Archives of Academic Emergency Medicine. 2023; 11(1): e11 OR I G I N A L RE S E A RC H The Effect of L-Citrulline Supplementation on Outcomes of Critically Ill Patients under Mechanical Ventilation; a Double-Blind Randomized Controlled Trial Mohammad Reza Asgary1, Sayid Mahdi Mirghazanfari2, Ebrahim Hazrati3, Vahid Hadi1, Mojgan Mehri Ardestani4, Faeze Bani Yaghoobi5, Saeid Hadi1∗ 1. Department of Health, School of Medicine, AJA University of Medical Sciences, Tehran, Iran. 2. Department of Physiology and Iranian Medicine, School of Medicine, AJA University of Medical Sciences, Tehran, Iran. 3. Trauma Research Center, AJA University of Medical Sciences, Tehran, Iran. 4. Department of Persian Medicine, Faculty of Medicine, AJA University of Medical Sciences, Tehran, Iran. 5. Instructor, Department of Military Nursing, Faculty of Nursing, AJA University of Medical sciences, Tehran, Iran. Received: October 2022; Accepted: December 2022; Published online: 1 January 2023 Abstract: Introduction: Effective parenteral and enteral amino acid replacement is crucial for critically ill patients with altered amino acid metabolism. This study aimed to assess the effects of l-citrulline supplementation on the clinical and labo- ratory outcomes in critically patients. Methods: This was a double-blind placebo-controlled randomized clinical trial. 82 critically ill patients who were expected to receive mechanical ventilation for more than 72 hours were selected. The patients were assigned to either a placebo or an intervention group. The patients in the placebo group received 10 gr of microcrystalline cellulose and the ones in the intervention group were given l-citrulline daily for 7 days. Serum levels of fasting blood sugar (FBS), lipid profile, hepatic enzymes, serum electrolytes, urea nitrogen, creatinine, and C-reactive protein (CRP) were evaluated before and after the intervention. Duration of invasive ventilation, intensive care unit (ICU) length of stay, ventilator-free days, and 28-day mortality rate were recorded and compared between groups. Re- sults: Eighty-two patients completed the trial. No statistically significant differences were observed between the two groups in terms of age (p = 0.46), sex (p = 0.49), body mass index (BMI) (p = 0.41), Sequential Organ Failure Assessment (SOFA) Score (p = 0.08), Clinical Pulmonary Infection Score (CPIS) score (p = 0.76), Acute Physiology and Chronic Health Evaluation (APACHE II) score (p = 0.58), risk factors (p = 0.13), ICU stay before randomization (p = 0.32), and reason of admission (p = 0.50) before the intervention. Citrulline group had a notable reduction in FBS (p = 0.04), total choles- terol (TC) (p = 0.02), low density lipoprotein (LDL-C) (p <0.001) and high-sensitivity CRP (hs-CRP) (p <0.001). Also, a significant increase in lactate dehydrogenase (LDH) concentration (p <0.001) was observed in the intervention group at the end of the trial. Total duration of invasive ventilation and the mean SOFA score on 7th day were significantly lower in the citrulline group compared to the control group. Moreover, a significant increase in days alive and ventilator-free days within 28 days after admission was found in the citrulline group at the end of the trial. Also, there were no signifi- cant differences between the groups in terms of mortality rate during intervention, serious adverse events, endotracheal intubation, the use of tracheotomy or non-invasive ventilation after extubation, length of ICU stay, ICU-free days at 28 days, and CPIS and APACHE II scores. For mortality, in the citrulline group, there was two deaths compared to eight deaths in the control group. This resulted in an absolute risk reduction (ARR) of 14.05% (95% CI: 0.39–27.71%) and a number needed to treat (NNT) of 7.1 (95% CI: 3.6–29.5), regarding mortality. Conclusion: The results of the present study demonstrated the probable positive effects of citrulline supplementation on lipid profile, hs-CRP levels, duration of invasive ventilation, and SOFA score. Also, l-citrulline consumption may increase the probability of survival without mechanical ventilation. Keywords:L-citrulline; critical illness; ventilation; intensive care units; treatment outcome; clinical trial Cite this article as: Asgary