Heart Rate Variability and 24 hour pH analysis in patients symptomatic for Acid Peptic Disease

Dissertation submitted for M.D. ( Branch V Physiology)

THE TAMIL NADU DR. M.G.R. MEDICAL UNIVERSITY

Department of physiology PSG Institute of Medical Sciences and Research Coimbatore 641004 April 2011

DECLARATION

I hereby declare that this dissertation entitled Heart Rate Variability and 24 hour pH analysis in patients symptomatic for Acid Peptic Disease was prepared by me under the guidance and supervision of Dr.M.Nagashree, Professor and Head, Department of Physiology, PSG IMS&R. This dissertation is submitted to Tamilnadu Dr. MGR Medical University in fulfillment of the university regulations for the award of MD Degree in Physiology.

Certificate

PSG INSTITUTE OF MEDICAL SCIENCE & RESEARCH PEELAMEDU, COIMBATORE 4.

CERTIFICATE

This is to certify that the dissertation work entitled Heart Rate Variability and 24 hour pH analysis in patients symptomatic for Acid Peptic Disease submitted by Dr.Subhashini.S, is work done by her during the period of study of her post graduation in Physiology from June 2008 to March 2010 in our institution. This work is done under the guidance of Dr.M.Nagashree Professor and Head, Department of Physiology, PSG IMS & R.

Dr.M.Nagashree Prof & Head Department of Physiology, PSG IMS & R.

Dr.S.Ramalingam Principal PSG IMS & R.

Acknowledgement

Acknowledgement

I owe a great many thanks to a great many people who helped and supported me in presenting this work. First of all, I express my thanks to Dr. S .Ramalingam, Principal, PSG Institute of Medical Sciences and Research, Coimbatore, for allowing me to do my dissertation in the PSG Medical institute. My deepest thanks to Dr .R. Nagashree M.D., Professor and Head, Department of Physiology, PSG Institute of Medical Sciences and Research, the Guide of the project for guiding me with attention and care. My deep sense of gratitude to Dr. R. Tharakeswari, Assistant Professor, Department of Physiology, PSGIMS&R, for her support and guidance in pH analysis. I also thank my faculty members Dr. T. Uma Maheswari, Professor, Dr.G.V.Latha Devi, Dr.P.Sathyavathi, Associate Professors, Dr.V.Kannan, Dr.N.Shuba, Assistant Professors, Department of Physiology, PSGIMS&R, without whom this project would have been a distant reality. My sincere thanks to Dr.K.P.Suresh, Scientist (Biostatistics), National Institute of Animal Nutrition & Physiology, Bangalore for reviewing the research methodology and statistical results of the study. I also want to express my grateful and sincere thanks to all my other colleagues and friends of my department as well as other departments in PSGIMSR for their appraisal and help that made this endeavor possible. My sincere thanks to PSGIMS&R, ethical and research committee for their approval and financial assistance.

Thanks and appreciation to the helpful people at PSG Institute of Medical Sciences and Research , for their support. I am heartily thankful to my parents and my husband, whose encouragement and support from the initial to the final level enabled me to complete this work. Lastly, I offer my regards and blessings to all those who supported me in any respect during the completion of the work. I pray and thank the all mighty for giving me the strength and health to do this work until its done.

Contents

CONTENTS

S.NO 1. 2. 3. 4. 5. 6. 7. 8. 9.

TITLE INTRODUCTION AIM AND OBJECTIVES REVIEW OF LITERATURE MATERIALS AND METHODOLOGY RESULTS DISCUSSION CONCLUSION BIBLIOGRAPHY ANNEXURES

Introduction

I. INTRODUCTION
Acid peptic disorders include a number of conditions whose pathophysiology is believed to be the result of damage from acid and peptic activity in gastric secretions. The two most common and well defined disease states under acid peptic disease are 1. Gastro esophageal reflux disease (GERD) 2. Peptic ulcer disease

Gastric esophageal reflux disease is defined as chronic symptoms of heart burn, acid regurgitation, or both, or mucosal damage, produced by the abnormal reflux of gastric contents into the esophagus 1 Reflux esophagitis refers to a subgroup of GERD patients with histopathologically demonstrated characteristic changes in the esophageal mucosa.

GERD is a highly prevalent gastrointestinal (GI) disorder, affecting the upper part of the gastrointestinal tract, and is one of the most common GI illnesses encountered in clinical practice. A population-based study, using a validated questionnaire, found that 58.7% of the population has heartburn or acid regurgitation at least once during the course of a year and that 19.8% experience symptoms at least once weekly2. It is estimated that approximately 50% of patients with typical reflux symptoms have erosive esophagitis.

The term GERD applies to a group of symptoms and signs with possible concomitant morphological changes, which arise as a consequence of pathological reflux of stomach and duodenal contents into the esophagus3

The motor control of the lower esophageal sphincter (LES) is critical for normal swallowing and emesis, as well as for the prevention of gastroesophageal reflux. Another important function of the esophagus is to prevent gastroesophageal reflux. It has always been assumed that some kind of barrier at the gastroesophageal junction was present to prevent back flow of stomach contents into the esophagus.

However, existence of a well defined lower esophageal sphincter (LES) was a matter of debate in the past. Although anatomists failed to find a well defined LES, a physiological sphincter was discovered on intraluminal pressure recordings4. It was also found that the LES relaxed on swallowing. These observations stimulated investigations into the neuromuscular basis of basal tonic closure and LES relaxation associated with or unassociated with swallowing.

GERD occurs when the normal antireflux barrier between the stomach and esophagus is impaired, either transiently or permanently. Therefore, defects in the esophagogastric barrier, such as incomplete, transient lower esophageal sphincter

relaxation, and hiatal hernia, are the primary factors involved in the development of GERD
4,5

. Symptoms develop when the offensive factors in the gastroduodenal contents,

such as acid, pepsin, bile acids, and trypsin, overcome several lines of esophageal defense, including esophageal acid clearance and mucosal resistance. As more components of esophageal defense break down, the severity of the reflux increases.

Gastroesophageal reflux disease can be divided into two groups, according to the presence of esophageal mucosal breaks 1. reflux esophagitis and 2. non-erosive reflux disease Almost all the reflux esophagitis with mucosal breaks are caused by the pathological reflux of acidic gastric contents to esophagus. Therefore, drugs that suppress gastric acid secretion effectively control reflux symptoms. On the other hand, almost 40% of nonerosive reflux disease are not caused by the reflux of gastric acid but by acid-unrelated mechanisms. Therefore, administration of proton pump inhibitors cures reflux symptom of only 50% of cases with non-erosive reflux diseases. The multi-pathogenesis of nonerosive reflux disease should be considered for the treatment of patients with reflux symptoms.5

Individuals with GERD have frequent, recurring, and prolonged episodes of reflux, usually at night5 It usually causes the typical esophageal symptoms with the most common heartburn (pyrosis) being the hallmark symptom of GERD, however the clinical manifestations of GERD can be misleading. Other manifestations include acid regurgitation and dysphagia6. Some patients with GERD have no standard symptoms while asthma, angina-like pain (NCCP - Non Cardiac Chest Pain) or pain in upper abdomen are all atypical presentations of GERD 6

Despite the fact that GERD is a common clinical problem, there is no diagnostic gold standard for this disease. 24-hour pH testing is useful to document acid exposure and allows for the correlation of symptoms to acid reflux events. Determining how long the pH

is lower than 4 (expressed as a percentage of total recording time) is the single most important parameter in these studies.

The autonomic control of gastrointestinal tract is mediated by the extrinsic parasympathetic and sympathetic nervous system and the intrinsic enteric nervous system. The parasympathetic input to the gut originates from the vagus and pelvic nerves from second through fourth sacral segments. The post-synaptic cholinergic neurons provide excitatory input to the gastrointestinal tract. The sympathetic system provides inhibitory input to the gastrointestinal tract.

Specifically, vagal and spinal afferent nerves convey sensory information from the gut to the central nervous system, and are sensitive to both mechanical and chemical stimuli. The central nervous system influences the enteric nervous system and vice versa, with a reciprocal interference activity.7 In some cases, disturbances of the vagal nervous system have been identified as cause of functional gastrointestinal diseases, although intestinal events can influence autonomic balance.8-12

Autonomic nervous activity is often described as being modified in oesophageal disease; indeed previous studies have shown that an altered sympatheticparasympathetic balance is common in patients with reflux oesophagitis and that a low resting vagal tone is strongly associated with increased oesophageal acid sensitivity.

