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Open Access

Peer-reviewed

Research Article

Preliminary analyses of tryptophan, kynurenine, and the kynurenine: Tryptophan ratio in plasma, as potential biomarkers for systemic chlamydial infections in koalas

  • Chien-Jung Chen ,

    Roles Conceptualization, Data curation, Formal analysis, Investigation, Methodology, Validation, Visualization, Writing – original draft, Writing – review & editing

    chien-jung.chen@sydney.edu.au

    Affiliation Sydney School of Veterinary Science, The University of Sydney, Sydney, New South Wales, Australia

    ⨯
  • Benjamin Kimble,

    Roles Conceptualization, Methodology, Validation, Writing – review & editing

    Affiliation Sydney School of Veterinary Science, The University of Sydney, Sydney, New South Wales, Australia

    ⨯
  • Astrid Van Aggelen ,

    Contributed equally to this work with: Astrid Van Aggelen, Shalini Fischer, Amber Gillett, Jackie Reed, Jodie Wakeman

    Roles Data curation, Methodology, Resources, Writing – review & editing

    Affiliation Port Macquarie Koala Hospital, Koala Conservation Australia, Port Macquarie, New South Wales, Australia

    ⨯
  • Shalini Fischer ,

    Contributed equally to this work with: Astrid Van Aggelen, Shalini Fischer, Amber Gillett, Jackie Reed, Jodie Wakeman

    Roles Data curation, Methodology, Resources, Writing – review & editing

    Affiliation Port Macquarie Koala Hospital, Koala Conservation Australia, Port Macquarie, New South Wales, Australia

    ⨯
  • Cheyne Flanagan,

    Roles Data curation, Resources, Writing – review & editing

    Affiliation Port Macquarie Koala Hospital, Koala Conservation Australia, Port Macquarie, New South Wales, Australia

    ⨯
  • Amber Gillett ,

    Contributed equally to this work with: Astrid Van Aggelen, Shalini Fischer, Amber Gillett, Jackie Reed, Jodie Wakeman

    Roles Data curation, Methodology, Resources, Writing – review & editing

    Affiliation Australia Zoo Wildlife Hospital, Wildlife Warriors, Beerwah, Queensland, Australia

    ⨯
  • Jackie Reed ,

    Contributed equally to this work with: Astrid Van Aggelen, Shalini Fischer, Amber Gillett, Jackie Reed, Jodie Wakeman

    Roles Data curation, Methodology, Resources, Writing – review & editing

    Affiliation Northern Rivers Koala Hospital, Friends of the Koala, East Lismore, New South Wales, Australia

    ⨯
  • Jodie Wakeman ,

    Contributed equally to this work with: Astrid Van Aggelen, Shalini Fischer, Amber Gillett, Jackie Reed, Jodie Wakeman

    Roles Data curation, Methodology, Resources, Writing – review & editing

    Affiliation Northern Rivers Koala Hospital, Friends of the Koala, East Lismore, New South Wales, Australia

    ⨯
  • Merran Govendir

    Roles Conceptualization, Funding acquisition, Project administration, Resources, Supervision, Writing – review & editing

    Affiliation Sydney School of Veterinary Science, The University of Sydney, Sydney, New South Wales, Australia

    ⨯

Abstract

Chlamydiosis is the major infectious disease responsible for significant morbidity and mortality in free-living koalas. Recently, it was reported that 28.5% of koalas infected with chlamydiosis were presented with no overt clinical signs. Identification and quantification of changes in plasma biomarkers’ fluctuations have the potential to enhance C. pecorum detection and facilitate the monitoring of therapeutic efficacy of antibiotics to treat this disease in koalas. Therefore, concentrations of the essential amino acid tryptophan, tryptophan’s metabolite kynurenine, and the kynurenine:tryptophan ratio were quantified by high-performance liquid chromatography in the plasma of clinically normal koalas (n = 35), koalas identified with chlamydial disease (n = 35) and koalas that had other non-chlamydial co-morbidities (n = 10). Results showed that there was a significant difference between the clinically normal versus diseased, and clinically normal versus ‘other’ (both p < 0.001) in kynurenine plasma concentrations and kynurenine:tryptophan ratio; and also between the clinically normal and diseased in tryptophan plasma concentrations (p = 0.001). Proposed reference ranges of tryptophan, kynurenine, and kynurenine:tryptophan ratio in koalas are: 4.27–10.4 μg/mL, 0.34–1.23 μg/mL, and 0.05–0.22, respectively. Proposed optimal cut-off points to differentiate between clinically normal and diseased are: ≤ 4.75 μg/mL (tryptophan), ≥ 0.88 μg/mL (kynurenine), and ≥ 0.12 (kynurenine:tryptophan); and ≤ 7.67 μg/mL (tryptophan), ≥ 1.18 μg/mL (kynurenine), and ≥ 0.16 (kynurenine:tryptophan) to differentiate between released/recovered and euthanised of the diseased/‘other’ koalas. Significant differences in haematological and biochemical analytes were in the plasma globulins between the clinically normal and diseased koalas (p = 0.01), and in alkaline phosphatase between the clinically normal and ‘other’ koalas (p = 0.03). Although these potential biomarkers, especially tryptophan, may not be specific for detecting C. pecorum from the rest of the population, kynurenine and the kynurenine:tryptophan ratio may have a role in identifying unhealthy koalas from the clinically normal ones, irrespective of the underlying cause.

Citation: Chen C-J, Kimble B, Van Aggelen A, Fischer S, Flanagan C, Gillett A, et al. (2024) Preliminary analyses of tryptophan, kynurenine, and the kynurenine: Tryptophan ratio in plasma, as potential biomarkers for systemic chlamydial infections in koalas. PLoS ONE 19(12): e0314945. https://doi.org/10.1371/journal.pone.0314945

Editor: Ping Song, Georgia State University, UNITED STATES OF AMERICA

Received: June 27, 2024; Accepted: November 18, 2024; Published: December 19, 2024

Copyright: © 2024 Chen et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

Data Availability: All relevant data are within the manuscript and its Supporting Information files.

Funding: WIRES (USYD217760). Winifred Violet Scott Charitable Foundation (USYD G218502). We confirm that the funders had no role in the study design, data collection and analysis, decision to publish, or preparation of the manuscript.

Competing interests: The authors have declared that no competing interests exist.

Introduction

Tryptophan (TRP), an essential amino acid, is active in the regulation of immune responses in humans and animals [1]. In mammals, TRP depletion can be due to one of four pathways with the major pathway being the metabolism of TRP to kynurenine (KYN), commonly referred as the TRP-KYN pathway, accounting for 95.0% of dietary TRP metabolism (90.0% in the liver and 5.00% outside the liver) [2]. The remaining 5.00% of TRP is metabolised as the serotonin synthesis pathway in the periphery and brain, in addition to other minor physiological pathways [3, 4]. The primary pathway of TRP depletion is activated as a response to stressors such as infection and inflammation [5, 6], oxidative stress [7, 8], and environmental toxicants [9, 10]. Therefore, the concentration of KYN (one of tryptophan’s metabolites) is expected to increase with depletion in the TRP concentration in times of stress and/or inflammation. In the clinically normal state, the TRP-KYN pathway is triggered by up-regulation of the enzyme tryptophan 2,3-dioxygenase (TDO2) in the liver, that converts TRP into formylkynurenine at a controlled rate [11, 12]. A diagram of the TRP-KYN metabolic pathway is found at https://journals.sagepub.com/doi/10.4137/IJTR.S12626 [13].

The pathophysiological significance of the KYN:TRP is that degradation of TRP and elevated KYN concentrations in the blood are associated with inflamed physiological states [3]. Inflammation induces the enzyme indoleamine 2,3-dioxygenase (IDO-1/IDO-2) by the inflammatory cytokine interferon gamma (IFN-γ), resulting in TRP biotransformation to active metabolites [3] such as KYN and melatonin (via the serotonin pathway). All three metabolites modulate immunity and induce anti-inflammatory responses [1]. Accumulation of KYN leads to cell cycle arrest of the effector T cells [14] and induces the differentiation of proinflammatory T helper type 17 cells [15]. Consequently, a significant increase in KYN metabolites, such as 3-hydroxykynurenine and quinolinic acid, are associated with neuro- [16, 17] and immune- [18–20] associated diseases, in humans and in animal models.

Chlamydiosis is the most prevalent infectious disease in koalas (Phascolarctos cinereus) [21]. This disease has devastating impacts on both individual koalas and the survival of whole free-living populations in South-East Queensland (QLD) and New South Wales (NSW) in Australia [21]. Depending on the site/s of infection, chlamydiosis can cause conjunctivitis and/or cystitis resulting in blindness, renal disease, infertility, and ultimately death [22, 23]. However, our recent retrospective study [24] reported that 28.5% of the infected koalas that are polymerase chain reaction (PCR) positive for chlamydial pathogens do not exhibit overt clinical signs, so establishing a reliable biomarker for detection of sub-clinical chlamydial infection, with Chlamydia pecorum (C. pecorum) being the predominant species, may allow for improved detection of disease onset and therapeutic monitoring of antibiotic treatment outcomes.

