- Email: [email protected]
Glutathione metabolism and antioxidant enzymes in children with down syndrome
GLUTATHIONE METABOLISM AND ANTIOXIDANT ENZYMES IN CHILDREN WITH DOWN SYNDROME ANNA PASTORE, BSC, PhD, GIULIA TOZZI, BSC, LAURA MARIA GAETA, BSC, ALDO GIANNOTTI, MD, ENRICO BERTINI, MD, GIORGIO FEDERICI, MD, MARIA CRISTINA DIGILIO, MD, AND FIORELLA PIEMONTE, BSC, PhD
Oxidative stress has been proposed as a pathogenic mechanism of atherosclerosis, cell aging, and neurologic disorders in Down syndrome. This study demonstrates a systemic decrease of all glutathione forms, including glutathionyl-hemoglobin, in the blood of children with Down syndrome. Furthermore, we obtained a disequilibrium, in vivo, between the antioxidant enzyme activities. (J Pediatr 2003;142:583-5)
own syndrome (DS) is the most common genetic cause of human mental retardation (prevalence of 1/700-800 live births), consisting of a trisomy of the 21st chromosome. The overexpression of the genes located on chromosome 21 is thought to underlie the pathogenesis of the neurologic, immunologic, endocrine, and biochemical abnormalities that are characteristics of the syndrome. Children often have congenital hearts defects, gastrointestinal anomalies, immunologic disorders, thyroid dysfunction, and diabetes.1 In DS, elevated levels of CuZn superoxide dismutase, increased lipid peroxidation, and oxidative damage to DNA were observed in patients.2-3 Reduced levels of homocysteine, methionine, S-adenosylhomocysteine and S-adenosylmethionine were also found in the plasma of patients with DS.4 Glutathione plays a fundamental role in the detoxification of free radicals.5 Total glutathione (totGSH) can be free or bound to proteins; free glutathione (freeGSH) is present mainly in its reduced form, which can be converted to oxidized glutathione (GSSG) during oxidative stress and can be reverted to the reduced form by glutathione reductase. Glutathione can also be bound to proteins, leading to the formation of glutathionylated proteins. Among them, glutathionyl-hemoglobin has growing biologic relevance for its potential use as a clinical marker of oxidative stress in human blood.6,7 Glutathione is also involved as a cosubstrate for a number of important enzymes, such as glutathione peroxidase (GPx), glutathione reductase (GR), and glutathione S-transferases (GST).5 GST expression is regulated by the cellular redox status and represents a sensor able to transmit the redox variation to the apoptosis machinery by modulating the stress kinase pathway.5 Disequilibrium between the antioxidant systems may contribute to an overproduction of reactive oxygen species and to the genesis and progress of neurodegeneration in several diseases.8,9 Given the implications of glutathione in neuronal cell death, we analyzed glutathione metabolism and superoxide dismutase (SOD), GPx, GR, and GST enzyme activities in blood from 46 children with DS. Furthermore, because of the recent use of glutathione bound to hemoglobin as a biochemical marker of oxidative stress, we performed cation-exchange chromatography analysis of glutathionyl-hemoglobin (GS-Hb) in patients with DS.
D
DS freeGSH GPx GR GSSG
Down syndrome Free glutathione Glutathione peroxidase Glutathione reductase Oxidized glutathione
GST Hb HPLC SOD totGSH
Glutathione S-transferase Hemoglobin High-pressure liquid chromatography Superoxide dismutase Total glutathione
From the Laboratory of Biochemistry, Molecular Medicine Unit, and Medical Genetics Unit, Children’s Hospital and Research Institute Bambino Gesù, Rome, Italy. Submitted for publication Sept 11, 2002; revision received Feb 7, 2003; accepted Feb 21, 2003. Reprint requests: Fiorella Piemonte, PhD, Molecular Medicine Unit, Children’s Hospital and Research Institute Bambino Gesù, Piazza S. Onofrio, 4, 00165 Rome Italy. E-mail: [email protected]. Copyright © 2003 Mosby, Inc. All rights reserved. 0022-3476/2003/$30.00 + 0
10.1067/mpd.2003.203
583
Table I. Blood concentrations of various forms of glutathione in patients with DS and in controls Group
Total GSH*
Free GSH*
GSSG/GSH
GS-Hb†
Control DS P
14.7 ± 4.8 10.3 ± 4.2 < .0001
7.9 ± 2.5 5.9 ± 2.2 < .0001
0.067 ± 0.018 0.050 ± 0.013 < .0001
2.65 ± 1.1 1.47 ± 0.6 < .0001
*Data are expressed as nmol/mg Hb. †Data are expressed as % of total Hb.
