Roles of catechol neurochemistry in autonomic function testing

Abstract

Catechols are a class of compounds that contain adjacent hydroxyl groups on a benzene ring. Endogenous catechols in human plasma include the catecholamines norepinephrine, epinephrine (adrenaline), and dopamine; the catecholamine precursor DOPA, 3,4-dihydroxyphenylglycol (DHPG), which is the main neuronal metabolite of norepinephrine; and 3,4-dihydroxyphenylacetic acid (DOPAC), which is the main neuronal metabolite of dopamine. In the diagnostic evaluation of patients with known or suspected dysautonomias, measurement of plasma catechols is rarely diagnostic but often is informative. This review summarizes the roles of clinical catechol neurochemistry in autonomic function testing.

INTRODUCTION

Catechols look like cats

The term “catechol” refers to a chemical structure in which there are adjacent hydroxyl groups on an aromatic ring. The word derives from the Malay word catechu, referring to an extract derived from a type of acacia tree. The chemical catechol itself is not an endogenous compound in humans. One way to remember the characteristic structure of catechols is to visualize the head of a cat (Figure 1). The two pointy ears correspond to the hydroxyl groups.

Figure 1: Catechols look like cats.

The benzene ring of catechols with its two adjacent hydroxyl groups resembles the head of a cat.

Several 3,4-dihydroxy catechols are found normally in human plasma (Figure 2). Measurement of some of these can be used to diagnose or provide informative information relevant to the diagnosis of several autonomic disorders [40].

Figure 2: Chemical structures of catechols found in human plasma.

Additional abbreviations: Cys = 5-S-cysteinyl; DOPAC = 3,4-dihydroxyphenylacetic acid; DHPG = 3,4-dihydroxyphenylglycol

Catecholamines are a subtype of catechols in which there is a hydrocarbon tail ending in an amine (−NH₂) group. The endogenous catecholamine in humans are dopamine, norepinephrine, and epinephrine. Since urine has an ammonia-like smell, to remember the chemical structure of catecholamines, visualize the entire cat, from head to tail, in its litterbox.

Sources and meanings of plasma levels of catechols

Human plasma normally contains at least 7 catechols. Three are the catecholamines DA, norepinephrine, and epinephrine. One is 3,4-dihydroxyphenylalanine (DOPA), which is the precursor of the catecholamines and the immediate product of the rate-limiting enzyme in catecholamine biosynthesis, tyrosine hydroxylase (TH). DOPA is converted to DA by the enzymatic action of L-aromatic-amino-acid decarboxylase (LAAAD).

3,4-Dihydroxyphenylglycol (DHPG, DOPEG) is the main intra-neuronal metabolite of norepinephrine (Figure 2), and 3,4-dihydroxyphenylacetic acid (DOPAC) is the main intra-neuronal metabolite of DA. Both are formed from the enzymatic action of monoamine oxidase (MAO) in the outer mitochondrial membrane. As will be seen, simultaneous measurement of the intra-neuronal metabolites with the parent compounds greatly enhances the diagnostic information compared to measuring the catecholamines alone.

It should be noted that circulating epinephrine is also converted to DHPG by MAO. In fact, when Irwin J. Kopin (for many years the supervisor and mentor of the first author of this review) and Julius Axelrod first reported that DHPG is an endogenous compound [84] they described DHPG as a metabolite of epinephrine. DHPG in human biological fluids, however, is only a minor metabolite of epinephrine, because in non-neuronal cells epinephrine (and DHPG) are efficiently and rapidly metabolized by catechol-O-methyltransferase (COMT) to form non-catechol O-methylated compounds (mainly 3-methoxy-4-hydroxyphenylglycol (MHPG), which Axelrod discovered [6] with Kopin. COMT is present in adrenomedullary chromaffin cells and non-catecholaminergic cells in the brain and sympathetic nervous system but not in catecholaminergic neurons.

Two other catechols typically found in human plasma are 5-S-cysteinylDOPA (Cys-DOPA) and 5-S-cysteinyl-DA (Cys-DA). These compounds are formed from covalent bonding of DOPA or DA with cysteine (or with glutathione, followed by enzymatic conversion of glutathione to cysteine).

The following briefly discusses the sources and meanings of plasma levels of some of the catechols found in human plasma (Figure 2).

Norepinephrine

Plasma norepinephrine is derived mainly from exocytotic release of norepinephrine from sympathetic noradrenergic nerves [59]. Most of released norepinephrine is taken back up into the neuronal cytoplasm via the Uptake-1 process mediated by the cell membrane norepinephrine transporter (NET). It was for discovering neuronal reuptake as a means to inactivate neurotransmitters that Julius Axelrod received his Nobel Prize in 1970. Because of the prominence of neuronal reuptake in terminating the actions of norepinephrine, only a small proportion of norepinephrine released from sympathetic noradrenergic nerves reaches the plasma unchanged. Drugs or disorders that inhibit Uptake-1 augment plasma norepinephrine for a given amount of norepinephrine release [66]. This means that high plasma norepinephrine levels considered in isolation may not specifically indicate increased sympathetic nerve traffic.

DA-beta-hydroxylase (DBH), which catalyzes the conversion of DA to norepinephrine, normally is localized to vesicles. The immunofluorescence micrograph in Figure 5 depicts beautifully this vesicular localization. In the image, both TH (red) and DBH (green) are present in the cytoplasm, the co-localization indicated by the yellow color. Against this background, the green punctate structures show DBH without TH, likely corresponding to vesicles. The formation of norepinephrine in sympathetic nerves depends on vesicular uptake of cytoplasmic DA via the vesicular monoamine transporter (VMAT) and intravesicular conversion of DA to norepinephrine via DBH.

Figure 5: Vesicular localization of DBH in a human sympathetic ganglionic neuron.

