(Calcium Chloride, Calcium Acetate and Calcium Ascorbate) i - J-Stage

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Institute for Foods & Kampo Medicines, 1443 Kami_Hachiman_cho,. Tokushima 770-8041, Japan.,Tel: +81 088- 602 -8468; Fax: +81 088- 655-. 3051; E-mail: ...
Yukari Ueda et al.: Pharmacokinetic Characterization of Calcium from Three Calcium Salts

Journal of Hard Tissue Biology 21[3] (2012) p291-298 © 2012 The Hard Tissue Biology Network Association Printed in Japan, All rights reserved. CODEN-JHTBFF, ISSN 1341-7649

Original Pharmacokinetic Characterization of Calcium from Three Calcium Salts (Calcium Chloride, Calcium Acetate and Calcium Ascorbate) in Mice Yukari Ueda and Zenei Taira Faculty of Pharmaceutical Sciences, Tokushima Bunri University, Tokushima, Japan (Accepted for publication, May 1, 2012) Abstract: Calcium is an essential mineral, and its deficiency causes several diseases such as osteoporosis. The absolute bioavailability of calcium using modern pharmacokinetic methods has not been determined even though the relative bioavailability of calcium from various calcium salts has been examined using classic kinetics and pharmacokinetics. The serum calcium concentrations of three calcium salts, calcium chloride, calcium acetate and calcium ascorbate, were measured at various times after intravenous (i.v.) and oral administrations in mice, and the pharmacokinetic behaviors of the salts were investigated using a noncompartmental model. The degree of dissociation of the calcium salts was determined based on the extent of freezing-point depression. The pharmacokinetic parameters, MRT, Vdss, CLtot and AUC for i.v. administration of calcium at 15 and 30 mg/kg from three calcium salts indicated that all three may undergo similar mechanisms of calcium metabolism. The pharmacokinetic process was linear due to a first-order reaction. The pharmacokinetic parameters of calcium after oral administration at 150 mg/kg indicated that the calcium absorption was significantly different among the three calcium salts. The absolute calcium bioavailability of calcium ascorbate and calcium acetate was 2.6 and 1.5-fold, respectively, greater than that of calcium chloride. The mean residence time, MRTab, for absorption of calcium from calcium ascorbate was longer than those from calcium chloride and calcium acetate. Furthermore, it was estimated that calcium absorbed by passing through the intestinal membrane was the dissociated form because of higher degrees of apparent dissociation for the three salts. The calcium absorbability from calcium ascorbate via the intestinal track is significantly higher than those of calcium chloride and calcium acetate. Keywords: Calcium bioavailability, Pharmacokinetics, Calcium chloride, Calcium acetate, Calcium ascorbate

Introduction Calcium is an essential mineral element, acting primarily as a component in bone, as well as playing various physiological control roles in cells, even when found at low levels.1,2) It has been shown that calcium deficit causes various diseases, including osteoporosis, hypertension, hypercholesterolemia, and cancer.3,4) The absorption of calcium takes place through both active and passive transport from the gut lumen after food intake.1,5-7) The calcium absorbability from diets or its elements is traditionally defined to be the quantity of calcium in the blood, urine or body compartments, particularly bone, after multiple administrations or ingestions over several days, and its extent is defined as absorption, fractional absorption, or nutrient bioavailability of calcium, measured usually by mass balance techniques.8-11) Studies

have shown that calcium absorbability is as low as 20–40% after calcium salt administration,9-12) and many modern diets do not provide the recommended levels of calcium (400–1,200 mg/day).12) Thus, dietary calcium supplements have been recommended for prevention of calcium related diseases, and various calcium salts including calcium carbonate and calcium lactate have been examined as calcium supplement sources.9-12) Using classic kinetics for several compartmental models, it has been shown that retention of calcium in bones changes during various physiological growth periods over a life span13-16) or during diseases13,17,18) in humans, and furthermore, calcium absorption is different depending on the calcium salt19-21). Neer et al.14) reported the analysis of the time-course of calcium concentration in serum or urine of humans after intravenous administration using a four compartmental kinetic model schema jointed in series. O’Brien et al.15) showed that net deficits in bone calcium balance occurred during pregnancy and lactation. Schulze et al.17) showed that rates of bone calcium deposition were lower than those in healthy

