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| United States Patent |
6,861,225 |
| Bertha , et al. |
March 1, 2005 |
Method and reagent kit for determining activity of 5-nucleotidase
Abstract
The invention relates to a method for determining activity of
5'-nucleotidase, in which a biological sample is incubated in a manner and under
conditions known per se with a nucleotide pentose monophosphate as substrate,
the liberated inorganic phosphate is converted into a colored complex by
treating it with ammonium molybdate and a reducing agent, color intensity is
measured by a known method, and from the measured value the activity of
5'-nucle-otidase or the amount of inorganic phosphate liberated within unit
time, as a figure proportional to the activity of 5'-nucleotidase, is calculated
with a known calculation and/or with the aid of a calibration curve. According
to the invention 5'-AMP, 5'-CMP, 5'-UMP, 5'-GMP, 5'-IMP and 5'-TMP are used as
nucleotide pentose monophosphates, and measurement is performed on all of these
six substrates. The invention also relates to a reagent kit for performing the
above method.
| Inventors: |
Bertha; Andras (Maria ter 4., H-1011
Budapest, HU); Tulok; Istvan (Dozsa Gyorgy u.23/b, H-2013 Pomaz,
HU) |
| Appl. No.: |
296136 |
| Filed: |
November 21, 2002 |
| PCT Filed: |
July 3, 2000 |
| PCT NO: |
PCT/HU00/00069 |
| 371 Date: |
November 21, 2002 |
| 102(e) Date: |
November 21, 2002 |
| PCT PUB.NO.: |
WO01/90403 |
| PCT PUB. Date: |
November 29, 2001 |
| Current U.S. Class: |
435/6; 435/7.1; 435/7.2
|
| Intern'l Class: |
C12Q 001/68; G01N033/53 |
| Field of Search: |
435/6,7.1,7.2 |
References Cited [Referenced
By]
Other References
Paglia et al. The Journal of Biological Chemistry, vol.
250, No. 20, pp. 7973-7979, 1975.*
El-Aaser, Abdel-basset Anwer, et
al., "Simultaneous Determination of 5'-Nucleotidase and Alkaline
Phosphatase Activities in Serum," Z. Klin. Chem. Klin. Biochem., vol. 13,
No. 10, pp. 453-459 (1975).
Parthasarathi, K., et al., "Effect of
Spike Disease on the 5'- and 3'-Nucleotidase Activities in Sandal Plants,"
Experientia, vol. 37, No. 5, pp. 448-449 (1981).
Wood, Raymond John,
et al., "Colorimetric Determination of Serum 5'-Nucleotidase without
Deproteinization," Clin. Chem., vol. 27, No. 3, pp. 464-465 (1981).
Fioretti, E., et al., "Spectrophotometric Assays for 5'-Nucleotidase,
Using IMP, GMP and CMP as Substrates," The Italian Journal of
Biochemistry, vol. 21, No. 3, pp. 103-112 (May-Jun. 1972).
Bodansky,
Oscar, et al., "5'-Nucleotidase," Advances in Clinical Chemistry, vol 11,
pp. 277-328 (1968). |
Primary Examiner:
Riley; Jezia
Attorney, Agent or Firm: Speckman Law Group PLLC,
Sleath; Janet, King; Victor N.
Claims
What we claim is:
1. A method for diagnosing the existence of a
malignant process and/or for monitoring results of a therapy applied in its
treatment by determining 5'-ribonucleotide phosphohydrolase activity, wherein a
biological sample is incubated with a nucleotide pentose monophosphate as
substrate, the liberated inorganic phosphate is converted into a colored complex
by treating it with ammonium molybdate and a reducing agent, color intensity is
measured, and from the measured value the activity of 5'-ribonucleotide
phosphohydrolase or the amount of inorganic phosphate liberated within unit
time, as a figure proportional to the activity of 5'-ribonucleotide
phosphohydrolase is calculated, wherein 5'-AMP, 5'-CMP, 5'-UMP, 5-GMP, 5'-IMP
and 5'-TMP are used as nucleotide pentose monophosphates, the measurement is
performed on all of these six substrates, and the obtained results are compared
with one another and with control values.
2. The method of claim 1,
wherein the biological sample is incubated with the substrate in a buffered
medium optionally in the presence of an inhibitor, and then, over ammonium
molybdate and reducing agent, a protein solubilizing reagent and a stabilizing
reagent are also added to the incubated mixture.
3. The method of claim
1, wherein the nucleotide pentose monophosphate substrates are used as aqueous
solutions with concentrations of 1-10 mmoles/l.
4. The method of claim
1, wherein whole blood, serum or a haemolysate is used as biological sample, and
incubation with the substrate is performed both in the presence and in the
absence of an alkaline phosphatase inhibitor.
5. A reagent kit for
performing a method of claim 1, comprising 5'-AMP, 5'-CMP, 5'-UMP, 5'-GMP,
5'-IMP and 5'-TMP as substrates together with conventional components of a
reagent kit applicable to determine 5'-ribonucleotide phosphohydrolase activity
via forming an inorganic phosphate, converting the resulting phosphate into a
colored complex and measuring color intensity by photometry.
