The case for the repeatability intra-class correlation as a metric of precision for salivary bioscience data: Justification, assessment, application, and implications.

Best practice standards for measuring analyte levels in saliva recommend that all biospecimens be tested in replicate with mean concentrations used in statistical analyses. This approach prioritizes minimizing laboratory-based measurement error but, in the process, expends considerable resources. We explore the possibility that, due to advances in salivary assay precision, the contribution of laboratory-based measurement error in salivary analyte data is very small relative to more important and meaningful variability in analyte levels across biological replicates (i.e., between different specimens). To evaluate this possibility, we examine the utility of the repeatability intra-class correlation (rICC) as an additional index of salivary analyte data precision. Using randomly selected subsamples (Ns=200 and 60) of salivary analyte data collected as part of a larger epidemiologic study, we compute the rICCs for seven commonly assayed salivary measures in biobehavioral research - cortisol, alpha-amylase, c-reactive protein, interlekin-6, uric acid, secretory immunoglobulin A, and testosterone. We assess the sensitivity of rICC estimates to assay type and the unique distributions of the underlying analyte data. We also use simulations to examine the bias, precision, and coverage probability of rICC estimates calculated for small to large sample sizes. For each analyte, the rICCs revealed that less than 5% of variation in analyte levels was attributable to laboratory-based measurement error. rICC estimates were similar across all analytes despite differences in analyte levels, average intra-assay coefficients of variation, and in the distributional properties of the data. Guidelines for calculating rICC are provided to enable investigators and laboratory staff to apply this metric and more accurately quantify, and communicate, the magnitude of laboratory-based measurement error in their data. By helping investigators scale measurement error relative to more scientifically meaningful variability between biological replicates, the application of the rICC has the potential to influence research strategies and tactics such that resources (e.g., finances, effort, number/volume of biospecimens) are allocated more efficiently and effectively.

Effects of nightmares on the cortisol awakening response: An ambulatory assessment pilot study.

Previous studies have reported an association between sleep-related factors such as sleep duration, sleep quality and time of awakening with the cortisol awakening response (CAR). Preliminary evidence suggests that frequent nightmares are associated with a blunted CAR. In the present pilot study we investigated the effect of acute nightmares on the CAR and the cortisol profile of the subsequent day using a within-subject ambulatory assessment study design. Thirty healthy volunteers who reported frequent nightmares enrolled in the study. Over a period of 14 days, salivary samples in addition to sleep-related and state variables were collected at home over the day. Salivary profiles of free cortisol and alpha amylase for up to two days following a nightmare were compared to those following a neutral dream. Overall, mood and health were decreased and the CAR was elevated following nightmares compared to neutral dreams. No differences were found for alpha amylase and for all variables over the remaining part of the day. Results suggest that sleep-related factors such as the occurrence of nightmares account for a certain amount of variance observed in the CAR. This has important methodological implications for CAR studies in general and might have even more relevance for studies in clinical populations suffering from chronic sleep disturbances.

Intubation Setting, Aspiration, and Ventilator-Associated Conditions.

BACKGROUND: Patients experience endotracheal intubation in various settings with wide-ranging risks for postintubation complications such as aspiration and ventilator-associated conditions. OBJECTIVES: To evaluate associations between intubation setting, presence of aspiration biomarkers, and clinical outcomes. METHODS: This study is a subanalysis of data from the NO-ASPIRATE single-blinded randomized clinical trial. Data were prospectively collected for 513 adult patients intubated within 24 hours of enrollment. Patients with documented aspiration events at intubation were excluded. In the NO-ASPIRATE trial, intervention patients received enhanced oropharyngeal suctioning every 4 hours and control patients received sham suctioning. Tracheal specimens for α-amylase and pepsin tests were collected upon enrollment. Primary outcomes were ventilator hours, lengths of stay, and rates of ventilator-associated conditions. RESULTS: Of the baseline tracheal specimens, 76.4% were positive for α-amylase and 33.1% were positive for pepsin. Proportions of positive tracheal α-amylase and pepsin tests did not differ significantly between intubation locations (study hospital, transfer from other hospital, or field intubation). No differences were found for ventilator hours or lengths of stay. Patients intubated at another hospital and transferred had significantly higher ventilator-associated condition rates than did those intubated at the study hospital (P = .02). Ventilator-associated condition rates did not differ significantly between patients intubated in the field and patients in other groups. CONCLUSIONS: Higher ventilator-associated condition rates associated with interhospital transfer may be related to movement from bed, vehicle loading and unloading, and transport vehicle vibrations. Airway assessment and care may also be suboptimal in the transport environment.

