Effect of Citrus Fruit (Sudachi) Juice on Absorption of Calcium

Home , Calcium citrate, Citric acid, Sudachi

Food Sci. Technol. Res., +, (+), ,1ῌ-*, ,**0

E#ect of Citrus Fruit (Sudachi) Juice on Absorption of Calcium from Whole Small Fish in Healthy Young Men

+ῌ , , + - . Yoshitaka NII , Takamasa OSAWA , Daisuke KUNII , Kazuhiro FUKUTA , Kentaro SAKAI , Maki KONDO and , Shigeru YAMAMOTO

+ Food Technology Division, Tokushima Prefectural Industrial Technology Center, ++ῌ, Nishibari, Saika-cho, Tokushima 11*ῌ2*,+, Japan , Department of International Public Health Nutrition, Institute of Health Biosciences, The University of Tokushima Graduate School, -ῌ+2ῌ+/ Kuramoto-cho, Tokushima 11*ῌ2/*-, Japan - Department of Nutrition and Health Promotion, Faculty of Human Life Science, Hiroshima Jogakuin University, .ῌ+-ῌ+ Ushita- Higashi, Higashi-ku, Hiroshima 1-,ῌ**0-, Japan . Faculty of Human Life Science, Shikoku University, +,-ῌ+ Ebisuno, Furukawa, Ojin-cho, Tokushima 11+ῌ++3,, Japan

Received July +, ,**/; Accepted January ,/, ,**0

Shirasuboshi (boiled and semi-dried whitebait) is a processed seafood that is abundant in calcium. It is eaten whole and commonly consumed in Japan. In this study, we examined the e#ect of Sudachi (Citrus sudachi Hort. ex. Shirai) juice on calcium, magnesium and phosphorus bioavailability in healthy young men. Dried shirasuboshi powder treated with distilled water (C) or sudachi juice (S,*) was prepared for use in two experimental diets, the control diet and the sudachi diet. Either S,* or C was added to a basal diet with a low calcium content (+2* mg/d). The basal diet and the two experimental diets were each consumed for 0 d by six healthy young men according to a randomized and crossover design. The apparent absorption and retention of calcium, magnesium and phosphorus from shirasuboshi were determined and were found to be signiﬁcantly higher in the sudachi diet than in the control diet. The apparent absorption and retention of calcium from the basal diet were found to be in negative balance. Our results indicate that shirasuboshi added to sudachi juice was associated with increased mineral bioavailability in healthy young men.

Keywords: ﬁsh, citrus fruit, calcium absorption, bone resorption, healthy young men

Introduction from shirasuboshi in vitro (Nii et al., ,**+). Furthermore, The importance of adequate calcium intake for the we examined the e#ect of sudachi juice on calcium, mag- development and maintenance of peak bone mass is well nesium and phosphorus bioavailability and bone metabo- established (Weaver and Liebman, ,**,). Insu$cient cal- lism in young male rats fed shirasuboshi diets (Nii et al., cium intake increases bone resorption, decreases bone ,**.). The present study was designed to examine the mass (Ensrud et al., ,***, Weaver and Liebman, ,**,)and e#ect of sudachi juice on calcium, magnesium and phos- increases the risk of osteoporosis after menopause phorus absorption in healthy young men who were pro- (Dawson-Hughes et al., +33*). A su$cient calcium intake vided with shirasuboshi diets in addition to a basal diet is therefore required to prevent bone loss. with a low calcium content. Although shirasuboshi (boiled and semi-dried whitebait) is a processed seafood that is eaten whole and com- Materials and Methods monly consumed in Japan, there is very little information Subjects Six healthy men who were students of on the availability of calcium from the bones of small ﬁsh Shikoku University volunteered to participate in this in humans (Hansen et al., +332, Uenishi et al., +332, Larsen study. The subjects were aged ,*./ῌ*./ y, with heights et al., ,***). of +1,.+ῌ1.. cm and weights of 01.,ῌ2.+ kg; their body , Sudachi (Citrus sudachi Hort. ex. Shirai) is a major citrus mass index values (kg/m ) were ,,.1ῌ,.,. They were fruit grown in Tokushima Prefecture, which is located on fully informed of the aims and purposes of the study. Shikoku Island in Japan. In this area there is a dietary The study complied with the code of ethics of the World habit of squeezing sudachi juice on shirasuboshi. We have Medical Association (Helsinki Declaration of +30. and previously found that sudachi juice solubilizes calcium revised in +323). Preparation of dried shirasuboshi powder Shirasuboshi ῌ To whom correspondence should be addressed. with a water content of -2.1 g/+** g was purchased from E-mail: [email protected] Yoshimi Kaisan Co., Ltd. (Komatsushima, Tokushima). 28 Y. NII et al.

