Bioavailability
of Phenylalanine and Aspartate From Aspartame Capsules and Solution
(20 mg/kg)
in
Thomas S. Burns, W. Wayne Stargel, and Aryeh Hurwitz Aspartame (L-aspartyl+-phenylalanine methyl ester) was given in capsules or solution to compare the bioavailability of its constituent amino acids, aspartate and phenylalanine. Twenty healthy subjects received a single 20 mg/kg dose of aspartame in capsules or solution in a randomized, crossover design. Plasma amino acid concentrations and the phenylalanine to large neutral amino acid ratios (Phe/LNAA) were determined. Plasma aspartate concentrations did not increase with either treatment. For plasma phenylalanine following capsule ingestion, there was a smaller peak plasma concentration (Cmax; 103.3 w 126.6 fimol/Lj, a longer time to peak concentration (tmax; 108.6 Y 36.6 minutes). but no significant difference in the area under the plasma concentration-time curve (AUCj (7,656 v 7,200 pmol - min/Lj when compared with solution ingestion. The maximum plasma Phe/LNAA ratio was smaller (0.16 v 0.19) with capsules. The changes for plasma tyrosine were similar to those seen with phenylalanine. There were no significant differences in the plasma concentrations of the other LNAAs between capsule and solution ingestion. Thus, given the small effect on phenylalanine Cmax and Phe/LNAA and no effect on the extent of absorption of phenylalanine, aspartame ingested in capsules at doses up to 20 mg/kg is a suitable dosage form for blinded clinical studies, provided that the slower rate of absorption of phenvlalanine from capsules is taken into account. . . @ 1990 by W.B. Saunders Company.
A
LTHOUGH THE DIPEPTIDE sweetener aspartame (L-aspartyl+phenylalanine methyl ester) is routinely consumed in solution in beverages, its sweet taste cannot be masked when it is ingested in solution. Therefore, blinded clinical studies with aspartame require the use of capsules. Upon ingestion, aspartame is hydrolyzed to equimolar quantities of aspartate, phenylalanine, and methanol. Aspartate is rapidly metabolized and thus the plasma aspartate concentrations are not significantly elevated following acute aspartame doses of 34 to 50 mg/kg body weight, whereas plasma phenylalanine concentrations may increase depending on dose.’ Methanol is also rapidly metabolized and blood concentrations are usually not detectable unless large bolus doses of aspartame (250 mg/kg) are administered.’ Aspartame ingestion may also increase the ratio of the plasma phenylalanine concentration to the sum of the plasma concentrations of the other large neutral amino acids (Phe/ LNAA).3 It has been proposed that increased plasma Phe/ LNAA ratios may increase brain phenylalanine uptake in humans.4 A previous study’ indicated that the plasma concentrations of aspartate and phenylalanine were significantly less when aspartame was ingested in capsules compared with solution. The reason given for the difference observed was a delay in absorption due to capsule disintegration and a slow dissolution rate of aspartame in the stomach and the intestinal lumen. The extent of absorption, as measured by the area under the plasma concentration-time curve (AUC), was also less with capsules. Presumably, because of its relatively low
solubility, aspartame administered at such large bolus doses is incompletely dissolved in the intestinal lumen. The subjects in the above study were given a single 3-g dose of aspartame (-50 mg/kg). This dose is equivalent to the amount of aspartame in approximately 6 L of aspartamesweetened beverage. Because phenylalanine is speculated to be associated with adverse reactions allegedly due to aspartame consumption, the rate and extent of phenylalanine absorption are of importance when using aspartame in capsules in clinical studies. The pharmacokinetic response of plasma phenylalanine was determined at an aspartame dose of 20 mg/kg, which is equivalent to the amount of aspartame contained in approximately 2.5 L of an aspartame-sweetened beverage ingested by a 70-kg individual. It is not likely that a single dose of such magnitude would be exceeded by an individual. The previous study’ showed that a single aspartame dose of approximately 50 mg/kg in capsules resulted in a decrease in phenylalanine bioavailability compared with aspartame in solution. An observation of equivalent phenylalanine bioavailability from capsules and solution with a lower dose of aspartame, ie, 20 mg/kg, would demonstrate the suitability of capsules for use in blinded clinical studies. The present investigation compared the plasma concentrations of aspartate and phenylalanine in healthy subjects following the ingestion of a single 20 mg/kg dose of aspartame in capsules or solution in a randomized, two-way crossover design. MATERIALS AND METHODS
