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Blood oxidative stress markers in Gaucher disease patients
Clinica Chimica Acta 364 (2006) 316 – 320 www.elsevier.com/locate/clinchim
Blood oxidative stress markers in Gaucher disease patients Fernanda M. Roversi, Luciano C. Galdieri, Bruno H.C. Grego, Fernanda G. Souza, Cecı´lia Micheletti, Ana Maria Martins, Vaˆnia D’Almeida * Department of Pediatrics, Universidade Federal de Sa˜o Paulo/Escola Paulista de Medicina (UNIFESP/EPM), Rua Napolea˜o de Barros, 925, 3rd floor, ZIP: 04024-002, Sa˜o Paulo, SP, Brazil Received 11 May 2005; received in revised form 26 July 2005; accepted 28 July 2005 Available online 24 August 2005
Abstract Background: Gaucher disease (GD) is the most common glycosphingolipidosis resulting in accumulation of glucoceramide. The most effective treatment for this disease is enzyme replacement therapy (ERT) which involves recombinant enzyme infusion. Enzymatic deficiency in GD patients may induce a cascade of events culminating in secondary effects such as the production of reactive oxygen species (ROS). We investigated the relationship between ROS and GD by analyzing blood oxidative stress markers in GD patients submitted to ERT at different stages during the treatment. Methods: Blood were collected before and just after enzyme infusion. Red blood cell catalase (CAT), glutathione peroxidase (GPx), superoxide dismutase (SOD) and total glutathione (tGSH), and plasma thiobarbituric acid reactive substances (TBARS) were assayed by spectrophotometry. Homocysteine concentrations and related polymorphisms were also studied. Control individuals matched for sex and age were also analyzed. Results: Concentrations of homocysteine and TBARS, and GPx enzyme activity were not different in ERT-treated GD patients. CAT activity was higher while SOD was lower in patients compared to controls. No variations in any of these parameters were found before and just after ERT. Regarding tGSH, a significant increase was observed in GD patients after infusion. Genotypic frequencies studied did not differ from controls or other Brazilian samples. Conclusion: ERT-treated GD patients show an improvement in antioxidant capacity, which is further increased just after recombinant enzyme infusion. D 2005 Elsevier B.V. All rights reserved. Keywords: Gaucher disease; Enzyme replacement therapy; Oxidative stress; Glutathione; Free radicals; Homocysteine
1. Introduction Gaucher disease (GD) is an inborn error of glycosphingolipid metabolism caused by deficient activity of the lysosomal enzyme glucocerebrosidase, an essential component of the phagocytic process. In healthy individuals, macrophages use glucocerebrosidase to break down dead cells and cellular debris, but when there is a deficiency of this enzyme, fats and
* Corresponding author. Tel.: +55 11 5539 0155x183; fax: +55 11 5572 5092. E-mail address: [email protected] (V. D’Almeida). 0009-8981/$ - see front matter D 2005 Elsevier B.V. All rights reserved. doi:10.1016/j.cca.2005.07.022
carbohydrates accumulate in the macrophage lysosomes in the reticuloendothelial system [1]. The macrophages expand to several times their normal size, and become known as Gaucher cells. They usually accumulate in the spleen, liver and bone marrow, but may also be found elsewhere. As Gaucher cells build up, organ dysfunction and damage result [2,3]. Three main forms of the disease have been identified and, classified as types I, II and III, depending on their effects in the central nervous system, their symptomatology and severity. Type I, the most common, is non-neuronopathic, and shows highly variable signs and symptoms and a variable course. The clinical onset of Gaucher disease type I may be observed at any age, but manifestations typically occur after childhood and, in
F.M. Roversi et al. / Clinica Chimica Acta 364 (2006) 316 – 320
some cases, not until adult life [2,3]. Types II and III are neuronopathic. All forms of the disease involve some degree of hepatosplenomegaly, anemia, thrombocytopenia, bone complications and abnormal energetic metabolism [2]. Since 1990’s, treatment of Gaucher disease has included enzyme replacement therapy (ERT) using mannose-terminated recombinant human glucocerebrosidase imiglucerase (Cerezyme\, Genzyme Corporation, Cambridge, MA). ERT has been shown to ameliorate systemic involvement in type I Gaucher disease and to enhance the quality of life [4–6]. Although not curative, ERT not only improves many clinical manifestations of Gaucher disease but actually prevents them if initiated early enough. No effective prevention or treatment of the neurological manifestation of the disease has been discovered yet. The enzymatic deficiency that occurs in many inborn errors of metabolism as well as the alterations induced by ERT may lead to a cascade of events culminating in secondary effects, such as production and release of reactive oxygen species (ROS). In fact, ROS seems to be involved in a large number of human diseases. There is an increasing amount of evidence showing that damage caused by free radicals is an important contributing factor in neurodegenerative, chronic-inflammatory, vascular and neoplasic disease [7,8]. Although ROS is produced constantly, it is deactivated by natural molecules denominated antioxidants that can prevent uncontrolled formation of free radicals and activated oxygen species, or inhibit their reactions with biological structures [8,9]. Primary antioxidant defense is provided by enzymes such as superoxide dismutase (SOD), catalase (CAT) and glutathione peroxidase (GPx). For proper removal of ROS from a biological system, there must be a balance between these 3 enzymes [8]. Non-enzymatic defense, which also plays a significant role in control of oxidative injury, includes dietary supplements such as antioxidant vitamins (A, C, E and Q), flavonoids, lycopenes, glutathione (GSH), quinines and uric acid, all of which are important for proper antioxidant defense [7–9]. ROS production and degradation are usually balanced in the organism. Oxidative stress increases when ROS levels, such as superoxide anion (O2& ), hydroxil radical (OH&), and peroxynitrite (ONOO ), exceed the cellular antioxidant defense capacity [8]. Other biological mechanisms contribute to ROS formation. For example, the auto-oxidation of sulphur amino acids such as homocysteine may lead to ROS formation in the presence of molecular oxygen [10,11]. In humans, normal concentration of plasma total homocysteine ranges from 5 to 15 Amol/l and several genetic, physiologic, pharmacologic, and pathological factors may affect these concentrations [10,12,13]. We investigated the relationship between ROS and GD by measuring variations in oxidative stress markers in blood samples of GD patients submitted to ERT at different stages of treatment. We also studied the relationship between homocysteine, GD and gene polymorphism in methylene tetrahydrofolate reductase (MTHFR), an important enzyme in homocysteine metabolism.
