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Annals of Clinical & Laboratory Science 38:120-125 (2008)
© 2008 Association of Clinical Scientists

Disturbance of Pro-oxidative/Antioxidative Balance in Allogeneic Peripheral Blood Stem Cell Transplantation

Ismail Sari1, Aysun Cetin2, Leylagul Kaynar3, Recep Saraymen2, Sibel Kabukcu Hacioglu1, Ahmet Ozturk4, Ismail Kocyigit3, Fevzi Altuntas3 and Bulent Eser3
1 Department of Hematology, Pamukkale University Faculty of Medicine, Denizli; 2 Departments of Biochemistry/Clinical Biochemistry, 3 Hematology, and 4 Biostatistics, Erciyes University Faculty of Medicine, Kayseri, Turkey

Address correspondence to Ismail Sari, M.D., Department of Hematology, Pamukkale University Faculty of Medicine, TR-20070, Denizli, Turkey; tel 90 258 211 8585/2332; fax 90 258 213 4922; e-mail hisari{at}pau.edu.tr, or hisari{at}gmail.com.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
High dose chemotherapy causes increased free radical formation and depletion of tissue antioxidants. Whether allogeneic hematopoietic stem cell transplantation (HSCT) has an effect on oxidative stress is uncertain. The aims of the study were to determine the effect of allogeneic HSCT on plasma concentrations of antioxidants and oxidative stress biomarkers, and to investigate their relationships with graft-versus-host disease (GVHD), conditioning regimens, and transplant-related mortality (TRM) in patients with hematological malignancies. Patients (n=25) undergoing allogeneic HSCT from HLA-matched sibling donors were enrolled in the study. Plasma oxidant and antioxidant status were measured at day -1 before transplantation and 30 days after HSCT. In both myeloablative (n=14) and non-myeloablative (n=11) transplant groups, the mean levels of plasma malondialdehyde (MDA) and nitric oxide (NO) increased after allogeneic HSCT (p <0.01), whereas superoxide dismutase (SOD), glutathione peroxidase (GSH-Px), and catalase (CAT) activities decreased compared with baseline values (p <0.01). No significant relationships were found between either the pretransplant or post-transplant mean levels of the oxidative stress parameters and the existence of graft-versus-host disease (GVHD), the type of conditioning regimen, or transplant related mortality (TRM). This study documents a significant disturbance of pro-oxidative/antioxidative balance in the plasma of patients undergoing allogeneic HSCT regardless of the intensity of the conditioning regimen.

Keywords: oxidative stress, free radicals, allogeneic stem cell transplantation


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Oxidative stress resulting from increased production of free radicals and reactive oxygen species (ROS) causes severe damage to biological macromolecules and dysregulation of normal metabolism and physiology [1,2]. Additionally, the ROS content is balanced by antioxidative systems, which include enzymes such as glutathione peroxidase (GSH-Px), superoxide dismutase (SOD), and catalase (CAT) [3–5]. Increased tissue levels of ROS and concomitant depletion of intra- and extra-cellular anti-oxidants are among the suspected mechanisms that may mediate post-HSCT damage to lung, liver, and other organs [6,7]. Therefore, oxidative imbalance has been viewed as a common denominator in the pathogenesis of treatment-related morbidity and mortality in high dose therapy (HDT) followed by hematopoietic stem cell transplantation (HSCT) [8,9].

The studies in which the oxidative stress was investigated have involved small numbers of patients and were associated with HDT and/or radiotherapy [4,5,10]. In this study, we aimed to investigate changes of oxidative stress parameters in patients with hematological malignancies, undergoing both myeloablative and nonmyeloablative allogeneic HSCT and to assess the relationship of HSCT with graft-versus-host disease (GVHD), conditioning regimens, and transplant-related mortality (TRM).


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Patients.  Between July 2003 and February 2007, 25 patients with hematological malignancies undergoing allogeneic HSCT from HLA-matched sibling donors were enrolled in the study. Seven of the patients were male and 18 were female, ranging in age from 19 to 61 yr, with a median age of 42. Ten patients were previously diagnosed to have acute myeloid leukemia, 9 had acute lymphoblastic leukemia, 3 had chronic myeloid leukemia, 2 had non-Hodgkin’s lymphoma, and the remaining patient had myelofibrosis. At the beginning of the study, informed consent about the investigation was obtained from the patients. General characteristics of the patients are summarized in Table 1Go.


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  		Table 1. General characteristics of the patients.
 
Chemotherapy regimens.  Two different chemotherapy regimens were used for conditioning. Fourteen patients who were prepared with the iv BuCy regimen for myeloablative stem cell transplantation initially received an iv dose of busulphan of 3.2 mg/kg/day for 4 days (from day -7 to day -4), followed by cyclophosphamide 60 mg/kg/day iv for 2 days (day -3 to day -2). Eleven patients who were prepared with the FluCy regimen for nonmyeloablative stem cell transplantation received fludarabine 25 mg/m2/day iv for 5 days (day -6 to day -2) and cyclophosphamide 10 mg/kg/day iv for 2 days (day -3 to day -2).