In this immunofluorescence micrograph green corresponds to DBH, red to TH, and yellow to co-localized DBH and TH. Punctate regions of green show vesicular localization of DBH without TH (Image courtesy of R. Isonaka).

Stressors, disorders, and drugs that increase sympathetic noradrenergic nerve traffic augment exocytotic release of norepinephrine, elevating the plasma norepinephrine concentration while tending to deplete tissue norepinephrine content. For instance, congestive heart failure is associated with markedly increased entry of norepinephrine into the venous drainage of the heart [30] and depletion of myocardial norepinephrine [21].

3,4-Dihydroxyphenylglycol (DHPG)

As illustrated in Figure 2, DHPG formed in noradrenergic neurons has two general sources. The first and main source is leakage of norepinephrine from vesicles into the cytoplasm. The second and under resting conditions minor source is reuptake of norepinephrine from the interstitial fluid via the NET. Blockade of ganglionic neurotransmission or of norepinephrine release therefore produces greater proportionate decreases in plasma norepinephrine than DHPG levels [16]. Because of neuronal reuptake of released norepinephrine, stimuli that increase exocytotic release of norepinephrine result in increases in plasma levels of both norepinephrine and DHPG [41].

Leakage of vesicular catecholamines is a passive process. Depletion of vesicular norepinephrine, such as from sympathetic noradrenergic denervation or decreased VMAT activity, therefore decreases plasma DHPG levels [32, 46]. The effect can be greater than for plasma norepinephrine levels because of compensatory increases in sympathetic nerve traffic, which would maintain release of the neurotransmitter.

Drugs or disorders that decrease NET activity increase plasma norepinephrine/DHPG ratios, because more of released norepinephrine is able to reach the plasma and less of released norepinephrine is taken back up into sympathetic noradrenergic nerves to produce DHPG [32, 111].

Stressors, disorders, and drugs that increase sympathetic noradrenergic nerve traffic increase plasma DHPG levels, due to reuptake of the released norepinephrine (and possibly increased catecholamine biosynthesis) [41].

DHPG formation from cytoplasmic norepinephrine is a 2-step process involving oxidative deamination catalyzed by MAO, to form the catecholaldehyde 3,4-dihydroxyphenylglycolaldehyde (DOPEGAL) and then aldehyde/aldose reductase (AR) to form the glycol. As a glycol, DHPG readily traverses cell membranes.

DA

In catecholaminergic cells dopamine is produced from the enzymatic action of LAAAD on cytoplasmic DOPA. Pyridoxal phosphate (vitamin B6) is a required co-factor for the reaction. Cytoplasmic DA levels normally are kept very low, due to efficient uptake of dopamine into storage vesicles and oxidative metabolism mediated by MAO (Figure 2). Plasma dopamine seems to be derived partly from exocytotic release from vesicles containing newly-synthesized norepinephrine [47]. There are also important dietary sources of dopamine (bananas stand out in this regard) [24, 63].

3,4-Dihydroxyphenylacetic acid (DOPAC)

Perhaps surprisingly, given efficient vesicular uptake of cytoplasmic dopamine, human plasma normally contains substantial DOPAC [40]. DOPAC formation reflects the actions of two enzymes in series, MAO and aldehyde dehydrogenase (ALDH). The intermediary metabolite, 3,4-dihydroxyphenylacetaldehyde (DOPAL) is highly toxic and is the focus of the “catecholaldehyde hypothesis” for the death of catecholamine neurons [57]. Levels of DOPAC, like dopamine, in plasma have important dietary sources [63]. Factors that inhibit vesicular uptake, augment vesicular leakage, decrease AR activity, or decrease DBH activity would be expected to increase plasma DOPAC/DHPG ratios.

As an acid, DOPAC is pumped actively out of cells [88]. DOPAC levels in the urine exceed by far those of other free (unconjugated) catechols and plasma DOPAC [76], probably because of active secretion of DOPAC formed from dopamine in renal parenchymal cells.

Epinephrine

Plasma epinephrine levels relatively straightforwardly reflect secretion of the adrenomedullary hormone. Interpreting epinephrine levels based on blood sampled from an arm vein requires consideration of removal of arterial epinephrine from the circulation in passage through tissues of the forearm and hand. Forearm or digital vasoconstriction augments local extraction of circulating catecholamines [68]. Epinephrine levels in plasma from antecubital venous blood underestimate epinephrine levels from arterial blood, with the extent of underestimation depending on the extent of local vasoconstriction.

DOPA

DOPA is the immediate product of the rate-limiting enzyme in catecholamine biosynthesis, and plasma DOPA levels are substantial and at least partly reflect tyrosine hydroxylation in sympathetic noradrenergic nerves [64, 86]. Plasma DOPA also has important dietary sources [63]. In addition, at least theoretically, plasma DOPA levels can be influenced by tyrosinase converting tyrosine to DOPA-quinone, followed by reduction of the quinone [33, 107].

Disorders or drugs that involve blockade of LAAAD or lack of availability of pyridoxal phosphate result in buildup of DOPA with respect to dopamine and other catecholamines [120, 121]. TH blockade or deficiency would be expected to decrease plasma levels of DOPA and catecholamines [64].

5-S-Cysteinyl-DOPA (Cys-DOPA)

Catechols are susceptible to spontaneous oxidation to form quinones. DOPA-quinone binds covalently with cysteine (or with glutathione, with subsequent enzymatic hydrolysis) to form 5-S-cysteinyl-DOPA (Cys-DOPA).

The plasma of healthy people contains Cys-DOPA; however, sources of plasma Cys-DOPA have not been formally studied. Tyrosinase in melanocytes acts on tyrosine and produces DOPA-quinone and consequently Cys-DOPA [101], and as a result plasma Cys-DOPA levels are high in metastatic melanoma [1].

