Li Wang (Presenter)
BC Children's Hospital
Bio: Li Wang MD, MSc, FRCPC, is a medical biochemist at the Children’s and Women’s Hospital of BC (Vancouver, Canada) and Clinical Assistant Professor of Pathology and Laboratory Medicine at University of British Columbia. Her areas of research are preeclampsia, pediatric toxicology & endocrinology, method development and point of care testing.
Authorship: Li Wang and Andy De Souza
Department of Pathology and Laboratory Medicine, Children's and Women's Health Centre of British Columbia, University of British Columbia, Vancouver, Canada
| Short Abstract Maternal tryptophan and kynurenine pathway metabolites have been found to be associated with the risk of pre-eclampsia. In order to explore the role of these metabolites in predicting pre-eclampsia, we aimed to establish a simple and rapid ultra performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) method to measure tryptophan, kynurenine, and kynurenic acid in both plasma and urine samples. Samples/deuterated internal standards were deproteinized with methanol and injected without derivatization (Acquity Xevo TQ MS, Waters Corporation). Validation of the method was based on Clinical and Laboratory Standards Institute protocol C62-A (Liquid Chromatography-Mass Spectrometry Methods). |
Long Abstract
Introduction:
Pre-eclampsia is a complex multifactorial disease involving genetic (both maternal and fetal) and environmental factors. It is the leading cause of maternal and perinatal mortality and morbidity, and is associated with a wide range of maternal, fetal, and neonatal complications [1]. The precise mechanisms underlying this disorder remain unknown, but disturbed placental function in early pregnancy, characterized by placental hypoxia, secretion of pro-inflammatory cytokines, and the release of angiogenic and antiangiogenic factors are the leading hypotheses [1,2].
Tryptophan is an essential amino acid for protein synthesis [3,4], whose major catabolic route is the kynurenine pathway. Tryptophan is converted to kynurenine by indoleamine 2,3-dioxygenase (IDO) [3], a rate-limiting enzyme widely distributed in peripheral tissues including placenta [5]. Kynurenine can then be further metabolized to kynurenic acid (KYNA) [3,4]. Tryptophan and its metabolites are of interest because they have been implicated in various pathological conditions associated with altered immune response [6,7]. Pregnancy is considered an inflammatory process and IDO has been shown to be involved in pre-eclampsia as both an immune modulator and an antioxidant [8,9]. Previous research has shown that IDO mRNA expression and IDO enzyme activity in the placenta were low in pre-eclamptic women near term (~35 weeks' gestation), and maternal kynurenine/tryptophan ratio, which reflects IDO activity, was decreased [8]. The placental IDO enzyme activity levels also inversely correlate with the severity of the symptoms of pre-eclampsia [9]. A recent study done earlier in pregnancy (~18 weeks' gestation) showed that elevated maternal plasma KYNA concentrations are associated with a substantially increased risk of pre-eclampsia in obese women; however, a decreased kynurenine/tryptophan ratio was not observed in this population. The differences in findings among these studies could be due to the gestational week at blood sampling, and warrant further investigation [10].
To address these aspects, we aimed to develop a simple and rapid method to measure tryptophan, kynurenine, and KYNA in both plasma and urine samples, after which we will measure these metabolites in normal pregnancy and pre-eclampsia as a pilot case control study.
Methods:
Plasma sample or urine sample with 1:5 dilution (total 60 µL) was mixed (1:2) with methanol (containing the internal standards: Tryptophan-d5 25 µM, KYNA-d5 0.1 µM and Kynurenine-d6 0.1 µM), vortexed, and incubated at -20˚C for 30 min to induce precipitation. The mixture was centrifuged at 13000 g for 10 min. Ten µL of sample was injected in the UPLC-MS/MS (Acquity Xevo TQ MS, Waters Corporation).
UPLC (Waters Acquity UPLC HSS T3 column) with initial conditions of 70:30 (0.12% formic acid in H2O:100% methanol) and a linear gradient to 30:70 in 2.75 min at 250 µL/min was performed. Total run time was 7 minutes. Electrospray ionization in positive and negative mode was utilized, and data collected in multiple reaction monitoring mode.
