Aqueous/micellar peroxyoxalate chemiluminescent detection for ion chromatography

This thesis covers the development of the traditionally fluorescent bis(8-quinolinol-5-sulfonic acid) magnesium (II) fluorophore as a chemiluminescent emitter. A brief description of luminescence spectroscopy and its application to analytical chemistry lays the foundation to the discussion of the results obtained herein. This includes the synthesis and identification of two so called ‘water soluble’ aryl oxamides 2,2’-oxalyl-bis(trifluoromethanesulfonyl) imino] ethylene-bis(N- methylpyridinium) trifluoromethane sulfonate (PETQ) and 2,2’-oxalyl-bis(trifluoromethanesulfonyl) imino]ethylene-bis(N-pyridinium) chloride (PETH), previously developed for the US navy as a possible emergency light source, yet the synthetic methodology were incomplete. The inconsistencies of the synthetic methods for PETQ and PETH were overcome with yields satisfactory for their preliminary analytical evaluation. The evaluation of these aryl oxamides, including 4,4’-oxalyI- bis[(trifluoromethanesulfonyl) imino]ethylene-bis(l-methyM-benzylpiperidinium) trifluoromethanesulfonate (BPTQ), 4,4’-oxalyl-bis [(trifluoromethylsulfonyl)imino] ethylene-bis(N-methylmorpholinium)trifluoromethanesulfonate (METQ) and the oxalate bis(2,4,6-trichlorophenyl) oxalate (TCPO) were performed with the peroxyoxalate chemiluminescent reaction using bis(8-quinolinol-5-sulfonic acid) magnesium (II) as the fluorophore. A univariate optimisation of this system resulted in 0,0082 mol 1-1 the detection limit of magnesium in the absence of cationic surfactants and 0.0041 mol 1-1 in their presence for the majority of these compounds. The oxamides were found to be insoluble in water with long ulrasonication periods required to dissolve the compound, with solvents such as acetonitrile preferred. The determination of other chemiluminescent metal-8HQS chelates to replace magnesium -8HQS in the peroxyoxalate were limited to Al (III), Cd (II), Ca (II), In (II) and Zn (II), unfortunately these metals all possessed poorer detection limits than those obtained using magnesium The base reaction conditions used for the flow injection system with chemiluminescent detection were transferred to an ion chromatographic configuration for the separation of magnesium from other cations on an exchange column. After a univariate and simplex optimisation of these conditions, the detection limit of magnesium was found to be 0.0411 mol 1-1 which was less than the limits that could be achieved with fluorescent detection, The further development of this reaction to incorporate the displacement of magnesium from Mg-EDTA by other metals that possessed a higher conditional stability constant than magnesium also proved to be problematic with interferences from not only EDTA but from the eluant (lactic acid) from the cation column. Using this system the detection limits of the displacing metals were found to be in the order of 10 mg 1-1 which was substantially less that what was observed when exactly the same configuration was used with fluorescent detection. The final component of the thesis entails the discussion of the background emission that results from the reaction of oxamides/oxalates with hydrogen peroxide. A detailed investigation into the reaction of TCPO and hydrogen peroxide in the presence of various additives, such as imidazole , heavy atoms and triethylamine illustrated the existence of a further intermediate in fee mechanism for this reaction. The species responsible for this emission was attributed to the degradation product 2,4,6-trichlorophenyi of TCPO, which was supported by the non-existent background present with the oxamides that do not contain this degradation product.

Bioanalytical experiments for the undergraduate laboratory : monitoring glucose in sports drinks

This paper describes two complementary bioanalytical experiments for analyzing the concentration of glucose in sports drinks. The first experiment is a spectrophotometric enzyme assay employing the enzymes glucose oxidase (GOx) and horseradish peroxidase (HRP). The glucose is oxidized by the GOx, producing hydrogen peroxide, which is the substrate for HRP. In the reduction of the H2O2 a chromogen is oxidized, causing a color change. In the partner experiment, the GOx is immobilized on a platinum electrode using a dialysis membrane. The hydrogen peroxide produced in the enzyme reaction is monitored amperometrically by oxidizing the hydrogen peroxide produced. The simple method of preparing the enzyme electrode is useful in demonstrating the important parameters in defining the response of enzyme electrodes. The same sports drinks are analyzed in both experiments. The two experiments together illustrate the advantage of bioanalysis in analyzing complex samples with minimal sample preparation.

