005 http://refhub.elsevier.com/S0141-8130(16)31614-2/sbref0005 http://refhub.elsevier.com/S0141-8130(16)31614-2/sbref0005 http://refhub.elsevier.com/S0141-8130(16)31614-2/sbref0005 http://refhub.elsevier.com/S0141-8130(16)31614-2/sbref0005 http://refhub.elsevier.com/S0141-8130(16)31614-2/sbref0005 http://refhub.elsevier.com/S0141-8130(16)31614-2/sbref0005 http://refhub.elsevier.com/S0141-8130(16)31614-2/sbref0005 http://refhub.elsevier.com/S0141-8130(16)31614-2/sbref0005 http://refhub.elsevier.com/S0141-8130(16)31614-2/sbref0005 http://refhub.elsevier.com/S0141-8130(16)31614-2/sbref0005 http://refhub.elsevier.com/S0141-8130(16)31614-2/sbref0005 http://refhub.elsevier.com/S0141-8130(16)31614-2/sbref0005 http://refhub.elsevier.com/S0141-8130(16)31614-2/sbref0005 http://refhub.elsevier.com/S0141-8130(16)31614-2/sbref0005 http://refhub.elsevier.com/S0141-8130(16)31614-2/sbref0010 http://refhub.elsevier.com/S0141-8130(16)31614-2/sbref0010 http://refhub.elsevier.com/S0141-8130(16)31614-2/sbref0010 http://refhub.elsevier.com/S0141-8130(16)31614-2/sbref0010 http://refhub.elsevier.com/S0141-8130(16)31614-2/sbref0010 http://refhub.elsevier.com/S0141-8130(16)31614-2/sbref0010 http://refhub.elsevier.com/S0141-8130(16)31614-2/sbref0010 http://refhub.elsevier.com/S0141-8130(16)31614-2/sbref0010 http://refhub.elsevier.com/S0141-8130(16)31614-2/sbref0010 http://refhub.elsevier.com/S0141-8130(16)31614-2/sbref0010 http://refhub.elsevier.com/S0141-8130(16)31614-2/sbref0010 http://refhub.elsevier.com/S0141-8130(16)31614-2/sbref0010 http://refhub.elsevier.com/S0141-8130(16)31614-2/sbref0010 http://refhub.elsevier.com/S0141-8130(16)31614-2/sbref0010 http://refhub.elsevier.com/S0141-8130(16)31614-2/sbref0010 http://refhub.elsevier.com/S0141-8130(16)31614-2/sbref0010 http://refhub.elsevier.com/S0141-8130(16)31614-2/sbref0010 http://refhub.elsevier.com/S0141-8130(16)31614-2/sbref0010 http://refhub.elsevier.com/S0141-8130(16)31614-2/sbref0010 http://refhub.elsevier.com/S0141-8130(16)31614-2/sbref0010 http://refhub.elsevier.com/S0141-8130(16)31614-2/sbref0010 http://refhub.elsevier.com/S0141-8130(16)31614-2/sbref0010 http://refhub.elsevier.com/S0141-8130(16)31614-2/sbref0010 http://refhub.elsevier.com/S0141-8130(16)31614-2/sbref0010 http://refhub.elsevier.com/S0141-8130(16)31614-2/sbref0010 http://refhub.elsevier.com/S0141-8130(16)31614-2/sbref0010 http://refhub.elsevier.com/S0141-8130(16)31614-2/sbref0010 http://refhub.elsevier.com/S0141-8130(16)31614-2/sbref0010 http://refhub.elsevier.com/S0141-8130(16)31614-2/sbref0010 http://refhub.elsevier.com/S0141-8130(16)31614-2/sbref0010 http://refhub.elsevier.com/S0141-8130(16)31614-2/sbref0015 http://refhub.elsevier.com/S0141-8130(16)31614-2/sbref0015 http://refhub.elsevier.com/S0141-8130(16)31614-2/sbref0015 http://refhub.elsevier.com/S0141-8130(16)31614-2/sbref0015 http://refhub.elsevier.com/S0141-8130(16)31614-2/sbref0015 http://refhub.elsevier.com/S0141-8130(16)31614-2/sbref0015 http://refhub.elsevier.com/S0141-8130(16)31614-2/sbref0015 http://refhub.elsevier.com/S0141-8130(16)31614-2/sbref0015 http://refhub.elsevier.com/S0141-8130(16)31614-2/sbref0015 http://refhub.elsevier.com/S0141-8130(16)31614-2/sbref0015 http://refhub.elsevier.com/S0141-8130(16)31614-2/sbref0015 http://refhub.elsevier.com/S0141-8130(16)31614-2/sbref0015 http://refhub.elsevier.com/S0141-8130(16)31614-2/sbref0015 http://refhub.elsevier.com/S0141-8130(16)31614-2/sbref0015 http://refhub.elsevier.com/S0141-8130(16)31614-2/sbref0015 http://refhub.elsevier.com/S0141-8130(16)31614-2/sbref0015 http://refhub.elsevier.com/S0141-8130(16)31614-2/sbref0015 http://refhub.elsevier.com/S0141-8130(16)31614-2/sbref0015 http://refhub.elsevier.com/S0141-8130(16)31614-2/sbref0015 http://refhub.elsevier.com/S0141-8130(16)31614-2/sbref0020 