Differential estimation of adrenaline, noradrenaline, dopamine, metanephrine and normetanephrine in urine

CLINICA CHIMICA ACTA

DIFFERENTIAL DOPAMINE,

G. L. MATTOK, Psychiatric

(Received

99

ESTIMATION METANEPHRINE

D. L. WILSON

AND

OF AND

ADRENALINE,

NORADRENALINE,

NORMETANEPHRINE

IN URINE*

R. A. HEACOCK**

Research Unit, University Hospital, Saskatoon, Saskatchewan (Canada) September

r6th, 1965)

SUMMARY

A method is described for the determination of the individual levels of adrenaline, noradrenaline, dopamine, metanephrine and normetanephrine in urine. The amines present in the urine are adsorbed on a weak cation exchange resin (Amberlite IRC-50) and the catecholamines then eluted by a boric acid solution tollowed by metanephrine and normetanephrine with dilute sulfuric acid. The amines in each fraction are oxidised by iodine to the corresponding aminochromes which are then converted to their sodium bisulfite addition compounds. The levels of each of the five amines are calculated from measurements of the absorbances, at several wavelengths, of the solutions of the bisulfite addition compounds. The average excretion rates of the free amines in urine, based on 41 determinations using this method are (in,ug/h) : A***, 0.04; N. 2.36: D, 8.~2; M, 1.25; NM, 8.55. Thecorrespondingexcretion rates (in ,ug/h) for the total amines (i.e. free plus conjugated) 27.20; M, 3.65; N.M. 17.48.

are: A, 0.06; N, 4.69; D,

INTRODUCTION

A voluminous literature exists on the determination of adrenaline (A) and noradrenaline (N) by chemical means (cJ review articles1-4) and in recent years a number of papers have appeared which have described either the determination of urinary dopamine (D)6-g, or urinary metanephrine (M) and normetanephrine (NM)10-12. However, as far as the authors are aware there is only one reference in the literature which describes an assay for the determination of A, N, D, M and NM levels in the same urine sample13. In this procedure the catecholamines were extracted from the urine and separated from their 03-methyl derivatives by adsorption on alumina; M * This investigation was supported by grants from the Department of Public Health (Saskatchewan) and the Department of National Health and Welfare (Ottawa). ** Present address: The Atlantic Regional Laboratory, The National Research Council, 141 I Oxford Street, Halifax, Nova Scotia, Canada. *** The following abbreviations will be used in this paper: A = adrenaline; N = noradrenaline; D = dopamine; M = metanephrine and NM = normetanephrine. Clin. Chim. Acta, 14 (1966)

99-107

100

G.

L.

MATTOK

et.

d.

and NM were subsequently extracted by an ion-exchange procedure. The total catecholamine content i.e. (A + N + D) was determined by the “ethylenediamine” procedure and the A and N levels estimated by the “lutin” method; the D level was obtained by difference r3. The M and NM were determined by a modified “lutin” procedurer4. Oesterling and Tser5-‘” tion of the total catecholamine

have recently described a method for the determinacontent of urine based on the adsorption in the ultra-

violet region of the aminochrome-sodium bisulfite addition compounds derived from these catecholamines. The aminochrome-sodium bisulfite addition products are pale-yellow crystalline solids18*20 which are quite stable in the solid state and in dilute solution (see Ref. z for a review of the chemical properties of these compounds). Solutions of these addition products, which are stable for several days at room temperature, exhibit a yellow fluorescence, but it is of a relatively low intensity. Difficulties are often encountered when using the standard fluorometric catecholamine assay procedures; these are due either to the inherent instability of the system in the case of the “lutin” method or to certain doubts about the specificity of the “ethylenediamine” reaction. It was decided, therefore, to attempt to modify Oesterling and Tse’s procedure to enable a differential estimation of the A, N and D content of urine to be made. Mattok and Wilson21 have recently described a simple procedure for the separation of the urinary catecholamines from their OS-methyl derivatives, based on the observation that boiic acid will elute A, N and D, but not M and NM, from a weak cation exchange resin. It has also been reported10,11~1~,22~2” that under certain conditions M and NM can be oxidised to substances, presumably aminochromes, which can be rearranged to substances with a “lutin”-type fluorescence. It should, therefore, be possible to obtain aminochrome-sodium bisulfite addition products from M and NM and to apply Oesterling and Tse’s basic procedure to the determination of these amines. The present paper describes a simple method for the determination of A, N, D, M and NM in the same urine sample. The analysis is based on: (a) the separation of ,4, N and D from M and NM by the ion-exchange chromatographic procedure described by Mattok and 1Vilson’l and (b) the ultraviolet absorption of the aminochrome-sodium bisulfite addition products15 lli at several wavelengths. Although attempts to include the determination of 3-methoxytyramine in the assay procedure have, so far, not been completely successful, it has been possible to assess its possible contribution to M and NM levels. METHOD

