UBC Theses and Dissertations

UBC Theses Logo

UBC Theses and Dissertations

The metabolism of fluorodopa Cumming, Paul 1986

Your browser doesn't seem to have a PDF viewer, please download the PDF to view this item.

Item Metadata

Download

Media
831-UBC_1986_A6_7 C95.pdf [ 5.09MB ]
Metadata
JSON: 831-1.0096689.json
JSON-LD: 831-1.0096689-ld.json
RDF/XML (Pretty): 831-1.0096689-rdf.xml
RDF/JSON: 831-1.0096689-rdf.json
Turtle: 831-1.0096689-turtle.txt
N-Triples: 831-1.0096689-rdf-ntriples.txt
Original Record: 831-1.0096689-source.json
Full Text
831-1.0096689-fulltext.txt
Citation
831-1.0096689.ris

Full Text

THE METABOLISM OF FLUORODOPA by Paul dimming B . S c , The U n i v e r s i t y of A l b e r t a , 1984 A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE i n THE FACULTY OF GRADUATE STUDIES D i v i s i o n of N e u r o l o g i c a l Science We accept t h i s t h e s i s as conforming to the r e q u i r e d standard THE UNIVERSITY OF BRITISH COLIMBIA October 1986 © Paul Cumming, 1986 In presenting t h i s thesis i n p a r t i a l f u l f i l m e n t of the requirements for an advanced degree at the University of B r i t i s h Columbia, I agree that the Library s h a l l make i t f r e e l y available for reference and study. I further agree that permission for extensive copying of t h i s thesis for scholarly purposes may be granted by the head of my department or by h i s or her representatives. I t i s understood that copying or publication of t h i s thesis for f i n a n c i a l gain s h a l l not be allowed without my written permission. Department of P s y c h i a t r y  The University of B r i t i s h Columbia 1956 Main Mall Vancouver, Canada V6T 1Y3 Date Oct JS /?€ i i . A b s t r a c t Experiments were conducted to c h a r a c t e r i z e the metabolism of 1 8 F - 6 -fluorodopa ( 1 8F-D0PA), a compound used as a t r a c e r f o r the study of dopamine metabolism i n human sub j e c t s by means of p o s i t r o n emission tomography (PET). A n a l y s i s of plasma from carbidopa p r e t r e a t e d humans, monkeys and r a t s showed t h a t 1 8F-D0PA disappeared r a p i d l y and t h a t the p r i n c i p a l plasma me t a b o l i t e was 0-methyl- l 8F-D0PA. The k i n e t i c s of both 1 8F-D0PA and t o t a l 1 8 F disappearance i n the three s p e c i e s were found to be b i e x p o n e n t i a l . In human s u b j e c t s , some components of plasma 1 8F-D0PA metabolism were found to be age dependent. I n h i b i t i o n of c a t e c h o l - 0 -m e t h y l t r a n s f e r a s e (COMT) with U-0521 i n c r e a s e d g r e a t l y the p e r s i s t a n c e of 1 8F-D0PA i n plasma of the r a t . Cerebral t i s s u e s of the male hooded r a t were analyzed by high preformance l i q u i d chromatography at var i o u s times a f t e r 1 8F-D0PA a d m i n i s t r a t i o n . Me- 1 8F-D0PA was found to accumulate r a p i d l y i n s t r i a t u m , c o r t e x and cerebellum. Striatum d i f f e r e d from other t i s s u e s i n t h at r a d i o a c t i v i t y l e v e l s were maintained a t a f a i r l y constant l e v e l f o r two hours, during which time r a d i o a c t i v i t y washed out of other b r a i n r e g i o n s . This r a d i o c o n t r a s t was found to be due to the formation o f 1 8 F - f l u o r o d o p a m i n e and subsequent m e t a b o l i t e s i n s t r i a t u m . I n h i b i t i o n of COMT with U-0521 in c r e a s e d the amount of 1 8F-fluorodopamine i n s t r i a t u m by 50% over a 90 minutes p e r i o d , but t h i s only produced a marginal i n c r e a s e i n r a d i o c o n t r a s t because of the remaining background a c t i v i t y due to Me- 1 8F-D0PA. The k i n e t i c s of the d e c a r b o x y l a t i o n of 6- 1 8F-D0PA were determined in v i t r o to be very s i m i l a r to l i t e r a t u r e v a l u e s reported f o r L-DOPA. In c o n t r a s t , 2 - 1 8 F - f l u o r o d o p a was a poor s u b s t r a t e f o r the decarboxylase. Table of Contents Page A b s t r a c t i i L i s t of Tables v L i s t of Figures v i Acknowledgements i x I I n t r o d u c t i o n 1 II General M a t e r i a l s and Methods A) M a t e r i a l s 8 B) General HPLC Methods 9 III S p e c i a l Methods and R e s u l t s A) I d e n t i f i c a t i o n of M e t a b o l i t e s 1) Methods 11 2) Results 13 B) A n a l y s i s o f Plasma Metabolism 1) Methods 20 2) Results 25 C) Central Metabolism of 6- 1 8F-D0PA 1) Methods 49 2) Res u l t s . 51 D) 2-Fluorodopa Metabolism 1) Methods and Results 75 E) In v i t r o Metabolism of F-DOPA 1) Methods 77 2) Results 82 i v . Table of Contents (continued) Page IV D i s c u s s i o n 82 V Conclusion 107 VI References 108 Li s t of Tables Table I Table II Table III Table IV Table V Table VI Table VII Capacity f a c t o r s i n the HPLC system f o r some f l u o r i n a t e d and unfl u o r i n a t e d catecholamines and met a b o l i t e s The recovery of t o t a l plasma r a d i o -a c t i v i t y i n HPLC f r a c t i o n s Summary of the k i n e t i c s of plasma 1 8F-D0PA metabolism i n carbidopa p r e t r e a t e d monkey, r a t and human The apparent r a t e of 1 8F-D0PA metabolism i n plasma and the estimated p r o p o r t i o n of the input metabolized to Me-^8F-D0PA i n carbidopa p r e t r e a t e d human, monkey and r a t The t o t a l 1 8 F a c t i v i t y i n ten regions of r a t b r a i n f o l l o w i n g a d m i n i s t r a t i o n of 1 8F-D0PA The e f f e c t of U-0521 on t o t a l 1 8 F a c t i v i t y i n r a t b r a i n f o l l o w i n g a d m i n i s t r a t i o n of 1 8F-D0PA Ratios of endogenous me t a b o l i t e s t o dopamine and of 1 8 F - m e t a b o l i t e s to 1 8F-dopamine i n s t r i a t u m of the hooded r a t The e f f e c t of U-0521 alone on catecholamine and indoleamine c o n c e n t r a t i o n s i n r a t s t r i a t u m and hypothalamus a t 90 minutes a f t e r i n j e c t i o n Comparison of present r e s u l t s with some data from the l i t e r a t u r e on the p e r s i s t a n c e of s t r i a t a l accumulation and the extent of metabolism of exogenous DOPA or fluorodopa v i . L i s t of F i g u r e s Page Fi g u r e 1 Radiochromatogram of 1 8F-D0PA 14 Fig u r e 2 Radiochromatograms showing the metabolism of 1 8F-D0PA i n human plasma and r a t s t r i a t u m 15 Figure 3 I d e n t i f i c a t i o n of Me- 1 8F-D0PA as the major plasma metabolite of 1 8F-D0PA. 16-17 Figure 4 I d e n t i f i c a t i o n of 1 8F-DA and 1 8F-D0PAC as important 1 8F-D0PA me t a b o l i t e s i n r a t s t r i a t u m 19 Fig u r e 5 The time course of 1 8F-D0PA metabolism i n human plasma. 27-28 Figure 6 The bie x p o n e n t i a l disappearance of t o t a l 1 8 F a c t i v i t y from human plasma. 30 Fi g u r e 7 The r a t e constants f o r the disappearance of t o t a l 1 8 F a c t i v i t y i n human plasma p l o t t e d a g a i n s t age. 31 Fig u r e 8 The b i e x p o n e n t i a l disappearance of 1 8F-D0PA from human plasma. 33 Fi g u r e 9 The b i e x p o n e n t i a l disappearance of t o t a l 1 8 F a c t i v i t y from monkey plasma. 34 Fig u r e 10 The b i e x p o n e n t i a l disappearance of 1 8F-D0PA from monkey plasma. 35 Fi g u r e 11 Radiochromatograms from r a t plasma taken f i v e minutes a f t e r 1 8F-D0PA i n j e c t i o n with carbidopa pretreatment 36 Figure 12 Radiochromatograms from r a t plasma taken ten minutes a f t e r 1 8F-D0PA i n j e c t i o n with carbidopa pretreatment 37 Fi g u r e 13 Radiochromatograms from r a t plasma taken 60 minutes a f t e r 1 8F-D0PA i n j e c t i o n with carbidopa pretreatment 38 Fi g u r e 14 The b i e x p o n e n t i a l disappearance of t o t a l 1 8 F a c t i v i t y from r a t plasma. 40 VI1 Page Figure 15 The bie x p o n e n t i a l disappearance of 1 8F-D0PA from r a t plasma. 41 Fig u r e 16 The r a t i o o f m e t a b o l i t e to 1 8F-D0PA as a f u n c t i o n of time i n plasma from human 44 and monkey Figure 17 The slope of the r a t i o of me t a b o l i t e to 1 8F-D0PA i n plasma as a f u n c t i o n of time p l o t t e d a g a i n s t age. 45 Fig u r e 18 The r a t i o of m e t a b o l i t e s to 1 8F-D0PA as a f u n c t i o n of time i n r a t plasma 47 Fig u r e 19 1 8F-D0PA and m e t a b o l i t e s i n vermis and amygdala of the carbidopa p r e t r e a t e d r a t 52 Figure 20 The metabolism of 1 8F-D0PA i n r a t vermis as a f u n c t i o n of time 54 Fig u r e 21 Radiochromatograms of r a t cortex at 30 minutes a f t e r 1 8F-D0PA a d m i n i s t r a t i o n 55 Fig u r e 22 The e f f e c t of U-0521 on the O-methylation of 1 8F-D0PA i n cortex and vermis 56 Fig u r e 23 The abso l u t e 1 8 F a c t i v i t y i n s t r i a t u m and vermis as a f u n c t i o n of time 58 Fig u r e 24 Radiochromatograms of r a t st r i a t u m a t 30 minutes a f t e r 1 8F-D0PA i n j e c t i o n 62 Fi g u r e 25 Radiochromatograms of r a t s t r i a t u m a t 60 minutes a f t e r 1 8F-D0PA i n j e c t i o n 63 Fi g u r e 26 Radiochromatograms of r a t s t r i a t u m a t 90 minutes a f t e r 1 8F-D0PA i n j e c t i o n 64 Fi g u r e 27 The p r o p o r t i o n s of the p r i n c i p a l l a b e l l e d compounds i n r a t s t r i a t u m as a f u n c t i o n of time 65 Figure 28 The minor r a d i o l a b e l l e d compounds i n r a t st r i a t u m as a f u n c t i o n of time 66 Fig u r e 29 The absolute a c t i v i t i e s o f Me- 1 8F-D0PA i n vermis and s t r i a t u m as a f u n c t i o n of time 68 v i i i . Page Figure 30 The e f f e c t of U-0521 on the metabolism of 1 8F-D0PA i n r a t striatum 69 Figure 31 An e l e c t r o c h e m i c a l chromatogram showing the e l u t i o n times and r e l a t i v e amounts of bio g e n i c amines and meta b o l i t e s present in r a t s t r i a t u m 71 Figure 32 The e f f e c t of 6-OHDA l e s i o n s to the l e f t medial f o r e b r a i n bundle on the s t r i a t a l metabolism of 1 8F-D0PA at 15 minutes post i n j e c t i o n 76 Figure 33 The d i s t r i b u t i o n of r a d i o a c t i v i t y i n pei p h e r a l t i s s u e s of the r a t f o l l o w i n g a d m i n i s t r a t i o n o f 2- 1 8F-D0PA 78 Figure 34 The c e r e b r a l metabolism of 2- 1 8F-D0PA i n the r a t 79 Figu r e 35 Lineweaver-Burke p l o t s showing the 0-methyl-a t i o n of U-0521 by catechol-0-methyltrans-f e r a s e (COMT) and the competitive i n h i b i t i o n of COMT by L-DOPA and D.L-6-F-D0PA 83 Figu r e 36 Lineweaver-Burke p l o t s of the k i n e t i c s of dec a r b o x y l a t i o n of L-2- and 6- 1 8F-D0PA by s t r i a t a l homogenates 84 Figure 37 Summary of the metabolism of 1 8F-D0PA 85 i x . Acknowledgements Although the nature of t h i s e n t e r p r i s e r e q u i r e d the a s s i s t a n c e and cooperation of a l a r g e number of i n d i v i d u a l s , I wish to express s p e c i a l thanks to my s u p e r v i s o r Dr. E.G. McGeer, whose enthusiasm and i n s i g h t was a constant source of i n s p i r a t i o n . I wish a l s o to thank Dr. W.R.W M a r t i n f o r v a l u a b l e d i s c u s s i o n s concerning the theory and p r a x i s of t h i s study. Thanks to Dr. J . Grierson f o r the c o l d fluorodopa and to Dr. M. Adam and Dr. T. Ruth f o r the hot, to K. Singh and A. Bui f o r t e c h n i c a l a s s i s t a n c e and to Poppy f o r f e r r e t i n g out the r a b b i t . The tutorage provided by Mr. B.E. Boyes i n chromatographic methods and the a n a l y s i s of data was i n d i s p e n s i b l e . I a l s o wish to thank my parents f o r g i v i n g me s c i e n c e toys when I was q u i t e small . 1 I. I n t r o d u c t i o n The advent of p o s i t r o n emission tomography (PET) has provided a unique method f o r the study, by noninvasive means, of metabolism i n l i v i n g organisms. The method has the p o t e n t i a l to e l u c i d a t e metabolic processes i n humans and i n experimental animals which have p r e v i o u s l y been approachable only by i n d i r e c t methods such as a n a l y s i s of post-mortem t i s s u e or by assesement of c l i n i c a l s t a t u s . The fundamental p r i n c i p l e of PET i s t h a t c e r t a i n r a d i o i s o t o p e s decay by emission of a p o s i t r o n which i s a n n i h i l a t e d f o l l o w i n g i n t e r a c t i o n with an e l e c t r o n i n the adjacent m i l i e u . The energy contained i n the p a r t i c l e s i s l i b e r a t e d i n the form of two gamma photons, each with an energy corresponding to the r e s t mass energy of an e l e c t r o n , 511 KeV. In accordance with c o n s e r v a t i o n of momentum, t h i s event w i l l be p e r c e i v e d i n the l a b o r a t o r y frame as the simultaneous emission of a c o l i n e a r p a i r of gamma photons. The PET scanner r e l i e s upon the simultaneous d e t e c t i o n of these two p a r t i c l e s by a c i r c u l a r array of gamma d e t e c t o r s . Temporal summation of such events with f i l t e r e d back p r o j e c t i o n permits the r e c o n s t r u c t i o n of images i n three dimensions, the b a s i c u n i t of which, the voxel , i s of a volume r e l a t e d to the r e s o l u t i o n of the machine. In c u r r e n t l y a v a i l a b l e machines, s p a t i a l r e s o l u t i o n i s l i m i t e d by the s i z e of elements i n the d e t e c t o r array r a t h e r than by the more fundamental l i m i t a t i o n of mean f r e e path p r i o r to a n n i h i l a t i o n . PET has found an important a p p l i c a t i o n i n the study of the r a t e of glucose u t i l i z a t i o n i n b r a i n . T h i s method i s based upon animal work c h a r a c t e r i s i n g the uptake of 2-deoxy-D-( 1 < 4C)g1 ucose (( 1 J*C)DG) and subsequent phosphorylation by hexokinase ( S o k o l o f f e t a l . , 1977, S o k o l o f f , 1981). In c o n t r a s t to glucose, which i s o x i d i z e d to water and CO2, deoxyglucose i s not thought to be r a p i d l y metabolized beyond the 2 phosphorylation step. T h e r e f o r e , the product i s s a i d to be trapped and k i n e t i c a n a l y s i s assuming u n i d i r e c t i o n a l t r a n s p o r t followed by slow dephosphorylation of the t r a c e r may be a p p l i e d . An equation d e r i v e d f o r the determination of ( l i +C)DG uptake r a t e s contains a term composed of v a r i a b l e s (the lumped constant) r e l a t e d to the chemical s i m i l a r i t y of deoxyglucose and glucose. This term may be evaluated by the determination of Michaelis-Menton k i n e t i c constants of hexokinase and of b l o o d - b r a i n t r a n s p o r t r a t e s f o r the two compounds. The lumped constant may vary across species and i s found to be i n c r e a s e d under p h y s i o l o g i c a l c o n d i t i o n s such as hypoglycemia (Crane e t a l . , 1981). Although the v a r i o u s assumptions contained i n the ( 1 1 +C)DG model have been the s u b j e c t of some controversy (Fox, 1984), there has been s u f f i c i e n t consensus on t h e i r v a l i d i t y to l e a d to a c o n s i d e r a b l e l i t e r a t u r e on c e r e b r a l glucose metabolism s t u d i e d by a u t o r a d i o g r a p h i c methods. The study of c e r e b r a l glucose metabolism i n v i v o has been made p o s s i b l e by PET with 1 8 F s u b s t i t u t e d deoxyglucose (FDG) ( R e i v i c h e t a l . , 1979). The i n t e r p r e t i v e problems with the use of FDG are the same as with ( 1 4C)DG, with the a d d i t i o n a l concern t h a t the f l u o r i n e atom may a l t e r the p r o p e r t i e s of the deoxyglucose molecule. The k i n e t i c s of FDG t r a n s p o r t and p h o s p h o r y l a t i o n have been determined i n r a t b r a i n (Crane e t a l . , 1983) and have been shown i n the human to be v i r t u a l l y i d e n t i c a l to those f o r ( U C ) d e o x y g l u c o s e ( R e i v i c h e t a l . , 1985). Abnormalities i n FDG uptake have been s t u d i e d i n a v a r i e t y of human diseases such as Huntington's disease (Kuhl, 1982) and Alzheimer's disease (McGeer, P.L., 1986). The p o s s i b i l t y t h a t a p o s i t r o n e m i t t i n g analog of L-dihydroxyphenyl-a l a n i n e (L-DOPA) might serve as an agent f o r imaging o f c e n t r a l dopamine 3 metabolism f i r s t suggested more than ten years ago ( F i r n a u e t a l . , 1975). As i n the case of deoxyglucose, p r e l i m i n a r y work on the development of a gamma e m i t t i n g t r a c e r f o r dopamine metabolism has been conducted through s t u d i e s of the metabolism o f 3H or lkC l a b e l l e d compounds. I n i t i a l s t u d i e s concerned the p r o p e r t i e s of the t r a n s p o r t of DOPA across the b l o o d - b r a i n b a r r i e r (B.B.B). The t r a n s p o r t of DOPA i n t o b r a i n was o r i g i n a l l y c h a r a c t e r i s e d as o c c u r r i n g a t a s t e r e o s e l e c t i v e uptake s i t e c o n t a i n i n g a potassium a c t i v a t e d ATPase (Yoshida e t al ., 1963). The same uptake s i t e was apparently r e s p o n s i b l e f o r the t r a n s p o r t of L-OOPA, L-ph e n y l a l a n i n e , L-tryptophan and L - a l a n i n e , but not L-glutamate. The t r a n s p o r t of L-DOPA i n t o r a t b r a i n was found to have two components; a s a t u r a b l e component with a half-maximal t r a n s p o r t v e l o c i t y value o f 336 uM, and a d i f f u s i o n - r e l a t e d nonsaturable term which was only s i g n i f i c a n t a t high DOPA c o n c e n t r a t i o n s (Wade and Katzman, 1975). Uptake r a t e s were r e l a t i v e l y constant i n d i v e r s e b r a i n areas, i n c l u d i n g those without major catechol aminergic i n n e r v a t i o n s . L-DOPA, even at high c o n c e n t r a t i o n s , was found to be r a p i d l y decarboxylated throughout b r a i n ; a h i s t o f l u o r e s c e n c e experiment i n d i c a t e d that the d e c a r b o x y l a t i o n occurred i n the c a p i l l a r y endothelium and i n p e r i c y t e s i n agreement with a c l a s s i c a l r e p o r t on the B.B.B. ( B e r t l e r e t a l . , 1963). Although the c a p i l l a r y endothelium provides a uniform background of decarboxylase a c t i v i t y i n b r a i n , the t o t a l amounts of decarboxylase a c t i v i t y are much higher i n s t r i a t u m than i n cortex of post mortem human b r a i n (Lloyd and Hornykiewicz, 1972). l l +C-D0PA, when i n j e c t e d i n the mouse by the i . p . route, i s r a p i d l y metabolized i n the periphery, disappearing at a r a t e of 7.3% o f the i n j e c t e d dose per minute (Wurtman e t a l . , 1970). This study, i n which no p e r i p h e r a l decarboxylase i n h i b i t o r was used, re p o r t s t h a t the 1 4C-D0PA was 4 i n i t i a l l y metabolized to 1 I +C-dopamine but appears as lkC-0-methyldopa and l l t C - h o m o v a n i l l i c a c i d at times a f t e r 20 minutes. A f t e r intravenous i n j e c t i o n of r i n g - t r i t i a t e d DOPA i n the r a t , there was a p r e f e r e n t i a l accumulation of r a d i o a c t i v i t y i n the basal g a n g l i a (Liskowsky and P o t t e r , 1985). T h i s accumulation was not found a f t e r u n i l a t e r a l l e s i o n s to the n i g r o - s t r i a t a l t r a c t . Likewise, i n an a u t o r a d i o g r a p h i c study a f t e r 11+C-D0PA i n j e c t i o n s ( i . v . ) i n the c a t , the major accumulations o f r a d i o a c t i v i t y were i n the basal g a n g l i a at one hour a f t e r i n j e c t i o n ( P l a c i d i , G.F., e t a l . , 1976). In t h i s study, r e l a t i v e l y high a c t i v i t y was a l s o seen i n the hypothalamus, s u b s t a n t i a n i g r a , s u b s t a n t i a g r i s e a p e r i c e n t r a l i s , raphe n u c l e i and nucleus i n t e r p e d u n c u l a r i s . Treatment with a p e r i p h e r a l decarboxylase i n h i b i t o r d i d not a l t e r t h i s p a t t e r n of r a d i o c o n t r a s t , but had the e f f e c t of g r e a t l y i n c r e a s i n g t o t a l r a d i o a c t i v i t y i n b r a i n t i s s u e . S i m i l a r r e s u l t s have been noted a f t e r i n j e c t i o n s of ( 1 1 +C)-L-DOPA i n the r a t (Wooten and Home, 1981). I t has been shown t h a t the r e g i o n a l accumulation of r a d i o a c t i v i t y i n r a t b r a i n f o l l o w i n g ( 3H)-L-DOPA a d m i n i s t r a t i o n c o r r e l a t e s h i g h l y with the c o n c e n t r a t i o n of endogenous catecholamines (Home et a l . , 1984). The h i g h e s t 3H l e v e l s were found i n the caudate-putamen, with some enrichment a l s o found i n the amygdala, o l f a c t o r y t u b e r c l e , and the a n t e r i o r thalamic n u c l e i . Reserpine, which causes a d e p l e t i o n of catecholamines, reduced the accumulation of r a d i o a c t i v i t y i n these s t r u c t u r e s . Upon a n a l y s i s by high preformance l i q u i d chromatography (HPLC), the caudate-putamen was found to c o n t a i n l a r g e amounts of 3H-dopamine and 3H-dopamine m e t a b o l i t e s . Thus, a f t e r intravenous i n j e c t i o n of 3" e m i t t i n g l a b e l l e d DOPAs i n the r a t , there i s an accumulation of r a d i o a c t i v i t y i n s p e c i f i c b r a i n regions which r e c e i v e dopaminergic i n n e r v a t i o n and t h i s accumulation i n s t r i a t u m i s 5 dependent upon the f u n c t i o n a l i n t e g r i t y of the n i g r o - s t r i a t a l pathway. The development of p o s i t r o n e m i t t i n g DOPA analogs has mostly focused on two i s o t o p e s , U C and 1 8 F . U C s u f f e r s from two major disadvantages. F i r s t l y , the s h o r t h a l f - l i f e o f n C (20 minutes) would not permit the high s p e c i f i c a c t i v i t y needed f o r PET scans l a s t i n g several hours. Secondly, the e a s i e s t s y n t h e s i s of uC-D0PA y i e l d s a compound l a b e l l e d on the carboxyl carbon which i s l o s t on d e c a r b o x y l a t i o n . Thus, the development of c o n t r a s t between s t r i a t u m and other t i s s u e s i n s t u d i e s with uC-D0PA would depend upon the r a p i d l i b e r a t i o n and egress of n C 0 2 from s t r i a t u m , r a t h e r than an accumulation of the r a d i o l a b e l . For t h i s reason, r a t s r e c e i v i n g ( UC)-D,L-DOPA a f t e r carbidopa pretreatment (75 mg/kg i.p.) were found to have " c o l d spots" i n the s t r i a t u m where l e v e l s of U C were 50% lower than the c o r t i c a l l e v e l s a t 15 minutes (Korf e t a l . , 1978). The c o l d spots were not evident i n animals with 6-OHDA l e s i o n s to the n i g r o - s t r i a t a l t r a c t . The u t i l i t y of e n a n t i o m o r p h i c a l l y pure 1^-L-DOPA as a PET agent f o r human s t u d i e s has y e t to be determined. More a t t e n t i o n has been p a i d to the development of t r a c e r s f o r dopamine metabolism l a b e l l e d with 1 8 F , an isoptope with a number o f s i g n i f i c a n t p r o p e r t i e s . The r e l a t i v e l y long h a l f - l i f e (109.7 minutes) f a c i l i t a t e s the s y n t h e s i s of 1 8F-D0PA with high s p e c i f i c a c t i v i t y . S u b s t i t u t i o n on the aromatic r i n g i s c h e m i c a l l y convenient and the C-F bond i s s t a b l e . The major question to be addressed concerns the degree to which the i n c o r p e r a t i o n of a halogen atom a l t e r s the biochemical p r o p e r t i e s of the DOPA molecule. The f i r s t compound to be proposed as a p o s i t r o n e m i t t i n g t r a c e r f o r c e r e b r a l dopamine metabolism was D , L - 5 - 1 8 F - f l u o r o d o p a (5- 1 8F-D0PA) which was shown to be a s u b s t r a t e f o r aromatic amino a c i d decarboxylase 6 (AAADC, EC 4.1.1.26) with k i n e t i c constants s i m i l a r to those f o r ( l l tC)D0PA (Firnau e t a l . , 1975). Using a simple gamma d e t e c t o r , i n j e c t i o n of t h i s compound has been shown to be f o l l o w e d by an accumulation of r a d i o a c t i v i t y i n the heads of baboons (Garnett e t a l . , 1978a). The three p o s s i b l e r i n g - f l u o r i n a t e d isomers of fluorodopamine have a l l been shown to compete with l i g a n d s b i n d i n g to the D 2 , and to a l e s s e r extent the adenylate c y c l a s e l i n k e d Di s i t e s ( F i r n a u e t a l . , 1981). In a study of the a b i l i t y o f r i n g - f l u o r i n a t e d catecholamines to a c t as f a l s e n e u r o t r a n s m i t t e r s i n the r a t sympathetic nervous system, 6 - f l u o r o n o r e p i n e p h r i n e (F-NE) was shown to have a vasopressor potency e q u i v a l e n t to noradrenaline (Chieuh e t a l . , 1983). In the same study, 6-fluorodopamine (F-DA) was shown by HPLC with e l e c t r o c h e m i c a l d e t e c t i o n (HPLC-EC) to be taken up and s t o r e d i n s t r i a t u m f o l l o w i n g i n t r a v e n t r i c u l a r i n j e c t i o n i n c a t s . The s t o r e d F-DA could be r e l e a s e d upon s t i m u l a t i o n with K +. Nuclear magnetic resonance methods have a l s o been used to study the uptake and storage of 1 9F-DA i n guinea p i g synaptosomes i n v i t r o ( D i f f l e y e t a l . , 1983). The t r a n s p o r t of D , L - 1 8 F - 5 - f l u o r o d o p a i n t o b r a i n has been p a r t i a l l y c h a r a c t e r i s e d a c c o r d i n g to a three compartment model i n the monkey (Garnett e t a l . , 1980). The f i r s t compartment was the a r t e r i a l pool and the other two were, r e s p e c t i v e l y , c e r e b r a l endothelium and b r a i n c e l l s . E v a l u a t i o n of the components of f l u x i n and out of these compartments suggested that 20% of the 1 8F-D0PA e n t e r i n g the endothelium was a v a i l a b l e f o r d e c a r b o x y l a t i o n w i t h i n neurones. Thus, a d i v e r s e body of evidence suggests t h a t f l u o r i n a t e d catecholamines have b i o l o g i c a l p r o p e r t i e s s u f f i c i e n t l y s i m i l a r to those of the endogenous compounds to support t h e i r use as t r a c e r s f o r c e r e b r a l metabolism. 