Oxidative Stress Assessment in Patients with Mucopolysaccharidosis I, II, and VI (PDF)

Genetics and Molecular Biology, 42, 1, 1-8 (2019) Copyright © 2019, Sociedade Brasileira de Genética. Printed in Brazil DOI: http://dx.doi.org/10.1590/1678-4685-GMB-2017-0334 Research Article Oxidative stress assessment by glutathione peroxidase activity and glutathione l...

Genetics and Molecular Biology, 42, 1, 1-8 (2019) Copyright © 2019, Sociedade Brasileira de Genética. Printed in Brazil DOI: http://dx.doi.org/10.1590/1678-4685-GMB-2017-0334 Research Article Oxidative stress assessment by glutathione peroxidase activity and glutathione levels in response to selenium supplementation in patients with Mucopolysaccharidosis I, II and VI José Araújo de Oliveira-Silva1, Joyce Umbelino PintoYamamoto2, Renata Bernardes de Oliveira1, Vaneisse Cristina Lima Monteiro1, Beatriz Jurkiewcz Frangipani1, Sandra Obikawa Kyosen1 , Ana Maria Martins1 and Vânia D’Almeida2 1 Centro de Referência em Erros Inatos do Metabolismo (CREIM), Universidade Federal de São Paulo, São Paulo, SP, Brazil. 2 Laboratório de Erros Inatos do Metabolismo (LEIM), Departmento de Psicobiologia, Universidade Federal de São Paulo, São Paulo, SP, Brazil. Abstract We assessed levels of plasma selenium (Se), selenoproteins and their change after Se supplementation in patients with mucopolysaccharidosis (MPS) types I, II and VI. This was done in a retrospective study of the medical records of 30 patients with MPS I (n=13), MPS II (n=9) and MPS VI (n=8) who were being treated with enzyme replacement therapy. As part of routine nutritional monitoring, Se levels were measured, revealing that 28 patients (93.3%) had values below the normal range. Therefore, they received supplementation for 12 months, and Se was measured af- ter 6 and 12 months. Glutathione peroxidase (GPx) activity, total glutathione (GSHt), oxidized glutathione (GSSG) and reduced glutathione (GSH) were measured at baseline and 6 months after Se supplementation. The mean GSHt at baseline was 7.90 ± 2.36 mmol/g Hb, and after Se supplementation it was 5.76 ± 1.13 mmol/g Hb; GSH/GSSG was 2.3 ± 1.16 at baseline and 0.58 ± 0.38 after supplementation. GPx activity was 16.46 ± 3.31 U/g Hb at baseline and 4.53 ± 4.92 U/g Hb after Se supplementation. The difference was shown to be statistically significant by paired t-test. In conclusion, our study demonstrated that oxidative stress parameters were altered by Se supplementation in pa- tients with MPS I, II and VI who were previously deficient in Se. Keywords: Mucopolysaccharidosis, oxidative stress, selenium, glutathione peroxidase. Received: Accepted: October 31, 2017; Accepted: April 30, 2018. Introduction zyme responsible for metabolizing the GAGs dermatan and Mucopolysaccharidoses (MPSs) are hereditary meta- heparan sulfate, and encompasses a spectrum of pheno- bolic diseases caused by the deficiency in the activity of the types that have been delineated into three separate diseases lysosomal enzymes responsible for the degradation of gly- based on clinical presentation that are not distinguishable cosaminoglycans (GAGs). The storage of non-degraded or biochemically: the severe form, Hurler syndrome (OMIM: partially degraded GAGs compromises both the structure 607014), and the attenuated forms Hurler-Scheie (OMIM: and function of cells and organs (Neufeld and Muenzer, 607015) and Scheie (OMIM 607016) (Neufeld, 2001). 2001). MPSs are classified into 11 syndromes according to MPS II, also known as Hunter syndrome (OMIM the deficient enzyme. The clinical manifestations are 309900), is an X-linked inherited trait, caused by mutations chronic and progressive, usually presenting a wide spec- in the IDS gene that encode the enzyme iduronate 2-sul- trum of severity depending on the enzyme deficiency (Neu- fatase (E.C. 3.1.6.13) leading to accumulation of dermatan feld and Muenzer, 2001; Wraith, 2006). and heparan sulfate in different organs and tissues (Neufeld MPS I is inherited in an autosomal recessive trait, and Muenzer, 2001). MPS VI, also known as Maroteaux- caused by mutations in the IDUA gene that encodes the en- Lamy syndrome (OMIM 253200), is inherited in an auto- zyme alpha-L-iduronidase (EC 3.2.1.76), a lysosomal en- somal recessive trait, caused by mutations in the ARSB gene that encodes the enzyme arylsulfatase B (E.C. 3.1.6.12), leading to accumulation of chondroitin sulfate in different Send correspondence to José Araújo de Oliveira Silva. Departa- mento de Pediatria, Universidade Federal de São Paulo, C.L. BOX organs and tissues (Neufeld and Muenzer, 2001). 