Coordination dynamics and coordination mechanism of a new type of anticoagulant diethyl citrate with [Ca.sup.2+] ions.
An anticoagulant must be added to dialysates to prevent blood solidification in vitro (in a dialysis machine). Sodium citrate ([Na.sub.3]Cit) is an important anticoagulant used in clinical settings [1-3]. However, using [Na.sub.3]Cit as an anticoagulant easily causes hypocalcemia and hypercalcemia [4, 5] because of the strong chelating ability of [Na.sub.3]Cit with [Ca.sup.2+] ions. Given this ability, the dissociation metabolism of the formed chelate CaCit in vivo takes 30 min. Using [Na.sub.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 , namely, diethyl citrate ([Et.sub.2]Cit). The anticoagulant mechanism of [Et.sub.2]Cit is based on the formation of [Ca.sup.2+] with [Et.sub.2]Cit. This formation decreases the [Ca.sup.2+] concentration in blood and inhibits prothrombin conversion into thrombin, thereby influencing the anticoagulant effect. The large steric effect of [Et.sub.2]Cit weakens the coordination of [Ca.sup.2+] ion compared with that of [Na.sub.3]Cit. Therefore, hypocalcemia and hypercalcemia can be avoided using [Et.sub.2]Cit as anticoagulant . 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.sub.2]Cit with [Ca.sup.2+] (Ca[Et.sub.2]Cit) is reportedly weaker than that of CaCit . At pH 7.4 and 37[degrees]C, the stability constants (K/s) are 1988 for CaCit and 231 for Ca[Et.sub.2]Cit. However, several problems remain unsolved when [Et.sub.2]Cit is used as an anticoagulant. These problems include the reaction kinetics of [Et.sub.2]Cit with [Ca.sup.2+] and coordination reaction mechanisms, as well as the composition and characterization of the complex. Accordingly, the coordination dynamics of [Et.sub.2]Cit and [Na.sub.3]Cit with [Ca.sup.2+], 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 CD[Cl.sub.3] as solvent and TMS as internal standard), Nicolet-170 SX type FT-IR spectrometer, D/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. [Et.sub.2]Cit was prepared in our laboratory (99.3% purity) .
2.2. Experimental Methods
2.2.1. Reaction Rate Constants of[Et.sub.2]Cit and[Na.sub.3]Cit with [Ca.sup.2+]. Ca[Cl.sub.2] and [Et.sub.2]Cit solutions (2.0mmol/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[degrees]C under stirring. The result was then compared with that of [Na.sub.3]Cit.
The linear regression equation of the calcium ion-selective electrode was y = 29x + 69 (where y is the electrode potential and x is -p([Ca.sup.2+]). The concentration of [Ca.sup.2+] [c([Ca.sup.2+])] at t time was also calculated. Given that Ca[Cl.sub.2] was mixed with [Na.sub.3]Cit or [Et.sub.2]Cit (1: 1) and that the reaction of [Ca.sup.2+] with [Na.sub.3]Cit or [Et.sub.2]Cit was equal in solution , the following reaction rate equation can be established using r to represent the reaction rate:
r = [kc.sup.n], (1)
where k is the reaction rate constant and n is the reaction order. Assuming that x is the amount of [Ca.sup.2+] substance concentration that disappeared at t time, that is, x = a - c ([Ca.sup.2+]), the following can be obtained by arranging formula (1):
r = - dc/dt = - d(a - x)/dt = dx/dt = k[(a - x).sup.n]. (2)
After logarithm on both sides we get
Log r = log (- d(a - x)/dt) = log k + n log (a - x) = log k + n log c. (3)
From the plot of x versus t, we can calculate the tangent slope of the curve dx/dt, which is the reaction rate of various points. Formula (3) shows a linear relationship between log r and log c([Ca.sup.2+]). In the diagram of log r on log c([Ca.sup.2+]), the slope of the straight line is the reaction order n, whereas the intercept is log k.
2.2.2. Effect of pH on Reaction Rate. The pH of the system was adjusted to 6.0,74, and 8.0. Then, the effect of pH on k and n was determined.
2.2.3. Synthesis of Diethyl Citrate Calcium Complex Crystal. About 1.665 g (15 mmol) of anhydrous Ca[Cl.sub.2] was completely dissolved in water. Then, 1.241 g (5 mmol) of [Et.sub.2]Cit 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[degrees] 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), [sup.1]H NMR, and ICP.
