Measurements of Leukotrienes in the Urine- Kumlin

7/28/2019 Measurements of Leukotrienes in the Urine- Kumlin

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Review article

Measurements of leukotrienes in the urine:strategies and applicationsKumlin M. Measurements of leukotrienand applieations.Allergy 1997: 52: J24-135. Munksga

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The biologic actions of the leukotrienes (LTs) C4,D4, and E4, collectively referred to as the cysteinyl-eontaining leukotrienes, support their putative roleas mediators of bronchial asthma (1, 2). Thus, theyall provoke plasma exudation, constriction of bron-chial smooth muscle, and increased mucus secre-tion in the airways (3-5). In addition, asthmaticsubjects show bronchial hyperresponsiveness tocysteinyl-leukotrienes (6,7), and these leukotrienesmay also enhance airway reactivity to histamine inpatients with asthma (8-1 0). Furthertnore , aspirin-intolerant asthmatics, i.e., asthmatic patients inwhom bronchoeonstriction is elicited by aspirin orother nonsteroidal anti-inflatnmatory drugs, werefound to be hyperresponsive to LTE4 (11), whereastheir reactivity to LTC4 and histamine was notdifferent from other asthmatics (12). D esensitiza-tion with aspirin attenuated the airway response toLTE4, whereas histamine sensitivity remained un-altered (If). It has also been shown both in vitro(13) and in vivo in asthmatics (14) that cysteinyl-leukotrienes are chemotactic toward eosinophilicgranulocytes, suggesting that they contribu te to therecruitment of eosinophils into the airways duringasthtnatic responses.Cysteinyl-leukotrienes are formed in the human

specimens from asthmatic subjects (17). Morerecently,//I vivo formation of cysteinyl-leukotrieneshas been documented in association with allergen-induced airway obstruction (18-20). A number ofantileukotriene drugs, which block cither the for-mation (biosynthesis inhibitors) or the biologicactions (receptor antagonists) of the cysteinyl-leukotrienes, have proved beneficial in attenuatingprovoeation-induced asthmatic reactions (21-25).Clinical improvement of asthma has been docu-mented in a long-term treatment study with abiosynthesis inhibitor on control of clinical asthma(26). For evaluation of the effects of such newantiasthmatic drugs and correlation of their effectswith the release and action of leukotrienes, thereis a great need for safe and reliable analyticprocedures to determine leukotriene formation //;

To follow endogenous pulmonary production ofcysteinyl-leukotrienes in association with airwayobstruction and allergic responses in asthma, wehave developed methods for analysis of LTE4 inthe urine (27). These tiiethods have been utilizedto monitor excretion of cysteinyl-leukotrienes inhealthy subjeets and patients suffering from differ-ent types of asthma, as well as in association with


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M easurem ent of urinary LTE4and summarize applications of activity originating from intravenously infused

ueh methods instudies of spontaneous or indueed [ 14,15- -12]-LTC4. Thecause of this discrepancy issthma and theeffeets of antileukotiiene drugs. not clear, but mayrelate to administration of the

substrate via a bolus injection (42), as opposed to, . . , J ^. * slow infusion (43,44), or the use of substrates withonitoring endogenous production of ,adioactivity in different parts of the moleeules.

