Antisera Raised Against Tetrahydrocannabinol in the

10 Antisera Raised Against Tetrahydrocannabinol in the Radioimmunoassay of Cannabinoids

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J. D. T E A L E , JACQUELINE M. CLOUGH, L. J. KING, and V. MARKS Department of Biochemistry, University of Surrey, Guildford, Surrey, United Kingdom P. L. WILLIAMS and A. C. MOFFAT Home Office Central Research Establishment, Aldermaston, Berkshire, United Kingdom

Radioimmunoassay (RIA), whether it is used for the measurement of peptides, steroids or drugs, has many attractions, particularly for the routine laboratory engaged in the analysis of large numbers of samples. The major attribute of RIA is its potential for the detection of picogram quantities of compound, often by direct analysis of untreated biological specimens. In the measurement of low molecular weight substances, such as drugs, RIA can provide results on many samples in a matter of hours. The use of automated procedures w i l l increase the speed of operation still further, as well as improving precision. The original RIA concept (1) was based on antibodies to a naturally immunogenic peptide (insulin) but it is possible to produce antibodies to any non-protein molecule by attachment to an immunogenic carrier molecule (2). Attention has turned recently to the development of RIA for drugs (3), particularly those drugs given in low doses and requiring a sensitive detection system. Considerable progress has been made over the past decade in the measurement of drugs by RIA. The main reason for the advance in this application of the technique is the improved development of the key reagents; antiserum and radioactive label. More effective meth0-8412-0488-8/79/47-098-155$05.00/0 © 1979 American Chemical Society Vinson; Cannabinoid Analysis in Physiological Fluids ACS Symposium Series; American Chemical Society: Washington, DC, 1979.

CANNABINOID ANALYSIS IN


156

PHYSIOLOGICAL FLUIDS

ods have l e a d t o t h e p r o d u c t i o n o f h i g h e r s p e c i f i c a c t i v i t y t r i t i a t e d d r u g s , w i t h a concommitant i n c r e a s e i n assay s e n s i t i v i t y . L i q u i d s c i n t i l l a t i o n counting i s used f o r c o u n t i n g t r i t i u m . This type of l a b e l i n g provides a tracer with s i m i l a r antibody-combining p r o p e r t i e s t o the n a t i v e d r u g and o b v i a t e s problems o f t e n encountered w i t h i o d i n a t e d drug d e r i v a t i v e , f o r example ( 4 ) . A n t i s e r u m p r o p e r t i e s e x e r t the g r e a t e s t i n f l u e n c e on a s s a y p e r f o r m a n c e , b o t h i n terms o f s e n s i t i v i t y and specificity. I t i s u s u a l t o aim f o r a h i g h a v i d i t y ; m o n o s p e c i f i c a n t i b o d y p r e s e n t i n the serum a t h i g h titre. Such a n t i b o d i e s w i l l b i n d a n t i g e n t i g h t l y r e s u l t i n g i n (1) s h o r t e r i n c u b a t i o n t i m e s , (2) m i n i m a l d i s t u r b a n c e i n a n t i g e n - a n t i b o d y b i n d i n g d u r i n g phase s e p a r a t i o n o f f r e e and a n t i b o d y - b o u n d f r a c t i o n s o f a n t i g e n , and (3) t h e s e n s i t i v e d i s p l a c e m e n t o f t r a c e r w i t h standard preparations. A high t i t r e antiserum although not e s s e n t i a l , permits the e s t a b l i s h m e n t of a s s a y c o n d i t i o n s and a n a l y s i s o f l a r g e numbers o f specimens u s i n g t h e same r e a g e n t s . The p r o b l e m o f a n t i serum s p e c i f i c i t y - the a b i l i t y t o b i n d a s i n g l e compound i n t h e p r e s e n c e o f many o t h e r s - r e m a i n s as y e t the u n s o l v e d m y s t e r y o f RIA. S p e c i f i c i t y can be i n f l u e n c e d t h r o u g h the p o i n t o f a t t a c h m e n t o f the a n t i gen t o c a r r i e r p r o t e i n d u r i n g immunogen p r o d u c t i o n . Then, f o l l o w i n g L a n d s t e i n e r s h y p o t h e s i s ( 5 ) , t h e a n t i b o d i e s produced f o l l o w i n g i m m u n i z a t i o n w i t h such a conjugate w i l l recognize t h a t p a r t of the antigen f u r t h e s t from i t s l i n k t o t h e c a r r i e r . However, even a f t e r s t r e n u o u s e f f o r t s t o produce a c o n j u g a t e d e s i g n e d to e l i c i t s p e c i f i c a n t i b o d i e s , g e n e t i c v a r i a t i o n w i t h i n t h e groups o f a n i m a l s immunized s t i l l makes t h e result uncertain. 1

