A simultaneous assay of two enzymes by a flow injection analytical (FIA) ... This FIA system has an additional channel for evaluation and correction for the error ...
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Simultaneous Assay of Aspartate Aminotransferase and Alanine Aminotransferase by Flow Injection Method Using Immobilized Enzyme Reactors Takeshi SUGAYA, Satoru NAITO,Shuhei YONEZAWA, Fujio MORIsHITAand Tsugio KOJIMA Department of Industrial Chemistry, Faculty of Engineering, Kyoto University, Kyoto 606
A simultaneous assay of two enzymes by a flow injection analytical (FIA)method using enzyme-immobilized opentubular reactors in parallel is described. Aspartate aminotransferase (AST) and alanine aminotransferase (ALT) were studied. This FIA system has an additional channel for evaluation and correction for the error resulting from pyruvate coexisting in sample sera. The results obtained by the FIA method corresponded well with those obtained by a conventional UV method. The recognition of the signal pattern from three channels of the FIA system will make possible the primary screening of diseases. Keywords
Aspartate reactor,
aminotransferase, simultaneous
alanine
aminotransferase,
flow injection analysis,
enzyme
immo bilized
assay, serum enzyme
Because the conditions of reactions can be controlled freely and strictly in a flow injection analysis (FIA), analysts are able to obtain satisfactorily reproducible results with many samples. These advantages show the applicability of the FIA to clinical analyses in which automated batch operations or air-segmented continuous flow systems have conventionally been used.' The authors have reported the determinations of many substrates involving biological samples by FIA methods using an enzyme-immobilized open-tubular reactor.2-6 There are two aminotransferases of clinical importance in sera; aspartate aminotransferase (AST) and alanine aminotransferase (ALT). AST is primarily in heart muscle, skeletal muscle and liver, and ALT exists mainly in liver. So, in myocardial infarction only the former level in serum rises greatly and both levels increase when the patient suffers from liver deseases. Generally ALT shows higher activity than AST in the acute phase of viral hepatitis, but the reverse is found in the recovering phase or in liver cirrhosis. Thus, the measurement and the comparison of the activities of both AST and ALT in sera are quite important for the diagnosis of diseases and the judgment of prognosis. Because of the clinical importance of the assay of these aminotransferases, various methods including the Skeggs type continuous flow analysis and various reagent kits have been proposed.' But there have been few applications of FIA to the assays of these enzymes. Yamane8 described an FIA method using pyruvate oxidase dissolved in the carrier solution and pointed out the serious error which resulted from pyruvate coexisting in sample sera. In this paper, AST and ALT
are assayed by an FIA method using immobilized enzymes, in which the endogenous pyruvate is corrected for. The simultaneous evaluation of both enzyme activities by an FIA system equipped with multiple reactors in parallel will also be described.
Experimental
Reagents The following enzymes were purchased: Lactate dehydrogenase (LDH, E.C. 1.1.1.27, from pig heart, 350 U/ mg-protein, Toyobo) and malate dehydrogenase (MDH, E.C.1.1.1.37, from pig heart, 1450 U/mgprotein, Boehringer-Mannheim-Yamanouchi). L-Aspartic acid, alanine, oxalacetic acid, a-ketoglutaraldehyde, 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide (Wako Pure Chemical Co.), NADH (Oriental Yeast Co.) and pyruvic acid (Sigma Chemical Co.) were also purchased and used without further purification. The pyruvate standards and the oxalacetate standards were prepared freshly before use. The AST standards and the ALT standards were prepared by dissolving the commercially available AST (E.C.2.6.1.1, from pig heart, about 200 U/mg, Boehringer-Mannheim-Yamanouchi) and ALT (E.C. 2.6.1.2, from pig heart, about 80 U/ mg, Boehringer-Mannheim-Yamanouchi), respectively, in a 0.1 M Tris buffer (pH 7.5) and assayed before use by UV methods using commercially available reagent kits (GOT-UV Test Wako and GPT-UV Test Wako, Wako Pure Chemical Co.). The activities were measured as the decrease in the absorbance of NADH at 340 nm
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with a spectrophotometer (Shimadzu, Model UV-200) and expressed according to Karmen et al.9 Control sera (Control Serum I and II, Wako Pure Chemical Co.) were purchased, dissolved in distilled water and assayed by the UV method immediately before use. The enzyme-immobilized reactors were prepared by the carbodiimide method or the glutaraldehyde-coupling method.10 The FIA systems The enzymatic reactions for the AST or ALT assay are as follows: L-aspartate+a-ketoglutarate oxalacetate+L-glutamate
(1-1)
oxalacetate+NADH+H* MPL L-malate+NAD+ L-alanine+a-ketoglutarate pyruvate+L-glutamate
(1-2)
pyruvate+NADH+H+ LL
(2-2)
L-lactate+NAD+,
(2-1)
Figure 1 (a) shows the schematic diagram of an FIA system for the assay of AST. A carrier solution was fed by a reciprocating pump (Sanuki Kogyo, Model SSP DM2M-1016), which was a 0.1 M Tris buffer solution (pH 7.5) containing 0.3 mM aspartate, 0.05 M aketoglutarate and 0.5 mM NADH. The sample solutions were injected through a loop injector (Japan
Fig. 1 Schematic diagram of FIA system for the measurement of enzyme activities: (a) The AST assay; (b) The ALT assay; (c) The simultaneous assays of AST and ALT. S1: 0.3 mM aspartate, 0.05 M a-ketoglutarate, 0.5 mM NADH, 0.1 M Tris buffer (pH 7.5); S2: 0.3 mM alanine, 0.05 M «ketoglutarate, 0.5 mM NADH, 0.1 M Tris buffer (pH 7.5); S3: 0.05 M a-ketoglutarate, 0.5 mM NADH, 0.1 M Tris buffer (pH 7.5); P: pump; I: injector; El: MDH-immobilized reactor; E2, E3: LDH-immobilized reactors; DP: deproteination column; FD: fluorometric detector.
