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Urinary amino acid excretion in phenylketonuric, hyperphenylalaninemic, and normal patients
474
March, 1971 T h e Journal o/ P E D I A T R I C S
Urinary amino acid excretion in p beny lketonu ric, byperp beny la lan i nemic, and normal patients The urinary excretion o[ amino acids was examined in phenylketonuric patients on and off low-phenylalanine diets, in hyperphenylalaninemie individuals, and in normal children. In phenylketonuric persons off diets, the pattern o[ amino add excretion suggested competitive inhibition o[ reabsorption of amino acids by the high filtered load o[ phenylalanine. Hyperphenylalaninemic patients had a generalized aminoaciduria, prompting the hypothesis that they are phenylketonuric individuals who are protected by their increased urinary excretion o[ phenflalanine.
David R. Lines, M.B.B.S., M.R.A.C.P., "x"and Harry A. Waisman, M.D., Ph.D. MADISON~
WIS.
THOUOa IT IS generally agreed that patients with phenylketonuria excrete more phenylalanine in their urine than do normal subjects, 1 renal excretion of other amino acids has been considered to be normal, as determined by paper chromatography? Since phenylalanine shares a common renal transport mechanism with many other amino acids, as demonstrated in Hartnup disease, 8 the high filtered load of phenylatanine could theoretically lead to impaired renal reabsorption of other amino acids. Efron and
From the Joseph P. Kennedy, Jr., Laboratories, University o] Wisconsin Medical Center. Supported in part by United States Public Health Service Grant HDO034I to the Joseph P. Kennedy, Jr., Laboratories. Reprint address: Dr. Walsman, Joseph P. Kennedy, Jr., Laboratories, University of Wisconsin Medical Center, 1300 University Ave., Madison, Wis. 53706. ~Rec~plent of United States Postdoctoral National Institutes of Health Fellowship F05 TW 1406. Present address: Department o[ Pediatrics, University of Adelaide, Adelaide, Australia.
Vol. 78, No. 3, pp. 474-480
associates 2 examined the renal reabsorption of amino acids in 2 phenylketonuric patients and one control subject. They showed a slight trend toward decreased reabsorption of most amino acids but concluded that it was not significant. If such a trend were real, it could account for the low plasma levels of amino acids other than phenylalanine reported by many investigators. ~, 4-7 This study was undertaken to investigate the renal excretion of amino acids in patients with classic phenylketonuria or hyperphenylalaninemia, as well as in appropriate control children of the same age. Hyperphenylalaninemic patients were included in this study because such individuals could possibly be "phenylketonuric" but partially protected by a renal tubular transport defect which caused them to lose phenylalanine in the urine. If a similar transport defect were present in the ileum, the plasma
Volume 78 Number 3
Urinary amino acid excretion
475
Table I. Age, weight, and serum phenylalanine levels of patients
Subjects
Phenylketonuric, off diets ( 11 ) Phenylketonuric, on diets ( 7) Hyperphenylalaninemie (7) Normal (7)
Mean age (yr.)
19:}~2 57A2 8 7
Median age (yr.)
Mean weight (Kg.)
15~}i2 43/i2 311,A_2 6aA2
*This value was not measured but derived f r o m the llterature. 21 All serum phenylalanine metrically31
concentration of phenylalanine would be further reduced. A less likely possibility would be that hyperphenylalaninemic patients could be carriers for phenylketonuria, who had a "supernormal" absorption of phenylalanine from the renal tubule and ileum. MATERIALS
AND METHODS
Four groups of patients were studied: (1) eleven untreated phenylketonuric children, (2) seven phenylketonuric subjects on lowphenylalanine diets, (3) seven hyperphenylalaninemic patients, and (4) seven normal control children. The ages, weights, and serum phenylalanine determinations were obtained at 6 month intervals, or more frequently, depending on the age of the child and the group to which he belonged. A carefully monitored low-phenylalanine diet was provided to those in the treated group. The patients in the other groups consumed a regular diet in their home or in an institution for the mentally retarded (9 of the phenylketonuric children off diets). Twentyfour-hour urine collections were obtained from all patients and stored in containers in a refrigerator with 2 ml. of 1N HC1 as a preservative. On arrival in the laboratory (which was within 24 hours of collection) the urine specimens were kept frozen (-70 ~ C.) until analyzed. The urine was filtered and its acidity checked before analysis by the method of Spackman and associates,8 using the long column of a Beckman-Spinco amino acid analyzer. For basic amino acids the urine was deaminated, reacidified, diluted, and
33.7 24.1 25.3 26.0
Mean serum phenylalanine (reg.~tO0 ml.)
33.9 11.4 16.7 1.4"
levels were measured spectrofluoro-
analyzed on the same instruments. Creatinine concentration in the urine was determined by the method of Peters2 RESULTS
Table I records the age, weight, and serum phenylalanine level in all patients investigated. The mean serum phenylalanine concentration is appropriate for the group. The excretion of each individual amino acid is expressed in 3 ways in Table II: milligrams per day, milligrams per kilogram per day, and milligrams per gram of creatinine. All identifiable ninhydrin-positive peaks have been included provided that at least 5 patients in each group excreted measurable quantities. Thus, phosphoserine, phosphoethanolamine, and taurine are included; aspartic acid, cystine, and many of the basic amino acids have been omitted, a-n-Butyric acid and a-amino-adipic acids could not be separated consistently and are therefore expressed as a combination. Tryptophan was not estimated because it is destroyed by the procedure involving exchange chromatography? ~ When the results are expressed as milligrams per day, most amino acids are excreted in larger quantities by phenylketonuric children, as compared to normal children. This is significant at the p < 0.10 level for glycine, isoleucine, and alanine, fi-Alanine excretion is significantly less than normal in untreated phenylketonuria, both in total daily output and expressed as per kilogram per day. As expected, excretion of phenylalanine is elevated in both phenylketonuria and hyperphenylalaninemia, no matter which way
A m i n o acid
*p < "~p < ~p < w <
0.20. 0.I0. 0.01. 0.001.
