An Improved Test for Phenylketonuria

Transcription

1 An Improved Test for Phenylketonuria Chauncey 0. Rupe and Alfred H. Free HENYLKETONURIA (phenylpyruvic oligophrenia) is a metabolic disorder first recognized by Foiling (1) approximately 25 years ago. He found that two mentally retarded children in. the same family excreted urine containing a substance which gave a green color with ferric chloride. This substance was identified as phenylpyruvic acid. It has subsequently been recognized that in phenylketonuria a block in the utilization of phenylalanine causes this amino acid to accumulate in the blood. The work of Wallace, Moldave and Meister (2) suggests that the deficiency in. the utilization of phenylalanine by the phenyilcetonuric may be due to the absence of at least one component of an enzyme system which effects the conversion of phenylalanine to tyrosine. With the increase of serum phenylalanine, an accompanying irreversible injury to the brain occurs which may or may not be due to direct toxic effect of phenylalanine. A portion of the phenylalanine is deaminated to phenylpyruvic acid which appears in the urine. Recent innovations in diet therapy indicate successful treatment of phenylpyruvic oligophrenia (3), and this development makes the early detection of the disorder an important practical procedure. The present report describes an improved and simplified test for phenylpyruvic acid in urine. It also presents results obtained with this test in practical performance studies both by experienced and inexperienced operators. From the Milee.Ames Research Laboratory, Elkhart, Indiana. Presented in part before the Division of Biological Chemistry at the 134th National Meeting of the American Chemical Society, Chicago, IllinoIs, September The authors would like to express their appreciation to Dr. Henry Baird UI of the Department of Pediatrics, Temple University School of Medicine, Philadelphia, for stimulating their interest in this subject and making available results of studies made with impregnated strips. The authors are indebted to Dr. Hans Meyer, Clinical Director, and Mr. Andy Webber, Chief Technologist, of the Fort Wayne State School, Fort Wayne, Indiana, for supplying the urine specimens from patients with phenylketonuria. Received for publication December 4,

2 406 RUPE & FREE Clinkal Ch.mhfry METHODS Strip Test. This test which is called PHENISTIX1 is in. the form of stiff strips of cellulose impregnated with ferric and magnesium ions plus cyclohexylsulfamic acid. The ferric ions react with phenylpy-ruvate to form a color. The magnesium ions minimize interference of color development by urine phosphates. The cyclohexylsulfamic acid provides a buffer system which establishes the ph in the range for optimal color formation. The procedure is to dip a strip in. the urine sample to be tested or to moisten the strip by pressing it against a wet diaper. The reaction on the strip is observed one-half minute later by comparison with a color chart on the bottle label. If the reaction is negative, the strip remains yellow and will most closely match the negative color block. If the reaction is positive, a gray to blue color develops, the intensity of which is proportional to the amount of phenylpyruvic acid present. Three positive color blocks on the chart represent approximately 15, 40, and 100 mg. of phenylpyruvic acid per 100 ml. of urine. Liquid Ferric Chloride Test. Various methods of carrying out a ferric chloride test in a liquid system have been described (4, 5). Each modification has its own sensitivity and specificity, depending upon the amounts of acid and ferric ions present in the reaction mixture, the character of the urine, and the time at which the reaction is read. This variable response of the ferric chloride tests is one of their main drawbacks. In. the present study two liquid ferric chloride methods have been used. The first procedure was one in which 4 drops of SN H2S04 were added to 5 ml. of urine and mixed; then 4 drops of 10% FeC13 were added and the color observed at about ten seconds after adding the FeC13. Besides, an alternative method was used in which 1 drop of 1% H2S04 was mixed with r ml. of urine; 2 drops of 5% FeC13 were then added and the color of the mixture observed after ten seconds. In some of the studies both of these methods were employed. Addition of Phenylpyruvate to Urine. For the study in which the performance of the strip test was compared with that of the liquid ferric chloride test, sodium phenylpyruvate was dissolved in water to make a solution containing 500 mg. of phenylpyruvate per 100 ml.. This stock solution or a dilution of it was added to normal urine to give concentrations of 8, 15, 40 or 100 mg. per 100 ml. In each case at least 80% of the volume of the unknown test solution was urine. PHENISTIX is a trademark of the Ames Company, Inc., Elkhart, Indiana..

