Preparation and tandem mass spectrometric analyses of deuterium-labeled cysteine-containing leukotrienes

Raftery, Mark J.; Thorne, Gareth C.; Orkiszewski, Ralph S.; Gaskell, Simon J.

doi:10.1002/bms.1200190804

Cited by 27 publications

(15 citation statements)

References 21 publications

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“…Thus, in spleen and liver almost all of the metabolism of LTC 4 appears to result from this enzyme activity rather than from GGT. 4 , LTD 4 , and LTE 4 from reactions run with homogenates from both wild type and GGT-deficient mice were analyzed by a FAB-MS/MS (20,21). Our studies revealed that the presumed LTC 4 , LTD 4 , and LTE 4 peaks produced the same [M ϩ H] ϩ ions and fragment ions as standard LTC 4 , LTD 4 , and LTE 4 (results not shown).…”

Section: Cleavage Of Substrates Used To Assay Ggt By Ggt-deficientmentioning

confidence: 82%

“…Tandem mass spectrometric analyses were performed using a VG ZAB-SEQ (VG Analytical, Manchester, UK) hybrid mass spectrometer of BEqQ configuration (B, magnetic sector; E, electric sector; q, radio frequency-only quadrupole; Q, quadrupole mass filter), equipped with a VG 11-250J data system. The methods employed were similar to those previously described (20,21). Briefly, ionization was accomplished by continuous flow FAB using xenon atoms (8 keV) for the primary atom beam.…”

Section: Methodsmentioning

confidence: 99%

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Metabolism of Leukotriene C4 in γ-Glutamyl Transpeptidase-deficient Mice

Carter

Wiseman

Orkiszewski

et al. 1997

Journal of Biological Chemistry

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LTC 4 -converting activity has a tissue distribution different from GGT with highest activity in spleen followed by small intestine, kidney, and pancreas and lower activity in liver and lung. The activity is membrane-bound and is inhibited by acivicin, a known inhibitor of GGT. The enzyme was partially purified from the small intestine of GGT-deficient mice by papain treatment and gel filtration chromatography. The partially purified fragment released by papain has an apparent molecular mass of 65-70 kDa and the same substrate specificity as the tissue homogenate. In addition to LTC 4 , S-decyl-GSH is also cleaved. GSH itself, oxidized GSH, and the synthetic substrates used to analyze GGT activity (␥-glutamyl-p-nitroanilide and ␥-glutamyl-4-methoxy-2-naphthylamide) are not substrates for this newly discovered enzyme. These data demonstrate that in addition to GGT at least one other enzyme cleaves LTC 4 in mice. To reflect this enzyme's preferred substrate, we suggest that it be named ␥-glutamyl leukotrienase.Peptidyl leukotrienes, cysteine-containing derivatives of arachidonic acid, are potent inducers of airway constriction, vasoconstriction, smooth muscle contraction, edema, and inflammation (1-4). LTC 4 1 is formed by conjugation of leukotriene A 4 with GSH (5) and is known to be cleaved by GGT, which removes the glutamyl moiety to form LTD 4 (6). LTC 4 conversion to LTD 4 has long been thought to be mediated solely by GGT (7,8). Recently, however, the existence of an activity termed GGT-rel has been identified in humans (9). GGT-rel shares an overall 40% amino acid sequence identity with human GGT and is capable of cleaving the ␥-glutamyl linkage of LTC 4 , but it is unable to hydrolyze synthetic substrates that are commonly used for assaying GGT. This activity has been reported to be absent in mice (9).The role of GGT in leukotriene metabolism is of interest because of the great potency of these compounds in responses to injury. However, the distribution of GGT activity and the sites of peptidyl leukotriene actions are not concordant. In the mouse, GGT tends to be expressed at very high levels in epithelia concerned with catabolism of GSH and reabsorption of its constituent amino acids (kidney and small intestine) and other ductular and secretory epithelia (pancreas and seminal vesicles) (7, 10 -12). It is characteristically low in organs in which leukotrienes may play a role in responses to injury (e.g. lung, heart, and lymphoid tissue (spleen)). In addition, the types of responses mediated by the peptidyl leukotrienes (vasoconstriction, bronchoconstriction, increases in vascular permeability, and mucus formation) do not occur in close proximity to sites where GGT is most abundant (13). Although there are reports of GGT activity in endothelium (14 -16), the discordance between peptidyl leukotriene targets and GGT activity raises the possibility that there are other LTC 4 -cleaving enzymes in the mouse.We have recently used homologous recombination to inactivate GGT in the mouse (12). Mice homozygous for th...