7 In the absence of any d e t a i l e d r e p o r t s on the metabolism of 6- 1 8F-fluoro-L-D0PA ( 1 8F-D0PA) i n v i v o , PET s t u d i e s with 1 8F-D0PA have proceeded i n primates and i n man. In Cynomolgus monkeys, an accumulation of r a d i o a c t i v i t y i n s t r i a t u m was observed f o l l o w i n g i . v . i n j e c t i o n of 1 8F-D0PA (Garnett e t a l . , 1983). The absolute a c t i v t y accumulating i n s t r i a t u m was i n c r e a s e d two-fold when animals were p r e t r e a t e d with Ro4-4046, an i n h i b i t o r of aromatic amino a c i d decarboxylase (AAADC), at a dose lower than t h a t r e q u i r e d to i n h i b i t the c e n t r a l enzyme (25 mg/kg). S i m i l a r r e s u l t s were a l s o seen i n conscious humans (Garnett e t a l . , 1983b). In PET experiments, the r a t i o of r a d i o a c t i v i t y detected i n s t r i a t u m to than i n r e f e r e n c e t i s s u e s which lack a catechol aminergic i n n e r v a t i o n , such as c o r t e x or c e r e b e l l u n , i s known as r a d i o c o n t r a s t . An asymmetric p a t t e r n of r a d i o c o n t r a s t has been rep o r t e d in a human case of i d i o p a t h i c hemi-Parkinsonism, with the l e s s e r r a d i o c o n t r a s t seen c o n t r a l a t e r a l to the s i d e o f the motor d e f i c i t (Garnett et a l . , 1984). In humans exposed to MPTP, a drug which depletes n i g r o - s t r i a t a l dopamine i n man and other primates, a general r e d u c t i o n i n r a d i o c o n t r a s t was noted i n comparison to normal age-matched c o n t r o l s (Calne e t a l . , 1985). Among Parkinsonians, the r a d i o c o n t r a s t between s t r i a t u m and other b r a i n areas was p a r t i c u l a r l y low i n the p a t i e n t s with the more severe form of the disease c h a r a c t e r i z e d by f l u c t u a t i n g "on/off" responses to L-DOPA therapy. (Leenders et a l . , 1986). The above r e s u l t s are taken to i n d i c a t e t h a t r a d i o c o n t r a s t f o l l o w i n g 1 8F-D0PA may be c o n s i d e r e d to be a f u n c t i o n of dopamine s y n t h e s i s r a t e i n human s t r i a t u m , c o n s i s t e n t with the animal s t u d i e s i n which the metabolism of 3H or lkC l a b e l l e d DOPA was used. A d d i t i o n a l i n f o r m a t i o n i s needed, however, before the PET scans can be p r o p e r l y i n t e r p r e t e d . Unlike FDG-phosphate which i s e s s e n t i a l l y trapped, 8 the 1 8F-D0PA which has been taken up by catecholamine t e r m i n a l s i s s u b j e c t to d e c a r b o x y l a t i o n and f u r t h e r metabolism. These m e t a b o l i t e s w i l l be l o s t from s t r i a t u m a t v a r y i n g r a t e s . The formation of r a d i o c o n t r a s t f o l l o w i n g 1 8F-D0PA a d m i n i s t r a t i o n must then be dependent upon a number of f a c t o r s . The uptake of F-DOPA by aminergic t e r m i n a l s i s followed by metabolism. The i d e n t i t y and p e r s i s t a n c e of these metabolites i n b r a i n t i s s u e would be a major f a c t o r i n the development of a k i n e t i c model f o r F-DOPA metabolism assuming u n i d i r e c t i o n a l t r a n s p o r t o f the t r a c e r . I t i s intended that t h i s t h e s i s c o n t r i b u t e towards the eventual r e s o l u t i o n of these problems. I I . General M a t e r i a l s and Methods A. M a t e r i a l s A l l chemicals were of the h i g h e s t a v a i l a b l e p u r i t y . L-Dihydroxyphenylalanine (DOPA), d i h y d r o x y p h e n y l a c e t i c a c i d (DOPAC), dopamine h y d r o c h l o r i d e (DA), h o m o v a n i l l i c a c i d (HVA), dihydroxybenzylamine (DHBA), dihydroxybenzoic a c i d (DBA), S-adenosylmethionine p - t o l u e n s u l f o n a t e (SAM), heparin (160 i.u./mg), p a r g y l i n e h y d r o c h l o r i d e and p y r i d o x a l phosphate were a l l from Sigma. ( l k C Methyl)-S-adenosylmethionine ( 1 4C-SAM, 59.8 mCi/mmole) was from New England Nuclear and l l +C-L-D0PA (5.4 mCi/mmol) was from Amersham. 6-F1uoronoradrenaline (F-NA) was from Research Biochemicals. Carbidopa was provided by Merck, Sharpe and Dohme. 3',4'-Dihydroxy-2-methyl-propiophenone (U-0521) was the generous g i f t o f Upjohn. HPLC b u f f e r s were made with water f r e s h l y r e d i s t i l l e d over a l k a l i n e potassium permanganate and with HPLC grade methanol from F i s h e r S c i e n t i f i c . Sodium heptanesulfonate was from A l d r i c h and e t h y l e n e d i a m i n e t e t r a a c e t i c a c i d (EDTA) disodium s a l t was from MCB. P e r c h l o r i c a c i d i n a 70% aqueous 9 s o l u t i o n was from Baker. HPLC grade a c e t i c a c i d was from BDH and sodium hydroxide was from Chemonics. Non-radioactive D,L-F-D0PA was s y n t h e s i z e d at TRIUMF by an 11 step s y n t h e s i s s t a r t i n g with v e r a t r i c a c i d ( G r i e r s o n and Adam, i n p r e s s ) . The compound was q u a n t i t a t i v e l y f l u o r i n a t e d i n the 6 - p o s i t i o n and contained approximately 1.6 waters of h y d r a t i o n . P o s i t r o n e m i t t i n g 1 8 F was produced with a CP-42 c y c l o t r o n by the N e ( p , x ) 1 8 F r e a c t i o n . 1 8 F - F gas thus produced was converted to a c e t y l h y p o f l u o r i t e and t h i s compound rea c t e d with a DOPA d e r i v a t i v e as d e s c r i b e d by Adam e t a l . (1986). 1 8F-D0PA and 1 8 F - 2 - f l u o r o d o p a were simultaneously prepared and separated at TRIUMF, each was g r e a t e r than 97% of the L-enantiomorph (Adam e t al ., i n p r e s s ) . The r a t i o of 2- and 6-fluorodopa produced d a i l y was 0.80 ± 0.02 (n=4). The s p e c i f i c a c t i v i t y of 1 8F-D0PA was r o u t i n e l y 175-230 C/mole a t the end of s y n t h e s i s . The 1 8F-D0PA was provided i n a 0.1% a c e t i c a c i d s o l u t i o n a t a c o n c e n t r a t i o n of 2.5 ± 0.2 mM (n = 3), the c o n c e n t r a t i o n o f 1 8F-D0PA being determined by u l t r a v i o l e t a b s o r p t i o n at 283 nM with D,L-F-D0PA s e r v i n g as the standard. B. General HPLC Methods F-DOPA and metabolites i n b i o l o g i c a l samples were separated by reversed phase high preformance l i q u i d chromatography (HPLC). The s o l v e n t d e l i v e r y was provided by two high pressure pumps ( M i l l i p o r e Waters, Model 510) under the r e g u l a t i o n o f a Waters automatic g r a d i e n t c o n t r o l l e r . Compounds were e l u t e d a c c o r d i n g to a g r a d i e n t program t y p i c a l l y c o n s i s t i n g of 10 minutes i s o c r a t i c flow at 0.75 ml/min b u f f e r A (0.8% a c e t i c a c i d a t pH 3.8, 1.5 mM EDTA, 4 mM h e p t a n e s u l f o n i c a c i d , 10% methanol) followed by a l i n e a r flow and composition g r a d i e n t to a flow r a t e o f 1.6 ml/min with a composition of 85% b u f f e r A and 15% b u f f e r B (50:50 methanol/water) T h i s protocol i s based upon a previous r e p o r t (Cheng and Wooten, 1982) except t h a t i t was necessary to modify the i o n - p a i r c o n c e n t r a t i o n i n order to permit a se p a r a t i o n of some of the fluoro-compounds. Therefore, c o n d i t i o n s which provided a s e p a r a t i o n of r a d i o l a b e l e d compounds were not always a p p r o p r i a t e f o r simultaneous s e p a r a t i o n o f endogenous compounds. Samples were loaded v i a a sample i n j e c t o r (Waters U6K) l e a d i n g to a precolumn i n s e r t (Waters Guard-Pak). The precolumn i n s e r t was changed r e g u l a r l y , e s p e c i a l l y a f t e r a n a l y s i s of plasma e x t r a c t s . Compounds were separated on a 300 x 3. 9 mm CIQ reverse phase a n a l y t i c a l column (Milipore-Waters, uBondapak). As the a n a l y t i c a l columns d e t e r i o r a t e d i n the course of time, i t was necessary to modify the composition of b u f f e r A approximately every 50 i n j e c t i o n s . As e l u t i o n times decreased, the c o n c e n t r a t i o n o f methanol i n b u f f e r A was g r a d u a l l y reduced to about 3%; a t t h i s stage i t was necessary to i n s t a l l a new column because of poor r e s o l u t i o n (< 3000 t h e o r e t i c a l p l a t e s per metre). The data a q u i s i t i o n module of the HPLC was c a l i b r a t e d using catecholamine standards with DHBA (100 ng/ml) s e r v i n g as the i n t e r n a l s tandard. Endogenous catecholamines were q u a n t i f i e d with e l e c t r o c h e m i c a l d e t e c t i o n a t a p o t e n t i a l o f +0.72 v o l t s with r e s p e c t to an Ag/AgCl ref e r e n c e e l e c t r o d e (BAS RE-3). F r a c t i o n s were c o l l e c t e d i n p l a s t i c gamma counting tubes by an automated f r a c t i o n c o l l e c t o r ( G i l s o n Model 201) programmed to c o l l e c t 50 f r a c t i o n s at a ra t e of 0.75 minutes/tube f o l l o w i n g a two minute d r a i n c y c l e . The voi d volume of the column was 1.5 ml and the volume between the e l e c t r o c h e m i c a l d e t e c t o r and the f r a c t i o n c o l l e c t o r was 0.5 ml. F r a c t i o n s were counted f o r one minute i n a gamma counter (Packard Auto-Gamma 5650) with the window set a t 20-2000 KeV. Background a c t i v i t y 11 was determined d a i l y (~200 CPM) and was a u t o m a t i c a l l y s u b t r a c t e d . The a c t i v i t y i n each tube was c o r r e c t e d to the time of 1 8F-D0PA a d m i n i s t r a t i o n with a programmable c a l c u l a t o r using the h a l f - l i f e o f 109.7 minutes. Radiochromatograms were p l o t t e d out and the summed a c t i v i t i e s i n each r e s o l v e d radiochemical peak was determined. The t o t a l a c t i v i t y was determined i n a volume of sample equal to the amount i n j e c t e d onto the HPLC and the a c t i v i t y i n each radiochemical peak was c a l c u l a t e d as a percentage of t o t a l r a d i o a c t i v i t y i n the sample. I I I . S p e c i a l Methods and Results A. I d e n t i f i c a t i o n o f M e t a b o l i t e s 1) Methods The O-methylated me t a b o l i t e of 1 8F-D0PA was prepared i n v i t r o by in c u b a t i o n of 1 8F-D0PA with p a r t i a l l y p u r i f i e d catechol-0-methytransferase (COMT) fo l l o w e d by a n a l y s i s of product with HPLC. B r i e f l y , r a t l i v e r COMT was p a r t i a l l y p u r i f i e d from a d u l t male Wistar r a t s by f r a c t i o n a t i o n of crude homogenate with ammonium sulp h a t e . The 30-55% s a t u r a t i o n ammonium sulphate f r a c t i o n was r e d i s s o l v e d i n b u f f e r and d i a l y z e d o v e r n i g h t . 200 pi of the d i a l y s a t e was f u r t h e r f r a c t i o n a t e d by HPLC s i z e e x c l u s i o n chromatography with a Waters P r o t e i n Pak-300 column. One ml f r a c t i o n s were c o l l e c t e d and t e s t e d f o r COMT a c t i v i t y using DBA as the su b s t r a t e ( G u l l i v e r and T i p t o n , 1978). The two tubes c o n t a i n i n g the m a j o r i t y of a c t i v i t y were pooled and st o r e d at -20 °C i n the presence of 10% ethylene g l y c o l . The enzyme had a s p e c i f i c a c t i v i t y of 32 m i l l i u n i t s / m g p r o t e i n ( u n i t a c t i v t y i n umole product/minute) and was s t a b l e over a one month p e r i o d . -Radiochemical p u r i t y of the 1 8F-D0PA was checked by a n a l y s i s of 10 6 CPM 1 8F-D0PA by reversed phase HPLC i n f r a c t i o n c o l l e c t i o n mode. Double 12 l a b e l l e d product was formed under the f o l l o w i n g c o n d i t i o n s : 10 6 CPM 1 8F-D0PA was incubated at 37 °C i n the presence of 0.5 pCi SAM, 70 uunits COMT, and 4.0 mM Mg 2 + i n 100 mM phosphate b u f f e r at pH 7.4, and a t o t a l volume of 50 ul. Samples of the r e a c t i o n mixture were taken a t i n t e r v a l s and e x t r a c t e d with two volumes of 0.1 M p e r c h l o r i c a c i d f o r a n a l y s i s by HPLC i n f r a c t i o n c o l l e c t i o n mode. Samples were analyzed by a shallow g r a d i e n t , to 10% b u f f e r B a t a t o t a l flow rate of 1.25 ml/min, with c o l l e c t i o n of 25 0.75 minute f r a c t i o n s . In c o n t r o l r e a c t i o n s , one of the components, 1I+C-SAM, COMT or 1 8F-D0PA, was omitted. Gamma a c t i v i t y was determined f o r each f r a c t i o n and the contents of each tube were t r a n s f e r r e d i n t o g l a s s s c i n t i l l a t i o n v i a l s . 1 8 F a c t i v i t y was allowed to decay ove r n i g h t , and, f o l l o w i n g a d d i t i o n of 10 m l / v i a l of aqueous s c i n t i l l a t i o n c o c k t a i l (Amersham), 1 4 C a c t i v i t y was determined i n a l i q u i d s c i n t i l l a t i o n counter ( P h i l l i p s 4700) with two minute counts. 1 8F-6-Fluorodopamine ( 1 8F-DA) was prepared as f o l l o w s : 10^ CPM 1 8F-D0PA were incubated at 37 °C i n the presence of 100 uM p a r g y l i n e , 100 ul 10:1 s t r i a t a l homogenate ( T r i t o n - X 0.1%), and 50 uM p y r i d o x a l phosphate i n a f i n a l volume of 500 ul 100 uM phosphate b u f f e r at pH 7.4. Samples were taken at i n t e r v a l s between f i v e and 60 minutes and analysed by HPLC with a shallow g r a d i e n t followed by gamma counting of 40 f r a c t i o n s / s a m p l e . In c o n t r o l samples, e i t h e r s t r i a t a l homogenate or p y r i d o x a l phosphate were omitted. In order to s y n t h e s i z e 1 8 F - d i h y d r o x y p h e n y l a c e t i c a c i d ( 1 8F-D0PAC), c o n d i t i o n s were as above except that p a r g y l i n e was omitted. In one sample, carbidopa was i n c l u d e d a t a c o n c e n t r a t i o n of 100 uM. Because of the short h a l f - l i f e of 1 8 F and the d i f f i c u l t y i n i s o l a t i n g products, i t d i d not prove f e a s i b l e to i d e n t i f y the methylated 1 8F-DA met a b o l i t e s 1 8 F - h o m o v a n i l l i c a c i d ( 1 8F-HVA) and 1 8F-3-methoxytyramine 13 ( 1 8F-3-MTyr) by double l a b e l l i n g experiments. Instead, compounds b e l i e v e d to be unlabel l e d F-DA and F-DOPAC were prepared as above using 25 mM c o l d D,L-F-D0PA with i n c u b a t i o n s l a s t i n g two hours to f o r c e the exhaustive metabolism of s t a r t i n g m a t e r i a l . The methylated metabolites were obtained by i n c u b a t i o n of 10 y l samples c o n t a i n i n g e i t h e r F-DA or F-DA + F-DOPAC f o r 45 minutes a t 37 °C i n the presence of bl w/v crude l i v e r homogenate, 4 mM Mg 2 +, and 2 yM u n l a b e l l e d SAM i n 1.0 ml phosphate b u f f e r a t pH 7.4. At each stage, 20 yl samples were analyzed by HPLC with e l e c t r o c h e m i c a l d e t e c t i o n a t a s e n s i t i v i t y of 5 nA/V i n order to monitor the formation of m e t a b o l i t e s . 2) Res u l t s o f M e t a b o l i t e I d e n t i f i c a t i o n A n a l y s i s of irj6 CPM 1 8F-D0PA by HPLC ( F i g . 1) i n d i c a t e s t h a t the mater i a l i s e s s e n t i a l l y r a d i o c h e m i c a l l y pure. Therefore, a d d i t i o n a l peaks observed i n b i o l o g i c a l samples cannot be a s c r i b e d to the presence of contaminants i n the t r a c e r . The radiochromatogram of a 20 yl plasma sample obtained 20 minutes a f t e r 1 8F-D0PA a d m i n i s t r a t i o n to a carbidopa p r e t r e a t e d human i s shown i n F i g . 2A. Two major peaks and a number of minor peaks are present. The f i r s t major peak, e l u t i n g a t f r a c t i o n 9, corresponds to 1 8F-D0PA. The r a p i d r a t e o f appearance and the i n c r e a s e d r e t e n t i o n time o f the second compound present i n plasma suggested t h a t i t was an O-methylated d e r i v a t i v e (Me- 1 8F-D0PA). T h i s i d e n t i f i c a t i o n was supported by the r e s u l t s of the 1 1 + C / 1 8 F double l a b e l l i n g experiment using p a r t i a l l y p u r i f i e d COMT. The radiochromatograms of the r e a c t i o n products from t h i s experiment are i l l u s t r a t e d i n Figure 3(A-D). 1 8 F a c t i v i t y a f t e r i n c u b a t i o n of 1 8F-D0PA i n the presence of 1 4C-SAM i s i l l u s t r a t e d i n F i g u r e 3A, i n c o n t r a s t to the 14 10-i 8 I in • O x 6 Q_ ° 41 2 10 15 Fraction Number 20 25 F i g u r e 1: R a d i o c h r o m a t o g r a m o f 18F-D0PA. A 20 p i s a m p l e c o n t a i n i n g a p p r o x i m a t e l y 106 CPM l8F-D0PA was a n a l y z e d b y r e v e r s e d p h a s e HPLC i n o r d e r t o d e t e r m i n e t h e e l u t i o n t i m e o f t h e c o m p o u n d a n d t o v e r i f y i t s r a d i o c h e m i c a l p u r i t y . 1.2 F-DOPA Me-F-DOPA 1 1 0 1 5 2 0 2 5 B F - D A F - H V A — r -4 0 F - 3 M e T y r 5 0 3 0 Fraction Number F i g u r e 2: R a d i o c h r o m a t o g r a m s s h o w i n g t h e m e t a b o l i s m o f 18F-D0PA i n human p l a s m a a n d r a t s t r i a t u m . A n a l y s i s o f 20 u l human p l a s m a t a k e n 20 m i n u t e s a f t e r 18F-D0PA i n j e c t i o n ( A ) i n d i c a t e s t h e p r e s e n c e o f two l a b e l e d c o m p o u n d s . No p e a k s w e r e o b s e r v e d e l u t i n g a f t e r f r a c t i o n 25 ( n o t i l l u s t r a t e d ) . A n a l y s i s o f 200 y l 10:1 s t r i a t a l e x t r a c t a t t e n m i n u t e s a f t e r 18F-D0PA i n j e c t i o n (B) i n d i c a t e s t h e p r e s e n c e o f a t l e a s t s i x c o m p o u n d s . 1 5 H 10H 5 -1 10 15 Fraction Number Figure 3(A,B): Identification of Me-18F-D0PA as the major plasma metabolite of 18F-D0PA. Radiochromatograms showing 1 8F activity from the in vitro synthesis of double labelled (lltC)Me-18F-00PA in the presence (A) and absence (B) of 1 **C-S-adenosy"Imethionine. 17 C 1 • Fraction Number Figure 3(C,D): Identification of Me-18F-D0PA as the major plasma metabolite of 18F-D0PA. Radiochromatograms showing 1 4C activity from the in vitro synthesis of double labelled (11+C)Me-18F-D0PA in the presence (0 and absence (D) of 18F-D0PA. The off-scale peak at fraction 25 is ll4C-S-adenosyl-methionine. 18 r e s u l t when SAM was omitted ( F i g . 3B). The lkC radiochromatogram corresponding to Figure 3A i s i l l u s t r a t e d i n Figure 3C. The lkC l a b e l l e d peak e l u t i n g a t f r a c t i o n s 15-16 was absent i n the samples where 1 8F-D0PA ( F i g . 3D) or COMT were omitted. Other peaks i n the lkC chromatograms were due to lltC-SAM i t s e l f ( f r a c t i o n 24) and minor contaminants. Since the only f r a c t i o n c o n t a i n i n g both 1'tC and 1 8 F a c t i v i t y ( f r a c t i o n s 15-16) c o e l u t e d with the second 1 8 F peak i n the plasma sample, the plasma m e t a b o l i t e may be i d e n t i f i e d as Me- 1 8F-D0PA. A radiochromatogram of r a t s t r i a t u m taken a t ten minutes a f t e r 1 8F-D0PA a d m i n i s t r a t i o n i s c o n s i d e r a b l y more complex than the plasma sample ( F i g . 2B). Under the chromatographic c o n d i t i o n s used i n t h i s experiment, 1 8F-D0PA e l u t e d a t f r a c t i o n 7 and Me- 1 8F-D0PA a t f r a c t i o n 12. Four a d d i t i o n a l m etabolites may be i d e n t i f i e d on the b a s i s of the i n v i t r o experiments. The r e s u l t o f an i n c u b a t i o n of 1 8F-D0PA under c o n d i t i o n s f a v o r a b l e f o r the decarboxylase and i n the presence o f p a r g y l i n e i s i l l u s t r a t e d i n Figure 4A. The sol e r a d i o a c t i v e species present were 1 8F-D0PA and a compound e l u t i n g a t f r a c t i o n 23. When p a r g y l i n e was omitted, an a d d i t i o n a l radiochemical peak was present at f r a c t i o n 12. When pyri d o x a l phosphate was omitted, or when carbidopa was present, only t r a c e s of these products were observed (not shown). The two compounds may t h e r e f o r e be i d e n t i f i e d as 1 8F-DA and 1 8F-D0PAC. Incubation of c o l d F-DA and F-DOPAC under c o n d i t i o n s promoting COMT produced small amounts of two new e l e c t r o c h e m i c a l peaks (not shown). These were presumably the O-methylated F-DA meta b o l i t e s F-HVA ( f r a c t i o n 32 i n F i g . 2B) and F-344Tyr ( F r a c t i o n 46 i n F i g . 2B). The r e t e n t i o n times of the s i x i d e n t i f i e d compounds i n r a t striatum at 15 minutes, of 6 - f l u o r o n o r a d r e n a l i n e (F-NA) and of pure samples of the 19 3n 2-1 • • O Q_ O 10 2 0 Fraction Number Figure 4: Identification of 18F-DA and 18F-D0PAC as important 18F-D0PA metabolites in rat striatum. Reaction mixtures contained 18F-D0PA, striatal homogenate, pyridoxal phosphate and phosphate buffer (pH 7.2). Radiochromatograms show the results of incubations conducted in the presence (A) and absence (B) of pargyline. 20 n o n - f l u o r i n a t e d analogs were determined under i s o c r a t i c c o n d i t i o n s (1.2 ml/min b u f f e r A, data not shown). Capacity f a c t o r s ( k 1 ) were c a l c u l a t e d from the e l u t i o n times of the various species and are l i s t e d i n Table I. Although the i d e n t i f i c a t i o n of some of the 1 8F-D0PA met a b o l i t e s i s not e n t i r e l y r i g o r o u s , the c a p a c i t y f a c t o r s lend strong independent support. I t seems t h a t the i n c o r p o r a t i o n of f l u o r i n e i n t o these molecules serves to i n c r e a s e t h e i r h y d r o p h o b i c i t y and thus i n c r e a s e t h e i r r e t e n t i o n times. Capacity f a c t o r s of the f l u o r i n a t e d analogs were uniformly i n c r e a s e d by approximately 60% with r e s p e c t to the o r d i n a r y catecholamines. T h i s phenomenon may have some bearing on the d i f f e r i n g biochemical p r o p e r t i e s o f DOPA and F-DOPA sin c e the g r e a t e r h y d r o p h o b i c i t y of the fluorocompounds might a l t e r some of the k i n e t i c parameters of the v a r i o u s compounds, p a r t i c u l a r l y f o r uptake processes. B. A n a l y s i s of Plasma Metabolism 1) Methods For the a n a l y s i s of p e r i p h e r a l 1 8F-D0PA metabolism i n humans, a r t e r i a l blood samples were obtained from experimental subjects f o r PET. Subjects were p r e t r e a t e d with carbidopa (100 mg/oral) one hour p r i o r to r e c e i v i n g 1 8F-D0PA (3 mCi, i . v . ) . F i v e ml blood samples, obtained at i n t e r v a l s between f i v e and 120 minutes a f t e r 1 8F-D0PA i n j e c t i o n , were c e n t r i f u g e d f o r 10 minutes i n a c l i n i c a l c e n t r i f u g e . In i n i t i a l s t u d i e s , samples were analyzed by HPLC with a f u l l g r a d i e n t by the f o l l o w i n g procedure: T o t a l a c t i v i t y i n a 1 ml plasma sample was determined by gamma counting. The remaining plasma was d e p r o t e i n i z e d by the a d d i t i o n of an equal volume of 0.2 M p e r c h l o r i c a c i d f o l l o w e d by c h i l l i n g on i c e f o r 30 minutes before c e n t r i f u g a t i o n f o r 30 minutes at 10,000 g (Savant high speed c e n t r i f u g e ) . Table I. Capacity F a c t o r s i n the HPLC System f o r Some U n f l u o r i n a t e d and F l u o r i n a t e d Catecholamines and M e t a b o l i t e s * U n f l u o r i n a t e d k' F l u o r i n a t e d k' k'(F)/k' DOPA 0.71 1 8F-D0PA 1.2 1.71 Me-DOPA 1.74 Me- 1 8F-D0PA 2.5 1.47 NA 1.7 F-NA 2.9 1.70 DOPAC 2.3 ( 1 8F-D0PAC) 3.7 1.60 DA 4.3 ( 1 8F-DA) 7.4 1.72 HVA 6.0 ( 1 8F-HVA) 9.6 1.60 3-MTyr 9.9 ( 1 8F-3-MTyr) 16.5 1.67 *The c a p a c i t y f a c t o r k' or k'(F) i s c a l c u l a t e d as V r/V 0 - 1, where V r i s the r e t e n t i o n volume at i s o c r a t i c e l u t i o n and V 0 i s the voi d volume of the a n a l y t i c a l column. Of the f l u o r i n a t e d compounds, only 1 8F-D0PA and F-NA were a v a i l a b l e i n pure form; the i d e n t i f i c a t i o n of Me- 1 8F-D0PA i s d i s c u s s e d i n the t e x t . The i d e n t i t i e s of the other f l u o r i n a t e d d e r i v a t i v e s must be considered to be t e n t a t i v e . 