Sudachi juice (pH ,.,, acidity 0..ῌ and Brix 1.2ῌ)was Table +. Energy and nutrient content in the basal, purchased from Tokushima City Agricultural Coopera- control and sudachi diets. tives (Tokushima). Citric acid and malic acid concentrations in the sudachi juice were 0* mg/mL and -./ mg/mL, respectively. To + kg of fresh shirasuboshi, ,** mL of sudachi juice was added to prepare the sudachi diet. Distilled water equivalent to the amount of sudachi juice was added to + kg of shirasuboshi to make the control diet. The shirasuboshi treated with sudachi juice or distilled water was immediately dried for +, hat2*. Dried shirasuboshi was ground to a powder using a food chop- per (DLC-XG; Conair Corp., Stamford, Conn., USA). Experimental design A basal diet with a low calcium were calculated using a computer program based on the content (+2* mg/d), mainly composed of lunch box food Standard Tables of Food Composition in Japan (Resources typically found on the market, was prepared and provid- Council, Science and Technology Agency, Japan, ,***) ed to all subjects (basal diet). The data of the basal diet and nutrition facts for each processed food. Citric acid was handled as a reference. The control diet was formu- and malic acid in sudachi juice were measured by high- lated by adding dried shirasuboshi powder treated with performance liquid chromatography (Organic Acid Anal- distilled water to the basal diet, and the sudachi diet was ysis System; Japan Spectroscopic Co., Ltd., Tokyo) formulated by adding dried shirasuboshi powder treated (Nakajima et al., +310). with sudachi juice to the basal diet. Each dosage of dried Statistical analysis All data are presented as mean shirasuboshi powder was formulated so that it included valuesΐstandard deviation. Statistical analyses were .** mg/d of calcium uniformly. Subjects were given performed using StatView /.* (SAS Institute, Cary, N.C., each of the three diets for 0 d according to a randomized USA). Fisher’s protected least significant di#erence test and crossover design. A su$cient interval (over + wk) was done after one-way analysis of variance to examine was set to eliminate interference in each diet. Consump- the di#erence in the control and sudachi diets and was tion of fruit juice and fruit-containing food products was considered statistically significant at p *.*/. prohibited, but water or beverages were allowed ad libitum during the study. Results Feces and urine were collected separately during the The energy, protein and sodium contents of the basal, last - d of each treatment period to determine apparent control and sudachi diets are shown in Table +. The absorption and retention of the minerals studied. Car- apparent absorption and retention of calcium (Table ,), mine was used as a marker, and feces were collected until magnesium (Table -) and phosphorus (Table .) from the complete excretion of the marker. Feces were weighed sudachi diet were significantly higher than from the con- and ground with a blender. trol diet. The apparent absorption and retention of calci- Analytical methods Feces and food samples were um from the basal diet were found to be in negative dried, and then ashed for ,. hat//* in a mu%e furnace balance. (FO-**; Yamato Scientific Co., Ltd., Tokyo) in which the temperature was gradually raised to //*. The ashed Discussion samples were dissolved in +ῌ hydrochloric acid. Urine Intestinal calcium absorption involves two processes: was directly analyzed with dilution in +ῌ hydrochloric transcellular, which is metabolically driven transport, acid. and a paracellular, or passive, process (Bronner, +332). Calcium and magnesium concentrations were deter- Active transport is important when calcium intake is low, mined by atomic absorption spectrophotometry (Z-2+**; and passive transport is predominant when calcium Hitachi, Ltd., Tokyo) with strontium added to the sample intake is adequate or high (Bronner and Pansu, +333). at a final concentration of -,*** mg/L. Phosphorus con- For most minerals, including calcium, magnesium and tent was determined by a colorimetric method using am- phosphate, the paracellular pathway appears to be the monium molybdenate (Fiske and Subbarow, +3,/). The predominant route of entry from the lumen to blood. apparent absorption and retention of calcium, magnesium Factors that determine paracellular mineral movement and phosphorus were calculated using the following and play a role in mineral absorption are solubility, intes- formulae: tinal permeability and sojourn time. The amount of mineral absorbed by the paracellular route is determined by Apparent absorption῎ῌ῏῕ῐ῎intakeῒfecal excretion῏ how much is solubilized in the intestinal lumen (Bronner, ῌ῎intake῏ῑ῔+** ῍Eqῌ (+) +332). Accordingly, the taking of highly soluble calcium into the digestive tract seems to be a nutritionally e#ec- Apparent retention῎ῌ῏῕ῐ῎intakeῒfecal excretion tive measure. ῒurinary excretion῏ It has been reported that calcium from casein-phospho- ῌ῎intake῏ῑ῔+** ῍Eqῌ (,) peptides or a combination of calcium carbonate, citric Energy, protein, fat and sodium contents in the diets acid and malic acid with high solubility increases calcium E#ect of Citrus Fruit on Ca Absorption 29

