Influence of cysteine and nitrate on the endogenous formation of N-nitrosamino acids

Letters, 34 (1987) 39-41 Elsevier Scientific Publishers Ireland Ltd. Cancer

39

INFLUENCE OF CYSTEXNE AND NITRATE FORMATION OF N-NITROSAMINO ACIDS

A.R. TRICKER

ON THE ENDOGENOUS

and R. PREUSSMANN

Institute of Toxicology Neuenheimer Feld 280,

and Chemotherapy, D-6900 Heidelberg

German (F.R.G.)

Cancer

Research

Centre,

Im

(Received 18 July, 1986) (Revised version received 30 September 1986) (Accepted 13 October 1986)

SUMMARY

The excretion of N-nitrosamino acids and nitrate in 24-h urine was studied over a continuous 45-day period for a single volunteer on a supplemented diet regime. Days l-10 (normal diet), 11-20 (normal diet + 600 mg nitrate/ day), 21-25 (normal diet), 26-30 (normal diet + 200 mg L-cysteine/day), 31-35 (normal diet), 36-40 (normal diet + 600 mg nitrate + 200 mg Lcysteinelday) and days 41-45 (normal diet). The presence of N-nitrosohydroxyproline (NHPRO) in human urine is reported for the first time as well as the influence of L-cysteine on the formation of N-nitrosothiazolidine-4carboxylic acid (NTCA) and N-nitroso-2-methylthiazolidine-Ccarboxylic acid (NMTCA) under a normal nitrate burden (75 mg/day) and a high nitrate burden (675 mg/day). After estimation of the amount of dietary nitrite consumed and the extent of nitrite formation by bacterial reduction of nitrate in the oral cavity, the total daily exposure to nitrite was calculated. From a total daily exposure of 7.2 mg nitrite/day from a typical European diet, 0.08% is required for the endogenous formation of N-nitrosamino acids found in urine. Under an increased nitrate burden (675 mg/day), the total exposure to nitrite was increased to 38 mg/day of which 0.14% was required for the endogenous formation on N-nitrosamino acids.

INTRODUCTION

Recently, NTCA and its 2-methyl derivative (NMTCA) have been found in human urine [ 1,2] in addition to the two previously knownN-nitrosamino acids, N-nitrosoproline (NPRO) and N-nitrososarcosine (NSAR) [3]. A possible pathway to the formation of NTCA and NMTCA in vivo is by nonenzymic conversion of cysteine and formaldehyde or acetaldehyde to thiazolidine-4-carboxylic acid and 2-methylthiazolidine-4-carboxylic acid, o 1987 Elsevier Scientific Publishers Ireland Ltd. 0304-3835/87/$03.50 Published and Printed in Ireland

40

respectively, followed by endogenous nitrosation .of these compounds [2]. However, until now, no studies using cysteine and subsequent monitoring of urine for NTCA and NMTCA have been reported to support this theory. The effect of cigarette smoking [4 ] and vitamins C and E [ 51 on the endogenous synthesis of both NPRO and sulphur-containingN-nitrosamino acids excreted in urine has been reported. NHPRO has not previously been determined in human urine although rat feeding studies show this compound to be 92.9 f 4.0% excreted, almost equally in urine and feces [6]. In the present paper, using a human volunteer on a supplemented diet regime, we have obtained results to show the effect of dietary nitrate burden on the endogenous formation of N-nitrosamino acids. The effect of L-cysteine intake on the formation of NTCA and NMTCA is also reported as well as conclusive evidence to validate their in vivo formation from cysteine and formaldehyde and acetaldehyde, respectively. The presence of NHPRO, not previously reported in human urine was also found in all but one 24-h urine samples over 45 days. MATERIALS

AND METHODS

Chemicals StandardN-nitroso compounds were synthesised by the following literature methods: N-nitroso-L-azetidinecarboxylic acid (NAzCA), NHPRO, NPRO and NSAR [7], NTCA [8]. NMTCA was prepared by synthesis of 2-methylthiazolidine4carboxylic acid [ 91, on nitrosation using an analogous method to the above [ 71. All other reagents (purchased from Merck, Darmstadt, or Sigma, F.R.G.) were of analytical grade and used without further purification. Ethereal diazomethane was prepared from N-nitrosomethylurea and distilled before use. Urine collection and Storage Urine (24-h) was collected in 2 II polyethylene containers containing 10 ml 20% ammonium sulphamate in 3.6 M sulphuric acid to prevent artefact formation. Samples were stored at -2O’C until analysis. Analysis of nitrate in urine Urinary nitrate was determined using a dual channel continuous flow analyser (Skalar Analytical Instruments, Breda, The Netherlands) equipped with an automatic sampler, a multichannel peristaltic pump for sample and reagent delivery and a reducing column for nitrate to nitrite conversion. Nitrate was determined after reduction to nitrite in an on-line column containing granulated copper cadmium followed by diazotisation with sulphanihunide and coupled with N-1( 1-naphthyl)ethylenediamine to form a coloured azo dye which was measured at 540 nm in a lo-mm flow cell. The

