596
Specialia
Gas chromatographic-mass superior cervical ganglia
spectrometric identification and formation in vivo
EXPERIENTIA 33/5
of epinine
in rat
F. H. FoppenZ, A. Liuzzi 2 a n d I. J. K o p i n
Laboratory o/Clinical Science, National Institute o/ Mental Health, 9000 RockviIle Pike Bethesda (Maryland 20014, USA), I November 7976 Summary. E p i n i n e was i d e n t i f i e d in r a t s u p e r i o r c e r v i c a l g a n g l i a b y a gas c h r o m a t o g r a p h i c - m a s s s p e c t r o m e t r i c m e t h o d . T h e d e u t e r a t e d m e t h y l g r o u p of i.v. a d m i n i s t e r e d labeled m e t h i o n i n e was i n c o r p o r a t e d i n t o e p i n i n e a t a slow rate, a l t h o u g h e p i n e p h r i n e - C D ~ was r a p i d l y formed. T h e s e results i n d i c a t e t h a t e p i n i n e f o u n d in t h e g a n g l i a is n o t a prec u r s o r of e p i n e p h r i n e . I n 1906, H a l l e 3 p r o p o s e d t h a t e p i n e p h r i n e m i g h t b e f o r m e d b y # - h y d r o x y l a t i o n of epinine, b u t t h e l a t t e r c o m p o u n d was n o t r e p o r t e d in a n y a n i m a l tissues u n t i l 1962, w h e n i t w a s f o u n d ill t h e p a r o t i d g l a n d of B u f o m a r i n u s 4. I n 1973, L a d u r o n 5 d e m o n s t r a t e d t h e p r e s e n c e of e p i n i n e ill b o v i n e a d r e n a l m e d u l l a a n d s h o w e d t h a t i t c o u l d be s y n t h e t i z e d in v i t r o f r o m d o p a m i n e b y t h e a c t i o n of a n N - m e t h y l t r a n s f e r a s e i s o l a t e d f r o m t h e a d r e n a l m e d u l l a ~ as well as in p e r f u s e d a d r e n a l s ~. A l t h o u g h n o r e p i n e p h r i n e is g e n e r a l l y c o n s i d e r e d t o be t h e p r e c u r s o r s-z0 of e p i n e p h r i n e , on t h e b a s i s of t h e s e o b s e r v a t i o n s , L a d u r o n p r o p o s e d t h a t e p i n e p h r i n e could also b e f o r m e d , b y a p a t h w a y i n v o l v i n g t h e N - m e t h y l a t i o n of d o p a m i n e t o epinine and subsequent fl-hydroxylation to epinephrine. E p i n e p h r i n e is p r e s e n t in t h e s u p e r i o r c e r v i c a l g a n g l i o n 11, p e r h a p s i n c h r o m a f f i n cells 12 w h i c h are s i m i l a r t o t h e p r i n c i p a l cell t y p e of a d r e n a l m e d u l l a ~3. Since c h r o m a f f i n or s m a l l i n t e n s e l y f l u o r e s c e n t (SIF) cells i n t h e s u p e r i o r c e r v i c a l g a n g l i a d i s a p p e a r d u r i n g t h e d e v e l o p m e n t Z L it was of i n t e r e s t t o d e t e r m i n e if e p i n i n e was p r e s e n t in g a n g l i a of n e o n a t a l r a t s a n d if its levels w o u l d decline d u r i n g d e v e l o p m e n t in t h e s a m e m a n n e r as do c h r o m a f f i n cells. T h e r e l a t i v e s y n t h e s i s r a t e s of e p i n e p h r i n e a n d e p i n i n e in n e w b o r n r a t s was e x a m i n e d b y d e t e r m i n i n g t h e r e l a t i v e r a t e of i n c o r p o r a t i o n of CD~ f r o m a d m i n i s t e r e d m e t h i o n i n e - C D a i n t o t h e N - m e t h y l g r o u p of t h e 2 c o m pounds.
Epinine-PFP CI CH4
Zivic Miller (Allison P a r k , Pa., U S A ) o n t h e 1 8 t h d a y of g e s t a t i o n a n d were h o u s e d i n d i v i d u a l l y u n t i l b i r t h of t h e litters. P u p s of d i f f e r e n t ages were killed b y d e c a p i t a t i o n a n d s u p e r i o r c e r v i c a l g a n g l i a were r e m o v e d , c l e a n e d a n d Acknowledgments. We wish to thank Dr Magda Claeys for helpful discussions. 2 A. Liuzzi, on leave from Consiglio Nazionale delle Ricerche, Laboratorio di Biologia Cellulare, Roma, Italy. 3 W.L. Halle, Beitr. chem. Physiol. Path. 8, 276 (1906). 4 F. Maerki, J. Axelrod and B. Witkop, Biochim. biophys. Aeta 58, 367 (1962). 5 P. Laduron, in: Frontiers in Catecholamine Research, p. 121. Eds. E. D. Usdin and S. Snyder. Pergamon Press, New York 1973. 6 P. Laduron, Arehs tilt. Pharmaeodyn. Th@r. 195, 197 (1972). 7 P. Laduron, P. van Gompel, .L Leysen and M. Claeys, NaunynSchmiedebergs Arch. Pharma. 286, 227 (1974). 8 H. Blaschko, J. Physiol, Lond., 96, 50P (1939). 9 J. Axelrod, Pharmac. Rev. 18, 95 (1966). 10 N. Kirschner, Pharmac. Rev. 77,350 (1959). i1 S.H. Koslow, N. Bjegovic and E. Costa, J. Neurochem. 2g, 227, 1
(1975).
