Abstract
Systemic sclerosis (SSc) is a progressive autoimmune connective tissue disease, leading to disability and often to a shorter life expectancy. Numerous reports indicate that the kynurenine pathway (KP) is dysregulated in systemic sclerosis (SSc). Therefore, this exploratory pilot study aimed to assess whether levels of tryptophan (TRP) and its selected metabolites within the KP correlate across different body fluids. Forty-one patients with SSc participated in the study. Blood, saliva, and morning urine samples were collected. The concentrations of TRP, kynurenine (KYN), 3-hydroxykynurenine (3-HK), quinolinic acid (QUIN), and kynurenic acid (KYNA) were measured. In addition, precursor/product ratios reflecting the activity of KP enzymes were calculated. In the cohort of patients comprising both men and women the significant correlations were observed between blood plasma and urine levels of KYN (ρ = 0.531, P < 0.001), as well as between the KYN/TRP (ρ = 0.774, P < 0.001), 3-HK/TRP (ρ = 0.549, P = 0.001), QUIN/TRP (ρ = 0.467, P = 0.002), and KYNA/TRP (ρ = 0.412, P < 0.008) ratios in patients with SSc. Correlation between plasma and saliva was limited to KYNA in female SSc (ρ = 0.526, P < 0.007) which suggests that saliva has limited diagnostic value for assessing KP activity in patients with SSc. In contrast, urine-based measurements were promising and support further development of diagnostic approaches based on urinary KP metabolites.
Keywords: Systemic sclerosis, Kynurenine, Plasma, Saliva, Urine
Introduction
In recent years, as our understanding of the role of kynurenines in pathophysiological processes in both the brain and periphery has deepened, interest in their diagnostic and prognostic significance has also grown [1]. It has been shown that tryptophan (TRP) metabolites formed along kynurenine metabolic pathway (KP), the primary route for TRP catabolism, possess diverse biological effects [1].
The term KP refers to a series of enzymatic reactions leading from TRP through kynurenine (KYN) to quinolinic acid (QUIN) (see Fig. 1). There are also two side branches within the kynurenine pathway, one leading to the synthesis of kynurenic acid (KYNA) and the other to the synthesis of xanthurenic acid. QUIN is a precursor which contributes to the de novo synthesis of nicotinamide adenine dinucleotide (NAD+), while KYNA is the final metabolite, which is primarily excreted in urine. Numerous KP metabolites act as aryl hydrocarbon receptor (AhR) agonists, including KYN, KYNA, and xanthurenic acid. KYNA plays a unique role.It acts not only on the AhR but also as an agonist of G protein-coupled receptor 35 (GPR35), a non-selective broad-spectrum antagonist of ionotropic glutamate receptors and antagonist of the alpha-7 nicotinic receptor [2]. It is intriguing that, unlike KYNA, QUIN acts as an agonist of the glutamatergic NMDA receptor. 3-Hydroxykynurenine (3-HK) is involved in redox processes in a concentration-dependent manner; in low concentrations it may act as an antioxidant, whereas at high concentrations it induces oxidative stress [1, 2].
Fig. 1.
A simplified diagram illustrating the metabolism of tryptophan (TRP) in the human body. The diagram highlights the substances (in boxes) whose levels were measured. Enzymes are written in italics. The meaning of the ratios indirectly determining the activity of individual enzymes or enzyme cascades has been visualized (vertical brown lines). Solid arrows indicate steps in the kynurenine pathway (KP); dashed arrows indicate other metabolic pathways
Systemic sclerosis (SSc) is an autoimmune connective tissue disease that occurs in 17.6 out of every 100,000 people. It is more common in women than in men (5:1) [3]. The essential feature of the disease is excessive fibrosis, which affects blood vessels and peripheral organs (i.e. skin, gastrointestinal tract, lower respiratory tract including the lungs, cardiac muscle, and kidney). SSc is a progressive, currently incurable disease that leads to multiorgan failure, patient disability and reduced life expectancy. The lack of an effective treatment is possibly due to uncertainty regarding the underlying causes of the pathological process, which are thought to involve genetic, immunological, and environmental factors [4].
Since collected data indicate that kynurenines modulate the activity of the immune system [5, 6], it can be expected that immune-mediated diseases are associated with alterations in KP activity. These changes can be assessed by measuring individual metabolites levels or metabolites ratios and correlated with disease severity and progression. Indeed, in our earlier publication, we demonstrated alterations in the level of kynurenines in the blood of affected individuals compared to healthy controls [7]. Since no early risk indicators have been identified that reliably predict the onset of SSc, it is reasonable to search for novel appropriate biological markers [8, 9]. Since blood testing is an invasive procedure, in this study we intend to check whether the measurement of TRP and kynurenines in non-invasive material, such as saliva and urine, reflects their levels in the blood.