I. Preparation of the Amber&e* column. Amberlite IRC-50 (3 g) is allowed to swell in water for 15 min and then transferred as a slurry to the column (50 ml buret ; i.d. I cm). The column is then washed with the following reagents in turn, using a drop rate of about 1.5 ml/min and adding each reagent when the preceding one has just passed completely into the resin : I N H,SO, (35 ml) ; water (25 ml) ; I A: NaOH (35 ml) ; water (25 ml) ; 0.4 IM phosphate buffer (pH 6.5, 25 ml). The column was left to stand for I h at some stage during the addition of the alkali to allow maximum swelling of the resin to occur. II. Preparation of wines. First morning samples of urine were used in the *

The

ion-exchange

resin was obtained

from

the Mallinckrodt

Chemical

Works

I,td.

ESTIMATION

development

OF URINARY

101

AMINES

of the method;

these

were collected

during the ro-h period

between

ro p.m. and 8 a.m. Estimations of the levels of free amines are obtained withfiltered urines. Estimations of the total (free and conjugated) amine levels are carriedout on urines which have been hydrolysed by dilute mineral acid. The optimum conditions for the hydrolysis of the conjugated amines were found to be: (i) adjustment of the urine to pH I with 3 N HCl; (ii) immersion of the flask containing the acidified urine in a bath of boiling water for 30 min and (iii) cooling in an ice bath. III. Extraction procedure. A sample (50 ml) of the urine, either before or after hydrolysis (adjusted to pH 5.5), is diluted with water (150 ml) and then added to the column, using a drop rate of 1.5 ml/min. When all the urine solution has passed onto the resin, the column is washed with water (25 ml). Boric acid solution (4%, 30 ml) is then added to the column; the first 3 ml of eluate is discarded and the next 25 ml (which contains the catecholamines) is collected. The remaining boric acid solution is allowed to pass onto the resin, the effluent was discarded and the resin was then washed with water (25 ml). M and h’M are then eluted with 2 N sulfuric acid (IO ml) followed by 0.01 N sulfuric acid (20 ml) ; the first 3 ml of effluent were discarded and the next 25 ml collected. IV. Formation of aminochtrome-sodium bisulfite addition cornpow&. The boric acid eluate is adjusted to pH 5.0 with 3 N HCl. Two samples (4 ml) of this solution contained in test tubes are treated with a solution of iodine in potassium iodide (0.02 N I, in 0.02 M KI, 0.5 ml) and the tubes then placed in a water bath at 30” for 5 and 15 min respectively. At the end of these times, the oxidations are stopped by the addition of alkaline sodium thiosulfate solution (0.05 N in 0.002 M sodium carbonate, 0.9 ml). A small quantity of solid sodium bisulfite (5 mg or less) is then added to each of the reaction mixtures. The sulfuric acid eluate is adjusted to pH 6.5 with solid potassium carbonate and the amines present oxidised for 5 min by the above procedure. The excess iodine is then removed by the addition of the sodium thiosulfate reagent (0.9 ml) and the bisulfite addition compounds formed by the addition of solid sodium bisulfite V. Absorbance measurements. Absorbances of the three solutions of the compounds obtained by the procedure described in section IV (which will be to as “final solutions”) are measured 5 min after the addition of the sodium

(5 mg). bisulfite referred bisulfite

on a Beckman DK-2 recording spectrophotometer. Measurements are made at 373 and 390 m,u; additional readings for the dopamine determination are taken at 355 rnp with the two final solutions obtained from the boric acid eluate. The reference solutions for these measurements are prepared by addition of water (1.4 ml) to samples

(4 ml) of each eluate.

