Waukegan, Ill. 60085). The reagent was prepared to yield incubation concentrations of substrates, cofactors, and activator in accordance with the optimized.
CLIN. CHEM.24/8, 1408-1413 (1978)
Chromatographic Separation and ContinuouslyReferenced, On-Line Monitoringof Creatine Kinase lsoenzymes by Use of an Immobilized-Enzyme Microreactor Mark S. Denton, William D. Bostick, Stan R. Dinsmore, and John E. Mrochek”
We describe a new concept in continuously referenced monitoring of the isoenzyme activities of creatine kinase (EC 2.7.3.2) after liquid.-chromatographic separation. After separation on a diethylaminoethyl-Sephacel column, the three isoenzymes of creatine kinase undergo a series of coupled enzyme reactions, ultimately resulting in the formation of ultraviolet-detectable NADPH. A major advantage of this detection system is the immobilized-enzyme microreactor (2 X 17 mm), which may be removed and stored refrigerated when not in use. A split-stream configuration allows self-blanking of endogenous ultraviolet-
absorbing constituents in authentic sera samples, which would otherwise make definitive diagnosis and quantitation difficult or impossible. This detection system is applicable to the automated analysis of creatine kinase isoenzymes in the clinical laboratory. Addftlonal Keyphrases heart disease column (DEAE-Sephacel) chromatography tems diagnostic aids fluorometry ants .
.
.
enzyme activity analytical sysintrinsic interfer#{149}
.
Isoenzyme separation and measurement has become important in diagnostic procedures with the discovery that various tissues express different isoenzyme contents. Thus, monitoring isoenzyme activities in serum provides a noninvasive method of identifying tissue damage. One enzyme for which isoenzyme measurement has proven clinical utility is creatine kinase (EC 2.7.3.2, CK). Three CK isoenzymes, commonly designated MM, MB, and BB, have been identified. The primary tissue source for each is skeletal muscle, myocardium, and smooth muscle, respectively. Numerous reports link CK-MB to myocardial infarction (1-4). Wagner et al. (5) evaluated four clinical measurements-electrocardiogram, lactate dehydrogenase LD1:LD2 isoenzyme ratio, total CK, and CK-MB-with regard to their sensitivity and specificity for the detection of myocardial infarction. Only CK-MB had a 0% incidence of false negatives and a reported specificity of 99%.
Chemical Oak Ridge, Author Received
Technology Division, Oak Ridge National Tenn. 37830. to whom inquires should be made. April 17, 1978; accepted May 22, 1978.
1408 CLINICALCHEMISTRY,Vol. 24, No. 8, 1978
Laboratory,
In addition
to the previously mentioned localization in specific tissues, CK is also normally present in serum; of the total, less than 5% is CK-MB (6); the remainder is usually CK-MM. The third isoenzyme, CK-BB, is not normally found in serum (7), but its detection has been reported in connection with acute brain damage (8), brain damage due to anoxia (9), renal damage (10), gastrointestinal carcinoma (11), and cerebrovascular damage (12). With these documented relationships to damage of various organs and the especially important relationship to myocardial infarction, monitoring of CK isoenzymes in the clinical laboratory has assumed major importance. In the past, electrophoresis, with subsequent electrochemical, fluorometric, or microcalorimetric detection, has been the method most commonly used for CK isoenzymes (13-18). This method, however, is highly operator-dependent, time-consuming, semiquantitative, and insensitive to low enzyme activities (19). Many isoenzymes, including the isoenzymes of CK (13, 14, 19-24), LD (25-29), acid phosphatase (30), and arylsulfatase (31), have been successfully separated by ion-exchange column chromatography. Results are obtained more rapidly than by electrophoresis, and direct quantitation of enzyme activity in column eluates is possible. Although column chromatography can yield excellent isoenzyme separation, it is not foolproof, as evidenced by recent critical comments concerning various commercial adaptations of chromatographic methods for the separation of CK isoenzymes (32, 33). This criticism has centered around the apparent poor resolution resulting from the collection of only a minimum number of eluate fractions. Column chromatography, as it is currently practiced for isoenzyme separations in authentic serum samples, requires the collection of fractions and the individual assay of these fractions by some kinetic method. Frequently, dilution of the sample by the eluent causes problems of sensitivity, and the number of fractions obtained will affect, to some extent, the degree of apparent peak overlap. Continuous, on-line monitoring of enzyme activities in the column effluent would save of isoenzymes
time and improve
the resolution
between
isoenzymes.
