Capillary zone electrophoretic analysis of isoenzymes

Disclosed herein is a methodology for analyzing isoenzymes using capillary zone electrophoresis ("CZE") techniques. Briefly, an isoenzyme-containing sample and a substrate capable of being catalyzed by said isoenzyme into a reaction product are introduced into a capillary column comprising a buffer. Most preferably, the buffer contains the substrate prior to introduction of the sample into such substrate-buffer. CZE separation techniques are applied to the column such that the isoenzymes are separated from each other into discrete zones. The separation techniques are terminated such that product is rapidly generated by the catalytic conversion of substrate by the isoenzymes, and accumulated, within each discrete zone, followed by detection of product. Information regarding the relative distribution of the isoenzymes can be derived from the relative distribution of the product.

FIELD OF THE INVENTION 
The present invention is directed to the analysis of samples in general, 
the analysis of samples by capillary zone electrophoresis in particular, 
and specifically the analysis of isoenzymes by capillary zone 
electrophoresis. In a particularly preferred embodiment, the invention is 
directed to the analysis of clinical isoenzymes by capillary zone 
electrophoresis. 
BACKGROUND OF THE INVENTION 
Enzymes are proteins having catalytic properties. A "catalyst" increases 
the rate of a particular chemical reaction without itself being consumed 
or permanently altered; at the end of a catalyzed reaction, the main 
reaction products have undergone transformation into new products, but the 
catalyst appears unchanged in form and quantity. Thus the presence of a 
small number of enzyme molecules in a reaction mixture involving a 
substrate can convert a greater number of the substrate molecules to 
products. Similarly, an increase in the amount of an enzyme in a sample, 
such as, for example, a clinical sample (e.g., serum, plasma, cerebro 
spinal fluid, urine), can be detected with great specificity because of 
the unique and characteristic effect that the enzyme has on the chemical 
reaction which it catalyzes. 
Isoenzymes are two or more enzymes which catalyze the same (or similar) 
specific reactions but which have different physical properties, e.g., 
electrophoretic mobility, resistance to chemical or thermal inactivation, 
etc. Thus, while in a class of isoenzymes, each will have the same 
catalytic function, subtle, yet detectable differences between each can be 
determined such that, in a sample material, the relative distribution of 
each isoenzyme having the same catalytic function can be determined. 
Analysis of the presence of enzymes from clinical samples is diagnostically 
valuable. Specific enzymes are associated with specific tissue sources. 
For example, the enzyme lactate dehydrogenase ("LDH") is found principally 
in the heart, liver, skeletal muscle and lymph nodes. Creatine kinase 
("CK") is found principally in skeletal muscle, brain, heart and smooth 
muscle. Cholinesterase ("CHE") is found principally in the liver. When 
these tissue sources are damaged (due to internal causes, such as disease, 
or external sources, such as alcohol), there is typically a release of the 
enzyme(s) associated therewith into the blood stream. Accordingly, a 
clinical sample can be analyzed and if elevated levels of, for example, 
LDH, are identified, possible damage to the associated tissues is evident. 
LDH has five isoenzymes. It is well known that certain diseases will cause 
a change in the relative distribution of LDH isoenzymes occurring in 
serum. LDH is a hydrogen transfer enzyme that catalyzes the oxidation of 
L-lactate to pyruvate with the mediation of nicotinamide adenine 
dinucleotide (NAD+) as the hydrogen acceptor. Thus, in the presence of 
LDH, L-lactate and NAD+ will be catalytically converted to pyruvate and 
NADH. NADH production can be measured and the amount can be correlated 
with the amount of LDH in the sample. Each LDH isoenzyme has a unique 
concentration in a given sample. Similarly each isoenzyme is capable of 
catalyzing the L-lactate/NAD+ reaction. Thus each isoenzyme will produce 
different amounts of NADH by such catalysis. 
Electrophoretic separation on agarose gels or cellulose acetate is a well 
known procedure used to demonstrate the presence of LDH isoenzymes in a 
sample. Typically, a clinical sample (e.g. serum) is inserted into a well 
in the gel surface and a voltage impressed across the gel. Since each 
isoenzyme has a unique electrophoretic mobility, the voltage separates the 
isoenzymes from each other. A reaction mixture is then layered over the 
separation medium and, following sufficient incubation, the NADH generated 
over the individual LDH zones is detected (typically by fluorescence when 
the NADH is excited by ultraviolet light). Thereafter, the gel patterns 
may be read directly by observing the relative intensities of the bands, 
or scanned by, e.g., color densitometer instruments, such that relative 
peak distribution can be obtained. Such an electrophoresis system is 
commercially available from Beckman Instruments, Inc. (Fullerton, Calif., 
USA) under the trademarks APPRAISE.RTM. and AGON.RTM.. 
