/ . Biochem., 78, 269-275 (1975)
Inhibition of Intestinal Absorption of Phenylalanine by Phenylalaninol Kiyokazu SHIMOMURA,* Tomiko FUKUSHIMA,* Tamotsu DANNO,* Kazuo MATSUMOTO,** Munetsugu MIYOSHI,** and Yoshio KOWA* *Safety Research Laboratory, **Research Laboratory of Applied Biochemistry, Tanabe Seiyaku Co., Ltd., Yodogawa-ku, Osaka, Osaka 532 Received for publication, February 12, 1975
Plasma phenylalanine and tyrosine levels in rats which had been orally administered L-phenylalaninol and L-phenylalanine were determined. Since these amino acid levels in rats administered L-phenylalanine solution containing L-phenylalaninol were significantly lower than those in rats administered L-phenylalanine alone, L-phenylalaninol appears to inhibit the intestinal absorption of L-phenylalanine. This effect was more potent than that of cycloleucine. L-Phenylalaninol inhibited the phenylalanine transport of everted sacs. The Km value of L-phenylalanine was 3.44xlO~3M and the Ki value of L-phenylalaninol was 7.69xlO"3M from Lineweaver-Burk plots. From these two curves, it appeared that L-phenylalaninol may competitively inhibit the intestinal transport of L-phenylalanine. The effects of L-phenylalanine, L-phenylalaninol and cycloleucine on the urinary excretions of Na+ and K+ in rats were also examined. Potassium excretion, which increased on oral administration of L-phenylalanine, was suppressed by the administration of L-phenylalaninol but not by administration of cycloleucine. L-Phenylalaninol alone enhanced Na+ excretion in urine. These results confirmed that L-phenylalaninol shows inhibitory effects as potent as those of cycloleucine on the intestinal absorption of L-phenylalanine.
Phenylketonuria arises from a congenital defeet in phenylalanine hydroxylase [EC 1.14.16.1] and causes mental retardation ( / ) . However, phenylalanine hydroxylase activity has recently been demonstrated in phenylketonuria (2). In order to find a therapeutic agent for phenylketonuria, various substances have been investigated for stimulation of the enzyme ( 3 - 5 ) or inhibition of the intestinal absorption of phenylalanine (5). Cycloleucine Vol. 78, No. 2, 1975
269
is useless because it suppresses the intestinal absorption not only of phenylalanine but also of other amino acid (7). If a substance which shows a specific inhibitory effect on the intestinal absorption of phenylalanine can be found, mental retardation due to excessive accumulation of phenylalanine might be prevented by oral administration of the substance, As a result of screening for compounds which inhibit the intestinal absorption of
270
K. SHIMOMURA, T. FUKUSHIMA, T. DANNO, K. MATSUMOTO, M. MIYOSHI, and Y. KOWA
phenylalanine, it was found that phenylalaninol showed such an effect. This paper deals with the inhibitory effect of phenylalaninol on intestinal absorption of phenylalanine in the phenylalanine load test and in the everted sac method. MATERIALS AND METHODS L-Leucyl-L-alanine was purchased from Nutritional Biochemicals Corporation. L-Phenylalaninol was synthesized in the Research Laboratory of Applied Biochemistry, Tanabe Seiyaku Co., Ltd. Other reagents used here were all chemically pure grades. Deionized distilled water obtained from a Yamato Auto Still, Model WA-510, was used to rinse all glassware and prepare all reagents. Phenylalanine Load Tests — Male Wistar rats weighing 200-280 g were fasted overnight and then injected with 40 mg/kg of pentothal sodium intraperitoneally. Blood samples were obtained from polyethylene canulae filled with heparin sodium solution (500 U/ml) and placed in the jugular vein. Thirty, 60 or 90 min after oral administration of phenylalaninol dissolved in 300 mg/10 ml of L-phenylalanine or pure water, plasma phenylalanine and tyrosine levels were determined by the fiuorometric methods of McCaman and Robins (8) and Wong et al. (9), respectively. Ten ml per kg of L-phenylalanine solution without L-phenylalaninol was administered orally as a control. Everted Sac Experiments — Male Wistar strain rats weighing 180—260 g were fasted overnight and killed by stunning and decapitation. The small intestine, from a point 34 cm under the pylorus, was quickly removed, rinsed, everted and tied into sacs 5—6 cm (450 —600 mg) in length according to the method of Wilson and Wiseman (10). Three sacs were prepared from each rat. L-Phenylalanine or L-tyrosine in Krebs-Henseleit bicarbonate buffer, pH 7.4, containing 20 mM glucose was placed within the sacs (0.5 ml) at the same concentration as the solution outside the sacs (5.0 ml). Incubation time was 1 hr and the temperature was 37° unless otherwise indicated. In some experiments, phenylalaninol was added at a 5 : 1 or 10 : 1 ratio with respect to the
