Note: Descriptions are shown in the official language in which they were submitted.
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Background of the Invention
1. Field of the Invention
This invention pertains to a fluorometric method for
determining the individual activities of isoenzymes and to an
5. electrophoretic method for separating creatine phosphokinase.
2. Description of the Prior Art
Fluorometric techniques for determining the presence of
isoenzymes are well known to those skilled in the art. A. L.
Sherwin et al., "Fluorescent Technique to Demonstrate Creatine
10. Phosphokinase Isozymes", Clinica Chemica Acta, 17: 245 to 249
(1967). The standard fluorometric technique employed by those
skilled in the art entails detecting the presence of the NADH,
NADPH, derivatives thereof, and mixtures thereof (hereinafter re-
ferred to as the "fluorometric product") in an electrophoretic
15. medium. H. Somer et al., "Demonstration of Serum Creatine Kinase
Isoenzyme by Fluorescence Technique", Clinica Chemica Acta, 40:
133 to 138 (1972); D. W. Moss et al., "Characterization of Tissue
Alkaline Phosphatases and their Partial Purification by Starch-Gel
Electrophoresis", Biochem. J., 81: 414 to 447 (1961); and F. R.
20. Elevitch, "Fluorometric Techniques in Clinical Chemistry", Little,
Brown and Company, Boston, Mass., (1973). Several problems are
inherent in detecting the fluorometric product in the electro-
phoretic medium. These problems include the presence of an albu-
min artifact (J. Goldberg, "Enzymes in Medicine", Vol. 7, No. 3,
25. 526 (1973); Helena Update, Vol. 18 (August, 1973); and R. Wong et
al., "Cellulose Acetate Electrophoresis of Creatine Phosphokinase
Isoenzymes in the Diagnosis of Myocardial Infraction", A. J. C.
P., 64: 209 to 216 (1975); the presence of beta-lipoprotein arti-
facts (C. R. Rowe et al., "Combined Isoenzyme Analysis in the
30. Diagnosis of Myocardial Injury: Application of Electrophoretic
Methods for the Detection and Quantitation of the Creatine Phos-
phokinase MB Isoenzyme", J. Lab. Clin. Med., Vol. 8, No. 4, 577
2 ~.
``` 1048910
to 590 (1972); the interference in fluorometric detection by
drugs (F. R. Elevitch, supra); and inaccuracies caused by the
diffusion of bands (Rowe et al, supra, and Wong et al, supra).
Another problem present in prior art fluorometric methods for
detecting fluorometric products is the well known fading problem,
i.e., the disappearance of some of the fluorescence of the bands
during drying. See H. Somer, et al, supra.
The detection of isoenzyme products in the substrate
medium is well known in the art of colorimetry. See S.S. Kind,
"Stable Test-Papers for Seminal Acid Phosphatase", Nature, Vol.
182, No. 4646 (Nov. 15, 1958); J. Clausen, "Immunochemical Tech-
niques for Identification and Estimation of Macromolecules",
North-Holland Publishing Co., Amsterdam (1969), and G.J. Bremer
and C.F. Singer, "An Introduction to Isoenzyme Technigues",
Academic Press, New York, N.Y. Fluorometric methods wherein
fluorometric products are detected in the substrate medium are also
known. See Ger. 2,136,880.
The diffusion and fading problems have both been a
significant defect in fluorometric determinations of isoenzymes
since the introduction of the fluorometric method for detecting
isoenzymes in about 1972.
The present invention has overcome the diffusion
problem via the discovery that by precipitating the fluorescent
product in filter paper or other suitable substrate medium, the
bands are localized and the sensitivity of the fluorometric
technique thereby increased. Further, the instant invention also
encompasses the discovery that the fading phenomenum can be
greatly curtailed by dehydrating the substrate medium via solvent
replacement.
