212 Pergamon Press Lfd 1979 Printed in Great Britain 0 International Saciety for Ncurocheniislry Ltd

Journol of Nuurochumrsrr) Vol. 33. pp 201 10

0022-3042/79/070 1-0201S0?.00/0

MASS FRAGMENTOGRAPHY OF PHENYLETHYLAMINE, m- A N D p-TYRAMINE A N D RELATED AMINES I N PLASMA, CEREBROSPINAL FLUID, URINE, A N D BRAIN F. KAROUM, H. NASRALLAH, S. POTKIN,L. CHUANG,J. MOYER-SCHWING, I. PHILLIPS and R. J. WYATT Laboratory of Clinical Psychopharmacology, National Institute of Mental Health, Saint Elizabeth’s Hospital, Washington, DC 20032, U.S.A. (Recriced I5 August 1978. Revised 7 Novrniher 1978. Accepted 9 Novrniher 1978)

Abstract-A mass fragmentographic method for the assay of phenylethylamine (PEA) and a number of related amines in several biological materials is described. The gas chromatographic column employed for this analysis is a 12ft 1/8in. 0.d. steel column packed with 0.5% OV,, + 2% SE54 + 1% O V z l 0 coated on 80jlOO mesh chromosorb W (HP). The mass spectral characteristics of these amines are illustrated, compared, and discussed. Of the various monoamines which could be measured, only PEA, m- and p-tyramine were detected in measurable quantities. Phenylethanolamine and p-octopamine were found in trace amounts in urine, plasma, cerebrosponal fluid, and rat brain. No diurnal variation in the urinary excretion of PEA, m- and p-tyramine was observed. Plasma concentration of PEA or p-tyramine did not significantly change 1 h after eating a breakfast. Furthermore, consuming 200 g of Cadbury milk chocolate containing about 1 mg of PEA, 0.1 mg of phenylethanolamine and lOmg of p-tyramine did not significantly alter urine excretions of these three amines. In the brain, as has been reported by others, we found that PEA and p-tyramine are not evenly distributed and that the highest concentrations are found in the hypothalamus and caudate. From the results obtained we concluded that PEA, m- and p-tyramine are probably produced from endogenous sources and that the direct contribution of diet to their urine excretion is small.

THE STRUCTURAL similarity between P-phenylethyl-

metric (SUZUKI & YAGI,1976, 1977) methods appear amine (PEA) and amphetamine as well as their simi- to be similar, while those reported by MOSNAIMrt larity in inducing behavioral responses in animals al. (1973) are considerably higher. & RIVA,1963; NAKAJIMA et al., 1964; (MANTEGAZZA Phenylethylamine is readly converted to p-tyramine RANDRUP & MUNKVAD, 1966; BRAESTRUP et a/., 1975; (BOULTON et al., 1974a; SILKAITIS & MOSNAIM, 1976; SABELLI et a/., 1975; MOJA ct al., 1976) has led to TALLMAN et al., 1976a) as well as 0- and m-tyramine the hypothesis of involvement of PEA in schizo- (BOULTONet al., 1 9 7 4 ~ BOULTON, ; 1976a, h). It also & REYNOLDS, 1976; can be converted to phenylethanolamine and octopphrenia (FISCHER,1975; SANDLER WYATTer al., 1977; POTKIN et al., 1978), depression amine by b-hydroxylation of PEA itself and of tyra& AXELROD, 1973). Because of these (FISCHERet a/., 1968; FISCHER et a/., 1972; SABELLXmine (SAAVEDRA & MOSNAIM,1974; SABELLIet a/., 1974) and other metabolic relationships, assays of PEA in biological medical disorders (SANDLER et a/., 1974). PEA is nor- materials ideally should include some of the above mally excreted in human urine (JEPSONet al., 1960; amines. OATES et al., 1963; SCHWEITZER et al., 1975) and is Our interest in PEA metabolism stems from the present in the brains of a number of animal species finding that platelet monoamine oxidase (MAO) (INWANGet al., 1973; MOSNAIM& INWANG,1973; which is of the B-type (enzyme with a high affinity SAAVEDRA, 1974a; WILLNERet al., 1974; DURDENet for PEA), is low in some chronic schizophrenics al., 1973; BOULTON et al., 1974b). The concentrations (WYATT& MURPHY,1976), particularly those with of PEA evaluated by radioenzymatic (SAAVEDRA,paranoid symptoms (POTKIN et a/., 1978). In this com1974a,h), mass spectrometric (WILLNERet a/., 1974; munication we describe a mass fragmentographic proDURDEN et al., 1973; BOULTON et al., 1974a; SLINGSBY ccdure capable of measuring PEA and other related & BOULTON,1976) and recently by a spectrofluoro- amines in plasma, urine, CSF and brain. Since in some clinical studies M A 0 inhibitors are used, the method described was developed so that pargyline (a Send reprint requests to Dr. F. Karoum. Abbreviations used: PEA, phenylethylamine; MF, mass commonly used M A 0 inhibitor) and its metabolites tragmentography; MID, multiple ion detection; AMU, would not interfere with the quantification of these atomic mass unit; MAO, monoamine oxidase. amines. 20 1

