Instrumentation
Nathan Gochman and Lemuel J. Bowie Veterans Administration Hospital, San Diego, Calif. 92161, and Department of Chemistry, University of California at San Diego, La Jolla, Calif. 92093
Automated Rate-Monitoring Systems for Enzyme Analyses While enzyme activities in body fluid samples are measured by a vari ety of techniques and instrumenta tion, an increasing percentage of these analyses is being performed by spe cialized rate-monitoring systems. This discussion will emphasize those ana lytical devices which have the fol lowing performance characteristics: • Sample pickup, dilution, and dis pensing capabilities are automated. • T h e rate of the enzyme reaction is continuously monitored by observa tion of a photometric change which occurs during, and is proportional to, the reaction. • T h e instruments have a data re duction system capable of direct re porting of reaction rates in interna tional units (μπιοί substrate consumed or product formed/minute) from absorbance vs. time data. T h e design specifications and oper ational features of the individual in struments differ substantially b u t have the following aspects in common: • Medium to high analytical throughput, about 40 samples/h or greater • Computation techniques for the detection of maximum linear rates in the presence of lag phases or nonlinearity • Sequential operation in which each procedure is completed for a group of samples, and the system is then adjusted for the next procedure. This report will not cover the in struments based on the centrifugal fast analyzer principle, but the reader may refer to the extensive review by Tiffany (1). These instruments do not provide automated sample aspiration and delivery into the photometric measuring system. A general discussion of automated reaction rate methods of analysis with descriptions of selected systems and a report on the principles of several electronic devices for rate determina tions were presented by M a l m s t a d t et al. (2, 3). Three commercially available sys tems which meet the performance specifications for enzymatic analyses
outlined above have been selected for extensive description: L K B kinetic analysis system (Model 2086), PerkinElmer KA-150 kinetic analyzer, and Beckman enzyme activity analyzer: System T R . L K B Kinetic Analysis S y s t e m — Model 2 0 8 6
T h e L K B reaction rate analyzer (Model 8600) was one of the earliest analytical systems specifically de signed to perform reaction rate moni toring for the determination of enzy matic activity. Introduced in the late 1960's, at t h a t point it incorporated some unique design features including slope detection routines and the use of a " s t a r t e r " reagent addition sequence. This semiautomated version under went only minor changes in the past decade and, with the recent addition of an integrated automatic sampler/ diluter assembly, is now marketed as the fully automated LKB-2086 kinetic analysis system (Figure 1). As indicated above, perhaps one of the most important design features was the incorporation of a second re agent dispenser for the addition of a starter reagent at some point in time subsequent to mixing serum with an incomplete substrate solution. This al lows certain undesirable side reactions to go to completion prior to the addi tion of the starter reagent and mea
surement of the enzymatic reaction rate of interest (4,5). This approach received approval, particularly in Eu ropean laboratories, and manufactur ers began to market two-vial reagent systems (incomplete substrate reagent plus starter reagent) for the analysis of many clinically important enzymes. A block diagram of the entire L K B in strument, complete with automatic sample handling system and HewlettPackard 9815 programmable calcula tor, is shown in Figure 2. On this system, samples are aspirat ed from the sample turntable and de livered, along with the first reagent, into disposable polystyrene cuvettes in aluminum racks. Each cuvette is se quentially moved through a dry heat temperature-controlled tunnel to allow side reactions to go to comple tion. Starter reagent preheated to the reaction temperature is then added, and mixing is accomplished by rapid rotation of the cuvette. T h e reaction rate is then monitored at the photom eter station for a period of 30 s to 5 min. T h e system is therefore capable of analyzing samples at a rate of u p to 100/h. Another important feature was the provision of a reaction rate calculator which had slope detection capabilities in addition to the usual recorder mode. T h e new kinetic analysis system has
Figure 1. LKB-2086 kinetic analysis system ANALYTICAL CHEMISTRY, VOL. 48, NO. 14, DECEMBER 1976 ·
1189A
Figure 2. Block diagram of LKB-2086 showing sample turntable and calculator/ printer module
a somewhat improved slope and error detection routine incorporated into software on the calculator/printer module. Measurement of the reaction rate can be started as early as 4 s after starter reagent addition, the time nec essary for complete mixing. Measure ments of absorbance are taken at 0.31-s intervals during the course of the reaction. However, for calculation purposes, only readings taken over longer, fixed intervals (generally 3 s) are used to determine slopes. At least four slopes (the gradient AA/At be tween two successive measuring points) are required to give an accept able result. Figure 3 demonstrates how this is accomplished. T h e calculator takes the first three intervals (for the LKB-8600) or the first four intervals (for the LKB-2086) and calculates the slopes, Λ(Λι = Δ Α ι / Δ ί ι , R2 = AA2/ At2, etc.), their mean value (Rm), and an estimate of scatter for each set of slopes. T h e calculator then determines
