The Catecholamines in Psychiatric and Neurologic Disorders

By C. Raymond Lake

The Catecholamines in Psychiatric and Neurologic Disorders focuses on the contributions of catecholamines (CA) in the modulation of blood pressure, stress and exercise, body movements, memory, learning, emotions, thought processing, appetite, and mediation of psychotropic drug action. The selection first elaborates on the techniques for the assessment and interpretation of catecholamine measurements in neuropsychiatric patients and catecholaminergic response to stress and exercise. Discussions focus on noradrenergic response to isometric exercise, isotonic exercise, effect of acceleration on sympathetic activity, techniques for sympathetic nervous system evaluation, and measurements of CA and their metabolites in cerebrospinal fluid. The text then takes a look at urinary CA in behavioral research on stress; CA in anxiety disorders and mitral valve prolapse; and interaction with neurotransmitters in normal subjects and in patients with selected neurologic diseases. The selection examines noradrenergic responses in postural hypotension, norepinephrine, alcohol, and alcoholism, and catecholamine metabolism in anorexia nervosa. Topics include cerebral catecholamine metabolism in anorexia nervosa; central nervous system norepinephrine and voluntary alcohol drinking; and overview of norepinephrine in selected pediatric disorders. The book is a dependable reference for neuropsychiatrists and readers interested in the contributions of catecholamines on psychiatric disorders.

• Language: English
• Publisher: Elsevier Science
• Release date: Oct 22, 2013
• ISBN: 9781483163123

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Preface

Central and peripheral catecholaminergic neurotransmission regulates many bodily functions. For example, one or more of the catecholamines (CA) are important in the modulation of blood pressure, responses to stress and exercise, body movements, memory, learning, mood, emotion, thought processing, appetite, and the mediation of psychotropic drug action. The study of CA has been approached from several directions. Animal research employing sophisticated histochemical and fluorescent antibody techniques has provided detailed anatomic and physiologic information about CA processes in the central and peripheral nervous systems, but since these techniques require tissue for analysis, parallel human studies have been impossible. Fortunately, advances in human CA assay methodology in the 1970s, including the development of complex radioenzymatic and high-pressure liquid chromatographic techniques which can measure CA in human plasma, have greatly increased our understanding of how the CA regulate movement and behavior. While early fluorimetric techniques could barely detect the change in plasma norepinephrine that occurs when people stand, these newer techniques are exquisitely sensitive. Our newfound ability to measure catecholamines and their metabolites in blood, urine, and cerebrospinal fluid has allowed us to make some basic observations: a brief period of exercise can increase plasma CA tenfold; CA regulate their own release through autoreceptors; the kidneys secrete CA.

Plasma norepinephrine (NE) levels from supine, resting subjects reflect basal sympathetic activity while sympathetic responsivity can be evaluated by comparing these basal levels with the NE values in plasma after a standardized five minute stand. An intact sympathetic nervous system responds to the stand with a doubling of its activity, which is paralleled by a 100 percent increase in circulating NE. A wider application of this approach involves examining these neurotransmitter mechanisms in various patient groups. For example, NE plasma measurements are now used in the diagnosis and treatment of neurologic patients with orthostatic hypotension. In addition, studying CA in schizophrenic patients may advance our understanding of this disease by helping to define homogeneous subgroups. Already there is consistent evidence that paranoid schizophrenics share CA abnormalities. Even if we cannot establish this as a cause-and-effect relationship, the identification of a reliable marker entirely secondary to the disease would be extremely valuable.

Investigating the mechanisms of action of drugs effective in the treatment of psychiatric conditions, especially schizophrenia and major affective disorders, has provided important information about abnormalities associated with the behavioral pathology. These drugs have major effects on CA metabolism, thus implicating abnormal catecholaminergic neurotransmission in the etiology of these disorders. Most recently, attention has focused on the sensitivity of CA receptors in affective disorders and the changes induced by antidepressant drugs which appear to normalize disturbed CA receptor sensitivity as mood improves.

New information that has accumulated about CA has led us to redefine our concepts of how they function in the body. The powerful techniques that have brought us this information are available for use in research and clinical settings. The goal of this volume is to bring together the most recent data published about the CA in neurologic and psychiatric disorders and instruct the reader in how best to use these new techniques.

