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. Author manuscript; available in PMC: 2026 Jan 19.
Published in final edited form as: Mol Genet Metab. 2023 Dec 10;141(3):108112. doi: 10.1016/j.ymgme.2023.108112

Impact of supplementation with L-citrulline/arginine after liver transplantation in individuals with Urea Cycle Disorders

Roland Posset 1, Sven F Garbade 1, Florian Gleich 1, Sandesh C S Nagamani 2, Andrea L Gropman 3, Friederike Epp 1, Nesrine Ramdhouni 1, Ann-Catrin Druck 1, Georg F Hoffmann 1, Stefan Kölker 1, Matthias Zielonka 1, on behalf of the Urea Cycle Disorders Consortium (UCDC) and the European registry and network for Intoxication type Metabolic Diseases (E-IMD) consortia study group
PMCID: PMC12812261  NIHMSID: NIHMS2129111  PMID: 38301530

Abstract

Objective

Liver transplantation (LTx) is an intervention when medical management is not sufficiently preventing individuals with urea cycle disorders (UCDs) from the occurrence of hyperammonemic events. Supplementation with L-citrulline/arginine is regularly performed prior to LTx to support ureagenesis and is often continued after the intervention. However, systematic studies assessing the impact of long-term L-citrulline/arginine supplementation in individuals who have undergone LTx is lacking to date.

Methods

Using longitudinal data collected systematically, a comparative analysis was carried out by studying the effects of long-term L-citrulline/arginine supplementation vs. no supplementation on health-related outcome parameters (i.e., anthropometric, neurological, and cognitive outcomes) in individuals with UCDs who have undergone LTx. Altogether, 52 individuals with male ornithine transcarbamylase deficiency, citrullinemia type 1 and argininosuccinic aciduria and a pre-transplant “severe” disease course who have undergone LTx were investigated by using recently established and validated genotype-specific in vitro enzyme activities.

Results

Long-term supplementation of individuals with L-citrulline/arginine who have undergone LTx (n=16) does neither appear to alter anthropometric nor neurocognitive endpoints when compared to their severity-adjusted counterparts that were not supplemented (n=36) after LTx with mean observation periods between four to five years. Moreover, supplementation with L-citrulline/arginine was not associated with an increase of disease-specific plasma arithmetic mean values for the respective amino acids when compared to the non-supplemented control cohort.

Conclusion

Although supplementation with L-citrulline/arginine is often continued after LTx, this pilot study does neither identify altered long-term anthropometric or neurocognitive health-related outcomes nor does it find an adequate biochemical response as reflected by the unaltered plasma arithmetic mean values for L-citrulline or L-arginine. Further prospective analyses in larger samples and even longer observation periods will provide more insight into the usefulness of long-term supplementation with L-citrulline/arginine for individuals with UCDs who have undergone LTx.

Keywords: Liver transplantation, urea cycle disorders, UCDC, E-IMD, L-citrulline/arginine supplementation

1. Introduction

Urea cycle disorders (UCDs), a group of rare inherited metabolic disorders with an estimated incidence of 1 in 35,000 to 52,000 newborns, are characterized by a high degree of phenotypic variability ranging from neonatal life-threatening hyperammonemic encephalopathy to attenuated forms that present with non-specific symptoms including recurrent vomiting, behavioral or psychiatric abnormalities and cognitive impairment with or without acute and/or chronic hyperammonemia (Summar et al., 2013; Ruegger et al., 2014; Kolker et al., 2015b; Kolker et al., 2015c; Nettesheim et al., 2017). Liver transplantation (LTx) is considered a therapeutic (and potentially curative) option, and is typically performed when medical management with protein intake restriction and nitrogen scavenging agents fails to prevent recurrent hyperammonemic events (HAEs) or when progressive hepatic disease evolves (Haberle et al., 2019). Clinical experiences demonstrate the positive effect of LTx on metabolic stability, prevention of recurrent HAEs, liberalization of protein intake, and discontinuation of nitrogen scavenging medications (Whitington et al., 1998; Morioka et al., 2005; Haberle et al., 2019; Sood et al., 2021). Whereas supplementation with L-citrulline or L-arginine is an important cornerstone of medical management by supporting residual ureagenesis in individuals with UCDs prior to a putative LTx (Haberle et al., 2019), the clinical rational for continued supplementation after LTx is controversially discussed since L-citrulline/arginine and supplementation is heterogeneously implemented in individuals who underwent LTx. Post-LTx, impairment of citrulline recycling in individuals with citrullinemia type 1 (CTLN1) and argininosuccinic aciduria (ASA) and impaired de novo synthesis of L-arginine in all types of UCDs except arginase 1-deficiency persist, rendering L-arginine an essential amino acid (Rabier et al., 1991), that might also account for neurocognitive deficits prior and post-LTx. This observation is an important factor as to why L-citrulline/arginine supplementation is considered even after transplantation.