Table II. Catalytic activity of antioxidant enzymes in patients with DS and in controls Group
SOD*
GPx*
SOD/GPx
GR*
GST*
Control DS P
847.5 ± 199 1133 ± 382 < .0001
45.9 ± 10.14 41.7 ± 6.90 < .05
19 ± 0.6 29 ± 1.0 < .0001
4.93 ± 1.08 5.05 ± 1.14 .67
3.92 ± 0.97 3.38 ± 1.05 .043
*Catalytic activities are expressed as U/g Hb.
Glutathione metabolism was studied in the blood of 46 children with DS (26 females and 20 males; mean age 6.7 ± 2.7 years) and in 64 healthy subjects (30 males, 34 females; mean age 5.1 ± 2.3 years). The reference sample group was established on the basis of a randomly selected group of healthy children who attended the outpatient clinic of our hospital for routine check-up. Informed consent was obtained from all subjects included in this study. The diagnosis of DS was made on the basis of the phenotype and confirmed cytogenetically. The study was performed according to the recommendations of the Ethics Committee of Children’s Hospital and Research Institute Bambino Gesù.
column (150 4.6 mm, 3-µm particle size). For glutathionylhemoglobin (Hb) analysis, the HPLC system was a ÅKTApurifier (Amersham Pharmacia Biotech, Uppsala, Sweden) 10XT (λ = 415 nm) with a 250 4.6 mm TSK-GEL CM2SW (Tosohaas Biosep GmbH, Stuttgart, Germany), column, equilibrated with 30 mmol/L Tris-buffer and 1.5 mmol/L sodium cyanide, pH 6.4 (Developer A). Hemoglobin forms were eluted from the column in 30 minutes with a gradient of 30 mmol/L Tris-buffer, 150 mmol/L sodium acetate and 1.5 mmol/L sodium cyanide, pH 6.4 (Developer B; 0-30 minutes, 30-80% B; 30-31 minutes, 80-100% B; 32-37 minutes, 100%B), at a flow rate of 0.9 mL/min. The glutathionylhemoglobin eluted as a distinct peak at 21.53 minutes and was calculated as the percentage of its peak height ratio to that of total Hb.
Glutathione Determinations
Antioxidant Enzyme Determinations
For free glutathione determination, 100 µL of fresh whole blood was immediately hemolyzed by adding 900 µL of ice-cold distilled water, and 100 µL of hemolysate was deproteinized by adding 200 µL of sulfosalicylic acid (12% by volume). The derivatization and chromatography procedures were performed as previously reported.10 For total glutathione determinations, 10 µL of hemolyzed sample was reduced in the presence of 0.19 mol/L NaBH4, deproteinized with 10% (w/v) sulfosalycilic acid, derivatized with 0.8 mmol/L monobromobimane (1:1 acetonitrile/H2O by volume) and subjected to reversed-phase chromatography. For GS-Hb determination, 100 µL of erythrocytes were hemolyzed by adding 300 µL of cold distilled water and stored at –80°C until chromatography was performed.11
SOD (E.C.1.15.1.1), GPx (E.C.1.11.1.9), and GR (E.C. 1.6.4.2) activities were spectrophotometrically assayed in erythrocytes with “Ransod,” “Ransel,” and “Glutathione Reductase Assay” kits, respectively, using a DU-640 spectrophotometer (Beckman Instruments Inc, Palo Alto, Calif ). GST (E.C. 2.5.1.18) activity was determined using 1-chloro2,4-dinitrobenzene as cosubstrate, as previously described.12
Chromatography
Hemolysates of patients with Down syndrome showed a 30% reduction of total glutathione and a 25% decrease of free glutathione, with respect to controls. When we analyzed the hemolysates by cation exchange HPLC, we obtained a 44% decrease of GS-Hb level in DS patients, compared with healthy subjects (Table I).
