Role of Antioxidants by Comparative Evaluation of Oxidative Stress in Heterozygous v/s Homozygous Sickle Cell Disease Patients: Research Protocol

Background: Red blood cells are sources for oxidants in Sickle cell anaemia, a genetic disorder of itself. Heme iron and oxygen in oxygenated hemoglobin have bonding interaction and association with electron transfer. In response, antioxidant has modus operandi to reduce oxidative stress and damage to RBC and tissue. The studies on biochemical indicators for oxidative stress in sickle cell would further enhance the understanding and present knowledge of effects of antioxidants on the status of iron metabolism and consequent relief to the sickle cell anaemia patients. Objectives: To evaluate oxidative stress and Antioxidant levels and iron indices factors in heterozygous and homozygous sickle cell disease patients and compare between them along with normal healthy control and Iron Deficiency anaemia Methodology: Comparative Cross-Sectional Study is designed to explore specific antioxidant levels and oxidative stress along with Iron Indices among sickle cell anaemia diagnosed cases attending/admitted in the department of Medicine, Paediatrics and Community Medicine at Original Research Article Wasnik et al.; JPRI, 33(60B): 2671-2679, 2021; Article no.JPRI.80094 2672 Jawaharlal Nehru Medical College and its Hospital, Sawangi, Meghe, along with age and sex matched healthy individuals from general population. Results: Oxidative stress as evaluated by stress markers may be higher among the Homozygous sickle cell disease than among the Heterozygous. Conclusion: Oxidative stress may be compounded in sickle cell diseases patients in conjugation with iron deficiency.


INTRODUCTION
Red blood cells are the sources of free radicals in Sickle cell anaemia. RBC's compact with redox-active haemoglobin having rich oxygen supply. The bonding intercommunication within heme iron and oxygen in oxygenated haemoglobin is connected with electron transfer. With these antioxidants help to reduce oxidative stress and Injury to RBC and tissue. Iron extricated by haemolysis is obtained for recirculation and therefore iron deficiency is less common, in turn iron stores increases owing to blood transfusion [1], but still some studies shows iron deficiency in untransmuted patients [2]. Iron deficiency anaemia patients reported increased oxidant activity [3]. Compounded iron deficiency in Sickle cell may lead to increase in fold of oxidant activity and decrease in antioxidant activity. Moreover, Iron indices like Ferritin and Transferrin plays an important role as metabolic antioxidant.
In view of the above observations, it is felt that the studies on biochemical markers of oxidative stress in sickle cell anaemia would further enhance the understanding and present knowledge of effects of antioxidants on the status of iron metabolism and consequent relief to the sickle cell anaemia patients. Hence it is proposed to study the relationship and bearing of levels of antioxidants in sickle cell heterozygous (AS) and sickle cell homozygous (SS) disease patients along with non -sickle cell iron deficiency anaemia cases in the region of Wardha and adjoining districts in Maharashtra.

HYPOTHESIS
Oxidative stress as evaluated by stress markers is more among the Homozygous (SS) Sickle Cell Disease (SCD) than among Heterozygous (AS). The association between oxidative stress among patients of Anaemia due to SCD and iron deficiency states without SCD. Whether there exists the higher level of oxidative stress among the individuals of homozygous SCD as compared with their heterozygous counterpart and among the normal healthy individuals.

MATERIALS AND METHODS
The current study is designed to explore specific antioxidant levels and oxidative stress among Sickle Cell Anaemia (SCA) cases. The study will explore the factors associated with antioxidant levels and oxidative stress among SCA cases, also the specific associated factors among homozygous and heterozygous SCA cases will be determined.

Study Design
The proposed hospital-based case control study will be conducted among the cases of homozygous SCD & heterozygous SCD, which will be compared among themselves and among the healthy controls with or without iron deficiency anaemia.

Study Settings
The current study will be conducted at Jawaharlal Nehru Medical College (JNMC) and Acharya Vinoba Bhave Rural hospital (AVBRH), Sawangi (Meghe) Wardha in Central India.

Study Participants
The JNMC and AVBR Hospital is a tertiary care super speciality health care centre, located approximately 80 km southwest to the city of Nagpur.

Selection of Cases
The hospital conducts weekly specialized clinic for patients of SCD. The clinic collects and maintains an appropriate database of registered cases. This database is evaluated and updated with regular interval. The information of each case of SCD consists of baseline biochemical characteristics including conformation by haemoglobin electrophoresis [4] and solubility test [5], done in Department of Biochemistry. Accordingly, the potential participants that is, homozygous (SS) SCD and heterozygous (AS) SCD will be identified from the database.
The stratified random sampling will be applied for selection of cases. The strata will be based on the type of SCD either homozygous (SS) or heterozygous (AS). Among the SCD cases based on their status of haemoglobin 50% will be selected from homozygous (SS) and remaining 50% will be selected from heterozygous (AS) SCD.