Cys-Dopamine

As for DOPA, dopamine in the cytoplasm spontaneously oxidizes to form dopamine-quinone and then Cys-dopamine. Human plasma normally contains scant Cys-dopamine, and the sources of plasma Cys-dopamine in humans are poorly understood. Dopamine infusion results in high Cys-dopamine levels [92].

Diagnostic abnormal patterns of plasma catechols

Because of the distinctive sources of plasma levels of catechols, in rare diseases specific, abnormal patterns of plasma catechols are diagnostic. The following discussion explains briefly the abnormal catechol patterns in DBH deficiency, LAAAD deficiency, and Menkes disease.

Dopamine-beta-hydroxylase (DBH) deficiency

DBH is required for synthesizing norepinephrine from dopamine. DBH deficiency is a rare cause of congenital neurogenic orthostatic hypotension. Because of the specific noradrenergic deficiency, patients with DBH deficiency have normal parasympathetic and sympathetic cholinergic functions. Thus, they have normal respiratory sinus arrhythmia and sweat normally.

DBH deficiency results in elevated plasma levels of dopamine and DOPAC and extremely low plasma levels of norepinephrine and DHPG [8]. In contrast with TH deficiency, DOPA levels are increased, and in contrast with LAAAD deficiency, dopamine and DOPAC levels are increased.

3,4-Dihydroxyphenylserine (droxidopa, Northera^™) is remarkably effective in the treatment of DBH deficiency [9, 93]. This is because droxidopa is converted to norepinephrine via L-aromatic-amino-acid decarboxylase (LAAAD) [11], which is expressed by parenchymal cells in body organs such as the liver and kidneys. More generally, L-DOPS can be useful to treat symptomatic neurogenic orthostatic hypotension related to norepinephrine deficiency [79, 80, 97].

L-Aromatic-Amino-Acid decarboxylase (LAAAD) deficiency

LAAAD deficiency is a rare pediatric congenital disease. Plasma, urine, and cerebrospinal fluid levels of DOPA are high with respect to dopamine, other catecholamines, and catechol metabolites [120, 121]. LAAAD is required for synthesizing serotonin, and so levels of serotonin and its deaminated metabolite 5-hydroxyindoleacetic acid are low.

Menkes disease

Menkes disease is a disease of copper metabolism that is transmitted as an X-linked recessive trait. DBH is a copper enzyme, and patients with Menkes disease all have neurochemical evidence of DBH deficiency. In at-risk newborns, the finding of high plasma DOPA/DHPG, DOPAC/DHPG, and dopamine/norepinephrine levels is diagnostic [56, 77]. This is important, because if diagnosed early enough, Menkes disease can be treated effectively by copper histidine injections [78].

Supportive abnormal patterns of plasma catechols

Measurement of plasma levels of catechols often provides supportive diagnostic or pathophysiologic information. This section presents some applications of clinical catechol neurochemistry in a variety of autonomic disorders, in approximate descending order of prevalence.

Diabetic autonomic neuropathy (DAN)

Symptoms and signs of DAN occur in several organ systems, including the cardiovascular, gastrointestinal, urogenital systems [36]. Autonomic neuropathy occurs commonly in diabetes mellitus and is associated with substantially increased morbidity and mortality [36, 95].

The finding of low plasma norepinephrine levels in a patient with DAN and orthostatic hypotension suggests that the orthostatic hypotension is related to sympathetic noradrenergic denervation. Comparing groups of diabetics with vs. without orthostatic hypotension from DAN in the late 1970s was one of the earliest applications of assay methods adequately sensitive and specific to quantify norepinephrine and epinephrine separately in human plasma. Patients with DAN were reported to have decreased plasma norepinephrine levels and normal epinephrine levels under resting conditions and attenuated orthostatic increments in plasma norepinephrine levels [19]. Bolus injection of the rapidly acting acetylcholinesterase inhibitor edrophonium resulted in blunted norepinephrine but intact epinephrine responses, consistent with decreased post-ganglionic sympathetic noradrenergic innervation [90]. Patients with symptomatic DAN have marked attenuation of plasma norepinephrine responses to pharmacologic alterations in blood pressure, indicating substantially decreased baroreflex-mediated changes in sympathetic noradrenergic outflows [28]. When plasma norepinephrine kinetics was evaluated by the tracer dilution technique, both plasma norepinephrine and epinephrine were decreased in patients with Type 1 diabetes with vs. without DAN, and since plasma norepinephrine clearance did not differ between the groups, the calculated appearance rate (spillover) of endogenous norepinephrine was decreased in the group with DAN [26].

Although overall, plasma norepinephrine levels are lower in diabetic patients with neurogenic orthostatic hypotension than without neurogenic orthostatic hypotension, it is thought that early in the development of autonomic neuropathy some patients with neurogenic orthostatic hypotension in the setting of diabetes have a hypernoradrenergic response to orthostasis [73]. Bases for this seemingly paradoxical finding are poorly understood.

Plasma DHPG/norepinephrine ratios are decreased in patients with DAN and neurogenic orthostatic hypotension [22]. An explanation for this pattern would be attenuated reuptake of released norepinephrine. Unfortunately, there is no published replication of this finding. Increased sympathetic noradrenergic nerve traffic would also be expected to increase DHPG/norepinephrine ratios; the relative increase in plasma DHPG would be smaller than that in norepinephrine because of the higher DHPG than norepinephrine concentration during supine rest.

Myocardial ¹²³I-metaiodobenzylguanidine- (¹²³I-MIBG)-derived radioactivity has been reported to be decreased in DAN [99, 126], but the results are difficult to interpret because of the possibility of concurrently decreased myocardial perfusion due to microangiopathy.