the slopes and scatter for the next set of slopes. This is repeated for the whole measurement period, and t h e printout gives the mean (Rm) of those four consecutive slopes which gave the least scatter and the enzyme activity in appropriate units. T h e system also prints a number of error messages to indicate conditions in which activity values may be under estimated such as substrate exhaus tion, when optical limits of the system have been exceeded, or when linearity criteria have not been met. P e r k i n - E l m e r K A - 1 5 0 Kinetic Analyzer
This system incorporates a number of additional features in its approach to automated enzymatic analysis. T h e instrument (Figure 4) is a self-con tained bench-top unit 54 in. long and 22 in. deep. In operation, unmeasured samples are placed in a 40-sample tray, then
Figure 3. Diagram of slope detection routine on LKB-2086
aspirated, diluted, and mixed with the first reagent of a two-reagent system as was discussed with the L K B sys tem. T h e diluted samples are preincubated, after which the starter reagent is added, and the mixture is aspirated into the photometer. T h e reaction rate is monitored, and the results are printed out on the calculator unit which occupies the housing on the right. These various steps are repre sented diagrammatically in Figure 5 (6). Among the unique features of the system is the use of hollow cathode lamps in conjunction with interference filters as the source of inherently monochromatic light. Emission lines at either 340 or 404 nm are selected depending on the enzyme reactions to be monitored. This approach obviates some of the problems associated with wavelength drift and stray light. In addition, the photometric detection system (employing silicon diodes) is capable of measuring changes in ab sorbance of as little as 5 Χ 1 0 - 6 A/s. This sensitivity makes possible micro sampling (10 μΐ) and the use of short measurement intervals (8.8 s). Figure 6 gives a detailed representa tion of the timing sequence subse quent to the addition of the starter re agent. After preincubation for a mini m u m of 6 min, during which nonspe cific side reactions are completed, the starter reagent is added. T h e solution is stirred and transferred to the pho tometer through a heat exchanger. A delay of 18 s is included to allow for transfer and thermal equilibration. In the case of creatine kinase assays, this time is extended by 96 s to allow for the completion of any lag phase present in the rate profile. T h e ab sorbance is then monitored and inte grated continuously during four equal
Figure 4. Perkin-Elmer Model KA-150 kinetic analyzer
1 1 9 0 A · ANALYTICAL CHEMISTRY, VOL. 48, NO. 14, DECEMBER 1976
Figure 5. Block diagram showing sample preparation and analysis sequence of Perkin-Elmer KA-150
Figure 6. Timing sequence for kinetic measurements on Perkin-Elmer KA-150
integrating periods of 2.2 s each. T h e average rate in the last quarter of this 8.8-s measuring period is compared to the average rate in the second quarter of the measuring period to detect any curvature in the reaction profile. Should the curvature be excessive or the absorbance out of range, the print out is in red with a warning message appended. T h e combination of these features makes this system capable of very rapid rates of analysis. Specimens can be analyzed at the rate of 150 tests/hResults from stat samples can be available in 8-10 min, depending on t h e test. Based on the system's ultramicro capabilities, it is especially useful for pediatric samples and has low reagent costs. Beckman Enzyme Activity Analyzer: System TR This system is a compact (26 in. long by 21 in. deep by 17 in. high) yet highly sophisticated reaction rate ana lyzer. Figure 7 shows the unit com 1192A
Figure 7. Beckman enzyme activity analyzer, System TR
plete with printer. An optional strip chart recorder can be added to provide an analog representation of the photo metric output. With this instrument, 50-μ1 samples are aspirated from a 20-sample capaci ty tray and diluted sixteenfold with substrate into a refillable cuvette. T h e system then monitors the absorbance continuously until the reaction has maintained linearity for 17 s. T h e computer then calculates the activity in the desired units and displays and prints these results. A schematic rep resentation of the entire system is shown in Figure 8. One of the most interesting features of this system is its approach to reac tion rate monitoring and slope detec tion. T h e linear portion of the reaction profile is automatically determined for each individual s'ample. T h e way in which this is accomplished is shown in Figure 9. T h e continuous monitoring of the absorbance as a function of time is shown in the recording (uppermost part of the figure) of a typical creatine
