For years, nutritionists and medical experts have recommended an intake of about mg of calcium per day for most people, and mg per day for males and females aged , a time period when bone accumulation is the greatest. However, scientists have gradually realized that calcium absorption diminishes as people get older.

For that reason, the National Institute of Health in the United States is now calling for intakes of mg per day for men aged 25 or older and females between the ages of , mg per day for women who have stopped menstruating, daily mg for women over the age of 65, and mg for males and females aged These higher intakes may do more than build better bones.

Scientific evidence suggests that calcium- rich diets may represent a drug-free way to keep high blood pressure under control; as many as one out of two individuals may be able to lower blood pressure by taking in extra calcium.

Lofty levels of calcium may also reduce the risk of colon cancer, probably because calcium binds with cancer-promoting bile acids in the large intes tine. Fortunately, calcium-rich diets don't seem to increase one's chances of developing kidney stones; in fact, they may actually reduce the risk!

The easiest way to consume enough calcium is to eat yoghurt; just three cups of the stuff gives you over mg of the important mineral, which is an adequate daily intake formost people.

Milk and calcium-fortified orange juice are also good; each supplies about mg per cup. Calcium-processed tofu and spinach are the other major calcium sources; tofu yields about mg per quarter-pound, and spinach provides mg per cup.

What about calcium supplements? If you're eating enough of the above foods, you don't need them, but experts are recommending that those who rely on calcium pills should take them with meals and in doses of mg or less. If you also take iron supplements, you should take the calcium and iron at different meals, since calcium can interfere with iron absorption.

Individuals with lactose intolerance tend to shy away from milk and yoghurt as their sources of calcium, but such persons can try some of the low-lactose or lactose-free dairy products currently on the market or rely on products which aid lactose digestion.

All in all, taking in enough calcium is a very bright idea for athletes who want to perform at their highest level. Counting Your Calcium: How Much Is Enough? Related Files PPendurance-nutrition-boning-up-on-calcium.

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Moreover, calcium supplementation from tuna bone was able to prevent lactation-induced bone loss in experimental animals Thus, fish bone is considered a good natural source of calcium supplementation.

Besides, vitamin D 3 is essential for the musculoskeletal development and bone formation. Since crude tuna head oil contains a high amount of vitamin D 3 and Omega-3 polyunsaturated fatty acids 18 , the latter of which, have been known to improve cognitive function and increase neuroplasticity, tuna fish oil could be one of the most effective and readily available sources of vitamin D supplement.

In addition, Omega-3, essential fatty acids, especially those from marine oils, could stimulate intestinal calcium absorption in vitro study as well as promote bone growth in young growing age 19 , Since females have greater risk of fragility fracture and osteoporosis than males, especially after menopause and becoming aging 5 , and since early bone health and peak bone mass in young adult potentially determine osteoporosis risk of females later in life 6 , we sought to develop calcium supplement products for females.

In the present study, we therefore performed all experiments only in female animals. It is well recognized that regular exercise provides several physiological and psychological benefits. Previous experiment showed that weight-bearing exercise, such as forced treadmill running, in ovariectomized rats helped prevent reduction in trabecular bone volume and bone mineral density 21 , and even increased bone volume and thickness, osteoblast activity, and improved dynamic bone formation The conceptual idea of this study is to provide a new form of calcium supplement from a natural source for use during childhood and adolescence, i.

Combining exercise with calcium supplementation may provide additive effect on loaded bone, particularly tibiae and femora. In this section, there were 2 calcium diet formulae, i.

Calcium-replete diet was prepared by adding CaCO3 into L to obtain a final of 0. Rats fed either L or S1 exhibited an increase in bone growth as they got older from the age of 6 to 15 weeks. Th as analyzed by bone histomorphometry, increase in mechanical property as analyzed by 3-point bending, and increases in cortical bone mineral density BMD , cortical bone mineral content BMC , cortical thickness Ct.

Th , cortical area Ct. A of metaphyseal and diaphyseal mid-shaft femur as analyzed by pQCT. However, trabecular BMD and BMC did not change with increase in age in S1 reflecting the concurrent increase in the size of bone. On the other hand, comparing adolescent rats fed low calcium diet for 2 weeks 6 week old L and young adult rats fed low calcium for 11 weeks 15 week old L , the week old L showed normal bone length and increased in bone weight, trabecular BMC, cortical BMC, Ct.

Th and Ct. Compared with corresponding S1 control, bones of rats after 2 and 11 week calcium insufficiency exhibited drastic reductions in bone mechanical properties, cortical BMD, cortical BMC, Ct. Th, and Ct. A of metaphyseal and diaphyseal mid-shaft femur. Bone impairment found in rats fed low calcium for 11 weeks was less severe than those fed low calcium for 2 weeks.

