Evaluation of different extraction approaches for the determination of phenolic compounds and their metabolites in plasma by nanoLC-ESI-TOF-MS.

Sample preparation is an important step for the determination of phenolic compounds in biological samples. Different extraction methods have been tested to determine phenolic compounds and their metabolites in plasma by nano-liquid chromatography coupled to electrospray ionisation-time-of-flight mass spectrometry (nanoLC-ESI-TOF-MS). The sample treatment optimisation was performed using commercial foetal bovine serum spiked with representative phenolic standards, namely naringenin, luteolin, verbascoside, apigenin, rutin, syringic acid and catechin. Different protein-precipitation conditions were evaluated as well as enzymatic digestion with trypsin and solid-phase extraction using different phases such as C-18, ABN and ENV+, working at different pH values. The optimum extraction procedure consisted of a previous protein-precipitation step using HCl 200 mmol/L in methanol for 2.5 h at 50 °C followed by a solid-phase extraction using C-18 cartridges at pH 2.5. This procedure was finally applied to the plasma of rats overfed with a phenolic-rich Lippia citriodora extract. These samples were analysed by nanoLC-ESI-TOF-MS, enabling the identification of five compounds previously found in the administered L. citriodora extract and one metabolite.

Type I interferon induces inhibitory 16-kD CCAAT/ enhancer binding protein (C/EBP)beta, repressing the HIV-1 long terminal repeat in macrophages: pulmonary tuberculosis alters C/EBP expression, enhancing HIV-1 replication.

We have previously observed that HIV-1 replication is suppressed in uninflamed lung and increased during tuberculosis. In vitro THP-1 cell-derived macrophages inhibited HIV-1 replication after infection with Mycobacterium tuberculosis. Suppression of HIV-1 replication was associated with inhibition of the HIV-1 long terminal repeat (LTR) and induction of ISGF-3, a type I interferon (IFN)-specific transcription factor. Repression of the HIV-1 LTR required intact CCAAT/enhancer binding protein (C/EBP) sites. THP-1 cell-derived macrophages infected with M. tuberculosis, lipopolysaccharide, or IFN-beta induced the 16-kD inhibitory C/EBPbeta isoform and coincidentally repressed HIV-1 LTR transcription. C/EBPbeta was the predominant C/EBP family member produced in THP-1 macrophages during HIV-1 LTR repression. In vivo, alveolar macrophages from uninflamed lung strongly expressed inhibitory 16-kD C/EBPbeta, but pulmonary tuberculosis abolished inhibitory C/EBPbeta expression and induced a novel C/EBP DNA binding protein. Therefore, in vitro, proinflammatory stimulation produces an IFN response inhibiting viral replication by induction of a C/EBPbeta transcriptional repressor. THP-1 cell-derived macrophages stimulated with type I IFN are similar to alveolar macrophages in the uninflamed lung in vivo. In contrast, the cellular immune response in active pulmonary tuberculosis disrupts this innate immunity, switching C/EBP expression and allowing high level viral replication.

Requirement for MAP kinase (ERK2) activity in interferon alpha- and interferon beta-stimulated gene expression through STAT proteins.

Activation of early response genes by interferons (IFNs) requires tyrosine phosphorylation of STAT (signal transducers and activators of transcription) proteins. It was found that the serine-threonine kinase mitogen-activated protein kinase (MAPK) [specifically, the 42-kilodalton MAPK or extracellular signal-regulated kinase 2 (ERK2)] interacted with the alpha subunit of IFN-alpha/beta receptor in vitro and in vivo. Treatment of cells with IFN-beta induced tyrosine phosphorylation and activation of MAPK and caused MAPK and Stat1 alpha to coimmunoprecipitate. Furthermore, expression of dominant negative MAPK inhibited IFN-beta-induced transcription. Therefore, MAPK appears to regulate IFN-alpha and IFN-beta activation of early response genes by modifying the Jak-STAT signaling cascade.

In vivo evidence of interaction between interferon-stimulated gene factors and the interferon-stimulated response element.

Constitutive and interferon-inducible DNase hypersensitive sites in vivo are located in interferon-stimulated gene promoters near sequences that specifically bind constitutive or interferon-inducible proteins in vitro. Induced sites and proteins are transient or maintained, depending on cell type. Interferon-stimulated gene transcription is transient or maintained in parallel.

Purification and cloning of interferon-stimulated gene factor 2 (ISGF2): ISGF2 (IRF-1) can bind to the promoters of both beta interferon- and interferon-stimulated genes but is not a primary transcriptional activator of either.