957, 04020-041 São Paulo, SP. Brazil. E-mail: Although biochemically distinct, MPS I, II and VI [email protected]. share some common clinical features such as hepatospleno- 2 Oliveira-Silva et al. megaly, joint stiffness, dysostosis multiplex and cardiac al- São Paulo, Brazil were recruited for this study. All patients terations. Patients with the severe forms of MPS I and II or their respective legal guardian read and signed an in- present cognitive impairment and neurodegeneration as formed consent form. The study was approved by the Eth- part of disease progression. Patients with MPS VI do not ics Committee of the Universidade Federal de São Paulo present cognitive impairment (Neufeld, and Muenzer, under registration number 0763/11. 2001). It is estimated that the incidence for this group of dis- Study design eases is 3.4 - 4.5 in 100,000 live births (Baehner et al., A retrospective evaluation of medical records of pa- 2005; Lin et al., 2009). Though there is no specific treat- tients with MPS I, II and VI. As part of the routine nutri- ment or cure for MPS, a range of possible treatments are be- tional monitoring of patients with MPS, serum lipids, total ing explored, including enzyme replacement therapy protein, albumin, glucose, vitamin B12, vitamin D, folic (ERT), which is currently only available for MPS I, MPS II, acid, zinc and Se are assessed annually. In a cross-sectional MPS IVA, MPS VI, and MPS VII (Saudubray et al., 2006; retrospective analysis, it was noticed that the majority of Rohrbach and Clarke, 2007; Hendriksz et al., 2015; FDA, patients had Se deficiency, and based on these results, we 2017). Previous studies have shown that ERT helps to re- measured GPx activity, total glutathione (GSHt), oxidized duce the accumulation of GAGs in the organs, promoting a glutathione (GSSG), and reduced glutathione (GSH) at reduction in spleen and liver size, an improvement in baseline (T0). Se supplementation was then given for six growth rates, in walked distance measured by the 6-minute months, and Se, GPx activity, GSHt and GSSG/GSH ratio walking test, and in functional capacity (Decker et al., were measured (T1). After another six months of Se sup- 2010; Giugliani et al., 2010). plementation (a total time of 12 months) Se levels were Some studies report that there is an increase in oxida- measured again (T2). tive stress in patients with MPSs, even in those receiving ERT, but the mechanisms of action remain largely un- Selenium supplementation known (Pereira et al., 2008; Tessitore et al., 2009; Jacques The selenium supplementation was based on the rec- et al., 2016). Oxidative stress is also common in neuro- ommended dietary allowance (RDA) according to the age degenerative and non-neurodegenerative lysosomal stor- of the patients (20-55 mg/day), a daily value that would age diseases and is associated with a variety of diseases, meet the mineral needs of 97-98% of the population (IOM including cancer and cardiovascular disease (Finkel and 2000). The Se was supplied to the patients through a com- Holbrook, 2000; Dutta et al., 2012). pounding pharmacy, formulated as syrup and was to be There are a variety of defense systems against oxida- taken daily. Adherence to treatment was checked tive stress, including non-enzymatic antioxidants, such as weekly/monthly. melatonin, estrogens, bilirubin, reduced glutathione The biochemical assays were carried out on freshly (GSH), polyphenols, and vitamins, in addition to antioxi- drawn blood samples and analyzed at the Laboratory of In- dant enzymes, such as superoxide dismutase (SOD), cata- born Errors of Metabolism (LEIM), Universidade Federal lase (CAT), and glutathione peroxidase (GPx) (Tietze, de São Paulo. 