Amorphous Calcium Salt Composition Bioavailability Evaluation in Chickens

Despite the market saturation with a wide range of calcium preparations in dietary supplements as well as in pharmaceuticals, calcium product safety and efficacy remain an item for further optimization. Bones and teeth mainly consist of calcium phosphate, but tablets and capsules are predominantly produced from calcium carbonate. On the other hand, in human food Ca 2+ is bound with a lot of anions—carbonates, sulphates, chlorides, phosphates, ci-trates, oxalates etc. It seems that traditional calcium formulations due its low bioavailability cannot provide all spectra of biological calcium-dependant effects and dietary calcium might be safer and more effective than traditional calcium supplements and medicines. To increase calcium salt efficacy a new formulation of calcium salts has been developed. It is a mixture (water suspension) of amorphous calcium carbonate, citrate, hydrocarbonate, as well as magnesium hydrocarbonate and hydrocitrate as stabilizers. The capacity of a cockerel’s duodenal mucosa to absorb Ca 2+ after peroral ingestion of the mentioned composition in vivo was as much as 126% higher in comparison with CaCO 3 suspension intake. Vitamin D 3 enhanced the amorphous antirachitic activity of calcium salts. The aim of the study was to compare original amorphous calcium salt composition bioavailability with Ca salts most often in food and medicines, as well as to evaluate the antirachitic activity of the mentioned composition in combination with vitamin D 3 in chickens. Amorphous


Introduction
Due to the dependence of a huge number of the organism's essential biochemical and physiological processes of calcium (Ca), the complex system of Ca metabolism regulation in mammals has been developed during evolution. This mechanism regulates Ca intestinal absorption, supporting the constant cations blood concentration in any physiological periods. In the case of inadequate Ca absorption from the gastrointestinal tract, stabile calcemia is supported on the account of the destruction of the skeleton due to hyperactivity of the parathyroid glands [1].
Despite the wide prevalence of calcium minerals in nature, there is a global problem of Ca deficiency for livestock and humans. Of great importance is not only Ca content in the diet, but also Ca bioavailability. Inadequate consumption of dairy products, excessive drinking water purification from minerals and growing popularity of ortho-phosphate containing light drinks are tending toward Ca deficiency [2]. But medicinal technological progress, prolongation of human life, and the increase of the number of patients with earlier incurable gastroenterological and endocrinological diseases promote the prevalence of secondary Ca deficiency.
Intestinal Ca absorption occurs through the transcellular and paracellular pathways. The first route comprises the entrance of Ca across the brush border membranes (BBM) of enterocytes through special epithelial Ca channels; Ca movement from BBM to basolateral membranes by binding proteins with high Ca affinity (as calbindin) and Ca extrusion into the blood. Plasma membrane Ca-ATPase and a sodium calcium exchanger are involved in the exit of Ca from enterocytes. Paracellular Ca transport includes the movement of the ions through transmembranes of tight junction structures and depends on the electrochemical gradient of the intestinal epithelium. There is evidence of crosstalk between both pathways in intestinal Ca transport. Under the impact of pathogenic factors (oxidative stress) due to the decrease in expression of several molecules of both pathways, Ca absorption is inhibited [3]. Vitamin D, more precisely, its active metabolite 1.25(OH) 2 D 3 or calcitriol, which is the principal promoter of Ca intestinal absorption [4], acts in the opposite direction-increases gene and protein expression, involved in both pathways.
The presence of two pathways of intestinal Ca absorption has an important biological meaning. The paracellular pathway provides a passive basic flow of Ca into the organism's internal environment. The transcellular pathway is active, energy dependent and provides "fine" Ca absorption regulation, its adaptation to the mineral content in the diet, bioavailability, organism physiological status etc. Wherein the effects on Ca intestinal absorption of other hormones-thyroid, parathyroid, growth hormones, estrogens, prolactin-are mediated via calcitriol synthesis [5].
The above algorithm of Ca absorption regulation implies an important practical conclusion. In the case of Ca deficiency, it is rational to stimulate both Ca intestinal transport pathways. So, the combination of Ca preparations and vitamin D became the "golden standard" for metabolic bone disease prevention and treatment [6] [7]. Journal of Biosciences and Medicines  [8]. The percentage content of Ca is minimal for lactate and gluconate. Ca absorption from calcium citrate is consistently significantly higher than from calcium carbonate by 27.2% on an empty stomach, and by 21.6% with meals [9], but calcium carbonate has better cost effectiveness [10].
A lot of natural compounds modulate Ca salt solubility. Thus, sodium gluconate can increase the solubility of calcium lactate [11]. Amino acids and bile components reduce Ca salt precipitation in the intestine, increasing ion bioavailability [12]. It should be noted that Ca from dairy products as well as high-calcium mineral waters have high absorbability [13].
It seems the search for new Ca containing substances with high bioavailability has reached a certain impasse. Food does not contain pure Ca compounds but is the mixture of a lot of chemicals. On the other hand, Ca from salt mixtures has higher bioavailability in comparison with mono salts. In addition, food Ca toxicity is less than that of pure Ca chemicals at the same dosage [14]. This allows us to conclude that to develop highly bioavailable Ca compounds we should return to nature. From this point of view, amorphous Ca salts seem promising.
Amorphous calcium carbonate (ACC) has the highest reported specific surface area of all current forms of calcium carbonate [15]. Although unstable under ambient conditions, ACC is surprisingly widespread in biology either as a precursor phase, which later transforms into more crystalline forms, or as a stabilized phase sometimes coexisting with crystalline polymorphs [16]. Biogenic ACC often contains stabilizers to avoid or delay unwanted crystallization. Magnesium is commonly present as one of the stabilizing agents [17]. A study on rats revealed that Ca absorption from ACC preparations was up to 40% higher than from calcium carbonate [18]. Recent technology allows the synthesis of a stable amorphous calcium salt, containing different anions. The method of preparation of the composition of amorphous Ca and Mg salts has been developed recently [19]. Water and inorganic additives: calcium carbonate, calcium citrate, calcium hydrocitrate, magnesium citrate were used to stabilize the ACC.
The aim of the study was to compare original amorphous calcium salt composition bioavailability with Ca salts most often in food and medicines, as well as to evaluate the antirachitic activity of the mentioned composition in combination with vitamin D 3 in chickens.