other amino acids. In all cases the buffers were kept isotonic by the addition of appropriate amounts of sodium chloride. Flasks of 25 ml capacity were initially flushed with 95% oxygen and 5% carbon dioxide and incubated in a water bath on a vibrating rack (55 oscillations/min). Phenylalanine and tyrosine were assayed by the methods {8,9) described above. Sacs were weighed after incubation then drained and reweighed. Sac weight and internal volume were calculated. The sac weight was determined after drying for 2 hr at 110°. Sodium and Potassium Excretions in Urine —Male Wistar strain rats weighing 200-280 g were used. Rats were fasted overnight and were divided into six groups as follows. I) LPhenylalanine 800 mg/kg, II) L-Phenylalanine 800 mg/kg plus phenylalaninol 200 mg/kg, III) L-Phenylalanine 800 mg/kg plus cycloleucine 200 mg/kg, IV) Phenylalaninol 200 mg/kg, V) Cycloleucine 200 mg/kg, VI) Pure water. LPhenylalanine was dissolved in water in a boiling water bath and the others were dissolved in water at room temperature. Individual rats were each placed in a metabolic cage and urine was collected for six hours after oral administration of these compounds. Sodium and potassium in the urine were assayed by the flame photometric method (11). RESULTS Figure 1 shows rat plasma phenylalanine levels after oral administration of L-phenylalanine (100, 200, or 300 mg/kg). The plasma phenylalanine level 1 hr after a load of 300 mg/kg of L-phenylalanine was approximately 2.5 times higher than the normal level and regained the initial level 3 hr after the load. When the absorption of L-phenylalanine from the intestinal tract in vivo is inhibited by some antagonist, the plasma phenylalanine levels of rats loaded with L-phenylalanine should be several times the normal level and the effects of the antagonist should be reflected by the variation of plasma phenylalanine levels. Three hundred mg of L-phenylalanine per kg body weight was administered and plasma phenylalanine levels at 30, 60, and 120 min after administration were determined. Figures / . Biochem.
PHENYLALANINOL AND PHENYLALANINE ABSORPTION
271
180
TIME (min) Fig. 1. Plasma phenylalanine levels after after oral administration of L-phenylalanine in rats. L-Phenylalanine was administered orally at zero time. Plasma samples were taken for the determination of phenylalanine. Results are shown as mean values with standard errors for 6 rats. D, 300mg/kg; A, 200 mg/kg; O, lOOmg/kg.
30
60 TIME (mm)
120
Fig. 2. Plasma phenylalanine levels after oral administration of L-phenylalanine and phenylalaninol in rats. 300 mg of L-phenylalanine per kg body weight alone or with 40-300 mg of phenylalaninol per kg body weight was administered orally at zero time. Results are shown as mean values for 6 rats, and brackets indicate the standerd errors. O, 300 mg/kg of L-phenylalanine; 300 mg/kg of L-phenylalanine plus 300 mg/kg of phenylalaninol ( • ) , 150 mg/kg of phenylalaninol ( • ) , 75 mg/kg of phenylalaninol ( A ) , or 40mg/kg of phenylalaninol (X); A, 300 mg/kg of phenylalaninol alone.
2 and 3 show rat plasma phenylalanine levels after loading both L-phenylalanine and phenylalaninol or both L-phenylalanine and cycloVol. 78, No. 2, 1975
30
60 TIME (min)
120
Fig. 3. Plasma phenylalanine levels after oral administration of L-phenylalanine and cycloleucine in rats. 300 mg of L-phenylalanine per kg body weight alone or with 100-200 mg of cycloleucine per kg body weight was administered orally at zero time. Results are shown as mean values for 6 rats, and brackets indicate the standard errors. 0 , 300 mg/kg of L-phenylalanine; 300 mg/kg of L-phenylalanine plus 200 mg/kg of cycloleucine ( A ) or plus 100 mg/kg of cycloleucine ( • ) .