6. The
reagent kit of claim 5, wherein the conventional components is selected from the
group consisting of: a buffer, a protein solubilizing reagent, a stabilizing
reagent, a color reagent, a phosphorous standard, and optionally an inhibitor
and/or a substrate other than a nucleotide pentose monophosphate.
Description
The invention relates to a method for determining the activity of
5'-nucleotidase (5'-ribonucleotide phosphohydrolase) in body fluids, tissue
preparates and/or cell elements. The invention also relates to a reagent kit
usable in the method according to the invention.
FIELD OF THE INVENTION
Of the metabolic disturbances which are coupled with, incidental to and
maintaining factors of malignant processes, too, changes in nucleotide
metabolism play an outstanding role. This explains why influencing of DNA/RNA
synthesis and of the nucleic acid-nucleotide metabolism closely connected to
these processes is one of the main goals of the interventions used in tumour
therapy.
BACKGROUND OF THE INVENTION
Examination of nucleic
acid-nucleotide metabolic processes has an outstanding importance in detecting
tumorous processes and in distinguishing benignant and malignant processes from
one another. Examination of the changes in nucleic acid metabolic processes is
indispensible from the aspects of monitoring tumour therapy, too, since in the
overwhelming majority of chemotherapeutical treatments the therapeutic effect
appears in artificially provoked disturbances of the nucleic acid-nucleotide
metabolism and in artificial damaging of DNA synthesis. These artificially
provoked changes are decisive factors of the effectiveness of tumour therapy,
however, they are also responsible for basic damages of healthy cells and
disturbances of cell and organ functions.
Under routine conditions the
determination of the activity of 5'-nucleotidase (5'-ribonucleotide
phosphohydrolase), an enzyme detected by Reis (Reis, J.: Uber die spezifische
Phosphatase der Nerwengewebe: Enzymologia 2, 110-115/1937/), is one of the most
appropriate methods. This enzyme catalyzes solely the hydrolysis of nucleotide
pentose monophosphates (5'-AMP, 5'-CMP, 5'-UMP, 5'-GMP, 5'-IMOP, 5'-TMP, dAMP,
dCMP, dUMP, etc.), yielding nucleotides and inorganic phosphate (Bodansky, O.,
Schwartz, M. K.: 5'-Nucleotidase; Adv. Clin. Chem. 11, 277-327/1968/), and can
be regarded as one of the most sensitive plasm membrane markers. Determination
of the activity of 5'-nucleotidase is used both for clinical purposes and in
experimental tests; in the former instance measurment is performed usually on
non-haemolysed serum and/or on a haemolysate, whereas in the latter instance
measurement can be performed on body fluids, on tissue preparates (e.g. on plasm
membranes) and/or on cell elements (e.g. on microsomes, cytoplasms, lysosomes)
(see: Solyom, A., Trans., E. G.: Enzyme Markers in Characterization of Isolated
Plasma Membranes: Enzyme 13, 329-372/1972/).
As 5'-nucleotidase is an
enzyme which splits specifically nucleotide pentose monophosphates, a widely
applied method for determining the activity of 5'-nucleotidase is to incubate
the biological sample with a nucleotide pentose monophosphate substrate and
then, after adding an appropriate colour developing reagent, to determine by
colorimetry (spectrophotometry) the amount of inorganic phosphate liberated
within unit time upon the effect of the enzyme. If the biological sample to be
examined also comprises components which interfere with 5'-nucleotidase enzyme
(for blood or serum such a component is alkaline phosphatase, for lysosome
membrane such components are the other sugar phosphates), incubation is
performed in the presence of an inhibitor capable of inhibiting their effects.
As colour developing reagent ammonium molybdate is applied in the presence of a
reducing agent, which is usually stannous chloride optionally together with a
hydrazine salt orascorbic acid, which reacts with the inorganic phosphate to
form an intense blue complex. Considering that ammonium molybdate precipitates
the proteins present in the biological sample, proteins are either removed
before performing the colour reaction, or proteins are kept in solution by
adding an appropriate solubilizing agent to the mixture. To our recent
knowledge, this latter is one of the most up-to-date calorimetric methods.
Utilizing this method, the sample requirement of the determination can even be
reduced to some hundredth milliliters, and the accuracy of the assay can be
increased considerably. Determination of the activity of 5'-nucleotidase on the
above principle is disclosed in detail, among others, in the following papers
and in further references cited therein: J. Clin. Chem. Clin. Biochem. 7, 18-25
(1969), 13,453-459 (1975), 15, 715-718 (1977), 18, 781-788 (1980); Clin. Chem.
23, 2311-2323 (1977)127, 464-465 (1981); Adv. Clin. Chem. 11, 277-332 (1968).
A common feature of all routine methods applied before to determine the
activity of 5'-nucleotidase on the above basis for diagnostic or general
experimental purposes is that enzyme activity has been measured only on a single
substrate. As substrate, most frequently adenosine-5'-monophosphate (5'-AMP),
less frequently cytidine-5'-monophosphate (5'-CMP) or inositol-5'-monophosphate,
or -in haemo-lysates-uridine-5'-monophosphate (5'-UMP) is applied.
