Exhaled breath microanalysis in veterinary medicine

Breath microanalysis is an investigative method that is of considerable potential for non- invasive monitoring of health status, and early detection of disease in veterinary medicine. The aim of this thesis was to investigate the potential use of breath analysis for assessment of gastrointestinal transit and in vivo lipid peroxidation in animals. Initial studies in this thesis describe the use of the 13C-octanoic acid breath test (13C-OBT) and the 13C-lactose-ureide (13C-LUBT) for assessment of gastric emptying and oro-caecal transit time in the dog. The results revealed that collection of exhaled breath could be performed quickly, easily, and with minimal disturbance of the animal. The data produced could be fitted by simple mathematical models derived for analysis of gastric emptying breath tests, and reproducibility was comparable to previous reports of this test in humans. Furthermore, the parameters of the 13C-OBT were significantly altered by increased test meal energy density, a condition known to delay the rate of gastric emptying. In this study, the recovery of 2H2O in saliva did not occur simultaneously with the recovery of 13CO2 in breath following ingestion of 2H and 13C-octanoic acid in a dog. This finding indicates that the post-gastric processing of 13C-octanoic acid imposes a delay on the recovery of breath 13CO2, and confirms that the 13C-OBT does not provide a real-time measurement of gastric emptying in the dog. The results of these studies have shown that the 13C-OBT and the 13C-LUBT are potentially useful methods for assessment of gut transit in dog, although further validation is necessary. Lipid peroxidation is increasingly associated with many pathological processes, and the breath pentane test has been described as a non-invasive method for in vivo assessment of lipid peroxidation in humans. In order to assess the application of the breath pentane test in animals, a system for cryogenic concentration of exhaled breath samples, and analysis by gas chromatography (GC) was developed. Good specificity for discrimination of ethane and pentane from other breath hydrocarbons was demonstrated. The assay was sensitive to 0.5ppb and 5ppb pentane and ethane, respectively. Inter and intra-assay variation were comparable to previous studies. The test was sensitive enough to detect pentane in the exhaled breath of horses. Intra-subject variability in pentane exhalation in a group of 5 horses was lower than previously reported in man. The "electronic nose" is a method of analysis of gas samples that could potentially be applied for measurement of breath pentane. In these studies an array of composite polymer sensors, sample presentation system and data analysis protocol was integrated to form an "electronic nose" system. Composite polymer sensors were constructed by depositing a layer of a polymer solution with suspended carbon black particles on the surface of inter-digitated gold electrodes. Initial work demonstrated that these sensors were sensitive to alterations in temperature and humidity and that their baseline resistance was dependant on the percentage carbon black particles suspended in the polymer layer. Further work demonstrated that whilst the electronic nose was capable of discriminating between specific compounds, the specificity of this instrument was not sufficient to permit application for analysis of breath hydrocarbons. However, the unique chemical "fingerprint" measurement provided by the electronic nose could in the future be used to address problems in veterinary clinical investigation that cannot be contended using current analytical methods. The results of these studies suggest that the microanalysis of exhaled breath is a simple and potentially useful investigative method in veterinary medicine and is worthy of further investigation

Effects of photoperiod extension on clock gene and neuropeptide RNA expression in the SCN of the Soay sheep

In mammals, changing daylength (photoperiod) is the main synchronizer of seasonal functions. The photoperiodic information is transmitted through the retino-hypothalamic tract to the suprachiasmatic nuclei (SCN), site of the master circadian clock. To investigate effects of day length change on the sheep SCN, we used in-situ hybridization to assess the daily temporal organization of expression of circadian clock genes (Per1, Per2, Bmal1 and Fbxl21) and neuropeptides (Vip, Grp and Avp) in animals acclimated to a short photoperiod (SP; 8h of light) and at 3 or 15 days following transfer to a long photoperiod (LP3, LP15, respectively; 16h of light), achieved by an acute 8-h delay of lights off. We found that waveforms of SCN gene expression conformed to those previously seen in LP acclimated animals within 3 days of transfer to LP. Mean levels of expression for Per1-2 and Fbxl21 were nearly 2-fold higher in the LP15 than in the SP group. The expression of Vip was arrhythmic and unaffected by photoperiod, while, in contrast to rodents, Grp expression was not detectable within the sheep SCN. Expression of the circadian output gene Avp cycled robustly in all photoperiod groups with no detectable change in phasing. Overall these data suggest that synchronizing effects of light on SCN circadian organisation proceed similarly in ungulates and in rodents, despite differences in neuropeptide gene expression

Effects of dietary supplementation with krill meal on serum pro-inflammatory markers after the iditarod sled dog race

