Peak AAA fatty acid homolog contaminants present in the dietary supplement l-Tryptophan associated with the onset of eosinophilia-myalgia syndrome.

The eosinophilia-myalgia syndrome (EMS) outbreak that occurred in the USA and elsewhere in 1989 was caused by the ingestion of Showa Denko K.K. (SD) L-tryptophan (L-Trp). "Six compounds" detected in the L-Trp were reported as case-associated contaminants. Recently the final and most statistically significant contaminant, "Peak AAA" was structurally characterized. The "compound" was actually shown to be two structural isomers resulting from condensation reactions of L-Trp with fatty acids derived from the bacterial cell membrane. They were identified as the indole C-2 anteiso (AAA1-343) and linear (AAA2-343) aliphatic chain isomers. Based on those findings, we utilized a combination of on-line HPLC-electrospray ionization mass spectrometry (LC-MS), as well as both precursor and product ion tandem mass spectrometry (MS/MS) to facilitate identification of a homologous family of condensation products related to AAA1-343 and AAA2-343. We structurally characterized eight new AAA1-XXX/AAA2-XXX contaminants, where XXX represents the integer molecular ions of all the related homologs, differing by aliphatic chain length and isomer configuration. The contaminants were derived from the following fatty acids of the bacterial cell membrane, 5-methylheptanoic acid (anteiso-C8:0) for AAA1-315; n-octanoic acid (n-C8:0) for AAA2-315; 6-methyloctanoic acid (anteiso-C9:0) for AAA1-329; n-nonanoic acid (n-C9:0) for AAA2-329; 10-methyldodecanoic acid (anteiso-C13:0) for AAA1-385; n-tridecanoic acid (n-C13:0) for AAA2-385; 11-methyltridecanoic acid (anteiso-C14:0) for AAA1-399; and n-tetradecanoic acid (n-C14:0) for AAA2-399. The concentration levels for these contaminants were estimated to be 0.1-7.9 µg / 500 mg of an individual SD L-Trp tablet or capsule The structural similarity of these homologs to case-related contaminants of Spanish Toxic Oil Syndrome (TOS) is discussed.

Structure determination of disease associated peak AAA from l-Tryptophan implicated in the eosinophilia-myalgia syndrome.

The eosinophilia-myalgia syndrome (EMS) outbreak of 1989 that occurred in the USA and elsewhere was caused by the ingestion of l-Tryptophan (L-Trp) solely manufactured by the Japanese company Showa Denko K.K. (SD). Six compounds present in the SD L-Trp were reported to be case-associated contaminants. However, "one" of these compounds, Peak AAA has remained structurally uncharacterized, despite the fact that it was described as "the only statistically significant (p=0.0014) contaminant". Here, we employ on-line microcapillary-high performance liquid chromatography-electrospray ionization mass spectrometry (LC-MS), and tandem mass spectrometry (MS/MS) to determine that Peak AAA is in fact two structurally related isomers. Peak AAA1 and Peak AAA2 differed in LC retention times, and were determined by accurate mass-LC-MS to both have a protonated molecular ion (MH+) of mass 343.239Da (Da), corresponding to a molecular formula of C21H30N2O2, and possessing eight degrees of unsaturation (DoU) for the non-protonated molecule. By comparing the LC-MS and LC-MS-MS retention times and spectra with authentic synthetic standards, Peak AAA1 was identified as the intermolecular condensation product of L-Trp with anteiso 7-methylnonanoic acid, to afford (S)-2-amino-3-(2-((S,E)-7-methylnon-1-en-1-yl)-1H-indol-3-yl)propanoic acid. Peak AAA2 was determined to be a condensation product of L-Trp with decanoic acid, which produced (S)-2-amino-3-(2-((E)-dec-1-en-1-yl)-1H-indol-3-yl)propanoic acid.

Effects and side effects associated with the non-nutritional use of tryptophan by humans.