12 M. Lempinen, Acta physiol, scand., Suppl. 62, 231 (1964). 13 R.E. Coupland, in: The natural history of the ehromaffin cell. Longmans, London 1965. 14 R.D. Ciaranello, D. Jacohowitz and J. Axelrod, J. Neurochem. 20, 799 (1973),
O II
Nr. 19
100
Materials and methods. P r e g n a n t r a t s were o b t a i n e d f r o m
C2F5--G-Ox/----k
CH, I
C2FS- CO - - ( (\ - ~' ) /'X'/---CH2-CH2-N- CC2F5 II II O L....._, O MH+
MW605
0
.•
. . . .
70
,.r.., . . . , - . , , , . , . , . . . . , . , , , , . . . . , , ' r
100
, ' " ' , .......... ' " ' , ' " " ' " ' " " '
150
I I,
.........................................I.......................................... I................. ,.........................!..........t............
h L
......... . ' . ' , ' " ' , ' " ' , " " , ' " ' " ' " 1
........
200
' ....
400
450
600
650
Fig. 1. Chemical ionization mass spectra of epinine-PFP Oil a Finnigan Model 1015C Mass Spectrometer using methane as reagent gas.
15. 5. 1977
Speeiatia
8 2
,.,.t
i1
j~ 6
I 4
3
2
1
Fig. 2. Typical GC-MS tracing, under chemical ionization conditions with methane as reagent gas, of pentafluoropropionic anhydride derivatives of cateeholamines in rat superior cervical ganglia and of added authenic deuterated cateeholamines. I Epinephrine-PFP, m/e 604; 2 E-D~-PFP, m/e 607; 3 norepinephrine-PFP, m/e 590; d NED3-PFP , m/e 593 ; 5 dopamine-PFP, m/e 592; 6 DA-D2-PFP, m/e 594; 7 epinine-PFP, m/e 606; 8 Ep-PFP, m/e 609. The retention time is given in min.
l//~
25-
~-
500
1,5
300 EPINEPHRINEEPHR
I0
n~ 200~
,5 --
o
I00
I
I
I
i
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O
--
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~
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_-I
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~ II0 DAYS
~
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OF AGE
Fig. 3. Catecholamine content of the superior cervical ganglia of rats at various ages. Results are mean values (-E SEM) for groups of 6-8 rats9 The levels of amines were determined by GC-MS. 15 F.H. Foppen, unpublished results. 16 M. Ebert, W. Havens, K. Powers, S. Markey aI~d t. J. Kopin, Neurosci. Meeting, New York 1976, Abstract.
597
cooled o n ice. I m m e d i a t e l y after dissection, ganglia f r o m 1 or 2 r a t s were h o m o g e n i z e d in ice cold 100 ~zl N He1. To a n a l i q u o t of t h e h o m o g e n a t e was a d d e d 100 ~1 of 0.1 N HC1, c o n t a i n i n g d e u t e r a t e d e p i n i n e (epinine-N-CD3), e p i n e p h r i n e (epinephrine-e-D~-fl-D1), n o r - e p i n e p h r i n e (nor-epinephrine-~-D~-/5-D1) a n d d o p a m i n e ( d o p a m i n e /%D~). T h e a m o u n t s of d e u t e r a t e d s t a n d a r d s a d d e d were a b o u t 1 0 % less t h a n t h e e x p e c t e d a m o u n t of t h e respect i v e e n d o g e n o u s c o m p o u n d s as d e t e r m i n e d p r e v i o u s l y w i t h o u t s t a n d a r d s . To d i m i n i s h losses, 50 ~1 of 0.1 N HC1 c o n t a i n i n g 0.5% ascorbic acid, 0.1% E D T A , a n d 0 . 0 5 % ~-methyl-dopamine, which did not interfere with the q u a n t i t a t i v e m e a s u r e m e n t s , was also added 9 T h e h o m o g e h a t e was c e n t r i f u g e d a t 10.000 • for 15 m i n a t + 4 ~ T h e s u p e r n a t a n t was e x t r a c t e d w i t h 500 ~zl e t h y l a c e t a t e a n d t h e organic l a y e r discarded. Carefully a v o i d i n g contamination with material from the acid-ethylacetate i n t e r p h a s e , t h e a q u e o u s f r a c t i o n was t r a n s f e r r e d to a 5 m l r o u n d - b o t t o m flask a n d freeze-dried. T h e d r y residue was dissolved in 50 ~1 a c e t o n i t r i l a n d t h e n t r e a t e d a t r o o m t e m p e r a t u r e w i t h 100 ~1 p e n t a f l u o r o p r o p i o n i c anh y d r i d e ( P F P A ) for 30 rain. A f t e r t r a n s f e r to a 3 m l conical c e n t r i f u g e t u b e , t h e s o l v e n t was e v a p o r a t e d in a s t r e a m of n i t r o g e n a n d t h e d r y residue was redissolved in 5-10 ~1 d r y acetonitril. A l i q u o t s (2-3 txl) were i n j e c t e d o n a G C - c o l u m n c o u p l e d to a m a s s s p e c t r o m e t e r 9 T h e colu m n , 150 c m • ram, was p a c k e d w i t h OV-17, 3 % o n A n a k r o m Q. T h e c o l u m n t e m p e r a t u r e was 160 ~ a n d t h e h e l i u m flow was 20 m l / m i n . T h e r e t e n t i o n t i m e s were a p p r o x i m a t e l y 1.5 rain for e p i n e p h r i n e - P F P , 2 m i n for i l o r e p i n e p h r i n e - P F P , 3.5 m i n for d o p a m i n e - P F P a n d 4 m i n for e p i n i n e - P F P . T h e c o l u m n e f f l u e n t was subjected to chemical ionization-mass spectrometry using m e t