Antioxidant supplementation for sickle cell disease

Objectives

This is a protocol for a Cochrane Review (intervention). The objectives are as follows:

To assess the efficacy and safety of antioxidant supplementation for improving health outcomes in people with SCD.

Background

See Appendix 1 for a glossary of terms.

Description of the condition

Sickle cell disease (SCD) refers to a group of genetic disorders characterized by the presence of an abnormal haemoglobin molecule called haemoglobin S (HbS). It arises from a point mutation in the gene encoding for the molecule with adenine substituted for thymine at the sixth amino acid codon of the globin chain (Ingram 1958). This leads to the substitution of glutamic acid to valine, an hydrophobic amino acid. In low‐oxygen concentrations, the hydrophobic motifs of this abnormal haemoglobin tetramer are exposed causing globin chains of different haemoglobin to bind together, forming polymers to hide their hydrophobic motifs (Sundd 2018). This polymerization of the HbS results in stiffening of the cell membrane, a change in red cell rheology and the formation of the notable crescent or sickle shape (Li 2017). The red blood cell is able to revert from this shape as oxygen tension improves, however, repeated cycles of sickling and unsickling affects the stability of the cell and the cell eventually irreversibly sickles and is destroyed by the spleen (Goodman 2004). Sickled cells quickly become stuck in the microcapillaries and block circulation to supplied tissues. This in turn exacerbates deoxygenation and further sickling, initiating a vicious cycle of events referred to as the 'sickle cell crises' (Li 2017; Sherman 1978). There is also evidence that haemolysis, within the microvessels, significantly contributes to a reduction in the life span of HbS red blood cells, this is likely from oxidative damage (lipid peroxidation) to the red blood cell membrane (Fasola 2007; Kato 2009). Irreversibly sickled cells may lyse spontaneously, releasing free haemoglobin and other cell contents into the circulation. These compounds trigger the generation of oxygen free radicals, reduced nitric oxide concentrations that have been linked to the vascular stability and observed physiologic effects of crises (Aslan 2000; Chirico 2012; Mockesch 2017; Silva 2013). These effects include: an increased rate of haemolysis; adhesion of red blood cells to the endothelial wall of capillaries; and adhesion of the red blood cells, white blood cells and platelets. Increased oxidative stress has also been linked with an increase in some complications of SCD, especially acute chest syndrome and pulmonary hypertension. Antioxidants are said to play a role in ameliorating this oxidative stress and improving the overall well‐being of an individual with SCD (Chirico 2012; Gizi 2011; Morris 2008). Inflammation and reactive oxygen species (ROS) are linked in many chronic diseases, including SCD. While inflammatory cells generate ROS, ROS at physiologic concentrations, can initiate intracellular signalling cascade that enhances pro‐inflammatory gene expression. However, when ROS are produced in excess, as may occur in SCD, it leads to oxidative stress, which in turn may worsen the clinical manifestation of SCD. It also implies that the simultaneous existence of low‐grade chronic inflammation and oxidative stress can exist in SCD (Biswas 2016). The aetiology of this chronic inflammation is multifactorial and involves an increase in the number of and phagocytic function of leukocytes, the elaboration of cytokine production (such as Interleukin 1β, TNF‐α, interleukin 6), and changes to the cell membrane (leading to the externalization of phosphatidyl serine and an increased expression of adhesion molecules both on the surface of the RBC (CD36, integrin‐α4β1) and on the endothelial cells (e.g. VCAM1, ICAM 1, E‐selectin, P‐selectin)) (Chies 2001; Conran 2018). This leads to increased vaso‐occlusion with resultant ischaemia. Following reoxygenation, a reperfusion injury occurs, thereby increasing the oxidative stress and the mopping up of nitric oxide by the free radicals, as well as further endothelial injury, the escalation of the chronic inflammatory state and vaso‐occlusion. These repetitive episodes of inflammation, ischaemia and reperfusion injury have been linked to recurrent painful crises as well as chronic organ damage (Ballas 2012; Brandow 2017; Conran 2018; Ware 2017).

Description of the intervention

The discovery of the antioxidant properties of vitamins C and E created opportunities for studies on the range of uses for vitamins and other antioxidants. Many antioxidants are now used for managing conditions such as diabetes, malignancies, hypertension and for neurological and heart diseases (Ginter 2014). They exert their effect by countering the production of dangerous oxidants.