The daily nutritional requirement for L-tryptophan (Trp) is modest (5 mg/kg). However, many adults choose to consume much more, up to 4-5 g/d (60-70 mg/kg), typically to improve mood or sleep. Ingesting L-Trp raises brain tryptophan levels and stimulates its conversion to serotonin in neurons, which is thought to mediate its actions. Are there side effects from Trp supplementation? Some consider drowsiness a side effect, but not those who use it to improve sleep. Though the literature is thin, occasional side effects, seen mainly at higher doses (70-200 mg/kg), include tremor, nausea, and dizziness, and may occur when Trp is taken alone or with a drug that enhances serotonin function (e.g., antidepressants). In rare cases, the "serotonin syndrome" occurs, the result of too much serotonin stimulation when Trp is combined with serotonin drugs. Symptoms include delirium, myoclonus, hyperthermia, and coma. In 1989 a new syndrome appeared, dubbed eosinophilia myalgia syndrome (EMS), and was quickly linked to supplemental Trp use. Key symptoms included debilitating myalgia (muscle pain) and a high peripheral eosinophil count. The cause was shown not to be Trp but a contaminant in certain production batches. This is not surprising, because side effects long associated with Trp use were not those associated with the EMS. Over 5 decades, Trp has been taken as a supplement and as an adjunct to medications with occasional modest, short-lived side effects. Still, the database is small and largely anecdotal. A thorough, dose-related assessment of side effects remains to be conducted.

Strain-dependent acute lung injury after intra-tracheal administration of a 'refined' aniline-denatured rapeseed oil: a murine model of the toxic oil syndrome?

Most attempts to reproduce the toxic oil syndrome in animals, either with case-related oils or with refined rapeseed oils, have been unsuccessful. An aniline-denatured rapeseed oil that was subsequently refined according to a protocol yielding relevant markers of "toxic oil" (oil RSO160401) had led to possibly relevant lesions following oral administration in mice. Therefore, in the present study, RSO160401 was subjected to a more extended in vivo testing. To try and maximize the response, BALB/c, DBA/2, A/J, and C57BL/6 mice were administered RSO160401 oil by a single intra-tracheal instillation (1ml/kg), with sacrifice 2 or 7 days post-exposure. Intra-tracheal administration led to a strain-dependent acute response: acute pulmonary damage in DBA/2 and A/J mice, and increases in blood eosinophilia in DBA/2 mice (6.5% vs 3.1% in controls). The pulmonary lesions regressed with time after exposure, being more complete in A/J than in DBA/2 mice. The observation of strain-dependent effects suggests that genetic susceptibility is an important factor in disease induction by the RSO160401 oil.

A heretofore undisclosed crux of eosinophilia-myalgia syndrome: compromised histamine degradation.

In contrast to early epidemiological evidence offering links between eosinophilia-myalgia syndrome (EMS) and microimpurities of L-tryptophan-containing dietary supplements (LTCDS), this account shows why reliance on a finite impurity from one manufacturer is both unnecessary and insufficient to explain the etiology of EMS. Excessive histamine activity has induced blood eosinophilia and myalgia (Greek: mys, muscle + algos, pain). Termination of the multiple actions of histamine is dependent on particular amine oxidases and histamine-N-methyltransferase. Histamine metabolism is rapid when these degradative reactions are operative. The latent effects of incurred histamine can be potentiated and aggravating when these mechanisms are impaired. Overloads of tryptophan supplements cause - among other relevant side-effects - an increased formation of formate and indolyl metabolites, several of which inhibit the degradation of histamine. Moreover, (non-EMS) subjects with hypothalamic-pituitary- adrenal (HPA) axis dysregulation have also manifested greatly increased sensitivities to incurred tryptophan and histamine. A final common pathway for syndromes characterized by eosinophilia with myalgia is now evident.

Toxic oil syndrome: survival in the whole cohort between 1981 and 1995.

