QUANTITATIVE DETECTION OF PHENYLALANINE AND TYROSINE IN WHOLE BLOOD

Abstract: Whole blood is analyzed for phenylalanine and tyrosine by first subjecting a specimen of the blood to the enzymatic action of phenylalanine ammonia lyase to selectively convert the two amino acids to trans-cinnamic and trans-coumaric acids, respectively, then analyzing the sample containing the converted acids by cation exchange chromatography.

Description: QUANTITATIVE DETECTION OF PHENYLALANINE AND TYROSINE IN WHOLE BLOOD This invention resides in methods for the detection of phenylketonuria and related diseases and disease conditions in mammalian blood. BACKGROUND OF THE INVENTION Various inherited disorders of amino acid catabolism can lead to brain defects and conditions of abnormal physical and metnal development. Prominent among these disorders are hyperphenylalaninemias, in which the conversion of phenylalanine to tyrosine is impaired by an enzyme deficiency. Hyperphenylalaninemias include classic phenylketonuria, as well as benign, transient and malignant hyperphenylalaninemias. Enzymes whose deficiency is responsible for these conditions are phenylalanine hydroxylase, dihydropteridine reductase, GTP cyclohydrolase, and 6-pyruvoyltetrahydro- biopterin synthase. Phenylketonuria (PKU), which is caused by a deficiency in phenylalanine hydroxylase, inevitably leads to mental retardation when left untreated. PKU and other inherited amino acid disorders can be treated by dietary management to prevent the brain defects and allow normal physical and mental growth. Treatment should begin as soon as possible after birth, however, and for this reason newborns are routinely screened for PKU and, in some cases, other amino acid disorders, by determinations of the levels of phenylalanine and tyrosine. The most common screening methods are a bacterial inhibition assay known as the Guthrie method, Guthrie, R. , et al. , "A simple phenylalanine method for detecting phenylketonuria in large populations of newborn infants," Pediatrics 1963:332, and a fluorometric procedure, Hill, J.B. , et al. , "An Automated Procedure for Blood Phenylalanine," Clin. Chem. 11:541-546 (1965). The bacterial inhibition assay is only semiquantitative, however, and not suitable for automated interpretation and data processing. The fluorometric procedure involves the binding of fluorophores to the amino acids, which gives rise to false positives, often necessitating repeat tests. SUMMARY OF THE INVENTION It has now been discovered that the levels of phenylalanine and tyrosine in a blood sample can be determined in a highly specific manner that does not require protein removal or the use of fluorescent dyes. This is achieved by subjecting the blood sample to the enzymatic action of phenylalanine ammoma lyase, then analyzing the treated sample by simple chromatographic techniques, particularly ion exchange chromatography. The enzymatic action converts phenylalanine and tyrosine to tra -cinnamic acid and trans-coumaric acid, respectively, by elimination of an amine. These acids are readily separable by chromatography on a medium capable of separating organic acids, the resulting fractions then being detected by conventional detection methods. The assay is highly specific for phenylalanine and tyrosine, allows for high assay throughput, and offers both accuracy and reliability. These and other features and advantages of the invention will become apparent from the description that follows. DETAILED DESCRIPTION OF THE INVENTION AND PREFERRED EMBODIMENTS The two amino acids and their conversion products resulting from the action of phenylalanine ammonia lyase are as follows: OH phenylalanine trans-cinnamic acid tyrosine trøns-coumaric acid Samples on which the enzymatic reactions are performed are generally whole blood rather than serum, and rather than other bodily fluids such as urine. While whole blood in any form can be used, samples of particular interest are those that are collected in dried form as blood spot specimens on an inert support. Common supports are various forms of filter paper, although any absorptive material that is chemically inert relative to the blood and its components can be used. Thus, various porous polymers other than paper can be used as well. When dried blood spots and other forms of dried blood are used, the specimen is converted to liquid form by being dissolved or suspended in an appropriate solution. Buffered aqueous solutions are preferred. Dried blood spot specimens are readily extracted from their inert supports by immersion of the support in the solution. Prior to exposure of the liquified specimen to the enzyme, the specimen may be treated to stabilize the specimen components other than phenylalanine and tyrosine, i.e. , to prevent endogenous enzymes from cleaving proteins in the specimen to their amino acid substituents. This is done by any of various conventional means. Preferred means are the addition of a proteolytic enzyme inhibitor