Hidden Xenobiotic Conjugates - ACS Symposium Series (ACS

12 Hidden Xenobiotic Conjugates 1

H. Wyman Dorough and John D. Webb

Downloaded by UNIV OF CALIFORNIA SANTA BARBARA on May 26, 2018 | https://pubs.acs.org Publication Date: January 24, 1986 | doi: 10.1021/bk-1986-0299.ch012

Graduate Center for Toxicology, University of Kentucky, Lexington,KY40546 With few exceptions, diagrammatic presentations of metabolic pathways for xenobiotics include a number of structures that are enclosed within brackets. This i s the means by which the metabolism chemist designates a proposed intermediate, or hidden metabolite, in that particular pathway. Often, the proposed intermediate is considered so transient in nature that i t s i s o l a t i o n would be impractical, if not impossible, and i t s reac t i v i t y so great that i t s occurrence as a terminal metabolite would be v i r t u a l l y inconceivable. When secondary metabolic processes are involved, these tran sient metabolites are referred to as hidden conjugates. These intermediate, transitory metabolites have their own chemical and toxicological properties and i t i s reasonable to assume that they may influence, at least in part, the toxic action of the parent xenobiotic.

One of the most intriguing facets associated with the study of xenobiotic metabolism i s the occasional discovery of metabolites whose pathways of formation defy l o g i c a l explanation. One of the least intriguing i s the report by a colleague that a major metabo l i t e was overlooked in one's own study. Each of these situations may arise from what might be termed "hidden" metabolites. These hidden metabolites are common to both primary and secondary b i o transformation processes, but this paper deals solely with the l a t t e r where the metabolite i s referred to as a hidden conjugate. Perhaps the most common reason for the existence of hidden conjugates, especially i n the past, i s the f a i l u r e of the a n a l y t i c a l methods employed to detect total xenobiotic residues present i n a sample. This problem has been solved to a great extent by the use of radiotracer techniques i n xenobiotic investigations. Once i t s existence is established, the a n a l y t i c a l chemist can usually isolate and identify the metabolite. This, however, does not mean that 1Current address: Department of Biological Sciences, Mississippi State University, Mississippi State, M S 39762 0097-6156/86/0299-0242S06.75/0 © 1986 American Chemical Society

Paulson et al.; Xenobiotic Conjugation Chemistry ACS Symposium Series; American Chemical Society: Washington, DC, 1986.


12. DOROUGH AND WEBB

Hidden Xenobiotic Conjugates

243

hidden conjugates did not occur i n vivo during the formation of the newly i d e n t i f i e d tissue residue or excretory product. One must recognize that conjugates are not always resistant to further meta bolism and that products recovered from the excreta do not represent each step in a metabolic pathway. It i s quite possible that the endocon moiety of a conjugate i s metabolized, sometimes extensively, before being voided from the body. The intermediates leading to the form excreted are hidden conjugates. The same i s true when a conjugate formed i n vivo i s subsequently deconjugated and the exocon conjugated i n a d i f f e r e n t manner before elimination from the body occurs. Hidden conjugates may also result from the use of animal model systems and incorrectly assuming that the model i s v a l i d for humans. Species d i f f e r i n their metabolic c a p a b i l i t i e s and extreme caution must be exercised i n extrapolating animal data to humans, or for that matter, from any one species to another. Just because a p a r t i cular conjugate i s not formed i n an animal model does not mean i t i s not formed i n humans. Too often, however, metabolism studies in humans, especially involving nontherapeutic chemicals, are limited or lacking completely and hidden conjugates (those formed s p e c i f i c a l l y in humans) may remain as such i n d e f i n i t e l y . F i n a l l y , hidden conjugates may occur simply because of chemical i n s t a b i l i t y during sample storage and handling. Failure to consider conjugate degradation i n a sample (tissue extract, b i l e , urine, etc.) and to take steps to prevent such degradation from occurring may cause the analyst to miss i t s presence or to identify the degradation products as being derived from i n vivo biotransformation. I t may well be that some of the very bizarre metabolic pathways proposed in the l i t e r a t u r e stem from attempts to explain from a biochemical viewpoint the formation of certain degradation products. While there may be additional causes for the existence of hidden conjugates, those mentioned above w i l l be addressed i n this paper. What we do not know can hurt us, and the author i s well aware that the most important hidden conjugates possibly have yet to be postulated. Analytical Problems When conjugates are present in a sample extract but are insen s i t i v e to the a n a l y t i c a l methods being u t i l i z e d , they are indeed hidden from the analyst. Such was the case in one of the f i r s t s c i e n t i f i c projects in which the present author participated. The study dealt with the s t a b i l i t y and recovery of insecticides from milk (1) and one of the compounds was the carbamate i n s e c t i cide carbaryl. These studies were being conducted i n 1959 just after the introduction of this material which was touted insect i c i d a l l y as the new DDT without the persistence problems. Interest i n the s t a b i l i t y and recovery of carbaryl from milk was generated by reports, some of which were later published (2), that carbaryl i n the diet of dairy cows at levels i n excess of 400 ppm for 2 weeks did not result i n carbaryl residues in the milk. These findings were based on a colorimetric method for 1-naphthol because at the time hydrolysis was the assumed sole pathway for carbaryl metabolism i n mammals (Figure 1). 1-Naphthol was measured in samples before and after alkaline treatment to hydrolyze carbaryl