Most antioxidants contain aromatic or phenolic rings that donate a hydrogen atom (H) to the free radicals, thereby neutralizing their oxidative effect and interrupting the auto‐oxidative chain reaction (Brewer 2011; Cao 2014). These antioxidants form free‐radical intermediates, e.g. alpha tocophenoxyl and ascorbyl radicals, for both vitamins E and C, respectively (Brewer 2011), which are further stabilized by other antioxidants or by resonance de‐localization of the electron within aromatic ring or formation of quinone structure (Nawar 1996). Additionally, lipoic acid, although not belonging to the phenolic group, also acts as an excellent hydrogen atom donor, especially in its dehydrogenated form (dihydrolipoic acid) (Castaneda‐Arriaga 2014).

Antioxidants, such as ascorbic acid and zinc, act by chelation or antagonism of transition metals such as iron and copper; neutralizing their capacity to generate free radicals (Brewer 2011; Marreiro 2017; Powell 2000). Glutamine serves as a precursor to key molecules like glutathione, which can scavenge ROS directly or indirectly by acting as a substrate for gluthathione peroxidase and glutathione‐S transferase, both of which are potent endogenous antioxidants (Masella 2005).

Antioxidants work through various mechanisms and at various sites and therefore may have a different clinical impact in SCD, e.g. vitamin C is hydrophilic and acts intracellularly while vitamin E is lipophilic acting on the membrane. Despite the overall anticipated benefits of antioxidants, in conditions in which a high level of ROS is required to induce apoptosis, it may become harmful. This variability in the functional need for antioxidants may also determine the outcome of antioxidant therapy in people with SCD. Other factors that affect antioxidant action include: solubility in the food matrix; pH; temperature; activation energy; the rate constant; and the oxidation‐reduction potential of the antioxidant (Brewer 2011; Kurutas 2016; Nawar 1996).

How the intervention might work

The production of ROS occurs in healthy individuals but they are neutralized by the natural antioxidant mechanisms in the body and the role of some genetic modifiers in regulating oxidant stress has been elucidated in the literature. One of these is the nuclear factor erythroid 2‐related factor 2(nrf‐2) that increases the transcription of several target genes responsible for the production of innate antioxidants (Da 2017; Pall 2015). In SCD there is an increase in free radical generation from the increased activity of many oxidases, Hbs auto‐oxidation, heme iron release and decreases in nitric oxide concentrations (Aslan 2000). Studies have also reported a reduction in the natural protective mechanisms, such as superoxide dismutase, glutathione peroxidase, catalase, haem oxygenase, glutathione, vitamin C, vitamin E, etc. (Antwi‐Boasiako 2019; Chirico 2012; Gizi 2011; Silva 2013). The reduction in these natural antioxidants may be related to the genetic modifiers referred to earlier. This shifts the balance towards increased circulation of free radicals resulting in increased haemolysis, endothelial damage, increased cell adhesion, hypercoagulability, vaso‐occlusion, altered gene expression via DNA methylation and histone modifications (Chirico 2012; Nur 2011). The use of antioxidants in SCD could reverse or limit the progression of tissue damage and haemolysis by clearing free radicals (Belini 2012; Gizi 2011). Antioxidants can also exert their effects in various other ways. For example, selenium can act through direct modification of the epigenome by modulating DNA methylation and histone modification so as to favour anti‐inflammatory processes; phenolic antioxidants (e.g. vitamin E) also act in part by increasing nrf2 and may exert some anti‐inflammatory effect through same mechanism; levels of gluthathione can also be increased by some other antioxidants through direct activation of enzymes involved in gluthathione synthesis (Da 2017; Pall 2015; Speckmann 2015).

In clinical trials, antioxidants have been shown to be useful as adjunct treatment in preventing tissue injury during cancer therapy and potentiating the anti‐tumor effects of chemotherapy (Thyagarajan 2018). The accumulation of free radicals and the consequent oxidation of biological molecules is considered one of the mechanisms in ageing; and the use of antioxidants in preventing age‐related organ damage is currently being explored (Fusco 2007).

