Pharmacokinetics of isofraxidin in rat plasma after oral administration of the extract of acanthopanax senticosus using HPLC with solid phase extraction method

High-performance liquid chromatography coupled with solid phase extraction method was developed for determination of isofraxidin in rat plasma after oral administration of Acanthopanax senticosus extract (ASE), and pharmacokinetic parameters of isofraxidin either in ASE or pure compound were measured. The HPLC analysis was performed on a Dikma Diamonsil RP(18) column (4.6 mm x 150 mm, 5 microm) with the isocratic elution of solvent A (acetonitrile) and solvent B (0.1% aqueous phosphoric acid, v/v) (A : B = 22 : 78) and the detection wavelength was set at 343 nm. The calibration curve was linear over the range of 0.156-15.625 microg/ml. The limit of detection was 60 ng/ml. The intra-day precision was 5.8%, and the inter-day precision was 6.0%. The recovery was 87.30+/-1.73%. When the dosage of ASE is equal to pure compound caculated by the amount of isofraxidin, it has been found to have two maximum concentrations in plasma while the pure compound only showed one peak in the plasma concentration-time curve. The determined content of isofraxidin in plasma after oral administration of ASE is the total contents of free isofraxidin and its precursors in ASE in vitro. The pharmacokinetic characteristics of ASE showed the priority of the extract and the properities of traditional Chinese medicine.

Development and validation of a ultra performance LC-ESI/MS method for analysis of metabolic phenotypes of healthy men in day and night urine samples

Ultra-performance LC coupled to quadrupole TOF/MS (UPLC-QTOF/MS) in positive and negative ESI was developed and validated to analyze metabolite profiles for urine from healthy men during the day and at night. Data analysis using principal components analysis (PCA) revealed differences between metabolic phenotypes of urine in healthy men during the day and at night. Positive ions with mass-to-charge ratio (m/z) 310.24 (5.35 min), 286.24 (4.74 min) and 310.24 (5.63 min) were elevated in the urine from healthy men at night compared to that during the day. Negative ions elevated in day urine samples of healthy men included m/z 167.02 (0.66 min), 263.12 (2.55 min) and 191.03 (0.73 min), whilst ions m/z 212.01 (4.77 min) were at a lower concentration in urine of healthy men during the day compared to that at night. The ions m/z 212.01 (4.77 min), 191.03 (0.73 min) and 310.24 (5.35 min) preliminarily correspond to indoxyl sulfate, citric acid and N-acetylneuraminic acid, providing further support for an involvement of phenotypic difference in urine of healthy men in day and night samples, which may be associated with notably different activities of gut microbiota, velocity of tricarboxylic acid cycle and activity of sialic acid biosynthesis in healthy men as regulated by circadian rhythm of the mammalian bioclock.

Metabolite profiling and pathway analysis of acute hepatitis rats by UPLC-ESI MS combined with pattern recognition methods

BACKGROUND & AIMS Metabolomics is comprehensive analysis of low-molecular-weight endogenous metabolites in a biological sample. It could enable mapping of perturbations of early biochemical changes in diseases and hence provide an opportunity to develop predictive biomarkers that could provide valuable insights into the mechanisms of diseases. The aim of this study was to elucidate the changes in endogenous metabolites and to phenotype the metabolic profiling of d-galactosamine (GalN)-inducing acute hepatitis in rats by UPLC-ESI MS. METHODS The systemic biochemical actions of GalN administration (ip, 400 mg/kg) have been investigated in male wistar rats using conventional clinical chemistry, liver histopathology and metabolomic analysis of UPLC- ESI MS of urine. The urine was collected predose (-24 to 0 h) and 0-24, 24-48, 48-72, 72-96 h post-dose. Mass spectrometry of the urine was analysed visually and via conjunction with multivariate data analysis. RESULTS Results demonstrated that there was a time-dependent biochemical effect of GalN dosed on the levels of a range of low-molecular-weight metabolites in urine, which was correlated with developing phase of the GalN-inducing acute hepatitis. Urinary excretion of beta-hydroxybutanoic acid and citric acid was decreased following GalN dosing, whereas that of glycocholic acid, indole-3-acetic acid, sphinganine, n-acetyl-l-phenylalanine, cholic acid and creatinine excretion was increased, which suggests that several key metabolic pathways such as energy metabolism, lipid metabolism and amino acid metabolism were perturbed by GalN. CONCLUSION This metabolomic investigation demonstrates that this robust non-invasive tool offers insight into the metabolic states of diseases.