3 Vol. 5 No TEST FOR PHENYLICETONURIA 407 EXPERI MENTAL Performance. To evaluate the performance of the strip test in comparison with the liquid ferric chloride test, a total of 1904 tests was carried out by ten persons experienced in clinical testing (technologists and biochemists) and by eleven persons inexperienced in clinical testing (librarians, secretaries and organic chemists). These tests were all carried out as tests on unknowns. Each operator was given a coded series of urines containing added phenylpyruvate to be tested with the strip test and the same urines, differently coded, to be tested with the liquid ferric chloride test. The results, presented in Table 1, indicate that the strip test is more accurate than the liquid ferric chloride test in the hands of experienced as well as inexperienced operators. Since different numbers of tests were carried out at each level of phenylpyruvate concentration with each test, the figures in the table refer to the percentage of results at a given concentration which yielded the designated reading with each test. The strip test not only gave fewer false trace reactions with negative urines but also gave fewer false negative reactions with urines containing very small amounts of phenylpyruvate. From Table 1 it wifi be seen that false negative results were obtained with the ferric chloride test at levels as high as 100 mg. phenylpyruvate per Table 1. TESTs DONE AS UNKNOWNS BY EXPERIENCED AND INEXPERIENCED OPERATORS ON URINES CoNrAnciwo KNoWN AMOuNTs OF ADDED PHENYLPYETJVIC Acm Results With Strip Te8t Amoent of phenplpynevo acid Readings-% of Totai Totci a,idsd 15 m./10o 40 mg.f mg./100 Number mg./100 mi. 0 Trace ml. vol. ml. of Test, Re8uits with Liquid Ferne Chloride Te8t 0 Readiuga-% of Total Trac. Small ModeroAs Large

4 408 RUPE & FREE Clinkal Ch.mistry 100 ml. of urine. One reason for the better performance of the strip test is that a color chart is provided with it whereas with the liquid test positive reactions are arbitrarily graded. Results indicate that at actual levels of phenylpyruvate of 15, 40, and 100 mg. per 100 ml., every single gradation from 0 to large was obtained with the liquid test. Although the majority of the evaluation of the performance of the strip test has been carried out with urine containing added phenylpyruvate, approximately 25 different urines from patients with phenylpyruvic oligophrenia also have been used in this study. Careful examination of these urines containing naturally excreted phenylpyruvic acid with the strip test and with the liquid ferric chloride test has failed to reveal any behavior different from that observed with urine containing added phenylpyruvate. Employment of urine containing naturally excreted phenylpyruvic acid in small-scale blind testing procedures gave results comparable to those indicated in. Table 1. Sensitivity. The data of Table 1 also indicate that the strip test possesses optimal sensitivity. It will be seen that in blind testing 8 mg. of phenylpyruvate per 100 ml. gave a positive reaction with the strip test in. 82% of the tests, whereas the liquid ferric chloride test (one of the more sensitive modifications) gave positive reactions with this concentration in only 60% of the tests. Both experienced and inexperienced operators were able to recognize 15 mg. of phenylpyruvate per 100 ml. of urine as a positive reaction with the strip test in 99.5% of the blind tests. Specificity. Information with regard to the specificity of the strip test has been obtained by two different procedures. One procedure has involved testing several hundred urine samples from general hospital patients. These patients presented a variety of diseases and were receiving a variety of medications. The second procedure has been to investigate specific substances which might possibly be expected to give a reaction with the strip test. Information on the specificity of the test as determined by these procedures is summarized in Table 2. Only phenylpyruvic acid wifi react to give the typical gray to blue color with the strip at 30 seconds. The typical color is given by p-hydroxyphenylpyruvic acid but it fades within two to five seconds. Very high concentrations of salicylate or phenothiazine metabolites may give an atypical pink color. Moderate or high concentrations of ketone bodies in the urine give a Bordeaux-