Correspondence to: Prof. Zenei Taira; Faculty of Pharmaceutical Sciences, Tokushima Bunri University, Tokushima 770-8514, Japan. Address: The Institute for Foods & Kampo Medicines, 1443 Kami_Hachiman_cho, Tokushima 770-8041, Japan.,Tel: +81 088- 602 -8468; Fax: +81 088- 6553051; E-mail: [email protected]

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J.Hard Tissue Biology Vol. 21(3):291-298, 2012 children, and the alterations in bone turnover contribute to reduced calcium chloride, and is significantly higher than that from calcium bone mass in girls with cystic fibrosis. Furthermore, Yergey et carbonate. Hanzlik et al. 12) demonstrated that after oral 19) al. measured fractional absorption of calcium carbonate using a administration in humans, calcium formate is superior to calcium dual-isotope approach. Cai et al.20) showed that the turnover rate carbonate and calcium citrate for the delivery of calcium. However, in the gastrointestinal tract was much slower for calcium ascorbate no one has examined absolute calcium bioavailability using than calcium acetate. modern pharmacokinetics, even though Yergey et al.19) indicated As reviewed by Wagner, 22) and Rowland and Tozer, 23) theoretically that the fractional absorption using dual-isotope pharmacokinetics is a kinetic method advanced from classical approaches are closely related to the concept of absolute kinetic theory applied to study drugs and toxins, and is useful for bioavailability defined from pharmacokinetics.22,23) diagnosis and therapeutic treatments. In pharmacokinetics, the To examine different calcium supplements, the absorbability kinetic processes of the drug in the body are simply divided into of various calcium salts has been studied, and results indicated five steps referred to as ADME, that is, absorption (A), distribution that food or its elements, like lactose, can enhance calcium (D), metabolism (M) and excretion (E), and assumed usually to absorption via a paracellular route in the intestine,26-28) but what 22,23) be a linear first-order reaction. The metabolic processes for a causes the different degrees of absorption between the calcium drug are defined using pharmacokinetic parameters including the salts is a topic of discussion. Several studies have suggested that area under the plasma concentration curve (AUC), bioavailability solubility plays a crucial role in intestinal absorption,29-31) but others (F), distribution of volume, and clearance (CL), based on a have suggested that solubility has little or no correlation with compartmental or non-compartmental model. When dose D of a bioavailability. 10,29-32) Different studies have indicated that a drug is given, the pharmacokinetic characterization of calcium cationic ion group may play a specific role in the enhancement of absorption should help to understand its therapeutic effects and intestinal calcium absorption. 33-35) Hanes et al. 36) noted that its use in treatment strategies because of the following relationship: absorption of calcium oxalate did not require dissociation in rats, F•D = AUC•CL. AUC is defined from the increment in the plasma but Suzuki & Hara37) concluded that an increased intracellular concentration after a drug dosing. calcium ion concentration in rat small-intestinal enterocytes due f to non-digestible saccharides caused increased calcium absorption. Cp (t)dt AUC(0- ∞ ) = (1) 0 The aim of our study was to compare the absolute calcium The calcium absorption is defined to be the quantity taken into bioavailability from a single dose of three water-soluble calcium blood through the dietary track as shown in Eq. (1). Although salts: calcium chloride, calcium acetate and calcium ascorbate, in AUC itself is a relative quantity in proportion to the administered mice, and examine the effect of the anions on the pharmacokinetics dose, and thus is a measure of the extent of calcium absorption of of absorption, disposition and elimination of calcium. various calcium salts, the absolute bioavailability (FA) is defined as AUCoral after an oral dose of Doral, normalized with AUCiv after Materials and Methods an intravenous (i.v.) dose of Div as follows:12,23-25) Chemicals Calcium chloride hexahydrate, calcium acetate hydrate and FA = (AUCoral/Doral)/(AUCiv/Div) (2) calcium ascorbate dihydrate were purchased from Wako Pure When a certain drug (B), dosed DB, is compared with a standard Chemical Industries Ltd. (Osaka, Japan). Other reagents were drug (A), dosed DA, the relative bioavailability (FR) is the quantity purchased from commercial sources and were of the highest grade indicating the equivalency between drugs A and B as follows: available. FR = (AUCB/DB)/(AUCA/DA) (3) Usually the standard drug (A) has established pharmacokinetics, Animals and pharmacokinetic procedures and is assumed to be temporarily absorbed across the digestive Seven- to ten-week-old male ddY mice, weighing 20–30 g, were truck, and is used to describe the bioequivalency of drug obtained from SLC Co., Ltd. (Shizuoka, Japan). Animals had free absorption. Although the absorption of a drug is usually discussed access to food (commercial diet, MF pellets, Oriental Animal 12,23-25) using the absolute bioavailability, the absolute bioavailability Foods Co., Osaka, Japan) and water during the experimental for calcium has been disregarded from classic kinetic and period. Animals fasted for 18–20 h prior to administration of the pharmacokinetic studies to date because of it being an endogenous calcium salt doses, but had free access to water. Solutions of substance, and the use of dual isotopic methods is required for calcium (1% w/v) were prepared for each calcium salt, and either pharmacokinetic studies.8) Normally AUC has been used as a 15 or 30 mg per kg of body weight (mg/kg) of calcium was 26-29) relative measure for the extent of calcium absorption. Tsugawa intravenously administered to the tail vein under weak ether et al. 28 showed that the calcium absorbability from calcium anesthesia. Blood (ca. 0.02 mL) was collected from the orbital ascorbate is almost comparable with, or higher than that from sinus at 0, 5, 15, 30, 45, 60, 90, 105 and 120 min. The blood

