Endogenous metabolite

The newly defined eicosatetraenoates (ETEs), 5-oxoETE and 5-oxo-15(OH)-ETE, share structural motifs, synthetic origins, and bioactions with leukotriene B4 (LTB4). All three eicosanoids stimulate Ca2+ transients and chemotaxis in human neutrophils (PMN). However, unlike LTB4, 5-oxoETE and 5-oxo-15(OH)-ETE alone cause little degranulation and no superoxide anion production. However, we show herein that, in PMN pretreated with granulocyte-macrophage or granulocyte colony-stimulating factor (GM-CSF or G-CSF), the oxoETEs become potent activators of the last responses. The oxoETEs also induce translocation of secretory vesicles from the cytosol to the plasmalemma, an effect not requiring cytokine priming. To study the mechanism of PMN activation in response to the eicosanoids, we examined the activation of mitogen-activated protein kinase (MAPK) and cytosolic phospholipase A2 (cPLA2). PMN expressed three proteins (40, 42, and 44 kDa) that reacted with anti-MAPK antibodies. The oxoETEs, LTB4, GM-CSF, and G-CSF all stimulated PMN to activate the MAPKs and cPLA2, as defined by shifts in these proteins' electrophoretic mobility and tyrosine phosphorylation of the MAPKs. However, the speed and duration of the MAPK response varied markedly depending on the stimulus. 5-OxoETE caused a very rapid and transient activation of MAPK. In contrast, the response to the cytokines was rather slow and persistent. PMN pretreated with GM-CSF demonstrated a dramatic increase in the extent of MAPK tyrosine phosphorylation and electrophoretic mobility shift in response to 5-oxoETE. Similarly, 5-oxoETE induced PMN to release some preincorporated [14C]arachidonic acid, while GM-CSF greatly enhanced the extent of this release. Thus, the synergism exhibited by these agents is prominent at the level of MAPK stimulation and phospholipid deacylation. Pertussis toxin, but not Ca2+ depletion, inhibited MAPK responses to 5-oxoETE and LTB4, indicating that responses to both agents are coupled through G proteins but not dependent upon Ca2+ transients. 15-OxoETE and 15(OH)-ETE were inactive while 5-oxo-15(OH)-ETE and 5(OH)-ETE had 3- and 10-fold less potency than 5-oxoETE, indicating a rather strict structural specificity for the 5-keto group. LY 255283, a LTB4 antagonist, blocked the responses to LTB4 but not to 5-oxoETE. Therefore, the oxoETEs do not appear to operate through the LTB4 receptor. In summary, the oxoETEs are potent activators of PMN that share some but not all activities with LTB4. The response to the oxoETEs is greatly enhanced by pretreatment with cytokines, indicating that combinations of these mediators may be very important in the pathogenesis of inflammation [1].

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Degranulation and O−2 Production [1]
GM-CSF, when incubated with PMN at optimal levels (200 pM, 20 min), increased the degranulating potencies of FMLP, PAF, and LTB4 up to 10-fold. It had even greater effects on 5-oxoETE, 5-oxo-15(OH)-ETE, and 5-HETE, converting these otherwise weak agents to powerful degranulators with respective potencies (LTB4 = 100) of 20, 10, and 2 (Fig. 1, A and B). Similar results occurred with oxidative metabolism. 5-OxoETE and LTB4 caused GM-CSF-treated PMN to produce O−2 (Fig. 1C) but only LTB4 stimulated this response in unprimed cells (data not shown). Hence, 5-oxo/OH-ETEs approach LTB4 in potency on GM-CSF-primed PMN yet, unlike LTB4, have little activity on unprimed cells. This suggests that 5-oxo/OH-ETEs do not utilize LTB4 receptors and is supported by studies using a LTB4 receptor antagonist. PMN incubated with 200 pM GM-CSF for 18 min and 0 or 1 µM LY 255283 for 2 min released, respectively, 12 ± 3% (mean net percentage of total cell enzyme released, ± S.E.; n = 6) or 1 ± 2% lysozyme in response to 3 nM LTB4 and 12 ± 2% or 11 ± 2% in response to 16 nM 5-oxoETE. 15-OxoETE and 15-HETE were inactive with or without GM-CSF. The priming response to GM-CSF required 7.5 min to develop, peaked at 20 min, and increased with increasing concentrations of GM-CSF between 2 and 200 pM. G-CSF primed PMN degranulation responses to FMLP, LTB4, PAF, 5-oxoETE, 5-oxo-15(OH)-ETE, and 5-HETE (Fig. 1D). Priming in response to G-CSF was more rapid (optimal within 5 min) but led to somewhat less prominent responses than priming by GM-CSF. Thus, 5-oxo/OH-ETEs powerfully stimulate PMN primed by hematopoietic cytokines and act through a structurally specific and LTB4 receptor-independent mechanism.

