Citrulline and kynurenine to tryptophan ratio: potential EED (environmental enteric dysfunction) biomarkers in acute watery diarrhea among children in Bangladesh

Two emerging biomarkers of environmental enteric dysfunction (EED) include plasma citrulline (CIT), and the kynurenine (KYN): tryptophan (TRP)/ (KT) ratio. We sought to investigate the plasma concentration of CIT and KT ratio among the children having dehydrating diarrhea and examine associations between concentrations of CIT and KT ratio with concurrent factors. For this analysis, we used cross-sectional data from a total of 102, 6–36 months old male children who suffered from non-cholera acute watery diarrhea and had some dehydration admitted to an urban diarrheal hospital, in Bangladesh. CIT, TRP, and KYN concentrations were determined at enrollment from plasma samples using ELIZA. At enrollment, the mean plasma CIT concentration was 864.48 ± 388.55 µmol/L. The mean plasma kynurenine, tryptophan concentrations, and the KT ratio (× 1000) were 6.93 ± 3.08 µmol/L, 33.44 ± 16.39 µmol/L, and 12.12 ± 18.10, respectively. With increasing child age, KYN concentration decreased (coefficient: − 0.26; 95%CI: − 0.49, − 0.04; p = 0.021); with increasing lymphocyte count, CIT concentration decreased (coef.: − 0.01; 95% CI: − 0.02,0.001, p = 0.004); the wasted child had decreased KT ratio (coef.: − 0.6; 95% CI: − 1.18, − 0.02; p = 0.042) after adjusting for potential covariates. The CIT concentration was associated with blood neutrophils (coef.: 0.02; 95% CI: 0.01, 0.03; p < 0.001), lymphocytes (coef.: − 0.02; 95% CI: − 0.03, − 0.02; p < 0.001) and monocyte (coef.: 0.06; 95% CI: 0.01, 0.11; p = 0.021); KYN concentration was negatively associated with basophil (coef.: − 0.62; 95% CI: − 1.23, − 0.01; p = 0.048) after adjusting for age. In addition, total stool output (gm) increased (coef.: 793.84; 95% CI: 187.16, 1400.52; p = 0.011) and also increased duration of hospital stay (hour) (coef.: 22.89; 95% CI: 10.24, 35.54; p = 0.001) with increasing CIT concentration. The morphological changes associated with EED may increase the risk of enteric infection and diarrheal disease among children. Further research is critically needed to better understand the complex mechanisms by which EED biomarkers may impact susceptibility to dehydrating diarrhea in children.

The co-pathogens (EAEC and rotavirus) isolated from baseline stool samples were not associated with citrulline, kynurenine, and tryptophan concentrations and the KT ratio at baseline ( Table 5).

