Measurement Of Plasma Catecholamines Research Articles

B-151 Development and Validation of Plasma Catecholamines by Mass Spectrometry

Abstract Background Our established method for measuring plasma catecholamines utilized a Shimadzu HPLC coupled to an ESA Coulochem III electrochemical detector. Chromatographic run times were long, resulting in slow turnaround times. Instrument maintenance was labor intensive, and the assay was prone to interferences. To overcome these limitations, we changed the method to liquid chromatography-tandem mass spectrometry (LC-MS/MS) measurements. Methods After the addition of a labeled internal standard for each analyte, samples are loaded onto 50 mg of activated alumina at pH 8.6, utilizing a Tris-HCl, sodium carbonate and EDTA buffer. After incubation the alumina is then washed with 50% acetonitrile followed by water. Elution off the alumina is performed with 2% acetic acid. Derivatization is then performed on the eluate utilizing acetaldehyde and a cyanoborohydride buffer. Following derivatization, the samples are analyzed for epinephrine, norepinephrine, and dopamine by LC-MS/MS using a Sciex 7500 coupled to a ThermoFisher Scientific TLX-4 equipped with Vanquish pumps. Chromatographic separation was obtained using a Phenomenex Kinetex 2.6 um BiPhenyl 50x3 mm analytical column. Results Runtimes were significantly shorter than with the previous method, and no interferences were observed. Assay validation consisted of: intra/inter assay precision, accuracy, recovery, linearity, carryover, limit of detection (LOD), and interferences. Three levels were assessed for intra assay precision and each of the analytes monitored yielded %CVs of less than 6%. Four levels were assessed for inter assay precision and each of the analytes monitored yielded %CVs of less than 10%. When using linear regression analysis to compare results with the previous clinical assay, the slope, y-intercept and R2 were the following; dopamine: 1.06, 2.74, 0.978; epinephrine: 0.905, 2.20, 0.989; norepinephrine: 0.919, 9.75, 0.953. Spike and recovery from five samples yielded a mean average recovery of 93.4% for dopamine, 100% for epinephrine and 98.1% for norepinephrine. For linearity 4 samples were diluted and the mean % difference from expected was -1.66% for dopamine, -0.25% for epinephrine and 1.78% for norepinephrine. Blanks were injected following the high standard and carryover was negligible. LOD was determined by running a low sample pool along with a blank over 20 assays. The LOD was determined to be less than the LLOQ. Sixty-two common drugs/vitamins along with varying levels of hemolysis, lipemia, and bilirubin were also tested to ensure no interferences were seen. Conclusions The LC-MS/MS method exceeded the performance of the historical Shimdazu HPLC system with ESA Coluochem III electrochemical detector method. We anticipate that the better analytical performance will improve the testing quality of our patients.

Re-evaluating the prevalence and factors characteristic of catecholamine secreting head and neck paragangliomas.

IntroductionWe sought to characterize the prevalence and factors characteristic of head and neck paragangliomas (HNPGLs) that secrete catecholamines to inform best practices for diagnosis and management.MethodsThis was a retrospective cohort study from 2000 to 2020 at a single‐institution tertiary centre. One‐hundred fifty‐two patients (182 tumours) with HNPGLs with at least one measurement of urine or plasma catecholamines and/or catecholamine metabolite levels prior to treatment were included. We differentiated and characterized those patients with increased level(s) of any nature and those with ‘clinically significant’ versus ‘clinically insignificant’ catecholamine production.ResultsThirty‐one (20.4%) patients had increased catecholamine and/or catecholamine metabolite levels. In most patients, these levels were ≤5‐fold above the upper limit of the reference range. Four of these 31 patients with increased levels were ultimately found to have an additional catecholamine secreting mediastinal paraganglioma or pheochromocytoma. Fourteen of 31 patients with HNPGL were deemed clinically significant secretors of catecholamines based on hyper‐adrenergic symptoms and/or profound levels of normetanephrines. This cohort was enriched for patients with paragangliomas of the carotid body or cervical sympathetic chain and those with SDHB genetic mutations. Ultimately, the prevalence of clinically significant catecholamine secreting Hangs was determined to be 9.2% and 7.7% based on a per‐patient and per‐tumour basis, respectively.ConclusionsThe rate of catecholamine excess in the current cohort of patients with HNPGLs was higher than previously reported. Neuroendocrine tumours of any anatomic subsite may secrete catecholamines, although not all increased laboratory level(s) are indicative of clinically significant catecholamine secretion causing symptoms or warranting adrenergic blockade.

