(13) Hultberg, S., Stockendahl, R., Arkiv Fysik 14, 565 (1959). (14) Langer, L. M., Moffatt, R . J. D., Phys. Rev. 82, 635 (1951). (15) Lanaer, L. M., Wortman, D. E., Ibid., 132,’ 324 (1963). (16) McGowan, F. K., Stelson, P. H., Ibid., 107, 1674 ’( 1957). (17) Merritt, J. S., Campion, P. J., Taylor, J. G. V., Can. J . Chem. 37, 1109 (1959). (18) Merritt, J. S., Taylor, J. G. V., Merritt. W. F.. CamDion.’ P. J.. ANAL. CHEM. 32, 310 ’( 1960j. (19) . . Mitchell, A. C. G.. Peacock, C. L., Phys. Rev. 75, 197 (1949). (20) Nuclear Data Sheets, National Academv of Sciences. National Research CoLncil, U.S.A. ’ (21) Osoba, J. S., Phys. Rev. 76, 345 (1949).
(22) Peacock, C. L., Mitchell, A. C. G., Ibid., 75, 1272 (1949). (23) Ricci, R. A., Physica 23, 693 (1957). (24) Rider, B. F., Peterson, J. P., General Electric Co. Rept. GEAP 4008 (1962). (25) Rider, B. F., Peterson, J. P., Ruiz, C. P., Nucl. Sci. Eng. 15, 284 (1963). (26) Sharpe, J., Wade, F., Atomic Energy Research Establishment Rept. AERE E/R 806 (1951). (27) Sliv, L. A,, Band, I. XI., “Coefficients of Internal Conversion of Gamma Radiation,” Academy of Sciences, USSR, Moscow, Leningrad, 1956; Rept. 57 ICC K1, Physics Department, University of Illinois, Urbana, Ill. (28) Tavendale, A. J., Ewan, G. T., Nucl. Instr. Methods 25, 185 (1963). (29) Taylor, J. G. V., Merritt, J. S., Chalk River Nuclear Laboratories, Chalk River. Ont.. unwblished data. 1962. I
.
(30) Taylor, J. G. V., llerritt, J. S., Bull. Am. Phys. SOC.Ser. 11 7, 352 (1962). (31) Taylor, J. G. V., Merritt, J. S., llisra, 8. C., Geiger, J. S., in press, &Vucl.Phus.
(32) Towniend, J., Owen, G. E., Cleland, M., Hughes, A. L., Phys. Rev. 74, 99 (1948). (33) Waggoner, 51. A., Ibzd., 82, 906 (1951). (34) Wapstra, A. H., Arkiv ~ y s i k 7, 275 (1954). (35) Williams, A., Campion, P. J., Intern. J . A p p l . Radiation Isotopes 14, 533
(1963). (36) Yoshizawa, Y., Nucl. Phys. 5, 122 (1958). RECEIVED for review November 16, 1964. Accepted January 8, 1965.
Coupled Reaction System for Determination of Transaminase Enzymes Application to Glutamic Oxaloacetic Tra nsa rninase G. P. HICKS’ and W. J. BLAEDEL’ University of Wisconsin, Madison, Wis. The determination of transaminase activity with a versatile coupled enzyme reaction system and the automation of the determination are presented. The dependence of instrument response upon enzyme activity is linear. The interferences associated with the determination of glutamic oxaloacetic transaminase (GOT) in 0.2-1111. samples of serum and tissue homogenates are studied. Elimination of the interferences through the use of gel filtration is demonstrated. The standard deviation is 2 3 Karmen units, or 2% relative, whichever i s greater, for samples containing 0 to 1600 Karmen units
of GOT.
T
HE
ANALYTICAL
APPLICATIONS Of
enzyme-catalyzed reactions can be greatly extended through the use of coupled reactions. Unfortunately, as the number of coupled reactions is increased, the overall specificity of the method decreases whereas the purity of the reagents required and the number of possible interferences generally increase. Thus, the interferences and limitations associated with coupled enzyme reactions must be studied carefully in any application to complex 1
2
Department of Medicine. Department of Chemistry.
