Determination of intracellular glutathione and glutathione disulfide using high performance liquid chromatography with acidic potassium permanganate chemiluminescence detection

Measurement of glutathione (GSH) and glutathione disulfide (GSSG) is a crucial tool to assess cellular redox state. Herein we report a direct approach to determine intracellular GSH based on a rapid chromatographic separation coupled with acidic potassium permanganate chemiluminescence detection, which was extended to GSSG by incorporating thiol blocking and disulfide bond reduction. Importantly, this simple procedure avoids derivatisation of GSH (thus minimising auto-oxidation) and overcomes problems encountered when deriving the concentration of GSSG from ‘total GSH’. The linear range and limit of detection for both analytes were 7.5 × 10−7 to 1 × 10−5 M, and 5 × 10−7 M, respectively. GSH and GSSG were determined in cultured muscle cells treated for 24 h with glucose oxidase (0, 15, 30, 100, 250 and 500 mU mL−1), which exposed them to a continuous source of reactive oxygen species (ROS). Both analyte concentrations were greater in myotubes treated with 100 or 250 mU mL−1 glucose oxidase (compared to untreated controls), but were significantly lower in myotubes treated with 500 mU mL−1 (p < 0.05), which was rationalised by considering measurements of H2O2 and cell viability. However, the GSH/GSSG ratio in myotubes treated with 100, 250 and 500 mU mL−1 glucose oxidase exhibited a dose-dependent decrease that reflected the increase in intracellular ROS.

Endogenous parathyroid hormone is associated with reduced cartilage volume in vivo in a population-based sample of adult women

Objectives Animal and in vitro studies suggest that parathyroid hormone (PTH) may affect articular cartilage. However, little is known of the relationship between PTH and human joints in vivo.

Design Longitudinal.

Setting Barwon Statistical Division, Victoria, Australia.

Participants 101 asymptomatic women aged 35–49 years (2007–2009) and without clinical knee osteoarthritis, selected from the population-based Geelong Osteoporosis Study.

Risk factors Blood samples obtained 10 years before (1994–1997) and stored at −80°C for random batch analyses. Serum intact PTH was quantified by chemiluminescent enzyme assay. Serum 25-hydroxyvitamin D (25(OH)D) was assayed using equilibrium radioimmunoassay. Models were adjusted for age, bone area and body mass index; further adjustment was made for 25(OH)D and calcium supplementation.

Outcome Knee cartilage volume, measured by MRI.

Results A higher lnPTH was associated with reduced medial—but not lateral—cartilage volume (regression coefficient±SD, p value: −72.2±33.6 mm3, p=0.03) after adjustment for age, body mass index and bone area. Further sinusoidal adjustment (−80.8±34.4 mm3, p=0.02) and 25(OH)D with seasonal adjustment (−72.7±35.1 mm3, p=0.04), calcium supplementation and prevalent osteophytes did not affect the results.

Conclusions A higher lnPTH might be detrimental to knee cartilage in vivo. Animal studies suggest that higher PTH concentrations reduce the healing ability of cartilage following minor injury. This may be apparent in the presence of increased loading, which occurs in the medial compartment, placing the medial cartilage at higher risk for injury.

The effect of short-term canola oil ingestion on oxidative stress in the vasculature of stroke-prone spontaneously hypertensive rats

Background: This study aimed to determine if 25 days of canola oil intake in the absence of excess dietary salt or together with salt loading affects antioxidant and oxidative stress markers in the circulation. A further aim was to determine the mRNA expression of NADPH oxidase subunits and superoxide dismutase (SOD) isoforms in the aorta of stroke-prone spontaneously hypertensive (SHRSP) rats.

Methods: Male SHRSP rats, were fed a defatted control diet containing 10% wt/wt soybean oil or a defatted treatment diet containing 10% wt/wt canola oil, and given tap water or water containing 1% NaCl. Blood was collected at the end of study for analysis of red blood cell (RBC) antioxidant enzymes, RBC and plasma malondialdehyde (MDA), plasma 8-isoprostane and plasma lipids. The aorta was removed and the mRNA expression of NOX2, p22phox, CuZn-SOD, Mn-SOD and EC-SOD were determined.

