Urinary leukotriene E, after exercise in children with asthma

challenge

Yoshiharu Kikawa, MD,* Takeshi Miyanomae, MD,** Yasuhiro Inoue, MD,** Masakazu Saito, MD,* Akio Nakai, MD,* Yousuke Shigematsu, MD,* Susumu Hosoi, MD,** and Masakatsu Sudo, MD* Fukui and K~oro. Japan To u.s.se.ss 7he role of sulfidopeptide leukotrienes in the puthogene.sis of esc~rcise-induc,ed u.cthmtr rEIA). the urinurx levels of leukotriene E, (LTE,). u metubolitc qj’ LTC‘, ond LTD, . \t’ere mctrsured by RIA before and after exercise in 13 children with EIA u17d IO he&h! children .Mu.s.s spectrometr\ wus used to confirm the presence of LTE, in urine und the specjficitv (11‘ rl7c RIA. There wus no .signiJicant difference in the urinur! LTE, Ie\,el.s before exercise between r/u, children nlth asthma and healthy children (109 121 to 26.51 versus 122 145 to l.Fb/ p~!mv 01 c.reutinine; median and range). Lirinao LTE, levels increased .significuntl~ after exercise in rhc children with EIA (from IO9 121 to 2651 to 196 [4O to 6551 pq 1mg (J/’ creutinine: median and range; p ~1 0.05) but not in the heulthy children. Thr children with trsthmu demon.strated ml .signijic~ant correlation between the LTE, level ufter exercise and the degree of bronchoconstriction. us revealed by the marimal percent full in thr peuk expirutoc /iota, ruts. Taken together with u recent study that pretreatment with a potent and selective LTD, antaRoni\; markedlv uttenuuted EIA, our findings suggest thut stdjidopeptide leukotrienes mu! pl~x .W~P role in the puthogenesis of this type of asthma with other fuctors also being in\&etl in determining the overall airwu~ iesponse. (J At.t.t.H(;Y CIW M~t:Yot. I992 :8Y: I I I! -V. I Key words: leukotrienc,

Exercise-induced asthma. suljidopeptide leukotrirne. bronc~hocon.stricrion. I:, . mass sprctromet~. selected ion monitoring. uirway reucti,-it)

Exercise is a common stimulus leading to bronchoconstriction in children with asthma. Currently. a major initiating stimulus for EIA is suggested to be respiratory water loss resulting in hyperosmolarity of the respiratory mucosa.’ This hyperosmolarity is believed to cause the release of bronchoconstrictive mediators, such as histamine.2, 3 The sulfidopeptide leukotrienes (LTC.,, LTD,, and LTE,) are another major group of bronchoconstrictive mediators, which are several hundred times more potent than histamine in both subjects with asthma and normal subjects.’ Increased levels of sulfidopeptide leukotrienes have been

From the *Department and the **Deptiment

of Pediatrics. Fukui Medical School, of Pediatrics. National Sanatorium

ami-Kyoro Hospital. Kyoto, Japan. Supported m part hy a gram-in-aid for scientitic Ministry of Education t)f Japan. Received for publication Revised Jan 13, 1992. Accepted for publication

April

3. 1991

Jan.

17. 1992.

research

Reprint requests: Yoshiharu Kikawa. MD, Department rics, Fuktn Medical School. Matsuoka. Fukui 910-I l:l/36510

Fukui. Minfrom

the

of Pedlat1. Japan.

Abbreviutions

ur,nar\

used

LTC,. LTD,. LTE,: Leukotrienes C,. D,. and ElA: Exercise-induced asthma PEFR: Peak expiratory flow rate HPLC: High-performance liquid

E.

chro-

matography SIM: BALF: 111: 1:

Selected

ion

monitormg

Bronchoalveolar lavaee fluid Mass to charge ratio

detected in both BALF and urine after bronchoconstriction induced by antigen challenge or during spontaneous asthma attacks.‘-’ Furthermore. Manning et al.“’ have recently found that pretreatment with a potent and selective LTD,-receptor antagonist markedly attenuated exercise-induced bronchoconstriction. However, conflicting results have been published regarding sullidopeptide leukotriene levels in BALF cpbtained from patients with EIA or hypcrventiiationinduced bronchoconstriction.“’ ” In humans. infused LTC, or inhaled LTD, undergoes rapid enzymatic conversion in the blood to LTE,. 1111

1112

Kikawa

TABLE

J ALLERGY CLIN

et al.