F. KAROUM rt ul.

202 MATERIALS A N D METHODS Materials und subjects

Ortho-tyramine was obtained through Dr. A. MANIAN, National Institute of Health, Rockville, MD, and m-tyramine was purchased from Vega-Fox Biochemicals, Tucson, AZ. Deuterated isomers of PEA (a-d,, /3-d2 or d9), phenylethanolamine (a-d,, P-dJ p-tyramine (a-d,, P-d,) and octopamine (a-d,, p-d,) were obtained from Merck Sharpe and Dohme, Canada Ltd., Quebec. All other chemicals and reagents were of the highest grades obtainable from commercial sources. Ten-hour fasting morning plasmas were collected from 15 healthy young adults (right males and seven females) working at the National Institute of Mental Health. The subjects had not exercised strenuously for the previous 24 h. In order to understand the effects of diet, bloods were collected again I h after eating a meal consisting of one egg, a piece of Canadian ham (approx 0.2 kg). a slice of bread, at least one slice of pineapple and a cup of coffee. Bloods were collected in 16 ml glass tubes (Becton-Dickinson No. 4798) containing 2 ml ACD (acid-citrate-dextrose, N I H Formula A) and placed on ice. The cells and platelets were removed by centrifugation at 3000 y for 10 min. Lumar cerebrospinal fluids were obtained from 15 neurological patients undergoing diagnostic spinal taps. Urines were collected from 15 normal young adult volunteers as follows: from 9 a.m. to 9 p m . one day and from 9 p.m. t o 9 a.m. the following day. The urines were collected in containers containing 5 ml 10% EDTA. Total volume was measured and aliquots taken and stored at -70°C until analyzed. In a separate experiment, to study the dietary contribution to urine PEA, five volunteers consumed 200g of Cadbury milk chocolate within a 2 h period. Urines were collected for 24 h beginning at the start of chocolate consumption. For whole brain analyses. six rats were decapitated by guillotine and their brains frozen on solid CO,. The brains were weighed and homogenized in 5 ml normal saline and centrifuged at 20,000y for 15 min. In a second experiment, six rats were decapitated by guillotine and their brains dissected into seven major areas according to the pro& IVERSEN (1966). The regions were cedure of GLDWINSKI frozen, weighed, homogenized (in 2 ml normal saline), and centrifuged as described for the whole brains. Method The amines (PEA, phenylethanolamine, 0-, m-, and p-tyramine) were extracted from the biological materials into ethyl acetate (Fisher Scientific Company, Fairlawn, NJ) before they were prepared for mass fragmentography The materials analyzed were 0.5 ml of plasma, cerebrospinal fluid (CSF), the clear supernatant obtained from the brain homogenates after centrifugation, or 0.2 ml of urine. These materials were diluted to a volume of 1 ml with water in 15 ml round bottom glass centrifuge tubes. To each sample, 5 or l o n g of deuterated isomers of PEA ( a d , , P-d2), phenylethanolamine (a-d,, P-d,) and p-tyramine (a-d,, P d 2 ) were added and mixed. A modification of the WILLNERet ul. (1974) procedure was employed for the extraction of PEA and other amines derivatives. Five milliliters of ethyl acetate were added to each tube followed by the addition of 1.5 ml phosphate buffer (prepared by mixing 3 parts 0.5 M - N ~ , P O , and 1 part 0.5 M-Na,HPO,), and the content was vortex-mixed immediately for 30 s, centrifuged for 5 min at 900 y. and 4 ml of the ethyl acetate

.

