Grouper bone nano-calcium and substitution long-chain triglyceride (LCT) into medium-chain triglyceride (MCT) enhanced calcium bioavailability and rat’s bone density

2.1Materials

This study utilized a calcium-deficient AIN-93G-MX mineral mix (Dyets, Pennsylvania, USA) with two types of calcium: (1) food grade synthetic calcium carbonate (CaCO₃) (Shuren Kechuang Food Additive Co., Lianyungang, Jiangsu, China) purchased from an online marketplace for the CCa feed group and (2) GBN for the G0-G100 feed groups. Table 1 provides a comparison of the nutrients from the two calcium sources. MCT (MCT-Max, CV. Segala, Bandung, Indonesia), LCT (Mazola Soya Bean Oil, PT. Sukadjaya Djaya, Bekasi, Indonesia), and other necessary materials for the rat feed manufacturer were acquired from the local market. Calcium, phosphorus, and alkaline phosphatase levels in rat serum were analyzed using Diasys Diagnostic System kit (Holzheim, Germany).

2.2Grouper bone nano-calcium production

The grouper bone nano-calcium production method followed [15]. The dried grouper (Epinephelus sp.) fish bone was autoclaved with 3×3 hour cycle then dried in a drying cabinet (50°) for 24 hours. The fish bone was crushed in a mortar, grounded, and sifted with a 80 mesh sieve. The coarse grouper bone was soaked in 1 N 1 : 5 HCl (Mallinckrodt, 7647-01-0) (w/v) and agitated with a magnetic stirrer for 1 hour and incubated in room temperature for 24 hours.

HCl was separated and removed via centrifugation at 3000 rpm, the particle was transferred into a glass beaker and diluted at a 1 : 5 (w/v) ratio with a 1 N NaOH solution (Merck, 1310-73-2). The sediment was subsequently hydrolyzed three times for 60 minutes at 100°C using a hotplate stirrer. After each cycle of hydrolysis, the supernatant was removed from the residue via centrifugation.

After mixing the hydrolysed sediment into the distilled water, the solution was neutralized using HCl 1 N (Mallinckrodt, 7647-01-0) until a pH range of 6.9–7.1 was attained. Following that, it was recentrifuged. The sediment was then transferred into a ceramic tray and desiccated in a cabinet dryer at 50°C for 15 to 18 hours. Following a one-minute refining period in a laboratory disc mill (Kawasaki T-100, Kobe, Japan), the desiccated sediment was sifted through a 203-mesh sieve (Haver & Boecker 59302 OELDE, Germany) and the resulting particle size was approximately 47.47 nm.

2.3Experimental animals and feed preparation

The Ethical Clearance Commission of the Integrated Research and Testing Institute, Universitas Gadjah Mada, has approved all animal experimentation procedures in this research (Certificate No. 00059/04/LPPT/II/2022). The experiment used 35 healthy female Sprague Dawley rats weighing between 40 and 65 g at the weaning age (3 weeks). In this study, female rats were utilized in consideration of the findings of [1], which indicate that the incidence of osteoporosis in elderly women is four times greater than that in elderly men.

All rats were housed in separate cages in a room temperature of 27°C with a 12 hours light-dark cycle. The feed standard corresponds to the composition of the AIN-93G feed [5] for rats before they reach maturity, and all feeds were composed of iso calcium, refers to the calcium content of each calcium source and iso fat. The feeds were (1) CCa group was an AIN-93G standard feed with CaCO₃ as the calcium source. (2) C0 group was an AIN-93G standard feed with calcium-deficient mineral mix; (3) G0 group used GBN as the calcium source with MCT:LCT 0 : 100% substitution, (4) G25 group used GBN as the calcium source with MCT:LCT 25 : 75% substitution, (5) G50 group used GBN as the calcium source with MCT:LCT 50 : 50% substitution, (6) G75 group used GBN as the calcium source with MCT:LCT 75 : 25% substitution, (7) G100 group used GBN as the calcium source with MCT:LCT 100 : 0% substitution.

The result from previous research in Table 1 shows that GBN has 30.73% calcium, whereas CaCO₃ contains 45.19% calcium. The AIN-93G feed formulation, which is an international standard, should contain 40.04% calcium in the form of CaCO₃. This calcium content is specifically present in 35 g of mineral mix. To provide similar calcium levels in both forms of calcium, an iso-calcium method was applied. The weight conversion was determined such that it equated to 35 g of mineral mixture. The CCa feed required approximately 11.025 g of CaCO₃ and 23.975 g of mineral mix lacking of calcium. Conversely, G0-G100 required 15.365 g of GBN and 19.639 g of calcium-free mineral mix. The content of each feed may be observed in Table 2.

