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Abstract

  1. Top of page
  2. Abstract
  3. Introduction
  4. Patients and methods
  5. Results
  6. Discussion
  7. Acknowledgment
  8. Conflict of interest
  9. References

The aim of this study was to establish the antioxidant status and oxidative stress in adult patients with chronic idiopathic thrombocytopenic purpura (ITP). Eighty-four patients diagnosed with chronic ITP were studied. Fifty-eight age-matched healthy subjects were selected as controls. Serum nitrogen monoxide ( NO), oxidized glutathione (GSSG), malondialdehyde (MDA), total antioxidant status (TAS), total oxidant status (TOS), superoxide dismutase(SOD), hydrogen peroxide enzyme (CAT), glutathione peroxidase (GSH-Px), glutathione (GSH) were evaluated by enzyme-linked immunosorbent assay (ELISA). It was found that serum SOD, CAT, GSH-Px, GSH, TAS levels were significantly lower in patients with chronic ITP than controls (all P < 0.05), while serum NO, GSSG, MDA, TOS values were significantly higher (P < 0.05). The number of platelet showed a negative correlation with NO, GSSG, MDA, TOS, respectively,while platelet number showed a positive correlation with SOD, CAT, GSH-Px, GSH, TAS. These findings suggested that oxidants were increased and antioxidants were decreased in patients with chronic ITP, these may be prominent factors in destructing the platelet membrane. The scavenging of oxygen radical provides a theoretical basis for the treatment of ITP patients.


Introduction

  1. Top of page
  2. Abstract
  3. Introduction
  4. Patients and methods
  5. Results
  6. Discussion
  7. Acknowledgment
  8. Conflict of interest
  9. References

Primary immune thrombocytopenia, previously referred to as idiopathic thrombocytopenic purpura (ITP) is an immune-mediated acquired disorder characterized by isolated thrombocytopenia, defined as a peripheral platelet count less than 100 × 109/l in the absence of any specific cause of the thrombocytopenia [1]. It is further classified according to its duration since diagnosis: newly diagnosed (<3 months), persistent (3–12 months) and chronic (>12 months) [2]. Oxidative stress is often defined as an imbalance of pro-oxidants and antioxidants, which can be quantified in humans with the redox state of serum GSH/GSSG. Serum GSH redox in humans becomes oxidized with age, in response to oxidative stress (chemotherapy, smoking) and in common diseases (diabetes mellitus type 2, cardiovascular diseases) [3, 4]. Oxidative stress is caused by an imbalance between the production of reactive oxygen and a biological system's inability to readily detoxify the reactive intermediates or easily repair the resulting damage. All forms of life maintain a reducing environment within their cells. This reducing environment is preserved by enzymes that maintain the reduced state through a constant input of metabolic energy [5]. Disturbances in this normal redox state can cause toxic effects through the production of peroxides and free radicals that damage all components of the cell, including proteins, lipids and DNA [6]. In humans, oxidative stress is involved in many diseases, such as atherosclerosis, Parkinson's disease, heart failure, myocardial infarction, Alzheimer's disease, fragile X syndrome and chronic fatigue syndrome (CFS), but short-term oxidative stress may also be important in prevention of ageing by induction of a process called mitohormesis [7]. ITP in adults is associated with infection of hepatitis C virus, HIV and other viruses, and Helicobacter pylori [8, 9], although the mechanism is not clear. It is still unknown how platelets are targeted by the host's immune system. Infection-related oxidative stress may induce disturbed immune response, and ongoing oxygen stress may be a significant factor in patients with chronic ITP in adult. In this study, serum SOD, MDA, TAC, TOS and other oxidant/antioxidant stress parameters were studied in patients with chronic ITP. Our purpose is to determine oxidant and antioxidant status in patients with chronic ITP in comparing their presence in healthy subjects and to detect the relationship between these parameters and platelet count.

Patients and methods

  1. Top of page
  2. Abstract
  3. Introduction
  4. Patients and methods
  5. Results
  6. Discussion
  7. Acknowledgment
  8. Conflict of interest
  9. References

This study, conducted from October 2011 to October 2012, was approved by the Ethics Committee of the Attached Hospital of Jining Medical College, and informed consent was obtained from each subject prior to the start of our study. Eighty-four patients with a diagnosis of chronic ITP were introduced from Department of Haematology of the Attached Hospital of Jining Medical College. Chronic ITP patients were enrolled with the criteria of persistent thrombocytopenia (<100 × 109/l) for at least 12 months and the absence of any other disease that may cause thrombocytopenia [1, 2]. None of these patients were receiving therapeutic immunomodulatory intervention such as intravenous human immunoglobulin administration, which targets the whole immune response, monoclonal anti-CD20 antibodies, Rituximab (Rituxan), cyclosporine and none received splenectomy prior to the start of our study. Fifty-eight age-matched healthy subjects were selected as controls. General Information of chronic ITP patients and healthy subjects were presented (See Table 1).

