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 (
An anticoagulant must be added to dialysates to prevent blood solidification
Our group has previously synthesized a new anticoagulant [
The stability of the complex of Et2Cit with Ca2+ (CaEt2Cit) is reportedly weaker than that of CaCit [
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,
All chemical reagents used were of analytical grade. Et2Cit was prepared in our laboratory (99.3% purity) [
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
After logarithm on both sides we get
From the plot of
The pH of the system was adjusted to 6.0, 7.4, and 8.0. Then, the effect of pH on
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.
The change in concentration of free Ca2+ ion [
Changes in Ca2+ concentration with reaction time in different systems: (a) Et2Cit-Ca2+ system and (b) Na3Cit-Ca2+ system.
The tangent slope
Plots of
The reaction rate equations of Et2Cit and Na3Cit with Ca2+ were as follows:
Given that
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
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 [
In a previous article [
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 [
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
Plots of concentration change of free Ca2+ ions with reaction time under different pH conditions: (a) Et2Cit and (b) Na3Cit.
Table
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 ( |
9 | 120 | 4571 |
reverse reaction rate constant ( |
0.04 | 0.52 | 19.80 |
Na3Cit-CaCl2 system | |||
reaction order ( |
2.16 | 2.44 | 3.0 |
|
101.98 | 102.46 | 104.83 |
|
60 | 289 | 13489 |
|
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,
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
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) |
Figure
XRD patterns of CaEt2Cit and CaCl2: (a) CaEt2Cit and (b) CaCl2.
The FT-IR spectra of Et2Cit and CaEt2Cit complex are shown in Figure 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. 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 [ 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. 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).
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) |
FT-IR spectra of Et2Cit and its complex CaEt2Cit: (a) Et2Cit and (b) CaEt2Cit.
The 1H NMR spectra of Et2Cit and CaEt2Cit were studied using CDCl3 as a solvent, and the results are shown in Figure The proton peaks of the ligand at At 2.70 ppm to 3.0 ppm, the two groups of Et2Cit quartets were –CH2C=O (Figure 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. The peak at
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) |
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)
Elemental analysis, ICP, XRD, FT-IR, and 1H NMR revealed that Et2Cit formed a 1 : 1 complex with Ca2+, that is, CaEt2Cit.
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
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
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
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.