MATERIALS AND METHODS Patients
For glutathione determination, the high-pressure liquid chromatography (HPLC) system was an Agilent Technologies 1100 (Agilent Technologies, Deutschland GmbH, Valdbronn, Germany) equipped with fluorescence detector G1321A (λexc=390 nm, λem=478 nm) and a Hypersil ODS 584 Pastore et al
Statistical Analysis Data are expressed as mean ± SD. Two-tailed nonparametric Mann-Whitney U test was used for comparison between groups. A value of P < .05 was considered statistically significant.
RESULTS
The Journal of Pediatrics • May 2003
We calculated SOD/GPx activity ratio in erythrocytes of patients with DS and evaluated if the decrease of blood glutathione concentrations in patients could interfere with the catalytic activities of two important glutathione-dependent antioxidant enzymes: glutathione transferase and glutathione reductase. We consistently found a 53% increase of SOD/GPx activity ratio in erythrocytes of children with DS, because of a 34% increase of SOD activity, which does not correspond to a comparable rise of GPx activity in patients. Furthermore, the catalytic activity of glutathione S-transferase was 13.8% decreased in children with DS, whereas glutathione reductase activity did not show any significant difference between patients and controls (Table II).
DISCUSSION Increased generation of reactive oxygen species has been reported in Down syndrome neurons in vitro, but the cause of the neurodegenerative process is still unknown.9 The degeneration of DS neurons is prevented, in vitro, by treatment, with free-radical scavengers and chemical analyses of urine and blood samples have reported elevated levels of two oxidative stress biomarkers (8-hydroxy-2-deoxyguanosine and malondialdehyde).2 In conditions of increased oxidative stress, GSH status is a critical factor in determining the loss of mitochondrial function and cell viability.13 Glutathione, oxidative stress and neurodegeneration are strictly related and brain glutathione deficiency has been demonstrated in some neurologic disorders.13,14 A decrease of GSH concentrations was found in the substantia nigra of patients in preclinical stages of Parkinson’s disease, low GSH levels have also been found in lymphoblasts from patients with familial Alzheimer’s disease, and we recently found a reduction of free GSH concentration in Friedreich’s ataxia.15-17 Even in Down syndrome, a decrease of GSH level, under a specific threshold, could contribute to cell loss and neurodegeneration. Lower GSH levels in a mouse model of Down syndrome (trisomy 16 mice) have been reported to cause spontaneous and mitochondria-mediated cell death.18 In this study, we analyzed the blood profile of total, free, and Hb-bound glutathione in children with DS and we consistently found a decrease of all glutathione parameters including the fraction of glutathione bound to Hb. A reduced synthesis could be responsible for GSH decrease in children with DS, but the increase of plasma cysteine, reported by Pogribna et al,4 seems to keep out this possibility; an increased consumption could also be involved, although we did not find any increase of the glutathione-related enzyme (glutathione S-transferase and glutathione reductase) activities. However many others enzymes implicated in the GSH-utilizing pathways should be studied. Furthermore, SOD and GPx activities showed a disequilibrium in the blood of DS children, with a significant increase of SOD/GPx ratio. This disequilibrium, together to the impairment of glutathione metabolism, may contribute to some of the pathologic features occurring in the Down syndrome phenotype.
