Urinary Leukotriene E4 Levels after Allergen and Exercise Challenge in Bronchial Asthma 1- 3

CHRISTINE M. SMITH, PANDORA E. CHRISTIE, RICHARD J. HAWKSWORTH, FRANCIS THIEN, and TAK H. LEE

The sulfidopeptide leukotrienes (LTC4 , LTD4 , and LTE4 ) constitute the activity previously referred to as the slow-reacting substance of anaphylaxis (SRS-A).They enhance microvascular permeability, increase nonspecific bronchial hyperresponsiveness, and are potent bronchoconstrictors (1). They therefore have considerable potential to contribute to the pathophysiologic features of asthma. The role of the sulfidopeptide leukotrienes in exercise-induced asthma is not yet established. The LTD4 antagonist LY 171883 causes a small but significant shift in the dose-response curve to cold-air hyperventilation challenge in subjects with asthma (2). More recently, Manning and coworkers (3) reported a more substantial effect of the LTD4 antagonist MK571 on the response to exercise, and Israel and colleagues (4) reported that a 5-lipoxygenase inhibitor (A-64077)attenuates responsiveness to cold-air hyperventilation challenge. The evidence from these antagonist studies suggests that the leukotrienes contribute to the bronchoconstriction induced by exercise. The leukotrienes are derived from arachidonic acid, which is released from membrane phospholipids via the 5-lipoxygenase pathway. The unstable intermediary epoxide, LTA4 , is enzymatically metabolized to LTC4 , which is converted in turn to LTD4 and then to LTE4 (1). LTE4 can be measured in urine, and urinary levels of LTE4 have been used to reflect systemic release of sulfidopeptide leukotrienes (5-9). LTE4 concentrations in urine are significantly increased in response to allergen challenge in asthmatic subjects (6-8) and in response to aspirin challenge in aspirin-sensitive asthmatic subjects (9). If leukotrienes are released in exercise-induced asthma, it might be possible to detect an increase in urinary levels of LTE4 after exercise.Wehavetherefore measured urinary LTE4 before and for 24 h after exercise challenge in six subjects with exercise-induced asthma. The results have been compared with those observed after allergen challenge in five asthmatics. Six subjects with exercise-induced asthma participated in the study. Their details are given in table 1. Medications were withheld for at least 12 h before challenge. The subjects ran on a treadmill, and the work load was increased over the first 3 min until the

SUMMARY Urinary leukatrlene E. (LTE.) concentrations _re measured In six asthmatic subjects after treadmill exercise, and in five asthmatic subJeeta after allergen challenge. Exercise and allergen challenge produced a 42 :t: 18% (mean ± SO) and 22 :t: 8% filII In FEV" respectively. The baseline concentration of urlnery LTE.ln subJecla challenged with exercise _s 64 (27 to 150)pg/mg creatinine (geometric mean and 95% confidence Interval), and In those challenged with allergen it was 36 (23 to 59) pglmg creatinine. Urinary LTE.concentrations did not change significantly In the 24 h after exercise. In contrast, there _s e mean 4-fold Increase In urinary LTE. during the 3 h after allergen chellenge. AM REV RESPIR DIS 1991; 144:1411-1413

TABLE 1 ANTHROPOMETRIC DATA FOR EACH OF THE SUBJECTS CHALLENGED, TOGETHER WITH BASELINE MEASUREMENTS OF FEV,. MAXIMAL PERCENT FALL IN FEV, AFTER CHALLENGE, AND THEIR CURRENT MEDICATIONS Subject No. Exercise 1 2 3 4 5 6

Age

Height

Baseline FEV,

% Fall in

Sex

(yr)

(em)

(L)

FEV,

Medications

Atopy

F F M M M M

18 22 29 25 24 23

170 180 176 188 188 190

3.10 3.03 3.92 3.42 3.89 4.31

52 66 17 48 25 45

AlbuteroI Albulerol Albuterol Albuterol Albuterol Albuterol Cromolyn beclomethasone