The book is divided into five sections: I, Stress; II, Neuropsychiatric Disorders; III, Pediatric Disorders; IV, Affective Disorders; and V, Schizophrenia. The opening chapter discusses the historical background of CA research, the development and application of technology currently available to measure CA, and the establishment of normal values, a necessary step in the determination of abnormal CA neurotransmission. Because CA measurement techniques are now so sensitive that they can pick up minute biological variation, sampling procedures are very important. This first chapter outlines how to obtain samples for measuring CA, discussing how procedures as simple as venipuncture and standing can have a profound influence on CA levels. The chapter also discusses how to stress subjects in a mild and reproducible fashion to measure the responsivity of CA systems. Section I then describes how physical stress (Chapter 2), emotional stress (Chapter 3), anxiety and minor medical illnesses (Chapter 4) alter CA in humans.

Section II discusses CA findings in selected neuropsychiatric disorders. Chapter 5 provides a general review of CA abnormalities and discusses current neuroanatomical interrelationships among central nervous system (CNS) CA pathways in a variety of neurologic diseases, including patients with Parkinsonism and Huntington’s chorea. Subsequent chapters (6, 7, 8, 9) detail specific abnormalities in illnesses in which CA metabolism has been more thoroughly studied. All diseases that cause postural hypotension lead to abnormal CA responses to standing, so CA levels are very helpful in pinpointing the correct diagnosis and instituting proper therapy. Chapter 6 explains how to differentiate two distinct types of neurologically based orthostatic hypotension by measuring plasma NE levels. In alcoholism and in anorexia nervosa (Chapters 7 and 8) there is compelling evidence of central abnormalities in CA metabolism which are involved in the pathophysiology of these disorders.

Section III consists of two chapters which deal with CA metabolism in neuropsychiatric disorders in children. CA have been implicated in the etiology of many childhood disorders, which are reviewed in this section. Since drugs that help alleviate hyperactivity in children release endogenous CA in the CNS, the role of the CA in hyperactivity is thoroughly discussed.

Sections IV and V review studies assessing sympathetic nervous system function and central CA neurotransmitter activity in depression and schizophrenia. In general terms, depression can be viewed as a disease involving deficient CA tone and schizophrenia as a disease of excessive CA activity. Chapters 11–15 detail CA synthesis, storage, release rate, and receptor sensitivity in these disorders. It is here where the new analytic techniques discussed at the beginning of this text promise to yield essential information about these important but poorly understood diseases.

In summary, although the CA have been the object of intense study since 1890 in animals and in tissue preparations, no appropriate animal model depicts most of the neurologic and psychiatric diseases in which CA are involved. Increasingly sensitive techniques that allow us to study CA metabolism in these patients have brought us conceptual advances in understanding these disorders and practical advances in using CA measurements to help treat these diseases. Assays of the CA, their enzymes, and metabolites are currently important in the management of many illnesses. This book provides background information about how the CA are involved in these diseases and practical information about how to use CA measurements to evaluate neurologic and psychiatric patients.

CRL and MGZ

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Techniques for the Assessment and Interpretation of Catecholamine Measurements in Neuropsychiatric Patients

C. Raymond Lake and Michael G. Ziegler

Publisher Summary

This chapter discusses the techniques for the assessment and interpretation of catecholamine (CA) measurements in neuropsychiatric patients. CA is a group of compounds widely recognized for their sympathomimetic properties. The most intensely researched are epinephrine (E), norepinephrine (NE), and dopamine (DA), which have a similar structure consisting of a benzene ring, adjacent ring hydroxyl groups at positions 3 and4, and a carbon side chain containing an amine group. The first CA assays were based on a measurable physiologic response to the application of CA. The simplest of these measured increase in blood pressure after injection of a compound. However, because a variety of substances besides CA can raise or lower blood pressure, this technique is nonspecific. The enzyme catechol-O-methyltransferase (COMT) transfers a methyl group from S-adenosylmethionine (SAM) to the 3 or 4 hydroxyl position of catechols. Interpretation of results from the COMT assay requires awareness of the biochemistry of the assays. Because calcium and other inhibitors of COMT activity are present in plasma in variable amounts, plasma from different sources is needed to be individually standardized.

Catecholamines (CA) are a group of compounds widely recognized for their sympathomimetic properties. The most intensely researched are epinephrine (E), norepinephrine (NE), and dopamine (DA), which have a similar structure consisting of a benzene ring, adjacent ring hydroxyl groups at positions 3 and 4, and a carbon side chain containing an amine group (Figure 1.1). One of the earliest references to CA, the structures of which were unknown at the time, was made in 1895 by Oliver and Schafer [78] who noted that an adrenal gland extract caused a pressor response in recipient animals. Two years later this pressor substance was identified as N-methyl-3,4-dihydroxyphenylethanolamine or E, also called adrenaline [1]. The similarities between the pressor effects of E and the stimulation of the sympathetic nerves, reported in 1901 [71], led 4 years later to the proposal that the neurotransmitter of the sympathetic nervous system (SNS) was an E-like substance [26]. It was not until 1946, however, that von Euler [35,36] isolated this substance, NE, which had been synthesized in 1904 [96], and identified it as the primary neurotransmitter of the SNS.