Glutathione Metabolism and Antioxidant Enzymes in Children With Down Syndrome
In conclusion, our findings show evidence of a significant decrease in vivo of all the glutathione parameters and an increase of SOD/GPx activity ratio in blood of DS children. However, when we measured the catalytic activities of GST, whose expression is under redox control, and GR, which is modulated by GSSG, we did not find any significant increase with respect to controls. Moreover, GS-Hb, which represents a biochemical marker of oxidative stress in blood, appears decreased in children with DS. Thus, our observations seem to exclude the presence of a systemic oxidative stress in children with DS, although we observed an imbalance of glutathione homeostasis in blood. Further studies are necessary to establish the possibility of an antioxidant imbalance in other tissues.
REFERENCES 1. Jannson U, Johansson C. Down syndrome and celiac disease. J Pediatr Gastroenterol Nutr 1995;21:443-5. 2. Jovanovic SV, Clements D, MacLeod K. Biomarkers of oxidative stress are significantly elevated in Down syndrome. Free Rad Biol Med 1998;25:1044-8. 3. Pastor MC, Sierra C, Doladé M, Navarro E, Brandi N, Cabré E, et al. Antioxidant enzyme and fatty acid status in erythrocytes of Down syndrome patients. Clin Chem 1998;44:924-9. 4. Pogribna M, Melnyk S, Pogribny I, Chango A, Yi P, James SJ. Homocysteine metabolism in children with Down syndrome: in vitro modulation. Am J Hum Genet 2001;69:88-95. 5. Hayes JD, McLellan LI. Glutathione and glutathione-dependent enzymes represent a coordinately regulated defense against oxidative stress. Free Rad Res 1999;31:273-300. 6. Niwa T, Naito C, Mawjood AHM, Imai K. Increased glutathionyl hemoglobin in diabetes mellitus and hyperlipidemia demonstrated by liquid chromatography/electrospray ionization-mass spectrometry. Clin Chem 2000;46:82-8. 7. Bursell SE, King GL. The potential use of glutathionyl hemoglobin as a clinical marker of oxidative stress. Clin Chem 2000;46:145-6. 8. Sun AY, Chen YM. Oxidative stress and neurodegenerative disorders. J Biomed Sci 1998;5:401-14. 9. Busciglio J, Yankner BA. Apoptosis and increased generation of reactive oxygen species in Down syndrome neurons in vitro. Nature 1995;378:776-9. 10. Pastore A, Piemonte F, Locatelli M, Lo Russo A, Gaeta LM, Tozzi G, et al. Determination of blood total, reduced, and oxidized glutathione in pediatric subjects. Clin Chem 2001;47:1467-9. 11. Pastore A, Mozzi AF, Tozzi G, Gaeta LM, Federici G, Bertini E, et al. Determination of glutathionyl-hemoglobin in human erythrocytes by cationexchange high-performance liquid chromatography. Anal Biochem 2003;312:85-90. 12. Habig WH, Jacoby WB. Assays for differentiation of glutathione transferases. Methods Enzymol 1981;77:398-405. 13. Schulz JB, Lindenau J, Seyfried J, Dichgans J. Glutathione, oxidative stress, and neurodegeneration. Eur J Biochem 2000;267:4904-11. 14. Dringen R. Metabolism and functions of glutathione in brain. Prog Neurobiol 2000;62:649-71. 15. Pearce RK, Owen A, Daniel S, Jenner P, Marsden CD. Alterations in the distribution of glutathione in the substantia nigra in Parkinson’s disease. J Neural Transm 1997;104:661-77. 16. Latorraca S, Cecchi C, Lantomasi T, Vincenzini MT, Liguri G, Sorbi S. Glutathione levels in lymphoblasts from patients with familial Alzheimer’s disease linked to presenilin genes [abstract]. Neurology 1998;50:314. 17. Piemonte F, Pastore A, Tozzi G, Tagliacozzi D, Santorelli FM, Carrozzo R, et al. Glutathione in blood of patients with Friedreich’s ataxia. Eur J Clin Invest 2001;31:1007-11. 18. Schuchmann S, Heinemann U. Diminished glutathione levels cause spontaneous and mitochondria-mediated cell death in neurons from trisomy 16 mice: a model of Down syndrome. J Neurochem 2000;74:1205-14.
