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An enzymatic assay of plasma phenylalanine and tyrosine for the detection and management of phenylketonuria
BlOCHEMICAL
MEDICINE
26, 211-221 (1981)
An Enzymatic Assay of Plasma Phenylalanine and Tyrosine for the Detection and Management of Phenylketonuria’ RONG-SEN
SHEN,
C. JOAN RICHARDSON,* BOBBYE M. ROUSE,* AND CREED W. ABELL
Division of Biochemistry, Department of Human Biological Chemistry and Genetics, *Department of Pediatrics, The University of Texas Medical Branch, Galveston. Texas 77550 Received
and
February 2, 1981
The primary methods currently in use for detection and management of phenylketonuria (PKU) are the Guthrie bacterial inhibition assay (1) and the spectrofluorometric method of McCaman and Robins (2). We have developed an enzymatic method for the simultaneous determination of phenylalanine (Phe) and tyrosine (Tyr) in plasma or serum (3) which offers certain advantages over both of these assays. This method uses yeast phenylalanine ammonia-lyase (EC 4.3.1.5) to catalyze the conversion of L-Phe to transcinnamic acid and r.,-Tyr to p-coumaric acid, which are then determined by differential spectrophotometry at 290 and 315 nm. The assay is fast, simple, and quantitative. The Guthrie assay, using dried blood samples, can be adapted to widescale screening of inborn errors of amino acid metabolism other than hyperphenylalaninemia. However, for the detection of PKU, our assay is more sensitive than the Guthrie inhibition assay, and because it is quantitative, eliminates the need for a follow-up test. Furthermore, by simultaneously measuring plasma Phe and Tyr levels, it makes possible differentiation of transient hypertyrosinemia from true hyperphenylalaninemia. The enzymatic method, therefore, should produce fewer false positives than the Guthrie test (4-6). At its present state of development, however, our method is best suited to the management of PKU. It requires a much smaller blood sample than the spectrofluorometric method. In our tests it has proved as specific and sensitive as the spectrofluorometric method for the measurement of ’ This
work was supported by March of Dimes Clinical Administrative
Research Grant
6-165. 211 0006-2944/81/050211-11$02.00/O Copyright 0 1981 by Academic Press. Inc. All rights of reproduction in any form reserved.
212
SHEN
ET AL.
Phe, and more specific for the measurement of Tyr. Its ability to achieve accurate simultaneous determinations of Phe and Tyr is a real advantage in PKU management. Since PKU patients lack the capacity to convert Phe to Tyr, a precursor for the biosynthesis of dopamine and norepinephrine, it is important to monitor plasma Tyr levels during treatment to ensure its adequate intake from the diet. In this paper we describe preliminary tests of this new method as a screen for PKU in 1084 newborn infants. We also present the rest&s of exploratory tests of this method in monitoring plasma Phe and Tyr levels of PKU patients during dietary treatment, in the diagnosis of classical PKU through the Phe-loading test, and in the detection of PKU heterozygosity. MATERIALS
AND METHODS
Enzyme. Phenylalanine ammonia-lyase (PAL) was isolated and purified from the yeast Rhodotorula glut&is according to the method of Fritz et al. (7). Enzyme preparations obtained in this laboratory had specific activities of greater than 2.0 units/mg protein. One unit is defined as that amount of protein which catalyzes the conversion of 1 pmole L-Phe to transcinnamic acid per minute at 30°C. Enzyme preparations obtained commercially (P-L Biochemicals, Inc., Milwaukee, Wise.) had specific activities of approximately 1.0 unit/mg protein. All PAL preparations were kept frozen at - 80°C and demonstrated no loss of activity for 12 months. Human subjects. The newborn subjects were delivered at The University of Texas Medical Branch Hospital in Galveston. The group of 1084 newborns screened for PKU by our enzymatic method represented approximately one-third of the infants born annually at the hospital. Most of the blood samples were collected during the first 5 months of the year. The phenylketonurics were outpatients of the Pediatric Department on low-Phe diets. Since 1976, we have collected 350 blood specimens from 31 patients to measure plasma Phe and Tyr levels. In addition, we have studied Phe-loading tests on 6 PKU patients and 3 normal individuals. We report data from 2 patients and 2 healthy individuals who are representative of other subjects in their respective groups. Relevant details of their medical histories are recorded below. Patient R.L.R. was a male fraternal twin. A Guthrie test at 3 days of age revealed blood Phe levels in excess of 20 mg/dl. A confirmation test carried out by our enzymatic method at 17 days of age revealed plasma Phe and Tyr levels of 50.0 and 1.2 mg/dl, respectively. He was placed on a low-Phe diet and responded well to dietary management. At 9 months of age (284th day), he was challenged with an oral load of Phe (180 mg Phe/kg body wtiday for 3 consecutive days).
