EP0471062A1

EP0471062A1 - Method to use a reaction by-product as a calibrator for enzymatic assays

Info

Publication number: EP0471062A1
Application number: EP91905984A
Authority: EP
Inventors: Kenneth J. Gruys; Harryl C. Ammons
Original assignee: Baxter Diagnostics Inc
Current assignee: Baxter Healthcare Corp
Priority date: 1990-03-01
Filing date: 1991-02-27
Publication date: 1992-02-19
Also published as: JPH05505108A; AU7493791A; EP0471062A4; CA2053868A1; WO1991013169A1

Abstract

Procédé d'utilisation d'un sous-produit de réaction en tant que calibreur dans un dosage enzymatique automatique. On utilise notamment à titre de sous-produit de réaction le pyruvate, en tant que norme d'étalonnage d'instrument afin de mesurer l'activité d'aminotransférase d'alanine. L'emploi de ce procédé élimine les erreurs dues au prélèvement par pipette imprécis et corrige les petites déviations optiques de la longueur d'onde d'intérêt, dans l'instrument. Les erreurs de prélèvement par pipette et la déviation optique non corrigée ont un effet négatif sur la précision du calcul de l'activité enzymatique. Dans le mode de fluorescence, l'étalonnage à l'aide d'une norme pyruvate élimine également la nécessité d'utiliser des normes NADH instables. On a démontré que la norme d'étalonnage pyruvate de l'invention est stable dans le test de stabilité de normes. On peut étendre ce procédé à un certain nombre d'autres dosages enzymatiques automatiques. Comme l'aminotransférase d'alanine, la précision du calcul d'activité pour d'autres enzymes peut être améliorée à l'aide de cette technique.Method of using a reaction byproduct as a calibrator in an automatic enzymatic assay. Pyruvate is used in particular as a reaction byproduct, as an instrument calibration standard for measuring the activity of alanine aminotransferase. The use of this method eliminates errors due to imprecise pipetting and corrects small optical deviations of the wavelength of interest in the instrument. The sampling errors by pipette and the uncorrected optical deviation have a negative effect on the precision of the calculation of the enzymatic activity. In fluorescence mode, calibration using a pyruvate standard also eliminates the need to use unstable NADH standards. The pyruvate calibration standard of the invention has been shown to be stable in the standards stability test. This process can be extended to a number of other automatic enzyme assays. Like alanine aminotransferase, the accuracy of activity calculation for other enzymes can be improved using this technique.

Description

Method to use a Reaction By-Product as a

Calibrator for Enzymatic Assays

BACKGROUND OF THE INVENTION

1. Field of the Invention

Method to use pyruvate as a calibrator for a diagnostic instrument measuring alanine aminotransferase activity (hereinafter ALT). Based on this method, similar calibration methods could be devised for other enzymes of clinical importance.

2. Description of the Prior Art

The most common means of assaying ALT employs a spectrophotometric assay which couples pyruvate production to the reaction catalyzed by lactic dehydrogenase (hereinafter LDH) as shown in Figure 1. Lactic dehydrogenase catalyzes the reduction of pyruvate with the concurrent oxidation of NADH. When NADH is oxidized, it loses adsorption at 340 nm and it is by loss of adsorption that one can monitor the original transaminase activity. This coupled assay holds true as a quantitative measure of alanine aminotransferase activity as long as the coupling enzyme is in large excess relative to ALT. This assures that the rate limiting factor for the oxidation of NADH is the rate that ALT produces pyruvate.

To calculate the activity of ALT in terms of μmol es pyruvate produced per minute per liter of sample, one mathematically converts the change in absorption at 340 nm per unit time to actual μmoles of reduced NADH per minute. This calculation is accomplished using a molar absorptivity constant for NADH at 340 nm that relates absorbance units to concentration. Since the relationship between NADH and pyruvate as shown in Figure 1 is 1:1, one μmole of NADH oxidized is equivalent to one μmole pyruvate (the pyruvate being produced by the action of ALT) reduced. Using this relationship and the sample dilution factor, one can obtain the ALT activity in the appropriate units. An automated diagnostic instrument designed to measure alanine aminotransferase activity incorporates some type of pipetting scheme to add reagents and sample into a reaction well. This reaction well could be an optical flow cell. When calculating transamlnase activity, the volumes delivered by the plpettors and resulting sample dilution factor are incorporated into the equation used for this calculation. Pipetting errors, therefore, Impact the accuracy of the transaminase activity calculation. From this it is apparent that a calibration procedure that takes into account pipetting inaccuracy would be desirable.

