Measurement of total body water (TBW) and total energy expenditure (TEE) using stable isotopes

Understanding the relationship between diet, physical activity and health in humans requires accurate measurement of body composition and daily energy expenditure. Stable isotopes provide a means of measuring total body water and daily energy expenditure under free-living conditions. While the use of isotope ratio mass spectrometry (IRMS) for the analysis of 2H (Deuterium) and 18O (Oxygen-18) is well established in the field of human energy metabolism research, numerous questions remain regarding the factors which influence analytical and measurement error using this methodology. This thesis was comprised of four studies with the following emphases. The aim of Study 1 was to determine the analytical and measurement error of the IRMS with regard to sample handling under certain conditions. Study 2 involved the comparison of TEE (Total daily energy expenditure) using two commonly employed equations. Further, saliva and urine samples, collected at different times, were used to determine if clinically significant differences would occur. Study 3 was undertaken to determine the appropriate collection times for TBW estimates and derived body composition values. Finally, Study 4, a single case study to investigate if TEE measures are affected when the human condition changes due to altered exercise and water intake. The aim of Study 1 was to validate laboratory approaches to measure isotopic enrichment to ensure accurate (to international standards), precise (reproducibility of three replicate samples) and linear (isotope ratio was constant over the expected concentration range) results. This established the machine variability for the IRMS equipment in use at Queensland University for both TBW and TEE. Using either 0.4mL or 0.5mL sample volumes for both oxygen-18 and deuterium were statistically acceptable (p>0.05) and showed a within analytical variance of 5.8 Delta VSOW units for deuterium, 0.41 Delta VSOW units for oxygen-18. This variance was used as “within analytical noise” to determine sample deviations. It was also found that there was no influence of equilibration time on oxygen-18 or deuterium values when comparing the minimum (oxygen-18: 24hr; deuterium: 3 days) and maximum (oxygen-18: and deuterium: 14 days) equilibration times. With regard to preparation using the vacuum line, any order of preparation is suitable as the TEE values fall within 8% of each other regardless of preparation order. An 8% variation is acceptable for the TEE values due to biological and technical errors (Schoeller, 1988). However, for the automated line, deuterium must be assessed first followed by oxygen-18 as the automated machine line does not evacuate tubes but merely refills them with an injection of gas for a predetermined time. Any fractionation (which may occur for both isotopes), would cause a slight elevation in the values and hence a lower TEE. The purpose of the second and third study was to investigate the use of IRMS to measure the TEE and TBW of and to validate the current IRMS practices in use with regard to sample collection times of urine and saliva, the use of two TEE equations from different research centers and the body composition values derived from these TEE and TBW values. Following the collection of a fasting baseline urine and saliva sample, 10 people (8 women, 2 men) were dosed with a doubly labeled water does comprised of 1.25g 10% oxygen-18 and 0.1 g 100% deuterium/kg body weight. The samples were collected hourly for 12 hrs on the first day and then morning, midday, and evening samples were collected for the next 14 days. The samples were analyzed using an isotope ratio mass spectrometer. For the TBW, time to equilibration was determined using three commonly employed data analysis approaches. Isotopic equilibration was reached in 90% of the sample by hour 6, and in 100% of the sample by hour 7. With regard to the TBW estimations, the optimal time for urine collection was found to be between hours 4 and 10 as to where there was no significant difference between values. In contrast, statistically significant differences in TBW estimations were found between hours 1-3 and from 11-12 when compared with hours 4-10. Most of the individuals in this study were in equilibrium after 7 hours. The TEE equations of Prof Dale Scholler (Chicago, USA, IAEA) and Prof K.Westerterp were compared with that of Prof. Andrew Coward (Dunn Nutrition Centre). When comparing values derived from samples collected in the morning and evening there was no effect of time or equation on resulting TEE values. The fourth study was a pilot study (n=1) to test the variability in TEE as a result of manipulations in fluid consumption and level of physical activity; the magnitude of change which may be expected in a sedentary adult. Physical activity levels were manipulated by increasing the number of steps per day to mimic the increases that may result when a sedentary individual commences an activity program. The study was comprised of three sub-studies completed on the same individual over a period of 8 months. There were no significant changes in TBW across all studies, even though the elimination rates changed with the supplemented water intake and additional physical activity. The extra activity may not have sufficiently strenuous enough and the water intake high enough to cause a significant change in the TBW and hence the CO2 production and TEE values. The TEE values measured show good agreement based on the estimated values calculated on an RMR of 1455 kcal/day, a DIT of 10% of TEE and activity based on measured steps. The covariance values tracked when plotting the residuals were found to be representative of “well-behaved” data and are indicative of the analytical accuracy. The ratio and product plots were found to reflect the water turnover and CO2 production and thus could, with further investigation, be employed to identify the changes in physical activity.

