Improved Methods for Analysis of N

Chapter 9

Improved Methods for Analysis of N-Nitroso Compounds and Applications in Human Biomonitoring B. Pignatelli, C. Malaveille, P. Thuillier, A. Hautefeuille, and H. Bartsch

Downloaded by TUFTS UNIV on June 5, 2018 | https://pubs.acs.org Publication Date: March 28, 1994 | doi: 10.1021/bk-1994-0553.ch009

International Agency for Research on Cancer, 150 Cours Albert-Thomas, 69008 Lyon, France

A sensitive group-selective method for analysis of total N-nitroso compounds (NOC) has been developed and applied to investigate their role in gastric cancer etiology. Total NOC in gastric juice (GJ) were not elevated in subjects with gastric precancerous conditions or omeprazole-induced achlorhydria. However, the presence of precursors of nitrosation-dependent direct mutagens and a lowered anti-oxidant defense and reduced nitrite scavenging-ability were found in GJ of subjects with elevated cancer risk. Unknown non-volatile NOC were prevalent among total NOC occurring in body fluids and some foods. An improved HPLC-photohydrolysis-colorimetry method was set up and validated with 28 reference NOC in order to separate and detect hitherto unknown NOC in biological fluids and food extracts. Humans a r e exposed t o a wide range o f n i t r o s a t a b l e compounds and n i t r o s a t i n g agents (1,2) which can r e a c t i n v i v o t o form c a r c i n o g e n i c N - n i t r o s o compounds (NOC). N i t r i t e , n i t r a t e and n i t r o s a t i n g agents can a l s o be s y n t h e s i z e d endogenously i n r e a c t i o n s mediated by b a c t e r i a , a c t i v a t e d macrophages o r o t h e r c e l l types through the enzyme n i t r i c o x i d e s y n t h a s e ( 3 - 7 ) . C a r c i n o g e n i c NOC formed i n t h e stomach have been suggested t o be i n v o l v e d i n g a s t r i c c a n c e r c a u s a t i o n . G a s t r i c a c h l o r h y d r i a p e r m i t s the c o l o n i z a t i o n o f the stomach by b a c t e r i a t h a t c o n v e r t n i t r a t e Xo n i t r i t e , which may l e a d t o i n t r a g a s t r i c f o r m a t i o n o f NOC ( 8 ) . P o s i t i v e c o r r e l a t i o n s between the presence o f p r e c a n c e r o u s c o n d i t i o n s o f the stomach, h i g h i n t r a g a s t r i c pH, e l e v a t e d b a c t e r i a l count and n i t r i t e c o n c e n t r a t i o n s have been found c o n s i s t e n t l y ( r e v i e w e d i n 9). However, group d e t e r m i n a t i o n o f t o t a l NOC i n human g a s t r i c j u i c e (GJ) by two methods (10-12) gave c o n t r a d i c t o r y r e s u l t s and the r e p o r t e d c o n c e n t r a t i o n s showed wide i n t e r - l a b o r a t o r y v a r i a t i o n s . As b o t h methods may o v e r e s t i m a t e o r u n d e r e s t i m a t e t h e t r u e NOC l e v e l s ( 1 2 - 1 4 ) , we have developed an improved a n a l y t i c a l

0097-6156/94/0553-0102$08.00/0 © 1994 American Chemical Society

Loeppky and Michejda; Nitrosamines and Related N-Nitroso Compounds ACS Symposium Series; American Chemical Society: Washington, DC, 1994.

9.

PIGNATELLI ET AL.