One of the key defense mechanisms against chlamydiosis is the depletion of endogenous TRP concentration to inhibit chlamydial development, known as TRP starvation [25]. Although chlamydial pathogens are known to uptake TRP as their main source of energy to survive, there are chlamydial species that have adapted to TRP starvation by transforming into nonreplicating but viable, persistent forms [26]. However, studies report that the interaction between TRP and the C. pecorum strain is unique compared to other Chlamydia spp., and to survive within a TRP-free environment, C. pecorum is equipped with TrpR (TRP operon repressor) which upregulates TRP biosynthesis when the TRP concentration is low [25, 27, 28]. Adequate amounts of TRP can be synthesised from KYN and anthranilic acid, and that this newly formed TRP binds to its cognate operator to stop transcription of the TRP operon when the desired concentration is reached [25].

Clinically healthy animals have innate amounts of TRP and KYN already in their blood, which are obtained from their diets [29]. Interspecies variation is evident in the intrinsic total TRP and KYN plasma concentrations, such as a TRP concentration of 12.9 ± 0.41 μg/mL (63.0 ± 2.0 μM) and KYN concentration of 0.45 ± 0.02 μg/mL (2.15 ± 0.12 μM) in clinically normal humans [30]; and clinically normal mice exhibit mean TRP concentrations of 26.9 μg/mL (131.5 μM) [31] and mean KYN concentrations of 0.35 μg/mL (1.68 μM) [32]. Studies have extensively investigated the KYN:TRP ratio with human neurological diseases [33] and lung cancer [34, 35]; more recently, a significant elevation in the KYN:TRP ratio has been associated with long COVID in humans [36–38].

To date, there has been no report on relationships between the KYN:TRP ratio and disease progression in animals. Studies thus far have explored variations in KYN:TRP ratio in response to stress in fish [39], chickens [40], and mice [41]; as well as rats with renal insufficiency [42–44], and cows with ketosis [45] and mastitis [46]. As chlamydial infections induce chronic inflammation in koalas [47], examining the KYN:TRP ratio may provide insights into the detection of subclinical disease and ways to enhance antibiotic treatments of C. pecorum. Since plasma TRP and KYN concentrations have not been quantified in clinically normal and diseased koalas, the aims of this study were to develop and validate an assay to quantify TRP and KYN concentrations in plasma of clinically normal and diseased koalas; to determine the reference ranges of TRP, KYN and the KYN:TRP ratio with clinically normal koalas; and to investigate the relationships in KYN, TRP, and the KYN:TRP ratio between clinically normal, diseased, and koalas with other co-morbidities associated with inflammation.

Materials and methods

Animals and housing

The study was approved by The University of Sydney Animal Ethics Committee protocol 2023/2259.

A total of 80 koalas were recruited opportunistically for this study with the animals housed individually according to normal husbandry procedures used at the Australia Zoo Wildlife Hospital (Beerwah, QLD, Australia), the Port Macquarie Koala Hospital (Port Macquarie, NSW, Australia), and the Friends of the Koala Hospital (East Lismore, NSW, Australia). The recruited koalas were provided with fresh food ad-lib (various Eucalyptus spp. leaves) and water, perching material, shelter from inclement weather and daily removal of faeces and old browse. Of the 80 animals, 35 diseased koalas were admitted to the hospitals for chlamydiosis during the study period, 10 koalas were hospitalised for other co-morbidities, and 35 clinically normal koalas had samples taken opportunistically for other studies (Fig 1).

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Fig 1. Summary of the koala groups recruited for this study.

https://doi.org/10.1371/journal.pone.0314945.g001

The health status of the koalas, either clinically normal, diseased with C. pecorum, or with other co-morbidities, were determined by veterinarians upon physical examination, and swabs for suspected cases of chlamydial infections were taken and confirmed by either PCR or loop-mediated isothermal amplification (LAMP) assay. For some koalas, blood was available for haematology and biochemistry. Koalas that were PCR/LAMP positive for C. pecorum, with clinical signs present, were categorised as ‘diseased’, and animals with other non-chlamydial co-morbidities, such as retrovirus, liver disease, joint infections, trauma, advanced alopecia and skin infection, leukaemia, and abdominal lymphoma were categorised as ‘other’.

Sample collection, handling and blood component quantification

All koalas initially were examined under general anaesthesia. Sedation was achieved using up to 3 mg/kg alfaxalone (Alfaxan®-CD, RTU; Jurox Pty Ltd, Rutherford, NSW, Australia) as an intramuscular injection, while general anaesthesia was maintained with 1–2% isoflurane in 100% oxygen delivered via face mask or cuffed 4–4.5 mm endotracheal tube. The duration of anaesthesia was between 15 and 30 minutes to perform abdominal ultrasonography and swabbing of both conjunctivae and the urogenital tract for all animals, with or without clinical signs. A definitive diagnosis of chlamydiosis was confirmed by a positive PCR or LAMP result.

Whilst under general anaesthesia, 5 mL of blood was collected from each koala via venipuncture from the cephalic vein with a 23 g needle, of which 2 mL was collected into potassium EDTA tube for routine haematological analysis, and the remaining 3 mL was collected in a lithium heparin tube and centrifuged into plasma for biochemical analyses, and TRP and KYN quantification.

After the single blood collection from each koala for this study, the koalas classified as ‘diseased’ and ‘other’ were treated by the respective veterinarians (authors A.V.A., S.F., A.G., J.R., J.W.), who administered antibiotics, anti-inflammatories, and/or analgesics as required. No animals were sacrificed for this study; however, some koalas were euthanised due to poor treatment responses. The decision to euthanise was made by the veterinarians attending their respective patients, independent to this study.

Quantification of blood haematological values and biochemical analyte concentrations were conducted by Veterinary Pathology Diagnostic Services at the Sydney School of Veterinary Science, and Vetnostics at Macquarie Park in Sydney, to determine whether hydration status and the selected haematological or biochemical analytes significantly affected KYN and TRP plasma concentrations. The haematological values of interest were the total white blood cell count (WBC), red blood cell count (RBC), haematocrit (HCT), neutrophil count (Neut), lymphocyte count (Lymph), and monocyte count (Mono). The biochemical analytes investigated were alkaline phosphatase (ALP), alanine transaminase (ALT), albumin (ALB), creatinine (CRE), globulins (Glob), total protein (TP), gamma-glutamyl transferase (GGT), and blood urea nitrogen (BUN).

Most freshly collected samples, and only a few frozen, were transported on dry ice from all the koala hospitals to The University of Sydney, where the samples were thawed, and then assayed for TRP and KYN quantification within 72 h of arrival. Other freshly collected samples were either delivered at 4˚C for a maximum of 48 h, then assayed, or stored at -20˚C before thawing at ambient room temperature (∼20°C) for analysis.

Tryptophan and kynurenine analysis in koala plasma

Tryptophan (catalogue number T0254) and KYN (catalogue number K8625), acetic acid (catalogue number 695092), trichloroacetic acid (TCA; catalogue number T6399), and theophylline (catalogue number T1633), as the internal standard (IS), were purchased from Sigma-Aldrich (Castle Hill, NSW, Australia). Ammonium acetate (catalogue number A16343.30) and methanol (MeOH; catalogue number A452) were purchased from Thermo Fisher Scientific (Macquarie Park, NSW, Australia). Purified water was retrieved from the Milli-Q water purification system (Merck Millipore, Burlington, MA, USA).

A reversed-phase high-performance liquid chromatography (HPLC) system from Shimadzu (Rydalmere, NSW, Australia) that included a LC-20AT system controller, a DGU-20A3 degassing unit, a SIL-20A HT auto sampler, a RF-10A XL fluorescence detector, a SPD-20A UV detector, and a CTO-20A column oven was used.

The separation method was modified from a previous method [48], and then optimised to suit the complexity of the koala plasma. Chromatographic separation was performed by Agilent Polaris C18-A, 5 μm, 150 mm × 4.6 mm column that was maintained at ambient room temperature (∼20˚C). The gradient mobile phases composed of A = 4.76% MeOH in 20mM ammonium acetate (pH 4.5) in water, and B = 80% MeOH in 20mM ammonium acetate (pH 4.5) in water with a flow rate of 1.2 mL/min. The initial mobile phase composition was maintained at 5% solvent B for 1 min (0–1 min), increased linearly from 5–50% solvent B in 6 mins (1–7 min), held for 1 min (7–8 min), then returned to the initial composition at 5% for 5 mins (8–13 min) for the chromatograph column equilibrium. The fluorescence detector was set at an excitation wavelength of 297 nm and an emission wavelength of 347 nm to detect TRP, with the UV detector at 360 nm to detect the KYN and the IS. The total run time for each sample was 13 min.

Sample preparation

Since endogenous TRP and KYN concentrations were detected in blank koala plasma, fresh calibration standards were prepared daily in phosphate buffered saline (PBS) rather than using blank koala plasma. Samples for the calibration curve were spiked with the TRP and KYN working solutions (500 μg/mL in water) from the stock solutions (2 mg/mL in MeOH) to achieve concentrations of 0.63, 1.25, 2.50, 5.00, 10.0, and 20.0 μg/mL. A stock solution of the IS (2 mg/mL theophylline) was prepared fresh in MeOH and was further diluted fresh to give a working solution of 500 μg/mL before analysis. The same TRP, KYN and IS stock solutions were used for the quality control (QC) samples of low, medium, and high concentrations (2.50, 5.00 and 20.0 μg/mL) for assay validation in koala plasma. To calculate the area under the curve (AUC) of TRP and KYN in QC samples, the endogenous concentrations were subtracted using the following equations: A protein precipitation extraction method was undertaken to separate the desired compounds from the matrix, by adding 50 μL of 10% TCA, 200 μL of MeOH with 7 μL of IS working solution into 100 μL of each PBS sample. The sample was vortexed then centrifuged at 20,854 × g for 10 min before injecting 10 μL of the supernatant into the HPLC system. This was repeated for a total of three times for each calibration and QC sample.

