Compliance and Genetic Variability Are Determinants of the Success of l -Arginine/ l -Citrulline Supplementation in High Altitude

Abstract

Pena, Eduardo, Samia El Alam, Juliane Hannemann, and Rainer Böger. Compliance and genetic variability are determinants of the success of l-arginine/l-citrulline supplementation in high altitude—A pilot study. High Alt Med Biol. 00:00–00, 2026.

Background:

Asymmetric dimethylarginine (ADMA) is a risk biomarker of high-altitude pulmonary hypertension (HAPH). ADMA is inactivated by dimethylarginine dimethylaminohydrolase (DDAH)1 and 2. Single nucleotide polymorphisms in these genes contribute to genetic predisposition for HAPH. Supplementation with l-arginine may reverse ADMA’s inhibitory effect and, thus, HAPH. We conducted a pilot study to test whether supplementation with l-arginine/l-citrulline (A/C) during chronic intermittent hypoxia (CIH) limits the increase in pulmonary arterial pressure (PAP) as compared to placebo.

Methods:

Twenty male volunteers were randomly assigned to receive 6 months of A/C or placebo; PAP was assessed by echocardiography.

Results:

l-Arginine increased in the A/C group at month 1, which subsided until month 6 due to poor compliance with the study supplement. Individuals with 2 or more alleles of a previously described DDAH1/2 haplotype showed a higher increase in ADMA during CIH. A/C caused a trend toward a diminished increase in PAP. However, these findings should be interpreted as exploratory, consistent with the low adherence to treatment in this pilot study. The increase in l-arginine blood levels was attenuated in carriers of a previously described ARG1/2 haplotype.

Conclusion:

Our data show that A/C supplementation may offer an opportunity to alleviate the altitude-induced increase in PAP; however, long-term compliance and genetic factors may affect outcome.

Keywords

chronic intermittent hypoxia echocardiography high altitude pulmonary hypertension nitric oxide single nucleotide polymorphisms

Introduction

Hypoxic pulmonary vasoconstriction (HPV) is a major mechanism contributing to high altitude pulmonary hypertension (HAPH) during chronic-intermittent hypobaric hypoxia (CIH) (Brito et al., 2018; Böger and Hannemann, 2020). Having been discovered as a physiological mechanism in the 1940s (von Euler and Liljestrand, 1946; Blakemore et al., 1955), we now know that signal transduction pertaining to endothelium-derived nitric oxide (NO) is involved. NO is a potent vasodilator; its reduced production and/or biological action in the hypoxic pulmonary vasculature have been demonstrated (Böger and Hannemann, 2020).

Asymmetric dimethylarginine (ADMA) is an endogenous inhibitor of NO synthesis (Böger et al., 2000, Böger et al., 2004); ADMA concentration is increased in individuals exposed to chronic hypobaric hypoxia (CH) or CIH (Lüneburg et al., 2017; Brito et al., 2018; Verratti et al., 2020). In a previous study, we demonstrated the progressive increase in ADMA plasma concentration during 6 months of exposure to CIH in a young, healthy, male Chilean population. We also found that ADMA is a biomarker predicting the risk of HAPH (Siqués et al., 2019). This finding offers an opportunity to assess an individual’s risk of HAPH before ascent to high altitude; it also has a therapeutic perspective: ADMA exerts its effects by competitively displacing l-arginine, the substrate for NO synthesis, from the endothelial NO synthase. Thus, elevation of l-arginine concentration reverses the inhibitory action of ADMA, restores physiological NO production, and reverses endothelial dysfunction. This has been shown in hypercholesterolemic individuals (Böger et al., 1998a) and in patients with cardiovascular diseases (Böger et al., 1998b, Böger and Ron, 2005). In addition, we have previously identified gene polymorphisms in two genes coding for the two major enzymes that metabolically degrade ADMA and thereby determine its concentration, dimethylarginine dimethylaminohydrolase 1 (DDAH1) and DDAH2, to be genetic determinants of the increase of ADMA at high altitude (Hannemann et al., 2021a).