I. Characteristics

of the

children

With

asthma

PEFR (% of predicted)

Child No.

Sex

I

F

9

60

2 3 4

F

12

52

M M

8 II

66 95 90

Age (yr)

5

F

7

6

F M F F

9 7

I 8 9

IO II 12

I3 Mean SD

F

F M M

51

with

Duration asthma

of (yr)

3 6 4 9 5 3

51

5

9

9 2 I 8 4 7

9 1.6

68 I9

5.1

7 9 9

8

and

the

healthy

children Healthy

98 55 44 % 65 56

I2

asthma

IMMUNOL. JUNE 1392

Therapy*

Child

No.

I 1, 3, 4

Sex

Age hr)

PEFR (% of predicted value)

M

12

M F

I, 2, 4

I 2 3 4

II3 120

I, 2, azelasfine

5

M

9 IO IO IO

I

6

8

II0

I, tranilast I, azelastine Ketotifen fumarate

7 8

M M F

8 7

II3 104

M

10

I28

F

9

1, 3, azelastine

.

9 IO

I

F

105 91

99

1, ketotifen fumarate I, oxatomide I, ketotifen fumarate 9.3 1.3

2.5

109 9

*f, Oral theophylline: 2. oral P-agonist; 3. inhaled P-agonist; 4. sodium cromoglycate.

metabolite that is mostly excreted in the urine within several hours of infusion or inhalation.13 Urinary LTE, levels therefore reflect an integrated form of systemic LTC, or LTD, release during a period of time and allow the detection of small quantities of leukotrienes. Furthermore, urine collection has the advantage of being a simple and noninvasive procedure. 14.” In our previous study, we analyzed urinary LTE, levels in children with EIA and suggested that an increase in urinary LTE, might be present in such children. However, that study lacked a comparison with control healthy children and had some methodologic problems with LTE, assay that led to low recovery from the urine samples. In addition, we only collected a urine sample immediately before exercise plus the first urine voided between I to 2 hours after exercise,‘” and the latter sample might have been obtained too early to reveal the maximal urinary LTE, concentration. In addition, the identity and purity of urinary LTE, have previously been checked solely by RIA after HPLC purification (also a problem in the studies by other workers). In contrast, the specificity of RIAs for prostaglandins and thromboxanes has usually been confirmed by gas chromatography-mass spectrometry . ” Unfortunately, conventional HPLC mass spectromehy, a method corresponding to gas chromatography-mass spectrometry, apparently lacks the sensitivity to check accurately the small amount of LTE, present in urine.” Therefore, we applied a new ionization method (mass spectrometry with ion spray) to a stable

the analysis of urinary LTE, levels” and confirmed the specificity of LTE, detection by our RIA. The aim of the current study was to confirm under more optimum experimental conditions whether sulfidopeptide leukotrienes are related to the pathogenesis of EIA, as well as to confirm the identity of urinary LTE, by mass

spectrometry.

METHODS Subjects Twenty-three children were studied, including I3 children with asthma with a history of EIA, positive skin test responsesto common allergensand elevatedIgE levels, and IO normal healthy children with no history of asthma or airway disease. Informed consent was obtained from these two groups of children and their parents. The characteristics of each group are presentedin Table I. None of our subjects had taken steroids or sodium chromoglycate for at least several months before the start of this investigation, with the exception of two children in the group with EIA who were taking disodium chromoglycate. Subjects were asked to abstain from the intake of any medications for I2 hours

before the study, and antihistamines,

like azelastine, were

withheld at least for 24 hours. This study was approved the Ethics Committees of both Fukui Medical School the National Sanatorium Minami-Kyoto Hospital.

by and

Exercise challenge Exercisewas undertaken on an electrically driven treadmill for 6 minutes with the speed set at 6 km/hr and the incline set at 10%. PEFR was measured with the Minato AS500 peak flow meter (Minato Ikagaku Instrument Co.,