Table ,. E#ect of sudachi juice on the apparent absorption and retention of calcium from the basal, control and sudachi diets in healthy young men.

Table -. E#ect of sudachi juice on the apparent absorption and retention of magnesium from the basal, control and sudachi diets in healthy young men.

Table .. E#ect of sudachi juice on the apparent absorption and retention of phosphorus from the basal, control and sudachi diets in healthy young men.

bioavailability in humans (Smith et al., +321, Miller et al., the absorption of soluble organic calcium complexes is +322, Dawson-Hughes et al., +33*, Heaney et al., +33.). controversial, we suggest that the solubilization of calci- Among several studies examining the e#ect of fruit juice um from shirasuboshi was increased by the addition of on calcium absorption (Mehansho et al., +323, Kohls, +33+, sudachi juice. Our results also indicated that the appar- Andon et al., +330), Mehansho et al. (+323) indicated that ent absorption of magnesium and phosphorus was in- citric acid, the major component of orange juice, en- creased by the sudachi diet. As magnesium and phos- hanced the bioavailability of calcium and iron. Moreo- phorus are absorbed by passive transport (Bronner, +332), ver, citrate may increase calcium bioavailability because the solubility of these minerals may be a determinant of soluble calcium-citrate complexes are formed instead of absorption. insoluble calcium salts such as carbonate or phosphate It has previously been found that a high oral intake of (Lacour et al., +331). The major organic acid component sodium chloride increases urinary excretion of calcium of sudachi juice is citric acid. (Breslau et al., +32,, Weaver et al., +333). In this experi- We found that the apparent absorption of calcium, ment, although all study diets contained a high level of magnesium and phosphorus from shirasuboshi treated sodium chloride, the urinary excretion of calcium, magne- with sudachi juice was increased in healthy young men sium and phosphorus was improved in the control and when the sample was served as part of a complete diet sudachi diets. The high protein quality of shirasuboshi with a low calcium content. For this reason, although may a#ect excretion of minerals by the kidney (Ginty, 30 Y. NII et al.

