cis-Vaccenic acid (CVA) at concentrations of 50 μM, 70 μM, and 100 μM stimulates the production of gamma globin and causes differentiation in K562, JK1, and transgenic mouse erythroid progenitor stem cells[2]. Additionally, the proportion of JK-1 cells positive for benzidine was raised by 50 μM of cis-Vaccenic acid[2]. cis-Vaccenic acid

---

cis-vaccenic acid at 50 μg/ml inactivated 67.9% of T5 phage after 30 min incubation at 37°C under light in Tris-Cu buffer (pH 7.4).[1]
cis-vaccenic acid (50 μM) induced differentiation of K562 cells, with >20% benzidine-positive cells at 48 h post-induction.[2]
cis-vaccenic acid (50 μM) increased γ-globin mRNA levels in JK-1 cells in a time-dependent manner, with significant increase evident at 48 h and peak at 96 h (approximately 8-fold increase relative to control by qRT-PCR). It also up-regulated β-globin gene expression at the same concentration.[2]
cis-vaccenic acid (50 μM) suppressed KLF1 expression in JK-1 cells to approximately 60% of control levels (P<0.05) as measured by qRT-PCR 24 h post-induction.[2]
cis-vaccenic acid (50 μM) increased fetal hemoglobin synthesis in JK-1 cells assessed by alkaline denaturation assay, with significantly higher HbF levels compared to control (P<0.05); Hemin (40 μM) was used as positive control but failed to induce significant HbF in JK-1 cells.[2]
cis-vaccenic acid (50 μM) increased the percentage of benzidine-positive JK-1 cells from approximately 5% (control) to approximately 30% at 72 h.[2]
cis-vaccenic acid (50 μM) induced BFUe colony formation in transgenic mouse bone marrow erythroid progenitor stem cells (TMbmEPSCs), increasing the percentage of BFUe colonies compared to control.[2]
cis-vaccenic acid (50 μM) increased γ-globin mRNA expression in TMbmEPSCs within 24 h of incubation, and elevated levels persisted up to 96 h.[2]
cis-vaccenic acid (50 μM) induced differentiation of TMbmEPSCs as assessed by benzidine-Giemsa staining, showing a higher percentage of reticulocytes at 24 h post-induction.[2]
Pre-differentiation of JK-1 cells with erythropoietin (2 U/ml) for 48 h before cis-vaccenic acid induction enhanced CVA-induced γ-globin gene expression, but simultaneous treatment with EPO and CVA suppressed γ-globin mRNA synthesis compared to CVA alone.[2]
Inhibition of Elovl5 (with 40 μM Cycloate) or Δ9-desaturase (with 10 μM Isoxyl) in JK-1 cells completely suppressed cis-vaccenic acid-induced γ-globin expression; exogenous addition of cis-vaccenic acid (50 μM) 48 h post-inhibition failed to rescue γ-globin expression.[2]
Inhibition of Elovl5 or Δ9-desaturase in TMbmEPSCs also diminished cis-vaccenic acid-induced γ-globin gene expression and BFUe forming capacity, except that Isoxyl treatment enhanced BFUe formation.[2]

Cell Differentiation Assay[2]
Cell Types: K562 cells
Tested Concentrations: 50 μM, 70 μM and 100 μM
Incubation Duration: 48 and 120 hrs (hours)
Experimental Results: Induced differentiation appeared to be concentration dependent in K562 cells with 50 μM CVA being the most effective concentration with more than 20% of the K562 cells showing positive for Benzidine stain after 48 h of incubation with CVA.

---

T5 phage inactivation test: T5 phage was exposed to test compounds in Tris-Cu buffer (pH 7.4) containing 1.21 g Tris-hydroxymethyl-aminomethane, 5.8 g NaCl, 75 mg CaCl2, and 0.1 mg CuSO4 per liter distilled water. Test compounds (final concentration 50 μg/ml) were dissolved in 300 mM NaOH solution and added to the phage suspension to give a final NaOH concentration of 30 mM. Equal volumes of test and control phage were incubated for 30 min at 37°C under light. After treatment, phage titers were determined by the agar layer method in three replicates. Data are expressed as percent plaque forming unit reduction compared to control.[1]
Cell culture and differentiation assay: K562 and JK-1 cells were maintained in RPMI 1640 medium supplemented with 20% fetal bovine serum and penicillin/streptomycin (100 U/ml penicillin, 200 μg/ml streptomycin). Cells were seeded at a density of 1.5×10^4 cells/ml and cultured in a humidified environment at 37°C with 5% CO2. Induction was carried out by adding cis-vaccenic acid at specified concentrations (50, 75, 100 μM) for varying time lengths. Viable cell count was performed using trypan blue staining. Accumulation of hemoglobinized cells was assayed using benzidine staining. Cell morphology was determined using cytospin preparations stained with benzidine-Giemsa and May Grumwald-Giemsa staining.[2]
RNA isolation and quantitative real-time PCR: RNA was isolated from cells using TRIZOL, treated with DNase I, and cleaned using an RNA purification kit. Complementary DNA was synthesized from equal amounts of total RNA with reverse transcription system using a combination of random primers and oligo dT. γ-globin, β-globin, and KLF1 gene expression was quantified using real-time PCR with GAPDH as the normalization control. PCR conditions: denaturation at 95°C for 15 s, annealing and extension at 60°C and 72°C for 1 min respectively for up to 50 cycles.[2]
Fetal hemoglobin assay: Cells were harvested by centrifuging at 450g, washed twice with phosphate-buffered saline (pH 7.2), and lysed using 10% saponin in PBS. The mixture was centrifuged at 300g for 10 min. Adult hemoglobin in the supernatant was denatured using Drabkin's reagent for 30 min and then precipitated out of the solution using saturated ammonium sulfate. Absorbance of fetal hemoglobin was then assayed at 415 nm using a spectrophotometer.[2]
Clonogenic assay (BFUe colony formation): TMbmEPSCs were cultured at a density of 5×10^3 cells in 35 mm petri dishes containing semisolid media: Isocov Modified Dulbecco Medium supplemented with 20% fetal bovine serum, 200 U/ml penicillin, 250 μg/ml streptomycin, 2 mM L-glutamine, and 0.9% methylcellulose at 37°C in 5% CO2 in a humidified environment. Plates were estimated daily for hemoglobinized colonies with large aggregates of 65 or more hemoglobinized cells (BFUe colonies).[2]
Isolation of bone marrow cells: Mouse bone marrow was flushed from the femurs of transgenic mice using 1× PBS. Bone marrow cells were washed twice with 1× PBS. Hematopoietic progenitor stem cells were enriched by plastic adherence and subsequently cultured at a density of 2×10^6 cells/ml in IMDM supplemented with 20% fetal bovine serum, 250 units/ml penicillin, and 200 μg/ml streptomycin in a humidified environment at 37°C with 5% CO2.[2]
Enzyme inhibition studies: To assess the role of fatty acid elongase 5 (Elovl5) and Δ9-desaturase, JK-1 cells and TMbmEPSCs were treated with Cycloate (40 μM) or Isoxyl (10 μM) respectively at the start of culture. cis-vaccenic acid (50 μM) was then added to induce γ-globin expression. In rescue experiments, cis-vaccenic acid (50 μM) was added 48 h post-inhibition, and γ-globin expression or BFUe colony formation was assayed 48 h later.[2]