Many clinically significant isoenzyme product patterns have been correlated 
with certain disease states. Again focusing on LDH, a normal LDH 
distribution pattern will evidence a "darker" band (or higher peak) for 
LDH-2 vis-a-vis LDH-1, whereas a serum sample obtained from an individual 
having acute myocardial infarction will evidence a darker (or higher) 
LDH-1 band vis-a-vis LDH-2, the so-called "flipped" LDH-1. 
As noted, isoenzymes have different physical properties, e.g., different 
electrophoretic mobilities. Accordingly, isoenzymes lend themselves to 
analysis by capillary zone electrophoresis ("CZE"). CZE is a technique 
which permits rapid and efficient separations of charged substances. In 
general, CZE involves introduction of a sample into a capillary tube, 
i.e., a tube having an internal diameter from about 5 to about 2000 
microns, and the application of an electric field to the tube. The 
electric potential of the field both pulls the sample through the tube and 
separates it into its constituent parts. Each constituent of the sample 
has its own individual electrophoretic mobility; those having greater 
mobility travel through the capillary tube faster than those with slower 
mobility. As a result, the constituents of the sample are resolved into 
discrete zones in the capillary tube during their migration through the 
tube. An on-line detector can be used to continuously monitor the 
separation and provide data as to the various constituents based upon the 
discrete zones. 
CZE can be generally separated into two categories based upon the contents 
of the capillary columns. In "gel" CZE, the capillary tube is filled with 
a suitable gel, e.g., polyacrylamide gel. Separation of the constituents 
in the sample is predicated in part by the size and charge of the 
constituents travelling through the gel matrix. In "open" CZE, the 
capillary tube is filled with an electrically conductive buffer solution. 
Upon ionization of the capillary, the negatively charged capillary wall 
will attract a layer of positive ions from the buffer. As these ions flow 
towards the cathode, under the influence of the electrical potential, the 
bulk solution (the buffer solution and the sample being analyzed), must 
also flow in this direction to maintain electroneutrality. This 
electroendosmatic flow provides a fixed velocity component which drives 
both neutral species and ionic species, regardless of charge, towards the 
cathode. Fused silica is principally utilized as the material for the 
capillary tube because it can withstand the relatively high voltage used 
in CZE, and because the inner walls of a fused silica capillary ionize to 
create the negative charge which causes the desired electroendosomatic 
flow. 
The inner wall of the capillaries used in CZE can be either coated or 
uncoated. The coatings used are varied and known to those in the art. 
Generally, such coatings are utilized in order to reduce adsorption of the 
charged constituent species to the charged inner wall. Similarly, uncoated 
columns can be used. In order to prevent such adsorption, the pH of the 
running buffer, or the components within the buffer, are manipulated. 
While the different electrophoretic mobilities of isoenzymes suggests the 
applicability of CZE analytical techniques, a practical problem exists as 
to the parameters of such testing. This is because in order to determine 
the presence of isoenzymes in a sample, it is necessary to rely upon their 
catalytic activity relative to a substrate. Thus, in the electrophoresis 
system described above, separation of the isoenzymes must take place 
before addition of the substrate. However, this is not possible with CZE 
because there is presently no practical way to "add" the substrate to the 
sample after the constituent parts thereof have been separated. 
What is needed, then, is a method for analyzing isoenzymes that exploits 
the speed and accuracy of CZE techniques. 
SUMMARY OF THE INVENTION 
The present invention satisfies at least the above need by providing a 
capillary zone electrophoresis method for the analysis of isoenzyme 
constituents to be separated comprising the steps of: 
a) introducing an isoenzyme-containing sample and a substrate capable of 
being catalyzed to a reaction product by said isoenzymes into a capillary 
tube containing therein a buffer; 
b) subjecting said sample to capillary zone electrophoresis techniques by 
applying an electric field to said capillary tube of sufficient voltage to 
allow for the separation of said sample into its isoenzyme constituent 
parts; 
c) terminating the application of said electric field; 
d) allowing a sufficient period of time to transpire to permit said 
isoenzymes to catalytically convert said substrate to a reaction product; 
e) moving said reaction product through said tube to a detection region; 
and 
f) detecting said reaction product. 
