Life Sciences, Vol. 47, pp. 2143-2153 Printed in the U.S.A.
Pergamon Press
A MORE S E N S I T I V E A N D S P E C I F I C RADIOENZYMATIC ASSAY FOR CATECHOLAMINES Brian
Kennedy
and M i c h a e l
G.
Ziegler
D i v i s i o n of Nephrology, D e p a r t m e n t of M e d i c i n e U n i v e r s i t y of C a l i f o r n i a San Diego M e d i c a l C e n t e r 225 D i c k i n s o n St, H-781-B, San Diego, CA 92103 (Received in final form October I, 1990) Summary This m o d i f i c a t i o n of the c a t e c h o l - O - m e t h y l t r a n s f e r a s e (COMT) b a s e d r a d i o e n z y m a t i c assay for n o r e p i n e p h r i n e (NE) and e p i n e p h r i n e (E) improves sensitivity, s e l e c t i v i t y and eliminates m a n y inhibitors of COMT. Prior to assay, s a m p l e s are e x t r a c t e d into h e p t a n e w i t h d i p h e n y l b o r a t e , then into d i l u t e acetic acid. This e x t r a c t i o n p r o c e d u r e has an e f f i c i e n c y of 78% for NE but less than 2% for Sadenosylmethionine (SAM). The e x t r a c t i o n p r o c e d u r e also e x c l u d e s c a l c i u m and o t h e r COMT inhibitors p r e s e n t in urine, p l a s m a and every tissue tested. This e l i m i n a t e s the r e q u i r e m e n t for individual s t a n d a r d i z a t i o n of tissue and u r i n e samples. S e n s i t i v i t y of the assay for NE and E is 10 and 6 pg/ml r e s p e c t i v e l y in I ml of plasma. The i n t r a a s s a y c o e f f i c i e n t s of v a r i a t i o n for NE and E are 4 and 13% and the interassay c o e f f i c i e n t s of v a r i a t i o n for NE and E are 10 and 16% in a h u m a n plasma sample c o n t a i n i n g low c a t e c h o l a m i n e levels. The assay p e r m i t s q u a n t i t a t i o n of p l a s m a E levels that w e r e u n d e t e c t a b l e in p r i o r assays. Catecholamine-O-methyl transferase (COMT) b a s e d r a d i o e n z y m a t i c assays are among the most sensitive methods for measuring c a t e c h o l a m i n e levels, but often they are not s e n s i t i v e e n o u g h to m e a s u r e E levels in human plasma. Another s h o r t c o m i n g of C O M T b a s e d r a d i o e n z y m a t i c assays is that their s e n s i t i v i t y and a c c u r a c y may be r e d u c e d by C O M T inhibitors p r e s e n t in many types of samples (1,2,3,4,5,6). Consequently, it is n e c e s s a r y to include internal s t a n d a r d s in such samples b e c a u s e r e d u c t i o n s in O - m e t h y l a t i o n due to i n h i b i t o r s may be m i s i n t e r p r e t e d as r e d u c e d c a t e c h o l a m i n e levels (7,8,9). The o r i g i n a l C O M T b a s e d r a d i o e n z y m a t i c m e t h o d that was d e v e l o p e d by E n g e l m a n et al in 1968 (10) u s e d 14C-SAM to d o n a t e a l a b e l e d m e t h y l g r o u p to c a t e c h o l a m i n e s as they w e r e O - m e t h y l a t e d . This m e t h o d has a s e n s i t i v i t y of 300 pg and was m a d e more s p e c i f i c and s e n s i t i v e by the a d d i t i o n of thin layer c h r o m a t o g r a p h y to s e p a r a t e the O - m e t h y l a t e d p r o d u c t s (11). Several refinements have b e e n m a d e to further increase the s e n s i t i v i t ~ of the assay. In 1973, Coyle & Henry (12) i n t r o d u c e d the use of H-SAM, w h i c h has a h i g h e r s p e c i f i c a c t i v i t y than 14C-SAM. P a l k o v i t s et al (13) were the first to scale d o w n the r e a c t i o n mixture. De C h a m p l a i n et al (14) i n c l u d e d E G T A in the r e a c t i o n m i x to c h e l a t e Ca m , the m a i n C O M T i n h i b i t o r p r e s e n t in h u m a n plasma. 0024-3205/90 $3.00 +.00 Copyright (c) 1990 Pergamon Press plc
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E v e n w i t h these modifications, the most s e n s i t i v e m e t h o d s have a s e n s i t i v i t y for E of about 20 pg/ml p l a s m a (7,15) from r e s t i n g humans o f t e n has E values b e l o w this.
generally Plasma
Several i n v e s t i g a t o r s have tried to increase the s e n s i t i v i t y of the C O M T r a d i o e n z y m a t i c assay by using p r e e x t r a c t i o n techniques to concentrate catecholamines and remove C O M T inhibitors. Weise & Kopin (16) and Ball (17) bound sample c a t e c h o l a m i n e s to alumina, and e l u t e d w i t h a small volume of acid. However, A1 +++ ions inhibit COMT and are c o e l u t e d w i t h the c a t e c h o l a m i n e s (3) , d i m i n i s h i n g the e f f i c a c y of this procedure. Gauchy et al (3) d e v e l o p e d an a l u m i n a method to concentrate catecholamines which avoided A1 ÷++ ion c o n t a m i n a t i o n but this p r o c e d u r e is too lengthy for r o u t i n e use. C a t e c h o l a m i n e s can be s e l e c t i v e l y e x t r a c t e d into lipid solvents by c h e l a t i n g agents (18). We have used this t e c h n i q u e to effect a 10 fold c o n c e n t r a t i o n of c a t e c h o l a m i n e s while r e m o v i n g all common i n h i b i t o r s of COMT. After the c a t e c h o l a m i n e s are e x t r a c t e d into h e p t a n e w i t h d i p h e n y l borate, they are m a d e m o r e polar by i o n i z a t i o n of their amine w i t h acid. This forces the c a t e c h o l a m i n e s out of the o r g a n i c layer into an aqueous solution where they are a s s a y e d by a modification of our previously published method (19). This t e c h n i q u e improves assay s e n s i t i v i t y 10 fold and e l i m i n a t e s common C O M T inhibitors. Methods Materials: 3H-SAM (15 Ci/mmol) was p u r c h a s e d from Amersham, 3HNE (40 Ci/mmol) was p u r c h a s e d from N e w E n g l a n d Nuclear. All other r e a g e n t s were of a n a l y t i c a l grade and were o b t a i n e d from Sigma, F i s h e r (organic solvents), C a l b i o c h e m (glutathione, d i t h i o t h r e i t o l ) Alltech Associates (Stripmix) or A l d r i c h (diphenylborinic acid e t h a n o l a m i n e ester, t e t r a o c t y l a m m o n i u m bromide) COMT Preparation C O M T was p r e p a r e d from rat liver using a m o d i f i c a t i o n of the m e t h o d s of A x e l r o d and T o m c h i c k (20) and N i k o d e j e v i c et al (21). All p r o c e d u r e s were p e r f o r m e d at 4°C. Briefly, livers from about 30 rats w e r e h o m o g e n i z e d in 1.5% KCI, c e n t r i f u g e d at 16,000 g for 15 min, and the r e s u l t i n g supernate s t r a i n e d through cheese cloth. The s u p e r n a t e was then a d j u s t e d to pH 5.3 w i t h 1 M acetic acid, and centrifuged. The s u p e r n a t e was a d j u s t e d to pH 6.8 w i t h 1 M sodium p h o s p h a t e b u f f e r pH 7.4. A m m o n i u m sulfate was slowly added (17.6 g/I00 ml supernate). This solution was c e n t r i f u g e d at 16,000 g for 10 min and more a m m o n i u m sulfate (16.2 g/100 ml supernate) was added to the r e s u l t i n g supernate. This s o l u t i o n was c e n t r i f u g e d and the s u p e r n a t e discarded. The pellet was r e s u s p e n d e d in a 30% s a t u r a t e d a m m o n i u m sulfate solution and was centrifuged. The s u p e r n a t e was a d j u s t e d to 48% s a t u r a t i o n with a m m o n i u m sulfate, centrifuged, and the s u p e r n a t e was discarded. The pellet was r e s u s p e n d e d in 10 mM tris pH 7.4 c o n t a i n i n g 0.1 mM d i t h i o t h r e i t o l . The s o l u t i o n was added to small columns c o n t a i n i n g S e p h a d e x G-25 (Pharmacia) w h i c h had b e e n c e n t r i f u g e d at 300 g to remove buffer. The s o l u t i o n was c e n t r i f u g e d out of the columns at 300 g, c o l l e c t e d and b r o u g h t to 10 mM g l u t a t h i o n e and I mM O - b e n z y l h y d r o x y l a m i n e . It was then frozen in aliquots at -70°C. The activity of the C O M T was tested by a s s a y i n g w a t e r blanks and c a t e c h o l a m i n e standards using incubation mix c o n t a i n i n g 1 to 30 B1 of COMT. The amount of C O M T w h i c h y i e l d e d the h i g h e s t sample to blank ratio was r o u t i n e l y used for assays. In the p r e s e n t studies 10 ~i COMT was used.
