Measurement of gastric emptying in man using deuterated octanoic acid.

Gamma scintigraphy is considered the gold standard for the measurement of gastric emptying in humans. Recently, it has been proposed that a [(13)C]octanoate breath test can be used as an alternative technique for measuring gastric emptying of the solid phase, but the results from the two methods are not directly equivalent since in the breath test the label is subject to post-absorptive processing and consequently the emptying functions cannot be observed directly. This work investigates an alternative stable isotope method using deuterated octanoate where the kinetics of redistribution between and elimination from the various body pools are much more easily modelled. Gastric emptying was studied in healthy human volunteers by simultaneous measurement using both [(13)C]octanoate and [(2)H]octanoate as well as gamma scintigraphy. Comparison of the gastric emptying functions from the deuterium method and scintigraphy indicated that the two methods gave equivalent results. The new method can therefore be used in populations considered too vulnerable to ionising radiation to allow gamma scintigraphy to be performed, or as a proxy gold standard in laboratories where scintigraphic methods are unavailable, allowing further comparisons with the breath test method to be made to validate the latter in different population groups.     

Vibrational spectra and normal coordinate analysis of isotopically labelled peroxyformic acid

Infrared spectra of peroxyformic acid (HCOOOH) and the six isotopic modifications H¹³COOOH, HC¹⁸OOOH, DCOOOH, HCOOOD, H¹³COOOD, DCOOOD have been recorded in an argon matrix in the spectral range 200–4000 cm⁻¹. Assignments of the observed absorption lines are presented for the available isotopic species. All fundamental vibrations except one have been identified. A general valence force field has been determined based on the measured vibrational frequencies and on an ab initio MP2/6-31G** force field.     

The loss of water from isotopically labelled heptanals on chemical ionization

The chemical ionization spectra of a series of deuterated heptanals has been studied using methane and isobutane. Loss of water from the [MH]⁺ ion follows the same pattern as in electron ionization; hydrogen (deuterium) is lost from all positions except C-1 and C-2. The reagent proton is found in the water lost only to the extent of 28% and this is confirmed using deuteriomethane. Possible mechanisms are discussed.     

Determination of the enrichment of isotopically labelled molecules by mass spectrometry

A general method for the determination of the enrichment of isotopically labelled molecules by mass spectrometry (MS) is described. In contrast to other published procedures, the method described here takes into account and corrects for measurement errors such as the contribution at M ? 1 due to loss of hydrogen or lack of spectral resolution and provides an uncertainty value for the determined enrichment. The general procedure requires the following steps: (1) evaluation of linearity in the mass spectrometer by injecting the natural abundance compound at different concentration levels, (2) determination of the purity of the mass cluster using the natural abundance analogue, (3) calculation of the theoretical isotope composition of the labelled compound using different tentative isotope enrichments, (4) calculation of ‘convoluted’ isotope distributions for the labelled compound taking into account the purity of the mass cluster determined with the natural abundance analogue and (5) comparison of the isotope distributions measured for the labelled compound with those calculated for different isotope enrichments using linear regression. The method was applied to a series of commercially available ¹³C‐ and ²H‐labelled compounds and to a suite of singly ¹³C‐labelled β₂‐agonist prepared in‐house both by gas chromatography (GC)–MS, GC–tandem MS (MS/MS) and liquid chromatography–MS/MS with satisfactory results. It was observed that the main uncertainty source for the isotope enrichment was the uncertainty in the purity of the measured cluster as determined with the natural abundance compound. Copyright © 2014 John Wiley & Sons, Ltd.     

The use of isotopically labelled water to monitor the ingress of water into adhesive joints

Joints bonded with carried and uncarried film adhesives have been exposed to tritiated water vapour at 50° for periods up to 6 weeks. Comparison of the water uptake pattern in the joints with that calculated from diffusion coefficients showed the interior of the joints to be relatively deficient in water. In the systems studied, there is no contribution to water ingress from wicking along the carrier-adhesive or adhesive-metal interfaces.     

Co-adsorption studies of ethene with isotopically labelled water and hydrogen on copper/silica

The coadsorption of ethene with isotopically labelled water and hydrogen has been studied at temperatures between 423 and 623 K in a flow system. Ethene exchange has been shown to be independent of hydrogenation. When water is presnet, hydrogenation is poisoned but exchange can occur.

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423–623 . . , .