DETECTION
AND
MANAGEMENT
OF PHENYLKETONURIA
213
Patient S. R., a female, was under dietary treatment since 7 weeks of age. A Phe loading test was given at 6 months of age (114,138, 128,109, and 152 mg Phe/kg body wt/day for 5 consecutive days). Subjects P.Q. and R.Q., both female, were healthy siblings from a family with two PKU children. Both P.Q. and R.Q. were on normal diets. Phe tolerance tests were given at ages of 11 (P.Q.) and 13 (R.Q.) years by feeding the subject with a high-protein diet equivalent to 175 (P.Q.) and 149 mg Phe/kg/day (R.Q.). Method of collecting blood samples. Blood from newborns was collected in two heparinized capillary tubes (80-(11 capacity) through heel puncture at the time of their discharge from the nursery. Simultaneously, filter paper disks were impregnated with blood according to the Guthrie procedure and these were mailed to the Texas Department of Health at Austin for the Guthrie test (1). Blood samples from the PKU patients and siblings of known PKU patients were collected in heparinized capillary or centrifuge tubes (0.15- to 0.4-ml capacity) at the Pediatric Department or were delivered from other sources by mail. Plasma was prepared by centrifugation using an Adams micro-hematocrit centrifuge (Clay-Adams, Parsippany , N. J.) or an Eppendorf Model 5413 centrifuge (Brinkman Instruments, Inc., Westbury, N. Y.). Method of assay. Details of the enzymatic method for determination of plasma concentrations of Phe and Tyr have been reported (3). Plasma levels of Phe and Tyr were routinely confirmed by a Beckman Model 121M automated amino acid analyzer (Beckman Instruments, Inc., Irvine, Calif.) at The University of Texas Medical Branch Cancer Center or by spectrofluorometry at The Texas Department of Health, Austin, Texas. For amino acid analysis, plasma (50 t.~l) was deproteinized with 4 vol of 3.75% sulfosalicylic acid in 0.3 N lithium citrate buffer (pH 2.2) and the mixture was separated by centrifugation (Eppendorf Model 5413 centrifuge). Phe and Tyr concentrations were expressed in milligrams per IO0 ml of plasma (mg/dl). Statistical analysis. An IBM 270/158 MBS computer performed sorting, cross-tabulation, and statistical analysis of data from 1084 newborn plasma samples. Correlations between results of the enzymatic method and amino acid analysis/spectrofluorometry were calculated by linear regression analysis using a Wang Sysytem 2200. RESULTS PKU
Screening
In a comparative study between our enzymatic method and the Guthrie test for PKU, a total of 1145 blood samples from newborn infants were collected. Sixty-one samples (5%) were rejected because insufficient material was collected. (This problem can easily be remedied by adding
214
SHEN
ET AL.
an additional 80-u.1 capillary tube for collection.) Among the remaining newborn infants screened by the enzymatic method (N= 1084). we detected 5 infants with plasma Phe levels greater than 8 mg/dl (8.02,8.35,8.50,8.91, and 9.34 mg/dl), and 2 infants with plasma Tyr levels greater than 8 mg/dl (9.58 and 10.86 mg/dl). In the same group of 1084, the Guthrie test produced no positives. Table 1 shows plasma Phe and Tyr levels in the newborn infants classified according to their amino acid concentrations. Table 2 shows plasma Phe and Tyr levels in newborn infants classified according to age at the time of testing. The majority of infants (82%) was tested within 72 hr of age. The mean and standard deviations of Phe and Tyr levels among the 1084 plasma samples assayed were found to be 1.98 +- 1.40 and 1.64 k 1.08 mg/dl, respectively. The mean plasma levels of Phe and Tyr in newborn infants are essentially unchanged during the first 3 days of life. In the 4-day-old infants, however, the mean plasma Tyr levels increase significantly (P < 0.001) and the mean plasma Phe levels increase slightly (P < 0.05). PKU
Management
In a period of 4 years, we analyzed 350 blood specimens from 31 PKU patients by our enzymatic method. More than half of these plasma samples (53%) were randomly analyzed by the amino acid analyzer for comparison. A small number of plasma samples (4.5%) were analyzed comparatively by spectrofluorometry. Results are presented in Table 3. Plasma Phe and Tyr concentrations determined by the enzymatic method agreed with those obtained by the amino acid analyzer. Results from our enzymatic method also agreed with data from the spectrofluorometric analyses of plasma Phe values, but not of Tyr values. Spectrofluorometry generally yielded two to threefold higher values of Tyr than the enzymatic method or amino acid analysis. Table 3 also indicates that agreement between the enzymatic and other methods is independent of the source of enzyme. Analysis
of Phe-Loading
Tests
Enzymatic assay of plasma Phe and Tyr levels during dietary treatment and Phe-loading test are shown in Figs. 1-3. The accumulation and reduction of Phe and Tyr in plasma were rapidly and precisely monitored by the enzymatic method, allowing easy assessment of the effects of dietary treatment and Phe-loading tests. DISCUSSION
Our enzymatic method requires only 40 ~1 of plasma or serum for assay in duplicate of both Phe and Tyr. This method is specific for LPhe and L-Tyr, and the entire procedure can be performed in 20 min per
635 369 59 16 5
12.00 2.00-4.00 4.00-6.00 6.00-8.00 >8.00
1.08 2.19 4.78 6.76 8.62
t * k k f
0.56 0.54 0.54 0.43 0.51
Mean f SD
’ Phe and Tyr levels are expressed as mg/dl.
No.
Amino acid levels
Phe
59 34 5 1.5 0.5
% Population
1.14 k 0.49 2.59 2 0.50 4.64 * 0.50 6.62 2 0.55 10.22
4 2
Mean ~?r SD
781 262 35
No.
