Note: Descriptions are shown in the official language in which they were submitted.
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USE OF SUCCINATE AS A DIAGNOSTIC TOOL
ground of the Invention
Many parasites that live in microaerophilic environments, such as the
vertebrate intestine, survive in the absence of oxygen by generating energy
through
the reduction of fumarate by NADH to form succinate. Energy in the form of ATP
is
obtained as follows:
HH
l I
p~ + ADP + HOOC-C=C-COON + NADH ~ NAD + HOOC-CH2-CHZ-COOH + ATP + H+
(Fumarate) (Succinate)
It has been found that other organisms will revert to a succinate
accumulating metabolism to generate energy when exposed to hypoxic
environments.
Such organisms include numerous invertebrates (both parasitic and
saprophytic), and
vertebrates, including fish dwelling at the bottom of lakes and deep diving
mammals
such as whales and seals. In addition, it has been reported that succinate
accumulates
in localized regions of mammalian tissues that are subjected to hypoxic
conditions.
For example, it has been reported that mammalian rat heart in vivo and rat
heart cells
in culture, will accumulate succinate when the cells are deprived of oxygen.
20 The metabolism of humans and most other higher animals is highly
dependent on the supply of adequate amounts of oxygen. Reduction in
availability of
oxygen may occur due to hypoxia or anoxia (reduced or absent oxygen
concentration
in the blood supplying a cell), to ischemia (reduced blood supply) or to a
combination
of ischemia and hypoxia. Such conditions result in a variety of metabolic
changes and
25 adaptations within the cell, some of which enhance the ability of the cell
to produce
energy despite reductions in available oxygen. Various tissues have differing
tolerance to hypoxia and ischemia. If the resulting oxygen deprivation is
sufficiently
profound and prolonged, cells will die. In a "heart attack" or myocardial
infarction,
heart muscle cells die due to reduced blood flow (ischemia) caused by a
blockage in
30 the artery supplying those cells. In this setting, the heart cells receive
insufficient
quantities of oxygen and other nutrients to meet the basic demands for
survival. In
some patients, however, blood supply may be reduced only to the point that
tissue
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function is impaired, but not to the point at which the cells die. Depending
on the
degree of ischemia, such conditions may exist transiently or may persist for
extended
periods. In the heart, this may result in reduced contractile function in
ischemic
tissues.
The response of myocardial tissue to hypoxic/ischemic conditions
varies dependent on the severity and duration of the hypoxia/ischemia. When
hypoxia/ischemia is severe and prolonged, myocyte cell death occurs and there
is no
recovery of contractile function of these cells. When hypoxia/ischemia is less
severe
myocytes may remain viable but exhibit depressed contractile function, which
some
10 suggest may be a protective mechanism whereby these cells attempt to reduce
their
oxygen demand.
Myocardial cells that remain viable but exhibit contractile dysfunction
can be categorized into two groups (referred to generically as "jeopardized
myocardium"). Myocardial tissue subjected to chronically low coronary blood
flow
exhibits a reversible decrease in the force of contraction, a phenomenon known
as
"hibernation". A different sort of contractile dysfunction, "stunning"
prevails
following reperfusion after brief periods of ischemia. Accordingly, stunning
typically
involves contractile dysfunction after restoration of blood flow (perfusion),
whereas
myocardial hibernation typically involves contractile dysfunction during
ongoing low
20 myocardial blood flow (perfusion). Both types of contractile dysfunction
are
reversible with the appropriate therapy.
The identification of hibernating myocardium or myocardial stunning
in patients is of considerable importance, since, upon revascularization,
these
conditions are generally reversible. Accordingly, screening patients that have
poor
myocardial function for the presence of hibernating myocardium or stunned
myocardium allows for a more accurate prediction of the likelihood of clinical
improvement following surgical intervention or other revascularization
improvement
therapies known to those skilled in the art (i.e., angioplasty,
chemotherapeutics, etc.).
Typically, a patient having less than 18% to 20% of the total left ventricle
comprising
30 hibernating myocardial tissue will have little or no improvement in heart
function
upon revascularization. However, patients having greater than 18% to 20% of
the
total left ventricle comprising hibernating myocardial tissue have a high
likelihood for
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improvement in heart function following revascularization, and therefore are
considered good candidates for successful surgical intervention or the
application of
other revascularization therapies.
