AMP, ADP, and ATP Concentrations Differentially Affected by Meat Processing, Manufacturing, and Nonmeat Ingredients.

Given its presence in a wide spectrum of soils relevant to food process hygiene, the biological metabolite adenosine triphosphate (ATP) is used as a target for surface hygiene assessments in food processing facilities. Yet, ample evidence demonstrates that ATP is depleted into adenosine di- (ADP) and monophosphate (AMP) homologs resulting in a loss of sensitivity for ATP-based hygiene assays. Yet, there are few studies that denote the degree of these shifts under routine processing conditions such as those encountered during various meat processing steps that may likely alter redox potential and adenosine profiles (e.g., tissue/cellular disruption, application of reducing additives, fermentation, or thermal treatment steps). In this study, meat samples were collected from homogenized beef tissue treated with nonmeat ingredients (sodium chloride, sodium nitrite, sodium erythorbate, natural smoke condensate, and sodium acid pyrophosphate) during manufacture at predetermined steps, and from retail meat products purchased from local markets. Concentrations of ATP, ADP, AMP, and AXP (sum concentration of all homologs) in a lab setting and in situ meat processing venues were determined and compared. Greater differences in AXP were seen during manufacture, where ADP generally comprised ∼90% as a mole fraction of AXP across all treatments, with the exception of the final cook step where AMP predominated. ATP concentrations averaged 2 log values lower than ADP and AMP. Adenosine profiles in retail samples followed similar trends with minimal ATP concentrations with ADP predominant in uncooked samples and AMP predominant in cooked samples. Resultingly, meat processing steps during product manufacture will alter AXP-reliant test sensitivities which should be considered when such technologies are utilized for hygiene verification in meat processing.

Quantities of Adenylate Homologues (ATP+ADP+AMP) Change over Time in Prokaryotic and Eukaryotic Cells.

Rapid assays for the assessment of the hygienic state of surfaces in food and medical industries include the use of technologies designed to detect the presence of the metabolite ATP. ATP is a critical metabolite and energy source for most living organisms; therefore, the presence of ATP can be an indicator of surface hygiene based on the presence of soil or food residues associated with inadequate cleaning. The concentrations of ATP vary based on an organism's metabolic state, thus potentially influencing the sensitivity of ATP-based assays. However, little has been published detailing the quantitative changes of ATP to the adenylate homologues ADP and AMP nor the quantitative and cumulative fate of these homologues over time as the metabolic state remains in flux. The objective of this study was to quantify the individual and cumulative (AXP) concentrations of these three adenylate homologues over defined time periods in selected eukaryotic tissue and prokaryotic cell cultures of significance to hygiene. ATP concentrations differed substantially across these selected variables of time and source. The 1- to 3-log reductions in ATP concentrations over time were highly affected by organism type. In general, ADP became the predominate adenylate in eukaryotic tissue, and AMP was the predominate adenylate in the prokaryotic cells at later time points in each study. Total AXP concentrations dropped in general, reflective primarily of the loss of ATP. The results of ATP-based techniques for hygiene surveillance will vary as a function of the amount of cellular material present and the metabolic state of such material.

The poppy seed defense: a novel solution.

A major toxicological challenge is distinguishing whether morphine in urine, in the absence of 6-monoacetylmorphine (6-MAM), originates from 'street' heroin use or poppy seed ingestion. Manufacturing byproducts from the synthesis of illicit heroin include those that originate from the reaction of acetic anhydride with the alkaloid impurity, thebaine, which undergoes skeletal rearrangement, resulting in compounds with a 2-(N-methylacetamido)ethyl side-chain. The hypothesis that the tertiary amide in this side-chain is resistant to endogenous hydrolysis was supported from in-vitro experiments; a glucuronide metabolite (designated 'ATM4G') was identified that may be used as a marker of 'street' heroin administration. Liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis for this metabolite was then performed on selected urine specimens from 22 known heroin users, these being negative on routine testing for 6-MAM by gas chromatography-mass spectrometry (GC-MS), using the generally applied reporting threshold of 10 ng/mL, but positive for the presence of morphine. Peaks corresponding to the retention time for the metabolite marker were clearly observed for 16 of the 22 samples, with variations of the ratios of its three dependent ions being within ± 30% of that produced in vitro. Conversely, 6-MAM was detected in only 3 samples, but at concentrations <1 ng/mL. Such a high frequency for the presence of the metabolite marker in urine, in the absence of 6-MAM, is noteworthy and suggests that detection of this metabolite may offer an important advance in forensic toxicology, allowing the development of a new and more definitive test for heroin abuse and thus a potential solution to the so-called 'poppy seed defense'.

