The cells were then washed with 1 ml of staining buffer, resuspended in 500 ll of PBS, and the relative cellular fluorescence which reflects cellular MT levels, was determined using Flow Cytometry. Samples probed with an irrelevant (isotypic) antibody (clone MOPC21, IgG1k, Sig- ma-Aldrich, MI, Italia) in conjunction with the fluorescein-conjugated secondary antibody served as staining controls. The MT-specific fluorescence of the lymphocytes population was determined by subtracting the MFI of the cells stained with the isotypic antibody from the MFI of the cells stained with the MT-specific antibody.

Results

Genotyping of +1245 A/G MT1a polymorphism

Plasma IL-6 determination (multiplex immunoassay)

N. 251 healthy old and very old female were screened for +1245 MT1a SNP. Table 1 shows the genotypes distribution and the allelic frequencies of old and nonagenarian subjects. Old and nonagenarian healthy subjects were in Hardy–Weinberg equilibrium (P > 0.05) when a Markov-chain random walk algorithm was used (Guo and Thompson 1992). No significant diff- erent distribution of 1245 MT1A genotypes and

Diluted plasma samples were assayed in duplicate using commercially available multiplex bead- based immunoassay kits. IL-6 concentration was simultaneously evaluated with five other soluble factors using simplex reagent kits (Bio-Rad Laboratories, USA). Assays were performed following the manufacturer’s instructions. The low standard curve was used for IL-6 (ranging from 3952.5 pg/ml to 0.24 pg/ml).

Table 1 +1245 MT1a genotypic and allelic frequency in old and nonagenarian healthy female

Distinct dyed beads, loaded with specific capture antibodies, were incubated with plasma samples and subsequently with fluorescently labelled detection antibodies. The entire testing procedure was performed using 96-well plates, and the formation of different sandwich immu- nocomplexes on distinct bead sets was measured and quantified using the Bio-Plex Protein Array

Genotype Old (N.151) Nonagenarians (N.100)* Lys/Lys 82.1% (124/151) 77% (77/100) Lys/Arg 16.6% (25/151) 20% (20/100) Arg/Arg 1.3% (2/151) 3% (3/100) Allele Lys 0.90 (273/302) 0.87 (174/200) Arg 0.10 (29/302) 0.13 (26/200)

*P > 0.05 when compared with old subjects by v2 test

Table 2 +647 MT1a genotypic and allelic frequency in old and nonagenarian healthy female

Genotype Old (N.151) Nonagenarians (N.100)* Asp/Asp 49.6% (75/151) 79% (79/100) Asp/Thr 43.7% (66/151) 16% (16/100) Thr/Thr 6.6% (10/151) 5% (5/100) Allele Asp 0.72 (216/302) 0.87 (174/200) Thr 0.28 (86/302)a 0.13 (26/200)b

AC and CC genotype (Asp/Thr and Thr/Thr) carriers for +647 MT1a polymorphism were grouped and referred as C+, while AA (Asp/Asp) genotype carriers were referred as C–.

*P < 0.0001 when compared with old subjects by v2 test P = 0.0002 (by Fisher’s exact test) a vs. b Odds Ratio = 2.164 (CI = 1.386–3.380)

Plasma Zn levels were lower in old C+ in comparison with C– ones (P < 0.05), while significant difference was observed in C+ genarians with respect to very old C– (Table 3). Concomitantly, MT levels and plasma concentrations were always higher in C+ carriers with respect to old C– (P < 0.05), whereas no significant were observed for these parameters C+ and C– nonagenarian subjects (Table However, an interesting point was the significant differences in intracellular zinc availability (tested by means of between C+ and C– carriers both in old and old age (P < 0.05) (Table 3). Such were also evident after the in vitro addition of donor (calculated by means of the between MFI detected before and after addition of NO donor and herein expressed Delta MFI) (see Material and methods). shown in Table 3, Delta was always lower in carriers with respect to C– ones both in old very old age, suggesting that C+ carriers may less zinc release by MT than C– ones and sequently low zinc ion availability for

alleles was observed between old and nonage- narian subjects (P > 0.05) by v2 test, suggesting that this polymorphism might not be associated to the longevity in old female.

Table 2 shows the genotypes distribution and the allelic frequencies of old and nonagenarian women for +647 MT1a SNP. Old and nonage- narian healthy subjects were in Hardy–Weinberg equilibrium (P > 0.05). Significant different distribution of 647 MT1A genotypes was observed between old and nonagenarian subjects (P < 0.0001) by v2 test. A significant increased frequency of A (Asp) allele was observed among nonagenarian subjects with respect 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 as favourable to the longevity in old female.

Table 3 Plasma zinc, zinc ion availability, IL-6 and MT detection for +647MT1a polymorphism

+647 MT1a Zp1 (MFI – MFI ) D MFI IL-6 (pg/ml) Zn (lM) MT (F.U.) min Old C+ carriers 77.89 ± 7.21* 13.83 ± 3.33* 34.71 ± 7.26* 12.10 ± 0.13* 42.28 ± Old C– carriers 48.13 ± 7.76 17.30 ± 6.57 28.83 ± 6.70 12.72 ± 0.15 23.63 ± 5.67 Nonagenarian C+ carriers 59.5 ± 8.4* 3.9 ± 0.06** 24.34 ± 3.99 11.14 ± 0.55 18.95 ± 4.12 Nonagenarian C– carriers 49.35 ± 7.3 8.5 ± 1.5 28.83 ± 6.70 10.45 ± 0.26 17.76 ± 4.25

old individuals (18.7%) (Table 5), suggesting that the haplotype (Arg/Asp) might be favourable for the longevity in old female.

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

Discussion