However, trabecular BMD was significantly decreased after 11 weeks but not after 2 weeks of low calcium diet. Magnitude of changes in bone microstructural parameters in L compared with corresponding S1 after 11 weeks of low calcium diet were greater than those after 2 weeks of low calcium diet.

Blood chemical analysis revealed that dietary calcium insufficiency for 2 and 11 weeks led to hypocalcemia lower levels of total calcium and ionized calcium , lower level of 25 OH D 3 , and higher levels of inorganic phosphate only in 11 weeks and hormone 1,25 OH 2 D 3 Table 1.

All rats were challenged with low calcium diet L for 2 weeks, thereafter, the effect of calcium supplementation from tuna bone denoted as S2 was compared with calcium carbonate denoted as S1 Fig.

Fractional calcium absorption in young rats fed extra calcium from tuna bone was significantly higher than that of rats fed CaCO 3 Rats fed tuna bone calcium and CaCO 3 had similar body weight that increased with age from 6 to 10 weeks.

Supplemental Table 4 showed no difference in tibial length or bone mechanical properties. pQCT analysis of the femoral metaphysis and mid-shaft diaphysis showed no difference in BMD or BMC of trabecular Tb. BMD and Tb. BMC , cortical and sub-cortical region Sub. BMD and Sub. BMC or cortical compartment Ct.

BMD and Ct. Th, Ct. A, periosteal perimeter and endosteal perimeter Ps. Pm and Es. Pm, respectively were not different Supplemental Table 4. Bone histomorphometry showed no difference in the microstructure, and osteoblast and osteoclast surface normalized with bone surface Fig.

However, rat fed calcium supplementation from tuna bone exhibited higher mineral apposition rate 2. Blood chemical analysis showed no difference in the levels of 25 OH D 3 , 1,25 OH 2 D 3 , and total calcium between these 2 treatments, whereas serum inorganic phosphate was lower in rats given tuna bone calcium supplementation Supplemental Table 4.

Tuna bone calcium supplementation resulted in higher fractional calcium absorption and bone formation rate than calcium carbonate supplementation. A Experimental design, all rats were challenged with low calcium diet 0. Extra calcium 0. Th, trabecular separation, Tb. Sp, trabecular number, Tb.

N, osteoclast surface normalized with bone surface, Oc. S1 , calcium supplementation in diet from CaCO 3 , S2 , calcium supplementation in diet from tuna bone. The timeline for age-matched rats fed with calcium-replete diet from CaCO 3 S1 is shown in Fig.

Timeline for rats on low calcium diet L and rats receiving 3 formulae of tuna calcium supplements after 2 weeks of low dietary calcium diet is shown in Fig. The diet ingredients were shown in Supplemental Tables 1 and 2. To find the optimal treatment duration, we performed a preliminary study in 4-week-old young growing female rats by investigating the alteration of femoral trabecular bone mineral density BMD by using an in vivo micro-computed tomography Skyscan It showed that tuna bone calcium supplementation in 3 formulae for 4 and 9 weeks was able to completely restored bone loss to age-matched calcium-replete diet S1.

Therefore, we chose 9 weeks calcium supplementation for this study Supplemental Fig. Calcium supplementation from tuna bone, tuna bone with tuna head oil and tuna bone with 25 OH D 3 enhanced fractional calcium absorption.

A Experimental design, 4-week female rats were received calcium-replete diet 0. Thee-day calcium balance study was conducted in 3 time points, i. The differences between five experimental groups were determined by one-way ANOVA followed by Tukey post hoc test. The three tuna bone calcium supplement formulae were tuna bone calcium S2 , tuna bone with tuna head oil added 25 OH D 3 S3 and tuna bone with commercial 25 OH D 3 S4.

Tuna bone calcium supplement S2 diet was prepared by mixing 0. It showed that rats in groups S2 , S3 and S4 had similar body weight to those of L and S1 Fig.

The 6-week-old rats fed low calcium for 2 weeks during adolescence 4—6 week old were found to upregulate the fractional calcium absorption that remained significantly higher than in the age-matched S1 group when calcium insufficiency continued for another 4 or 9 weeks The week-old rats that received 4 weeks of calcium supplementation of S2 , S3 and S4 showed even higher fractional calcium absorption than those of L Fig.