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Section: Cleavage Of Substrates Used To Assay Ggt By Ggt-deficientmentioning

confidence: 82%

Section: Methodsmentioning

confidence: 99%

Metabolism of Leukotriene C4 in γ-Glutamyl Transpeptidase-deficient Mice

Carter

Wiseman

Orkiszewski

et al. 1997

Journal of Biological Chemistry

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“…The low-energy CID spectrum displayed in Figure 2a was generated in the linear ion trap instrument from protonated LTC 4 [14,16,25]. Scheme 1a shows the fragmentation pattern of these familiar ions.…”

Section: Protonated Ltc 4 (5 6) Isomermentioning

confidence: 99%

Structural analysis of leukotriene C₄isomers using collisional activation and 157 nm photodissociation

Devakumar

O'Dell

Walker

et al. 2008

J. Am. Soc. Mass Spectrom.

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The fragmentation of 5-hydroxy-6-glutathionyl-7,9,11,14-eicosatetraenoic acid [leukotriene C 4 or LTC 4 (5, 6)] and its isomeric counterpart LTC 4 (14, 15) were studied by low and high-energy collisional induced dissociation (CID) and 157 nm photofragmentation. For singly charged protonated LTC 4 precursors, photodissociation significantly enhances the signal intensities of informative fragment ions that are very important to distinguish the two LTC 4 isomers and generates a few additional fragment ions that are not usually observed in CID experiments. The ion trap enables MS n experiments on the fragment ions generated by photodissociation. Photofragmentation is found to be suitable for the structural identification and isomeric differentiation of cysteinyl leukotrienes and is more informative than low or high-energy CID. We describe for the first time the structural characterization of the LTC 4 (14, 15) isomer by mass spectrometry using CID and 157 nm light activation methods. (J

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“…The product ion spectrum of LTC 4 ( Fig. 2A) is characterized mainly by fragment ions originating from the glutathione part of the molecule (22). All major fragment ions observed in this product ion spectrum at m/z 128, 143, 179, 210, 254, and 272 are also found with similar relative abundance in the corresponding spectra obtained from the samples of the patients (Fig.…”

Section: Resultsmentioning

confidence: 79%

“…To confirm the identification of this signal as LTC 4 , a precursor ion scan for m/z 272 was performed, and the resulting spectra showed the ion at m/z 624 as the most abundant signal. The precursor ion scan for m/z 272 was selected because this fragment has been reported as an abundant fragment ion of LTC 4 generated by fission of the sulfur bridge, with retention of the sulfur atom at the fatty acid part and charge retention at the glutathionyl part, using fast atom bombardment (22) or electrospray ionization (23), as indicated in Fig. 1.…”