cis-vaccenic acid at 50 μM did not significantly alter cell viability or proliferation of JK-1 cells as assessed by trypan blue staining over 96 h (cell growth rate comparable to uninduced controls). Higher concentrations of cis-vaccenic acid (above 50 μM) affected JK-1 cell viability significantly (P<0.05).[2]
In K562 cells, sub-toxic concentrations of cis-vaccenic acid (50, 75, 100 μM) were used based on previous studies, with 50 μM being the most effective for differentiation without toxicity.[2]
Copper ion (Cu2+) at the concentration used (0.1 mg/L) in the Tris-Cu buffer showed no toxicity toward the host bacteria (E. coli) in the T5 phage inactivation assay.[1]

[1]. Inactivation of T5 phage by cis-vaccenic acid, an antivirus substance from Rhodopseudomonas capsulata, and by unsaturated fatty acids and related alcohols. FEMS Microbiol Lett. 1991 Jan 1;61(1):13-7.

[2]. Cis-vaccenic acid induces differentiation and up-regulates gamma globin synthesis in K562, JK1 and transgenic mice erythroid progenitor stem cells. Eur J Pharmacol. 2016 Apr 5;776:9-18.

Cis-octadecenoic acid is the cis isomer of octadecenoic acid and is the conjugate acid of cis-octadecenoate (1-). Octadecenoic acid is a metabolite found or produced in Escherichia coli (strains K12 and MG1655). Cis-octadecenoic acid has also been reported in Brasilia brasiliensis, Oscillatoria spp., and other organisms with relevant data. See also: cod liver oil (partial); krill oil (partial); oleic acid (note moved to).

---

cis-vaccenic acid is the predominant antiviral compound from Rhodopseudomonas capsulata, and this was the first study reporting inactivation of a non-enveloped virus (T5 bacteriophage) by unsaturated fatty acids.[1]
cis-vaccenic acid is an 18-carbon n-7 monounsaturated fatty acid biosynthesized in humans by hepatic fatty acid elongase 5 (Elovl5). It is converted to conjugated linoleic acid (9-cis,11-trans-octadecenoic acid) by Δ9-desaturase. cis-vaccenic acid has been shown to regulate the mTORC2-Akt-FOXO1 pathway through control of its synthesis by Elovl5.[2]
cis-vaccenic acid induces fetal hemoglobin (HbF) synthesis and has potential as a pharmacological therapeutic inducer for sickle cell anemia and beta-thalassemia. The gamma globin induction is associated with suppression of KLF1, which in turn down-regulates BCL11A. Inhibition of Elovl5 or Δ9-desaturase abolishes the gamma globin-inducing effects, suggesting that cis-vaccenic acid indirectly modulates γ-globin expression, likely via a downstream metabolite.[2]
cis-vaccenic acid acts preferentially on relatively early erythroid precursors, altering progenitor stem cell globin transcription. It induced differentiation of JK-1 cells to the erythroid lineage as assessed by benzidine-Giemsa staining, and erythropoietin was required for optimal CVA-induced γ-globin induction.[2]

C18H34O2

282.255

506-17-2

trans-Vaccenic acid;693-72-1;cis-Vaccenic acid-d13

5282761

Colorless to light yellow liquid

0.9±0.1 g/cm3

398.2±11.0 °C at 760 mmHg

14-15ºC(lit.)

295.0±14.4 °C

0.0±2.0 mmHg at 25°C

1.467

7.7

1

2

15

20

234

0

CCCCCC/C=C\CCCCCCCCCC(=O)O

UWHZIFQPPBDJPM-FPLPWBNLSA-N

InChI=1S/C18H34O2/c1-2-3-4-5-6-7-8-9-10-11-12-13-14-15-16-17-18(19)20/h7-8H,2-6,9-17H2,1H3,(H,19,20)/b8-7-

(Z)-octadec-11-enoic 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)

DMSO : ≥ 50 mg/mL (177.02 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.

---

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.)