Changes in sympathetic nervous system activity after renal denervation: results from the randomised Oslo RDN study

Purpose Sympathetic nervous system (SNS) over-activity is associated with essential hypertension. Renal sympathetic denervation (RDN) possibly lowers office- and ambulatory blood pressure (BP) in patients with treatment-resistant hypertension (TRH). We aimed to assess the effect of RDN compared to drug adjustment on SNS activity among patients with TRH by measuring plasma catecholamines and heart rate variability (HRV) during stress tests. Materials and methods Patients with TRH were randomised to RDN (n = 9) or Drug Adjustment (DA) (n = 10). We measured continuous HRV and beat-to-beat-BP using FinaPres® and obtained plasma catecholamines during standardised orthostatic- and cold-pressor stress tests (CPT) before- and six months after randomisation. Results CPT revealed no differences between groups at baseline in peak adrenaline concentration (69.3 pg/mL in the DA group vs. 70.0 pg/mL in the RDN group, p = 0.38) or adrenaline reactivity (Δ23.1 pg/mL in the DA group vs. Δ29.3 pg/mL in the RDN group, p = 0.40). After six months, adrenaline concentrations were statistically different between groups after one minute (66.9 pg/mL in the DA group vs. 55.3 pg/mL in the RDN group, p = 0.03), and six minutes (62.4 pg/mL in the DA group vs. 50.1 pg/mL in the RDN group, p = 0.03). There was a tendency of reduction in adrenaline reactivity after six months in the RDN group (Δ26.3 pg/mL at baseline vs. Δ12.8 pg/ml after six months, p = 0.08), while it increased in the DA group (Δ13.6 pg/mL at baseline vs. Δ19.9 pg/mL after six months, p = 0.53). We also found a difference in the Low Frequency band at baseline following the CPT (667µs2 in the DA group vs. 1628µs2 in the RDN group, p = 0.03) with a clear tendency of reduction in the RDN group to 743µs2 after six months (p = 0.07), compared to no change in the DA group (1052µs2, p = 0.39). Conclusion Our data suggest that RDN reduces SNS activity after six months. This finding warrants investigation in a larger study. Clinical Trial Number registered at www.clinicaltrials.gov: NCT01673516

In Matrix Derivatization Combined with LC-MS/MS Results in Ultrasensitive Quantification of Plasma Free Metanephrines and Catecholamines.

Plasma-free metanephrines and catecholamines are essential markers in the biochemical diagnosis and follow-up of neuroendocrine tumors and inborn errors of metabolism. However, their low circulating concentrations (in the nanomolar range) and poor fragmentation characteristics hinder facile simultaneous quantification by liquid chromatography and tandem mass spectrometry (LC-MS/MS). Here, we present a sensitive and simple matrix derivatization procedure using propionic anhydride that enables simultaneous quantification of unconjugated l-DOPA, catecholamines, and metanephrines in plasma by LC-MS/MS. Dilution of propionic anhydride 1:4 (v/v) in acetonitrile in combination with 50 μL of plasma resulted in the highest mass spectrometric response. In plasma, derivatization resulted in stable derivatives and increased sensitivity by a factor of 4–30 compared with a previous LC-MS/MS method for measuring plasma metanephrines in our laboratory. Furthermore, propionylation increased specificity, especially for 3-methoxytyramine, by preventing interference from antihypertensive medication (β-blockers). The method was validated according to international guidelines and correlated with a hydrophilic interaction LC-MS/MS method for measuring plasma metanephrines (R2 > 0.99) and high-performance liquid chromatography with an electrochemical detection method for measuring plasma catecholamines (R2 > 0.85). Reference intervals for l-DOPA, catecholamines, and metanephrines in n = 115 healthy individuals were established. Our work shows that analytes in the subnanomolar range in plasma can be derivatized in situ without any preceding sample extraction. The developed method shows improved sensitivity and selectivity over existing methods and enables simultaneous quantification of several classes of amines.

3117 “A Gut Feeling”: Autonomic Instability and Dysbiosis in Connective Tissue Disease

INTRODUCTION: Ehlers-Danlos Syndrome (EDS) is a connective tissue disease in which mutations in certain genes result in a disruption of normal collagen. There are several subtypes of the disease. Among the various subtypes which exist, hypermobility is the most common. Many of these patients can have co-existing gastrointestinal symptoms, which are often times overlooked by practitioners. CASE DESCRIPTION/METHODS: A 23 year old female presented with chest tightness, daily palpitations, and diarrhea which had been persistent for three days. Her past medical history was notable for spinal stenosis and lactose intolerance. She denied any associated abdominal pain, fever, or chills. Her family history was non contributory. One month later, she continued to have diarrhea and heartburn. She endorsed symptoms of mucorrhea with associated bright red blood per rectum, lower abdominal pain, and fecal urgency. A thorough physical examination revealed hyper flexible joints and greater than normal mobility of her fingers. After genetic testing, she was later diagnosed with Ehlers-Danlos Type 3. DISCUSSION: Up to 57% of patients with EDS have coexisting functional bowel disorders.1 Chronic gastrointestinal discomfort was reported in 86% of patients with Ehlers-Danlos – hyper mobile subtype.2 Gastrointestinal complications can include gastro esophageal reflux disease, hiatal hernia, gastritis, gastric emptying, irritable bowel syndrome (found in 62% of patients), rectal evacuatory dysfunction, and delayed colonic transit.1,2,4 Symptoms are thought to occur as a result of bidirectional signaling between gut bacteria and the brain. Enterochromaffin cells are not only accessible by enteric microbiota but also have contact with afferent and efferent nerve terminals acting as “transducers” between the gut and brain,8 able to secrete various compounds, including serotonin. One study showed that pretreatment of animals with probiotic formulation consisting of Lactobacillus helveticus R0052 and Bifidobacterium longum R0175 attenuated hypothalamic pituitary axis and autonomic nervous system activities, which were assessed through the measurement of plasma cortisol and catecholamines.10 In our patient, she was treated with a course of Rifaximin for IBS-D and was placed on a bowel regimen including a probiotic. At a four-week follow up, her symptoms had improved.

