Measurement of Leukotrienes in Humans

MARIA KUMLIN

Experimental Asthma and Allergy Research, The National Institute of Environmental Medicine, Karolinska Institutet, Stockholm, Sweden

	INTRODUCTION

TOP INTRODUCTION MEASUREMENT OF LEUKOTRIENES IN... MEASUREMENT OF OTHER URINARY... BASAL LEVELS OF URINARY... URINARY LTE4 IN ASTHMA... CONCLUSIONS REFERENCES

This article focuses on strategies to appreciate in vivo release of the cysteinyl-leukotrienes (LTs; LTC₄, LTD₄, and LTE₄) and presents some results obtained by such measurements. Whereas analyses of leukotriene formation in isolated tissues and cells primarily reflect ex vivo biosynthesis, measurement of intact leukotrienes or metabolites thereof in different biological fluids may be used as an index of in vivo production of the primary leukotrienes.

	MEASUREMENT OF LEUKOTRIENES IN BIOLOGICAL FLUIDS

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Analysis of cells and compounds in induced sputum seems to be a promising way to gain extended knowledge about mediator release in the airways in response to challenge or in patients with chronic asthma (1). However, so far no data are available on leukotriene levels in induced sputum. Bronchoalveolar lavage (BAL) or nasal lavage (NAL) can been used to monitor eicosanoid and leukotriene release into the airways under basal conditions and in response to challenge (2). Endobronchial challenge with allergen or aspirin led to increased levels of leukotrienes in the BAL fluid (7, 8). Leukotriene measurement in blood, serum, or plasma is associated with several problems, and most importantly, the circulating levels of leukotrienes are below the detection limit of most assays developed to date (9, 10). Measurement of leukotrienes in urine, however, has several advantages. Among them are the non invasive sampling technique that allows frequent collection of samples; the fact that the pattern of metabolites in urine most likely reflects whole body production; and that there is no risk of artifactual ex vivo formation of metabolites during sampling, which may be a major problem when measuring arachidonic acid metabolites in, e.g., blood. However, comprehensive metabolic studies are required to find the right metabolite as a suitable target for analysis, and liver and kidney disorders affect urinary excretion of leukotrienes, which may lead to misinterpretation of the results (11, 12). The pulmonary metabolism of LTC₄ in vitro results in a rapid, almost exclusive formation of LTE₄ with no further conversion, and thus LTE₄ seems to be the end product of cysteinyl-LT metabolism in the lung (13). Studies of the in vivo metabolism of cysteinyl-LTs generally have shown that about 5% of leukotrienes supplied exogenously, intravenously, or by inhalation is recovered as intact LTE₄ in the urine (reviewed in Reference 14). Metabolism of bronchoconstrictive doses of inhaled LTC₄ or LTE₄ in patients with asthma strongly supports the use of urinary LTE₄ as an index specifically reflecting cysteinyl-LT release in the airways of patients with asthma (15). Thus, urinary LTE₄ is considered the best target for analysis in order to appreciate in vivo cysteinyl-LT production. However, a peak increase in urinary LTE₄, as a result of provoked release of leukotrienes, is generally fairly short lasting and in order to trap such a transient increase, collection of urine at short intervals is necessary (16). This is particularly illustrated by the peak increase in urinary LTE₄ detected 1.5 h after inhalation of LTC₄ or LTE₄ in patients with asthma with levels almost back to baseline already after 3.5 h (15).

The usefulness of different analytical methods for measurement of cysteinyl-LTs and other eicosanoids is discussed in Reference 17. Immunoassays are regarded as fast and sensitive methods suitable for analysis of eicosanoids, and they have been extensively used for this purpose (18). To monitor whole body production of cysteinyl-LTs, urinary LTE₄ was determined with an enzyme immunoassay (EIA; Cayman Chemical, Ann Arbor, MI) which has been extensively validated for use in unextracted urine samples (16). A fast and specific purification strategy using an immunoaffinity resin has been described (19) and appears to be a promising alternative to the most common laborious procedure with purification by reversed phase high-performance liquid chromatography (RP-HPLC) prior to immunoassay. An aliquot of all urine samples is routinely analyzed for content of creatinine, with commercially available kits (Sigma, St. Louis, MO), to allow corrections for diuresis variations.

