A new method to evaluate anti-allergic effect of food component by measuring leukotriene B4 from a mouse mast cell line

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Abstract

Leukotrienes (LTs), chemical mediators produced by mast cells, play an important role in allergic symptoms such as food allergies and hay fever. We tried to construct an evaluation method for the anti-LTB 4 activity of chemical substances using a mast cell line, PB-3c. PB-3c pre-cultured with or without arachidonic acid (AA) was stimulated by calcium ionophore (A23187) for 20 min, and LTB 4 production by the cells was determined by HPLC with UV detection. LTB 4 was not detected when PB-3c was pre-cultured without AA. On the other hand, LTB 4 production by PB-3c pre-cultured with AA was detectable by HPLC, and the optimal conditions of PB-3c for LTB 4 detection were to utilize the cells pre-cultured with 50 µM AA for 48 h. MK-886 (5-lipoxygenase inhibitor) completely inhibited LTB 4 production, but AACOCF 3 (phospholipase A 2 inhibitor) slightly increased LTB 4 production, suggesting that LTB 4 was generated from exogenous free AA through 5-lipoxygenase pathway. We applied this technique to the evaluation of the anti-LTB 4 activity of food components. PB-3c pre-cultured with 50 µM AA for 48 h was stimulated with A23187 in the presence of 50 µM soybean isoflavones (daidzin, genistin, daidzein, and genistein), equol, quercetin, or kaempferol. Genistein, equol, quercetin, and kaempferol strongly inhibited LTB 4 production without cytotoxicity. These results suggest that a new assay system using PB-3c is convenient to evaluate LTB 4 inhibition activity by food components. This method could be utilized for elucidation of the mechanisms of LTB 4 release suppression by food components such as flavonoids and the structure–activity relationship.

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The high affinity receptor for immunoglobulin E (designated Fc epsilon RI) is the member of the antigen (Ag) receptor superfamily responsible for linking pathogen-or allergen-specific IgEs with cellular immunologic effector functions. This review provides background information on Fc epsilon RI function combined with more detailed summaries of recent progress in understanding specific aspects of Fc epsilon RI biology and biochemistry. Topics covered include the coordination and function of the large multiprotein signaling complexes that are assembled when Fc epsilon RI and other Ag receptors are engaged, new information on human receptor structures and tissue distribution, and the role of the FcR beta chain in signaling and its potential contribution to atopic phenotypes.

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Juan A. Rivera, Alasdair M Gilfillan (2009)

Mast cell mediator release represents a pivotal event in the initiation of inflammatory reactions associated with allergic disorders. These responses follow antigen-mediated aggregation of immunoglobulin E (IgE)-occupied high-affinity receptors for IgE (Fc epsilon RI) on the mast cell surface, a response which can be further enhanced following stem cell factor-induced ligation of the mast cell growth factor receptor KIT (CD117). Activation of tyrosine kinases is central to the ability of both Fc epsilon RI and KIT to transmit downstream signaling events required for the regulation of mast cell activation. Whereas KIT possesses inherent tyrosine kinase activity, Fc epsilon RI requires the recruitment of Src family tyrosine kinases and Syk to control the early receptor-proximal signaling events. The signaling pathways propagated by these tyrosine kinases can be further upregulated by the Tec kinase Bruton's tyrosine kinase and downregulated by the actions of the tyrosine Src homology 2 domain-containing phosphatase 1 (SHP-1) and SHP-2. In this review, we discuss the regulation and role of specific members of this tyrosine kinase network in KIT and Fc epsilon RI-mediated mast cell activation.

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Evidence for lack of absorption of soy isoflavone glycosides in humans, supporting the crucial role of intestinal metabolism for bioavailability.

Beth L. Zimmer, Kenneth Setchell, R. M. Brown … (2002)

The isoflavones daidzein and genistein occur naturally in most soyfoods, conjugated almost exclusively to sugars. Controversy exists regarding the extent of bioavailability of isoflavone glycosides, and the mechanism of intestinal absorption of isoflavones in humans is unclear. Evidence from intestinal perfusion and in vitro cell culture studies indicates that isoflavone glycosides are poorly absorbed, yet isoflavones are bioavailable and appear in high concentrations in plasma, irrespective of whether they are ingested as aglycones or glycoside conjugates. The objective was to determine whether isoflavone glycosides are absorbed from the intestine intact and reach the peripheral circulation unchanged. Plasma was collected at timed intervals before and after healthy adults ingested 50 mg of one of the isoflavone beta-glycosides (daidzin or genistin) or 250 mL soymilk containing mainly isoflavone glycosides. Electrospray ionization mass spectrometry was used to detect daidzin and genistin after solid-phase extraction of these conjugates from plasma. Bioavailability of isoflavones was confirmed by gas chromatography-mass spectrometry analysis. Specific and sensitive electrospray mass spectrometry failed to detect even traces of daidzin or genistin in plasma collected 1, 2, and 8 h after their ingestion as pure compounds or in a soyfood matrix. However, plasma was enriched in isoflavones that were hydrolyzable with a combined beta-glucuronidase and sulfatase enzyme preparation. Isoflavone glycosides are not absorbed intact across the enterocyte of healthy adults, and their bioavailability requires initial hydrolysis of the sugar moiety by intestinal beta-glucosidases for uptake to the peripheral circulation.