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Secretory Vesicle Mobilization [1]
In gradients of disrupted PMN, surface alkaline phosphatase localized to fractions 4-6 while latent alkaline phosphatase distributed bimodally to fractions 5-7 and 10-13. PMN exposed to 500 nM 5-oxoETE had increased exocytosis of secretory vesicles, as evidenced by raised surface and lowered latent alkaline phosphatase activity (Fig. 2). GM-CSF (200 pM), G-CSF (10 nM), and LTB4 (10 nM), but not 500 nM 15-OxoETE, had these same actions. Hence, 5-oxoETE mimics FMLP, PAF, and LTB4 in translocating secretory vesicles to the cell surface. We also examined unfractionated cavitates. In agreement with fractionation studies, cavitates from PMN exposed to GM-CSF, G-CSF, LTB4, 5-oxoETE, or 5-HETE had low latent (Fig. 3), elevated surface, and unchanged total alkaline phosphatase activities (data not shown). The responses to these stimuli were concentration dependent with ED50 values of 0.02, 1, 4, 20, and 200 nM, respectively. Again, 15-OxoETE (Fig. 3) and 15-HETE (data not shown) were inactive and LY 255283 blocked LTB4 but not 5-oxoETE or 5-HETE (Table I). We conclude that 5-oxo/OH-ETEs mobilize secretory vesicles by a structurally specific and LTB4 receptor-independent mechanism. Priming is not needed for this effect.

Arachidonic Acid Release [1]
PMN (2 × 107/ml) in Ca2+-free Hanks' buffer were incubated with 300,000 dpm/ml [14C]arachidonic acid for 30 min (37°C), washed in Ca2+-free Hanks buffer (2.5 mg/ml BSA) and then Ca2+-free Hanks' buffer, and resuspended in Hanks' buffer (1.4 mM Ca2+). Suspensions (500 µl) were incubated (37°C) for 15 min, treated with 0-2000 pM GM-CSF for 20 min, stimulated for 0.25 min, and treated with 10 mg/ml BSA (final concentration) to remove free arachidonic acid from cells. After 4.75 min, suspensions were centrifuged (12,000 × g; 5 s; 20°C) to isolate 400 µl of supernatant fluid. Pellets plus the lower 100 µl of supernatants were treated with 100 µl of 0.1% Triton X-100; reaction tubes were washed with 100 µl of 0.1% Triton X-100, and suspensions and washing fluids were pooled. The latter pools as well as the upper supernatants were separately counted for radioactivity. Results are expressed as the percentage of total cellular radiolabel released by stimulated cells minus that released by cells that were treated identically (including GM-CSF exposure) but incubated with BSA instead of 5-oxoETE.

