ALDH2; Human Endogenous Metabolite
4-Hydroxynonenal targets multiple cellular components through its electrophilic nature. It is a potent inhibitor of acetaldehyde dehydrogenase 2 (ALDH2). The compound forms covalent adducts with proteins, nucleic acids, and membrane lipids, modifying their function. It regulates various signaling pathways and plays a role in cancer through mitochondrial mechanisms. As a uremic toxin, it is actively transported into kidneys via organic ion transporters (especially OAT3).

In addition to being an inhibitor of ALDH2, 4-hydroxynonenal is also a substrate for ALDH2; at low concentrations, the inhibition of ALDH2 by 4-hydroxynonenal is reversible, but becomes irreversible above 10 μM. 4-To control its own synthesis and improve cellular defenses against oxidative stress, 4-hydroxynonenal can trigger antioxidant defense mechanisms[1]. 4-The byproduct of lipid peroxidation, 4-hydroxynonenal, is genotoxic and mutagenic to bacteria, viruses, and mammalian cells. All four DNA bases are reacted with, but to varying degrees of efficiency: G > C > A > T. The most reliable biomarker of 4-Hydroxynonenal's genotoxic effects is 4-Hydroxynonenal-dG, and these adducts are mostly identified in nucleus DNA. The p53 mutation caused by 4-hydroxynonenal-dG is a well-known illustration of the etiological significance of 4-hydroxynonenal-dG in human malignancies. 4-Hydroxynonenal-dG adducts were shown to form preferentially at codon 249's third base in the p53 gene. This resulted in gene mutation and altered a number of biological processes, such as differentiation, apoptosis, cell cycle arrest, and DNA repair[1].
In vitro, 4-Hydroxynonenal functions as a potent inhibitor of ALDH2. It modulates many signaling processes through the formation of covalent adducts with nucleophilic functional groups. The compound plays an important role in cancer through mitochondrial mechanisms. It is widely used as a biomarker of lipid peroxidation and oxidative/nitrosative stress. Its effects are concentration-dependent, with different biological outcomes at low versus high concentrations.

The expression levels of NADPH oxidase 1 (NOX1), inducible nitric oxide synthase (iNOS), and 4-Hydroxynonenal (4-HNE) are measured in mouse brain tissue 24 hours after fluid percussion injury (FPI). Both wild-type (Nrf2+/+) and Nrf2-deficient (Nrf2-/-) mice exhibit increased expression of 4-Hydroxynonenal following 15 psi injury (moderate injury) in comparison to uninjured Nrf2+/+ and Nrf2-/- mice. Comparing Nrf2-/-KO mice to correspondingly damaged and uninjured Nrf2+/+ WT animals, the expression level of 4-hydroxynonenal is much higher in these animals, in line with the iNOS result[2].
In vivo, 4-Hydroxynonenal is involved in various pathological conditions. As a uremic toxin, it can cause uremic syndrome, which may affect any part of the body and can cause nausea, vomiting, loss of appetite, and weight loss. It is actively transported into the kidneys via organic ion transporters (especially OAT3). The compound plays a role in oxidative stress-related diseases and cancer. Its levels in tissues and body fluids serve as biomarkers of oxidative stress.

The cell-free assay for 4-Hydroxynonenal typically involves measuring its inhibitory activity against purified ALDH2. Enzyme activity is monitored spectrophotometrically by measuring NADH production at 340 nm. Inhibition is assessed by incubating ALDH2 with varying concentrations of 4-Hydroxynonenal and measuring residual activity. The compound can also be detected and quantified using HPLC or mass spectrometry. Adduct formation with proteins can be assessed using immunoblotting with specific antibodies against 4-HNE-protein adducts.

