Serum dopamine β-hydroxylase as an index of sympathetic function

Pergemon Press

Life Sciences Vol. 14, pp " 1593-1604 Printed in û.S .A .

!lINIRBVI$9P SSBDM DOPA?IINE ß-HYDRO%YLASS AS AN INDES OF SYl~ATBETIC FQNCTION Laurence Gaffen School of l~iedicina, Flieders Dniversity of South Australia, S .A . (Received is final form 11 Jaauasy 1974)

5Wî2 .

.

Quantitative nathods of aseeseing sympathetic function in sins would be of considerable value in a vide variety of physiological and pathological states .

Yet, despite the comiderable progress in our present uaderatanding

of adrenergic neurones at the cellular level, these advances have not been readily tramlated to studies of the intact sympathetic nervous systes . Bzteasive clinical use has been wade of functional cardiovascular parsseters and there is as eaeasive literature on urinary and plasma catecholaoinas and their aetabolites but these physiological and biochemical indices are subject to numerous aethodological and interpretative difficulties that are yell recognised . In the period 1967 to 1969, studies on isolated organ demonstrated that specific proteins were released together with catecholaninee by stisulation of the adreml sedans and sympathetic neurones (1, 2, 3) . These proteins were called chromogranins because they were identified se soluble comtituents of the granulated vesicular organelles involved in catecholamine synthesis, storage and secretion.

Oae of the

protein

released van the ensyme dopamiae . ß-hydrozylasa (DBH) (BC 1 .14 .17 .1) that catalyses the conversion of dopaii.ae to noradreaaline is the catacholaniee storage vesicles of peripheral and central adreeergic neurones and adrenal medullary chromaffin cells.

In 1971, DBH emyuatic activity van detected

is human serum (4, 5) and the partially purified eerun ea :yme van found to have the ema co-factor requirements, kinetics and electrophoretic properties 1593

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Serum D8H and Sympathetic Function

ae the tissue enzyme (6) .

Vol. 14, No . 9

This rained the posgibility that circulating DBH

levels might constitute a more useful rode: of sympathetic function in the intact organism than assay of the sympathetic transmitter itself . Properties of DBH DBH is a copper-containing sized function ozidaee that catalyses hydroxylation of the ß carbon in the side chain of phenglethylamine derivatives (7, 8) .

The enzyme has bees tentatively characterized as a

tetrameric glycoprotein with 1 or 2 active sites, each containing one copper molecule that undergoes cyclic oxidation and reduction (9) . molecular weight of close to 290,000.

It has a

IInder dissociating conditions, four

eubunits of molecular weight 75,000 have bees reported (10) but it ie uncertain whether the units are identical or catalytically active .

Enaymatic

activity may depend upon the co-operative action of two or even all four aubunits, and upon sugar residues that comprise 4x of the molecule (9) .

Amino-

acid analysis has revealed that DBH ie a highly acidic protein, rich in glutamate and aepartate, and deficient in methionine and half-cyatine (11, 12) . The ensyae ezhibits a broad pH optimum in acid media down to pH 5 .5 and changes in pH between 4 - 5 and 8 - 11 inactivate DBH without significantly altering its molecular weight (12) . The enzyme requires solecular orygen and a reducing agent as cofactors.

Aacorbate is generally used to reduce the enzyme in vitro but can

be replaced by dimethyltetrahydropterine (13) .

Pterines may therefore play a

similar regulatory role to that proposed in the first rate-limiting step is catecholamine synthesis catalysed by tyrosine hydrozylase .

Unlike tyrosine

hydrorylase, however, DBH ie not inhibited by catecholamines, although it ie sensitive to a wide range of chelatiag agents and to an excess of copper ions (14) .

Non-specific inhibitors containing

sulphydryl groups have made

the activity of the enzyme difficult to assay is tissue homogenates without

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Serum DBS and Sympathetic Function

1595

partial purification, but these endogenous inhibitors can be neutralized with N-athylmaleimide, p-hydrozy mercuribenzoate or controlled addition of copper . IIp to 50Z of the DBH enzymic activity in chromaffia vesicles can be readfly aolubiliaed by osmotic lysis whereas only lOx of the eazyma in nerve vesicles is soluble under these conditions (15) .

Host neuronal DBH appears

to be firmly membrane bound is vesicles with electron-dense cores of both the large and small granular variety (16,

17) .