Why it is important to do this review

SCD is the most common monogenic disorder and a major public health concern globally (Johnson 2016). It is estimated that more than 300,000 babies are born annually with the disease, with the majority of these being in sub‐Saharan Africa where up to 2% of the population are affected (with the carrier state in some countries being as high as 30%) (WHO 2010). The high prevalence of HbS also mirrors the prevalence of malaria in this region (Macharia 2018; Piel 2010; Piel 2013). Although recent interventions have improved the survival of people with SCD, early childhood mortality in low‐income countries is still as high as 50% (Grosse 2011). SCD is characterized by the frequent punctuation of the steady state by crises (pain, sequestration, haemolysis or aplastic) and long‐term complications can follow due to vaso‐occlusion and haemolysis. Frequently encountered complications include stroke, retinopathy, avascular necrosis (especially that of femur), nephropathy, pulmonary hypertension, etc. (Macharia 2018; Sawe 2018). Many interventions (including antioxidants) are being evaluated to improve the clinical outcome of people with SCD. To date, antioxidants have not been widely accepted as a treatment for managing SCD, given the conflicting evidence in clinical trials. However, they could be a low‐cost and accessible add‐on treatment for people with SCD and help to improve quality of life (QoL). Therefore, it is important to review and synthesize the high‐quality evidence that is available on the effect of antioxidants on clinical outcomes and the QoL of people with SCD.

Objectives

To assess the efficacy and safety of antioxidant supplementation for improving health outcomes in people with SCD.

Methods

Criteria for considering studies for this review

Types of studies

All randomised controlled trials (RCTs) and quasi‐RCTs. Cross‐over RCTs will be included; however, only results from the first phase of each trial will be analysed if no 'washout' period is included in the trial design. A washout phase is designed to limit any potential residual treatment effects and as there is no consensus on duration, we have not defined a minimum duration for the washout (Elbourne 2002).

Types of participants

Individuals with SCD (including, but not limited to, homozygous sickle cell anemia (HbSS), HbS‐beta thalassaemia, sickle cell–hemoglobin C (HbSc)), irrespective of age, gender, genotype, disease severity, co‐morbidities or other concomitant drug therapies.

Types of interventions

Antioxidants (see 'Description of the intervention') compared to placebo or no treatment. We will also include trials that compare different types of antioxidants to each other, and those with co‐interventions (if they are the same across treatment arms).

Types of outcome measures

Primary outcomes

1. Frequency of crisis

2. Severity of pain (as reported by trial authors)

3. QoL of participants living with SCD and their caregivers (using a validated form, e.g. the SF36 questionnaire)

Secondary outcomes

1. Adverse effects (as reported by included trials)

2. Frequency of hospitalisation

3. Frequency of sickle cell‐related complications (e.g. chronic organ damage, avascular necrosis, priapism)

4. Haemoglobin status

5. Laboratory markers of haemolysis and inflammation

Search methods for identification of studies

We will search for all relevant published and unpublished trials without restrictions on language, year or publication status.

Electronic searches

The Cochrane Cystic Fibrosis and Genetic Disorders Group's Information Specialist will conduct a search of the Group's Haemoglobinopathies Trials Register for relevant trials using the following terms: (sickle cell OR (haemoglobinopathies AND general) AND (antioxidants OR vitamin e OR vitamin C OR vitamin a OR ascorbic acid OR zinc OR Glutamine OR glutathione OR Retinoid OR Selenium OR Micronutrients OR Beta‐carotene OR Edetate sodium).

The Haemoglobinopathies Trials Register is compiled from electronic searches of the Cochrane Central Register of Controlled Trials (CENTRAL) (updated each new issue of¬the Cochrane Library) and weekly searches of MEDLINE. Unpublished work is identified by searching the abstract books of five major conferences: the European Haematology Association conference; the American Society of Hematology conference; the British Society for Haematology Annual Scientific Meeting; the Caribbean Public Health Agency Annual Scientific Meeting (formerly the Caribbean Health Research Council Meeting); and the National Sickle Cell Disease Program Annual Meeting. For full details of all searching activities for the register, please see the relevant section of the Cochrane Cystic Fibrosis and Genetic Disorders Group's website.

We will search the following trial registries:

• US National Institutes of Health Ongoing Trials Register Clinicaltrials.gov (www.clinicaltrials.gov);

• World Health Organization International Clinical Trials Registry Platform (WHO ICTRP) (apps.who.int/trialsearch).

For details of our search strategies, please see the appendices (Appendix 2).

Searching other resources

We will check the bibliographies of included studies and any relevant systematic reviews identified for further references to relevant trials. We will also contact trial authors, experts and organisations in the field to obtain additional information on relevant and ongoing trials.

Data collection and analysis

We will employ the standard methods of the Cochrane Cystic Fibrosis and Genetic Disorders Group and refer to the methods in the Cochrane Handbook of Systematic Reviews of Interventions (Higgins 2011a).

Selection of studies

One author (ABB) will check for and remove duplicates and enter potentially eligible trials into Covidence (Covidence 2019). Two review authors (ABB and AAO) will independently assess abstracts and, if necessary, the full text of trials to determine which trials satisfy the inclusion criteria. We will resolve discrepancies by discussion and consensus with a third review author (JO). We will present the results in a PRISMA flow diagram.