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Spectroscopic Co., Model VL-611, the volume of the sample loop: 5 µl), and AST included in the samples catalyzed the transfer reaction of an amino group between a-ketoglutarate and aspartate (1-1). While the sample band passed through an MDH-reactor incubated at 36° C, oxalacetate produced by the reaction (1-1) was reduced, and NADH was consumed correspondingly at the same time (1-2). The decrease in the NADH concentration was measured with a fluorometric detector (Schoeffel, Model FS-970, ex. 340 nm. em. 470 nm), after removal of interference from blank signal by protein in the sample with a deproteination column (Toyopearl AF-blue 650 MH, 4.6 mm i.d.XS cm). The flow configuration of the FIA system for the ALT assay had to be modified in order to correct for the endogenous pyruvate in the sample. Figure 1 (b) shows the schematic diagram of an FIA system for the assay of ALT, which has two channels. A carrier solution flowing through channel (i) was fed by a reciprocating pump (Sanuki Kogyo, Model SSP DM2M-1016), which consisted of a 0.1 M Tris buffer solution (pH 7.5) containing 0.3 mM alanine, 0.05 M a-ketoglutarate and 0.5 mM NADH. The other carrier solution flowing through channel (ii) was fed by a reciprocating pump (Sanuki Kogyo, Model SSP DM2M-1024), the composition of which was the same as that for channel (i) except that one substrate, alanine, was not contained. The sample solutions were injected separately through two loop injectors (Japan Spectroscopic Co., Model VL-611, the volume of the sample loop: 5 µl, Rheodyne, Model 7000, the volume of the sample loop: 3 µl) at an appropriate interval to avoid overlap of the bands. While the sample bands passed through each LDH-reactor incubated at 37° C, the reactions (2-1) and (2-2) took place successively in channel (i); on the other hand, only the reaction (2-2) took place in channel (ii), that is, only the endogenous pyruvate in the sample was reduced. After the confluence of carrier solutions, each sample band flowed first into a deproteination column and then a detector. The ALT level corrected for the endogenous pyruvate can be evaluated from the signals on the two channels. Figure 1 (c) shows the schematic diagram of a threechannel FIA system for the simultaneous assays of AST and ALT. Each channel was assigned to either the AST measurement, the ALT measurement, or the endogenous pyruvate measurement. A set of three peaks was obtained after three shots of the same sample through different injectors at appropriate intervals.
Results
and
Discussion
AST assay Figure 2 (a) shows an FIA-gram obtained by injecting 5 sl of the sample sera or standard solutions. A blank signal was observed on injecting water. The
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Table
Fig.
2
FIA-gram
i.d.X400
cm.
for the AST assay.
MDH-reactor:
1
Comparison
of the determinations
for sample
sera
0.28 mm
Fig. 4 FIA record for the ALT assay. LDH-reactor, 0.25 mm i.d.X660 cm. i and ii indicate the peaks from channels (i) and (ii), respectively. The activities of ALT standards were determined by the UV method. Fig. 3 Correlation curves for the evaluation of enzyme activities: (a) the AST assay; (b) the ALT assay.