Importance of sympathetic nervous system activity during left ventricular functional recovery and its association with in-hospital complications in Takotsubo syndrome.

The relationship between activation of the sympathetic nervous system (SNS) and improvement of left ventricular (LV) function and how this correlates with clinical outcomes are not fully explored in Takotsubo syndrome (TS). The purpose of this study is to evaluate the relationship between activation of the SNS and LV function improvement and how this correlates with clinical outcomes in TS. Patients with TS were retrospectively identified. Patients were divided into two groups according to the timing of LV function improvement: < 1month (S group) and ≥ 1month (L group). Activation of the SNS was assessed by plasma catecholamine measurement and Iodine-123 meta-iodobenzylguanidine (I123-MIBG) scintigraphy. In-hospital complications included heart failure, cardiogenic shock, the use of invasive or noninvasive ventilation, life-threatening arrhythmia, cerebrovascular event and all-cause death. A total of 90 patients with TS were enrolled. Of these, 39 patients were in the S group and 51 in the L group. There were no significant differences between the two groups in clinical demographics. The L group was characterized by enhanced SNS activation, including higher levels of catecholamines and lower late heart-mediastinum ratio followed by higher washout rate in I123-MIBG scintigraphy, compared with the S group. In-hospital complications were increased in the L group (56% vs. 33.3%, p = 0.03), including higher rates of heart failure (45% vs. 23%, p = 0.03) and in-hospital death (8.0% vs. 0%, p = 0.03). In patients with TS, high activity of the SNS was observed in patients with delayed LV function recovery, which was associated with in-hospital adverse outcomes.

Plasma catecholamine levels in the early stages of treatment-naïve Parkinson’s disease

Rationale: Parkinson's disease (PD) is a neurodegenerative disorder with predominant involvement of catecholamine-producing neurons of the central and peripheral nervous system. Taking into account the relative availability and low costs of plasma catecholamine measurements, it is worthwhile to study these parameters as biomarkers of the early stages of PD.Aim: To determinate whether plasma levels of dopamine (DA), norepinephrine (NE), L-3,4-dihydroxyphenylalanine (DOPA) and dihydroxyphenylacetic acid (DOPAC) in patients with early stages of PD are related with akinetic-rigid and tremor-dominant variants and to compare the results to healthy volunteers.Materials and methods: This was an observational cross-sectional cohort study performed from 2012 to 2015. The main study group included unselected outpatients who attended the Republican Consultative and Diagnostic Center of Movement Disorders and Botulinotherapy (Kazan, Russia) with newly diagnosed early PD (Hoehn and Yahr stages I and II, 1967), of various ages and both genders, who had not been given any specific antiparkinsonian treatment. The control group included healthy volunteers with no clinical signs of PD (they could have other chronic diseases of the non-extrapyramidal origin). Plasma catecholamine levels were measured by gas liquid chromatography.Results: One hundred and thirty (130) treatment-naïve patients with newly diagnosed PD (mean age 59.34 ± 8.42 years, male gender 45.38%) were enrolled into the main study group. The control group included 56 healthy volunteers matched for age and gender. The distribution of various PD forms and stages was as follows: PD tremor-dominant variant 56.9%, PD akinetic-rigid variant 43.1%; PD stage I 76.9%, PD stage II 23.1%. Irrespective of the variant and stage, the PD patients demonstrated decreased NE levels, compared to the controls (95% confidence intervals 124–216 and 248–428 pg/mL, respectively, р &lt; 0.026). DOPA plasma level was reduced only in the patients with akinetic-rigid PD variant (р = 0.017), while DOPAC level in the patients with PD stage II (р = 0.008). The average DA:NE:DOPA:DOPAC ratio was 1:32:105:64 in the control group, 1:62:238:88 in the patients with PD tremor-dominant variant (the difference is significant for NE and DOPA, р &lt; 0.05), and 1:29:96:32 in those with PD akinetic-rigid variant (p &gt; 0.05). In the healthy controls the changes in DOPA levels account for 84% of the DA and NE variability; no correlation between DOPAC and other catecholamines was found. On the contrary, in the PD patients regardless of the stage and the disease variant, DOPAC levels directly correlated with DA (p &lt; 0.04). The PD tremor-dominant variant patients demonstrated a direct correlation between plasma NE and DOPA levels (p &lt; 0.05).Conclusion: The results obtained on absolute and relative parameters catecholamine turnover in the patients with early PD stages support the hypothesis on different pathophysiology of the tremor-dominant and akinetic-rigid variants of PD.