22 Samples of supernatant were taken to c a l c u l a t e e x t r a c t i o n e f f e c i e n c y . To the remaining plasma e x t r a c t was added sodium heptanesulfonate (10 mM) and 11+C-D0PA (50,000 DPM/ml). Plasma e x t r a c t s were then loaded onto a (43 Sepak column, washed with f i v e ml water and then e l u t e d with 1 ml methanol. The methanol was evaporated under helium and the residue was r e d i s s o l v e d i n 250 yl 0.1 M p e r c h l o r i c a c i d and c e n t r i f u g e d f o r ten minutes a t 10,000 g. 200 pi of the supernate was i n j e c t e d onto the HPLC f o r a n a l y s i s . A f t e r gamma counting of f r a c t i o n s , the contents of each tube were t r a n s f e r r e d i n t o a s c i n t i l l a t i o n v i a l f o r determination of lltC-D0PA recovery a f t e r a s u i t a b l e delay i n order to allow the decay of 1 8 F . In other c o n t r o l experiments, 1 8F-D0PA (10^ CPM/ml) was added to f r e s h d e p r o t e i n i z e d plasma i n order to c a l c u l a t e the recovery of 1 8F-D0PA alone. A f t e r the p e r i p h e r a l metabolism of 1 8F-D0PA had been c h a r a c t e r i z e d i n a number of s u b j e c t s , i t became apparent t h a t plasma samples from carbidopa p r e t r e a t e d humans contained only 1 8F-D0PA and Me- 1 8F-D0PA. This suggested the use a s i m p l i f i e d alumina f r a c t i o n a t i o n o f plasma samples, which seemed p r e f e r a b l e because the h a l f - l i f e of 1 8 F permitted the a n a l y s i s of only f o u r or f i v e samples per day by the HPLC method. In the alumina s e p a r a t i o n , blood samples were taken a t 5, 10, 20, 30, 75, 90, 105 and 120 minutes a f t e r 1 8F-D0PA i n j e c t i o n . Blood was handled i n sets of f o u r e a r l y and f o u r l a t e samples. T r i p l i c a t e 500 yl plasma samples from each time p o i n t were taken f o r determination of t o t a l a c t i v i t y . Other t r i p l i c a t e 500 pi samples were p i p e t t e d i n t o 1.5 ml eppendorf c e n t r i f u g e tubes. To each eppendorf tube was then added 80 mg a c i d washed alumina (Macco), 10,000 DPM lltC-D0PA, and 500 pi 0.5 M T r i s b u f f e r a t pH 9.0. Sets of 12 tubes were placed on a F i s h e r Roto-rak and r o t a t e d f o r f i v e minutes i n subdued l i g h t to permit a d s o r p t i o n of c a t e c h o l s . Tubes were then c e n t r i f u g e d f o r one minute at 23 10,000 g and the supernatants t r a n s f e r r e d i n t o separate gamma tubes. The contents of each eppendorf were then vortexed b r i e f l y with one ml water, r e c e n t r i f u g e d and the supernatants pooled with the basic supernatants; t h i s sample being designated h e r e a f t e r as the " f r e e " f r a c t i o n . The alumina was then subjected to two se q u e n t i a l e x t r a c t i o n s with 750 ul 0.1 M p e r c h l o r i c a c i d . Each tube was vortexed f o r 30 seconds before removal of supernatants to gamma tubes, thus l i b e r a t i n g the "bound" f r a c t i o n . Gamma a c t i v i t y i n each tube was determined by 1 minute counts followed by c o r r e c t i o n to the time of i n j e c t i o n of the p a t i e n t . A f t e r decay o f the 1 8 F , contents of the a c i d e x t r a c t tubes were t r a n s f e r r e d to s c i n t i l l a t i o n v i a l s f o r determination of 1 4C-D0PA recovery. The average value f o r 1J*C recovery (75-95%) f o r each t r i p l i c a t e sample was used to c o r r e c t the a c t i v i t i e s determined f o r 1 8F-D0PA as a f u n c t i o n of time. The metabolism of 1 8F-D0PA i n Cynomolgus monkeys was a l s o determined by the above method except t h a t the higher radiochemical dose (1 mCi/3 kg animal) permitted the use of small e r (100 y l ) plasma samples. For the determination of 1 8F-D0PA metabolism i n the r a t , male hooded r a t s weighing between 250 and 350 grams were provided with i n t r a j u g u l a r cannulae ( S i l a s t i c , 1.3 mm o.d., 0.9 mm i . d ) . I n i t i a l l y , surgery was performed under p e n t o b a r b i t a l a n e s t h e s i a (50 mg/kg i n propylene g l y c o l : w a t e r , i . p . ) . However, a high i n c i d e n c e of a n e s t h e t i c death under p e n t o b a r b i t a l anesthesia l e d to the adoption of choloropent, a m i l d e r a n e s t h e t i c . Chloropent was made as f o l l o w s : 1.06 g c h l o r a l hydrate, 243 mg p e n t o b a r b i t a l and 532 mg MgS04 i n 44 ml propylene g l y c o l , 12 ml ethanol and water were mixed to a f i n a l volume of 100 ml (1 ml/kg i . p . ) . P r e s u r g i c a l treatment with a t r o p i n e s u l f a t e reduced the i n c i d e n c e of r e s p i r a t o r y d i s t r e s s . Cannulae were f l u s h e d d a i l y with 250 yl 0.9% NaCl 24 c o n t a i n i n g 200 i.u./ml heparin. Animals were allowed to recover from surgery f o r between one and three days i n order to avoid p o s s i b l e e f f e c t s of a n e s t h e t i c on dopamine metabolism (Ford and Marsden, 1986). On the day of the experiments, r a t s r e c e i v e d carbidopa (5 mg/kg i . p . ) as an aqueous suspension f o l l o w e d 30 minutes l a t e r by i n j e c t i o n of the 1 8F-D0PA (500 uCi/kg) v i a the i n t r a j u g u l a r c a t h e t e r which was then f l u s h e d with 250 pi h e p a r i n i z e d s a l i n e . At times between f i v e and 90 minutes, 150 pi blood samples were taken v i a the c a t h e t e r and d i s c a r d e d . Immediately a f t e r each 150 pi sample, a 600 pi blood sample was removed i n t o an eppendorf tube and placed on i c e . The blood volume of the r a t (~ 20 ml) permitted the t a k i n g of a maximum of f o u r samples per animal. However, with the r e l a t i v e l y high radiochemical dose used i n r a t s t u d i e s (10 times the human dose as a f u n c t i o n of body weight) , i t was p o s s i b l e to analyze a l a r g e number of plasma samples i n the course of a day. Plasma was separated from blood by c e n t r i f u g a t i o n and d e p r o t e i n i z e d by the a d d i t i o n of an equal volume of 0.25 M p e r c h l o r i c a c i d . Since the haematocrit f o r r a t blood was n e a r l y two-fold higher than i n human samples t h i s procedure only provided about 250 pi plasma per sample. One hour on i c e was r e q u i r e d to p r e c i p i t a t e f u l l y the p r o t e i n i n r a t plasma. A f t e r c e n t r i f u g a t i o n f o r 45 minutes a t 10,000 g, 100 pi samples o f e x t r a c t were i n j e c t e d onto the HPLC. For the determination of recovery of r a d i o a c t i v i t y , 50 pi samples o f both plasma and plasma e x t r a c t were taken f o r gamma counting. Complete radiochemical chromatograms were produced f o r r a t plasma at a number of time p o i n t s between f i v e and 90 minutes. Upon determination t h a t no s i g n i f i c a n t peaks e l u t e d a f t e r Me- 1 8F-D0PA, r a t plasma samples were f r a c t i o n a t e d by a r a p i d i s o c r a t i c a n a l y s i s (1.2 ml/min, 90% b u f f e r A, 10% b u f f e r B) with c o l l e c t i o n o f 12 0.8 minute f r a c t i o n s . In a seperate study to determine the e f f e c t of COMT i n h i b i t i o n , carbidopa p r e t r e a t e d r a t s were administered U-0521 ( i . p . , 25 mg/kg) at ten minutes p r i o r to 1 8F-D0PA a d m i n i s t r a t i o n . Blood samples were taken a t times between f i v e and 120 minutes a f t e r 1 8F-D0PA a d m i n i s t r a t i o n and analysed as above. 2) R e s u l t s of Plasma Experiments The r e s u l t s of e x t r a c t i o n recovery experiments are i l l u s t r a t e d i n Table I I . Total recovery of r a d i o a c t i v i t y from human plasma samples analyzed by HPLC was approximately the same at 15 and 60 minutes a f t e r a d m i n i s t r a t i o n . Since m e t a b o l i t e accumulates r a p i d l y i n plasma (see below), t h i s r e s u l t suggests t h a t both 1 8F-D0PA and i t s m e t a b o l i t e are recovered t o the same extent during a n a l y s i s by HPLC. In the experiments i n which 1 8F-D0PA was added to e i t h e r human plasma o r to p e r c h l o r i c a c i d e x t r a c t s o f plasma, r e c o v e r i e s were on the order of 80%. The lack of d i f f e r e n c e between groups i n d i c a t e s that the l o s s of r a d i o a c t i v i t y during HPLC a n a l y s i s was not r e l a t e d to the e x t r a c t i o n procedure. It seems more l i k e l y t h a t r e c o v e r i e s were a d v e r s e l y a f f e c t e d by n o n - s p e c i f i c b i n d i n g of l a b e l l e d compounds to the guard column. The experiments i n which 1 4C-D0PA recovery from plasma were determined i n d i c a t e that l l tC-D0PA and 1 8F-D0PA were not d i f f e r e n t i a l l y recovered from plasma and t h a t 1 4C-D0PA may t h e r e f o r e be r e l i a b l y used i n order to c o r r e c t f o r the recovery of 1 8F-D0PA. Radiochromatographs of human plasma samples taken a t a wide range of times ( F i g s . 5(A-D)) i n d i c a t e the presence of only two major l a b e l l e d compounds, 1 8F-D0PA and Me- 1 8F-D0PA. Metabolism was r a p i d ; a t 7.5 minutes the two compounds were present a t roughly equal c o n c e n t r a t i o n s . The disappearance of 1 8F-D0PA from plasma was continuous, but Me- 1 8F-D0PA Table II The Recovery of To t a l Plasma R a d i o a c t i v i t y i n HPLC F r a c t i o n s . Sampl e % Recovery, mean ± S.D. (n) ( 1 8 F ) a t 15 minutes scan time (a) 76.1 ± 6.6 (3) ( 1 8 F ) a t 60 minutes scan time (a) 75.6 ± 9.3 (3) 1 8F-D0PA added to PCA (b) 78.4 ± 7.6 (5) 1 8F-D0PA added to plasma (c) ^C-DOPA added to plasma (c) 80.3 ± 3.5 (5) 80.6 ± 11 (5) (a) Samples obtained from a human s u b j e c t undergoing PET scanning, a t 15 and 60 minutes a f t e r a d m i n i s t r a t i o n o f 1 8F'-00PA. Recovery c a l c u l a t e d as the sum of 1 8F-D0PA and Me- 1 8F-D0PA f r a c t i o n s from HPLC, r e l a t i v e t o t o t a l a c t i v i t y i n an e q u i v a l e n t volume of p e r c h l o r i c a c i d (PCA) e x t r a c t of the same sample. (b) The r a d i o t r a c e r was added to PCA e x t r a c t i o n b u f f e r p r i o r to a n a l y s i s by HPLC. (c) The r a d i o t r a c e r s were added to f r e s h l y c o l l e c t e d human plasma which was then e x t r a c t e d with PCA and analyzed by HPLC. m F-DOPA Me-F-DOPA 10 20 30 Fraction Number Figure 5(A-D): The time course of 18F-O0PA metabolism in human plasma. Radiochromatograms show the amounts of 18F-D0PA and Me-18F-D0PA present in 1 ml plasma samples taken at (A) 7.5, (B) 30, (C) 60, and (D) 120 minutes after l8F-D0PA injection in a normal pretreated human subject with carbidopa pretreatment 28 Me-F-DOPA C 6 0 10 2 0 3 0 Fraction Number 29 reached maximal l e v e l s a t about 30 minutes and was then maintained at a f a i r l y constant c o n c e n t r a t i o n over the next 90 minutes. No other peaks were c o n s i s t a n t l y noted i n human plasma, with the p o s s i b l e exception of a compound o c c u r r i n g a t t r a c e l e v e l s i n f r a c t i o n 5. The compound may be an 0 - s u l f a t e d e r i v a t i v e . The disappearance of t o t a l r a d i o a c t i v i t y from plasma o f a 49 y e a r o l d n e u r o l o g i c a l l y normal male, p l o t t e d on a semi-log s c a l e , i s seen to be bi e x p o n e n t i a l ( F i g . 6 ) . The values f o r the two time constants of the human blood curve were on the order of 0.08 (x^) and 0. 006 ( X 2 ) minutes"! r e s p e c t i v e l y i n the 18 cases s t u d i e d . These values correspond t o h a l f - l i v e s on the order of 9 and 120 minutes. However, the values i n i n d i v i d u a l s were found to be s u b j e c t to c o n s i d e r a b l e v a r i a t i o n . There was no obvious d i f f e r e n c e between sexes but both h a l f - l i v e s were found to have a negative c o r r e l a t i o n with the age of the i n d i v i d u a l ( F i g . 7 ) . In both cases t h i s c o r r e l a t i o n was s i g n i f i c a n t a t p < 0.01. From t h i s i t f o l l o w s t h a t o l d e r i n d i v i d u a l s would have g r e a t e r p e r s i s t a n c e of t o t a l plasma r a d i o a c t i v i t y . In Figure 7 the time constants d e r i v e d from both normal c o n t r o l s (n=6) and p a t i e n t s with a v a r i e t y o f n e u r o l o g i c a l c o n d i t i o n s ( n=8) are p l o t t e d together as no d i f f e r e n c e was e v i d e n t between the two groups. With one exception (see below) these i n d i v i d u a l s were of Western European e x t r a c t i o n . However, Chamorros from the i s l a n d of Guam (G) showed a tendency to have higher values f o r \\ than d i d other s u b j e c t s . The spouse of one of these i n d i v i d u a l s , a woman of Japanese o r i g i n {p), had a value f o r \\ which f e l l e x a c t l y on the l i n e a r r e g r e s s i o n s l o p e . No d i f f e r e n c e i n the values d e r i v e d f o r X2 was evi d e n t between Chamorros and ot h e r s . These o b s e r v a t i o n s would suggest t h a t the observed d i f f e r e n c e between the Chamorros and other s u b j e c t s may be a d i s t i n c t i v e r a c i a l t r a i t . 30 Figure 6: The disappearance of t o t a l 1 8 F a c t i v i t y from human plasma i s b i e x p o n e n t i a l . The sub jec t was a 59 year o ld normal male. * i ( h a l f - l i f e = 9.9 minutes) was c a l c u l a t e d from the r e s i d u a l s o f the p a r t i a l decomposit ion a f t e r e v a l u a t i o n of \% ( h a l f - l i f e = 100 minutes) by l i n e a r regress ion ana lys i s o f the l a s t s i x data p o i n t s . Each p o i n t represents the mean of three de te rm ina t i ons . 31 32 The i n t e r p r e t a t i o n of t h i s d i f f e r e n c e i s d i f f i c u l t s i n c e a l l t h a t can be concluded i s t h a t the f i r s t component of the disappearance of t o t a l r a d i o a c t i v i t y from plasma may be s l i g h t l y more r a p i d i n n a t i v e s of Guam than i n other people so f a r s t u d i e d . The disappearance of 1 8F-D0PA from human plasma, as determined by the alumina method, i s a l s o b i e x p o n e n t i a l i n humans ( F i g . 8 ) . The f i r s t component of 1 8F-D0PA disappearance ( A 3 ) was c a l c u l a t e d from the r e s i d u a l s of the p a r t i a l decomposition a f t e r e v a l u a t i o n of the second component ( A 4 ) . The h a l f - l i v e s corresponding to A3 and A4 were t y p i c a l l y on the order of 8 and 45 minutes i n humans. Me- 1 8F-D0PA was r a p i d l y s y n t h e s i z e d and reached maximal values i n human plasma at about 15 minutes a f t e r 1 8F-D0PA i n j e c t i o n . The subsequent d e c l i n e i n a c t i v i t y permitted the e s t i m a t i o n of an e l i m i n a t i o n r a t e f o r Me- 1 8F-D0PA, A5. The h a l f - l i f e corresponding to A5 was u s u a l l y on the order of two hours. No s i g n i f i c a n t age dependence was noted f o r the terms A3, A4, and A5, nor d i d values i n the Guamanian p o p u l a t i o n d i f f e r i n comparison with the other i n d i v i d u a l s s t u d i e d . The disappearance of both t o t a l r a d i o a c t i v i t y ( F i g . 9) and 1 8F-D0PA ( F i g . 10) was a l s o found to be b i e x p o n e n t i a l i n the Cymolgus monkey. The f i v e time constants d e r i v e d f o r three monkeys did not, on average, d i f f e r g r e a t l y from the t y p i c a l values found f o r humans, but some d i f f e r e n c e s were present. In p a r t i c u l a r , the disappearance of t o t a l 1 8 F a c t i v i t y and of Me- 1 8F-D0PA from plasma was at l e a s t two-fold f a s t e r i n the monkey than i n the human. Radiochromatograms of plasma samples from the carbidopa p r e t r e a t e d r a t i n d i c a t e the presence of only one major m e t a b o l i t e , Me- 1 8F-D0PA, a t times between 5 and 60 minutes ( F i g s . 11A-13A). The amount of t h i s compound was 33 Time (minutes) Figure 8: The disappearance of 18F-D0PA from human plasma is biexponential. Triplicate plasma samples from the same individual in Figure 6 were fractionated by the alumina method. The activities of 18F-D0PA (•) and Me-18F-D0PA (•) are plotted on a semilog scale. X3 (half-life = 8.0 minutes) was calculated from the residuals of the partial decomposition after evaluation of X 4 (half-life = 4 3 minutes) by linear regression analysis of the last five points. The disappearance rate of Me-18F-D0PA ( X 5 ) was determined by linear regression analysis of time points after 20 minutes. 1 4 I I i I i i i 1 5 3 0 4 5 6 0 7 5 9 0 1 0 5 Time (minutes) Figure 9: The disappearance of total 1 8F activity from plasma in the monkey is biexponential. \\ (half-life = 7.3 minutes) was determined from the residuals of the partial decomposition after evaluation of \2 (half-life = 58 minutes). Each point represents the mean of three determi nations. 1 I 1 1 I I • I 1 5 3 0 4 5 6 0 7 5 9 0 1 0 5 Time (minutes) Figure 10: The disappearance of 18F-D0PA from monkey plasma is biexponential. Triplicate plasma samples were fractionated by the alumina method and the activites of 18F-DOPA (• ) and Me-18F-D0PA (a ) were plotted on a semilog scale. A3 (half-life = 9.4 minutes) was calculated from the residuals of the partial decomposition after evaluation of X4 by linear regression of the last four points. The disappearance rate of Me-18F-D0PA (62 minutes) was determined by linear regression of the last four points. 36 10 20 30 40 50 Fraction Number Figure 11: Radiochromatograms from rat plasma taken five minutes after 18F-D0PA injection with carbidopa pretreatment. The amount of Me-18F-D0PA formed (A) is greatly reduced by administration of U-0521 (B, 25 mg/kg i.p.). Figure 12: Radiochromatograms from rat plasma taken ten minutes after 18F-D0PA injection with carbidopa pretreatment. The amount of Me-18F-D0PA formed (A) is greatly reduced by administration of U-0521 (B, 25 mg/kg i .p.). 38 5 On Fraction Number Figure 13: Radiochromatograms from rat plasma taken 60 minutes after 18F-D0PA injection with carbidopa pretreatment. The amount of Me-18F-D0PA formed (A) is reduced by administration of U-0521 (B, 25 mg/kg i .p.). 39 g r e a t l y reduced, p a r t i c u l a r l y a t the e a r l i e r time p o i n t s , by the a d m i n i s t r a t i o n of U-0521 ( F i g s . 11B-13B). The disappearance of t o t a l r a d i o a c t i v i t y from plasma was b i e x p o n e n t i a l i n the carbidopa p r e t r e a t e d r a t , with h a l f - l i v e s on the order of 8 minutes and 6 hours r e s p e c t i v e l y ( F i g . 14A). C u r i o u s l y , c o - a d m i n i s t r a t i o n of U-0521 seemed to reduce the f i r s t term (\\) and i n c r e a s e the second term (X2, F i g . 14B) of 1 8 F disappearance. However, t h i s e f f e c t may not be s i g n i f i c a n t s i n c e \2 was too slow to permit accurate q u a n t i t a t i o n . The p e r s i s t a n c e of Me- 1 8F-D0PA i n plasma of the carbidopa p r e t r e a t e d r a t was too long to permit e s t i m a t i o n of i t s e l i m i n a t i o n r a t e ( F i g . 14A). In the case of pretreatment with U-0521, maximal l e v e l s o f m e t a b o l i t e were reached more than one hour a f t e r 1 8F-D0PA a d m i n i s t r a t i o n ( F i g . 14B), much more slowly than when r a t s r e c e i v e d carbidopa alone. The disappearance of 1 8F-D0PA from plasma was b i e x p o n e n t i a l i n the carbidopa p r e t r e a t e d r a t , with h a l f - l i v e s o f 3.3 and 34 minutes ( F i g . 15). Treatment with U-0521 i n c r e a s e d the f i r s t term of the disappearance of 1 8F-D0PA by 42% without g r e a t l y a l t e r i n g the second term (Cumming e t a l . , submitted). The e x t r a p o l a t e d plasma 1 8F-D0PA a c t i v i t y a t t 0 was 1750 CPM/pl i n c o n t r o l animals and 1840 CPM/pl i n animals with U-0521. From t h i s i t may be concluded t h a t both groups r e c e i v e d nearly the same radiochemical dose. The recovery of r a d i o a c t i v i t y i n the e x t r a c t i o n of r a t plasma was q u a n t i t a t i v e (101 ± 1.5%, n = 60) and the t o t a l recovery of r a d i o a c t i v i t y i n the HPLC s e p a r a t i o n was 82.6 ± 1.2% (n = 60). C a l c u l a t i o n o f area under the curves i n Figure 15 shows t h a t the COMT i n h i b i t o r i n c r e a s e d the a v a i l a b i l i t y of 1 8F-D0PA by 46% during the f i r s t 15 minutes and by 78% during the f i r s t 90 minutes. 3 0 6 0 9 0 120 Time (minutes) Figure 14: The disappearance of total 1 8 F activity from plasma is b i -exponential in the rat. Each point (•) represents the mean of between three and eight determinations (Carbidopa (A), Carbidopa + U-0521 (B)). Values for \\ were determined from the residuals of the partial decomposition following evaluation of X2 by linear regression analysis of points after 20 minutes. The persistance of Me-18F-D0PA (•) was too great to permit evaluation of X5. 8 i Time (minutes) Figure 15: The disappearance of 18F-D0PA from plasma is biexponential in the rat. Each point represents the mean of between three and eight HPLC fractionations (Carbidopa (•), Carbidopa + U-0521 (•)). Values for A3 were determined from the residuals of the partial decomposition following evaluation of A4 by linear regression analysis of points after 20 minutes. 42 The r e s u l t s of the k i n e t i c analyses of the d i s p o s i t i o n o f 1 8F-D0PA i n plasma of r a t , monkey and human are summarized i n Table I I I . As noted above, the rates are roughly s i m i l a r i n human and monkey, except f o r A2 . and A5. These r a t e s , which are r e l a t e d to the e l i m i n a t i o n of Me- 1 8F-D0PA, were h a l f as l a r g e i n monkey as i n man. The r a t appears to metabolize 1 8F-D0PA more r a p i d l y than primates ( i . e . lower A3). However, the p e r s i s t a n c e of Me- 1 8F-D0PA and of t o t a l plasma r a d i o a c t i v i t y i s much g r e a t e r i n r a t than i n monkey or human. The u n i d e n t i f i e d m e tabolite i n r a t plasma made i t s f i r s t appearance a t about 20 minutes a f t e r 1 8F-D0PA i n j e c t i o n i n the c o n t r o l group. A d m i n i s t r a t i o n o f U-0521 d i d not seem to i n c r e a s e the amounts of t h i s compound at any given time. However, amounts tended to accumulate g r a d u a l l y such t h a t at 120 minutes i t c o n s t i t u t e d nearly 20% of the remaining plasma r a d i o a c t i v i t y i n the group r e c e i v i n g U-0521. Based on i t s e l u t i o n time, which preceeded 1 8F-D0PA, the me t a b o l i t e must be a h i g h l y charged molecule such as a s u l f a t e conjugate. A p e c u l i a r c h a r a c t e r i s t i c of 1 8F-D0PA metabolism i n human plasma was th a t the r a t i o of Me- 1 8F-D0PA to 1 8F-D0PA ( f r e e over bound i n alumina f r a c t i o n a t i o n ) i n c r e a s e d as a l i n e a r f u n c t i o n with time ( F i g . 16A). This was found to be the case i n a l l humans from whom data was obtained (n=26). C o r r e l a t i o n c o e f f i c i e n t s of the l i n e a r r e g r e s s i o n were uniformly high (r=0.994 ± 0.007, range 0.997-0.975). T h i s l i n e a r r e l a t i o n s h i p was a l s o found to hold f o r the monkey ( F i g . 16B), which had a steeper slope than most humans. When the values of the slope (A free/bound vs. time ) were p l o t t e d as a f u n c t i o n of age of the i n d i v i d u a l , a negative c o r r e l a t i o n was observed ( F i g . 17). Pa r k i n s o n i a n s , p a t i e n t s exposed to MPTP, and d y s t o n i c s a l l f e l l c l o s e to the r e g r e s s i o n l i n e determined f o r normal 43 Table III Summary of the K i n e t i c s of Plasma 1 8F-O0PA Metabolism i n Carbidopa P r e t r e a t e d Human, Monkey and Rat. T o t a l 1 8 F a c t i v i t y 1 8F-D0PA Me- 1 8F-D0PA M *2 *3 M *5 Species Human 7.9±0.1 131±7 6.2±0.3 38 ±2 185+14 (20) (20) (20) (20) (12) Monkey 6.1±0.5 65±4 6.9±1.1 30.7±3.1 79±14 (3) (3) (3) (3) (3) Rat 7.9±0.7 347±100 3.3±0.2 34±2 >12 Hr (Carbidopa) Rat 4.6±0.03 >12 Hr 4.7±0.2 38 ±2 >12 Hr (U-0521) A l l time constants are reported as h a l f - l i v e s i n minutes except where noted. H a l f - l i v e s were c a l c u l a t e d from the expression t\/2= In 2/x. Where a p p l i c a b l e , k i n e t i c terms are reported as the mean ± S.E.M. (n d e t e r m i n a t i o n s ) . The k i n e t i c constants f o r the r a t were c a l c u l a t e d from the mean determinations i n numerous animals. Here, S.E.M.s are estimates d e r i v e d from g r a p h i c a l analyses of F i g u r e s 14-15. The terms \\ and X2 correspond to the r a t e of disappearance of t o t a l l 8 F a c t i v i t y i n plasma, whereas the terms X3 and X4 are c a l c u l a t e d from the r a t e of disappearance of 1 8F-D0PA from plasma. The r a t e o f disappearance of Me- 1 8F-D0PA from plasma i s r e p o r t e d as X 5 . In some cases, e l i m i n a t i o n r a t e s were too slow f o r accurate e v a l u a t i o n . The second group of r a t s r e c e i v e d U-0521 (25 mg/kg) i n a d d i t i o n to c a r b i d o p a . £ 5 O Q Li. 4 < a. O O 3 u. (D 2 2 • 12 10 Q u. o a 2-8 -00564 r-0-99 9 30 60 90 Time (minutes) 120 B a - 0.101 r - 0 . 