The aim of the study is to determine whether the levels of TRP and its selected metabolites formed along KP correlate in different compartments, i.e., blood, urine, and saliva. This study aims to evaluate whether kynurenine levels measured in non-invasive biological samples can reflect KP activity in patients with SSc.
Materials and methods
Systemic sclerosis (SSc) patients
The study protocol was approved by the Ethics Committee of the Medical University of Lublin, Poland (protocol number KE-0254/220/10/2023). Both male and female patients hospitalized at the Department of Rheumatology and Connective Tissue Diseases of the University Hospital (USK4) in Lublin, Poland, treated between October 2023 and October 2025 for SSc, were included in the study. The written informed consent was obtained from all the subjects. The diagnosis was made by a physician in accordance with the 2013 American College of Rheumatology/European League Against Rheumatism (ACR/EULAR) criteria for SSc [10]. The exclusion criteria were as follows: pregnancy or lactation; major surgery (including joint surgery) within 8 weeks prior to the study; rheumatic autoimmune disease other than SSc, including rheumatoid arthritis, systemic lupus erythematosus, mixed connective tissue disease, polymyositis, dermatomyositis, primary Sjögren syndrome; active infection, evidence of malignant disease, or malignancies diagnosed within the previous 5 years; a history of alcohol, drug, or chemical abuse within 1 year prior to the study.
Materials
Blood, morning urine, and saliva samples were collected from patients who were instructed to refrain from eating for 12 h. Blood and urine samples were collected in accordance with routine hospital procedures. Venous blood samples from the median antecubital vein were collected into vacutainer tubes with EDTA as an anticoagulant. Plasma was separated by centrifugation at 1200 × g for 15 min at 4 °C within 30 min of blood collection and stored at –80 °C until analysis. Saliva samples were collected using Salivette (Sarstedt Ag & Co, Nuembrecht, Germany). Saliva was collected according to the manufacturer's instruction, which recommend avoiding eating, drinking, smoking, or brushing teeth for at least 30 min prior to sample collection. Samples were immediately frozen at − 80 °C and stored until further analysis.
Determination of tryptophan and kynurenines
The levels of tryptophan (TRP), kynurenine (KYN), 3-hydroxykynurenine (3-HK), quinolinic acid (QUIN), and kynurenic acid (KYNA) were measured using liquid chromatography—mass spectrometry (LC–MS). 25 µL of stable-isotope labeled internal standard mixture was added to 200 µL of plasma, saliva and urine, samples were vortexed. Then cold (− 20 °C) methanol:ethanol (1:1 v/v) was added, 575 µL to plasma and saliva samples, and 375 µL to urine samples. Samples were vortexed for 30 s, stored at − 20 °C, and centrifuged at 16 000 g for 20 min at 4 °C. Supernatants were collected and filtered by 0.2 µm syringe filters (Titan3™ regenerated cellulose). Then samples were diluted 1:1 with 0.1% formic acid and subjected to LC/MS analysis, a triple quadrupole mass analyzer (Agilent 6470 LC/TQ) coupled to an ultrahigh performance liquid chromatograph (Agilent Infinity II 1290 HPLC) was used. Method limits of quantitation (LOQ) and limits of detection (LOD) are presented in Table 1. The intra- and interday precision of the method was below 3.1 and 5.6%, respectively.
Table 1.
Method limits of quantitation (LOQ) and limits of detection (LOD)
| Unit | Plasma [LOQ] |
Saliva [LOQ or LOD*] |
Urine [LOQ] |
|
|---|---|---|---|---|
| Tryptophan (TRP) | µg/mL | 5 | 0.01 | 0.5 |
| Kynurenine (KYN) | ηg/mL | 200 | 0.5 | 50 |
| 3-Hydroxykynurenine (3-HK) | ηg/mL | 0.5 | 1* | 50 |
| Quinolinic acid (QUIN) | ηg/mL | 20 | 2.5* | 500 |
| Kynurenic acid (KYNA) | ηg/mL | 0.5 | 0.4 | 50 |
An asterisk does not indicate statistical significance; anasterisk indicates that the values marked are LOD values. This is indicated inthe column header “LOQ or LOD*”. You can add note under the table: * indicatesan LOD value.
