Contaminants in Commercial Preparations of Melatonin

Contaminants in Commercial Preparations of Melatonin

1094 LETrERS searc hers in the c linic al liter ature ' and so ld commercially . Since our data coll ection , an additional stra in of B. burgdorferi...

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1094 LETrERS

searc hers in the c linic al liter ature ' and so ld commercially . Since our data coll ection , an additional stra in of B. burgdorferi, stra in 50772 , has been adde d to increase the se nsitivi ty of the BAT in ear ly Lyme disease (LD) .2 This information was released before our manuscript was submitted, and we referenced the report in our article. We did not discuss the theoretical reas ons for the increased sensitiv ity of the BAT with the addi tion of strai n 50772 because our study was meant to be a " real world" eva lua tion of the clinica l tests (BAT, enzyme -link ed immunosorbent assay [ELI SA l, and immunofluorescen ce ass ay [lFAD that were avail able for the diagn osis of LD at the time the study sa mples were obtained. The use of two strains of B. burgdorferi (297 and 50772) in bas ic science ex pe rimen ts has helped further the unde rstanding o f the humoral response to LD . For clin ical use , however , it has increased the complexity and cost of the BAT. Wh ether the use of both stra ins will result in wides pread clinical app lications of thi s assay rem ains doubtful. For ins tance, we know that B. burgdorferi has multiple anti genic variants.' Despite many common anti gen s, will a BAT assay incorporating only two strains be adeq uate for the detect ion of both early and late B. burgdorferi infections in all the loc atio ns in the United Stat es in which LD occ urs? In addition , the BAT depends on comparison of the relative killin g rates of normal human seru m with those in possibl e LD cases. Wh at, however , is "norm al human seru m',? When the BAT is performed with use of live organ ism s, the result s vary according to the source of the " normal seru m." Therefore , if mult iple laboratories chose to perform the BAT, se rum would need to be continually restandardi zed against kno wn LD sam ples . During the past 40 years, immunologic testing has moved away from whol e bacteri al ce ll serologic assays becau se of diffi cu lties in ma intaining appropriate organisms, lack of standardization of methods, and major problem s with automation. Researchers ho pe that the use of a new method, flow cytome try, will overcome the se probl em s , but its efficacy remai ns to be determined . In addition, " real world" factors such as expense, turnaround time , and ability to detect une xp ected antibiotics in serum sam ples must be con sidered . In short, sim ply running the BAT assay against 57 serum sam ples from patients with early LD and 145 sam ples fro m pat ien ts with other illne sses does not produce resul ts that will help clinicians determine the usefulness of this test in co mparison with the two step protocol currently recommended by the Centers for Di sease Con trol and Prev en tion (CDC).· In agre em ent with the statement by Callister and colleagues , our study showed that neither the ELISA nor the IFA is specific enough to be used alone for the serologic diagnosis of LD . The Sections of Infecti ou s Disea se and Rheumatology at Gundersen Luther an Med ical Center advoc ate that posit ive resul ts of these tests should usually be confmned by LD Western blot assay, as recommended by the CDC.· Althoug h not without problem s , thi s two -test protocol has been show n to be sensitive and speci fic in untreated LD patients.' Currently, we cannot recomme nd the BAT for c linical use unt il clinical studies have pro ved its superio rity to exi sting testing protocol s. In conclu sion , we ma intain that eve n wi th an improved sensitivity, the BAT should be carefully co mpared with a standard two-test protocol (IFA and ELISA followed by Western blot assay if positive) in a clini cal trial before it is acce pted as a diag nostic test for

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LD in hum an s. Ideall y, such a study should be done in multiple ce nters located in are as ende mic for LD. Until result s of suc h a study prove oth erwise, we do not ag ree that the BAT is the "gold standard'? for the serologic diagnosis of LD . William A. Agger, M.D. Secti on o f Infectiou s Disease Kay L. Case, M.T ., S.1. Department of Immunology and Mi crobiology Gundersen Lutheran Med ical Ce nter La Crosse , W isconsin REFERENCES I. Callister SM, Schell RF, Lim LCL, Jobe DA. Case KL. Bryant GL, ct al. Detection of borreliacidal antibodies by now cytomeiry: an accurate, highly specific serodiagnostic test for Lyme disease. Arch Intern Med t994;154:1625-1632 2. Callister SM, Jobe DA, Schell RF, Pavia CS, Lovrich SD. Sensitivity and specificity of the borreliacidaJ-anlibody test during early Lyme disease: a 'gold standard'? Clin Diagn Lab Immunol 1996;3:399-402 3. Mathiesen DA, Oliver JH Jr, Kolben CPo Tullson ED, Johnson BJ. Campbell GL, ct al. Genetic heterogeneity of Borrelia burgdorferi in the United States. J Infect Dis 1997;175:98-107 4. Association of State and Territorial Public Health Laboratory Dircc tors, Centers for Disease Control and Prevention. Recommendations. Proceedings of Second National Conference on Serologic Diagnosis of Lyme Disease; Dearborn (MI). Washington (DC): Association of State and Territorial Public Health Laboratory Directors, 1995; pp 1-7 5. Ledue TB, Collins MF, Craig WY. New laboratory guidelines for serologic diagnosis of Lyme disease: evaluation of the two-test protocol. J Clin Microbiol 1996;34:2343-2350