Antibody

Production

A t t e m p t s t o produce a n t i s e r a f o r use i n t h e s p e c i f i c measurement o f THC by RIA (6-9) p r o v i d e an example of the i n c o n s i s t e n c i e s i n the i n d u c t i o n of a n t i b o d i e s to s m a l l m o l e c u l e s . A t the U n i v e r s i t y o f S u r r e y s e v e r a l d i f f e r e n t T H C - p r o t e i n c o n j u g a t e s have been p r o duced u s i n g r e a c t i o n s i n v o l v i n g t h e p h e n o l i c h y d r o x y 1 group o f THC. F i g u r e 1 l i s t s t h e p r o d u c t s formed. R a b b i t s and sheep were immunized w i t h t h e s e c o n j u g a t e s . None o f t h e r a b b i t s produced a n t i b o d i e s w h i c h bound H-THC, b u t some s e r a d i d e x h i b i t b i n d i n g o f r a d i o i o d i n a t e d THC-copolymer. T h i s copolymer i s shown i n F i g u r e 2. T h i s b i n d i n g was n o t i n h i b i t e d by s t a n d a r d THC b u t c o u l d be d e c r e a s e d w i t h u n l a b e l e d THC-polymer. 3

Vinson; Cannabinoid Analysis in Physiological Fluids ACS Symposium Series; American Chemical Society: Washington, DC, 1979.

TEALE E T AL.

Antisera in Radioimmunoassay

157


10.

Figure 1. Conjugation reactions used in the production of THC-bovine serum albumin conjugates. Ptn is the symbol for the protein.

I t would seem t h a t r a b b i t a n t i s e r a c o n t a i n e d a n t i b o d i e s c a p a b l e o f r e c o g n i z i n g o n l y THC d e r i v a t i z e d at the hydroxyl. The same c o n j u g a t e s , when i n j e c t e d i n t o sheep,



158

CANNABINOID ANALYSIS IN PHYSIOLOGICAL FLUIDS

THC-hemi succ inate-GLAT

Figure 2.

The hemisuccinate derivative of THC conjugated to a synthetic polymer (MW 30,000) of GLAT in the ratio 36:24:35:5

J

d i d produce a n t i b o d i e s c a p a b l e o f b i n d i n g H-THC. T a b l e 1 shows t h e r e s p o n s e t o each immunogen. Of t h e 7 a n i m a l s r e s p o n d i n g , o n l y 2 produced a n t i s e r a o f s u f f i c i e n t t i t r e f o r p r a c t i c a l RIA u s e . On t h e b a s i s o f t h e s e r e s u l t s , T H C - p r o t e i n c o n j u g a t e s would appear t o be o f low i m m u n o g e n i c i t y and o t h e r r e s e a r c h g r o u p s , u s i n g s i m i l a r methods, have r e p o r t e d t h e same c o n c l u sions . One sheep, immunized by i n t r a m u s c u l a r i n j e c t i o n of T H C - h e m i s u c c i n a t e - a l b u m i n c o n j u g a t e , produced o u r a s s a y a n t i s e r a . F i g u r e 3 shows t h e v a r i a t i o n i n a n t i body t i t r e d u r i n g t h e i m m u n i z a t i o n s c h e d u l e . U s i n g s e r a c o l l e c t e d a t peaks i n t i t r e , a RIA system has been d e v e l o p e d . The c u r r e n t a s s a y i s s e n s i t i v e t o 50 p i c o g r a m pure THC, c o r r e s p o n d i n g t o 1.5 ng/ml i n plasma and 1 ng/ml i n u r i n e . F i g u r e 4 shows a t y p i c a l s t a n d a r d c u r v e c o r r e l a t i n g t h e p e r c e n t a g e o f 3n-THC a n t i b o d y - b o u n d w i t h t h e p r e s e n c e o f i n c r e a s i n g amounts of pure THC. A s s a y methodology has been p u b l i s h e d i n d e t a i l (10). A l t h o u g h r a i s e d a g a i n s t THC, t h e a s s a y a n t i s e r u m i s n o t s p e c i f i c f o r t h a t compound, b u t b i n d s s e v e r a l c a n n a b i n o i d s t o v a r y i n g d e g r e e s . An example o f t h e u n p r e d i c t a b l e n a t u r e o f a n t i s e r u m s p e c i f i c i t y i s demon-


10.

