Effects of l‐arginine supplementation in patients with sickle cell disease: A systematic review and meta‐analysis of clinical trials

Abstract Background and Aims Previous studies have shown that supplementation of some amino acids such as l‐arginine or its precursors could exert beneficial effects in patients with sickle cell disease (SCD). The objective of this study is to systematically review the literature to assess the effect of arginine administration on the clinical and paraclinical parameters of patients with SCD. Methods Four online databases of PubMed, Web of Sciences, Scopus, and Embase were selected for systematic search. Eligible studies were clinical trials that evaluated the effect of arginine usage in patients with SCD. Effects sizes were calculated using weighted mean difference (WMD) and Hedge's g and they were pooled using random‐effects modeling with Hartung–Knapp adjustment. Additional analyses were also conducted. Results Twelve studies containing detail of 399 patients with SCD were found to be eligible. The data synthesis showed that l‐arginine significantly increased the level of NO metabolites (Hedge's g: 1.50, 0.48–1.82, I 2: 88%) and hemoglobin F (WMD: 1.69%, 0.86–2.52, I 2: 0%) and significantly decreased systolic blood pressure (WMD: −8.46 mmHg, −15.58 to −1.33, I 2: 53%) and aspartate transaminase (Hedge's g: −0.49, −0.73 to −0.26, I 2: 0%). However, there were no significant effects on hemoglobin, reticulocyte, malondialdehyde and diastolic blood pressure, and alanine transaminase. Conclusion Our meta‐analysis showed that l‐arginine use for SCD could be beneficial, increase hemoglobin F and exert blood pressure‐lowering and hepatoprotective properties. However, for a firm conclusion and widespread use of l‐arginine for these patients, more studies are needed.


| INTRODUCTION
Sickle cell disease (SCD), a group of multisystem autosomally recessive inherited hemoglobin disorders, is caused by a point mutation in the gene encoding β chains of hemoglobin. 1,2 Although there is no accurate estimate of the global prevalence of SCD, it has been reported that nearly 6 million neonates are born each year with SCD, more than 300,000 with the homozygous genotype of the disease, worldwide. 3 The mutated gene gives rise to the production of dysfunctional chains and therefore, the formation of a dysfunctional hemoglobin, HbS. This hemoglobin possesses a lower affinity for oxygen and is liable to polymerization upon response to some triggers such as low oxygen tension, imbalance of acid-base homeostasis, and alteration in the temperature which results in highly inflexible, fragile, and adhesive erythrocytes. 4 These abnormal erythrocytes contribute to the main features of this multisystem disorder, premature destruction of the red cells, hemolysis and the ensuing anemia, and vaso-occlusion. 5 Substances such as arginase 1 and asymmetric dimethylarginine which are released from ruptured red cells target the synthesis of nitric oxide (NO) and its downstream pathway which has an important role in the pathophysiology of the course of SCD. 1,6 Previous studies have shown that supplementation of some amino acids such as L-arginine or its precursors could exert beneficial effects in patients with SCD, during both the steady state and the crisis. [7][8][9][10] This amino acid is considered to be a conditionally essential amino acid, particularly under certain circumstances in patients with a catabolic state or in the presence of an acute stressor. 11 L-arginine contains a nitrogenous group of guanidine which involves binding to NO synthase and serves as a substrate for this enzyme for NO production. 11,12 In a randomized placebo-controlled clinical trial, participants in the intervention group received L-arginine at a dose of 500 mg daily for over 4 months. 13 The study showed a significant increase in the level of NO metabolites in those on L-arginine compared with patients in the placebo group. Besides, this study demonstrated that this dietary supplement provided an analgesic effect. In addition to these effects, it has also been reported that L-arginine could have antioxidant, cardioprotective, hepatoprotective, and immunomodulatory properties and improve blood pressure control, metabolic profile components, and response to the therapeutic effects of hydroxyurea. 7,[14][15][16][17][18] Despite this body of evidence regarding the beneficial effects, safety, and tolerability of supplemental arginine in patients with SCD, no consensus has been reached on its use; besides, no meta-analysis has been conducted on the issue. The objective of this study is to systematically review the effect of L-arginine administration on the clinical and paraclinical parameters of patients with SCD.

| Databases and search strategy
The protocol of this study was registered in the International Prospective Register of Systematic Reviews (PROSPERO) with the registration number CRD42022355733. In this review, we used Preferred Reporting Items for Systematic reviews and Meta-Analysis (PRISMA) guideline version 2020. 19 For the literature review, four online databases were selected, PubMed, Web of Sciences, Scopus, and Embase. For the search strategy, we used two groups of words related to arginine and SCD.
The words for arginine were "Arginine", "L-arginine", "ArginMax", "AAKG", "Argicap", and "Argitall". The words considered for SCD were "Sickle cell", "SCD", "Sickle cell syndrome", "Sickle cell anemia", "hemoglobin S disease", "HBS disease", and "Sickling disorder". A combination of these terms was searched in the title, abstract, and keywords of the articles. We conducted our search twice; our primary search was done on April 24, 2022, and then was updated on January 31, 2023. The search strategies that we used in the online databases are provided in the Supplementary material. Furthermore, we checked the reference list and citations of records considered eligible to identify additional relevant documents. Respective main articles of eligible conference proceedings/abstracts were searched manually to retrieve the complete data.