Table 1. General comparison between chronic idiopathic thrombocytopenic purpura (ITP) group and the normal control group
VariablesITP patients(n = 84)Healthy subjects (n = 58)P-value
  1. The above measurements are routine examination before the experiment, the number of platelets < 0.05; BMI, Body Mass Index; RBC, red blood cell; WBC, white blood cell.

Age (years)36.3 ± 10.537.0 ± 10.80.23
Men [patients number (%)]31 (36.9)18 (45.0)0.36
Women [patients number (%)]53 (63.1)24 (55.0)0.44
BMI (Kg/m2)21.0 ± 2.924.3 ± 2.40.28
RBC (×1012/l)4.3 ± 1.24.2 ± 1.00.67
Hb(g/l)123 ± 19124 ± 230.39
Platelet (×109/l)52.5 ± 40.1195.0 ± 32.20.003
WBC (×109/l)7.6 ± 3.57.8 ± 2.70.57
Heart rate (beats/min)82 ± 1683 ± 120.30
Body temperature(℃c)37.6 ± 0.937.3 ± 0.90.89
Respiratory rate (beats/min)18 ± 317 ± 40.63

Measurement of NO, GSSG, MDA, TOS, TAS, SOD, GSH, CAT, GSH-Px, GSH

An in vitro enzyme-linked immunosorbent assay kit (ELISA; Sigma-Aldrich) for quantitatively detecting human GSH in serum was used to detect the concentrations of NO, GSSG, MDA, TOS, TAS, SOD, CAT, GSH-Px. The Stop Solution from GSH ELISA kit changes the colour from blue to yellow, and the light absorption was measured at 450 nm using a spectrophotometer. To measure the concentration of GSH in the samples, this GSH ELISA Kit includes a set of calibration standards, which were assayed in parallel, and a standard curve of optical density versus GSH concentration was generated after the measurement. The concentration of GSH in the samples was then calculated by the equation deduced from the standard curve. The detailed assay procedures are as follows:

  1. Serum - used a serum separator tube and allowed samples to clot for 30 min before pelleting the blood samples by centrifugation for 10 min at 3000 g. Removed serum and assayed immediately or aliquoted and store samples at −20 or −80 °C. Avoid repeated freezing–thawing cycles.
  2. Prepared all reagents before starting assay procedure. It is recommended that all standards and samples be added in duplicate to the microelisa stripplate.
  3. Added standard: Set standard wells, testing sample wells. Added 50 μl standard to standard well.
  4. Added sample: Added testing sample of 10 μl then add 40 μl of sample diluent to testing sample well; blank well does not add anything.
  5. Added 100 μl HRP-conjugate reagent to each well, cover with an adhesive strip and incubate for 60 min at 37 °C.
  6. Aspirated reactive mixtures from each well and washed, repeating the process four times for a total of five washes. Washed by filling each well with Wash Solution (400 μl) using a squirt bottle, manifold dispenser or autowasher. Complete removal of liquid at each step was essential to good performance. After the last wash, remove any remaining washed solution by aspirating or decanting. Invert the plate and blot it against clean paper towels.
  7. Added chromogen solution A 50 μl and chromogen solution B 50 μl to each well. Gently mix and incubate for 15 min at 37 °C. Protect from light.
  8. Added 50 μl Stop Solution to each well. The colour in the wells should change from blue to yellow. If the colour in the wells is green or the colour change does not appear uniform, gently tap the plate to ensure thorough mixing.
  9. Read the optical density (OD) at 450 nm using a microtiter plate reader within 15 min. The same methodology was used to detect NO, GSSG, MDA, TOS, TAS, SOD, CAT, GSH-Px.

Statistical analysis

All data were analysed using the Statistical Package for the Social Sciences (SPSS) software, Statistics 17.0 (SPSS Inc., Chicago, IL, USA), and the data were presented as mean ± standard error of the mean (SEM). Statistical differences between the two groups were evaluated by analysis with Student's t-test. A P-value <0.05 was considered statistically significant.

