sAA Activity Research Articles

Multiple time courses of salivary alpha-amylase and dimensions of affect in adolescence.

Previous research has illustrated associations among daily experiences, emotions and stress-responding physiological systems. Recently, investigators have examined salivary alpha-amylase (sAA), a surrogate marker of the autonomic nervous system, and its associations with affect. The current study examined associations among affective valence, arousal and sAA across three different time courses at the momentary, daily and inter-individual level to understand varying influences of adolescents' daily emotional experiences on sAA reactivity and diurnal sAA activity. Adolescents (N=82) provided salivary samples and diary reports of affect and experiences five times a day for three consecutive days. They also completed self-report questionnaires on trait affect. Findings from multilevel growth curves demonstrated that adolescents in our sample displayed typical sAA diurnal rhythms with levels dropping 30 min after waking and then increasing across the day to a peak in the late afternoon. Within person momentary experiences of high arousal positive affect were associated with momentary sAA reactivity. Prior day experiences of high arousal negative affect were associated with a greater amylase awakening response (i.e., greater decrease) and flatter slopes the next day. Trait positive affect was also associated with flatter sAA slopes. Our findings suggest that both affective arousal and valence should be accounted for when examining differences in sAA reactivity and diurnal patterns. Further, our results indicated that emotion-physiology transactions among adolescents occur over varying time scales for salivary alpha-amylase as well as cortisol.

Caffeine administration does not alter salivary α-amylase activity in young male daily caffeine consumers

BackgroundTo follow up on a recent report from our lab [Hum Psychopharmacol 25:359–367, 2010.] we examined the effects of caffeine on salivary α-amylase (sAA) activity in response to an engaging, non-stressful task in healthy young males (age 18–30 yrs) who consumed caffeine on a daily basis. Using a placebo-controlled, double-blind, between-subjects design, 45 men received either placebo, 200 mg or 400 mg of caffeine (Vivarin®). Participants then rested for 20 minutes, and performed a 20-minute computerized air traffic controller-like task that was cognitively engaging but not stressful. Saliva samples (assayed for sAA and cortisol), blood pressure, and heart rate were taken before (baseline) and 15 minutes after the computerized task.ResultsSystolic and diastolic blood pressure and sAA activity increased across the laboratory session (F’s > 9.20, p’s < 0.05); salivary cortisol levels decreased (F = 16.17, p < 0.05). There were no main effects for caffeine administration on sAA, salivary cortisol, or cardiovascular measures, and caffeine did not interact with the task to alter these measures.ConclusionsLaboratory administered caffeine does not alter sAA activity, even when sAA activity is stimulated by participating in a cognitively engaging task. These data demonstrate that caffeine administration does not affect sAA activity, at least in healthy young men who regularly consume caffeine. Results support recent findings that basal caffeine levels in habitual caffeine users are not associated with basal sAA activity and that daily caffeine intake and diurnal sAA activity are not related.

Low baseline salivary alpha-amylase in drug-naïve patients with short-illness-duration first episode major depressive disorder

BackgroundAltered monoamine neurotransmission accompanied by hypothalamic–pituitary–adrenal axis dysfunction and autonomic nervous system hyperactivity have been associated with major depressive disorder (MDD). Salivary α-amylase (sAA) is indicative of autonomic activation and reflects central noradrenergic activity. Scarce studies on sAA in MDD produce confounded results and no data is available regarding baseline sAA activity. MethodsThe basal, non-stimulated sAA activity was studied in this cross-sectional case-control study on 20 non-late-life adult, short-illness-duration first-episode, treatment-naïve MDD patients and in 20 age- and sex-matched healthy controls. Depressed patients showed a basal score in the Hamilton Rating Scale for Depression (HAMD-17) higher than 20. ResultsThe sAA was significantly lower in depressed individuals as compared to controls (p=0.011). In post hoc analysis significantly lower sAA was present in melancholic MDD (p=0.016) as related to controls whereas no difference was seen between non-melancholic MDD patients and controls. The sAA activity was not significantly correlated neither with duration nor the severity of depressive symptoms as measured by the total HAMD-17 score. LimitationsThe current study is limited by its cross-sectional design, small sample size, and factors related to saliva sampling methodology. ConclusionLow baseline sAA levels were found in MDD in basal, non-stimulated conditions. The study provides no support for elevated sAA in drug-naïve patients with short-illness-duration first episode MDD. The results support the evidence for decreased central noradrenergic transmission in MDD when sAA activity is considered indicative of central noradrenergic function.

