Bioinorganic Chemistry and Applications

Volume 2013 (2013), Article ID 354736, 8 pages

http://dx.doi.org/10.1155/2013/354736

Research Article

Coordination Dynamics and Coordination Mechanism of a New Type of Anticoagulant Diethyl Citrate with Ca²⁺ Ions

Jin Han,¹ Jun-Fa Xue,² Meng Xu,² Bao-Song Gui,¹ Li Kuang,² and Jian-Ming Ouyang²

¹Department of Nephrology, The Second Hospital of Xi'an Jiaotong University, Xi'an 710004, China

²Institute of Biomineralization and Lithiasis Research, Jinan University, Guangzhou 510632, China

Received 30 July 2013; Revised 14 November 2013; Accepted 29 November 2013

Academic Editor: Francesco Paolo Fanizzi

Copyright © 2013 Jin Han et al. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

Abstract.

Diethyl citrate (Et₂Cit) is a new potential anticoagulant. The coordination dynamics and coordination mechanism of Et₂Cit with Ca²⁺ ions and the effect of pH on the complex were examined. The result was compared with that for the conventional anticoagulant sodium citrate (Na₃Cit). The reaction order (n) of Et₂Cit and Na₃Cit with Ca²⁺ was 2.46 and 2.44, respectively. The reaction rate constant (k) was 120 and 289 L·mol⁻¹·s⁻¹. The reverse reaction rate constant () was 0.52 and 0.15 L·mol⁻¹·s⁻¹, respectively. It is indicated that the coordination ability of Et₂Cit with Ca²⁺ was weaker than that of Na₃Cit. However, the dissociation rate of the calcium complex of Et₂Cit was faster than that of Na₃Cit. Increased pH accelerated the dissociation rate of the complex and improved its anticoagulant effect. The Et₂Cit complex with calcium was synthesized and characterized by elemental analysis, XRD, FT-IR, ¹H NMR, and ICP. These characteristics indicated that O in –COOH and C–O–C of Et₂Cit was coordinated with Ca²⁺ in a bidentate manner with 1 : 1 coordination proportion; that is, complex CaEt₂Cit was formed. Given that CaEt₂Cit released Ca²⁺ more easily than Na₃Cit, a calcium solution was not needed in intravenous infusions using Et₂Cit as anticoagulant unlike using Na₃Cit. Consequently, hypocalcemia and hypercalcemia were avoided.

1. Introduction

An anticoagulant must be added to dialysates to prevent blood solidification in vitro (in a dialysis machine). Sodium citrate (Na₃Cit) is an important anticoagulant used in clinical settings [1–3]. However, using Na₃Cit as an anticoagulant easily causes hypocalcemia and hypercalcemia [4, 5] because of the strong chelating ability of Na₃Cit with Ca²⁺ ions. Given this ability, the dissociation metabolism of the formed chelate CaCit in vivo takes 30 min. Using Na₃Cit also negatively affects the maintenance of coagulation stability of high-risk hemorrhage patients in vivo, which easily causes complications such as hypocalcemia during or after dialysis.

Our group has previously synthesized a new anticoagulant [6], namely, diethyl citrate (Et₂Cit). The anticoagulant mechanism of Et₂Cit is based on the formation of Ca²⁺ with Et₂Cit. This formation decreases the Ca²⁺ concentration in blood and inhibits prothrombin conversion into thrombin, thereby influencing the anticoagulant effect. The large steric effect of Et₂Cit weakens the coordination of Ca²⁺ ion compared with that of Na₃Cit. Therefore, hypocalcemia and hypercalcemia can be avoided using Et₂Cit as anticoagulant [7]. The frequency of blood gas analyses can also be lessened by repeatedly taking the venous blood of patients to monitor serum calcium levels, which can help relieve the pain of patients and the workload of nurses.