Results

  1. Top of page
  2. Abstract
  3. Introduction
  4. Patients and methods
  5. Results
  6. Discussion
  7. Acknowledgment
  8. Conflict of interest
  9. References

Test results of GSH, NO, GSSG, MDA, TOS, TAS, SOD, CAT, GSH-Px

Compared to healthy subjects, patients with chronic ITP showed significantly decreased levels of SOD, CAT, GSH-Px, GSH, TAS, (SOD, = 10.08, < 0.05; CAT, = 5.82, < 0.05; GSH-Px, = 10.32, < 0.05; GSH, = 8.93, < 0.05; TAS, = 3.42, < 0.05) in the peripheral blood (Table 2), but concentrations of NO, GSSG, MDA, TOS significantly increased (NO, = 12.30, < 0.05; GSSG, = 8.27, < 0.05; MDA, = 6.81, < 0.05; TOS, = 13.62, < 0.05). The difference between chronic ITP patients and healthy subjects was statistically significant (Fig. 1, Table 3).

Table 2. Peripheral blood SOD, CAT, GSH-Px and GSH levels
GroupCase (n)SOD (U/l)CAT(U/l)GSH-Px(U/ml)GSH (um)TAS (mm)
Healthy subjects58456.22 ± 120.5018.20 ± 4.62420.07 ± 60.218.95 ± 1.091.93 ± 0.64
Idiopathic thrombocytopenic purpura84210.84 ± 48.0610.47 ± 2.21215.74 ± 29.304.42 ± 1.631.02 ± 0.26
t value10.085.8210.328.933.42
P-value0.0040.0090.0020.0050.010
Table 3. Peripheral blood NO, GSSG, MDA and TOS levels
GroupCase (n)NO (um)GSSG (um)MDA (um)TOS (um)
Healthy subjects5821.84 ± 3.422.67 ± 0.761.15 ± 0.3015.92 ±  2.37
Idiopathic thrombocytopenic purpura8430.50 ± 6.245.20 ± 1.301.82 ± 0.4924.81 ± 5.03
t value12.308.276.8113.62
P-value0.0010.0060.0080.001
image

Figure 1. Expression (mean ± SEM) of NO,GSSG,MDA,TOS were studied by ELISA in control subjects and ITP patients in active disease phase without any treatment (platelet count <100 × 109/l). *Significantly different (P<0.05) using unpaired 2-tail t test. Comparisons between control subjects and patients with ITP wre made under the same experimental conditions separately.

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Correlations between platelet count and NO, GSSG, MDA, TOS, SOD, CAT, GSH-Px, GSH, TAS

The correlation between contents of oxidant/antioxidant stress parameters and platelet count was assessed in patients with chronic ITP. Significant negative correlations were found between platelet count and NO (R = −0.6422,= 0.0012), GSSG (R = −0.7794, = 0.0007), MDA (R = −0.8326, = 0.0002), TOS (R = −0.8315, = 0.0002), respectively (Fig. 2 F,G,H,I). Meanwhile, significant positive correlations existed between platelet count and SOD (R = 0.8186, = 0.0003), CAT (R = 0.8657, = 0.0001), GSH-Px (R = 0.8321, = 0.0002), GSH (R = 0.7795, = 0.0006), TAS (R = 0.7711, = 0.0007), respectively (Fig. 2A,B,C,D,E).

image

Figure 2. Correlations between platelet number and NO, GSSG, malondialdehyde (MDA), TOS, SOD, CAT, GSH-Px, GSH, TAS. The number of platelets showed a negative correlation with NO (F) (R = −0.6422, = 0.0012), GSSG (G) (R = −0.7794, = 0.0007), MDA (H) (R = −0.8326,= 0.0002), TOS (I) (R = −0.8315, = 0.0002) respectively,while platelet number showed a positive correlation with SOD (A) (R = 0.8186,= 0.0003), CAT (B) (R = 0.8657, = 0.0001), GSH-Px (C) (R = 0.8321, = 0.0002), GSH (D) (R = 0.7795, = 0.0006), TAS (E) (R = 0.7711,= 0.0007) respectively. R: correlation coefficient of nonparametric Spearman's rank.