Most preferably, the substrate is included within the buffer prior to 
introduction of the sample into the capillary tube. In accordance with the 
present in invention, the isoenzymes in the sample are separated from each 
other using CZE techniques, followed by termination of the electric field. 
This separates the isoenzymes in the sample into discrete zones within the 
buffer. As such, product formation arising from the catalytic conversion 
of the substrate by the isoenzymes will take place within these discrete 
zones. Application of an electric field following this period allows these 
product zones to continue moving through the capillary to a detection 
region, where the product zones are resolved by, e.g., peaks of various 
widths and height. By analyzing such peaks, a clinician can determine the 
relative distribution of each isoenzyme within the sample such that 
clinical evaluations can be obtained.

DETAILED DESCRIPTION OF PREFERRED EMBODIMENTS 
Typically, extremely small quantities of an enzyme are all that is needed 
to catalyze a Substrate (S).fwdarw.Product (P) reaction. Thus, while an 
enzyme itself may not be detectable (due to the small quantities typically 
present in a sample), the amount of reaction product catalyzed by the 
enzyme can be detected and used as an indicator of the relative amount of 
enzyme present in the sample. The catalytic activity of an enzyme, in the 
presence of substrate, is nearly instantaneous. Accordingly, product will 
be created rapidly until either the reaction is terminated, substrate 
depletion occurs (i.e. all of the substrate is converted to product) or 
the reaction is reversed (i.e. product is converted to substrate by 
manipulation of, e.g., the reaction medium pH, temperature or conditions). 
In order to effectively measure the presence of specific isoenzymes in a 
sample based upon the conversion of substrate into reaction product, it is 
necessary to separate the isoenzymes from each other before addition of 
substrate. State again, heretofore if substrate and isoenzymes were 
co-mingled before separation of the isoenzymes, product would be rapidly 
produced, thus making the desired "separation" unattainable. 
Applicants have discovered, however, that the isoenzyme-containing sample 
and a buffer-containing substrate can be co-mingled "prior" to separation 
via CZE techniques by utilization of the present methodology. The present 
methodology is initiated by introducing an isoenzyme-containing sample and 
a substrate capable of being catalytically converted to a reaction product 
by said isoenzymes into a capillary column comprising a buffer. Most 
preferably, the substrate is included within the buffer ab initio. Sample 
introduction can be accomplished by, e.g., the electrokinetic injection 
method (i.e., where a short application of an electric field is applied to 
the column such that a sample "plug" is drawn into the capillary column) 
or by pressure injection (i.e., where pressure is used to "drive" a sample 
plug into the capillary column). Those skilled in the art will fully 
understand these two injection approaches; either may be used with the 
present invention. Reference is now made to FIG. 1 for elucidation of the 
methodology. 
FIG. 1A represents the post-sample-plug introduction into the capillary 
column. The sample-plug 10 is pulled into the column 100, where it 
co-mingles with the buffer comprising substrate 50; this leads to the 
catalytic conversion of substrate into product 20 within the region of the 
plug. 
Following introduction of the plug into the capillary, the isoenzyme 
constituents are separated based upon their individual electrophoretic 
mobilities. This is represented in FIG. 1B where three isoenzymes zones, 
12, 14 and 16, are separated from one another. Within each zone, some 
product will be formed during the traverse of the zones through the 
column; to the extent that the isoenzymes have a faster (relative) 
electrophoretic mobility than the product originally formed during initial 
sample introduction, then such original product will, in effect, proceed 
behind the isoenzymes. 
As those skilled in the art appreciate, one can achieve faster speeds 
through a capillary by increasing the voltage applied thereto, and 
vice-versa. In the presently disclosed protocol, however, a period of time 
is required where the sample is not traversing the column. During this 
so-called "park" period, reaction product is rapidly accumulated within 
the separated zones. Thus, the application of the electric field to the 
column, and the time of such application, cannot be such that the 
separated sample constituents will reach the detection area of the CZE 
instrument prior to the required "park" period. By increasing or 
decreasing the voltage applied to the column, the time of sample 
transition can be adjusted such that the park period can be initiated 
prior to a point where sample has reached the detection region of the 
instrument. Most preferably, the park period begins when the sample is 
approximately half-way through the column, although this is not critical. 