TEALE ET AL.

TABLE Conjugate


159


1

Immunogenicity

in

Sheep

THC D e r i v a t i v e C o n j u g a t e d t o BSA

Number Responding Number Immunized

Chlorocarbonate (1C l i n k t h r o u g h h y d r o x y l ) Hemisuccinate (4C l i n k t h r o u g h h y d r o x y l ) Hemiadipate (6C l i n k t h r o u g h h y d r o x y l ) Hemisebacate (10C l i n k t h r o u g h h y d r o x y l ) Azo-PABA (Phenyl l i n k para t o hydroxyl) Mannich (1C l i n k p a r a t o h y d r o x y l ) Acid (1C l i n k p a r a t o h y d r o x y l ) Valerate (5C l i n k p a r a t o h y d r o x y l )

2/3 4/10 1/3 0/5 0/5 0/1 0/2 0/2

Total

7/29 (24%)

s t r a t e d by T a b l e s 2 and 3. T a b l e 2 shows a c o m p a r i s o n of t h e c r o s s - r e a c t i v i t y p a t t e r n s o f two a n t i s e r a . A n t i s e r u m 133Y/30/9 was produced by us and R41/11 by P r o f . M. C a i s , Department o f C h e m i s t r y , T e c h h i o n I n s t i t u t e o f Technology, H a i f a , I s r a e l . R41/11 was r a i s e d i n a r a b b i t against A - T H C - l l - o i c a c i d conjugated t o BSA and i s o f h i g h t i t r e . (This demonstrates t h a t i t i s p o s s i b l e t o produce THC a n t i b o d i e s r e l a t i v e l y e a s i l y and i n s m a l l e r a n i m a l s p e c i e s ) . However, i n s p i t e o f the use o f d i f f e r e n t conjugates g i v e n t o d i f f e r e n t animal species, the c r o s s - r e a c t i v i t y patterns are a l m o s t i d e n t i c a l . Plasma samples from a c a n c e r p a t i e n t under t h e r a p e u t i c THC t r e a t m e n t , were a s s a y e d w i t h t h e two a n t i s e r a and s i m i l a r r e s u l t s were obt a i n e d (Table 3 ) . Assay o f u r i n e specimens from t h e same p a t i e n t , however, i n d i c a t e d l o w e r l e v e l s (approxi m a t e l y h a l f ) w i t h R41/11. From t h e r e s u l t s o f t h e d i f f e r e n t r e s e a r c h groups 8



1:500

1 : 1000

1:1500

TITRE

h

5

1

2

WEEKS

2

2

mg CONJUGATE INJECTED

2

2

Φ1

3

SHEEP S133Y

2

I

Figure 3. Immunization schedule and antibody titre in a sheep injected intramuscularly with THC-hemisuccinate-BSA in the amounts shown. Titre is defined as the reciprocal of the dilu tion of antiserum that bound 50% of the H-THC hbel.

2.5

ANT I-THC PRODUCTION


•
r *ι r

ο ο

Ο

C/5

Χ

Hi

> >

S ο

w

>

>

10.

TEALE ET AL.


161

ASSAY STANDARD CURVE FOR THC

Antiserum final d i l u t i o n : 1:2^00 Label: 260 pg H-THC Incubation medium: 0.1M phosphate buffer, pH 7.k. + 0,2% bovine γ - g l o b u l i n ,


3

I

« .