A relatively
inexpensive method of such continuous monitoring for enzyme activity is needed, to make column chromatography more amenable to direct use in the clinical laboratory. Here we present, in a preliminary manner, an enzyme-detection concept that is applicable to automated direct monitoring of actual clinical samples. The detection system includes a microreactor containing immobilized enzymes for the enzyme-coupled reactions. The advantages include: (a) decreased cost and improved storage stability for the continuously added substrate mixture without soluble enzymes present; (b) the small size of the microreactor enables it to be located immediately before the sample moni-
toring side of the dual-beam monitoring system without causing increased band spreading; (c) the dual-beam system lends itself to highly desirable “self-blanking” where the sample, in the absence of enzyme-generated chromophore, serves as its own reference; and (d) quantitation is facilitated. Although the isoenzymes of creatine kinase were investigated here, the system theoretically is applicable to any enzyme capable of generating a detectable product within reasonable reaction times by use of enzyme-coupled indicating reactions.
Materials and Methods Samples CPK Isotrol (Sigma Chemical Co., St. Louis, Mo. 63178), containing all three CK isoenzymes, was used as an enzyme reference. Statzyme Control Serum (Worthington Biochemical Corp., Freehold, N. J. 07728) served as a reference sample. All reference standards were reconstituted according to the manufacturers’ recommendations (except for “CPK Isotrol,” which was diluted sixfold more than recommended) and injected without further preparation. Sera from patients referred for CK isoenzyme determination were assayed for CK isoenzymes by electrophoresis on agarose gel, and auquots were frozen for later analysis
in our laboratory.
Reagents
The substrate and cofactors-creatine phosphate, ADP, NADP, AMP, imidazole, and N-acetyl-L-cysteme-were all obtained from Sigma, as were the two indicator enzymes, co-immobilized on agarose beads. The two other substrates used in this study were Mg acetate (Fisher Scientific Co., Fairlawn, J. 07410) and glucose N.
(Pfanstiehl Laboratories, Waukegan, Ill. 60085). The reagent was prepared to yield incubation concentrations of substrates, cofactors, and activator in accordance with the optimized conditions described by Szasz and Bernt (34). Instrumentation
for Liquid-Chromatography
Figure 1 shows a schematic of the component system for high-performance liquid chromatography. The three reagent reservoirs can be controlled either manually or automatically, via timer and solenoid valve. An Instrument Minipump (Laboratory Data Control Div. of
REAGENT RESERVOIR BUFFER A
BUFFER B
BUFFER C O.4OMNoCI
IRIS pH 7.4
pH 7.4
pM 7.4
SUBSTRATE CREATINE GLUCOSE ADP
- -
NAOP
SPECTROPHOIOMETER
AMP IMIOAZOLE
(340 ,,m) CYSTEINE
‘-10
WASTE
M5Ac
Fig. 1. Schematic for the chromatographic separation and continuously referenced monitoring of CK isoenzymes with a microreactor containing immobilized enzymes. M,
mol/liter
Milton Roy Corp., Riviera Beach, Fla. 33404) delivered the step gradient at 20 mi/h through the six-port injection valve and column, both fabricated here. This column effluent plus a 2-ml/h additional substrate flow make up the total 22-ml/h system flow. A Polystaltic Pump (Buchler Instruments, Fort Lee, N. J. 07024) pulled equal volumes of this total flow through 8-id sample and reference flow-through cells (Ultra-MicroCells; Helma Cells Inc., Jamaica, N. Y. 11424). This peristaltic pump was also used to feed the substrates to a T-mixer (equipped with a micro-stirring bar) between the analytical column and temperature-controlled delay coil. The T-mixer and stream splitter were made inhouse and are of approximately the same dimensions. Each was fabricated from 2.4-mm i.d. (3.0-mm o.d.) glass tubing. The central mixing chamber contains approximately 0.1 ml. One-eighth inch Teflon tubing was inserted well into each leg of the “T” and sealed in place with heat-shrink tubing. The micro-stirring bars were constructed from 2-mm lengths of 20-gauge wire which were sealed within capillary glass tubing. A simple DB-G Grating Spectrophotometer (Beckman Instruments, Inc., Fullerton, Calif. 92634) monitoring the 340-nm wavelength was used for absorbance measurements; a FS 970 LC Fluorometer (Schoeffel Instrument Corp., Westwood, J. 07675) or a modified ORNL flow-fluorometer (35) was used for fluorescence studies. N.
