J Nutr Sci Vitaminol, 50, 177-183, 2004

Japanese Citrus Fruit (Sudachi) Juice Is Associated with Increased Bioavailability of Calcium from Whole Small Fish and Suppressed Bone Resorption in Rats

Yoshitaka NIT1, Kazuhiro FUKUTA1, Kentaro SAKAI2 and Shigeru YAMAMOTO3

1Food TechnologyDivision , TokushimaPrefectural Industrial TechnologyCenter, Tokushima770-8021, Japan 2Department of Nutrition and Health Promotion, Faculty of Human Life Science,Hiroshima Jogakuin Univer sity, Hiroshima 732-0063, Japan 3Department of Nutrition, School of Medicine,The University of Tokushima, Tokushima770-8503, Japan (Received October 11, 2003)

Summary Shirasuboshi (boiled and semi-dried whitebait) is a processed fish food that con tains abundant calcium. It is eaten whole and commonly consumed in Japan. In this study, the effect of sudachi (Citrus sudachi) juice on calcium, magnesium and phosphorus bioavail ability, and bone metabolism in rats was examined. After 14 d of diets low in calcium and phosphorus, male Sprague-Dawley rats were fed shirasuboshi diets containing dried shira suboshi powder treated with 20% (S20) or 40% (S40) sudachi juice, or distilled water (C) (0.5% Ca; 0.3% P) for 14 d. The apparent absorptions and retentions of calcium, magne sium and phosphorus from shirasuboshi were determined. Bone formation was calculated by measuring serum osteocalcin, and bone resorption by measuring urinary pyridinoline and deoxypyridinoline. The apparent absorption and retention of calcium and magnesium in the S20 group were significantly higher than in the C and S40 groups. Although serum osteo calcin was not affected by the addition of sudachi juice, the urinary pyridinoline and deoxy pyridinoline concentrations in the S40 group were significantly lower than in the C and S20 groups. Our results indicate that sudachi juice added to shirasuboshi was associated with increased calcium bioavailability and suppressed bone resorption in rats. Key Words calcium-absorption, citrus fruit, fish, bone resorption, rats

The importance of adequate calcium intake for the solubilizing dietary calcium. development and maintenance of peak bone mass is Sudachi (Citrus sudachi) is a major citrus fruit grown well established (1). Insufficient calcium intake increas in Tokushima Prefecture, which is located on Shikoku es bone resorption (1, 2), decreases bone mass (3) and Island in Japan. There is a dietary habit of squeezing increases the risk of osteoporosis after menopause (4). sudachi juice on shirasuboshi in this area. The major A sufficient calcium intake is therefore required to pre organic acid component of sudachi juice is citric acid. vent bone loss. For these reasons, promoting diets high Previously, we found that sudachi juice solubilizes cal in calcium and exploring new ways of increasing the cium from shirasuboshi in vitro (11). Furthermore, we bioavailability of dietary calcium are very important. observed solubilization of calcium by sudachi juice fol Shirasuboshi (boiled and semi-dried whitebait) is a lowing in vitro peptic and peptic-pancreatic digestions processed fish food that is eaten whole and commonly of shirasuboshi (11). These results suggest that sudachi consumed in Japan. Although whole small fish with juice treatment may enhance the bioavailability of cal bones are potentially a good source of dietary calcium, cium from shirasuboshi. The present study was therefore bone calcium is unavailable for absorption due to its designed to examine the effect of sudachi juice on cal incorporation in insoluble hydroxyapatite crystals (5). cium, magnesium and phosphorus bioavailability, and There is very little information on the availability of cal bone metabolism in young male rats fed shirasuboshi cium from the bones of small fish (5, 6), and methods to diets. efficiently increase calcium availability from small fish MATERIALS AND METHODS have not been fully examined. It is thought that calcium must be ionized in order to Preparation of dried shirasuboshi powder. Shirasuboshi be absorbed in the intestine (7). Several reports have were purchased from Yoshimi Kaisan Co., Ltd. (Komat dealt with the significant effects of citric acid and citric sushima, Tokushima, Japan). The content of water in acid salts on the bioavailability of calcium (8, 9). the shirasuboshi was 38.7g/100g. Sudachi juice (pH Mehansho et al. (10) demonstrated that CCM (a combi 2.2) was purchased from Tokushima City Agricultural nation of calcium carbonate, citric acid and malic acid) Cooperatives (Tokushima, Japan). The concentrations of in orange juice enhances the bioavailability of calcium. citric acid and malic acid in the sudachi juice were These results suggest that citric acid is very effective in 60mg/mL and 3.5mg/mL, respectively. To 100g of

177 178 Nu Y et al.