h a n e as r e a g e n t gas. T h e m a s s s p e c t r o m e t e r was a F i n n i g a n M o d e l 1015C i n t e r f a c e d w i t h a P r o g r a m m a b l e M u l t i p l e I o n M o n i t o r ( P R O M I M ) . T h e /3-hydroxylated c a t e c h o l a m i n e d e r i v a t i v e s were m o n i t o r e d b y t h e i r m o s t a b u n d a n t f r a g m e n t s : m / e 604 a n d 607 for e p i n e p h r i n e P F P a n d e p i n e p h r i n e - D a - P F P a n d m / e 590 a n d 593 for norepinephrine-PFP and norepinephrine-D3-PFP. The o t h e r a m i n e s were e s t i m a t e d f r o m t h e i r M H + ions: m / e 606 a n d 609 for e p i n i n e - P F P a n d e p i n i n e - D 3 - P F P a n d m / e 592 a n d 594 for d o p a m i n e - P F P a n d d o p a m i n e D2-PFP. A standard curve was prepared using known a m o u n t s of epinine, e p i n e p r i n e , n o r e p i n e p h r i n e , d o p a m i n e a n d t h e i r d e u t e r a t e d h o m o l o g u e s 15. W h e n h y d r o l y s e d p a r t i a l l y , n o r e p i n e p h r i n e - P F P , c a n also give rise to a res p o n s e a t m / e 606. U n d e r t h e c o n d i t i o n s of t h e a s s a y t h i s was n o t o b s e r v e d . W h e n t h e d i s u b s t i t u t e d d e r i v a t i v e was m a d e b y p a r t i a l h y d r o l y s i s of t h e f u l l y s u b s t i t u t e d d e r i v ative, its r e t e n t i o n t i m e o n t h e gas c h r o m a t o g r a p h w a s s l i g h t l y longer (by 10-15 sec) t h a n t h a t of e p i n i n e - P F P so t h a t t h e 2 c o m p o u n d s c o u l d b e a d e q u a t e l y d i s t i n guished. T h e i d e n t i f i c a t i o n of e p i n i n e w a s c o n f i r m e d b y s i m i l a r gas c h r o m a t o g r a p h i c a n d m a s s s p e c t r o m e t r i c beh a v i o r of t h e t r i f l u o r o a c e t i c d e r i v a t i v e s of epinine, e p i n i n e N-CD a a n d t h e e n d o g e n o u s a m i n e . I n a n o t h e r e x p e r i m e n t , 24 n e w b o r n r a t s r e c e i v e d i.p. 2 m l / k g of a saline s o l u t i o n c o n t a i n i n g 5 m g / m l m e t h i o nine-CD~. T h e r a t s were killed 20 m i n later, t h e s u p e r i o r c e r v i c a l g a n g l i a r e m o v e d , a n d g r o u p s of 16 ganglia f r o m 8 a n i m a l s pooled, h o m o g e n i z e d , a n d t h e e p i n i n e a n d e p i n e p h r i n e a n a l y z e d b y GC-MS as d e s c r i b e d a b o v e , b u t w i t h o u t a d d i t i o n of t h e d e u t e r a t e d c o m p o u n d s . T h e carcasses of t h e a n i m a l s were h o m o g e n i z e d in 0.1 N HC1, a n d t h e r e l a t i v e e n r i c h m e n t of t h e m e t h i o n i n e w i t h CD 3 d e t e r m i n e d b y GC MS 16 Epinephrine-bitartrate, norepinephrine-tartrate-HCt and d o p a m i n e - H C 1 were p u r c h a s e d f r o m C a l b i o c h e m , Los Angeles, Cal., U S A ; epinine-HC1, f r o m A l d r i c h C h e m i c a l
598
Speeialia
Co., Inc., Milwaukee, Wisc., U S A ; methionine-CD3, epinephrine-e-D2-fi-D1, norepinephrine-a-D2-fl-D 1 and dopamine-~-D 2 were o b t a i n e d f r o m Merck, Sharp and D o h m e , Kirkland, Quebec, Canada. OV-17, 3% on A n a k r o m Q, 90-100 mesh, from Analabs, N o r t h H a v e n , Conn., U S A ; trifluoracetic a n h y d r i d e and pentafluoropropionic a n h y d r i d e f r o m Pierce Chemicals, Rockford, II1., U S A . E p i n i n e - N - C D 3 was generously d o n a t e d b y J a n s e n F a r m a c e u t i c a (Beerse, Belgium) and a - m e t h y l d o p a m i n e was the gift of Dr J. Daly, N I A M D , N I H . Results and discussion. E p i n i n e in the superior cervical ganglia of rats was d e t e c t e d and identified b y GC-MS. T h e mass spectra of t h e P F P d e r i v a t i v e s of epinine and epinine-CDa are shown in figure 1. The a m o u n t s of epinine and o t h e r amines present in t h e h o m o g e n a t e s of superior cervical ganglia were d e t e r m i n e d from the relat i v e p e a k heights of t h e unlabelled c o m p o u n d s and t h e added d e u t e r a t e d s t a n d a r d s at a p p r o p r i a t e r e t e n t i o n times for t h e gas c h r o m a t o g r a p h i c columns and m/e in t h e mass spectra. A t y p i c a l t r a c i n g f r o m the GC-MS recording, after injection of an aliquot of t h e d e r i v a t i v e s
Relative enrichment with CDa of epinine and epinephrine after administration of methionine-CDa Relative enrichment Methionine (carcass) Epinephrine (SCG) Epinine (SCG)
26 4- 3% 19 4- 4.8 0.74 =L 0.12
Newborn rats received 10 txg/g methionine-CDa i.p. The rats were killed after 20 mira The relative enrichments with CD3 of the indicated compounds were determined by GC-MS.
