Oncogenic KRAS-Induced Protein Signature in the Tumor Secretome Identifies Laminin-C2 and Pentraxin-3 as Useful Biomarkers for the Early Diagnosis of Pancreatic Cancer.

KRAS mutations characterize pancreatic cell transformation from the earliest stages of carcinogenesis, and are present in >95% of pancreatic ductal adenocarcinoma (PDAC) cases. In search of novel biomarkers for the early diagnosis of PDAC, we identified the proteins secreted by the normal human pancreatic cell line (HPDE) recently transformed by inducing the overexpression of the KRASG12V oncogene. We report a proteomic signature of KRAS-induced secreted proteins, which was confirmed in surgical tumor samples from resected PDAC patients. The putative diagnostic performance of three candidates, Laminin-C2 (LAMC2), Tenascin-C (TNC) and Pentraxin-3 (PTX3), was investigated by ELISA quantification in two cohorts of PDAC patients (n = 200) eligible for surgery. Circulating levels of LAMC2, TNC and PTX3 were significantly higher in PDAC patients compared to the healthy individuals (p < 0.0001). The Receiver Operating Characteristics (ROC) curve showed good sensitivity (1) and specificity (0.63 and 0.85) for LAMC2 and PTX3, respectively, but not for TNC, and patients with high levels of LAMC2 had significantly shorter overall survival (p = 0.0007). High levels of LAMC2 and PTX3 were detected at early stages (I−IIB) and in CA19-9-low PDAC patients. In conclusion, pancreatic tumors release LAMC2 and PTX3, which can be quantified in the systemic circulation, and may be useful in selecting patients for further diagnostic imaging.

Differential role of Interleukin-1 and Interleukin-6 in K-Ras-driven pancreatic carcinoma undergoing mesenchymal transition.

K-Ras mutations are a hallmark of human pancreatic adenocarcinoma (PDAC) and epithelial-mesenchymal-transition (EMT) is a driver of progression. Oncogenic K-Ras causes the constitutive activation of NF-kB and the switch-on of an inflammatory program, which further fuels NF-kB and STAT3 activation. In this study we investigated how inflammatory pathways triggered by oncogenic K-Ras are regulated in human pancreatic cancer cells with distict epithelial or mesenchymal phenotype. Our results demonstrate that in cells with epithelial features, K-Ras driven inflammation is under the control of IL-1, while in cells undergoing EMT, is IL-1 independent. In pancreatic tumor cells with EMT phenotype, treatment with IL-1R antagonist (Anakinra) did not inhibit inflammatory cytokine production and tumor growth in mice. In these cells IL-6 is actively transcribed by the EMT transcription factor TWIST. Targeting of mesenchymal pancreatic tumors in vivo with anti-IL-6RmAb (RoActemra) successfully decreased tumor growth in immunodeficient mice, inhibited the inflammatory stroma and NF-kB-p65 and STAT3 phosphorylation in cancer cells. The results confirm that IL-1 is an important driver of inflammation in epithelial pancreatic tumors; however, tumor cells undergoing EMT will likely escape IL-1R inhibition, as IL-6 is continuously transcribed by TWIST. These findings have implications for the rational targeting of inflammatory pathways in human pancreatic cancer.

Effects of dipeptidyl peptidase-4 (DPP-4) inhibition on angiogenesis and hypoxic injury in type 2 diabetes.

AIMS: We examined whether, in diabetic Ob/Ob mice, the dipeptidyl peptidase-4 (DPP-4) inhibitor (PKF275-055), an antihyperglycemic drug, that inhibits the biological inactivation of SDF-1 (stromal cell-derived factor-1), may increase endothelial progenitor cells (EPCs) mobilization and incorporation, which, in turn, may regenerate capillaries and reduce myocardial ischemia induced by strenuous exercise. MAIN METHODS: Half of sixteen control and Ob/Ob mice and eight Ob/Ob mice treated with PKF275-055 for four weeks underwent a forced swim protocol. Oral glucose tolerance, circulating EPCs, capillary ultrastructure and density, hypoxic areas and SDF-1 localization in myocardium were measured. KEY FINDINGS: Ob/Ob mice were glucose intolerant, had a significant low number of circulating EPCs and myocardial capillaries compared to lean controls. The DPP-4 inhibitor significantly improved their glucose tolerance, doubled the number of circulating EPCs, stimulated the formation of functional vessels and SDF-1 localization in the endothelium of myocardial capillaries and arterioles. Cardiac hypoxia after forced swim in Ob/Ob mice was significantly reduced when they were treated with the DPP-4 inhibitor. SIGNIFICANCE: DPP-4 inhibition may re-establish an adequate capillary network in the myocardium of diabetic Ob/Ob mice by the mobilization and SDF-1-mediated incorporation of EPCs and, consequently, reducing the susceptibility to myocardial ischemic injury provoked by strenuous exercise.