Does sympathetic dysfunction occur before denervation in pure autonomic failure?

Pure autonomic failure (PAF) is a rare sporadic disorder characterized by autonomic failure in the absence of a movement disorder or dementia and is associated with very low plasma norepinephrine (NE) levels-suggesting widespread sympathetic denervation, however due to its rarity the pathology remains poorly elucidated. We sought to correlate clinical and neurochemical findings with sympathetic nerve protein abundances, accessed by way of a forearm vein biopsy, in patients with PAF and in healthy controls and patients with multiple systems atrophy (MSA) in whom sympathetic nerves are considered intact. The abundance of sympathetic nerve proteins, extracted from forearm vein biopsy specimens, in 11 patients with PAF, 8 patients with MSA and 9 age-matched healthy control participants was performed following a clinical evaluation and detailed evaluation of sympathetic nervous system function, which included head-up tilt (HUT) testing with measurement of plasma catecholamines and muscle sympathetic nerve activity (MSNA) in addition to haemodynamic assessment to confirm the clinical phenotype. PAF participants were found to have normal abundance of the NE transporter (NET) protein, together with very low levels of tyrosine hydroxylase (TH) (P<0.0001) and reduced vesicular monoamine transporter 2 (VMAT2) (P<0.05) protein expression compared with control and MSA participants. These findings were associated with a significantly higher ratio of plasma 3,4-dihydroxyphenylglycol (DHPG):NE in PAF participants when compared with controls (P<0.05). The finding of normal NET abundance in PAF suggests intact sympathetic nerves but with reduced NE synthesis. The finding of elevated plasma ratio of DHPG:NE and reduced VMAT2 in PAF indicates a shift towards intraneuronal NE metabolism over sequestration in sympathetic nerves and suggests that sympathetic dysfunction may occur ahead of denervation.

Influences of Experimental Air Pollution on Human Sympathetic Nerve Traffic and Norepinephrine Metabolism: A Double Blind, Randomized, Twofold Crossover Study

BackgroundMuch of the evidence linking air pollutant exposure with changes in human cardiovascular autonomic regulation relied on epidemiological studies, exposure estimates, and indirect autonomic nervous system measurements. We tested the hypothesis that in healthy older individuals, experimental exposure to fine particles increases sympathetic nervous system activity and more so with addition of ozone.Material and MethodsEighteen healthy participants (age &gt;50 years) completed all study visits and were included in the final analysis. Participants were exposed to clean air (‘placebo’), ultrafine particles (UFP, 50μg/m3), and combination of UFP+ozone (250 ppb) for 3 hours combined with intermittent bicycle ergometer training in a randomized, three‐period, cross‐over, double‐blind fashion. Three hours following exposure, respiration, ECG, blood pressure, and muscle sympathetic nerve activity (MSNA) were continuously recorded at supine rest, during deep breathing, and during a Valsalva maneuver. Venous blood samples for plasma catecholamine measurements were taken at baseline. Induced sputum was obtained at the end of each study day.ResultsCombined exposure to ozone and UFP but not UFP alone caused a significant increase in sputum neutrophils and circulating leucocytes. We did not detect significant effects on blood pressure or heart rate. Resting MSNA was 47±12 with clean air, 47±14 with UFP, and 45±14 bursts/min with UFP+ozone. Maximum MSNA during Valsalva phase IIb was similar. Yet, with UFP+ozone plasma norepinephrine concentrations were significantly increased with concomitant reduction in the dihydroxyphenylglycol (DHPG)/norepinephrine ratio. Respiratory sinus arrhythmia and Valsalva ratio were unaffected by experimental air pollution.ConclusionExposure to ultrafine particles with or without ozone does not elicit clinically relevant changes in central sympathetic or parasympathetic activity in healthy older subjects. The paradoxical norepinephrine increase with UFP+ozone may be explained by reduced peripheral norepinephrine uptake and metabolism.Support or Funding InformationMM was funded by a grant from the German Research Foundation (Deutsche Forschungsgemeinschaft, DFG, awarded to JJ: JO 284/8‐1). The German Aerospace Center (DLR) supported JT, KH, and MM (50 WB1117, 50WB1517).

Sympathetic activity and early mobilization in patients in intensive and intermediate care with severe brain injuries: a preliminary prospective randomized study.