However, the effect of L-citrulline/arginine supplementation on health-related clinical and biochemical long-term outcome parameters in individuals with UCDs who have undergone LTx has not been investigated thus far mainly due to the lack of a severity-adjusted non-supplemented control cohort. Recently, we had developed a novel genotype-specific classification system based on in vitro residual enzymatic activities to determine phenotypic severity for (male) ornithine transcarbamylase-deficiency (mOTC-D), CTLN1 and ASA (Zielonka et al., 2019a; Zielonka et al., 2020; Scharre et al., 2022), which account for approximately 80% of individuals with UCDs who have undergone LTx (Yu et al., 2015; Ziogas et al., 2021). By applying this novel severity-adjusted classification system, we systematically evaluated the effect of L-citrulline/arginine supplementation on the biochemical response, annual frequency of hospitalizations, growth as well as the neurocognitive outcome in individuals with a “severe” disease course who have undergone LTx in a pediatric study sample from the US, Canada and Europe that is systematically followed by the Urea Cycle Disorders Consortium (UCDC) and the European registry and network for Intoxication type Metabolic Diseases (E-IMD).

2. Materials and Methods

2.1. Eligibility criteria and study sample

The combined cohort of individuals with UCDs longitudinally followed by the UCDC and E-IMD registries has been published (Posset et al., 2019a; Posset et al., 2019b) with a particular focus on OTC-D, CTLN1 and ASA, the three most prevalent UCDs (Zielonka et al., 2019a; Zielonka et al., 2020; Scharre et al., 2022). The data models of both registries have been described previously (Seminara et al., 2010; Summar et al., 2014; Kolker et al., 2015a; Posset et al., 2019a). Only individuals with confirmed diagnosis of mOTC-D, CTLN1 and ASA who had undergone LTx and on whom information on NH4+max was available for the first HAE (initial NH4+max) were included in the present analyses. Requirements set forth by the International Committee of Medical Journal Editors were all met. All procedures were in accordance with the ethical standards of the Helsinki Declaration of 1975, as revised in 2013. Written informed consent was given by the probands or their legal representatives before the enrollment to this study. Data were retrieved from the UCDC and E-IMD electronic databases as previously described (Posset et al., 2020b). Combined and comparative data analysis of both databases was performed according to the three principles Interoperability, Representativeness and Severity-adjustment (Posset et al., 2023).

2.2. Stratification of individuals with male OTC-D (mOTC-D), CTLN1, and ASA who had undergone LTx according to their underlying disease severity

Individuals with mOTC-D, CTLN1 and ASA who had undergone LTx were categorized according to previously described models to reliably predict individual disease severity based on the pathogenic variant(s) in OTC, ASS1 or ASL (Zielonka et al., 2019a; Zielonka et al., 2020; Scharre et al., 2022). To this end, individuals with residual enzymatic OTC, ASS1 or ASL activities as assessed by the recently described genotype-specific in vitro system below the threshold values of 4.3% (for mOTC-D), 8.1% (for CTLN1) and 7.9% (for ASA), respectively, were classified as “severe”, whereas individuals with residual enzymatic activities equal to or above these threshold values were classified as having an “attenuated” disease form. Given the strong correlation of NH4+max with residual enzymatic activities (Zielonka et al., 2019a; Zielonka et al., 2020; Scharre et al., 2022), post-hoc posterior simulation was applied by using the mathematical correlation functions of NH4+max with residual enzymatic activities (i.e. OTC, ASS1, ASL) to determine estimated NH4+max values corresponding to the respective enzymatic threshold values for phenotypic differentiation. For this purpose, 200 predicted NH4+max values from fitted generalized additive models (GAM) were drawn and the following NH4+max values identified (50th centile): 564 μmol/L for OTC-D (corresponding to residual enzymatic OTC activity of 4.3%), 303 μmol/L for CTLN1 (corresponding to residual enzymatic ASS1 activity of 8.1%) and 131 μmol/L for ASA (corresponding to residual ASL activity of 7.9%; Supplementary Figure 1). Subsequently, NH4+max as surrogate marker for residual enzymatic OTC, ASS1 or ASL activity was used for group assignment of individuals to their underlying phenotypic severity to increase the number of included individuals.

2.3. Cornerstones and strategy for data analysis

For subsequent analyses, only individuals who underwent LTx with a “severe” disease course were included. To evaluate the impact of supplementation with L-citrulline/arginine, individuals were divided into a supplemented and non-supplemented group. Individual observations periods were defined starting with the first post-transplant visit. Assignment to the respective treatment group (supplemented vs. non-supplemented group) was carried out at the same time point. The end of specific individual observation periods was defined if supplementation was stopped (in the supplemented group) or if supplementation with L-citrulline/arginine was commenced (in the non-supplemented group) during the post-transplant disease course. Moreover, patients were only included in subsequent analyses if 1.) at least two post-transplant regular follow-up visits were available, 2.) treatment information (L-citrulline/arginine supplementation vs. no supplementation) was available for each follow-up visit, 3.) the individual observation period for each patient was at least 12 months and 4.) no switch between the two treatment groups occurred (from non-supplemented to supplemented or vice versa).

