NOTE: Since this study in 2001 better co factors for Glutathione Peroxidase and reduced Glutathione (in the form of Setria Glutathione) have been added to the formula to increase GHPX and GHX. At the time this study was done the supplements for Glutathione available were large molecules that was very difficult to absorb. SOD1 could not be inhibited at the time of this study and that remains the case today. But modern formulas contain Superoxide scavengers that prevent damage by excess hydrogen peroxide providing an even greater improvement in the antioxidant enzyme triad.
The results of this study proved that utilizing natural substances to upregulate CAT and GPHX while avoiding the upregulation of SOD1 significantly restored the antioxidant enzyme triad in Down syndrome. The elevation of CAT and GHPX enables the antioxidant to function far more normally by elevating them to equal, as much as possible, the levels of SOD1. See Epigenics and Targeted Nutrional Intervention under Research on this site.
THE MIGUID STUDY
Available online www.jocpr.com Journal of Chemical and Pharmaceutical Research, 2015, 7(2):324-331 Research Article ISSN : 0975-7384 CODEN(USA) : JCPRC5 Antioxidant activity in Egyptian children with Down syndrome before and after nutritional supplementation 1* 2 2 2 Nagwa A. Meguid , Eman R. Zaki , Yasser E. Shahein , Ragaa R. Hamed , Rasha A. 213 Guneidy , Mona Anwar and Ola S. Mohamed 1 Department of Research on Children with Special Needs, National Research Centre, Giza, Egypt 2 Department of Molecular Biology, National Research Centre, Giza, Egypt 3 Department of Biochemistry, Faculty of Pharmacy, Al-Azhar University, Cairo, Egypt _____________________________________________________________________________________________ ABSTRACT Down syndrome (DS) is a chromosome abnormality with specific clinical symptoms and mental deficiencies caused by trisomy of chromosome 21. Although the genetic changes cannot be cured, control of the associated symptoms may improve the patients’ quality of life. Evidences indicate oxidative stress may play a role in some of the degenerative changes seen in DS. The present study aimed to evaluate red blood cell antioxidant capacity and redox cycle enzymes activities before and after nutritional supplementation, in an attempt to protect DS children from premature degeneration. The study included 21 children with DS, their age ranged from 1 month to 5 years received mixture of nutritional supplements (formula X) for six months and 20healthy matched children served as controls received placebo. Reduced glutathione (GSH), in addition to antioxidant enzymes activities[superoxide dismutase (SOD), glutathione peroxidase (GPx), catalase (CAT), glutathione reductase (GR), and the glutathione-S- transferase (GST)] were assessed to all participants. Before nutritional supplementation, GSH and GST levels were significantly low, while SOD levels in DS children were significantly high compared to controls. After 6 months’ supplementation, significant high levels were observed in the activity of GPx and CAT with elevated GPx/GR ratio and reduced SOD/(CAT + GPx) ratio among DS children. Supplementation of DS children with formula X is important at early years of life, as it may protect against harmful oxidative damage. Keywords: Down syndrome, Antioxidant enzymes, Egyptian, children, Nutritional supplementation. _____________________________________________________________________________________________ INTRODUCTION Down syndrome (DS) is the most common genetic cause of human mental retardation with a frequency of approximately 1 in 600 to 1,000 live births in Egypt.The disease is associated with mental retardation, immune system disorders, and autoimmune processes. It is further characterized by increased incidence of heart defects, gastrointestinal anomalies, and malignancies. Typical manifestations of DS are premature aging and Alzheimer disease as early as ages 30-40 years. There is a primary defense system against oxidative stress, mediated by sequential enzymatic reactions. In the first – step of the process, CuZn-superoxide dismutase (SOD1) catalyzes the dismutation of O2 to H2O2. Glutathione peroxidase (GPx) and catalase (CAT) then independently convert H2O2 to water . Any increase in SOD1catalytic activity, therefore, produces an excess of H2O2 that must be efficiently neutralized by either GPx or CAT.