BackgroundPatients who experience severe brain injuries are at risk of secondary brain damage, because of delayed vasospasm and edema. Traditionally, many of these patients are kept on prolonged bed rest in order to maintain adequate cerebral blood flow, especially in the case of subarachnoid hemorrhage. On the other hand, prolonged bed rest carries important morbidity. There may be a clinical benefit in early mobilization and our hypothesis is that early gradual mobilization is safe in these patients. The aim of this study was to observe and quantify the changes in sympathetic activity, mainly related to stress, and blood pressure in gradual postural changes by the verticalization robot (Erigo®) and after training by a lower body ergometer (MOTOmed-letto®), after prolonged bed rest of minimum 7 days.MethodsThirty patients with severe neurological injuries were randomized into 3 groups with different protocols of mobilization: Standard, MOTOmed-letto® or Erigo® protocol. We measured plasma catecholamines, metanephrines and blood pressure before, during and after mobilization.ResultsBlood pressure does not show any significant difference between the 3 groups. The analysis of the catecholamines suggests a significant increase in catecholamine production during Standard mobilization with physiotherapists and with MOTOmed-letto® and no changes with Erigo®.ConclusionsThis preliminary prospective randomized study shows that the mobilization of patients with severe brain injuries by means of Erigo® does not increase the production of catecholamines. It means that Erigo® is a well-tolerated method of mobilization and can be considered a safe system of early mobilization of these patients. Further studies are required to validate our conclusions.Trial registrationThe study was registered in the ISRCTN registry with the trial registration number ISRCTN56402432. Date of registration: 08.03.2016. Retrospectively registered.

123I-MIBG Scintigraphy in the Subacute State of Takotsubo Cardiomyopathy

ObjectivesThe study sought to investigate adrenergic activity in patients with takotsubo cardiomyopathy (TTC). BackgroundTTC is a specific type of reversible heart failure possibly caused by excessive catecholamine stimulation of the myocardium. Scintigraphic iodine-123–meta-iodobenzylguanidine (mIBG) imaging of the heart and measurement of plasma catecholamines can be used to assess adrenergic activity in vivo. The authors hypothesized that sympathetic nerve activity is increased in the subacute state of TTC, and this study used cardiac mIBG imaging and plasma levels of norepinephrine and epinephrine as markers to assess this hypothesis. MethodsIn this study, 32 patients with TTC and 20 controls were examined at admission and again on follow-up with echocardiography, mIBG scintigraphy, and plasma catecholamine measurements. ResultsEjection fraction (EF) was initially 36 ± 9% but increased to >60% (p = 0.0004) in all patients with TTC. In the control subjects EF was initially higher (51 ± 11%; p = 0.0004) than in the patients with TTC. However, EF of the patients with TTC exceeded that of the control subjects on follow-up (56 ± 8%; p = 0.0007). The mIBG imaging showed a lower late (4-h) heart-to-mediastinum ratio (H/Mlate) (2.00 ± 0.38) and a higher washout rate (WR) (45 ± 12%) in the subacute state of TTC, both when compared with follow-up (H/Mlate: 2.42 ± 0.45; p = 0.0004; WR: 33 ± 14%; p = 0.0004) and when compared with the control group in the subacute state (H/Mlate: 2.34 ± 0.60, p = 0.035; WR: 33 ± 19%, p = 0.026). On follow-up, no differences in mIBG parameters were observed between the TTC and control groups (H/Mlate: 2.41 ± 0.51, p = 0.93; WR: 30 ± 13%, p = 0.48) group. In the TTC group, plasma epinephrine levels were elevated in the subacute state (Log2[epinephrine]: 6.13 ± 1.04 pg/ml), both when compared with follow-up (5.25 ± 0.62 pg/ml; p = 0.0004) and when compared with the control group in the subacute state (5.46 ± 0.69 pg/ml; p = 0.044), and these levels remained elevated in the TTC group on follow-up compared with the control group (4.56 ± 0.95 pg/ml; p = 0.014). No significant differences in plasma norepinephrine levels were observed. ConclusionsThe present study supports a possible role of adrenergic hyperactivity in TTC.

