Procedures for monitoring the low-phenylalanine diet in treatment of phenylketonuria

T h e Journal of P E D I A T R I C S

609

Proceduresfor monitoring the low-phenylalanine diet in treatment of phenylketonuria Treatment of phenylketonuria consists of providing an intake of phenylalanine high enough to meet the nutritional needs of a growing child without exceeding his limited capacity to utilize it. This balance can be achieved by considering serum phenylalanine levels, urinary phenylalanine excretion, and urinary orthohydroxyphenylacetic acid excretion in relation to the phenylalanine intake. Practical procedures are recommended [or obtaining these data. During periods o[ [ebrile illness, phenylalanine deficiency, or phenylalanine excess, characteristic changes occur in the relationships between intake of phenylalanine and the biochemical parameters. Interpretation of these changes is the basis on which dietary alterations are made.

Helen K. Berry, M.A.,* Barbara Umbarger, M.A., and Betty S. Sutherland, M.D. CINCINNATI,

OHIO

T R E A T M E N T Of phenylketonuria by restriction of dietary intake of phenylalanine is well established. The major function of the metabolic team in monitoring the lowphenylalanine diet is to correlate biochemical, dietary, and clinical data in such a way that prompt specific recommendations can be made to meet the patient's current nutritional needs. Phenylalanine intake is carefully considered in relation to the patient's serum phenylalanine level and his urinary excretion of phenylalanine and orthohydroxyphenylacetic acid. This information is the basis for dietary changes which are then translated into appropriate menu plans. Because of the frequency of testing needed for From the Children's Hospital Research Foundation, Department o[ Pediatrics, College o[ Medicine o[ the University o[ Cincinnati. Supported in part by Grant No. HD-00324 from the United States Public Health Service, National Institutes o[ Health. ~Address, The Children's Hospital Research Foundation, Elland & Bethesda Avenues, Cincinnati, Ohio 45229.

proper maintenance of the treatment, simple practical micromethods for collecting and testing blood and urine specimens were devised. In this paper we present methods we have found useful in biochemical monitoring of the treatment of phenylketonuria. Details of patient management are presented elsewhere. 1 C O L L E C T I O N OF S P E C I M E N S Blood from a finger or heel puncture, collected in microhematocrit tubes, was used for measuring serum phenylalanine levels. Specimens were obtained daily during the first week of treatment, then weekly until the end of the first month, and thereafter at intervals of 2 weeks for 6 months. Periods between collection of blood specimens were then lengthened. Blood specimens were usually obtained at midmorning about 2 hours after a meal. After the treatment diet was well established, parents of patients obtained

6 10

Berry, Umbarger, and SutherlmM

the blood specimens and mailed them to the laboratory in a protective package. Filter paper urine specimens were requested daily during the first month of treatment and subsequently at least twice weekly for the first year. Both blood and urine specimens were requested more frequently during periods of illness. Records of daily intake of phenylalanine in the form of Lofenalac :~' and protein-containing foods were kept by the mother. These were mailed with the urine specimens. METHODS

The paper chromatographic procedure for serum phenylalanine described earlier was modified by using butanol-acetic acid-water (120-30-30) as solvent.-' Following deproteinization with 95 per cent alcohol, serum amino acids were resolved in the solvent overnight and developed with ninhydrin. Strips of standard size were cut from the dry filter paper specimens as described earlier. 3 These were used to prepare chromatograms for measurement of urinary phenylalanine and urinary orthohydroxyphenylacetic acid. The phenylalanine chromatogram was resolved in butanol acetic acid-water solvent and developed with ninhydrin as for serum phenylalanine. Orthohydroxyphenylacetic acid was resolved overnight in a solvent of butanolethanol-concentrated anunonium hydroxide (120-30-30). The dry chromatogram was sprayed with 2, 6-dichloroquinonechlorimide reagent (I per cent solution in 95 per cent ethanol). ~ After 5 minutes, the chromatogram was sprayed with an aqueous soIution of 0.5 per cent sodium tetraborate. Orthosubstituted phenolic derivatives produced blue spots. As little as 0.2 /'~g of orthohydroxyphenylacetic acid (Rf 0.72) was visualized with this reagent. Quantitative measurements were made by using both maxim u m density readings obtained from a photovolt densitometer with a 570 mt.~ filter and the area of the spot. Known amounts of orthohydroxyphenylacetic acid (0.5 to 2.5 ":Mead Jolm~on Labo~atmies, Divisi(m ,)t Mead J(,hus~:. & Company. Evansville, Ind.

