Bernard Fruteau de Laclos

Eosinophil-rich human polymorphonuclear leukocyte preparations characteristically release leukotriene C4 on ionophore A23187 challenge

Blood samples were obtained from a group of 20 patients with hypereosinophilia (greater than or equal to 1500 eosinophils/mm3). The polymorphonuclear leukocytes (PMNLs) were prepared from blood treated with ethylenediaminetetra-acetic acid by successive dextran sedimentation of the red blood cells, separation of mononuclear leukocytes and PMNLs on Ficoll-Paque, and ammonium chloride treatment of the PMNL fraction. The eosinophil content of the final PMNL preparations ranged from 15% to 75%, as assessed by Wright-stained smears, and the remaining leukocytes were predominantly neutrophils with only 3% to 5% mononuclear cells. The eosinophil-rich PMNL preparations as well as PMNL preparations from normal volunteers were incubated under various conditions and the arachidonic acid metabolites were analyzed by reverse-phase high-performance liquid chromatography. The synthesis of 5-lipoxygenase products was strongly stimulated by the ionophore A23187 in both normal and eosinophil-rich PMNL preparations. Whereas the normal PMNL preparations, which were eosinophil poor, produced 10 to 25 times more leukotriene B4 than leukotriene C4, the eosinophil-rich PMNL preparations characteristically released leukotriene C4 in equal or up to 20 times greater amounts than leukotriene B4.

Studies on the mechanism of formation of the 5S,12S-dihydroxy-6,8,10,14(E,Z,E,Z)-icosatetraenoic acid in leukocytes

Incubation of peripheral blood leukocytes with arachidonic acid (and ionophore A23187) led to the formation of leukotriene B4, delta 6-trans-leukotriene B4, delta 6-trans-12-epi-leukotriene B4, 5-hydroxy-icosatetraenoic acid, 12-hydroxy-icosatetraenoic acid and of 5S,12S-dihydroxy-6,8,10,14-(E,Z,E,Z)-icosatetraenoic acid (5S,12S-DiHETE). Incubation of leukocytes with leukotriene A4 resulted in the formation of leukotriene B4 and of its two delta 6-trans-isomers but not of the 5S, 12S-DiHETE. 18O2 labeling experiments have shown that the hydroxyl groups at C5 and C12 in the 5S,12S-DiHETE are derived from molecular oxygen. The tetraacetylenic analog of arachidonic acid was found to be potent inhibitor of the formation of the 5S,12S-DiHETE whereas it potentiated the synthesis of the 5-hydroxy acid and of leukotriene B4. Addition of the 12-hydroxy-icosatetraenoic acid to leukocytes, or of the 5-hydroxy-icosatetraenoic acid to a suspension of platelets caused the formation of the 5S,12S-DiHETE. It is concluded that the 5S,12S-DiHETE is not derived from leukotriene A4 but is a product of the successive reactions of arachidonic acid with two lipoxygenases of different positional specificities.

Leukotrienes: biosynthesis, metabolism, and analysis

Publisher Summary This chapter discusses biosynthesis, metabolism, and analysis of leukotrienes (LTs). The discovery of the LTs further emphasized the complexity of the metabolism of arachidonic acid in mammalian cells. As the initial report of the occurrence of 5-HETE in rabbit peritoneal polymorphonuclear leukocyte, an impressive number of novel structures has been described and their mechanisms of formation have been elucidated in most cases. Chemical synthesis has made possible pharmacological studies on the LTs, which show potent biological activities, and assay methods are at present available. The presence in a single cell type of several metabolic pathways of arachidonic acid suggests complex regulation mechanisms for the synthesis of the various bioactive metabolites. Although their potent pharmacological properties have been clearly demonstrated, only the measurement of the synthesis of these substances under normal and various pathological situations will permit their regulatory functions in the body to be ascertained. Such studies will require a better knowledge of the in vivo metabolism of the LTs and the development of assay methods for stable plasma or urinary metabolites. Thus, it appears that a new field of biomedical investigation has just been opened.

