Control of oxidative reactions of hemoglobin in the design of blood substitutes: role of the Vc, NAC, TEMPO and their reductant system

Abstract

Oxidative reactions of hemoglobin (Hb) are still a serious problem for Hb-based blood substitute development. Although varieties of antioxidant strategies have been suggested, this in vitro study examined the ability of the ascorbate, N-Acetyl-L-Cysteine (NAC), 4-hydroxy-2, 2, 6, 6-tetramethylpiperidine-1-oxygen free radicals (TEMPO) and their reductant system in preventing Hb oxidation. The content of ferric Hb is monitored in the process of vitamin C (Vc), NAC, TEMPO and their reductant system. The results suggest that ascorbate is effective in reducing ferryl Hb, and TEMPO with Vc/NAC could obviously shorten the reaction time, but it does not play the role of Met-Hb reductases. It demonstrates that TEMPO did little to recover Hb under oxidative stress.

Keywords::

• Met-Hb
• NAC
• reduction
• TEMPO
• Vc

Introduction

Blood transfusion is a lifesaving intervention (Reddy and Leitman 2013). But blood transfusion has many inherent side effects and dangers such as the risk of exposure to blood-borne pathogens (Chen et al. 2009) and inaccurate cross-matching (Ramasubramanian and Anthony 2004). Although the incidence is less than one in 1.5 million transfusions (McCullough 2003), it remains the leading direct cause of deaths resulting from blood transfusion. Due to the biological limitations, side effects and logistical constraints of blood transfusions, it is important to develop various blood substitutes. Although no single product can imitate all properties of blood, substantial progress has been made, especially in the development of hemoglobin-based oxygen carriers (HBOCs).

However, it is believed that the side effects caused by the administration of HBOCs might be linked with the Hb pro-oxidant potential, and its pressor effect, particularly in subjects with compromised antioxidant status (Natanson et al. 2008). To possess respiratory function, heme iron must be in its ferrous (Fe²⁺) state that is protected in the red blood cell (RBC) environment by sophisticated enzymatic and nonenzymatic antioxidant defense systems. But these systems cannot fully protect heme against oxidation. And approximately 3% of total Hb is in its ferric (Fe³⁺) form that is unable to transport oxygen and carbon dioxide (Misra and Fridovich 1972, Wever et al. 1973). Aging of the RBCs leads to the weakening of their antioxidant systems, which results in excessive Hb oxidation and formation of reactive oxygen and heme species (Bunn and Forget 1986) that oxidize the membranal lipids causing RBC hemolysis and release of free Hb into plasma. Plasma does not have as sophisticated reducing/antioxidant powers as RBCs, except for the presence of a few exogenous and endogenous antioxidants such as ascorbate, urate, reduced glutathione, alpha-tocopherol, beta-carotene, bilirubin and albumin in micromolar concentrations (Frei et al. 1989, Halliwell and Gutteridge, 1990). They are strong reducing agents and potent antioxidants that act together.

The spontaneous oxidation of plasma-free Hb produces superoxide anion (O₂^•−). Hence the iron is oxidized to the ferric state (Misra and Fridovich, 1972) .

And the O₂^•− could induce the production of much more reactive oxygen species (ROS) (Fridovich 1978, Halliwell et al. 2000, Weiland et al. 2004). These super oxygen anions have high reaction activity and toxicity, which could cause oxidative stress response in the process of hemoglobin (Hb) delivering oxygen to tissues (Figure 1). Under the normal condition, there are superoxide dismutase (SOD), catalase (CAT) and Met-Hb reductase (Lu et al. 2003) in the red blood cells. Not only does the redox system reduce side effects on Hb, but it also controls the content of Met-Hb. Because the materials of clinical trial product are pure poly-Hb without these enzymes, the products are more easily oxidized.

Figure 1. The oxidation of Hb and the tissue damaging of ROS.