From the Department of Clinical Research, The NutraSweet Company, Deerjeld, IL; and the Division of Clinical Pharmacology, The University of Kansas Medical Center, Kansas City, KS. Supported by a grant from The NutraSweet Company, Deerfield, IL. Address reprint requests to Aryeh Hurwitz, MD. Division of Clinical Pharmacology, The University of Kansas Medical Center. Kansas City, KS 66103. @ 1990 by W.B. Saunders Company. 0026-0495/90/3911-0016$03.00/0 1200
The study protocol was approved by the University of Kansas College of Health Sciences and Hospital Human Subjects Committee. The study population consisted of 20 healthy subjects, 10 men and 10 women, from whom informed written consent was obtained. The subjects ranged in age from 18 to 36 years (mean, 26.0 f 3.7 years). Their body weights ranged from 45 to 94 kg (mean, 65.8 -t 12.8 kg). The subjects were assigned to a treatment sequence by means of a computer-generated random assignment schedule. An overnight fast of at least 10 hours preceded the administration of aspartame. On 1 study day, the subjects ingested 300 mL of unsweetened beverage Metabolism, Vol39,
No 11 (November), 1990: pp 1200- 1203
ASPARTAME IN CAPSULES AND SOLUTION
plus a sufficient number of capsules, each containing 300 mg of aspartame, so as to provide a dose as close as possible to 20 mg/kg. On the other study day, the subjects ingested 300 mL of beverage in which the same amount of aspartame as given in capsules was dissolved. The subjects also ingested empty capsules with the solution. The 2 study days were separated by a washout period of at least 1 week. Heparinized blood samples for plasma amino acid analyses were obtained before dosing and at 15, 30,45,60, 90, 120, 150, 180,240, 360, and 480 minutes after ingestion. Plasma was immediately separated from cellular elements by low-speed centrifugation and stored at - 70°C until analyzed. Samples were prepared for analysis by precipitating the proteins with 5% sulfosalicylic acid in pH 2.2 lithium citrate buffer (0.2 mol/L) and centrifugation (17,000 x g for 15 minutes) to remove proteins. The resulting supernatant was then passed through a 0.2-pm syringe filter, and the filtrate was stored frozen at -20°C until analyzed. The concentrations of 28 amino acids were measured using a Beckman 6300 Amino Acid Analyzer (Beckman Instruments, Palo Alto, CA) with an on-line Spectra-Physics SP4200 Computing Integrator (Spectra-Physics, San Jose, CA). Plasma total tryptophan was determined by the fluorometric method of Denckla and Dewey’ as modified by Bloxam and Warren.’ The unsweetened beverage was cherry-flavored Kool-Aid mix (General Foods, White Plains, NY), which was reconstituted according to package instructions. The aspartame-filled capsules were provided by G.D. Searle (Skokie, IL). Mean (-rSD) plasma concentrations of each of 29 amino acids were determined at each time point. For each subject the apparent peak plasma concentration (Cmax) and time to peak concentration (tmax) were determined by inspection of the individual plasma concentration-time curves. The AUC was calculated by the trapezoidal rule,8 with the fasting plasma concentration at 0 time as the base of the trapezoid. The AUC was evaluated at 4, 6, and 8 hours after aspartame ingestion. The AUC was not determined beyond 8 hours, because plasma amino acid concentrations were expected to have returned to baseline values by then. The ratio of plasma phenylalanine concentration to the sum of the plasma concentrations of the other large neutral amino acids (Phe/LNAA) was determined for each subject at each time point. The other LNAAs were isoleucine (Ile), leucine (Leu), methionine (Met), tryptophan (Trp), tyrosine (Tyr), and valine (Val). Statistical analyses comparing solution to capsules were performed using ANOVA for a two-period crossover study. The paired Student’s t test was used for changes over time within a study arm. Statistical analyses were performed using SAS, Version V, as implemented on an IBM 308 1. All statistical testing was performed at the two-tailed, (Y5 0.05, level of significance. RESULTS Mean (*SD) plasma phenylalanine concentrations following ingestion of aspartame in capsules or solution at 20 mg/kg are given in Fig 1, with relevant parameters in Table 1. When aspartame was ingested in solution, plasma phenylalanine concentration increased significantly from a mean baseline of 65.7 t 13.6 pmol/L to a high mean value of 118.9 2 28.6 timol/L at 30 minutes after dosing. The mean AUC for plasma phenylalanine from 0 to 4 hours was 6,012 k 1,866 wmol . min/L. Mean AUC(O-6) and AUC(O-8) values were 6,864 + 3,078 and 7,200 k 4,236 Hmol - min/L, respectively. When aspartame was ingested in capsules, plasma phenylalanine concentrations increased significantly from a mean baseline value of 67.1 * 9.9
1201
160.
Phenylalanine
140.
120.