EIA-induced

Tokyo. Japan) before exercise and at 5. IS, and 30 minutes after exercise. The best of three consecutive PEFR readings at each point in time was used for the subsequent analysis. All procedures were undertaken at ambient room temperature and humidity. The bronchial response to exercise was calculated as the percent fall in PEFR, and the maximal pcrccnt I’d11 was expressed as: Preexercise PEFR value lowest postexercise PEFR value x 100 Preexercise PEFR value

Collection

of urine samples

The children were requested to pass urine immediately before and every 2 hours after exercise (until going to bed). However, although all the children managed to pass urine immediately before exercise. they provided the first postexercise urine sample at 3 to 6 hours after exercise, and only tive children with asthma and three healthy children managed to pass a second postexercise urine sample (at 6 to 9 hours after exercise). The exact times of collection were recorded for all the children. All urine samples were collected into tubes containing 0.2 ml of the oxygen-radical scavenger. 4-hydroxytetramethyl-piperidinooxy-free radical (0. I moliL) and 0. I ml of IO N NaOH. and were stored at - 60” C until later use.

RIA for urinary

LTE,

Urine samples were tested in duplicate. Samples were allowed to thaw immediately before the assay, and a I ml aliquot was taken to measure the creatinine level. [‘HI-LTE, (6000 disintegrations per minute; Amersham, Arlington Heights, Ill.) was added to 20 ml of urine as an internal standard. and the urine was centrifuged at 45.000 g for IO minutes at 4” C after adjustment of the pH to 5.4 with acetic aid. The supematant was tirst applied to a Sep-Pak C,, cartridge (Millipore Corp.. Milford. Mass.), which was preconditioned with IO ml of methanol, 5 ml of distilled water, and then 5 ml of 0.5% ethylenediaminetetraacetic acid. The cartridge was washed with IO ml of distilled water and then 5 ml of methanol/ water (40:60), after which leukotrienes were eluted with 3 ml of methanol.“’ The 3 ml methanol fraction was again applied to a Sep-Pak Light HN, cartridge I Millipore Corp.). After the cartridge was washed with 6 ml of methanol. the leukotrienes were eluted in I2 ml of ;L 0.5% acetic acid/methanol solution (vol/vol). Then the sample was carefully evaporated to about 100 pl under a stream of nitrogen gas and adjusted to 200 pJ with 65% methanol. The sample was then injected into a C,, reversephase HPLC column (Nacalai Tesque, Kyoto, Japan) and cluted through an HPLC system (Millipore Corp.) at a constant how rate of I mlimin, with a mixture of methanol/water/acetic acid (65:35:0. I) adjusted to pH 4.9 with ammonia. Fractions having the same elution time as [‘HjLTE, were collected. and the immunoreactive LTE, content was determined by RIA. The RIA was performed as described in the manufacturer’s instructions, with an LTC,/LTD,ILTE, [‘H] assay kit (Amersham). The manufacturer’s instructions state that

urinary LTEd excretior.

1113

their antiserum has the following cross-reactivmca I: IX: :. 100%; LTD,, 49%; and LTE,. 40%. A standard soiution of LTE, was dissolved in the same HPLC solvent uith the samples to compensate for loss by evaporation during processing for the RIA. Both the standard solution .md the fractions obtained from HPLC were evaporated in v~ua. and the precipitates were resuspended in the unmunoassa! buffer. LTE, was used as the radioligand. and the ~ar~lples were quantitied against a standard LTE, curve. All ITtS. values were corrected for recovery and expressed Ijs pica grams per milligrams of creatinine. The precisioii 01 the overall procedure was checked by repeating the analysis on the same urine samples pooled from different patients. both with and without the addition of a known amount ol \vr~thetic LTE, immediately before extraction.‘. Addition 01’ 200 pg/ml of LTE, produced an Increase of IT? :. 2 Y pg ml after analysis of the urine. The intra-assa) and mterassay coefficients of variation were 7% and 9%. rcxpecti\ cl>. for pooled urine samples from healthy adult\.

