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Low-molecular-mass metal complexes in the mouse brain.

ABSTRACT: The presence of labile low-molecular-mass (LMM, defined as <10 kDa) metal complexes in cells and super-cellular structures such as the brain has been inferred from chelation studies, but direct evidence is lacking. To evaluate the presence of LMM metal complexes in the brain, supernatant fractions of fresh mouse brain homogenates were passed through a 10 kDa cutoff membrane and subjected to size-exclusion liquid chromatography under anaerobic refrigerated conditions. Fractions were monitored for Mn, Fe, Co, Cu, Zn, Mo, S and P using an on-line ICP-MS. At least 30 different LMM metal complexes were detected along with numerous P- and S- containing species. Reproducibility was assessed by performing the experiment 13 times, using different buffers, and by examining whether complexes changed with time. Eleven Co, 2 Cu, 5 Mn, 4 Mo, 3 Fe and 2 Zn complexes with molecular masses <4 kDa were detected. One LMM Mo complex comigrated with the molybdopterin cofactor. Most Cu and Zn complexes appeared to be protein-bound with masses ranging from 4-20 kDa. Co was the only metal for which the "free" or aqueous complex was reproducibly observed. Aqueous Co may be sufficiently stable in this environment due to its relatively slow water-exchange kinetics. Attempts were made to assign some of these complexes, but further efforts will be required to identify them unambiguously and to determine their functions. This is among the first studies to detect low-molecular-mass transition metal complexes in the mouse brain using LC-ICP-MS.

SUBMITTER: McCormick SP

PROVIDER: S-EPMC3608428 | biostudies-literature | 2013 Mar

REPOSITORIES: biostudies-literature

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Low-molecular-mass metal complexes in the mouse brain.

McCormick Sean P SP Chakrabarti Mrinmoy M Cockrell Allison L AL Park Jinkyu J Lindahl Lora S LS Lindahl Paul A PA

Metallomics : integrated biometal science 20130301 3

The presence of labile low-molecular-mass (LMM, defined as <10 kDa) metal complexes in cells and super-cellular structures such as the brain has been inferred from chelation studies, but direct evidence is lacking. To evaluate the presence of LMM metal complexes in the brain, supernatant fractions of fresh mouse brain homogenates were passed through a 10 kDa cutoff membrane and subjected to size-exclusion liquid chromatography under anaerobic refrigerated conditions. Fractions were monitored for ...[more]

PMID: 23443205

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Project description:Fluorescence-based chelators are commonly used to probe labile low-molecular-mass (LMM) metal pools in the cytosol of eukaryotic cells, but such chelators destroy the complexes of interest during detection. The objective of this study was to use chromatography to directly detect such complexes. Towards this end, 47 batches of cytosol were isolated from fermenting S. cerevisiae yeast cells and passed through a 10 kDa cut-off membrane. The metal contents of the cytosol and resulting flow-through solution (FTS) were determined. FTSs were applied to a size-exclusion LC column located in an anaerobic refrigerated glove box. The LC system was coupled to an online inductively-coupled-plasma mass spectrometer (ICP-MS) for detection of individual metals. Iron-detected chromatograms of cytosolic FTSs from WT cells exhibited 2-4 major species with apparent masses between 500-1300 Da. Increasing the iron concentration in the growth medium 40-fold increased the overall intensity of these peaks. Approximately 3 LMM cytosolic copper complexes with apparent masses between 300-1300 Da were also detected; their LC intensities were weak, but these increased with increasing concentrations of copper in the growth medium. Observed higher-mass copper-detected peaks were tentatively assigned to copper-bound metallothioneins Cup1 and Crs5. FTSs from strains in which Cup1 or the Cox17 copper chaperone were deleted altered the distribution of LMM copper complexes. LMM zinc- and manganese-detected species were also present in cytosol, albeit at low concentrations. Supplementing the growth medium with zinc increased the intensity of the zinc peak assigned to Crs5 but the intensities of LMM zinc complexes were unaffected. Phosphorus-detected chromatograms were dominated by peaks at apparent masses 400-800 Da, with minor peaks at 1000-1500 Da in some batches. Sulfur chromatograms contained a low-intensity peak that comigrated with a glutathione standard; quantification suggested a GSH concentration in the cytosol of ca. 13 mM. A second LMM sulfur peak that migrated at an apparent mass of 100 Da was also evident.