We therefore hypothesized that dietary supplementation with l-arginine might help to prevent the increase in pulmonary arterial pressure during prolonged exposure to high altitude. However, there are several drawbacks to such a strategy: (a) l-arginine has a very short plasma half-life of only about 1 hour (Bode-Böger et al., 1998; Tangphao et al., 1999). This means that it must be administered in many, repeated doses per day to allow for sustained elevation of plasma l-arginine concentration. (b) About 6 g of l-arginine are consumed by an ordinary Western diet every day. To induce a relevant elevation of plasma l-arginine concentration beyond this “baseline” level, several grams of l-arginine powder must be ingested. (c) l-arginine has a bitter taste that undermines efforts to administer high oral doses and that specifically impairs compliance of those who are supposed to ingest this remedy for a prolonged period of time.

We have previously shown that a combination of l-arginine plus l-citrulline significantly extends the plasma half-life of l-arginine, as l-citrulline does not undergo pre-systemic elimination by intestinal arginases (as does l-arginine), and it is converted gradually to l-arginine in the kidneys (Ratner and Petrack, 1953). Preparing a mixture of l-arginine and l-citrulline has allowed us to add ingredients that overcome l-arginine’s bad taste, allowing to administer the desired, high daily dosages over prolonged periods of time.

We performed the present clinical pilot study to test whether administration of an oral l-arginine/l-citrulline (A/C) supplement is feasible and succeeds in sustained elevation of circulating l-arginine concentration in the conditions of a remote high-altitude environment. We also tested participant follow-up and the logistical feasibility of blood sampling, preservation, and transport to a clinical laboratory in due time as well as echocardiographic analyses of cardiac function in a remote high-altitude setting. Echocardiographic variables are considered exploratory and hypothesis-generating, consistent with the pilot nature of this study in interpreting the functional impact of the dietary supplement.

Patients and Methods

Study participants and study protocol

We included 20 healthy male army draftees; none of them had been exposed to high altitude before. Written informed consent was obtained from all study participants before the study. The protocol was approved by the Ethical Committee of Universidad de Tarapacá, Arica, Chile (N°08/2023).

During the study period of 6 months, the participants adhered to a shift regimen of 5 days at high altitude (HA; 3,550 m) followed by 2 days of recovery at sea level (SL). Their daily routine at high altitude comprised moderate physical activity and a standardized diet (3,000 kcal daily). The supplements were handed to the participants in containers that contained supply for 1 month each, that is, each participant received six containers with supplements during the study. Compliance was expressed as a percentage of unopened containers returned by participants relative to the total supplied.

Measurements were taken at baseline (month 0; complete investigation incl. echocardiography), month 1 (sampling of dried blood spots only), and month 6 (complete investigation incl. echocardiography).

Preparation and administration of supplements

Dietary supplements contained a mixture of l-arginine and l-citrulline (2 daily doses of 3 g of l-arginine plus 1.5 g of l-citrulline) or placebo (2 daily doses of lactose placebo powder). The supplement for each month was filled into plastic containers and protected from moisture by sealing. Allocation of study participants to l-arginine/l-citrulline or to placebo was blinded.

Analysis of l-arginine and its metabolites by UPLC-MS/MS in dried blood spots

l-arginine, l-citrulline, l-ornithine, ADMA, and SDMA were quantified in dried blood spots using an ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) method previously developed and validated in our laboratory (Schwedhelm et al., 2007). Extraction of analytes from dried blood spots was performed as published before (Hannemann et al., 2020). Briefly, dried blood from three punched-out circles (5 mm each) was dissolved in 300 µl extraction buffer. 25 µl of the extract solution was diluted in 100 µl of methanol to which stable isotope-labelled internal standards had been added. Subsequently, the compounds were converted into their butyl ester derivatives. Quantification of analytes was performed on a Waters UPLC-MS/MS platform (Xevo TQ-S cronos, Waters GmbH, Eschborn, Germany) applying an ACQUITY UPLC BEH C₁₈ column (2.1 × 50 mm, 1.7 μm, Waters GmbH) for chromatographic separation. The coefficient of variation for the quality control samples was below 6% for all compounds.