317
2. Methods 2.1. Subjects 2.1.1. Sample The sample consisted of the following (two) groups: (1) an experimental group of Gaucher disease patients (N = 10), and (2) a control group consisting of healthy individuals who voluntarily agreed to participate after receiving detailed information of the aims of the study (N =25). At the time of blood collection control subjects did not present any symptoms of infectious disease nor other pathologies that could produce changes in oxidative stress parameters. Gaucher disease patients (3 males and 7 females) were aged from 6 to 52 y at the time of first blood collection, and had been in ERT for at least 2 y (2 infusions per month of 15 to 120 U/kg depending on age and level of commitment). In this sense, they will be referred as ERT-treated GD patients throughout this study. This study was carried out at the Inborn Errors of Metabolism Center (local acronym CREIM) of the Pediatrics Department — Universidade Federal de Sa˜o Paulo/Escola Paulista de Medicina (local acronym UNIFESP/EPM). This study involved Gaucher disease patients from the Hereditary Metabolic Diseases Clinic — UNIFESP/EPM. 2.1.2. Approval by Medical Research and Ethical Committee The research protocols and consent forms as well as the investigation were ethically and scientifically approved by the Medical Research and Ethical Committee of UNIFESP/ EPM (Proc. # 0309/03). 2.2. Procedures 2.2.1. Collection and processing of blood samples Two samples were collected from each patient, one just before the beginning of an enzyme infusion session and another at the end of the session. Six months after this first analysis, another blood sample was taken following the same protocol. Venous blood was collected in tubes containing heparin and in other tubes containing EDTA, before and after the treatment (infusion of recombinant enzyme). Red blood cells (RBC) and plasma from heparin tubes were separated by centrifugation, and plasma was immediately used for lipid peroxidation analysis while washed RBC were stored at 80 -C until processing of antioxidant enzyme assays. Leucocytes and plasma from EDTA tubes were separated by centrifugation and used for homocysteine analysis while leucocytes were used for DNA extraction and polymorphism analysis. Serum creatinine levels were measured before and after enzyme infusion in order to confirm that ERT did not produce hemodillution, which would affect biochemical parameters. 2.2.2. Analysis of plasma TBARS To access lipid peroxidation we analyzed the levels of thiobarbituric acid reactive substances (TBARS) in plasma by
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colorimetric assay (k = 535 nm) [14]. The results were expressed as nanomole of malondyaldehyde per milliliter (Dmol/ml). 2.2.3. Antioxidant assays Hemolysates were prepared by washing and lysing RBC samples, and then used for analyses of antioxidant enzymes, SOD, GPx and CAT. Portions of these RBC samples were mixed with HClO4 2 mol/l EDTA 4 mmol/l, and used for analyses of total glutathione (tGSH).
Blood DNA Purification kit, by Amersham Biosciences Company (Piscataway, NJ), following the manufacturer’s manual. 2.2.5.2. C667T and A1298C polymorphisms analysis of MTHFR gene. C667T and A1298C polymorphisms analysis of MTHFR gene were based on polymerase chain reaction (PCR) technique and restriction fragment length polymorphism (RFLP) analysis [19–21]. 2.3. Statistical analysis
2.2.3.1. Enzymes. The CAT assay was carried out following the method described by Adamo et al. [14]. Hemolysate samples were used and a decrease in absorbance at 230 nm was observed. The activity values were expressed as units per milligram of hemoglobin (U/mg Hb). GPx activity levels were measured using the method described by Sies et al. [15]. Absorbance at 340 nm was observed and the activity was expressed as U/g Hb. SOD was assayed using the method described by McCord and Fridovich [16]. The decrease in absorbance at 550 nm was observed and the activity was calculated as U/mg Hb.
The results were presented as mean T standard deviation (SD). The Student’s t test for independent samples was used to compare the two groups: ERT-treated GD patients and control subjects. Differences between oxidative stress markers before and just after an enzyme replacement therapy session were analyzed using Student’s t test for dependent samples. The level of significance for both statistical analyses was set at p < 0.05.
2.2.3.2. Glutathione. Total GSH assay was carried out using Tietze’s method [17]. Total GSH levels were obtained spectrophotometrically at 412 nm and the results expressed as Amol/g Hb.
