Normetanephrine, catecholamine

Fio. 51. Dependence of retention of catecholamines on volume percent acetonitrile in hetaeric chromatography. The ehient is water-acetonitrile at the volume percent indicated containing 0.2% (v/v) sulfuric acid and 0.1% (w/v) sodium dodecyl sulfote. The catecholamines separated are noradrenaline (NA), adrenaline (A). L-3,4-dihydroxyphenylalanine (LD), normetanephrine (NMA), dopamine (DA), metadrenaline (MA), and 3-methoxytyramine (MDA). Column 5- tm octadecyl silica treated with triroethylchlorosilane, 125 X 5 mm i.d. Reprinted with permission from Knox and Jurand (223). ... 

Pheochromocytoma is a tumor of the adrenal medulla or sympathetic ganglion cells. The tumor secretes catecholamines, especially norepinephrine and epinephrine. The patient in the case study at the beginning of the chapter had a left adrenal pheochromocytoma that was identified by imaging. In addition, she had elevated plasma and urinary norepinephrine, epinephrine, and their metabolites, normetanephrine and metanephrine. 

Oxidation of fi-(4-Hydroxy-3-methoxyphenyl)ethylamines The j3-(4-hydroxy-3-methoxyphenyl)ethylamines [i.e. the catecholamine 03-methyl ethers, metanephrine (18) and normetanephrine (19)] are known to be among the major in vivo metabolites of... 

A sensitive spectrophotometric method based on the strong absorption of the aminochrome-sodium bisulfite addition products (see Section IV, F) at ca. 350 m/x. has been described recently by van Espen128and Oesterling and Tse 277-278 for determining total catecholamines. While not as sensitive as the fluorimetric procedures, this method is considerably more sensitive than the older colorimetric methods based on the visible absorption peak of the aminochromes. Also, it does not have many of the disadvantages (e.g. costly equipment and unstable blanks) often associated with fluorimetric techniques. The basic procedure can be satisfactorily applied to the differential determination of mixtures of adrenaline, noradrenaline, dopamine, metanephrine, and normetanephrine.178... 

Degradation of catecholamines The catecholamines are inacti vated by oxidative deamination catalyzed by monoamine oxidase (MAO), and by O-methylation carried out by catechol-O-methyl-transferase (COMT, Figure 21.15). The two reactions can occur in either order. The aldehyde products of the MAO reaction are axi dized to the corresponding acids. The metabolic products of these reactions are excreted in the urine as vanillylmandelic acid, metanephrine, and normetanephrine. 

The most well known of the naturally occurring phenethylamine derivatives (Table I) are the transmitters of the sympathetic nervous system, epinephrine, norepinephrine, and dopamine. All these compounds are 3,4-dioxygenated in the aromatic nucleus and are collectively known as the catecholamines. Norepinephrine is the transmitter of most sympathetic postganglionic fibers, dopamine is the predominant transmitter of the mammalian extrapyramidal system and of several mesocortical and mesolimbic neuronal pathways, and epinephrine is the major hormone of the adrenal medulla (363). The literature that has accumulated on the action of these compounds in higher animals is enormous. Metanephrine and normetanephrine are known from animals as deactivated metabolites of epinephrine and norepinephrine that result from the action of the enzyme catechol O-methyltransferase (364). 

The effect of released norepinephrine wanes quickly, because -90% is transported back into the axoplasm by a specific transport mechanism (norepinephrine transporter, NAT) and then into storage vesicles by the vesicular transporter (neuronal reuptake). The NAT can be inhibited by tricyclic antidepressants and cocaine. Moreover, norepinephrine is taken up by transporters into the effector cells (extraneuronal monoamine transporter, EMT). Part of the norepinephrine undergoing reuptake is enzymatically inactivated to normetanephrine via catecholamine O-methyltransferase (COMT, present in the cytoplasm of postjunctional cells) and to dihydroxymandelic acid via monoamine oxidase (MAO, present in mitochondria of nerve cells and postjunctional cells). 