It is possible that differing

degrees of incorporation of the enzyme into vesicle mesbraaes reflects different stages in the maturation of the vesicles (17) .

In three

independent studies, no differences were found in the molecular size, amiaoacid composition, co-factors, tinetice or immimological identity of the soluble and bound forms of the enzyme, thereby excluding the ezisteace of ieoenzymae

(11,

18, 19) . Release of DBfl

In the adrenal medulla, there is strong biochemical sad morphological evidence that chromaffia vesicles discharge their soluble contents by a process of azocytosis that involves temporary fusion of vesicle and cell membrane (1, 15) .

The evidence is similar but less compelling for

sympathetic nerves (2, 3,

15, 17) .

In the adrenal medulla, catecholamiaes

and macromolecules such as DBH era released is the same proportions that exist in the intact vesicle, whereas in sympathetically iaaervated tissues, the proportions of vesicle constituents preeant both in nerve terminsle and released from them have yet to be quantitatively established (15,

17) .

Catecholaminea and macromolecules released from the adrenal medulla appear to be cleared equally well into the circulation whereas in sympathetically imfnrvated tiasues,noradreasline but not DBH is rapidly inactivated by pre- sad post-juactional uptake processes (20) .

While DBH

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does not appear to be specifically takes up after release, passage into the circulation of as large a molecule as DHH say vell be retarded by capillary barriers aad an undetermined proportion of the easyme released from nerves nay enter the circulation through lymphatic channels .

Aaother difficulty in

establishing the proportions of vesicle coastitueats released from sympathetic nerves arises from the fact that both large aad wall graaular vesicles contain DBH aad noradreasline .

If the too vesicle typee represent different

stages of maturity, the large vesicles may undergo a transition to the smaller type vesicles due to loss of vesicle proteins including DBH during repeated participation of the vesicle in ezocytosie (17) .

An alternative interpret-

ation that is also consistent with the evüence that DBH is released by eucytosis is that the two vesicle types have different functions, the large vesicles releasing proteins and the smaller transmitter (2, 15) . Sources and Turnover of Serin DBH in Animale The relative contributions of the adrenal medulla and of sympathetic nerves to circulating dopamine ß-hydrorylase have only been e:ained in rate . These results need to be interpreted cautiously since the DBH activity of rat sera is close to the limits of sensitivity of the assay.

Sympathetic

stresses such as forced mobilization produced moderate increases in serum DBH that were abolished by chemical sympathectomy with 6-hydroaydopsmine (21) .

6-Hydrozydopamine alone, in doses that did not affect the adrenal medulla, caused a 25S reduction in servo DBH, vhareas adreaslectomy caused no change in basal levels and the response to stress persisted (22) .

While no allowance

vas made in these studies for compensatory changes in sympathetic nerve and adrenal activity they do suggest that sympathetic nerves make the major quantitative contribution to serum dopamine ß-hydrozylase levels as they do to circulating catecholamines .

Increased sersmt DBH activity has also been

reported in svim stressed rate (23) .

Turnover of serum DBH has been estimated

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Serum D8H and Sympathetic Function

125 -labelled ovine DBH into lambs . by steady state intravenous infusion of 1 Labelled easyme vas cleared from the circulation with a half life of 2.8 hours sad the turnover vas calculated as 10 Ug/kg body weight/hour (20) .

A similar

half life has beor calculated for endogenous DBH in swim stressed rate (23) . Aseaye of Hum an Serves DBH Eüsynrttio assays Whor high levels of activity are presort, such as in the adrenal medulla, DBH activity can be measured radiametrically using vaiiforcly labelled tritiated tyraaine followed by solvent earactian and counting of labelled octopsmine (7) .

!lore sensitive radiometric and photonetric assays capable

of measuring DBH activity in plasma have bem described recently that use unlabelled tyramiae as substrate.

In the radiometric assay, octopaninn

formed from tyrsmine is incubated with a second ensyce, phenylethylamiae N-methyl transferase, and the methyl donor S-adenosylmethioninraethy1 14 C to foes syaephrine- 14C that is ehor separated by solvmt Baraction and counted (4, 5) .

For routine clinical purposes, phorylethylamian has bem found to

havB sate advantages over tyramine ae a substrata in this assay (24) .