Salivary alpha amylase activity in human beings of different age groups subjected to psychological stress.

Salivary alpha-amylase (sAA) has been proposed as a sensitive non-invasive biomarker for stress-induced changes in the body that reflect the activity of the sympathetic nervous system. Though several experiments have been conducted to determine the validity of this salivary component as a reliable stress marker in human subjects, the effect of stress induced changes on sAA levelin different age groups is least studied. This article reports the activity of sAA in human subjects of different age groups subjected to psychological stress induced through stressful video clip. Differences in sAA level based on sex of different age groups under stress have also been studied. A total of 112 subjects consisting of both the male and female subjects, divided into two groups on basis of age were viewed a video clip of corneal transplant surgery as stressor. Activity of sAA from saliva samples of the stressed subjects were measured and compared with the activity of the samples collected from the subjects before viewing the clip. The age ranges of subjects were 18-25 and 40-60years. The sAA level increased significantly in both the groups after viewing the stressful video. The increase was more pronounced in the younger subjects. The level of sAA was comparatively more in males than females in the respective groups. No significant change in sAA activity was observed after viewing the soothed video clip. Significant increase of sAA level in response to psychological stress suggests that it might act as a reliable sympathetic activity biochemical marker in different stages of humanbeings.

Cortisol, alpha amylase, and daily stressors in spouses of persons with mild cognitive impairment.

This study examines the effects of daily primary objective stressors (behavioral problems exhibited by persons with mild cognitive impairment) and subjective stressors (unpleasant marital interactions) on the diurnal cortisol pattern and the diurnal pattern of salivary alpha-amylase (sAA) of spousal care partners. Thirty spouse care partners (age 59-85 years) participated in a 7-day diary study and submitted saliva samples on 4 consecutive study days, totaling 406 valid samples. Results from multilevel models revealed that daily objective stressors were associated with elevated cortisol levels and a flatter slope but were not associated with sAA activity. Conversely, unpleasant marital interactions were associated with flatter sAA slopes but not associated with cortisol activity. Furthermore, daily levels of sAA moderated variations in cortisol in the presence of a primary objective stressor. The utility of our research has implications for advancing scientific understanding of biosocial processes and the development of stress prevention and intervention strategies.

Effects of methamphetamine on the noradrenergic activity biomarker salivary alpha-amylase

BackgroundMethamphetamine (METH) potently activates the sympathetic nervous system (SNS) by increasing central and peripheral norepinephrine (NE). Salivary α-amylase (sAA) is a biomarker of SNS activation that correlates with plasma NE levels. The purpose of this study was to determine the impact of METH on sAA activity and whether changes in sAA activity were correlated with subjective effects ratings. MethodsNon-treatment seeking METH-dependent volunteers (N=8) participated in this within-subjects laboratory-based study. Volunteers received randomly administered intravenous METH (0mg, 30mg) and sAA activity, cardiovascular measures and subjective ratings were assessed at baseline (−15min) and five post-METH time points (10, 20, 30, 45, and 60min). ResultsMETH (30mg) increased sAA activity over time. sAA activity significantly correlated with diastolic blood pressure following 0mg METH and systolic blood pressure following 30mg METH. Subjective ratings (ANY EFFECT, HIGH, GOOD, STIMULATED, LIKE, WLLING TO PAY) highly correlated with sAA over five post-METH time points (N=40; r's=0.543–0.684, p's<0.001). Age, body mass index and METH amount received on a mg/kg basis were significantly associated with sAA activity. Multiple linear regression analysis indicated sAA activity remained a significant predictor of subjective ratings following METH after controlling for these factors. ConclusionsThe NE peripheral biomarker sAA activity is associated with METH's subjective effects.

Measurements of salivary alpha amylase and salivary cortisol in hominoid primates reveal within-species consistency and between-species differences.

