12(S)-Hydroxy-5,8,10 (Z,E,E)-heptadecatrienoic acid (HHT) is preferentially metabolized to its 12-keto derivative by human erythrocytes in vitro.

The metabolism of [1-14C]-labelled 12 (S)-hydroxy-5,8,10 (Z,E,E)-heptadecatrienoic acid (HHT) by crude 15-hydroxyprostaglandin dehydrogenase (PGDH) fractions from swine kidney and human erythrocytes has been investigated. HPLC radiochromatography analysis revealed that HHT was extensively converted into three metabolites by swine kidney cytosol in the presence of NAD+. They were identified by combined GLC mass spectrometry as 12-keto-5,8,10 (Z,E,E)-heptadecatrienoic acid (KHT), 12-keto-5,8 (Z,E)-heptadecadienoic acid and 12 (RS)-hydroxy-5,8 (Z,E)-heptadecadienoic acid, respectively. In contrast, HHT was metabolized only to the 12-keto derivative by human erythrocyte cytosol supplemented with NADP+, and HHT turnover was found to be enhanced severalfold when compared to prostaglandins E2 (PGE2) or F2 alpha. Since PGE2 was also converted only into 15-keto-PGE2, and no metabolism of KHT was detected with NADPH, there is probably no 15-ketoprostaglandin delta 13-reductase activity in human erythrocytes. Biosynthetic KHT (0.5-5 microM) inhibited the aggregation of human platelets to almost all agonists, probably by raising intracellular cAMP. KHT (between 0.01 and 1 microM) also induced the chemotaxis of human polymorphonuclear leukocytes. Among other still unrecognized effects, these biological activities of KHT may be of physiological significance with respect to its presumably exclusive formation in the blood. The potential use of KHT for monitoring thromboxane synthase activity in vivo is discussed.

Hecker M ，Ullrich V 《-》

12-L-hydroxy-5,8,10-heptadecatrienoic acid (HHT) is an excellent substrate for NAD+-dependent 15-hydroxyprostaglandin dehydrogenase.

12-L-hydroxy-5,8,10-heptadecatrienoic acid (HHT) was found to be an excellent substrate for NAD+ dependent 15-hydroxyprostaglandin dehydrogenase from porcine kidney. Kcat/Km value of HHT was comparable to that of prostaglandin E although HHT is not a prostanoic acid derivative. Product of enzyme catalyzed oxidation of HHT was identified as 12-keto-5,8,10-heptadecatrienoic acid by gas chromatography-mass spectrometry. The fact that HHT is an excellent substrate for 15-hydroxyprostaglandin dehydrogenase suggest that HHT may have profound unrecognized biological actions and its inactivation may be via oxidation of the hydroxyl group.

Liu Y ，Yoden K ，Shen RF ，Tai HH ... -  《BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS》

Identification and characterization of bioactive metabolites of 12-hydroxyheptadecatrienoic acid, a ligand for leukotriene B4 receptor 2.

12(S)-hydroxyheptadecatrienoic acid (12-HHT) is a bioactive fatty acid synthesized from arachidonic acid via the cyclooxygenase pathway and serves as an endogenous ligand for the low-affinity leukotriene B4 receptor 2 (BLT2). Although the 12-HHT/BLT2 axis contributes to the maintenance of epithelial homeostasis, 12-HHT metabolism under physiological conditions is unclear. In this study, 12-keto-heptadecatrienoic acid (12-KHT) and 10,11-dihydro-12-KHT (10,11dh-12-KHT) were detected as 12-HHT metabolites in the human megakaryocytic cell line MEG01s. We found that 12-KHT and 10,11dh-12-KHT are produced from 12-HHT by 15-hydroxyprostaglandin dehydrogenase (15-PGDH) and prostaglandin reductase 1 (PTGR1), key enzymes in the degradation of prostaglandins, respectively. The 15-PGDH inhibitor SW033291 completely suppressed the production of 12-KHT and 10,11dh-12-KHT in MEG01s cells, resulting in a 9-fold accumulation of 12-HHT. 12-KHT and 10,11dh-12-KHT were produced in mouse skin wounds, and the levels were significantly suppressed by SW033291. Surprisingly, the agonistic activities of 12-KHT and 10,11dh-12-KHT on BLT2 were comparable to that of 12-HHT. Taken together, 12-HHT is metabolized into 12-KHT by 15-PGDH, and then 10,11dh-12-KHT by PTGR1 without losing the agonistic activity.

