|Year : 2015 | Volume
| Issue : 4 | Page : 153-158
Zinc and malondialdehyde levels among Egyptian patients with acute myeloid leukemia and their relation with disease phenotype and genotype
Inas A Asfour, Maryse S Ayoub, Amro M.S. El-Ghammaz MD , Ibtesam M Khalifa
Clinical Hematology and Bone Marrow Transplantation Unit, Department of Internal Medicine, Ain Shams University, Cairo, Egypt
|Date of Submission||22-Jun-2015|
|Date of Acceptance||23-Jun-2015|
|Date of Web Publication||23-Nov-2015|
Amro M.S. El-Ghammaz
37 Mohamed Korayem Street, Nasr City, 11371 Cairo
Source of Support: None, Conflict of Interest: None
Objectives This study was conducted to evaluate serum zinc and plasma malondialdehyde (MDA) levels in de-novo acute myeloid leukemia (AML) before and after induction chemotherapy and their relation with AML phenotype and genotype.
Materials and methods Twenty-five AML patients were subjected to serum zinc evaluation using flame atomic absorption spectrophotometry and plasma MDA evaluation using colorimetric method at day 1 before induction chemotherapy and at day 21 after induction chemotherapy.
Results Pretreatment MDA levels were higher in patients in comparison with controls (P = 0.03). Pretreatment zinc levels differed significantly compared with post-treatment levels (P = 0.005). The percentage of bone marrow infiltration by blasts at diagnosis correlated inversely with zinc levels (P = 0.011) and positively with MDA levels (P = 0.041). Finally, pretreatment MDA levels were higher among patients harboring adverse cytogenetics (P = 0.004).
Conclusion The elevated plasma MDA status at diagnosis in AML patients correlates with a higher tumor burden in the bone marrow and adverse cytogenetic risk.
Keywords: acute myeloid leukemia, genotype, malondialdehyde, phenotype, zinc
|How to cite this article:|
Asfour IA, Ayoub MS, El-Ghammaz AM, Khalifa IM. Zinc and malondialdehyde levels among Egyptian patients with acute myeloid leukemia and their relation with disease phenotype and genotype. Egypt J Haematol 2015;40:153-8
|How to cite this URL:|
Asfour IA, Ayoub MS, El-Ghammaz AM, Khalifa IM. Zinc and malondialdehyde levels among Egyptian patients with acute myeloid leukemia and their relation with disease phenotype and genotype. Egypt J Haematol [serial online] 2015 [cited 2017 Jun 25];40:153-8. Available from: http://www.ehj.eg.net/text.asp?2015/40/4/153/170190
| Introduction|| |
Acute myeloid leukemia (AML) is a very heterogeneous disorder characterized by the accumulation of somatically acquired genetic changes in hematopoietic progenitor cells that alter normal mechanisms of self-renewal, proliferation, and differentiation  . Excessive production of reactive oxygen species and/or a deficiency in antioxidant pathways can lead to oxidative stress, a state that has been observed in several hematopoietic malignancies including AML  .
Zinc is an essential micronutrient for human health because of its structural and biochemical functions influencing growth and affecting multiple aspects of the immune system  . Moreover, zinc is involved in the protection against oxidative stress  . Zinc status has been associated with cancer in epidemiological studies  . The mechanisms by which zinc deficiency increases the risk for cancer are still unclear and understudied  . For example, it has been suggested that intracellular mobile reactive zinc activates metal-responsive transcription factors, or interacts directly with intracellular signaling molecules (e.g. RAS), to modulate the expression of zinc-responsive genes and to regulate specific signal transduction pathways  . Moreover, zinc acts as a cofactor for several DNA repair pathways (e.g. p53)  . Finally, zinc inhibits nuclear factor-κB, which is constitutively activated in many cancer cells, through induction of A-20  . It has been suggested that AML is susceptible to antioxidant enzymes and essential element alterations, including zinc alterations, and that these alterations in leukemia patients were mostly dependent on tumor activity  .