However, fractional calcium absorption returned to the basal level after 9 weeks of calcium supplementation, i. Immunostaining of PMCA1, a calcium transporter expressed at basolateral membrane of enterocytes, which is responsible for pumping calcium out of the cell, showed increase in the protein expression in L compared with S1 , and the expression was compromised by calcium supplementation with or without 25 OH D 3 Fig.

As expected, urinary calcium excretion was significantly decreased in L Fig. While 4 weeks of calcium supplementation did not change urinary calcium excretion in L Fig. Serum level of 25 OH D 3 was consistently lower in L compared with the age-matched S1 Table 2.

Similarly, the week-old B group show a significantly lower level of 25 OH D 3 when compared with S1. However, 25 OH D 3 level of the week-old S2 was not different from that of S1.

Supplementation of tuna head oil added 25 OH D 3 in S3 significantly increased the serum level of 25 OH D 3 in both and week old rats when compared with the corresponding L groups. Animals fed low calcium diet with or without calcium supplementation had higher serum level of 1,25 OH 2 D 3 at both 10 and 15 weeks of age compared with age-matched S1.

Low calcium diet also resulted in hypocalcemia and hyperphosphatemia. Calcium supplementation from S2 , S3 and S4 restored the level of ionized calcium and inorganic phosphate to the level seen in S1 , but the total calcium levels of S2 and S4 were still lower than that of S1 Table 2.

Femoral length of L group was not different from calcium-replete diet S1 , suggesting that calcium insufficiency in this study did not impair bone linear growth.

Rats fed tuna bone calcium supplement from 3 formulae also had similar bone length to L and S1 Table 2. Bones from L had lower dry and ash weight indicating lower mineral content.

Bone weight after calcium supplementation was fully restored to that of S1 Table 2. Representative coronal images of the micro-computed tomography scanning of femur at the age of 10 weeks and 15 weeks, i.

At the age of 10 weeks, L exhibited a drastically reduced metaphyseal trabecular bone area and mid-shaft cortical shell, whereas calcium supplementation in S2 , S3 and S4 groups resulted in greater trabecular bone area and thickening of the mid-shaft cortical shell. At the age of 15 weeks, bones of every group were longer than those of the week-old animals and the effects of calcium supplementation were similar to those seen in the 10 week old animals Supp Fig.

Representative scanning images of the cross-sectional section of metaphyseal distal femur were shown in Fig. BMD and BMC of total tissue TOT; including trabecular and cortical compartments , trabecular and cortical compartments in L were drastically reduced when compared with S1.

As seen in Fig. A and Ct. Th were also lower than those of S1. The thinning of Ct. A in L apparently resulted from reduction in Ct.

Pm without any change in Ct. All 3 tuna bone calcium supplement formulae i. BMD, TOT. BMC, Tb. BMD, Tb. BMC, Ct. A, and Ct. Th with an exception of Ct. Pm and Ct. However, Tb. BMC of S2 , S3 and S4 were still significantly lower than S1 , while Ct. BMD of S3 and S4 was significantly higher than that of S1 Fig.

Figure 3 C depicted scanning images of the cross-sectional sections of femoral midshaft diaphysis. As expected, L had lower Ct. BMD, Ct. A, Ct. Pm, and calcium supplementation from S2 , S3 and S4 restored these parameters to those of S1 Fig. Even though the cortical shell of both femoral metaphysis and diaphysis was thicker after calcium supplementation, bone growth in width was compromised as Ct.

Pm were significantly lower than those of S1 Fig. N and Tb. Th, and increase in Tb. Tuna bone calcium supplementation from S2 , S3 and S4 completely restored all values to the levels of S1 Fig. All three formulae of calcium supplementation were able to lower the maximum displacement from the level seen in L , but only the reduction in S4 was significantly different from that of L.

Changes in strain showed a similar trend to that of maximum displacement Fig. Calcium supplementation from tuna bone, tuna bone with tuna head oil added 25 OH D 3 and tuna bone with 25 OH D 3 mitigated calcium insufficiency-induced osteoporosis.

Four-week female rats were received calcium-replete diet 0. A Representative images of three-dimensional reconstruction of metaphyseal distal femur cross-sectional view , Scale bars, 1 mm, B bone mineral density and content BMD and BMC, respectively of total bone TOT , trabecular Tb and cortical compartment Ct.

Cortical area Ct. A thickness Ct. Th , periosteal perimeter Ct. Pm and endosteal perimeter Ct. Pm , all parameters were analyzed by pQCT at distal metaphyseal femur, C representative images of three-dimensional reconstruction of mid-shaft diaphyseal femur cross-sectional view , Scale bars, 1 mm, and D cortical bone parameters analyzed by pQCT at mid-shaft femur.

