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  • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
  • C12Q1/48—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving transferase
  • C12Q1/52—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving transferase involving transaminase
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
  • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
  • C12Q1/26—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving oxidoreductase
  • C12Q1/32—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving oxidoreductase involving dehydrogenase
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
  • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
  • C12Q1/48—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving transferase

Definitions

• ALT alanine aminotransferase activity
• LDH lactic dehydrogenase
• Lactic dehydrogenase catalyzes the reduction of pyruvate with the concurrent oxidation of NADH.
• NADH lactic dehydrogenase
• 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.
• 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.
• 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.
• 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
• 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 ⁇ M) 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.
• 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.
• 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.
• 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.
• the amount of NADH oxidized would account for approximately 80% of the original 0.125 mM NADH used in the reaction mixture described herein.
• 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.
• Reagents for ALT activity measurements and pyruvate calibration on a clinical chemistry analyzer were purchased from Clba-Corning.
• ALT reagents In-house manufactured ALT reagents (PandexTM ALT reagents A and B together mixed with diluted sample in the ratio of 1:1:2 is composed of 12.5 mM ⁇ -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 H 2 O).
• 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 PandexTM fluorescence microtiter plate reader with filters appropriate for monitoring NADH fluorescence was used to test equivalency between NADH and the pyruvate calibrator.
• a 1 mM concentration standard was prepared in an idential manner except for half of the sodium pyruvate being added to the flask.
• Table 1 gives the A 340 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.
• ⁇ 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.
• 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.
• 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 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.
• 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,
• Example 3 is a study to test the equivalency between the pyruvate calibration procedure and a NADH standard curve when calibrating a fluorescence spectrophotometer.
• NADH based calibrator 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.
• AFU fluorescence units
• 4 uL of pyruvate calibrator + 36 uL blank buffer or 40 uL blank buffer were added to 20 uL each of PandexTM 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.
• 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.
• 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.
• AST aspartate aminotransferase
• malate dehydrogenase replaces LDH
• aspartate replaces alanine
• oxaloacetate replaces pyruvate, as shown in Figure 5.
• a similar calibration method is devised where oxaloacetate replaces pyruvate as an instrument calibrator.
• 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.

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