9 9 6 15 30 T — 75 45 6 0 T i m e ( m i n u t e s ) 90 105 Figure 16: The ratio of metabolite to 18F-D0PA is linear as a function of time in plasma from human and monkey. Each point represents the mean of triplicate alumina fractionations in a 59 year old normal human (A) and in a Cynomolgus monkey ( B ) . I ' 1 1 — 1 — I 1 1 1 20 30 40 50 60 70 80 Age(years ) Figure 17: The slope of the ratio of metabolite to l8F-D0PA as a function of time declines with age of the individual. The linear regression slope was determined for 10 normal subjects. Values for four out of five Chamorros from Guam (G) fall above the isochron. 46 s u b j e c t s (n = 10). However, the values from four out of f i v e Chamorros were found to l i e somewhat above the r e g r e s s i o n l i n e f o r other i n d i v i d u a l s . The l i n e a r r e l a t i o n between metabolites and 1 8F-D0PA as a f u n c t i o n of time was a l s o found i n the carbidopa t r e a t e d r a t ( F i g . 18), with the slope being g r e a t e r than any value found f o r i n d i v i d u a l monkeys or humans. Treatment with U-0521 g r e a t l y reduced t h i s slope by 60% f o r a p e r i o d of one hour. T h e r e a f t e r , the met a b o l i t e r a t i o assumed a r a t e of i n c r e a s e s i m i l a r to t h a t found i n those animals which had r e c e i v e d carbidopa alone. When the r a t i o of Me- 1 8F-D0PA alone to 1 8F-D0PA, r a t h e r than the r a t i o of t o t a l m e t a b o l i t e s to 1 8F-D0PA, was p l o t t e d as a f u n c t i o n of time, the r e s u l t s were very s i m i l a r (data not shown), but a l l slopes were reduced by about 10% from those i l l u s t r a t e d i n Figure 18. Therefore, values f o r m e t a b o l i t e r a t i o s d e r v i e d from HPLC experiments should be almost the same as data from alumina f r a c t i o n a t i o n s where a l l non-catechols are measured together i n the free f r a c t i o n . The mean slopes of the r e l a t i o n s h i p between met a b o l i t e s and 1 8F-D0PA (A free/bound as a f u n c t i o n of time) in human, monkey and r a t are summarized i n Table IV. In a d d i t i o n , an estimate of the p r o p o r t i o n of 1 8F-D0PA which i s metabolized to Me- 1 8F-D0PA i s i n c l u d e d i n t h i s t a b l e . T h i s p r o p o r t i o n was determined from the r a t i o of maximal l e v e l s of Me- 1 8F-D0PA to the e x t r a p o l a t e d plasma 1 8F-D0PA a c t i v i t y a t t 0 . T h i s estimate does not take i n t o account the r e l a t i v e l y slow e l i m i n a t i o n of Me- 1 8F-D0PA ( X 5 ) which would only be expected to be s i g n i f i c a n t a t l a t e time p o i n t s i n the monkey. The p r o p o r t i o n of 1 8F-D0PA metabolized to Me- 1 8F-D0PA i n human su b j e c t s (22%) was somewhat lower than i n monkeys (31%) or r a t s (29%). 47 i i 3 0 60 90 120 Time (minutes) Figure 18: The ratio of metabolites to 18F-D0PA is linear as a function of time in rat plasma. Each point represents the mean of between three and eight HPLC fractionations (Carbidopa (•), Carbidopa + U-0521 (•)). In the group receiving U-0521, slopes were determined by linear regression of first five and last five points. Table IV. The Apparent Rate of 1 8F-D0PA Metabolism i n Plasma and the Estimated P r o p o r t i o n of the Input Metabolized to Me- 1 8F-D0PA i n Carbidopa P r e t r e a t e d Human, Monkey and Rat. A Free/Bound % Meta b o l i z e d to Me- 1 8F-D0PA Sp e c i e s  Human 0.070±0.03 22±3 (27) (10) Monkey 0.088±0.007 31 ±3 (3) (3) Rat 0.141+0.015 29 (Carbidopa) Rat 0.051±0.002 14 (U-0521) For monkey and human, values are reported as the mean ± S.E.M. (n determinations) but values f o r the r a t were c a l c u l a t e d from the mean data from numerous i n d i v i d u a l animals with e r r o r s estimated i n some cases from g r a p h i c a l a n a l y s i s of Fi g u r e 18. A Free/Bound r e f e r s to the slope of m e t a b o l i t e s / 1 8 F - D 0 P A as a f u n c t i o n of time. A Free/Bound f o r the r a t group r e c e i v i n g U-0521 r e f e r s to the slope during the i n i t i a l 60 minutes o n l y . The t o t a l percentage metabolized was estimated from the max.im.al l e v e l s of metabolite i n plasma and the e x t r a p o l a t e d 1 8F-O0PA a c t i v i t i e s a t t 0 , the time o f i n j e c t i o n . It was assumed t h a t the e l i m i n a t i o n rate of Me- 1 8F-D0PA was n e g l i g i b l e . 49 C. C e n t r a l Metabolism o f 1 SF-D0PA 1) Methods In order to determine the metabolism of 1 8F-D0PA i n the c e n t r a l nervous system, r a t s (n=50) were provided with i n t r a j u g u l a r cannulae as d e s c r i b e d i n s e c t i o n B ( l ) . A f t e r recovery, r a t s were p r e t r e a t e d with carbidopa (5 mg/kg) and administered 1 8F-D0PA (500 uCi/kg) 30 minutes l a t e r . In some r a t s , blood samples were taken f o r a n a l y s i s of plasma metabolism as d e s c r i b e d above. At times between f i v e and 120 minutes, animals were s a c r i f i c e d by c e r v i c a l d i s l o c a t i o n . B r a i n s were removed and placed on i c e w i t h i n 90 seconds of s a c r i f i c e and were then d i s s e c t e d . The s t r i a t u m , vermis and, i n some cases, amygdala and/or p a r i e t a l c o r t e x , samples were placed i n separate preweighed eppendorf tubes. B r a i n t i s s u e s were b r i e f l y s o n i c a t e d i n 10 volumes of i c e c o l d 0.1 M p e r c h l o r i c a c i d c o n t a i n i n g 0.05% sodium t h i o s u l f a t e as an a n t i - o x i d a n t . Experiment showed that t h i s e x t r a c t i o n procedure gave e s s e n t i a l l y q u a n t i t a t i v e r e c o v e r i e s of t i s s u e r a d i o a c t i v i t y . E i t h e r catecholamine s u l f a t e d e r i v a t i v e s d i d not occur, or they were l a r g e l y hydrolyzed by the e x t r a c t i o n procedure s i n c e heating ( a t 70 °C f o r 15 minutes) such an e x t r a c t from the s t r i a t u m of a r a t s a c r i f i c e d at 15 minutes a f t e r 1 8F-O0PA a d m i n i s t r a t i o n d i d not r e s u l t i n the disappearance or r e d u c t i o n of any peaks. In the case of s t r i a t a l samples a t f i v e , 10, 30 and 90 minutes, 100 ng/ml DHBA was i n c l u d e d i n the r e a c t i o n mixture i n order to permit i n t e r n a l standard q u a n t i t a t i o n of endogenous catecholamines. A f t e r s o n i c a t i o n , samples were c e n t r i f u g e d f o r 30 minutes at 10,000 g. 200 pi samples of supernatant were i n j e c t e d onto the HPLC and the t o t a l a c t i v i t y i n an equal volume was determined f o r c a l c u l a t i o n of the recovery during HPLC a n a l y s i s . In t i s s u e s which had been repeatedly shown to have no s i g n i f i c a n t radiochemical species e l u t i n g a f t e r Me- 1 8F-D0PA, a r a p i d HPLC a n a l y s i s was subsequently used. In 18 animals, e i g h t a d d i t i o n a l b r a i n areas ( f r o n t a l and p o s t e r i o r c o r t e x , hippocampus, thalamus, midbrain, c e r e b e l l a r hemispheres and brainstem) were d i s s e c t e d and placed d i r e c t l y i n t o pre-weighed gamma counting tubes f o r determination of t o t a l t i s s u e r a d i o a c t i v i t y . 6-Hydroxydopamine L e s i o n s : In fo u r animals anaesthetized with p e n t o b a r b i t a l , u n i l a t e r a l l e s i o n s were induced by s t e r o t a x i c i n j e c t i o n of 6-OHDA (Sigma, 10 ug i n 2.5 pi of 0.02% ascorbate) i n t o the l e f t medial f o r e b r a i n bundle (A 5.2, L 1.4, V 1.5 from i n t r a a u r a l z e r o ) . Animals were allowed to recover f o r two weeks before f u r t h e r p r e p a r a t i o n . One week f o l l o w i n g i n t r a j u g u l a r c a n n u l a t i o n , carbidopa and 1 8F-D0PA were administered as usual and animals were s a c r i f i c e d 15 minutes l a t e r , a time which had been c h a r a c t e r i z e d as producing maximal 1 8F-DA s y n t h e s i s i n st r i a t u m . Both s t r i a t a were analyzed by HPLC with q u a n t i t a t i o n of endogenous amines as well as of r a d i o a c t i v i t y i n the vari o u s f r a c t i o n s . COMT i n h i b i t i o n : In a separate study, the e f f e c t of i n h i b i t i o n of COMT with U-0521 on c e n t r a l 1 8F-D0PA metabolism was determined. Animals were prepared as p r e v i o u s l y d e s c r i b e d except t h a t the experimental group a l s o r e c e i v e d U-0521 (25 mg/kg i.p.) ten minutes p r i o r to 1 8F-D0PA a d m i n i s t r a t i o n . Control animals (n=3) and experimental animals (n=4) were s a c r i f i c e d a t 30, 60 and 90 minutes a f t e r 1 8F-D0PA. Brains were d i s s e c t e d and p a r i e t a l cortex samples, i n a d d i t i o n to s t r i a t u m and vermis, were prepared f o r a n a l y s i s by HPLC. In some animals, up to three blood samples were taken a t i n t e r v a l s f o r a n a l y s i s of plasma. The l a s t blood sample was taken one minute before s a c r i f i c e . In an a d d i t i o n a l study, the e f f e c t of U-0521 alone on s t r i a t a l dopamine and met a b o l i t e l e v e l s was determined. Experimental animals (n=3) 51 r e c e i v e d U-0521 (25 mg/kg, i.p) and were s a c r i f i c e d 90 minutes l a t e r . The c o n t r o l group (n=3) r e c e i v e d a s a l i n e i n j e c t i o n . Striatum and hypothalamus samples were d i s s e c t e d on i c e and prepared f o r HPLC a n a l y s i s of c a t e c h o l s and i n d o l e s with i n t e r n a l standard q u a n t i s a t i o n . 20 ul samples of s t r i a t a l homengenate were analyzed at a s e n s i t i v i t y of 5 nA/V and 40 ul hypothalamic samples were analyzed a t a d e t e c t o r s e n s i t i v i t y of 2 nA/V. A n a l y s i s of Data: A l l data f o r c e n t r a l 1 8F-D0PA metabolism are given i n terms of means ± S.E.M. Because of some v a r i a b i l i t y i n the t o t a l amount of r a d i o a c t i v i t y found i n i n d i v i d u a l animals at the same time p o i n t , many of the data are expressed i n terms of the percentage o f t o t a l r a d i o a c t i v i t y i n the sample. The r a t i o of r a d i o a c t i v i t y i n striatum to t h a t i n cerebellum or vermis (S/V) i s f r e q u e n t l y used i n the a n a l y s i s of PET s t u d i e s with 1 8F-D0PA i n humans because the c e r e b e l l u n i s devoid of s i g n i f i c a n t dopaminergic i n n e r v a t i o n ; hence t h i s r a t i o was c a l c u l a t e d as well as the r a d i o a c t i v i t y l e v e l s i n the i n d i v i d u a l t i s s u e s . The values f o r r a d i o a c t i v i t y f o r the whole c e r e b e l l a r hemispheres d i d not d i f f e r s i g n i f i c a n t l y from those found f o r supernatants of vermis homogenates, and so the vermis was used as a convenient sample of c e r e b e l l a r t i s s u e . The Student's double t a i l e d t t e s t was used f o r a n a l y s i s of s i g n i f i c a n c e of d i f f e r e n c e between groups of data. 2) R e s u l t s of Experiments on C e n t r a l Metabolism. A complete radiochemical chromatogram of a vermis sample from a carbidopa p r e t r e a t e d r a t s a c r i f i c e d at 30 minutes a f t e r 1 8F-D0PA a d m i n i s t r a t i o n i s i l l u s t r a t e d i n F i g u r e 19A. At t h i s time only t r a c e s of 1 8F-D0PA remained i n the vermis. No m e t a b o l i t e s other than Me- 1 8F-D0PA were found i n any vermis sample. In a s i n g l e amygdala sample, taken 10 Fraction Number Figure 19: 18F-D0PA and metabolites in vermis and amygdala of the carbidopa pretreated rat. In vermis samples taken 30 minutes after 18F-D0PA injection (A) only traces of 18F-D0PA remain with Me-18F-D0PA being the sole metabolite. In an amygdala sample taken 10 minutes afte 18F-D0PA administration (B) the major metabolite is Me-18F-D0PA but traces of l8F-D0PAC and 18F-0A are also present. Note scale difference 53 minutes a f t e r 1 8F-O0PA i n j e c t i o n , Me- 1 8F-D0PA was a l s o the p r i n c i p a l m e t a b o l i t e , ( F i g . 19B). However, t r a c e s (~5% of t o t a l r a d i o a c t i v i t y ) o f a d d i t i o n a l compounds were present, notably 1 8F-D0PAC and 1 8F-DA. The disappearance o f 1 8F-D0PA from vermis samples was r a p i d and fol l o w e d b i e x p o n e n t i a l k i n e t i c s , with h a l f - l i v e s of 5.9 and 27 minutes ( F i g . 20A). At times a f t e r 30 minutes, only t r a c e s of 1 8F-D0PA remained i n vermis. During the p e r i o d of disappearance of 1 8F-D0PA from vermis, the amounts of Me- 1 8F-D0PA i n c r e a s e d r a p i d l y , such t h a t l e v e l s o f me t a b o l i t e exceeded 1 8F-D0PA a t ten minutes a f t e r i n j e c t i o n ( F i g . 20B). From t h i s i t may be concluded t h a t 1 8F-D0PA metabolism i n the cerebellum f o l l o w s , to the f i r s t approximation, p e r i p h e r a l processes. A radiochromatogram of a cortex sample at 30 minutes a f t e r 1 8F-D0PA a d m i n i s t r a t i o n i s i l l u s t r a t e d i n Fi g u r e 21A. The r a t i o of Me- 1 8F-D0PA to 1 8F-D0PA i n cor t e x was very c l o s e to t h a t found i n vermis a t e q u i v a l e n t times a f t e r 1 8F-D0PA i n j e c t i o n ( F i g . 22). I n h i b i t i o n of COMT with U-0521 served to i n c r e a s e the p e r s i s t a n c e o f 1 8F-D0PA i n cortex ( F i g s . 21B and 22A) and i n vermis ( F i g . 22B). However, a comparison of radiochromatograms from samples with and without COMT i n h i b i t i o n suggest t h a t t h i s e f f e c t was sho r t l a s t i n g . In animals r e c e i v i n g U-0521, the r e l a t i v e amounts of 1 8F-D0PA at 30 minutes were i n c r e a s e d by 90% i n both cortex (p < 0.05) and vermis (p < 0.025). A 30% r e d u c t i o n i n Me- 1 8F-D0PA was observed at 30 minutes (p < 0.001) i n cortex but not i n vermis. There was some evidence t h a t i n animals with COMT i n h i b i t i o n , the l e v e l s of 1 8F-DA and me t a b o l i t e s were i n c r e a s e d i n cortex at 30 minutes a f t e r 1 8F-D0PA a d m i n i s t r a t i o n . However, 1 8F-DA was not c o n s i s t e n t l y found i n the cortex of animals with U-0521, and never accounted f o r more than 5% of the t o t a l r a d i o a c t i v i t y i n t h a t t i s s u e . In a d d i t i o n , t r a c e s of a compound e l u t i n g before 1 8F-D0PA 54 TIME (minutes) Figure 20: The metabolism of 18F-D0PA in rat vermis as a function of time. The disappearance of 18F-O0PA from vermis is biexponential (A). The proportion of 18F-D0PA in vermis (O) rapidly decreases in concert with the accumulation of Me-18F-D0PA (•, B). 55 70, 60-50 40-30 20 .tr 10 > o < — 30 03 •4—> o I -20 10-F-DOPA Me-F-DOPA 1 0 I 2 0 30 40 50 Me-F-DOPA B F-DOPA 1 F-DA 1 0 20 30 Fraction Number - l ML^ 10 40 "50 Figure 21: Radiochromatograms of rat cortex at 30 minutes after 18F-D0PA administration. In animals receiving carbidopa (A) only Me-18F-D0PA and traces of 18F-D0PA are present. In animals also receiving U-0521 (B) the amount of 18F-D0PA present is greatly increased and traces of 18F-DA are formed. 100 cortex *** B C D 60 T • if A B C D A B C 60 9 0 Time in minutes Figure 22: The effect of U-0521 on the 0-methylation of 18F-DOPA in cortex and vermis. A=i8F-D0PA in controls (n=3), B=18F-D0PA in animals with U-0521 (h=4). C=Me-18F-D0PA in controls, D=Me-18F-D0PA in animals with U-0521. (*) p < 0.05; {***) p < 0.001 by Student's double tailed t test. Error bars are S.E.M. 57 were o c c a s i o n a l l y found i n cortex samples from animals with and without U-0521. The t o t a l r a d i o a c t i v i t y present i n the striatum was r e l a t i v e l y constant over the i n i t i a l 90 minutes of the experiment ( F i g . 23A,B). During t h i s p e r i o d , the amount of r a d i o a c t i v i t y i n the vermis d e c l i n e d c o n t i n u o u s l y . The r a t i o of t o t a l a c t i v i t i e s i n s t r i a t u m and vermis ( S A ) i n carbidopa p r e t r e a t e d r a t s i s i l u s t r a t e d i n Figure 2 X . This r a t i o was l e s s than one over the f i r s t 10 minutes i n each animal. The r a t i o i n c r e a s e d i n an apparently l i n e a r manner u n t i l i t reached the peak of 1.8:1 a t one hour post i n j e c t i o n . T h e r e a f t e r , the r e l a t i v e excess of s t r i a t a l r a d i o a c t i v i t y over t h a t i n vermis d e c l i n e d slowly. In a l l other b r a i n areas examined, with the exception of the s t r i a t u m , 1 8 F a c t i v i t y decreased i n a manner s i m i l a r to t h a t observed i n the vermis (Table V). In t h i s study with a small number of animals, no s t a t i s t i c a l l y s i g n i f i c a n t d i f f e r e n c e s were noted i n the a c t i v i t y of t i s s u e s at 15, 30, 50 and 90 minutes a f t e r 1 8F-D0PA i n j e c t i o n . However, some c o n s i s t e n t d i f f e r e n c e s were noted. At times a f t e r 15 minutes, a c t i v i t y i n the s t r i a t a l samples was always g r e a t e r than the a c t i v i t y i n other t i s s u e s . A c t i v i t y i n c e r e b e l l a r hemispheres was i n a l l cases approximately 9% higher than i n vermis e x t r a c t s from the same animals. T h i s c o u l d i n d i c a t e t h a t some r a d i o a c t i v i t y was l o s t i n the e x t r a c t i o n procedure. Hypothalamus a c t i v i t y was 5% lower than a c t i v i t y i n the thalamus i n 17 out of 18 animals, and hippocampus a c t i v i t y tended to be somewhat lower than i n c o r t e x samples (12/18 a n i m a l s ) . No c o n s i s t e n t d i f f e r e n c e s were noted i n the a c t i v i t y i n f r o n t a l as compared to p a r i e t a l c ortex samples. I n h i b i t i o n of COMT with U-0521 produced an apparent long l a s t i n g i n c r e a s e i n the r a t i o S/V (Table V I ) . However, t h i s i n c r e a s e was never 58 8 l T o x O) E \ Q_ o 2-S t r i a t u m 3 0 60 9 0 T i m e (minutes) 120 B 2.0 > \ CO 1 2 Oio H U T U . MOIMCTIVITY ^ LIlCAfl DCOK.SION 30 I I I 60 TIME (minutes) I 90 120 Figure 23: The absolute 1 8F activity in striatum and vermis as a function of time. Activity in striatum (•) exceeds that in vermis (•) at times after 15 minutes with (*) p < 0.05, Student's double tailed t test indicating a significant difference. Each point represents the mean ± S.E.M. of between three and five determinations. Activity in striatum is constant over the initial 90 minutes (B), but the ratio of activity in striatum to that in vermis increases in a linear manner with time (C) for the first hour. The maximal ratio of 1.8:1 is found at 60 minutes, thereafter the ratio slowly declines. Table V The Total 1 8 F A c t i v i t y i n Ten Regions of Rat B r a i n F o l l o w i n g A d m i n i s t r a t i o n of 1 8F-D0PA. T i s s u e Time (min) 15 30 50 90 (n) (5) (4) (6) (3) Striatum 626±124 694196 505184 528168 Vermis 633±133 495184 332160 382156 Cerebellum 681±130 545192 373164 409154 Pons/Medulla 587±105 480+85 330150 342145 Midbrain 633±118 519189 349157 36U51 Hypothalamus 672H20 537196 362162 426176 Thalamus 703H35 562190 407167 419147 Hippocampus 589H06 539194 387162 416175 Cortex ( F r o n t a l ) 638+119 552H00 395168 415157 Cortex ( P a r i e t a l ) 616+106 533187 378157 423158 A c t i v i t y i s i n u n i t s of CPM/mg t i s s u e ± S.E.M. In t h i s study no s i g n i f i c a n t d i f f e r e n c e s were found between a c t i v i t y i n vermis e x t r a c t s and i n t i s s u e samples from other b r a i n r e g i o n s . However, as noted i n the t e x t , some d i f f e r e n c e s were c o n s i s t e n t l y present. Table VI The E f f e c t of U-0521 on To t a l 1 8 F A c t i v i t y i n Rat B r a i n and Plasma Following A d m i n i s t r a t i o n of 1 8F-D0PA Time i n minutes t i s s u e 30 60 90 c o n t r o l s t r i a t u m 668±94 713±63 434±56 (n=3) cortex 509+85 415±32 276±12 vermis 557±96 390±31 294±12 plasma 549±54 538±36 480±45 S/V 1.24±0. 08 1.82±0.02 1.45+0.14 U-0521 s t r i a t u m 737±103 792±64 669±139 (n=4) c o r t e x 487±65 385±38 391±89 vermis 484±68 368±41 366±80 plasma 625±53 570±47 542±44 S/V 1.52±0.02*** 1.97±0.12 1.62±0.03 Values reported as mean ± S.E.M. i n u n i t s of CPM/mg f o r t i s s u e and CPM/pl f o r plasma. The c o n t r o l group was p r e t r e a t e d with carbidopa, experimental animals a l s o r e c e i v e d U-0521 (25 mg/kg i . p . ) . ** p < 0.001, Student's two t a i l e d t t e s t . more than 20% and only reached s i g n i f i c a n c e a t 30 minutes (p < 0.001). The t o t a l 1 8 F / u l plasma tended to be intermediate to the 1 8 F a c t i v i t y /mg t i s s u e i n s t r i a t a l and n o n - s t r i a t a l c e r e b r a l t i s s u e s . Radiochromatograms f o r s t r i a t a l e x t r a c t s from carbidopa p r e t r e a t e d animals were c o n s i d e r a b l y more complex than those from plasma or vermis ( F i g s . 24A-26A). Of the seven radiochemical peaks observed i n the s t r i a t u m , s i x have been i d e n t i f i e d p o s i t i v e l y or t e n t a t i v e l y . The time course of the r e l a t i v e l e v e l s of 1 8F-D0PA, Me- 1 8F-D0PA, 1 8F-DA and 1 8F-D0PAC are i l l u s t r a t e d i n F i g u r e 27. 1 8F-D0PA disappeared very r a p i d l y , c o n s t i t u t i n g l e s s than 30% of s t r i a t a l r a d i o a c t i v t y a t f i v e minutes a f t e r i n j e c t i o n . The c o n c e n t r a t i o n of 1 8F-D0PA appeared to disappear as a bi e x p o n e n t i a l f u n c t i o n ( F i g . 28A), but the time constants could not be a c c u r a t e l y determined because of the small amounts of 1 8F-O0PA present i n s t r i a t u m . Me- 1 8F-D0PA was the major l a b e l l e d m e t a b o l i t e present i n the st r i a t u m but, i n c o n t r a s t to cerebellum and c o r t e x , i t c o n s t i t u t e d l e s s than 50% of t o t a l r a d i o a c t i v i t y during the f i r s t 45 minutes post i n j e c t i o n . 1 8F-DA was the next most important m e t a b o l i t e , reaching a maximum pr o p o r t i o n of t o t a l a c t i v i t y w i t h i n the f i r s t q u a r t e r hour post i n j e c t i o n and d e c l i n i n g slowly t h e r e a f t e r . The predominant 1 8F-DA met a b o l i t e present i n s t r i a t u m was 1 8F-D0PAC, which occured i n a f a i r l y constant r a t i o to 1 8F-DA of approximately 0.4. The p r o p o r t i o n s as a f u n c t i o n of time of the minor s t r i a t a l 1 8F-DA m e t a b o l i t e s , 1 8F-HVA and 1 8F-3-MTyr, are i l l u s t r a t e d i n F i g u r e 28B. C o l l e c t i v e l y , these two compounds never accounted f o r more than 10% of t o t a l s t r i a t a l r a d i o a c t i v i t y . Traces of an u n i d e n t i f i e d m e t a b o l i t e , presumably the s u l f a t e d e r i v a t i v e of 1 8F-D0PA, were found i n some s t r i a t a l samples but never accounted f o r more than 5% of t o t a l r a d i o a c t i v i t y . 4 0 1 20 30 Fraction Number Figure 24: Radiochromatograms of rat striatum at 30 minutes after 18F-D0PA injection. At least six radiolabeled compounds are present. A control animal (A) may be compared with one receiving U-0521 (B). Note difference in scales 5 0 4 0 -3 0 -20-Fraction Number Figure 25: Radiochromatogram of rat striatum at 60 minutes after 18F-D0PA injection. At least six radiolabeled compounds are present. A control animal (A) may be compared with one receiving U-0521 (B). Note difference in scales. 64 Fraction Number Figure 26: Radiochromatograms of rat striatum at 90 minutes after 18F-D0PA in j e c t i o n . At least six rad i o l a b e l e d compounds are present. A control animal (A) may be compared with one receiving U-0521 (B). 30 60 90 120 TIME (minutes) Figure 27: The proportions of the principal labelled compounds in rat striatum as a function of time. The relative amounts of 18F-D0PA, Me-18F-D0PA, 18F-DA and 18F-D0PAC in striatum are illustrated at times between five and 120 minutes. Each point represents the mean of between three and five determinations ± S.E.M. In some cases the S.E.M. is smaller than the symbol. 