Tyr .-~-
%
0.4 0.2
72 24 3
Population
TABLE 1 PLASMA Phe AND Tyr LEVELS IN NEWBORN INFANTS CLASSIFIEDACCORDINGTO THEIR AMINO ACID CONCENTRATIONS”
i’l
2 2 s x
%
2
E
$J
216
PLASMA
SHEN ET AL. TABLE 2 Phe AND Tyr LEVELS IN NEWBORN INFANTS CLASSIFIED ACCORDING TO AGE
AT
TIME
OF TESTING”
Percentage of population
Age
(days)
No.
1 2 3 24* Overall
19 320 545 200 1084
Phe 2.11 1.95 1.93 2.15 1.98
t f t iz +
TY~ l.27b 1.44 1.37 1.43 1.40
1.47 1.53 1.56 2.03 1.64
-c 0.636 2 0.91 f 1.03 -+ 1.37 2 1.08
2 30 50 18 loo
’ Phe and Tyr levels are expressed as mg/dl. * Mean 2 SD. * P values between 4-day-old and other groups: Phe, 3 day vs 4 day, P < 0.05; Tyr, 2 day vs 4 day and 3 day vs 4 day, P < 0.001.
specimen. It requires only one drop of blood, so the blood specimen can be collected in a microcentrifuge tube after skin puncture and then mailed to the laboratory. We routinely receive blood specimens by mail; some are collected by mothers. The method is currently being adapted for use with blood samples dried on filter paper. The sensitivity of our method is equivalent to the method of automated amino acid analysis. We have assayed 350 plasma and serum specimens from 31 PKU patients undergoing dietary management. Approximately 12% of these specimens have Phe values below 1 mg/dl, and 37% have TABLE COMPARISON
OF ENZYMATIC
Methods compared b vs Y) Phe PAL vs AAA PAL vs SF Tyr PAL vs AAA PAL vs SF
3
ANALYSIS (PAL) WITH AMINO SPECTROFLUOROMETRY (SF)
Number analysis
ACID ANALYSIS (AAA) AND
Regression equation (mg/dl)
130 606 190 19”
y=o.9151x y=l.O5lOx y=O.9262x y=O.975Ox
0.10 0.25 0.21 1.56
0.9759 0.9868 0.9761 0.9813
123 60” 183 14”
y = 1.0784x - 0.15 y=o.9464.r + 0.12 y=O.9839x y = 1.4536x + 1.02
0.9632 0.9912 0.9818 0.5350
” Indicates PAL prepared in our laboratory. b Indicates commercially available PAL.
+ + + +
Correlation coefficient
DETECTION
AND MANAGEMENT
35q g30d ‘g2 25‘E 20c3 if iI a
OF PHENYLKETONURIA
1 : i ;I f
217
i-
.1
4l-
15lo5o-
Age in days FIG. 1. Plasma Phe and Tyr levels of a phenylketonuric child (R.L.R.) during dietary treatment and Phe-loading test. PAL indicates analysis by the enzymatic method and AAA indicates amino acid analysis. The oral load of Phe was performed on 284 days of age for 3 consecutive days. Points under PAL represent the means of at least three determinations: points under AAA are a single determination.
Tyr values below 1 mg/dl. Paired t test indicates that our method yields slightly, but not significantly (P > 0.05), lower Phe values (0.85 5 0.17 mg/dl vs 0.91 k 0.20 mg/dl, N = 22) than does the amino acid analyzer but Tyr values are essentailly the same (0.79 + 0.18 vs 0.75 + 0.18 mg/ dl, N = 68) by both methods. Our results suggest that the enzymatic method provides a sensitive indicator of elevated Phe levels. In the comparative screening study, we detected seven infants with threshold high levels of Phe and Tyr who were not Guthrie positives.* A larger scale study would be required to ’ According to our calculations, a plasma Phe level of 9.09 mg/dl as determined by our method is equivalent to a whole-blood Phe level of 4.0 mg/dl as measured by the Guthrie method. Since the mean hematocrit reading in the blood of these infants (N = 679) was 56, 1 ml of whole blood yielded approximately 0.44 ml plasma. Assuming that all the blood Phe occurs in the plasma, a Guthrie blood level of 4.0 mg/dl is equivalent to our plasma level of 9.09 mg/dl.
218
SHEN ET AL.
o--0Tyr by PAL
0
1
2
3
4
5
Days
FIG. 2. Twenty-four-hour levels of Phe and Tyr in plasma of a PKU patient during Phe-loading test. Arrow and P indicate daily challenge of an oral load of Phe. Bloods were collected 24 hr after Phe loading. PAL indicates analysis by the enzymatic method, AAA indicates amino acid analysis, and SF indicates analysis by spectrofluorometry. Points under PAL represent the means of at least three determinations. Points under AAA and SF are a single determination.
adequately compare the two methods, since our sample contained only a small number of infants in the threshold range and none with Phe levels in the PKU range. The mean plasma levels of Phe and Tyr in newborn infants remained unchanged during the first 3 days of life, but increased thereafter. This finding may explain why 10% of PKU infants are missed by screening during the first 3 days of life (f&8). The rise in plasma Phe and Tyr levels in 4-day-old infants, which probably coincides with increased consumption of milk, ensures a more accurate detection of PKU. Other investigators have found the mean serum Phe levels in newborn infants to be 1.85 (9) and 2.08 mg/dl (10). The mean plasma Phe value found in this study (1.98 mg/dl) agrees well with these earlier reports. While the mean serum Tyr level in newborn infants has been previously reported to be 3.21 (9) and 2.50 mg/dl (11). our values were considerably lower (1.64 mg/dl). The higher mean serum Tyr values reported in the literature may be attributed to the use of l-nitroso-Znaphthol, which reacts with compounds other than Tyr. The enzymatic method has proved most valuable in monitoring the dietary management of PKU patients. During the treatment of 31 PKU
DETECTION
AND MANAGEMENT
012
4
8
OF PHENYLKETONURIA
12
16
20
219
24
Hours after Phe loading FIG. 3. Plasma Phe and Tyr levels after an oral load of Phe in two normal siblings (P.Q. and R.Q.) from a family with two PKU children. Plasma Phe and Tyr levels were determined by the enzymatic method at the time of Phe loading (0 hr) and subsequently (1,2,4, and 24 hr). Each point is the mean of at least three determinations.