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Yukari Ueda et al.: Pharmacokinetic Characterization of Calcium from Three Calcium Salts

Figure 1. Time course of plasma calcium concentration after i.v. calcium administration of 15 or 30 mg/kg of body weight of calcium chloride (a), calcium acetate (b) or calcium ascorbate (c). Calcium salt solutions (1%) were intravenously administered and blood was collected at 0, 5, 15, 30, 45, 60, 90, 105 and 120 min. Open circles refer to plasma calcium concentrations after administration of 30 mg/kg of body weight, closed circles refer to calcium administration of 15 mg/kg of body weight, and open squares are the plasma calcium concentrations of the control mice. Data points represent the mean ± SD (n = 6).

Figure 2: Time course of plasma calcium concentration after oral administration (125 mg/kg of body weight) of calcium chloride (a), calcium acetate (b) or calcium ascorbate (c). Calcium salt solutions (1%) were orally delivered to the duodenum and blood was collected at 0, 5, 15, 30, 45, 60, 90, 105 and 120 min. Open circles refer to plasma calcium concentration after oral administration of one of the three calcium salts, and open squares are the plasma calcium concentration of the control mice. For detailed conditions see Figure 1.

protocols conformed to the guide for the institutional animal care and use of Tokushima Bunri University, Tokushima, Japan.

samples were centrifuged at 6,000 rpm for 2 min at room temperature (25 ± 1°C). The plasma calcium concentration was measured spectroscopically at 620 nm using a microplate reader model 680 (Bio-Rad Laboratories Inc., Orlando, FL, USA) and a calcium diagnostic kit (code 437-58201) from Wako Pure Chemical Industries Ltd., Osaka, Japan. For oral administration, calcium (150 mg/kg) was delivered to the duodenum using a stainless steel intubation needle and a 1.0 mL syringe (29Gx1/2) under weak ether anesthesia. The plasma calcium concentration was determined using the same method as described above. All