In patients with Type 1 diabetes, rigorous insulin treatment is associated with hypoglycemia unawareness and blunted plasma epinephrine responses to insulin [71]. Such treatment lowers the threshold for an adrenomedullary counter-regulatory response [38]. Avoidance of insulin-induced hypoglycemia ameliorates both the hypoglycemia unawareness and blunted plasma epinephrine responses [81]. Pancreatic islet cell transplantation reverses both hypoglycemia unawareness and blunted plasma epinephrine responses [81]. Data have been inconsistent about whether concurrent autonomic neuropathy contributes to hypoglycemia unawareness [12, 81]. Therapy emphasizing preventing hypoglycemia reverses hypoglycemia unawareness in patients with diabetic autonomic neuropathy, despite only marginal improvement of epinephrine responses [35].

Postural tachycardia syndrome (POTS)

Postural tachycardia syndrome (POTS) is a heterogeneous disorder manifested by orthostatic intolerance with excessive heart rate response upon standing in the absence of orthostatic hypotension. The condition is far more common in women than in men, for unknown reasons. POTS patients often have increased arterial [42] and arm venous [37] plasma norepinephrine and epinephrine levels during supine rest; however, the condition seems to be heterogeneous, and whether the increased SNS and SAS outflows are primary or secondary remains poorly understood. Cardiac norepinephrine spillover is increased in POTS, without decreased cardiac NET activity [50].

Parkinson disease with orthostatic hypotension (PD+OH)

A substantial minority (30–40%) of patients with Parkinson disease have orthostatic hypotension (PD+OH) [110, 127]. Plasma norepinephrine is normal in PD+OH [53], although below that in PD without orthostatic hypotension [55]. As in other forms of neurogenic orthostatic hypotension, PD+OH patients have attenuated orthostatic increments in plasma norepinephrine levels [44]. Plasma levels of catechols do not effectively distinguish PD+OH from the parkinsonian form of multiple system atrophy (MSA-P). Consistent with denervation supersensitivity in PD+OH, the patients have augmented pressor responses to infused norepinephrine [100]; however, baroreflex-sympathoneural failure can also explain this finding.

Dementia with Lewy bodies (DLB)

There is accumulating evidence that dementia with Lewy bodies (DLB) is a form of autonomic synucleinopathy. About ½ of DLB patients have orthostatic hypotension [124]. Plasma norepinephrine levels have been reported to be decreased during supine rest, with attenuated orthostatic increments in norepinephrine levels compared to healthy controls [104]. Plasma levels of other catechols have not yet been formally studied during supine rest or orthostasis.

Multiple system atrophy (MSA)

In 1977, Ziegler, Lake, and Kopin divided primary chronic autonomic failure into 2 forms—idiopathic orthostatic hypotension and the Shy-Drager syndrome [133]. These investigators reported that idiopathic orthostatic hypotension involves a sympathetic nervous system lesion, with low plasma norepinephrine levels, whereas the Shy-Drager syndrome involves central neurodegeneration, with normal plasma norepinephrine levels. Idiopathic orthostatic hypotension is now called pure autonomic failure (PAF), and the Shy-Drager syndrome is now called MSA. In confirmation of generally intact SNS innervation in MSA, we reported normal plasma levels of catechols and metanephrines in this disease [61]. Rarely, MSA involves cardiac sympathetic noradrenergic denervation [23]; even in this case plasma levels of catechols were normal during supine rest (unpublished observations).

Pure autonomic failure (PAF)

Pure autonomic failure (PAF) manifests with symptomatic neurogenic orthostatic hypotension. Plasma levels of norepinephrine, DHPG, epinephrine, and dopamine are decreased [47, 53], although there are occasional patients with normal plasma norepinephrine levels, possibly related to decreased plasma clearance of norepinephrine [106]. During orthostasis there is usually an attenuated orthostatic increment in plasma norepinephrine levels; however, an orthostatic decrease in plasma norepinephrine clearance can result in normal orthostatic increases in plasma norepinephrine [98]. Patients who have clinically diagnosed PAF with normal norepinephrine levels may have MSA that has not yet evolved to the overt motor stage.

Takotsubo cardiopathy

Takotsubo cardiopathy refers to an acute, reversible form of heart failure, usually in post-menopausal women, that is brought on by emotional distress or other factors associated with acute release of catecholamines [3, 108]. The name, takotsubo, refers to the shape of a Japanese ceramic pot for catching octopi (Figure 6). Patients with takotsubo cardiopathy have a lack of contraction or even a ballooning out of the left ventricular apex and normal basal contraction during systole, giving the heart the appearance of a takotsubo. Plasma levels of norepinephrine, DHPG, and especially epinephrine are markedly increased [130].

Figure 6: Takotsubo cardiopathy.

During diastole (a) the heart appears normal, but during systole (b) the absence of apical left ventricular contraction and presence of basal contraction give the heart the appearance of a takotsubo (c).

Pheochromocytoma

Pheochromocytomas are rare tumors of neural crest origin where the tumor cells synthesize catecholamines. The tumors usually are in the adrenal gland although can be located elsewhere and usually are non-malignant. Although rare, pheochromocytomas are important clinically, because they constitute a potentially surgically curable form of hypertension and because if left undetected they can produce sudden, unexpected hypertensive paroxysms in response to seemingly minor manipulations such as anesthesia induction. Pheochromocytoma patients can have orthostatic hypotension, possibly from decreased vascular responses to norepinephrine [117].

In patients with symptoms suggestive of pheochromocytoma (hypertensive episodes, orthostatic hypotension, pallor, sweating, headache), measurement of plasma levels of free (unconjugated) metanephrines (O-methylated metabolites of norepinephrine and epinephrine) provides a highly sensitive screening test [89] that is superior to measurement of plasma levels of the parent catecholamines themselves. This is because chromaffin cells express COMT, whereas sympathetic noradrenergic neurons do not. Within the tumor cells, catecholamines undergo continuous production and metabolism relatively independently of sympathetic nerve traffic, whereas plasma levels of epinephrine and especially of norepinephrine depend on sympathetic nerve traffic. In patients with positive plasma metanephrines, clonidine-suppression testing may be done, to separate pheochromocytoma from much more common conditions such as emotional distress [13]. Failure to suppress plasma catecholamines or metanephrines is a positive test, distinguishing pheochromocytoma from hypernoradrenergic hypertension [58].

