Analysis of L-tryptophan for the etiology of eosinophilia-myalgia syndrome

Morbidity and Mortality Weekly Report, August 31, 1990

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-trytophan 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<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.sub.24.H.sub.26.N.sub.4.O.sub.4], indicating that peak 97 contained two tryptophan molecules and an additional [C.sub.2.H.sub.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.

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 unindentified, 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 [micro-gram]/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

[1] CDC. Eosinophilia-myalgia syndrome - New Mexico. MMWR 1989;38:765-7.

[2] CDC. Eosinophilia-myalgia syndrome and L-tryptophan-containing products - Nex Mexico, Minnesota, Oregon, and New York, 1989. MMWR 1989;38:785-8.

[3] 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.

[4] 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.