Endogenous Metabolite

In cells treated with eicosapentaenoic acid (EPA; 100 μM; 24 hours), the phosphorylated form of C/EBPβ was significantly visible, while it was hardly noticeable in control and OA or LA-treated U937 cells [1]. H-Ras and N-Ras mRNA levels significantly increased after 1, 3, and 24 hours and continued after 1 to 3 hours. Eicosapentaenoic acid has no effect on the levels of K-Ras mRNA [1].

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Previous studies have suggested that dietary alpha-linolenic acid (ALA) increases the levels of omega-3 long-chain polyunsaturated fatty acids (ω-3 LC-PUFAs) in vivo, but the conversion procedure and the genes involved remain poorly understood. In the present work, we designed diets containing various concentrations of ALA and eicosapentaenoic acid (EPA) to feed to mice. Dietary ALA increased the ALA levels in the body in a linear manner and also increased the ω-3 LC-PUFA concentration, but higher ALA intake (above 5%) had no additional effect on ω-3 LC-PUFA levels in vivo. Dietary ALA at a moderate level increased the expression of genes such as Fads1, Fads2, and Elovl5, but higher levels of dietary ALA (above 5%) inhibited their expression in the liver. Further studies demonstrated that the converted EPA could also inhibit the expression of these genes in a concentration-dependent manner, which illustrated that Fads1, Fads2, and Elovl5 were the key genes involved in the conversion of ALA to ω-3 LC-PUFAs. Endogenous ω-3 LC-PUFA biosynthesis from ALA was affected by substrate level, gene expression, and product inhibition [1].

Two hundred forty-one studies were identified, of which 28 met the above inclusion criteria and were therefore included in the subsequent meta-analysis. Using a random effects model, overall standardized mean depression scores were reduced in response to omega3 LC-PUFA supplementation as compared with placebo (standardized mean difference = -0.291, 95% CI = -0.463 to -0.120, z = -3.327, p = 0.001). However, significant heterogeneity and evidence of publication bias were present. Meta-regression studies showed a significant effect of higher levels of baseline depression and lower supplement DHAEPA ratio on therapeutic efficacy. Subgroup analyses showed significant effects for: (1) diagnostic category (bipolar disorder and major depression showing significant improvement with omega3 LC-PUFA supplementation versus mild-to-moderate depression, chronic fatigue and non-clinical populations not showing significant improvement); (2) therapeutic as opposed to preventive intervention; (3) adjunctive treatment as opposed to monotherapy; and (4) supplement type. Symptoms of depression were not significantly reduced in 3 studies using pure DHA (standardized mean difference 0.001, 95% CI -0.330 to 0.332, z = 0.004, p = 0.997) or in 4 studies using supplements containing greater than 50% DHA (standardized mean difference = 0.141, 95% CI = -0.195 to 0.477, z = 0.821, p = 0.417). In contrast, symptoms of depression were significantly reduced in 13 studies using supplements containing greater than 50% EPA (standardized mean difference = -0.446, 95% CI = -0.753 to -0.138, z = -2.843, p = 0.005) and in 8 studies using pure ethyl-EPA (standardized mean difference = -0.396, 95% CI = -0.650 to -0.141, z = -3.051, p = 0.002). However, further meta-regression studies showed significant inverse associations between efficacy and study methodological quality, study sample size, and duration, thus limiting the confidence of these findings. Conclusions: The current meta-analysis provides evidence that EPA may be more efficacious than DHA in treating depression. However, owing to the identified limitations of the included studies, larger, well-designed, randomized controlled trials of sufficient duration are needed to confirm these findings [1].

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Myopia is a leading cause of visual impairment and blindness worldwide. However, a safe and accessible approach for myopia control and prevention is currently unavailable. Here, we investigated the therapeutic effect of dietary supplements of omega-3 polyunsaturated fatty acids (ω-3 PUFAs) on myopia progression in animal models and on decreases in choroidal blood perfusion (ChBP) caused by near work, a risk factor for myopia in young adults. We demonstrated that daily gavage of ω-3 PUFAs (300 mg docosahexaenoic acid [DHA] plus 60 mg eicosapentaenoic acid [EPA]) significantly attenuated the development of form deprivation myopia in guinea pigs and mice, as well as of lens-induced myopia in guinea pigs. Peribulbar injections of DHA also inhibited myopia progression in form-deprived guinea pigs. The suppression of myopia in guinea pigs was accompanied by inhibition of the "ChBP reduction-scleral hypoxia cascade." Additionally, treatment with DHA or EPA antagonized hypoxia-induced myofibroblast transdifferentiation in cultured human scleral fibroblasts. In human subjects, oral administration of ω-3 PUFAs partially alleviated the near-work-induced decreases in ChBP. Therefore, evidence from these animal and human studies suggests ω-3 PUFAs are potential and readily available candidates for myopia control [2].