Catecholamines can be variously oxidized or methylated. Extracellular epinephrine is O-methylated [via liver catechol-O-methyltransferase (COMT)] to 3-methoxyepinephrine (metanephrine) which can thence be oxidized [via monoamine oxidase (MAO)] to 3-methoxy-4-hydroxy-mandelic aldehyde and thence to 3-methoxy-4-hydroxyphenylglycol (MHPG) and 3-methoxy-4-hydroxy-mandelic. acid (VMA). Similarly, extracellular norepinephrine is O-methylated [via liver COMT] to 3-methoxynorepinephrine (normetanephrine) which can be oxidized [via MAO] to 3-methoxy-4-hydroxy-mandelic... 

Four catecholamines have been found in increased amounts in the urine after injury (W2), metanephrine, normetanephrine, iV-methyl-metanephrine, and 3-methoxytyramine. The JV-methylmetanephrine probably indicates an increased production of epinephrine (Cll). The increase in norepinephrine production is relatively greater than that of epinephrine, suggesting an active release of hormone from sympathetic nerve tissue as well as from the adrenal medulla. 

Catechol-0-methyltransferase (COMT) is responsible for the second major pathway of catecholamine metabolism, catalyzing 0-methylation of dopamine to methoxytyramine, norepinephrine to normetanephrine, and epinephrine to metanephrine. COMT is not present in monoamine-producing neurons, which contain exclusively MAO, but is present along with MAO in most extraneuronal tissue. The membrane-bound isoform of COMT, which has high affinity for catecholamines, is especially abundant in adrenal chromaffin cells. As a result of the preceding and other differences in the expression of metabolizing enzymes, catecholamines produced at neuronal and adrenal medullary locations follow different neuronal and extraneuronal pathways of metabolism (Figure 29-5). 

Procedure The test is best performed in the morning after an overnight fast. The patient remains recumbent throughout the entire procedure. A forearm venous cannula is placed for baseline and 3-hour blood sampling during the procedure. After at least 20 minutes of supine rest, a baseline blood sample is drawn in a heparinized tube. Clonidine, 4.3 pg/kg of body weight, is then given orally, and a repeat blood sample is drawn 3 hours later. The samples are analyzed for plasma catecholamines, with plasma normetanephrine measurement also recommended. 

Additional markers of catecholamine overproduction have been employed to improve the biochemical detection of neuroblastomas. Free dopamine may be abnormal in urine from neuroblastoma patients with VMA and HVA excretion. Combined testing for VMA, HVA, and dopamine may therefore improve tumor detection, and in 1993 an international consensus report on neuroblastoma diagnosis added dopamine to the Hst of acceptable measurements to document the adrenergic nature of the tumor. Plasma measurements of dopamine and L-dopa, the amino acid precursor of dopamine, may also have clinical value and allow the alternate use of plasma. Measurement of methylated metabolites, especially normetanephrine, has also been explored. When urinary normetanephrine, metanephrine, methoxytyra-mine, dopamine, norepinephrine, VMA, and HVA were measured, clinical sensitivity for detection of neuroblastomas was 97% to 100% when results of normetanephrine testing were coupled either with VMA in the infants or with HVA in children greater than age 1. Even with an extended panel of catecholamines and metabolite measurements, a low incidence of nonsecreting tumors continues to be identified and should be considered in the interpretation of a negative test result. 

Isolated deficiencies of MAO A and B are extremely rare and are associated with distinct clinical and neurochemical phenotypes. Deficiency of MAO A is associated with a behavioral disorder characterized by increased aggressiveness. Plasma and urinary levels of deaminated metabolites of catecholamines are severely decreased, whereas levels of normetanephrine and metanephrine are increased. An increased ratio of plasma normetanephrine to DHPG has therefore been proposed to provide a sensitive marker for the deficiency state. In contrast, deficiency of MAO B is associated with a mild phenotype, the only biochemical alteration is increased urinary excretion of phenylethyiamine. 

In contrast to the catecholamines, measurements of urinary metanephrines and VMA are still based in some routine laboratories on the early spectrophotometric assays developed by Pisano, Crout, and others in the late 1950s and early 1960s. Despite subsequent development of a variety of preanalytical cleanup and extraction procedures, these assays remain susceptible to analytical interference. They are also restricted to measurements in urine. Another limitation for spectrophotometric or fiuorometric assays of urinary metanephrines is that these methods do not allow separate (fractionated) measurements of normetanephrine and metanephrine. 