Columns All CK isoenzyme separations were performed on a glass column (4 X 70 mm) fabricated with Teflon inlet and outlet fittings. DEAE-Sephacel Beaded-Cellulose
Ion Exchange Support (Pharmacia, Uppsala, Sweden) was slurry packed by use of dilute buffer. The microreactor column (2 X 17 mm) containing hexokinase (EC 2.7.1) and glucose-6-phosphate dehydrogenase (EC 1.1.1.49), co-immobilized on agarose, was fabricated of Teflon tubing, with a porous Teflon frit and glass-wool CLINICAL CHEMISTRY,
Vol.
24, No. 8. 1978
1409
plug installed agarose.
at the downstream
end to contain
the
Eluents
are potential interferences, are not affected by passage through the microreactor; therefore, effective selfblanking is possible.
Results and Discussion
CK isoenzymes were separated by discontinuous elution with 30 mmol/liter tris(hydroxymethyl)methylamine buffer (Ultrol; Calbiochem, La Jolla, Calif. 92037), pH 7.4, containing NaCl solution, either (a) 0.02 mol/liter (MM eluent), (b) 0.15 mol/liter (MB eluent), or (c) 0.4 mol/liter (BB eluent). The salt content of the eluent stream was monitored with a conductivity meter (cat. no. 4988; Leeds & Northrup Co., North Wales, Pa. 19454) located between the stream-splitter and the reference cell. After each run, the system was thoroughly flushed with the dilute buffer for about 10 minutes, while the effluent was being monitored with the conductivity meter.
Isoenzyme
Analysis
The detection system is based on the enzymic conversion of a reagent mixture by CK isoenzymes to intermediate products (equation 1), with subsequent conversion by the coupled reactions (equations 2 and 3) to the monitored product, NADPH. Creatine
+ ADP
phosphate
CK -b’
creatine
+
ATP
(1)
HK
ATP + D-glucose
-
ADP + D-glucose-6-phosphate
D-Glucose-6-phosphate
+ NADP
(2)
GPDH -
NADPH (3)
+ D-glucose--lactone-6-phosphate
NADPH
is formed in amounts proportional to the hydrolyzed creatine phosphate and may be monitored by its absorbance at 340 nm or its native fluorescence (Xex 363 nm; Xem = 440 nm). The sites of these reactions can easily be traced through the schematic in Figure 1. =
The substrate and cofactor mixture (CP, ADP, NADP, AMP, imidazole, N-acetyl-L-cysteine, and Mg acetate) is added to the column effluent by means of a mixing tee. This mixture then flows through a delay coil mm) immersed in a 37 #{176}C water bath, where ATP is formed (equation 1). The resulting stream is subsequently divided, half going through the reference cell and half passing through the microreactor with its immobilized HK and GPDH (equations 2 and 3). D-Glucose-6-phosphate, formed in an amount equivalent to the ATP of equation 1, is rapidly and quantitatively converted to the lactone in the presence of the cofactor (-7
NADP. This reaction generates NADPH, which is monitored in the sample cell. The contribution of ultraviolet-absorbing constituents endogenous to complex body fluids is effectively subtracted from the sample beam by passage through the reference beam. Such nonspecific ultraviolet-absorbing constituents, which 1410 CLINICALCHEMISTRY,Vol. 24, No. 8, 1978
Originally, the separation medium chosen for studies isoenzyme separations was DEAE-Glycophase/ CPG-250, 5-10 m particle size (sample provided courtesy of Corning Glass Works, Corning Biological Products Dept., Medfieid, Mass. 02052) because of its noncompressibility, small particle size, and inertness toward sensitive biological macromolecules (28, 36). This medium, with DEAE groups bonded to a glycerol monolayer, which in turn is covalently bound to controlled-pore glass, provides good separations, but it is not now commercially available. We found that the readily available, inexpensive DEAE-Sephacel beaded cellulose ion-exchanger (Pharmacia) gave excellent