Wainwright S, Falck J R, Yadagiri P, Powell W S
Endocrine Laboratory, Royal Victoria Hospital, Montreal, Quebec, Canada.
Biochemistry. 1990 Oct 30;29(43):10126-35. doi: 10.1021/bi00495a017.
We have previously shown that porcine polymorphonuclear leukocytes (PMNL) reduce leukotriene B4 (LTB4) to 10,11-dihydro-LTB4, 10,11-dihydro-12-epi-LTB4, and 10,11-dihydro-12-oxo-LTB4 [Wainwright et al. (1990) Biochemistry 29, 1180-1185]. We have now demonstrated that 12(S)-hydroxy-5,8,10,14-eicosatetraenoic acid [12(S)-HETE] is metabolized by a similar pathway in porcine PMNL. 12(S)-HETE was metabolized to two products that were identified by gas chromatography-mass spectrometry and nuclear magnetic resonance spectroscopy as 12-hydroxy-5,8,14-eicosatrienoic acid (10,11-dihydro-12-HETE) and 12-oxo-5,8,14-eicosatrienoic acid (10,11-dihydro-12-oxo-ETE). Derivatization of 12-hydroxy-5,8,14-eicosatrienoic acid with (R)-(+)-alpha-methoxy-alpha-(trifluoromethyl)phenylacetic acid, followed by chromatography on a silicic acid column, enabled the resolution of 12R and 12S stereoisomers, which were identified by cochromatography with synthetic standards. Incubation of 12(S)-HETE with PMNL for various times revealed that the stereochemistry of the 12-hydroxyl group of 12-hydroxy-5,8,14-eicosatrienoic acid was initially the same as that of 12(S)-HETE. However, after 40 min, 30% of the 12-hydroxy-5,8,14-eicosatrienoic acid had the opposite configuration at C12. 13-Hydroxy-9,11-octadecadienoic acid (13-HODE) was metabolized in a similar fashion by porcine PMNL to 13-hydroxy-9-octadecenoic acid (11,12-dihydro-13-HODE) and 13-oxooctadecenoic acid (11,12-dihydro-13-oxo-ODE). The apparent Km values for the reduction of 12-HETE, LTB4, and 13-HODE were 0.21, 0.28, and 2.22 microM, respectively. All three substrates had the same apparent Vmax [0.029 pmol min-1 (10(6) cells)-1]. Competition experiments between LTB4 and 12-HETE indicated that they were metabolized by the same pathway. Various structurally related compounds were metabolized by porcine PMNL in the order LTB4 = 6-trans-LTB4 greater than 12-epi-6-trans,8-cis-LTB4 greater than 12-epi-6-trans-LTB4 greater than 12-HETE greater than LTB5 greater than 15-HETE = 13-HODE much greater than 5-HETE greater than 9-HODE greater than 20-hydroxy-LTB4 greater than 12-hydroxy-5,8,10-heptadecatrienoic acid. Prostaglandins E2 and F2 alpha were not metabolized to any detectable products by porcine PMNL.
我们之前已经表明，猪多形核白细胞（PMNL）可将白三烯B4（LTB4）还原为10,11-二氢-LTB4、10,11-二氢-12-表-LTB4和10,11-二氢-12-氧代-LTB4 [温赖特等人（1990年）《生物化学》29卷，1180 - 1185页]。我们现在已经证明，12(S)-羟基-5,8,10,14-二十碳四烯酸[12(S)-HETE]在猪PMNL中通过类似途径代谢。12(S)-HETE代谢为两种产物，通过气相色谱 - 质谱联用和核磁共振光谱鉴定为12-羟基-5,8,14-二十碳三烯酸（10,11-二氢-12-HETE）和12-氧代-5,8,14-二十碳三烯酸（10,11-二氢-12-氧代-ETE）。用(R)-(+)-α-甲氧基-α-(三氟甲基)苯乙酸对12-羟基-5,8,14-二十碳三烯酸进行衍生化，然后在硅酸柱上进行色谱分离，能够分离出12R和12S立体异构体，通过与合成标准品共色谱法进行鉴定。将12(S)-HETE与PMNL孵育不同时间表明，12-羟基-5,8,14-二十碳三烯酸12-羟基的立体化学最初与12(S)-HETE相同。然而，40分钟后，30%的12-羟基-5,8,14-二十碳三烯酸在C12处具有相反的构型。13-羟基-9,11-十八碳二烯酸（13-HODE）在猪PMNL中以类似方式代谢为13-羟基-9-十八碳烯酸（11,12-二氢-13-HODE）和13-氧代十八碳烯酸（11,12-二氢-13-氧代-ODE）。还原12-HETE、LTB4和13-HODE的表观Km值分别为0.21、0.28和2.22微摩尔。所有三种底物具有相同的表观Vmax [0.029皮摩尔·分钟-1（10^6个细胞）-1]。LTB4和12-HETE之间的竞争实验表明它们通过相同途径代谢。各种结构相关的化合物在猪PMNL中按以下顺序代谢：LTB4 = 6-反式-LTB4 > 12-表-6-反式,8-顺式-LTB4 > 12-表-6-反式-LTB4 > 12-HETE > LTB5 > 15-HETE = 13-HODE >> 5-HETE > 9-HODE > 20-羟基-LTB4 > 12-羟基-5,8,10-十七碳三烯酸。前列腺素E2和F2α未被猪PMNL代谢为任何可检测到的产物。
