Bradykinin mediated gastrointestinal edema as a cause of abdominal attacks in patients with hereditary angioedema due to C1-inhibitor deficiency.

Hereditary angioedema (HAE) due to C1-inhibitor (C1-INH) deficiency is a rare hereditary disease characterized by recurrent subcutaneous or submucosal angioedema due to uncontrolled bradykinin production caused by C1-INH dysfunction. Submucosal gastrointestinal swellings provoking abdominal attacks are common and mimic acute abdomen, thus constituting a diagnostic challenge. We aimed to investigate the difficulties in diagnosing abdominal attacks in patients with C1-INH-HAE and to assess the diagnostic value of medical history, the course of the attack, abdominal imaging, and treatment efficacy. The retrospective analysis of diagnostic problems and treatment complications of abdominal attacks in 274 patients with C1-INH-HAE were performed. The value of history, laboratory findings, prodromal symptoms and course of attacks and imaging were assessed. Abdominal attacks were confirmed in 274 of the 322 patients (85%; 190 women and 84 men; age, 4-70 years). In 49% of cases, the abdominal attack was the first and the only symptom for years. The simultaneous presence of marginal erythema (45% of cases), subcutaneous edema (30%), and pharyngo-laryngeal edema (10%) facilitated the diagnosis of an abdominal attack due to C1-INH-HAE. Abdominal attacks manifested with recurrent acute abdominal symptoms lasting 2 to 5 days. The disease course was characterized by the phase of progressive prodromal symptoms followed by peak symptoms and spontaneous symptom resolution. Abdominal imaging often revealed abundant ascites and limited bowel edema. In 60 cases (22%), the diagnostic difficulties resulted in exploratory laparotomy, which was inconclusive in 48 patients (80%). The attacks usually subsided within 2 hours from the administration of recommended drugs (plasma-derived C1-INH, recombinant C1-INH or icatibant). We conclude that recurrent abdominal attacks lasting a few days and resolving spontaneously were common symptoms of C1-INH-HAE. Abdominal imaging revealed transitional fluid or bowel edema. The effectiveness of recommended drugs as plasma-derived C1-INH, recombinant C1-INH or icatibant confirmed the diagnosis.

Effect of small molecules on cell reprogramming.

The essential idea of regenerative medicine is to fix or replace tissues or organs with alive and patient-specific implants. Pluripotent stem cells are able to indefinitely self-renew and differentiate into all cell types of the body which makes them a potent substantial player in regenerative medicine. The easily accessible source of induced pluripotent stem cells may allow obtaining and cultivating tissues in vitro. Reprogramming refers to regression of mature cells to its initial pluripotent state. One of the approaches affecting pluripotency is the usage of low molecular mass compounds that can modulate enzymes and receptors leading to the formation of pluripotent stem cells (iPSCs). It would be great to assess the general character of such compounds and reveal their new derivatives or modifications to increase the cell reprogramming efficiency. Many improvements in the methods of pluripotency induction have been made by various groups in order to limit the immunogenicity and tumorigenesis, increase the efficiency and accelerate the kinetics. Understanding the epigenetic changes during the cellular reprogramming process will extend the comprehension of stem cell biology and lead to potential therapeutic approaches. There are compounds which have been already proven to be or for now only putative inducers of the pluripotent state that may substitute for the classic reprogramming factors (Oct3/4, Sox2, Klf4, c-Myc) in order to improve the time and efficiency of pluripotency induction. The effect of small molecules on gene expression is dosage-dependent and their application concentration needs to be strictly determined. In this review we analysed the role of small molecules in modulations leading to pluripotency induction, thereby contributing to our understanding of stem cell biology and uncovering the major mechanisms involved in that process.

Optimizing of MALDI-ToF-based low-molecular-weight serum proteome pattern analysis in detection of breast cancer patients; the effect of albumin removal on classification performance.