Salivary alpha amylase (sAA) is the most abundant enzyme in saliva. Studies in humans found variation in enzymatic activity of sAA across populations that could be linked to the copy number of loci for salivary amylase (AMY1), which was seen as an adaptive response to the intake of dietary starch. In addition to diet dependent variation, differences in sAA activity have been related to social stress. In a previous study, we found evidence for stress-induced variation in sAA activity in the bonobos, a hominoid primate that is closely related to humans. In this study, we explored patterns of variation in sAA activity in bonobos and three other hominoid primates, chimpanzee, gorilla, and orangutan to (a) examine if within-species differences in sAA activity found in bonobos are characteristic for hominoids and (b) assess the extent of variation in sAA activity between different species. The results revealed species-differences in sAA activity with gorillas and orangutans having higher basal sAA activity when compared to Pan. To assess the impact of stress, sAA values were related to cortisol levels measured in the same saliva samples. Gorillas and orangutans had low salivary cortisol concentrations and the highest cortisol concentration was found in samples from male bonobos, the group that also showed the highest sAA activity. Considering published information, the differences in sAA activity correspond with differences in AMY1 copy numbers and match with general features of natural diet. Studies on sAA activity have the potential to complement molecular studies and may contribute to research on feeding ecology and nutrition.

Salivary Alpha-Amylase Activity and Salivary Flow Rate in Young Adults

The secretion of salivary alpha-amylase (sAA) is more associated with psychoneuroendocrinological response to stress than with the flow rate and age. The aim of this cross sectional study is to build an explanatory model based on patterns of relationship between age 20-39 in resting and stimulated saliva under no stressful condition in healthy volunteers. Both resting and stimulated saliva were collected from 40 subjects. The sAA values were log-transformed, the normality assumption was verified with the Shapiro-Wilk test and the reliability of the measurements was estimated by the Pearsons’ r correlation coefficient. The estimated model was based on the theory of the Linear Mixed Models. Significant mean changes were observed in flow rate and sAA activity between resting and stimulated saliva. The final model consists of two components, the first revealed a positive correlation between age and sAA while the second one revealed a negative correlation between the interaction of age × flow rate in its condition (resting or stimulated saliva), with sAA. Both flow rate and age influence sAA activity.

Salivary α-amylase response to endotoxin administration in humans

Salivary α-amylase (sAA) is a digestive enzyme that plays also an important role in mucosal immunity. Secretion of the sAA is largely under the control of the autonomic nervous system and increases in sAA activity have repeatedly been observed in response to various stressors. The present study aimed at investigating whether and to what extent sAA activity levels are affected during systemic inflammation. Fourteen healthy male volunteers received intravenous injections of either bacterial endotoxin or placebo at two different occasions in a randomized and double-blinded manner. sAA activity was monitored over a period of 6h together with inflammatory markers, plasma norepinephrine (NE) and salivary cortisol levels, vital parameters, and state anxiety. Endotoxin administration elicited a transient inflammatory response reflected by increases in body temperature, whole blood cell counts, and circulating levels of interleukin (IL)-6. The immune changes were accompanied by a transient increase in sAA activity, elevations in salivary cortisol and plasma NE concentrations, as well as increases in heart rate and state anxiety. Although sAA and plasma NE responses showed distinct time courses, a significant positive correlation over the total observation period was found. Whether the observed sAA response is driven by an increase in sympathetic activity or more generally reflects inflammation induced changes in sympathetic-parasympathetic balance remains to be elucidated.

Salivary alpha-amylase: a new non-invasive biomarker for assessment of pain perception in epileptic children

The aim of this study was to evaluate pain perception in epileptic children during an invasive procedure as the collection of venous blood through salivary alpha-amylase (sAA) activity determination, and to compare it with that of healthy children. In the study 23 children, 12 with epilepsy and 11 healthy controls were enrolled. From all children of both groups, one sample of saliva was collected through a non-invasive device, 15 min before (t 0), during (t 1), and 15 min later (t 2) blood withdrawal, and sAA activity was then determined through a kinetic-colorimetric assay. A statistically significant difference (p < 0.001) was found at t 2 between the sAA activity in the two groups, suggesting that epileptic children have an increased sensitization to pain, while at t 0 the difference was at the limit of statistical significance and at t 1 no statistically significant difference was found indicating that in both groups the venipuncture equally induced a state of stress. Our data suggest that sAA activity could represent a new objective and non-invasive biomarker for the assessment of pain perception in epileptic children.

