On the role of PGD2 metabolites as markers of mast cell activation in asthma

Abstract: Prostaglandin D2 (PGD2) is the major cyclooxygenase metabolite of arachidonic acid released after stimulation of mast cells. Prostaglandin D2, itself is a very unstable compound, therefore, quantitative determination of PGD2 is an unreliable indicator of its own endogenous production. However, quantification of metabolites of PGD2 can be used as an objective indices of PGD2 production and hence mast cell activation in vivo. The aim of this thesis was to investigate the feasibility of measuring the primary urinary metabolite of PGD2, 9[alpha],11ß-PGF2 with enzyme immunoassay (EIA). This strategy represents a simple, non- invasive approach to following temporal changes in mast cell activation. Despite the many advantages of EIA, it can suffer from a lack of selectivity, therefore measurements of 9[alpha],11ß-PGF2 in urine made by EIA were compared with values obtained by negative ion chemical ionisation gas chromatography-mass spectrometry (NCI GC-MS), the gold standard method. Levels of 9[alpha],11ß-PGF2, in urine samples measured by NCI GC-MS were consistently lower than those obtained by EIA. NCI GC-MS analysis revealed the presence of two additional dinor compounds, shorter metabolites of 9[alpha],11ß-PGF2 in the urine. One of the compounds was identical to 9[alpha],11ß-2,3-dinor-PGF2 which was generated by ß-oxidation of 9[alpha],11ß-PGF2 and identified by electron impact (EI GC-MS). Thus, urinary 9[alpha],11ß-PGF2 concentrations measured by EIA represent the sum of three PGD2 metabolites. For convenience sake, the metabolites are collectively referred to as 9[alpha],11ß-PGF2 in the subsequent studies. Urine samples collected at 3 h intervals over a 24 h period from a group of healthy volunteers displayed no diurnal variation in the urinary excretion of 9[alpha],11ß-PGF2. There was a 3-fold increase in the urinary excretion of 9[alpha],11ß-PGF2 after allergen-induced bronchconstriction in nine atopic asthmatics. This challenge was considered a positive control since it is unambiguous that mast cell activation occurs during the early phase of allergen- induced airway obstruction. Histamine-induced bronchcoconstriction did not result in an increase in the levels of 9[alpha],11ß-PGF2 demonstrating that PGD2 was not formed as a consequence of the bronchoconstriction per se. Moreover, bronchial challenge with lysine- aspirin in eight aspirin-intolerant asthmatics elicited bronchoconstriction and was accompanied by a significant increase in the urinary excretion of 9[alpha],11ß-PGF2. Challenge with a higher dose of aspirin produced an even greater increase in 9[alpha],11ß-PGF2 levels, indicating a dose-dependent release of PGD2 during aspirin-induced bronchconstriction. The pattern of mediator release during the early (EAR) and late asthmatic response (LAR) to allergen was investigated by subjecting twelve mild atopic asthmatics to allergen challenge. Within one hour of the maximal bronchoconstrictor response, there was a significant increase in the urinary concentrations of the mast cell markers, 9[alpha],11ß-PGF2 and N-methylhistamine, urinary metabolite of histamine, and the end product of the cysteinyl-leukotrienes, leukotriene (LT)E4. Levels of all three mediators were also significantly elevated above baseline during the LAR. Urinary levels of eosinophil protein X (EPX), a marker of eosinophil activation, remained unaltered during both the EAR and LAR. It appears that there was diurnal variation in the urinary excretion of EPX. Increased airway fluid osmolarity in the lower airways as a result of exercise, has been suggested to trigger mast cell activation and subsequent bronchconstriction in a subset of asthmatics. Twelve subjects with a history of exercise-induced bronchconstriction (EIB), exercised on a stationary bicycle ergometer for 5 minutes. Seven of the subjects (responders) experienced bronchconstriction, whereas, the pulmonary function of the remaining five subjects (non-responders) remained stable. The urinary excretion of 9[alpha],11ß-PGF2 in the responder group increased significantly compared to the nonresponders at 30 and 90 minutes after exercise. The urinary excretion of LTE4 and N-methylhistamine was not significantly different between the two groups at either time point after exercise, although there was a tendency for elevated levels of N-methylhistamine in the responder group. A 3 h exposure of ten healthy volunteers to swine house dust resulted in bronchial hyperresponsiveness (BHR) to methacholine as assessed the day after the exposure, but no acute bronchconstriction. The BHR was associated with marked elevations of urinary LTE4, levels peaking 46 h after the commencement of the exposure. Levels of LTE4 measured in nasal lavage seven hours after the exposure were also significantly increased. Urinary 9[alpha],11ß-PGF2 excretion was also significantly elevated above pre-challenge levels following the exposure to swine dust. These data imply a putative causal link between these mediators and the development of BHR. In conclusion the findings from this work support that measurements of 9[alpha],11ß-PGF2, encompassing 9[alpha],11ß-2,3-dinor-PGF2 in unextracted urine by enzyme immunoassay represents a rapid, sensitive and sufficiently specific approach to monitoring activation of the PGD2 pathway, thereby providing a valuable analytical tool to further explore the role of the mast cell in a