The extrinsic autonomic nervous system appears to integrate functions in anatomically discrete areas of the gastrointestinal tract. Even though the role of GERD in the production of reflux esophagitis has been well established in the literature , the causative mechanisms in GERD have not been entirely identified and continue to attract the interest of researches.

Impaired gastric emptying is frequently associated with cardiovagal neuropathy and small-fiber dysfunction. Morphological changes in the vagus nerve are reported in some, but not all studies.

Power spectrum analysis of cardiovascular signal variability, and in particular of the RR period (heart rate variability, HRV), is a widely used methodology for investigating autonomic neural regulation in health and disease that can quantify the sympathovagal balance modulating the sinus node pacemaker13. In some cases, it can also quantify the neural regulation of other organs or apparatuses.

In numerous abnormal conditions, such as essential arterial hypertension, acute myocardial infarction and heart failure, the sympathovagal balance may be altered in basal conditions14. However, a reduced responsiveness to an excitatory stimulus is the most common feature that characterizes numerous pathophysiological states. The attenuation of an oscillatory pattern can also reflect an altered target function, thus providing important prognostic markers.

The resting autonomic control of the heart is reflected in the beat to-beat fluctuations of the heart rate or the RR interval in the electrocardiogram (ECG). These variations in milliseconds of the duration of one cardiac cycle from the other is known as heart rate variability (HRV) and is traditionally expressed in statistical measures of time-domain analysis and as measures of spectral power under frequency-domain analysis. These measures are reproducible when obtained under resting conditions13 implying constancy of the resting cardiac sympathetic and parasympathetic modulation. In the last decade, there has been renewed interest in the functional relationship between the gastrointestinal tract , the oesophagus in particular and the cardiovascular system.14-17

In the past, the purported link between upper gastrointestinal and cardiovascular diseases was defined as the Roemheld gastrocardiac syndrome, where an irritant esophagogastric stimulus is able to induce not only chest pain, but also cardiac dysrhythmias.16 Among the reasons for this increased interest is the evidence of a particular subset of dysrhythmic patients with negative cardiologic examination and symptoms of gastroesophageal reflux disease (GERD). There is clinical evidence showing the role of the esophagus in triggering cardiac dysrhythmias.16-18

Autonomic modulation of cardiac function can be considered as the efferent response resulting from the integration, in the central nervous system, of multiple viscerosensorial signals from peripheral organs, such as the gastrointestinal wall.

The esophagus and the heart share a strictly related autonomic innervation, with many afferent visceral fibres rising from the distal esophagus that constitute the afferent neural pathway through which esophageal events can influence cardiac autonomic behaviour.19-22

Normally, the tonic LES contraction is the essential element of the antireflux barrier mentioned above and it prevents the occurrence of reflux episodes in excessive number. The autonomic LES insufficiency generates TLESRs -Transient Lower Esophageal Sphincter Relaxations, which may be the reason for both physiologic and most of pathologic reflux episodes 23-27

Heart rate variability analysis helps to identify possible autonomic imbalance in patients with gastroesophageal reflux disease and help us to understand the pathophysiology of gastroesophageal reflux disease.

Aim & Objective

II. AIM AND OBJECTIVE

Although autonomic alterations are observed in patients with gastroesophageal reflux disease, the pattern of autonomic imbalance remains to be explored.

Aim:
The aim of our study is to investigate a possible relationship between gastroesophageal reflux and autonomic imbalance in patients symptomatic for acid peptic disease using spectral analysis of HRV.

Objective:
1. To identify the reflux positive group from the patients who are symptomatic for acid peptic disease using 24 hour pH analysis. 2. To compare heart rate variability analysis between reflux positive and reflux negative group. 3. To study the type of autonomic imbalance in patients who are positive for acid reflux. 4. To study the parasympathetic influence in gatro esophageal reflux disease and its role in the pathophysiology of acid reflux.

Review of Literature

III. REVIEW OF LITERATURE

Gastroesophageal reflux disease (GERD) is a clinical condition that results from the reflux of gastric contents into the esophagus. The pathophysiology of GERD is multifactorial with the disease ultimately related to the balance between factors tending to damage (or sensitize) the esophageal mucosa and those tending to preserve it (a competent esophagogastric junction (EGJ) and normal esophageal acid clearance (esophageal and gastric motility-dependent).

GERD can be caused by malfunction of esophageal, lower esophageal sphincter (LES) and the stomach defense mechanisms. Most common factor in etiology of GERD is disturbed LES functions. The LES is defined as a zone of elevated intraluminal pressure at the esophagogastric junction.

Motor LES and the proximal stomach dysfunction, acting as whole unit, results in increased number and duration of reflux episodes. Dysfunction of the LES occurs via one of several mechanisms including the most common vagal dependent increased number of transient LES relaxations or as the second mechanism, permanent decrease in LES pressure with lost high pressure zone (HPZ) causing that each increase of intra abdominal pressure overcomes LES resulting in reflux episode.

Mechanism of (non-deglutive) Transient Lower Esophageal Sphincter Relaxation ( TLESR )

Gastric distension

Gastric distension, particularly just below the cardia, is the strongest stimulus for generating TLESRs TLESRs
29 28

. This is likely the mechanism for the postprandial increase in

30

, and probably plays a role in the postprandial increase in reflux frequency

This is consistent with the view that function of TLESRs is gastric venting by belching 31.

Pharyngeal stimulation

Data regarding the effect of pharyngeal stimulation on TLESRs are less uniform. Pharyngeal intubation
32

liquid installation33, and light stroking34 have all been

demonstrated to induce lower esophageal sphincter relaxations without swallowing. However, lower esophageal sphincter relaxations after pharyngeal stimulation are generally lower amplitude and shorter duration than those associated with gastric distension. Whether they represent "sub-threshold" or "incomplete" swallows35or TLESRs or both is a matter of definition.

Esophageal stimulation There are even fewer data linking esophageal stimulation to TLESRs 36.

Cortical afferents

Voluntary belching (particularly by adolescent males) suggests the possibility of cortical control over TLESRs, although it is possible that the effect is indirect, mediated by effects on intragastric pressure.

Neural pathways

The hypothetical neural pathways involved in the TLESR were recently diagrammed and explained by Mittal et al37. Afferents from stomach, esophagus, and pharynx are carried in the vagus nerve (and possibly in the glossopharyngeal nerve from the pharynx) to the medullary dorsal motor nucleus of the vagus (DMV), (either directly or) via the nucleus tractus solitarius. Efferents to the LES from the dorsal motor nucleus of the vagus are also carried in the vagal nerve. Phenomena in the pharynx and crural diaphragm linked to TLESRs are probably mediated by efferent fibers from the nucleus ambiguus.

Studies in which lower esophageal sphincter and esophageal pH have been monitored simultaneously have challenged the traditional concept that most reflux episodes result from weak steady state lower esophageal sphincter tone. Instead, the majority of reflux episodes have occurred during 5-35 second abrupt periods of complete sphincter relaxation usually against a background of normal sphincter tone.

This distinctive pattern of relaxation, which has been named "transient lower esophageal sphincter relaxation," occurs independently of swallowing.38 It depends on vagal

pathways and the hindbrain for its patterning and control, but many of these aspects are still not well understood.

Transient relaxations of the LES are considered to be an important contributory mechanism of reflux. Absent basal LES pressure is another mechanism, which accounts for about one-fourth of the reflux episodes in patients with severe reflux esophagitis. During long-lasting inappropriate relaxations, swallows often produce deglutitive contraction waves that die out in the upper esophagus, suggesting that reflux often occurs during periods of inhibition of both LES tone and peristaltic esophageal activity.39

The genesis of the resting lower esophageal sphincter pressure is not fully understood, but the following major factors have been implicated 40-42 1. hormonal influences 2. neural influences. 3. myogenic characteristics of sphincter muscle and 4. mechanical factors which relate wall tension to intraluminal pressure.43

Currently neurohormonal influences are considered to be the main determinants of tonic esophageal sphincter closure
40-41

. The tonic neural activity is believed to play a key

role in this regard. The hormonal influences are thought to be exerted indirectly by way of

cholinergic excitatory neurons. For example, gastrin, which has been proposed as the major determinant of LESP 44-45 , is shown to act by stimulating cholinergic neurons 46-47

The cholinergic neurons in the LES are also believed to be kept tonically active by fibers in the vagus nerves. Recently it has been reported that a portion of resting lower esophageal sphincter pressure may also be dependent upon tonic -adrenergic activity.48 Recent in vitro studies have also shown that sphincter muscle possesses several unique characteristics which may play an important role in the genesis of LESP in intact animals49,50.