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P r e p a r a t i o n of Plasma, U r i n e a n d T i s s u e S a m p l e s P l a s m a was c o l l e c t e d by v e n i p u n c t u r e of resting, r e c u m b e n t n o r m a l and u r e m i c subjects. T h e h e p a r i n i z e d b l o o d was k e p t o n ice, c e n t r i f u g e d , a n d the r e s u l t i n g p l a s m a was r e m o v e d and f r o z e n at - 7 0 ° C u n t i l assay. A f t e r c o l l e c t i o n , u r i n e was p u t o n ice and f r o z e n at - 7 0 ° C u n t i l assay. When sufficient plasma was available 1 ml was a s s a y e d to a c h i e v e m a x i m a l s e n s i t i v i t y . T i s s u e s to be a s s a y e d w e r e d i s s e c t e d from f r e s h l y s a c r i f i c e d rats. T h e y w e r e t h e n weighed, put in a tube and I ml of ice c o l d 0.1 M T r i s b u f f e r pH 7 was added. T i s s u e s w e r e then h o m o g e n i z e d for 10 - 30 s e c o n d s u s i n g a P o l y t r o n ( B r i n k m a n Corp.). Tissue h o m o g e n a t e s w e r e then c e n t r i f u g e d at 5000 g for 10 m i n and the s u p e r n a t e was t r a n s f e r r e d to a n e w tube and f r o z e n at - 7 0 ° C u n t i l assay. Borate Extraction Procedure A n N H 4 b u f f e r was m a d e in a fume h o o d by b r i n g i n g the pH of c o n c e n t r a t e d NH40H to 8.5 w i t h c o n c e n t r a t e d HCI. T h i s s o l u t i o n was diluted with water until the concentration of N H 4 w a s 2 M. Diphenylborinic acid ethanolamine ester (0.2% wt/vol) and 0.5% (wt/vol) d i s o d i u m E D T A w e r e then added. This s o l u t i o n was h e a t e d and s t i r r e d u n t i l all c o m p o n e n t s w e r e fully d i s s o l v e d . A h e p t a n e s o l u t i o n was m a d e in a fume h o o d by a d d i n g I% (vol/vol) o c t a n o l to heptane. T e t r a o c t y l a m m o n i u m b r o m i d e (0°25% wt/vol) was then a d d e d and the s o l u t i o n was h e a t e d and s t i r r e d u n t i l all c o m p o n e n t s w e r e fully dissolved. One h a l f ml of N H 4 b u f f e r was a d d e d to 5 to 2000 B1 samples, f o l l o w e d by 2 ml of the h e p t a n e solution. S a m p l e s w e r e then s h a k e n for 5 m i n and c e n t r i f u g e d at a b o u t 3000 g for 3 min. A b a t h of e t h a n o l w i t h dry ice was p r e p a r e d w h i c h was d e e p e n o u g h to c o v e r o n l y the a q u e o u s l a y e r of the sample tubes. The tubes w e r e then i m m e r s e d u n t i l the a q u e o u s l a y e r a p p e a r e d s o l i d l y frozen. The o r g a n i c l a y e r was then d e c a n t e d into a n o t h e r set of 13 x 100 m m p o l y p r o p y l e n e tubes. One ml of o c t a n o l and 0.1 ml of 0.08 N a c e t i c a c i d w e r e then a d d e d to this o r g a n i c supernate. S a m p l e s w e r e then s h a k e n for 5 m i n and c e n t r i f u g e d at 3000 g for 3 min. Afterwards, s a m p l e s w e r e p l a c e d in a - 7 0 ° C f r e e z e r for a b o u t 4 m i n or in a dry i c e - e t h a n o l b a t h u n t i l the a q u e o u s p e l l e t froze. C a r e was t a k e n to e n s u r e that o n l y the a q u e o u s layer froze. An a l u m i n u m s c r e e n w i t h a m e s h size of a b o u t 2 m m was then p l a c e d on the r a c k of s a m p l e s and the r a c k i n v e r t e d to s i m u l t a n e o u s l y d e c a n t the o r g a n i c s u p e r n a t e w h i l e the s c r e e n r e t a i n e d the p e l l e t s in the tubes. The samples w e r e p l a c e d in an ice w a t e r b a t h and any r e m a i n i n g o r g a n i c l a y e r was a s p i r a t e d from the f r o z e n pellets. P e l l e t s w e r e t h a w e d and a s s a y e d by the C O M T r a d i o e n z y m a t i c m e t h o d or s t o r e d at - 7 0 ° C u n t i l assay. COMT Radioenzymatic Assay Procedure Tubes containing pellets from b o r a t e e x t r a c t e d samples were t h a w e d and p l a c e d in an ice b a t h p r i o r to the a d d i t i o n of 100 ~i of i n c u b a t i o n mix. For u n e x t r a c t e d samples, 100 pl of s a m p l e was p i p e t t e d into 13 x 100 m m p o l y p r o p y l e n e tubes p r i o r to a d d i t i o n of i n c u b a t i o n mix. I n c u b a t i o n m i x for e a c h s a m p l e c o n t a i n e d 85 ~i buffer, 10 ~i p a r t i a l l y p u r i f i e d COMT, 5 ~i 3H-SAM (15 Ci/mmol), .06 m g g l u t a t h i o n e and .013 mg d e s f e r o x a m i n e . The b u f f e r s o l u t i o n c o n t a i n e d 2 M tris, 20 m M EGTA, and .09 M M g C I 2. A f t e r m i x was added, s a m p l e s w e r e i n c u b a t e d at 25°C for 90 min, p l a c e d on ice, then 200 B1 of I M b o r i c a c i d pH 10 w i t h 2.5% E D T A ( d i s o d i u m salt)
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was added, followed by 250BI of KaHPO 4 (I g/ml water). Fifty ~i of c a t e c h o l a m i n e cold c a r r i e r (I mg of n o r m e t a n e p h r i n e and m e t a n e p h r i n e /ml . 0 1 N HCI) were then added followed by 5 ml of w a t e r saturated 3:2 t o l u e n e : i s o a m y l alcohol. Samples were a g i t a t e d in a m e c h a n i c a l shaker for 5 min then c e n t r i f u g e d at 3000 g for 5 min. Next, samples w e r e p l a c e d in a dry ice-ethanol bath until the aqueous layer was frozen. Each supernate was d e c a n t e d into a 13 x 100 mm polypropylene tube and 250 ~i of 0.1 N acetic acid was added. Samples w e r e then shaken for 5 min and c e n t r i f u g e d for 5 min. Samples were then r e t u r n e d to the dry ice-ethanol bath. A f t e r the aqueous layer was frozen a screen was firmly p r e s s e d on top of the tubes and the rack was inverted to decant the supernatant. Five ml of w a t e r s a t u r a t e d 3:2 t o l u e n e : i s o a m y l alcohol was then added to each tube and samples were shaken and c e n t r i f u g e d as above. Samples were r e t u r n e d to the dry-ice ethanol bath until the aqueous layer was frozen. A f t e r p l a c i n g the screen on top of a rack of frozen sample tubes, the rack was inverted to decant the u n f r o z e n o r g a n i c layer. The aqueous layer was kept frozen for up to 3 days at -70°C before the assay was continued. After thawing, the c o n t e n t s of each tube were d r a w n into a 500 ~I H a m i l t o n syringe. E a c h tube was then r i n s e d w i t h 100 ~i of ethanol and the rinse was also d r a w n into the syringe. E i g h t e e n syringes were s i m u l t a n e o u s l y spotted o n t o f l u o r e s c e n t silica gel TLC plates (Baker # 7010-04) using an AIS m u l t i s p o t t e r and a h a i r d r y e r to enhance e v a p o r a t i o n of solvents. Plates were developed in a tank c o n t a i n i n g 105 ml of a 2:16:3 m i x t u r e of ethylamine:chloroform:ethanol. Normetanephrine and m e t a n e p h r i n e spots w e r e l o c a t e d under u l t r a v i o l e t light and o u t l i n e d w i t h a pencil. The spots were then sprayed w i t h a 1:2 m i x t u r e of S t r i p m i x : a c e t o n e in order to make each spot come off the plate as a unit w h e n s c r a p e d with a single edged razor blade. A f t e r scraping, spots w e r e p l a c e d in 7 ml c o u n t i n g vials. Two m o l a r a m m o n i u m sulfate s o l u t i o n (0.9 ml) was then added to each vial. The open vials w e r e slowly shaken for 5 min and then 50 ~i of 4% sodium periodate (wt/vol) was added. Exactly 5 min later 50 ~i of 10% g l y c e r o l was added to each sample, followed by 200 ~i of 10 M acetic acid. Finally, 200 ~i of s c i n t i l l a t o r and 4 ml of toluene were added. Samples w e r e then capped, shaken for 5 min and c o u n t e d in a liquid s c i n t i l l a t i o n counter. Results The assay g e n e r a t e d a linear increase in dpm w h e n up to 10,000 pg NE and E w e r e assayed. Above 10,000 pg the rate at which d i s i n t e g r a t i o n s per minute (dpm) were g e n e r a t e d g r a d u a l l y d e c r e a s e d (Fig I). A typical plasma sample from a resting, r e c u m b e n t subject c o n t a i n i n g 260 pg/ml NE and 13 pg/ml E had an intraassay c o e f f i c i e n t of v a r i a n c e of 4% for NE and 13% for E when 0.5 ml of the sample was a s s a y e d after b o r a t e e x t r a c t i o n (Table I). W h e n a s s a y e d w i t h o u t b o r a t e e x t r a c t i o n the c o e f f i c i e n t of v a r i a t i o n for a 100 ~i aliquot of this sample was 6% for NE and E levels were u n d e t e c t a b l e . The i n t e r a s s a y c o e f f i c i e n t of v a r i a t i o n for this sample w h e n e x t r a c t e d was 10% for NE and 16% for E. U n e x t r a c t e d samples had an interassay CV of 15% for NE.