4-hydroxy-2, 2, 6, 6-tetramethylpiperidine-1-oxygen free radicals (TEMPO) acts as a free-radical scavenger to prevent the damage inflicted by free radicals. To simulate the function of SOD and CAT in this study, TEMPO was added into the solution of ascorbate/NAC and Hb (Buehler et al. 2000, Lu et al. 2003). If it was confirmed that TEMPO not only has the function of SOD and CAT, but also the function of Met-Hb reductase, it would have all the effects of the reduced enzyme system in red cell. In our opinion, the hydroxylamine is more hydrophobic to easily approach the bag of heme. Hence,we speculate that the reduction rate of the hydroxylamine is higher than that of ascorbate and Met-Hb. Because the hydroxylamine is more hydrophobic,it approaches the heme-binding pocket of the Hb more easily. We thus assume that TEMPO could speed up the reaction. But our results suggest that this is not an issue.

This research explores the effects of ascorbate and NAC alone, and their reduction order in Hb solution. Furthermore, the purpose of the present study was to evaluate the actual role of vitamin C (Vc) and NAC in the control of Hb oxidation in the absence and presence of TEMPO.

Experimental

Reagents and instrument

Human umbilical cord blood was obtained from Tianjin Stem Cell Gene Engineering Company. NAC and TEMPO were purchased from Aladdin Reagent co. Ltd. (Shanghai, China). Vc was obtained from Tianxin Fine Chemical Engineering Development Center (Tianjin, China). Other chemicals used were of analytical grade and the solutions were prepared with deionized water. Spectrophotometry was conducted using SHIMADZU UV-2550, and the Dissolved Oxygen Analyzer (DO-813) was purchased from Chengdu Instrument and Meter Co., Ltd.

Methods

• (1) Preparation of Hb from human placentas (Feng-Juan 2007).

• (2) Determination of the concentration of Hb by the method of Evelyn and Malloy (Yang Cheng Min and Zeng Min 2000).

• (3) Spectrophotometry assays.

As we all know, Hb has three states: Oxy-Hb, Deoxy-Hband Met-Hb, respectively. The percentage of three components can be expressed as a function of the measured absorbance by solving the system of simultaneous equations (Yong-Ning 2001):

1.8814 × Oxy-Hb% + 1.4773 × Deoxy-Hb%+ 0.8419 × Met-Hb% = A₅₄₀/A₅₂₃

1.2539 × Oxy-Hb% + 1.8095 × Deoxy-Hb%+ 0.616 l × Met-Hb% = A₅₅₄/A₅₂₃

2.0201 × Oxy-Hb% + 1.3345 × Deoxy-Hb%+ 0.5473 × Met-Hb% = A₅₇₆/A₅₂₃

Y = Oxy-O₂Hb/(Oxy-Hb + Deoxy-Hb),

where A is absorbance and Y is oxy-Hb saturation; Oxy-Hb%, Deoxy-Hb% and Met-Hb% are the percentage of Oxy-Hb, Red-Hb and Met-Hb; and 523 nm, 540 nm, 554 nm and 576 nm are the four wavelengths to be chosen. Based on the above equation, the absorbances were determined at different times. And then, a graph illustrating Met-Hb (oxy-Hb saturation) with time is obtained.

Results and discussion

The order of the reductions function

It is well known that Vc and NAC (Simoni et al. 2009), especially the Vc, are the common reductants to Met-Hb. But there is no report about the reaction order. In this study, a combination of statistics was developed by determining the oxygen content, Met-Hb and oxygen saturation in the process of reaction. The result is shown in Figure 2.

Figure 2. The variation curve of oxygen content, Met-Hb and oxygen saturation in the process of reaction (200 min). Assay conditions: (A) instead of Hb solution with water and adding 200 times Vc or 1000 times NAC; (B) Hb is diluted with 0.2 M saline phosphate buffer, pH 7.4, to obtain 0.075 g/dL Hb solution; (C) condition as described in (A).

This figure indicates that the oxygen content, oxygen saturation and Met-Hb content were declined in turn. It was also noticed in this figure that the process of Vc or NAC restoring Met-Hb can be subdivided into three parts: the dissolved oxygen in the water is depleted firstly; then, the oxygen bound with Hb is cleaned up; and Met-Hb is reduced finally. As shown in Figure 2, it can also be seen that the reductive effect of Vc is higher than that of NAC. When Vc is added, the reaction time is shorter, and the final content of Met-Hb is lower than that of NAC.