Isolation of DNA from dried blood and genotyping

DNA was isolated from dried blood spots using a salting-out procedure as reported previously (Hannemann et al., 2021b). DNA concentration was determined using a nanophotometer NP60 (Implen, Munich, Germany). Genotyping of single nucleotide polymorphisms (SNPs) was performed using single-tube human TaqMan SNP Genotyping Assays (Thermofisher Scientific, Dreieich, Germany) as described before (Hannemann et al., 2021a). We genotyped two SNPs in the ARG1 gene (rs2246012 and rs2781667), ARG2 gene (rs3742879 and rs2759757), and DDAH2 gene (rs805304 and rs2272592), and three SNPs in the DDAH1 gene (rs1241321, rs480414, and rs233112), respectively, after having excluded SNPs that were in strong linkage disequilibrium with each other by checking the LDLink database of the National Cancer Institute, USA (https://ldlink.nci.nih.gov/). In order to test for the combined effects of DDAH1 and DDAH2 on ADMA concentration and the combined effects of ARG1 and ARG2 on l-arginine concentration, we constructed haplotypes combining the inactive alleles of both in each of these gene pairs, as described previously (Hannemann et al., 2021a, 2021b).

Echocardiography

Echocardiography was performed by trained cardiologists according to the standards set forth in the 2010 guideline of the American Society of Echocardiography (Rudski et al., 2010). Left ventricular ejection fraction (LVEF) was measured using the standard Simpson method. The diameter and respiratory variability of the inferior vena cava was measured to estimate right atrial pressure (RAP). Right ventricular pressure gradient (RVPG) was calculated from the peak velocity of the tricuspid regurgitation using the simplified Bernoulli equation; systolic pulmonary artery pressure (sPAP) was calculated as RVPG + RAP (Yock and Popp, 1984; Rudski et al., 2010). Mean pulmonary artery pressure (mPAP) was calculated from the formula: mPAP = (0.61 × sPAP) + 2 (Chemla et al., 2004). Due to small numbers of echocardiographic investigations at month 6, no formal statistical analyses were performed on echocardiographic data.

Calculations and statistical analyses

From biomarker concentrations measured, we calculated the l-arginine/ADMA ratio, a measure for NO synthase substrate availability, as well as the l-citrulline/l-arginine ratio, a surrogate measure of NOS catalytic activity, and the l-ornithine/l-arginine ratio, a measure of the arginase catalytic activity. Data are presented as mean ± standard deviation for all continuous variables. We analyzed the data by fitting a mixed model as implemented in GraphPad Prism. This mixed model uses a compound symmetry covariance matrix and is fit using Restricted Maximum Likelihood. In the absence of missing values, this method gives the same p values and multiple comparisons tests as repeated measures analysis of variance (ANOVA). In the presence of randomly missing values, the results can be interpreted like repeated measures ANOVA. All statistical analyses were performed using SPSS (version 25; IBM Corporation, Armonk, NY, USA) and GraphPad Prism (version 6.01, GraphPad Software, San Diego, CA, USA). For all tests, p < 0.05 was considered statistically significant after applying the Geisser Greenhouse correction for multiple testing.

Results

Baseline characteristics of the study population

We recruited 20 healthy, male study participants aged 20.0 ± 1.8 years. They had a mean body mass index of 24.8 ± 3.6 kg/m² and no concomitant chronic or acute diseases. All study participants had cardiac dimensions and function parameters within the normal range of healthy individuals in the baseline echocardiographic assessment with no significant differences between both groups (Table 1). Concentrations of l-arginine and its metabolites in dried blood spots were within previously published normal ranges (Table 1).

Table 1.