DNA isolation and quantitative DNA methylation analysis of C/EBPβ and H-Ras CpG islands [4]
Genomic DNA from control U937 cells or U937 grown for 24 hours with 100 µM OA or 100 µM Eicosapentaenoic Acid/EPA was extracted using FlexiGene DNA Kit. EMBOSS and MethPrimer on-line software programs were used to identify potential CpG islands for C/EBPβ, N-Ras, and H-Ras genes. DNA methylation levels were quantified for human C/EBPβ (MePH25981-3A) and H-Ras (MePH14574-1A) using Methyl-Profiler qPCR Primer Assay. qRT-PCR program was performed as indicated in the manual instructions. Analysis of DNA methylation status of CpG islands was carried out using restriction enzyme digestion (DNA Methylation Enzyme kit MeA-03) followed by SYBR Green-based real time PCR detection as previously described. The relative amount of each DNA fraction (methylated and unmethylated) was calculated using ΔCt method.

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 Bisulfite modification of genomic DNA and sequencing [4]
Genomic DNA was obtained from U937 cells, control or grown for 24 h with 100 µM OA or 100 µM Eicosapentaenoic Acid/EPA, using FlexiGene DNA Kit. The bisulfite reaction to determine DNA methylation status was performed as previously described. The DNA fragments covering N-Ras CpG island (−29/+171) and H-Ras CpG island B (640/882) were amplified by PCR using the following primers. N-Ras: for, 5′-AAAGTTTTATTGATTTTTGAGATATTAGTA-3′; rev, 5′-TTTAAACAAATTTAAAACCACAACC-3′. H-Ras: for, 5′-AGTTTTTTGTGGTTGAAAGATGTT-3′; rev, 5′-ACACCCAAATTAAAAACTACTAAATC -3′. The PCR products were cloned into pCR2.1 TOPO and six clones randomly picked from each of two independent PCRs were sequenced using T7 primer at the Genechron-Ylichron Laboratory.

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 Chromatin immunoprecipitation [4]
ChIP assays were performed on U937 cells (about 106), control or grown with 100 µM OA or Eicosapentaenoic Acid/EPA for 24 hours, using the EZ-Chip kit. Cells were cross-linked and cell lysates sonicated until chromatin fragments became 200–1.000 bp in size. Mouse RNAPII 8WG16 monoclonal antibody MMS-126R or rabbit p53 antibody #928 were used for immunoprecipitation. Mouse or rabbit IgG were used as a negative control. After immunoprecipitation, recovered chromatin samples were subject to qRT-PCR with Brilliant SYBR Green qPCR Master Mix. In RNAPII assays the H-Ras sequences amplified were within i) exon 1 (1/+135), ii) intron 1 region C (+136/+639), iii) CpG island B (+640/+882), iv) intron 1 region D (+883/+1167), and v) exon 2 (+1168/+1331). The following primers were used. i) Exon 1: for, 5′-TGCCCTGCGCCCGCAACCCGAG-3′; rev, 5′-CGTTCACAGGCGCGACTGCC-3′. ii) Intron 1 region C: for, 5′-GTGAACGGTGAGTGCGGGCA-3′; rev, 5′-CGCGCCGCGCGTATTGCTGC-3′. iii) CpG island B: for, 5′-CCTGTTCTGGAGGACGGTAA-3′; rev, 5′-GTCGGCAGAAAGGCTAAAGG-3′. iv) Intron 1 region D: for, 5′-TCAGATGGCCCTGCCAGCAG-3′; rev, 5′-TCCTCCTACAGGGTCTCCTG-3′. v) Exon 2: for, 5′CAGGAGACCCTGTAGGAGGA-3′; rev, 5′-GGATCAGCTGGATGGTCAGC-3′. In p53 assays the sequence containing the p53 element of CpG island B was amplified using the following primers: for, 5′-CGCTCAGCAAATACTTGTCGG-3′; rev, 5′-TTACCGTCCTCCAGAACAGG-3′. Data were analyzed quantitatively according to the formula 2−Δ[C(IP)−C(input)]−2−Δ[C(control IgG)−C(input)].