Table 1. Composition of experimental diets.

1C , the diet containing dried shirasuboshi powder treated with distilled water; S20 , the diet containing dried shirasuboshi powder treated with 20% sudachi juice; S40, the diet containing dried shirasuboshi powder treated with 40% sudachi juice .2 Containing (g/100g): protein, 63.6; fat, 5.2; carbohydrate, 0.2; (mg/100g): calcium , 750; magnesium, 190; phospho rus, 950. 3Prepared according to AIN -93G formulation except for calcium and phosphorus .

fresh shirasuboshi, 20 or 40mL of sudachi juice was with the Guideline for the Care and Use of Laboratory added to make the experimental diets. Distilled water Animals at the University of Tokushima. was added to an equivalent amount of sudachi juice to Metabolism studies. During the periods of 11-14 d make the control diet. The shirasuboshi treated with and 25-28 d, the rats were placed in metabolic cages sudachi juice or distilled water was then dried for 12h at (Try Tec Co., Ltd., Osaka, Japan). The food intake was 80•Ž. Dried shirasuboshi were ground to a powder using measured, and feces and urine were collected separately a food cutter (DLC-XG; Conair Corp., Stamford, CN, USA) . for each period for the determination of apparent Animals and diets. Male Sprague-Dawley rats, 3 wk absorption and retention. The 0.1% carmine was added of age (Japan SLC Co., Ltd., Hamamatsu, Shizuoka, to each diet as a marker, and the feces were collected Japan) were preliminarily maintained on a stock diet until the marker was all excreted. Feces collected were (MF; Oriental Yeast Co., Ltd., Tokyo, Japan) for 7 d, and dried for 24h at 105•Ž, weighed, and ground with a the rats were then fed ad libitum a low-calcium blender. Contaminants, including hairs, were removed

(0.01%), low-phosphorus (0.15%) diet for 14 d (Table by filtering through an 80-mesh stainless steel sieve . 1). The rats were then divided into three groups of six Urine was collected in a glass flask containing 1 N rats each. The three groups were fed ad libitum the hydrochloric acid, and filtered with No. 7 filter paper shirasuboshi diets containing dried shirasuboshi powder (Toyo Roshi Co., Ltd., Tokyo, Japan). treated with 20% (S20) or 40% (S40) sudachi juice, or Analytical methods. At the end of the study period , distilled water (C) (0.5% Ca; 0.3% P) for 14 d (Table 1) . the rats were anesthetized by ethyl ether and killed, and The diets were based on the AIN-93 G formulation (12) . blood samples and femurs were collected. Serum was The dried shirasuboshi powder was the main source of aliquoted after centrifugation (1, 500•~g) for 15min at protein, calcium and phosphorus in each diet, and the 4•Ž and stored at -40•Ž until analysis. Feces, right total level of fat was adjusted to 7% (w/w) with soybean femurs and food samples were dried, and then ashed for oil. The contents of energy, protein, fat, fiber, calcium 24h at 550•Ž in a muffle furnace (FO300; Yamato Sci and phosphorus were constant in each diet. The rats entific Co., Ltd., Tokyo, Japan). The temperature was were housed individually in stainless steel cages at gradually raised until 550•Ž. The ashed samples were room temperature (24•Ž) and 55% humidity with a dissolved in 1% hydrochloric acid. 12h light-dark cycle. All rats were given distilled water Calcium and magnesium concentrations were deter ad libitum. The animals were maintained in accordance mined by atomic absorption spectrophotometry (Z Effect of Citrus Fruit on Calcium Absorption 179

Table 2. Body weight gain, food intake and food efficiency in rats fed low Ca and P diet or dried shirasuboshi diet.

Values are means•}SD, n=18 in (A) and n=6 per group in (B).