Previous methods for instrument calibration rely on using standardized optical filters that have constant absorbance values at the wavelength of interest. This assures that the instrument is optically correct and that using the molar absorptivity constant to convert absorbance units to μmoles NADH will be valid and accurate. This only addresses the optical characteristics of the instrument, however, and does not correct for pipetting errors.

In addition to instrumentation based on absorption, detection of activity can also be done using a fluorimeter, since NADH is fluorescent. Brooks & olken, An Automated Fluorometric Method for Determination of Lactic Dehvdrooenase in Serum, 11 Clin. Chem. 748 (1965). While fluorescence adds increased sensitivity, It does not have a universal molar absorptivity constant to relate fluorescence units to concentration of NADH. Because of this, one needs to generate a standard curve of fluorescence units versus NADH concentration whenever the assay is run. Unfortunately, NADH standards are not suitably stable and would require frequent restandardization. Lowry & Passonneau, "A Flexible System of Enzymatic Analysis", Chap 1, p 3-20, Academic Press, New York (1972). This makes NADH based standards impractical for clinical diagnostics with an automated instrument. 3. Summary of the Invention

A known analytical concentration of pyruvate is used as a substitute for an optical filter or NADH as the calibrator for an instrument in a diagnostic assay. The diagnostic assay of interest, in this case, is the measurement of alanine aminotransferase activity. Pyruvate is introduced to the ALT reagents in the same manner as a sample and is rapidly converted to lactate with concurrent oxidation of NADH (Figure 1). Since the stoicheometry of pyruvate turnover to NADH oxidation is 1:1, the difference in absorbance or fluorescent units between this solution and a sample that does not contain pyruvate is an exact analytical measure relating absorbance or fluorescence to NADH concentration. This relationship then is used to calculate the original enzymatic activity of ALT. The advantages to this invention include the elimination of pipetting inaccuracy as a negative effector in activity determination, the elimination of precise optical calibration, and in the case of fluorescence detection, replacement of unstable NADH standards with a stable pyruvate standard.

DETAILED DESCRIPTION OF THE DRAWINGS

For a better understanding of the invention as well as other objects and further features thereof, reference is made to the following detailed disclosure of this invention taken in conjunction with the accompanying drawings wherein:

Figure 1 shows the reaction scheme involved in the measurement of alanine aminotransferase.

Figure 2 shows the stability data for pyruvate calibrators.

Figure 3 shows a comparison of standard curves relating fluorescence units to NADH concentration either directly or with the pyruvate calibrator.

Figure 4 demonstrates the equivalency of using either NADH standards or the pyruvate calibrator in the fluorescence mode by way of slope values (fluorescence units per μM) over a period of two weeks.

Figure 5 shows the reaction scheme involvement in the measurement of aspartate aminotransferase. Figure 6 shows reaction schemes for pyruvate klnase, creatine kinase and glycerol kinase.

DETAILED DESCRIPTION/BEST MODE

If one introduces a known analytical amount of pyruvate in place of the sample using the normal assay scheme (i.e. same reagents in reagent pipettors, same volumes, etc.), the high level of LDH present will convert all of the pyruvate and therefore an equivalent amount of NADH within a very short time. The difference in absorbance from a blank that contains no pyruvate to a pyruvate standard will be an exact analytical measure that relates absorbance or fluorescent units to concentration of NADH. Since the pyruvate standard is delivered to the reagents in the same manner as the samples, pipetting inaccuracy will no longer negatively impact the accuracy of the ALT activity calculation. In addition, since this type of calibration eliminates the need to use the molar absorptivity constant to calculate μmoles of NADH (this is so because it is the pyruvate standard that is now relating absorbance units to μmoles of NADH), precise optical calibration of the instrument is not required. In the fluorescence mode, calibration with a pyruvate standard also eliminates having to use NADH standards for calibration.