0 50 100

ι

200

I

500

Amount of THC (pg) added to assay tube

Figure 4. Standard curve constructed using the conditions shown. Displacement of H-THC from antibody binding sites by increasing amounts of THC in assay buffer is measured. 3

t h r o u g h o u t t h e w o r l d , t h e r e f o r e , i n f o r m a t i o n has a c c u m u l a t e d on t h e immunogenic p r o p e r t i e s o f v a r i o u s THCp r o t e i n c o n j u g a t e s . W h i l e a n t i b o d y p r o d u c t i o n t o THC can now be v i r t u a l l y g u a r e n t e e d , t h e s p e c i f i c i t y o f t h e p r o d u c t a l m o s t c e r t a i n l y w i l l n o t be r e s t r i c t e d t o THC.


162



A p p l i c a t i o n s o f RIA A l t h o u g h o u r RIA system i s n o t T H C - s p e c i f i c , i t i s s p e c i f i c f o r t h e 3 - r i n g e d c a n n a b i n o i d n u c l e u s and c a n be used as a s e n s i t i v e and r a p i d c a n n a b i n o i d d e t e c t i o n system. T h i s RIA system has been used f o r the measurement o f THC c r o s s - r e a c t i n g c a n n a b i n o i d (THC-CRC) l e v e l s i n 1002 u r i n e specimens c o l l e c t e d from v a r i o u s s o u r c e s . F o r t h i s purpose t h e c r o s s - r e a c t i v i t y w i t h m e t a b o l i t e s i s b e n e f i c i a l s i n c e THC i s n o t e x c r e t e d unchanged ( 1 1 ) . F i g u r e 5 shows the number o f samples from each s o u r c e and t h e p e r c e n t a g e p o s i t i v e i n each group. The 82 cont r o l specimens were t a k e n from h o s p i t a l i n - p a t i e n t s f o r r o u t i n e b i o c h e m i c a l a n a l y s e s . The 740 samples r e c e i v e d f o r r o u t i n e drug a n a l y s i s a t a l o c a l h o s p i t a l d u r i n g the p e r i o d s December 1974 t o F e b r u a r y 1975 and May 1975 and J u l y 1975 were s c r e e n e d f o r THC-CRC. These samples o r i g i n a t e d from two h o s p i t a l a d d i c t i o n t r e a t m e n t c l i n i c s (A and B) and two i n d e p e n d e n t t r e a t m e n t c e n t e r s (C and h o s t e l ) . A l s o i n c l u d e d were m i s c e l l a n e o u s specimens from o t h e r s o u r c e s , such as g e n e r a l m e d i c a l d e p a r t m e n t s , p s y c h i a t r i c c l i n i c s and g e n e r a l p r a c t i t i o n e r s ' s u r g e r i e s . I n a d d i t i o n 172 specimens t a k e n from new d e t a i n e e s a t a j u v e n i l e d e t e n t i o n c e n t e r ( s u b j e c t s a r r i v i n g d i r e c t l y from home). TABLE 2 Cross-reactivities of pure cannabinoids sera raised against different THC-protein in different species. Antiserum

with

133Y/30/9

Species

two anticonjugates

RM/11

Sheep

Rabbit 0

(Ptn)NH-CO-(CHj Immunogen

Final titre

1:2400

Cannabinoid

1:18000 % cross-reactivity

A -THC 9

11-hydroxy-A -THC 9

8,11-dihydroxy-A -THC 9

11-carbomethoxy-A -THC 8

100

100

160

100

150

200

46

1l-carboxy-A -THC methyl ether 8

Cannabi nol

66

11-hydroxy-cannabi nol

6.3

3.2

Cannabidiol

3.2

2.0


10.

TEALE ET AL.

163

Antisera in Radioimmunoassay TABLE

3

THC-CRC levels in plasma and urine specimens from a cancer patient undergoing therapeutic treatment with TEC. Levels were measured using the two antisera described in Table 2.