| Eligibility criteria
We restricted our research to trial studies that evaluated the effect of supplemental L-arginine in patients with SCD. The eligible trials were on human subjects with an established diagnosis of SCD. We excluded studies in which a combination of arginine with other amino acids or supplements was administrated to the patients. Single-arm studies, studies with a control group consisting of normal healthy individuals, nonrandomized trials, and noninferiority trials were not excluded from the study. There was no restriction on the age of the participants, the genotype of SCD (homozygous form or others), the setting of the patients (outpatient or inpatient), language, country, or publication year. Conference proceedings/abstracts, editorial papers, letters to the editor, and correspondences were removed in the screening rounds in the case that they presented the same findings; otherwise, they were included in the case that they met other eligibility criteria.

| Selection process and data extraction
Two authors (A. S. and N. D. E.) conducted this systematic search. The records found upon the search in the databases were exported to Endnote library (Clarivate Analytics) version X9. After the removal of duplicate records, these two authors completed the first round of screening based on the titles and abstracts of the identified records. Then, full texts of the remaining records were retrieved and screened by two authors (A. S. and N. D. E.), independently. Disagreements between the authors were resolved by discussion. Interrater reliability was measured by both percent agreement and Cohen's Κ. 20,21 We designed an Excel spreadsheet for data extraction. These authors (Ehsan T. and Erfan T.) were responsible for the process.
The following data were extracted from each study: first author, publication year, country, study design, the sample size in each group of the study, characteristics of the participants, percentage of males, age of the participants, the genotypic subtype of SCD, dosage, route and duration of administration of arginine and endpoints of each study and adverse events occurred in the course of study.

| Risk of bias assessment
Based on the design of the included clinical trial, we used Cochrane tools for risk of bias assessment, revised Cochrane risk of bias tool for randomized trials (RoB2), and risk of bias in nonrandomized studies of interventions (ROBINS-I). 22,23 The risk assessment was conducted by two authors (F. H. and E. R.), separately.

| Certainty of evidence assessment
Assessment of certainty of the evidence was done by two authors (F. H. and E. R.) using the Grading of Recommendations, Assessment, Development and Evaluation (GRADE) approach, separately. 24,25 Using this approach, the quality of a body of evidence for each outcome was rated as one of the following four grades: high, moderate, low, or very low. This rating was determined by assessing the outcomes and its quantitative analysis against several factors including the presence of risk of bias, inconsistency of the results, indirectness of the evidence, imprecision, publication bias, large magnitude of an effect, dose-response gradient and effect of plausible residual confounding.  26 The effects sizes of arginine use on NO metabolites concentration, hemoglobin, hemoglobin F (HbF), reticulocyte, malondialdehyde level, blood pressure level, both systolic and diastolic blood pressure (SBP and DBP), aspartate transaminase (AST), and alanine transaminase (ALT) were estimated. These effects were pooled using weighted mean difference (WMD) for hemoglobin, Hb, SBP, and DBP. For the rest of the variables, Hedge's g statistic was used. Then, random effects modeling, the DerSimonian-Laird model with standard-error adjustment for the overall effect size of Hartung-Knapp, was applied. 27 Standard deviations (SD) were measured from standard errors of the mean or confidence intervals (CI) by use of appropriate formulas when they were not reported in the articles. 28 The r value as a correlation coefficient was measured using pre-and postadministration of arginine SDs and SD of change when available. Interstudy heterogeneity was assessed using I 2 statistics; we also calculated the 95% CIs for I 2 . 29 Sensitivity analyses and subgroup analyses were also performed. We used Egger's test for the presence of potential publication bias.

| Characteristics of the included studies
Characteristics of the 12 records in this systematic review and metaanalysis are provided in Table 1. All the included studies were trials, some of them with a randomized design (six studies). All studies except the one that was conducted by Elias et al., 8   Data from six trials were used for the quantitative analysis of changes in the concentration of NO, 7,8,13,[30][31][32] five for changes in hemoglobin, 8,9,[33][34][35] three for changes in HbF, 8,9,33 three for changes in reticulocytes, 8,33,34 four for changes in malondialdehyde level, 9,14,32,35 three for changes in SBP and DBP, 9,10,34 and three for changes in AST and ALT. 7,9,34 The results of the quality assessment with ROBINS-I and RoB2 for included randomized and nonrandomized trials are there was at least one study with a high risk of bias based on the Cochrane tools for risk of bias assessment we used in this review.

| Additional analyses
As shown in Supporting Information: Figure 1, sensitivity analyses for the changes in the concentrations of NO metabolites, hemoglobin, reticulocyte, malondialdehyde, DBP, and ALT did not result in any significant differences. However, for changes in HbF, SBP, and AST, the pooled effect sizes were sensitive to the "leave-one-out" of the studies (Supporting Information: Figure 1). The potential publication bias in this study was assessed using Egger's test. The test showed that the estimation of none of the pooled effect sizes was significantly affected by a considerable publication bias.
F I G U R E 1 PRISMA 2020 flowchart of study identification, screening, and selection process.
T A B L E 1 Characteristics of the studies included in this systematic review and meta-analysis.

