Xenobiotica the fate of foreign compounds in biological systems

ISSN: 0049-8254 (Print) 1366-5928 (Online) Journal homepage: http://www.tandfonline.com/loi/ixen20

Metabolism of 3, 4-Dihydroxyphenylalanine, its Metabolites and Analogues in vivo in the Rat: Urinary Excretion Pattern B. L. Goodwin, C. R. J. Ruthven, G. S. King, M. Sandler & B. G. S. Leask To cite this article: B. L. Goodwin, C. R. J. Ruthven, G. S. King, M. Sandler & B. G. S. Leask (1978) Metabolism of 3, 4-Dihydroxyphenylalanine, its Metabolites and Analogues in vivo in the Rat: Urinary Excretion Pattern, Xenobiotica, 8:10, 629-651, DOI: 10.3109/00498257809069575 To link to this article: http://dx.doi.org/10.3109/00498257809069575

Published online: 22 Sep 2008.

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Date: 17 March 2016, At: 00:24

XENOBIOTICA,

1978, VOL. 8, NO. 10, 629-651

Metabolism of 3, 4-Dihydroxyphenylalanine,its Metabolites and Analogues in vivo in the Rat: Urinary Excretion Pattern B. L. GOODWIN, C. R. J. RUTHVEN, G. S. K I N G and M. SANDLER

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Bernhard Baron Memorial Research Laboratories and Institute of Obstetrics and Gynaecology, Queen Charlotte’s Hospital for Women, Goldhawk Road, London W6 OXG, U.K.

and B. G. S. LEASK Belvidere Hospital, London Road, Glasgow G31 4PG, U.K. 1. The metabolism and interrelationships of orally and intraperitoneally administered L-dopa, related amino acids and their metabolites have been studied. 2. Amino acids were decarboxylated. N-Methyldopa formed dopamine but not epinine. D-Dopa was absorbed from the intestine and metabolized by a series of reactions which resulted in greater decarboxylation than was observed after L-dopa. Transamination was a minor pathway. 3. m-Hydroxylated phenylpyruvic acids were poorly reduced, but vanilpyruvic acid was reduced fairly readily. Lactic acids were largely unchanged. Lactic and pyruvic acids formed phenylethylamines and their metabolites. Small amounts of phenylpyruvic acids may be decarboxylated to phenylacetic acids. 4. Glycine conjugates were formed from phenylacetic acids, a partially reversible change. 3,4-Dihydroxyphenylaceticacid was metabolized to homovanillic and m-hydroxyphenylacetic acids, especially when given orally. Little 3-hydroxy-4-methoxyphenylacetic acid was oxidized to 3,4-dihydroxyphenylacetic acid but some increase in m-hydroxyphenylacetic acid excretion was observed. 5. 2-Phenylethanol analogues were largely converted to the corresponding acids. 3,4-Dihydroxyphenylethanol was partially m-0-methylated before oxidation. 6. /I-Phenylethylamine analogues were oxidized mainly to phenylacetic acids, but a variable amount of analogous phenylethanol was also formed, especially from m-tyramine. Dopamine was 0-methylated, a process not readily reversible. It was also p-dehydroxylated following oral and intraperitoneal administration but not after oral neomycin; biliary excretion of amines may be involved in this sequence of events. N-Methylated amines were oxidized less readily than the parent amine. 7. Differences in route of administration resulted in quantitative changes in degradation pathways, an effect deriving, to some extent, from p-dehydroxylation and 0-methylation in the gut.

Introduction Numerous studies of 3,4-dihydroxy-~-phenylalanine (L-dopa) metabolism in the rat are recorded in the literature (Gey & Pletscher, 1964; Sourkes et al., 1964; Kuruma, Rartholini & Pletscher, 1970; Borud, Midtvedt & Gjessing, 1972; Osumi, Wada & Fujiwara, 1972; Shindo, Komai & Kawai, 1973 a ; Shindo et al., 1973 b ; Cheng & Fung, 1975); the related compound, 3-methoxytyrosine, quantitatively a minor precursor of L-dopa, has also been investigated (Bartholini, Kuruma & Pletscher, 1970, 1971, 1972; Chalmers et al., 1971; Bartholini & Pletscher, 1972). Even though the major metabolic routes are defined, the interrelationships of the minor degradation pathways of L-dopa, its metabolites and analogues are not well understood.

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T h e present work was designed to examine the metabolic pathways of amines and amino acids related to L-dopa (with the exception of the noradrenaline pathway, which has been intensively investigated by others). Technical difficulties precluded the study of amino acids and conjugates as such, apart from some glycine conjugates. Pyruvic acids did not run well in our gas chromatographic systems, so that they could only be examined qualitatively. Urinary metabolites were detected and identified by paper chromatography, and determined quantitatively by gas chromatography (g.1.c.). These metabolites were in turn administered to rats to identify their metabolites; it thus became possible to piece together an interconnected network of metabolic pathways. A preliminary account of certain aspects of this work was presented by Goodwin et a2. (1971).

Experimental Materials Certain ' transamination series ' metabolites of aromatic acids, m-hydroxyphenylacetic acid, vanilpyruvic and isovanilpyruvic acids (abbreviations and trivial names are listed in Table 1) were prepared from their analogous benzaldehydes by the azlactone synthesis (Sealock, Speeter & Schweet, 1951) as modified by Shaw, Armstrong and McMillan (1956). m-Hydroxyphenyllactic, vanillactic and isovanillactic acids were prepared by reducing pyruvic acids with sodium amalgam in water (Matsuura & Cahnmann, 1959). Because the azlactone synthesis failed with protocatechualdehyde, 3,4-dihydroxyphenylpyruvic and 3,4-dihydroxyphenyllacticacids were synthesized from &3,4-diacetoxyphenyl-a-acetamidoacrylic acid (Harington & Randall, 1931). Isohomovanillic acid was prepared by oxidation of its analogous pyruvic acid with peroxide in alkali (Shaw, McMillan & Armstrong, 1958). 3-Hydroxy-4-methoxyphenylethanoland m- and p-tyrosol were prepared by reduction of their analogous acids with LiAlH, (Nystrom & Brown, 1947). N-Methyldopa was synthesized by condensing vanillin with creatinine (Deulofeu & Guerrero, 1942). N Methyl-m-tyramine was prepared from m-sympatol by catalytic hydrogenation with palladium catalyst in the presence of perchloric acid (Rosenmund & Karg, 1942). N,NDimethyl-m-tyramine was prepared by condensation of m-methoxyphenethyl bromide with dimethylamine and removal of the 0-methyl group (cf. Amundsen, Krantz & Sanderson, 1955). 4-0-Methyldopa was prepared by hydrolysing the analogous azlactone to an a-acetamidocinnamic acid, followed by reduction with sodium amalgam and water, and acid hydrolysis (cf. Shaw et al., 1958). Isohomovanillic acid, m- and p-hydroxyphenylacetic acids were converted to their glycine conjugates by heating with thionyl chloride and shaking a solution of the resulting acid chloride with a concentrated solution of glycine at alkaline p H (cf. Baum, 1885). m-Tyramine and ~L-3-methoxytyrosinewere kindly donated by Dr. A. Pletscher, Hoffmann-La Roche Ltd., Basle, Switzerland. Dopac, 4-hydroxy-3methoxyphenylethanol, 3,4-dihydroxyphenylethanol, m-hydroxyphenylacetic and homovanillic acids, D- and L-dopa, dopamine HCl, 3-methoxytyramine HCl, 4-0-methyldopamine HCl, DL-WZ-tyrOSine and pentafluoropropionic (PFP) anhydride of the purest grade available were obtained from commercial sources. Animal experiments Each compound under test was administered (100 mg/kg body weight) orally or intraperitoneally to four male Wistar rats (approx. 200 g); urine was collected over 6 M HC1 (0.5 ml) in metabolism cages for a period of 24 h and stored at - 20" until assay. I n the neomycin-dopamine experiments, neomycin was administered orally (100 mg /kg) on four successive days, and dopamine injected intraperitoneally (100 mg/kg) after the final dose. Determination of urinary metabolites All samples were hydrolysed overnight with 0.1 ml of a sulphatase-glucuronidase preparation (suc d'Helix pomatia, Industrie Biologique FranGaise, Clichy, France) except in the determination of amines or catechols when hydrolysis was carried out in 0.1 M HC1 at 100" for 20 min. Urine was qualitatively examined for phenolic compounds by two-dimensional paper chromatography, using Whatman No. 52 paper, pattern A. Amines were chromatographed as described previously (Sandler et al., 1971). Monophenolic acids and alcohols were extracted from acidified urine into ethyl acetate and chromatographed, using isopropanolammonia soln. (sp. gr. 0.88)-water (8 : 1 : 1) and benzene-acetic acid-water (125 : 72 : 3),