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,-1000
o
o
Z ~o
FIG. 1 Disintegrations per minute generated in the radioenzymatic assay by borate extracted I ml samples of dialysed plasma containing increasing amounts of NE and E. Values are the mean of duplicate determinations varying by ( 15% at dpm > 200. Note logarithmic scales.
100
Q
0.1 0 .0 1
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0.1 1 10 100 NANOGRAMS CATECHOLAMINE
TABLE I.
CHARACTERISTICS OF A COMT BASED RADIOENZYMATIC ASSAY WITH AND WITHOUT BORATE EXTRACTION OF SAMPLE UNEXTRACTED
Maximum Sample Volume (ml) Sensitivity (pg/ml) Intraassay CV (%) Interassay CV (%)
0.1
I
NE
85
10
E
60
6
NE
6
E
_a
4 13
NE E
Linear range
(pg) (pg/ml)
EXTRACTED
NE NE
15b
10
-
16
8.5 - 10,000 85 - 100,000
10 - 10,000 10 - 10,000
Sensitivity defined as pg NE or E/ml generating twice blank dpm Intrassay coefficient of variation (CV) based on 10 determinations of a plasma sample containing about 260 pg NE and 13 pg E/ml Interassay C V b a s e d on 10 extractions of the same sample used to perform Intraassay CV. Linear range, when expressed as pg NE/ml is based on 100 B1 samples for unextracted assay and 1000 B1 samples for extracted assay a = Undetectable b = Determination not performed because E levels undetectable
T h e s e n s i t i v i t y of t h i s a s s a y v a r i e s c o n s i d e r a b l y d e p e n d i n g u p o n the s p e c i f i c a c t i v i t y o f 3H-SAM, a n d t h e a c t i v i t y a n d p u r i t y of the COMT employed. T y p i c a l l y , b l a n k v a l u e s for N E a n d E a r e a b o u t 100 dpm, w i t h 1 0 0 0 p g s t a n d a r d s in h u m a n p l a s m a g e n e r a t i n g roughly 2 0 , 0 0 0 d p m for N E a n d 3 0 , 0 0 0 d p m f o r E. Using a I ml sample and t h e c r i t e r i o n o f t w i c e b l a n k as the l o w e r l i m i t o f s e n s i t i v i t y , t h e s e p a r a m e t e r s g e n e r a t e a s e n s i t i v i t y of 10 p g / m l f o r N E a n d 6 p g / m l f o r E. W h e n s a m p l e s a r e n o t s u b j e c t e d to b o r a t e e x t r a c t i o n b l a n k s a v e r a g e a b o u t 60 d p m a n d 1000 p g s t a n d a r d s of N E a n d E generate about 14,000 and 20,000 dpm respectively. S i n c e o n l y 0.1 ml of p l a s m a m a y b e used, the a s s a y w i t h o u t b o r a t e e x t r a c t i o n h a s a s e n s i t i v i t y of a b o u t 85 p g / m l for N E a n d 60 p g / m l f o r E.
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The borate e x t r a c t i o n m i n i m i z e s the influence of several types of inhibitors of O - m e t h y l a t i o n by COMT. The c a l c i u m ion is a potent inhibitor of COMT. CaCI 2 added to plasma to increase Ca ~ c o n c e n t r a t i o n by 25 mM had no effect on the d p m g e n e r a t e d by borate e x t r a c t e d samples. However, this c o n c e n t r a t i o n of CaCI 2 d e c r e a s e d dpm in u n e x t r a c t e d samples by about 80%. Borate e x t r a c t i o n also reduces inhibition of O - m e t h y l a t i o n caused by h i g h c o n c e n t r a t i o n s of tissue. Liver, at a c o n c e n t r a t i o n of 7.5 mg/ 100 ~i sample, inhibited m e t h y l a t i o n of NE by 53% in the u n e x t r a c t e d assay but had no effect on m e t h y l a t i o n after borate extraction. Liver contains about 25 ~g SAM/g wet w e i g h t (22,23) which can compete w i t h the 3H-SA~4 in the assay. Increasing c o n c e n t r a t i o n s of SAM in the range found in several rat tissues (24) i n t e r f e r e d with the r a d i o e n z y m a t i c assay of u n e x t r a c t e d samples and r e d u c e d product generated (fig 2). In contrast, SAM had no d e t e c t a b l e effect f o l l o w i n g borate extraction. Over 98% of 3H-SAM added to a plasma sample was e x c l u d e d by borate extraction.
EXTRACTED
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FIG 2 Disintegrations per minute generated from I ng NE added to samples of buffer containing increasing concentrations of Sadenosylmethionine (SAM) and assayed with or without borate extraction. Values are the mean of duplicate determinations varying by < I0%.
Borate e x t r a c t i o n also m i n i m i z e s i n h i b i t i o n of COMT by normal p l a s m a (Fig 3). F o l l o w i n g borate e x t r a c t i o n of p l a s m a samples containing 1000 pg NE, dpm g e n e r a t e d were roughly double that g e n e r a t e d by u n e x t r a c t e d samples. The c o e f f i c i e n t of v a r i a t i o n was also r e d u c e d by more than 50% following borate extraction. Dpm were also i n c r e a s e d in u n a l t e r e d human plasma samples (Fig 3). The b o r a t e p r e e x t r a c t i o n removes inhibitors p r e s e n t in uremic plasma. W i t h o u t borate e x t r a c t i o n 1000 pg c a t e c h o l a m i n e s t a n d a r d s g e n e r a t e one third less dpm when added to uremic p l a s m a than when added to normal plasma. F o l l o w i n g borate extraction, standards generated as many dpm in uremic p l a s m a as in e x t r a c t e d normal plasma. I n c r e a s i n g volumes of uremic plasma p r o g r e s s i v e l y r e d u c e d the d p m g e n e r a t e d by added c a t e c h o l a m i n e s (Fig 4). However, after borate e x t r a c t i o n even volumes as large as I ml c a u s e d no r e d u c t i o n in dpm. W i t h o u t borate e x t r a c t i o n the dpm g e n e r a t e d by urine were not linear above 5 N1 urine (Fig 5). F o l l o w i n g borate e x t r a c t i o n the dpm i n c r e a s e d linearly.
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Radioenzymatic Catecholamine Assay
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I
40000-
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~ g 2oooo. co
=
.Oo
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0 2000-
EXTRACTED
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1500. FIG 3 Disintegrations per minute generated by 100 B1 of plasma from 8 resting, recumbent normal humans that was either borate extracted or unextracted. A) I ng NE added to each sample before assay. B) No catecholamines added. Values are means of duplicate determinations varying by < 10% at dpm > 200.
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FIG 4 DPM generated from I ng NE and E added to samples containing increasing volumes of uremic plasma and assayed with or without borate extraction. Values are the mean of duplicates and are corrected for dpm generated by endogenous catecholamines.
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10 25 50 100 250 1000 UREMIC PLASMA VOLUME (UL}
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FIG 5 DPM 3H-normetanephrine generated from increasing volumes of human urine assayed with or without borate extraction. Values are the mean of duplicates varying by < 15%.
2150
Radioenzymatic Catecholamine Assay
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7 • E~CTED 6
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FIG 6 Crossover into A) NE and B) E bands on the thin layer chromatography plate by I ng of 17 related compounds. Results were calculated by dividing the cpm crossing over into the NE or E band by the cpm generated by I ng standards of NE or E and multiplying by 100. Values are the mean ± SEM of triplicate determinations. * P < .05 vs extracted by Student's t-test. The borate extraction improved the specificity of the r a d i o e n z y m a t i c a s s a y for NE and E (Fig 6). T h e c r o s s o v e r i n t o the NE and E bands by several of the 17 compounds tested was significantly lower following borate extraction. A b o u t 1.4% of E c r o s s e s o v e r i n t o the NE b a n d and 0.6% of NE c r o s s e s o v e r i n t o the E b a n d for b o t h b o r a t e e x t a c t e d and u n e x t r a c t e d s a m p l e s . Borate e x t r a c t i o n e l i m i n a t e d o v e r 90% of n o r m e t a n e p h r i n e , m e t a n e p h r i n e and 3-methoxytyramine a d d e d to s a m p l e s . T h e r e c o v e r y of 3H-NE a d d e d to I ml p l a s m a s a m p l e s was 77.8 ± .7% following borate extraction. R e c o v e r y d r o p p e d to 71.7 ± .7% w h e n the s a m p l e v o l u m e was i n c r e a s e d to 2 ml. I n d e p e n d e n t l y c h a n g i n g the v o l u m e s of the NH 4 buffer, h e p t a n e s o l u t i o n , .08 M a c e t i c acid, or octanol o v e r a b o u t a 4 fold r a n g e o n l y s l i g h t l y affected the e x t r a c t i o n e f f i c i e n c y of 3H-NE by the b o r a t e m e t h o d . A f t e r the b o r a t e e x t r a c t i o n , s a m p l e s are i n c u b a t e d w i t h 3H-SAM to g e n e r a t e 3 H - m e t a n e p h r i n e f r o m E a n d 3 H - n o r m e t a n e p h r i n e f r o m NE. T h e s e 3 H - m e t a b o l i t e s are t h e n e x t r a c t e d f r o m the a q u e o u s l a y e r into a l i p i d layer. T h e a d d i t i o n of 250 mg K2HPO 4 /250 ~i w a t e r at the ~nd of the 90 m i n i n c u b a t i o n w i t h ~H-SAM i n c r e a s e d the r e c o v e r y of H - m e t a n e p h r i n e by 15% and of 3 H - n o r m e t a n e p h r i n e by 17%.