What is critical is that the park period must begin before any isoenzyme 
sample has reached the detection region. 
Reference is now made to FIG. 1C, which illustrates the park period. During 
this period, the applied voltage is terminated--this has the effect of 
substantially stopping the flow of all materials through the capillary 
column. I.e., each separated isoenzyme is "parked" in a specified zone. 
This, then, allows for the catalytic conversion of substrate into product 
within these discrete zones. During the park period, product is rapidly 
produced and accumulated within these zones. 
Another factor which is of import to the park period is the time for 
parking. The amount of time for parking is dependent upon the amount of 
detectable reaction product likely to be produced within the discrete 
isoenzyme zones. This is a function of at least two factors, the amount of 
substrate available, and the physiological amount of the enzyme available. 
The park time is limited principally by the amount of substrate present in 
the buffer via the affects of substrate depletion. I.e., if the park time 
is too long and there is insufficient amount of substrate in the buffer, 
the Substrate.fwdarw.Product reaction will terminate. Accordingly, there 
should be sufficient substrate within the buffer to prevent substrate 
depletion. This can be avoided by adding at least twice the K.sub.m value 
for a given substrate to the buffer, where K.sub.m is equal to the 
substrate concentration at which the initial reaction rate is half 
maximal. Such K.sub.m values can be determined using the techniques 
described in, for example, Biochemistry, 2nd Ed. Chpt. 8, p. 193, 
Lehninger, Ed. (Worth Publishers, Inc. 1975). Preferably, at least three 
times K.sub.m is used, and most preferably at least five times K.sub.m. 
For example, the K.sub.m for lactate is about 10.0 mM in an LDH enzyme 
system; most preferably, then, the concentration of lactate in the buffer 
is about 50.0 mM. 
Detectable product is of import because it is necessary to have a 
product-signal to noise (or "background") ratio of greater than at least 
about 2:1, preferably greater than about 50:1 and most preferable greater 
than about 300:1. While background noise varies typically from instrument 
to instrument, a level of about 100 .mu.A is achievable and acceptable. 
The signal is also predicated, in part, by the unit activity of the 
enzyme. The unit activity of the enzyme (designated herein as "U"), is a 
relative value given to the enzyme based upon specific operating 
conditions. For example, at 25.degree. C., pH 7.2, the unit activity for 
the NADH.fwdarw.NAD.sup.+ conversion in an LDH enzyme reaction is 1.0; at, 
25.degree. C., pH 8.7, the unit activity of NAD.sup.+ .fwdarw.NADH 
conversion is approximately 0.1. Specific unit activity values for various 
enzymes is typically determined by the vendor and printed on the label. As 
such, these values are considered to be within the purview of the skilled 
artisan. 
In order to determine a minimum park time period, the following 
transformant can be utilized (throughout this disclosure, the symbol 
".cndot." represent a multiplication symbol): 
##EQU1## 
where: A is the desired absorbance value of the product; 
K is the molar extinction coefficient of the product; e.g., the absorbance 
value of 1.0M of product across a 1.0 cm path length at the maximum 
absorbance wavelength of the product; 
L is the measuring path length through the capillary (e.g., the internal 
diameter of the capillary tube); 
U is the unit activity value of the enzyme, at temperature T under 
specified conditions; 
IU is a constant, 10.sup.-6 /min, the enzyme activity necessary to convert 
one micromole of substrate to product, per minute, at temperature T; 
MWp is the molecular weight of the product; 
T.sub.1 is the running temperature of the system; and 
T is the enzyme calibration temperature. 
Typically, T is equal to 25.degree. C. The molar extinction coefficient of 
particular species are readily determined and are considered to be within 
the purview of the skilled artisan. 
The catalytic activity of enzymes are typically affected by a variety of 
factors, e.g., temperature and pH. For example, by increasing the 
temperature of the reaction mixture, the kinetics of the catalytic 
reaction will increase, and by lowering the temperature, the kinetics 
decrease. For the majority of enzymes, such kinetics will increase up to 
about 47.degree. C., at which point such temperatures have a deleterious 
affect upon, inter alia, the enzyme. Generally, adjusting the pH of the 
reaction mixture can drive the catalytic reaction either forward 
(S.fwdarw.P) or backwards (P.fwdarw.S). Accordingly, it should be readily 
apparent that notwithstanding the transformant of Equation 1, the minimum 
park time can be decreased by, e.g., increasing the running temperature, 
T.sub.1. This is of import in analytical settings where the time of 
analysis is a consideration. 