Calcium supplementation from tuna bone, tuna bone with tuna head oil added 25 OH D 3 and tuna bone with 25 OH D 3 improved bone microstructure impairments and mechanical properties.

Experimental design was similar to Fig. The experimental design to evaluate the effect of impact exercise and calcium supplementation on calcium and bone metabolism was shown in Fig.

Animals that performed voluntary running exercise with calcium supplementation from tuna bone EB showed the same body weight gain as those of the exercise without supplementation group EL , and the sedentary with and without calcium supplementation groups SB and SL , respectively Fig.

EL and EB also demonstrated similar exercise performance as shown by cumulative running distance Fig. Similar to the experimental design in the first part, in this experiment, week and week old groups received calcium supplementation for 6 weeks and 12 weeks, respectively Fig.

Tuna bone supplemented B groups exhibited lower fractional calcium absorption compared to age-matched control groups whether they were sedentary or voluntary exercise group In the 12 week old group, exercise had no effect on fractional calcium absorption in calcium insufficient L group, whereas in B group that were fed adequate calcium diet, exercise significantly decreased calcium absorption As for the 18 week old groups, exercise had no effect on fractional calcium absorption whether with or without calcium supplementation Fig.

Calcium supplementation had a tendency to increase urinary calcium excretion in sedentary group, but the increase reached a statistical significance only in the exercise group 5. Calcium supplementation suppressed fractional calcium absorption and increased urinary calcium excretion in both sedentary and exercise groups.

A Experimental design, 4-week female rats were challenged with low calcium diet for 2 weeks, thereafter, rats were randomly divided into 2 sets, calcium repletion diet 0. Each set, rats were randomly sub-divided into 2 groups, voluntary running exercise rats were housed in cage-equipped with running wheel for 12 weeks or sedentary rats were housed in cage-equipped with running wheel but wheel was locked for 12 weeks , B body weight, C cumulative running distance, D relative fractional calcium absorption at rats on age of 12 weeks and 18 weeks, and E 3-day urinary calcium excretion.

SL , sedentary with low calcium diet, SB , sedentary with calcium supplementation, EL , exercise with low calcium diet, EB , exercise with calcium supplementation. Representative 3D images of CT scanning of distal femur longitudinal section was shown in Fig. Neither calcium supplementation from tuna bone nor running exercise had effect on bone length Table 3.

On the other hand, it was clearly shown that calcium supplementation with or without exercise increased bone weight and improved bone microstructure by alleviating all calcium insufficiency-associated osteoporotic features.

Interestingly, running exercise without calcium supplementation L significantly increased Tb. Th of the diaphyseal mid-shaft femur. Neither calcium supplement nor exercise had effect on Ct. BMD of metaphyseal region. The overall data from pQCT strongly suggested that calcium supplementation had greater benefits to bone than exercise.

Result from two-way ANOVA indicates that calcium supplement had effect on both metaphyseal and diaphyseal midshaft, while exercise had effect on bone predominately at the diaphyseal midshaft. Moreover, the effect of calcium supplementation and exercise had interaction on Ct.

BMD of diaphyseal midshaft. Data on blood chemical analysis showed that calcium supplementation in sedentary group, but not in the exercise group, significantly increased serum levels of total calcium and inorganic phosphate Table 3.

From the ultra-high resolution µCT analysis Fig. Sp in both sedentary and exercise groups. SB exhibited a higher Tb. Th than SL. Calcium supplementation resulted in a lower trabecular connectivity density Conn. D in sedentary 8. Calcium supplementation with exercise increased Conn.

D more than calcium supplement without exercise Neither calcium supplementation nor exercise had effect on the degree of anisotropy. From the mechanical property analysis, exercise was found to significantly increase these mechanical parameters only in calcium deficient L groups, but not in calcium supplemented B groups.

In contrast, tuna bone calcium supplementation, whether with or without exercise, significantly increased the maximum load and stiffness, and increased yield load in the sedentary group Fig.

Voluntary running exercise improved bone microstructure and mechanical property in calcium insufficiency-induced osteoporosis. A Representative images of three-dimensional reconstruction of metaphyseal distal femur longitudinal view , B representative images of three-dimensional reconstruction of metaphyseal distal femur upper panels and diaphyseal mid-shaft femur lower panels , Scale bars, 1 mm, C bone microstructure analyzed by ultra-high resolution micro-computed tomography at distal femur, D mechanical properties analyzed by 3-point bending apparatus at femoral mid-shaft.