October 1965

/~g) were run with each chromatogram for comparison. RESULTS

Phenylalanine concentration in serum and phenylalanine and orthohydroxyphenylacetic acid excretions in urine were measured routinely in specimens from phenylketonuric patients treated at Children's Hospital Metabolic Clinic. In a preliminary study we asked mothers to collect urine specimens three times daily for several weeks to determine fluctuations of urinary metabolites. As a rule excretions of phenylalanine and orthohydroxyphenylacetic acid showed only small variations during the day. Each mother then collected urine specimens about the same time cach day. If more than one specimen was tested each day, the results were averaged. The following examples illustrate the relations between phenylalanine intake, phenylalanine in serum, and excretions of phenylalanine and orthohydroxyphenylacetic acid in urine which were observed during treatment of phenylketonuric patients. The periods are arbitrarily chosen to facilitate interpretation of the graphs. The child whose biochemical guides are shown in Fig. 1 was 8 months old when her case was diagnosed as phenylketonuria. "File illustration begins several weeks after treatment was begun. Phenylalanine intake during Period 1 was 150 to 250 mg. per day. Phenylalanine excretion was low (0 to 60 /~g per milliliter), and traces of orthohydroxyphenylacetic acid were detected on two occasions. During Period 2 the phenylalanine intake remained relatively constant, but phenylalanine excretion rose to 75 p.g per milliliter. In Period 3 phenylalanine excretion continued to increase to 125/~g per milliliter, and orthohydroxyphenylacetic acid appeared in the urine in larger amounts. This rise in urinary excretion of metabolites was transient. It was followed by very low excretion of phenylalanine (Period 4), but no significant change in phcnylalanine intake was noted. Serum phenylalanine concentration was not measured during Periods 3 and 4. Period 5 represented an episode of vomit-

Volume 67 Number 4

Monitoring the low-phenylalanine diet

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Fig. 1. Biochemical guides used in adjustment of phenylalanine intake for patient T. F. Urine phenylalanine, urine orthohydroxyphenylacetic acid, and serum phenylalanine. Note the increased excretion of phenylalanine and orthohydroxyphenylacetic acid and increased serum phenylalanine on January 26, following period of low phenylalanine intake. Excess phenylalanine probably resulted from tissue breakdown. ing accompanied by eruption of an acute dermatitis. Phenylalanine intake dropped sharply as a result of the vomiting and refusal to eat. Phenylalanine in serum was 10 rag. per cent when measured 2 days following onset of the acute episode and coincided with appearance of orthohydroxyphenylacetic acid in the urine, in spite of a very low intake of phenylalanine (75 to 150 mg. per day). We interpreted this series of events as breakdown of tissue secondary to phenylalanine deficiency. Our speculation was supported by the fact that, at the end of Period 5, increasing the intake of phenylalanine beyond the previous level resulted in decrease of serum phenylalanine, to 5 mg. per cent, together with decrease in urinary phenylalanine excretion and disappearance of orthohydroxyphenylacetic acid from the urine. In

reviewing these data, it appeared that the low excretion of phenylalanine in the urine during Period 4 should have warned us of impending phenylalanine deficiency and emphasized the need for more frequent serum phenylalanine determinations. During Period 6, urinary metabolites and serum phenylalanine levels were 2 to 3 mg. per cent. Fig. 2 shows the course of a child whose phenylketonuria was diagnosed when she was 11 months old, for whom Lofenalac supplied the total phenylalanine intake for 4y2 months. Our contact with the patient began in January, 1962, when she was 16 months old. Intake of phenylalanine from natural foods was increased immediately. In Period 1, serum phenylalanine values were 3 to 7 rag. per cent. Excretion of phenylalanine was 20 to 65 /xg per milliliter. Orthohydroxy-

6 1 2 Berry, Umbarger, and Sutherland

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Fig. 9. Biochemical monitoring of treatment of Patient S. O. Note the variation in excretion of urinary metabolites with relatively constant intake of phenylalanine. This period was characterized by repeated illness during which maintenance of biochemical control was difficult. phenylacetic acid was not detected. Although intake of phenylalanine was high throughout the period, serum phenylalanine levels did not increase appreciably and the case was considered under control. In Period 2 increased excretion of phenylalanine was noted and orthohydroxyphenytacetic acid was detected in urine. A respiratory infection occurred in Period 3. During the acute febrile phase of the illness, phenylalanine intake dropped, but orthohydroxyphenylacetic acid was still demonstrable in the urine. Period 4 repre~nts the course of the respiratory illness, characterized by intermittent failure of appetite with consequent decrease in phenylalanine intake, marked elevation of urinary phenylalanine and orthohydroxyphenylacetic acid excretion, and rise in serum phenylalanine to 12 mg. per cent. The increased excretion of phenylalanine and orthohydroxyphenylacetic acid persisted throughout the