THC-CRC

Plasma samples


133Y/30/9 1 2 3 4 5 6 7 8

(ng/ml) R41/11

0 176 204 320 220 296 164 160

0 192 196 316 228 304 174 188

96 492 472

56 240 304

U r i n e samples 1 2 3

F i g u r e 5 shows t h a t none o f t h e c o n t r o l specimens c o n t a i n e d c a n n a b i n o i d s . A p p r o x i m a t e l y 30% o f s p e c i mens from h o s p i t a l t r e a t m e n t c l i n i c s were p o s i t i v e f o r THC-CRC, w h i l e 50-60% o f t h e samples from i n d e p e n d e n t c e n t e r s were p o s i t i v e . The i n c i d e n c e o f p o s i t i v e specimens i n t h e m i s c e l l a n e o u s group was l o w e r ( 1 3 % ) . Only 3 o f t h e 172 d e t e n t i o n c e n t e r samples c o n t a i n e d THC-CRC and each inmate a d m i t t e d s u b s e q u e n t l y t o havi n g smoked c a n n a b i s i n t h e 48 h o u r s p r i o r t o h i s a r r i v a l a t the detention center. The 740 specimens r e c e i v e d f o r r o u t i n e d r u g a n a l y s i s were d i v i d e d i n t o groups a c c o r d i n g t o t h e l e v e l o f THC-CRC d e t e c t e d . F i g u r e 6 shows t h e d a t a p a t t e r n . V a l u e s o f 10 yg/1 o r l e s s were c l a s s e d as n e g a t i v e . 35% o f t h e specimens were p o s i t i v e and h a l f o f t h e s e contained very high l e v e l s . The a s s a y was a l s o used f o r t h e measurement o f THC-CRC l e v e l s i n plasma and 24-hour u r i n e specimens c o l l e c t e d f o l l o w i n g t h e smoking o f a c i g a r e t t e i m p r e g n a t e d w i t h 5 mg p u r e THC by each o f 4 v o l u n t e e r s ( 1 2 ) . F i g u r e 7 shows t h e plasma l e v e l s d e t e c t e d i n each o f the v o l u n t e e r s . As a c o m p a r i s o n t h e plasma THC l e v e l i n a car driver f a t a l l y injured i n a t r a f f i c accident (13) was measured. The d r i v e r ' s plasma l e v e l o f


164


350 y g / l c a n be compared w i t h a peak v a l u e o f 70 y g / l i n one v o l u n t e e r i m m e d i a t e l y a f t e r smoking when sub j e c t i v e f e e l i n g s o f m i l d e u p h o r i a were r e p o r t e d . Fig u r e 8 shows t h e u r i n a r y THC-CRC l e v e l s i n t h e v o l u n t e e r s . The d r i v e r was found t o have a u r i n a r y l e v e l o f 1215 y g / l . % positive


0

0

29

hospital clinic in-patients A

33

clinic Β

66

52

clinic C

hostel

33

15

hospital others psychiatric wa rd

2

detention centre

Figure 5. Proportion of urine samples positive for THC-CRC according to the origin of the sample. Hatched areas indicate positive THC-CRC specimens.

The s c r e e n i n g o f p o s t mortem specimens from o t h e r f a t a l l y i n j u r e d d r i v e r s , f o l l o w i n g the o r i g i n a l case, was u n d e r t a k e n w i t h t h e c o o p e r a t i o n o f c o r o n e r s and p a t h o l o g i s t s t h r o u g h o u t E n g l a n d and Wales. Between J u l y 1976 and December 1976 b l o o d samples from 50 c a s e s were r e c e i v e d , 40 i n v o l v i n g c a r d r i v e r s and 10 motor cyclists. The upper p a r t o f F i g u r e 9 shows t h e s p e c i mens grouped a c c o r d i n g t o t h e v i c t i m ' s age, and i n t h e


10.

TEALE ET

AL.


479

37

165

112

71


50

25

0-10

10-20 Urinary

Figure 6.

20-50

THC-CRC

50-100

>100

(μς/1)

THC-CRC in 740 urine specimens received for routine drug analysis from patients known or suspected to be taking drugs

