Urinary Excretion of Hippuric Acid After Administration of Sodium Benzoate (Biological Monitoring 1)

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1 June Urinary Excretion of Hippuric Acid After Administration of Sodium Benzoate (Biological Monitoring 1) (Received August 2, 1990) Toshiko FUJII*1, Toyonori OMORI*2, Toyohiro TAGUCHI*3 and Masana OGATA*3 (*1Kawasaki College of Allied Health Professions: 316, Matsushima, Kurashiki, Okayama, Japan; *2Sho-o Health Center: Sho-o-cho, Katsuta-gun, Okayama Japan; *3Department of Public Health, Okayama University Medical School: Shikata-cho, Okayama Japan) Six female students were orally given 175 and 350 ml of a soft drink containing 190 mg/ L of sodium benzoate (BA-Na) and three male volunteers were also orally given BA-Na solution at a dosage of 20 mg/kg body weight. Urinary hippuric acid (HA) and creatinine excreted by the subjects given either the soft drink or the BA-Na solution were determined simultaneously by high performance liquid chromatography (HPLC) from the end of the administration until 4 or 5 hr later, at appropriate intervals. In the case of soft drink administration, the concentration of HA attained a maximum in urine taken from 0 to 30 min and recovered to the predose level after 3 hr. The molar ratios of HA excreted in the 0-3 hr urine samples to the administered dose were estimated to be 66 r86%, by subtracting the amount of HA excreted in the predose period on the day of the administration. In the case of BA-Na solution administration, the concentration of HA attained a maximum in the 0-1 hr urine samples and did not recover to the predose level within 5 hr. The molar ratio of HA excreted in the 0-5 hr urine samples to the administered dose was estimated to be about 89%, by subtracting the amount of HA excreted in the predose period on the day of the administration. Based upon these findings, it is suggested that the amount of absorbed BA-Na in human subjects can be estimated by determining the amount of HA excreted in the urine. Key words: urinary excretion; hippuric acid; oral administration; sodium benzoate; biological monitoring; soft drink; high performance liquid chromatography Introduction The purpose of this study was to examine whether the amount of benzoic acid (BA) absorbed from the intestine in human subjects can be estimated by determining urinary HA levels. In the experiment the urinary excretion of HA following the oral administration of a soft drink containing BA-Na and a BA-Na solution was determined. HA in the urine is derived mainly from benzoic acid and other related compounds which are ingredients of some fresh foods. On the other hand, BA and BA-Na are legally permitted as preservatives in particular foods, e. g., caviar, soft drinks, etc. Urinary HA levels increase after the intake of foods or drinks containing BA or BA-Na. Urinary HA levels also increase when toluene is inhaled and/or absorbed via skin by subjects. Urinary HA has been utilized for estimating the amount of toluene to which industrial workers are exposed, by biological monitoring. The American Conference of Governmental Industrial Hygienists (ACGIH) proposed biological exposure indices (BETS) for urinary metabolites of toluene. The BET value of urinary HA in expressed as a metabolite concentration adjusted for creatinine in the urine. Creatinine is a metabolic product of skeletal muscles, and its excretion rate reflects the lean body mass of the subject. Under usual circumstances, the effects of diet, state of hydration, and diuresis on excre-

