ω‐Oxidation of cysteine‐containing leukotrienes by rat‐liver microsomes

Abstract

Leukotriene E4 was metabolized to two polar products by rat liver microsomes. These products were characterized by physico-chemical and chemical techniques. The chemical structures, (SS, 6R)-5,20-dihydroxy-6S-cysteinyl-7,9-trans-ll,l4-cis-icosatetraenoic acid (w-hydroxy-leukotriene E4) and (SS, 6R)-S-hydroxy-6S-cysteinyl-7,9-fruns-11,14-cis-icosatetraen-l,20-dioic acid (w-carboxy-leukotriene E,) suggested that leukotriene E4 was transformed by an o-hydroxylase and o-hydroxyleukotriene E dehydrogenase in sequence. N-Acetylleukotriene E4 was also transformed by these enzymes, but at a rate six times lower than leukotriene E4. The products formed from N-acetylleukotriene E4 were characterized as being N-acetyl-o-hydroxy-leukotriene E4 and N-acetyl-w-carboxy-leukotriene E4. Other substrates were 11 -trans-leukotriene E4 and N-acetyl-1 1 -transleukotriene E4. In contrast, leukotrienes C4 and D4 were not converted into o-oxidized metabolites. The leukotriene E o-hydroxylase reaction required NADPH and molecular oxygen as cofactors, and was most rapidly catalyzed by liver microsomes. Liver cytosol, fortified with NAD' , converted o-hydroxyleukotriene E4 and N-acetyl-o-hydroxy-leukotriene E4 into o-carboxy metabolites. Microsomes contained at least 18 times less w-hydroxy-leukotriene E dehydrogenase activity than did cytosol. Liver microsomes supplemented with acetylcoenzyme A converted w-hydroxy and o-carboxy-leukotriene E4 into the corresponding N-acetyl derivatives. The novel enzyme, leukotriene E o-hydroxylase, which is described here is distinct from a previously described leukotriene B w-hydroxylase based on substrate competition and kinetic data.Previous investigations in our and other laboratories have shown that cysteine-containing leukotrienes [2] are extensively metabolized both in vitro and in vivo (reviewed in [3]). Following administration of tritium-labeled leukotriene C,, a biliary and two fecal metabolites in the rat (N-acetylleukotriene E4 and N-acetyl-11-trans-leukotriene E4 [4, 5]), a biliary metabolite in the guinea pig (leukotriene D4 [6]) and a biliary and urinary compound in pig and monkey and a urinary metabolite in man (leukotriene E4 [7 -91) were characterized. During the course of these and other (e.g. [lo, 111) investigations it was found that more polar metabolites were also formed from LTC. The relative amount of the latter compounds increased with time following LTC administration. Since a large Correspondence to L. Orning, Institutionen for Fysiologisk Kemi, Karolinska Institutet, S-104 01 Stockholm, SwedenAbbreviations. RP-HPLC, reverse-phase high-performance liquid chromatography; FAB-MS, fast-atom-bombardment mass spectrometry; GC-MS, gas-liquid chromatography/mass spectrometry; Me,Si, trimethylsilyl; LTB4, leukotriene B4; LTC4, leukotriene C4; LTD4, leukotriene D4; LTE4, leukotriene E4; N-Ac-LTE4, Nacetylleukotriene E4; 11 -trans-LTC4, 1 1-trans-leukotriene C4; 11-trans-LTE4, 1 1-trans-leukotriene E4; N-Ac-11 -trans-LTE4, N-acetyl-1 1-trans-leukotriene Ed; w-OH-LTE4, w-hydroxy-leukot...

Halothane metabolism

“…Never1 heless, at apparent LTE4NAc concentrations of 7.5 suggesting that the observed impairment in vivo of LTE4NAc w-oxidation in rats 18 h after halothane exposure might also be true for both LTE4 and LTB4. The data obtained here, indicating a single P450 isozyme acting on LTB4 and LTE4 as substrates, are in contrast to earlier reports [2] suggesting that LTB4 w-hydroxylase and LTE4 w-hydroxylase were distinct enzymes based on apparent absence of inhibition of metabolism of either substrate in the presence of the other. We do not yet have an explanation for this discrepancy.…”

“…We do not yet have an explanation for this discrepancy. However, the preparation method of microsomal fractions from hepatocytes (our experiments) or microsomal membranes from whole liver tissue [2], as well as the substrate concentrations used, may affect enzymatic activity. Alternatively, leukotriene o-hydroxylases present in distinct cells of the liver may have characteristics largely different from those in hepatocytes separated from the reminder of hepatic cells.…”

Hepatic uptake and metabolic disposition of leukotriene B4 in rats

“…1), LTB4 undergoes extensive intrahepatic metabolism via w-oxidation and ,-oxidation from the w-end (Fig. 2), as described in studies using isolated hepatocytes, liver microsomes and cytosol [10,12,[42][43][44][45][46][47]. The extensive hepatocellular catabolism of LTB4, which proceeds faster than the comparable catabolism of cysteinyl LTs [43,45], is probably responsible for the lower total recovery of radioactivity from infused [3H8]LTB4 (Table 2).…”

Impaired degradation of leukotrienes in patients with peroxisome deficiency disorders.

“…-LTB4 using rat liver microsomes and cytosol (17,52). The RP-HPLC fractions containing these standards were collected and evaporated under reduced pressure.…”

“…The liver represents the main organ for the uptake, metabolic inactivation, and biliary elimination of LTs and their metabolites ( 10-1 3). The metabolic inactivation of LTs is mediated by w-oxidation and subsequent 3-oxidation from the w-end of LTB4 (14)(15)(16), LTE4, and N-acetyl-LTE4 (17)(18)(19)(20)(21). Administration ofradiolabeled LTC4 and LTE4 in human subjects leads to cw-and (3-oxidation products which are excreted into bile and urine (22)(23)(24).…”