In-depth glycoproteomic characterization of γ-conglutin by high-resolution accurate mass spectrometry.

The molecular characterization of bioactive food components is necessary for understanding the mechanisms of their beneficial or detrimental effects on human health. This study focused on γ-conglutin, a well-known lupin seed N-glycoprotein with health-promoting properties and controversial allergenic potential. Given the importance of N-glycosylation for the functional and structural characteristics of proteins, we studied the purified protein by a mass spectrometry-based glycoproteomic approach able to identify the structure, micro-heterogeneity and attachment site of the bound N-glycan(s), and to provide extensive coverage of the protein sequence. The peptide/N-glycopeptide mixtures generated by enzymatic digestion (with or without N-deglycosylation) were analyzed by high-resolution accurate mass liquid chromatography-multi-stage mass spectrometry. The four main micro-heterogeneous variants of the single N-glycan bound to γ-conglutin were identified as Man2(Xyl) (Fuc) GlcNAc2, Man3(Xyl) (Fuc) GlcNAc2, GlcNAcMan3(Xyl) (Fuc) GlcNAc2 and GlcNAc 2Man3(Xyl) (Fuc) GlcNAc2. These carry both core ß1,2-xylose and core α1-3-fucose (well known Cross-Reactive Carbohydrate Determinants), but corresponding fucose-free variants were also identified as minor components. The N-glycan was proven to reside on Asn131, one of the two potential N-glycosylation sites. The extensive coverage of the γ-conglutin amino acid sequence suggested three alternative N-termini of the small subunit, that were later confirmed by direct-infusion Orbitrap mass spectrometry analysis of the intact subunit.

Neuroprotective effects of toll-like receptor 4 antagonism in spinal cord cultures and in a mouse model of motor neuron degeneration.

Sustained inflammatory reactions are common pathological events associated with neuron loss in neurodegenerative diseases. Reported evidence suggests that Toll-like receptor 4 (TLR4) is a key player of neuroinflammation in several neurodegenerative diseases. However, the mechanisms by which TLR4 mediates neurotoxic signals remain poorly understood. We investigated the role of TLR4 in in vitro and in vivo settings of motor neuron degeneration. Using primary cultures from mouse spinal cords, we characterized both the proinflammatory and neurotoxic effects of TLR4 activation with lipopolysaccharide (activation of microglial cells, release of proinflammatory cytokines and motor neuron death) and the protective effects of a cyanobacteria-derived TLR4 antagonist (VB3323). With the use of TLR4-deficient cells, a critical role of the microglial component with functionally active TLR4 emerged in this setting. The in vivo experiments were carried out in a mouse model of spontaneous motor neuron degeneration, the wobbler mouse, where we preliminarily confirmed a protective effect of TLR4 antagonism. Compared with vehicle- and riluzole-treated mice, those chronically treated with VB3323 showed a decrease in microglial activation and morphological alterations of spinal cord neurons and a better performance in the paw abnormality and grip-strength tests. Taken together, our data add new understanding of the role of TLR4 in mediating neurotoxicity in the spinal cord and suggest that TLR4 antagonists could be considered in future studies as candidate protective agents for motor neurons in degenerative diseases.

Secretome analysis of multiple pancreatic cancer cell lines reveals perturbations of key functional networks.

The cancer secretome is a rich repository in which to mine useful information for both cancer biology and clinical oncology. To help understand the mechanisms underlying the progression of pancreatic cancer, we characterized the secretomes of four human pancreatic ductal adenocarcinoma (PDAC) cell lines versus a normal counterpart. To this end, we used a proteomic workflow based on high-confidence protein identification by mass spectrometry, semiquantitation by a label-free approach, and network enrichment analysis by a system biology tool. Functional networks significantly enriched with PDAC-dysregulated proteins included not only expected alterations within key mechanisms known to be relevant for tumor progression (e.g., cell-cell/cell-matrix adhesion, extracellular matrix remodeling, and cytoskeleton rearrangement), but also other extensive, coordinated perturbations never observed in pancreatic cancer. In particular, we highlighted perturbations possibly favoring tumor progression through immune escape (i.e., inhibition of the complement system, deficiency of selected proteasome components within the antigen-presentation machinery, and inhibition of T cell cytoxicity), and a defective protein folding machinery. Among the proteins found concordantly oversecreted in all of our PDAC cell lines, many are reportedly overexpressed in pancreatic cancer (e.g., CD9 and Vimentin), while others (PLOD3, SH3L3, PCBP1, and SFRS1) represent novel PDAC-secreted proteins that may be worth investigating.