separation of CK isoenzymes. Furthermore, it demonstrated a lower pressure drop and improved stability over other exchangers tested. A 4 X 70 mm analytical column demonstrated adequate separation and a low back pressure, which precluded the need for high-performance liquid-chromatography equipment. The microreactor column (2 X 17 mm), comprised of co-immobilized enzymes on agarose beads [unlike the soluble enzymes used by Chang et al. (28)], performs similarly to larger reactor columns described by Cremonesi et al. (37). This microreactor, which was used in the final reaction stages (see equations 2 and 3), should not be confused with the so-called post-column enzyme reaction bed used by Chang et al. (28). They added soluble enzymes with substrates, while the inert (nonporous spherical sodium silicate glass beads) post-column performed the same function as the delay coil shown in Figure 1. The initial selection of analytical column dimensions was determined by injecting 72-Ml (external loop) samples of partly purified CK isoenzymes and following the degree of separations on various columns by use of single-beam detection of fluorescence or absorbance. With these samples, three chromatographic peaks were detected by their fluorescence. However, it was noted that, with human control serum (Worthington Statzyme Control), three peaks were also observed without the addition of reagents (38) (see Figure 2). These endogenous, fluorescent constituents of serum elute with or near the CK isoenzymes. Although not as intense as the background fluroescence, this phenomenon (three peaks) was also noted with ultraviolet absorbance detection when reconstituted commercial (Statzyme) serum was injected without reagents and the eluate was of
monitored by use of a static solution of dilute buffer in the reference beam. With sera having CK activities only slightly above the normal range, two endogenous peaks were obtained, as shown in Figure 3B. Figure 3C shows the actual CK-activity profile for this sample (with electrophoretically confirmed CK-MB isoenzyme). It is obvious that nonspecific background peaks, as in Figure 3B (static-reference serum blank), would lead
RELATIVE CONDUCTIVITY OF ELUTION GRADIENT-...
B ‘ii 0
0
a
Iii 0 U) lii
‘Si ‘I
a 0
z
0
4
IL
a
-J
STATIC
REFERENCE
0
a
‘Si >
SPLIT
4
‘C ‘Si
REAG;NT;LANKS
a
0
I B. SERUM BLANKS I
30
0 TIME.
30
n,in
Fig. 3. On-line photometric monitoring of column effluents reagent blanks (no sample injected); B. serum blanks (no reagent added); C, corrected serum CK-lsoenzyme activity profile (total activIty 167 U/liter). The presence of isoenzyme MB (second peak) was confirmed by electrophoresis TIME.
mi,,.
Fig. 2. Separation of unidentified, fluorescent constituents endogenous to human control serum (no reagent present) M, mol/liter;
PIPES, 1,4-piperazine diethane sulfonic acid buffer
to gross overestimation of CK-MB isoenzyme and possible false-positive diagnosis of myocardial infarction. Similarly, Coolen and Herbstman (39) report a
nonprotein fluorescent artifact that interferes with the detection of CK-MB isoenzyme. Aleyassine et al. (40)
0
‘C
z C
a a
S C
note the presence of endogenous fluorescing material in serum from patients with chronic renal failure which
has an electrophoretic mobility similar to that of CK-BB isoenzyme, thus making results ambiguous. [Ed. note: see also Clin. Chem. 24, 1084] Fluorescent artifacts that could interfere with the detection of lactate dehydrogenase FEC 1.1.1.27) isoenzymes have also been observed by McKenzie and Henderson (41). Finally, Bostick et al. (31) report endogenous fluorescence in urine and serum that interferes with the identification of isoenzymes in column eluates by the use of fluo-
rogenic
umbelliferone-labeled
substrates.