Assay validation

The assay analytical conditions were defined for specificity, linearity, the lower limit of detection (LLOD), the lower limit of quantitation (LLOQ), and the analyte stability and recovery from the samples, with the assay validated for accuracy and precision [49]. The specificity was determined by identifying the peaks and retention times of the compounds of interest in plasma samples. The TRP, KYN, and the IS peaks were consistently observed with the retention times of ≈ 6.10 mins (fluorescence detection), ≈ 3.40 mins (UV detection), and ≈ 7.00 mins (UV detection), respectively.

Linearity was evaluated by analysing the prepared calibration solutions of TRP and KYN in PBS at concentrations of 0.63, 1.25, 2.50, 5.00, 10.0, and 20.0 μg/mL to generate a universal calibration curve (average of the three linear regressions) in the form of y = ax + b.

Sensitivity was based on the LLOD and the LLOQ values, which were calculated using the formulae [50]: where σ = the standard deviation of the y-axis intercepts and X = the average of the calibration curve gradients.

A weighting factor (1/x) was applied to ensure that the observations were not overfitted, especially at the lower concentrations.

The intra- and inter-day accuracy and precision was determined using the same TRP and KYN QC concentrations of 2.50, 5.00, and 20.0 μg/mL in triplicates across three days. Accuracy (%) was calculated using the formula [49]: Precision (CV %) was obtained by dividing the standard deviation by the mean estimated concentration, multiplied by 100 [49]. The accuracy and precision of the calculated values at each concentration was within ± 15% of the expected value [49].

The stability of TRP and KYN in the koala plasma was investigated under the following storage conditions in duplicates with the IS spiked at 10 μg/mL: ambient room temperature (∼20˚C) for 5 h; 4˚C for 2, 7, 9 and 14 days; and -20˚C for 3, 8, 15, 29 and 43 days. The mean ‘stable’ concentration obtained was within ± 15% of the initial concentration [49].

The recovery of TRP and KYN was calculated by pre-spiking the TRP and KYN concentrations to 2.50, 5.00, and 20.0 μg/mL, while the concentration of IS was 10 μg/mL in the blank plasma. Identical TRP, KYN and IS concentrations were vortexed into the Milli-Q water as a separate batch to calculate the average recovery percentage with the equation: All samples in the plasma and water followed the sample preparation and extraction methods described previously, which were then analysed as triplicates.

Statistical analyses

All data analyses were performed in jamovi (version 1.6, Australia) at https://www.jamovi.org/ [51]. Descriptive statistics, such as counts and percentages, were conducted to explore the data distribution of the four variables, namely sex (male/female), maturity (juvenile/adult [≥ 2 years of age]), location (NSW/QLD), and koala health status (clinically normal/diseased/‘other’). One-way analysis of variance (ANOVA), coupled with Dwass-Steel-Critchlow-Fligner pairwise comparison, were conducted to determine the statistical significance between the hydration haematological values and biochemical analytes concentrations, and the clinically normal/diseased/‘other’ groups. Pearson’s correlation analysis was performed to investigate the association between KYN:TRP ratio and some of the haematological (WBC, Mono, Lymph) and biochemical (ALP, ALT, ALB, CRE, GGT, Glob) analytes. The Shapiro-Wilk test was used to investigate the normality of the data. The Welch’s t-test and the Mann-Whitney U (non-parametric) test were used to investigate significant differences between the variables and KYN, TRP, and KYN:TRP ratios. For all statistical analyses, the level of significance was set at p < 0.05.

Reference ranges of clinically normal koalas

The 95% reference interval (reference range) was calculated with the equation conventionally used for data with a normal distribution as (mean ± 1.96 × SD), where 1.96 = z-score, and SD is the standard deviation [52, 53]. For non-parametric datasets, log transformations or Box-cox transformation were used to transform into parametric datasets before calculating the reference intervals [54]. Data transformations and reference interval calculations were performed on the MedCalc Statistical Software (version 22.013, Belgium) at https://www.medcalc.org/ [55].

Sensitivity, specificity, and predictive values

The sensitivity, specificity, positive predictive value (PPV), and negative predictive value (NPV) of TRP and KYN concentrations, and the KYN:TRP ratio were calculated using conventional formulae corresponding to the cells in Table 1 [56]:

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Table 1. Two-by-two table for calculating the sensitivity, specificity, NPV and PPV.

https://doi.org/10.1371/journal.pone.0314945.t001

Receiver Operating Characteristic (ROC) analyses

The optimal cut-off points for KYN, TRP, and the KYN:TRP ratio were determined based on the Youden’s index [57], which favours the cut-off point with the best trade-off between sensitivity and specificity, using the Receiver Operating Characteristic (ROC) analysis [58]. All ROC curves and the optimal cut-off points were performed in jamovi.

Results

Assay validation

The accuracy and precision of the estimated KYN and TRP concentrations in intra-day and inter-day QC samples, spiked at 2.5, 5 and 20 μg/mL, are within ± 15% (S1 Table). The calculated LLOD and LLOQ values were 0.05 μg/mL and 0.15 μg/mL for KYN, and 0.03 μg/mL and 0.08 μg/mL for TRP, respectively.

The stabilities of KYN and TRP were evaluated at ambient room temperature (∼20˚C) over 5 h, 4˚C over 14 days, and -20˚C over 43 days, with both analytes determined to be unstable after 2 h at ambient temperature, 14 days at 4˚C, and 29 days at -20˚C.

The high recovery of KYN, obtained from concentrations of 2.50, 5.00 and 20.0 μg/mL, were 94.9 ± 8.85%, 107 ± 15.3%, and 99.9 ± 14.3%, respectively. Comparably, the recovery of TRP, with the same concentrations, yielded 83.8 ± 7.24%, 100 ± 4.38%, and 96.6 ± 5.83%, respectively.

Koala demographics

A total of 80 koalas were recruited, comprising of 48.8% males and 51.3% females. Of these, 90.0% were adults and 10.0% were juveniles (< 2 years of age). Geographically, 80.0% were located in NSW and 20.0% in QLD. The proportion of clinically normal (43.8%) and diseased koalas with chlamydiosis (43.8%) were identical, while 12.5% of the koalas had ‘other’ co-morbidities (see S2 Table for demographic information, details on specific ‘other’ presentations, and the KYN and TRP concentrations).

Observations in kynurenine and tryptophan concentrations

There was no significant difference in the potential biomarkers (KYN, TRP and KYN:TRP ratio) between clinically normal or diseased males and females.

A significant univariable difference was observed in the TRP concentrations between the clinically normal juvenile and adult koalas (juveniles had higher TRP concentrations than adults) when calculated using the Welch’s t-test (p = 0.04) as presented in S3 Table. However, there was no difference between the KYN, TRP, or KYN:TRP ratio and the maturity within the diseased or ‘other’ koalas.

There were no differences in KYN and TRP concentrations, and the KYN:TRP ratio, between NSW and QLD in the clinically normal group, nor in the diseased group.

A summary of the median and range of KYN, TRP, and the KYN:TRP ratios in clinically normal, diseased, and ‘other’ koalas are presented in Table 2. There is a significant difference between the KYN and KYN:TRP in clinically normal versus diseased, and clinically normal versus ‘other’ (all p < 0.001) but not between diseased versus ‘other’ in KYN and the KYN:TRP ratio (Fig 2). There is a significant difference between the clinically normal versus diseased in TRP (p = 0.001) only.

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Fig 2. Box and whiskers plot of KYN, TRP, and KYN:TRP ratio in clinically normal (n = 35), diseased (n = 35) and ‘other’ koalas (n = 10).

(A) KYN concentrations. (B) TRP concentrations. (C) KYN:TRP ratio. The outlier in the diseased group had both eyes and UGT clinical signs with mammary mass, and the outlier in the ‘other’ group had leukaemia. * = statistically significant.

https://doi.org/10.1371/journal.pone.0314945.g002

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Table 2. Summary of the median and range of TRP, KYN, and the KYN:TRP ratios in clinically normal, diseased, and ‘other’ koalas.

https://doi.org/10.1371/journal.pone.0314945.t002

Haematological values and biochemical analytes.

The haematological values and biochemical analytes concentrations (S4 Table) showed no significant differences in the haematological values and biochemical analytes except for Glob between the clinically normal and diseased koalas (p = 0.01), and in ALP between the clinically normal and ‘other’ koalas (p = 0.03) (S5 Table). There were no statistical differences in the haematological values and biochemical analytes concentrations associated with hydration status between the clinically normal, diseased, and ‘other’ koalas.

There was no correlation between KYN:TRP ratio and some other biochemical and haematological inflammatory biomarkers when Pearson’s correlation analysis was conducted with koalas in the diseased group, and separately with clinically normal koalas. When analysed with koalas in the diseased or ’other’ groups together, correlations were observed between KYN:TRP ratio and WBC (Pearson’s r value of 0.805, p < 0.001); KYN:TRP and Lymph (0.671, p < 0.001); and KYN:TRP and ALT (0.621, p = 0.001).