244

XENOBIOTIC CONJUGATION CHEMISTRY

to 1-naphthol and the d i f f e r e n c e was taken as the c o n c e n t r a t i o n of c a r b a r y l per s e . Our s t u d i e s demonstrated t h a t the l a c k of r e p o r t e d r e s i d u e s was not a f u n c t i o n o f c a r b a r y l i n s t a b i l i t y and r e c o v e r y , and added s u p p o r t to the r e p o r t s t h a t c a r b a r y l r e s i d u e s were not p r e s e n t i n the m i l k of cows f e d the i n s e c t i c i d e i n the d i e t . S e v e r a l y e a r s l a t e r , the problem was a g a i n a d d r e s s e d u s i n g c a r b a r y l r a d i o l a b e l e d w i t h carbon-14 and l i q u i d s c i n t i l l a t i o n c o u n t ing as the method o f r e s i d u e d e t e c t i o n (3). The r e s u l t s o b t a i n e d ( F i g u r e 2) were t y p i c a l o f those o b s e r v e d f o r hundreds o f c h e m i c a l s a f t e r carbon-14 a n a l y s i s by s c i n t i l l a t i o n c o u n t i n g became commonly used i n l a b o r a t o r i e s around the w o r l d . R a d i o a s s a y o f the whole m i l k sampled 6 hr a f t e r treatment (3 mg/kg, o r a l ) showed t h a t 950 ppb **C-carbaryl e q u i v a l e n t s were p r e s e n t ( F i g u r e 2 ) . The c o l o r i m e t r i c method f o r c a r b a r y l used i n the p r e v i o u s s t u d i e s (2) i n d i c a t ed t h a t o n l y 3% of the t o t a l r a d i o c a r b o n was c a r b a r y l or o t h e r meta b o l i t e s which responded to the f l u o b o r a t e chromogenic r e a g e n t . This study and many o t h e r s made i t c l e a r t h a t the carbamate i n s e c t i c i d e s are e x t e n s i v e l y m e t a b o l i z e d by o x i d a t i v e and c o n j u g a t i o n mechanisms. Numerous h i d d e n m e t a b o l i t e s would s t i l l be p r e s e n t i f the a n a l y t i c a l m e t h o d o l o g i e s were l i m i t e d to d e t e c t i o n o f the p a r e n t m o l e c u l e . The use o f r a d i o i s o t o p e s i n x e n o b i o t i c m e t a b o l i s m s t u d i e s has l a r g e l y e l i m i n a t e d the problem o f c e r t a i n m e t a b o l i t e s t o t a l l y e s c a p i n g d e t e c t i o n . A word o f c a u t i o n , however, i s n e c e s s a r y i f t o t a l a c c o u n t a b i l i t y i s to be a c h i e v e d . Most p r o t o c o l s c a l l f o r the use of a r a d i o a c t i v e compound h a v i n g the l a b e l e d atom a t a n o n - l a b i l e s i t e on the m o l e c u l e . The advantages o f t h i s approach i s a p p a r e n t , but t h e r e a r e some d i s a d v a n t a g e s . For example, what i f t h a t l a b i l e p o r t i o n o f the m o l e c u l e i s a r e a c t i v e i n t e r m e d i a t e t h a t r e a c t s w i t h m a c r o m o l e c u l e 8 i n the c e l l to induce n e c r o s i s , c a r c i n o g e n e s i s , or some o t h e r t o x i c e f f e c t ? One concern o f the author i n t h i s r e g a r d i s the assumed f a t e o f the carbamate moiety ( F i g u r e 1) a f t e r b e i n g c l e a v e d from the a l c o h o l / p h e n o l • I f the carbamic a c i d i s immediate ly degraded i n v i v o to carbon d i o x i d e and methylamine, t h e n , t h e r e i s no a p p a r e n t cause f o r a l a r m . U n f o r t u n a t e l y , t h i s has not been a d e q u a t e l y demonstrated u s i n g a v a r i e t y o f carbamate c h e m i c a l s . In f a c t , the o p p o s i t e i s i n d i c a t e d by s t u d i e s u s i n g c e r t a i n carbamates r a d i o l a b e l e d i n the c a r b o n y l and N-methyl p o s i t i o n s . There i s almost a1way8 r a d i o c a r b o n i n t i s s u e s which i s d e r i v e d s o l e l y from the carbamate p o r t i o n of the m o l e c u l e . This i s u s u a l l y a t t r i b u t e d to i n c o r p o r a t i o n o f r a d i o a c t i v e carbon d i o x i d e i n t o normal b i o c h e m i c a l pathways to y i e l d n a t u r a l l y o c c u r r i n g c e l l components. In some cases the l e v e l s o f such r e s i d u e s are q u i t e h i g h (4) and i t may be unwise to c o n s i d e r a l l the m e t a b o l i t e s as innocuous n a t u r a l c e l l u l a r chemicals • 1