In order to simplify the process of preparing the rat’s feed, the feed was manufactured in quantities of 999,986 grammes, which was then rounded up to 1 kilogramme and its multiples, based on the rat’s feed needs. Each morning, the rats were given an amount of food that was equal to 10% of their body weight (The calcium requirement for rats is 0.110 g CaCO₃ for CCa and 0.153 g GBN for G0-G100 in 10 g of feed according to the AIN-93G standard). The feeds were stored in a refrigerator at 4°C before use. Distilled water was provided ad libitum.

2.4Acclimatization phase and maintenance phase in In vivo experiment

The acclimatization period was carried out for 7 days. Every rat was given AIN-93G feed. After the acclimatization phase, 35 rats were randomly separated into seven feed groups and housed in labelled individual cages.

On the eighth day, the customized feed for each feed group was administered. Every four weeks, the rat was anesthetized using ketamine-xylazine (60 mg/kg BW) to obtained blood sample from the orbital sinus using microhematocrit. The rat’s blood was centrifuged for 15 minutes at 10,000 rpm to get rat serum. Rat serum was used to measure the calcium (Calcium AS FS), phosphorus (Phosphat FS), and alkaline phosphatase (ALP IFOC FS) using kits from Diasys Diagnostic System (Holzheim, Germany). The results of the serum and kit reactions will be calibrated using aspectrophotometer.

After 12 weeks, all rats were weighed, blood samples were collected, and they were sacrificed with ketamine-xylaizine (100 mg/kg BW). Rat femur and tibiae bones were collected and then cleansed of any remaining flesh. The tibiae bones were submerged for 24 hours in 10% PBS formalin to make histological preparations for histomorphometric tests followed [16] and [17] methods. The rat femur bone was cleaned and then dried for two hours in a cabinet dryer (50°C). The femurs of rats were weighed, measured for length and diameter, and then divided into two groups. The left femur group was used to test the bone’s mechanical strength using a universal testing machine (UTM) (Zwick Z0.5, Ulm, Germany) with three-bending test followed [6] and [18] methods. The right femurs were used for bone density testing used a Micro-Computed Tomography (μCT) (Phoenix V|tome|x s240, Waygate Technologies, Wunstorf, Germany) followed [19] methods. Figure 1 depicts the flowchart for this in vivo research.

Fig. 1

In vivo experimental flowchart.

2.5Statistical analysis

All data (excuding μCT data) were analyzed using a one-factor Completely Randomized Design (CRD), which consisted of 7 groups in feed composition, each with 5 replicates. The data were evaluated for diversity (ANOVA), and if the findings indicated a significant difference, the Duncan Multiple Range Test was performed (DMRT). If the two best values were not substantially different from one another, the two data were re-tested using the Independent T-Test differentiating test. The data were processed using the SPSS application (IBM SPSS 26 version, IL, USA) according to [20].

3.1In vivo nutrient bioavailability

3.1.1Calcium level of rat’s serum

The aim of the calcium test in rat serum was to determine how much GBN was effectively absorbed during the digestion of feed. Calcium concentrations in rat serum in response to different feed are shown in Fig. 2. According to Fig. 2, serum calcium levels at the beginning of maintenance for all feed groups were almost similar, ranging between 10.2–11 mg/dL. At 4th week, blood calcium levels decreased in all feeds, particularly the C0 therapy. At week 8, serum calcium levels were reasonably steady between 9.3 and 10 mg/dL, while the C0 therapy had shown 25% reduction. At week 8, the serum calcium concentrations in the G100 and CCa groups were almost identical. In the 12^th week, the G0–G75 therapy group repeatedly reduced blood calcium levels by 5–10%, but the C0 feed group demonstrated a serum calcium reduction of almost 30%. The C0 feed group which provided calcium-deficient feed for 12 weeks, the blood calcium level continued to decline, indicating the remodelling of calcium in the bones, which might cause the bones shrink and brittle. It will be evaluated in the rat femur bone’sstrength test.

Fig. 2

Calcium levels in rat serum every 4 weeks for 12 weeks of maintenance. Various feed formulas: CCa: standard diet AIN-93G; C0: standard calcium deficient feed (without calcium); G0: GBN + MCT:LCT 0:100%; G25: GBN + MCT:LCT 25:75%; G50: GBN + MCT 50:50%; G75: GBN + MCT:LCT 75:25%; G100: GBN + MCT:LCT 100:0%.