354
ANALYTICAL CHEMISTRY
samples if full advantage of the enzyme analysis is to be realized. This paper presents a coupled reaction system for the determination of transaminases utilizing the reactions shown below:
+ CYKGXT keto acid + glutamate Glutamate + N i i D $ a K G + NADH, + XH4+ NADH, + dye-ox $ NAD + dye-red colorless^ G
Amino acid
(1)
GDH
(2)
PMS
(blue)
(3)
I n Reaction 1, a transaminase enzyme catalyzes the transfer of an amino group from an amino acid to CY-ketoglutarate (aKG), converting a K G to glutamate and the amino acid to a keto acid. I n Reaction 2, the oxidation of glutamate by nicotinamide adenine dinucleotide (NAD) to give aKG, reduced NAD (NADH,), and NH4+ is catalyzed by glutamic dehydrogenase (GDH). Finally in the third reaction, NADH, reduces an oxidized dye (dyeox) to give NAD and reduced dye (dye-red) in the presence of phenazine methosulfate (PMS). When the concentrations of the amino acid, a K G , XAD, GDH, dye-ox and PMS are not
rate-limiting, the rate of dye reduction is proportional to the activity of the transaminase enzyme. The reduction of the blue dye is measured as the decrease in absorbance a t 600 mp. The amino acid required depends upon the transaminase to be determined. For determination of the clinically important glutamic oxaloacetic transaminase (GOT), aspartic acid : ’s required. Alanine as a substrate permits the determination of glutamic pyruvic transaminase (GPT). G X T is a term used in this paper to denote a general transaminase. The determination of transaminase activity in tissues and biological fluids is of considerable interest and is the basis for important routine tests in the clinical laboratory (IO). Although most work has been limited to only a few transaminase systems, studies have shown that many transaminases are present in animal tissues (3, 7 ) . I n this paper, the coupled reaction system described above is applied to the determination of GOT and, in principle, should permit the determination of many other transaminase activities simply by changing the amino acid substrate. The automation of the determination is described. Interferences in human serum and tissue homogenates are studied and eliminated through the use of gel filtration.
1.0 ml/min
37 'C
TRANSAMINASE
Aspartic Acid GOT Sample
Figure 1,
Equipment for the determination of GOT activity
140 ml. of phosphate buffer, adjust to pH 7.4 with 1N NaOH, and dilute to 200 ml. with phosphate buffer. The reagent is stable for at least 1 month in a refrigerator. D Y E SOLUTION. Dissolve 34 mg. of 2,6-dichlorophenolindophenol (grade I, Sigma) in 100 ml. of distilled water. This solution is stable for 1 EXPERIMENTAL week at room temperature. TRANSAMINASE REAGENT.Add 200 Apparatus. An instrument has mg. of NAD (grade 111, Sigma), 4 ml. been described which makes it possible of oKG, 6 mg. of PMS (Sigma), and to automate the determination of GOT 20 ml. of dye solution to 80 ml. of (2, 6). A schematic outline is given phosphate buffer. The reagent is evacin Figure 1. Reagents. PHOSPHATE BUFFER. uated with an aspirator before use t o remove dissolved air. It is stable for Mix 420 ml. of 0.1M N a 2 H P 0 4 and 1 day a t room temperature. 80 ml. of 0.1M K H z P 0 4 to give 500 GDH. Process 5 ml. of G D H ml. of O . 1 M buffer, p H 7.4. (Sigma, type 11, in 50% glycerol, conASPARTICACID. Add 10.65 grams taining 20 mg. of protein per ml.) by the of L-aspartic acid (Sigma grade, Sigma gel filtration procedure described later. Chemical Co., St. Louis, Ma.) to 90 Procedure. An enzyme sample ml. of phosphate buffer, adjust the pH (serum or tissue homogenate) diluted to 7.4 with 1N NaOH, and dilute to with G D H and buffered amino acid 200 ml. with phosphate buffer. Stored solution is metered a t a constant in a refrigerator, the reagent is stable rate (1.0 ml. per minute) and mixed for a t least 1 month. with a reagent stream as shown in oKG. Add 2.94 grams of a-ketoFigure 1. T h e reagent stream, also glutaric acid (free acid, Sigma) to
The method presented in this paper has been used routinely to continuously monitor transaminase activities in column efluents during the separation of isoenzymes on ion exchange gels (4, and also to study the distribution of transaminases in tissue homogenates.