I
I
I
I
1
i1
I
I
I
I
I I Aspartate
I
I
I
I
I
I
3
4
5
6
7
8
I
2
I
TIME, hours Fig 1. Mean (k SD) plasma phenylalanine and aspartate concentrations (Pmol/Lj in 20 normal adults ingesting aspartame at 20 mglkg in solution (O-0) or capsules f-j.
pmol/L to a high mean value of 95.3 i 13.8 pmol/L at 2.5 hours after dosing. The mean AUC values for 4, 6, and 8 hours were 5,022 * 1,782, 6,702 ? 2,634, and 7,656 + 3,120 rmol - min/L, respectively. The changes in plasma phenylaTable 1. Pharmacokinetic and Aspartate
Parameters
for Plasma Phenylalanine
for 20 Subjects Ingesting Aspartame
at 20 mg/kg
in Solution or Capsules Phenylalanine Parameter
Baseline (pmol/L)
Solution 65.7 (13.6)
High mean (bmol/L) Cmax (pmol/L) tmax (min) A UC O-4 h (pm01. min/L) AUC O-6 h (fimol. min/L) AUCO-8h (firnol. min/L)
Capsules
Aspartate Solution
67.1 (9.9)
Capsules
5.1
5.3
(2.1)
95.3”
5.7
(2.3) 5.9
(28.6)
(13.8)
(2.4)
(2.4)
126.6
103.3’
7.6
8.7
(27.4)
(14.4)
(3.3)
36.6
108.6”
110.4
(3.3) 94.8
(11.4)
(49.8)
(168.0)
(1 11.6)
118.9
6,012
5,022
-48
(1,866) 6,864
(1.782) 6,702
(384) -108
(372) -12
(3.076) 7,200
(2,634) 7,656
1510) -186
(5881 -144
(4,236)
(3.120)
(654)
(756)
NOTE. Values are means (SD). *Significantly different from solution (p -c 0.05).
54
BURNS, STARGEL, AND HURWITZ
1202
lanine concentrations from baseline were significantly larger at 15, 30, 45, and 60 minutes after dosing and significantly smaller at 150, 180, and 240 minutes after dosing when aspartame was ingested in solution compared with capsules. There were no significant differences between the solution and capsules for the mean 4-, 6-, and 8-hour AUC values. The individual peak plasma phenylalanine concentrations without regard to time (Cmax) were also determined along with the corresponding times to reach Cmax (tmax) (Table 1). The mean Cmax value for aspartame in solution was 126.6 f 27.4 pmol/L, compared with 103.3 f 14.4 pmol/L for aspartame in capsules. The corresponding tmax values were 36.6 + 11.4 minutes (solution) and 108.6 + 49.8 minutes (capsules). For aspartame ingested in solution compared with capsules, the mean Cmax value was significantly larger and the mean tmax value was significantly shorter. The mean plasma Phe/LNAA ratios are shown in Fig 2. When aspartame was ingested in solution, the plasma Phe/ LNAA ratio increased significantly from a mean baseline value of 0.10 + 0.01 to a high mean value of 0.18 2 0.03 at 30 minutes after dosing. When aspartame was ingested in capsules, the mean baseline ratio was 0.10 + 0.01 and the high mean ratio was 0.15 * 0.03 at 90 minutes after dosing. The mean of the individual maximum Phe/LNAA ratios was significantly greater and occurred significantly earlier for aspartame ingested in solution (0.19 t 0.03 at 43.5 * 16.1 minutes) compared with capsules (0.16 f 0.03 at 126.7 i 95.8 minutes). The 4-, 6-, and 8-hour Phe/LNAA AUC values did not differ significantly between solution and capsule ingestion. The mean plasma aspartate concentrations are shown in Fig 1 and relevant parameters in Table 1. Plasma aspartate concentrations did not increase significantly from baseline following the ingestion of aspartame in either solution or capsules. Also, there were no significant differences in the mean Cmax and tmax values, nor were the AUC values for 4, 6, and 8 hours different between solution and capsules (Table 1). Plasma concentrations
of tyrosine
increased
over baseline
0.30 1
Fig 2. Mean (_cSDi values for the ratio of plasma phenylalanine concentration to the sum of the plasma concentrations of the other large neutral amino acids (Phe/LNAA) in 20 normal adults ingesting aspartame at 20 mglkg in solution (-1 or capsules w--w.