leucine. Plasma phenylalanine level 30 min after the administration of L-phenylalanine were approximately 6 times higher (12.5±0.9 mg/dl) than the normal value and decreased to one-half of the maximal level after 60 min, gradually returning to the normal level after 120 min. Simultaneous administration of phenylalanine and phenylalaninol yielded significant lower plasma phenylalanine levels 30 and 60 min after loading than administration of phenylalanine alone. The ascent ratio of plasma phenylalanine level 30 min after loading determined from the dose of phenylalaninol and the sigmoid dose-response curves, which showed inverse relationships, was obtained as described in Fig. 4. Plasma phenylalanine levels of normal rats administered 300 mg/kg of phenylalaninol alone were normal and showed no significant variation. The administration of water alone gave similar results. Cycloleucine, which inhibited the intestinal absorption of phenylalanine (5), and L-phenylalanine were administered orally at same time. In this case, plasma phenylalanine levels 30 min
272
K. SHIMOMURA, T. FUKUSHIMA, T. DANNO, K. MATSUMOTO, M. MIYOSHI, and Y. KOWA 100
10
20 40 100 200 300 PHENYLALANINOL (mg/kg)
Fig. 4. Dose-response curve for phenylalaninol. This was carried out simultaneously with the experiments in Fig. 2. The vertical axis indicates the percentage level of plasma phenylalanine, with the plasma phenylalanine level 30 min after oral administration of L-phenylalanine taken as 100. Results are shown as mean values for 6 rats.
after administration were approximately 3 times higher than normal and the inhibitory effect of cycloleucine corresponded to that of 40 mg/ kg of phenylalaninol. Figure 5 shows plasma tyrosine levels when both L-phenylalanine and phenylalaninol were administered simultaneously. Plasma tyrosine levels 30 or 60 min after loading 300 mg/kg of L-phenylalanine alone were enhanced approximately 3.2-fold over the normal value and decreased to 2.5 times the normal value. This pattern differs from that of plasma phenylalanine but suggests that phenylalanine loaded orally is converted to tyrosine by liver phenylalanine hydroxylase. Plasma tyrosine levels 30 and 60 min after the simultaneous administration of L-phenylalanine and 300 mg/kg of phenylalaninol were 2.5±0.3 and 2.4+0.2 mg/dl, respectively, and these levels were significantly (/>
Inhibition of Intestinal Absorption of Phenylalanine by Phenylalaninol Kiyokazu SHIMOMURA,* Tomiko FUKUSHIMA,* Tamotsu DANNO,* Kazuo MATSUMOTO,** Munetsugu MIYOSHI,** and Yoshio KOWA* *Safety Research Laboratory, **Research Laboratory of Applied Biochemistry, Tanabe Seiyaku Co., Ltd., Yodogawa-ku, Osaka, Osaka 532 Received for publication, February 12, 1975
Plasma phenylalanine and tyrosine levels in rats which had been orally administered L-phenylalaninol and L-phenylalanine were determined. Since these amino acid levels in rats administered L-phenylalanine solution containing L-phenylalaninol were significantly lower than those in rats administered L-phenylalanine alone, L-phenylalaninol appears to inhibit the intestinal absorption of L-phenylalanine. This effect was more potent than that of cycloleucine. L-Phenylalaninol inhibited the phenylalanine transport of everted sacs. The Km value of L-phenylalanine was 3.44xlO~3M and the Ki value of L-phenylalaninol was 7.69xlO"3M from Lineweaver-Burk plots. From these two curves, it appeared that L-phenylalaninol may competitively inhibit the intestinal transport of L-phenylalanine. The effects of L-phenylalanine, L-phenylalaninol and cycloleucine on the urinary excretions of Na+ and K+ in rats were also examined. Potassium excretion, which increased on oral administration of L-phenylalanine, was suppressed by the administration of L-phenylalaninol but not by administration of cycloleucine. L-Phenylalaninol alone enhanced Na+ excretion in urine. These results confirmed that L-phenylalaninol shows inhibitory effects as potent as those of cycloleucine on the intestinal absorption of L-phenylalanine.