Assessment of Salivary Catalase, a-Amylase, and Cotinine Levels in Chronic Smokers: A Comparative Study.

INTRODUCTION: One of the common practices observed in many parts of the world is smoking, of which tobacco forms an important constituent which is burned and inhaled. Smoking is known to have potential effect on body's immune system, antioxidants level, and salivary cotinine levels. Hence, we planned the present study to evaluate the impact of cigarette smoke on salivary anti-oxidant levels and cotinine levels in smokers and nonsmokers. MATERIALS AND METHODS: The present study included assessment of salivary parameters of smokers and nonsmokers. A total of 400 subjects were analyzed, of which 200 were active smokers and 200 were nonsmokers. Unstimulated salivary samples were taken and assessment of a-amylase levels was done using biochemical kit and spectrophotometer. Assessment of salivary catalase (CAT) activity was done using Luck method. For the determination of cotinine levels, Bioassay Technology Laboratory kit was used using enzyme-linked immunosorbent assay (ELISA) technique. After the assessment of levels of all the salivary parameters, all the data were recorded, compiled, and analyzed. RESULTS: a-Amylase in smokers and nonsmokers group was found to be 206.25 and 169.85 U/mL respectively. Nonsignificant results were obtained while comparing the salivary a-amylase levels among the two study groups. Nonsignificant results were obtained while comparing the salivary CAT levels among the smokers and nonsmokers group. We observed statistically significant results while comparing mean cotinine levels among smokers group and nonsmokers group. CONCLUSION: Alteration in cotinine levels occurs in smokers in comparison to nonsmokers. CLINICAL SIGNIFICANCE: Smoking can cause harmful effect on the oral mucous membrane by altering salivary defense components.

Differential effects of eating and drinking on wellbeing-An ecological ambulatory assessment study.

INTRODUCTION: Various behaviors, such as physical activity and sleep, have been shown to have stress-reducing and beneficial effects on wellbeing. In contrast, the rewarding effects of eating and drinking have rarely been investigated, particularly using ecologically valid approaches, and little is known about mediating mechanisms on the biological level. METHODS: Seventy-seven healthy young adults completed items on eating and drinking as well as momentary wellbeing (measured by mood, energy, tension, stress and fatigue levels) on an iPod touch 5×/day for 4 consecutive days. With each entry, a saliva sample was collected for the later assessment of cortisol, alpha-amylase and salivary flow rate as markers of neuroendocrine and autonomic activity, respectively. RESULTS: Hierarchical linear models showed better momentary wellbeing on various scales after the consumption of juice, coffee and alcohol. Having a snack predicted lower fatigue levels. In contrast, consuming high-fat food resulted in impaired wellbeing. With regard to affect-induced eating as well as biomarkers, only a few associations emerged as significant. However, autonomic activity partially mediated the alcohol-stress association. CONCLUSION: These findings corroborate previous reports that dietary behaviors could have rewarding effects, but also challenge the assumption of a general mood-enhancing and stress-relieving effect of certain foods. Findings on biomarkers provide first insights into underlying biological mechanisms. Research on further assumed mechanisms (reward-associated brain networks) and moderators (hedonic overeating) is highly warranted. Moreover, implications for addiction research are discussed.

Effects of VA Facility Dog on Hospitalized Veterans Seen by a Palliative Care Psychologist: An Innovative Approach to Impacting Stress Indicators.