The CCa feed group was able to maintain constant blood calcium levels of around 10 mg/dL for a period of 12 weeks. The G100 feed group had the best GBN feed compared to the other GBN groups, which, despite a fall in blood calcium levels at week 8, continued to rise until 12^th week.

3.1.2Phosphorous level of rat’s serum

The pattern of phosphorus levels in rat serum in Fig. 3 was almost identical to that of calcium level in Fig. 2; all feed groups began with serum phosphorus levels of around 7 mg/dL. The phosphorus level serum decreased in all calcium CaCO₃ and GBN feed groups, notably in the C0 group at 4 and 8 weeks. Even though the adjusted feed in group C0 had a calcium deficiency and did not diminish the demand for phosphorus in the feed, it nonetheless affected the phosphorus level serum of rats, which declined with time. This suggests that calcium deficiency impacts phosphorus bioavailability. The blood phosphorus levels of rats were similarly impacted by the calcium-deficient feed, as shown by [21] study.

Fig. 3

Phosphorous levels in rat serum every 4 weeks for 12 weeks of maintenance. Various feed formulas: CCa: standard diet AIN-93G; C0: standard calcium deficient feed (without calcium); G0: GBN + MCT:LCT 0:100%; G25: GBN + MCT:LCT 25:75%; G50: GBN + MCT 50:50%; G75: GBN + MCT:LCT 75:25%; G100: GBN + MCT:LCT 100:0%.

At the end of the maintenance period (after 12 weeks), all phosphorus levels in the calcium source of CaCO₃ and GBN feed were comparable to their baseline values. This did not happen in the C0 feed group, where phosphorus levels kept descending until they were 32.6% lower at the end of the maintenance period than they were at the beginning. In addition to phosphorus from the mineral mix, feed G0–G100 also includes natural phosphorus from GBN so that phosphorus level serum can be kept consistently high for the 12 weeks of maintenance.

According to [22], sufficient calcium consumption without phosphorus intake will lead to phosphorus insufficiency inside the body. G0–G100 feed groups also get phosphorus through mineral mix, and GBN contains natural phosphorus, allowing normal blood phosphorus levels to be maintained for 12 weeks.

3.1.3Alkaline phosphatase level of rat’s serum

Alkaline Phosphatase/ALP is an enzyme that is extensively expressed in cells of mineralized tissue and plays a crucial role in the development of hard tissues. Intestinal, placenta, liver, and bone are the four primary sites of ALP in the human body [23]. ALP serum level is a particular indicator of bone activity. A rise in ALP suggests that the parathyroid gland is responsible for maintaining calcium levels in the blood when calcium levels is below normal which trigger the remodelling of bone calcium. Bone ALP is physiologically bound to the membrane of osteoblast cells, and only a little quantity is discharged into the serum. Increasing ALP serum level indicating an increase in bone resorption [24–26].

ALP level in rat serum in response to different feed are shown in Fig. 4. The C0 feed group had considerably greater blood ALP levels than those other feeds. C0 ALP serum level was the highest and substantially distinct from those of other feed groups. It suggests that only the C0 feed group, which began in the 4^th week, suffered bone remodelling. Bone remodelling is the release of calcium from bones in response to low blood calcium levels; hence, calcium stores in the bones must be replenished to fulfil the body’s calcium requirements in order to maintain homeostasis [27]. This condition will lead to osteopenia or even osteoporosis if it is not promptly treated with an adequate calcium intake.

Fig. 4

Alkaline Phosphatase levels in rat serum every 4 weeks for 12 weeks. Various feed formulas: CCa: standard diet AIN-93G; C0: standard calcium deficient feed (without calcium); G0: GBN + MCT:LCT 0:100%; G25: GBN + MCT:LCT 25:75%; G50: GBN + MCT 50:50%; G75: GBN + MCT:LCT 75:25%; G100: GBN + MCT:LCT 100:0%.

3.2Physical parameter and mechanical strength of rat’s femur bone

3.3Rat’s tibiae histomorphometry

Bone volume fraction in the 2D field is defined as the area of bone (Bone Volume) relative to the overall area of the ROI (Tissue Volume) [17], which is displayed as a percentage (%) in Table 4. The greater the bone volume percentage, the thicker or broader the microstructure of the cross-section of the bone.