http://refhub.elsevier.com/S0141-8130(16)31614-2/sbref0020 http://refhub.elsevier.com/S0141-8130(16)31614-2/sbref0020 http://refhub.elsevier.com/S0141-8130(16)31614-2/sbref0020 http://refhub.elsevier.com/S0141-8130(16)31614-2/sbref0020 http://refhub.elsevier.com/S0141-8130(16)31614-2/sbref0020 http://refhub.elsevier.com/S0141-8130(16)31614-2/sbref0020 http://refhub.elsevier.com/S0141-8130(16)31614-2/sbref0020 http://refhub.elsevier.com/S0141-8130(16)31614-2/sbref0020 http://refhub.elsevier.com/S0141-8130(16)31614-2/sbref0020 http://refhub.elsevier.com/S0141-8130(16)31614-2/sbref0020 http://refhub.elsevier.com/S0141-8130(16)31614-2/sbref0020 http://refhub.elsevier.com/S0141-8130(16)31614-2/sbref0020 http://refhub.elsevier.com/S0141-8130(16)31614-2/sbref0020 http://refhub.elsevier.com/S0141-8130(16)31614-2/sbref0020 http://refhub.elsevier.com/S0141-8130(16)31614-2/sbref0020 http://refhub.elsevier.com/S0141-8130(16)31614-2/sbref0020 http://refhub.elsevier.com/S0141-8130(16)31614-2/sbref0020 http://refhub.elsevier.com/S0141-8130(16)31614-2/sbref0020 http://refhub.elsevier.com/S0141-8130(16)31614-2/sbref0020 http://refhub.elsevier.com/S0141-8130(16)31614-2/sbref0020 http://refhub.elsevier.com/S0141-8130(16)31614-2/sbref0020 http://refhub.elsevier.com/S0141-8130(16)31614-2/sbref0020 http://refhub.elsevier.com/S0141-8130(16)31614-2/sbref0020 http://refhub.elsevier.com/S0141-8130(16)31614-2/sbref0020 http://refhub.elsevier.com/S0141-8130(16)31614-2/sbref0020 http://refhub.elsevier.com/S0141-8130(16)31614-2/sbref0020 http://refhub.elsevier.com/S0141-8130(16)31614-2/sbref0020 http://refhub.elsevier.com/S0141-8130(16)31614-2/sbref0020 http://refhub.elsevier.com/S0141-8130(16)31614-2/sbref0020 http://refhub.elsevier.com/S0141-8130(16)31614-2/sbref0020 http://refhub.elsevier.com/S0141-8130(16)31614-2/sbref0020 http://refhub.elsevier.com/S0141-8130(16)31614-2/sbref0025 http://refhub.elsevier.com/S0141-8130(16)31614-2/sbref0025 http://refhub.elsevier.com/S0141-8130(16)31614-2/sbref0025 http://refhub.elsevier.com/S0141-8130(16)31614-2/sbref0025 http://refhub.elsevier.com/S0141-8130(16)31614-2/sbref0025 http://refhub.elsevier.com/S0141-8130(16)31614-2/sbref0025 http://refhub.elsevier.com/S0141-8130(16)31614-2/sbref0025 http://refhub.elsevier.com/S0141-8130(16)31614-2/sbref0025 http://refhub.elsevier.com/S0141-8130(16)31614-2/sbref0025 http://refhub.elsevier.com/S0141-8130(16)31614-2/sbref0025 http://refhub.elsevier.com/S0141-8130(16)31614-2/sbref0025 http://refhub.elsevier.com/S0141-8130(16)31614-2/sbref0025 http://refhub.elsevier.com/S0141-8130(16)31614-2/sbref0025 http://refhub.elsevier.com/S0141-8130(16)31614-2/sbref0025 http://refhub.elsevier.com/S0141-8130(16)31614-2/sbref0025 http://refhub.elsevier.com/S0141-8130(16)31614-2/sbref0025 http://refhub.elsevier.com/S0141-8130(16)31614-2/sbref0025 http://refhub.elsevier.com/S0141-8130(16)31614-2/sbref0025 http://refhub.elsevier.com/S0141-8130(16)31614-2/sbref0025 http://refhub.elsevier.com/S0141-8130(16)31614-2/sbref0025 http://refhub.elsevier.com/S0141-8130(16)31614-2/sbref0025 http://refhub.elsevier.com/S0141-8130(16)31614-2/sbref0025 http://refhub.elsevier.com/S0141-8130(16)31614-2/sbref0025 http://refhub.elsevier.com/S0141-8130(16)31614-2/sbref0025 http://refhub.elsevier.com/S0141-8130(16)31614-2/sbref0025 http://refhub.elsevier.com/S0141-8130(16)31614-2/sbref0025 http://refhub.elsevier.com/S0141-8130(16)31614-2/sbref0025 http://refhub.elsevier.com/S0141-8130(16)31614-2/sbref0025 http://refhub.elsevier.com/S0141-8130(16)31614-2/sbref0025 http://refhub.elsevier.com/S0141-8130(16)31614-2/sbref0025 http://refhub.elsevier.com/S0141-8130(16)31614-2/sbref0025 http://refhub.elsevier.com/S0141-8130(16)31614-2/sbref0025 http://refhub.elsevier.com/S0141-8130(16)31614-2/sbref0025 