Six-week-old male C57BL/6J mice were used. After one week of acclimatization with free access to standard mouse chow (commercial diet, 17.14% of energy from fat, 5.05 g/100 g) and water, the mice were randomly divided into nine groups each containing six mice and fed ALA series diets (1, 2.5, 5, or 7.5 wt%), 5% ALA and EPA series diets (0.25, 0.5, 1 wt%), EPA diet (2 wt%), or the control diet (Ctl diet: depleted in ω-3 PUFA) for seven weeks. The diet ingredients were shown in ESI Tables S1 and S2.† All animals were maintained in barrier cages and fed with the appropriate special diet restricted to 10 g per mouse per day.[3]

Metabolism / Metabolites
The known human metabolites of eicosapentaenoic acid include juniper acid.

[1]. Martins, J.G., EPA but not DHA appears to be responsible for the efficacy of omega-3 long chain polyunsaturated fatty acid supplementation in depression: evidence from a meta-analysis of randomized controlled trials. J Am Coll Nutr, 2009. 28(5): p. 525-42.

[2]. Dietary ω-3 polyunsaturated fatty acids are protective for myopia. Proc Natl Acad Sci U S A. 2021 Oct 26;118(43):e2104689118.

[3]. Endogenous omega-3 long-chain fatty acid biosynthesis from alpha-linolenic acid is affected by substrate levels, gene expression, and product inhibition. RSC Adv., 2017, 7, 40946-40951.

[4]. Eicosapentaenoic acid activates RAS/ERK/C/EBPβ pathway through H-Ras intron 1 CpG island demethylation in U937 leukemia cells. PLoS One. 2014 Jan 13;9(1):e85025.

All-cis-5,8,11,14,17-icosapentaenoic acid (ACA) is an eicosapentaenoic acid with five cis double bonds at positions 5, 8, 11, 14, and 17. It has multiple functions, including as a nutritional supplement, micronutrient, anti-tumor drug, antidepressant, Daphnia galeata metabolite, mouse metabolite, cholesterol-lowering drug, and fungal metabolite. It is an eicosapentaenoic acid and also an omega-3 fatty acid. It is the conjugate acid of all-cis-5,8,11,14,17-icosapentaenoate. It is an important polyunsaturated fatty acid in fish oil and a precursor to the prostaglandin-3 and thromboxane-3 families. A diet rich in eicosapentaenoic acid (EPA) can lower blood lipid levels, reduce the incidence of cardiovascular disease, prevent platelet aggregation, and inhibit the conversion of arachidonic acid into thromboxane-2 and the prostaglandin-2 family. Eicosapentaenoic acid has been reported to be found in Mortierella alpina, Tornabea scutellifera, and other organisms with relevant data. The free fatty acid (FFA) form of EPA is eicosapentaenoic acid (Icosapent). Eicosapentaenoic acid is a polyunsaturated long-chain fatty acid found in fish oil, with 20 carbon atoms and 5 double bonds, possessing potential supplemental, anti-inflammatory, antithrombotic, immunomodulatory, anti-angiogenic, and chemopreventive activities. After consuming EPA, the free form of eicosapentaenoic acid is incorporated into cell membrane phospholipids and replaces arachidonic acid. It can inhibit the conversion of arachidonic acid into thromboxane and prostaglandin E2 (PGE2). Following oral administration of eicosapentaenoic acid (EPA-FFA), EPA-FFA can prevent and inhibit colon cancer and reduce polyp formation and growth through mechanisms not yet fully elucidated. Eicosapentaenoic acid is an important polyunsaturated fatty acid found in fish oil. It is a precursor to the prostaglandin-3 and thromboxane-3 families. A diet rich in EPA can lower blood lipid levels, reduce the incidence of cardiovascular disease, prevent platelet aggregation, and inhibit the conversion of arachidonic acid to thromboxane-2 and the prostaglandin-2 family. See also: Ethyl eicosapentaenoate (active ingredient); Fish oil (active ingredient). Eicosapentaenoic acid (subclass)...see more...

