Stopped-flow kinetic determination of glucose and lactate with immobilized enzymes

Analytica Chimica Acta, 159 (1984) 337-342 Elsevier Science Publishers B.V., Amsterdam - Printed in The Netherlands

Short Communication

STOPPED-FLOW KINETIC DETERMINATION LACTATE WITH IMMOBILIZED ENZYMES

OF GLUCOSE AND

R. Q. THOMPSONa and S. R. CROUCH* Department

of Chemistry,

Michigan State

University,

East Lansing, MI 48824

(U.S.A.)

(Received 4th November 1983)

Summary. Kinetic procedures are described for the measurement of lactate and glucose with enzymes immobilized in the observation cell of a stopped-flow spectrophotometer. Kinetic data are obtained by two-point or multipoint methods with data acquisition over any time range desired. Glucose was quantified in a fixed-time mode with a linear range of O-10 mM. Lactate was quantified in the range O-50 PM by obtaining the slope of the absorbance/time profile between 10 and 20 s after each reaction was initiated. Results for these two substrates in control sera are presented.

Immobilized enzymes have become popular as reagents for quantifying substrates in recent years because they combine the selectivity and sensitivity of aqueous enzymes with the convenience and low cost of reusable reagents [l]. Substrate determinations have been based on immobilized enzyme electrodes [l-4], open-tubular reactors in continuous flow manifolds [5-71, packed-bed reactors [8, 91, and enzymes immobilized on pipet tips [lo] and stirring bars [ll]. Most of these determinations are based on single-point, fixed-time kinetic methods [ 121. Consequently, instrument response can be affected by any enzyme lag period so that nonzero intercepts and non-linear calibration plots can result. Single-point fixed-time methods are also inherently less accurate than multipoint methods. A stopped-flow instrument which allows continuous monitoring of immobilized enzymecatalyzed reactions has been described [ 131. The enzyme is immobilized on the inside of a nylon tube, which is inserted into the observation cell of a commercial stopped-flow instrument. The nylon insert serves as a combined observation cell and immobilized enzyme reactor and hereafter is referred to as the cell-reactor. After the proper reagents have been mixed and forced into the cell-reactor, the progress of the reaction can be monitored directly by spectrophotometry. The determination of fixed-time absorbance changes and linear regression slopes of absorbance/time plots over any time range from milliseconds to minutes is possible. The initial time of data acquisition can be nonzero and set to a value subsequent to any lag phase. Reactions occur under static aPresent address: Department of Chemistry, Oberlin College, Oberlin, OH 44074, 0003-2670/84/$03.00

0 1984 Elsevier Science Publishers B.V.

U.S.A.

338

conditions and, therefore, are influenced by substrate diffusion [ 131. This is also advantageous, because diffusional limitations often extend the linear range for substrate concentration. This communication describes evaluation of the system for kinetic methods for two clinically important substrates, glucose and lactate. Fixed-time conditions were used for the glucose procedure whereas the lactate results were obtained from rates calculated by linear regression of the absorbance/time data. The instrument allows calculation of the initial reaction rate by two-point methods (fixed-time, variabletime) or multi-point methods [ 121. Results for glucose and lactate in control sera are reported. The Trinder method [ 14,151 for glucose with glucose oxidase was adopted. The reactions involved are D-D-Glucose + O2 + Hz0 s 2HZ02 + DCPS + AAP -Dye

6 -Gluconolactone + HZOz + 4Hz0

where DCPS is 2,4-dichlorophenolsulfonate, and AAP is 4-aminoantipyrine. The absorbance of the dye product at 505 nm was monitored. Lactate was quantified by measuring at 340 nm the absorbance of flNADH (p-nicotinamide adenine dinucleotide, reduced form), produced in the reaction Lactate + p-NAD deh~~~g~n~se~Pyruvate + P-NADH An alkaline solution completion.