l o w e r p a r t o f t h e d i a g r a m t h e specimens have been grouped a c c o r d i n g t o t h e r e g i o n a l s o u r c e . I t w i l l be n o t e d t h a t 70% o f t h e specimens came from s u b j e c t s un der 30 y e a r s o f age and t h a t n e a r l y h a l f came from one a r e a o f E n g l a n d . The samples were r e c e i v e d from a t o t a l o f 24 d i f f e r e n t p a t h o l o g i s t s . The THC-CRC l e v e l s i n t h e 5 p o s i t i v e c a s e s (2 o f them b e i n g m o t o r c y c l i s t s ) f e l l w i t h i n a moderate 25-65 y g / l r a n g e , w h i c h c o u l d n o t be c o n s i d e r e d t o have e x e r t e d t h e same d e g r e e o f i n t o x i c a t i o n and, t h e r e f o r e , i n f l u e n c e on d r i v i n g c a p a b i l i t y as t h e v e r y h i g h l e v e l s o b s e r v e d i n t h e o r i g i n a l c a s e . The group o f specimens a n a l y z e d c a n n o t be c o n s i d e r e d r e p r e s e n t a t i v e f o r s e v e r a l r e a s o n s , be cause o n l y 7 c a s e s were r e c e i v e d from t h e most h i g h l y p o p u l a t e d a r e a - t h e South E a s t o f E n g l a n d . However, the s t u d y c o n s t i t u t e s an example o f RIA use i n epidemiological studies.




166

Figure 7. Plasma THC-CRC in four healthy volunteers who began smoking a cigarette impregnated with 5 mg THC at zero time. Subjects 1 and 2 were cigarette smokers; subject 3, a cigar smoker; and subject 4, a pipe smoker. The level detected in a post morten specimen from a car driver was 350 pg/L.



10.

T E A L E ET

AL.

0-24

Figure 8.

167


hr

24-48 hr

48 hr

+

Urinary THC-CRC in the same subjects described in Figure 7

H i g h P r e s s u r e L i q u i d Chromatography

(HPLC) and

RIA

Work has r e c e n t l y been u n d e r t a k e n a t t h e Home O f f i c e C e n t r a l R e s e a r c h E s t a b l i s h m e n t a t A l d e r m a s t o n on a p r o c e d u r e f o r t h e s p e c i f i c measurement o f THC and i t s m e t a b o l i t e s . F o l l o w i n g an i n i t i a l HPLC s e p a r a t i o n s t a g e , t h e RIA i s used as a s e n s i t i v e q u a n t a t a t i v e method f o r s c r e e n i n g column e l u a n t s . HPLC s e p a r a t i o n was p e r f o r m e d on a r e v e r s e phase column system - h i g h l y p o l a r compounds b e i n g e l u t e d f i r s t f o l l o w e d by substances o f d e c r e a s i n g p o l a r i t y . Methanol e x t r a c t s o f plasma o r h y d r o l y z e d u r i n e samples were chromatographed. F i g u r e 10 shows chromatograms o f a sample o f u r i n e c o l l e c t e d from a r a b b i t f o l l o w i n g an i n t r a v e n o u s dose o f 100 yg C-THC. The u r i n e was s u b j e c t e d t o 14


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CANNABINOID ANALYSIS IN PHYSIOLOGICAL

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20


10

YÏ/M/M

15-19 20-24 25-29 30-34 35-39 40-44 45-49

50+

Age group 30L

J

20

SE

YZZZZZw sw

NW

NE

UK area

Figure 9. Post mortem blood specimens from 50 drivers, received during July 1976-December 1976 and 24 centers. (Upper) Specimens grouped according to the victims age. (Lower) Specimens grouped according to regional source. (SE = South East England; SW = South West; M = Midhnds; NW = North West; NE = North East; W = Wales). Hatched areas represent specimens positive for THC-CRC.

a l k a l i n e h y d r o l y s i s before separation since cannabinoid compounds from u n t r e a t e d u r i n e were e l u t e d i n a s i n g l e peak c o n c u r r e n t l y w i t h h i g h l y p o l a r m a t e r i a l . I n t h e d i a g r a m , t h e p l a i n l i n e r e p r e s e n t s t h e amount o f r a d i o a c t i v i t y i n t h e column e l u e n t due t o C - l a b e l e d canna1 4



10.

T E A L E E T AL.

