Transfusion is an important medical practice of fl uid resuscitation, and this service is commonly used for patients with acute massive blood loss, burns, anemia, surgery, malignancies, and severe trauma. It has an irreplaceable role in signifi cantly improving oxygen supply, promoting coagulation, and saving lives. The concentrates of red blood cells (RBCs) during in vitro storage undergoes a series of biochemical and morphological changes, i. e., the RBCs storage lesions [1],[2], which leads to reduced survival rate of RBCs. Therefore, it is necessary to improve the storage quality and prolong the storage time, and these have become the hot research topics in recent years. Energy depletion and oxidative damage are the key factors of RBCs lesion during the storage [3],[4]. To reduce oxidative damage, some antioxidants were added into the additive solution. Currently, the endogenous antioxidants such as vitamin E [5], vitamin C [6],[7], glutathione (GSH) [8],[9] and N-acetylcysteine [10], and some exogen ous phenolic compounds, such as propofol [11], have been reported. The addition of most of these antioxidants could improve the storage quality of RBCs to a certain extent but could not completely counteract the strong oxidative damage. This is because there are many kinds of free radicals, and the single free radical scavenger cannot effectively block the polymorphic chain reaction. To solve this problem, the protective effects of vitamin C [10],[12], vitamin E [5],[12], cysteine [13] and their combination [10] were studied. All of these have achieved some antioxidative effects by scavenging hydroxyl radicals, stabilizing cell membrane, and scavenging H2 O2 , respectively. However, vitamin C can be easily oxidized by itself, and the RBCs were further damaged by generated free radicals. In addition, the additives of additive solution, vitamin C, cysteine, and glutathione (GSH) are all water-soluble, and so they can be easily dissolved, but it’s diffi cult for them to enter RBCs. In contrast, as a liposoluble antioxidant, vitamin E can enter the cell, but is diffi cult to dissolve in the additive solution. The study of additive solution has never stopped, and also the damage of RBCs during storage has not been substantially improved.

Melatonin (MT), also known as N-acetyl-5-methoxy tryptamine, is a hormone synthesized and secreted by pineal gland [14]. It has a wide range of physiological functions and influences biological clock, regulates immune system, and has antioxidant actions that can act as a direct free radical scavenger or by increasing the expression and activity of endogenous antioxidant enzymes [15],[16]. In addition, MT can exert antioxidant effects in the membrane as well as cytosol because of its lipophilic and hydrophilic properties. In view of this, MT is speculated to be a better supplement for storage of RBCs. Some studies have shown that the pro- and antioxidative properties of MT depended on the type of cells, redox state, as well as experimental conditions [17]. The human serum MT concentration is lower during the day (10–20 pg/mL) and significantly higher at night (30–120 pg/mL), which reaches its peak at about 3:00 am [18]. The general hypnotic-curing oral dose of melatonin is 1 to 3 mg, and the blood drug concentration achieves more than 1.9 ng/mL after 1 hour. M.R. Şekeroğlu et al. [11] have shown that 500 pg/ mL MT could prevent the accumulation of malondialdehyde (MDA) and protect GSH, glutathione peroxidase and superoxide dismutase levels, but did not affect the catalase levels of RBCs. M. Allegra et al. [19] have shown that MT not only prevents MDA production, but also prevents damage of RBCs caused by MDA. Some studies have reported the limited antioxidative activity of MT [20] or even provide evidence for its pro-oxidative properties [21],[22],[23],[24]. 

Aim of this study — to investigate the long-term preservation effects of low hypnotic drug concentration of MT on the morphology, aggregation, oxidative stress and metabolism of glucose in stored RBCs.

This study was approved by the Ethics Committee of Baotou Medical College. Six healthy volunteers aged 18–23 years were recruited, and informed consent form was obtained before blood donation. 200 mL of whole blood was collected from each volunteer. After depletion of white blood cells, the packed RBCs were resuspended in 50 mL MAP solution (citric acid 0.20 g, sodium citrate dihydrate 1.50 g, glucose 7.93 g, sodium dihydrogen phosphate 0.94 g, sodium chloride 4.97 g, adenine 0.14 g, mannitol 14.57 g in each 1000 mL additive solution). The RBCs were gently mixed and then evenly distributed in 2 blood bags, the main component of the blood bag is 2-ethylhexyl ester (DEHP) plasticized polyvinyl chloride (PVC) (Shandong Weigao Group medical polymer products Co., Ltd, Shandong, China). MT was added in one bag (MT group) to reach a final whole blood concentration of 150 pg/mL, and an equal volume of MT solvent was added in the other bag (control group). These RBCs were then stored at 4 ± 2 °С. At shelf-life of 0, 7, 14, 21, 28, 35, and 42 days, 5 mL of blood sample was taken from each bag after gently mixing the suspension.

RBCs suspension of 50 μL was used to push the blood smear, and then was stained by Giemsa. The morphology of RBCs was observed under a microscope (×100) and the number of deformed RBCs was counted in each 1000 RBCs.