MR, Mirghazanfari SM, Hazrati E, Hadi V, et al. The Effect of L-Citrulline Supplementation on Outcomes of Critically Ill Patients under Mechanical Ventilation; a Double-Blind Randomized Controlled Trial. Arch Acad Emerg Med. 2023; 11(1): e11. https://doi.org/10.22037/aaem.v11i1.1774. ∗Corresponding Author: Saeid Hadi; Department of Health, Aja University of Medical Sciences, Fatemi Street, Tehran, Iran. P. O. Box: 1416643931, Tel: + This open-access article distributed under the terms of the Creative Commons Attribution NonCommercial 3.0 License (CC BY-NC 3.0). Downloaded from: https://journals.sbmu.ac.ir/aaem/index.php/AAEM/index MR. Asgary et al. 2 1. Introduction Providing the appropriate nutrition therapy in an intensive care unit (ICU), especially for critically ill patients, is essential to improve the metabolic and clinical outcomes (1). Critical illnesses, such as major trauma and sepsis are characterized by a high level of stress/inflammation. Regulation of amino acid metabolism is altered by neuroendocrine changes and cytokine effects due to stress and inflammation, respectively (2). The 2019 European Society for Clinical Nutrition and Metabolism (ESPEN) guidelines recommend nutrition ther- apy to all critically ill patients staying in the ICU for more than 48 hours (1). Catabolic conditions in critically ill and injured patients cause an increase in energy expenditure and muscle wasting (3, 4). Muscle protein degradation and loss of contractile protein can affect the diaphragm, which is the main inspiratory muscle. People with respiratory problems stay on ventilators for prolonged periods (5). For critically ill and injured patients, amino acids are cru- cial for nutritional and metabolic support (6). Citrulline, which has gained attention recently, is included in these immune-nutrition (IMN) formulas, along with arginine, n- 3 fatty acids, glutamine, antioxidants, and nucleic acids (7). As our knowledge of altered amino acid metabolism in such patients increases, it is crucial to develop more effective par- enteral and enteral amino acid replacement products. Fol- lowing trauma and surgery, arginine and citrulline levels ap- pear to decrease in critically ill patients (7-10). Moreover, these amino acids are reversely correlated with levels of cy- tokines and inflammatory markers (11). A growing body of evidence shows that cytokines can contribute to the emer- gence of critical illness (12). Cytokines are strongly related to higher disease severity in these states, while the persistence in the spread of the cytokinesis is associated with improve- ment in multiple organ failure (MOF) (13). Orally ingested L-citrulline can result in the biosynthesis of L-arginine and, L-citrulline (14) ,and also enhance arginine bioavailability in the circulation, which acts as a precursor for nitric oxide (NO) formation (15). NO level could regulate vasodilation, blood flow, and muscle oxygenation (16, 17). L-citrulline has been found to be more effective than L- arginine supplementation for formation of NO, which can be due to the fact that arginine might be metabolized by arginase and transformed to urea and ornithine, making it less available to nitric oxide synthase for producing NO and citrulline(18)(19). 98/218/895 556, Fax: + 98/218/8984 861, Email: s.hadinu@yahoo.com, ORCID: https://orcid.org/0000-0003-2770-7084. Several studies have examined the effects of L-arginine sup- plementation on ICU patients using arginine-rich IMN for- mulas, including other compounds (20-22). Based on a re- cent study on critical care, citrulline plasma levels are ex- tremely low in patients with sepsis and even lower in those with acute respiratory distress syndrome (ARDS) (12). Ac- cording to one viewpoint, plasma citrulline levels could be linked with better vision in critically ill patients. Most im- portantly, plasma citrulline levels are low in the majority of these patients (23-25). Low plasma citrulline levels are re- lated to poor prognosis (24, 25). Plasma citrulline and C- responsive protein (CRP) fixations are conversely connected (24-26). Although several clinical studies have demonstrated the favourable effects of citrulline on critically ill patients (27, 28), it is yet not confirmed to be effective as a solitary treat- ment and further controlled clinical trials are needed to ex- amine the effects of citrulline on clinical outcomes, such as the respiratory capacity and the duration of ventilation in these patients. Therefore, the aim of the current study was to evaluate the effect of oral L-citrulline supplementation on FBS, lipid profile, hepatic enzymes, serum electrolytes, urea nitrogen, creatinine, CRP, duration of invasive ventilation, ICU length of stay, ventilator-free days, and 28-day mortal- ity rate in ventilated intensive care unit patients. 