EXPERIENTIA33]5
derived from the h o m o g e n a t e of s y m p a t h e t i c ganglia to which d e u t e r a t e d standards had been added, is shown in figure 2. N o r e p i n e p h r i n e is t h e m a j o r c a t e c h o l a m i n e present in s y m p a t h e t i c ganglia (figure 3). The a m o u n t of norepinephrine present progressively increases w i t h age as t h e ganglia and the animal grow in size. D o p a m i n e levels are highest at birth, decline during t h e n e x t few days, and t h e n increase slightly as the ganglia become larger. The a m o u n t s of t h e N - m e t h y l a t e d d e r i v a t i v e s of these catecholamines are considerably lower. E p i n e p h r i n e levels are h i g h e s t at b i r t h b u t its levels are m a i n t a i n e d only slightly below those found at b i r t h for at least 30 days. Thus the r e l a t i v e a m o u n t of epinephrine c o m p a r e d to norepinephrine and ganglia size decreases w i t h age, from 10% of norepinephrine at b i r t h to less t h a n 1% at 30 days of age. Epinine, the N - m e t h y l a t e d d e r i v a t i v e of dopamine, is present in v e r y small amounts. Its level is highest a t birth, at which t i m e its levels are a b o u t half those of epinephrine, and r a p i d l y declines during the first 4 days of life. T h e a m o u n t of epinephrine p r e s e n t is usually less t h a n o n e - t e n t h those of d o p a m i n e (figure 3). 20 min after i.p. injection of m e t h i o n i n e labelled w i t h d e u t e r i u m on the m e t h y l group (CDa), labelled epinephrine and epinine are found in the superior cervical ganglia. The r e l a t i v e enr i c h m e n t of epinephrine w i t h CD a approaches t h a t of m e t h i o n i n e found in t h e carcass, b u t t h a t of epinine is o n l y 4 % of epinephrine (table). The results of these e x p e r i m e n t s establish t h a t epinine is p r e s e n t in the superior cervical ganglia and can be formed in vivo. T h e a m o u n t s of epinine present, however, are v e r y small, and even at their highest are less t h a n half those of epinephrine. The r e l a t i v e l y low e n r i c h m e n t w i t h CD a after injection of methionine-CDa indicates t h a t t h e r a t e of epinine f o r m a t i o n in t h e ganglia is v e r y slow compared to t h a t of epinephrine and t h a t f o r m a t i o n of epinephrine f r o m epinine is at m o s t a m i n o r p a t h w a y .
I n v i t r o b i n d i n g of c i t r i n i n t o s e r u m p r o t e i n 1 C. D a m o d a r a n ~
Department o/Biological Sciences, Madurai University, Madurai 625 027 (India), 20 April 7976 Summary. I n v i t r o s t u d y shows t h a t t h e m y c o t o x i n citrinin binds to h u m a n serum albumin. T h e binding of toxins to p l a s m a proteins and s u b s e q u e n t biological a c t i v i t y has been observed earlier 3. Such observations h a v e been m a d e w i t h t h e food-borne m y c o toxins, aflatoxin and o c h r a t o x i n a-*. The p r e s e n t comm u n i c a t i o n is a r e p o r t on t h e b i n d i n g of t h e m y c o t o x i n citrinin to h u m a n s e r u m protein. Material and method. P u r e crystalline citrinin (isolated f r o m cultures of Aspergillus candidus or Penicillium eitrinum) was used in this study. H u m a n s e r u m was centrifuged o u t f r o m pooled blood samples of h e a l t h y a d u l t v o l u n t e e r s and used fresh. 3 different p r e p a r a t i o n s were t h e n o b t a i n e d : a) 1 m l of serum was dialysed at 4-7 ~ o v e r n i g h t against 1 1 of b a r b i t o n e - s o d i n m b a r b i t o n e buffer (ionic s t r e n g t h - 0 . 0 5 ; p H 8.6) and t h e dialysed s e r u m was r e a d y for electrophoresis; b) 0.2 m l of chlorof o r m solution of citrinin (200 ~g) was t a k e n in a small b e a k e r and dried. To t h e d r y toxin, 1 m l of s e r u m was a d d e d and i n c u b a t e d a t 37 ~ for 2 h w i t h i n t e r m i t t e n t gentle agitation. This was t h e n dialysed and k e p t r e a d y
for electrophoresis; c) a solution of citrinin was p r e p a r e d in b a r b i t o n e buffer (200 ~zg/ml) j u s t before electrophoretic run. P a p e r electrophoresis was carried out on a strip (10 • 30 cm) of W h a t m a n No. 3 paper: 10 V1 each of a), b) and c) was spotted separately b u t alongside and placed for electrophoretic run in b a r b i t o n e buffer a t 180 V and 5 m A for 31/2 h at r o o m t e m p e r a t u r e (29-31 ~
1 Part of Ph.D. thesis, University of Madras, 1973. 2 Acknowledgment is due to Dr E. R, B. Shanmugasundaram (University of Madras) and Dr J. Jayaraman (MaduraiUniversity) for kind guidance. 3 R. Truhaut, in: Transport function of plasma proteins, p. 147. Ed. P. Desgrez and P. M. De Traverse. Elsevier Publ. Co., Amsterdam, London and New York 1966. 4 V.N. Viswanatha Rao, K. Valmikinathan and N. Verghese, Bioehim. biophys. Aeta 765, 288 (1968). 5 R.D. Wet and S. S. Lee, Experientia 27, 458 (1971). 6 F.S. Chu, Arch. biochem. Biophys. 747, 359 (1971).