2 178 J. Food Hyg. Soc. Japan Vol. 32, No. 3 tion rate of creatinine are relatively insignificant. Diurnal variation is minimal. Thus, normalization of the concentration of HA with respect to creatinine is optimal, because both HA and creatinine are filtered by the glomeruli. The correlation coefficient obtained for toluene concentration in the air and HA concentration in workers' urine corrected for creatinine and/or specific gravity is higher than that for toluene concentration and uncorrected HA concentration. In the present study, the concentration of HA in the urine was determined by the HPLC procedure which was established by Ogata and Taguchi for the quantitative and simultaneous determination of urinary metabolites and creatinine. Materials and Methods 1. Materials HA, creatinine, sodium 1-decanesulfonate (for ion-pair chromatography) and BA-Na (food additive grade) were obtained from Tokyo Kasei Co., Tokyo. Acetonitrile and methanol (HPLC grade) were obtained from Wako Pure Chemical Industries Co., Osaka. Coke light (Sanyo Coca Cola Bottling Co., Hiroshima) was used as a soft drink containing BA-Na. Reference compounds (1 mg/ml) were dissolved in 50% (v/v) methanol. 2. Experimental procedure The procedure was explained to all subjects and this study was conducted with informed consent. After intake of toast and tea for breakfast, six healthy female students, averaging 21 years of age, were orally given 175 ml and 350 ml of the soft drink containing BA-Na on two separate days. And three healthy male volunteers, averaging 35 years of age, maintaining their usual intake of foods, were orally given BA-Na at a dosage of 20 mg per kilogram body weight dissolved in 200 ml of water. The predose urine samples were collected just before the administration. Then the urine samples were collected for 4 or 5 hr at the appropriate elapsed times (30 min and/or 1 hr intervals, i. e., in the case of soft drink administration, 0-30 min, min, 1-2, 2-3 and 3-4 hr, and in the case of BA-Na solution administration, 0-1, 1-2, 2-3, 3-4, and 4-5 hr) following the administration. Upon the collection of urine, the specific gravity of urine was determined by refractometry and the volume of urine was measured immediately. Urine specimens were stored at - 20C until analysis and were diluted 100-fold with 50 (v/v)% methanol. Twenty microliter aliquots of the diluted samples were used for HPLC analysis. 3. High performance liquid chromatography Analysis of urinary metabolites: The liquid chromatograph used was a Tosoh system, incorporating a UV 8010 variable-wavelength detector and an AS-8000 autoinjector. The detector was connected to a Hitachi D-2500 chromatointegrator. A stainless-steel column (4. 6 mmq5 X 100 mm) packed with octadecyl-silanized silica gel (TSK gel, ODS-80 TM CTR, 5, um, Tosoh Co. ) and equipped with a jacket for temperature control was used throughout the investigation. The flow rate was 0. 7 ml/min, producing a pressure of kg/cm2 in the separation procedure. The column temperature was 30C. The eluate was monitored at a wavelength of 225 nm. To separate urinary creatinine and HA, a mixed solution of [20 mm potassium dihydrogen phosphate (ph 3. 3) containing 3 mm sodium 1-decanesulfonate]/acetonitrile (85/ 15) was used as a mobile phase. Determination of BA-Na in the soft drink: The liquid chromatograph used was the same instrument as in the above analysis. In this analysis, a stainless-steel column (4.6mmX 150 mm) packed with the same gel as in the above experiment was used. The flow rate was 0. 7 ml/min, producing a pressure of 99 kg/cm2 in the separation procedure. The mobile phase used was the same as that used in the above experiment. Results 1. Elution pattern For the determination of HA and creatinine in the urine: HPLC chromatograms of HA and creatinine are shown in Fig. 1. The retention times for HA and creatinine peaks were and min. HA was observed in the predose urine. HA and creatinine were well separated and could be determined in the samples without interferance. For the determination of BA-Na in the soft drink: HPLC chromatograms of HA, creatinine and BA-Na in the standard solution are shown