SUMMARY OF THE INVENTION
-
In one particular aspect the present invention
provides an improved electrophoretic technique for separating
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creatine phosphokinase into its constituent isoenzymes of the type
wherein a sample containing creatine phosphokinase is applied to
an electrophoretic medium, said electrophoretic medium is placed
into an electrophoretic cell having located therein an electro-
phoretic buffer in contact with two electrodes, and said isoenzymes
are separated by applying a direct electrical current to the electro-
phoretic medium, wherein the improvement comprises using as said
electrophoretic buffer one comprising (a) an acid having a formula
HOOC(CH2) CH(NH2)C00H wherein n is an integer from about 1 to about
4, (b) tris(hydroxymethyl)aminomethane, and (c) water, said
electrophoretic buffer having a pH range of about 7 to about 8.5
at 23C and an ionic strength of about 0.02 to about 1.5.
BRIEF DESCRIPTION OF THE DR~I~ING
Figure 1 is a comparison of the electrophoretic
separation of creatine phosphokinase obtained when using a tris-
aspratate buffer vs. a barbital buffer.
DESCRIPTION OF THE PREFERRED EMBODIMENTS
The method of the present invention can be used to
measure the concentration of any isoenzyme capable of directly
reacting to produce a fluorescent product or of being part of a
reaction sequence which produces a fluorescent product. Exemplary
enzymes having isoenzymes capable of fulfilling the above require-
ment include creatine phosphokinase, lactate dehydrogenase,
jl/ -4-
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glucose-6-phosphate dehydrogenase, hexokinase, malate dehydroge-
nase, isocitrate dehydrogenase, aldolase, and alcohol dehydroge-
nase. Because of their common use in clinical laboratories, crea-
tine phosphokinase and lactate dehydrogenase are preferably used
5. in the present invention.
The general analysis of isoenzymes can be broken down
into three basic steps. Step 1 is the placement of the sample
containing an enzyme onto a support means and physically separat-
ing the enzyme into its various isoenzyme components by any of the
10. various separation means well known to those skilled in the art.
Step 2 is the location of each isoenzyme component. This consists
of a set of chemical reactions catalyzed by the isoenzyme compon-
ents to produce a detectable chemical product, such as a fluores-
cent product, the quantity of which is in proportion to the amount
15. of isoenzyme present. Step 3 is a quantitation of each isoenzyme
component. This is accomplished by scanning either the support
means or the substrate means with a fluorometer or densitometer
to quantitate the chemical product produced by each isoenzyme
component.
20. Various means of separating isoenzymes are well known
to those skilled in the art. These methods include gel eIectro-
phoresis (commonly referred to simply as "the electrophoretic
separation method"), thin-layer gel chromatography, thin-layer
electrophoresis, and thin-layer isoelectric focusing. The tech-
25. nique most commonly used in clinical laboratories at present, and
therefore the technique most preferably used with this invention's
improved method for detecting fluorometric products, is the elec-
trophoretic separation method. In general, the electrophoretic
separation of isoenzymes is accomplished by applying a sample,
30. such as human serum, to an electrophoretic medium. The electro-
phoretic medium is placed into a chamber known as an eIectro-
phoretic ceIl which contains an aqueous buffer known as an
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electrophoretic buffer. The electrophoretic medium is placed in
direct contact with the electrophoretic buffer and in turn the
electrophoretic buffer is in direct contact with two separate
electrodes (a cathode and an anode). The electrodes are connected
5. with a direct current power source whereby the electrphoretic
process takes place when direct current is applied to the elec-
trophoretic medium. In order to adequately separate the various
isoenzyme constituents, the electrophoretic process usually takes
place at an established electrical potential, for example, from
10. about 50 to about 500 volts, and for a sufficient length of time,
for example, from about 0.1 to about 2 hours.
The location of each isoenzyme component requires con-
tacting the separated isoenzymes with reagents capable of reacting
to produce a detectable product. One method well known to those
15. skilled in the art encompasses contacting the separated isoenzymes
with a substrate medium containing said reagents. The substrate
medium is prepared by incorporating the specific chemical sub-
strate required for the specific isoenzyme system into a stabiliz-
ing medium such as filter paper, chromatography paper, cellulose
20. acetate membrane, agarose gel, etc. The preferred stabilizing
medium for use in the present invention is filter paper and chro-
matography paper, the latter being the stabilizing medium of
choice. To the stabilizing medium is usually added a substrate
buffer, metal ions as activators, specific reactants or sub-
25. strates, and a co-enzyme, i.e., NAD, NADP, derivatives thereof,
and mixtures thereof, which serves as a fluorogenic substrate.