and hydrazine were used to drive the reaction to

Experimental Reagents. All solutions were prepared in distilled-deionized water. The glucose stock solution was prepared by dissolving 2.000 g of anhydrous /I-n-glucose and 0.50 g of benzoic acid in about 0.75 1 of water and then diluting to exactly 1 1; standard solutions were prepared by appropriate dilution of the stock, which was stable I’or a month at 4°C. The method of Barham and Trinder [ 151 was used to prepare a solution of 2,4-dichlorophenolsulfonate. A portion (2 ml; ca. 0.2 mmol) of this solution, 10 mg (1670 U) of peroxidase (Type II, Sigma), and 10 mg (ca. 0.05 mmol) of 4aminoantipyrine were dissolved in 75 ml of 0.1 M phosphate buffer, pH 6.4, and the solution was diluted to exactly 1 1 with the pH 6.4 buffer; this reagent was prepared daily. Purified L-lactic acid (0.960 g, Grade L-X, Sigma) and 0.125 ml of concentrated sulfuric acid were added to 500 ml of water and the resulting solution was diluted to exactly 1 1 with water. This stock was diluted appropriately to give the desired standard solutions. The stock was stable for two weeks; standards were prepared daily. For the preparation of fi-NAD reagent, 10.0 g of tris(hydroxyamino)-

339

methane, 13.0 g of hydrazine sulfate, and 2.0 g of ethylenediaminetetraacetic acid were dissolved in water, the pH of the solution was adjusted to 9.6 with sodium hydroxide, and the solution was then diluted to 1.0 1 with water; this buffer was stable for one week. In 40 ml of this buffer was dissolved 0.300 g of fl-NAD (Grade III, Sigma) and the solution was diluted to exactly 50.0 ml to give a 8.5 mM P-NAD solution; this reagent was prepared daily. Serum samples. The control sera (Monitrol and Pathotrol, Dade Division, American Hospital Supply) were reconstituted according to manufacturer’s instructions. For glucose, protein precipitation was used. To a centrifuge tube, 1.00 ml of the serum, 1.50 ml of a 20 g 1-l solution of barium hydroxide, and 1.40 ml of a 20 g 1-i solution of zinc sulfate were added in that order. The solution was thoroughly mixed and then centrifuged for 3 min. The supernatant liquid was used directly in the measurement step. For lactate, 1.00 ml of the control serum was diluted to exactly 0.1 1 with water, and the resulting solution was used in the measurement step. Enzyme immobilization. The enzymes, glucose oxidase (Type II, Sigma) and beef muscle lactate dehydrogenase (Type X, Sigma), were immobilized on Type 6 nylon tubing (0.1 cm i.d., Portex Ltd.) by a procedure adapted from that described by Morris et al. [16]. The steps are summarized in Table 1. The tubing was washed with water between steps, and the triethyloxonium tetrafluoroborate (TOTFB) was rinsed out of the nylon with cold methanol after step 1. A 10 mg ml-’ solution of glucose oxidase was prepared for use in step 4; the lactic dehydrogenase suspension was used directly as purchased. The completed reactor was filled with buffer (pH 6.4 phosphate) and stored at 4°C when not in use. Short (1.75 cm) segments of the tubing were cut for use in the stopped-flow observation cell as required. Instrumentation. The design and operation of the stopped-flow instrument and filter photometer have been described in detail [13]. For the fixed-time measurements, the photometer output voltage was acquired by a sample-and-hold circuit [17] thirty seconds (VI) and sixty seconds (V,) after the reaction had been initiated (flow stopped). The change in absorbance (AA) over the 30-s interval was taken as AA = log (VI/V,). This assumes no drift in the 100% T level during the interval. The times were chosen as a compromise between sample throughput and errors arising from mixing effects and enzyme lag periods. TABLE 1 Summary of steps in the enzyme-immobilization

procedure

Step

Reagent

Duration

Temp. (“C)