2. Methods This was a randomized clinical trial performed on in- tensive care unit (ICU) patients admitted to Imam Reza Hospital affiliated with the AJA University of Medical Sci- ences, Tehran, Iran, from December 21, 2021 to May 27, 2022. The Research Ethics Committee of AJA Uni- versity of Medical Sciences approved the study proto- col (IR.AJAUMS.REC.1400.269). We registered the present trial on the Iranian Registry of Clinical Trials website (http://www.irct.ir, identifier: IRCT20210920052530N1). All patients’ families provided written informed consent after a full explanation of the study. 2.1. Participants This study was performed on adult critically ill patients. Only subjects that were under invasive ventilation either through intubation or tracheotomy tube and required ventilation for at least 72 hours after study entry and expected to survive and remain in the ICU for at least 96 hours after were in- cluded. The study exclusion criteria were as follows: less than 18 years old, pregnancy, previous allergy to citrulline or arginine, a history of gastrointestinal disease, digestive tract surgery, intestinal obstruction, paralytic ileus, intestinal is- chemia, septic patients, and hyperthyroidism. This open-access article distributed under the terms of the Creative Commons Attribution NonCommercial 3.0 License (CC BY-NC 3.0). Downloaded from: https://journals.sbmu.ac.ir/aaem/index.php/AAEM/index 3 Archives of Academic Emergency Medicine. 2023; 11(1): e11 2.2. Randomization and allocation Random allocation software (RAS) was used for randomiza- tion. During the first 24 hours of invasive ventilation, the sub- jects were randomly assigned to each group in a centralized, blinded manner by means of an assignment sequence that was generated by computer. In addition, the Acute Physiol- ogy, and Chronic Health Evaluation (APACHE) II (score di- chotomized as ≤ 15 or > 15) was used to assess severity of disease at the time of inclusion. Random block sizes were uti- lized to balance the list. Furthermore, investigators, patients, and research staff did not know the group of the participants. 2.3. Study protocol Participants in placebo (n=42) and Citrulline (n=40) groups received 10 gram/day Microcrystalline cellulose and L- Citrulline powder (Karen Pharma & Food Supplement Co., Iran) for 7 days, respectively. Blood samples were drawn be- fore and after the 7-day intervention. Acute-phase proteins (APPs) as immunology factors, complete blood count (CBC), blood urea nitrogen (BUN), creatine, albumin, glycemic sta- tus, lipid profile, and liver function were determined before and after the intervention. Demographic characteristics, physiological variables, and other clinical and laboratory data were collected. Data on the days with mechanical ventilation were collected within 28 days. Enteral feeding started for all patients within 24-48 hours of admission in hospital. All subjects received the same Hos- pital Prepared Enteral Formulation (HPF), which contained 42.8% carbohydrates, 16.6% protein, and 34.2% fat. 2.4. Measurements Clinical outcomes The number of days free from mechanical ventilation for at least 48 consecutive hours and alive is defined as ventilator- free days within the first 28 days. If patients were discharged prior to the end of study period, they are considered as alive without mechanical ventilation. Also, for subjects who died, the ventilator-free days are omitted. Data on all-cause mortality, ICU-free days, and mechanical ventilation duration at 28 days plus both Sequential Organ Failure Assessment (SOFA) score and Clinical Pulmonary In- fection Score (CPIS) on the first and seventh days were mea- sured in addition to other outcomes. The severity