Quantitation of Gut Microbiota-Derived Indole

Application Note Academia

Quantitation of Gut Microbiota‑Derived Indole Metabolites by Ion Pairing dMRM LC/QQQ

Authors Abstract Jiye Cheng1,2, Carolina B. Livi3, and Siddarth Venkatesh1,2 The surge of interest in the role of the human gut microbiota in health and disease research has created a demand for quantifying community-derived metabolites 1 Edison Family Center for representing diverse chemical classes. Agilent has developed a highly reproducible Genome Sciences and and robust ion-pair reversed-phase (IP-RP) dynamic multiple reaction monitoring Systems Biology (dMRM) method that provides efficient separation of over 200 central carbon 2 Center for Gut Microbiome metabolites. This Application Note focuses on extending the IP-RP dMRM method to and Nutrition Research, the measurement of tryptophan and indole metabolites that have been associated Washington University in with effects on host metabolic and immunologic pathways. The extended IP-RP St. Louis, St. Louis, MO dMRM method enables sensitive detection of these compounds across a wide 63108 USA dynamic range in relevant biological samples. 3 Agilent Technologies, Inc. Santa Clara, 95051 USA Introduction Some indole metabolites are Experimental endogenous ligands of the aryl The gut microbiota, composed of tens hydrocarbon receptor (AhR), which Sample preparation of trillions of microbes belonging to all mediates anti‑inflammatory and Indole-3-acetamide, indole‑3‑acetic three domains of life, but dominated anti‑microbial effects through effects acid, indole-3-butyric acid, by members of bacteria, produces on intra-epithelial γδ T cells, regulatory indole‑3‑carboxaldehyde, many classes of metabolites that have T cells, and cytokine production, indole‑3‑carboxylic acid, indole‑3‑lactic wide‑ranging effects on host biology.1–4 including IL-22.9,10 As a case in point, acid, indole‑3-propionic acid, The indole catabolites of the essential microglial cells use tryptophan‑derived indole‑3‑pyruvic acid, indoxyl sulfate, amino acid tryptophan constitute an indoxyl-3-sulfate in an Ahr-dependent tryptophol, kynurenic acid, kynurenine, exemplary class of microbial metabolites manner to ameliorate the symptoms quinolinic acid, xanthurenic acid, and that confer beneficial effects upon of experimental autoimmune 5-hydroxytryptophan were purchased host physiology. encephalomyelitis.11 A recent study from Sigma-Aldrich and prepared at a demonstrated that Lactobacillus reuteri Dietary tryptophan has multiple fates concentration of 1 mg/mL in methanol. is a producer of indole-3-lactic acid, when ingested: Standards were combined into three which activates AhR, and leads to mixtures where each mixture contained • Incorporation into peptides and reprogramming of CD4+ intraepithelial no compounds whose molecular weights proteins lymphocytes into CD8aα cells.12 were within two daltons of each other. • Catabolism by the host to kynurenine Interestingly, patients with Crohn’s The final concentration of each standard and serotonin through the action of disease and ulcerative colitis also have in the mixture was 100 µg/mL mixture. the rate-limiting enzymes tryptophan reduced fecal levels of tryptophan and 2,3-dioxygenase and tryptophan indole-3-acetic acid, and increased Instrumentation 13,14 hydroxylase, respectively levels of kynurenine. Tryptamine • Agilent 1290 Infinity II LC activates the epithelial GPCR 5-HT4 • Transformation by members of the • Agilent 6470A triple quadrupole receptor and increases colonic anion gut microbiota to various catabolites, LC/MS including tryptamine, indole‑3- flux and secretion in a cAMP-dependent 15 MS parameters, chromatographic acetic acid, indole‑3‑propionic acid, manner. conditions, and gradient were taken indole‑3‑lactic acid, and indole5,6 While our understanding of gut from a metabolomics dMRM method microbiota-derived metabolites The latter metabolite is further sulfated (described in the Agilent Quick Start is accruing, assays for accurate in the liver to indoxyl sulfate to enhance Guide, “MassHunter Metabolomics quantification of metabolites across its eventual clearance. Dynamic MRM Database and Method”, diverse chemical classes are not widely Indole metabolites modulate host G6412-90006 Revision A, 5991‑6467EN) available. Recently, Agilent developed biology though diverse mechanisms an IP-RP dMRM method that Adding new compounds to the of action. For example, Clostridium provides efficient separation of over Agilent metabolomics dMRM method sporogenes metabolizes tryptophan 200 metabolites representing diverse to indole-3-propionic acid, which is an Agilent's dynamic multiple reaction chemical classes, many a part of central endogenous ligand for the pregnane X monitoring (dMRM) uses retention carbon metabolism across organisms. receptor (PXR), which promotes time scheduling of MRM transitions to This Application Note describes steps gut barrier integrity by inducing the allow more metabolites to be analyzed for extending the IP-RP dMRM method expression of tight junction proteins such in a single run without sacrificing to cover a panel of indole metabolites as Occludin, Zonulin-1, E-cadherin, and data quality. Therefore, to add new of general interest, including a Claudin-7.7 Similarly, indole acrylic acid compounds to the metabolomics dMRM microbial‑derived subset. produced by some Peptostreptococcus method, the retention time of new species has anti-inflammatory effects; compounds must first be determined, treatment of human peripheral blood then compound-specific acquisition mononuclear cells with this compound parameters can be optimized. leads to reduced IL-6 and IL-1 secretion in response to LPS stimulation.8