· ANALYTICAL CHEMISTRY, VOL. 48, NO. 14, DECEMBER
1976
kinase (CPK) assay. Note t h a t there is a "lag" phase in which the reaction rate initially proceeds slowly, followed by a more rapid linear change of ab sorbance vs. time. T h e middle portion of the figure shows the recorder out put from an electronic differentiator circuit which is also incorporated into the system. This circuitry provides the first derivative (dA/dt) of the absorb ance as a function of time. Note t h a t this first derivative becomes constant at the completion of the lag phase. In addition, since the first deriva tive (dA/dt) is nothing more than the value of the slope of the absorbance vs. time curve, the height of this "pla t e a u " is linearly related to enzyme ac tivity. T h e bottom panel in the figure shows the output from a second dif ferentiator which gives the second de rivative of the absorbance vs. time (d2A/dt2). Note t h a t the second deriv ative approaches a value of zero as the reaction rate becomes constant. This signal will remain zero as long as the rate is constant. Therefore, it is possi-
not account for oxidation of N A D H due to other pyridine nucleotide-dependent dehydrogenases. For exam ple, glutamate dehydrogenase which catalyzes the following reaction: α-ketoglutarate + N H J + N A D H + H + — glutamate + NAD+ + H 2 0
Figure 8. Block diagram of Beckman TR showing printer and recorder
340 nm by the following reaction se quence: tt-ketoglutarate
+ aspartate
AST
• oxaloacetate + glutamate oxaloacetate + N A D H + H+ malate
—>-
malate + NAD+
dehydrogenase
Figure 9. Diagram demonstrating use of A vs. time, dA/dt vs. time, and d2A/dt2 vs. time to detect linear phase of reac tion containing lag phase and to deter mine enzyme activity levels
ble to very simply define criteria for linearity in terms of a single parame ter (d2A/dt2). T h e inclusion of these two differentiator circuits obviates the need for complex computer manipula tion of analog or digital data to deter mine the linear phase and slope of a nonlinear reaction profile. Another very useful feature of this system is the ability to utilize dual sample-dual beam spectrophotometry in reaction rate analysis. This ap proach allows for automatic subtrac tion of activity due to the presence of undesired reactions which occur si multaneously with the reaction of in terest. T h e usefulness of this tech nique has been demonstrated very elo quently for the transaminases by Rodgerson and Osberg (7). Figure 10 shows a typical progress curve for as p a r t a t e aminotransferase (AST) which is assayed spectrophotometrically at
Currently, most single-vial commer cial substrate formulations contain added excess lactate dehydrogenase (LDH) to accelerate the conversion of endogenous pyruvate to lactate which oxidizes some N A D H in the process. This is represented in t h e upper lefthand corner of the figure. Although this prevents the measurement of a superimposed rate due to LDH-dependent oxidation of N A D H , it does
T h u s , although the reaction profile becomes linear as seen to the right of the figure, the measured rate is a com posite of the rate due to aspartate am inotransferase plus t h a t due to other dehydrogenases. This kind of problem is not obviated even in techniques employing a starter reagent, since they are usually triggered by the addition of α-ketoglutarate. However, if one places sample plus the complete re agent in the sample cell and sample plus blank reagent (containing no as partate) in the reference cell, oxida tion of N A D H by other dehydroge nases is easily corrected for. With the T R the correction is automatically ac complished since the instrument can be programmed to pick up two sam ples simultaneously and to deliver these samples to separate cuvettes along with two separate reagents. Under these conditions, the net rate observed is represented by the dashed line in the figure. T o minimize the possibility of false ly low results for very high activity samples, this system also prints warn ings whenever the following conditions occur: the final absorbance exceeds a specified limit; the change in absorb ance during the measurement period is greater than a preset limit; and the rate exceeds a certain value (in U/l.). From the previous discussion it is
Figure 10. Diagram demonstrating contribution of reactions catalyzed by other de hydrogenases in assay of aspartate aminotransferase (AST)
1194 A · ANALYTICAL CHEMISTRY, VOL. 48, NO. 14, DECEMBER 1976
obvious t h a t each of the different re action rate analyzers has its advan tages for specific laboratory require ments. T h e LKB-2086 system is a moderately priced system ($27,000) t h a t offers discrete sample handling capabilities (no flow through cells) with relatively high rates of analysis (up to 100/h). T h e Perkin-Elmer KA150 is also moderately priced ($27,000) and is capable of very high rates of analysis (up to 150/h). In addition, its microsampling capabilities are very useful for certain analyses. T h e Beckman T R system is a relatively inex pensive ($16,000) instrument with highly sophisticated slope and error detection capabilities. Its throughput, however, is significantly lower (up to 45/h). All three instruments represent completely automated systems capa ble of continuous monitoring of reac tion rates. T h e choice of any individu al system for a particular laboratory setting will be dictated by the special requirements of each laboratory. With regard to automated enzymat ic analysis, some special concerns are worth mentioning. T h e importance of accurate estimation and regulation of intracuvette sample temperatures has been well recognized (8). Manufactur ers have responded by not only pro viding better methods of temperature
