Automated fluorometric determination of tyrosine in blood

ANALYTICAL

BIOCHEMISTRY

Automated

21, 227-234

Fluorometric Determination in Blood1j2 NORMAN

Department

(1967)

of Pharmacology, Chapel

J. HOCHELLA

University Hill, North

Received

of Tyrosine

of North Carolina

March

Carolina .%76l4

School

of Medicine,

23, 1967

It has been proposed by several workers (l-4) that analyses of blood levels of tyrosine, when used in conjunction with values for blood phenylalanine, are useful as an aid in differentiating phenylketonurics from nonphenylketonurics. In order to further the study of this phenomenon and of the metabolism of tyrosine, it seemed advantageous to develop an automated procedure for tyrosine utilizing the AutoAnalyzer, which could be used in conjunction with the automated phenylalanine procedure of Hill et al. (5). The procedure to be described is rapid and reliable and can measure levels of tyrosine of the order of 0.1 pg/ml of solution, concentrations present in eluates of the blood spots on filter paper used in screening newborn infants for phenylketonuria. The automated procedure is a modification of the manual fluoromet.ric method of Waalkes and Udenfriend (6). The manual procedure is based on the Gerngross reaction (7)) and in the Waalkes and Udenfriend modification of this reaction, tyrosine is coupled to 1-nitroso-2-naphthol in the presence of nitrous acid to form a fluorescent compound. In contrast to the manual method in which excess 1-nitroso-2-naphthol is removed by extraction with methylene dichloride, in the automated procedure a reaction with sodium metabisulfite is used to accomplish the same result. The fluorescent tyrosinenitrosonaphthol derivative is excited at 460 rnp :Ind measured at 570 m/-c. REAGENTS

Dilzlent

delphia,

solution: 8 gm of Surfactant 705 (Rohm and Haas Co., PhilaPa.) is melted in an Erlenmeyer flask placed in a boiling water

‘This investigation was supported dren’s Bureau, Department of Health, *This paper was presented in part N. Y., October 17-19, 1966. ’ Technicon Instruments Corporation,

by grant 12:HS, Project #236 from the ChilEducation, and Welfare. at the 1966 Technicon Symposium, Nclw York, Ardsley, 227

N. Y.

228

NOR&MT\

J.

HOCHELLA

bath and is mixed with 250 ml of dimethyl sulfoxide (Matheson, Coleman & Bell, East Rutherford, N. J.) This is added to 750 ml of distilled H,O. Recipient solution: 8 gm of Surfactant 705 and 250 mg of 1-nitroso-2naphthol [Fisher Scientific Co., Fair Lawn, N. J., reagent grade, recrystallized from methanol (S)] are melted together in a boiling water bath until the 1-nitroso-2-naphthol is completely dissolved; 250 ml of dimethyl sulfoxide is added and the mixture is poured into 750 ml of distilled H,O. Nitrous acid: 98 ml of 1:5 nitric acid (Fisher) (v/v) plus 2 ml of 2.5% sodium nitrite (Fisher certified) (w/v). So&urn metabisuljite: Na,&O, (Baker and Adamson, Morristown, N. J.), 15% (w/v). The diluent and recipient solutions are stable for at least a month at room temperature. The nitrous acid and sodium metabisulfite solutions are stable for at least a week at room temperature, but should be prepared fresh weekly. All reagents were kept in brown glass bottles. AUTOMATION

The flow diagram is shown ing from the dialyzer is mixed passes through a double-coil exit from the heating bath Sodium metabisulfite solution stream is mixed and passes

in Figure 1. The recipient stream after exitwith nitrous acid solution. The stream then heating bath regulated at 7O”C, and upon is cooled in a water-jacketed mixing coil. is then added to the reagent stream. The to the fluorometer. In the fluorometer the

AU TRANSMISSION TUBING FROM THE PUMPING TUBES :;M;;METER IS ACIDFLEX ,

T O THE

:;&;‘s;i

@?i%L%

;rhwr

TABISULFITE T O SAMPLER II WASH RECEPTACLE

EXCITATION FILTERS 2Aond EMISSION FILTER 28-12

FLUOROMETER

FIG.

1. Flow

diagram

for

RECORDER

automated

fluorometric

tyrosine

assay.

478

AUTOMATED

TYROSINE

ASSAY

229

excitation filters used are the 2A + 47B and the emission filter is a 2A-12. Samples are run at a rate of 30/hr at a 1 to 1 ratio of sample to wash. PREPARATION

OF

STANDARDS

The procedure was standardized with L-tyrosine (Mann Research Laboratories, Inc., New York, N. Y.). A weighed quantity was dissolved in 1.0 ml of 0.5 N hydrochloric acid and was then diluted to an appropriate level, which was of the order of 0.5-2.0 pg/ml. Standards may be poured into AutoAnalyzer cups and kept frozen until use or may be preserved at room temperature by adding a small amount of chloroform to the vessels containing the standard solution. If chloroform is used, care must be taken to prevent it from entering the AutoAnalyzer as it readily attacks acrylic dialyzer plates. PREPARATION