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Discussion

  1. Top of page
  2. Abstract
  3. Introduction
  4. Patients and methods
  5. Results
  6. Discussion
  7. Acknowledgment
  8. Conflict of interest
  9. References

Immune thrombocytopenic purpura (ITP) is a common autoimmune disorder resulting in isolated thrombocytopenia. ITP can present either alone (primary) or in the setting of other conditions (secondary) such as infections or altered immune states. ITP is associated with a loss of immune tolerance to platelet antigens and a phenotype of accelerated platelet destruction and impaired platelet production [10]. Although the aetiology of ITP remains unknown, complex dysregulation of the immune system is observed in ITP patients. Antiplatelet antibodies mediate rapid clearance from the circulation in large part via the reticuloendothelial (monocytic phagocytic) system [11]. In addition, cellular immunity is perturbed and T cell and cytokine profiles are significantly shifted towards a type 1 and Th17 proinflammatory immune response [12]. The precise mechanism of the immune dysfunction, however, is generally not known.

Until recently, no diagnostic criteria have been established, and the diagnosis is based on excluding other causes of thrombocytopenia. A recent report [1] from an international working group established a platelet count threshold of less than 100 × 109/l for diagnosing ITP, down from the previous threshold of 150 × 109/l. The panel also recommended using the term ‘immune’ rather than ‘idiopathic’ thrombocytopenia, emphasizing the role of underlying immune mechanisms. The 2011 American Society of Haematology's evidence-based guidelines for the treatment of ITP present the most recent authoritative diagnostic and therapeutic recommendations [13]. ITP is considered to be primary if it occurs in isolation and secondary, if it is associated with an underlying disorder. In adults, ITP tends to be chronic, presenting with a more indolent course than in childhood, and unlike childhood ITP, infrequently following a viral infection [2].

Oxidative stress, defined as ‘the imbalance between oxidants and antioxidants in favour of the oxidants, potentially leading to damage’ has been associated with several autoimmune diseases, such as colon malignancies, multiple sclerosis, neurodegenerative diseases, psoriasis, vitiligo and alopecia areata [14-17]. Oxidative damage may be involved in the pathogenesis of these autoimmune diseases. Under some conditions, increase in oxidants and decrease in antioxidants cannot be prevented, and the oxidative/antioxidative balance shifts towards the oxidative status. In response to oxidative stress, living organisms have developed an antioxidant defence, which prevents the harmful effects of free radical overproduction. Although free radicals act as a part of the defence system of the body in appropriate conditions, they may cause tissue damage when inappropriately produced [18]. The antioxidant defence system of the body eliminates these harmful effects. Oxidant stress appears when the free radical formation rate exceeds the antioxidant defence mechanism capacity. ITP has characteristics of an immune disease [19-21]. Increased oxidative stress is thought to have a role in the pathogenesis of autoimmune disorders because of its contribution to inflammation and its role in apoptotic cell death, in addition to decreasing immune system functions [22]. Zhang et al. reported that gene expression and molecular-oxidative stress presented as causative factors for chronic ITP in children [23, 24]. The numbers of the patient/control groups entered the study, however are small, but ongoing oxygen stress may play an important part in the immune pathogenesis in patients with chronic ITP, and the specific mechanism is still unclear. But, the exact triggering event remains elusive. A direct link between platelets in ITP and oxidative stress has not yet been addressed. Kamhieh-Milz et al. [25] found that the intracellular platelet antioxidant capacity (AOC) of ITP patients in the active phase was drastically reduced, with significantly high mean fluorescence intensity values. Higher hydroperoxide glutathione peroxidase (GPx) activity was observed in both active phase and remission in comparison with healthy controls, with greater activity observed in active ITP than remission. However, this study included only 36 ITP patients at the active phase (n = 24) and remission (n = 12), the number of patients seem to be small. Furthermore, GPX can effectively remove free radicals by catalytic glutathione GSH in vivo to protect the cells against oxidative damage, and increased GPx seems likely to be contradictory with the reduced AOC in this literature, the oxidant and antioxidant systems in patients with ITP need an in-depth study. Akbayram et al. [26] found that increased MDA, TOS and OSI, and decreased TAC levels were found in children with acute and chronic ITP. However, the association of oxidant status and antioxidant capacity in adult chronic ITP is not very clear until now. In general, the consumption of apples or apple juice as well as oranges, grapefruit and cruciferous vegetables, sources of large amounts of tested derivatives, has beneficial effects on platelets under oxidative stress [27], but the detailed mechanism is not very clear. Antibodies binding to membrane lipids and platelet destruction may play a role in lipid peroxidation in ITP. The platelet destruction and bleeding may play significant role on elevation of lipid peroxidation and reduction in antioxidant capacity in patients with ITP, further studies on oxidant and antioxidant status of ITP are also needed to confirm these results [28].