Tumor-conditioned macrophages secrete migration-stimulating factor: a new marker for M2-polarization, influencing tumor cell motility.

Tumor-associated macrophages (TAMs) are key orchestrators of the tumor microenvironment directly affecting neoplastic cell growth, neoangiogenesis, and extracellular matrix remodeling. In turn, the tumor milieu strongly influences maturation of TAMs and shapes several of their features. To address the early macrophage (M) differentiation phase in a malignant context, we mimicked a tumor microenvironment by in vitro coculturing human blood monocytes with conditioned media from different cancer cell lines. Only 2 out of 16 tumor cell lines induced M differentiation due to secreted M-CSF isoforms, including high molecular mass species. A global gene profiling of tumor-conditioned M was performed. Comparison with other datasets (polarized M1-M, M2-M, and TAMs isolated from human tumors) highlighted the upregulation of several genes also shared by TAM and M2-polarized M. The most expressed genes were selenoprotein 1, osteoactivin, osteopontin, and, interestingly, migration-stimulating factor (MSF), a poorly studied oncofoetal isoform of fibronectin. MSF (present in fetal/cancer epithelial and stromal cells but not in healthy tissues) was never identified in M. MSF production was confirmed by immunohistochemistry in human TAMs. MSF was induced by M-CSF, IL-4, and TGFbeta but not by proinflammatory stimuli. RNA and protein analysis clearly demonstrated that it is specifically associated with the M2 polarization of M. Tumor-conditioned M-derived MSFs strongly stimulated tumor cell migration, thus contributing to the motile phenotype of neoplastic cells. In conclusion, MSF is a new molecule associated with the M2 polarization of M and expressed by TAMs. Its biological function may contribute to M-mediated promotion of cancer cell invasion and metastasis.

Effects of cigarette smoking on the human urinary proteome.

In this pilot study we used a proteomic approach to compare the urinary protein patterns of healthy smokers and non-smokers. Proteins were resolved by two-dimensional gel electrophoresis and identified by mass spectrometry. The relative abundance of three inflammatory proteins (S100A8, inter-alpha-trypsin inhibitor heavy chain 4, CD59) and that of two isoforms of pancreatic alpha amylase was significantly higher in smokers. Zinc-alpha-2-glycoprotein was the only protein down-regulated in smokers. Its abundance was significantly correlated with urinary glucocorticoids. Most of the proteins identified may be non-specific biomarkers of tobacco effects, since they are involved in inflammatory responses associated with several diseases. Of greater interest are the changes in abundance of pancreatic alpha amylase and zinc-alpha-2-glycoprotein, which after proper validation, might be candidate biomarkers of diseases resulting from exposure to tobacco smoke. The data also show for the first time that smoking can affect the expression profile of urinary proteins.

Proteasomal processing of albumin by renal dendritic cells generates antigenic peptides.

The role of dendritic cells (DC) that accumulate in the renal parenchyma of non-immune-mediated proteinuric nephropathies is not well understood. Under certain circumstances, DC capture immunologically ignored antigens, including self-antigens, and present them within MHC class I, initiating an autoimmune response. We studied whether DC could generate antigenic peptides from the self-protein albumin. Exposure of rat proximal tubular cells to autologous albumin resulted in its proteolytic cleavage to form an N-terminal 24-amino acid peptide (ALB1-24). This peptide was further processed by the DC proteasome into antigenic peptides that had binding motifs for MHC class I and were capable of activating syngeneic CD8+ T cells. In vivo, the rat five-sixths nephrectomy model allowed the localization and activation of renal DC. Accumulation of DC in the renal parenchyma peaked 1 wk after surgery and decreased at 4 wk, concomitant with their appearance in the renal draining lymph nodes. DC from renal lymph nodes, loaded with ALB1-24, activated syngeneic CD8+ T cells in primary culture. The response of CD8+ T cells of five-sixths nephrectomized rats was amplified with secondary stimulation. In contrast, DC from renal lymph nodes of five-sixths nephrectomized rats treated with the proteasomal inhibitor bortezomib lost their capacity to stimulate CD8+ T cells in primary and secondary cultures. These data suggest that albumin can be a source of potentially antigenic peptides upon renal injury and that renal DC play a role in processing self-proteins through a proteasome-dependent pathway.

