Frederik J. Zijlstra

Determination of leukotrienes and prostaglandins in [14C] arachidonic acid labelled human lung tissue by high-performance liquid chromatography and radioimmunoassay

A liquid chromatographic method for the determination of 14C-labelled prostaglandins, leukotrienes and other lipoxygenase products formed by human lung tissue is described. In this paper we report our problems identifying these substances when 3H- or 14C-labelled compounds are compared with measurements of the mass by absorption or radioimmunoassay. Furthermore, some preliminary results of [14C] arachidonic acid labelled human lung tissue, stimulated by the Ca-ionophore A23187, show that, of the lipoxygenase products, mostly leukotriene B4 like compounds are formed and less leukotriene C4, E4 and D4. Relatively large amounts of hydroxyeicosatetraenoic acids are present. The main cyclooxygenase products are thromboxane B2, 6-ketoprostaglandin F1 alpha and prostaglandin D2.

Acute anti-inflammatory effects of aspirin and dexamethasone in rats deprived of endogenous prostaglandin precursors

Paw oedema, induced by carrageenan, was potentiated in normal rats by arachidonic acid and bishomo‐γ‐linoleic acid, but not by 5,8,11‐eicosatrienoic acid. The latter is not an endogenous prostaglandin precursor, but replaces the other two in essential fatty acid deficient (EFAD) rats. Carrageenan oedema was partially suppressed in these EFAD rats. Aspirin exhibited equal suppression of carrageenan oedema in both normal and EFAD rats, despite the fact that, in the latter, prostaglandins are of negligible importance. The anti‐inflammatory effect of dexamethasone was also identical in both normal and EFAD rats. The view that interference with the prostaglandin‐system explains the acute anti‐inflammatory effects of the two drugs, is discussed, in relation to the present results.

Eicosanoid levels in bronchoalveolar lavage fluid of young female smokers and non‐smokers

Abstract. To evaluate indicators of inflammatory changes in the airways of young smokers we have measured the levels of several eicosanoids in bronchoalveolar lavage (BAL) fluid of 18 female smokers (age 33 ±2 years) and 9 female non‐smokers (age 29 ± 2 years) who were hospitalized for treatment not related to any pulmonary disease. In each BAL specimen the following eicosanoids were determined by radioimmunoassay: prostaglandin (PG) E2; PGF2x; 9α, 1 lβ‐PGF2, a metabolite of PGD2; 6‐keto PGF1x, a metabolite of prostacyclin; thromboxane (Tx) B2, a metabolite of TxA2; the 5‐lipoxygenase products 5‐hydroxy‐eicosa‐tetraenoic acid (HETE), leukotriene (LT) B4 and LTC4; the 12‐lipoxygenase product 12‐HETE; and the 15‐lipoxygenase product 15‐HETE. The concentrations of the cyclooxygenase products (pg ml‐1) in the BAL fluid of the non‐smokers were: PGE2 15.4±1.9, PGF2x, 7.6±1.0, 9α,11β‐PGF2 8.7±1.8, TxB2 8.8 ± 1.3, and 6‐keto PGF1α only 1.5±0.8. The concentration of the lipoxygenase products were: 15‐HETE 781 ± 200,12‐HETE 193 ± 33,5‐HETE 14.0 ± 3.1, LTC49.5±3.1, LTB46.2± 1.4. BAL fluid from smokers contained two‐ to three‐fold higher levels of TxB2 and PGF2α (P < 0.05). The levels of TxB2 and PGF2α were positively correlated to the number of package years (rs= 0.55 and rs= 0.65, P<0.02). The concentrations of 5‐, 12‐ and 15‐HETE tended to be higher in BAL fluid from smokers, but this was not significant. We conclude that in BAL fluid from females who denied pulmonary symptoms, the concentration of 12‐ and 15‐lipoxygenase products is 15 — 100‐fold higher than that of 5‐lipoxygenase or cyclooxygenase products. In these subjects smoking is associated with higher concentrations of bronchoconstrictive cyclooxygenase products and a tendency towards higher levels of 5‐, 12‐ and 15‐HETE, probably reflecting inflammatory changes in the airways. Therefore, these results indicate that smoking induces formation of the metabolites that may play a role in the development of chronic airways obstruction especially when inflammatory changes become more pronounced after greater exposure to tobacco smoke.

The use of essential fatty acid deficient rats to study pathophysiological roles of prostaglandins. Comparison of prostaglandin production with some parameters of deficiency

In a retrospective study on essential fatty acid deficient, (EFAD) rats used to study pathophysiological roles of prostaglandins (PGs) slight increases in the linoleic acid content of the diet were found to gradually restore the depressed growth rate and to increase the reduced endogenous PG production. These apparently poorly deficient animals had a serum triene tetraene (ω9:ω6) ratio much higher than the value of 0.4 used as a criterion for EFA deficiency by nutritionists. Changes in body weight, serum ω9∶ω6 and platelet PG production were not correlated with each other. Feeding rats on a diet containing <0.1 mg/g/linoleic acid led to decreasing platelet PG production as the degree of EFA deficiency increased. At this high level of deficiency, a serum ω9∶ω6 ratio of 6 or over was achieved. This high ratio may be taken as anindicator of the degree of EFA deficiency required for studies of PG deprivation, but PG production by the tissue investigated or by plalets should preferentially be measured.