2 • Step 1. To enable us to find the Results and discussion reproducibility (RSD <5% from triplicate retention time of the new indole determinations) of both retention time metabolites to be added to the The microbiota-derived indole and peak area response including at the dMRM method, we modified the metabolites examined were well resolved limit of quantitation, which was below dMRM method to a scan method. using this modified IP-RP dMRM 100 nM for all metabolites tested. • Step 2. We optimized the MRM method (Figure 1). There was excellent transition, fragmentor, and collision energy parameters of the Table 1. Tryptophan and indole compounds added to dMRM method.

indole metabolites using Agilent Compound Retention Time Fragmentor MRM Collision Energy (V) MassHunter Optimizer. We used Indole-3-acetamide 12.1 110 173.1 & 130.0 17 Optimizer’s injection mode for all Indole-3-acetic acid 16.8 70 174.1 & 130.0 5 new compounds. Indole-3-butyric acid 17.9 140 202.1 & 158.1 17

• Step 3. When Optimizer was finished Indole-3-carboxaldehyde 14.9 140 144.0 & 115.0 33

optimizing the MRM acquisition Indole-3-carboxylic acid 15.3 100 160.0 & 116.0 17

parameters, we imported the Indole-3-lactic acid 16.5 140 204.1 & 158.0 17

retention time, MRM transitions, Indole-3-propionic acid 16.7 130 188.1 & 59.1 13

fragmentor, and collision energy Indole-3-pyruvic acid 17.3 90 202.1 & 174.0 5

parameters into the original Indoxyl sulphate 16.4 100 212.0 & 80.0 25

metabolomics dMRM method, and Tryptophol 15.9 80 160.1 & 116.0 17 saved it to a new file name. Kynurenic acid 15.5 80 188.0 & 144.0 13

4 ×10 Indole-3-carboxaldehyde 2.0 Indole-3-acetamide 1.8 Indole-3-carboxylic acid 1.6

1.4

Indole-3-acetic acid 1.2 Indole-3-propionic acid 1.0 Counts Indole-3-lactic acid 0.8 Indoxyl Indole-3-pyruvic acid sulfate Indole-3-butyric acid 0.6

0.4

0.2 Tryptophol

0 10 11 12 13 14 15 16 17 18 19

Figure 1. Composite dMRM chromatogram of microbial-derived indole metabolites.

3 The calibration curves for all the ×103 Kynurenic acid ×103 Indole-3-lactic acid 500 y = 53.532x – 5905.8 14 y = 1.4821x + 192.03 standards showed excellent linearity R2 = 0.9968 R2 = 0.9996 400 12 (R2 >0.999) and wide dynamic range 10 300 8 (>three orders of magnitude). Figure 2 200 6 Response Response 4 shows examples of calibration curves for 100 2 six representative metabolites. 0 0 0 2,000 4,000 6,000 8,000 10,000 0 2,000 4,000 6,000 8,000 10,000 Concentration (ng/mL) Concentration (ng/mL) Conclusion ×103 ×103 Indoxyl sulphate 25 Indole-3-butyric acid This Application Note describes a 20 y = 2.0275x + 226.37 y = 2.7781x + 272.26 20 R2 = 0.999 R2 = 1 modification to extend an existing 15 IP‑RP dMRM metabolomics method 15 10 to detect and measure a wider panel 10 Response 5 Response of important microbial metabolites. 5 0 0 Reliable quantitation of the added 0 2,000 4,000 6,000 8,000 10,000 0 2,000 4,000 6,000 8,000 10,000 tryptophan and indole metabolites was Concentration (ng/mL) Concentration (ng/mL) achieved using the modified Agilent ×103 ×103 IP-RP dMRM method. The analytical 18 Indole-3-propionic acid 8 Tryptophol 16 y = 1.9236x + 273.64 7 y = 0.4824x – 77.271 sensitivity, linearity, and dynamic range 14 R2 = 0.9998 6 R2 = 0.9997 12 5 of measuring tryptophan and indole 10 4 metabolites show that this method is 8 6 3 applicable for microbiome applications in Response 4 Response 2 2 1 biological samples. 0 0 0 2,000 4,000 6,000 8,000 10,000 0 5,000 10,000 15,000 20,000 See the following recently published Concentration (ng/mL) Concentration (ng/mL) study that uses this method to measure Figure 2. Calibration curves of for selected compounds. the levels of indole metabolites in biologic samples: Gehrig, J. L. et al. Effects of Microbiota‑Directed Foods in Gnotobiotic Animals and Undernourished Children. Science 2019, 365, eaau4732.

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