2.4. Biochemical and clinical variables

The following numerical biochemical and clinical variables were included: 1) Peak plasma ammonium concentration at initial hyperammonemic decompensation (NH4+max), 2) annual frequency of hospitalizations after transplantation (i.e., time between the first visit after LTx and the last study visit) including causes of hospital admissions, and 3) weighted arithmetic mean values for plasma concentrations of L-citrulline, L-arginine, L-leucine, L-isoleucine, and L-valine. Anthropometric data of European probands were compared to growth charts from the UK, since the ethnic background of the European study cohort corresponds well to that of individuals in the UK (Cole et al., 2011; Kolker et al., 2015c). For North American probands, growth charts from the Center for Disease Control and Prevention (https://www.cdc.gov/growthcharts/cdc_charts.htm) were applied. Z-scores for body height and weight were calculated at each scheduled longitudinal research follow-up visit. Preterm infants (< 37th pregnancy week), and z-scores < −3 or > 3 were excluded from data analyses. Motor abnormality was used as superordinate dichotomous variable (yes/no) including several motor variables (dystonia, chorea, ataxia, spasticity, abnormal gross or fine motor function, delayed milestones, muscular hypotonia or hypertonia) analogous to a previous study (Posset et al., 2016). Cognitive outcome was assessed using cognitive standard deviation scores (SDS) at the most recent study visit. Cognitive SDS was calculated applying full scale IQ values from Wechsler Abbreviated Scale of Intelligence, Wechsler Intelligence Scale for Children, and Wechsler Preschool and Primary Scale of Intelligence. Results of the mental developmental index, and cognitive scale from the Bayley Scales of Infant Development, and the general adaptive composite from the Adaptive Behavior Assessment System were implemented as previously described (Buerger et al., 2019; Posset et al., 2019b).

2.5. Comparative analysis of L-citrulline/arginine supplementation on evidence-based and validated health-related outcome parameters

Subsequently, the effect of L-citrulline/arginine supplementation was systematically compared to the non-supplemented control cohort. The following validated and previously published outcome parameters were used: 1.) linear growth (Posset et al., 2020a), 2.) neurological outcome (Posset et al., 2016), and 3.) cognitive function (Posset et al., 2019b; Zielonka et al., 2019a; Zielonka et al., 2020; Scharre et al., 2022). Moreover, a comparative analysis of L-citrulline/arginine supplementation with regard to the annual frequency and causes of hospitalizations after LTx was performed. Clinical endpoints were investigated to the latest possible time point during each patient’s individual observation period.

2.6. Statistical analysis

R language version 4.3.1 was used to compute statistical analysis. As descriptive statistics, mean, standard deviation, median or inter-quartile range were reported, unless stated otherwise. Exact Wilcoxon-Mann-Whitney test in package ‘coin’ (package version 1.4.2) was used to compare a continuous response variable between supplemented and non-supplemented individuals. We report effect size of Wilcoxon-Mann-Whitney computed by with z as Wilcoxon-Mann-Whitney test statistic and n is total sample size (Fritz et al., 2012). Two-way contingency tables were analyzed with Boschloo’s test (R package ‘Exact’, version 3.2). Boschloo’s test is known to be powerful even in small samples sizes below five observations in a cell. No a priori formulated hypotheses were tested, and therefore all p-values and CIs are reported as descriptive measures.

3. Results

3.1. Description of the overall study cohort

Overall, 52 individuals (mOTC: n=18; CTLN1: n=19; ASA: n=15) with a severe phenotype who had undergone LTx were included in this analysis with 16 individuals receiving L-citrulline/arginine supplementation (mOTC: n=6; CTLN1: n=5; ASA: n=5) and 36 individuals having no supplementation (mOTC: n=12; CTLN1: n=14; ASA: n=10). Mean observation periods corresponded to 3.9 years in the supplemented and 5.7 years in the non-supplemented group. Baseline characteristics as well as detailed descriptive measures of subsequent analyses are depicted separately in Supplementary Tables 1 and 2.

3.2. Annual frequency and causes of hospitalizations did not differ between the supplemented and non-supplemented group

To investigate whether individuals without long-term L-citrulline/arginine supplementation after LTx have a higher risk of hospitalizations when compared to their supplemented counterparts, we studied the annual frequency and causes of hospitalizations in both groups. Interestingly, neither annual frequency (non-supplemented group: 0.3 mean hospitalizations per year of observation vs. supplemented group: 0.6 mean hospitalizations per year of observation; p=0.79; effect size: 0.04; Figure 1A) nor causes of hospitalizations (Figure 1B) differed between both groups.

Figure 1. Supplementation of L-citrulline/arginine is not associated with altered annual frequency and causes of hospitalizations after LTx.

Figure 1.