The stability of the complex of Et₂Cit with Ca²⁺ (CaEt₂Cit) is reportedly weaker than that of CaCit [8]. At pH 7.4 and 37°C, the stability constants (’s) are 1988 for CaCit and 231 for CaEt₂Cit. However, several problems remain unsolved when Et₂Cit is used as an anticoagulant. These problems include the reaction kinetics of Et₂Cit with Ca²⁺ and coordination reaction mechanisms, as well as the composition and characterization of the complex. Accordingly, the coordination dynamics of Et₂Cit and Na₃Cit with Ca²⁺, as well as the influencing factors, were studied. The underlying coordination principle was also proposed.

2. Materials and Methods

2.1. Instruments and Reagents

The instruments used were as follows: CHN–O– rapid type element analyzer (Foss-Heraeus Company), Bruker AM 500 nuclear magnetic resonance (NMR) spectrometer (with CDCl₃ as solvent and TMS as internal standard), Nicolet-170 SX type FT-IR spectrometer, /max 2400 (Rigaku) X-ray diffractometer, inductively coupled plasma emission spectrometry (ICP) system (PE Company, USA), PHS-3C pH meter (Shanghai Precision & Scientific Instrument Co., Ltd., China), and sodium chloride injection system (Wuhan Binhu Double-Crane Pharmaceutical Co., Ltd., China).

All chemical reagents used were of analytical grade. Et₂Cit was prepared in our laboratory (99.3% purity) [6].

2.2. Experimental Methods

2.2.1. Reaction Rate Constants of Et₂Cit and Na₃Cit with Ca²⁺

CaCl₂ and Et₂Cit solutions (2.0 mmol/L) were prepared and mixed. A calcium-ion-selective electrode was used to determine the change in electrode potential of the mixed solution with reaction time at pH 7.4 and 37°C under stirring. The result was then compared with that of Na₃Cit.

The linear regression equation of the calcium ion-selective electrode was (where is the electrode potential and is –p(Ca²⁺). The concentration of Ca²⁺ [(Ca²⁺)] at time was also calculated. Given that CaCl₂ was mixed with Na₃Cit or Et₂Cit (1 : 1) and that the reaction of Ca²⁺ with Na₃Cit or Et₂Cit was equal in solution [7], the following reaction rate equation can be established using to represent the reaction rate:

where is the reaction rate constant and is the reaction order. Assuming that is the amount of Ca²⁺ substance concentration that disappeared at time, that is, (Ca²⁺), the following can be obtained by arranging formula (1):

After logarithm on both sides we get

From the plot of   versus , we can calculate the tangent slope of the curve , which is the reaction rate of various points. Formula (3) shows a linear relationship between and (Ca²⁺). In the diagram of on (Ca²⁺), the slope of the straight line is the reaction order , whereas the intercept is .

2.2.2. Effect of pH on Reaction Rate

The pH of the system was adjusted to 6.0, 7.4, and 8.0. Then, the effect of pH on and was determined.

2.2.3. Synthesis of Diethyl Citrate Calcium Complex Crystal

About 1.665 g (15 mmol) of anhydrous CaCl₂ was completely dissolved in water. Then, 1.241 g (5 mmol) of Et₂Cit was slowly trickled under stirring. The pH was adjusted to 7.0 after obtaining a colorless and transparent solution. The solution was sealed with a plastic wrap having holes and then placed in an oven at 37°C for slow volatilization and crystallization. The precipitated colorless, needle-like crystals were filtered, washed with anhydrous ethanol, dried, and characterized. The methods of characterization included elemental analysis, X-ray powder diffraction (XRD), Fourier-transform infrared spectroscopy (FT-IR), ¹H NMR, and ICP.

3. Results and Discussion

3.1. Reaction Rate Equation of Et₂Cit and Na₃Cit with Ca²⁺

The change in concentration of free Ca²⁺ ion [(Ca²⁺)] with in reaction system of Et₂Cit and Na₃Cit with CaCl₂ is shown in Figure 1. A rapid decrease in (Ca²⁺) was observed with prolonged from 0 s to 30 s. This finding indicated that Et₂Cit or Na₃Cit was rapidly coordinated with Ca²⁺. At , (Ca²⁺) decreased from 1.0 mmol/L to 0.49 mmol/L in the Na₃Cit system and from 1.0 mmol/L to 0.87 mmol/L in the Et₂Cit system. (Ca²⁺) slowly decreased when , indicating that the system was in a dynamic equilibrium of complexation dissociation.