585
Oxidative stress has been proposed as a pathogenic mechanism of atherosclerosis, cell aging, and neurologic disorders in Down syndrome. This study demonstrates a systemic decrease of all glutathione forms, including glutathionyl-hemoglobin, in the blood of children with Down syndrome. Furthermore, we obtained a disequilibrium, in vivo, between the antioxidant enzyme activities. (J Pediatr 2003;142:583-5)
own syndrome (DS) is the most common genetic cause of human mental retardation (prevalence of 1/700-800 live births), consisting of a trisomy of the 21st chromosome. The overexpression of the genes located on chromosome 21 is thought to underlie the pathogenesis of the neurologic, immunologic, endocrine, and biochemical abnormalities that are characteristics of the syndrome. Children often have congenital hearts defects, gastrointestinal anomalies, immunologic disorders, thyroid dysfunction, and diabetes.1 In DS, elevated levels of CuZn superoxide dismutase, increased lipid peroxidation, and oxidative damage to DNA were observed in patients.2-3 Reduced levels of homocysteine, methionine, S-adenosylhomocysteine and S-adenosylmethionine were also found in the plasma of patients with DS.4 Glutathione plays a fundamental role in the detoxification of free radicals.5 Total glutathione (totGSH) can be free or bound to proteins; free glutathione (freeGSH) is present mainly in its reduced form, which can be converted to oxidized glutathione (GSSG) during oxidative stress and can be reverted to the reduced form by glutathione reductase. Glutathione can also be bound to proteins, leading to the formation of glutathionylated proteins. Among them, glutathionyl-hemoglobin has growing biologic relevance for its potential use as a clinical marker of oxidative stress in human blood.6,7 Glutathione is also involved as a cosubstrate for a number of important enzymes, such as glutathione peroxidase (GPx), glutathione reductase (GR), and glutathione S-transferases (GST).5 GST expression is regulated by the cellular redox status and represents a sensor able to transmit the redox variation to the apoptosis machinery by modulating the stress kinase pathway.5 Disequilibrium between the antioxidant systems may contribute to an overproduction of reactive oxygen species and to the genesis and progress of neurodegeneration in several diseases.8,9 Given the implications of glutathione in neuronal cell death, we analyzed glutathione metabolism and superoxide dismutase (SOD), GPx, GR, and GST enzyme activities in blood from 46 children with DS. Furthermore, because of the recent use of glutathione bound to hemoglobin as a biochemical marker of oxidative stress, we performed cation-exchange chromatography analysis of glutathionyl-hemoglobin (GS-Hb) in patients with DS.
D
DS freeGSH GPx GR GSSG
Down syndrome Free glutathione Glutathione peroxidase Glutathione reductase Oxidized glutathione
GST Hb HPLC SOD totGSH
Glutathione S-transferase Hemoglobin High-pressure liquid chromatography Superoxide dismutase Total glutathione
From the Laboratory of Biochemistry, Molecular Medicine Unit, and Medical Genetics Unit, Children’s Hospital and Research Institute Bambino Gesù, Rome, Italy. Submitted for publication Sept 11, 2002; revision received Feb 7, 2003; accepted Feb 21, 2003. Reprint requests: Fiorella Piemonte, PhD, Molecular Medicine Unit, Children’s Hospital and Research Institute Bambino Gesù, Piazza S. Onofrio, 4, 00165 Rome Italy. E-mail: [email protected]. Copyright © 2003 Mosby, Inc. All rights reserved. 0022-3476/2003/$30.00 + 0
10.1067/mpd.2003.203
583
Table I. Blood concentrations of various forms of glutathione in patients with DS and in controls Group
Total GSH*
Free GSH*
GSSG/GSH
GS-Hb†
Control DS P
14.7 ± 4.8 10.3 ± 4.2 < .0001
7.9 ± 2.5 5.9 ± 2.2 < .0001
0.067 ± 0.018 0.050 ± 0.013 < .0001
2.65 ± 1.1 1.47 ± 0.6 < .0001
*Data are expressed as nmol/mg Hb. †Data are expressed as % of total Hb.