Two criteria must be met, however, in order for a pyruvate calibration standard to work. First, the reaction catalyzed by LDH in the presence of pyruvate needs to occur relatively fast under the conditions of the assay and second, the pyruvate standard solution must be stable so that the analytical concentration does not change over an extended period of of time. The second criteria is of particular significance, since stability of pyruvate and α-ketoacids in general can be a source of error. R. Von Korff, Purlty and Stability of Pyruvate and α-ketoglutarate. 13 Meth. Enzymol. 519 (1969). In general, the pyruvate calibration standard is made by the process comprising admixing sodium acetate, trihydate, sodium azide, and sodium pyruvate and water until all solids dissove and adjusting the pH of the admixture from between about 4.0 to 6.0, with the optimum pH of the final mixture being about 5.5. This calibration standard is stable over an extended period of time.

It should be noted that the final dilution of pyruvate standard in the assay solution at 1:20, as described is not critical, but is in line with the ratios used in the present ALT assays. Other manufacturers final dilution is from 1:10 to 1:15 and could easily be substituted. With the 2 mM pyruvate standard, the amount of NADH oxidized would account for approximately 80% of the original 0.125 mM NADH used in the reaction mixture described herein. Of importance with this is that the final concentration of pyruvate not exceed the NADH concentration. If this were to occur, all 340 nm absorption due to NADH would disappear as well as the relationship of absorbance dlffernece to NADH concentration.

Example l

In this example, the materials and instrumentation used for all studies are described along with the procedure for manufacturing the pyruvate calibration standard and the method to calculate its analytical concentration. In addition, the data and results addressing stability and reaction rate issues are presented.

A. Materials and Instrumentation

Sodium acetate trihydrate and sodi um azide were purchased from

Aldrich Chemical Company. Sodium pyruvate was purchased from

Boehringer Mannheim Biochemicals. Sterile filtration was done using a 1 L, 0.2 urn pore size Nalgene Disposable Filter (Nalge Company).

Reagents for ALT activity measurements and pyruvate calibration on a clinical chemistry analyzer were purchased from Clba-Corning.

In-house manufactured ALT reagents (Pandex™ ALT reagents A and B together mixed with diluted sample in the ratio of 1:1:2 is composed of 12.5 mM α-ketoglutarate, 0.125 mM NADH, 0.30 M L-alanine, 1.5 units/mL LDH, and 30 mM tris, pH 7.8) were used both for the concentration determination of the pyruvate calibrator and equivalency testing of NADH fluorescence as a function of concentration. Water used in all experiments and for reagent preparation was obtained through an in-house deionized system that was further purified with a Millipore, Milli-Q Water System purifi er (milli-Q H₂O).

Spectral measurements for stability studies and to determine pyruvate calibrator concentration were done using a Hewlett Packard diode array spectrophotometer (model 8452A)/HP 9000 series 300 computer along with the manufacturer's Chemstatlon software. Alanine transamlnase activity measurements were done using a Gilford SBA 300 automated clinical chemistry analyzer. A Pandex™ fluorescence microtiter plate reader with filters appropriate for monitoring NADH fluorescence was used to test equivalency between NADH and the pyruvate calibrator.

B. Preparation of Pyruvate Calibration Standard (1 & 2 mM)

For a 2 mM concentration and 1 L size volume; 0.680 g (0.005 moles) sodium acetate trihydrate, 0.200 g sodium azide, and 0.224 g (0.002 moles) of sodium pyruvate were weighed together in a 1 L flask. To this was added 950 mL of milli-Q H₂O and a stir bar. The solution was then stirred on a magnetic stlrrer until all solids were dissolved. The pH was adjusted to 5.5 ± 0.1 with 1 N HCL and brought to 1.0 L final volume. The final solution was sterile filtered, divided into appropriate volumes, and stored at 2-8°C.

A 1 mM concentration standard was prepared in an idential manner except for half of the sodium pyruvate being added to the flask.