SUMMARY OF THE INVENTION
In our examinations performed to detect
malignant processes and to monitor the efficiacy of tumour therapy the activity
of 5'-nucleotidase in one and the same biological sample has been measured in
parallel on six different substrates, i.e. on adenosine-5'-monophosphate
(5'-AMP), cytidine-5'-monophosphate (5'-CMP), uridine-5'-monophosphage (5'-UMP),
guanosine-5'-monophosphate (5'-GMP), inositol-5'-monophosphate (5'-IMP) and
thymidine-5'-monophosphate (5'-TMP). The activity of nonspecific phosphatase in
the biological sample has been measured on .beta.-glycerophosphate substrate,
and this was taken as correction. As it was expectable on the basis of the
references cited above, we have observed that although the 5'-nucleotidase
activity values measured on different nucleotide pentose monophosphates
quantitatively differ from one another, on healthy and sick individuals there is
a strict correlation between the activity values measured on different
substratres. This is because under physiological conditions a dynamic
equilibrium state of nucleic acid-nucleotide metabolism processes exists in the
serum as a common metabolic pool. Again, as it follows from the literature, we
have observed that the 5'-nucleotidase activity values measured on the above six
substrates in sera of healthy and sick individuals differ from one another to
such an extent which cannot be attributed to the differences in the chemical
natures of the substrates, but result from physiologic processes. However, we
have found, unexpectedly, that the above strict correlation changes upon
therapy, and considerable differences appear on the basis whether the therapy
has exerted its effects on the purine metabolism, on the pyrimidine metabolism,
or on the thymidine metabolism. Thus, based on the results of activity
measurements performed on the above six different substrates, it can be decided
whether the applied therapy is effective, whether it exerts its effects on the
field intended to be influenced, and whether it induces undesirable cell damages
or not. This recognition forms the basis of our invention.
Based on the
above, the invention relates to a method for diagnosing the existence of a
malignant process and/or for monitoring the results of a therapy applied in its
treatment by determining 5'-nucleotidase activity, in which a biological sample
is incubated in a manner and under conditions known per se with a nucleotide
pentose monophosphate as substrate, the liberated inorganic phosphate is
converted into a coloured complex by treating it with ammonium molybdate and a
reducing agent, colour intensity is measured by a known method, and from the
measured value the activity of 5'-nucleotidase or the amount of inorganic
phosphate liberated within unit time, as a figure proportional to the activity
of 5'-nucleotidase, is calculated with a known calculation and/or with the ais
of a calibration curve. According to the invention 5'-AMP, 5'-CMP, 5-UMP,
5'-GMP, 5'-IMP and 5'-TMP are used as nucleotide pentose monophosphates, the
measurement is performed on all of these six substrates, and the obtained
results are compared with on another and with control valves.
Since upon
a chemotherapeutic treatment well observable changes in the activity of
5'-nucleotidase appear on all the six different substrates even after a short
period (24 hours), a well-founded decision can be brought on the maintenance,
modification or termination of therapy within a shorter period of time and with
less ambiguities. Here we remark, for the purpose of comparison, that a change
which may serve as a basis for decision can be observed with most of the tumour
markers only after about 1 week of treatment.
DETAILED DESCRIPTION OF
THE INVENTION
When the activity of 5'-nucleotidase is measured in
parallel on all of the above six substrates and the results are summarized, much
more reliable diagnosis can be set up on the condition of the individual and on
the occurrence of liver and bone processes than it could be obtained when the
measurement would be performed only on a single substrate. This may provide a
valuable assistance to the early recognition of malignant processes.
Based on the consequential interconnections of biochemical processes,
activity measurements of 5'-nucleotidase on the above six substrates may replace
enzyme activity determinations which are less frequently used in experimental
and clinical practice, since they are much more complicated and are difficult to
perform as a routine test, if at all. These replacement or substitution
possibilities are e.g. as follows: 5'-CMP nucleotidase.fwdarw.cytidine
kinase.fwdarw.cytidine deaminase.fwdarw.cytidine triphosphate synthetase; 5'-GMP
nucleotidase.fwdarw.guanosine deaminase, 5'-IMP nucleotidase.fwdarw.IMP
dehydrogenase; 5'-TMP nucleotidase.fwdarw.thymidine kinase{character
pullout}thymidine synthetase.
Of the reactants required in the
measurements, of the reaction conditions and of the method of calculation
detailed information can be found in the literature, among others in the papers
cited above. As an example, in the following we give the composition of a
reagent system applicable for measurements without protein removal, which proved
to be particularly preferred, and also describe how it should be used in
measurements performed on serum samples, together with the method of
calculation, without, however, limiting the usability of the method of the
invention to the reagent system and to the conditions disclosed below.
The reagents to be used are as follows:
(1) Buffer/Activator
Solution:
75-400 mmoles/l of TRIS, 5.0-10.0 mmoles/l of MgCl.sub.2 or
2.0-5.0 mmoles/l of MnCl.sub.2, 1.0-5.0 mmoles/l of KCl; the pH of the solution
being adjusted to 9-9.5 with a 3-6 M aqueous HCl solution.
(2)
Inhibitor:
An inhibitor should be used only when the biological sample
comprises a substance which interferes with the determination of the activity of
5'-nucleotidase. For blood and serum such a substance is alkaline phosphatase,
which can be inhibited with a solution containing 10-20 g/l of L-cysteine or
L-glycine or an equivalent amount of a L-cysteine- or L-glycine-salt, or 50-250
mg/l of Concavalin A, the solvent being the buffer described above. When the
measurements are performed on lysosome membranes, a solution of 5-50 mmoles/l of
L(+)-tartarate in the above buffer is used to inhibit the sugar phosphates
present.
