Zinc determination by Induction Coupled Plasma Mass Spectrometry (ICP-MS): Plasma samples and standard were diluted with a diluent containing the following reagents: 0.1% triton, to maintain a stable emulsion, the diluted sample and 0.15% HNO3, to ensure that trace elements are maintained in and to aid washout of these elements from samples. External calibration solutions Zn (blank to 2,000 ppb) were prepared by serial dilution of parent 1,000 ppm stock Italia, Milano, Italy), using the same diluent used to dilute the samples. Measurement of zinc was performed with a Thermo XII ICP-MS (Thermo Electron Corporation, MA, USA). The instrument was operated 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 nebulizer (Burgener, Ontario, Canada) was used as this device does not block during aspiration of clinical samples. The instrument was in standard mode (nonCCT), which is the preferred method for acquiring data for Zn66. The instrument was operated using 1400 W RF, 1.10 l/min nebulizer gas flow, 0.70 l/min auxiliary gas flow, 13.0 l/min cool gas flow, 70 ms integration time, 30 s sample uptake and 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). Defrosted PBMCs were subdivided into three equal aliquots of minimum 2x105 cells. One aliquot was incubated with 20 μM zinpyr-1 (ZP-1) (Neurobiotex, Galveston, Texas, USA) for 30 min at 37°C, 5% CO2 in HEPES buffered zinc-free RPMI medium containing 1 mM EDTA. The addition of membrane impermeable zinc chelator EDTA was used to remove possible interferences due to free zinc in the medium and adsorbed to the cell membrane. Another aliquot was always incubated in the same conditions plus 50 μM N,N,N-tetrakis (2-pyridylmethyl) ethylenediamine (TPEN) (Sigma-Aldrich, Milan, Italy), to detect the autofluorescence of the zinc-free ZP-1 probe, which means the Minimum of Mean Fluorescence intensity (MFI) for ZP-1 fluorescence min (Burdette et al. 2001). A further aliquot was incubated in the same condition as the first aliquCarriers with respect to C ones, both in old and very old age, suggest that C+ carriers may have less zinc release by MT than C ones, resulting in low zinc ion availability for alleles between old and nonagenarian subjects (P > 0.05) by v2 test. This suggests that this polymorphism might not be associated with longevity in old females. Table 2 shows the genotype distribution and allelic frequencies of old and nonagenarian women for +647 MT1a SNP. Old and nonagenarian healthy subjects were in Hardy-Weinberg equilibrium (P > 0.05). A significantly different distribution of 647 MT1A genotypes was observed between old and nonagenarian subjects (P < 0.0001) by v2 test. A significantly increased frequency of A (Asp) allele was observed among nonagenarian subjects compared to old ones [P = 0.0002 (by Fisher's exact test) a vs. b odds ratio = 2.164 (CI = 1.386-3.380)], suggesting that the AA (Asp/Asp) genotype might be considered favorable for longevity in old females. Table 3 shows plasma zinc, zinc ion availability, IL-6, and MT detection for +647MT1a polymorphism.

The +647 MT1a Zp1 (MFI-MFI) D MFI IL-6 (pg/ml) Zn (lM) MT (F.U.) min for old C+ carriers is 77.89 - 7.21* 13.83 - 3.33* 34.71 - 7.26* 12.10 - 0.13* 42.28; for old C carriers, it is 48.13 - 7.76 17.30 - 6.57, 28.83 - 6.70, 12.72 - 0.15, and 23.63 - 5.67; for nonagenarian C+ carriers, it is 59.5 - 8.4* 3.9 - 0.06** 24.34 - 3.99, 11.14 - 0.55, and 18.95 - 4.12; and for nonagenarian C carriers, it is 49.35 - 7.3, 8.5 -1.5, 28.83 - 6.70,10.45 - 0.26, and 17.76 -4.25 old individuals (18.7%) (Table 5). This suggests that the haplotype (Arg/Asp) might be favorable for longevity in old females.

AG and GG genotype (Lys/Arg and Arg/Arg) carriers for +1245 MT1a polymorphism were grouped and referred to as G+, while AA (Lys/Lys) genotype carriers were referred to as G. With regard to this MT1a polymorphism, no significant differences in all parameters considered (plasma zinc, zinc ion availability, Delta MFI, IL-6, and MT) were observed between G+ and G carriers both in old and very old individuals (Table 4). However, there was a trend for more zinc ion bioavailability in old G+ carriers, suggesting that this polymorphism might not affect either the inflammatory status or zinc ion bioavailability.

The screening of two polymorphisms in the human metallothionein gene 1a coding region from old and very old healthy females has revealed their involvement in longevity for the first time. In particular, the polymorphism corresponding to A/C (Asp/Thr) transition at the 647 nt position seems to be more involved than the polymorphism corresponding to A/G (Lys/Arg) transition at the 1,245 nt position. Indeed, for the first SNP, a significant increment of AA (Asp/Asp) genotype (P < 0.0001) and Asp allele (P = 0.0002, odds ratio = 2.164, CI = 1.386-3.380) occurs in nonagenarian subjects compared to old ones. For the second SNP, instead, no significant different distribution of genotypes and alleles between old and nonagenarian subjects is observed. These findings clearly show that these polymorphisms are differently involved in longevity, at least in women, where the presence of the genotype Asp/Asp is crucial for successful aging.