The lower esophageal sphincter is innervated by both parasympathetic (vagus) and sympathetic (primarily splanchnic) nerves; however, the vagal pathways are the ones that are essential for reflex relaxation of the lower esophageal sphincter (LES), such as that which occurs during transient LES relaxations. Vagal afferent sensory endings from the distal esophagus and LES terminate in the hindbrain nucleus tractus solitarius. The preganglionic motor innervation of the LES arises from the dorsal motor nucleus of the vagus. Together these nuclei comprise the dorsal vagal complex within which there is a neural network coordinating reflex control of the sphincter.

Vagal efferent preganglionic neurons to the gastrointestinal tract are organized viscerotopically in the dorsal motor nucleus of the vagus. Stimulation of the dorsal motor nucleus of the vagus caudal to the opening of the fourth ventricle results in relaxations, whereas stimulation in the rostral portion of the nucleus evokes contractions of the LES.

Few details are known about the neural circuitry that links sensory information from the stomach and esophagus within the nucleus tractus solitarius to these separate populations of neurons within the dorsal motor nucleus of the vagus.

The motor vagal preganglionic output is primarily cholinergic, which ultimately stimulates excitatory or inhibitory motor neurons that control the smooth muscle tone. Excitatory neurons evoke muscarinic receptor-mediated muscle contraction. Inhibitory neurons evoke nitric oxide or vasoactive intestinal polypeptide mediated relaxation of the lower esophageal sphincter. However, other neurotransmitters are found in vagal preganglionic neurons, including norepinephrine/dopamine and nitric oxide. A

subpopulation of nitric oxide synthase containing vagal preganglionic neurons innervate the upper gastrointestinal tract and mediate relaxation.

The neurotransmitters and circuitry controlling lower esophageal sphincter pressure are important to characterize, because part of the dorsal vagal complex is outside of the blood brain barrier and is a potential target for pharmacologic intervention in the treatment of such disorders as gastroesophageal reflux disease.51

The very first scientific investigation of mechanism of food transport through the esophagus was conducted in Berlin in 1883 by a German professor, Kronecker and his medical student, Samuel Meltzer. Meltzer swallowed a balloon attached to a tube whose other end was attached a manometer to record pressure changes in the esophagus during swallowing. Professor Kronecker noted that when Meltzer swallowed a liquid bolus, the

bolus was felt to enter the stomach without any pressure changes in the esophagus being recorded. Some pressure changes were noted only late in swallow. Kronecker and Meltzer52 concluded that the tongue pumped the bolus into the esophagus which served primarily as a passive conduit for the transport of the bolus to the stomach. However, Kronecker and Meltzers conclusion that esophagus serves only as passive conduit during gravity assisted food bolus transport did not explain other known observations regarding the swallow associated esophageal transport of food. For example, the esophageal transport of food to the stomach in recumbent or head down position, as in many animals and sometimes in man, remained unexplained.

Ingelfinger53observed on fluoroscopy that swallowed barium bolus in normal volunteers in a head-down position is carried up the esophagus into the stomach by a peristaltic wave. More recent studies of simultaneously recorded intraluminal pressures and movement of barium during a swallow resolved the apparent controversy between Kronecker and Meltzers and Ingelfingers findings. These studies showed that the leading edge and bulk of barium is indeed transported by gravity in the upright position. However, the tail of the liquid bolus in the upright posture, or bulk of the bolus against gravity, is propelled through the esophagus by peristaltic contraction. Thus, although barium bolus may reach the stomach soon after swallowing due to transport by gravity, it may take up to 10 minutes to complete the peristaltic sequence and complete esophageal transport of food into the stomach 54

The existence of lower esophageal sphincter has been topics of speculation for a long time. However, there was a general sense that a pressure barrier at the gastroesophageal junction must exist. Anatomists could not detect a sphincter like structure or at least, a circular muscle thickening, at the lower end of the esophagus. The absence of any landmarks of LES made it difficult to study its morphology. Finally, manometric studies of Code and Butin55 (Butin et. al., 1953) and Ingelfinger et al56 beginning in 1950s identified a high pressure zone that relaxed on swallowing, suggesting the presence of a functional LES at the gastroesophageal junction and received general acceptance. However, some specialized morphological features that characterize the LES were described.57

If there is a functional lower esophageal sphincter, how is its basal tone on relaxation regulated? Prevalent view at that time was that smooth muscles were innervated by the parasympathetic and sympathetic nerves and the parasympathetic nerves acted by releasing acetylcholine while the sympathetic nerves exerted their effects via the release of norepinephrine. It was thought that tonic cholinergic influence due to tonic vagal activity kept the LES closed. However, effects of vagal stimulation and vagotomy yielded contradictory and confusing results.58

However, this proposal was also not well substantiated by studies of stimulation and sectioning of the sympathetic innervation and the enigma of the LES persisted. A kind of breakthrough occurred in early 1970s when it was proposed that it was not the tonic extrinsic nerve (vagal or sympathetic) activity that was involved in LES closure.59 Instead, it was the tonic excitation of the intramural cholinergic neuron by circulating gastrin that

kept the LES closed. However, this suggestion did not stand the test of time. So far, the mechanism of LES relaxation has remained elusive.

Myogenic basis of LES tone

In order to demonstrate specialized nature of the muscle at the gastroesophageal junction, Biancani and colleagues60 performed pressure diameter curves in the LES and found that the sphincter muscle showed steeper tension-diameter curves as compared to the esophageal body muscle. Christensen et. al. (1973) generated 2 mm thick strips of the esophagus at the gastroesophageal junction. He found that these strips had mechanical properties and behavior that was different from the esophageal body circular muscle. These strips developed spontaneous tone and had steeper length tension curves than those from the esophageal body muscles.61

The presence and the contribution of the myogenic tone to LES pressure in vivo was demonstrated by performing LES pressure by manometry, administering the sodium channel blocker tetrodotoxin and providing cardiorespiratory support to the anesthetized opossums. These studies showed that the doses that abolished neural responses in the LES did not affect LES pressure, providing strong evidence for myogenic basis of the LES tone.62

Subsequent studies focused on the cellular basis of myogenic tone. Asoh (Asoh and Goyal, 1978) found that the sphincter muscle shows a continuous electrical spike activity

that is not seen in the esophageal body (EB).63 Harnett et al., 2005 studied the signaling pathway for tonic contraction of LES smooth muscles.64 Szymanski et al. (2002) reported that the composition of the contractile proteins differed in LES and EB smooth circular muscle and suggested that composition of the contractile proteins contributed to the tonic behaviour of the sphincter muscle.65

Neural control of LES

The presence of the myogenic tone allowed better assessment of excitation as well as inhibition of the LES by parasympathetic (vagus) and sympathetic nerves. Overall, the sympathetic nerves were not found to exert a major effect on LES tonic contraction and relaxation. A systematic study of the effect of vagus nerve suggested that the vagus nerve exerts a tonic inhibitory effect on the sphincter pressure so that vagotomy was found to cause LES contraction and vagal efferent stimulation caused frequency dependent LES relaxation. The tonic cholinergic excitatory effect was demonstrated by the effect of atropine in causing basal LES hypotension and suppression of reflex contractions of the LES. It now appears that the vagus nerve provides both inhibitory and excitatory innervation to the LES.66

Nature of the neurotransmitters in the LES

The vagal preganglionic fibers innervate the LES smooth muscle via the postganglionic myenteric neurons. The vagal preganglionic fibers to the excitatory and the inhibitory myenteric neurons are cholinergic. On the inhibitory neurons, they exert their

effects via both nicotinic and muscarinic receptors. The postganglionic excitatory neurons may excite the sphincter by releasing acetylcholine and substance P. The nature of the inhibitory neurotransmitter had been elusive. Tttrup et. al. (1991) reported that NO, as had been reported in some other gastrointestinal sphincter, may also be the inhibitory neurotransmitter in the LES67 and Yamato (1992) showed that nitric oxide was involved in swallow induced LES relaxation.68 A definitive evidence for neuronal nitric oxide synthase (nNOS) as the enzymatic source of nitric oxide (NO) in LES relaxation associated with swallowing in mice was presented by Sivarao.69

Distinct excitatory and inhibitory vagal projection from the CNS to the LES

Rossiter and colleagues (1991) investigated whether the excitatory and inhibitory vagal pathways to the LES were represented by distinct preganglionic neurons in the brain stem.70 They showed that the inhibitory and excitatory pathway preganglionic neurons were located separately in the caudal and the rostral parts of the dorsal motor nucleus of vagus respectively.