Currently, positron emission tomography (PET) using a blood flow
tracer and '8F-fluorodeoxyglucose {FDG) is often employed to identify
hibernating
and stunned myocardium and to triage patients for revascularization procedures
such
as bypass. This is a two-step process in which a blood flow scan of the heart
is
obtained to identify underperfused myocardium. A second scan is then obtained
using
FDG which is taken up by myocardial cells in proportion to cellular glucose
uptake.
Non-viable cells and scar tissue take up little or no FDG. Hibernating
myocardium
accumulates large amounts of FDG relative to the corresponding tissue
perfusion.
Stunned myocardium takes up FDG, but in small amounts relative to tissue
perfusion.
Thus, by comparison of the perfusion and FDG scans, heart tissue can be
classified as
normal, scar, hibernating or stunned. Both hibernating and stunned myocardium
are
considered "jeopardized". -
Although reduced blood oxygen content (hypoxia) is easily diagnosed
using widely available techniques, the clinical diagnosis of tissue ischemia
and
infarction remains a vexing and important problem. In the heart, ischemia
affects
millions of people annually and represents the largest single cause of death
in the
United States. 5-7 million patients are seen annually in emergency departments
in the
U.S. for complaints of chest pain which may or may not be on the basis of
cardiac
ischemia. A number of enzyme-based blood tests (e.g. CPK isoenzymes) exist
which
are effective in diagnosing the presence of myocardial infarction (cell death
due to
ischemia). Unfortunately, many hours may be required for blood levels of these
25 substances to become elevated after an infarction. This results in a costly
delay in
diagnosis and institution of treatment, frequently requiring patients to be
admitted to
the hospital for extended monitoring and blood sampling.
An even more vexing problem is the detection of ischemic events
which do not result in actual infarction. Many patients experience transient
episodes
30 of cardiac ischemia which may or may not be associated with chest pain
(angina). In
patients presenting for emergency department evaluation with ischemia but who
have
not (yet) actually experienced cardiac cell death, diagnosis is notoriously
difficult and
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inaccurate. Blood levels of cardiac enzymes are typically normal in such
patients.
Electrocardiogram (EKG) is negative or non-diagnostic in most patients.
Nonetheless, a large number of such patients, especially those with so-called
unstable
angina, will progress to actual myocardial infarction, typically within the
next few
5 weeks after initial presentation to the emergency room. In an attempt to
avoid
sending such high-risk patients home, most emergency physicians have adopted a
policy of admitting patients suspected of unstable angina to the hospital for
observation and further testing. 70% of such patients are eventually proven to
have
no heart disease at all. This necessarily cautious practice results in excess
health care
costs of billions of dollars annually. Yet, despite this very careful pattern
of medical
practice, approximately 8% of all chest pain patients sent home from emergency
departments go on to have a heart attack within the next four weeks. Such
events
represent a frequent cause of potentially preventable death from heart disease
and
constitute 25% of all malpractice settlements against emergency physicians.
Positron emission tomography, is the most accurate technique available
in detecting the presence of hibernating myocardial tissues, but suffers the
disadvantage that it costs $2,000 to $3,000 per test and is available in only
a few
centers. In addition the equipment required to conduct PET scans costs
approximately
3-8 million dollars per facility. However, it has recently been reported that
the use of
even highly expensive means of assessing cardiac blood flow such as nuclear
scanning of the heart (charges of $1,000 - $2,000 per test) resulted in a net
reduction
of cost to the health-care system for managing chest pain patients.
Accordingly, a less
expensive but accurate method of detecting cardiac ischemia, especially in the
absence of acute infarction, will result in more appropriate management and
treatment
25 of these patients, will save lives and will reduce health-care costs. Thus,
a diagnostic
test for detecting viable ischemic cells, that is accurate and relatively
inexpensive is
desired.
Along with heart disease and stroke, cancer is a major cause of death
and disability. Much cancer mortality is directly related to the difficulty in
detecting
30 the presence of a malignancy in a given patient. Current cancer screening
methods are
highly dependent on the detection of masses or lumps and on patient symptoms,
e.g.
pain. Unfortunately, these signs are absent in the early stages of most
malignancies.