Interferon-stimulated gene factor 2 (ISGF2) was purified from HeLa cells treated with alpha interferon. The factor, a single polypeptide of 56 kilodaltons (kDa), bound both to the central 9 base pairs of the 15-base-pair interferon-stimulated response element (ISRE) that is required for transcriptional activation of interferon-stimulated genes and to the PRD-I regulatory element of the beta interferon gene. ISGF2 was a phosphoprotein, and dephosphorylation in vitro reduced its DNA-binding activity. However, conditions that changed the amount of ISGF2 did not change the phosphorylated isoforms in vivo. ISGF2 in unstimulated cells existed in trace amounts and was induced by both alpha interferon and gamma interferon as well as by virus infection. Plasmid-bearing Escherichia coli clones encoding ISGF2 were selected with antibody against purified ISGF2. Sequence analysis revealed that the ISGF2 protein was the same as that encoded by the cDNA clone IRF-1, which has been claimed to activate transcription of interferon genes. We show that transcription of the ISGF2 gene was induced by alpha interferon, gamma interferon, and double-stranded RNA. However, ISGF2 was neither necessary nor sufficient for induced transcription of the beta interferon gene, while the factor NF kappa B was clearly involved.

Endothelial interferon regulatory factor 1 cooperates with NF-kappa B as a transcriptional activator of vascular cell adhesion molecule 1.

Transcription of the vascular cell adhesion molecule 1 (VCAM-1) gene in endothelial cells is induced by lipopolysaccharide and the inflammatory cytokines interleukin-1 beta and tumor necrosis factor alpha (TNF-alpha). Previous studies have demonstrated that tandem binding sites for the inducible transcription factor NF-kappa B are necessary but not sufficient for full cytokine-mediated transcriptional activation. Herein, we demonstrate that full cytokine-induced accumulation of VCAM1 transcript requires protein synthesis. We report the definition of a functional regulatory element in the VCAM1 promoter interacting with the transcriptional activator interferon regulatory factor 1 (IRF-1). DNA-protein binding studies with endothelial nuclear extracts revealed that IRF-1 is cytokine inducible and binds specifically to a consensus sequence motif located 3' of the TATA element. We have identified heterodimeric p65 and p50 as the NF-kappa B species binding to the VCAM1 promoter in TNF-alpha-activated endothelial cells. Experiments with recombinant proteins showed that p50/p65 and high-mobility-group I(Y) protein cooperatively facilitated the binding of IRF-1 to the VCAM1 IRF binding site and that IRF-1 physically interacted with p50 and with high-mobility-group I(Y) protein. Transient transfection assay in endothelial cells showed that overexpressed IRF-1 resulted in superinduction of TNF-alpha-stimulated transcription. Site-directed mutations in the IRF binding element decreased TNF-alpha-induced activity and totally abolished superinduction. Cotransfection assays in P19 embryonal carcinoma cells revealed that IRF-1 synergized with p50/p65 NF-kappa B to activate the VCAM1 promoter or heterologous promoter constructs bearing isolated VCAM1 NF-kappa B and IRF binding motifs. Cytokine inducibility of VCAM1 in endothelial cells utilizes the interaction of heterodimeric p50/p65 proteins with IRF-1.

Assessment of the stability of proanthocyanidins and other phenolic compounds in cranberry syrup after gamma-irradiation treatment and during storage.

Shelf life of commercial cranberry syrup irradiated with gamma radiation at a rate of 5 kGy and stored for 6 months at 25 °C and 60% relative humidity (RH) and under accelerated stability conditions was investigated. High-performance liquid chromatography coupled to electrospray ionisation quadrupole-time-of-flight mass spectrometry (HPLC-ESI-QTOF-MS) was used to characterise cranberry syrup. Afterwards, these compounds were quantified by HPLC-ESI-QTOF-MS and 4-dimethylaminocinnamaldehyde (DMAC) assay. A significant increase in the content of procyanidin B isomer 1 (from 4.4 to 7.0 µg/ml) and procyanidin A2 (from 83 to 93 µg/ml) was observed after irradiation and compared with the non-irradiated syrup. Procyanidin B isomers and prodelphinidin were stable at 25 °C during the first month of storage, whereas quercetin and some derivatives remained constant for 3 months of storage at this temperature. In short, after gamma-irradiation in dose of 5 kGy, most compounds were highly stable for a month at 25 °C.

Involvement of two regulatory elements in interferon-gamma-regulated expression of human indoleamine 2,3-dioxygenase gene.