8100; Hitachi, Ltd., Tokyo, Japan) with strontium add Table 3. The apparent absorption and retention of cal ed to the sample at a final concentration of 3,000mg/L. cium, magnesium and phosphorus in rats fed low Ca Phosphorus was determined by a colorimetric method and P diet. using ammonium molybdenate (13). The apparent ab sorption and retention of calcium, magnesium and phosphorus were calculated by the following formulae: Apparent absorption (%)=[(intake-fecal excretion)/

(intake)]•~100, and apparent retention (%)=[(intake -fecal excretion-urinary excretion)/(intake)]•~100 . Values are means•}SD, n=18. Citric acid and malic acid in sudachi juice were mea sured by high-performance liquid chromatography

(Organic Acids Analytic System; Japan Spectroscopic dex (SSI), which is an indicator of whole bone strength Co., Ltd., Tokyo, Japan) (14). (16-18). Biochemical analysis. The concentrations of cal In vitro solubilization of calcium from dried shirasuboshi

cium, magnesium and inorganic phosphorus in serum powder. To 20mL of Ultra Pure Water (Milli-Q SO; Mil were measured by a colorimetric method using assay lipore Corp., Tokyo, Japan), 1g of dried shirasuboshi

kits for each mineral (Calcium C, Magnesium B and powder was added and incubated on a shaker for 1h at Phosphor C; wako Pure Chemical Industries, Ltd.,. 37•Ž. Samples were centrifuged (18,000•~g) for 10 min

Osaka, Japan). at 4•Ž and the amount of calcium in the supernatant The concentrations of calcitonin and osteocalcin in was determined by atomic absorption spectrophotome

serum were measured by enzyme immunoassay (Penin try (Z-8100; Hitachi, Ltd.). The amount of solubilized sula Laboratories, Inc., San Carlos, CA, USA and Bio calcium was expressed as a percentage of the total cal medical Technologies Inc., Stoughton, MA, USA). Uri cium in the dried shirasuboshi powder.

nary pyridinoline and deoxypyridinoline were mea Statistical analysis. All data are presented as mean sured according to the method of Fujimoto et al. (15). values•}standard deviation. Statistical analyses were The urine samples were hydrolyzed in 6mol/L sodium performed using StatView 5.0 (SAS Institute, Cary, NC, chloride for 18h at 107•Ž in a heat block (DTU-2B; USA). Fisher's protected least significant difference TAITEC Co., Koshigaya, Saitama, Japan). (PLSD) test was done after one-way ANOVA to examine Assessment of bone length, bone mass and bone mineral the effect of diet, and was considered statistically signif densities. The lengths of the right femurs from the icant at p<0.05.

proximal neck to the distal condylar surfaces were mea RESULTS sured with a digital caliper. The right femurs were dried for 24h at 105•Ž and asked at 550•Ž in a muffle fur Body weight gain, food intake and food efficiency nace (FO300; Yamato Scientific Co., Ltd.) and the dry There were no significant differences in body weight weights and ash weights were determined. gain, food intake or food efficiency between rats fed the The left femoral diaphysis was scanned by peripheral low-calcium, low-phosphorus diet or the shirasuboshi

quantitative computed tomography (pQCT) (XCT Re diets (Table 2). search SA+; Stratec Medizintechnik GmbH, Pforzhein, Apparent absorption and retention of calcium, magnesium Germany) with 120•~120•~460ƒÊm voxel size, and to and phosphorus tal and cortical bone mineral contents (BMC) and den The apparent absorptions and retentions of calcium, sities (BMD) were analyzed as well as strength strain in magnesium and phosphorus in the rats fed the low-cal 180 NII Y et al.

cium, low-phosphorus diet are shown in Table 3. Apparent absorptions and retentions of calcium, magnesium and phosphorus in the rats fed the shira suboshi diets are shown in Figs. 1 and 2. The apparent absorption of calcium in the S20 group was signifi cantly higher than in the C and S40 groups, and the apparent retention of calcium in the S20 group was sig nificantly higher than in the C group. The apparent absorption and retention of magnesium in the S20