The balance of oxidative/antioxidative of individuals can be evaluated by measuring the status of each oxidative/antioxidative of serum. To obtain parameters summarizing the various single oxidants/antioxidants, total antioxidant status (TAS) and total oxidant status (TOS) can be determined. TAS is composed of antioxidant capacity of total protein (85%; mainly albumin), uric acid, bilirubin, carotenoids, tocopherol and ascorbic acid [29]. All antioxidants or the total antioxidant status (TAS) is often used to estimate the overall antioxidative status. Likewise, total oxidant status (TOS) is measured to determine a patient's overall oxidation state [30].

In our study, serum levels of NO, GSSG, MDA, TOS were statistically significantly higher, and serum SOD, CAT, GSH-Px, GSH, TAS levels were found to be statistically significantly lower in patients with chronic ITP than those in the control group (all < 0.05). These mean oxygen free radicals increased and antioxidant enzyme for clearing oxygen free radicals decreased in the serum of patients with chronic ITP. Significant negative correlations were also found between platelet count and NO, GSSG, MDA, TOS, respectively (all < 0.05). Meanwhile, significant positive correlations existed between platelet count and SOD, CAT, GSH-Px, GSH, TAS, respectively (all < 0.05). On the basis of these findings, it is suggests that oxidative stress may have an effect on the structural and functional damage of platelets and on the mechanism of thrombocytopenia in chronic ITP. Consequently, oxidative stress is thought to play a role in thrombocyte damage.

Acknowledgment

  1. Top of page
  2. Abstract
  3. Introduction
  4. Patients and methods
  5. Results
  6. Discussion
  7. Acknowledgment
  8. Conflict of interest
  9. References

This study was supported by Nature Science Foundation of Shandong Province (Grant Number: ZR2010HL038). Science and Technology Development Projects of Jining City (Grant Number: 2012jnjc16).

