Formation of EBT
J. Agric. Food Chem., Vol. 44, No. 5, 1996 1229
suggests that an addition of one proton to the EBT
molecule was involved in the decomposition.
The pH of gastric fluid in humans is much lower than
that in rats. EBT will decompose much more rapidly
in humans than in rats. This difference should be
considered when animal feeding data are used to
explain EBT metabolism in humans.
EBT was formed from the reaction of L-tryptophan
and acetaldehyde under acidic conditions. The rate of
EBT formation decreased with increasing pH and
decreasing temperature, and the presence of excess
acetaldehyde increased EBT formation to a lesser extent
than did the presence of excess L-tryptophan.
[L-tryptophan], a compound associated with eosinophilia-
myalgia syndrome. Bull. Environ. Contam. Toxicol. 1992,
4
8, 679-687.
Hill, R. H.; Gaudill, S. P.; Philen, R. M. Contaminants in
L-tryptophan associated with eosinophilia myalgia syn-
drome. Arch. Environ. Contam. Toxicol. 1993, 25, 134-142.
Ito, J .; Hosaki, Y.; Torigoe, Y.; Sakimoto, K. Identification of
substances formed by decomposition of peak E substance
in tryptophan. Food Chem. Toxicol. 1992, 30, 71-81.
Love, L. A.; Rader, J . I.; Crofford, L. J .; Raybourne, R. B.;
Principato, M. A.; Page, S. W.; Trucksess, M. W.; Smith, M.
J .; Dugan, E. M.; Turner, M. L.; Zelazonski, E.; Zelazonski,
P.; Sternberg, E. M. Pathological and immunological effects
of ingesting L-tryptophan and 1,1′-ethylidenebis[L-tryp-
tophan] in Lewis rats. J . Clin. Invest. 1993, 91, 804-811.
Lund, D. B. Heat processing. In Principles of Food Science.
Part II. Physical Principles of Food Preservation; Fennema,
O. R., Ed.; Dekker: New York, NY, 1975; Chapter 3.
Mayeno, A. N.; Lin, F.; Foote, C. S.; Loegering, D. A.; Ames,
M. M.; Hedberg, C. W.; Gleich, G. J . Characterization of
“peak E,” a novel amino acid associated with the eosino-
philia-myalgia syndrome. Science 1990, 250, 1707-1708.
Ogawa, Y.; Adachi, J .; Tatsuno, Y. Accumulation of 1-methyl-
tetrahydro-â-carboline-3-carboxylic acid in blood and organs
of rat. A possible causative substance of eosinophilia-
myalgia syndrome associated with ingestion of L-tryptophan.
Arch. Toxicol. 1993, 67, 290-293.
In the commercial process that resulted in the pro-
duction of EMS-associated L-tryptophan, the only unit
operation that was performed under acidic conditions
was the cation-exchange chromatography. Therefore,
it is likely that EBT was formed during the absorption
stage of the cation-exchange operation. At the concen-
trations of L-tryptophan (0.24 M) and acetaldehyde (9.1
-
5
×
10 M) found in the commercial process, the maxi-
mum amount of EBT formed in solution was 0.27 µg/
mL (5.6 ppm with respect to the initial L-tryptophan
concentration) at pH 1.75 and 25 °C. This value is only
1
about /10 of that observed by Sakimoto and Torigoe
Sakimoto, K.; Torigoe, Y. Study of mechanism of peak E
substance formation in process for manufacture of L-
tryptophan. Report submitted to U.S. Food and Drug
Administration, April 1992.
(
1992) in the cation-exchange process, indicating that
the rate of EBT formation in a column matrix differs
from that in solution.
Silver, R. M.; Ludwicka, A.; Hampton, M.; Ohba, T.; Bingel,
S. A.; Smith, T.; Harley, R. A.; Maize, J .; Hehes, M. P. A
Murine model of the eosinophilia myalgia syndrome induced
by 1,1′-ethylidenebis[L-tryptophan]. J . Clin. Invest. 1994, 93,
1473-1480.
Slutsker, L.; Hoesly, F. C.; Miller, L.; Williams, L. P.; Watson,
J . C.; Fleming, D. W. Eosinophilia-myalgia syndrome as-
sociated with exposure to tryptophan from a single manu-
facturer. J AMA, J . Am. Med. Assoc. 1990, 264, 213-217.