After electrophoresis, the electrophoretic medium, as
weIl known to those skilled in the art, is placed into a chamber
and the substrate medium is placed in direct contact with the sur-
30. face of the electrophoretic medium. The eIectrophoretic and sub-
strate media are incubated at a predetermined temperature and
time. Although the incubation temperature and time are not
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critical, the incubation process used in the present invention
preferably takes place at a temperature of about 2Q to about
50C., more preferably at a temperature`of about 35 to about 39C.
and preferably for a period of time from about 30 to about 90
minutes and more preferably for a period of time from about 45 to
about 75 minutes. During incubation, the chemical substrate dif-
fuses into the electrophoretic gel where the isoenzyme reacts
upon the substrate to produce, i.e., catalyze, the formation of
specific fluorometric products. The fluorometric products formed
10. in the zones where isoenzymes are located are free to diffuse
back into the substrate medium. Proteins, lipids, and bound
drugs do not diffuse any appreciable amount into the substrate
medium because of their large physical size, i.e., molecular
weight. After termination of the incubation phase, the substrate
15. medium is separated from the electrophoretic medium.
Detection of the fluorescent product is accomplished by
exciting the fluorescent product with ultraviolet light in the
range of 300 to 400 nm with maximum absorbance at 340 to 350 nm.
The fluorescent product fluoresces with a visible light at 400 to
20. 500 nm with a maximum fluorescence about 460 nm. Detection of the
visible fluorescent light can be done visually with a fluorescent
densitometer or a fluorometer. Another detection technique which
can also be employed comprises cutting out the areas of the sub-
strate medium which contain the fluorescent product and placing
25. each section into an aqueous solution to redissolve and elute the
fluorescent product from the substrate medium. Once this has
taken place the eluted fluorescent product can be measured by ab-
sorbance, fluorescence, or by a chemical assay method. The a-
mount and position of the fluorescent product detected is pro-
30. portional to the isoenzymes located in the~electrophoretic mediumand therefore to the amount of isoenzymes in the sample being
assayed.
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Two other exemplary methods for detecting the fluoro-
metric product are the photographic reproduction technique and
the photographic negative scanning technique. The former is a
qualitative method which involves visually examining a picture
5. taken of the substrate medium. The latter is a quantitative
method which entails densitometrically analyzing the negative
of a photograph taken of the substrate medium.
The improved fluorometric detection technique within
the scope of the present invention entails modifying the above
10. isoenzyme analysis steps so that prior to the fluorometric detec-
tion step the fluorometric product is precipitated in situ in the
substrate medium. The precipitation of the fluorescent product
can be brought about by the use o~ a reagent selected from a group
consisting of a composition comprising from about 90 to 100, pre-
15. ferably 100, weight percent alcohol per unit volume, the alcoholcontaining from one to six, preferably from one to three, carbon
atoms, and from zero to 10, preferably zero, weight percent urea
per unit volume; ketones containing from three to eight carbon
atoms, preferably from three to five carbon atoms; an inorganic
20- salt solution comprising from about 90 to about 99.9, preferably
from about 98 to about 99, percent of a solvent selected from a
group consisting of water and inert polar organic solvents, pref-
erably from the group consisting of alcohol containing one to six
carbon atoms and ketones containing three to eight carbon atoms,
25- and more preferably from the group consisting of alcohols con-
taining one to three carbon atoms, and from about 0.01 to about
10, preferably from about 1 to about 2, percent of an inorganic
salt having a formula R(Y)2, wherein R is selected from the group
consisting of Pb , Ca 2, Sr 2, and Ba 2 and wherein Y is selected
30. from a group consisting of Cl , N03 , and C104 ; and mixtures
thereof. Other exemplary inert polar organic solvents include
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dimethylformamide and dimethylacetamide. The sole criteria that
the inert polar organic solvents must satisfy is that they must
be able to solubilize the inorganic salt. The preferred reagent
is isopropanol. The above reagents can be used along with a poly-
5- mer addition, e.g., polypropylene glycol, polyethylene glycol,
etc., to enhance precipitation and control surface characteris-
tics (i.e., to prevent warping or wrinkling) of the substrate
medium.
