A Metalloproteomics Study on the Association of Mercury With Breast Milk in Samples From Lactating Women in the Amazon Region of Brazil

Abstract

This study aimed to identify metalloproteins that lose their metal ions in the presence of mercury (Hg) and bind to Hg in breast milk samples collected from the riverine population of the Madeira River, a tributary of the Amazon River. Initially, total Hg was determined from the hair of lactating women to identify individuals who were contaminated followed by a proteomic analysis of breast milk samples through two-dimensional polyacrylamide gel electrophoresis after acetone precipitation. The presence of Hg in the obtained protein spots was determined by cold vapor atomic absorption spectrometry and cold vapor atomic fluorescence spectrometry. These determinations indicated the presence of Hg in one protein spot, which was then characterized through electrospray ionization tandem mass spectrometry. Based on searches in the UniProt database, this protein spot was identified as lysozyme C.

Metalloproteomics Approach to Analyze Mercury in Breast Milk and Hair Samples of Lactating Women in Communities of the Amazon Basin, Brazil

“…The transient signal is measured in series by a high-sensitivity cell followed by a low-sensitivity cell. At the end of this analysis, there was no residue from the analyzed hair in the Bboat^ [18].…”

“…Finally, the sample was decanted again and the water was discarded; this procedure was repeated three times. After the washings, the sample was transferred to a clean container and completely dried at room temperature [18].…”

“…The process of protein fractionation to obtain the proteome of the breast milk samples was carried out by two-dimensional electrophoresis (2D-PAGE) using procedures described by Santos et al 2015 and Pozzi et al 2015 [18,19]. The separation programs for the first dimension (isoelectric focusing-IEF) and second dimension (SDS-PAGE) lasted 4.5 and 5.5 h, respectively.…”

“…The assembly of digestion tubes was placed in the digester block with warming until complete mineralization of the sample was achieved (clear extract after heating at 80°C for around two hours). Then, the acid extracts were transferred to 5 mL volumetric flasks and filled to volume with ultrapure water (18.2 MΩ cm −1 ) [18]. The certificate standard of muscle protein DORM 4-NRC containing 410 ± 55 μg kg −1 and certificate standard of liver protein DOLT 4-NRC containing 2.580 ± 220 μg kg −1 of total mercury were used to validate the mineralization method.…”

“…The concentrations of total mercury in the acid extracts of breast milk samples, pellets and protein spots were determined by graphite furnace atomic absorption spectrometry (GFAAS) [18,[23][24][25][26]. For this, an analytical curve was prepared with Titrisol Merck® mercury aqueous standard solutions in the concentration range of 0.10 to 2.00 μg L −1 , and the absorbance values of standard solutions and samples were measured with a Shimadzu atomic absorption spectrometer model AA-6800.…”