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

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 (k re) 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.


Introduction
An anticoagulant must be added to dialysates to prevent blood solidification in vitro (in a dialysis machine). Sodium citrate (Na 3 Cit) is an important anticoagulant used in clinical settings [1][2][3]. However, using Na 3 Cit as an anticoagulant easily causes hypocalcemia and hypercalcemia [4,5] because of the strong chelating ability of Na 3 Cit with Ca 2+ ions. Given this ability, the dissociation metabolism of the formed chelate CaCit in vivo takes 30 min. Using Na 3 Cit 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 (Et 2 Cit). The anticoagulant mechanism of Et 2 Cit is based on the formation of Ca 2+ with Et 2 Cit. This formation decreases the Ca 2+ concentration in blood and inhibits prothrombin conversion into thrombin, thereby influencing the anticoagulant effect. The large steric effect of Et 2 Cit weakens the coordination of Ca 2+ ion compared with that of Na 3 Cit. Therefore, hypocalcemia and hypercalcemia can be avoided using Et 2 Cit 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 Et 2 Cit with Ca 2+ (CaEt 2 Cit) 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 CaEt 2 Cit. However, several problems remain unsolved when Et 2 Cit is used as an anticoagulant. These problems include the reaction kinetics of Et 2 Cit with Ca 2+ and coordination reaction mechanisms, as well as the composition and characterization of the complex. Accordingly, the coordination dynamics of Et 2 Cit and Na 3 Cit with Ca 2+ , as well as the influencing factors, were studied. The underlying coordination principle was also proposed.
All chemical reagents used were of analytical grade. Et 2 Cit was prepared in our laboratory (99.3% purity) [6]. 3 Cit with Ca 2+ . CaCl 2 and Et 2 Cit 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 Na 3 Cit.

Reaction Rate Constants of Et 2 Cit and Na
The linear regression equation of the calcium ionselective electrode was = 29 + 69 (where is the electrode potential and is -p(Ca 2+ ). The concentration of Ca 2+ [ (Ca 2+ )] at time was also calculated. Given that CaCl 2 was mixed with Na 3 Cit or Et 2 Cit (1 : 1) and that the reaction of Ca 2+ with Na 3 Cit or Et 2 Cit 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 Ca 2+ substance concentration that disappeared at time, that is, = − (Ca 2+ ), 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 log and log (Ca 2+ ). In the diagram of log on log (Ca 2+ ), the slope of the straight line is the reaction order , whereas the intercept is log .

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.