http://refhub.elsevier.com/S0141-8130(16)31614-2/sbref0025 http://refhub.elsevier.com/S0141-8130(16)31614-2/sbref0025 http://refhub.elsevier.com/S0141-8130(16)31614-2/sbref0025 http://refhub.elsevier.com/S0141-8130(16)31614-2/sbref0025 http://refhub.elsevier.com/S0141-8130(16)31614-2/sbref0025 http://refhub.elsevier.com/S0141-8130(16)31614-2/sbref0025 http://refhub.elsevier.com/S0141-8130(16)31614-2/sbref0025 http://refhub.elsevier.com/S0141-8130(16)31614-2/sbref0030 http://refhub.elsevier.com/S0141-8130(16)31614-2/sbref0030 http://refhub.elsevier.com/S0141-8130(16)31614-2/sbref0030 http://refhub.elsevier.com/S0141-8130(16)31614-2/sbref0030 http://refhub.elsevier.com/S0141-8130(16)31614-2/sbref0030 http://refhub.elsevier.com/S0141-8130(16)31614-2/sbref0030 http://refhub.elsevier.com/S0141-8130(16)31614-2/sbref0030 http://refhub.elsevier.com/S0141-8130(16)31614-2/sbref0030 http://refhub.elsevier.com/S0141-8130(16)31614-2/sbref0030 http://refhub.elsevier.com/S0141-8130(16)31614-2/sbref0030 http://refhub.elsevier.com/S0141-8130(16)31614-2/sbref0030 http://refhub.elsevier.com/S0141-8130(16)31614-2/sbref0030 http://refhub.elsevier.com/S0141-8130(16)31614-2/sbref0030 http://refhub.elsevier.com/S0141-8130(16)31614-2/sbref0030 http://refhub.elsevier.com/S0141-8130(16)31614-2/sbref0030 http://refhub.elsevier.com/S0141-8130(16)31614-2/sbref0030 http://refhub.elsevier.com/S0141-8130(16)31614-2/sbref0030 http://refhub.elsevier.com/S0141-8130(16)31614-2/sbref0030 http://refhub.elsevier.com/S0141-8130(16)31614-2/sbref0030 http://refhub.elsevier.com/S0141-8130(16)31614-2/sbref0035 http://refhub.elsevier.com/S0141-8130(16)31614-2/sbref0035 http://refhub.elsevier.com/S0141-8130(16)31614-2/sbref0035 http://refhub.elsevier.com/S0141-8130(16)31614-2/sbref0035 http://refhub.elsevier.com/S0141-8130(16)31614-2/sbref0035 http://refhub.elsevier.com/S0141-8130(16)31614-2/sbref0035 http://refhub.elsevier.com/S0141-8130(16)31614-2/sbref0035 http://refhub.elsevier.com/S0141-8130(16)31614-2/sbref0035 http://refhub.elsevier.com/S0141-8130(16)31614-2/sbref0035 http://refhub.elsevier.com/S0141-8130(16)31614-2/sbref0035 http://refhub.elsevier.com/S0141-8130(16)31614-2/sbref0035 http://refhub.elsevier.com/S0141-8130(16)31614-2/sbref0035 http://refhub.elsevier.com/S0141-8130(16)31614-2/sbref0040 http://refhub.elsevier.com/S0141-8130(16)31614-2/sbref0040 http://refhub.elsevier.com/S0141-8130(16)31614-2/sbref0040 http://refhub.elsevier.com/S0141-8130(16)31614-2/sbref0040 http://refhub.elsevier.com/S0141-8130(16)31614-2/sbref0040 http://refhub.elsevier.com/S0141-8130(16)31614-2/sbref0040 http://refhub.elsevier.com/S0141-8130(16)31614-2/sbref0040 http://refhub.elsevier.com/S0141-8130(16)31614-2/sbref0040 http://refhub.elsevier.com/S0141-8130(16)31614-2/sbref0040 http://refhub.elsevier.com/S0141-8130(16)31614-2/sbref0040 http://refhub.elsevier.com/S0141-8130(16)31614-2/sbref0040 http://refhub.elsevier.com/S0141-8130(16)31614-2/sbref0040 http://refhub.elsevier.com/S0141-8130(16)31614-2/sbref0040 http://refhub.elsevier.com/S0141-8130(16)31614-2/sbref0040 http://refhub.elsevier.com/S0141-8130(16)31614-2/sbref0040 http://refhub.elsevier.com/S0141-8130(16)31614-2/sbref0040 http://refhub.elsevier.com/S0141-8130(16)31614-2/sbref0040 http://refhub.elsevier.com/S0141-8130(16)31614-2/sbref0040 http://refhub.elsevier.com/S0141-8130(16)31614-2/sbref0040 http://refhub.elsevier.com/S0141-8130(16)31614-2/sbref0040 http://refhub.elsevier.com/S0141-8130(16)31614-2/sbref0040 http://refhub.elsevier.com/S0141-8130(16)31614-2/sbref0040 http://refhub.elsevier.com/S0141-8130(16)31614-2/sbref0040 http://refhub.elsevier.com/S0141-8130(16)31614-2/sbref0040 http://refhub.elsevier.com/S0141-8130(16)31614-2/sbref0045 http://refhub.elsevier.com/S0141-8130(16)31614-2/sbref0045 http://refhub.elsevier.com/S0141-8130(16)31614-2/sbref0045 