Contaminants in Commercial Preparations of Melatonin To the Editor; Th e outbreak of the eosinophilia-my alg ia syndrome (EMS) that occurred in 19 89 affected more than 1,500 people and res ulted in at least 37 death s. I Subsequent reports ind icated that the EMS was triggered by the co nsumption of a co ntaminated batch o f the dietary suppleme nt i.-try prophan (t -T rp). Epidemiologic studies showed that at least six contam inants in the L- Trp we re connected to the illness.' One such compound, identified as "peak E" (Fig. I A), received co nside rable att ention as possibly the prim ary etiologic agent of the disease.' Th e EMS epidemic clearly indicated that subs tances sold and used as nutritional supplements req uire much stricte r quali ty co ntro l and regulation." In regard to dietary suppleme nts, Wurtman' further sugges ted that " another accident [ep idemi c] is waiting to happen ." In the pas t seve ral ye ars , the naturally occ urring hormone me laton in,.another indole ring-containing compound, has found widespread use in the se lf-treatment of sleep disorders and sym ptoms of jet lag," In 199 5, an est ima ted 20 million new US consume rs used melatonin. Moreover, becau se this compound is regulated by the Dietary Supplem ent Health and Education Act, it is so ld "over the counter," and its safe ty and e fficacy are not evaluated by the US Food and Dru g Administratio n.

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Mayo Clin Proc, November 1997, voln

LEITERS

Although the potent ial beneficia l and deleterious effects of melatonin continue to be debated, a recent report noted that "several randomly purchased samples of melatonin ...contained unknown matter.' ''' Because melatonin is structurally similar to t-Trp (both compounds contain the indole ring functionality). we investigated the nature of the contaminants in commercial preparations of melatonin with use of on-line high-performance liquid chromatographytandem mass spectrometry (LC-MS/MS). We found sever al contaminants in the commercial preparations of melatonin. presumably formed during the manufacturing process. Struc tural characterization by LC-MS/MS revealed the presence of a formaldehyde-melatonin condensation product (Fig. I B) and two structural isomers of hydroxymelatonin (Fig. I C and D ). Of interest, an EMS-related contaminant in L- Trp referred to as " peak C" has been proposed as a 5-hydroxytryptophan isomer.' Of most importance, however, a melatonin contaminant (Fig. 1 E) was identified as a structural analogue of the peak E (Fig. I A) present in EMS-related. caseimplicated t.-Trp. The melatonin contaminant was initially characterized structurally by LC-MS/MS, and we subsequently synthesized this compound and confirmed its structure by nuclear magnetic resonance studies. The LC-MS/MS of synthetic material was identical to the authentic contaminant found in commercial preparations of melatonin. In all three of the commerc ial preparations we analyzed. the peak E analogue constituted from 0.1 to 0.5% of the unmod ified melatonin. In comparison, the amount of peak E found in contaminated i.-Trp is much lower. from 0.05 to 0. 1% of the parenl t-Trp, The fact that no cases of EMS-like diseases have been repo rted in subjects ingesting melatonin is perhaps due to differences in dosage. A typical regimen of melatonin for alleviation of jet lag is 5 mg/day for 7 days.? In contrast. the reported daily intake of L- Trp in patients who contracted EMS ranged from 3 to 15 g/day taken for a period of several months.' The identification of four contami nants in commerci al preparations of melatonin that are also found as LTrp analogues in the contaminated dietary supplement shown to cause EMS is cause for concern. In particular. hydroxymelatonin (Fig. I C and D) and the peak E-melatonin compound (Fig. I E) are analogues of known EMS-related i.-Trp components. peak C and peak E. These findings should raise serious questions about the consequences of long-term ingestion of melatonin preparations containing such impurities.

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Silver RM. Unraveling the eosi nophilia-mya lgia syndrome [ed itorial]. Arch Derma tol 1991;127:12 14-1216 Wurtman R. Prese nted at Annual Surnmer Toxico logy Forum, Session 7: Free Ami no Acids ; 1990 Ju1 19; Aspen (CO) Lamberg L. Melatonin potentia lly useful but safet y. efficacy rema in uncertain. JAMA 1996;276:1011- 1014 Kamb ML, Murphy JJ, Jones JL, Caston JC. ederlof K, Homey LF, et al. Eosinophilia -myalg ia syndro me in L-tryptophan-exposed patients. JAMA 1992;267:77-82

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Swygert LA, Maes EF, Sewe ll LE. Miller L, Falk H, Kilbo urne EM, et al. Eosinophilia-myalgia syndrome: results of national surveillance. JAMA 1990;264:1698-1703 Hill RH Jr, Caudill SP, Philen RM, Bailey SL. Flanders WD, Driskell WJ. et al. Contaminants in L-tryptophan assoc iated with eosinophilia myalgi a syndro me. Arch Environ Contam Toxicol 1993;25:134- 142 Mayeno AN, Lin F, Foote CSt Loege ring DA, Ames MM , Hedberg CW, et al. Charac terization of"peak E," a novel ami no acid associated with eosinophilia-myalgia syndrome . Scienc e 1990;250 :1707-1708

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Brian L. Williamson, Ph.D. Andrew J. Tom linson, Ph.D. Stephen Naylor, Ph.D. Gerald J. Gleich, M.D. Mayo Clinic Rochester Rochester. Minnesota

REFERENCES

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Fig. I. Chemical structures of contam inants detected in eosi nophiliamyalgia syndrome (EMS)· associated t -tryp tophan (t -Trp) and in commercial preparations of melatonin. " Peak E" co ntaminant from EMS-associated t -Trp (A ). Forma ldehyde-melatonin con densati on product (8 ) , structural isomers of hydroxymelatonin (C and D), and peak E analogue (E) detected in random samp les of melatonin.

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