References

  1. Top of page
  2. Abstract
  3. Introduction
  4. Patients and methods
  5. Results
  6. Discussion
  7. Acknowledgment
  8. Conflict of interest
  9. References
  • 1
    Rodeghiero F, Stasi R, Gernsheimer T et al. Standardization of terminology, definitions and outcome criteria in immune thrombocytopenic purpura of adults and children: report from an international working group. Blood 2009;113:238693.
  • 2
    Thota S, Kistangari G, Daw H, Spiro T. Immune thrombocytopenia in adults: an update. Cleve Clin J Med 2012;79:64150.
  • 3
    Jones DP. Redefining oxidative stress. Antioxid Redox Signal 2006;8:186579.
  • 4
    Udupa AS, Nahar PS, Shah SH, Kshirsagar MJ, Ghongane BB. Study of comparative effects of antioxidants on insulin sensitivity in type 2 diabetes mellitus. J Clin Diagn Res 2012;6:146973.
  • 5
    Doria E, Buonocore D, Focarelli A, Marzatico F. Relationship between human aging muscle and oxidative system pathway. Oxid Med Cell Longev 2012;2012:830257.
  • 6
    Singh S, Greene RM, Pisano MM. Arsenate-induced apoptosis in murine embryonic maxillary mesenchymal cells via mitochondrial-mediated oxidative injury. Birth Defects Res A Clin Mol Teratol 2010;88:2534.
  • 7
    Birben E, Sahiner UM, Sackesen C, Erzurum S, Kalayci O. Oxidative stress and antioxidant defense. World Allergy Organ J 2012;5:919.
  • 8
    Arnold DM, Bernotas A, Nazi I et al. Platelet count response to H. pylori treatment in patients with immune thrombocytopenic purpura with and without H. pylori infection: a systematic review. Haematologica 2009;94:8506.
  • 9
    Li Z, Nardi MA, Karpatkin S. Role of molecular mimicry to HIV-1 peptides in HIV-1-related immunologic thrombocytopenia. Blood 2005;106:5726.
  • 10
    Johnsen J. Pathogenesis in immune thrombocytopenia: new insights. Hematology Am Soc Hematol Educ Program 2012;2012:30612.
  • 11
    Ho WL, Lee CC, Chen CJ et al. Clinical features, prognostic factors, and their relationship with antiplatelet antibodies in children with immune thrombocytopenia. J Pediatr Hematol Oncol 2012;34:612.
  • 12
    Ji L, Zhan Y, Hua F et al. The ratio of Treg/Th17 cells correlates with the disease activity of primary immune thrombocytopenia. PLoS One 2012;7:e50909.
  • 13
    Neunert C, Lim W, Crowther M, Cohen A, Solberg L Jr, Crowther MA. The American Society of Hematology 2011 evidence-based practice guideline for immune thrombocytopenia. Blood 2011;117:4190207.
  • 14
    Rachisan AL, David BA, Cainap S, Miu N, Andreica M, Samasca G. Immunological manifestations in type I diabetic children. Roum Arch Microbiol Immunol 2012;71:959.
  • 15
    Jrah-Harzallah H, Ben-Hadj-Khalifa S, Almawi WY, Maaloul A, Houas Z, Mahjoub T. Effect of thymoquinone on 1,2-dimethyl-hydrazine-induced oxidative stress during initiation and promotion of colon carcinogenesis. Eur J Cancer 2013;49:112735.
  • 16
    Lee DH, Gold R, Linker RA. Mechanisms of oxidative damage in multiple sclerosis and neurodegenerative diseases: therapeutic modulation via fumaric acid esters. Int J Mol Sci 2012;13:11783803.
  • 17
    Ramadan R, Tawdy A, Abdel HayR, Rashed L, Tawfik D. The antioxidant role of paraoxonase 1 and vitamin e in three autoimmune diseases. Skin Pharmacol Physiol 2013;26:27.
  • 18
    Kuppusamy UR, Dharmani M, Kanthimathi MS, Indran M. Antioxidant enzyme activities of human peripheral blood mononuclear cells exposed to trace elements. Biol Trace Elem Res 2005;106:2940.
  • 19
    Liu F, Wu C, Yang X et al. Polarization and apoptosis of T cell subsets in idiopathic thrombocytopenic purpura. Cell Mol Immunol 2005;2:38792.
  • 20
    Liu F, Wu CL, Xiao H, Yang XM, Lv XW, Chen Q. Effect of protein kinase C on T lymphocyte proliferation and apoptosis in acute idiopathic thrombocytopenic purpura. Acta Haematol 2006;116:17380.
  • 21
    Jin CQ, Liu F, Dong HX et al. Type 2 polarized immune response holds a major position in Epstein-Barr virus-related idiopathic thrombocytopenic purpura (EBV-ITP). Int J Lab Hematol 2012;34:16471.
  • 22
    Agarwal D, Dange RB, Vila J, Otamendi AJ, Francis J. Detraining differentially preserved beneficial effects of exercise on hypertension: effects on blood pressure, cardiac function, brain inflammatory cytokines and oxidative stress. PLoS One 2012;7:e52569.
  • 23
    Zhang B, Lo C, Shen L et al. The role of vanin-1 and oxidative stress-related pathways in distinguishing acute and chronic pediatric ITP. Blood 2011;117:456979.
  • 24
    Imbach P. Oxidative stress may cause ITP. Blood 2011;117:44056.
  • 25
    Kamhieh-Milz J, Bal G, Sterzer V, Kamhieh-Milz S, Arbach O, Salama A. Reduced antioxidant capacities in platelets from patients with autoimmune thrombocytopenia purpura (ITP). Platelets 2012;23:18494.
  • 26
    Akbayram S, Dogan M, Akgun C et al. The association of oxidant status and antioxidant capacity in children with acute and chronic ITP. J Pediatr Hematol Oncol 2010;32:27781.
  • 27
    Saluk-Juszczak J. A comparative study of antioxidative activity of calcium-D-glucarate, sodium-D-gluconate and D-glucono-1,4-lactone in a human blood platelet model. Platelets 2010;21:63240.
  • 28
    Polat G, Tamer L, Tanriverdi K, Gurkan E, Baslamisli F, Atik U. Levels of malondialdehyde, glutathione and ascorbic acid in idiopathic thrombocytopaenic purpura. East Afr Med J 2002;79:4469.
  • 29
    Altindag O, Erel O, Soran N, Celik H, Selek S. Total oxidative/anti-oxidative status and relation to bone mineral density in osteoporosis. Rheumatol Int 2008;28:31721.
  • 30
    Nechifor MT, Niculite CM, Urs AO et al. UVA irradiation of dysplastic keratinocytes: oxidative damage versus antioxidant defense. Int J Mol Sci 2012;13:1671836.