HPLC method for preliminary analysis of constituents in rat blood after oral administration of the extract of Acanthopanax senticosus

An HPLC with SPE method has been developed for analysis of constituents in rat blood after oral administration of the extract of Acanthopanax senticosus (ASE). The plasma sample was prepared by SPE method equipped with Oasis HLB cartridge (3cc, 60 mg). The analysis was performed on a Dikma Diamonsil RP(18) column (4.6 mmx150 mm, 5 microm) with the gradient elution of solvent A (ACN) and solvent B (0.1% aqueous phosphoric acid, v/v) and the detection wavelength was set at 270 nm. The calibration curve was linear over the range of 0.156-15.625 microg/mL. The LOD was 60 ng/mL. The intraday precision was less than 5.80%, and the interday precision was less than 6.0%. The recovery was (87.30 +/- 1.73)%. As a result, 19 constituents were detected in rat plasma after oral administration of the ASE, including 11 original compounds in ASE and eight metabolites, and three of the metabolites originated from syringin in ASE. Six constituents were identified by comparing with the corresponding reference compounds.

A rapid and sensitive UPLC-ESI MS method for analysis of isofraxidin, a natural antistress compound, and its metabolites in rat plasma

Isofraxidin is one of the main bioactive constituents in the root of Acanthopanax senticosus, which has antifatigue, antistress, and immuno-accommondating effects. In this study, an ultraperformance LC (UPLC)-ESI MS method was developed for analyzing isofraxidin and its metabolites in rat plasma. The analysis was performed on a UPLC coupled with ESI MS (quadropole MS tandem TOF MS). The lower LOD (LLOD) for isofraxidin was 0.25 ng/mL, the intraday precision was less than 10%, the interday precision was less than 10%, and the extraction recovery was more than 80%. Isofraxidin and two metabolites (M1 and M2) were detected in rat plasma after oral administration of isofraxidin, and the molecular polarities of M1 and M2 were both increased compared to isofraxidin. The metabolites were identified as 5,6-dihydroxyl-7-methoxycoumarin and 5-hydroxyl-6,7-dimethoxycoumarin when subjected to parent ion spectra, product ion spectra, and extract mass and element composition analyses.

Determination of urinary thymidine glycol using affinity chromatography, HPLC and post-column reaction detection : a biomarker of oxidative DNA damage upon kidney transplantation

Reactive oxygen species are generated during ischaemia-reperfusion of tissue. Oxidation of thymidine by hydroxyl radicals (HO) leads to the formation of 5,6-dihydroxy-5,6-dihydrothymidine (thymidine glycol). Thymidine glycol is excreted in urine and can be used as biomarker of oxidative DNA damage. Time dependent changes in urinary excretion rates of thymidine glycol were determined in six patients after kidney transplantation and in six healthy controls. A new analytical method was developed involving affinity chromatography and subsequent reverse-phase high-performance liquid chromatography (RP-HPLC) with a post-column chemical reaction detector and endpoint fluorescence detection. The detection limit of this fluorimetric assay was 1.6 ng thymidine glycol per ml urine, which corresponds to about half of the physiological excretion level in healthy control persons. After kidney transplantation the urinary excretion rate of thymidine glycol increased gradually reaching a maximum around 48 h. The excretion rate remained elevated until the end of the observation period of 10 days. Severe proteinuria with an excretion rate of up to 7.2 g of total protein per mmol creatinine was also observed immediately after transplantation and declined within the first 24 h of allograft function (0.35 + 0.26 g/mmol creatinine). The protein excretion pattern, based on separation of urinary proteins on sodium dodecyl sulphate-polyacrylamide gel electrophorosis (SDS-PAGE), as well as excretion of individual biomarker proteins, indicated nonselective glomerular and tubular damage. The increased excretion of thymidine glycol after kidney transplantation may be explained by ischaemia-reperfusion induced oxidative DNA damage of the transplanted kidney.