Dopa Metabolism in Rat

63 1

Table 1. Abbreviations and trivial names of compounds Abbreviation Trivial name

Chemical name

-

2-(3,4-Dihydroxyphenyl)ethanol 3-(3,4-Dihydroxyphenyl)lacticacid 3-(3,4-Dihydroxyphenyl)pyruvic acid N,N-Dimethyl-m-tyramine 2-(m-Hydroxyphenyl)-N,N-dimethylethylamine 3 -(3,4-Dihydroxyphenyl)alanine Dopa Dopac 3,4-Dihydroxyphenylaceticacid Dopamine 2-( 3,4-Dihydroxyphenyl)ethylamine 2 - (3,4- Dihydroxyphenyl) -N-me thylEpinine ethylamine 2-(4-Hydroxy-3 -methoxyphenyl)ethanol HMPE 1-(4-Hydroxy-3-methoxyphenyl)glycol HMPG Homovanillic acid 4-Hydroxy-3-methoxyphenylaceticacid HVA 4-Hydroxy-3-methoxyphenylacetylglycine HVAGly 2-(3-Hydroxy-4-rnethoxyphenyl)ethanol isoHMPE Isohomovanillic acid 3-Hydroxy-4-methoxyphenylaceticacid isoHVA 3-Hydroxy-4-methoxyphenylacetylglycine isoHVAGly 4-O-methyldopa, isovanilalanine 3-(3-Hydroxy-4-methoxyphenyl)alanine Isovanillactic acid 3-(3-Hydroxy-4-methoxyphenyl)lacticacid isoVLA Isovanilpyruvic acid 3-(3-Hydroxy-4-methoxyphenyl)pyruvic isoVPA acid 3 -(4-Hydroxy-3 -methoxyphenyl) alanine 3-Methoxytyrosine 3 -(3,4-Dihydroxyphenyl)-N-methylalanine N-Methyldopa N-Methyl-m-tyramine 2-(m-Hydroxyphenyl)-N-methylethylamine m-Hydroxyphenylacetic acid mHPAA m-Hydroxyphenylacetylglycine mHPAAGly 3-(m-Hydroxyphenyl)lactic acid mHPLA mHPPA 3-(m-Hydroxyphenyl)pyruvic acid 3MT 3-Methoxytyramine 2-(4-Hydroxy-3 -me thoxyphenyl) e thylamine 4MT 4-0-Methyldopamine 2- (3-Hydroxy-4-methoxyphenyl)ethylamine 9-Hydroxyphenylacetic acid pHPAA pHPAAGly p-Hydroxyphenylacetylglycine 2- (m-Hydroxypheny1)ethylamine m-Tyramine 3 -(m-Hydroxypheny1)alanine m-Tyrosine 2-(m-Hydroxyphenyl)ethanol m-Tyrosol 2-(p-Hydroxyphenyl)ethanol p-Tyrosol 3 -(4-Hydroxy-3 -methoxyphenyl)lactic acid VLA Vanillactic acid 3-(4-Hydroxy-3 -methoxyphenyl)pyruvic VPA Vanilpyruvic acid acid

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DHPE DHPLA DHPPA

as developing solvents. For the detection of catechol acids, the basic solvent system was replaced by n-butanol-acetic acid-water (4 : 1 : 1). Phenolic compounds were detected by spraying with a diazotized sulphanilamide reagent modified from that described by Block, Durrum and Zweig (1958). Sulphanilamide (10 mg) in 1 ml of 1 M HCl was shaken with 7 ml n-butanol to give a single phase. This soln. was then shaken with 1 ml 5 % (w/v) aq. NaNO,, the mixture allowed to separate and the chromatogram sprayed with the upper phase, followed by 10% (w/v) aq. Na,CO,%. Phenolic compounds yielded azo dyes, of a colour depending on the structure of the phenol. Pyruvic acids were detected as their analogous quinoxalinols, by a method based on that of Nielson (1962) using t.1.c. on silica gel. Five per cent of a 24 h volume of urine was

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632

B . I,. Goodwin et al.