cysteinyl-containing leukotrienes Nevertheless, asimilar proportion, i.e., about 3-5%Several biologic fluids may cotne into consideration of inhaled LTD4 (20, 46),2-6% of infused LTC4for monitoring endogenous production of cysteinyl- (43, 44), and 7^8% of injeeted LTE4 (45) wereleukotrienes. The release of mediators into nasal recovered asintaet LTE4 in the urine. Tluis, regard-secretions can be analyzed in nasal lavages after less of isotope labeling and route of administration,antigen challenge (28, 29). Local production in the it appears that about 5% of exogenously suppliedairways canalso beestimated bymeasuiements in leukotrienes arerecovered as urinary LTE4.bronchial lavage fluid (30-33), ormediator release More importantly, in none of these studies wasin themueosa of the airways can be estimated by LTC4orLTD4 recovered inthe urine, and A^-acetyl-analysis in nasopharyngeal or traeheobronchial LTE4, the major metabolite in rodents, was foundsecretions (34, 35). Monitoring circulating meta- to be only a tninor tnetabolite in tnan (43-45).bolites in whole blood or plasma is another After allergen-ehallenge. uiinary 16-carboxytetranoi-approach tomeasure in vivo production, but circu- dihydro-LTE4 was detected by specific radioimmuno-lating levels of LTE4 seem to be too low to be assay (RIA), but LTE4 was still found to be thereliably detected (36, 37). However, all these major urinary leukotriene derivative up to3 h afterstrategies may encounter difficulty; e.g., irreproduei- theallergen-indueed airway obstruction (48). Inte-ble sample collection and artifactual formation of restingly, inhalation ofbronchoconstrictive doses ofcompounds during sampling. Furthermore, inva- LTC4 or LTE4 in asthmatics altered urinary excre-sive techniques arealways associated with certain tion of LTE4 in a dose-dependent fashion (47).risks and can be used to obtain only a relatively Again, however, about 7% of inhaled LTC4waslimited number of samples. Urine, on the other recovered asurinary LTE4 (47). Tliese data stronglyhand, is a biologic fluid that is easy to eollect support the use of urinary LTE4 as a marker ofwithout any significant risk of artifactual c.\vivo leukotriene biosynthesis in the airways of asthma-formation of metabolites. Thepattern of metabo- tics. However, further studies are required tolites in urine probably refleets a whole-body pro- eharacterize fully the //; vivo metabolism of theduetion, with a fewexceptions where thekidney is eysteinyl-leukotrienes in man.known to be a main producer of a certain com- Maelouf et al. eoncluded from a study on thepound, such as intact urinary prostaglandin (PG)E2 metabolism of LTC4 that LTE4 is the best para-andTXB,. Production of the eysteinyl-leukotrienes meter for short-time /// vivo release, whereas theby thehuman kidney andurinary tract has not yet w- and (3-oxidized metabolites may better reflectbeen eompletely aseertained. Nevertheless, as indi- chronic changes in cysteinyl-leukotriene productioncated below, urine has been found useful for meas- (49). However, theusefulness of measurements ofuring whole-body production of the cysteinyl- co- and |3-oxidized metabolites ofLTE4 in theurineleukotrienes (18-20, 33, 38-41). needs further evaluation. For example, we must

One important aspeet in the seleetion of com- discover whether disease processes alter the nor-pounds tomeasure is knowledge of the metabolism mal excretion pattern of the compounds. It hasof the cysteinyl-leukotrienes. Metabolic studies been suggested that allergen challenge of asthma-have shown that LTE4 is the end metabolite of ties may affect thetransfer of formed leukotrienes,cysteinyl-leukotrienes in human lung invitro (16). leading to longer duration of elevated leukotrieneA few studies have focused on the //; vivo metabolic levels in theairways (50), a condition which, how-fate inman of LTC4, LTD4, or LTE4, administered ever, was not reflected as altered urinary excretion,intravenously (42-45) or byinhalation (46, 47), or At the moment, the most important factors toendogenously formed after allergen challenge (48). consider appear to be liver and kidney diseases thatMost radioactivity administered was at all times clearly alter the excretion of metabolites althoughrecovered in urine and bile, although with //; i/vo produetion is unchanged (39, 51). Normalsomewhat different relative distributions. Orning variations in diuresis are routinely corrected foret al.recovered about 50% of administered radio- by expressing the results in relation to urinaryactivity in urine after intravenous injection of creatinine levels (27).[5,6,7,8,ll,12,14,15--^H,s]-LTC4(42), whereas, in two Taken together, the results obtained to date


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cysteinyl-leukotrienes and thus appears to be thebest parameter to select for analysis.Methods for analysis of nrinary LTE4For several applications, including clinical trials ofnew antileukotriene drugs, it is of the utmostimportance to have sensitive, specific, and simplemethods for analysis of urinary LTE4. Immuno-assays utilizing polyclonal or monoclonal, virtuallyspecific, LTE4 antibodies, together with radioactivetracer (RIA) or enzyme-linked tracer (enzymeimmunoassay; EIA) have so far been used.However, though sensitive and relatively easy toperform, immunoassays may have methodologicalproblems. For instance, structurally similar com-pounds may cross-react with the antibody, andsubstances in the biologic matrix may nonspecifi-cally interfere with the antigen-antibody inter-action. Most data on immunoreactive urinary LTE4have so far been generated with the combinationof reversed-phase high-performance liquid chroma-tography (RP -HPL C) and immunoassay, in attemptsto increase the specificity of the assay (33, 38, 40,52).We have validated a simpler strategy using EIAor RIA analysis of serially diluted, unextractedurine samples, and compared the results withmeasurements of the same samples after solid-phase extraction (SPE) and R P- H PL C separation.Fractions at the retention time of authentic LTE4were assayed directly with RIA (20, 27) with amouse monoclonal antibody directed against LTD4with around 51% cross-reactivity to LTE4 (Adv.Magn. Inc., Boston, MA, U SA ). Alternatively, theLTE4 fractions were pooled, evaporated, andassayed for content of LTE4 with EIA (27) with arabbit polyclonal antiserum specific for LTE4 andacetylcholine esterase-linked tracer (CaymanChemical Company, Ann Arbor, MI, US A). Lossesduring purification were estimated by the use oftritium-labeled leukotrienes. For RIA measure-ments, 'H-labeled LTC4 was employed as internalstandard, whereas, in the case of EIA analysis, thehomologous tracer ^H-labeled LTE4 could be used.Considerable losses of leukotrienes during thepurification were documented with the internalstandards (Fig. 1). However, there was no signifi-cant difference between the recoveries of - H-labeled LTC4 and ^H-labeled LTE4 in SPE andRP-H PLC (Fig. 1; 27). The cross-reactivities of thedifferent antibodies were also carefully determined(27).Comparisons were then made, on the one hand,between analyses of unextracted samples with RIA