Phosphoserine Phosphoethanolamine Taurine Threonine Serine Glutamine Glutamic acid Glycine Alanine Valine Cystathionine Methionine Isoleucine Leucine Tyrosine Phenylalanlne Lyslne 1-methyl histidine Histidine 3-methyl histidine /?-alanine ~-amino-n-butTric + a-amino-adiplc
0.214
0.427* 0.784~ 0.359 0.623 1.73 0.062t 1.51 0.477 0.067 0.118 0.151" 0.165 0.206 0.337 4,49w 0.422 3.63 1.17 0.326 0.059 t
0.1 I0
8.26
17.3 27.8 14.1 23.8 65.4 2.20 60.7 t 18.6" 2.39 4,91 6.24* 6.19t 6.99 12.6 168.6w 17.1 24.4 41.7 13.7 1.58w
4.06
11.3
49.3 106 41.9 76.85 197t 7.15 193 56.1w 8.10" 11.6 18.3t 19,1t 26.6t 38.9 t 539w 42.6t 57,7 138 36.8 9.16
25.9
0.237 0,540 1.38 0.381 0,752 2.56~ 0.036 # 1,29 0.632 e 0.096 0.037 0'.439 0.232t 0.187 0.285 2.45w 0..604 0.314 2.767 0.584 0.181 0.169
10.3 10.5~ 28.5 9.48 16.1 58.8 0.843 29.6 13.0 2.31 1.95 5.03t 6.46t 5.27 6,63 58.4t 15.6 14.3 50.9 19.0 3.82 3.39
27.0t
108" 175 72.9* 154t 592w 6.95 180t 130t 24.5t 8.66 139t 33.2w 23.5 52.0t 409w 92.3t 36.2 382 85.25 28.1
36.8 r
3.53
16.7 51.3 14.3 18.6 43.8 3.05 31.6 12.0 2,15 2.12 1.70 2.26 5.24 10.8 4.77 13.30 32.4 37.4 14.5 4.06
6.50
0.161
0.705 1.90 0.603 0.732 1.80 0.112 1.26 0.488 0.081 0.084 0.080 0.096 0.194 0.365 0.181 0.490 1.03 1.55 0.501 0.229
0.23
10.9
44.6 94.8 38.0 47.5 115 7.59 84.0 30,7 4.91 5.85 5.05 6.37 12.05 24.4 11.9 30.4 55.7 75,6 29.6 16.5
16.8
5.58
10.7~ 13.0 13.5 25.8 ~ 40,.5 1.42" 38.4 10.7 1.85 -18.4t 3.78 6.76 3.89t 36.0~ 21.4 -22.8 6.54 11.I
5.50
0.331"
0.58I 0.485* 0.731 1.32t 2.18 0.085 2.08* 0.56 0.115 -1.01t 0.231" 0.368t 0.191t 1.52t 0.817" -2.15 0.395 0.656
0.275
47,0-~
70.6* 83.0 104.8" 197w 3195 10.1 305:~ 84.1~ 15.8~ -148t 33.85 55.4w 29 241t 141.0t --253"I 53.4 "~ 88.6
41.5 r
Patients with Patients with _ Patients iwith phenylketonuria hyperphenylalaninemia Normal c h i l d r e n phenylketonuria on diets off diets mg./Kg./ I mg./Gm. I mg./Kg, mg./Gm, mg./Kg./ mg./Gm, mg./Kg./ reg.~Gin. day creatinine reg.~day day . creatinine reg.~day day creatinine reg.~day day ereatinine reg.~day
T a b l e I I . E x c r e t i o n of aaaaino acids in p h e n y l k e t o n u r i c p a t i e n t s off diets, p h e n y l k e t o n u r i c p a t i e n t s o n diets, a n d h y p e r p h e n y l a l a n i n e m i c p a t i e n t s c o m p a r e d to c o n t r o l subjects
Volume 78 Number 3
it is expressed, at highly significant levels. The hyperphenylalaninemic patients excrete greater quantities of methionine and leucine per day but less phosphoethanolamine than do normal children (p < 0.10). On a per kilogram per day basis, the phenylketonuric patients off diets excrete taurine and glutalnic acid (p < 0.10) in addition to the fi-alanine mentioned above. Examined this way the only amino acid these individuals excrete in significantly larger amounts is phenylalanine (p < 0.001). In the hyperphenylalaninemic group, glutamine, isoleucine, histidine (all at p < 0.10), and alanine (p < 0.20) are excreted in increased amounts in addition to phenylalanine (p < 0.001). When excretion is expressed in milligrams per gram of creatinine, more differences become evident. In the children with phenylketonuria off diets the following amino acids are excreted at higher levels: phenylalanine and alanine (both at p < 0.001), serlne (p < 0.01), glutamine, methionine, isoleucine, leucine, tyrosine, lysine (all at p < 0.10), and valine (p < 0.20). In the hyperphenylalaninemic patients, when expressed on a milligrams per gram of creatinine basis, the following ninhydrin-positive substances are excreted in excess: phenylatanine, glutamine, isoleucine (all at p < 0.001), 3-methyl histldine (p < 0.01), glycine, alanine, valine, methionine, tyrosine, lysine, histidine, and a-amino-n-butyric and a-amino-adipic combined (all at p < 0.10), and phosphoserine, phosphoethanolamine, and threonine (all at p < 0.20). When one compares excretion of amino acids in phenylketonuric patients still on lowphenylalanine diets with normal control subjects, there is a lower daily excretion of phosphoethanolamine, tyrosine (both at p < 0.10), and glutamic acid (p < 0.20), which might be expected because the patients are generally younger and smaller than the normal children (Table I). However, in addition to phenylalanine, methionine (p < 0.10) and serine (p < 0.20) are excreted in increased quantities. On a per kilogram basis, leucine (p < 0.10) glycine, isoleucine,
Urinary amino acid excretion
4 77
lysine, and a-amino-adipic combined (all at p < 0.20) and phenylalanine, serine, methionine, and tyrosine (all at p < 0.10) are hyperexcreted. Patients with phenylketonuria on low-phenylalanine diets excrete most ninhydrin-positive substances at a greater rate than do normal children when compared as a function of creatinine, i.e., phosphoserine, serine, leucine (all at p < 0.001), glutamine, glycine, alanine, isoleucine (all at p < 0.01), valine, methionine, phenylalanine, lysine, histidine, and a-amino-adipic and o~-amlno-nbutyric combined (all at p < 0.10), phosphoethanolamine, threonine, and 3-methyl histidine (all at p < 0.2). In addition, glutamic acid, tyrosine, and /?