Otherwise, H2O2 reacts with O2, producing OH , which is one of the most active reactive oxygen species. Thus the activity of the first-step (SOD1) and second-step (GPx, CAT) antioxidant enzymes must be balanced to prevent cell damage. Genetic over-expression of anti-oxidant enzymes leads to over-consumption of their enzymatic substrates and over-production of their metabolic end-products. In DS individuals, an elevated level of SOD is a leading factor to increased lipid peroxidation and oxidative damage to DNA . Several evidences suggested that oxidative stress may be involved in the premature neuronal degeneration, the cerebral cortex from fetuses with DS was found to have increased SOD activity. Also, cortical neurons from fetuses with DS have an increased concentration of intracellular oxygen-derived free radicals and increased lipid peroxidation  and in vitro fetal neurons in DS show increased apoptotic degeneration. Glutathione (GSH) plays a fundamental role in numerous metabolic and biochemical reactions such as DNA synthesis and repair, protein synthesis, prostaglandin synthesis, amino acid transport and enzymes activation. Thus, every system in the body can be affected by the state of the glutathione system, especially the immune system, the gastrointestinal system and the nervous system.GSH is also considered as an antioxidant as it has an important role in detoxification of free radicals. GSH appears to be decreased in the blood of DS children[3,4]. Glutathione S-transferase (GST) is a family of isoenzymes serving a major role in the biotransformation of many reactive compounds; catalyze the conjugation of GSH with a wide variety of organic peroxides to form more water- soluble compounds for more efficient elimination of toxic radicals . The aim of this study was to evaluate of the effect of mixture of nutritional supplements on the activity of the antioxidant enzymes in DS children which may protect against harmful systemic reactive oxygen species. This may be of great importance in the management of this disorder. EXPERIMENTAL SECTION Participants The present study included 21 patients with DS (males and females) aged 1 month to 5 years. All cases had trisomy 21 karyotype. All patients were selected from the attendants of the Children with Special Needs clinic, National Research Centre, Giza, Egypt. A comparable 20 healthy children were selected and enrolled in the study as control group. DS children were divided into two age categories: Group 1 included 10 cases less than one year and group 2 included 11 cases from 1-5 years. Ethics approval and consent: A written consent was obtained from the parents of each participant, according to the guidelines of the ethical committee of the National Research Centre.
The supplement studied was Formula X consisted of three parts, a daily supplement containing (vitamins, minerals, amino acids and related substances) (Table 1), an enzyme supplement (Table 2), and a night-time supplement (Table 3). Nutrients shown were based on 8.2 gm dose (Table 4). Formula X supplement was from International Nutrition, Inc., Baltimore, MD, U.S. DS children received the formula daily according to body weight for six months, while the control children received placebo.
Sample Collection and Analytical Procedures Venous blood samples were collected in EDTA tubes (1 mg EDTA/1 mL blood) and blood was centrifuged immediately after withdrawn. The erythrocytes were washed three times with a saline solution (9g/l) and hemolyzed by the addition of an equal volume of ice-cold distilled water to yield a 50-percent hemolysate. The erythrocyte lysate was divided into aliquots of 500 μL each and then frozen at -20oC. Hemoglobin was determined in the whole blood samples using a kit obtained from Sigma Chemicals Co. GSH were measured in hemolysate using the Saville method;buffers were prepared according to Gomori . GST activity was assayed with the aromatic substrate 1- chloro-2,4-dinitrobenzene (CDNB) and co-substrate GSH as described by Habdous et al.. GR activity was assayed and expressed in terms of micromoles of NADPH oxidized/g Hb/minute as described by Zanetti. GPx activity was assayed as described by Paglia and Valentine, using GSH as a substrate.CAT was assayed at 37°C with H2O2 as a substrate according to Aebi. SOD activity was assayed by using the RANSEL kit. All spectrophotometeric measurements were performed in a Shimadzu UV-2401 PC spectrophotometer.
Table 1: Daily Supplement Formula
Units Vitamin A (beta-carotene) 3000 IU Vitamin A (palmitate) Vitamin D3 Vitamin E Biotin Folic acid Niacinamide Pantothenic acid Vitamin B1 Vitamin B12 Vitamin B2 Vitamin B6 Vitamin C Minerals Calcium (citrate) Choline bitartrate Chromium Iodine Magnesium Manganese Molybdenum Potassium 15mg Ingredients Vitamins 5000 IU 75 IU 400 IU 0.2 mg 800 mcg 125 mg 45mg 55mg 90 mcg 45mg 35mg 1000 mg 25mg 800 mg 75 mcg 7 mcg 150 mg 1.5 mg 75 mcg Selenium Zinc 10mg Acetyl-L-carnitine 45mg Amino Acids and related substances 45 mcg L-Citrulline L-Glutathione L-Histidine alpha-Ketoglutaric acid L-Methionine 50mg 70 mg 150 mg 25 mg 500 mg L-Ornithine L-Proline L-Serine L-Tryptophan 50mg 100 mg 100 mg 150 mg L-Tyrosine Betaine hydrochloride Bioflavonoids Bromelain 5mg 100 mg 60 mg 150 mg Coenzyme Q10 Inositol 75mg Paba 75mg Papain 5mg 30mg Taurine Alpha-Lipoic acid 200 mg 45mg
Table 2: Daily Enzyme Supplement Formula
Table 3: Night-time Supplement Formula Ingredients Units alpha-Amylase 25 mg Cellusase 1 mg Lactase 1 mg Lipase 25 mg Ingredients Units Vitamin B6 2.5 mg L-Ornithine 150 mg L-Tryptophan 125 mg.