Discussion
Comparisons to the body of existing literature are challenging since this study is the first to explore the effects of dehydrating diarrhea on CIT and KT ratio as potential novel indicators of alterations in systemic inflammation and EED.
Age, lymphocyte count (a measure of systemic inflammation and immunological function), and wasting were the three primary groups into which concomitant covariates that were reliably related with citrulline, kynurenine, and tryptophan concentrations and the KT ratio tended to cluster 37,38 . In our study we also found that with increasing child age, kynurenine concentration decreased, increasing lymphocyte count, and CIT concentration decreased. Some evidence suggested that tryptophan and citrulline were positively correlated with household wealth (e.g., socioeconomic status index and the presence of domestic animals in a household) 39 . But in our study, we could not observe any association with the presence of domestic animal and citrulline or kynurenine, as most of the enrolled children were from urban site. Citrulline, kynurenine, and tryptophan concentrations and the KT ratio, on the other hand, were only weakly significantly correlated with markers of anthropometry, IYCF (Infant and child feeding) practices, and maternal features, as other research suggests 12,40,41 , but not all 12,42 of the previous literature. There were only weak correlations in the current study between the KT ratio, citrulline, kynurenine, and tryptophan concentrations, and the indicators of water, sanitation, and hygiene (WASH). Recent community-based randomized controlled efficacy trials of three WASH interventions have been performed. These studies were predicated on the idea that fecal-oral pollution in the presence of subpar WASH causes EED [43][44][45][46] . In Bangladesh, WASH interventions reduced intestinal permeability and inflammation in infants as young as 3 months old when compared to controls, but they were later linked to higher EED biomarkers in infants as old as 28 months [47][48][49] . However, none of the three trials found a correlation between the WASH interventions and children's growth, suggesting the interventions may not have been sufficiently intense to have a significant influence 43-46 . In the present study, citrulline was negatively associated with concurrent concentrations of lymphocytes and the Kynurenine was negatively associated with basophil count. The relationship between intestinal injury and repair and systemic inflammation was further supported by the favorable associations of citrulline with neutrophil and monocyte count 9 . However, somewhat unexpectedly, we did not observe a significant association between blood leukocyte concentrations (as immune biomarkers) and tryptophan, or the KT ratio. Decreased citrulline and kynurenine levels have been reported during infections in animal models 13 , likely due to increased IDO1 activity during inflammation 50  www.nature.com/scientificreports/ maintenance of T-cell homeostasis in healthy individuals, although such data have not, to our knowledge, been reported previously. This is why the negative correlation between citrulline and kynurenine levels and lymphocyte and basophil count observed in the current study was not entirely unexpected. A study conducted among rural Laotian children found, that the KT ratio was not associated with blood leukocyte concentrations (lymphocytes, monocytes, neutrophils, eosinophils, and basophils) but tryptophan level is positively associated with Lymphocyte count 51 . Confirmation of this hypothesis would require further work with more specific characterization of systemic inflammation. We did not find any association of pathogens (EAEC and rotavirus) isolated from the fecal samples of our study children. Similar findings were observed among the children in Zambia, the EED severity score was significantly higher among asymptomatic controls compared to cases with rotavirus diarrhea (p = 0.02) 26 . Another study analyzed a total of 16 age-paired stool samples: 8 diarrheal samples positive for one diarrhoeagenic E. coli pathotype and 8 stool samples from healthy children and they observed almost similar findings: arginine levels were similar in both groups, but citrulline levels were higher in healthy samples 52 . However, impacts from the metabolism of citrulline or tryptophan may also play a role in important pathways. These essential metabolites might be less readily available in children due to changed host or microbial metabolism or signaling, higher needs, or both. We also found that lower plasma tryptophan levels are not surprisingly associated with biomarkers     53 . This information might be useful to identify mechanisms and signaling molecules involved in the crosstalk between EED biomarkers and the isolation of diarrhoeagenic E. coli and other pathogens in the stool.
In patients with inflammatory bowel illnesses, HIV patients, and those who are critically ill, plasma citrulline, which is virtually solely produced by the enterocytes, is a marker of enterocyte mass 54 . It could be a quantitative biomarker of small intestine mass integrity in a group with tropical enteropathy and HIV-associated villous atrophy in Zambia that correlates with crypt depth and xylose absorption 55 . In our study, we found almost 68% of children had rotaviral diarrhea. Viral infections damage small bowel enterocytes and cause low-grade fever and watery diarrhea without blood 56 . Plasma Citrulline levels reflect enterocyte mass and children with viral watery diarrhea have acute enterocyte volume loss due to damage small bowel enterocytes. However, its application in the context of children with dehydrating diarrhea is yet to be explored. In our analysis, we found that raised citrulline levels increased stool output among dehydrated hospitalized children and subsequently caused prolonged hospital stays. This might be due to the children who had higher citrulline levels had increased school output and had worse hospital outcomes due to more acute intestinal damage in dehydrating diarrhea. From a systematic review and meta-analysis 57 , citrulline levels are strongly negatively correlated with intestinal disease Table 4. Concurrent predictors associated with citrulline, kynurenine, and tryptophan concentrations and the KT ratio at baseline with blood leukocyte concentrations (lymphocytes, monocytes, neutrophils, eosinophils, and basophils) as a marker of systemic inflammation. Coef.: coefficient; 95% CI: 95% confidence interval; *Models adjusted for age at enrollment. Values represent the beta coefficient and 95% CI from linear regression models. For continuous predictors, this represents the change in outcome corresponding to a oneunit increase in the predictor.  Table 5. Association of citrulline, kynurenine, and tryptophan concentrations and the KT ratio at baseline with isolated pathogens in stool culture. Coef.: coefficient; 95% CI: 95% confidence interval; *Models adjusted for age at enrollment. Values represent the beta coefficient and 95% CI from linear regression models.  www.nature.com/scientificreports/ severity with regard to enteropathies (coeliac disease, tropical enteropathy, mucositis, acute rejection in intestinal transplantation, but not Crohn's disease), and strongly positively correlated with small bowel length in patients with short bowel syndrome. Citrulline levels were 10 μmol/L lower compared to controls, and citrulline cut-off levels have an overall sensitivity and specificity of 80%. According to these findings, citrulline may be a sign of acute intestine damage or intestinal insufficiency 57 . The importance of plasma citrulline as a biomarker of gut mass, where plasma citrulline levels are predictive of intestinal healing, has also been highlighted by studies in children with short bowel syndrome 58,59 . Overall, further studies are recommended as exploratory EED biomarkers among children with dehydrating diarrhea which is essential to foster the hypothesis and to understand completely the clinical relevance of citrulline among the diarrhea patients for better hospital outcome. This analysis has several limitations. A primary limitation of this study is the lack of clear diagnostic criteria for EED. We used cross-sectional data and had a small sample size; therefore, we are unable to draw any conclusions about the causality of the link between EED biomarkers and dehydration acute diarrhea. Based on univariate relationships with the outcome of diarrheal disease, data-driven selection of factors to include in adjusted models may be more susceptible to unmeasured confounding. Moreover, we did not analyze fecal markers of intestinal inflammation in the trial for our cost constraints. As the data were collected from hospital-admitted male children of an urban hospital in Bangladesh, results may not be generalizable for all. Nevertheless, the present study provided new insights into the role of CIT and KT ratios with dehydrating diarrhea and their hospital outcome.
We provide the first estimates, to our knowledge, of the association between dehydrating watery diarrhea and EED biomarkers. Our findings not only identify the predictors of EED biomarkers among children having diarrhea but also stress the urgent requirement for EED measures to be included in upcoming research projects in order to better understand the intricate correlations between diarrheal disease and EED biomarkers. Future research examining the association between EED biomarkers and diarrheal disease should therefore utilize longitudinal follow-ups.