Fig. 1. Effects of sudachi juice on the apparent absorp group were significantly higher than in the C and S40

tion of calcium (A), magnesium (B) and phosphorus groups. Serum concentrations of calcium, magnesium and phospho (C) in rats fed the shirasuboshi diets. Values are means•}SD, n=6 per group. Values were considered rus significantly different at * p<0.05 or ** p<0.01. There were no significant differences in serum cal cium or inorganic phosphorus concentrations among the dietary groups (Table 4). However, the serum mag nesium concentration in the S40 group was signifi cantly lower than the S20 group. Biochemicalmarkers for bone remodeling The serum calcitonin concentration in the S40 group was significantly higher than in the C group (Table 5). The serum osteocalcin, which is a marker for bone for mation, was not affected by the addition of sudachi juice to the diet. In addition, the urinary pyrydinoline and deoxypyridinoline, which are markers for bone resorp tion, were significantly lower in the S40 group than in Fig. 2. Effects of sudachi juice on the apparent retention

of calcium (A), magnesium (B) and phosphorus (C) in the C and S20 groups.

rats fed the shirasuboshi diets. Values are means•}SD, Bone and ash weight, and length and mineral contents of the

n=6 per group. Values were considered significantly right femur different at * p<0.05 or **p<0.01 Bone and ash weights, lengths, and mineral contents of the right femur were not significantly different among the dietary groups (Table 6). Table 4. Effects of sudachi juice on serum biochemical pQCT measurements of the left femur indices in rats fed on dried shirasuboshi diets. Neither total nor cortical BMC and BMD, nor stress strain index of the left femur were statistically different among the dietary groups (Table 7). The effect of sudachi juice on the solubilization of calcium from dried shirasuboshi powder The amount of solubilized calcium from the dried shirasuboshi powder was measured in vitro. The amount of solubilized calcium was significantly higher in the dried shirasuboshi powder treated with 20% or 40% Values are means•}SD, n=6 per group. Values in a row sudachi juice compared with distilled water, and signifi with different superscripts are significantly different at cantly more calcium was solubulized by 40% sudachi p<0.05. juice compared to 20% (Fig. 3).

Table 5. Effects of sudachi juice on biochemical markers for bone remodeling.

Values are means•}SD, n=6. Values in a row with different superscripts are significantly different at p<0.05.

. Effect of Citrus Fruit on Calcium Absorption 181

Table 6. Right femoral measurements of bone weight, length and mineral contents.

Values are means•}SD, n=6 per group.

Table 7. Left femoral measurements with peripheral quantitative computed tomography (pQCT).

Values are means•}SD, n=6 per group. * BMC , bone mineral contents; BMD, bone mineral densities; SSI, strength strain index.

magnesium and phosphorus bioavailability from exper imental diets for 14 d, after rats were fed a calcium and phosphorus-restricted diet for 14 d. Experimentally, low calcium feeding has been accepted as a method of increasing bone resorption (19). We observed significantly higher apparent absorption of calcium and magnesium in the S20 group than in either the C or the S40 groups. Calcium absorption is influenced by the type of calcium salt and by other food components. Nicar and Pak (20) suggest that the high solubility of calcium citrate is responsible for an increased absorption of calcium. When an appropriate

Fig. 3. Effect of sudachi juice on in vitro solubilization of mixture of calcium hydroxide and citric acid are sus calcium from dried shirasuboshi powder treated with pended in water, a metastably supersaturated solution 20% or 40% sudachi juice, or distilled water. Values are of calcium citrate is formed, keeping calcium in a solu means•}SD, n=6 per group. Values were considered ble form at a much higher concentration than is possi significantly different at *** p<0.001. ble from a solid preparation of tricalcium dicitrate (21). In the intestine, calcium citrate or a mixture of calcium citrate and calcium malate has been shown to enhance calcium absorption, compared to calcium carbonate DISCUSSION (8). Moreover, citrate may increase calcium bioavail It is apparent that when calcium intake is adequate, ability because soluble calcium-citrate complexes are differences in bioavailability, as from increased solubili formed instead of insoluble calcium salts such as car zation, play no or only a minor role in the amount of bonate or phosphate. In several studies examining the calcium that is absorbed or deposited in the skeleton effect of fruit juice on calcium absorption, Newell and (7). We therefore performed the comparison between Miller (22) reported the beneficial influence of orange each diet under the condition for raising the calcium juice on children's growth, and Mehansho et al. (10) requirement. In this study, we examined the calcium, indicated that orange juice enhanced the bioavailability 182 NII Y et al.