F u r t h e r M e t a b o l i s m of

Conjugates

Conjugates o f x e n o b i o t i c s e l i m i n a t e d i n the u r i n e and f e c e s of exposed animals n o r m a l l y p r o v i d e the b a s i s upon which secondary m e t a b o l i c pathways are p r o p o s e d . In many c a s e s , the mechanism o f f o r m a t i o n o f the c o n j u g a t e i s r e a d i l y a p p a r e n t . Such i s the case w i t h p h e n o l s which are e x c r e t e d as s u l f a t e and g l u c u r o n i d e conju gates. The b i o c h e m i c a l donors are known, the enzymes are w e l l c h a r a c t e r i z e d and the s u b s t r a t e s p e c i f i c i t i e s of these enzymes are g e n e r a l l y w e l l e s t a b l i s h e d . A l t h o u g h t h i s does not r u l e out the




245

O 0-(!-NHCH

o


CO.

RAT

ORAL LD, 50 mg/Kg

RAT

610

2

ORAL

LD. 50 mg/Kg

2500

CARBARYL

NH CH

1-NAPHTHOL

Figure 1. Hydrolytic pathway of metabolism of the insecticide carbaryl. Once considered the only route of metabolism, radio tracer studies later showed that oxidation, hydrolysis and con jugation reactions resulted in over a dozen metabolites being formed by some organisms.

0-&-NHCH,

3Mg/Kg O R A L TO COW

O

28 PPB CARBARYL RESIDUES

1

950 PPB ^C-CARBARYL EQUIVALENTS

Figure 2. Hidden residues i n cow's milk as a result of the f a i l u r e of colorimetric analysis to detect t o t a l **C-carbaryl equivalents present i n sample. 1