Thus
we
considered it necessary to design an automated, dualbeam photometric detection system to provide unambiguous peak identification in the presence of the
complex background matrices of authentic body-fluid samples.
Once analytical
tion of such a continuously
olet-monitoring cating
of the CK isoenzymes was our efforts on the optimiza-
separation
effected, we concentrated
referenced,
on-line
ultravi-
system. The first attempt involved lo-
the microreactor
column
directly
reference and sample compartments,
between
the
in series. This
configuration was based on the assumption that the short delay between cells would be small relative to peak
width, enabling electronic compensation. However, because derivative-type chromatograms were obtained, which would require sophisticated electronic compensation, the alternative split-stream configuration (Figure 1) was chosen, thus utilizing a system that could be adapted to any commercial or component system.
TIME
laA)
Fig. 4. (Left) Chromatographic separation of the three CK isoenzymes in a reference sample (CPK lsotrol). (Right) Chromatogram is corrected for the effect of reagent blank
With this mode, it is quite simple to split the flow evenly with a T-splitter before the sample (and microreactor) and reference
cells. Two
channels
of the peristaltic
pump were used to pull the combined flow of column effluent plus reagent feed through both sample and reference cells at equivalent rates. Figure 3B illustrates a comparison of serum blanks (no substrate added) run with the split-stream and the static-reference system. As illustrated, even with 8-M’ flow cells, the background absorbance has been well blanked. Figure 3C illustrates the same patient’s serum with reagents added and use of the split-stream configuration. Tailing after the last chromatographic peak was noted with CK Isotrol standards as well as patient sera (see Figures 4 and 5, respectively). Because we did not see this phenomenon in serum blanks, we examined reagent blanks (no sample, but with the normal step-gradient present). As shown in Figure 3A, the absorbance of the reagent blank decreases in proportion to increases in the salt concentration
of the eluent.
To observe this inverse rela-
tionship, it was necessary
for reagents and step-gradient
CLINICALCHEMISTRY,Vol. 24, No. 8, 1978
1411
With the present system, efficient isoenzyme separations have been made on a very small (4 X 70 mm) analytical column packed with a readily available beaded cellulose anion-exchanger. This results in a very low-pressure system, thus avoiding the cost of more sophisticated “high-performance” equipment. Also, the utility of a very small (2 X 17 mm) immobilized-enzyme microreactor column has been demonstrated. This system affords two advantages: the sample dilution and subsequent band-broadening that accompanies the use of soluble enzymes and relatively long (4.8 X 600 mm in reference 28) reaction bed columns is minimized here, and the reusable microreactor can be stored in the refrigerator between runs. The high cost of isoenzyme assays is thereby greatly reduced and reactor viability
0 ‘C U, U’ ).1
z
4 a
a 0
U)
a
4
prolonged. Isolation of the substrate-cofactor TIME
(,nIU)
Fig. 5. Chromatographic separation of CK isoenzymes in the serum of a patient with electrophoretically confirmed CK-MB (total activity 745 U/liter) as well as the microreactor to be present. This suggests some interaction of the NaCl of the eluting solvent and the reagent mixture on the agarose column. The background can be easily compensated for, because it is constant and reproducible, unlike the variable serum background. Compensation can be done either by sub-
tracting the reagent blank drift from the uncorrected chromatogram, as shown in Figures 4 and 5, or by placing a duplicate agarose column, without immobil-
ized enzymes, upstream of the reference cell. Regardless of separation or detection capabilities demonstrated on reference samples, the true test of any useful clinical system must rely on its performance with authentic clinical samples. We found that, even with split-stream analysis, and hence some material loss, the use of 8-gil Suprasil-Quartz Ultra-Micro (1-mm aperture X 10-mm pathlength) flow-through cells yielded sufficient sensitivity for the measurement of both normal
and above-normal
CK isoenzyme activities.