The United States is home to 23 million veterans. In many instances, veterans with serious illness who seek healthcare at the VA receive care from a palliative care service. Animal-assisted intervention (AAI) is gaining attention as a therapeutic stress reducing modality; however, its effects have not been well studied in veterans receiving palliative care in an acute care setting. A crossover repeated-measures study was conducted to examine the effects of an animal-assisted intervention (AAI) in the form of a therapy dog on stress indicators in 25 veterans on the palliative care service at the VA Eastern Colorado Healthcare System in Denver, CO. Veterans had a visit from a therapy dog and the dog's handler, a clinical psychologist (experimental condition) and an unstructured visit with the clinical psychologist alone (control condition). Blood pressure, heart rate, and the salivary biomarkers cortisol, alpha-amylase, and immunoglobulin A were collected before, after, and 30-minutes after both the experimental and control conditions. Significant decreases in cortisol were found when the before time period was compared to the 30-minutes after time period for both the experimental ( p = 0.007) and control condition ( p = 0.036). A significant decrease in HR was also found when the before time period was compared to the 30-minutes after time period for both the experimental ( p = 0.0046) and control ( p = 0.0119) condition. Results of this study supported that a VA facility dog paired with a palliative care psychologist had a measurable impact on salivary cortisol levels and HR in veterans.

A new nano-optical sensor thin film cadmium sulfide doped in sol-gel matrix for assessment of α-amylase activity in human saliva.

A novel, simple, sensitive and precise spectrofluorimetric method is developed for measuring the activity of the α-amylase enzyme in human saliva. The remarkable quenching of the luminescence intensity at 634 nm of nano CdS doped in a sol-gel matrix by various concentrations of maltose (produced from the reaction of the enzyme with the starch substrate) was successfully used as an optical sensor for the assessment of α-amylase activity. The calibration plot was achieved over the concentration range 4.8 × 10(-10) to 1.2 × 10(-5) mol L(-1) maltose with a correlation coefficient of 0.999 and a detection limit of 5.7 × 10(-11) mol L(-1). The method was used satisfactorily for assessment of the α-amylase activity in a number of human saliva samples collected from various healthy volunteers.

Utility of a salivary biosensor for objective assessment of surgery-related stress.

PURPOSE: To evaluate the clinical utility of a salivary α-amylase (sAA) biosensor for assessing oral surgery-related stress responses and the differential effect of the personality trait of pain catastrophizing. PATIENTS AND METHODS: A prospective cohort study was conducted in 76 healthy subjects who underwent elective removal of their third molars. Along with subjects' self-reports of anxiety and pain, biosensor-facilitated measurements of sAA levels were obtained at multiple time points during the preoperative consult, surgery, and postsurgical follow-up visits. In addition, subjects completed the Pain Catastrophizing Scale at baseline. Mixed-effect regression models examined changes in sAA levels and self-report ratings within and across visits and the contribution of pain catastrophizing. RESULTS: The sAA levels were lower during surgery and postsurgical follow-up compared with the consult visit (P < .01). The sAA levels decreased during the surgery visit (P < .05) and did not change during the consult or follow-up visits. Individuals who reported greater helplessness to pain manifested higher sAA levels during the surgery visit (P < .05). Self-reported anxiety ratings were highest during the surgery visit, and pain ratings were highest during the follow-up visit. CONCLUSIONS: The sAA levels did not show the predicted increases during the surgery visit compared with the consult and postsurgical follow-up visits or increases during the surgery visit. However, individuals who reported responding to pain with helplessness had higher sAA levels in anticipation of surgery, providing proof of concept for the value of point-of-care assessments of surgery-induced stresses and the differential effect of personality traits.

Assessment of salivary amylase as a stress biomarker in pregnant patients.

