Unbiased high-throughput screening of reactive metabolites on the linear ion trap mass spectrometer using polarity switch and mass tag triggered data-dependent acquisition

Constant neutral loss (CNL) and precursor ion (PI) scan have been widely used for the in vitro screening of glutathione conjugates derived from reactive metabolites, but these two methods are only applicable to triple quadrupole or hybrid triple quadrupole mass spectrometers. Additionally, the success of CNL and PI scanning largely depends on structure and CID fragmentation pathways of GSH conjugates. In the present study, a highly efficient methodology has been developed as an alternative approach for high-throughput screening and structural characterization of reactive metabolites using the linear ion trap mass spectrometer. In microsomal incubations, a mixture of glutathione [GSH, gamma-glutamyl-cystein-glycin] and the stable-isotope labeled compound [GSX, gamma-glutamyl-cystein-glycin-(13)C2-(15)N] was used to trap reactive metabolites, resulting in formation of both labeled and unlabeled conjugates at a given isotopic ratio. A mass difference of 3.0 Da between the natural and labeled GSH conjugate (mass tag) at a fixed isotopic ratio constitutes a unique mass pattern that can selectively trigger the data-dependent MS(2) scan of both isotopic partner ions, respectively. In order to eliminate the response bias of GSH adducts in the positive and negative mode, a polarity switch is executed between the mass tag-triggered data dependent MS(2) scan, and thus ESI- and ESI+ MS(2) spectra of both labeled and nonlabeled GSH conjugates are obtained in a single LC-MS run. Unambiguous identification of glutathione adducts was readily achieved with great confidence by MS(2) spectra of both labeled and unlabeled conjugates. Reliability of this method was vigorously validated using several model compounds that are known to form reactive metabolites. This approach is not based on the appearance of a particular product ion such as MH(+) - 129 and anion at m/z 272, whose formation can be structure-dependent and sensitive to the collision energy level; therefore, the present method can be suitable for unbiased screening of any reactive metabolites, regardless of their CID fragmentation pathways. Additionally, this methodology can potentially be applied to triple quadrupole or hybrid triple quadrupole mass spectrometers.     

Effects of the application of fertilisers and trace elements on the cobalt content of herbage cut for conservation

The effects of the application of fertilisers, especially N and trace elements, on the content of Co in mixed grassland herbage and its main constituent species, have been studied. Data have been obtained from field experiments on three mineral soils and on a deep acid peat. The herbage was cut at the silage stage of growth three or four times a year for at least three successive years. Without added Co, N applications increased Co content in herbage on peat but reduced it on mineral soils mainly by eliminating clover which had a higher Co content than grasses. Applying 0.5 kg ha^?1 Co as cobalt sulphate increased herbage Co content, the increase being greater on peat than on mineral soils. These increased levels were unaffected by N applications on mineral soils but were further increased by applied N on peat. The number of years during which the Co additions were effective in producing herbage containing at least 0.1 mg Co kg^?1 dry matter varied on the different soils, ranging from barely three years on one mineral soil to over five years on peat. Without added Co the last cuts in October usually had the highest Co content and the first cuts in June the lowest. With added Co, the seasonal effects were more variable, but the Co content in the herbage frequently decreased from the first to the last cuts.     

Mitochondrial DNA Fragmentation as a Molecular Tool to Monitor Thermal Processing of Plant‐Derived,Low‐Acid Foods,and Biomaterials