C. Analytical Concentration Determination of Pyruvate Calibrator The actual analytical concentration of pyruvate was determi ned by lactic dehydrogenase dependent turnover of pyruvate to lactate with the concurrent oxidation of NADH (1:1 stoicheometry). This was calculated using the A_340nm difference between a buffer blank and the pyruvate calibrator, and the molar extinction coefficient of NADH (6.22 mM^-1 cm^-1). To 360 uL of blank buffer (30 mM tris, 0.02% sodium azide, pH 7.8) was added 40 uL of the pyruvate calibrator followed by 200 uL each of Pandex™ ALT A and B reagents in a 1.0 mL semi-micro cuvette (1 cm path length). After 5 minutes of reaction time at 25 °C, the absorption at 340 nm was recorded (the spectrophotometer was zeroed with blank buffer). The average of three replicates was accepted as the absorbance value. This procedure was repeated using 40 uL of blank buffer to get an average blank value. Concentration of the pyruvate calibrator was calculated using the formula below.

Pyruvate calibrator concentration (mM) =

(Average A_340nm blank - Average A_340nm calibrator) X 20 / 6.22 Illustrated below is an example calculation:

With an average A_340nm blank = 0.790 and an average A340nm calibrator = 0.159, pyruvate concentration (mM) = (0.790 - 0.159) X 20 / 6.22 - 2.03 mM

D. Kinetics of Pyruvate Turnover with ALT Reagents

In a 1 mL cuvette was added in order 360 uL blank buffer, 40 uL or pyruvate calibrator (1 or 2 mM), and 200 uL each of Pandex^™ ALT A and B. For blank measurements, 40 uL of blank buffer was substituted for the pyruvate calibrator. After addition of all reagents, the reaction was allowed to progress at 25 °C. The absorbance at 340 nm was measured at one, three, and nine minutes. For each time point the average of four replicates was accepted as the A₃₄₀ value.

Table 1 gives the A₃₄₀ difference from blank and pyruvate calibrators of 1 and 2 mM as a function of time. The data indicates that for both pyruvate calibrator concentrations the reaction is greater than 98% complete within the first minute and 100% by three minutes. This satlfies the criteria for a rapid reaction rate.

Δ A_340nm is the difference between blank and sample absorbance values at 340nm. Reactions were run according to the procedure outlinedd in the Example section.

E. Pyruvate Calibrator Stability Study

Pyruvate calibrators (1 and 2 mM) were prepared and their analytical concentration determined as described above. These solutions were then stored at 2-8 °C, room temperature, and 37 °C.

Periodically, the solutions were reanalyzed for their pyruvate content. Data were collected for 76 days at 2-8°C, room temperature, and 56 days at 37°C.

Figure 2 shows the stability data for pyruvate calibrators of approximately 1 and 2 mM concentration. As can be seen, there is no loss in the level of pyruvate at any of the temperatures for the length of the study period. In addition, in a separate study, a 2 mM calibrator has shown no loss in pyruvate content after 200 days when stored at 2 - 8 °C. These results then meet the second criteria of suitable stability of the pyruvate calibrator.

Example 2

Example 2 details a study aimed at testing the hypothesis that the procedure for using the pyruvate calibration standard will eliminate much of the error due to pipetting inaccuracy.

A. Pyruvate Calibrator Performance wi th an Automated Instrument

The Gilford SBA 300 automated clinical chemistry analyzer was programed to calculate a NADH conversion factor from the delta absorbance change that occurs via the reaction described above. This was done using 5, 10, 20, and 30 uL of the pyruvate calibrator, as delivered by the sample pipettor, with 0.5 mL of Gilford ALT reagent delivered by the reagent pipettor. After 5 minutes of reaction time at 25 °C, the absorption at 340 nm was recorded. The average of four replicates was accepted as the absorbance value. The formula for calculating the conversion factor is shown below,

Pyruvate calibrator conversion factor =

Δ Abs_340nm X total volume (mL) / umol pyruvate - mM^-1 Illustrated below is an example cal cul ation

With an average Δ Abs_340nm = 0.1162 from 5 uL of 2.05 mM pyruvate calibrator (0.01025 umol) plus 0.5 mL ALT reagent, conversion factor = 0.1162 X 0.505 / 0.01025 - 5.73 mM^-1 Normally, as directed by the instrument manufacturer, the molar extinction coefficient is used alone as the NADH conversion factor on the assumption that the pipette is delivering accurately. The value used by the Gilford SBA 300 is 6.30 mM^-1.