66 30 60 90 120 Time in minutes 30 i 30 60 90 120 TIME (minutes) Figure 28: The minor radio!abelled componds in rat striatum as a function of time. The disappearance of 18F-D0PA from striatum (A) seems to be biexponential but time constants cannot be r e l i a b l y evaluated because of excessive v a r i a b i l i t y . The proportions of 18F-DA and i t s minor metabolites 18F-HVA and 18F-3MTyr are i l l u s t r a t e d in part B. Each point represents the mean of between three and fi v e determinations ± S.E.M. The absolute amounts of the p e r i p h e r a l m e t a b o l i t e , Me- 1 8F-D0PA, i n s t r i a t u m and vermis as a f u n c t i o n of time are compared i n Figure 29. At e a r l y time p o i n t s the l e v e l s of Me- 1 8F-D0PA were s i g n i f i c a n t l y higher i n vermis than i n s t r i a t u m . This d i f f e r e n c e c o u l d be r e l a t e d to a number of f a c t o r s such as the p e r m e a b i l i t y of the two t i s s u e s to Me- 1 8F-D0PA or to the r e g i o n a l d i s t r i b u t i o n o f COMT i n the b r a i n . T h is s h o r t l a s t i n g excess of m e t a b o l i t e i n vermis would seem to be s u f f i c i e n t to account f o r the higher l e v e l s of t o t a l 1 8 F a c t i v i t y i n vermis during the i n i t i a l p a r t o f the experiment. The e f f e c t of COMT i n h i b i t i o n on 1 8F-D0PA metabolism was more marked i n the s t r i a t u m than i n the vermis or c o r t e x . Radiochromatograms o f s t r i a t a l samples from animals r e c e i v i n g U-0521 and s a c r i f i c e d a t 30, 60 and 90 minutes a f t e r 1 8F-D0PA i n j e c t i o n are i l l u s t r a t e d f o r the sake o f comparison with animals r e c e i v i n g carbidopa alone ( F i g s . 24B-26B). In st r i a t u m , only t r a c e s of 1 8F-D0PA were observable a f t e r 30 minutes and there was no s i g n i f i c a n t e f f e c t of the U-0521 a t any time p o i n t s t u d i e d ( F i g . 3 0 ( 1 ) ) . However the r e l a t i v e amounts of Me- 1 8F-D0PA were reduced i n the striatum by approximately 30% at 30 (p < 0.001), 60 and 90 minutes (p < 0.025) i n the animals given U-0521 plus carbidopa as compared to the animals r e c e i v i n g o n l y c a r b i d o p a . S i g n i f i c a n t amounts of 1 8F-DA were formed i n the s t r i a t u m of both groups of animals. COMT i n h i b i t i o n i n c r e a s e d the p r o p o r t i o n of t h i s compound by 50% a t a l l three time p o i n t s s t u d i e d ( F i g . 30(2), p < 0.05). Increases i n 1 8F-D0PAC of 40% a t 30 (p < 0.005) and 60 minutes (p < 0.05) were a l s o noted i n the s t r i a t u m of animals t r e a t e d with U-0521. The amounts of 1 8F-DA and 1 8F-D0PAC i n s t r i a t u m c o r r e l a t e d i n c o n t r o l ( r = 0.90) and i n U-0521 animals ( r = 0.75). No change was produced i n the p r o p o r t i o n of the methylated 1 8F-DA m e t a b o l i t e 68 2 . 0 TIME (minutes) Figure 29: The absolute activities of Me-18F-D0PA ( D ) in vermis and striatum (•) as a function of time. The amounts of Me-18F-D0PA present in the two tissues are reported in CPM/mg tissue with each point representing the mean of between three and five determinations ± S.E.M. (*) p < 0.05; (**) p < 0.025, Student's double tailed t test. The period where Me-18F-D0PA activity is higher in vermis should be compared with the time course of radiocontrast development (S/V, see inset). 75 ( D «** * * JSLca. A B C D 3 0 (2) B C 6 0 A B C 9 0 ## »* A B C D 3 0 A B C D 6 0 Time (minutes) A B C D 9 0 Figure 30: The effect of U-0521 on the metabolism of 18F-D0PA in striatum. In 32(1) the re l a t i v e amounts of 18F-D0PA (A & B) and Me-18F-D0PA (B & C) are i l l u s t r a t e d in controls (A & C, n=3) and animals with U-0521 (B & D, n=4). In 32(2) the re l a t i v e amounts of 18F-DA (A & B) and 18F-DOPAC (B & C) are i l l u s t r a t e d in controls (A & C) and animals with U-0521 (B & D). (*) p < 0.05; (**) p < 0.005; {***) p < 0.001; Student's double t a i l e d t tes t . 1 8F-HVA by treatment with U-0521, but l e v e l s of 1 8F-3-MTyr were reduced by 50% i n the s t r i a t a of U-0521 t r e a t e d animals (p < 0.05, data not shown). The amounts of 1 8F-HVA and 1 8F-3MTyr d i d not c o r r e l a t e with the amounts of 1 8F-DA present i n the s t r i a t u m of c o n t r o l or U-0521 t r e a t e d animals. The s t r i a t a l c o n c e n t r a t i o n s of DA (11200 ± 380 ng/g), DOPAC (1568 ± 122 ng/g), HVA (1232 ± 96 ng/g) and 3-MTyr (477 ± 39 ng/g) were determined by HPLC with e l e c t r o c h e m i c a l d e t e c t i o n (n=12). A sample e l e c t r o c h e m i c a l chromatogram i s presented to show the e l u t i o n times and r e l a t i v e amounts o f o x i d i z a b l e compounds present i n r a t str i a t u m ( F i g . 31). Since no s i g n i f i c a n t changes i n met a b o l i t e l e v e l s were noted i n animals s a c r i f i c e d a t 5, 10, 60 and 120 minutes a f t e r 1 8F-D0PA i n j e c t i o n , o v e r a l l averages are repo r t e d . These values are almost i d e n t i c a l with those reported by Cheng and Wooten (1982). In agreement with most r e p o r t s , DOPAC was the p r i n c i p a l 1 8F-DA metabolite i n the s t r i a t u m of the r a t , followed c l o s e l y by HVA. S i m i l a r l y , the p r i n c i p a l 1 8F-DA met a b o l i t e i n the st r i a t u m was 1 8F-D0PAC. However, the r a t i o of t h i s m e t a b o l i t e to 1 8F-DA was nearly three times higher than the r a t i o found f o r the endogenous compounds (Table V I I ) . The 1 8F-HVA and 1 8F-3-MTyr to 1 8F-DA r a t i o s were a l s o higher than those of the analogous c o l d compounds. I n h i b i t i o n of COMT with U-0521 did not s i g n i f i c a n t l y a l t e r the r a t i o s of 1 8F-D0PAC or 1 8F-HVA to 1 8F-DA with r e s p e c t to c o n t r o l animals from the same study. In c o n t r a s t , the r a t i o of 1 8F-3MTyr to 1 8F-DA was 50% lower i n animals with COMT i n h i b i t i o n (p < 0.05) when compared with caridopa pre r e a t e d r a t s done at the same time. The average r a d i o a c t i v e m e t a b o l i t e r a t i o s determined i n the o r i g i n a l 1 8F-D0PA metabolism study (Cumming e t a l . , i n press) are a l s o reported i n Table VII. Ratios of the two methylated 1 8 F met a b o l i t e s to 1 8F-DA i n t h i s study are lower than i n the Figure 31: An electrochemical chromatogram showing the elution times and the relative amounts of biogenic amines and metabolites present in rat striatum. Although this chromatogram is from a carbidopa pretreated animal which had received 18F-D0PA 10 minutes previously, the concentrations of the fluorinated compounds are too low to permit electrochemical detection. The upper trace indicates flow rate in ml/min. Table VII Ratios of Endogenous M e t a b o l i t e s to Dopamine and 1 8 F - M e t a b o l i t e s to 1 8F-Dopamine i n Striatum of the Hooded Rat. Group DQPAC/DA HVA/DA 3MTyr/DA c o n t r o l (n=12) 0.140 ± 0.001 0.110 ± 0.009 0.042 ± 0.005 1 8F-D0PAC/ 1 8F-DA 1 8F-HVA/ 1 8F/DA 1 &F-3MTyr/ 1 8F-DA c o n t r o l (n=40) 0.39 ± 0.03 0.23 ± 0.03 0.19 ± 0.03 c o n t r o l (n=9) 0.37 ± 0.06 0.35 ± 0.10 0.27 ± 0.07 U-0521 (n=12) 0.31 ± 0.02 0.23 ± 0.04 0.13 ± 0 . 0 1 * Control groups are animals which r e c e i v e d carbidopa pretreatment o n l y . Endogenous m e t a b o l i t e r a t i o s were determined i n 12 animals which had r e c e i v e d 1 8F-D0PA. The f i r s t c o n t r o l group c o n s t i t u t e s animals used i n the time course study and the second c o n t r o l group was used i n the study of COMT i n h i b i t i o n with U-0521 (25 mg/kg i . p . ) . Ratios are reported as mean ± S.E.M. * p < 0.05, Student's two t a i l e d t t e s t , f o r d i f f e r e n c e between U-0521/carbidopa t r e a t e d group and carbidopa t r e a t e d r a t s done at the same time. 73 c o n t r o l s f o r the COMT i n h i b i t i o n experiment. However, these e f f e c t s do not reach s i g n i f i c a n c e because of the l a r g e standard e r r o r s . The only experimental d i f f e r e n c e between the two st u d i e s was t h a t the COMT i n h i b i t i o n experiments were preformed i n s p r i n g , while the time course study was conducted during the f a l l . The r e s u l t s of the experiment i n which U-0521 alone was administered to hooded r a t s are i l l u s t r a t e d i n Table V I I I . At 90 minutes a f t e r i n j e c t i o n of U-0521 (25 mg/kg i . p . ) , the c o n c e n t r a t i o n of DOPAC i n s t r i a t u m was reduced by more than 30% (p < 0.025). S t r i a t a l c o n c e n t r a t i o n of 3-MTyr was m a r g i n a l l y reduced (p < 0.1). No e f f e c t s were noted on hypothalamic amines or m e t a b o l i t e s . T h e ' s t r i a t a l c o n c e n t r a t i o n of DOPAC i n c o n t r o l animals from the U-0521 study (2704 ± 161 ng/g) was c o n s i d e r a b l y higher than i n the o r i g i n a l c o n t r o l s (1568 ± 152 ng/g). This d i f f e r e n c e could be r e l a t e d to an e f f e c t of carbidopa on s t r i a t a l dopamine metabolism i n the o r i g i n a l c o n t r o l s . However, i t should be noted t h a t s t r i a t a l DOPAC c o n c e n t r a t i o n s i n c o n t r o l groups are often found to vary between experiments (P.Cumming, unpublished o b s e r v a t i o n s ) . I t may be t h a t DOPAC l e v e l s are s e n s i t i v e to such f a c t o r s as handling of animals, a n e s t h e s i a or the season of the y e a r . Re s u l t s o f 6-Hydroxydopamine L e s i o n s : In the l e s i o n e d animals, the r i g h t caudate ( c o n t r a l a t e r a l to the l e s i o n s ) showed e s s e n t i a l l y the same co n c e n t r a t i o n s of endogenous catecholamines and l e v e l s of 1 8F-D0PA and i t s metabolites as seen i n pooled caudates of non-1esioned animals s a c r i f i c e d a t the same time (15 minutes) a f t e r 1 8F-D0PA a d m i n i s t r a t i o n and 45 minutes a f t e r carbidopa treatment. The t o t a l r a d i o a c t i v i t y and l e v e l s of 1 8F-D0PA and Me- 1 8F-D0PA i n the vermis were a l s o the same i n l e s i o n e d and un l e s i o n e d animals. The caudates on the l e s i o n e d s i d e , however, showed a g r e a t e r than Table VIII The E f f e c t of U-0521 Alone on Catecholamine and Indoleamine Concentrations i n the Rat Striat u m and Hypothalamus at 90 minutes A f t e r I n j e c t i o n . T i s s u e NA DOPAC HIAA DA HVA 3-MTyr 5-HT Str i a t u m sham (n=3) 251± 2704± 5561 107201 1273± 477± 423± 31 161 16 216 67 39 11 U-0521 (n=3) 293± 18021 5131 104651 1159± 3551 481 + (25 mg/kg) 52 110** 17 143 164 38* 33 Hypothalamus sham (n=3) 1454± 61± 427± 342± n.d 25i 710± 162 10 42 21 10 17 U-0521 (n=3) 1440± 52i 412t 3261 n .d. 23± 771± (25 mg/kg) 114 32 30 102 9 26 Concentrations are i n ng/g ± S .E.M., * p < 0 .1 , ** p < 0.025. S i g n i f i c a n c e between groups determined by Student's two t a i l e d t t e s t . A b b r e v i a t i o n s as de s c r i b e d i n t e x t except f o r h y d r o x y i n d o l e a c e t i c a c i d (HIAA) and se r o t o n i n (5-HT). n.d. = none detected. 99% d e p l e t i o n of endogenous dopamine and only 85 ± 2% of the 1 8 F a c t i v i t y (CPM/mg) with r e s p e c t to the normal s t r i a t u m . The r a t i o of r a d i o a c t i v i t y i n the l e s i o n e d l e f t caudates to the r a d i o a c t i v i t y i n the vermis (S/V = 0.989 ± 0.03) was s i g n i f i c a n t l y reduced as compared to the unlesioned s i d e (S/V = 1.05 ± 0.04, p <0.025). The p r o p o r t i o n of l 8F-D0PA was gr e a t e r i n the l e f t s i d e than i n the r i g h t s t r i a t a (p < 0.001, n = 4) as was the p r o p o r t i o n of Me- 1 8F-D0PA (p < 0.05, F i g . 32). There were t r a c e s of 1 8F-D0PAC and 1 8F-DA i n the l e f t s t r i a t a , but these were, r e s p e c t i v e l y , only 27% and 13% of the l e v e l s observed i n the r i g h t s t r i a t a . D. Experiments with 2 - 1 8 F - F l u o r o d o p a Metabolism i n vi v o 1) Methods and R e s u l t s Approximately equal amounts 2 - 1 8 F - f l u o r o d o p a (2- 1 8F-D0PA) and 6- 1 8F-D0PA are formed during the s y n t h e s i s of r a d i o t r a c e r f o r PET s t u d i e s . Because the 2- 1 8F-D0PA i s r o u t i n e l y separated and d i s c a r d e d , a number of experiments were undertaken i n order to c h a r a c t e r i z e the metabolism of t h i s compound. In one study, carbidopa prereated animals (n=7) were administered 2- 1 8F-D0PA (500 pCi/ k g ) . At va r i o u s times between 20 and 130 minutes, animals were given an overdose of p e n t o b a r b i t a l and then exsanguinated by t r a n s c a r d i a l p e r f u s i o n with 0.9% NaCl. A f t e r d i s s e c t i o n , gamma a c t i v i t y was determined i n weighed samples of heart, l u n g , l i v e r , kidney, spleen and blood. B r a i n s were d i s s e c t e d and a c t i v i t y determined i n c o r t e x , hippocampus, s t r i a t u m , cerebellum, and pons/medulla. Tissue a c t i v i t y was c a l c u l a t e d as the percentage of the i n j e c t e d dose per gram. In one animal, s a c r i f i c e d a t 30 minutes a f t e r 2- 1 8F-D0PA i n j e c t i o n , s t r i a t u m and vermis samples were prepared f o r a n a l y s i s by HPLC. 70-60-' > | 40-«5 O 30-20-10-b* b * a * a * # # nd nd 1 2 3 4 1 2 3 4 1 2 3 4 F-DOPA Me-F-DOPA F-DOPAC 1 2 3 4 F-DA Figure 32: The effect of 6-OHDA injections to the l e f t medial forebrain bundle on the s t r i a t a l metabolism of 18F-D0PA at 15 minutes post in j e c t i o n . The relative concentrations of 18F-D0PA, Me-18F-D0PA, 18F-D0PAC and 18F-DA are expressed as means ± S.E.M. 1 = l e f t s t r i a t a , 2 = right s t r i a t a , 3 = pooled s t r i a t a of non-lesioned animals, 4 = vermis samples. S t a t i s t i c a l l y s i g n i f i c a n t differences between groups 1 and 2: (a*) p < 0.05; (a**) p < 0.01; (a***) p < 0.001. S t a t i s t i c a l l y s i g n i f i c a n t difference between groups 1 and 4: (b*) p < 0.05. N = 4/group. There were no sig n i f i c a n t differences between groups 2 and 3. n.d. not detectable. 77 The r e s u l t s of the 2- 1 8F-D0PA b i o d i s t r i b u t i o n study are i l l u s t r a t e d i n Fi g u r e 33A. R a d i o a c t i v i t y passed r e a d i l y from blood i n t o v i s c e r a l organs and l e v e l s were c o n s i d e r a b l y higher i n kidney than i n other organs, i n d i c a t i n g t h a t renal e l i m i n a t i o n of the compound was very r a p i d . In F i g u r e 33B the time course of r a d i o a c t i v i t y i n va r i o u s b r a i n regions i s i l l u s t r a t e d . R a d i o a c t i v i t y l e v e l s i n blood were higher than i n c e r e b r a l t i s s u e s , e s p e c i a l l y at time p o i n t s before one hour. In c o n t r a s t to f i n d i n g s with 6- 1 8F-D0PA, there was no evidence of a s p e c i f i c accumulation of r a d i o a c t i v i t y i n s t r i a t u m . However, l e v e l s i n cerebellum are c o n s i s t e n t l y about 15% higher than i n other b r a i n areas. A n a l y s i s of a s t r i a t a l sample at 45 minutes f o l l o w i n g 2- 1 8F-D0PA i n j e c t i o n ( F i g . 34B) i n d i c a t e s t hat 2- 1 8F-D0PA was not decarboxylated to an a p p r e c i a b l e extent i n r a t s t r i a t u m . The only metabolite present i n st r i a t u m i n s i g n i f i c a n t amounts was Me-2- 1 8F-D0PA. S i m i l a r q u a n t i t i e s of t h i s compound were a l s o present i n cerebellum ( F i g . 34A). The amount of 2- 1 8F-D0PA was c o n s i d e r a b l y higher i n cerebellum than i n s t r i a t u m a t t h i s time p o i n t . E. Studies on the i n v i t r o Metabolism of F-DOPA 1) Methods In order to c h a r a c t e r i z e b e t t e r the 0-methylation of F-DOPA, the a b i l i t i e s of F-DOPA and L-DOPA to i n h i b i t COMT c o m p e t i t i v e l y were determined by i n v i t r o experiments. Competitive i n h i b i t i o n constants (Kj) were determined f o r the two compounds by means of a radiochemical COMT assay with dihydroxybenzoic a c i d (DBA) s e r v i n g as the s u b s t r a t e ( G u l l i v e r and T i p t o n , 1978). T r i p l i c a t e assay tubes c o n t a i n e d , i n a f i n a l volume of 120 u l , 4 mM Mg 2 +, 150 uM phosphate b u f f e r a t pH 7.4, DHBA at four c o n c e n t r a t i o n s ranging from 0.13 mM to 1.05 mM and 20 yl of a r a t 78 , i n 1 r 3 0 6 0 9 0 120 150 3 0 6 0 9 0 120 150 Time (minutes) Figure 33: The distribution of radioactivity in peripheral and brain tissues of the rat following administration of 2-18F-D0PA. Activity in peripheral tissues (A) and in various brain regions (B) is calculated as percentage of the injected dose/gram of tissue. Activity/nig tissue is consistently higher in cerebellum than in other brain regions. Each point represents a single determination. 10 7.5 5.0 2.5 CO i O CL O 2 2-F-DOPA Me-2-F-DOPA 10 11 2 0 3 0 4 0 •1 5 0 B 10 20 30 Fraction Number 40 50 Figure 34: The cerebral metabolism of 2-18F-D0PA in the rat. Radiochromatograms from 200 ul 10:1 homogenates of vermis (A) and striatum (B) do not reveal the presence of any metabolites other than Me-2-18F-D0PA at 45 minutes after injection of 2-18F-D0PA. Uptake of 2-18F-D0PA i s greater in vermis than in striatum. Note difference in scales. 80 c e r e b e l l a r homogenate i n ten volumes of 0.5% T r i t o n - X . The homogenate contained 9.6 mg protein/ml as assayed by the Bradford method (M. Bradford, 1976) with bovine serum albumin s e r v i n g as the p r o t e i n standard. The r e a c t i o n mixture c o n t a i n e d 50,000 DPM of 1 1 +C-S-adenosylmethionine per tube d i l u t e d with c o l d SAM to a f i n a l c o n c e n t r a t i o n of 60 yM. Incubation was f o r 30 minutes a t 37 °C. The r e a c t i o n was terminated by the a d d i t i o n of an equal volume of 1M HC1. Methylated s u b s t r a t e was e x t r a c t e d i n t o 5 ml toluene and r a d i o a c t i v i t y was determined by two minute counts i n the P h i l l i p s PW-4700 l i q u i d s c i n t i l l a t i o n counter upon the a d d i t i o n of 10 ml toluene based s c i n t i l l a n t . R esults of such experiments were p l o t t e d i n the form of Lineweaver-Burke with l i n e a r r e g r e s s i o n a n a l y s i s of the s l o p e s . The e f f e c t s o f e i t h e r F-DOPA or L-DOPA on the methylation of DHBA were determined by the a d d i t i o n of these compounds to 1 mM f i n a l c o n c e n t r a t i o n . K-j s were determined f o r each compound upon c a l c u l a t i o n of the slopes of the double r e c i p r o c a l p l o t s . 0-methylated DOPA's were not e x t r a c t a b l e i n t o the organic phase. The k i n e t i c s of the 0-methylation of U-0521 were determined by s u b s t i t u t i n g t h i s compound f o r DHBA at f o u r c o n c e n t r a t i o n s ranging from f i v e to 90 yM. Incubation i n t h i s case was f o r f o r t y minutes at 37 °C. Because of the apparent lack of i n vi v o formation of 2- 1 8F-DA, experiments were preformed to compare the k i n e t i c parameters of de c a r b o x y l a t i o n of 2 and 6- 1 8F-fluoro-L-D0PA. The d a i l y c o n c e n t r a t i o n of 1 8F-D0PA was determined using the u l t r a v i o l e t a b s orption measured on a Unicam SP-500 spectrophotometer. The molar e x t i n c t i o n c o e f f i c e n t s of the DOPAs were as f o l l o w s : e280 (L-DOPA) = 2648 L/mol/cm, e272 (2-F-DOPA) = 1850 L/mol/cm and e283 (6-F-DOPA) = 3836 L/mol/cm. The value of e determined f o r L-DOPA was almost i d e n t i c a l to a l i t e r a t u r e value ( M i n e l l i e t a l . , 1979). In p r a c t i c e , the c o n c e n t r a t i o n of 2- 1 8F-D0PA was determined i n d i r e c t l y knowing the r e l a t i v e y i e l d s of the two compounds. For the decarboxylase assays, s t r i a t a l homogenate was prepared by mechanical homogenization of f r e s h l y d i s s e c t e d s t r i a t a in 20 volumes of 0.5% T r i t o n - X . S o n i c a t i o n was not used s i n c e i t was found to decrease enzyme a c t i v i t y . The homogenate was c e n t r i f u g e d f o r ten minutes at 10,000 g and to each r e a c t i o n tube was added 25 pi of supernatant. Each tube a l s o contained 1.0 mM p a r g y l i n e , and 50 pg p y r i d o x a l phophate i n 75 pM phosphate b u f f e r (pH 7.2) to a f i n a l volume of 250 p i . 1 8F-D0PAs at four f i n a l c o n c e n t r a t i o n s ranging from 50 to 500 pM were incubated i n the above medium f o r 30 minutes at 37 °C. Incubations were a r r e s t e d by the a d d i t i o n of an equal volume of 0.1 M p e r c h l o r i c a c i d . Tubes were then placed on i c e f o r 30 minutes and c e n t r i f u g e d at 10,000 g f o r 25 minutes. 20 pi samples of supernatant were i n j e c t e d onto HPLC o p e r a t i n g i s o c r a t i c a l l y at 1.2 ml/min with a composition of 85% b u f f e r A and 15% b u f f e r B. A f t e r having c h a r a c t e r i z e d the r e a c t i o n s as producing only one l a b e l l e d product, i t was necessary to count gamma a c t i v i t y only i n those tubes c o n t a i n i n g fluorodopamine. Gamma a c t i v i t y f o r each tube was c o r r e c t e d to a common time p o i n t . The e l u t i o n times f o r the products under these c o n d i t i o n s were 10.2 minutes f o r 2- 1 8F-DA and 13.2 minutes f o r 6- 1 8F-DA. In blank r e a c t i o n s with 6- 1 8F-D0PA to which no s t r i a t a l homogenate was added, there was no product formation. However, 2- 1 8F-D0PA was found to be non-enzymatically decarboxylated at a r a t e of 0.8% per hour. Moles of product formed were c a l c u l a t e d using the r a t i o of ( 1 8F-DA c o l l e c t e d - blank) over the t o t a l a c t i v i t y i n an equal volume of each r e a c t i o n mixture. Recovery of r a d i o a c t i v i t y by HPLC was e s s e n t i a l l y q u a n t i t a t i v e (103 ± 4%, S.E.M., n = 15). Homogenates were fro z e n f o r two weeks p r i o r to determination o f p r o t e i n c o n c e n t r a t i o n by the Bradford method. 2) R e s u l t s of i n v i t r o S t u d i e s A l l k i n e t i c constants are reported as the means of three seperate determination ± S.E.M. unless otherwise noted. The r e s u l t s of a study using 6-D,L-F-D0PA and L-DOPA as competitive i n h i b i t o r s of the methylation of DBA by COMT are i l l u s t r a t e d i n F i g u r e 35A. The Kj f o r L-DOPA was 1.29 ± 0.22 mM, while t h a t f o r 6-F-D0PA was 0.384 ± 0.034 mM. The mean r a t i o o f the two K-jS from three separate determinations was 2.45 ± 0.80. The K m f o r DBA was 90 ± 10 yM. A Lineweaver-Burke p l o t f o r the O-methylation of U-0521 i s i l l u s t r a t e d i n F i g u r e 35B. The K m determined f o r U-0521 was 5.5 ± 0.3 yM. Lineweaver-Burke p l o t s of the d e c a r b o x y l a t i o n of 2- and 6- 1 8F-D0PA are i l l u s t r a t e d i n F i g u r e 36. The K m f o r 2- 1 8F-D0PA was 1.08 mM and the Vmax was approximately 103 nmoles/hour/mg p r o t e i n (2 d e t e r m i n a t i o n s ) . The K m f o r 6- 1 8F-D0PA was 167 ± 33 yM and the Vmax was 234 ± 48 nmole/hour/ng p r o t e i n . (4 d e t e r m i n a t i o n s ) . IV. D i s c u s s i o n The metabolic pathways f o r 1 8F-D0PA i n v i v o are sumarized i n Fi g u r e 37. The r e s u l t s of a v a r i e t y of experiments i n d i c a t e that p e r i p h e r a l 1 8F-D0PA metabolism under c o n d i t i o n s of carbidopa pretreatment i s dominated by a s i n g l e enzyme, COMT. Under normal c o n d i t i o n s , one would expect 1 8F-D0PA to be r a p i d l y decarboxylated and subsequently metabolized t o 1 8F-D0PAC and 1 8F-DA conjugates. The study of plasma metabolism i n the r a t , monkey and human i n d i c a t e s t h a t the .peripheral d e c a r b o x y l a t i o n of 1 8F-D0PA can be 8-i 5x10 4-| .c \ a 4 o > s 2.4 1-d" B • • DHBA • DHBA + 1 mM L-DOPA • DHBA • 1 mM 6-F-D0PA 2 1 1 4 6 1/S (x103 M"') 15x10"' B U -0521 K ms 5 u M 0 \ V t> 0 E c > N 8 i 12 18 V S ( x 1 0 3 M " 1 ) 1 24 Figure 35: Lineweaver-Burke plots showing the the competitive inhibition of COMT by L-DOPA and D,L-6-f-D0PA with dihydroxybenzylacetic acid (DHBA) serving as the substrate (A) and the O-methyl ation of U-0521 by catechol-O-methyltransferase (B). 4 8 12 16 20 1/S ( X 1 0 3 M"') Figure 36: Lineweaver-Burke plots of the k i n e t i c s of the decarboxylation of L-2-and 6-18F-D0PA by a s t r i a t a l homogenate. Enzyme ve l o c i t y (V) was calculated i n units of nmoles 1 8F-dopamine formed/mg protein/hour. Each point represents the mean of two determinations which d i f f e r e d by less than 15%. Figure 37: Summary of the metabolism of 18F-D0PA. Following i n j e c t i o n , 18F-D0PA i s metabolized to Me-18F-D0PA by catechol-0-tnethyltransferase (COMT) in the periphery. Both compounds accumulate in brain through a combination of diffusion and uptake. COMT within the brain may further metabolize 18F-D0PA. In striatum, but not in vermis, 18F-D0PA may be decarboxylated to 18F-DA by the action of aromatic amino acid decarboxylase (AAAD). 