Modulation of basal LES tone

The myogenic tone of the LES is modulated by a variety of neurohormonal influences. Inhibitory and excitatory nerves exert a tonic influence on LES pressure in vivo . Many hormones and neurotransmitters can modify LES pressure. Nicotine, -adrenergic agonists, dopamine, cholecystokinin, secretin, vasoactive intestinal polypeptide (VIP), calcitonin gene-related peptide, adenosine, prostaglandin E, nitric oxide donors such as

nitrates and inhibitors of phosphodiesterase-5 reduce LES pressure. On the other hand, muscarinic M2 and M3 receptor agonists, -adrenergic agonists, gastrin, substance P, and prostaglandin F2 cause LES contraction.

LES reflexes

Swallow associated LES Relaxation

The LES response to swallowing consists of relaxation of the LES tone, which is followed sometimes by a rebound contraction. The monitoring of the LES relaxation requires avoidance of the artifacts related to the movement of the abdominal esophagus into the chest. The relaxation starts within seconds of swallowing and lasts 5 to 8 minutes. The relaxation is part of the deglutitive inhibition and is mediated by the vagal inhibitory pathway and the postganglionic myenteric neurons that act by releasing nitric oxide.

Isolated LES Relaxation

Relaxation of the LES without esophageal peristalsis may occur during belching, vomiting and the so called transient LES relaxation (TLESR) .71 TLESRs are evoked by gastric distension and stimulation of abdominal vagal afferents. Sang and colleagues investigated brain stem neurons involved in isolated LES relaxation and swallow reflex.72-73 Isolated LES relaxation is a vagovagal reflex that involves only the inhibitory pathway . This is in contrast to swallow reflex that involves both inhibitory and excitatory pathways. Reflex LES relaxation is augmented by phosphodiesterase-5 inhibitors such as sildenafil that increase cyclic guanosine monophosphate (cGMP) in the sphincter muscle, and is inhibited by GABA-B agonists such as baclofen.

24 hour pH analysis:

Gastroesophageal reflux is a physiologic phenomenon that occurs in normal individuals, especially in the postprandial period and upright position. Although reflux frequency in controls is not normally distributed, if nonparametric analysis of normal data using the 95th percentile is used, normal values are remarkably similar for controls in different geographic areas.

Johnson and DeMeester proposed a scoring system,74 which is used more commonly and incorporates six variables as outlined below,

Variable Time pH < 4 (%) Total period Supine period Upright period Duration of longest episode (min) Number of episodes Total Longer than 5 min < 50 <3 < 4.2 < 1.2 < 6.3 < 9.2

Normal

Heart rate variability analysis in GERD The measurement of beat-to-beat heart rate variability (HRV) has provided a window through which we can noninvasively investigate the effect of visceral sensory stimuli originating from the gastrointestinal tract on autonomic outflow to the heart 75

There are complex interactions between the sympathetic and parasympathetic nervous system inputs to the sinus node. The concept of "sympathovagal balance" reflects the autonomic state resulting from the sympathetic and parasympathetic influences.

In most physiological conditions, sympathetic and vagal activities modulating heart period undergo a reciprocal regulation, leading to the concept of sympathovagal balance. This pattern can be indirectly quantified by computing the spectral powers of the oscillatory components corresponding to respiratory acts (high frequency) and to vasomotor waves (low frequency) present in heart rate variability.

In most physiological conditions, the activation of either sympathetic or vagal outflow is accompanied by the inhibition of the other (therefore the concept of "balance," as a horizontal beam pivoted at its center). This is true for reflexes arising not only predominantly from the arterial baroreceptive areas but also from the heart. For instance, the stimulation of cardiac sympathetic afferents induces reflex sympathetic excitation and vagal inhibition, whereas the opposite effect is elicited by stimulating cardiac vagal

afferents; this reciprocal reflex organization, alluding to a synergistic design, was demonstrated by recording the activity of single sympathetic or vagal efferent fibers isolated from the same mixed nerve impinging upon the heart 76.

It is now widely known that the variability of heart period, usually indicated as HRV, and of arterial pressure can also be described as the sum of elementary oscillatory components, defined by their frequency and amplitude. Various algorithms can be used to extract from the tachogram the characteristics of the rhythmic components embedded in its variability. Most studies have relied on either the Fast Fourier transform autoregressive approach
78-79 77

or an

Autoregressive algorithms can automatically furnish the

number, center frequency, and associated power of oscillatory components.

In the power spectral analysis, three components are highly evident: a high frequency (HF) at 0.33 Hz (corresponding to respiratory activity), a low frequency (LF) at 0.09 Hz (usually corresponding to vasomotor waves), and a very low frequency (VLF) around 0 Hz. Under normal conditions, the VLF component cannot be properly assessed with short time series but only with longer periods of uninterrupted data.

The power of each individual spectral component is expressed graphically by its area in absolute units (ms2). However, because the absolute values of spectral components are highly correlated to variance (corresponding to total power), some further indexes focusing mainly on the fractional distribution of power and independent of the absolute values of variance are also necessary. This is accomplished by calculating the LF-to-HF

ratio or LF and HF in normalized units (nu). These are obtained by dividing LF or HF components by the total power from which the VLF has been subtracted (to minimize the influence of noise and slow trends affecting mainly the VLF) and multiplying by 100. Although the sum of LFnu and HFnu should approximate 100 nu, it usually falls short of this value because of the presence of smaller components.

Numerous data, collected in various experimental conditions involving human and animal studies,78 support the assumptions that 1. the respiratory rhythm of heart period variability (HF) is a marker of vagal modulation (an issue widely accepted). 2. the rhythm corresponding to vasomotor waves and present in heart period and arterial pressure variability (LF) is a marker of sympathetic modulation of, respectively, heart period and vasomotion and 3. the reciprocal relation existing in the R-R variability spectrum between LFnu and HFnu is a marker of the state of the sympathovagal balance modulating sinus node pacemaker activity . The two main rhythms, one a marker of excitation and intrinsic in sympathetic activation (LF) and the other a marker of inhibition and quiet and linked to vagal predominance (HF), would be organized, in physiological conditions, in a reciprocal manner. This could also be viewed as a widespread code signaling the balance between excitation and inhibition.

Power spectral analysis of HRV includes three parameters: low-frequency power (LF), high-frequency power (HF), and the LF/HF power ratio. Vagal activity is the major contributor to the HF component, and both sympathetic and vagal activities contribute to the LF component. The LF/HF power ratio reflects the balance between autonomic nervous functions.80

Several studies have demonstrated associations between pathological acid reflux and changes in HRV among susceptible patients
81-83

However, these studies entailed a

series of short term and complicated physiological tests. Traditional ambulatory pH monitoring itself causes significant discomfort, which limits its application to investigation of delicate autonomic functional changes. Therefore, no intervention such as mechanical, electrical or chemical stimulation of esophageal receptors has been performed to assess the influence of esophageal afferents on the dynamic nature of autonomic function measurements.

Several studies have indicated that parasympathetic dysfunction is highly prevalent in patients with pathological reflux.84-85 Some reports also found a decreased sympathetic function or a generalized autonomic decline in patients with GERD.86-88 The principal mechanism of GE reflux is mediated through afferent stimuli from the gastric fundus to the sensory nucleus in the medulla and then the efferent signals for transient lower esophageal sphincter relaxation (TLESR) The observed autonomic dysfunction is supposed to cause intrinsic inhibitory reflex disturbances, abnormal fundal accommodation and gastric emptying and consequently, an increased number of TLESR.