activities indicated for the AST standards were determined by the UV method. The samples can be injected at 3-min intervals without remarkable overlap of the bands despite the band broadening within the deproteination column. Satisfactorily reproducible results were obtained without interference from protein in sera. Figure 3 (a) shows the correlation curve derived from the FIA-gram in Fig. 2. The activities seen along the abscissa were determined by a UV method using a reagent kit. The ordinate indicates the net peak height corrected for the blank. There is good correlation between the AST assays obtained by the authors' FIA method and those obtained by a conventional UV method. The correlation curve is linear in the range 0 to 240 U/ 1 and can be used as a calibration curve. Table 1 (a) shows results of the assays of AST in sera by the two methods. The results obtained by both
methods agreed with each other satisfactorily. These results exhibit the usefulness of the authors' method, considering the normal AST level in human serum (5 to 40 U/ 1). ALT assay Figure 4 shows an FIA-gram obtained by injecting 5 and 3 µl of samples alternately into channel (i) and (ii), respectively. The left member of each peak pair passed through channel (i) and the right one through channel (ii). The repeatablility was also examined by three injections of 2 mM pyruvate solution into each channel. As seen on the left side of Fig. 4, satisfactory results were obtained (relative standard deviation: Ca. 1%). The pyruvate standards were injected to set the calibration curves to correct for the endogenous pyruvate, as seen on the right side in Fig. 4. Figure 5 shows the calibration curves, both of which are linear in the range 0 to 2 mM pyruvate. The contribution of pyruvate in samples to the peak height in channel (i) could be
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Fig. 5 Calibration curves for pyruvate channel (i) or channel (ii).
flowing
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estimated from the corresponding peak height in channel (ii) by using these calibration curves. Figure 3 (b) shows the correlation curve for the ALT assay, in which the ordinate indicates the net peak height in FIAgrams and the abscissa is the activity determined by a conventional UV method. It is linear in the range 0 to 150 U/ l and reflects the satisfactory correlation between the two methods. Table 1 (b) compares the results of the assay of ALT in sera by the two methods. In the FIA method, the ALT activities were determined by using the correlation curve seen in Fig. 3 (b) as a calibration curve, after subtracting the contribution of the endogenous pyruvate. The ALT activities measured by the FIA method agreed with those by the UV method. The normal ALT level in human serum is 0 to 30 U/1. The serum pyruvate level is generally below 0.1 mM but comes up to several mM in a few cases. The FIA method is thus found to be quite useful, in light of the above results.
Fig. 6 FIA record for the simultaneous assays of AST and ALT. MDH-reactor, 0.25 mm i.d.X830 cm; LDH-reactor, 0.25 mm i.d.X690 cm. i, ii and iii indicate the peaks from channels (i), (ii) and (iii), respectively.
method in which the sample has to be diluted into less than 250 U/l. In addition, it was difficult to assay for the serum sample(II) containing 1.4 mM pyruvate by the UV method. It seems that the consumption of NADH by excess pyruvate in the sample results in underestimation of the enzyme levels. This FIA method enabled the samples to be analyzed without dilution and regardless of the endogenous pyruvate. The recognition of the peak pattern from three channels will make easy and possible not only the evaluation of individual levels of AST and ALT but also a primary screening for clinical check.
References
Simultaneous assays of AST and ALT Figure 6 shows an FIA-gram obtained by injecting each 5 µl sample into three channels of the FIA system seen in Fig. 1 (c). The right member of each peak set passed through channel (i) for the AST measurement, the middle through channel (ii) for the ALT measurement and the left through channel (iii) for the endogenous pyruvate measurement. The correlation curves for the AST and the ALT assays are linear in the ranges 0 to 1100 U/ l and 0 to 570 U/ 1, respectively. The calibration curves for pyruvate in channel (ii) and (iii) are also linear in the range 0 to 2 mM pyruvate. Since serum samples of patients suffering from the different diseases were not available, three serum samples were prepared by spiking with AST, ALT and/ or pyruvate standards; they can be considered to be representative of sera in liver disease(I), in high endogenous pyruvate(II) and in myocardial infarction(III). Table 1(c) shows the results of the assays of AST and ALT in sera obtained by the FIA method and by a conventional UV method. The results suggest that the FIA method has a wider dynamic range than the UV
1. J. Ruzicka and E. H. Hansen, Anal. Chim. Acta,106, 207 (1979). 2. T. Kojima, Y. Hara and F. Morishita, Bunseki Kagku, 32, E l Ol (1983). 3. F. Morishita, Y. Hara and T. Kojima, Bunseki Kagaku, 33, 643 (1984). 4. Y. Nishikawa, F. Morishita and T. Kojima, Bunseki Kagaku, 35, 575 (1986). 5. F. Morishita, Y. Nishikawa and T. Kojima, Anal. Sci., 2, 411 (1986). 6. S. Yonezawa, F. Morishita and T. Kojima, Anal Sci., 3, 553 (1987). 7. T. Murachi, Kagaku no Ryoiki, Vol. 34, p. 721, Nankodo, Tokyo (1980). 8. T. Yamane, Yamanashi Daigaku Kyoiku Gakubu Kenkyu Hokoku, 32, 52 (1981). 9. A. Karmen, F. Wroblewski and J. S. La Due, J. Clin. Invest., 34,126 (1955). 10. J. E. Dixon, F. E. Stolzenbach, J. A. Berenson and N. 0. Kaplan, Biochem. Biophys. Res. Commun., 52, 905 (1973). (Received July 6, 1988) (Accepted September 5, 1988)
