Reprinted from THe JourNAL of LABORATORY AND CLINICAL MbeIcing, St. Louis. Vol. 55, No, 3, Pages 491-496, March, 1960. (Printed in the U. S. A.) (Copyright © 1960 by The C. V. Mosby Company) AN ENZYMATIC SPECTROPHOTOMETRIG METHOD FOR THE DETERMINATION OF PHENYLALANINE IN BLOOD Bert N. La Du, M.D., Pu.D., anp Parrrcia J. Micuarn, B.A, Betuerspa, Mp. HERE is a need tor a simple rapid method to measure phenylalanine in serum or plasma, not only as an aid in the diagnosis of phenylketonuria, but also in the evaluation of the effectiveness of a diet low in phenylalanine in the treatment of patients with this condition. The method most commonly used to measure phenylalanine in plasma is that of Udenfriend and Cooper,: in which plasma is deproteinized and treated with Streptococcus faecalis to decarboxylate i-phenylalanine to phenylethylamine. The latter is then determined colorimet- rically after reaction with methyl orange. Although this method has been used successfully by many workers,” it requires relatively large amounts of plasma and takes several hours to perform each set of analyses. Furthermore, extremely careful technique is necessary to avoid an appreciable and variable blank from methyl orange. Another method for the determination of serum phenylalanine is that of Berry,” by paper chromatography. This method, although simple, has limitations in quantitative analysis inherent in any method which depends upon estimation of the intensity of colored spots on paper chromatograms. Scr, CHCOOH Sc. C—COOH Sp 9s Snake venom TL-amino acid oxidase L-phenylalanine phenylpyruvie acid H SN beg ee « 2 "I , oO NZ B SN 0 0 K Dek —b=0 H Borate-enol complex of phenylpyruvic acid Arsenate borate From the National Institute of Arthritis and Metabolic Diseases, National Institutes of Health, U. S. Public Health Service, Bethesda, Md. Received for publication June 22, 1959, 491 492 LA DU AND MICHAEL J. Lab. & Clin. Med. A simple rapid method is described whieh permits the analysis of plasma L-phenylalanine in 0.1 ml. of serum without deproteinization. The method uti- lizes L-amino acid oxidase from snake venom to oxidize L-phenylalanine to phenylpyruvic acid. In the presence of arsenate and borate ions the resulting a-keto acid is rapidly converted to an enol-borate complex which has a high ab- sorption in ultraviolet light. Catalase is added to protect the a-keto acid from peroxide formed in the oxidative deamination by L-amino acid oxidase. Enol-borate complexes have been employed by Knox and Pitt® to determine p-hydroxyphenylpyruvie acid oxidase activity in mammalian liver preparations. More recently these complexes have been used to measure the activity of several enzymes whieh either form or break down the aromatic a-keto acids related to phenylalanine, tyrosine, tryptophan, and histidine.” METHOD Reagents.— 1. Phosphate buffer: 0.2M sodium phosphate buffer, pH 6.5. 2. Arsenate-phosphate buffer: 2.0M sodium arsenate was dissolved in the 0.2M phosphate buffer, and the final pH was readjusted to 6.5 with dilute HCl. 3. Borate-arsenate reagent: 1.0M borate, dissolved in 2.0M arsenate, pIl 6.5, (61.8 Gm. borie acid + 624 Gm. sodium arsenate [Na,HAsO,-7H.0]) was adjusted to pH 6.5 with HCl and made up to 1 L. 4, Snake venom L-amino acid oxidase (the venom of Crotalus adamanteus*): a suspen- sion of dried venom in water was made which contained 10 mg. per milliliter. This was ecen- trifuged, and the clear supernatant so.ution was removed and used. The enzyme solution is kept at 0 to 5° ©, and remains active with little loss of activity for several days. The dry venom is stored in the refrigerator and maintains high enzyme activity for several months. The activity of the L-amino acid oxidase preparation can be assayed by using 0.1 ml. of the standard phenylalanine solution in place of serum in the directions given below. 