-alanine are excreated in larger if not significant quantities. The only amino acid excreted in lesser quantities is taurine. If one compares hyperphenylalaninemic patients and phenylketonuric patients on diets with those off diets, again the most striking results are seen when output is expressed as per gram of creatinlne. The hyperphenylalaninemic children excrete the following substances at greater levels than patients with phenylketonuria off diets: a-amino-n- butyric and a-amino-adipic and a-amino-n-butyric combined (p < 0.01), taurine, threonine, serine, alanine, methionine, isoleucine, 3methyl histidine (all at p < 0.01), phosphoethanolamine, valine, and lysine (all at p < 0.20). Phenylketonuric patients on diets also have a significantly greater aminoaciduria than phenylketonuric patients off diets when the excretion is expressed in milligrams per gram of creatinine: serine and o~-amino-butyric and o~-amino-adipic combined (p < 0.001), methionine (p < 0.01), threonine, glutamine, alanine, valine, isoleucine, leucine, and lysine (all at p < 0.10), and phosphoserine, phosphoethanolamine, and glycine (all at p < 0.20). DISCUSSION
The excretion of amino acids in patients with elevated plasma phenylalanine has been compared to that of normal patients and expressed in several ways. The most striking
478
Lines and Waisman
differences are noted when compared on the basis of excretion per gram of creatinine. This method of expressing excretion is time honored but has been criticized by numerous authors. 1~-14 In this study the statistical significance increases when excretion is expressed as milligrams per gram of creatinine, but this increased excretion is also apparent when expressed as milligrams per kilogram per day. It should be pointed out that the average weight for age shown in Table I represents a range in growth patterns; the weights of phenylketonuric children on a low-phenylalanine diet are at the ninetyseventh percentile, those of the phenylketonurlc patients off diets (many of whom are severely retarded) are well below the third percentile, the average weight of hyperphenylalaninemic patients is at the fiftieth percentile, while that of the normal group is equal to the ninetieth percentile. I t should also be pointed out that all but one of the mothers who collected urine specimens from the normal children were trained pediatric nurses, whereas none of the mothers in the other 3 groups had such training. One might therefore assume that the home urine collection in the normal group would be more complete than in the 3 patient groups. Thus, in this study, expression of excretion as a function of creatinine might be the most accurate parameter. When phenylketonuric patients off diets, who have the greatest urinary excretion of phenylalanine, are compared with normal control patients, serine, glutamine, alanine, valine, methionine, isoleucine, leucine, and tyrosine are all excreted in significantly greater amounts (as is lysine). In addition, histidine is also excreted in l a n e amounts when compared with normal, but the difference is not statistically significant (p < 0.4, > 0.2) due to marked variation of excretion by normal subjects. Threonine excretion per gram of creatinine is modestly, but not significantly, elevated. Serine, glutamine, alanine, v~iline, methionlne, isoleucine, leucine, tyrosine, phenylalanine, histidine, and threonine are the amino acids excreted in increased amounts in
The Journal o~ Pediatrics March 1971
Hartnup disease, together with tryptophan (which was not measured because it is destroyed by cation exchange resins1~ These amino acids are believed to share a common transport mechanism in the renal tubulel~; the increased phenylalanine in the glomerular filtrate could therefore competitively block the renal reabsorption of serine, glutamine, alanine, valine, methionine, isoleucine, Ieucine, tyrosine, histidine, and threonine and could account for the increased urinary excretion found. r The increased excretion of !ysine requires some other explanation. Christensen 15 has shown that phenylalanine inhibits uptake of lysine by the Ehrlich ascites cell but at a concentration of phenylalanine above that found in this study. The increased excretion of these amino acids could be a cause of the lower plasma levels of many amino acids that have been found in phenylketonuria3, 4-7 In hyperphenylalaninemic patients, when excretion of these amino acids is expressed as a function of creatinine, the following amino acids are excreted in significantly increased amounts: phenylalanine, serine, glutamine, glycine, alanine, valine, methionine, isoleucine, tyrosine, lysine, histidine, 3-methyl histidine, a-amino-n-butyric and o~-amino-adipic acids, phosphoserine, phosphoethanolamine, and threonine. In addition, all other amino acids with the exception of 1-methyt histidine are excreted in increased but not in statistically significant amounts. This pattern of excretion approximates a type of nonspecific renal aminoaciduria which Scriver 1Gdescribes as being due to inhibition of amino acid transfer. If these hyperphenylalaninemic patients did have such a nonspecifie renal tubular arninoaciduria, they might be "phenylketonuric" patients whose blood levels are kept below 20 mg. per cent by their excessive loss of phenylaIanine in the urine. It is noteworthy that the phenylalanine excretion per gram of creatinine for the hyperphenylalaninemie group is not statistically less than that "~The low fl-alanine excretion persists when expressed as per g r a m of creadnlne. T h e low excretion could be explained by the aversion to m e a t that m a n y of our phenylketonuric patients have when taken off diets. M e a t is the m a i n source of fl-alanlne.
