INTRODUCTION

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LIPOXINS (LX) ARE TETRAENE-CONTAINING EICOSANOIDS that are generated by lipoxygenase transformation of arachidonic acid. Multiple routes of LX biosynthesis have been recognized that involve single cell types or cell-to-cell interactions (reviewed in Ref. 22). LX possess potent bioactions that may regulate key mechanisms of the immunoinflammatory reaction. In particular, LXA₄ inhibits polymorphonuclear neutrophil (PMN) adhesion to human endothelial cells by reducing P-selectin expression (14) and blocks IL-6, IL-8, and metalloproteinase release (9, 23). Both LXA₄ and B₄ stimulate monocytic cell migration and adhesion to laminin by using distinct signal transduction pathways (12, 19). LX and their recently discovered 15R epimers, aspirin-triggered LX (4), possess potent anti-inflammatory activity in vivo (25, 26), and temporal biosynthesis of LX, concurrent with spontaneous resolution, has been observed during exudate formation (10). LXA₄ may contribute to the resolution of the inflammatory response by stimulating phagocytosis of apoptotic neutrophils (8).

Release of inflammatory mediators and multicellular activation can be observed during strenuous exercise. In particular, increased production of cytokines, chemokines, and arachidonic acid metabolites, as well as platelet-PMN activation and interactions, have been documented during maximal exertion (11, 13, 15, 27). However, no appreciably clinical signs of inflammation are commonly denoted after exercise, suggesting that homeostatic mechanisms may limit the extent and duration of the exercise-induced inflammatory response.

Because LX are potently anti-inflammatory and because potential LX-forming multicellular interactions occur during strenuous exercise, we evaluated urinary excretion of LXA₄ and related compounds in healthy volunteers.

In this report, we show that maximal physical exertion is accompanied by a rapid increase in the urinary excretion of iLXA₄, which is likely to reflect in vivo cell-to-cell interactions and to represent a defense mechanism against stress-induced inflammation.

	METHODS

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Subjects and study design. Nine healthy subjects, six men and three women, aged 29.3 ± 3.5 yr, were recruited for this study. They were all nonsmokers and free from drugs for at least 2 wk. Urine samples were collected after an overnight fast. Volunteers were then asked to rest for at least 30 min in supine position. Exercise was started on an electrically braked cycle ergometer (Cardioline ERG 602) with an initial workload of 25 W for 2 min that was incremented by 25 W every 2 min. Heart rate and blood pressure were constantly monitored, and the exercise was terminated when the heart rate approached the theoretical maximal heart rate (93-98%), which was calculated according to the formula 220 age (yr). In two subjects, the test was interrupted, respectively, at 80 and 85% of the theoretical maximal heart rate because of muscular exhaustion. Maximal workload was 146.1 ± 44.9 W. Exercise duration was 13.3 ± 2.8 min. Urine and blood samples were collected after a recovery interval of 3 min from the interruption of the test and after 6 and 24 h.

Extractions and reversed-phase-high-performance liquid chromatography. LX were extracted from 5 ml of urine and suspended with 100 µl of methanol according to a recently published protocol (18). Extraction recovery was assessed by exogenously added prostaglandin (PG) B₂ measurements with a dual pump reversed-phase-HPLC gradient system equipped with a 996 photodiode array detector (Waters, Milford, MA) and a Waters Symmetry C₁₈, 3 µm, 2.1 ×150-mm column. Postrun analysis was performed with the Millennium 32 chromatography manager. PGB₂ recovery was determined by reporting the peak area of the sample PGB₂ to a calibration curve constructed by injecting increasing amounts of authentic PGB₂ (Cascade Biochem, Reading, UK). Quantitation of the previously described urinary tetraene (18) was obtained by comparing the peak area of the tetraene with a standard curve constructed by injecting increasing amounts of authentic LXA₄.

LXA₄ ELISA. Immunoreactive LXA₄ (iLXA₄) levels were determined by using the LXA₄ ELISA kit, kindly provided by Neogen (Lexington, KY), as previously described (18). Briefly, 20 µl of extracted urine in methanol were taken to dryness and suspended with 150 µl of ELISA extraction buffer. Fifty microliters were used for iLXA₄ ELISA measurements in duplicate, as indicated by the manufacturer.