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Screening tests which look for masses (e.g. mammograms or chest x-rays) are
notoriously inaccurate. In addition, they only screen certain portions of the
body. It
is impractical to x-ray the entire body to screen for cancer. Some blood tests
for
detecting cancer have been developed and a few (e.g. prostate specific antigen
(PSA),
5 carcinoembryonic antigen (CEA)) have reached clinical use. These blood
tests,
however, each test for only one or a few types of cancer. No existing blood
test
permits screening for a wide variety of cancers.
An additional problem in medical practice is the detection of cancer
recurrence in patients who have already undergone some therapy. Tests such as
x-ray
or CT scan are particularly problematic in this setting. A blood marker
applicable for
a wide variety of tumor types would be of clinical benefit in monitoring
patients for
cancer recurrence and for guiding therapy.
The present invention is based on the surprising discovery that
localized tissue of an animal subjected to hypoxic/ischemic conditions can be
detected
15 by measuring the concentration of succinate in an animal's peripheral blood
and
comparing the detected succinate levels to the basal succinate levels of the
appropriate
control group (i.e., whether the succinate levels exceed a "threshold" value).
The
technique claimed here permits an inexpensive, widely available diagnostic
tool for
identifying hypoxia/ischemia in patients at risk for, or suffering from,
myocardial
20 disease, a stroke, or cancer, and can be used to assess the likelihood of
patient benefit
from bypass surgery, angioplasty, pharmaceutical therapies or other therapies
that
restore or enhance blood and/or oxygen supply to the myocardial tissues.
in accordance with one embodiment, a diagnostic method for detecting
a disease state, characterized by a localized hypoxic/ischemic cell population
or other
25 cells with altered metabolisms similar to hypoxic/ischemic cells in an
animal, is
described. Disease states that are characterized by a localized
hypoxic/ischemic cell
populations or other cells with altered metabolisms similar to
hypoxic/ischemic cell
populations include but are not limited to cancer (presence of hypoxic tumor
cells),
cardiovascular disease (presence of ischemic, hibernating or stunned
myocardial cells)
30 and stroke (presence of ischemic brain cells). The diagnostic method
comprises the
step of determining the concentration of succinate in a bodily fluid sample of
said
animal and optionally comparing the succinate concentration to the baseline
succinate
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levels established for the applicable gender and age group. In one preferred
embodiment the bodily fluid sample used to measure the succinate concentration
is
blood.
S Summary of the Invention
The present invention is directed to measuring succinate levels in
bodily fluids as a diagnostic indicator for the presence of localized ischemic
tissue or
regions of hypoxia that are indicative of the existence of jeopardized
myocardium, the
risk of stroke, or the presence of cancer cells. The method comprises the
steps of
obtaining a bodily fluid sample from the individual and determining if the
concentration of succinate is greater than the clinical threshold value. This
procedure
can be used in conjunction with other standard techniques (such as PET) and
can be
used as a first screen to determine if additional diagnostic testing is
warranted or the
procedure can be used directly to determine treatment strategy.
1S
Brief Description of the Drawings
Figs. lA-1F are graphic representations of succinate concentration
verses time in the rat heart perfusion test. Rat hearts were excised from the
rat, the
blood was rinsed out of the heart and the heart was then hooked-up via the
aorta to a
perfusion apparatus which circulated a glucose-buffer solution through the
heart in a
predetermined OZ-Nz-COZ mixture at 37°C as follows: Fig. lA, gas
mixture 70%OZ /
2S% NZ / S% COZ; Fig. 1B, gas mixture 80%OZ / 1S% Nz l S% COZ; Fig. 1C, gas
mixture 9S%OZ l S% COz; Fig. 1D, gas mixture 70%OZ / 2S% NZ / S% COZ; Fig. lE,
gas mixture 9S%OZ / S% COZ; Fig. 1F, gas mixture 9S%OZ / S% COz.
2S Figs. 2A and 2B are graphic representations of the data collected
during the fumarate reduction assay. Perfused rat heart tissue was assayed
anaerobically for fumarate reductase in mitochondria) preparations. Fumarate
reductase was detected spectrophotometrically by measuring the anaerobic
oxidation
of NADH in the presence and absence of fumarate. Preliminary results from
analysis
30 of two separate homogenized rat myocardium samples (Figs. 2A and 2B)
indicate the
presence of a fumarate reductase.