3. Results and Discussion
3.1. Reaction Rate Equation of [Et.sub.2]Cit and [Na.sub.3]Cit with [Ca.sup.2+]. The change in concentration of free [Ca.sup.2+] ion [c([Ca.sup.2+])] with t in reaction system of [Et.sub.2]Cit and [Na.sub.3]Cit with Ca[Cl.sub.2] is shown in Figure 1. A rapid decrease in c([Ca.sup.2+]) was observed with prolonged t from 0 s to 30 s. This finding indicated that [Et.sub.2]Cit or [Na.sub.3]Cit was rapidly coordinated with Ca. At t = 30 s, c([Ca.sup.2+]) decreased from 1.0 mmol/L to 0.49 mmol/L in the [Na.sub.3]Cit system and from 1.0 mmol/L to 0.87 mmol/L in the [Et.sub.2]Cit system. c([Ca.sup.2+]) slowly decreased when t > 120 s, indicating that the system was in a dynamic equilibrium of complexation dissociation.
The tangent slope (dx/dt) of points on the curve, that is, the reaction rate r of each point formula (2), can be obtained according to Figure 1. In the diagram of log r versus log c([Ca.sup.2+]) (Figure 2), the slope of the line was the reaction order n (formula (3)). The intercept of the line was log k in Figure 2, as shown in the following:
[Et.sub.2]Cit-Ca system: n = 2.46; log k = 2.06, so k = 120; [Na.sub.3]Cit-Ca system: n = 2.44; log k = 2.46, so k = 289. (4)
The reaction rate equations of [Et.sub.2]Cit and [Na.sub.3]Cit with [Ca.sup.2+] were as follows:
[Et.sub.2]Cit-Ca system : r = [ka.sup.2.46] = [120a.sup.2.46], [Na.sub.3]Cit-Ca system : r = [ka.sup.2.44] = [289a.sup.2.44]. (5)
Given that k can directly reflect the reaction rate, (5) shown that the complexation rate of [Na.sub.3]Cit with [Ca.sup.2+] was faster than that of [Et.sub.2]Cit.
The anticoagulant mechanism of [Na.sub.3]Cit and [Et.sub.2]Cit was based on the combination of calcium ion ([Ca.sup.2+]) in serum, as well as the reduced concentration of free [Ca.sup.2+] in plasma that disturbed the blood clotting process from reaching the anticoagulation effect in vitro [9-11]. However, the strong coordination ability of [Na.sub.3]Cit, particularly as an anticoagulant, can coordinate a large number of [Ca.sup.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 [12-15]. Therefore, calcium is needed to be replenished in the anticoagulation process of [Na.sub.3]Cit . Meanwhile, calcium citrate [CaCit] can dissociate during the metabolism and release [Ca.sup.2+] after entering the body in the dialysis process. Additionally, hypercalcemia easily ensued in patients with presupplementary [Ca.sup.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:
k[(a - x).sup.n] = [k.sub.re][x.sup.n]. (6)
The above equation can be written as follows :
[x.sup.n]/[(a - x).sup.n] = k/[k.sub.re] = [K.sub.s], (7)
where k is the reaction rate constant, [k.sub.re] is the inverse reaction rate constant, and [K.sub.s] is the complex stability constant.
In a previous article , the [K.sub.s] values of Ca[Et.sub.2]Cit and CaCit were 231 and 1988 at pH 7.4 and 37[degrees]C, respectively, and the k values in the coordination reaction of [Et.sub.2]Cit and [Na.sub.3]Cit with [Ca.sup.2+] were 120 and 289 L x [mol.sup.-1] x [s.sup.-1], respectively. According to (7), [k.sub.re] of [Et.sub.2]Cit and [Na.sub.3]Cit with [Ca.sup.2+] in the coordination reaction were 0.52 and 0.15 L-[mol.sup.-1]-[s.sup.-1], respectively. Thus, the rate of decomposition and release of [Ca.sup.2+] was faster for Ca[Et.sub.2]Cit than for CaCit. The above results indicated that [Et.sub.2]Cit can complex with [Ca.sup.2+] and reduce the free [Ca.sup.2+] concentration during anticoagulation; thus, anticoagulation can be achieved. Meanwhile, the complexing ability of [Et.sub.2]Cit with [Ca.sup.2+] was weaker than that of [Na.sub.3]Cit. After [Et.sub.2]Cit coordinated with [Ca.sup.2+], the [Ca.sup.2+] releasing rate of Ca[Et.sub.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.sub.2]Cit as anticoagulant during dialysis unlike using [Na.sub.3]Cit. Thus, the occurrence of hypercalcemia can be avoided using [Et.sub.2]Cit 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 . In , 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.sub.3]Cit was a strong base-weak acid salt, 1 mol of [Na.sub.3]Cit contained 3 mol of carboxylate (CO[O.sup.-]), wherein [Na.sub.3]Cit was alkaline. Therefore, when [Na.sub.3]Cit was used as an anticoagulant, the blood pH decreased and metabolic alkalosis likely ensued.