Smith, M. J .; Mazzola, E. P.; Farrell, T. J .; Sphon, J . A.; Page,
S. W.; Ashley, D.; Sirimanne, S. R.; Hill, R. H.; Needham,
L. L. 1,1′-Ethylidenebis-(L-tryptophan), structure determi-
nation of contaminant “97”-implicated in the eosinophilia-
myalgia syndrome (EMS). Tetrahedron Lett. 1991, 32, 991-
994.
ACKNOWLEDGMENT
I am grateful to Drs. Samuel Page, Mitchell Smith,
Terryl Farrell, and J oseph Betz for providing technical
information and to Dr. Steven Gendel for helpful com-
ments and suggestions about the manuscript.
LITERATURE CITED
Adachi, J .; Yamamoto, K.; Ogawa, Y.; Ueno, Y.; Mizoi, Y.;
Tatsuno, Y. Endogenous formation of 1-methyl-1,2,3,4,-
tetrahydro-â-carboline-3-carboxylic acid in man as the pos-
sible causative substance of eosinophilia-myalgia syndrome
associated with ingestion of L-tryptophan. Arch. Toxicol.
1
991, 65, 505-509.
Toyo’oka, T.; Yamazaki, T.; Tanimoto, T.; Sato, K.; Sato, M.;
Toyoda, M.; Ishibashi, M.; Yoshihira, K.; Uchiyama, M.
Characterization of contaminants in EMS-associated L-
tryptophan samples by high-performance liquid chromatog-
raphy. Chem. Pharm. Bull. 1991, 39, 820-822.
Adachi, J .; Naito, T.; Ueno, Y.; Ogawa, Y.; Ninomiya, I.;
Tatsuno, Y. Metabolism and distribution in the rat of peak
E substance, a constituent in L-tryptophan product impli-
cated in eosinophilia-myalgia syndrome. Arch. Toxicol.
1
993a , 67, 284-289.
Wakabayashi, K.; Ochiai, M.; Saito, H.; Tsuda, M.; Suwa, Y.;
Nagao, M.; Sugimura, T. Presence of 1-methyl-1,2,3,4-
tetrahydro-â-carboline-3-carboxylic acid, a precursor of mu-
tagenic nitroso compound, in soya sauce. Proc. Natl. Acad.
Sci. U.S.A. 1983, 80, 2912-2916.
Adachi, J .; Ueno, Y.; Ogawa, Y.; Hishida, S.; Yamamoto, K.;
Ouchi, H.; Tatsuno, Y. Acetaldehyde-induced formation of
1
-methyl-1,2,3,4-tetrahydro-â-carboline-3-carboxylic acid in
rats. Biochem. Pharmcol. 1993b, 45, 935-941.
Belongia, E. A.; Hedberg, C. W.; Gleich, G. J .; White, K. E.;
Mayeno, A. N.; Loegering, D. A.; Dunnette, S. L.; Pirie, P.
L.; MacDonald, K. L.; Osterholm, M. T. An investigation of
the cause of the eosinophilia-myalgia syndrome associated
with tryptophan use. N. Engl. J . Med. 1990, 323, 357-365.
Brehm, W. J .; Lindwall, H. Carboline syntheses. J . Org. Chem.
Yamaoka, K. A.; Miyasaka, N.; Inuo, G.; Saito, I.; Kolb, J -P.;
Fujita, K.; Kashiwazaki, S. 1,1′-Ethylidenebis(tryptophan)
(peak E) induces functional activation of human eosinophils
and interleukin 5 production from T lymphocytes: associa-
tion of eosinophilia-myalgia syndrome with a L-tryptophan
contaminant. J . Clin. Immunol. 1994, 14, 50-60.
1
950, 15, 685-694.
Calabrese, E. J . Principles of Animal Extrapolation; Wiley:
New York, 1983; pp 46-47.
X
Received for review J uly 7, 1995. Accepted March 13, 1996.
Dixon, M. The effect of pH on the affinities of enzymes for
substrates and inhibitors. Biochem. J . 1953, 155, 161.
Driskell, W. L.; Ashley, D. L.; Grainger, J .; Sirimanne, S. R.;
Mazzola, E. P.; Page, S. W.; Needham, L. L.; Hill, R. H.
Identification of decomposition products of 1,1′-ethylidenebis-
J F950421D
X
Abstract published in Advance ACS Abstracts, May
1, 1996.