of illness was evaluated using APACHE II on the day of admission Blood sample collection Fasting blood samples (10 mL) were taken at baseline (day 0) and at the end of the trial (day 7) early in the morning and after an overnight fast. Serum was immediately separated by centrifugation at 3400 rpm for 3 minutes. Serum samples were stored at -80°C until assayed. Laboratory investigation CRP was measured via the method of agglutination of la- tex particles on the slide (ENISON Co kits). Albumin was assessed using a commercially available enzyme-linked im- munosorbent assay (ELISA) kit (Abcam). BUN was deter- mined using enzymatic methods. Alanine transaminase (ALT), aspartate transaminase (AST), and lactate dehydro- genase (LDH) were measured based on the method recom- mended by the IFCC (International Federation of Clinical Chemistry). Sample size and statistical methods The minimum sample size was determined as 30 subjects in each group. The calculation is based on the mean ±SD of hs- CRP determined by Barkhidarian et al. (19) considering α = 0.05 and power of 80%. Considering a possible dropout of 35%, 40 subjects were included in each group. Data were presented as mean ± SD. We used the Kolmogrov- Smirnov test to examine the normal distribution of vari- ables. Log transformation was conducted for nonnormally distributed variables. Independent sample t-test and paired Student’s t-test were employed to identify the effect of the in- tervention on outcome variables. The chi-square test was ap- plied to compare the relative or absolute frequency. Analysis of covariance (ANCOVA) was used for comparisons between the two groups post-intervention after adjusting for baseline values. Paired t-test was used to compare differences from baseline to post-intervention period within groups. Absolute risk reduction (ARR) and the number needed to treat (NNT) were also calculated (29). The Statistical Pack- age for the Social Sciences (SPSS, version 19; Chicago, IL) was utilized to carry out the statistical analysis, and a p-value of < 0.05 was regarded statistically significant. 3. Results 3.1. Baseline characteristics of studies cases From December 21, 2021 to May 27, 2022, a total of 90 pa- tients were randomly assigned to the intervention (n=45) and placebo (n=45) groups. After randomization, there was drop out throughout the investigation and finally 40 subjects in the citrulline group and 42 subjects in the placebo group completed the trial. 5 and 3 participants in the citrulline and placebo groups, respectively, were excluded after randomiza- tion due to the poor adherence to the intervention (Figure 1). Table 1 shows the baseline characteristics of the patients in the L-citrulline group and the placebo group. The mean age of the participants in the L-citrulline and the placebo groups were 52.2 ± 18.4 and 49.9 ± 19.0 years, respectively. All base- line characteristics of study participants were well balanced between groups and no statistically significant differences were observed between the two groups in terms of age (p = 0.46), sex (p = 0.49), BMI (p = 0.41), SOFA score (p = 0.08), This open-access article distributed under the terms of the Creative Commons Attribution NonCommercial 3.0 License (CC BY-NC 3.0). Downloaded from: https://journals.sbmu.ac.ir/aaem/index.php/AAEM/index MR. Asgary et al. 4 CPIS score (p = 0.76), APACH II score (p = 0.58), risk factors (p = 0.13), ICU stay before randomization (p = 0.32), and reason of admission (p = 0.50) before the intervention. 3.2. Comparing the outcomes of intervention Laboratory parameters Among the laboratory variables, as presented in Table 2, cit- rulline group had a notable reduction in FBS (p = 0.04), to- tal cholesterol (TC) (p = 0.02), low density lipoprotein (LDL- C) (p <0.001) and high-sensitivity CRP (hs-CRP) (p <0.001). Also, a significant increase in LDH concentration (p <0.001) was observed in the intervention group compared to the con- trol group after adjusting for baseline values at the end of the trial. Non-significant differences were found between groups in term of albumin (p = 0.58), triglyceride (TG) (p = 0.68), high density lipoprotein (HDL-C) (p = 0.59), AST (p = 0.22), ALT (p = 0.75), and BUN (p = 0.40). Serum concentrations of creatine (p = 0.01) significantly decreased during 7 days in patients re- ceiving citrulline, while such effect was not observed in the placebo group. Considering that no adverse side effects were reported, citrulline was well tolerated. Disease severity and disposition As demonstrated in Table 3, the total duration of invasive ventilation and the mean SOFA score on the 7th day were significantly lower in the citrulline group compared to the control group. Moreover, a significant increase in days alive and ventilator-free days within 28 days after admission was found after citrulline supplementation. Also, there were no significant differences between the groups in terms of mor- tality rate during intervention, serious adverse events, endo- tracheal intubation, the use of tracheotomy or non-invasive ventilation after extubation, length of ICU stay, ICU-free days at 28 days, and CPIS and APACHE II scores. Regarding mor- tality, in the citrulline group, there was two deaths compared to eight deaths in the control group. This resulted in an ab- solute risk reduction (ARR) of 14.05% (95% CI: 0.39–27.71%) and a number needed to treat (NNT) of 7.1 (95% CI: 3.6–29.5), regarding mortality. 4. Discussion Based on the obtained findings, L-citrulline supplementa- tion significantly decreased the serum levels of FBS, LDL-C, TC, and hs-CRP, duration of invasive ventilation, and SOFA score. Also, serum LDH levels and days alive and ventilator- free days within 28 days after admission were significantly in- creased by L citrulline supplementation. However, there was no difference in terms of other assessed variables after L cit- rulline supplementation. As mentioned above, serum levels of LDL-C and TC in the in- tervention group were lower than the placebo group. Based on a prior study that was conducted on type 2 diabetes mellitus patients, an eight-week supplementation with L- Citrulline powder showed a remarkable improvement on glu- cose homeostasis, some lipid profiles, and inflammatory biomarkers, which is consistent with our results (30). Also, the efficacy of L-citrulline supplementation on lipid profile was assessed in numerous animal models. For example, Kudo et al. investigated the effect of nine weeks of 1 gr/kg L-citrulline administration on the serum level of TG in rats. In this study, authors concluded that L-citrulline powder does not affect serum levels of TG (2). Moreover, notable reduction in cholesterol levels without affecting TG concentration were detected as a result of L-citrulline (0.5 g/kg) supplementa- tion for 11 weeks in rats with high-fat diet (31). The plau- sible hypolipidemic effects of L-citrulline may justify liver lipid metabolism improvement. L-citrulline can particularly suppress the expression of sterol regulatory element-binding protein 1 (SREBP-1). SREBP-1 expression is decreased with activation of adenosine monophosphate-activated protein kinase alpha (AMPKalpha) (32). AMPK is a cell strength sen- sor that merges different physiological indicators for power balance restoration. Specifically, it induces silencing of SREBP-1 cleavage, its target gene expression, and the nuclear translocation. This results in the reduction of lipid accumula- tion and synthesis of fatty acids in hepatocytes (33). Further- more, it has been demonstrated that intervention with cit- rulline stimulates visceral fat lipolysis. Hormone-sensitive li- pase phosphorylation and down-regulation of glyceroneoge- nesis might be induced by L-citrulline, which leads to accel- erated fatty acid launch from the adipose tissue (34). More- over, it is known that the phosphorylation and activation of AMPKα in various tissues are raised through NO produced by L-citrulline (35, 36). Not finding notable variation in other lipid profiles can be justified by various factors such as small sample size, insufficient dosage, and short intervention in- terval. In contrast with the study that found a significant increase in serum LDH levels, an animal study performed by Villareal et al. demonstrated a decrease in the expression of LDH after L- citrulline supplementation (37). Regarding hepatic enzymes including ALT and AST, in opposition to our results that rec- ommended no remarkable effect of L-citrulline, a clinical trial done by