Estimating community drug abuse by wastewater analysis.

BACKGROUND: The social and medical problems of drug abuse are a matter of increasing global concern. To tackle drug abuse in changing scenarios, international drug agencies need fresh methods to monitor trends and patterns of illicit drug consumption. OBJECTIVE: We tested a sewage epidemiology approach, using levels of excreted drug residues in wastewater, to monitor collective use of the major drugs of abuse in near real time. METHODS: Selected drug target residues derived from use of cocaine, opiates, cannabis, and amphetamines were measured by mass spectrometry in wastewater collected at major sewage treatment plants in Milan (Italy), Lugano (Switzerland), and London (United Kingdom). The amounts of drug residues conveyed to the treatment plants, reflecting the amounts collectively excreted with urine, were used to estimate consumption of the active parent drugs. RESULTS: Reproducible and characteristic profiles of illicit drug use were obtained in the three cities, thus for the first time quickly revealing changes in local consumption (e.g., cocaine consumption rose significantly on weekends in Milan). Profiles of local drug consumption based on waste-water measurements are in line with national annual prevalence estimates. CONCLUSIONS: Patterns and trends of drug abuse in local communities can be promptly monitored by this tool, a convenient new complement to more complex, lengthy survey methods. In principle, searching the sewage for excreted compounds relevant to public health issues appears to have the potential to become a convenient source of real-time epidemiologic information.

Mass spectrometric analysis of illicit drugs in wastewater and surface water.

Residues of illicit drugs have been recently found in urban wastewater and surface water. Their levels reflect the amount of drugs collectively excreted by consumers and can therefore be used to estimate drug abuse. An overview of the most widely used illicit drugs and of the analytical methods used for their detection in wastewater and surface water is presented here. Solid-phase extraction and high performance liquid chromatography-tandem mass spectrometry are the techniques that have been used for these investigations. Instrumental conditions and fragmentation patterns of illicit drugs and their metabolites are described.

Illicit drugs, a novel group of environmental contaminants.

It is now well established that residues from therapeutic drugs consumed by humans can end up, through the sewage system, in the surface water of populated areas. Given that the global production of major illicit drugs is comparable to that of widely used pharmaceuticals, we tested for the presence of drugs of abuse (cocaine, opioids, amphetamines and cannabis derivatives), some related opioid pharmaceuticals (codeine and methadone) and/or their metabolites in Italian and British surface waters. Having identified residues of all major drugs of abuse in raw and treated urban wastewater, we now measured their levels in several rivers and lakes by a selective multi-residue assay based on liquid chromatography-tandem mass spectrometry. Recoveries in surface water were generally higher than 80%, with overall variability of the method lower than 10%. LODs were generally lower than 0.2 ng/L, and LOQs were lower than 0.6 ng/L, with few exceptions. Many of the tested substances were found in both rivers and lakes, at concentrations ranging from high pg/L to high ng/L, with loads in rivers in the range of tenths to hundreds of grams per day. Our data indicate that residues of drugs of abuse have become widespread surface water contaminants in populated areas. Since most of these residues still have potent pharmacological activities, their presence in the aquatic environment may have potential implications for human health and wildlife.

Identification and measurement of illicit drugs and their metabolites in urban wastewater by liquid chromatography-tandem mass spectrometry.

Residues of illicit drugs and their metabolites that are excreted by humans may flow into and through wastewater treatment plants. The aim of this study was to develop a method for the determination of cocaine, amphetamines, morphine, cannabinoids, methadone, and some of their metabolites in wastewater. Composite 24-h samples from urban treatment plants were enriched with deuterated internal standards before solid-phase extraction. High-pressure liquid chromatography tandem mass spectrometry with multiple reaction monitoring was used for quantitation. Recoveries were generally higher than 80%, and limits of quantifications were in the low nanograms-per-liter range for untreated and treated wastewater. The overall variability of the method was lower than 10% for untreated and 5% for treated wastewater. The method was applied to wastewater samples coming from two treatment plants in Italy and Switzerland. Quantification ranges were found to be 0.2-1 microg/L for cocaine and its metabolite benzoylecgonine, 80-200 ng/L for morphine, 10 ng/L for 6-acetylmorphine, 60-90 ng/L for 11-nor-9-carboxy-Delta9-tetrahydrocannabinol, 10-90 ng/L for methadone and its main metabolite 2-ethylidene-1,5-dimethyl-3,3-diphenylpyrrolidine, and lower than 20 ng/L for amphetamines. As previously reported for cocaine, this method could be useful to estimate and monitor drug consumption in the population in real time, helping social scientists and authorities to combat drug abuse.