Malondialdehyde (MDA) is a major lipid peroxidation product that is mutagenic and tumorigenic  . Normally, MDA is quickly oxidized to acetate or malonate and then to carbon dioxide through Kreb's cycle. If it is accumulated in excess, MDA can combine with different serum proteins and cell membrane components to form altered determinants  . Moreover, it can interact with DNA and inhibit the biosynthesis of the DNA, RNA, and proteins. Notably, the chemical structure of MDA closely resembles that of carcinogenic compounds such as glyceraldehyde and β-propiolactone  . MDA was used as a surrogate marker of oxidative damage to tissues  . Plasma MDA levels has been reported to be significantly high in patients with various types of cancer, including both hematological malignancies and solid tumors (e.g. breast, lung, oral, and cervical cancer) ,,, . In this study, we assessed serum zinc and plasma MDA levels in adult de-novo AML patients before and after receiving treatment induction to study their status in AML and their relation with AML phenotype and genotype.
| Materials and methods|| |
Twenty-five adult de-novo AML patients and 15 age-matched and sex-matched healthy controls were enrolled in the study. Patients receiving supportive antioxidant medications were excluded from the study. Both the patient and control groups were almost of same socioeconomic status with similar diet habits. Patient characteristics are summarized in [Table 1].
Treatment plan and follow-up
All patients were treated with '3+7' induction chemotherapy protocol, comprising doxorubicin (25 mg/m 2 ) on days 1-3 and cytosine arabinoside (100-200 mg/m 2 ) on days 1-7  . Those patients with acute promyelocytic leukemia received all-trans retinoic acid daily (45 mg/m 2 /day) in addition to idarubicin (12 mg/m 2 ) on days 2, 4, 6, and 8  . The patients received normal nutrition with healthy diet throughout the study period.
Sample collection and storage
Patients were subjected to blood sampling for evaluation of serum zinc and plasma MDA at day 1 before initiation of induction chemotherapy. Another sampling was performed at day 21 for surviving patients. Two 3 ml peripheral venous blood samples were collected and those with signs of hemolysis were discarded. Serum and plasma were prepared for zinc and MDA evaluation, respectively, and were stored at -80°C until the day of analysis.
Determination of serum zinc level
Serum zinc measurement was performed using the spectrophotometric method using flame atomic absorption spectrometer (Perkin-Elmer Model 400; Perkin-Elmer Inc., Shelton, Connecticut, USA). Serum samples were diluted with deionized water in a ratio of 1 : 5. The analysis was performed against standards prepared in glycerol to approximate the viscosity characteristics of the diluted samples. The experimental condition of atomic absorption spectrometer for determination of zinc was as follows: wavelength, 213.9 nm; band width, 0.7 nm; 30 mA; and air-acetylene flame gas  .
Determination of plasma MDA level
MDA level of the plasma was measured using colorimetric technique. The basic principle is the reaction of one molecule of MDA and two molecules of thiobarbituric acid (TBA) to form a red MDA-TBA complex. TBA solution (Sigma Diagnostics, St Louis, Missouri, USA) was prepared as 0.67% solution and its pH was adjusted to 7.2 using 0.1 mol/l NaOH. A volume of 0.5 ml of plasma was added to 2.5 ml of 10% trichloroacetic acid (TCA) (Sigma Diagnostics) in a 10 ml centrifuge tube and then the mixture was incubated in a boiling water bath for 15 min, followed by cooling under tap water and centrifugation at 3000 rpm for 10 min (to precipitate proteins). A volume of 2 ml of the supernatant was added to 1 ml of TBA solution, followed by boiling in water bath for 15 min and then cooling of the mixture under tap water. The optical density of the developed color was then measured at 535 nm absorbance with a spectrophotometer, reflecting MDA concentration in plasma  .
Descriptive statistical analysis of the main characteristics of patients was performed (mean, SD, range, and number and percentage). Variables in two or more groups were compared using the unpaired t-test, one-way analysis of variance, or χ2 -test as appropriate. Paired t-test was used to compare quantitative variables in the same group. Spearman's correlation coefficients were used to determine correlation between variables. Statistical significance was determined at level 0.05. All P values were two sided. Standard computer program SPSS for Windows, version 16.0 (SPSS Inc., Chicago, Illinois, USA), was used for data entry and analysis.