Darabi et al. indicated a statistically significant decrease in ALT levels following L-citrulline supplementation (38). Also, regarding kidney function, contrary to our results that presented no favourable effect by L-citrulline, a recent animal study by Hashemi et al. concluded that L-citrulline could significantly reduce the levels of BUN and creatinine in rats (39). The discrepancies between aforementioned find- ings may result from the different nature of the studies and dissimilar dosage and duration of L-citrulline supplementa- tion. This open-access article distributed under the terms of the Creative Commons Attribution NonCommercial 3.0 License (CC BY-NC 3.0). Downloaded from: https://journals.sbmu.ac.ir/aaem/index.php/AAEM/index 5 Archives of Academic Emergency Medicine. 2023; 11(1): e11 Our study exhibited that L-citrulline powder notably de- creased the duration of invasive ventilation. This finding is consistent with the results of a study conducted by Lauter- bach et al. in 2018, which measured the effectiveness of L-Citrulline supplementation in treating pulmonary hyper- tension. This study finally proposed that treatment with L- citrulline may be an alternative and potentially useful treat- ment in the prevention or treatment of chronic pulmonary hypertension in infants (40). Also, an animal study inves- tigated the efficacy of L-citrulline on alveolar development and lung condition in newborn rats. The results of this research indicated that L-Citrulline therapy prevents lung damage caused by hyperoxia and high blood pressure in newborn rats (41). Furthermore, L-Citrulline may be a new therapeutic alternative for inhaled NO and, prevent bron- chopulmonary dysplasia. In addition, oral L-citrulline, as a precursor of NO, improved symptoms of sickle cell dis- ease in children and reduced pulmonary blood pressure after surgery for congenital heart disease (42). Orally ingested L- citrulline can also enhance arginine bioavailability in the cir- culation and act as a precursor for nitric oxide (NO•) forma- tion (15). NO• levels could regulate vasodilation, blood flow, and muscle oxygenation (16, 17). Although no side effects were previously reported for L-citrulline, oral administration of L-arginine in high doses could result in nausea, vomit- ing, diarrhea, headache, flushing, and numbness, owing to intestinal and hepatic conversion of L-arginine to ornithine and urea. Orally ingested L-citrulline can mainly have a more beneficial effect compared to L-arginine (43). The SOFA score evaluates blood pressure and the function of neurological system, blood, liver, and kidney. A higher SOFA score indicates a higher chance of mortality (44). According to the findings of the present study, the SOFA score in the group that received L-citrulline was notably lower in compar- ison to the control group. Based on previous investigations, L-Citrulline therapy induced an anti-inflammatory profile and obviously protected against kidney dysfunction through improving the function of glomerulus and its associated tubule (45). In addition, L-citrulline amino acid consump- tion exerts beneficial effects on cardiometabolic health, glu- cose homeostasis, and protein and lipid metabolism through direct and indirect pathways (46-48). Moreover, L-citrulline intake may truncate the required time for complete myocar- dial depolarization and repolarization. Thus, L-citrulline in- gestion transiently modifies myocardial blood supply and improves the energy supply required for faster recovery in restoring ATP-dependent ionic exchanges. Taking this sup- plement can even control blood pressure in people with hy- pertension. The findings of a systematic review and meta- analysis showed that L-citrulline supplementation reduced the systolic blood pressure by 4 mmHg (49). Hs-CRP, a measurable protein in the blood, increases in the body during systematic inflammation. Measurement of this index in the blood is used to determine the risk of cardio- vascular diseases and other inflammatory-related complica- tions. Based on our results, L-Citrulline supplementation significantly decreased hs-CRP levels. In line with our find- ings, Abbaszadeh et al. found a notable drop in hs-CRP lev- els in patients who received 10 g L-citrulline daily for 10 days (50). Also, another study demonstrated that an increase in L- citrulline concentrations caused a considerable reduction in hs-CRP production in obese rats with diabetes (51). Several in vitro and in vivo models proposed that L-citrulline supple- mentation caused a remarkable reduction in inflammation by inhibiting the expression of NF-kB as an important tran- scription factor involved in the inflammatory response (52). 