Table 4: Percentage of Each Consumed Listed Nutrient (Nutrients shown are based on 8.2 gm dose) Percentage of each Listed Nutrient Up to 20 lbs (≤ 9 kgs) 17 % 21 – 40 lbs (10 – 18 kgs) 33 % 41 – 60 lbs (19 – 27 kgs) 50 % 61 – 80 lbs (28 – 36 kgs) 75 % Over 80 lbs (>37 kgs) 100 %
Statistical analysis Statistical analyses were performed using the Statistical Package for Social Sciences (SPSS/Windows Version 9.05, SPSS Inc., Chicago, IL, USA).Student’s t-test was used; data were expressed as mean ± SE, p-values less than 0.05 were considered statistically significant. The present study showed the following results before taking formula X, significant increase of SOD activity in DS patients (p<0.01), significant decrease of GST and GSH activities in DS patients (p<0.01) compared to controls. On the other hand no change in GPx, GR and CAT levels were detected. After taking formula X, for six months level of GSH, SOD, GR, and GST showed no statistical difference. However, CAT and GPx level is significantly increased (p<0.05, p<0. 01 respectively) (Table 5) (Figure1,2). Controls Down syndrome (pre) p1 Down syndrome (post) p2 1800 1600 1400 1200 1000 800 600 400 200 U/gHb
Table 5: Mean Levels of Investigated parameters in Controls and DS Children Each value represents the mean±SE; controls (n=20); Down syndrome (n=21) P1:DS versus control; P2: DS (Pre) versus DS (Post) p >0.05=nonsignificant; p<0.05=significant* Group SOD (U/gHb) CAT (KU/g Hb) GPx (U/g Hb) GR (U/g Hb) GST (U/g Hb) GSH (μmol/g Hb) Controls 705±65 391± 56.4 19±0.6 2.92±0.235 6.60±0.69 10.3±4.2 Down syndrome (pre) 1472 ±171 427±51.4 23.2 ±2.32 2.99± 0.337 2.28±0.334 7.05 ±0.652 p1 * <0.01 >0.05 >0.05 >0.05 * <0.01 * <0.01 Down syndrome (post) 1510±196 650±87 45.4±5.44 2.79± 0.342 1.96±0.25 7.59±0.532 p2 >0.05 * <0.05 * <0. 01 >0.05 >0.05 >0.05
A significant difference was observed in CAT between group 1 and group 2 in DS patients (p<0.05). On the other hand, no statistically significant differences were present neither in the activities of the other enzymes tested nor in the level of RBCs GSH (Table 6).
The calculated data of the enzymatic activity in DS groups SOD/GPX 63.4 and SOD/GPX + CAT 3.27 were decreased to 33.3 and 2.17 respectively after supplementation. These ratios almost approach the normal values (37 and 1.27 respectively) however; GPX/GR ratio is increased from 7.78 to 16.29 after supplementation (Table 7).
Supplementations may be one of the most promising treatment methods for DS patients, but no scientifically proven diet or drug is yet available. Nutritional therapies were proposed for DS and usually focusing on one or two items, mixtures that included vitamins, hormones and enzymes were advocated by many authors. MSB Plus(Nutri-Chem Labs) and NuTriVene-D, (International Nutrition, Inc.) are formulas have more than 40 ingredients, most of them the same or similar, but with differences in dosage. Various formulas from other companies have not been as popular and reviewed by Roizen. Measures of oxidative damage in DS patients during the early years of life demonstrated increased lipid and DNA oxidation biomarkers as compared with their non-DS siblings .
In the present study, the concentration of GSH was significantly low in DS groups, compared to controls. The deficiency of GSH observed in DS patients may be attributed to increased oxidation to GSSG, increased degradation or decreased synthesis of GSH. Pogribna et al. reported that plasma GSH levels were significantly low in the DS children that may reflect an increase of oxidative stress due to over expression of the superoxide dismutase gene located on chromosome 21.Also over expression of Cystathionine beta synthase in DS, located on chromosome 21 indirectly lead to reduction of methionine and regeneration of cysteine which is the rate limiting amino acid for glutathione synthesis . However, supplementation of DS patients with Formula X containing L-glutathione (150 mg) and the amino acids methionine (50 mg) and α- ketoglutaric acid (500 mg) required for GSH synthesis did not elevated the reduced GSH level in RBCs.