http://refhub.elsevier.com/S0141-8130(16)31614-2/sbref0045 http://refhub.elsevier.com/S0141-8130(16)31614-2/sbref0045 http://refhub.elsevier.com/S0141-8130(16)31614-2/sbref0045 http://refhub.elsevier.com/S0141-8130(16)31614-2/sbref0045 http://refhub.elsevier.com/S0141-8130(16)31614-2/sbref0045 http://refhub.elsevier.com/S0141-8130(16)31614-2/sbref0045 http://refhub.elsevier.com/S0141-8130(16)31614-2/sbref0045 http://refhub.elsevier.com/S0141-8130(16)31614-2/sbref0045 http://refhub.elsevier.com/S0141-8130(16)31614-2/sbref0045 http://refhub.elsevier.com/S0141-8130(16)31614-2/sbref0045 http://refhub.elsevier.com/S0141-8130(16)31614-2/sbref0050 http://refhub.elsevier.com/S0141-8130(16)31614-2/sbref0050 http://refhub.elsevier.com/S0141-8130(16)31614-2/sbref0050 http://refhub.elsevier.com/S0141-8130(16)31614-2/sbref0050 http://refhub.elsevier.com/S0141-8130(16)31614-2/sbref0050 http://refhub.elsevier.com/S0141-8130(16)31614-2/sbref0050 http://refhub.elsevier.com/S0141-8130(16)31614-2/sbref0050 http://refhub.elsevier.com/S0141-8130(16)31614-2/sbref0050 http://refhub.elsevier.com/S0141-8130(16)31614-2/sbref0050 http://refhub.elsevier.com/S0141-8130(16)31614-2/sbref0050 http://refhub.elsevier.com/S0141-8130(16)31614-2/sbref0050 http://refhub.elsevier.com/S0141-8130(16)31614-2/sbref0050 http://refhub.elsevier.com/S0141-8130(16)31614-2/sbref0050 http://refhub.elsevier.com/S0141-8130(16)31614-2/sbref0050 http://refhub.elsevier.com/S0141-8130(16)31614-2/sbref0050 http://refhub.elsevier.com/S0141-8130(16)31614-2/sbref0050 http://refhub.elsevier.com/S0141-8130(16)31614-2/sbref0050 http://refhub.elsevier.com/S0141-8130(16)31614-2/sbref0050 http://refhub.elsevier.com/S0141-8130(16)31614-2/sbref0050 http://refhub.elsevier.com/S0141-8130(16)31614-2/sbref0050 http://refhub.elsevier.com/S0141-8130(16)31614-2/sbref0050 http://refhub.elsevier.com/S0141-8130(16)31614-2/sbref0055 http://refhub.elsevier.com/S0141-8130(16)31614-2/sbref0055 http://refhub.elsevier.com/S0141-8130(16)31614-2/sbref0055 http://refhub.elsevier.com/S0141-8130(16)31614-2/sbref0055 http://refhub.elsevier.com/S0141-8130(16)31614-2/sbref0055 http://refhub.elsevier.com/S0141-8130(16)31614-2/sbref0055 http://refhub.elsevier.com/S0141-8130(16)31614-2/sbref0055 http://refhub.elsevier.com/S0141-8130(16)31614-2/sbref0055 http://refhub.elsevier.com/S0141-8130(16)31614-2/sbref0055 http://refhub.elsevier.com/S0141-8130(16)31614-2/sbref0055 http://refhub.elsevier.com/S0141-8130(16)31614-2/sbref0055 http://refhub.elsevier.com/S0141-8130(16)31614-2/sbref0055 http://refhub.elsevier.com/S0141-8130(16)31614-2/sbref0055 http://refhub.elsevier.com/S0141-8130(16)31614-2/sbref0055 http://refhub.elsevier.com/S0141-8130(16)31614-2/sbref0055 http://refhub.elsevier.com/S0141-8130(16)31614-2/sbref0055 http://refhub.elsevier.com/S0141-8130(16)31614-2/sbref0055 http://refhub.elsevier.com/S0141-8130(16)31614-2/sbref0055 http://refhub.elsevier.com/S0141-8130(16)31614-2/sbref0055 http://refhub.elsevier.com/S0141-8130(16)31614-2/sbref0055 http://refhub.elsevier.com/S0141-8130(16)31614-2/sbref0055 http://refhub.elsevier.com/S0141-8130(16)31614-2/sbref0055 http://refhub.elsevier.com/S0141-8130(16)31614-2/sbref0055 http://refhub.elsevier.com/S0141-8130(16)31614-2/sbref0055 http://refhub.elsevier.com/S0141-8130(16)31614-2/sbref0055 http://refhub.elsevier.com/S0141-8130(16)31614-2/sbref0055 http://refhub.elsevier.com/S0141-8130(16)31614-2/sbref0055 http://refhub.elsevier.com/S0141-8130(16)31614-2/sbref0055 http://refhub.elsevier.com/S0141-8130(16)31614-2/sbref0055 http://refhub.elsevier.com/S0141-8130(16)31614-2/sbref0055 http://refhub.elsevier.com/S0141-8130(16)31614-2/sbref0055 http://refhub.elsevier.com/S0141-8130(16)31614-2/sbref0060 http://refhub.elsevier.com/S0141-8130(16)31614-2/sbref0060 