Milk lactate determination with a rotating bioreactor based on an electron transfer mediated by osmium complexes incorporating a continuous-flow/stopped-flow system

The high sensitivity that can be attained using a bienzymatic system and mediated by the redox polymer [Os(bpy)₂ClPyCH₂NHpoly(allylamine)] (Os-PAA), has been verified by on-line interfacing of a rotating bioreactor and continuous-flow/stopped-flow/continuous-flow processing. When the hydrogen peroxide formed by LO_x layer reaches the inner layer, the electronic flow between the immobilized peroxidase and the electrode surface produces a current, proportional to lactate concentration. The determination of lactate was possible with a limit of detection of 5 nmol l⁻¹ in the processing of as many as 30 samples per hour. This arrangement allows working in undiluted milk samples with a good stability and reproducibility. Horseradish peroxidase [EC 1.11.1.7] and Os-PAA were covalently immobilized on the glassy carbon electrode surface (upper cell body), lactate oxidase [EC 1.1.3.x] was immobilized on a disk that can be rotated.

Antioxidant enzyme activities of iron-saturated bovine lactoferrin (Fe-bLf) in human gut epithelial cells under oxidative stress

Chemoprevention by dietary constituents in the form of functional food has emerged as a novel approach to control inflammatory diseases and cancers. Recently we reported for the first time that iron content is a critical determinant in the anti-tumour activity of bovine milk lactoferrin (bLf). We therefore wanted to evaluate the chemo-preventative efficacy of Apo-bLF and 100% iron-saturated bLF (Fe-bLF) on hydrogen peroxide (H2O 2)-induced colon carcinogenesis, and their influence on antioxidant enzyme activities within colon carcinogenesis. This was undertaken through observing how oxidative stress induced by H2O2 alters antioxidant enzyme activity within HT29 colon cancer cells, and then observing changes in this activity by treatments with the different antioxidants ascorbic acid (AA), Apo-bLF and Fe-bLF. All antioxidant enzymes (catalase, glutathione peroxidase (GPx), glutathione reductase (GR), glutathione-s-transferase (GsT) and superoxide dismutase (SOD)) appeared to be increased within HT29 cells, even prior to H2O2 exposure, and all enzymes showed significant decreased activity when cells were treated with the antioxidants AA, Apo-bLF or Fe-bLF, with or without H2O2 exposure. The results indicate that all three antioxidants have the ability to scavenge ROS, lower antioxidant enzyme activities within already excited states, and possibly allow colon cancer cells to be overcome by oxidative stress that would normally be prevented, perhaps leading to damage and potential apoptosis of the cancer cells. In conclusion, the anti-oxidative effects of Apo-bLF and Fe-bLf studied for the first time, show dynamic changes that may allow for necessary protection from imbalanced oxidative conditions, and potential at reducing the ability of cancer cells to protect themselves from oxidative stress states.

Effect of selenium-saturated bovine lactoferrin (Se-bLF) on antioxidant enzyme activities in human gut epithelial cells under oxidative stress