Differences between Takotsubo cardiomyopathy and reverse Takotsubo cardiomyopathy associated with subarachnoid hemorrhage

BackgroundBoth Takotsubo cardiomyopathy (TTC) and reverse TTC (r-TTC) are characterized by reversible regional wall motion abnormalities of the heart unrelated to coronary artery pathology. It remains unclear whether and/or how r-TTC differs from TTC. Subarachnoid hemorrhage (SAH) is occasionally causative of TTC/r-TTC, and this study was conducted to detect possible differences between TTC and r-TTC associated with SAH. MethodsA single-center retrospective study was conducted on 328 consecutive SAH patients. They routinely underwent transthoracic echocardiography (TTE), ECG, and measurements of plasma catecholamines and other cardiac biomarkers within 24h of admission. Demographic, echocardiographic, electrocardiographic and neurochemical profiles were compared between patients with TTC and r-TTC. The diagnosis of TTC/r-TTC was based on the revised Mayo Clinic Criteria. ResultsData of 21 SAH-induced TTC and 10 SAH-induced r-TTC patients admitted between January 2009 and December 2014 were analyzed. The patients with r-TTC were significantly younger than those with TTC (51.8±10.9 vs. 63.5±14.4years, p=0.04). The former exhibited significantly higher plasma epinephrine levels than the latter (809±710 vs. 380±391pg/mL, p=0.04). Plasma norepinephrine levels did not differ significantly (2421±1374 vs. 1724±1591pg/mL, p=0.25). No significant differences were observed in other demographic/physiologic variables, echocardiographic parameters, frequency of ECG abnormalities, and 90-day mortality. Moreover, none of the patients who underwent a follow-up TTE exhibited morphologic change from one type to the other. ConclusionsThe pathomechanisms in TTC and r-TTC may not be identical: however, distinguishing the two conditions may not have great importance from the standpoint of clinical management and prognostication.

Abstract 14124: A Marked Increase in Myocardial Oxidative Stress Associated With Sympathetic Hyperactivity is Related to Transient Myocardial Dysfunction in Patients With Takotsubo Cardiomyopathy

Introduction: Sympathetic hyperactivity is considered as one of the most likely pathogenic mechanism in takotsubo cardiomyopathy (TC). However, little is known about the relationship between sympathetic hyperactivity and myocardial oxidative stress (OS). Hypothesis: We hypothesized that cardiac sympathetic hyperactivity was associated with cardiac myocardial OS, and OS related to transient myocardial dysfunction in patients with TC compared with acute anteroseptal myocardial infarction (AMI). Methods: In 8 TC patients and 8 AMI patients (NYHA 3), ECG, echocardiography, cardiac catheterization and serial measurements of plasma catecholamines, urinary (U) 8-hydroxy-2’-deoxyguanosine (8-OHdG) as a marker of OS and plasma CPK were performed for two weeks from onset. Results: Blood sampling from coronary sinus (CS) and aorta (Ao) at 1st day showed that norepinephrine (NE) levels and 8-OHdG level in CS were significantly higher than those in Ao and peripheral blood, indicating that an increase in myocardial OS was associated with cardiac sympathetic hyperactivity. NE levels in peripheral blood were peaked out at 1st day and 2-times higher in TC than in AMI within a week, while the max CPK value in TC was significantly lower than that in AMI. Dual scintigraphy of 201Tl and 123I-meta-iodobenzylguanidine was performed at 7th day showed that cardiac sympathetic nervous injury was much larger than myocardial perfusion damage in all TC patients. U-8OHdG level (ng/mg Cr; normal range:&lt;10.0) in TC was elevated in parallel with the profile of plasma NE level (1st day: 65.6±24.5, 4th day: 19.1±8.3, 7th day: 16.4±9.1), while it was decreased to almost normal range after percutaneous coronary intervention for left anterior descending artery in AMI (1st day: 24.3±13.8, 4th day: 9.8±3.4, 7th day: 12.3±2.7). Interestingly, difference in serum 8-OHdG levels (CS vs Ao) in TC increased proportionally with wall motion score (WMS) as an index of LV dysfunction (R=0.93, p&lt;0.001), while difference in serum NA levels (CA vs Ao) in TC did not. U-8OHdG level in TC was positively correlated with WMS at each day (1st day, 7th day). Conclusions: These findings suggest that myocardial OS induced by cardiac sympathetic hyperactivity may play a critical role in transient LV dysfunction in TC.

Sympathetic Activity and Early Mobilization in Patients with Severe Brain Injuries: A Preliminary Randomized Study

Aim: Patients who experienced severe brain injuries, in particular SAH, are at risk of secondary brain damages, because of delayed vasospasm and edema. Traditionally, these patients are kept in prolonged bed rest, to maintain adequate cerebral blood flow. However, the data supporting this assumption are limited. On the other hand, it is well known that prolonged bed rest carries out important morbidities, including cardiovascular, respiratory, and musculoskeletal. Our hypothesis is that early gradual mobilization is safe in those patients. The aim of this study was to quantify the changing in sympathetic activity, mainly related to stress, in gradual postural changes (Erigo) and in leg movements alone (Motomed) after prolonged bed rest and its effect on blood pressure. Methods: A total of 30 patients with severe neurologic injuries were randomized in three groups, according to the protocol of mobilization used after a period of bed rest of minimum 7 days: Standard protocol, Motomed protocol, or Erigo protocol. We measured plasma catecholamines and blood pressure before, during, and after mobilization. Results: Our statistical analysis of the blood pressure does not show any significant difference in the three groups. We can just observe a tendency of higher prevalence of hypotension events in the group of patients mobilized by physiotherapists (Standard protocol). The statistical analysis of the catecholamines suggests a significant increase in catecholamine production during mobilization with physiotherapists and with Motomed and no changes with Erigo, in comparison with premobilization data. The increasing production of catecholamines is an indirect sign of stress. It means that, if we compare these three methods of mobilization, Erigo is the best tolerated. In contrast to this, the most important change in catecholamine production was recorded during Motomed mobilization, and with all the types of catecholamines we measured. This can potentially complicate SAH, but prevent orthostatic problems in other categories of patients. Conclusions: This preliminary randomized study shows that the mobilization of patients with severe brain injuries by means of Erigo does not increase the production of catecholamines. It can be considered a safe system to early mobilization of these patients. Erigo avoids stress and reduces the risk of complications connected to prolonged bed rest, especially in patients after SAH. Further studies are required to validate our conclusion.