OF

SAMPLES

Blood obtained by finger or heel stick is placed on Schleicher and Schuel14 number 903 filter paper and is dried at room temperature similar to the procedures of Hill et al. (5,9) or Clark and Rice (10). The center of a dried spot is punched out with a s/s in. paper punch. The spot is then placed in an AutoAnalyzer cup to which has been added 1.0 ml of distilled water. After 0.5 hr the paper disc is removed and the eluted sample in the cup is run through the analytical system. Wet blood measured with a micropipet and diluted with distilled water can be run directly without deproteinization. DISCUSSION

The automation of the manual method of Waalkes and Udenfriend (6) posed several problems. In the manual method, excess l-nitroso-%-naphthol was removed by means of a solvent extraction with methylene dichloride; no tubing satisfactory for pumping methylene dichloride was available. This difficulty was circumvented by reacting excess l-nitroso2-naphthol with sodium metabisulfite in a manner similar to the procedure of Black (ll), in which sodium bisulfite was used for this purpose. A second serious problem in automating the manual method (6) was that the I-nitroso-2-naphthol precipitated on the walls of the AutoAnalyzer tubing. In the manual method, ethanol was the solvent used to keep the 1-nitroso-2-naphthol in solution. In the present study, dimethyl sulfoxide was found to be a much better solvent for I-nitroso-2-naphthol and prevented precipitation of the l-nitroso-2-naphthol and other products throughout the course of the reaction. In addition, it was found advantageous to include a detergent in the system. A large number of 4 Carl

Schleicher

and Schuell

Co.,

Keene,

N. H.

230

NORMAPi

J.

HOCHELLA

detergents was tried in this system and the one found to be most satisfactory was Rohm and Haas Surfactant 705. The concentrations of all reagents, the dimethyl sulfoxide, and the detergent were carefully studied to find optimum concentrations for maximum sensitivity. It was found, using the aforementioned reagents and conditions for fluorometry, that the sensitivity obtained was still not sufficient to measure quantities of tyrosine of the order of 0.1 ,ag/ml of solution. The necessary sensitivity was achieved by either of two separate procedures. In the first, a cuvet as seen in Figure 2 was prepared and used. This cuvet

FIG.

exciting

2. Diagram and emitted

of silvered light.

fluorometer

flow

cuvet

used

to increase

intensity

of

is a standard flow cuvet, such as is ordinarily used, that has been silvered on the outer surface (12). After it has been silvered it is spray-coated with several coats of flat black paint to protect the silver. When the paint is dry, windows are cut in the silver and coating of paint at right angles to match the excitation and emission windows in the flow door of the fluorometer. The cuvet is then attached to the appropriate tubes in the fluorometer and is taped firmly in place with black plastic electrician’s tape. It is very important that this type of cuvet be prevented from any movement as this would change its optical characteristics and cause shifts in the baseline, A standard General Electric F4T4-BL tube (G. K. Turner

AUTOMATED

TYROSINE

131

ASSAY

Associates lamp 110-850) was used as a light source with the silvered ouvet. This arrangement gives about a 5-fold increase in sensitivity ovel that obtained with an unsilvered cuvet. In the second alternative procedure, the additional sensitivity was obtained by using a General Electric F4T5-B (Turner lamp 110-853) blue tube (in a Turner 110-856 lamp adaptor) as a light source in the fluorometer module and a piece of quartz or Pyrex glass tubing (6 mm o.d., unsilvered) as a flow cuvet. Levels of sensitivity obtained with either combination described above are comparable, and the individual investigator may choose the one that seems most appropriate for his particular needs. VARIABLE TRANSFORMER

FLOW

RATE

CONSTANT

Ciht$nc2nter 0

FIG. 3. Diagram

of heating-bath

temperature

control

by variable

transformer.

Still higher sensitivity can be obtained by using a combination of a General Electric F4T5-B (Turner lamp 110-853) blue tube plus a silvered cuvet. However, with this combination the blank fluorescence is increased to such a degree that the baseline is raised off scale and cannot be returned to an acceptable level without going to a lower slit setting where the increased sensitivity advantage is thus lost. Another problem encountered was temperature control of the heating bath. It was noted that the ordinary on and off fluctuations of the thermoregulator of the heating bath produced fluctuations in the baseline and peak heights obtained with standards. It was found that the temperature of the heating bath could be controlled better by the use of a variable transformer (Variac, General Radio Company, Cambridge, Mass.) as outlined in Figure 3. In this procedure, reagents were run through the AutoAnalyzer, and after the coils of the heating bath were completely filled with reagents the plug of the heating bath was transferred from the house current to the Variac. By trial and error, a setting was found on the Variac such that the heat entering the heating bath exactly balanced the heat being lost to the environment and particularly the stream

232

NORMAN

J.

HOCHELLA

THERMOSTATIC

VARIABLE

TRANSFORMER

REPLICATE FIG.

variable

4. Comparison

of replicate

-40 >

TYROSINE

standards

li!