169


20

40

60

Elution volume (ml)

Figure 10. Radiochromatogram and radioimmunochromatogram of THC metabolites in hydrolyzed rabbit urine. Urine was collected following an iv dose of 100 ixg C-THC. Fractions collected from HPLC separation of hydrolyzed urine were screened for C radioactivity (plain lines) and THC-CRC (hatched peaks). The elution volume for pure CBN is indicated. 14

14

b i n o i d s . The h a t c h e d peaks r e p r e s e n t THC-CRC l e v e l s measured by t h e RIA. The l a s t peak o f c a n n a b i n o i d s e l u t e d c o i n c i d e d w i t h t h e e l u t i o n volume o f p u r e cannabinol. THC and c r o s s - r e a c t i n g m e t a b o l i t e s were measured i n e x t r a c t s o f plasma samples t a k e n from a v o l u n t e e r a t t i m e d i n t e r v a l s a f t e r smoking 10 mg p u r e THC. F i g ure 11 shows t h e peaks o f THC-CRC measured i n plasma samples t a k e n b e f o r e smoking and a t 2, 12, 24 and 34 m i n u t e s a f t e r smoking. The c o n t r o l plasma specimen i s seen t o c o n t a i n r e s i d u a l c a n n a b i n o i d s o f h i g h p o l a r i t y and t h e s e a r e p r o b a b l y due t o t h e p r e v i o u s use o f c a n n a b i s by t h e v o l u n t e e r w h i c h was o n l y r e v e a l e d a f t e r t h e r e s u l t s shown were o b t a i n e d . I n t h e samples t a k e n a f t e r smoking, THC c a n be seen t o d e c r e a s e p r o g r e s s i v e l y and m e t a b o l i t e s o f v a r i o u s p o l a r i t i e s i n c r e a s e and d e c r e a s e o v e r t h e time o f s a m p l i n g . The peak o f h i g h p o l a r i t y compounds remained r e l a t i v e l y


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CANNABINOID ANALYSIS IN PHYSIOLOGICAL

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constant. A l s o i n c l u d e d i n the diagram are the e l u t i o n p o i n t s o f samples o f t h e few r e f e r e n c e compounds a v a i l a b l e i n a p u r i f i e d form. The e l u t i o n volumes i n each c a s e were u n c o r r e c t e d f o r t h e volume o f sample a p p l i e d t o t h e column. T h e r e f o r e t h e f i r s t peaks e l u t e d show v a r i a t i o n i n e l u t i o n volume.

8.11-Oi-OM-THC

Φ THC-n-OIC ACM) ^

C8N^

M,

R

10

34 minutes

20 Elution

30

40

50

60

vo 1 unie ( m l )

Figure 11. Radioimmunochromatograms of plasma samples taken from a volun teer before, and 2,12, 24, and 34 min after smoking 10 mg THC. Sample extracts of 1.5-6.0 mL were injected and the elution volumes are uncorrected.

A u r i n e specimen, c o l l e c t e d from t h e same v o l u n t e e r 60 m i n u t e s a f t e r smoking, was s u b j e c t e d t o a l k a -


10.

T E A L E ET

AL.


171

l i n e h y d r o l y s i s . The h y d r o l y s a t e was chromatographed under d i f f e r e n t column c o n d i t i o n s t o t h o s e used f o r t h e plasma a n a l y s e s . F i g u r e 12 shows the r e s u l t a n t r a d i o immunochromatogram w i t h m a i n l y 2 peaks o f THC-CRC, t h e second peak b e i n g e l u t e d a t t h e e l u t i o n volume o f pure c a n n a b i n o l .


6r

Figure 12. Radioimmunochromatogram of a urine sample taken from a volunteer 60 min after smoking 10 mg THC. The urine was hydrolyzed before HPLC separation. The elution volume for CBN was 29 mL.

The n a t u r e o f most o f t h e compounds s e p a r a t e d from t h e s e samples i s unknown and t h i s emphasizes the advantage o f a b r o a d s p e c i f i c i t y a n t i s e r u m i n t h e det e c t i o n o f u r i n a r y THC m e t a b o l i t e s . I n d e e d , none o f t h e m e t a b o l i t e s (11-hydroxy-THC, 8,11-dihydroxy-THC and T H C - l l - o i c a c i d ) known t o c r o s s - r e a c t w i t h t h e RIA a n t i s e r u m , a r e seen t o be p r e s e n t i n t h e h y d r o l y z e d u r i n e sample ( F i g u r e 1 2 ) . These compounds as shown i n F i g u r e 11 have e l u t i o n volumes g r e a t e r t h a n 40 m l . On t h e b a s i s o f t h e s e r e s u l t s an RIA s p e c i f i c f o r t h e s e compounds would n o t have found any d e t e c t a b l e m a t e r i a l i n t h e u r i n e . To d e v e l o p the c o m b i n a t i o n o f HPLC and RIA f u r t h e r , an a n t i s e r u m e x h i b i t i n g c o m p l e t e c r o s s r e a c t i v i t y w i t h a l l m e t a b o l i t e s would be o f g r e a t e s t