Considering that one [Et.sub.2]Cit molecule only had one -CO[O.sup.-], the possibility of causing alkalosis was significantly reduced when [Et.sub.2]Cit was used as anticoagulant. With increased pH from 6.0 to 8.0, free c([Ca.sup.2+]) decreased faster in the system (Figure 3) because increased pH benefited the ionization of -OH and -COOH of [Et.sub.2]Cit or [Na.sub.3]Cit, which in turn benefited the coordination with [Ca.sup.2+].
Table 1 shows the reaction rate constants k of [Et.sub.2]Cit and [Na.sub.3]Cit with Ca[Cl.sub.2], as well as the complex dissociation rate [k.sub.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.sub.2]Cit and [Na.sub.3]Cit with Ca[Cl.sub.2] was influenced by pH because [H.sup.+] inhibits the ionization of the active H of -COOH in [Et.sub.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, [k.sub.re] of the [Et.sub.2]Cit-Ca[Cl.sub.2] system increased from 0.04 to 19.8, whereas [k.sub.re] of the [Na.sub.3]Cit-Ca[Cl.sub.2] system increased from 0.03 to 6.79. The dissociation rate of the complex for the coordination of [Et.sub.2]Cit and [Na.sub.3]Cit with calcium under an alkaline condition was faster than that under an acidic condition. Therefore, the pH increase of anticoagulants such as [Et.sub.2]Cit and [Na.sub.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.
3.3. Research on [Et.sub.2]Cit and Ca Complexes
3.3.1. Elemental Analysis and Ca Content as Determined by ICP. To further study the coordination of [Et.sub.2]Cit with [Ca.sup.2+], the complex of [Et.sub.2]Cit with [Ca.sup.2+] was synthesized. Its composition was analyzed using elemental analysis and ICP, and the results are shown in Table 2. [Et.sub.2]Cit was found to form the complex of Ca[Et.sub.2]Cit with [Ca.sup.2+] in 1:1 coordination ratio. Therefore, the experimental value was consistent with the theoretical value.
3.3.2. XRD Analysis. Figure 4 is the XRD pattern of Ca[Cl.sub.2] and Ca[Et.sub.2]Cit crystals. The diffraction peaks of Ca[Cl.sub.2] appeared at d = 5.97, 2.78, 3.03, 4.28, and 2.90 [Angstrom] (Figure 4(a)), whereas the diffraction peaks of the complex appeared at d = 6.99 and 3.02 [Angstrom].
3.3.3. FT-IR Analysis. The FT-IR spectra of [Et.sub.2]Cit and Ca[Et.sub.2]Cit complex are shown in Figure 5. The wavenumbers of the main absorption peaks are shown in Table 3 .
(1) The peak at 3430 [cm.sup.-1] was due to the stretching vibration of the hydroxyl group in the Ca[Et.sub.2]Cit complex, which red shifted by approximately 50 [cm.sup.-1] more than that of [Et.sub.2]Cit (3480 [cm.sup.-1]), indicating a hydrogen bond.
(2) The carbonyl absorption peak (C=O) of Ca[Et.sub.2]Cit split into two peaks, which were 1709 and 1624 [cm.sup.-1], respectively, indicating two different coordination environments in carbonyl. The position of both peaks red-shifted by approximately more than 30 and 110 [cm.sup.-1] compared with the carbonyl absorption peaks of [Et.sub.2]Cit at 1736 [cm.sup.-1]. This finding indicated that the carbonyl of [Et.sub.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 .