LBY-N6C automatic blood rheometer was used to detect the aggregation of RBCs.

RBCs suspension of 1 mL was centrifuged at 3000 rpm for 5 minutes. Then 200 μL of supernatan t was taken out and diluted with deionized water. The diluted supernatant was used for detecting the absorbance at 540 nm by multifunction microplate reader (Thermo 3001). The absorbance of 50 times diluted hemolysate was used as reference, and the relative hemolysis rate of each sample was calculated by using the following formula:

Relative hemolysis rate =sample absorbance /reference absorbance.

The concentration of MetHb, glucose and lactic acid was detected by the Radiometer ABL90 blood gas analyzer.

The pH of blood sample was determined by using the Mettler Toledo pH meter. The concentration of MDA was detected according to the MDA kit instructions (S0131, Beyotime Institute of Biotechnology). The ATP level in RBCs was determined according to the ATP detection kit (S0027, Beyotime Institute of Biotechnology).

Statistical analyses. GraphPad Prism 5.02 statistical software was used for data statistics and analysis. Measurement data was expressed as means ± standard deviation (x ± s), and two-way Analysis of Variance (ANOVA) was used to do statistical analysis. P < 0.05 represents a statistically signifi cant difference.

The blood smear was used to observe the morphology of RBCs. As shown in Figure 1A, with the prolonged storage time, the morphology of RBCs changes from smooth double concave disk to acanthocyte, smooth spherical and acanthus erythrocyte in both control group and MT group. Statistical analysis (Figure 1B) showed that the number of deformed RBCs was affected by both storage time (p < 0.0001) and MT concentration (p < 0.0001), and there was interaction between them (p < 0.0001). The number of deformed RBCs in MT group was signifi - cantly less than that in the control group on days 21, 28, 35 and 42 (p < 0.0001).

The relative hemolysis rate refl ects the destruction of RBCs. The results (Figure 3) showed that the relative hemolysis rate was affected by both storage time (p < 0.0001) and melatonin concentration (p < 0.01), and there was interaction between them (p < 0.01). Compared with the control group, the relative hemolysis r ate of MT group was signifi - cantly lower on day 42 (p < 0.0001).

The RBC aggregation index w as detected by LBYN6C automatic blood rheometer. As shown in Figure 2, the RBC aggregation index was gradually increased in both control and MT groups during the storage of RBCs. Statistical analysis (Figure 2) showed that both storage time (p < 0.0001) and melatonin concentration (p < 0.01) affect the aggregation index, but there is no interaction between them (p > 0.05). The aggregation index of MT group is lower than control group on days 35 (p = 0.0602) and 42 (p = 0.0542), but there is no signifi cant difference.

MDA is one of the fi nal products of polyunsaturated fatty acids peroxidation in the cells, and MDA level is commonly used as a marker to refl ect oxidative stress and the antioxidant status. This study showed (Figure 3A) that both storage time (p < 0.0001) and melatonin concentration (p < 0.0001) affect the MDA, and there is also interaction between them (p < 0.01). Compared with the control group, the relative hemolysis rate of MT group was signifi cantly lower on days 35 (p < 0.05) and 42 (p < 0.0001).

MetHb is formed by reversi ble oxidation of heme iron (Fe2+) to ferric state (Fe3+). It is a reactive molecule, which can further increase oxidative stress and cause osmotic fragility and intravascular hemolysis. As shown in Figure 3B, both storage time (p < 0.0001) and melatonin concentration (p = 0.0002) affect the MetHb, but there is no interaction between them (p > 0.05). The MetHb of MT group was signifi cantly lower than control group on day 42 (p < 0.0001).

Glucose in the additive solution was considered as the main energy source of RBCs. Our results (Figure 4A) showed that the glucose concentration decreased wi th storage time (p < 0.0 001) but could not be affected by MT concentration (p > 0.05), and there is no interaction between them (p > 0.05).

Lactic acid is the end product of the anaerobic oxidation of glucose. During storage, lactic acid was accumulated, and its concentration was increased with storage time (p < 0.0001) but could not be affected by melatonin concentration (p > 0.05), and there is no interaction between them (p > 0.05) (Figure 4B).

ATP is the direct energy source for the life activities of RBCs. As shown in Figure 4C, the concentration of ATP was gradually decreased with storage time (p < 0.0001) but could not be affected by melatonin concentration (p > 0.05), and there is no interaction between them (p > 0.05).