It is also desirable to dehydrate the substrate medium
10. to increase the intensity of the fluorescence of the fluorescent
product. The dehydration can be accomplished by evaporation of
the water from the substrate medium or preferably by solvent re-
placement such as alcohol and ether replacement of water.
To eliminate the previously referred to undesirable
15. fading phenomenum, it is preferred to combine the precipitation
and dehydration into a one step process by placing the substrate
medium into alcohol for a short period of time. At this point
the fluorescent product can be detected by fluorescence, but to
aid handling, the alcohol is preferably allowed to evaporate which
20- results in a dried substrate medium containing a localized, rela-
tively non-fading, fluorescent product.
Although there are many suitable electrophoretic buf-
fers known to those skilled in the art which can be used in the
electrophoretic isoenzyme separation step described above, when
25- one is electrophoretically separating creatine phosphokinase into
its constituent isoenzymes it is preferred to use a particular
electrophoretic buffer comprising (a) an acid having a formula
HOOC(CH2)nCH(NH2)COOH wherein n is an integer from about one to
about four, preferably from about one to about two, (b) tris-
30- (hydroxymethyl)aminomethane, and (c) water, preferably distilled
or deionized water and mixtures thereof. This electrophoretic
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buffer has a pH of about 7 to about 8.5, preferably about 7.2 to
about 7.7, and more preferably 7.5, at 23C., and an ionic
strength of about 0.02 to about 1.5, preferably about 0.03 to
about 0.07, and more preferably about 0.05.
5- There are several reasons why the aforesaid electropho-
retic buffer is preferred for use in electrophoretically separat-
ing creatine phosphokinase isoenzymes. One reason is that the
use of barbital buffers, or other buffers similar to barbital
buffers, produce an application artifact. The application arti-
10. fact results from the creatine phosphokinase enzyme known as CPK3
being electrophoretically located at the trench sample applica-
tion point, thereby producing an outline of the application area
when the electrophoretic medium is scanned densitometrically.
Also, these prior art buffers electrophoretically separate the
15. creatine phosphokinase isoenzyme known as CPKl into an area where-
in serum albumin is present. This type of separation interferes
with the enzymatic action of the CPKl isoenzyme. B. A. Nealson
et al., J. Clin. Pathol., 28 (10): 834 to 836 (1975). Further,
the pH of prior art buffers is in the range of about 7.5 to 9,
20- while the optimum pH for the determination of creatine phospho-
kinase isoenzymes is 6.8. In contrast with the above disadvan-
tages, by using the above described buffer, the sample artifact
is eliminated because the CPK3 isoenzyme is located entirely to
the cathode side of the sample application point. Also, the in-
25- terference from serum albumin with the CPKl isoenzyme is also
eliminated because this creatine phosphokinase isoenzyme is lo-
cated entirely to the anode side of the area occupied by the
serum albumin. Further, although the pH titration curve for the
above described buffer is similar to the barbital buffer curve,
30. the lower pH of the above buffer as compared to the high pH of
the barbital buffer enables one to use less substrate buffer in
1~
1~4~9~0
order to lower the pH of the electrophoretic medium to the opti-
mal pH range for creatine phosphokinase isoenzymes.
The following examples are provided for the purpose of
further illustration only and are not intended to be limitations
5. of the disclosed invention.
Example 1
Analysis of Creatine Phosphokinase Isoenzymes by Fluorometric
Detection of Reduced NAD.
