Intestinal microbial metabolite

Intestinal microbial metabolites are a risk factor for cardiovascular diseases, and phenylacetylglutamine (PAGln) is a newly discovered intestinal metabolite in the latest study. In addition, elevated plasma PAGln concentration was associated with increased mortality and hospitalization rates in patients with heart failure (HF). However, the mechanism of PAGln leading to increased HF mortality is unclear. The present study was performed to investigate whether the PAGln deteriorated the susceptibility of ventricular arrhythmias (VAs) in the setting of HF [1].

PAGln increased the susceptibility of VAs in HF mice by activating the TLR4/AKT/mTOR signaling pathway. PAGln promoted the activation of cardiac inflammation and fibrosis and deteriorated cardiac function in HF mice. Moreover, PAGln extended APD90, shortened the ERP/APD90 and increased the incidence of VAs following HF in isolated heart perfusion. Mechanistically, PAGln significantly enhanced the activation of the TLR4/AKT/mTOR signaling pathway in vivo and in vitro [1].

Biochemical Measurements[2] At inclusion, blood was taken by venous puncture for measurement of hemoglobin (grams per deciliter), albumin (grams per liter), C-reactive protein (milligrams per liter), cholesterol (milligrams per deciliter), calcium (milligrams per deciliter), phosphate (milligrams per deciliter), biointact parathyroid hormone (nanograms per liter), and creatinine (milligrams per deciliter), all measured using standard laboratory techniques. The eGFR was calculated using the CKD-EPI equation. We also had ancillary data available on free serum levels of p-cresyl sulfate determined as p-cresol with a dedicated gas chromatography-mass spectrometry method, allowing comparison with a protein-bound solute. Additionally, serum levels of Phenylacetylglutamine (PAG) were quantified by ultraperformance liquid chromatography-tandem mass spectrometry. For sample preparation, 50 μl serum or urine, 50 μl solution of milli-Q (MQ) water:MeOH:0.01 N sodium hydroxide (75:20:5 vol/vol/vol), 20 μl internal standard mixture (Phenylacetylglutamine (PAG)-d5), and 150 μl acetonitrile were thoroughly mixed in 96-well Ostro Plates (Waters). After separation by a positive pressure manifold, supernatants were collected in 2-ml collection plates. Subsequently, the organic phase was removed by a gentle stream of nitrogen for 30 minutes at 40°C. After dilution with 1000 μl MQ water, 5 μl final solution was injected on the ultraperformance liquid chromatography-tandem mass spectrometry system. Chromatographic separation was performed on an Acquity CSHFluoroPhenyl Column (50×2.5 mm; 1.7-μm particle size; Waters). The mobile phase, delivered at a flow rate of 0.5 ml/min at 40°C, consisted of a gradient of 0.1% formic acid in MQ water (A) and MeOH (B). The gradient was as follows: starting with 3% B, there was a subsequent increase to 16% B within 1 minute followed by an increase to 80% B within 3 minutes and thereafter, an increase to 95% B within 30 seconds for a duration of 1 minute, after which the initial 3% B was reintroduced with equilibration for a duration of 3.5 minutes before the next injection. Ionization of Phenylacetylglutamine (PAG) and the corresponding isotopologue (internal standard) was achieved in negative mode. The following multiple reaction monitoring transitions were used for quantification: Phenylacetylglutamine (PAG) 263→145 and Phenylacetylglutamine (PAG)-d5 268→145. Limit of detection and limit of quantification (LOQ) were 0.06 and 0.18 μM for Phenylacetylglutamine (PAG). For analysis, solute levels below the LOQ were treated as the average value of the limit of detection and the LOQ. The total, within–run, between–run, and between–day method imprecisions according to the National Committee for Clinical Laboratory Standards EP5-T guideline were 3.92%, 1.61%, 2.69%, and 2.02%, respectively, and the mean recovery was 97%. We also sampled 24-hour urinary collections when available at the time of inclusion to calculate renal clearance and 24-hour urinary excretion of Phenylacetylglutamine (PAG). Collections were considered complete when 24-hour urinary creatinine excretion was within 2 SDs (range =0.7–1.8 g) of the mean creatinine excretion for the geographic region of this study derived from the INTERSALT Study. Assuming steady-state conditions and negligible nonrenal clearance, 24-hour urinary excretion of Phenylacetylglutamine (PAG) was considered an indirect estimate of 24-hour intestinal uptake of Phenylacetylglutamine (PAG). Furthermore, protein intake was calculated according to the formula by Maroni et al. using 24-hour urinary urea nitrogen excretion and body weight.

H9C2 cell culture and Ang II-induced hypertrophy[2] H9C2 cell were plated in 6 well plates at a 1 × 106 /ml density and cultured in Dulbecco's Modified Eagle Medium, supplemented with penicillin, streptomycin, and 10 % FBS. Ang II was dissolved in PBS and stimulation of Ang II at the concentration of 1 μM induced H9C2 hypertrophy. CCK-8 kits detected H9C2 cell viability at variable concentrations of 0, 25,50,100,200,400 μM Phenylacetylglutamine (PAGIn). In addition, TAK-242, a TLR4 inhibitor, was dissolved in 10 % dimethyl sulfoxide (DMSO) and added to cells at the concentration of 10 μM and Phenylacetylglutamine (PAGIn) at the concentration of 100 μM intervened H9C2 cell. Subsequent experiments were carried out 24 h later.