Reference ranges of KYN, TRP, and the KYN:TRP ratio in clinically normal koalas

The proposed reference ranges of KYN, TRP, and the KYN:TRP ratio in clinically normal koalas are 0.34–1.23 μg/mL, 4.27–10.4 μg/mL, and 0.05–0.22, respectively. Concentrations of KYN, TRP, and the KYN:TRP ratios of all koalas, along with the hypothesised reference ranges, are presented in S1 Fig.

Sensitivity, specificity and predictive values

The sensitivity, specificity, PPV and NPV of the proposed KYN, TRP, and KYN:TRP ratio reference ranges were calculated using the conventional formulae, with the reference ranges yielding high specificity and PPV across the three biomarkers (Table 3).

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Table 3. Sensitivity, specificity, and predictive values of the proposed KYN, TRP, and KYN:TRP ratio references ranges for screening diseased koalas.

https://doi.org/10.1371/journal.pone.0314945.t003

Receiver Operating Characteristic (ROC) analyses of KYN, TRP, and the KYN:TRP ratio

The optimal cut-off points of KYN, TRP and the KYN:TRP ratio are shown in Table 4. These values were derived from the clinically normal, diseased, and ‘other’ koalas with the combined ROC curves presented in S2 Fig. Further ROC analysis was performed to decide cut-off points of KYN, TRP and the KYN:TRP ratio for survival outcome (i.e., released versus euthanised) of the koalas in the diseased and ‘other’ groups in S3 Fig.

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Table 4. Optimal cut-off points and its sensitivity, specificity, and predictive values of the proposed KYN, TRP, and KYN:TRP ratio in clinically normal versus diseased/‘other’, and released/recovered versus euthanised in diseased/‘other’ koalas.

https://doi.org/10.1371/journal.pone.0314945.t004

Discussion

This is the first study to investigate TRP and KYN concentrations, and the KYN:TRP ratio in koala plasma between clinically normal and general disease states. This was a pilot study, and therefore reference ranges and the optimal cut-off points for KYN, TRP and the KYN:TRP ratio in the plasma are proposed, however, a larger sample size would increase the confidence of these ranges.

An accurate and precise assay to detect and quantify KYN and TRP concentrations was optimised for koala plasma. Other studies have developed reversed-phase HPLC assays for the same purpose in human plasma [59, 60] and serum [48, 61–64], however, modifications requiring UV detection of KYN, and fluorescence detection of both TRP and the IS, were necessary to overcome the interference of the endogenous peaks inherent in the plasma of clinically normal koalas [65, 66]. Additionally, developing an assay that could analyse both substrates simultaneously was critical due to their instability after two hours at ambient room temperature (∼20˚C).

Although TRP and KYN blood concentrations have been quantified in many species [30, 31, 46, 67–69], and the KYN:TRP in others [39, 41, 70], there are no studies proposing reference ranges for these potential biomarkers within the biological matrix of any animal species.

In comparison to the human plasma TRP reference range of 8.76–15.2 μg/mL (42.9–74.4 μmol/L) [71], the proposed koala plasma TRP reference range is lower at 4.27–10.4 μg/mL. The lower range in the koala could be due to their diet that is restricted to certain Eucalyptus spp. leaves known to be low in TRP content (1.10% and 3.60% in young and mature leaves, respectively) [72]; compared to human dietary sources of TRP such as the cow milk protein, α-lactalbumin, containing around 5.80% TRP [73].

The human plasma KYN reference range of 0.25–0.85 μg/mL (1.20–4.10μM) [74] is slightly lower than the proposed plasma KYN reference range at 0.34–1.23 μg/mL in koalas. As the main source of endogenous KYN concentration is synthesised in the liver predominantly by tryptophan 2,3-dioxygenase (TDO2) [75], perhaps higher KYN concentrations in koalas could be the result of greater liver activity due to their needs of metabolising plant secondary metabolites (PSMs) from the diet. Koalas have some very active hepatic metabolism pathways as a result of high cytochrome P450 monooxygenases (CYP) content in their livers [76], most noticeably CYP2C [77]. Another possibility for koalas having higher endogenous KYN concentrations than humans is due to their lower metabolic rate [78, 79], which may result in slower breakdown/excretion of KYN.

As a result of higher KYN and lower TRP plasma concentrations detected in clinically normal koalas when compared to humans, it is expected that the proposed KYN:TRP ratio reference range of 0.05–0.22 in clinically normal koalas is greater than in healthy humans of < 0.06 (calculated from the human TRP and KYN reference ranges above in μg/mL).

A common characteristic of all three potential biomarkers was high specificity (range of 94.3–97.1%) and low sensitivity (28.6–40.0%); in addition to high PPV (87.5–93.3%) and relatively lower NPV (57.6–61.8%) (Table 3). Altogether, high specificity and high PPV indicate that the proposed reference ranges have high probabilities of avoiding false positives and identifying true positives [80], when the koalas deemed positive are indeed diseased. Conversely, there is a relatively higher probability that the infected koalas could obtain KYN:TRP ratios within the proposed reference range (false negatives) with low sensitivity and low NPV. Practically, this implies that if a koala has a low KYN:TRP ratio, it cannot be discounted for being diseased and should be monitored, with the KYN:TRP ratio repeated later. However, if a high ratio is calculated, there is a high probability that inflammation is present.

For screening chlamydial infections, higher specificity can prevent unnecessary psychological stress and costly treatments [81]. This is reflected in previous studies for screening chlamydial infections in humans, where the reported specificity has been higher than the sensitivity [82–84]. In koalas, minimising the possibility of false positives is critical to prevent unnecessary stress, cost, and the need for invasive treatment procedures under general anaesthesia. However, many koalas with sub-clinical infections could be missed if relying solely on an elevated KYN:TRP ratio.

The ROC analyses were conducted on the TRP and KYN concentrations, and the KYN:TRP ratio in clinically normal versus diseased and ‘other’ koalas; and also on the potential biomarkers of released/recovered koalas versus euthanised of the diseased and ‘other’ koalas. For both, the optimal cut-off points demonstrated good sensitivity, specificity, and predictive values for both KYN and the KYN:TRP ratio but not for TRP (Table 4). As AUCs of 0.70 or above are acceptable, and values above 0.80 considered having excellent accuracy [85], only the KYN and KYN:TRP ratio optimal cut-off points should be considered. Therefore, koalas with KYN concentrations of ≥ 0.88 μg/mL and/or KYN:TRP ratios of ≥ 0.12 have high probabilities of being diseased or ‘other’. Further, koalas with KYN concentrations of ≥ 1.18 μg/mL and/or KYN:TRP ratios of ≥ 0.16 have greater chances of being euthanised due to disease than being released. While the ROC analysis is an attempt to determine the survivability of infected koalas, more comprehensive data, such as the severity of infections, should be collected and considered for greater confidence.

Tryptophan plasma concentrations

Although plasma TRP concentrations were significantly higher in the clinically normal than the diseased koalas (Fig 2B and S6 Table), the concentration could be dependent on the stage of infection due to the nearly complete TrpR of C. pecorum pathogens, whereby TRP biosynthesis is upregulated by the pathogen when the concentration is low [25, 27, 28]. However, to date, TRP threshold and the change in TRP concentration upon the activation of the TrpR (whether it surpasses or maintains its threshold concentration) are undefined. Furthermore, there are 11 koalas, 10 in the diseased group and 1 from the ‘other’ group with TRP concentrations below the proposed reference range (< 4.24 μg/mL). Of these 11 koalas, the five were euthanised, five diagnosed with chlamydiosis were later released, and one had trauma and on recovery was released (S2 Table). As anticipated, koalas with the TRP concentrations below the proposed reference range were predominantly in the cohort afflicted by the disease. However, a low TRP concentration is not a convincing indication of disease severity, consequently, considering TRP alone as a potential biomarker for C. pecorum is not recommended.

Kynurenine concentrations and the KYN:TRP ratio

Higher concentrations of KYN and the KYN:TRP ratio are associated with koalas in the diseased and ‘other’ groups in comparison to the clinically normal. Expectedly, a narrow distribution was observed in the KYN:TRP ratio of the clinically normal, followed by the diseased, then the ‘other’ group with the widest distribution (Table 2 and Fig 2C). The anomaly for KYN and the KYN:TRP ratio in the ‘other’ group was the same koala (K62) with leukaemia, while the other anomaly for the KYN:TRP ratio in the diseased group (K39) was positive for C. pecorum, from swabs collected from the conjunctiva and UGT, with a mammary mass. Although a significantly higher KYN:TRP ratio does not consistently indicate the presence of neoplasia, as demonstrated in K74 with abdominal lymphoma (KYN:TRP of 0.28). Conversely, there was no statistical significance in KYN, TRP, and the KYN:TRP ratio between diseased and the ‘other’ koalas, suggesting that the potential biomarkers may not be specific for C. pecorum infections; though they may indicate the severity of inflammation as both koalas diagnosed with neoplastic disease were later euthanised.