Chromatograms for two successive samples from a patient suspected of having experienced a myocardial infarction are shown in Figures 3C and 5. The increased CK-MB, previously also found by electrophoresis, are readily apparent in these chromatograms. The total CK activities supplied with the samples, 167 and 745 U/liter, respectively, were verified by means of kinetic assay with a centrifugal analyzer (42). Furthermore, measurements of peak areas obtained by using chromatographic separation and the self-referencing detection system for the Statzyme reference serum agreed well with values reported by the manu-
facturer. To verify retention times and further eliminate the possibility of system response to nonspecific sample components, partly purified Eby prior chromatographic separation (42)] CK-MB and CK-BB were used to supplement these samples of control sera. The expected increases in absorbance at the appropriate elution positions were observed. 1412 CLINICALCHEMISTRY,Vol. 24, No. 8, 1978
mixture
from the indicating enzymes allows this mixture to be frozen, a condition that is too harsh for hexokinase and glucose-6-phosphate dehydrogenase in solution. The major advantage of the system is the continuously referenced detection, which eliminates ambiguous or false-positive peaks from appearing with isoenzymes in
complex matrices and consequently
allows isoenzyme
separation and detection in authentic clinical samples without extensive sample preparation. Further studies are desirable to demonstrate the coupling of two or more self-referencing, on-line detection systems to multiple, specific microreactors. An additional stream splitter might be used downstream of the analytical column and thus, with separate and specific substrate addition and micro-reactors (if necessary for enzyme-coupled reactions), simultaneously detect several different isoenzyme systems.
We gratefully
the courtesy of Dr. Burton Goodge, Ft. Hospital, Knoxville, Tenn., in providing serum samples and electrophoretic data on them. This research was sponsored jointly by the National Cancer Institute under Interagency Agreement 40-279-71 and the Division of Biomedical and Environmental Sciences, U.S. Department of Energy, under contract W7405-eng-26 with the Union Carbide Corp. acknowledge
SandersPresbyterian
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enzyme in cerebrospinal fluid in a case of brain damage. Clin. Chem. 22, 1405 (1976). 9. Itano, M., The detection of CPK-BB in serum. Am. J. Clin. Pat hol. 65, 351 (1976). 10. Galen, R. S., Creatine kinase isoenzyme BB in serum of renaldisease patients. Clin. Chem. 22, 120 (1976). 11. Lederer, W. H., and Gerstbrein, H. L., Creatine kinase isoenzyme BB activity in serum of a patient with gastric cancer. Clin. Chem. 22, 1748 (1976).
12. Coolen, R. B., Pragay, D. A., and Chilcote, M. E., The occurrence of the brain (BB) isoenzyme of serum creatine kinase (CK) in different diseases as determined by quantitative electrophoresis and ion exchange column chromatography. Clin. Chem. 21,976 (1975). 13. Wong, P. C.-P., and Smith, A. F., Comparison of 3 methods of analysis of the MB isoenzyme of creatine kinase in serum. Clin. Chim. Acta 65,99 (1975). 14. Lum, G., and Levy, A. L., Chromatographic and electrophoretic separation of creatine isoenzyme compared. Clin. Chem. 21, 1601 (1975). 15. Smith, A. R., Separation of tissue and serum creatine kinase isoenzymes on polyacrylamide gel slabs. Clin. Chim. Acta 39, 351 (1972). 16. Konttinen, A., and Hannu, S., Determination of serum creatine kinase isoenzymes in myocardial infarction. Am. J. Cardiol. 29,817 (1972). 17. Elevitch, F. R., Isoenzymes. In Fluorometric Techniques in Clinical Chemistry, Little, Brown, and Co., Boston, Mass., 1973, p 244. 18. Roe, C. R., Limbird, L. E., Warner, G. S., and Nerenberg, S. T.,
separation and measurement of lactate dehydrogenase Clin. Chem. 22, 468 (1976). 26. Kudirka, P. J., Schroeder, R. R., Hewitt, T. E., and Toren Jr., E. Automated isoenzymes.