mixed with 4 ml of 674 (w/v) aq. Na2S, previously adjusted to p H 1, followed by 2.5 ml of a 10% (w/v) aq. soln. of o-phenylenediamine in HC1 at p H 2 and finally 1.2 ml of 1 M HC1 was added. The o-phenylenediamine had previously been recryst. 3 times from chloroform. The mixture was heated for 45 min at 90", cooled, and extracted 3 times with 1 vol. ethyl acetate. The extract was washed twice with water, once with 2% (w/v) aq, NaHCO,, twice more with water and then evaporated to dryness in vacuo. Five per cent of the residue was chromatographed two-dimensionally on silica gel G plates, using as the developing solvents (a) the organic phase from benzene-ethanol-ammonia s o h . (sp. gr. 0.88) (10 : 3 : 1) mixture and (b) chloroform-acetic acid (9 : 1). Quinoxalinols were detected by spraying with 0.4% (w/v) fast red G G salt (a stabilized diazotized p-nitroaniline obtained from R. A. Lamb, Alperton, Middx.) in 1 M HCI. Arylpyruvic acids gave grey-brown spots which appeared in about 30 min and then faded over several hours. Quantitative assay procedures for acids as trimethylsilyl (TMS) ether/esters or methyl ester/TMS ethers, and for alcohols and catecholamines as pentafluoropropionyl (PFP) derivatives have already been described (Karoum et al., 1969; Karoum, Ruthven & Sandler, 1971 ; Wong, Ruthven & Sandler, 1973; Goodwin, Rtlthven & Sandler, 1974.) For monophenolic m i n e assays, the hydrolysate was adjusted to p H 5 and passed through a 1 cm diameter column of recycled Dowex 50 x 8 cation exchange resin, hydrogen form (2 g ) . The soln. was washed in with water and, after a further wash, amines were eluted with 20 ml of 1 : 1 mixture of 6 M HC1 and ethanol. The eluate was taken to dryness, residual HCl removed under high vacuum and 0.75 M borate buffer, p H 11 (2 ml) added. The buffer was extracted twice with 25 ml portions of ethyl acetate, the extract evaporated to dryness and the residue dissolved in 5 ml methanol. A 50 pl portion was derivatised after evaporating the solvent, by heating with 100 p1 PFP at 65" for 1 h. After removing excess reagent in a stream of N2, the residue was dissolved in a suitable volume (0.1-1.0 ml) of dry ethyl acetate containing 0.5 pg/ml of a-lindane as an internal standard. PFP-amines were determined by g.1.c. on a 365 cm column of 10% SE 54 on 80-100 mesh Chromosorb W HP at 200°, using N, as carrier gas and electron capture detection. The precision of the g.1.c. methods was assessed by examining pairs of replicate results selected at random from the experimental data, after excluding very low values (see below). When the difference between each member of a set of nine pairs for each type of compound was expressed as a percentage of the mean, the mean percentage difference was 8.7 for acids, 9.9 for amines and 8.7 for alcohols. There was little difference between different compounds in each class. With the exception of compounds identified by mass spectrometry, the identity of metabolites was accepted only after confirmation by co-chromatography on a two-dimensional paper chromatogram; the Whatman No. 52 paper used gave a high resolution, and the colour reaction, which was specific for phenolic compounds and which gave a structurally related colour, enabled an unambiguous identification of each compound to be made. Urinary amines and alcohols could be resolved from other urinary components on a macro g.1.c. column. However, a capillary column was necessary to permit adequate separation of the urinary acids, because compared with amines and alcohols the number of acids present in urine in large amount was much greater. The lower concentration at which the accuracy diminishes is equivalent to an excretion of less than 200 pg/24 h for acids (i.e. equiv. to 1% of the dose of a compound under investigation), 1 pg/24 h for amines, and an intermediate value for alcohols. Because of these factors, results have in most cases been corrected to two significant figures. m-Tyramine in urine from animals receiving 4-0-methyldopamine or 4-0-methyldopa could not be determined in this way because of the presence of an overlapping peak on thc g.1.c. traces. This problem was overcome using g.1.c.-mass fragmentography with a 1:{, OV17 column at 150", monitoring ions at m/e 253 and m / e 266 (King et al., 1974). A homologous compound, p-hydroxyamphetamine, was added as an internal standard to correct for differences between injections and to permit accurate quantification of mtyramine. This standard compound was monitored simultaneously at m / e 280. These measurements were performed with an LKB 9000s GC-MS and standard LKR multiple ion detector/peak matcher. Excellent linearity was obtained using internal standard down to levels of 1 ng of m-tyramine-PFP injected on to the column. Epinine was sought by g.1.c.-mass spectrometry in rat brain and urine extracts which had been processed for catecholamines. PFP derivatives were chromatographed on a 305 cm 3% SE52 column at 170", and peaks were examined at m / e 428, 442 and 472. The limit of sensitivity was about 10 ng per brain and 25 ng/24 h sample of urine. The rats under study had been injected intraperitoneally with pargyline (100 mg/kg) 24 h before an intraperitoneal injection of L-dopa (100 mg/kg) to prevent deamination of amines. One hour later they were sacrificed by decapitation. Brains were rapidly removed and coarsely minced into 0.1 M HCI (5 ml). The mince was then homogenized, first in the HCI and

Dopa Metabolism i~ Rat

63 3

then again after adding 60% (w/v) HClO, (0.2 ml). The homogenate was centrifuged at about 35 000 g for 15 min and the supernatant extracted for catecholamines by adsorption of alumina as described for urine by Wong et al. (1973).

Results T h e urinary excretion of metabolites after administration of amino acids and amines is shown in Tables 2 and 3 respectively. Compounds formed from phenylacetic acid analogues are presented in Table 4, from pyruvic and lactic acids in Table 5 and from alcohols in Table 6. on

OM UOQCW2-LM-

COOW

M~Q-ct+,-~H-cooW

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OM

OCM,

DHPL A

dM

dM

D-DOPA

no

VLA

9CM,

DHPPA

NMCMi

AM-

CM,-CM

-COOH

OM

N-METHYLDOPA

t MOeCH,-CH,-NMCM,

4+

MO~CMt-CI(,-NWt

on

DOPA M INE

EPlNlNE

n o ~ C W , - C O O M

on

-4 MO

\ 1

CH,-CWO

Mopcnz-cM,on

on DHPE

/

MOP cn,-coOH

OW,

.1

P

cM1-cooM

1 CUIO

p w , - c n , - N n ,

m-T V RAM I N E

on

DOPAC

nvp.

-

OM

mHPAA

n

o -

9 cn,-cH,on

O W

HMPE

01 1

CM,-COOH

OM

iroHVA

Figure 1. Metabolic pathways of dopa.

+More than 5% dose is metabolized by this pathway. +Some, but less than 5 % dose is metabolized by this pathway.

- - -b The results suggest this pathway, but either the observed metabolites have not unambiguously eliminated other possible pathways, or else the amount of metabolite measured leaves doubt about the administered compound being the source. -+-b Blocked. Abbreviations are defined in Table 1.

B. L. Goodwin et al.

634

OH

dCH,

Oh

mHPLA

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t

I

1

L-DOPA

3-METHOXYTYROSINE

OH

DOPAMINE

3MT

m-TY R A M INE

++

OW,

OH

DOPAC

HVACLY

dH

DHPE

Figure 2. Metabolic pathways of 3-methoxytyrosine. For the significance of the arrows see Figure 1 . Abbreviations are defined in Table 1.

T h e metabolic pathways responsible for these reactions are demonstrated in Figures 1-4. Normal excretion values for these compounds, where detectable, are shown in Table 7.