BACKGROUND: Chronic stress during pregnancy has been associated with worsened maternal and fetal outcomes. Acute stress immediately before spinal anaesthesia for caesarean section may contribute to hypotension. Therefore objective measures of acute stress may help identify women at risk of adverse outcomes. Salivary alpha-amylase is a stress biomarker that has so far been poorly investigated during pregnancy. The reference change value is the difference between two sequential results that must be exceeded for a change to be considered clinically relevant. Our first aim was to determine if salivary alpha-amylase increased in pregnant patients when subjected to the stress of transfer to the operating room. Our second aim was to determine if changes in salivary alpha-amylase were likely to be clinically significant by measuring reference change value in healthy volunteers. METHODS: In 15 pregnant patients undergoing planned caesarean section under spinal anaesthesia, salivary alpha-amylase, systolic blood pressure, heart rate, and immediate anxiety were measured on the morning of surgery on the ward and again in the operating room. The reference change value was calculated from 18 healthy volunteers. RESULTS: A median 220% increase in salivary alpha-amylase activity (P=0.0015) and a 17% increase in systolic blood pressure (P=0.0006) were observed between the ward and operating room. No changes of immediate anxiety or heart rate were observed. Reference change value was ±76% in volunteers and 13 of the 15 pregnant patients had a salivary alpha-amylase increase greater than the reference change value. CONCLUSION: When pregnant women are taken to the operating room, a clinically and statistically significant increase in salivary alpha-amylase was observed. Further studies are required to define its clinical usefulness.

Alternative method for quantification of alfa-amylase activity.

A modification of the sensitive agar diffusion method was developed for macro-scale determination of alfa-amylase. The proposed modifications lower costs with the utilisation of starch as substrate and agar as supporting medium. Thus, a standard curve was built using alfa-amylase solution from Aspergillus oryzae, with concentrations ranging from 2.4 to 7,500 U.mL-1. Clear radial diffusion zones were measured after 4 hours of incubation at 20 A degrees C. A linear relationship between the logarithm of enzyme activities and the area of clear zones was obtained. The method was validated by testing alpha-amylase from barley at the concentrations of 2.4; 60; 300 and 1,500 U.mL-1. The proposed method turned out to be simpler, faster, less expensive and able to determine on a macro-scale alpha-amylase over a wide range (2.4 to 7,500 U.mL-1) in scientific investigation as well as in teaching laboratory activities.

Alternative method for quantification of alfa-amylase activity / Alternativa para quantificação de alfa-amilase

A modification of the sensitive agar diffusion method was developed for macro-scale determination of alfa-amylase. The proposed modifications lower costs with the utilisation of starch as substrate and agar as supporting medium. Thus, a standard curve was built using alfa-amylase solution from Aspergillus oryzae, with concentrations ranging from 2.4 to 7,500 U.mL-1. Clear radial diffusion zones were measured after 4 hours of incubation at 20 °C. A linear relationship between the logarithm of enzyme activities and the area of clear zones was obtained. The method was validated by testing α-amylase from barley at the concentrations of 2.4; 60; 300 and 1,500 U.mL-1. The proposed method turned out to be simpler, faster, less expensive and able to determine on a macro-scale α-amylase over a wide range (2.4 to 7,500 U.mL-1) in scientific investigation as well as in teaching laboratory activities.

Modificações foram propostas ao método sensível de difusão em ágar para a macrodeterminação de alfa-amilase. As modificações propostas diminuem os custos, com a utilização de amido como substrato e ágar como meio solidificante. Assim, foi construída uma curva padrão utilizando uma solução de alfa-amilase de Aspergillus oryzae com concentrações variando de 2,4 a 7.500 U.mL-1. Em seguida, as zonas claras de difusão radial foram mensuradas depois de 4 horas de incubação a 20 °C. Foi obtida uma relação linear entre o logaritmo da atividade enzimática e os diâmetros das zonas claras. O método foi validado utilizando-se soluções de alfa-amilase de cevada nas concentrações de 2,4; 60; 300 e 1.500 U.mL-1. O método tornou-se mais simples, rápido, com baixo custo e passível de ser utilizado para macrodeterminação de alfa-amilase em ampla faixa (2,4 a 7.500 U.mL-1) na investigação científica e para fins didáticos em aulas práticas.