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Fig. 3 is a graphic representation of the percent jeopardized
myocardium. yg. the Ln [succinate] in whole blood samples obtained from human
subjects. Approximately 70 samples of whole blood from patients and 1 S
samples of
whole blood from volunteers (the controls) were analyzed for succinate
concentration.
S Individuals were also analyzed by PET scanning for the presence of
jeopardized
myocardium
Detailed Description of the Invention
The present invention is directed to a novel diagnostic procedure for
determining the presence of ischemic tissues or living tissues subject to
hypoxic
conditions in an animal or human. The method is based on the discovery that
eukaryotic cells of an individual exposed to hypoxic and or ischemic
conditions will
release succinate, leading to detectable elevated levels of succinate in the
bodily
fluids of the animal. Accordingly, an increase in succinate levels over the
basal levels
of succinate found in healthy individuals is a diagnostic indicator that the
individual is
suffering from one or more diseases that are characterized by localized tissue
ischemia, hypoxia or other similar metabolic derangements. Such diseases
include,
but are not limited to, myocardial disease, strokes, and carcinomas.
Surprisingly, a
localized region of hypoxia {i.e., such as ischemic myocardial tissue) can
result in a
significant increase in blood succinate levels.
The present invention utilizes the concentration of succinate in bodily
fluids (including, but not limited to, whole blood, plasma, serum, urine and
saliva) to
detect the presence of cells that have an altered metabolism, caused by, or
similar to
that resulting from cells exposed to localized hypoxic or ischemic conditions.
Measurements of succinate levels provide a simple, inexpensive screen for
detecting
myocardial hibernation and/or stunning, myocardial infarction, cerebral
ischemia
and/or infarction and ischemia/infarction of other body tissues. Furthermore,
the
concentration of succinate in the bodily fluids is directly correlated with
the severity
of these conditions. The diagnostic technique described in the present
invention
30 permits the detection of cells having an altered cellular metabolism and
has potential
for broad application in clinical medicine.
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In one embodiment the method is used to determine the succinate
levels in a warm blooded vertebrate species as a diagnostic tool for screening
for
diseases characterized by cells that exhibit altered metabolic functions.
Three areas of
clinical applicability are immediately evident: myocardial ischemia/infarct,
cerebral
S ischemia/infarct and cancer.
Myocardial ischemia leads to heart failure, myocardial infarction and
death. Current methods of detecting heart disease lack sensitivity and
specificity. In
the situation where an individual has severe ischemia and potentially has
hibernating
myocardium, current techniques for selecting patients for coronary artery
bypass are
10 imperfect. The best available technique, positron emission tomography, is
reasonably
accurate but costs $2,000 to $3,000 per test and is available in only a few
centers. The
technique described in the present invention permits inexpensive, widely
available
assessment of the likelihood of a patient benefiting from bypass surgery. For
example, in individuals suffering from blockage or reduced blood flow to the
15 myocardial tissues, scar tissue as well as hibernating and/or stunned
tissue will be
present. Hibernating tissue is tissue that is chronically ischemic, oxygen
starved and
is "struggling" to remain viable. Using standard analytical techniques it is
difficult to
distinguish scar tissue from hibernating tissue. However, once an adequate
blood and
oxygen supply is restored to hibernating tissues (i.e., by standard coronary
artery
20 bypass surgery) the hibernating tissue responds well and there is a high
success rate
with recovery of contractile function. However, the recovery rate after bypass
surgery
in patients without hibernating myocardial tissue is significantly lower. The
presence
of hibernating myocardial tissue can be detected by elevated succinate levels.
Therefore the measurements of succinate levels in an individual can predict
those
25 patients that will benefit from bypass surgery or other revascularization
therapies.
Cerebral ischemia and stroke are major causes of death and disability.
The only successful therapy for stroke involves use of thrombolytic drugs, a
high-risk
procedure. Prior to administering therapy, a firm diagnosis must be
established within
3 hours of stroke onset. There is currently no objective technique available
for rapidly
30 identifying and diagnosing brain ischemia. Such a simple tool might prove
to be
extremely important, particularly in emergency care patients who exhibit
symptoms of
a stroke. If within several hours of the attack, drugs, having the ability to
lyse clots or
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having anti-clotting activity, are administered to the patient, the patient's
life may be
saved. However, if the stroke results from a hemorrhage rather than a blood
clot, the
patient may be placed in a more precarious state. By providing a simple, rapid
and
objective means of identifying brain ischemia/infarction, the technique
disclosed
5 herein may permit rapid and confident administration of thrombolytic therapy
and
more appropriate triage of patients towards or away from this high-risk
procedure.
