Identification and quantification of phytoprostanes and phytofurans of coffee and cocoa by- and co-products.

Phytoprostanes (PhytoPs) and phytofurans (PhytoFs) are isoprostanoids that result from the peroxidation of α-linolenic acid and are biomarkers of oxidative stress in plants and humans. These compounds exhibit several interesting biological activities (e.g. neuroprotection and anti-inflammatory activities). The aim of this research was to add value to coffee pulp (CP), cocoa husk (CH) and cocoa pod husk (CPH) by identifying and quantifying PhytoPs and PhytoFs by liquid chromatography-tandem mass spectrometry. The contents of PhytoPs and PhytoFs in CP, CH, and CPH were, respectively, 654.6, 474.3 and 179.9, and 543.2, 278.0 and 393.8 ng per g dry weight (dw). The main PhytoP found in CP (171.37 ng per g dw) and CPH (37.12 ng per g dw) was 9-epi-9-F1t-PhytoP, while ent-9-L1t-PhytoP was the most abundant in CH (109.78 ng per g dw). The main PhytoF found in all sources was ent-16(RS)-13-epi-ST-Δ14-9-PhytoF, at 196.56, 126.22, and 207.57 ng per g dw in CP, CH, and CPH, respectively. We provide the first complete profile of PhytoPs and PhytoFs for these agro-residues, which could be used in the functional food industry for enriching food or as nutritional supplements.

Increased levels of prostaglandin E-major urinary metabolite (PGE-MUM) in chronic fibrosing interstitial pneumonia.

BACKGROUND: Dysregulation of the prostaglandin E2 (PGE2) signaling pathway has been implicated in interstitial pneumonia (IP) pathogenesis. Due to the unstable nature of PGE2, available detection methods may not precisely reflect PGE2 levels. We explored the clinical usefulness of measuring stable prostaglandin E-major urinary metabolite (PGE-MUM) with respect to pathogenesis and extent of chronic fibrosing IP (CFIP), including idiopathic pulmonary fibrosis (IPF), as PGE-MUM is reflective of systemic PGE2 production. METHODS: PGE-MUM was measured by radioimmunoassay in controls (n = 124) and patients with lung diseases (bronchial asthma (BA): n = 78, chronic obstructive pulmonary disease (COPD): n = 33, CFIP: n = 44). Extent of lung fibrosis was assessed by fibrosing score (FS) of computed tomography (CT) (FS1-4). Immunohistochemical evaluation of COX-2 was performed to find PGE2 producing cells in IPF. Human bronchial epithelial cells (HBEC) and lung fibroblasts (LFB) were used in in vitro experiments. RESULTS: Compared to control, PGE-MUM levels were significantly elevated in CFIP. PGE-MUM levels were positively correlated with FS, and inversely correlated with %DLCO in IP (FS 1-3). COX-2 was highly expressed in metaplastic epithelial cells in IPF, but lower expression of EP2 receptor was demonstrated in LFB derived from IPF. TGF-ß induced COX-2 expression in HBEC. CONCLUSIONS: PGE-MUM, elevated in CFIP, is a promising biomarker reflecting disease activity. Metaplastic epithelial cells can be a source of elevated PGE-MUM in IPF.

GC-ECNICI-MS/MS of eicosanoids as pentafluorobenzyl-trimethylsilyl (TMS) derivatives: Evidence of CAD-induced intramolecular TMS ether-to-ester rearrangement using carboxy-18O-labelled eicosanoids and possible implications in quantitative analysis.