Pharmacokinetic calculations Calcium pharmacokinetic parameters were calculated using the MOMENT software program, 24) for moment analysis of the increment of plasma calcium concentration-time curves after administration of calcium. The calculations were based on statistical moment theory using an iterative least-squares method. The plasma clearance (CLiv), mean residence time (MRTiv) and 293

J.Hard Tissue Biology Vol. 21(3):291-298, 2012

Figure 3: Degree of dissociation (a) of three calcium salts, calcium chloride (a), calcium acetate (b), or calcium ascorbate (c), at various concentrations. The extent of freezing-point depression, —”Tf (°C), was determined with a Beckmann thermometer, followed by obtaining the period of constant temperature after the heat of fusion was dissipated. Each value was determined in triplicate. All solutions were prepared at the following molar concentrations: 150, 125, 100, 50, 25 and 0 mM. The result is that in dilute electrolyte solution. —”Tf is directly proportional to the molality concentration (mB) of the solution according to Eq. (8). The degree of dissociation (a) of calcium salts was measured using Eq (8).

Table 1. Pharmacokinetic parameters of Ca2+ in mice after i.v. or oral administration of saline solution of either calcium chloride, calcium acetate or calcium ascorbate.

1. i.v. Administration Salts

CaCl2 CaAc2 CaAs2

Dose (mg/kg) 15 30 15 30 15 30

AUCiv (µg•min/mL)

MRTiv (min)

CL (mL/min/kg)

1484.5 ± 41.0 2870.6 ± 90.8 1507.9 ± 128.4 2637.2 ± 121.6 1193.9 ± 101.7 2711.3 ± 154.2

29.3 ± 1.3 33.0 ± 1.1 29.1 ± 2.0 30.0 ± 2.4 30.4 ± 1.0 32.5 ± 1.2 30.8 ± 2.1

10.1 ± 0.3 10.5 ± 0.3 10.0 ± 0.9 11.4 ± 0.5 12.6 ± 1.1 11.0 ± 0.6 10.9 ± 1.1

Means

Vdss (mL/kg) 296.5 ± 8.5 345.1 ± 15.8 289.9 ± 7.4 340.9 ± 24.7 383.6 ± 20.9 359.2 ± 34.7 335.9 ± 38.6

in the body compartment following the intravenous administration and MRT oral is the mean residence time in all compartments following the oral administration. The elimination kinetic constant (kel) and the absorption kinetic constant (kab) in the compartment model are related to following relationships: MRTiv = 1/kel and MRToral = 1/kel + 1/kab, respectively.24) Thus the mean residence time (MRTab) in the absorption track is as follows:

volume of distribution (Vdss) at steady-state after intravenous (i.v.) administration of the dose (D iv) were determined using the equations described by Yamaoka et al.24) as follows: Vdss = Div • MRTiv/AUCiv (4) CLiv = Vdss/MRTiv (5) The absolute bioavailability (FA) is calculated using Eq. (2). AUC (0- ∞ ) was calculated as follows:

MRTab = 1/kab AUC (0- ∞ ) = AUC (0-τ) + Cp (τ)/ λ (6)

(7)

Measurement of the degree of dissociation of the calcium salts To measure the degree of dissociation of the calcium salts, based on their colligative properties in dilute solution, the extent of freezing-point depressionΔTf (°C) was determined with a Beckmann thermometer, calibrated from +1 to –6°C. Each value was determined in triplicate. All solutions were prepared in molar

Where τ is the time of the last plasma concentration (Cp) and λ is the apparent elimination rate constant calculated as the slope of the plasma concentration at the time τ after semilogarithmic transformation. AUC (0-τ) was calculated using the trapezoidal rule with linear interpolation. MRTiv is the mean residence time 294

Yukari Ueda et al.: Pharmacokinetic Characterization of Calcium from Three Calcium Salts 2. Oral administration

Salts

Dose Tmax Cmax (mg/kg) (min) (µg/mL)