patients, we have consistently observed a high correlation coefficient between our method and the established methods, such as amino acid analysis and spectrofluorometry, over a broad range of Phe (0.6 to 50 mgidl) and Tyr (0.3 to 4 mg/dl). These results indicate that our method is reliable and suitable for the dietary management of PKU. Furthermore, because the method is time efficient, it allows plasma levels of Phe and Tyr to be monitored frequently. Our method can also be used in conjunction with Phe-loading for the diagnosis of classical PKU and the detection of PKU heterozygotes. Simultaneous determination of Phe and Tyr allows the construction of both Phe and Tyr clearance curves from plasma. Since Phe is metabolized to Tyr, this information can be important. For example, we found that two healthy siblings from a family with two PKU children responded differently when they were challenged with an oral dose of Phe (Fig. 3):
220
SHEN ET AL.
one had a faster reduction of Phe accompanied by a slight accumulation of Tyr, while the other had a slower reduction of Phe and Tyr levels unchanged. In an unrelated PKU patient, when Phe was orally administered, the plasma Phe levels increased linearly for 5 days, whereas Tyr levels remained relatively unchanged (Fig. 2). Since the extent of conversion of Phe to Tyr depends on the activity of liver Phe hydroxylase, which is absent in classical PKU, and partially reduced in hyperphenylalaninemia and PKU heterozygosity, analyzing the accumulation of both amino acids may provide a sensitive means of discriminating PKU disease status. Our enzymatic method, therefore, allows considerable information to be extracted from the Phe-loading test. The tests of the enzymatic method reported here demonstrate that it is convenient and reliable for clinical applications and offers certain advantages over the most widely used methods of Phe and Tyr determination. Further development of the assay for wider availability and use is in progress. SUMMARY An enzymatic method for the determination of plasma phenylalanine (Phe) and tyrosine (Tyr) in newborns and phenylketonurics is described. The method uses yeast phenylalanine ammonia-lyase to catalyze the conversion of L-Phe to transcinnamic acid and L-Tyr to p-coumaric acid, which are then determined by differential spectrophotometry at 290 and 315 nm. This method, requiring only one drop of blood, is simple, quantitative, and time efficient, and it has been adopted for the management of phenylketonuria (PKU). It can be used for monitoring plasma Phe and Tyr levels of PKU patients during dietary treatment, for diagnosis of classical PKU through the Phe-loading test, and for detection of PKU heterozygotes. A total of 350 plasma samples collected from 31 PKU patients over a 4 year period were analyzed and yielded results similar to those obtained by automated amino acid analysis. In an exploratory study, 1084 newborn infants were screened for PKU by our enzymatic method and the Guthrie test. In this group we detected 5 infants with Phe levels greater than 8 mg/dl plasma and 2 infants with Tyr levels greater than 8 mg/dl plasma, while the Guthrie test on the same subjects gave no positives. ACKNOWLEDGMENTS We thank Melanie L. Nobel for collecting blood specimens from PKU patients, Jane Sonnenberg and Richard L. Eberle for running the enzymatic assays, and Horace Kelso for running the amino acid analyzer. We thank Constance B. Denney for her critical evaluation of this manuscript.
DETECTION
AND MANAGEMENT
OF PHENYLKETONURIA
221
REFERENCES I. 2. 3. 4. 5. 6. 7. 8. 9. IO. Il.
Guthrie, R., and Susi, A., Pediatrics 32, 338 (1963). McCaman, M. W., and Robins, E., J. Lab. C/in. Med. 59, 885 (1962). Shen, R. S., and Abell, C. W., Science 197, 665 (1977). Hsia, D. Y. Y., and Holtzman, N. A., in “Medical Genetics” (V. A. McKusick and R. Clairborne, Eds.), p. 237. H. P. Publishing, New York, 1973. Belton, N. R.. Crombie, J. D., Robins, S. P., Stephen, R.. and Farquhar, J. W.. Arch. Dis. Child. 48, 472 (1973). Holtzman. N. A., Meek, A. G., and Mellits, E. D.. 1. Amer. Med. Assoc. 229, 667 (1974). Fritz, R. R., Hodgins, D. S., and Abell, C. W., J. Biol. Chem. 251, 4646 (1976). Hsia. Y. E., N. Engl. J. Med. 301, 553 (1979). Hsia, D. Y. Y.. Litwack, M., O’Flynn, M., and Jakovcic, S.. N. Engl. J. Med. 267, 1067 (1962). Hsia, D. Y. Y., Berman, J. L., and Slatis, H. M., /. Amer. Med. Assoc. ~$8, 131 (1964). Wang, P. W. K.. O’Flynn, M. E., and Inouye, T., C/in. Chem. 10, 1098 (1964).