Methodology
Study design, study setting, and study population. The current analyses are based on data from a subsample of 102 participants enrolled in the VS002A clinical trial 60 . The VS002A trial is a randomized, doubleblind, two-cell superiority clinical trial comparing WHO-ORS and amino acid-based ORS: VS002A conducted in children, presenting with non-bloody acute non-cholera diarrhea with some dehydration in the Dhaka Hospital of icddr,b situated in Dhaka, capital of Bangladesh. The Dhaka Hospital is the largest diarrheal disease hospital in the world. An average of 250 patients are treated in the hospital each day. Details about the study site have been reported elsewhere 61 .
In brief, the study was conducted among 312 male (to facilitate separate collection of urine and stool) children who were 6-36 months old presenting with non-cholera acute watery diarrhea (onset ≤ 48 h) and some dehydration, admitted to Dhaka Hospital of icddr,b in between June 2021 and September 2022.
Children were eligible to participate in the study if they were 6-36 months, duration of diarrhea ≤ 48 h, some dehydration (judged clinically according to the "Dhaka method") 62 , and written informed consent was obtained by either parent/guardian. Children were excluded from the study if they presented with any of the following: Severe malnutrition (Weight-for-length WLZ/weight-for-height WHZ/ weight-for-age WAZ < − 3 or presence of nutritional edema), patients with cholera, bloody diarrhea, presence of systemic illness (e.g. Pneumonia, tuberculosis, enteric fever, meningitis, etc.), any congenital anomaly or disorder (e.g. diagnosed inborn error of metabolism, congenital cardiac disease, seizure disorders, hypothyroidism, Down's syndrome, etc.), a requirement of additional intravenous fluids after being provided with an IV for 4 h on admission if severely dehydrated, has documentation of taking antibiotics and/or antidiarrheal within the last 48 h before hospitalization. The study protocol is described in detail 60 .
For this study, the sample was restricted to 102 enrolled children based on the availability of tests done for biomarkers in the laboratory except for the weekly holidays from June 2021 to March 2022. To date, as there is no data available for this type of study, we estimated to collect first one-third of patients' EED biomarker data from the total enrolled children (n = 312) admitted in icddr,b Dhaka Hospital with dehydrating diarrhea based on our study budget and cost (Fig. 1, Supplementary file 1).