Pharmaceuticals in the environment in Italy: causes, occurrence, effects and control.

BACKGROUND, AIM AND SCOPE: Environmental contamination by pharmaceuticals is an emerging issue. Until recently, information on medicinal substances released into the environment was scant, but several studies have now been published. Data are, however, usually scattered and a systematic approach to this subject is generally lacking. Moreover, because of differences in the prevalence of diseases, treatment habits and options, or simply for market reasons, the pollution profile can differ significantly in different countries. The aim of this work is to review the papers dealing with environmental contamination by pharmaceuticals in Italy, with the aim of providing a comprehensive view on a national scale. METHODS: Papers related to environmental contamination by pharmaceuticals in Italy were reviewed, in order to offer a comprehensive view of this subject. Topics included analysis, occurrence, monitoring, modelling, treatment, control of the emissions, and ecotoxicological effects of pharmaceuticals in the environment. RESULTS AND CONCLUSION: The literature suggests that pharmaceuticals are widespread contaminants, entering the environment from a myriad of scattered points. Patients, in case of drugs for human use, or animals for veterinary drugs, are the main sources of contamination. Pharmaceuticals can be ranked according to environmental loads, predicted by multiplying sales figures by the rate of metabolism in man or animals. Priority pharmaceuticals, i.e. the molecules of concern for the environment, can be measured in waste and surface water by liquid chromatography-tandem mass spectrometry, and the loads detected are generally comparable to the predicted ones. Pharmaceuticals are designed to stimulate a response in humans and animals at low doses, with a very specific target, so the implications for human health and the environment need to be assessed. In vitro and in vivo studies suggest that pharmaceutical principles, taken singularly or in combinations, and concentrations close to those detected in the environment, may have ecotoxicological effects. The sewage system is an important point in the control of contamination, but sewage treatment plants are not able efficiently to abate a substantial part of water-borne pharmaceuticals. Several variables play a role, however, in the processes of waste water treatment, and could be specifically adjusted to improve the efficiency of drug abatement, mitigating the potential environmental hazards. RECOMMENDATION AND PERSPECTIVE: Pharmaceuticals in the environment are becoming a subject of global concern, with potential environmental consequences. Further knowledge of the causes, occurrence and effects of drugs as environmental pollutants is necessary for a better understanding of this ecological issue, as well as to improve abatement strategies, and to mitigate subtle environmental consequences.

Proteome analysis for the identification of in vivo estrogen-regulated proteins in bone.

Estrogen deficiency results in a reduced bone mass, which can be prevented by treatment with estrogens. This study used a proteomic approach for the first time to obtain a global perspective of estrogens' effects on whole-bone proteins. Bone proteome profiles were examined in three groups of mice: (1) sham-operated with normal ovarian functions, (2) ovariectomised and (3) ovariectomised with estrogen replacement therapy. Bone proteins extracted from the humerus were separated by 2-DE and visualised by CBB colloidal staining. Spot detection and quantification was done by image analysis. Differentially expressed proteins were identified by MS and database search, using peptide mass fingerprint and peptide sequence analysis. Differential expression analysis in the three experimental groups showed significant changes for 14 proteins. These included proteins related to bone metabolism, cytoskeleton components and energy metabolic pathways. Our data suggest that some proteins related to cytoskeleton and to energy pathways, such as tropomyosins, aconitase 2 and enolase beta, might be new molecular targets responsive to the effects of estrogen. Differentially expressed proteins identified in this model may offer a useful starting point for elucidating novel aspects of the pleiotropic effects of estrogens on bone.

Cocaine in surface waters: a new evidence-based tool to monitor community drug abuse.