Low dietary calcium intakes and poor 25 OH D 3 status are common findings in children living in developing countries. Low dietary calcium intakes are typically observed as a consequence of a diet deficient in dairy products and high in phytates and oxalates which reduce calcium bioavailability.

Childhood and adolescence are the critical period of bone development and mineralization, and are the period for maximizing genetically predetermined peak bone mass.

Peak bone mass and subsequent bone losses are important determinants of osteoporosis later in life. Thus, maximizing peak bone mass in early life, a period of relatively high plasticity of the skeleton in response to physical forces, is advocated as a way to protect against osteoporotic fractures later in life.

It was shown that daily calcium intake was associated with mineral acquisition in adolescents in a dose response manner Thus, inadequate calcium intake during this critical period could reduce bone accrual and impede bone growth, which, in turn, reduced bone size.

This study aimed to investigate whether calcium supplementation as well as impact exercise was able to ameliorate bone defects caused by inadequate calcium intake in young growing rats. According to the previous literatures, 4—6 weeks old in rats is considered young adult, which is a proxy of adolescence in human 30 , Creedon and Cashman 32 showed in young growing female rats fed low calcium diet between aged of 5—8 weeks that 5-week-old rats fed 0.

Therefore, 4-week old young growing female rats was used in this study and was challenged with 0. Herein, we demonstrated that the body could undergo significant adaptation to low calcium intake by reducing renal calcium excretion and increasing fractional intestinal calcium absorption through enhanced 1,25 OH 2 D 3 production.

Generally, bone mass and strength increased with age As expected, the week-old rats in the present study had higher bone mass at both cortical and trabecular regions compared to the 6-week old rats Table 1.

At the site of trabecular metaphysis, higher bone mass was detected by bone histomorphometry but not pQCT technique, which was probably due to the lower sensitivity of the pQCT technique. Two weeks of dietary calcium insufficiency led to a drastic reduction in trabecular BMC, cortical BMD and cortical BMC in both adolescent and young adult rats.

The reduction in trabecular BMC with no change in trabecular BMD in the adolescent 6-week-old rats was probably due to a parallel decrease in bone size and trabecular bone as shown by lower dry weight and ash weight Table 1.

The week old young adult rats that remained on low calcium diet for another 11 weeks showed clear impairment of bone growth as seen in all bone-related parameters except for bone length.

Considering the impact of 2 and 11 weeks of low calcium diet on 6- and week old rats, respectively, on trabecular bone, reduction in trabecular bone volume in 6-week old low calcium rats was due mostly to decrease in trabecular thickness.

On the other hand, the drastic reduction in bone volume in week old low calcium rats was due to a decrease in trabecular thickness as well as a huge reduction in trabecular number.

This study confirmed previous reports that inadequate calcium intake did not impair bone growth in length in young growing female rodents 34 , young adult male rodents 35 , and prepuberty girls However, it did impair the microscopic structure of bone that clearly compromise the mechanical properties and function.

Calcium supplements derived from natural sources are generally considered more beneficial than purified calcium carbonate from inorganic sources 37 , 38 , 39 , There are two important advantages for tuna bone as a calcium source.

First, calcium in tuna bone is a form of hydroxyapatite providing both calcium and phosphorus in an appropriate or optimal ratio , the nanocrystal of which is naturally formed in skeletal tissues. Second, certain nutrients in natural products present in bone have been shown to improve the calcium absorption.

For example, collagen in bone can enhance calcium absorption as shown in the in vivo 37 and in vitro experiments Calcium in the form of protein complex is also easily dissolved and released by protein digestion in the stomach rendering it ready for absorption in the small intestine.

In agreement with our previous study, fish bone calcium supplement exhibited greater bioavailability than calcium carbonate Unlike with CaCO 3 supplement, the higher fractional calcium absorption induced by tuna bone supplements did not lead to the extra absorbed calcium being excreted in the urine, but was retained to form new bone as shown by higher mineral apposition rate and bone formation marker Fig.

However, we failed to observe any difference in bone static microstructure parameters after 4-week supplementation, only dynamic change, i. Longer supplementation could have more pronounced effects on bone structure.

Herein, we showed that calcium supplementation from tuna bone did not suppress 1,25 OH 2 D 3 production, nor did it compromise intestinal calcium absorption.

It further enhanced the fractional calcium absorption and mitigated negative body calcium balance as shown by the restoration of serum ionized calcium and almost all bone-related parameters to the control levels Figs.

We found a modest additive effect of calcium supplementation together with 25 OH D 3 on cortical BMD of the distal femur as compared to the effect of calcium supplementation alone Fig. Although, 25 OH D 3 supplementation with tuna head oil increased the serum level of 25 OH D 3 , but the level of hormone 1,25 OH 2 D 3 did not change Table 2.