C., High-pressure liquid hydrogenase isoenzyme.
chromatographic
and chromatography
on DEAE-Sephadex
A-50 compared
in
the estimation of creatine kinase isoenzymes. Clin. Chem. 21, 381 (1975). 21, Morin, L. G., Improved separation of creatine kinase cardiac isoenzyme in serum by batch fractionation. Clin. Chem. 22, 92 (1976).
22. Morin, L. B., Evaluation of current methods for creatine kinase isoenzyme fractionation. Clin. Chem. 23, 205 (1977). 23. Klein, B., Foreman, J. A., Jeunelot, C. L., and Sheehan, J. E., Separation of serum creatine kinase isoenzymes by ion-exchange column chromatography. Clin. Chem. 23, 504 (1977). 24. Mercer, D. W., Separation of tissue and serum creatine kinase isoenzymes by ion-exchange column chromatography. Clin. Chem. 20, 36 (1974). 25. Busby, M. G., Kudirka, P. J., Carey, R. N., and Toren Jr., E. C.,
de-
G., and Varat, M. A., Use of a column
for measurement
of lactate
dehydrogenase
(1976). mination (1976).
of optimum
chromatography.
acetate
method
34. Szasz, W. G., and Bernt, E., Creatine
35. Thacker,
titation
of lactate
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Application
Med. 80,577 (1972). 19. Mercer, P. W., and Varat, M. A., Detection of cardiac-specific creatine kinase isoenzyme in sera with normal or slightly increased total creatine kinase activity. Clin. Chem. 21, 1088 (1975). 20. Yasmineh, W. G., and Hanson, N. Q., Electrophoresis on cellulose
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(1976).
29. Mercer, D. W., Vasudevan,
Combined isoenzyme analysis in the diagnosis of myocardial injury: of electrophoretic methods for the detection and quanof the creatine phosphokinase MB isoenzyme. J. Lab. Clin.
chromatographic
Clin. Chem. 22, 471 (1976). 27. Mercer, D. W., Simultaneous separation of serum creatine kinase and lactate dehydrogenase isoenzymes by ion-exchange column chromatography. Clin. Chem. 21, 1102 (1975). 28. Chang, S. H., Gooding, K. M., and Regnier, F. E., High performance liquid chromatography of proteins. J. Chromatogr. 125, 103
reaction
conditions.
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L. H., Improved miniature flow fluorometer for liquid J. Chromatogr. 136, 213 (1977). 36. Regnier, F. E., and Noel R., Glycerolpropyl bonded phases in the stain exclusion chromatography of biological macromolecules. J. Chromatogr. Sci. 14, 316 (1976). 37. Cremonesi, P., and Bovara, R., High performance enzyme reactors. Biotechnol. Bioeng. 18, 1487 (1976). 38. Eadie, J. S., An automated system for the separation and detection of creatine phosphokinase by high-pressure liquid chromatography and on-line fluorometry. ORNL (unpublished data), 1976. 39. Coolen, R. B., and Herbstman, R., Artifactual brain (BB) creatine kinase (CK) isoenzyme in kidney transplant and renal dialysis patients. Clin. Chem. 23, 1142 (1977). 40. Aleyassine, H., Tonks, D. B., and Kaye, M., Natural fluorescence in serum of patients with chronic renal failure not to be confused with creatine kinase-BB isoenzyme. Clin. Chem. 24, 492 (1978). 41. McKenzie, D., and Henderson, A. R., An artifact in lactate dehydrogenase isoenzyme patterns, assayed by fluorescence, occurring in the serum of patients with end-stage renal disease requiring maintenance haemodialysis. Clin. Chim. Acta 70, 333 (1976). 42. Bostick, W. D., and Mrochek, J. E., Evaluation with the Centrifugal Fast Analyzer of a chemical activation procedure for creatine kinase MB isoenzyme. Clin. Chem. 23, 1633 (1977).
CLINICALCHEMISTRY,Vol. 24, No. 8, 1978 1413