(3) Substrates:
As substrates 5'-AMP, 5'-CMP, 5'-UMP,
5'-GMP, 5'-IMP and 5'-TMP are used as 1-10 mmolar solutions in demineralized
water. .beta.-Glycerophosphate or disodium-phenylphosphate should also be used
as substrate in a solution of the same concentration. The latter two substrates
are two known substrates of the determination of nonspecific phosphatase
activity; this activity should also be determined in order to determine
accurately the activity of 5'-nucleotidase.
(4) Protein Solubilizing
Reagent:
Neat (concentrated) formic acid or propionic acid of the
highest purity grade.
(5) Stabilizing Reagent:
A 1:9 to 4:6 v/v
mixture of glycerol and water, or a 0.5-5 w/w % aqueous sorbitol solution, or a
0.25-2.5 w/w % aqueous mannitol solution, or a 1:2 to 1:1 v/v mixture of
n-propanol and water.
It is advisable to dissolve a small amount
(0.001-0.01 w/w %) of sodium azide in the stabilizing reagent in order to
increase its storability.
(6) Colour Reagent:
A 5-10 g/l
solution of ammonium molybdate in demineralized water.
(7) Reducing
Solution:
A 1-5 g/l solution of SnCl.sub.2 in 1 M aqueous hydrochloric
acid, or a 1-5 g/l solution of ascorbic acid in demineralized water.
(8)
Phosphorous Standard Solution:
A 1.61 mmol/l solution of inorganic
phosphate, which is diluted in series.
Measurement is performed as
follows:
An assay reagent for determining the amount of inorganic
phosphate is prepared by admixing the protein solubilizing reagent in a
volume/volume ratio of
4:1 to 1:1 with the aqueous glycerol solution, or
3:1 to 1:1 with the aqueous sorbitol solution, or
5:1 to 1:2
with the aqueous mannitol solution, or
7:1 to 4:1 with the aqueous
n-propanol solution,
and 15-25% by volume, related to the total volume
of the final mixture, of the colour reagent is added to the resulting mixture.
The components listed in the first five lines of Table 1 are admixed
with one another in the ratios as indicated in the Table, and the resulting
mixture is incubated at 37.degree. C. for 60 minutes. Thereafter the further
components listed in Table 1 are added to the mixture, the resulting mixture is
stirred, then allowed to stand for 15 minutes, and thereafter the absorbance of
the solution is measured at 620-720 nm. The column in Table 1 with the heading
"5'-ND+ALP" relates to measurements where the activity of 5'-nucleotidase is
measured together with the activity of alkaline phosphatase (i.e. no inhibitor
is used), whereas the column with the heading "5'-ND" relates to measurements
performed in the presence of an inhibitor for alkaline phosphatase. The activity
of nonspecific phosphatase appears in the results of both measurements.
Nonspecific phospatase activity is determined separately on
.beta.-glycerophosphate substrate in the presence of an inhibitor; the resulting
value is subtracted from the previous ones to obtain the activity of
5'-nucleotidase+alkaline phosphatase or the activity of 5'-nucleotidase,
respectively.
Calculation is performed as follows:
(a)
Calculation of extinction differences (.DELTA.E values):
.beta.-glycerophosphate: E.sub.sample -E.sub.blind
=.DELTA.E.sub..beta.-glycerophosphate
other substrates: E.sub.sample
-E.sub.sample blind =.DELTA.E.sub.5'-AMP, 5'-CMP, 5'-UMP, 5'-GMP, 5'-TMP, 5'-IMP
standard: E.sub.standard -E.sub.reagent blind =.DELTA.E.sub.standard
(b) Calculation of enzyme activities on the basis of the above .DELTA.E
values: ##EQU1##
wherein
Standard cc. is the concentration of
the standard, and
K is a constant calculated from the following formula:
##EQU2##
(c) Calculation of individual enzyme activities or combined
activities from the above activity data:
Nonspecific phosphatase
activity=the activity measured on .beta.-glycerophosphate 5'-Nucleotidase
activities measured on different nucleotide pentose monophosphates=5-ND
activity-nonspecific phosphatase activity
5'-Nucleotidase+alkaline
phosphatase activity=5'-ND+ALP-nonspecific phosphatase activity
Alkaline
phosphatase activity=5'-ND+ALP-5-ND-nonspecific phosphatase activity
If
the biological sample is other than serum, the measurement is performed in the
same manner, with the difference that sometimes no inhibitor is necessary, or
other inhibitor is used. The calculations are also performed as given above.