Statistics
The obtained results were examined for normal distribution with Shapiro–Wilk test. Since the results do not follow a normal distribution, the data are presented as the median, minimum (min), maximum (max) and interquartile range (IQR). Accordingly, the correlation was calculated using Spearman’s rho rank correlation test. The actual P values are reported in tables, while P values shown in bold indicate statistical significance after adjusting for the false discovery rate (FDR) at the P < 0.05 level. The statistical sensitivity of the study was calculated. For a two-sided correlation analysis with α = 0.05 and n = 41, the study provides approximately 80% power to detect moderate-to-strong correlations (approximately ρ ≥ 0.42). However, results of subgroup analyses, particularly in male patients (n = 15), should be interpreted cautiously and considered exploratory.
Results
Study participants
General characteristics of study participant is presented in Table 2. A total of 41 patients diagnosed with SSc by a rheumatologist participated in the study, including 26 women and 15 men. The average age was 54 (43–65) years and the mean disease duration since the onset was 5 (2–13) years. Average BMI was 24.03 (22.77–27.80). Twelve patients (29.3%) had a limited, while 29 (70.7%) diffuse systemic SSc.
Table 2.
General characteristics of systemic sclerosis patients
| SSc patients | ||||
|---|---|---|---|---|
| Result/median | Min–max | IQR (Q1 – Q3) | ||
| Number of subjects | 41 | – | – | |
| Sex [female/male] | 26/15 | – | ||
| Age | 54.0 | 43–65 | ||
| Clinical data | ||||
| SSc localized/systemic | 12/29 | – | – | |
| BMI | 24.03 | 22.77–27.80 | ||
| C-Reactive Protein (CRP) | [mg/dL] | 0.19 | 0.05–0.77 | |
| Creatinine | [mg/dL] | 0.65 | 0.64–0.66 | |
| Estimated glomerular filtration rate (eGFR) | [mL/min] | 90.0 | 81.5–90.0 | |
| Plasma content | ||||
| Tryptophan (TRP) | [µg/mL] | 11.15 | 4.86–18.02 | 9.27–13.17 |
| Kynurenine | [µg/mL] | 0.76 | 0.43–2.61 | 0.65–0.93 |
| 3-Hydroxykynurenine (3-HK) | [µg/mL] | 0.013 | 0.007–0.042 | 0.011–0.0185 |
| Quinolinic acid (QUIN) | [µg/mL] | 0.059 | 0.026–0.333 | 0.049–0.096 |
| Kynurenic acid | [µg/mL] | 0.006 | 0.002–0.127 | 0.004–0.009 |
| Saliva content | ||||
| Tryptophan (TRP) | [µg/mL] | 0.085 | 0.017–4.772 | 0.049–0.235 |
| Kynurenine (KYN) | [µg/mL]* | 0.0012 | 0.0001–0.033 | 0.0009–0.0029 |
| Kynurenic acid (KYNA) | [µg/mL] | 0.0017 | 0.0001–0.050 | 0.0008–0.0029 |
| Urine content | ||||
| Tryptophan (TRP) | [µg/g] | 7.30 | 1.43–31.27 | 4.83–10.73 |
| [µg/mL] | 7.45 | 1.44–32.05 | 4.9–10.95 | |
| Kynurenine (KYN) | [µg/g] | 1.08 | 0.11–45.39 | 0.43–2.49 |
| [µg/mL] | 1.10 | 0.11–45.8 | 0.43–2.56 | |
|
3-Hydroxykynurenine (3HK) |
[µg/g] | 0.20 | 0.03–4.68 | 0.09–0.43 |
| [µg/mL] | 0.21 | 0.03–4.72 | 0.10–0.44 | |
| Quinolinic acid (QUIN) | [µg/g] | 3.70 | 0.53–25.14 | 1.98–6.10 |
| [µg/mL] | 3.75 | 0.53–25.65 | 2.02–6.26 | |
| Kynurenic acid | [µg/g] | 1.48 | 0.08–7.73 | 0.74–2.54 |
| [µg/mL] | 1.50 | 0.08–7.80 | 0.74–2.62 | |
Data are presented as median, minimum (min), maximum (max) and inter-quantile range (IQR). *Results higher than zero were calculated
Concerning disease related antibodies, 75.6% of participants were positive for Scl-70 antibodies, 15.0% had positive anti-ACA antibodies, 12.2% had anti-PM/Scl antibodies, and 7.3% had anti-SS-A antibodies. Antibodies targeting SS-B, Sm, RNP/Sm and AMA-M2 appeared in 2 out of 41 patients. Antibodies targeting RNP, a-Ku, Jo-1 was detected in 1 out of 41 subjects.
Patients were treated with the following drugs, cyclosporine (65.85%), hydroxychloroquine (65.85%), methotrexate (63.41%), mycophenolate mofetil (60.98%), corticosteroids (46.34%), azathioprine (14.63%), intravenous immunoglobulins (IVIg) (2.44%). They also received, amlodipine (75.61%), pentoxifylline (56.10%), angiotensin-converting enzyme inhibitors (iACE) (29.27%), sildenafil (17.07%), sartan (8.11%).