+

24 4

182 8

3.61 0.51

42 18

32 34 33 22 23 28 6

187 173 179 170 170 176 7

3.40 3.65 5.00 2.91 3.08 3.61 0.83

22 15 15 25 35 22 8

Mean SO Allergen 7 8 9 10 11 Mean SO

M M M

F F

heart rate was 75070 of the predicted maximal value. The subjects continued to exercise at this intensity for a further 6 min. The inspired air was delivered cool and dry. Dry medical air was delivered to a 1000-Lreservoir (P.K. Morgan, Kent, UK). As the subject inhaled, air passed from the reservoir through a heat exchanger and was delivered via a two-way valve and face mask. The mean inspired air temperature measured at the inspiratory port of the valve was 15.2 ± 1.6° C. Measurements of FEV, were made in triplicate and before and at I, 3, S, 7, 10, and 15 min after exercise, and then at 5-min intervals until the minimal value had been recorded. Subjects recovered spontaneously, and refrained from taking bronchodilators for at least 6 h after the challenge. Five subjects were challenged with allergen. Their

Albuterol Albuterol Albuterol

+ + + +

+ + + + +

details are given in table 1. All asthma medications were withheld for at least 12 h before the study. Allergen extracts (Dome Hollister-Stier, Seattle, WA) were diluted in Coca's solution. The allergen

(Receivedin originalform September 25, 1990and in revised form July 8, 1991) , From the Department of Allergy and Allied Respiratory Disorders, Guy's Hospital, London, United Kingdom. 2 Supported by the National Asthma Campaign (UK). 3 Correspondence and requests for reprints should be addressed to Prof. T.H. Lee, M.D. ER.C.P., Department of Allergy and Allied Respiratory Disorders, 4th Floor, Hunt's House, Guy's Hospital, London SEI 9RT, UK. 1411

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1412

chosen was that which gave the largest weal and flare response on skin prick testing with a battery of common aeroallergens. After baseline measurements of FEV" subjects inhaled five breaths of Coca's solution. If the decrease in FEV 1 was less than 5010, allergen challenge was performed. The value measured after Coca's solution was taken and the baseline value was used to calculate the percent fall in FEV,. The starting concentration of allergen was that which just failed to elicit a weal and flare response on skin-prick testing. The allergen was delivered from a Hudson nebulizer linked to a breath-activated dosimeter (DCPB; Guy's Hospital, London). Air was delivered to the nebulizer at a pressure of 149 kPa (20 Ib/inch') for 0.6 s from the start of each inspiration. Each dose was delivered in five breaths, and the FEV, was then measured every 5 min for 15 min. If the fall in FEV, was less than 150/0, twofold increasing concentrations of aIlergen were inhaled until a greater than 150/0 fall in FEV I was achieved. The FEV 1 was then measured at 5-min intervals to 20 min, then at 30 min and I h and hourly until