5. Catalase: crystalline beef liver catalase,t diluted 1:5 with 0.2M phosphate buffer, pH 6.5. The enzyme solution is kept at 0 to 5° C. and can be used for several weeks. 6. Standard phenylalanine solution: L-phenylalanine, 1 «M per milliliter (0.1 ml. con- tains 16.5 ag). 7, Blood serum: Blood is drawn and allowed to clot. (Some samples of heparinized plasma develop turbidity during analysis which makes them unsuitable for this determination.) Procedwure.— Three 1.2 ml. quartz Beckman cuvettes with a 1 em. light path are used for the determinations, The additions to each of the cuvettes are as follows: O By Ey PO, buffer, 0.2M, pH 6.51: 0.39 0.39 0.39 Arsenate-phosphate, pH 6.5: 0.5 — = 1M borate in 2M arsenate: _ 0.5 0.5 Catalase, 1:5 dilution: 0.01 0.01 0.01 Venom, 10 mg. per milliliter: 0.10 0.10 0.10 (Serum added later.) The contents are mixed and the duplicate experimental cuvettes, E, and FE., are read against the control cuvette, C,, at 308 mu, in the Beckman spectrophotometer.§ If the addi- *From the Ross Allen Reptile Institute. Silver Springs, Fla. 7From the Worthington Biochemical Corporation, Freehold, N. J. tIf more than 0.1 ml. of serum is to be added, the volume of phosphate buffer must be reduced proportionately. §Model DU with ultraviolet attachment. Nahas > MEASUREMENT OF PHENYLALANINE IN PLASMA AND BLOOD 493 umber tions have been made correctly, this reading should be approximately zero. Then 0.1 ml. of serum is added to cuvettes C,, E,, and E,, mixed, and readings are taken at 1 or 2 minute intervals for 10 minutes at 308 mu, to be certain that the enzyme activity is adequate, and that the reaction is essentially complete, as indicated by finding no further change in the optical density reading. Final readings are made at 308, 330, and 350 my at this time. Calculations.— Readings are taken at 330 and 350 mu, as well as at 308 mu, to correct for the ab- sorption of the keto acids derived from tyrosine and tryptophan, since the latter 2 amino acids are also oxidized by the venom L-amino acid oxidase. ‘The correction applied is based upon the relative absorption of the enol-borate complexes of these keto acids at 308, 330, and 350 mz: RELATIVE OPTICAL DENSITY READINGS AT DIFFERENT WAVELENGTHS (mp) KETO ACIDS OF 308 330° 350 X = tyrosine x 0.60X 0.06X Y¥ = phenylalanine Y 0.10Y 0 Z = tryptophan 0.852, 1.41Z Z Let the reading at 308 mp — A; 330 mu = B; 350 my = C Then: A= X + ¥ + 0,852 B= 0.6X + 0.1Y + 1417 C = 0.06X + Z Solving these equations gives: X = 2.38B — 0.238A — 3.16C (1) Y = L234 ~ 226R + 2.15C (2) Z = 1.19C - 0.14B + 0.014A = C -— 0,06x (3) The absorption at 308 my caused by phenylpyruvie acid is given by solving for Y in equation (2): Y 0.031 * 10 = gg phenylalanine per milliliter of serum (if 0.1 ml. of serum were analyzed). One microgram phenylalanine under these conditions of 1.1 ml., ete, at 308 mu reads 0.031 O.D. units. x By the same means, solving for X in equation (1) and dividing: $045 X10 = ug tyrosine per milliliter of plasma (1 wg tyrosine under these conditions reads 0.045 at 308 Z m#); and, similarly, using equation (3): 0.033 * 10 = ug tryptophan per milliliter of serum (1 wg of tryptophan reads 0.033 at 350 mz). RESULTS AND DISCUSSION Spectficity—The L-amino acid oxidase of snake venom catalyzes the oxida- tion of a number of amino acids, However, only 4 of these are present in ap- preciable quantities in serum which yields a-keto acids with highly absorbing enol-borate complexes; these are phenylalanine, tyrosine, tryptophan, and his- tidine. Fortunately, histidine is not oxidized under the experimental conditions described above, and the relative contribution of each of the other 3 amino acids can easily be determined from the absorption of the combined complexes at 3 different wavelengths. Therefore, this method permits the simultaneous deter- mination of phenylalanine, tyrosine, and tryptophan in serum. If the value of phenylalanine