5. Limitations Among the limitations of this study was the short duration of the follow-up period as well as the budget restrictions, which prevented us from examining the complete profile of inflam- matory biomarkers. Moreover, our participants were ill pa- tients over 18 years old, and consequently, our findings may not apply to people of other ages and health conditions. Fi- nally, although several potential confounders were consid- ered in the statistical analysis, some other unknown con- founders could affect the obtained results and lead to bias. 6. Conclusions In conclusion, our finding proposed that L-citrulline supple- mentation decreased the serum levels of FBS, LDL-C, TC, and hs-CRP, duration of invasive ventilation, and SOFA score. On the other hand, serum LDH levels and days alive and ventilator-free days within 28 days after admission signifi- cantly increased with L citrulline supplementation. Although obtained findings suggested that L citrulline supplementa- tion could improve some clinical outcomes in critically ill pa- tients, further well-designed clinical trials with larger sam- ple size and longer follow-up period are required to verify its favourable effects in ventilated intensive care unit patients. 7. Declarations 7.1. Acknowledgments We are sincerely grateful to the patients who participated in our research. 7.2. Conflict of interest The authors declare that they have no conflicts of interest. 7.3. Fundings and supports The Research Vice Chancellor of AJA University of Medical Sciences, Tehran, Iran, financially supported this research This open-access article distributed under the terms of the Creative Commons Attribution NonCommercial 3.0 License (CC BY-NC 3.0). Downloaded from: https://journals.sbmu.ac.ir/aaem/index.php/AAEM/index MR. Asgary et al. 6 and provided the drugs (Grant Number 97001782). 7.4. Authors’ contribution MRA, SH, SMM, EH and VA studied concept and designed the study. MRA, EH and FBY collected data. MRA, SH, SMM, MMA and VH analyzed and interpreted data. MRA drafted whole of the manuscript. All authors read and approved the final version of manuscript. 7.5. Ethics approval The protocol of the study was approved by the Research Ethics Committees of AJA University of Medical Sciences (IR.AJAUMS.REC.1400.269). 7.6. Consent for publication All participants provided written informed consent. 7.7. 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Downloaded from: https://journals.sbmu.ac.ir/aaem/index.php/AAEM/index 9 Archives of Academic Emergency Medicine. 2023; 11(1): e11 Table 1: Comparing the baseline characteristics of studied groups Characteristic L-citrulline Placebo P value (n = 40) (n = 42) Age (year) Mean ± SD 52.5 ± 18.4 49.9 ± 19.0 0.46 Gender Male 21 (52.5) 26 (59.09) 0.49 Female 19 (47.5) 16 (40.91) Body mass index (kg/m2) Mean ± SD 27.2 ± 8.0 26.7 ± 9.1 0.4 Disease severity Sequential Organ Failure Assessment 8.2 ± 0.5 8.4 ± 0.55 0.08 Clinical Pulmonary Infection Score 3.4 ± 1.6 3.1 ± 1.0 0.76 APACHE II score 16.77 ± 4.3 17.3 ± 4.6 0.58 ICU stay before randomization (day) Median (IQR) 1 (0-2) 1 (0-1) 0.32 Comorbidities and risk factors Cardiovascular disease 3 (7.5) 3 (7.1) 0.13 Diabetes Mellitus 15 (37.5) 20 (47.6) Hypertension 24 (60.0) 31 (73.8) Neurological diseases 1 (2.5) 1 (2.3) Respiratory disease 13 (32.5) 10 (23.8) Reason for admission Pneumonia 14 (35.0) 15 (35.7) Surgical 10 (25.0) 9 (21.4) Chronic Obstructive Pulmonary Disease 5 (12.5) 7 (16.6) 0.5 Acute Respiratory Distress Syndrome 5 (12.5) 6 (14.2) Stroke 2 (5.0) 2 (4.7) Trauma 4 (10.0) 5 (11.9) Data are presented as mean ± standard deviation (SD), frequency (%), or median (interquartile range; IQR). APACHE II score: Acute Physiology and Chronic Health Evaluation. II Score; ICU: intensive care unit. This open-access article distributed under the