Phenylketonuria arises from a congenital defeet in phenylalanine hydroxylase [EC 1.14.16.1] and causes mental retardation ( / ) . However, phenylalanine hydroxylase activity has recently been demonstrated in phenylketonuria (2). In order to find a therapeutic agent for phenylketonuria, various substances have been investigated for stimulation of the enzyme ( 3 - 5 ) or inhibition of the intestinal absorption of phenylalanine (5). Cycloleucine Vol. 78, No. 2, 1975
269
is useless because it suppresses the intestinal absorption not only of phenylalanine but also of other amino acid (7). If a substance which shows a specific inhibitory effect on the intestinal absorption of phenylalanine can be found, mental retardation due to excessive accumulation of phenylalanine might be prevented by oral administration of the substance, As a result of screening for compounds which inhibit the intestinal absorption of
270
K. SHIMOMURA, T. FUKUSHIMA, T. DANNO, K. MATSUMOTO, M. MIYOSHI, and Y. KOWA
phenylalanine, it was found that phenylalaninol showed such an effect. This paper deals with the inhibitory effect of phenylalaninol on intestinal absorption of phenylalanine in the phenylalanine load test and in the everted sac method. MATERIALS AND METHODS L-Leucyl-L-alanine was purchased from Nutritional Biochemicals Corporation. L-Phenylalaninol was synthesized in the Research Laboratory of Applied Biochemistry, Tanabe Seiyaku Co., Ltd. Other reagents used here were all chemically pure grades. Deionized distilled water obtained from a Yamato Auto Still, Model WA-510, was used to rinse all glassware and prepare all reagents. Phenylalanine Load Tests — Male Wistar rats weighing 200-280 g were fasted overnight and then injected with 40 mg/kg of pentothal sodium intraperitoneally. Blood samples were obtained from polyethylene canulae filled with heparin sodium solution (500 U/ml) and placed in the jugular vein. Thirty, 60 or 90 min after oral administration of phenylalaninol dissolved in 300 mg/10 ml of L-phenylalanine or pure water, plasma phenylalanine and tyrosine levels were determined by the fiuorometric methods of McCaman and Robins (8) and Wong et al. (9), respectively. Ten ml per kg of L-phenylalanine solution without L-phenylalaninol was administered orally as a control. Everted Sac Experiments — Male Wistar strain rats weighing 180—260 g were fasted overnight and killed by stunning and decapitation. The small intestine, from a point 34 cm under the pylorus, was quickly removed, rinsed, everted and tied into sacs 5—6 cm (450 —600 mg) in length according to the method of Wilson and Wiseman (10). Three sacs were prepared from each rat. L-Phenylalanine or L-tyrosine in Krebs-Henseleit bicarbonate buffer, pH 7.4, containing 20 mM glucose was placed within the sacs (0.5 ml) at the same concentration as the solution outside the sacs (5.0 ml). Incubation time was 1 hr and the temperature was 37° unless otherwise indicated. In some experiments, phenylalaninol was added at a 5 : 1 or 10 : 1 ratio with respect to the
other amino acids. In all cases the buffers were kept isotonic by the addition of appropriate amounts of sodium chloride. Flasks of 25 ml capacity were initially flushed with 95% oxygen and 5% carbon dioxide and incubated in a water bath on a vibrating rack (55 oscillations/min). Phenylalanine and tyrosine were assayed by the methods {8,9) described above. Sacs were weighed after incubation then drained and reweighed. Sac weight and internal volume were calculated. The sac weight was determined after drying for 2 hr at 110°. Sodium and Potassium Excretions in Urine —Male Wistar strain rats weighing 200-280 g were used. Rats were fasted overnight and were divided into six groups as follows. I) LPhenylalanine 800 mg/kg, II) L-Phenylalanine 800 mg/kg plus phenylalaninol 200 mg/kg, III) L-Phenylalanine 800 mg/kg plus cycloleucine 200 mg/kg, IV) Phenylalaninol 200 mg/kg, V) Cycloleucine 200 mg/kg, VI) Pure water. LPhenylalanine was dissolved in water in a boiling water bath and the others were dissolved in water at room temperature. Individual rats were each placed in a metabolic cage and urine was collected for six hours after oral administration of these compounds. Sodium and potassium in the urine were assayed by the flame photometric method (11). RESULTS Figure 1 shows rat plasma phenylalanine levels after oral administration of L-phenylalanine (100, 200, or 300 mg/kg). The plasma phenylalanine level 1 hr after a load of 300 mg/kg of L-phenylalanine was approximately 2.5 times higher than the normal level and regained the initial level 3 hr after the load. When the absorption of L-phenylalanine from the intestinal tract in vivo is inhibited by some antagonist, the plasma phenylalanine levels of rats loaded with L-phenylalanine should be several times the normal level and the effects of the antagonist should be reflected by the variation of plasma phenylalanine levels. Three hundred mg of L-phenylalanine per kg body weight was administered and plasma phenylalanine levels at 30, 60, and 120 min after administration were determined. Figures / . Biochem.
