Maximizing Product Formation and Minimizing Degradation: A Look at Buffering Conditions and Post-Reaction Degradation for the In-Line Jaffe Reaction with Capillary Electrophoresis

Creatinine levels in blood serum are typically used to assess renal function. Clinical determination of creatinine is often based on the Jaffe reaction, in which creatinine in the serum reacts with sodium picrate, resulting in a spectrophotometrically quantifiable product. Previous work from our lab has introduced an electrophoretically mediated initiation of this reaction, in which nanoliter plugs of individual reagent solutions can be added to the capillary and then mixed and reacted. Following electrophoretic separation of the product from excess reactant(s), the product can be directly determined on column. This work aims to gain a detailed understanding of the in-capillary reagent mixing dynamics, in-line reaction yield, and product degradation during electrophoresis, with an overall goal of improving assay sensitivity. One set of experiments focuses on maximizing product formation through manipulation of various conditions such as pH, voltage applied, and timing of the applied voltage, in addition to manipulations in the identity, concentration, and pH of the background electrolyte. Through this work, it was determined that dramatic changes in local voltage fields within the various reagent zones lead to ineffective reagent overlapping. Use of the software simulation program Simul 5 enabled visualization of the reaction dynamics within the capillary, specifically the wide variance between the electric field intensities within the creatinine and picrate zones. Because of this simulation work, the experimental method was modified to increase the ionic strength of the creatinine reagent zone to lower the local voltage field, thus producing more predictable and effective overlap conditions for the reagents and allowing the formation of more Jaffe product. As second set of experiments focuses on controlling the post-reaction product degradation. In that vein, we have systematically explored the importance of the identity, concentration, and pH of the background electrolyte on the post-reaction degradation rate of the product. Although prior work with borate background electrolytes indicated that product degradation was probably a function of the ionic strength of the background electrolyte, this work with a glycine background electrolyte demonstrates that degradation is in fact not a function of ionic strength of the background electrolyte. As the concentration and pH of the glycine background increased, the rate of degradation of product did not change dramatically, whereas in borate-buffered systems, the rate of Jaffe product degradation increased linearly with background electrolyte concentration above 100.0 mM borate. Similarly, increasing pH of the glycine background electrolyte did not result in a corresponding increase in product degradation, as it had with the borate background electrolyte. Other general trends that were observed include: increasing background electrolyte concentration increases peak efficiency and higher pH favors product formation; thus, it appears that use of a background electrolyte other than borate, such as glycine, the rate of degradation of the Jaffe product can be slowed, increasing the sensitivity of this in-line assay.

Analysis of lorazepam and its 30-glucuronide in human urine by capillary electrophoresis: evidence for the formation of two distinct diastereoisomeric glucuronides

Lorazepam (LOR) is a 3-hydroxy-1,4-benzodiazepine that is chiral and undergoes enantiomerization at room temperature. In humans, about 75% of the administered dose of LOR is excreted in the urine as its 30-glucuronide. CE-MS with negative ESI was used to confirm the presence of LOR-30-glucuronide in urines that stemmed from a healthy individual who ingested 1 or 2 mg LOR, whereas free LOR could be detected in extracts prepared from enzymatically hydrolyzed urines. As the 30-glucuronidation reaction occurs at the chiral center of the molecule, two diastereoisomers can theoretically be formed, molecules that can no longer interconvert. The stereoselective formation of LOR glucuronides in humans and in vitro was investigated. MEKC analysis of extracts of the nonhydrolyzed urines suggested the presence of the two different LOR glucuronides in the urine. The formation of the same two diastereoisomers was also observed in vitro employing incubations of LOR with human liver microsomes in the presence of uridine 5'-diphospho-glucuronic acid as coenzyme. The absence of other coenzymes excluded the formation of phase I or other phase II metabolites of LOR. Both results revealed a stereoselectivity, one diastereoisomer being formed in a higher amount than the other. After enzymatic hydrolysis using beta-glucuronidase, these peaks could not be detected any more. Instead, LOR was monitored. Analysis of the extracts prepared from enzymatically hydrolyzed urines by MEKC in the presence of 2-hydroxypropyl-beta-CD revealed the enantiomerization process of LOR (observation of two peaks of equal magnitude connected with a plateau zone). The data presented provide for the first time the evidence of the stereoselectivity of the LOR glucuronidation in humans.