Salivary alpha‐amylase changes promoted by sustained exposure to affective pictures

We studied the changes in salivary alpha-amylase (sAA) and other psychophysiological indices (heart rate, skin conductance, and corrugator supercilii activity) elicited by sustained exposure to affective pictures. Thirty-nine subjects viewed five blocks of pictures depicting mutilations, human attack, neutral scenes, sport/adventure, and erotica. Each block comprised 12 pictures of the same content. Saliva samples were collected before and after each block of pictures. The results showed that mutilation pictures promoted the greatest increase in sAA activity and output, as well as greater corrugator supercilii activity than pleasant pictures. Skin conductance response did not differ among high arousal picture contents. Changes in sAA varied with the affective valence but not with the arousal ratings of the pictures. Our results point to sAA as an index directly related to the unpleasantness elicited by sustained exposure to affective stimuli.

Salivary Nitric Oxide and Alpha-Amylase as Indexes of Training Intensity and Load

This study examined the variation in salivary nitric oxide (NO), alpha-amylase (sAA) and serum markers of muscle injury during 21 weeks of training in elite swimmers. Samples of saliva and blood were collected once a month during 5 months from 11 male professional athletes during their regular training season. The variation in each marker throughout the 21 weeks was compared with the dynamics of training volume, intensity and load. Unstimulated whole saliva was assessed for NO and sAA whereas venous blood was assessed for lactate dehydrogenase, creatine kinase, and γ-glutamyltransferase. Nitric oxide and sAA showed a proportional response to the intensity of training. However, whereas the concentration of NO increased across the 21 weeks, the activity of sAA decreased. Similar variations in the concentration of NO and the markers of muscle injury were also observed. The higher concentration of NO might be attributed to changes in haemodynamics and muscle regenerative processes. On the other hand, autonomic regulation towards parasympathetic predominance might have been responsible for the decrease in sAA activity. These findings provide appealing evidence for the utilization of salivary constituents in sports medicine to monitor training programmes.

51. Salivary alpha-amylase response to endotoxin administration in humans

Salivary alpha-amylase (sAA) has been proposed as a non-invasive biomarker for stress-related activation of the sympathetic nervous system (SNS). However, there is still much debate concerning the validity and reliability of sAA as a surrogate measure of SNS activity. Since acute systemic inflammation leads to pronounced activation of the SNS, we investigated in a double-blind, randomized and placebo-controlled crossover study the influence of intravenous lipopolysaccharide (0.4 ng/kg body weight) or placebo injection on sAA activity in healthy male subjects. Endotoxin administration induced significant increases in sAA activity, plasma norepinephrine (NE) concentration, heart rate, and state anxiety. Both sAA activity and plasma NE reached maximum levels at the peak of the inflammatory response (2 h post injection) and rapidly returned to baseline whereas heart rate further increased and then gradually normalized until six hours post injection. Most importantly, sAA activity was significantly correlated with circulating NE levels but not with other markers of SNS activity. Together, these findings demonstrate that immunogenic stress is associated with a significant increase in sAA activity, but whether this reflects SNS activity remains to be elucidated.

Acute stress responses in salivary alpha-amylase predict increases of plasma norepinephrine

Current biobehavioral research increasingly employs salivary alpha-amylase (sAA) as a surrogate marker for sympathetic nervous system (SNS) activity. While different lines of evidence point to the validity of this assumption, the literature is inconsistent with regard to associations of sAA with well-established SNS indicators, such as plasma norepinephrine (NE) or epinephrine (E). Small samples as well as application of different stress paradigms might be responsible. This study therefore set out to examine the relation between stress-induced sAA activity with NE and E by exposing a larger and less constrained sample to an effective stress protocol. Sixty-six healthy participants (mean age 24.30±4.24 yrs), including n=40 women, n=26 men, n=18 oral contraceptive (OC) users, and n=15 habitual smokers, were recruited and subjected to the Trier Social Stress Test (TSST). Saliva and blood samples were taken at four time points throughout the experiment for later analysis of sAA activity and NE/E concentration, respectively. As expected, sAA, NE, and E showed significant increases in response to the acute stress induction (all p<0.001). Regression analyses (controlling for age, BMI, sex, smoking and OC) revealed that stress responses in sAA significantly predicted stress responses in NE (r=0.326; p=0.025). Interestingly, stress responses in E predicted NE to a lesser extend (β=0.265; p=0.064). E responses showed no association with sAA (β=0.265; p=0.064). Higher sAA levels were found in habitual smokers (F=4.27; p=0.043) and in individuals with lower BMI (F=2.81; p=0.099). In conclusion, current data clearly show an association between stress responses of sAA and plasma NE. This relationship is stronger than the association of norepinephrine and epinephrine responses, thus placing the predictive power of sAA well within the expected range for different SNS markers.