Thoracic aortic coarctation (TAC) was used to construct an animal model of HF in mice. Intraperitoneal injection of PAGln for 4 weeks intervened in HF mice. The concentration of PAGln was quantitatively determined by liquid chromatography-tandem mass spectrometry. Cardiac function was assessed by echocardiography; assessment of cardiac electrophysiological indexes was measured by electrocardiogram (ECG) and programmed electrical stimulation in isolated cardiac perfusion. Masson was stained for collagen deposition, and wheat germ agglutinin (WGA) was stained for the cross-sectional area of the myocytes. The qRT-PCR and Western Blotting were used to determine target gene expression in vivo and in vitro [1].

[1]. Phenylacetylglutamine increases the susceptibility of ventricular arrhythmias in heart failure mice by exacerbated activation of the TLR4/AKT/mTOR signaling pathway. Int Immunopharmacol. 2023 Mar:116:109795.
[2]. Microbiota-Derived Phenylacetylglutamine Associates with Overall Mortality and Cardiovascular Disease in Patients with CKD. J Am Soc Nephrol. 2016 Nov;27(11):3479-3487.

C14H17NO4.NA+

286.27888

286.106

C, 54.55; H, 5.28; N, 9.79; Na, 8.03; O, 22.36

104771-87-1

28047-15-6 (free acid); 104771-87-1 (sodium)

23663405

Solid powder

565.5ºC at 760mmHg

295.8ºC

1.26E-13mmHg at 25°C

2.304

2

4

7

20

343

1

C1=CC=C(C=C1)CC(=O)N[C@@H](CCC(=O)N)C(=O)[O-].[Na+]

JABIYIZXMBIFLT-PPHPATTJSA-M

InChI=1S/C13H16N2O4.Na/c14-11(16)7-6-10(13(18)19)15-12(17)8-9-4-2-1-3-5-9;/h1-5,10H,6-8H2,(H2,14,16)(H,15,17)(H,18,19);/q;+1/p-1/t10-;/m0./s1

sodium (2-phenylacetyl)-L-glutaminate

sodium phenylacetyl glutamine; 104771-87-1; Antineoplaston AS2-5; Antineoplaston AS 2-5; Sodium (S)-5-amino-5-oxo-2-(2-phenylacetamido)pentanoate; sodium phenylacetylglutaminate; I5X366518D; sodium (2-phenylacetyl)-L-glutaminate; Antineoplaston AS 2-5

2934.99.03.00

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

DMSO: > 10 mM

Note: Listed below are some common formulations that may be used to formulate products with low water solubility (e.g. < 1 mg/mL), you may test these formulations using a minute amount of products to avoid loss of samples.

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Injection Formulation 1: DMSO : Tween 80： Saline = 10 : 5 : 85 (i.e. 100 μL DMSO stock solution → 50 μL Tween 80 → 850 μL Saline)
*Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH ₂ O to obtain a clear solution.
Injection Formulation 2: DMSO : PEG300 ：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL DMSO → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline)
Injection Formulation 3: DMSO : Corn oil = 10 : 90 (i.e. 100 μL DMSO → 900 μL Corn oil)
Example: Take the Injection Formulation 3 (DMSO : Corn oil = 10 : 90) as an example, if 1 mL of 2.5 mg/mL working solution is to be prepared, you can take 100 μL 25 mg/mL DMSO stock solution and add to 900 μL corn oil, mix well to obtain a clear or suspension solution (2.5 mg/mL, ready for use in animals).

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Injection Formulation 4: DMSO : 20% SBE-β-CD in saline = 10 : 90 [i.e. 100 μL DMSO → 900 μL (20% SBE-β-CD in saline)]
*Preparation of 20% SBE-β-CD in Saline (4°C，1 week): Dissolve 2 g SBE-β-CD in 10 mL saline to obtain a clear solution.
Injection Formulation 5: 2-Hydroxypropyl-β-cyclodextrin : Saline = 50 : 50 (i.e. 500 μL 2-Hydroxypropyl-β-cyclodextrin → 500 μL Saline)
Injection Formulation 6: DMSO : PEG300 : castor oil : Saline = 5 : 10 : 20 : 65 (i.e. 50 μL DMSO → 100 μLPEG300 → 200 μL castor oil → 650 μL Saline)
Injection Formulation 7: Ethanol : Cremophor : Saline = 10： 10 : 80 (i.e. 100 μL Ethanol → 100 μL Cremophor → 800 μL Saline)
Injection Formulation 8: Dissolve in Cremophor/Ethanol (50 : 50), then diluted by Saline
Injection Formulation 9: EtOH : Corn oil = 10 : 90 (i.e. 100 μL EtOH → 900 μL Corn oil)
Injection Formulation 10: EtOH : PEG300：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL EtOH → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline)

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Oral Formulation 1: Suspend in 0.5% CMC Na (carboxymethylcellulose sodium)
Oral Formulation 2: Suspend in 0.5% Carboxymethyl cellulose
Example: Take the Oral Formulation 1 (Suspend in 0.5% CMC Na) as an example, if 100 mL of 2.5 mg/mL working solution is to be prepared, you can first prepare 0.5% CMC Na solution by measuring 0.5 g CMC Na and dissolve it in 100 mL ddH2O to obtain a clear solution; then add 250 mg of the product to 100 mL 0.5% CMC Na solution, to make the suspension solution (2.5 mg/mL, ready for use in animals).

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Oral Formulation 3: Dissolved in PEG400
Oral Formulation 4: Suspend in 0.2% Carboxymethyl cellulose
Oral Formulation 5: Dissolve in 0.25% Tween 80 and 0.5% Carboxymethyl cellulose
Oral Formulation 6: Mixing with food powders

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Note: Please be aware that the above formulations are for reference only. InvivoChem strongly recommends customers to read literature methods/protocols carefully before determining which formulation you should use for in vivo studies, as different compounds have different solubility properties and have to be formulated differently.

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 (Please use freshly prepared in vivo formulations for optimal results.)