Systematic Review of L-Citrulline Supplementation Effect in Patients with Sickle Cell Disease

Background and Aim: Preliminary studies indicated that L-Citrulline supplementation is safe and could be beneficial in patients with SCD [1]. The objective of this study is to systematically review the literature to assess the effect of L-Citrulline administration on both oxidative stress measures and clinical outcomes in patients with SCD. Method: A search was performed in four online databases (PubMed, Web of Science (Clarivate), CINAHL (EBSCO) and EMBASE (Elsevier) and the search engine Google Scholar [2] up to Dec. 2024. Results: Three studies involving a total of 153 patients with SCD were deemed eligible for this search. All 3 studies showed that oral L-Citrulline supplementation significantly increased plasma arginine levels (P value <0.05). Two of the studies showed improved other oxidative stress measures with L-Citrulline supplementation. One study reported improved well-being in all participants. Adverse events were rare, mild, and transient. Conclusion: Our systematic review showed that oral L-Citrulline supplementation is safe in patients with SCD and could be beneficial in decreasing oxidative stress and improving quality of life. However, more studies are needed for a definitive conclusion on using L-Citrulline for these patients. Title Page: Title: Systematic Review of L-Citrulline Supplementation Effect in Patients with Sickle Cell Disease Authors: Zahra Al Khuridah, MD University of Illinois Chicago Corresponding author: Address: 840 S. Wood St., MC 856 Pediatrics, University of Illinois Chicago, Chicago, IL. 60612 Phone: +1(872) 257-7050 Fax: +(202) 914-0636 Email: [email protected] Rashid Halloway, MPH, MSc University of Illinois Chicago Tina Griffin, MLIS University of Illinois Chicago Lewis Hsu, MD, PhD University of Illinois Chicago Word Count:

184 words Main text: 2402 Tables, figures and supporting files: This manuscript contains 2 tables, 1 figure, and 1 supporting information file. Short running title: Systematic Review of L-Citrulline Supplementation Effect in Patients with Sickle Cell Disease

Sickle Cell Disease Citrulline Safety Adverse Effect Therapeutic Nutritional Supplement Arginine Abbreviations key: | NO | Nitric Oxide | | VOE | Vaso-occlusive episode | | PROSPERO | Prospective Register of Systematic Reviews | | PRISMA | Preferred Reporting Items for Systematic Reviews and Meta-Analyses | | IV | Intravenous | | ADMA | Asymmetric dimethylarginine | | SD | Standard Deviation | Title: SYSTEMATIC REVIEW OF L-CITRULLINE SUPPLEMENTATION EFFECT IN PATIENTS WITH SICKLE CELL DISEASE Zahra Al Khuridah, Rashid Halloway, Tina Griffin, Lewis L. Hsu University of Illinois Chicago, USA

Background and Aim: Preliminary studies indicated that L-Citrulline supplementation is safe and could be beneficial in patients with SCD [1]. The objective of this study is to systematically review the literature to assess the effect of L-Citrulline administration on both oxidative stress measures and clinical outcomes in patients with SCD.

A search was performed in four online databases (PubMed, Web of Science (Clarivate), CINAHL (EBSCO) and EMBASE (Elsevier) and the search engine Google Scholar [2] up to Dec. 2024.

Three studies involving a total of 153 patients with SCD were deemed eligible for this search. All 3 studies showed that oral L-Citrulline supplementation significantly increased plasma arginine levels (P value stress measures with L-Citrulline supplementation. One study reported improved well-being in all participants. Adverse events were rare, mild, and transient.

Our systematic review showed that oral L-Citrulline supplementation is safe in patients with SCD and could be beneficial in decreasing oxidative stress and improving quality of life. However, more studies are needed for a definitive conclusion on using L-Citrulline for these patients.