Results:
1. Linearity: Calibrations were performed using standards as shown below. Nine triplicates were used to establish the linear calibration equation. The corresponding correlation coefficients (R2) are 0.9901 for tryptophan, 0.9976 for kynurenine and 0.9899 for KYNA. The regression line equation for each analyte was: tryptophan (y=0.0097x+0.0022), kynurenine (y=0.4624x-0.0003) and KYNA (y=0.1310x+0.0006).
Calibrator concentrations (µM):
Tryptophan: 0 25 50 75 100 125 150 175 200
Kynurenine: 0 0.5 1 1.5 2 2.5 3 3.5 4
KYNA: 0 0.05 0.1 0.15 0.2 0.25 0.3 0.35 0.4
2. Limit of Quantitation: The limit of quantitation was determined to be for 1 µmol/L, 50 nmol/L and 20 nmol/L for tryptophan, kynurenine and KYNA, respectively.
3. Precision: Intra- and inter-day precision for tryptophan, kynurenine and KYNA were evaluated according to the requirements of CLSI guidelines on bioanalytical method validation. Intra-day variation was assessed by six replicate determinations of three concentrations (QC1, QC2, and QC3) over the tested range. Throughout these concentration ranges, the mean intra-day assay precision was below 10%. The inter-day precision of the method was determined by analyzing replicates of these QC samples for 20 days, and the mean inter-day precision was below 15%.
Conclusions:
In the present study, a simple and rapid UPLC-MS/MS method for the determination of tryptophan and its metabolites (kynurenine and KYNA) in plasma and urine samples has been developed and validated. This method is suitable for research uses that include appropriate control subjects.
References & Acknowledgements:
1. Keszthelyi D, Troost FJ, Masclee AA. Understanding the role of tryptophan and serotonin metabolism in gastrointestinal function. Neurogastroenterol Motil. 2009 Dec; 21(12):1239-49.
2. Schwarcz R, Bruno JP, Muchowski PJ, Wu HQ. Kynurenines in the mammalian brain: when physiology meets pathology. Nat Rev Neurosci. 2012 Jul; 13(7):465-77.
3. Yamazaki F, Kuroiwa T, Takikawa O, Kido R. Human indolylamine 2,3-dioxygenase. Its tissue distribution, and characterization of the placental enzyme. Biochem J. 1985 Sep; 230(3):635-8.
4. Steegers EA, von Dadelszen P, Duvekot JJ, Pijnenborg R. Pre-eclampsia. Lancet. 2010 Aug; 376(9741):631-44.
5. Laresgoiti-Servitje E. A leading role for the immune system in the pathophysiology of preeclampsia. J Leukoc Biol. 2013 Aug; 94(2):247-57.
6. Schröcksnadel K, Wirleitner B, Winkler C, Fuchs D. Monitoring tryptophan metabolism in chronic immune activation. Clin Chim Acta. 2006 Feb; 364(1-2):82-90.
7. Wang Y, Liu H, McKenzie G, Witting PK, Stasch JP, Hahn M, et al. Kynurenine is an endothelium-derived relaxing factor produced during inflammation. Nat Med. 2010 Mar; 16(3):279-85.
8. Kudo Y, Boyd CA, Sargent IL, Redman CW. Decreased tryptophan catabolism by placental indoleamine 2,3-dioxygenase in preeclampsia. Am J Obstet Gynecol. 2003 Mar; 188(3):719-26.
9. Nishizawa H, Suzuki M, Pryor-Koishi K, Sekiya T, Tada S, Kurahashi H, Udagawa Y. Impact of indoleamine 2,3-dioxygenase on the antioxidant system in the placentas of severely pre-eclamptic patients. Syst Biol Reprod Med. 2011 Aug; 57(4):174-8.
10.Nilsen RM, Bjørke-Monsen AL, Midttun O, Nygård O, Pedersen ER, Ulvik A, et al. Maternal tryptophan and kynurenine pathway metabolites and risk of preeclampsia. Obstet Gynecol. 2012 Jun; 119(6):1243-50.
Acknowledgements: Thank you to Dr. Graham Sinclair for his kind technical support on tandam mass spectrometry. Thank you to all of my collaborators:Drs. K.S Joseph, Laura Magee, Peter von Dadelszen and Amy Metcalfe for their encouragement and support.
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