Ethical consideration.
The study was conducted following the Declaration of Helsinki of 1975, revised in 1983, and was approved by the Institutional Review Board (IRB) of icddr,b (protocol no PR-17028). The IRB of icddr,b comprises the Research Review Committee (RRC) and Ethical Review Committee (ERC). This study was registered as a clinical trial (www. Clini calTr ials. gov; NCT04677296. Registered on December 21, 2020). All methods were performed in accordance with the relevant guidelines and regulations. https:// clini caltr ials. gov/ ct2/ show/ NCT04 677296? term= NCT04 67729 6& draw= 2& rank=1. Written informed consent was obtained from the parents or legal guardians of every child.
Data collection. Case record form. Information on maternal and paternal education, household socioeconomic and demographic characteristics, infant and young child feeding (IYCF) practices (breastfeeding, and formula feeding), and vaccination were collected via a structured interview at the time of enrollment. A thorough clinical history and physical examination were done, and body weight and height were measured. For children, weight-for-age (WAZ), length-for-age (LAZ), and weight-for-length (WHZ) Z-scores were calculated according to the WHO growth standards at enrollment 63  www.nature.com/scientificreports/

Variable of interest.
Based on a comprehensive literature review, previous descriptive studies, and the availability of data in our investigation, several variables were considered explanatory variables. The outcome variables for this study were the level of gut health biomarkers: citrulline and KT ratio among the children having dehydrating diarrhea.
Household data. During hospital visits, detailed household data were obtained. The household demographic variables included type of floor (cemented or non-cemented), handwashing practices (before nursing or preparing baby food; after cleaning a child), access and the main source of drinking water (tube well water and non-tube well water), water treatment method (boiled or not), sanitation facilities (improved toilet facility for disposal of human fecal waste available or not), and the use of handwashing materials (water with soap or without soap). Parenteral education (illiterate or below primary level or primary and above), household size (number of children < 5 years of age), family income.
Feeding history. Breastfeeding referred to the breastfed baby or not. And whether the child was offered formula milk or not.
Vomiting and fever. Many of the components, such as vomiting (≥ 3 times/ day), and fever on admission (measured at least 38 degrees Celsius) were assessed.
Vaccination status. Defined as not vaccinated/vaccinated (age appropriate)/partially vaccinated, which can only be retrospectively assessed.
Measurement of stool output and hospital outcome. Stool output was measured from 4 h up to 120 h after randomization. We analyzed the data on total stool output (gram) till caseation of diarrhea (passage of soft or formed stool/no stool for 12 h) in the hospital, total duration of hospital stays in hours, and outcome of the child: usual discharge/treatment failure/DORB (Discharge on risk bond)/ dropout.  Baseline blood biochemistry. Around 5 ml of venous blood samples were collected at enrolment and 24 h after the enrollment to determine serum electrolytes and blood glucose. We also performed a complete blood count, and serum creatinine, for all 102 patients on admission following standard microbiological laboratory procedures in the Dhaka Hospital, icddr,b 68 .

