Quantitation of Human Metallothionein Isoforms in Cells, Tissues, and Cerebrospinal Fluid by Mass Spectrometry.

Metallothioneins (MTs) are a family of small, highly conserved, cysteine-rich metal-binding proteins that are important for zinc and copper homeostasis, protection against oxidative stress, and buffering against toxic heavy metals. Individual human MT isoforms are candidate biomarkers for heavy metal toxicity, and selected cancers and neurodegenerative diseases. The similar antigenicity of human MT-1 and MT-2 isoforms precludes development of antibody-based assays for their individual quantitation. Metal-based MT quantitation methods do not directly measure MT isoforms. A bottom-up mass spectrometry-based approach solves these problems by exploiting the unique masses and chromatographic properties of the acetylated N-terminal tryptic peptides of MT isoforms. These unusually hydrophilic 20- to 21-residue peptides contain five invariant cysteines. Strong cation exchange chromatography separates them from bulk internal tryptic peptides. Reversed-phase chromatography further separates them from more hydrophobic peptides of similar mass. Absolute quantitation is obtained by adding MT peptide standards alkylated with 15N-iodoacetamide to biological samples alkylated with 14N-iodoacetamide. Accurate quantitation is enhanced by dimethyl sulfide treatment to reverse oxidation of the N-terminal methionine. Originally optimized for measuring MT isoforms in cell lines, the method has been adapted to quantify MT isoforms in brain tissue and cerebrospinal fluid. The method can also be adapted for relative quantitation of MT isoforms between matched biological samples. It cannot be used to measure human MT-4 because of an arginine at position 4. Except for this type of limitation, the method is applicable to MT quantitation in many other species.

Separation of proteins including metallothionein in cerebrospinal fluid by size exclusion HPLC and determination of trace elements by HR-ICP-MS.

A method to study the protein binding patterns of trace elements in human cerebrospinal fluid (CSF) is described. Proteins in CSF samples were separated by size exclusion chromatography combined with high performance liquid chromatography (SEC-HPLC). The column was calibrated to separate proteins in the molecular weight range 6-70 kDa. Fractions were then analyzed off-line for trace elements using high resolution inductively coupled plasma mass spectrometry (HR-ICP-MS). We were able to accurately determine more than 10 elements of clinical interest in the CSF fractions. Results are presented for Cd, Mn, Fe, Pb, Cu and Zn. The total concentrations of 16 trace elements in human plasma and CSF are also presented. The method was able to differentiate the relative contribution of metallothionein and other proteins towards metal binding in human CSF.

Axonal damage induced by cerebrospinal fluid from patients with relapsing-remitting multiple sclerosis.

The importance of axonal damage in multiple sclerosis (MS) has been recently stressed in proton magnetic resonance spectroscopy and pathological studies, but the exact mechanism producing this damage is unknown. The aim of our study was to ascertain whether soluble mediators present in the cerebrospinal fluid (CSF) of patients with relapsing-remitting MS could induce neuron injury in culture. Different biochemical and cytochemical parameters were determined in primary embryonal rat neuron cultures following 8 days of exposure to CSF. Cytotoxic activity was evaluated with a blue formazan production colorimetric assay. Morphological and immunocytochemical studies performed with antibodies against beta-tubulin revealed neuritic fragmentation, axonal damage and cellular shrinkage indicating apoptosis. Detection of apoptosis was carried out using the fluorescent DNA-binding dye Hoechst 33342, as well as by a Terminal deoxynucleotidyl transferase-mediated dUTP Nick End-Labeling assay. We observed that soluble factors in CSF from patients with "aggressive" MS i.e, those with poor recovery after relapses, induced neurite breakdown and neuronal apoptosis in cultures. Neuron injury is not related with blood-brain barrier dysfunction nor with IgG index. Interestingly, CSF from patients with "non-aggressive" MS i.e., relapsing-remitting patients with a good recovery after relapses, did not induce any damage. In conclusion, we report that CSF from patients with aggressive MS bears soluble mediators that induce axonal damage and apoptosis of neurons in culture. These mediators can be present during the first attack of the disease, and the neuronal damage caused could be related to the functional deficit of these MS patients.