PHENYLALANINOL AND PHENYLALANINE ABSORPTION
271
180
TIME (min) Fig. 1. Plasma phenylalanine levels after after oral administration of L-phenylalanine in rats. L-Phenylalanine was administered orally at zero time. Plasma samples were taken for the determination of phenylalanine. Results are shown as mean values with standard errors for 6 rats. D, 300mg/kg; A, 200 mg/kg; O, lOOmg/kg.
30
60 TIME (mm)
120
Fig. 2. Plasma phenylalanine levels after oral administration of L-phenylalanine and phenylalaninol in rats. 300 mg of L-phenylalanine per kg body weight alone or with 40-300 mg of phenylalaninol per kg body weight was administered orally at zero time. Results are shown as mean values for 6 rats, and brackets indicate the standerd errors. O, 300 mg/kg of L-phenylalanine; 300 mg/kg of L-phenylalanine plus 300 mg/kg of phenylalaninol ( • ) , 150 mg/kg of phenylalaninol ( • ) , 75 mg/kg of phenylalaninol ( A ) , or 40mg/kg of phenylalaninol (X); A, 300 mg/kg of phenylalaninol alone.
2 and 3 show rat plasma phenylalanine levels after loading both L-phenylalanine and phenylalaninol or both L-phenylalanine and cycloVol. 78, No. 2, 1975
30
60 TIME (min)
120
Fig. 3. Plasma phenylalanine levels after oral administration of L-phenylalanine and cycloleucine in rats. 300 mg of L-phenylalanine per kg body weight alone or with 100-200 mg of cycloleucine per kg body weight was administered orally at zero time. Results are shown as mean values for 6 rats, and brackets indicate the standard errors. 0 , 300 mg/kg of L-phenylalanine; 300 mg/kg of L-phenylalanine plus 200 mg/kg of cycloleucine ( A ) or plus 100 mg/kg of cycloleucine ( • ) .
leucine. Plasma phenylalanine level 30 min after the administration of L-phenylalanine were approximately 6 times higher (12.5±0.9 mg/dl) than the normal value and decreased to one-half of the maximal level after 60 min, gradually returning to the normal level after 120 min. Simultaneous administration of phenylalanine and phenylalaninol yielded significant lower plasma phenylalanine levels 30 and 60 min after loading than administration of phenylalanine alone. The ascent ratio of plasma phenylalanine level 30 min after loading determined from the dose of phenylalaninol and the sigmoid dose-response curves, which showed inverse relationships, was obtained as described in Fig. 4. Plasma phenylalanine levels of normal rats administered 300 mg/kg of phenylalaninol alone were normal and showed no significant variation. The administration of water alone gave similar results. Cycloleucine, which inhibited the intestinal absorption of phenylalanine (5), and L-phenylalanine were administered orally at same time. In this case, plasma phenylalanine levels 30 min
272
K. SHIMOMURA, T. FUKUSHIMA, T. DANNO, K. MATSUMOTO, M. MIYOSHI, and Y. KOWA 100
10
20 40 100 200 300 PHENYLALANINOL (mg/kg)
Fig. 4. Dose-response curve for phenylalaninol. This was carried out simultaneously with the experiments in Fig. 2. The vertical axis indicates the percentage level of plasma phenylalanine, with the plasma phenylalanine level 30 min after oral administration of L-phenylalanine taken as 100. Results are shown as mean values for 6 rats.
after administration were approximately 3 times higher than normal and the inhibitory effect of cycloleucine corresponded to that of 40 mg/ kg of phenylalaninol. Figure 5 shows plasma tyrosine levels when both L-phenylalanine and phenylalaninol were administered simultaneously. Plasma tyrosine levels 30 or 60 min after loading 300 mg/kg of L-phenylalanine alone were enhanced approximately 3.2-fold over the normal value and decreased to 2.5 times the normal value. This pattern differs from that of plasma phenylalanine but suggests that phenylalanine loaded orally is converted to tyrosine by liver phenylalanine hydroxylase. Plasma tyrosine levels 30 and 60 min after the simultaneous administration of L-phenylalanine and 300 mg/kg of phenylalaninol were 2.5±0.3 and 2.4+0.2 mg/dl, respectively, and these levels were significantly (/>