The bone volume fraction revealed that the G100 feed group had the greatest bone volume fraction (28.27%), which did not vary from the CCa and G75 feed groups. The microstructure of the large bone reflects the bone’s strength, which is also proportional to bone density. [6] reported 12–16% for tibiae bone volume percentage which were lower than in this study. This was probably because the rats they used were younger than the ones used in this study. In addition, the rats used in their research were post-lactating rats, which bones experienced remodelling and a decrease in bone volume fraction during lactation.

Trabeculae are microstructures of cancellous/spongy bone shaped like spicules. The trabeculae serve as a structural support that provides substantial strength without substantially increasing the bone’s mass. If the periosteum covers the surface of the compact bone, the endosteum covers the trabecular surface of the cancellous/spongy bone [27]. Trabecular thickness is the average thickness of the trabeculae in 2D or 3D, measured in mm or μm [19]. The trabeculae thickness in this study as shown in Table 4.

The trabeculae thickness in the G100 group was not substantially different from the CCa and G75 feed group, similar to the bone volume percentage. The trabecular thickness in this study was higher than the tibial trabecular thickness in [6] research.

Histomorphometric trabecular thickness was calculated using tibial histology photo files which were opened using Image Rasher software. The trabecular thickness was determined by drawing 5 lines at random and measuring their length (in μm units) to determine the trabecular thickness. This calculation was subjective because the starting point of the trabecular line was selected at randomly, as was the positioning of the line drawing, making it hard to standardise. To assure its validity, the histomorphometric measurement of trabecular thickness should be confirmed by other techniques, such as the μCT method.

3.4Micro-computed tomography (μCT) analysis of rat femur bones

The samples tested using μCT were 3 rat femur samples from 3 selected feed groups: CCa, C0, and G100. The CCa sample represents the AIN-93G feed group which utilises a synthetic CaCO₃ calcium source, the C0 sample represents the calcium deficiency feed group, and G100 was the best treatment and was significantly different from the other four GBN + MCT:LCT feed groups based on the Duncan Test (against the five G0–G100 feed groups), especially on calcium, phosphorus and ALP levels, as well as on measurements of femur weight and strength.

The aim of this test is to validate the microstructure of the bone tissue tested using the μCT instrument. The appearance of the femur bone sample using μCT can be observed in Fig. 5. The VOI or volume studied in this study was the area of the proximal epiphysis up to the epiphyseal plate as the boundary of the spongy bone, which is 8.5 mm high from the proximal epiphysis.

Fig. 5

3D structure of femur bone sample reconstructed image from µCT (a) CCa, (b) C0, dan (c) G100.

Trabeculae in the femur are most commonly found in the spongy bone, because this is where the most bone growth occurs during the growth period. In old age, indicators of bone loss are also easiest to detect in the spongy bone. The μCT data analysis process was carried out by trans-section scanning/cross-section of the bone using the SkyScanTM CT-analyzer software program. The pixel size resolution was 14.25μm, resulting in 597 layers with a height of 8.5 mm.

3.5Structure linear density

Struture linear density or also known as trabecular number (Tb.N) is the number of trabeculars per unit length [19]. The number of trabeculae is the inverse of the average distance from the central axis of the structure [30]. The higher the number of linear density structures implies the greater the number of trabeculae in the VOI, thus the spongy bone microstructure will be stronger. Based on Table 5, it was known that the linear structure density of the G100 femur sample is the highest compared to other samples.

CCa femur had fewer spongy/trabecular structures but were thicker than the other 2 samples. Meanwhile, the G100 femur had greater number of spongi/trabecular but the structure was thinner. Meanwhile, the C0 femur, although it has a thinner spongy/trabecula structure thickness than the CCa femur, apparently has a greater number of spongi/trabecula structures than the CCa femur.

3.6Structure separation

Structural separation can be defined as trabecular separation (Tb.Sp), which is the area of the cavity between the trabeculae measured in 3D [19]. The Tb.Sp value is calculated using the same method as Tb.Th but the cavity part is considered as a non-bone part, so it can be said that Tp.Sp is the thickness of the cavity in the bone [30].

Based on Table 5, it was known that the structure separation was the smallest in the G100 femur and the highest in the CCa sample. Based on calculations of structure separation, structure linear density, and structure thickness, it can be seen that CCa has the thickest trabecula thickness compared to the other two femurs, but it turned out that the number of trabeculae was the smallest, resulting in the largest cavity compared to the other two femurs. Even the G100 femur had slimmer trabeculae than CCa, but it had greater number of trabeculae and resulting in the lowest structure separation to the other two femurs.

