Prostaglandin E-Major Urinary Metabolite Predicts Relapse in Patients With Ulcerative Colitis in Clinical Remission.

INTRODUCTION: Prostaglandin E-major urinary metabolite (PGE-MUM) is a novel biomarker reflecting endoscopic activity in ulcerative colitis (UC). However, there are no studies investigating the efficacy of PGE-MUM as a biomarker for predicting relapse. We investigated whether PGE-MUM can predict clinical relapse of UC. METHODS: The measurement of PGE-MUM and endoscopic evaluation were performed in 70 patients with UC in clinical remission. The optimal cutoff values predicting relapse and relapse-free rate were analyzed. RESULTS: Sixteen patients (22.9%) relapsed during the 12-month follow-up. The median PGE-MUM value of relapsed patients at entry was significantly higher than that of patients in clinical remission (P = 0.008). The cutoff value of PGE-MUM predicting future relapse was 25.2 µg/g Cr by receiver-operating characteristic (ROC) analysis, and the area under the ROC curve was 0.721 (95% confidence interval: 0.556-0.886). The relapse-free rate of patients with PGE-MUM ≥25.2 µg/g Cr was significantly lower than that in patients with PGE-MUM <25.2 µg/g Cr (log-rank test: P < 0.001). The ROC analysis of UC patients with disease duration more than 1-8 years showed that duration of more than 5 years had the largest area under the ROC curve 0.821 (95% confidence interval: 0.583-1.000) and that the optimal cutoff value was 26.3 µg/g Cr. DISCUSSION: PGE-MUM is a reliable biomarker for predicting future relapse, particularly in UC patients with long-disease duration.

Measurement of prostaglandin metabolites is useful in diagnosis of small bowel ulcerations.

BACKGROUND: We recently reported on a hereditary enteropathy associated with a gene encoding a prostaglandin transporter and referred to as chronic enteropathy associated with SLCO2A1 gene (CEAS). Crohn's disease (CD) is a major differential diagnosis of CEAS, because these diseases share some clinical features. Therefore, there is a need to develop a convenient screening test to distinguish CEAS from CD. AIM: To examine whether prostaglandin E major urinary metabolites (PGE-MUM) can serve as a biomarker to distinguish CEAS from CD. METHODS: This was a transactional study of 20 patients with CEAS and 98 patients with CD. CEAS was diagnosed by the confirmation of homozygous or compound heterozygous mutation of SLCO2A1. We measured the concentration of PGE-MUM in spot urine by radioimmunoassay, and the concentration was compared between the two groups of patients. We also determined the optimal cut-off value of PGE-MUM to distinguish CEAS from CD by receiver operating characteristic (ROC) curve analysis. RESULTS: Twenty Japanese patients with CEAS and 98 patients with CD were enrolled. PGE-MUM concentration in patients with CEAS was significantly higher than that in patients with CD (median 102.7 vs 27.9 µg/g × Cre, P < 0.0001). One log unit increase in PGE-MUM contributed to 7.3 increase in the likelihood for the diagnosis of CEAS [95% confidence interval (CI) 3.2-16.7]. A logistic regression analysis revealed that the association was significant even after adjusting confounding factors (adjusted odds ratio 29.6, 95%CI 4.7-185.7). ROC curve analysis revealed the optimal PGE-MUM cut-off value for the distinction of CEAS from CD to be 48.9 µg/g × Cre with 95.0% sensitivity and 79.6% specificity. CONCLUSION: PGE-MUM measurement is a convenient, non-invasive and useful test for the distinction of CEAS from CD.

Amyloid precursor protein promotes endoplasmic reticulum stress-induced cell death via C/EBP homologous protein-mediated pathway.

The accumulation of unfolded or misfolded proteins in the endoplasmic reticulum (ER) is known to activate the ER, which is termed ER stress. Here, we demonstrated that amyloid precursor protein (APP) is a novel mediator of ER stress-induced apoptosis through the C/EBP homologous protein (CHOP) pathway. Expression of APP mRNA was elevated by tunicamycin- or dithiothreitol-induced ER stress. The levels of C83 and APP intracellular domain (AICD) fragments, which are cleaved from APP, were significantly increased under ER stress, although the protein level of full-length APP was decreased. Cellular viability was reduced in APP-over-expressing cells, which was attenuated by treatment with a gamma-secretase inhibitor, N-[N-(3,5-difluorophenacetyl)-L-alanyl]-S-phenylglycine t-butyl ester (DAPT). Cellular viability was also reduced in AICD-FLAG-over-expressing cells. The mRNA and protein levels of CHOP, an ER stress-responsive gene, were remarkably increased by APP over-expression, which was attenuated by treatment with DAPT. CHOP mRNA induction was also found in AICD-FLAG-over-expressing cells. Cell death and CHOP up-regulation by ER stress were attenuated by APP knockdown. Data obtained with a luciferase assay and chromatin immunoprecipitation assay indicated that AICD associates with the promoter region of the CHOP gene. In conclusion, ER stress-induced APP undergoes alpha- and gamma-secretase cleavage and subsequently induces CHOP-mediated cell death.