BACKGROUND AND OBJECTIVE: In 1981, toxic oil syndrome (TOS) appeared in Spain, affecting more than 20,000 persons and causing over 2500 deaths to date. Previous studies have addressed mortality only by gender and age. We analyzed possible prognostic factors in the survival of the cohort. METHODS: The study period was 1 May 1981 to 31 December 1995 (31 December 1995 was the cut-off date for survivors). The study population consisted of the entire cohort. Overall mortality and TOS-related deaths were studied. Kaplan-Meier method and Cox regression were used in the analyses. RESULTS: Among the 20,084 subjects in the cohort, 12,164 (60.6%) were women, and 7917 (39.4%) were men. Of the 1799 deaths, 958 (53.3%) were women, and 841 (46.71%) were men; of the 356 TOS-related deaths, 234 (65.7%) were women, and 122 (34.3%) were men. TOS was the leading cause of death among subjects <40 years of age. Among the TOS-related deaths, the shortest survival times were for women and subjects <40 years of age. The major disease manifestations had the highest relative risks (RR) (liver disease, RR 3.83; pulmonary infection, RR 1.54; motor neuropathy, RR 2.24; pulmonary hypertension, RR 3.19; and eosinophilia, RR 1.14.). CONCLUSIONS: The major clinical manifestations showed worse prognosis for overall and TOS-related mortality. Application of these results to the survivors will help clarify the validity of these conclusions.

Eosinophilia-myalgia syndrome case-associated contaminants in commercially available 5-hydroxytryptophan.

Recently, 5-hydroxy-L-tryptophan (5-OHTrp) has been promoted as an alternative to banned L-tryptophan as a dietary supplement. It has been claimed to help alleviate obesity, insomnia, depression, and headaches. However, eosinophilia-myalgia syndrome (EMS)-like symptoms have also been associated with ingestion or exposure to 5-OHTrp. HPLC-UV analysis of EMS-implicated 5-OHTrp revealed the presence of peak X, described as case-implicated. We show that peak X is actually a family of contaminants with the same molecular weight (234 Da) and similar HPLC retention times. We also demonstrate that all eight samples of commercially available 5-OHTrp analyzed by HPLC-MS contained three or more contaminants of the peak X family. The significance of these findings is discussed.

Rapid HPLC screening method for contaminants found in implicated L-tryptophan associated with eosinophilia myalgia syndrome and adulterated rapeseed oil associated with toxic oil syndrome.

In 1981 a massive food-borne epidemic, termed the toxic oil syndrome (TOS), occurred in Spain. Eight years later a closely related disease, the eosinophilia myalgia syndrome (EMS), was reported in the USA with many additional cases being reported worldwide. Although EMS was linked to the ingestion of contaminated L-tryptophan and TOS to aniline denatured rapeseed oil, the etiological agent(s) responsible for both diseases remains unknown. However, contaminants in both the oil and the dietary supplement are believed to have triggered these diseases, and there has been much speculation that a common contaminant may have caused both epidemics. In this report, methods for the facile preparation and HPLC analysis of EMS-implicated L-tryptophan and adulterated rapeseed oil samples associated with TOS are described which allow a direct comparison between the contaminants of both foodstuffs. A combination of solvent and solid phase extraction methods are demonstrated along with the application of C18 reversed-phase high-performance liquid chromatography (RP-HPLC) coupled with on-line UV and MS detection. These methods have allowed us to determine for the first time, based upon this work, that there are no detectable common contaminants that possess a UV response, between EMS implicated L-tryptophan and TOS implicated rapeseed oil samples.

Structural characterization of contaminants found in commercial preparations of melatonin: similarities to case-related compounds from L-tryptophan associated with eosinophilia-myalgia syndrome.