Figure 2.
metered a t 1.0 ml. per minute, contains optimum concentrations of all of the other components necessary for reaction. The resultant reaction stream is split into two delay lines, one a t 37" C. and the other at 18' C. After a delay of two minutes, the reaction stream in each line flows through a photometer cell. The absorbance difference between the cells is measured and recorded by a differential filter photometer and is proportional t o the GOT concentration. Provisions are made in the photometer and amplifier for setting zero and for calibrating with a standard sample so that the recorder chart will read directly in any desired enzyme units. Samples are introduced automatically a t the rate of 20 per hour by a sample turntable. [A detailed description of instrument use can be found elsewhere (2, 51.1 SAMPLEPURIFICATION BY ROUTINE Enzyme samples GEL FILTRATIOS. (serum or tissue homogenate) are passed through a dextran gel column to remove low-molecular-weight substances which interfere in the transaminase determinations. The column procedure has been described ( 5 ) . Routinely, 5.0 ml. of the specimen (serum or homogenate) is put through a 11- X 2-cm. column of Sephadex G-50 (coarse, block form) which has been equilibrated with phosphate buffer. The 5.0-ml. specimen is quantitatively collected in a 20-ml. fraction and diluted t o 25 ml. according to a simple procedure ( 5 ) . The fraction routinely collected in this manner quantitatively contains the enzyme activity in phosphate buffer free from low-molecular&eight interferences (below about 10,000) as will be shown later. The preparation requires only a few minutes and one technician can process many samples simultaneously ( 5 ) . The gel technique is modifiable to process very small samples, but this particular study required that many experiments be performed on each sample, and it was therefore more convenient to process large sample volumes. G D H reagent is also purified by Sephadex separation, using the procedure described above for the enzyme samples.
Recording of a series of diluted serum samples VOL. 37, NO. 3, MARCH 1965
e
355
1
I
I
300
450
I
150 mg N A D p e r
0
600 G X T REAGENT
lOOml
I
I
2
4
mg PMS per 100ml
1
6 GXT
I
8 REAGENT
ib
2.01A s par tic
KKG
OY
I
I
Asportic 0.2 GDH 0.5
0.4 LO
ml
Figure 3.
per
I
0.6 1.5
I
0.8
l!O
2.0
2.5
0 2
4
6
ml d K G per 100ml G X T
8
IO
REAGENT
S A M P L E CUP
Dependence of response on NAD, PMS, GDH, aKG, and aspartic
SAMPLE SIZE. Routine samples are prepared by diluting 1.0 ml. of serum processed by gel filtration (0.2 ml. of original serum sample) Kith 1.5 ml. of G D H , 0.5 nil. of L-aspartic acid, and 2.0 nil. of phosphate buffer. Tissue homogenates are diluted into the activity range of serum samples before use. When the volumes of samples or other reagents are varied, enough buffer is added to bring the final volume in the sample cup to 5.0 ml. BLANK.The instrument baseline is set by running a blank sample, prepared by mixing 1.5 ml. of GDH, 0.5 ml. of aspartic acid, and 3.0 ml. of phosphate buffer. The blank sample takes into account any transaminase activity contained as contamination in the GDH. RESULTS
Figure 2 is a record of the type of response obtained with the enzyme analyzer. From right to left, a baseline was established with only buffer in the sample cup. Sext, a blank sample was introduced, and a t steady state, the instrument was set to zero. After setting the blank, a series of four dilutions on the same serum sample was run. The original serum sample contained 1200 Karmen units of GOT per ml. as determined by an accepted routine colorimetric procedure (6, 8). The first dilution (corresponding to 0.067 ml. of the original serum, or to 400 Karmen units in a 0.2-ml. sample) was used for calibration, and was run longer than the other dilutions, to permit setting the sensitivity to give a reading of about 0.4 chart unit. For this sample, the chart reading is constnnt within about 0.01 chart unit, cor356
-
ANALYTICAL CHEMISTRY
responding to 20 Karmen units in a 0.2-ml. serum sample. Finally, the remaining three dilutions were run for 3 minutes each, a time selected for routine determinations, which was generally long enough just to reach steady state. The chart reading corresponding to each sample was therefore taken at the end of its 3-minute interval ( 5 ) . h plot of chart readings (ordinates) against the known values of the diluted serum samples gives a straight-line working curve that extrapolates through the origin within experimental error. Tbe standard deviation of the five individual points (including the origin) from the straight line is 1 2 3 Karmen units of GOT, which may be regarded as a measure of the precision of the procedure for samples ranging from 0 to 1600 units. This is a relative standard deviation of about 2% for samples in the upper third of the range. This precision compares with that obtained for other applications of this enzyme analyzer (2, 5 ) . STUDY