terms of the Creative Commons Attribution NonCommercial 3.0 License (CC BY-NC 3.0). Downloaded from: https://journals.sbmu.ac.ir/aaem/index.php/AAEM/index MR. Asgary et al. 10 Table 2: Comparing the laboratory variables between the L-citrulline-treated (n=40) and placebo (n=42) groups before and one week after intervention Factors Baseline After intervention P value Albumin L-citrulline 3.67 ± 0.51 3.31 ± 0.54 0.58 Placebo 3.61 ± 0.44 3.38 ± 0.51 0.67 FBS (mg/dl) L-citrulline 106.93 ± 15.28 93.23 ± 9.79 0.04 Placebo 106.96 ± 16.28 100.89 ± 13.94 0.57 TG (mg/dl) L-citrulline 185.50 ± 44.34 136.41 ± 32.86 0.68 Placebo 164.44 ± 53.77 155.61 ± 44.12 0.52 TC (mg/dl) L-citrulline 181.23 ± 41.00 163.86 ± 40.21 0.02 Placebo 200.00 ± 34.06 190.77 ± 31.84 0.01 LDL-C (mg/dl) L-citrulline 118.78 ± 34.78 105.97 ± 37.22 <0.001 Placebo 128.73 ± 34.82 120.48 ± 35.65 0.01 HDL-C (mg/dl) L-citrulline 35.01 ± 11.68 37.28 ± 11.43 0.59 Placebo 33.49 ± 7.70 34.78 ± 7.78 0.35 Hs-CRP (mg/L) L-citrulline 3691.50 ± 644.67 2026.62 ± 246.82 <0.001 Placebo 3964.23 ± 241.09 3360.44 ± 486.36 0.01 AST (U/l) L-citrulline 88.43 ± 22.48 117.98 ± 34.07 0.22 Placebo 63.23 ± 24.14 117.63 ± 47.97 0.97 ALT (U/l) L-citrulline 66.54 ± 34.61 107.34 ± 29.21 0.75 Placebo 46.41 ± 18.79 135.22 ± 38.41 0.84 LDH (U/l) L-citrulline 63.93 ± 24.51 99.66 ± 31.37 <0.001 Placebo 79.04 ± 32.75 117.42 ± 33.23 0.01 BUN (mg/dl) L-citrulline 50.63 ± 24.42 47.25 ± 13.08 0.40 Placebo 42.07 ± 15.02 44.05 ± 13.69 0.99 Creatinine (mg/dl) L-citrulline 1.19 ± 0.56 0.78 ± 0.31 0.01 Placebo 1.12 ± 0.23 0.96 ± 0.52 0.13 Data are presented as mean ± standard deviation (SD). FBS: fasting blood sugar; TG: triglyceride; TC: Total Cholesterol; LDL-C: low density lipoprotein; HDL-C: high density lipoprotein; hs-CRP: high-sensitive C-reactive protein; AST: aspartate aminotransferase; ALT: alanine aminotransferase; LDH: lactate dehydrogenase; BUN: blood urea nitrogen. This open-access article distributed under the terms of the Creative Commons Attribution NonCommercial 3.0 License (CC BY-NC 3.0). Downloaded from: https://journals.sbmu.ac.ir/aaem/index.php/AAEM/index 11 Archives of Academic Emergency Medicine. 2023; 11(1): e11 Table 3: Comparing the clinical outcomes (after 28 days) and disease severity between the L-citrulline-treated (n=40) and placebo (n=42) groups Variables L-citrulline Placebo P value Mortality rate during intervention 2 (5.0) 8 (19.0) 0.61 Serious adverse events 1 (2.5) 2 (4.5) 0.82 Endotracheal intubation 40 (100.0) 41 (97.5) 0.99 NIV after extubation 13 (32.5) 16 (38.0) 0.39 All-Cause Mortality 7 (17.5) 9 (21.5) 0.85 Duration of invasive ventilation (hour) 112.5 ± 59 139 ± 61.35 0.04 Days alive and ventilator-free 6.6 ± 1.6 4 ± 1.25 < 0.001 Length of ICU stay (days) 7 ± 5.5 8 ± 5.5 0.41 ICU-free days 2.1 ± 1.75 2.0 ± 1.7 0.79 SOFA score 1th day 8.2 ± 0.5 8.4 ± 0.55 0.08 7th day 6.1 ± 0.6 7.5 ± 1.2 < 0.001 P value 0.08 0.38 CPIS score 1th day 3.4 ± 1.6 3.1 ± 1.0 0.31 7th day 2.9 ± 1.1 2.7 ± 0.9 0.37 P value 0.73 0.96 APACHE II score 1th day 16.77 ± 4.3 17.3 ± 4.6 0.59 7th day 22.9 ± 7.6 22.0 ± 7.6 0.59 P value 0.82 0.96 Data are presented as mean ± standard deviation (SD) or frequency (%). Abbreviations: ICU: intensive care unit; SOFA: Sequential Organ Failure Assessment; CPIS: Clinical Pulmonary Infection Score; APACHE II score: Acute Physiology and Chronic Health Evaluation II Score; NIV: non-invasive ventilation. Adverse events were considered serious when they required intensive care procedures (use of vasopressors, haemodialysis, central venous catheterization, cardiac pacing, or tube thoracostomy) or surgery, and events that prolonged hospitalization or resulted in persistent or major disability or incapacity. This open-access article distributed under the terms of the Creative Commons Attribution NonCommercial 3.0 License (CC BY-NC 3.0). Downloaded from: https://journals.sbmu.ac.ir/aaem/index.php/AAEM/index MR. Asgary et al. 12 Figure 1: Flowchart of patient enrollment. This open-access article distributed under the terms of the Creative Commons Attribution NonCommercial 3.0 License (CC BY-NC 3.0). Downloaded from: https://journals.sbmu.ac.ir/aaem/index.php/AAEM/index Introduction Methods Results Discussion Limitations Conclusions Declarations References