Cells and Bioassays [1]
We isolated PMN (>95% PMN, < 5 platelets/100 PMN, no red blood cells) from normal human blood. To assay degranulation, 1.3 × 106 PMN in 0.5 ml of Hanks' buffer were incubated at 37°C for 20 min, exposed to BSA ± cytokine for 0-40 min, treated with 5 µg/ml cytochalasin B for 3 min, challenged for 5 min, placed on ice, and centrifuged (12,000 × g; 0.2 min; 4°C) to obtain supernatants that were assayed for LDH, lysozyme, and β-glucuronidase. Results are reported as net enzyme release, i.e. the percentage of total cell enzyme released by stimulated PMN minus that released by unstimulated but otherwise identically treated (including cytokine exposure) PMN. None of the stimuli or reagents used here caused net release of cytosolic LDH. Hence, observed enzyme release reflected degranulation, not cell lysis. To assay O−2 production, 1 × 107 PMN in 1 ml of Hanks' buffer containing 50 nM cytochrome c were incubated at 37°C for 20 min, exposed to GM-CSF for 17 min and 5 µg cytochalasin B for 3 min, and stimulated while being monitored (550 nm) with a split-beam spectrophotometer. Data were corrected for the responses of PMN stimulated in the presence of 50 µg/ml superoxide dismutase and reported as superoxide dismutase-inhibitable changes in optical density. We assayed secretory vesicle mobilization by the method of Borregaard et al. 2-4 × 108 PMN in 5 ml of Hanks' buffer were challenged at 37°C for 20 min, transferred to 4°C cavitation buffer (5 ml), and further processed at 4°C. Cells were subjected to N2 cavitation, freed of nuclei by low speed centrifugation, normalized for protein and LDH, and centrifuged through Percoll discontinuous gradients to obtain 19 fractions that were enriched with markers for cytosol (LDH, fractions 1-3), endoplasmic reticulum (NADPH-dependent cytochrome c reductase, fractions 1-7), plasmalemma (surface alkaline phosphatase and [14C]concanavalin A-labeled cell surface glycoprotein, fractions 4-6), secretory vesicles (total minus surface alkaline phosphatase, peaks at fractions 5-7 and 10-13), light Golgi (UDP-[3H]galactose:N-acetylglucosamine galactosyltransferase, fractions 4-6), secondary granules (vitamin B12-binding protein, fractions 12-14), heavy Golgi (UDP-[3H]galactose:N-acetylglucosamine galactosyltransferase, fractions 12-14), and primary granules (β-glucuronidase, fractions 16-18), as detailed elsewhere. Alkaline phosphatase was assayed by incubating (30 min; 37°C) 60 µl of a Percoll gradient fraction in 640 µl of a pH 10 solution of 50 mM 2-amino-2-methylpropanol, 0.4% Triton X-100, 14 mM MgCl2, and 1.6 mg of p-nitrophenol phosphate. Reactions were centrifuged (12,000 × g; for 5 min; 20°C) to remove Percoll, supernatants were quantitated for p-nitrophenol by measuring absorbance at 410 nm, and data were converted to rates of p-nitrophenol formed (nmol/30 min). Surface alkaline phosphatase was assayed identically except the reaction mixtures did not contain Triton X-100. Latent alkaline phosphatase is the total minus surface alkaline phosphatase. During secretory vesicle mobilization, latent alkaline phosphatase falls while surface alkaline phosphatase rises. To assay secretory vesicle mobilization more easily, 5 × 107 PMN in 2.5 ml of Hanks' buffer were incubated with stimuli at 37°C for 20 min, resuspended in 4°C cavitation buffer, subjected to N2 cavitation, and centrifuged (200 × g; 4 min; 4°C) to remove nuclear debris. Postnuclear supernatants were assayed for total, surface, and latent alkaline phosphatase activity. Results are given as the percentage of the total alkaline phosphatase activity that is latent.

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Gel Electrophoresis and Immunoblotting [1]
PMN, 5 × 106, were incubated in Hanks' buffer (0.5 ml) at 37°C for 20 min, stimulated, centrifuged (12,000 × g; for 10 s; 20°C), and suspended in 0.25 ml of buffer (50 mM dithiothreitol, 25 mMβ-mercaptoethanol, 10 mM Tris, 1 mM EDTA, 2 mM diisopropyl fluorophosphate, 25 µg/ml pepstatin A, 40 µg/ml leupeptin, 1 mM phenylmethylsulfonyl fluoride, 25 mM NaF, 200 µM NH3VO4, and 2% SDS, pH 7.5). The samples were immediately placed in a boiling water bath for 5 min before storing at −20°C. For MAPK, 50-µl samples (equal to 1 × 106 PMN) were resolved by SDS-polyacrylamide (12%) gel electrophoresis (60 mAmp, run 5-6 h to 14 cm) and electrotransferred (18 h, 10 mAmp) to nitrocellulose membranes. Membranes were blocked for 1.5 h with 5% Carnation nonfat milk, reacted for 1.5 h with 1 µg/ml anti-ERK-1 antibody, washed five times (10 mM Tris and 0.1% Tween, 100 mM NaCl, pH 7.5), incubated with horseradish peroxidase-linked anti-IgG antibody, and analyzed by ECL. For tyrosine-phosphorylated MAPK, samples (4 × 106 cells in 50 µl) were resolved by SDS-polyacrylamide (11%) gel electrophoresis (50 mAmp, run 8 h) and electrotransferred (6 h, 25 mV) to polyvinyl difluoride membranes. The membranes were prepared, blocked, and treated with anti-ERK-1-Y(PO4) and alkaline phosphatase-linked anti-rabbit IgG as recommended by the supplier of the PhosphoPlus antibody kits. For cPLA2, samples (1 × 106 cell eq) were resolved by SDS-polyacrylamide (7.5%) gel electrophoresis (22 mAmp, run 5 h to 14 cm) and transferred (60 mAmp, 12 h) to nitrocellulose membranes. Membranes were blocked with 5% milk, washed three times in 0.2% Tween in 50 mM phosphate-buffered normal saline, blotted with anti-cPLA2 antibody, incubated with horseradish peroxidase-linked anti-rabbit IgG for 2 h, and analyzed by ECL.