Content of tryptophan (TRP) and its metabolites
TRP, KYN, 3-HK, QUIN, and KYNA were found in the blood plasma and urine of all patients. TRP and KYNA were found in the saliva of all patients, while KYN was found in 12 women and 5 men. No quantifiable amounts of 3-HK and QUIN were found in saliva of any of the subjects. Detailed results are presented in Table 2.
The correlation between plasma level of TRP, its metabolites and value of indicators of kynurenine pathway enzyme activity
Correlations between disease duration, erythrocyte sedimentation rate (ESR), C-reactive protein (CRP) and plasma concentrations of the tested substances were analyzed, together with correlations of markers indirectly reflecting enzyme activity in the two branches of the kynurenine pathway (KP), leading to quinolinic acid (QUIN) and kynurenic acid (KYNA), respectively. Based on measurements of the levels of individual substances tested, the following ratios were calculated: KYN/TRP, 3-HK/TRP, QUIN/TRP, KYNA/TRP, 3-HK/KYN, KYNA/KYN and QUIN/3-HK. It was found that, TRP levels are negatively correlated with CRP. KYN/TRP, 3-HK/TRP and QUIN/TRP ratio correlated with CRP (Table 3). The analyzed substances and the values of indicators of kynurenine pathway enzyme activity did not correlate with disease duration or ESR (Table 3).
Table 3.
Correlation between blood level of TRP, level of TRP, its metabolites and value of indicators of kynurenine pathway enzyme activity in the total investigated population of patients with systemic sclerosis
| Compound/ratio | Correlation | |||||
|---|---|---|---|---|---|---|
| Disease duration | Erythrocyte sedimentation rate | C-reactive protein | ||||
| ρ | P value | ρ | P value | ρ | P value | |
| Tryptophan (TRP) | 0.016 | 0.924 | − 0.412 | 0.008 | − 0.415 | 0.008 |
| Kynurenine (KYN) | − 0.015 | 0.927 | 0.102 | 0.531 | 0.249 | 0.121 |
| 3-Hydroxykynurenine (3-HK) | 0.069 | 0.673 | 0.287 | 0.073 | 0.301 | 0.059 |
| Quinolinic acid (QUIN) | 0.247 | 0.124 | 0.168 | 0.300 | 0.312 | 0.049 |
| Kynurenic acid (KYNA) | 0.377 | 0.016 | − 0.043 | 0.792 | 0.119 | 0.463 |
| KYN/TRP ratio | 0.055 | 0.736 | 0.364 | 0.021 | 0.500 | 0.001 |
| 3-HK/TRP ratio | 0.059 | 0.717 | 0.394 | 0.012 | 0.385 | 0.014 |
| QUIN/TRP ratio | 0.204 | 0.208 | 0.290 | 0.070 | 0.460 | 0.003 |
| KYNA/TRP ratio | 0.363 | 0.022 | 0.112 | 0.492 | 0.298 | 0.062 |
| 3-HK/KYN ratio | 0.026 | 0.873 | 0.280 | 0.081 | 0.128 | 0.431 |
| KYNA/KYN ratio | 0.426 | 0.006 | − 0.135 | 0.407 | − 0.135 | 0.407 |
| QUIN/3-HK ratio | 0.184 | 0.257 | − 0.120 | 0.461 | − 0.100 | 0.540 |
The correlation was calculated using Spearman’s rho rank correlation test. P values shown in bold indicate statistical significance after adjusting for the false discovery rate (FDR) at the P < 0.05 level
The correlation between saliva and plasma, and urine and plasma
Table 4 presents the results of the analysis, which was conducted without sex differentiation.
Table 4.