3 June 1991 Urinary HA of Subjects Given BA-Na 179 Fig. 1. High performance liquid chromatograms (A) A standard solution of authentic HA and creatinine. Concentrations of HA and creatinine were 1 mg/ml each. (B) The predose urine of a subject. (C) The 0-30 min urine of the subject given soft drink containing BA-Na. HA: hippuric acid; cre: creatinine in Fig. 2. The retention times for HA, creatinine and BA-Na peaks were 5. 17, and min, respectively. The concentration of BA-Na in the soft drink was estimated to be 190 mg/l. 2. Time course of urinary HA excreted by subjects As shown in Tables 1 and 2, urinary HA is represented as the observed concentration, adjusted for the specific gravity of urine (1. 020) and excretion rate. Changes in the concentration of urinary HA normalized with respect to creatinine (g/g creatinine) are shown in Fig. 3. In the case of soft drink administration urinary HA showed a maximal concentration in the 0-30 min urine and then declined gradually and recovered to its predose level in the 2-3 hr urine. In the case of BA-Na solution administration, HA showed a maximal concentration in the 0--1 hr urine and then declined gradually, but did not recover to the predose level in the 4-5 hr urine. 3. Relationship between the increased amount of urinary HA and the administered dose In the case of soft drink administration, the molar ratios of HA excreted in the 0-3 hr urine to the amount of BA-Na consumed in the soft Fig. 2. High performance liquid chromatogram of the standard solution a: HA; b: creatinine; c: BA-Na drink were estimated to be 66% (175 ml) and 86% (350 ml), assuming that the predose excretion rates of urinary HA were mg/min (175 ml) and mg/min (350 ml), respectively. On the other hand, the molar ratio of HA excreted in the 0-5 hr urine of the subjects given the BA-Na solution to the administered dose was estimated to be about 89%, assuming that the predose excretion rate of urinary HA was mg/min. Discussion In Japan, average daily intakes of BA per capita have been reported to be 10.9mg, mg and 1.987mg. These reports indicate that large proportions of BA intake come from food additives added to processed foods, especially seasonings and beverages, when the respective daily intakes from fresh foodstuffs are compared with those from processed foodstuffs. HA in normal urine increases to some extent after the intake of foods or drinks containing BA and its precursors (chlorogenic acid, quinic acid, etc.). The intake of BA or BA-Na is supposed to increase greatly when beverages containing them are consumed. The cumulative urinary excretion of HA after the administration of BA-Na can be estimated by subtracting the amount of HA excreted in the predose urine sample. We have shown that maximum excretion rates of urinary HA were observed in 30-60

4 180 J. Food Hyg. Soc. Japan Vol. 32, No. 3 Table 1. Urinary Excretion of HA from Subjects Given Soft Drink Containing BA-Na (A) A dosage of 175 ml of a soft drink containing 33 mg of BA-Na (B) A dosage of 350 ml of a soft drink containing 66 mg of BA-Na The number of subjects was six. Urinary excretion of HA is expressed as mean +S. D. The urine samples are collected for 4 hrs after administration, and figures on the left side of parentheses () indicate the period of collection of individual samples. *1: The measured concentration of urinary HA (g/l) *2: The concentration of urinary HA adjusted for specific gravity (g/l) =The measured concentration of urinary HAx1. 0d -1 [where: d=the measured specific gravity, =the mean level of specific gravity in urine of adults)] *3: Excretion rate (mg/min) The volume of urine (ml) x The measured concentration of urinary HA (mg/ml) The elapsed time during which the urine was collected (min) Table 2. Urinary Excretion of HA from Subjects Given BA-Na Solution BA-Na was give orally to each subject at a dosage of 20 mg per kilogram body weight in 200 ml of water. The number of subjects was three. Urinary excretion of HA is expressed as mean S. D. The urine samples were collected for 5 hrs after administration, and figures on the left side on the parentheses () indicate the period of collection of individual samples. *1,*2,*3 The same as in Table 1.