A seafood-based supplement from krill, rich in omega-3 phospholipids and proteins was tested on a group of dogs competing in the 2016 Iditarod dog sled race to investigate the effects of krill meal on exercise-induced inflammation and muscle damage in comparison to a control group. A single team of 16 dogs received 8% krill meal for 5 weeks prior to the start of race, while another team of 16 dogs received no supplementation. Ten dogs of the treatment and 11 dogs of the control group finished the race and their blood was analyzed for omega-3 index, inflammation (CRP) and muscle damage (CK). The omega-3 index of the krill meal-fed dogs was significantly higher at the beginning of the race (mean 6.2% in the supplemented vs 5.2% in the control group, p < .001). CRP concentrations increased from 7.05 ± 2.27 to 37.04 ± 9.16 μg/ml in the control and from 4.26 ± 0.69 to 16.56 ± 3.03 μg/ml in the treatment group, with a significant difference between the groups (p < .001). CK activity was increased from 90.75 ± 8.15 IU/l to 715.90 ± 218.9 IU/l in the control group and from 99.55 ± 12.15 to 515.69 ± 98.98 in the supplemented group, but there were no differences between groups (p = .266). The results showed that krill meal supplementation led to significantly higher omega-3 index, which correlated with lower inflammation and a tendency for reduced muscle damage after this long-distance sled dog competition. However, these results need to be confirmed by more controlled studies, since it was a field study and effects of race speed or other performance-related factors such as fitness and musher skill on the results cannot be excluded

Seasonal and daytime variation in multiple immune parameters in humans: evidence from 329,261 participants of the UK Biobank Cohort

Seasonal disease outbreaks are perennial features of human infectious disease but the factors generating these patterns are unclear. Here we investigate seasonal and daytime variability in multiple immune parameters in 329,261 participants in UK Biobank, and test for associations with a wide range of environmental and lifestyle factors, including changes in day length, outdoor temperature and vitamin D at the time the blood sample was collected. Seasonal patterns were evident in lymphocyte and neutrophil counts, and CRP, but not monocytes, and these were independent of lifestyle, demographic and environmental factors. All the immune parameters assessed demonstrated significant daytime variation that was independent of confounding factors. At a population level, human immune parameters vary across season and across time of day, independent of multiple confounding factors. Both season and time of day are fundamental dimensions of immune function that should be considered in all studies of immuno-prophylaxis and disease transmission

Methionine restriction restores a younger metabolic phenotype in adult mice with alterations in fibroblast growth factor 21.

Methionine restriction (MR) decreases body weight and adiposity and improves glucose homeostasis in rodents. Similar to caloric restriction, MR extends lifespan, but is accompanied by increased food intake and energy expenditure. Most studies have examined MR in young animals; therefore, the aim of this study was to investigate the ability of MR to reverse age-induced obesity and insulin resistance in adult animals. Male C57BL/6J mice aged 2 and 12 months old were fed MR (0.172% methionine) or control diet (0.86% methionine) for 8 weeks or 48 h. Food intake and whole-body physiology were assessed and serum/tissues analyzed biochemically. Methionine restriction in 12-month-old mice completely reversed age-induced alterations in body weight, adiposity, physical activity, and glucose tolerance to the levels measured in healthy 2-month-old control-fed mice. This was despite a significant increase in food intake in 12-month-old MR-fed mice. Methionine restriction decreased hepatic lipogenic gene expression and caused a remodeling of lipid metabolism in white adipose tissue, alongside increased insulin-induced phosphorylation of the insulin receptor (IR) and Akt in peripheral tissues. Mice restricted of methionine exhibited increased circulating and hepatic gene expression levels of FGF21, phosphorylation of eIF2a, and expression of ATF4, with a concomitant decrease in IRE1α phosphorylation. Short-term 48-h MR treatment increased hepatic FGF21 expression/secretion and insulin signaling and improved whole-body glucose homeostasis without affecting body weight. Our findings suggest that MR feeding can reverse the negative effects of aging on body mass, adiposity, and insulin resistance through an FGF21 mechanism. These findings implicate MR dietary intervention as a viable therapy for age-induced metabolic syndrome in adult humans

Susceptibility to the common cold virus is associated with day length.