RNA interference silencing of DRAL affects processing of amyloid precursor protein.

In a previous study, we reported that Alzheimer's disease-associated presenilin-2 interacts with a LIM-domain protein, namely, DRAL/FHL2/SLIM3. In this study, we investigated whether DRAL modifies the metabolism of the amyloid precursor protein (APP). We used small interfering RNA (siRNA) to knockdown DRAL in COS7 and HEK293 cells that stably overexpress APP695. We found that the knockdown was accompanied by a decrease in the amount of secreted alpha-secretase-cleaved APP and the membrane-bound C-terminal fragment C83 and an increase in the amount of secreted beta-amyloid peptide (Abeta) from the cells. We also found that in addition to a disintegrin and metalloprotease (ADAM)-17, DRAL binds to ADAM-10. Thus, DRAL may be involved in the processing of APP through the alpha-secretase pathway.

Effects of anoxia and hypoxia on amyloid precursor protein processing in cerebral microvascular smooth muscle cells.

Cerebral amyloid angiopathy (CAA) is characterized by the degeneration of cerebral microvascular smooth muscle cells (MV-SMC) and the replacement of normal vessel wall components by beta-amyloid (Abeta) protein. Little is known regarding the mechanisms of SMC degeneration in CAA. The effects of anoxia on the metabolism of the amyloid precursor protein (APP) were studied to investigate the MV-SMC response to anoxic stress and its possible role in the pathogenesis of CAA. MV-SMC exposed to chronic anoxia (24-48 hours) showed a decrease in expression of the 2 putative alpha-secretase enzymes, mature TACE (TNFalpha-converting enzyme) and ADAM10 (a disintegrin and metalloprotease). A concomitant decrease in the alpha-secretase cleavage products sAPPalpha and C83 was observed. Investigation of mRNA expression showed an increase in TACE and a sharp decrease in ADAM10 at 24 hours. Exposing MV-SMC to hypoxia (1% O2) revealed a different pattern of expression with no significant change in TACE protein, but an increase in TACE mRNA occurring at a later time point (48 hours). There was no change in ADAM10 mRNA expression, but a reduction in mature ADAM10 with a parallel increase in immature ADAM10 protein. These results demonstrate a requirement for oxygen in the regulation of the alpha-secretase pathway during APP metabolism.

The isoprostanes: novel prostaglandin-like products of the free radical-catalyzed peroxidation of arachidonic acid.

The isoprostanes (IsoPs) are a unique series of prostaglandin-like compounds formed in vivo from the free radical-catalyzed peroxidation of arachidonic acid. This review summarizes our current knowledge regarding these compounds. Novel aspects of the biochemistry and bioactivity of IsoPs are detailed and methods by which these compounds are analyzed are discussed. A considerable portion of this review deals with the utility of measuring IsoPs as markers of oxidant injury in human diseases particularly in association with risk factors that predispose to atherosclerosis, a condition in which excessive oxidative stress has been causally implicated.

Cyclosporine-induced coronary artery constriction--dissociation between thromboxane release and coronary vasospasm.