GC-MS and GC-MS/MS of pentafluorobenzyl (PFB) ester trimethylsilyl (TMS) ether (PFB-TMS) derivatives of hydroxylated long-chain fatty acids including arachidonic acid metabolites, the eicosanoids, in the electron-capture negative-ion chemical ionization (ECNICI) mode are the most sensitive and accurate approaches to quantify carboxyl groups-containing compounds in complex biological fluids such as plasma and urine. Under ECNICI conditions, PFB-TMS derivatives of eicosanoids ionize to form very few ions, with the carboxylates [M-PFB]- being typically the most intense. Less intense ions may be additionally formed by consecutive neutral loss (NL) of trimethylsilanol (TMSOH, 90Da) groups ([M-PFB-(TMSOH)n]-). By using [1,1-18O2]- and [1,ω-18O2]-eicosanoids, we studied ion processes following collisionally activated dissociation (CAD) of the precursor ions [M-PFB]-. We found that CAD resulted in formation of product ions due to NL of a TMS18OH (92Da) group in monocarboxylic and of a PFB18OH (200Da) group in dicarboxylic eicosanoids. TMS18OH NL implies an intra-molecular transfer of the TMS group from hydroxyl groups to their carboxylate anions [M-PFB]-. From a mechanistic point of view, this rearrangement may explain formation of unique product ions in GC-MS/MS of eicosanoids under ECNICI conditions. From the quantitative point of view, quantification by GC-MS/MS of product ions due to [M-PFB-(TMSOH)n]- and [M-PFB-TMS18OH-(TMSOH)n-1]-would reveal incorrect data, if [1,1-18O2]-eicosanoids are used as internal standards and if no correction for the 18O-loss is performed. In 18O-labelled dicarboxylic eicosanoids, such as the major urinary metabolite (MUM) of E prostaglandins, i.e., [1,ω-18O2]-PGE-MUM), no TMS ester/TMS ether rearrangement was observed. Yet, 18O-loss occurred upon CAD of [M-PFB]- due to NL of PFB18OH (200Da). In both cases the extent of 18O-loss needs to be determined and considered for accurate quantification of monocarboxylic acids such as 8-isoprostaglandin F2α (8-iso-PGF2α) and dicarboxylic eicosanoids such as PGE-MUM.

Inhibition of ovulation in the gonadotropin-primed immature rat by exogenous prostaglandin E2.

In the past two decades there have been innumerable reports that prostaglandins (PGs) are essential for mammalian ovulation. However, we have recently found that a relatively low dose of 0.03 mg indomethacin (INDO) sc to PMSG/hCG-primed immature Wistar rats can significantly reduce ovarian PG levels without inhibiting the control ovulation rate of 60+ ova/rat (1-3). In view of this information, the present study was an effort to duplicate the earlier reports that PGs can reverse the "inhibitory" effect of INDO on ovulation. In control animals, which received PMSG and hCG only, the ovulation rate was 63.8 +/- 4.5 ova/rat. This rate was reduced to 4.1 +/- 1.1 ova/rat when the animals were injected with 1.0 mg INDO at 3 h after hCG. In no instance was this inhibition reversed when the animals were treated with 1.0 mg of PGE2 or PGF2 alpha, or a combination of both prostanoids in either a single dose at 3 h after hCG, or in 4x doses at 2-h intervals beginning at 3 h after hCG. Furthermore, in animals that did not receive INDO, the ovulation rate in PGE2-treated animals was reduced to 20.0 +/- 6.7 ova/rat, and in animals treated with PGE2 and PGF2 alpha (combined) it was reduced to 19.4 +/- 6.5 ova/rat. In summary, not only did the PGs fail to reverse the anti-ovulatory effect of INDO, PGE2 actually suppressed the ovulation rate.

Release of arachidonic acid metabolites during acute pancreatitis in pigs.