Volume 78 Number 3
for the phenylketonuric children off diets, although their blood levels are reduced by half (Table I). Also, the hyperphenylalaninemic patients excrete significantly more of the ninhydrin-positive substances when compared to phenylketonuric children off diets, again suggesting a renal tubular transport defect (Table I I ) . If such a defect were duplicated in the ileum, as is common in other renal tubular transport defects, the patient would have a lower absorption of dietary phenylalanine and be further protected. The urinary excretion pattern of phenylketonuric patients on diets indicates a general loss of amino acids. When expressed as a function of creatinine, all amino acids except taurine are lost in increased amounts, most of them at significant levels. What is the cause of this nonspecific generalized aminoaciduria? In 1954 Rose and associates 17 showed that a man fed a synthetic amino acid mixture required an increased intake of calories to avoid negative nitrogen balance. In 1969 Anderson and Linkswiler18 showed that adult males fed an amino acid mixture excreted more urinary amino acids than when fed casein containing the same amounts of the essential amino acids. The treated phenylketonuric children receive most of their amino acids as a hydrolysate of casein in the form of Lofenalac. Each patient had a lower calorie intake per kilogram than that recommended by the National Research Council Recommended Daily Allowances~9 for his particular age. The phenylketonuric patients on diets received an average 17 calories per kilogram (range of 5 to 25) less than that recommended for their age. Thus, the increased amino acid loss in the treated phenylketonuric individuals may be explained by the combination of the dietary source of their amino acids and a low calorie intake. (It should be emphasized that all of the hyperphenylalaninemic patients were on normal diets at the time of this investigation.) This urinary loss of amino acid in the treated phenylketonuric children is significant when compared to that in those off diets. The highly significant loss of serine was also
Urinary amino acid excretion
4 79
found by Anderson and Linkswiler ~s in their adults fed synthetic diets. The increased urinary excretion of glutamine in phenylketonuric children off diets may explain the low blood levels found in phenylketonuria, which Perry and associates 2~ suggest may be important in causing mental retardation. Such increased excretion of glutamine was found in hyperphenylalaninemia, and in phenylketonuria on diet in greater quantities still, suggesting that urinary loss of glutamine is not the mechanism by which mental retardation arises in untreated phenylketonuria. SUMMARY
The urinary excretion of amino acids has been examined in phenylketonuric patients on low-phenylalanine diets, phenylketonuric children on normal diets, hyperphenylalaninemic individuals, and normal children. When expressed as a function of creatinine, statistically significant abnormal patterns of amino acid excretion were found. In the phenylketonuric patients on normal diets a pattern of amino acid excretion reminiscent of Hartnup disease is seen and could be due to competitive inhibition of reabsorption of those amino acids by the highly filtered load of phenylalanine. Treated phenylketonuric patients have a generalized aminoaciduria probably due to the highly modified diet they receive, ttyperphenylalanlnemic patients have a generalized aminoaciduria suggesting a renal tubular lesion of amino acid reabsorption. However, concurrent clearance studies are necessary before it can be decided whether hyperphenylalaninemic individuals are phenylketonuric but protected by increased urinary excretion of phenylalanine. The authors would like to express their appreciation to Barbara Johnson for help with the dietary data, Carol Bergdoll for aid in calculation of much of the data, Robert Colwell for the blood phenylalanine levels, and the parents of the children for the collection of 24 hour urine specimens. REFERENCES
1. Berry, I-I. K., Umbarger, B., and Livlngston, B.: Excretion of phenylalanlne by normal
480
2.
3. 4.
5. 6.
7. 8.
9.
10.
11.
Lines and Waisman
children and by patients with phenylketonuria, J. PEDIAT. 63: 954, 1963. Efron, M. L., Kang, E. S., Visakorpl, J., and Fellers, J. X.: Effect of elevated pIasma phenylalanine levels on other amino acids in phenylketonuric and normal subjects, J. PEDIAT. 74: 399, 1969. Striver, C. R.: The human biochemical genetics of amino acid transport, Pediatrics 44: 348, 1969. Benirsehke, K., Brownhill, L., Efron, M. L., and Hoefnagel, D.: Phenylketonuria associated with Klinefelter's syndrome, J. Ment. Defic. Res. 6: 44, 1962. Biekel, H., and Griiter, W.: Phenylketonuriec mlt normalen Intelligenzquotienten, Z. Kinderheilk. 79: 509, 1957. Linnewech, .F., and Ehrlich, M.: Die Renalen und prarenalen StSrungen des Aminosaiiren Stoffwechsels bei Phenylalaninarmer Ernahrung, Klin. Wschr. 38: 904, 1960. Linnewech, F , and Ehrlich, M.: Zur Pathogenese des Schwachsinns bei Phenylketonurle, Klin. Wschr. 40: i6, 1962. Spackman, D. H., Stein, W. H., and Moore, S.: Automatic recording apparatus for use in the chromatography of amino acids, Anal. Chem. 30: 1t90, 1958~ Peters, J. H., The determination d creatinine and creatine in blood and urine with the photoelectric colorimeter, J. Biol. Chem. 146: 179, 1942. Cusworth, D. C., and Dent, C. E.: Renal clearances of amino acids in normal adults and in patients with aminoaciduria, Biochem. J. 74: 550, t960. Bergstedt, J.: O'Brien, D., and Lubehenco, L.: Interrelationships in the urinary excretion of creatine, creatinine, free alpha-amino
The ]ournal o[ Pediatrics March 1971
12.
13. 14. 15. 16.
17.
i8.
19.
20.
21.
acid nitrogen, and total nitrogen in premature infants, J. PEDIAT. 56: 635, 1960. Edwards, O. M., Bayliss, R. I. S., and Milien, S.: Urinary creatinine excretion as an index of the completeness of 24 hour urine collections, Lancet 2:1165, 1969. Zorab, P. A., Clark, S., and Harrison, A.: Creatinine excretion, Lancet 2: 1254, 1969. Bailey, R. R., and de Wardener, H. E.: Creatinine excretion, Lancet l: 145, i970. Christensen, H. N.: A transport system serving for mono- and diamino acids, Proc. Nat. Acad. Sci. U.S.A. 51: 337, 1964. Striver, C. R.: Use of human genetic variation to study membrane transport of amino acids in kidney, Amer. J. Dis. Child. 117: 4, 1969. Rose, W. C., Coon, M. J., and Lambert, G. M.: The amino acid requirements of man. VI. The role of the caloric intake, J. Biol. Chem. 210: 331, 1954. Anderson, H. L., and Linkswiler, H.: Effect of source of dietary nitrogen on plasma concentration and urinary excretion of amino acids of men, J. Nutr. 99: 91, 1969. National Academy of Sciences--National Research Council Recommended Dietary AIiowances, Publication No. 1694~ Washington, D. C., 1968. Perry, T. L., Hansen, S., Tischler, B., Bunting, R., and Diamond, S.: Glutamine depletion in phenylketonuria: A possible cause of the mental defect, New Eng. J. Med. 282: 761, 1970. McCaman, M. W., and Robins, E.: Fluorimetric method for the determination of phenylalanine in serum, J. Lab. Clin. Med. 59: 885, 1962.