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Discussion This p r o c e d u r e i n c r e a s e d s e n s i t i v i t y and specificity, removed i n h i b i t o r s and d e c r e a s e d v a r i a b i l i t y in the c a t e c h o l a m i n e assay. A f t e r b o r a t e extraction, c a t e c h o l a m i n e s t a n d a r d s in h u m a n p l a s m a and s e v e r a l types of tissue O - m e t h y l a t e as r a p i d l y as s t a n d a r d s in water. It is thus not n e c e s s a r y to run internal c a t e c h o l a m i n e s t a n d a r d s to c o r r e c t for e n d o g e n o u s i n h i b i t i o n of COMT. H u m a n p l a s m a c o n t a i n s about 2.5 mM c a l c i u m concentration to inhibit COMT. CaCI 2 added inhibit the assay f o l l o w i n g borate extraction.
(25), a s u f f i c i e n t to p l a s m a did not
S e v e r a l rat tissues c o n t a i n c o n c e n t r a t i o n s of SAM w h i c h are high e n o u g h to inhibit C O M T (24). Tissues w i t h h i g h e n d o g e n o u s SAM levels r e d u c e the e f f e c t i v e specific a c t i v i t y of the 3H-SAM in the i n c u b a t i o n m i x thereby r e d u c i n g dpm generated. For example, liver c o n t a i n s about 25 Bg SAM/g tissue (22) and the COMT assay incubation m i x c o n t a i n s about 0.2 ~g 3H-SAM. An u n e x t r a c t e d 100 ~i sample of liver at 75 mg t i s s u e / m l should c o n t a i n about 0.185 ~g SAM and should reduce dpm g e n e r a t e d by about 50% r e l a t i v e to sample containing no e n d o g e n o u s SAM. We found that liver h o m o g e n a t e r e d u c e d counts by 53% r e l a t i v e to an identical sample that was b o r a t e extracted. The borate e x t r a c t i o n e l i m i n a t e s m o r e than 98% of SAM, so even a d d i t i o n of 4 Bg SAM to samples had no effect on the assay. R e m o v a l of SAM allows assay of large sample volumes, and for many tissues makes i n c l u s i o n of internal standards unnecessary. COMT inhibitors present in uremic p l a s m a and urine are e f f e c t i v e l y e x c l u d e d by the borate procedure. U r e m i c p l a s m a can inhibit the u n e x t r a c t e d assay by more than 50%. In contrast, borate e x t r a c t e d u r e m i c p l a s m a did not inhibit the assay. A d u l t humans excrete about I-2 liter of urine a day (26) w h i c h c o n t a i n s about 5 mmol of Ca ++ (27). Urine also c o n t a i n s several c a t e c h o l a m i n e metabolites. It is such a p o w e r f u l i n h i b i t o r of COMT that C O M T b a s e d assays have rarely been used to m e a s u r e urine catecholamines. B o r a t e e x t r a c t e d urine did not inhibit the assay. Urine samples as large as 100 B1 may be assayed w i t h o u t internal s t a n d a r d s f o l l o w i n g borate extraction. Borate extraction d i m i n i s h e d i n t e r f e r e n c e by several c o m p o u n d s structurally r e l a t e d to NE and E. This increase in s p e c i f i c i t y a p p e a r s to be due to the s e l e c t i v i t y of the b o r a t e e x t r a c t i o n for NE and E. For example, the borate extraction efficiency for catecholamines was 78% but for the 3 c a t e c h o l a m i n e metabolites, normetanephrine, metanephrine, and 3 - m e t h o x y t y r a m i n e it was less than 10%. In a d d i t i o n to the b o r a t e e x t r a c t i o n procedure, 2 other changes improve the assay method. P o t a s s i u m p h o s p h a t e (250 ~g in 250 ~i H20) added just after incubation w i t h ~H-SAM increases e x t r a c t i o n e f f i c i e n c y of 3H-metanephrine and 3H-normetanephrine by at least 15% w i t h o u t a f f e c t i n g b l a n k values. Also, after the aqueous layer is frozen, instead of i n d i v i d u a l l y a s p i r a t i n g s u p e r n a t e s we n o w firmly hold a screen on the top of a rack c o n t a i n i n g as m a n y as 90 sample tubes and invert the rack. This p r o c e d u r e s i m u l t a n e o u s l y decants the s u p e r n a t e t h e r e b y e l i m i n a t i n g v a r i a n c e due to s e q u e n t i a l removal of supernate.
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The most striking improvement of this assay over prior r a d i o e n z y m a t i c c a t e c h o l a m i n e assays is increased s e n s i t i v i t y when large sample volumes are assayed. Previous assays f r e q u e n t l y could not d e t e c t E in normal r e s t i n g human p l a s m a samples. This assay should p e r m i t i n v e s t i g a t i o n into the p h y s i o l o g y of low levels of adrenomedullary E secretion. The borate e x t r a c t i o n eliminates i n h i b i t i o n of the assay by calcium, SAM, urine and u r e m i c plasma. This can d i m i n i s h variability and d e c r e a s e s the chance that v a r i a b i l i t y due to inhibitors might be m i s i n t e r p r e t e d as v a r i a b i l i t y in c a t e c h o l a m i n e levels. Removal of inhibitors yields the p r a c t i c a l a d v a n t a g e that internal standards do not need to be run w i t h each sample. Acknowledqements The authors a c k n o w l e d g e Bain to the d e v e l o p m e n t NIH Grant HL 35924.
the important c o n t r i b u t i o n s of Mr. Robert of this assay. This w o r k was s u p p o r t e d by
References I 2 3 4 5 6 7 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19. 20. 21.
22.
D.R. Q U I R A M & R.M. WEINSHILBOUM, J N e u r o c h e m 27, 1197-1203 (1976) N BEN-JONATHAN & J.C. PORTER, E n d o c r i n o l o g y 98, 1497-1507 (1976) C GAUCHY, J.P. TASSIN, J. GLOWINSKI & A. CHERAMY, J N e u r o c h e m 26, 471-480 (1976) H HORTNAGL, C.R. BENEDICT, D.G. G R A H A M E - S M I T H & B. McGRATH, Br J Clin Pharmac 4, 553-558 (1977) S D E M A S S I E U X , L. CORNEILLE, S. LACHANCE, & S. CARRIERE, Clin C h i m Acta 115, 377-391 (1981) J L. Jr. IZZO, M.S. IZZ0, R.H. STERNS, & R.B. FREEMAN, Trans Am Soc Artif Intern Organs 28, 604-607 (1982) J D. PEULER & G.A. JOHNSON, Life Sci 21, 625-636 (1977) M.J. SOLE & M.N. HUSSAIN, B i o c h e m Med 18, 301-307 (1977) J.J.M.L. HOFFMANN, J.J. WILLEMSEN, T. THIEN & T.J. BENRAAD, Clin C h i m A c t a 125, 319-327 (1982) K.ENGELMAN, B. PORTNOY & W. LOVENBERG, Am J Med Sci 255, 259268 (1968) K. E N G E L M A N & B. PORTNOY, Circ Res 26, 53-57 (1970) J . T . C O Y L E & D. HENRY, J N e u r o c h e m 21, 61-67 (1973) M. PALKOVITS, M. BROWNSTEIN, J.M. S A A V E D R A & J. AXELROD, Brain Res 77, 137-149 (1974) J. DeCHAMPLAIN, L. FARLEY, D. C O U S I N E A U & M.R. van AMERINGEN, Circ Res 38, 109-114 (1976) L. BAUCE, J.A. T H O R N H I L L , K.E. C O O P E R & W.L. VEALE, Life Sci 27, 1921-1928 (1980) V.K. WEISE & I.J. KOPIN, Life Sci 19, 1673-1686 (1976) S.G. BALL, N.S. OATS & M.R.LEE, Clin Sci 55, 167-173 (1978) F.SMEDES, J.C. KRAAK, & H. POPPE, J C h r o m a t o g r a p h y 231, 2539 (1982) M.G. ZIEGLER, L.C. W O O D S O N & B. KENNEDY, Quantitative Analysis of Catecholamines and Related Compounds A.M. K r s t u l o v i c (ed) 263-280. H a l s t e d Press, New York (1986) J. A X E L R O D & R. TOMCHICK, J Biol C h e m 233, 702-705 (1958) B. NICODEJEVIC, S. SENOH, J.W. DALY & C.R. CREVELING, J P h a r m a c o l Exper Ther 174, 83-93 (1970) R.J. B A L D E S S A R I N I ,G. S T R A M E N T I N O L I & J.F. LIPINSKI, Arch Gen P s y c h i a t 36, 303-309 (1979)
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23. 24. 25. 26.