It may be briefly mentioned in this context that for measurements of LTB₄ or its metabolites, no methods for routine analysis of urine is available to date, partly because of limited knowledge of the whole body metabolism and excretion of breakdown products of this leukotriene. Intact LTB₄ may be assayed in BAL and NAL fluids (4, 6).

	MEASUREMENT OF OTHER URINARY MARKERS AND MEDIATORS OF AIRWAY INFLAMMATION

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Mediators or metabolites thereof other than cysteinyl-LTs may serve as additional indicators of specific cell activation and inflammatory activity, particularly in the airways. The primary cyclooxygenase product of mast cells is prostaglandin D₂ (PGD₂). The first urinary metabolite of PGD₂ to appear, namely 9,11-PGF₂, is analyzed as an index of PGD₂ formation, and thereby mast cell activation, with an enzyme immunoassay (Cayman Chemical) (20). Methyl-histamine as another marker of mast cells, and eosinophil protein X (EPX) as a marker of eosinophil activation, are both measured with radioimmunoassays (Pharmacia, Uppsala, Sweden) (21). All results are expressed as nanograms or micrograms per millimole of creatinine in the urine.

	BASAL LEVELS OF URINARY LTE4

TOP INTRODUCTION MEASUREMENT OF LEUKOTRIENES IN... MEASUREMENT OF OTHER URINARY... BASAL LEVELS OF URINARY... URINARY LTE4 IN ASTHMA... CONCLUSIONS REFERENCES

To date, knowledge about levels of urinary LTE₄ under basal conditions in healthy subjects and patients with asthma is limited. A few reports in the literature describe basal levels in small groups of individuals. Absolute values are, however, difficult to compare since different methods have been employed by different investigators. In a group of nine healthy nonasthmatic subjects a mean basal level of LTE₄ in urine was 14.9 ± 5.4 ng/mmol of creatinine when samples were collected every third hour during 24 h and no diurnal variation was seen (16). Similarly, no diurnal variation was seen in either normal (n = 5) or asthmatic (n = 8) individuals (22). However, conflicting data concerning levels of urinary LTE₄ over 24 h in patients with nocturnal asthma have been published. In nine patients with nocturnal exacerbations urinary LTE₄ was higher at night (9:00 P.M. to 9:00 A.M.; mean of 35.16 [95% CI, 28.77-42.85] ng/ mmol of creatinine) as compared with daytime values (23.12 [17.18-30.06] and 25.18 [21.03-30.13]) (23). No significant increase could, however, be documented from day to night in a group of 12 patients with nocturnal asthma (10.7 ± 1.5 versus 14.9 ± 2.8 ng/mmol of creatinine, p = 0.08) (24). In both groups, however, the levels at all time points were higher as compared with controls and asthmatic subjects without nocturnal attacks (23, 24). Generally, no difference has been documented between baseline urinary LTE₄ in nonasthmatic and atopic asthmatic subjects (16, 23, 25), with the exception of a study in which a significantly higher urinary level of LTE₄ was seen in patients with asthma as compared with normal controls (12.4 ± 6.7 versus 9.5 ± 4.3 ng/mmol of creatinine) (22). Moreover, in a more recent study, Westcott and coworkers could show that basal urinary levels of LTE₄ did not differ significantly between healthy adults (9.0 ± 0.8 ng/mmol of creatinine, n = 14) and healthy children, age 3-12 yr (12 ± 1, n = 20) (19).

An interesting observation is the significantly higher basal levels of urinary LTE₄ in aspirin-intolerant patients with asthma as compared with aspirin-tolerant patients with asthma (16, 26). In contrast, no difference was seen between urinary basal levels of the PGD₂ metabolite 9,11-PGF₂ in the aspirin-intolerant patients with asthma as compared with other patients with asthma (20). One explanation for this discrepancy may be that the basal excretion of the two metabolites is derived from different cell sources. The cysteinyl-LTs may be released from, e.g., eosinophils under basal conditions whereas the PGD₂ metabolite is at all times primarily derived from the mast cells.