4 2 2 3 % 58+23%Fig. 1. Purification of urine samples. Results obtained by EIAor RfA analysis of unpurified samples were compared withmeasurements after two steps of purification. Tritium-labeledleukotrienes (^H-labeled LT Q and ^H-labeled LTE4) were usedas internal standards. Recoveries were measured after each stepof purification. Results of immunoassay analyses were finallyrecovery. Means SD.

immunoassays. More than 80 individual urine sam-ples were analyzed with EIA and RIA in parallel.Although RIA analysis consistently gave highervalues, which in part was due to the use of a differentantibody, a very good correlation between theresults was obtained (r^-0.81). Furthermore, in twosets of experiments, analysis in unextracted andpurified samples was compared. First, RIA measure-ments were applied on a total of more than 40samples collected before and after bronchial provo-cation (23). Although the average amounts of immu-noreactivity detected in the purified samples weresomewhat lower than values obtained in unpurifiedsamples, the differences did not reach statisticalsignificance. Rather, a similar ratio bet-ween pre-and postchallenge levels of urinary LTE4 wasobtained when analyzing unpurified or purified sam-ples (23). Similar comparisons were performed inanother study where a number of samples coUeetedhourly during allergen provocation were analyzedwith EIA before and after purification (27). Again,values obtained by analysis of HPLC-fractions cor-responding to the retention time of LTE4 were at alltime points somewhat lower than the valuesobtained by direct analysis of unextracted samples.However, the difference between baseline andpostchallenge samples were the same regardless ofwhether or not the samples were purified (27).Furthermore, when a larger number of urine sam-ples were analyzed before and after purification andthe results were validated according to Bland &Altman's measure of repeatability (53), there wasan excellent correlation between results obtainedwith the different assay procedures (27).


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addition of preservatives. For example, the influ-ence of an antioxidant on the stability of LTE4 wasinvestigated with urine and buffer samples spikedwith -^H-labeled LTE4. Samples were either storedwithout additions or supplied with the antioxidant4-hydroxy-TEMPO free radical to a final concen-tration of 1 mM, and the pH was adjusted to 9 withNaOH before freezing. Aliquots of the sampleswere withdrawn after t, 8, 30, and 60 days, andrecovery of intact ''H-labeled LTE4 was determined.The stability of LTE4 in urine at -20C was notimproved by addition of the antioxidant and pHadjustment (27).One important aspect of urinary ETE4 analysesis the procedure for collection of the urine samples.We have found that the peak increase in urinaryETE4, as a result of provoked release of leuko-trienes, generally is fairly short-lasting (20, 23, 27).In order to trap such an instantaneous release,collection of urine with short intervals is necessary.In our bronchial provocation studies, we usedhourly collected samples. If samples are collectedwith longer time intervals, the leukotrienesexcreted into the urine will be diluted and may thusescape detection. It is of interest that, in oneparticular study (25), we were unable to detect asignificant postehallenge inerease in urinary ETE4when an average value was ealculated for the foursamples collected hourly after the maximal airwayobstruction, whereas a significant increase wasdocumented when the highest concentration in the1- or 2-h postehallenge sample was selected indi-vidually (25). Tliis point was further illustrated ina study by Christie et al., which showed a peakincrease in urinary LTE4 1.5 h after inhalation ofLTC4 or LTE4 in asthmatics, with levels almost backto baseline after 3.5 h (47).Gas chromatography-mass spectrometry (GC-MS), which is a highly sensitive and specificmethod, may be an alternative for measurementsof urinary LTE4. However, derivatization pro-eedures used for other eicosanoids before GC-MScannot be used for cysteinyl-containing leuko-trienes. Recently, an alternative GC-MS methodwas described where the cysteinyl-leukotrieneswere subjected to catalytic reduction and desulfuri-zation before conversion into the pentafluorobenzyl(PFB) ester trimethylsilyl (TMS) ether derivatives(54). Analysis with GC-MS was subsequently per-formed with selected ion monitoring (SIM) usingstable isotope-labeled LTE4 analogs as internalstandards. Interestingly, the values obtained withquantitative GC-MS for basal urinary LTE4 inhealthy subjects (52) correlated very well withvalues reported from immunoassay analyses (22,