1 2 3 4

0.1 M TOTFB in CH,Cl, 0.1 M Diaminobutane in pH 9.4 HCO;-buffer 5% (w/v) Glutaraldehyde in pH 8.0 phosphate-buffer Enzyme in pH 6.85 phosphate buffer

3 min 4.5 h 1.5 h 18 h

22 22 22 4

340

The lactate data were acquired at 2 Hz by a microcomputer [13]. The photometer voltage corresponding to 100% T was taken as the first data point acquired. Linear regression of the absorbance/time data between 10 and 20 s gave reliable estimates of the initial slopes. A minicomputer (PDPS/e, Digital Equipment Corporation) was used to calculate the absorbance values and to perform the regression. Results and discussion Glucose. Glucose standards and the DCPS/AAP/peroxidase reagent were mixed rapidly and pushed into the cell-reactor by the stopped-flow instrument. The decrease in light intensity reaching the detector was monitored, and the change in absorbance was determined as described above. A blank was tested between each sample because the Trinder reagents and product adsorbed strongly to the nylon, producing some carry-over. Thus, the sample throughput was limited to a maximum of thirty samples per hour. Figure 1 shows the glucose calibration graph; each data point represents five separate AA measurements taken over a four-day period with the same glucose oxidase reactor. The error bars represent the ranges of the five measurements. Linear least-squares statistics for the data are included in the figure legend. When the reactor was stored at room temperature for five days, a decrease in activity of about 8% was observed. The linear range of the method, O-10 mM, covers the normal levels found in human blood serum (3.8-5.9 mM [lS]). The Michaelis constant (K,) of glucose for aqueous glucose oxidase is about 0.09 M [ 191, and, thus, the linear range extends beyond 0.1 K,. The reason for the abnormally large linear region is most likely due to diffusional effects on the overall reaction rate [ 131.

f

I

I 6

I

2

[Gl”cose]

(mt.4)

8

I 0

;

I

:

20

:

40

[Lactate]

:

50

,

80

(PM)

Fig. 1. Glucose calibration plot. Error bars represent range of values obtained at each concentration. Least-squares statistics are AA = (87 * 4) C(M) - 0.01 * 0.01 with Syx = 1.04 X lo-’ and r = 0.998. Fig. 2. Lactate calibration plot. Error bars represent ranges obtained at each concentration. Data at highest two lactate concentrations were excluded in linear regression calculations. Least-squares statistics are AA/At (s-l) = (18 f 3) C(M) + (8 f 6) 6-l with S,, = 2.2 X loss 6-l and r = 0.996.

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Three control sera were assayed. During the protein precipitation treatment, the samples were diluted 1:3.9; the resulting concentrations were still within the linear range. Table 2 compares the concentrations found by the proposed method with those given on the data sheets accompanying the samples. The largest difference occurred with samples of low glucose content. This error is possibly due to the presence of ascorbic acid, gentisic acid, and other reducing agents, which can interfere with peroxidase-catalyzed reactions [20,21]. Lactate. Sets of lactate standards were tested with an immobilized lactic dehydrogenase reactor incorporated in the stopped-flow instrument. The fl-NAD reagent mixed with water was used as a blank, and tested at the end of each set of standards. Sample carry-over was small with the lactate dehydrogenase system, apparently because little adsorption of reagents or products on the nylon occurred. Thus, the processing of 90 samples per hour was possible. Five rate measurements were made at each concentration over a fourday period with the same reactor and the rates are plotted versus lactate concentrations in Fig. 2. Good linearity was obtained up to a concentration of 40 PM; least-squares statistics are included in the figure legend. The intercept may have been caused by a slow nonenzymatic reaction, which was not compensated by the reagent blank. The errors in the rate data are most likely due to fluctuations in the reactor activity. Sixty percent of the original enzyme activity remained after seven days of heavy use at room temperature. The linear range of the method, O-50 PM, extends to about 0.025 K, (lactate) [13]. The linear range is not as wide as in the glucose determination because diffusional limitations are not as extensive. Lactic dehydrogenase is a less active enzyme than glucose oxidase, and the mass transport may be increased in the lactate study by coulombic attraction between the positively-charged nylon and the lactate anion. Serum assays were done on 1:lOO dilutions of the reconstituted controls TABLE 2 Results for glucose and lactate in serum control samples Serum type

No. of detns.