I. Electrophoretic Techni~ue
10. 1. Electrophoretic buffer: To prepare the electrophoretic
buffer, dissolve the following reagents in one liter of
distilled water.
a) 6.7 grams tris(hydroxymethyl)aminomethane
b) 6.0 grams DL-aspartic acid
15. 2. Agarose gel: To prepare an agarose geI, one gram of
agarose is dissolved in 100 ml of electrophoretic buf-
fer with heat. Apply 12 ml of the warm buffered aga-
rose solution to a 2-3/4 x 6-1/2 inch sheet of Mylar
brand polyester film (Mylar is a trademark of E. I. du
20. Pont de Nemours & Co., Wilmington, Delaware) which has
been coated with a hydrophilic resin subbing. Place
into the warm agarose solution four 1/2 x 1/32 inch
steel rods in a linear fashion across the width and ap-
proximately three inches from the end of the Mylar
25. sheet. After the agarose solution has solidified to
a gel, the four steel rods are removed with a magnet
thereby forming four slots in the gel matrix which can
be used to contain the sample specimens for electro-
phoretic separation. The gel surface is gently blotted
30. with a sheet of Whatman No. 2 chromatographic paper
before the sample specimens are applied to the gel.
11
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3. Sample Specimen:
a) Serum from a freshly clotted blood
b) Body fluid, such as pleural fluid
c) Tissue extracts
5, 4. Sample Application: Apply 5 ~1 of sample to each slot
in the gel with a microliter syringe. Place the gel
into an electrophoretic cell which contains electro-
phoretic buffer.
5. Electrical Parameters: Perform the electrophoretic
10. separation at 150 volts for a duration of 30 minutes.
II. Subs rate Overlay Techni~ue
1. Substrate: Use any commercially available creatine
phosphokinase reagent.
2. Substrate Overlay
15. a) Saturate a 2-3/4 x 4 inch sheet of Whatman No. 542
chromatograph paper with 1.5 ml substrate solution.
b) After electrophoresis has been terminated, remove
the agarose gel from the electrophoretic cell and
place it into a small plastic incubation box.
20. With a sheet of Whatman No. 2 chromatography paper,
gently blot the surface by laying the substrate
overlay paper onto the gel surface in a rolling
motion to avoid entrapment of air bubbles between
the gel surface and the substrate overlay paper.
25. c) Place a lid on the plastic incubation box and incu-
bate the combination gel substrate overlay paper
at 37C. for 60 minutes.
3. Precipitate and Dehydration
Precipitation and dehydration of the reduced NAD is ac-
30, complished by removing the substrate overlay paper from
the gel surface and immerging the substrate overlay
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paper into 100 ml of 2-propanol for a period of five
minutes. Then placing the overlay onto a paper towel,
it is allowed to dry.
III. Detection of Fluorescence
5. 1. Excitation Source
Expose the dried substrate overlay paper to a source of
ultraviolet light in the spectral region of 300 to 400
nm.
2. Fluorescent Detection
10. a) Direct visual observation
b) Scanning fluorescent densitometer
c) Fluorometer
d) Photographic reproduction
Example 2
15. Analysis of Lactate Dehydrogenase Isoenzymes by Fluorometric
Detection of Reduced NAD.
I. 'Electrophoretic Technique
1. Electrophoretic'buffer: To prepare the electrophoretic
buffer, dissolve the following reagents in one liter of
20. distilled water.
a) 2.6g sodium barbital
b) 1.15g citric acid
c) 3.5g tris-(hydroxymethyl)-amino methane
2. Agarose gel - See Example 1
25. 3. Sample Specimen - See Example 1
4. Sample Application - See Example 1
5. Electrical P'arameters - Perform the electrophoretic
separation at 150 volts for a duration of 45 minutes.
II. Substrate Overlay Technique
30. 1. a) 30 mg L-lithium lactate
b) 15 mg ~-Nicotinamide adenine dinucleotide (NAD)
c) 1.5 ml electro~horetic buffer
1~4~910
2. Substrate Overlay - See Example l
3. Precipitation and Dehydration - See Example l
III. Detection of Fluorescence - See Example l
Other enzymes, for example, glucose-6-phosphate dehydro-
5. genase, hexokinase, malate dehydrogenase, isocitrate: dehydroge-
nase, aldolase, and alcohol dehydrogenase can be fluorometrically
analyzed in a manner analogous to that.set forth in examples l
and 2.