Mass spectrometry-based analysis of the serum proteome allows identifying multi-peptide patterns/signatures specific for blood of cancer patients, thus having high potential value for cancer diagnostics. However, because of problems with optimization and standardization of experimental and computational design, none of identified proteome patterns/signatures was approved for diagnostics in clinical practice as yet. Here we compared two methods of serum sample preparation for mass spectrometry-based proteome pattern analysis aimed to identify biomarkers that could be used in early detection of breast cancer patients. Blood samples were collected in a group of 92 patients diagnosed at early (I and II) stages of the disease before the start of therapy, and in a group of age-matched healthy controls (104 women). Serum specimens were purified and analyzed using MALDI-ToF spectrometry, either directly or after membrane filtration (50 kDa cut-off) to remove albumin and other large serum proteins. Mass spectra of the low-molecular-weight fraction (2-10 kDa) of the serum proteome were resolved using the Gaussian mixture decomposition, and identified spectral components were used to build classifiers that differentiated samples from breast cancer patients and healthy persons. Mass spectra of complete serum and membrane-filtered albumin-depleted samples have apparently different structure and peaks specific for both types of samples could be identified. The optimal classifier built for the complete serum specimens consisted of 8 spectral components, and had 81% specificity and 72% sensitivity, while that built for the membrane-filtered samples consisted of 4 components, and had 80% specificity and 81% sensitivity. We concluded that pre-processing of samples to remove albumin might be recommended before MALDI-ToF mass spectrometric analysis of the low-molecular-weight components of human serum Keywords: albumin removal; breast cancer; clinical proteomics; mass spectrometry; pattern analysis; serum proteome.

Evaluation of glycosylation and malonylation patterns in flavonoid glycosides during LC/MS/MS metabolite profiling.

Flavonoid conjugates constitute several classes of plant phenolic secondary metabolites including many isomeric compounds differing in the hydroxylation pattern and substitution of their rings with different groups such as alkyls, acyls or sugars. These compounds occur in plant tissues mainly as glycosides and in many cases it is necessary to have reliable and detailed information concerning the structure of these natural products. Our results were obtained using leaf extracts of Arabidopsis thaliana and Lupinus angustifolius in which different glycosides of flavones, flavonols and isoflavones are present. Analysis of collision-induced dissociation (CID)/MS/MS spectra of protonated [M + H](+), sodiated [M + Na](+) or deprotonated [M - H](-) molecules recorded during HPLC runs may bring needed information in this respect. However, registration of mass spectra of [M + Na](+) ions with a good efficiency is possible only after post-column addition of a sodium acetate solution to the LC column eluate. The retention of sodium cation on the saccharidic parts of the molecule is observed after the CID fragmentation. In many cases, the location of this cation on the glycan attached to C-3 hydroxyl group of flavonol led to assignment of its structure. Additionally, the determination of the structure of the aglycone and of the sequence of the glycan part was made possible through the CID data obtained from the [M + H](+) and [M - H](-) ions. CID spectra show a different order of sugar elimination from hydroxyl groups at C-3 and C-7 in flavonol glycosides isolated from A. thaliana leaves and give sufficient information to discriminate flavonoid O-diglycosides from flavonoid di-O-glycosides.

Profiling isoflavone conjugates in different organs of Lupinus exaltatus Zucc.

The profiles of isoflavone conjugates in extracts obtained from different parts of Lupinus exaltatus Zucc. grown in Mexico were compared using HPLC-UV and HPLC-ESI/MSn. Collision-induced dissociation-MSn experiments were performed using an ion trap analyser during HPLC-ESI/MS analyses. Nineteen isoflavone conjugates were identified in samples obtained from air-dried roots, leaves, stems and inflorescences of lupin plants. It was possible to determine the structures of the studied compounds on the basis of the MS recorded. The compounds identified were di- and mono-glucosides of genistein and 2'-hydroxygenistein with a different pattern of C- and O-glycosylation. Some glucosides were acylated with malonic acid. It was not possible to establish the glycosylation sites on the basis of MS alone; however, it was possible to differentiate isoflavone C- and O-glucosides. The highest levels of isoflavones and their conjugates were detected in roots and the lowest in stems. Free aglycones were identified in roots and inflorescences but they were not found in stems and leaves.

Secondary metabolites in in vitro cultured plants of the genus Drosera.

Extracts from plantlets of different species of the genus Drosera, grown as in vitro cultures, were evaluated for the level of phenolic secondary metabolites from the group of naphthoquinones and flavonols. The profiles of natural products in the extracts obtained from different species were monitored by HPLC with UV detection at 260 and 330 nm. On the basis of the data obtained, Drosera binata, the species with the highest amount of plumbagin, was selected for further studies. The most effective method of extraction of quinones was established and the composition of phenolic secondary metabolites in the tissues was determined. For the identification of phenolic compounds, HPLC-UV and HPLC-ESI/MS were applied.

Profiling of flavonoid conjugates in Lupinus albus and Lupinus angustifolius responding to biotic and abiotic stimuli.