Measurement of catecholamines in rat and mini-pig plasma and urine by liquid chromatography-tandem mass spectrometry coupled with solid phase extraction.

A tandem mass spectrometry method combined with an ion-pair chromatographic separation after weak cation exchange solid phase sample extraction for epinephrine (E), norepinephrine (NE) and dopamine (DA) has been developed. Two surrogate matrixes for plasma and urine as well as stable isotope labeled internal standards were utilized for quantitation. The observed dynamic range of E, NE and DA was 0.025-100ng/ml for plasma, and 0.25-1000ng/ml for urine with a r(2) regression coefficient >0.99. Extraction recoveries were greater than 60% and the lower limit of quantitation was 25pg/ml for all three analytes in plasma. This method provided excellent sensitivity and selectivity for use with small sample volumes (≤25uL), enabling high-throughput pharmacodynamic animal model development and screening of adverse effects.

Study of Heart Rate Variability and Stress Markers in Basketball Players Submitted to Selective Loads Periodization System

The decrease in the performance of athlete is often associated with an imbalance between workload and recovery period. Thus, it is very important to implement tools which can assist in the quantifying the effects of workloads, so that the maximum performance of the athlete is reached. In this context, we know little about the influence of selective load periodization system (SLPS) on cardiac autonomic control and the effects on stress markers already known. Thus, the aim of this study was to investigate if the application of SLPS promoted alterations in autonomic modulation of heart rate variability (HRV), as well as same stress markers. Therefore, sixteen male basketball players (mean ± SE: age 23.3 ± 1.0 years; mass 87.5 ± 3.5 kg; height 194  2 cm) were submitted to SLPS and evaluated before and after a competition period. The HRV was evaluated by a spectral analysis of the time series composed of R-R intervals obtained in the supine position and during a tilt test. The evaluation of stress markers consisted of measuring plasma catecholamines, cortisol and free testosterone. The results demonstrated that the training load used during the competition period did not cause significant changes in the autonomic modulation of HRV. This affirmation is supported by the absence of change in oscillations of low frequency (LF: 0,04-0,15Hz), that corresponding to sympathetic and parasympathetic modulations, and high frequency (HF:0,15-05Hz), that corresponding only to parasympathetic modulations of HRV. Additionally, no changes were observed in plasma concentrations of catecholamines, free testosterone, cortisol and, consequently, in testosterone/cortisol ratio, when pre-competition and post-competition values were compared. In summary, our findings suggest that the use of SLPS in basketball athletes presented balance between workloads and recovery periods. However, further investigations are needed, including in other sports, so that we can evaluate the effects of SLPS on cardiac autonomic modulation and stress markers evaluated in this study.

Cardiac wall motion abnormality after bleeding from vertebral artery aneurysms.

Lesions in the medulla oblongata may be causally associated with cardiac wall motion abnormality (WMA). Although subarachnoid hemorrhage (SAH) patients occasionally develop WMA, the relationship between aneurysmal locations and the frequency of WMA has rarely been investigated. The objective of this study was to evaluate whether the frequency of WMA was higher after the rupture of vertebral artery (VA) aneurysms than that of non-VA aneurysms. We performed a retrospective chart analysis of 244 SAH patients who underwent transthoracic echocardiography and plasma catecholamine measurements. The frequencies of WMA and electrocardiographic (ECG) abnormalities were compared among patients classified by the location of aneurysms. Multivariate regression analysis was conducted to identify variables correlated with WMA. Furthermore, the relationship between plasma catecholamine levels and aneurysmal locations was evaluated. The frequency of WMA was significantly higher in patients with VA aneurysms than in those with non-VA aneurysms (45 vs. 22%, p=0.01). However, there was no significant difference in the frequency of ECG abnormalities. Multivariate regression analysis showed that VA aneurysms (OR, 3.317; 95% CI, 1.129-9.745), poor-grade SAH (OR, 2.733; 95% CI, 1.320-5.658) and concomitant hydrocephalus (OR, 3.658; 95% CI, 1.690-7.917) correlated with WMA. There were no significant intergroup differences in plasma catecholamine levels. VA aneurysms are close to several medullary nuclei that integrate autonomic inputs. A transient deformation and ischemia of the medulla oblongata caused by the mechanical stress related to the rupture of a VA aneurysm and/or a concomitant hydrocephalus may be responsible for the disproportionately high frequency of WMA.

