(690e) Synergistic Effect of Polyamines Against Lipid Oxidation As in Vitro Model Aging System

Polyamines are aliphatic compounds with two or more primary amino groups in the molecule, and are often found in fermented foods such as natto and yogurt. In recent years, the polyamines have attracted much attention in preventive effects on aging-related diseases such as atherosclerosis and Alzheimer's disease, as well as life-extending effects.¹ However, the detailed working mechanism of polyamines for this effect remains unclear.

Biological membranes are composed of lipids such as phospholipids and cholesterol, and the oxidation of biological membrane has recognized to lead to the atherosclerosis and aging. Its biological membrane oxidation is thought to proceed by the radical chain reaction mechanism.² First, water-soluble radicals (R•) in the blood around the biological membrane react with oxygen into peroxyl radicals (ROO•), which are then transferred to the lipid phase. These peroxyl radicals attack lipids (LH) constituting biological membranes, resulting in lipid radicals (L•). These lipid radicals combine with oxygen to form highly reactive peroxyl radicals (LOO•). This peroxyl radical attacks another lipid continuously, leading to a chain reaction of oxidation. The aging is considered to progress due to damage to biological membranes and accumulation of lipid peroxide (LOOH) during this radical chain oxidation of lipid. In neurodegenerative diseases such as the Alzheimer's disease and Parkinson's disease, which are aging-related diseases, the lipid peroxide as oxidative stress markers have been reported to be increased.³

Phenolic antioxidants, such as vitamin E (V_EH), is known to suppress the oxidation of biological membrane lipids.⁴ For example, low concentrations of α-tocopherol, V_EH, in plasma have been found in patients with the Alzheimer's disease associated with the aging,⁵ suggesting a close link between aging and lipid oxidation. Therefore, the life extension effect of the polyamines seems to be due to their high ability to suppress lipid oxidation. Toro-Funes et al. reported the antioxidant effect of polyamines when spermine was added alone to soybean oil.⁶ Løvass et al. reported that when spermine and spermidine were added to tocopherol-containing fish oil, the antioxidant effect increased with increasing the added amount.⁷ However, there are only a few reports on the antioxidant effect of polyamines on lipid oxidation, and the mechanism of this antioxidant effect of polyamines on lipids is still unclear.

In this study, as the first step for verification of lifespan-extending effect by polyamines, we investigated how polyamines act against oxidative stress in vitro homogeneous lipid model system. We performed the oxidation experiments for a co-addition of polyamines and phenolic antioxidants and the combination effects were discussed with synergistic effects in different combinations of antioxidants.

Experimental

Methyl linoleate was used as a model lipid. Spermine (Spe), spermidine (Spd), and putrescine (Put) were used as polyamines. Vitamin E (a-tocopherol) was used as a general phenolic antioxidant within an organism. Oxidation experiments were carried out according to the Rancimat method using a Rancimat apparatus. The reaction tube was filled with 3.0 g of model lipids and the experiments were performed under accelerated conditions of 120°C and aeration of air with a flow rate of 20 L/h. The progression of oxidation was evaluated by collecting the volatile oxidation products formed by the oxidation in pure water and then measuring the electrical conductivity in the collected solution. The amount of antioxidant added to the model lipid was 0, and 7.0 µmol when the oxidant was added alone. For co-addition of V_EH and polyamines, the added amounts of both antioxidants were set at 7.0 µmol.

Results and discussion

The change in electrical conductivity when Spe is used as polyamine is shown in Fig. 1. In the absence of antioxidants, the electrical conductivity increased immediately after the heating started, indicating that the oxidation of lipid proceeded rapidly. In contrast, when polyamine, Spe, was added alone, the rapid rise of electrical conductivity was slightly delayed. The period during which the change in the conductivity is small until the rise, that is, the period during which oxidation proceeds slowly, is called an induction period (IP). In the case of V_EH alone, the IP was longer than that of Spe alone. This indicated that Spe itself has only a small suppression effect of oxidation and VEH has greater effect of oxidation suppression than Spe. The IP was twice as long as that of V_EH alone when V_EH was co-added with Spe. This suggests that Spe can synergistically increase the suppression effect of oxidation by combined with V_EH.

To quantitatively analyze this synergistic effect, the sum of IPs for Spe alone (IP_Spe) and V_EH alone (IP_VEH), IP_sole = IP_Spe + IP_VEH, is calculated. IP_coadd in the case of co-addition and IPsole are shown in Table 1. For comparison, similar experiments were performed for different polyamines, Spd and Put, and the obtained IPs are also shown in Table 1. When Put was used as a polyamine, the values of both IPs were comparable, suggesting that there was no synergistic effect. On the other hand, when Spd was used, IP_coadd under co-addition conditions was larger, considering that a synergistic effect by Spd addition has appeared. In the case of Spe, the synergistic effect of co-addition was greater than that in case of Spd. The difference between these three polyamines is the number of secondary amine groups in the structure, which indicates that the greater the number of secondary amine groups, the greater the synergistic effect. The suppression mechanism of lipid oxidation involving this secondary amine group is discussed. Since the antioxidant effect of polyamines themselves is small, the reaction of the V_EH with LO₂• radicals into a stable radical (V_E•) is considered to occur mainly as antioxidation reaction (Equation (1)). Then, the secondary amine group of polyamines donates hydrogen to V_E• (Equation (2)), which results in the regeneration of V_EH, and the antioxidantion reaction of Eq. (1) again proceeds. We believe that the synergistic effect of oxidative suppression of polyamines was expressed by such an indirect reaction to lipid oxidation.

Conclusion

Polyamine synergistically protects against oxidative stress by supplementing and regenerating other phenolic antioxidants. When compared with other polyamines, the synergistic effect was thought to be closely related to secondary amine groups of polyamines.

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