Metabolism of amino acids (Table 2) With the exception of 3-methoxytyrosine, amino acid decarboxylation proceeded briskly, confirming the results of Ferrini and Glasser (1964). Thus, high urinary concentrations of analogous amines and their metabolites were clearly demonstrated whichever route of administration was adopted. Even 3-methoxytyrosine was decarboxylated to a minor extent, traces of decarboxylation products, including 3-methoxytyramine, being detected. After a dose of 1 mg/kg of 3-methoxytyrosine, Bartholini et al. (1972) noted that the proportion of 3-methoxytyrosine excreted as homovanillic acid was twice as great as that observed here. Whether this finding is due to a difference in strain of rat or some other factor is unclear. More dopamine is excreted after administration of D- or L-dopa by either oral or parenteral route than after administration of dopamine, in agreement with the findings of Murphy and Sourkes (1961) and Sourkes et al. (1964); however,

Dopa Metabolism iz Rat cn,o+2-c-coon

0

I1

635 ++ cn,o 1

0

on

on

I cn,-cn-cmn

an

isoVPA

isoVLA

y2 no-Q-cn,--

cnl I

+- -

coon

on

dn

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L-DOPA

isoVANlLALANlNE

1 no-Q-cnt

-coon

on

dn

6n D OPAC

4 MT

4

m-TVRAMINE

1

c n , o~cn,-coon

c c n , o ~ c ~ , - c nc + o CH,o-Q--cn2

on

OH

-cn,on

on i soHMPE

isoHVA

1 cn,o~cn~-co~n-cn,-cW~

n o e c n , - cn,on

on

on I

soHVAGLY

Figure 3 .

D HP E

Metabolic pathways of 4-0-methyldopa.

For significance of the arrows see Figure 1. Abbreviations are defined in TabIe 1.

the percentage conversion of n-dopa to dopamine and other metabolites was appreciably higher than these workers noted. T h e formation of lactic acids from their analogous amino acids proceeded poorly except in the case of 3-methoxytyrosine which, apart from vanillactic acid, also yielded a significant amount of 3,4-dihydroxyphenyllactic acid. T h e greater effectiveness of the transamination pathway for 3-methoxytyrosine may be due to the relative inefficiency of the decarboxylation pathway. These results are compatible with those obtained by Maeda and Shindo (1976) in rat liver. Vanillactic acid is also found in urine from human subjects receiving L-dopa therapy, the conversion being increased many-fold when decarboxylation is prevented by the concomitant administration of peripheral decarboxylase inhibitors (Sander et a/., 1974 a). Examination of urine by t.1.c. demonstrated qualitatively that m-tyrosine and 4-0-methyldopa yield analogous pyruvic acids. m-Tyrosine-treated rats excreted a 30-100-fold greater amount of dopamine than controls. This increase, of about 40-100 pg/24 h (Table 2), exceeds the amount capable of being released through displacement of stores by a false transmitter (Bertler & Rosengren, 1959; Anton & Sayre, 1964). T h e conversion of m-tyrosine to dopa in adrenal and liver has been described (Tong, D’Iorio & Benoiton, 1971)) perhaps the source of the extra dopamine. These findings agree with those of Sourkes, Murphy and Rabinovitch (1961) who also noted increased dopamine excretion after m-tyrosine administration to rats, but are

B. L. Goodwin et al.

636 OH

I

W H , -CH -COOH

mHPPA

mHPLA

t.c

NH z I

Nli 2 I p C H 2 - C H -COOH

+

-.. m - T Y ROS I N E

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L- D O PA

1 m- TY RAMIN E

OOPAM I N E

p,, -CHO

T YRAMl N E

OH

N,N- D IMETH YL m-TYRAMINE

0

cH,-cooH

2-” OH

/

OH

rn - T Y ROSOL

mHPAA

mHPAGLY

DOPAC

Figure 4. Metabolic pathways of m-tyrosine. For significance of the arrows see Figure 1. Abbreviations are defined in Table 1.

quite different from observations in man (Sandler et a/., 1975), where no evidence could be obtained for the conversion of m-tyrosine to dopamine. After intraperitoneal administration of 3-methoxytyrosine, a small but significant increase in dopamine excretion was observed. However, its oral administration led to an output ten times greater, suggesting that some demethylation in the gut had taken place. Bartholini and Pletscher (1972) found that 0.054% of intraperitoneally administered 3-methoxytyrosine (1 mg/kg) was excreted as dopamine, whereas we found that 0.15% was so excreted in 24 h.

Dopa Metabolism in Rat

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Experiments designed to detect the N-methylated amine, epinine, in urine and brain after L-dopa administration failed to reveal its presence. From the evidence of our g.1.c.-mass spectrometry studies, N-methyldopa does not undergo decarboxylation to epinine, even to a minor extent, confirming the classical findings of Blaschko (1942). However, large amounts of dopamine were present in the urine, whilst only traces of dopamine metabolites were detected by paper chromatography. This pattern suggests that N-demethylation and decarboxylation occur in the kidney, followed by immediate excretion of much of the resulting dopamine. Moritani et al. (1954) demonstrated that an enzyme in rabbit kidney demethylates N-methylamino acids inchding N methyldopa. T h e present results are consistent with the presence of a similar renal enzyme in the rat. Metabolism of amines (Table 3) All the amines tested were converted primarily to the analogous phenylacetic acids, although there was a small degree of conversion to the phenylethanol analogues. I n the case of dopamine, there was some conversion to the p dehydroxylated acid and alcohol and, to a greater extent, to their 3-0-methyl analogues. Dehydroxylation or 0-methylation occurred with the parent amine, as well. N-Methyl-m-tyramine and m-tyramine were deaminated to a comparable extent but N,N-dimethyl-m-tyramine yielded smaller amounts of deaminated metabolite. Metabolism of carboxylic acids (Tables 4 and 5) Ring-substituted phenylacetic acids formed compounds which co-chromatographed on paper with and gave the same colour reaction as their glycine conjugates. These conjugates could not be assayed by g.l.c., so that their spot intensity on paper was used as a semi-quantitative measure of the amount present. This approach indicated that about 1% of each administered acid was converted to its glycine conjugate. These glycine conjugates were in turn partially hydrolysed in vivo. For example, about 5 yo of administered isohomovanilloglycine or p-hydroxyphenylacetylglycine were excreted as their parent acids. T h e reduction of pyruvic to lactic acids was comparatively inefficient with the exception of vanilpyruvic acid. 3,4-Dihydroxyphenyllactic acid was converted fairly efficiently to vanillactic acid but vanillactic acid was not formed to any large extent from 3,4-dihydroxyphenylpyruvic. Thus, the formation of vanillactic acid from 3,4-dihydroxyphenyllacticacid proceeds by direct methylation and not by oxidation to 3,4-dihydroxyphenylpyruvicacid, methylation and subsequent reduction. Vanillactic acid is also excreted in man after L-dopa therapy (Sandler et al., 1974 a), a process facilitated by peripheral decarboxylase inhibition, and the rat seems to behave similarly. T h e amount of vanillactic acid formed from 3-methoxytyrosine was comparable with that formed from vanilpyruvic acid, suggesting that the amino acid is readily converted t o vanilpyruvic acid. T h e reduction of the other pyruvic acids was too inefficient to provide information about transamination of their corresponding amino acids by examination of the lactic acids. T h e oxidation of lactic to pyruvic acids and their subsequent transamination appeared to proceed easily, with the possible exception of vanillactic acid to vanilpyruvic acid, although no conclusion can be drawn in this case because of the inefficient decarboxylation of 3-methoxytyrosine. After administration of vanilpyruvic acid or vanillactic acid, there X.B.