There is currently no available generalized blood test for cancer. All
existing tests are specific to only a few tumor types, and may require biopsy
for
diagnosis. Testing for one of these types does not eliminate other tumor
types.
Accordingly, there is a need for a simple generalized assay to screen for the
presence
of tumor cells. It has also been reported that many, if not all solid tumors,
lack
adequate vascularization, and therefore have hypoxic regions. In general
cancer cells
have abnormal metabolisms that are similar to that seen in ischemic tissues
even when
the cancer cells are well oxygenated. As originally postulated by Otto Warburg
in
15 1926, there is support that many, if not all, cancer cells metabolize
anaerobically.
Accordingly it is anticipated that many tumor cells will produce succinate as
a by-
product of their altered metabolism. Surprisingly, this localized production
of
succinate by solid tumors can be detected in the peripheral blood and used as
an initial
screen for tumors.
In accordance with one embodiment, the measurement of succinate
levels in a vertebrate's bodily fluids can be used in accordance with the
present
invention for detecting the presence of cancer in a patient. The technique
claimed
here may prove useful in routine screening of patients for cancer, searching
for tumor
recurrence, and in assessing response of tumors to therapy. In accordance with
one
25 embodiment, patients can be screened for cancerous cells merely by means of
determining succinate levels in peripheral blood.
It is well established that exercising muscle is also capable of
functioning anaerobically (lactate production). Therefore, muscle diseases
relating to
metabolic defects may also produce high succinate levels and thus can be
diagnosed
30 by determining succinate levels in peripheral blood. This would constitute
a broad
and general method for determining any hypoxic/ischemic metabolism in human
tissues.
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Succinate is a common metabolite and methods for detecting and
quantitating succinate levels have been described in the art and are familiar
to those
skilled in the art. One of the first methods for quantitative determination of
succinic
acid was a gravimetric procedure in which succinic acid was precipitated by
silver
ions and the precipitate was weighed. In a later improvement, gravimetry was
replaced by titration of the excess silver ions.
Methods based on ion-exchange chromatography are more reliable
than gravimetric assays. Lawson et al. (Acta (Wien) 1961, {pp. 415-419) were
able to
separate a large number of organic acids on a weak exchanger with formic acid
as
eluant. In addition, a relatively reliable method for detecting succinic acid
was
developed by Dimotaki-Kourakou (Ann. Falsif. Expert. Chim. 54, 70-83 ( 1961
)). In
this method, the organic acids of the sample are bound to a strongly alkaline
ion-
exchanger. After elution with an ammonium carbonate solution, the interfering
organic acids are oxidized and the remaining succinate is titrated by silver
nitrate/potassium rhodanide after extraction with ether. However, the method
is very
cumbersome, taking at least 15 hours to complete.
A gas-chromatographic method for detecting succinate in biological
materials has also been developed (Chromatographia 12, 22-24 (1979)). Succinic
acid
in cerebrospinal fluid is methylated with a sulphuric acid/methanol mixture.
The
method takes at least 20 hours. The main disadvantage is that a large sample
volume
is needed for the assay.
A method has been described using high-performance liquid
chromatography for determination of succinic acid in citrus fruits (J.Sci.
Food Agric.
34, 1285-1288 (1983)). A preliminary separation of organic acids on ion-
exchange
columns is necessary to remove interfering substances. Chromatography on a
propylamine column is very effective because of the relatively sharp peak that
is
obtained, but the level of detection for succinic acid is about 0.005%. The
coefficient
of variation is relatively poor in the lower concentration range.
The first enzymatic method for determination of succinic acid was
suggested by Bernath et al. (Succinic Dehydrogenase, in: S.P. Colowick, N. O.
Kaplan (eds.), Methods in Enzymology, Vol. V, Academic Press, New York 1962,
pp.
597-614) succinic acid was converted to fumaric acid by means of the enzyme
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succinate dehydrogenase. Pires et al. (Z. Lebensm. Unterscuh. Forsch. 143, 96-
99
(1970)) isolated a succinate dehydrogenase from Ascaris suum which oxidizes
succinic acid to fumaric acid in the presence of tripotassium cyanoferrate
(III). This
enzyme is free from the enzymes of the citric acid cycle, and interference in
the
5 measurement due to the presence of other organic acids is therefore
generally
excluded.