http://refhub.elsevier.com/S0141-8130(16)31614-2/sbref0060 http://refhub.elsevier.com/S0141-8130(16)31614-2/sbref0060 http://refhub.elsevier.com/S0141-8130(16)31614-2/sbref0060 http://refhub.elsevier.com/S0141-8130(16)31614-2/sbref0060 http://refhub.elsevier.com/S0141-8130(16)31614-2/sbref0060 http://refhub.elsevier.com/S0141-8130(16)31614-2/sbref0060 http://refhub.elsevier.com/S0141-8130(16)31614-2/sbref0060 http://refhub.elsevier.com/S0141-8130(16)31614-2/sbref0060 http://refhub.elsevier.com/S0141-8130(16)31614-2/sbref0060 http://refhub.elsevier.com/S0141-8130(16)31614-2/sbref0060 http://refhub.elsevier.com/S0141-8130(16)31614-2/sbref0060 http://refhub.elsevier.com/S0141-8130(16)31614-2/sbref0060 http://refhub.elsevier.com/S0141-8130(16)31614-2/sbref0060 http://refhub.elsevier.com/S0141-8130(16)31614-2/sbref0060 http://refhub.elsevier.com/S0141-8130(16)31614-2/sbref0060 http://refhub.elsevier.com/S0141-8130(16)31614-2/sbref0060 http://refhub.elsevier.com/S0141-8130(16)31614-2/sbref0060 http://refhub.elsevier.com/S0141-8130(16)31614-2/sbref0060 http://refhub.elsevier.com/S0141-8130(16)31614-2/sbref0060 http://refhub.elsevier.com/S0141-8130(16)31614-2/sbref0060 http://refhub.elsevier.com/S0141-8130(16)31614-2/sbref0060 http://refhub.elsevier.com/S0141-8130(16)31614-2/sbref0060 http://refhub.elsevier.com/S0141-8130(16)31614-2/sbref0060 http://refhub.elsevier.com/S0141-8130(16)31614-2/sbref0060 http://refhub.elsevier.com/S0141-8130(16)31614-2/sbref0060 http://refhub.elsevier.com/S0141-8130(16)31614-2/sbref0060 http://refhub.elsevier.com/S0141-8130(16)31614-2/sbref0060 http://refhub.elsevier.com/S0141-8130(16)31614-2/sbref0060 http://refhub.elsevier.com/S0141-8130(16)31614-2/sbref0060 http://refhub.elsevier.com/S0141-8130(16)31614-2/sbref0060 http://refhub.elsevier.com/S0141-8130(16)31614-2/sbref0060 http://refhub.elsevier.com/S0141-8130(16)31614-2/sbref0060 http://refhub.elsevier.com/S0141-8130(16)31614-2/sbref0060 http://refhub.elsevier.com/S0141-8130(16)31614-2/sbref0060 http://refhub.elsevier.com/S0141-8130(16)31614-2/sbref0060 http://refhub.elsevier.com/S0141-8130(16)31614-2/sbref0060 http://refhub.elsevier.com/S0141-8130(16)31614-2/sbref0065 http://refhub.elsevier.com/S0141-8130(16)31614-2/sbref0065 http://refhub.elsevier.com/S0141-8130(16)31614-2/sbref0065 http://refhub.elsevier.com/S0141-8130(16)31614-2/sbref0065 http://refhub.elsevier.com/S0141-8130(16)31614-2/sbref0065 http://refhub.elsevier.com/S0141-8130(16)31614-2/sbref0065 http://refhub.elsevier.com/S0141-8130(16)31614-2/sbref0065 http://refhub.elsevier.com/S0141-8130(16)31614-2/sbref0065 http://refhub.elsevier.com/S0141-8130(16)31614-2/sbref0065 http://refhub.elsevier.com/S0141-8130(16)31614-2/sbref0065 http://refhub.elsevier.com/S0141-8130(16)31614-2/sbref0065 http://refhub.elsevier.com/S0141-8130(16)31614-2/sbref0065 http://refhub.elsevier.com/S0141-8130(16)31614-2/sbref0065 http://refhub.elsevier.com/S0141-8130(16)31614-2/sbref0065 http://refhub.elsevier.com/S0141-8130(16)31614-2/sbref0065 http://refhub.elsevier.com/S0141-8130(16)31614-2/sbref0065 http://refhub.elsevier.com/S0141-8130(16)31614-2/sbref0065 http://refhub.elsevier.com/S0141-8130(16)31614-2/sbref0065 http://refhub.elsevier.com/S0141-8130(16)31614-2/sbref0065 http://refhub.elsevier.com/S0141-8130(16)31614-2/sbref0065 http://refhub.elsevier.com/S0141-8130(16)31614-2/sbref0065 http://refhub.elsevier.com/S0141-8130(16)31614-2/sbref0065 http://refhub.elsevier.com/S0141-8130(16)31614-2/sbref0065 http://refhub.elsevier.com/S0141-8130(16)31614-2/sbref0065 http://refhub.elsevier.com/S0141-8130(16)31614-2/sbref0065 http://refhub.elsevier.com/S0141-8130(16)31614-2/sbref0065 http://refhub.elsevier.com/S0141-8130(16)31614-2/sbref0065 http://refhub.elsevier.com/S0141-8130(16)31614-2/sbref0065 