Specialia
Gas chromatographic-mass superior cervical ganglia
spectrometric identification and formation in vivo
EXPERIENTIA 33/5
of epinine
in rat
F. H. FoppenZ, A. Liuzzi 2 a n d I. J. K o p i n
Laboratory o/Clinical Science, National Institute o/ Mental Health, 9000 RockviIle Pike Bethesda (Maryland 20014, USA), I November 7976 Summary. E p i n i n e was i d e n t i f i e d in r a t s u p e r i o r c e r v i c a l g a n g l i a b y a gas c h r o m a t o g r a p h i c - m a s s s p e c t r o m e t r i c m e t h o d . T h e d e u t e r a t e d m e t h y l g r o u p of i.v. a d m i n i s t e r e d labeled m e t h i o n i n e was i n c o r p o r a t e d i n t o e p i n i n e a t a slow rate, a l t h o u g h e p i n e p h r i n e - C D ~ was r a p i d l y formed. T h e s e results i n d i c a t e t h a t e p i n i n e f o u n d in t h e g a n g l i a is n o t a prec u r s o r of e p i n e p h r i n e . I n 1906, H a l l e 3 p r o p o s e d t h a t e p i n e p h r i n e m i g h t b e f o r m e d b y # - h y d r o x y l a t i o n of epinine, b u t t h e l a t t e r c o m p o u n d was n o t r e p o r t e d in a n y a n i m a l tissues u n t i l 1962, w h e n i t w a s f o u n d ill t h e p a r o t i d g l a n d of B u f o m a r i n u s 4. I n 1973, L a d u r o n 5 d e m o n s t r a t e d t h e p r e s e n c e of e p i n i n e ill b o v i n e a d r e n a l m e d u l l a a n d s h o w e d t h a t i t c o u l d be s y n t h e t i z e d in v i t r o f r o m d o p a m i n e b y t h e a c t i o n of a n N - m e t h y l t r a n s f e r a s e i s o l a t e d f r o m t h e a d r e n a l m e d u l l a ~ as well as in p e r f u s e d a d r e n a l s ~. A l t h o u g h n o r e p i n e p h r i n e is g e n e r a l l y c o n s i d e r e d t o be t h e p r e c u r s o r s-z0 of e p i n e p h r i n e , on t h e b a s i s of t h e s e o b s e r v a t i o n s , L a d u r o n p r o p o s e d t h a t e p i n e p h r i n e could also b e f o r m e d , b y a p a t h w a y i n v o l v i n g t h e N - m e t h y l a t i o n of d o p a m i n e t o epinine and subsequent fl-hydroxylation to epinephrine. E p i n e p h r i n e is p r e s e n t in t h e s u p e r i o r c e r v i c a l g a n g l i o n 11, p e r h a p s i n c h r o m a f f i n cells 12 w h i c h are s i m i l a r t o t h e p r i n c i p a l cell t y p e of a d r e n a l m e d u l l a ~3. Since c h r o m a f f i n or s m a l l i n t e n s e l y f l u o r e s c e n t (SIF) cells i n t h e s u p e r i o r c e r v i c a l g a n g l i a d i s a p p e a r d u r i n g t h e d e v e l o p m e n t Z L it was of i n t e r e s t t o d e t e r m i n e if e p i n i n e was p r e s e n t in g a n g l i a of n e o n a t a l r a t s a n d if its levels w o u l d decline d u r i n g d e v e l o p m e n t in t h e s a m e m a n n e r as do c h r o m a f f i n cells. T h e r e l a t i v e s y n t h e s i s r a t e s of e p i n e p h r i n e a n d e p i n i n e in n e w b o r n r a t s was e x a m i n e d b y d e t e r m i n i n g t h e r e l a t i v e r a t e of i n c o r p o r a t i o n of CD~ f r o m a d m i n i s t e r e d m e t h i o n i n e - C D a i n t o t h e N - m e t h y l g r o u p of t h e 2 c o m pounds.
Epinine-PFP CI CH4
Zivic Miller (Allison P a r k , Pa., U S A ) o n t h e 1 8 t h d a y of g e s t a t i o n a n d were h o u s e d i n d i v i d u a l l y u n t i l b i r t h of t h e litters. P u p s of d i f f e r e n t ages were killed b y d e c a p i t a t i o n a n d s u p e r i o r c e r v i c a l g a n g l i a were r e m o v e d , c l e a n e d a n d Acknowledgments. We wish to thank Dr Magda Claeys for helpful discussions. 2 A. Liuzzi, on leave from Consiglio Nazionale delle Ricerche, Laboratorio di Biologia Cellulare, Roma, Italy. 3 W.L. Halle, Beitr. chem. Physiol. Path. 8, 276 (1906). 4 F. Maerki, J. Axelrod and B. Witkop, Biochim. biophys. Aeta 58, 367 (1962). 5 P. Laduron, in: Frontiers in Catecholamine Research, p. 121. Eds. E. D. Usdin and S. Snyder. Pergamon Press, New York 1973. 6 P. Laduron, Arehs tilt. Pharmaeodyn. Th@r. 195, 197 (1972). 7 P. Laduron, P. van Gompel, .L Leysen and M. Claeys, NaunynSchmiedebergs Arch. Pharma. 286, 227 (1974). 8 H. Blaschko, J. Physiol, Lond., 96, 50P (1939). 9 J. Axelrod, Pharmac. Rev. 18, 95 (1966). 10 N. Kirschner, Pharmac. Rev. 77,350 (1959). i1 S.H. Koslow, N. Bjegovic and E. Costa, J. Neurochem. 2g, 227, 1
(1975).