The TRP, KYN and KYN:TRP did not appear to be distorted due to hydration status, nor were there any obvious differences in hepatic and/or renal function between the clinically normal, diseased, and ‘other’ groups. While a significant increase in Glob in the diseased group (p = 0.01) and a reduction in ALP in the ‘other’ when compared to the clinically normal koalas (p = 0.03) were observed, and even though changes in these biomarkers could occur in dehydrated animals, elevated concentrations of CRE and BUN are more convincing dehydration markers [86, 87]. A possible factor for the fluctuation of Glob is likely due to the body’s response to inflammation in the diseased, as many globulins are well-established inflammatory markers [88, 89]. Whereas, the decline in ALP concentration has been attributed to dietary magnesium deficiency [90] as a consequence of loss of appetite from co-morbidities such as leukaemia, abdominal lymphoma, and trauma in the ‘other’ koalas. Although there was no significant difference in ALB concentration and the health status of the koalas, TRP is known to bind exclusively to ALB in the plasma; whereby 10.0–15.0% of the total plasma TRP existing in an unbound state in rats [91, 92], and only 6% in kangaroos [68]. While the unbound proportion of TRP is undefined in the koala, this study exclusively quantified the total plasma TRP concentrations and did not distinguish between TRP bound to plasma proteins and the unbound form.

Of the inflammatory biomarkers analysed, the Pearson’s correlation analysis determined a strong positive relationship between KYN:TRP ratio and WBC (0.805, p < 0.001), Lymph (0.671, p < 0.001), and ALT (0.621, p = 0.001) for the koalas in diseased or ‘other’ groups. This was expected as the activation of the TRP-KYN pathway, accompanied with an elevation in WBC, Lymph and ALT, are common responses to systemic and/or liver inflammation [93, 94]; though no correlations were evident for the koalas in the diseased group only, nor the clinically normal only, which indicated KYN:TRP to be a promising non-specific inflammatory biomarker in koalas.

One of the limitations of this study was the sample size; specifically in demographic variables, such as number of juvenile (n = 6), samples from a QLD wildlife hospital (n = 16), and ‘other’ with non-chlamydial diseases (n = 10). As koala samples are collected opportunistically, a larger sample size was restricted due to exhaustion of resources. As a result of limited access to koalas due to their endangered status, opportunistic sampling was used in this study. Consequently, while associations between KYN, TRP, and KYN:TRP and chlamydial infection were observed, the causal relationships between these biomarkers and the infection have yet to be established. Another limitation is that the haematology and biochemistry results were only available for 35 and 36 koalas, respectively, from a total of 80 koalas. This can also be resolved with additional increased time and resource allocations to ensure completeness of data. The final limitation was that some diseased koalas may have had elevated endogenous glucocorticoid levels. As a response to stressors, like other species, koalas will increase glucocorticoid production, such as cortisol, from their hypothalamic-pituitary-adrenal (HPA) axis [95]. Endogenous glucocorticoids have anti-inflammatory properties and can induce tryptophan 2,3-dioxygenase (TDO2), thereby increase TRP depletion and elevate the KYN concentration [96, 97]. Only one of the recruited koalas was administered exogenous prednisolone to treat UGT infections (K17), yet there was no significant decrease in TRP concentration or increase in KYN concentration when compared to the other diseased koalas. Even with the study design assisting to minimise the effect of glucocorticoids on KYN concentrations by collecting blood samples under general anaesthesia, the various amount of stress endured by the koalas may have impacted the results. Even though other anti-inflammatory drugs, such as non-steroidal anti-inflammatory drugs (NSAIDs), could also affect TRP and KYN blood concentrations [98], these drugs were not administered prior to blood collection from the recruited koalas.

Further investigation on these potential biomarkers, along with serotonin and quinolinic acid as these metabolites are also indicators of inflammation [99], in a larger sample size with the disease severity documented would draw more robust conclusions. This can also determine whether there is a change in the KYN:TRP during treatment and whether these TRP metabolites have the potential to be reliable prognostic markers for antibiotic treatment success for chlamydiosis in this species. Examination of the KYN:TRP may also be helpful to monitor response to treatment for other non-infectious, inflammatory conditions, such as an animal with arthritis response to non-steroidal anti-inflammatory agents. Correlation analysis could also be conducted between the KYN:TRP ratio and other inflammatory biomarkers, including the acute phase proteins such as C-reactive protein (CRP) and serum amyloid A protein [100], cancer antigen 125 (CA-125), and erythrocyte sedimentation rate (ESR) [101], to determine the strength of the associations. A novel strategy to improve antibiotic efficacy against chlamydial pathogen is oral supplementation of TRP, which may reactivate dormant chlamydial pathogens into their metabolically active states and preventing them to revert to their dormant state [102]. Antibiotics are efficacious against metabolically active pathogens; however, it is also recognised that they can also induce dormancy [103]. Therefore, exploring the treatment efficacy of administering TRP supplementation with antibiotics against dormant chlamydial pathogens could be a new avenue for research.

Conclusion

This is the first study to successfully detect and quantify plasma KYN and TRP concentrations in koala plasma, and to investigate the associations between clinically normal, diseased, and koalas with other co-morbidities. The koala reference ranges of KYN, TRP, and the KYN:TRP ratio are also proposed, in addition to the optimal cut-off points to distinguish between clinically normal versus diseased and released versus euthanised of diseased koalas to suggest the chances of recovery. The study concluded that although the potential biomarkers may not be specific for detecting C. pecorum from the rest of the population, this study strengthened the level of confidence in the capability of KYN and the KYN:TRP ratio to identify unhealthy koalas from the clinically normal, regardless of the cause. Until further studies demonstrate high confidence in detecting C. pecorum pathogens with biomarkers, PCR still remains the golden standard to identify chlamydial presence in koalas.

Supporting information

S1 Table. Estimated plasma KYN and TRP concentrations of the QC samples as triplicates spiked with KYN and TRP at different concentrations (2.50, 5.00, 20.0 μg/mL), with the accuracy (%) and precision (%) of each intra-day, and the mean ± SD for the inter-day results across three days.

https://doi.org/10.1371/journal.pone.0314945.s001

(PDF)

S2 Table. Demographic information and the KYN and TRP concentrations of the koalas recruited (n = 80).

https://doi.org/10.1371/journal.pone.0314945.s002

(PDF)

S3 Table. Univariable difference of the KYN, TRP, and KYN:TRP ratio between clinically normal juveniles (n = 6) versus adults (n = 29).

Non-normally distributed data was analysed using non-parametric test. 1 = reference category; α = non-normal distribution; β = normal distribution; SD = standard deviation. Statistically significant value is bolded.

https://doi.org/10.1371/journal.pone.0314945.s003

(PDF)

S4 Table. Haematological values and biochemical analytes concentrations in clinically normal (n = 11), diseased (n = 20), or ‘other’ (n = 5) koalas.

Reference ranges: WBC = 2.6–9.8 × 109/L; RBC = 2.9–4.4 × 1012/L; HCT = 0.31–0.45 L/L; Neut = 0.6–6.6 × 109/L; Lymph = 0.5–3.8 × 109/L; Mono = < 1.2 × 109/L; ALP = 30–564 U/L; ALT = 5–21 U/L; ALB = 34–50 g/L; CRE = 80–150 μmol/L; Glob = 18–30 g/L; TP = 58–73 g/L; GGT = 6–17 U/L; BUN = 1–8 mmol/L. Reference ranges were retrieved from Canfield et al. [104] and Vetnostics. Abbreviations: WBC = white blood cells; RBC = red blood cells; HCT = haematocrit test; Neut = neutrophils; Lymph = lymphocytes; Mono = monocytes; ALP = alkaline phosphatase; ALT = alanine transaminase; ALB = albumin; CRE = creatinine; Glob = globulins; TP = total protein; GGT = gamma-glutamyl transferase; BUN = blood urea nitrogen. Information is not available for blank cells. Values outside the reference range are in red.

https://doi.org/10.1371/journal.pone.0314945.s004

(PDF)

S5 Table. Pairwise comparison of the biochemical analytes between clinically normal, diseased, and ‘other’ koalas.

1 = reference category; SD = standard deviation. Statistically significant value is bolded.

https://doi.org/10.1371/journal.pone.0314945.s005

(PDF)

S6 Table. Univariable results showing the KYN, TRP, and KYN:TRP ratio between clinically normal (n = 35) versus diseased (n = 35), clinically normal versus ‘other’ (n = 10), and diseased versus ‘other’ groups.

1 = reference category; α = non-normal distribution; β = normal distribution; SD = standard deviation. Statistically significant value is bolded.

https://doi.org/10.1371/journal.pone.0314945.s006

(PDF)

S1 Fig. Scatterplot of KYN concentrations (reference range: 0.34–1.23 μg/mL), TRP concentrations (reference range: 4.27–10.4 μg/mL), and KYN:TRP ratios (reference range: 0.05–0.22) in clinically normal, diseased, and ‘other’ koalas.

(A) KYN concentrations. (B) TRP concentrations. (C) KYN:TRP ratios. Each point represents an individual koala. The green lines define the reference range interval. The outlier for KYN and the KYN:TRP ratio in the ‘other’ group is the same koala with leukaemia. The outlier for the KYN:TRP ratio in the ‘diseased’ group had both eyes and UGT clinical signs with mammary mass.

https://doi.org/10.1371/journal.pone.0314945.s007

(TIF)

S2 Fig. ROC curves of the KYN, TRP and KYN:TRP ratio in clinically normal, diseased, and ‘other’ koalas.

Abbreviations: KYN = kynurenine; TRP = tryptophan; concen = concentration.

https://doi.org/10.1371/journal.pone.0314945.s008

(TIF)

S3 Fig. ROC curves of the KYN, TRP and KYN:TRP ratio in the released and euthanised koalas of the diseased and ‘other’ groups.

