Abstract: Low Density Lipoprotein Cholesterol (LDL-Ch) is the main risk factor for atherogenesis. The efficacy of cardio-protective supplements may therefore be judged based on their ability to reduce LDL-Ch concentration in the blood. Additional dietary Calcium (Ca) decreases blood Total Ch (TCh) concentration. Thus researchers evaluated the influence of additional dietary Ca in form of fossil shells (Aragonite) on total Ch (TCh), LDL-Ch, High Density Lipoprotein Ch (HDL-Ch), Tiglycerols (TG) and Non-esterified Fatty Acids (NEFA) concentrations in chickens. Thirty 21 weeks old Barred Plymouth Rock roosters were fed diets containing 0, 2 or 4% Aragonite flour from 21-53 weeks of age. At 21, 25, 29, 34, 37, 42, 46, 51 and 53 weeks of age, birds were individually weighed and TCh, LDL-Ch, HDL-Ch, TG and NEFA levels in blood were measured. Body weights and NEFA levels were similar among treatments. Treated males showed significantly lower TCh, LDL-Ch and TG levels but higher HDL-Ch concentration than control males. However, these parameters were similar between treated males. Thus, additional dietary Ca in form of Aragonite flour did not affect the nutritional status of the birds when fed at 2 or 4% but beneficially altered levels of TCh, LDL-Ch, HDL-Ch and TG in blood.

INTRODUCTION

The role of blood plasma Total Cholesterol (TCh) as a factor in stimulating atherogenesis is now widely recognized by health experts. Concentration of TCh in the blood is strongly influenced by the quantity and quality of fat in the diet. For this reason, most previous studies focused on the effects of diet on blood TCh concentrations. However, recently attention has shifted to individual lipoprotein Ch fractions, i.e., low density lipoprotein Ch (LDL-Ch) and high density lipoprotein Ch (HDL-Ch). This has been prompted by the discovery that most of the Tch in the blood is contained in the LDL-Ch fraction and that it is this component that has the most undesirable effects on health. Though LDL-Ch provides cells with their Ch requirements, its excessive supply causes atherosclerosis (Castelli et al., 1986; Ohlsen and Rogers, 2004a; Stanford, 2005) whereas high HDL-Ch levels are cardio-protective, independent of other blood lipid measures (Castelli et al., 1977; Ohlsen and Rogers, 2004b). On this basis, LDL-Ch levels or ratio of LDL-Ch to HDL-Ch have been used as summary measures of risks of atherosclerosis.

Although, diagnoses and treatment for cardiovascular diseases have advanced the most important treatment is prevention and a major preventive measure is averting hypercholesterolemia. Daily dietary supplementation with Calcium (Ca) from conventional Ca carbonates has been reported to modify blood lipid profiles towards low TCh and LDL-Ch concentrations in humans (Denke et al., 1993; Reid, 2004; Ditscheid et al., 2005), rats (Vaskonen et al., 2002; Sun et al., 2004), rabbits (Renaud et al., 1983; Hsu and Culley, 2006) and goats (Hines et al., 1985). The mechanisms by which it achieves this are attributed to the mineral’s propensity to bind with fatty acids (Grundy and Denke, 1990) and bile acids (Lupton et al., 1994; Ditscheid et al., 2005) in the gut which interferes with their absorption into the blood circulatory system. Thus, a reduction in the absorption of fats, especially saturated fatty acids, decreases not only blood Tch but also LDL-Ch concentrations (Grundy and Dunke, 1990; Vaskonen, 2003) whereas a decrease in the re-absorption of bile acids induces an increase in the removal of Ch from the circulatory system by the liver for conversion to bile acid (Vaskonen et al., 2002). These physiological processes cause an overall reduction in TCh and LDL-Ch levels in the blood.

Fossil shells (Aragonite) flour is a Ca carbonate of marine origin with 38% Ca (Finkelstein et al., 1993). The biological availability of its Ca is similar to most conventional Ca carbonates (Ross et al., 1984). This compound is commercially available in Japan (GaiaTec Co., Inc., Kagoshima, Japan) and may be used as a possible therapeutic agent for cardio-protection. However, because its origin (i.e., marine animal/plant fossils vs mineral deposits) and chemical structure are different from most conventional Ca sources (Ajakaiye et al., 1996), its physiological effects on individual plasma lipoprotein Ch profiles may also be different but scientific evidence for this conclusion is not readily available in the literature.