Results: In the absence of salt, canola oil reduced RBC SOD and glutathione peroxidase, and increased total cholesterol and LDL cholesterol compared with soybean oil. RBC glutathione peroxidase activity was significantly lower in both the salt loaded groups compared to the soybean oil only group. In addition, RBC MDA and plasma HDL cholesterol were significantly higher in both the salt loaded groups compared to the no salt groups. Plasma MDA concentration was higher and LDL cholesterol concentration lower in the canola oil group loaded with salt compared to the canola oil group without salt. The mRNA expression of NADPH oxidase subunits and SOD isoforms were significantly reduced in the canola oil group with salt compared to canola oil group without salt.

Conclusion: In conclusion, these results indicate that canola oil reduces antioxidant status and increases plasma lipids, which are risk factors for cardiovascular disease. However, canola oil in combination with salt intake increased MDA, a marker of lipid peroxidation and decreased NAPDH oxidase subunits and aortic SOD gene expression.

Differential effects of dietary canola and soybean oil intake on oxidative stress in stroke-prone spontaneously hypertensive rats

Background: Canola oil shortens the life span of stroke-prone spontaneously hypertensive (SHRSP) rats compared with rats fed soybean oil when given as the sole dietary lipid source. One possible mechanism leading to the damage and deterioration of organs due to canola oil ingestion is oxidative stress. This study investigated the effect of canola oil intake on oxidative stress in this animal model.
Method: Male SHRSP rats, were fed a defatted control diet containing 10% wt/wt soybean oil or a defatted treatment diet containing 10% wt/wt canola oil, and given water containing 1% NaCl. Blood pressure was measured weekly. Blood was collected prior to beginning the diets and at the end of completion of the study for analysis of red blood cell (RBC) antioxidant enzymes, RBC and plasma malondialdehyde (MDA), plasma 8- isoprostane and plasma lipids.
Results: Canola oil ingestion significantly decreased the life span of SHRSP rats compared with soybean oil, 85.8 ± 1.1 and 98.3 ± 3.4 days, respectively. Systolic blood pressure increased over time with a significant difference between the diets at the 6th week of feeding. Canola oil ingestion significantly reduced RBC superoxide dismutase, glutathione peroxidase and catalase activities, total cholesterol and low-density lipoprotein cholesterol compared with soybean oil. There were no significant differences in RBC MDA concentration between canola oil fed and soybean oil fed rats. In contrast, plasma MDA and 8-isoprostane concentration was significantly lower in the canola oil group compared to the soybean oil group.
Conclusion: In conclusion, canola oil ingestion shortens the life span of SHRSP rats and leads to changes in oxidative status, despite an improvement in the plasma lipids.

The effect of cocoa supplementation on hepatic steatosis, reactive oxygen species and LFABP in a rat model of NASH

Background: Non alcoholic steatohepatitis is hypothesised to develop via a mechanism involving fat accumulation and oxidative stress. The current study aimed to investigate if an increase in oxidative stress was associated with changes in the expression of liver fatty acid binding protein in a rat model of non alcoholic steatohepatitis and whether cocoa supplementation attenuated those changes.

Methods: Female Sprague Dawley rats were fed a high fat control diet, a high fat methionine choline deficient diet, or one of four 12.5% cocoa supplementation regimes in combination with the high fat methionine choline deficient diet.

Results: Liver fatty acid binding protein mRNA and protein levels were reduced in the liver of animals with fatty liver disease when compared to controls. Increased hepatic fat content was accompanied by higher levels of oxidative stress in animals with fatty liver disease when compared to controls. An inverse association was found between the levels of hepatic liver fatty acid binding protein and the level of hepatic oxidative stress in fatty liver disease. Elevated NADPH oxidase protein levels were detected in the liver of animals with increased severity in inflammation and fibrosis. Cocoa supplementation was associated with partial attenuation of these pathological changes, although the severity of liver disease induced by the methionine choline deficient diet prevented complete reversal of any disease associated changes. Red blood cell glutathione was increased by cocoa supplementation, whereas liver glutathione was reduced by cocoa compared to methionine choline deficient diet fed animals.

