Arachidonic acid derived lipid mediator

15-hydroxyeicosatetraenoic acid (15-HETE) is an arachidonic acid derived lipid mediator which can originate both from 15-lipoxygenase (15-LOX) activity and cyclooxygenase (COX) activity. The enzymatic source determines the enantiomeric profile of the 15-HETE formed. 15-HETE is the most abundant arachidonic acid metabolite in the human lung and has been suggested to influence the pathophysiology of asthma. Mast cells are central effectors in asthma, but there are contradictory reports on whether 15-HETE originates from 15-LOX or COX in human mast cells. This prompted the current study where the pathway of 15-HETE biosynthesis was examined in three human mast cell models; the cell line LAD2, cord blood derived mast cells (CBMC) and tissue isolated human lung mast cells (HLMC). Levels and enantiomeric profiles of 15-HETE and levels of the downstream metabolite 15-KETE, were analyzed by UPLC-MS/MS after stimulation with anti-IgE or calcium ionophore A23187 in the presence and absence of inhibitors of COX isoenzymes. We found that 15-HETE was produced by COX-1 in human mast cells under these experimental conditions. Unexpectedly, chiral analysis showed that the 15(R) isomer was predominant and gradually accumulated, whereas the 15(S) isomer was metabolized by the 15-hydroxyprostaglandin dehydrogenase. We conclude that during physiological conditions, i.e., without addition of exogenous arachidonic acid, both enantiomers of 15-HETE are produced by COX-1 in human mast cells but that the 15(S) isomer is selectively depleted by undergoing further metabolism. The study highlights that 15-HETE cannot be used as an indicator of 15-LOX activity for cellular studies, unless chirality and sensitivity to pharmacologic inhibition is determined. [3]

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Endogenous metabolites are those that the Kyoto Encyclopedia of Genes and Genomes has identified as substrates or products of the roughly 1900 metabolic enzymes that are encoded in our genomes. Numerous studies have conclusively shown that many of these metabolites are hazardous [1].

To determine the importance of peroxisomes and mitochondria in hydroxyeicosatetraenoic acid (HETE) oxidation in vivo, urinary excretion of 12- and 15-HETE was measured in eight patients with a peroxisome deficiency disorder (Zellweger syndrome) showing normal mitochondrial beta-oxidation capacity, in three patients with a defect of mitochondrial long-chain fatty acid oxidation (long-chain acyl-CoA dehydrogenase deficiency), and in eight healthy subjects. 12- and 15-HETE were identified and quantified by gas chromatography/negative ion chemical ionization-mass spectrometry and specific RIA. The free compounds were found exclusively in the urine of peroxisome-deficient subjects (12-HETE: median 26 pg/mL, range 17-36 pg/mL; 15-HETE: median 40 pg/mL, range 29-61 pg/mL), whereas both compounds were below the detection limit (< 0.5 pg/mL) in the urine of patients with defective mitochondrial long-chain fatty acid oxidation and normal subjects (p < 0.002). These results implicate that peroxisomes are the main cellular organelle responsible for HETE oxidation in vivo. Analysis of HETE excretion in urine represents an additional new specific diagnostic tool in patients with Zellweger syndrome. [1]

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Identification of 12- and 15-HETE. [1]
In the investigation of HETE as their hydrogenated TMS/PFB derivatives all regioisomeric HETE give rise to an intense [M - PFB] fragment ion at m/z 399. Using capillary GC the HETE positional isomers are separated in the order of the position of the oxidized carbon atom, i.e. the derivative of 3-hydroxyeicosanoic acid elutes before the corresponding derivative of 4-hydroxyeicosanoic acid, and so forth. Although separation is not complete for positional isomers near the central part of the carbon chain, it is sufficient to allow a specific detection of the four lipoxygenase-derived species oxidized at positions 5, 8, 12, and 15. This is demonstrated in the upper trace of Figure 1 showing the single ion monitoring analysis atm/z 399 of a standard mixture containing similar amounts of 5-, 8-, 12-, and 15-HETE (Fig. 1a). 12- and 15-HETE were isolated by HPLC from the urine of healthy subjects and patients with ZS syndrome or LCAD deficiency, as described above.

In urine samples from healthy control subjects and patients with LCAD 12- and 15-HETE could not be detected. Figure 1b shows a representative spectrum from a healthy control patient, documenting the lack of urinary 12- and 15-HETE excretion. In contrast, the presence of 12- and 15-HETE in urine samples was verified in all eight ZS patients by GC/NICI-MS as exemplary shown in Figure 1, c and d, respectively. Because reproducibility of the GC retention times is very high (within day reproducibility ±1 s) this method provides a specific identification of the lipoxygenase-derived HETE and thus confirms the presence of 12- and 15-HETE in urine of ZS patients.

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Quantification of 12- and 15-HETE. [1]
The urinary concentrations of 12- and 15-HETE for the patients with a peroxisome deficiency disorder, patients with LCAD deficiency, and normal subjects were determined by RIA and are given in Table 1. Both HETE were present in the urine from ZS patients at concentrations in the picogram/mL range. In contrast, analyses of urine samples from LCAD-deficient patients and normal subjects showed that both 12- as well as 15-HETE were below the detection limit in these groups.