after both solution and capsules, reflecting the conversion of phenylalanine to tyrosine. The mean Cmax value was significantly larger for aspartame ingested in solution (88.9 + 2.2 v 78.9 + 1.6 Mmol/L), whereas the mean tmax value was significantly smaller (50.4 k 36.0 v 98.4 f 89.4 minutes) compared with aspartame ingested in capsules. The mean AUC values for 4, 6, and 8 hours did not differ significantly between solution and capsules. The mean plasma Tyr/ LNAA ratios did not show any meaningful changes following capsules or solution. The mean of the individual maximum Tyr/LNAA ratios was not significantly different for solution (0.13 k 0.02) and capsules (0.12 + 0.02), but did occur significantly earlier (62.2 + 28.5 v 108 f 85.5 minutes) following aspartame in solution. The plasma concentrations of the branched chain amino acids (Ile, Leu, and Val) and Trp did not differ significantly between aspartame ingested in solution or in capsules. The LNAA ratios for Ile, Leu, Val, and Trp showed small decreases following solution compared with capsules at time points of 1 hour or less after aspartame ingestion. These decreases were attributable to the increased plasma Phe concentration associated with aspartame in solution in the denominator of the ratios at these early time points. The means of the individual maximum ratios for Ile, Leu, Val, and Trp were not significantly different for solution and capsules. Plasma concentrations of the other amino acids did not change significantly following aspartame ingestion in either solution or capsules. DISCUSSION
The comparison of solution and capsule formulations showed a delay in phenylalanine absorption from aspartame ingested in capsules due, initially, to capsule disintegration time. However, the most important factor contributing to the absorption delay was probably the dissolution time in the stomach and intestinal lumen of aspartame, which has relatively poor solubility in aqueous solution. Gastric emptying time differences due to the presence of capsules and undissolved aspartame also may have been a factor. These factors were most likely responsible for the differences in plasma phenylalanine Cmax and tmax values and Phe/ LNAA ratios observed in the present study. Although the mean plasma phenylalanine Cmax values (126.6 v 103.3 Mmol/L) and the mean peak Phe/LNAA ratios (0.19 v 0.16) were statistically significantly greater following ingestion of aspartame in solution compared with capsules, the differences between the solution and capsule mean values were relatively small. For comparison purposes, the reported normal postprandial plasma phenylalanine concentration range is 60 to 120 pmol/L.’ Thus, peak plasma phenylalanine concentrations, a measure of the rate of phenylalanine absorption, following aspartame given as either capsules or solution, generally remained within the reported postprandial range. The pronounced tmax difference (36.6 v 108.6 minutes) reflected the delay in absorption of phenylalanine from aspartame capsules. The AUC values, a measure of the extent of phenylalanine absorption, were not significantly
ASPARTAME
1203
IN CAPSULES AND SOLUTION
different for capsules and solution. Thus, although there was some reduction in the rate of absorption of phenylalanine from capsules compared with solution, the extent of absorption did not differ significantly. Following aspartame doses much larger’ than the 20 mg/kg used in this study, the extent of absorption may differ due to incomplete aspartame dissolution from capsules. It is our opinion that the extent of phenylalanine absorption (AUC) is the more appropriate pharmacokinetic parameter for evaluating putative adverse effects. This opinion is supported by the phenylketonuria which indicates that sustained, elevated plasma literature,““’ concentrations of phenylalanine (2 1200 pmol/L) are associated with neurotoxicity. However, transient, elevated plasma concentrations resulting from large loading doses of aspartame’3”5 do not produce any adverse effects, including cognitive or behavioral effects. The plasma concentrations of aspartate from a 20 mg/kg dose of aspartame were not significantly increased by ingestion of the aspartame in either solution or capsules. This is
consistent with previous studies’ that showed that plasma aspartate concentrations were not significantly affected by acute aspartame doses ranging from 34 to 50 mg/kg. In conclusion, given the small effect on phenylalanine Cmax and Phe/LNAA and no effect on the extent of phenylalanine absorption from aspartame ingested in capsules compared with solution, aspartame in capsules is a suitable dosage form for blinded clinical studies at single doses up to 20 mg/kg. However, the investigator must take into account that peak plasma phenylalanine concentrations following aspartame ingestion in capsules occur later than comparable doses ingested in solution.
ACKNOWLEDGMENT
We wish to thank Dr David K. Rassin, The University
of Texas Medical Branch at Galveston, TX, for the amino acid analyses and Dr Dennis Haack, Statistical Consultants, Inc. Lexington, KY, for the statistical
analyses.
REFERENCES
1. Stegink LD: Aspartame metabolism in humans: Acute dosing studies, in Stegink LD, Filer LJ Jr (eds): Aspartame: Physiology and Biochemistry. New York, NY, Marcel Dekker, 1984, pp 509-553 2. Stegink LD, Brummel MC, McMartin K, et al: Blood methanol concentrations in normal adult subjects administered abuse doses of aspartame. J Toxicol Environ Health 7:218-290, 1981 3. Caballero B, Mahon BE, Rohr FJ, et al: Plasma amino acid levels after single-dose aspartame consumption in phenylketonuria, mild hyperphenylalaninemia, and heterozygous state for phenylketonuria. J Pediatr 109:668-67 1, 1986 4. Pardridge WM: Potential effects of the dipeptide sweetener aspartame on the brain, in Wurtman RJ, Wurtman JJ (eds): Nutrition and the Brain, vol 7. New York, NY, Raven, 1986, pp 199-241 5. Stegink LD, Filer LJ Jr, Bell EF, et al: Plasma amino acid concentrations in normal adults administered aspartame in capsules or solution: Lack of bioequivalence. Metabolism 36:507-512, 1987 6. Denckla WD, Dewey HK: The determination of tryptophan plasma, liver, and urine. J Lab Clin Med 69:160-169, 1967