This was possibly due to fact that calcium supplementation alone was able to effectively correct hypocalcemia so that there was no stimulator to enhance conversion of 25 OH D 3 to 1,25 OH 2 D 3.

Therefore, we did not detect any change in serum 1,25 OH 2 D 3 level in the group receiving calcium supplement with 25 OH D 3 in S3 group. Interestingly, calcium supplementation with or without 25 OH D 3 restored trabecular and cortical BMD, cortical thickness and cortical area to the normal values, but did not restore trabecular BMC, or cortical periosteal and endosteal perimeters of distal femur and midshaft femur Fig.

The mechanical properties were also restored to normal level through compensatory increases in cortical thickness and area Figs. These findings indicate that transient inadequate calcium intake during childhood potently compromised bone accrual and bone growth in width, and these bone defects were long-lasting and could not be restored even with calcium supplementation during the period of adolescence to young adulthood.

Our results were in partial agreement with the previous study in rats which showed that low calcium intake through adolescence had a nonreversible, deleterious effect on peak bone mass, i. As mention earlier, dietary calcium intake was not directly associated with bone length, but rather controlled by a complex interaction of systemic and local factors, i.

An impairment of bone growth in width was highly significant because for bones of the same length, if one was twice as wide as the other, that bone would be eight times stronger Although calcium supplementation and the resulted increase in cortical thickness could restore bone strength to a normal level, its smaller diameter may make it more susceptible to fracture if exposed to risks brought on by inadequate calcium intake or menopause later in life.

It is well accepted that impact exercise or mechanical loading promoted osteogenesis, bone angiogenesis and increase in bone strength in both rodents and human 46 , 47 , 48 , 49 , Moreover, aerobic exercise was reported to upregulate serum 1,25 OH 2 D 3 and intestinal calcium absorption in young human and rodents 51 , 52 but was found to have no effect on calcium absorption in another human study In the present study, we found that 6 week running exercise in 6 week old rats led to a slight, but significant reduction in fractional calcium absorption when rats got to 12 weeks of age if they had received tuna bone calcium supplement, but not if they received low calcium diet.

However, this suppressive effect was not observed if running protocol continued for another 6 weeks Fig. Thus, the effect of aerobic exercise on intestinal calcium absorption varied with body calcium status and exercise duration.

We further demonstrated that voluntary running exercise increased both trabecular and cortical BMD, cortical thickness, and improved mechanical properties in low calcium intake condition, but not in adequate calcium condition Table 3 and Fig.

The benefit of exercise is usually region specific, i. In contrast, the effect of calcium supplementation is more generalized and not restricted to a particular region Herein, we used femur and tibia to evaluate the possibility of combined benefits of exercise and calcium supplementation Fig.

We found that the effect of running exercise was predominately seen on cortical-related parameters, which may be explained by the fact that exercise increased periosteal bone formation with no effect on endosteal bone formation Calcium supplement on the other hand, benefited both trabecular metaphysis and diaphysis.

We failed to see additive effect of exercise and calcium supplementation on bone-related parameters. It appeared that, tuna bone calcium supplementation alone was enough to alleviate the negative calcium balance in low calcium-fed group and maximized bone accrual in young adults.

Bass and co-workers in a randomized controlled trial in 7—year-old boys, found some additive effects of exercise and calcium supplement on BMC of femur, whereby calcium supplement alone or exercise alone showed only a tendency to increase femoral BMC.

Furthermore, this additive effect was region specific, i. Similar finding was also report in randomized controlled trial study in girls In partial agreement with the previous study in children 56 which reported an interaction between calcium supplementation and physical activity in cortical bone i.

It was unclear whether the exercise-induced bone benefits during adulthood were retained and could reduce fracture risk in old age. Studies in human found that soccer players had higher BMD that was maintained several years after retirement from the sport.

However, when they aged over 60 years, the fracture incidence in elderly former soccer players was not different from that of control Similar result was also found in young rat study On the contrary, other studies reported that exercise in youth was associated with a greater quality and strength in bones at older age 59 , Thus, long-term benefits of exercise in youth on the prevention of osteoporotic later in life is still debatable.

In conclusions, inadequate calcium intake during childhood and adolescence caused impairments of bone calcium accrual and appositional bone growth bone growth in diameter , but did not impair linear bone growth, and this impairment persisted into young adulthood in rats. Calcium supplementation from a natural source such as tuna bone had greater bioavailability than that of calcium carbonate.