Project description:Nutrient iron entering the blood binds transferrin (TFN)d, which delivers iron to cells in the body. In healthy individuals, ?30% of TFN is iron-bound while the remainder is unbound (apo-TFN). TFN saturates the plasma of individuals with iron-overload diseases such as hereditary hemochromatosis, prompting release of a poorly-defined low-molecular-mass (LMM) iron species called non-transferrin-bound iron (NTBI). An experiment was devised to directly detect NTBI in plasma of iron-deficient pigs and to assess the role of the liver which is known to bind NTBI. Catheters were surgically installed in the portal vein (PV) and either the caudal vena cava or the cranial vena cava. After the animals recovered, 57Fe II ascorbate was injected into the stomach via a feeding tube. Blood was removed through the catheters before and after injection; plasma became 57Fe-enriched after injection. 57Fe-enriched plasma was passed through a 10 kDa cutoff membrane and the flow-through solution (FTS) was subjected to size-exclusion liquid chromatography (LC). The eluent flowed into an ICP-MS where 56Fe and 57Fe were detected. Low-intensity iron peaks with masses of 400-1600 Da were observed, but none became enriched in 57Fe after injection. Rather, the injected 57Fe bound to apo-TFN. Viewed naively, this implies that nutrient-derived 57Fe in healthy mammals passes from the intestines to apo-TFN without first entering the blood as a LMM intermediate. In this case, nutrient iron exported from intestinal enterocytes of healthy individuals may quickly bind apo-TFN such that LMM iron species do not accumulate in blood plasma. Some 57Fe from the FTS may have adsorbed onto the column. In any event, the LMM iron species in plasma that eluted from the column must have originated from iron stored within the body, perhaps in macrophages - not directly from nutrient iron absorption. The liver absorbed and released LMM iron species, but the effect was modest, consistent with its role as a dynamic iron buffer. Passage through the liver also altered the distribution of different forms of TFN present in the PV.

Project description:BackgroundToenails are a promising matrix for chronic metal exposure assessment, but there are currently no standard methods for collection and analysis. Questions remain about sample mass requirements and the extent to which metals measured in this matrix are representative of chronic body burden.ObjectiveThis study proposes a method to maximize sample conservation for toenail metals analysis using inductively coupled plasma mass spectrometry (ICP-MS). We demonstrate the reliability of an ~25 mg toenail sample (typically 1-2 clippings) for metals analysis and evaluate the intra-individual variability of multiple metals in this matrix over time in men from the Gulf Long-term Follow-up (GuLF) Study.MethodsToenail samples from 123 GuLF Study participants were collected at two visits 3 years apart and analyzed for 18 elements using ICP-MS. Participants with samples exceeding 200 mg at the first visit (n = 29) were selected for triplicate sub-sample analysis. Kendall's coefficient of concordance (W) was used to assess sub-sample reliability and Spearman's correlation coefficients (ρ) were used to evaluate fluctuations in elemental concentrations over time.ResultsResults were not reported for Cd, Co, Mo, Sb, and V (detected in <60% of the samples). There was strong agreement among triplicate samples (Kendall's W: 0.72 (Cu)-0.90 (Cu)) across all elements evaluated, moderate correlations of elemental concentrations (Spearman's ρ: 0.21-0.42) over 3 years for As, Ca, Cr, Fe, Pb, Mn, and Zn, and strong correlations (>0.50) for Se, Cu, and Hg.Impact statementThis toenail reliability study found that a low-mass (~25 mg) toenail sample (1-2 clippings) is suitable for the determination of most elements using ICP-MS and helps to increase the analytical capacity of limited toenail biospecimens collected in cohort studies. The results highlight differences in the suitability of toenails for chronic metal exposure assessment by element and underscore the need to consider intra-person variability, especially when comparing results across studies. We also provide recommendations for analytical standardization and the partitioning of the total collected toenail sample into multiple analytic sub-samples for future studies using toenail biospecimen for multiple assays.

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Low-molecular-mass metal complexes in the mouse brain.

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Low-molecular-mass metal complexes in the mouse brain.

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