Determination of the ethylene oxide adduct S-(2-hydroxyethyl)cysteine by a fluorometric HPLC method in albumin and globin from human blood

A new method has been developed for the quantification of 2-hydroxyethylated cysteine resulting as adduct in blood proteins after human exposure to ethylene oxide, by reversed-phase HPLC with fluorometric detection. The specific adduct is analysed in albumin and in globin. After isolation of albumin and globin from blood, acid hydrolysis of the protein and precolumn derivatisation of the digest with 9-fluorenylmethoxycarbonylchloride, the levels of derivatised S-hydroxyethylcysteine are analysed by RP-HPLC and fluorescence detection, with a detection limit of 8 nmol/g protein. Background levels of S-hydroxyethylcysteine were quantified in both albumin and globin, under special consideration of the glutathione transferase GSTT1 and GSTM1 polymorphisms. GSTT1 polymorphism had a marked influence on the physiological background alkylation of cysteine. While S-hydroxyethylcysteine levels in "non-conjugators" were between 15 and 50 nmol/g albumin, "low conjugators" displayed levels between 8 and 21 nmol/g albumin, and "high conjugators" did not show levels above the detection limit. The human GSTM1 polymorphism had no apparent effect on background levels of blood protein 2-hydroxyethylation.

Combining HPLC-DAD and ICP-MS data for improved analysis of complex samples: Classification of the root samples from Cortex moutan

A combined data matrix consisting of high performance liquid chromatography–diode array detector (HPLC–DAD) and inductively coupled plasma-mass spectrometry (ICP-MS) measurements of samples from the plant roots of the Cortex moutan (CM), produced much better classification and prediction results in comparison with those obtained from either of the individual data sets. The HPLC peaks (organic components) of the CM samples, and the ICP-MS measurements (trace metal elements) were investigated with the use of principal component analysis (PCA) and the linear discriminant analysis (LDA) methods of data analysis; essentially, qualitative results suggested that discrimination of the CM samples from three different provinces was possible with the combined matrix producing best results. Another three methods, K-nearest neighbor (KNN), back-propagation artificial neural network (BP-ANN) and least squares support vector machines (LS-SVM) were applied for the classification and prediction of the samples. Again, the combined data matrix analyzed by the KNN method produced best results (100% correct; prediction set data). Additionally, multiple linear regression (MLR) was utilized to explore any relationship between the organic constituents and the metal elements of the CM samples; the extracted linear regression equations showed that the essential metals as well as some metallic pollutants were related to the organic compounds on the basis of their concentrations

Esi-isät ja tiedonhankinta : Ennustus sukuma-nyamwezi-yhteisössä Luoteis-Tansaniassa

Tutkielmassa tarkastellaan esi-isien merkitystä sukuma-nymawezi-alueen ennustajientyössä. Työ perustuu kenttätyömateriaaliin, joka on pääasiassa kerätty neljän kylän muodostamalta hallinnolliselta alueelta, Isakasta, joka sijaitsee sukuma-nyamwczi-alueen kes-kiosissa. Sukumat ja nyamwezit ovat Tansanian suurimmat kansat ja muodostavat kulttuurisesti hyvinkin yhtenäisen alueen. Isakan alue, jossa kenttätyö on tehty, on nopeasti kasvava keskus, jonka kasvun ovat mahdollistaneet erinomaiset liikenneyhteydet, joiden vaikutuksesta alueelle on muuttanut useiden kansojen edustajia lähinnä vaurastumisen toivossa. Mm. ennustajia alueella on runsaasti juuri tästä syystä. Tutkielmassa esitellään erilaisia näkökulmia ennustukseen ja esi-isien merkitykseen Isakassa. Ennustuksen asiantuntijoiden, ennustajien, lisäksi näitä näkökulmia tarjoavat alueen perinteisten tanssirituaalien johtajat sekä länsimaisen terveydenhuollon ja valtion kulttuuriviraston edustajat. Nämä eri tahot edustavat usein jopa vastakkaisia näkemyksiä ennustuksen merkitykseen yhteisössä. Tutkielmassa tarkastellaan kuitenkin sitä, miten nämä toisistaan poikkeavat näkemykset yhdistyvät yksilöiden (isakalaisten naisten) kohdalla erilaisiksi kokonaisuuksiksi, joissa erilaiset näkemykset täydentävät toisiaan. Isakassa ennustajat selvittävät erilaisten ennustusmenetelmien avulla asiakkaittensa ongelmat ja tarvittavat hoitomuodot. Ennustuksen lisäksi he tarjoavat usein asiakkailleen myöstarvittavan hoidon. Lähes poikkeuksetta ennustus tapahtuu esi-isien tai muiden henkienavulla. Ennustuksessa voidaan käyttää henkipossessiota, jolloin henki kertoo asiakkaanongelmat ennustajan ruumiin kautta. Myös käytettäessä muita ennustusmenetelmiä kuin henkipossessiota, on esi-isien merkitys suuri, sillä ennustajan kykyjen nähdään olevannäiltä peräisin. Tutkielmassa esi-isien ja mahdollisten muiden henkien merkitystä ennus-tustyössä valotetaan henkipossessioteorioiden avulla ja tarkastellaan mm. sitä, miten esi-isät kommentoivat yhteisön jäsenten elämää "historian äänenä". Sukuma-nyamwezi-kosmologiassa esi-isät ja noituus nähdään mahdollisina sairauksien j amuiden ongelmien aiheuttajina. Erityisesti näissä tapauksissa virallisen (länsimaisen) terveydenhuollon palveluja ei nähdä riittävinä. Tutkielmassa korostetaankin sitä, miten isakalaisille ennustajien ja virallisen terveydenhuollon palvelut näyttäytyvät toisiaan täydentävi-nä vaihtoehtoina etsittäessä ratkaisuja sairauksien tai muiden ongelmien alkuperään ja tar-vittavaan hoitoon. Avainsanat: Tansania; sukuma-nyamwczi; ennustus; esi-isät ja muut henget; noituus