[Device for measuring salivary alpha-amylase--application for pain assessment].

The salivary enzyme alpha-amylase is an established non-invasive marker of psychological and physiological stress. Since there are positive correlations between salivary alpha-amylase secretion and sympathetic parameter increment during stress, salivary alpha-amylase might be an indicator of sympathetic-adrenal-medullary (SAM) system and central sympathetic activity In contrast, it is well known that sympathetic neural activity is significantly involved in development and deterioration of pain symptoms as a part of pain disorders. Additionally, it has been suggested that various painful stimuli activate SAM system. Taking these data together, salivary alpha-amylase might be an indirect indicator of pain-induced central sympathetic tone. Furthermore, a hand-held device, which is able to measure the activity of salivary alpha-amylase easily and quickly, was developed recently, and we can try to evaluate the condition of pain at the outpatient clinic and the bedside. Though the actual utility of this measuring device for clinical use is unclear, it is hoped that the measuring device of salivary alpha-amylase is one of helpful methods for pain assessment in the future.

Biologic cost of caring for a cancer patient: dysregulation of pro- and anti-inflammatory signaling pathways.

PURPOSE: Caring for a family member with cancer is a psychologically demanding experience. However, it remains unclear whether the distress that caregiving provokes also takes a physiologic toll on the body. This study observed familial caregivers of patients with brain cancer for a year after diagnosis and tracked changes in neurohormonal and inflammatory processes. PATIENTS AND METHODS: Eighteen caregivers (age 50.4 +/- 3.5 years) and 19 controls (age 50.2 +/- 2.6 years) were assessed four times during a year (before and after radiotherapy, as well as 6 weeks and 4 months thereafter). Salivary biomarkers of hypothalamus-pituitary-adrenal axis and sympathetic nervous system (SNS) activity were collected, and blood was drawn for assessment of the systemic inflammatory markers C-reactive protein (CRP) and interleukin-6 (IL-6). Blood was also used to monitor in vitro IL-6 production by endotoxin-stimulated leukocytes and expression of mRNA for pro- and anti-inflammatory signaling molecules. RESULTS: Caregivers showed marked changes over time in diurnal output of salivary amylase, a marker of SNS activity, whereas secretions in controls were stable during follow-up. Cortisol output was similar in caregivers and controls. During the year, caregivers showed a profound linear increase in systemic inflammation, as indexed by CRP. At the same time, they displayed a linear decline in mRNA for anti-inflammatory signaling molecules and diminished in vitro glucocorticoid sensitivity. CONCLUSION: These preliminary data show that familial caregivers of patients with cancer experience marked changes in neurohormonal and inflammatory processes in the year after diagnosis. These changes may place them at risk for morbidity and mortality from diseases fostered by excessive inflammation.

Effect of an intervention aimed at reducing the risk of allergic respiratory disease in bakers: change in flour dust and fungal alpha-amylase levels.

INTRODUCTION: We evaluated the effect on exposure of an intervention programme, which focused on risk education and providing information on good work practices. This intervention programme was enrolled as part of a Dutch covenant in the flour processing industry (industrial bakeries, flour mills, ingredient producers). METHODS: Data from several measurement surveys collected pre- and post-intervention were used to evaluate changes in exposure over time. All datasets contained personal measurements analysed for flour dust and fungal alpha-amylase contents, and contextual information was available on process characteristics, work practice, and use of control measures. RESULTS: Changes in exposure over time varied substantially between sectors and jobs. For bakeries a modest downward annual trend of -2% was found for flour dust and -8% for amylase. For flour mills the annual trend for flour dust was -12%; no significant trend was observed for amylase. For ingredient producers results were generally non-significant but indicated a reduction in flour dust exposure and increase in fungal alpha-amylase exposure. Modest increase in use of control measures and proper work practices were reported in most sectors, especially the use of local exhaust ventilation and decreased use of compressed air. CONCLUSIONS: The magnitude of the observed reductions in exposure levels indicates that the sector-wide intervention strategy implemented during the covenant period had a limited overall effect. This indicates that a more rigorous approach is needed to substantially decrease the exposure levels to flour dust and related allergens and, respectively, the prevalence of associated occupational diseases.