12 M. Lempinen, Acta physiol, scand., Suppl. 62, 231 (1964). 13 R.E. Coupland, in: The natural history of the ehromaffin cell. Longmans, London 1965. 14 R.D. Ciaranello, D. Jacohowitz and J. Axelrod, J. Neurochem. 20, 799 (1973),
O II
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100
Materials and methods. P r e g n a n t r a t s were o b t a i n e d f r o m
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.........................................I.......................................... I................. ,.........................!..........t............
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200
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400
450
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Fig. 1. Chemical ionization mass spectra of epinine-PFP Oil a Finnigan Model 1015C Mass Spectrometer using methane as reagent gas.
15. 5. 1977
Speeiatia
8 2
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j~ 6
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Fig. 2. Typical GC-MS tracing, under chemical ionization conditions with methane as reagent gas, of pentafluoropropionic anhydride derivatives of cateeholamines in rat superior cervical ganglia and of added authenic deuterated cateeholamines. I Epinephrine-PFP, m/e 604; 2 E-D~-PFP, m/e 607; 3 norepinephrine-PFP, m/e 590; d NED3-PFP , m/e 593 ; 5 dopamine-PFP, m/e 592; 6 DA-D2-PFP, m/e 594; 7 epinine-PFP, m/e 606; 8 Ep-PFP, m/e 609. The retention time is given in min.
l//~
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Fig. 3. Catecholamine content of the superior cervical ganglia of rats at various ages. Results are mean values (-E SEM) for groups of 6-8 rats9 The levels of amines were determined by GC-MS. 15 F.H. Foppen, unpublished results. 16 M. Ebert, W. Havens, K. Powers, S. Markey aI~d t. J. Kopin, Neurosci. Meeting, New York 1976, Abstract.
597
cooled o n ice. I m m e d i a t e l y after dissection, ganglia f r o m 1 or 2 r a t s were h o m o g e n i z e d in ice cold 100 ~zl N He1. To a n a l i q u o t of t h e h o m o g e n a t e was a d d e d 100 ~1 of 0.1 N HC1, c o n t a i n i n g d e u t e r a t e d e p i n i n e (epinine-N-CD3), e p i n e p h r i n e (epinephrine-e-D~-fl-D1), n o r - e p i n e p h r i n e (nor-epinephrine-~-D~-/5-D1) a n d d o p a m i n e ( d o p a m i n e /%D~). T h e a m o u n t s of d e u t e r a t e d s t a n d a r d s a d d e d were a b o u t 1 0 % less t h a n t h e e x p e c t e d a m o u n t of t h e respect i v e e n d o g e n o u s c o m p o u n d s as d e t e r m i n e d p r e v i o u s l y w i t h o u t s t a n d a r d s . To d i m i n i s h losses, 50 ~1 of 0.1 N HC1 c o n t a i n i n g 0.5% ascorbic acid, 0.1% E D T A , a n d 0 . 0 5 % ~-methyl-dopamine, which did not interfere with the q u a n t i t a t i v e m e a s u r e m e n t s , was also added 9 T h e h o m o g e h a t e was c e n t r i f u g e d a t 10.000 • for 15 m i n a t + 4 ~ T h e s u p e r n a t a n t was e x t r a c t e d w i t h 500 ~zl e t h y l a c e t a t e a n d t h e organic l a y e r discarded. Carefully a v o i d i n g contamination with material from the acid-ethylacetate i n t e r p h a s e , t h e a q u e o u s f r a c t i o n was t r a n s f e r r e d to a 5 m l r o u n d - b o t t o m flask a n d freeze-dried. T h e d r y residue was dissolved in 50 ~1 a c e t o n i t r i l a n d t h e n t r e a t e d a t r o o m t e m p e r a t u r e w i t h 100 ~1 p e n t a f l u o r o p r o p i o n i c anh y d r i d e ( P F P A ) for 30 rain. A f t e r t r a n s f e r to a 3 m l conical c e n t r i f u g e t u b e , t h e s o l v e n t was e v a p o r a t e d in a s t r e a m of n i t r o g e n a n d t h e d r y residue was redissolved in 5-10 ~1 d r y acetonitril. A l i q u o t s (2-3 txl) were i n j e c t e d o n a G C - c o l u m n c o u p l e d to a m a s s s p e c t r o m e t e r 9 T h e colu m n , 150 c m • ram, was p a c k e d w i t h OV-17, 3 % o n A n a k r o m Q. T h e c o l u m n t e m p e r a t u r e was 160 ~ a n d t h e h e l i u m flow was 20 m l / m i n . T h e r e t e n t i o n t i m e s were a p p r o x i m a t e l y 1.5 rain for e p i n e p h r i n e - P F P , 2 m i n for i l o r e p i n e p h r i n e - P F P , 3.5 m i n for d o p a m i n e - P F P a n d 4 m i n for e p i n i n e - P F P . T h e c o l u m n e f f l u e n t was subjected to chemical ionization-mass spectrometry using m e t h a n e as r e a g e n t gas. T h e m a s s s p e c t r o m e t e r was a F i n n i g a n M o d e l 1015C i n t e r f a c e d w i t h a P r o g r a