Reaction Rate Equation of Et 2 Cit and Na
The change in concentration of free Ca 2+ ion [ (Ca 2+ )] with in reaction system of Et 2 Cit and Na 3 Cit with CaCl 2 is shown in Figure 1. A rapid decrease in (Ca 2+ ) was observed with prolonged from 0 s to 30 s. This finding indicated that Et 2 Cit or Na 3 Cit was rapidly coordinated with Ca 2+ . At = 30 s, (Ca 2+ ) decreased from 1.0 mmol/L to 0.49 mmol/L in the Na 3 Cit system and from 1.0 mmol/L to 0.87 mmol/L in the Et 2 Cit system. (Ca 2+ ) slowly decreased when > 120 s, indicating that the system was in a dynamic equilibrium of complexation dissociation. 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 log versus log (Ca 2+ ) (Figure 2), the slope of the line was the reaction order (formula (3)). The intercept of the line was log in Figure 2, as shown in the following: The reaction rate equations of Et 2 Cit and Na 3 Cit with Ca 2+ were as follows: Given that can directly reflect the reaction rate, (5) shown that the complexation rate of Na 3 Cit with Ca 2+ was faster than that of Et 2 Cit.
The anticoagulant mechanism of Na 3 Cit and Et 2 Cit was based on the combination of calcium ion (Ca 2+ ) in serum, as well as the reduced concentration of free Ca 2+ in plasma that disturbed the blood clotting process from reaching the anticoagulation effect in vitro [9][10][11]. However, the strong coordination ability of Na 3 Cit, particularly as an anticoagulant, can coordinate a large number of Ca 2+ 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][13][14][15]. Therefore, calcium is needed to be replenished in the anticoagulation process of Na 3 Cit [16]. Meanwhile, calcium citrate [CaCit] can dissociate during the metabolism and release Ca 2+ after entering the body in the dialysis process. Additionally, hypercalcemia easily ensued in patients with presupplementary Ca 2+ . 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, re is the inverse reaction rate constant, and is the complex stability constant.
In a previous article [8], the values of CaEt 2 Cit and CaCit were 231 and 1988 at pH 7.4 and 37 ∘ C, respectively, and the values in the coordination reaction of Et 2 Cit and Na 3 Cit with Ca 2+ were 120 and 289 L⋅mol −1 ⋅s −1 , respectively. According to (7), re of Et 2 Cit and Na 3 Cit with Ca 2+ in the coordination reaction were 0.52 and 0.15 L⋅mol −1 ⋅s −1 , respectively. Thus, the rate of decomposition and release of Ca 2+ was faster for CaEt 2 Cit than for CaCit. The above results indicated that Et 2 Cit can complex with Ca 2+ and reduce the free Ca 2+ concentration during anticoagulation; thus, anticoagulation can be achieved. Meanwhile, the complexing ability of Et 2 Cit with Ca 2+ was weaker than that of Na 3 Cit. After Et 2 Cit coordinated with Ca 2+ , the Ca 2+ releasing rate of CaEt 2 Cit 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 Et 2 Cit as anticoagulant during dialysis unlike using Na 3 Cit. Thus, the occurrence of hypercalcemia can be avoided using Et 2 Cit as an anticoagulant.

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 Na 3 Cit was a strong base-weak acid salt, 1 mol of Na 3 Cit contained 3 mol of carboxylate (COO − ), wherein Na 3 Cit was alkaline. Therefore, when Na 3 Cit was used as an anticoagulant, the blood pH decreased and metabolic alkalosis likely ensued.
Considering that one Et 2 Cit molecule only had one -COO − , the possibility of causing alkalosis was significantly reduced when Et 2 Cit was used as anticoagulant. With increased pH from 6.0 to 8.0, free (Ca 2+ ) decreased faster in the system (Figure 3) because increased pH benefited the ionization of -OH and -COOH of Et 2 Cit or Na 3 Cit, which in turn benefited the coordination with Ca 2+ . Table 1 shows the reaction rate constants of Et 2 Cit and Na 3 Cit with CaCl 2 , as well as the complex dissociation rate re 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 Et 2 Cit and Na 3 Cit with CaCl 2 was influenced by pH because H + inhibits the ionization of the active H of -COOH in Et 2 Cit molecule, as well as changing the course of coordination reaction. Thus, the reaction rate constant and reaction order changed.
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, re of the Et 2 Cit-CaCl 2 system increased from 0.04 to 19.8, whereas re of the Na 3 Cit-CaCl 2 system increased from 0.03 to 6.79. The dissociation rate of the complex for the coordination of Et 2 Cit and Na 3 Cit with calcium under an alkaline 4 Bioinorganic Chemistry and Applications  condition was faster than that under an acidic condition. Therefore, the pH increase of anticoagulants such as Et 2 Cit and Na 3 Cit 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.

Elemental Analysis and Ca Content as Determined by ICP.
To further study the coordination of Et 2 Cit with Ca 2+ , the complex of Et 2 Cit with Ca 2+ was synthesized. Its composition was analyzed using elemental analysis and ICP, and the results are shown in Table 2. Et 2 Cit was found to form the complex of CaEt 2 Cit with Ca 2+ in 1 : 1 coordination ratio. Therefore, the experimental value was consistent with the theoretical value. Figure 4 is the XRD pattern of CaCl 2 and CaEt 2 Cit crystals. The diffraction peaks of CaCl 2 appeared at = 5.97, 2.78, 3.03, 4.28, and 2.90Å (Figure 4(a)),    whereas the diffraction peaks of the complex appeared at = 6.99 and 3.02Å.