Table II. Catalytic activity of antioxidant enzymes in patients with DS and in controls Group
SOD*
GPx*
SOD/GPx
GR*
GST*
Control DS P
847.5 ± 199 1133 ± 382 < .0001
45.9 ± 10.14 41.7 ± 6.90 < .05
19 ± 0.6 29 ± 1.0 < .0001
4.93 ± 1.08 5.05 ± 1.14 .67
3.92 ± 0.97 3.38 ± 1.05 .043
*Catalytic activities are expressed as U/g Hb.
Glutathione metabolism was studied in the blood of 46 children with DS (26 females and 20 males; mean age 6.7 ± 2.7 years) and in 64 healthy subjects (30 males, 34 females; mean age 5.1 ± 2.3 years). The reference sample group was established on the basis of a randomly selected group of healthy children who attended the outpatient clinic of our hospital for routine check-up. Informed consent was obtained from all subjects included in this study. The diagnosis of DS was made on the basis of the phenotype and confirmed cytogenetically. The study was performed according to the recommendations of the Ethics Committee of Children’s Hospital and Research Institute Bambino Gesù.
column (150 4.6 mm, 3-µm particle size). For glutathionylhemoglobin (Hb) analysis, the HPLC system was a ÅKTApurifier (Amersham Pharmacia Biotech, Uppsala, Sweden) 10XT (λ = 415 nm) with a 250 4.6 mm TSK-GEL CM2SW (Tosohaas Biosep GmbH, Stuttgart, Germany), column, equilibrated with 30 mmol/L Tris-buffer and 1.5 mmol/L sodium cyanide, pH 6.4 (Developer A). Hemoglobin forms were eluted from the column in 30 minutes with a gradient of 30 mmol/L Tris-buffer, 150 mmol/L sodium acetate and 1.5 mmol/L sodium cyanide, pH 6.4 (Developer B; 0-30 minutes, 30-80% B; 30-31 minutes, 80-100% B; 32-37 minutes, 100%B), at a flow rate of 0.9 mL/min. The glutathionylhemoglobin eluted as a distinct peak at 21.53 minutes and was calculated as the percentage of its peak height ratio to that of total Hb.
Glutathione Determinations
Antioxidant Enzyme Determinations
For free glutathione determination, 100 µL of fresh whole blood was immediately hemolyzed by adding 900 µL of ice-cold distilled water, and 100 µL of hemolysate was deproteinized by adding 200 µL of sulfosalicylic acid (12% by volume). The derivatization and chromatography procedures were performed as previously reported.10 For total glutathione determinations, 10 µL of hemolyzed sample was reduced in the presence of 0.19 mol/L NaBH4, deproteinized with 10% (w/v) sulfosalycilic acid, derivatized with 0.8 mmol/L monobromobimane (1:1 acetonitrile/H2O by volume) and subjected to reversed-phase chromatography. For GS-Hb determination, 100 µL of erythrocytes were hemolyzed by adding 300 µL of cold distilled water and stored at –80°C until chromatography was performed.11
SOD (E.C.1.15.1.1), GPx (E.C.1.11.1.9), and GR (E.C. 1.6.4.2) activities were spectrophotometrically assayed in erythrocytes with “Ransod,” “Ransel,” and “Glutathione Reductase Assay” kits, respectively, using a DU-640 spectrophotometer (Beckman Instruments Inc, Palo Alto, Calif ). GST (E.C. 2.5.1.18) activity was determined using 1-chloro2,4-dinitrobenzene as cosubstrate, as previously described.12
Chromatography
Hemolysates of patients with Down syndrome showed a 30% reduction of total glutathione and a 25% decrease of free glutathione, with respect to controls. When we analyzed the hemolysates by cation exchange HPLC, we obtained a 44% decrease of GS-Hb level in DS patients, compared with healthy subjects (Table I).