Dynamic high-resolution computer simulation of isotachophoretic enantiomer separation and zone stability

The development of electrophoretic computer models and their use for simulation of electrophoretic processes has increased significantly during the last few years. Recently, GENTRANS and SIMUL5 were extended with algorithms that describe chemical equilibria between solutes and a buffer additive in a fast 1:1 interaction process, an approach that enables simulation of the electrophoretic separation of enantiomers. For acidic cationic systems with sodium and H3 0(+) as leading and terminating components, respectively, acetic acid as counter component, charged weak bases as samples, and a neutral CD as chiral selector, the new codes were used to investigate the dynamics of isotachophoretic adjustment of enantiomers, enantiomer separation, boundaries between enantiomers and between an enantiomer and a buffer constituent of like charge, and zone stability. The impact of leader pH, selector concentration, free mobility of the weak base, mobilities of the formed complexes and complexation constants could thereby be elucidated. For selected examples with methadone enantiomers as analytes and (2-hydroxypropyl)-β-CD as selector, simulated zone patterns were found to compare well with those monitored experimentally in capillary setups with two conductivity detectors or an absorbance and a conductivity detector. Simulation represents an elegant way to provide insight into the formation of isotachophoretic boundaries and zone stability in presence of complexation equilibria in a hitherto inaccessible way.

Sampling strategies for capillary isoelectric focusing with electroosmotic zone mobilization assessed by high-resolution dynamic computer simulation

The impact of initial sample distribution on separation and focusing of analytes in a pH 3–11 gradient formed by 101 biprotic carrier ampholytes under concomitant electroosmotic displacement was studied by dynamic high-resolution computer simulation. Data obtained with application of the analytes mixed with the carrier ampholytes (as is customarily done), as a short zone within the initial carrier ampholyte zone, sandwiched between zones of carrier ampholytes, or introduced before or after the initial carrier ampholyte zone were compared. With sampling as a short zone within or adjacent to the carrier ampholytes, separation and focusing of analytes is shown to proceed as a cationic, anionic, or mixed process and separation of the analytes is predicted to be much faster than the separation of the carrier components. Thus, after the initial separation, analytes continue to separate and eventually reach their focusing locations. This is different to the double-peak approach to equilibrium that takes place when analytes and carrier ampholytes are applied as a homogenous mixture. Simulation data reveal that sample application between two zones of carrier ampholytes results in the formation of a pH gradient disturbance as the concentration of the carrier ampholytes within the fluid element initially occupied by the sample will be lower compared to the other parts of the gradient. As a consequence thereof, the properties of this region are sample matrix dependent, the pH gradient is flatter, and the region is likely to represent a conductance gap (hot spot). Simulation data suggest that sample placed at the anodic side or at the anodic end of the initial carrier ampholyte zone are the favorable configurations for capillary isoelectric focusing with electroosmotic zone mobilization.

Impact of Taylor-Aris diffusivity on analyte and system zone dispersion in CZE assessed by computer simulation and experimental validation.

Application of pressure-driven laminar flow has an impact on zone and boundary dispersion in open tubular CE. The GENTRANS dynamic simulator for electrophoresis was extended with Taylor-Aris diffusivity which accounts for dispersion due to the parabolic flow profile associated with pressure-driven flow. Effective diffusivity of analyte and system zones as functions of the capillary diameter and the amount of flow in comparison to molecular diffusion alone were studied for configurations with concomitant action of imposed hydrodynamic flow and electroosmosis. For selected examples under realistic experimental conditions, simulation data are compared with those monitored experimentally using modular CE setups featuring both capacitively coupled contactless conductivity and UV absorbance detection along a 50 μm id fused-silica capillary of 90 cm total length. The data presented indicate that inclusion of flow profile based Taylor-Aris diffusivity provides realistic simulation data for analyte and system peaks, particularly those monitored in CE with conductivity detection.