alone is desired, the correction for absorption eaused by the keto acids of tyrosine and tryptophan would appear to be a disadvantage of the method. This is partly compensated for by the commercial availability of the 494 LA DU AND MICHAEL J. Lab. & Clip, Med. enzyme, its stability, and its high activity. Furthermore, the ease with which both phenylalanine and tyrosine can be measured is of special interest in some studies on phenylketonuria; for example, in the detection of the heterozygous carrier of this trait by the ratio between the levels of phenylalanine and tyro- sine in the blood after the administration of phenylalanine as a tolerance test.® The wide range of substrates which can be used with this enzyme has made it posstble to extend this method to measure a number of other aromatie amino acid analogues and antimetabolites, such ag p-fluorophenylalanine, m-tyrosine, monoiodotyrosinc, mononitrotyrosine, and 8-2-thienylalanine. These compounds are also rapidly oxidized by the L-amino acid oxidase of snake venom, and the resulting keto acid products ean be measured as enol-borate complexes.” 1.000 o PHENYLALANINE ALONE o @ RECOVERY FROM PLASMA «800 +600 «400 -200 OPTICAL DENSITY, 308 m. t l I J 10 20 30 MICROGRAMS OF PHENYLALANINE Fig. 1.—Graph of the optical density at 308 mz versus concentration of phenylalanine when determined in Standard solutions and when added to normal plasma. The ‘linearity between optical density and phenylalanine concentration is demonstrated. Reproducibility and Accuracy.—The method described is well suited to measuring the elevated blood level of phenylalanine found in untreated phenyl- ketonuric¢ individuals and to following the effectiveness of a diet low in phenylal- anine in lowering the serum level in these individuals. The specificity of the absorption spectrum of the phenylpyruvie acid enol-borate complex and the ease and rapidity of the analysis permit a large number of determinations to be carried out within a short period of time. Fig. 1 demonstrates the linear relationship between phenylalanine concentration and optical density change between 2 and 30 ng analyzed alone and the reeovery of phenylalanine added to normal plasma in this range. These results are in agreement with the theo- retic values expected. However, the precision of the analytic method is lower in analyses of phenylalanine in normal serum than when the values are elevated as in serum of an individual with phenylketonuria. In normal serum the con- tributions of tyrosine and tryptophan to the reading at 308 my are appreciable. Even though duplicate pairs of analyses in a series of normal serum samples analyzed on suceessive days rarely disagreed by more than 7 per cent, larger aliquots of serum would be desirable if the method were to be used to measure, with high accuracy, the endogenous phenylalanine, tyrosine, and tryptophan levels. Attempts to carry out the analyses directly on 0.2 or 0.3 ml. of serum have bec only partly successful, since a turbidity often occurs. For this reason, None MEASUREMENT OF PHENYLALANINE IN PLASMA AND BLOOD 495 several methods were tested to deproteinize the sample before analysis. Preliminary experiments have been made using perchlorie acid precipitation followed by neutralization and the removal of KC10,, as described by Segal, Blair, and Wyngaarden,! before cnzymatie assay of blood pyruvate. This procedure for deproteinization appears to be satisfactory in eliminating the turbidity problem, and the solution which results can be used directly in the enzymatic assay. Phenylalanine Levels in Normal and Phenylketonuric Individuals —-The serum phenylalanine levels of 30 adult blood bank donors in a nonfasting con- dition were found to have an average value of 1.55 mg. per cent (range, 0.84 to 2.64 mg. per cent). It is probable that the level in normal subjects in a fast- ing condition is somewhat lower than these values. In a smaller number of analyses in laboratory workers in a fasting condition, the group average is slightly lower than the above value. The level of phenylalanine in a group of 10 phenylketonuric individuals on a regular diet was found to have an average value of 36.8 mg. per cent (range, 26.2 to 59.1 mg. per cent). These values in normal and in phenylketonurie people are in good agreement with those in the literature? 