TABLE 1
5'-
ND + Sample Reagent
Solution ALP 5'-ND blind Standard blind
Buffer/ 0.125 ml 0.125 ml 0.125 ml -- --
activator
De- 0.075 ml 0.050 ml 0.075 ml -- --
mineralized
water
Inhibitor -- 0.025 ml -- -- --
Sample 0.025 ml 0.025 ml -- -- --
(serum)
Substrate 0.025 ml 0.025 ml -- -- --
Assay 0.750 ml 0.750 ml 0.750 ml 0.750 ml 0.750 ml
reagent
De- -- -- -- 0.125 ml 0.250 ml
mineralized
water
Sample -- -- 0.025 ml -- --
(serum)
Substrate -- -- 0.025 ml -- --
Standard -- -- -- 0.125 ml --
Reducing 0.125 ml 0.125 ml 0.125 ml 0.125 ml 0.125 ml
solution
From the 5'-nucleotidase activity values obtained on the
individual substrates in the above series of measurements the following
biological conclusions can be drawn:
Decision on a status: The figure
obtained upon summarizing the six measured activity values denotes a
disease-free state up to 54-65 U/l, provided that at the same time alkaline
phosphatase activity is 10-25 U/l. If alkaline phosphatase activity is above 50
U/l and at the same time the sum of 5'-nucleotidase activity values is 54-65
U/l, an indication of malignant bone processes arises. If the sum of
5'-nucleotidase activities is above 70 U/l, this is a sure indication of sick
state. If at the same time alkaline phosphatase activity is above 35 U/l, this
is a sure indication of the presence of liver damages. For malignant liver
tumours both the sum of 5'-nucleotidase activity values and the alkaline
phosphatase activity are 4-10 times higher than the normal values.
Evaluation of a therapy: A change in activity measured on 5'-AMP, 5'-GMP
and 5'-IMP substrates indicates that the tumour therapy has intervened in the
purine metabolism, whereas a change in activity measured on 5'-CMP, 5'-UMP and
5'-TMP substrates indicates an intervention in the pyrimidine metabolism.
Unchanged values indicate that the therapy is useless. A decrease in activity
indicates always that the therapy exerted a beneficial intervention in the
processes. It occurs in numerous cases that upon the applied chemotherapy the
decrease in 5'-nucleotidase activities is so pronounced that both the activities
measured separately on the individual substrates and the sum of these activities
are lower than 10 U/l, even more, sometimes negative figures can also be
obtained for 5-nucleotidase activities. This arises from the fact that the
therapy attacks nucleotide metabolism, but does not influence nonspecific
phosphatase activity values; a negative activity value is obtained when a higher
nonspecific phosphatase activity value should be subtracted from a lower 5'-ND
activity value. When 5'-nucleotidase activity values are lower than those
obtained for healthy controls, this is already an indication of cytotoxic
damages of healthy cells with rapid nucleotide metabolisms (primarily of
intestinal mucosa, bone marrow and erythrocites), particularly when such a
change occurs at all 5'-nucleotidase activities measured on all the six
substrates. In such instances therapy should be modified or stopped.
The
invention also relates to a reagent kit usable for the method of the invention,
which comprises 5'-AMP, 5'-CMP, 5'-UMP, 5'-GMP, 5'-IMP and 5'-TMP as substrates
beside the conventional components of a reagent kit applicable to determine
5'-nucleotidase activity via forming an inorganic phosphate, converting the
resulting phosphate into a coloured complex and measuring colour intensity by
photometry.
The conventional components of the reagent kit may be those
utilized for the known methods, e.g. for those disclosed in the publications
cited above. A preferred reagent kit may comprise the following conventional
components:
buffer,
protein solubilizing reagent,
stabilizing reagent,
colour reagent,
reducing agent,
phosphorous standard,
further, if required,
inhibitor,
and/or
a substrate other than a nucleotide pentose monophosphate (such
as .beta.-glycerophosphate).
Preferred representatives of the above
conventional components are those listed above at the exemplified description of
the method of the invention. The reagent kit may comprise the conventional
components either as solutions with the above-indicated concentrations, or as
more concentrated solutions to be diluted before use, or as neat chemicals.
In the following, as an example, results of 5'-nucleotidase activity
measurements performed on serum samples are given, together with the conclusions
which can be drawn therefrom. The amounts of the individual substances and the
assay conditions were always those as given in Table 1. The accurate
compositions of the individual reagents used in the measurements were as
follows:
(1) Buffer/activator solution: 75.0 mmoles/l of TRIS.HCl, 5.0
mmoles/l of MgCl.sub.2, 5.0 mmoles/l of KCl: pH: 9.3.
(2) Inhibitor:
0.29 g of L-cysteine HCl dissolved in 20.0 ml of the buffer/activator solution.
(3) Substrates: 8.0 mmoles/l solutions of 5'-AMP, 5'-CMP, 5'-UMP,
5'-GMP, 5'-IMP, 5'-TMP and .beta.-glycerophosphate in demineralized water.
(4) Protein solubilizing reagent: Neat (concentrated) formic acid of the
highest analytical purity grade.
(5) Stabilizing reagent: 300 ml of
glycerol of the highest analytical purity grade+700 ml of demineralized
water+0.05 g of NaN.sub.3.
(6) Colour reagent: 8.0 g/l solution of
ammonium molybdate in demineralized water.
(7) Reducing solution: 0.20 g
of SnCl.sub.2 dissolved in 100 ml of 1.0 M aqueous hydrochloric acid.
(8) Phosphorous standard solution: "Dyalab".sup.(R) type inorganic
phosphate solution with a concentration of 1.61 mmoles/l (for further
thousandfol dilution)
Assay reagent: 200 ml of component (4) admixed
with 200 ml of component (5) and 100 ml of component (6).
