Haplotype analysis

Using the acquired allele data, +647 MT1a and +1245 MT1a, two loci haplotypes were estimated by the expectation-maximisation algorithm using the Arlequin software package. To test for departures from Hardy–Weinberg a Markov- chain random walk algorithm was used (Guo and Thompson 1992) also implemented in Arlequin.

PBMC recovery and storage

Twenty-five microlitres of PCR mixture was composed of genomic DNA samples (200 ng), 300 lmol/l dNTPs, 2 mM MgCl , 1· Taq poly- 2 merase buffer, 10 pmol of each primer, 2.5 U of Taq DNA polymerase (Eppendorf, Germany).

Individual blood samples from old healthy female were drawn in plastic syringe containing heparin. PBMCs were obtained by ficoll-hypaque

(gradient density d = 1,077) (Biochrom AG, Berlin, Germany) and centrifuged at 1,500g for 15 min, at 20(cid:2)C. All samples presented viability higher than 90% assessed by Tripan blue dye exclusion. Aliquots of cells (2 · 106 cells) were frozen in FCS containing 10% of DMSO under liquid nitrogen until use. Before using, the cells were gently defrozen and counted.

Zinc determination by Induction Coupled Plasma Mass Spectrometry (ICP-MS)

plasma samples and standard were diluted with a diluent containing the following re- 0.1% triton, to maintain a stable emulsion the diluted sample and 0.15% HNO , to 3 that trace elements are maintained in and to aid washout of these elements samples. External calibration solutions Zn (blank to 2,000 ppb), were pre- by serial dilution of parent 1,000 ppm stock Italia, Milano, Italy), using the same dil- used to dilute the samples. Measurement of zinc was performed with a Thermo XII ICP-MS (Thermo Electron Corporation, MA, USA). The instrument was oper- with a Peltier cooled impact bead spray single piece quartz torch (1.5 mm i.d. together with Xi interface cones and a 100 autosampler (CETAC Technol- Omaha, Nebraska, USA). A Burgener nebuliser (Burgener, Ontario, Canada) was as this device does not block during aspira- of clinical samples. The instrument was in standard mode (nonCCT), which is preferred method for acquiring data for Zn66. instrument was operated using 1400 W RF 1.10 l/min nebuliser gas flow, 0.70 l/min gas flow, 13.0 l/min cool gas flow, 70 ms time, 30 s sample uptake 35 s wash time

Flow Cytometric (FC) analysis of zinc ion availability with Zinpyr-1

‘‘Zinc free’’ RPMI medium was obtained by treatment of RPMI with 5% chelex 100 (Sigma- Aldrich, MI, Italia). Defrozen PBMCs were sub- divided into three equal aliquots of minimum 2 · 105 cells. One aliquot was incubated with 20 lM zinpyr-1 (ZP-1) (Neurobiotex, Galveston, Texas, USA) for 30 min at 37(cid:2)C, 5% CO2 in HEPES buffered ‘‘zinc free’’ RPMI medium containing 1 mM EDTA. The addition of mem- brane impermeable zinc chelator EDTA was used to remove possible interferences due to free zinc in the medium and adsorbed to the cell mem-

Another aliquot was always incubated in the same conditions plus 50 lM N,N¢,N¢-tetrakis (2-pyridylmethyl) ethylenediamine (TPEN) (Sig- ma–Aldrich, Milan, Italy), in order to detect the autofluorescence of the zinc-free ZP-1 probe, which means the ‘‘Minimum of Mean Fluores- cence intensity (MFI )’’ for ZP-1 fluorescence min (Burdette et al. 2001).

Further aliquot was incubated in the same condition of the first aliquot plus 500 lM dye- thilamine NOnoate acetoxymethylated (AcOM DEA/NO) (Calbiochem, VWR International, Milan, Italy) in order to detect the release of intracellular free zinc from MT (Misra et al. 1996). In fact, AcOM-DEA/NO is a cell-per- meable acetoxymethylated diazeniumdiolate compound that donates NO ‘‘intracellularly’’ following the action of intracellular esterases (Saavedra et al. 2000). After incubation, the aliquots were immediately read by the Flow Cytometer (Coulter Epics XL, USA). After selecting lymphocyte populations according to the FW and SS scatter, the Mean Fluorescence Intensity (MFI) for ZP-1 was detected (excita-

Defrozen PBMCs (0.2 · were treated with 0.3% paraformaldehyde and stored at 4(cid:2)C for 2 days prior to processing. As previously reported (Yurkow and Makhijani 1998) for MT determination, samples were centrifuged (800g, 5 min), the resulting supernatants aspirated, and the cells resuspended in a permeabilisation buf- fer (1 ml) containing 3% foetal bovine serum (FBS), 0.1% sodium azide, and 0.1% saponin in

System (Bio-Rad Laboratories, USA). Values presenting a coefficient of variation beyond 10% were discarded before the final data analysis. Data were analysed using the Bio-Plex Manager software version 3.0 (Bio-Rad Laboratories, USA). Values were expressed as pg/ml.

phosphate-buffered saline (PBS, pH 7.4) for 1 h at room temperature.

Cells were then washed (3 · 3 ml) with stain- ing buffer (3% FBS, 0.1% sodium azide in PBS, pH 7.4) and centrifuged again as indicated above. The resulting supernatants were aspirated and the cells were incubated with MT specific antibody [1 lg/105 cells/200 ll of monoclonal mouse anti- horse Metallotionein clone E9 (Dakocytomation, Denmark)] for 30 min at room temperature. After treatment with the primary antibody, cells were washed (2 · 1 ml) with staining buffer and incubated for 1 h at room temperature with a FITC-conjugated secondary antibody (Becton Dickinson, USA).

Statistical analysis

Statistics were calculated by using the SPSS soft- ware for windows 2000. Binary variables were checked by Fisher’s exact test or v2 test, which was also used to detect the differences in the genotypic or allelic frequencies of the MT1a polymorphisms and to check the differences in allele distribution from Hardy–Weinberg’s equi- librium in old and nonagenarian subjects. Odds ratios (ORs) were determined as relative risk for longevity. Paired student’s t-test and one-way Anova test with Turkey as ‘‘post hoc’’ test were also used. Differences were considered significant when P < 0.05.