41

sensitivity range was 2-250 by dilution. Analysis

of N-nitrosamino

ppm. Higher concentrations

were determined

acids in urine

A sample aliquot of urine (30 ml) was acidified with 1 ml 20% ammonium sulphamate in 3.6 M sulphuric acid and spiked with NAzCA (250 ng in 250 ~1 acetone, internal standard). Urine sample applied to a column containing 16 g Extrelut 20 (Merck, Darmstadt, F.R.G.) suppor’ted by a glass frit and a 0.5 cm bed of anhydrous sodium sulphate. After 20 min, the column was eluted with ethyl formate (150 ml) and the eluent collected in a 250-ml round-bottomed flask containing 0.5 ml NH,OH-MeOH (10% NH,) to prevent acid decomposition and the solvent removed in vacua at 30°C. The residue was suspended in acetone (5 ml) using sonic bath agitation and transferred to a 20-ml reaction vial. Acetone suspension derivatised with excess ethereal diazomethane solution at ambient temperature for 30 min. Final sample concentrate of 500 ~1 obtained by gentle evaporation in a stream of nitrogen. The extract was analysed using gas chromatography-thermal energy analysis (GC-TEA) for NAzCA, NPRO, NSAR, NTCA and NMTCA. Immediately following analysis, the sample vials were opened and the solvent gently removed in a stream of nitrogen. The residue was suspended in 250 ~1 acetonitrile and derivatised using 250 ~1 N,O-bis-(trimethylsilyl)trifluoroacetamide (BSTFA) for 30 min at 40°C. The sample was then reanalysed for NHPRO. When the standard compounds were simultaneously added to urine samples at a concentration of 1 11811,the recoveries were as follows: NAzCA (internal standard), 79% (SD. 5.5, n = 5); NSAR, 95% (S.D. 2.5, n = 5); NPRO, 90% (S.D. 1.7, n = 5); NTCA, 86% (S.D. 2.1, n = 5); NMTCA, 84% (S.D. 2.8, n = 5) and NHPRO, 57% (S.D. 4.7, n = 5). Analysis

of urinary N-nitrosamino

acids by GC-TEA

An aliquot (5 ~1) was analysed by GC-TEA using a 1 m X 2 mm glass column packed with 10% OV 17 on Supelcoport (So-100 mesh). Helium was used as a carrier gas (30 ml/min) and the injection port temperature was maintained at 180°C. The oven was maintained at 90°C for 1 min and then programmed to 210°C at 8”C/min. The retention times (in min) of the methylated derivatives (methylated and silylated for NHPRO) were as follows: NSAR 6.2, NAzCA (internal standard) 9.1, NPRO 10.8, NTCA 11.9, NHPRO 13.0 and NMTCA 14.9. The level of detection was determined as 0.5 ng for all standard compounds. Quantification was made by comparison of peak areas to those obtained from known concentrations of the appropriate standard compounds. Experimental

The healthy

protocol

male volunteer

(26 years, 70 kg body wt), a smoker (lo-15

42

I

diet for 5 days (days 41-45) 2.45 6.09 (3.9-9.8) (1.7-3.6) 8.85 (5.4-10.9)

1.53 (0.8-2.6)

15.8 (ll.l-

1.53 (0.8-2.9)

1.30 (0.6-1.7)

18.7)

‘Sum of all five nitrosamino acids. Detection limit 0.5 pg/l. ND, not detected. bUrinary excretion of NO;, detection limit 0.1 mg/l. ‘600 mg NO;/day taken as 3 X 200 mg NO; dissolved in 50 ml water 1 h prior to each meal. d200 mg L-cysteine/day taken 3~ a day using a standard solution in water (3x 50 ml of a standard 150 ml water) with each meal. e600 mg NO;/day and 200 mg L-cysteine/day taken as described above.