Measurement of urinary LTE4spectrometric procedures demand eunibersomeand time-consuming purification of the samples.Although GC-MS is an important tool to quantifyurinary LTE4 reliably and may serve as a "goldstandard" for calibration of other analytic methods,it is unlikely that GC-MS will become useful forhigh-output routine purposes. Another sophisti-cated teehnique, capillary electrophoresis, wasrecently employed for measurements of eieosa-noids, among them LTE4 and its metabolites (60).However, the sensitivity of the electrophoresis waseven less than that of HPLC with UV detection formeasurements of leukotrienes, and thus was about100 times less sensitive than the immunoassays.Basal levels of urinary LTE4Tlie procedure with imm unoassay analysis of unex-tracted urine samples, stored without preservatives,has been applied to samples collected during clini-cal experimental studies. First, UrE4 was measuredby EIA in urine samples collected every third hour,for a period of 24 h, from a group of healthynonasthmatic subjects without diurnal variation inpeak expiratory flow. In this group of nonasthmaticsubjects, there were no signs of diurnal variation inthe excretion of urinary LTE4 (27). In line with thisobservation, Asano et al. eould not identify diurnalvariation in the excretion of LTE4 in either normalsubjects or a group of patients with asthma (61).On the other hand, it was recently reported thatpatients experiencing nocturnal exacerbation showedincreased excretion of urinary LTE4 in samplescollected from 9 pm to 9 am (62). There was a linearcorrelation between morning dip in lung functionand urinary excretion of LTE4 (62). However, in astudy by Wenzel et al., the dil'ferenee from day tonight in urinary LTE4 in patients with nocturnalasthma was not significant, but the levels weresignificantly higher than in the controls in samplescollected between 11 pm and 7 am, as well asbetween 3 pni and 11 pm (63). Treatment with the5-lipoxygenase inhibitor Zileuton showed signifi-cant attenuation of urinary LTE4 in the asthmaticpatients together with a trend to improvement inlung function (63).The mean baseline level of LTE4 in morningurine samples (at 6 am) from the healthy, non-asthmatic subjeets was not different from a groupof atopic asthmatics (27). Basehne values given inthe literature for asthmatic and nonasthmatic sub-jects vary considerably, mainly because of varyingassay procedures. Nevertheless, the studies gener-ally show no significant discrepancies betweenbaseline urinary LTE4 in healthy nonasthmatic and


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Measurement of urinary LTE4An increased excretion of urinary LTE4 after ehallenge was oral or by inhalation (20, 23, 58, 66,allergen-induced airway obstruction was initially 73, 75, 76); in one ease, it was as much as sevenfolddocumented by Taylor et al. (18), and later con- (67). Interestingly, inhaled PG E, was shown tofirmed by us and others (19, 20, 48, 69, 71 , 72). prevent aspirin-induced bronchoeonstriction as wellBronchial provocation by cumulative ehallenge as to attenuate the aspirin-induced rise in urinarywith increasing doses of specific allergen in atopic LTE4 excretion (76). However, baseUne levels of