3


246


p o s s i b i l i t y t h a t s u l f a t e and g l u c u r o n i d e c o n j u g a t e s i n the e x c r e t a were not formed by some u n u s u a l , and unknown, pathway, t h e r e i s c l e a r l y no need to s u s p e c t t h a t such i s the c a s e . I t i s when a c o n j u g a t e s does not f i t w i t h i n known pathways of b i o c h e m i c a l s y n t h e s i s t h a t the m e t a b o l i s m chemist t u r n s to h y p o t h e t i c a l i n t e r m e d i a t e s to e x p l a i n h i s / h e r f i n d i n g s . The soundness o f the h y p o t h e t i c a l pathways v a r y , but i n most i n s t a n c e s i t i s p u r e l y c r e a t i v e c h e m i s t r y w i t h a dash of l o g i c thrown i n to g i v e some sense o f l e g i t i m a c y to the proposed scheme. N o n e t h e l e s s , i t i s o f t e n these h y p o t h e t i c a l i n t e r m e d i a t e s , or unknown c o n j u g a t e s f o r purposes of t h i s p a p e r , t h a t are e v e n t u a l l y i s o l a t e d , i d e n t i f i e d , and c o n s e q u e n t l y , removed from the l i s t o f h i d d e n c o n j u g a t e s . The s o u r c e o f c o n j u g a t e s p r e v i o u s l y c o n s i d e r e d as h i d d e n has f o r the most p a r t been the b i l e . A c l a s s i c a l example i s the p r o g r e s s i v e e s t a b l i s h m e n t of the pathway l e a d i n g to the presence of 1n a p h t h y l m e r c a p t u r i c a c i d i n the u r i n e o f mammals t r e a t e d w i t h n a p h t h a l e n e ( 5 , 6)• I n i t i a l l y , i t was known o n l y t h a t 1-naphthyl m e r c a p t u r i c a c i d was the major m e t a b o l i t e o f n a p h t h a l e n e and t h a t i t was formed from an a c i d - l a b i l e p r o d u c t v o i d e d i n the u r i n e . T h i s product was e v e n t u a l l y i d e n t i f i e d as n a p h t h y l p r e m e r c a p t u r i c a c i d ( F i g u r e 3) and there i s some e v i d e n c e t h a t the N - a c e t y l c y s t e i n e s u b s t i t u e n t may be a t the number 2 p o s i t i o n o f the n a p h t h a l e n e r i n g r a t h e r than the number 1 p o s i t i o n as shown i n F i g u r e s 3 and 4 ( 7 ) . Over the y e a r s , i t s f o r m a t i o n from the g l u t a t h i o n e c o n j u g a t e o f n a p h t h a l e n e ( i s o l a t e d from the b i l e ) has been w e l l documented. As demonstrated i n F i g u r e 4, the epoxide o f n a p t h a l e n e must be gene r a t e d by the cytochrome P-450 system p r i o r to i t s r e a c t i o n w i t h g l u t a t h i o n e . While we have i s o l a t e d the i n t a c t g l u t a t h i o n e c o n j u gate from r a t b i l e and from S-15 l i v e r enzymes to which g l u t a t h i o n e was added, o n l y those p r o d u c t s marked w i t h an a s t e r i s k i n F i g u r e 4 were d e t e c t e d i n the u r i n e o f i n t a c t r a t s . Almost 60% of the Cn a p h t h a l e n e dose was v o i d e d i n the u r i n e and almost 70% of t h i s was as the p r e m e r c a p t u r i c a c i d . These i n v i t r o and i n v i v o s t u d i e s w i t h r a t s are used i n our l a b o r a t o r y as p o s i t i v e c o n t r o l s f o r g l u t a t h i o n e c o n j u g a t i o n when a s s e s s i n g s i m i l a r c o n j u g a t i o n r e a c t i o n s w i t h o t h e r substrates (8). There are many o t h e r examples of documented g l u t a t h i o n e c o n j u gates o f x e n o b i o t i c s where the f i n a l p r o o f was g a i n e d by a n a l y s i s of the b i l e . One which i n v o l v e s a compound r e c o g n i z e d by almost every one and where g l u t a t h i o n e c o n j u g a t i o n i s o f d e f i n i t e t o x i c o l o g i c a l importance i s o u t l i n e d i n F i g u r e 5. The compound i s the w i d e l y used a n a l g e s i c acetaminophen ( N - h y d r o x y a c e t a n i l i d e ) which a t h i g h doses ha8 been shown to cause l i v e r n e c r o s i s i n man, r a t s and o t h e r mam m a l s . M i t c h e l l and coworkers (9) demonstrated t h a t acetaminophen was b i o a c t i v a t e d to a r e a c t i v e i n t e r m e d i a t e ( F i g u r e 5,A) by the cytochrome P-450 system. T h i s h y p o t h e t i c a l i n t e r m e d i a t e ( N - a c e t y l p - q u i n o n e i m i n e ) was proposed as the h e p a t o t o x i n and i t was f u r t h e r proposed t h a t at low doses the i n t e r m e d i a t e was d e t o x i f i e d by r e a c t i o n w i t h g l u t a t h i o n e . While t h e r e a r e d i f f e r e n c e s of o p i n i o n as to the mechanism o f f o r m a t i o n o f the r e a c t i v e i n t e r m e d i a t e ( 1 0 ) , i t s i d e n t i t y as N - a c e t y l - p - q u i n o n e i m i n e has been c o n f i r m e d ( 1 1 ) . That the i n t e r m e d i a t e was d e t o x i f i e d by g l u t a t h i o n e c o n j u g a t i o n was sup p o r t e d by the i s o l a t i o n of a c e t a m i n o p h e n - g l u t a t h i o n e ( F i g u r e 5,B) from the b i l e o f r a t s (12). These f i n d i n g s s u p p o r t the p r o p o s a l (9) t h a t h i g h doses o f acetaminophen d e p l e t e h e p a t i c g l u t a t h i o n e and,