Sickle Cell Disease (SCD) affects about 100,000 Americans and about 7 million individuals worldwide [3], making it the most common inherited blood disorder. Early understanding of this “first molecular disease” focused on the abnormal polymerization of the sickle hemoglobin, and the abnormal fluid mechanics [4]. More recent understanding of pathophysiology includes a complex interplay of hemolysis, oxidant stress, inflammation, and thrombosis, with abnormal interaction of red blood cells with endothelium, platelets, and leukocytes [5]. Blood stasis and vaso-occlusion cause pathophysiologic vascular changes that are still being elucidated [6], [7]. Nitric oxide (NO) pathways have drawn the interest of SCD investigators because NO depletion could play a major role in vaso-occlusive episodes (VOE) in SCD [8]. NO is a vasodilator that also controls platelet aggregation, and as such prevents sickle cell erythrocytic adhesions to the vascular endothelium. L-Citrulline is a non-essential amino acid for humans, because it is produced by the liver and intestine. It also occurs naturally in many foods, notably watermelon and cucumber. L-Citrulline acts as a precursor of the amino acid L-arginine, a key substrate of NO [1], [10]. L-Citrulline supplementation is associated with a dose-dependent increase in L-Arginine level, and the increased L-arginine bioavailability subsequently increases NO concentration [9]. Many studies of healthy athletes have administered L-Citrulline as single oral doses of 8 grams and dose-ranging studies have administered single doses up to 15 grams, as reviewed by Gonzalez and colleagues [11]. L-Citrulline has been used safely in lifelong treatment of urea cycle disorders starting in newborns [12]. Thus, supplementation of L-Citrulline could offer therapeutic benefits in sickle cell disease as a potential nitric oxide substrate [1],[7]. Building upon evidence of safety and tolerability of L-Citrulline supplementation in other human studies, the objective of this study is to systematically review the literature to assess the effect of L-Citrulline administration (oral and intravenous) in patients with SCD. We focus our primary outcome on oxidative stress measures. Our secondary outcome is the effect of citrulline administration on the clinical course 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 CRD42024622190. A systematic literature review was designed and reported according to standards laid out by the Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) Statement and the Cochrane Handbook for Systematic Reviews [13]. A search of four online databases was performed; PubMed, Web of Science (Clarivate), CINAHL (EBSCO) and EMBASE (Elsevier) and a search engine Google Scholar as recommended by Bramer [2]. Vocabulary choice included terms that were limited to sickle cell disease or disorder or related hemoglobin typing and citrulline terms, which included alternate spellings and synonyms. Relevant controlled vocabulary from each database were applied in addition to searching by title, abstract and keywords. Searches included all dates up to December 20, 2024. Searches did not limit by study type or language. All results from the search were uploaded into Covidence (Covidence.org. Melbourne, Australia). Full search strategies with scope notes are provided in the Appendix. Eligibility Criteria: We included only studies involving human subjects that studied the effect of oral or intravenous L-Citrulline supplementation on patients aged 5 year or older with SCD of any genotype irrespective of the setting of the patients (outpatient or inpatient), language, country, or publication year. Study Selection: Publications identified from the database searches were screened by the authors according to eligibility criteria above. Screening was performed as a two-step process; in the first step titles and abstracts of all unique results of the search were screened for relevance in accordance with the agreed upon criteria. In the second step, full-text versions of all publications which were deemed relevant in the first step were assessed for relevance, again in accordance with the predetermined inclusion and exclusion criteria. Both steps of the screening were performed by two reviewers (Z.A. & R.H.) working independently. Any differences between the two researchers were resolved via discussion. In the event of an impasse, a third reviewer (L.L.H.) resolved any conflicts. Data Extraction: Data from the included publications were independently entered into a standardized predesigned extraction template in Covidence by both researchers. All extracted data was exported to Excel. Once both primary reviewers had completed data extraction, they met to resolve any differences and achieve consensus. Analysis: Extracted data was analyzed via both narrative summary and qualitative synthesis. Cohen’s Kappa was used to assess inter-rater reliability. Given the heterogeneity of the data and study designs, a meta-analysis was not conducted.