Referring to FIG. 1D, after the park period, voltage is again applied to 
the capillary column sufficient to move the product through the column to 
the detection region. Alternatively, external pressure (i.e., as in the 
pressure-injection protocol), can be applied to the column to effectuate 
the same results. As noted, once the substrate and the enzyme interact 
(e.g., upon injection of the sample into the capillary), product will be 
formed. Thus, there will be a region of product, typically behind the 
separated isoenzyme zones, which will be detected. This region, depicted 
as 30 in FIG. 1D, is referred to herein as a "sample shock". The sample 
shock is generally comprised of the majority of product formation 
throughout the analysis but is not attributed to the discrete isoenzyme 
zones; it is attributed to the non-separated isoenzymes in toto. This 
region is typically segregated visually upon review of the resulting 
electropherogram due to the higher (relative) peak attributed thereto 
vis-a-vis the product peaks resulting from the isoenzymes. 
EXAMPLES 
The following examples directed to preferred embodiments of the invention 
disclosed herein are not intended, nor should they be construed, as 
limiting the disclosure, or the claims to follow: 
I. MATERIALS AND METHODS 
A. Capillary Electrophoresis Procedures 
Capillary electrophoresis of samples was performed on a Beckman 
Instruments, Inc. high performance capillary electrophoresis system 
(Beckman Instruments, Inc., Fullerton, Calif., U.S.A., Model No. 357575). 
Data analysis was performed on System Gold.TM. software (Beckman 
Instruments, Inc.). The aforementioned capillary electrophoresis system 
contains built-in 200, 206, 214, 280 and 340 nm narrow-band filters for 
on-line detection and quantification. Electrophoresis was performed in 
uncoated fused silica tubes having 75 .mu.m i.d. and 25 cm long (Polymicro 
Technologies, Phoenix, Ariz. Product No. TSPO 75375). Prior to analysis, 
the column was filled with Substrate Buffer. The detection window is 
located approximately 6.5 cm from the column outlet. 
LDH Isoenzyme Samples were placed on the inlet tray and introduced into the 
capillary by the electrokinetic method by applying 3 kV to the column for 
about 1 second. Isoenzymes were separated using a column voltage gradient 
of about 300 volts/cm for about 2 minutes. After the park period, the 
product was moved to the detection window using a voltage gradient of 
about 300 volts/cm. 
For the Examples, the resulting product, NADH, was detected at 340 nm. 
Analysis was conducted at ambient temperature (25.degree. C.). 
B. Substrate Buffer 
All chemicals were at least of ACS grade. As noted, it is most preferred 
that the substrate be included within the buffer and that such substrate 
buffer be within the capillary column prior to the introduction of sample 
to the column. 
The Substrate Buffer utilized was Beckman Dri-Stat.RTM. LD-L Reagent 
(Beckman Instruments, Inc., part no. 270230132-A) which was diluted 1 part 
reagent to 4 parts distilled water. To 80 ml of the diluted reagent was 
added 329 mg NAD.sup.+ and 278 .mu.l of Lactic Acid, which are substrates 
for the LDH isoenzymes. The pH of the Substrate Buffer was adjusted to 8.7 
by drop-wise addition of 1N NaOH. 
C. LDH Isoenzyme Samples 
The LDH Isoenzyme Samples comprised tris-hydroxymethyl amino methane 
buffer, pH 8.4, to which was added 1.0 IU/ml each of the five purified 
human LDH isoenzymes, designated herein as LD-1, LD-2, LD-3, LD-4 and LD-5 
(Sigma Chemical Co., St. Louis, Mo., part nos. L-3632; L-3757; L-3882; and 
L-6508 for LD-1; LD-2; LD-3; and LD-5, respectively; Aalto-Scientific 
Ltd., San Diego, Calif., part no. LD-4, P for LD-4), and 10.0 IU/ml each 
of LD-1 and LD-5 purified bovine isoenzymes (Boehringer Mannheim, GmbH, 
Germany, part nos. 106 984 and 106 992, respectively). 