to the specimen. Such inhibitors are well known, and those most convenient for use in this method will be readily apparent to those skilled in the art. Examples of such inhibitors are ethylenediaminetetraacetic acid, aprotenin, diisopropyl fluorophosphate, and phenylmethylsulfonyl fluoride. Combinations of inhibitors can be used to further effect. When the specimen is a dried blood spot specimen, the inhibitor is conveniently included in the solution used to extract the specimen from the support. The enzyme phenylalanine ammonia lyase is readily available from suppliers of laboratory chemicals. The strength of the enzyme as it is used in the practice of this invention can vary and is not critical to this invention. In most cases, best results will be achieved by adding enzyme at enzyme strengths in the range of about 20 to about 500 units per liter, preferably about 100 to about 300 units per liter. The enzyme is conveniently added as a buffer solution, the buffer selected to provide a pH that is compatible with the enzyme reaction and conducive with high enzyme activity. For phenylalanine ammoma lyase, a preferred pH range of the specimen solution with the enzyme added is about 8 to about 10, and more preferably from about 8 to about 9. The enzyme reaction is permitted to occur for sufficient time and under appropriate conditions to achieve conversion of at least substantial amounts of both phenylalanine and tyrosine to trans-cinnamic acid and tra/w-coumaric acid, respectively. The optimal time and conditions will be readily apparent to those skilled in the use of these enzymes. The actual amount converted is not critical, although the amounts of each converted will be proportional to the amounts present. Any degree of conversion that will supply sufficient product to be detected will suffice, and preferably sufficient amounts of each product to serve as an indication of the relative amounts originally present. Preferably, at least about 90% will be converted. Prior to performing a chromatographic separation of the sample to detect the levels of the trøλw-coumaric and trans-cinnamic acids produced by the enzymatic reactions, a stopping agent to quench the activity of the enzyme is preferably added. An acidic stopping agent will not only quench the enzyme but also lower the pH of the mixture to a level that is compatible with the chromatographic medium. Any conventional acid, preferably a strong mineral acid, can be used. The strength of the acid will determine the final pH, whose optimal value will depend on the chromatographic medium. A final pH within the range of about 1 to about 4 is preferred, most preferably from about 2 to about 3. The chromatographic medium itself can be any such medium and configuration capable of separating organic acids by ion exchange. Conventional cation exchange columns, readily available from suppliers of analytical equipment and materials, can be used. The functional groups on the column can range widely, including sulfonic acid, carboxylic acid, phenol, and substituted amino groups. Groups in hydrogen form (i.e. , with hydrogen ion occupying the functional sites on these groups) are preferred. Appropriate mobile phases for the separation will also be readily apparent to those skilled in the art. In preferred embodiments, the mobile phase is aqueous with a pH maintained at a value within the range of about 2 to about 3 with acid and an appropriate buffer. Common acids can be used, such as sulfuric acid, perchloric acid, phosphoric acid, or nitric acid. Cosol vents can also be included, such as acetonitrile, methanol, tetrahydrofuran, and isopropanol. While gradient elutions can be used, the invention can readily be performed with isocratic elution (t ' .e. , use of a mobile phase of unchanging composition and pH throughout the separation). Detection and quantitation of the trαns-coumaric and trans-cinnamic acid fractions in the eluate is done by any means capable of differentiating these acids from the mobile phase itself. A preferred method is ultraviolet light absorptivity, particularly for its absorptivity by double bonds. Selection of the appropriate wavelength will be readily apparent to those skilled in the use of UV detection. Quantitation is readily achieved by conventional integration techniques. The following example is offered solely for purposes of illustration. EXAMPLE This is an example of one presently preferred procedure for the practice of this invention. A dried blood spot specimen collected on filter paper from a newborn infant according to standard procedures of the National Committee for Clinical Laboratory Standards (NCCLS) is punched to obtain a circle having a diameter of 3/8 inch (0.95 cm). The circle is placed in a 10-mL vial, and 700 μL of an aqueous elution reagent containing 0.5% ethylenediaminetetraacetic acid is added to elute the blood components from the filter paper. Elution is permitted to proceed for thirty minutes with periodic mixing. A