in
7. Bloxam DL, Warren WH: Error in the determination of tryptophan by method of Denckla and Dewey. A revised procedure. Anal Biochem 60:621-625, 1974
8. Gibaldi M, Perrier D: Pharmacokinetics. New York, NY, Marcel Dekker, 1982, pp 445-449 9. Stegink LD, Filer LJ Jr, Baker GL: Repeated ingestion of aspartame-sweetened beverage: Effect on plasma amino acid concentrations in normal adults. Metabolism 37:246-25 1, 1988 10. Levy HL, Waisbren SE: Effects of untreated maternal phenylketonuria and hyperphenylalaninemia on the fetus. N Engl J Med 309:1269-1274,1983 11. Bickel H, Gerrard J, Hickmans EM: The influence of phenylalanine intake on the chemistry and behaviour of a phenylketonuric child. Acta Paediatr 43:64-77, 1954 12. Blaskovics ME, Nelson TL: Phenylketonuria and its variations: A review of recent developments. Calif Med 115:42-57, 1971 13. Stegink LD, Filer LJ Jr, Baker GL: Plasma and erythrocyte concentrations of free amino acids in adult humans administered abuse doses of aspartame. J Toxicol Environ Health 7:291-305, 1979 14. Lieberman HR, Caballero B, Emde GG, et al: The effects of aspartame on human mood, performance, and plasma amino acid levels, in Wurtman RJ, Ritter-Walker E (eds): Dietary Phenylalanine and Brain Function. Boston, MA, Birkhauser, 1988, pp 196-200 15. Ryan-Harshman M, Leiter LA, Anderson GH: Phenylalanine and aspartame fail to alter feeding behavior, mood and arousal in men. Physiol Behav 39:247-253, 1987
of Phenylalanine and Aspartate From Aspartame Capsules and Solution
(20 mg/kg)
in
Thomas S. Burns, W. Wayne Stargel, and Aryeh Hurwitz Aspartame (L-aspartyl+-phenylalanine methyl ester) was given in capsules or solution to compare the bioavailability of its constituent amino acids, aspartate and phenylalanine. Twenty healthy subjects received a single 20 mg/kg dose of aspartame in capsules or solution in a randomized, crossover design. Plasma amino acid concentrations and the phenylalanine to large neutral amino acid ratios (Phe/LNAA) were determined. Plasma aspartate concentrations did not increase with either treatment. For plasma phenylalanine following capsule ingestion, there was a smaller peak plasma concentration (Cmax; 103.3 w 126.6 fimol/Lj, a longer time to peak concentration (tmax; 108.6 Y 36.6 minutes). but no significant difference in the area under the plasma concentration-time curve (AUCj (7,656 v 7,200 pmol - min/Lj when compared with solution ingestion. The maximum plasma Phe/LNAA ratio was smaller (0.16 v 0.19) with capsules. The changes for plasma tyrosine were similar to those seen with phenylalanine. There were no significant differences in the plasma concentrations of the other LNAAs between capsule and solution ingestion. Thus, given the small effect on phenylalanine Cmax and Phe/LNAA and no effect on the extent of absorption of phenylalanine, aspartame ingested in capsules at doses up to 20 mg/kg is a suitable dosage form for blinded clinical studies, provided that the slower rate of absorption of phenvlalanine from capsules is taken into account. . . @ 1990 by W.B. Saunders Company.
A
LTHOUGH THE DIPEPTIDE sweetener aspartame (L-aspartyl+phenylalanine methyl ester) is routinely consumed in solution in beverages, its sweet taste cannot be masked when it is ingested in solution. Therefore, blinded clinical studies with aspartame require the use of capsules. Upon ingestion, aspartame is hydrolyzed to equimolar quantities of aspartate, phenylalanine, and methanol. Aspartate is rapidly metabolized and thus the plasma aspartate concentrations are not significantly elevated following acute aspartame doses of 34 to 50 mg/kg body weight, whereas plasma phenylalanine concentrations may increase depending on dose.’ Methanol is also rapidly metabolized and blood concentrations are usually not detectable unless large bolus doses of aspartame (250 mg/kg) are administered.’ Aspartame ingestion may also increase the ratio of the plasma phenylalanine concentration to the sum of the plasma concentrations of the other large neutral amino acids (Phe/ LNAA).3 It has been proposed that increased plasma Phe/ LNAA ratios may increase brain phenylalanine uptake in humans.4 A previous study’ indicated that the plasma concentrations of aspartate and phenylalanine were significantly less when aspartame was ingested in capsules compared with solution. The reason given for the difference observed was a delay in absorption due to capsule disintegration and a slow dissolution rate of aspartame in the stomach and the intestinal lumen. The extent of absorption, as measured by the area under the plasma concentration-time curve (AUC), was also less with capsules. Presumably, because of its relatively low