Baseline Characteristics of the Study Population

Parameter	All	Arg/Cit	Placebo	p
Demographic and anthropometric measures
Number of participants	20	10	10	n.a.
Height [cm]	171 ± 6	171 ± 4	172 ± 8	0.726
Weight [kg]	73.0 ± 13.4	67.4 ± 8.7	78.5 ± 15.3	0.067
BMI [kg/m²]	24.8 ± 3.6	23.1 ± 2.5	26.5 ± 3.8	0.036
Echocardiographic parameters
LVEF [%]	66.9 ± 9.1	62.7 ± 5.9	71.1 ± 10.0	0.037
TAPSE [mm]	21.3 ± 2.1	21.0 ± 2.3	21.6 ± 1.8	0.529
Transtricuspid gradient [mm Hg]	4.9 ± 2.9	3.6 ± 1.1	5.8 ± 3.6	0.144
sPAP [mm Hg]	9.6 ± 3.0	8.7 ± 1.0	10.4 ± 3.8	0.260
mPAP [mm Hg]	7.9 ± 1.8	7.3 ± 0.6	8.3 ± 2.3	0.260
L-Arginine metabolite concentrations [µmol/l]
l-Arginine	21.3 ± 5.3	20.2 ± 6.8	22.4 ± 3.2	0.361
l-Citrulline	21.5 ± 5.1	21.5 ± 5.3	21.5 ± 5.1	0.998
l-Ornithine	25.7 ± 5.2	23.7 ± 4.4	27.7 ± 5.4	0.087
ADMA	0.358 ± 0.053	0.354 ± 0.046	0.362 ± 0.061	0.733
SDMA	0.426 ± 0.060	0.409 ± 0.041	0.443 ± 0.072	0.217

Data are mean ± standard deviation. p Values relate to statistical differences between the Arg/Cit and placebo groups, as assessed by one-way ANOVA.

ADMA, asymmetric dimethylarginine; BMI, body mass index; LVEF, left ventricular ejection fraction; mPAP, mean pulmonary arterial pressure; SDMA, symmetric dimethylarginine; sPAP, systolic pulmonary arterial pressure, TAPSE, tricuspid annular plane systolic excursion.

Effects of high altitude exposure and l-arginine supplementation on blood metabolite concentrations

ADMA concentration increased slightly but significantly, with no significant difference between both intervention groups during exposure to CIH (0.36 ± 0.06 to 0.41 ± 0.08 µmol/l; p = 0.0015), whereas SDMA concentration was significantly reduced (0.44 ± 0.06 to 0.36 ± 0.06 µmol/l; p = 0.0011; Fig. 1a and b).

FIG. 1.

Concentrations of (a) asymmetrical dimethylarginine (ADMA) and (b) symmetric dimethylarginine (SDMA) in dried blood spots taken at baseline, after 1 month, and after 6 months of study duration in the l-arginine/l-citrulline-supplemented group (A) and in the placebo group (P). Each dot represents one study participant; boxes represent median and interquartile range, with whiskers extending to the range of measurements. **p < 0.01 for comparisons of time points of measurement for both treatment groups combined.

In month 1 of the study, we observed a significant 60% increase in mean l-arginine concentration in A/C-supplemented participants, whereas no such effect was noted in those allocated to placebo (Fig. 2a). However, this effect in the A/C-supplemented group was lost over time, so that l-arginine concentrations in both groups were similar in month 6 of the study. Mean l-citrulline concentration slightly increased in the A/C-supplemented group (21.5 ± 5.1 to 26.2 ± 6.8 µmol/l; p < 0.01; Fig. 2b), whereas l-ornithine concentration showed a steep, highly significant increase in both groups in months 1 and 6 (Fig. 2c).

FIG. 2.

Concentrations of (a) l-arginine, (b) l-citrulline, and (c) l-ornithine in dried blood spots taken at baseline, after 1 month, and after 6months of study duration in the l-arginine/l-citrulline-supplemented group (A) and in the placebo group (P). Each dot represents one study participant; boxes represent median and interquartile range, with whiskers extending to the range of measurements. *p < 0.05, **p < 0.01 for comparison with baseline concentrations.