| Certainty of evidence
The certainty of the evidence was very low for four outcomes estimated in our study, change in concentration of NO metabolites, reticulocyte, malondialdehyde, and DBP. The overall certainty for changes in the levels of hemoglobin, HbF, SBP, ALT, and AST was rated as low. Therefore, for none of the outcomes, the certainty of the evidence was moderate or high. The main two reasons for rating down the evidence across different outcomes were the risk of bias and imprecision (Table 2). Besides, there was not any factor to upgrade the quality of the evidence for each of the estimated outcomes. of L-arginine and the production of citrulline and NO. Therefore, the concentration of L-arginine seems to be tightly related to production and thus, the concentration of NO. 39 Consistently, our study found a positive association between L-arginine supplementation and serum nitrogen oxides; There was a statistically significant increase in nitrogen oxides level (Hedge's g of 1.50 with 95% CI of 0. 48-1.82) following the use of L-arginine. In a population-based study on 2771 individuals, the dietary intake of L-arginine was measured using a semiquantitative questionnaire. 40 In this study, it was reported higher intake of L-arginine was associated with higher concentrations of serum NO compounds. Therefore, not only the use of L-arginine as a separate medicinal medication or product but also, the intake of diets with higher arginine content might bring the positive effects shown in our study for SCD patients; however, concerning the dysregulated The analyses showed that L-arginine could lead to a higher level of HbF in patients with SCD. Augmentation of HbF synthesis is among the most effective measures for these patients. In a cohort study on 3764 patients with SCD, risk factors for early death were investigated. 41 In this study, it was shown that higher levels of HbF were associated with a higher rate of survival. Besides, HbF could help to prevent complications occurring as a result of the sickling phenomenon of altered hemoglobin, successive vaso-occlusion, and abnormal blood viscosity. 42,43 The main therapeutic effects of hydroxyurea on morbidity and mortality of patients with SCD are believed to be exerted through its enhancing effect on the level of HbF. 44 Therefore, L-arginine supplementation could also result in positive outcomes.

| Adverse events
The included studies also showed that this effect of L-arginine on the HbF level could be additive to the effect of hydroxyurea and could further increase the HbF level in those who are on hydroxyurea. In the study conducted by Temiye et al., the study participants were on hydroxyurea therapy before the trial and then, L-arginine was given to them. The study reported that there was a mean increase of about 32% in the level of HbF in these patients. 9 A similar finding was observed in Elias et al. work 8 ; the participants were taking hydroxyurea for more than 1 year and then they were assigned to either receive L-arginine or not. After the 12 weeks of trial, the patients in the intervention group had a significant increase in the level of HbF.
Therefore, it seems that L-arginine may have the potential to be used as an adjunct to improve the induction of a high level of HbF.
Significant decreases in levels of AST observed in the study could be indicative of the hepatoprotective effects of L-arginine. Liver individuals without a history of a medical illness were selected and their liver functions were, then, measured. 48 The serum concentrations of liver enzymes were shown to be higher in the SCD patients than in the control group. Consistently, in another cross-sectional study, only onequarter of steady-state patients had normal levels of liver enzymes. 49 The dysfunction and abnormality in the liver enzymatic activity could further proceed following occlusion of the vessels and sinusoids and subsequent ischemia, and extreme destruction of the red cells with injurious products within a crisis. 50  given to the participants in the included studies, we were not able to use meta-regression to explore whether this different dosing could be a source of heterogeneity in the overall estimates. Fourth, we also investigated potential publication bias in this study and the statistical analyses showed no potential publication bias; however, since there was a small number of studies in each analysis, the results of the publication bias test may not be reliable. Fifth, the certainty of the body of evidence for all the outcomes estimated in this study was either low or very low mainly owing to significant limitations in the included studies and imprecision. Therefore, our study showed statistically significant beneficial effects for treatment with L-arginine without serious adverse effects with low and very low-quality of evidence. Therefore, its findings should be approached with caution, and well-developed and adequately powered trials are required to consolidate them.
T A B L E 2 Certainty of evidence for each outcome estimated in this systematic review and meta-analysis using the GRADE approach. supervision; writing-original draft; writing-review and editing.

ACKNOWLEDGMENTS
The authors would like to express special thanks to Dr. Hamed Movahed for improving the use of English in this work.

CONFLICT OF INTEREST STATEMENT
The authors declare no conflict of interest.

DATA AVAILABILITY STATEMENT
The data sets generated during and/or analyzed during the current study are available from the corresponding author on reasonable request.

TRANSPARENCY STATEMENT
The lead author Erfan Taherifard affirms that this manuscript is an honest, accurate, and transparent account of the study being reported; that no important aspects of the study have been omitted; and that any discrepancies from the study as planned (and, if relevant, registered) have been explained.