characterization of n nitrosomethyl urea in nitrosated fermented

7/31/2019 Characterization of N Nitrosomethyl Urea in Nitrosated Fermented

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C h a r a c t e r i z a t i o n o f N-( N it r o s o m e t h y l )u r e a i n N i t ro s a t e d F e r m e n t e d

F i s h P r o d u c t s

Dajun Deng,* Tong Li, Hong Ma, Ruming Wang, Liankun Gu, and J ing Zhou

Departm ent of Can cer Etiology, Beijing Instit ute for Cancer Research a nd School of Oncology,Beijing Medical U niversity, 100034 China

To characterize chemical carcinogens in acidic-nitrosated fish sauce sample, N-nitrosamides in thesampl e wer e separ ated by t wo kinds of r ever sed- phase HPLC columns, and with detection byphotolysis-pyrolysis-therm al energy ana lyzer. A strong chromatographic peak at tR 12 or 4.5 min,same as that for N-(nitrosomethyl)urea (NMU), was obtained on PRP-1 or C 18 HPLC column fromfish sauce sample with 10 mM trifluoroacetic acid as the basic mobile; acetonitrile, as organic modifierafter the sample was nitrosated by 5 mmol/L of sodium nitrite (final concentration) at 37 C andpH 2.0 for 1 h . No response a bove tR could be observed from the nitrosated sam ple in th e detectionsystem wit hout photolysis. Such a peak could not be obtained f r om the unnitr osated f ish sauceeither. These results indicated tha t th e component was NMU. Fur thermore, this component, NMU,could also be detected in the ni tr osat ed h uman gastr ic juice sample spiked with fish sau ce. Theforma tion of NMU in th e samp le was pH- and nitr ite-dependent . This paper pr ovides direct evidencethat NMU formation could occur in fish sauce from the high-risk area for stomach cancer and inthe fish sauce spiked human gastric juice during nitrosation under simulated gastric conditions.

K e y w o r d s : Nitrosomethylurea; fish products; nitrosation; human; gastric juice; stomach cancer;nitrosamide; nitroso compounds; nitrite

INTRODUCTION

Stomach carcinoma is still the lead ing cause of can cerdeath in China a nd th e second one in the world. Sincethe i ntr agastr i c N-nitrosamide formation may play animpor t ant r ole in the et iology of gastr ic car cinomas(Mir vish, 1983), mu ch at tent ion h as been paid to thechar a cter ization of n at ur al N- nitr osamides i n humanenvir onment. Many N-nitrosamines can be sensitivelyan alyzed by the t herm al energy an alyzer (TEA) detectorrelying on th erma l cleavage of the N-N bond t o producea n itrogen oxide (NO) radical. Unlike N-nitrosamines,

N-nit r osamides a nd r elated compunds typically r ear -ran ge on pyrolysis to yield m olecular nitrogen instea dof nitrogen oxide. A photolysis device was assembledby Shuker and Tannembaum (1983) in which N-nitro-samides were cleaved photolytically by U V irra diationto produce nitrogen oxide. HP LC-photolytic interface-T E A m e t h od s w er e e s t a bl is h e d t o d e t ect p r e ci se ly

N-nitrosam ides an d other nonvolatile NOC (Conboy an dHotchkiss, 1989). We set up a selective and sen sitiveHPLC-P H P S-chemiluminescence met hod for N-nitro-sami des (Chen et al ., 1991; Li and Deng, 1995). Theestablishment of sensitive methods for determining of

N-nitrosamides ma kes it pr acticable to detect t he t raceamount of N-nitr osamides in t he envir onment. Somepr ogr ess was made in the investigat ion of t he na tur a l

N-nit r osamides in t h e past year s. I t was r epor ted tha tt h e l ev el of t ot a l N-nitr osamides in huma n fastinggastric juice samples from high-risk areas correlatedwith t he sever ity of gastr i t is a nd was si gni ficantlyhigher th an th at from low-risk a reas (Zhang et a l., 1991;

Deng et a l., 1995, 1997). However, little is kn own a boutt h e d e t a il ed ch e m ica l s t r u ct u r e s of t h e n a t u r a l N-nitr osamides, al though Mende et al . ( 1994) r epor t edt h a t I n dia n n a s a l s n u ff con t a in e d low a m ou n t s of

N-nitrosopyrrolidin-2-one. Fish sa uce is a liquid prod-uct of sma ll ma rine fish an d sodium chloride (7:3). Themain species of fishes a re Sardinella aurita (Val.) an d