Compliance of study participants with supplement intake

Under the conditions of a fully voluntary study participation with no infrastructural means of maintaining compliance of study participants at the high-altitude site during the 6 months’ duration of this pilot trial, 18 individuals agreed to provide dried blood spots for metabolite measurements at month 1, but only 12 participants (60% of the initial study group of 20 participants) participated in the 6 months’ follow-up investigation, which included clinical parameters and echocardiography.

Based on quantification of l-arginine in dried blood spots, even for those who attended the follow-up investigations, we observed decreasing blood l-arginine concentration in three out of six A/C-supplemented participants (month 6).

Consistent with the above, at the end of the study, the mean adherence in the A/C group was 80.6%. Specifically, three participants consumed all six containers (100% adherence), one participant consumed five containers (83.3%), and two participants consumed three containers (50%). Overall, 66.7% of participants in this group achieved adherence ≥80%. In contrast, the placebo group showed a mean adherence of 66.7%. Within this group, three participants consumed all six containers (100%), one participant consumed four containers (66.7%), and two participants consumed three containers (50%). Consequently, only 50% of participants in the placebo group reached adherence ≥80%.

Association of arginase gene polymorphisms with blood l-arginine and l-ornithine concentrations

None of the ARG1 SNPs were significantly associated with l-arginine blood concentration in A/C-supplemented participants, nor in those allocated to placebo. However, homozygous carriers of the minor allele of ARG1 rs2781667 showed the highest increase in l-ornithine/l-arginine ratio during A/C supplementation (Supplementary Figs. S1 and S2). For the ARG2 SNPs, homozygous carriers of the major allele of both, rs3759757 and rs3742879 had the highest l-ornithine/l-arginine ratio at month 6 in the A/C-supplemented group; this was significant for the ARG2 SNP rs3759757 (Supplementary Figs. S3 and S4).

To assess the influence of both arginase genes on l-arginine and l-ornithine blood concentrations during arginine supplementation, we constructed a previously published ARG1/2 haplotype consisting of the minor allele of ARG1 rs2246012 and the major allele of ARG2 rs3759757 (Hannemann et al., 2021b). Study participants supplemented with A/C who carried two or more of these alleles had a steeper increase in l-ornithine blood concentration and an attenuated increase in l-arginine blood concentration, whilst the time course of both metabolites showed no differences in the placebo group (Fig. 3).

FIG. 3.

(a) and (c) Time course of l-arginine and l-ornithine blood concentration at baseline (sea level), 1 month, and 6 months of chronic intermittent hypoxia in participants carrying either 0–1 active allele or 2 or more active alleles of the combined ARG1/ARG2 haplotype. (b) and (d) l-Ornithine/l-arginine ratio (Orn/Arg Ratio) at baseline, 1 month, and 6 months of chronic intermittent hypoxia in participants carrying either 0–1 active allele or 2 or more active alleles of the combined ARG1/ARG2 haplotype. (a) and (b) display results from the l-arginine/l-citrulline-supplemented group; (c) and (d) display results from the placebo group.

None of the ARG1 nor ARG2 SNPs showed significant associations with l-citrulline, ADMA, or SDMA blood concentrations (data not shown).

Association of DDAH gene polymorphisms with blood ADMA concentrations

SNPs in the DDAH1 and DDAH2 genes were not significantly associated with ADMA blood concentrations in this study. However, a previously described DDAH1/DDAH2 haplotype (Hannemann et al., 2021a) was significantly associated with ADMA concentration: Carriers of two or more alleles of the haplotype had a significantly higher ADMA concentration at 6 months of CIH exposure, whereas carriers of less than two alleles showed no significant increase in ADMA (Fig. 4).

FIG. 4.

ADMA concentration in dried blood spots at baseline and at 6 months of chronic intermittent hypoxia in carriers of 0–1 active allele or 2 or more active alleles of the combined DDAH1/DDAH2 haplotype. Data represent median and interquartile range, with whiskers extending to the range of measurements. **p < 0.01 for comparison with baseline values.