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ANALYSIS

IN PHYSIOLOGICAL

FLUIDS


benefit. I n summary, RIA has two a p p l i c a t i o n s i n t h e s c r e e n i n g o f plasma and u r i n e specimens f o r c a n n a b i s use. D i r e c t a n a l y s i s o f a sample g i v e s an i n d i c a t i o n o f t h e p r e s e n c e o f c a n n a b i n o i d s on a s e m i - q u a n t i t a t i v e basis. Q u a n t i t a t i o n may t h e n be expanded f u r t h e r and more s p e c i f i c a l l y f o l l o w i n g HPLC s e p a r a t i o n . The i d e n t i f i c a t i o n o f THC and i t s m e t a b o l i t e s i n e s t a b l i s h e d p a t t e r n s c o u l d form the b a s i s f o r an e s t i m a t e o f b o t h t h e q u a n t i t y o f THC absorbed and t h e time o f intake. ACKNOWLEDGEMENTS We a r e g r a t e f u l t o t h e Cancer R e s e a r c h Campaign f o r f i n a n c i n g t h i s r e s e a r c h . We a l s o thank NIDA and P r o f e s s o r R. Mechoulam f o r pure c a n n a b i n o i d s , P r o f e s s o r M. C a i s f o r a sample o f h i s a n t i s e r u m , and P r o f e s s o r J . W. Thompson f o r plasma and u r i n e samples from THC smokers. Anti-THC a n t i s e r u m i s a v a i l a b l e from G u i l d h a y , Department o f B i o c h e m i s t r y , U n i v e r s i t y o f S u r r e y , G u i l d f o r d , S u r r e y , UK. REFERENCES (1)

Y a l o w , R . S . and B e r s o n , S . A., J. Clin. Invest. 39, 1157 (1960). (2) B u t l e r , V . P., Adv. Immunol. 17, 255 ( 1 9 7 3 ) . (3) L a n d o n , J. and M o f f a t , A . C., The Analyst 101, 225 (1976). (4) R o b i n s o n , J. D., et. al. "Radioimmunoassay in Clinical. B i o c h e m i s t r y " , E d . , C . P a s t e r n a k , London Press, Heyden, 1 975, p . 1 1 3 . (5) L a n d s t e i n e r , K., "The Specificity of Serological R e a c t i o n s " , Dover P ress, New Y o r k , 1 9 6 2 . (6) G r o s s , S . J., S o a r e s , J. R., Wong, S - L . R . and S c h u s t e r , R . E., Nature 252, 581 ( 1 9 7 4 ) . (7) T s u i , P . T., Kelly, Κ. Α., Ponpipom, M . M., Strahilevitz, M . and Sehon, A . H., Can. J. Bio chem. 52, 252 ( 1 9 7 4 ) . (8) Van V u n a k i s , H . and Levine, L., "Immunoassays f o r Drugs S u b j e c t t o A b u s e " , S . J. M u l e , I . S u n s h i n e , M . Braude and R . E. Willette, Eds., CRC P r e s s , C l e v e l a n d , O h i o , 1974, p . 2 3 . (9) C a i s , M., D a n i , S., J o s e p h y , Y., M o d i a n o , Α., G e r s h o n , Η. and Mechoulam, R., FEBS Letters 55, 257 (1975). (10) T e a l e , J. D., Forman, E . J., K i n g , L. J., Piall. Ε . M. and M a r k s , V., J. Pharm. Pharmac. 27, 465 (1975).


10. TEALE ET AL.

(11)

(12) (13)

Hollister L. and G r e e n , Pharmac. Teale, J. Marks, V., Teale, J. (1976). r


E., Kanter, S . L. Board, R. D. D . E., Res. Commun. Chem. Path. 8, 579 (1974). D., F o r m a n , E. J., King, L. J. a n d Lancet, ii, 553 (1974). D . a n d Marks, V., Lancet, i, 884


RECEIVED December 12, 1978.


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Antisera Raised Against Tetrahydrocannabinol in the

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