Methods for Analysis of N-Nitroso Compounds

103


p r o c e d u r e and a p p l i e d i t t o p a t i e n t s w i t h and w i t h o u t p r e c a n c e r o u s c o n d i t i o n s o f t h e stomach, who l i v e i n a r e a s w i t h c o n t r a s t i n g g a s t r i c c a n c e r r i s k and t o v o l u n t e e r s b e f o r e and a f t e r treatment f o r i n h i b i t i o n o f g a s t r i c a c i d s e c r e t i o n . As a s c o r b i c a c i d , a known a n t i - o x i d a n t and i n h i b i t o r o f n i t r o s a t i o n , p r o t e c t s a g a i n s t g a s t r i c c a n c e r ( 1 5 - 1 7 ) , t h e r e l a t i o n s h i p s between p r e c a n c e r o u s c o n d i t i o n s o f stomach and g a s t r i c l e v e l s o f n i t r i t e , t o t a l NOC and a s c o r b i c a c i d were a l s o i n v e s t i g a t e d . Combined a p p l i c a t i o n o f t h e methods f o r t h e a n a l y s i s o f t o t a l NOC, v o l a t i l e n i t r o s a m i n e s (VNA) and N - n i t r o s a m i n o a c i d s (NAA), r e v e a l e d a h i g h p r e v a l e n c e o f u n known NOC i n human body f l u i d s and i n some food e x t r a c t s . HPLCp h o t o h y d r o l y s i s - c o l o r i m e t r y method f o r t r a c e a n a l y s i s o f NOC was v a l i d a t e d and c a n be used i n c o m b i n a t i o n w i t h b a c t e r i a l m u t a g e n i c i t y a s s a y s f o r f u r t h e r i d e n t i f i c a t i o n o f h i t h e r t o unknown g e n o t o x i c NOC. Improved Method f o r Group-Selective Analysis of T o t a l NOC i n Human Body F l u i d s As humans a r e exposed t o a v a r i e t y o f NOC p r e c u r s o r s , h i t h e r t o u n known n o n - v o l a t i l e NOC (and t h e i r m e t a b o l i t e s ) a r e e x p e c t e d t o o c c u r i n body f l u i d s . We have m o d i f i e d p r e v i o u s methods (10-12) to d e v e l o p an improved g r o u p - s e l e c t i v e method f o r t h e d e t e r m i n a t i o n o f t o t a l NOC i n GJ ( 1 8 ) . I t i s based on t h e i r c h e m i c a l d e n i t r o s a t i o n w i t h HBr and c h e m i l u m i n e s c e n c e d e t e c t i o n o f t h e r e l e a s e d n i t r i c o x i d e u s i n g a t h e r m a l energy a n a l y s e r (TEA). Sequential a d d i t i o n o f CH C00H and HBr p e r m i t s t o d i s t i n g u i s h NOC from a c i d l a b i l e TEA r e s p o n s i v e compounds (TAC). The G J sample, t r e a t e d w i t h sulphamic a c i d t o destroy n i t r i t e , i s i n j e c t e d d i r e c t l y i n t o r e f l u x i n g e t h y l a c e t a t e c o n t a i n i n g e i t h e r CH^COOH - 0.1% ( v / v ) HC1 for determining TAC o r HBr f o r t h e s i m u l t a n e o u s d e t e r m i n a t i o n o f TAC and NOC. The amount o f n i t r i c o x i d e r e l e a s e d i n each case i s measured by a TEA d e t e c t o r ( F i g u r e 1 ) . The d i f f e r e n c e between t h e two d e t e r m i n a t i o n s r e p r e s e n t s t h e c o n c e n t r a t i o n o f t o t a l NOC i n the sample. The amount o f TAC and (TAC + NOC) c a n be c a l c u l a t e d u s i n g n i t r i t e and n i t r o s o d i p h e n y l a m i n e a s r e s p e c t i v e s t a n d a r d s . The TAC c o n c e n t r a t i o n c a n g r e a t l y i n f l u e n c e t h e r e s p o n s e o f t o t a l T E A - r e s p o n s i v e compounds. The p r e s e n c e o f up t o 6% w a t e r i n e t h y l a c e t a t e o r o f n i t r a t e up t o 1000 y m o l / l d i d n o t a f f e c t t h e d e t e r minations. The method was adapted f o r a n a l y s i n g aqueous samples c o n t a i n i n g h i g h c o n c e n t r a t i o n s o f n i t r a t e ( e . g . , human u r i n e ) (19). N i t r a t e was removed by an anion-exchange p r o c e d u r e w i t h o u t a s i g n i f i c a n t l o s s o f v a r i o u s added r e f e r e n c e NOC and u n i d e n t i f i e d u r i n a r y NOC. T h i s method i s h i g h l y s e l e c t i v e f o r NOC, r e p r o d u c i b l e ( c o e f f i c i e n t o f v a r i a t i o n from t r i p l i c a t e measurements 5-10%), s e n s i t i v e ( d e t e c t i o n l i m i t 0.01 y m o l / l ) , r e q u i r e s o n l y a few ml o f sample and no p r i o r e x t r a c t i o n . S u i t a b l e t e c h n i q u e s f o r s t a b i l i z i n g human G J and u r i n e s a m p l e s , and t h e i n f l u e n c e o f time and s t o r a g e c o n d i t i o n s on NOC c o n c e n t r a t i o n s have been e s t a b l i s h e d (18,19). The absence o f a r t e f a c t u a l n i t r o s a t i o n due t o sample h a n d l i n g has been v e r i f i e d . The method d i s t i n g u i s h e s NOC from most o t h e r T E A - r e s p o n s i v e s p e c i e s s u c h a s p s e u d o n i t r o s i t e s , n i t r o s o t h i o l s , a l k y l n i t r i t e s and i s a l s o a p p l i c a b l e t o t h e a n a l y s i s o f f a e c e s ( 2 0 ) and food e x t r a c t s ( 2 1 ) . 3