12.

D O R O U G H A N D WEBB

I Downloaded by UNIV OF CALIFORNIA SANTA BARBARA on May 26, 2018 | https://pubs.acs.org Publication Date: January 24, 1986 | doi: 10.1021/bk-1986-0299.ch012

247


Glycine

CO-NH-CH -COOH 2

H S - C H - C H - N H - C O - C H - C H - C H ( N H ) - C O O H 2

2

Cysteine

I

2

2

Glutamic Acid

Glutathione

C O - N H - C H - C O O H 2

S - C H - C H - N H - C O - C H C H - C H ( N H ) - C O O H 2

2

2

2

H

S - ( 1 , 2 - d i h y d r o - 2 - h y d r o x y - 1 - n a p h t h y l ) glutathione Naphthalene-GSH conjugate COOH S - C H

ι

2

COOH

- C H - N H - C O - C H

3

S - C H - C H - N H - C 0 - C H 2

H

N-acetyl-S-(1,2,-dihydro-2h y d r o x y - 1 - n a p h t h y l ) cysteine l-Naphthyl premercapturic

acid

N-acetyl-S-(l-naphthyl) cysteine l-naphthyl mercapturic acid

Figure 3. Glutathione conjugation of naphthalene in mammals is a classic example of in vivo conjugate formation (Nap-GSH) that is not evident by the nature of metabolites (mercapturic acids) recovered from the urine.


3


2

2

CO-NH-CH -COOH

EPOXIDATION

2

NH

2

2

2

-GLYCINE

HYDROLYSIS

ACIDIFIED URINE

H

DEHYDRATION

^NH,

OH

S-CH,-CN-CO0H H-CO-CH3

T*^ H

ACID

1-and/or 2QLUCURONIDE

S-CHj-CH-COOH

1-and/or 2-NAPHTHOL

8ULFATE

1-and/or 2-

O-SULFATE

1-NAPHTHYL PREMERCAPTURIC

CONJUGATION^

Figure 4. In v i t r o studies and i d e n t i f i c a t i o n of b i l i a r y meta bolites were necessary to establish the l i k e l y metabolic reac tions involved in the i n vivo conversion of naphthalene into those metabolites (see asterisks) excreted i n the urine.

1-NAPHTHYL MERCAPTURIC ACID

2

S-CH -CH-COOH NH-CO-CHo

2

S-CH -CH-CO-NH-CH -COOH

2

S-CH -CH NH-CO-CH -CH -CH(NH )-COOH

NAPHTHALENE


c/3 H 73 -

URINE 4'H0S0 0-3PBA

B

ENTEROHEPATIC

Rat)

HR

3PBA - ^ i ^ T G L U C U R O N I D E S

(3PBA)

Urine

0-24

Bile

(Intact

10 M g / K g , ip

PHENOXYBENZOIC

C-OH

4'0GLU-3PBA



260


BILE

COMPONENTS

ISORH AMNETIN Weakly

Mutagenic

Figure 14. Hidden conjugates of quercetin, a naturally-occurring mutagen, are the glucuronide and sulfate derivative of isorhamnet i n . These products are not present i n the excreta of mammals but are b i l i a r y metabolites.