Unrestricted 0.42 (ND-0.7)

with 600 mg NO; and 200 mg L-cysteine/day= (days 36-40) 15.5 123.0 260.4 (50.4-185) (143-461) (10.0-24.3)

6.50 (4.3-9.7)

with 200 mg L-cysteine/dayd (days 26-30) 2.16 9.08 13.1 (1.9-2.6) (4.7-13.2) (9.8-15.7)

diet for 5 days (days 31-35) 5.19 2.17 (1.8-2.4) 3.4-6.7)

Diet supplemented 0.98 (0.6-1.2)

Unrestricted 0.61 (ND-1.2)

Diet supplemented 0.62 (ND-1.1) (21.2-67.4)

solution

in

57.4 (24.3-102)

290.0 (136-490)

57.4 (21.5-79.8)

48.4

of 200 mg L-cysteine

17.8 (13.2-19.6)

415.5 (266-654)

17.8 (12.2-20.9)

26.3 (23.0-80.1)

44

cigarettes/day) ate a normal European diet throughout the experimental period but avoided the consumption of alcohol and nitrite cured meat products. The volunteer was not undergoing any form of medical treatment and took no medicines of any kind 2 weeks prior to or during the experimental period. On days l-10 of the study, the volunteer ate an unrestricted diet with the above exceptions. On days 11-20, the diet was supplemented with 600 mg nitrate/day, 3 times 200 mg in 50 ml water 1 h prior to meals. On days 21-25, the volunteer returned to a normal diet. On days 26-30, the diet was supplemented with 200 mg L-cysteine/day, taken 3 times a day with meals (50-ml aliquots from a standard solution of 200 mg L-cysteine in 150 ml water). The volunteer returned to a normal diet for days 31-35. On days 36-40, the volunteer’s diet was supplemented with 600 mg nitrate and 200 mg L-cysteine/day as described above prior to returning to a normal diet for days 40- 45. Twenty-four-hour urine samples were collected over the whole of the experimental period and analysed for nitrate and iV-nitrosamino acids as described above. RESULTS AND DISCUSSION Neglecting the availability of endogenously nitrosatable pharmaceuticals and food contaminants such as pesticide and herbicide residues, the daily intake of nitrosatable compound classes in a typical European diet consist of: amides in the form of oligopeptides (approx. 100 g/day), guanidines (approx. 1 g/day), primary amines and amino acids (approx. 100 mg/day), aryl amines, secondary amines and ureas (approx. l-10 mg/day) [lo]. For a typical European diet, nitrate and nitrite exposure of 75 mg and 3.3 mg/day can be calculated [ 111. Dietary nitrate is lost by excretion in urine (approx. 65--7O%), feces (approx. 10%) and sweat (approx. 2%/day) [12]. From the residual nitrate, approx. 20% is reduced by bacteria in the oral cavity to nitrite [ 131. Thus, an average for the total available nitrite can be calculated as being 7.2 mg/day for a normal diet and 38 mg/day for a diet supplemented with a 600 mg/day nitrate burden as used in our study. These figures assume that the previous stated losses for nitrate in sweat and feces occur whilst the mean level excreted in urine as determined was used, of the unaccounted nitrate, 20% was assumed to be bacterially reduced increasing the dietary available nitrite (3.3 mg/day) to give a total available nitrite burden of 7.2 mg/day (normal diet) and 38 mg/day for a high nitrate burden diet. The mean levels and range of N-nitrosamino acids corrected for recovery and nitrate found in urine during the experimental period are shown in Table 1. For days l-10, the concentrations of NSAR, NPRO, NTCA, NMTCA and nitrate are in agreement with previous findings for N-nitrosamino acids [14,15] and nitrate [12] in urine. The concentration of NHPRO and total amounts of the 5 N-nitrosamino acids ranged from 1 .O to