Expected (mM)

Found (mM)

Monitrol I Pathotrol Monitrol II

5 6 9

5.67 12.8 13.4

4*1 13.3 f 0.3 13.2 f 0.5

Lactate Monitrol II Monitrol I Monitrol I

6 5 6

1.8 2.2 3.1

2.1 f 0.2 2.2 f 0.4 3.2 f 0.6

Glucose

342

with water in order to adjust the sample concentrations to within the linear range. Table 2 shows that the lactate concentrations in all three samples were accurately determined. The very slight positive errors may result from the small amount of soluble lactate dehydrogenase present in the sera. The ability to follow absorbance versus time directly inside the reactor allows most of the kinetic methods classified previously [12] to be implemented. Potential errors caused by induction periods can be readily avoided. With a greater degree of automation, it should be feasible to improve the throughput of the system and make the method more practical. In addition, the stopped-flow system can be used for fundamental studies [ 131, such as determinations of Michaelis constants or the effects of inhibitors. This work was partially supported by National Science Foundation grant no. CHE 79-26490 and by a Union Carbide Summer Fellowship (RQT). REFERENCES 1 P. W. Carr and L. D. Bowers, Immobilized Enzymes in Analytical and Clinical Chemistry, Wiley, New York, 1980. 2 G. G. Guilbault and M. H. Sadar, Act. Chem. Res., 12 (1979) 344. 3 A. Attiyat and G. D. Christian, Analyst (London), 105 (1980) 154. 4 W. J. Blaedel and R. C. Engstrom, Anal. Chem., 52 (1980) 1691. 5 L. D. Bowers and P. W. Carr, Anal. Cbem., 48 (1976) 544A. 6 C. Horvath and H. Pederson, Advances in Automated Analysis, Technicon Int. Congress, 1976, Vol. 1, Mediad, Tarrytown, NY, 1977, pp. 86-95. 7 J. R&iEka and E. H. Hansen, Anal. Chim. Acta, 106 (1979) 207. 8 C. H. Spink, CRC Crit. Rev. Anal. Chem., 9 (1980) 1. 9 B. Danielsson, B. Mattiasson and M. Mosbach, Pure Appl. Chem., 51(1979) 1443. 10 P. V. Sundaram and S. Jarayaman, Clin. Chim. Acta, 94 (1979) 309. 11 J.-C. W. Kuan, S. S. Kuan and G. C. Guilbault, Anal. Chim. Acta, 100 (1978) 229. 12 H. L. Pardue, Clin. Chim., 12 (1977) 2189. 13 R. Q. Thompson and S. R. Crouch, Anal. Chim. Acta, 144 (1982) 155. 14 P. Trinder, Ann. Clin. Biochem., 6 (1969) 24. 15 D. Barham and P. Trinder, Analyst (London), 97 (1972) 142. 16 D. L. Morris, J. Campbell and W. E. Hornby, Biochem. J., 147 (1975) 593. 17 H. V. Malmstadt, C. G. Enke and S. R. Crouch, Electronics and Instrumentation for Scientists, Benjamin/Cummings, Menlo Park, CA, 1981, pp. 386-389. 18 N. W. Tietz, Fundamentals of Clinical Chemistry, W. B. Saunders, Philadelphia, PA, 1976, p_ 1213. 19 V. Linek, P. Benes, J. Sinkule, 0. Holecek and V. Maly, Biotech. Bioeng., 22 (1980) 2515. 20 R. H. White-Stevens, Clin. Chem., 28 (1982) 578. 21 J. A. Lott and K. Turner, Clin. Chem., 21(1975) 1754.