12T

4-Hydroxy-3methoxyphenylethanol

0.2 (0.1-0.3)

0.3 (0.25-0.4)

0.55 (0.5465)

0.4 (0.2546)

0.35 (0.25-0.45)

Vanillactic acid

0.4 (0.3-0.5)

21 (20-2 3)

4.7 (4.3-5 '0)

3.3 (24-3.4)

Homovanillic acid

1.5 (0.95-2.2)

1.55 (1.2-1.8)

2.3 (1'9-2.85)

1.05 (0.85-1.25)

0.35 (0.3-0.41

0.3 (0,25-0.3)

-

-

0.1 (0.03-0.17)

6.3 (4.3-7.3)

0

9.4 (6'5-1 1.5)

5.1 (4.7-5.6)

4.9 (3'3-5'6)

-

16.5 (145-19.5)

0.28 0.05 (0.17-0.39) (0.04-0.07)

-

-

-

-

-

(0.05-0.2)

0.1

0.9 (0.45-1.35)

0

I

0.17 (0.10-0.27)

Oral

0.15

IP

1.1 (0.75-1.5)

-

0.9 (0.8-1.1)

1.9 (1.4-2'4)

Oral

IP

-

-

-

-

-

(0.3-0.7)

0.5

-

-

-

-

0

0.20 0.60 (042-0.33) (0.36-1.17)

Oral

DL-m-Tyrosine

Each figure represents a mean of

DL-4-0DL-3-Methoxytyrosine Methyldopa

-

-

-

2.3 (1'7-3.0)

IP

DL-NMethyldopa

2.0 (1*6-2.25)

0.8 0.8 (0*65-1,05) (0.65-1.05)

2.7 (24-3.2)

0.5 (0.2-0.8)

0.95 (0.3-2.0)

20.5 (19-22)

19 (16.0-21 '5)

4.4 (4.0-5.1)

IP

26 15.1 (12.8-1 6.6) (19.5-32.5)

Oral

57 (49-71)

IP

L-Dopa

3 -Methoxytyramine

3,4-Dihydroxyphenylethanol

0.15 (0-0.6)

3.5 (3.0-4.0)

Dopac

3,4-Dihydroxyphenyllactic acid

62 (59-68)

Oral

D-Dopa

Dopamine

Metabolite

Route administered

Compound administered

Table 2. Metabolism of amino acids administered to rats Figures express excretion of total (free plus conjugated) metabolites as a percentage of the dose excreted in 24 h. 4 results, with the range in parentheses.

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.-2. m rr

b

&

cc

OI w

N t.l Li

%

(68-89)

80

0.2 (0.10-0.25)

0.2

60 (26-70)

-

2.4 (1'7-2.8)

1.65 (1.4-2'25)

-

2.1 (1'45-2'55)

1.3 (0.95-1.60)

34 (30-37)

0.45 (04-0.45)

0

3-1 (2'8-3.3)

61 (50-71)

0.45 (0.35-0.55)

0.6 (0.35-1.0)

1.8 (1'2-2'45)

0.30 0.26 (0.23-0.40) (0.20-0.30)

-

-

+

-

t By paper chromatography; 1by g.1.c.-mass spectrometry; -not values are too small to be reported.

4-Hydroxy-3methoxyphenylglycol ( d 2 4 h)

m-Hydroxyp henyllactic acid

nz-Hydroxyphenylpyruvic acid

nz-Hydroxyphenylacetic acid

m-Tyramine

3-Hydroxy-4methoxyp henylethanol

-

-

-

Isovanillactic acid

-

Isovanilpyruvic acid

-

-

-

-

+

-

-

-

Isohomovanillic acid

4-0-Methyldopamine

-

-

-

-

+

-

-

L

-

0.35 (0.32-0.40)

0-t

+

(17-22.5)

19.0

16.0 (9.1-21.5)

+ present,

46 (29-60)

but not quantitated.

(18-42)

31

0.10 0.22 0.1 1 (0.10-0.12) (0.17-0.29) (0.054.15)

0.5 (0.3-0.7)

2.2 (1.3-3.9)

0.1 5 (0.04-0.20)

determined;

-

-

-

-

-

-

-

-

-

Downloaded by [Flinders University of South Australia] at 00:24 17 March 2016

-

1.9 (1.35-2.7)

-

-

-

-

-

Where no range is given,

-

2.8 (2.05-4'6)

-

-

-

-

+

\D

w

cn

18 (8.5-26.5)

16.5 (3.3-33)

IP

IP

4-0-Methyldopamine

22.3 (17.7-26.7)

11 (8.9-13'3)

-

0

1.4 (1.2-1.65)

34.5 (2543)

3.9 (1'85-6.4)

-

1.1 (0-9-1.5)

-

-

-

0.15 (0.05-0.30)

0.65 (0-5-0.8)

0.60 0.09 0 .02 (0.29-0.80) (0.05-0.13) (0.01-0.03)

Oral

3-Methoxytyramine

0.25 1.o (0.18-0.30) (0.75-1.15)

1.9 (14-2.7)

2.1 (0.95-2.5)

23 (6.7-3 5)

6.7 (243-10.3)

IP

0.57 (0.30-0.73)

1.95 (0.45-2.5)

17 (3.O-3 5)

8.4 (54-12.4)

Oral+

Dopamine

0.35 0.25 (0.30-0.40) (0.13-0.39)

26 (23-29)

Homovanillic acid

4-Hydroxy-3methoxyp henylethanol

2.6 (2.1-3.0)

3-Methoxytyramine

2.0 (1.65-2'2)

23 (20-28.5)

Dopac

3,4-Dihydroxyp henylethanol

7.5 (6.0-10.5)

Oral

Dopamine

Metabolite

Route administered

Compound administered

-

0

-

-

-

-

-

-

IP

-

IP

-

-

0.2 (0.1-0.25)

0-02 (0.01-0.05)

IP

N,NN-Methyl Dimethyl m-tyramine m-tyramine

-

-

0.4 (0.15-0.95)

0.02 (0-0.06)

Oral

rn-Tyramine

Table 3. Metabolism of amines administered to rats Figures expressed as in Table 2.