Our preliminary work has revealed that vitamin D receptor (VDR) activation is protective against cisplatin induced acute kidney injury (AKI). Ferroptosis was recently reported to be involved in AKI. Here in this study, we investigated the internal relation between ferroptosis and the protective effect of VDR in cisplatin induced AKI. By using ferroptosis inhibitor ferrostatin-1 and measurement of ferroptotic cell death phenotype in both in vivo and in vitro cisplatin induced AKI model, we observed the decreased blood urea nitrogen, creatinine, and tissue injury by ferrostatin-1, hence validated the essential involvement of ferroptosis in cisplatin induced AKI. VDR agonist paricalcitol could both functionally and histologically attenuate cisplatin induced AKI by decreasing lipid peroxidation (featured phenotype of ferroptosis), biomarker 4-hydroxynonenal (4HNE), and malondialdehyde (MDA), while reversing glutathione peroxidase 4 (GPX4, key regulator of ferroptosis) downregulation. VDR knockout mouse exhibited much more ferroptotic cell death and worsen kidney injury than wild type mice. And VDR deficiency remarkably decreased the expression of GPX4 under cisplatin stress in both in vivo and in vitro, further luciferase reporter gene assay showed that GPX4 were target gene of transcription factor VDR. In addition, in vitro study showed that GPX4 inhibition by siRNA largely abolished the protective effect of paricalcitol against cisplatin induced tubular cell injury. Besides, pretreatment of paricalcitol could also alleviated Erastin (an inducer of ferroptosis) induced cell death in HK-2 cell. These data suggested that ferroptosis plays an important role in cisplatin induced AKI. VDR activation can protect against cisplatin induced renal injury by inhibiting ferroptosis partly via trans-regulation of GPX4[3].
For in vitro cellular assays, cells are treated with 4-Hydroxynonenal at various concentrations (typically 1-100 μM) for defined periods. Oxidative stress markers are measured using fluorescent probes. Protein carbonylation and 4-HNE adduct formation are assessed by immunoblotting. Cell viability is evaluated using MTT or LDH release assays. Mitochondrial function is assessed using JC-1 staining or Seahorse assays. Signaling pathway activation is evaluated by western blotting using phospho-specific antibodies.

Increased methylglyoxal (MG) formation is associated with diabetes and its complications. In zebrafish, knockout of the main MG detoxifying system Glyoxalase 1, led to limited MG elevation but significantly elevated aldehyde dehydrogenases (ALDH) activity and aldh3a1 expression, suggesting the compensatory role of Aldh3a1 in diabetes. To evaluate the function of Aldh3a1 in glucose homeostasis and diabetes, aldh3a1-/- zebrafish mutants were generated using CRISPR-Cas9. Vasculature and pancreas morphology were analysed by zebrafish transgenic reporter lines. Corresponding reactive carbonyl species (RCS), glucose, transcriptome and metabolomics screenings were performed and ALDH activity was measured for further verification. Aldh3a1-/- zebrafish larvae displayed retinal vasodilatory alterations, impaired glucose homeostasis, which can be aggravated via pdx1 silencing induced hyperglycaemia. Unexpectedly, MG was not altered, but 4-hydroxynonenal (4-HNE), another prominent lipid peroxidation RCS exhibited high affinity with Aldh3a1, was increased in aldh3a1 mutants. 4-HNE was responsible for the retinal phenotype via pancreas disruption induced hyperglycaemia and can be rescued via l-Carnosine treatment. Furthermore, in type 2 diabetic patients, serum 4-HNE was increased and correlated with disease progression. Thus, our data suggest impaired 4-HNE detoxification and elevated 4-HNE concentration as biomarkers but also the possible inducers for diabetes, from genetic susceptibility to the pathological progression[4].
In vivo animal studies for 4-Hydroxynonenal typically involve models of oxidative stress, inflammation, or cancer. The compound can be administered via various routes including intravenous or intraperitoneal injection. Tissue levels of 4-HNE are measured using HPLC or mass spectrometry. Biomarkers of oxidative stress and inflammation are assessed in blood and tissues. The compound's effects on disease progression (tumor growth, organ damage) are monitored.

Metabolism / Metabolites
Uremic toxins often accumulate in the blood due to overeating or poor kidney filtration. Most uremic toxins are metabolic waste products that are normally excreted through urine or feces.
Pharmacokinetic properties of 4-Hydroxynonenal include a molecular weight of 156.22 g/mol and molecular formula C9H16O2. As a reactive aldehyde, it has a short half-life in biological systems due to rapid metabolism and adduct formation. It is transported into kidneys via organic ion transporters (OAT3). The compound is typically handled as a solution in organic solvents and stored at low temperatures. Its reactive nature requires careful handling and storage to prevent degradation.

Toxicity Summary
Uremic toxins, such as 4-hydroxynonenal, 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 the KLOTHO protein. 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 reduced due to elevated local ROS levels (A7869).
The toxicity profile of 4-Hydroxynonenal is related to its ability to form covalent adducts with cellular macromolecules. As a uremic toxin, it contributes to the pathology of uremic syndrome. High concentrations can cause cytotoxicity through oxidative stress and mitochondrial dysfunction. The compound is implicated in various diseases including cancer, neurodegenerative disorders, and cardiovascular disease. Standard safety precautions should be followed when handling this compound.