3.1. Indices of oxidative stress and oxidative membrane damage in Gaucher disease
2.2.4. Plasma total homocysteine assay Plasma total homocysteine concentrations were obtained by the method described by Pfeiffer et al. [18] using high performance liquid chromatography (HPLC) with fluorimetric detection and isocratic separation. Homocysteine concentrations were given as Amol/l (normal range 5 –12 Amol/l). 2.2.5. Molecular analysis 2.2.5.1. Genomic DNA extraction. Genomic DNA extraction from lymphocytes was performed using the GFX Genomic
3. Results and discussion
GPx activity levels did not differ in ERT-treated GD patients compared with controls, irrespective of treatment (Table 1). Superoxide dismutase activity was significantly lower in ERT-treated GD patients, while catalase activity was significantly higher when compared to controls, before and just after the ERT session (Table 1). When we compared antioxidant enzyme activity levels before and after infusion, we did not observe significant alterations in the first analysis or after 6 months of ERT. Plasma TBARS levels did not differ between controls and patients (Table 1). Comparison of TBARS levels before and after infusion did not reveal significant alterations on the first analysis or after 6 months. Total glutathione after ERT did not significantly differ
Table 1 Levels of antioxidant enzymes (catalase, superoxide dimutase and glutathione peroxidase), plasma TBARS, total glutathione and plasma total homocysteine in controls subjects and Gaucher disease patients Control (N = 27)
Gaucher disease patients First analysis (N = 9)
Catalase (U/mg of Hb) Superoxide dismutase (U/mg of Hb) Glutathione peroxidase (U/g Hb) TBARS (Dmol/mL) Total glutathione (Amol/gHb) Homocysteine (Amol/L)
82.6 T 25.7 17.0 T 3.6 10.2 T 6.9 1.4 T 0.8 5.3 T 1.9 9.9 T 3.6
After 6 months (N = 8)
Before
After
Before
After
102.6 T 19.8* 12.4 T 3.9* 9.9 T 2.3 1.8 T 1.1 6.7 T 2.8 8.9 T 2.6
111.7 T 30.7* 10.4 T 1.4* 9.2 T 1.4 1.1 T 0.04 7.1 T 2.6** 8.3 T 2.5
113.5 T 13.4* 14.9 T 3.3* 10.9 T 2.8 1.1 T 0.6 6.6 T 1.7 NA
108.3 T 9.9* 13.5 T 3.3* 11.1 T 4.5 0.94 T 0.4 7.1 T1.8** NA
Gaucher patients were analyzed twice during a period of 6 months. ‘‘Before’’ and ‘‘After’’ mean before enzyme infusion and just after the end of ERT session. All values are mean T S.D. * Statistical significant differences ( p < 0.05) between Gaucher disease patients and controls; ** different from Gaucher disease patients before enzyme infusion; Na=not accessed.
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from control values (Table 1). Nevertheless, glutathione levels just after enzyme infusion were significantly higher at both time points studied (Table 1) compared to levels before infusion. The data from the present study showed that in ERTtreated GD patients, the activity of two antioxidant enzymes was altered: superoxide dismutase levels were significantly lower and catalase levels were significantly higher, indicating a situation of oxidative stress imbalance. However, there was no increase in plasma lipid peroxides accessed by TBARS, one of the indices of membrane dysfunction caused by oxidative stress. On the other hand, low levels of SOD activity could result in higher levels of superoxide anion in ERT-treated Gaucher disease patients, which in turn, could lead to other oxidative processes such as DNA or protein damage [8]. Another possible explanation for decreased SOD activity could be a response from the whole organism to impaired generation of superoxide anion by monocytes and macrophages from GD patients. This phenomenon was observed in vitro [24,25] and may occur in vivo with GD patients. If GD patients produce less superoxide anion, a decrease in SOD activity could be an adaptation to these differences. Recently, inhibition of NADPH oxidase by glucocerebrosides was described [26] using various forms of a cell-free assay system, suggesting that assembly of the respiratory burst oxidase phagocitic cells may be a possible target of the pathologic actions of glucocerebrosides [26]. Interestingly, all these studies were performed using in vitro systems and we observed decreased SOD levels measuring enzyme activity on RBC of GD patients before and just after an ERT session. Although we observed reduction in enzyme activity, our sample included only patients in ERT for at least 2 y and the enzyme replacement treatment did not change this situation. In all these in vitro studies [24 – 26], the authors believe that ERT could restore superoxide production and improve functions of monocytes and macrophages leading GD patients to show more resistance to severe bacterial infection. Patients analyzed in the present study showed decreased SOD activity but did not present elevated index of infections (data not shown). In fact, the antioxidant enzymes analyses just after ERT showed the same pattern observed before treatment: decreased SOD and increased catalase activities and unchanged GPx. No alterations were observed in TBARS assay just after the infusion. This situation confirms that even after 2 y of ERT some oxidative stress related parameters are altered and these alterations were confirmed after the sixmonth interval. No significant alterations were observed in total glutathione levels before enzyme replacement infusion in ERTtreated GD patients. On the other hand, total glutathione levels just after infusion were significantly higher in patients than in controls or in patients before enzyme infusion. These increases indicate that activation of antioxidant defense mechanisms may be induced by ERT.