Study of short polystyrene monolith-fritted micro-liquid chromatography columns for analysis of neutral and basic compounds.

This study details the effects of poly(styrene-divinylbenzene) (PS-DVB) frits in micro-HPLC columns for the separation of neutral and basic compounds. The procedure comprised the optimization of separations with only monolith or conventional fritted columns followed by method transference to short monolith-fritted columns. It was observed that a superior separation was achieved with the new columns compared to silica-fritted-packed columns once triethylamine (TEA) was added in small percentages. The separation of basic and neutral compounds was achieved in fast analysis times in the isocratic mode.

Link between biological signaling and increased enantioseparations of acids using glycopeptide antibiotics.

The vancomycin analog A82846B has been shown to provide excellent selectivity as a chiral recognition agent for some acidic test analytes in capillary electrophoresis (CE) and high-performance liquid chromatography (HPLC). In both modes A82846B outperforms vancomycin as a chiral selector. A82846B has enhanced antibacterial activity data in comparison to vancomycin, which is probably due to the increased dimerization constant of over 100 in magnitude in comparison to vancomycin. The link between the electrophoretic and chromatographic separations observed and the biological activity of A82846B is discussed. Dimerization of A82846B in solution was proposed as a theory as to why A82846B gave such enhanced separations for acidic racemates. Further literature and experimental studies support the theory.

Quantification of the sensitivity increase of a micro-high-performance liquid chromatography-electrospray ionization mass spectrometry system with decreasing column diameter.

This study details the sensitivity achieved with capillary columns when used with a micro-HPLC-electrospray ionization MS system. It is comprised of two sections, the first is the comparative study of three columns, one of narrow-bore diameter and two of capillary diameter. The second section compares three columns of decreasing diameter in the capillary scale. All the experiments achieved enhanced sensitivity using capillary columns. The increase in the experimental MS response ranged from -20% to +20% compared to the UV experimental response when decreasing the internal diameter of the columns used. When comparing the experimental MS response to the maximum theoretical UV response achievable, the increase in response ranged from 40 to 50%.

Effect of ionic strength on perfusive flow in capillary electrochromatography columns packed with wide-pore stationary phases.

The use of wide-pore stationary phases in capillary electrochromatography has shown exceptional increases in separation efficiency in conjunction with high electroosmotic flow. These effects are due to the perfusive flow mechanism which is primarily controlled by the ionic strength of the mobile phase. Good correlation between calculated values of electrochemical double-layer thickness and efficiency data have also been obtained. Reduced plate height values of <0.5 have been observed with pore sizes of 4000 A. In addition, electroosmotic flow mobility twice that of 3 microm Spherisorb ODS-1 has been obtained.

Influence of the unpacked section on the chromatographic performance of duplex strong anion-exchange columns in capillary electrochromatography.

This work describes initial investigations of strong anion-exchange (SAX) packing materials for capillary electrochromatography (CEC). The use of SAX phases in CEC is theoretically appealing for the analysis of negatively charged species. The reversed direction of the electroosmotic flow (EOF) generated by SAX phases (in comparison to reversed phases and strong cation-exchange phases) means that negative species can migrate with the EOF, not against it, hence the analysis times, of such species should be decreased and efficiencies improved. Duplex CEC columns (the standard for instruments using UV detection) consist of a packed and an unpacked section. Using common reversed-phase packing materials the direction of the EOF in both sections is co-linear, however when normal fused-silica capillaries are packed with SAX material the direction of the EOF in the two sections oppose one another. It has been shown, using conventional duplex CEC columns and fully packed CEC-MS columns that the opposing direction of EOF causes a massive degradation in column performance. Consequentially, it is demonstrated that if the EOF in the open section of the duplex SAX column can be controlled via pH or capillary derivatisation then good, reproducible CEC can be performed on anionic species using SAX packed CEC columns.

Electrochromatography.

Although Mould and Synge were credited with the first demonstration of the use of electroosmotic flow to drive a liquid in a thin-layer chromatography system, it was the work of Knox and Grant that extensively detailed much of both the theoretical and practical implications of capillary electrochromatography. This review is aimed at giving a very broad outline of the progression of this technique since the late 1980s up to the present time, including some of the current limitations. Examples will be given of the analysis of a wide variety of materials including neutral, acidic, basic and chiral pharmaceutical compounds, along with some of the instrumentation used for this technique.

Comparison of aqueous and non-aqueous capillary electrochromatography for the separation of basic solutes.