SALVATORE M. A. CAMPO, AMBROGIO CAPRIA, FABIOLA ANTONUCCI, GENNARO MARTINO, ANGELA CIAMEI, PAOLO MARIA ROSSINI, ENRICO BOLOGNA AND DARIO CANNATA 89 evaluated autonomic nervous system function in patients with gastroesophageal reflux disease. Based on clinical criteria, 28 consecutive patients with no history of heart, metabolic, or neurologic disease reporting with upper gastrointestinal symptoms typical of gastroesophageal reflux underwent esophageal manometry, ambulatory 24-hour pH study with electrocardiographic monitoring, power spectral analysis of heart rate variability, and cardiovascular tests. A decrease of sympathetic function occurred only in the reflux group (p<0.05) supported by the lower increase of systolic/diastolic blood pressure at sustained handgrip. Decreased sympathetic function in patients with gastroesophageal reflux disease was inversely correlated with total time reflux. In these patients, decreased sympathetic function would have caused dysfunction of intrinsic inhibitory control with increased transient spontaneous loweresophageal sphincter relaxations, thus resulting in gastroesophageal reflux disease.

YI-CHIA LEE , HSIU-PO WANG , LIAN-YU LIN , KAI-JEN CHUANG , HAN-MO CHIU ,MING-SHIANG WU , MING-FONG CHEN , JAW-TOWN LIN 90 investigated the autonomic functional changes in association with intra-esophageal pH under ambulation. Over the 24-h period, patients with pathological reflux had lower average LF and HF powers than patients with functional heartburn, but the LF/HF power ratios were similar for the two patient groups. Stratified data according to waking and sleeping times, showed a significantly higher HF power but lower LF/HF power ratio during sleeping time regardless of diagnosis. In the regression analysis, esophageal pH was positively associated with

change (not basal tone) of both LF and HF powers during waking, but only with change of HF power during sleeping time. The significant associations between pH values and changes in HRV decreased gradually with time. The LF/HF power ratio did not alter significantly with pH.

BAUT U, DOBREK , LASKIEWICZ J, THOR PJ

91

studied autonomic nervous system

(ANS) disturbances in GERD patients. 23 persons (healthy volunteers and GERD patients) took part in the examination. 24-h-esophageal pH-metry and the resting, "deep breathing" (DB) test, as a short-term measurement of heart rate variability, were performed in every person. The results proved that the GERD patients have abnormal low values of the basic components that make up the HRV spectrum (LF and HF obtained from rest record and HF from record during DB). They demonstrated the evidence of functional ANS disturbances, which may be responsible for the changing the HRV parameters of the frequency domain analysis in GERD patients. The disturbances mentioned above are supposed to influence the normal modulation of the vagus nerve, which plays an important role in the maintaining the physiological LES function.

HARTLEY ET AL.

92

postulated, that presence of gastric emptying disorders, determined

by autonomic regulation disturbances, allow to regard GERD as a disease primarily related to the gut motility disorders. Decreased parasympathetic activity is linked with an increased TLESRs frequency
93

Moreover LES pressure can be lowered pharmacologically using

anticholinergic drugs which is another fact contributing to the role of decreased parasympathetic activity in the pathogenesis of the esophageal reflux disease 94

Research made by Cunningham et al.

95

confirmed former reports of the presence of the

functional disturbances in patients with GERD related to vagal regulation of the lower esophageal sphincter tension, esophageal peristalsis and gastric emptying, which in many ways contribute to the onset of reflux disease. Researchers estimated the presence of autonomic neuropathy by performing in a group of 48 patients deep breathing test and the tilt test with calculating HR changes as a 30/15 ratio and measuring a drop in systolic pressure. They did not encounter any differences in autonomic nervous system activity in patients with different degree of inflammatory process in the esophagus, by that proving those changes are among etiologic factors, and not results of the existence of inflammatory changes in the esophagus.

Q ZHANG, A LEHMANN, R RIGDA, J DENT, R H HOLLOWAY

96

Investigated the

effect of baclofen on TLESRs and postprandial gastroesophageal reflux in patients with reflux disease. They showed that Baclofen reduced the rate of TLESRs by 40% from 15 (13.818.3) to 9 (5.813.3) per three hours (p<0.0002) and increased basal lower oesophageal sphincter pressure. Baclofen also significantly reduced the rate of reflux episodes by 43% from 7.0 (4.012.0) to 4.0 (1.59) per three hours (median (interquartile range); p<0.02). In patients with reflux disease, the GABAB agonist baclofen significantly inhibits gastroesophageal reflux episodes by inhibition of TLESRs. These findings suggest that GABAB agonists may be useful as therapeutic agents for the management of reflux disease.

D. HONG, M. KAMATH, S. WANG, J. TABET, G. TOUGAS AND M. ANVARI

97

assessed the integrity of the vagal nerve afferent pathways in patients with

gastroesophageal reflux disease (GERD). Seven GERD patients (4 males and 3 females, mean age 39 8 years) were studied. Twelve healthy volunteers (11 males and 1 female, mean age 32 8 years) were used as the control group. Cortical evoked potentials were measured. Electrical stimulation of the esophageal mucosa was performed through a custom-built device. Latencies and N2/P2 amplitude were measured. Reproducible cortical evoked potentials were obtained from all subjects. The stimulation threshold for GERD patients to electrical esophageal stimulation was 5.1 1.5 mA compared to 7.8 2.0 mA in healthy volunteers (p <0.05). There was no difference in peak latencies or N2/P2 amplitude between GERD patients and controls. They showed that GERD patients have a normal vagal nerve function, but they exhibit a decreased threshold for esophageal perception. The mechanism responsible for increased esophageal sensitivity observed in GERD patients is still undetermined and warrants further study.

CHIEN-LIN CHEN, WILLIAM C ORR

98

studied the Autonomic responses to heartburn

induced by esophageal acid infusion. Total of 12 GERD patients (six male, six female; mean age 37.2 2.7 years) and 12 controls (five male, seven female; mean age 32.8 2.2 years) were studied. The study protocol included a 20-min water infusion (6 mL/min), and 20-min acid infusion (0.1 N HCl, 6 mL/min). Spectral analysis of heart rate variability (HRV) was used to assess autonomic functioning. The measured HRV indices included the power in the low frequency (LF) band (0.040.15 Hz) reflecting

sympathetic tone, and power in the high frequency (HF) band (0.150.5 Hz) reflecting vagal tone, and the LF/HF ratio as an indicator of sympathovagal balance.

Methodology

IV. METHODOLOGY
The study was conducted in the department of physiology, PSG IMS & R, after obtaining clearance from the Instuitional Human Ethics Committee (IHEC) and Department of Clinical Research and Bioethics (DCRB). Informed consent was obtained from the subjects before the study.

The study group included symptomatic patients with acid peptic disease who attended gastroenterology OP at PSG IMS & R and referred for 24 hour pH monitoring.

Classic symptoms of GERD are heartburn, defined as a retrosternal burning discomfort, and acid regurgitation. Symptoms often occur after meals and can increase when a patient is recumbent. Other ancillary symptoms seen in typical reflux are dysphagia, odynophagia, and belching. Atypical GERD symptoms include chest pain, asthma, cough, hoarseness, sore throat, globus, and repetitive throat clearing.

The selection criteria for the study group included

Inclusion criteria: 1. Age groups of 20 60 years (Both male and female) 2. Patients with symptoms of acid peptic disease 3. Patients referred for 24 hour pH study. 4. Those with BMI greater than 18.5 and less than 25. 5. Those not suffering from systemic hypertension and diabetes mellitus.

Exclusion criteria: 1. Age group <20 and >60 years 2. Subjects on drugs like proton pump inhibitors, histamine receptor antagonists and

antacids for the past 5 days will not be included. 3. Patients on drugs with a known anticholinergic activity and adrenergic blocking drugs for the past 2 months were excluded. 4. Patients who are on drugs affecting cardiac rhythymicity for past 2 months. 5. Those with BMI more than 25 and less than 18.5. 6. Those suffering from systemic hypertension and diabetes mellitus.

After selecting the study group according to the selection criteria, written informed consent was obtained. A detailed history taking and a complete physical examination was done and a complete record was obtained for future verification.

Anthropometry:
Body mass index All the participants were measured for height and weight. Height was measured in cms as the study participants stood in their upright position using the Height measuring scale. The weight was measured using weighing machine. From this BMI was obtained by dividing weight in Kg by square of the height in meters. BMI of 18.5 to 24.9 were graded as normal. 25 29.9 were graded as overweight and more than 30 as obese. Only the participants with normal BMI were included in the study.

