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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

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 [13]. 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.

354736.fig.001
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:

354736.fig.002
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 [911]. 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 [1215]. 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+.

fig3
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.

tab1
Table 1: Reaction rate constant and reaction order of Et2Cit and Na3Cit with Ca2+ ions.

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.

tab2
Table 2: Elemental analysis data and Ca content measured by the ICP 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.

354736.fig.004
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).

tab3
Table 3: Wavenumber of the main absorption peaks of FT-IR spectra of Et2Cit and its complex CaEt2Cit.
354736.fig.005
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.

tab4
Table 4: Absorption peak section and its assignment of the 1H NMR spectra of Et2Cit and CaEt2Cit.
fig6
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.

354736.fig.007
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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