Correlation between level of TRP, its metabolites and value of indicators of kynurenine pathway enzyme activity in the whole investigated population of patients with systemic sclerosis
| Compound/ratio | Correlation | |||
|---|---|---|---|---|
| Between saliva and plasma | Between urine and plasma | |||
| ρ | P value | ρ | P value | |
| Tryptophan (TRP) | − 0.113 | 0.489 | 0.2235 | 0.166 |
| Kynurenine (KYN) | nd | nd | 0.493 | 0.001 |
| 3-Hydroxykynurenine (3-HK) | nd | nd | 0.317 | 0.046 |
| Quinolinic acid (QUIN) | nd | nd | 0.313 | 0.049 |
| Kynurenic acid (KYNA) | 0.301 | 0.059 | − 0.114 | 0.482 |
| KYN/TRP ratio | nd | nd | 0.774 | 0.001 |
| 3-HK/TRP ratio | nd | nd | 0.549 | 0.001 |
| QUIN/TRP ratio | nd | nd | 0.467 | 0.002 |
| KYNA/TRP ratio | 0.123 | 0.451 | 0.412 | 0.008 |
| 3-HK/KYN ratio | nd | nd | 0.304 | 0.056 |
| KYNA/KYN ratio | nd | nd | 0.135 | 0.408 |
| QUIN/3-HK ratio | nd | nd | 0.292 | 0.067 |
The correlation was calculated using Spearman’s rho rank correlation test. P-values shown in bold indicate statistical significance after adjusting for the false discovery rate (FDR) at the P < 0.05 level. nd means not determined
Neither content of TRP and KYNA nor KYNA/TRP ratio examined in saliva correlate with plasma. The levels of KYN in urine correlated with the corresponding levels in blood plasma. The following enzyme activity ratios were found to correlate in urine and plasma: KYN/TRP, 3-HK/TRP, QUIN/TRP, and KYNA/TRP.
Table 5 presents the results of the analysis conducted in the group of female study participants. A significant correlation has been demonstrated between KYNA levels in blood plasma and saliva. In women, no correlations were observed between salivary enzyme activity indicators and plasma levels. In urine, KYN levels showed a correlation with plasma concentrations. In women’s urine, correlations with plasma levels were observed for KYN/TRP ratio.
Table 5.
Correlation between level of TRP, its metabolites and value of indicators of kynurenine pathway enzyme activity in female patients with systemic sclerosis
| Compound/ratio | Correlation | |||
|---|---|---|---|---|
| Between saliva and plasma | Between urine and plasma | |||
| ρ | P value | ρ | P value | |
| Tryptophan (TRP) | − 0.121 | 0.565 | 0.269 | 0.194 |
| Kynurenine (KYN) | nd | nd | 0.587 | 0.002 |
| 3-Hydroxykynurenine (3-HK) | nd | nd | 0.090 | 0.669 |
| Quinolinic acid (QUIN) | nd | nd | 0.170 | 0.419 |
| Kynurenic acid (KYNA) | 0.526 | 0.007 | − 0.017 | 0.936 |
| KYN/TRP ratio | nd | nd | 0.735 | 0.001 |
| 3-HK/TRP ratio | nd | nd | 0.280 | 0.175 |
| QUIN/TRP ratio | nd | nd | 0.481 | 0.015 |
| KYNA/TRP ratio | 0.241 | 0.246 | 0.382 | 0.059 |
| 3-HK/KYN ratio | nd | nd | 0.193 | 0.355 |
| KYNA/KYN ratio | − 0.121 | 0.694 | 0.017 | 0.936 |
| QUIN/3-HK ratio | nd | nd | − 0.170 | 0.417 |
The correlation was calculated using Spearman’s rho rank correlation test. P-values shown in bold indicate statistical significance after adjusting for the false discovery rate (FDR) at the P < 0.05 level. nd means not determined
Table 6 presents the results of the analysis conducted in the group of male study participants. In men's saliva, neither the levels of the tested substances nor the enzyme activity indices correlated with the plasma levels. In male urine, a correlation was observed between the following plasma enzyme activity indicators: KYN/TRP, 3-HK/TRP, and QUIN/3-HK ratio.
Table 6.
Correlation between level of TRP, its metabolites and value of indicators of kynurenine pathway enzyme activity in male patients with systemic sclerosis.
