August 1987 251

Metabolism of , Potassium Citrate, Sodium Citrate and 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 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 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 14Cradioactivity 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. Mogi et al.8). The concentration of 14C-citrate The amounts of urinary citrates in the potas- and/or its metabolites was calculated assuming sium salt (CK) administered group and sodium that the weight of whole blood accounts for 1/13 salt (CNa) administered group were much higher of the body weight. than those in the control group, citric acid (CH) Experiment 4. Distribution of 14C-citrate and/or administered group, and calcium citrate (CCa) its metabolites in tissues administered group. Volumes of urine were Twelve rats were divided into 6 groups of 2 also increased in the CK and CNa group (data each and orally given 6 kinds of samples as not shown). The apparent excretion rates show- described in Experiment 3. One rat in each ed similar trends. group was killed after 30 minutes and the other Time courses of the urinary excretion rate per rat was killed after 24 hours. At 30 minutes hour are illustrated in Fig. 2. The urinary ex- and 24 hours after administration, blood samples cretion rate of citrates reached the maximum were collected from the jugular vein into a after 0-2 hours in the CH and CK groups, after heparinized syringe under light diethylether 2-4 hours in the CNa group, and after 4 6 anesthesia. Then, the liver, kidneys, lungs, sto- hours in the CCa group. the excretion pattern mach, small intestine, and large intestine (in- of the CK administered group was somewhat cluding their contents) were removed immedi- different from that of the CNa administered ately. They were each homogenized with 10 group; the excretion started sooner and con-

Table 1. Body Weight and Urinary Excretion in 24 Hours

Abbreviations: CH, citric acid; CK, tripotassium citrate; CNa, trisodium citrate; CCa, calcium citrate; HD, high dose 1) Results are represented as mean±SEM for three rats. Urinary excretion 2) Apparent excretion rate [%] x100 Dose administered August 1987 Metabolism of Citrates in the Rat 255

Fig. 2. Urinary excretion of citric acid as a function of time Urinary excretion of citric acid is indicated as the percentage of citric acid excreted in urine per one hour with respect to the orally administered citric acid dose. Each column represents the mean of 3 animals and the vertical bar indicates the SEM. Abbreviations: CH, citric acid; CK, tripotassium citrate; CNa, trisodium citrate; CCa, calcium citrate

Table 2. Excretion of 14C Radioactivity in Urine, eces and Expiratory 14C02 in 24 Hours

Abbreviations: CH, citric acid; CK, tripotassium citrate; CNa, trisodium citrate; CCa, calcium citrate; LD, low dose; HD, high dose tinued for longer in the CK group than the CNa salts was excreted as 14C02 within 24 hours. group. Fecal excretion was very low. In urine, 3.8 to Experiment 2. Expiratory 14C02 and urinary 11.20 of the administered 14C-labeled citric acid and fecal excretions of 14C-cit- and its salts was excreted. Total 14C-citrate rate and/or its metabolites and/or its metabolites excreted in expired air, Table 2 indicates the expiratory 14C02 and urine, and feces accounted for 88.1 to 94.30 of urinary and fecal excretions of 14C-citrate and/ the dose within 24 hours and 96.4 to 99.5% with- or its metabolites relative to the amount of in 48 hours. administered 14C-citric acid and its salts over 24 The proportion of urinary excretion was or 48 hours. In all the groups, 76.7 to 88.9% higher in the rats given the high dose than in the of administered 14C-labeled citric acid and its rats given the low dose. The urinary excretion 256 J. Food Hyg. Soc. Japan Vol. 28, No. 4

●CH(LD) ▲CK(LD) □CNa(LD) ○CCa(LD)

Fig. 3-A, Changes of the expiratory 14C radioactivity as a function of time Abbreviations: CH, citric acid; CK, tripotassium citrate; CNa, trisodium citrate; CCa, calcium citrate; LD, low dose (37.78 mg/kg)

▲CK(HD) □CNa(HD)

Fig. 3-B. Changes of the expiratory 14C radioactivity as a function of time Abbreviations: CK, tripotassium citrate; CNa, trisodium citrate; HD, high dose (1889 mg/kg) rate in Table 2 is a little different from the More than 80% was excreted as expiratory 14CO2 apparent excretion rate in Table 1 for the fol- in the rats given the low dose while the expi- lowing two reasons. 1) Citrate and/or its meta- ratory 14CO2 increased almost linearly for 10 bolites were measured in Experiment 2 but only hours in the rats given the high dose. The pat- citrate was measured in Experiment 1. 2) The terns of expiratory 14CO2 excretion showed no total of endogenous and exogenous (administer- significant difference among the groups given ed) citrates was measured in Experiment 1 but the same dose. only the exogenous (administered) citrate was Experiment 3. Blood concentration of 14C-cit- measured in Experiment 2. There was no signi- rate and/or its metabolites as a ficant difference among the groups given the function of time same dose. Time courses of blood concentration of 14C- Time courses of the proportion of expiratory citrate and/or its metabolites are shown in Fig. 14CO 2 with respect to administered 14C-citric 4-A, B. The proportion of 14C radioactivity to acid and its salts are shown in Fig. 3-A, B. administered 14C amount was taken as the blood August 1987 Metabolism of Citrates in the Rat 257