5 June 1991 Urinary HA of Subjects Given BA-Na 181 Fig. 3. Time course of urinary HA Excretion The concentration*' of urinary HA normalized with respect to creatinine following three different doses of BA-Na was determined (mean value and standard deviation represented by vertical bars). The closed squares (O) and the closed circles (O) indicate data observed after the administration of soft drink containing 33 mg and 66 mg of BA-Na, respectively. The closed triangles (A) indicates data observed after the administration of BA-Na at a dosage of 20 mg per kilogram body weight in 200 ml of water. The data plotted at time 0 are endogenously derived HA in the predose urine. *1: The concentration of urinary HA normalized with respect to creatinine (g/g creatinine) =The measured concentration of urinary HA (g/l)/the measured concentration of urinary creatinine (g/l) min urine and 0-1 hr urine after the administrations of soft drink and BA-Na solution, respectively. The rates were proportional to the two doses following the administration of the soft drink during 60 min after administration. The biological half-time of urinary HA was estimated from the excretion rate to be about 40 min. In addition, the molar ratios of the increased amounts of urinary HA to the administered dose were calculated to be relatively large, i. e., the cumulative urinary excretion of HA after the administration reached more than 66% of the administered dose of BA-Na, when HA was expressed as the corresponding amount (equivalent) to BA-Na. Thus, we think that biological dose monitoring can be applied to determine the absorbed amount of BA-Na in the human body. Changes in the urinary excretion of HA following the intake of several kinds of soft drinks containing BA or BA-Na have already been reported by Michitsuji et al. Our results resemble theirs. In our case, the urinary concentration of HA normalized with respect to creatinine was easily determined by simultaneous analysis of HA and creatinine, which is important for biological monitoring. In conclusion, estimation of the absorbed amount of BA-Na in human subjects can be done by determining the amount of urinary HA. The measurement of urinary HA may be useful for the biological monitoring of BA-Na consumed in not only soft drinks, but also foods, although the measureable dose is limited to the range where a linear dose-response relationship is observed. Further investigation is necessary on the effects of some constituents of foods, e. g, dietary fiber, etc, on the absorption rate of BA-Na. This approach is also useful for minimizing the error in the normal value of urinary HA applied in the field of industrial hygiene. References 1) Nagayama, T., Nishijima, M., Yasuda, K., Saito, K., Kamimura, H., Ibe, A., Ushiyama, H., Naoi, Y., Nishima, T.: J. Food Hyg. Soc. Japan 27, (1986). 2) American Conference of Governmental Industrial Hygienists: "Documentation of the Biological Exposure Indices for ", p. 11-v13, 29-33, 35-39, 47^-49 (1989), ACGIH Inc., Ohio. 3) American Conference of Governmental Industrial Hygienists: "Documentation of the Threshold Limit Values and Biological Exposure Indices", p. BEI-xix (88) BEI-xxii (88) (1988), ACGIH, Cincinnati, Ohio. 4) Araki, S., Aono, H., Yokoyama, K., Murata, K.: Arch. Environ. Health 41, 216^-221 (1986). 5) Araki, S., Aono, H., Murata, K.: ibid. 41, 171

6 182 J. Food Hyg. Soc. Japan Vol. 32, No (1986). 6) Taguchi, T.: Okayama-Igakkai-Zashi, 102, (1990). 7) Ogata, M., Taguchi, T.: Int. Arch. Occup. Environ. Health 61, (1988). 8) Araki, S.: Ind. Health 11, (1973). 9) Toyoda, M., Ito, Y., Isshiki, K., Onishi, K., Kato T., Kamikura, M., Shiroishi, Y., Harada, Y., Hukasawa, Y., Yokoyama, T., Yoneda, M. Iwaida, M.: J. Jpn. Soc. Nutr. Food Sci. 36, (1983). 10) Toyoda, M., Ito. Y., Isshiki, K., Onishi, K., Kato, T., Kamikura, M., Shiroishi, Y., Harada, Y., Hukasawa, Y., Yokoyama, T., Yoneda, M., Hirayama, Y., Yamamoto, Y., Fujii, M., Iwaida, M.: ibid. 36, (1983). 11) Yomota, C., Isshiki, K., Kato, T., Kamikura, M., Shiroishi, Y., Nishijima, M., Hayashi, H., Hukasawa, Y., Yokoyama, T., Yoneda, M., Moriguchi, H., Uchiyama, H., Shiro, T., Ito, Y.: ibid. 41, (1988). 12) Michitsuji, H., Ohara, A., Yamaguchi, K., Fujiki, K.: Shojinkai Igakushi 26, (1987).

RITONAVIRI COMPRESSI RITONAVIR TABLETS. Final text for addition to The International Pharmacopoeia (July 2012)

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