Abbreviations: KYN = kynurenine; TRP = tryptophan; concen = concentration.

https://doi.org/10.1371/journal.pone.0314945.s009

(TIF)

References

  1. 1. Kanova M, Kohout P. Tryptophan: A Unique Role in the Critically Ill. Int J Mol Sci. 2021;22(21). Epub 2021/11/14. pmid:34769144; PubMed Central PMCID: PMC8583765.
  2. 2. Badawy AA. Kynurenine Pathway of Tryptophan Metabolism: Regulatory and Functional Aspects. Int J Tryptophan Res. 2017;10:1178646917691938. Epub 2017/05/05. pmid:28469468; PubMed Central PMCID: PMC5398323.
  3. 3. Bender DA. Biochemistry of tryptophan in health and disease. Molecular aspects of medicine. 1983;6(2):101–97. pmid:6371429
  4. 4. Badawy AA. Tryptophan Metabolism: A Versatile Area Providing Multiple Targets for Pharmacological Intervention. Egypt J Basic Clin Pharmacol. 2019;9. Epub 2019/05/21. pmid:31105983; PubMed Central PMCID: PMC6520243.
  5. 5. Wu W, Nicolazzo JA, Wen L, Chung R, Stankovic R, Bao SS, et al. Expression of tryptophan 2, 3-dioxygenase and production of kynurenine pathway metabolites in triple transgenic mice and human Alzheimer’s disease brain. PloS one. 2013;8(4):e59749. pmid:23630570
  6. 6. Bai M, Liu H, Xu K, Oso AO, Wu X, Liu G, et al. A review of the immunomodulatory role of dietary tryptophan in livestock and poultry. Amino Acids. 2017;49:67–74. pmid:27778165
  7. 7. Gonzalez Esquivel D, Ramirez-Ortega D, Pineda B, Castro N, Rios C, Perez de la Cruz V. Kynurenine pathway metabolites and enzymes involved in redox reactions. Neuropharmacology 2017;112 331–45.
  8. 8. Mao X, Lv M, Yu B, He J, Zheng P, Yu J, et al. The effect of dietary tryptophan levels on oxidative stress of liver induced by diquat in weaned piglets. Journal of animal science and biotechnology. 2014;5:1–7.
  9. 9. Farmahin R, Crump D, Kennedy SW. Sensitivity of avian species to the aryl hydrocarbon receptor ligand 6-formylindolo [3, 2-b] carbazole (FICZ). Chemico-Biological Interactions. 2014;221:61–9. pmid:25093689
  10. 10. Song J, Clagett-Dame M, Peterson RE, Hahn ME, Westler WM, Sicinski RR, et al. A ligand for the aryl hydrocarbon receptor isolated from lung. Proceedings of the National Academy of Sciences. 2002;99(23):14694–9. pmid:12409613
  11. 11. Shimizu T, Nomiyama S, Hirata F, Hayaishi O. Indoleamine 2,3-dioxygenase. Purification and some properties. J Biol Chem. 1978;253(13):4700–6. Epub 1978/07/10. pmid:26687
  12. 12. Ball HJ, Sanchez-Perez A, Weiser S, Austin CJ, Astelbauer F, Miu J, et al. Characterization of an indoleamine 2,3-dioxygenase-like protein found in humans and mice. Gene. 2007;396(1):203–13. Epub 2007/05/15. pmid:17499941.
  13. 13. Jones SP, Guillemin GJ, Brew BJ. The kynurenine pathway in stem cell biology. Int J Tryptophan Res. 2013;6:57–66. Epub 2013/10/05. pmid:24092986; PubMed Central PMCID: PMC3782398.
  14. 14. Brown J, Robusto B, Morel L. Intestinal dysbiosis and tryptophan metabolism in autoimmunity. Frontiers in immunology. 2020;11:1741. pmid:32849620
  15. 15. Riaz F, Wei P, Pan F. Fine-tuning of regulatory T cells is indispensable for the metabolic steatosis-related hepatocellular carcinoma: A review. Frontiers in Cell and Developmental Biology. 2022;10. pmid:35912096
  16. 16. Albert PR, Benkelfat C, Descarries L. The neurobiology of depression—revisiting the serotonin hypothesis. I. Cellular and molecular mechanisms. The Royal Society; 2012. p. 2378–81.
  17. 17. Oxenkrug GF. Tryptophan–kynurenine metabolism as a common mediator of genetic and environmental impacts in major depressive disorder: the serotonin hypothesis revisited 40 years later. The Israel journal of psychiatry and related sciences. 2010;47(1):56. pmid:20686200
  18. 18. Choi S-C, Brown J, Gong M, Ge Y, Zadeh M, Li W, et al. Gut microbiota dysbiosis and altered tryptophan catabolism contribute to autoimmunity in lupus-susceptible mice. Science translational medicine. 2020;12(551):eaax2220. pmid:32641487
  19. 19. Brown R, Ozaki Y, Datta S, Borden E, Sondel P, Malone D. Implications of interferon-induced tryptophan catabolism in cancer, autoimmune diseases and AIDS. Kynurenine and Serotonin Pathways: Progress in Tryptophan Research. 1991:425–35.
  20. 20. Mondanelli G, Iacono A, Carvalho A, Orabona C, Volpi C, Pallotta MT, et al. Amino acid metabolism as drug target in autoimmune diseases. Autoimmunity Reviews. 2019;18(4):334–48. pmid:30797943
  21. 21. Gonzalez-Astudillo V, Allavena R, McKinnon A, Larkin R, Henning J. Decline causes of Koalas in South East Queensland, Australia: a 17-year retrospective study of mortality and morbidity. Scientific Reports. 2017;7(1):42587. pmid:28218272
  22. 22. Polkinghorne A, Hanger J, Timms P. Recent advances in understanding the biology, epidemiology and control of chlamydial infections in koalas. Vet Microbiol. 2013;165:214–23. pmid:23523170
  23. 23. Phillips S, Robbins A, Loader J, Hanger J, Booth R, Jelocnik M, et al. Chlamydia pecorum gastrointestinal tract infection associations with urogenital tract infections in the koala (Phascolarctos cinereus). PloS one. 2018;13(11):e0206471. pmid:30383822.
  24. 24. Chen CJ, Casteriano A, Green AC, Govendir M. A retrospective study on antibacterial treatments for koalas infected with Chlamydia pecorum. Sci Rep. 2023;13(1):12670. Epub 2023/08/05. pmid:37542093; PubMed Central PMCID: PMC10403558.
  25. 25. Islam MM, Jelocnik M, Huston WM, Timms P, Polkinghorne A. Characterization of the In Vitro Chlamydia pecorum Response to Gamma Interferon. Infection and Immunity. 2018;86(4):10.1128/iai.00714-17. pmid:29358337
  26. 26. Akers JC, Tan M. Molecular mechanism of tryptophan-dependent transcriptional regulation in Chlamydia trachomatis. Journal of bacteriology. 2006;188(12):4236–43. pmid:16740930
  27. 27. Bachmann NL, Fraser TA, Bertelli C, Jelocnik M, Gillett A, Funnell O, et al. Comparative genomics of koala, cattle and sheep strains of Chlamydia pecorum. BMC Genomics. 2014;15(1):667. pmid:25106440
  28. 28. Mojica S, Huot Creasy H, Daugherty S, Read TD, Kim T, Kaltenboeck B, et al. Genome sequence of the obligate intracellular animal pathogen Chlamydia pecorum E58. J Bacteriol. 2011;193(14):3690. Epub 2011/05/17. pmid:21571992; PubMed Central PMCID: PMC3133325.
  29. 29. Yao K, Fang J, Yin YL, Feng ZM, Tang ZR, Wu G. Tryptophan metabolism in animals: important roles in nutrition and health. Front Biosci (Schol Ed). 2011;3(1):286–97. Epub 2011/01/05. pmid:21196377.
  30. 30. Badawy AA, Dougherty DM. Assessment of the Human Kynurenine Pathway: Comparisons and Clinical Implications of Ethnic and Gender Differences in Plasma Tryptophan, Kynurenine Metabolites, and Enzyme Expressions at Baseline and After Acute Tryptophan Loading and Depletion. Int J Tryptophan Res. 2016;9:31–49. Epub 2016/08/23. pmid:27547036; PubMed Central PMCID: PMC4981220.
  31. 31. Allegri G, Costa CV, Bertazzo A, Biasiolo M, Ragazzi E. Enzyme activities of tryptophan metabolism along the kynurenine pathway in various species of animals. Farmaco. 2003;58(9):829–36. Epub 2003/09/19. pmid:13679176.
  32. 32. Gál EM, Sherman AD. Synthesis and metabolism of L-kynurenine in rat brain. J Neurochem. 1978;30(3):607–13. Epub 1978/03/01. pmid:681935
  33. 33. Yan J, Kothur K, Mohammad S, Chung J, Patel S, Jones HF, et al. CSF neopterin, quinolinic acid and kynurenine/tryptophan ratio are biomarkers of active neuroinflammation. eBioMedicine. 2023;91. pmid:37119734
  34. 34. Mandarano M, Orecchini E, Bellezza G, Vannucci J, Ludovini V, Baglivo S, et al. Kynurenine/Tryptophan Ratio as a Potential Blood-Based Biomarker in Non-Small Cell Lung Cancer. Int J Mol Sci. 2021;22(9). Epub 2021/05/01. pmid:33922388; PubMed Central PMCID: PMC8122814.
