Tetranor PGDM, an Abundant Urinary Metabolite Reflects Biosynthesis of Prostaglandin D₂ in Mice and Humans^*

Prostaglandin (PGD₂)² is formed from PGH₂, a cyclooxygenase (COX) product of arachidonic acid by the action of either a lipocalin (L)-like or hemopoietic (H) PGD synthase (1). Both enzymes may form PGD₂ in vitro, but it is unclear which PGDS enzyme predominates under varied conditions in vivo. Suppression of PGD₂ has been implicated in the bronchoconstriction of aspirin-evoked asthma (2, 3), and release of PGD₂ mediates the facial flushing and vascular instability of systemic mastocytosis (4). PGD₂ relaxes vascular smooth muscle cells in vitro and its release by dermal dendritic cells contributes to the facial flushing, which complicates administration of the hypolipidemic drug, niacin (5). PGD₂ mediates its effects via activation of D prostanoid receptors (DPs). DP1, a member of the prostanoid family of G protein-coupled receptors, mediates the vasorelaxant and bronchodilator effects (6, 7), whereas DP2, a G protein-coupled receptor of the formylmethionylleucylphenylalanine receptor subfamily regulates Th1 and Th2 switching in lymphocytes (8) and is also expressed on eosinophils and basophils (9).

Recent interest in PGD₂ has been prompted by the use of DP1 blockade as an adjunct to niacin therapy (10) and by the potential role of PGD₂ and its metabolites in the resolution of inflammation (11). However, DP1 is expressed on human platelets, and its activation in vitro results in a cyclic AMP-dependent inhibition of platelet function (12, 13). Nothing is known about the formation of PGD₂ or the consequences of its inhibition in hyperlipidemic patients. Aside from a potential role in cardiovascular disease, PGD₂ may be of importance in the resolution of inflammation. A metabolite of PGD₂, 15-deoxy-Δ^12,14-PGJ₂, has been postulated to activate peroxisome proliferator-activated receptor γ (14) and promote resolution of an inflammatory infiltrate (11). However, it remains to be determined by physicochemical methodology whether formation of 15-deoxy-Δ^12,14-PGJ₂ is indeed augmented during the resolution of human inflammation, and, although it can activate PPARγ, the concentrations required are unlikely to be attained in vivo (15).

Attempts to assess the biosynthesis of PGD₂ have been constrained by a paucity of commercially available, sensitive, and specific methodology. Aside from asthma and mastocytosis (2-4), little information on biosynthesis of PGD₂ in humans has been acquired. Given the evanescence of primary PGs, biosynthesis is classically estimated by measurement of metabolites (16, 17). However, despite deletion of both DPs and PGDS enzymes, no metabolites of PGD₂ have been reported in mouse, preventing assessment of biosynthetic response to experimental manipulation in that species. Initial attempts at commercial assay development in humans have focused on 11β-PGF_2α (18) and 2,3-dinor-11β-PGF_2α. Both are formed as minor urinary metabolites in monkeys and in a human volunteer following infusion of radiolabeled PGD₂ (19). Indeed, paired analysis of 11β-PGF_2α by gas chromatography and mass spectrometry (GC/MS), and the commercially available immunoassay revealed poor concordance in the urine of patients with asthma (20, 21). Quantitative analysis of the major F-ring metabolite identified in urine after PGD₂ infusion in a volunteer, 9α, 11β-dihydroxy-15-oxo-2,3,18,19-tetranorprost-5-ene-1,20-dioic acid (19) has been reported in human plasma and urine (22) and reflects nicely the marked augmentation of PGD₂ biosynthesis in systemic mastocytosis (4).

Here, we report the identification of a novel D-ring metabolite formed from infused PGD₂, 11,15-dioxo-9α-hydroxy-, 2,3,4,5-tetranorprostan-1,20-dioic acid (tetranor PGDM) as an abundant endogenous metabolite of PGD₂ in both human and mouse urine. The formation from PGD₂ of its F-, J-, and D-ring products is depicted in Fig. 1. Gene manipulations in mice and pharmacological studies in humans implicate COX-1 as the major source of PGD₂ as reflected by this metabolite in urine.