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p

a

3

tr

pl

54 (43-64)

1.1 (0.75-1 * 5 )

7.5 (5'5-9.1)

-

+t:

-

38 (3443)

0.3

-

54 (48-62)

1.5 (0.9-2.0)

-

0.3 (0.254.35)

-

0.13 1.35 (0.06-0.23) (0.48-3915)

-

-

-

56 (41-63)

(O.SS-0.8)

0.65

4.1 (3.0-5'3)

0.85 (0.55-1.2)

-

-

-

61 (49-78)

0.4 (0.4-0.45)

3-1 (2.2-3'7)

0.76 (0.52-0.90)

-

-

-

-

(0.354.6)

0.5

2.8 (2'1-3.6)

0.22s

1.05 (0.97-1 40)

27 (23-2 9)

15.2 (10*0-19.5)

-

-

-

-

3.45 (2-4-4.1)

41 (26-48)

c

6-5 (3.0-10- 5)

-

L

-

- not determined;

-

5 .o (3.2-6.5)

52 (45-57)

5.0 (4.0-6.9)

-f Animals pretreated with neomycin; 1by paper chromatography; $ by g.1.c.-mass spectrometry; tated. Where no range is given, values are too small to be reported.

4-Hydroxy- 3methoxyphenylglycol (CLgP4 h)

m-Tyrosol

m-Hydroxyphenylacetic acid

-

N-Methyl-mt yramine

-

3-Hydroxy-4methoxyphenylethanol

1.08 (0.94-1.22)

+I

Isohomovanillic acid

m-Tyramine

-

4-0-Methyldopamine

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1*2 (0.8-1.5)

(13'5-18) 16

-

+ present, but not quanti-

(0-75-1.95) 1.45

(30-43) 39

4.6 (3.5-6.3)

642

B. L. Goodzuin et al.

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was a relatively large excretion of homovanillic acid compared with that of 3-methoxytyramine. The oxidation of 3,4-dihydroxyphenylpyruvic acid appeared to be slower than that of m-hydroxyphenyllactic acid and isovanillactic acid. After administration of the other pyruvic and lactic acids investigated, a substantial excretion of the analogous phenylethylamine and its metabolites was seen, implying that the corresponding amino acids were produced as intermediates. Metabolism of alcohols (Table 6) These compounds were largely, although not quantitatively, converted to the analogous acid, presumably via the aldehyde. Only a small proportion of 3,4-dihydroxyphenylethanol was converted to 4-hydroxy-3-methoxyphenylethanol, and the reverse reaction was, similarly, inefficient. I t is theoretically possible for aldehydes to be transaminated to amines, although such a reaction does not appear to have been described. T o test this possibility, urine from rats receiving 4-hydroxy-3-methoxyphenylethanol and m-tyrosol were examined for amines by paper chromatography, but in the event, no evidence of any analogous amine was obtained. Thus, the aldehydes formed during oxidation of these alcohols are not significantly transaminated. Formation of 4-O-methylated metabolites I n human subjects receiving L-dopa, some isohomovanillic acid is formed, the urinary concentration being about 10-200/, that of homovanillic acid (unpublished results; Ebinger & Adriaenssens, 1973). Isohomovanillic acid could not be measured at these concentrations by the capillary g.1.c. method of Karoum and Sandler (1971) because of the similarity of its retention time to that of homovanillic acid, but two-dimensional paper chromatography provided a sensitive method for distinguishing between the two isomers. Although they overlapped, homovanillic acid gave a violet colour with diazotized sulphanilamide, and isohomovanillic acid an orange pigment about ten times more intensely coloured than the equivalent amount of homovanillic acid pigment. This property allowed HVAlisoHVA ratios as high as 50 : 1 to be detected visually. Urine samples from rats receiving L-dopa, dopac or dopamine contained just detectable amounts of isohomovanillic acid, thus pointing to a ratio of HVA/ isoHVA of between 20 and 50. Hence, 4-O-methylation in the rat appears to be an inefficient procedure, in contrast with conditions in vitro where 4-Omethylation takes place more readily (Creveling et al., 1972). Amine excretion was too small to enable an unambiguous decision to be made by paper chromatography that no 4-O-methyldopamine was formed from dopamine. If any is produced, however, it cannot be more than about 2yoof the methoxytyramine formed. Nor has it been possible in human subjects receiving L-dopa to provide any evidence for the formation of 4-O-methyldopamine (unpublished results). EfSect of the route of administration T h e excretion of m-hydroxylated catechol metabolites differed quantitatively depending on route of administration. Many catechols undergo p-dehydroxylation when exposed to gut flora (Scheline, 1968), a degradation step demonstrated in man, where oral dopa is converted to m-tyramine (Sandler et al., 1971) and m-hydroxyphenylacetic acid (Sandler et al., 1969); a significant decrease in this conversion occurs after oral neomycin administration. We

+t -

+t

-

Isohomovanillic acid

Isohomovanilloylglycine

~

p-Hydroxyphenylacetylglycine

p-Hydroxyphenylacetic acid

m-Hydroxyphenylacetylglycine

m-Hydroxyphenylacetic acid

-

2.2 (1.7-2.7)

+t

75 (64-88)

0.4 (0.25-0.6)

IP

-

+t

5.1 (4.3-5.8)

IP

+t

79 (72-87)

-

1.o (0.85-1 -25)

IP

+t

5t

-

IP

Isohomovanillic Isohomovanilloyl- m-Hydroxyphenyl- p-Hydroxyphenylacid glycine acetic acid acetylglycine

t By paper chromatography; - not determined; + present, but not quantitated.

-

-

2.7 1 .o (0-5-4.3) (0.5-1.95)

-

-

+t

105.5 (70-114)

16.0 (12.9-19.4)

28.5 (20-34)

Homovanillic acid

Homovanilloylglycine

1.3 (0.3-3.0)

IP

81 (66-96)

IP

Homovanillic acid

53 (35-66)

Oral

Dopac

Dopac

Metabolite

Route administered

Compound administered

Table 4. Metabolism of phenylacetic acids in rats Figures are expressed as in Table 2.

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4-Hydroxy-3-methoxyphenylethanol

Vanillactic acid

0.15 (0.10-0-19)

0.2 (0.1-0.3) 0.15 (0.10-0-27)

6.4 (5.3-6.7)

-

-

Vanilpyruvic acid

5.3 (5.1-5.4)

0.59 (0.53-0.71)

0.1 (0-1-0-15)

8.1 (7.6-8.7)

0.4 (0-1-0.6)

3,4-Dihydroxyphenylethanol

68 (61-77)

Homovanillic acid

2.0 (1 1-2.8)

3,4-Dihydroxyphenyllacticacid

3.8 (3.0-4.6)

0.35 (0.25-0-50)

9.1 (8.2-10.2)

Dopac

3.55 (2.65-4-65)

3-Methoxytyramine

25 (17.5-33)

15.8 (13.1-1 7.8)

+t

5.0 (4.5-5.5)

92 (88-96)

+t

(1'6-2'1)

1.7

0.06 (0.04-0*10)

-

-

0.04 (0.03-0.05)

0.75 (0.25-1.4)

-

-

0-5 (0-1.5)

1-9 (1.5-2.6)

-

3,4-Dihydroxy- 3,4-Dihydroxy- Vanilpyruvic Vanillactic phenylpyruvic phenyllactic acid acid acid acid

Dopamine

Metabolite

Compound administered

-

-

-

-

-

-

-

0.05

0.3 (0.2-0'45)

-

0.03

Isovanillactic acid

1-4 (0*05-3*0)

0.3 (0'2-0.6)

(0.2-1.1)

0.5

0-2 (0.15-0.3)

Isovanilpyruvic acid

m-Hydroxy- m-Hydroxyphenylpyruvic phenyllactic acid acid

Table 5. Metabolism of lactic and pyruvic acids in rats, administered intraperitoneally Figures are expressed as in Table 2.