Figure 1.1 Chemical structures of the three most common biogenic catecholamines: dopamine, norepinephrine, and epinephrine.

DA, first synthesized in 1910 [4], was shown by Blaschko [6] in 1939 to be an intermediate compound in the synthesis of NE and E, and currently is known to function as a central nervous system (CNS) neurotransmitter. DA may be involved in some types of schizophrenia (see Chapter 15), in major affective disorders (see Chapter 13), and certainly is important in blood pressure and neuroendocrine regulation. Peripheral actions are less clearly established.

By 1950 a fluorometric assay technique had been developed which attempted to estimate CA in the range found in human plasma [73]. There have been many subsequent developments in CA methodology, not all of which are reviewed in this chapter. The reader interested in a more detailed account of developments in CA methodology should consult references 49 and 53. Our goals are to briefly discuss current assay techniques, metabolism and function, and to review the state-of-the-art techniques for SNS evaluation in normal subjects and patients suffering from various neuropsychiatric disorders.

One principal area of interest is the diagnostic utility of plasma CA measurements in the clinical evaluation of many diseases which have similar clinical pictures but different etiologies. For example, neurologic disorders of the CNS and peripheral neuropathies can produce the same autonomic symptoms, but their plasma NE levels are different (see Chapter 6). Supraventricular tachycardias can be caused by CA or by an intrinsic cardiac defect. CA levels can also help predict the success of β-blocker therapy. Many current theories of etiology of the functional psychoses involve CA (see Chapters 13 through 15).

An indication of the increasing interest in CA is the number of articles on the topic in the medical literature. CA articles listed in the Index Medicus have increased nearly fivefold since 1960, a trend that appears to be continuing into the 1980s with more than 1045 papers published in the first half of 1984 (Figure 1.2). Since 1960, more than 35,000 articles about CA have been published with interest initially focused on E, then, beginning in the late 1960s, on NE and more recently, on DA.

Figure 1.2 Number of catecholamine articles published each year in journals indexed by Index Medicus from 1960 through mid-1984.

CATECHOLAMINE ASSAY TECHNIQUES

Interest in measuring CA dates to the late 1800s but it took approximately 75 years to develop assays sufficiently sensitive and specific to reliably estimate plasma levels. Some of the major areas in which advancements in the development of CA assays were made include the following:

Sensitivity

Because the CA are present in plasma in picogram quantities, only an extremely sensitive assay can detect them.

Specificity

CA (NE, E, and DA) are chemically similar to amines, amino acids, and fluorescent compounds that can be falsely measured as CA.

Interfering compounds

Human plasma contains proteins that bind CA, cations and purines that inhibit their enzymatic conversion, and lipids that interfere with their extraction.

Stability

CA are labile at physiologic pH and room temperature and are easily oxidized.

In vitro CA formation

CA in vivo are metabolized to glucuronides and sulfated compounds. When samples are stored at acidic pH to prevent oxidation, these CA conjugates can be hydrolyzed back to the parent CA and increase apparent blood levels of the free (nonconjugated) compounds.

Protein binding

CA are capable of binding to proteins, and the bound component may be measured by some assays but not by others.

A chronologic summary of some of the developments of CA measurement methodology follows.

Bioassays

The first CA assays were based on a measurable physiologic response to the application of CA. The simplest of these measured increase in blood pressure after injection of a compound. However, because a variety of substances besides CA can raise or lower blood pressure, this technique is nonspecific.

Colorimetry

In 1856, Vulpian [104] noted that aqueous extracts of the adrenal gland acquired a rosy color when exposed to sunlight. This color reaction formed the basis for some of the original chemical methods used to measure CA levels. These assays are also nonspecific. One technique reported plasma venous levels of CA as high as 10 μg/ml. (Normal supine, resting NE is approximately 250 to 300 pg/ml, E is 30 to 50 pg/ml, and DA is reported from 50 to 400 pg/ml.)

Fluorimetry

The CA phenolic group fluoresces with an excitation maximum at 285 nm and emission maximum at 325 nm. Fluorescence can be enhanced by chemical derivitization with trihydroxyindole or ethylenediamine. Anton and Sayre [2] thoroughly evaluated the variables involved in the fluorometric assay and concluded that only DA would interfere markedly with the assay for NE and E. Assays based on their technique accurately measure large increases in CA levels following stress but, typically, give artifactually high supine, resting (baseline) levels of CA. The method of Renzini et al [88] provides more accurate measurements of plasma CA levels following both stress and at rest. Miura et al’s [74] adaptation of this assay compares favorably with radioenzymatic techniques. However, in other articles by this group [74], basal CA levels measured by their fluorescent technique were quite low.

