Isolation of mutagenic β-carboline derivatives after nitrite treatment of maillard reaction mixtures and analysis of these compounds from foodstuffs and human urine

Article abstract of DOI:10.1271/bbb.69.2232

Mixtures of carbohydrate decomposition products and L-tryptophan were incubated at pH 7.0 and 37°C for 4 weeks. These mixtures exhibited mutagenic activity toward S. typhimurium TA 100 without metabolic activation after a nitrite treatment at pH 4.0. Four β-carboline derivatives were isolated as premutagens from mixtures of methylglyoxal and furfural. These premutagens were also found to be contained in daily foodstuffs and human urine samples.

Full text of DOI:10.1271/bbb.69.2232

Biosci. Biotechnol. Biochem., 69 (11), 2232–2235, 2005
Note
Isolation of Mutagenic ꢀ-Carboline Derivatives after Nitrite
Treatment of Maillard Reaction Mixtures and Analysis
of These Compounds from Foodstuffs and Human Urine
1
;y
2
1
Shuichi MASUDA, Hisayuki KANAMORI, and Naohide KINAE
1
Department of Food and Nutritional Sciences, Graduate School of Nutritional and Environmental Sciences and
st
COE Program in the 21 Century, University of Shizuoka, 52-1 Yada, Suruga-ku, Shizuoka 422-8526, Japan
2
Hiroshima Prefectural Institute of Public Health and Environment Center, 1-6-29 Minami-cho, Minami-ku,
Hiroshima 734-0007, Japan
Received May 2, 2005; Accepted July 28, 2005
Mixtures of carbohydrate decomposition products
ꢀ
glucosone, furfural, methylglyoxal or glyoxal) and
0.05 mol of L-tryptophan was dissolved in 500 ml of a
0.1 M phosphate buffer solution at pH 7.0 and kept at
and L-tryptophan were incubated at pH 7.0 and 37 C
for 4 weeks. These mixtures exhibited mutagenic
activity toward S. typhimurium TA 100 without meta-
bolic activation after a nitrite treatment at pH 4.0. Four
ꢀ
37 C for 4 weeks. This solution (100 ml) was then
mixed with 200 ml of a 0.2 M acetate buffer solution at
pH 4.0 and 100 ml of a 0.3 M sodium nitrite solution.
ꢀ
-carboline derivatives were isolated as premutagens
ꢀ
from mixtures of methylglyoxal and furfural. These
premutagens were also found to be contained in daily
foodstuffs and human urine samples.
The solution was kept at 37 C for 1 hr, before 100 ml of
a 0.3 M ammonium sulfate solution was added to stop the
reaction. The resulting solution was lyophilized. The
lyophilized concentrate was used as sample for sub-
mission to a mutagenic assay essentially as reported by
Key words: ꢀ-carboline; Maillard reaction; nitrite; mu-
tagenic activity
5
)
Ames et al. The numbers of revertant colonies given in
figures are averages for three plates. 4NQO was used as
a positive control (0.3 mg/plate).
Several reaction mixtures of carbohydrates and amino
acids have shown mutagenic activities toward certain
þ
Figure 1 shows the mutagenic activity (Net His
1
,2)
bacteria and/or mammalian cells.
The browning
revertants/200 mg of a sample (the lyophilized Maillard
reaction mixture after a nitrite treatment)/plate) toward
S. typhimurium TA 100 without metabolic activation.
Each sample of the carbohydrate decomposition prod-
ucts and L-tryptophan showed no mutagenic activity
before the nitrite treatment. However, the mixtures of
furfural, methylglyoxal and glyoxal after the nitrite
treatment each showed strong mutagenic activity. In
particular, the reaction mixture containing furfural
exhibited the strongest mutagenic activity after the
nitrite treatment.
reaction mixture consisting of L-ascorbic acid and L-
tryptophan showed DNA-damaging and mutagenic
effects on S. typhimurium TA 100 without metabolic
activation. We have identified ꢀ-carboline derivatives
3
)
from the reaction mixtures. We have also reported that
-carboline derivatives, which were formed in the
mixtures of 2-furaldehyde and L-tryptophan, showed
ꢀ
4
)
mutagenic activity after a nitrite treatment. Such other
carbohydrate decomposition products as 3-deoxy-D-
glucosone, furfural, methylglyoxal, and glyoxal are
formed in the Maillard reaction. However, the formation
of ꢀ-carboline derivatives in the reaction mixtures of
these carbohydrate decomposition products with L-
tryptophan has not been reported. The extent of human
exposure to ꢀ-carboline derivatives remains undeter-
mined.
We then determined the products after the reaction of
methylglyoxal or furfural with L-tryptophan. The lyophi-
lized samples were extracted with methanol, and the
resulting methanol extracts were analyzed by HPLC,
using an SPD-6AV UV detector (Shimadzu Co., Ltd.,
Kyoto, Japan), an L-7100 intelligent pump (Hitachi Ltd.,
Tokyo, Japan), and a L-7300 column oven (Hitachi Ltd.,
Tokyo Japan). The UV wavelength was 254 nm. Each
sample was chromatographed in a YMC-Pack A-314
column (6.0 mm i.d. and 300 mm in length; YMC Co.,
We report in this paper the isolation of ꢀ-carboline
derivatives as premutagens from the reaction mixtures
of some carbohydrate decomposition products and L-
tryptophan. Furthermore, we describe the detection of
these premutagens in daily foodstuffs and urine samples.
A mixture of 0.1 mol of a carbohydrate (3-deoxy-D-
ꢀ
Ltd., Kyoto, Japan) at 40 C, the flow rate being 0.8 ml/
min. The mobile phase was a mixture of methanol and
y
To whom correspondence should be addressed. Tel: +81-54-264-5526; Fax: +81-54-264-5528; E-mail: masudas@u-shizuoka-ken.ac.jp