(3) The absorption peak of the symmetric stretching vibrations of (C-O-C) in C-O-C of [Et.sub.2]Cit was at 1100 [cm.sup.-1]. However, the peak split into two in the complex, that is, at 1081 and 1041 [cm.sup.-1], respectively. This phenomenon was ascribed to one of the three C-O-C groups of the [Et.sub.2]Cit molecular complex with [Ca.sup.2+], in which C-O-C absorption was bimodal and red shifted.
(4) The peak at 2982 [cm.sup.-1] was the absorption peak of the methyl hydrocarbon of Ca[Et.sub.2]Cit. It did not significantly change compared with the absorption peak of [Et.sub.2]Cit methyl hydrocarbon (2986 [cm.sup.-1]).
3.3.4. [sup.1]HNMR. The [sup.1]HNMR spectra of [Et.sub.2]Cit and Ca[Et.sub.2]Cit were studied using CD[Cl.sub.3] 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 [delta] = 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 CD[Cl.sub.3] solution.
(2) At 2.70 ppm to 3.0 ppm, the two groups of [Et.sub.2]Cit quartets were -[CH.sub.2]C=O (Figure 6(b)). -[CH.sub.2]C=O groups occurred in different chemical environments, that is, 1,3-[Et.sub.2]Cit and 1,5-[Et.sub.2]Cit. The physical and chemical properties of the two isomers were very similar, so the two peaks did not significantly differ. After Ca[Et.sub.2]Cit was generated, the chemical environment of [Et.sub.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.sub.2]Cit and 1,5-[Et.sub.2]Cit 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 -C[H.sub.2]- shifted from 2.85 ppm to 2.91 ppm, and then to 2.81 ppm to 2.94 ppm after [Et.sub.2]Cit coordinated with calcium. This finding was due to the O in -OC[H.sub.2] that coordinated with Ca, consistent with the IR spectra.
(4) The peak at S = 4.0 ppm was assigned to -O[CH.sub.2] of -COO[CH.sub.2][CH.sub.3] (Figure 6(c)). Compared with [Et.sub.2]Cit ([delta] = 4.13 ppm to 4.20 ppm), this peak of the complex ([delta] = 4.12 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 -OC[H.sub.2] from H after the O atom in -OC[H.sub.2] coordinated with Ca. Thus, the total electron density of H increased, and the absorption peaks moved to a high field.
Elemental analysis, ICP, XRD, FT-IR, and [sup.1]H NMR revealed that [Et.sub.2]Cit formed a 1: 1 complex with [Ca.sup.2+], that is, Ca[Et.sub.2]Cit.
3.3.5. Coordination Mechanism. The above results showed that [Ca.sup.2+] was coordinated with [Et.sub.2]Cit. O in -COO and C-O-C of [Et.sub.2]Cit was coordinated with [Ca.sup.2+] in bidentate ligand. Two kinds of -OC[H.sub.2]C[H.sub.3] had different chemical environments in the crystals, that is, 1,3-Ca[Et.sub.2]Cit and 1,5-Ca[Et.sub.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.sub.2]Cit with [Ca.sup.2+] are shown in Figure 7.
We rule out the possible coordination of hydroxyl group of [Et.sub.2]Cit based on the reason that the FT-IR (Figure 5) and [sup.1]H NMR spectra (Figure 6) have confirmed that one of the carbonyl of [Et.sub.2]Cit was coordinated with the calcium ion. When one -COOH and one -COOC[H.sub.2]C[H.sub.3] in [Et.sub.2]Cit were coordinated with calcium ion, the -OH group and the coordinated Ca ion were separated on opposite sides of the center C atom of [Et.sub.2]Cit (Figure 7); thus -OH cannot coordinate with calcium ion because of the space steric hindrance.