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3.2. Concentrations of plasma total homocysteine and molecular analysis of MTHFR gene The concentrations of plasma total homocysteine were not significantly different in ERT-treated GD patients compared with controls (Table 1). Nor were there significant differences between values obtained before and after the infusion. Genotypic distribution of GD patients for the 2 most common mutations in methylene tetrahydrofolate reductase gene is shown in Table 2. The genotypic frequencies found did not differ from controls or those found in larger Brazilian samples studied by our group [22,23]. When total plasma homocysteine in ERT-treated GD patients and controls was compared, no significant alterations were observed. This parameter was analyzed only once due to difficulties in obtaining large amounts of blood. The molecular data concerning homocysteine metabolism presented no variations in Gaucher patients compared to the Brazilian population [22,23]. It is interesting to note that all changes in oxidative stress parameters studied were not related to age or duration of the illness. Another point was that ERT did not change protein concentrations since creatinine levels were the same before and after infusion. A number of recent studies have shown that lysosomes are in fact highly susceptible to free radical oxidative stress in brain and other tissues and organs [27]. This evidence led us to believe that any imbalance in antioxidant defense could produce appropriate conditions for causing oxidative damage. The inherent toxicity of aggregated material in Gaucher patients lysosomes may be a fundamental component in the pathophysiology of the disease, contributing to the fullfeature disease these patients present. This is particularly interesting in relation to Gaucher patients since they have lysosome damage and their levels of important antioxidant enzymes differ from those of healthy individuals. However, we also found higher total GSH levels just after enzyme infusion. This fact points to another important consequence of ERT, since it shows that enzyme infusion may, for a short time at least, lead to an improvement in antioxidant defense. It would be interesting to monitor patients during the interval between both infusions, since ROS is not proposed here as the cause of disease but rather as involved in progression of the symptoms, perhaps in conjunction with several other events. If an improvement in antioxidant defense could be brought about by ERT, more biochemical analysis (e.g. levels
Table 2 Genotypic distribution of C677T and A1298C polymorphism of the enzyme methylene tetrahydrofolate reductase gene (%) in Gaucher disease patients Gaucher disease patients
Homozygote CC Heterozygote CT Homozygote TT
C677T (N = 10)
A1298C (N = 6)
70.0 30.0 –
33.3 50.0 16.7
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of other antioxidants) should be performed in these patients to enable carers to plan more strategies to support them.
4. Conclusion Our study showed an alteration in reactive oxygen species production, considering the high catalase levels and low levels of superoxide dismutase observed in the blood of ERT-treated GD patients, and the increase in total glutathione levels just after enzymatic replacement therapy. The data presented here suggest that GD patients treated by ERT have an improvement in the antioxidant capacity, which is further increased immediately after recombinant enzyme infusion.
Acknowledgments This research was supported by Conselho Nacional de Desenvolvimento Cientı´fico e Tecnolo´gico (CNPq), Brazil (Proc. # 473924/2003-0), Fundac¸a˜o de Amparo a` Pesquisa do Estado de Sa˜o Paulo (FAPESP), Brazil (Proc. # 97/1870-4 and 01/11366-9), Associac¸a˜o Fundo de Incentivo a` Psicofarmacologia (AFIP) and Genzyme do Brasil. The authors are grateful for technical assistance provided by Jose´ Adelmo de Barros e Maria Aparecida de Oliveira. The valuable support of Dr. Nestor A. Chamoles and Dra. Elvira Ponce is greatly appreciated.
References [1] Beutler E, Grabowski G. Gaucher disease. In: Scriver CR, Beaudet AL, Sly WS, et al, editors. Metabolic and molecular bases of inherited disease. New York’ McGraw Hill; 2001. [2] Enderlin C, Vogel R, Conaway P. Gaucher disease. Am J Nurs 2003; 103:50 – 60. [3] Grabowski GA. Gaucher disease: lessons from a decade of therapy. J Pediatr 2004;144:S15 – 9. [4] Sly WS. Enzyme replacement therapy for lysosomal storage disorders: successful transition from concept to clinical practice. Mol Med 2004; 101:100 – 4. [5] Futerman AH, Sussman JL, Horowitz M, Silman I, Zimran A. New directions in the treatment of Gaucher disease. Trends Pharmacol Sci 2004;25:147 – 51. [6] Grabowski GA, Hopkin RJ. Enzyme therapy for lysosomal storage disease: principles, practice, and prospects. Annu Rev Genomics Hum Genet 2003;4:403 – 36. [7] Halliwell B. Free radicals, antioxidants, and human disease: curiosity, cause or consequence? Lancet 1994;344:721 – 4. [8] Halliwell B, Gutteridge JM. Free radicals in biology and medicine. 3rd ed. Oxford’ Oxford University Press; 1999. [9] Chaudie`re J, Ferrari-Iliou R. Intracellular antioxidant: from chemical to biochemical mechanisms. Food Chem Toxicol 1999;37:49 – 962.