1969). The activity of GPx is dependent on selenium, which is an essential mineral in the diet due to the require- Blood concentrations of Selenium, GSH/GSSG, and ment for selenocysteine in some selenoproteins. GPx pro- GPx activity motes protection against reactive oxygen species (ROS) Plasma concentrations of Se were determined by hy- and reactive nitrogen induced cell damage. Because of its dride generation atomic absorption spectrometry (HG antioxidant activity, there has been a great deal of interest in AAS) according to the method of Hao et al. (1996) using a the study of Se and GPx (Halliwell and Gutteridge, 2007; Hitachi® Z-500 spectrometer. The results were expressed Tinggi, 2008). as mg/L. Our study aims to determine the levels of plasma sele- Total glutathione and GSH concentrations were ana- nium, oxidative stress status evaluated by the ratio of re- lyzed by high performance liquid chromatography (HPLC) duced glutathione to oxidized glutathione, GPx activity and through fluorescence detection and isocratic elution. The the response to Se supplementation in patients with MPSs I, method used was that developed by Pfeiffer et al. (1999), II and VI selected by convenience sampling. with slight modifications: column C18 Luna (5 mm, 150 mm 4.6 mm), mobile phase (0.06 M sodium acetate, 0.5% Materials and Methods acetic acid, pH 4.7 (adjusted with acetic acid, 2% methanol) and a flow rate of 1.1 mL/min. The retention time was 9 Subjects minutes for GSH. Patients with MPS I, II and VI who were receiving For GSSG quantification, the method previously de- weekly ERT at the Reference Center for Inborn Errors of scribed for measuring erythrocyte GSH was used (Galdieri Metabolism (CREIM), Universidade Federal de São Paulo, et al., 2007), but without adding the reducing agent. Selenoproteins in MPS I, II and VI 3 The determination of the erythrocyte GPx was per- Selenium status before and after supplementation in formed in aliquots of the material obtained from the pa- MPSs patients tients and spectrophotometrically analyzed using reduced As part of the routine nutritional monitoring of MPS nicotinamide adenine dinucleotide phosphate (NADPH) as patients, Se levels were measured in 30 patients, showing a marker of the glutathione peroxidase activity. The reac- that 28 patients (93.33%) were deficient with a mean of tion is based on the reduction of tert-butyl hydroperoxide 35.7 ± 10.0 mg/L, and two were within the normal level (52 by glutathione peroxidase, which uses NADPH to provide the reducing power in this reaction (Wahllander et al., ± 1.13 mg/L) considering the laboratory reference value 1979). The results were expressed as mmol/g Hb for GSHt, (46-143 mg/L). Thus, Se supplementation was given, ad- GSSG, and GSH; for GPx they are given as U/g Hb. justed to the required RDA according to age (T0). Selenium levels were measured after 6 (T1) and 12 Statistical analysis months (T2). During this period, few patients were lost to follow-up due to transfer to another center, and the number The quantitative data were evaluated for their internal of patients was therefore reduced in the later phases, mean- consistency by the researchers before being included in the ing that only 24 patients underwent sample collection at T1 analysis. All data were presented as mean ± standard devia- and 27 at T2. At T1, 21 out of 24 patients (87.5%) were in tion (SD). the normal Se level range (mean: 59.20 ± 15.6 mg/L) and 3 Differences in continuous variables, such as Se, GSH, (12.5%) were below normal values (mean 33.67 ± 6.3 GSSG, and GPx before and after supplementation were mg/L). At T2, the mean Se level was 44.63 ± 16.6 mg/L, evaluated by Tukey’s multiple comparison test and, the de- with 13 out of 27 patients (54.1%) having values below nor- pendencies between variables were calculated using the mality, and the difference between T0 vs. T1, and T1 vs. T2 Pearson or Spearman coefficient of correlation. Also, the was statistically significant by Tukey’s multiple compari- regression coefficient was calculated, and a 95% confi- son test (Figure 1). Spearman’s correlation coefficient was dence interval (CI) for net misclassification was calculated not significant (p = 0.23) between T0 and T1, but was statis- using the bootstrap method with SPSS version 22.0 (IBM tically significant between T1 and T2 (p = 0.035). SPSS Statistics, New York, United States). Total glutathione, GSH/GSSG and GPx in MPSs The significance of the differences between GPx, patients GSHt, and the GSH/GSSG ratio before and after Se supple- mentation was assessed by Student’s t-test and Paired t-test Total glutathione (GSHt), reduced glutathione, and using Prism 5.0, GraphPad (San Diego, CA) software. A oxidized glutathione ratio (GSH/GSSG), as well as GPx ac- level of p