Deca pt eru s m ar u ad si (T. & S.). The f ishes a r e com-pletely liquidized after fermentation for 1-2 ye a r s . I t

is consu med da ily (about 30 mL/capita) by resident s a sa traditional seasoning in the Chinese southeast coast,the highest r isk ar ea f or stomach cancer in Chi na. I tis direct mutagenic in prokaryocytes and mammaliancells and human gastric mucosal cells, and carcinogenicto glandular stomach of Wistar rats after being nitro-sated in the simulated gastr ic condition (Deng et al .,1991; Zhang et al ., 1991; Chen et al ., 1992). Recentepidemiological stu dies showed furth er th at the intak eof fish sau ce is a high-risk factor for gastr ic car cinogen-esis for the local residents (Cai et al., 1993; Ye et al.,1994). However, the specific chemicals ultimat ely re-sponsible for th e carcinogenicity of nitrosat ed fish sa ucear e st i ll un known, al though n itr osabl e amines can bedetected in fish sauce (Zhang et a l., 1993). In th is paper,NMU in nitr osated f ish sauce was identif i ed by t woHPLC m ethods connected with a postcolumn PHPSdevice an d TEA.

MATERIALS AND METHODS

C h e m i c a l s a n d S a m p l e s . S ta nda r d N-nitrosamide NMUwas purchased from Sigma Chemical Co. (N-4766); acetoni-trile, from BDH, En gland. Both acetone and dichlorometha newere analytical reagents an d purified by redistillation. Fishsauce samples were collected from Changle County, FujianP r ovince , the a r e a with the highest m or ta lity for stom a ch

* Author to whom cor r e sponde nce should be a ddr e ssed(telephone +8610-66162978; fax +8610-66175832; e-mailden [email protected] et).

202 J. Agric. Food Chem. 1998, 46, 202205

S0021-8561(97)00628-6 CCC: $15.00 1998 American Chemical SocietyPublished on Web 01/19/1998


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cancer (more th an 120/105 in m a le s a fte r a ge -a djusted) inC hina ( Zha ng e t a l ., 1984). Hum a n fa sting ga str ic juicesamples were collected by gastric tube or fibroendoscope andanalyzed for the total amount s ofN-nitrosamides (Deng et al.,1997). The gastric juice samples without N-nitrosamides werepooled and s tored at -20 C for furth er st udy ofN-nitrosamideformation from fish sauce.

N i t r o s a t i o n a n d P u r i f i c a t i o n o f F i s h S a u c e a n d G a s -tric Ju ice Sam ples (Den g et al. , 1991; Zhan g et al. , 1991).A 10 mL fish sau ce sample or mixture of gastr ic juice sampleand fish sauce (1/1, v/v) was adjusted to pH 2.0 with 6 N HCl,nitrosated with 5 mm ol/L of sodium nitrite (the final concen-tr a tion) a t 37 C f or 1 h a nd the n stoppe d by 50 m m ol/L of sulfamic acid (th e final concent ra tion). Different concentr at ionof nitrite an d pH values were also used in the experiments t ostudy relationships between the nitrosamide formation andconcentra tion of nitrite or hydrogen ion. Samples were treat edwith cr ysta l sodium chlor ide a nd tungstophosphor ic a c id,ce ntr ifuge d, fi ltr a te d to se pa r a te pr ote ins, a nd fur the r e x-tracted by acetone/dichlorometha ne (1:5). Then, th e extractswere evaporated, an d dry residues were dissolved in 2 m L of10 m M tr ifluor oa ce tic a cid just be for e use . The a bsoluterecovery of the procedures for NMU was about 50%.

C h r o m a t o g r a p h y . R e ve r se d- pha se HP LC wa s use d toseparate NOC. Trifluoroacetic acid (10 mM) was u sed as thebasic mobile; acetonitrile, a s organic modifier with gradiente lution. The f low r a t e wa s 1. 0 m L/m in. C olum ns we r e aHamilton PRP-1 polymeric analytical (150 4.1 mm i.d., 10m m pa r ticle siz e) a nd gua r d colum ns. The Wa te r s HP LCsystem consisted of one 680 aut omated gra dient controller, two510 pump s, and two Rheodyne injectors (7125 before and 7012after column, th e latter one was used to monitor the workingsta tu s of detector rapidly) (Li and Deng, 1995). In add ition, aChrompac chromsphere C18 analytical (100 3 m m i. d. , 5mm particle size) column was used.

