Polyamines (putrescine, spermidine, and spermine) are a family of molecules that derive from ornithine through a decarboxylation process. They are essential for cell growth and proliferation, stabilization of negative charges of DNA, RNA transcription, translation, and apoptosis. Recently, it has been demonstrated that exogenously administered spermidine promotes longevity in yeasts, flies, worms, and human cultured immune cells. Here, using a cross-sectional observational study, we determined whole-blood polyamines levels from 78 sex-matched unrelated individuals divided into three age groups: Group 1 (31–56 years, n = 26, mean age 44.6– 6.07), group 2 (60–80 years, n = 26, mean age 68.7– 6.07), and group 3 (90–106 years, n = 26, mean age 96.5– 4.59). The total content of polyamines is significantly lower in groups 2 and 3 compared to group 1 ( p = 3.6 · 10 - 12). Interestingly, this reduction is mainly attributable to the lower putrescine content. Group 2 displays the lowest levels of spermidine and spermine. On the other hand, nona/centenarians (group 3) display a significantly higher median relative percentage content of spermine with respect to total polyamines, compared to the other groups (13.2% vs. 14.1% vs. 30.6%, p = 6.0 · 10 - 4 ). 

 The study population was divided into three age groups: Group 1 (31–56 years, n = 26, 13 males and 13 females, mean age 44.6 – 6.07), group 2 (60–80 years, n = 26, 13 males and 13 females, mean age 68.7 – 6.07), and group 3 (90–106 years, n = 26, 13 males and 13 females, mean age 96.5 – 4.59). A total of 3 mL of whole blood was collected from each subject during fasting, in a K2EDTA-containing vacutainer. The study protocol was approved by the Joint Ethical Committee ( JEC) University of Camerino-Azienda ASUR Marche ZT-10 Camerino, in accordance with the Declaration of Helsinki in its revised edition and with international and local regulatory requirements. Precolumn derivatization and high-performance liquid chromatography separation of polyamines Direct determination of polyamines in biological fluids is difficult because they are small aliphatic molecules that do not exhibit any structural features that would allow their sensitive spectral detection without derivatization. Thus, 100 lL of whole blood (previously treated with 5% trichloroacetic acid (TCA) to allow protein precipitation) and 1 mL of standard polyamines stock solutions (1 mg/mL) were subjected to a derivatization process with dansyl chloride according to Seiler.11 After incubation at 40C for 45 min with dansyl chloride, mixtures were filtered through a 0.45-lm regenerated cellulose-membrane (ALBET-LabScience, Dassel/Relliehausen, Germany) and injected into a highperformance liquid chromatography (HPLC) apparatus (Agilent 1100) for analysis. The separation was executed on a Supelcosil LC 18 RP column (5 lm; 4.6 · 250 mm) maintained at 35C. 

 The derivative polyamines were identified from the comparison of retention times characteristic of standard polyamines (see Figure S1A)(Supplementary Data are available at www.liebertonline/rej/), to that obtained from whole blood samples (Fig. S1B). The relative content was quantified according to the linear regression obtained from each calibration curve (Fig. S2). The detected polyamines total content is reported in Fig. 1. According to previous evidence,10 we noticed a significant reduction for total polyamines content in groups 2 and 3 compared to group 1 (Kruskal–Wallis H = 26.42, degrees of freedom [df] 2, p = 1.8 · 10 - 6 ). Notably, the lower total polyamines content detected in groups 2 and 3 was mainly attributable to the lower putrescine level compared to age group 1 (data not shown). In fact, groups 2 and 3 displayed negligible levels of putrescine ( < 0.013 nmol/mg) for the majority of subjects studied (26/26 for group 2 and 18/26 for group 3, respectively), whereas 20/26 of subjects in group 1 showed relevant levels of putrescine (0.149–0.441 nmol/mg). Spermidine levels were significantly different across the three age groups (Fig. 2), with group 3 displaying levels similar to group 1, whereas group 2 subjects showed lower levels compared to the other two groups (Kruskal–Wallis H = 44.19, df 2, p = 2.5 · 10 - 10). 