Association of inducible nitric oxide synthase with asthma severity, total serum immunoglobulin E and blood eosinophil levels

Background Nitric oxide is released by immune, epithelial and endothelial cells, and plays an important part in the pathophysiology of asthma. Objective To investigate the association of inducible nitric oxide synthases (iNOS) gene repeat polymorphisms with asthma. Methods 230 families with asthma (842 individuals) were recruited to identify and establish the genetic association of iNOS repeats with asthma and associated phenotypes. Serum nitric oxide levels in selected individuals were measured and correlated with specific genotypes. Multiple logistic regression analysis was performed to determine the effect of age and sex. Results A total of four repeats—a (CCTTT)n promoter repeat, a novel intron 2 (GT)n repeat (BV680047), an intron 4 (GT)n repeat (AFM311ZB1) and an intron 5 (CA)n repeat (D17S1878)—were identified and genotyped. A significant transmission distortion to the probands with asthma was seen for allele 3 of the AFM311ZB1 gene (p = 0.006). This allele was also found to be significantly associated with percentage blood eosinophils (p<0.001) and asthma severity (p = 0.04). Moreover, it was functionally correlated with high serum nitric oxide levels (p = 0.006). Similarly, the promoter repeat was found to be associated with serum total immunoglobulin (Ig)E (p = 0.028). Individuals carrying allele 4 of this repeat have high serum IgE (p<0.001) and nitric oxide levels (p = 0.03). Conclusion This is the first study to identify the repeat polymorphisms in the iNOS gene that are associated with severity of asthma and eosinophils. The functional relevance of the associated alleles with serum nitric oxide levels was also shown. Therefore, these results could be valuable in elucidating the role of nitric oxide in asthma pathogenesis.

Femoral impaction grafting in revision total hip arthroplasty

Between 1987 and 1999, 540 revision total hip replacements in 487 patients were performed at our institution with the femoral impaction grafting technique with a cemented femoral stem. All patients were prospectively followed for 2-15years post-operatively with no loss to follow-up. 494 hips remained successfully in situ at an average 6.7years. The ten year survival rate was 98.0% (95% CI 96.2 to 99.8) with aseptic loosening as the endpoint and 84.2% (95% CI 78.5 to 89.9) for re-operation for any reason. Indication for surgery and the use of any kind of reinforcement significantly influenced outcome (p<0.001). This is the largest known series of revision THR with femoral impaction grafting and the results support continued use of this technique.

Scaling digital radiographs for templating in total hip arthroplasty using conventional acetate templates independent of calibration markers

We describe a scaling method for templating digital radiographs using conventional acetate templates independent of template magnification without the need for a calibration marker. The mean magnification factor for the radiology department was determined (119.8%, range117%-123.4%). This fixed magnification factor was used to scale the radiographs by the method described. 32 femoral heads on postoperative THR radiographs were then measured and compared to the actual size. The mean absolute accuracy was within 0.5% of actual head size (range 0 to 3%) with a mean absolute difference of 0.16mm (range 0-1mm, SD 0.26mm). Intraclass Correlation Coefficient (ICC) showed excellent reliability for both inter and intraobserver measurements with ICC scores of 0.993 (95% CI 0.988-0.996) for interobserver measurements and intraobserver measurements ranging between 0.990-0.993 (95% CI 0.980-0.997).

Acetabular impaction grafting in total hip replacement

Background and purpose: Acetabular impaction grafting has been shown to have excellent results, but concerns regarding its suitability for larger defects have been highlighted. We report the use of this technique in a large cohort of patients with the aim of better understanding the limitations of the technique. Methods: We investigated a consecutive group of 339 cases of impaction grafting of the cup with morcellised impacted allograft bone for survivorship and mechanisms for early failure. Results: Kaplan Meier survival was 89.1% (95% CI 83.2 to 95.0%) at 5.8 years for revision for any reason, and 91.6% (95% CI 85.9 to 97.3%) for revision for aseptic loosening of the cup. Of the 15 cases revised for aseptic cup loosening, nine were large rim mesh reconstructions, two were fractured Kerboull-Postel plates, two were migrating cages, one medial wall mesh failure and one impaction alone failed. Interpretation: In our series, results were disappointing where a large rim mesh or significant reconstruction was required. In light of these results, our technique has changed in that we now use predominantly larger chips of purely cancellous bone, 8-10 mm3 in size, to fill the cavity and larger diameter cups to better fill the mouth of the reconstructed acetabulum. In addition we now make greater use of i) implants made of a highly porous in-growth surface to constrain allograft chips and ii) bulk allografts combined with cages and morcellised chips in cases with very large segmental and cavitary defects.

