title: Synthesis and characterization of mercapturic acid (N-acetyl-L-cysteine)-aflatoxin B1 adduct and its quantitation in rat urine by an enzyme immunoassay.
abstract: A simple and sensitive indirect noncompetitive enzyme immunoassay to quantitate mercapturic acid-aflatoxin B1 (AFB1) adduct in rat urine is reported. A novel procedure was developed for in vitro synthesis of an immunogen, bovine serum albumen-glutathione-aflatoxin B1 (BSA-GSH-AFB1) using 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride. Sulphydryl group's analysis confirmed the conjugation of-SH groups to AFB1. Thin-layer chromatography and spectral analysis (absorption, fluorescence, and Fourier transform infrared) of the conjugates further confirmed the formation of the adducts. Polyclonal antibodies specific to mercapturic acid-AFB1 adduct were produced against BSA-GSH-AFB1. The assay was found to be linear in the range of 100 pg-100 ng of the analyte (y = a + bx). A 50% displacement of BSA-GSH-AFB1 antibodies was achieved at an inhibitory concentration (IC50) of 11.9 ng GSH-AFB1 (r2 = 0.98) and 1.22 ng N-acetyl-L-cysteine (NAC)-AFB1 (r2 = 0.98). Spiking 5 microg/mL of reference standard to the control rat urine showed a recovery of 98 +/- 2%. The immunoassay was validated in a rodent model exposed to a single oral dose of 1 mg/kg body mass of pure AFB1. The excretion of NAC-AFB1 adduct was quantitated at the end of 24 h. The concentration of the NAC-AFB1 adduct excreted in urine as determined by the immunoassay was found to be in the range of 3.22-5.97 microg/mg creatinine. The present method may find wide application as a biochemical tool in molecular epidemiological and intervention studies with respect to human exposure to dietary aflatoxins.
authors.affiliation: Osmania University, University College of Science, Department of Biochemistry, Hyderabad-500 007, India.
authors.forename: Sujatha
authors.initials: S
authors.lastname: Nayak
authors.forename: Penmatsa
authors.initials: P
authors.lastname: Tanuja
authors.forename: Rao Beedu
authors.initials: RB
authors.lastname: Sashidhar
mesh_terms: D000111:Acetylcysteine / Q000138:chemical synthesis / Q000652:urine*; D016604:Aflatoxin B1 / Q000138:chemical synthesis / Q000276:immunology / Q000633:toxicity / Q000652:urine*; D000818:Animals; D000917:Antibody Formation; D002417:Cattle; D002855:Chromatography, Thin Layer; D005506:Food Contamination / Q000032:analysis; D005978:Glutathione / Q000737:chemistry; D006801:Humans; D007124:Immunoenzyme Techniques / Q000379:methods* / Q000592:standards / Q000706:statistics & numerical data; D008297:Male; D010047:Ovalbumin / Q000737:chemistry; D011817:Rabbits; D051381:Rats; D011916:Rats, Inbred F344; D012015:Reference Standards; D012680:Sensitivity and Specificity; D012710:Serum Albumin, Bovine / Q000737:chemistry; D017550:Spectroscopy, Fourier Transform Infrared
chemical_list: D012710:Serum Albumin, Bovine; D010047:Ovalbumin; D016604:Aflatoxin B1; D005978:Glutathione; D000111:Acetylcysteine