5 Vol. 5, No TEST FOR PHENYLKETONURIA 409 Table 2. Srzcincx rv or Bmw TEST Reacts with Strip Test Do.. Not React with Strip Test phenylpyruvic acid phenyllactie acid phenylacetic acid p-hydroxy-phenylpyruvic acid (fleeting) phenylalanine pyruvie acid salicylates (high concentrations give pink color) lactic acid tyrosine phenothiacine metabolitee (high concentrations give tryptophane pink color) homogentisic acid acetoacetic thymol toluene acid acetyl-p-amino-phenol assorted baby powders assorted baby oils assorted baby antisepties miscellaneous constituents from a large number of random urines from hospital patients and healthy subjects red color with liquid ferric chloride tests. In fact some modifications of the liquid ferric chloride test for phenylpyruvic acid are exactly the same as the ferric chloride test (Gerhardt s) for acetoacetic acid. However, with the concentrations of acetoacetic acid in urine which are encountered in moderate to severe ketosis, no color is produced with the strip test. The specificity of the strip test greatly exceeds that of the liquid ferric chloride test since with the latter test a relatively large number of urines from either healthy subjects or hospital patients give colors which make the recognition of phenylpyruvic acid difficult or even impossible. Influence of Urinary Acidity. The acidity or alkalinity of a urine sample has much to do with the reactivity of phenylpyruvic acid in liquid ferric chloride tests. Such tests which do not involve acidification of the urine will give false negative tests with alkaline urine samples containing phenylpyruvic acid, simply because the ferric ions precipitate as hydroxide. Acid urines or weakly buffered samples may become too acid with some of the modifications of the ferric chloride test so that the usual color with phenylpyruvic acid does not appear. Studies with the strip test have been carried out with urines covering the ph range from 5 to 8.5 and involving differing specific gravities from dilute to concentrated samples. In no instance was

6 410 RUPE & FREE Clinical Ch.misfry there any indication that the strip test did not react accurately with either negative or positive samples. Influence of Urinary Phosphate. Phosphate occurs in practically all urine samples and its concentration varies greatly. To a dilute urine, containing a minimal amount of phosphate, were added amounts of NaH2PO4 to supply 0.02%, 0.04%, 0.08% and 0.16% of phosphorus (expressed as % P). This corresponds approximately to the physiologic range of phosphate concentration encountered in human urine. Different amounts of phenylpyruvate were added to all of the urines, and they were tested with the strip test and with the liquid ferric chloride test. With the strip test, all samples gave accurate results. With the liquid ferric chloride test, the urine containing 0.16% phosphorus gave a false negative result with a concentration of phenylpyruvate as high as 100 mg. per 100 ml. of urine. The sensitivity of this test was markedly decreased in the sample of urine containing 0.08% phosphorus. The results of this study demonstrate the efficacy of the magnesium ions in the strip test. Variability in Laboratory Procedures. Most current texts dealing with clinical urinalysis do not describe a test for phenylketonuria. Among those texts that do mention the test most describe it in a very superficial manner and indirectly imply that the exact procedure employed is of no great consequence. Experiments were carried out which employed different tests described in texts, the principal variants being the amount and type of acid and the amount of ferric chloride. It was found that the testing procedure has a great influence on the sensitivity of the test. Excess of either ferric chloride or sulfuric acid causes poor color development and rapid fading. An insufficient amount of either reagent may result in no color development or may decrease the intensity of the color formed. Variability in the Excretion of Phenylpyruvic Acid. Serial urine samples were collected from two patients with phenylpyruvic oligophrenia. Results obtained in testing these urines are shown in Table 3. There is a considerable variation in the concentration of phenyl. pyruvic acid in different samples. This concentration appears to bear some relationship to the specific gravity of the urine. Miscellaneous Observations. A variety of other properties of the strip test has been studied. The sticks appear to be nontoxic since no recognizable effect was observed when the test portion of 50 strips per rat were administered orally to white rats in one feeding by grinding the sticks together with the rats food ration. High con-

7 Vol. 5, No. 5, 1959 TEST FOR PHENYLKETONURIA 411 Table 3. TEsTs ON SERIAL U ann SAMPLES FROM Two PATIEi!rs witu PHENn.KroNunIA Result, with Results with liquid ferric Time Specific gravity strip test chloride test Patient A 7:00 A.M Over 100 mg.% Large 9:00 A.M mg.% Large 11:00 A.M mg.% Medium 3:00 P.M mg.% Large 5:00 P.M mg.% Large 7:00 P.M mg.% Large Patient B 7:00 A.M mg.% Medium 9:00 &.M mg.% Medium 11:00 A.M Trace Small 1:30 P.M Trace Small 3:30 P.M Trace Negative 5:00 P.M mg.% Small 8:00 p.m mg.% Medium 10:00 A.M. (Next Day) mg.% Small centrations of reducing agents such as ascorbic acid in the urine which may interfere with the liquid ferric chloride test do not present any problem with the strip test. The stability of the strip test is an important property. Short-time stability studies suggest that strips kept in the container in. which they are supplied will have a laboratory-shelf life of several years. In contrast, several proponents of the liquid ferric chloride tests recommend that the solution employed be freshly prepared. DISCUSSION Now that successful treatment for patients with phenylketonuria is available, the early detection of this abnormality becomes increasingly important. The need for a quick simple test which can be carried out with equal efficiency in either the laboratory, physician s office, well-baby clinics or at home has been emphasized by Centerwall (6), Baird (7), and Berry, Sutherland, Guest and Warkany (8). Centerwall has suggested that in the well-baby clinic all infants in. the early months of life should be screened for phenylketonuria by placing a drop of 10% solution of Fe013 upon the wet diaper. Baird, the first to use an impregnated strip to test for phenylpyruvate, applied the test to collected urine specimens and to the wet diaper. He states that a positive strip reaction is so easily recognized that the test can be entrusted to most mothers for home testing. Berry and her