OF
REAGENT C O N D I T I O N S
The effect of the concentration of each component upon the rate of dye reduction a t constant enzyme activity was studied to determine the optimum conditions for each reagent. In each case, all components except the one under study were a t the following concentrations, 400 mg. of NAD (except 200 mg. for G D H and aspartic acid studies), 6 mg. of PMS, and 4.0 ml. of a K G in 100 ml. of transaminase reagent: and 0.5 ml. of aspartic acid and 1.5 ml. of
G D H in 5.0-ml. sample cup. *ill samples contained 1.0 ml. of Sephadex prepared serum (0.2 ml.) which had 350 Karmen units (6, 8) of GOT. The results are shown in Figure 3. A nonrate-limiting region of concentration was obtained for the aspartic acid, YAD, GDH, and PAIS. The response goes through a maximum for a K G , which is a substrate for Reaction 1 and a product in Reaction 2. A maximum rate is obtained when the rate of Reaction 1 is maximum with a minimum amount of product inhibition of Reaction 2. Even though the a K G concentration may limit the rate of Reaction 1, linearity is still obtained with the system because the a K G is recycled by Reaction 2 and its concentration does not change during the measurement. The same argument would hold true for the NAD in Reactions 2 and 3, but high concentrations are required to inhibit Reaction 3. The optimum concentrations given in the section on reagents were selected from the data in Figure 3 and are shown by the arrows. The linearity between the recorder response and enzyme activity is evidence that the reagent conditions are optimal. For routine enzyme analysis, ?;AD and G D H were set a t slightly suboptimal concentrations (circled in Figure 3) to conserve these reagents, a condition which did not adversely affect the linearity of the response. STUDY
OF
INTERFERENCES
When the coupled reaction system was used to determine transaminase activity in samples which had not been processed by gel filtration, a response was obtained even when the amino acid substrate was omitted from the sample cup. Such residual activity has been found in variable amounts in all serum samples and tissue homogenates studied. It can, therefore, be regarded as a general interference which must be removed by gel filtration prior to the assay of transaminase as described in this paper. Several experiments were performed to study the nature of this residual activity and to test for other interferences. Five-milliliter aliquots of a serum pool-made by pooling 200 serum specimens from patients with cancer, most of whom were being treated uith chemotherapy-were frozen. Xliquots were thawed and used in the following studies. The serum pool had 14 units (Karmen) of GOT (0,a), which falls within the normal range. ,1 pathological serum pool (PSP) was used because it seemed best suited for interferences that could be expected in clinical applications. Removal of Residual Activity by Gel Filtration. Five milliliters of the
cn
'g
-
-----
ALL REAGENTS NO
P
"\
D ' o/*\
--eC
ASPARTIC
1.6
-k-+-
SMS. NO ArDorlk
Purified G O T ,
All R c o g c n t r
Temperature, 'C
Figure 5. Effect of heating GDH on the responses
Milliliters
Collected
From Column
Figure 4. Separation of GOT activity from small molecular size fraction on Sephadex G-50
P S P were p u t through a gel column identical to t h a t described for the routine procedure and they were followed by 45 ml. of the phosphate buffer. Twenty-five 2-ml. fractions were collected from t h e column. Each fraction was assayed for G O T as previously described, using 0.4 ml. of each fraction as t h e sample. T h e results are shown in Figure 4. Two peaks of apparent GOT activity were eluted from the column. Because in gel filtration the substances of large molecular size (LMS)-protein for example-are eluted first, the first peak is the actual GOT activity. The second peak, which contains substances of small molecular size (SMS), is not GOT activity. Also, as shown in Figure 4 by the dotted line, the SMS peak was obtained equally well when aspartic acid was omitted from the sample cups in the assay, showing that the SMS peak was not GOT. The LMS fractions required aspartic acid in the assay to give activity. When purified GOT (3.034 ammonium sulfate-ketoglutarate-maleate suspension, Sigma Chemical Co.) was added to the SMS or LMS fractions, lOOyo of the added GOT activity was recovered. Thus, neither peak contained inhibitors. Study of the Dependence of Activity on Various Reagents.