Taken together, more data on basal levels in larger groups of individuals must be obtained before any reference values for urinary LTE₄ in humans can be settled.

	URINARY LTE4 IN ASTHMA AND AIRWAY INFLAMMATION

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Several trigger factors in asthma have now been correlated with increased urinary excretion of LTE₄, both in studies of provoked asthma and as a result of acute spontaneous asthmatic attacks (28).

Allergen

A number of studies have documented increased levels of urinary LTE₄ during the early phase of allergen induced airway obstruction (28, 32, 33). There has, however, been a debate in the literature as to whether cysteinyl-LTs are also involved in the late phase of the asthmatic response in dual responders. To investigate the mechanisms behind the early asthmatic reaction (EAR) and late asthmatic reaction (LAR) to allergen, we examined the pattern of mediator release during both these phases of the asthmatic reaction after allergen challenge (21). Twelve subjects with atopic asthma underwent bronchial provocation with allergen. They all experienced an early, severe drop in their FEV₁ values, with at least 25% followed by a more protracted decrease during the late asthmatic response, occurring about 6-8 h after allergen challenge. Values had returned to baseline when measured 24 h after the provocation (Figure 1). Urinary LTE₄ was monitored hourly before and up to 12 h after the allergen challenge and was shown to increase significantly during both EAR and LAR, with levels not returning to normal until after 24 h [Figure 1 (21)]. The data indicate that leukotrienes also contribute to the LAR, which was further supported by the results of pharmacological intervention (34). The EAR as well as the LAR to allergen was significantly attenuated by pretreatment with the leukotriene receptor antagonist zafirlukast, alone or in combination with the histamine antagonist loratidine. The combined treatment gave even better protection against allergen-induced airway obstruction than any of the drugs administered alone.

View larger version (18K): [in this window] [in a new window]   Figure 1.   Time course of allergen-induced early- and late-phase bronchoconstriction (top) and urinary excretion of LTE4 in 12 subjects with atopic asthma (bottom). Pulmonary function is expressed as percentage of post-diluent FEV1 and urinary LTE4 is expressed as nanograms per millimole of creatinine, and both were monitored up to 24 h after allergen inhalation. The arrow indicates the time point at which allergen challenge was commenced. Each point represents the group mean ± SE.

Aspirin

Among aspirin-intolerant patients with asthma, bronchoconstriction is elicited by aspirin and other nonsteroidal antiinflammatory drugs (NSAIDs). Increased urinary excretion of LTE₄ as well as 9,11-PGF₂ after aspirin-induced airway obstruction has been documented (20, 26, 35). An even more pronounced increase in the urinary excretion was observed when the patients tolerated higher doses of aspirin after pretreatment with the leukotriene receptor antagonist MK-0679, indicating a dose-dependent release in response to aspirin challenge (35). Interestingly, it was documented that the long-acting ₂-agonist salmeterol attenuated the lysine-aspirin- induced increase in urinary LTE₄ as well as a PGD₂ metabolite (PGD-M) in parallel with a protective effect of salmeterol on lysine-aspirin-induced bronchoconstriction (36).

Exercise

Some controversy remains about the causative mediators in the bronchoconstrictive responses to exercise in some patients with asthma. A study was performed with analysis of urinary mediator excretion in association with exercise-induced bronchoconstriction (EIB) (37). Seven of 12 subjects with mild asthma and a history of EIB responded to 5 min of exercise on a stationary bicycle ergometer at 80% maximum work load with a more than 15% fall in FEV₁. Urinary excretion of the PGD₂ metabolite 9,11-PGF₂ was significantly higher 30 and 90 min after exercise in these subjects as compared with the remaining five nonresponders, indicative of mast cell activation during the EIB. However, no increased urinary excretion of LTE₄ was observed in either group and no significant difference was seen between the responders and the nonresponders in this study (37). This observation confirms the results of other studies, in which no increased leukotriene production could be documented although a significant attenuation of the EIB was achieved by pretreatment with leukotriene receptor antagonist (38) or biosynthesis inhibitor (41). In contrast to a study of exercise-induced asthma in children (42), a more recent study documented for the first time increased urinary LTE₄ after EIB in a group of 21 adult patients with asthma (43). Together, all these data strongly suggest that leukotrienes are important mediators also of EIB.