     

Adsorption of octanoic acid at the water/oil interface

Fats are widely present in a large variety of food and represent the main source of energy for the body. In the current study we investigate the behaviour of fatty acids at liquid–liquid interfaces, mimicking some steps of the very complex digestion process. Octanoic acid is used as an example of middle chain fatty acids. For the oil phase we choose sunflower oil as an industrial product and hexane as pure oil.The influence of the fatty acid concentration and the pH of the aqueous phase on the interfacial tension is determined by profile analyse tensiometry (PAT), which allows to examine the way of adsorption and transition of the fatty acids from one phase to the other. Predominantly, the pH affects the dissociation and thereby the strength of the hydrophilic character of the fatty acid. The adsorption behaviour indicates the different interfacial activity of the studied octanoic acid.     

Simultaneous determination of stable isotopically labelled L-histidine and urocanic acid in human plasma by stable isotope dilution mass spectrometry.

A capillary gas chromatographic-mass spectrometric method for the simultaneous determination of stable isotopically labelled L-histidine (L-[3,3-2H2,1',3'-15N2]histidine, L-His-[M + 4]) and urocanic acid ([3-2H,1',3'-15N2]urocanic acid, UA-[M + 3]) in human plasma was developed using DL-[2,3,3,5'-2H4,2'-13C,1',3'-15N2]histidine (DL-His-[M + 7]) and [2,3,5'-2H3,2'-13C,1',3'-15N2]urocanic acid (UA-[M + 6]) as internal standards. L-Histidine and urocanic acid were derivatized to alpha N-(trifluoroacetyl)-imN-(ethoxycarbonyl)-L-histidine n-butyl ester and imN-(ethoxycarbonyl)urocanic acid n-butyl ester. Quantification was carried out by selected ion monitoring of the molecular ions of the respective derivatives of L-His-[M + 4], DL-His-[M + 7], UA-[M + 3] and UA-[M + 6]. The sensitivity, specificity, precision and accuracy of the method were demonstrated to be satisfactory for measuring plasma concentrations of L-His-[M + 4] and UA-[M + 3] following administration of trace amounts of L-His-[M + 4] to humans.     

Mapping phosphorylation sites: a new strategy based on the use of isotopically labelled DTT and mass spectrometry

Phosphoproteomics, nowadays, represents a front line in functional proteomics as testified by the number of papers recently appearing in the literature. In an attempt to improve and simplify the methods so far suggested we have set up a simple isotope-coded approach to label and quantitate phospho-Ser/-Thr residues in protein mixtures. First of all, after appropriate oxidation of cysteine/cystine residues followed by tryptic hydrolysis, we have optimised and simplified the beta-elimination reaction to get the corresponding alkene moiety from the phosphate esters. This was achieved by (a) separating the elimination reaction from the addition reaction, (b) the use of Ba(OH)(2) as alkali reagent and (c) its further elimination by the simple addition of solid CO(2) to the peptide mixture. The Michael reaction was then performed, after the removal of BaCO(3) by centrifugation, by adding dithiothreitol (DTT) to the peptide mixture. Finally, the direct purification of the modified phosphopeptides was performed on a thiol-sepharose column. The availability of fully deuterated DTT, introducing a 6 Da difference with respect to the non-deuterated species, allows quantitation of the differential extent of signalling modification when analysed by matrix-assisted laser desorption/ionisation mass spectrometry (MALDI-MS) and liquid chromatography/mass spectrometry. The entire procedure has been set up by using bovine alpha-casein, and resulted in the identification of all the phosphorylated tryptic peptides, including the tetraphosphorylated peptides, which escaped all previously reported procedures     

Effectiveness of isotopically labelled and non‐isotopically labelled internal standards in the gas chromatography/mass spectrometry analysis of sulfur compounds in wines: use of a statistically based matrix comprehensive approach

The effectiveness of isotopically and non‐isotopically labelled internal standards in reducing matrix‐induced effects is evaluated. The question is addressed in the quantitative analysis by gas chromatography/mass spectrometry (GC/MS) of dimethyl sulphide, chosen as a typical example of volatile sulphur compounds, in wine matrices. When matrix/run effects are not cancelled out the use of a variance component model (VCM) to handle the linear calibrations obtained by regression technique is successful. The method implies the estimation of an overall calibration straight line, which properly takes into account the uncertainty due to different matrices, the calibration run and the measurement error, making the use of an isotopically labelled internal standard not necessary. The obtained results show that the benefits of lowering times and costs for routine analyses compensate for the small increase in uncertainty in the concentration values obtained in the regression analysis and the slight increase in the detection limit. Copyright © 2009 John Wiley & Sons, Ltd.     