N-Nitrosamide Detec tor (Chen et al. , 1991; Deng et al. ,1995). A Hitachi 056 recorder was u sed. The detector wascomposed of a photolysis device, a pyrolysis apparatus, a seriesof cold traps (two ice-water ones, two dry ice-acetone ones,and a liquid nitrogen-ethanol one), and a Model 510 TEA.This detector could detect N-nitrosamides an d distinguishN-nitrosamides from other kinds of NOC by controlling theworking st atu s of photolysis device. With t he postcolumnphotolysis, both N-nitr osa m ides a nd other NOC c ould bedetected with high sensitivity (1-10 ng). Without photolysis,the response for N-nitrosamides decreased from 100 to 84%;for other kinds of NOC, only 0-32%.

RESULTS

A st r ong chr omatogr aphic peak ( 19.8 mol/L) wasobtained at tR 12 min from nitrosated fish sauce whenit was separ ated by PRP -1 columns (Figure 1A-b). Astronger peak could be seen with nitrosated fish sauces p i k e d w i t h N M U a t t h e s a m e tR (Figure 1A-c). Theheight of the peak in the u nspiked nitrosated fish sa uced e cr e a s ed b y 9 2% w h en t h e p h ot ol ys is d e vi ce w a sswitched off (Figure 1A-a). The peak was n ot detectedin unnitr osat ed fish sauce. A peak at tR 4. 5 min anda n ot h e r a t tR 5.5 min were observed on chromsphereC18 column fr om the concentr a ted chr omatogr aphicfraction of nitrosated fish sauce collected from tR 11 minto tR 13 min on P RP-1 colum n (Figure 1B-b). A strongerp e a k a t tR 4.5 min could also obtain from the fractionspiked with NMU at the same position (Figure 1B-c).N o p ea k a t tR 4.5 or 5.5 min could be detected in theunspiked fraction without photolysis (Figure 1B-a).

The same peak at tR 12 min could a lso be detected ona P RP-1 column from the n itrosated mixture of huma nfasting gastric juice samples and fish sau ce (1/1, v/v).The formation of the peak (NMU) was pH-dependentin two independent exper iments (Table 1) . Of NMU,

3.04, 1.57, an d 0.33 mol/L was det ected in t he mixtur etrea ted with 10.0, 5.0, and 2.5 mm ol/L of sodium nitr ite.

DISCUSSION

Weisburger et al. (1980) reported that several fishconsumed pr edominantly in J apan wer e muta genic inS . t y p h i m u r i u m , and car cinogenic for t he glandul arstomach of Wistar rats after incubation with nitrite. Itwas r epor ted tha t 16-31 mg/kg of methylurea could bedetected in dried, salted bonito fish a fter nitrosat ion a nddenitrosation, though n o methylurea could be detectedi n t h e fi sh d ir e ct l y ( Mi r vi sh e t a l ., 1 97 8). F u r t h e rstudies showed tha t m ethylurea was synthesized th rough

F i g u r e 1 . Chromatograms obtained by HPLC-photolysis-pyrolysis-TEA from fish sauce. Fish sau ce was nitr osated by5 m m ol/L of sodium n itr i te a t 37 C, pH 2.0, for 1 h a ndextracted with acetone/dichloromethan e and concentra ted fivetimes. NMU and other NOC were fractionat ed on PRP-1 orC18 column using an acetonitrile gradient, as indicated: (a)without photolysis; (b and c) with photolysis; (A-a and A-b)from nitrosated fish sauce; (A-c) from NMU (40 ng)-spikednitr osa te d fish sa uce ; ( B -a a nd B -b) f r om the conde nse d

fraction between tR 1 1 m in a n d tR 13 min of nitrosated fishsau ce on PRP-1 column ; (B-c) the NMU-spiked fraction. Thechemical marked by arrow was NMU, 20 L injection.

T a b l e 1 . F o r m a t i o n o f N M U in H u m a n F a s t i n g G a s t r i cJ u i c e S p i k e d b y F i s h S a u c e ( 1 : 1 ) a f t e r N i t r o s a t i o n w i t h 8

m g p e r 1 0 m L o f S o d i u m N i t r i t e a t V a r i o u s p H V a l u e s

concentra tion (M)a ofNMU formed at various pH values

1.0 2.0 3.0 4.0 5.0 6.0 7.0

expt 1 3.68 5.22 3.35 0.86 BDLb BDL NDb

expt 2 5.16 4.09 3.44 BDL BDL ND BDL

a Concentra tions of NMU were calculated from chromat ogramon PRP-1 and corrected with the recovery (50%). b BDL, belowdetection limits; ND not determined.