The absorbance

at 450 rnp is subtracted

from the

absorbances at 355, 373 and 390 m,u to give the true absorbances, at these wavelengths, due to the bisulfite addition compounds. VI. Calibrations. The above extraction and appropriate oxidation procedures are applied to a series of solutions of any one of the amines (up to 2 rug/ml) in urine. The calibration curve is obtained by plotting the absorbances (due to added amine) at 355, 373, 390 m,u against the concentrations of added amine. The slopes of the straight lines give the coefficients* for the amine at each wavelength. This procedure is repeated for each amine using 5 and 15 min oxidation periods. A typical calibration is shown in Fig. I. * Absorbance/ml of final solution/pg of amine added

to urine.

Clin. Chim.

Acta,

14

(1966)99-10,

102

G. L. ?vIATTOK et al.

0.14 15 min

0 /

0.12

-

0.10

-

*.** J’ f

DOPAMINE Fig. I. Dopamine

(pg/ml

final solution)

calibrations

at 355 rnp using 5 and I5-min oxidation

periods.

VII. Calculations. The method separates the five amines into two fractions i.e., the catecholamines and a combined metanephrine and normetanephrine fraction. (I) Catecholamines. (a) Doeamine (0). The difference in absorbances at 355 rnp of solutions of the aminochrome-bisulfite addition compounds, obtained from the boric acid eluate after 5 and 15 min oxidation, is a direct measure of the D level (see DISCUSSION). The concentration of D is obtained by dividing this difference in absorbance by the difference in the slopes of the two dopamine calibration curves at 355 rnp (see Table I). (b) Adrenaline (A) and Noradrenaline (N). The contribution of D to the total absorbances of the catecholamine fraction at a given wavelength is equal to the product of the concentration of D and its coefficient at that wavelength. The absorbances, at 373 and 390 m,u, due to A and N, are obtained by subtraction of the contribution of D to the absorbances of the reaction mixture (15 min oxidation) at these wavelengths. Let A and

hr = concentrations

I,,, and I,,, a, and n, a, and n,

= coefficients*

Then

solution

&/ml) of A and N resp. due to (A + N) at 373 and 3go rnp resp. = calculated absorbances = coefficients* for A and N resp. at 373 m,u. for A and N resp. at 3go mp.

I,,,

= a,A + n,N

(I)

I 390

= a,A + n,N

(4

of equations A

1

N

=

and

(I) and (2) for A and N gives:

nzIa73 w2

n1IS9O

-

wl

a&,,

-

a,I,,,

w2

-

wh

* Absorbance/ml

of final solution/pg

Chim.

14 (1966) 99-107

Clin.

Acta,

(3)

(4) of amine added to urine.

ESTIMATION

OF URINARY

Substitution

103

AMINES

of the appropriate

coefficients

(see Table

I) in equations

(3) and (4)

gives A

= 0.803 I,,,

-

0.412

I,,,

N

=

-

0.865

I,,,

0.687

I,,,

;z;

(2) MetanePhrine (M) and normetanephtrine (NM). The calculation of the levels of these amines is based on the absorbances at 373 and 390 m,u of the solution of the bisulfite addition compounds obtained from the sulfuric acid eluate. These values are substituted in formulae derived for M and NM which are similar to those obtained

for A and N. The final equations

are

M = 0.798 I,,,

-

0.428 I,,,

(7)

= 0.988 I,,,

-

1.171

(8)

NM

I,,,

M and NM are the concentrations

where

&g/ml) of M and NM respectively.

RESULTS

The coefficients

Calibrations. shown in Table TABLE

at the various

wavelengths

are

I

CALIBRATION Amine

COEFFICIENTS*

Coeficients*

Oxidation time (min)

A N D

373 mp

390 mlu

5 I5 5 I5 5 I5

3.72 3.72 4.90 4.90 2.85 4.58

4.28 4.28 4.03 4.03 2.29 3.75

3.40 3.40 2.04 2.04 1.36 2.OI

5 5

3.30 3.36

4.09 2.79

3.46 I.50

* Absorbance/ml TABLE

at

355 m/J

M NM

of final solution/pg

of amine added to urine.

II

ANALYSES

Amount

OF

MIXTURES

OF

AUTHENTIC

added (pg)

A

N

D

0.95 0.95

0.98 0.98

0.96

0.95 0.95 0.95

for each amine

I.