EXPERIMENTAL PROCEDURES

RESULTS

Discovery of Tetranor PGDM—While measuring tetranor PGEM, the major urinary PGE metabolite in mouse urine (26), four other major peaks, apparently tetranor PGEM isomers, were observed in the mass chromatogram (Fig. 2A, lower panel). Because PGD₂ and PGE₂ are themselves structural isomers (27), it was surmised that some of these peaks might originate from PGD₂. When PGD₂ was infused into mice, two of these four peaks were increased dramatically (Fig. 2B, lower panel), consistent with the hypothesis that they corresponded to the analogous D-ring tetranor metabolites. Following addition of authentic [²H₆]tetranor PGDM to mouse urine, the deuterated compound coeluted with the endogenous material (Fig. 2, A and B). There are four chromatographic peaks for tetranor PGDM methoxyamine derivatives. However, the two smaller peaks were variably detectable. Mass spectral analysis of human urine also revealed the existence of tetranor PGDM, which differed from mouse only in its abundance relative to tetranor PGEM (Fig. 2C). Co-injection of extracts of urine from mice infused with PGD₂ with extracts of human urine indicated that the endogenous peaks corresponding to tetranor PGDM in human urine corresponded to the peaks increased dose dependently by PGD₂ infusion in mice (supplemental Fig. S1).

Product ion analysis of the [²H₆]tetranor PGDM at m/z 391 gave rise to a series of major fragment ions with m/z values of 373, 342, 313, 267, 182, 164, and 142 (Fig. 3A), virtually identical to the product ion spectrum obtained from endogenous tetranor PGDM (m/z 385); m/z 367, 336, 307, 261, 182, 164, and 142 (Fig. 3B). The differences in m/z values between these two groups were either 0 or 6 mass units reflecting fragments with or without deuterium, again consistent with the original hypothesis. The transitions m/z 391 > 336 and m/z 385 > 336 are the same as those for tetranor PGEM, facilitating an integrated approach to lipidomic analysis (28). The assay was highly reproducible, and the D-ring metabolite was chemically stable at -20 °C and -80 °C (supplemental Fig. S2).

A study was performed to determine whether tetranor PGDM might activate the DP1 receptor on human platelets (12, 13). However, unlike PGD₂ itself, tetranor PGDM did not inhibit platelet aggregation (supplemental Fig. S3).

HPLC-MS/MS Analysis of Urinary 2,3-Dinor-11β-PGF_2α and 11β-PGF_2α—A representative selected reaction monitoring chromatogram of 11β-PGF_2α in human urine is shown in Fig. 4A. Transitions characteristic of [²H₄]11β-PGF_2α (m/z 357→197) and 11β-PGF_2α (m/z 353→193) are shown in the upper and lower panel, respectively. A detectable endogenous chromatographic peak that co-eluted with spike was absent from most human urine samples. Some peaks eluted close to, but not coincident with the internal standard, as in Fig. 4A. These peaks were further confirmed to be distinct from endogenous 11β-PGF_2α by addition of synthetic exogenous standards at the time of analysis. We surmise that these peaks represent F₂-isoprostanes that would have the potential to compete with 11β-PGF_2α in an immunoassay and also may not have separated from endogenous 11β-PGF_2α under the elution conditions utilized in a GC/MS assay (29). Although 2,3-dinor-11β-PGF_2α was readily detectable in human urine, it required a long LC program (28) to achieve separation from interfering compounds. A representative selected reaction monitoring chromatogram of 2,3-dinor-11β-PGF_2α in human urine is shown in Fig. 4B. Urinary 2,3-dinor-11β-PGF_2α and 11β-PGF_2α were both below the limits of detection (∼1 ng/mg Cre) in mouse urine (data not shown).

