Human Aflatoxin Albumin Adducts Quantitatively Compared by ELISA, HPLC with Fluorescence Detection, and HPLC with Isotope Dilution Mass Spectrometry

Abstract

Essential to the conduct of epidemiologic studies examining aflatoxin exposure and the risk of heptocellular carcinoma, impaired growth, and acute toxicity has been the development of quantitative biomarkers of exposure to aflatoxins, particularly aflatoxin B 1 . In this study, identical serum sample sets were analyzed for aflatoxin-albumin adducts by ELISA, high-performance liquid chromatography (HPLC) with fluorescence detection (HPLC-f), and HPLC with isotope dilution mass spectrometry (IDMS). The human samples analyzed were from an acute aflatoxicosis outbreak in Kenya in 2004 (n = 102) and the measured values ranged from 0.018 to 67.0, nondetectable to 13.6, and 0.002 to 17.7 ng/mg albumin for the respective methods. The Deming regression slopes for the HPLC-f and ELISA concentrations as a function of the IDMS concentrations were 0.71 (r 2 = 0.95) and 3.3 (r 2 = 0.96), respectively. When the samples were classified as cases or controls, based on clinical diagnosis, all methods were predictive of outcome (P < 0.01). Further, to evaluate assay precision, duplicate samples were prepared at three levels by dilution of an exposed human sample and were analyzed on three separate days. Excluding one assay value by ELISA and one assay by HPLC-f, the overall relative SD were 8.7%, 10.5%, and 9.4% for IDMS, HPLC-f, and ELISA, respectively. IDMS was the most sensitive technique and HPLC-f was the least sensitive method. Overall, this study shows an excellent correlation between three independent methodologies conducted in different laboratories and supports the validation of these technologies for assessment of human exposure to this environmental toxin and carcinogen. (Cancer Epidemiol Biomarkers Prev 2008;17(7):1653 -7)

Aflatoxin exposure during the first 1000 days of life in rural South Asia assessed by aflatoxin B1-lysine albumin biomarkers

“…To address this data gap, a quantitative isotope dilution mass spec-trometry method was deployed to measure the aflatoxin B 1 -lysine albumin adduct at biologically critical times in mothers during pregnancy and in the first thousand days of life in their children. An initial analysis of aflatoxin-albumin adducts in a pooled sample from one thousand 6–7 year old children in Nepal found 56.5 pg aflatoxin B 1 -lysine/mg albumin, a level comparable to that detected in very nutritionally compromised children in West Africa when the different method of analyses are considered (Gong et al, 2004; McCoy et al, 2008). This biomarker observation prompted a more detailed examination using samples already analyzed for its plasma proteome that had collected during the first and third trimester of pregnancy of women enrolled in a randomized micronutrient intervention clinical trial in Sarlahi, Nepal (Cole et al, 2013; Scholl et al, 2012).…”

“…The development of mechanistically based biomarkers, such as the aflatoxin B 1 -lysine albumin adduct, has helped to overcome the inherent difficulties of assessing aflatoxin exposure by measuring dietary constituents. These biomarkers have the added advantage of being able to integrate exposures over the course of many weeks or months due to the long half-lives of these biomarkers in plasma (McCoy et al, 2005, 2008). Research from our laboratory has also demonstrated the long-term stability of aflatoxin albumin biomarkers in stored samples where we have found these adducts to be stable for up to 25 years (Scholl and Groopman, 2008).…”

“…All of the serum or plasma samples were analyzed using a minor variation of the method reported by McCoy and colleagues (McCoy et al, 2008). The sample (200 μL) was mixed with an internal standard (10 μL × 0.1 ng AFB 1 -D4-lys/mL) and Pronase solution (250 μL, 13 mg/mL PBS), and incubated for 18 h at 37 °C.…”

Developing biomarkers of human exposure to mycotoxins

“…Although considerable success has been gained by measuring aflatoxin DNA and protein adducts using immunoassay methods, method development continues in this field, with the development of isotope dilution tandem mass spectrometry (IDMS) methods for detection of both urinary aflatoxin DNA adducts and serum albumin adducts (Egner et al, 2006;McCoy et al, 2008). Mass spectrometry provides higher specificity than other methods because the detection focuses on the signature fragmentation pattern of the aflatoxin adduct (Egner et al, 2006).…”

Visual and microplate detection of aflatoxin B2 based on NaCl-induced aggregation of aptamer-modified gold nanoparticles

“…Analytical techniques such as thin-layer chromatography [6], high-performance liquid chromatography (HPLC) [7][8][9][10], and enzyme-linked immunosorbent assay (ELISA) have been available for the qualitative and quantitative analysis of AFB2 [11]. Although HPLC is the most commonly used method, because it offers high sensitivity and selectivity, it has some limitations in terms of costs incurred by the sophisticated equipment and the methods are not cost-effective, requiring a relatively long analysis time, so they are neither readily available in developing countries nor capable of on-site detection.…”