Our study showed high level of SOD activity in DS patients compared to controls. Similar results was observed by Pastore et al. who reported elevated SOD level 50-percent higher than normal in a variety of DS cells and tissues, including erythrocytes, B- and T-lymphocytes, and fibroblasts. No significant difference in SOD activity level was observed between the two groups of DS patients. These data are consistent with the study conducted by Muchova et al. who found that SOD activity in DS patients did not change significantly with age. In the present work, supplementation with Formula X for 6 months did not significantly affect the SOD level in DS children. (SEE NOTATION AT THE BEGINNING OF THE ARTICLE).
CAT activity level was high in DS patients compared to controls but with no significant difference, significant increase in CAT activity levels in DS patients was noticed after supplementation with Formula X. Significant high levels of CAT activity were observed in group1 DS compared to group 2; this is consistent with Casado and López-Fernández who reported high CAT activity levels in newborns and low activity levels in children between 1-9 years. McElroy et al. reported that CAT is the only antioxidant increased in activity with progressing gestational age. In our study, no significant difference was observed in GPx activity level in DS children compared to controls or between both groups of DS patients. These results are in agreement with the study conducted by Muchova and Shawkywho reported that the oxidative imbalance in trisomic cells is age-dependent and depends on the ratio of SOD to (CAT + GPx) rather than on absolute amounts of these antioxidant enzymes. An imbalance in these enzymes may have adverse effects on cell membranes via the indiscriminate oxidation of susceptible molecules such as polyunsaturated fatty acids (PUFAs). De Haan et al. and Percy et al. ,reported that this alteration in this ratio is representing an important determinant of cellular damage, because changes in this quotient correlate well with an increase in lipid damage. The erythrocytes of DS children, adolescents, and adults exhibited systemic increases in SOD, SOD/GPx or the SOD/(GPx +CAT) activity ratio. This disequilibrium may contribute to some of the pathologic features occurring in the DS phenotype . Meguidet al. reported an increase in whole- blood GPx level in DS patients (8 month-3years) with complete trisomy, which caused by abnormal cell division during development of the ovum or sperm or during fertilization and represented by 95% of all cases, and translocations (partial trisomy 21), which caused by break off the extra chromosome 21 and become attached to another chromosome representing by 3%- 4%. The balance in the SOD/GPx ratio is an important determinant of cellular aging and increased with the severity of the stress. The ratio of SOD/(GPx+CAT) in our study was decreased after supplementation with formula X for six months in DS children from 3.27 to 2.17,. In contrast to our results, Ellis et al. reported non-significant effect of only antioxidant supplementation on SOD or GPx activities or on the SOD/GPx ratio in DS infants under age seven months.
In a study by Antila et al.  applied 15-25 mcg selenium/kg/day to seven DS patients (ages 1-54 years) resulted in a 25-percent increase in GPx activity and a 24-percent reduction in the SOD/GPx ratio. Anneren et al.  reported that oral selenium supplementation to DS patients reduced the rate of infections and had immunoregulatory effects in DS subjects.
Our results showed non-significant difference in GR activity between DS patients and controls before and after nutritional supplementation. Concentration of GSH is partly regulated by GPx and GR activity. The decreased activity of GR together with the increased GPx activity could lead to a reduced GSH/GSSG ratio during the aging process and could explain age-associated decrease in blood GSH levels .In a study conducted by Erden-Inal et al. , they reported that glutathione redox system is affected in the first years of life and aging periods in healthy individuals. Also, Pastor et al found that GR catalytic activity decreased with age in DS groups, with significant differences between the young and old age groups. However, neither Muchova nor Pastore  found significant differences in GR catalytic activity in DS compared to controls, our results are in agreements with these latter findings.
No gender difference in SOD, GPx, CAT, GR, GST, or total GSH were detected in DS children, in our study supporting the results of Muchova et al. Casado and López-Fernández  and Shawky et al..On the other hand, Anneren et al reported higher GPx level in girls than in boys with DS .
Nutritional supplementation with Formula X at early ages may play an important role in protecting against oxidative damage in Ds patients. Further studies are needed with large sample size and for long duration, as it may be too optimistic to expect subtle physiological improvements to be translated into detectable physical and mental changes.
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