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

Abstract

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).

Manganese and iron species in Sprague–Dawley rats exposed with MnCl2∙4H2O (i.v.)

“…Furthermore, both groups of controls (−Mn) and exposed animals (+Mn (i.v)) showed consistent extraction ratios of Mn or Fe for each tissue extraction (Mn: Table 1, Iron: Table C electronic supplement). These values were additionally in accordance with earlier found extraction efficiencies based on the same quality controlled method [37]. Mn tissue extraction of both groups ranged from 23 to 34% (median: 28.0%; Ref.…”

“…Both, ICP-AES and SEC-ICP-MS ascertained congruent Mn concentrations, whereas exclusively in serum Fe showed an up to 65% higher concentration when using the hyphenated system. The slightly higher Fe values (increase b 7%) in brain found by ICP-MS [37] and remarkable higher Fe concentration in serum measured by SEC-ICP-MS could be explained by poly-atomic interferences of 56 Fe ( 40 Ar-16 O + , 40 Ca- 16 O + and 37 Cl-18 O-1 H + ) [40]. Argon as well as chlorine from eluent-buffer are abundantly present and may contribute to the elevated Fe levels detected by SEC-ICP-MS.…”

“…Comparability between the ICP-AES and -MS was recently checked by analyzing identical rat brain and liver extracts (n = 3) with both detectors [37]. Concurrent Mn and Fe results were found with ICP-AES and -MS in both tissue extracts, whereas ICP-MS showed slightly higher Fe values (increase b 7%) in brain.…”

“…The specifically developed, quality controlled extract preparation method used cryogenic conditions ("+N method") under inert gas atmosphere and was recently published [37]. It provided high extraction efficiency of native Mn-and Fe-species and species stability was improved even after storage for 1 month.…”

“…Characterizations of Mnand Fe-species were performed with size exclusion chromatography online coupled to inductively coupled plasma-mass spectrometry equipped with a dynamic reaction cell (SEC-ICP-MS). Both, the specifically developed sample preparation under cryogenic conditions and the analytical methods ICP-AES/SEC-ICP-MS were previously evaluated and quality controlled with regard to extraction efficiency, species stability and long-term storage of labile native Mn species in rat sample matrices [37].…”