A Manual Fluorometric Paper Disc Method for Detecting Phenylketonuria

Transcription

1 A Manual Fluorometric Paper Disc Method for Detecting Phenylketonuria Bernard Searle, Milutin B. Mijuskovic, Daniel Widelock, and Bernard Davidow A method is described for a spectrophotofluorometricdetermination of phenylalanine. The amino acid is extracted from a spot of blood which is found to be stable when dried on filter paper. The method is relatively simple and the analysis may be completed on the day the sample is received. THE MICROBIOLOGIC PROCEDURE of Guthrie and Susi (1) is probably the most widely used screening technic for detecting phenylketonuria. The method of McCaman and Robins (2) and its modifications by Wong et al. (3) and Hsia et at. (4), as well as that of La Du and Michael (5) are the chemical procedures generally used as confirmatory tests. Hill et at. (6) have adapted the McCaman procedure to the AutoAnalyzer for determining phenylalanine in blood and in filter paper discs saturated with blood. More recently Bourdillon and Vanderlinde (7), using autoclaved blood discs, increased the sensitivity of the Hill procedure. Autoclaving the blood filter paper discs coagulated the proteins and obviated the need for the dialyzer required in the automated procedure. After autoclaving, the phenylalanine may be eluted with water while the denatured proteins remain bound to the filter paper discs. The method described in this paper will enable other workers to perform the test for phenylalanine on the material eluted from the blood disc without the use of automated equipment. Hill showed that blood discs stored at room conditions are stable for at least 27 days. Our own experiments showed blood discs to be stable for at least 3 months if stored in the dark at 5#{176} in a desiccator over silica gel. However, less exacting conditions may be adequate for storage of the discs. From the Bureau of Laboratories, New York City Department of Health, New York, N. Y The authors are indebted to Florence Treibis, Senior Statistician, New York City Department of Health, for the statistical evaluation of the results. Received for publication Dec. 16, 1966; accepted for publication Mar. 13,

2 622 SEARLE ft AL. Clinical Chemistry it has been our experience that on aging, blood I)rote!1)s undergo a partial hydrolysis with a resulting rise in pheiy1alaiiine level, particularly at summer room temperatures (85#{176} F.). Thus, falsely elevated levels may be found in some whole blood samples which are not aimlyzed immediately. For this reason, a simple chemical method was developed to utilize filter paper discs saturated with blood which when dry are relatively stable. The method described employs the solutions used in the McCarnan technic. Equipment Materials and Methods An Amninco-Bowman spectrophotofluorometer having a 1P21 potted photomultiplier tube was used in this study. A Corning filter No was placed in the position provided in the phototube housing. The excitation wave length was set at 390 nm. Reagents All reagents were the same as those used iii the MeCaman and Robins procedure (2). Standards Blood filter paper disc standards* having concentrations of 4, 8, and 16 mg. phenylalanine per 100 ml. of blood were prepared from human transfusion blood. Recently outdated blood was found satisfactory. The first step is to analyze a small sample of the blood as received for its phenylalanme content by means of the McCaman and Robins proce- (lure (2). A stock solution of phenylalanine (Mann Research Labs) having a concentration of 10 mg./ml. in physiologic saline (0.85%, w/v) is prepared. The known standards are prepared by adding a calculated volume of the phenylalanine stock solution to tile required amount of transfusion blood. Table 1 shows the quantities of the phenylalanine stock solution to be added, assuming that the small sample of blood on analysis gave a value of 1 mg. phenylalanine per 100 ml. of blood. A portion of each blood standard is aspirated into a 5-mi. pipet and then allowed to fall dropwise onto Schicicher and Schuell filter paper No. 903, each drop covering a different area of the paper. A separate sheet of filter paper is used for each of the 3 blood standards. The spots will he approximately 1/, in. in diameter. Tile paper should be held horizontally anti should not touch a solid surface while the blood is being *Stassdardsm.e available commercially from the Difco Co., Detroit, Mich. and Becton Dickinson Co., Rutherford, N. J.

3 Vol. 13, No. 8, 1967 PHENYLKETONURIA 623 Table 1. QUANTITY OF PHENYLALANINE SOLUTION REQUIRED TO IREPARE STANDARDS USING BLOOD CONTAINING 1 MG. PHENYLALANINN/100 ML. BLOOD Phenylatanine sot ulion Sinodard (mq./100 ml.) (10 mg/mi. online) Whole blood dropped on it. (A wide mesh wire screen drying rack is suitable.) The treated filter paper is allowed to dry in a clean area and then a 1/44fl hole punch is used to cut out the center of each spot. Autoclaving of Blood Paper Disc Standards It is best to place the discs of each concentration into Sej)arate beakers in such a manner that the discs do not touch each other. Cover the beakers with a sufficient layer of large size filter paper which is held in place with a rubber band to prevent condensed steam from failing upon the paper discs when the autoclave is opened. The discs are then autoclaved for 3 mm. at 15 lb. pressure. Prolonged autoclaving should be avoided as destruction of phenylalanine occurs when the heating time is increased. The discs are stored in the cold and protected from light and moisture. Preparation of Standard Curve and Determination of Unknown The autoclaved discs containing the standards and the unknown specimen are placed into individual 75 X 10 mm. Pyrex test tubes. Tube 1 is the blank containing a biood-free paper disc. Tubes 2, 3, and 4 contam the standard blood discs and Tubes 5 and 6 contain one disc each of the unknown specimen (Table 2). Triple distilled water, 0.35 ml., is then added to each tube and the tubes are placed in a rack and shaken in a mechanicai shaker for 15 mm. As indicated in Table 2, a 0.1-mi. aliquot is taken from each tube containing the paper disc amid 1)laCed in a similar tube. The remaining reagents are added in the order as shown in this table. After the addition of the copper reagent, the tubes are weli shaken. They are then ailowed to stand for 15 mm. before taking fluorornetric readings. The instrument is calibrated by setting the 8 mg./100 ml. standard at 50% of full scale. All other readings of the standards are made with the instrument set at the same sensitivity level. The fluorescent value obtained for the blank disc is subtracted from the fluorescent value of the other tubes. A plot of the fluorometric readings vs. concentration of phenylaianine using square-ruled paper will