  35. 35. Suzuki Y, Suda T, Furuhashi K, Suzuki M, Fujie M, Hahimoto D, et al. Increased serum kynurenine/tryptophan ratio correlates with disease progression in lung cancer. Lung Cancer. 2010;67(3):361–5. Epub 2009/06/03. pmid:19487045.
  36. 36. Badawy AA. The kynurenine pathway of tryptophan metabolism: a neglected therapeutic target of COVID-19 pathophysiology and immunotherapy. Biosci Rep. 2023;43(8). Epub 2023/07/24. pmid:37486805; PubMed Central PMCID: PMC10407158.
  37. 37. Lionetto L, Ulivieri M, Capi M, De Bernardini D, Fazio F, Petrucca A, et al. Increased kynurenine-to-tryptophan ratio in the serum of patients infected with SARS-CoV2: An observational cohort study. Biochim Biophys Acta Mol Basis Dis. 2021;1867(3):166042. Epub 2020/12/19. pmid:33338598; PubMed Central PMCID: PMC7834629.
  38. 38. Cihan M, Doğan Ö, Ceran Serdar C, Altunçekiç Yıldırım A, Kurt C, Serdar MA. Kynurenine pathway in Coronavirus disease (COVID-19): Potential role in prognosis. J Clin Lab Anal. 2022;36(3):e24257. Epub 2022/01/30. pmid:35092710; PubMed Central PMCID: PMC8906035.
  39. 39. Wish J, Bulloch P, Oswald L, Halldorson T, Raine JC, Jamshed L, et al. Kynurenine to tryptophan ratio as a biomarker of acute stress in fish. Chemosphere. 2022;288:132522. pmid:34648784
  40. 40. van Hierden YM, Koolhaas JM, Korte SM. Chronic increase of dietary L-tryptophan decreases gentle feather pecking behaviour. Applied Animal Behaviour Science. 2004;89(1–2):71–84.
  41. 41. Clarke G, Grenham S, Scully P, Fitzgerald P, Moloney RD, Shanahan F, et al. The microbiome-gut-brain axis during early life regulates the hippocampal serotonergic system in a sex-dependent manner. Mol Psychiatry. 2013;18(6):666–73. Epub 2012/06/13. pmid:22688187.
  42. 42. Pawlak D, Tankiewicz A, Buczko W. Kynurenine and its metabolites in the rat with experimental renal insufficiency. J Physiol Pharmacol. 2001;52(4 Pt 2):755–66. Epub 2002/01/12. pmid:11785771.
  43. 43. Saito K, Fujigaki S, Heyes MP, Shibata K, Takemura M, Fujii H, et al. Mechanism of increases in L-kynurenine and quinolinic acid in renal insufficiency. Am J Physiol Renal Physiol. 2000;279(3):F565–72. Epub 2000/09/01. pmid:10966936.
  44. 44. Pawlak D, Tankiewicz A, Matys T, Buczko W. Peripheral distribution of kynurenine metabolites and activity of kynurenine pathway enzymes in renal failure. J Physiol Pharmacol. 2003;54(2):175–89. Epub 2003/07/02. pmid:12832720.
  45. 45. Luo Z, Yong K, Du Z, Huang Y, Zhou T, Ma L, et al. Association between Tryptophan Metabolism and Inflammatory Biomarkers in Dairy Cows with Ketosis. Metabolites. 2023;13(3):333. pmid:36984773
  46. 46. Bochniarz M, Kocki T, Dąbrowski R, Szczubiał M, Wawron W, Turski WA. Tryptophan, kynurenine, kynurenic acid concentrations and indoleamine 2,3-dioxygenase activity in serum and milk of dairy cows with subclinical mastitis caused by coagulase-negative staphylococci. Reproduction in Domestic Animals. 2018;53(6):1491–7. pmid:30066969
  47. 47. Higgins DP, Hemsley S, Canfield PJ. Association of uterine and salpingeal fibrosis with chlamydial hsp60 and hsp10 antigen-specific antibodies in Chlamydia-infected koalas. Clin Diagn Lab Immunol. 2005;12(5):632–9. Epub 2005/05/10. pmid:15879024; PubMed Central PMCID: PMC1112079.
  48. 48. Widner B, Werner ER, Schennach H, Fuchs D. An HPLC method to determine tryptophan and kynurenine in serum simultaneously. Adv Exp Med Biol. 1999;467:827–32. Epub 2000/03/18. pmid:10721136.
  49. 49. ICH. Bioanalytical method validation and study sample analysis M10. Switzerland: ICH, 2022.
    • 50. Taleuzzaman M. Limit of Blank (LOB), Limit of Detection (LOD), and Limit of Quantification (LOQ). Organic and Medicinal Chemistry International Journal. 2018;7(5):1–5.
    • 51. jamovi. 1.6 ed2021.
    • 52. Whitley E, Ball J. Statistics review 2: samples and populations. Crit Care. 2002;6(2):143–8. Epub 2002/05/02. pmid:11983040; PubMed Central PMCID: PMC137296.
    • 53. Bland M. An Introduction to Medical Statistics: Oxford University Press; 2015.
      • 54. Shine B. Use of routine clinical laboratory data to define reference intervals. Ann Clin Biochem. 2008;45(Pt 5):467–75. Epub 2008/08/30. pmid:18753418.
      • 55. MedCalc Statistical Software. 22.013 ed. Belgium: MedCalc Software Ltd; 2023.
        • 56. Parikh R, Mathai A, Parikh S, Chandra Sekhar G, Thomas R. Understanding and using sensitivity, specificity and predictive values. Indian J Ophthalmol. 2008;56(1):45–50. Epub 2007/12/26. pmid:18158403; PubMed Central PMCID: PMC2636062.
        • 57. Youden WJ. Index for rating diagnostic tests. Cancer. 1950;3(1):32–5. Epub 1950/01/01. pmid:15405679.
        • 58. Greiner M, Pfeiffer D, Smith RD. Principles and practical application of the receiver-operating characteristic analysis for diagnostic tests. Prev Vet Med. 2000;45(1–2):23–41. Epub 2000/05/10. pmid:10802332.
        • 59. Zhao J, Gao P, Zhu D. Optimization of Zn2+-containing mobile phase for simultaneous determination of kynurenine, kynurenic acid and tryptophan in human plasma by high performance liquid chromatography. J Chromatogr B Analyt Technol Biomed Life Sci. 2010;878(5–6):603–8. Epub 2010/01/28. pmid:20102795.
        • 60. Myint AM, Kim YK, Verkerk R, Scharpé S, Steinbusch H, Leonard B. Kynurenine pathway in major depression: evidence of impaired neuroprotection. J Affect Disord. 2007;98(1–2):143–51. Epub 2006/09/06. pmid:16952400.
        • 61. Laich A, Neurauter G, Widner B, Fuchs D. More rapid method for simultaneous measurement of tryptophan and kynurenine by HPLC. Clin Chem. 2002;48(3):579–81. Epub 2002/02/28. pmid:11861457.
        • 62. Hervé C, Beyne P, Jamault H, Delacoux E. Determination of tryptophan and its kynurenine pathway metabolites in human serum by high-performance liquid chromatography with simultaneous ultraviolet and fluorimetric detection. J Chromatogr B Biomed Appl. 1996;675(1):157–61. Epub 1996/01/12. pmid:8634758.
        • 63. Vignau J, Jacquemont MC, Lefort A, Imbenotte M, Lhermitte M. Simultaneous determination of tryptophan and kynurenine in serum by HPLC with UV and fluorescence detection. Biomed Chromatogr. 2004;18(10):872–4. Epub 2004/12/08. pmid:15584004.
        • 64. Krcmova L, Solichova D, Melichar B, Kasparova M, Plisek J, Sobotka L, et al. Determination of neopterin, kynurenine, tryptophan and creatinine in human serum by high throuput HPLC. Talanta. 2011;85(3):1466–71. Epub 2011/08/03. pmid:21807211.
        • 65. Kimble B, Li KM, Govendir M. Quantitation of meloxicam in the plasma of koalas (Phascolarctos cinereus) by improved high performance liquid chromatography. J Vet Sci. 2013;14(1):7. pmid:23388431
        • 66. Chen CJ, Gillett A, Booth R, Kimble B, Govendir M. Pharmacokinetic Profile of Doxycycline in Koala Plasma after Weekly Subcutaneous Injections for the Treatment of Chlamydiosis. Animals. 2022;12(3):250. Epub 2022/02/16. pmid:35158574; PubMed Central PMCID: PMC8833767.
        • 67. Fuller RW, Roush BW. Binding of tryptophan to plasma proteins in several species. Comp Biochem Physiol B. 1973;46(2):273–6. Epub 1973/10/15. pmid:4202192.
        • 68. Fellows FCI, Hird FJR. A comparative study of the binding of l-tryptophan and bilirubin by plasma proteins. Archives of Biochemistry and Biophysics. 1982;216(1):93–100. pmid:6808929
        • 69. Fujigaki S, Saito K, Takemura M, Fujii H, Wada H, Noma A, et al. Species differences in L-tryptophan-kynurenine pathway metabolism: quantification of anthranilic acid and its related enzymes. Arch Biochem Biophys. 1998;358(2):329–35. Epub 1998/10/24. pmid:9784247.