While

the double mayors radiometric assay is uadoubtadly sensitive and accurate in e~erienced heads, careful control of assay conditions is necessary, particularly since substrate conemtrations are not optical for both easynatic steps . In the spectrophotametric assay, vmlabellBd tyramine is comrerted to ottopaniae, the octopamiae forced is then ozidised with periodate to parahydrorybmsaldehyds,,aad its absorbante at 330 nn measured (25) .

This

assay ie stapler than the radioeetric assay and is sufficiently sensitive and reproducible to usay dopamine ß-hydro:ylase activity is 10 yl of human serum! with a coefficient of variation of less than 10Z .

In aortal populations

aarvm DBH activity varies fraa undetectablB levels in 2 - 5x of the population over a range of several hundred units (defined in a number of different ways

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Serum DBH and Sympathetic Function

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depending on the assay employed) . Radiai~mrunoaaaaya Not all the DBH released into the circulation is necessarily still active enzyme .

DBH has no known function in blood and its secretion may

represent one mechanism of ridding nerve terni.nals of vesicular protein, for azoas

lack well developed protein degrading ae well as synthesizing

aystema.

Ia 1972, as imeunoassay was described that was independent of the

enzyaes's catalyctic activity (17) .

The assay was based upon competition

between 1125 -labelled DBH and unlabelled enzyme for a limited masher of antibody binding sites attached to a solid phase support . and homologous immunoassays

Both heterologous

using ovine, bovine and human enzyme have now

been developed using this principle (17,26,27) .

In a study of 34 normal

subjects using the heterologous bovine assay, sera: values had a unimodal distribution and varied over a 3-fold range from 12 to 23 Ug protein/ml (corrected for an attenuation of cross reactivity of approzinately 172-fold) (26) .

No correlation was found between enzymatic and imauaological activity,

the proportion of active to imotmoreactive enzyme varying between 0.3x and 30 .7x in different individuals (26) .

Similar serum DBH protein concentr-

ations have been reported with the homologous immmoaseay (27), and a significant correlation was found between the enzymatic and immmoassays of individual sera at teat (27) but not with ezercise (28) .

In both studies,

a considerable proportion of circulating DBH was found to be enzyuatically inactive .

This raises the question of to what eztent the enzyme is

inactivated before, during and after release and to what degree subunits of the eazyae remain imm~moreactive . Suoaa Serum DBH Açtivity in Normal and Diaaase States Ags Serus DBH activity increases markedly in children for the first two or three years of life (24,29) .

In one study it was reported to continue to

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Serum DHH and Sympathetic Function

1599

rise throughout adulthood (29), but the larger and amore controlled study raoealed very little change after aiz years of age (24) . Ia the largest study yet made of a normal population, the frequency distribution of DBH activity in 180 unrelated children aged 6 to 12 years shoved a pronounced skew to the right with approximately 5x having negligible activity .

A similar distribution was noted in a eeaple of 227 unrelated

adults (30) .

A significant correlation has been found between the servo DBH

activity of siblings (31) and a very high correlation coefficient in mono sygotic twins (32) .

This suggests that inheritable factors are important

determinants of serum dopamine ß-hydrozylase activity affecting either the total amount of DBH released or the proportion that reoaine active or the rate of clearance of the ensyna from the blood. Ses No differences have.heeâ .natabüahed.betveen the sewn is any age group (24, 29, 30) . Pkys{oZogiaal Stress Tha study of serua DBH activity changes is acute and chronic ezperimeatal stress situations is small groups of human subjects has been hindered by the wide range of normal serum DBH activity values encountered . Nevertheless a ausber of mall group studies have been reported on seam DBH activity changes in response to acute physiological etraseee .

Transient

increases in mean DBH activity were found is response to a cold preaeor test and during ezercise on a bicycle .

These changes were small compared to the

haeodyaamic responses sad ao change is DBH activity could be detected in response to tilting (33) .

Ia a second study the mean earvm DBH activity was

higher at 10 ° than at 40° at rent whereas during ezarcise a spell increase was found at 40° but not at 10 ° (34) .

In a third study,

and inconsistent increases in aervm DBH activity (35) .

ezerciee caused sash In the largest study

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Serum DBH and Sympathetic Function

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on 34 normal subjects a highly significant wean increase in serum DBH activity vas found with ezercise (36) .