DOROUGH AND WEBB


CH*

CH* Hamster


261

* Rat

Liver 3,2

-DIMETHYL-4-

AMINOBIPHENYL (DMAB)

Hydrolysis BLADDER

NITROSO-DMAB

(HAMSTER) BLADDER CANCER HAMSTER

Urine

ONLY

^HYDROXY D M A B - Nl GLUCURONIDE Bile (Rat

INTESTINAL A Hamster)

HAMSTER

CANCER AND R A T

INTESTINE £H~ Hydrolysis

ULTIMATE

CARCINOGEN

NITROSO-DMAB Figure 15. As shown here with DMAB, conjugates may serve as procarcinogens whose s i t e s of carcinogenicity d i f f e r among species because of d i s s i m i l a r routes of transport and excretion.



262


Species D i f f e r e n c e s As p o i n t e d out i n the p r e v i o u s p a r a g r a p h , s p e c i e s h a n d l e x e n o b i o t i c s d i f f e r e n t l y and t h i s may p l a y a major r o l e i n the s e l e c t i v e t o x i c i t y o f t e n observed among d i f f e r e n t a n i m a l s . Hidden c o n j u g a t e s which a r e s p e c i e s s p e c i f i c a r e o f p a r t i c u l a r importance when humans are one of the s p e c i e s b e i n g c o n s i d e r e d . Many t o x i c a n t s a r e j u s t too hazardous to t e s t i n human8 and the m e t a b o l i c pathways must be p r e d i c t e d u s i n g data o b t a i n e d w i t h v a r i o u s a n i m a l m o d e l s . I r o n i c a l l y , the p o i n t b e i n g made i s b e s t e x p l a i n e d u s i n g a c h e m i c a l commonly used as a c l i n i c a l t o o l i n human medicine ( F i g u r e 1 6 ) . D i b r o m o s u l f o p h t h a l e i n (DBSP) i s used i n humans to a s s e s s o r g a n i c a n i o n t r a n s p o r t i n the l i v e r because a n i m a l s t u d i e s u n e q u i v o c a l l y showed t h a t i s was e x c r e t e d unchanged i n the b i l e and was not m e t a b o l i z e d by the l i v e r . While t h e r e a r e s e v e r a l c l i n i c a l advan tages o f t e s t c h e m i c a l s h a v i n g these c h a r a c t e r i s t i c s , a very o b v i o u s one i s t h a t the compound s h o u l d be u s e f u l i n d i s t i n g u i s h i n g f a c t o r s a f f e c t i n g h e p a t i c t r a n s p o r t from those i n t e r f e r i n g w i t h c o n j u g a t i o n . Based on the f i n d i n g s o f M e i j e r e t a l . ( 2 6 ) , DBSP can no l o n g e r be c o n s i d e r e d as e x h i b i t i n g i n humans those c h a r a c t e r i s t i c s j u s t de s c r i b e d f o r r a t s and o t h e r t e s t a n i m a l s . T h e i r r e s u l t s show t h a t i n humans about 25% of the b i l i a r y "DBSP" i s a c t u a l l y a m e t a b o l i t e . The i d e n t i t y has not been c o n f i r m e d , but t h e r e i s s t r o n g e v i d e n c e that the p r o d u c t i s a g l u t a t h i o n e c o n j u g a t e o f DBSP. I f a n i m a l data are to be used as the b a s i s f o r p r e d i c t i n g x e n o b i o t i c m e t a b o l i s m , or l a c k t h e r e o f , when unwarranted by t o x i c i t y c o n s i d e r a t i o n s , and l a t e r i t i s d i s c o v e r e d t h a t humans d i f f e r s i g n i f i c a n t l y from the a n i m a l model, what must be the s i t u a t i o n w i t h the hundreds o f t o x i c a n t s tested i n animals only? There i s no p e r f e c t system f o r e l i m i n a t i n g the p o s s i b i l i t y o f h i d d e n c o n j u g a t e s i n man as a r e s u l t o f h a v i n g to r e l y upon a n i m a l e x p e r i m e n t a t i o n . However, the p o t e n t i a l f o r such s h o u l d be kept i n mind and human data g e n e r a t e d when p o s s i b l e ( c a s e s o f p o i s o n i n g , e t c ) so t h a t a b e t t e r d a t a base w i l l e v e n t u a l l y be established • Conjugate