Isolation of ꢀ-Carboline Derivatives in Maillard Reaction Mixtures, Foodstuffs and Human Urine
2233
Fig. 1. Mutagenic Activities of Reaction Mixtures from the Carbonyl Compounds and L-Tryptophan.
DG, 3-deoxy-D-glucosone; Fur, furfural; MG, methylglyoxal; GO, glyoxal; Trp, L-tryptophan; 4NQO, 4-nitroquinoline 1-oxide. Each
sample was the lyophilized Maillard reaction mixture after a nitrite treatment. 4NQO was used as a positive control.
ꢁ
3
water (45/55, v/v), which was isocratically eluted.
The HPLC analysis enabled us to isolate 3,4-dinitro-
activities of these compounds were increased by the
nitrite treatment under acidic conditions.
1
and 1-methyl-1,2,3,4-tetrahydro-9H-pyrido[3,4-b]in-
-methyl-9H-pyrido[3,4-b]indole-3-carboxylic acid (I)
We next quantified the premutagens in three daily
foodstuffs. We lyophilized 100 ml of soy sauce and
300 ml of beer and extracted the residue with methanol.
Bean paste (50 g) was concentrated to dryness under
reduced pressure and then applied to a Sephadex LH-20
column. Elution was carried out with methanol at a flow
rate of 45 ml/hr, the eluate being separated into 3–4
fractions by measuring the optical density at 254 nm.
The eluate corresponding to the retention time of each
standard compound was fractionated and rechromato-
graphed under the same conditions. Table 2 shows the
content of premutagens in soy sauce, bean paste and
beer. Compounds I, II and IV were respectively con-
tained in amounts of 1724.1, 5268.9, 293.2 mg/100 ml in
soy sauce. The respective contents of compounds II, III
and IV were 253.8, 1226.0, 232.8 mg/100 g in bean
paste, while the concentrations of these compounds in
beer were lower than in two other foodstuffs.
dole-3-carboxylic acid (II) from the solution of meth-
ylglyoxal and L-tryptophan (I, 2.6 mg/200 mg of sample;
0
II, 7.8 mg/200 mg of sample). 1,2,3,4-Tetrahydro-1-(5 -
hydroxymethylfuryl)-9H-pyrido[3,4-b]indole-3-carbox-
ylic acid (III) and 1,2,3,4-tetrahydro-1-furyl-9H-pyri-
do[3,4-b]indole-3-carboxylic acid (IV) were identified
from the reaction mixture of furfural and L-tryptophan
(III, 18.6 mg/200 mg of sample; IV, 8.7 mg/200 mg of
sample). These ꢀ-carboline derivatives have been
identified in some Maillard reaction mixtures, which
had been respectively treated with L-ascorbic acid and
3
)
4)
L-tryptophan, and 2-furaldehyde and L-tryptophan, as
6
)
well as in soy sauce. We examined the mutagenicity of
these products by a nitrite treatment under acidic
conditions. They all showed high mutagenic activity
þ
(
I, 3100; II, 12100; III, 28750; IV, 7700 Net His revs./
mg) after the nitrite treatment (Table 1). Compounds II
and III exhibited equal mutagenicity to that of the
reference compound. The nitrite treatment of these ꢀ-
carboline derivatives might have produced the N-nitroso
compounds. These results indicate that compounds I, II,
III and IV were formed in the reaction mixtures of the
carbohydrate decomposition products such as furfural or
methylglyoxal and L-tryptophan, and that the mutagenic
We also investigated the contents of these premuta-
gens in the urine of subjects taking a normal diet. The
experimental procedure used in this study was approved
by the Research Ethics Committee of University of
Shizuoka. Urine samples were collected for 18 hr after
consumption of the diet. Acetic acid was added to each
sample to adjust to pH 3.0, before filtrating thorough an
Amberlite XAD-2 column and washing the resin in