[1]. Zhong H, et al. Role of lipid peroxidation derived 4-hydroxynonenal (4-HNE) in cancer: focusing on mitochondria. Redox Biol. 2015;4:193-9.
[2]. Csala M, et al. On the role of 4-hydroxynonenal in health and disease. Biochim Biophys Acta. 2015 May;1852(5):826-38.
[3]. Bhowmick S, et al. Traumatic brain injury-induced downregulation of Nrf2 activates inflammatory response and apoptotic cell death. J Mol Med (Berl). 2019 Nov 22.
[3]. Cell Death Dis. 2020 Jan 29;11(1):73. doi: 10.1038/s41419-020-2256-z.
[4]. Redox Biol. 2020 Oct;37:101723. doi: 10.1016/j.redox.2020.101723.

4-Hydroxynonenal is an enal composed of a non-2-ene molecule with a carbonyl group at position 1 and a hydroxyl group at position 4. It is a human metabolite. It is both a hydroxy aldehyde and an enal, and specifically 4-hydroxynonenal. 4-Hydroxynonenal is a uremic toxin. Based on 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 and/or protein-bound compounds, such as phenols; and 3) larger so-called medium molecules, such as β2-microglobulin. Long-term exposure to uremic toxins can lead to various diseases, including kidney damage, chronic kidney disease, and cardiovascular disease. 4-Hydroxynonenal (HNE) is one of the major end products of lipid peroxidation and has been shown to participate in signal transduction; current evidence suggests that it can affect cell cycle events in a concentration-dependent manner. Glutathione S-transferases (GSTs) regulate HNE production during lipid peroxidation by reducing hydrogen peroxide and converting it into glutathione conjugates, thereby affecting intracellular HNE concentration. Overexpression of α-GSTs in cells leads to decreased HNE homeostasis, and these cells are resistant to apoptosis induced by lipid peroxidation inducers such as H₂O₂, UVA, superoxide anion, and pro-oxidoxogens, suggesting that the apoptotic signaling of these inducers is transduced through HNE. Cells that can more rapidly expel HNE from the intracellular environment are relatively more resistant to H₂O₂, UVA, superoxide anion, pro-oxidoxogens, and HNE-induced apoptosis, suggesting that HNE may be a common factor in the mechanism of oxidative stress-induced apoptosis. Transfection of adherent cells with HNE-metabolized GSTs leads to transformation of these cells due to HNE depletion. (A3295)
4-Hydroxynonenal (4-HNE) is an α,β-unsaturated hydroxyalkenal that serves as a marker of lipid peroxidation and oxidative/nitrosative stress. It is a potent inhibitor and substrate of ALDH2. 4-HNE modulates signaling processes through covalent adduct formation with proteins, nucleic acids, and membrane lipids. It plays an important role in cancer through mitochondrial mechanisms and is recognized as a uremic toxin. This compound is widely used in oxidative stress research and as a biomarker.

C₉H₁₆O₂

156.22

156.115

C, 69.19; H, 10.32; O, 20.48

75899-68-2

4-Hydroxynonenal-d3;148706-06-3

5283344

Colorless to light yellow liquid

0.9±0.1 g/cm3

275.6±23.0 °C at 760 mmHg

115.2±15.2 °C

0.0±1.3 mmHg at 25°C

1.460

1.85

1

2

6

11

119

0

CCCCCC(/C=C/C=O)O

JVJFIQYAHPMBBX-FNORWQNLSA-N

InChI=1S/C9H16O2/c1-2-3-4-6-9(11)7-5-8-10/h5,7-9,11H,2-4,6H2,1H3/b7-5+

4-hydroxy-2E-nonenal

4 Hydroxynonenal; HNE;4-Hydroxynonenal; 4-Hydroxy-2-nonenal; 75899-68-2; 4-HNE; 4-hydroxynon-2-enal; (E)-4-hydroxynon-2-enal; trans-4-Hydroxy-2-nonenal; 4-Hydroxy-2,3-nonenal;

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Note: Please store this product in a sealed and protected environment (e.g. under nitrogen), avoid exposure to moisture and light.

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 (~640.12 mM)

Solubility in Formulation 1: ≥ 2.5 mg/mL (16.00 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 25.0 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 2: ≥ 2.08 mg/mL (13.31 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 3: ≥ 2.08 mg/mL (13.31 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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 (Please use freshly prepared in vivo formulations for optimal results.)