Visuaalis-spatiaalisten työmuistivalmiuksien yhteys (esi)matemaattisiin taitoihin ja merkitys osana matemaattisilta taidoiltaan heikkojen lasten ja nuorten kognitiivista profiilia

Tämän itsenäisistä osatutkimuksista koostuvan tutkimussarjan tavoitteena oli pyrkiä täydentämään kuvaa matemaattisilta taidoiltaan heikkojen lasten ja nuorten tiedonkäsittelyvalmiuksista selvittämällä, ovatko visuaalis-spatiaaliset työmuistivalmiudet yhteydessä matemaattiseen suoriutumiseen. Teoreettinen viitekehys rakentui Baddeleyn (1986, 1997) kolmikomponenttimallin ympärille. Työmuistikäsitys oli kuitenkin esikuvaansa laajempi sisällyttäen visuaalis-spatiaaliseen työmuistiin Cornoldin ja Vecchin (2003) termein sekä passiiviset varastotoiminnot että aktiiviset prosessointitoiminnot. Yhteyksiä työmuistin ja matemaattisten taitojen välillä tarkasteltiin viiden eri osatutkimuksen avulla. Kaksi ensimmäistä keskittyivät alle kouluikäisten lukukäsitteen hallinnan ja visuaalis-spatiaalisten työmuistivalmiuksen tutkimiseen ja kolme jälkimmäistä peruskoulun yhdeksäsluokkalaisten matemaattisten taitojen ja visuaalis-spatiaalisten työmuistitaitojen välisten yhteyksien selvittämiseen. Tutkimussarjan avulla pyrittiin selvittämään, ovatko visuaalis-spatiaaliset työmuistivalmiudet yhteydessä matemaattiseen suoriutumiseen sekä esi- että yläkouluiässä (osatutkimukset I, II, III, IV, V), onko yhteys spesifi rajoittuen tiettyjen visuaalis-spatiaalisten valmiuksien ja matemaattisen suoriutumisen välille vai onko se yleinen koskien matemaattisia taitoja ja koko visuaalis-spatiaalista työmuistia (osatutkimukset I, II, III, IV, V) tai työmuistia laajemmin (osatutkimukset II, III) sekä onko yhteys työmuistispesifi vai selitettävissä älykkyyden kaltaisella yleisellä päättelykapasiteetilla (osatutkimukset I, II, IV). Tutkimussarjan tulokset osoittavat, että kyky säilyttää ja käsitellä hetkellisesti visuaalis-spatiaalista informaatiota on yhteydessä matemaattiseen suoriutumiseen eikä yhteyttä voida selittää yksinomaan joustavalla älykkyydellä. Suoriutuminen visuaalis-spatiaalista työmuistia mittaavissa tehtävissä on yhteydessä sekä alle kouluikäisten esimatemaattisten taitojen hallintaan että peruskoulun yhdeksäsluokkalaisten matematiikan taitoihin. Matemaattisilta taidoiltaan heikkojen lasten ja nuorten visuaalis-spatiaalisten työmuistiresurssien heikkoudet vaikuttavat kuitenkin olevan sangen spesifejä rajoittuen tietyntyyppisissä muistitehtävissä vaadittaviin valmiuksiin; kaikissa visuaalis-spatiaalisen työmuistin valmiuksia mittaavissa tehtävissä suoriutuminen ei ole yhteydessä matemaattisiin taitoihin. Työmuistivalmiuksissa ilmenevät erot sekä alle kouluikäisten että kouluikäisten matemaattisilta taidoiltaan heikkojen ja normaalisuoriutujien välillä näyttävät olevan kuitenkin jossain määrin yhteydessä kielellisiin taitoihin viitaten vaikeuksien tietynlaiseen kasautumiseen; niillä matemaattisesti heikoilla, joilla on myös kielellisiä vaikeuksia, on keskimäärin laajemmat työmuistiheikkoudet. Osalla matematiikassa heikosti suoriutuvista on näin ollen selvästi keskimääräistä heikommat visuaalis-spatiaaliset työmuistivalmiudet, ja tämä heikkous saattaa olla yksi mahdollinen syy tai vaikeuksia lisäävä tekijä heikon matemaattisen suoriutumisen taustalla. Visuaalis-spatiaalisen työmuistin heikkous merkitsee konkreettisesti vähemmän mentaalista prosessointitilaa, joka rajoittaa oppimista ja suoritustilanteita. Tiedonkäsittelyvalmiuksien heikkous liittyy nimenomaan oppimisnopeuteen, ei asioiden opittavuuteen sinänsä. Mikäli oppimisympäristö ottaa huomioon valmiuksien rajallisuuden, työmuistiheikkoudet eivät todennäköisesti estä asioiden oppimista sinänsä. Avainsanat: Työmuisti, visuaalis-spatiaalinen työmuisti, matemaattiset taidot, lukukäsite, matematiikan oppimisvaikeudet