1*™ obtained by other methods. The wide difference in the levels of normal and phenylkctonurie individuals makes the analysis of serum phenylalanine a valuable diagnostic procedure. SUMMARY A simple enzymatic spectrophotometric method has been described for the quantitative determination of phenylalanine in serum. The method is based upon the measurement of the absorption of the enol-borate complex of pheny]- pyruvic acid generated enzymatically from phenylalanine by L-amino aeid oxi- dase of snake venom. For elevated serum levels of phenylalanine the method is rapid, specific, and precise. It should be useful as a confirmatory test in the diagnosis of suspected phenylketonuria and in the evaluation of the effectiveness of a dict low in phenylalanine. The values obtained by this method agree well with those in the literature obtained by other techniques. Tyrosine and tryp- tophan are also determined by this method, and a suitable modification of the method is deseribed which should allow an accurate estimation of these amino acids and phenylalanine in normal serum. REFERENCES 1. Udenfriend, 8. and Cooper, J. R.: Assay of L-Phenylalanine as Phenylethylamine After Enzymatic Decarboxylation; Application to Isotopie Studies, J. Biol, Chem. 203: 953, 1958. . Meister, A., Udenfriend, 8., and Bessman, 8. P.: Diminished Phenylketonuria in Phenyi- pyruvic Oligophrenia After Administration of 1-Glutamine, 1-Glutamate or L- Asparagine, J. Clin. Invest. 35: 619, 1956. 3. Hsia, D. Y.-Y., Driscoll, K. W., Troll, W., and Knox, W. E.: Detection by Phenylalanine Tolerance Tests of Heterozygous Carriers of Phenylketonuria, Nature, London 178: 1239, 1956. 4. Hsia, D. Y.-Y., and Paine, R. S.: Phenylketonuria: Detection of the Heterozygous Carrier, J. Ment. Deficiency Res. 1: 53, 1957, . Hsia, D. Y.-Y.: Phenylketonuria: The Phenylalanine-Tyrosine Ratio in the Detection of the Heterozygous Carrier, J. Ment. Deficiency Res, 2: 8, 1958. tw oO 496 LA DU AND MICHAEL J. Lab. & Clin, Med. March, 1960 . Knox, W. E., and Messinger, E, C.: The Detection in the Heterozygote of the Meta- bolic Effect of the Recessive Gene for Phenylketonuria, Am. J. Human Genet. 10: 53, 1958. . Berry, H. K.: Paper Chromatographic Method for Estimation of Phenylalanine, Proc. Soc. Exper. Biol. & Med. 95: 71, 1957. . Knox, W. E., and Pitt, B. M.: Enzymatic Catalysis of the Keto-Enol Tautomerization of Phenylpyruvie Acids, J. Biol. Chem. 225: 675, 1957. 9, Lin, E. C. C., Pitt, B. M., Civen, M., and Knox, W. L.: The Assay of Aromati¢ Amino Acid Transaminations and Keto Acid Oxidation by the Enol Borate-Tautomerasc Method, J. Biol. Chem. 233: 668, 1958. . La Du, B. N., and Michael, P. J.: A New Assay for Analogues and Antimetabolites of Tyrosine and Phenylalanine. In preparation. . Segal, S., Blair, A. E., and Wyngaarden, J. B.: An Enzymatic Spectrophotometric Method for the Determination of Pyruvie Acid in Blood, J. Las. & CLIN. Merb. 48: 137, 1956. . Hsia, D. Y.-Y., and Driscoll, K. W.: Detection of the Heterozygous Carriers of Phenyl- ketonuria, Lancet 2: 1337, 1956. . Jervis, G. A., Block, R. J., Bolling, D., and Kanze, E.: Chemical and Metabolie Studies on Phenylalanine. II. The Phenylalanine Content of the Blood and Spinal Fluid in Phenylpyruvie Oligophrenia, J. Biol. Chem. 134; 105, 1940. . Bickel, H., Gerrard, J., and Hickmans, EK. M.: The Influence of Phenylalanine Intake on the Chemistry and Behavior of a Phenylketonuric Child, Acta paediat. 43: 64, 1954.