transferred. The final pH of the extraction mixture was between 1 I and 13. The extraction was repeated once more with another 5 ml of ethyl acetate and 5 ml of the organic phase transferred and combined with that of the first extract. The combined ethyl acetate extracts were evaporated in uacuo; the dried residues were dissolved in 0.3 ml 1 N-HCI, mixed with 5 ml ethyl acetate for 30 s, and centrifuged. The organic phase was then aspirated off. Twotenths of a milliliter of the aqueous phase was transferred into 1 ml microflex tubes (Kontes Glass Company. Vineland, NJ) dried under a gentle stream of N,, and the amines converted to their pentafluoropropionate derivaet a/., 1975). After evaporating the acylating tives (KAROUM agent under N 2 , the derivatives were reconstituted in 15pl of ethylacetate. and 1 or 2 PI of this solution were injected into the gas chromatographic column. T o correct for the contribution of non-deuterated amines arising from the deuterated amine isomers (the deuterated amines used were between 97% and 98% enriched), we included in each batch of analyses a tube containing 1 ml of water plus 5 ng of the deuterated amines. This sample was carried through the entire procedure. To ascertain that the glass tubes used were also free of adsorped PEA, two blank tubes containing 1 ml of water were included. Triplicates of pooled biological materials prepared as described above were also included in each batch of analyses. T o two of these triplicates, 2 and 4 n g of the non-deutcrated amines were added, respectively. These pooled samples were used to construct a standard curve to quantify thc amines in the biological samples. Puru-octopamine and para-tyramine in brain were analyzed by a direct method, since the extraction procedure described above was not suitable for the assay of the low concentrations of these amines in rat brain tissues. In this method, 1OOpl of the clear supernatant obtained from the brain homogenate were introduced into 1 ml microflex tubes, followed by 5 ng of deuterated p-tyramine and octopamine. The mixture was evaporated under a gentle stream of N, and the amines in the dried residue converted to the pentafluoropropionate derivatives. The non-deuterated amines were quantified by directly comparing their peak heights with the appropriate deuterated standards. For the quantification of PEA, phenylethanolamine, and p-tyramine in the Cadbury chocolate, duplicates of 1, 2, 3, 4, and 5 g of the chocolate were melted at 50°C in 5 ml 0.1 N-HCI, and 50 ng deuterated PEA, phenylethanolamine, and p-tyramine were added. The suspensions thus obtained were centrifuged at 800 y for 5 min, the fatty and solid layers were aspirated off, and 1 ml aliquots of the aqueous phases were transferred and centrifuged at 12,000g for 10 min. O n e hundred microliters of the supernatant were analyzed in duplicate for PEA, m- and p-tyramine. The mean concentrations of these three amines as calculated from the various analyses were selected to reflect their concentrations in the chocolate used. Massfrogmentography ( M F ) . M F was carried out on a Model 3000D Finnigan gas chromatograph quadrupole mass spectrometer. Separation of the amines was achieved on a 12 ft lj8 in. 0.d. stainless steel column packed with 2% SE54 + 1% OVzl0 coated a mixture of 0.5% OV,, on 80/100 mesh W (HP) support (Pierce Chemical Company, Rockford, IL) (see Fig. 1). The oven temperature was maintained isothermally at 174°C. The fragments selected for M F are summarized in Table I . The flow of the helium carrier gas was maintained at approx 35 ml/min.

+

203

Noncatecholic biogenic amines Glassware. Because PEA adsorbs t o heated glass, special care was taken to ensure that all glassware was free from adsorbed PEA. To achieve this, we rinsed all tubes with 0.1 N-HCI and then sonicated them in an ‘RBS’ cleaning solution (Pierce Chemical Company, Rockford, IL) for 30min. The tubes were then dried at 50°C after several rinses with distilled water. In our experience, washing with dichromate sulfuric mixture was not helpful, and often caused the appearance of spurious peaks. Quunrificurion. All peak heights were corrected for equal recoveries from those of the appropriate deuterated et al., 1975). The isomers as previously described (KAROUM peak heights of the non-deuterated amines measured in the blank sample (sample which contains only deuterated amines) were then subtracted from those of the appropriate test samples. The amount of the amines present in the biological materials was measured by comparing their peak heights with those of the appropriate non-deuterated internal standards as determined from their standard curves. For the assay of wtyramine in urine the deuterated p-tyramine was used as the reference standard.