8 h. The protocols were approved by the Guy's Hospital Ethical Committee, and all subjects gave informed consent. Urine was coIlected between zero and 3, 3 and 6, 6 and 12, and 12 and 24 h after the challenges; 50-ml aliquots weretaken from each collection period. To these aliquots, 4·hydroxy-TEMPO (Sigma Chemical, St Louis, MO) was added to a final concentration of I mM, and the pH was adjusted to 9.0 with NaOH to stabilize the LTE•. The samples were coded and stored at - 70 0 C until analysis. The analysis was performed blinded, with samples from the allergen and exercisechallenges being analyzed in random order. Stored urine samples were thawed, and a lO-ml aliquot of each sample was taken. The pH was adjusted to 3.5 to 3.8 with NaOH, and the sample was centrifuged at 10,000 g for 10min at 4 0 C. The supernatants were removed, and 4,000 dpm of ['H]LTE. (39.3 Ci/mmol; NEN-DuPont, Boston, MA) were added. The sample was then loaded directly onto a 10-l1m precolumn 3.4 mm x 4.5 em (Ultrasil ODS; Beckman Instruments; Fullerton, CA), which was isolated from the analytical column. The precolumn was washed at 2 ml/min for 8 min with a phosphate buffer (0.10/0 NaH,PO. at pH 3.8) and then for 12 min with a methanol.phosphate buffer in the proportions 50:50 (vol:vol) at a flow rate of 2 mllmin. The sample was then retrogradely eluted via an automatic switching valve onto a lfl-um reversed-phase analytical column 4.5 mm x 25 em (Ultrasil ODS; AItex, Los Altos, CA) that had been equilibrated in 62010 methanol:37.8010 water:O.1OJo acetic acid:O.l010 EDTA (vol:vol) at pH 5.6. The LTE. was eluted at I ml/min. After RP-HPLC, the activity in 250 I!I of each fraction was counted, and the peak of radioactivity containing the tritiated internal standard was identified. The remaining 750 IIIofthose fractions containing the [3H]LTE., and of the two fractions eluting before and after the peak, were dried under vacuum and resuspended in 250 I!Iof 10 mM TrisHCI buffer (pH, 7.4) containing 0.15 M NaCI and 0.1010 gelatin. The concentrations of immunoreactive LTE. wereassessed by radioimmunoassay, using synthetic LTE. (0.01 to 10 ng/ml) to construct the standard curve. The immunoreactivity in the fractions eluting before and after the peak of internal standard were used to calculate mean background immunoreactivity. The recovery ofLTE. was 74 ± 9070 (mean ± SD). The intraassay and interassay coefficients of variation were 8% and 15010, respec-

tively, and sensitivity of the assay was 8 pg. The intrasubject coefficient of variation was 52%. This was calculated from measurements taken from 17 subjects on 2 different days at the same time of day. The airway response to challenge with exercise and allergen was calculated as the percent fall in FEV 1 from the baseline value. All statistics used for comparison of urinary LTE. concentrations were performed using loglO-transformed data, and values are given as the geometric mean and 95010 confidence interval (6, 7). The relationship between the quantity of LTE. in the urine with time before and after challenge was analyzed using repeated measures analysis of variance (AN OVA). When the ANOVA revealed a significant difference, this was further analyzed using the Newman-Keuls test of means.

Baseline values for FEV, in the two groups were not significantly different by unpaired t test (table 1). The mean (± 1 SD) percent fall in FEV, after exercise was 42 ± 18070, which was significantly greater than that after allergen challenge (22 ± 8%) when compared by unpaired t test (p < 0.05). Baseline LTE. concentrations in those subjects challenged with exercise was 64 (27 to 150) pg/mg creatinine (geometric mean and 950/0 confidence interval), and in those challenged with allergen it was 36 (23 to 59) pg/mg creatinine. These values were not significantly different by unpaired t test. All subjects challenged with allergen had increased urinary concentrations of LTE. in the 3 h after challenge (figure 1). The levels increased by a mean 4-fold (range, 2- to 8-fold) from baseline values, and were significantly higher (P < 0.001) than at any other time point. The levels,though still slightly elevated at 6 h after challenge, were not significantly different. Samples from four of the five subjects wereavailable for the 12-and 24-h time points, and urinary LTE. in these samples had all returned to baseline values. In contrast, there was no significant change in urinary LTE. in the 24 h after exercise (figure 1). Urinary LTE. remained static in Subjects I, 3, 4, and 5. In Subject 2, levels were elevated at 6 h, but returned to baseline values 1000 _.....-[J-.....

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**** The finding that allergen challengeis associated with a mean 4-fold increase in concentrations of urinary LTE4 confirms those of a number of recent publications (6-8). The coefficient of variation in our assay was52%. Taking 2 SD as the limit of variability, measurements in the absence of provocation can be expected to vary by 104%, or a factor of approximately 2. The rise in urinary LTE. after allergen challenge was at least 2-fold in all five subjects studied, and occurred consistently in the sample taken from zero to 3 h. It is therefore unlikely that the rise detected after allergen challenge was due to random variability of urinary concentrations of LTE•. Using the same assay, we did not demonstrate consistent or significant changes in urinary LTE. after exercise challenge, even though the response was more severethan that induced by allergen. The pattern of change in urinary LTE4 after exercise challenge was less consistent than that after allergen challenge. In four of the sixsubjects, urinary LTE. remained stable over the 24-h period. In Subject 2, who had the most severe response to exercise, there was an almost 3-fold rise in urinary LTE4 from baseline levels in the sample collected 3 to 6 h after challenge. However, in Subject 6, who also had a severe response to exercise, levels fell by almost 3-fold in the samples collected 3 to 6 and 6 to 12 h after challenge, but they had returned to baseline by the next morning. Regardless of these fluctuations exercise was not associated with a rise in urinary LTE. in five of the six subjects studied. This might suggest that the sulfidopeptide leukotrienes are not important mediators contributing to the. mechanism of exercise-induced asthma and that other mediators, or some other mechanism, is responsible. For example, it has been suggested that the mech1000