27.
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R. PASCALE, R. GARCEA, L. DAINO, S. FRASSETTO, M. RUGGIU, L. PIRISI, G. S T R A M E N T I N O L I & F. FEO, Res C o m m Sub A b u s e ~, 321324 (1984) R.J. B A L D E S S A R I N I & I.J. KOPIN, J N e u r o c h e m 13, 769-777 (1966) F.L. S T U T Z M A N & D.S. AMATUZIO, A r c h B i o c h e m 39, 271-275 (1952) R. W E I T Z M A N & C.R. K L E E M A N ,Clinical Disorders of Fluid and ElectrolFte Metabolism, M . H . M a x w e l l and C.R. K l e e m a n (eds.) 531-654. McGraw-Hill, N e w Y o r k (1980) S.D. B H A N D A R K A R & B.E.C. NORDIN, Br Med J !, 145-147 (1962)
Pergamon Press
A MORE S E N S I T I V E A N D S P E C I F I C RADIOENZYMATIC ASSAY FOR CATECHOLAMINES Brian
Kennedy
and M i c h a e l
G.
Ziegler
D i v i s i o n of Nephrology, D e p a r t m e n t of M e d i c i n e U n i v e r s i t y of C a l i f o r n i a San Diego M e d i c a l C e n t e r 225 D i c k i n s o n St, H-781-B, San Diego, CA 92103 (Received in final form October I, 1990) Summary This m o d i f i c a t i o n of the c a t e c h o l - O - m e t h y l t r a n s f e r a s e (COMT) b a s e d r a d i o e n z y m a t i c assay for n o r e p i n e p h r i n e (NE) and e p i n e p h r i n e (E) improves sensitivity, s e l e c t i v i t y and eliminates m a n y inhibitors of COMT. Prior to assay, s a m p l e s are e x t r a c t e d into h e p t a n e w i t h d i p h e n y l b o r a t e , then into d i l u t e acetic acid. This e x t r a c t i o n p r o c e d u r e has an e f f i c i e n c y of 78% for NE but less than 2% for Sadenosylmethionine (SAM). The e x t r a c t i o n p r o c e d u r e also e x c l u d e s c a l c i u m and o t h e r COMT inhibitors p r e s e n t in urine, p l a s m a and every tissue tested. This e l i m i n a t e s the r e q u i r e m e n t for individual s t a n d a r d i z a t i o n of tissue and u r i n e samples. S e n s i t i v i t y of the assay for NE and E is 10 and 6 pg/ml r e s p e c t i v e l y in I ml of plasma. The i n t r a a s s a y c o e f f i c i e n t s of v a r i a t i o n for NE and E are 4 and 13% and the interassay c o e f f i c i e n t s of v a r i a t i o n for NE and E are 10 and 16% in a h u m a n plasma sample c o n t a i n i n g low c a t e c h o l a m i n e levels. The assay p e r m i t s q u a n t i t a t i o n of p l a s m a E levels that w e r e u n d e t e c t a b l e in p r i o r assays. Catecholamine-O-methyl transferase (COMT) b a s e d r a d i o e n z y m a t i c assays are among the most sensitive methods for measuring c a t e c h o l a m i n e levels, but often they are not s e n s i t i v e e n o u g h to m e a s u r e E levels in human plasma. Another s h o r t c o m i n g of C O M T b a s e d r a d i o e n z y m a t i c assays is that their s e n s i t i v i t y and a c c u r a c y may be r e d u c e d by C O M T inhibitors p r e s e n t in many types of samples (1,2,3,4,5,6). Consequently, it is n e c e s s a r y to include internal s t a n d a r d s in such samples b e c a u s e r e d u c t i o n s in O - m e t h y l a t i o n due to i n h i b i t o r s may be m i s i n t e r p r e t e d as r e d u c e d c a t e c h o l a m i n e levels (7,8,9). The o r i g i n a l C O M T b a s e d r a d i o e n z y m a t i c m e t h o d that was d e v e l o p e d by E n g e l m a n et al in 1968 (10) u s e d 14C-SAM to d o n a t e a l a b e l e d m e t h y l g r o u p to c a t e c h o l a m i n e s as they w e r e O - m e t h y l a t e d . This m e t h o d has a s e n s i t i v i t y of 300 pg and was m a d e more s p e c i f i c and s e n s i t i v e by the a d d i t i o n of thin layer c h r o m a t o g r a p h y to s e p a r a t e the O - m e t h y l a t e d p r o d u c t s (11). Several refinements have b e e n m a d e to further increase the s e n s i t i v i t ~ of the assay. In 1973, Coyle & Henry (12) i n t r o d u c e d the use of H-SAM, w h i c h has a h i g h e r s p e c i f i c a c t i v i t y than 14C-SAM. P a l k o v i t s et al (13) were the first to scale d o w n the r e a c t i o n mixture. De C h a m p l a i n et al (14) i n c l u d e d E G T A in the r e a c t i o n m i x to c h e l a t e Ca m , the m a i n C O M T i n h i b i t o r p r e s e n t in h u m a n plasma. 0024-3205/90 $3.00 +.00 Copyright (c) 1990 Pergamon Press plc
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E v e n w i t h these modifications, the most s e n s i t i v e m e t h o d s have a s e n s i t i v i t y for E of about 20 pg/ml p l a s m a (7,15) from r e s t i n g humans o f t e n has E values b e l o w this.
generally Plasma
Several i n v e s t i g a t o r s have tried to increase the s e n s i t i v i t y of the C O M T r a d i o e n z y m a t i c assay by using p r e e x t r a c t i o n techniques to concentrate catecholamines and remove C O M T inhibitors. Weise & Kopin (16) and Ball (17) bound sample c a t e c h o l a m i n e s to alumina, and e l u t e d w i t h a small volume of acid. However, A1 +++ ions inhibit COMT and are c o e l u t e d w i t h the c a t e c h o l a m i n e s (3) , d i m i n i s h i n g the e f f i c a c y of this procedure. Gauchy et al (3) d e v e l o p e d an a l u m i n a method to concentrate catecholamines which avoided A1 ÷++ ion c o n t a m i n a t i o n but this p r o c e d u r e is too lengthy for r o u t i n e use. C a t e c h o l a m i n e s can be s e l e c t i v e l y e x t r a c t e d into lipid solvents by c h e l a t i n g agents (18). We have used this t e c h n i q u e to effect a 10 fold c o n c e n t r a t i o n of c a t e c h o l a m i n e s while r e m o v i n g all common i n h i b i t o r s of COMT. After the c a t e c h o l a m i n e s are e x t r a c t e d into h e p t a n e w i t h d i p h e n y l borate, they are m a d e m o r e polar by i o n i z a t i o n of their amine w i t h acid. This forces the c a t e c h o l a m i n e s out of the o r g a n i c layer into an aqueous solution where they are a s s a y e d by a modification of our previously published method (19). This t e c h n i q u e improves assay s e n s i t i v i t y 10 fold and e l i m i n a t e s common C O M T inhibitors. Methods Materials: 3H-SAM (15 Ci/mmol) was p u r c h a s e d from Amersham, 3HNE (40 Ci/mmol) was p u r c h a s e d from N e w E n g l a n d Nuclear. All other r e a g e n t s were of a n a l y t i c a l grade and were o b t a i n e d from Sigma, F i s h e r (organic solvents), C a l b i o c h e m (glutathione, d i t h i o t h r e i t o l ) Alltech Associates (Stripmix) or A l d r i c h (diphenylborinic acid e t h a n o l a m i n e ester, t e t r a o c t y l a m m o n i u m bromide) COMT Preparation C O M T was p r e p a r e d from rat liver using a m o d i f i c a t i o n of the m e t h o d s of A x e l r o d and T o m c h i c k (20) and N i k o d e j e v i c et al (21). All p r o c e d u r e s were p e r f o r m e d at 4°C. Briefly, livers from about 30 rats w e r e h o m o g e n i z e d in 1.5% KCI, c e n t r i f u g e d at 16,000 g for 15 min, and the r e s u l t i n g supernate s t r a i n e d through cheese cloth. The s u p e r n a t e was then a d j u s t e d to pH 5.3 w i t h 1 M acetic acid, and centrifuged. The s u p e r n a t e was a d j u s t e d to pH 6.8 w i t h 1 M sodium p h o s p h a t e b u f f e r pH 7.4. A m m o n i u m sulfate was slowly added (17.6 g/I00 ml supernate). This solution was c e n t r i f u g e d at 16,000 g for 10 min and more a m m o n i u m sulfate (16.2 g/100 ml supernate) was added to the r e s u l t i n g supernate. This s o l u t i o n was c e n t r i f u g e d and the s u p e r n a t e discarded. The pellet was r e s u s p e n d e d in a 30% s a t u r a t e d a m m o n i u m sulfate solution and was centrifuged. The s u p e r n a t e was a d j u s t e d to 48% s a t u r a t i o n with a m m o n i u m sulfate, centrifuged, and the s u p e r n a t e was discarded. The pellet was r e s u s p e n d e d in 10 mM tris pH 7.4 c o n t a i n i n g 0.1 mM d i t h i o t h r e i t o l . The s o l u t i o n was added to small columns c o n t a i n i n g S e p h a d e x G-25 (Pharmacia) w h i c h had b e e n c e n t r i f u g e d at 300 g to remove buffer. The s o l u t i o n was c e n t r i f u g e d out of the columns at 300 g, c o l l e c t e d and b r o u g h t to 10 mM g l u t a t h i o n e and I mM O - b e n z y l h y d r o x y l a m i n e . It was then frozen in aliquots at -70°C. The activity of the C O M T was tested by a s s a y i n g w a t e r blanks and c a t e c h o l a m i n e standards using incubation mix c o n t a i n i n g 1 to 30 B1 of COMT. The amount of C O M T w h i c h y i e l d e d the h i g h e s t sample to blank ratio was r o u t i n e l y used for assays. In the p r e s e n t studies 10 ~i COMT was used.