Gut health biomarkers.
On admission and 24 h after enrolment of the 102 children, plasma CIT and KT ratios were measured to assess the absorptive functions of the intestine and gut inflammation. The tests were done at the icddr,b laboratory, using the serum collected for routine blood testing, therefore no extra amount of blood or venipuncture was required. To separate the plasma, blood samples were centrifuged at 4000 rotations per minute (rpm) for 10 min. Quantitative analyses of plasma CIT concentrations and KT ratios are performed at icddr,b using the Enzyme-linked immunosorbent assay (ELISA) method.

Determination of citrulline and K/T ratio with enzyme-linked immunosorbent assay (ELISA).
Citrulline, tryptophan (Immusmol, Bordeaux, France), and kynurenine (Immusmol, Bordeaux, France) levels were determined using commercially available kits following the manufacturer's instructions. The measurement ranges for kynurenine and tryptophan were from 0.5 to 50 μmol/L and 15 to 150 μmol/L respectively. All samples yielded measurable results and are included in the data presented. The KT ratio was obtained by dividing the plasma concentration of KYN (μmol/L) by the TRP concentration (μmol/L) and multiplying the quotient by 1000 16,17 . The methods to determine CIT and KT ratios are described in detail elsewhere 69,70 .
Statistical analysis. We reported the child, parental, and household-level characteristics by using mean and standard deviation (SD) for continuous variables and frequency as a percentage for categorical variables to summarize the data. Raw data of EED biomarkers were subsequently normalized by log transformation. To examine the relations between biomarkers of EED at baseline and associated predictors, potential factors associated with citrulline, kynurenine, and tryptophan concentrations and the KT ratio were examined with linear regression; analyses were adjusted for age, breastfeeding status, wasting, lymphocyte count, maternal education, WASH and co-pathogen (EAEC and rotavirus) isolated from stool sample at enrollment; dependent variables were the EED biomarkers. In addition, to find the association between hospital outcome with EED biomarkers among diarrheal children we used a linear regression model where the outcome variables were duration of hospitalization (hours) till diarrhea ceased and total stool output during hospitalization in gram. Minimally adjusted separate models controlled for child age were used during the analysis (due to the small sample size, n = 102). To detect multicollinearity, the variance inflation factor (VIF) was calculated, and no variable producing a VIF value > 5 was found in the final model. The criterion of significance was set at 0.05, and 95% confidence intervals were calculated to determine the directions and strength of the effects. All data were analyzed using version 15.0 IC of STATA (College Station, TX, USA: Stata Corp LLC).
Informed consent. Written informed consent was obtained from all the participants involved in this study.

Data availability
The dataset analyzed during the current study are not publicly available due to the data of this manuscript has been obtained from a clinical trial with a huge data set that deals with several objectives. The submitted manuscript deals with one of the objectives of that data set. This data set contains some personal information of the study patients (such as name, date of birth, admission date, month, and area of residence). However, during taking the consent from the patients, it has been ensured that their personal information of them will not be disclosed, but, the study results will be published. Thus, the availability of this whole data set in the manuscript, the supplemental files, or a public repository will open all the personal information of the patients that should not be disclosed; additionally, this will disclose other important information that is yet to be published. Thus, the policy of our center (icddr,b) is that we should not make the availability of the whole data set in the manuscript, the supplemental files, or a public repository. However, part of the data set related to this manuscript is available upon request and readers may contact Ms. Armana Ahmed (aahmed@icddrb.org) of the Research Administration & Strategy of icddr,b to request the data (http:// www. icddrb. org/). www.nature.com/scientificreports/ the tables, and wrote the manuscript's initial draft. P.K.B., B.N., A.G., M.M., and T.A. have critically revised the manuscript for essential intellectual content; T.A. gave final approval for the version to be published. Every author was sufficiently involved in the research to take on public responsibility for content-related sections. All authors have read and agreed to the published version of the manuscript.

Funding
This study was funded by Entrinsic Bioscience Inc. The funder had no role in study design, data collection, analysis, decision to publish, or preparation of the manuscript.