Interferon (IFN)-gamma-induced expression of indoleamine 2,3-dioxygenase (IDO) gene is implicated in the antimicrobial and antiproliferative effects of IFN-gamma in cell cultures. Earlier studies identified a 96 base pair (bp) regulatory region upstream of the IDO gene that conferred IFN-gamma response to the chloroamphenicol acetyltransferase (CAT) gene linked to herpesvirus thymidine kinase promoter. The IFN-gamma-responsive region was further narrowed to a 67 bp fragment by 3' deletion. This 67 bp fragment contains several sequence elements of potential interest, including a 14 bp sequence homologous to the ISRE sequence found in IFN-alpha-inducible genes and two palindromic sequences (PE I and PE II) homologous to the GAS sequence identified in IFN-gamma-inducible genes. Site-directed mutagenesis studies showed that IFN-gamma-induced expression of IDO-CAT constructs involved cooperation between two elements: the ISRE homolog and the PE II (but not PE I). Either element alone with its flanking sequence was inadequate in conferring an IFN-gamma response to CAT reporter gene. Two IFN-gamma-regulated protein factors interacting with these two elements were identified. The factor binding to the ISRE region was induced with a slower kinetics, required new protein synthesis, and reacted with antibodies to IRF-1. The factor interacting with the PE II region appeared rapidly after treatment with IFN-gamma independently of new protein synthesis, and its binding to DNA probe was blocked by antibodies to p91 factor, reported to bind to GAS element.(ABSTRACT TRUNCATED AT 250 WORDS)

Determinants of organ malfunction or death in patients with intra-abdominal sepsis. A discriminant analysis.

One hundred and six patients found at operation to have intra-abdominal sepsis were prospectively followed up to determine the incidence of organ malfunction and death. These outcomes were correlated with age, preexisting disease, underlying cause of sepsis, shock, nutritional status, and alcoholism. Organ malfunction occurred in 31 patients (29%), 19 (61%) of whom died. Two (3%) of 75 patients without organ malfunction died. Discriminant analysis revealed a significantly increased risk of death in patients with shock at any time, age greater than 65 years, alcoholism, bowel infarction, or malnutrition. A discriminant equation based only on preoperative variables correctly assigned the outcome of death or survival in 97 (92%) of the patients based on probabilities derived from this analysis. At present, this information is primarily of interest for researchers comparing outcomes in groups of patients, but with additional refinements it may become clinically useful for individual patients.

Distribution characteristics of mitoxantrone in a patient undergoing hemodialysis.

The pharmacokinetic profile of mitoxantrone in a patient undergoing hemodialysis is described. Significant characteristics of our patient included lymphoma with liver involvement, tumor lysis syndrome, renal and hepatic failure. Combination chemotherapy consisted of mitoxantrone, vincristine, and cyclophosphamide. Mitoxantrone plasma samples were obtained prior to dosing and at 0, 0.25, 0.5, 0.75, 1.0, 1.5, 2.0, 2.5, 3.0, 3.5, 4.0, 4.5, 5.5, 7.0, and 12 h after the intravenous infusion of a 17-mg dose over 20 min. Serum concentrations were determined by high-performance liquid chromatography. The serum concentration versus time curve was consistent with a three-compartment model. However, rebounds in serum drug concentrations were detected during the last portion of dialysis and after its completion. The gamma elimination half-life could not be determined due to the continued detection of rebounds in drug concentrations throughout the postdialysis sampling period. The alpha and beta distribution phases did not appear to be affected by hemodialysis. The peak mitoxantrone concentration fell within the reported range. Mitoxantrone does not appear to be eliminated by hemodialysis, and dose adjustments are not needed in patients undergoing this procedure.

Control of postcarotid endarterectomy hypotension with baroreceptor blockade.

The reported incidence of hypotension after carotid reconstruction ranges from 21 to 50 percent. To evaluate baroreceptor dysfunction, 283 carotid reconstructions were studied. During 181 consecutive reconstructions, a transcutaneous Teflon catheter was positioned intraoperatively at the carotid bifurcation for postoperative injection of lidocaine as necessary. Of 283 reconstructions, 210 (74 percent) were not followed by hypotension. In the remaining 73 (26.5 percent), hypotension (systolic blood pressure less than 100 mm Hg) was seen. In 14 of the 73 cases of hypotension, vasoactive drugs, atropine, or a fluid bolus was administered with variable responses. In 27 of the 73, no treatment was instituted. In the remaining cases of hypotension, treatment consisted of 1 to 2 ml of lidocaine administered through the catheter. A prompt increase in systolic blood pressure from 93 to 139 mm Hg (means) within 30 minutes of treatment occurred in 29 of the 32 cases (91 percent). These data currently support routine catheter placement.