March, 1971 T h e Journal o/ P E D I A T R I C S
Urinary amino acid excretion in p beny lketonu ric, byperp beny la lan i nemic, and normal patients The urinary excretion o[ amino acids was examined in phenylketonuric patients on and off low-phenylalanine diets, in hyperphenylalaninemie individuals, and in normal children. In phenylketonuric persons off diets, the pattern o[ amino add excretion suggested competitive inhibition o[ reabsorption of amino acids by the high filtered load o[ phenylalanine. Hyperphenylalaninemic patients had a generalized aminoaciduria, prompting the hypothesis that they are phenylketonuric individuals who are protected by their increased urinary excretion o[ phenflalanine.
David R. Lines, M.B.B.S., M.R.A.C.P., "x"and Harry A. Waisman, M.D., Ph.D. MADISON~
WIS.
THOUOa IT IS generally agreed that patients with phenylketonuria excrete more phenylalanine in their urine than do normal subjects, 1 renal excretion of other amino acids has been considered to be normal, as determined by paper chromatography? Since phenylalanine shares a common renal transport mechanism with many other amino acids, as demonstrated in Hartnup disease, 8 the high filtered load of phenylatanine could theoretically lead to impaired renal reabsorption of other amino acids. Efron and
From the Joseph P. Kennedy, Jr., Laboratories, University o] Wisconsin Medical Center. Supported in part by United States Public Health Service Grant HDO034I to the Joseph P. Kennedy, Jr., Laboratories. Reprint address: Dr. Walsman, Joseph P. Kennedy, Jr., Laboratories, University of Wisconsin Medical Center, 1300 University Ave., Madison, Wis. 53706. ~Rec~plent of United States Postdoctoral National Institutes of Health Fellowship F05 TW 1406. Present address: Department o[ Pediatrics, University of Adelaide, Adelaide, Australia.
Vol. 78, No. 3, pp. 474-480
associates 2 examined the renal reabsorption of amino acids in 2 phenylketonuric patients and one control subject. They showed a slight trend toward decreased reabsorption of most amino acids but concluded that it was not significant. If such a trend were real, it could account for the low plasma levels of amino acids other than phenylalanine reported by many investigators. ~, 4-7 This study was undertaken to investigate the renal excretion of amino acids in patients with classic phenylketonuria or hyperphenylalaninemia, as well as in appropriate control children of the same age. Hyperphenylalaninemic patients were included in this study because such individuals could possibly be "phenylketonuric" but partially protected by a renal tubular transport defect which caused them to lose phenylalanine in the urine. If a similar transport defect were present in the ileum, the plasma
Volume 78 Number 3
Urinary amino acid excretion
475
Table I. Age, weight, and serum phenylalanine levels of patients
Subjects
Phenylketonuric, off diets ( 11 ) Phenylketonuric, on diets ( 7) Hyperphenylalaninemie (7) Normal (7)
Mean age (yr.)
19:}~2 57A2 8 7
Median age (yr.)
Mean weight (Kg.)
15~}i2 43/i2 311,A_2 6aA2
*This value was not measured but derived f r o m the llterature. 21 All serum phenylalanine metrically31
concentration of phenylalanine would be further reduced. A less likely possibility would be that hyperphenylalaninemic patients could be carriers for phenylketonuria, who had a "supernormal" absorption of phenylalanine from the renal tubule and ileum. MATERIALS
AND METHODS
Four groups of patients were studied: (1) eleven untreated phenylketonuric children, (2) seven phenylketonuric subjects on lowphenylalanine diets, (3) seven hyperphenylalaninemic patients, and (4) seven normal control children. The ages, weights, and serum phenylalanine determinations were obtained at 6 month intervals, or more frequently, depending on the age of the child and the group to which he belonged. A carefully monitored low-phenylalanine diet was provided to those in the treated group. The patients in the other groups consumed a regular diet in their home or in an institution for the mentally retarded (9 of the phenylketonuric children off diets). Twentyfour-hour urine collections were obtained from all patients and stored in containers in a refrigerator with 2 ml. of 1N HC1 as a preservative. On arrival in the laboratory (which was within 24 hours of collection) the urine specimens were kept frozen (-70 ~ C.) until analyzed. The urine was filtered and its acidity checked before analysis by the method of Spackman and associates,8 using the long column of a Beckman-Spinco amino acid analyzer. For basic amino acids the urine was deaminated, reacidified, diluted, and
33.7 24.1 25.3 26.0
Mean serum phenylalanine (reg.~tO0 ml.)
33.9 11.4 16.7 1.4"
levels were measured spectrofluoro-
analyzed on the same instruments. Creatinine concentration in the urine was determined by the method of Peters2 RESULTS
Table I records the age, weight, and serum phenylalanine level in all patients investigated. The mean serum phenylalanine concentration is appropriate for the group. The excretion of each individual amino acid is expressed in 3 ways in Table II: milligrams per day, milligrams per kilogram per day, and milligrams per gram of creatinine. All identifiable ninhydrin-positive peaks have been included provided that at least 5 patients in each group excreted measurable quantities. Thus, phosphoserine, phosphoethanolamine, and taurine are included; aspartic acid, cystine, and many of the basic amino acids have been omitted, a-n-Butyric acid and a-amino-adipic acids could not be separated consistently and are therefore expressed as a combination. Tryptophan was not estimated because it is destroyed by the procedure involving exchange chromatography? ~ When the results are expressed as milligrams per day, most amino acids are excreted in larger quantities by phenylketonuric children, as compared to normal children. This is significant at the p < 0.10 level for glycine, isoleucine, and alanine, fi-Alanine excretion is significantly less than normal in untreated phenylketonuria, both in total daily output and expressed as per kilogram per day. As expected, excretion of phenylalanine is elevated in both phenylketonuria and hyperphenylalaninemia, no matter which way
A m i n o acid
*p < "~p < ~p < w <
0.20. 0.I0. 0.01. 0.001.
