Serum indoleamine 2,3 dioxygenase and tryptophan and kynurenine ratio using the UPLC-MS/MS method, in patients undergoing peritoneal dialysis, hemodialysis, and kidney transplantation

Figures & data

Figure 1. (a) MRM chromatograms and calibration curves of 100 mg mL⁻¹ of tryptophan analyzed using UPLC-ESI-MS/MS. (b) MRM chromatograms and calibration curves of 100 mg mL⁻¹ of kynurenine analyzed using UPLC-ESI-MS/MS.

Table 1. Method parameters for tryptophan and kynurenine using UPLC-ESI-MS/MS.

Analyte	Abb.	Quantification transition (m/z)	Confirmatory transition (m/z)	Cone voltage (V)	Collision energy (V)	Retention time (min)
Tryptophan	Trp	205	188, 146, 118, 91	20	10, 16, 25, 35	4.42
Kynurenine	Kyn	209	192, 146, 118, 91	20	8, 15, 28, 35	3.42

Table 2. Analytical parameters of UPLC-MS/MS method validation.

No.	Compounds	CV%^a	CV%^b	Recovery (%)^b	Stability CV%^c	Accuracy (%)^d	Calibration equations	r^2
1	Kynurenine	0.016	0.112	96.8–99.8	0.71	96.3–99.7	y = 33205.2x + 2985.9	0.999310
2	Tryptophan	0.011	0.082	97.9–99.4	1.48	97.5–99.7	y = 53474.9x + 7118.1	0.998461

^a Intra-day repeatability.

^b Inter-day reproducibility and recovery from once a day of three consecutive days.

^c Stability were measured by standard solutions over three consecutive days.

^d Accuracy represents the ratio of the measured concentration to the theoretical concentration from medium concentration value of the calibration range.

Figure 2. Serum IDO concentrations between the groups.

Figure 3. Serum tryptophan concentrations between the groups.

Figure 4. Serum kynurenine concentrations between the groups.

Figure 5. Serum kynurenine to tryptophan ratio between the groups.

Table 3. Comparison of demographic and characteristics of the patients and healthy controls (mean ± SD).

Parameters	PD patients	KT patients	HD patients	Controls	p Values
Number of patients, n	20	21	19	21	NS
N (male/female)	9/11	13/8	10/9	13/8	NS
Age (years)	51.6 ± 9.69	47.33 ± 9.97	47.47 ± 15.27	48.80 ± 6.63	NS
BMI (kg/m^2)	26.85 ± 5.15	28.33 ± 5.55	25.58 ± 4.44	28.09 ± 4.53	NS
Systolic BP (mmHg)	128.25 ± 21.47	126.66 ± 11.1	122.35 ± 18.21	121.66 ± 13.72	NS
Diastolic BP (mmHg)	80.25 ± 11.97	77.61 ± 7.0	75.88 ± 11.21	77.61 ± 10.44	NS
BUN (mmol/L)	89.83 ± 33.4^a^,^b^,^c	25.25 ± 12.52	62.98 ± 24.18	12.22 ± 2.58	<0.001
Cr (μmol/L)	6.10 ± 2.58^a^,^c	1.55 ± 0.61	7.95 ± 2.60	0.86 ± 0.14	<0.001
Total cholesterol (mg/dL)	196.15 ± 46.44^a	145.94 ± 56.97^c	174.94 ± 52.99^c	202.76 ± 42.87	0.01
LDL-C (mg/dL)	110.85 ± 43.34^a	77.76 ± 42.42^c	110.33 ± 50.45	125.7 ± 36.37	0.01
HDL-C (mg/dL)	46.25 ± 15.49^a^,^b	31.41 ± 8.76^c	36.68 ± 10.32^c	46.61 ± 11.55	0.01
Triglycerid (mg/dL)	195.25 ± 108.3	183.82 ± 125.74	139.63 ± 70.11	162.19 ± 85.24	NS
TAS (μmol Trolox equiv./L)	1.42 ± 0.65^a^,^c	3.21 ± 1.34	2.82 ± 0.74	3.69 ± 2.15	<0.05
TOS (mmol H2O2 equiv./L)	5.63 ± 0.85^a^,^b^,^c	3.82 ± 1.91	4.18 ± 2.26	3.14 ± 0.95	<0.05
IDO (ng/mL)	43.77 ± 26.19^c	31.01 ± 16.36^b^,^c	41.2 ± 34.59^c	25.65 ± 22.03	<0.05
KYN (μmol/L)	5.18 ± 1.4^a^,^b^,^c	3.45 ± 1.15^b	4.06 ± 0.86	2.20 ± 0.72	<0.01
TRP (μmol/L)	25.31 ± 5.49^a^,^c	36.23 ± 5.21^b^,^c	25.96 ± 4.03^c	38.1 ± 4.99	<0.001
KYN/TRP ratio	0.206 ± 0.039^a^,^b^,^c	0.097 ± 0.034^b^,^c	0.159 ± 0.040^c	0.058 ± 0.019	<0.01

^a vs transplantation group.

^b vs HD group.

^c vs control group.

BMI: body mass index; BP: blood pressure; BUN: blood urea nitrogen; Cr: creatinine; LDL-C: low-density lipoprotein cholesterol; HDL-C: high-density lipoprotein cholesterol; TAS: total antioxidant status; TOS: total oxidant status; IDO: indoleamine 2,3-dioxygenase; KYN: kynurenine; TRP: tryptophan; KYN/TRP ratio: kynurenine to tryptophan ratio; NS: nonsignificant.

Table 4. The correlations of BUN and creatinine with gender, IDO, KYN, TRP, KYN/TRP ratio (n = 81).

	BUN		Creatinine
	r	p	r	p
Gender	−0.372	0.001	−0.364	0.001
IDO	0.245	0.027	0.392	<0.001
KYN	0.617	0.000	0.545	<0.001
TRP	−0.656	0.000	−0.680	<0.001
KYN/TRP ratio	0.779	0.000	0.738	<0.001

BUN: blood urea nitrogen; IDO: indoleamine 2,3-dioxygenase; KYN: kynurenine; TRP: tryptophan; KYN/TRP ratio: kynurenine to tryptophan ratio.