Separation and quantitation of leukotrienes by reversed-phase high-performance liquid chromatography

Verhagen, Jan; Walstra, Pauline; Veldink, Gerrit A.; Vliegenthart, Johannes F.G.; Bruynzeel, P.L.B.

doi:10.1016/0262-1746(84)90097-0

Cited by 50 publications

(6 citation statements)

References 11 publications

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“…Chromatographic purification of fatty acid hydroperoxides as reference compounds. Fatty acid hydroperoxides were further purified by reversed-phase HPLC (12). The isomeric LOX products were analyzed on a Spherisorb ODS-2 column (5 µm, 4.6 × 250 mm) (Knauer, Berlin, Germany) using an ultraviolet (UV) detector (variable wavelength detector, Knauer) set to 234 nm for detection of conjugated dienes.…”

Section: Methodsmentioning

confidence: 99%

Analysis of lipoxygenase‐derived fatty acid hydroperoxides by electrospray ionization tandem mass spectrometry

Schneider

Schreier

Herderich

1997

Lipids

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A rapid method is described for the identification of fatty acid hydroperoxides by electrospray ionization-tandem mass spectrometry coupled to liquid chromatography without any derivatization required prior to analysis. Localization of fatty acid hydroperoxides in complex mixtures was achieved by monitoring the loss of hydrogen peroxide using constant neutral loss scanning. In the presence of 5 mM NH4OAc in methanol-water, adduct ions [M + NH4]+ were formed almost exclusively, directly revealing the molecular mass of the thermolabile hydroperoxides. In addition, low energy collision-induced dissociation of precursor ions [M + NH4]+ led to characteristic product ions from both the 9- and 13-regioisomers. Thus, electrospray ionization-tandem mass spectrometry provides a straightforward approach for the study of the regioselectivity of lipoxygenase catalysis without any derivatization step required prior to analysis.

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Section: Methodsmentioning

confidence: 99%

Analysis of lipoxygenase‐derived fatty acid hydroperoxides by electrospray ionization tandem mass spectrometry

Schneider

Schreier

Herderich

1997

Lipids

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“…Isocratic elution was carried out using tetrahydrofuran/methanol/water/acetic acid, 25 : 30 : 45 : 0.1 (by vol. ), which had been adjusted to pH 5.5 with ammonia [16]. The aqueous phase contained 0.1% (w/v) EDTA, which improves the recovery of the sulfidopeptide leukotrienes [17].…”

Section: Reversed-phase Hplc Analysismentioning

confidence: 99%

Evidence for lipoxin formation by bovine polymorphonuclear leukocytes via triple dioxygenation of arachidonic acid

et al. 1988

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Incubation of bovine polymorphonuclear leukocytes (PMNs) with arachidonic acid leads to the formation of four lipoxins. The same lipoxins are also formed upon incubation of bovine PMNs with S(S)-hydroperoxy-6truns-8,1 l,ICciseicosatetraenoic acid, S-hydroxy-6-rrans-8,11,14-cis-eicosatetraenoic acid, S(S)-hydroperoxy, 15(S)-hydroxy-6,13-rrans-8,ll -cis-eicosatetraenoic acid or S(s), 1 S(S)-dihydroxy-6,13-fruns-8,11 -cis-eicosatetraenoic acid. A 5,6-epoxide as intermediate in lipoxin formation in the bovine PMN is highly improbable because the 5-hydroxy compounds are as good substrates as the 5-hydroperoxy compounds. Moreover, the two main lipoxins were found to coelute with the two lipoxins produced via a triple dioxygenation of arachidonic acid by soybean lipoxygenase-I . Hence the bovine PMN is the first cell for which evidence is presented that the formation of lipoxins proceeds mainly via triple dioxygenation and not via 15-hydroxy-leukotriene A., as is proposed for human and porcine PMNs.