Phosphoserine Phosphoethanolamine Taurine Threonine Serine Glutamine Glutamic acid Glycine Alanine Valine Cystathionine Methionine Isoleucine Leucine Tyrosine Phenylalanlne Lyslne 1-methyl histidine Histidine 3-methyl histidine /?-alanine ~-amino-n-butTric + a-amino-adiplc
0.214
0.427* 0.784~ 0.359 0.623 1.73 0.062t 1.51 0.477 0.067 0.118 0.151" 0.165 0.206 0.337 4,49w 0.422 3.63 1.17 0.326 0.059 t
0.1 I0
8.26
17.3 27.8 14.1 23.8 65.4 2.20 60.7 t 18.6" 2.39 4,91 6.24* 6.19t 6.99 12.6 168.6w 17.1 24.4 41.7 13.7 1.58w
4.06
11.3
49.3 106 41.9 76.85 197t 7.15 193 56.1w 8.10" 11.6 18.3t 19,1t 26.6t 38.9 t 539w 42.6t 57,7 138 36.8 9.16
25.9
0.237 0,540 1.38 0.381 0,752 2.56~ 0.036 # 1,29 0.632 e 0.096 0.037 0'.439 0.232t 0.187 0.285 2.45w 0..604 0.314 2.767 0.584 0.181 0.169
10.3 10.5~ 28.5 9.48 16.1 58.8 0.843 29.6 13.0 2.31 1.95 5.03t 6.46t 5.27 6,63 58.4t 15.6 14.3 50.9 19.0 3.82 3.39
27.0t
108" 175 72.9* 154t 592w 6.95 180t 130t 24.5t 8.66 139t 33.2w 23.5 52.0t 409w 92.3t 36.2 382 85.25 28.1
36.8 r
3.53
16.7 51.3 14.3 18.6 43.8 3.05 31.6 12.0 2,15 2.12 1.70 2.26 5.24 10.8 4.77 13.30 32.4 37.4 14.5 4.06
6.50
0.161
0.705 1.90 0.603 0.732 1.80 0.112 1.26 0.488 0.081 0.084 0.080 0.096 0.194 0.365 0.181 0.490 1.03 1.55 0.501 0.229
0.23
10.9
44.6 94.8 38.0 47.5 115 7.59 84.0 30,7 4.91 5.85 5.05 6.37 12.05 24.4 11.9 30.4 55.7 75,6 29.6 16.5
16.8
5.58
10.7~ 13.0 13.5 25.8 ~ 40,.5 1.42" 38.4 10.7 1.85 -18.4t 3.78 6.76 3.89t 36.0~ 21.4 -22.8 6.54 11.I
5.50
0.331"
0.58I 0.485* 0.731 1.32t 2.18 0.085 2.08* 0.56 0.115 -1.01t 0.231" 0.368t 0.191t 1.52t 0.817" -2.15 0.395 0.656
0.275
47,0-~
70.6* 83.0 104.8" 197w 3195 10.1 305:~ 84.1~ 15.8~ -148t 33.85 55.4w 29 241t 141.0t --253"I 53.4 "~ 88.6
41.5 r
Patients with Patients with _ Patients iwith phenylketonuria hyperphenylalaninemia Normal c h i l d r e n phenylketonuria on diets off diets mg./Kg./ I mg./Gm. I mg./Kg, mg./Gm, mg./Kg./ mg./Gm, mg./Kg./ reg.~Gin. day creatinine reg.~day day . creatinine reg.~day day creatinine reg.~day day ereatinine reg.~day
T a b l e I I . E x c r e t i o n of aaaaino acids in p h e n y l k e t o n u r i c p a t i e n t s off diets, p h e n y l k e t o n u r i c p a t i e n t s o n diets, a n d h y p e r p h e n y l a l a n i n e m i c p a t i e n t s c o m p a r e d to c o n t r o l subjects
Volume 78 Number 3
it is expressed, at highly significant levels. The hyperphenylalaninemic patients excrete greater quantities of methionine and leucine per day but less phosphoethanolamine than do normal children (p < 0.10). On a per kilogram per day basis, the phenylketonuric patients off diets excrete taurine and glutalnic acid (p < 0.10) in addition to the fi-alanine mentioned above. Examined this way the only amino acid these individuals excrete in significantly larger amounts is phenylalanine (p < 0.001). In the hyperphenylalaninemic group, glutamine, isoleucine, histidine (all at p < 0.10), and alanine (p < 0.20) are excreted in increased amounts in addition to phenylalanine (p < 0.001). When excretion is expressed in milligrams per gram of creatinine, more differences become evident. In the children with phenylketonuria off diets the following amino acids are excreted at higher levels: phenylalanine and alanine (both at p < 0.001), serlne (p < 0.01), glutamine, methionine, isoleucine, leucine, tyrosine, lysine (all at p < 0.10), and valine (p < 0.20). In the hyperphenylalaninemic patients, when expressed on a milligrams per gram of creatinine basis, the following ninhydrin-positive substances are excreted in excess: phenylatanine, glutamine, isoleucine (all at p < 0.001), 3-methyl histldine (p < 0.01), glycine, alanine, valine, methionine, tyrosine, lysine, histidine, and a-amino-n-butyric and a-amino-adipic combined (all at p < 0.10), and phosphoserine, phosphoethanolamine, and threonine (all at p < 0.20). When one compares excretion of amino acids in phenylketonuric patients still on lowphenylalanine diets with normal control subjects, there is a lower daily excretion of phosphoethanolamine, tyrosine (both at p < 0.10), and glutamic acid (p < 0.20), which might be expected because the patients are generally younger and smaller than the normal children (Table I). However, in addition to phenylalanine, methionine (p < 0.10) and serine (p < 0.20) are excreted in increased quantities. On a per kilogram basis, leucine (p < 0.10) glycine, isoleucine,
Urinary amino acid excretion
4 77
lysine, and a-amino-adipic combined (all at p < 0.20) and phenylalanine, serine, methionine, and tyrosine (all at p < 0.10) are hyperexcreted. Patients with phenylketonuria on low-phenylalanine diets excrete most ninhydrin-positive substances at a greater rate than do normal children when compared as a function of creatinine, i.e., phosphoserine, serine, leucine (all at p < 0.001), glutamine, glycine, alanine, isoleucine (all at p < 0.01), valine, methionine, phenylalanine, lysine, histidine, and a-amino-adipic and o~-amlno-nbutyric combined (all at p < 0.10), phosphoethanolamine, threonine, and 3-methyl histidine (all at p < 0.2). In addition, glutamic acid, tyrosine, and /?