The application of the enzyme succinyl-CoA synthetase (succinate
thiokinase) for determining succinic acid is another enzymatic procedure used
to
quantitate succinate. Williamson et al. (Assays of Intermediates of the Citric
Acid
cycle and Related Compounds by Fluorometric Enzyme Methods, in: S.P. Colowick,
N.O. Kaplan (eds.), Methods in Enzymology, vo. XIII, Academic Press, New York
1969, pp. 434-513) describe a procedure using succinyl-CoA synthetase from
Escherichia coli. This enzyme converts succinic acid to activated succinate in
the
presence of ATP and CoA, to form ADP. ADP can be measured via the PK/LDH
system using techniques known to those skilled in the art. The reliability
ofxhe assay
depends strongly on the purity of the succinyl-CoA synthetase. The Michaelis
constant of this enzyme toward succinate is relatively high (K~, ca. 10-3 mol/
1 ), so that
a relatively high activity has to be used for the assay. Since succinyl-CoA
synthetase
is often accompanied by a myokinase, interference is observed if the sample
material
20 contains ATP (tissues and biological materials). This problem can be
avoided if the
measurement of succinic acid is carned out using the GTP (ITP)-dependent
enzyme
succinyl-CoA synthetase from animal sources. A kit for detecting succinate via
the
succinyl-CoA method is commercially available from Boehringer Mannheim.
The present invention provides a method for detecting a disease state,
25 characterized by a localized hypoxic/ischemic cell population or tissues
comprising
cells exhibiting metabolisms similar to those of hypoxic/ischemic cells. The
method
comprises the step of determining the concentration of succinate in a bodily
fluid
sample, such as blood. The method allows for a simple blood test that is
capable of
detecting the presence of tumor cells, ischemic brain cells, ischemic
myocardial cells
30 and hibernating/stunned myocardial cells and other ischemic/hypoxic
tissues, or other
cells with altered metabolisms similar to those states.
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One embodiment of the present invention comprises a kit for use in
detecting succinate levels in vertebrate bodily fluids. The kit comprises an
enzyme
that reacts with succinate to produce a detectable reactant, wherein the
concentration
of the reactant can be determined and can be correlated with the concentration
of the
succinate substrate. The detection scheme used to quantitate the reactant
should be
sensitive enough to determine the presence of succinate as low as 5 nmoles.
Example 1
Rat Heart Perfusion Studies
Succinate levels in the perfusate were determined according to Kmetec and
Bueding using Ascaris succinoxidase and coupling to the tetrazolium, dye, INT
(Kmetec, Anal. Biochem., 16, 474 (1966)).
Enzymatic Succinate Assay with Int
Reagents:
1. Succinate Std. - 270 2 mg. 270.2 mg of disodium succinate was made to 10
ml (O.1M soln.) in water. Then, 0.1 ml of the O.1M soln. was diluted to 100 ml
(to
produce a 0.1 mm solution) in water. For standard curve use 0.05, 0.1 and 0.2
ml of
diluted sole.
2. 0.2% Gelatin - 20mg of Knox gelatin made to 10 ml in water.
3. 0.2% INT- 20mg Sigma p-Iodonitrotetrazolium violet (also available from
Pierce Biochemicals) made to 10 ml. in water.
Procedure
Control Cuvette Exptl. Cuvette
0.1 ml. Tris buffer, 0.4M (pH=8.5) "
0.2 ml gelatin "
0.33 ml INT "
0.33 ml H20 0.33 ml, sample + H20
Zero Reading
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0.04 ml Ascaris succinate dehydrogenase is added to each Cuvette and the
Cuvette incubated at 37°C in dark until no further increase occurs at
OD 540 mp.
Range = 0.005-0.05 pmoles; O.OOSpmoles reads about 0.060 after subtraction of
blank
(without succinate).
S Note: If ppt. forms upon addn. of INT to sample, the following procedure is
employed: 0.05 ml sample + 0.08 ml Tris (0.4M; pH 8,5) +lml 0.2% INT-Spin at
15,000 RPM for 30 min. Assay aliquot of supernatant for succinate. This
procedure
results in lower, but linear readings and should be adjusted accordingly.