Cycle threshold (Ct) increase, quantifying plant‐derived DNA fragmentation, was evaluated for its utility as a time‐temperature integrator. This novel approach to monitoring thermal processing of fresh, plant‐based foods represents a paradigm shift. Instead of using quantitative polymerase chain reaction (qPCR) to detect pathogens, identify adulterants, or authenticate ingredients, this rapid technique was used to quantify the fragmentation of an intrinsic plant mitochondrial DNA (mtDNA) gene over time‐temperature treatments. Universal primers were developed which amplified a mitochondrial gene common to plants (atp1). These consensus primers produced a robust qPCR signal in 10 vegetables, 6 fruits, 3 types of nuts, and a biofuel precursor. Using sweet potato (Ipomoea batatas) puree as a model low‐acid product and simple linear regression, Ct value was highly correlated to time‐temperature treatment (R² = 0.87); the logarithmic reduction (log CFU/mL) of the spore‐forming Clostridium botulinum surrogate, Geobacillus stearothermophilus (R² = 0.87); and cumulative F‐value (min) in a canned retort process (R² = 0.88), all comparisons conducted at 121 °C. D₁₂₁ and z‐values were determined for G. stearothermophilus ATCC 7953 and were 2.71 min and 11.0 °C, respectively. D₁₂₁ and z‐values for a 174‐bp universal plant amplicon were 11.3 min and 9.17 °C, respectively, for mtDNA from sweet potato puree. We present these data as proof‐of‐concept for a molecular tool that can be used as a rapid, presumptive method for monitoring thermal processing in low‐acid plant products.     

42—THE ESTIMATION OF CROSS-LINKS IN WOOL FROM THE EXTENT OF SWELLING IN FORMIC ACID

An account is given of the application of a gravimetric method for measuring the extent of swelling of wool in formic acid to a series of wool samples containing decreasing amounts of randomly distributed disulphide bonds. In accordance with the Flory-Rehner equation, a linear relation was observed between disulphide content and V 5/3, where V is the volume of dry wool expressed as a fraction of the volume of swollen wool. This calibration curve was then used in conjunction with swelling data to assess the number of cross-links introduced on the treatment of wool with formaldehyde and other bifunctional reagents. The method is particularly useful for ranking in order relatively high levels of cross-linking.     

Correction to Bodner et al. (2000).

Reports an error in the original article by G. E. Bodner et al, ( Journal of Experimental Psychology: Learning, Memory, and Cognition, 2000 [Mar], Vol. 26[2], 267–293). The parameter estimates and goodness-of-fit (G2) statistics for the fits of the generate-recognize and direct-retrieval models to the data from Exps 1, 2, and 4 were inaccurate because of errors in the frequency data used to perform the model fits. The pertinent portion of Table 7 is provided and includes the correct parameter estimates and G2 values for these experiments. Information updates for page 283 (the second full paragraph of the right-hand column) and the Discussion section on pages 285–286 (i.e., the discussion of multinomial modeling) are also provided. (The following abstract of this article originally appeared in record 2000-07237-001). Application of the process-dissociation procedure has shown that conceptual encoding episodes do not lead to automatic influences of memory on purportedly data-driven indirect tests of memory. Using 2 variants of the process-dissociation procedure with the word-stem completion task, the procedure is shown to underestimate automatic influences of memory when prior encoding includes a conceptual component. The underestimation is attributed to an awareness of… (PsycINFO Database Record (c) 2010 APA, all rights reserved)     

Investigations of deep resistivity structures at the Wairakei geothermal field

The Wairakei geothermal field was the proving ground for the use of electrical resistivity methods for geothermal exploration. At this site it was first demonstrated that a large contrast in resistivity existed between geothermal ground and the cold surroundings. Within the top 500 m of the geothermal field, low-resistivity (5–10 Ωm) reflects the effects of both the hot saline water in the pore spaces and the conductive rock-matrix. The first surveys at Wairakei used a Wenner array (a ∼550 m) to measure the resistivity values along tracks throughout the field; contour maps of the resistivities were used to estimate the lateral extent of the geothermal waters at a few hundred metres depth. In the late 1960s the Wenner array was superseded by the Schlumberger array (AB/2 = 500 m and 1000 m), which enabled deeper penetration and better definition of the extent of the geothermal waters. These early surveys showed that the bounds of the geothermal waters were often sharp, leading to the concept that a ‘resistivity boundary’ could be defined for New Zealand's liquid-dominated geothermal fields. As new methods of measuring electrical structure with greater precision became available, Wairakei was often chosen as the testing ground.     