MATERIALS AND METHODS Patients
For glutathione determination, the high-pressure liquid chromatography (HPLC) system was an Agilent Technologies 1100 (Agilent Technologies, Deutschland GmbH, Valdbronn, Germany) equipped with fluorescence detector G1321A (λexc=390 nm, λem=478 nm) and a Hypersil ODS 584 Pastore et al
Statistical Analysis Data are expressed as mean ± SD. Two-tailed nonparametric Mann-Whitney U test was used for comparison between groups. A value of P < .05 was considered statistically significant.
RESULTS
The Journal of Pediatrics • May 2003
We calculated SOD/GPx activity ratio in erythrocytes of patients with DS and evaluated if the decrease of blood glutathione concentrations in patients could interfere with the catalytic activities of two important glutathione-dependent antioxidant enzymes: glutathione transferase and glutathione reductase. We consistently found a 53% increase of SOD/GPx activity ratio in erythrocytes of children with DS, because of a 34% increase of SOD activity, which does not correspond to a comparable rise of GPx activity in patients. Furthermore, the catalytic activity of glutathione S-transferase was 13.8% decreased in children with DS, whereas glutathione reductase activity did not show any significant difference between patients and controls (Table II).
DISCUSSION Increased generation of reactive oxygen species has been reported in Down syndrome neurons in vitro, but the cause of the neurodegenerative process is still unknown.9 The degeneration of DS neurons is prevented, in vitro, by treatment, with free-radical scavengers and chemical analyses of urine and blood samples have reported elevated levels of two oxidative stress biomarkers (8-hydroxy-2-deoxyguanosine and malondialdehyde).2 In conditions of increased oxidative stress, GSH status is a critical factor in determining the loss of mitochondrial function and cell viability.13 Glutathione, oxidative stress and neurodegeneration are strictly related and brain glutathione deficiency has been demonstrated in some neurologic disorders.13,14 A decrease of GSH concentrations was found in the substantia nigra of patients in preclinical stages of Parkinson’s disease, low GSH levels have also been found in lymphoblasts from patients with familial Alzheimer’s disease, and we recently found a reduction of free GSH concentration in Friedreich’s ataxia.15-17 Even in Down syndrome, a decrease of GSH level, under a specific threshold, could contribute to cell loss and neurodegeneration. Lower GSH levels in a mouse model of Down syndrome (trisomy 16 mice) have been reported to cause spontaneous and mitochondria-mediated cell death.18 In this study, we analyzed the blood profile of total, free, and Hb-bound glutathione in children with DS and we consistently found a decrease of all glutathione parameters including the fraction of glutathione bound to Hb. A reduced synthesis could be responsible for GSH decrease in children with DS, but the increase of plasma cysteine, reported by Pogribna et al,4 seems to keep out this possibility; an increased consumption could also be involved, although we did not find any increase of the glutathione-related enzyme (glutathione S-transferase and glutathione reductase) activities. However many others enzymes implicated in the GSH-utilizing pathways should be studied. Furthermore, SOD and GPx activities showed a disequilibrium in the blood of DS children, with a significant increase of SOD/GPx ratio. This disequilibrium, together to the impairment of glutathione metabolism, may contribute to some of the pathologic features occurring in the Down syndrome phenotype.
Glutathione Metabolism and Antioxidant Enzymes in Children With Down Syndrome
In conclusion, our findings show evidence of a significant decrease in vivo of all the glutathione parameters and an increase of SOD/GPx activity ratio in blood of DS children. However, when we measured the catalytic activities of GST, whose expression is under redox control, and GR, which is modulated by GSSG, we did not find any significant increase with respect to controls. Moreover, GS-Hb, which represents a biochemical marker of oxidative stress in blood, appears decreased in children with DS. Thus, our observations seem to exclude the presence of a systemic oxidative stress in children with DS, although we observed an imbalance of glutathione homeostasis in blood. Further studies are necessary to establish the possibility of an antioxidant imbalance in other tissues.