SUbject 1 Subject 2 Subject 3 Subject 4 Subject 5 Subject 6

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at 12 and at 24 h. In Subject 6, urinary LTE. fell at 6 and at 12 h, but returned to baseline at 24 h.

24

10 +-'~"""..,...,~~-r~~¥~~"" 24 12 o 3

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Fig. 1. Values for concentrations of urinary leukotriene E. measured before and at 0-3,3-6,6-12, and 12-24 h after challenge with exercise (left panel) or allergen (right penel).

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anism of exercise-induced asthma is clue to vascular engorgement associated with reactive hyperemia as airways rapidly rewarm at the end of exercise, rather than that the constriction is associated with mediator release (to). However, this conclusion is not consistent with the observation that the leukotriene antagonist MK-571effectively attenuates the response to exercise(3), and that the 5-lipoxygenase inhibitor A64077 significantly decreases responsiveness to cold-air hyperventilation challenge (4). This apparent discrepancy raises the matter of the difficulty of interpreting mechanistic events occurring in the lung based on measurements of mediator concentrations measured in the peripheral circulation or in the urine. Where a positive finding can be useful, a negative finding may be misleading and difficult to interpret. It is possible that the respiratory heat and water loss associated with exercise results in a highly localized stimulus causing high local concentrations that are not detectable once diluted into the systemic circulation. Unlike inhalational challenges where the aerosol penetrates beyond the proximal airways, the stimulus associated with hyperventilation is limited to the proximal airways and progressively diminishes in the peripheral airways as the inspired air becomes conditioned (10). The finding of Pliss and coworkers (11)that hyperventilation challenge in humans results in a mean 5-fold increase in SRS-A (LTC., D., E.) in bronchoalveolar lavage fluid indicates that localized increases in sulfidopeptide leukotrienes are induced by hyperpnea. Taylor and colleagues (6) have also suggested that the failure to detect increased levels of urinary LTE. after nasal allergen challenge, despite the fact that high levels ofleukotrienes can be detected in nasal washes and tear fluid, may be due to the fact that localized increases do not alter whole-body LTE. excretion over 24 h. The release of sulfidopeptide leukotrienes may also be more prolonged after allergen challenge than after exercise challenge. Under these circumstances the total release of leukotrienes after allergen challenge may be greater than that after exercise. However,there was no difference in the rate of recovery after allergen and exercise (56 ± 9 and 64 ± 37 min, respectively), making it unlikely that more prolonged release of leukotrienes after allergen can explain the difference between the detection of LTE. in the urine after allergen and exercise challenge.