The COMT Radioenzymatic Assay

The enzyme catechol-O-methyltransferase (COMT) transfers a methyl group from S-adenosylmethionine (SAM) to the 3 or 4 hydroxyl position of catechols. In 1958, Axelrod and Tomchick [3] published a method to purify rat liver COMT, showed that divalent cations stimulated enzymatic activity, and demonstrated the wide range of catechols methylated by this enzyme. Ten years later, Engelman et al [28] developed a double isotopic method for converting catechols to their ¹⁴C-labeled derivatives. The ¹⁴C-labeled E and NE are converted by oxidation to radiolabeled vanillin, which is extracted into toluene and counted by liquid scintillation spectroscopy. The sensitivity of this assay was much greater than that of currently available fluorescent or bioassay techniques, but was limited by the specific activity of the ¹⁴C-SAM used as a tracer methyl donor.

E is converted to metanephrine and NE to normetanephrine by COMT. Engleman and Portnoy [27] separated these compounds by thin-layer chromatography before converting them to vanillin. The assay’s sensitivity is enhanced by use of ³H-SAM* [79].

COMT will methylate most catechols including both the small catechol molecules and large molecules such as dobutamine. This is a disadvantage if the methylated products are not separated, but when the assay is combined with the appropriate separation techniques, a variety of CA can be measured [22]. In the COMT assay for CA, plasma samples can either be analyzed directly, or after plasma proteins are precipitated with perchloric acid. The sample is incubated with the enzyme COMT, the methyl donor ³H-SAM, ethyleneglycol-bis-(β-aminoethyl ether)-N,N¹,-tetraacetic acid (EGTA) to bind inhibiting calcium, and magnesium to stimulate enzyme activity. During incubation in a buffer solution, radiolabeled O-methylated derivatives are formed, which are then extracted into a lipid solvent. The labeled amines are back-extracted into an acid layer and then separated by thin-layer chromatography. The β-hydroxylated compounds can be oxidized to ³H-vanillin, which is extracted into toluene and counted by scintillation spectroscopy. Non-β-hydroxylated substituents such as DA must be extracted into a slightly more polar solution, which gives higher blank levels.

Interpretation of results from the COMT assay requires awareness of the biochemistry of the assays. Because calcium and other inhibitors of COMT activity are present in plasma in variable amounts, plasma from different sources may need to be individually standardized. Many catechol drugs, such as isoproterenol, isoetharine, dobutamine, and apomorphine, can competitively inhibit the assay. Derivatives of the antihypertensive drug α-methyldopa, such as α-methyldopamine, can also interfere. Aluminum is a potent inhibitor of the COMT enzyme, and patients with renal failure may have high blood levels of aluminum. Because the samples are separated by thin-layer chromatography, very high levels of one compound may cause some cross-over contamination into the chromatographic band of another compound, thus artifactually elevating levels of the adjacent compound. This elevation can be corrected by subtracting the cross-over. Despite problems with this assay, the COMT technique is less disturbed by interfering substances than the old fluorometric methods and is sufficiently sensitive and reliable to provide useful information about plasma CA in most circumstances. In fact, variability within the assay itself is usually less than variability caused by different sample collection techniques, a subject discussed in the section, Techniques for Sympathetic Nervous System Evaluation, later in this chapter.

The PNMT Radioenzymatic Assay

In the body, phenylethanolamine-N-methyltransferase (PNMT) converts NE into E by transfer of a methyl group from SAM to the primary amine of NE [47,69]. The enzyme can be partially purified from bovine adrenal glands. In the test tube radiolabeled ³H-SAM is used as a methyl donor to convert NE to radiolabeled E, which can be isolated and counted by scintillation spectroscopy. The enzyme is fairly specific for β-hydroxylated phenylethylamines and does not appreciably label DA or further metabolize E. The PNMT technique is the most reliable assay available for measuring plasma NE levels and can accommodate large numbers of samples easily [69]. Some laboratories have experienced difficulty in adequately purifying PNMT, so the technique has been used less widely than the COMT method.