Qualitative and quantitative composition of flavonoid and isoflavonoid glycosides as well as free aglycones in lupin seedlings (roots and aerial parts) grown under different light conditions or responding to infection with Pleiochaeta setosa, a fungus causing brown leaf spot, were monitored by liquid chromatography with UV and/or mass spectrometric detection. Both physical and biotic factors affected flavonoid and isoflavonoid levels in lupin tissues. Fungal infection evoked significant increase in the amounts of genistein, 2'-hydroxygenistein, and their prenylated derivatives that are thought to function as lupin phytoalexins. Effect on quantitative changes of glycosylated flavonoids and isoflavonoids in the roots and aerial parts was less significant. Moreover, different light conditions applied during seedling growth caused relative changes of flavonoid and isoflavonoid conjugates composition, especially in the leaves of white lupin plants. The chemical structures of flavonoid and isoflavonoid conjugates present in Lupinus angustifolius were elucidated. In addition to genistein and 2'-hydroxygenistein glycosides, flavonol conjugates were identified in leaves, while the composition of root isoflavonoids was similar to that of L. albus reported earlier.

Profiling changes in metabolism of isoflavonoids and their conjugates in Lupinus albus treated with biotic elicitor.

Liquid chromatography with ultraviolet and mass spectrometric detection was applied to monitor changes in profiles of isoflavonoid glycosides and free isoflavonoid aglycones in Lupinus albus L. Four isoflavonoid aglycones, fourteen isoflavonoid glycosides, four flavonol glycosides and flavone glycoside were identified in lupin tissue after LC/ESI/MS analyses. An elicitor preparation from purified yeast cell wall was used to inject the shoots of 3-week old seedlings or to infiltrate the cut lupin leaves. Qualitative and quantitative changes of isoflavonoids were measured at different time points after elicitation. In elicited lupin seedlings increased amounts of prenylated isoflavone aglycones were identified. The concentrations of glycosidic conjugates of isoflavones present in plant tissue were less affected.

Identification of 6-furfuryladenine (kinetin) in human urine.

In contrast to the current view of kinetin (K, N(6)-furfuryladenine) as an unnatural and synthetic cytokinin, recently it has been identified in plant DNA and plant extract. Here we describe identification of K in human urine using chromatography/mass-spectrometry analysis for the first time. The amount of kinetin in urine taken from unhealthy patients lung carcinoma was established to be 0.5 ng in 20 ml and a 100-fold reduced amount in healthy subjects. Since this rare base is a potential source of structural constrains it has to be removed from DNA by enzymatic DNA-repair reactions. It seems that the presence of kinetin in human is linked to oxidative damage processes.

Identification of photodegradation products of nilvadipine using GC-MS.

Nilvadipine (NV) photodegradation products have been analysed with gas chromatography-mass spectrometry (GC-MS). The photodegradation was carried out in the conditions recommended in the first version of the document issued by the International Conference on Harmonization (ICH), currently in force in the studies of photochemical stability of drugs and therapeutic substances. Methanol solutions of NV were irradiated with a high-pressure UV lamp - type HBO 200. The maximum intensity at the wavelength lambda = 365 nm was achieved by applying the interference filter and Wood's filter. Using the Reinecke salt as a chemical actinometer, apparent quantum yields of photodegradation were obtained, which after extrapolation to the zero time of irradiation gave the actual quantum yield ((phi = 7.58 x 10(-5). The structure of three nilvadipine photodegradation products was established, after mass spectra analysis of compounds registered during GC-MS carried out of irradiated nilvadipine solutions. The quantitative results of GC-MS analyses enabled to determination of the kinetic parameters of NV photodegradation, calculated from the dependence In c =f(t). Quantitatively the process was described with the calculated rate constant of decomposition (k), decomposition time of 50% of the compound (t0.5) and decomposition time of 10% of the compound (t0.1). The exposure of nilvadipine to UV light was found to lead to aromatization of the DHP ring and elimination of the HCN molecule.

Application of mass spectrometry for identification and structural studies of flavonoid glycosides.

Mass spectrometry is an important tool for the identification and structural determination of flavonoid glycosides. The advantages of mass spectrometry are high sensitivity and possibilities of hyphenation with liquid chromatographic methods for the analysis of mixtures of compounds. Different desorption ionization methods allow the analysis of underivatized glycosides. A review of mass spectrometric techniques applied to the identification and structural studies of flavonoid glycosides is presented.