Optimized methods for fungal alpha-amylase airborne exposure assessment in bakeries and mills.

BACKGROUND: In order to enable reproducible and comparable exposure measurements of fungal alpha-amylase (alpha-amylase) in different laboratories and countries, the entire procedure from sampling of airborne dust to measuring extracted samples (including standards and the used enzyme) immunoassays must be standardized. The aim of this study was to establish optimal elution and assay conditions. METHODS: A parallel sampler was used for simultaneous collection of 10 samples of inhalable dust in bakeries and mills in Germany, England, the Netherlands and Spain. Three enzyme-immunoassays (EIAs) for detection of fungal alpha-amylase based on monoclonal antibodies or polyclonal antibodies were used for the measurement of the parallel-sampled filters (n=432) extracted using several methods. The results were analysed by regression analysis of variance. Additional filters (n=54) were extracted and analysed using two EIAs to investigate the storage stability of the extracts. RESULTS: Although alpha-amylase concentrations correlated well (r> or =0.88), differences were found between the EIAs in the sensitivity and nominal values (up to a mean factor 5.8). The best elution medium for airborne filters (phosphate-buffered saline 'PBS' with 0.05% Tween-20) led to 1.2 to two times higher alpha-amylase allergen yields than extraction in PBS only, while higher Tween-20 concentrations decreased the extracted alpha-amylase yield. During storage of frozen dust/filter extracts for 3-4 months at -20 degrees C, a loss of approximately 40% of measurable alpha-amylase was observed, which could be partially prevented by addition of 0.1% casein to the medium directly after extraction. CONCLUSION: Although the effects of only a few of many possible causes of variation were investigated, for these factors a clear choice could be made with regard to optimal elution conditions and the use of validated EIAs with calibrated standards, thus making significant progress towards a completely standardized procedure for airborne alpha-amylase measurements.

Fed-batch optimization of alpha-amylase and protease-producing Bacillus subtilis using Markov chain methods.

A stoichiometry-based model for the fed-batch culture of the recombinant bacterium Bacillus subtilis ATCC 6051a, producing extracellular alpha-amylase as a desirable product and proteases as undesirable products, was developed and verified. The model was then used for optimizing the feeding schedule in fed-batch culture. To handle higher-order model equations (14 state variables), an optimization methodology for the dual-enzyme system is proposed by integrating Pontryagin's optimum principle with fermentation measurements. Markov chain Monte Carlo (MCMC) procedures were appropriate for model parameter and decision variable estimation by using a priori parameter distributions reflecting the experimental results. Using a simplified Metropolis-Hastings algorithm, the specific productivity of alpha-amylase was maximized and the optimum path was confirmed by experimentation. The optimization process predicted a further 14% improvement of alpha-amylase productivity that could not be realized because of the onset of sporulation. Among the decision variables, the switching time from batch to fed-batch operation (t(s)) was the most sensitive decision variable.

24-hour intragastric pH measurement in the assessment of duodenogastric reflux.

In this study we compared the results of measurement of duodenogastric reflux using 24-hour intragastric pH monitoring and nasogastric aspiration with analysis of bile acid and alpha amylase concentrations. Eight patients were studied at least 5 months after biliary and/or gastric surgery. The correlation coefficient of pH and bile acid concentration was 0.36 (p less than 0.001) and of pH and amylase concentration was 0.48 (p less than 0.001). If a pH greater than 4.0 was taken to indicate duodenogastric reflux this had a sensitivity of 84% compared with either high bile acid or amylase concentration. Bile acid concentration has been the "standard" method of measuring duodenogastric reflux. pH monitoring is relatively simple to perform, causes little patient distress, and can measure episodes of duodenogastric reflux over a continuous 24-hour period.