Recently, the laboratory evaluated the hypocholesterolemic potential of Aragonite flour in chickens. The results from these studies showed that additional dietary Ca in form of Aragonite flour had indeed Tch-lowering actions in both male broilers (Kanyinji et al., 2010) and breeding Barred Plymouth Rock males (Kanyinji and Maeda, 2010). However, in these studies, no attempts were made to measure levels of individual lipoprotein Ch fractions of TCh (i.e., LDL-Ch and HDL-Ch) in the blood. Since LDL-Ch fraction is the main atherogenic agent (Kannel et al., 1986; Isles and Paterson, 2000; Ohlsen and Rogers, 2004a) whereas HDL-Ch is cardioprotective (Miller and Miller, 1977; Castelli et al., 1986; Ohlsen and Rogers, 2004b), the health benefits gained from the hypocholesterolemic characteristics of Aragonite can therefore, best be judged based on its influence on levels of LDL-Ch and HDL-Ch. Moreover, Ohlsen and Rogers (2004a) argued that the risks of coronary heart diseases cannot be evaluated based on levels of TCh alone but also on levels of individual lipoprotein Ch fractions.

Therefore, the present research sought to evaluate the influence of feeding 2 or 4% additional Ca in form of Aragonite flour to breeding Barred Plymouth Rock males on levels of TCh and its individual lipoprotein fractions in the blood. Additionally because high dietary Ca in the diet precipitates lipids in the gut thereby inhibiting their digestion and absorption despite lipids being essential for normal growth and development (Hulan et al., 1984), researchers endeavored to establish the effects of additional dietary Ca on the nutritional status of the birds fed additional dietary Ca by measuring body weights and levels of Non-Esterified Fatty Acids (NEFA).

MATERIALS AND METHODS

Animals, diets and experimental design: The birds used in this study were managed according to the guidelines outlined by the Hiroshima University Animal Ethics Committee. The chickens, diets and details of the design of the experiment are as described in Kanyinji and Maeda (2010). Briefly, thirty 21 weeks old Barred Plymouth Rock roosters were divided into three groups based on their body weights and put in individual cages under one house. The birds were fed ad libitum breeders diet (Nishinihon Feed Co. Ltd., Okayama, Japan) fortified with 0, 2 or 4% Aragonite flour (GaiaTec Co., Inc., Kagoshima, Japan) as source of additional dietary Ca until 53 weeks of age. At 21, 25, 29, 34, 37, 42, 46, 51 and 53 weeks of age, body weights of individual males were recorded and a 1~2 mL blood sample from each bird was also collected into 15x85 mm Borosilicate disposable culture test tubes (CAT No. 14-961-28 Fisher brand, Thermo Fisher Scientific Inc., MA, USA) treated with heparin (Novo-Heparin, Mochida Pharmaceutical Co., Ltd., Tokyo, Japan). The samples were then centrifuged at 1,000xg for 15 min at 5°C and the plasma obtained was stored frozen at -20°C until analysis.

Laboratory analysis: Frozen plasma samples of all males were thawed at room temperature (25°C) for determination of levels of TCh, LDL-Ch, HDL-Ch, Triglycerols (TG) and NEFA. These parameters were measured from a 20 μmL plasma sample of each bird at each sampling age using an automated Beckman Coulter AU480 instrument (Beckman Coulter Inc., Fullerton, CA, USA).

Data analysis: The data were analyzed as repeated measure data using PROC MIXED of SAS (2003) based on the mathematical model:

Where:

Age of the birds at which blood was sampled was used as repeated measure. Means were compared using Tukey test (SAS, 2003). Differences among means with p<0.05 were accepted as representing statistically significant differences. However, differences among means with 0.05<p<0.10 were accepted as representing statistical tendency to differ.