 Our data are consistent with already published data obtained by HPLC determination in whole blood from human volunteers.14 In their work, Soda et al. reported that a long-term oral intake of polyamine-rich diet causes an increase in blood polyamine levels (especially in spermine concentration). On the contrary, they demonstrated that a short-term supplementation of polyamine-rich food does not increase blood polyamine levels. This observation allows us to consider our data likely unaffected by the wide variability of polyamine content among foods, and in any case randomly affected. On the other hand, we are aware that polyamine concentrations in the blood can vary considerably among healthy individuals, as observed by others.3,15,16 Several factors can contribute to this variability; polyamines produced in the intestinal lumen are quickly absorbed by blood because of the highly microvascularized intestinal mucosa, and gut microbiota are also an efficient source of polyamines.17,18 Here we found that age-related decrease in polyamine levels is attributable to a very significant reduction in putrescine content, whereas spermidine and spermine are significantly enriched in whole blood of nona/centenarians. Interestingly, high putrescine levels were reported in several age-related diseases such as cancer,1,10 Parkinson disease,19 pancreatitis,20 and ischemia.21 Notably, spermidine and spermine concentrations were markedly decreased in Alzheimer disease.22 On the other hand, spermidine and spermine were found to ameliorate some pathological conditions.

 Spermidine supplementation improves protein utilization efficiency and ameliorates trauma effects on amino acid levels23; it has also been suggested that oral spermidine administration could be a useful treatment for multiple sclerosis.24 Spermine confers protection against lethal sepsis partly by attenuating sepsis- and high-mobility group protein B1 (HMGB1)-induced inflammatory responses,25 and it has been proven to reduce infarction and neurological deficit following middle cerebral artery occlusion.26 The underlying mechanisms of age-related alterations in the relative content of whole blood polyamines observed in this study are not fully understood at present. Given the observational nature of this study, it is very difficult to explain whether the changes in the polyamines levels between the three age groups are due to a decrease in cellular biosynthesis of polyamines (e.g., reduced cellular mitotic rate) or due to a reduction in metabolic uptake from the diet. Nevertheless, our data add new insight about the relevance of polyamines in human longevity. A distinct role of the three physiological polyamines has been proposed to regulate the different cellular functions in which this class of molecule is involved.27 Cell proliferation and apoptosis are strictly related to polyamine intracellular levels, which are regulated by multiple mechanisms affecting their synthesis, degradation, uptake, and excretion. The enzymes involved in these biochemical processes are finely regulated; thus, an increase of one of the three natural polyamines must have a biological meaning.

 Many investigations seem to converge to the observation that rapidly growing prokaryotes mainly contain putrescine and spermidine, whereas slowly growing eukaryotes mainly contain spermidine and spermine.27 The relationship between polyamine content and rate of cell proliferation is also supported by the hypothesis that the reduced polyamine biosynthetic capacity of aged mammals can account for the slower course of some tumors in elderly patients.10 Aging is a biological phenomenon significantly influenced by both genetic and environmental factors. Gene-related regulatory systems (evolutionarily conserved) also control diapause-like states allowing the postponed reproduction in response to adverse environmental conditions.28 Spermidine has been shown to act synergistically with resveratrol (a natural polyphenol found in grapes, red wine, berries, knotweed, peanuts, and other plants, and a known sirtuin 1 [SIRT1] activator) in inducing autophagy through convergent deacetylation and acetylation reactions in the cytosol and in the nucleus.29 Polyamines (spermidine, in particular) could modulate the cell cycle in response to environmental changes (i.e., oxidative stress, food limitation, and crowding), induce autophagy, and eventually increase life span.30 Notably, methionine cycle and polyamine pathway are interdependent (Fig. 6),27 and high homocysteine levels (marker of folate deficiency) have been associated with cardiovascular disorders.31 Recently, a surprisingly higher content of plasmatic homocysteine in centenarians compared to randomly selected donors has been reported.

32 The authors suggest the existence of a protective mechanism (probably gene related), allowing a long survival despite the high value of homocysteinemia. Cellular resistance to homocysteine, as a key mechanism for successful aging, has been also proposed by others.33 In this scenario, the relative increase of spermidine in the blood of nona/centenarians we observed might be viewed in contrast with high levels of homocysteine, unless compensatory mechanisms related to vitamin intake (e.g., folate) or to the enzymatic activities of polyamine pathway (in particular ornithine decarboxylase [ODC] and S-adenosylmethionine decarboxylase) are implied.34 Clearly, the present work should be considered a preliminary investigation, and further studies are warranted to fully understand the role of polyamines in determining life span. Also, longitudinal and nutritional studies might suggest potential therapeutic approaches to sustain healthy aging and to increase human life span. In conclusion, our study extends our knowledge on biochemical determinants of human longevity and confirms the relevance of polyamines in biological processes.