Exploratory investigation of atmospheric total suspended particulate matter and heavy metals in urban air

Atmospheric concentration of total suspended particulate matter (TSP) and associated heavy metals are a great concern due to their adverse health impacts and contribution to stormwater pollution. This paper discusses the outcomes of a study which investigated the variation of atmospheric TSP and heavy metal concentrations with traffic and land use characteristics during weekdays and weekends. Data for this study was gathered from fifteen sites at the Gold Coast, Australia using a high volume air sampler. The study detected consistently high TSP concentrations during weekdays compared to weekends. This confirms the significant influence of traffic related sources on TSP loads during weekdays. Both traffic and land use related sources equally contribute to TSP during weekends. Almost all the measured heavy metals showed high concentration on weekdays compared to weekends indicating significant contributions from traffic related emissions. Among the heavy metals, Zn concentration was the highest followed by Pb. It is postulated that re-suspension of previously deposited reserves was the main Pb source. Soil related sources were the main contributors of Mn.

Simulation of ankle joint forces to optimize total ankle replacement design

When compared with other arthoplasties, Total Ankle Joint Replacement (TAR) is much less successful. Attempts to remedy this situation by modifying the implant design, for example by making its form more akin to the original ankle anatomy, have largely met with failure. One of the major obstacles is a gap in current knowledge relating to ankle joint force. Specifically this is the lack of reliable data quantifying forces and moments acting on the ankle, in both the healthy and diseased joints. The limited data that does exist is thought to be inaccurate [1] and is based upon simplistic two dimensional discrete and outdated techniques.

Phenolics in sugar cane juice : potential degradation by hydrogen peroxide and Fenton's reagent

The presence of colour in raw sugar plays a key role in the marketing strategy of the Australian raw sugar industry. Some sugars are relatively difficult to decolourise during refining and develop colour during storage. A new approach that might result in efficient and cost-effective colour removal during the sugar manufacturing process is the use of an advanced oxidation process (AOP), known as Fenton oxidation, that is, catalytic production of hydroxyl radicals from the decomposition of hydrogen peroxide using ferrous iron. As a first step towards developing this technology, this study determined the composition of colour precursors present in the juice of cane harvested by three different methods. The methods were harvesting cane after burning, harvesting the whole crop with half of the trash extracted and harvesting the whole crop with no trash extracted. The study also investigated the degradation at pH 3, 4 and 5 of a phenolic compound, caffeic acid (3,4–dihydroxycinnamic acid), which is present in sugar cane juice, using both hydrogen peroxide and Fenton’s reagent. The results show that juice expressed from whole crop cane has significantly higher colour than juices expressed from burnt cane. However, the concentrations of phenolic acids were lower in the juices expressed from whole crop cane. The main phenolic acids present in these juices were p-coumaric, vanillic, 2,3–dihydroxybenzoic, gallic and 3,4–dihydroxybenzoic acids. The degradation of caffeic acid significantly improved using Fenton’s reagent in comparison to hydrogen peroxide alone. The Fenton oxidation was optimum at pH 5 when up to ~86 % of caffeic acid degraded within 5 min.

Phenolics in sugar cane juice : potential degradation by hydrogen peroxide and Fenton’s reagent

The presence of colour in raw sugar plays a key role in the marketing strategy of the Australian raw sugar industry. Some sugars are relatively difficult to decolourise during refining and develop colour during storage. A new approach that might result in efficient and cost-effective colour removal during the sugar manufacturing process is the use of an advanced oxidation process (AOP), known as Fenton oxidation, that is, catalytic production of hydroxyl radicals from the decomposition of hydrogen peroxide using ferrous iron. As a first step towards developing this technology, this study determined the composition of colour precursors present in the juice of cane harvested by three different methods. The methods were harvesting cane after burning, harvesting the whole crop with half of the trash extracted and harvesting the whole crop with no trash extracted. The study also investigated the degradation at pH 3, 4 and 5 of a phenolic compound, caffeic acid (3,4–dihydroxycinnamic acid), which is present in sugar cane juice, using both hydrogen peroxide and Fenton’s reagent. The results show that juice expressed from whole crop cane has significantly higher colour than juices expressed from burnt cane. However, the concentrations of phenolic acids were lower in the juices expressed from whole crop cane. The main phenolic acids present in these juices were p-coumaric, vanillic, 2,3–dihydroxybenzoic, gallic and 3,4–dihydroxybenzoic acids. The degradation of caffeic acid significantly improved using Fenton’s reagent in comparison to hydrogen peroxide alone. The Fenton oxidation was optimum at pH 5 when up to ~86% of caffeic acid degraded within 5 min.