Differences in Salivary Alpha-Amylase and Cortisol Responsiveness following Exposure to Electrical Stimulation versus the Trier Social Stress Tests

BackgroundCortisol is an essential hormone in the regulation of the stress response along the HPA axis, and salivary cortisol has been used as a measure of free circulating cortisol levels. Recently, salivary alpha-amylase (sAA) has also emerged as a novel biomarker for psychosocial stress responsiveness within the sympathetic adrenomedullary (SAM) system.Principal FindingsWe measured sAA and salivary cortisol in healthy volunteers after exposure to the Trier Social Stress Test (TSST) and electric stimulation stress. One hundred forty-nine healthy volunteers participated in this study. All subjects were exposed to both the TSST and electric stimulation stress on separate days. We measured sAA and salivary cortisol levels three times immediately before, immediately after, and 20 min after the stress challenge. The State (STAI-S) and Trait (STAI-T) versions of the Spielberger Anxiety Inventory test and the Profile of Mood State (POMS) tests were administered to participants before the electrical stimulation and TSST protocols. We also measured HF, LF and LF/HF Heart Rate Variability ratio immediately after electrical stimulation and TSST exposure. Following TSST exposure or electrical stimulation, sAA levels displayed a rapid increase and recovery, returning to baseline levels 20 min after the stress challenge. Salivary cortisol responses showed a delayed increase, which remained significantly elevated from baseline levels 20 min after the stress challenge. Analyses revealed no differences between men and women with regard to their sAA response to the challenges (TSST or electric stimulations), while we found significantly higher salivary cortisol responses to the TSST in females. We also found that younger subjects tended to display higher sAA activity. Salivary cortisol levels were significantly correlated with the strength of the applied electrical stimulation.ConclusionsThese preliminary results suggest that the HPA axis (but not the SAM system) may show differential response patterns to distinct kinds of stressors.

Salivary alpha-amylase activity—An indicator of relaxation response in perioperative patients

The psychological stress of most perioperative patients has been reduced by many interventions. In order to evaluate the effects of these interventions, biomarkers are being considered as objective assessment tools in addition to the subjective assessment tools. It has been thought that salivary α-amylase (sAA) activity is associated with sympathetic nervous system activity, which reflects psychological stress. We examined the relationship between sAA, self-reported data, and autonomic nervous system activity to assess the reliability of sAA as a marker for relaxing response in surgery-related stress. The sAA, the State anxiety, Visual Analogue Scale (VAS) of pain, and heart rate variability were used to assess prior to and after the relaxation on the day before surgery, and on postoperative days (POD) 1, 2, 3, and 7. A total of 75 patients participated and 301 relaxing response readings were obtained. We found that the sAA significantly decreased depending upon the pain and anxiety by relaxation, but they were unrelated to sympathetic nervous system activity. There was weak correlation between the change in sAA, and anxiety and VAS pain. Thus, sAA appears to be an easy-to-use, non-invasive and good marker for relaxing response within a short period in surgery-related stress patients.

Evaluation of Sympathetic Nerve Activity using Salivary ^|^alpha;-Amylase Activity (sAA) Level in Different Educational Setting of Autistic Children

This study investigated whether salivary α-amylase activity (sAA) can be a useful index for evaluating sympathetic nervous system (SNS) function of autistic children in an educational setting. Subjects were 13 autistic children who were enrolled at a special needs education school. We measured the children's sAA activity and heart rate (HR) before and after their daily group and individual study sessions. The results showed that there was no significant difference in sAA or HR measured before and after each session. However, the variance of sAA values was significantly smaller after individual study sessions compared with that before the sessions. On the other hand, no significant difference was observed in the mean or variance of HR values between before and after each session. These results indicate that, in individual study sessions, teachers accurately recognize the arousal level of individual students and adjust the level of learning to meet each student's needs. Our findings also suggest that sAA values can be a useful indicator of functional changes in the SNS that are difficult to evaluate using HR values.