To determine the pyruvate calibrator performance as compared to the calibration procedure normally used, an ALT standard with an approximate activity of 80 units/mL at 37 °C was assayed using the same pipetting schemes and Gilford ALT reagent as for calibration.

The background rate produced from blanks were subtracted from sample rates with each pipetting scheme so that activity calculated from

Δ AbS_340nm was due only from the ALT present in the sample itself. The actual activity calculation was then done either by the normal use of the extinction coefficient, or with the pyruvate calibrator determined NADH conversion factor. The ALT standard and blank were run in replicates of 4 at 37 °C. After the addition of sample and reagent to the reaction cup, a lag time of three minutes was used to assure steady-state kinetics in the ensuing 45 second read time. The left portion of Table 2 shows how the conversion factor varies as a function of sample pipetting volume and is in reality an indicator of the pipetting accuracy of the SBA 300 Clinical Chemistry analyzer. If pipetting volumes were exact, then the calculated conversion factor would be the same for each and should be close to the reported coefficient of 6.22 mM^-1 (the value used by Gilford, as is shown in the table, is 6.30). While this basically holds true for volumes of 10, 20, and 30 uL, the 5 uL setting is significantly off from the reported value. It is in this situation that a pyruvate calibrator theoretically should give superior results to normal optical calibration. The right portion of Table 2, showing the calculated ALT values for a 80 unlt/L standard, supports this conclusion. For the pyruvate calibrator conversion, all pipetting volumes give almost exactly the same unlts/L value. This is evident in the standard deviation of the means for the pyruvate and Gilford unit/L calculations. It is particularly striking to note the 5 uL volume sample where the Gilford calculation is excessively low, whereas with the pyruvate calculation is in excellent agreement with the other values.

Table 2 *

* Reactions and calculations were done according to procedures in the Example section Example 3

Example 3 is a study to test the equivalency between the pyruvate calibration procedure and a NADH standard curve when calibrating a fluorescence spectrophotometer.

A. Pyruvate Calibrator Performance with a Fluorescence Instrument

In the fluorescence mode, since there is not the luxury of a molar extinction coefficient, the use of a NADH based calibrator is essential to convert fluorescence units (AFU) to NADH concentration. Using a microtiter based assay, 4 uL of pyruvate calibrator + 36 uL blank buffer or 40 uL blank buffer were added to 20 uL each of Pandex™ ALT reagents to make 80 uL total volume. The reaction was allowed to proceed for 5 minutes at room temperature after which the AFU was recorded. This procedure is essentially the same as above for calibrator concentration determination, except for total volume in the microtlter fluorescence assay being 1/10 of an absorbance mode determination. For performance comparison, an AFU vs. NADH standard curve was produced using known amounts of NADH in the range of 0 to 135 uM with the same buffer components and volumes as above. The slope values for AFU vs. NADH and AFU vs. pyruvate calibrator were then compared for equivalency. This was done over a period of 2 weeks. The formula for calculating slope is shown below.

Pyruvate calibrator slope = (AFU_blank = AFU_calibrator)

X 20 / uM pyruvate cal i brator

Illustrated below is an example calculation:

With AFU_blank = 13500, - AFU_calibrator = 5000

and pyruvate calibrator = 2050 uM, slope =

(13500 - 5000) X 20 / 2050 = 82.9

Figure 3 shows that slope values generated either by an NADH standard curve or by the 2 mM pyruvate calibrator in the fluore- scence mode are essentially equivalent. In this particular case the slope value for the NADH standard curve is 84.5 whereas It is 81.1 for the pyruvate calibrator. The equivalency of these two procedures was tested five different times over a period of two weeks. As can be seen in Figure 4, excluding day 10, both slope values are within error of each other for each time tested. The discrepancy in day 10 is likely a result from an error in pipetting or in manufacturing of the standard NADH solution. The equivalency demonstrated here shows that a pyruvate standard can replace NADH standards in a fluorescence based assay.