1413

It is possible that mediators can become ''trapped'' in areas of poor perfusion, and that they are further metabolized to the more polar metabolites of LTE., which would not coelute with LTE. during RP-HPLC (12). Anderson and coworkers (13), measuring plasma histamine levels after challenge with exercise,reported that in those subjects whose response was associated with increased plasma histamine, administration of the 112agonist terbutaline resulted in a "flushing out" of histamine, with plasma levels being higher than those measured immediately after exercise.They suggested that terbutaline may have had a relaxant effect on venular endothelial cells and vascular smooth muscle, thereby increasing flow to areas previously constricted either by local mediator release or hypoxia. This may be a feature of exercise-inducedasthma, where airway cooling may further influence local vascular tone. We did not compare responses to the two stimuli in the same group of patients. However, it is unlikely that heterogeneity of mechanisms or differences between the groups can explain the inability to detect LTE. in the urine after exercise. The pattern of changes in urinary LTE. in those subjects challenged with allergen were typical ofthose reported in three other studies (6-8). In these studies, a mean increase in urinary LTE. of 3- to 4-fold is consistently found in the first 3 h after challenge. No such increase was found in the group challenged with exercise despite the fact that five of the six subjects were atopic. Given the consistency with which urinary levelsof LTE. rise after alJergen challenge, it is likely that urinary LTE. levels would have risen in these atopic subjects if they had been challenged with allergen. In conclusion, we have shown that urinary LTE. concentrations are significantly elevated after allergen challenge, but not after exercise challenge. Because the leukotriene antagonist MK571 effectively attenuates the response to exercise, and leukotriene levels in BAL fluid are significantly increased after hyperventilation challenge, it is likely that the sulfidopeptide leukotrienes contribute to the pathophysiologic aspects of asthma. We suggest that the inability to detect elevated levels of LTE. in urine after exercise may be due to the fact that the stimulus is highly localized and/or that changes in bronchial or pul-

monary blood flow in response to airway cooling and hypoxia may modulate the absorption of these locally produced mediators into the systemic circulation.

References 1. Holtzman MJ. Arachidonic acid metabolism: implications of biological chemistry for lung function and disease. Am Rev Respir Dis 1991; 143: 188-203. 2. Israel E, Juniper EF, Callaghan JT, et al. Effect ofa leukotriene antagonist LYI71883 on coldair-induced bronchoconstriction in asthmatics. Am Rev Respir Dis 1989; 140:1348-53. 3. ManningPJ, WatsonRM, MargolskeeDJ, Williams VC, Schwartz Jl, O'Byrne PM. Inhibition of exercise-inducedbronchoconstriction by MK 571, a potent leukotriene Ds-receptor antagonist. N Engl J Med 1990; 323:1717-23. 4. Israel E, Damarkarian R, Rosenberg M, et al. The effects of a 5-lipoxygenase inhibitor on asthma induced by cold air. N Engl J Med 1990; 323:1740-4. 5. Tagari P, Ethier D, Carry M, et al. Measurement of urinary leukotrienes by reversed-phase liquid chromatography and radioimmunoassay. Clin Chem 1989; 35:388-91. 6. Taylor GW, Black P, Turner N, Taylor I, Maltby NH, Fuller RW, Dollery CT. Urinary leukotriene E. after antigen challenge and in acute asthma and allergic rhinitis. Lancet 1989; 1:584-8. 7. Manning PJ, Rockach J, Malo J-L, et al. Urinary leukotriene E.levels during early and late asthmatic responses. J Allergy Clin Immunol 1990; 86:211-20. 8. Sladek K, Dworski R, Fitzgerald GA, et al. Allergen-stimulated releaseof thromboxane A, and leukotriene E. in humans: effect of indomethacin. Am Rev Respir Dis 1990; 141:1441-5. 9. Christie P, Tagari P, Ford-Hutchinson AW, et at. Urinary leukotriene E. concentrations increase after aspirin challenge in aspirin-sensitiveasthmatic subjects. Am Rev Respir Dis 1991; 143:1025-9. 10. Gilbert lA, Fouke JM, McFadden ER. Heat and water flux in intrathoracic airwaysand exerciseinduced asthma. J Appl Physiol1987; 63:1681-91. 11. Pliss LB, Ingenito EP, Ingram RH, Pichurko B. Assessment of bronchoalveolar cell and mediator response to isocapnic hyperpnea in asthma. Am Rev Respir Dis 1990; 142:73-8. 12. SalaA, VoeldN, Madouf J, Murphy RC. I..eukotriene E. elimination and metabolism in normal human subjects. J Biol Chem 1990; 265:21771-8. 13. Anderson So, Bye PTP, Schoeffel RE, Seale JP, Taylor KM, Ferris L. Arterial plasma histamine levelsat rest, and after exercisein patients with asthma: effects of terbutaline aerosol. Thorax 1981; 36:259-67.