m m a b l e M u l t i p l e I o n M o n i t o r ( P R O M I M ) . T h e /3-hydroxylated c a t e c h o l a m i n e d e r i v a t i v e s were m o n i t o r e d b y t h e i r m o s t a b u n d a n t f r a g m e n t s : m / e 604 a n d 607 for e p i n e p h r i n e P F P a n d e p i n e p h r i n e - D a - P F P a n d m / e 590 a n d 593 for norepinephrine-PFP and norepinephrine-D3-PFP. The o t h e r a m i n e s were e s t i m a t e d f r o m t h e i r M H + ions: m / e 606 a n d 609 for e p i n i n e - P F P a n d e p i n i n e - D 3 - P F P a n d m / e 592 a n d 594 for d o p a m i n e - P F P a n d d o p a m i n e D2-PFP. A standard curve was prepared using known a m o u n t s of epinine, e p i n e p r i n e , n o r e p i n e p h r i n e , d o p a m i n e a n d t h e i r d e u t e r a t e d h o m o l o g u e s 15. W h e n h y d r o l y s e d p a r t i a l l y , n o r e p i n e p h r i n e - P F P , c a n also give rise to a res p o n s e a t m / e 606. U n d e r t h e c o n d i t i o n s of t h e a s s a y t h i s was n o t o b s e r v e d . W h e n t h e d i s u b s t i t u t e d d e r i v a t i v e was m a d e b y p a r t i a l h y d r o l y s i s of t h e f u l l y s u b s t i t u t e d d e r i v ative, its r e t e n t i o n t i m e o n t h e gas c h r o m a t o g r a p h w a s s l i g h t l y longer (by 10-15 sec) t h a n t h a t of e p i n i n e - P F P so t h a t t h e 2 c o m p o u n d s c o u l d b e a d e q u a t e l y d i s t i n guished. T h e i d e n t i f i c a t i o n of e p i n i n e w a s c o n f i r m e d b y s i m i l a r gas c h r o m a t o g r a p h i c a n d m a s s s p e c t r o m e t r i c beh a v i o r of t h e t r i f l u o r o a c e t i c d e r i v a t i v e s of epinine, e p i n i n e N-CD a a n d t h e e n d o g e n o u s a m i n e . I n a n o t h e r e x p e r i m e n t , 24 n e w b o r n r a t s r e c e i v e d i.p. 2 m l / k g of a saline s o l u t i o n c o n t a i n i n g 5 m g / m l m e t h i o nine-CD~. T h e r a t s were killed 20 m i n later, t h e s u p e r i o r c e r v i c a l g a n g l i a r e m o v e d , a n d g r o u p s of 16 ganglia f r o m 8 a n i m a l s pooled, h o m o g e n i z e d , a n d t h e e p i n i n e a n d e p i n e p h r i n e a n a l y z e d b y GC-MS as d e s c r i b e d a b o v e , b u t w i t h o u t a d d i t i o n of t h e d e u t e r a t e d c o m p o u n d s . T h e carcasses of t h e a n i m a l s were h o m o g e n i z e d in 0.1 N HC1, a n d t h e r e l a t i v e e n r i c h m e n t of t h e m e t h i o n i n e w i t h CD 3 d e t e r m i n e d b y GC MS 16 Epinephrine-bitartrate, norepinephrine-tartrate-HCt and d o p a m i n e - H C 1 were p u r c h a s e d f r o m C a l b i o c h e m , Los Angeles, Cal., U S A ; epinine-HC1, f r o m A l d r i c h C h e m i c a l
598
Speeialia
Co., Inc., Milwaukee, Wisc., U S A ; methionine-CD3, epinephrine-e-D2-fi-D1, norepinephrine-a-D2-fl-D 1 and dopamine-~-D 2 were o b t a i n e d f r o m Merck, Sharp and D o h m e , Kirkland, Quebec, Canada. OV-17, 3% on A n a k r o m Q, 90-100 mesh, from Analabs, N o r t h H a v e n , Conn., U S A ; trifluoracetic a n h y d r i d e and pentafluoropropionic a n h y d r i d e f r o m Pierce Chemicals, Rockford, II1., U S A . E p i n i n e - N - C D 3 was generously d o n a t e d b y J a n s e n F a r m a c e u t i c a (Beerse, Belgium) and a - m e t h y l d o p a m i n e was the gift of Dr J. Daly, N I A M D , N I H . Results and discussion. E p i n i n e in the superior cervical ganglia of rats was d e t e c t e d and identified b y GC-MS. T h e mass spectra of t h e P F P d e r i v a t i v e s of epinine and epinine-CDa are shown in figure 1. The a m o u n t s of epinine and o t h e r amines present in t h e h o m o g e n a t e s of superior cervical ganglia were d e t e r m i n e d from the relat i v e p e a k heights of t h e unlabelled c o m p o u n d s and t h e added d e u t e r a t e d s t a n d a r d s at a p p r o p r i a t e r e t e n t i o n times for t h e gas c h r o m a t o g r a p h i c columns and m/e in t h e mass spectra. A t y p i c a l t r a c i n g f r o m the GC-MS recording, after injection of an aliquot of t h e d e r i v a t i v e s
Relative enrichment with CDa of epinine and epinephrine after administration of methionine-CDa Relative enrichment Methionine (carcass) Epinephrine (SCG) Epinine (SCG)
26 4- 3% 19 4- 4.8 0.74 =L 0.12
Newborn rats received 10 txg/g methionine-CDa i.p. The rats were killed after 20 mira The relative enrichments with CD3 of the indicated compounds were determined by GC-MS.