Section: Resultsmentioning

confidence: 99%

Synthesis and metabolism of leukotrienes in γ-glutamyl transpeptidase deficiency

Mayatepek

Okun

Meißner

et al. 2004

Journal of Lipid Research

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Leukotrienes (LTs) are active lipid mediators derived in the 5-lipoxygenase pathway. LTC 4 , the primary cysteinyl LT, is cleaved by ␥ -glutamyl transpeptidase (GGT), resulting in LTD 4 . We studied the synthesis and metabolism of LTs in three patients with GGT deficiency. LTs were analyzed in urine, plasma, and monocytes after HPLC separation by enzyme immunoassays, radioactivity detection, and electrospray tandem mass spectrometry. Analysis of LTs in urine revealed increased concentrations of LTC 4 (12.8-17.9 nmol/mol creatinine; controls, Ͻ 0.005 nmol/mol creatinine), whereas LTE 4 was below the detection limit ( Ͻ 0.005 nmol/ mol creatinine; controls, 32.2 ؎ 8.6 nmol/mol creatinine). In plasma of one patient, LTC 4 was found to be increased (17.3 ng/ml; controls, 9.6 ؎ 0.4 ng/ml), whereas LTD 4 and LTE 4 were below the detection limit ( Ͻ 0.005 ng/ml). LTB 4 was found within normal ranges. In contrast to controls, the synthesis of LTD 4 and LTE 4 in stimulated monocytes was below the detection limit ( Leukotrienes (LTs) constitute a group of biologically highly active lipid mediators derived from 20-carbon polyunsaturated fatty acids, predominantly arachidonic acid via the 5-lipoxygenase pathway (1-3). They include the cysteinyl LTs LTC 4 , LTD 4 , and LTE 4 , representing biologically active constituents of the long-known "slow-reacting substance of anaphylaxis" and the dihydroxyeicosatetraenoate LTB 4 .The biosynthesis of LTs is limited to a few types of human cells, including mast cells, eosinophils, basophils, and macrophages. The synthesis of LTs is initiated by cell activation with the release of arachidonic acid from membrane phospholipid by the action of cytosolic phospholipase A 2 . Arachidonic acid then binds to the 5-lipoxygenase-activating protein and is presented to 5-lipoxygenase (4). Calcium-dependent activation of 5-lipoxygenase converts arachidonate via 5-hydroperoxyeicosatetraenoate to 5,6-epoxide LTA 4 , which is unstable and is catalyzed to LTB 4 (5, 6). Alternatively, the conjugation of LTA 4 with glutathione at carbon 6 is mediated by LTC 4 synthase, resulting in the formation of LTC 4 , the primary cysteinyl LT (7). LTC 4 is known to be cleaved by ␥ -glutamyl transpeptidase (GGT), which removes the glutamyl moiety to form LTD 4 (1). LTC 4 conversion to LTD 4 has long been thought to be mediated solely by GGT. The cleavage of glycine from LTD 4 yields LTE 4 (8).Some years ago, a human gene was cloned that appeared to direct the cleavage of LTC 4 . This enzyme was termed ␥ -glutamyl transpeptidase-related (GGT-rel) (9). GGT-rel shares an overall 40% amino acid sequence identity with human GGT and is capable of cleaving the ␥ -glutamyl linkage of LTC 4 , but it is unable to hydrolyze synthetic substrates that are commonly used to assay GGT. GGT-rel is not expressed in the mouse (9).Recently, mice deficient in GGT were developed and used in LT metabolism studies (10-12). These studies un-

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Preparation and tandem mass spectrometric analyses of deuterium-labeled cysteine-containing leukotrienes

Cited by 27 publications

References 21 publications

Metabolism of Leukotriene C4 in γ-Glutamyl Transpeptidase-deficient Mice

Metabolism of Leukotriene C4 in γ-Glutamyl Transpeptidase-deficient Mice

Structural analysis of leukotriene C₄isomers using collisional activation and 157 nm photodissociation

Synthesis and metabolism of leukotrienes in γ-glutamyl transpeptidase deficiency

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Preparation and tandem mass spectrometric analyses of deuterium-labeled cysteine-containing leukotrienes

Cited by 27 publications

References 21 publications

Metabolism of Leukotriene C4 in γ-Glutamyl Transpeptidase-deficient Mice

Metabolism of Leukotriene C4 in γ-Glutamyl Transpeptidase-deficient Mice

Structural analysis of leukotriene C4isomers using collisional activation and 157 nm photodissociation

Synthesis and metabolism of leukotrienes in γ-glutamyl transpeptidase deficiency

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Structural analysis of leukotriene C₄isomers using collisional activation and 157 nm photodissociation