Salivary Alpha-Amylase Activity, a New Biomarker in Heart Failure?

Salivary α-amylase activity is an increasingly investigated biomarker for the activation of the autonomic nervous system. Autonomic imbalance is associated to several diseases, one of which is heart failure, and the aim of the present study was to test if salivary α-amylase activity might be a new biomarker in patients with chronic heart failure. Methods: In this pilot study, 48 elderly men (range 59-89 years), 24 patients with established chronic heart failure in NYHA class I to III, and 24 controls were included. In all participants, saliva was collected for three consecutive days at three time points (at awakening, 30 minutes later and in the late afternoon), and blood was sampled for analysis of NT-proBNP. Results: Within the whole group of participants, a statistically significant positive correlation between morning salivary α-amylase activity levels and serum NT-proBNP could be found, which was strongest for the measurement taken 30 minutes after awakening, as well as a significant negative correlation of awakening α-amylase activity levels with arterial blood pressure. Within the control group separately, higher daily salivary α-amylase activity output correlated with increasing levels of NT-proBNP, while within the patients, the strongest association of α-amylase activity measures were found to be a negative correlation with blood pressure. Conclusions: Our data supports the idea that sAA activity has the potential as a non-invasive index of adrenergic activity in specific pathological conditions, though for heart failure in particular the results were merely modest, which was likely due to the specific intake of beta-receptor blocking drugs by all patients. Due to the large variability of sAA activity levels, we expect a greater potential for monitoring its changes over time, which could prove a valuable surrogate biomarker for cardiovascular diseases, including heart failure.

Salivary alpha amylase and cortisol levels in children with global developmental delay and their relation with the expectation of dental care and behavior during the intervention

The purpose of this study was to analyze the alpha-amylase (sAA) and cortisol levels in children with Global developmental delay (GDD) before and after dental treatment and its association with the children's behavior during treatment. The morning salivary cortisol levels and activity of sAA of 33 children with GDD were evaluated before and after dental treatment and were compared to 19 healthy children. The behavior of children with GDD during dental care was assessed by the Frankl scale. Children with GDD showed lower levels of sAA activity than healthy children, but this result was not significant. The salivary cortisol levels were similar between GDD and healthy children. GDD children showed increased levels of sAA (but not cortisol) prior to the dental treatment as compared to the post-treatment phase. GDD children who showed less favorable behavior during dental care had higher levels of sAA and salivary cortisol than GDD children with more favorable behavior, but only the sAA results were significant. In conclusion, GDD children show hyperactivity of the SNS-axis in anticipation of dental treatment which indicates the need for strategies to reduce their anxiety levels before and during dental care.

Diagnostic Performance of Initial Salivary Alpha-Amylase Activity for Acute Myocardial Infarction in Patients with Acute Chest Pain

BackgroundTo rule out acute myocardial infarction (AMI) in chest pain patients constitutes a diagnostic challenge to emergency department (ED) physicians. Study ObjectivesTo evaluate the diagnostic value of measuring salivary alpha-amylase (sAA) activity for detecting AMI in patients presenting to the ED with acute chest pain. MethodssAA activity was measured in a prospective cohort of 473 consecutive adult patients within 4 h of onset of chest pain. Comparisons were made between patients with a final diagnosis of AMI and those with non-AMI. Univariate analysis and multiple logistic regression model were used to identify independent clinical predictors of AMI. ResultsInitial sAA activity in the AMI group (n = 85; 266 ± 127.6 U/mL) was significantly higher than in the non-AMI group (n = 388; 130 ± 92.8 U/mL, p < 0.001). sAA activity levels were also significantly higher in patients with ST elevation AMI (n = 53) compared to in those with non-ST elevation AMI (n = 32) (300 ± 141.1 vs. 210 ± 74.1 U/mL, p < 0.001). The area under the receiver operating characteristic curve of sAA activity for predicting AMI in patients with acute chest pain was 0.826 (95% confidence interval [CI] 0.782–0.869), with diagnostic odds ratio 10.87 (95% CI 6.16–19.18). With a best cutoff value of 197.7 U/mL, the sAA activity revealed moderate sensitivity and specificity as an independent predictor of AMI (78.8% and 74.5%). ConclusionsHigh initial sAA activity is an independent predictor of AMI in patients presenting to the ED with chest pain.