AUCoral (µg•min/mL)

CaCl2 CaAc2 CaAs2

150 150

30 45

94.5 ± 1.2 103.6 ± 1.2

150

15

100.8 ± 1.2

MRToral (min)

F (%)

813.0 ± 187.6 1137.4 ± 225.1

46.6 ± 1.8 45.0 ± 2.6

5.7 ± 1.3 8.6 ± 1.7

2007.6 ± 159.9

64.7 ± 3.8

14.8 ± 1.2

Dose means calcium quantity from each calcium salt. The pharmacokinetic parameters of calcium were calculated from increment of plasma calcium concentration over the mean control level of calcium after i.v. or oral administration of calcium salts, as shown in Figs. 1 and 2. Each value represents the mean ± S.D. (n = 5). CaCl2 = calcium chloride, CaAc2 = calcium acetate and CaAs2 = calcium ascorbate.

value after i.v. calcium administration of 30 mg/kg was 1.96-fold (mean) higher than after i.v. calcium administration of 15 mg/kg (p < 0.05). This suggests that the pharmacokinetic process is linear in the calcium dosing range of 15 to 30 mg/kg.

concentrations, 150, 125, 100, 50, 25 and 0 mM. The result is that in a dilute electrolyte solution, Δ Tf is directly proportional to the molality concentration (mB) of the solution according to Eq. (8) below:38) Δ Tf = i•Kf•mB (8) where the cryoscopic constant (Kf) is 1.86°C for an aqueous solution, which is dependent on the properties of the solvent. The van’t Hoff factor i accounts for the practical number of individual particles (typically ions) formed by a compound in solution. Thus if N is the total number of particles furnished by one molecule of the solute, for example N = 3 for calcium chloride in an aqueous solution, the degree of dissociation (a) is as follows:38) a = (i – 1)/(N – 1) (9)

Pharmacokinetic analysis after oral administration Figure 2 shows the time-course of plasma calcium concentration after oral administration of 150 mg of calcium/kg from an aqueous solution of one of the three calcium salts. Insert Figure 2 here The pharmacokinetic parameters of calcium were calculated from the increment in the plasma calcium concentration over the mean control level of calcium after oral administration, as shown in Figure 2, and summarized in Table 1. The absolute bioavailability values (FA’s) of calcium from dosing with the three calcium salts were calculated by comparing the AUC after oral administration with the AUC after i.v. administration of 30 mg of calcium per kg of body weight, as shown in Eq. (2). The results indicate that after administration of calcium from calcium chloride, calcium acetate or calcium ascorbate, the plasma calcium concentration reaches a maximum (Cmax) of 94.5, 103.6, and 100.8 µg/mL at Tmax, respectively. The FA value for calcium from calcium ascorbate (14.8%) was significantly higher than those for calcium chloride and calcium acetate (5.7 and 8.6%, respectively) (p < 0.05). The quantities absorbed into the circulating blood (F A•D oral = AUC oral•CL iv ), 24) 8.9 mg/kg, 12.4 mg/kg and 21.9 mg/kg, respectively, demonstrates that calcium exhibits a linear absorption behavior in mice after oral administration of calcium chloride, calcium acetate or calcium ascorbate.

Statistical analysis Data are presented as the mean value ± SD. A parameter was considered to be significantly different when the p values were < 0.05 using a Student’s t-test. Results Pharmacokinetic analysis after i.v. administration As shown in Figure 1, the plasma calcium concentrations in mice decreased to the control level two hours after i.v. calcium administration of 15 or 30 mg/kg from the aqueous solution of calcium chloride, calcium acetate or calcium ascorbate. The pharmacokinetic parameters of calcium were calculated from the increment in the plasma calcium concentration over the mean control level of calcium (8.08 ± 0.07 mg/dL) after i.v. administration, as shown in Figure 1, and summarized in Table 1. Insert Figure 1 and Table 1 here The results indicate that the following pharmacokinetic parameters, MRT, CL and Vdss, of calcium for each calcium salt were not significantly different between the calcium doses of 15 and 30 mg/kg (p < 0.05). These parameters were also not significantly different among the three calcium salts. Thus the pharmacokinetic behavior of calcium in the mice was not affected by the partner anions in the calcium salts. Furthermore, the AUC