Figure 1: Changes in Ca²⁺ concentration with reaction time in different systems: (a) Et₂Cit-Ca²⁺ system and (b) Na₃Cit-Ca²⁺ system.

The tangent slope of points on the curve, that is, the reaction rate of each point formula (2), can be obtained according to Figure 1. In the diagram of   versus  (Ca²⁺) (Figure 2), the slope of the line was the reaction order (formula (3)). The intercept of the line was in Figure 2, as shown in the following:

Figure 2: Plots of -(Ca²⁺) in different systems: (a) Et₂Cit-Ca²⁺ system and (b) Na₃Cit-Ca²⁺ system.

The reaction rate equations of Et₂Cit and Na₃Cit with Ca²⁺ were as follows:

Given that can directly reflect the reaction rate, (5) shown that the complexation rate of Na₃Cit with Ca²⁺ was faster than that of Et₂Cit.

The anticoagulant mechanism of Na₃Cit and Et₂Cit was based on the combination of calcium ion (Ca²⁺) in serum, as well as the reduced concentration of free Ca²⁺ in plasma that disturbed the blood clotting process from reaching the anticoagulation effect in vitro [9–11]. However, the strong coordination ability of Na₃Cit, particularly as an anticoagulant, can coordinate a large number of Ca²⁺ ions in the blood. This phenomenon can lead to the low serum concentration of calcium in patients, as well as to hypocalcemia and all kinds of complications [12–15]. Therefore, calcium is needed to be replenished in the anticoagulation process of Na₃Cit [16]. Meanwhile, calcium citrate [CaCit] can dissociate during the metabolism and release Ca²⁺ after entering the body in the dialysis process. Additionally, hypercalcemia easily ensued in patients with presupplementary Ca²⁺. Therefore, the incidence of hypocalcemia and hypercalcemia can be reduced if we can reduce the coordination ability of anticoagulant.

The reaction rate was equal to the inverse reaction rate when the reaction reached equilibrium, as shown in the following:

The above equation can be written as follows [17]:

where is the reaction rate constant, is the inverse reaction rate constant, and is the complex stability constant.

In a previous article [8], the values of CaEt₂Cit and CaCit were 231 and 1988 at pH 7.4 and 37°C, respectively, and the values in the coordination reaction of Et₂Cit and Na₃Cit with Ca²⁺ were 120 and 289 L·mol⁻¹·s⁻¹, respectively. According to (7), of Et₂Cit and Na₃Cit with Ca²⁺ in the coordination reaction were 0.52 and 0.15 L·mol⁻¹·s⁻¹, respectively. Thus, the rate of decomposition and release of Ca²⁺ was faster for CaEt₂Cit than for CaCit. The above results indicated that Et₂Cit can complex with Ca²⁺ and reduce the free Ca²⁺ concentration during anticoagulation; thus, anticoagulation can be achieved. Meanwhile, the complexing ability of Et₂Cit with Ca²⁺ was weaker than that of Na₃Cit. After Et₂Cit coordinated with Ca²⁺, the Ca²⁺ releasing rate of CaEt₂Cit was faster than that of CaCit. Therefore, the occurrence of hypocalcemia in patients can be avoided. Moreover, only a small amount of calcium or none at all was needed using Et₂Cit as anticoagulant during dialysis unlike using Na₃Cit. Thus, the occurrence of hypercalcemia can be avoided using Et₂Cit as an anticoagulant.

3.2. Effect of pH on Reaction Rate

At present, the main dialysates in clinical practice are bicarbonate and acetic dialysis liquid. The pH of acetate dialysate is generally controlled to remain at 6.0 to 7.2 [18]. In [19], the pH range of the dialysate is 5.3–8.2. At the entrance of the dialysis machine, the pH of a patient’s whole blood was between 7.15 and 7.4, whereas the pH of the exports of the dialysis machine was between 6.2 and 7.4.