Cancer and many chronic inflammatory diseases are associated with increased amounts of reactive oxygen species (ROS). The potential cellular and tissue damage created by ROS has significant impact on many disease and cancer states and natural therapeutics are becoming essential in regulating altered redox states. We have shown recently that iron content is a critical determinant in the antitumour activity of bovine milk lactoferrin (bLF). We found that 100% iron-saturated bLF (Fe-bLF) acts as a potent natural adjuvant and fortifying agent for augmenting cancer chemotherapy and thus has a broad utility in the treatment of cancer. Furthermore, we also studied the effects of iron saturated bLF's ability as an antioxidant in the human epithelial colon cancer cell line HT29, giving insights into the potential of bLF in its different states. Thus, metal saturated bLF could be implemented as anti-cancer neutraceutical. In this regard, we have recently been able to prepare a selenium (Se) saturated form of bLF, being up to 98% saturated. Therefore, the objectives of this study were to determine how oxidative stress induced by hydrogen peroxide (H₂O₂) alters antioxidant enzyme activity within HT29 epithelial colon cancer cells, and observe changes in this activity by treatments with different antioxidants ascorbic acid (AA), Apo (iron free)-bLF and selenium (Se)-bLF. The states of all antioxidant enzymes (glutathione peroxidase (GPx), glutathione reductase (GR), glutathione-s-transferase (GsT), catalase and superoxide dismutase (SOD)) demonstrated high levels within untreated HT29 cells compared to the majority of other treatments being used, even prior to H₂O₂ exposure. All enzymes showed significant alterations in activity when cells were treated with antioxidants AA, Apo-bLF or Se-bLF, with and/or without H₂O₂ exposure. Obvious indications that the Se content of the bLF potentially interacted with the glutathione (GSH)/GPx/GR/GsT associated redox system could be observed immediately, showing capability of Se-bLF being highly beneficial in helping to maintain a balance between the oxidant/antioxidant systems within cells and tissues, especially in selenium deficient systems. In conclusion, the antioxidative defence activity of Se-bLf, investigated in this study for the first time, shows dynamic adaptations that may allow for essential protection from the imbalanced oxidative conditions. Because of its lack of toxicity and the availability of both selenium and bLF in whole milk, Se-bLF offers a promise for a prospective natural dietary supplement, in addition to being an immune system enhancement, or a potential chemopreventive agent for cancers.

Use of silicone hydrogel contact lenses by Australian optometrists

Background: Prior to the launch of silcone hydrogel (Si-H) materials in Australia in 1999, only 1.6 per cent of lenses were prescribed on a continuous wear basis.

Methods: One thousand surveys were distributed randomly to practitioners in Australia during January 2000, 2001 and 2002 (total surveys 3,000). Each anonymous survey requested data about the next 10 patients fitted with contact lenses, including date, age, gender, new fitting or refitting, lens material type, lens design, frequency of replacement, modality of wear, uses per week and care regimen.

Results: Twenty per cent (599) were returned, reporting data on 5,976 fittings. A total of 710 fittings used Si-H contact lenses (11.9 per cent), which represented 18.6 per cent of all soft lens fittings. During the three years, the proportion of practitioners prescribing Si-H lenses increased from 42.2 to 52.5 per cent. In 2000, 43.8 per cent were daily wear, which decreased to 32.2 per cent by 2002. The solution system of choice for daily wear lenses was multipurpose solutions (98.4 per cent); the only alternative was hydrogen peroxide systems. Continuous wear represented 11.7 per cent of all fittings, ofwhich 85.7 per cent were Si-H, 3.0 per cent RGP lenses and 11.3 per cent conventional hydrogels. For continuous wear, 79.0 per cent of fittings were to existing wearers, whereas for daily wear, 59.4 per cent were existing wearers. More males were prescribed Si-H continuous wear contact lenses, while females were more likely to be prescribed Si-H on a daily wear basis.

Discussion: Si-H contact lenses were introduced to the Australian market as a continuous wear contact lens, yet many practitioners use this product for daily wear with multipurpose solutions. By 2002, more practitioners were prescribing Si-H contact lenses for continuous wear than in 2000, suggesting a growing confidence in that product for that mode of wear. A comparison with an earlier survey shows there is an increase in continuous wear from 1.6 per cent to 11.7 per cent over a five-year period.

Contact lens prescribing in the Australian states and territories 2001

Background: This study was conducted to ascertain current preferences for contact lens prescribing in the Australian states and territories.

Methods: One thousand questionnaires were randomly distributed to proportionate samples of optometrists in each state of Australia. We requested details of the first 10 patients fitted with contact lenses after receipt of the questionnaire.