asthmatics was followed by a significant, twofold urinary LTE4 in these aspirin-sensitive asthmaticsincrease in urinary eoncen tration of LTE4 (20). In were not affected by PG Ej inhalation,anothe r study, after a single dose of allergen, the Another type of trigger in asthma is exercise,postchallenge urinary LTE4 values increased by causing exercise-induced bronchocotistriction. Studiesabout 40% above prechallenge baseline, and the of exereise-induced asthmatic responses have pro-peak excretion occurred in the first two hourly duced somewhat contradictory results regardingsamples, collected after the induced bronchial the role of leukotrienes. It has not been possibleobstruction (25). A correlation between the airway to document increased excretion of LTE4 afterresponse and urinary exeretion of LTE4 could be exercise-induced airway obstruction (57, 78), withseen when the area under the curve representing the exception of one report on elevated urinaryairway obstruction was com pared with the net LTE4 levels after exercise ehallenge in a group ofrelease of urinary LTE4 (25). Studies repo rted in children (79). Therefore, at present, the role ofthe literature have been perfonned according to cysteinyl-leukotrienes in this type of asthma eandifferent protocols; nevertheless, the increase in not be determined solely on the basis of analysesurinary LTE4 after allergen challenge has been of urinary LTE4. However, since several differentshown to be between two- and fivefold in a number antileuko triene drugs provide significant protec -of independent reports (18 -20 , 57). Tlie end-point tion in exercise-induced asthma, it has been sug-in most provocation protocols has been a decrease gested that leukotrienes mediate also this type ofin FE"V, by 20% ; thus, approximately the same airway obstruction (80 -83 ). In fact, since exercise-degree of bronchoeonstriction was elicited in all induced airway obstruction is relatively brief, thepatien ts. brief stimulation by exereise may be insufficient toIt is still not clear how the release of leukotrienes generate enough leukotrienes detectab le abovein the airways is regulated. In studies of leukotriene basal levels. Tliis further suggests that the durationreceptor antagonists (see also above), we could of the ehallenge eontributes to the amount of LTE4show an even more pronounced increase in urinary excreted in the urine (see discussion of allergenLTE4 when higher doses of allergen were tolerated above).by the patients in the presence of the receptor It should also be mentioned in this context thatantagonist (which attenuated the airway obstruction) airway obstruction induced by histamine or metha-(20). Tliis would point to a dose-dependent release choline, two compounds commonly used to assessof leukotrienes. W hether there is a relation to airway reactivity, does not lead to increased excre-the tiine-span over which the cells are activated tion of urinary LTE4, as shown by us and others (20,and whether repeated bursts of activation of the 84). Hence, there are no indications that leuko-leukotriene-producing cells by several ehallenges trienes are second messengers in the smooth-muscleare involved in the meehanism is currently under contraction induced by histamine or methacholine.investigation. On the other hand, airway obstruction induced by

Eurthennore, inhalation of lysine-aspirin in aspirin- platelet-activating factor (PAF) was followed byintolerant pa tients, leading to a significant airway augmented LTE4 levels in the urine (84), and a roleobstruction, was shown to be assoeiated with for leukotrienes in PAF-induced bronchial obstruc-increased excretion of urinary LTE4 (20). This was tion was further supported by the beneficial effectexclusive for the intolerant patients, whereas aspirin- of leukotriene recep tor antagonists on the broncho-toleran t asthmatics did not react with either bron- constrictor effect of inhaled PAF (85, 86).chial obstruction or increased excretion of urinary Whether urinary LTE4 is increased also in associ-LTE4 in response to inhaled lysine-aspirin (20). ation with late-phase asthmatic responses has alsoIncreased urinary LTE4 excretion in aspirin-sensitive been unclear. A limited number of bronchial provo-subjects has been confirmed by others using oral cation studies have considered the role of leuko-as well as inhaled routes of aspirin administration trienes in the late asthmatie response. Generally, a(58, 66, 67 ,13-16). How ever, the oral challenge is prolonged elevation of the urinary levels of LTE4often associated with severe systemie reactions has been doeumented, rather than a significant(77). Although absolute values vary between the distinct second peak of increase (18,1 9, 22, 48, 5 5 -


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Kumlincontinuous excretion of leukotrienes previouslyformed during the early response is not possible todetermine at the mom ent, and contradictory resultshave been presented (19, 55, 56, 69). How ever, therapid m etabolic clearance of cysteinyl-Ieukotrienes(43, 45) rather supports de novo formation ofleukotrienes also during the late-phase response.Recent data from our laboratory further support arelease of cysteinyl-Ieukotrienes, as well as PGD2,during the late asthmatic response to allergenchallenge in dual responders (87), and this wascorroborated by results from pharmacologic inter-vention (88). These latter data confirm earherreports on significant attenuation of the late-phaseresponse by antileukotriene drugs, which in turnstrongly supports the view that leukotrienes alsocontribute to the late phase of antigen-inducedairway obstruction (21, 22, 69).