T e f l o n tubing

\ I /

r

a

p

ΝΟ· NO:

ill

3

> NOx + hv

TEA

600nm

Signal

Vacuum

chamber

Reaction

> NOt • Ο,

D e t e c t i o n of

Νθ·

Gas filter

Regulatory valve

80/150°C

0

2

|OZONATOR|

0

Photomultiplier tube & electronics

Time

Filter INTEGRATOR

I

APPARATUS

F i g u r e 1. Scheme f o r t h e g r o u p - s e l e c t i v e method f o r the de t e r m i n a t i o n o f t o t a l NOC.

T

Ûlilïû

Injection port (Teflon septum)

Constant Heating pressure mantle device Chemical denitrosation >N-NO > > Ν · * ΝΟ·

Helium

v.

High-efficiency low-volume condenser

Traps containing 6 mol/l N a O H


9.

PIGNATELLI ET AL.

Methods for Analysis of N-Nitroso Compounds

105


Levels of NOC, Direct Genotoxins, and their Precursors i n GJ of Patients v i t h and without Precancerous Lesions o f the Stomach and L i v i n g i n Areas v i t h Contrasting G a s t r i c Cancer Risk. A s t u d y was performed t o examine whether e l e v a t e d r i s k o f g a s t r i c c a n c e r i s a s s o c i a t e d w i t h h i g h l e v e l s o f t o t a l NOC, t h e i r p r e c u r s o r s and n i t r o s a t i o n - d e p e n d e n t d i r e c t g e n o t o x i n s i n GJ ( 2 2 ) . P a t i e n t s (N = 207) were c l a s s i f i e d a c c o r d i n g t o h i s t o l o g i c a l l y c o n f i r m e d d i a g n o s i s o f t h e stomach, c o u n t r y o f o r i g i n and pH o f t h e i r GJ ( T a b l e I ) . They were l i v i n g i n t h r e e a r e a s w i t h up t o 8 - f o l d v a r i a t i o n s i n g a s t r i c c a n c e r r i s k : one i n C o l o m b i a ( N a r i n o ) , where the g a s t r i c c a n c e r i n c i d e n c e i s among the h i g h e s t i n t h e w o r l d , and two o t h e r s i n t h e UK and F r a n c e , where r i s k s a r e lower. C o n c e n t r a t i o n s o f t o t a l NOC (18) and d i r e c t g e n o t o x i c a c t i v i t y (23) were d e t e r m i n e d i n GJ o f f a s t i n g s u b j e c t s b e f o r e and after i n vitro nitrosation. N i t r i t e was a n a l y z e d c o l o r i m e t r i c a l l y (24). I n agreement w i t h e a r l i e r f i n d i n g s , an a c i d i c g a s t r i c pH (< 4 ) was s t r o n g l y and s i g n i f i c a n t l y a s s o c i a t e d w i t h n o r m a l g a s t r i c mucosa o r moderate g a s t r i t i s . Among p a t i e n t s w i t h o u t g a s t r i c p r e c a n c e r o u s l e s i o n s , GJ w i t h e l e v a t e d pH was seen much more f r e q u e n t l y i n C o l o m b i a , as compared t o t h e o t h e r c o u n t r i e s ( T a b l e I ) . Colombian p a t i e n t s w i t h p r e c a n c e r o u s l e s i o n s had the h i g h e s t p r e v a l e n c e o f e l e v a t e d g a s t r i c pH ( T a b l e I ) . The n i t r i t e c o n c e n t r a t i o n s i n GJ ( r a n g e 1-472 umol/1) were h i g h e s t f o r C o l o m b i a n p a t i e n t s w i t h p r e c a n c e r o u s l e s i o n s and f o r s u b j e c t s w i t h GJ o f pH>4 ( F i g u r e 2 A ) . I n c o n t r a s t , l e v e l s o f t o t a l NOC (range < 0.01 - 8.0 y m o l / l ) d i d n o t d i f f e r between c o u n t r i e s n o r between GJ samples grouped a c c o r d i n g t o h i s t o p a t h o l o g i c a l d i a g n o s i s o r a c c o r d i n g t o a pH below o r above 4 ( F i g u r e 2B). T o t a l NOC l e v e l s increased with o r i g i n a l n