Seasonal rhythms are endogenous timing mechanisms that allow animals living at temperate latitudes to synchronize their physiology to the seasons. Human viral respiratory disease is prevalent in the winter at temperate latitudes, but the role of endogenous mechanisms in these recurring annual patterns is unclear. The Common Cold Project is a repository of data describing the experimental viral challenge of 1,337 participants across the seasons of the year. We report a secondary analysis of these data to investigate if susceptibility to the common cold is associated with day length. The majority of the participants (78%) showed signs of infection but only 32% developed clinical signs of disease, and the probability of infection was significantly higher in longer day lengths (summer), but the disease was more likely in short (winter) day lengths. The persistence of winter disease patterns in experimental conditions supports the role of endogenous seasonality in human susceptibility to viral infection

Population-level seasonality in cardiovascular mortality, blood pressure, BMI and inflammatory cells in UK Biobank

Introduction: The risk of mortality from cardiovascular disease (CVD) is higher in wintertime throughout the world, but it is not known if this reflects annual changes in diet or lifestyle, or an endogenous photoperiodic mechanism that is sensitive to changes in daylength. Methods: Phenotypic data on cardiometabolic and lifestyle factors were collected throughout a 4 year time period from 502,642 middle-aged participants in UK Biobank. To assess the impact of seasonal environmental changes on cardiovascular risk factors, we linked these data to the outdoor temperature and day length at the time of assessment. Self-reported information on physical activity, diet and disease status were used to adjust for confounding factors related to health and lifestyle. Results: Mortality related to CVD was higher in winter, as were risk factors for this condition including blood pressure, markers of inflammation and BMI. These seasonal rhythms were significantly related to day length after adjustment for other factors that might affect seasonality including physical activity, diet and outdoor temperature. Conclusions: The risk of CVD may be modulated by day length at temperate latitudes, and the implications of seasonality should be considered in all studies of human cardiometabolic health

Adverse metabolic and mental health outcomes associated with shiftwork in a population-based study of 277,168 workers in UK biobank

Background: Reported associations between shiftwork and health have largely been based on occupation-specific, or single sex studies that might not be generalizable to the entire working population. The objective of this study was to investigate whether shiftwork was independently associated with obesity, diabetes, poor sleep, and well-being in a large, UK general population cohort. Methods: Participants of the UK Biobank study who were employed at the time of assessment were included. Exposure variables were self-reported shiftwork (any shiftwork and night shiftwork); and outcomes were objectively measured obesity, inflammation and physical activity and self-reported lifestyle, sleep and well-being variables, including mental health. Results: Shiftwork was reported by 17% of the 277,168 employed participants. Shiftworkers were more likely to be male, socioeconomically deprived and smokers, and to have higher levels of physical activity. Univariately, and following adjustment for lifestyle and work-related confounders, shiftworkers were more likely to be obese, depressed, to report disturbed sleep, and to have neurotic traits. Conclusions: Shiftwork was independently associated with multiple indicators of poor health and wellbeing, despite higher physical activity, and even in shiftworkers that did not work nights. Shiftwork is an emerging social factor that contributes to disease in the urban environment across the working population

Association of disrupted circadian rhythmicity with mood disorders, subjective wellbeing, and cognitive function: a cross-sectional study of 91 105 participants from the UK Biobank

Background: Disruption of sleep and circadian rhythmicity is a core feature of mood disorders and might be associated with increased susceptibility to such disorders. Previous studies in this area have used subjective reports of activity and sleep patterns, but the availability of accelerometer-based data from UK Biobank participants permits the derivation and analysis of new, objectively ascertained circadian rhythmicity parameters. We examined associations between objectively assessed circadian rhythmicity and mental health and wellbeing phenotypes, including lifetime history of mood disorder. Methods: UK residents aged 37–73 years were recruited into the UK Biobank general population cohort from 2006 to 2010. We used data from a subset of participants whose activity levels were recorded by wearing a wrist-worn accelerometer for 7 days. From these data, we derived a circadian relative amplitude variable, which is a measure of the extent to which circadian rhythmicity of rest–activity cycles is disrupted. In the same sample, we examined cross-sectional associations between low relative amplitude and mood disorder, wellbeing, and cognitive variables using a series of regression models. Our final model adjusted for age and season at the time that accelerometry started, sex, ethnic origin, Townsend deprivation score, smoking status, alcohol intake, educational attainment, overall mean acceleration recorded by accelerometry, body-mass index, and a binary measure of childhood trauma. Findings: We included 91 105 participants with accelerometery data collected between 2013 and 2015 in our analyses. A one-quintile reduction in relative amplitude was associated with increased risk of lifetime major depressive disorder (odds ratio [OR] 1·06, 95% CI 1·04–1·08) and lifetime bipolar disorder (1·11, 1·03–1·20), as well as with greater mood instability (1·02, 1·01–1·04), higher neuroticism scores (incident rate ratio 1·01, 1·01–1·02), more subjective loneliness (OR 1·09, 1·07–1·11), lower happiness (0·91, 0·90–0·93), lower health satisfaction (0·90, 0·89–0·91), and slower reaction times (linear regression coefficient 1·75, 1·05–2·45). These associations were independent of demographic, lifestyle, education, and overall activity confounders. Interpretation: Circadian disruption is reliably associated with various adverse mental health and wellbeing outcomes, including major depressive disorder and bipolar disorder. Lower relative amplitude might be linked to increased susceptibility to mood disorders