0.98 I.47

AMINES

Amount M

NM

ADDED

N

D

0.86 0.89

0.90 0.96

0.97

I.09

0.48

I.10

1.09

I.03

I.10

0.96

0.55

0.55 I.09

0.92 1.06

URINE

recovered

(iugl A

1.10

TO

1.07 1.72

9/, A

M

NM

I.22

0.40

0.98 0.89

0.88

108

0.98

0.93 0.45

0.55 1.01

97 106

90 94

N 92 98

IO9 II7

D

M

NM

84

89 81

III 81

IOI

85 83

gg 92

IOI

Recovery of amines added to urine. Mixtures of known amounts of the various amines were added to urine samples, which were then processed by the bisulfite method, as described above. The urines were assayed without the addition of authenClin. Chim.

Acta,

14 (1966) 99-107

G.

104 tic compounds, and the amounts amounts found in the recovery Table II.

L. ikIATTOK et. a/.

originally present were subtracted from the total experiments. Some typical results are shown in

Amine levels in abrine. Urines were obtained from miscellaneous psychiatric patients and staff at the University Hospital (Saskatoon). The average levels and ranges of the five amines in 41 different urine specimens are shown in Table III. TABLE

III

ANALYSES

OF

OVERNIGHT

Average* A N D M

NM

URINES.

AVERAGE

LEVELS

OF

AMINES

Total (pglh) MU.** Average*

iwax-.**-

0.06 4.69

0.q

1.q

8.22

IO.95 23.25

27.20

2.35 21.60 56.80

1.25 8.55

6.80 17.80

3.65 ‘7.48

13.30 58.00

2.36

* 41 Samples ** Minimum values were ca. 3

IO-~

in all cases except total D

(10.20)

and total NM (4.82)

3-Methoxytyramine. 3-Methoxytyramine was eluted from the column by the sulfuric acid, but the chromatographic procedure did not efficiently separate it from M and NM. The effects of pH and reaction time on the oxidation of j-methoxytyramine, M and NM by iodine were not sufficiently different to permit the direct determination of 3-methoxytyramine. The absorbances and wavelengths of maximum absorption of solutions of the bisulfite addition compounds obtained from 3-methoxytyramine and normetanephrine were very close. It is probable, therefore, that any 3-methoxytyramine present in a urine would be largely included in the value obtained for normetanephrine. DISCUSSION

A concentration

and partial

separation

of the biogenic

amines

in urine

is

obtained by adsorbing them on a weak cation exchange resin (Amberlite IRC-50) followed by elution of (i) A, N and D with 4% aqueous boric acid and (ii) M and NM with dilute sulfuric acid21. A sample of the urine (50 ml) is diluted with water (150 ml) and then added to the column. It was found that this dilution was necessary to prevent variations in the recoveries of the amines due to a breakdown in the resin capacity by salts present in the urine. Since the dilution step was incorporated into the assay procedure excellent reproducibility has been obtained and the recoveries of each of the amines are 80% or better. The separation of the amines into catecholamine and combined M and NM fractions is essential for a differential determination of the five individual amines. Further, this separation is also desirable even if only the catecholamine levels are required since M and NM, which are present in urine at higher levels than the catecholamines, are oxidised by iodine (and probably by some other reagents) to a small extent under the conditions used for the oxidation of the catecholamines in many assay procedures. A further advantage of eluting the cateClin. Chim. Acta,

14 (1966)

99-107

cholamines as their boric acid complexes is that these complexes are very stable and resistant to oxidatiun, but may be readily decomposed by acid to regenerate the catechalamine. The amines (e.g. A and M shown in Fig. 2) in each fraction are oxidiscd by iodine to the corresponding aminochromes (e.g. I), which are then converted to their sodium bisulfitc addition compounds (e.4. II). These bisulfite addition compounds exhibit maximum absorption in the ultraviolet region near to 360 rnp (cJ.~~$~~).The catecholamines, which are present as borate complexes in the boric acid cluate, are rcgenerated when this solution is adjusted to pH 5.0 and they arc then oxidised at this PH. Ikpamint is not as readily oxidised at pH 5.0 as the other catecholamines and this property is used as a basis for the direct determination of dopamint. Under the conditions used, maximum oxidation of adrenaline and noradrenaline by iodine is obtained after 5 min at 30”, whereas dopamine requires about 15 min at that temperature for maximum oxidation. Therefore, when two aliquots of a solution containing a misturc of the three catec~~~larnin~s are oxidiscd for 5 and 15 min respectively, any differcncc in absorbance of the final solutions at a given wavelength is a measure of the U present in the mixture. Maximum increase in absorbance occurs at 355 mp and this wavelength was, therefore, selected for the dopamine assay. The M and NM in the sulfuric acid eluate, are oxidised by iodine at pH 6.5 to the corresponding ami~lochromes. The oxidation was carried otlt at pN 6.5 since, in practice, it was easier to attain this pH and more reproducible results were obtained than at a pH of 7.2 used b\: liandrup2”. Oxidation of the Q-methyl derivatives is accompanied by demctthyl&on. The bisulfitc addition compounds are formed instantaneously.

