Enhanced extract preparation of native manganese and iron species from brain and liver tissue

To date, no reference method for the extraction of labile Mn species from biological tissues is published which provides sufficient extraction efficiency combined with monitoring speciation. Here, an extraction method is reported using cryogenic conditions (+N) under inert gas atmosphere. Fresh brain and liver tissues were used, then stored either 1 day (+N) or 1 month in N(2liq) (+N 1 m) to evaluate degradation effects during long-term storage. Both attempts were compared to a previous extraction method (-N) using neither N(2liq) nor storage ability. Mn and Fe concentrations in extracts and pellets were determined with inductively coupled plasma (ICP)-atomic emission spectroscopy (AES) and compared to acid digests of the same sample. Element ratios of extracts/digest indicated the extraction efficiency, which was increased from 17% (-N) to 26% (+N) for Mn in brain or from 28% (-N) to 44% (+N) in liver extracts. For Fe species, the increase was only from 40% (-N) to 44% (+N) in brain but from 64% (-N) to 74% (+N) in liver. Size exclusion chromatography (SEC)-ICP-mass spectrometry (MS) was employed to screen for Mn and Fe species pattern in extracts. In brain, surplus extracted Mn (+N, +N 1 m) was assigned to organic Mn species, mainly from the 0.7-4 kDa fraction, while in the liver, it was seen in the 70-80 kDa fraction. Fe speciation was similar for -N and +N methods in brain extracts. In liver, higher amounts of Fe species were extracted from the 140-160 kDa fraction. Storage at -196 °C for 1 month did neither affect Mn speciation in brain nor in liver extracts. Fe species pattern showed a negligible shift (≤5%) from 140-160 to 70-80 kDa fraction in liver extracts stored 1 month in N(2liq).