http://refhub.elsevier.com/S0141-8130(16)31614-2/sbref0065 http://refhub.elsevier.com/S0141-8130(16)31614-2/sbref0065 http://refhub.elsevier.com/S0141-8130(16)31614-2/sbref0065 http://refhub.elsevier.com/S0141-8130(16)31614-2/sbref0065 http://refhub.elsevier.com/S0141-8130(16)31614-2/sbref0065 http://refhub.elsevier.com/S0141-8130(16)31614-2/sbref0070 http://refhub.elsevier.com/S0141-8130(16)31614-2/sbref0070 http://refhub.elsevier.com/S0141-8130(16)31614-2/sbref0070 http://refhub.elsevier.com/S0141-8130(16)31614-2/sbref0070 http://refhub.elsevier.com/S0141-8130(16)31614-2/sbref0070 http://refhub.elsevier.com/S0141-8130(16)31614-2/sbref0070 http://refhub.elsevier.com/S0141-8130(16)31614-2/sbref0070 http://refhub.elsevier.com/S0141-8130(16)31614-2/sbref0070 http://refhub.elsevier.com/S0141-8130(16)31614-2/sbref0070 http://refhub.elsevier.com/S0141-8130(16)31614-2/sbref0070 http://refhub.elsevier.com/S0141-8130(16)31614-2/sbref0070 http://refhub.elsevier.com/S0141-8130(16)31614-2/sbref0070 http://refhub.elsevier.com/S0141-8130(16)31614-2/sbref0070 http://refhub.elsevier.com/S0141-8130(16)31614-2/sbref0070 http://refhub.elsevier.com/S0141-8130(16)31614-2/sbref0070 http://refhub.elsevier.com/S0141-8130(16)31614-2/sbref0070 http://refhub.elsevier.com/S0141-8130(16)31614-2/sbref0070 http://refhub.elsevier.com/S0141-8130(16)31614-2/sbref0070 http://refhub.elsevier.com/S0141-8130(16)31614-2/sbref0070 http://refhub.elsevier.com/S0141-8130(16)31614-2/sbref0070 http://refhub.elsevier.com/S0141-8130(16)31614-2/sbref0070 http://refhub.elsevier.com/S0141-8130(16)31614-2/sbref0070 http://refhub.elsevier.com/S0141-8130(16)31614-2/sbref0070 http://refhub.elsevier.com/S0141-8130(16)31614-2/sbref0070 http://refhub.elsevier.com/S0141-8130(16)31614-2/sbref0070 http://refhub.elsevier.com/S0141-8130(16)31614-2/sbref0070 http://refhub.elsevier.com/S0141-8130(16)31614-2/sbref0070 http://refhub.elsevier.com/S0141-8130(16)31614-2/sbref0075 http://refhub.elsevier.com/S0141-8130(16)31614-2/sbref0075 http://refhub.elsevier.com/S0141-8130(16)31614-2/sbref0075 http://refhub.elsevier.com/S0141-8130(16)31614-2/sbref0075 http://refhub.elsevier.com/S0141-8130(16)31614-2/sbref0075 http://refhub.elsevier.com/S0141-8130(16)31614-2/sbref0075 http://refhub.elsevier.com/S0141-8130(16)31614-2/sbref0075 http://refhub.elsevier.com/S0141-8130(16)31614-2/sbref0075 http://refhub.elsevier.com/S0141-8130(16)31614-2/sbref0075 http://refhub.elsevier.com/S0141-8130(16)31614-2/sbref0075 http://refhub.elsevier.com/S0141-8130(16)31614-2/sbref0075 http://refhub.elsevier.com/S0141-8130(16)31614-2/sbref0075 http://refhub.elsevier.com/S0141-8130(16)31614-2/sbref0075 http://refhub.elsevier.com/S0141-8130(16)31614-2/sbref0075 http://refhub.elsevier.com/S0141-8130(16)31614-2/sbref0075 http://refhub.elsevier.com/S0141-8130(16)31614-2/sbref0075 http://refhub.elsevier.com/S0141-8130(16)31614-2/sbref0075 http://refhub.elsevier.com/S0141-8130(16)31614-2/sbref0075 http://refhub.elsevier.com/S0141-8130(16)31614-2/sbref0075 http://refhub.elsevier.com/S0141-8130(16)31614-2/sbref0075 http://refhub.elsevier.com/S0141-8130(16)31614-2/sbref0075 http://refhub.elsevier.com/S0141-8130(16)31614-2/sbref0075 http://refhub.elsevier.com/S0141-8130(16)31614-2/sbref0075 http://refhub.elsevier.com/S0141-8130(16)31614-2/sbref0075 http://refhub.elsevier.com/S0141-8130(16)31614-2/sbref0075 http://refhub.elsevier.com/S0141-8130(16)31614-2/sbref0075 http://refhub.elsevier.com/S0141-8130(16)31614-2/sbref0075 http://refhub.elsevier.com/S0141-8130(16)31614-2/sbref0075 http://refhub.elsevier.com/S0141-8130(16)31614-2/sbref0075 http://refhub.elsevier.com/S0141-8130(16)31614-2/sbref0075 http://refhub.elsevier.com/S0141-8130(16)31614-2/sbref0075 http://refhub.elsevier.com/S0141-8130(16)31614-2/sbref0075 http://refhub.elsevier.com/S0141-8130(16)31614-2/sbref0075 