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 absorp- tion 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 white- bait) 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 concentra- tions 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 min- eral 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.).