| Compound/ratio | Correlation | |||
|---|---|---|---|---|
| Between saliva and plasma | Between urine and plasma | |||
| ρ | P value | ρ | P value | |
| Tryptophan (TRP) | − 0.157 | 0.576 | 0.325 | 0.237 |
| Kynurenine (KYN) | nd | nd | 0.346 | 0.206 |
| 3-Hydroxykynurenine (3-HK) | nd | nd | 0.532 | 0.041 |
| Quinolinic acid (QUIN) | nd | nd | 0.475 | 0.074 |
| Kynurenic acid (KYNA) | − 0.057 | 0.840 | 0.339 | 0.216 |
| KYN/TRP ratio | nd | nd | 0.864 | 0.001 |
| 3-HK/TRP ratio | nd | nd | 0.879 | 0.001 |
| QUIN/TRP ratio | nd | nd | 0.518 | 0.048 |
| KYNA/TRP ratio | − 0.225 | 0.420 | 0.557 | 0.031 |
| 3-HK/KYN ratio | nd | nd | 0.593 | 0.020 |
| KYNA/KYN ratio | nd | nd | 0.014 | 0.960 |
| QUIN/3-HK ratio | nd | nd | 0.732 | 0.002 |
The correlation was calculated using Spearman’s rho rank correlation test. P-values shown in bold indicate statistical significance after adjusting for the false discovery rate (FDR) at the P < 0.05 level. nd means not determined
Discussion
In our previous work, we demonstrated that SSc patients exhibit alterations in KP activity. Specifically, a decrease in TRP and an increase in KYN/TRP and KYNA/TRP ratios in plasma were observed in both women and men as compared to healthy controls. In addition, an increase in plasma KYN level and a decrease in the KYNA/KYN ratio were observed in men [7]. These findings are consistent with results published previously [11–18]. In the current study, the results were not compared with a control group comprising healthy patients; instead, the target was focused on the correlation between KP activity and the underlying determinants of the disease. Notably, a negative correlation was observed between TRP and CRP. This finding is consistent with previous reports of reduced TRP in SSc compared with a control group of healthy individuals [11, 12, 14, 15, 17]. A new aspect is the correlation with typical clinical markers of inflammation. We demonstrate positive correlations between the KYN/TRP and 3-HK/TRP and QUIN/TRP ratio and CRP, which further supports the assumption that the main branch of the KP pathway is activated by the inflammatory process; this is consistent with the widely accepted consensus regarding the activation of indoleamine 2,3-diooxygenase (activity reflected by KYN/TRP ratio) by inflammatory factors. Collectively, these results indicate that TRP metabolism disturbances accompany the disease, although they do not determine whether there is a causative relationship. There is strong evidence that TRP metabolites may influence the development of pathological conditions and serve as markers of such diseases, particularly those involving pronounced immune system activation. Since SSc is a progressive disease that usually develops slowly in its early stages, diagnosis is usually difficult until clear clinical symptoms appear. However at that time pathological changes are already advanced. Currently, there is no reliable early marker for the disease [8, 9, 18]. Therefore determination of TRP metabolites formed on KP and a full clarification of their relevance in the pathogenesis of SSc may lead to early diagnosis improvement and hopefully to the identification of new therapeutic targets. It should be emphasized that very recently, Mathew et al. (2026), in a comprehensive review article, proposed the use of a combination of galantamine-memantine, with or without N-acetylcysteine in the treatment of rheumatic diseases, as such combination affects the activity of KP [19].
Blood tests are inconvenient for patients, as they are invasive and require a visit to a dedicated laboratory for sample collection. They are also painful and involve risks associated with injections. This is why alternative methods should be investigated. The collection of both urine and saliva represents non-invasive sampling approaches. While urine analysis is well established and widely utilized in routine clinical diagnostics, saliva-based testing remains less commonly implemented in standard practice. In this study, we compared the levels of TRP and its four metabolites measured in plasma with the results obtained in saliva and urine. TRP and kynurenines were determined using tandem mass spectrometry (LC–MS/MS). Labeled substances were used as standards, which enabled precise and selective determination of the tested compounds.
TRP and its four metabolites were detected in blood and urine in quantities sufficient to determine their levels in all 41 studied patients, indicating that the applied method demonstrates adequate sensitivity. Neither 3-HK nor QUIN was detected in the saliva of any of the examined subjects, which may reflect either the absence of these metabolites in saliva or its presence at concentrations below the analytical detection limit. The detection of KYN in saliva in only 41.5% of our patients is unexpected. Considering the relatively low detection limit of the analytical method, a higher detection rate would be anticipated, suggesting potential biological variability, matrix-related effects, or other factors influencing salivary KYN levels. This may indicate that KYN is not a permanent component of saliva. It cannot be ruled out that the presence of KYN in saliva results from its local production in the oral cavity, which is activated, for example, by an ongoing inflammatory process in the oral cavity [20, 21]. It should be noted that the patients included in the study did not report any oral complaints and were undergoing no dental therapy at the time of saliva sampling. Furthermore, no symptoms of dry mouth were observed in the study cohort, and none of the patients had any difficulty collecting sufficient saliva for the saliva sampler.
