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.

Fig. 1

Phosphorus chromatograms of brain FTS and various phosphorus-containing compounds. A, brain FTS; B, IP₆; C, ATP; D, ADP; E, AMP, and F, HPO₄²⁻. Red lines in Figures 1 - 8 are simulations representing the LMM species listed in Table 1. The blue line is the overall simulation.

Fig. 2

Sulfur chromatograms of brain FTS and various S-containing compounds. A, brain FTS; B, glutathione, GSH; C, oxidized glutathione, GSSG. The authentic glutathione solution contained some GSSG, while the GSSG solution contained a contaminant.

Fig. 3

Cobalt chromatograms of brain FTS and other Co species. A, brain FTS; B, cyanocobalamin; and C, aqueous Co. The three lowest MM simulations in the figure are considered a single species (Co_aq) in Table 1.

Fig.4

Copper chromatograms of brain FTS and other Cu species. A, brain FTS ×15; B, brain FTS ×1; C, Cu^II(EDTA); and D, aqueous Cu.

Fig.5

Zinc chromatograms of brain FTS. A, brain FTS ×16; B, brain FTS ×1; C, Zn^II(TPEN); and D, aqueous Zn.

Fig. 6

Iron chromatograms of brain FTS. A, brain FTS ×15; B, brain FTS ×1; C, Fe^II(ATP); and D, aqueous Fe.

Fig.7

Manganese chromatograms of brain FTS and other Mn species. A, brain FTS; B, Mn^II(EDTA); and C, aqueous Mn.

Fig.8

Molybdenum chromatograms of brain FTS and molybdopterin extract from xanthine oxidase. A, brain FTS; and B, molybdopterin. The intense peak in B with MM ~1.5 kDa was not assigned.