Mass spectrometry The speciticity of the RIA for urinary LTE., was checked by comparing the RIA data with the mass spectromctry data obtained in tive assays. The portion of the tirst urmc sample obtained after exercise that was surplus to the requirements of the RIA described above was used. The samples for both assays were 60 to 80 ml of urine pooled from two to three children with asthma. These five urine samples wcrc extracted and injected into the HPLC column in the sdmc way as described above. An aliquot\of the column eluare was analyzed by RIA. A nonradioactive deuterated analog (11. LTE,. [‘H,)LTE; (70 ng; Caymann, Ann Arbor, Mich. I was added as an internal standard to another aliquot of the eluate. which was then diluted with distilled water to elicit a meth. anol concentration of about 40% and applied to a Sep-Pak C;, cartridge that had been preconditioned as described above. The cartridge was washed with 5 mi 01’ 40% methanol, after which LTE, was elutcd with ( ml 01 methanol. This 3 ml methanol fraction was then carefully evaporated to about IO0 p.1 under a stream 01 nitrogen gas. HPLC was performed with a Waters 625 LC system (Mu. lipore Corp.). The column used was an Aquapore 12. I mm inside diameter by IOU mm; Applied Biosystcm. Foster City, Calif.). The mobile phase was acetonitnle-distilled water-acetic acid (50:50:0.5) with a How rate of 200 $imin. and 40 ulimin was introduced into the ion source to elicit a split ratio of 5 : I Analysis was perfommed ai room temperature with a S&x API III triple quadripole mash spectrometer (Thornhill. Ontario. Canada) equipped with an atmospheric pressure ion source. The interface electrode voltage was S kV for monitoring positive ions. i? positive ion-spray spectrum of LTE, was obtained with Ii)0 ng 01 synthetic LTE, and revealed a base peak at rn’ I +tO as the only ion of significant Intensity. corresponding to 11wb plus hydrogen’ ( Fig. I ). Mass spectrometric analysts was lxrformed both in the scanning mode from rn. I X0 to ml I 600, and the SIM mode at m/z 440 and m / 144. corrcsponding to mass plus hydrogen. of [‘H:~lTt~. l+ak area\

1114

Kikawa et al

J ALLERGY CLlrY IMMUNOL JUNE 1992

440.1

100 ’

0 V

751

p

50

y

25

P

.3 5 az

!

o-

Lt.

250

I

L

300

IA

h

350

400 450 m/z

500

550

600

FIG. 1. Positive ion mass spectrum of authentic LTE,. for these two ions were measured automatically by the data system. A calibration curve in the SIM mode was constructed by injecting IO p,I of various standard mixtures of LTE, and (‘H,]LTE,. The LTE, content of the HPLC elute was calculated with the Scicx API 111data system and was converted to the LTE, concentration in urine (picograms per milligrams of creatinine) after correction for radioactive rccovely. Statistical

analysis

Linear regression analysis by the least-squaresmethod was performed to compare the urinary LTE, valuesmeasured by mass spectrometry (SIIM mode) and RIA. These data. except for urinary LTE, Icvels. are expressed as the mean ? standard deviation. The unpaired Student’s I test was used to compare the maximal percent fall in PEFR and the recovery of radioactive LTE, from the urine between children with asthma and healthy children. Some authors have reported that the urinary LTE, levels from certain Populationsdo not have a normal distribution.” Since our data also supported this finding, urinary LTE., levels are expressedas the median and range in this articlc. Algebraic means were also calculated for comparison with the data in the previous studies. The LTE.,levels in children with asthma and healthy children were compared with Mann-Whitney U rank-sum test. and the changes in LTE, levelswith exercisewere compared with Wilcoxon’s signedrank test. A Bonferroni correction was applied to multiple comparisons. The level of significance was set asp < 0.05. RESULTS All except one of the children with asthma developed mild and transient bronchoconstriction that resolved without specific therapy, whereas the remaining child developed severe bronchoconstriction that