Ethics Statement
All experimental procedures were approved by the local Animal Ethics Committee.

Amorphous Calcium Salt Composition (ACaSC)
ACaSC was prepared using our previously developed method [19] [20]. ACaSC is a thick white suspension and contains calcium carbonate, calcium citrate, calcium hydrocitrate and magnesium citrate. The total calcium content in the mixture was 50 mg/g of calcium and magnesium-0.64 mg/g. ACaSC pH (diluted with water 1:1) is 7.53.

Experimental Design and Animals
Lohmann Brown cockerels were used for the laboratory investigations. Chickens At the end of the experiment the cockerels were weighted and sacrificed by decapitation, in accordance with the recommendations of the European Convention [21] for the euthanasia of experimental animals. Experiment 1. 30-day-old chickens with a body weight of 289.3 ± 29.5 g were randomized in 7 experimental groups of 7 heads each. Ca absorption in chicken duodenum was studied in vivo. 18  At the age of 4 weeks venous blood sampling was done, the chickens were decapitated, the parathyroid glands were extirpated and weighted, the femurs collected for subsequent analysis. Parathyroid relative weight was calculated as a gland and body weight ratio. In blood serum calcium, phosphorus concentration and alkaline phosphatase activity were measured. The gravity of defatted and dried femur was measured; also, calcium and phosphorus content in bone was analyzed.
Three days before the decapitation of the birds, the daily balance of minerals in the chickens' bodies was evaluated. For 3 days, the food eaten every 24 h as well as the birds' litter were controlled (weighted, dried and analyzed for Ca content).

Laboratory Determinations
At the end of the experiment, Fe, Mg and Ca concentrations in organs and tissues were estimated using a flame atomic absorption spectrophotometer (Perkin-Elmer, model AAnalyst 700) [22]. Duodenal mucosa were homogenized in a solution of ice-cold tris-buffer (pH 7.4) using a glass homogenizer and a motor-driven Teflon pestle at 3000 rpm for 1 min to yield a 20% (w/v) homogenate.
Proteins were denatured with trichloroacetic acid (120 g/l) and removed by centrifugation for 10 min at 600 g.
The liver samples were dried at 1050 C to constant weight, ashed in a muffle furnace at 6500 C, and subsequently dissolved in 6 N-HCl.
Blood serum was analyzed for Fe, Mg, Ca and alkaline phosphatase performed on ILAB 300+ analyzer (Instrumentation Laboratory, USA).

Statistical Analysis
All statistics were performed using the software Statistica 7. Results of investigated parameters are presented as means ± SE. Multiple group comparison was done using one-way ANOVA and Post-hoc Tukey HSD test.