Early surgical excision versus conventional therapy in patients with 20 to 40 percent burns. A comparative study.

Using the records of 72 patients treated at the University of Washington Burn Center, this study compared the results of early surgical excision (by 14 days postburn) and autografting to those of autografting after spontaneous separation and bedside debridement of burn eschar. Excised patients had shorter hospitalizations and lower rates of burn wound sepsis and serious burn wound contamination, and less use of potentially toxic antibiotics (p less than 0.05) than did the prognostically equivalent group treated before the introduction of early excision. Excised patients required more blood transfusions (p less than 0.05), but did not differ significantly from controls in rates of mortality or other inpatient complications, in the number of operations performed, or in the adjusted hospital costs. Evaluation of patients treated over the entire study period for more shallow burns indicated no concurrent change in other aspects of burn care which might account for the observed results. We conclude that early excision and grafting in young, otherwise healthy patients with 20 to 40 percent total body surface area burns that are likely to heal within 3 weeks is more effective than the more traditional management of slow wound separation and debridement.

Postburn delirium associated with use of intravenous lorazepam.

A case of delirium with persecutory delusions is reported, which occurred three days after a previously healthy male suffered burns over 30% of his total body surface area. Routine evaluation was negative for other etiology of delirium, and the symptoms spontaneously remitted within seven hours with no sequella. We found that the benefits and potential side effects of intravenous lorazepam are similar to but of longer duration than those of intravenous diazepam. The effects of intravenous lorazepam should be recognized in the differential diagnosis of postburn delirium in patients given this medication.

Seeking a stable future: perspectives on population policy. The legal approach: women's rights as human rights.

PIP: As it has grappled with issues of population policy, the international community has emphasized that women's reproductive rights are human rights. Scholars have also acknowledged that the right to reproductive health care exists within the scope of international human rights treaties and conventions and that gender equality, nondiscrimination, and freedom from government interference in marriage and family life are also guaranteed. Further protections extend to counseling and health information and referral. The Programme of Action of the 1994 International Conference on Population and Development continues this trend by emphasizing the importance of human rights for attaining population and development objectives, calling on governments to focus their efforts on improving the quality of life for individuals, and endorsing the notion that reproductive rights are universal human rights. Reproductive health care options are also influenced by sovereign laws that restrict availability of contraception, sterilization, or abortion. However, universal rights and unrestricted access must be complemented by other factors controlled by domestic laws to guarantee reproductive choice. Such laws cover issues like marriage age, divorce, marital property, child support, maternity benefits, day care, sex discrimination, eligibility for insurance, confidentiality, spousal consent, rape, and sexual abuse. Countries must modify restrictive national laws and promote laws protecting women's rights.^ieng

Phenylpropanoids and their metabolites are the major compounds responsible for blood-cell protection against oxidative stress after administration of Lippia citriodora in rats.

Lippia citriodora (lemon verbena) has been widely used in folk medicine for its pharmacological properties. Verbascoside, the most abundant compound in this plant, has protective effects associated mostly with its strong antioxidant activity. The purpose of this study was to test the effect of L. citriodora extract intake on the antioxidant response of blood cells and to correlate this response with the phenolic metabolites found in plasma. For this purpose, firstly the L. citriodora extract was characterized and its radical scavenging activity was measured by 2,2-diphenyl-1-picrylhydrazyl (DPPH) assay. Then, catalase (CAT), glutathione peroxidase (GPx), and glutathione reductase (GRed) activities were determined in lymphocytes, erythrocytes, and neutrophils isolated from rats after acute intake of L. citriodora. Phenolic metabolites were analyzed in the same plasma samples by HPLC-ESI-TOF-MS. Myeloperoxidase (MPO) activity in neutrophils, which has been proposed as a marker for inflammatory vascular damage, was also determined. After L. citriodora administration, the antioxidant enzymes activities significantly accelerated (p<0.05) while MPO activity subsided, indicating that the extract protects blood cells against oxidative damage and shows potential anti-inflammatory and antiatherogenic activities. The main compounds found in plasma were verbascoside and isoverbascoside at a concentration of 80±10 and 57±4 ng/ml, respectively. Five other metabolites derived from verbascoside and isoverbascoside were also found in plasma, namely hydroxytyrosol, caffeic acid, ferulic acid, ferulic acid glucuronide, and homoprotocatechuic acid, together with another eight phenolic compounds. Therefore, the phenylpropanoids verbascoside and isoverbascoside, as well as their metabolites, seem to be the responsible for the above-mentioned effects, although the post-transcriptional activation mechanism of blood-cell antioxidant enzymes by these compounds needs further investigation.