required treatment and could not undergo spirometry after exercise. None of the healthy children developed any bronchoconstriction. The urinary LTE, levels before and after exercise, the maximal percent fall in PEFR, and the timing of urine collection are presented in Tables IIA and IIB for the children with asthma and healthy children, respectively. There was no significant difference between the children with asthma and healthy children with regard to the urinary LTE, levels before exercise (109 121 to 2651 pglmg of creatinine for children with asthma versus 122 145 to 1561 pg/mg of creatinine for healthy children, median and range) and the time at which the first and second postexercise urine samples were collected (first sample, 4.6 _C I .2 hours for children with asthma versus 4.8 t 1 hour for healthy children; second sample. 6.8 2 0.8 hours for children with asthma versus 7 + 0.8 hours for healthy children; mean -C SD). Also, there was no significant difference in the radioactive LTE, recovery from the urine samples obtained before and after exercise from both groups of children (group with ElA: 62.2% + 9% [before], 66.2% + 7.2% [first], and 66.2% -C 3.2% [second]; healthy group: 62.6% ? 6.7% [before], 65% -C 5.7% [first], and 69% ? 0.8% [second]; mean + SD. (Individual radioactive LTE, recovery data are not presented.) The urinary LTE, level increased in eight of the 13 children with EIA and was highest in patient No. 8 after exercise (the child who could not undergo spirometry because of severe EIA). The increase in the children with asthma was 1.5-fold (0.6 to 9.3-fold) from 109 (2 I to 265) pg/mg of creatinine before cx-

VOLUME 89 YUMBER f

EIA-Induced

urinary

LTEa excretlof.

1115

IIA. Urinary LTE, levels, maximal percent fall in PEFR, and the timing of urine collection after exercise in the children with asthma

TABLE

Timing Urinary

LTE,

level

(pglmg

After Child

No. I

2 3 4 5 6 7 x Y IO II I2 I3 MeaIl

SD Median Maximum Minimum

Before

exercise

21 I IO 50 61 69 12.5 265 145 142 I79 IO!, I01 s3 I IO

109 -‘65_ 21

of 1st and 2nd urine collection (hr) after exercise -.. -.. -.

creatinine) exercise

1st

2nd

196 X8 50 40 104 70 537 655 365 267 429 370 51 248

44 .-. 23 I8 --

196

44

655 40

72 IX

Maximal % fall in PEFR

46 72 41

1st

31 3 30 17 64 54 73 NA” so ‘Y 51 32 s3 -11

.: 7 0 0 0 .-I I) 0 5.5 \ i t 3s 4.6

13

I.1

.-

2nd

< ‘,

7 0 ‘1 8

:I x

*NA. Not available because ol‘ acute asthma.

ercise to 196 (40 to 655) pg/ mg of creatinine in the tirst urine sample after exercise (median and range; n = 13). There was a 2.2-fold increase in the algebraic mean, and the difference was statistically significant ( p < 0.05). Urinary LTE, levels subsequently decreased to 44 ( 18 to 72) pgl mg of creatinine in the second urine sample after exercise (median and range; n = 5). However, there was no significant correlation between the level in the first postexercise urine sample and the maximal percent fall in PEFR in the children with EIA. Urinary LTE, levels in the healthy children demonstrated no significant changes, being 122 (45 to 156) pg/ mg of creatinine before exercise (n = lo), 104 (22 to 186) pg/mg of creatinine in the first postexercise urine sample (n = 10). and 128 (51 to 137) pg i mg of creatinine in the second postexercise urine sample after exercise (n = 3) (median and range). The identity of urinary LTE, was not confirmed in any of the five urine samples by the mass spectra, probably because of the presence of impurities and small quantity of LTE,. However, the presence of LTE, was demonstrated in all five urine samples by typical chromatograms in the SIM mode. The calibration curve was linear over the range monitored by the SIM mode. Urinary LTE, levels determined in the SIM

mode, together with levels determined by RIA to allow a comparison of these two assay methods, are illustrated in Fig. 2. Linear regression analysis revealed a good correlation of the values obtained by RIA with values obtained by SIM mode mass spectromctry (Y. 0.88; X, 32.0). DISCUSSKIN We found a significant increase of urinary LTE, levels after exercise in the children with EIA. but not in the healthy children. Our results arc somewhat in conflict with those of Broide et al.” who found LTC, to be undetectable in BALF from patients with EIA. However, Pliss et al. ” obtained results more consistent with our results; they demonstrated a small but significant increase of sultidopeptide leukotrienes in BALF from patients with asthma after hyperventilation-induced bronchoconstriction. In addition, two other research groups have also detected LTE., and, to a lesser extent, LTD, (but not LTC,), in BALI: obtained from patients with asthma during acute asthma or after allergen inhalation.5. ’ Thus, the differences between our findings and those of Broide et al.” cannot simply be explained by the use of urine versus BALF. All these studies (including our study). except

1116

Kikawa et al.