Organic Dust

Exposure of healthy subjects to swine house dust caused increased bronchial responsiveness to methacholine, without acute bronchoconstriction (44). Previous studies have suggested a relationship between increased bronchial responsiveness and mast cell mediator release (45). Moreover, there are some indications of improvements in increased airway reactivity during treatment with antileukotriene drugs (46). A study was thus initiated with the aim of determining whether activation of the cysteinyl-LT pathway occurs after exposure to swine house dust and, if so, if release of the cysteinyl-LTs is associated with the development of the increased bronchial responsiveness. Ten nonasthmatic subjects were exposed to swine house dust for 3 h in the morning while weighing pigs in a piggery. An acute inflammatory reaction was elicited in the airways, as assessed by increased number of cells and release of cytokines in NAL fluid (6). Bronchial responsiveness to methacholine was determined 5 d before and the day after the exposure (Day 1 and Day 3), and a significantly increased bronchial reactivity to methacholine was seen after the exposure to swine house dust (Figure 2). Urine was collected at hourly interval during the days of methacholine challenge and also on the day of exposure (Day 2). There was no change in the urinary levels of LTE₄ during Day 1 or 3. However, after exposure to swine house dust on Day 2, the urinary excretion of LTE₄ increased significantly in the afternoon [Figure 2 (6)]. Moreover, a significantly elevated level of interleukin 8 (IL-8) was accompanied by an increased number of neutrophils in NAL fluid. Both LTE₄ and LTB₄ were also significantly increased in NAL fluid 7 h after the exposure to swine house dust. Together the results suggest that cysteinyl-LTs may contribute to the development of increased bronchial responsiveness and that increased urinary LTE₄ may reflect the inflammatory reaction in the airways after inhalation of organic dust.

View larger version (16K): [in this window] [in a new window]   Figure 2.   (Left panel ) Change in bronchial responsiveness to methacholine after exposure to swine house dust as determined 5 d before exposure (preexposure day; open symbols) and after 3 h on the day of exposure (filled symbols). Data are expressed as geometric mean. (Right panel ) Mean and individual urinary levels of LTE4 in samples collected on the preexposure day (Day 1), day of exposure (Day 2), and postexposure day (Day 3). Open bars show group mean (± SE, n ± 10) values in samples collected before methacholine challenges (Day 1 and Day 3) or exposure to swine house dust (Day 2) and solid bars show group mean peak values of samples collected after the respective challenges. A significant increase in the excretion of LTE4 was seen in the afternoon of the swine house dust exposure day (**p < 0.01).

	CONCLUSIONS

TOP INTRODUCTION MEASUREMENT OF LEUKOTRIENES IN... MEASUREMENT OF OTHER URINARY... BASAL LEVELS OF URINARY... URINARY LTE4 IN ASTHMA... CONCLUSIONS REFERENCES

It is now well established that urinary LTE₄ may serve as an index of in vivo whole body production of cysteinyl-LTs. Reports are accumulating on different pathological events, including asthmatic attacks, in which increased urinary LTE₄ is indicative of ongoing inflammatory activity in the airways or other organs. Furthermore, the urinary PGD₂ metabolite 9, 11-PGF₂ appears to be a most sensitive marker of mast cell activation.

	Footnotes

Correspondence and requests for reprints should be addressed to Maria Kumlin, B.M., Ph.D., Experimental Asthma and Allergy Research, The National Institute of Environmental Medicine, Karolinska Institutet, P.O. Box 210, S-171 77 Stockholm, Sweden. E-mail: maria.kumlin{at}imm.ki.se

Acknowledgments: Supported by the Medical Research Council (14X-13047, 14X-9071, 99P-12754), the Heart-Lung Foundation, the Foundation for Health Care Sciences and Allergy Research (Vårdalstiftelsen), the Association against Asthma and Allergy, and the Karolinska Institutet.

	References

TOP INTRODUCTION MEASUREMENT OF LEUKOTRIENES IN... MEASUREMENT OF OTHER URINARY... BASAL LEVELS OF URINARY... URINARY LTE4 IN ASTHMA... CONCLUSIONS REFERENCES

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