Comparative Mediatorlipidomic Profiling Of Engineered Experimental Animals

The powerful approach of transgenics (TG), namely, deletion and overexpression of a gene product coupled with lipidomics, can give valuable insights into the role of select pathways in disease processes. We recently used the mediator lipidomics approach to evaluate transgenic rabbits overexpressing human 15-lipoxygenase (LOX) type 1 in their leukocytes.11 We can take a lipidomic snapshot of cell activation and examine the difference between the transgenic and the nontransgenic rabbits, where the key enzyme is not overproduced, but rather is in its normal state, to evaluate the impact of overexpression of a key enzyme in a pathway. In this case, 15-LOX overexpression leads to enhanced LXA4, as well as enhanced 5,15-diHETE formation with reduced leukotriene B4 (LTB4) formation (Figure 12.4). Because LTB4 is a potent chemoattractant and LXA4 is a counter-regulatory anti-inflammatory within the eicosanoid family, the relationship between these mediators and the overproduction of LXA4 is a key index to appreciating the overall role of the 15-LOX type 1 in inflammation. In short, overexpression of this enzyme yields upreg-ulation of its pathway products, such as LXA4, in these transgenic rabbits that also displayed a generally reduced inflammation and protection from tissue damage.

353.5

351.5 xy

lxaj,

15-PGDHQ NAD"1"^,

Inactive

NSAIDs i-J I-• Indomethacin

353.5

351.5 xy

HO OH

i5-OXO-lxa4,

NADH 15-PGDHQ +H

NAD+ HO OH

13,14-dihydro-LXA4

Inactive

Figure 12.3

Lipoxin local inactivation route: LC-MS chromatograms of LXA4 further metabolites. The initial step in LXA4 (m/z351.5, retention time 13.3 min) inactivation is dehydrogenation of the 15-hydroxyl group catalyzed by an enzyme that was first characterized as 15-hydroxyprostaglandin dehydrogenase (15-PGDH) to yield 15-oxo-LXA4 (m/z 349.5, retention time 11.6 min). A multifunctional eicosanoid oxidoreductase (LTB4DH/PGR), which has been named both leukotriene B412-hydroxydehydrogenase and 15-oxoprostaglandin 13-reductase as result of independent findings that the enzyme can convert these substrates, catalyzes the reduction of the 13,14 double bond of 15-oxo-LXA4 to give 13,14-dihydro-15-oxo-LXA4(m/z351.5, retention time 12.1 min). This product then serves as a substrate for the 15-hydroxy/oxo-eicosanoid oxidoreductase, which catalyzes the reduction of the C15 oxo-group to give 13,14-dihydro-LXA4 (m/z 353.5, retention time 17.5 min). Neither 15-oxo-LXA4 nor 13,14-dihydro-LXA4 binds to the LXA4 receptor and, unlike the parent compound, they do not inhibit the generation of reactive oxygen species in human neutrophils.8

100 n

100 n

LXA4

5, 15-diHETE ^

LTB4 i

H

Im

100 80 60 40 20

100 120 140 160 180 200 220 240 260 280 300 320 m/z B

-h2o

-115

m/z 251

m/z 251

-H2O (M-H)-

233 -2H,O (M-H)-

251 2712

-CO2

115

1 11,,.

100 140

180

220

260

300

m/z

C

Lipoxin Biosynthesis

-COOH

15-Lipoxygenase

Arachidonic Acid

COOH 15-H(p)ETE

5-Lipoxygenase

-COOH

COOH

Hydrolysis

HQ QH

-COOH Lipoxin A4

Figure 12.4 Mediator-lipidomic profiling of engineered experimental animals. Leukocytes were isolated from both 15-LOX type 1 transgenic rabbits and nontransgenic rabbits and incubated with ionophore A23187 (15 mM, 20 min, 37"C). Products were extracted and identified.7 (A) LC profiles from 15-LOX type 1 transgenic rabbits. (B) MS/MS spectrum of 5,15-diHETE with diagnostic ions as indicated. (C) MS/MS spectrum of LXA4 with diagnostic ions as indicated.

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