Echocardiographic assessment of cardiac function after CIH exposure

In our exploratory analysis of echocardiographic data, we observed no significant change in mean LVEF from month 0 to month 6 in either of the two groups (Fig. 5a). However, we noted trends toward a higher increase in parameters of right ventricular pressure in the placebo group as compared to the A/C group (Fig. 5b–d). Whilst we performed no formal test for statistical significance due to missing data, there was a clear and uniform trend toward an alleviated increase in right ventricular pressure in the A/C group.

FIG. 5.

Echocardiographic parameters of left and right ventricular function and pulmonary artery pressure at baseline and at 6 months of chronic intermittent hypoxia. (a) Left ventricular ejection fraction (LVEF), (b) tricuspid annular plane systolic extension (TAPSE), (c) tricuspid regurgitation peak pressure gradient (TRPG_max), (d) systolic pulmonary artery pressure (sPAP). Due to the exploratory nature of this analysis, data shown represent mean only to account for small numbers of observations in some of the variables, and no formal statistical testing was performed.

Incidence of acute mountain sickness

The Lake Louise Score was 2.8 ± 2.1 immediately after the first ascent to high altitude with no significant difference between both groups. It was 2.2 ± 2.3 after 1 month and declined to 1.2 ± 1.7 after 6 months (Table 2).

Table 2.

Lake Louise Score of Acute Mountain Sickness

Group	Day 1	Month 1	Month 6	p
Total Cohort	2.8 ± 2.1 (20)	2.2 ± 2.3 (18)	1.2 ± 1.7 (12)	0.164
Arg/Cit Group	3.1 ± 2.2 (10)	3.3 ± 3.1 (8)	0.8 ± 0.8 (6)	0.213
Placebo Group	2.4 ± 2.0 (10)	1.3 ± 0.8 (10)	1.5 ± 2.3 (6)	0.247
p	0.758	0.241	0.223

Data represent mean ± standard deviation of the Lake Louise Score at the indicated times of investigation at high altitude. Arg, l-arginine; Cit, l-citrulline. p Values refer to mixed models analysis for rows and columns, respectively.

Discussion

We performed this pilot study to assess conditions that affect the conduct and outcome of an interventional trial under the conditions of high-altitude exposure. Although direct results from this study are difficult to interpret due to the small number of participants, our principal findings are as follows: 1.

Supplementation of study participants with A/C during high altitude exposure is feasible and bears promising potential as a measure of prevention of increased PAP and, consequently, HAPH.

The major factors influencing variability of outcome data are genetic variability between study participants and limited compliance of participants with intake of study product(s).

Adherence of study participants to present at follow-up visits strongly depends on measures of study management to maintain contact, send reminders, and clearly define appointments in line with individual work schedules.

Biological plausibility and interindividual variability

Studies have assessed HPV, which results in an increased PAP and, when extended over prolonged periods of time, may lead to pulmonary hypertension (Siqués et al., 2019; Hannemann et al., 2020). For example, HAPH may be considered a cause of long-term morbidity and mortality among workers at high altitude in the Andean region, who often maintain an intermittent regimen of several days of work at high altitude, followed by some days of recovery at SL (Brito et al., 2018; Yang et al., 2025). Our group has previously reported the prevalence of manifest HAPH to be as high as 9% among long-term high-altitude mining workers in Chile (Brito et al., 2018). Given the high number of workers undergoing such CIH work schedules (Brito et al., 2007; Reveco et al., 2026), this is also a matter of health concern for the Andean countries’ health systems, as follow-up cost and loss of work ability are enormous (Reveco et al., 2026).

In previous studies our group has identified upregulation of the endogenous inhibitor of NO synthesis, ADMA, to be a prospective marker of the risk of HAPH (Siqués et al., 2019). Amongst other experimental proof, a causal involvement of ADMA in HPV was suggested by enhanced pulmonary arterial vasoconstriction in DDAH1 k.o. mice (Leiper et al., 2007; Wang et al., 2019). Reversely, DDAH1-overexpressing mice showed decreased HPV as compared to wild-type littermates (Bakr et al., 2013). As we have shown before that the biological effects exerted by ADMA can be overcome by increasing availability of l-arginine (Böger, 2004; Böger, 2007), dietary supplementation of l-arginine might be a promising approach to primary prevention of HAPH in those at risk. It offers the advantage of being well tolerated, not being a chemical drug, and thus being well acceptable to consumers (Naito et al., 2025). However, as l-arginine has a short half-life, we previously demonstrated that prolongation of its half-life by adding l-citrulline—which is not a substrate of intestinal arginases and, after intestinal absorption, is slowly converted to l-arginine in the kidneys—helps to maintain l-arginine concentration in blood at an elevated level over 24-hours (Schwedhelm et al., 2008; Morita et al., 2014).