illness, in spite of intake of phenylalanine less than that of Period 1, when blood phenylalanine levels had been 3 to 7 mg. per cent. The pattern of illness with increased excretion of urinary metabolites was repeated in Period 5. It appeared that biochemical control was difficult to maintain during periods of illness. In Period 6, phenylalanine intake was adjusted downward to 275 to 300 mg. per day. Urinary phenylalanine excretion was then in the range from 25 to 75 ~g. per milliliter and serum phenylalanine levels were between 3 and 7 mg. per cent. Fig. 3 shows data from a ease diagnosed as phenylketonuria when the infant was 48 hours of age. Treatment was begun elsewhere at the age of 2 weeks. Lofenalac served as the sole dietary source of phenylalanine during the first 4 months of life. Our contact with this patient began at 5 ~ months of age. She had been hospitalized several

Volume 67 Number4

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times for refractory anemia and failure to thrive. Phenytalanine intake from Lofenalac alone was calculated as approximately 100 mg. per day, a value much lower than the requirement for a child of this age. Serum phenylalanine was below 0.5 rag. per cent when the patient was first seen here (Period 1). Phenylalanine intake was increased immediately to 200 to 300 mg. per day by addition of natural foods to the diet. Serum phenylalanine levels, however, remained below 1 mg. per cent during Period 2. The child was hospitalized (Period 3) and placed on a high phenylalanine intake. Serum phenylalanine levels were measured daily and after 5 days on a regular diet serum phenylalanine

rose to 20 mg. per cent. Treatment with Lofenalac was then resumed. Phenylalanine intake was adjusted between 200 and 300 mg. per day during Period 4 and serum phenylalanine was between 0.5 and 3 mg. per cent. Phenylalanine excretion during this period was below 25 txg per milliliter. In Period 5, urinary phenylalanine excretion increased, though there were no significant changes in intake of phenylalanine. Period 6 marked the onset of a febrile illness characterized by elevation of phenylalanine excretion to 100 /xg per milliliter. The immediate onset of fever was accompanied by loss of appetite and rise in serum phenylalanine to 6 mg. per cent. Serum phenylalanine rose to 10 mg.

6 14

Berry, Umbar~er, and Sutherland

October 1965

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per cent during the illness and urinary phenylalanine ranged from 100 to 250 /xg per milliliter. Brief illnesses occurred in Period 7, but serum phenylalanine levels generally were 4 to 6 mg. per cent. Urine phenylalanine excretion was elevated in Period 8 and was followed by hospitalization (Period 9) for a respiratory infection. Biochemical control as indicated by serum phenylalanine concentrations between 3 and 7 mg. per cent was somewhat easier to maintain during illness in this child. Fig. 4 represents a ease diagnosed as phenylketonuria in an infant at 8 months. Treatment with Lofenalac was begun at age

9 months. Serum phenylalanine dropped to 3 mg. per cent within 6 days after withdrawal of protein foods (Period 1). Orthohydroxyphenylacetic acid excretion decreased rapidly, while urinary phenylalanine excretion decreased more slowly. After the first week of treatment, phenylalanine intake was increased to 200 to 300 mg. per day (Period 2). The child was exposed to chicken pox in Period 3. Our earlier experience had shown that serum phenylalanine levels were likely to rise during a febrile illness, and that the addition of phenylalanine to the diet during such an episode resulted in decrease of serum phenylalanine levels rather than in-

Volume 67 Number 4

crease as would be expected. Accordingly, when we observed the increased excretion of urinary phenylalanine at the end of Period 3, phenylalanine intake was increased to 350 mg. per day. As the chicken pox erupted, intake was increased to 400 mg. per day (Period 4). Urine specimens were tested daily and serum specimens weekly. The increased intake of phenylalanine in Period 4 was accompanied by excretion of phenylalanine in the range 30 to 60/zg per milliliter; serum levels remained low. It was not possible to maintain the high phenylalanine intake, however. A failure of appetite (Period 5) with consequent decreased intake of phenylalanine was followed by several days of increased excretion of urinary orthohydroxyphenylacetic acid. A brief illness occurred in Period 6, and serum phenylalanine measured at the peak of the illness was 11 mg. per cent. In Period 7, the mother became careless about recording dietary intake of phenylalanine. Urine specimens were sent irregularly or not at all. Very high urinary levels of phenylalanine and orthohydroxyphenylacetic acid were observed and serum phenylalanine rose to 23 mg. per cent. The child was not ill during the period, and the lack of biochemical control reflected failure of dietary control from excess phenylalanine in the diet. Following such a period the treatment regimen had to be reinstated with the same care as was required in Period 1. DISCUSSION In general, when phenylalanine intake of these patients (aged 4 ~ to 16 months) ranged from 200 to 300 mg. per day, phenylalanine excretion was 0 to 50/xg per milliliter. Serum phenylalanine levels were under 2 mg. per cent. We suggest that this represented a deficient intake of phenylalanine. When phenylalanine intake was in the range of 275 to 350 mg. per day, serum phenylalanine levels were in the range of 3 to 7 mg. per cent, and urinary phenylalanine was in the range of 25 to 75 ~g per milliliter. Orthohydroxyphenylacetic acid was not excreted at these levels of serum phenylalanine. We considered this a safe