solubility, aspartame administered at such large bolus doses is incompletely dissolved in the intestinal lumen. The subjects in the above study were given a single 3-g dose of aspartame (-50 mg/kg). This dose is equivalent to the amount of aspartame in approximately 6 L of aspartamesweetened beverage. Because phenylalanine is speculated to be associated with adverse reactions allegedly due to aspartame consumption, the rate and extent of phenylalanine absorption are of importance when using aspartame in capsules in clinical studies. The pharmacokinetic response of plasma phenylalanine was determined at an aspartame dose of 20 mg/kg, which is equivalent to the amount of aspartame contained in approximately 2.5 L of an aspartame-sweetened beverage ingested by a 70-kg individual. It is not likely that a single dose of such magnitude would be exceeded by an individual. The previous study’ showed that a single aspartame dose of approximately 50 mg/kg in capsules resulted in a decrease in phenylalanine bioavailability compared with aspartame in solution. An observation of equivalent phenylalanine bioavailability from capsules and solution with a lower dose of aspartame, ie, 20 mg/kg, would demonstrate the suitability of capsules for use in blinded clinical studies. The present investigation compared the plasma concentrations of aspartate and phenylalanine in healthy subjects following the ingestion of a single 20 mg/kg dose of aspartame in capsules or solution in a randomized, two-way crossover design. MATERIALS AND METHODS
From the Department of Clinical Research, The NutraSweet Company, Deerjeld, IL; and the Division of Clinical Pharmacology, The University of Kansas Medical Center, Kansas City, KS. Supported by a grant from The NutraSweet Company, Deerfield, IL. Address reprint requests to Aryeh Hurwitz, MD. Division of Clinical Pharmacology, The University of Kansas Medical Center. Kansas City, KS 66103. @ 1990 by W.B. Saunders Company. 0026-0495/90/3911-0016$03.00/0 1200
The study protocol was approved by the University of Kansas College of Health Sciences and Hospital Human Subjects Committee. The study population consisted of 20 healthy subjects, 10 men and 10 women, from whom informed written consent was obtained. The subjects ranged in age from 18 to 36 years (mean, 26.0 f 3.7 years). Their body weights ranged from 45 to 94 kg (mean, 65.8 -t 12.8 kg). The subjects were assigned to a treatment sequence by means of a computer-generated random assignment schedule. An overnight fast of at least 10 hours preceded the administration of aspartame. On 1 study day, the subjects ingested 300 mL of unsweetened beverage Metabolism, Vol39,
No 11 (November), 1990: pp 1200- 1203
ASPARTAME IN CAPSULES AND SOLUTION
plus a sufficient number of capsules, each containing 300 mg of aspartame, so as to provide a dose as close as possible to 20 mg/kg. On the other study day, the subjects ingested 300 mL of beverage in which the same amount of aspartame as given in capsules was dissolved. The subjects also ingested empty capsules with the solution. The 2 study days were separated by a washout period of at least 1 week. Heparinized blood samples for plasma amino acid analyses were obtained before dosing and at 15, 30,45,60, 90, 120, 150, 180,240, 360, and 480 minutes after ingestion. Plasma was immediately separated from cellular elements by low-speed centrifugation and stored at - 70°C until analyzed. Samples were prepared for analysis by precipitating the proteins with 5% sulfosalicylic acid in pH 2.2 lithium citrate buffer (0.2 mol/L) and centrifugation (17,000 x g for 15 minutes) to remove proteins. The resulting supernatant was then passed through a 0.2-pm syringe filter, and the filtrate was stored frozen at -20°C until analyzed. The concentrations of 28 amino acids were measured using a Beckman 6300 Amino Acid Analyzer (Beckman Instruments, Palo Alto, CA) with an on-line Spectra-Physics SP4200 Computing Integrator (Spectra-Physics, San Jose, CA). Plasma total tryptophan was determined by the fluorometric method of Denckla and Dewey’ as modified by Bloxam and Warren.’ The unsweetened beverage was cherry-flavored Kool-Aid mix (General Foods, White Plains, NY), which was reconstituted according to package instructions. The aspartame-filled capsules were provided by G.D. Searle (Skokie, IL). Mean (-rSD) plasma concentrations of each of 29 amino acids were determined at each time point. For each subject the apparent peak plasma concentration (Cmax) and time to peak concentration (tmax) were determined by inspection of the individual plasma concentration-time curves. The AUC was calculated by the trapezoidal rule,8 with the fasting plasma concentration at 0 time as the base of the trapezoid. The AUC was evaluated at 4, 6, and 8 hours after aspartame ingestion. The AUC was not determined beyond 8 hours, because plasma amino acid concentrations were expected to have returned to baseline values by then. The ratio of plasma phenylalanine concentration to the sum of the plasma concentrations of the other large neutral amino acids (Phe/LNAA) was determined for each subject at each time point. The other LNAAs were isoleucine (Ile), leucine (Leu), methionine (Met), tryptophan (Trp), tyrosine (Tyr), and valine (Val). Statistical analyses comparing solution to capsules were performed using ANOVA for a two-period crossover study. The paired Student’s t test was used for changes over time within a study arm. Statistical analyses were performed using SAS, Version V, as implemented on an IBM 308 1. All statistical testing was performed at the two-tailed, (Y5 0.05, level of significance. RESULTS Mean (*SD) plasma phenylalanine concentrations following ingestion of aspartame in capsules or solution at 20 mg/kg are given in Fig 1, with relevant parameters in Table 1. When aspartame was ingested in solution, plasma phenylalanine concentration increased significantly from a mean baseline of 65.7 t 13.6 pmol/L to a high mean value of 118.9 2 28.6 timol/L at 30 minutes after dosing. The mean AUC for plasma phenylalanine from 0 to 4 hours was 6,012 k 1,866 wmol . min/L. Mean AUC(O-6) and AUC(O-8) values were 6,864 + 3,078 and 7,200 k 4,236 Hmol - min/L, respectively. When aspartame was ingested in capsules, plasma phenylalanine concentrations increased significantly from a mean baseline value of 67.1 * 9.9
1201
160.