RESULTS

The molecular ions, relative retention times, and recommend fragments for measuring PEA and several amine derivatives are summarized in Table 1. Included in this table is information on pargyline and one of its metabolites, benzylamine. As can be seen in Table 1 the gas chromatographic column employed TABLE1. MASSTO

CHARGE RATIO

(mle) OF

Amine Phenylethylamine (PEA) Phenylethanolamine (PEOA) N-methyl-PEA N-methyl-PEOA p-met hoxy-PEA a-methyl-PEOA jnorephedrine) N-methyl-a-methyl-PEOA (ephedrine) o-Tyramine (o-TY) m-Tyramine (m-TY) p-Tyramine (p-TY) N-methyl-p-TY p-hydroxy-PEOA (p-octopamine) m-hydroxy-PEOA (norphenylephrine or m-octopamine) N-methyloctopamine (synephrine) N-methyl-m-hydroxy-PEOA (phenylephrine) a-Methyloctopamine a-Methylsynephrine Amphetamine Pargylinet Benzylamine

is highly selective and is capable of separating the m- and p-isomers of tyramine and octopamine as well as the nor- and N-methylated derivatives of several amines. Figure 1 illustrates the separation of 6 closely related amines. Figures 2 and 3 illustrate the multiple ion detection of PEA in plasma and the hypothalamus, and p-tyramine in the urine. The spectra of a number of structurally related amines are shown in Figs. 4-10. Plasma concentrations of PEA and p-tyramine after overnight fasting and 1 h after eating, as well as CSF concentrations of these two amines, are summarized in Table 2. Neither fasting nor eating significantly altered plasma PEA or p-tyramine. The CSF concentrations of PEA and p-tyramine appear t o be comparable to plasma, but both these media show wide variability between subjects. Table 3 shows the normal urinary excretion of PEA, m- and p-tyramine in adult volunteers, and after consuming 200g of Cadbury milk chocolate which contained a total amount of 1.15mg PEA, 0.12mg phenylethanolamine and 10 mg p-tyramine. Normal urine excretions for the three amines from 9 p.m. to 9 a.m. on one day, and 9 a.m. to 9 p.m. the following were not statistically different from each other, thus suggesting that the urinary excretion of these three amines are not diurnally regulated. However, other collection intervals must be examined before we can

FRAGMENTS OF PHENYLETHYLAMINE DERIVATIVES SELECTED FOR t o N DETECTION

Molecular ion of derivatized compound Mt

m/e of Fragment (1st/2nd choice)

267 429 28 1 443 297 443

104191 2531266 1041190 2791253 1211297 1901266

Mt-[NH2COC,F;] Mt-[CH2NHCOC2F;] Mt-[NHCH,COCZFJ M -[M -HOCOC,F;] Mi-[CH2NHCOC2F;] CHCH3NHCOC2F:

1.o 1.14 1.18 1.27 2.22 1.08

457

2041150

CHCH3NCH,COC,F:

1.25

429 429 429 443 59 1

266 266 266 2661190 4281415

Mt-[NH2COC2F;] M t-[NH2COC2F;] Mt-[NH3COC2F;] M t-[CH3NHCOC2F;] Mt-[NHZCOC2F;]

1.29 1.81 2.06 2.36 2.32

59 1

4281415

Mt-[NH2COC2F;]

1.98

605

42814 I 5

M t-[CH3NHCOC2F;]

2.62

605

1901267

CHCH3NHCOC2F:

2.10

605 619 28 1 159 243

190 2041150

CHCH,NHCOC,F: CHCH,NCH,COC,F: M t-[NH2COCzF,]

2.03 2.15 0.98 0.67 0.74

118/190

Structure of 1st choice fragment

-

2531134

-

Mt

Relative* retention time

* PEA relative retention time is arbitrarily set to 1. Other retention times are relative t o PEA. The retention time of PEA is approx 2.5min. t Pargyline does not form any derivative with pentafluoropropionic anhydride.

F. KAROUMc't

204

I

100

23

a1

of specific individuals were 0.996, 0.886 and 0.967 for PEA, m- and p-tyramine, respectively. The slightly lower correlation for rn-tyramine is probably due to the absence of an internal deuterated standard (deuterated p-tyramine was used to correct for m-tyramine recoveries).