Monitoring the low-phenylalanine diet

6 15

range for serum phenylalanine concentrations. When phenylalanine intake was 350 to 450 rag. per day, the dietary excess of phenylalanine was eliminated in the urine without causing marked increase in serum phenylalanine levels. This balance between intake, blood level, and urinary excretion of phenylalanine was not maintained during illness. The onset of a febrile illness was usually preceded by an increased excretion of phenylalanine, in spite of a fairly stable phenylalanine intake. The acute phase of the illness was usually characterized by loss of appetite and, frequently, vomiting with consequent decrease in phenylalanine intake. This was followed, in turn, by a brief period of increased phenylalanine excretion, increased levels of phenylalanine in blood, and excretion of orthohydroxyphenylacetic acid. The excess phenylalanine present in serum and urine during an acute illness probably represented phenylalanine from accelerated protien catabolism. From the concentrations of phenylalanine and orthohydroxyphenylacetic acid in the urine, a reliable estimate of phenylalanine level in serum could be made, provided the child was not ill at the time. Individual patients differed in the amount of phenylalanine required to maintain serum phenylalanine levels in the safe range. When urine phenylalanine concentration was consistently less than 1 ~g in urine eluted from the standard size filter paper strip (approximately 50 IA), phenylalanine intake was too low. Serum phenylalanine concentration usually increased within a short time if the intake remained low. The rise in serum phenylalanine was usually very brief, however, since protein reserves in infants are not large. In acute phenylalanine deficiency, very low serum phenylalanine concentrations are observed (less than 1 mg. per cent). Episodes of fever and respiratory symptoms occurred frequently. Orthohydroxyphenylacetie acid was not usually detected in urine specimens from well children until blood phenylalanine levels were at least 8 mg. per cent. This metabolite was almost always present in varying amounts during periods of ill-

6 1 6 Berry, Umbarger, and Sutherland

ness. I n earlier stages of our treatment program we did not recognize the significance of orthohydroxyphenylacetic acid excretion and as a result we did not often obtain serum specimens during the acute phase of an illness. Later we observed that serum phenylalanine concentrations were usually raised during illness, although rarely to levels greater than 10 to 12 mg. per cent. Measurement of urinary excretion of orthohydroxyphenylacetic acid was particularly useful to us, since its presence in urine signaled an increase in phenylalanine concentration in serum. Whether the increase resulted from illness, excessive intake of phenylalanine, or the transient stage of tissue breakdown associated with phenylalanine deficiency had to be determined. T h e requirements for phenylalanine vary with different patients and are subject to change with age and physiological state. Biochemical control of phenylketonuria goes hand in hand with dietary control, for if the biochemical results are not available there is no rational basis for making the required dietary adjustments. Changes in phenylalanine intake are made on the basis of the individual patient's ability to tolerate phenylalanine in excess of that required for growth;

October 1965

that is, to eliminate any dietary excess by excretion in the urine without increase in serum levels or production of abnormal metabolites. Serum phenylalanine measurements at frequent intervals are necessary for checking the effect of adjustments in the diet. Urinary phenylalanine and orthohydroxyphenylacetic acid excretions provide a continuing measure of the ability or inability of the child to eliminate excess phenylalanine. Increase in urine phenylalanine is frequently the first sign of an approaching illness. Urinary orthohydroxyphenylacetic acid excretion warns that the capacity of the child to utilize or eliminate phenylalanine has been exceeded, and serum phenylalanine levels have increased. REFERENCES

1. Umbarger, B., Sutherland, B. S., and Berry, H . K . : Advances in the management of phenylketonuric patients. Submitted for publication to J. A. M. A. 2. Berry, H. K. : Use of micromethod for phenylalanine in management of phenylketonuric patients, Clin. Chem. 8: 172, 1962. 3. Berry, H. K., Umbarger, B., and Livingston, B.: Excretion of phenylalanine by normal children and by patients with phenylketonuria, J. PEDIAT.63: 954, 1963. 4. Block, R. J., Durrum, E. L., and Zweig, C.: A manual of paper chromatography and paper electrophoresis, ed. 2, New York, 1958, Academic Press, Inc.