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Analysis of L-Tryptophan for the Etiology of Eosinophilia-Myalgia
Syndrome
Eosinophilia-myalgia syndrome (EMS) has been associated with
consumption of L-tryptophan-containing products (LTCPs) (1,2) and
most
strongly associated with consumption of LTCPs produced by one
manufacturer (3-5). Epidemiologic and laboratory investigations
have
suggested that the implicated LTCPs were contaminated (3-5). To
further
examine this hypothesis, CDC and the Food and Drug Administration
(FDA)
conducted additional laboratory studies. This report summarizes
preliminary data that indicate that implicated LTCPs were
contaminated
with the di-tryptophan aminal of acetaldehyde (DTAA).
The laboratory investigation determined that case-associated
L-tryptophan (LT) cultures were negative for bacteria and viruses
and
that endotoxin levels were not elevated in case-associated LT (6).
Analysis of case-associated LT for 37 elements identified none at
toxicologically significant concentrations.
Fifty lots of LT produced between March 1985 and June 1989 by
the
implicated manufacturer were analyzed by high-performance liquid
chromatography (HPLC). Thirteen lots were linked to EMS cases;
other
lots were considered as controls because no link with cases could
be
identified. Several HPLC peaks (called peaks 97, 100, and 200) were
identified that were predictive of case-associated LT lots. Amounts
of
peak 97 in LT lots from the implicated manufacturer increased
dramatically between March and June 1989. Based on a Wilcoxon
rank-sum
test, peak 97 was the single most predictive peak (p less than
0.0001)
of case-associated LT lots. A bivariate plot of peaks 100 and 200
was
as predictive as peak 97. HPLC analysis of samples exchanged
between
CDC and the Mayo Clinic revealed that peak 97 is likely the same as
peak E (5).
HPLC was used to isolate peak 97 from case-associated LT lots.
Proton nuclear magnetic resonance indicated that peak 97 was a
tryptophan derivative with its characteristic aromatic indole
protons
and aliphatic protons but with an unusual doublet at a chemical
shift
of 2.2 ppm. HPLC combined with atmospheric pressure ionization/mass
spectrometry/mass spectrometry determined that peak 97 had a
molecular
weight of 434. High-resolution fast-atom bombardment mass
spectrometry
determined the exact mass of peak 97 to be 434.2020 corresponding
to a
molecular formula of C 24H 26N 4O 4, indicating that peak 97
contained
two tryptophan molecules and an additional C 2H 2. These data
suggested
that peak 97 was the DTAA (Figure 1A).
With LT as a standard, the concentration of peak 97 was
estimated
at 0.01% in a typical case-associated LT lot. Scientists at the
implicated manufacturer independently arrived at the same proposed
structure (R. Hinds, personal communication). Confirmation of this
structure by synthesizing DTAA is in progress. In addition to peaks
97,
100, and 200, ongoing investigation is directed at
1-methyl-1,2,3,4-tetrahydro-beta-carboline-3-carboxylic acid (MTCA)
(Figure 1B) and bacitracin, detected in LT lots from the implicated
manufacturer (6). MTCA could be produced from the breakdown of DTAA
or
independently formed.
Reported by: Sciex, Div of MDS Health Group, Ontario, Canada.
National
Institute of Arthritis and Musculoskeletal and Skin Diseases,
National
Institutes of Health. National Institute of Mental Health, Alcohol,
Drug Abuse and Mental Health Administration. Center for Food Safety
and
Applied Nutrition, Food and Drug Administration. Center for
Infectious
Diseases; Center for Environmental Health and Injury Control, CDC. Editorial Note
Editorial Note: The epidemiologic association of peak 97 and any
other
particular compound with EMS indicates that these compounds may
either
be the causative agent(s) or marker(s) for a different, as yet
unidentified, causative agent in case-associated LT lots. Based on
an
average daily dose of 2 g of LT for a 70-kg person and a 30-day
delay
before onset of EMS, the total dose of peak 97 is approximately 90
ug/kg. The toxic properties of the aminals are not well defined;
however, the suspected decomposition products, the beta-carbolines,
exhibit a variety of biologic properties (7).
The full definition of biologic and toxic effects of the
contaminants can be determined only in an animal model for EMS. A
joint
National Institute of Mental Health/National Institutes of
Health/FDA/CDC study has recently reproduced EMS-like changes in
rats
(E.M. Sternberg, personal communication). Synthesizing these
contaminants and testing them in the new rat model may help to
clarify
their relationship to the etiology and pathogenesis of EMS.
Continuing
studies include analyzing additional LT lots, identifying and
synthesizing contaminants, and attempting to associate changes in
the
manufacturing process with these contaminants.
References
CDC. Eosinophilia-myalgia syndrome--New Mexico. MMWR
1989;38:765-7.
CDC. Eosinophilia-myalgia syndrome and L-tryptophan-containing
products--New Mexico, Minnesota, Oregon, and New York, 1989. MMWR
1989;38:785-8.
Slutsker L, Hoesly FC, Miller L, Williams P, Watson JC, Fleming
DW.
Eosinophilia-myalgia syndrome associated with exposure to
tryptophan
from a single manufacturer. JAMA 1990;264:213-7.
Belongia EA, Hedberg CW, White KE, MacDonald KL, Osterholm MT.
Epidemiology of eosinophilia-myalgia syndrome in Minnesota.
Presented
at the 39th Annual Epidemic Intelligence Service Conference, CDC,
Atlanta, April 25, 1990.
Belongia EA, Hedberg CW, Gleich GJ, et al. An investigation of
the
cause of the eosinophilia-myalgia syndrome associated with
tryptophan
use. N Engl J Med 1990;323:357-65.
Needham L, Page S. Chemical analysis of LTCPS and related
samples.
Conference on Eosinophilia-Myalgia Syndrome, Los Alamos National
Laboratory, Los Alamos, New Mexico, June 12, 1990.
Buckholtz NS. Neurobiology of tetrahydro-beta-carbolines. Life
Sci
1980;27:893-903.
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