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(A) Plot illustrates annual frequency of hospitalizations after LTx for individuals with (n=16) or without (n=36) L-citrulline/arginine supplementation. Data are shown as mean (bullet point), upper and lower whiskers correspond to standard deviation. P=0.79, effect size: 0.04; Wilcoxon-Mann-Whitney Test. (B) Relative frequency of causes for unscheduled hospital admission in the respective cohort (supplemented or non-supplemented group). Causes were balanced between both groups and included in descending order of frequency for the infections, GI problems, others, rejections, and biochemical alterations. The category “GI problems” summarizes vomiting, gastroesophageal reflux disease, constipation, gastritis, diarrhea, abdominal pain/distension, eosinophilic enteritis, GI bleeding, gastrocutaneous fistula, pneumatosis intestinalis, colitis etc. The category “biochemical alterations” includes neutropenia, abnormal liver function tests, hyperkalemia, elevated transaminases etc. The category “others” comprises a.) either isolated symptoms such as throat pain, oral aphthous ulcerations, stridor, difficulties in breathing, nasal bleeding secondary to interventions, post-transplant lymphoproliferative disease, allergic reactions such as rash and anaphylaxis, psychiatric disorders (e.g. aggressiveness, behavioral problems), kidney injury, seizures etc., or b.) a combination of reasons without one clear cardinal symptom leading to hospitalization such as fever and electrolyte imbalance, dumping syndrome and mild rejection, vomiting and coughing, vomiting and lethargy, etc. A combination of reasons with one clearly identifiable cardinal symptom leading to the hospitalization were assigned to the respective and specific categories according to the author’s assessment. GI, gastrointestinal. For descriptive characteristics see Supplementary Table 2.

3.3. L-citrulline/arginine supplementation does not appear to be associated with altered growth and weight development after LTx

Recently, we have shown that LTx might be associated with a more favorable growth outcome, which is likely (at least partially) due to normalization of plasma branched-chain amino acids (BCAA) concentration after LTx (Posset et al., 2020a). To study whether favorable growth and weight development after LTx are also associated with the supplementation of L-citrulline/arginine, we compared anthropometric parameters between the supplemented (n=10) and non-supplemented (n=20) groups. In both groups, mean observation periods (supplemented group: 4.5 years; non-supplemented group: 5.6 years; p=0.30), and weighted mean arithmetic plasma BCAA concentrations (supplemented group vs. non-supplemented group: L-valine 189 μmol/l vs. 222 μmol/l; L-leucine 99 μmol/l vs. 118 μmol/l; L-isoleucine 54 μmol/l vs. 65 μmol/l; each p>0.05; Table 1) were balanced thereby reducing the effect of potential confounders. Importantly, at the end of the observation period, growth (mean z-score: supplemented group: −0.09 vs. non-supplemented group: −0.45; p=0.25; effect size: 0.21; Figure 2) and weight (mean z-score: supplemented group: 0.19 vs. non-supplemented group: −0.08; p=0.60; effect size: 0.10; Suppl. Figure 2) of individuals who underwent LTx were independent from L-citrulline/arginine supplementation. Moreover, plasma L-citrulline and L-arginine concentrations did not differ between the supplemented (mean L-citrulline/arginine for OTC-D: 10/46 μmol/l; mean L-citrulline/arginine for CTLN1: 427/76 μmol/l; mean L-citrulline/arginine for ASA: 160/54 μmol/l) and non-supplemented group (mean L-citrulline/arginine for OTC-D: 2/38 μmol/l; mean L-citrulline/arginine for CTLN1: 437/50 μmol/l; mean L-citrulline/arginine for ASA: 143/51 μmol/l; each p>0.05; Table 1).

Table 1.

Weighted mean arithmetic plasma concentrations of BCAAs, L-citrulline and L-arginine as well as dosages of supplemented amino acids in the study sample

L-valine
Mean, SD; n 		L-leucine
Mean, SD; n 		L-isoleucine
Mean, SD; n
Reference range 142–278 76–168 38–94
Supplemented group 189, 39; 10 99, 29; 10 54, 12; 10
Non-Supplemented group 222, 58; 20 118, 29; 20 65, 18; 20
p-value 0.16 0.23 0.13
OTC-D CTLN1 ASA
L-citrulline
Mean, SD; n 		L-arginine
Mean, SD; n 		L-citrulline
Mean, SD; n 		L-arginine
Mean, SD; n 		L-citrulline
Mean, SD; n 		L-arginine
Mean, SD; n
Reference range 17–49 45–125 17–49 45–125 17–49 45–125
Supplemented group 10, 10, 3 46, 17, 3 427, 26, 4 76, 48, 4 160, 33, 5 54, 22, 5
Non-Supplemented group 2, 4, 6 38, 13, 5 437, 124, 11 50, 19, 11 143, 31, 9 51, 15, 9
p-value 0.29 0.51 0.89 0.37 0.35 0.74
Dosages for the supplemented group [mg/kg/d] 151, 83, 6 n/a n/a 144, 46, 2 n/a 128, 25, 3
Dosages for the supplemented group [g/m 2 /d] n/a n/a n/a 4.8, 1.8, 4 n/a 2.2, 0.3, 4
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Reference ranges were defined according to (Duran, 2008) for children in the age range of 10 to 18 years and are shown in μmol/l. P-values were calculated using Wilcoxon-Mann-Whitney Test, p-values < 0.05 were considered significant, n refers to number of patients included in each group. Plasma BCAA concentrations are well within the normal range after LTx independent from the L-citrulline/arginine supplementation. Of note, supplementation with L-citrulline/arginine did not lead to increased weighted mean arithmetic plasma concentrations of L-citrulline or L-arginine in the investigated UCD subtypes when compared to the non-supplemented cohort (OTC-D, CTLN1, and ASA; each p>0.05). Despite supplementation, reduced weighted mean arithmetic plasma L-citrulline concentrations were found in OTC-D, whereas borderline low weighted mean arithmetic plasma L-arginine concentrations were observed in CTLN1 and ASA. Dosages for the supplemented groups were calculated as arithmetic mean values per disease and are illustrated either as mg/kg/d (for all individuals with OTC-D and for individuals with CTLN1 and ASA with a body weight below 20 kg) or g/m2/d (for individuals with CTLN1 and ASA with a body weight above 20 kg), according to (Haberle et al., 2019). ASA, argininosuccinic aciduria; BCAAs, branched-chain amino acid(s); CTLN1, citrullinemia type 1; LTx, liver transplantation; n/a, not applicable; OTC-D, ornithine transcarbamylase-deficiency.