In the dialysis process, the pH values of different dialysates varied. The acidities of different anticoagulants also differed. Therefore, the pH of blood in the dialysis process also changed. Considering that Na₃Cit was a strong base-weak acid salt, 1 mol of Na₃Cit contained 3 mol of carboxylate (COO⁻), wherein Na₃Cit was alkaline. Therefore, when Na₃Cit was used as an anticoagulant, the blood pH decreased and metabolic alkalosis likely ensued.

Considering that one Et₂Cit molecule only had one –COO⁻, the possibility of causing alkalosis was significantly reduced when Et₂Cit was used as anticoagulant. With increased pH from 6.0 to 8.0, free (Ca²⁺) decreased faster in the system (Figure 3) because increased pH benefited the ionization of –OH and –COOH of Et₂Cit or Na₃Cit, which in turn benefited the coordination with Ca²⁺.

(a)

(b)

Figure 3: Plots of concentration change of free Ca²⁺ ions with reaction time under different pH conditions: (a) Et₂Cit and (b) Na₃Cit.

Table 1 shows the reaction rate constants of Et₂Cit and Na₃Cit with CaCl₂, as well as the complex dissociation rate when the pH values of the system were 6.0, 7.4, and 8.0. The reaction rate and dissociation rate of the complex were found to accelerate with increased pH. The reaction rate of Et₂Cit and Na₃Cit with CaCl₂ was influenced by pH because H⁺ inhibits the ionization of the active H of –COOH in Et₂Cit molecule, as well as changing the course of coordination reaction. Thus, the reaction rate constant and reaction order changed.

Table 1: Reaction rate constant and reaction order of Et₂Cit and Na₃Cit with Ca²⁺ ions.

pH
6.0
7.4
8.0

Et₂Cit-CaCl₂ system

    reaction order ()
2.03
2.46
2.73

    stability constants ()
100.93
102.06
103.06

    rate constant ()/L·mol⁻¹·s⁻¹
9
120
4571

    reverse reaction rate constant ()/L·mol⁻¹·s⁻¹
0.04
0.52
19.80

Na₃Cit-CaCl₂ system

    reaction order ()
2.16
2.44
3.0

101.98
102.46
104.83

    /L·mol⁻¹·s⁻¹
60
289
13489

    /L·mol⁻¹·s⁻¹
0.03
0.15
6.79

Within pH 6.0–8.0, the pH increase accelerated the dissociation rate of the complex. With increased pH from 6.0 to 8.0, of the Et₂Cit-CaCl₂ system increased from 0.04 to 19.8, whereas of the Na₃Cit-CaCl₂ system increased from 0.03 to 6.79. The dissociation rate of the complex for the coordination of Et₂Cit and Na₃Cit with calcium under an alkaline condition was faster than that under an acidic condition. Therefore, the pH increase of anticoagulants such as Et₂Cit and Na₃Cit and dialysis under alkaline conditions achieved the purpose of anticoagulation and avoided the occurrence of dialysis acidosis, thereby improving the survival rate and quality of life.

3.3. Research on Et₂Cit and Ca Complexes

3.3.1. Elemental Analysis and Ca Content as Determined by ICP

To further study the coordination of Et₂Cit with Ca²⁺, the complex of Et₂Cit with Ca²⁺ was synthesized. Its composition was analyzed using elemental analysis and ICP, and the results are shown in Table 2. Et₂Cit was found to form the complex of CaEt₂Cit with Ca²⁺ in 1 : 1 coordination ratio. Therefore, the experimental value was consistent with the theoretical value.

Table 2: Elemental analysis data and Ca content measured by the ICP of complex CaEt₂Cit.

C%
H%
Ca%

EA results
41.55 (41.64)*
5.73 (5.55)
—

ICP result
—
—
13.68 (13.93)

The value in bracket was theoretical value, which is calculated according to the formula of complex CaEt₂Cit.

3.3.2. XRD Analysis

Figure 4 is the XRD pattern of CaCl₂ and CaEt₂Cit crystals. The diffraction peaks of CaCl₂ appeared at , and 2.90 Å (Figure 4(a)), whereas the diffraction peaks of the complex appeared at and 3.02 Å.