[10] Selhub J. Homocysteine metabolism. Annu Rev Nutr 1999;19:217 – 46. [11] Bleich S, Degner D, Sperling W, Bonsch D, Thurauf N, Kornhuber J. Homocysteine as a neurotoxin in chronic alcoholism. Prog Neuropsychopharmacol Biol Psychiatry 2004;28:453 – 64. [12] Miner SES, Evrovski J, Cole DEC. Clinical chemistry and molecular biology of homocysteine metabolism: an update. Clin Biochem 1997; 30:189 – 201. [13] Shaw GM, Velie EM, Schaffer DM. Is dietary intake of methionine associated with a reduction in risk for neural tube defect—affected pregnancies? Teratology 1997;56:295 – 9. [14] Adamo AM, Llesuy LF, Pasquini JM, Boveris A. Brain chemiluminescence and oxidative stress in hyperthyroid rats. Biochem J 1989; 263:273 – 7. [15] Sies H, Koch OR, Martino E, Boveris A. Increased biliary glutathione disulfide release in chronically ethanol treated rats. FEBS Lett 1979; 103:287 – 90. [16] McCord J, Fridovich I. Superoxide dismutase: an enzymic function for erythrocuprein. J Biol Chem 1969;244:6049 – 55. [17] Tietze F. Enzymic method for quantitative determination of nanogram amounts of total and oxidized glutathione: applications to mammalian blood and other tissues. Anal Biochem 1969;27:502 – 22. [18] Pfeiffer CM, Huff DL, Gunter EW. Rapid and accurate HPLC assay for plasma total homocysteine and cysteine in a clinical laboratory setting. Clin Chem 1999;45:290 – 2. [19] Frosst P, Blom HJ, Milos R, et al. A candidate genetic risk factor for vascular disease: a common mutation in methylenetetrahydrofolate reductase. Nature Genet 1995;10:111 – 3. [20] Goyette P, Frosst P, Rosenblatt DS, Rozen R. Seven novel mutations in the methylenetetrahydrofolate reductase gene and genotype/phenotype correlations in severe methylenetetrahydrofolate reductase deficiency. Am J Hum Genet 1995;56:1052 – 9. [21] van der Put NMJ, Gabree¨ls F, Stevens BEM, et al. A second common mutation in the methyleletetrahydrofolate reductase gene: an additional risk factor for neural-tube defects? Am J Hum Genet 1998; 62:1044 – 51. [22] Morelli VM, Lourenco DM, D’Almeida V, et al. Hyperhomocysteinemia increases the risk of venous thrombosis independent of the C677T mutation of the methylenetetrahydrofolate reductase gene in selected Brazilian patients. Blood Coagul Fibrinolysis 2002;13:271 – 5. [23] Perez AB, D’Almeida V, Vergani N, de Oliveira AC, de Lima FT, Brunoni D. Methylenetetrahydrofolate reductase (MTHFR): incidence of mutations C677T and A1298C in Brazilian population and its correlation with plasma homocysteine levels in spina bifida. Am J Med Genet 2003;119A:20 – 5. [24] Liel Y, Rudich A, Nagauker-Shriker O, Yermiyahu T, Levy R. Monocyte dysfunction in patients with Gaucher disease: evidence for interference of glucocerebroside with superoxide generation. Blood 1994;83(9):2646 – 53. [25] Marodi L, Kaposzta R, Toth J, Laszlo A. Impaired microbicidal capacity of mononuclear phagocytes from patients with type I Gaucher disease: partial correction by enzyme replacement therapy. Blood 1995;86(12):4645 – 9. [26] Moskwa P, Palicz A, Paclet MH, et al. Glucocerebroside inhibits NADPH oxidase activation in cell-free system. Biochim Biophys Acta 2004;1688(3):197 – 203. [27] Bahr BA, Bendiske J. The neuropathogenic contributions of lysosomal dysfunction. J Neurochem 2002;83:481 – 9.
Blood oxidative stress markers in Gaucher disease patients Fernanda M. Roversi, Luciano C. Galdieri, Bruno H.C. Grego, Fernanda G. Souza, Cecı´lia Micheletti, Ana Maria Martins, Vaˆnia D’Almeida * Department of Pediatrics, Universidade Federal de Sa˜o Paulo/Escola Paulista de Medicina (UNIFESP/EPM), Rua Napolea˜o de Barros, 925, 3rd floor, ZIP: 04024-002, Sa˜o Paulo, SP, Brazil Received 11 May 2005; received in revised form 26 July 2005; accepted 28 July 2005 Available online 24 August 2005
Abstract Background: Gaucher disease (GD) is the most common glycosphingolipidosis resulting in accumulation of glucoceramide. The most effective treatment for this disease is enzyme replacement therapy (ERT) which involves recombinant enzyme infusion. Enzymatic deficiency in GD patients may induce a cascade of events culminating in secondary effects such as the production of reactive oxygen species (ROS). We investigated the relationship between ROS and GD by analyzing blood oxidative stress markers in GD patients submitted to ERT at different stages during the treatment. Methods: Blood were collected before and just after enzyme infusion. Red blood cell catalase (CAT), glutathione peroxidase (GPx), superoxide dismutase (SOD) and total glutathione (tGSH), and plasma thiobarbituric acid reactive substances (TBARS) were assayed by spectrophotometry. Homocysteine concentrations and related polymorphisms were also studied. Control individuals matched for sex and age were also analyzed. Results: Concentrations of homocysteine and TBARS, and GPx enzyme activity were not different in ERT-treated GD patients. CAT activity was higher while SOD was lower in patients compared to controls. No variations in any of these parameters were found before and just after ERT. Regarding tGSH, a significant increase was observed in GD patients after infusion. Genotypic frequencies studied did not differ from controls or other Brazilian samples. Conclusion: ERT-treated GD patients show an improvement in antioxidant capacity, which is further increased just after recombinant enzyme infusion. D 2005 Elsevier B.V. All rights reserved. Keywords: Gaucher disease; Enzyme replacement therapy; Oxidative stress; Glutathione; Free radicals; Homocysteine
1. Introduction Gaucher disease (GD) is an inborn error of glycosphingolipid metabolism caused by deficient activity of the lysosomal enzyme glucocerebrosidase, an essential component of the phagocytic process. In healthy individuals, macrophages use glucocerebrosidase to break down dead cells and cellular debris, but when there is a deficiency of this enzyme, fats and
* Corresponding author. Tel.: +55 11 5539 0155x183; fax: +55 11 5572 5092. E-mail address: [email protected] (V. D’Almeida). 0009-8981/$ - see front matter D 2005 Elsevier B.V. All rights reserved. doi:10.1016/j.cca.2005.07.022
carbohydrates accumulate in the macrophage lysosomes in the reticuloendothelial system [1]. The macrophages expand to several times their normal size, and become known as Gaucher cells. They usually accumulate in the spleen, liver and bone marrow, but may also be found elsewhere. As Gaucher cells build up, organ dysfunction and damage result [2,3]. Three main forms of the disease have been identified and, classified as types I, II and III, depending on their effects in the central nervous system, their symptomatology and severity. Type I, the most common, is non-neuronopathic, and shows highly variable signs and symptoms and a variable course. The clinical onset of Gaucher disease type I may be observed at any age, but manifestations typically occur after childhood and, in