Figure 2. Favorable linear growth outcome after LTx is independent from the supplementation with L-citrulline/arginine.

Figure 2.

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Boxplot illustrating height SDS for individuals with (n=10) or without (n=20) L-citrulline/arginine supplementation, each at last observation after LTx. Mean observation periods between first and last observation after LTx corresponds to 4.51 years for the supplemented cohort and 5.64 years for the non-supplemented control cohort. Growth outcome parameters at last observation after LTx are well within the normal range and do not differ between the supplemented and non-supplemented cohort. P=0.25; effect size: 0.21; Wilcoxon-Mann-Whitney Test. Data are shown as median (black thick line) and mean (triangle), length of the box corresponds to the interquartile range (IQR), upper and lower whiskers correspond to max. 1.5 × IQR, each point represents an outlier. LTx, liver transplantation. For descriptive characteristics see Supplementary Table 2.

3.4. Prevalence of motor abnormalities does not differ between the supplemented and non-supplemented cohorts

We next studied the prevalence of motor abnormalities in the L-citrulline/arginine supplemented (n=13) and non-supplemented cohort (n=16) during a balanced observation period of approximately three years in mean after LTx (supplemented group: 3.1 years; non-supplemented group: 3.7 years; p=0.24). No difference in the proportion of motor abnormalities was found between both groups (supplemented group: n=5/13; non-supplemented group: n=7/16; p=1.00; Figure 3).

Figure 3. Prevalence of motor abnormalities does not differ between the supplemented and non-supplemented cohorts.

Figure 3.

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Boxplot illustrating the presence and absence of motor abnormality for individuals with a severe phenotype after LTx. Bright shading corresponds to the presence, dark shading corresponds to the absence of motor abnormalities at last observation. Proportion of motor abnormalities (supplemented: n=5/13; non-supplemented: n=7/16; p=1.00, Boschloo’s Exact Test) is unaffected by the supplementation with L-citrulline/arginine when compared to the non-supplemented control cohort. This finding could also be corroborated in both cohorts (supplemented: n=5/9; non-supplemented: n=4/10; p=1.00, Boschloo’s Exact Test) at last observation prior to transplantation. For descriptive characteristics see Supplementary Table 2.

3.5. Cognitive function does not appear to be associated with the supplementation of L-citrulline/arginine

Since recent data suggested that individuals with a severe phenotype who underwent LTx did not perform better in cognitive testing when compared to their medically managed and severity-adjusted counterparts (unpublished data), we wondered whether supplementation with L-citrulline/arginine after transplantation might be associated with cognitive performance. To this end, we investigated supplemented (n=8) and non-supplemented (n=28) individuals after LTx with equal mean observations periods of approximately 2.5 years (supplemented group: 2.2 years; non-supplemented group: 3.0 years; p=0.49) until cognitive testing and found no difference in the cognitive outcome between both groups (cSDS in supplemented group: −1.86; cSDS in non-supplemented group: −2,36; p=0.24; effect size: 0.20; Figure 4).

Figure 4. Supplementation with L-citrulline/arginine does not differ with regard to the cognitive outcome when compared to the non-supplemented control cohort.

Figure 4.

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Boxplot depicting cognitive SDS at last follow-up testing after LTx of individuals with (n=8) or without (n=28) L-citrulline/arginine supplementation. Data are shown as median (black thick line) and mean (triangle), length of the box corresponds to the IQR, upper and lower whiskers correspond to max. 1.5 × IQR, each point represents an outlier. P=0.24; effect size: 0.20; Wilcoxon-Mann-Whitney Test. For descriptive characteristics see Supplementary Table 2.