8 412 RUPE & FREE Clinical Chemistry associates have found that phenylpyruvate is sufficiently stable so that urine containing phenylpyruvate can be taken up by ifiter paper, dried and then tested by adding a drop of FeC!3 solution to the paper at a later date. Mothers of newborn infants were given several pieces of absorbent paper and instructed to moisten the paper with the infant s urine, dry it and return it for testing. These three methods, even though they differ somewhat in procedure, emphasize the importance of early detection and the need for a simple reliable test for phenylketonuria. The present strip test has a number of obvious advantages which make it suitable for a variety of uses. Centerwall and Centerwall (9) suggested that a proportion of urine samples from untreated patients known to have phenylketonuria is negative to the liquid ferric chloride test. The data obtained in the present study on two patients with phenylpyruvic oligophrenia show a definite variability in the concentration of phenylpyruvic acid which appears to bear a relation to the concentration or dilution of the urine. The diet of the patient or severity of the metabolic disorder may also have a significant influence on the presence or absence of a positive test for phenylpyruvic acid in the urine. Employment of liquid ferric chloride tests, which under some conditions as indicated above may give false negative results, may account for some negative tests in patients with phenylketonuria. One possible difficulty that may arise with any simple qualitative test is the tendency of some users to overread or attempt to recognize smaller amounts of the substance in question than may be possible. This leads to occurrences where some negative samples erroneously are read as slightly positive. It may happen with the strip test for phenylketonuria as with other tests. Since the strip test is more sensitive than most laboratory tests one can recommend that overreading be avoided without getting unduly concerned about missing significant amounts of phenylpyruvic acid. SUMMARY A simple dip-and-read strip test for detection of phenylketonuria is described. This test contains ferric and magnesium ions and an acid buffer. It is called PHENISTIX. When the strip is moistened with urine it gives a gray to blue color with samples which contain phenylpyruvic acid, the intensity being proportional to the concentration of this constituent. A color chart provides for standardization of results and reproducible reports. Comparison of the perform-

9 Vol. 5, No. 5, 1959 TEST FOR PHENYLKETONURIA 413 ance of the test with conventional laboratory procedures has been carried out on over 1900 samples as tests on unknowns. The new test is reliable for the recognition of phenylketonuria and has some advantages over conventional laboratory procedures. REFERENCES i. Polling A., Z. Physiol. Chem. 227, 169 (1934). 2. Wallace, H. W., Moldave, K., and Meister, A., Proc. Boo. Exper. Biol. 4 Med. 94, 632 (1957). 3. Armstrong, M. D., and Tyler, F. H., J. Clin. Int est. 34, 565 (1955). 4. Levinson, S. A., and MacPate, R. P., Clinical Laboratory Diagnosis. 5th Ed. Philadelphia, Lea and Febiger, 1956, pp Hepler, 0. E., Manual o.f Clinical Laboratory Methods (4th Ed.). Springfield, Charles C Thomas, 1953, p Centerwall, W. B., J. A. M. A. 165, 392 (1957). 7. Baird, H. W., J. Pediat. 52, 715 (1958). 8. Berry, H. K., Sutherland, B., Guest, G. M., and Warkany, J.,.1. A. M. A. 167, 2189 (1958). 9. Centerwall, W. B., and Centerwall, S. A., Early Diagnosis and Management of Phenyl. ketonuria. Scientific exhibit presented at Annual Session of American Medical Association (June 23-27) 1958.