The reduction of Vc and NAC with TEMPO without de-oxygenation

Due to the lack of reductant, the Met-Hb content and oxygen saturation do not change with time, when nothing or only TEMPO is added in control experiment. When Vc is added, the Met-Hb content is decreased quickly (Figure 4A). But it is very different when both TEMPO and reductant are present.

Generally, reduction of TEMPO yields hydroxylamine by Vc (or NAC). Hydroxylamine is easily oxidized by oxygen in solution. The chemical reaction is shown in Figure 3. So the oxygen saturations of Hb decline, when TEMPO and Vc/NAC are added to the solution (Figures 4B and 5B). But TEMPO makes the starting time of Hb decline earlier. This indicates that TEMPO could speed up the reaction of cleaning up the dissolved oxygen. On the other hand, the three slopes of oxygen saturations of Hb are similar in Figures 4B and 5B. This means that there is no obvious difference in the reaction rate of reducing bound oxygen in this case. Furthermore, the Met-Hb content reduced more quickly with 0.05 g/dL or 0.01 g/dL of TEMPO and 0.02 g/dL of Vc than only with Vc. The rate of Met-Hb reduction is nearly similar whatever the TEMPO concentration is, and that of NAC is the same (Figure 5A). This suggests that TEMPO is not the catalyst of this process.

Figure 3. The reaction of ascorbate with TEMPO.

Figure 4. The variation curve of Met-Hb and oxygen saturation in the process of Vc and TEMPO without de-oxygenation. Assay condition: Hb is diluted with 0.2 M saline phosphate buffer, pH 7.4, to obtain 0.075 g/dL Hb solution and the pH of VC solution is adjusted to 7.4 before use. Oxygen is isolated in the whole process.

Figure 5. The variation curve of Met-Hb and oxygen saturation in the process of NAC and TEMPO without de-oxygenation. Assay condition as described in Figure 4.

The reduction of Vc and NAC with TEMPO after de-oxygenation

In this group of experiments, the oxygen dissolved in Hb solution is removed by degassing. Because the oxy-Hb releases bound oxygen to maintain a balance, the oxygen saturation decreased in Hb solution (shown in Figure 6B). Furthermore, the reaction rate of scavenging bound oxygen increased markedly. This means that TEMPO speeded up the second step obviously after de-oxygenation in this case.

Figure 6. The variation curve of Met-Hb and oxygen saturation in the process of Vc and TEMPO after de-oxygenation. Assay condition: remove oxygen dissolved in Hb solution within 30 min before adding the reductant or the mixture and other conditions are as described in Figure 3.

The changes of Met-Hb content are shown in Figures 6A and 7A. As soon as the reductant is added, the bound oxygen is cleaned up quickly. So the drop points of Met-Hb reducing curve are obviously earlier as shown in Figures 6A and 7A than in Figures 4A and 5A. On the other hand, the slopes of Met-Hb reducing curve are very similar, be it with reductant-TEMPO or with reductant only. This means that the TEMPO has an effect on the reduction of Met-Hb after de-oxygenation.

Figure 7. The variation curve of Met-Hb and oxygen saturation in the process of NAC and TEMPO after de-oxygenation. Assay condition as described in Figure 4.

Conclusion

The results of this study demonstrated the reaction order of Vc and NAC. They also show that the TEMPO could speed up the reaction of cleaning up oxygen, but it does not have the function of Met-Hb reductase. This work suggests that the designing of a Vc/NAC-TEMPO, HBOCs system could be a preferred option over any Hb antioxidation strategy developed so far.

Acknowledgments

This report would not have been possible without the support and substantive contributions of Liu Jiaxin and Wang Hong, the Senior Research Scientists at Institute of Blood Transfusion, Chinese Academy of Medical Sciences, Chengdu, P. R. China. I would like to thank Li Shen and Zhou Wentao who gave me a lot of help in the experiment.

Declaration of interest

The authors report no declarations of interest. The authors alone are responsible for the content and writing of the paper.

The work is supported by The National High Technology Research and Development Program (“863”Program) of China (N0.2012AA021903) and The National Science Foundation of China (No.21202117/B020703).