In one study, serum DBH vas measured both by

as enzymatic and a radio{+~+++~. aeay in ai.z normal adults whose heads and feet were 19oarsed briefly in iced water.

Imimoreactive earum DBH rose in all

subjects during and after the stress by an average of SOZ but the changes in serer DHH

activity were inconsistent and of smaller magnitude . None of these studies were directly comparable with regard to assay

aethods, duration and severity of the sympathetic stress and the timing of eamples~and it is difficult at this early stage to draw firm conclusions about the validity of serum DBH as as rode: of sympathetic activation . Nevertheless, one criticism that can be made is that the stresses employed in the various studies may have bees too acute and the sampling too limited in duration to validly assess the usefulness of serum DBH as as integrative rode: of sympathetic function over periods of hours or eves days .

Another

criticism is that little account has been taken of variations in serum DBH activity within ae well ae between individuals .

While the variability in

individuals measures at the sane tine over a period of weeks is small and within

the coefficient of variation of the assay (< 10x), wide fluctuations

of between 20 and 100x have been observed in subjects studied over a 24 hour cycle during which they perforaed their normal routines (37) .

Ajthough there

were considerable differences in timing, the highest levels tended to occur during the afternoon and the lowest in the late evening and early morning. Similar diurnal fluctuations were observed in a group of hospital patients with diegaoaes unrelated to autonomic disorders (37) . Ser~ DBH 0hang-es in Disesee _States The pathological changes in serum DBH reported thus far are even more difficult to evaluate at the present time than the physiologically induced changes .

Serum DBH enzymatic activity has reported to be increased

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Serum DBH sad Sympathetic Pvnotion

in soae patients with nnuroblaatoma (38), Huntington's chorea (39), autoaomal dominant torsion dyetonia (40) and phaeochroswcytoma (35), and decreased in some patients with familial dysautonoai.a (24), Dorm's syndrome (32), Parünaon's disease (39), paraplegia and quadriplegia (41) .

Fer of these

studies had adequate statistical and clinical controls and at the present stage it seems unlikely that isolated determinations of seraa DBH activity rill ba useful diagnostically .

Even in the rare conditions of torsion

dystonia and familial dysautononia, the vide range of variation in the morsel population

and within individuals necessitates cautious interpretation of

abnoroally high or low values respectively .

Longitudinal studies of changes

in aerum~DBH activity in individuals, on the other head, may yell be useful in folloring the course of various diseases and their response to therapy but this retains to be investigated . The narrow range of im~moreactive DBH protein in normal populations offers some prospect that immmoaesay of DBH will be a more useful method clinically .

In the one clinical aeries reported thus far with this method,

both plasma catecholamines and DBH protein ware significantly elevated in patients with essential hypertension (42), whereas in phaeochro®ocytoma patieata catecholanine levels only were raised (43) .

In patients without

tusiours there were positive correlations between resting basal 'diastolic' BP and both catecholanine and DBH plasma levels .

The effects of acute

sympathetic ganglion bloclrade on BP, plasma noradrenalina and DBH were also studied (44) .

There were significant falls in all three paratleters within

30 miss . that persisted for at least tyro hours.

These results suggest that

increased sympathetic activity contributed to the elevated blood pressure in the patients with essential hypertension .

On the other hand, the

dissociation of catecholamine sad DBH secretion in phaeochraaocytona supports the hypotheses (45) that release of catecholaaiaes frog the tuoour occurs by diffusion from an ezcase pool of aerly synthesized catecholamiaes that bypasses

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Serum DBH and Sympathetic Function

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the normal storage aad secretion mechanism rather than by the normal process of ezocytoaie of catecholaaine storage vesicles . Conclusions The wide variation in the proportions of active and inactive DBH in the circulation appears to be primarily geaetically determined .

Nevertheless

environmental and disease processes may induce changes in either enzymatic activity or immuaoreactivity that could be used as an integrative iodez of sympathetic function, particularly in individuals studied over a period . Until the relationship between serum DBH enzymatic activity and immmoreactivity ie better understood, it would be advisable in future studies to measure serum enzyme levels by both methods aad to correlate them with other indices of sympathetic function such as plasma catecholaoinea and haeaodynasic responses .

Turnover studies are needed to evaluate the contribution of

changes in clearance as well as release to circulating levels of the enzyme . Befereacea 1.

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Serum DBH and Symp~rthetic Function

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