Patients with pheochromocytoma often have high plasma norepinephrine/DHPG ratios [15, 27]. This is because plasma DHPG is derived importantly from the metabolism of norepinephrine in sympathetic noradrenergic nerves [41], whereas high circulating norepinephrine levels in pheochromocytoma are due to norepinephrine release from the tumor. False-negative results can arise in the setting of an epinephrine-secreting tumor, since plasma DHPG can be increased from extraneuronal uptake and metabolism of epinephrine.

Pseudopheochromocytoma

In patients with symptoms suggestive of pheochromocytoma, measurement of plasma free (unconjugated) metanephrines (normetanephrine and metanephrine) provide a highly sensitive screening test [89]. In pseudopheochromocytoma the patients have paroxysmal episodes of hypertension as in pheochromocytoma and less commonly have hypotension [85] but do not actually have the tumor [94]. Plasma levels of dopamine sulfate have been reported to be high in pseudopheochromocytoma, without concomitant increases in plasma levels of free (unconjugated) norepinephrine, epinephrine, or dopamine [85]. In our series, compared with controls, patients with pseudopheochromocytoma had normal plasma concentrations of norepinephrine and elevated epinephrine during supine rest [114].

Familial amyloidotic polyneuropathy

In familial amyloidotic polyneuropathy, amyloid deposition is due to extracellular deposition of the mutated protein, transthyretin, in a variety of organs and tissues—especially the liver, heart, gut, and peripheral nerves. When presenting mainly as sensory, autonomic, and motor polyneuropathy, the term familial amyloid polyneuropathy has been used, and when presenting mainly as cardiomyopathy, familial amyloid cardiomyopathy has been used. A mixed form also can occur. The disease is transmitted as an autosomal dominant trait. The amyloid fibrils are extracellular, and the neurotoxic mechanism producing length-dependent polyneuropathy is poorly understood.

Patients with familial amyloidotic polyneuropathy can have disabling orthostatic hypotension, associated with low plasma norepinephrine levels and a blunted orthostatic increment in plasma norepinephrine [18, 119]. Treatment with the norepinephrine precursor L-threo-dihydroxyphenylserine (L-DOPS) improves orthostatic intolerance in these patients [18].

Type III hereditary sensory and autonomic neuropathy (HSAN III, familial dysautonomia, Riley-Day syndrome)

HSAN III, or familial dysautonomia (FD), is a rare autosomally recessively transmitted disease in which virtually all the patients are of Ashkenazic origin. The cause is a splicing mutation of the gene, Ikappa-B-kinase-associated protein, or IBKAP. FD is syndromic, involving decreased thermal pain sensation, absence of overflow tears [70]and of lingual fungiform papillae [116], orthostatic hypotension [96], orthopedic problems, afferent baroreflex failure [102], and episodes of “crises” of vomiting and extreme swings in blood pressure and heart rate. There is arrested development of sympathetic noradrenergic innervation [43, 105], and because of this FD patients have an abnormal pattern of plasma catechols reminiscent of that in DBH deficiency [67, 129], with attenuated plasma norepinephrine responses to orthostasis [132] and elevated DOPA/DHPG ratios [5, 48].

Type IV hereditary sensory and autonomic neuropathy (HSAN IV)

HSAN IV is a rare genetic disease manifesting with pain insensitivity and anhidrosis. It is also called congenital insensitivity to pain with anhidrosis. The disease results from mutation of the NKTR1 gene. This interferes with the development of nerve growth factor-dependent neurons including sympathetic noradrenergic and cholinergic neurons. HSAN IV patients have extremely low plasma norepinephrine levels and blunted plasma norepinephrine responses to orthostasis, yet they do not have orthostatic hypotension [103]. Plasma epinephrine levels are normal and increase during orthostasis. The dissociation between SNS and SAS outflows in HSAN IV resembles that in other diseases of development of sympathetic noradrenergic innervation and points to hormonal or autocrine-paracrine determinants of adrenomedullary epinephrine release. One may speculate that denervation supersensitivity [17] involving increased numbers of alpha-adrenoceptors [25] coupled with orthostatic increases in circulating epinephrine levels may be sufficient to prevent orthostatic hypotension.

NET deficiency

Loss of function of the gene encoding the cell membrane norepinephrine transporter (NET) is an exceedingly rare cause of POTS [111]. Decreased NET activity would be expected to be associated with a high norepinephrine/DHPG ratio, due to decreased reuptake of released norepinephrine. This is the pattern found with tricyclic anti-depressants [29, 31, 41] and the NET inhibitors duloxetine [128] and atomoxetine [10].

Autoimmune autonomic ganglionopathy (AAG)

There are a variety of dysautonomias where links with autoimmunity have been described. For almost all of these, the exact bases for these links and whether they are causal remain unproven. An exception is AAG, in which autonomic failure results from circulating antibodies to the neuronal nicotinic receptor mediating ganglionic neurotransmission [49].

A few abnormalities in the pattern of plasma catechols can distinguish AAG from PAF [52]. One can conceptualize these differences in terms of intact post-ganglionic sympathetic noradrenergic neurons in AAG and loss of these neurons in PAF. Both AAG and PAF involve low plasma norepinephrine levels; however, plasma levels of DOPA, DOPAC, and DHPG have been reported to be lower in PAF than in AAG [52]. Based on our ongoing database of AAG and PAF patients the following parameters are strongest in differentiating the two forms of autonomic failure: DOPA (higher in AAG), DHPG/norepinephrine (higher in AAG), and the orthostatic absolute and fractional increments in plasma norepinephrine (greater in AAG) (unpublished observations).