Peripheral blood stem cells.  PBSC donors were mobilized with filgrastim at 10 µg/kg/d for 5 days. PBSC collection was initiated by leukapheresis on the fifth day of filgrastim treatment and was continued until a sufficient number of CD34+ stem cells was obtained. The targeted cell number for nonmyeloablative transplantation was at least 1x107 CD34+ cells/kg. The targeted PBSC number for myeloablative transplantation was at least 5x106 CD34+ cells/kg.

Specimen collection.  Blood samples were drawn from antecubital veins following overnight fasting and collected in heparinized polypropylene tubes. To evaluate the effect of stem cell transplantation on prooxidant status over time, baseline values of each subject were determined from samples drawn on day -1 (before HSCT). Patients received allogeneic HSCT on day 0. Other blood samples were obtained on day +30 after allogeneic HSCT. After immediate centrifugation (100 x g, 10 min, +4°C), plasma samples were stored at –70°C until analysis.

Malondialdehyde.  The levels of MDA in plasma were assessed spectrophotometrically by the method of Ohkawa et al [11]. MDA, an end product of lipid peroxidation, was determined by the thiobarbituric acid reaction, in which MDA couples to thiobarbituric acid to form a pink chromogen that has maximum absorbance at 532 nm. Results were expressed as µmol/L. The within-run CV of replicates was 2.7% (n = 10) and the run-to-run CV of replicates was 3.4% (n = 10).

Nitric oxide.  NO is synthesized from L-arginine by NO synthase. Since NO is highly labile, its direct measurement in biological samples is difficult. NO is rapidly converted to stable end-products, nitrite (NO2–) and nitrate (NO3–) ions, which can serve as indicators of NO production. After enzymatic reduction of nitrate to nitrite by nitrate reductase, nitrite concentrations were quantified by a colorimetric assay based on the Griess reaction [12]. Results were expressed as µmol/L. The within-run CV of replicates was 2.0% (n = 10) and the run-to-run CV of replicates was 3.1% (n = 10).

SOD activity.  Plasma SOD activity was measured spectrophotometrically as described by Sun et al [13]. The method is based on inhibition of nitro blue tetrazolium (NBT) reduction by a xanthine–xanthine oxidase system as a superoxide generator. Reduction of NBT to blue formazan was determined at 560 nm. One unit of SOD activity was defined as the amount of enzyme causing 50% inhibition of the NBT reduction rate. SOD activity was expressed as U/ml. The within-run CV of replicates was 3.0% (n = 10) and the run-to-run CV of replicates was 4.1% (n = 10).

CAT activity.  Plasma CAT activity was determined as described by Aebi [14] by monitoring the reduction rate of H2O2 spectrophotometrically at 240 nm. One unit of catalase activity was defined as 1 mmol H2O2 utilized/min. Enzyme activity was expressed as K/ml. The within-run CV of replicates was 3.1% (n = 10) and the run-to-run CV of replicates was 3.4% (n = 10).

GSH-Px activity.  Plasma GSH-Px activity was measured spectrophotometrically by the method of Paglia and Valentina [15]. The enzyme reaction was initiated by addition of H2O2 and the rate of NADPH oxidation was followed at 340 nm. One unit of GSH-Px activity was defiined as the amount of enzyme that oxidizes 1 mmol of NADPH/min. Results were expressed as U/L. The within-run CV of replicates was 3.7% (n = 10) and the run-to-run CV was 4.3% (n = 10).

Statistics.  Statistical analyses were performed using the Statistical Package for Social Sciences (SPSS) for Windows 13.0 program. Normality tests (Shapiro Wilks) showed that all distributions were skewed. Values were reported as mean ± SD and range. Pre-transplatation and post-transplantation data were compared using the Wilcoxon signed-ranks test. The Mann-Whitney U test was used to assess the relationships between oxidative stress parameters and GVHD, conditioning regimens, ot TRM. A p value <0.05 was deemed significant.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
In both the myeloablative (n = 14) and non-myeloablative (n = 11) transplant groups, there were statistically significant increases in the mean levels of the plasma oxidative stress biomarkers MDA and NO after allogeneic PBSCT (p <0.01). Additionally, statistically significant decreases were found in the activities of the plasma antioxidant enzymes SOD, GSH-Px, and CAT, compared to the baseline values in both groups (p <0.01). Moreover, there were statistically significant differences between the mean values of pre- and post-transplant oxidative stress parameters for the combined data of the entire group of 25 patients. Changes in oxidative stress parameters of the subjects, before and after transplantation, are listed in Table 2Go and shown in Figs. 1Go–5GoGoGoGo.


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  		Table 2. The effect of allogeneic HSCT on oxidative stress parameters.
 

Figure 1
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  		Fig. 1. Plasma MDA levels pre- and 30 days post-HSCT.

 

Figure 2
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  		Fig. 2. Plasma MDA levels pre- and 30 days post-HSCT.

 

Figure 3
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  		Fig. 3. Plasma SOD activities pre- and 30 days post-HSCT.