4 624 SEARLE ft AL. Clinical Chemistry Table 2. SYSTEM FOR PHENYLALANINE DETERMINATION* Discs Tube no. Aliquot Buffert Ninhydrin, 0.55% Leucytalanine H,O Copper reagent Tot at volume (nil.) Blank, no blood Blood standards 4mg/lOOm! mg/lOOm! mg./100 ml Unknown Testi Test Heat the aliquots prepared from discs (see text), ninhydrin, leucyl-alanine, and water for 2 hr. in a water bath before adding the copper reagent. t Prepared from discs (see Preparation of Standard Curve and Determination of Unkiwwn). Buffer is 0.3 M sodium succinate, ph 5.8. give a straight line over the physiologic range (2). It will be noticed from Table 2 that Tube 5 does not contain any leucyl-alanine. Since phenylalanine has negligible fluorescence in the absence of leucyl-alanine, any significant fluorescence found can only be due to some other component in the blood. The tube without leucyl-alanine was included to make certain that the fluorescence of the unknown was truly that of phenylalanine and not due to drug therapy, diet, hormones, etc. Accuracy and Precision The accuracy and precision of the recovery of phenylalanine from blood paper discs were determined at concentrations ranging from 2 to 50 mg. per 100 ml. After preliminary analysis of blood, phenylalanine was added to bring the total concentration to the amounts shown in the first column in Table 3. Ten paper-disc samples prepared from the same blood were run at each concentration level. The standards were prepared in the same way and from the same blood as the discs used for the recovery experiment. The amounts of phenylalanine recovered are shown in the second column of the table. These differ from the known levels because of statistical variation. Discussion The chemical method described has several advantages over those presently in use. The main advantage is the stability of the sample and the convenience of shipping a dry, stable sample to a central analytic lal)oratory. The shipment of the paper disc in a waterproof glassine envelope involves no special precaution. Tile collection of blood on filter

5 Vol. 13. No PHENYLKETONURIA 625 Table 3. REC OVERY OF PHENYLALANINE FROM BLOOD STA NDARD Discs Phenylalanine in discs (mg./j00 ml.) Phenylalanine recovered Mean *.S.D.* (mg./100 ml.) Range (mg./w0 ml.) ± ± ± ± ± ± ± ± ± * S.D. =,_f)2 where N (number of samples ren at each concentration) equals 10. paper offers less difficulty than the collection of blood in microcapillary tubes. The technic of autociaving the blood filter paper discs eliminates the necessity of using trichloroacetic acid as described by McCaman to precipitate the proteins. In the automated method (6), a dialyzer removes the protein but also reduces the amount of available phenylalanine. It is obvious that the use of the protein-free eluate of the filter paper disc would circumvent the use of the dialyzing module in the automation procedure, thus increasing the sensitivity of the method. References 1. Guthrie, B., and Susi, A., A simple phenylalanine method for detecting phenylketonuria in large populations of newborn infants. Pediatrics 32, 338 (1963). 2. McCaman, M. w., and Robins, E., F]uorimetric method for the determination of phenyl. alanine in serum. J. Lab. Gun. Med. 59, 885 (1962). 3. Wong, P. W. K., O Flynn, M. E., and Inouye, T., Micromethods for measuring phenylalanine and tyrosine in serum. GUn. Chem. 10, 1098 (1964). 4. ilsia, D. Y., Berman, J. L., and Slatis, H. L., Screening newborn infants for phenylketonuria. J. Am. Med. Asso. 188, 131 (1964). 5. La Du, B. N., and Michael, P. J., An enzymatic spectrophotometric method for the determination of phenylalanine in blood. J. Lab. Clin. Med. 55, 491 (1960). 6. Hill, J. B., Summer, G. K., Pender, M. W., and Roszel, N. 0., An automated procedure for blood phenylalanine. Clin. Chem. 5, 541 (1965). 7. Bourdillon, J., and Vanderlinde, B. E., An improved screening procedure for blood plienylalanine. Public Health Rep. 11, 991 (1966).