Autoimmunity-associated autonomic failure with sympathetic denervation (AAD)

Acquired pandysautonomia is an uncommon cause of neurogenic orthostatic hypotension, and autoimmunity-associated pandysautonomia is rare. Beginning with the report by Young et al. [131] in 1969, several cases have been described of acquired pandysautonomia with loss of post-ganglionic, non-myelinated nerves [4], including a variant of the Guillain-Barré syndrome [125]. The acute tachycardia and hypertension that can occur in Guillain-Barré syndrome are associated with elevated plasma norepinephrine levels [2].

We recently reported a case of autoimmunity-associated autonomic failure in a young adult woman with arthritis followed by pandysautonomia, early parasympathetic recovery, and persistent sympathetic denervation. Multiple positive autoimmune markers justified diagnosing a form of autoimmunity-associated autonomic failure. AAD in this patient was associated with extremely low plasma levels of norepinephrine, DHPG, dopamine, DOPAC, epinephrine, and DOPA [54]. Levels of all these catechols increased between the initial and 1.5 year follow-up visits, suggesting partial sympathetic re-innervation.

Evoked changes in plasma catechol levels

Orthostatic norepinephrine responses to identify neurogenic orthostatic hypotension

Orthostatic hypotension occurs relatively commonly and increases in prevalence in aging populations. Orthostatic hypotension often reflects effects of drugs, dehydration, hypovolemia, or chronic debility [20]. Less commonly orthostatic hypotension is neurogenic, due to decreased reflexive systemic vasoconstriction during orthostasis. neurogenic orthostatic hypotension characterizes a variety of autonomic disorders, including diabetic autonomic neuropathy, PD+OH, MSA, DLB, and the prototype of primary chronic autonomic failure, PAF.

Plasma norepinephrine levels normally approximately double within 5 minutes of standing up from lying supine [87]. In neurogenic orthostatic hypotension the orthostatic increment in plasma norepinephrine is attenuated. The extent to which this blunting reflects baroreflex-sympathoneural failure vs. sympathetic noradrenergic denervation has not been determined formally. The result can be a false negative when plasma norepinephrine clearance declines during orthostasis [98] and false positive when the testing ends before about 3 minutes [39]. On the other hand, an increment in plasma norepinephrine less than 60% after 5 minutes of orthostasis supports a diagnosis of neurogenic orthostatic hypotension.

Orthostatic norepinephrine in POTS

POTS patients often have high plasma norepinephrine levels during orthostasis [118]. High orthostatic norepinephrine levels are associated with higher heart rates and blood pressures [37]. Considering that the POTS patients with high orthostatic norepinephrine had increased plasma renin activity and decreased aldosterone levels, one may speculate that a form of adrenocortical insufficiency interferes with renal sodium reabsorption, decreasing blood volume and compensatorily recruiting SNS outflows [118].

Tilt-evoked sympathoadrenal imbalance to identify autonomically mediated syncope

Tilt-evoked autonomically mediated syncope (also called neurally mediated syncope, neurocardiogenic syncope, reflex syncope, and vasovagal or vasodepressor syncope) is associated with substantial proportionate increases in plasma epinephrine levels [62, 123] that are larger than concurrent increases in norepinephrine levels, a phenomenon called sympathoadrenal imbalance [7, 34, 51, 82, 122]. Similar findings have been noted in syncope evoked by lower body negative pressure [75]. Sympathoadrenal imbalance is associated with tilt-induced syncope even in healthy adults [123] and therefore does not of itself indicate an abnormality of SNS or SAS functions so much as a centrally evoked neuroendocrine pattern [20].

Drug tests using plasma catechols as dependent measures

Infusion of the sympathomimetic amine tyramine increases release of endogenous norepinephrine [14] and thereby increases blood pressure. Patients with sympathetic noradrenergic denervation therefore have blunted vasoconstrictor and pressor responses to tyramine [83]. Tyramine infusion increases plasma norepinephrine levels [60, 109] (Figure 7). Larger increases in plasma DHPG than norepinephrine levels probably reflect tyramine-induced displacement of norepinephrine from vesicular stores into the cytoplasm [115]. In PAF and PD+OH, plasma norepinephrine and DHPG responses to tyramine are attenuated, as one would expect from sympathetic noradrenergic denervation and thereby decreased neuronal uptake of tyramine. Although neurochemical responses to tyramine were reported to be attenuated in patients with “neuropathic” POTS [74], the results were probably influenced artifactually by dopamine contamination of the infusate [72]. Attenuation of the plasma norepinephrine response to tyramine was part of the discovery process leading to identification of POTS from inherited NET deficiency [111].

Figure 7: The “cheese effect.”.

In the setting of blockade of MAO-A or both MAO-A and MAO-B, ingested tyramine can reach the liver, the systemic circulation, and sympathetic noradrenergic nerves. Displacement of vesicular NE can then evoke NE release, resulting in paroxysmally increased blood pressure and plasma NE.

Here is why if you were taking an MAO inhibitor, you wouldn’t want to attend a wine and cheese party. Patients on a MAO inhibitor are susceptible to hypertensive paroxysms after ingestion of tyramine-containing foodstuffs such as red wine and hard cheese. The latter is the basis for naming the condition “the cheese effect.” Ordinarily, relatively little of ingested tyramine makes its way to the bloodstream because of an effective “gut-blood barrier,” which includes a variety of enzymes, one of which is MAO. Patients taking an MAO inhibitor have a relatively permissive gut-blood barrier for dietary substances that normally would be broken down by MAO in the gut or liver. In the setting of MAO inhibition dietary tyramine can penetrate the gut-blood barrier and reach sympathetic nerves. Once inside the nerves, tyramine in the cytoplasm gets taken up into the vesicles by way of the vesicular monoamine transporter (VMAT), and inside the vesicles tyramine accelerates leakage of norepinephrine from the vesicles, possibly by alkalinizing the vesicles and decreasing the hydrogen ion gradient required for concentrating norepinephrine in the vesicles. Norepinephrine then builds up in the cytoplasm and can go through the NET to reach the fluid surrounding the cells, or it can exit the cell from vesicles that are porated at the membrane surface. By these mechanisms, norepinephrine is delivered to its receptors on cardiovascular cells, and the blood pressure and the force of the heartbeat increase in a manner correlated with the increase in plasma norepinephrine levels. In people taking an MAO inhibitor, such as for depression, ingestion of tyramine can produce a paroxysmal increase in blood pressure or evoke a dangerously abnormal heart rhythm.