i t r i t e concentrations at a greater rate i n a c i d i c GJ than i n those w i t h pH > 4.. The d a t a t o g e t h e r suggest t h a t a c i d - c a t a l y s e d n i t r o s a t i o n c o n t r i b u t e s a t l e a s t as much as o t h e r n i t r o s a t i o n pathways t o i n t r a g a s t r i c NOC f o r m a t i o n . I n v i t r o n i t r o s a t i o n o f GJ w i t h e x c e s s NaNÛ2 ( a t pH 1.5 f o r 60 min a t 37°C) i n c r e a s e d t h e c o n c e n t r a t i o n o f t o t a l NOC up t o s e v e r a l t h o u s a n d - f o l d , w i t h a maximum o f 1330 y m o l / l ( F i g u r e 2 C ) . H i g h NOC l e v e l s a f t e r i n v i t r o n i t r o s a t i o n were n o t a s s o c i a t e d w i t h h i g h e r g a s t r i c c a n c e r r i s k and t h e NOC c o n c e n t r a t i o n i n n i t r o s a t e d GJ i n c r e a s e d w i t h t h e o r i g i n a l pH o f GJ o n l y f o r French samples ( F i g u r e 2C). These f i n d i n g s suggest t h a t b o t h t h e l e v e l and p r o b a b l y t h e n a t u r e o f some o f t h e s u b s t a n c e s i n GJ a r e d i f f e r e n t between F r e n c h and Colombian s u b j e c t s . A f t e r a c i d c a t a l y s e d n i t r o s a t i o n , a l l GJ from F r a n c e and C o l o m b i a e x h i b i t e d g e n o t o x i c a c t i v i t y t h a t was more e l e v a t e d i n GJ w i t h an o r i g i n a l pH above 4 than i n a c i d i c G J ; t h e h i g h e s t g e n o t o x i c i t y was seen i n C o l o m b i a n GJ ( F i g u r e 2D). Thus, g e n o t o x i c i t y o f n i t r o s a t e d GJ was dependent on i t s o r i g i n a l pH and p a t i e n t s from t h e h i g h e s t g a s t r i c c a n c e r r i s k a r e a had t h e h i g h e s t l e v e l s o f p r e c u r s o r s o f n i t r o s a t i o n dependent g e n o t o x i n s i n t h e i r G J . The r e s u l t s o f t h i s s t u d y do n o t support t h e i d e a t h a t i n t r a g a s t r i c t o t a l NOC l e v e l s a r e e l e v a t e d i n s u b j e c t s w i t h p r e c a n c e r ous stomach c o n d i t i o n s o r i n those l i v i n g i n a h i g h - r i s k a r e a f o r stomach cancer but a r e c o n s i s t e n t w i t h i n t r a g a s t r i c a l l y formed


106

NITROSAMINES AND RELATED ΛΤ-NITROSO COMPOUNDS


Table I .

D i s t r i b u t i o n of Patients According to t h e i r Gastric pH, Country of O r i g i n & H i s t o l o g i c a l Diagnosis

D i a g n o s i s (Group)

Country o f origin

Normal g a s t r i c mucosa or superficial gastritis (I)

UK France Colombia

Reflux g a s t r i t i s ( I I )

UK

Diffuse i n t e r s t i t i a l ( I I I ) gastritis

No

X of gastric juice samples v i t h pH < 4 > 4

18 27 32

89 85 56

11 15 44

9

78

22

UK France

12 17

92 71

8 29

Chronic atrophic g a s t r i t i s v i t h or vithout i n t e s t i n a l metaplasia or dysplasia (IV)

UK France Colombia

17 37 38

53 52 29

47 48 71

Total

UK France Colombia

56 81 70

77 66 41

23 34 59

Table I I . Influence of Treatment v i t h Omeprazole on G a s t r i c ^ A c i d i t y and B a c t e r i a l F l o r a i n Healthy Volunteers

pH

b

c

Total

bacterial counts (x 10 Y m l ) 0

Treatment

Ni trate-reducing bacterial.counts (x l O Y m l )

None/placebo

1.9 (1.3-5.8)

Improved Methods for Analysis of N

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