●CH(LD) ▲CK(LD) △CK(HD) ■CNa(LD) □CNa(HD) ○CCa(LD)

Fig. 4-A. Changes of the 14C radioactivity in Fig. 4-B. Changes of the "C radioactivity in blood as a function of time blood as a function of time Each point is the mean for 3 rats and Each point is the mean for 3 rats and the vertical bar indicates the SEM. the vertical bar indicates the SEM. Abbreviations: CH, citric acid; CK, tri- Abbreviations: CK, tripotassium citrate; potassium citrate; CNa, trisodium citrate; CNa, trisodium citrate; HD, high dose CCa, calcium citrate; LD, low dose (37.78 (1889 mg/kg) mg/kg) relative). concentration here. Experiment 4. Distribution of 14C-citrate and/ Fig. 4-A shows the time course of blood con- or its metabolites in tissues centration in the rats given the low dose. Blood The tissue distributions of 14C-labeled citrate concentration reached the maximum after 15 and/or its metabolites at 30 minutes and 24 hours minutes in the citric acid (CH(LD)) administer- after oral administration are, indicated in Table ed group, after 1 hour in the potassium citrate 3. The values are the proportion of 14Cradio- (CK(LD)) and sodium citrate (CNa(LD)) admin- activity in tissues to the administered amount of 4C-labeled compound. Distribution in tissues istered groups, and after 4 hours in the calcium citrate (CCa(LD)) administered group. There was higher after 30 minutes than after 24 hours was no significant difference between CK(LD) in all the rats. 14C-Citrates remaining in the and CNa(LD). gastrointestinal tract amounted to 37.6 to 47.2% Time courses of the blood concentration in the in the rats given the low dose, and 61.5 to rats given the high dose are shown in Fig. 4-B. 64.3% in the rats given the high dose, while less Blood concentration increased during the ex- than 3% remained in the gastrointestinal tract perimental period (over 4 hours) in both the in all the rats. The total 14C radioactivity in potassium salt (CK(HD)) administered group blood, liver, kidney, lung, stomach, small in- and the sodium citrate (CNa(HD)) administered testine and large intestine amounted to 41.6 to group. The concentration was slightly higher in 51.0% after 30 minutes and 2.0 to 2.4% after 24 the CK(HD) group than in the CNa(HD) group. hours in the rats given the low dose, while the The maximum values were 1.0 to 1.5% in all corresponding values were 64.6 to 66.8% after the groups (these values are not absolute, but 30 minutes and 2.0 to 3.8% after 24 hours in the 258 J. Food Hyg. Soc. Japan Vol. 28, No. 4

rats given the high dose. creted in urine (Table 2). However, the in- There was no clear difference in the pattern crease of urinary citrate excretion was milder in of distribution among the groups given the same the rats given citric acid and calcium citrate dose. In the rats given the high dose, the dis- (Table 1). Accordingly, the metabolism of potas- tribution in the gastrointestinal tract was higher sium and sodium citrates might differ somewhat but that in the blood was lower than those of from that of citric acid and calcium citrate at rats given the low dose after 30 minutes. There the high dose. was no significant difference in the patterns of Second, urinary citrate excretion reached distribution among all groups after 24 hours. maximum after 0 to 2 hours in the rats given citric acid and potassium citrate, after 2 to 4 Discussion hours in the rats given sodium citrate, and after 4 to 6 hours in the rats given calcium citrate Citric acid is a tricarboxylic acid, and at (Fig. 2). Blood concentration of citrate and/or physiological pH is more than 90% ionized its metabolites reached the maximum after 15 Citrate levels in the plasma of humans and other minutes in the rats given citric acid, after 1 hour mammals range between 0.05 and 0.3mM in the rats given potassium citrate and sodium Together with the liver, the kidney is the most citrate, and after 2 hours in the rats given cal- important site for the regulation of plasma cit- cium citrate (Fig. 4-A). Thus, it is considered rate, as well as of other tricarboxylic acid cycle that the order of absorption rates of citric acid metabolites. More than 90% of blood citrate and its salts is as follows: citric acid > potassium is ultrafilterable, passing readily through the glo- citrate >_sodium citrate > calcium citrate. merulus12). In man, 5 to 30% of filtered citrate Third, expiratory 14CO2 excretion increased is excreted12), whereas in rats and dogs the almost linearly for 10 hours in the rats given the amount is much smaller, averaging 3 to 7% in high dose, but reached a plateau of more than rats12). The proximal tubule reabsorbs between 80% within 6 hours in the rats given the low 70 and 95% of filtered citrate and returns it to dose (Fig. 3-A, B). Blood concentration of the blood or metabolizes it12). A large propor- citrate and/or its metabolites increased during tion of citrate is converted to CO2 and H2O12). 4 hours in the rats given the high dose, but Many factors alter citrate excretion, parti- reached the maximum within 2 hours in those cularly acid-base conditions, blood citrate levels, given the low dose. In a comparison of the gastrointestinal citrate absorption, magnesium distributions of citrate and/or its metabolites at and hormones12). In at least one instance, the 30 minutes after administration of the high dose change in citrate excretion occurs within min- and the low dose, the distribution in the gastro- utes12). The urinary excretion of citrate may be intestinal tract was higher but that in blood was increased following an oral load of citric acid or lower in the former case (Table 3). Therefore it alkaline citrate, although other investigators is suggested that the more citric acid and its salts have not found this13). The object of this study were administered orally, the slower was their was to investigate the metabolism of citric acid absorption rate. and its potassium, sodium and calcium salts. Fourth, expiratory excretion of 14CO2reached Experiments were performed 1) to determine 76.7 to 88.9% within 24 hours in all cases (Table whether there is any difference in the meta- 2). The excretion in expired air, urine, and bolism of citric acid and its alkaline salts and 2) feces during 24 or 48 hours amounted to 88.1 to to compare the metabolism of potassium citrate 99.5% of the administered dose (Table 2). The and sodium citrate. We obtained several inter- levels of citrate and/or its metabolites after 24 esting results as follows. hours were low (Table 3). Therefore it is suggest- First, the excretion of urinary citrate was ed that citric acid, potassium citrate, sodium apparently increased when potassium and so- citrate and calcium citrate are not accumulated dium citrate were administered (Table 1). The in the body but are metabolized and excreted greater the amount of citrate administered, the mainly in expired air and urine. more citrate and/or its metabolites were ex- In conclusion, we studied the metabolism of August 1987 Metabolism of Citrates in the Rat 259