Preparation of Ascaris Succinoxidase
Preparation of Succinoxidase - Modified from Kmetec & Beuding, J. Biol.
Chem. 236, 584 (1961).
Reagents -
a. 0.04M Tris, pH=8.5 containing 3.6 units of catalase per ml
b. 20% Sucrose + 0.04 M Tris, pH 8.5
c. 1.8% Sodium deoxycholate
d. Saturated (NH4)Z S04
40 g. of fresh or frozen Ascaris muscle is minced and homogenized with 4
volumes (160 ml) of 0.04M Tris containing 3.6 units of catalase per ml.
Homogenate
was centrifuged at 6,000 x g for 10 minutes. Enzyme remains in supernate, do
not
take fluffy layer. The supemate is then centrifuged at 100,000 x g for 1.5
hours.
Clear supernate was discarded. The residue was transferred with 2 volumes
(80m1) of
20% sucrose-Tris solution. As much of the transparent glycogen layer on the
bottom
of the tube was left behind. It may be necessary to homogenize the particulate
fraction to obtain a suspension. The suspension was recentrifuged at 144,000 x
g for
1.5 hours. Washed particles were resuspended in the original volume (40 ml) of
sucrose-Tris and frozen overnight. The volume was adjusted so that 1 ml. of
suspension was equivalent to 1 gram of muscle (40 ml). Actual volume was 43
ml.
To this was added 8.5 ml of 1.8% Na deoxycholate slowly over a i 5 minute
period. If
volume is different, deoxycholate added is adjusted accordingly. The mixture
was
incubated in the cold for two hours then frozen overnight. The volume is
measured
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and saturated (NH4)2 S04 added slowly to 25% saturation. The mixture was then
centrifuged at 10,000 RPM for 20 minutes and the supernatant was taken up to
55%
saturation and spun at 10,000 RPM for 20 minutes. Residue of the SS% fraction
was
taken up in 18 ml of sucrose-Tris solution. The enzyme is stored frozen in
test tubes
with 0.3 ml per tube.
Results
Rat hearts were excised from the rat, the blood was rinsed out of the heart
and
the heart was then hooked-up via the aorta to a perfusion apparatus which
circulated a
glucose-buffer solution through the heart in a predetermined 02-NZ-COZ mixture
at
37°C. Samples of the perfusate were collected, usually every 15
minutes. The hearts
were maintained beating for varying time periods up to six hours. After
finishing the
collection of the last perfusate sample, heart was frozen and subsequently
homogenized, centrifuged and assayed anaerobically for fumarate reduction in
1 S mitochondria) preparations. Fumarate reduction was detected
spectrophotometrically
by measuring the anaerobic oxidation of NADH in the presence and absence of
fumarate (referred to as fumerate reductase).
In all instances examined, succinate levels in the perfusate continued to
increase until the hearts became erratic in their beats whereupon the
succinate levels
20 tend to decrease (See Figs. lA-1F). The gas mixtures utilized in the rat
heart
perfusion test comprise OZ levels of 95% O2, $0% OZ and 70% 02. Considerable
amounts of succinate were formed at all of these OZ levels. Lower
concentrations of
OZ are expected to produce even higher concentrations of succinate and such
conditions may be more physiologically relative since the usual concentration
of OZ in
25 the lung would be about 20%. Although in some experiments there was an
indication
that small quantities of succinate were present at zero time, it took 10 to 15
minutes
for the surgery and setting up. Therefore, it can not be determined whether or
not
there is at times a small pool of succinate present endogenously.
Preliminary results from analysis of the homogenized rat myocardium
30 indicate the presence of a fumarate reductase. (See Figs. 2A and 2B). Since
oxygen
can not be excluded completely, some NADH reductase activity could be present
in
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the control. In addition, succinate was quantitated from heart mitochondria
incubated
anaerobically with fumarate plus NADH.
Example 2
S Screening Human Blood Samples for Succinate Levels
Numerous whole blood samples from patients have been analyzed for
succinate including normal healthy "controls". Approximately 70 samples of
whole
blood from patients and 1 S samples of whole blood from volunteers (the
controls)
were analyzed for succinate concentration. Individuals were also analyzed by
PET
10 scanning for the presence of jeopardized myocardium after first obtaining
peripheral
blood samples. The blood samples of 22 patients were analyzed in blinded
fashion for
succinate level after the source patients' hearts had been PET scanned. After
the
succinate levels were determined the blood sample succinate levels were then
correlated with the corresponding PET scan data and the percent of the left
ventricle
1S involved in hibernation or stunning (jeopardized) was compared to the
succinate
blood levels.
