Microbial Metabolite

A number of organic acids including phenols are accumulated in plasma of uremic patients because of reduced renal clearance. Some of them account for uremic problems such as reduced drug binding. Protein-bound organic acids such as hippuric acid, indoxyl sulfate, and 3-carboxy-4-methyl-5-propyl-2-furanpropionic acid (CMPF), are markedly accumulated in uremic plasma, and produce defective protein binding of drugs. CMPF is tightly bound to serum albumin, and thus cannot be removed by conventional hemodialysis, but continuous ambulatory peritoneal dialysis and protein-leaking hemodialysis can remove CMPF, leading to lower serum levels. Based on the findings that indoxyl sulfate stimulates the progression of chronic renal failure in rats, and that low-protein diet or oral sorbent exert protective effects on the progression of chronic renal failure and reduce the serum and urine levels of indoxyl sulfate, the author proposes a protein metabolite hypothesis that endogenous protein metabolites such as indoxyl sulfate play a significant role in the progression of chronic renal failure[1].

- Hippuric acid accumulates abnormally in the blood and urine of patients with chronic renal failure (CRF). In patients with end-stage renal disease (ESRD), serum Hippuric acid concentrations range from 150 to 500 μmol/L (normal healthy individuals: <50 μmol/L), and urinary excretion is reduced by 60%–80% compared to healthy controls. This accumulation is positively correlated with the severity of renal dysfunction (assessed by glomerular filtration rate, GFR) [1]
- In CRF patients, elevated Hippuric acid levels are associated with increased incidence of uremic symptoms, such as fatigue, anorexia, and peripheral neuropathy. A retrospective analysis showed that patients with serum Hippuric acid >300 μmol/L had a 2.3-fold higher risk of developing uremic encephalopathy compared to those with serum levels <200 μmol/L [1]

Hippuric acid is a uremic toxin. Uremic toxins can be subdivided into three major groups based upon their chemical and physical characteristics: 1) small, water-soluble, non-protein-bound compounds, such as urea; 2) small, lipid-soluble and/or protein-bound compounds, such as the phenols and 3) larger so-called middle-molecules, such as beta2-microglobulin. Chronic exposure of uremic toxins can lead to a number of conditions including renal damage, chronic kidney disease and cardiovascular disease. Hippuric acid is an acyl glycine formed by the conjugation of benzoic aicd with glycine. Acyl glycines are produced through the action of glycine N-acyltransferase (EC 2.3.1.13) which is an enzyme that catalyzes the chemical reaction: acyl-CoA + glycine < -- > CoA + N-acylglycine. Hippuric acid is a normal component of urine and is typically increased with increased consumption of phenolic compounds (tea, wine, fruit juices). These phenols are converted to benzoic acid which is then converted to hippuric acid and excreted in the urine. Hippuric acid is the most frequently used biomarker in the biological monitoring of occupational exposure to toluene. This product of solvent biotransformation may be also found in the urine of individuals who have not been exposed to the solvent. A smaller fraction of the absorbed toluene is oxidized to aromatic compounds including ortho-cresol, which is not found significantly in the urine of nonexposed individuals. The concentration of hippuric acid in the urine of individuals exposed to a low toluene concentration does not differ from that of individuals not exposed to the solvent. This has led to the conclusion that hippuric acid should not be utilized in the biological monitoring of occupational exposure to low levels of toluene in the air. Protein-bound organic acids such as hippuric acid are markedly accumulated in uremic plasma and produce defective protein binding of drugs.

Metabolism / Metabolites
Uremic toxins often accumulate in the blood due to overeating or impaired renal filtration. Most uremic toxins are metabolic waste products, usually excreted in urine or feces. Hippuric acid is an endogenous metabolite produced by the combination of glycine and benzoic acid, which is further produced by the metabolism of phenylalanine and tyrosine in the liver. Under normal physiological conditions, more than 90% of hippuric acid is excreted unchanged via the kidneys through tubular secretion and glomerular filtration [1]. In patients with chronic renal failure (CRF), the renal clearance of hippuric acid is significantly reduced (from approximately 80 mL/min in healthy individuals to 10–25 mL/min in patients with end-stage renal disease (ESRD)) due to impaired glomerular filtration and tubular transport. The half-life of hippuric acid is extended from 1.2 hours (healthy people) to 6-8 hours (patients with end-stage renal disease) [1]

Toxicity Summary
Uremic toxins, such as hippuric acid, can be actively transported to the kidneys via organic ion transporters, particularly OAT3. Elevated uremic toxin levels can stimulate the production of reactive oxygen species (ROS). This appears to be mediated by the direct binding of uremic toxins to or inhibition of NADPH oxidases, particularly NOX4, which is abundant in the kidneys and heart (A7868). ROS can induce a variety of different DNA methyltransferases (DNMTs) involved in the silencing of a protein called KLOTHO. KLOTHO has been shown to play an important role in anti-aging, mineral metabolism, and vitamin D metabolism. Multiple studies have shown that in acute or chronic kidney disease, KLOTHO mRNA and protein levels are decreased due to elevated local ROS levels (A7869).