All procedures were carried out in accordance with the ethical standards of the Ethical Committee of Faculty of Medicine of Ain Shams University and with the Helsinki Declaration of 1975, as revised in 2000. Informed consent was obtained from all participants included in the study.
| Results|| |
Comparison of serum zinc and plasma MDA levels in patients and controls
At day 21, 15 patients (60%) were still alive and were available for withdrawal of day 21 blood samples. Although serum zinc levels at day 1 were lower in patients than in controls, the difference was statistically insignificant [mean in μg% = 104.2 ± 18.1 (range = 57-135) vs. 113.3 ± 17.3 (range = 85-140), respectively; P = 0.125]. At day 21, there was statistically insignificant difference in serum zinc levels between patients and controls [mean in μg% = 120.1 ± 16.3 (range = 90-155) vs. 113.3 ± 17.3, respectively; P = 0.278]. Plasma MDA levels were significantly higher in patients at day 1 compared with controls [mean in μmol/l = 3.9 ± 1.2 (range = 2.03-6.27) vs. 3.1 ± 0.7 (range = 1.73-4.45), respectively; P = 0.03]. In contrast, there was no significant difference in plasma MDA levels at day 21 between patients and controls [mean in μmol/l = 3.5 ± 0.8 (range = 2.36-4.87) vs. 3.1 ± 0.7, respectively; P = 0.149].
Comparison of serum zinc and plasma MDA levels in patients at day 1 and at day 21
Serum zinc levels were significantly lower at day 1 than at day 21 (mean in μg% = 103.9 ± 17.6 vs. 120.1 ± 16.3, respectively; P = 0.005). In contrast, there was no significant difference in plasma MDA levels at day 1 and at day 21 (mean in μmol/l = 3.7 ± 1 vs. 3.5 ± 0.8, respectively; P = 0.684).
Relation of serum zinc and plasma MDA levels at day 1 with phenotypic and genotypic features
On correlating pretreatment serum zinc and plasma MDA levels with age, total leukocytic count, hemoglobin, platelets, absolute blast count in peripheral blood, serum albumin, serum uric acid, serum lactate dehydrogenase, and percentage of bone marrow (BM) infiltration by blasts, only percentage of BM infiltration by blasts correlated inversely with serum zinc levels (r = −0.5, P = 0.011) and positively with plasma MDA levels (r = 0.41, P = 0.041). On comparing serum zinc and plasma MDA levels in relation to sex, extramedullary infiltration, French-American-British subtype, aberrant cluster of differentiation expression, karyotype, and cytogenetic risk groups, there was statistically significant difference only in plasma MDA levels among cytogenetic risk groups (P = 0.004). There was a tendency towards significance between MDA levels in different karyotype groups (P = 0.060) ([Table 2]).
|Table 2 Comparison of serum zinc and plasma MDA levels at day 1 in relation to phenotypic and genotypic features |
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Relation of serum zinc levels with plasma MDA levels at day 1 and at day 21
There was a tendency towards an inverse correlation between serum zinc and plasma MDA levels at day 1 (r = -0.375, P = 0.065). In contrast, there was no correlation between them at day 21 (r = 0.132, P = 0.639).
| Discussion|| |
Trace elements such as zinc may play a role in the protection against oxidative stress in blood cells. The levels of antioxidants in hematological malignancies before and after exposure to antineoplastic agents were contradictory  . In the present study, we found no statistically significant difference in serum zinc between patients and controls either before or after induction chemotherapy. These results are in agreement with those of Olaniyi et al.  . In contrast, other studies found significantly higher serum zinc in controls than in cases ,, . This difference can be attributed to the relatively lower number of patients in our study and to the inclusion of patients with acute lymphoblastic leukemia and/or chronic myeloid leukemia in addition to AML in the other studies ,, . It should be noted that our study and the former one were conducted on African patients, whereas the latter studies were performed on Asian patients. A possible impact of geographical factors on zinc status in different populations has been previously reported  .
On comparing the serum zinc levels at day 1 and at day 21, they were significantly higher at day 21. Our results are in agreement with the results of Sanaat et al.  . A similar finding has been observed in a study performed on patients undergoing hematopoietic stem cell transplantation, in which serum zinc level significantly increased by about 30% after conditioning chemotherapy compared with its level before conditioning chemotherapy  . Collectively, these findings can be attributed to zinc mobilization from tissues in response to chemotherapy  . In contrast, other studies reported an insignificant change in serum zinc and even its decrease after receiving chemotherapy, but it should be noted that these studies were performed on children and many of them had associated malnutrition , . We found an inverse correlation between serum zinc at day 1 and percentage of BM infiltration by blasts. In a previous study, serum zinc level correlated negatively with the absolute peripheral blast count  . It has been suggested that changes in metabolism and proliferative rate would affect mitochondrial reactive oxygen species output in leukemic blasts and in turn they might influence the levels of antioxidants  . In another study, serum zinc level significantly correlated with age in acute leukemia patients and it differed significantly among male and female patients  .