The analysis of basic compounds by capillary electrochromatography (CEC) on silica-based materials using conventional HPLC stationary phases has failed to address the problem of severe peak tailing and non-reproducible chromatography. Several new generation stationary phases were evaluated using aqueous and non-aqueous mobile phases. The best results were obtained in the aqueous mode using Waters Symmetry Shield RP-8, a material in which the residual silanol groups were shielded by an octylcarbamate function. For comparison, experiments were carried out using unmodified silica.

Resolution of positional isomers by capillary electrochromatography.

Electroseparations have been very successful in increasing efficiencies and reducing analysis times. The analytical technique originally applied to open tube capillaries (capillary electrophoresis) has been used as a basis to develop renewed interest in electrochromatography. This paper describes the use of capillary electrochromatography to separate two positional isomers and describes a comparison between gas chromatography (GC), capillary electrochromatography (CEC) and nano-HPLC. The resolution of these isomers is quite crucial, since one of the isomers is the impurity in a pharmaceutically active drug.

Enantiomeric separations by capillary electrochromatography using a macrocyclic antibiotic chiral stationary phase.

Racemic mixtures of tryptophan and dinitrobenzoyl leucine have been successfully resolved by capillary electrochromatography (CEC) using the macrocyclic antibiotic teicoplanin, covalently bonded to a 5 microns silica support. Modification of a previously published packing procedure was required to pack reliable capillaries, capable of performing enantiomeric separations. Good levels of enantioselectivity were obtained in all cases, with optimised separations being performed in less than 6 min. Retention times, resolution and reproducibility are discussed.

Analysis of acidic compounds using capillary electrochromatography.

Capillary electrochromatography, CEC, is a hybrid of CE and HPLC and is rapidly gaining interest as a potential complementary technique. This paper provides an overview of literature concerning the separation of acidic compounds by CEC which fall into three distinct groups. These groups are those performed using capillaries packed with novel or unique stationary phases designed for CEC, and a smaller group where standard HPLC stationary phases packings such as ODS has been used. The third group involves the use of surface coated capillaries. This paper reviews the separation of acidic compounds by CEC and also includes a number of novel applications to illustrate the separation approaches and the analytical performance possible.

Interleukin-1 beta induces the synthesis of adenylyl cyclase in Swiss 3T3 fibroblasts and MG-63 osteosarcoma cells.

In this study, the longer term (> or = 6 hours) effect of interleukin-1 beta on adenylyl cyclase activity was investigated in Swiss 3T3 and MG-63 cells. No change was evident after 6 hours but after 1, 7 or 15 day incubations a significant increase in basal (1.5- 2 fold) and NaF-stimulated (2-4 fold) adenylyl cyclase activity was observed in interleukin-1 beta pre-treated cell membranes compared with non pre-treated controls. The response to forskolin, a direct stimulus of adenylyl cyclase, was also significantly enhanced, indicating that the effect of interleukin-1 beta was targeted to the enzyme itself. This action of interleukin-1 beta was blocked by co-incubation with cycloheximide, an inhibitor of protein synthesis, which demonstrated that de novo protein synthesis was required. It is concluded that interleukin-1 induces the expression of adenylyl cyclase in 3T3 and MG-63 cells, leading to enhanced activation by NaF and forskolin.

Capillary zone electrophoresis in pharmaceutical and biomedical analysis.

Capillary zone electrophoresis is a technique that is rapidly gaining popularity in the pharmaceutical industry because of the wide range of compounds applicable to the technique. These range from small biologically active molecules through to large nucleotides and proteins. This review outlines the historical development of the technique and some of the basic theory of capillary zone electrophoresis. This is followed by an explanation of some of the adaptations now being used to enable the separation of neutral molecules as well as charged species, and ways of achieving chiral selectivity.

Sarcomatoid carcinoma of the urinary bladder.

Sarcomatoid carcinoma of the urinary bladder is a rare malignancy of an aggressive nature usually seen in patients in the seventh to eighth decade of life. A case of sarcomatoid carcinoma of the bladder in a 37 year old male presenting with gross haematuria is reported. A potency-preserving radical cystoprostatectomy with ileal conduit was performed.

Transferrin receptor expression in primary superficial human bladder tumours identifies patients who develop recurrences.

A group of 65 patients with superficial bladder carcinoma was followed for 2 years and tumour recurrence rate was correlated both with transferrin receptor status of the initial primary tumour and with the results of voided urine cytology. Nine of 24 patients with transferrin receptor negative tumours had recurrences compared with 30 of 41 patients with transferrin receptor positive tumours. This difference was highly significant. Urine cytology at presentation was also predictive of further tumour formation: of 30 patients who were transferrin receptor positive and had positive urine cytology, 25 developed recurrences.