Stereoselectivity of the aliphatic hydroxylation of 6-n-propylchromone-2-carboxylic acid in rat and guinea pig

Following administration of 6-n-propylchromone-2-carboxylic acid (6-n-PCCA) (500 mumol/kg) to male rats, three metabolic products were detected and isolated from the 0-24 h urine. All were identified as resulting from oxidation exclusively along the 6-n-propyl moiety. Some 66% of the dose was excreted in the 0-24 h urine, 55% of which was 6-PCCA, with 15% as (6-1'-hydroxypropyl)chromone-2-carboxylic acid (6-1'-HPCCA), 22% as 6-(2'-hydroxypropyl)chromone-2-carboxylic acid (6-2'-HPCCA), and 4% as 6-3'-carboxypropyl)chromone-2-carboxylic acid (6-3'-CPCCA). Derivatization of the methyl esters of the hydroxylated metabolites with S-alpha-methoxy-alpha-(trifuloromethyl)-phenylacetyl chloride (Mosher's reagent) allowed the evaluation of urinary enantiomeric composition by HPLC and assignment of their absolute configurations by NMR. This was found to be 90:10 (R/S) for 6-2'-HPCCA, and 7:93 (R/S) for 6-1'-HPCCA. When rats were dosed with the racemic 1'- and 2-hydroxy metabolites; no stereoselective metabolism or excretion was observed. Administration of 6-n-PCCA to male guinea pigs revealed that this species was unable to metabolise this compound.     

The interaction of zinc(II) and phytic acid with soya bean glycinin

Binding of phytic acid to Zn(II) free glycinin was not observed in 0.5 M KCl at pH 6.2. The addition of varying quantities of phytic acid to a Zn(II)- glycinin system at pH 6.2 ([KCl]=0.5 M ) initially resulted in binding of phytate and increased binding of Zn(II) to glycinin probably as a phytate-Zn(II)-glycinin complex. Further addition of phytate resulted in precipitation of zinc and protein. Bovine serum albumin also showed increased affinity for Zn(II) owing to the presence of phytic acid. Phytic acid-Zn(II) precipitates have a capacity of removing glycinin from solution, presumably by surface adsorption. The presence of soluble Zn(II) inhibits this adsorption. Bovine serum albumin is removed from solution by phytic acid-Zn(II) precipitates only when soluble zinc is present.     

Acoustically active lipospheres containing paclitaxel: a new therapeutic ultrasound contrast agent

RATIONALE AND OBJECTIVES: Paclitaxel-carrying lipospheres (MRX-552) were developed and evaluated as a new ultrasound contrast agent for chemotherapeutic drug delivery. METHODS: Paclitaxel was suspended in soybean oil and added to an aqueous suspension of phospholipids in vials. The headspace of the vials was replaced with perfluorobutane gas; the vials were sealed, and they were agitated at 4200 rpm on a shaking device. The resulting lipospheres containing paclitaxel were studied for concentration, size, acute toxicity in mice, and acoustic activity and drug release with ultrasound. Lipospheres containing sudan black dye were produced to demonstrate the acoustically active liposphere (AAL)-ultrasound release concept. RESULTS: Acoustically active lipospheres containing paclitaxel had a mean particle count of approximately 1 x 10(9) particles per mL and a mean size of 2.9 microns. Acute toxicity studies in mice showed a 10-fold reduction in toxicity for paclitaxel in AALs compared with free paclitaxel. The AALs reflected ultrasound as a contrast agent. Increasing amounts of ultrasound energy selectively ruptured the AALs and released the paclitaxel. CONCLUSIONS: Acoustically active lipospheres represent a new class of acoustically active drug delivery vehicles. Future studies will assess efficacy of AALs for ultrasound-mediated drug delivery.