http://refhub.elsevier.com/S0141-8130(16)31614-2/sbref0075 http://refhub.elsevier.com/S0141-8130(16)31614-2/sbref0075 http://refhub.elsevier.com/S0141-8130(16)31614-2/sbref0075 http://refhub.elsevier.com/S0141-8130(16)31614-2/sbref0075 http://refhub.elsevier.com/S0141-8130(16)31614-2/sbref0075 http://refhub.elsevier.com/S0141-8130(16)31614-2/sbref0075 http://refhub.elsevier.com/S0141-8130(16)31614-2/sbref0075 http://refhub.elsevier.com/S0141-8130(16)31614-2/sbref0080 http://refhub.elsevier.com/S0141-8130(16)31614-2/sbref0080 http://refhub.elsevier.com/S0141-8130(16)31614-2/sbref0080 http://refhub.elsevier.com/S0141-8130(16)31614-2/sbref0080 http://refhub.elsevier.com/S0141-8130(16)31614-2/sbref0080 http://refhub.elsevier.com/S0141-8130(16)31614-2/sbref0080 http://refhub.elsevier.com/S0141-8130(16)31614-2/sbref0080 http://refhub.elsevier.com/S0141-8130(16)31614-2/sbref0080 http://refhub.elsevier.com/S0141-8130(16)31614-2/sbref0080 http://refhub.elsevier.com/S0141-8130(16)31614-2/sbref0080 http://refhub.elsevier.com/S0141-8130(16)31614-2/sbref0080 http://refhub.elsevier.com/S0141-8130(16)31614-2/sbref0080 http://refhub.elsevier.com/S0141-8130(16)31614-2/sbref0080 http://refhub.elsevier.com/S0141-8130(16)31614-2/sbref0080 http://refhub.elsevier.com/S0141-8130(16)31614-2/sbref0080 http://refhub.elsevier.com/S0141-8130(16)31614-2/sbref0080 http://refhub.elsevier.com/S0141-8130(16)31614-2/sbref0080 http://refhub.elsevier.com/S0141-8130(16)31614-2/sbref0080 http://refhub.elsevier.com/S0141-8130(16)31614-2/sbref0080 http://refhub.elsevier.com/S0141-8130(16)31614-2/sbref0080 http://refhub.elsevier.com/S0141-8130(16)31614-2/sbref0080 http://refhub.elsevier.com/S0141-8130(16)31614-2/sbref0080 http://refhub.elsevier.com/S0141-8130(16)31614-2/sbref0080 http://refhub.elsevier.com/S0141-8130(16)31614-2/sbref0080 http://refhub.elsevier.com/S0141-8130(16)31614-2/sbref0080 http://refhub.elsevier.com/S0141-8130(16)31614-2/sbref0080 http://refhub.elsevier.com/S0141-8130(16)31614-2/sbref0080 http://refhub.elsevier.com/S0141-8130(16)31614-2/sbref0080 http://refhub.elsevier.com/S0141-8130(16)31614-2/sbref0080 http://refhub.elsevier.com/S0141-8130(16)31614-2/sbref0080 http://refhub.elsevier.com/S0141-8130(16)31614-2/sbref0080 http://refhub.elsevier.com/S0141-8130(16)31614-2/sbref0080 http://refhub.elsevier.com/S0141-8130(16)31614-2/sbref0080 http://refhub.elsevier.com/S0141-8130(16)31614-2/sbref0080 http://refhub.elsevier.com/S0141-8130(16)31614-2/sbref0080 http://refhub.elsevier.com/S0141-8130(16)31614-2/sbref0080 http://refhub.elsevier.com/S0141-8130(16)31614-2/sbref0080 http://refhub.elsevier.com/S0141-8130(16)31614-2/sbref0080 http://refhub.elsevier.com/S0141-8130(16)31614-2/sbref0080 http://refhub.elsevier.com/S0141-8130(16)31614-2/sbref0080 http://refhub.elsevier.com/S0141-8130(16)31614-2/sbref0080 http://refhub.elsevier.com/S0141-8130(16)31614-2/sbref0080 http://refhub.elsevier.com/S0141-8130(16)31614-2/sbref0080 http://refhub.elsevier.com/S0141-8130(16)31614-2/sbref0080 http://refhub.elsevier.com/S0141-8130(16)31614-2/sbref0080 http://refhub.elsevier.com/S0141-8130(16)31614-2/sbref0080 http://refhub.elsevier.com/S0141-8130(16)31614-2/sbref0080 http://refhub.elsevier.com/S0141-8130(16)31614-2/sbref0080 http://refhub.elsevier.com/S0141-8130(16)31614-2/sbref0080 http://refhub.elsevier.com/S0141-8130(16)31614-2/sbref0080 http://refhub.elsevier.com/S0141-8130(16)31614-2/sbref0080 http://refhub.elsevier.com/S0141-8130(16)31614-2/sbref0080 http://refhub.elsevier.com/S0141-8130(16)31614-2/sbref0080 http://refhub.elsevier.com/S0141-8130(16)31614-2/sbref0080 http://refhub.elsevier.com/S0141-8130(16)31614-2/sbref0080 http://refhub.elsevier.com/S0141-8130(16)31614-2/sbref0080 http://refhub.elsevier.com/S0141-8130(16)31614-2/sbref0080 