Nitrosamide in Nitrosated Fish Sauce J. Agric. Food Chem., Vol. 46, No. 1, 1998 203


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5-oxocreat inine 5-oxime a nd 1-methyl-5-oxohydra nt oin5-oxime dur ing nitr osation of creat inine (Mirvish et a l.,1982, 1993). Piacek-Llanes and Tannenbaum (1982)also reported that the t reatm ent of fresh a nd dried favabeans with low concentra tions of nitrite un der simu latedgastr ic conditions results in t he p roduction of a powerfulactivated mut agen, which was strongly suggested to bea N-nitrosourea. Fish sau ce, th e fermen ted fish productconsumed t r adit i onally in t he highest r isk a r ea forstomach cancer i n China, was suggested to be one of the major causal factors of gastric cancer in the localpopulation (Deng et al., 1991; Zhang et al., 1991, 1993;Cai et a l., 1993; Ye et a l., 1994). Creatinine a nd othernitrosable amines can be detected in fish sau ce (Zha nget al., 1993). Here, we reported that a str ong peak witht h e s a m e tR as NMU (12 min by PRP-1 and 4.5 min byC18 columns) was observed on the chromatogram fromnitrosated fish sauce by two different reversed HPLCcolumn s, conn ected with a sensitive an d selective detec-tor for N-nit r osamides. I n addition, l ike N-nitrosa-mides, i ts r esponse i n the detector decr eased almostcompletely when the photolysis device was switched off.These results indicated that the component was NMU.The chemical structure of the product peak at tR 5.5 min

obtai ned on C18 column is un known. Like st an dar dN-nitrosamides, the peak at tR 5.5 min did not appearw it h ou t p h ot ol ys is . T h is s u gg es t s i t i s a n ot h e r N-nitrosamide.

NMU is directly carcinogenic to the glandu lar stom-ach of experimental animals (Tatematsu et al., 1993).I t ca n b e s yn t h e s iz ed ch e m ica l ly fr om n i t r it e a n dmethylurea both in acidic condition in vitro (pH 1-3)and in the stomach of rats, guinea pig in vivo (Yama-moto et al., 1987). We found t ha t t he forma tion of NMUin human fasting gastric juice mixed with fish sauce (1:1) was pH- dependent as well. I t was r epor ted that t hesynth esis of NMU at pH 6-7 could be catalyzed by somebacteria isolated from gastric juice of subjects at the

high-risk area for gast ric cancer (Pa n et al., 1995). Theresidents in the studied high-risk area, Changle County,consumed f ish sauce daily and wer e exposed to highlevels of nitra te an d nitrite. Chronic atr ophic gastritis,achlorhydria, an d intra gastric colonization of bacteriawere common a mong the residents (Zhang et al., 1984;Cai et a l., 1993; Ye et a l., 1994). Pigna telli et al. (1993)reported th at the level of nitrite in fast ing gastric juicei n C o l u m b i a w a s u p t o 4 7 2 mol/L. Mar agos et al .(1991) reported the level of nitrite in pig gastric juicewas increased to 2.l mmol/L with the combined treat-ment of 6 mmol of nitrate an d cimetidine. The level ofnitrite in fasting gastric juice in Changle County wasu p t o 1 0 0 m o l / L , a n d t h e c o n t e n t o f n i t r a t e i n t h evegetables consumed by the local r esidents was up to

16 mmol/kg (Zhan g et al., 1984). We observed t ha t 3.04,1.57, and 0.33 mol/L of NMU wer e detect ed in thehuman f ast ing gastr ic jui ce samples mixed with thesame volume of fish sa uce af ter tr eatment wit h 10.0,5.0, a nd 2.5 mm ol/L of sodium nitrite, respectively.Thus, NMU may also be synthesized in their stomachsand be responsible for t he h igh risk for gastric cancer.

ABBREVIATIONS USED

HPLC, high-per for ma nce l iquid chr omat ogr aphy;NMU, N-nitr osometh yl ur ea; NOC, N- nitr oso com-pounds; PHPS, photol ysi s-pyr olysis; TEA, ther malenergy analyzer; tR, retention time.

ACKNOWLEDGMENT

We g r a t efu l ly a ck n ow le d ge h e lp w it h t h e P R P -1column provided by Dr. W. C. You from Beijing Institutefor Can cer Research.

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Received for r eview Ju ly 21, 1997. Revised man uscript receivedOctober 29, 1997. Accepted N ovember 1, 1997.X This work wassuppor te d by Gr a nt 39470780 f r om the Na tiona l Na tur a lScience Foundation of China and by Grant 854871900 fromthe Beijing Municipal Commission for Science and Technology.

JF9706282

X Abstract published in Advance ACS Abstracts, December15, 1997.

Nitrosamide in Nitrosated Fish Sauce J. Agric. Food Chem., Vol. 46, No. 1, 1998 205

characterization of n nitrosomethyl urea in nitrosated fermented

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