2234
S. MASUDA et al.
Table 1. Determination of Mutagenic ꢀ-Carbolines in Maillard Reaction Mixtures
Mutagenic activity toward TA 100,
þ
Maillard reaction
mixture
Concentration
(mg/200 mg of sample )
(
Net His revs./mg)
Product
ꢁ
Untreated
Treated with nitrite
I
MG + Trp
MG + Trp
Fur + Trp
Fur + Trp
2.6
7.8
0
0
3100
12100 (10,400)
28750 (22,750)
7700
6
4
)
ꢁꢁ
ꢁꢁ
II
)
III
IV
18.6
8.7
0
2400
MG, methylglyoxal; Fur, furfural; Trp, L-tryptophan.
ꢁ
,
ꢁ
Samples were obtained by lyophilizing Maillard reaction mixtures after a nitrite treatment.
Values in parentheses were cited from references.
ꢁ
,
ꢀ
HPLC conditions: A UV wavelength of 254 nm was used. Each sample was chromatographed in a YMC-Pack A-314 column at 40 C. The flow rate was 0.8 ml min.
The mobile phase was a mixture of methanol and water (45/55, vv), which was eluted isocratically.
revealed that these products were present in daily
Table 2. Content of ꢀ-Carbolines in Daily Foodstuffs and Urine
Samples
foodstuffs and in human urine. These results indicate
the possibility that the reaction might be developed in
food-making processes and in vivo. Therefore, these
premutagens would play an important role in the
induction of gastric cancer in Japan. We need to identify
the products from the reaction between the premutagens
and nitrite, and further details will be reported in the
near future.
Contents
Sample
I
II
III
IV
Soy sauce
Bean paste
Beer
1724.1
<0.1
52.1
5268.9
253.8
12.2
ND
1226.0
<0.1
293.2
232.8
5.1
Foodstuffs
Urine
Subject A
ND
ND
66.7
328.9
36.2
ND
ND
ND
ND
6.8
ND
4.5
B
C
D
37.1
26.6
References
201.2
3.6
Contents: Foodstuffs (mg/100 ml or 100 g)
Urine (mg/l)
HPLC conditions: A UV wavelength of 254 nm was used. Each sample
ND, not detected.
1) Cuzzoni, M. T., Stoppini, G., Gazzani, G., and Mazza,
P., Effect of storage on mutagenicity, absorbance and
content of furfural in the ribose-lysine Maillard system.
Food Chem. Toxicol., 27, 209–213 (1989).
ꢀ
was chromatographed in a YMC-Pack A-314 column at 40 C. The flow rate
was 0.8 ml/min. The mobile phase was a mixture of methanol and water
2
)
Glosl, S., Wagner, K. H., Draxler, A., Kaniak, M.,
Lichtenecker, S., Sonnleitner, A., Somoza, V.,
Erbersdobler, H., and Elmadfa, I., Genotoxicity and
mutagenicity of melanoidins isolated from a roasted
glucose–glycine model in human lymphocyte cultures,
intestinal Caco-2 cells and in Salmonella typhimurium
strains TA98 and TA102 by applying the Ames test.
Food Chem. Toxicol., 42, 1487–1495 (2004).
(
45/55, v/v), which was eluted isocratically.
methanol. The methanol extract was evaporated under
reduced pressure to give a sample for the HPLC
analysis. Table 2 shows the contents of premutagens in
the urine samples. Compound II was contained at the
highest concentration (36.2–328.9 mg/l). Compounds I
and IV were each contained at a low concentration,
while compound III could not be detected in any
sample.
3
)
Kanamori, H., Morimoto, K., Kinae, N., and Tomita, I.,
Mutagenicity of the reaction products of L-ascorbic acid
and L-tryptophan. Yakugaku Zasshi (in Japanese), 101,
596–604 (1981).
Several ꢀ-carboline derivatives other than the pre-
mutagens isolated in our study have been found in
4) Kanamori, H., Kinae, N., Saito, M., and Tomita, I.,
Isolation and identification of 1-furyl-ꢀ-carboline deriv-
atives that are mutagenic after nitrite treatment. Chem.
Pharm. Bull., 32, 1980–1986 (1984).
7)
8)
9)
10)
tobacco tar, sake, alcoholic beverages, fruits, and
cheese.11) These ꢀ-carboline derivatives, such as norhar-
man, harman, harmol and harmalol, exhibited mutagenic
5
)
McCann, J., Spingarn, N. E., Kobori, J., and Ames,
B. N., Detection of carcinogens as mutagens: Bacterial
tester strains with R factor plasmids. Proc. Natl. Acad.
Sci. U.S.A., 72, 979–983 (1975).
1
2)
activity after a nitrite treatment. We isolated four
premutagens from the reaction mixtures of carbohydrate
decomposition products of methylglyoxal and furfural
6
)
Wakabayashi, K., Ochiai, M., Saito, H., Tsuda, M.,
Suwa, Y., Nagao, M., and Sugimura, T., Presence of 1-
methyl-1,2,3,4-tetrahydro-ꢀ-carboline-3-carboxylic acid,
a precursor of mutagenic nitroso compounds, in soy
with L-tryptophan kept under mild conditions (pH 7.0,
ꢀ
3
7 C), and have demonstrated that these premutagens
showed mutagenicity after a nitrite treatment. We also