A total of 250 studies were found in the four databases and Google Scholar search engine: 78 records from Embase, 42 records from Web of Science, 28 from PubMed, and 2 from CINAHL. The first 100 records from a search of Google Scholar were also included. After removing 87 duplicate records, 162 studies were screened based on the title/abstract, 150 studies were deemed irrelevant and excluded. Twelve studies were assessed for eligibility based on full texts, 9 studies excluded; 4 due to wrong study design, 2 because they were abstract and not articles, 2 due to wrong outcomes, and 1 due to wrong indication. The remaining 3 studies evaluated the effect of L-Citrulline in SCD patients. Characteristics of the included studies: All three included studies were clinical trials [1,10,17]. One was a prospective cohort trial [1], the other two were randomized-controlled cross-over trials [10,17]. One study was conducted in the United States [1], and two studies were conducted in Tanzania [10,17]. The three included studies limited participation to children and adolescents. All studies were conducted in outpatient settings. All studies used oral L-Citrulline supplementation. The duration of supplementation was 28 days to 4 months (Table 1). Pooled effects of L-Citrulline supplementation: All three studies showed decreased oxidative stress by rising serum levels of Arginine, ornithine, and/or global arginine bioavailability, along with decreased asymmetric dimethylarginine (ADMA) level in two studies. Meta-analysis was precluded by the heterogeneity of study designs, but citrulline administration was associated with a rise in plasma arginine in all of the studies (Table 2). Adverse events: No serious adverse events were reported. In one of the studies, observed adverse events included acid reflux, chest tightness, diarrhea, fever without source, muscle spasm, and vomiting [10]. All these adverse events were self-limited. Each of these studies concluded that L-Citrulline supplementation is generally safe. Analysis: The inter-rater reliability for the primary reviewers was found to be fair, with a Cohen’s Kappa of 0.32. As previously stated, data were limited and heterogenous, which made performing a meta-analysis unfeasible. Additional information from excluded studies: One publication was excluded from the systematic review set because the outcomes were focused on plasma citrulline level while our outcome of interest is oxidative stress measures and patient clinical course. It was a Phase 1 study with no control arm of intravenous (IV) L-Citrulline ([18] Majumdar 2018). Eight adolescents with SCD received IV L-Citrulline supplementation (four were at steady state and four during a sickle cell pain episode). In one of the four infused during steady state, “diastolic blood pressure transiently dropped > 20% from baseline during the first 30 min of drug infusion but then normalized within 1 hour without any intervention.” Drowsiness was noted in 6 out of 8 participants in that Phase 1 study. All adverse effects appeared to resolve without treatment and no participants dropped out early due to adverse effects from the L-Citrulline.

This systematic review assesses the effect of short-term L-Citrulline supplementation in patients with SCD. SCD is a potentially life-threatening condition that puts a major burden on patients, their families and the healthcare system. Multimodal therapies are needed to improve patients’ well-being and quality of life [14]. Most SCD disease modifying therapies, such as hydroxyurea, when prescribed, require frequent clinical visits and lab monitoring to ensure patient safety and well-being, and to minimize the risks of adverse events [15]. L-Citrulline as a naturally occurring amino acid, could possibly provide a safer and less burdensome therapeutic option to patients with SCD. L-Citrulline supplementation has already been safely used in other diseases, such as urea cycle disorders, in individuals as young as neonates [12]. Based the studies included in this review, L-Citrulline supplementation has the promise of providing therapeutic benefits to patients with SCD with potentially no major or significant adverse effects, and less burdensome monitoring requirements. A total of three studies containing 153 patients with SCD were included in our review [1, 10, 17]. There was a statistically significant increase in plasma arginine level following L-Citrulline supplementation observed in all three studies. Two of the three studies also showed improvements in other oxidative measures of stress including Ornithine, Dimethylarginine, and Global arginine bioavailability. There are some limitations to our review. First, the modest sample size might have reduced the statistical power to detect the effect of L-Citrulline supplementation in patients with SCD. There is also a risk of overestimation of effect given the limited number of studies included. Future studies with larger sample sizes are needed to derive a more accurate assessment of the effects of L-citrulline supplementation in sickle cell disease. Second, the participants in all three included studies were children and adolescents. While sickle cell disease has historically been a disease of childhood, recent improvements in standards of care combined with new therapeutic agents have led to a rise in life expectancy [16]. In order to match this new reality and fully develop a clearer understanding of the potential benefits of L-citrulline supplementation across all age groups, further studies which also include adult patients must be undertaken. Third, most of the study participants were from Tanzania which likely limits the generalizability of the data due to differences in health systems and dietary patterns. Multisite studies in different countries should be undertaken to better control for this. Lastly, two of the studies used L-Citrulline as a part of ready-to-use food supplement. This presents the potential for underlying synergistic effects from this type of combination. Studies which rely on only citrulline supplementation in adequately nourished populations as their intervention are required to better assess its true effect.

