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August 1987 251 Metabolism of Citric Acid, Potassium Citrate, Sodium Citrate and Calcium Citrate in the Rat (Received December 13, 1986) Yumiko NAKAMURA,Yasuhide TONOGAI, Sumiko TSUJI and YOshio ITo (National Institute of Hygienic Sciences, Osaka Branch; 1-1-43, Hoenzaka, Higashi-ku, Osaka, Japan) The metabolism of citric acid and potassium, sodium and calcium salts of citrate in rats was investigated by single-dose oral administration. The per capita daily intake of citric acid and its salts in Japanese was estimated to be 37.78 mg/kg by Ito et al., so 37.78 mg/kg was chosen as the low dose and 1,889mg/kg (50 times more) as the high dose. The urinary excretion of citrate, expiratory carbon dioxide (C02), and the blood concentration and tissue distribution of citrate and/or its metabolites were investigated in fasting rats. A large proportion of admin- istered citric acid and its salts was excreted within 24 hours, i.e., 76.7 to 88.9% as expiratory C02, 3.8 to 11.2% in urine, and only a small amount in feces. In the rats given the high dose of potassium or sodium citrate, the absorption rate of citrate decreased while the urinary ex- cretion increased. The times at which the urinary excretion of citrate and the blood concentra- tion of citrate and/or its metabolites reached the maximum were different for citric acid and its salts. The absorption rates of citric acid and its salts were considered to be in the following order: citric acid > potassium citrate ? sodium citrate > calcium citrate. The distribution in the gastrointestinal tract was 37.6 to 64.3% after 30 minutes but less than 2.7% after 24 hours. Therefore, it is clear that a large proportion of administered citric acid and its salts is absorbed, metabolized and excreted mainly as C02, and that the difference in metabolism between citric acid and its alkaline salts is negligible. Key words: metabolism; citric acid; potassium citrate; sodium citrate; calcium citrate; urinary citrate; expiratory carbon dioxide; 4C-radioactivity Introduction Thus, we investigated the metabolism of citric acid, potassium citrate, sodium citrate and cal- Citric acid is an intermediary substance in cium citrate in the rat, in order to elucidate oxidative metabolism, being a component of the possible differences in metabolism between po- tricarboxylic acid cycle. Citric acid and its salts tassium citrate and sodium citrate. occur in many foods, and are normal metabolites in the body1),2). Citric acid and its salts are Materials and Methods widely used as food additives in Japan. For example, citric acid is used as an acidulant, Citric acid and tripotassium citrate were pur- sodium citrate as a seasoning, and calcium cit- chased from Ishizu Pharmaceutical Co., Ltd. rate as a dietary supplement3). Recently it has (Osaka), trisodium citrate from Nakarai Chem- been suggested that the percentage of sodium icals, Ltd. (Kyoto), and calcium citrate from salts in food additives is too high. The Ministry Yoneyama Chemical Industries Ltd. (Osaka). of Health and Welfare of Japan recommends the They were all of guaranteed reagent grade. [1,5- replacement of sodium salts with other salts such 14C]Citric acid (specific activity 64.7 pCi/nmol), as potassium salts from the viewpoint of the Aquasol-2 and Protosol® were purchased from prevention of hypertension. Potassium citrate New England Nuclear (Boston, Mass). Other has been permitted as a food additive in the reagents were of guaranteed grade. United States of America1),4) but not in Japan. A system consisting of a Shimadzu LC-6A, 252 J. Food Hyg. Soc. Japan Vol. 28, No. 4 Apparatus: Shimadzu LC-6A Detection: UV 210 nm Column: Shimadzu SCR-101(H) 7.9 mmc x 3O cm Mobile phase: 5 mM H2SO4 Flow rate: 1.0 ml/min Scheme 1. Procedure for analysis of citric acid by HPLC Shimadzu SPD-6AV and Shimadzu SCR-101(H) citrate, and calcium citrate were each dissolved (7.9mm 5x30cm) (Shimadzu Seisakusho, Kyoto) or suspended in water, and the following sam- was used for the analysis of urinary citrate by ples (high dose) were prepared: citric acid, 1889 HPLC. 14C-Radioactivity was counted with an mg/5 ml/kg; potassium citrate, 1889 mg/5 ml/kg; Aloka liquid scintillation spectrometer, model sodium citrate, 1889 mg/7.5 ml/kg; calcium cit- LSC-673 (Aloka Co., Ltd., Tokyo). rate, 1889 mg/7.5 ml/kg. Male Sic: Wister rats (Shizuoka Laboratory Fifteen rats were divided into 5 groups of 3 Animal Center) were acclimated in an air-con- each. Rats were housed individually in meta- ditioned room for more than 1 week before use. bolic cages after oral administration of the sam- Rats were housed in a Metabolica® CO-2 system