Isolation of ꢀ-Carboline Derivatives in Maillard Reaction Mixtures, Foodstuffs and Human Urine
sauce. Proc. Natl. Acad. Sci. U.S.A., 80, 2912–2916 tions. Alcohol Clin. Exp. Res., 12, 679–682 (1988).
1983).
Pfau, W., and Skog, K., Exposure to beta-carbolines
norharman and harman. J. Chromatogr. B Analyt.
Technol. Biomed. Life Sci., 802, 115–126 (2004).
2235
(
10) Kartal, M., Altun, M. L., and Kurucu, S., HPLC method
for the analysis of harmol, harmalol, harmine and
harmaline in the seeds of Peganum harmala L. J. Pharm.
Biomed. Anal., 31, 263–269 (2003).
7
)
)
8
Tomita, I., Kinae, N., Nakamura, Y., Takenaka, H.,
Kanamori, H., Hashizume, H., and Yokoyama, T.,
Mutagenicity of various Japanese foodstuffs treated
with nitrite. II. Directly-acting mutagens produced from
N-containing compounds in foodstuffs. IARC Sci. Publ.,
11) Herraiz, T., and Sanchez, F., Presence of tetrahydro-
beta-carboline-3-carboxylic acids in foods by gas chro-
matography–mass spectrometry as their N-methoxy-
carbonyl methyl ester derivatives. J. Chromatogr. A,
765, 265–277 (1997).
5
7, 33–41 (1984).
12) Lin, J. K., Wu, S. S., and Chen, J. T., Mutagenicities of
nitrosated carboline derivatives. Proc. Natl. Sci. Counc.
Repub. China B, 10, 280–286 (1986).
9)
Bosin, T. R., and Faull, K. F., Harman in alcoholic
beverages: pharmacological and toxicological implica-