of calcium and iron. resorption. We postulate that this is due to the presence Although 40% sudachi juice results in much greater of organic acids such as citric acid in sudachi juice, but calcium solubilization than water or 20% sudachi juice, further studies are required to determine if other com this was not reflected in the apparent calcium absorp ponents are involved. tion or other indices. For this reason, we think that the In the 520 group calcium absorption was signifi calcium absorption observed in this study cannot be cantly enhanced, yet bone resorption was not sup explained by calcium solubilization alone. Although the pressed. On the other hand, in the S40 group, bone absorption of soluble organic calcium complexes is con resorption was significantly suppressed, but calcium troversial, it is unlikely that the soluble calcium-citrate absorption was not enhanced. The component of suda complexes formed by addition of sudachi juice at a level chi juice, especially the quantity of citric acid, greatly of 40% would have a negative effect on absorption. differs in the diets with S20 and S40 groups. In the diet There may be other unidentified factors related to suda with S40 group, the citric acid from dried shirasuboshi chi juice that explain the poor calcium absorption in the powder treated with sudachi juice is included further S40 group compared to the S20 grop. than the diet with S20 group. Lacour et al. (8) reported There may also exist an optimal ratio of citric acid to that the urinary excretion of the calcium is increased in solubilized calcium for maximum calcium absorption. the presence of high amounts of citric acid. However, it Pak and colleagues found the most efficient calcium may be a condition that bone metabolism in the S40 absorption was obtained with a calcium to citrate molar group was balanced according to bone resorption effect ratio of 1.25, based on a range of molar ratios from by sudachi juice in this study. Although the apparent 0.67 to 1.5 (21). Our data show that a ratio of 20% absorption and retention of calcium were significantly sudachi juice to shirasuboshi in the diet increased appar increased by adding sudachi juice to shirasuboshi, bone ent absorption and retention the most. mineral content and bone mineral density in rats were Intestinal calcium absorption involves two processes: not influenced. This may be due to the short duration of transcellular, metabolically driven transport and a the experiment. If the experimental period were to be paracellular, passive process (23). Favus and Pak (24) prolonged, enhanced bone metabolism due to the demonstrated that soluble calcium complexes are intake of sudachi-treated shirasuboshi might become absorbed in the, duodenum by a passive paracellular apparent. process, and ionic calcium is easily absorbed in the Muhlbauer and Li (30) reported that several com small intestine and the colon. For most minerals includ mon vegetables in the human diet alter the bone metab ing calcium, the paracellular pathway appears to be the olism in rats. It is therefore plausible that citrus fruits predominant route of entry from lumen to blood. The such as sudachi may affect bone metabolism as well. We amount of mineral absorbed by the paracellular route is have shown that the intake of sudachi-treated shira determined by how much is solubilized in the intestinal suboshi is associated with suppressed bone resorption in lumen (23). rats. A similar effect may be expected from other citrus The results of our experiment suggest that the solubi fruit juices, if this effect is due to the citric acid in suda lization of magnesium and phosphorus is increased by chi juice. If such an effect also happens in humans, add 20% sudachi juice. In rats, the calcium and phosphorus ing sudachi juice to shirasuboshi meals could be an content of the diet influences magnesium homeostasis effective way to decrease the risk of osteoporosis. These (25). Brink et al. (26) speculated that magnesium solu results suggest that a dietary habit of squeezing sudachi bility was a determinant of magnesium absorption. juice on shirasuboshi affects the mineral absorption from Magnesium is extremely important in skeletal metabo whole small fish as the traditional Japanese diet style. lism and there is a growing appreciation that magne sium deficiency may be a cause of osteoporosis (27). Acknowledgments Calcitonin lowers serum calcium by decreasing bone The authors thank Ms. Kaori Yamamoto (ELKCorpo resorption in osteoclastic cells, and it is therefore a ration, Tokyo, Japan) for her help with pQCT analysis, potent inhibitor of osteoclastic resorption. Serum calci Ms. Kimiko Nii for her technical assistance, and Dr. tonin was significantly increased in rats in the S40 Jonathan Siekmann for help with the manuscript. This group, suggesting that bone resorption was suppressed study was supported by a grant from the Regional Sci in osteoclasts. Biochemical markers of bone metabolism ence Promoter Program of the Japan Science and Tech have also been used to determine qualitative changes in nology Corporation. bone turnover (28). We measured serum osteocalcin, which is released from osteoblasts as the biochemical REFERENCES marker of bone formation. We also measured urinary 1) Creedon A, Cashman KD. 2001. 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