RESULTS

The mean body weights recorded at 21, 25, 29, 34, 37, 42, 46, 51 and 53 weeks of age are shown in Fig. 1a. Initially, there was a tendency (p = 0.08) to decline in body weights in all treatment groups but from 25 weeks of age onwards, they increased with time (p = 0.02). No differences (p = 0.98) in this parameter were observed between treated males and those in the control group nor between 2 and 4% groups (p = 0.73).

Concentration of NEFA in treated birds and those fed control diet are shown in Fig. 1b. Initially, levels of NEFA in all treatment groups were high but declined significantly with time (p = 0.05) until 25 weeks of age after which they stabilized. However, no differences (p = 0.62) were observed between treated birds and those fed control diets at all ages. Likewise, NEFA levels between birds fed diets with 2 or 4% Aragonite flour were similar (p = 0.91).

Blood TCh levels in Barred Plymouth Rock roosters fed diets with or without additional Ca source between 21 and 53 weeks of age are shown in Fig. 2a. From 25 weeks onwards, males receiving additional Ca (i.e., 2 or 4% additional Ca groups) exhibited lower blood total Ch levels than birds fed control diet (p = 0.01) but no differences (p = 0.80) were observed between 2 and 4% additional Ca-fed groups during this period. TCh levels were not affected (p = 0.97) by the age of the birds at which sampling was done.

Figure 2b shows the LDL-Ch levels in the birds that received diets with or without additional Ca source. In all the treatment groups, mean LDL-Ch levels for the whole trial period averaged 87.9, 78.8 and 78.3 mg dL^-1 for the control, 2 and 4% additional Ca-fed groups, respectively. This implies that LDL-Ch levels were at least 10% lower in the birds that received additional Ca source in the diet than those fed control diet. The LDL-Ch levels for treated birds (i.e., 2 or 4% additional Ca-fed groups) were different (p = 0.001) from those of males in the control group but no differences (p = 0.89) were observed between the 2 and 4% additional Ca source-fed males. Moreover, the age at which blood sampling was conducted did not significantly affect (p = 0.99) LDL-Ch levels.

Figure 2c shows the HDL-Ch levels in males that received diets with additional Ca source and those fed control diet. In all the treatment groups, mean HDL-Ch levels between 21 and 53 weeks of age averaged 26.0, 28.7 and 28.9 mg dL^-1 for the control, 2 and 4% additional Ca source-fed males, respectively, implying that HDL-Ch levels were also at least 10% higher in the birds that received diets with additional Ca compared to those fed control diet.

Fig. 1:	a) average body weight and b) Non-Esterified Total Fatty Acids (NEFA) levels in blood of Barred Plymouth Rock males fed diets containing 0 (control), 2 and 4% additional Ca in form of Aragonite flour sfrom 21-53 weeks of age

The HDL-Ch values for treated birds were significantly different from those of the control group (p = 0.001, control vs. 2 or 4% additional Ca fed groups). A decline in LDL-Ch and increase in HDL-Ch consequently changed the ratio of LDL-Ch to HDL-Ch (LDL-Ch:HDL-Ch) in treated birds to 2.8:1 and 2.7:1 for 2 and 4% additional Ca-fed groups, respectively from 3.4:1 for males fed control diets. The LDL-Ch:HDL-Ch ratios for both 2 and 4% additional Ca-treated males were significantly different (p = 0.001) from that of control group.

However, no differences in HDL-Ch levels were observed between 2 and 4% additional Ca-fed birds (p = 0.67) or LDL-Ch to HDL-Ch ratios (p = 0.93). Additionally, the age at which blood sampling was done did not influence HDL-Ch levels (p = 0.60).

Levels of TG in males that received diets with or without additional Ca source are shown in Fig. 2d. Mean TG levels between 21 and 53 weeks of age were 34.1, 32.2 and 32.0 mg dL^-1 for males fed control, 2 and 4% additional Ca-fortified diets, respectively.