F.M. Roversi et al. / Clinica Chimica Acta 364 (2006) 316 – 320
some cases, not until adult life [2,3]. Types II and III are neuronopathic. All forms of the disease involve some degree of hepatosplenomegaly, anemia, thrombocytopenia, bone complications and abnormal energetic metabolism [2]. Since 1990’s, treatment of Gaucher disease has included enzyme replacement therapy (ERT) using mannose-terminated recombinant human glucocerebrosidase imiglucerase (Cerezyme\, Genzyme Corporation, Cambridge, MA). ERT has been shown to ameliorate systemic involvement in type I Gaucher disease and to enhance the quality of life [4–6]. Although not curative, ERT not only improves many clinical manifestations of Gaucher disease but actually prevents them if initiated early enough. No effective prevention or treatment of the neurological manifestation of the disease has been discovered yet. The enzymatic deficiency that occurs in many inborn errors of metabolism as well as the alterations induced by ERT may lead to a cascade of events culminating in secondary effects, such as production and release of reactive oxygen species (ROS). In fact, ROS seems to be involved in a large number of human diseases. There is an increasing amount of evidence showing that damage caused by free radicals is an important contributing factor in neurodegenerative, chronic-inflammatory, vascular and neoplasic disease [7,8]. Although ROS is produced constantly, it is deactivated by natural molecules denominated antioxidants that can prevent uncontrolled formation of free radicals and activated oxygen species, or inhibit their reactions with biological structures [8,9]. Primary antioxidant defense is provided by enzymes such as superoxide dismutase (SOD), catalase (CAT) and glutathione peroxidase (GPx). For proper removal of ROS from a biological system, there must be a balance between these 3 enzymes [8]. Non-enzymatic defense, which also plays a significant role in control of oxidative injury, includes dietary supplements such as antioxidant vitamins (A, C, E and Q), flavonoids, lycopenes, glutathione (GSH), quinines and uric acid, all of which are important for proper antioxidant defense [7–9]. ROS production and degradation are usually balanced in the organism. Oxidative stress increases when ROS levels, such as superoxide anion (O2& ), hydroxil radical (OH&), and peroxynitrite (ONOO ), exceed the cellular antioxidant defense capacity [8]. Other biological mechanisms contribute to ROS formation. For example, the auto-oxidation of sulphur amino acids such as homocysteine may lead to ROS formation in the presence of molecular oxygen [10,11]. In humans, normal concentration of plasma total homocysteine ranges from 5 to 15 Amol/l and several genetic, physiologic, pharmacologic, and pathological factors may affect these concentrations [10,12,13]. We investigated the relationship between ROS and GD by measuring variations in oxidative stress markers in blood samples of GD patients submitted to ERT at different stages of treatment. We also studied the relationship between homocysteine, GD and gene polymorphism in methylene tetrahydrofolate reductase (MTHFR), an important enzyme in homocysteine metabolism.
317
2. Methods 2.1. Subjects 2.1.1. Sample The sample consisted of the following (two) groups: (1) an experimental group of Gaucher disease patients (N = 10), and (2) a control group consisting of healthy individuals who voluntarily agreed to participate after receiving detailed information of the aims of the study (N =25). At the time of blood collection control subjects did not present any symptoms of infectious disease nor other pathologies that could produce changes in oxidative stress parameters. Gaucher disease patients (3 males and 7 females) were aged from 6 to 52 y at the time of first blood collection, and had been in ERT for at least 2 y (2 infusions per month of 15 to 120 U/kg depending on age and level of commitment). In this sense, they will be referred as ERT-treated GD patients throughout this study. This study was carried out at the Inborn Errors of Metabolism Center (local acronym CREIM) of the Pediatrics Department — Universidade Federal de Sa˜o Paulo/Escola Paulista de Medicina (local acronym UNIFESP/EPM). This study involved Gaucher disease patients from the Hereditary Metabolic Diseases Clinic — UNIFESP/EPM. 2.1.2. Approval by Medical Research and Ethical Committee The research protocols and consent forms as well as the investigation were ethically and scientifically approved by the Medical Research and Ethical Committee of UNIFESP/ EPM (Proc. # 0309/03). 2.2. Procedures 2.2.1. Collection and processing of blood samples Two samples were collected from each patient, one just before the beginning of an enzyme infusion session and another at the end of the session. Six months after this first analysis, another blood sample was taken following the same protocol. Venous blood was collected in tubes containing heparin and in other tubes containing EDTA, before and after the treatment (infusion of recombinant enzyme). Red blood cells (RBC) and plasma from heparin tubes were separated by centrifugation, and plasma was immediately used for lipid peroxidation analysis while washed RBC were stored at 80 -C until processing of antioxidant enzyme assays. Leucocytes and plasma from EDTA tubes were separated by centrifugation and used for homocysteine analysis while leucocytes were used for DNA extraction and polymorphism analysis. Serum creatinine levels were measured before and after enzyme infusion in order to confirm that ERT did not produce hemodillution, which would affect biochemical parameters. 2.2.2. Analysis of plasma TBARS To access lipid peroxidation we analyzed the levels of thiobarbituric acid reactive substances (TBARS) in plasma by
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colorimetric assay (k = 535 nm) [14]. The results were expressed as nanomole of malondyaldehyde per milliliter (Dmol/ml). 2.2.3. Antioxidant assays Hemolysates were prepared by washing and lysing RBC samples, and then used for analyses of antioxidant enzymes, SOD, GPx and CAT. Portions of these RBC samples were mixed with HClO4 2 mol/l EDTA 4 mmol/l, and used for analyses of total glutathione (tGSH).
