Endogenous metabolite

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.

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.

15(S)-HETE is an optically active form of 15-HETE having 15(S)-configuration.. It has a role as a human metabolite and a mouse metabolite. It is a conjugate acid of a 15(S)-HETE(1-). It is an enantiomer of a 15(R)-HETE.

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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]

C20H32O3

320.47

320.235

C, 74.96; H, 10.07; O, 14.98

54845-95-3

15(S)-HETE-d8;84807-87-4; 54845-95-3; 339046-14-9 (sodium)

5280724

Colorless to light yellow 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-VAEKSGALSA-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-/m0/s1

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

AL 12959; 15(S)-HETE; 54845-95-3; 15S-HETE; Icomucret [INN]; (5Z,8Z,11Z,13E,15S)-15-hydroxyicosa-5,8,11,13-tetraenoic acid; Icomucret [USAN:INN]; AL-12959; AL-12959; Icomucret

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.)