The effect of perch access during pullet rearing and egg laying on physiological measures of stress in White Leghorns at 71 weeks of age

Egg laying strains of chickens have a strong motivation to perch. Providing caged chickens with perches allows them to perform their natural perching behavior and also improves their musculoskeletal health due to exercise. Little is known about the effect of perch access for hens on physiological measures of stress. Our hypothesis was that denying chickens access to perches would elicit a stress response. The objective of this study was to determine the effect of perch access during all or part of life cycle on physiological homeostasis in caged 71-wk-old White Leghorn hens. A total of 1,064 chicks were assigned randomly to cages with and without perches (n = 14 pullet cages/perch treatment) on day of hatch. As pullets aged, chicks were removed from cages to provide more space. At 17 wk of age, 324 chickens in total were assigned to laying cages consisting of 4 treatments with 9 replicates per treatment. Treatment 1 chickens never had access to perches during their life cycle. Treatment 2 chickens had access to perches only from 17 to 71 wk of age (laying phase). Treatment 3 chickens had access to perches only from hatch to 16.9 wk of age (pullet phase). Treatment 4 chickens always had access to perches during their life cycle. At 71 wk of age, chickens were sampled for measurement of plasma catecholamines (epinephrine, norepinephrine, and dopamine) and corticosterone; blood serotonin and Trp; fluctuating asymmetry of shank length and width; and adrenal weight. Only shank width differed among treatments. Chickens with previous exposure to perches during the pullet phase had wider shanks than chickens without access to perches (P = 0.006), suggesting that early perching promoted skeletal development. These results suggest that a stress response was not elicited in 71-wk-old White Leghorn hens that always had access to perches compared with hens that never had access to perches during all or part of their life cycle.

Probiotic gut effect prevents the chronic psychological stress‐induced brain activity abnormality in mice

A probiotic formulation (Lactobacillus helveticus R0052 and Bifidobacterium longum R0175 combination, Probio'Stick(®) ) displays anxiolytic-like activity and reduces apoptosis in the lymbic system in animal models of depression. Based on the hypothesis that modulation of gut microbiota by this probiotic formulation has beneficial effects on brain activity in stress conditions, we report a set of probiotic-evoked physiological, cellular, and molecular events in the brain of Probio'Stick(®) pretreated mice submitted to chronic psychological stress. Water avoidance stress (WAS) was applied or not (sham). Hypothalamic-pituitary-adrenal (HPA) axis and autonomic nervous system (ANS) responses to the chronic stress were assessed through plasma corticosterone and catecholamine measurements. Specific markers for neuronal activity, neurogenesis, and synaptic plasticity were used to assess brain activity. In addition, gut permeability and tight junction (TJ) proteins levels were also determinated. We observed that a pretreatment with the probiotic formulation attenuated HPA axis and ANS activities in response to WAS, and reduced cFos expression in different brain areas but Lactobacillus salivarius (a negative control) treatment was ineffective on these parameters. Moreover, probiotic pretreatment prevented the WAS-induced decrease hippocampal neurogenesis and expression changes in hypothalamic genes involved in synaptic plasticity. These central effects were associated with restoration of TJ barrier integrity in stressed mice. These data suggest that chronic stress-induced abnormal brain plasticity and reduction in neurogenesis can be prevented by a pretreatment with the Probio'Stick(®) formulation, suggesting that probiotics modulate neuroregulatory factors and various signaling pathways in the central nervous system involved in stress response.

Cyclic Vomiting Associated With Excessive Dopamine in Riley-day Syndrome

To analyze the neurochemical profile during the recurrent attacks of nausea and vomiting in patients with Riley-day syndrome. One of the most disabling features of patients with Riley-day syndrome are recurrent attacks of severe nausea/retching/vomiting accompanied by hypertension, tachycardia, and skin flushing, usually triggered by emotional or other stresses. We monitored blood pressure and heart rate and measured plasma catecholamines during typical dysautonomic crises triggered by emotionally charged situations. For comparison, measurements were repeated at follow-up after the symptoms had resolved and the patients were feeling calm and well. During a typical attack, patients were hypertensive and tachycardic. In all patients, circulating levels of norepinephrine (P < 0.002) and dopamine (P < 0.007) increased significantly. Activation of dopamine receptors in the chemoreceptor trigger zone may explain the cyclic nausea/retching/vomiting of patients with Riley-day syndrome.