Similarly to our findings, TRP, KYN and KYNA were previously detected in the saliva of healthy volunteers and patients with periodontitis, Sjögren's disease and schizophrenia [20, 22, 23]. The presence of both 3-HK and QUIN in saliva was described by Kurgan et al. (2022). In this study, saliva was collected by spitting, whereas we used the commercially available saline collection system. In addition, a different sample preparation procedure was used; samples were derivatized prior to liquid chromatography–mass spectrometry detection [20]. Presence of QUIN in measurable amount in saliva of healthy controls and schizophrenics was communicated by Yin et al. (2025), who used the chromatographic method (HPLC/MS) and labelled standards [22]. The procedure for salivary collection is not described in detail; it is known that saliva was obtained via passive drooling. Park et al. (2025) also reported the presence of QUIN in the saliva of patients with Sjögren's disease [23]. However, the procedure of saliva collection was not specified, and, importantly, quantitative measurements were performed using enzyme-linked immunosorbent assay kits. The procedure of saliva collection may affect the results of the measurement of the substances present in saliva. In 2024, Mortazavi et al. reviewed salivary collection, transportation, preparation, and storage methods widely used for scientific purposes, identifying as many as 24 most common procedures [24]. We used a commercially available system to collect saliva and strictly followed provided instructions to enable standardized collection of material, which allows the comparison of results achieved by other researchers.
Using the obtained results, correlations were analyzed between metabolite levels in plasma and saliva, as well as between plasma and urine. Similarly, correlations were examined for indicators that indirectly reflect the efficiency of substrate-to-metabolite conversion, which may act as indirect markers of enzymatic activity within the pathway. Based on the level of TRP and KP metabolites, the KYN/TRP ratio was calculated, which characterizes KP activity at its initial stage and is catalyzed by the constitutive enzyme tryptophan 2,3-dioxygenase and/or the inducible enzyme indoleamine 2,3-diooxygenase. In addition, the 3-HK/KYN ratio, which indirectly reflects kynurenine 3-monooxygenase activity, was calculated, as well as the QUIN/3-HK ratio, which comprises two consecutive enzymatic steps catalyzed by kynureninase and 3-hydroxyanthranilate 3,4-dioxygenase. Finally, the QUIN/TRP ratio was calculated as a general measure of the activity of the main KP branch. The activity of the KP side branch, leading to the formation of KYNA via a one-step enzymatic reaction was also analyzed. The KYNA/KYN ratio was determined as a measure of kynurenine aminotransferase(s) enzyme activity, and the KYNA/TRP ratio was determined as a broader measure of the KP side branch that leads to the formation of KYNA. Although this pathway represents only approximately 1–2% of KYN conversion, it remains functionally relevant, as its end product, KYNA, interacts with multiple molecular targets and may exert distinct physiological effects compared with other kynurenine pathway metabolites. Furthermore, KYNA is a final metabolite that is excreted unchanged mainly in urine, unlike QUIN, which is further metabolized by quinolinate phosphoribosyl transferase, a key rate-limiting enzyme in de novo NAD+ biosynthetic pathway, converting quinolinic acid to nicotinic acid mononucleotide. KYN and 3-HK are also metabolized, and therefore their measurement reflects their current levels.
In SSc patients, regardless of sex, no correlation was found between TRP and KYNA levels and KYNA/TRP ratio in saliva and blood plasma. The only exception was the KYNA correlation, which reached statistical significance in women. The correlation with KYN was not calculated due to the presence of KYN in the saliva of less than 50% of the patients studied. In contrast, all the metabolites tested were detected in the urine, which enabled the calculation of several correlations. It was found that only KYN correlates with its blood level in whole cohort and females. KYN/TRP ratio correlated across the whole study population of SSc patients, regardless of gender indicating activation of the main TRP conversion pathway towards NAD+. 3-HK/TRP, QUIN/TRP and KYNA/TRP ratios correlated in the whole population. Furthermore, correlations of QUIN/3-HK were observed in men; however, these results should be interpreted with caution due to the limited sample size.
The correlation between TRP and KP pathway metabolites in saliva and plasma was studied in generalized periodontitis patients [20]. Similarly to our study, no correlation was found in TRP level. In addition, no correlations were reported in 3-HK and QUIN levels which were not assessed in our study. In contrast to our findings, a negative correlation between KYN and TRP, and no correlation for KYNA levels, have been reported.
The lack of correlation between TRP levels in saliva and urine compared with plasma is not unexpected, as TRP is a multifunctional amino acid, and its distribution across different body compartmentsis likely influenced by its diverse physiological roles. In the first place, it is a protein-building amino acid, also metabolized in the KP (90%) and serotonin (5%) pathways. It is also a precursor of indoles, but these processes occur in the gut microbiome [25]. The lack of correlation between KYNA in urine and plasma is surprising, as KYNA is mainly excreted through the kidneys in urine. It should be emphasized that in the course of SSc, the kidneys may be affected by the disease process [26]; however in our cohort, only 3 out of 41 patients had an eGFR value less than 60 mL/min, and the results analyzed are expressed on gravidity basis. Whether the lack of correlation between urinary and plasma KYNA levels is a specific feature of SSc remains to be determined and requires further investigation. This topic seems particularly interesting because in a previous publication we demonstrated that plasma content of KYNA negatively correlate with eGFR and in patients using ACE inhibitors the level of KYNA in blood plasma is significantly higher [7]. Thus, the role of the kidneys in the maintenance of KP metabolites level in the blood and urine deserves closer consideration.