Determination of particle concentration in the breathing zone for four different types of office ventilation systems

Many factors affect the airflow patterns, thermal comfort, contaminant removal efficiency and indoor air quality at individual workstations in office buildings. In this study, four ventilation systems were used in a test chamber designed to represent an area of a typical office building floor and reproduce the real characteristics of a modern office space. Measurements of particle concentration and thermal parameters (temperature and velocity) were carried out for each of the following types of ventilation systems: a) conventional air distribution system with ceiling supply and return; b) conventional air distribution system with ceiling supply and return near the floor; c) underfloor air distribution system; and d) split system. The measurements aimed to analyse the particle removal efficiency in the breathing zone and the impact of particle concentration on an individual at the workstation. The efficiency of the ventilation system was analysed by measuring particle size and concentration, ventilation effectiveness and the Indoor/Outdoor ratio. Each ventilation system showed different airflow patterns and the efficiency of each ventilation system in the removal of the particles in the breathing zone showed no correlation with particle size and the various methods of analyses used.

Determining the zone of reflection for posterior corneal surface comparison phakometry

Although comparison phakometry has been used by a number of studies to measure posterior corneal shape, these studies have not calculated the size of the posterior corneal zones of reflection they assessed. This paper develops paraxial equations for calculating posterior corneal zones of reflection, based on standard keratometry equations and equivalent mirror theory. For targets used in previous studies, posterior corneal reflection zone sizes were calculated using paraxial equations and using exact ray tracing, assuming spherical and aspheric corneal surfaces. Paraxial methods and exact ray tracing methods give similar estimates for reflection zone sizes less than 2 mm, but for larger zone sizes ray tracing methods should be used.

Effect of clear zone widths on run-off-road crash outcomes

Current guidelines on clear zone selection and roadside hazard management adopt the US approach based on the likelihood of roadside encroachment by drivers. This approach is based on the available research conducted in the 1960s and 70s. Over time, questions have been raised regarding the robustness and applicability of this research in Australasia in 2010 and in the Safe System context. This paper presents a review of the fundamental research relating to selection of clear zones. Results of extensive rural highway statistical data modelling suggest that a significant proportion of run-off-road to the left casualty crashes occurs in clear zones exceeding 13 m. They also show that the risk of run-off-road to the left casualty crashes was 21% lower where clear zones exceeded 8 m when compared with clear zones in the 4 – 8 m range. The paper discusses a possible approach to selection of clear zones based on managing crash outcomes, rather than on the likelihood of roadside encroachment which is the basis for the current practice. It is expected that this approach would encourage selection of clear zones wider than 8 m when the combination of other road features suggests higher than average casualty crash risk.

