Leukotrienes detection

The detection of reactions mediated by specific IgE to agents triggering anaphylaxis may be achieved by means of serological methods serum-specific IgE, or by means of cellular tests which determine the release of basophil mediators (leukotrienes and histamine) or by means of the analysis of basophil expression markers, a technique known as the basophil activation test (BAT). 

Lipoxygenases catalyse the regio-specific and stereoselective oxygenation of unsaturated fatty acids. The mammalian enzymes have been detected in human platelets, lung, kidney, testes and white blood cells. The leukotrienes, derived from the enzymatic action of the enzyme on arachidonic acid, have effects on neutrophil migration and aggregation, release of lysosomal enzymes, capillary permeability, induction of pain and smooth muscle contraction (Salmon, 1986). 

This assay procedure has been applied to various haptens having a primary amino group, including leukotriene C4 (VI), T4 (P2), substance P (P2), endothelin (P2), and AGII (G2) (for structures, see Fig. 5A and 5C). These noncompetitive assays were 10-300-fold more sensitive than corresponding competitive assays (G2). The minimal detectable concentrations described in these papers were as follows 2 pg/ml ( 0.3 fmol/assay) for leukotriene C4, 14 pg/ml ( 0.2 fmol/assay) for T4, 6 pg/ml ( 0.4 fmol/assay) for substance P, and 20 pg/ml ( 0.8 fmol/assay) for endothelin. 

Lipids are made up of many classes of very different molecules that all show solubility properties in organic solvents. Mass spectrometry plays a key role in the biochemistry of lipids. Indeed, mass spectrometry allows not only the detection and determination of the structure of these molecules but also their quantification. For practical reasons, only the fatty acids, acylglycerols and bile acids are discussed here, although other types of lipids such as phospholipids, [253-256] steroids, [257-259] prostaglandins, [260] ceramides, [261,262] sphingolipids [263,264] and leukotrienes [265,266] have been analysed successfully by mass spectrometry. Moreover, the described methods will be limited to those that are based only on mass spectrometry, even if the majority of these methods generally are coupled directly or indirectly with separation techniques such as GC or HPLC. A book on the mass spectrometry of lipids was published in 1993. [267]... 

The numerous biochemical substances that may be considered with regard to hepatic encephalopathy or ascites have been outlined in detail, (s. tabs. 15.2 16.5) Similarly, an extensive synopsis of pathogenetically effective biochemical factors can be drawn up for HRS as well. All of them ultimately interfere — directly or indirectly - with the renal retention or excretion of sodium and/ or the balance between vasodilation and vasoconstriction. RAAS is markedly activated. (5, 28, 33, 41, 62, 64, 65) (s. tab. 17.1) Vasodilative factors under discussion include bilirubin, bile acids, nitrogen monoxide (NO), false neurotransmitters, calcitonin peptide (25) as well as platelet-activating factor (RAF). In more recent studies, considerably higher plasma values of the vasoconstrictor leukotrienes (C4 and D4) (41) and endothehn 1 and 3 (42) were detected. 

In connection with the work on the relationship between chemical structure and anti-inflammatory activity, the effect of ursolic acid, betulin, betulinic acid and erythrodiol on a system of chronic dermal edema and cellular proliferation caused by repeated administration of TPA has recently been examined [89], This experimental model of chronic inflammation has considerable selectivity for corticosteroids and leukotriene synthesis inhibitors. Erythrodiol and ursolic acid were significantly effective and also reduced the neutrophil infiltration detected by MPO activity. The lupane derivatives, betulin and betulinic acid, despite their possible steroid-like mechanism of action [47], were not effective in the chronic model. This result could mean that a six-member E ring of the pentacyclic structure is necessary for the activity against a multiple dose of TPA. The data confirm that a hydroxyl group at the C-28 position is important for the activity, as is also true in the case of erythrodiol, and it may explain the anti-inflammatory effect of this compound in each of the methods. 

Enzymic lipid peroxidation reactions are a common reaction in mammalian cells as response to stress from outside. In contrast to plants mammalian cell membranes contain arachidonic acid instead of linolenic acid. The latter is known to generate by LPO processes physiological potent compounds prostaglandines, prostacyclines, thromboxanes and leukotrienes [221]. In addition a great number of other LPO products derived not only from arachidonic acid but also from linoleic acid have been detected in blood, especially low density lipoprotein (LDL) and tissue samples of humans [222-227]. These are increased especially in diseases combined with cell degradation [228], indicating that cell death in plant and mammalian cells is connected with a similar cascade of events. 

The role of LTB as an inflammato mediator is supported by studies showing high amounts of LTB. in the skin chan r fluid of involved skin from psoriatics ° and in gouty effusions. In contrast, LTC. and LTD, but not LTB, are detected by bioassay and HPLC in carrageenan-induced pleurisy in rats. Leukotrienes may also be involved in the modulation of pain in inflanmatory lesions. In the rat paw, LTB. causes... 

Fig. 11.5.4. HPLC separation of leukotriene standards. Chromatographic conditions column, 5 im ODS (250x4.6 mm I.D.) coupled to a guard column packed with Corasil (ODS) pellicular packing mobile phase, methanol-water-acetic acid-ammonium hydroxide (67 33 0.08 0.04), pH 6.2 flow rate, 1 ml/min detection, UV at 280 nm. The peaks shown include two non-enzymatically formed d -/rans-leukotriene B4 (diHETE) isomers of leukotriene B4. Reproduced from Metz et al. (1982), with permission.

An alternative strategy for detection, where cellular systems or enzymatic fluxes are being investigated, is the use of radioactively labelled arachidonic acid or glutathione, which can function as precursor molecules and facilitate detection of the synthesised radio-labelled leukotrienes. The labelled molecules can be detected either directly by a radioactive flow monitor, or indirectly by fraction collection and scintillation counting. 

Historical At the beginning of the 1930 s U. S. von Euler and M. W. Goldblatt independently detected a substance with activity in smooth musculature in seminal fluid, the structure of this compound was elucidated by Beigstrdm in 1958-1964. Further key data 1968 total synthesis by Corey, beginning of the 1970 s elucidation of the mechanism of action of nsaid (nonsteroidal anti-inflammatory drugs), 1975 thromboxane, 1976 prostacyclin, 1979 leukotrienes, 1982 Nobel Prize for Medicine awarded to Vane, Samuelsson, and Bergstrom. ... 

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