A metabolite-profiling approach allows the identification of new compounds from Pistacia lentiscus leaves.

Pistacia lentiscus L., commonly known as Mastic tree or lentisk, is a Mediterranean evergreen shrub widely used in traditional medicine to treat such diseases as eczema, diarrhoea, and throat infections. Furthermore, other properties are currently attributed to P. lentiscus, such as antioxidant capacity, hepatoprotective action, and anti-inflammatory effects. High-performance liquid chromatography with diode array coupled to electrospray ionization mass spectrometry (HPLC-ESI-QTOF-MS) was used for the comprehensive characterization of methanol extract from P. lentiscus leaves. After the optimisation of the HPLC-ESI-QTOF-MS method and the use of the negative ionization mode, 46 different compounds were identified, 20 of which were tentatively characterized for the first time in P. Lentiscus leaves. The majority of the compounds were quantified. Flavonoids, phenolic acids and their derivatives were the most abundant compounds, those with the highest concentrations being myricetin glycoside (6216.13 mg/kg of plant), catechin (3354.78 mg/kg of plant), ß-glucogallin (2214.461 mg/kg of plant), and quercitrin gallate (1160 mg/kg of plant). The importance of the knowledge of plants is increasing and our study may help in the future to formulate nutraceutical preparations and will provide the basis for new investigation into activities of the various compounds found in P. lentiscus.

A metabolite-profiling approach to assess the uptake and metabolism of phenolic compounds from olive leaves in SKBR3 cells by HPLC-ESI-QTOF-MS.

Olive leaves, an easily available natural low-cost material, constitute a source of extracts with significant antitumor activity that inhibits cell proliferation in several breast-cancer-cell models. In this work, a metabolite-profiling approach has been used to assess the uptake and metabolism of phenolic compounds from an olive-leaf extract in the breast-cancer-cell line SKBR3 to evaluate the compound or compounds responsible for the cytotoxic activity. For this, the extract was firstly characterized quantitatively by high-performance liquid chromatography coupled to electrospray ionization-quadrupole time-of-flight mass spectrometry (HPLC-ESI-QTOF-MS). Then, SKBR3 cells were incubated with 200 µg/mL of the olive-leaf extract at different times (15 min, 1, 2, 24, and 48 h). A metabolite-profiling approach based on HPLC-ESI-QTOF-MS was used to determine the intracellular phenolic compounds, enabling the identification of 16 intact phenolic compounds from the extract and four metabolites derived from these compounds in the cell cytoplasm. The major compounds found within the cells were oleuropein, luteolin-7-O-glucoside and its metabolites luteolin aglycone and methyl-luteolin glucoside, as well as apigenin, and verbascoside. Neither hydroxytyrosol nor any of its metabolites were found within the cells at any incubation time. It is proposed that the major compounds responsible for the cytotoxic activity of the olive-leaf extract in SKBR3 cells are oleuropein and the flavones luteolin and apigenin, since these compounds showed high uptake and their antitumor activity has been previously reported.

Application of nanoLC-ESI-TOF-MS for the metabolomic analysis of phenolic compounds from extra-virgin olive oil in treated colon-cancer cells.

Crude phenolic extracts (PE) have been obtained from naturally bearing Spanish extra-virgin olive oil (EVOO) showing different polyphenol families such as secoiridoids, phenolic alcohols, lignans, and flavones. EVOO-derived complex phenols (especially from the Arbequina variety olive) have been shown to suppress cell growth of SW480 and HT29 human colon adenocarcinoma cell lines. Inhibition of proliferation by EVOO-PE Arbequina variety extract was accompanied by apoptosis in both colon-cancer-cell lines and a limited G2M cell-cycle arrest in the case of SW480 cells. The metabolized compounds from EVOO-PE in culture medium and cytoplasm of both cell lines were analyzed using nano-liquid chromatography (nanoLC) coupled with electrospray ionization-time-of-flight-mass spectrometry (ESI-TOF-MS). The results showed many phenolic compounds and their metabolites both in the culture medium as well as in the cytoplasm. The main compounds identified from EVOO-PE were hydroxylated luteolin and decarboxymethyl oleuropein aglycone.