control (e.g., direct contact heating/ cooling of cuvettes), but also by pro viding some indication of the intracu vette temperature. Work in this laboratory (9) with an aqueous temperature indicating tech nique has revealed substantial differ ences in temperature equilibration times with a variety of automated en zyme analyzers. T h e importance of being able to de tect aberrant results due to nonlinear ities in reaction rate profiles has also become increasingly more obvious. Again, manufacturers have responded by incorporating error detection rou tines aimed at detection of excessive lag phases, substrate exhaustion prior to time of rate monitoring, reagent de terioration, kinetic nonlinearities, and optical problems associated with cer tain samples (e.g., turbidity, lipemia, very high activity). T h e different sys tems have varying capabilities to de tect these problems, and even some of the most sophisticated cannot alert the operator when certain problems occur simultaneously. One example is the problem associated with discrimi nating between substrate exhaustion and true low activity in lipemic or ic teric samples. In addition to the systems described here, there are a number of general
purpose automated systems, designed to do analysis of both enzyme and substrates. These systems may incor porate some of t h e principles de scribed above for reaction rate moni toring. Also, a variety of semiautomated rate analyzers are available which have continuous monitoring and slope detection capabilities. In this report, we have described a few examples of highly sophisticated and moderately priced instruments which are specifi cally designed to do reaction rate monitoring with special emphasis on the application to automated enzy matic analysis. References (1) T. O. Tiffany, Crit. Rev. Clin. Lab. Sci., 5,129 (1974). (2) H. V. Malmstadt, E. A. Cordos, and C. J. Delaney, Anal. Chem., 44 (12), 26A (1972). (3) H. V. Malmstadt, C. J. Delaney, and E. A. Cordos, ibid., ρ 79Α. (4) A. Karmen, J. Clin. Invest., 34,131 (1955). (5) R. J. Henry, N. Chiamori, O. J. Golub, and S. Berkman, Am. J. Clin. Pathol., 34, 381 (1960). (6) J. G. Atwood and J. L. DiCesare, Clin. Chem., 21,1263 (1975). (7) D. O. Rodgerson and I. M. Osberg, ibid., 20,43 (1974). (8) G. N. Bowers, Jr., H. U. Bergmeyer, and D. W. Moss, ibid., 19, 268 (1973). (9) L. Bowie, F. Esters, J. Bolin, and N. Gochman, ibid., 22, 449 (1976).
Taking Things Apart and Putting Things Together
ACS Centennial Seals
ACS Centennial Plate
ACS Playing Cards
ACS Centennial Tie
1 1/2 inch blue and gold ACS Centennial seal. Ideal for business correspondence or greeting cards. Roll of 2,000 for $10.00 200 for 1.00
10 3/4 inch blue and white plate with gold rim featuring ACS Cen tennial seal. Made of top-quality Pickard china. $20.00
Blue and gold bridge set with ACS Cen tennial seal design. Case included. $5.00
Necktie with ACS Cen tennial date and emblem pattern. Speci fy blue or red back ground. $6.50
J
Books Dept./American Chemical Society 1155 16th St., N.W., Wash., D.C. 20036
ACS Centennial Medallion
A Century of Chemistry
ACS Centennial Mug
ACS Centennial Emblem
1 1/2 inch Franklin Mint proof-quality circu lar medallion with ACS Centennial seal front, and chemical symbols reverse. Housed in pro tective plastic holder. Limited edition, gold on silver $20.00 sterling silver 15.00 bronze 7.00
An illuminating portrait of the growth and activ ities of the ACS in the context of a century that saw two World Wars, the Great Depression, the Cold War, the Space Race, and the environ mental movement. 468 pages (1976) clothbound $15.00
The perfect accessory for office, den, or bar— 4 3/4 inch high pewtercolored noggin with ACS Centennial seal made of Armetale® to keep beer or ale ice cold. $8.50
Blue, white, and di amond-shaped emblem commemorating the Centennial is available with pin back for women members, or as a lapel pin for men. $2.00
· ANALYTICAL CHEMISTRY, VOL. 48, NO. 14, DECEMBER
D ACS Mug ($8.50 each) D ACS Plata ($20.00 each) D ACS Seals roll Of 2,000 ($10.00) 200for$1.00 D ACS Emblem ($2.00 each) women's pin men's lapel pin α ACS Cards ($5.00 set) D Check enclosed for $
• ACS Tie ($6.50 each) blue tie red tie α ACS Medallion gold on silver ($20.00) sterling silver ($15.00) bronze ($7.00) case ($3.00) α A Century of Chemistry (clothbound $15.00) D Taking Things Apart (paperback $1.75)
All orders postpaid in U.S. and Canada.
Name
.
,
,
Ad d ress Clty_
presentation case 3.00
1196A
An absorbing narrative of what chemistry is all about; case histories tell the fascinating stories of cyclamates, DDT, insulin, anesthet ics, penicillin, polio vaccine, and more. Pro fusely illustrated with four-color designs and photographs from the ACS Centennial Exhibit. 128 pages (1976) paperback $1.75
State
1976
', ,
, Zip
.