Cell assays. [3]
CBMC were primed with IL-4 (10 ng/mL) for 4 days before experiment and exposed one day pior to IgE (1 μg/mL). HLMC were only preincubated with IgE 1 μg/mL O/N. Before the assay, cells were washed with PBS and resuspended in PIPES-BSA (9.2 mM PIPES, 139.7 mM NaAc, 0.6 mM CaCl2, 1.1 mM MgCl2 and 0.2% BSA). Cells were used at 200,000 per sample. Before stimulation by IgE-receptor cross-linking or calcium ionophore A23187, cells were incubated 15 min in the absence or presence of inhibitors, which included FR122047 (selective COX-1 inhibitor, 1 μM), etoricoxib (selective COX-2 inhibitor, 1 μM), indomethacin 10 μM, (nonselective COX inhibitor, 10 μM) 15-PGDH inhibitor Cay10397 (10 μM). Inhibitor concentrations were chosen based on our previous experience and data from the literature. LAD2 cells were stimulated by the addition of 1 μM A23187 for 30 min. CBMC and HLMC were washed and stimulated by addition of 2 μg/mL of anti-IgE for 30 min. For exogenous addition of 15-HETE and 15-KETE the lipid mediator was added to the cells at 40 ng/mL. After the assay, cells were spun down and supernatant stored at -80 °C until solid phase extraction (SPE) and analysis by ultra-performance liquid chromatography coupled to tandem mass spectrometry (UPLC-MS/MS). Bronchi were dissected immediately before the experiment and put directly in PIPES-BSA with or without 15-LOX inhibitor (3 μM BLX3887 or COX-1/2 inhibitor Indomethacin 10 μM). Supernatant was frozen after 30 min of incubation at 37 °C, 5% CO2.

Patients. [2]
Excretion of 12- and 15-HETE was studied in eight patients with a peroxisome deficiency disorder (ZS), three patients with a defect of mitochondrial long-chain fatty acid oxidation (LCAD deficiency), and eight healthy control subjects. All patients with ZS exhibited the characteristic clinical and biochemical abnormalities described for ZS. Specific biochemical analyses in these patients included very long-chain fatty acids (>C22) in plasma and fibroblasts as well as plasma bile acid intermediates and de novo plasmalogen biosynthesis in cultured fibroblasts. Mitochondrialβ-oxidation activity was assayed in cultured fibroblasts using[1-14C]palmitic acid and found to be in the range of normal subjects. The biochemical characteristics of these patients have been published already in detail. All patients with a defect of mitochondrial long-chain fatty acid oxidation had LCAD deficiency as measured in cultured skin fibroblasts.

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Urine extraction and purification. [2]
Five-milliliter aliquots of urine were labeled with 3 500 dpm each of [3H]-12-HETE and[3H]-15-HETE. The samples were acidified to pH 4.5 by addition of 0.1 M HCl, mixed, and pumped slowly through activated Sep-Pak C18 cartridges. The cartridges were washed with 50 mL of distilled H2O, and HETE were eluted with 5 mL of 90% aqueous methanol containing 1 mM 4-hydroxy-2,2,6,6-tetramethylpiperidine-N(1)-oxyl and 0.5 mM EDTA. The eluates were evaporated to dryness under reduced pressure and resuspended in 30% acetonitrile and 70% water acidified to pH 3.4 with phosphoric acid.

[1]. Endogenous toxic metabolites and implications in cancer therapy. Oncogene. 2020 Aug;39(35):5709-5720.

[2]. 12- and 15-hydroxyeicosatetraenoic acid are excreted in the urine of peroxisome-deficient patients: evidence for peroxisomal metabolism in vivo. Pediatr Res. 1996 Jan;39(1):146-9.

[3]. COX-1 dependent biosynthesis of 15-hydroxyeicosatetraenoic acid in human mast cells. Biochim Biophys Acta Mol Cell Biol Lipids. 2021 May;1866(5):158886.

15(R)-HETE is an optically active form of 15-HETE having 15(R)-configuration. It is a conjugate acid of a 15(R)-HETE(1-). It is an enantiomer of a 15(S)-HETE.
15R-hydroxy-5Z,8Z,11Z,13E-eicosatetraenoic acid has been reported in Bos taurus with data available.