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Drug Indications
EPA can be used to lower triglyceride levels in patients with hypertriglyceridemia. In addition, EPA can exert a therapeutic effect by reducing the severity of disease in patients with cystic fibrosis and may play a similar role in patients with type 2 diabetes, slowing the progression of diabetic nephropathy.
FDA Label
Treatment of Familial Adenomatous Polyposis

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Mechanism of Action
EPA's anti-inflammatory, antithrombotic, and immunomodulatory effects may be related to its role in the physiology and biochemistry of eicosates. Most eicosates are produced by the metabolism of omega-3 fatty acids, particularly arachidonic acid. These eicosates, such as leukotriene B4 (LTB4) and thromboxane A2 (TXA2), can stimulate leukocyte chemotaxis, platelet aggregation, and vasoconstriction, thus promoting thrombosis and atherosclerosis. On the other hand, EPA is metabolized into leukotriene B5 (LTB5) and thromboxane A3 (TXA3), both of which promote vasodilation, inhibit platelet aggregation and leukocyte chemotaxis, thus having anti-atherosclerotic and antithrombotic effects. EPA's triglyceride-lowering effect stems from its inhibition of lipogenesis and promotion of fatty acid oxidation. Fatty acid oxidation of EPA mainly occurs in mitochondria. EPA is a substrate for prostaglandin intraperoxide synthases 1 and 2. It also appears to affect and bind to carbohydrate response element-binding protein (ChREBP), as well as to a fatty acid receptor (G protein-coupled receptor) called GP40.

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Pharmacodynamics
Eicosate derivatives are chemical messengers derived from 20-carbon polyunsaturated fatty acids and play a crucial role in immune and inflammatory responses. Both 20-carbon omega-6 fatty acids (arachidonic acid) and 20-carbon omega-3 fatty acids (EPA) are present in cell membranes. During inflammatory responses, arachidonic acid and EPA are metabolized by cyclooxygenase and lipoxygenase to produce eicosate derivatives. Increased omega-3 fatty acid intake increases EPA levels in cell membranes and decreases arachidonic acid levels, leading to an increased proportion of EPA-derived eicosate derivatives. The physiological responses induced by arachidonic acid-derived eicosate derivatives differ from those induced by EPA-derived eicosate derivatives. Generally, EPA-derived eicosate derivatives are less effective than arachidonic acid-derived eicosate derivatives in inducing inflammation, vasoconstriction, and coagulation.

C20H30O2

302.451

302.224

10417-94-4

Eicosapentaenoic Acid-d5;1197205-73-4;Eicosapentaenoic acid ethyl ester;86227-47-6;Eicosapentaenoic Acid sodium;73167-03-0

446284

Colorless to light yellow liquid

0.9±0.1 g/cm3

439.3±24.0 °C at 760 mmHg

-54--53ºC(lit.)

336.0±18.0 °C

0.0±2.3 mmHg at 25°C

1.513

6.23

1

2

13

22

398

0

CC/C=C\C/C=C\C/C=C\C/C=C\C/C=C\CCCC(O)=O

JAZBEHYOTPTENJ-JLNKQSITSA-N

InChI=1S/C20H30O2/c1-2-3-4-5-6-7-8-9-10-11-12-13-14-15-16-17-18-19-20(21)22/h3-4,6-7,9-10,12-13,15-16H,2,5,8,11,14,17-19H2,1H3,(H,21,22)/b4-3-,7-6-,10-9-,13-12-,16-15-

(5Z,8Z,11Z,14Z,17Z)-icosa-5,8,11,14,17-pentaenoic acid

EPA. Icosapent, Eicosapentaenoic acid; Timnodonic acid; Icosapent; 10417-94-4; Icosapentaenoic acid; EPA; cis-5,8,11,14,17-Eicosapentaenoic acid; Icosapento; Eicosapentaenoic acid, Timnodonic acid, Vascepa, Epadel, EPAX

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)

Ethanol :≥ 100 mg/mL (~330.63 mM)
DMSO : ≥ 30 mg/mL (~99.19 mM)

Solubility in Formulation 1: ≥ 2.5 mg/mL (8.27 mM) (saturation unknown) in 10% EtOH + 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 25.0 mg/mL clear EtOH stock solution to 900 μL of corn oil and mix evenly.
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 (6.88 mM) in 10% DMSO + 40% PEG300 + 5% Tween80 + 45% Saline (add these co-solvents sequentially from left to right, and one by one), suspension solution; with ultrasonication.
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 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 (6.88 mM) in 10% DMSO + 90% (20% SBE-β-CD in Saline) (add these co-solvents sequentially from left to right, and one by one), suspension solution; with ultrasonication.
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 4: ≥ 2.08 mg/mL (6.88 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 corn oil and mix evenly.

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