Interpretation of a biochemical test result as normal or abnormal depends on availability of valid reference intervals (see Chapter 16). For tests of a single analyte, such as VMA, it can be expected that at least 2.5% of patients without pheochromocytomas will have values for the analyte above the upper reference limit and 2.5% below the lower reference limit. Up to a 5% incidence of false-positive results might be expected for tests of pairs of analytes, such as norepinephrine and epinephrine in tests of urinary or plasma catecholamines or normetanephrine and metanephrine in tests of plasma free or urinary fractionated metanephrines. False-positive rates usually, however, tend to be higher than expected this is likely due to reduced control over sampling conditions and sources of interference or differences in clinical characteristics of reference and patient populations. 

Reference intervals for plasma and urinary catecholamines and catecholamine metabolites also differ according to sex and age. Females have lower plasma concentrations of epinephrine and metanephrine than males. Similarly, 24-hour urinary outputs of catecholamines and metanephrines are lower in women than men for epinephrine this difference remains significant when values are normalized for creatinine excretion Plasma levels of norepinephrine and normetanephrine increase with advancing age in adults, whereas plasma levels of epinephrine and metanephrine are little affected. Age-related increases in 24-hour urinary outputs of norepinephrine and normetanephrine have also been reported,but not consistently by all studies. In general, the influences of age... 

Normetanephrine andmetanephrine are metabolic products of norepinephrine and epinephrine, respectively, and are formed by the action of catechol-0-methyltransferase without deamination. As a result of active neuronal reuptake and deamination of norepinephrine, normetanephrine normally represents <5% of the total norepinephrine excretion products in urine. Metanephrine, however, even with its lower urinary concentration relative to normetanephrine, represents a major excretion product of epinephrine. The metanephrines are excreted in both conjugated and unconjugated forms. Unlike the catecholamines, total metanephrine excretion is not significantly influenced by diet. As a result, the total metanephrines are routinely measured after acid hydrolysis or sulfatase pretreatment. 

Coward and Dunlop (C17) found four catecholamines to be excreted in increased quantities after surgery, viz., metanephrine, normetanephrine, iV-methylmetanephrine, and 3-methoxytyramine. The A-methylmeta-nephrine probably reflects increased adrenaline formation, and the 3-me-thoxytyramine levels probably represent the increased homovanillic acid secretion after stress. Of the metanephrine and normetanephrine, the lat-... 

Fig. 4.4.12. Separation of a mixture of acidic and basic catecholamines on a LiChrosorb RP-8 column (0.28x25 cm). Eluent Water containing 0.02 M citrate (pH 2.5)/l% propanol/NaCIQ4 (0.08 M)/0.3% sodium dodecyl sulphate. Peaks DHMA, 3,4-dihydroxymandelic acid VMA, vanilmandelic acid HGA, 2,5-dihydroxyphenylacetic acid DOPAC, 3,4-dihydroxyphenylacetic acid 5-HIAA, 5-hydroxyindole-3-acetic acid HVA, homovanillylmandelic acid E, epinephrine NE, norepinephrine N-Syn, norsyneph-rine Syn, synephrine Dopa, 3,4-dihydroxyphenylalanine NM, normetanephrine MN, metanephrine Isopren, isoprenaline 3-H-Tyrm, dopamine Tyrm, tyramine 3-M-Tyrm, 3-methoxytyramine. Reprinted from Ref. 21 with permission.

From tyrosine, a series of enzymatic reactions including ring hydroxyla-tion, deciurboxylation, deamination, and D-methylation are responsible for over 20 majof metabolites of neurochemical interest. All of the above metabolites retain at least an electroactive phent nudeus, and most provide enhanced detectability via vanillyl or catechol functional groups. Table III provides cyclic voltammctric data in 90% 0.1 M citrate (pH 4)/10% methanol for a series of these metabolites. Substituent effects are evident. Gener-aUy the catecholamines with their OKlihydroxyphenyl structures are easiest to oxidize, followed by vanillyl dmvatives such as normetanephrine and vanillylmandelic acid. Simple phenols such as tyrosine remain the most difficult to oxidize in this series. 