Stored RBCs are considered as the main source of transfusion therapy. The quality of stored RBCs depends on the composition of additive solution and their storage. However, the additive solution can only delay the aging of RBCs but cannot prevent their aging. In other words, RBCs undergo a series of changes in morphology, function and biochemistry, and these changes become more and more obvious with prolonged storage time. To reduce or delay the damage to RBCs during storage, it is necessary to find effective blood preservation additives or formulations. This has become a hot research topic in blood conservation study. In mammals, MT not only regulates circadian rhythm, seasonal rhythm, vascular tone, and suppression of cancer, but also is an effective antioxidant both in in vivo and in vitro [25],[26]. As a strong hydroxyl radical scavenger, its scavenging capacity is 4 times that of GSH and 14 times that of mannitol [27],[28]. As an effective lipophilic antioxidant, the scavenging activity of peroxyl alkyl radical is 2 times that of vitamin E [28]. Moreover, M. Allegra et al. have showed that MT can directly remove free radicals produced in RBCs [19]. The serum concentration of MT is generally less than 120 pg/ml (about 66 pg/mL of whole blood concentration), and the hypnotic blood drug concentration is more than 1900 pg/ mL (about 1045 pg/mL of whole blood concentration). In this study, the protective effects of 150 pg/mL (whole blood concentration) of MT on RBCs were observed during long-term storage of RBCs.

Firstly, the morphology of RBCs was observed at different storage time points. As shown in Figure 1A, there were regular degenerative changes in the morphology of RBCs from smooth double concave disc to spiny to smooth ball in both groups, and more and more RBCs were aggregated together in the blood smear and were gradually increased with RBCs aggregation index. In addition, with the continuous destruction of RBCs, the relative hemolysis rate of RBCs was also increased. Compared with control group, the number of deformed RBCs and relative hemolysis rate of MT group decreased significantly at the end storage stage. The aggregation index of MT group was lower than that of control group on days 35 (p = 0.0602) and 42 (p = 0.0542), but the difference is not statistically significant, it might be related to the small number of samples. From the above results, we can preliminarily infer that low concentration of MT has protective effects on the long-term stored RBCs. Next, the effect of MT on the oxidative stress and glucose metabolites of stored RBCs were measured at different storage time.

Oxidative stress is one of the important factors of storage lesion. To further disclose the protective mechanism of MT, MDA and MetHb, the indicator of oxidative stress, were detected. MDA is considered as one of the most important products of membrane lipid peroxidation and indirectly reflects the degree of oxidative damage [23]. MetHb is a hemoglobin but cannot transport O2 . MetHb occurs when oxyhemoglobin in a ferrous (Fe2+) state is transformed into a ferric (Fe3+) state. Low levels of MetHb (< 3 %) are always present in the circulation, but it markedly increases after exposure to certain pathological conditions. It also acts as an important oxidative marker of RBCs. The contents of MDA and MetHb were gradually increased with the storage time in both the groups, and this might be because of the decrease of reductive substances (such as NADPH) in RBCs. NADPH is produced by pentose phosphate pathway, and is the coenzyme of glutathione reductase, which is responsible for the reduction of oxidized glutathione (GSSG) to reduced GSH. At low temperature, pentose phosphate pathway slows down in the storage of RBCs, leading to decreased NADPH and GSH. GSH is an essential component of antioxidative stress, and decreased GSH was speculated to have correlation with increased MDA and MetHb in this study. However, the MDA and MetHb level in MT group were lower than that of the control group, indicating that low concentration of MT could reduce oxidative stress and protect the stored RBCs.

Glycolysis is regarded as the main energy source of RBCs. This pathway breaks down glucose to produce ATP and lactic acid. Accumulation of lactic acid decreases the pH of the storage solution. In our study, the concentration of glucose and ATP were gradually decreased, and the lactic acid were gradually accumulated with the increasing storage time in both groups, however, no statistically significant differences were observed between control and MT group. Therefore, we speculated that the protective effect of low concentration of MT mainly through its antioxidant function of MT, but also could affect glucose metabolism of stored RBCs, which might strengthen the pentose phosphate pathway while reducing glycolysis and energy consumption by some unknown mechanism to improve the survival environment of stored RBCs.

A. Krokosz et al. [17] had revealed that the prolonged incubation of RBCs at 0.02 to 3 mM MT for up to 96 hours at 37 °C induced a progressive destruction of erythrocytes. In order to avoid the possible damage of high concentrations of MT on RBCs, low concentrations (150 pg/mL) of MT on stored RBCs were initially observed in our experiment. The results showed that MT not only protects the morphology of RBCs, improves the hemolysis and aggregation index, but also reduces the oxidative stress. It is worth mentioning, at the middle and end of storage, the consumption rate of glucose and ATP, and the accumulation rate of lactose in MT group were all lower than that in control group, although no statistically significant differences were observed, the trends of these 3 related indicators remained consistent. In recent year, some research showed that increased MT signaling has relation with glucose metabolism in some types of cells [29],[30]. Therefore, MT was speculated to strengthen the pentose phosphate pathway while reducing glycolysis and energy consumption by some unknown mechanism to improve the survival environment of stored RBCs in our study. In the further research, the concentration of MT should be expanded to observe the effects on glucose metabolism of stored RBCs. These will provide a theoretical basis for the development of more effective RBCs storage solution.