Measurements
were performed for the following cases:
On healthy individuals and on
individuals with clinically diagnostized tumours before treatment. The results
are listed in Table 2.
For monitoring CMF
(cyclophosphamide-methoxtrexate-5-fluorouracyl) therapy of patients with
mammalian tumours after surgery. The results are listed in Tables 4 and 5.
For monitoring methotrexate (a folic acid-antagonizing agent) therapy of
patients with osteosarcoma. The results are listed in Table 7.
For
simplifying purposes In Table 2 and in all of the subsequent tables only the
symbols of the respective substrates were indicated; these refer, however,
always to the 5'-nucleotidase activity values measured on the given substrates.
From the data of Table 2 it appears that, with the exception of
lymphoma+bone manifestation, 5'-nucleotidase activity values measured on the six
substrates increased considerably on patients suffering from the examined
malignomes in comparison to the healthy control values. The sum of
5'-nucleotidase activity values (.SIGMA.5'-ND) showed the most pronounced
increase for lung tumours and or lymphomas coupled with liver manifestations.
The increase of alkaline phosphatase activity, compared to the change in
5'-nucleotidase activity, was the most pronounced for lymphomas coupled with
bone manifestations.
TABLE 2
Enzyme activity, U/l .+-. standard deviation
Groups tested 5'-AMP 5'-CMP 5'-UMP 5'-GMP 5'-IMP 5'-TMP
.SIGMA.5'-ND Alk. ph.
Healthy control 6.5 10.1 8.4 4.1 3.3 8.5 40.9
13.2
n = 157 .+-.1.2 .+-.2.2 .+-.1.6 .+-.1.1 .+-.0.9 .+-.1.6
.+-.1.43 .+-.4.4
Mammalian carcinoma 23.45 50.92 34.8 13.76 10.6 27.87 161.4
45.7
n = 341 .+-.4.1 .+-.9.8 .+-.4.7 .+-.4.3 .+-.3.3 .+-.5.8
.+-.5.33 .+-.7.8
Ovarian carcinoma 47.56 95.87 67.1 33.2 13.7 30.4 287.8
65.5
n = 189 .+-.7.8 .+-.9.1 .+-.6.9 .+-.4.4 .+-.3.5 .+-.6.1
.+-.6.3 .+-.11.2
Thyroid carcinoma 34.66 70.3 45.4 22.3 17.8 45.1
235.56 57.6
n = 76 .+-.7.2 .+-.9.7 .+-.5.9 .+-.4.4 .+-.5.2 .+-.9.5
.+-.6.98 .+-.12.3
non-Hodgkin lymphoma 23.5 46.8 34.5 11.6 9.11 18.6
144.11 23.4
n = 45 .+-.5.6 .+-.8.4 .+-.5.1 .+-.6.3 .+-.2.2 .+-.3.2
.+-.5.13 .+-.4.4
Hodgkin disease 34.6 80.6 57.5 21.2 10.7 15.5 220.1
43.3
n = 36 .+-.3.9 .+-.12.7 .+-.7.4 .+-.5.2 .+-.1.9 .+-.3.2
.+-.5.71 .+-.6.2
Melanoma malignans 10.8 23.7 15.8 5.7 8.8 23.2
88.01 41.3
n = 1567 .+-.2.3 .+-.3.8 .+-.2.9 .+-.0.89 .+-.1.5 .+-.4.3
.+-.2.61 .+-.6.8
Lung tumour 67.7 123.4 78.3 34 23 34 360.4
62.2
n = 76 .+-.13.4 .+-.19.4 .+-.13.5 .+-.5.9 .+-.3.9 .+-.4.5
.+-.10.15 .+-.11.2
Lymphoma + bone 9.2 10.8 8.2 5.4 3.3 9.8 45.8
167.6
manifestation, n = 23 .+-.2.3 .+-.1.7 .+-.1.1 .+-.0.9 .+-.0.2 .+-.1.8
.+-.1.33 .+-.23
Lymphoma + liver 89.7 189.7 102.6 45.8 34.9 34.6 497.3
56.7
manifestation, n = 76 .+-.19 .+-.23 .+-.31 .+-.12 .+-.8.9 .+-.4.6
.+-.16.41 .+-.11.2
As it appears from the data of Table 3, strict correlations
can be found between the 5'-nucleotidase activity values-belonging to the
individual substrates, as well as between the activity measured on 5'-AMP and
the activity measured on .beta.-glycerophosphate which indicates nonspecific
phosphatase activity, both for the patients suffering from the examined
malignomes and being before treatment and for the healthy controls.