FT-IR Analysis.
The FT-IR spectra of Et 2 Cit and CaEt 2 Cit 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 CaEt 2 Cit complex, which red shifted by approximately 50 cm −1 more than that of Et 2 Cit (3480 cm −1 ), indicating a hydrogen bond.
(2) The carbonyl absorption peak (C=O) of CaEt 2 Cit 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 Et 2 Cit at 1736 cm −1 . This finding indicated that the carbonyl of Et 2 Cit 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 Et 2 Cit 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 Et 2 Cit molecular complex with Ca 2+ , 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 CaEt 2 Cit. It did not significantly change compared with the absorption peak of Et 2 Cit methyl hydrocarbon (2986 cm −1 ).

1 H NMR .
The 1 H NMR spectra of Et 2 Cit and CaEt 2 Cit were studied using CDCl 3 as a solvent, and the results are shown in Figure 6. The absorption peaks of 1 H NMR are shown in Table 4.
(1) The proton peaks of the ligand at = 7.26 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 CDCl 3 solution.
(2) At 2.70 ppm to 3.0 ppm, the two groups of Et 2 Cit quartets were -CH 2 C=O (Figure 6(b)). -CH 2 C=O groups occurred in different chemical environments, that is, 1,3-Et 2 Cit and 1,5-Et 2 Cit. The physical and chemical properties of the two isomers were very similar, so the two peaks did not significantly differ. After CaEt 2 Cit was generated, the chemical environment of Et 2 Cit 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-Et 2 Cit and 1,5-Et 2 Cit coordinated with calcium ions, the property difference of the two formed complexes increased compared with those of the original two ligands.
(  Thus, the total electron density of H increased, and the absorption peaks moved to a high field.

Coordination Mechanism.
The above results showed that Ca 2+ was coordinated with Et 2 Cit. O in -COO and C-O-C of Et 2 Cit was coordinated with Ca 2+ in bidentate ligand. Two kinds of -OCH 2 CH 3 had different chemical environments in the crystals, that is, 1,3-CaEt 2 Cit and 1,5-CaEt 2 Cit. 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 Et 2 Cit with Ca 2+ are shown in Figure 7.
We rule out the possible coordination of hydroxyl group of Et 2 Cit based on the reason that the FT-IR ( Figure 5) and 1 H NMR spectra ( Figure 6) have confirmed that one of the carbonyl of Et 2 Cit was coordinated with the calcium ion. When one -COOH and one -COOCH 2 CH 3 in Et 2 Cit were coordinated with calcium ion, the -OH group and

Conclusion
The coordination dynamics and effect of Et 2 Cit and Na 3 Cit pH on Ca 2+ in saline water were studied. In 37 ∘ C saline water, the coordination dynamics equations of Et 2 Cit and Na 3 Cit with Ca 2+ were = 120 2.46 and = 289 2.44 , respectively. The reverse reaction rate constants ( re 's) of coordination with CaCl 2 were 0.52 and 0.15 L⋅mol −1 ⋅s −1 for Et 2 Cit and Na 3 Cit, respectively. The dissociation rate of Ca 2+ of CaEt 2 Cit was faster than that of CaCit. The increased pH accelerated the dissociation of the complex. With increased pH from 6.0 to 8.0, re of Et 2 Cit-CaCl 2 increased from 0.04 to 19.80, which was beneficial in improving the anticoagulant effect. Et 2 Cit and Ca 2+ were coordinated to form a 1 : 1 complex, and O atoms in -COOH and C-O-C of Et 2 Cit were coordinated with Ca 2+ in bidentate ligand. Et 2 Cit was able to coordinate with Ca 2+ , and its release capacity of Ca 2+ was stronger than that of Et 2 Cit. 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 Na 3 Cit. Overall, Et 2 Cit was a better anticoagulant than Na 3 Cit.