Statistics. Data are shown as means ± SD. The overall impact of exercise on iLXA₄ levels was evaluated by using one-way ANOVA with repeated measures. Comparisons between measurements at individual time points were analyzed with Wilcoxon's signed rank test. P < 0.05 was considered statistically significant. All calculations were made with the computer program Stat-View II (Abacus Concepts, Berkley, CA).

	RESULTS

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Urinary excretion of iLXA₄ during exercise was monitored by using a recently developed extraction method coupled to a commercially available ELISA kit (18). Exercise increased systolic blood pressure and heart rate from 115.6 ± 4.1 to 186.7 ± 5.3 mmHg and from 74.4 ± 11.6 to 171.2 ± 3.2 beats/min, respectively. It was also associated with a time-dependent variation in iLXA₄ excretion (P = 0.0016; 1-way ANOVA). In particular, a significant increment in urinary iLXA₄ was observed at the end of the exercise (0.061 ± 0.023 vs. 0.113 ± 0.057 ng/mg creatinine; P = 0.028; Wilcoxon's test). These levels returned to baseline within 6 h to increase again, although at a lesser extent, after 24 h (P = 0.05; Wilcoxon's test; Fig. 1). Notably, seven of the volunteers showed an increment that ranged from 17 to 383% above basal levels, whereas two of them did not display significant variations. No significant correlations were denoted between iLXA₄ levels at all time points and either exercise duration, maximal workload, or percentage of theoretical maximal heart rate.

View larger version (24K): [in this window] [in a new window]   Fig. 1.   Impact of strenuous exercise on urinary immunoreactive lipoxins A4 (iLXA4) levels. Nine healthy volunteers (each symbol represents 1 volunteer) were subjected to exercise, as described in METHODS. Urine samples were collected immediately before (basal), after the exercise (A.E.), and also 6 and 24 h after the test. Results are expressed as ng/mg creatinine corrected for PGB2 recovery. Individual determinations are shown as dot-connecting lines. Means ± SD for each time point are depicted by the bars and were 0.061 ± 0.023 (basal), 0.113 ± 0.057 (A.E.), 0.061 ± 0.025 (6 h), and 0.09 ± 0.039 ng/mg (24 h). P = 0.0016 (ANOVA for all groups). P = 0.028 (Wilcoxon's test for basal vs. A.E.).

Because in a recent report (18) our laboratory showed that a LXA₄-related material bearing the physical properties of a LXA₄ metabolite, including the typical tetraene structure, is excreted with human urine, we investigated the relationship between the ELISA-measured iLXA₄ and the amount of the urinary tetraene in the volunteers of this study. To this end, urinary tetraene was measured by RP-HPLC as previously described (18), and the values obtained were plotted as a function of the correspondent ELISA readings of iLXA₄, expressed as nanograms per milliliter. As shown in Fig. 2, a strong linear correlation (r = 0.988) was observed between the iLXA₄ ELISA readings and the RP-HPLC-based quantitations of the putative LXA₄ urinary metabolite, although individual ELISA measurements were ~60-fold lower than RP-HPLC quantitations of the tetraene in the corresponding sample. Also, materials eluted in the section of the chromatogram corresponding to the retention time of authentic LXA₄ were collected, concentrated, and subjected to ELISA. However, they did not give appreciable readings (results not shown). These results confirm the LXA₄-related nature of the urinary tetraene and indicate that urinary iLXA₄ is not native to LXA₄ but is rather a LXA₄-derived product.

View larger version (13K): [in this window] [in a new window]   Fig. 2.   Regression analysis of reversed-phase (RP)-HPLC quantitation of the urinary tetraene vs. iLXA4 measurements. Not all values from the exercise study were included because in some cases the RP-HPLC resolution of the tetraene was not optimal. r = 0.988; n = 27.