Dopamine was produced by incubating liver microsomes of rabbits with p- and m-tyramine, noradrenaline, and normetanephrine by incubation with w-octopamine, and adrenaline by incubation with p- and w-methyloctop-amine/" The injection of labelled tyramine as well as of labelled octopamine in the intact animal (rat) caused the appearance of labelled noradrenaline and normetanephrine in the urine/ As tyramine is also hydroxylated to octopamine, it is possible that the production of noradrenaline from tyramine not only takes place via its conversion to dopamine, but also via its conversion to octopamine. The possibility that octopamine is produced by the hydroxylation of tyrosine to hydroxyphenylserine with ensuing decarboxylation is under discussion. The production of noradrenaline from 3,4-dihydroxyphenylserine, 192-201) amino-acid so far not discovered in the mammal could be demonstrated both in organ extracts and in intact animals. Finally, a transamination of 3-hydroxy- or 3,4-dihydroxyphenylpyruvate to the corresponding amino-acids (m-tyrosin, dopa), and their decarboxylation to m-tyramine and dopamine was observed in intact animals (cats). For the time being it is impossible to determine the importance of the means of formation of catecholamines which have been referred to here. Of the above-mentioned precursor substances, p- and m-tyramine,( octopamine, ... 

Indirect effects of drugs on catecholamines have ako resulted in takotsubo syndrome. In one case there was transient typical ballooning of the left ventricular apex during systole following the use of cocaine, thought to have been due to inhibition of catecholamine reuptake [22ft]. A 43-year-old woman who took an overdose of venlafaxine, an inhibitor of serotonin and noradrenaline reuptake, developed a takotsubo cardiomyopathy and there was an increase in urinary normetadrenaline (normetanephrine) concentration [23 ]. 

The mechanism by which the guaiacolamines are oxidised to the amino-chromes is of interest. Whilst the possibility of a demethylation reaction to give the corresponding catecholamine directly prior to its oxidation cannot be entirely ruled out, it is probably not always the case. The mechanism proposed for the periodate oxidation of guaiacol to o-benzoquinone [136, 137] can easily be adapted to explain the oxidation of metanephrine and normetanephrine to adrenochrome and noradrenochrome respectively, as shown in Scheme 6.1. 

Metanephrine and normetanephrine are deactivated O-methyl metabolites of epinephrine and norepinephrine, produced by the action of catechol O-methyl transferase. Both metanephrine and normetanephrine are produced at low concentrations from normal metabolism of catecholamines by the sympathetic nervous system, but their concentration can be greatly elevated by catecholamine-producing tumors, such as pheochromocytomas. Pheochromocytomas are rare malignancies that often present with symptoms of catecholamine excess, most prominently h5q)ertension. When used to identify pheochromocytoma, therefore. 

Catecholamines can be taken up by certain non-neuronal tissues. This uptake involves a process which is different from those described above. Histochemical studies have shown, for example, in the rat heart, that following perfusion with media containing very high concentrations of NA (S-50 intense fluorescence due to NA is present not only in adrenergic nerves but also in the cardiac muscle and other cells. A similar extraneuronal accumulation of NA has been observed in the secretory parenchyma of salivary glands, and in vascular smooth muscle, when these tissues are exposed to high concentrations of NA. This fluorescence rapidly disappears if the tissues are exposed to amine-free media. The uptake and the wash-out are inhibited by normetanephrine and phenoxybenzamine. 

FIGURE 18 Separation of catecholamines and interfering compounds. Ultrasphere C-18 column, 25 cm x 4.6 mm, mobile phase 10% methanol, 90% 0.1 M potassium phosphate, pH 3.0, 0.2 rr M sodium octylsulfonate, at 0.72 V vs Ag/AgCI reference electrode, sample size 20 /xl. Peaks (1) Ascorbic acid, (2) Dihydroxyphenylglycol, (3) Norepinephrine (4) Epinephrine, (5) Hydroxymethoxyphenylglycol, (6) Dihydroxybenzylamine, (7) Normetanephrine, (8) Dopamine, and (9) Dihydroxybenzylamine. [Reprinted from permission from Beckman/Altex Scientific.]... 

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