HPLC analysis of plant sterol oxidation products

Increased interest in the cholesterol-lowering effect of plant sterols has led to development of plant sterol-enriched foods. When products are enriched, the safety of the added components must be evaluated. In the case of plant sterols, oxidation is the reaction of main concern. In vitro studies have indicated that cholesterol oxides may have harmful effects. Due their structural similarity, plant sterol oxidation products may have similar health implications. This study concentrated on developing high-performance liquid chromatography (HPLC) methods that enable the investigation of formation of both primary and secondary oxidation products and thus can be used for oxidation mechanism studies of plant sterols. The applicability of the methods for following the oxidation reactions of plant sterols was evaluated by using oxidized stigmasterol and sterol mixture as model samples. An HPLC method with ultraviolet and fluorescence detection (HPLC-UV-FL) was developed. It allowed the specific detection of hydroperoxides with FL detection after post-column reagent addition. The formation of primary and secondary oxidation products and amount of unoxidized sterol could be followed by using UV detection. With the HPLC-UV-FL method, separation between oxides was essential and oxides of only one plant sterol could be quantified in one run. Quantification with UV can lead to inaccuracy of the results since the number of double bonds had effect on the UV absorbance. In the case of liquid chromatography-mass spectrometry (LC-MS), separation of oxides with different functionalities was important because some oxides of the same sterol have similar molecular weight and moreover epimers have similar fragmentation behaviour. On the other hand, coelution of different plant sterol oxides with the same functional group was acceptable since they differ in molecular weights. Results revealed that all studied plant sterols and cholesterol seem to have similar fragmentation behaviour, with only relative ion abundances being slightly different. The major advantage of MS detection coupled with LC separation is the capability to analyse totally or partly coeluting analytes if these have different molecular weights. The HPLC-UV-FL and LC-MS methods were demonstrated to be suitable for studying the photo-oxidation and thermo-oxidation reactions of plant sterols. The HPLC-UV-FL method was able to show different formation rates of hydroperoxides during photo-oxidation. The method also confirmed that plant sterols have similar photo-oxidation behaviour to cholesterol. When thermo-oxidation of plant sterols was investigated by HPLC-UV-FL and LC-MS, the results revealed that the formation and decomposition of individual hydroperoxides and secondary oxidation products could be studied. The methods used revealed that all of the plant sterols had similar thermo-oxidation behaviour when compared with each other, and the predominant reactions and oxidation rates were temperature dependent. Overall, these findings showed that with these LC methods the oxidation mechanisms of plant sterols can be examined in detail, including the formation and degradation of individual hydroperoxides and secondary oxidation products, with less sample pretreatment and without derivatization.