II. EXAMPLES 
Example I-Park Period Minimum 
To evaluate minimum park periods, an analysis was conducted using the 
bovine LD-1 and LD-5 isoenzymes (hereinafter BLD-1 and BLD-5, 
respectfully). Under the experimental conditions described herein, the 
enzyme has approximately one-tenth of the unit activity in the S.fwdarw.P 
direction (for the specified bovine LD, the desired direction of the 
system is P.fwdarw.S, and in this direction, the unit value is 10). 
Accordingly, the value for U is 1. A desired absorbance value for the 
aforementioned system is between about 0.002 and about 0.006 Absorbance 
Units; for this Example, a desired value for A was 0.005. The molecular 
weight of NADH is 665.4, and the molar extinction coefficient for NADH is 
6300. The measuring path length for the aforementioned system was 0.0075 
cm. Thus, a predicted minimum park time is calculated as follows (for ease 
of presentation, unit values are not included): 
##EQU2## 
Thus, a predicted minimum park time necessary to achieve a definitive 
signal to noise ratio for this system is about 90 sec. Reference is now 
made to FIGS. 2-5. 
The electropherogram of FIG. 2 was generated with a 0 second park time, 
i.e., the analysis continued uninterrupted from introduction of the sample 
through detection. As is apparent, the resolution between the peaks and 
the sample shock ("SS") is less than ideal, as would be expected under 
these conditions. In FIG. 3, a park period of 60 seconds was utilized, 33% 
less than the predicted minimum. While the resolution is an improvement 
over that of the no-park condition, it is not ideal. 
The electropherogram of FIG. 4 was achieved using a 90 second park period. 
In this electropherogram, the resolution is improved over that of FIG. 3, 
particularly between the BLD-5 and SS peaks. Accordingly, by following the 
predicted minimum park period for this system of 90 seconds, good 
resolution between the peaks was achieved. 
The electropherogram of FIG. 5 was achieved using a 180 second park period, 
twice that of the predicted minimum. Note that the resolution thereof is 
substantially similar to that of the resolution obtained in the 
electropherogram of FIG. 4. This too would be expected in that once a 
minimum park period is utilized for obtaining a desired absorbance peak, 
the amount of product produced does not dramatically affect the 
resolution. 
The foregoing indicates that the park time minimum transformant of Equation 
1 can be utilized to accurately determine a minimum park time necessary to 
achieve a sufficient signal for between-peak resolution and 
signal-to-noise ratio. 
Example II-Procedure Validation 
For Example II, the human LDH Isoenzyme Sample was utilized. For this 
example, about 0.003 Absorbance Units was desired. All other values were 
the same as in Example 1, except that U was 0.1 (one-tenth of the 
specified P.fwdarw.S value of 1.0). Accordingly, a predicted minimum park 
time is derived as follows: 
##EQU3## 
Note that if it was desirable to decrease this minimum park period, 
T.sub.1 (running temperature) could be increased. For example, if T.sub.1 
was 37.degree. C., then t.sub.min =0.67 minutes. 
As noted, t.sub.min is a minimum park period. Thus, it is possibl to 
increase this period of time, provided that substrate depletion does not 
occur. This can be prevented by utilizing at least twice the K.sub.m 
amount for the substrate. For these examples, approximately five times the 
value of K.sub.m was utilized, thus ensuring that the park time could be 
significantly increased without substrate depletion. Thus, the "limiting" 
factor on the park period, under these parameters, is primarily a function 
of the desired time for each analytical run. This time period can vary 
substantially, depending on the arena of analysis. In a clinical setting, 
this period can be dictated by the particular needs of the investigator. 
For Example II, the actual park period utilized was well in excess of the 
predicted minimum, i.e., about 10 minutes. The electropherogram results 
for the analysis are presented in FIGS. 6 and 7. 
As those in the art appreciate, CZE electrophoresis represent a 
"first-in-last-out" arrangement such that the first constituent to be 
detected, designated in FIGS. 6 and 7 as "LD-1", appears to be the last 
zone to be analyzed. The relevance of this is with respect to the "first" 
peak, designated as "SS" for "sample shock". As noted, immediately upon 
injection of the LDH Isoenzyme Sample into the Substrate buffer, the 
catalytic reaction begins, such that NADH is formed. This, of course, 
takes place irrespective of separation of the isoenzymes. As the 
substrate, enzymes and product proceed through the capillary, separation 
of the isoenzymes will begin such that defined "fronts" develop. 