The use of microcalorimetry to assess the size exclusion properties of carbon molecular sieves

Carbon molecular sieves (CMSs) for separating gaseous mixtures have been prepared through chemical vapour deposition (CVD) of benzene on activated carbon fibres (ACFs) obtained from Nomex aramid fibres. The effect of the CVD treatment on the porous texture of the ACFs has been followed by immersion calorimetry into liquids of different molecular sizes (dichloromethane, benzene and cyclohexane). Both the kinetics of the immersion process and the equilibrium immersion enthalpies have been studied. Besides, the derived specific surface areas accessible to the different liquids employed have been calculated. The results show that the carbon deposition has taken place mainly at the pore entrances and the CVD treatment has succeeded to introduce selectivity without a significant loss of capacity.     

Enzymatic detoxification of jojoba meal and effect of the resulting meal on food intake in rats

When defatted jojoba meal is used as animal food, it causes food-intake reduction and growth retardation. Detoxification procedures by chemical, microbiological, and solvent extraction methods are reported by several authors. Here we report a successful detoxification of jojoba meal using enzymes. We establish reaction conditions that yield new meal which has the same nutritional qualities in proteins as the original meal. The enzymatic reaction gives rise to one major compound to which the structure of an amide is assigned on the basis of IR, 1H and 13C NMR spectra. The effect of the resulting jojoba meal on the food intake in rats is checked. In contrast, the detoxified meal containing the amide derivatives shows no toxicological activity since rats receiving oral administration of the obtained meal show normal growth. Thus, it is expected that this meal could be used as an animal feed ingredient.     

Study of selected ions in the ammonia desorption chemical ionization mass spectra of peracetylated gentiobiose and two isotopically labelled peracetylated gentiobioses

The ammonia desorption chemical ionization (NH₃-DCI) mass spectra of peracetylated gentiobiose (1) and two isotopically labelled gentiobioses (2 and 3) were examined. Compound 2 is labelled with trideuteroacetyl groups in the non-reducing moiety and 3 with trideuteroacetyl groups in the reducing moiety. It is shown that the [M + NH₄ – 42]⁺ ion is not formed direct from [M + NH₄]⁺ by loss of ketene but appears to be formed by way of a nucleophilic acyl substitution reaction resulting in a neutral species which complexes with NH₄⁺. The disaccharides undergo cleavage at either side of the glycosidic oxygen joining the two sugar residues, a process which is accompanied by addition of H or CH₃CO to afford neutral species which complex with NH₄⁺. The structures of the ions resulting from H transfer have been inferred by comparison of their mass-analysed ion kinetic energy (MIKE) spectra with MIKE spectra of the [M + NH₄]⁺ ions of compounds of established structure. A ring fragmentation reaction of 1, 2 and 3 is reported.     

Application of thermospray liquid chromatography-mass spectrometry to the simultaneous quantification of tracer concentrations of isotopically labelled carbamazepine epoxide and steady-state levels of carbamazepine and carbamazepine epoxide

A thermospray high-performance liquid chromatography-mass spectrometry method for the separation and quantification of tracer concentrations of isotopically labelled carbamazepine epoxide ([15N, 13C]CBZE) in the presence of steady-state levels of the anticonvulsant carbamazepine (CBZ) and its epoxide metabolite (CBZE) has been developed. The technique does not require derivatization, demonstrates little or no thermal degradation of the analytes, provides increased specificity not available from conventional high-performance liquid chromatography, and has a detection limit of 500 pg for CBZE on-column. The method, incorporating d4-CBZ and d4-CBZE as internal standards, allows precise and accurate determination of the analytes with good reproducibility and stability.     

Microfluidic wound-healing assay to assess the regenerative effect of HGF on wounded alveolar epithelium

We present a microfluidic epithelial wound-healing assay that allows characterization of the effect of hepatocyte growth factor (HGF) on the regeneration of alveolar epithelium using a flow-focusing technique to create a regular wound in the epithelial monolayer. The phenotype of the epithelial cell was characterized using immunostaining for tight junction (TJ) proteins and transmission electron micrographs (TEMs) of cells cultured in the microfluidic system, a technique that is reported here for the first time. We demonstrate that alveolar epithelial cells cultured in a microfluidic environment preserve their phenotype before and after wounding. In addition, we report a wound-healing benefit induced by addition of HGF to the cell culture medium (19.2 vs. 13.5 μm h(-1) healing rate).     