SMS
The S M S peak was collected as a single 10-ml. fraction (35 t o 45 ml. in Figure 4) from several aliquots of P S P and used to determine the reagents required for the S M S fraction to give a response. T h e a m o u n t of S M S sample in t h e sample cup was varied with certain components omitted from the transaminase reagent or sampie cup. With all reagents included, a linear response with sample size was obtained. KO significant response was obtained without G D H or XAD, and only a very small response was obtained with no a K G . As shown in Figure 4, about the same response could be obtained with
or without aspartic acid in the sample cup. The requirements for a K G , G D H , and NAD strongly suggest that the SMS response involves some enzymic reactions, as opposed to some nonspecific chemical activity which reduces the dye or some reagent component. Such a response could be explained by the presence of substrates in the SMS peak which react with enzymes in the G D H . The requirement for a K G suggests that some of the response is transamination. The G D H does have a small amount of GOT activity, but only enough to account for a small fraction of the response with the SMS peak. Other transaminases have not been identified in the G D H reagent. The small response with no a K G could come from direct react ion of G D H with substrates. G D H is known to react with many different substrates (9) some of which might be contained in serum or tissue samples. While, in this particular case, the interfering enzymic activity was in a reagent (GDH), it is worth noting that similar variable blank activity could come from substrates and enzymes contained in the serum or tissue homogenates used as samples. Residual activity has also been observed in other procedures ( I ) . The presence of a residual activity in the transaminase method presented here demonstrates the limitations (loss of specificity and purity of reagents required) which are frequently associated with the direct application of coupled enzyme reactions. Gel filtration is a useful means of extending the applications of coupled enzyme reactions by greatly reducing these limitations. Effect of H e a t on GDH Reagent. Because G D H was the only source of enzymes in the study of the dependence of S M S activity of various reagents, further studies were made to evaluate t h e requirement of G D H . G D H solutions which had been heated
a t various temperatures for 10 minutes were used in two ways: t o assay the activity of purified G O T (Sigma) according t o the routine procedure described; and to assay the response of 1.2 ml. of SMS fraction as described in the SMS reagent study, but with the aspartic acid omitted from the sample cups. In both cases, the amount of G D H added was limiting so that any decrease in G D H activity would result in a proportional decrease in the overall rate. The results are shown in Figure 5. No attempt was made to run blanks for each point, so the slight residual activity above 65" C. does not mean that the G D H activity was not completely destroyed. That the heat destruction curves were essentially the same in each series shows that the G D H activity itself was required in both the GOT and SMS assays. That is, if some of the response from the SMS fraction had been due to some activity (enzymic) contained in the G D H which was not G D H or did not require G D H , the heat destruction for SMS would have been different, and the SMS response might have persisted, even after the destruction of GDH. ABBREVIATIONS
GDH GOT
= =
GPT
=
GXT aKG LMS NAD
= = = =
NADHz PMS PSP ShfS
= = = =
glutamic dehydrogenase glutamic oxaloacetic transaminase glutamic pyruvic transaminase general transaminase a-ketoglutarate large molecular size nicotinamide adenine dinucleotide reduced NAD phenazine methosulfate pathological serum pool small molecular size ACKNOWLEDGMENT
The authors gratefully acknowledge the technical assistance of Mrs. Lois E. Posnanski. VOL. 37, NO. 3, M A R C H 1965
357
LITERATURE CITED
(1) Amador, E., Wacker, W. E. C., Clin. Chem. 8, No. 4, 343 (1962).
(2) Blaedel, W. J., Hicks, G. P., ANAL. CHEM.34, 388 (1962). (3) Cammarata, P. S., Cohen, P. P., J. Biol. Chem. 187, 439 (1950). (4) Hicks, G. P., Ndevac, G. N., unpublished data, Department of Medicine, University of Wisconsin, 1964.
( 5 ) Hicks, G. P., Updike, S. J., Anal.
Biochem. in press. (6) Karman, A., J. Clin. Invest. 34, 131
(1955). ( 7 ) Meister, A,, Advan. Enzymol. 16, 185
(1955). (8) Sigma Chemical Company, St. Louis, Mo., Tech. Bull. No. 505, 1958. (9) Struck, J. J., Sizer, I. W., Arch. Biochem. Biophys. 86, 260 (1960).
(10) Wilkinson, J. H., "An Introduction to Diagnostic Enzymology,' ' Williams and Wilkins, Baltimore, 1962. RECEIVEDfor review October 19, 1964. Accepted December 31, 1964. Work supported by grants No. AT (11-1)-1082 from Atomic Energy Commission, KO. IN-35-D from the American Cancer Society, and No. G-72-3 from National Institutes of Health, United States Public Health Service.
Preconcentration of Trace Elements by Precipitation Ion Exchange FOUAD TERA, R. R. RUCH, and G. H. MORRISON Department of Chemistry, Cornell University, Ithaca, N .
b A new approach to preconcentration of trace elements, precipitation ion exchange, is demonstrated whereby matrix and traces are sorbed on a cation exchange column, the matrix is precipitated by concentrated HCI, and traces are then eluted with additional HCI. The separation is based on the insolubility of the matrix and the low selectivity of the traces in 12.2M HCI. Many trace elements were found to be concentrated in greater than 90% yields, matrix-free or with only small amounts of matrix from NaCI, KCI, BaCl2, SrCI2, and AgCI.
Y.
plicability of the approach is governed by .two main conditions. First, the matrix must be insoluble or only sparingly soluble in the eluent to prevent early breakthrough, and, second, the trace elements must have a very low distribution coefficient (preferably D,