 

Figure 4
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  		Fig. 4. Plasma CAT activities pre- and 30 days post-HSCT.

 

Figure 5
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  		Fig. 5. Plasma GSH-Px activities pre- and 30 days post-HSCT.

 
The changes in oxidative stress parameters were evaluated in relation to the existence of GVHD, the type of conditioning regimen (myeloablative or nonmyeloablative), and transplant-related mortality (TRM). No significant relationships were found between the pre- and post-transplant mean levels of any oxidative stress parameter and GVHD existence, type of conditioning regimen, or TRM.


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
It is well known that chemotherapy and radiation therapy are associated with increased formation of reactive oxygen species and depletion of critical plasma and tissue antioxidants [3–5,7,16,17]. It has been shown that oxidative imbalance occurs in patients undergoing high-dose chemotherapy with autologous hematopoietic stem cell support because of the routine use of chemotherapeutic agents as conditioning regimens. Previous studies of oxidative stress and antioxidant conditions after high-dose chemotherapy involved a limited number of patients [8,10,18]. Durken et al [10] assessed antioxidant parameters in 8 HSCT patients (3 autologous, 5 allogeneic) who received high-dose chemotherapy as a conditioning regimen. They measured the total radical antioxidant parameter of plasma (TRAP), which measures the overall capacity of human plasma to inhibit radical-induced lipid peroxidation in vitro. They found significant inverse correlation with nontransferrin binding iron [NTBI] and TRAP, indicating a pro-oxidant state. Cetin et al [8] documented a pro-oxidant state in patients conditioned with chemo-radiotherapy followed by autologous HSCT. Compared with baseline levels, they found significant increases in erythrocyte iron, zinc, MDA, glutathione peroxidase, and SOD at day -1. White et al [18] also demonstrated changes in the plasma antioxidant system of subjects just prior to HSCT. The major novel findings of their study were reduction in plasma glutathione peroxidase activity and elevation of the plasma concentration of gamma-tocopherol, a nonenzymatic antioxidant, in subjects before HSCT, compared to controls. These studies all showed that oxidative stress increases after high-dose chemotherapy whether or not stem cell transplantation is performed.

Current knowledge is lacking regarding the oxidative stress status of patients after allogeneic HSCT independent from the conditioning regimen. Therefore, in the present study, plasma oxidant and antioxidant parameters were measured at day -1 before transplantation (after conditioning regimen) and at day 30 after HSCT. We demonstrated that the levels of plasma oxidative stress biomarkers were markedly increased, whereas the antioxidant enzyme activities were significantly decreased regardless of the intensity of the conditioning regimen. Hence, our results indicate that high-dose chemotherapy is not the only factor that causes increased oxidative stress post-HSCT.

GVHD results from reactivity of donor immunocompetent cells vs host tissues. Its pathogenesis involves co-stimulatory molecules, cytokines, free radicals, and oxidative stress products [19]. There are few data regarding oxidant status in GVHD in either humans or animals [19,20]. Amer et al [20] studied the oxidative status of red and white blood cells (polymorphonuclear leukocytes and lymphocytes) during the development of GVHD in a mouse model. They demonstrated that all blood cells were under oxidative stress and that treatment with the antioxidants N-acetylcysteine (NAC) and a vitamin E derivative (tocopherol) reduced oxidative stress, both in vitro and in vivo. However, they did not find any benefit of the antioxidant treatment on survival.

Studies have also reported increased tissue levels of ROS and depletion of intra- and extra-cellular antioxidants during post-HSCT damage to lung, liver, and other organs [6,7,21,22]. NO, an important short-lived free radical involved in oxidative stress, was found to be increased in GVHD in both humans [23] and animals [24]. But the effect of blocking nitric oxide production in GVHD is still controversial [25]. In this study, we did not find any relationship between either pre- or post-transplant mean levels of oxidative stress parameters and the existence of GVHD or TRM, possibly because of the small number of relevant subjects.

In conclusion, this study revealed substantial disturbance of the pro-oxidative/antioxidative balance of plasma parameters of patients undergoing allogeneic HSCT regardless of intensity of the conditioning regimen. It is clear that prospective trials in a larger number of patients are needed in order to elucidate the pathobiology and chemistry of these processes, their relationship with GVHD, and the clinical efficacy of antioxidant therapy after HSCT.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

  1. Knight JA. Free radicals: their history and current status in aging and disease. Ann Clin Lab Sci 1998;28:331–346.[Abstract]
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  19. Colombo AA, Alessandrino EP, Bernasconi P, Arcese GW, Rabusin M, Bacigalupo A, Bernasconi C. N-acetylcysteine in the treatment of steroid-resistant acute graft-versus-host-disease: preliminary results. Transplantation 1999;68:1414–1416.[Medline]
  20. Amer J, Weiss L, Reich S, Shapira MY, Slavin S, Fibach E. The oxidative status of blood cells in a murine model of graft-versus-host disease. Ann Hematol 2007;86:753–758.[Medline]
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