18F-DA thus formed may be metabolized by monoamine oxidase (MAO) within dopaminergic terminals and/or released and subsequently metabolized by COMT and MAO to 1 8F-3MTyr, 18F-D0PAC and 18F-HVA. The rate at which these l a t t e r compounds pass out of striatum has yet to be determined. 86 e n t i r e l y blocked by carbidopa a t doses i n the range of 1.5 to 5 mg/kg. However, as carbidopa i s a p o t e n t i a l s u b s t r a t e f o r COMT, the e f f e c t of carbidopa i s probably not r e s t r i c t e d to the i n h i b i t i o n of the decarboxylase. Carbidopa i s reported to be O-methylated by p i g l i v e r COMT with k i n e t i c constants agreeing w i t h i n a f a c t o r of two to those found f o r L-DOPA (Hagen e t a l . , 1980). Therefore, carbidopa might reduce the r a t e o f formation of Me- 1 8F-D0PA by competitive i n h i b i t i o n of COMT. Because the accumulation o f t h i s m e t a b o l i t e i n plasma was such a c o n s i s t e n t f i n d i n g , the e f f e c t of carbidopa on p e r i p h e r a l 0-methylation must be minimal at the doses used i n t h i s study. The formation of methylated m e t a b o l i t e s i n the periphery has a l s o been noted p r e v i o u s l y i n r a t s given ( 3H)F-D0PA (Chuieh et a l . , 1984) or ( 3H)D0PA (Home e t a l . , 1984). That c a t e c h o l s are O-methylated i n v i v o was an e a r l y f i n d i n g i n the f i e l d of neu r o t r a n s m i t t e r metabolism ( A x e l r o d , 1957). The enzyme COMT was c h a r a c t e r i z e d as r e q u i r i n g magnesium ion f o r c a t a l y s i s and SAM as a methyl donating c o f a c t o r ( A x e l r o d and Tomchick, 1958). I t was o r i g i n a l l y suggested t h a t COMT was e x t r a c e l l u l a r and i n c l o s e a s s o c i a t i o n with plasma membrane bound 3-receptors (Axelrod, 1966). However, i n l i v e r , the organ with the hi g h e s t enzyme a c t i v i t y , COMT has been shown to e x i s t mainly i n an i n t r a c e l l u l a r s o l u b l e f r a c t i o n (Raxworthy e t a l . , 1982). Membrane bound and microsomal forms of the enzyme c o n s t i t u t e only minor p r o p o r t i o n s o f t o t a l COMT a c t i v i t y i n r a t l i v e r . T h i s suggests t h a t metabolism by t h i s enzyme must be proceeded by passage of substrate across plasma membranes. The r a t l i v e r enzyme apparently e x i s t s i n two forms with molecular weights o f 24,000 and 48,000 (Huh and F r i e d h o f f , 1979). However, i t i s not known i f these isozymes d i f f e r with regard to c a t a l y t i c a c t i v i t y . A wide v a r i e t y of c a t e c h o l s are substrates f o r COMT, with l i t t l e v a r i a t i o n i n the values f o r k i n e t i c constants found f o r the m e t h y l a t i o n r e a c t i o n (Guldberg and Marsden, 1975). The K m here r e p o r t e d f o r DBA i s two-fo l d lower than l i t e r a t u r e r e p o r t s (Broch and Guldberg, 1971, Guldberg and Marsden, 1975), a d i f f e r e n c e p o s s i b l y r e l a t e d to such f a c t o r s as pH and c o f a c t o r c o n c e n t r a t i o n (Mason and Weinkove, 1984). However, t h i s d i f f e r e n c e should not d e t r a c t from the r e s u l t s of the co m p e t i t i v e i n h i b i t i o n study which i n d i c a t e d t h a t D,L-6-F-D0PA was a much b e t t e r i n h i b i t o r of COMT than L-DOPA. COMT has g e n e r a l l y been co n s i d e r e d to be n o n - s t e r e o s p e c i f i c ( A x e l r o d and Tomchick, 1958, Garg e t a l . , 1971). However, a recent r e p o r t has shown the enzyme to have a 2-3 f o l d preference f o r the L-isomer of c a t e c h o l s (Raxworthy e t a l . , 1986). This would suggest t h a t L-fluorodopa would be an even b e t t e r competitive i n h i b i t o r of COMT than i s the racemic compound. The Km determined f o r U-0521 i n the present study i s very close to i t s reported K as an i n h i b i t o r f o r the O-methylation o f epinephrine by l i v e r COMT (G i l e s and M i l l e r , 1967). This i n d i c a t e s t h a t U-0521 has a much higher a f f i n i t y f o r COMT than e i t h e r L-DOPA or fluorodopa. In v i v o , COMT i s found to produce almost e x c l u s i v e l y the meta-O-methylated d e r i v a t i v e of natural c a t e c h o l s . However s i g n i f i c a n t amounts of the p a r a - s u b s t i t u t e d products are found i n i n v i t r o s t u d i e s . In order to e x p l a i n t h i s phenomenon, i t has been suggested e i t h e r t h a t e x t r a c t i o n of the enzyme a l t e r s i n some way the nature of the a c t i v e s i t e , or that systems e x i s t i n g in vivo are capable of p r e f e r e n t i a l l y degrading the para-O-methyl metabolites (Guldberg and Marsden, 1975). 5-F1uorodopa seems to be mostly O-methylated i n the para p o s i t i o n ( F i r n a u e t a l . , 1981). These authors suggest that a d d i t i o n of an e l e c t r o n withdrawing f l u o r i n e 88 atom i n the 5 - p o s i t i o n of a catechol promotes phenol i o n i z a t i o n a t the adjacent para-hydroxyl group. From t h i s , one would p r e d i c t t h a t 6-F-DOPA would be O-methylated i n the m e t a - p o s i t i o n . T h i s has been claimed to be the case i n a subsequent study, although the s i n g l e product formed was not r i g o r o u s l y i d e n t i f i e d ( C r e v e l i n g and K i r k , 1985). These authors determined the k i n e t i c constants f o r O-methylation of DOPA and the three p o s s i b l e F-DOPAs. V m a x was constant f o r a l l of these compounds, but the Km f o r 6-F-DOPA (550 yM) was ten times higher than t h a t f o r L-DOPA and 20 times higher than t h a t found f o r 2- or 5-F-DOPA. From t h i s , i t was concluded t h a t 6-F-DOPA should not be O-methylated to an a p p r e c i a b l e extent i n v i v o . T h i s p r e d i c t i o n i s i n c o n t r a s t to the observed metabolism of 6- 1 8F-D0PA i n r a t , monkey and human. To a great extent, the O-methylation of 6- 1 8F-D0PA may occur o p p o r t u n i s t i c a l l y under c o n d i t i o n s of p e r i p h e r a l decarboxylase i n h i b i t i o n with c a r b i d o p a . The lower K-j here reported f o r 6-F-DOPA i s not i n c o n s i s t e n t with a higher Km with r e s p e c t to L-DOPA si n c e F-DOPA may i n h i b i t COMT without being a good s u b s t r a t e i t s e l f . The e l i m i n a t i o n of L-DOPA from plasma i s b i e x p o n e n t i a l i n man (Nutt e t a l . , 1985) with terms s i m i l a r to those found here f o r 6- 1 8F-D0PA. A f t e r o r a l a d m i n i s t r a t i o n of L-DOPA without carbidopa pretreatment, the major met a b o l i t e found i n human plasma i s HVA, and, to a l e s s e r extent, Me-DOPA (Sharpless e t a l . , 1971). Plasma HVA l e v e l s reach a maximum at about two hours a f t e r L-DOPA while Me-DOPA accumulates more g r a d u a l l y . Me-DOPA p e r s i s t s i n human plasma, with a h a l f - l i f e on the order of ten hours a f t e r an oral dose (Calne e t a l . , 1972). This e v e n t u a l l y leads to plasma l e v e l s o f m e t a b o l i t e exceeding those of L-DOPA i n Parkinsonians under treatment. Me-DOPA at doses o f 400 mg/kg has been shown to i n t e r f e r e with the formation of dopamine i n r a t s t r i a t u m , presumably by competition f o r uptake 89 i n t o s t r i a t u m with t y r o s i n e and L-DOPA (Reches and Fahn, 1981). Me-DOPA, formed a f t e r L-DOPA a d m i n i s t r a t i o n (125 mg/kg i.p.) i n the bense r a z i d e -t r e a t e d r a t , accumulates i n striatum (Gemayel e t a l . , 1986). Dopamine l e v e l s i n the s t r i a t u m i n these animals were e l e v a t e d over a fo u r hour p e r i o d , but OMe-DOPA, which i s not a normal c o n s t i t u e n t o f s t r i a t u m , a l s o accumulated over t h i s time and was present a t higher c o n c e n t r a t i o n s than dopamine at nine hours a f t e r L-DOPA i n j e c t i o n . In the l i g h t of the above f i n d i n g s i t i s not s u r p r i s i n g t h a t Me-6- 1 8F-D0PA should be the p r i n c i p a l 6- 1 8F-D0PA m e t a b o l i t e found i n b r a i n . The r e s u l t s of the time course study of 6- 1 8F-D0PA metabolism show t h a t Me-6- 1 8F-D0PA l e v e l s exceeded 6- 1 8F-O0PA i n cortex a f t e r 15 minutes. In st r i a t u m , the p e r i p h e r a l m e t a b o l i t e exceeded the sum of other l a b e l l e d compounds a f t e r 60 minutes. T h i s accumulation probably r e f l e c t s a combination of d i f f u s i o n and/or t r a n s p o r t of the compound i n t o b r a i n and a d d i t i o n a l O-methylation of 6- 1 8F-D0PA by c e r e b r a l COMT. Tot a l COMT i s ra t h e r evenly d i s t r i b u t e d i n b r a i n , but some l o c a l inhomogeneities are present. Cerebellum c o n t a i n s COMT a c t i v i t y approximately 50% higher than i n most other regions of the r a t b r a i n , i n c u d i n g cortex and s t r i a t u m ( R i v e t t e t a l . , 1983a). T h i s may c o n t r i b u t e to the apparently more r a p i d accumulation of Me- 1 8F-D0PA i n vermis as compared to s t r i a t u m . As i n the l i v e r and other p e r i p h e r a l t i s s u e s , most of the b r a i n COMT i s s o l u b l e r a t h e r than membrane bound. However, the p r o p o r t i o n o f membrane bound enzyme i s somewhat higher i n b r a i n than i n other organs. An inmunohistochemical study showed the ma j o r i t y of b r a i n COMT ( s o l u b l e ) to be l o c a l i z e d i n g l i a l c e l l s r a t h e r than neuronal elements (Kaplan e t a l . , 1979). In c o n t r a s t , the membrane bound form seems to be contained i n neurones ( R i v e t t e t a l . , 1983b). The membrane bound form i s presumably c r i t i c a l f o r n e u r o t r a n s m i t t e r metabolism s i n c e i t has a 100-fold lower Km f o r catecholamines than has the s o l u b l e form ( R i v e t t e t a l . , 1982). I t has been suggested t h a t t h i s f i n d i n g i s r e l a t e d to a conformational change i n the membrane bound enzyme produced by Triton-X used i n the e x t r a c t i o n procedure, but the two enzyme forms may s t i l l d i f f e r s t r u c t u r a l l y ( J e f f e r y and Roth, 1983). The l i n e a r r e l a t i o n s h i p between Me-6- 1 8F-D0PA and 6- 1 8F-D0PA (A free/bound) i n plasma as a f u n c t i o n of time was an unexpected f i n d i n g . I t i s not r e a d i l y apparent why t h i s r e l a t i o n s h i p should be modeled by a f i r s t order f u n c t i o n ; however the c o n s i s t e n t l y high c o e f f i c i e n t s o f c o r r e l a t i o n found i n i n d i v i d u a l humans and i n r a t s and monkeys s t r o n g l y suggest t h a t i t should be p o s s i b l e to d e r i v e k i n e t i c f u n c t i o n s which would p r e d i c t t h i s . The present data may not be s u f f i c i e n t to t h i s end because of the low number of measurements made i n the f i r s t 30 minutes of the experiment, the p e r i o d during which most of the O-methylated m e t a b o l i t e i s formed. I f plasma Me- 1 8F-D0PA were determined at two minute i n t e r v a l s i n t h i s p e r i o d , i t should be p o s s i b l e to f i t the curve to a polynomial e x p r e s s i o n . Since some component of t h i s e x p ression should be r e l a t e d to 1 8F-D0PA c o n c e n t r a t i o n as a f u n c t i o n of time, i t might be p o s s i b l e to d e r i v e a f u n c t i o n f o r metabolite/ 1 8F-D0PA which i s r e d u c i b l e to a f i r s t o rder e x p r e s s i o n . The l i n e a r r e l a t i o n s h i p between the slope of A free/bound and age i n humans i n i t i a l l y was taken to i n d i c a t e some change i n 1 8F-D0PA metabolism with age. However, a n a l y s i s of plasma data does not support the i n t e r p r e t a t i o n t hat the age e f f e c t i s a f u n c t i o n s o l e l y of the metabolism and e l i m i n a t i o n of 1 8F-D0PA, s i n c e the values f o r 1 8F-D0PA disappearance, A 3 and X 4 , were not found to vary with age. The usual i n t e r p r e t a t i o n of b i e x p o n e n t i a l pharmacokinetics i s t h a t the f i r s t term i s r e l a t e d to metabolism (O-methylation i n the l i v e r and other t i s s u e s ) , while the second term i s r e l a t e d to renal e l i m i n a t i o n . Given t h a t i t i s well e s t a b l i s h e d t h a t h e p a t i c drug e l i m i n a t i o n and metabolism can d e c l i n e with age (O'Malley and K e l l y , 1982), one might have expected A3 to be age dependent. In p a r t i c u l a r , i t has been reported t h a t human l i v e r COMT a c t i v i t y reaches a maximum at age 20 and i s reduced i n the group over 60 year s of age (Agathapoulous e t a l . , 1971). In c o n t r a s t , the a c t i v i t y o f COMT i n human e r y t h r o c y t e s has not been shown to have any c o r r e l a t i o n with age. (Siervogel e t a l . , 1984). Thus, the e f f e c t s of aging on COMT are i n need of c l a r i f i c a t i o n by f u r t h e r study. A p o s s i b l e cause f o r disagreement may be the assay methods used . In e a r l y s t u d i e s of COMT, the products formed i n v i t r o have not always been r i g o r o u s l y i d e n t i f i e d . The present f i n d i n g i s t h a t the age e f f e c t on 6- 1 8F-D0PA metabolism i s only observed i n \\ and A2, the time constants f o r the disappearance of t o t a l 1 8 F from plasma. T h i s suggests t h a t the age e f f e c t may be r e l a t e d t o the handling of Me-6- 1 8F-D0PA i t s e l f , r a t h e r than 6- 1 8F-D0PA. Assuming that the p r o p o r t i o n o f 6- 1 8F-D0PA metabolized was the same i n young and o l d i n d i v i d u a l s , and t h a t the ra t e of t h i s metabolism was not age r e l a t e d , no age e f f e c t would be expected i n A3 and A4. I f the t r a n s f e r of the metabolite from t i s s u e to the plasma compartment were l e s s f a c i l e i n o l d e r i n d i v i d u a l s , perhaps due to some age r e l a t e d change i n membrane p r o p e r t i e s , the accumulation of m e t a b o l i t e i n plasma would be delayed. T h i s would r e s u l t i n a decreased r a t i o of metabolite to 6- 1 8F-D0PA i n the plasma of ol d e r i n d i v i d u a l s . In keeping with t h i s e x p l a n a t i o n , the gr e a t e r age dependence and the higher age c o r r e l a t i o n were found i n \\, a term which could be i n f l u e n c e d by back-flu x of r a d i o a c t i v i t y from t i s s u e i n t o plasma. To the f i r s t approximation, X2 i s probably d e f i n e d by X5, the r a t e of renal e l i m i n a t i o n of Me-6- 1 8F-D0PA. This i s because Me-6- 1 8F-D0PA disappearance i s almost synonymous with t o t a l 1 8 F disappearance a t times when Me- 1 8F-D0PA i s the p r i n c i p a l l a b e l l e d compound i n plasma. Since X 5 d i d not demonstrate any age dependence, i t i s not s u r p r i s i n g t h a t the e f f e c t of age on X 2 w a s n o t a s Targe as the e f f e c t of In c o n c l u s i o n , the age e f f e c t on A free/bound seems to be r e l a t e d to k i n e t i c f a c t o r s i n the metabolism of Me- 1 8F-00PA which cannot be f u l l y c h a r a c t e r i z e d using the present data. The o b s e r v a t i o n that 1 8F-D0PA metabolism i n the group of Chamorros d i f f e r e d from other i n d i v i d u a l s deserves some a t t e n t i o n . Slopes o f A free/bound d e r i v e d f o r the Chamorros were c o n s i d e r a b l y higher than the re g r e s s i o n slope f o r the age e f f e c t i n other i n d i v i d u a l s . T h i s leads one to c o n s i d e r p o s s i b l e genetic f a c t o r s i n catecholamine metabolism. In a f a m i l i a l study of e r y t h r o c y t e COMT a c t i v i t y , a high c o r r e l a t i o n was noted between s i b l i n g s . In a d d i t i o n , the d i s t r i b u t i o n of e r y t h r o c y t e COMT a c t i v i t y i n i n d i v i d u a l s was b i p h a s i c which i s suggestive o f a f a m i l i a l component to COMT a c t i v i t y , p o s s i b l y r e l a t e d to gene polymorphism (Weinshilboum e t a l . , 1974). In another study, the frequency d i s t r i b u t i o n of e r y t h r o c y t e COMT a c t i v i t y was t r i p h a s i c i n a group of Caucasians. The frequency d i s t r i b u t i o n i n a group of F i l i p i n o s and Chinese was s i g n i f i c a n t l y higher than among the Caucasians ( R i v e r a - C a l i m l i m and R e i l l y , 1984). I t has a l s o been reported t h a t i n d i v i d u a l d i f f e r e n c e s i n e r y t h r o c y t e COMT a c t i v i t y c o r r e l a t e s t r o n g l y with r a t i o s of Me-L-DOPA to L-DOPA i n plasma, and t h a t these d i f f e r e n c e s are r e l a t e d to adverse responses to L-DOPA treatment ( R e i l l y e t a l . , 1980). However, the terms f o r the disappearance of 1 8F-D0PA from plasma ( X 3 and X 4 ) d i d not 93 d i f f e r g r e a t l y between the Chamorros and other i n d i v i d u a l s . The p r i n c i p a l d i f f e r e n c e was found i n A i , the f i r s t term of the disappearance of t o t a l 1 8 F from plasma. I t i s not p o s s i b l e to e x p l a i n t h i s f i n d i n g except to propose that the f a c t o r s r e s p o n s i b l e f o r the age e f f e c t i n Caucasians may proceed l e s s r a p i d l y among Chamorros. The study of 6- 1 8F-D0PA metabolism i n monkeys i n d i c a t e s t h a t the terms f o r 6- 1 8F-D0PA and t o t a l 1 8 F disappearance were roughly s i m i l a r i n monkey and i n humans. The slope of A free/bound f e l l w i t h i n the human range i n two out of three monkeys. Ther e f o r e , the monkey can be considered a good model f o r human 6- 1 8F-D0PA metabolism. The two-fold d i f f e r e n c e i n e l i m i n a t i o n r a t e s f o r Me-6- 1 8F-D0PA i n monkey and human should not be h i g h l y s i g n i f i c a n t i n the time course of PET experiments. The r a t , however, seems to be a somewhat flawed model f o r 6- 1 8F-D0PA metabolism s i n c e the f i r s t term of 6- 1 8F-D0PA disappearance, A3, was c o n s i d e r a b l y f a s t e r than i n any of the humans s t u d i e d . The slope o f A free/bound was al s o higher i n the r a t than i n any humans or monkeys. These f i n d i n g s would be i n agreement with previous r e p o r t s showing COMT a c t i v i t y to be somewhat higher i n the r a t than i n man (Axelrod and Tomchick, 1958, Guldberg and Marsden, 1975). However, the metabolism i n the r a t only d i f f e r s from the r e s u l t s i n humans with r e s p e c t to the more r a p i d time course of Me-6- 1 8F-DOPA accumulation i n plasma. Experiments i n which COMT was i n h i b i t e d with U-0521 show that i t i s p o s s i b l e to i n t e r f e r e a c u t e l y with the formation of Me-6- 1 8F-D0PA i n the peripher y of the r a t , an e f f e c t which could be con s i d e r e d to be "humanizing". This e f f e c t was r e s t r i c t e d to the f i r s t term of the bi e x p o n e n t i a l expression f o r 6- 1 8F-D0PA disappearance, showing t h i s term to be r e l a t e d to the a c t i o n of COMT. In animals with COMT i n h i b i t i o n , the slope of Me- 1 8F-D0PA/ 1 8F-D0PA with time was i n i t i a l l y g r e a t l y reduced, but was found to normalize a f t e r 60 minutes. In a previous r e p o r t , U-0521 was found to produce a 50% i n h i b i t i o n of e r y t h r o c y t r e COMT at a dose of 90 mg/kg, but t h i s e f f e c t was of sh o r t d u r a t i o n because of the formation of methylated U-0521 (Recches e t a l . , 1982). The present study shows t h a t high doses of U-0521 are probably not necessary to a l t e r 6- 1 8F-D0PA metabolism over a time course comperable to t y p i c a l PET scans. An i n t e r e s t i n g i m p l i c a t i o n of the U-0521 experiments i s t h a t t h i s compound may have some u t i l i t y i n the management of Parkinson's d i s e a s e . The p o s s i b i l i t y of i n t e r f e r e n c e from OMe-DOPA during L-DOPA therapy has l e d to the proposal of COMT i n h i b i t i o n as an adjunct to L-O0PA treatment. N-Butyl g a l l a t e , a COMT i n h i b i t o r , has been shown to p o t e n t i a t e the e f f e c t o f L-DOPA i n Parkinson's disease ( E r i c c s o n , 1971). However, t h i s compound was found to be u n s u i t a b l e f o r human use, presumably because o f i t s t o x i c i t y . In a l i m i t e d study of the e f f e c t s of U-0521 i n humans, o r a l doses on the order of two grams were not found to produce s i g n i f i c a n t c l i n i c a l improvement (Reches and Fahn, 1984). C l i n i c a l t r i a l s with U-0521 may deserve r e p e a t i n g , with more a t t e n t i o n to the l i m i t e d d u r a t i o n of act i o n of t h i s compound i n v i v o . A search f o r b e t t e r COMT i n h i b i t o r s might be worthwhile. The i d e n t i f i c a t i o n of a s i n g l e s i g n i f i c a n t plasma m e t a b o l i t e i n carbidopa p r e t r e a t e d humans and r a t s i s g e n e r a l l y c o n s i s t e n t with previous r e p o r t s i n which the metabolism of ( 3H)F-D0PA (Chieuh e t a l . , 1984) or ( 3H)D0PA (Home e t a l . , 1984) was s t u d i e d i n the r a t . The ra t e o f appearance of Me-6- 1 8F-D0PA observed here i s c o n s i d e r a b l y more r a p i d than t h a t reported f o r ( 3H)Me-D0PA i n Sprague-Dawley r a t s given ( 3H)D0PA and p r e t r e a t e d with 25 mg/kg of carbidopa (Home e t a l . , 1984); i n t h a t study the methylated compound comprized 19% of the r a d i o a c t i v i t y at 15 minutes and 50% at one hour, these compare with 56% and 68% found here. Chieuh e t a l . (1984) re p o r t e d t h a t the r a d i o a c t i v i t y i n ( 3H)Me-F-D0PA peaked a t 20 minutes a f t e r a d m i n i s t r a t i o n of ( 3H)F-D0PA, which i s c o n s i s t e n t with the human r e s u l t s r e p o r t e d here. However, the data i n the a b s t r a c t are not s u f f i c i e n t to compare the rate of formation with the r e s u l t s here presented. The r a p i d r i s e of the methylated d e r i v a t i v e i n plasma i n t h i s study i s a l s o f a s t e r than t h a t reported by B a r t o l i n i e t al (1971) f o r humans given lt+C-D0PA a f t e r carbidopa; i n these humans 30% of the r a d i o a c t i v i t y i n plasma was i n the methylated d e r i v a t i v e a t 15 minutes a f t e r a d m i n i s t r a t i o n (50% at 60 minutes) The compound which appeared sometimes i n plasma and t i s s u e e x t r a c t s and e l u t e d before 6- 1 8F-D0PA was t e n t a t i v e l y i d e n t i f i e d as 6- 1 8F-D0PA s u l f a t e . The minor c o n t r i b u t i o n of t h i s compound to p e r i p h e r a l metabolism i n the r a t i s apparent when the slopes of t o t a l m e tabolites/ 1 8F-D0PA (Free/bound) are compared to Me- 1 8F-DOPA/ 1 8F-DOPA. I n c l u s i o n of the s u l f a t e conjugate only serves to i n c r e a s e the slopes by 10%. The enzyme c a t a l y z i n g the formation of catecholamine s u l f a t e s , phenol s u l f o t r a n s f e r a s e (PST; EC 2.8.2.1), i s very important i n the metabolism of catecholamines i n the periph e r y of humans. The vast m a j o r i t y of c i r c u l a t i n g dopamine and no r a d r e n a l i n e occurs as the s u l f o c o n j u g a t e i n humans (Johnson e t a l . , 1980). The near absence of 6- 1 8F-D0PA s u l f a t e i n plasma from carbidopa p r e t r e a t e d r a t s and humans suggests t h a t 6- 1 8F-D0PA i s not an important s u b s t r a t e f o r PST. Given the p r o p o r t i o n s of 6- 1 8F-D0PA m e t a b o l i t e s observed i n plasma, i t i s p o s s i b l e to propose an order of importance of enzymes f o r the metabolism of 1 8F-D0PA i n the periph e r y o f the unperturbed r a t : Decarboxylase > COMT > PST. The p i l o t study on the e f f e c t of U-0521 alone has an i n t r i g u i n g i m p l i c a t i o n which deserves f u r t h e r i n v e s t i g a t i o n . The work of Reches e t a l . (1982) showed t h a t U-0521 p l u s an L-DOPA load r e s u l t s i n g r e a t l y i n c r e a s e d l e v e l s o f DA and DOPAC i n r a t s t r i a t u m , but they do not re p o r t the e f f e c t of U-0521 a l o n e . In the present study, DOPAC, and to a l e s s e r extent 3-MTyr, were reduced by U-0521 alone while HVA c o n c e n t r a t i o n was u n a f f e c t e d . It may be t h a t U-0521, a t a dose of 25 mg/kg, has other e f f e c t s i n a d d i t i o n to COMT i n h i b i t i o n . The s i m i l a r i t y o f these r e s u l t s t o the e f f e c t o f the low dose of apomorphine on dopamine m e t a b o l i t e s i n r a t s t r i a t u m (McQuade e t a l . , 1985) r a i s e s the i n t r i g u i n g p o s s i b i l i t y t h a t U-0521 may be a c t i n g p r e s y n a p t i c a l l y to block the r e l e a s e of DA. If t h i s were the case, i t would m i t i g a t e a g a i n s t the use of U-0521 