Table 3.. Distribution of 14C Radioactivity

Abbreviations: CH, citric acid; CK, tripotassium citrate; CNa, trisodium citrate; CCa, Calcium citrate; LD, low dose; HD, high dose citric acid and its potassium, sodium and cal- experiment and Dr. Sekizawa (Division of In- cium salts in rats, and found that a large propor- formation on Chemical Safety, National Insti- tion of orally administered citric acid or its salts tute of Hygienic Sciences, Tokyo) for making is absorbed, metabolized, and excreted mainly available his research data. as expiratory CO2. No significant difference in References metabolism of citric acid and its salts was found. Studies on the safety of citric acid and its salts 1) FDA in USA: SGOGS-81, contract No. FDA have been carried out from various points of 223-75-2004, 1-23 (1977). view1)14),. The WHO15) concluded that citric 2) Thunberg, T.: Physiol. Rev. 33, 1-12 (1953). acid and its calcium, potassium and sodium 3) Federation of Food Additives: "The Japanese salts do not constitute a significant toxicological Standards of Food Additives" 4th Edition, p. hazard to man and evaluated the acceptable 17-39 (1979). daily intake for man as "not limited". Our 4) FDA in USA: Federal Register 46, 834-839 (1983). 5) Committee of Research for Daily Intake of Food results confirm that potassium, sodium and cal- Additives: "The Study about Daily Intake of Food cium salts of citric acid are metabolized similar- Additives in the 60th Year of Showa" (in press). ly to citric acid when used at levels that are now 6) Adler, S., Anderson, B., Zemolel, L.: Am. J. current. It is suggested that potassium citrate is Physiol. 220, 986-990 (1971). not a hazard to man at the level currently 7) Jessop, N. S., Scaife, J. R.: Biochem. Soc. Trans. ingested from foods. 13, 1222-1223 (1985). 8) Mogi, M., Nakao, M., Goto, Y., Ito, T., Matsu- Acknowledgements ki, Y.: Iyakuhin Kenkyu, 17, 714725 (1986). 9) Suzuki, T., Tazaki, K., Nakahama, H.: "Princi- We thanks Dr. Tanaka (Head of the Division ples of Physiology III" p. 2 (1972), Kyoritsu Shup- of Biological Chemistry and Reference Stand- pan, Tokyo. 10) Swille, P. 0., Smith, L. H., Robertson, W. G., ards) and Dr. Takahashi (Head of the Division Vahlensieck, W.: "Urolithiasis and Related Cli- of Medical Chemistry) in The National Institute nical Research" p. 181'188 (1985), Plenum Press, of Hygienic Sciences for their support of this Mew York & London. work. We also thank the staff of the Division 11) Simpson, D. P.: Am. J. Physiol. 244, F223-F234 of Microbiology and Pharmacy in our labora- (1983). tory for assistance in carrying out the animal 12) Swille, P. 0., Smith, L. H., Robertson, W. G. 260 J. Food Hyg. Soc. Japan Vol. 28, No. 4

Vahlensieck, W.: "Urolithiasis and Related Cli- 14) Food and Drug Research Lab., Inc.: "Specific nical Research" p.173-180 (1985),Plenum Press, Literature Reviews on Generally Recognized as New York & London. Safe (GRAS) Food Ingredients-Citrates" PB223- 13) Swille, P. 0, Smith, L. H., Robertson, W. G., 850 (1973). Vahlensieck, W.: ibid. p. 525-532 (1985),Plenum 15) WHO: "WHO Food Additives Series No. 5" p. Press, New York & London. 170-172 (1974).