4. Discussion

Applying the recently established genotype-specific classification system for early prediction of disease severity for UCDs, we were able perform a comparative and descriptive analysis of the effect of L-citrulline/arginine supplementation in individuals with OTC-D, CTLN1 and ASA having a severe phenotype and who have undergone LTx. The present study has four main results: (1) Annual frequency and causes of hospitalizations after LTx do not differ between the supplemented and non-supplemented groups. (2) Supplementation of L-citrulline/arginine does not increase weighted mean arithmetic plasma concentration of L-citrulline or L-arginine when compared to the non-supplemented control cohort. (3) Individuals after LTx have normal growth parameters independent from the supplementation with L-citrulline/arginine. (4) Supplementation with L-citrulline/arginine does neither appear to be associated with reduced frequency of motor abnormality nor with an altered cognitive long-term outcome when compared to the non-supplemented control cohort.

4.1. Annual frequency and causes of hospitalizations after LTx do not differ between the supplemented and non-supplemented cohorts

Although LTx is associated with sustained metabolic stability, prevention of the recurrence of hyperammonemic decompensations, liberalization of protein intake restriction as well as cessation of nitrogen scavenging therapy in UCDs (Whitington et al., 1998; Kayler et al., 2002; Morioka et al., 2005; Kim et al., 2013; Kido et al., 2017; Haberle et al., 2019; Kido et al., 2021; Sood et al., 2021), individuals who have undergone LTx are confronted with the risk of unscheduled hospitalizations due to complications associated with the immunosuppressive therapy or impaired transplant function. The present study reveals, that the number of hospitalizations per year of observation and the causes for hospital admission after LTx do not differ between individuals receiving L-citrulline/arginine supplementation and the non-supplemented group. Causes of hospitalizations included infections, gastrointestinal problems, acute and chronic rejections, biochemical alterations and various other reasons (see Figure 1), reflecting and confirming previously published results from larger observational LTx cohort studies (Yu et al., 2015; Ziogas et al., 2021).

4.2. Favorable growth after LTx does not appear to be associated with supplementation of L-citrulline/arginine

Symptomatic individuals with UCDs under long-term medical management suffer from impaired postnatal linear growth, which might be independent from the degree of (iatrogenic) protein restriction and may rather be associated with disease-severity, reduced to borderline plasma BCAA concentrations under acute and chronic hyperammonemic conditions, and reduced BCAA concentrations due to nitrogen-scavenging agents such as sodium phenylbutyrate (Burrage et al., 2014; Zielonka et al., 2018; Zielonka et al., 2019b; Posset et al., 2020a; Probst et al., 2020). Interestingly, a positive effect of L-arginine supplementation on growth of individuals with OTC-D was postulated (Nagasaka et al., 2006), and a favorable effect of LTx on growth was identified recently (unpublished data). To evaluate effects of supplementation of L-citrulline/arginine in individuals who had undergone LTx, we studied biochemical and anthropometric parameters (growth and weight) in individuals receiving supplementation with L-citrulline/arginine and found no difference in comparison to their non-supplemented counterparts with regard to anthropometric parameters that were well within the normal range at the end of the observation period. Intriguingly, supplementation with L-citrulline or L-arginine – as currently performed – was not associated with higher weighted mean arithmetic plasma amino acid concentrations of respective amino acids. Moreover, reduced weighted mean arithmetic plasma L-citrulline concentrations (for OTC-D) as well as borderline low mean arithmetic plasma L-arginine concentrations (for CTLN1 and ASA) were observed, suggesting that in the light of normalized plasma BCAA concentrations and cessation of nitrogen-scavenging agents after LTx, supplementation of L-citrulline/arginine appears to have a subordinate role for maintaining favorable growth outcome in individuals with UCDs after LTx.

4.3. Supplementation with L-citrulline/arginine does not appear to be associated with neurocognitive outcome(s) after LTx