Selection of Controls
The participants in the control group will be selected among the apparently healthy individuals accompanying with patients other than SCD Clinic, such persons will be matched for the age and sex of the case group individuals (as above). A baseline screening of potential controls will be conducted with routine physical examination and primary investigations consisted of hemogram, urine routine, liver function test and kidney function tests. The stratified random sampling for selection of control will be based on the haemoglobin status. Among the controls 50% individuals will be selected with iron deficiency anaemia and remaining 50% with normal haemoglobin concentration.

Sample Size Estimation
The study size is calculated on the formula n=4pq/E 2 Where p is prevalence of oxidative stress among SCD (22.5%) [6].,q is 100-p, E is anticipated relative precision (20% of p, that is 20*22.5/100=4.5).
Therefore, the sample of n= 4x22.5x77.5/4.5 2 =344. As there are three groups, each group will be consisting of 115 participants.
Accordingly, the case group will be consisting of 240 participants with 120 having homozygous SCD and remaining 120 with heterozygous SCD.
The control group will consist of 240 participants with 120 having iron deficiency anaemia and remaining 120 without IDA.

Inclusion Criteria
Following is the inclusion and exclusion criteria that will be applied for while selecting the potential participants in each of the Case and Control groups.
Inclusion criteria for cases: currently on the register of SCD Clinic with documented haemoglobin electrophoresis report.
Exclusion criteria for cases 1) Case of SCD admitted for ailments other than routine blood transfusion. (As they will be biased to have higher oxidative stress) 2) Case of SCD with its complications such as acute crisis 3) History of recent blood transfusion 3 months preceding the enrolment. 4) Case of SCD presented as apparently healthy but detected illness during the baseline screening, infectious diseases presented with acute febrile illness or concomitant chronic non communicable diseases such as Ischemic Heart Diseases, diabetes mellitus, hypertension etc.

Method of Collection of Data
After due consent from the study subject, 4ml venous blood will be collected in plain bulb and allowed to stand for 30 min on the table. After 30 minutes the blood sample in plan bulb shall be centrifuged at 3000rpm for 15 min. Separated serum will be stored at -20 0 c and analysed within two weeks of storage. Collected serum shall be used to estimate following parameters:

Statistical Analysis
The information on the source documents will be digitized in MS excel and further analysed using SPSS version 17.0. The quantitative variables will be analysed by percentages, mean & SD etc. The qualitative variables will be analysed by proportions. The comparative analysis will be performed by individual values with degree of dispersion with 95% confidence interval and the appropriate test of significance with 5% level of significance. The factors associate with oxidative stress will be analysed in the individual groups and compared with bivariate analysis using appropriate test of significance. The quantification of association will be determined by intergroup odds ratio. The logistic regression analysis will be performed to determine the role of independent association of the key factors as determined in the bivariate analysis.

EXPECTED OUTCOMES
Oxidative stress as evaluated by stress markers is more among Homozygous (SS) sickle cell disease than in sickle cell Heterozygous (AS) disease patients. Association of oxidative stress in Sickle cell anaemia and iron deficiency states without sickle cell disease.

DISCUSSION
Polymerization of deoxygenated red blood cells results in a cascade of ischemic injury, inflammation, and vaso-occlusion crises. Cumulative effects of these increases reactive oxygen species (ROS), in turn reflecting increased intensity towards symptoms of SCD with generation of vicious circle [21].
Oxidative stress has a role to play in the pathophysiology of SCA and intervention aimed at increasing the antioxidant capacity of these patients may be beneficial [22]. Striking increased levels of superoxide dismutase and lipid peroxidation are found in both sickle cell carrier and diseases [23].
Normally iron stores is reflected by Ferritin, a metabolic antioxidant produced during iron metabolism. When need arises, they are released by apoferritin shell and binds with transferrin. Transferrin is another metabolic antioxidant, transports iron towards erythropoietic cells. Iron is generally 25 to 30 % saturated with transferrin. Additionally, iron status is constituted by serum iron and total iron binding capacity [24].
In India suggestion towards iron deficiency needs an attention with its caution supplementation in sickle cell anemia, in light of iron status of the body already under the stress due to haemolysis [25]. Also, iron deficiency anaemia seems to be more in heterozygous than homozygous Sickle cell disease [26]. Owing to higher iron content in the reticuloendothelial, sickle cell children show comparatively higher ferritin considering healthy children [27].

CONCLUSION
Oxidative stress may be compounded in sickle cell diseases patients in conjugation with iron deficiency.

CONSENT AND ETHICAL APPROVAL
As per international standard or university standard guideline participant consent and ethical approval will be collected and preserved by the authors.