J. ALLERGY CLIN

TABLE IIB. Urinary LTE, levels, maximal after exercise in the healthy children

Urinary

LTE,

level

(pglmg

percent

No.

Before

exercise

132 143 156 141 45 1,47 85 74 II 94 I3

I19 106 I51 138 186 102 83 22 58 77 105

Median Maximum Minimum

122 I56 45

104 186 22

collection

exercise

1st

I 2 3 4 5 6 7 8 9 IO Mean SD

of urine

Timing of 1st and 2nd urine collection (hr) after exercise

creatininel

After Child

fall in PEFR, and the timing

IMMUNOL JUNE 1992

the study of Broide et al. ,‘I analyzed the BALF or urine by RIA with antibodies with a relatively high cross-reactivity to LTE, (40% or 64%).‘. ‘. I2 In contrast, Broide et al. ” used an antibody highly specific to LTC, (cross-reactivity to LTE4, 8%). These investigators might therefore have missed the production of LTC, in BALF because it was not detected by their specific antibody after its rapid metabolism to LTD, and LTE,. Another possible explanation is that there may be different subpopulations of subjects with asthma,’ in some of whom sulfidopeptide leukotrienes are not released during EIA. The urinary LTE, level in our children with asthma before exercise were similar to that found in adults with asthma before allergen inhalation or in a stable state.6. *. 9 The increase of urinary LTE, after exercise was 2.2-fold for the algebraic mean and 1.5fold for the median in the entire group with EIA and 3.6-fold for the algebraic mean in those with increased urinary LTE, levels. It was as high as 9.3-fold in the child with the most dramatic increase. These changes were much less marked than the mean sixfold or twelve, fold increase noted by some authors in adults with an isolated early asthmatic response after allergen inhalation”, 9 but were comparable to the mean 2. lfold or 2.6-fold increases reported by other investigators in adults with an isolated early asthmatic response or adults with both an early and late asthmatic response after allergen inhalation.‘, ”

Maximal in

2nd

137 51 128 105 128 137 51

% fall

PEFR

1st

5 0 6 2 3 0 7 6 8 4 4 3 -

6 5.5 3 4 5 4.5 5.5 6 3.5 5 4.8 1.0 -

2nd

8 6 7 7.0 0.8

-

Urinary LTE, levels increased in 8! 13 children with asthma and l/10 healthy children, but the other live children with asthma (38%) demonstrated no increase. In this respect, our findings differ from the findings of consistent increase after allergen challenge but are similar to findings of Westcott et al.2’ who demonstrated no increase during the 12 hours after allergen inhalation in 2/7 (28%) adults with an isolated early asthmatic response. The following explanations can be suggested: First, the release of sulfidopeptide leukotrienes may not occur during EIA in some subpopulations with asthma, as mentioned above. Second, most of the LTE, might be rapidly converted in the liver to oxidative metabolites, like 16-carboxytetranordihydro-LTE,, the existence of which has been demonstrated in urine from LTC,-infused monkeys but not in human urine.22 Thus, an increase of LTE, might possibly have been missed in five of the children with asthma and nine of the healthy children. This second possibility cannot be explored until an assay method becomes available for these metabolites. Third, urinary LTE, concentrations in these children might not have reflected the release of sulfidopeptide leukotrienes because of the collection of urine samples outside the time of excretion of high levels of LTE,. LTC, release into BALF or increase of sultidopeptide leukotrienes in blood might well miss detection unless samples are taken at the peak time of release or increase of sulfidopeptide leukotrienes. since they

‘.‘CILUME 89 hll:MBER 6

EIA-Induced

LTEL,in Urine Mass Spectrometric Value PS Per mg i creatinine

LTE.: excrel~

:-

1117

600 500

r Y = 0.88 X - 32 r = 0.95 l

400 t ’