Figure 4: XRD patterns of CaEt₂Cit and CaCl₂: (a) CaEt₂Cit and (b) CaCl₂.

3.3.3. FT-IR Analysis

The FT-IR spectra of Et₂Cit and CaEt₂Cit complex are shown in Figure 5. The wavenumbers of the main absorption peaks are shown in Table 3 [20].

(1)The peak at 3430 cm⁻¹ was due to the stretching vibration of the hydroxyl group in the CaEt₂Cit complex, which red shifted by approximately 50 cm⁻¹ more than that of Et₂Cit (3480 cm⁻¹), indicating a hydrogen bond.(2)The carbonyl absorption peak (C=O) of CaEt₂Cit split into two peaks, which were 1709 and 1624 cm⁻¹, respectively, indicating two different coordination environments in carbonyl. The position of both peaks red-shifted by approximately more than 30 and 110 cm⁻¹ compared with the carbonyl absorption peaks of Et₂Cit at 1736 cm⁻¹. This finding indicated that the carbonyl of Et₂Cit was coordinated with the calcium ions and was consistent with the change in the carbonyl characteristic absorption peak before and after coordination, as reported in [20].(3)The absorption peak of the symmetric stretching vibrations of (C–O–C) in C–O–C of Et₂Cit was at 1100 cm⁻¹. However, the peak split into two in the complex, that is, at 1081 and 1041 cm⁻¹, respectively. This phenomenon was ascribed to one of the three C–O–C groups of the Et₂Cit molecular complex with Ca²⁺, in which C–O–C absorption was bimodal and red shifted.(4)The peak at 2982 cm⁻¹ was the absorption peak of the methyl hydrocarbon of CaEt₂Cit. It did not significantly change compared with the absorption peak of Et₂Cit methyl hydrocarbon (2986 cm⁻¹).

Table 3: Wavenumber of the main absorption peaks of FT-IR spectra of Et₂Cit and its complex CaEt₂Cit.

Et₂Cit/cm⁻¹
3480 (OH)*
2986 (CH₂, CH₃)
1732 (O–C=O)
1100 (C–O–C)

CaEt₂Cit/cm⁻¹
3430 (OH)
2982 (CH₂, –CH₃)
1709, 1624 (O–C=O)
1081, 1041 (C–O–C)

Figure 5: FT-IR spectra of Et₂Cit and its complex CaEt₂Cit: (a) Et₂Cit and (b) CaEt₂Cit.

3.3.4. ¹H NMR

The ¹H NMR spectra of Et₂Cit and CaEt₂Cit were studied using CDCl₃ as a solvent, and the results are shown in Figure 6. The absorption peaks of ¹H NMR are shown in Table 4.

(1)The proton peaks of the ligand at and 6.28 ppm disappeared, indicating that –COOH participated in the coordination reaction. Meanwhile, the hydrogen in –OH group is very active; it can be easily dissociated and be partially or entirely substituted by deuterium in CDCl₃ solution.(2)At 2.70 ppm to 3.0 ppm, the two groups of Et₂Cit quartets were –CH₂C=O (Figure 6(b)). –CH₂C=O groups occurred in different chemical environments, that is, 1,3-Et₂Cit and 1,5-Et₂Cit. The physical and chemical properties of the two isomers were very similar, so the two peaks did not significantly differ. After CaEt₂Cit was generated, the chemical environment of Et₂Cit changed and resulted in obvious dispersion and specificity of the two peaks of 2.70 ppm from 3.0 ppm. This result indicated that after 1,3-Et₂Cit and 1,5-Et₂Cit coordinated with calcium ions, the property difference of the two formed complexes increased compared with those of the original two ligands.(3)At 2.70 ppm to 3.0 ppm, the H peaks of –CH₂– shifted from 2.85 ppm to 2.91 ppm, and then to 2.81 ppm to 2.94 ppm after Et₂Cit coordinated with calcium. This finding was due to the O in –OCH₂ that coordinated with Ca, consistent with the IR spectra.(4)The peak at  ppm was assigned to –OCH₂ of –COOCH₂CH₃ (Figure 6(c)). Compared with Et₂Cit  ppm to  4.20 ppm, this peak of the complex  ppm to  4.17 ppm shifted to a high field. This finding indicated the weakening of the induction effect of attracting electrons of O in –OCH₂ from H after the O atom in –OCH₂ coordinated with Ca. Thus, the total electron density of H increased, and the absorption peaks moved to a high field.