http://refhub.elsevier.com/S0141-8130(16)31614-2/sbref0080 http://refhub.elsevier.com/S0141-8130(16)31614-2/sbref0080 http://refhub.elsevier.com/S0141-8130(16)31614-2/sbref0080 http://refhub.elsevier.com/S0141-8130(16)31614-2/sbref0080 http://refhub.elsevier.com/S0141-8130(16)31614-2/sbref0080 http://refhub.elsevier.com/S0141-8130(16)31614-2/sbref0080 http://refhub.elsevier.com/S0141-8130(16)31614-2/sbref0080 http://refhub.elsevier.com/S0141-8130(16)31614-2/sbref0080 http://refhub.elsevier.com/S0141-8130(16)31614-2/sbref0080 http://refhub.elsevier.com/S0141-8130(16)31614-2/sbref0080 http://refhub.elsevier.com/S0141-8130(16)31614-2/sbref0080 http://refhub.elsevier.com/S0141-8130(16)31614-2/sbref0080 http://refhub.elsevier.com/S0141-8130(16)31614-2/sbref0080 http://refhub.elsevier.com/S0141-8130(16)31614-2/sbref0080 http://refhub.elsevier.com/S0141-8130(16)31614-2/sbref0080 http://refhub.elsevier.com/S0141-8130(16)31614-2/sbref0080 http://refhub.elsevier.com/S0141-8130(16)31614-2/sbref0080 http://refhub.elsevier.com/S0141-8130(16)31614-2/sbref0080 http://refhub.elsevier.com/S0141-8130(16)31614-2/sbref0080 http://refhub.elsevier.com/S0141-8130(16)31614-2/sbref0080 http://refhub.elsevier.com/S0141-8130(16)31614-2/sbref0080 http://refhub.elsevier.com/S0141-8130(16)31614-2/sbref0080 http://refhub.elsevier.com/S0141-8130(16)31614-2/sbref0080 http://refhub.elsevier.com/S0141-8130(16)31614-2/sbref0080 http://refhub.elsevier.com/S0141-8130(16)31614-2/sbref0085 http://refhub.elsevier.com/S0141-8130(16)31614-2/sbref0085 http://refhub.elsevier.com/S0141-8130(16)31614-2/sbref0085 http://refhub.elsevier.com/S0141-8130(16)31614-2/sbref0085 http://refhub.elsevier.com/S0141-8130(16)31614-2/sbref0085 http://refhub.elsevier.com/S0141-8130(16)31614-2/sbref0085 http://refhub.elsevier.com/S0141-8130(16)31614-2/sbref0085 http://refhub.elsevier.com/S0141-8130(16)31614-2/sbref0085 http://refhub.elsevier.com/S0141-8130(16)31614-2/sbref0085 http://refhub.elsevier.com/S0141-8130(16)31614-2/sbref0085 http://refhub.elsevier.com/S0141-8130(16)31614-2/sbref0085 http://refhub.elsevier.com/S0141-8130(16)31614-2/sbref0085 http://refhub.elsevier.com/S0141-8130(16)31614-2/sbref0085 http://refhub.elsevier.com/S0141-8130(16)31614-2/sbref0085 http://refhub.elsevier.com/S0141-8130(16)31614-2/sbref0085 http://refhub.elsevier.com/S0141-8130(16)31614-2/sbref0085 http://refhub.elsevier.com/S0141-8130(16)31614-2/sbref0085 http://refhub.elsevier.com/S0141-8130(16)31614-2/sbref0085 http://refhub.elsevier.com/S0141-8130(16)31614-2/sbref0085 http://refhub.elsevier.com/S0141-8130(16)31614-2/sbref0085 http://refhub.elsevier.com/S0141-8130(16)31614-2/sbref0085 http://refhub.elsevier.com/S0141-8130(16)31614-2/sbref0085 http://refhub.elsevier.com/S0141-8130(16)31614-2/sbref0085 http://refhub.elsevier.com/S0141-8130(16)31614-2/sbref0085 http://refhub.elsevier.com/S0141-8130(16)31614-2/sbref0090 http://refhub.elsevier.com/S0141-8130(16)31614-2/sbref0090 http://refhub.elsevier.com/S0141-8130(16)31614-2/sbref0090 http://refhub.elsevier.com/S0141-8130(16)31614-2/sbref0090 http://refhub.elsevier.com/S0141-8130(16)31614-2/sbref0090 http://refhub.elsevier.com/S0141-8130(16)31614-2/sbref0090 http://refhub.elsevier.com/S0141-8130(16)31614-2/sbref0090 http://refhub.elsevier.com/S0141-8130(16)31614-2/sbref0090 http://refhub.elsevier.com/S0141-8130(16)31614-2/sbref0090 http://refhub.elsevier.com/S0141-8130(16)31614-2/sbref0090 http://refhub.elsevier.com/S0141-8130(16)31614-2/sbref0090 http://refhub.elsevier.com/S0141-8130(16)31614-2/sbref0090 http://refhub.elsevier.com/S0141-8130(16)31614-2/sbref0090 http://refhub.elsevier.com/S0141-8130(16)31614-2/sbref0090 http://refhub.elsevier.com/S0141-8130(16)31614-2/sbref0090 http://refhub.elsevier.com/S0141-8130(16)31614-2/sbref0090 http://