Characteristics of leukotriene (LT) and hydroxy eicosatetraenoic acid (HETE) synthesis in human leukocytes in vitro : Effect of arachidonic acid concentration

Suspensions of human blood leukocytes and platelets were stimulated with the ionophore A23187 in the presence of increasing concentrations of arachidonic acid. Nine metabolites of the 5-, 12- and 15-lipoxygenase and the cyclooxygenase pathways were analyzed by high performance liquid chromatography in order to study the rate-limiting steps and other characteristics of their biosynthesis. The data indicate that the LTA4 hydrolase and the LTA4 glutathione transferase activities are respectively the limiting factors in the synthesis of LTB4 and LTC4. At high substrate concentrations (5 X 10(-5)M and above), the 5-lipoxygenase activity was inhibited whereas the synthesis of the 15-HETE (15-lipoxygenase product) increased linearly up to the highest concentration tested (3 X 10(-4)M). The data also indicate that contrary to 5-HETE and LTs, 15-HETE is not formed upon stimulation with the ionophore, but only following incubation with exogenous arachidonic acid (20 microM and above), pointing out major differences in the synthesis of 5- and 15-lipoxygenase products.

Conditions for the formation of the oxo derivatives of arachidonic acid from platelet 12-lipoxygenase and soybean 15-lipoxygenase.

Three carbonyl compounds derived from arachidonic acid have recently been characterized in human platelets, namely, 12-ketoeicosatetraenoic acid and two isomeric 12-oxododecatrienoic acids. The conditions for the synthesis of these compounds and for the synthesis of analogous products from soybean lipoxygenase, i.e., 15-ketoeicosatetraenoic acid and 15-oxopentadecatetraenoic acids, were compared with regard to the role of oxygen and fatty acid availability, and heme catalysis. Using platelet homogenates or soybean lipoxygenase and arachidonic acid as a substrate, it was found that the establishment of anaerobic conditions during the incubation was mandatory only for the synthesis of 15-oxopentadecatetraenoic acids. Anaerobic conditions, however, greatly increased the formation of 15-ketoeicosatetraenoic acid and, to a lesser extent, of 12-oxododecatrienoic acids. On the other hand, 12-hydroperoxyeicosatetraenoic acid (12-HPETE) was transformed into 12-ketoeicosatetraenoic acid and 12-oxododecatrienoic acids by platelet homogenates or soybean lipoxygenase. This transformation was increased when the incubation was performed in anaerobic conditions and in the presence of a fatty acid substrate of the enzyme. These data suggest that oxygen deprivation and excess fatty acid could play a stimulatory role in the synthesis of 12-oxo compounds by platelets. Finally, we have compared the heme-catalyzed generation of the 12-oxo and 15-oxo derivatives from their hydroperoxide precursors: whereas 12-oxododecatrienoic acids and 12-ketoeicosatetraenoic acid were formed in the proportion of 8.5: 1.5 from 12-HPETE incubated with hematin (150 nM), 15-ketoeicosatetraenoic acid was the only carbonyl compound generated from 15-HPETE in the same conditions, emphasizing the unique reactivity of the 12-HPETE.

Calcium mobilization and right-angle light scatter responses to 12-oxo-derivatives of arachidonic acid in neutrophils: evidence for the involvement of the leukotriene B4 receptor

The biological activities of two carbonyl compounds derived from arachidonic acid, (5Z,8Z,10E,14Z)-12-keto-5,8,10,14-eicosatetraeno ic acid (12-OxoETE) and (5Z,8Z,10E)-12-oxo-5,8,10-dodecatrienoic acid (12-OxoDTrE) were investigated. The ability of these compounds to induce a mobilization of calcium and to trigger a right-angle scatter response in isolated peripheral blood human neutrophils was determined. The two compounds induced a rapid and dose-dependent increase in the concentration of cytoplasmic free calcium; these effects were clearly detectable at concentrations greater than or equal to 10(-8) M. Pre-exposure of neutrophils to leukotriene B4 completely abolished the calcium mobilization induced by 12-OxoDTre and 12-OxoETE, while pre-exposure of the cells to the carbonyl compounds only slightly reduced the response to subsequent stimulation of neutrophils by leukotriene B4. The carbonyl compounds also induced a decrease in right-angle light scatter and these effects were abolished by pretreatment of neutrophils with leukotriene B4. These data demonstrate that 12-OxoETE and 12-OxoDTrE show significant agonist activities towards human neutrophils and strongly suggest that their mechanisms of action involve the leukotriene B4 binding sites or a common activation sequence.