A l t e r a t i o n D u r i n g S t o r a g e and

Handling

Of a l l the p o t e n t i a l causes o f h i d d e n c o n j u g a t e s , p r o b a b l y none i s as common as c h e m i c a l changes which o c c u r d u r i n g s a m p l i n g , s t o r a g e and a n a l y s i s . Spontaneous c l e a v a g e o f t e n o c c u r s w i t h s u l f a t e and g l u c u r o n i d e c o n j u g a t e s , enzymes are not always as s u b s t r a t e - s p e c i f i c as s t a t e d , and many components o f b i o l o g i c a l e x t r a c t s are p o t e n t i n h i b i t o r s of most enzyme systems used i n c o n j u g a t e i d e n t i f i c a t i o n . These and many more problems e x i s t which may l e a d to the i n c o r r e c t i d e n t i f i c a t i o n of x e n o b i o t i c conjugates. T h i s was v i v i d l y demonstrated i n a study by D i c k i n s o n e t a l . (27) u s i n g the e s t e r g l u c u r o n i d e o f v a l p r o i c a c i d (VPA) as a model compound ( F i g u r e 17). With the c o n j u g a t e c o n t a i n e d i n f r e s h l y c o l l e c t e d r a t b i l e h a v i n g a pH o f 8.2, the enzyme B - g l u c u r o n i d a s e c l e a v e d over 98% o f the c o n j u g a t e to y e i l d f r e e VPA. T h i s was e q u a l to the a l k a l i t r e a t m e n t which was used to e f f e c t maximum h y d r o l y s i s of the c o n j u g a t e w i t h o u t a l t e r a t i o n of the a g l y c o n e . Storage o f the b i l e under a v a r i e t y o f c o n d i t i o n s and f o r v a r y i n g p e r i o d s o f time changed the r e s u l t s d r a m a t i c a l l y . R e s u l t s o f one o f these t e s t s a r e p r e s e n t e d i n F i g u r e 17. When the pH o f the b i l e was a d j u s t e d to pH



DIBROMOSULFOPHTHALEIN Br


263


(DBSP)

0

OH

LIVER FUNCTION TEST

BILE

BILE 100%

75%DBSP 25% [ D B S P - M E T A B O L I T E ]

DBSP

DERIVEO?

GSH

Figure 16. Hidden metabolites may be the consequence of using one species as a model for another. With DBSP, humans were recently reported to d i f f e r from that assumed to be the case based on e a r l i e r studies with r a t s . O II (CH CH CH ) CHC-0-GLUCURONIDE 2

2

2

2

GLUCURONIDE

OF

VALPROIC

ACID

(VPA-GLU) ALKALI IN

VPA-GLU

FRESH

RAT BILE nU ft O

B-GLUCURONIDAS

^-FREE

VPA

98%

^-FREE

VPA

98%

VPA

5%

ALKALI VPA-GLU

OF

BILE

37°C,3HR,pH9 B-QLUCURONIDAS^

VPA +

6 Unknown

Esters

labile

to

FREE

alkaline

hydrolysis

Figure 17. Chemical a l t e r a t i o n during storage and handling of samples may mask the presence of certain conjugates. For exam ple, 8torage of b i l e containing VPA-Glu resulted i n almost com plete loss of glucuronidase-sensitive substrate after just 3 hrs.




264

9 and held for 3 hr at 37°C, only 5% of the VPA-glucuronide was enzymatically cleaved to free VPA. Alkaline treatment, however, s t i l l cleaved 98% of the conjugate. Further analysis of the "VPA" generated by alkaline hydrolysis showed that the glucuronidase re sistant conjugates were s t r u c t u r a l isomers formed by migration of the xenobiotic acyl group around the pyranose ring of the exocon; these products were not substrates for B-glucuronidase. Conse quently, such metabolites would normally be considered as conjugates other than glucuronides. Proper sample preparation and analysis are e s s e n t i a l in a l l studies of xenobiotic metabolism. With known standards of the free metabolites and often with just a radioactive unknown, i t i s cus tomary to test their s t a b i l i t y under conditions of a n a l y s i s . The same caution i s not always possible with conjugates, but much can be done to improve the v a l i d i t y of conjugate i d e n t i f i c a t i o n . Not only w i l l greater caution in sample handling improve the chances of correctly i d e n t i f y i n g conjugates, i t w i l l also reduce the endless hour8 of time spent by metabolism chemists i n inventing bizarre pathways to j u s t i f y the formation of metabolites which never existed in the f i r s t place. Literature Cited 1.

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RECEIVED August 19, 1985


Hidden Xenobiotic Conjugates - ACS Symposium Series (ACS

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