Accordingly, the faster (relative) isoenzymes will react with substrate in 
regions different than slower isoenzymes. As demonstrated in Example I, 
without "parking" the sample, a continuum of product will form, defined by 
a generally small front caused by the faster isoenzymes through to 
generally larger front caused by the slower isoenzymes. This phenomenon is 
represented by the dashed line of FIG. 6 and comports with a consideration 
of the electrophoretic mobilities of isoenzymes as they simultaneously 
travel and catalyze the Substrate.fwdarw.Product reaction. As should be 
appreciated, merely advancing the isoenzyme-containing sample through the 
substrate buffer will not, in and of itself, evidence useful data relative 
to the isoenzyme relationships, as is shown by the dashed line portion of 
FIG. 6. 
By "parking" the LDH Isoenzyme Sample-Substrate Buffer solution, however, 
the separated isoenzymes will principally catalyze the 
Substrate.fwdarw.Product reaction in the defined isoenzyme zones such that 
product, in this case NADH, is rapidly generated at the locus of each 
isoenzyme-separated fragment. Therefore, product is accumulated within 
these zones. Thus, when "parking" is discontinued, the concentrated 
product zones and the sample shock continuum continue through the 
capillary and are detected as a Sample Shock continuum including peaks 
which represent the effect of concentrated product production within the 
discrete zones. Accordingly, and as is shown in FIG. 6, it is possible to 
avoid the product-continuum via the methodology disclosed herein--good 
separation of the NADH product was achieved relative to each of the five 
LDH isoenzymes in the LDH Isoenzyme Sample. 
For the LDH Isoenzyme Sample, following electrokinetic injection, 7.5 kV 
was applied to the column for 2 minutes. This voltage and period of time 
is sufficient to separate the isoenzymes, although higher or lower 
voltages can be used in conjuction with shorter or longer, respectively, 
periods of time. 
After the 2 minute separation period, the voltage was terminated, i.e., 
initiation of the "park" period began. Thus, the separated isoenzymes 
were, in essence, stationary within discrete zones. As noted, these zones 
are co-mingled with Substrate Buffer so that within each zone, product is 
generated based upon the catalytic reaction taking place within each zone. 
Following the park period, 7.5 kV was applied to the capillary such that 
the product moved from the stationary discrete zones to the detection 
window. Because the final "peak" (i.e., the sample shock) was detected 
within 4 minutes of injection, the total analytical time, including the 
park period, was about 14 minutes. 
Another observation derived from FIG. 6 is that after the 10 minute park 
period, product diffusion is not a limitation under these parameters. This 
is of value because the park period utilized was ten times that of the 
minimum park period value derived. 
The electropherogram of FIG. 7 was derived from the same data which led to 
the electropherogram of FIG. 6. In essence, the dashed-line portion of 
FIG. 6 has been subtracted from the Figure; this portion, as noted, is 
attributed to total sample shock over the course of the entire analytical 
run. Thus; by removing this portion from the electropherogram, FIG. 7 
represents a baseline corrected analysis of the 5 LDH isoenzymes. 
An alternative approach to the termination of the electric field to 
effectuate the park period is utilization of a "reverse pulse" protocol. 
Under such a protocol, the polarity of the CZE system is reversed, such 
that the discrete zones are pulled in a reverse direction back towards the 
area of sample injection. The reverse pulse protocol can be utilized in 
situations where it is desirable to utilize park periods in excess of 
about 60 minutes, or where product diffusion from the discrete zone may be 
a concentration. I.e., by simultaneously "pulling and pushing" the zones 
through the capillary, the zones do not remain in a discrete region during 
the product formation period. Methodologies for reversing the polarity of 
CZE systems are known to those skilled in the art and are not set forth in 
detail herein. 
The foregoing data demonstrates that isoenzymes can be analyzed in a CZE 
format utilizing the methodology disclosed herein. It is to be understood 
that the foregoing Examples are not to be construed as limiting the 
invention. The disclosed methodology is applicable to isoenzyme systems 
other than those set forth in the Examples. Additionally, the methodology 
is not limited to the specified high performance capillary electrophoresis 
system utilized herein. Furthermore, the methodology is not to be 
construed as limited to analysis of clinical samples in that the 
methodology has broader applications to non-clinical samples comprises 
enzymes and/or isoenzymes. As such, the foregoing Detailed Description and 
Examples are not intended, nor are they to be construed, as a limitation 
on the disclosed methodology or the claims to follow. Modifications and 
equivalents which are within the purview of the skilled artisan are 
considered to be included within the scope of the invention as claimed.