TABLE 3
Correlation pairs Correlation
5'-AMP 5'-CMP 0.9943
5'-AMP 5'-UMP 0.9728
5'-AMP 5'-GMP 0.9234
5'-AMP 5'-IMP 0.9656
5'-AMP 5'-TMP 0.9878
5'-CMP 5'-UMP 0.9456
5'-CMP 5'-GMP 0.9678
5'-CMP 5'-IMP 0.9489
5'-CMP 5'-TMP 0.8978
5'-UMP 5'-GMP 0.8989
5'-UMP 5'-IMP 0.9323
5'-UMP 5'-TMP 0.9562
5'-GMP 5'-IMP 0.8978
5'-GMP 5'-TMP 0.9545
5'-AMP .beta.-glycerophosphate 0.8978
The results of the measurements applied to monitor the therapy
of patients subjected to mammalian tumourectomy and then to chemotherapy are
given in Tables 4 and 5. Table 4 summarizes the data of successful cases,
whereas Table 5 summarizes the data of cases resistant to CMF-therapy. Data
obtained after therapy change are also given in Table 5. In therapy change the
patients were subjected to Cis-platin therapy, and 5'-nucleotidase activities
were measured 1 day after the first treatment (*) and 1 day after terminating
the therapy (**). In all other cases measurements were performed 24 hours after
the actual chemotherapeutic treatment. For comparison purposes, values measured
on healthy controls are also given.
TABLE 4
5'-nucleotidase activity, U/l .+-. standard deviation
5'-AMP 5'-CMP 5'-UMP 5'-GMP 5'-IMP 5'-TMP
.SIGMA.5'-ND
Healthy control 6.5 10.1 8.4 4.1 3.3 8.5 40.9
.+-.1.2 .+-.2.2 .+-.1.6 .+-.1.1 .+-.0.9 .+-.1.6 .+-.1.43
Sick, before CMF 29.4 34.7 11.8 7.9 7.6 19.8 112.2
.+-.3.7 .+-.7.8 .+-.3.4 .+-.1.8 .+-.1.1 .+-.3.7 .+-.3.5
After 1 series of CMF 11.6 3.4 4.6 2.3 3.2 4.5
29.6
.+-.2.1 .+-.0.9 .+-.1.2 .+-.0.8 .+-.0.9 .+-.1.4 .+-.1.2
After 3 series of CMF 3.4 2.3 2.1 -2.4 -2.8 1.1
1.28
.+-.1.1 .+-.0.9 .+-.0.9 .+-.0.1 .+-.0.8 .+-.0.03
.+-.0.63
After 6 series of CMF 0.7 -1.7 2.2 -3.3 1.1 -3.4
-4.4
.+-.0.04 .+-.0.3 .+-.0.7 .+-.0.8 .+-.0.1 .+-.0.7
.+-.0.44
From the data the following conclusions can be drawn:
5'-nucleotidase activities measured on 5'-CMP, 5'-GMP, 5'-IMP and 5'-TMP
substrates decreased considerably already after the first series of CMF
treatments. After the third series of CMF treatments considerably great
decreases could be observed in 5'-nucleotidase activities measured on 5'-AMP,
5'-GMP and 5'-IMP substrates. After the sixth series of CMF treatments
considerable decreases appeared in 5'-nucleotidase activities measured on
5'-AMP, 5'-CMP, 5'-GMP and 5'-IMP substrates. For drug combinations the effect
exerted on the point of attack is difficult to define, owing to the different
points of attack of the individual components. Thus in such instances a
comparison to the status before treatment serves as guidance. The measured data
show that the drug combination applied exerts its therapeutic effect in two
phases, via the consequences exerted on the purine and pyrimidine metabolism
processes.
TABLE 5
5'-nucleotidase activity, U/l .+-. standard deviation
5'-AMP 5'-CMP 5'-UMP 5'-GMP 5'-IMP 5'-TMP
.SIGMA.5'-ND
Healthy control 6.5 10.1 8.4 4.1 3.3 8.5 40.9
.+-.1.2 .+-.2.2 .+-.1.6 .+-.1.1 .+-.0.9 .+-.1.6 .+-.1.43
Sick, before CMF 39.6 72.4 45.8 23.7 18.9 24.7 225.11
.+-.8.67 .+-.11.3 .+-.10.9 .+-.7.8 .+-.3.5 .+-.6.2
.+-.8.05
After 1 series of CMF 26.7 68.9 40.2 19.8 16.4 21.1
193.1
.+-.5.3 .+-.10.2 .+-.9.9 .+-.6.9 .+-.4.6 .+-.5.2
.+-.7.01
After Cis-platin (*) 20.3 43.2 26.5 4.2 2.3 8.9 115.4
.+-.3.4 .+-.8.1 .+-.5.7 .+-.1.1 .+-.0.9 .+-.1.8 .+-.3.5
After Cis-platin (**) 4.1 3.6 4.4 -1.6 1.1 1.2
12.8
.+-.1.3 .+-.0.9 .+-.1.5 .+-.0.8 .+-.0.6 .+-.0.3 .+-.1.2
After an ineffective CMF treatment no significant changes
could be observed in the 5'-nucleotidase activities measured on the individual
substrates; therefore therapy was continued with Cis-platin. On the basis of the
results we have observed that significant changes in 5'-nucleotidase activities
belonging to the individual substrates occurred already in the first stage of
Cis-platin treatment (24 hours after treatment; marked by * in the Table);
considerable decreases could be observed primarily on substrates participating
in the purine metabolism. The values measured on the first day following
termination of Cis-platin treatment (marked by ** in the Table) indicate that
the changes in purine metabolism are followed in time by changes in pirimidine
metabolism, with the remark that the changes in the two metabolic processes are
superimposed. Our tests revealed the ineffectiveness of CMF therapy already 5
days before the measurement with diagnostic tumor marker CA 15-3 (which also
indicated ineffectiveness), thus the patients could be saved of a five days'
superfluous CMF treatment.