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“…Identification was based on co-elution with synthetic LTB 4, on UV spectroscopy and on comparison with a corresponding RP-HPLC system [5]. The amounts of LTB, released were found to be dependent on the concentrations of arachidonic acid added to PMN (see Table).…”

mentioning

confidence: 99%

“…The supernatant fractions were extracted for leukotrienes using C18 reverse-phase extraction columns. The amounts of LTB4 were determined by reverse-phase high-performance liquid chromatography (RP-HPLC) on a Nucleosil 5C18 column with a mobile phase consisting of tetrahydrofuran-methanolwater-acetic acid (25:30:45:0.1; pH = 5.5) at a flow rate of 1 ml/min [5]. UV absorbance was measured at 280 nm.…”

mentioning

confidence: 99%

Production of leukotriene B4 by polymorphonuclear leukocytes during phagocytosis ofStaphylococcus aureus

1986

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Polymorphonuclear leukocytes (PMN) play an important role in the host defense against invading microorganisms and in the cellular response in inflammatory reactions. Specifically stimulated PMN convert arachidonic acid into prostaglandins and leukotrienes [1,2]. Of these aracbidonic acid metabolites, leukotriene B4 (LTB4) is thought to play a major role as a soluble mediator in inflammatory reactions. This compound binds via a receptor to PMN, induces adherence to vascular endothelium and is a potent chemotactic agent [3]. Therefore, LTB4 can act as an amplification system for the attraction of PMN to the area of inflammation. Most in vitro studies have focussed on the formation of LTB4 by PMN upon stimulation with soluble stimuli such as the calcium-ionophore A23187 in the presence of exogenous arachidonic acid. Fewer data are available on LTB4 release during the phagocytic process of PMN. In the present study, we investigated the release of LTB4 by human PMN during phagocytosis of S. aureus and/or during incubation with arachidonic acid.PMN were isolated by dextran sedimentation, differential density centrifugation, and NH4C1 lysis of contaminating red blood cells [4]. S. aureus was prepared and opsonized in 10% human pooled serum as previously described [4]. Arachidonic acid (5,8,1 I, 14-eicosatetraenoic acid) was stored as a stock solution of 10 mg/ml in n-hexane. Just before starting the experiment, the solvent was evaporated rapidly under nitrogen. The acid was redissolved in a 0.1 M KOH solution in ethanol and again evaporated to give the potassium salt. This material was dissolved in 50 mM Tris-HCl buffer (pH = 7.5). 12 x l0 T PMN were incubated with arachidonic acid in the presence or absence of S. aureus for I0 min at 37~ in a total volume 4 ml. The supernatant fractions were extracted for leukotrienes using C18 reverse-phase extraction columns. The amounts of LTB4 were determined by reverse-phase high-performance liquid chromatography (RP-HPLC) on a Nucleosil 5C18 column with a mobile phase consisting of tetrahydrofuran-methanolwater-acetic acid (25:30:45:0.1; pH = 5.5) at a flow rate of 1 ml/min [5]. UV absorbance was measured at 280 nm.When human PMN were incubated with arachidonic acid, and the extracted supernatant fractions subjected to RP-HPLC, a major metabolite was identified at LTB4. Identification was based on co-elution with synthetic LTB 4, on UV spectroscopy and on comparison with a corresponding RP-HPLC system [5]. The amounts of LTB, released were found to be dependent on the concentrations of arachidonic acid added to PMN (see Table). By using concentrations lower than 100/zM arachidonic acid, no detectable amounts of LTB4 were released by the cells. After stimulation of PMN with S. aureus (ratio 1:25), no detectable amounts of arachidonic acid metabolites were released by the neutrophils. However, when low amounts of arachidonic acid were present during the incubation of PMN with bacteria, LTB4 release could be detected (see Table). These concentrations of arachidonic acid by themselve...

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Separation and quantitation of leukotrienes by reversed-phase high-performance liquid chromatography

Cited by 50 publications

References 11 publications

Analysis of lipoxygenase‐derived fatty acid hydroperoxides by electrospray ionization tandem mass spectrometry

Analysis of lipoxygenase‐derived fatty acid hydroperoxides by electrospray ionization tandem mass spectrometry

Evidence for lipoxin formation by bovine polymorphonuclear leukocytes via triple dioxygenation of arachidonic acid

Production of leukotriene B4 by polymorphonuclear leukocytes during phagocytosis ofStaphylococcus aureus

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