REFERENCES 1. Jannson U, Johansson C. Down syndrome and celiac disease. J Pediatr Gastroenterol Nutr 1995;21:443-5. 2. Jovanovic SV, Clements D, MacLeod K. Biomarkers of oxidative stress are significantly elevated in Down syndrome. Free Rad Biol Med 1998;25:1044-8. 3. Pastor MC, Sierra C, Doladé M, Navarro E, Brandi N, Cabré E, et al. Antioxidant enzyme and fatty acid status in erythrocytes of Down syndrome patients. Clin Chem 1998;44:924-9. 4. Pogribna M, Melnyk S, Pogribny I, Chango A, Yi P, James SJ. Homocysteine metabolism in children with Down syndrome: in vitro modulation. Am J Hum Genet 2001;69:88-95. 5. Hayes JD, McLellan LI. Glutathione and glutathione-dependent enzymes represent a coordinately regulated defense against oxidative stress. Free Rad Res 1999;31:273-300. 6. Niwa T, Naito C, Mawjood AHM, Imai K. Increased glutathionyl hemoglobin in diabetes mellitus and hyperlipidemia demonstrated by liquid chromatography/electrospray ionization-mass spectrometry. Clin Chem 2000;46:82-8. 7. Bursell SE, King GL. The potential use of glutathionyl hemoglobin as a clinical marker of oxidative stress. Clin Chem 2000;46:145-6. 8. Sun AY, Chen YM. Oxidative stress and neurodegenerative disorders. J Biomed Sci 1998;5:401-14. 9. Busciglio J, Yankner BA. Apoptosis and increased generation of reactive oxygen species in Down syndrome neurons in vitro. Nature 1995;378:776-9. 10. Pastore A, Piemonte F, Locatelli M, Lo Russo A, Gaeta LM, Tozzi G, et al. Determination of blood total, reduced, and oxidized glutathione in pediatric subjects. Clin Chem 2001;47:1467-9. 11. Pastore A, Mozzi AF, Tozzi G, Gaeta LM, Federici G, Bertini E, et al. Determination of glutathionyl-hemoglobin in human erythrocytes by cationexchange high-performance liquid chromatography. Anal Biochem 2003;312:85-90. 12. Habig WH, Jacoby WB. Assays for differentiation of glutathione transferases. Methods Enzymol 1981;77:398-405. 13. Schulz JB, Lindenau J, Seyfried J, Dichgans J. Glutathione, oxidative stress, and neurodegeneration. Eur J Biochem 2000;267:4904-11. 14. Dringen R. Metabolism and functions of glutathione in brain. Prog Neurobiol 2000;62:649-71. 15. Pearce RK, Owen A, Daniel S, Jenner P, Marsden CD. Alterations in the distribution of glutathione in the substantia nigra in Parkinson’s disease. J Neural Transm 1997;104:661-77. 16. Latorraca S, Cecchi C, Lantomasi T, Vincenzini MT, Liguri G, Sorbi S. Glutathione levels in lymphoblasts from patients with familial Alzheimer’s disease linked to presenilin genes [abstract]. Neurology 1998;50:314. 17. Piemonte F, Pastore A, Tozzi G, Tagliacozzi D, Santorelli FM, Carrozzo R, et al. Glutathione in blood of patients with Friedreich’s ataxia. Eur J Clin Invest 2001;31:1007-11. 18. Schuchmann S, Heinemann U. Diminished glutathione levels cause spontaneous and mitochondria-mediated cell death in neurons from trisomy 16 mice: a model of Down syndrome. J Neurochem 2000;74:1205-14.
585