Our systematic review showed that L-Citrulline supplementation is safe and could be beneficial in decreasing oxidative stress and improving quality of life in patients with SCD with no significant adverse events. However, results are limited by heterogeneity of data and the small sample of studies included. Further research is needed to understand the potential of L-Citrulline supplementation as a possible therapeutic agent for patients with SCD. Conflict of Interest Disclosures Z.A., R.H., and T.G. declare no affiliation with commercial or industrial entities related to the present study. L.L.H. is scientific consultant for Asklepion Pharmaceuticals and research funding from Sanofi for clinical research in sickle cell disease.

The study was supported by internal educational funds at the University of Illinois Chicago. All authors participated in the conception and design of the study and in the data analysis and editing.

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Br J Haematol, 184: 634-636. https://doi.org/10.1111/bjh.15124 Figure 1 PRISMA Flow Diagram | Authors, Ref | Title | Country | Study Design | Participants’ Mean age in years (SD) | Sex (% female) | L-Citrulline Intervention | ||| | Route of Administration | Dose | Frequency | Duration | |||||| | Waugh et al. [1] | Oral citrulline as arginine precursor may be beneficial in sickle cell disease: early phase two results. | United States of America | Prospective cohort study | 14.2 | 20% | Oral | 0.09 to 0.13 g/k/d | Twice daily | 28 days | | Cox et al. [10] | Ready-to-use food supplement, with or without arginine and citrulline, with daily chloroquine in Tanzanian children with sickle-cell disease: a double-blind, random order crossover trial. | Tanzania | Randomized control trial | 10.01 (1.25) | 40% | Oral | 0.1 g/kg/d | Twice daily | 4 months | | Marealle et al. [17] | A pilot study of a non-invasive oral nitrate stable isotopic method suggests that arginine and citrulline supplementation increases whole-body NO production in Tanzanian children with sickle cell disease. | Tanzania | Randomized control trial–nested pilot study | 11.06 (1.23) | 30% | Oral | 0.1 g/kg/d | Twice daily | 10.7 weeks | SD: Standard deviation TABLE 2 Study outcomes | Plasma Arginine | Ornithine Level | ADMA | Global Arginine Bioavailability | |||||| | Authors | Ref. | Mean | SD | Mean | SD | Mean | SD | Mean | SD | | Waugh et al. | 1 | 127.00 | 18.00 | |||||| | Cox et al. | 10 | 53.72 | 47.69 | -9.51 | 0.001 | 15.02 | ||| | Marealle et al. | 17 | 130.16 | 115.65 | 101.05 | 71.00 | 0.95 | 0.25 | 0.86 | 0.23 | ADMA: Asymmetric dimethylarginine Information & Authors Information Version history Copyright This work is licensed under a Non Exclusive No Reuse License.

Authors Metrics & Citations Metrics Article Usage 1246views 311downloads Citations Download citation Zahra Al Khuridah, Rashid Halloway, Tina Griffin, et al. Systematic Review of L-Citrulline Supplementation Effect in Patients with Sickle Cell Disease. Authorea. 01 August 2025. DOI: https://doi.org/10.22541/au.175404797.70233011/v1 DOI: https://doi.org/10.22541/au.175404797.70233011/v1 If you have the appropriate software installed, you can download article citation data to the citation manager of your choice. Simply select your manager software from the list below and click Download. For more information or tips please see 'Downloading to a citation manager' in the Help menu.