MEDICINE
26, 211-221 (1981)
An Enzymatic Assay of Plasma Phenylalanine and Tyrosine for the Detection and Management of Phenylketonuria’ RONG-SEN
SHEN,
C. JOAN RICHARDSON,* BOBBYE M. ROUSE,* AND CREED W. ABELL
Division of Biochemistry, Department of Human Biological Chemistry and Genetics, *Department of Pediatrics, The University of Texas Medical Branch, Galveston. Texas 77550 Received
and
February 2, 1981
The primary methods currently in use for detection and management of phenylketonuria (PKU) are the Guthrie bacterial inhibition assay (1) and the spectrofluorometric method of McCaman and Robins (2). We have developed an enzymatic method for the simultaneous determination of phenylalanine (Phe) and tyrosine (Tyr) in plasma or serum (3) which offers certain advantages over both of these assays. This method uses yeast phenylalanine ammonia-lyase (EC 4.3.1.5) to catalyze the conversion of L-Phe to transcinnamic acid and r.,-Tyr to p-coumaric acid, which are then determined by differential spectrophotometry at 290 and 315 nm. The assay is fast, simple, and quantitative. The Guthrie assay, using dried blood samples, can be adapted to widescale screening of inborn errors of amino acid metabolism other than hyperphenylalaninemia. However, for the detection of PKU, our assay is more sensitive than the Guthrie inhibition assay, and because it is quantitative, eliminates the need for a follow-up test. Furthermore, by simultaneously measuring plasma Phe and Tyr levels, it makes possible differentiation of transient hypertyrosinemia from true hyperphenylalaninemia. The enzymatic method, therefore, should produce fewer false positives than the Guthrie test (4-6). At its present state of development, however, our method is best suited to the management of PKU. It requires a much smaller blood sample than the spectrofluorometric method. In our tests it has proved as specific and sensitive as the spectrofluorometric method for the measurement of ’ This
work was supported by March of Dimes Clinical Administrative
Research Grant
6-165. 211 0006-2944/81/050211-11$02.00/O Copyright 0 1981 by Academic Press. Inc. All rights of reproduction in any form reserved.
212
SHEN
ET AL.
Phe, and more specific for the measurement of Tyr. Its ability to achieve accurate simultaneous determinations of Phe and Tyr is a real advantage in PKU management. Since PKU patients lack the capacity to convert Phe to Tyr, a precursor for the biosynthesis of dopamine and norepinephrine, it is important to monitor plasma Tyr levels during treatment to ensure its adequate intake from the diet. In this paper we describe preliminary tests of this new method as a screen for PKU in 1084 newborn infants. We also present the rest&s of exploratory tests of this method in monitoring plasma Phe and Tyr levels of PKU patients during dietary treatment, in the diagnosis of classical PKU through the Phe-loading test, and in the detection of PKU heterozygosity. MATERIALS
AND METHODS
Enzyme. Phenylalanine ammonia-lyase (PAL) was isolated and purified from the yeast Rhodotorula glut&is according to the method of Fritz et al. (7). Enzyme preparations obtained in this laboratory had specific activities of greater than 2.0 units/mg protein. One unit is defined as that amount of protein which catalyzes the conversion of 1 pmole L-Phe to transcinnamic acid per minute at 30°C. Enzyme preparations obtained commercially (P-L Biochemicals, Inc., Milwaukee, Wise.) had specific activities of approximately 1.0 unit/mg protein. All PAL preparations were kept frozen at - 80°C and demonstrated no loss of activity for 12 months. Human subjects. The newborn subjects were delivered at The University of Texas Medical Branch Hospital in Galveston. The group of 1084 newborns screened for PKU by our enzymatic method represented approximately one-third of the infants born annually at the hospital. Most of the blood samples were collected during the first 5 months of the year. The phenylketonurics were outpatients of the Pediatric Department on low-Phe diets. Since 1976, we have collected 350 blood specimens from 31 patients to measure plasma Phe and Tyr levels. In addition, we have studied Phe-loading tests on 6 PKU patients and 3 normal individuals. We report data from 2 patients and 2 healthy individuals who are representative of other subjects in their respective groups. Relevant details of their medical histories are recorded below. Patient R.L.R. was a male fraternal twin. A Guthrie test at 3 days of age revealed blood Phe levels in excess of 20 mg/dl. A confirmation test carried out by our enzymatic method at 17 days of age revealed plasma Phe and Tyr levels of 50.0 and 1.2 mg/dl, respectively. He was placed on a low-Phe diet and responded well to dietary management. At 9 months of age (284th day), he was challenged with an oral load of Phe (180 mg Phe/kg body wtiday for 3 consecutive days).