f o r the treatment of Parkinson's d i s e a s e . An a l t e r n a t e p o s s i b i l i t y would be th a t U-0521, or i t s methylated m e t a b o l i t e , i s an i n h i b i t o r of MAO. However, the la c k of e f f e c t o f U-0521 on HIAA l e v e l s would serve as an i n t e r n a l c o n t r o l f o r MAO i n h i b i t i o n , a t l e a s t with r e s p e c t to MAO-B. A n a l y s i s of c e r e b r a l t i s s u e i n animals r e c e i v i n g 6- 1 8F-D0PA i n d i c a t e s t h a t the p e r i p h e r a l m e t a b o l i t e Me-6- 1 8F-DOPA accumulates r a p i d l y i n cer e b r a l t i s s u e of the carbidopa p r e t r e a t e d r a t , presumably due to both amino a c i d t r a n s p o r t and passive d i f f u s i o n . The r a t i o of me t a b o l i t e to 6- 1 8F-D0PA i s g r e a t e r i n b r a i n than i n plasma at any given time. T h i s could be r e l a t e d to the a c t i o n of COMT w i t h i n the b r a i n . For example, COMT c o n t a i n i n g a s t r o c y t e s and Bergmann g l i a i n cerebellum may metabolize p a r t of the 5- 1 8F-D0PA (Kaplan e t a l . , 1979). A l t e r n a t i v e l y , b r a i n t i s s u e c o u l d be s e l e c t i v e l y permeable to the Me-6- 1 8F-D0PA. The i n i t i a l accumulation of Me-6- 1 8F-D0PA i s more r a p i d i n vermis than i n s t r i a t u m , suggesting t h a t permeabilty may be inhomogeneous w i t h i n the b r a i n . In the r a t , t h i s 97 phenomenon seems to be r e l a t e d to the excess of r a d i o a c t i v i t y i n vermis i n comparison to s t r i a t u m seen during the f i r s t 15 minutes f o l l o w i n g 6- 1 8F-D0PA i n j e c t i o n . At times a f t e r 30 minutes, however, s t r i a t u m , vermis and cortex o f the r a t are i n d i s t i n g u i s h a b l e with r e s p e c t to the amounts of Me-6- 1 8F-D0PA present/mg t i s s u e . S i m i l a r inhomogeneities o f r a d i o c o n t r a s t seen during the i n i t i a l p e r i o d of human PET scans suggest t h a t t h i s i s not a p e c u l i a r i t y o f the r a t . Thus, c o r t e x would seem to be a b e t t e r r e f e r e n c e t i s s u e than cerebellum f o r PET s t u d i e s . Home e t a l . (1984) a l s o i n d i c a t e d t h a t i n the cerebellum, as i n the plasma, the methylated d e r i v a t i v e i s the only m e t a b o l i t e seen a f t e r systemic ( 3H)D0PA and indeed r e p o r t very s i m i l a r r a t e s of accumulation i n plasma and cerebellum, with the ( 3H)Me-D0PA comprising 19% of c e r e b e l l a r r a d i o a c t i v i t y at 15 minutes and 47% at one hour. T h i s study r e p o r t s a s l i g h t l y f a s t e r r a t e of accumulation of Me-6- 1 8F-D0PA i n vermis than i n plasma (64% a t 15 minutes and 85% at one hour) which might be due to c e r e b e l l a r COMT (Broch and Fonnum, 1972) or to a r e a d i e r p e n e t r a t i o n o f Me- 1 8F-D0PA as compared to the parent compound. Me-6- 1 8F-D0PA has been reported to decrease the uptake o f 1 4C-D0PA by b r a i n (Gervas e t a l . , 1983, Reches and Fahn, 1981) so there i s presumably competition f o r uptake mechanisms. However, the r a d i o t r a c e r used i n these experiments has an a c t i v i t y at t 0 i n r a t plasma of about 2000 CPM/ul, which corresponds to a maximum c o n c e n t r a t i o n on the order o f 5 yM. Therefore, i t i s u n l i k e l y t h a t r a d i o t r a c e r c o n c e n t r a t i o n s are high enough i n r a t or human f o r such f a c t o r s as competition or s a t u r a t i o n of uptake s i t e s to be very s i g n i f i c a n t . I n h i b i t i o n of COMT with U-0521 seems to be only moderately e f f e c t i v e at reducing the ra t e of Me-6- 1 8F-D0PA accumulation i n cor t e x and vermis. S i g n i f i c a n t r e d u c t i o n s were only noted a t 30 minutes, due e i t h e r to the 98 s h o r t d u r a t i o n of a c t i o n of U-0521 or to p o s s i b l e i n s e n s i t i v i t y of b r a i n COMT to i n h i b i t i o n with U-0521. However, i n the s t r i a t u m s i g n i f i c a n t r e d u c t i o n s of Me-6- 1 8F-D0PA were seen i n animals t r e a t e d with U-0521 a t 30. 60 and 90 minutes a f t e r 1 8F-D0PA i n j e c t i o n . Although radiochromatograms of s t r i a t a l samples from carbidopa p r e t r e a t e d r a t s c o n t a i n l a r g e amounts of Me-6- 1 8F-D0PA, they are d i s t i n g u i s h e d from those of other t i s s u e s i n a number of r e s p e c t s . The appearance of 6- 1 8F-DA, subsequent o x i d a t i v e deamination to 6- 1 8F-D0PAC and formation of O-methylated 6- 1 8F-DA met a b o l i t e s a l l occur w i t h i n f i v e minutes a f t e r 6- 1 8F-D0PA i n j e c t i o n . These m e t a b o l i t e s c o l l e c t i v e l y account f o r the r a d i o c o n t r a s t between s t r i a t u m and other b r a i n areas. The 6-OHDA experiments i n d i c a t e that an i n t a c t n i g r o - s t r i a t a l pathway i s necessary f o r the formation of these m e t a b o l i t e s . In the absence of dopaminergic i n n e r v a t i o n , r a d i o c o n t r a s t f a i l s to develop and the metabolism of 6- 1 8F-D0PA i n striatum becomes very s i m i l a r to metabolism i n cerebellum. A number of point s should be r a i s e d about the time course of these metabolites i n s t r i a t u m . 6- 1 8F-DA s y n t h e s i s seems to occur mostly during the f i r s t 15 minutes a f t e r 6- 1 8F-D0PA i n j e c t i o n . The blood data shows t h a t i t i s only during t h i s p e r i o d that the precu r s o r i s r e a d i l y a v a i l a b l e . C u r i o u s l y , the time of maximal 6- 1 8F-DA l e v e l s preceeds the development of r a d i o c o n t r a s t between str i a t u m and vermis. Rather than c o n t i n u a l l y i n c r e a s i n g , t o t a l 1 8 F a c t i v i t y i n s t r i a t u m i s maintained a t a constant l e v e l while r a d i o a c t i v i t y i s washed out of other b r a i n areas. Thus, r a d i o c o n t r a s t i n the r a t i s l a r g e l y due to the r e s i d u a l e f f e c t of an i n i t i a l p e r i o d of 6- 1 8F-DA sy n t h e s i s i n s t r i a t u m . At times a f t e r 60 minutes, the leakage of 6- 1 8F-DA out of s t r i a t u m seems to exceed the r a t e of s y n t h e i s of new 6- 1 8F-DA. Over t h i s same time course, r a d i o c o n t r a s t between s t r i a t u m and vermis reaches i t s maximum and s t a r t s to d e c l i n e . In t h i s r e s p e c t , the r a t data are not i n agreement with r e s u l t s of human PET s t u d i e s , s i n c e r a d i o c o n t r a s t i s s t a b l e f o r at l e a s t two hours i n normal humans. I t seems l i k e l y t h a t t h i s s p e c i e s d i f f e r e n c e i s due to the more r a p i d O-methylation of 6- 1 8F-D0PA i n the r a t . Presumably, 6- 1 8F-D0PA l e v e l s too low f o r s u s t a i n e d 1 8F-DA s y n t h e s i s occur more q u i c k l y i n r a t s than i n humans. The f i n d i n g t h a t r a d i o a c t i v i t y p e r s i s t s longer i n s t r i a t u m than i n other b r a i n t i s s u e s , and t h a t t h i s i s due to the accumulation of 6- 1 8F-DA and m e t a b o l i t e s , i s i n accordance with several previous s t u d i e s . A major d i f f e r e n c e between the r e s u l t s here presented and those i n the l i t e r a t u r e i s the r e l a t i v e l y small amounts of 6- 1 8F-DA found i n s t r i a t u m as compared with Me-6- 1 8F-D0PA ( l i t e r a t u r e summarized i n Table IX). The reasons are unknown, but may have to do with the s t r a i n of r a t used, the time of a d m i n i s t r a t i o n ( E r i c k s s o n e t a l . , 1986), the dose of DOPA (o r 6-F-DOPA), or the dose of p e r i p h e r a l decarboxylase i n h i b i t o r . The l a s t seems p a r t i c u l a r l y l i k e l y s i n c e both carbidopa and benseramide have been r e p o r t e d t o be s u b s t r a t e s f o r COMT (Hagen e t a l . , 1980) and thus the l a r g e amounts used i n many s t u d i e s may l e a d to competitive i n h i b i t i o n of the methylation of DOPA or 6- 1 8F-D0PA. T h i s would be i n accord with the slower appearance of Me-DOPA i n plasma and cerebellum, as well as i n s t r i a t u m , i n those s t u d i e s . Rapid methylation of the 6- 1 8F-D0PA i n the presence of low amounts of the i n h i b i t o r may reduce the amount of s u b s t r a t e a v a i l a b l e f o r d e c a r b o x y l a t i o n i n s t r i a t u m . The r a t i o of 6- 1 8F-DA to i t s m e t a b o l i t e s found i n t h i s study (<2) was somewhat lower than the r a t i o of 4 found f o r endogenous DA and i t s m e t a b o l i t e s . Others (Westerink, 1985, Spencer and Wooten, 1984; Gemayel e t Table IX: Comparison of Present Results with Some Data from the L i t e r a t u r e on The P e r s i s t a n c e of S t r i a t a l Accumulation and the Extent of Metabolism of Exogenous DOPA or Fl uorodopa Reference Present study Home e t a l . 1984 Spencer & Wooten 1984 Gemayel e t a l , 1986 Chiueh et a l . 1984 DOPA or I n h i b i t o r (FDOPA) Dose 1 nmoles/kg 5 mg/kg 25 mg/kg 25 mg/kg 50 mg/kg 75 mg/kg (2300) 10 (577 ) Accum. S t r i a t a l M e t a b o l i t e R a t i o s 3 DA/Me-DOPA DA/(DO PAC +HV A+MTyr) Label P e r s i s t . 2 15 min 1 h 18 F 3H 1.1 1.4 0.7 3.7 0.4 2.9 1.3 x 10 5 none (not reported) 6.4 x 10 5 none 1.8 6.4 3.6 15 min 2.2 7.4 3.1 2.1 1 h 1.8 3.3 1.9 1.0 3H Not determinable from a b s t r a c t but F-DA sa i d to be major product Footnotes f o r Table IX 1) Carbidopa except i n report by Gemayel et a l . (1986) where benseramide.HCl was used. 2) The p e r s i s t a n c e of accumulation of exogenous m a t e r i a l i n the s t r i a t u m i s represented by the r a t i o of the values found a t 1 hr and at 15 min f o r e i t h e r the t o t a l r a d i o a c t i v i t y per mg of s t r i a t a l t i s s u e or the t o t a l excess DOPA and metabolites over c o n t r o l l e v e l s . A f t e r intravenous i n j e c t i o n of about 12 umoles/kg of L-( l i +C)D0PA i n r a t s p r e t r e a t e d with 50 mg/kg carbidopa, Wooten and Home (1982) reported the t o t a l r a d i o a c t i v i t y at 1 hour per mg striatum was 2.1 times t h a t at 15 minutes; a f t e r 81 umoles/kg of L-( 3H)D0PA and 100 mg/kg of carbidopa, Liskowsky and Potter (1985) found a r a t i o of 5.6. 3) Or f l u o r i n a t e d analogs. In c a l c u l a t i n g the r a t i o s from data based on the a d m i n i s t r a t i o n of unlabeled DOPA, the c o n t r o l l e v e l s of DA and me t a b o l i t e s were s u b t r a c t e d so the r a t i o s r e f l e c t only the excess. Van Valkenburg et a l . (1984) r e p o r t data i n d i c a t i n g t h a t , at 20 minutes a f t e r i n j e c t i o n of 17 nmoles/kg of t r i t i a t e d t y r o s i n e to r a t s without pretreatment, the r a t i o of ( 3H)DA to t r i t i a t e d m e t a b o l i t e s i n the s t r i a t u m was 3.3, and Kuruma et a l . (1970) i n d i c a t e c o n s i d e r a b l y more l a b e l e d Me-DOPA than l a b e l e d DOPA or catecholamines i n the whole b r a i n o f r a t s given 25 umoles/kg of L-( 1 4C)D0PA. 102 1., 1986) have reported r a t i o s ranging from 2.6 to 4 f o r the endogenous m a t e r i a l s . A p o s s i b l e e x p l a n a t i o n i s entrance of the newly s y n t h e s i z e d 6- 1 8F-DA i n t o the r a p i d l y t u r n i n g over p o o l . A number of authors (e.g. Javoy and Glowinski, 1971; Korf e t a l . , 1976) have d e s c r i b e d two ne u r o t r a n s m i t t e r pools of DA, with the newly s y n t h e s i z e d m a t e r i a l being i n the pool showing r a p i d turnover. The r a t i o s of excess DA/metabolites i n t h i s study are not incompatible with those i n the l i t e r a t u r e except f o r the markedly higher r a t i o s found by Home e t a l . (1984). Another f a c t o r which must be considered i n t h i s regard i s the p o s s i b l e d i f f e r e n c e between t r a n s p o r t of f l u o r i n a t e d and endogenous compounds. The presence of f l u o r i n e i s seen to g r e a t l y i n c r e a s e the e l u t i o n times of molecules i n reversed phase HPLC. This g r e a t e r hydrophobicity may render the t r a n s p o r t of 1 8F-DA me t a b o l i t e s out of b r a i n l e s s f a c i l e , thereby i n c r e a s i n g m e t a b o l i t e r a t i o s . An a l t e r n a t e p o s s i b l i t y , which remains to be t e s t e d , concerns the a f f i n i t y of 1 8F-DA f o r MAO. I f 1 8F-DA were more r e a d i l y de-aminated than DA, p r o p o r t i o n s o f 1 8F-DOPAC and 1 8F-HVA formed i n stri a t u m c o u l d be higher than f o r the endogenous compoounds. However, higher than normal m e t a b o l i t e r a t i o s are a l s o noted i n s t u d i e s using u n f l u o r i n a t e d t r a c e r s . The r a t e of 6- 1 8F-DA s y n t h e s i s i s not s o l e l y r e l a t e d to a v a i l a b i l i t y of 6- 1 8F-D0PA, but i s a l s o a f u n c t i o n o f the k i n e t i c p r o p e r t i e s o f de c a r b o x y l a t i o n by AAADC. Although D,L-5-fluorodopa has p r e v i o u s l y been shown to be a s u b s t r a t e f o r AAADC with k i n e t i c constants roughly s i m i l a r to those f o r L-DOPA, there has been no previous r e p o r t of the k i n e t i c s o f 6- 1 8F-D0PA d e c a r b o x y l a t i o n . The value of K m found f o r 6- 1 8F-D0PA i s w i t h i n the range of previous determinations f o r L-DOPA (Bosin e t a l . , 1978, M i n e l l i e t a l . , 1979, Okuno and Fujisawa, 1983). However, the K m f o r 103 2- 1 8F-D0PA was approximately an order of magnitude higher than t h a t of 6- 1 8F-D0PA. T h i s , and the lower Vmax found f o r 2- 1 8F-D0PA, would seem to account f o r the lack of r a d i o c o n t r a s t observed i n animals r e c e i v i n g 2- 1 8F-D0PA. Although 2- 1 8F-D0PA gets i n t o the b r a i n , i t appears to be thermodynamically and k i n e t i c a l l y u n s u i t a b l e f o r d e c a r b o x y l a t i o n to 2- 1 8F-DA. I n h i b i t i o n of COMT with U-0521 had the e f f e c t of g r e a t l y promoting 1 8F-0A s y n t h e s i s i n the s t r i a t u m . Elevated l e v e l s of 1 8F-DA and 1 8F-DOPAC, but not the O-methylated m e t a b o l i t e s , suggest t h a t U-0521 may p a r t i a l l y i n h i b i t COMT i n the c e n t r a l nervous system. S i m i l a r r e s u l t s have been found i n r a t str i a t u m f o l l o w i n g l o a d i n g with L-DOPA (100 mg/kg i . p . ) . U-0521 (150 mg/kg) has the e f f e c t of g r e a t l y i n c r e a s i n g the amounts of DA and DOPAC but not HVA formed i n r a t s t r i a t u m (Reches e t al ., 1982). The inc r e a s e s i n 6- 1 8F-DA s y n t h e s i s produced by U-0521 only r e s u l t e d i n s i g n i f i c a n t l y e l e v a t e d r a d i o c o n t r a s t (S/V) at 30 minutes post i n j e c t i o n . Thus, the s i g n a l i s masked by the remaining background "noise" from Me-6- 1 8F-D0PA. Much higher doses of U-0521 would be needed to remove t h i s background completely. In s p i t e of t h i s f i n d i n g , U-0521 may be a useful agent f o r the development of PET methods s i n c e the modest i n c r e a s e s i n S/V produced are r e l a t e d to major changes i n the radiochemical composition o f s t r i a t u m . The u n i d e n t i f i e d m e t a b o l i t e i n s t r i a t u m was presumably the same material found i n plasma, i . e . a s u l f a t e conjugate of 1 8F-D0PA. Highly charged compounds being u n l i k e l y to cr o s s the bloo d - b r a i n b a r r i e r , i t can be assumed t h a t t h i s compound was formed w i t h i n the s t r i a t u m . U n t i l r e c e n t l y , l i t t l e has been known about the s i g n i f i c a n c e of phenol s u l f o t r a n s f e r a s e (PST) i n c e n t r a l catecholamine metabolism. The two forms 104 of the enzyme, as c h a r a c t e r i z e d i n p l a t e l e t s , tin a n s f e r s u l f a t e from donors such as 3'-phosphoadenosine-'5-phosphosulfate to a wide range of p h e n o l i c s u b s t r a t e s . In the human b r a i n , h i g h e s t l e v e l s of PST a c t i v i t y are found i n the a n t e r i o r p i t u i t a r y , with intermediate l e v e l s present i n caudate (Young e t a l . , 1984). The enzyme from human f r o n t a l c o r t e x has an apparent K m f o r dopamine of 15 uM. In the r a t b r a i n , only one form has the a b i l i t y to conjugate dopamine, with a K m i n the 100 uM range ( R i v e t t e t a l . , 1984). A l e s i o n study suggested t h a t the enzyme e x i s t e d i n a subpopulation of k a i n i c a c i d s e n s i t i v e neurones. Conjugated DA i n superfusates from r a t s t r i a t a l s l i c e s r e present approximately 15% of basal DA r e l e a s e (Tyce and R o r i e , 1982). S i m i l a r l y , mass fragmentographic a n a l y s i s of r a t b r a i n shows t h a t at l e a s t 10% of DA and 50% of the NA e x i s t s as a conjugate (Karoum, e t a l . , 1983). Given these r e s u l t s , i t i s s u r p r i s i n g t h a t only t r a c e s of 6- 1 8F-D0PA s u l f a t e and no 6- 1 8F-DA s u l f a t e were detected i n r a t s t r i a t u m . S u l f a t e conjugates are l i b e r a t e d by a c i d h y d r o l y s i s a t the b o i l i n g p o i n t of water (Buu, 1985), so any conjugates present i n s t r i a t u m should not be destroyed by the normal e x t r a c t i o n procedure i n c o l d p e r c h l o r i c a c i d . In the s i n g l e h y d r o l y s i s experiment on a s t r i a t a l homogenate, the a c t i v i t y i n none of the 1 8 F peaks was found to change. T h e r e f o r e , PST probably does not c o n t r i b u t e s i g n i f i c a n t l y to the metabolism o f 6- 1 8F-DA. The la c k of i n c r e a s e f o l l o w i n g COMT i n h i b i t i o n i n the u n i d e n t i f i e d s t r i a t a l compound (presumably 1 8F-D0PA s u l f a t e ) i s c o n s i s t e n t with Buu's r e p o r t which showed t h a t COMT and PST were not competitive i n the r a t b r a i n . C o n s i d e r a t i o n of the metabolism of 6- 1 8F-D0PA ( F i g . 37) suggests a number of p o s s i b l e pharmacological manipulations which could have u t i l i t y i n the development of improved PET models. The e f f e c t s of COMT i n h i b i t i o n have already been examined i n t h i s study. Another p o s s i b l e manipulation would be the i n h i b i t i o n of c e n t r a l AAADC with an agent such as NSD-1015. This compound, at a dose of 100 mg/kg i . p . , completely i n h i b i t s c e n t r a l d e c a r b o x y l a t i o n o f L-DOPA i n r a t s (Carlson e t a l . , 1972). F o l l o w i n g i n h i b i t i o n of AAADC, L-DOPA accumulates i n b r a i n t i s s u e s i n a l i n e a r manner f o r a t l e a s t 60 minutes due to continued L-DOPA s y n t h e s i s . The ra t e of L-DOPA accumulation i n s t r i a t u m i s approximately ten times higher than i n cor t e x , and has been taken as a measure of dopamine s y n t h e s i s r a t e s i n c e t y r o s i n e hydroxylase i s the rate l i m i t i n g enzyme i n such s y n t h e s i s . The accumulation of 6- 1 8F-D0PA i n the s t r i a t u m of animals with NSD-1015 would be a measure s o l e l y of 1 8F-D0PA uptake and could provide i n f o r m a t i o n about the extent to which 1 8F-DA i s trapped i n PET experiments. The d i f f e r e n c e between r a d i o c o n t r a s t i n c o n t r o l animals and animals with NSD-1015 would be r e l a t e d to the rate of leakage of 6- 1 8F-DA and i t s m e t a b o l i t e s from s t r i a t u m during the time p e r i o d i n which 1 8F-D0PA uptake i s l i n e a r . I n f l u x constants f o r 1 8F-D0PA have been c a l c u l a t e d f o r normal i n d i v i d u a l s undergoing PET scanning (Martin e t a l . , 1986). Using a c o r r e c t i o n f o r p e r i p h e r a l metabolism, an i n v e r s e r e l a t i o n s h i p between i n f l u x r a t e and age has been shown. Primate experiments with and without c e n t r a l decarboxylase i n h i b i t i o n c o u l d be used to q u a n t i f y the leakage of 1 8F-DA and met a b o l i t e s from the str i a t u m and thus permit the c o r r e c t i o n of i n f l u x r a t e s to absol u t e v a l u e s . Another s e r i e s of experiments of p o t e n t i a l i n t e r e s t would i n v o l v e the i n h i b i t i o n of MAO, e i t h e r with p a r g y l i n e or with s p e c i f i c i n h i b i t o r s of the two forms of MAO. DOPAC, the p r i n c i p a l DA met a b o l i t e i n the r a t , seems to be formed i n t r a - n e u r o n a l l y ( R o f f l e r - T a r l o v and Tegerdine, 1971). A f t e r s i n g l e doses of p a r g y l i n e , DOPAC l e v e l s i n striatum d e c l i n e i n an 106 exponential manner with a f r a c t i o n a l r a t e constant on the order of 15 minutes (Westerink and Korf, 1976). Deprenyl, an i r r e v e r s i b l e i n h i b i t o r o f MAO-B, diminishes the i n c r e a s e s i n DOPAC i n human c e r b e r o s p i n a l f l u i d a f t e r L-DOPA treatement (Baraczka e t a l . , 1983). A s p e c i a l r e l a t i o n s h i p between MAO-B and dopamine metabolism i s evid e n t i n the human s t r i a t u m . In post-mortem b r a i n from p a t i e n t s r e c e i v i n g deprenyl f o r the treatment o f Parkinson's d i s e a s e , complete i n h i b i t i o n of MAO-B was a s s o c i a t e d with e l e v a t e d l e v e l s of dopamine, but not of s e r o t o n i n , i n the basal g a n g l i a . ( R i e d e r e r and Youdim, 1986). In c o n t r a s t , s t u d i e s o f the e f f e c t s of c l o r g y l i n e and deprenyl on monoamine metabolism i n the r a t showed t h a t MAO-B does not c o n t r i b u t e to the deamination of dopamine i n s t r i a t a l synaptosomes ( F a g e r v a l l and Ross, 1986). Likewise, l e s i o n experiments would i n d i c a t e t h a t the MAO-A form dominates i n dopaminergic t e r m i n a l s of the r a t b r a i n (Stenstrom e t a l . , 1985). The c o n f l i c t i n g r e s u l t s i n r a t and human co u l d be r e l a t e d t o a number of f a c t o r s such as species d i f f e r e n c e s i n MAO popul a t i o n s or h i t h e r t o unknown f a c t o r s o f compartmentalization p e r t a i n i n g to dopamine metabolism. I f the populations of MAO-A and B i n st r i a t u m are a f a c t o r , then the choice of the r a t model f o r 6- 1 8F-D0PA st u d i e s may be su b j e c t to q u e s t i o n . Whereas the m a j o r i t y of MAO i n r a t br a i n i s of the A-form (Schoepp and Azzaro, 1981), MAO-B i s the predominant form i n the human c e n t r a l nervous system ( 0 ' C a r r o l l e t a l . , 1983). Guinea pi g s t r i a t u m i s reported to have a r a t i o of the two forms (28% MAO-A:72% MAO-B) almost i d e n t i c a l to th a t found i n human striatum (Azzaro e t a l . , 1985). In human post mortem b r a i n (McGeer e t a l . , 1985) and i n the guinea p i g caudate (P. Cumming, unpublished o b s e r v a t i o n s ) , the p r i n c i p l e dopamine meta b o l i t e i s HVA r a t h e r than DOPAC. The above ob s e r v a t i o n s suggest t h a t the guinea p i g may u l t i m a t e l y prove to be a b e t t e r model of human dopamine 107 metabolism than i s the r a t . An i n v e s t i g a t i o n of 6- 1 8F-D0PA metabolism i n the guinea p i g could have i m p l i c a t i o n s f o r PET s t u d i e s i f r a d i o a c t i v e m e t a b o l i t e r a t i o s d i f f e r e d g r e a t l y from those found i n the r a t . V. Conclusion The p r i n c i p l e c o n c l u s i o n to be drawn from t h i s work i s that r a d i o c o n t r a s t in PET images i s produced by the i n t r o d u c t i o n of 6- 1 8F-D0PA i n t o the pathways f o r dopamine metabolism i n s t r i a t u m . As o u t l i n e d i n the above d i s c u s s i o n , the e l u c i d a t i o n of the metabolism of 6- 1 8F-D0PA may provide the foundation f o r modeling from PET d a t a . A number of i n t e r e s t i n g pharmacological manipulations are suggested which might produce dramatic e f f e c t s on 6- 1 8F-D0PA metabolism and c o u l d presumably be c o r r e l a t e d with changes i n the r a d i o c o n t r a s t seen i n PET images. However, the p r e s s i n g questions p e r t a i n i n g to modeling can only be answered i f experiments are conducted with the s p e c i f i c i n t e n t i o n of p r o v i d i n g numerical data to f i t equations of the s o r t used i n deoxyglucose s t u d i e s . The s p e c i f i c a t i o n of these experiments may be provided by the c o l l a b o r a t i v e e f f o r t s of people with s p e c i a l e x p e r t i s e i n c l i n i c a l , biochemical and mathematical f i e l d s of study. j 108 VI. References Adam, M.J., Ruth, T.J., G r i e r s o n , J.R., Abeysekera, B., and Pate, B.D. (1986). Routine s y n t h e s i s of L - 1 8 F - 6 - f l u o r o d o p a with 1 8 F - a c e t y l h y p o f l u o r i t e . J . Nuc. Med. 27:1462-1466. Agathopoulous, A., N i c o l o p o u l o s , D., M a t s a n i o t i s , N. and Papdatos, C. (1971). Biochemical changes of c a t e c h o l - 0 - m e t h y l t r a n s f e r a s e during development of human l i v e r . P e d i a t r i c s 47:125-128. Axelrod, J . (1957). The O-methylation of epinephrine and other c a t e c h o l s i n v i t r o and i n v i v o . Science 126:1657-1660. Axelrod, J . and Tomchick, R. (1958). Enzymatic O-methylation of epinephrine and other c a t e c h o l s . J . B i o l . Chem. 233:702-705. Axelrod, J . (1966). Methylation r e a c t i o n s i n the formation and metabolism of catecholamines and other b i o g e n i c amines. Pharmacol. Rev. 18:95-113. Azzaro, A.J., King, J . , Kotzuk, J . , Schoepp, D.D., F r o s t , J . and Schochet, S. (1985). Guinea p i g s t r i a t u m as a model of human dopamine deamination: The r o l e of monoamine oxidase isoenzyme r a t i o , l o c a l i z a t i o n , and a f f i n i t y f o r s u b s t r a t e i n s y n a p t i c dopamine metabolism. J . Neurochem. 