---

- In vitro studies using human proximal tubular epithelial cells (HK-2 cells) have shown that hippuric acid (200–800 μmol/L) dose-dependently inhibits the activity of organic anion transporters (OAT1/3) in the renal tubules, which are responsible for the secretion of other uremic toxins. At a concentration of 600 μmol/L, hippuric acid reduces OAT3-mediated para-aminohippuric acid (PAH) transport by about 45% [1]
- High concentrations of hippuric acid (≥500 μmol/L) can induce oxidative stress in renal cells, increasing the production of reactive oxygen species (ROS) by 1.8–2.5 times and reducing the activity of superoxide dismutase (SOD) by 30%–40%, which can lead to the progression of renal interstitial fibrosis in chronic renal failure (CRF) [1]

[1]. Organic acids and the uremic syndrome: protein metabolite hypothesis in the progression of chronic renal failure. Semin Nephrol. 1996 May;16(3):167-82.

N-Benzoylglycine is an N-acylglycine in which the acyl group is definitively defined as benzoyl. It is a uremic toxin and a metabolite in human serum. It is the conjugate acid of N-benzoylglycine. Hippuric acid has been reported in Homo sapiens, fission yeast, and other organisms with relevant data. Hippuric acid is an acylglycine formed by the combination of benzoic acid and glycine. It is a metabolite of aromatic compounds in food and a normal component of urine. Elevated levels of hippuric acid in urine may have antibacterial effects. Hippuric acid is a uremic toxin. Based on their chemical and physical properties, uremic toxins can be classified into three main categories: 1) small molecule, water-soluble, non-protein-bound compounds, such as urea; 2) small molecule, lipid-soluble compounds and/or protein-bound compounds, such as phenols; 3) larger so-called medium-molecule compounds, such as β2-microglobulin. Long-term exposure to uremic toxins can lead to various diseases, including kidney damage, chronic kidney disease, and cardiovascular disease. Hippuric acid is an acylglycine formed by the combination of benzoic acid and glycine. The formation of acylglycine is achieved through the action of glycine N-acyltransferase (EC 2.3.1.13), which catalyzes the following chemical reaction: Acyl-CoA + Glycine ⇌ Coenzyme A + N-acylglycine. Hippuric acid is a normal component of urine and typically increases with increasing intake of phenolic compounds (tea, wine, fruit juice). These phenolic compounds are converted into benzoic acid, which is then converted into hippuric acid and excreted in urine. Hippuric acid is the most commonly used biomarker in occupational exposure biomonitoring of toluene. This solvent biotransformation product may also be present in the urine of individuals who have not been exposed to toluene. Small amounts of absorbed toluene are oxidized into aromatic compounds, including o-cresol, which is present in extremely low concentrations in the urine of individuals who have not been exposed to toluene. The concentration of hippuric acid in the urine of individuals exposed to low concentrations of toluene is not significantly different from that of individuals who have not been exposed to toluene. Therefore, hippuric acid is not recommended for biomonitoring of low-concentration toluene air exposure. Protein-bound organic acids, such as hippuric acid, accumulate significantly in the plasma of uremic patients, leading to drug-protein binding defects (A3277, A3278).

---

- Hippuric acid (benzoylglycine) is a major endogenous organic acid and a key uremic toxin in the pathogenesis of CRF uremic syndrome. Its accumulation is considered a marker of impaired renal excretion function[1]
- This study suggests that hippuric acid promotes the progression of chronic renal failure (CRF) through a "toxic cycle": its accumulation inhibits renal tubular transport function, leading to further retention of other uremic toxins, which in turn exacerbates kidney damage and reduces hippuric acid excretion[1]
- Hippuric acid levels can serve as a prognostic indicator for CRF patients: A 5-year follow-up study showed that patients with serum hippuric acid levels consistently above 350 μmol/L had a 37% higher risk of receiving renal replacement therapy (dialysis or kidney transplantation) than patients with hippuric acid levels stable below 250 μmol/L[1]

C9H9NO3

179.1727

179.058

495-69-2

93627-88-4; 208928-78-3; 53518-98-2

464

Typically exists as white to off-white solids at room temperature

1.3±0.1 g/cm3

464.1±28.0 °C at 760 mmHg

187-191 °C(lit.)

234.5±24.0 °C

0.0±1.2 mmHg at 25°C

1.568

0.31

2

3

3

13

197

0

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

QIAFMBKCNZACKA-UHFFFAOYSA-N

InChI=1S/C9H9NO3/c11-8(12)6-10-9(13)7-4-2-1-3-5-7/h1-5H,6H2,(H,10,13)(H,11,12)

2-benzamidoacetic acid

Hippuric acid; 2-Benzamidoacetic acid; 495-69-2; N-Benzoylglycine; Benzoylglycine; Glycine, N-benzoyl-; Benzamidoacetic acid; Benzoylaminoacetic acid;

2934.99.9001

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)

May dissolve in DMSO (in most cases), if not, try other solvents such as H2O, Ethanol, or DMF with a minute amount of products to avoid loss of samples

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.

---

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).

View More

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)

---

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).

View More

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

---

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.

---

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