-alanine are excreated in larger if not significant quantities. The only amino acid excreted in lesser quantities is taurine. If one compares hyperphenylalaninemic patients and phenylketonuric patients on diets with those off diets, again the most striking results are seen when output is expressed as per gram of creatinlne. The hyperphenylalaninemic children excrete the following substances at greater levels than patients with phenylketonuria off diets: a-amino-n- butyric and a-amino-adipic and a-amino-n-butyric combined (p < 0.01), taurine, threonine, serine, alanine, methionine, isoleucine, 3methyl histidine (all at p < 0.01), phosphoethanolamine, valine, and lysine (all at p < 0.20). Phenylketonuric patients on diets also have a significantly greater aminoaciduria than phenylketonuric patients off diets when the excretion is expressed in milligrams per gram of creatinine: serine and o~-amino-butyric and o~-amino-adipic combined (p < 0.001), methionine (p < 0.01), threonine, glutamine, alanine, valine, isoleucine, leucine, and lysine (all at p < 0.10), and phosphoserine, phosphoethanolamine, and glycine (all at p < 0.20). DISCUSSION
The excretion of amino acids in patients with elevated plasma phenylalanine has been compared to that of normal patients and expressed in several ways. The most striking
478
Lines and Waisman
differences are noted when compared on the basis of excretion per gram of creatinine. This method of expressing excretion is time honored but has been criticized by numerous authors. 1~-14 In this study the statistical significance increases when excretion is expressed as milligrams per gram of creatinine, but this increased excretion is also apparent when expressed as milligrams per kilogram per day. It should be pointed out that the average weight for age shown in Table I represents a range in growth patterns; the weights of phenylketonuric children on a low-phenylalanine diet are at the ninetyseventh percentile, those of the phenylketonurlc patients off diets (many of whom are severely retarded) are well below the third percentile, the average weight of hyperphenylalaninemic patients is at the fiftieth percentile, while that of the normal group is equal to the ninetieth percentile. I t should also be pointed out that all but one of the mothers who collected urine specimens from the normal children were trained pediatric nurses, whereas none of the mothers in the other 3 groups had such training. One might therefore assume that the home urine collection in the normal group would be more complete than in the 3 patient groups. Thus, in this study, expression of excretion as a function of creatinine might be the most accurate parameter. When phenylketonuric patients off diets, who have the greatest urinary excretion of phenylalanine, are compared with normal control patients, serine, glutamine, alanine, valine, methionine, isoleucine, leucine, and tyrosine are all excreted in significantly greater amounts (as is lysine). In addition, histidine is also excreted in l a n e amounts when compared with normal, but the difference is not statistically significant (p < 0.4, > 0.2) due to marked variation of excretion by normal subjects. Threonine excretion per gram of creatinine is modestly, but not significantly, elevated. Serine, glutamine, alanine, v~iline, methionlne, isoleucine, leucine, tyrosine, phenylalanine, histidine, and threonine are the amino acids excreted in increased amounts in
The Journal o~ Pediatrics March 1971
Hartnup disease, together with tryptophan (which was not measured because it is destroyed by cation exchange resins1~ These amino acids are believed to share a common transport mechanism in the renal tubulel~; the increased phenylalanine in the glomerular filtrate could therefore competitively block the renal reabsorption of serine, glutamine, alanine, valine, methionine, isoleucine, Ieucine, tyrosine, histidine, and threonine and could account for the increased urinary excretion found. r The increased excretion of !ysine requires some other explanation. Christensen 15 has shown that phenylalanine inhibits uptake of lysine by the Ehrlich ascites cell but at a concentration of phenylalanine above that found in this study. The increased excretion of these amino acids could be a cause of the lower plasma levels of many amino acids that have been found in phenylketonuria3, 4-7 In hyperphenylalaninemic patients, when excretion of these amino acids is expressed as a function of creatinine, the following amino acids are excreted in significantly increased amounts: phenylalanine, serine, glutamine, glycine, alanine, valine, methionine, isoleucine, tyrosine, lysine, histidine, 3-methyl histidine, a-amino-n-butyric and o~-amino-adipic acids, phosphoserine, phosphoethanolamine, and threonine. In addition, all other amino acids with the exception of 1-methyt histidine are excreted in increased but not in statistically significant amounts. This pattern of excretion approximates a type of nonspecific renal aminoaciduria which Scriver 1Gdescribes as being due to inhibition of amino acid transfer. If these hyperphenylalaninemic patients did have such a nonspecifie renal tubular arninoaciduria, they might be "phenylketonuric" patients whose blood levels are kept below 20 mg. per cent by their excessive loss of phenylaIanine in the urine. It is noteworthy that the phenylalanine excretion per gram of creatinine for the hyperphenylalaninemie group is not statistically less than that "~The low fl-alanine excretion persists when expressed as per g r a m of creadnlne. T h e low excretion could be explained by the aversion to m e a t that m a n y of our phenylketonuric patients have when taken off diets. M e a t is the m a i n source of fl-alanlne.