Blood DNA Purification kit, by Amersham Biosciences Company (Piscataway, NJ), following the manufacturer’s manual. 2.2.5.2. C667T and A1298C polymorphisms analysis of MTHFR gene. C667T and A1298C polymorphisms analysis of MTHFR gene were based on polymerase chain reaction (PCR) technique and restriction fragment length polymorphism (RFLP) analysis [19–21]. 2.3. Statistical analysis
2.2.3.1. Enzymes. The CAT assay was carried out following the method described by Adamo et al. [14]. Hemolysate samples were used and a decrease in absorbance at 230 nm was observed. The activity values were expressed as units per milligram of hemoglobin (U/mg Hb). GPx activity levels were measured using the method described by Sies et al. [15]. Absorbance at 340 nm was observed and the activity was expressed as U/g Hb. SOD was assayed using the method described by McCord and Fridovich [16]. The decrease in absorbance at 550 nm was observed and the activity was calculated as U/mg Hb.
The results were presented as mean T standard deviation (SD). The Student’s t test for independent samples was used to compare the two groups: ERT-treated GD patients and control subjects. Differences between oxidative stress markers before and just after an enzyme replacement therapy session were analyzed using Student’s t test for dependent samples. The level of significance for both statistical analyses was set at p < 0.05.
2.2.3.2. Glutathione. Total GSH assay was carried out using Tietze’s method [17]. Total GSH levels were obtained spectrophotometrically at 412 nm and the results expressed as Amol/g Hb.
3.1. Indices of oxidative stress and oxidative membrane damage in Gaucher disease
2.2.4. Plasma total homocysteine assay Plasma total homocysteine concentrations were obtained by the method described by Pfeiffer et al. [18] using high performance liquid chromatography (HPLC) with fluorimetric detection and isocratic separation. Homocysteine concentrations were given as Amol/l (normal range 5 –12 Amol/l). 2.2.5. Molecular analysis 2.2.5.1. Genomic DNA extraction. Genomic DNA extraction from lymphocytes was performed using the GFX Genomic
3. Results and discussion
GPx activity levels did not differ in ERT-treated GD patients compared with controls, irrespective of treatment (Table 1). Superoxide dismutase activity was significantly lower in ERT-treated GD patients, while catalase activity was significantly higher when compared to controls, before and just after the ERT session (Table 1). When we compared antioxidant enzyme activity levels before and after infusion, we did not observe significant alterations in the first analysis or after 6 months of ERT. Plasma TBARS levels did not differ between controls and patients (Table 1). Comparison of TBARS levels before and after infusion did not reveal significant alterations on the first analysis or after 6 months. Total glutathione after ERT did not significantly differ
Table 1 Levels of antioxidant enzymes (catalase, superoxide dimutase and glutathione peroxidase), plasma TBARS, total glutathione and plasma total homocysteine in controls subjects and Gaucher disease patients Control (N = 27)
Gaucher disease patients First analysis (N = 9)
Catalase (U/mg of Hb) Superoxide dismutase (U/mg of Hb) Glutathione peroxidase (U/g Hb) TBARS (Dmol/mL) Total glutathione (Amol/gHb) Homocysteine (Amol/L)
82.6 T 25.7 17.0 T 3.6 10.2 T 6.9 1.4 T 0.8 5.3 T 1.9 9.9 T 3.6
After 6 months (N = 8)
Before
After
Before
After
102.6 T 19.8* 12.4 T 3.9* 9.9 T 2.3 1.8 T 1.1 6.7 T 2.8 8.9 T 2.6
111.7 T 30.7* 10.4 T 1.4* 9.2 T 1.4 1.1 T 0.04 7.1 T 2.6** 8.3 T 2.5
113.5 T 13.4* 14.9 T 3.3* 10.9 T 2.8 1.1 T 0.6 6.6 T 1.7 NA
108.3 T 9.9* 13.5 T 3.3* 11.1 T 4.5 0.94 T 0.4 7.1 T1.8** NA
Gaucher patients were analyzed twice during a period of 6 months. ‘‘Before’’ and ‘‘After’’ mean before enzyme infusion and just after the end of ERT session. All values are mean T S.D. * Statistical significant differences ( p < 0.05) between Gaucher disease patients and controls; ** different from Gaucher disease patients before enzyme infusion; Na=not accessed.