A new rapid immunoinhibition pancreatic amylase assay: Diagnostic value for pancreatitis

A new rapid immunoinhibition pancreatic amylase assay was compared to total amylase and lipase in an unbiased sample of 1005 emergency department patients with suspicion of pancreatitis, of which 55 had a final diagnosis of pancreatitis. Imprecision of the assays for both amylases (less than 2.5%) were better than for lipase (less than 6.1%). Correlation (R2) of pancreatic amylase with total amylase was 0.991 but only 0.789 with lipase. Using Receiver Operator Characteristics analysis, the best diagnostic cutoff point for all three enzymes was near the upper limit of the reference interval. With pancreatic amylase, sensitivity, specificity, and predictive values for positive and negative results are, respectively, 85.5, 92.5, 39.8, and 99.1%; we found similar values for lipase but poorer values (78.2, 92.0, 36.1, and 98.7%) for total amylase. Tests combination did not improve the diagnostic performance significantly. In the diagnosis of pancreatitis, pancreatic amylase (p = 0.037) and lipase (p = 0.049) had better diagnostic performance than total amylase. The correct diagnosis of pancreatitis could be achieved in 47 instead of 43 patients with either pancreatic amylase or lipase as opposed to total amylase among 1005 patients in this study. We conclude that pancreatic amylase and lipase are incrementally better diagnostic tools than total amylase for the diagnosis of pancreatitis.

Determination of free testosterone fraction of human serum by gel bead dialysis.

The gel bead dialysis technique is an application of the steady-state gel filtration method. Preswollen gel beads are used as microdialysers. The gel is incubated for 3.5 h at 22 degrees C with 2.5 mL of serum and 3H-testosterone. During incubation, the equilibrium between free and protein-bound fractions is reached and the concentration of testosterone in the bead dialysate is equal to the free testosterone concentration in the incubation medium surrounding the beads. After incubation, the gel is separated from the medium by using a rigid filter and the radioactivity within the gel is counted. The effect on the assay of serum dilution and incubation time were studied. Within-batch imprecision (CV) was 3% (N = 30) and between-batch imprecision computed from lyophilized quality controls run over a one year-period, was 7%, 13% and 17% for free testosterone fractions (free over total) of 0.043, 0.032 and 0.021 respectively. An excellent correlation between this technique and the equilibrium dialysis method was found (r = 0.944, N = 59). Reference intervals were determined for men, women and pregnant women; mean +/- SD were 0.0216 +/- 0.0067 (N = 20), 0.0106 +/- 0.0020 (N = 20) and 0.0077 +/- 0.0017 (N = 19) respectively. Patients suffering from polycystic ovary syndrome had significantly higher free testosterone ratio (mean of 0.0285 +/- 0.0074, N = 20) than normal females.

Characterization of Epidermal Lipoxygenase Expression in Normal Human Skin and Tissue-Engineered Skin Substitutes

Lipoxygenases (LOXs) are enzymes likely to be involved in corneocyte lipid envelope formation and skin barrier function. In humans, mutations in epidermis-type lipoxygenase 3 (eLOX-3) and 12R–lipoxygenase (12R-LOX) genes are associated with autosomal recessive congenital ichthyosis (ARCI), whereas deletion of these genes in mice causes epidermal defects. LOXs also represent a matter of interest in psoriasis as well as in cancer research. However, their expression as well as the exact role of these enzymes in normal human skin have not been fully described. Our goal was to characterize the expression of epidermal LOXs in both normal human skin and Tissue-Engineered Skin Substitutes (TESS) and to consider TESS as a potential model for LOX functional studies. Staining for epidermal differentiation markers and LOXs was performed, in parallel, on normal human skin and TESS. Our results showed similar expression profiles in TESS when compared with native skin for e-LOX3, 12R-LOX, 12S-lipoxygenase (12S-LOX), and 15-lipoxygenase 2 (15-LOX-2) but not for 15-lipoxygenase 1 (15-LOX-1). Because of their appropriate epidermal differentiation and LOX expression, TESS represent an alternative model for future studies on LOX function.