Increased levels of MDA in patients with AML were first reported by Stocks et al.  . They attributed this phenomenon to an increased availability of the substrate (i.e. polyunsaturated fatty acids) in the red cells  . However, a more recent study attributed this finding to the presence of excess numbers of myeloid cells, which are the major source of free radicals  . Another explanation is the state of oxidative stress present in malignant leukemic cells  . It is worthy of mention that it has been reported that mean plasma levels of MDA are significantly high in AML patients at relapse  . In the present study, we found a statistically significantly higher level of plasma MDA in patients than in controls at day 1, which differed from the results of Olaniyi et al.  . This difference can be attributed to the use of enzyme-linked immunosorbent assay for MDA assessment in the other study  . However, our result was in concordance with many studies performed in nonhematological neoplasms ,, . There was no statistically significant difference between levels of MDA in patients at day 1 and at day 21 in our study, which is in agreement with the results of Esfahani et al.  . In contrast, another study has reported contradictory results in which the levels of MDA were significantly higher at diagnosis than after chemotherapy  . This difference can be attributed to the measurement of erythrocytic rather than plasma MDA levels and to the later time point at which the postchemotherapy samples have been collected in the study (sixth week)  .
We found a statistically significant positive correlation between plasma MDA levels at day 1 and percentage of BM infiltration by blasts. To our knowledge, no study has determined the correlation between MDA levels and AML phenotypic features. As regards genotypic features, although the differences in MDA levels among karyotype groups did not reach significance, they were significantly higher in patients with unfavorable cytogenetics in comparison with those in patients with intermediate and favorable cytogenetics. A similar finding has been observed in chronic lymphocytic leukemia patients in whom significant increases in MDA were observed in patients with unfavorable cytogenetic aberrations (17p and 11q deletions) in comparison with the favorable 13q deletion and trisomy 12 groups  . However, because of the small number of patients in unfavorable and favorable risk groups in our study, such finding should be interpreted with caution.
Many studies found an inverse correlation between serum zinc and plasma MDA levels whether in benign or malignant conditions ,,, . Other studies failed to declare any correlation ,,, . In our study, there was a tendency towards a significant inverse correlation between serum zinc and plasma MDA levels in AML patients at day 1. In conclusion, there is a state of high oxidative stress and cellular damage in de-novo AML patients at diagnosis as evidenced by the elevated plasma MDA level. Moreover, treatment of AML patients using chemotherapy causes elevation of serum zinc levels possibly because of tissue mobilization by chemotherapy. Furthermore, pretreatment serum zinc and plasma MDA levels can represent surrogate markers for the percentage of BM infiltration by blasts and in turn for the tumor burden inside the BM. Finally, a higher MDA level at diagnosis and in turn a higher oxidative stress, either as triggering factors or as results, are associated with the occurrence of adverse cytogenetics. Large studies are needed to evaluate the impact of pretreatment plasma MDA levels on AML outcome.
Concept: Inas A. Asfour, Maryse S. Ayoub; design: Inas A. Asfour, Maryse S. Ayoub; definition of intellectual content: Inas A. Asfour, Maryse S. Ayoub; literature search: Amro M.S. El-Ghammaz, Ibtesam M. Khalifa; clinical studies: Inas A. Asfour, Maryse S. Ayoub, Amro M. Sedky El-Ghammaz, Ibtesam M. Khalifa; experimental studies: Inas A. Asfour, Maryse S. Ayoub, Amro M.S. El-Ghammaz, Ibtesam M. Khalifa; data acquisition: Amro M.S. El-Ghammaz, Ibtesam M. Khalifa; data analysis: Inas A. Asfour, Maryse S. Ayoub, Amro M.S. El-Ghammaz, Ibtesam M. Khalifa; statistical analysis: Amro M.S. El-Ghammaz, Ibtesam M. Khalifa; manuscript preparation: Inas A. Asfour, Amro M.S. El-Ghammaz; manuscript editing: Inas A. Asfour, Amro M.S. El-Ghammaz; and manuscript review: Inas A. Asfour, Maryse S. Ayoub, Amro M.S. El-Ghammaz, Ibtesam M. Khalifa.
Financial support and sponsorship
Conflicts of interest
There are no conflicts of interest.
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[Table 1], [Table 2]