Decreased concentrations of L-citrulline and L-arginine have been recently reported to be associated with dementia by targeted metabolomic analyses and the degree of impaired L-arginine signaling reflected severity of cognitive impairment (Fleszar et al., 2019). Moreover, administration of L-arginine suppressed cognitive decline and behavioral depression by counteracting cerebral oxidative stress response and reducing neuronal excitotoxic cell stress in senescence-accelerated SAMP10-mice (Pervin et al., 2021). Consistently, L-arginine supplementation improved cognitive function in hypertensive frail older adults (Mone et al., 2022). Intriguingly, it was recently reported that also L-citrulline administration was associated with improved spatial memory in a murine model of Alzheimer’s disease as assessed by the Morris water maze test (Martinez-Gonzalez et al., 2021). However, even though administration of L-arginine led to a significant increase in plasma L-arginine concentrations (Atzler et al., 2016), it was not associated with improved cognitive performance in a cohort of healthy adults (Schonhoff et al., 2018). The present study reveals that supplementation of L-citrulline/arginine in individuals with UCDs who have undergone LTx did not appear to alter the neurocognitive outcome (motor and cognitive function) when compared to the non-supplemented control cohort. This observation might be explained by the fact that the positive effect of L-arginine administration on cognitive function mainly relies on interfering with impaired L-arginine/NO signaling underlying vascular pathology in Alzheimer disease, other forms of dementia and cardiovascular diseases. Notably, impaired L-arginine/NO signaling is not supposed to be a pathophysiological hallmark in UCDs except for ASA (Erez et al., 2011). In the present study, the effect of L-arginine supplementation could not be assessed in ASA on an individual level due to low data density with regard to cognitive testing in the databases. Moreover, cognitive impairment in UCDs is thought to be (mainly) caused by the devastating impact of hyperammonemic encephalopathy at initial disease manifestation and therefore might be a neurological long-term sequela of NH4+max and/or subsequent hyperammonemic decompensations prior to LTx, that appears not to be sufficiently addressed by LTx and subsequent L-citrulline/arginine supplementation as currently performed. Furthermore, ammonia-independent pathomechanisms due to tissue-specific neuronal dysfunction of the ASL protein have been identified potentially contributing to the neurodegenerative disease course observed in ASA (Erez et al., 2011; Baruteau et al., 2017; Lerner et al., 2019; Lerner et al., 2021). Of note, ASS1, the enzyme deficient in CTLN1, has recently been reported to exert RNA-binding properties involved in post-transcriptional gene regulation and thus, pathogenic ASS1 variants might interfere with RNA-metabolism potentially underlying progressive cognitive decline in CTLN1 even in the absence of hyperammonemic episodes after LTx (Castello et al., 2012). However, it remains unclear whether higher weighted mean arithmetic plasma concentrations of L-citrulline and/or L-arginine might be associated with improved cognitive outcome (at least for ASA), which remains subject to future systematic prospective and controlled clinical trials.

4.4. Limitations and directions for future research

This study has some inherent limitations. Firstly, effect sizes of each subanalyses are expectedly low which along with the relatively low number of included probands leads to low statistical power of the respective subanalyses, which is a general limitation in rare disease research. Therefore, the results of this pilot study need to be confirmed in larger prospective controlled clinical trials with even longer observation periods. Secondly, important covariates including presence, severity and duration of coma during hyperammonemic decompensation(s) as well as epilepsy could not be considered for phenotypic severity classification due to low data density and quality of entered data. Given the lack of appropriate disease prediction models, further UCD subtypes could not yet be included in the present analysis and remain subject to future research. Furthermore, blood pressure as further outcome parameter (in ASA) could not be assessed due to low data density. Information on indication to supplement or not to supplement with L-citrulline/arginine as well as on the proband’s adherence to the supplementation were not systematically captured in both databases. In perspective, future analyses might replace NH4+max as surrogate parameter for residual enzymatic activity once genetic testing is carried out for all individuals with UCDs followed by determination of in vitro residual enzymatic activity. Importantly, future severity-adjusted prospective clinical trials will need to prove whether supplementation of L-citrulline/arginine resulting in therapeutic plasma levels is beneficial for the long-term management of individuals with UCDs after LTx.

5. Conclusion

This severity-adjusted and comparative pilot study for mOTC-D, CTLN1 and ASA, i.e., the three most prevalent UCD subtypes, shows that supplementation of L-citrulline/arginine after LTx – as currently performed – is not associated with higher plasma concentrations of L-citrulline or L-arginine when compared to the non-supplemented control cohort. Growth parameters after LTx are within the normal range independent from the supplementation with L-citrulline/arginine. Moreover, the neurocognitive long-term outcome in individuals who have undergone LTx does not appear to be associated with L-citrulline/arginine supplementation as reflected by the unaltered frequency of motor abnormalities and cognitive SDS at last observation.