DISCUSSION

1.0 2.0 3.04.0 5.06.0 MINUTE Fiti. 1. Total ion chromatogram of the pentafluoropropionate (PFP) derivative of (1) phenylethylamine (PEA), (2) phenylethanolamine, (3)o-tyramine, (4) m-tyramine, (5) p-tyramine,( 6 )hydroxyphenylethanolamine (p-octopamine). Vertical scale represents total ion intensity as a percentage of that produced by PEA.

state this conclusively. The 24h output of these amines also was not significantly changed after chocolate consumption. Brain concentrations of PEA and p-tyramine in both the whole brain and brain parts are summarized in Table 4. The highest concentrations were observed in the caudate and hypothalamus. The recoveries of PEA, phenylethanolamine, mand p-tyramine from plasma, CSF and urine by the extraction procedure described ranged between 70% and 90%. Similar recoveries were also observed when 0.5 ml of the clear supernatant obtained from brain tissue homogenates was used. Other amines listed in Table 1 were found to be extractable into ethyl acetate with recoveries rangng between 40 and 70%. Recoveries were estimated by comparing the responses to 5 ng of the amine when derivatized directly and after extracting it from plasma or urine. The amine always was added to one of duplicate samples and the same volume of the derivatized amine in ethyl acetate was always injected. The difference in responses between the duplicate biological sample divided by that of the directly derivatized arnine gives an approximate evaluation of the amine extractability into ethyl acetate. In order to determine the day to day reliability of the assay, urines from 12 individuals were divided into two duplicate batches. The 12 samples in each batch were coded separately so that knowledge of the results of the first day's analysis would not effect the results of the second day. The intraclass correlations which are sensitive to systematic day to day shifts in the assay as well as to changes in the relationship

The selection of the gas chromatographic column for mass fragmentography was made after surveying over ten different stationary phases. The phases were tested when coated on solid support alone or in combination with one or two other phases. The following criteria were used for the selection: an arrangement that separates most of the amines listed in Table I ; an arrangement that produces symmetrical and welldefined peaks of the biogenic amines of interest; and finally, an arrangement that produces peaks from the biological materials that are the same as thc peaks produced by authentic compounds as judged from multiple ion detection (MID). Multiple ion detection is a technique whereby the mass spectrometer is focused simultaneously on two or more fragments of the arnine in question, and the intensity ratios of the fragments against one another determined and compared with those of the authentic compound. The closeness in the ratios between the biological material and the authentic standard offer two important pieces of information. First, it confirms the identity and purity of the peak corresponding to the biological compound, and second, it establishes the specificity of the fragments for mass fragmentography. The 12 ft 0.5% OV,, 2% SE54 1% OV,,, column satisfied all of the above criteria. The selectivity of this column is illustrated in Table 1 and Fig. 1. Besides being very selective in its chromatographic properties, the column offers a relatively brief analysis time of approx 8 min. The purity of the peaks corresponding to plasma and hypothalamus PEA is illustrated in Fig. 2a and 2b. The ratios of the two fragments selected for MID in both the biological materials and authentic PEA are very similar, thus signifying the authenticity of PEA measured. Similar diagnostic studies by MID were carried out to ascertain the purity of PEA, ni- and p-tyramine in plasma and other biological media analyzed (see Fig. 3). The spectra shown in Fig. 4-10 illustrate some general fragmentation patterns of PEA derivatives. As can be seen from Table 1, several of these amines share common fragments, thus making it mandatory that they are separated from each other by the gas chromatograph. N-Methylated and a-methylated amines always had a fragment with an atomic mass unit (AMU) of 190, corresponding to CH2NCH3COC2F, and CHCH,NHCOC2F5, respectively. These fragments are not specific and should only be used when fragments with higher AMU values cannot be employed because of their

+

+

205

Noncatecholic biogenic amines

MID FOR PEA IN PLASMA 2 ng PEA STANDARD

PLASMA

m / e 91

1 I

I

I'

Injection

I I

0

1

2

3

4 0 Minuter

1

1

2

3

4

5

MID FOR PEA I N RAT HYPOTHALAMUS RAT HYPOTHALAMUS

m/e 104 m / a 91 I

m/r 91

1.19

0

1

2 ng PEA STANDARD

2

3

4

5 Minutes

0

1

2

3

4

5

FIG. 2a,b. The multiple ion chromatograms (MID) of the P F P derivatives of PEA in plasma and hypothalamus and of authentic PEA.