EXPERIENTIA33]5
derived from the h o m o g e n a t e of s y m p a t h e t i c ganglia to which d e u t e r a t e d standards had been added, is shown in figure 2. N o r e p i n e p h r i n e is t h e m a j o r c a t e c h o l a m i n e present in s y m p a t h e t i c ganglia (figure 3). The a m o u n t of norepinephrine present progressively increases w i t h age as t h e ganglia and the animal grow in size. D o p a m i n e levels are highest at birth, decline during t h e n e x t few days, and t h e n increase slightly as the ganglia become larger. The a m o u n t s of t h e N - m e t h y l a t e d d e r i v a t i v e s of these catecholamines are considerably lower. E p i n e p h r i n e levels are h i g h e s t at b i r t h b u t its levels are m a i n t a i n e d only slightly below those found at b i r t h for at least 30 days. Thus the r e l a t i v e a m o u n t of epinephrine c o m p a r e d to norepinephrine and ganglia size decreases w i t h age, from 10% of norepinephrine at b i r t h to less t h a n 1% at 30 days of age. Epinine, the N - m e t h y l a t e d d e r i v a t i v e of dopamine, is present in v e r y small amounts. Its level is highest a t birth, at which t i m e its levels are a b o u t half those of epinephrine, and r a p i d l y declines during the first 4 days of life. T h e a m o u n t of epinephrine p r e s e n t is usually less t h a n o n e - t e n t h those of d o p a m i n e (figure 3). 20 min after i.p. injection of m e t h i o n i n e labelled w i t h d e u t e r i u m on the m e t h y l group (CDa), labelled epinephrine and epinine are found in the superior cervical ganglia. The r e l a t i v e enr i c h m e n t of epinephrine w i t h CD a approaches t h a t of m e t h i o n i n e found in t h e carcass, b u t t h a t of epinine is o n l y 4 % of epinephrine (table). The results of these e x p e r i m e n t s establish t h a t epinine is p r e s e n t in the superior cervical ganglia and can be formed in vivo. T h e a m o u n t s of epinine present, however, are v e r y small, and even at their highest are less t h a n half those of epinephrine. The r e l a t i v e l y low e n r i c h m e n t w i t h CD a after injection of methionine-CDa indicates t h a t t h e r a t e of epinine f o r m a t i o n in t h e ganglia is v e r y slow compared to t h a t of epinephrine and t h a t f o r m a t i o n of epinephrine f r o m epinine is at m o s t a m i n o r p a t h w a y .
I n v i t r o b i n d i n g of c i t r i n i n t o s e r u m p r o t e i n 1 C. D a m o d a r a n ~
Department o/Biological Sciences, Madurai University, Madurai 625 027 (India), 20 April 7976 Summary. I n v i t r o s t u d y shows t h a t t h e m y c o t o x i n citrinin binds to h u m a n serum albumin. T h e binding of toxins to p l a s m a proteins and s u b s e q u e n t biological a c t i v i t y has been observed earlier 3. Such observations h a v e been m a d e w i t h t h e food-borne m y c o toxins, aflatoxin and o c h r a t o x i n a-*. The p r e s e n t comm u n i c a t i o n is a r e p o r t on t h e b i n d i n g of t h e m y c o t o x i n citrinin to h u m a n s e r u m protein. Material and method. P u r e crystalline citrinin (isolated f r o m cultures of Aspergillus candidus or Penicillium eitrinum) was used in this study. H u m a n s e r u m was centrifuged o u t f r o m pooled blood samples of h e a l t h y a d u l t v o l u n t e e r s and used fresh. 3 different p r e p a r a t i o n s were t h e n o b t a i n e d : a) 1 m l of serum was dialysed at 4-7 ~ o v e r n i g h t against 1 1 of b a r b i t o n e - s o d i n m b a r b i t o n e buffer (ionic s t r e n g t h - 0 . 0 5 ; p H 8.6) and t h e dialysed s e r u m was r e a d y for electrophoresis; b) 0.2 m l of chlorof o r m solution of citrinin (200 ~g) was t a k e n in a small b e a k e r and dried. To t h e d r y toxin, 1 m l of s e r u m was a d d e d and i n c u b a t e d a t 37 ~ for 2 h w i t h i n t e r m i t t e n t gentle agitation. This was t h e n dialysed and k e p t r e a d y
for electrophoresis; c) a solution of citrinin was p r e p a r e d in b a r b i t o n e buffer (200 ~zg/ml) j u s t before electrophoretic run. P a p e r electrophoresis was carried out on a strip (10 • 30 cm) of W h a t m a n No. 3 paper: 10 V1 each of a), b) and c) was spotted separately b u t alongside and placed for electrophoretic run in b a r b i t o n e buffer a t 180 V and 5 m A for 31/2 h at r o o m t e m p e r a t u r e (29-31 ~
1 Part of Ph.D. thesis, University of Madras, 1973. 2 Acknowledgment is due to Dr E. R, B. Shanmugasundaram (University of Madras) and Dr J. Jayaraman (MaduraiUniversity) for kind guidance. 3 R. Truhaut, in: Transport function of plasma proteins, p. 147. Ed. P. Desgrez and P. M. De Traverse. Elsevier Publ. Co., Amsterdam, London and New York 1966. 4 V.N. Viswanatha Rao, K. Valmikinathan and N. Verghese, Bioehim. biophys. Aeta 765, 288 (1968). 5 R.D. Wet and S. S. Lee, Experientia 27, 458 (1971). 6 F.S. Chu, Arch. biochem. Biophys. 747, 359 (1971).