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from control values (Table 1). Nevertheless, glutathione levels just after enzyme infusion were significantly higher at both time points studied (Table 1) compared to levels before infusion. The data from the present study showed that in ERTtreated GD patients, the activity of two antioxidant enzymes was altered: superoxide dismutase levels were significantly lower and catalase levels were significantly higher, indicating a situation of oxidative stress imbalance. However, there was no increase in plasma lipid peroxides accessed by TBARS, one of the indices of membrane dysfunction caused by oxidative stress. On the other hand, low levels of SOD activity could result in higher levels of superoxide anion in ERT-treated Gaucher disease patients, which in turn, could lead to other oxidative processes such as DNA or protein damage [8]. Another possible explanation for decreased SOD activity could be a response from the whole organism to impaired generation of superoxide anion by monocytes and macrophages from GD patients. This phenomenon was observed in vitro [24,25] and may occur in vivo with GD patients. If GD patients produce less superoxide anion, a decrease in SOD activity could be an adaptation to these differences. Recently, inhibition of NADPH oxidase by glucocerebrosides was described [26] using various forms of a cell-free assay system, suggesting that assembly of the respiratory burst oxidase phagocitic cells may be a possible target of the pathologic actions of glucocerebrosides [26]. Interestingly, all these studies were performed using in vitro systems and we observed decreased SOD levels measuring enzyme activity on RBC of GD patients before and just after an ERT session. Although we observed reduction in enzyme activity, our sample included only patients in ERT for at least 2 y and the enzyme replacement treatment did not change this situation. In all these in vitro studies [24 – 26], the authors believe that ERT could restore superoxide production and improve functions of monocytes and macrophages leading GD patients to show more resistance to severe bacterial infection. Patients analyzed in the present study showed decreased SOD activity but did not present elevated index of infections (data not shown). In fact, the antioxidant enzymes analyses just after ERT showed the same pattern observed before treatment: decreased SOD and increased catalase activities and unchanged GPx. No alterations were observed in TBARS assay just after the infusion. This situation confirms that even after 2 y of ERT some oxidative stress related parameters are altered and these alterations were confirmed after the sixmonth interval. No significant alterations were observed in total glutathione levels before enzyme replacement infusion in ERTtreated GD patients. On the other hand, total glutathione levels just after infusion were significantly higher in patients than in controls or in patients before enzyme infusion. These increases indicate that activation of antioxidant defense mechanisms may be induced by ERT.
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3.2. Concentrations of plasma total homocysteine and molecular analysis of MTHFR gene The concentrations of plasma total homocysteine were not significantly different in ERT-treated GD patients compared with controls (Table 1). Nor were there significant differences between values obtained before and after the infusion. Genotypic distribution of GD patients for the 2 most common mutations in methylene tetrahydrofolate reductase gene is shown in Table 2. The genotypic frequencies found did not differ from controls or those found in larger Brazilian samples studied by our group [22,23]. When total plasma homocysteine in ERT-treated GD patients and controls was compared, no significant alterations were observed. This parameter was analyzed only once due to difficulties in obtaining large amounts of blood. The molecular data concerning homocysteine metabolism presented no variations in Gaucher patients compared to the Brazilian population [22,23]. It is interesting to note that all changes in oxidative stress parameters studied were not related to age or duration of the illness. Another point was that ERT did not change protein concentrations since creatinine levels were the same before and after infusion. A number of recent studies have shown that lysosomes are in fact highly susceptible to free radical oxidative stress in brain and other tissues and organs [27]. This evidence led us to believe that any imbalance in antioxidant defense could produce appropriate conditions for causing oxidative damage. The inherent toxicity of aggregated material in Gaucher patients lysosomes may be a fundamental component in the pathophysiology of the disease, contributing to the fullfeature disease these patients present. This is particularly interesting in relation to Gaucher patients since they have lysosome damage and their levels of important antioxidant enzymes differ from those of healthy individuals. However, we also found higher total GSH levels just after enzyme infusion. This fact points to another important consequence of ERT, since it shows that enzyme infusion may, for a short time at least, lead to an improvement in antioxidant defense. It would be interesting to monitor patients during the interval between both infusions, since ROS is not proposed here as the cause of disease but rather as involved in progression of the symptoms, perhaps in conjunction with several other events. If an improvement in antioxidant defense could be brought about by ERT, more biochemical analysis (e.g. levels
Table 2 Genotypic distribution of C677T and A1298C polymorphism of the enzyme methylene tetrahydrofolate reductase gene (%) in Gaucher disease patients Gaucher disease patients
Homozygote CC Heterozygote CT Homozygote TT
C677T (N = 10)
A1298C (N = 6)
70.0 30.0 –
33.3 50.0 16.7
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of other antioxidants) should be performed in these patients to enable carers to plan more strategies to support them.
4. Conclusion Our study showed an alteration in reactive oxygen species production, considering the high catalase levels and low levels of superoxide dismutase observed in the blood of ERT-treated GD patients, and the increase in total glutathione levels just after enzymatic replacement therapy. The data presented here suggest that GD patients treated by ERT have an improvement in the antioxidant capacity, which is further increased immediately after recombinant enzyme infusion.
Acknowledgments This research was supported by Conselho Nacional de Desenvolvimento Cientı´fico e Tecnolo´gico (CNPq), Brazil (Proc. # 473924/2003-0), Fundac¸a˜o de Amparo a` Pesquisa do Estado de Sa˜o Paulo (FAPESP), Brazil (Proc. # 97/1870-4 and 01/11366-9), Associac¸a˜o Fundo de Incentivo a` Psicofarmacologia (AFIP) and Genzyme do Brasil. The authors are grateful for technical assistance provided by Jose´ Adelmo de Barros e Maria Aparecida de Oliveira. The valuable support of Dr. Nestor A. Chamoles and Dra. Elvira Ponce is greatly appreciated.
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