It should be noted that selective MAO-B inhibitors are less likely than MAO-A inhibitors or mixed MAO-A/B inhibitors to produce a cheese effect, because enzymatic breakdown of ingested tyramine in the gut occurs especially via MAO-A (Figure 7).

Isoproterenol infusion increases plasma norepinephrine levels, probably via occupation of beta-2 adrenoceptors on sympathetic nerves and increased SNS outflow in response to decreased total peripheral resistance [65]. In patients with neuroimaging evidence of cardiac sympathetic denervation (e.g., PAF, PD+OH), isoproterenol infusion results in markedly attenuated increments in plasma norepinephrine levels, while tachycardia responses are intact [115].

Effects of clonidine, yohimbine, or ganglion blockade on plasma norepinephrine levels can identify hypernoradrenergic hypertension, in which there is an augmented contribution of SNS outflows to blood pressure [45, 58, 91].

Yohimbine and ganglion blockade have also been used as neuropharmacologic probes to distinguish among neurogenic orthostatic hypotension syndromes [112, 113]. In patients with generalized sympathetic noradrenergic denervation, yohimbine produces less than expected increments and ganglion blockade less than expected decrements in blood pressure. Although responses of plasma norepinephrine levels to yohimbine and ganglion blockade do not distinguish among patient groups with vs. without sympathetic noradrenergic denervation, pressure responses are related to plasma norepinephrine at baseline, consistent with dependence of those pressure responses on releasable norepinephrine in sympathetic nerves.

Pseudopheochromocytoma patients have been reported to have markedly augmented plasma epinephrine responses to glucagon injection and exaggerated blood pressure responses to pharmacologic manipulations of sympathoneural release of norepinephrine [114]. Beta blockers are contraindicated in pheochromocytoma, because of unopposed alpha-adrenoceptor stimulation by circulating norepinephrine. Whether beta-blockers are harmful in pseudopheochromocytoma is unknown. Treatment with a combined alpha- and beta-blocker such as labetalol would seem prudent.

Conclusions

Measurement of plasma levels of catechols is diagnostic in some rare diseases (e.g., DBH deficiency, LAAAD deficiency, Menkes disease) but often can aid the diagnosis and differential diagnosis of more common autonomic disorders. An attenuated orthostatic response of plasma norepinephrine levels helps establish that orthostatic hypotension is neurogenic. The occurrence of sympathoadrenal imbalance is sufficiently characteristic of autonomically mediated syncope to include serial blood sampling via an indwelling vascular catheter for plasma catechols as part of provocative tilt table testing [38]. By “provocative” we are not referring to administering a drug to provoke a positive test result (neurally mediated hypotension or a fainting reaction). Instead of 3–5 minutes of upright tilting to detect neurogenic orthostatic hypotension, the patient undergoes upright tilting for up to about 40 minutes to see if prolonged tilting yields a positive test result.

Low plasma DHPG/norepinephrine ratios characterize conditions associated with decreased NET activity. The pattern of plasma catechols distinguishes PAF from AAG. Extremely low plasma levels of catechols occur in AAD.

Clinical assays of plasma catechols for diagnostic purposes are available without charge in selected patients as a public service by the CLIA-certified clinical neurochemistry laboratory of the Clinical Neurocardiology Section in intramural NINDS.

Figure 3: Sympathoneural sources of plasma levels of catechols.

Abbreviations: ALDH = aldehyde dehydrogenase; AR = aldehyde/aldose reductase; Cys-DA = 5-S-cysteinyldopamine; Cys-DOPA = 5-S-cysteinylDOPA; DA = dopamine; DBH = dopamine-beta-hydroxylase; DHPG = dihydroxyphenylglycol; DOPAC = dihydroxyphenylacetic acid; DOPAL = dihydroxyphenylacetaldehyde; DOPEGAL = dihydroxyphenylglycolaldehyde; LAAAD = L-aromatic-amino-acid decarboxylase; MAO = monoamine oxidase; NE = norepinephrine; NET = cell membrane norepinephrine transporter; TH = tyrosine hydroxylase; VMAT = vesicular monoamine transporter.

Figure 4: Irwin J. (“Irv”) Kopin and Julius (“Julie”) Axelrod.

The photo on the left was taken after Axelrod’s Nobel Prize was announced in 1970 (the third person is Dr. Fred Goodwin). The photo on the right shows Julie and Irv many years later in 2003.

TABLE 1:

Plasma catechol patterns in various diseases.