Use of alkaline Comet assay to assess DNA repair after m-THPC-PDT

Photodynamic therapy (PDT) with Photofrin has already been authorized for certain applications in Japan, the USA and France, and powerful second-generation sensitizers such as meta-(tetrahydroxyphenyl) chlorin (m-THPC) are now being considered for approval. Although sensitizers are likely to localize within the cytoplasm or the plasma membrane, nuclear membrane can be damaged at an early stage of photodynamic reaction, resulting in DNA lesions. Thus, it is of critical importance to assess the safety of m-THPC-PDT, which would be used mainly against early well-differentiated cancers. In this context, m-THPC toxicity and phototoxicity were studied by a colorimetric MTT assay on C6 cells to determine the LD50 (2.5 microg/ml m-THPC for 10 J/cm2 irradiation and 1 microg/ml for 25 J/cm2 irradiation) and PDT doses inducing around 25% cell death. Single-cell electrophoresis (a Comet assay with Tail Moment calculation) was used to evaluate DNA damage and repair in murine glioblastoma C6 cells after LD25 or higher doses for assays of PDT. These results were correlated with m-THPC nuclear distribution by confocal microspectrofluorimetry. m-THPC failed to induce significant changes in the Tail Moment of C6 cells in the absence of light, whereas m-THPC-PDT induced DNA damage immediately after irradiation. The Tail Moment increase was not linear (curve slope being 43 for 0-1 microg/ml m-THPC and 117 for 1-3 microg/ml), but the mean value increased with the light dose (0, 10 or 25 J/cm2) and incubation time (every hour from 1 to 4 h) for an incubation with m-THPC 1 microg/ml. However, cultured murine glioblastoma cells were capable of significant DNA repair after 4 h, and no residual DNA damage was evident after 24-h post-treatment incubation at 37 degrees C. An increase in the light dose appeared to be less genotoxic than an increase in the m-THPC dose for similar toxicities. Our results indicate that m-THPC PDT appears to be a safe treatment since DNA repair seemed to not be impaired and DNA damage occurred only with lethal PDT doses. However, the Comet assay cannot give us the certainty that no mutation, photoadducts or oxidative damage have been developed so this point would be verified with another mutagenicity assay.     

Pyrrolidinyl peptide nucleic acid homologues: effect of ring size on hybridization properties

The effect of ring size of four- to six-membered cyclic β-amino acid on the hybridization properties of pyrrolidinyl peptide nucleic acid with an alternating α/β peptide backbone is reported. The cyclobutane derivatives (acbcPNA) show the highest T(m) and excellent specificity with cDNA and RNA.     

Application of isothermal and isoperibolic calorimetry to assess the effect of zinc on cement hydration

The amount of zinc in the clinker or in the secondary raw materials has been increasing in recent years. Zinc can get to Portland cement from solid waste or tires which are widely used as a fuel for burning in a rotary kiln. The aim of this work was to determine the effect of zinc on Portland cement hydration. This effect was studied by isothermal and isoperibolic calorimetry. Both calorimetry methods are suitable for measurements during the first days of hydration. Isoperibolic calorimetry monitors hydration process in real-life conditions, while isothermal calorimetry does it at a defined chosen temperature. Zinc was added to the cement in the form of two soluble salts of Zn(NO₃)₂, ZnCl₂ and a poorly soluble compound ZnO. The concentration of zinc added was chosen as 0.05, 0.1, 0.5 and 1 mass%. The results show that increasing amounts of zinc ions in cement pastes lead to hydration retardation and reduce both the maximum temperature and the maximum heat flow due to the retarding effect of zinc. The newly formed compounds during hydration were identified by X-ray diffraction method.     

A preliminary study to assess the effect of some seawater components on the speciation of plutonium

Since Pu(IV) and Pu(V) exhibit very different sediment sorption behaviour, the transport of Pu in the aquatic environment is dependant upon oxidation state and the rate of interconversion between the species. A number of laboratory experiments have been carried out to determine possible parameters which influence the rate of Pu redox reactions and the extent of sorption by suspended particulate in the marine environment. Results suggest that, although the initial sorption of Pu(IV) did not appear to be dependant upon the major cations present in seawater, the sorption of Pu(V) was decreased in the presence of Ca²⁺ and Mg²⁺ ions. Both the rates of oxidation of dissolved Pu(IV) and reduction of dissolved Pu(V) increased with increasing suspended particulate concentration.