24 Hour pH analysis:
After an overnight fast, patients were presented to the gastrointestinal investigation unit. They had been asked to discontinue acid suppressing medications (PPI) 5 days prior to the study, histamine receptor antagonists 3 days prior to the study, and prokinetic agents 24 h before the test. Antacids were stopped at midnight the evening before evaluation. Patients were also asked to abstain from alcohol and tobacco after 10 PM the previous evening. Medications required for other conditions were continued but drugs with a known anticholinergic activity were withheld for 24 hours prior to testing. Patients receiving adrenergic blocking drugs were excluded.

Before testing a patient, the pH monitor was tested by immersing the electrode in solutions of different pH. With the patient sitting in an upright position, the electrode is gently inserted into the better nostril. The patient is asked to sip water as the electrode is advanced. The electrode is advanced for about 55-60 cm until it reaches the stomach. The

pH values are continuously displayed on the monitors LCD screen. Once the electrode reaches the stomach the monitor will show a pH value below 4. After a small period of acclimation, the electrode is gently pulled out. Once the electrode begins to cross the lower esophageal sphincter ( LES ), the pH value will rise above 6. The electrode is pulled by 5 cm and taped.

Now the tip of pH-metry is positioned 5 cm above the upper border of LES. The position of LES was also manometrically determined before the pH study. The pH

electrode is connected to Naik-II pH Monitor; version II (RED TECH). Naik-II is a portable, dual channel data logger for recording pH from the digestive system, especially esophagus and the stomach.

It is used in conjunction with sterilized pH electrodes approved by Regulatory agencies. The reference electrode was placed on chest surface. Both electrodes were

calibrated before the examination in standard buffer solutions (pH = 1 and pH = 7). Once the electrode is positioned, data recording begins. The number of hours to be recorded is entered. During the study, patients were instructed to press event keys as per, following 1. meals 2. Body position changes (supine vs. upright). 3. Pain.

The patients were instructed to maintain a dairy noting down times and related activities such as sleeping, meals, pain and other events.

Patients were instructed to follow their daily routine but they were not allowed to smoke and they were instructed to avoid consumption of food and beverages of pH<4. The next day the study was completed, the catheter was removed and the recording was stored on the PC and analyzed using the GiPc pH software. A 24 hour plot of pH data is obtained. The reflux of stomach content was considered as a drop of pH < 4 in the distal esophagus. Standard parameters were taken under consideration.

HRV analysis:
The following parameters were recorded in the study group 1. Resting heart rate. 2. Resting blood pressure. 3. 5 minute ECG recording in resting supine position and HRV analysis. The guidelines provided by the task force on HRV analysis were followed.The patients, after their 24 hour pH analysis were brought to the research lab, department of Physiology. They were asked to lie down with eyes open for 20 minutes in silent.

At the end of 20 minutes of quiet supine rest, blood pressure was recorded in the supine position, using a manual sphygmomanometer. Systolic and diastolic blood pressure was measured with a calibrated manometer from the right arm and recorded to the nearest 2 mm Hg. Blood pressure was defined as the points of appearance and disappearance of Korotokoff sounds respectively.

ECG was recorded continuously for a period of 5 minutes. ECG was recorded by placing three disposable adhesion electrodes on the limb in the pattern of lead II configuration. Baseline electrocardiograms were obtained from all subjects and those with abnormal baseline ECG were excluded.

ECG was recorded using the computerized Physiograph (NIVIQURE Digital ECG recorder) in lead II for a period of 5 minutes. RR intervals were obtained after

clearance of noise and baseline fluctuations by digital filters. Those who had ectopic beats were excluded from the study. The data was filtered using digital notch filters with a sampling rate of 1024 samples per second. The inbuilt software selected the RR peaks and these RR intervals which were obtained as time points were then fed into a Microsoft excel sheet and RR intervals were copied to a notepad file. The Tachogram

The resting autonomic activity was assessed by measuring the 5 minute Heart rate variability (HRV) and HRV indices determined are 1. Time-domain parameters (standard deviation of normal normal intervals [SDNN] and Root mean square of successive differences [RMSSD] and 2. Frequency-domain parameters (low frequency power [LF], high frequency power [HF] and LF/HF power ratio). HRV analysis was done by feeding this RR interval notepad file to the HRV analysis software, version 1.1 from Biomedical Signal Analysis group, Department of Applied Physics, University of Kuopio, Finland. To analyze data lengths of 5 minutes, 256 seconds of RR interval data was taken from the tachogram and interpolated at 4 Hz to get 1024 points. Power spectral analysis was done by Fast Fourier transformation ( FFT ) after detrending and removal of the mean from the data points. Hanning window was applied to

prevent spectral leakage. The power spectral density was obtained by Welchs periodogram, using window width of 512 data points with an overlap of 256 points. The results were expressed in ms2/Hz. Two frequency bands of interest were obtained from the power spectrum of R-R intervals: LF (0.030.15 Hz) and HF (0.16 0.40 Hz). The area subtended by each spectral band was computed by numerically integrating the power therein contained. In addition, the normalized areas under both LF and HF bands were derived by dividing the integrated power within each band by the total power contained in the entire spectrum. Power spectrum of HRV

PC display of the HRV analysis software version 1.1. The LF/HF ratio was expressed as the ratio of the normalized areas, calculating the ratio of the percentage power contained in the LF band to that in the HF band (LF/HF ratio). Mean heart rate, LF/HF power ratio, individual LF and HF area power, and normalized area were individually measured in all subjects for all experimental conditions.

Report sheet

Statistical analysis
Descriptive statistical analysis has been carried out in the present study. Results on continuous measurements are presented on Mean SD (Min-Max) and results on categorical measurements are presented in Number (%). Significance is assessed at 5 % level of significance. Student t test ( two tailed, independent) has been used to find the significance of study parameters on continuous scale between two groups Inter group analysis) on metric parameters. Statistical software: The Statistical software namely SAS 9.2, SPSS 15.0, Stata 10.1, MedCalc 9.0.1 ,Systat 12.0 and R environment ver.2.11.1 were used for the analysis of the data and Microsoft word and Excel have been used to generate graphs, tables etc.

Results

V. RESULTS
47 patients who were symptomatic for acid peptic disease were subjected to 24 hour pH analysis. 23 patients were reflux positive and 24 patients were reflux negative. 5 minute ECG was taken for all the 47 patients and HRV analysis was done.

The HRV parameters ( Time domain measures and the frequency domain measures) were compared between the reflux positive and reflux negative group.

HRV analysis was done and compared between the reflux positive and reflux negative group to understand the exact relationship between gastroesophageal reflux disease and the influence of autonomic nervous system.

Following comparisons were done 1. Comparison of baseline characteristics of the reflux positive and reflux negative group. 2. Comparison of Resting Heart rate and blood pressure of reflux positive and reflux. 3. Comparison of frequency domain measures of the reflux positive and reflux negative group. 4. Comparison of time domain measures of the reflux positive and reflux negative group.

ANALYSIS BETWEEN REFLUX POSITIVE AND REFLUX NEGAITVE GROUP 1. Baseline characteristics of the reflux positive and reflux negative group Results are expressed as Mean + SD

Age Age of the reflux positive was 36.04 + 4.49 and the reflux negative was 36.83+ There was no significant difference between ages with p value 0.546. 4.42.

BMI BMI of the reflux positive was 22.48 + 0.79 and the reflux negative was 22.04 + 1.16. There was no significant difference between BMI with p value 0.140.

Weight Weight of the reflux positive was 55.35 + 2.48 and the reflux negative was 56.21 +1.74. There was no significant difference in weight between these groups with p value 0.174.

2. Resting Heart rate and blood pressure of reflux positive and reflux negative group

Resting Heart rate (RHR) RHR of the reflux positive was 75.13+2.49 and that of reflux negative was 77.08+2.81. There was significant difference in the resting heart rate with p value of 0.016.

Systolic blood pressure (SBP) SBP of the reflux positive was 121.92+5.40 and the reflux negative was 122.92+5.27. There was no significant difference between systolic BP with p value 0.361.

Diastolic blood pressure (DBP) DBP of the reflux positive group was 78.26+4.10 and the reflux negative group was 79.33+4.20. There was no significant difference between diastolic BP with p value 0.381.

3. Comparison of frequency domain measures of the reflux positive and reflux negative group Low power frequency ( LF ) LF of the reflux positive was 292.85+51.65 and the reflux negative was 247.51+63.54. There was significant difference between the LF powers with a p value of 0.01.