ples. Distilled water was administered orally to (Sugiyamagen Iriki Co. Ltd., Tokyo) after the the 3 rats of the control group. Urine was administration of 14C-containing citrates. Rats collected at constant intervals, and the total weighing 150200 g were used in the following amount of citrate in urine was analyzed by the experiments. method summarized in Scheme 1. Briefly, urine The rats were fasted overnight before use and was treated with 10 volumes of 6% perchloric the experiments were carried out in the fasting acid6i and centrifuged. The supernatant was state; water was allowed ad libitum. adjusted to pH 8 9 with 30% potassium hydro- Ito et al. estimated the per capita daily in- xide and hydrolyzed at 80C for 15 minutes. take of citrate of Japanese to be 37.78 mg/kg. Then, the solution was filtered and the filtrate Thus, the doses for oral administration (cal- was assayed by the HPLC method. The con- culated as citric acid) were set at 37.78 mg/kg for ditions of HPLC were as follows: the low dose and 1889 mg/kg (50 times the low Apparatus: Shimadzu LC-6A dose) for the high dose. Detector: Shimadzu SPD-6 AV UV 210 nm Experiment 1. Time course of urinary excre- Column: Shimadzu SCR-101(H) 7.9 mmq tion of citrate x 30cm Citric acid, tripotassium citrate, trisodium Mobile phase: 5 mM sulfuric acid August 1987 Metabolism of Citrates in the Rat 253 Fig. 1. Diagram of the apparatus for the estimation of expiratory 14C02 Experiment 2. Expiratory 14CO2, and urinary ml of the 4C02-trapping solution was sampled at and fecal excretions of 14C-cit- constant intervals, and the 14C-radioactivity was rate and/or its metabolites measured according to the method of Mogi et [1,5-14C]Citric acid was mixed with equivalent al. amounts of potassium hydroxide, sodium hydro- Feces and urine were collected individually xide, and calcium hydroxide to prepare the for 24 or 48 hours after oral administration. potassium, sodium, and calcium salts of [1,5- To 50ccl of urine were added 1 ml of water, 14C]citric acid, respectively. The 14C-labeled 5 ml of methanol and 10 ml of Aquasol-2, and citric acid and its salts were diluted with the the 14C radioactivity in urine was measured with corresponding non-labeled citric acid and its a scintillation counter using an external stand- salts, and the following samples for oral admin- ardization method. Feces were homogenized istration were prepared. Low dose: citric acid, with 10 volumes of water. A suitable amount 37.78 mg/5 pCi/5.5 ml/kg; potassium citrate, 37.78 of the homogenate (containing about 3 mg of mg/5 iCi/5.5 ml/kg; sodium citrate, 37.78 mg/5 feces) was solubilized with 2 ml of Protosol®-n- ;iCi/5.5 ml/kg; calcium citrate, 37.78 mg/5uCi/ butanol (1:1, v/v) mixture, and decolorized with 5.5 ml/kg. High dose: potassium citrate, 1889 0.5 ml of 30% hydrogen peroxide, then 0.6 ml mg/5 pCi/5.5 mi/kg; sodium citrate, 1889 mg/7.5 of 1 N hydrochloric acid and 10 ml of Aquasol- ;uCi/8.25 ml/kg. Rats were housed in metabolic 2® were added, and the 14C radioactivity was cages (Fig. 1) after orall administration of a 14C- measured as described above. containing sample. Air flow was 1 L/min, and Experiment 3. Blood concentration of 14C-cit- the expiratory 14CO2 was trapped in 200 ml of rate and/or its metabolites as a methylcellosolve-ethanolamine (1:1, v/v). One function of time 254 J. Food Hyg. Soc. Japan Vol. 28, No. 4 The administered samples were prepared by volumes of water. The 14C radioactivity in a the method described in Experiment 2. The dose suitable amount of each homogenate (contain- of samples for oral administration (calculated as ing 10 - 20 mg of tissues) was measured by the citric acid) was as follows. Low dose: citric same method as in the case of the fecal analysis acid, 37.78 mg/10 pCi/6 ml/kg; potassium citrate, described in Experiment 2. 37.78 mg/10 pCi/6 ml/kg; sodium citrate, 37.78 mg/10 pCi/6 ml/kg; calcium citrate, 37.78 mg/10 Results ; tCi/6 ml/kg. High dose: potassium citrate, 1889 mg/10iCi/6 mi/kg; sodium citrate, 1889 mg/15 Experiment 1. Time course of urinary excre- 1iCi/9 ml/kg. tion of citrate The experiment was carried out in triplicate. Amounts of urinary citrate excreted during 24 Rats were housed in metabolic cages (Fig. 1) hours after oral administration of citric acid after administration of the 14C-containing sam- and its salts at the high dose are indicated in ples. Blood samples were collected periodically Table 1. Apparent excretion rate, defined as the from the tip of the tail, and the 14C radioac- ratio of excretion to administration, is also in- tivity was measured according to the method of dicated in Table 1.

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