Concerning pharmacologic regulation of leuko-triene formation, a few studies on inhaled steroids,(3-agonists, and sodium cromoglycate have concen-trated on the relationship between effects onallergen-provoked airway obstruction and increasedurinary LTE4 excretion (56, 89, 90). With all threedrugs, a significant attenuation of the airwayresponse was observed, whereas the allergen-induced increase in urinary LTE4 was not affected.This may reflect differences between in vitro andin vivo actions of glucocorticoids on leukotrienebiosynthesis and also that the pharmacologicactions of [3-agonists and sodium cromoglycate donot primarily involve interference with leukotrieneformation or action. There are several other pos-sible explanations of these findings which cannotbe elaborated on here. However, a less cumber-some method for measurement of urinary LTE4, asdescribed in this chapter, might give better oppor-tunities to elucidate further the effect of currentasthma therapy on the leukotriene pathway. Con-sidered together, however, the current data supportthe view that glucocorticosteroids have no signifi-cant direct effects on leukotriene formation in asthma.Effects of leukotriene biosynthesisinhibitors and leukotriene receptorantagonists on induced increase in urinaryThe effects of two specific leukotriene receptorantagonists (ICI 204, 219 and MK-0679) andone leukotriene biosynthesis inhibitor (the FLAP-inhibitor BAY xlOO5) on airway response andurinary LTE4 were evaluated in bronchoprovoca-tion studies (20, 23, 25). The trials followed adouble-blind, placebo-controlled, crossover design.

presence of the leukotriene receptor antagonistAccolate (ICI 204, 219; 20 mg, 2 h before start ofprovocation), the amount of allergen required toprovoke a 20% drop in FEV,, i.e. the PD20 forallergen, was significantly increased. In line withthis increase in allergen dose, there was a meanfivefold postchallenge increase in urinary LTE4 inthe presence of the leukotriene receptor antago-nist, as compared to a twofold increase during theplacebo session (20). This supports dose- and per-haps also exposure-time-dependent influences onthe leukotriene formation.Since it was clear that aspirin-intolerant asthma-tics had a higher basal excretion of LTE4, it was ofinterest to see whether this group of asthmaticswould benefit from treatment with a leukotrienereceptor antagonist. The patients were given either750 mg of MK-0679 or placebo 1 h before inhalation challenge with increasing doses of lysine-aspirin (23). Pretreatment with MK-0679 caused adistinct rightward shift of the dose-response rela-tionship for all subjects, three out of eight failingto produce a 20% decrease in FEV, after inhala-tion of the highest possible aspirin-dose. UrinaryLTE4 was determined in hourly collected samplesby RIA. It was confirmed that the bronchoconstrictorresponse to aspirin was again associated withincreased levels of urinary LTE4, with peak concen-tration in the first hourly collected samples aftermaximal airway response. As was seen for ICI204,219 in the allergen challenge, the urinary LTE4levels were found to be higher after pretreatmentwith MK-0679, in association with the augmentedPD20 for lysine-aspirin (23), indicating a similardose-dependence for aspirin-induced leukotrienerelease as for allergen-provoked release (see above).In neither of these studies did the leukotrieneantagonists cause significant elevation of basalurinary LTE4 (20, 23). Partial inhibition of aspirin-induced asthma was reported by Christie et al. forthe leukotriene receptor antagonist SK&F 104353in an oral challenge study (91), and a role for 5-lipoxygenase products in aspirin-induced asthmawas corroborated in a 3-week trial with the leuko-triene biosynthesis inhibitor Zileuton (92) andmore recently with the 5-lipoxygenase inhibitorZD2138, where the inhibition of the aspirin-induced fall in FEVi was associated with substan-tial inhibition of leukotriene production (75).Next, bronchial provocation with a single pre-determined PD20 for allergen was performed in thepresence or absence of the leukotriene biosynthesisinhibitor BAY xlOO5 (25). The airway response tothe inhaled dose of allergen was significantly inhib-ited on the day when BAY xlOO5 (750 mg) was


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the reaction. The calculated mean inhibition of themaximal drop in FEV, was approximately 65%.Basal urinary excretion of LTE4 was not signifi-cantly different on the two study days. The netrelease (postchallenge level minus prechallengelevel) of LTE4 was significantly less during the BAYxlOO5 session than the placebo session. Tlie inhibi-tions of the airway response and postchallengeincrease in urinary LTE4 were 73% and 88%,respectively, and thus correlated very well (Fig. 3)(25).To date, data have been published on the cor-relation between the effects of leukotriene bio-synthesis inhibitors on airway response, excretionof urinary LTE4, and ionophore-stimulated ex vivoformation of LTB4 in whole blood. The results fromtwo studies performed with Zileuton and MK-886indicated that an inhibition of up to almost 100%of ex vivo LTB4 formation does not correlatedirectly with the effect of the drugs on the airwayresponse to allergen (69, 71). Although the effectof Zileuton on the airway response was nonsignifi-cant for the group as a whole, some correlationbetween inhibition of the early asthmatic responseand urinary LTE4 excretion was obtained for theindividual subjects (71). Nasser et al. could notdocument any protection against allergen-inducedasthmatic responses by the 5-lipoxygenase inhibitorZD2138, despite 82% inhibition of LTB4 atid 52%inhibition of urinary LTE4 excretion (72). On theother hand, Friedman et al. could document asignificant correlation only between plasma levelsof MK-886 and the attenuation of ex vivo LTB4production (69). Together with our own data onBAY xlOO5, the results obtained so far suggestthat a high degree of in vivo inhibition of pul-monary leukotriene production is necessary toobtain a significant effect on the airway responseby the leukotriene biosynthesis inhibitor. Moreover,