On-line HPLC/electrospray ionization-tandem mass spectrometry (LC/ESI-MS/MS) in conjunction with NMR has been successfully employed to identify and structurally characterize seven contaminants found in three different commercial preparations of melatonin. Six of these contaminants were identified as analogues of impurities found in contaminated L-tryptophan (an over-the-counter dietary supplement) associated with the eosinophilia-myalgia syndrome (EMS) epidemic that occurred in the United States during 1989. In particular, our studies identified two compounds with MH+ = 249 to be hydroxymelatonin isomers. Four other compounds with MH+ = 477 were identified as melatonin-formaldehyde condensation products. These compounds are structural analogues of L-tryptophan contaminants, namely, 'peak C' and 'peak E' that were previously implicated as etiological agents causing EMS. It has been reported that melatonin consumption has resulted in eosinophilia in some humans taking high doses of this supplement. Although there has not been a major outbreak of EMS-like symptoms from consumption of melatonin, this study clearly suggests that tighter control and regulation of nutritional supplements sold and used as drugs is necessary.

Eosinophilia-myalgia syndrome and tryptophan production: a cautionary tale.

An epidemic of a new disease, termed eosinophilia-myalgia syndrome, occurred in the USA in 1989. This syndrome was linked to the consumption of L-tryptophan manufactured by a single company utilizing a fermentation process. All the findings indicate that the illness was probably triggered by an impurity formed when the manufacturing conditions were modified. This outbreak highlights the need for close monitoring of the chemical purity of biotechnology-derived products, and for rigorous testing of such products following any significant changes to the manufacturing process.

Toxic oil syndrome and eosinophilia-myalgia syndrome: May 8-10, 1991, World Health Organization meeting report.

In May 1991, researchers and clinicians from throughout the world met at a workshop sponsored by the Regional Office for Europe of the World Health Organization in collaboration with the Fondo de Investigación Sanitaria, Spain, and the U.S. Food and Drug Administration, National Institutes of Health, National Institutes of Mental Health, and Centers for Disease Control and Prevention to share information about two very similar diseases--toxic oil syndrome and eosinophilia-myalgia syndrome. In this paper the interpretation of conference proceedings is presented, current knowledge of the two disorders is summarized, and some possible areas for future research are mentioned. Toxic oil syndrome and eosinophilia-myalgia syndrome have many similarities. Both are related to consumer products that were presumed to be safe but have been found to have numerous trace contaminants, many of which remain to be identified, including the etiologic agents of both disorders. Both illnesses affect patients clinically by causing intense, incapacitating myalgias and a marked peripheral eosinophilia. Other rheumatologic manifestations are common in both, including arthralgias, sicca syndrome, scleroderma-like skin changes, carpal tunnel syndrome, and joint contractures. No clinical or laboratory feature has been found to be pathognomonic of either disease, and accurate diagnosis rests on the clinical judgment of the attending physician. Deaths have occurred in both diseases, and the cumulative mortality for each is approximately 2.5% for the first 2 years. Long-term complications include pulmonary hypertension, peripheral neuropathies, and joint contractures. Although treatment with corticosteroids has resulted in significant symptomatic relief in persons with either disorder, it does not alter the clinical course or long-term outcome. Research into the etiologic agents, preferred treatments, and ways to avoid similar problems in the future is needed.

Reduced severity of eosinophilia-myalgia syndrome associated with the consumption of vitamin-containing supplements before illness.

OBJECTIVE: To determine if the severity of subacute symptoms in eosinophilia-myalgia syndrome (EMS) was affected by medical history or use of nutritional supplements other than tryptophan before illness. DESIGN AND STUDY POPULATION: A case-control study was conducted of EMS cases systematically sampled from all those reported to a statewide surveillance system in California in 1989. Excluding two previous EMS-related deaths, interviews were completed in 73% (57/78) of the eligible case patients sampled. MAIN OUTCOME MEASURES: The severity of any myalgia(s), dyspnea, or walking impairment during each of the first 3 months of EMS was quantified by means of self-reported integer scores ranging from 0 (asymptomatic) to 10 (severe symptoms). Case patients in the top tercile of combined, unweighted monthly scores were defined as having severe symptoms. RESULTS: All interviewees (57 of 57) had consumed supplemental tryptophan before illness; 89% (51/57) were female. The odds of severe symptoms were not significantly associated with gender, age, previous antidepressant use, or cumulative amounts of supplemental tryptophan consumed before or after EMS onset (P > .1). Previous consumption of any multivitamin(s), however, was associated with significantly lower odds of severe symptoms (adjusted odds ratio, 0.05; 95% confidence limits, 0.007, 0.4; P = .006). CONCLUSIONS: The consumption of multivitamin-containing supplements before EMS appears to have modified the severity of subacute symptoms in this sample of cases from California.