High power frequency HF of the reflux positive was 637.29+221.97 and the reflux negative was 455.94+215.98. There was significant difference between the HF powers with a p value of 0.007.

LF/HF ratio LF/HF of the reflux positive was 0.87+0.57 and the reflux negative was 1.57+0.98. There was significant difference between LF/HF ratios with p value of 0.007.

Low power frequency in normalised units ( LF nu ) LF nu of the reflux positive was 41.97+16.78 and the reflux negative was 55.35+16.72. There was significant difference between LF nu with p value of 0.009.

High power frequency in normalised units ( nu ) HF nu of the reflux positive was 58.02+16.78 and the reflux negative was 44.65+16.72. There was significant difference between HF nu with p value of 0.009.

4. Comparison of time domain measures of the reflux positive and reflux negative group Mean RR Mean RR of the reflux positive was 0.94+0.13 and the reflux negative was 0.85+0.14. There was significant difference between the Mean RR of both the groups with p value of 0.032.

RMSSD RMSSD of the reflux positive was 55.82+10.32 and the reflux negative was 49.05+9.71. There was significant difference between RMSSD of the study population with a p value of 0.025. NN 50 NN 50 of the reflux positive was 77.81+16.91 and the reflux negative was 65.33+14.31. There was significant difference between NN 50 of the study population with a p value of 0.009

pNN50 pNN50 of the reflux positive was 23.42+8.4 and the reflux negative was 17.61+6.03. There was significant difference between pNN50 of the study population with a p value of 0.009.

Baseline characteristics of the reflux positive and reflux negative group

Parameters

Group

Mean SD

P value

Reflux Positive Age Reflux Negative Reflux Positive BMI Reflux Negative Reflux Positive Weight Reflux Negative

36.044.49 0.546 (NS) 36.834.42

22.480.79 0.140 (NS) 22.041.16

55.352.48 0.174 (NS) 56.211.74

Resting heart rate and blood pressure of the reflux positive and reflux negative group

Parameters

Group

Mean SD

P value

Reflux Positive RHR Reflux Negative

75.132.49 0.016* 77.082.81

Reflux Positive SBP (mm Hg) Reflux Negative

121.45.40 0.361 (NS) 122.925.27

Reflux Positive DBP (mm Hg) Reflux Negative

78.264.10 0.381(NS) 79.334.20

Comparison of Frequency domain measures of reflux positive and reflux negative group

Parameter

Group

Mean SD

P value

Reflux Positive LF (nu) Reflux Negative

41.9716.78 0.009** 55.3516.72

Reflux Positive HF(nu) Reflux Negative

58.0216.78 0.009** 44.6516.72

Reflux Positive LF/HF ratio Reflux Negative

0.870.57 0.005** 1.570.98

Reflux Positive LF in ms Reflux Negative

292.8551.65 0.01** 247.5163.54

Reflux Positive HF in ms Reflux Negative

637.29221.97 0.007** 455.94215.98

Comparison of time domain measures of reflux positive and reflux negative group

Parameter

Group

Mean SD

P value

Reflux Positive Mean RR Reflux Negative

0.940.13 0.032* 0.850.14

Reflux Positive RMSSD Reflux Negative

55.8210.32 0.025* 49.059.71

Reflux Positive NN 50 Reflux Negative

77.8116.91 0.009** 65.3314.31

Reflux Positive pNN50 Reflux Negative

23.428.44 0.009** 17.616.03

Chart 1 Comparison of LF nu between reflux positive and reflux negative

80 70 60

LF (nu)

50 40 30 20 10 0 Reflux Negative Reflux Positive

Chart 2 Comparison of LF power between reflux positive and reflux negative

400 350

LF in ms#

300 250 200 150 100 50 0 Reflux Negative Reflux Positive

Chart 3 Comparison of HF nu between reflux positive and reflux negative

80 70 60
HF(nu)

50 40 30 20 10 0 Reflux Negative Reflux Positive

Chart 4 Comparison of HF power between reflux positive and reflux negative

1200 1000

HF in ms#

800 600 400 200 0 Reflux Negative Reflux Positive

Chart 5 Comparison of LF/HF ratio between reflux positive and reflux negative

3 2.5
LF/HF ratio

2 1.5 1 0.5 0 Reflux Negative Reflux Positive

Chart 6 Comparison of mean RR between reflux positive and reflux negative

1.2 1

mean RR

0.8 0.6 0.4 0.2 0 Reflux Negative Reflux Positive

Chart 7 Comparison of RMSSD between reflux positive and reflux negative

70 60

RMSSD#

50 40 30 20 10 0 Reflux Negative Reflux Positive

Chart 8 Comparison of NN50 between reflux positive and reflux negative

90 80 70 60 50 40 30 20 10 0 Reflux Negative Reflux Positive

Chart 9 Comparison of pNN50 power between reflux positive and reflux negative

NN 50#
pNN 50#

35 30 25 20 15 10 5 0 Reflux Negative Reflux Positive

VI. DISCUSSION
Gastroesophageal reflux disease (GERD) or acid reflux disease is defined as chronic symptoms or mucosal damage produced by the abnormal reflux of stomach acid to the esophagus.99

Acid reflux irritates the walls of the esophagus, inducing a secondary peristaltic contraction of the smooth muscle, and may produce the discomfort or pain known as heartburn. Evidence indicates up to 36% of otherwise healthy individuals experience heartburn at least once a month. Most episodes of acid reflux are asymptomatic.

Heartburn is the most common symptom of GERD. In some patients it may be accompanied by other GERD symptoms, such as regurgitation of gastric contents into the mouth, chest pain and difficulty swallowing. Pulmonary manifestations, such as asthma, coughing, or intermittent wheezing and vocal cord inflammation with hoarseness, occur in some GERD patients. In addition, acid can be regurgitated into the lungs in some GERD patients, causing wheezing or cough. Acid refluxed into the throat can cause sore throat. If acid reaches the mouth, it can dissolve enamel of the teeth.

Only a minority of patients develop complications of GERD. These complications include breaks in the lining of the esophagus (esophageal erosions), esophageal ulcer, and narrowing of the esophagus (esophageal stricture). In some patients, the normal esophageal lining or epithelium may be replaced with abnormal (Barrett's) epithelium. This condition (Barrett's esophagus) has been linked to cancer of the esophagus and must be carefully

watched. Lung (pulmonary) aspiration, asthma and inflammation of the vocal cords or throat may also be caused by GERD.

After a meal, the lower esophageal sphincter (LES) usually remains closed. When it relaxes at an inappropriate time, it allows acid and food particles to reflux into the esophagus. Secondary peristalsis returns approximately 90% of the acid and food to the stomach. Once peristalsis ends, the LES closes again. The remaining acid in the esophagus is neutralized by successive swallows of saliva, which is alkaline in nature, and then cleared into the stomach.

The pathophysiology of GERD is associated with dysfunction of the various mechanisms called the anti-reflux barrier. Lately, the disturbances of the autonomic nervous system have been stressed in the pathogenesis of GERD.

Transient Lower Esophageal Sphincter Relaxation ( TLESR) in the absence of a swallow is an important mechanism responsible for gastroesophageal reflux events. The TLESR is a vagally mediated reflex event that is triggered by intra esophageal or intragastric stimuli or both.

Transient, inappropriate relaxation of the LES appears to be the most important LES mechanism for gastroesophageal reflux. Unlike the brief (3 to 10 seconds in duration), appropriate LES relaxations that accompany primary peristalsis, so-called transient LES

relaxations do not occur during normal peristalsis and last for up to 30 seconds. In normal individuals, brief episodes of gastroesophageal reflux (called physiologic reflux) occur almost exclusively through the mechanism of transient LES relaxation. Transient LES relaxations can occur spontaneously, after primary peristalsis, or with failed peristalsis.

In transient LES relaxation there is no swallow (absent pharyngeal wave) and no activity in the body of the esophagus. For no apparent reason, LES pressure collapses to O at which point the esophageal pH records an episode of acid reflux.

In Transient LES relaxation after primary peristalsis - there is a swallow (evidenced by a pharyngeal wave) followed by peristalsis in the body of the esophagus. The LES relaxes appropriately during the primary peristaltic sequence and, when the peristaltic wave reaches the LES, the muscle contracts appropriately. Rather than returning to baseline pressure as is normal, however, the LES again relaxes completely after the primary peristaltic sequence at which time acid reflux occurs (indicated by the drop in esophageal pH).