Analysis of the correlation between the computed values of KP enzyme activity indicators shows a particularly high values of KYN/TRP, QUIN/TRP and 3-HK/TRP correlation. All these metabolites constitute the main branch of KP. Our finding points to the conclusion that urinary analysis for TRP and kynurenines accurately reflects processes occurring in the body along the main KP pathway, leading to the formation of QUIN, which subsequently enters the NAD+ biosynthetic pathway. Notably, a significant correlation involving the KYNA/TRP ratio has been demonstrated, however, its importance is difficult to interpret due to the correlation between KYNA and age [27].
Since we demonstrated sex differences in KP activity in our previous publication [7], we also performed a correlation analysis stratified by sex. The cohort of women consisted of 26 subjects, and the cohort of men consisted of 15 subjects. In women, but not in men, a significant correlation was found between KYNA levels in saliva and plasma. All other correlations did not reach statistical significance regardless of sex. In women's urine, a correlation to plasma levels was found in KYN, as well as in the KYN/TRP ratio. In the urine and plasma of men, a correlation was observed between the ratios of KYN/TRP, 3-HK/TRP, and QUIN/3-HK. However, it should be noted that the number of men in the study was relatively small.
In summary, only salivary KYNA levels correlate with plasma level in women, but not in men, suggesting that saliva is not an optimal diagnostic material, at least in patients with SSc. In contrast, urine appears to be a more suitable diagnostic matrix, since the KYN/TRP ratios correlate positively with plasma values in SSc patients, regardless of their sex.
Limitations
The study was conducted on patients with SSc; however a control group of healthy volunteers matched for sex and age was not included. Therefore, the results of this study should not be generalized. The sensitivity of the method used did not allow for quantitative measurement of 3-HK and QUIN in saliva collected using commercially available sampler. Thus, further efforts should be undertaken to modify the method of detecting TRP metabolites formed on KP in this material. Due to the relatively small sample size, particularly when divided by gender, weaker correlations may have remained undetected. Therefore, results should be interpreted cautiously.
Conclusions
Although saliva is a convenient material that can be easily obtained and stored by the examined person, it has limited utility for assessing systemic KP activity, at least in patients with SSc. However, analysis of TRP and KP metabolite content in urine is a promising potential marker that reflects the activity of two branches of KP. The results of our study indicate that it is worthwhile to conduct further simultaneous analyses of KP activity in blood and urine under various pathological conditions, as currently available measurement methods are sufficiently sensitive for such assessments. It appears reasonable to develop and validate a set of simple diagnostic tests suitable for use in clinical laboratories and potentially outside of healthcare providers. Importantly, efforts to develop method of KYN detection using a smartphone are already underway [28]. This research was carried out using artificial saliva [28], which even more clearly emphasizes the need to identify and validate correlations between the levels of substances in blood, urine and saliva under authentic conditions.
Acknowledgements
This study was conducted within the framework of the Polish Clinical Scholars Research Training (P-CSRT), a Harvard Medical School Postgraduate Medical Education program implemented in collaboration with the Medical Research Agency of Poland. The content of the manuscript has not previously been presented at conferences. All authors take full responsibility for the integrity and accuracy of all aspects of this report. AI was not used to analyse the results or write the paper. No part of this report is copied, published elsewhere, or generated by AI.
Author contributions
M.T-K.: Conceptualization, data curation, formal analysis, investigation, project administration, validation, visualization, writing—original draft preparation, writing—review and editing; A.W.: Conceptualization, investigation chromatographic analysis, validation, writing – review & editing; A.S.: Conceptualization, investigation, chromatographic analysis, validation, writing – review & editing; E.F. Conceptualization, investigation, chromatographic analysis, resources, data curation, validation, supervision, writing – review & editing; J.P–T.: Conceptualization, funding acquisition, collection of clinical data and biological samples, resources, data curation, validation, supervision, writing – review & editing.
Funding
None. John Paul II Catholic University of Lublin, Poland has an agreement that cover the article processing charge (APC).
Data availability
The datasets generated during and/or analyzed during the current study are available from the corresponding author on reasonable request.
Declarations
Conflict of interest
The authors declare no conflict of interest.
Footnotes
Publisher's Note
Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
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Associated Data
This section collects any data citations, data availability statements, or supplementary materials included in this article.
Data Availability Statement
The datasets generated during and/or analyzed during the current study are available from the corresponding author on reasonable request.