The toxic oil syndrome (TOS, 1981): from the disease towards a toxicological understanding of its chemical aetiology and mechanism.

In Spain early in May 1981, 20,000 people became ill with a severe acute respiratory illness. The eosinophilia and subsequent myalgia, scleroderma and muscle wasting indicated a unique disease entity. Epidemiological evidence linked the disease with the consumption of oils containing "refined" aniline denatured rape seed oil. Ten years after the explosive appearance of this disease (approximately 350 deaths and over 1000 in the chronic phase) the clinical and pathological description is now well established. The aetiological agent(s) in the food oil are unknown and the mechanism(s) of pathogenesis are uncertain. There is no experimental animal model. A new disease, Eosinophilia Myalgia Syndrome (EMS) which appeared late in 1989 in the USA, is due to the consumption of impure 1-tryptophan. There may be similarities between the diseases and the aetiological agents for TOS and EMS: possibilities for future research will be discussed. Underlying the time lag for solution of this problem is a lack of knowledge of the basic biology involved.

3-(Phenylamino)alanine, a novel aniline-derived amino acid associated with the eosinophilia-myalgia syndrome: a link to the toxic oil syndrome?

The eosinophilia-myalgia syndrome (EMS) is an inflammatory disease that occurred in epidemic proportions in the United States during 1989. Cases of EMS were also reported in Europe and elsewhere. Clinically, EMS resembles the Spanish toxic oil syndrome. EMS has been associated with ingestion of manufactured L-tryptophan and, more specifically, with lots of tryptophan that contained the trace contaminant 1,1'-ethylidenebis(tryptophan) (EBT). Another trace contaminant ("peak UV-5") has been reported, but the strength of its association with EMS has not been demonstrated. Herein we report independently that peak UV-5 is 3-(phenylamino)alanine (PAA). Patients with EMS ingested significantly greater amounts of both PAA and EBT than did control tryptophan users. PAA is chemically similar to 3-phenylamino-1,2-propanediol, an aniline derivative isolated from samples of oil that were consumed by persons in whom the toxic oil syndrome developed. The discovery of an aniline-derived contaminant in tryptophan raises the possibility that EMS and toxic oil syndrome may have a common etiologic trigger.

[Toxic oil syndrome--an example of an exogenously-induced autoimmune disease]. / Das Toxische-Ol-Syndrom ("toxic oil syndrome")--Beispiel einer exogen induzierten Autoimmunerkrankung.

In 1981 epidemic poisoning with adulterated cooking oil occurred in Spain, affecting more than 20,000 people. The condition caused has since become known as the toxic oil syndrome (TOS). About 10-15% of the patients with acute symptoms developed a chronic disease with scleroderma-like skin manifestations, polyneuropathy and myositis. While the acute phase of the TOS was characterized by eosinophilia and elevated IgE, the chronic stage involved humoral autoimmune phenomena, such as antinuclear and antinucleolar antibodies, in many cases. In women with the chronic phase of TOS there was a possible prevalence of HLA-DR3 and HLA-DR4. The recently characterized eosinophilia-myalgia syndrome (EMS), which is thought to have been induced by contaminated L-tryptophan preparations, is similar to the TOS in some particulars. Understanding of the toxicological, immunological and genetic pathways leading to these diseases might give us some insight into the pathogenesis of spontaneously occurring autoimmune diseases, such as systemic scleroderma.