MID FOR TYRAMINE IN URINE

m h 253 ' 3'8

F. K A R ~ UetM a / .

206

PHENYLETHYLAMINE I00 ) 200 x 2 0 1

low intensities. They can be employed, however, when the concentrations of the amines in the media to be analyzed are high. Another fragment which should be avoided whenever possible is that corresponding to 91AMU. This fragment corresponds to a tropylium ion which could arise from several benzylic compounds. This precaution is particularly relevant to the analysis of PEA in urine. In the CSF and brain, results obtained by focusing on either mle 104 or 91 for PEA were found to be comparable. We attribute the similarity of results to the high selectivity of the gas chromatographic

39

,

PARA, METHOXY-PHENYLETHYLAMINE !OO ) 170 x 20 45 I

It

I00

ri I

N-METHY L,PHENYLETHANOLAMI NE

I2l

150

200

2m

300

FIG.4. The partial spectra of the PFP derivatives of PEA and p-methoxy PEA. Note the two prominent fragments with rnjr values of 91 (base peak) and 104 in PEA, corresponding, respectively, to a tropolium ion and the product of an 2, /Icarbon cleavage of the molecule (this cleavage is associated with carbon hydrogen transfer). Similar fragmentation in p-methoxy PEA produces ions 121 and 134. The intensities of ions with atomic mass units (AMU) over 200 (for PEA) and 170 (for p-rnethoxy PEA) are multiplied by 20. Left hand vertical scale represents the percentage of the ion intensity against the base peak. The horizontal scale represents the AMU of the ion.

N-METHYLTYRAMINE ) 270 x 20

157 200

250

300

153

rgo

40

i

I

250

200

FIG.5. The partial mass spectra of the PFP derivative of N-methyl, phenylethanolamine. Notice ions 190 (CH2NCH3COC2F,) (characteristic of a-methylated and N-methylated amines) and 279 (produced by the loss of the fi-hydroxyl radical of the molecule). The production of ion 279 is associated with hydrogen atom transfer from adjacent carbon atoms. See Fig. 4 for interpretation of vertical and horizontal scales.

P-TYRAMI NE

125 I50

200

250

PHENYLETHANOLAMIN E

I00

11

I00

150

200

250

FIG.6. The partial mass spectra of the PFP derivative of N-methyl tyramine, p-tyramine and phenylethanolamine. Notice the presence of ions 266 in all three amines. The intensities of ions over 270 AMU in N-methyl tyramine are multiplied by 20. See Fig. 4 for interpretation of vertical and horizontal scales.

Noncatecholic biogenic amines

100

OCTOPAMI NE

200

250

100

300

350

NORPHENYLEPHRINE

250

3 k

300 )

PHENYLEPHRINE

’9

ALPHA- METHYL-OCTOPAY IN€

7 400

N-M ETHYL-OCTOPAMINE

100

200 x 20

400

i- t2,

260

)

207

55

30011 20

FIG.7. The partial mass spectra of the PFP derivative of p-hydroxyphenylethanolamine (octopamine), m-hydroxyphenylethanolamine (norphenylephrine) and N-methyl octopamine. Notice the presence of ions 267, 415 and 428 in all three spectra. See Fig. 4 for interpretation of vertical and horizontal scales. The intensities of ions with A M U wlues over 300 are multiplied by 20.

126

iso

200

2%

FIG. 9. The partial mass spectra of the PFP derivatives of N-methyl, rn-hydroxyphenylethanolamine (phenylephrine) [this spectrum is almost identical to that corresponding to N-methyl, p-hydroxyphenylethanolamine (N-methylsynephrine), and p-hydroxyphenylethanolamine (r-methyloctopamine)]. Notice the presence of fragment 190 in both spectra. See Fig. 4 for interpretation of vertical and horizontal scales.

N-METHY L-PHENYLETHYLAMINE I00 ) 200 I 20 25 ALPHA- METHYL-SYNEPHRI NE > 2 2 0 x 20

p“

80 100

150

200

250

NOREPHEDRINE

L

)

220

1-190

I204

)I

lo2() r

too

FIG. 8. The partial mass spectra of the PFP derivatives of N-methyl PEA and a-methyl PEA (norephedrine). Notice the dissimilarities of the two spectra. The only common ion in both spectra has an m/e value of 190. The intensities of ions over 200 A M U are multiplied by 20 and 10 for N-methyl P E A and a-methyl PEA, respectively. See Fig. 4 for interpretation of vertical and horizontal scales.

150 200 EPHEDRINE ) 2

250

0 x 10

is-A

121 150

200

250

300

FIG. 10. The partial mass spectra of the PFP derivatives of a-methyl, N-methyl p-hydroxyphenylethanolamine (a-methylsynephrine) and cx-methyl, N-methyl PEA (eph204 corresponding to edrine). Notice ions CH3CHNCH3COCzFS.