Phenylalanine
140.
120.
I
I
I
I
1
i1
I
I
I
I
I I Aspartate
I
I
I
I
I
I
3
4
5
6
7
8
I
2
I
TIME, hours Fig 1. Mean (k SD) plasma phenylalanine and aspartate concentrations (Pmol/Lj in 20 normal adults ingesting aspartame at 20 mglkg in solution (O-0) or capsules f-j.
pmol/L to a high mean value of 95.3 i 13.8 pmol/L at 2.5 hours after dosing. The mean AUC values for 4, 6, and 8 hours were 5,022 * 1,782, 6,702 ? 2,634, and 7,656 + 3,120 rmol - min/L, respectively. The changes in plasma phenylaTable 1. Pharmacokinetic and Aspartate
Parameters
for Plasma Phenylalanine
for 20 Subjects Ingesting Aspartame
at 20 mg/kg
in Solution or Capsules Phenylalanine Parameter
Baseline (pmol/L)
Solution 65.7 (13.6)
High mean (bmol/L) Cmax (pmol/L) tmax (min) A UC O-4 h (pm01. min/L) AUC O-6 h (fimol. min/L) AUCO-8h (firnol. min/L)
Capsules
Aspartate Solution
67.1 (9.9)
Capsules
5.1
5.3
(2.1)
95.3”
5.7
(2.3) 5.9
(28.6)
(13.8)
(2.4)
(2.4)
126.6
103.3’
7.6
8.7
(27.4)
(14.4)
(3.3)
36.6
108.6”
110.4
(3.3) 94.8
(11.4)
(49.8)
(168.0)
(1 11.6)
118.9
6,012
5,022
-48
(1,866) 6,864
(1.782) 6,702
(384) -108
(372) -12
(3.076) 7,200
(2,634) 7,656
1510) -186
(5881 -144
(4,236)
(3.120)
(654)
(756)
NOTE. Values are means (SD). *Significantly different from solution (p -c 0.05).
54
BURNS, STARGEL, AND HURWITZ
1202
lanine concentrations from baseline were significantly larger at 15, 30, 45, and 60 minutes after dosing and significantly smaller at 150, 180, and 240 minutes after dosing when aspartame was ingested in solution compared with capsules. There were no significant differences between the solution and capsules for the mean 4-, 6-, and 8-hour AUC values. The individual peak plasma phenylalanine concentrations without regard to time (Cmax) were also determined along with the corresponding times to reach Cmax (tmax) (Table 1). The mean Cmax value for aspartame in solution was 126.6 f 27.4 pmol/L, compared with 103.3 f 14.4 pmol/L for aspartame in capsules. The corresponding tmax values were 36.6 + 11.4 minutes (solution) and 108.6 + 49.8 minutes (capsules). For aspartame ingested in solution compared with capsules, the mean Cmax value was significantly larger and the mean tmax value was significantly shorter. The mean plasma Phe/LNAA ratios are shown in Fig 2. When aspartame was ingested in solution, the plasma Phe/ LNAA ratio increased significantly from a mean baseline value of 0.10 + 0.01 to a high mean value of 0.18 2 0.03 at 30 minutes after dosing. When aspartame was ingested in capsules, the mean baseline ratio was 0.10 + 0.01 and the high mean ratio was 0.15 * 0.03 at 90 minutes after dosing. The mean of the individual maximum Phe/LNAA ratios was significantly greater and occurred significantly earlier for aspartame ingested in solution (0.19 t 0.03 at 43.5 * 16.1 minutes) compared with capsules (0.16 f 0.03 at 126.7 i 95.8 minutes). The 4-, 6-, and 8-hour Phe/LNAA AUC values did not differ significantly between solution and capsule ingestion. The mean plasma aspartate concentrations are shown in Fig 1 and relevant parameters in Table 1. Plasma aspartate concentrations did not increase significantly from baseline following the ingestion of aspartame in either solution or capsules. Also, there were no significant differences in the mean Cmax and tmax values, nor were the AUC values for 4, 6, and 8 hours different between solution and capsules (Table 1). Plasma concentrations
of tyrosine
increased
over baseline
0.30 1
Fig 2. Mean (_cSDi values for the ratio of plasma phenylalanine concentration to the sum of the plasma concentrations of the other large neutral amino acids (Phe/LNAA) in 20 normal adults ingesting aspartame at 20 mglkg in solution (-1 or capsules w--w.