Results: One hundred and seventy-eight completed questionnaires were returned, detailing contact lens fits to 1,611 patients. The mean age of the patient group was 32.1 $pL 13.0 yrs (65 per cent female). For Australia as a whole: 53 per cent of patients were existing wearers, the remainder were new fits; 93 per cent of new fits were with soft lenses, of which seven per cent were for extended wear. Of the refits, 89 per cent were soft lenses and 18 per cent for extended wear. The lens material of first choice was mid-water-content (62 per cent of all soft lens fits). Only eight per cent of all soft fits were for lenses that were not replaced on a planned basis, with two weeks being the replacement interval of choice in all states and territories. The majority of rigid lenses were prescribed using mid-Dk materials (50 per cent). Analysis of solution prescribing indicates that multi-purpose products were the most common regimens for planned replacement soft lenses. The percentage of hydrogen peroxide prescribed increased as lens replacement became less frequent. By state or territory: practitioners in Tasmania prescribed more extended wear than those in any other state (p = 0.007) and practitioners in Queensland prescribed more daily disposable contact lenses than those in any other state (p = 0.009).

Conclusions: Non-planned replacement lenses are now rarely prescribed to patients. Extended-wear lenses and rigid lenses continue to be prescribed more to existing contact lens wearers than to new patients. The impact of soft multifocal lens designs on contact lens prescribing is very small, ranging from 2.6 per cent in Queensland to 4.7 per cent in Victoria, despite 20 per cent of patients being more than 45 years of age.

Trends in Australian contact lenses prescribing 2000

Background:This study was conducted to ascertain current modes of contact lens prescribing in Australia.

Methods:One thousand questionnaires were randomly distributed to proportionate samples of optometrists in each state of Australia. The profession fits the majority of contact lenses in Australia and the sample represents 55 per cent of all optometrists. We requested details of the first 10 patients fitted with contact lenses after receipt of the questionnaire.

Results:We received 224 completed questionnaires, detailing contact lens fits to 2,230 patients. The mean age of the patient group was 32.3 ± 12.9 years and 65 per cent of these were female. Sixty per cent of patients were existing wearers, the remainder being new fits. The data indicated that 94 per cent of new fits were with soft lenses, of which six per cent were for extended wear. For refits 83 per cent were soft lenses and 24 per cent were extended wear. It was clear that the lens of first choice was mid-water-content (52 per cent of all soft lens fits). Only nine per cent of all soft fits were for lenses which were not replaced on a planned basis. The majority of rigid lenses were prescribed using mid-Dk materials (71 per cent). Analysis of solution prescribing indicates that multipurpose products were the most common regimens for planned replacement soft lenses. The percentage of hydrogen peroxide prescribed increased as lens replacement became less frequent.

Conclusions:Non-planned replacement lenses are now rarely prescribed to patients. Extended wear lenses and rigid lenses are prescribed more to existing contact lens wearers. The impact of multifocal lens designs on contact lens prescribing is very small, namely, five per cent of soft contact lenses and eight per cent of rigid lenses, despite 20 per cent of patients being over 45 years of age.

The influence of genetic background on the induction of oxidative stress and impaired insulin secretion in mouse islets

Aims/hypothesis We determined whether high-glucose-induced beta cell dysfunction is associated with oxidative stress in the DBA/2 mouse, a mouse strain susceptible to islet failure.

Materials and methods Glucose- and non-glucose-mediated insulin secretion from the islets of DBA/2 and control C57BL/6 mice was determined following a 48-h exposure to high glucose. Flux via the hexosamine biosynthesis pathway was assessed by determining O-glycosylated protein levels. Oxidative stress was determined by measuring hydrogen peroxide levels and the expression of anti-oxidant enzymes.

Results Exposure to high glucose levels impaired glucose-stimulated insulin secretion in DBA/2 islets but not C57BL/6 islets, and this was associated with reduced islet insulin content and lower ATP levels than in C57BL/6 islets. Exposure of islets to glucosamine for 48 h mimicked the effects of high glucose on insulin secretion in the DBA/2 islets. High glucose exposure elevated O-glycosylated proteins; however, this occurred in islets from both strains, excluding a role for O-glycosylation in the impairment of DBA/2 insulin secretion. Additionally, both glucosamine and high glucose caused an increase in hydrogen peroxide in DBA/2 islets but not in C57BL/6 islets, an effect prevented by the antioxidant N-acetyl-l-cysteine. Interestingly, while glutathione peroxidase and catalase expression was comparable between the two strains, the antioxidant enzyme manganese superoxide dismutase, which converts superoxide to hydrogen peroxide, was increased in DBA/2 islets, possibly explaining the increase in hydrogen peroxide levels.