Review : Surfers Paradise foreshore redevelopment masterplan - urban plaza zone

The Centre for Subtropical Design has prepared this submission to assist the Gold Coast City Council to finalise a plan and detailed design guidelines for the Urban Plaza Zone of Surfers Paradise Foreshore Redevelopment Masterplan which will create a public open space ‘alive’ with the quality appropriate to a place which is both a local centre and an international destination. This review has been informed by the two over-arching values identified as characteristics of a subtropical place and people’s connection to it:  A sense of openness and permeability, and  Engagement with the natural environment. The existing qualities of the foreshore area proposed as the Urban Plaza Zone, reflect these subtropical place values, and are integral to the Surfers Paradise identity:  Seamless visual and spatial access to the beach and sea,  Permeable interface between beach and built zones provided by beach planting and shade to sand by Pandanus,  A shade zone mediating beach and linear promenade, road and commercial zones, enabling a variety of social and visual experiences, on soft and hard finishes, and  A lively, constantly moving shared road and pedestrian way catering for events and day to day activities with visual access to beach and shaded areas. The Centre for Subtropical Design commends the Gold Coast City Council on preparing a plan for a public open space that is a contemporary departure from the adhoc basis of development that has occurred, in that it will make this area more accessible. However, the proposed plan seems to be working too hard in terms of ‘program’. While providing an identifiable interruption in the linear extent of the Foreshore, the lack of continuity of design in terms of both hardscaping (such as perpendicular paving elements) and softscaping (such as tree selections) may contribute to a lack of definition for the entire Foreshore as a place that mediates, along its length, between sea and land. Providing a hard edge to a beach character of soft and planted transitional elements needs to balance the proposed visual and physical barrier with the need for perceived and actual easy access. The Surfers Paradise identity needs strengthening through attention to planting for shade, materials, particularly selection of paving colours, and stronger delineation of the linear nature of the Foreshore. The Urban Plaza zone is an appropriate interruption to the continuous planting, however the link from the commercial zone overtakes the public and beach zone. A more seamless transition from shop to sea, better integration of the roadway and pedestrian zone and improved physical transition from concrete to sand is recommended. Built form solutions must be robust and designed with the subtropical design principles and the Surfers Paradise identity as underpinning parameters for a lasting and memorable public open space.

Zonal chondrocyte subpopulations reacquire zone-specific characteristics during in Vitro redifferentiation

Background: If chondrocytes from the superficial, middle, and deep zones of articular cartilage could maintain or regain their characteristic properties during in vitro culture, it would be feasible to create constructs comprising these distinctive zones. ----- ----- Hypothesis: Zone-specific characteristics of zonal cell populations will disappear during 2-dimensional expansion but will reappear after 3-dimensional redifferentiation, independent of the culture technique used (alginate beads versus pellet culture).----- ----- Study Design: Controlled laboratory study.----- ----- Methods: Equine articular chondrocytes from the 3 zones were expanded in monolayer culture (8 donors) and subsequently redifferentiated in pellet and alginate bead cultures for up to 4 weeks. Glycosaminoglycans and DNA were quantified, along with immunohistochemical assessment of the expression of various zonal markers, including cartilage oligomeric protein (marking cells from the deeper zones) and clusterin (specifically expressed by superficial chondrocytes).----- ----- Results: Cell yield varied between zones, but proliferation rates did not show significant differences. Expression of all evaluated zonal markers was lost during expansion. Compared to the alginate bead cultures, pellet cultures showed a higher amount of glycosaminoglycans produced per DNA after redifferentiation. In contrast to cells in pellet cultures, cells in alginate beads regained zonal differences, as evidenced by zone-specific reappearance of cartilage oligomeric protein and clusterin, as well as significantly higher glycosaminoglycans production by cells from the deep zone compared to the superficial zone.----- ----- Conclusion: Chondrocytes isolated from the 3 zones of equine cartilage can restore their zone-specific matrix expression when cultured in alginate after in vitro expansion.

Theraplay : staying in life's performance zone with play - Homo ludens: Man the player - Homo Cogito: Man the thinker

The paper introduces an update on a therapy originally developed by Jernberg (1979) for the American Head Start Programme for use with children and parents. Theraplay was based on John Bowlby's (1969) attachment theory and was started by Jernberg in Chicago. The present use of theraplay is for young adults to deal with negative mood and mood disorders resulting from stress, anxiety or depression. It is for the sad, lonely, sluggish, miserable or melancholic amongst us as well as the worried well.

Motivation : staying in the achievement zone with personal integrity

This paper was presented at orientation, to a select group of Business Faculty First Year students accepted into the "Corporate Partners in Excellence Programme" (CPIE). It discusses some of the strategies for continuing as high performing students in University studies whilst at the same time maturing into successful, ethical professionals with a social and environmental conscience.