45

2.4 (mean + S.D. 1.44 f 0.45) and 9.9 to 19.1 (15.4 it 3.3) pg/day, respectively. On increasing the nitrate burden by 600 mg/day to 675 mg/day the nitrite burden was increased from 7.2 to 38 mg/day. The mean levels of urinary N-nitrosamino acids were significantly increased as follows; NSAR 0.42 to 0.98, NPRO 2.09 to 15.3, NTCA 4.92 to 34.8, NMTCA 6.83 to 77.0, NHPRO 1.44 to 16.2 and the total amounts of the 5 N-nitrosamino acids from 15.4 to 159.8 pg/day. A corresponding increase in urinary nitrate was also found. NSAR was found in 7/10 normal urine samples (days l-10) and in 9/10 samples when the nitrate (and consequently the nitrite) burden was increased. Under a high nitrate burden the concentration of urinary NSAR was increased by a factor of 2.3. By comparison, the concentrations of the other N-nitrosamino acids and the total of the 5 N-nitrosamino acids was increased by approximately lo-fold. On returning to a normal unrestricted diet with a normal nitrate burden (days 21-25 and 41-45), the levels of both urinary nitrate and IV-nitrosamino acids were slightly elevated on the first day but returned to normal after 24 h. This can be explained by retention of nitrate in the body giving rise to a slight increase in available nitrite on days 21 and 40. Normal was defined as within the mean range occurring on days l-10 of the study. Supplementing the diet with 200 mg L-cysteine/day (days 26- 30) caused a 2-fold increase in the levels of urinary NTCA and NMTCA, the urinary excretion of the other 3 N-nitrosamino acids was not affected. On increasing the nitrate burden to 600 mg/day (days 36-40), the urinary concentration of NSAR, NPRO and NHPRO increased by an equivalent amount as in the absence of 200 mg L-cysteine/day supplement (days 11-20). However, the mean concentrations of urinary NTCA and NMTCA, under a simultaneous high nitrate burden were significantly increased from 34.8 to 123.3 and 77.0 to 260.4 pg/day, respectively. Whilst this reflects the role of cysteine on NTCA and NMTCA formation, the second possible role of cysteine as a nitrite scavenger via its interaction with nitrite to form Snitrosocysteine cannot be directly determined from these results. However, this may be reflected in the decreased urinary excretion of nitrate (days 36- .40 as compared to days 11-20) whilst the endogenous formation and excretion of NSAR, NPRO and NHPRO remains unchanged. The effect of cysteine on the nitrosation of naturally occurring amino acids in the diet and thiazolidine carboxylic acids formed by the reaction of cysteine with formaldehyde or acetaldehyde is currently under investigation. Under gastric conditions, two nitrite molecules are required to form a nitrosating species via the following reaction; NO; + H’ + HN02

2HN0,

= NzOj + H,O

Thus, on calculation of the total available nitrite and analysis of the daily excretion of N-nitrosoamino acids and nitrate, the percentage of available nitrite used in the endogenous formation of N-nitrosoamino acids can be

46 TABLE 2 PERCENTAGE OF AVAILABLE NITRITE USED IN THE ENDOGENOUS NITROSATION FORMATION OF N-NITROSAMINO ACIDS Diet regime

?&NO; used

Normal unrestricted diet Diet supplemented with 600 mg NO;/day Diet supplemented with 200 mg L-cysteine/day Diet supplemented with 600 mg NO; and 200 mg L-cysteine/day

0.08 0.14 0.13 0.20

*

calculated as shown in Table 2. Calculations were made using molar concentrations to account for both the molecular weight difference between the total available nitrite expressed in mg/day and the excreted N-nitrosamino acids in @g/day and the molecular weight differences of the individual Nnitrosamino acids. Under normal gastric conditions, 0.08% of the total available nitrite is used in the endogenous formation of N-nitrosamino acids, this figure is increased to 0.14% under a high nitrate burden. On supplementing the diet with 200 mg L-cysteine/day, a slight increase in the percentage of the accountable loss of nitrite was observed. However, this result indicates that the formation of NTCA and NMTCA is more dependent on the availability of both formaldehyde and acetaldehyde and not the availability of nitrite. Whilst the availability of exogenous N-nitrosamino acids was not determined in the diet, it is known that NSAR, NPRO, NTCA and NHPRO are found primarily in nitrite cured meat products [ 161. The avoidance of these products during the experimental period thus reduced the exogenous exposure to a negligible level compared to the level of these compounds formed by endogenous nitrosation. Nitrosation of dipeptide without imino amino acids under in vivo conditions to yield N-nitrosoiminodialkanoic acids [ 171 has been shown to occur; by a similar calculation, approx. 0.26% of the total available nitrite is used in the endogenous nitrosation of dipeptides to yield N-nitrosoiminodialkanoic acids. Thus on a quantitative basis, less than 0.5% of the total available nitrite can be accounted for during the endogenous nitrosation of simple dipeptides and amino acids to form N-nitroso derivatives. ACKNOWLEDGEMENT

The authors thank Dr. B. Spiegelhalder for useful discussion on the bacterial conversion of nitrate to nitrite in the oral cavity. REFERENCES 1 Ohshima, H., Friesen, M., O’Neill, I.K. and Bartsch, H. (1983) Presence in human urine of a new N-nitroso compound, N-nitrosothiaxolidine-4-carboxylic acid. Cancer Letters, 20, 183-190.

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