Another issue that must be considered in studying A/C supplementation in HAPH-prone individuals is the genetic makeup of study participants. Our previous data showed that DDAH1 and DDAH2 SNPs, which affect enzyme activity and/or expressional regulation of these enzymes, have a strong association not only of blood ADMA concentration but also of HAPH risk (Hannemann et al., 2021a). Specifically, in this study two SNPs in the DDAH1 and DDAH2 genes (rs233112 and rs805304) were significantly associated with ADMA concentration and HAPH incidence. Therefore, our present data, although generated in a very small group of participants in this pilot study, is consistent with our previous finding by showing that a DDAH1/DDAH2 haplotype combining these two SNPs was significantly associated with high ADMA concentration after 6 months of CIH. Our present study also supports the prior finding that SNPs in the arginase genes affect individuals’ responses to l-arginine supplementation (Hannemann et al., 2021b). These data, whilst preliminary and not suited to prove a genetic determination of treatment response, allow to generate the hypothesis that individuals carrying the DDAH1/2 haplotype might experience a greater increase in ADMA at high altitude and benefit more from l-arginine supplementation, whilst carriers of the ARG1/2 haplotype might have an attenuated response to l-arginine supplementation. These hypotheses need to be confirmed in larger, well-controlled trials.

Notably, we observed that l-ornithine concentration showed a significant increase during high altitude exposure, independently of the allocation to treatment groups. It is well known that arginase-2 is induced in hypoxia; arginase converts l-arginine into l-ornithine and thereby deprives NO synthase from its substrate (Caldwell et al., 2018; Hannemann et al., 2021b). Our data show that A/C supplementation increased blood levels of both, l-arginine and l-citrulline, whilst the altitude-induced increase in L-ornithine did override the static levels of l-arginine and l-citrulline in the placebo group. As a net effect, the balance between l-arginine and l-ornithine, as expressed by the l-ornithine/l-arginine concentration ratio in blood, was maintained stable in the A/C-supplemented group, whereas it showed a steep, significant increase in the placebo group.

Compliance as a central determinant of interpretability

A key finding of this study is the progressive reduction in adherence to the study supplement throughout follow-up, which fundamentally shaped the interpretability of our results. High initial adherence likely enabled the detection of short-term biological effects, such as early increases in l-arginine and l-citrulline. However, as the study advanced, participants’ willingness not only to consume the supplements but also to attend scheduled follow-up assessments declined substantially. This decline resulted in incomplete data at the 6-month endpoint, limiting the robustness of cardiopulmonary and metabolic readouts and compelling us to interpret long-term outcomes with considerable caution. Consequently, our findings warrant a clear distinction between biologically plausible short-term responses observed under adequate adherence and inconclusive long-term effects, which cannot be attributed to a lack of physiological efficacy but rather to insufficient and inconsistent exposure to the intervention. Therefore, based on the above it is important to highlight that in a definite trial, adherence needs to be potentiated with intervention strategies such as simplifying the treatment plan, improving patient knowledge, and organizing social support (Schnorrerova et al., 2025).