Fig. 2:	a) Total Cholesterol (TCh); b) Low density Lipoprotein Cholesterol (LDL-Ch); c) high density cholesterol (HDL-Ch) and d) Triglycerols (TG) levels in blood of Barred Plymouth Rock males fed diets containing 0 (control), 2 and 4% additional Ca in form of Aragonite flour from 21-53 weeks of age

Statistical analysis showed that additional Ca in the diets reduced TG levels (p = 0.001) in treated birds (2 or 4% additional Ca-fed birds vs control) when compared to the control. However, no differences (p = 0.79) were observed when TG levels for 2% additional Ca-fed males were compared to those that received feed with 4% Aragonite flour. As was the case with TCh, LDL-Ch and HDL-Ch levels, the age at which blood samples were collected did not influence (p = 0.99) TG levels in the birds.

DISCUSSION

The results described in this study agreed with the suggestion by Grundy and Denke (1990) that dietary induced changes in plasma TCh levels yield essentially reciprocal alterations in individual lipoproteins. They have also demonstrated that the hypocholesterolemic characteristics of additional dietary Ca in form of Aragonite flour not only reduced levels of TCh in Barred Plymouth Rock males but also modified the lipoprotein Ch fractions towards lower LDL-Ch and higher HDL-Ch levels which improved the LDH-Ch to HDL-Ch ratio. Furthermore, this study permitted the validation of the relationship between the level of Ca in the diet and level of lipoprotein Ch fractions. For example, in spite of similarities in macronutrient compositions between control diet and diets with 2 or 4% additional Ca tabulated by Kanyinji and Maeda (2010), plasma LDL-Ch and TG levels significantly reduced while HDL-Ch levels increased in birds fed diets with additional Ca source compared to those in the control group. This suggested that macronutrients could not be responsible for the observed differences in lipoprotein Ch fractions.

The control-treated groups differences in lipoprotein Ch fractions in the present study concurred with observations of Denke et al. (1993) and Reid et al. (2002) in humans with regard to additional dietary Ca-plasma LDL-Ch and HDL-Ch changes. Thus, these results suggested a beneficial influence of additional Ca in the form of Aragonite flour not only on the TCh levels but also on LDL-Ch and HDL-Ch levels. The decrease in TCh and LDL-Ch levels in this study could be due to several effects attributed to high Ca intake by the chickens such as a reduction in fatty acid absorption, resulting most likely from the formation of insoluble Ca-fatty soaps in the gut (Denke et al., 1993; Reid, 2004; Ditscheid et al., 2005). According to Grundy and Denke (1990) and Vaskonen (2003), decreased absorption of fat especially of saturated fatty acids, reduces both TCh and LDL-Ch concentrations.

Another suggested Ch-lowering mechanism of additional dietary Ca in the present study is with regards to the mineral’s propensity to bind and precipitate bile acids in the gut (Lupton et al., 1994; Welberg et al., 1994; Shahkhalili et al., 2001). Though there did not measure fecal bile acids, precipitation of bile acids by additional Ca would reduce the intestinal concentrations of bile acids available for fat digestion as well as that which is supposed to return to the liver via the entero-hepatic circulation. Thus, additional dietary Ca changed the fat/Ch uptake and metabolism as well as bile acid feedback mechanism. Decrease in the bile acids returning to the liver induced an increase in the conversion of Ch to bile acids by the liver (Fuchs, 2003). This mechanism is known at least from the lipid-lowering effects of cholestyramine and other bile acid binding resins (Witztum, 1996; Ohlsen and Rogers, 2004b). Thus, the interruption of the re-absorption of bile acids into the entero-hepatic circulation by additional Ca modified the hepatic metabolism of Ch. A high bile acid biosynthesis induced by treatment with additional Ca may have caused an increase in demand for Ch by the liver and the liver responds to this by enhancing the synthesis of Ch and uptake of LDL-Ch via the LDL receptors with the overall effect of reduced plasma TCh and LDL-Ch levels.