Table 4: Absorption peak section and its assignment of the ¹H NMR spectra of Et₂Cit and CaEt₂Cit.

Et₂Cit/ppm
1.24~1.31 (–CH₃)
2.80–2.97 (–CH₂CO)
4.13~4.30 (–OCH₂)
7.26, 6.28 (–OH)

CaEt₂Cit/ppm
1.22~1.28 (–CH₃)
2.77–2.98 (–CH₂CO)
4.12~4.17 (–OCH₂)

(a)

(b)

(c)

Figure 6: ¹H NMR spectra of Et₂Cit and its complex CaEt₂Cit. (a₁, b₁, and c₁) are Et₂Cit; (a₂, b₂, and c₂) are CaEt₂Cit. (a) Total spectra, (b) section, (c) section.

Elemental analysis, ICP, XRD, FT-IR, and ¹H NMR revealed that Et₂Cit formed a 1 : 1 complex with Ca²⁺, that is, CaEt2Cit.

3.3.5. Coordination Mechanism

The above results showed that Ca²⁺ was coordinated with Et₂Cit. O in –COO and C–O–C of Et₂Cit was coordinated with Ca²⁺ in bidentate ligand. Two kinds of –OCH₂CH₃ had different chemical environments in the crystals, that is, 1,3-CaEt₂Cit and 1,5-CaEt₂Cit. However, their proportions were still difficult to ascertain because of their similar physical and chemical properties. Based on the above characterization results, two kinds of coordination of Et₂Cit with Ca²⁺ are shown in Figure 7.

Figure 7: Schematic of the coordination of Ca²⁺ ion with two isomers of anticoagulant Et₂Cit. The asterisk * shows the center C atom of Et₂Cit.

We rule out the possible coordination of hydroxyl group of Et₂Cit based on the reason that the FT-IR (Figure 5) and ¹H NMR spectra (Figure 6) have confirmed that one of the carbonyl of Et₂Cit was coordinated with the calcium ion. When one –COOH and one –COOCH₂CH₃ in Et₂Cit were coordinated with calcium ion, the –OH group and the coordinated Ca ion were separated on opposite sides of the center C atom of Et₂Cit (Figure 7); thus –OH cannot coordinate with calcium ion because of the space steric hindrance.

4. Conclusion

The coordination dynamics and effect of Et₂Cit and Na₃Cit pH on Ca²⁺ in saline water were studied. In 37°C saline water, the coordination dynamics equations of Et₂Cit and Na₃Cit with Ca²⁺ were and , respectively. The reverse reaction rate constants (’s) of coordination with CaCl₂ were 0.52 and 0.15 L·mol⁻¹·s⁻¹ for Et₂Cit and Na₃Cit, respectively. The dissociation rate of Ca²⁺ of CaEt₂Cit was faster than that of CaCit. The increased pH accelerated the dissociation of the complex. With increased pH from 6.0 to 8.0, of Et₂Cit-CaCl₂ increased from 0.04 to 19.80, which was beneficial in improving the anticoagulant effect. Et₂Cit and Ca²⁺ were coordinated to form a 1 : 1 complex, and O atoms in –COOH and C–O–C of Et₂Cit were coordinated with Ca²⁺ in bidentate ligand. Et₂Cit was able to coordinate with Ca²⁺, and its release capacity of Ca²⁺ was stronger than that of Et₂Cit. Thus, it did not require an intravenous infusion of calcium when used as an anticoagulant, thereby avoiding hypocalcemia and hypercalcemia that can be caused by Na₃Cit. Overall, Et₂Cit was a better anticoagulant than Na₃Cit.

Acknowledgments

This work was supported by the National Natural Science Foundation of China (30871164) and the Scientific and Technological International Cooperation Project of Xi’an Jiaotong University of China.

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