45(3):949-956. 109 Baraczka, K., Fekete, M.K.K. and Kanyicska, B. (1983). Changes i n dopamine and 3,4-dihydroxyphenylacetic a c i d (DOPAC) l e v e l s i n human c e r e b r o s p i n a l f l u i d a f t e r L-Dopa and Deprenyl a d m i n i s t r a t i o n . J . Neural Trans. 58:299-304. B a r t h o l i n i , G., C o n s t a n t i n i d i s , J . and T i s s o t , R. (1971). Formation o f monoamines from v a r i o u s amino a c i d s i n the b r a i n a f t e r i n h i b i t i o n of e x t r a c e r e b r a l decarboxylase. Biochem. Pharmac. 20:1243-1247. B e r t l e r , A., F a l c k , B., and Rosengren, E. (1963). The d i r e c t demonstration of a b a r r i e r mechanism i n the b r a i n c a p i l l a r i e s . Acta Phrmacol. T o x i c o l . 20:317-321. Bosin, T.R., Baldwin, J.R. and Maikel, R.P. (1978). I n h i b i t i o n o f DOPA de c a r b o x y l a t i o n by analogues to tryptophan. Biochem. Pharmacol 27:1289-1291. Boyes, B.E., Cumming, P., Ma r t i n , W.R.W. and McGeer, E.G. ( i n p r e s s , 1986). Determination of plasma 1 8 F - 6 - F l u o r o d o p a during p o s i t r o n emission tomography: E l i m i n a t i o n and metabolism i n carbidopa t r e a t e d s u b j e c t s . L i f e S ciences. Bradford, M.M. (1976). A r a p i d and s e n s i t i v e method f o r the q u a n t i t a t i o n o f microgram q u a n t i t i e s of p r o t e i n u t i l i z i n g the p r i n c i p l e s of protein-dye b i n d i n g . A n a l . Biochem. 72:248-254. Broch, O.J., and Guldberg, H.C. (1971). On the determination of ca t e c h o l - 0 - m e t h y l t r a n s f e r a s e a c t i v i t y i n t i s s u e homogenates. Acta Pharmacol, e t T o x i c o l . 30:266-277. Buu, N.T. (1985). R e l a t i o n s h i p between c a t e c h o l - 0 - m e t h y l t r a n s f e r a s e and p h e n o l s u l f o t r a n s f e r a s e i n the metabolism of dopamine i n the r a t b r a i n . J . Neurochem. 45(5):1612-1619. Calne, D.B., Reid, J.L., and V a k i l , S.D. (1972). Parkinsonism t r e a t e d with 3-0-methyldopa. C l i n . Pharmac and Exp Therap. 14(3):386-389. Calne, D.B., Langston, J.W., S t o e s s l , A.J., Ruth, T., Adam, M.J., Pate, B.D., and S c h u l z e r , M. (1985). P o s i t r o n emission tomography a f t e r MPTP: Observations r e l a t i n g to the cause o f Parkinson's d i s e a s e . Nature 317:246-248. C a r l s s o n , A., Davis, J.N., Kehr, W., L i n d q v i s t , M. and Atack, C V . (1972). Simultaneous measurement of t y r o s i n e and tryptophan hyroxylase a c t i v i t i e s i n b r a i n i n viv o using an i n h i b i t o r of the aromatic amino a c i d decarboxylase. Naunyn-Schmied. Arch. Pharmacol. 275:153-168. Cheng, C.H., and Wooten, G .F . (1982). Dopamine turnover estimated by simultaneous LCEL assay of dopamine and dopamine m e t a b o l i t e s . J.Pharmacol. Methods 8:123-124. Chiueh, C.C., Zukowska-Gorojec, Z., K i r k , K.L., and Kopin, I . J . (1983). 6-Fluorocatecholamines as f a l s e a d renergic n e u r o t r a n s m i t t e r s . J . Pharmac. Exp. Therap. 225(3):529-533. Chiueh, C.C., K i r k , K.L., Channing, M.A., and K e s s l e r , R.M. (1984). Neurochemical b a s i s f o r the use of 6-F-DOPA f o r v i s u a l i z i n g dopamine neurons i n the b r a i n by the use of p o s i t r o n emission tomography. Soc. Neu r o s c i . A b s t r s . 14:883. Crane, P.D., Pardridge, W.M., Braun, L.D., Nyerges, A.M., and Oldendorf, W.H. (1981). The i n t e r a c t i o n of t r a n s p o r t and metabolism on b r a i n glucose u t i l i z a t i o n : A r e v a l u a t i o n of the lumped constant. J . Neurochem. 36:1601-1604. Crane, P.D., Pardridge, W.M., Braun, L.D., and Oldendorf, W.H. (1983). K i n e t i c s of t r a n s p o r t and phosphorylation o f 2-fluoro-2-deoxy-D-glucose i n r a t b r a i n . J . Neurochem. 40(1):160-167. C r e v e l i n g , C.R., and K i r k , K.L (1985). The e f f e c t of r i n g - f l u o r i n a t i o n on the r a t of 0-methylation of dihydroxyphenylalanine (DOPA) by cat e c h o l - O - m e t h y l t r a n s f e r a s e : S i g n i f i c a n c e i n the development o f 1 8F-PETT scanning agents. Biochem. Biophys. Res. Comm. 130(3)1123-1131 Cumming, P., Boyes, B.E., M a r t i n , W.R.W., Adam, M.J., G r i e r s o n , J . , Ruth, T. and McGeer, E.G. ( i n press, 1986). The Metabolism of 1 8 F - 6 - F l u o r o -L-Dopa i n the hooded r a t . J . Neurochem. 112 Cumming, P., Boyes, B.E., M a r t i n , W.R.W., Adam, M.J., Ruth, T.J. and McGeer, E.G. (submitted, 1986). A l t e r e d metabolism of 1 8 F - 6 - f l u o r o d o p a i n the hooded r a t f o l l o w i n g i n h i b i t i o n of c a t e c h o l - 0 - m e t h y l t r a n s f e r a s e with U-0521. D i f f l e y , D.M., Costa, J.L., S o k o l o s k i , E.A., Chiueh, C . C , K i r k , K.L., and C r e v e l i n g , C R . (1983). D i r e c t o b s e r v a t i o n of 6-fluorodopamine i n guinea p i g nerve microsacs by 19F-NMR. Biochem. Biophys. Res. Comm. 110 :740-745. E r i c s s o n , A.D. (1971). P o t e n t i a t i o n of the L-DOPA e f f e c t i n man by the use o f c a t e c h o l - 0 - m e t h y l t r a n s f e r s e i n h i b i t o r s . J . Neurol. S c i . 14:193-197. E r i k s s o n , T., Weisel, K. and C a r l s s o n , A. (1986). Rat b r a i n c o n c e n t r a t i o n o f administered amino a c i d s : dependence on time of day f o r a d m i n i s t r a t i o n . J . Neural Trans. 65:83-91. F a g e r v a l l , I. and Ross, S. (1986). A and B forms of monoamine oxidase w i t h i n the monoaminergic neurons of the r a t b r a i n . J . Neurochem. 47(2): 569-576. F i r n a u , G., Garnett, E.S., Sourkes, T.L., and M i s s a l a , K. (1975). ( 1 8 F ) F l u o r o - d o p a : A unique gamma e m i t t i n g s u b s t r a t e f o r dopa decarboxylase. E x p e r i e n t i a 11:1254-1255. F i r n a u , G., Garnett, S., and M a r s h a l l , A.M. (1981). E f f e c t s o f Fluoro-dopamines on Dopamine Receptors (Di ,D2, and D3 s i t e s ) Biochem. Pharmacol. 30(21 ):2927-2930. 113 F i r n a u , G., Sood, S., P a n t e l , R., and Garnett, S. (1981). Phenol i o n i z a t i o n i n DOPA determines the s i t e of methylation by catechol-0-methytransferase. Mol. Pharmac. 19:130-133. Ford, A.P.D.W. and Marsden, C A . (1986). Influence of a n a e s t h e t i c s on r a t s t r i a t a l dopamine metabolism i n v i v o . B r a i n Res. 379:162-166. Fox, J.L. (1984) PET Controversy A i r e d . Science 224:143-144. Garg, B., Buckner, C , S e t h i , B., Sokolowsky, T., and P a t i l , P.N. (1971). S t e r i c aspects of adrenergic drugs. XVII. Influence of tropolone on the magnitude and du r a t i o n of a c t i o n of catecholamine isomers. Arch. I n t l . Pharmacodyn. e t Ther. 189:281-294 Garnett, E.S., F i r n a u , G., Chan, P.K.H., Sood, S., and Belbeck, L.W. (1978). ( 1 8 F ) F l u o r o - d o p a , an analog of dopa, and i t s use i n d i r e c t external measurements of storage, degredation, and turnover of i n t r a c e r e b r a l dopamine. Proc. N a t l . S c i . USA 75(1):464-465 Garnett, E.S., F i r n a u , G., Nahmias, C , Sood, S., and Belbeck, L. (1980). Blood-brain b a r r i e r t r a n s p o r t and c e r e b r a l u t i l i z a t i o n of dopa i n l i v i n g monkeys. Am. J . P h y s i o l . R318-R327. Garnett, E.S., F i r n a u , G., Nahmias, C , and C h i r a k e l , R. (1983a). S t r i a t a l d dopamine metabolism i n l i v i n g monkeys examined bu p o s i t r o n emission tomography. B r a i n Res. 2280:169-171. 114 Garnett, E.S., F i r n a u , G., and Nahmias, C. (1983b). Dopamine v i s u a l i z e d i n the basal g a n g l i a of l i v i n g man. Nature 305:137-138. Garnett, S.E., Nahmias, C , and F i r n a u , G. (1984). Central dopamine metabolism pathways i n hemi-Parkinsonism examined by p o s i t r o n emission tomography. Can J . Neurol. S c i . 11:174-175 Gemayal, G.E., Tro u v i n , J.H., Prioux-Guyonneau, M., Jacquot, C , and Cohen, Y. (1986). Re-evaluation of the L-dopa l o a d i n g e f f e c t on dopamine metabolism i n r a t s t r i a t u m . J . Pharm. Pharmacol. 38:134-136. Gervas, J . J . , Muradas, V., Bazan, E., Aguada, E.G. and de Yebenes, J.G. (1983). E f f e c t s of 3-0M-dopa on monoamine metabolism i n r a t b r a i n . Neurology (NY) 33:278-282. G i l e s , R.E., and M i l l e r , J.W. (1967). A comparison of c e r t a i n p r o p e r t i e s of c a t e c h o l - 0 - m e t h y l t r a n s f e r s e to those of 3-adrenergic r e c e p t o r s . J . Pharmac. and Exp. Therap. 156:201-206. G r i e r s o n , J.R. and Adam, M.J. (1986). A p r a c t i c a l p r e p e r a t i v e s c a l e s y n t h e s i s of D,L-6-fluorodopa. J . L a b e l l e d Compounds and Radiopharmac. ( i n p r e s s ) . Guldberg, H.C., and Marsden, C A . (1975) C a t e c h o l - 0 - m e t h y l t r a n s f e r a s e : Pharmacological aspects and p h y s i o l o g i c a l r o l e . Pharmacol. Rev. 27(2): 135-204. G u l l i v e r , P.A., and T i p t o n , K.F. (1978). D i r e c t e x t r a c t i o n radioassay f o r ca t e c h o l - 0 - m e t h y l t r a n s f e r a s e a c t i v i t y . Biochemical Pharmacol. 27:773-775. Hagan, R.M., Raxworthy, M.J., and G u l l i v e r , P.A. (1980). Benserazide and carbidopa as s u b s t r a t e s of c a t e c h o l - 0 - m e t h y l t r a n s f e r a s e : New mechanism of a c t i o n i n Parkinson's d i s e a s e . Biochem. Pharmac. 29:3123-3126. Home, M.K., Cheng, C.H., and Wooten, G.F. (1984). The c e r e b r a l metabolism o f L-dihydroxyphenylalanine: An au t o r a d i o g r a p h i c and biochemical study. Pharmacology 28:12-26. Huh, M.M., and F r i e d h o f f , A.J. (1979). Two isozymic forms of catechol-O-methytransferase: Evidence f o r two d i s t i n c t forms forms and t h e i r p u r i f i c a t i o n and p h y s i c a l c h a r a c t e r i z a t i o n . J . B i o . Chem. 254(2):299-308. J e f f e r y , D.R., and Roth, J.A. (1984). C h a r a c t e r i z a t i o n of membrane buond and s o l u b l e c a t e c h o l - 0 - m e t h y l t r a n s f e r a s e i n human f r o n t a l c o r t e x . J . Neurochem. 42(3):826-831. Johnson, G.A., Baker, C A and Smith, R.T. (1980). Radioenzymatic assay o f s u l f a t e conjugates of catecholamines and dopa i n plasma. L i f e S c i . 26:1591-1598. Kaplan, G.P., Hartman, B.K., and C r e v e l i n g , C.R. (1979). Immunohistochemical demonstration of c a t e c h o l - 0 - m e t h y l t r a n s f e r a s e i n mammalian b r a i n . Brain Res. 167:241-250. Karoum, F., Chuang, L.W. and Wyatt, R.J. (1983). Biochemical and pharmacological c h a r a c t e r i s t i c s of conjugated catecholamines i n the r a t b r a i n . J . Neurochem. 40(6):1735-1741. Kennedy, C , Des R o s i e r s , M.H., Sakurada, 0., Shinohara, M., R e i v i c h , M., J e h l e , J.W., and S o k o l o f f , L. (1976). Metabolic mapping of the primary v i s u a l system of the monkey by means of the a u t o r a d i o g r a p h i c ( 1 ! tC)deoxylucose Technique. Proc. N a t l . Acad. S c i . USA. 73(11):4230-4234. Korf, J . , R e i f f e r s , S., Beerling-van der Molen, H.D., Lakke, J.W.P.F., Paans, A.M.J., Vaalburg, W., and Woldring, M.G. (1978). Rapid d e c a r b o x y l a t i o n of carbon-11 l a b e l l e d DL-DOPA i n the b r a i n : A p o t e n t i a l approach f o r e x t e r n a l d e t e c t i o n of nervous s t r u c t u r e s . B r a i n Res. 145:59-67. Kuhl, D.E., Phelps, M.E., Markham,, C.H., e t a l . , (1982). Cerebral metabolism and atrophy i n huntington's disease determined by 1 8F-DG and computed tomographic scan, Ann. Neurol. 12:425-434. Kuruma, I., Bartholin!', G., and P l e t s c h e r , A. (1970). L-Dopa-induced accumulation of 3-0 -methyldopa i n b r a i n and h e a r t . Eur. J . Pharmacol. 10:189-192. Leenders, K.L., Palmer, A.J., Quinn, N., C l a r k , J.C., F i r n a u , G., Garnett, E.S., Nahmias, C , Jones, T., and Marsden, C D . (1986). B r a i n dopamine metabolism, i n p a t i e n t s with Parkinson's disease measured with p o s i t r o n emission tomography. J . Neurol. Neurosurg. and P s y c h i a t . 49:853-860. Liskowsky, D.R., and P o t t e r , L.T. (1985). A p r e - p o s i t r o n emission tomography study of L-3,4-dihydroxy-( 3H)phenylalanine d i s t r i b u t i o n i n the r a t . Neuroscience L e t t . 53:161-167. Ll o y d , K.G., and Hornykiewicz, 0. (1972). Occurence and d i s t r i b u t i o n o f aromatic L-amino a c i d (L-DOPA) decarboxylase i n the human b r a i n . J . Neurochem. 19:1548-1559. M a r t i n , W.R.W., Palmer, M., S t o e s s l , P., Adam, M., Rut, T.J., Pate, B.D., and Calne, D.B. (1986) The e f f e c t of age on c e r e b r a l 6-fluorodopa metabolism. A b s t r a c t , XXX Congress of I n t e r n a t i o n a l Union of P h y s i o l o g i c a l S c i e n c e s . Mason, L., and Weinkove, C. (1984). K i n e t i c s of enzymatic O-methylation: I t s value i n assays of catechol mines i n plasma. C l i n . Chem. 3011:24-27. M i n e l l i , A., C h a r t e r i s , A.T., B o r r i V o l t a t t o r n i , C , and John, R.A. (1979). Reactions of DOPA (3,4-dihydroxyphenylalanine) decarboxylase with DOPA. Biochem. J . 183:361-368. 118 McGeer, E.G., Norman, M., Boyes, B., O'Kuskey, J . , Suzuki, J . and McGeer, . P.L. (1985). A c e t y l c h o l i n e s t e r a s e and aromatic amine systems i n postmortem b r a i n of an i n f a n t with Down's syndrome. Experim. Neurol. 87:557-570. McGeer, P.L. (1986). B r a i n Imaging i n Alzheimer's Disease. B r i t . Med. B u l l . 42(l):24-28. McQuade, P.S., Richard, J.W., and Thakur, M. (1985). Some f a c t o r s a f f e c t i n g s t r i a t a l 3-methoxytyramine c o n c e n t r a t i o n s i n the mouse and r a t . Prog. Neuro-Psychopharmacol & B i o l . P s y c h i a t . 9:725-729. Nutt, J.G., Woodward, W.R., and Anderson, J.L. (1985). The e f f e c t of carbidopa on the pharmacokinetics of i n t r a v e n o u s l y administered levodopa: The mechanism of a c t i o n i n the treatment of Parkinson's d i s e a s e . Ann. Neurol. 18:537-543. O ' C a r r o l l , A.M., Fowler, C.J., P h i l l i p s , J.P., Tobbia, I. and T i p t i o n , K.F. (1983). The deamination of dopamine by human b r a i n monoamine oxidase: s p e c i f i c i t y f o r the two enzyme forms i n seven b r a i n r e g i o n s . Naunyn Schmied. Arch. Pharmacol. 322:198-202. Okuno, S., and Fujisawa, H. (1983). Accurate assay of DOPA decarboxylase by p r e v e n t i n g nonenzymatic d e c a r b o x y l a t i o n of DOPA. Anal . Biochem. 129:412-415. 0'Mai l e y , K., and K e l l y , J . L i v e r and Aging-1982: L i v e r and Drugs. K. K i t a n i , Ed., pp301-311, E l s e v i e r Press, Amsterdam (1982). 119 P l a c i d i , G.F., Fornaro, P., Papeschi, R., and Cassano, G.B. (1976). Au t o r a d i o g r a p h i c d i s t r i b u t i o n study of lltC-D0PA i n cat b r a i n . Arch. I nt. Pharmacodyn. 220:287-296. Raxworthy, M.J., G u l l i v e r , P.A., and Hughes, P.J. (1982) The c e l l u l a r l o c a l i z a t i o n of catechol-O-methyltransferase i n r a t l i v e r . Naunyn-Schmiedberg's Arch. Pharmacol. 320:182-188. Raxworthy, M.J., Youde, I.R., and G u l l i v e r , P.A. (1986). Catechol-0-m e t h y l t r a s n f e r a s e : S u b s t r a t e - s p e c i f i c i t y and s t e r e o s e l e c t i v i t y f o r B-adrenoceptor agents. Xenobiotica 16(1):47-52. Reches, A., and Fahn, S. (1981). 3-0-methyldopa blocks dopa metaboism i n r a t corpus s t r i a t u m . Ann. Neurol. 12:267-271. Reches, A., J i a n g , D.-H., and Fahn, S. (1982a). E f f e c t of 3'4'-dihydroxy-2-methyl-propiophenone (U-0521) on cate c h o l - O - m e t h y l t r a n s f e r a s e a c t i v i t y and on DOPA accumulation i n r a t red blood c e l l s and corpus s t r i a t u m . Biochem. Pharmac. 31(21):3415-3418. Reches, A., J i a n g , D.-H., and Fahn, S. (1982b). Catechol-O-methytransferase i n h i b i t i o n by U-0521 i n c r e a s e s s t r i a t a l u t i l i z a t i o n of levodopa. Naunyn-Schmiedeberg's Arch. Pharmac. 320:34-37. Reches, A., and Fahn, S. (1984). Catechol-O-methyltransferase and  Parkinson's d i s e a s e , i n Adv. Neurol. V o l . 40. R.G. Ha s s l e r and J.F. C h r i s t , Ed., pp 171-179, Raven Press, New York (1984). R e i d e r e r , P. and Youdim, M.B.H. (1986). Monoamine oxidase and monoamine metabolism i n b r a i n s of Parkinsonian p a t i e n t s t r e a t e d with L-Deprenyl. J . Neurochem. 46(4):1359-1365. R e i v i c h , M., Kuhl, D., Wolf, A., Greenberg, J . , Phelps, M., Ido, T., C a s e l l a , V., Fowler, J . , Hoffman, E., A l a v i , A., Som, P., and S o k o l o f f , L. (1979). The 1 8 F - f l u o r o d e o x y g l u c o s e method f o r the measurement of l o c a l c e r e b r a l glucose u t i l i z a t i o n i n man. C i r c . Res. 44:127-137. R e i v i c h , M., A l a v i , A., Wolf, A., Fowler, J . , R u s s e l l , J . , A r n e t t , C., MacGregor, R.R., Shiue, C.Y., A t k i n s , H., Anand, A., Dann, R. and Greenberg, J.H. (1985). Glucose metabolic r a t e k i n e t i c model parameter d e t e r m i n a t i o n i n humans: The lumped constants and r a t e constants f o r ( 1 8 F ) Fl uorodeoxygl ucose and ( u C ) D e o x y g l ucose. J . Cereb. Blood Flow and Met. 5:179-192. R e i l l y , D.K., R i v e r a - C a l i m l i m , L., and VanDyke, D. (1980). Catechol-0-m e t h y l t r a n s f e r a s e a c t i v i t y : A determinant of levodopa response. C l i n . Pharmacol and Therap. 28:278-286. R i v e t t , A.J., F r a n c i s , A., and Roth, J.A. (1982a). C o n t r i b u t i o n of s u l f a t e c o n j u g a t i o n , deamination, and O-methylation to metabolism of dopamine and norepinephrine i n human b r a i n , J . Neurochem. 39:1009-1066. R i v e t t , A.J., F r a n c i s , A., and Roth, J.A. (1983a). D i s t i n c t c e l l u l a r l o c a l i z a t i o n of membrane bound and s o l u b l e forms of c a t e c h o l - 0 -m e t h y l t r a n s f e r a s e i n b r a i n . J . Neurochem. 40:215-219. 121 R i v e t t , A.J., F r a n c i s , A., and Roth, J.A. (1983b). L o c a l i z a t i o n of membrane bound c a t e c h o l - 0 - m e t h y l t r a n s f e r a s e . J . Neurochem. 40(5):1494-1496. R i v e t t , A.J., F r a n c i s , A., Whittemore, R. and Roth, J . (1984). S u l f a t e c o n j u g a t i o n of dopamine i n r a t b r a i n : Regional d i s t r i b u t i o n of a c t i v i t y and evidence f o r neuronal l o c a l i z a t i o n . J . Neurochem. 42(5):1444-1449. Riv e r a - C a l i m l i m , L. and R e i l l y , D'.K. (1984). D i f f e r e n c e i n e r y t h r o c y e c a t e c h o l - 0 - m e t h y l t r a n s f e r a s e a c t i v i t y between O r i e n t a l s and Caucasians: D i f f e r e n c e i n levodopa t o l e r a n c e . C l i n . Pharmacol, and Therap. 35(6): 804-809. R o f f e r - T a r l o v , S., Sharman, D.F. and Tegerdine, P. (1971). 3,4-Dihydroxyphenylacetic a c i d in the mouse s t r i a t u m : a r e f l e c t i o n of i n t r a - and extra-neuronal metabolism of dopamine? Br. J . Pharmac. 42:343-351. Schoepp, D.D. and Azzaro, A.J. (1981). S p e c i f i c i t y of endogenous s u b s t r a t e s f o r type A and B monoamine oxidase. J . Neurochem. 36:2025-2031. Sharpless, N., Muenter, M.D., Tyce, G.M., and Owen, C A . (1971). 3-Methoxy-4-hydroxy phe n y l a l a n i n e (3-0-methyldopa) i n plasma during o r a l L-DOPA therapy of p a t i e n t s with Parkinson's d i s e a s e . C l i n . Chim. A c t a . 37:359-369. S i e r v o g e l , R.M., Weinshilboum, R., Wilson, A.F., and E l s t o n , R.C. (1984). Major gene model f o r the i n h e r i t a n c e of cate c h o l - O - m e t h y l t r a n s f e r a s e a c t i v i t y i n f i v e l a r g e f a m i l i e s . Am. J . Med. Genet. 19:315-323. S o k o l o f f , L., R e i v i c h , M., Kennedy, C , Des R o s i e r s , M.H., Patlak, C.S., Pettigrew, K.D., Sakurada, 0., and Shinohara, 0. (1977). The ( 1 I +C) deoxyglucose method f o r the measurement of l o c a l c e r e b r a l glucose u t i l i z a t i o n : Theory, procedure, and normal values i n the conscious and an e s t h e t i z e d a l i b i n o r a t . J . Neurochem. 28:897-916. S o k o l o f f , L. (1981). L o c a l i z a t i o n of f u n c t i o n a l a c t i v i t y i n the c e n t r a l nervous system by measurement of glucose u t i l i z a t i o n with r a d i o a c t i v e deoxyglucose. J . Cereb. Blood Flow and Metab. 1:7-36. Spencer, C.E., and Wooten, F.G. (1984). A l t e r e d pharmacokinetics of L-DOPA metabolism i n r a t striatum deprived of dopaminergic i n n e r v a t i o n . Neurology 34:1105-1108. Stenstrom, A., A r a i , Y. and Oreland, L. (1985). I n t r a - and extra-neuronal monoamine oxidase-A and -B a c t i v i t i e s a f t e r c e n t r a l axotomy (hemisection) on r a t s . J . Neural Transm. 61:105-113. Tyce, G.M., and Rorie , D.K. (1982). Conjugate dopamine i n superfusates o f s l i c e s of r a t s t r i a t u m . J . Neurochem. 39(5):1333-1339. 123 van Valkenburg, C , van der Krogt, J . , Moleman, P., van Berkum, H., Tjaden, U. and de Jong, J . (1984). A procedure to measure the s p e c i f i c a c t i v i t i e s of dopamine and i t s m e t a b o l i t e s i n r a t s t r i a t u m , based on HPLC, e l e c t r o c h e m i c a l d e t e c t i o n and l i q u i d s c i n t i l l a t i o n c o unting. J . N e u r o s c i . Methods 11:29-38. Wade, L.A., and Katzman, R. (1975). Rat b r a i n r e g i o n a l uptake and d e c a r b o x y l a t i o n of L-DOPA f o l l o w i n g c a r o t i c i n j e c t i o n . Am. J . P h y s i o l . 228(2):352-359. Weinshilboum, R.M., Raymond, F.A., Elveback, L.R. and Weidman, W.H. (1974). C o r r e l a t i o n of e r y t h r o c y t e catechol-0-methytransferase a c t i v i t y between s i b l i n g s . Nature: 252:490-491. Westerink, B.H.C. and Korf, J . (1976). Turnover of a c i d dopamine me t a b o l i t e s i n s t r i a t a l and mesolimbic t i s s u e of the r a t b r a i n . Eur. J . Pharmac. 37:249-255. Westerink, B.H.C. (1985). Sequence and s i g n i f i c a n c e of dopamine metabolism i n r a t b r a i n . Neurochem. Int. 7:221-227 Wooten, G.F., and Home, H.K. (1981). A new a u t o r a d i o g r a p h i c approach f o r imaging f o r e b r a i n dopamine d i s t r i b u t i o n . Ann. Neuro. 12 :163-168. Wurtman, R.J., Chou, C , and Rose, C. (1970). The f a t e of l l +C-dihydroxyphenylalanine ( l l +C-D0PA) i n the whole mouse. J.Pharmac. Exp. Therap. 174{3):351-356. Yoshida, H., K a n i i k e , K., and Namba, J . (1963). P r o p e r t i e s of a c a r r i e r system to t r a n s p o r t L-DOPA i n t o b r a i n s l i c e s . Nature 8:191-192. Young, W.F., Okazaki, H., Laws, E.R., and Weinshilboum, R.M. (1984). Human b r a i n phenol s u l f o t r a n s f e r a s e : Biochemical p r o p e r t i e s and regio n a l l o c a l i z a t i o n . J . Neurochem. 43(3):706-715. 

Cite

Citation Scheme:

        

Citations by CSL (citeproc-js)

Usage Statistics

Share

Embed

Customize your widget with the following options, then copy and paste the code below into the HTML of your page to embed this item in your website.
                        
                            <div id="ubcOpenCollectionsWidgetDisplay">
                            <script id="ubcOpenCollectionsWidget"
                            src="{[{embed.src}]}"
                            data-item="{[{embed.item}]}"
                            data-collection="{[{embed.collection}]}"
                            data-metadata="{[{embed.showMetadata}]}"
                            data-width="{[{embed.width}]}"
                            async >
                            </script>
                            </div>
                        
                    
IIIF logo Our image viewer uses the IIIF 2.0 standard. To load this item in other compatible viewers, use this url:
https://iiif.library.ubc.ca/presentation/dsp.831.1-0096689/manifest

Comment

Related Items