        • 70. Geisler S, Lytton SD, Toan NL, Nghia TH, Nam NM, Hung HV, et al. Neopterin levels and Kyn/Trp ratios were significantly increased in dengue virus patients and subsequently decreased after recovery. International Journal of Infectious Diseases. 2020;91:162–8. pmid:31821895
        • 71. Yamamoto H, Kondo K, Tanaka T, Muramatsu T, Yoshida H, Imaizumi A, et al. Reference intervals for plasma-free amino acid in a Japanese population. Ann Clin Biochem. 2016;53(Pt 3):357–64. Epub 2015/04/02. pmid:25829462.
        • 72. Journet ARP, Cochrane PM. Free amino acids in the leaf tissue of Eucalyptus blakelyi. Phytochemistry. 1978;17(10):1789–90.
        • 73. Heine WE, Klein PD, Reeds PJ. The importance of alpha-lactalbumin in infant nutrition. J Nutr. 1991;121(3):277–83. Epub 1991/03/01. pmid:2002399.
        • 74. Bizjak DA, Stangl M, Börner N, Bösch F, Durner J, Drunin G, et al. Kynurenine serves as useful biomarker in acute, Long- and Post-COVID-19 diagnostics. Frontiers in Immunology. 2022;13. pmid:36211365
        • 75. Knox WE, Mehler AH. The conversion of tryptophan to kynurenine in liver: I. The coupled tryptophan peroxidase-oxidase system forming formylkynurenine. Journal of Biological Chemistry. 1950;187(1):419–30.
        • 76. Ngo S, Kong S, Kirlich A, McKinnon RA, Stupans I. Cytochrome P450 4A, peroxisomal enzymes and nicotinamide cofactors in koala liver. Comparative Biochemistry and Physiology Part C: Pharmacology, Toxicology and Endocrinology. 2000;127(3):327–34. pmid:11246504
        • 77. Liapis P, Pass GJ, McKinnon RA, Stupans I. Characterisation of tolbutamide hydroxylase activity in the common brushtail possum, (Trichosurus vulpecula) and koala (Phascolarctos cinereus): inhibition by the eucalyptus terpene 1,8-cineole. Comp Biochem Physiol C Toxicol Pharmacol. 2000;127(3):351–7. Epub 2001/03/15. pmid:11246507.
        • 78. Dawson TJ, Hulbert AJ. Standard metabolism, body temperature, and surface areas of Australian marsupials. Am J Physiol. 1970;218(4):1233–8. Epub 1970/04/01. pmid:5435424.
        • 79. McNab BK. The comparative energetics of neotropical marsupials. Journal of comparative physiology. 1978;125(2):115–28.
        • 80. Trevethan R. Sensitivity, Specificity, and Predictive Values: Foundations, Pliabilities, and Pitfalls in Research and Practice. Front Public Health. 2017;5:307. Epub 2017/12/07. pmid:29209603; PubMed Central PMCID: PMC5701930.
        • 81. Maxim LD, Niebo R, Utell MJ. Screening tests: a review with examples. Inhal Toxicol. 2014;26(13):811–28. Epub 2014/09/30. pmid:25264934; PubMed Central PMCID: PMC4389712.
        • 82. Bassiri M, Hu H, Domeika M, Burczak J, Svensson L, Lee H, et al. Detection of Chlamydia trachomatis in urine specimens from women by ligase chain reaction. Journal of Clinical Microbiology. 1995;33(4):898–900. pmid:7790456
        • 83. Cook RL, St. George K, Lassak M, Tran N, Anhalt JP, Rinaldo CR. Screening for Chlamydia trachomatis infection in college women with a polymerase chain reaction assay. Clinical infectious diseases. 1999;28(5):1002–7. pmid:10452625
        • 84. Gaydos CA, Howell MR, Quinn TC, Gaydos JC, McKee Jr KT. Use of ligase chain reaction with urine versus cervical culture for detection of Chlamydia trachomatis in an asymptomatic military population of pregnant and nonpregnant females attending Papanicolaou smear clinics. Journal of clinical microbiology. 1998;36(5):1300–4. pmid:9574695
        • 85. Hosmer DW, Lemeshow S. Area under the ROC curve. Applied Logistic Regression. 2000:160–4.
        • 86. Braun JP, Lefebvre HP, Watson AD. Creatinine in the dog: a review. Vet Clin Pathol. 2003;32(4):162–79. Epub 2003/12/05. pmid:14655101.
        • 87. Lane MR, Lowe A, Vukcevic J, Clark RG, Madani G, Higgins DP, et al. Health Assessments of Koalas after Wildfire: A Temporal Comparison of Rehabilitated and Non-Rescued Resident Individuals. Animals (Basel). 2023;13(18). Epub 2023/09/28. pmid:37760263; PubMed Central PMCID: PMC10525633.
        • 88. Hashash JG, Koutroumpakis F, Anderson AM, Rivers CR, Hosni M, Koutroubakis IE, et al. Elevated serum globulin fraction as a biomarker of multiyear disease severity in inflammatory bowel disease. Ann Gastroenterol. 2022;35(6):609–17. Epub 2022/11/22. pmid:36406970; PubMed Central PMCID: PMC9648529.
        • 89. Smith GS, Hall RL, Walker RM. 6—Applied Clinical Pathology in Preclinical Toxicology Testing. In: Haschek WM, Rousseaux CG, Wallig MA, editors. Handbook of Toxicologic Pathology (Second Edition). San Diego: Academic Press; 2002. p. 123–56.
          • 90. Lum G. Significance of low serum alkaline phosphatase activity in a predominantly adult male population. Clin Chem. 1995;41(4):515–8. Epub 1995/04/01. pmid:7720239.
          • 91. Madras BK, Cohen EL, Messing R, Munro HN, Wurtman RJ. Relevance of free tryptophan in serum to tissue tryptophan concentrations. Metabolism. 1974;23(12):1107–16. pmid:4427559
          • 92. Ruddick JP, Evans AK, Nutt DJ, Lightman SL, Rook GA, Lowry CA. Tryptophan metabolism in the central nervous system: medical implications. Expert Rev Mol Med. 2006;8(20):1–27. Epub 2006/09/01. pmid:16942634.
          • 93. Gabay C, Kushner I. Acute-Phase Proteins and Other Systemic Responses to Inflammation. New England Journal of Medicine. 1999;340(6):448–54. pmid:9971870
          • 94. Oh RC, Hustead TR, Ali SM, Pantsari MW. Mildly Elevated Liver Transaminase Levels: Causes and Evaluation. Am Fam Physician. 2017;96(11):709–15. pmid:29431403.
          • 95. Charalambous R, Simonato T, Peel M, Narayan EJ. Physiological Stress in Rescued Wild Koalas (Phascolarctos cinereus) Being Held in a Rehabilitation Sanctuary: A Pilot Study. Animals. 2021;11(10):2864. pmid:34679883
          • 96. Danesch U, Hashimoto S, Renkawitz R, Schütz G. Transcriptional regulation of the tryptophan oxygenase gene in rat liver by glucocorticoids. J Biol Chem. 1983;258(8):4750–3. Epub 1983/04/25. pmid:6187742.
          • 97. Hirota T, Hirota K, Sanno Y, Tanaka T. A New Glucocorticoid Receptor Species: Relation to Induction of Tryptophan Dioxygenase by Glucocorticoids. Endocrinology. 1985;117(5):1788–95. pmid:2864234
          • 98. Edwards SR, Mather LE. Diclofenac increases the accumulation of kynurenate following tryptophan pretreatment in the rat: a possible factor contributing to its antihyperalgesic effect. Inflammopharmacology. 2003;11(3):277–92. Epub 2004/03/24. pmid:15035810.
          • 99. Moffett JR, Els T, Espey MG, Walter SA, Streit WJ, Namboodiri MA. Quinolinate immunoreactivity in experimental rat brain tumors is present in macrophages but not in astrocytes. Exp Neurol. 1997;144(2):287–301. Epub 1997/04/01. pmid:9168830.
          • 100. Fey G, Hocke G, Wilson D. Cytokines and acute phase response of the liver. Arias I, Boyer J, Fausto N, Jacoby W, Schachter D, Shafritz D, editors. New York, USA: Raven Press, Ltd.; 1994. 113–43 p.
            • 101. Park ST, Lee SW, Kim MJ, Kang YM, Moon HM, Rhim CC. Clinical characteristics of genital chlamydia infection in pelvic inflammatory disease. BMC Womens Health. 2017;17(1):5. Epub 20170113. pmid:28086838; PubMed Central PMCID: PMC5237214.
            • 102. Singla M. Role of Tryptophan supplementation in the treatment of Chlamydia. Med Hypotheses. 2007;68(2):278–80. Epub 2006/10/19. pmid:17045416.
            • 103. Hogan RJ, Mathews SA, Mukhopadhyay S, Summersgill JT, Timms P. Chlamydial persistence: beyond the biphasic paradigm. Infect Immun. 2004;72(4):1843–55. pmid:15039303; PubMed Central PMCID: PMC375192.
            • 104. Canfield PM, O’Neill ME, Smith EF. Haematological and biochemical reference values for the koala (Phascolarctos cinereus). Aust Vet J. 1989;66(10):324–6. Epub 1989/10/01. pmid:2818360.