calibrating solution (100 μL) containing 100 μM each of phenylalanine and tyrosine is placed in one well of a microtiter plate. The dried blood spot eluate and eluates similarly prepared from other dried blood spots that are being analyzed simultaneously are placed in other wells in amounts of 80 μL each. An enzyme reagent containing 200 units per liter of phenylalanine ammoma lyase in a 1.0 M tris-(hydroxymethyl)aminomethane buffer is added to each well of the microtiter plate in amounts of 20 μL per well, including the well containing the calibrating solution. The pH of the enzyme reagent is one that will result in a reaction pH of 8.5 when added to wells containing the dried blood spot eluates. The contents of each well are mixed, and incubated for fifteen minutes at 37 °C, followed by addition of 20 μL of a stopping reagent consisting of 0.65 M phosphoric acid. The contents of each well are again mixed, and the microtiter plate is transfered to a high- performance liquid chromatography (HPLC) automatic sampler, where analysis is initiated. The separation column is a capillary column that is either 100 mm in length and 7.8 mm in internal diameter or 30 mm in length and 4.6 mm in internal diameter. One example of a separation column that is suitable for this stage of the procedure is the AMINEX ® HPX-87H column, packed with divinylbenzene-styrene copolymer particles of a particle size 9 μm in diameter, with 8% crosslinkage and sulfonic acid groups in hydrogen form. Such a column is commercially available from Bio-Rad Laboratories, Inc., Hercules, California, USA. A mobile phase consisting of 0.004 M sulfuric acid containing 15% acetonitrile (by weight) is used at a flow rate of 0.5 mL/min and a temperature of 60°C. The injection volume is 10 μL, and the injection interval is less than three minutes. Detection is performed at a wavelength of 285 nm. A tyrosine peak elutes at a retention time of 1.6 minutes, followed by a phenylalanine peak at a retention time of 2.1 minutes. The foregoing is offered primarily for purposes of illustration. It will be readily apparent to those skilled in the art that the operating conditions, materials, procedural steps and other parameters of the invention may be further modified or substituted in various ways from what is described above without departing from the spirit and scope of the invention.

Claims: WE CLAIM: 1. A method for determining the content of phenylalanine and tyrosine in blood, comprising: (a) incubating a sample of said blood in a solution of phenylalanine ammonia lyase to produce a reaction mixture in which phenylalanine in said sample has been converted to trans-cinnamic acid and tyrosine in said sample has been converted to tr ns-coumaric acid; (b) passing said reaction mixture through a cation exchange column to elute therefrom tran^-cinnamic acid and trans-coumaric acid as distinct fractions; and (c) quantifying said fractions as representative of the levels of tn s-cinnamic acid and trans-coumaric acid in said sample. 2. A method in accordance with claim 1 in which said sample is a dried blood spot specimen on an inert support, and (a) comprises immersing said support in a liquid solution to extract blood from said dried blood spot specimen into said liquid solution, and adding said phenylalanine ammonia lyase to said liquid solution. 3. A method in accordance with claim 2 in which said liquid solution contains a proteolytic enzyme inhibitor. 4. A method in accordance with claim 2 in which said liquid solution with said phenylalanine ammonia lyase added is at a pH of from about 8 to about 10. 5. A method in accordance with claim 1 further comprising acidifying said liquid solution subsequent to (a) and before (b). 6. A method in accordance with claim 1 in which (b) comprises detecting eluting fractions by ultraviolet light absorption. 7. A method in accordance with claim 1 in which said cation exchange column has functional sites occupied by hydrogen ion. 8. A method in accordance with claim 1 in which (b) comprises passing said reaction mixture through said cation exchange column at a pH of about 1 to about 3 in the presence of acetonitrile. 9. A method in accordance with claim 1 in which said cation exchange column has functional sites occupied by hydrogen ion, and (b) comprises passing said reaction mixture through said cation exchange column at a pH of about 2 to about 3 in the presence of acetonitrile. 10. A method in accordance with claim 1 in which (b) is performed without prior removal of protein from said sample.

Inventors: BARTLING DONALD J, BINDER STEVEN R, ESSIEN HERBERT DI, VLASENKO SERGEY, BARTLING, DONALD, J., BINDER, STEVEN R., ESSIEN, HERBERT +DI, VLASENKO, SERGEY
Applicants: BARTLING DONALD J, BINDER STEVEN R, BIO RAD LABORATORIES, ESSIEN CHRIS & HM, VLASENKO SERGEY, BARTLING, DONALD, J., BINDER, STEVEN R., BIO-RAD LABORATORIES, INC., ESSIEN, CHRIS +HM, VLASENKO, SERGEY
Application year: 1998
Main classification: C12N15
IPC codes: C12Q1