Vol. 47, No. 23, 1990
Radioenzymatic Catecholamine Assay
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P r e p a r a t i o n of Plasma, U r i n e a n d T i s s u e S a m p l e s P l a s m a was c o l l e c t e d by v e n i p u n c t u r e of resting, r e c u m b e n t n o r m a l and u r e m i c subjects. T h e h e p a r i n i z e d b l o o d was k e p t o n ice, c e n t r i f u g e d , a n d the r e s u l t i n g p l a s m a was r e m o v e d and f r o z e n at - 7 0 ° C u n t i l assay. A f t e r c o l l e c t i o n , u r i n e was p u t o n ice and f r o z e n at - 7 0 ° C u n t i l assay. When sufficient plasma was available 1 ml was a s s a y e d to a c h i e v e m a x i m a l s e n s i t i v i t y . T i s s u e s to be a s s a y e d w e r e d i s s e c t e d from f r e s h l y s a c r i f i c e d rats. T h e y w e r e t h e n weighed, put in a tube and I ml of ice c o l d 0.1 M T r i s b u f f e r pH 7 was added. T i s s u e s w e r e then h o m o g e n i z e d for 10 - 30 s e c o n d s u s i n g a P o l y t r o n ( B r i n k m a n Corp.). Tissue h o m o g e n a t e s w e r e then c e n t r i f u g e d at 5000 g for 10 m i n and the s u p e r n a t e was t r a n s f e r r e d to a n e w tube and f r o z e n at - 7 0 ° C u n t i l assay. Borate Extraction Procedure A n N H 4 b u f f e r was m a d e in a fume h o o d by b r i n g i n g the pH of c o n c e n t r a t e d NH40H to 8.5 w i t h c o n c e n t r a t e d HCI. T h i s s o l u t i o n was diluted with water until the concentration of N H 4 w a s 2 M. Diphenylborinic acid ethanolamine ester (0.2% wt/vol) and 0.5% (wt/vol) d i s o d i u m E D T A w e r e then added. This s o l u t i o n was h e a t e d and s t i r r e d u n t i l all c o m p o n e n t s w e r e fully d i s s o l v e d . A h e p t a n e s o l u t i o n was m a d e in a fume h o o d by a d d i n g I% (vol/vol) o c t a n o l to heptane. T e t r a o c t y l a m m o n i u m b r o m i d e (0°25% wt/vol) was then a d d e d and the s o l u t i o n was h e a t e d and s t i r r e d u n t i l all c o m p o n e n t s w e r e fully dissolved. One h a l f ml of N H 4 b u f f e r was a d d e d to 5 to 2000 B1 samples, f o l l o w e d by 2 ml of the h e p t a n e solution. S a m p l e s w e r e then s h a k e n for 5 m i n and c e n t r i f u g e d at a b o u t 3000 g for 3 min. A b a t h of e t h a n o l w i t h dry ice was p r e p a r e d w h i c h was d e e p e n o u g h to c o v e r o n l y the a q u e o u s l a y e r of the sample tubes. The tubes w e r e then i m m e r s e d u n t i l the a q u e o u s l a y e r a p p e a r e d s o l i d l y frozen. The o r g a n i c l a y e r was then d e c a n t e d into a n o t h e r set of 13 x 100 m m p o l y p r o p y l e n e tubes. One ml of o c t a n o l and 0.1 ml of 0.08 N a c e t i c a c i d w e r e then a d d e d to this o r g a n i c supernate. S a m p l e s w e r e then s h a k e n for 5 m i n and c e n t r i f u g e d at 3000 g for 3 min. Afterwards, s a m p l e s w e r e p l a c e d in a - 7 0 ° C f r e e z e r for a b o u t 4 m i n or in a dry i c e - e t h a n o l b a t h u n t i l the a q u e o u s p e l l e t froze. C a r e was t a k e n to e n s u r e that o n l y the a q u e o u s layer froze. An a l u m i n u m s c r e e n w i t h a m e s h size of a b o u t 2 m m was then p l a c e d on the r a c k of s a m p l e s and the r a c k i n v e r t e d to s i m u l t a n e o u s l y d e c a n t the o r g a n i c s u p e r n a t e w h i l e the s c r e e n r e t a i n e d the p e l l e t s in the tubes. The samples w e r e p l a c e d in an ice w a t e r b a t h and any r e m a i n i n g o r g a n i c l a y e r was a s p i r a t e d from the f r o z e n pellets. P e l l e t s w e r e t h a w e d and a s s a y e d by the C O M T r a d i o e n z y m a t i c m e t h o d or s t o r e d at - 7 0 ° C u n t i l assay. COMT Radioenzymatic Assay Procedure Tubes containing pellets from b o r a t e e x t r a c t e d samples were t h a w e d and p l a c e d in an ice b a t h p r i o r to the a d d i t i o n of 100 ~i of i n c u b a t i o n mix. For u n e x t r a c t e d samples, 100 pl of s a m p l e was p i p e t t e d into 13 x 100 m m p o l y p r o p y l e n e tubes p r i o r to a d d i t i o n of i n c u b a t i o n mix. I n c u b a t i o n m i x for e a c h s a m p l e c o n t a i n e d 85 ~i buffer, 10 ~i p a r t i a l l y p u r i f i e d COMT, 5 ~i 3H-SAM (15 Ci/mmol), .06 m g g l u t a t h i o n e and .013 mg d e s f e r o x a m i n e . The b u f f e r s o l u t i o n c o n t a i n e d 2 M tris, 20 m M EGTA, and .09 M M g C I 2. A f t e r m i x was added, s a m p l e s w e r e i n c u b a t e d at 25°C for 90 min, p l a c e d on ice, then 200 B1 of I M b o r i c a c i d pH 10 w i t h 2.5% E D T A ( d i s o d i u m salt)
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Radioenzymatic Catecholamine Assay
Vol. 47, No, 23, 1990
was added, followed by 250BI of KaHPO 4 (I g/ml water). Fifty ~i of c a t e c h o l a m i n e cold c a r r i e r (I mg of n o r m e t a n e p h r i n e and m e t a n e p h r i n e /ml . 0 1 N HCI) were then added followed by 5 ml of w a t e r saturated 3:2 t o l u e n e : i s o a m y l alcohol. Samples were a g i t a t e d in a m e c h a n i c a l shaker for 5 min then c e n t r i f u g e d at 3000 g for 5 min. Next, samples w e r e p l a c e d in a dry ice-ethanol bath until the aqueous layer was frozen. Each supernate was d e c a n t e d into a 13 x 100 mm polypropylene tube and 250 ~i of 0.1 N acetic acid was added. Samples w e r e then shaken for 5 min and c e n t r i f u g e d for 5 min. Samples were then r e t u r n e d to the dry ice-ethanol bath. A f t e r the aqueous layer was frozen a screen was firmly p r e s s e d on top of the tubes and the rack was inverted to decant the supernatant. Five ml of w a t e r s a t u r a t e d 3:2 t o l u e n e : i s o a m y l alcohol was then added to each tube and samples were shaken and c e n t r i f u g e d as above. Samples were r e t u r n e d to the dry-ice ethanol bath until the aqueous layer was frozen. A f t e r p l a c i n g the screen on top of a rack of frozen sample tubes, the rack was inverted to decant the u n f r o z e n o r g a n i c layer. The aqueous layer was kept frozen for up to 3 days at -70°C before the assay was continued. After thawing, the c o n t e n t s of each tube were d r a w n into a 500 ~I H a m i l t o n syringe. E a c h tube was then r i n s e d w i t h 100 ~i of ethanol and the rinse was also d r a w n into the syringe. E i g h t e e n syringes were s i m u l t a n e o u s l y spotted o n t o f l u o r e s c e n t silica gel TLC plates (Baker # 7010-04) using an AIS m u l t i s p o t t e r and a h a i r d r y e r to enhance e v a p o r a t i o n of solvents. Plates were developed in a tank c o n t a i n i n g 105 ml of a 2:16:3 m i x t u r e of ethylamine:chloroform:ethanol. Normetanephrine and m e t a n e p h r i n e spots w e r e l o c a t e d under u l t r a v i o l e t light and o u t l i n e d w i t h a pencil. The spots were then sprayed w i t h a 1:2 m i x t u r e of S t r i p m i x : a c e t o n e in order to make each spot come off the plate as a unit w h e n s c r a p e d with a single edged razor blade. A f t e r scraping, spots w e r e p l a c e d in 7 ml c o u n t i n g vials. Two m o l a r a m m o n i u m sulfate s o l u t i o n (0.9 ml) was then added to each vial. The open vials w e r e slowly shaken for 5 min and then 50 ~i of 4% sodium periodate (wt/vol) was added. Exactly 5 min later 50 ~i of 10% g l y c e r o l was added to each sample, followed by 200 ~i of 10 M acetic acid. Finally, 200 ~i of s c i n t i l l a t o r and 4 ml of toluene were added. Samples w e r e then capped, shaken for 5 min and c o u n t e d in a liquid s c i n t i l l a t i o n counter. Results The assay g e n e r a t e d a linear increase in dpm w h e n up to 10,000 pg NE and E w e r e assayed. Above 10,000 pg the rate at which d i s i n t e g r a t i o n s per minute (dpm) were g e n e r a t e d g r a d u a l l y d e c r e a s e d (Fig I). A typical plasma sample from a resting, r e c u m b e n t subject c o n t a i n i n g 260 pg/ml NE and 13 pg/ml E had an intraassay c o e f f i c i e n t of v a r i a n c e of 4% for NE and 13% for E when 0.5 ml of the sample was a s s a y e d after b o r a t e e x t r a c t i o n (Table I). W h e n a s s a y e d w i t h o u t b o r a t e e x t r a c t i o n the c o e f f i c i e n t of v a r i a t i o n for a 100 ~i aliquot of this sample was 6% for NE and E levels were u n d e t e c t a b l e . The i n t e r a s s a y c o e f f i c i e n t of v a r i a t i o n for this sample w h e n e x t r a c t e d was 10% for NE and 16% for E. U n e x t r a c t e d samples had an interassay CV of 15% for NE.