Furthermore, the lipid-lowering mechanism attributed to additional Ca could also have been due to the mineral’s capability to influence adipocyte activity. About 99% of Ca in the body is stored in the extracellular spaces and intracellular cytosolic soluble Ca mediates many metabolic pathways including platelet aggregation and insulin resistance. Calcitropic hormones such as parathyroid hormone and 1, 25-hydroxy vitamin D3, regulate intracellular Ca concentration (Ca²⁺). Low dietary Ca intake for example, stimulates high levels of parathyroid hormone and 1, 25-hydroxy vitamin D3 which in turn stimulate high levels of intracellular (Ca2+) in adipocytes that promote lipogenesis while inhibiting lipolysis (Kelly and Gimble, 1998; Zemel et al., 2000). In contrast, high dietary Ca intake depresses levels of parathyroid hormone and 1, 25-hydroxy vitamin D3 which results in low levels of intracellular [Ca²⁺] leading to inhibition of lipogenesis and stimulation of lipolysis (Zemel, 2002, 2003). Thus, the Ca level in the diet may determine whether adipocytes store or break down fats. In the present study, it is possible that increased lipolysis usually resulting from Ca-rich diets favored lipid mobilization in birds that received additional Ca and may explain the observed reduction in TG levels. Additionally, it has been noted that an increase in intracellular (Ca²⁺) in hepatocytes stimulates microsomal triacylglycerol transfer protein that has been implicated in the formation and secretion of very low density lipoproteins (Cho et al., 2005). On this understanding, it is likely that ingested additional Ca in treated birds suppressed increase in hepatocellular (Ca²⁺) thereby inhibiting the formation and secretion of very low density lipoproteins that elevate TG and LDL-Ch levels.

The levels of some of the blood metabolites indicate the nutritional status of an animal (Russell and Wright, 1983). For example, in ruminants, plasma concentrations of NEFA have been related to their growth (Ellenberger et al., 1989) and growth depends on the nutritional plane of the animal. The effects of feed intake on plasma NEFA levels (Grummer, 1995; O’Doherty and Crosby, 1998) have been investigated and results have shown that this parameter increases during periods of energy restriction. In the present study, no differences in NEFA concentrations were detected between treated birds and those fed control diet. Though feed intake was not measured, lack of differences in NEFA levels may at least suggest that the energy status of birds in all treatment groups was similar. Since body weight is also another good indicator of the nutritional status of animals (Sheehan et al., 1977; Juarez-Reyes et al., 2004), this supposition is supported by lack of significant differences in body weights between treated birds and those fed control diets. It also implies that additional dietary Ca source did not significantly change the availability of macronutrients required for growth.

In all treatment groups, NEFA levels were high at 21 weeks of age but declined with time before stabilizing at lower levels from 25 weeks of age onwards. Hershock and Vogel (1989) stated that stress as well as dietary influences may affect TG, NEFA and TCh levels in humans and animals. Interestingly, the occurrence of high NEFA levels in the present study corresponded with the time the males had just been transferred into their individual cages which led to fights between neighboring males. It was also at the time during which the males were being prepared for another study on sperm parameters which entailed clipping their tail feathers and feathers around the cloaca and training them for semen collection by massage. Thus, in the absence of data on feed intake in the present experiment, it can only be speculated that high initial NEFA levels were due to stress induced by the described activities and as these activities ceased, NEFA levels also declined.

CONCLUSION

Additional dietary Ca in diets fed to Barred Plymouth Rock males at 2 or 4% as Aragonite flour had no adverse effects on the nutritional status of the birds compared to those fed control diet but beneficially decreased not only blood TCh but also LDL-Ch and TG levels. A reduction in TCh and LDL-Ch levels resulted in an increase in the HDL-Ch fraction in treated birds which improved the LDL-Ch to HDL-Ch ratio. Since chickens are recognized as suitable animal models for studies on the comparative biochemistry of Ch metabolism and transport in humans (Chandler et al., 1979; Castillo et al., 1996), these results provide a reason to encourage trials of Aragonite flour in humans for possible health benefits.

ACKNOWLEDGEMENTS

The first researcher was supported by the Government of Japan through MEXT: Ministry of Education, Cultural, Sports, Science and Technology (Monbukagakusho). Both researchers are grateful to Dr Sugino for his help in the analysis of samples, Prof. Lawrence. M. Liao for proof reading this manuscript and GaiaTec Co., Inc., Kagoshima, Japan for kindly providing the Aragonite flour.