Microbial Metabolite; Endogenous Metabolite

Phenylacetylglutamine is a microbial metabolite found in the colon that is produced by fermenting amino acids. It is created through the nearly complete microbial conversion of phenylalanine, which leads to the glutamine conjugation of phenylacetic acid [1].

Colonic microbial metabolism substantially contributes to uremic solute production. p-Cresyl sulfate and indoxyl sulfate are the main representatives of solutes of microbial origin and also, protein-bound solutes, exhibiting high protein-binding affinity and dependence on tubular secretion. Phenylacetylglutamine is another microbial metabolite with high dependence on tubular secretion but low protein-binding affinity. The relevance of such solutes is unknown. Therefore, we prospectively followed 488 patients with CKD stages 1-5 and a measurement of serum phenylacetylglutamine by liquid chromatography-mass spectrometry. In a subgroup, we determined 24-hour urinary excretion as a surrogate of intestinal uptake as well as renal clearance of phenylacetylglutamine. We performed outcome analysis for mortality (51 events) and cardiovascular disease (75 events). Serum phenylacetylglutamine level correlated with 24-hour urinary excretion (rho=0.55; P<0.001) and clearance of phenylacetylglutamine (rho=-0.76; P<0.001). Phenylacetylglutamine clearance also correlated with eGFR (rho=0.84; P<0.001). Furthermore, serum phenylacetylglutamine level associated with mortality (hazard ratio per 1-SD increase, 1.77; 95% confidence interval, 1.22 to 2.57; P=0.003) and cardiovascular disease (hazard ratio, 1.79; 95% confidence interval, 1.32 to 2.41; P<0.001) after adjustment for age, sex, presence of diabetes mellitus, prior cardiovascular disease, and eGFR. Thus, serum phenylacetylglutamine level is elevated in patients with more advanced CKD and determined by intestinal uptake and renal clearance, and it is not fully accounted for by differences in eGFR. High serum phenylacetylglutamine level is a strong and independent risk factor for mortality and cardiovascular disease, suggesting the relevance of microbial metabolism and/or tubular dysfunction in CKD, irrespective of protein binding [1].

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Phenylacetylglutamine increases the susceptibility of ventricular arrhythmias in heart failure mice by exacerbated activation of the TLR4/AKT/mTOR signaling pathway. [2]
PAGln (Phenylacetylglutamine) increases the susceptibility to ventricular arrhythmias in heart failure mice. [2]
PAGln (Phenylacetylglutamine) deteriorates cardiac dysfunction in heart failure mice. [2]
PAGln worsens cardiac pathological structure remodeling and cardiac inflammation. [2]
PAGln exacerbates maladaptive cardiac remodeling via enhanced activation of the TLR4/AKT/mTOR signaling pathway.[2]

Biochemical Measurements[1]
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.

Male 8 weeks old C57Bl/6 mice were housed in the laboratory animal center of the cardiovascular research institute of Wuhan University, at 12 h light/dark cycles at 22 ± 2 °C, with free access to food and water. Animal experiments were performed according to the Guide for the Care and Use of Laboratory Animals published by the US National Institute of Health and were approved by the appropriate authorities. The mice were randomly divided into 4 groups (24 per group) for treatment, namely, the Sham group, sham operation without phenylacetylglutamine (PAGln) intervention; Sham +phenylacetylglutamine (PAGln) group, sham operation with phenylacetylglutamine (PAGln) intervention; HF group, TAC surgery without phenylacetylglutamine (PAGln) intervention; and HF + phenylacetylglutamine (PAGln) group, TAC surgery with phenylacetylglutamine (PAGln) intervention. TAC surgery created overpressure-induced HF mice, as described previously. Briefly, after anesthetizing the mice with 3 % pentobarbital sodium at 40 mg/kg, a 27G needle was used to ligate the thoracic aorta. The sham surgery was only operated through thoracotomy without ligation. The mice, after the operation, start to intervene within 24 h through intraperitoneal injectionphenylacetylglutamine (PAGln) solution (100 mg/kg/d) for consecutive 4 weeks. [2]

[1]. Microbiota-Derived Phenylacetylglutamine Associates with Overall Mortality and Cardiovascular Disease in Patients with CKD. J Am Soc Nephrol. 2016 Nov;27(11):3479-3487.
[2]. 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.

N(2)-phenylacetyl-L-glutamine is an a N(2)-phenylacetylglutamine having L-configuration. It has a role as a human metabolite. It is a conjugate acid of a N(2)-phenylacetyl-L-glutaminate.
Phenylacetylglutamine has been reported in Homo sapiens with data available.

C13H16N2O4

264.2771

281.089

C, 59.08; H, 6.10; N, 10.60; O, 24.22

28047-15-6

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

92258

White to off-white solid powder

1.4±0.1 g/cm3

646.6±55.0 °C at 760 mmHg

85-87?C

344.9±31.5 °C

0.0±2.0 mmHg at 25°C

1.595

-1.38

3

4

7

19

338

1

O([H])C([C@]([H])(C([H])([H])C([H])([H])C(N([H])[H])=O)N([H])C(C([H])([H])C1C([H])=C([H])C([H])=C([H])C=1[H])=O)=O

JFLIEFSWGNOPJJ-JTQLQIEISA-N

InChI=1S/C13H16N2O4/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)/t10-/m0/s1

(2-phenylacetyl)-L-glutamine

Phenylacetyl-L-glutamine; NSC 203800; NSC-203800; Phenylacetylglutamine; 28047-15-6; PHENYLAC-GLN-OH; Phenylacetyl L-Glutamine; (S)-5-Amino-5-oxo-2-(2-phenylacetamido)pentanoic acid; Phenylacetyl-L-glutamine; alpha-N-Phenylacetyl-L-glutamine; L-Glutamine, N2-(phenylacetyl)-; NSC203800 PA-L-Glutamine;

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 : ~100 mg/mL (~378.39 mM)
H2O : ~25 mg/mL (~94.60 mM)

Solubility in Formulation 1: ≥ 2.08 mg/mL (7.87 mM) (saturation unknown) in 10% DMSO + 40% PEG300 + 5% Tween80 + 45% Saline (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 20.8 mg/mL clear DMSO stock solution to 400 μL PEG300 and mix evenly; then add 50 μL Tween-80 to the above solution and mix evenly; then add 450 μL normal saline to adjust the volume to 1 mL.
Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH₂ O to obtain a clear solution.

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Solubility in Formulation 2: ≥ 2.08 mg/mL (7.87 mM) (saturation unknown) in 10% DMSO + 90% (20% SBE-β-CD in Saline) (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 20.8 mg/mL clear DMSO stock solution to 900 μL of 20% SBE-β-CD physiological saline solution and mix evenly.
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.

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Solubility in Formulation 3: ≥ 2.08 mg/mL (7.87 mM) (saturation unknown) in 10% DMSO + 90% Corn Oil (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 20.8 mg/mL clear DMSO stock solution to 900 μL of corn oil and mix evenly.

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Solubility in Formulation 4: 50 mg/mL (189.19 mM) in PBS (add these co-solvents sequentially from left to right, and one by one), clear solution; with ultrasonication.

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