Measurement of urinary LTE4determination of inhibition of ex vivo formation ofLTB4 may well correlate with the drug plasma level,but not necessarily with inhibition of enzymaticactivity in target cells in the lutig.Conclusions on methodological aspectsand further applications of m easuremen tsof urinary LTE4We have validated a simple strategy for tneasure-nients of urinary LTE4 and successfully applied thisprocedure in some clinical studies of antileuko-triene drugs. In summary, there was no diurnalvariation in urinary LTE4 excretion in healthysubjects, and we could document that aspirin-intolerant asthmatics as a group had higher basallevels of urinary LTE4, whereas there was nodifference betweeti other asthmatics and normalcontrols. Furthermore, lysine-aspirin-induced air-way obstruction in aspirin-intolerant asthmaticsresulted in increased levels of urinary LTE4. Like-wise, allergen challetige in atopies caused increasedurinary excretion of LTE4, whereas airway ob-struction induced by histamine did not cause anyalteration in urinary LTE4. Furthermore, the leuko-triene biosynthesis inhibitor BAY xlOO5 inhibitedpostchallenge increase in urinary LTE4, as well asimmediate airway response to a single dose ofinhaled allergen.

Tlie methodological coticlusions that emergefrom the published data are as follows. First,immunoassay of serially diluted unextracted urinesamples is a reliable means to measure in vivoproduction of cysteinyl-leukotrienes. Purificationof the samples causes substantial losses of leuko-trienes and does not necessarily improve the assay.Second, inhibition of postchallenge increase inurinary LTE4 appears to correlate with inhibitionof allergen-induced airway obstructioti, whereas

Fig. 3. InhibilBAY xlOO5 inhibited allergen-induced :(AUC) for change in FEV ,, as compare on by 73% (left panel, elosed bar) when expt:atment session (open bar). BAY xlOO5 likewise


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Kumlin

inhibition ofexvivo formation of LTB4 correlates choline and histamine in normal and asthmatic subjectswith plasma concentration of drugs ra the r than Eur Respir J I988;l:9l3-17.inhibition of airway response. Third, measurements ' j,.jg"g ^^ th 'alr'waesôns'iv*eneLio'\'ii t'a'mine' iiof basal urinary LTE4 alone may be a poor index siibje'cts wi'th asthnTa' ^nd iToTmars titijec s.*J Aller'gy^Cliiito assess the in vivo effects of different doses of Immunol ]988;82:654-60.leukotriene biosynthesis inhibitors on pulmonary 9 Jacques CAJ, Spur BW Johnson M, Lee TH. Tlie mecha-production ofcysteinyl-Ieukotrienes. It is proposed "'' "i ''LTE4-induced histamine hyperresponsiveness intha t bronchial provocati on studies with allergen, L"'Xo''lfrTptiar iLicMl 1991 ^ ^ âspirin, or PAF preferentially be used in at te mp ts IQ O'Hickey SP, Hawksworth RJ Fong CY Arm JR Spur BWto establish the effective doses of biosynthesis Lee TH. Leukotrienes C4, D4 and E4 enhance histamineinhibi tors. responsiveness in asthmatic airways. Am Rev Respir Dis

Taken together, thesensi tive and ra ther simple i99l;l44;l053-7.method we describe for analysis of excretion of '^ - '"^ ' ' O'Hickey SR Spur BW Lee TH. Airway respon-LTE4 in human urine may be useful in further rsÎ^lri inlcrd Ts\hU" A nrReT Rer p'i r" D r^chnical trials with an ti-leuko triene drugs. Since 148-53.many disorders, such as other pulmonary diseases 12. Christie PE, Schmitz-Schumann M, Spur BW Lee TH(33, 93, 94), atopic diseases (95), anaphylact ic Airway responsiveness to leukotriene C4 (LTC4), leuko-reactions (70), cardiovascular disorders (96, 97) , tnene E4 (LTE4) and histamme in aspirm-sensitive asth-and autoimmune diseases (98-100), have been jj slsadVcs' ' Nieves AL'"' Krauss AH-P Woodward DF.associated with altered excretion of urinary LTE4, ' Comparison of leukotriene B4 and D4 effects on humanthis method may be applicable in a broad range of eosinophil and neutrophil motility in vitro. J Leukoc Bioexperimental clinical studies. 1994;55:]83-91.