Data extraction and management

Two review authors (BAA and OEO) will independently extract data using a standard data extraction form in Covidence (Covidence 2019). We will attempt to translate any trials reported in a non‐English language before assessment by contacting Cochrane's Task Exchange (Task Exchange 2019).

We will collect data on:

1. participant characteristics;

2. trial characteristics and design;

3. interventions and comparator;

4. outcome data ‐ reported separately for each outcome.

We will refer to chapter 7 of the Cochrane Handbook of Systematic Reviews for Interventions (Higgins 2011b). We will resolve discrepancies by discussion and achieve consensus with a third review author (OO). When data are incomplete, we will contact the authors of the included trials to request further information and clarification. When we identify multiple publications from a single trial, we will group these references together. We will enter the extracted data into the Review Manager (RevMan) software for analysis (RevMan 2014).

Assessment of risk of bias in included studies

Two review authors (BAA and OEO) will use the risk of bias tool, described in the Cochrane Handbook of Systematic Reviews for Interventions, to assess the risk of bias across six domains for each trial (Higgins 2011c). These domains are:

1. sequence generation;

2. allocation concealment;

3. blinding (self‐reported and objective);

4. incomplete outcome data;

5. selective reporting, and

6. other potential sources of bias.

We will assess each domain as having either a 'low', 'unclear' or 'high' risk of bias, as per the guidance in chapter 8 of the Cochrane Handbook for Systematic Reveiws of Interventions (Higgins 2011c). We aim to resolve discrepancies by discussion and achieve consensus with a third review author (OO). We will enter the data into the ’Risk of bias’ tables in the 'Characteristics of included studies' table.

If a trial describes the randomisation and allocation processes, including concealment from the researchers and at least two review authors deem these to be adequate, then we will consider the trial to have an overall low risk of bias. When these processes are inadequate or unclear, we will deem the trial as having an overall high risk of bias or unclear risk of bias, respectively.

We will not exclude trials on the basis of risk of bias, but will perform a sensitivity analysis to explore the synthesis of evidence with variable quality.

Measures of treatment effect

For dichotomous outcomes, we will record the number of participants with the event and the number of participants analysed in each group. We will calculate a pooled estimate of the treatment effect for each outcome across trials using a risk ratio (RR) with 95% confidence intervals (CI), where appropriate. We will present absolute treatment effects with number needed to treat for an additional beneficial (NNTB) or harmful outcome (NNTH) for all outcome measures regardless of statistical significance. For continuous outcomes, we will record the mean change from baseline and its standard deviation (SD) for each group or mean post‐treatment values and SD or standard error (SE) for each group. We will calculate a pooled estimate of the treatment effect for each outcome using the mean difference (MD) with 95% CIs or the standardised mean difference (SMD) with 95% CIs, depending on the variability of outcome measures (Deeks 2011).

Unit of analysis issues

The unit of analysis in a trial with a parallel group design will be the individual participant.

We will review any trial with a cross‐over design. If a 'washout' period is included in the trial design and investigators perform an appropriate paired analysis, then we will include the effect estimate of the intervention for each outcome in a meta‐analysis using the generic inverse‐variance method (Higgins 2011d). If a 'washout' period is not included or investigators do not analyse the data appropriately (i.e. paired analyses), we will include data from the first phase of the cross‐over and analyse these as if the trial had a parallel group design (Deeks 2011).

Dealing with missing data

If the reported trial data are insufficient or unclear for our purposes, we will contact the trial author(S) or sponsor(S) (or both) to request data and additional information to complete our assessment. We will assess whether investigators have performed an ITT analysis and report the number of participants missing from each trial arm, where possible (Higgins 2011d).

Assessment of heterogeneity

We will assess heterogeneity between trials using the Chi² and I² statistics and by visual inspection of the overlap in CIs on the forest plots (Higgins 2003). Regarding the Chi² test, a P value of less than 0.1 is of interest. Regarding the I² statistic, we will define our interpretation as in chapter 9 of the Cochrane Handbook for Systematic Reviews of Interventions (Deeks 2011):

• 0% to 40%: no heterogeneity;

• 30% to 60%: may represent moderate heterogeneity;

• 50% to 90%: may represent substantial heterogeneity;

• 75% to 100%: considerable heterogeneity.

Assessment of reporting biases

We will minimise reporting bias from the non‐publication of trials or selective outcome reporting by using a broad search strategy, searching trial registries and contacting regulatory agencies. If we identify 10 or more trials we will create a funnel plot to assess for publication bias (Sterne 2011).