Comparative Levels of Endogenous Metabolites of PGD₂ Metabolites—In human urine, the comparative levels of tetranor PGDM and 2,3-dinor-11β-PGF_2α were 1.5 ± 0.3 ng/mg Cre and 0.6 ± 0.1 ng/mg Cre (p < 0.01), respectively, whereas 11β-PGF_2α was usually below the limits of detection (Fig. 5A). Tetranor PGDM was the only metabolite detectable in mouse urine at 8.1 ± 1.3 ng/mg Cre (Fig. 5B).

Enzymatic Contributions to the Biosynthesis of PGD₂—Both lipocalin (L)-like or hemopoietic (H) PGD synthase may form PGD₂ in vitro, but it is unclear which PGDS enzyme predominates under varied conditions in vivo. The enzymatic contributions to the biosynthesis of PGD₂ were examined by L-PGDS and H-PGDS mutant mouse models. Deletion of either PGDS significantly reduced biosynthesis of PGD₂. Tetranor PGDM was suppressed ∼30% on average by deletion of L-PGDS (p < 0.05) and 80% on average by deletion of H-PGDS (p < 0.0001, Fig. 5C). Deletion of H-PGDS had a significantly greater (p < 0.001) impact on urinary tetranor PGDM than did deletion of L-PGDS in mice. Cross-regulation of PGDS enzymes differed in response to gene deletion. Expression of H-PGDS was up-regulated in the aorta of L-PGDS KOs. By contrast, compensatory up-regulation of L-PGDS was not observed in H-PGDS KOs (supplemental Fig. S4).

The comparative contribution to metabolite excretion of the COX enzymes in mice was addressed by comparing the impact of genetic manipulation of COX-1 and COX-2. Both KO and KD of COX-1 suppressed urinary tetranor PGDM by ∼90%, whereas deletion of COX-2 failed to alter significantly metabolite excretion (Fig. 5D).

In humans, aspirin at a dose of 325 mg inhibits both COXs (30), whereas rofecoxib (25 mg) and celecoxib (200 mg) inhibit COX-2 selectively (31). Corresponding to the results in mice, inhibition of both COX-1 and COX-2 by aspirin, but not COX-2 alone by rofecoxib, depressed tetranor PGDM (Fig. 6A). Similar results were obtained with urinary 2,3-dinor-11β-PGF_2α (Fig. 6B). Both acute and chronic dosing with a second COX-2 inhibitor, celecoxib, also failed to depress tetranor PGDM (Fig. 6C).

Aspirin decreased urinary tetranor PGDM from 1.71 ± 0.21 ng/mg Cre to 0.86 ± 0.07 ng/mg Cre (p < 0.001, Fig. 7A) and 2,3-dinor-11β-PGF_2α decreased from 0.63 ± 0.11 ng/mg Cre to 0.32 ± 0.04 ng/mg Cre (p < 0.01, Fig. 7B). This dose of aspirin inhibits COX enzymes incompletely, as reflected by comparable suppression of prostaglandin E₂ metabolite (tetranor PGEM) and prostacyclin metabolite (PGIM). By contrast, urinary isoprostanes, as reflected by the major F₂ isoprostane in urine, 8,12-iso-iPF_2α-VI is unaltered by this regimen (supplemental Fig. S5).

Excretion of PGD₂ Metabolites in PGD₂-infused Mice—The comparative disposition of PGD₂ was examined by infusion of exogenous PGD₂. Urinary metabolites of PGD₂ increased dose dependently in response to the infusion (Fig. 8A). The levels of tetranor PGDM, 2,3-dinor-11β-PGF_2α and 11β-PGF_2α attained after the maximal dose (500 μg) of PGD₂ were 2498 ± 792 ng/mg Cre, 809 ± 346 ng/mg Cre, and 207 ± 67 ng/mg Cre, respectively (Fig. 8B). Roughly 3-10% of infused PGD₂ was excreted as the tetranor PGDM metabolite in mouse urine. The fractional conversion to each metabolite appeared to be uninfluenced by dose.