Example 4

The aspartate aminotransferase (hereinafter AST), which is clinically important in monitoring both heart and liver dysfunction, is assayed in a very similar manner as the previously disclosed ALT assay. With the AST assay, malate dehydrogenase replaces LDH, aspartate replaces alanine, and oxaloacetate replaces pyruvate, as shown in Figure 5. A similar calibration method is devised where oxaloacetate replaces pyruvate as an instrument calibrator.

The similarities between the assays for ALT and AST are such that preparation of the oxaloacetate calibration standard at 1 or 2 mM concentration would be idential (excluding differences in grams oxaloacetate necessary to give 0.002 or 0.001 moles) to that described in Example 1 for pyruvate calibrator. In addition, the analytical concentration determination of a oxaloacetate calibrator would be carried out in a similar manner as described for the pyruvate calibrator in Example 1. The mathematical formula used for the calculation would be the same. The only exceptions to the procedure would be to substitute AST reagents for ALT reagents.

Example 5

ALT is only one example of an enzyme whose activity is measured by NADH reduction via the coupling to LDH activity. Other enzymes that are of clinical importance that also utilize the NADH/LDH couple are pyruvate kinase, creatlne kinase, and glycerol kinase. In the latter two cases, the NADH/LDH couple is extended by including pyruvate klnase. The assay schemes for these three enzymes are shown in Figure 6. The pyruvate calibrator that was manufactured for ALT as described in Example 1 will work equally well as a calibrator when assaying for these other enzymes with their appropriate reagents.

The invention has been described in detail with particular reference to the above embodiments. It will be understood, however, that variations and modifications can be effected within the spirit and scope of the invention.

Claims

We Cl aim:

1. A method of assaying a sample for enzyme activity using standard reagents and a reaction by-product of enzymatic activity as a calibration standard comprising:

a. adding a known amount of said reaction by-product to said reagents to form a solution;

b. measuring the change in absorbance or fluorescence units as a result of the reaction of said reaction by-product and standard reagents;

c. determining the difference in absorbance or fluorescence units between said solution and a blank solution that does not contain said by-product;

d. relating said difference in absorbance or fluorescence units to NADH concentration;

e. adding sample to said standard reagents;

f. measuring the change in absorbance or fluorescence units resulting from said enzymatic activity from said enzymatic activity in said sample per unit time;

g. determining the change in NADH concentration resulting from said enzymatic activity in said sample per unit time;

h. using said relationship between absorbance or fluorescence units to NADH concentration to determine enzyme activity in said sample based on the change in NADH concentration per unit time.

2. The method of Claim 1 wherein the enzyme is a transaminase enzyme.

3. The method of Claim 1 wherein the enzymatic by-product is an α-ketoacid.

4. A method of assaying a sample for alanine aminotransferase enzyme activity using standard reagents and pyruvate as a calibration standard comprising:

a. adding a known amount of pyruvate to said reagents to form a solution; b. measuring the change in absorbance or fluorescence units as a result of the reaction of pyruvate and standard reagents;

c. determining the difference in absorbance or fluorescence units between said solution and a blank solution that does not contain pyruvate;

d. relating said difference in absorbance or fluorescence uni ts to NADH concentration;

e. adding sample to said standard reagents;

f. measuring the change in absorbance or fluorescence units resulting from said alanine aminotransferase activity in said sample per unit time;

g. determining the change in NADH concentration resulting from said alanine aminotransferase activity in said sample per unit time; h. using said relationship between absorbance or fluorescence units to NADH concentration to determine alanine aminotransferase activity in said sample based on the change in NADH concentration per unit time.

5. A stable pyruvate calibration standard made by the process comprising:

Admixing buffer and a salt of pyruvate or pyruvic add and water until all solids dissolve; adjusting the pH of said admixture from between about 4.0 - 6.0.

6. The calibration standard of Claim 5 wherein the pH is about 5.5.

7. The calibration standard of Claim 5 wherein the standard is stable.

8. The calibration standard of Claim 5 wherein the buffer is sodium acetate and sodium azide.