M

Formation and metancphrinc Fig.

z.

of arninochromc-sodium (>I).

bisulfite

addition

compounds

from adrenaline

(~1)

Since dopamine is determined directly the calculation of the levels of the other amines is resolved into essentially the analysis of a pair of two component mixtures; this simplifies the calculations and increases the accuracy of the results. Calculations using the final equations (5-8) are conveniently presented on standardized forms. The stability of the aminochrome-bisulfite compounds, together with their characteristic fluorescence suggested that fluorometri~ assay procedures for A, N and I) might be developed. The excitation and fluorescence wavelengths for maximum Auorescencc, which have not previously been reported in the literature, for the Clin. ChiWz.Acta, “$

(1966)

‘,‘)--107

G. L. MATTOK et al.

106

aminochrome-sodium bisulfite addition compounds derived initially from the catecholamines are: A, (excitation, 370 mp; fluorescence, 520 m,u) ; N, (excitation, 385 m,u; fluorescence, 520 m,u) and D, (excitation, 390 rnpu; fluorescence, 520 mp). However, preliminary experiments indicated that the intensity of the fluorescence obtained from these compounds was relatively low and, furthermore, the fluorescence intensities of mixtures were not additive. This approach was, therefore, abandoned in favor of the procedure

described

above, based on ultraviolet

spectroscopic

measure-

ments. The urine samples used in the development of this assay procedure were overnight samples (IO p.m.-8 a.m.) obtained from miscellaneous psychiatric patients and staff members of the University Hospital in Saskatoon. The overnight samples were used because of the difficulties often encountered in collecting 24-h urine samples from psychiatric patients. As far as the authors are aware, there are no previous publications which give both the free and total amine levels in urine for all five biogenic amines, estimated on one overnight sample. It is not possible, therefore, to make a direct comparison between the amine levels reported in this paper and other investigations. However, the values shown in Table III are of the same order as those reported in the literature. An indication of the 24-h excretion values can be obtained from the hourly excretion rates shown in Table III, although it is realised that this does not take into account the variations in excretion rate over the 24-h period. A comparison of these “24-h” values and some literature values is shown in Table IV; only a few of the A and N references are given while the list of references to D/M and MN determinations is more complete. The free noradren-

COMPARISONOF REPORTEDEXCRETIONRATES OF SOMEBICGENICAMINESIN URINE ~__~~~ ~__~ _~~ Total amines (,ug/z# h) Free amines (,ugcg/2qh) .___ Ref. M NM M NM Ref. N D A I 16.4 9

57 55 25

=97 I99

3o

29.5

205

21.2

100-200

3’6 * These rates were estimated

This paper* 8 ‘4

88 220

II

*o-30 I64

7 ‘7

330

from values obtained

110

with overnight

420 176 30-70 245 192 640

This paper* 26

‘3 12 II 25

_____.~~

urines.

aline and dopamine and free and total metanephrine values obtained during the course of this investigation are well within the ranges quoted in the literature8~11-13~24-26. The free adrenaline value is lower than some reported previously8124; however, this may be because in the present investigation it was obtained with an overnight urine. The normetanephrine values found by this procedure are higher than those reported by present in the urine would be some other authors11-13~25~26. A ny 3-methoxytyramine largely included in the NM value determined by the present method. However, when these amines are assayed by the “lutin” procedure, 3-methoxytyramine would not affect the determination of M and NM, but it would possibly interfere with the estimation of D. No attempt has been made, at this stage, to evaluate the clinical significance of the results; it is expected that this aspect of the problem will be published elsewhere. Clin. Chim.