300 1

I

0

FIG. 2. Linear correlation trometry in the selected

urinary

is demonstrated ion monitoring

1

I

100 200 300 400 500 600 LTE4 in Urine (RIA Value) (pg per mg creatinine > between mode and

are rapidly metabolized to LTE, or its subsequent metabolites. However, urinary LTE, is stable, and urinary LTE, levels can reflect an integrated form of systemic LTE, release during a period of time, but not an extent of leukotrienes release at one point of time.“. ” The timing of urine collection thus does not need to be so exact as that of BALF or blood collection, but it still appeared important to rule out the third possibility. The excretion of urinary LTE, was maximal in the second urine samples collected at 2 hours after allergen inhalation (first urine at I hour) in one study,’ in the first urine samples collected 2 to 3 hours after allergen inhalation in another study.’ and in the second urine samples collected at 6 hours after aspirin provocation (first urine at 3 hours) in a third study.‘J Thus. there is some variation in the timing of the excretion of urine with the maximal LTE, concentration under different experimental conditions. In our study, the first urine sample was collected between 3 and 6 hours after exercise, with a mean of 4.6 ? I .2 hours in the group with EIA and 4.8 t 1 hour in the healthy group. The urinary excretion of LTE, was maximal between 3 and 6 hours after exercise, at least in all the children with asthma who gave a second urine sample. All the reported data (including our data) are fairly compatible with the finding of Maltby et al.’ ’ that LTE, is mainly excreted in the first 4 hours after the intravenous infusion of LTC,. Thus, the timing of the urine collection in our study was unlikely to be too early. If anything. it may have been late in the healthy children. like patient No. 1, who gave a first postexercise urine sample at 6 hours after exercise.

urinary LTE, that measured

level measured by RIA.

by mass

spec-

In such cases, the urinary LTE, concentration might be reduced by the urine produced after the excretion of that with the maximal LTE, concentration. thereby missing an actual increase in sulfidopeptide Icukotriene release. However. since the times of urine collection in our study were similar in the children with asthma and in healthy children, the dilutional effect should have been nearly equal in both groups. Therefore, even if the urinary LTE, concentrations of the healthy children would have been higher with more optimum timing of urine collection, those of the children with asthma would also have been higher and would still have demonstrated a significant increase in comparison with the healthy children. Thus, the timing of urine collection is unlikely to unduly infuence our conclusion that release of sulfidepeptide leukotrienes may bc increased in children with asthma and with EIA, unless the metabolic clearance of these leukotrienes differ between children with asthma and healthy children. ‘,’ Urinary LTE., levels demonstrated about a threefold decrease in 415 children with asthma providing a second urine sample after exercise, but no such decrease was observed in the healthy children. nor has it been reported by other authors after antigen challenge.‘. ” This phenomenon may possibly be related to the refractory period. known as the time with a lower response to an identical exercise load performed within I hour after exercise.’ Depletion of mediators, such as histamine. however. does not appear to be the c.~planation for refractoriness.“ We failed to demonstrate any significant correlation

1118

Kikawa et al.

between the changes of urinary L.TE, levels after exercise and airway obstruction. This result is consistent with results of three other studies of BALF or urine from patients with acute asthma, hypcrventilation-induced bronchoconstriction. or allergen-induced asthma.‘. ‘. ” However. a significant correlation was found in two studies of urine from patients with allergen-induced asthma. x.1)Thus, these data on the rclationship between release of sulfidopeptide leukotrienes and airway obstruction remain conflicting. Another factor, like airway reactivity, in addition 10 the release of sulfidopeptide leukotrienes, might be important in determining the overall degree of airway obstruction, and leukotrienes may cause EIA only in subjects with asthma with a relatively high reactive airway. For example. child No. 5 had the largest maximal percent fall in PEFR in the group with EIA despite a relatively small increase of the urinary LTE, level. Children like this child might be more sensitive to sulfidepeptide Ieukotrienes or have greater airway reactivity than the children with asthma. with a smaller maximal percent fall in PEFR despite a greater increase of the urinary LTE, level. Furthermore, only healthy child No. 5, among the healthy children, demonstrated a fourfold increase of the urinary LTE, level but almost no change in PEFR. This result is not consistent with our conclusion that sulfidopeptide leukotrienes may play some role in the pathogenesis of EIA and appears to suggest that the increase of urinary LTE, in this child is exercise related rather than bronchoconstriction. Our explanation for this child is that leukotriene release may not be necessarily related directly to bronchoconstriction. This healthy child might be quite insensitive to sulfidepeptide leukotrienes. and the level of sensitivity to these leukotrienes or the airway reactivity might vary not only among children with asthma but also among healthy children. In conclusion, we confirmed our earlier finding that urinary LTE, excretion is increased after exercise, at least in part of the subpopulation of children with asthma and with EIA. This study was done under improved experimental conditions with healthy children as a control group and thorough evaluation of the method for determining urinary LTE,.‘” To our present knowledge, this is the first use of mass spectrometry to check the identity of LTE, in urine samples. Airway reactivity (especially the response to sulfidepeptide leukotrienes) also appears to be an important factor in the development of EIA. Further experiments are necessary to investigate the bronchial responses to inhaled sulfidopeptide leukotrienes, and the measurement of LTE, metabolites in urine is also required to determine more reliably the extent of sulfidopeptide leukotriene release.