Food and Indoor Air Isolated Bacillus Non-Protein Toxins: : Structures, Physico-Chemical Properties and Mechanisms of Effects on Eukaryotic Cells

We report here the structures and properties of heat-stable, non-protein, and mammalian cell-toxic compounds produced by spore-forming bacilli isolated from indoor air of buildings and from food. Little information is available on the effects and occurrence of heat-stable non-protein toxins produced by bacilli in moisture-damaged buildings. Bacilli emit spores that move in the air and can serve as the carriers of toxins, in a manner similar to that of the spores of toxic fungi found in contaminated indoor air. Bacillus spores in food cause problems because they tolerate the temperatures applied in food manufacture and the spores later initiate growth when food storage conditions are more favorable. Detection of the toxic compounds in Bacillus is based on using the change in mobility of boar spermatozoa as an indicator of toxic exposure. GC, LC, MS, and nuclear magnetic resonance NMR spectroscopy were used for purification, detection, quantitation, and analysis of the properties and structures of the compounds. Toxicity and the mechanisms of toxicity of the compounds were studied using boar spermatozoa, feline lung cells, human neural cells, and mitochondria isolated from rat liver. The ionophoric properties were studied using the BLM (black-lipid membrane) method. One novel toxin, forming ion channels permeant to K+ > Na+ > Ca2+, was found and named amylosin. It is produced by B. amyloliquefaciens isolated from indoor air of moisture-damaged buildings. Amylosin was purified with an RP-HPLC and a monoisotopic mass of 1197 Da was determined with ESI-IT-MS. Furthermore, acid hydrolysis of amylosin followed by analysis of the amino acids with the GS-MS showed that it was a peptide. The presence of a chromophoric polyene group was found using a NMR spectroscopy. The quantification method developed for amylosin based on RP-HPLC-UV, using the macrolactone polyene, amphotericin B (MW 924), as a reference compound. The B. licheniformis strains isolated from a food poisoning case produced a lipopeptide, lichenysin A, that ruptured mammalian cell membranes and was purified with a LC. Lichenysin A was identified by its protonated molecules and sodium- and potassium- cationized molecules with MALDI-TOF-MS. Its protonated forms were observed at m/z 1007, 1021 and 1035. The amino acids of lichenysin A were analyzed with ESI-TQ-MS/MS and, after acid hydrolysis, the stereoisomeric forms of the amino acids with RP-HPLC. The indoor air isolates of the strain of B. amyloliquefaciens produced not only amylosin but also lipopeptides: the cell membrane-damaging surfactin and the fungicidal fengycin. They were identified with ESI-IT-MS observing their protonated molecules, the sodium- and potassium-cationized molecules and analysing the MS/MS spectra. The protonated molecules of surfactin and fengycin showed m/z values of 1009, 1023, and 1037 and 1450, 1463, 1493, and 1506, respectively. Cereulide (MW 1152) was purified with RP-HPLC from a food poisoning strain of B. cereus. Cereulide was identified with ESI-TQ-MS according to the protonated molecule observed at m/z 1154 and the ammonium-, sodium- and potassium-cationized molecules observed at m/z 1171, 1176, and 1192, respectively. The fragment ions of the MS/MS spectrum obtained from the protonated molecule of cereulide at m/z 1154 were also interpreted. We developed a quantification method for cereulide, using RP-HPLC-UV and valinomycin (MW 1110, which structurally resembles cereulide) as the reference compound. Furthermore, we showed empirically, using the BLM method, that the emetic toxin cereulide is a specific and effective potassium ionophore of whose toxicity target is especially the mitochondria.