BACKGROUND: Cocaine use seems to be increasing in some urban areas worldwide, but it is not straightforward to determine the real extent of this phenomenon. Trends in drug abuse are currently estimated indirectly, mainly by large-scale social, medical, and crime statistics that may be biased or too generic. We thus tested a more direct approach based on 'field' evidence of cocaine use by the general population. METHODS: Cocaine and its main urinary metabolite (benzoylecgonine, BE) were measured by mass spectrometry in water samples collected from the River Po and urban waste water treatment plants of medium-size Italian cities. Drug concentration, water flow rate, and population at each site were used to estimate local cocaine consumption. RESULTS: We showed that cocaine and BE are present, and measurable, in surface waters of populated areas. The largest Italian river, the Po, with a five-million people catchment basin, steadily carried the equivalent of about 4 kg cocaine per day. This would imply an average daily use of at least 27 +/- 5 doses (100 mg each) for every 1000 young adults, an estimate that greatly exceeds official national figures. Data from waste water treatment plants serving medium-size Italian cities were consistent with this figure. CONCLUSION: This paper shows for the first time that an illicit drug, cocaine, is present in the aquatic environment, namely untreated urban waste water and a major river. We used environmental cocaine levels for estimating collective consumption of the drug, an approach with the unique potential ability to monitor local drug abuse trends in real time, while preserving the anonymity of individuals. The method tested here--in principle extendable to other drugs of abuse--might be further refined to become a standardized, objective tool for monitoring drug abuse.

Long-term vitamin E supplementation fails to reduce lipid peroxidation in people at cardiovascular risk: analysis of underlying factors.

BACKGROUND: Antioxidant supplementation with vitamin E had no effect in the prevention of cardiovascular diseases (CVD) in three recent large, randomized clinical trials. In order to reassess critically the role of vitamin E in CVD prevention, it is important to establish whether these results are related to a lack of antioxidant action. METHODS: We examined the in vivo antioxidant effect of vitamin E (300 mg/day for about three years) in 144 participants in the Primary Prevention Project (females and males, aged >/= 50 y, with at least one major CV risk factor, but no history of CVD). Urinary 8-epi-PGF2alpha (isoprostane F2alpha-III or 15-F2t-isoP), a validated biomarker of lipid peroxidation, was measured by mass spectrometry. RESULTS: Urinary excretion of 8-epi-PGF2alpha [pg/mg creatinine, median (range)] was 141 (67-498) in treated and 148 (76-561) in untreated subjects (p = 0.10). Taking into account possible confounding variables, multiple regression analysis confirmed that vitamin E had no significant effect on this biomarker. Levels of 8-epi-PGF2alpha were in the normal range for most subjects, except smokers and those with uncontrolled blood pressure or hyperglycemia. CONCLUSIONS: Prolonged vitamin E supplementation did not reduce lipid peroxidation in subjects with major cardiovascular risk factors. The observation that the rate of lipid peroxidation was near normal in a large proportion of subjects may help explain why vitamin E was not effective as an antioxidant in the PPP study and was ineffective for CVD prevention in large scale trials.

Identification of metabolites from type III F2-isoprostane diastereoisomers by mass spectrometry.

F(2)-isoprostanes (F(2)-iPs) are prostaglandin (PG)-like products of non-enzymatic free radical-catalyzed peroxidation of arachidonic acid that are now widely used as indices of lipid peroxidation in vivo. Knowledge of the metabolic fate of F(2)-iPs in vivo is still scant, despite its importance for defining their overall formation and biological effects in vivo. Type III F(2)-iPs, which are diastereoisomers of cyclooxygenase-derived PGF(2alpha), may be metabolized through the pathways of PG metabolism. We therefore studied the in vitro metabolism of eight synthetic Type III F(2)-iP diastereoisomers in comparison with PGF(2alpha). We used gas chromatography-mass spectrometry and high performance liquid chromatography-electrospray-tandem mass spectrometry for structural identification of metabolites formed after incubation of the various compounds with isolated rat hepatocytes. PGF(2alpha) was metabolized to several known products, resulting from a combination of beta-oxidation, reduction of Delta(5) and/or Delta(13) double bonds, and 15-OH oxidation, plus other novel products deriving from conjugation with taurine of PGF(2alpha) and its metabolites. Of the eight F(2)-iP diastereoisomers, some were processed similarly to PGF(2alpha), whereas others showed peculiar metabolic profiles according to specific stereochemical configurations. These data represent the first evidence of biodegradation of selected Type III F(2)-iP isomers other than 8-epi-PGF(2alpha), through known and novel pathways of PGF(2alpha) metabolism. The analytical characterization of these products may serve as a basis for identifying the most significant products formed in vivo.