Results and Discussion
The aim of the first experiment was to compare the impact of different ingested Ca salts on Ca content in duodenal mucosa as well as in blood serum and in the liver. ACaSC specific activity was compared with Ca chloride, which is characterized by high Ca bioavailability, as well as with calcium carbonate -the most commonly used Ca salt in food and medicines but having low solubility and bioavailability. In dose 1.2 mg Ca all tested preparations did not induce statistically significant changes neither in the duodenal mucosa, nor the blood serum ( Table 2 and Table 3). However, induced Ca accumulation in the liver was found by all formulations, but it was the same in all groups.  Ca content in the liver of animals of the 7th group did not differ from that of groups 2 -4, the calcemia level tended to increase, but not significantly. Attention is drawn to the fact of the decrease of iron concentration in blood serum in animals of group 6 (received calcium carbonate), although this fact was not observed in groups 5 (calcium chloride) and 7 (ACaSC) ( Table 1). The probable mechanism of such a phenomenon is a lumenal event: calcium carbonate with phosphates and iron ions form an insoluble complex, which decreases Fe bioavailability [23] and can cause iron deficiency anaemia [24]. From the point of view of practical application, ACaSC has an advantage in comparison with calcium chloride, which is not indicated for long term use.
In the second experiment antirachitic efficacy of ACaSC per os, as well as in combination with vitamin D 3 , was tested. At the age of 4 weeks the rachitic chickens' body weight was 54% of the control. The birds had an untidy appearance, a soft and deformed beak. Hypocalcemia and hyposphatemia were detected in the blood serum, the activity of alkaline phosphatase increased more than 2.5 times; hyperplasia of parathyroid glands was found: its relative weight exceeded the control 10 times (Table 4). The ash content in the femur was significantly less in comparison with the control (Table 5). This is the result of impaired bone mineralization due to primary calcium and vitamin deficiency as well as secondary hyperparathyroidism [1].
Under the influence of diet enrichment with calcium carbonate manifestations of rachitic in Group 3 weakened (increased blood serum Ca, diminished alkaline Тable 4. Impact of calcium preparations and vitamin D 3 peroral ingestion on biochemical indices of blood serum and parathyroid gland relative weight of chickens with rickets.  phosphatase activity and parathyroid relative gland weight), but did not disappear completely. This was achieved using calcium carbonate in combination with cholecalciferol (Group 4). Based on body weight gain during the experiment, chicken groups may be arranged as follows: control/Group 1 = Group 6 > Group 4 > Group 5 > Group 3 > Group 2 (Table 5).
How did rachitic chickens (Group 5) respond to the administration of ACaSC, which has a higher than calcium carbonate bioavailability? All tested indices show, that ACaSC efficacy was better than for CaCO 3 (Group 3), but worse than for CaCO 3 in combination with vitamin D 3 (Group 4). Complete normalization of all analyzed indices was achieved in chickens of Group 6, received ACaSC and vitamin D 3 . It seems logical, because vitamin D functions are not limited by influence on calcium intestinal absorption regulation [25].
In this experiment the different severity of rickets in bird groups is apparent: between the "classical" picture of D-avitaminosis and the healthy birds (Control). Ingestion of calcium carbonate, vitamin D 3 and ACaSC to varying degrees normalized tested parameters. In general, the final outcomes, i.e. antirachitic efficacy, was practically the same in two groups receiving calcium carbonate + vitamin D 3 (Group 4) and ACaSC + vitamin D 3 (Group 6).
A calcium balance study did not identify significant differences between Ca accumulation only in the chickens of groups 4 and 6 ( Table 6).
Ii case ACaSC was used without vitamin D, its specific activity exceeded CaCO 3 in all tested physiological and biochemical parameters. This fact coincides with information, that in a double-blind randomized crossover clinical trial in postmenopausal women, it was shown that fractional Ca absorption of amorphous Ca was doubled on average compared to that of crystalline calcium carbonate [26]. The differences between CaCO 3 and ACaSC biological activity in our experiment were "smoothed" by vitamin D.
Nevertheless, there is evidence of an amorphous calcium salt composition advantage in comparison with crystalline calcium carbonate. It was shown that Table 6. The effect of calcium salts and vitamin D 3 administration on chicken feed consumption and calcium daily balance.
impacting blood level of phosphates. In case ACaSC is used without vitamin D, its specific activity excels CaCO 3 in all tested physiological and biochemical parameters. No less important is the ACaSC formulation: the fluid product is more suitable than calcium carbonate tablets and capsules for use by children and seniors.
The current paper contains only the initial part of the wider experiment's data. In the nearest future the information about combination's of ACaSC and vitamin D ricket's curative efficacy will be presented.

Conclusion
The studied composition of amorphous calcium salts seems prospective for new health food and pharmaceutical development. The product has a significantly higher bioavailability than calcium carbonate, suitable formulation for use by children and seniors. Combination with vitamin D increases ACaSC antirachitic effect. There is a reason to suppose that ACaSC together with some phytochemicals can provide significant osteoprotective activity in children and adults, including pregnant women.