DETECTION
AND
MANAGEMENT
OF PHENYLKETONURIA
213
Patient S. R., a female, was under dietary treatment since 7 weeks of age. A Phe loading test was given at 6 months of age (114,138, 128,109, and 152 mg Phe/kg body wt/day for 5 consecutive days). Subjects P.Q. and R.Q., both female, were healthy siblings from a family with two PKU children. Both P.Q. and R.Q. were on normal diets. Phe tolerance tests were given at ages of 11 (P.Q.) and 13 (R.Q.) years by feeding the subject with a high-protein diet equivalent to 175 (P.Q.) and 149 mg Phe/kg/day (R.Q.). Method of collecting blood samples. Blood from newborns was collected in two heparinized capillary tubes (80-(11 capacity) through heel puncture at the time of their discharge from the nursery. Simultaneously, filter paper disks were impregnated with blood according to the Guthrie procedure and these were mailed to the Texas Department of Health at Austin for the Guthrie test (1). Blood samples from the PKU patients and siblings of known PKU patients were collected in heparinized capillary or centrifuge tubes (0.15- to 0.4-ml capacity) at the Pediatric Department or were delivered from other sources by mail. Plasma was prepared by centrifugation using an Adams micro-hematocrit centrifuge (Clay-Adams, Parsippany , N. J.) or an Eppendorf Model 5413 centrifuge (Brinkman Instruments, Inc., Westbury, N. Y.). Method of assay. Details of the enzymatic method for determination of plasma concentrations of Phe and Tyr have been reported (3). Plasma levels of Phe and Tyr were routinely confirmed by a Beckman Model 121M automated amino acid analyzer (Beckman Instruments, Inc., Irvine, Calif.) at The University of Texas Medical Branch Cancer Center or by spectrofluorometry at The Texas Department of Health, Austin, Texas. For amino acid analysis, plasma (50 t.~l) was deproteinized with 4 vol of 3.75% sulfosalicylic acid in 0.3 N lithium citrate buffer (pH 2.2) and the mixture was separated by centrifugation (Eppendorf Model 5413 centrifuge). Phe and Tyr concentrations were expressed in milligrams per IO0 ml of plasma (mg/dl). Statistical analysis. An IBM 270/158 MBS computer performed sorting, cross-tabulation, and statistical analysis of data from 1084 newborn plasma samples. Correlations between results of the enzymatic method and amino acid analysis/spectrofluorometry were calculated by linear regression analysis using a Wang Sysytem 2200. RESULTS PKU
Screening
In a comparative study between our enzymatic method and the Guthrie test for PKU, a total of 1145 blood samples from newborn infants were collected. Sixty-one samples (5%) were rejected because insufficient material was collected. (This problem can easily be remedied by adding
214
SHEN
ET AL.
an additional 80-u.1 capillary tube for collection.) Among the remaining newborn infants screened by the enzymatic method (N= 1084). we detected 5 infants with plasma Phe levels greater than 8 mg/dl (8.02,8.35,8.50,8.91, and 9.34 mg/dl), and 2 infants with plasma Tyr levels greater than 8 mg/dl (9.58 and 10.86 mg/dl). In the same group of 1084, the Guthrie test produced no positives. Table 1 shows plasma Phe and Tyr levels in the newborn infants classified according to their amino acid concentrations. Table 2 shows plasma Phe and Tyr levels in newborn infants classified according to age at the time of testing. The majority of infants (82%) was tested within 72 hr of age. The mean and standard deviations of Phe and Tyr levels among the 1084 plasma samples assayed were found to be 1.98 +- 1.40 and 1.64 k 1.08 mg/dl, respectively. The mean plasma levels of Phe and Tyr in newborn infants are essentially unchanged during the first 3 days of life. In the 4-day-old infants, however, the mean plasma Tyr levels increase significantly (P < 0.001) and the mean plasma Phe levels increase slightly (P < 0.05). PKU
Management
In a period of 4 years, we analyzed 350 blood specimens from 31 PKU patients by our enzymatic method. More than half of these plasma samples (53%) were randomly analyzed by the amino acid analyzer for comparison. A small number of plasma samples (4.5%) were analyzed comparatively by spectrofluorometry. Results are presented in Table 3. Plasma Phe and Tyr concentrations determined by the enzymatic method agreed with those obtained by the amino acid analyzer. Results from our enzymatic method also agreed with data from the spectrofluorometric analyses of plasma Phe values, but not of Tyr values. Spectrofluorometry generally yielded two to threefold higher values of Tyr than the enzymatic method or amino acid analysis. Table 3 also indicates that agreement between the enzymatic and other methods is independent of the source of enzyme. Analysis
of Phe-Loading
Tests
Enzymatic assay of plasma Phe and Tyr levels during dietary treatment and Phe-loading test are shown in Figs. 1-3. The accumulation and reduction of Phe and Tyr in plasma were rapidly and precisely monitored by the enzymatic method, allowing easy assessment of the effects of dietary treatment and Phe-loading tests. DISCUSSION
Our enzymatic method requires only 40 ~1 of plasma or serum for assay in duplicate of both Phe and Tyr. This method is specific for LPhe and L-Tyr, and the entire procedure can be performed in 20 min per
635 369 59 16 5
12.00 2.00-4.00 4.00-6.00 6.00-8.00 >8.00
1.08 2.19 4.78 6.76 8.62
t * k k f
0.56 0.54 0.54 0.43 0.51
Mean f SD
’ Phe and Tyr levels are expressed as mg/dl.
No.
Amino acid levels
Phe
59 34 5 1.5 0.5
% Population
1.14 k 0.49 2.59 2 0.50 4.64 * 0.50 6.62 2 0.55 10.22
4 2
Mean ~?r SD
781 262 35
No.
Tyr .-~-
%
0.4 0.2
72 24 3
Population
TABLE 1 PLASMA Phe AND Tyr LEVELS IN NEWBORN INFANTS CLASSIFIEDACCORDINGTO THEIR AMINO ACID CONCENTRATIONS”
i’l
2 2 s x
%
2
E
$J
216
PLASMA
SHEN ET AL. TABLE 2 Phe AND Tyr LEVELS IN NEWBORN INFANTS CLASSIFIED ACCORDING TO AGE
AT
TIME
OF TESTING”
Percentage of population
Age
(days)
No.