Excessive prolongation of the bleeding time by aspirin in essential thrombocythemia is related to a decrease of large von Willebrand factor multimers in plasma

Abstract Patients with essential thrombocythemia (ET), who frequently have bleeding complications, may manifest an excessive prolongation of the bleeding time (BT) after ingestion of aspirin (ASA). The reason for this excessive prolongation of the BT is unknown, but it is attributed to qualitative platelet defects. Since patients with ET may also have acquired abnormalities of plasma and platelet von Willebrand factor (vWF), we questioned whether the excessive prolongation of the BT by ASA was related to changes in either plasma or platelet vWF. To that end, we studied BT and plasma and platelet vWF in ten ET patients, ten patients with reactive thrombocytosis (RT), and ten normal individuals, both before and after administration of 500 mg ASA for 7 days. In a second study, the effect of DDAVP infusion on plasma vWF in relation to the BT was studied in ten normal individuals and ten ET patients after treatment with 100 mg ASA for 3 days. In the first study, treatment with ASA resulted in a significant prolongation of the BT in normal subjects, RT patients, and ET patients. However, in five ET patients an excessive (>2 SD) prolongation of the BT by ASA was observed. Although ASA induced no direct changes in either plasma or platelet vWF levels in either normal subjects, RT patients, or ET patients, all five ET patients who showed an excessive prolongation of the BT by ASA had significantly decreased levels of large vWF multimers in plasma. In the second study, infusion with DDAVP resulted in a significant increase in plasma large vWF multimers, paralleled by a normalization of (excessively) prolonged BT. Our data suggest that in ET inhibition of platelet function by ASA in the presence of concurrently decreased levels of large vWF multimers in plasma may have provoked the excessive BT prolongation.

Time Dependent Production of Cytokines and Eicosanoids by Human Monocytic Leukaemia U937 Cells; Effects of Glucocorticosteroids

In the present study the human monoblast cell line U937 has been used as a model to study the function of human mononuclear phagocytes in asthma. The kinetics of the production of eicosanoids and cytokines, which are thought to play a role in the pathogenesis of asthma, were studied. In addition, the effects of glucocorticosteroids were investigated, as these drugs are of great importance for the treatment of asthmatic patients. After stimulation with phorbol-12 myristate acetate (PMA) for 24 h, U937 cells were cultured in the absence or presence of lipopolysaccharide (LPS: 1 and 5 microg ml(-1)) and glucocorticosteroids (budesonide, fluticasone propionate and prednisolone: 10(-11), 10(-9) and 10(-7) M) for 96 h. The production of interleukin-1beta (IL-1beta), interleukin-6 (IL-6), prostaglandin E2 (PGE2) and thromboxane B2 (TxB2) gradually increased in time after stimulation with LPS, whereas the transient production of tumor necrosis factor alpha (TNF-alpha) reached its maximum between 6 and 12 h. Interferon-gamma (IFN-gamma), interleukin-10 (IL-10) and leukotriene B4 (LTB4) were not detectable. All three glucocorticosteroids (budesonide, fluticasone propionate and prednisolone) completely inhibited the production of both eicosanoids and cytokines. The production of eicosanoids was more sensitive to these glucocorticoids than the production of cytokines. The observed differences in the kinetics of the production of eicosanoids and cytokines stress the importance of time course experiments in studies on the effect of drugs on mononuclear cells.

Nicotinic acid inhibits thromboxane synthesis in platelets

The formation of thromboxane A2 by phospholipase A2 in rat platelets is inhibited by nicotinic acid. The synthesis of PGE2 and PGF2α is increased. Nicotinic acid inhibits collagen-induced aggregation in rat platelets.

Accumulation of blood platelets in carrageenin rat paw oedema. Possible role in the inflammatory process

The accumulation of blood platelets in the carrageenin-induced paw oedema in rats was studied, using51Cr-labeled platelets. A maximum in the accumulation was seen after 4 hours, followed by a decline after 5–6 hours. During the first 6 hours of the oedema formation, changes in blood volume were small. A comparison was made with the accumulation of both125J-albumin as a measure of extravasation and125J-fibrinogen as an indication of blood clotting. When platelet aggregation was measured during the first 6 hours of the oedema formation, the lag time between the addition of collagen and the beginning of the aggregation was increased. No change in platelet serotonin was seen. These data support the idea that platelets participate in the inflammatory process.

The effects of leukotrienes, thromboxane A2 and phospholipase A2 on human, porcine and guinea pig lung parenchyma

A comparison was made of the contractions, induced by LTD4, histamine and phospholipase A2 in parenchymal strips of guinea pig (GPLP), porcine and human lung in a cascade superfusion system. The effects of LTD4 and phospholipase A2 on the release of TxA2 in these tissues and of TxA2, 5-HT and acetylcholine on the GPLP were also determined.In the GPLP strip, the LTC4-induced contractions are due for±80% to the release of TxA2 and for±20% to the direct effect of LTC4.The guinea pig tissue displayed the highest sentivity towards all substances, except to the contraction induced by histamine, which was most effective in the porcine tissue. Low activities wer found in the human tissue in all tests. The reason for these effects may be a difference in activities or number of cell types which participate in the reactions leading to the contractions.

Formation by phospholipase A2 of prostaglandins and thromboxane A2-like activity in the platelets of normal and essential fatty acid deficient rats. Comparison with effect on human and rabbit platelets

When rat platelets are incubated with phospholipase A2, thromboxane A2-like activity and prostaglandins are formed. The amounts are approximately similar, whether aggregation is induced after the incubation or not. No aggregation is observed when the platelets are incubated with phospholipase A2. In the platelets of essential fatty acid deficient rats, only small amounts of thromboxane A2-like acitivity and prostaglandins are formed. No formation of these substances occurs when human and rabbit platelets are incubated with phospholipase A2. The results indicate that formation of thromboxane A2-like activity enhances aggregation in rat platelets, but that aggregation is not induced.