Volume 78 Number 3
for the phenylketonuric children off diets, although their blood levels are reduced by half (Table I). Also, the hyperphenylalaninemic patients excrete significantly more of the ninhydrin-positive substances when compared to phenylketonuric children off diets, again suggesting a renal tubular transport defect (Table I I ) . If such a defect were duplicated in the ileum, as is common in other renal tubular transport defects, the patient would have a lower absorption of dietary phenylalanine and be further protected. The urinary excretion pattern of phenylketonuric patients on diets indicates a general loss of amino acids. When expressed as a function of creatinine, all amino acids except taurine are lost in increased amounts, most of them at significant levels. What is the cause of this nonspecific generalized aminoaciduria? In 1954 Rose and associates 17 showed that a man fed a synthetic amino acid mixture required an increased intake of calories to avoid negative nitrogen balance. In 1969 Anderson and Linkswiler18 showed that adult males fed an amino acid mixture excreted more urinary amino acids than when fed casein containing the same amounts of the essential amino acids. The treated phenylketonuric children receive most of their amino acids as a hydrolysate of casein in the form of Lofenalac. Each patient had a lower calorie intake per kilogram than that recommended by the National Research Council Recommended Daily Allowances~9 for his particular age. The phenylketonuric patients on diets received an average 17 calories per kilogram (range of 5 to 25) less than that recommended for their age. Thus, the increased amino acid loss in the treated phenylketonuric individuals may be explained by the combination of the dietary source of their amino acids and a low calorie intake. (It should be emphasized that all of the hyperphenylalaninemic patients were on normal diets at the time of this investigation.) This urinary loss of amino acid in the treated phenylketonuric children is significant when compared to that in those off diets. The highly significant loss of serine was also
Urinary amino acid excretion
4 79
found by Anderson and Linkswiler ~s in their adults fed synthetic diets. The increased urinary excretion of glutamine in phenylketonuric children off diets may explain the low blood levels found in phenylketonuria, which Perry and associates 2~ suggest may be important in causing mental retardation. Such increased excretion of glutamine was found in hyperphenylalaninemia, and in phenylketonuria on diet in greater quantities still, suggesting that urinary loss of glutamine is not the mechanism by which mental retardation arises in untreated phenylketonuria. SUMMARY
The urinary excretion of amino acids has been examined in phenylketonuric patients on low-phenylalanine diets, phenylketonuric children on normal diets, hyperphenylalaninemic individuals, and normal children. When expressed as a function of creatinine, statistically significant abnormal patterns of amino acid excretion were found. In the phenylketonuric patients on normal diets a pattern of amino acid excretion reminiscent of Hartnup disease is seen and could be due to competitive inhibition of reabsorption of those amino acids by the highly filtered load of phenylalanine. Treated phenylketonuric patients have a generalized aminoaciduria probably due to the highly modified diet they receive, ttyperphenylalanlnemic patients have a generalized aminoaciduria suggesting a renal tubular lesion of amino acid reabsorption. However, concurrent clearance studies are necessary before it can be decided whether hyperphenylalaninemic individuals are phenylketonuric but protected by increased urinary excretion of phenylalanine. The authors would like to express their appreciation to Barbara Johnson for help with the dietary data, Carol Bergdoll for aid in calculation of much of the data, Robert Colwell for the blood phenylalanine levels, and the parents of the children for the collection of 24 hour urine specimens. REFERENCES
1. Berry, I-I. K., Umbarger, B., and Livlngston, B.: Excretion of phenylalanlne by normal
480
2.
3. 4.
5. 6.
7. 8.
9.
10.
11.
Lines and Waisman
children and by patients with phenylketonuria, J. PEDIAT. 63: 954, 1963. Efron, M. L., Kang, E. S., Visakorpl, J., and Fellers, J. X.: Effect of elevated pIasma phenylalanine levels on other amino acids in phenylketonuric and normal subjects, J. PEDIAT. 74: 399, 1969. Striver, C. R.: The human biochemical genetics of amino acid transport, Pediatrics 44: 348, 1969. Benirsehke, K., Brownhill, L., Efron, M. L., and Hoefnagel, D.: Phenylketonuria associated with Klinefelter's syndrome, J. Ment. Defic. Res. 6: 44, 1962. Biekel, H., and Griiter, W.: Phenylketonuriec mlt normalen Intelligenzquotienten, Z. Kinderheilk. 79: 509, 1957. Linnewech, .F., and Ehrlich, M.: Die Renalen und prarenalen StSrungen des Aminosaiiren Stoffwechsels bei Phenylalaninarmer Ernahrung, Klin. Wschr. 38: 904, 1960. Linnewech, F , and Ehrlich, M.: Zur Pathogenese des Schwachsinns bei Phenylketonurle, Klin. Wschr. 40: i6, 1962. Spackman, D. H., Stein, W. H., and Moore, S.: Automatic recording apparatus for use in the chromatography of amino acids, Anal. Chem. 30: 1t90, 1958~ Peters, J. H., The determination d creatinine and creatine in blood and urine with the photoelectric colorimeter, J. Biol. Chem. 146: 179, 1942. Cusworth, D. C., and Dent, C. E.: Renal clearances of amino acids in normal adults and in patients with aminoaciduria, Biochem. J. 74: 550, t960. Bergstedt, J.: O'Brien, D., and Lubehenco, L.: Interrelationships in the urinary excretion of creatine, creatinine, free alpha-amino
The ]ournal o[ Pediatrics March 1971
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acid nitrogen, and total nitrogen in premature infants, J. PEDIAT. 56: 635, 1960. Edwards, O. M., Bayliss, R. I. S., and Milien, S.: Urinary creatinine excretion as an index of the completeness of 24 hour urine collections, Lancet 2:1165, 1969. Zorab, P. A., Clark, S., and Harrison, A.: Creatinine excretion, Lancet 2: 1254, 1969. Bailey, R. R., and de Wardener, H. E.: Creatinine excretion, Lancet l: 145, i970. Christensen, H. N.: A transport system serving for mono- and diamino acids, Proc. Nat. Acad. Sci. U.S.A. 51: 337, 1964. Striver, C. R.: Use of human genetic variation to study membrane transport of amino acids in kidney, Amer. J. Dis. Child. 117: 4, 1969. Rose, W. C., Coon, M. J., and Lambert, G. M.: The amino acid requirements of man. VI. The role of the caloric intake, J. Biol. Chem. 210: 331, 1954. Anderson, H. L., and Linkswiler, H.: Effect of source of dietary nitrogen on plasma concentration and urinary excretion of amino acids of men, J. Nutr. 99: 91, 1969. National Academy of Sciences--National Research Council Recommended Dietary AIiowances, Publication No. 1694~ Washington, D. C., 1968. Perry, T. L., Hansen, S., Tischler, B., Bunting, R., and Diamond, S.: Glutamine depletion in phenylketonuria: A possible cause of the mental defect, New Eng. J. Med. 282: 761, 1970. McCaman, M. W., and Robins, E.: Fluorimetric method for the determination of phenylalanine in serum, J. Lab. Clin. Med. 59: 885, 1962.