Evoked Biosynthesis of PGD₂—LPS evokes a systemic inflammatory response in humans that is accompanied by regulated expression of both COX isozymes ex vivo and augmented biosynthesis of thromboxane A₂ and prostacyclin (25). LPS induced a mean increase in both tetranor PGDM (1.49 ng/mg Cre at baseline to 2.15 ng/mg Cre at 2 h and 4.36 ng/mg Cre at 4 h after administration, Fig. 9A) and correspondingly in 2,3-dinor-11β-PGF_2α from 0.6 ng/mg Cre to 1.2 ng/mg Cre and 2.1 ng/mg Cre, respectively (Fig. 9B). The peak pyrexial response to LPS (from an average 36.6 to 37.8 °C) occurred ∼4 h after administration (Fig. 9C). The alterations in urinary tetranor PGDM and 2,3-dinor-11β-PGF_2α in response to LPS occur in a highly coordinated manner (Fig. 9D). Both metabolites fell after the inflammatory response and were not significantly different from basal levels during the resolution phase (8-10 h after LPS).

Oral administration of niacin, 600 mg, evoked an intense flush in both volunteers, primarily involving the face and upper part of the body. Flushing was most pronounced during the first hour after dosing and had dissipated after ∼2-3 h. Both urinary tetranor PGDM and 2,3-dinor-11β-PGF_2α were elevated by niacin (Fig 10, A and B), peaking 2-3 h and falling to basal levels 5-6 h after dosing. Again, reflecting the distinction from isoprostanes, urinary 8,12-iso-iPF_2α-VI was unaltered by niacin administration (Fig. 10C).

DISCUSSION

PGD₂ is the predominant COX product of mast cells and contributes to the cutaneous flushing and hemodynamic dys-regulation that characterizes excessive mast cell activation (2, 4). Mast cells are evident in the potentially vulnerable shoulder region of human atherosclerotic plaques (32), and mast cell depletion retards atherogenesis in mice (33). Mast cells have recently been implicated in aortic aneurysm formation (34). Other cells of relevance to atherogenesis, among them macrophages, platelets, and leukocytes, may also generate PGD₂ (35, 36). Studies of L-PGDS also implicate PGD₂ in cardiovascular biology. Levels are induced in endothelial cells by laminar shear (37) and are elevated in the circulation after angioplasty (38) and in angina (39). However, lipocalins, such as L-PGDS, may subserve diverse biological functions (40); thus these observations only indirectly implicate PGD₂.

Previous attempts to study biosynthesis of PGD₂ have relied particularly on commercially available assays of 11β-PGF_2α. This compound can be formed from PGD₂ in vitro by bovine PGF synthase (18) and was the earliest metabolite detectable by Liston and Roberts after administration of radiolabeled PGD₂ in a human (19) and itself retains biological activity, causing bronchoconstriction when inhaled by humans (41). The hemodynamic instability in patients with systemic mastocytosis is thought to reflect metabolism of PGD₂, and other vasodilator D-ring metabolites, to vasoconstrictor F-ring metabolites, such as 11β-PGF_2α. However, although endogenous concentrations of 11β-PGF_2α have been reported in human urine, there has been poor concordance between estimates based on immunoassay and GC/MS. Here, we failed to detect urinary 11β-PGF_2α using a more specific approach, HPLC-MS/MS. This raises the possibility that cross-contaminating substances, particularly F₂ isoprostanes (29), may have confounded, in some instances, analysis of 11β-PGF_2α by GC/MS. However, we did identify endogenous 2,3-dinor-11β-PGF_2α in human urine. Liston and Roberts detected predominantly F-ring metabolites in the 39% of administered radioactivity recovered after administration of radiolabeled PGD₂ to a volunteer. The most abundant of these metabolites was 9α, 11β-dihydroxy-15-oxo-2,3,18,19-tetranorprost-5-ene-1,20-dioic acid (22). Surprisingly, they failed to detect tetranor PGDM; indeed the detected D-ring metabolites accounted for <4% of administered radioactivity. It is unknown whether this reflected a feature of this particular individual's metabolism or technical factors. In the present studies tetranor PGDM was detected in the urines of all mice and humans studied under basal conditions. Indeed the levels in urine exceeded not only 2,3-dinor-11β-PGF_2α, but also the reported levels of 9α, 11β-dihydroxy-15-oxo-2,3,18,19 -tetranorprost-5-ene-1,20-dioic acid in healthy humans (22). Furthermore, tetranor PGDM is thus far uniquely detectable in mouse urine, affording the possibility of integrating estimates of biosynthesis with experimental manipulations in mice in which elements of the PGD₂ synthesis/response system have been genetically manipulated. Future studies may reveal biological activity for this metabolite, but unlike PGD₂ itself, it failed to inhibit platelet aggregation, an effect transduced via DP1 (12, 13).