Supplementary Material

Supplementary Material

Acknowledgements

All UCDC and E-IMD sites contributed to the datasets of the longitudinal studies used in this publication. Principal investigators and personnel with key contributions are listed as UCDC and E-IMD consortia study group members. The Urea Cycle Disorders Consortium (UCDC; U54HD061221) is a part of the National Institutes of Health (NIH) Rare Disease Clinical Research Network (RDCRN), supported through collaboration between the Office of Rare Diseases Research (ORDR), the National Center for Advancing Translational Science (NCATS), the Eunice Kennedy Shriver National Institute of Child Health and Human Development (NICHD), and the National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK). The Urea Cycle Disorders Consortium is also supported by the O’Malley Foundation and the Kettering Fund. Members of the UCDC include the following, some of whom have been listed as authors on this manuscript: Nicholas Ah Mew, Matthias R. Baumgartner, Gerard Berry, Susan A. Berry, Margo Breilyn, Lindsay Burrage, Curtis Coughlin, George A. Diaz, Gregory Enns, Can Ficicioglu, Renata C. Gallagher, Andrea Gropman, Cary O. Harding, Georg F. Hoffmann, Laura Konczal, Christina Lam, Cynthia Le Mons, Shawn E. McCandless, Sandesh CS Nagamani, Roland Posset, Andreas Schulze, Jennifer Seminara, Tamar Stricker, Derek Wong, and Greta Wilkening. Furthermore, we gratefully acknowledge the following study coordinators – Saima Ali, Kim Bardillon, Kia Bryan, Liora Caspi, Kiaira Coles, Sara Elsbecker, Debbie Fu, Florian Gleich, Seishu Horikoshi, Elijah Kravets, Genya Kisin, Rhonda Jones, Ursula Kuhn, Jennifer Phillip, Thu Quan, Kara Simpson, Julia Sloan, Tamar Stricker, Hayden Vreugdenhil, Ashley Wilson, and Melissa Zerofsky – and study neuropsychologists – Fabienne Dietrich Alber, Christopher Boys, David Breiger, Benjamin Goodlett, Elizabeth Kerr, Casey Krueger, Eva Mamak, Ami Norris-Brilliant, David Schwartz, Yuri Shishido, Arianna K. Stefanatos, Rachel Tangen, and Magdalena E. Walter. We would also like to acknowledge the contributions of (former) longitudinal study PIs: Mark L. Batshaw, Stephen Cederbaum, Annette Feigenbaum, Douglas S. Kerr, Brendan Lee, Uta Lichter-Konecki, Margretta R. Seashore, Marshall L. Summar, and Cynthia Le Mons. Moreover, we thank Dr. Christophe Chardot, Dr. Carmen Capito, and Dr. Florence Lacaille for their contribution. In particular, we are indebted to all our UCD individuals and their families for their trust, patience and participation in both longitudinal registry studies for many years.

Funding Source

The Urea Cycle Disorders Consortium (UCDC; U54HD061221) is part of the National Institutes of Health (NIH) Rare Disease Clinical Research Network (RDCRN), supported through collaboration between the Office of Rare Diseases Research (ORDR), the National Center for Advancing Translational Science (NCATS), the Eunice Kennedy Shriver National Institute of Child Health and Human Development (NICHD), and the National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK). The Urea Cycle Disorders Consortium is also supported by the O’Malley Foundation, the Rotenberg Family Fund, the Dietmar Hopp Foundation, the Kettering Fund, and the National Urea Cycle Disorders Foundation. In addition, support for neuropsychological testing is provided by a NIH grant for Intellectual and Developmental Disability Research Centers (U54HD090257). This work was also supported in part by the Clinical Translational Core at Baylor College of Medicine which is supported by the IDDRC grant number P50 HD103555 from the Eunice Kennedy Shriver National Institute of Child Health and Human Development. The E-IMD patient registry has received funding by the European Union (E-IMD; EAHC no 2010 12 01; coordinator: Stefan Kölker), in the framework of the Health Programme. After the end of the EU funding period the E-IMD patient registry has been sustained by funding from the Kindness-for-Kids Foundation (Munich, Germany), and the Kettering Fund. This work was supported by a Trainee Research Fellowship Award 2022–2023 provided by the UCDC. S.K., and G.F.H. received funding from the Dietmar Hopp Foundation for coordinating the study “Newborn Screening and Metabolic Medicine 2020 (NBS2025),” which included individuals with UCDs.

Declaration of competing interest

RP receives consultancy fees from Immedica Pharma AB. SK received funding from Immedica Pharma AB for the European Post-Authorization Registry for Ravicti® (glycerol phenylbutyrate) oral liquid in partnership with the E-IMD (RRPE) (EU PAS Register no. EUPAS17267; http://www.encepp.eu/). GFH received lecture fees from Swedish Orphan Biovitrum GmbH. AS is paid consultant and member of advisory boards for HZNP Canada Ltd, ModernaTX Inc., and Recordati Rare Diseases Canada Inc. AS is site PI on clinical trials sponsored by ModernaTX Inc., Ultragenyx Canada Inc., and iECURE Inc. The sponsors have in no way influenced the design, conductance, analysis and report of the present study. All other authors declare that they have no conflict of interest.

Abbreviations

ASA

argininosuccinic aciduria

ASL

argininosuccinate lyase

ASS1

argininosuccinate synthetase 1

BCAA(s)

branched-chain amino acid(s)

cSDS

cognitive standard deviation score

CTLN1

citrullinemia Type 1

UCD(s)

urea cycle disorder(s)

E-IMD

European registry and network for Intoxication type Metabolic Diseases

IQR

interquartile range

LME

linear mixed effect regression models

LTx

liver transplantation

(m)OTC-D

(male) OTC-deficiency

NH4+max

initial peak plasma ammonium concentration

OTC

ornithine transcarbamylase

SDS

standard deviation score

UCDC

Urea Cycle Disorders Consortium

Footnotes

Financial disclosure

All authors declare that they have no financial relationships relevant to this article to disclose.

Clinical Trial Registration

The E-IMD registry is registered on the German Clinical Trials Register (https://www.drks.de), whereas the UCDC database is recorded at the US National Library of Medicine (https://clinicaltrials.gov). Data sets generated and analyzed during the current study are not publicly available due to existing data protection laws. Ownership and availability of data is subject to the members of the UCDC and E-IMD consortia study group and their consent upon request.

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