Cyclosporine influences vascular tone, including that of coronary arteries. But its effect on myocardial prostanoid release, which may contribute to a drug-induced coronary and/or myocardial dysfunction, remains unknown. We used the isolated perfused rat heart to study the effect of cyclosporine on both the mechanical function parameters and myocardial prostanoid release into the effluent by ELISA. Cyclosporine (5 microM) induced an increase of perfusion pressure from 40 +/- 3 to 73 +/- 4 mm Hg within 60 minutes (p < 0.001), reflecting an increase of coronary tone. Cyclosporine did not affect heart rate but contractility (+dp/dtmax) tended to decrease, although not significantly. The drug's effect on coronary tone was rapidly reversible upon withdrawal. Cyclosporine perfusion resulted in an increase of thromboxane B2 liberation from 236 +/- 150 to 1321 +/- 354 pg/ml effluent (p < 0.001), whereas the 6-keto-prostaglandin F1 alpha release was unaffected. The vehicle cremophor did not change any of these parameters. Neither inhibition of myocardial prostanoid formation with acetylsalicylic acid nor thromboxane receptor blockade prevented the cyclosporine-induced increase of perfusion pressure. However, perfusion with nitroglycerin or the voltage-sensitive calcium channel antagonist nifedipine in addition to cyclosporine were able to prevent the increase of perfusion pressure. This is the first time it has been demonstrated that cyclosporine induces an acute release of the prostanoid thromboxane within the myocardium. Despite the resulting imbalance in favor of the vasoconstrictive prostanoid, a dependency of the cyclosporine-induced increase of coronary tone on this imbalance was excluded. Conversely, nitric oxide donation or calcium channel blockade were able to prevent the negative effect of the drug on coronary tone, supporting the concept of endothelium-dependent and/or myogenic mechanism of cyclosporine toxicity on the coronary vascular bed.

Enhancement of cell proliferation and prostaglandin biosynthesis by 1,8-dihydroxyanthraquinone in the rat large intestine.

The effects of 1,8-dihydroxyanthraquinone (DHAQ), a stimulant laxative named danthron, on cell kinetics and prostaglandin (PG) biosynthesis in the gastrointestinal tract were investigated in male 8-week-old F344 rats divided into three groups, each consisting of 10 animals. The animals in groups one, two and three were respectively given diets supplemented with 0%, 0.1% and 0.2% DHAQ for 24 days. PGE2 levels in the colorectal mucosa were significantly (P < 0.05 and 0.001) elevated after DHAQ treatment and showed some evidence of a dependence of DHAQ dose, consistent with the plasma PGE2 levels. BrdU-labeling indices in the large intestinal epithelium were also significantly (P < 0.01) increased, although the other portions of the gut such as the stomach and small intestine were not significantly affected. Excretion of the main urinary metabolite of PGE (PGE-MUM) was significantly (P < 0.001 or 0.01) increased whereas the urinary PGE2 concentration and total PGE2 excretion were not changed. Thus the results of the present study clearly indicate enhancement of cell proliferation by DHAQ in the large intestine epithelia, correlated with increased PGE2 levels in the large intestinal mucosa as well as the plasma, and possible support for the conclusion that quantitative analysis of urinary PGE-MUM, but not PGE2 itself, offer a useful approach for biomonitoring exposure to such stimulant laxatives.

Mass spectrometric identification of urinary and plasma metabolites of 6-(6'-carboxyhexyl)-7-n-hexyl-1,3-diazaspiro-[4-4]-nonan-2,4-dione, a new cytoprotective agent.

The compound IBI-P-01028, or R,S-cis-6-(6'-carboxyhexyl)-7-trans-n-hexyl-1,3-diazaspiro-[4-4]-nona n-2,4- dione, is a new cytoprotective agent under development. To study the metabolites of this compound in laboratory animals, we administered it to dogs and rats, and analyzed extracts from dog and rat urine, and from dog plasma, by GC-MS. The metabolic profiles were different in the rat and dog. In the dog (plasma and urine), one metabolite was found, and in the rat urine two other metabolites were found. The unmetabolized drug was found only in the dog plasma and urine.

Mass spectrometric identification of urinary and plasma metabolites of 9-hydroxy-19,20-bis-nor-prostanoic acid (rosaprostol).

9-Hydroxy-19,20-bis-nor-prostanoic acid (Rosaprostol) is an antiulcer compound with antisecretory and cytoprotective action. We studied the metabolites of Rosaprostol found in human plasma and in human and rat urine. Sixteen different metabolites were tentatively identified on the basis of their mass spectra. Two presumed metabolites were synthesized. To clarify the identities of some of them, deuterated Rosaprostol was administered to rats and mass spectra of the deuterated and protonated metabolites were examined. Rosaprostol is metabolized following three metabolic pathways leading, combined, to oxidized compounds with a lower number of carbons than the parent drug.

Use of [3H]trimetoquinol as a radioligand in human platelets: interaction with putative endoperoxide/thromboxane A2 receptor sites.