Formation of urinary tetranor PGDM is altered by modulation of PGD₂ biosynthesis in both mice and humans. Deletion of either H- or L-PGDS enzymes suppresses incompletely its excretion in mice. Deletion of H-PGDS had a significantly greater impact on urinary tetranor PGDM than did deletion of L-PGDS. This may reflect in part distinctions in cross-regulation of the synthases: whereas LPGDS was not up-regulated in HPGDS KOs, compensatory up-regulation of HPGDS was observed in aortic tissue in LPGDS KOs. Genetic manipulation in mice and pharmacological studies in humans both indicate that COX-1 is the dominant source of PGD₂, as reflected by this metabolite, under physiological conditions. Consistent with these observations, very low doses of aspirin that favor inhibition of COX-1 may induce asthma in susceptible subjects (42, 43). This phenomenon is thought to reflect both suppression of PGD₂, together with endoperoxide rediversion to augment leukotriene biosynthesis (20). Experiments in vitro indicate that both COX enzymes have the capacity to contribute to PGD₂ production. For example, PGD₂ appears to derive in roughly equal amounts from COX-1 and COX-2 in liver macrophages under basal and LPS-stimulated conditions (44), whereas in mast cells, by contrast, PGD₂ is initially derived from secretory phospholipase A₂ and COX-1 followed by sustained formation by cytoplasmic phospholipase A₂ and COX-2 (45). The availability of a urinary tetranor PGDM as a biomarker of PGD₂ biosynthesis will permit definition of the relative contribution of the two enzymes to its formation in response to experimental or disease-related perturbation in humans and mice.

There is considerable interest in the importance of PGD₂ and its metabolites both in the mediation and resolution of inflammation (11, 46). A tractable model of an acute inflammatory response in humans is to administer LPS, which evokes transient flu-like symptoms with pyrexia and a hemodynamic response. We have previously shown that both COX-1 and COX-2 are expressed ex vivo coincident with this symptomatic response and that biosynthesis of both thromboxane and prostacyclin, as reflected by their major urinary metabolites, is markedly augmented coincident with increased expression of the enzymes (25). Deletion of H-PGDS undermines resolution of the delayed type hypersensitivity evoked by injection of methylated bovine serum albumin in mice (47). Here, we show that tetranor PGDM increased markedly during the inflammatory response to LPS in humans and that this response is highly correlated with the less abundant 2,3-dinor-11β-PGF_2α. However, rather than a further increase, both metabolites fell during the resolution phase.

Morrow and colleagues have previously demonstrated that niacin evokes facial flushing by prompting release of PGD₂ by dermal dendritic cells (5). Given that niacin is the only currently available hypolipidemic drug that elevates high density lipoprotein (48), it is hoped that co-administration of a DP1 antagonist might reduce this complication and enhance compliance. However, DP1 activation elevates cAMP and inhibits aggregation of human platelets (49), raising concern that it might function as an endogenous modulator of platelet activation in vivo, much like PGI₂ (50). Urinary tetranor PGDM reflects the increase in biosynthesis evoked by niacin and should facilitate elucidation of the role of PGD₂ in cardiovascular disease.

In summary, here we report a novel, abundant, D-ring urinary PGD₂ metabolite 11,15-dioxo-9α-hydroxy-2,3,4,5-tetranor-prostan-1,20-dioic acid, tetranor PGDM, which is detectable in mouse and human urine. Analysis of this compound reflects modulated biosynthesis of PGD₂ in both species and will complement the use of genetic and pharmacological probes in the further elucidating the biology of PGD₂ in vivo.