Vol. 47, No. 23, 1990
Radioenzymatic Catecholamine Assay
,-1000
o
o
Z ~o
FIG. 1 Disintegrations per minute generated in the radioenzymatic assay by borate extracted I ml samples of dialysed plasma containing increasing amounts of NE and E. Values are the mean of duplicate determinations varying by ( 15% at dpm > 200. Note logarithmic scales.
100
Q
0.1 0 .0 1
2147
0.1 1 10 100 NANOGRAMS CATECHOLAMINE
TABLE I.
CHARACTERISTICS OF A COMT BASED RADIOENZYMATIC ASSAY WITH AND WITHOUT BORATE EXTRACTION OF SAMPLE UNEXTRACTED
Maximum Sample Volume (ml) Sensitivity (pg/ml) Intraassay CV (%) Interassay CV (%)
0.1
I
NE
85
10
E
60
6
NE
6
E
_a
4 13
NE E
Linear range
(pg) (pg/ml)
EXTRACTED
NE NE
15b
10
-
16
8.5 - 10,000 85 - 100,000
10 - 10,000 10 - 10,000
Sensitivity defined as pg NE or E/ml generating twice blank dpm Intrassay coefficient of variation (CV) based on 10 determinations of a plasma sample containing about 260 pg NE and 13 pg E/ml Interassay C V b a s e d on 10 extractions of the same sample used to perform Intraassay CV. Linear range, when expressed as pg NE/ml is based on 100 B1 samples for unextracted assay and 1000 B1 samples for extracted assay a = Undetectable b = Determination not performed because E levels undetectable
T h e s e n s i t i v i t y of t h i s a s s a y v a r i e s c o n s i d e r a b l y d e p e n d i n g u p o n the s p e c i f i c a c t i v i t y o f 3H-SAM, a n d t h e a c t i v i t y a n d p u r i t y of the COMT employed. T y p i c a l l y , b l a n k v a l u e s for N E a n d E a r e a b o u t 100 dpm, w i t h 1 0 0 0 p g s t a n d a r d s in h u m a n p l a s m a g e n e r a t i n g roughly 2 0 , 0 0 0 d p m for N E a n d 3 0 , 0 0 0 d p m f o r E. Using a I ml sample and t h e c r i t e r i o n o f t w i c e b l a n k as the l o w e r l i m i t o f s e n s i t i v i t y , t h e s e p a r a m e t e r s g e n e r a t e a s e n s i t i v i t y of 10 p g / m l f o r N E a n d 6 p g / m l f o r E. W h e n s a m p l e s a r e n o t s u b j e c t e d to b o r a t e e x t r a c t i o n b l a n k s a v e r a g e a b o u t 60 d p m a n d 1000 p g s t a n d a r d s of N E a n d E generate about 14,000 and 20,000 dpm respectively. S i n c e o n l y 0.1 ml of p l a s m a m a y b e used, the a s s a y w i t h o u t b o r a t e e x t r a c t i o n h a s a s e n s i t i v i t y of a b o u t 85 p g / m l for N E a n d 60 p g / m l f o r E.
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Catecholamine Assay
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The borate e x t r a c t i o n m i n i m i z e s the influence of several types of inhibitors of O - m e t h y l a t i o n by COMT. The c a l c i u m ion is a potent inhibitor of COMT. CaCI 2 added to plasma to increase Ca ~ c o n c e n t r a t i o n by 25 mM had no effect on the d p m g e n e r a t e d by borate e x t r a c t e d samples. However, this c o n c e n t r a t i o n of CaCI 2 d e c r e a s e d dpm in u n e x t r a c t e d samples by about 80%. Borate e x t r a c t i o n also reduces inhibition of O - m e t h y l a t i o n caused by h i g h c o n c e n t r a t i o n s of tissue. Liver, at a c o n c e n t r a t i o n of 7.5 mg/ 100 ~i sample, inhibited m e t h y l a t i o n of NE by 53% in the u n e x t r a c t e d assay but had no effect on m e t h y l a t i o n after borate extraction. Liver contains about 25 ~g SAM/g wet w e i g h t (22,23) which can compete w i t h the 3H-SA~4 in the assay. Increasing c o n c e n t r a t i o n s of SAM in the range found in several rat tissues (24) i n t e r f e r e d with the r a d i o e n z y m a t i c assay of u n e x t r a c t e d samples and r e d u c e d product generated (fig 2). In contrast, SAM had no d e t e c t a b l e effect f o l l o w i n g borate extraction. Over 98% of 3H-SAM added to a plasma sample was e x c l u d e d by borate extraction.
EXTRACTED
50000-
4°°°°I Z ~ 30000-
~
o 20000.
z
\
~
x
UNEXTRACTED
10000.
O|
0
I
I
I
0.5
1
1.5
I
I
2 2.5 SAM (UG)
I
I
I
3
3.5
4
FIG 2 Disintegrations per minute generated from I ng NE added to samples of buffer containing increasing concentrations of Sadenosylmethionine (SAM) and assayed with or without borate extraction. Values are the mean of duplicate determinations varying by < I0%.
Borate e x t r a c t i o n also m i n i m i z e s i n h i b i t i o n of COMT by normal p l a s m a (Fig 3). F o l l o w i n g borate e x t r a c t i o n of p l a s m a samples containing 1000 pg NE, dpm g e n e r a t e d were roughly double that g e n e r a t e d by u n e x t r a c t e d samples. The c o e f f i c i e n t of v a r i a t i o n was also r e d u c e d by more than 50% following borate extraction. Dpm were also i n c r e a s e d in u n a l t e r e d human plasma samples (Fig 3). The b o r a t e p r e e x t r a c t i o n removes inhibitors p r e s e n t in uremic plasma. W i t h o u t borate e x t r a c t i o n 1000 pg c a t e c h o l a m i n e s t a n d a r d s g e n e r a t e one third less dpm when added to uremic p l a s m a than when added to normal plasma. F o l l o w i n g borate extraction, standards generated as many dpm in uremic p l a s m a as in e x t r a c t e d normal plasma. I n c r e a s i n g volumes of uremic plasma p r o g r e s s i v e l y r e d u c e d the d p m g e n e r a t e d by added c a t e c h o l a m i n e s (Fig 4). However, after borate e x t r a c t i o n even volumes as large as I ml c a u s e d no r e d u c t i o n in dpm. W i t h o u t borate e x t r a c t i o n the dpm g e n e r a t e d by urine were not linear above 5 N1 urine (Fig 5). F o l l o w i n g borate e x t r a c t i o n the dpm i n c r e a s e d linearly.
Vol. 47, No. 23, 1990
Radioenzymatic Catecholamine Assay
2149
I
40000-
W.
A---]
~ g 2oooo. co
=
.Oo
10000
0 2000-
EXTRACTED
UNEXTRACTED
~]
1500. FIG 3 Disintegrations per minute generated by 100 B1 of plasma from 8 resting, recumbent normal humans that was either borate extracted or unextracted. A) I ng NE added to each sample before assay. B) No catecholamines added. Values are means of duplicate determinations varying by < 10% at dpm > 200.