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PMN Ca2+ Depletion and Pertussis Toxin Treatment [1]
PMN, 6 × 106/ml, were incubated at 37°C for 90 min in Ca2+-free Hanks' buffer containing 1 µM EGTA and 1 µM Fura-2 AM, washed twice, incubated (6 × 106 cells/ml) in Hanks' buffer containing 0 or 1.4 mM Ca2+ at 37°C for 20 min and then challenged. These Ca2+-depleted PMN, if incubated with no Ca2+, fail to alter cytosolic Ca2+ upon challenge by Ca2+-mobilizing agonists, but if incubated with 1.4 mM Ca2+, mount full Ca2+ transient responses. For pertussis toxin studies, PMN (1.8 × 107/ml) in Ca2+-free buffer were incubated with 4 µg/ml pertussis toxin or BSA at 37°C for 120 min, treated with 1.4 mM Ca2+ for 20 min, and challenged for 1-5 min. These PMN have 60% lower Ca2+ transient and G protein activation responses to 5-HETE, perhaps because of the extensive incubation period. Following challenge, PMN suspensions were centrifuged (12,000 × g; for 10 s; 20°C) and processed as described for MAPK.

[1]. 5-Oxo-eicosanoids and hematopoietic cytokines cooperate in stimulating neutrophil function and the mitogen-activated protein kinase pathway. J. Biol. Chem. 1996, 271(30), 17821-17828.

15-OxoETE is an oxoicosatetraenoic acid having (5Z,8Z,11Z,13E) double bond stereochemistry, and an oxo group in position 15. It has a role as a human metabolite. It is functionally related to an icosa-5,8,11,13-tetraenoic acid. It is a conjugate acid of a 15-oxo-ETE(1-).
(5Z,8Z,11Z,13E)-15-oxoicosa-5,8,11,13-tetraenoic acid has been reported in Arabidopsis thaliana with data available.
See also: 15-Keto-5,8,11,13-eicosatetraenoic acid (annotation moved to).

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In conclusion, we stress four findings. First, structure-activity relations for 5-oxoETE, 5-oxo-15(OH)-ETE, 5-HETE, 15-OxoETE, and 15-HETE in activating MAPK and cPLA2, in translocating secretory vesicle, and in degranulating PMN paralleled each other as well as those previously established for eliciting Ca2+ transients, chemokinesis, and aggregation responses. This suggests that all stimulatory effects of 5-oxo/OH-ETEs use a single recognition system. Second, pertussis toxin blocked MAPK responses to LTB4 and 5-oxo/OH-ETEs, whereas LY 255283 inhibited diverse responses to LTB4 without influencing those to 5-oxo/OH-ETEs (Fig. 7, Table I). The 5-oxo/OH-ETE recognition system likely involves a G protein- and Ca2+-linked receptor distinct from LTB4 receptors. Third, Ca2+-depleted PMN responded to 5-oxo/OH-ETEs with MAPK mobility shifts (Fig. 9). These data are the first to indicate that 5-oxo/OH-ETEs act on Ca2+-independent as well as Ca2+-dependent pathways, both of which are initiated by G proteins. Fourth, GM-CSF greatly enhanced the effects of 5-oxo/OH-ETEs on degranulation, O−2 production, and MAPK activation and arachidonic acid release (Figs. 1 and 6, 7, 8). 5-Oxo/OH-ETEs thus co-operate with hematopoietic cytokines to stimulate MAPK, cPLA2, and functional responses. [1]

C20H30O3

318.45

318.219

81416-72-0

5280701

Colorless to light yellow liquid

1.0±0.1 g/cm3

495.0±45.0 °C at 760 mmHg

267.3±25.2 °C

0.0±2.7 mmHg at 25°C

1.506

4.74

1

3

14

23

428

0

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

YGJTUEISKATQSM-USWFWKISSA-N

InChI=1S/C20H30O3/c1-2-3-13-16-19(21)17-14-11-9-7-5-4-6-8-10-12-15-18-20(22)23/h4-5,8-11,14,17H,2-3,6-7,12-13,15-16,18H2,1H3,(H,22,23)/b5-4-,10-8-,11-9-,17-14+

(5Z,8Z,11Z,13E)-15-oxoicosa-5,8,11,13-tetraenoic acid

81416-72-0; 5,8,11,13-Eicosatetraenoic acid, 15-oxo-, (E,Z,Z,Z)-; DTXSID50904327; 15-Keto-5,8,11,13-eicosatetraenoic acid; DTXCID101333485; 15-Kete; 15-KETE (15-oxo-ETE); 15-Ketoeicosa-6E,8Z,11Z,13E-tetraenoic 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.

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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).

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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)

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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).

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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

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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.

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