Tuna bone calcium supplement with tuna head oil containing 25 OH D 3 and Omega-3 did not show any additive effect on fractional calcium absorption nor bone-related parameters compared with calcium supplement alone. In addition, both calcium supplementation from tuna bone and impact exercise i.

Although, the present study provides supportive evidence for future use of calcium supplements from natural sources and exercise as therapeutic approaches or intervention for ameliorating bone impairment caused by low calcium intake, the data were mainly from animal studies.

Therefore, these approaches need to be confirmed in young adolescent volunteers who consume relatively low calcium intake before being translated for clinical uses. Moreover, whether the beneficial outcomes of calcium supplementation together with exercise last until late adulthood or disappear after cessation of such intervention requires future investigations in both animals and human subjects.

Three-week old Sprague—Dawley female rats were purchased from Nomura Siam International Co. Ltd, Bangkok, Thailand. Animals were fed with low calcium diet 0. Low calcium diet CE-2; calcium presented as a CaCO 3 was purchased from CLEA, Japan. Calcium-replete diet or calcium supplement was made by adding an extra 0.

The extra calcium came from either calcium carbonate S1 or from tuna bone powder S2. As for low calcium diet L used in this experiment, the kibbles were produced without adding any extra calcium and served as the control group.

Food ingredients of L , S1 and S2 were analyzed based on AOAC method Supplemental Tables 1 and 2. Animal were fed ad libitum with free access to reverse osmosis RO water. The detail of experimental designs and animal groups were described in following sections.

The present research was approved by the Animal Care and Use Committee IACUC , Faculty of Science, Mahidol University. Animal Protocol number MUSC Central Animal Facility, Faculty of Science, Mahidol University has been international accredited by AAALAC.

All experiments and methods used in the present study were performed in accordance with relevant regulations, and the ARRIVE Animal Research: Reporting of In Vivo Experiments guideline.

To test whether inadequate dietary calcium intake in growing period caused impairment of bone growth in length and width, and whether bone impairment was more severe if calcium insufficiency lasted until young adulthood.

As for S1 group, rats fed with calcium-replete diet 0. As for L group, rats fed with low calcium diet 0. Femora and tibiae were cleaned of adhering tissues, wrapped in normal saline-soaked gauze and kept frozen at —20 °C until analysis. The extra 0. After rats had been fed calcium sufficient diet for 4 weeks, intestinal fractional calcium absorption and urinary calcium excretion were investigated.

Louis, MO on 6- and 1-day before termination to examine dynamic bone formation. Tibiae were used for bone microstructural analysis by bone histomorphometry. Please note that, animal used in Experiment 2 was different set of animals used in Experiment 1.

The objectives of this experiment were i to investigate the adaptive changes of intestinal calcium transport in response to low calcium diet with and without subsequent calcium supplementation, ii to test whether calcium supplementation was able to fully alleviate bone impairment caused by low calcium intake during young growing period, and iii to investigate possible additive effects of 25 OH D 3 supplement together with tuna calcium.

Eight 4-week female rats were fed with either 0. As for S3 , 25 OH D 3 was undetectable in refined tuna head oil because it was removed during the refining process. Therefore, we added 25 OH D 3 to the refined tuna head oil before giving to S3 rats. Rats were daily given soy bean oil with or without 25 OH D 3 or tuna head oil approximately 0.

Regarding to nutrition labeled, this small supplemental volume provided 2. Intestinal fractional calcium absorption and urinary calcium excretion were measured before calcium supplementation and after 4- and 9-weeks of calcium supplementation.

Body weight was recorded every 2 weeks. All rats were fed with designated diet for 11 weeks. Tibiae were subjected to bone microstructural analysis by bone histomorphometry.

Importantly, rats used in S1 and L groups here were the same set of animals used in Experiment 1, while animals in groups S2 , S3 and S4 were independent from previous Experiments please see Supplemental Table 3 for summarize of animal used.

Calcium replete diet used in Exp 4 was similar to diet in group S2 in Exp 3. All rats were housed in cage-equipped with running wheel circumference of cm and equipped with the electronic counter model , Lafayette Instrument Company, Lafayette, IN, USA.

For the exercise groups, rats had free access to running wheel, whereas the sedentary groups were housed in cages with locked running wheels. The voluntary running program was modified from the method of Lapmanee et al.