Tandem solid-phase extraction of atrazine ozonation products in water.

The preconcentration of aqueous solutions containing atrazine degradation products was investigated using solid-phase extraction on octadecyl and cation-exchanger silica phases. The retention and elution steps were studied and evaluated separately in order to define and optimize the critical experimental parameters involved. A strategy which combines sequentially both phases is proposed to fractionate compounds into two groups of increasing polarities: firstly, the native pesticide, hydroxyatrazine and most chlorotriazines on octadecyl support, and secondly monodealkylated hydroxytriazines, ammeline and ammelide on cation-exchanger. This tandem procedure was successfully applied for analysing and quantifying atrazine ozonation products and its efficiency demonstrated using [U-ring 14C]-labelled atrazine experiments.

Application of mass spectrometry to structural identification of flavonoid monoglycosides isolated from shoot of lupin (Lupinus luteus L.).

Flavonoid glycosides constitute important group of plant secondary metabolites. This class of natural products play significant role in different physiological processes. A new methodological approach where mass spectrometric techniques are applied to structural studies of this class of compounds is presented. Four flavonoid O-monoglycosides and one C-monoglycoside were isolated from green parts of lupin (Lupinus luteus L.). Several different mass spectrometric techniques were applied to structural elucidation of isolated compounds. Desorption ionization mass spectrometry was used for registration of mass spectra of intact and derivatized (permethylated) flavonoid glycosides. In some cases electron impact mass spectra of permethylated compounds were also recorded. Methylated samples after methanolysis and further derivatization of free hydroxyl groups (methylation or acetylation) were analyzed with gas chromatography-mass spectrometry. Combined information drawn from the registered mass spectra enabled us to define molecular mass, structure of aglycones and sugars, and positions of glycosidic bonds on the aglycon. Structures of four flavonoid monoglycosides were elucidated as follows: genistein 7-O-glucoside (1), genistein 4'-O-glucoside (2), 2'-hydroxygenistein 7-O-glucoside (3), and apigenin or genistein 8-C-glycoside (5). For the fourth O-glycoside (4) only molecular mass and masses of the aglycone and sugar were estimated.

Stability of diclofenac sodium in the inclusion complex with beta-cyclodextrin in the solid state.

The aim of this study was to characterize the thermal stability of diclofenac sodium both alone and in the inclusion complex with beta-cyclodextrin in the solid state, by determination of the number of the products of its decomposition, which were identified by GC-MS. The molar ratio of diclofenac sodium in the inclusion complex with beta-cyclodextrin was 1:1. The decomposition of diclofenac sodium both alone and in inclusion complex with beta-cyclodextrin occurred according to the first-order reaction. The HPLC of the samples thermostated at 80 degrees C gave five products of decomposition, which were identified by GC-MS. Diclofenac sodium in the inclusion complex with beta-cyclodextrin was more thermally stable. Thermal decomposition of diclofenac sodium leads to formation of five products, of which 4-chloro-10H-9-acridinone had not been reported previously in the literature.

[Latex allergy in health services workers]. / Alergia na lateks u pracowników sluzby zdrowia.

The studies were performed in 20 workers from the Health Service (13 women and 7 men in the age 25-57) suffering from hand urticaria (6 persons) and hand dermatitis (14 persons) suspected of the allergy to latex gloves. In all patients the familial and personal predispositions to allergy were evaluated by the anamnesis, the estimation of total IgE serum level and the skin prick tests (SPT) with inhalant allergens. The latex allergy was diagnosed by SPT and contact test with standardized extract of the natural latex allergen in the concentration 1000 PNU/ml (Nexter-Allergopharma) and by estimation of specific to latex IgE serum level. In addition to this, contact tests with glove's material as well European standard contact allergens (Hermal) were done and the one with antiseptic substances to which the patient was exposed at his work. The allergy type I to latex gloves was confirmed in all 6 cases with contact urticaria. The SPT with standard extract of the natural latex was more valuable than latex specific IgE in the serum. Contact allergy (type IV) to latex gloves was confirmed in 10 from 14 suspected cases. In the next 4 the allergy to antiseptic substances was the reasons of the illness. The allergy to latex gloves appears more often in women. No case showed the familial predisposition to allergy and only 4 patients additionally suffered from the allergy to pollen and mites. Moreover in both groups of patients we showed the presence of the additional contact allergy to different allergens (to metals and antiseptic substances).

Secretion of stress-related proteins by suspension-cultured Lupinus albus cells.