The degree of the dissociation of calcium salts To confirm the effect of the degree of dissociation (α) of calcium salts on their absorbability, we measured the extent of freezingpoint depression Δ Tf (°C) in dilute calcium solution, and the degree of dissociation (α) of each calcium salt was calculated using Eq. (8). 295

J.Hard Tissue Biology Vol. 21(3):291-298, 2012 Insert Figure 3 here absorption of calcium in the intestine. Generally, it is believed As shown in Figure 3, a change in the degree of dissociation that dietary calcium sources are absorbed as its free or ionized linearly depended on the calcium concentration (mB) from 25 to form from the intestine, and thus the extent of absorption depends 150 mM in aqua solution as follows, on their solubility in aqueous solution. We found that the intestinal 3 α = -2.02×10 mB + 0.924, r = 0.998 (10) absorbability of dissociated calcium might be superior to calcium α = -0.61×103 mB + 0.923, r = 0.934 (11) from non-dissociated salts because the degrees of apparent 3 α = -0.46×10 mB + 0.917, r = 0.936 (12) dissociation of the three salts are relatively high at 0.67, 0.84 and The linear functions were obtained by the least-squares 0.87 for calcium chloride, calcium acetate and calcium ascorbate calculation with high correlation coefficients (r) (n = 3). Thus the at 125 mM, respectively. Furthermore, Suzuki & Hara37) showed degrees of intrinsic dissociation (α0) were found to be 0.92 ± 0.00, that an increase in intracellular calcium ion concentration due to 0.92 ± 0.01 and 0.92 ± 0.01 for calcium chloride, calcium acetate non-digestible saccharides causes an increase in calcium and calcium ascorbate, respectively, at the calcium concentration absorption. Thus, further studies are needed to determine for each mB = 0 M. The values are not significantly different from each calcium salt whether it is absorbed from the intestine as its free or other. However, the apparent α values (0.67 ± 0.00, 0.84 ± 0.01 ionized form. It will be particularly important to clarify which and 0.87 ± 0.01, respectively) were different at the orally factors are responsible for the long transit time of calcium through administered calcium concentration of 125 mM. the small intestine. In conclusion, as shown in this study, pharmacokinetic methods Discussion should prove to be more effective for studying acute absorption The serum calcium concentration of three calcium salts, calcium following single dosing even for endogenous nutrients like chloride, calcium acetate and calcium ascorbate, were measured calcium, while the traditional balance method may be suitable for at various times after i.v. and oral administrations in mice, and studying chronic absorption following multiple doses. the pharmacokinetic behaviors of the salts were investigated using Furthermore, pharmacokinetic methods may also be useful for a non-compartmental model, in which the FA, AUC, MRT, Vdss the study of other nutritional elements. Thus further studies are and CLtot values of calcium were calculated. The pharmacokinetic needed to elucidate calcium transport mechanisms and their link parameters, MRT, Vdss and CLtot for i.v. administration of calcium to calcium bioavailability. at 15 and 30 mg/kg from the three calcium salts showed that the calcium metabolism might be physiologically similar among the Acknowledgements three salts (p < 0.05). Furthermore, the AUC for each salt was The study was supported in part by research grants from the proportional to the dose, and therefore the pharmacokinetic process Ministry of Education, Culture, Sports, Science and Technology, may be linear due to a first-order reaction. On the other hand, the Japan. We acknowledge the experimental assistance of Yoshio pharmacokinetic parameters of calcium after oral calcium Sagara, Hiromi Shiraishi and Hiroki Asaji from the Department administration of 150 mg/kg of body weight indicated that the of Pharmaceutical Sciences. calcium absorption was significantly different between the three calcium salts. 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