In summary, the concentrations of succinate in the blood of patients
not only agreed well with the degree of myocardial hibernation, but also with
another
myocardial deficiency referred to as "stunning". Plotting the percent
jeopardized
20 myocardium. ~. the Ln [succinate] gives a more easily interpreted curve
(see Fig. 3).
Clinically up to 18 percent jeopardized myocardium is considered as
"insignificant"
(noted by horizontal line in Fig. 3). A level of 1 S.2 nmoles of succinate/ml
of
peripheral blood was found to give the best separation between groups and was
used
as the "threshold" level of succinate. Individuals having succinate levels of
less than
2S 1 S.2 nmoles/ml were considered "negative" for purposes of this study (as
noted by the
vertical line in Fig. 3).
As demonstrated in Fig. 3, the sensitivity of blood succinate as an
indicator of jeopardized myocardium (91%), as well as the specificity (86%)
and
accuracy (89%) place blood succinate levels as a viable diagnostic tool, at
30 approximately the same level as PET scanning. All of the data obtained are
in
agreement with the results described in Fig. 3, and these findings agree well
with the
rat heart perfusion experiments described in Example 1.
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To determine the "normal" blood levels of succinate in humans, the
blood succinate levels of 15 "healthy" individuals were determined. The blood
succinate levels ranged from 7.9 to 19.9 nmoles/ml in these individuals, with
an
average value of 15.2 nmoles/ml. The succinate levels in all "healthy"
individuals
were all very low in succinate, with 3 exceptions, all of whom were older
persons.
The higher levels of succinate in these individuals may be due to slower rates
of blood
flow to their hearts. Therefore it is anticipated that higher threshold values
may need
to be set for more elderly patients to account for higher overall succinate
levels in
those patients.
10 Numerous whole blood samples have been analyzed for succinate
levels in addition to the samples that generated the data of Figure 3, and the
data
generated from these additional samples were always consistent with each other
and
with the data of Figure 3.
To determine the succinate blood levels, whole blood samples were
first treated and decolorized in accordance with the following procedure:
Extraction of Succinate from Whole Blood
Mix 1.0 ml blood* and O.Sml 3.0 N HZS04. Vortex and allow to stand
for 10 min at room temperature. Spin for 20 min at 20,000 rpm.
20 Carefully remove supernatant and adjust pH to approx. 7.5 with 2.0 M
KOH. Place the neutralized fraction on ice for 10 min. A precipitate forms.
Spin for
min. at 20,000 rpm.
Remove the supernatant and add to it Norit A activated charcoal
powder, 30 mg/ml of 2rd spin supernatant. Spin for 30 minutes at 20,000 rpm
for the
third time.
Carefully remove supernatant and again add Norit A, 30 mg/ml of 3rd
spin supernatant. Spin again fox 30 minutes at 20,000 rpm.
Remove the supernatant and assay for enzymatic succinate with INT,
using 0.3 ml of final supernatant (Ref: Kmetec., Anal. Biochem. 16, 474
(1966)).
Modification: Yee & Cohen, Anal. Biochem. 22, 530 (1968).
*Note: If whole blood is clotted, add 0.4 M EDTA, 0.02 ml/ml blood,
and homogenize before adding HZS04.
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Two blood samples which were examined had very high succinate
levels, but no apparent cardiac problem. Upon further investigation it was
determined
that one of the patients had lymphoma (a blood cancer). The second blood
sample
came from a woman whose breast cancer had been removed surgically one year
5 earlier. The surgeon thought he had removed the whole tumor. PET scanning
indicated a recurrence at the margin of the prior surgical resection. This
finding was
reinforced by the high concentrations of succinate found in the patient's post-
operative blood, and subsequently by pathology. Therefore, it appears likely
that
succinate blood levels may increase with the occurrence of any tumor in the
cells of
10 the body, regardless of type or location. In accordance with one embodiment
of the
invention a simple blood test is used to screen for cancer. The test comprises
determining the concentration of succinate in the sample to determine if the
concentration is greater than the clinical threshold. This procedure
constitutes a
unique and excellent diagnostic tool.