To assess for selective reporting, we will compare trial protocols (if available) with the reported outcomes in the trial publication. If a trial protocol is unavailable, we will compare the methods section and outcomes reported in the results section. We will record information on the sponsors and funding sources for trials and conflicts of interest of authors in order to assess for external bias.

Data synthesis

When possible we plan to combine trials in meta‐analyses (Deeks 2011). We plan to analyse data using the fixed‐effect model; however, if there is at least substantial heterogeneity (I² ≥ 50%) between the identified trials, we will use the random‐effects model for analysis. If there are insufficient data available for any meta‐analysis, we will report any available information narratively.

Subgroup analysis and investigation of heterogeneity

If we identify and include sufficient trials and if we identify at least substantial heterogeneity, we will investigate possible causes through subgroup analyses.

1. Types of antioxidants

2. Age of participants (children up to 18 years versus adults)

3. Trial location (comparing the effect of race and nationality on the outcome of the trial)

4. Types of treatment (steady‐state treatment versus acute‐care treatment)

Sensitivity analysis

If we perform any meta‐analyses with sufficient trials, we will conduct sensitivity analyses to assess for the effect of the overall risk of bias by including or excluding those trials with an overall high risk of bias. We will also assess the effect of including or excluding cross‐over trials (Deeks 2011).

Summary of findings table

Two authors (OO and JO) will independently use the five GRADE considerations (trial limitations, consistency of effect, imprecision, indirectness, and publication bias) to assess the quality of the body of evidence identified for seven prespecified outcomes that are relevant to clinicians and consumers (Schünemann 2011a). We will use methods and recommendations described in section 8.5 and chapter 12 of the Cochrane Handbook for Systematic Reviews of Interventions (Higgins 2011c; Schünemann 2011b), employing GRADEpro GDT software (GRADEpro GDT 2015). We will justify all decisions to downgrade the quality of trials in footnotes, and make comments to aid readers' understanding of the review where necessary.

We will report a 'Summary of findings' table for each of the following outcomes using the GRADEPRO software:

1. frequency of crisis;

2. severity of pain;

3. QoL of participants living with SCD and their caregivers;

4. adverse effects (as reported by included trials)

5. frequency of hospitalisations;

6. frequency sickle cell‐related complications;

7. haemoglobin status.

History

Protocol first published: Issue 4, 2020

Appendices

Appendix 1. Glossary of terms

Term	Definition
Polymerization	Arrangement of haemoglobin within the red blood cell as long parallel fibres
Microcapillaries	The microvessels that carry blood from arterioles to venules. They are lined by the endothelial walls and they serve as an exchange site between the intravascular compartment and the interstitium
Lyse	Break down
peroxidation	Loss of free electron to free radicals
neutralizing	Removing the toxic effect
resonance delocalization	Release of free electron from a covalent bond
cell signaling	Transfer of information from receptors on the cell surface to the nucleus for gene expression
gene expression	Process by which nucleotide sequence is being used for protein synthesis

Appendix 2. Search methods – electronic searches

Database/ Resource	Strategy
ClinicalTrials.gov	[Advanced search] CONDITION OR DISEASE: sickle cell OTHER TERMS: antioxidant OR oxidative OR vitamin e OR vitamin C OR vitamin a OR ascorbic acid OR zinc OR Glutamine OR glutathione OR Retinoid OR Selenium OR Micronutrients OR Beta‐carotene OR Edetate sodium OR EDTA STUDY TYPE: Interventional Studies (Clinical Trials)
WHO ICTRP	[Advanced Search] CONDITION: sickle cell INTERVENTION: antioxidant OR oxidative OR vitamin e OR vitamin C OR vitamin a OR ascorbic acid OR zinc OR Glutamine OR glutathione OR Retinoid OR Selenium OR Micronutrients OR Beta‐carotene OR Edetate sodium OR EDTA RECRUITMENT STATUS: All

Contributions of authors

Abiola B Bolarinw (ABB) and Olabisi Oduwole (OO) conceived the review. ABB, JO and OO designed the protocol. ABB wrote the first draft of the protocol. All authors reviewed and critically appraised the draft of this protocol.

Sources of support

Internal sources

• No sources of support supplied

External sources

• National Institute for Health Research, UK

  This systematic review was supported by the National Institute for Health Research, via Cochrane Infrastructure funding to the Cochrane Cystic Fibrosis and Genetic Disorders Group.

Declarations of interest

All authors: none known.

New