1 2 3 24* Overall
19 320 545 200 1084
Phe 2.11 1.95 1.93 2.15 1.98
t f t iz +
TY~ l.27b 1.44 1.37 1.43 1.40
1.47 1.53 1.56 2.03 1.64
-c 0.636 2 0.91 f 1.03 -+ 1.37 2 1.08
2 30 50 18 loo
’ Phe and Tyr levels are expressed as mg/dl. * Mean 2 SD. * P values between 4-day-old and other groups: Phe, 3 day vs 4 day, P < 0.05; Tyr, 2 day vs 4 day and 3 day vs 4 day, P < 0.001.
specimen. It requires only one drop of blood, so the blood specimen can be collected in a microcentrifuge tube after skin puncture and then mailed to the laboratory. We routinely receive blood specimens by mail; some are collected by mothers. The method is currently being adapted for use with blood samples dried on filter paper. The sensitivity of our method is equivalent to the method of automated amino acid analysis. We have assayed 350 plasma and serum specimens from 31 PKU patients undergoing dietary management. Approximately 12% of these specimens have Phe values below 1 mg/dl, and 37% have TABLE COMPARISON
OF ENZYMATIC
Methods compared b vs Y) Phe PAL vs AAA PAL vs SF Tyr PAL vs AAA PAL vs SF
3
ANALYSIS (PAL) WITH AMINO SPECTROFLUOROMETRY (SF)
Number analysis
ACID ANALYSIS (AAA) AND
Regression equation (mg/dl)
130 606 190 19”
y=o.9151x y=l.O5lOx y=O.9262x y=O.975Ox
0.10 0.25 0.21 1.56
0.9759 0.9868 0.9761 0.9813
123 60” 183 14”
y = 1.0784x - 0.15 y=o.9464.r + 0.12 y=O.9839x y = 1.4536x + 1.02
0.9632 0.9912 0.9818 0.5350
” Indicates PAL prepared in our laboratory. b Indicates commercially available PAL.
+ + + +
Correlation coefficient
DETECTION
AND MANAGEMENT
35q g30d ‘g2 25‘E 20c3 if iI a
OF PHENYLKETONURIA
1 : i ;I f
217
i-
.1
4l-
15lo5o-
Age in days FIG. 1. Plasma Phe and Tyr levels of a phenylketonuric child (R.L.R.) during dietary treatment and Phe-loading test. PAL indicates analysis by the enzymatic method and AAA indicates amino acid analysis. The oral load of Phe was performed on 284 days of age for 3 consecutive days. Points under PAL represent the means of at least three determinations: points under AAA are a single determination.
Tyr values below 1 mg/dl. Paired t test indicates that our method yields slightly, but not significantly (P > 0.05), lower Phe values (0.85 5 0.17 mg/dl vs 0.91 k 0.20 mg/dl, N = 22) than does the amino acid analyzer but Tyr values are essentailly the same (0.79 + 0.18 vs 0.75 + 0.18 mg/ dl, N = 68) by both methods. Our results suggest that the enzymatic method provides a sensitive indicator of elevated Phe levels. In the comparative screening study, we detected seven infants with threshold high levels of Phe and Tyr who were not Guthrie positives.* A larger scale study would be required to ’ According to our calculations, a plasma Phe level of 9.09 mg/dl as determined by our method is equivalent to a whole-blood Phe level of 4.0 mg/dl as measured by the Guthrie method. Since the mean hematocrit reading in the blood of these infants (N = 679) was 56, 1 ml of whole blood yielded approximately 0.44 ml plasma. Assuming that all the blood Phe occurs in the plasma, a Guthrie blood level of 4.0 mg/dl is equivalent to our plasma level of 9.09 mg/dl.
218
SHEN ET AL.
o--0Tyr by PAL
0
1
2
3
4
5
Days
FIG. 2. Twenty-four-hour levels of Phe and Tyr in plasma of a PKU patient during Phe-loading test. Arrow and P indicate daily challenge of an oral load of Phe. Bloods were collected 24 hr after Phe loading. PAL indicates analysis by the enzymatic method, AAA indicates amino acid analysis, and SF indicates analysis by spectrofluorometry. Points under PAL represent the means of at least three determinations. Points under AAA and SF are a single determination.
adequately compare the two methods, since our sample contained only a small number of infants in the threshold range and none with Phe levels in the PKU range. The mean plasma levels of Phe and Tyr in newborn infants remained unchanged during the first 3 days of life, but increased thereafter. This finding may explain why 10% of PKU infants are missed by screening during the first 3 days of life (f&8). The rise in plasma Phe and Tyr levels in 4-day-old infants, which probably coincides with increased consumption of milk, ensures a more accurate detection of PKU. Other investigators have found the mean serum Phe levels in newborn infants to be 1.85 (9) and 2.08 mg/dl (10). The mean plasma Phe value found in this study (1.98 mg/dl) agrees well with these earlier reports. While the mean serum Tyr level in newborn infants has been previously reported to be 3.21 (9) and 2.50 mg/dl (11). our values were considerably lower (1.64 mg/dl). The higher mean serum Tyr values reported in the literature may be attributed to the use of l-nitroso-Znaphthol, which reacts with compounds other than Tyr. The enzymatic method has proved most valuable in monitoring the dietary management of PKU patients. During the treatment of 31 PKU
DETECTION
AND MANAGEMENT
012
4
8
OF PHENYLKETONURIA
12
16
20
219
24
Hours after Phe loading FIG. 3. Plasma Phe and Tyr levels after an oral load of Phe in two normal siblings (P.Q. and R.Q.) from a family with two PKU children. Plasma Phe and Tyr levels were determined by the enzymatic method at the time of Phe loading (0 hr) and subsequently (1,2,4, and 24 hr). Each point is the mean of at least three determinations.