In Transient LES relaxation during failed peristalsis there is a swallow evidenced by the pharyngeal wave, and peristalsis proceeds to the proximal esophagus. The peristaltic sequence fails, however, and does not advance beyond the proximal esophagus. Nevertheless, the LES relaxes in anticipation of a bolus that never arrives, and acid reflux follows. In some patients with GERD, the resting pressure in the LES is so weak that the

sphincter does not pose an effective barrier to reflux. LES pressure remains at or near 0, and the esophageal pH records multiple episodes of acid reflux.

Acid reflux can be associated with a sudden increase in abdominal pressure in a patient with a weak LES. Normally, increases in abdominal pressure are attended by commensurate increases in LES pressure. This mechanism ordinarily prevents gastroesophageal reflux during coughing, sneezing, and straining. In patients whose LES is weak, however, sudden increases in abdominal pressure may not be accompanied by similar elevations in LES pressure. The abrupt rise in abdominal pressure exceeds the rise in LES pressure, and acid is propelled into the esophagus as evidenced by the drop in esophageal pH.

The Lower Esophageal Sphincter (LES) is a thickened ring of tonically contracted circle of smooth muscle that generates 2-4 cm high pressure zone at gastroesophageal junction and serves as a mechanical barrier between stomach and esophagus. The right crus of the diaphragm encircle the LES by the phreno-esophageal ligament and thus provide additional mechanical support. Both structures generate a high pressure zone in the distal esophagus ( 15 mmHg to 30 mmHg above gastric pressure). Failure of one or both of these complimentary structures may lead to incompetence of the anti reflux barrier resulting in pathological gastroesophageal reflux.

Originally gastroesophageal reflux was thought to occur across a hypotensive LES. However, the LES basal pressure is variable in patients with GERD, and in most cases

within normal limits100. However patients with erosive esophagitis or Barretts esophagus demonstrate a significantly lower mean LES basal pressure than patients with non erosive reflux disease ( NERD)100. In patients with NERD the mean LES basal pressure is similar to normal controls. For most patients with GERD , the predominant mechanism of gastroesophageal reflux is TLESR of an otherwise normal LES101.

Physiologic reflux occurs mainly in an upright posture, post prandially102. This reflux is facilitated by episodes of inappropriate transient LES relaxation. TLESRs are spontaneous, abnormally prolonged episodes of LES relaxation, which are not preceded by a swallow or peristalsis. TLESR is a neural reflex, mediated through the brain stem, and the vagus nerve is its efferent pathway103. Gastric distension and pharyngeal stimulation have been demonstrated to elicit such relaxation. This is considered as the only mechanism of reflux in people without GERD and in most of those with GERD. GERD patients demonstrate prolonged post prandial receptive relaxation of the fundus leading to a delayed emptying of the proximal stomach.104

Since the first description of heartburn nearly 1800 years ago, our understanding of the pathophysiology of heartburn and esophageal pain has grown beyond the simple concept of acid induced mucosal injury. As the intricate neuroanatomy of esophageal innervation has become better defined, it is apparent that the pathophysiology of reflux may help in the treatment of GERD.

The association between pathological acid reflux and HRV changes have been demonstrated in several studies. Studies have shown that altered visceral perception and lower pain thresholds in patients with symptomatic gastroesophageal reflux disease (GERD) and noncardiac chest pain. Autonomic changes associated with the perception of heartburn in patients with GERD are poorly understood.

The HRV examination seems to be the best non-invasive method to evaluate the disturbances of ANS. Frequency domain analysis of HRV is considered to be the best semiquantitative method of ANS activity evaluation.

We observed the simultaneous decrease of LF component and increase of HF in the reflux positive patients. We demonstrated the evidence of functional ANS disturbances, which may be responsible for changing the HRV parameters of the frequency and time domain analysis in GERD patients. The disturbances mentioned above are supposed to influence the normal modulation of the vagus nerve, which plays an important role in maintaining the physiological lower esophageal sphincter function.

Other authors also showed impairment of ANS function in patients with GERD. Those authors, using the method of short term recording showed significant reduction of LF and HF in GERD patients in comparison with healthy individuals 105. Campo et al. observed reduced activity of sympathetic system and the total duration of reflux106Stimulation of esophagus with hydrochloric acid caused significant increase of parasympathetic

component HF and decrease of LF/HF during acid provocation and return to initial status directly after the infusion107

Pirtniecks et al. found the ANS disorders, mainly of the parasympathetic system, in patients with so called nonspecific esophageal motility disorders108

The esophagus and the heart share common spinal and vagal innervations. The latter give rise to number of esophagocardiac reflexes. One of these is a vagally mediated inhibitory reflex in which esophageal distension results in a decreased heart rate. Clinical studies illustrate the presence of an altered esophagocardiac reflex in noncardiac chest pain. Balloon distension of the esophagus resulted in an attenuated inhibitory esophagocardiac reflex in patients with noncardiac chest pain.109 In contrast using power spectral Heart Rate Variability as a measure of the vagal outflow of the heart, Tougas et al. demonstrated an increase in vagal activity in acid sensitive patients with noncardiac chest pain.

Our study gives a solid and strong evidence of altered cardiac autonomic activity in patients with gastro esophageal reflux.The key role has been attributed to parasympathetic dysfunction, which may adversely affect motor activity of this area by increasing transient LES relaxation number and impairing LES pressure, esophageal acid clearance and motility of the proximal stomach.

CONCLUSION

VII. CONCLUSION
Despite the limitations of the study, to our knowledge, this is the first study to investigate the cardiac autonomic innervations in patients with acid peptic disease in Indian subcontinent by means of HRV parameters.

The esophagus, LES and stomach can be envisioned as single functional unit controlled by neuro-hormonal factors. The abnormalities that contribute to GERD can start in any component of this unit, resulting particularly from disturbances in their control system. It is extremely important to identify factors and mechanisms leading to functional failure of this system so that causative therapy can be effectively applied.

The results show that the parasympathetic component of HRV is increased and sympathetic component is decreased in patients with gastroesophageal reflux. In the view of presented study results, disturbances of the parasympathetic branch of the autonomic nervous system can be observed in patients with gastroesophageal reflux disease. It can be presumed that disturbances mentioned above are not simply just the results of inflammatory process. They can be regarded as essential in the pathogenesis of gastroesophageal disease as they can be involved in TLESRs generation, which is a major contributor to the development of GERD.

In patients with GERD, transient LES relaxation is the most frequent, but not necessarily the exclusive, cause of reflux. However we believe that the role of sympathetic

component (LF) in the disease pathogenesis is also possible and cannot be omitted. It seems that this topic needs further studies in order to find answers to all the remaining questions.

Most patients with reflux disease donot produce more gastric acid than normal people. It is because of reflux of the acid into the esophagus. The most effective drugs currently available are those that inhibit acid production.

Understanding the pathophysiology of the gastroesophageal reflux will help in the development of drugs that will enhance lower esophageal sphinter function, and thus prevent reflux. Control of transient lower esophageal sphincter relaxation by drug therapy is an attractive future option for management of supraesophageal complications of reflux disease. In addition, recent studies on the effect of baclofen, a known GABA agonist, in inhibiting TLESRs should prompt randomized control trials in patients with GERD.

Annexures

CONSENT FORM Heart Rate Variability And 24 hour pH analysis in patients symptomatic for Acid Peptic Disease MD Thesis Research Project 2008 - 2011

Department of physiology PSG IMS&R

I, _____________________, have been explained about the purpose of the research study on pH and HRV analysis. I have received verbal information on the above study and on the possible benefits, risks and discomforts of ECG recording. I have been given chance to discuss the study and ask questions.

By signing this form I give my consent to take part in this study and I am aware that my participation is entirely voluntary. I agree that my personal data, including data relating to my physical or mental health condition may be used for this study. I have been assured that the information from this study, if published in scientific journals or presented at scientific meetings, will not reveal my identity. Truly, ______________________ (Signature of the subject) Name: Date & Time: (To be filled by the subject) ______________________ (Signature of the researcher) Witness: 1. 2.

QUESTIONNAIRE

Name Age :

Sex Occupation Income Address

: : : :

Height Weight :

Present complaints

Duration of the symptoms:

Are you currently taking any prescription or non-prescription medications?

Do you have an ongoing or chronic illness?