A suspension culture of white lupin cells has been established, and proteins of the exocellular matrix analysed. Based on homologies of N-terminal amino-acid sequences, three stress- or defence-related proteins: acidic class III chitinase, polygalacturonase-inhibiting protein, and germin/oxalate oxidase, secreted by lupin cell culture, were identified.

Immunotropic activity of lupin seeds extracts and fractions from Lupinus angustifolius and Lupinus albus.

The results demonstrated immunotropic activity of seeds extracts and fractions from Lupinus angustifolius and Lupinus albus. Plaque forming cells (PFC) number to SRBC (sheep red blood cells) were elevated by an extract from Lupinus angustifolius and lowered by extracts from Lupinus albus. All preparations obtained from Lupinus angustifolius reduced the number of rosette forming cells (E-RFC). These preparations suppressed also the intensity of graft versus host reaction (GVHR) in case when the donors were treated. Lupinus albus extract suppressed GVH reaction when recipients were treated. Lupin extracts stimulated draining popliteal lymph nodes in PLN assay.

The toxicity of seed extracts and their fractions from Lupinus angustifolius L. and Lupinus albus L.

Seed extracts obtained from Lupinus albus and Lupinus angustifolius by treatment with 48% ethanol contained ca. 10% alkaloids (on a dry weight basis) and were non-toxic. Their acute toxicity (LD50) in the mouse is > 4000 mg kg-1 body wt. After fractionation, the extract from L. angustifolius seeds afforded several fractions with differing toxicities (LD50 750-4000 mg kg-1 body wt.). None of the fractions tested in vitro were toxic. The results obtained showed that, in spite of the alkaloids, other low-molecular-weight constituents present significantly modified the toxicity of the lupin extracts.

Phenolic compounds isolated from bitter lupine seeds and their inhibitory effects on germination and seedling growth of lettuce.

The phenolic acids, including 4-hydroxybenzoic and 4-hydroxycinnamic acids, and their derivatives, such as 6,7-dihydroxycoumarin and 1,2-dihydroxybenzene, were isolated from bitter lupine seeds and were identified using gas chromatography-mass spectrometry. These compounds inhibited lettuce seed germination in the first 24 hr after sowing, but after 72 hr germination was comparable with that of the control. However, very strong suppression of seedling growth, especially the roots, was observed for higher concentrations of the lupine seed fractions containing phenolic acids. Effects observed in the lettuce germination bioassays were compared with those produced by nine pure phenolic acids previously identified in the mixture. The ethyl acetate fraction from lupine seed extract inhibited seedling growth as effectively as pure 1,2-dihydroxybenzene, the strongest inhibitor of the pure phenolic compounds studied. The possible reason for this could be the synergistic effect created in the mixture of phenolic compounds isolated from the extract.

Urinary organic acid profiles in fatty Zucker rats: indications for impaired oxidation of butyrate and hexanoate.

The urinary excretion of 45 organic acids, monitored by gas-liquid chromatography, was compared in fatty (fa/fa) and lean (Fa/?) Zucker rats maintained on a chemically simplified diet. At the age of 6, 16, and 22 weeks, fatty rats excreted more of the various organic acids than their lean counterparts. However, the greatest difference was in the excretion of ethylmalonate, even when excretion data were normalized to body weight. The next highest excretion difference was in adipate and an unknown compound, and the third highest in pyruvate. A second group of rats examined at 7 weeks also excreted an excess of these four acids, as well as glucuronate and indole-3-acetate. The excessive excretion of ethylmalonate and adipate, which is characteristic of human genetic defects in short- and medium-chain fatty acid oxidation, suggested that the oxidation of butyrate and hexanoate might be impaired in the fatty rat. Thus, as a test of their capacity to oxidize medium- and short-chain fatty acids, two groups of fatty and lean rats were transferred to diets enriched with either trioctanoylglyceride, a medium-chain triglyceride (MCT), or sodium butyrate, a short-chain fatty acid. Both lean and fatty rats on the MCT diet, but only the lean rats on the butyrate-enriched diet, increased their excretion of adipate. However, on both the MCT and butyrate diet, ethylmalonate excretion increased only in lean rats, almost reaching amounts found previously in fatty rats. These results suggest that the fatty rat has an impairment of the beta-oxidation of butyrate and hexanoate, a defect that might increase intracellular concentrations of butyryl-CoA, the optimal primer for the synthesis of long-chain fatty acids.