The coordination dynamics and effect of [Et.sub.2]Cit and [Na.sub.3]Cit pH on [Ca.sup.2+] in saline water were studied. In 37[degrees]C saline water, the coordination dynamics equations of [Et.sub.2]Cit and [Na.sub.3]Cit with [Ca.sup.2+] were r = [120a.sup.2.46] and r = [289a.sup.2.44], respectively. The reverse reaction rate constants (fcre's) of coordination with Ca[Cl.sub.2] were 0.52 and 0.15 L x [mol.sup.-1] x [s.sup.-1] for [Et.sub.2]Cit and [Na.sub.3]Cit, respectively. The dissociation rate of [Ca.sup.2+] of Ca[Et.sub.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, kre of [Et.sub.2]Cit-Ca[Cl.sub.2] increased from 0.04 to 19.80, which was beneficial in improving the anticoagulant effect. [Et.sub.2]Cit and [Ca.sup.2+] were coordinated to form a 1: 1 complex, and O atoms in -COOH and C-O-C of [Et.sub.2]Cit were coordinated with [Ca.sup.2+] in bidentate ligand. [Et.sub.2]Cit was able to coordinate with [Ca.sup.2+], and its release capacity of [Ca.sup.2+] was stronger than that of [Et.sub.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.sub.3]Cit. Overall, [Et.sub.2]Cit was a better anticoagulant than [Na.sub.3]Cit.
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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Jin Han, (1) Jun-Fa Xue, (2) Meng Xu, (2) Bao-Song Gui, (1) Li Kuang, (2) Jian-Ming Ouyang (2)
(1) Department of Nephrology, The Second Hospital of Xi'an Jiaotong University, Xi'an 710004, China
(2) Institute of Biomineralization and Lithiasis Research, Jinan University, Guangzhou 510632, China
Correspondence should be addressed to Bao-Song Gui; firstname.lastname@example.org and Jian-Ming Ouyang; email@example.com
Received 30 July 2013; Revised 14 November 2013; Accepted 29 November 2013
Academic Editor: Francesco Paolo Fanizzi
Table 1: Reaction rate constant and reaction order of [Et.sub.2]Cit and [Na.sub.3]Cit with [Ca.sup.2+] ions. pH 6.0 74 8.0 [Et.sub.2]Cit-Ca [Cl.sub.2] system reaction order (n) 2.03 2.46 2.73 stability constants [10.sup.0.93] [10.sup.2.06] [10.sup.3.06] ([K.sub.s]) rate constant (k)/L x 9 120 4571 [mol.sup.-1] x [s.sup.-1] reverse reaction rate 0.04 0.52 19.80 constant ([k.sub.re])/L x [mol.sup.-1] x [s.sup.-1] [Na.sub.3]Cit-Ca [Cl.sub.2] system reaction order (n) 2.16 2.44 3.0 [K.sub.s] [10.sup.198] [10.sup.246] [10.sup.4.83] k/L x [mol.sup.-1] 60 289 13489 x [s.sup.-1] [k.sub.re]/L x 0.03 0.15 6.79 [mol.sup.-1] x [s.sup.-1] Table 2: Elemental analysis data and Ca content measured by the ICP of complex Ca[Et.sub.2]Cit. 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 Ca[Et.sub.2]Cit. Table 3: Wavenumber of the main absorption peaks of FT-IR spectra of [Et.sub.2]Cit and its complex Ca[Et.sub.2]Cit. [Et.sub.2]Cit/ 3480 (OH) * 2986 1732 1100 [cm.sup.-1] ([CH.sub.2], (O-C=O) (C-O-C) [CH.sub.3]) Ca[Et.sub.2]Cit/ 3430 (OH) 2982 1709, 1624 1081, 1041 [cm.sup.-1] ([CH.sub.2], (O-C=O) (C-O-C) -[CH.sub.2]) Table 4: Absorption peak section and its assignment of the [sup.1]H NMR spectra of [Et.sub.2]Cit and Ca[Et.sub.2]Cit. [Et.sub.2] 1.24-1.31 2.80-2.97 4.13-4.30 Cit/ppm (-[CH.sub.3]) (-[CH.sub.2]CO) (-O[CH.sub.2]) Ca[Et.sub.2] 1.22-1.28 2.77-2.98 4.12-4.17 Cit/ppm (-[CH.sub.3]) (-[CH.sub.2]CO) (-O[CH.sub.2]) [Et.sub.2] 7.26, 6.28 Cit/ppm (-OH) Ca[Et.sub.2] Cit/ppm
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|Title Annotation:||Research Article|
|Author:||Han, Jin; Xue, Jun-Fa; Xu, Meng; Gui, Bao-Song; Kuang, Li; Ouyang, Jian-Ming|
|Publication:||Bioinorganic Chemistry and Applications|
|Date:||Jan 1, 2014|
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