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-

-

-

0

0

m-Hydroxyphenyllactic acid

45 (43-52)

t By t.1.c. ; 1by g.1.c.-mass spectrometry; be reported.

4-Hydroxy - 3-methoxyphenylglycol ( d 2 4 h)

m-Tyrosol

- not determined;

-

0.2 (0.20-0.25)

-

-

m-Hydroxyphenylpymvic acid

0.25 (0.1 5-0-31)

0.8

m-Hydroxyphenylacetic acid (0.7-1.1)

0.07 (0.06-0.08)

1.7

0.06 (0~05-0~08)

-

-

-

-

-

-

(1 4-2.35)

m-Tyramine

3-Hydroxy-4-methoxyp henylethanol

Isovanillactic acid

Isovanilpyruvic acid

Isohomovanillic acid

4-0-Methyldopamine

I

0

1.7 (14-1.9)

-

0

5-9 (4.3-7-4)

0.091

-

3.45 (2.75-4.2)

0-5 (0-0-9)

+t

13.5 (9.5-1 6.5)

9.9 (6.6-13.2)

-

0.5 (0.35-0-65)

79.5 (54.5-88.5)

+t

3.9 (3.1-4.4)

6.7 (4.5-7.9)

-

-

-

-

-

-

-

-

Where no range is given, values are too small to

0.15 (0.11-0.19)

0

6-8 (3.5-8.6)

0.061

+ present, but not quantitated.

-

0

1.7 (1-3-2.6)

0.06 0.14 (0*05-0*10) (0-07-0.28)

44 (22-61)

+t

9.4 (5.6-1 3.3)

5.0 (3.0-8.7)

0.35 0.15 (0-17-0.54) (0.1 1-0-22)

2.6 (1-9-3-4)

+t

19.5 (5 '9-27)

9.3 (1.25-15.7)

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-

-

-

-

-

4-Hydroxy - 3-me thoxyphenylglycol (pg /24 h)

p-Tyrosol

t By paper

chromatography; - not determined.

51 (41-69)

-

-

-

p-Hydroxyphenylacetic acid

20.6 (13-26) -

40 (32-45)

0.12 (0.09-0.1 5) -

1.4 (1.2-1-65)

ot

m-Tyrosol

0.5 (0.35-0.7) -

I

-

-

-

-

-

-

-

0.6 (04-0.7)

-

m-Tyramine

47 (43-51) 13 (10.5-14.8)

0.65 (0~55-0~80) 0.75 (0.55-0.85)

m-Tyrosol

nz-Hydroxyphenylacetic acid

-

3-Hydroxy-4-methoxyphenylethanol

0-t 92 (71-102) 16.3 (13-22.5)

0.8 (0.45-1 '1) 2.3 (2.0-2.9)

4-Hydroxy-3 3-Hydroxy-4methoxyphenylethanol methoxyphenylethanol

-

15 (11.5-20)

-

14.5 (13-17.5) 8.9 (7.9-10)

3,4-Dihydroxyphenylethanol

Isohomovanillic acid

4-Hydroxy-3 -methoxyphenylethanol

Homovanillic acid

3-Methoxytyramine

3,4-Dihydroxyphenylethanol

Dopac

Metabolite

Compound administered

Table 6. Metabolism of alcohols in rats, administered intraperitoneally Figures are expressed as in Table 2.

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(77-88) 21.5 (12-27) -

82

p-Tyrosol

o\ o\

P

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Dopa Metabolism in R a t

647

have now shown that a similar reaction is observed in the rat given neomycin before dopamine ingestion. m-Hydroxy metabolite excretion dropped to about 5 yl of the value obtained after dopamine administration alone. Unexpectedly large amounts of urinary m-hydroxy metabolites, particularly m-tyramine, were even observed after intraperitoneal injection of D- or L-dopa, or dopamine. T h e neomycin evidence suggests that a catechol precursor of m-tyramine possesses a facilitated mode of transport into the gut lumen, presumably by biliary excretion; compounds o f a molecular weight as low as 300 can be eliminated by such a mechanism in the rat (Abdel Aziz et al., 1971; Smith, 1973). Although dopa and 4-0-methyldopa undergo hepatic acetylation and biliary secretion in rats (Mathieu et al., 1976; Tyce, 1971), we were unable to identify N-acetyldopamine in urine unequivocally because of technical difficulties. It is o f interest that we failed to detect significant concentrations of dopa metabolites in bile samples obtained from patients on L-dopa therapy (Sandler et al., 1974 b); however, in man, the biliary excretion mechanism can only deal with compounds having a molecular weight greater than about 500 (Smith, 1973). T h e excretion of 3-methoxytyramine after oral administration of dopamine plus neomycin was smaller than that after dopamine alone, presumably implying that some methylation of dopamine is effected by neomycin-sensitive gut flora. T h e urinary excretion of m-tyramine was far smaller when DL-m-tyrosine was administered orally than when it was given intraperitoneally ; however, there was no increase in the excretion of m-hydroxyphenylactic acid or rn-tyrosol after intraperitoneal administration compared with oral administration. This observation suggests that D-m-tyrosine is not absorbed from the gut, and that after intraperitoneal administration, D-m-tyrosine, like n-dopa, is readily racemized and decarboxylated by the kidney, the amine so formed then being excreted without entering the general circulation.

Discussion The observation that more dopamine is excreted after administration of Ldopa than after dopamine points to considerable direct decarboxylation of L-dopa in the kidney with the dopamine so formed being excreted without entering the general circulation. The substantial conversion of D-dopa to dopamine with a small accompanying output of dopamine metabolites suggests that racemization too occurs in the kidney. These findings are consistent with the known presence there of D-amino acid oxidasc and transaminases (Negelein & Rrorriel, 1939; Lin & Knox, 1958). T h e failure to detect epinine after L-dopa administration is in agreement with the findings of Schiimann and Brodde (1976). However, Foppen, Liuzzi and Kopin (1977) appear to have demonstrated this amine in sympathetic ganglia, so that L-dopa administration does not necessarily drive the reaction sufficiently to cause an overspill with liberation into the urine. Although an enzyme capable of catalysing the N-niethylation reaction has been demonstrated in rabbit lung (Axelrod, 1962), its presence in other species has not yet been demonstrated unequivocally. T h e metabolism of D-dopa was quantitatively similar both on oral and intraperitoneal administration, suggesting that its gastro-intestinal absorption is relatively efficient. There has been some controversy about the mechanism of L-dopa absorption by gut (e.g. Wade, Mearrick & Morris, 1973); our present data suggest that, in addition to active transport of L-dopa, there may be a diffusion process for absorption of both D- and L-dopa, able to handle relatively large amounts of compound. Shindo et al. (1973 a, b, c) noted a more efficient absorption of L-dopa than D-dopa, a difference reflected in output of urinary