Yasukawa K ，Okuno T ，Ogawa N ，Kobayashi Y ，Yokomizo T ... -  《-》

Identification of 12-keto-5,8,10-heptadecatrienoic acid as an arachidonic acid metabolite produced by human HL-60 leukemia cells.

An unusual cyclooxygenase-derived metabolite of arachidonic acid has been shown to be produced by N,N-dimethylformamide (DMF)-induced, terminally differentiated human HL-60 promyelocytic leukemia cells and to a much lesser extent by untreated cells. Biochemical evidence in conjunction with gas chromatography/mass spectrometry and liquid chromatography/thermospray mass spectrometry analyses indicates that the product is 12-keto-5,8,10-heptadecatrienoic acid (KHT). Both KHT and 12-hydroxy-5,8,10-heptadecatrienoic acid (HHT) were produced when arachidonic acid was incubated with cell lysates obtained from differentiated HL-60 granulocytes. Indomethacin and the thromboxane synthetase inhibitor UK-38485 inhibited the production of both metabolites, whereas ethacrynic acid inhibited only the production of KHT. In 100,000 g supernatant fractions, obtained from either untreated or differentiated cells, KHT was produced when HHT was used as substrate. The addition of exogenous NAD, but not NADP, to incubations caused a significant increase in the production of KHT coincident with a decrease in the level of HHT. These data suggest that, in both differentiated and undifferentiated HL-60 cells, an NAD-dependent enzyme, apparently 15-prostaglandin dehydrogenase (15-PGDH), is expressed and catalyzes the conversion of HHT to KHT. In differentiated HL-60 cells, this metabolite is produced from arachidonic acid through a multi-enzymatic process involving the activities of cyclooxygenase, thromboxane synthetase and 15-PGDH. The production of KHT from arachidonic acid in undifferentiated HL-60 cells is probably limited, therefore, by the virtual absence of cyclooxygenase activity in these cells.

Agins AP ，Thomas MJ ，Edmonds CG ，McCloskey JA ... -  《BIOCHEMICAL PHARMACOLOGY》

Mass determination of 15-hydroxyprostaglandin dehydrogenase from human placenta and kinetic studies with (5Z, 8E, 10E, 12S)-12-hydroxy-5,8,10-heptadecatrienoic acid as substrate.

NAD(+)-dependent 15-hydroxyprostaglandin dehydrogenase catalyzes the first step in the metabolism of prostaglandins which is usually associated with physiological inactivation. A highly purified homogenous enzyme preparation from human placenta was used to determine the molecular mass and lack of quaternary structure of the enzyme. Furthermore we have examined enzyme kinetics of the purified enzyme with (5Z,8E,10E,12S)-12-hydroxy-5,8,10-heptadecatrienoic acid (HHT) an equimolar coproduct of thromboxane biosynthesis. Using gel electrophoresis and gel filtration on FPLC, we could estimate a molecular mass of 28 +/- 1 kDa, indicating that the enzyme consists of one single protein chain. The exact molecular mass of the monomer was calculated by matrix-assisted laser desorption/ionization mass spectrometry to 28740 +/- 30 Da. (5Z,8E,10E)-12-oxo-5,8,10-heptadecatrienoic acid (oxo-HT) could be identified as the only product obtained from the enzymatic reaction with HHT. Quantification of this metabolite was achieved by gas chromatography/tandem mass spectrometry. The calculated enzyme kinetic constants for the formation of the metabolic product [Km (HHT) = 9.68 microM, Vi = 12.78 mU/micrograms] were in agreement with those determined for NADH formation (Km = 7.65 microM, Vi = 11.79 mU/micrograms). This demonstrates that HHT shows high affinity to the enzyme which is comparable to prostaglandin E2 (PGE2). As the product oxo-HT is a potent inhibitor of platelet aggregation, dehydrogenation of HHT might represent a biological activation step.

Höhl W ，Stahl B ，Mundkowski R ，Hofmann U ，Meese CO ，Kuhlmann U ，Schlegel W ... -  《european journal of biochemistry》