14. Laitinen LA, Laitinen A, Haahtela T, Vilkka V Spur BWLee TH. Leukotriene E4 and granulocytic infiltration intoasthmatic airways. Lancet 1993;341:989-90.Acknowledgments 15. MacGlashan DWJ, Schleimer RR Peters SR et al. Gener-

Tliese studies were supported by grants from the Swedish ^' '" ' leukotrienes by purified human lung mast cells.Medical Research Council (projects 14X-09071 03X-217) the J Chn Invest 1982;70:747-51.Swedish Association Against Asthma and Allergy (A&A), the 1^. Kumlin M, Dahlen S-E. Character.st.es of formation andSwedish Heart Lung Foundation, Vardalstiftelsen, the Swedish ^'''^^'"' metabolism of leukotrienes m the chopped humanSociety of Medicine, Stiftelsen Lars Hiertas Minne, Stiftelsen '""8- Biochim Biophys Acta f990;1044:201-10.Ragnhild ochEinar Lundstroms Minne, the Institute of 17. Dahlen S-E, Hansson G, Hedqvist R Bjorck T Granstrom EEnvironmental Medicine, the Swedish Environment Protection Dahlen B. Allergen ehallenge of lung tissue from asthma-Board (312049), and Karolinska Institutet '"^^ "^ ' ' " '^ bronchial contraction that correlates with the^ ' release of leukotrienes C4, D4 and E4.Proc Natl Acad Sci

USA 1983;80:1712-16.18. Taylor GW Taylor I, Black R et al. Urinary leukotriene E4after antigen challenge and inacute asthma and allergic

1. Arm J, Lee T Sulphidopeptide leukotrienes in asthma. Clin rhinitis. Lancet 1989;l:584-8.Sci 1993;84:501-10. 1' - Sladek K Dworski R Fitzgerald GA, et al. Allergen-

2. Dahlen S-E. Leukotrienes as mediators of airway obstruc- stimulated release of thromboxane Aj and leukotriene E4tion andbronchial hyperresponsiveness. In: Page C ' " humans. Effects of indomethacin. Am Rev Respir DisGardiner P, editors. Airway hyperresponsiveness: is it really 1990;]41:1441-5.important for asthma? Oxford: Blackwell Scientific, 1993: 20. Kumlin M, Dahlen B, Bjorck T, Zetterstrom O, Granstrom E,188-205. Dahlen S-E. Urinary excretion of leukotriene E4 and

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5. Marom Z, Shelhamer JH, Bach MK, Morton DR, 22. Rasmussen JB, Eriksson L-O, Margolskee DJ, TagariRKaliner M. Slow-reacting substances, leukotrienes C4 and Williams VC, Andersson K-E. Leukotriene D4 receptorD 4, increase the release of mucus from human airways in blockade inhibits the immediate and late broncho-vitro. Am Rev Respir Dis 1982;126:449-51. constrictor responses to inhaled antigen inpatients with

6. Arm JR O'Hickey SP, Hawksworth RJ, et al. Asthmatic asthma. J Allergy Clin Immunol 1992;90:193-20f.airways have a disproportionate hyperresponsiveness to 23. Dahlen B, Kumlin M, Margolskee D.I, et al. The leuko-LTE4, as compared with normal airways, but not to LTC4, triene-receptor antagonist MK-0679 blocks airway obstruc-LTD4, methaeholine, and histamine. Am Rev Respir Dis tion induced by inhaled lysine-aspirin in aspirin-sensitive1990;142:ll]2-18. asthmatics. Eur Respir J 1993;6:I018-26.


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Measurement of usurgery. Coron Artery Dis 1993:4:899-904. 1992:19:252-8.98. Fauler J, Thon A. Tsikas D, von der Hardt H, Froiich JC. 100. Hackshaw KV, Yuhong SHI, Brandwein SR. Jones K.Enhaneed synthesis of cysteinyl leukotrienes in juvenile Westcott JY. A pilot study of Zileuton, a novel selectiverheumatoid arthritis. Arthritis Rheum 1994;37:93-7. 5-lipoxygenase inhibitor, in patients with systemic lupus

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Measurements of Leukotrienes in the Urine- Kumlin