J ALLERGY CLIN. IMMUNOL JUNE 1992

We thank

Dr.

Fumihiko

Kurimoto

Bio-clinical-laboratories.

Inc..

for

the

of

Mitsubishi

KIA.

Drs.

Yuka Yoshihisa

and Yasuko Ogawa of the Biotechnology

Umeda L&oratorios

of Takara

analysis.

and

Miss

Co.,

L&i..

Chiemi

for the mass

Mizumoto

for

Research

spectrometo her

sccrctarial

assistance.

REFERENCES I. Lee TH.

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Effects of a thromboxane-receptor antagonist, BAY u 3405, on prostaglandin Cl,- and exercise-induced bronchoconstriction Helgo Magnussen, MD,* Silke Boerger,* Anne Rose Baunack, MD** Grosshansdorf.

Kerstin

Templin,*

Hamburg.

and

and

Wuppertul,

German\

In the pathogenesis of exercise-induced bronchoconsrriction (E/R). prostaglandin D. (PC;/) ! mu! play a role as (I newly generated. mast cell-derived mediator. As the bronc,hoc~o,lstric,t~,t yffects of PGD, are predominantly mediated via stimulution of thromboxane receptors III I/U, lung. we studied u novel, orally effective, thromboxane-receptor untagonist. BAY u .ely. Incrausing dosages of PGD, were inhaled to establish dose-response curves thut ullowed determinutl~nr (t/ the provocutive concentration necessary to decrease FEV. by ut least 20% (PC..,,) and IO increuse specific airway resistance (SR,) by IOOqo (PC,,,,). EIH was metcsured as 11 mcl.rimtrl fulllincrease in postexertional FEV,ISR,. after bicycle exercise cmd cold-air breclthinq. Prechullenge lung-function values were similar on uil four occusions. BAY u 340.5 did no! ~,/I,.I/ tmy effect on resting bronchial tone. After placebo, the geometric meuns (SD) of PC,,, trnti Pi.‘:.. were 0.0380 (2.6) and 0.0264 (2.4) mglml, increasing to 0.554 (5.Y) and 0.143 18.1) mginrr ufter BAY u 340.5 (p = O.ooO2). Mean (SD) maximal postexertional decrease in FEV onrl increase in SR,, after placebo was 29.4% (16.4%) und 2809 (135vc). and qfier RAY II 3405. 31.44 (18.1%) and 379% (281%) (not significant). No clinical& relevant BAY u 3405~relate,1 side effects were observed. From these results we conclude that BAY u 340.5 is high!\ eflkcri~ c rn attenuuting PGD,-induced bronchoconstriction. However, BAY u 3405 does not modultrt~ rolr 01 EIB. suggesting that the mast cell-derived mediutor, PGD, . does not play un important the puthogenesis of exercise-induced asthma. (J ALLERGY CLI.V IHMINOI. 1992:X9:1 I IY-26 I Key words: BAY u 3405, ptrtirnts with nsthma

From the *Krankenhaus Grosshansdorf, Thoracic Surgery. LVA Freie- und **Bayer AC. Institute for Clinical Germany. Supported by a grant Received

for publication

thromboxune-receptor

Center for Pneumology and Hansestadt Hamburg, and Research D. Wuppertal,

antugonist.

PGD,

Bayer

AG.

Sept. 6. 1991.

Wuppertal,

ererci.\c~

chtrli~tl~t~

Revised Jan. 17. 1993. Accepted for publication Jan. 74. 1992. Reprint requests: H. Magnussen, MD, Krankenhaus Wiihrendamm

from

chullenge,

80. 2070 Gmsshansdorf.

Gro~shansdorf

Grmrn;u~~

Germany. l/l/36759

1119

.