The PNMT assay has several other advantages over the COMT-based radioenzymatic technique. The PNMT assay is more specific for NE than is the COMT technique because PNMT has much greater substrate specificity than does COMT. Because PNMT is not inhibited by aluminum, CA can be preconcentrated on alumina prior to assay and the assay can be made extremely sensitive when large volumes of plasma (1-20 ml) are concentrated on alumina and eluted into 0.1 ml of 0.1 to 0.3 N HC1. The same preconcentration step eliminates inhibiting substances and allows the assay to be easily standardized for tissue or plasma samples without fear that standardization will vary widely from sample to sample. Henry et al’s [47] technique for purifying radiolabeled E from other radioactive contaminants without using chromatography makes the PNMT method one of the most rapid NE assay systems available.

The PNMT assay is performed by shaking the plasma sample with alumina to adsorb CA and then washing the alumina and eluting CA with a small volume of acid. The acid eluate is incubated with PNMT and ³H-SAM to form ³H-E from the NE. The ³H-E is then adsorbed onto alumina, which is washed three times to remove contaminating ³H-SAM. The ³H-E is eluted from the alumina and any remaining ³H-SAM is precipitated prior to scintillation counting of the ³H-E product [69].

Liquid Chromatographic Assay Techniques

Modern liquid chromatographic techniques, called high-pressure or high-performance liquid chromatography (HPLC), separate CA or metabolites and internal standards into sharp peaks. After separation, CA can be detected by native fluorescence, by fluorescence of their chemical derivatives, or by electrochemical detection. The resolving power of this technique makes it desirable for research applications.

Separation Techniques

Early HPLC separations used cation exchange materials that were relatively inexpensive and offered separation efficiencies of 1,000 plates per meter. Newer 5- to 10-μm reverse-phase and cation exchange materials increase the efficiency to greater than 25,000 plates per meter and allow shorter columns, which shorten the time needed to perform the same analysis. Reverse-phase columns can be used directly, but have most frequently been modified using soap chromatography with the addition of sodium octylsulfate or sodium heptylsulfonate to the mobile phase. The hydrophobic, anionic detergents are strongly adsorbed to the stationary phase, in effect transforming it into a cation exchange column. This transformation creates a versatile column suitable for the separation of neutral and anionic substances as well as CA.

Fluorescence Detection

Once CA are separated by HPLC, a sensitive detection system is necessary to quantify the small amounts present in human plasma. Natural fluorescence of the amines requires several nanograms for detection. Derivatized fluorescence techniques can greatly enhance the sensitivity of detection. CA can be derivatized by trihydroxyindole, ethylenediamine, ninhydrin, fluorescamine, or O-phthalaldehyde to greatly enhance their fluorescence. Derivatization may be done precolumn or postcolumn, the latter having recently been automated. Yui and Kawai [114] compared the sensitivity of these detection techniques and concluded that postcolumn derivitization with trihydroxyindole is the most sensitive and specific detection system.

Electrochemical Detection

CA are unstable in solution because they oxidize easily, a characteristic used to advantage in electrochemical detection in which the solution containing CA is passed by a carbon electrode with an electrical potential in the range of +600 mV. The CA are oxidized to orthoquinones, and the resulting electric current passing across the electrode is proportional to the amount of CA present. The electrochemical potential of the CA group is similar to that of uric acid, which is present in 10,000-fold higher concentrations than the biogenic amines. For these reasons, the amines need to be separated prior to electrochemical detection by isolation on alumina and then separation by HPLC. This process can provide detection limits in the range of 25 pg/ml [114], which makes the system, when performing optimally, applicable to the measurement of human plasma CA levels. Because all CA are detected with equal sensitivity, this technique is more applicable to research than is fluorescence or derivitization, which are variably sensitive to NE, E, and DA.

Gas Chromatography

CA are polar, nonvolatile, and unstable molecules, and thus are unsuitable for use in gas chromatographic procedures. However, they are small enough to allow volatile derivatives to be made by chemical techniques. These derivatives can be separated by gas chromatography. As with any chromatographic technique, CA must be detected after separation, which is accomplished by flame ionization detection, electron capture, or mass spectroscopy. Flame ionization is not sufficiently sensitive to detect the low concentrations of CA in plasma. Electron capture is approximately 2000 times more sensitive but lacks specificity. Mass spectroscopy has a sensitivity similar to that of electron capture detection but is much more specific. Gas chromatography with mass spectroscopy (GCMS) is extremely accurate because it can be standardized by deuterated CA, which differ slightly in molecular weight from the compounds being measured. Because of this, GCMS provides a reference standard against other less rigorous procedures and has been used, for example, to verify the accuracy of the PNMT radioenzymatic assay [117]. However, the technique is so time-consuming and expensive that it is not suitable for routine CA