patients, we have consistently observed a high correlation coefficient between our method and the established methods, such as amino acid analysis and spectrofluorometry, over a broad range of Phe (0.6 to 50 mgidl) and Tyr (0.3 to 4 mg/dl). These results indicate that our method is reliable and suitable for the dietary management of PKU. Furthermore, because the method is time efficient, it allows plasma levels of Phe and Tyr to be monitored frequently. Our method can also be used in conjunction with Phe-loading for the diagnosis of classical PKU and the detection of PKU heterozygotes. Simultaneous determination of Phe and Tyr allows the construction of both Phe and Tyr clearance curves from plasma. Since Phe is metabolized to Tyr, this information can be important. For example, we found that two healthy siblings from a family with two PKU children responded differently when they were challenged with an oral dose of Phe (Fig. 3):
220
SHEN ET AL.
one had a faster reduction of Phe accompanied by a slight accumulation of Tyr, while the other had a slower reduction of Phe and Tyr levels unchanged. In an unrelated PKU patient, when Phe was orally administered, the plasma Phe levels increased linearly for 5 days, whereas Tyr levels remained relatively unchanged (Fig. 2). Since the extent of conversion of Phe to Tyr depends on the activity of liver Phe hydroxylase, which is absent in classical PKU, and partially reduced in hyperphenylalaninemia and PKU heterozygosity, analyzing the accumulation of both amino acids may provide a sensitive means of discriminating PKU disease status. Our enzymatic method, therefore, allows considerable information to be extracted from the Phe-loading test. The tests of the enzymatic method reported here demonstrate that it is convenient and reliable for clinical applications and offers certain advantages over the most widely used methods of Phe and Tyr determination. Further development of the assay for wider availability and use is in progress. SUMMARY An enzymatic method for the determination of plasma phenylalanine (Phe) and tyrosine (Tyr) in newborns and phenylketonurics is described. The method uses yeast phenylalanine ammonia-lyase to catalyze the conversion of L-Phe to transcinnamic acid and L-Tyr to p-coumaric acid, which are then determined by differential spectrophotometry at 290 and 315 nm. This method, requiring only one drop of blood, is simple, quantitative, and time efficient, and it has been adopted for the management of phenylketonuria (PKU). It can be used for monitoring plasma Phe and Tyr levels of PKU patients during dietary treatment, for diagnosis of classical PKU through the Phe-loading test, and for detection of PKU heterozygotes. A total of 350 plasma samples collected from 31 PKU patients over a 4 year period were analyzed and yielded results similar to those obtained by automated amino acid analysis. In an exploratory study, 1084 newborn infants were screened for PKU by our enzymatic method and the Guthrie test. In this group we detected 5 infants with Phe levels greater than 8 mg/dl plasma and 2 infants with Tyr levels greater than 8 mg/dl plasma, while the Guthrie test on the same subjects gave no positives. ACKNOWLEDGMENTS We thank Melanie L. Nobel for collecting blood specimens from PKU patients, Jane Sonnenberg and Richard L. Eberle for running the enzymatic assays, and Horace Kelso for running the amino acid analyzer. We thank Constance B. Denney for her critical evaluation of this manuscript.
DETECTION
AND MANAGEMENT
OF PHENYLKETONURIA
221
REFERENCES I. 2. 3. 4. 5. 6. 7. 8. 9. IO. Il.
Guthrie, R., and Susi, A., Pediatrics 32, 338 (1963). McCaman, M. W., and Robins, E., J. Lab. C/in. Med. 59, 885 (1962). Shen, R. S., and Abell, C. W., Science 197, 665 (1977). Hsia, D. Y. Y., and Holtzman, N. A., in “Medical Genetics” (V. A. McKusick and R. Clairborne, Eds.), p. 237. H. P. Publishing, New York, 1973. Belton, N. R.. Crombie, J. D., Robins, S. P., Stephen, R.. and Farquhar, J. W.. Arch. Dis. Child. 48, 472 (1973). Holtzman. N. A., Meek, A. G., and Mellits, E. D.. 1. Amer. Med. Assoc. 229, 667 (1974). Fritz, R. R., Hodgins, D. S., and Abell, C. W., J. Biol. Chem. 251, 4646 (1976). Hsia. Y. E., N. Engl. J. Med. 301, 553 (1979). Hsia, D. Y. Y.. Litwack, M., O’Flynn, M., and Jakovcic, S.. N. Engl. J. Med. 267, 1067 (1962). Hsia, D. Y. Y., Berman, J. L., and Slatis, H. M., /. Amer. Med. Assoc. ~$8, 131 (1964). Wang, P. W. K.. O’Flynn, M. E., and Inouye, T., C/in. Chem. 10, 1098 (1964).