F. KAROUM et a/.

208

TABLE 2, CtKLBRoSPINAL FLUID (CSF) A N D PLASMA CONCtNTRATIONS* OF PEA p-TYRAMINL Description (n)

PEA (range)

+

Lumbar CSF (15) Plasma after fasting overnight (15) Plasma 1 h after eatingt (15)

AND

p-Tyramine (range)

0.6 0.1 (0-1.5) 0.64 f 0.05 (0.5-1.1)

0.79 f 0.25 (0-3.5) 0.68 k 0.09 (0.41.9)

0.57 k 0.13 (0.1-1.8)

0.80 f 0.12 (0.5-1.7)

* The results are expressed means f S.E.M.in ng of amine/ml

of C S F or plasma.

t The meal consisted of one egg, a slice of Canadian ham (approx 0.2 kg), a slice of bread, coffee, milk, and pineapple. tTest comparisons of plasma amines while fasting and 1 h after eating were not statistically significant. LXCRETION TAHLF3. URINAKY

(MEAN

Description (n) 12 h Excretion* ( I 5 )

12 h Excretion* (15)

s.E.M.) OF P H E N Y L E T H Y L A M I N (PEA), ~ (pg/TIME PERIOD)

PEA PEA (range)

Time 9 p.m.-9 a.m. 9 a.m.-9 p.m.

24 h Excretion (15) 24 h Excretion after 200 g of chocolate? (5)

3.4 2 0.4 (1.5-7) 3.7 f 0.4 (0.5-6) 7.1 f 0.5 (5.7-1 1.2) 6.6 f 0.9 (4.5- 8.9)

m- A N D p-TYRAMINE BY

A D U L T VOLUNTEERS

Unconjugated m-Tyramine (range)

Unconjugated p-Tyramine (range) 292 k 60

38.9 f 5.9 ( 1 5-60) 47.6 f 7.1 (23-104) 86.5 8.4 (40-123) 37.6 f 15.7$ ( I 0- 64.9)

(1 12-960)

308 f 41 (52-670) 608 k 90 (209-1 564) 268 i: 99$ (86656)

* The differences between the two 12 h excretions of the amines were not statistically significant (t test). t The total amounts of PEA, phenylethanolamine and p-tyramine in the 200g of Cadbury milk chocolate consumed were, respectively, I. I5 mg. 0. I 2 mg and 10 mg. $ The amines excretions after chocolate were not significantly different from the base line levels of the same subjects (see text). TABLE 4. RAT BRAIN

(MEAN

f S.E.M.)

REGIONAL DISTRIBUTION OF PHENYLETHYLAMINE AND

PEA Description (n) Whole brain (6) Cortex (6) Medulla (6) Midbrain (6) Cerebellum (6) Hippocampus (6) Caudate (6) Hypothalamus (6)

P-TYRAMINt

p-Tyramine

ng/g

pmol/g

ng/g

Pmok

8.1 1.3 4.5 & 1.9 4.1 1.7 2.9 f 1.1 6.8 f 1.3 7.7 f 2.3 30.7 2.7 54.5 f 7

66.9 f 10.7 37.2 f 15.7 33.8 k 14.0 23.9 & 9.1 56.2 & 10.8 63.6 f 1.9 253.7 22.3 450.4 f 57.8

4.2 k 0.64 1.8 f 0.6 1.1 k 0.3 0.75 & 0.38 1.0 & 0.1 4.0 f 1.0 13.6 f 5.8 13.9 f 3.0

30.6 k 4.7 13.14 k 4.4 8.0 & 2.2 5.47 f 2.7 7.3 f 0.7 29.2 k 7.3 99.3 k 42.3 101.5 f 21.9

column employed. Fragment m/e 91 is currently being used to study the effects of two types of M A 0 inhibitors (clorgyline and pargyline) on PEA concentration in the rat brain. Fragment m/e 104, although desirable, could not be used in rat brain because the above M A 0 inhibitors or their metabolites possess a fragment with 104AMU in their spectra. Although these peaks have close but different retention times from PEA, their high concentrations elevate the base line of the recorder pen, thus distorting the PEA peak when the mass spectrometer is focused on m/e 104. Fortunately, when m/e 91 is used for mass fragmentography of brain PEA of rats given M A 0 inhibitors,

the base line elevation of the recorder is considerably less serious than when m/e 104 is used, thus allowing the easy quantification of PEA. It is interesting to note that the second major fragment in the spectra of N-methylphenylethanolamine (Fig. 5 ) corresponds to m/e 279. This fragment probably originates from the loss of an acylated 8-hydroxyl radical of the side chain (M HOCOC2F5) (see Table 1). This cleavage is associated with a transfer of a hydrogen atom, possibly from either the GL or 8 carbon. The method as described is ideally suited for the analysis of moderate numbers of samples. Twenty to