1000. o~ mo i~:
-.
__~J°
600
UNEXTRACFED
z x ~~ . . ~ E X ~30000
~
ZO000
xx~
10000
EXTRACTED
T RACTED
FIG 4 DPM generated from I ng NE and E added to samples containing increasing volumes of uremic plasma and assayed with or without borate extraction. Values are the mean of duplicates and are corrected for dpm generated by endogenous catecholamines.
NE EXTRACTED
"
Ol
5
l
i
I
I
I
I
10 25 50 100 250 1000 UREMIC PLASMA VOLUME (UL}
lOOT
Nm oo
60 :~m 40 ~
20
./..
XTRACTED
O,r '~
-.
i
o 1'o 2'o 3'0 ;o 5'0 6'o ¢o do ¢o 1oo pl URINE / 100 ~*! SAMPLE
FIG 5 DPM 3H-normetanephrine generated from increasing volumes of human urine assayed with or without borate extraction. Values are the mean of duplicates varying by < 15%.
2150
Radioenzymatic Catecholamine Assay
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7 • E~CTED 6
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~
N n
FIG 6 Crossover into A) NE and B) E bands on the thin layer chromatography plate by I ng of 17 related compounds. Results were calculated by dividing the cpm crossing over into the NE or E band by the cpm generated by I ng standards of NE or E and multiplying by 100. Values are the mean ± SEM of triplicate determinations. * P < .05 vs extracted by Student's t-test. The borate extraction improved the specificity of the r a d i o e n z y m a t i c a s s a y for NE and E (Fig 6). T h e c r o s s o v e r i n t o the NE and E bands by several of the 17 compounds tested was significantly lower following borate extraction. A b o u t 1.4% of E c r o s s e s o v e r i n t o the NE b a n d and 0.6% of NE c r o s s e s o v e r i n t o the E b a n d for b o t h b o r a t e e x t a c t e d and u n e x t r a c t e d s a m p l e s . Borate e x t r a c t i o n e l i m i n a t e d o v e r 90% of n o r m e t a n e p h r i n e , m e t a n e p h r i n e and 3-methoxytyramine a d d e d to s a m p l e s . T h e r e c o v e r y of 3H-NE a d d e d to I ml p l a s m a s a m p l e s was 77.8 ± .7% following borate extraction. R e c o v e r y d r o p p e d to 71.7 ± .7% w h e n the s a m p l e v o l u m e was i n c r e a s e d to 2 ml. I n d e p e n d e n t l y c h a n g i n g the v o l u m e s of the NH 4 buffer, h e p t a n e s o l u t i o n , .08 M a c e t i c acid, or octanol o v e r a b o u t a 4 fold r a n g e o n l y s l i g h t l y affected the e x t r a c t i o n e f f i c i e n c y of 3H-NE by the b o r a t e m e t h o d . A f t e r the b o r a t e e x t r a c t i o n , s a m p l e s are i n c u b a t e d w i t h 3H-SAM to g e n e r a t e 3 H - m e t a n e p h r i n e f r o m E a n d 3 H - n o r m e t a n e p h r i n e f r o m NE. T h e s e 3 H - m e t a b o l i t e s are t h e n e x t r a c t e d f r o m the a q u e o u s l a y e r into a l i p i d layer. T h e a d d i t i o n of 250 mg K2HPO 4 /250 ~i w a t e r at the ~nd of the 90 m i n i n c u b a t i o n w i t h ~H-SAM i n c r e a s e d the r e c o v e r y of H - m e t a n e p h r i n e by 15% and of 3 H - n o r m e t a n e p h r i n e by 17%.
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Discussion This p r o c e d u r e i n c r e a s e d s e n s i t i v i t y and specificity, removed i n h i b i t o r s and d e c r e a s e d v a r i a b i l i t y in the c a t e c h o l a m i n e assay. A f t e r b o r a t e extraction, c a t e c h o l a m i n e s t a n d a r d s in h u m a n p l a s m a and s e v e r a l types of tissue O - m e t h y l a t e as r a p i d l y as s t a n d a r d s in water. It is thus not n e c e s s a r y to run internal c a t e c h o l a m i n e s t a n d a r d s to c o r r e c t for e n d o g e n o u s i n h i b i t i o n of COMT. H u m a n p l a s m a c o n t a i n s about 2.5 mM c a l c i u m concentration to inhibit COMT. CaCI 2 added inhibit the assay f o l l o w i n g borate extraction.
(25), a s u f f i c i e n t to p l a s m a did not
S e v e r a l rat tissues c o n t a i n c o n c e n t r a t i o n s of SAM w h i c h are high e n o u g h to inhibit C O M T (24). Tissues w i t h h i g h e n d o g e n o u s SAM levels r e d u c e the e f f e c t i v e specific a c t i v i t y of the 3H-SAM in the i n c u b a t i o n m i x thereby r e d u c i n g dpm generated. For example, liver c o n t a i n s about 25 Bg SAM/g tissue (22) and the COMT assay incubation m i x c o n t a i n s about 0.2 ~g 3H-SAM. An u n e x t r a c t e d 100 ~i sample of liver at 75 mg t i s s u e / m l should c o n t a i n about 0.185 ~g SAM and should reduce dpm g e n e r a t e d by about 50% r e l a t i v e to sample containing no e n d o g e n o u s SAM. We found that liver h o m o g e n a t e r e d u c e d counts by 53% r e l a t i v e to an identical sample that was b o r a t e extracted. The borate e x t r a c t i o n e l i m i n a t e s m o r e than 98% of SAM, so even a d d i t i o n of 4 Bg SAM to samples had no effect on the assay. R e m o v a l of SAM allows assay of large sample volumes, and for many tissues makes i n c l u s i o n of internal standards unnecessary. COMT inhibitors present in uremic p l a s m a and urine are e f f e c t i v e l y e x c l u d e d by the borate procedure. U r e m i c p l a s m a can inhibit the u n e x t r a c t e d assay by more than 50%. In contrast, borate e x t r a c t e d u r e m i c p l a s m a did not inhibit the assay. A d u l t humans excrete about I-2 liter of urine a day (26) w h i c h c o n t a i n s about 5 mmol of Ca ++ (27). Urine also c o n t a i n s several c a t e c h o l a m i n e metabolites. It is such a p o w e r f u l i n h i b i t o r of COMT that C O M T b a s e d assays have rarely been used to m e a s u r e urine catecholamines. B o r a t e e x t r a c t e d urine did not inhibit the assay. Urine samples as large as 100 B1 may be assayed w i t h o u t internal s t a n d a r d s f o l l o w i n g borate extraction. Borate extraction d i m i n i s h e d i n t e r f e r e n c e by several c o m p o u n d s structurally r e l a t e d to NE and E. This increase in s p e c i f i c i t y a p p e a r s to be due to the s e l e c t i v i t y of the b o r a t e e x t r a c t i o n for NE and E. For example, the borate extraction efficiency for catecholamines was 78% but for the 3 c a t e c h o l a m i n e metabolites, normetanephrine, metanephrine, and 3 - m e t h o x y t y r a m i n e it was less than 10%. In a d d i t i o n to the b o r a t e e x t r a c t i o n procedure, 2 other changes improve the assay method. P o t a s s i u m p h o s p h a t e (250 ~g in 250 ~i H20) added just after incubation w i t h ~H-SAM increases e x t r a c t i o n e f f i c i e n c y of 3H-metanephrine and 3H-normetanephrine by at least 15% w i t h o u t a f f e c t i n g b l a n k values. Also, after the aqueous layer is frozen, instead of i n d i v i d u a l l y a s p i r a t i n g s u p e r n a t e s we n o w firmly hold a screen on the top of a rack c o n t a i n i n g as m a n y as 90 sample tubes and invert the rack. This p r o c e d u r e s i m u l t a n e o u s l y decants the s u p e r n a t e t h e r e b y e l i m i n a t i n g v a r i a n c e due to s e q u e n t i a l removal of supernate.
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The most striking improvement of this assay over prior r a d i o e n z y m a t i c c a t e c h o l a m i n e assays is increased s e n s i t i v i t y when large sample volumes are assayed. Previous assays f r e q u e n t l y could not d e t e c t E in normal r e s t i n g human p l a s m a samples. This assay should p e r m i t i n v e s t i g a t i o n into the p h y s i o l o g y of low levels of adrenomedullary E secretion. The borate e x t r a c t i o n eliminates i n h i b i t i o n of the assay by calcium, SAM, urine and u r e m i c plasma. This can d i m i n i s h variability and d e c r e a s e s the chance that v a r i a b i l i t y due to inhibitors might be m i s i n t e r p r e t e d as v a r i a b i l i t y in c a t e c h o l a m i n e levels. Removal of inhibitors yields the p r a c t i c a l a d v a n t a g e that internal standards do not need to be run w i t h each sample. Acknowledqements The authors a c k n o w l e d g e Bain to the d e v e l o p m e n t NIH Grant HL 35924.
the important c o n t r i b u t i o n s of Mr. Robert of this assay. This w o r k was s u p p o r t e d by
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