Example 3
10. In order to demonstrate the ability of various buffers
to approach the optimum pH for the determination of creatine
phosphokinase isoenzyme, 8 ml of the electrophoretic buffers of
examples l and 2 were .separately mixed with l ml of creatine
phosphokinase substrate and the pH of the mixture was measured.
15. The results derived from these experiments are listed in Table I.
14
1~4~910
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m ~ ,~
o ~ o I
o P~
O ~1 ~
.~ ~ ~
h U
o ~a oO oO
~C ~ ~ D
H C~ H .~
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~ U~ O
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~o 0~ 1~ ~ ~
m ;Y; ~ ~ o
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m ~ ~ m
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1~48910
Table I clearly indicates that the electrophoretic buf-
fer wi.thin the scope of this invention, as exemplified by the
electrophoretic buffer of Example 1, more closely approximates
the desirable pH of the creatine phosphokinase:substrate than does
. the prior art electrophoretic buffer of Example 2.
Example 4
The creatine:phosphokinase enzyme present in a sample of
human serum was electrophoretically separated under an electrical
current of 1.4 volts per centimeter for 30 minutes. This separa-
10. tion was performed in both a tris-aspartic buffer and a barbital
buffer. The barbital buffer comprised 5.1 grams per weight sodium
diethyl barbituate and 0.92 grams per weight diethyl barbituric
acid. The tris-aspartic buffer employed was the electrophoretic
buffer of Example 1. .The locations of the various isoenzymes are
15. shown graphically in Figure 1 and numerically in Table II.
The encircled neg.ative signs in Figure 1 represent the
cathode electrode and the encircled positive signs represent the
anode electrodes. The metric scale in Figure 1 is employed to
measure the distance that the isoenzymes traveled from the sample
20. trench (sample application point) as well as the wi.dth of the
various areas depicted in Figure 1. CPKl, CPK2, and CPK3 are the
three isoenzymes present in creatine phosphokinase and albumin is
a protein also present in human serum.
Figure 1 clearly indicates that when creatine phospho-
25. kinase is electrophoretically separated using the barbital buffer,the CPK3 isoenzyme is present on both sides of the sample trench,
thereby producing an application artifact. However, when crea-
tine:phosphokinase is electrophoretically separated using the
tris-aspartic buffer, the~CPK3 isoenzyme is only located on the
30. cathode side of the sample trench, thereby eliminating the sample
artifact mentioned above.
~6
~)4891
Figure 1 also clearly depicts that when creatine phos-
phokinase is electrophoretically separated using the barbital
buffer, the area occupied by the albumin overlaps the area occu-
pied by the CPKl isoenzyme. This overlap, as noted above, inter-
5. feres with the enzymatic action of the CPKl isoenzyme. Becausethere is no overlap present when creatine phosphokinase is elec-
trophoretically separated using the tris-aspartic buffer this
interference is totally eliminated.
Table II depicts in numerical form the same information
10. conveyed in Figure 1. The millimeters referred to in Table I
correspond to the metric scale shown in Figure 1.
Figure I and Tahle II clearly indicate that the electro-
phoretic buffer within the scope of this invention, as exemplified
by the;electrophoretic buffer of Example 1, is able to eliminate
15. both the trench artifact and the albumin interference present in
the prior art barbital buffer electrophoretic separation.
While various methods and reagents have been described
in detail herein, it should be appreciated that changes and modi-
fications may be made without departing from the spirit of the
20. inven~ion. For example, while the use of tris-aspartic buffer
has been described above as being capable of improving the elec-
trophoretic separation of creatine phosphokinase wherein the
creatine phosphokinase isoenzymes are detected via the present
invention's fluorometric technique, it should be clear said tris-
25. aspartic buffer can also be employed in the electrophoretic sep-
aration of creatine phosphokinase wherein the creatine phospho-
kinase isoenzymes are detected via prior art fluorescent and
non-fluorescent techniques.
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