STANDARD

run

with

either

a thermoregulator

or a

transformer.

of moving reagents. By this arrangement a steady constant temperature of 70°C was maintained. As can be seen in Figure 4, the upper set of tyrosine standards is not uniform. It has a standard deviation of 1.7 fluorescence units. This set of standards was run with a regular thermoregulator controlled heating bath set at 70”. The lower set of standards, on the other hand, is much more uniform and has a standard deviation of 0.38 fluorescence unit. This lower set was run with the heating bath controlled with a Variac as described to a temperature of 70”. RESULTS

A typical standard curve is given in Figure 5. Replicate samples, as shown in the center and right side of the figure, were reproducible at all concentrations studied. At the left hand of the figure a steady state is shown as obtained with a standard solution of tyrosine at 1.5 pg/ml. This steady state represents a recorder deflection of 84 fluorescence units. At 30 samples per hour, 1: 1 ratio of sample to wash, the peak heights obtained with discrete samples were about 76% of the steady-state value. The system exhibited very good washout characteristics. A series of 100 blood samples obtained from normal newborns and dried as spots on filter paper were assayed for their tyrosine content. All of them had phenylalanine levels below 3.5 mg/lOO ml. The mean tyrosine value was 2.0 mg/lOO ml with a range of 1.1 mg/lOO ml to 3.7 mg/lOO ml and a standard deviation of 0.6. A series of 19 blood spots obtained from newborns with levels of phenylalanine above 3.5 mg/lOO ml had a mean

AUTOMATED

TYROSINE

TYROSINE

ASSAY

pg/ml

---2--l

J FIG.

5. Steady state, reproducibility

of replicates, and linearity

of tyrosine standard

curve.

of 15.3 mg/lOO ml, a range of 1.2 mg/lOO ml to 35.2 mg/lOO ml, and a standard deviation of 11.8. These values are in agreement with blood tyrosine levels reported by Wong et al. (4) for newborns. TABLE 1 Recovery of Tyrosine Added in Varying Amounts to the Same Whole Blood Sample Blood

tyroaine,

pg/ml

Tyrosine

19.1 19.1 19.1 19.1

added,

&ml

Tymdna

40.5 75.5 115.9 156.4

found,

33.2 65.9 105.5 155.5

pg/ml

Per cent recovery

86.3 87.0 90.8 99.3

Table 1 shows the amount of tyrosine recovered after being added to whole blood and then spotted and dried on filter paper. The average recovery was 91%. A recovery experiment was performed in which a diluent solution was prepared containing 1 pg tyrosine/ml. A series of 19 samples was assayed first with the ordinary diluent solution and then with tyrosine in the diluent. Recovery of the added tyrosine was 99.5% with a standard deviation of 10.6%. An amino acid standard solution5 containing 30 different amino acids which had been prepared for use in standardizing an amino acid analyzer *Galaxy

Chemical

Company,

Closter, N. J.

234

NORMAN

J.

HOCHELLA

was assayed for its tyrosine content. Recovery of tyrosine was 100% of the t.heoretical value, indicating that none of the other common amino acids interferes with this analytical procedure for tyrosine. ACKNOWLEDGMENTS The help and consultation investigation, and the editing fully acknowledged.

of Dr. John B. of this manuscript

Hill by

throughout Dr. Paul

the course of this L. Munson, are grate-

REFERENCES 1. ANDERSON, J. A., GRAVEM, H., ERTEL, R., AND FISCH, R., J. Pediat. 61, 603 (1962). 2. HSIA, D. Y. Y., LITWACK, M., O’FLYNN, M., AND JAKOVCIC, S., New Engl. J. Med. 267, 1067 (1962). 3. BREMER, H. J., TOSBERG, P., AND HBNSCHER, U., Ann. Paediat. 206, 12 (1966). 4. WONG, P. W. Ii., O’FLYNN, M. E., AND INOUYE, T., Clin. Chem. 10, 1098 (1964). 5. HILL, J. B., SUMZVIER, G. K., PENDER, M. W., AND ROSZEL, N. O., C&n. Chem. 11, 541 (1965). 6. W.~.~LKES, T. P., AND UDENFRIEND, S., J. Lab. Clin. Med. 50, 733 (1957). 7. GERN~ROSS, O., Voss, K., AND HERFELD, H., Chem. Ber. 66, 435 (1933). 8. M.\SSIN, M., AND LINDENBERG, A. B., Bull. Sot. Chim. Biol. 39, 1201 (1957). 9. HILL, J. B., SUM~~ER, G. K., SHAVENDER, E. F., SCURLETIS, T. D., ROBIE, W. A., MADDRY, L. G., MATHESON, M. S., AND BROOKS, M. F., “Proceedings of the 1965 Technicon New York Symposium on Automation in Analytical Chemistry,” p. 404. Technicon Instruments Corp., Ardsley, New York. 10. CLARK, P. T., AND RICE, J. D., JR., Am. J. Clin. Path. 4f+ 486 (1966). 11. BLACK, I. A., Soil Sci. 51, 387 (1941). 12. “Handbook of Chemistry and Physics” (Hodgman, C. D., Weast, R. C. and Wallace, C. W., eds.), 35th ed., pp. 2989, 2996-7. Chemical Rubber Publishing Co., Cleveland, Ohio, 1953-1954.