+

Noncatecholic biogenic amines

209

thirty samples can conveniently be processed and cal- trace amines including p-octopamine, originate from the intestine, because sterilization of the gut comculated within 2 days. Of the various amines that could be derived from pletely reduces these amines’ excretion. DEQUATTRO (1967), on the other hand, reported the the hydroxylation of PEA (BOULTONer al.. 1974u.h) & SJOERDSMA only m- and p-tyramine were detected in measurable opposite finding and therefore suggested that p-tyramounts in urine. These two isomers of tyramine were amine excreted in the urine is endogenously propreviously detected and quantified in human (JEPSON duced. In a study in which neomycin was given to et a/., 1960; PERRY & SCHROEDER,1963; COWARD et Parkinsonian patients on L-DOPA, no change in the al., 1964; KING et a/., 1974; SLINGSBY& BOULTON, excretion of p-hydroxyphenylacetic acid (KAROUM. 1976) and rat (BOULTONet a/., 1 9 7 4 ~ )urines. They 1970), the major deaminated metabolite of p-tyrhave also been found in various animal tissues includ- amine, was observed, thus indirectly excluding the gut ing the brain (PHILIPS et a/., 1974; BOULTONet a/., as the source of urinary p-tyramine. The most logical 1974a, b ; BOULTON, 1976a, b). We are unable to detect source of p-tyramine must then be decarboxylation o-tyramine or phenylethanolamine in urine, plasma, of tyrosine and hydroxylation of PEA. Urine ni-tyror CSF at the sensitivity limit of the method. Thus amine, in contrast to p-tyramine, could easily be detheir excretion corresponds to less than 0.1 pg/24 h rived from certain food containing L-DOPA through in urine, and their concentrations in plasma and CSF dehydroxylation, as has been well established from are less than 50pg/ml. Excretions of o-tyramine in the analysis of urines of Parkinsonian patients on urines of man and rats were found by KING et al. L-DOPA therapy (SANDLER et a/., 1969, 1971). In this (1974) to be less than 1 psi24 h. The excretion of m- connection it is worth mentioning that ingestion of and p-tyramine vary considerably between subjects, L-DOPA did not increase urine excretion of p-tyrahence the different means in the excretions in Table 3. mine (BOULTON et al., 1972) or p-hydroxyphenylacetic The excretion rate of m- and p-tyramine in man acid (CALNE et a/., 1969). is comparable to those reported in the literature (JEPNo rn-tyramine could be detected in the CSF at SON er al., 1960; PERRY & SCHROEDER, 1963; COWARD the sensitivity of the method; thus, the concentration er al., 1964; VOGELet al., 1967; KING et al., 1974; is less than 50pg/ml. As far as we are aware, plasma & BOULTON,1976). SLINGSBY and CSF concentrations of PEA. ni- and p-tyramine As shown in Table 2, the concentrations of the have not been reported before. We are of the opinion. three amines measured did not significantly change however, that these two media are not ideal for 1 h after eating. Whether the amine excretion would measuring abnormal PEA production or metabolism, change if other types of food were consumed or if because of the wide ranges we observed between northe subjects were studied for longer periods of time mals. Thus, unless PEA production is very abnormal has yet to be determined. Furthermore, as shown in (e.g. as in phenylketonuria) one may not be able to Table 3, consumption of 200g of chocolate did not observe moderate deviations from the norm. Urine significantly increase urinary PEA excretion. Excre- analysis, on the other hand, may prove to be more tions of m- and p-tyramine were not significantly useful than either plasma or CSF. In this connection changed. From these two observations, it appears that it is worth mentioning that the wide variations in the dietary contribution of PEA, tn- and p-tyramine plasma or CSF PEA concentrations are unlikely to is minimal, and therefore suggest that the observed be attributed to the method or to decomposition on concentrations of these amines reflect endogenous storage. Variation in the concentration on repeated sources. It is, however, also possible that these amines analysis of the same sample of plasma (in some cases are rapidly metabolized within the body, thus making 3 months apart) was found to be within lo”