Acta,

14 (1966) 99-107

ESTIMATION

This addition

OF URINARY

method,

AMINES

based

compounds,

107

on absorbances

has several advantages

of the

aminochrome-sodium

over existing

procedures.

bisulfite

Most existing

methods are for the assay of adrenaline and noradrenaline or dopamine or metanephrine and normetanephrine in urine. Those methods which include the determination of M and NM involve two extraction procedures, one to remove the catecholamines (usually alumina) and another for M and NM (usually an ion-exchange resin). In the “bisulfite” method, described in this paper, all the chromatography is carried out on one column and the amines are separated into two fractions. Elution of the catecholamines as their boric acid complexes has its own advantages from the point of view of stability, as indicated above; the aminochrome-sodium bisulfite addition compounds are also quite stable and the timing of the various steps is, therefore, not critical as is the case with methods which are based on the fluorescence of the lutins. The only step in the bisulfite method which must be accurately timed is the 5 min oxidation of the catecholamines, for the dopamine determination. Furthermore, compensation for substances which might interfere with the absorbance measurements is fairly straightforward whereas it is often difficult to assess the contribution of interfering substances in fluorescence measurements. ACKNOWLEDGMENT

The authors Psychiatric

are grateful

Research

to members

Unit for their

of the clinical

co-operation

and nursing

in obtaining

staff of the

the urine samples.

REFERENCES I S. UDENFRIEND, Fluorescence Assay in Biology and Medicine, Academic Press Inc., New York, 1962, Chap. 5. 2 R. A. HEACOCK, Advan. Heterocyclic Chem., 5 (1965) 205. 3 U. S. VON EULER, Pharmacol. Rev., II (1959) 262. 4 H. WEIL-MALHERBE, Pharmacol. Rev., II (1959) 278. 5 A. CARLSSON, Pharmacol. Rev., II (1959) 300. 6 F. BISCHOFF AND A. TORRES, Clin. Chem., 8 (1962) 370. 7 U. S. VON EULER, U. HAMBERG AND S. HEL~NER, B&hem. J., 4g (1951) 655. 8 B. D. DRUJAN, T. L. SOURKES, D. S. LAYNE AND G. F. MURPHY, Can. J. Biochem. Physiol..

37 (1959) 1’53. 9 V. J. UUSPKK, Ann. Med. Exptl. Biol. Fenniae (Helsinki), 41 (1963) 194. IO J. HKGGENDAL, Acta Ph.ysysiol.&and., 56 (1962) 258. II S. BRUNJES, D. WYBENGA AND V. J. JOHNS, Clin. Chem., IO (1964) I. 12 K. TANIGUCHI, Y. KAKIMOTO AND M. D. ARMSTRONG, J. Lab. C&z. Med., 64 (1964) 469. 13 H. WEIL-MALHERBE, 2. Klin. Chem., 2 (1964) 21. 14 E. R. B. SMITH AND H. WEIL-MALHERBE, J. Lab. Clin. Med., 60 (1962) 212. 15 R. L. TSE AND M. J. OESTERLING, Clin. Chim. Acta, 4 (1959) 307. 16 M. J. OESTERLING AND R. L. TSE, Federation Proc., 18 (1959) 296. 17 M. Jo OESTERLING AND R. L. TSE, Am. J. Med. Technoi., ;7 (1961) 112. 18 M. J. OESTERLING, R. L. TSE AND H. M. HOLMES, Federation Proc., 21 (1962) 192. 19 J. VAN ESPEN, Pharm. Acta Helv., 33 (1958) 207. 20 M. J. OESTERLING AND R. L. TSE, Clin. Chim. Acta, 8 (1963) 393. 21 G. L. MATTOK AND D. L. WILSON, Anal. Biochem., 11 (1965) 575. 22 A. BERTLER, A. CARLSSON AND E. ROSENGREN, Clin. Chim. Acta, 4 (1959) 456. 23 A. RANDRUP, C2in. Chim. Acta, 6 (1961) 584. 24 J. G. WISWELL, G. E. HURWITZ, V. CORONHO, D. H. L. BING AND D. L. CHILD, J. C&n. Emdo&n. Metab., 23 (1963) 1102. 25 0. KRAUPP, H. BERNHEIMER AND D. PAPISTAS, CZin. Chim. Acta, 6 (1961) 851. 26 K. YOSHINAGA, C. ITOH, H. ISHIDA, T. SATO AND Y. WADA, Tohoku J. Eq%l. Med., 74 (1961) 105; Chem. Abstr., 56 (1962) 658. 27 A. BARBEAU, A. M. A. Arch. Neural. Psychiat.,

4 (1961) 97. Clin. Chim. Acta, 14 (1966) w-107