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Recently, it was reported that isolated rat liver and kidney peroxisomes convert 12-HETE to 8-hydroxyhexadecatrienoic acid, confirming a peroxisomalβ-oxidation. However, mitochondrial function might also be abnormal in peroxisomedeficient cells of patients. Additionally, in vitro data also point to mitochondria as potential sites of β-oxidation of 12- and 15-HETE. This could induce doubt as to whether HETE oxidation is possible only on the peroxisomal pathway in men. We therefore studied ZS patients showing a normal mitochondrial β-oxidation capacity in fibroblasts. Because these patients are peroxisome-deficient but have normal mitochondrial enzymatic activity, their urinary excretion of 12- and 15-HETE implicates a failure to convert both HETE to oxidative metabolites. Further evidence that the β-oxidation process of these HETE occurs in the peroxisomes rather than in the mitochondria was obtained from urine analyses of LCAD-deficient patients. As opposed to the 12- and 15-HETE excretion by ZS patients, the mitochondrial long-chain fatty acid β-oxidation-deficient patients showed, in analogy to normal subjects, no detectable amounts of these HETE. Thus, patients with LCAD deficiency are able to oxidize both HETE, indicating that in vivo this oxidative process does not take place in the mitochondria. All together, these results make it likely that in vivo oxidation of both HETE occurs mainly in the peroxisomes and not in the mitochondria. Lack of HETE oxidation in peroxisome-deficient patients resulting in urinary excretion of 12- and 15-HETE represents an additional new specific diagnostic tool in patients with ZS. Because these HETE possess a diversity of important biologic properties, impaired degradation and inactivation of these potent mediators may be of pathophysiologic relevance in the course of the disease. [2]

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It is well recognized that many metabolic enzymes play essential roles in cancer cells in producing building blocks such as nucleotides, which are required in greater amounts due to their increased proliferation. On the other hand, the significance of enzymes in preventing the accumulation of their substrates is less recognized. Here, we outline the evidence and underlying mechanisms for how many metabolites normally produced in cells are highly toxic, such as metabolites containing reactive groups (e.g., methylglyoxal, 4-hydroxynonenal, and glutaconyl-CoA), or metabolites that act as competitive analogs against other metabolites (e.g., deoxyuridine triphosphate and l-2-hydroxyglutarate). Thus, if a metabolic pathway contains a toxic intermediate, then we may be able to induce accumulation and poison a cancer cell by targeting the downstream enzyme. Furthermore, this poisoning may be cancer cell selective if this pathway is overactive in a cancer cell relative to a nontransformed cell. We describe this concept as illustrated in selenocysteine metabolism and other pathways and discuss future directions in exploiting toxic metabolites to kill cancer cells. [1]

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This study shows the importance of applying interventions with selective pharmacologic tools and using chiral analysis when assessing eicosanoid pathways. Although 15-LOX is known to be the main enzymatic pathway for the generation of 15-HETE in airway epithelium, this was not the case in the three different human mast cells upon IgE-receptor activation or calcium ionophore stimulation. In contrast, COX-1 was the enzymatic source of 15-HETE in the three mast cell preparations of this study. This demonstration, which agrees with findings in the mast cell line HMC-1, has a general relevance, i.e. measuring 15-HETE without pharmacologic interventions and in the absence of chiral analysis does not prove that appearance of 15-HETE is an indicator of 15-LOX activity. This is particularly relevant if the measurement of 15-HETE has been made with an 15(S)-HETE selective immunoassay since the presence of S-isomer does not exclude the presence of R-isomer. The results also raise the possibility that some therapeutic effects of NSAIDs might relate to reduce levels of 15-HETE and 15-KETE in addition to the established effects on prostaglandin biosynthesis. Another important implication of this study is that the appearance of a particular compound should not only be related to upstream effects but also to downstream metabolism. We thus could explain that the excess of 15(R)-HETE was because the 15-PGDH catalysed stereoselective metabolism of 15(S)-HETE camouflaged that the two stereoisomers in fact were formed in parallel. Finally, some of the authors have previously published clear evidence that human CBMC when activated by exogenous arachidonic acid or mannitol indeed release 15-HETE in 15-LOX catalysed reactions. This is not a contradiction of the current results but rather underpin how stimulus-dependent biosynthesis of eicosanoids is. Exogenous arachidonic acid thus being a particularly effective stimulus for activation of the 15-LOX. It is interesting to note that the same signaling molecule 15-HETE may be produced along two different pathways, suggesting that it has important cellular functions that remain to uncover.[3]

C20H32O3

320.47

320.235

C, 74.96; H, 10.07; O, 14.98

83603-31-0

54845-95-3; 339046-14-9 (sodium)

5283169

Liquid

1.0±0.1 g/cm3

487.7±45.0 °C at 760 mmHg

262.8±25.2 °C

0.0±2.8 mmHg at 25°C

1.514

5.22

2

3

14

23

392

1

CCCCC[C@H](/C=C/C=C\C/C=C\C/C=C\CCCC(=O)O)O

JSFATNQSLKRBCI-UDQWCNDOSA-N

InChI=1S/C20H32O3/c1-2-3-13-16-19(21)17-14-11-9-7-5-4-6-8-10-12-15-18-20(22)23/h4-5,8-11,14,17,19,21H,2-3,6-7,12-13,15-16,18H2,1H3,(H,22,23)/b5-4-,10-8-,11-9-,17-14+/t19-/m1/s1

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

CTK3E6555; DTXSID50390600; 83603-31-0; DTXCID60341461; (15S)-15-hydroxyicosa-5,8,11,13-tetraenoic acid; CBiol_001765; KBioGR_000057; KBioSS_000057;

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.)