3.7Discussions

The objective of the four-weekly calcium serum examination in rats is to evaluate the bioavailability of calcium derived from different feed groups. The AIN-93G feed was able to maintain stable calcium levels for 12 weeks, whereas the G0-G100 nano-calcium grouper demonstrated that the greater the percentage of MCT, the greater the bioavailability of calcium in rat serum. Similarly, serum phosphorus levels in rats exhibited a similar trend to serum calcium levels. In contrast, alkaline phosphatase (ALP) levels exhibited an inverse relationship with calcium and phosphorus. ALP is extensively secreted by osteoblast cells during mineralization and calcification of bone tissue [27, 32].

The results of in vivo research showed that among 5 GBN feeds with various percentages of MCT substitution for LCT. The independent T-Test statistical test was then carried out on CCa and G100 to test the differences between the two, whether they were purely not significantly different or whether they were actually significantly different between the two. The results of the T-Test test showed that G100 feed was significantly different from CCa feed in testing bioavailability of calcium, bone strength/ultimate strength and bone volume fraction (BV/TV) % by histomorphometry. Thus, calcium sourced from GBN plus MCT:LCT 100 : 0% was superior to synthetic calcium carbonate (CaCO₃) in calcium and phosphorus bioavailability, bone strength/ultimate strength and bone volume fraction (BV/TV) % by histomorphometry.

The VOI section examination of bone density did not reveal the true state of a bone density. According to [18] if the length of the bone is considered 100%, the transect position of the femur in the distal part is at positions 15, 17, and 20% of the length of the femur, midway at 40, 42.5, and 45%, and proximal at 64, 66.5, and 69% of the length of the femur.

[6] research revealed that calcium carbonate (CaCO₃) and tuna bonepowder, as calcium supplements, increased the bone mineral density (BMD) of the mother rat. On the contrary, bone histomorphometry demonstrated that the maternal bone microstructure was beneficially impacted by tuna bone. This was evidenced by enhanced bone formation, reduced bone resorption, and increased bone volume.

The advantage of the GBN calcium source, apart from its nano size, GBN also contains various amino acids [15] which support the absorption of fish bone nano-calcium to pass through the transcellular pathway for active absorption.

The findings of [33] study indicated that Protein Bonito Hydrolysate (PBH-Ca) enhanced rat growth, increased the apparent calcium absorption and quantity of bone trabeculae, stabilised serum calcium and phosphorus levels, also inhibited ALP activity, reduced bone trabeculae separation, accelerated rat bone growth, and elevated BV/TV and femoral BMD. PBH-Ca has considerable potential as an ingredient in a calcium-rich feed.

The mechanism of MCT’s role in calcium absorption is still debated. [34] assume that MCT basically does not directly increase calcium absorption, but MCT intake improves fat absorption in the intestinal tract. This is thought to increase Vitamin D absorption, where increasing Vitamin D absorption will increase calcium absorption.

According to [27], the bone mineralization process begins with osteoblast cells secreting type I collagen, several glycoproteins and proteoglycans. Some of these factors, especially osteocalcin and certain glycoproteins, bind Ca^2 + with high affinity, increasing local concentrations of calcium ions. Osteoblasts also release very small membrane-enclosed matrix vesicles containing alkaline phosphatase and other enzymes. These enzymes hydrolyze PO^4 - ions from various matrix macromolecules, creating high concentrations of these ions locally. High concentrations of Ca^2 + and PO^4 - ions cause calcified nanocrystals to form in and around the matrix vesicles. The crystals grow and mineralize further with the formation of small masses of calcium hydroxyapatite [Ca₁₀(PO₄)₆(OH)₂], which surround the collagen fiber and all other macromolecules. Eventually the hydroxyapatite masses coalesce as a solid, fused bone matrix when matrix calcificationis complete.

The presence of natural protein in calcium supplements is very important to help osteoblast cells convert calcium into hydroxyapatite crystals. The mineralization process aims to thicken the trabeculae or cortical bone or to replace calcium lost due to osteoclast cell activity if calcium breakdown has previously occurred.

3.8Conclusion

Administration of grouper bone nano-calcium with 100% MCT substitution for LCT has been shown to increase calcium bioavailability and bone density in rats. It beneficially affected the bioavailability of calcium, was letting the rat’s bones to develop properly, had high density, and been strong throughout the growth phase.