Limitations

Considering this drawback of our pilot study, we have refrained from plotting standard deviations and performing statistical tests for echocardiographic outcome variables. Nonetheless, our current data present a promising picture of the potential effect of the dietary intervention on the pulmonary circulation. We observed no trend of any differences in LEVF, neither over time of high-altitude exposure nor between treatment groups. By contrast, the tricuspid annular plane systolic excursion trended to increase more in A/C-supplemented individuals than in those allocated to placebo. Most intriguingly, the maximal tricuspid regurgation pressure (TRPG_max) showed a steep increase during high altitude exposure in the placebo group (in fact, it almost tripled), whereas this increase was attenuated in the A/C group. The same was true for systolic and mPAP, which both increased more strongly in the placebo group than in the A/C group. As mentioned above, we interpret these differences between treatment groups with considerable caution. Nonetheless, the observed trends provide a reasonable basis to anticipate that a future, adequately powered interventional trial may have the potential to demonstrate a preventive effect on PAP elevation and, possibly, on HAPH incidence in individuals exposed to CIH. Finally, we did not assess systemic blood pressure because the study enrolled young, healthy, normotensive participants and was not powered to detect systemic hemodynamic changes. For this reason, our conclusions are restricted to pulmonary responses. Nonetheless, it is important to acknowledge that prior evidence indicates a link between ADMA, high-altitude exposure, and systemic blood pressure regulation, as demonstrated by Verratti et al. (2020), which further supports the physiological relevance of the ADMA–NO pathway in hypoxic environments.

Implications for future trials

Our findings, which align with previous observations from our group and others, highlight several methodological considerations that should guide the design of future interventional trials. Beyond informing sample size calculations, this pilot study underscores the need for active, continuous participant engagement to maintain adherence—an essential determinant of interpretable outcomes. We therefore recommend that a dedicated study team member remain available throughout the entire study period to maintain regular contact with participants, not only at scheduled follow-ups but also between visits, to enhance compliance and retention. Furthermore, coherent echocardiographic data require consistent acquisition across time; relying on clinical personnel who must balance routine duties with research tasks is insufficient. Instead, future trials should incorporate an experienced echocardiographer as part of the study team to ensure standardized image acquisition by the same observer and to guarantee availability for all planned assessments. These measures will strengthen data completeness, reduce variability, and ultimately improve the reliability of long-term outcomes in CIH intervention research.

Footnotes

Acknowledgments

The authors gratefully thank Alina Haack for excellent technical assistance, Natalia Bazaes for her collaboration in the project, and the Chilean Army for their logistical support during fieldwork.

Author Disclosure Statement

No competing financial interests exist.

Funding Information

This work was in large part funded by the co-authors’ academic institutions and partially supported by the project funded by FIC GORE-TARAPACÁ (grant BIP-400187737-0). In addition, both research groups are joint members of the DECIPHER Institute for Research on Pulmonary Hypoxia, which was initiated with funds provided by the German Federal Ministry of Education and Research. RB is the recipient of an ERC Advanced Grant by the European Research Council (grant 101096706–NO PRESSURE). Views and opinions expressed are, however, those of the author(s) only and do not necessarily reflect those of the European Union or the European Research Council. Neither the European Union nor the granting authority can be held responsible for them.

Supplemental Material

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Supplementary Material

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Compliance and Genetic Variability Are Determinants of the Success of l -Arginine/ l -Citrulline Supplementation in High Altitude—A Pilot Study

Abstract

Background:

Methods:

Results:

Conclusion:

Keywords

Introduction

Patients and Methods

Study participants and study protocol

Preparation and administration of supplements

Analysis of l-arginine and its metabolites by UPLC-MS/MS in dried blood spots

Isolation of DNA from dried blood and genotyping

Echocardiography

Calculations and statistical analyses

Results

Baseline characteristics of the study population

Effects of high altitude exposure and l-arginine supplementation on blood metabolite concentrations

Compliance of study participants with supplement intake

Association of arginase gene polymorphisms with blood l-arginine and l-ornithine concentrations

Association of DDAH gene polymorphisms with blood ADMA concentrations

Echocardiographic assessment of cardiac function after CIH exposure

Incidence of acute mountain sickness

Discussion

Biological plausibility and interindividual variability

Compliance as a central determinant of interpretability

Limitations

Implications for future trials

Footnotes

Acknowledgments

Author Disclosure Statement

Funding Information

Supplemental Material

References

Supplementary Material