Conclusions/interpretation Chronic high glucose culture caused an impairment in glucose-stimulated insulin secretion in DBA/2 islets, which have a genetic predisposition to failure, and this may be the result of oxidative stress.

Novel approaches to process bamboo plants into fibres and their UV-blocking property

Currently viscose production methods are primarily used to process bamboo into commercial textile fibres. However, viscose methods use large quantities of chemicals and hence the process is not considered as environmentally friendly. The process also fails to retain bamboo’s inherent unique properties such as ultraviolet (UV) screening and antibacterial functions. Hence, it is necessary to design an effective and more eco-friendly manufacturing method that would also retain the unique properties of raw bamboo plant into the fibres. In this research, bamboo was processed using new methods involving thermo mechanical treatments such as ultra-sonication, shaker milling and boiling with continuous stirring. Sodium hydroxide, hydrogen peroxide, enzyme and water were used separately in this process and their effects on fibre processing were compared. The morphology and UV shielding ability were analysed before and after processing. It was demonstrated that bamboo can be processed into fibres using only water and ball milling without the aid of any hazardous chemicals. The combination of mild acid hydrolysis and ultrasonic treatment with hydrogen peroxide was effective in the fibre separation and provided better appearance of fibres.

Glutathione peroxidase-1 reduces influenza A virus-induced lung inflammation

Oxidative stress caused by excessive reactive oxygen species production is implicated in influenza A virus–induced lung disease. Glutathione peroxidase (GPx)-1 is an antioxidant enzyme that may protect lungs from  such damage. The objective of this study was to determine if GPx-1 protects the lung against influenza A virus–induced lung inflammation in vivo.

Human corneal epithelial cell shedding and fluorescein staining in response to silicone hydrogel lenses and contact lens disinfecting solutions

A pilot study was conducted to evaluate human corneal epithelial cell shedding in response to wearing a silicone hydrogel contact lens/solution combination inducing corneal staining. The nature of ex vivo collected cells staining with fluorescein was also examined. A contralateral eye study was conducted in which up to eight participants were unilaterally exposed to a multipurpose contact lens solution/silicone hydrogel lens combination previously shown to induce corneal staining (renu® fresh™ and balafilcon A; test eye), with the other eye using a combination of balafilcon A soaked in a hydrogen peroxide care system (Clear Care®; control eye). Lenses were worn for 2, 4 or 6 hours. Corneal staining was graded after lens removal. The Ocular Surface Cell Collection Apparatus was used to collect cells from the cornea and the contact lens. In the test eye, maximum solution-induced corneal staining (SICS) was observed after 2 hours of lens wear (reducing significantly by 4 hours; p < 0.001). There were significantly more cells collected from the test eye after 4 hours of lens wear when compared to the control eye and the collection from the test eye after 2 hours (for both; n = 5; p < 0.001). The total cell yield at 4 hours was 813 ± 333 and 455 ± 218 for the test and control eyes, respectively (N = 5, triplicate, p = 0.003). A number of cells were observed to have taken up the fluorescein dye from the initial fluorescein instillation. Confocal microscopy of fluorescein-stained cells revealed that fluorescein was present throughout the cell cytoplasm and was retained in the cells for many hours after recovery from the corneal surface. This pilot study indicates that increased epithelial cell shedding was associated with a lens-solution combination which induces SICS. Our data provides insight into the transient nature of the SICS reaction and the nature of fluorescein staining observed in SICS.

Recent insights into microbial catalases: isolation, production and purification

Catalase, an oxidoreductase enzyme, works as a detoxification system inside living cells against reactive oxygen species formed as a by-product of different metabolic reactions. The enzyme is found in a wide range of aerobic and anaerobic organisms. Catalase has also been employed in various analytical and diagnostic methods in the form of biosensors and biomarkers in addition to its other applications in textile, paper, food and pharmaceutical industries. New applications for catalases are constantly emerging thanks to their high turnover rate, distinct evolutionary origin, relatively simple and well-defined reaction mechanisms. The following review provides comprehensive information on isolation, production and purification of catalases with different techniques from various microbial sources along with their types, structure, mechanism of action and applications.