The success and effectivity of therapy is
also reflexted by the fact that the initial strict correlation between the
5'-nucleotidase activity values measured on different substrates changes.
Correlation values calculated after a successful therapy are listed in Table 6
below, where the pre-therapy values are also given for comparison purposes.
TABLE 6
Correlation
Correlation Before After
pairs chemotherapy chemotherapy
5'-AMP 5'-CMP 0.9856 0.6767
5'-AMP 5'-UMP 0.8997 0.4561
5'-AMP 5'-GMP 0.7845 0.3267
5'-AMP 5'-IMP 0.8967 0.4519
5'-AMP 5'-TMP 0.7897 0.4687
5'-CMP 5'-UMP 0.9897 0.5639
5'-CMP 5'-GMP 0.7868 0.2345
5'-CMP 5'-IMP 0.8887 0.2689
5'-CMP 5'-TMP 0.6767 0.1123
5'-UMP 5'-GMP 0.8978 0.3493
5'-UMP 5'-IMP 0.8889 0.0567
5'-UMP 5'-TMP 0.8992 0.3635
5'-GMP 5'-IMP 0.9981 0.4684
5'-GMP 5'-TMP 0.9767 0.4781
5'-IMP 5'-TMP 0.8898 0.4356
The data of Table 6 show that the initial strict correlation
between the 5'-nucleotidase activities measured on the different substrates has
ceased, which also reflects that measurements on different substrates enable one
to separately detect and evaluate the individual biological processes proceeding
during therapy.
Determination of 5'-nucleotidase activity with the six
above substrates provides irreplaceably useful pieces of information in those
cases, too, when chemotherapy has no direct influence on the nucleotide
metabolism. A good example of this situation is the methothrexate treatment of
osteosarcomatic patients. Methothrexate is a substance with folic acid
antagonizing effect, it has no direct effect on the nucleotide metabolism, thus
every change in nucleotide metabolism which occurs upon methothrexate treatment
indicates a de novo nucleotide synthesis, i.e. a damaging of healthy cells. Any
damages occurring incidentally upon treatment have steadily serious
consequences; such a consequence may be e.g. a reduction of cellular and humoral
immunity, which may also induce formation of a second tumour.
Changes in
5'-nucleotidase activities measured on osteosarcomatic patients treated with
12.0 g doses of methothrexate are summarized in Table 7.
From the data
of Table 7 the following conclusions can be drawn:
The results of
5'-nucleotidase activity determinations performed on the six substrates applied
to examine the changes in pyrimidine and purine metabolism show that, in the
first period, the treatment affects the pyrimidine metabolism. The differences
between the two metabolic pathways are particularly pronounced 44 hours after
treatment. It also appears from the data of Table 7 that considerable
differences can be observed between the individual substrates even within one
and the same metabolic pathway. This phenomenon is unequivocally due to the
differences between the biochemical functions of the individual substrates. Upon
monitoring the therapy according to the method of the invention it becomes
possible to stop or change the therapy before the detected disturbances would
lead to more serious changes as late consequences.
TABLE 7
5'-nucleotidase activity, U/I
Pvrimidine Purine
metabolism metabolism
Time 5'-AMP 5'-CMP 5'-UMP 5'-GMP 5'-IMP 5'-TMP
0 min. -8.39 5.33 5.05 3.34 3.77 1.56
15 min. -3.12 2.21 0.14 0.57 -0.2 -6.75
30 min. -11.87 -2.19 1.92 -2.19 2.98 -6.89
1 hour -5.05 2.41 1.2 -2.1 1.34 -1.01
2 hours -4.84 5.97 2.77 2.84 -3.49 -2.49
3 hours -0.5 8.6 4.05 7.61 5.68 2.41
4 hours -3.63 6.76 4.19 0.92 -0.28 -3.98
6 hours -10.32 -1.63 -1.85 -5.48 -2.7 -3.7
12 hours -1.71 -0.15 1.48 1.98 3.69 -4.49
21 hours -2.56 6.04 5.83 -0.99 0.35 -4.84
44 hours 31.38 4.69 7.89 1.99 -12.95 -9.46
69 hours 7.96 18.42 14.93 13.16 13.01 8.6
90 hours 6.83 13.09 15.16 3.49 10.1 3.63
162 hours -12.6 6.97 29.53 -6.33 -1.99 -1.35
186 hours 1.41 7.53 12.16 14.94 6.54 0.35
210 hours 5.76 4.48 7.39 3.91 -3.91 0.92
234 hours 8.61 3.48 10.88 8.68 5.47 6.11
258 hours 3.99 2.42 2.64 0.22 0.43 -5.82
330 hours 9.25 7.47 2.99 8.46 10.53 -2.7
358 hours 9.67 5.47 4.9 5.97 -2.28 2.56
402 hours 5.18 8.03 9.59 8.24 6.04 5.4
Although in the above the method and reagent kit according to
the invention have been disclosed in connection with diagnostic use, the scope
of the invention is not limited to this type of use. The method and reagent kit
can also be utilized with excellent results for research purposes, e.g. to
detect the mechanisms of certain malignant processes, to test known or new
anti-cancer agents and to examine the mechanism of their effects.
* * * * *
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