B. L. Goodwin et al.

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648

metabolites. They also observed, in contrast with our own findings which show only marginal differences, that metabolite excretion is greater after intraperitoneal than oral administration although their total metabolite recovery was smaller than we report here. Differences between strains of rats may be responsible. Although phenylethanolamines are converted mainly to their glycol analogues in rat brain, /3-phenylethylamines are oxidized largely to analogous acids (Breese, Chase & Kopin, 1969), perhaps because the glycols are conjugated with sulphate (Wong, 1975) and this protected from further catabolism (Waterbury, 1974). I n our hands, the administration of P-phenylethylamines, their precursors or 2-phenylethyl alcohols including p-tyrosol led to urinary elimination of the acids in preference to the alcohols, in agreement with the observations of Waterbury (1974), who administered 4-hydroxy-3-methoxyphenylethanolto rats. These findings suggest that the whole organism behaves in a similar manner to the brain in oxidizing the intermediate aldehyde rather than reducing it. T h e ratio of urinary acid to alcohol was considerably less when the intermediate aldehyde was generated from an alcohol rather than an amine precursor, suggesting that either a small percentage of administered alcohol is not presented to the relevant dehydrogenase before excretion, or that the large amount of administered alcohol saturates the electron transport system briefly and drives the aldehyde-alcohol equilibrium towards the alcohol, or both processes together.

Table 7. Excretion of dopa metabolites in control rat Mean of 4 results.

Compound Dopamine Homovanillic acid Dopac Vanillactic acid 4-Hydroxy-3-methoxyphenylglycol

m-Hydroxyphenylacetic acid

3-Methoxytyramine

Total (free plus conjugated) Approx. equiv. to yo of a 20 mg dose of test compound in a 200 g rat (pg/24 h) 1.5 (1-2) 30 (21-40) 90t (70-1 00) 130t (90-1 90) 30 (21-3 6) 100 (80-1 20) 4

0.01 0.15 0.45

0.65 0.2

0.5 0.02

(0-14) m-Tyramine Isohomovanillic acid

0-25 (0-1 ) 3 (2-4)

0.001 0.02

t Tests with another g.1.c. column indicate that these peaks were not primarily dopac or vanillactic acid, but the values are included here to give a valid base-line comparison. The following compounds were not detected : 4-hydroxy-3-methoxyphenylethano1, m-tyrosol, p-tyrosol, 4-O-methyldopamine, 3,4-dihydroxyphenylethanol, 3-hydroxy-4methoxyphenylethanol, 3,4-dihydroxyphenyllacticacid, 3-hydroxy-4-methoxyphenyllactic acid, m-hydroxyphenylpyruvic acid, isovanilpyruvic acid and vanilpyruvic acid.

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After administration of relatively large amounts of its amino acid or amine precursors (excluding noradrenaline and adrenaline), 4-hydroxy-3-methoxyphenylglycol output was not increased to more than twice endogenous excretion values. (The concentration of 4-hydroxy-3-methoxymandelic acid was not measured because of technical problems encountered at low concentrations). T h e finding is similar to that in man during L-dopa therapy (Calne et al., 1969) although the actual mechanism is unclear. Thus, although most of the administered dopa and dopamine was recovered as deaminated metabolites, only a very small proportion arose from P-hydroxylated amines. This may be due to P-hydroxylation being the rate-limiting step. Pogrund, Drell & Clark (1961) have demonstrated the formation of phenylacetic acids after 3,4-dihydroxyphenylpyruvicacid and m-hydroxyphenylpyruvic acid administration, but they were not able to show that the analogous amines were formed at the same time, which left the mechanism unresolved. T h e direct conversion of 3,4-dihydroxyphenylpyruvic acid to L-dopa in rats, particularly in liver and kidney, has recently been demonstrated by Maeda and Shindo (1977). T h e decarboxylation of 3-methoxytyramine after administration of vanillactic or vanilpyruvic acids merely reflects this fact rather than poor transamination of vanilpyruvic acid. Bartholini et al. (1972) also noted the relatively large excretion of homovanillic acid compared with that of 3-methoxytyramine after vanillactic acid administration, and suggested that vanilpyruvic acid is decarboxylated and oxidized to homovanillic acid. Our present finding is consistent with that hypothesis, although the homovanillic acid might also derive from limited decarboxplation of the amino acid in conjunction with efficient transamination of the pyruvic acid. T h e low conversion of 3-methoxytyrosine to dopamine is consistent with the finding by Maeda and Shindo (1976) that demethylation does not occur in rat liver. Even so, excretion was greater than after 3-methoxytyramine administration, suggesting that some 3-methoxytyrosine is converted to dopa before decarboxylation. Any conversion of m-tyramine to dopamine was at best marginal, indicating that direct hydroxylation of the amine does not occur and that virtually all the increase in dopamine excretion observed after m-tyrosine administration arises via 1,-dopa formation. This in vivo finding is unexpected, for Daly, Inscoe and Axelrod (1965) found that rat liver microsomes readily form dopamine from m-tyramine. Orally-administered L-dopa and dopamine were 0-methylated more efficiently than their intraperitoneal counterparts, to judge from the excretion of homovanillic acid. There are at least two possible reasons for this: there may be 0-methylating activity in gut flora; after intestinal absorption these compounds are transported by portal vein to the liver, which has high catechol O-methyltransferase activity. I n contrast, intravenous isoprenaline in man is more efficiently methylated than when administered orally (Morgan et al., 1969 a ; Morgan, Ruthven & Sandler, 1969 b). After a small dose of dopamine in rats, Williams, Babuscio and Watson (1960) found that the HVAIdopac ratio was higher than in the experiments reported here; this effect may well be dosedependent. There is some evidence that the ratio decreases with increasing L-dopa dosage (Calne et al., 1969), perhaps from a relative decrease of available methyl groups (Wurtman, Chou & Rose, 1970 a ; Wurtman et al., 1970 b). When 3,4-dihydroxyphenylethanol, 4-hydroxy-3-methoxypheny1ethano1, homovanillic acid and dopac were formed from administered L-dopa or dopamine, homovanillic acid and dopac were excreted in about equal amount, whereas the excretion of 3,4-dihydroxyphenylethanol was greater than that of 4-hydroxy-3methoxyphenylethanol. This finding suggests that dopac is more readily

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0-methylated than 3,4-dihydroxyphenylethanol. After intraperitoneal injection of dopac, a higher proportion of this acid is excreted relative to homovanillic acid, an effect even more marked after oral administration, Thus, exogenous dopac may not be as accessible to methylating enzymes as that formed endogenously. This finding may, however, be a dose effect. T h e small amount of 3-methoxytyramine excreted after dopamine administration suggests that niethylation of dopamine does not contribute significantly to the observed ratio of the acids when dopamine is administered.

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