Conclusion: These findings suggest a potential role for liver fatty acid binding protein and NADPH oxidase in the development of non alcoholic steatohepatitis. Furthermore, cocoa supplementation may have be of therapeutic benefit in less sever forms of NASH.

The impact of bullying on health care administration staff : reduced commitment beyond the influences of negative affectivity

Background: Investigations of workplace bullying in health care settings have tended to focus on nurses or other clinical staff. However, the organizational and power structures enabling bullying in health care are present for all employees, including administrative staff.

Purposes: The purpose of this study was to specifically focus on health care administration staff and examine the prevalence and consequences of workplace bullying in this occupational group.

Methodology/Approach: A cross-sectional study was conducted based on questionnaire data from health care administration staff who work across facilities within a medium to large health care organization in Australia. The questionnaire included measures of bullying, negative affectivity (NA), job satisfaction, organizational commitment, well-being, and psychological distress. The three hypotheses of the study were that (a) workplace bullying will be linked to negative employee outcomes, (b) individual differences on demographic factors will have an impact on these outcomes, and (c) individual differences in NA will be a significant covariate in the analyses. The hypotheses were tested using t tests and analyses of covariances.

Findings: A total of 150 health care administration staff completed the questionnaire (76% response rate). Significant main effects were found for workplace bullying, with lower organizational commitment and well-being with the effect on commitment remaining over and above NA. Main effects were found for age on job satisfaction and for employment type on psychological distress. A significant interaction between bullying and employment type for psychological distress was also observed. Negative affectivity was a significant covariate for all analyses of covariance.

Practice Implications: The applications of these results include the need to consider the occupations receiving attention in health care to include administration employees, that bullying is present across health care occupations, and that some employees, particularly part-time staff, may need to be managed slightly differently to the full-time workforce.

Reduced food access due to a lack of money, inability to lift and lack of access to a car for food shopping : a multilevel study in Melbourne, Victoria

Objective: To describe associations between demographic and individual and arealevel socio-economic variables and restricted household food access due to lack of money, inability to lift groceries and lack of access to a car to do food shopping.
Design: Multilevel study of three measures of restricted food access, i.e. running out of money to buy food, inability to lift groceries and lack of access to a car for food shopping. Multilevel logistic regression was conducted to examine the risk of each of these outcomes according to demographic and socio-economic variables.
Setting: Random selection of households from fifty small areas in Melbourne, Australia, in 2003.
Subjects: The main food shoppers in each household (n 2564).
Results: A lack of money was significantly more likely among the young and in households with single adults. Difficultly lifting was more likely among the elderly and those born overseas. The youngest and highest age groups both reported reduced car access, as did those born overseas and single-adult households. All three factors were most likely among those with a lower individual or household socio-economic position. Increased levels of area disadvantage were independently associated with difficultly lifting and reduced car access.
Conclusions: In Melbourne, households with lower individual socio-economic position and area disadvantage have restricted access to food because of a lack of money and/or having physical limitations due difficulty lifting or lack of access to a car for food shopping. Further research is required to explore the relationship between physical restrictions and food access.

Hip adductor muscle strength is reduced preceding and during the onset of groin pain in elite junior Australian football players

Groin pain is a condition with a high prevalence in young Australian football players. It is considered that early identification of this condition allows for optimal management. Eighty-six players from two elite under-age Australian football sides were screened weekly for hip adductor muscle strength, using a hand-held dynamometer and for the onset of groin pain. The maximum variation in the average hip adductor muscle strength values of the sample was a 2.6% decrease from baseline in week 7 of the study. Twelve players (14% of the sample studied) reported groin pain for two consecutive weeks and were considered to have an onset of groin injury. The mean hip adductor muscle strength of these players was decreased significantly from baseline by an average of 11.75 ± 2.50% at the week of pain onset (F = 264.76 (1,11), p < 0.001), and 5.82 ± 5.16% in the week preceding the onset of pain (F = 14.03 (1,10), p = 0.004). These results confirm that hip adductor muscle strength is decreased both preceding and during the onset of groin injury in elite under-age Australian footballers.