	DISCUSSION

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Evidence indicates that physical stress is associated with the release of inflammatory mediators and with potentially prothrombotic cell-to-cell interactions (11, 13, 15, 27). However, it is generally accepted and proved by a number of clinical studies that physical exercise is beneficial for the cardiovascular system, particularly in pathological conditions (24). To identify novel mechanisms that may counterbalance the effects of inflammatory mediators released during exercise, we measured urinary levels of the potent anti-inflammatory LXA₄. We observed a significant increase in iLXA₄ urinary excretion after strenuous exercise in nine healthy volunteers (Fig. 1). Moreover, we established that the material recognized by the anti-LXA₄ antibody of the ELISA assay is a tetraene, which is likely to represent a LXA₄ metabolite (Fig. 2). Together, these results indicate that strenuous exercise may induce LX biosynthesis and further metabolism. In fact, LX bear the typical trait of autacoids, because they are rapidly formed on cell stimulation, act locally, and are rapidly metabolized and inactivated. Thus it is unlikely that exercise-induced urinary excretion of LX-related compounds may originate by an enhanced release of preformed, stored material.

Platelet and PMN may be the cellular sources of LX during exercise. Indeed, strenuous physical exertion is associated with the rapid formation of in vivo platelet/PMN aggregates and activation of both cell types (11). Earlier studies have shown that LX are formed during coincubations of platelet with PMN, because platelet 12-lipoxygenase functions as a LX-synthase, being able to convert PMN-derived leukotriene A₄ into LXA₄ and B₄ (7, 17). This biosynthetic pathway also occurs in vivo after atherosclerotic plaque rupture by coronary angioplasty, when nanogram amounts of LXA₄ are formed (2). Thus the increase in urinary excretion of iLXA₄ after exercise may be consistent with transcellular metabolic exchanges between activated platelets and PMN, although the contribution of additional sources cannot be excluded, because transcellular exchange-generating LX may also occur in the urinary tract (1). Along these lines, from the present results, it is difficult to firmly establish the anatomic district(s) involved in LX biosynthesis during physical exercise. Whether it reflects renal production, muscle release, or the summation of whole body vascular stress remains to be determined. Also, it is not clear whether the smaller increment in urinary iLXA₄ observed 24 h postexercise has a similar cellular and regional origin as that denoted immediately after the exercise.

That a LXA₄-derived material (i.e., metabolite) appears in urine immediately at the end of strenuous exercise may be justified by the fact that in vivo LXA₄ formation can be very rapid, because it can be observed within 10 s from angioplasty (2). Moreover, >60% of LXA₄ is metabolized by peripheral blood monocytes within 30 s (21). Consistently, a rapid postexercise increment in the urinary levels of metabolites of other arachidonic acid-derived eicosanoids, namely thromboxane B₂ and prostacyclin, has been documented (16, 28).

An increase in LX biosynthesis during exercise may have relevant pathophysiological implications. LX function as stop signals during inflammation, and their role in the resolution of the inflammatory response has been recently elucidated (10). Moreover, LX possess vasodilatory properties (3, 5, 6) and inhibit PMN adherence to microvasculature by downregulating P-selectin expression (20). Thus LX production in the course of physical exercise may, on one side, counterbalance the action of exercise-induced proinflammatory mediators and, on the other, may represent one of the mechanisms of the long-term beneficial effect of physical exercise on the cardiovascular system. In this respect, it has to be pointed out that LX generated on physical exercise are also potentially proresolving in local cellular damage; thus they may contribute to limit the extent of exercise-associated microtrauma. Whether the capability to produce higher LXA₄ levels should be regarded as predictive of a lower incidence of vascular diseases remains to be established. In this respect, the variable extent among our volunteers of iLXA₄ increase in response to exercise, independently from exercise duration, maximal workload, or percentage of theoretical maximal heart rate, as well as the finding that in two of the subjects no significant variation was appreciated (Fig. 1) is intriguing and warrants further investigation.

In conclusion, this study represents the first in vivo evidence of an increment in iLXA₄ excretion in a nonpathological state in humans. The present results may contribute to a better understanding of the inflammatory reaction during maximal exercise and confirm that the methodology recently developed in our laboratory (18) can be successfully applied to human studies.