Briefly, on 1st week of exercise training, the running distance was monitored daily by reading from electronic counter display. Rats performed exercise and were given calcium sufficient diet from tuna bone for 12 weeks during which running distance was recorded every 2 weeks. Intestinal fractional calcium absorption and urinary calcium excretion were determined twice i.

was prepared as previously reported Briefly, after removing the remaining meat from tuna bone frames Katsuwonus pelamis , backbones were crushed into small pieces and placed in 0. Small pieces of bone were further incubated with protease enzymes for 60 min, after which the mixture was heated to 90—95 °C and maintained at this range of temperature for 60 min to inactivate the enzymes.

Thereafter, they were crushed in a fluidized bed opposed jet mills model AFG; Hosokawa Alpine Aktiengesellschaft, Augsburg, Germany. Tuna head oil was extracted with in-house protocol Thai Union Group, PCL.

Tuna head oil used in this study contained For plasma ionized calcium analysis, blood was drawn with a commercial sterile heparinized syringe model REF; BD, Diagnostics, Phymouth, UK. Plasma ionized calcium was determined by ion-selective electrode model Stat Profile CCX; Nova Biomedical, Waltham, MA under an anaerobic condition.

For serum collection, whole blood was allowed to clot at room temperature and was centrifuged model D; Kendro Laboratory Products, Hanau, Germany at × g for 10 min at 4 °C. Clotted blood samples were used to determine total serum calcium and serum inorganic phosphate by o -cresolphthalein complexone method model Dimension RxL analyzer, Dade Behring, Marburg, Germany.

Bone turnover markers i. ACF1, Immunodiagnostic Systems, AZ, USA , CTX-1 Cat ACF1, Immunodiagnostic Systems, AZ, USA , 25 OH D 3 Cat KRR, DIAsource, Belgium and 1,25 OH 2 D 3 Cat MBS, MyBiosource, USA.

Dehydrated tibiae were then embedded in methyl methacrylate resin in a posterior end down position, and incubated at 42 °C until the resin became fully polymerized. Undecalcified bone specimens were longitudinally cut into 7-µm-thick sections with a microtome equipped with tungsten carbide knife model RM; Leica, Nussloch, Germany.

Imaging analysis was performed under a light microscope using the computer-assisted Osteomeasure system Osteometric Inc. The region of interest was secondary spongiosa the trabecular part of proximal tibia at 2 mm distal to the epiphyseal plate , which was analyzed to obtain static parameters, i.

Th, µm , trabecular separation Tb. Sp, µm , trabecular number Tb. As for the dynamic parameters, undecalcified bone specimens were longitudinally cut into µm-thick sections, deplastinated, dehydrated and were covered by standard cover slip.

Bone mineral density BMD and mineral content BMC were analyzed at femoral distal metaphyseal region and diaphyseal mid-shaft. Cortical microstructure i. A , cortical periosteal perimeter Ct. Pm , and endosteal perimeter Ct. Bones were scanned with 50 kV X-ray tube, current of 0. As for the metaphyseal region, measured parameters were averaged from three points of volume of interests VOI that were at 2, 2.

Three-dimensional trabecular metaphyseal microstructure was analyzed by ultra-high resolution micro-computed tomography model UHR U-CT, Milabs, Utrecht, Netherlands. Th, mm , and trabecular separation Tb. Sp, mm analyzed by Imalytics Preclinical software version Femora were tested by 3-point bending technique model ; Instron, Norwood, MA, USA.

Femoral length was recorded before the mechanical test. Tests were conducted on the mid-diaphysis of the femora, which was placed on two supports 16 mm apart and with the femoral anterior margins facing downward toward the actuator.

All mechanical parameters of each femur were automatically generated by Instron BlueHill Software version 3. Rats were housed in individual metabolic cage Techniplast, Venice, Italy for 3 days to collect fecal pellets and urine.

Fecal pellets were dried in an oven at 80 °C for 3 days and then ashed at °C overnight in a muffle furnace model ; Thermolyne, Dubuque, IA. Fecal dry and ash weight were recorded. Fecal ash was dissolved in acid solution and underwent microwave digestion at °C, W for 35 min Microwave acid digestion system, Ethos Up, Milestone Inc, CT.

Feces and urine calcium contents were determined by flame atomic absorption spectrometry PerkinElmer, MA, USA. where, calcium intake is amount of calcium intake from diet over 3-day balance study, fecal calcium excretion is the amount of calcium presented in feces over 3-day balance study.

Duodenal segments were fixed overnight in 0. After being embedded in paraffin, the specimens were cut longitudinally into 4-µm sections which were later incubated at 95 °C for 20 min in sodium citrate buffer solution pH 6. After being washed with 0.

Sections were mounted with slow fade Diamond antifade mounting medium containing DAPI Cat S, Invitrogen visualized under a fluorescent microscope model eclipse Ni-U; Nikon.

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