Diagnostic	Note	Reference
DBH deficiency	High DA, DOPAC	[8]
	Low NE, DHPG
HSAN III (FD)	High DOPA/DHPG	[5]
Menkes disease	High DOPA/DHPG	[56]
	High DOPAC/DHPG
	High DA/NE
LAAAD deficiency	High DOPA/DA	[120, 121]
Supportive
Diabetic autonomic neuropathy	Low DHPG/NE	[22]
POTS	High NE upright	[37]
	High NE, EPI during supine rest	[42]
PD+OH	Low DHPG/NE	[53]
DLB	Low NE, blunted orthostatic change	[104]
PAF	Low NE, DHPG	[53, 133]
Takotusubo cardiopathy	High EPI	[130]
Pseudopheo	High EPI after glucagon	[114]
Familial amyloid polyneuropathy	Low NE, blunted orthostatic change	[119]
HSAN IV	Low NE	[103]
NET deficiency	High NE/DHPG	[111]
Autonomically mediated syncope	High EPI/NE before syncope	[51]
Pheo	High NE	[27]
	High plasma metanephrines	[89]
	Failure of NE suppression clonidine	[13, 69]
Not helpful
MSA	Normal NE, DHPG	[53, 133]

Abbreviations: DA = dopamine; EPI = epinephrine; NE = norepinephrine

TABLE 2:

Sample collection and handling for plasma catechol assays

1. INTERFERENCES (“Garbage in, garbage out”): Several medications are catechols and may interfere with results of the catechols assay. These include alpha-methyldopa, alpha-methyl-para-tyrosine, isoproterenol, dobutamine, levodopa, and carbidopa. In addition, dihydroxyhydrocinnamic acid, a catechol metabolite of caffeic acid, can occur at high plasma concentrations in people who have recently ingested caffeinated or even decaffeinated coffee. Medications: Many medications affect the synthesis, storage, release, uptake, or metabolism of catechols. For these reasons all medications the patient is taking and the time of last dosing should be listed on an accompanying patient information sheet. It is best if the sample be obtained in the morning after an overnight fast (water and non-caffeinated soft drinks are permissible). Diet: A recent meal can have a major influence on some catechols. Therefore, the influence of diet should be controlled for wherever possible. Posture and stress: Catecholamines are sensitive to posture and any kind of stress. Therefore, samples should be collected after the subject has been at supine rest (head on pillow) for at least 15 minutes and collected using an indwelling intravenous (IV) catheter inserted at least 15 minutes before sampling. If the room is hot or cold this should be noted. For orthostatic changes in plasma levels of catechols the upright sample should be obtained after the patient has been upright for 5 minutes. If hypotension or orthostatic symptoms require a shorter period of upright posture, this should be noted on the patient information sheet.

2. BLOOD COLLECTION: The patient should be in the supine position for at least 15 minutes before and during collection of the blood sample. The sample may be drawn through an indwelling butterfly IV or an IV cannula (normal saline to keep the line patent) via a 3-way stopcock and then transferred to a collection tube containing heparin as an anticoagulant (e.g., a green-top Vacutainer tube). It is preferable but not essential to draw the sample without a tourniquet. Sample volume: At least 1.2 mL of plasma is preferred, ideally provided in duplicate to allow for repeat assay if required. Anticoagulants: Blood samples should be collected into heparinized tubes (sodium or lithium heparin). Note: Although some investigators use EDTA as an anticoagulant and glutathione as an antioxidant, this is unnecessary as long as all other storage procedures below are followed.

3. SAMPLE PROCESSING: The blood sample (at least 5 mL) should be stored on ice until centrifuged (preferably at 4 degrees centigrade) to separate the plasma within 2 hours of blood collection. The plasma should be transferred to plastic tubes clearly marked with the date and patient ID using a permanent marker and frozen immediately (e.g., on dry ice). The sample should be stored at −70 degrees centigrade or colder. Catechols in plasma stored at −20 degrees centigrade are not stable. Plasma should be transferred into small polypropylene tubes, because glass or polystyrene tubes tend to crack at low temperatures or during subsequent centrifugations. For diagnostic assays the sample tubes must be labeled with three forms of personal identifying information (e.g., last and first names, date of birth, and medical chart number). All sample tubes with patient identification labels should be stored in a locked freezer until shipped to the assay laboratory.

4. SPECIMEN SHIPPING: It is mandatory to notify personnel to receive the shipped sample by phone, fax, or e-mail about the shipment and agree in advance on who will receive the shipment when it arrives. Plasma samples should be shipped frozen (e.g., on dry ice in a styrofoam container). Samples should be placed in a sealed styrofoam box with sufficient dry ice (preferably block ice with crushed ice to fill the gaps) to last at least 2 days. Samples should be labeled as HUMAN PLASMA: NON-INFECTIOUS. Before the samples are sent, confirmation must be received by phone, fax, or e-mail that appropriate personnel will be available to receive the shipment. All samples should be shipped accompanied by a patient information sheet.

Abbreviations:

AAD

autoimmunity-associated autonomic failure with sympathetic denervation

AAG

autoimmune autonomic ganglionopathy

ALDH

aldehyde dehydrogenase

ANS

autonomic nervous system

AR

aldehyde/aldose reductase

COI

chronic orthostatic intolerance

COMT

catechol-O-methyltransferase

Cys-Dopamine

5-S-cysteinyldopamine

Cys-DOPA

5-S-cysteinylDOPA

DAN

diabetic autonomic neuropathy

DBH

dopamine-beta-hydroxylase

DHPG

3,4-dihydroxyphenylglycol

DHPR

dihydropteridine reductase

DLB

dementia with Lewy bodies

DOPAC

3,4-dihydroxyphenylacetic acid

DOPAL

3,4-dihydroxyphenylacetaldehyde

DOPEGAL

3,4-dihydroxyphenylglycolaldehyde

FD

familial dysautonomia

HSAN

hereditary sensory and autonomic neuropathy

LAAAD

L-aromatic-amino-acid decarboxylase

MAO

monoamine oxidase

MSA

multiple system atrophy

MSA-P

parkinsonian form of multiple system atrophy

NET

cell membrane norepinephrine transporter

OI

orthostatic intolerance

PAF

pure autonomic failure

PD+OH

Parkinson disease with orthostatic hypotension

POTS

postural tachycardia syndrome

SAI

sympathoadrenal imbalance

SAS

sympathetic adrenergic system

SNS

sympathetic noradrenergic system

TH

tyrosine hydroxylase

VMAT

vesicular monoamine transporter