after both solution and capsules, reflecting the conversion of phenylalanine to tyrosine. The mean Cmax value was significantly larger for aspartame ingested in solution (88.9 + 2.2 v 78.9 + 1.6 Mmol/L), whereas the mean tmax value was significantly smaller (50.4 k 36.0 v 98.4 f 89.4 minutes) compared with aspartame ingested in capsules. The mean AUC values for 4, 6, and 8 hours did not differ significantly between solution and capsules. The mean plasma Tyr/ LNAA ratios did not show any meaningful changes following capsules or solution. The mean of the individual maximum Tyr/LNAA ratios was not significantly different for solution (0.13 k 0.02) and capsules (0.12 + 0.02), but did occur significantly earlier (62.2 + 28.5 v 108 f 85.5 minutes) following aspartame in solution. The plasma concentrations of the branched chain amino acids (Ile, Leu, and Val) and Trp did not differ significantly between aspartame ingested in solution or in capsules. The LNAA ratios for Ile, Leu, Val, and Trp showed small decreases following solution compared with capsules at time points of 1 hour or less after aspartame ingestion. These decreases were attributable to the increased plasma Phe concentration associated with aspartame in solution in the denominator of the ratios at these early time points. The means of the individual maximum ratios for Ile, Leu, Val, and Trp were not significantly different for solution and capsules. Plasma concentrations of the other amino acids did not change significantly following aspartame ingestion in either solution or capsules. DISCUSSION
The comparison of solution and capsule formulations showed a delay in phenylalanine absorption from aspartame ingested in capsules due, initially, to capsule disintegration time. However, the most important factor contributing to the absorption delay was probably the dissolution time in the stomach and intestinal lumen of aspartame, which has relatively poor solubility in aqueous solution. Gastric emptying time differences due to the presence of capsules and undissolved aspartame also may have been a factor. These factors were most likely responsible for the differences in plasma phenylalanine Cmax and tmax values and Phe/ LNAA ratios observed in the present study. Although the mean plasma phenylalanine Cmax values (126.6 v 103.3 Mmol/L) and the mean peak Phe/LNAA ratios (0.19 v 0.16) were statistically significantly greater following ingestion of aspartame in solution compared with capsules, the differences between the solution and capsule mean values were relatively small. For comparison purposes, the reported normal postprandial plasma phenylalanine concentration range is 60 to 120 pmol/L.’ Thus, peak plasma phenylalanine concentrations, a measure of the rate of phenylalanine absorption, following aspartame given as either capsules or solution, generally remained within the reported postprandial range. The pronounced tmax difference (36.6 v 108.6 minutes) reflected the delay in absorption of phenylalanine from aspartame capsules. The AUC values, a measure of the extent of phenylalanine absorption, were not significantly
ASPARTAME
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IN CAPSULES AND SOLUTION
different for capsules and solution. Thus, although there was some reduction in the rate of absorption of phenylalanine from capsules compared with solution, the extent of absorption did not differ significantly. Following aspartame doses much larger’ than the 20 mg/kg used in this study, the extent of absorption may differ due to incomplete aspartame dissolution from capsules. It is our opinion that the extent of phenylalanine absorption (AUC) is the more appropriate pharmacokinetic parameter for evaluating putative adverse effects. This opinion is supported by the phenylketonuria which indicates that sustained, elevated plasma literature,““’ concentrations of phenylalanine (2 1200 pmol/L) are associated with neurotoxicity. However, transient, elevated plasma concentrations resulting from large loading doses of aspartame’3”5 do not produce any adverse effects, including cognitive or behavioral effects. The plasma concentrations of aspartate from a 20 mg/kg dose of aspartame were not significantly increased by ingestion of the aspartame in either solution or capsules. This is
consistent with previous studies’ that showed that plasma aspartate concentrations were not significantly affected by acute aspartame doses ranging from 34 to 50 mg/kg. In conclusion, given the small effect on phenylalanine Cmax and Phe/LNAA and no effect on the extent of phenylalanine absorption from aspartame ingested in capsules compared with solution, aspartame in capsules is a suitable dosage form for blinded clinical studies at single doses up to 20 mg/kg. However, the investigator must take into account that peak plasma phenylalanine concentrations following aspartame ingestion in capsules occur later than comparable doses ingested in solution.
ACKNOWLEDGMENT
We wish to thank Dr David K. Rassin, The University
of Texas Medical Branch at Galveston, TX, for the amino acid analyses and Dr Dennis Haack, Statistical Consultants, Inc. Lexington, KY, for the statistical
analyses.
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