We have recently shown that trimetoquinol [1-(3,4,5-trimethoxybenzyl)-6,7-dihydroxy-1,2,3,4- tetrahydroisoquinoline; TMQ] is a potent and stereoselective [R(+)-isomer greater than S(-)-isomer] antagonist of aggregation induced by thromboxane A2 and stable epoxymethano PGH2 analogues (U46619; U44069) in human platelets. The present study was undertaken to characterize the pharmacological specificity of binding sites for racemic- [3H]TMQ in washed human platelets. Specific binding of [3H]TMQ, determined by addition of 100 microM R(+)-TMQ, accounted for 12-26% of the total binding at 1.0 microM. Saturation data suggested two binding sites with apparent dissociation constants of 2.8 nM and 1.4 microM for the high and low affinity binding sites, respectively. Maximal binding densities (pmol/mg protein) were 2.3 and 42.9 for the respective high and low affinity sites. The optical isomers of TMQ, cis and trans-isomers of 13-azaprostanoic acid (13-APA), and U46619 inhibited [3H]TMQ specific binding to the low affinity site. R(+)-TMQ was 32-fold more potent than S(-)-TMQ at inhibiting [3H]TMQ binding, and the stereoselective potency difference and concentrations needed to inhibit binding were similar to those required for inhibition of U46619-induced platelet aggregation and serotonin secretion. Similarly, trans-13-APA was 90-fold more potent than cis-13-APA as a competitor of [3H]TMQ binding. U46619 also inhibited TMQ binding at concentrations (IC50 = 0.4 microM) similar to those required for aggregation (EC50 = 0.16 microM) and secretion (EC50 = 0.23 microM). At 1 mM, arachidonic acid inhibited [3H] TMQ binding by only 25%. Our studies indicate that [3H]TMQ interacts with specific binding sites with characteristics indicative of putative endoperoxide/thromboxane A2 receptors in human platelets.

How the sauna affects the endocrine system.

The sauna induces changes in the secretion of hormones, some similar to changes induced in any other stress situation and others characteristic of exposure to the sauna. Noradrenaline is usually the only catecholamine raised by the sauna in people accustomed to it. The secretion of the antidiuretic hormone is increased and the renin-angiotensin-aldosterone system is activated. The concentrations of the growth hormone and prolactin, in particular, secreted from the anterior pituitary are increased in the circulation. The concentration of the immunoreactive beta-endorphin in blood may also increase which may reflect the feeling of pleasure or, on the other hand, discomfort induced by the sauna. The views on the effects of the sauna on the secretion of the ACTH and cortisol are partly contradictory, probably due to differing ways of taking the sauna bath. In Finnish sauna takers the concentration of cortisol in blood is not usually increased. The changes induced by the sauna in various hormone concentrations in the circulation are, however, normalized within a couple of hours after the heat stress.

Production of prostanoids by rheumatic synovial cells in vitro: effects of anti-inflammatory drugs on arachidonic acid metabolism.

To evaluate the role of prostanoids in rheumatoid arthritis the effects of anti-inflammatory drugs on prostanoid concentrations and their ratios were studied in a primary culture of adherent synovial cells from patients with rheumatoid arthritis. Cells from rheumatoid synovium have a great capacity for prostanoid production. PGE2 is the main prostanoid but synovial cells are also capable of producing 6-keto-PGF1 alpha and PGF2 alpha. There were also very low TxB2 concentrations in the culture medium after incubation. All nonsteroidal anti-inflammatory drugs used (diclofenac, indomethacin and tolfenamic acid) reduced markedly, in concentrations achieved therapeutically (greater than or equal to 0.13 mumol/l), the production of all the prostanoids from endogenous substrate. There were no differences in the efficacity of the drugs. Hydrocortisone was needed for higher concentrations to inhibit PGE2, 6-keto-PGF1 alpha and PGF2 alpha production. TxB2 formation remained almost unaltered. After the drug incubation there were also clear alterations in the ratios between these prostanoids, which may have therapeutic importance. It is suggested that this kind of synovial cell culture can be used for testing the effects and mechanisms of different anti-inflammatory drugs in standardized cell culture conditions.

A photoaffinity label for the thromboxane A2/prostaglandin H2 receptor in human blood platelets.

A photoactive iodoarylazide derivative (I-APA-PhN3) of the competitive thromboxane A2/prostaglandin H2 (TXA2/PGH2) antagonist 13-azaprostanoic acid is evaluated. Upon photoactivation, the compound was found to inhibit specifically and irreversibly human platelet aggregation induced by the TXA2/PGH2 mimetic U46619. In receptor-binding studies using [3H]U46619, I-APA-PhN3 exhibited an IC50 of 300 nM for inhibition of U46619 binding. Photoactivation of I-APA-PhN3 resulted in an irreversible 58% reduction in specific binding of U46619. This compound and its corresponding ratio-iodinated form will prove to be useful tools for the isolation and purification of the TXA2/PGH2-binding protein in human platelets.