The pancreatic release of arachidonic acid metabolites was studied in a porcine model of acute pancreatitis. In situ isolation of the pancreatic gland enabled selective collection of pancreatic venous blood, pancreatic lymph, and ascites fluid. Three experimental groups were studied: 1) control (n = 9); 2) hemorrhagic pancreatitis induced by injection of 5% bile salt (sodium taurocholate) into the pancreatic duct (n = 10); and 3) edematous pancreatitis induced by injection of free fatty acid (FFA) into the pancreatic artery (n = 10). Determinations of cyclooxygenase metabolites were performed by radioimmunoassay; lipoxygenase metabolites (LTC4, LTD4) were measured by radioimmunoassay after purification by high-performance liquid chromatography. Prostaglandin (PG)F1 alpha, thromboxane B2, and PGF2 alpha concentrations were almost doubled in the lymph of the FFA group during pancreatitis, as were PGF1 alpha levels in pancreatic venous blood. However, concentrations of cyclooxygenase metabolites remained unchanged in the control group and in the bile salt group. Concentrations of LTC4 and LTD4 in lymph and ascites fluid of both pancreatitis groups increased from about 50 pg/ml to a mean level of 600 pg/ml at 6 h. Leukotriene concentrations in the control group were consistently below 50 pg/ml. The results of this study indicate that above all LTC4 and LTD4 are released from the organ and that these arachidonic acid metabolites may be also involved in the events following acute pancreatitis contributing to the systemic effects of the disease.

Eicosanoid release as laboratory indicator of biocompatibility.

Biocompatibility evaluation of extracorporeal devices requires the establishment of sensitive indicators of blood cells/surface interactions. Among others, arachidonic acid derivatives, such as prostaglandins and thromboxanes, play an important role in the cell control systems. Hence, the release of eicosanoids during blood exposure to dialyzer membranes was investigated. Experiments included in vitro incubation of human blood with flat membranes (FM), as well as ex vivo perfusion of hollow fiber membranes (HFM) with blood from healthy volunteers in single-pass fashion. In both models, a significant release of prostaglandin E2 (PGE2) and thromboxane B2 (TXB2) was detected. In addition, the amount of eicosanoid release depended on the type of membrane tested. After a 10-min FM incubation with fresh blood, plasma concentrations of TXB2 and PGE2 were pronounced by polycarbonate when compared to Cuprophan and polyacrylonitrile. During 10 min of open loop perfusion of HFM, polymethylmethacrylate was the most active biomaterial, whereas the reactivity of Cuprophan was significantly lower. Among HFM, Hemophan was by far the less active. These results indicate that the release of eicosanoids represents a sensitive parameter of blood cells/membrane reactivity. Thus, the question arises as to whether or not the extracorporeal process of cyclooxygenase activity could contribute to the clinical side effects of chronical hemodialysis.

Postischemic cerebral microvascular responses to norepinephrine and hypotension in newborn pigs.

We examined the effects of 20 minutes' cerebral ischemia on cerebral microcirculatory responses to topical norepinephrine and systemic hypotension in three groups (sham-operated control, 2-3 hours postischemia, and 24 hours postischemia) of anesthetized newborn pigs equipped with closed cranial windows. Cerebral ischemia may eliminate the prostanoid vasodilator system from the cerebral circulation. Norepinephrine (10(-4) M) decreased pial arteriolar diameters similarly in all three groups (27%, 28%, and 21%, respectively), but only the sham-operated group exhibited pial arteriolar dilation in response to hypotension (28% at 33 mm Hg). Two-three and 24 hours after cerebral ischemia, hypotension decreased pial arteriolar diameters (21% and 17%, respectively). In sham-operated piglets, norepinephrine and hypotension increased cortical periarachnoid cerebrospinal fluid prostanoid concentrations. However, neither norepinephrine nor hypotension altered cerebral prostanoid production 2-3 or 24 hours after cerebral ischemia. Therefore, we conclude that after cerebral ischemia, autoregulatory pial arteriolar dilation in response to hypotension is absent, while vasoconstriction in response to norepinephrine is intact.

Radioimmunoassays for prostaglandin metabolites.

Radioimmunoassays were developed for the main plasma and urinary metabolites of PGF2alpha in the human, viz., 15-keto-13,14-dihydro-PGF2alpha and 5alpha, 7alpha-dihydroxy-11-ketotetranorprosta-1,16-dioic acid. The methods allowed assay of unextracted samples and had a sensitivity of 5 pg and 2 pg, respectively. The assays were evaluated by comparison with mass spectrometric data. Normal levels of these metabolites were estimated in a number of subjects. Levels in plasma and urine of the metabolites were followed after injection or during infusion of PGF2alpha and during administration of various antiinflammatory drugs.