,**-). normal postmenopausal women. J. Bone Miner. Metab., +,, 11ῌ We have shown that intake of sudachi-treated shirasu- 2+. +33+ boshi is associated with increased bioavailability of calci- Kohls, K. ( ). Calcium bioavailability from calcium fortified food products. J. Nutr. Sci. Vitaminol., -1, -+3ῌ-,2. um, magnesium and phosphorus from whole small fish in Lacour, B., Tardivel, S. and Druëke,T. (+331). Stimulation by citric # healthy young men. If this e ect was mainly due to acid of calcium and phosphorus bioavailability in rats fed a sudachi juice, a similar e#ect may be expected from other calcium-rich diet. Miner. Electrolyte. Metab., ,-, 13ῌ21. citrus fruit juices. These results suggest that adding Larsen, T., Thilsted, S.H., Kongsbak, K. and Hansen, M. (,***). sudachi juice to shirasuboshi could be an e#ective way of Whole small fish as a rich calcium source. Br. J. Nutr., 2-, +3+ῌ obtaining calcium and magnesium for those worried +30. about decreased intake from their usual diets. Mehansho, H., Kanerva, R.L., Hudepohl, G.R. and Smith, K.T. (+323). Calcium bioavailability and iron-calcium interaction in orange juice. J. Am. Coll. Nutr., 2, 0+ῌ02. Acknowledgments This study was supported by a grant from Miller, J.Z., Smith, D.L., Flora, L., Slemenda, C., Jiang, X. and the Regional Science Promoter Program of the Japan Science Johnston, C.C. Jr. (+322). Calcium absorption from calcium and Technology Corporation. carbonate and a new form of calcium (CCM) in healthy male and female adolescents. Am. J. Clin. Nutr., /2, +,3+ῌ+,3.. References Nakajima, M., Ozawa, Y. and Tamura, Z. (+310). A highly e$cient Andon, M.B., Peacock, M., Kanerva, R.L. and De Castro, J.A. (+330). carboxylic acid analyzer and its application. J. Chromatogr., Calcium absorption from apple and orange juice fortified with +,-, +,3ῌ+-2. calcium citrate malate (CCM). J. Am. Coll. Nutr., +/, -+-ῌ-+0. Nii, Y., Sakai, K. and Yamamoto, S. (,**+). E#ects of citrus fruit Breslau, N.A., McGuire, J.L., Zerwekh, J.E. and Pak, C.Y.C. (+32,). juices on solubilization of calcium from shirasuboshi (boiled The role of dietary sodium on renal excretion and intestinal and semi-dried whitebait). Presented at +1th International Con- absorption of calcium and on vitamin D metabolism. J. gress of Nutrition, Vienna, Austria, Aug ,1ῌ-+,p.-20ῌ-21. Endocrinol. Metab., //, -03ῌ-1-. Nii, Y., Fukuta, K., Sakai, K. and Yamamoto, S. (,**.). Japanese Bronner, F. (+332). Calcium absorption ῌ a paradigm for mineral citrus fruit (sudachi) juice is associated with increased bio- absorption. J. Nutr., +,2, 3+1ῌ3,*. availability of calcium from whole small fish and suppressed Bronner, F. and Pansu, D. (+333). Nutritional aspects of calcium bone resorption in rats. J. Nutr. Sci. Vitaminol., /*, +11ῌ+2-. absorption. J. Nutr., +,3, 3ῌ+,. Resources Council, Science and Technology Agency, Japan. Dawson-Hughes, B., Dallal, G.E., Krall, E.A., Sadowski, L., (,***). “Standard tables of food composition in Japan. /th ed.” Sahyoun, N. and Tannenbaum, S. (+33*). A controlled trial of Printing Bureau, Ministry of Finance, Tokyo. the e#ect of calcium supplementation on bone density in Smith, K.T., Heaney, R.P., Flora, L. and Hinders, S.M. (+321). postmenopausal women. N. Engl. J. Med., -,-, 212ῌ22-. Calcium absorption from a new calcium delivery system Ensrud, K.E., Duong, T., Cauley, J.A., Heaney, R.P., Wolf, R.L., (CCM). Calcif. Tissue Int., .+, -/+ῌ-/,. Harris, E. and Cummings, S.R. (,***). Low fractional calcium Uenishi, K., Ezawa, I., Kajimoto, M. and Tsuchiya, F. (+332). Calci- absorption increases the risk for hip fracture in women with um absorption from milk, fish (pond smelt, sardine) and vege- low calcium intake. Ann. Intern. Med., +-,, -./ῌ-/-. tables (komatsuna-green, jew’s marrow, saltwort) in Japanese Fiske, C.H. and Subbarow, Y. (+3,/). The colorimetric determina- young women. Nippon Eiyo Syokuryo Gakkaishi, /+, ,/3ῌ,00 (in tion of phosphorus. J. Biol. Chem., 00, -1/ῌ.**. Japanese). Ginty, F. (,**-). Dietary protein and bone health. Proc. Nutr. Soc., Weaver, C.M., Proulx, W.R. and Heaney, R. (+333). Choices for 0,, 201ῌ210. achieving adequate dietary calcium with a vegetarian diet. Hansen, M., Thilsted, S.H., Sandstro¨m, B., Kongsbak, K., Larsen, Am. J. Clin. Nutr., 1* (Suppl.), /.-S-/.2S. T., Jensen, M. and Sῌrensen, S.S. (+332). Calcium absorption Weaver, C.M. and Liebman, M. (,**,). Biomarker of bone health from small soft-boned fish. J. Trace Elem. Med. Biol., +,, +.2ῌ+/.. appropriate for evaluating functional foods designed to reduce Heaney, R.P., Saito, Y. and Orimo, H. (+33.). E#ect of casein risk of osteoporosis. Br. J. Nutr., 22 (Suppl. ,), S,,/-S,-,. phosphopeptide on absorbability of co-ingested calcium in