Thromboxane B.sub.2 assay for coronary artery thrombosis

This invention is directed to the use of thromboxane as an early indicator for coronary artery thrombosis (acute myocardial infarction), and assays for detecting thromboxane B.sub.2 or a metabolite of TXB.sub.2 or TXA.sub.2.

FIELD OF THE INVENTION 
This invention is directed to the use of thromboxane B.sub.2 or a 
metabolite of TXB.sub.2 or TXA.sub.2 as an early indicator for coronary 
artery thrombosis (acute myocardial infarction), and assays for detecting 
thromboxane B.sub.2 or a metabolite of TXB.sub.2 or TXA.sub.2. 
BACKGROUND OF THE INVENTION 
Activation of blood components such as lymphocytes, granulocytes, 
monocytes, and platelets is associated with the release of thromboxane 
A.sub.2 and other metabolites of arachidonic acid. Thromboxane A.sub.2 
(TXA.sub.2) is formed from metabolism of arachidonic acid by the enzyme 
cyclooxygenase via cyclic endoperoxide intermediates. TXA.sub.2, the major 
cyclooxygenase product in the platelet, is a vasoconstrictor and potent 
stimulus to platelet aggregation in vitro. 
Platelets express a high synthetic capacity for thromboxane A.sub.2 
(TXA.sub.2) and therefore this product may have clinical diagnostic 
significance. However, TXA.sub.2 itself is chemically unstable with a half 
life of less than one minute. For this reason the stable breakdown product 
thromboxane B.sub.2 (TXB.sub.2) and its primary metabolite 2,3 
dinor-TXB.sub.2 are usually measured by RIA when large numbers of samples 
are required. 
FitzGerald et al., "Analysis of Prostacyclin and Thromboxane Biosynthesis 
in Cardiovascular Disease," Circulation 67(6):1174 (1983) focuses on the 
methods that have been used for measuring prostacyclin and thromboxane 
A.sub.2 in human biological fluids. After reviewing the difficulties 
associated with invasive analysis techniques for measuring prostacyclin 
and thromboxane A.sub.2, the authors assert that the measurement of 
urinary metabolites represents the only noninvasive approach to 
quantitation of endogenous prostacyclin and thromboxane A.sub.2 
biosynthesis. In conclusion, the report states that further research is 
needed to define the relationship of tissue-specific, capacity-related 
indexes to endogenous production rates of prostacyclin and thromboxane 
A.sub.2. 
In thrombotic events such as deep vein thrombosis (Foegh et al., "Urine 
i-TXB.sub.2 in Renal Allograft Rejection," Lancet II:431-434 (1981)) and 
pulmonary embolism (Klotz et al.. "Urinary Excretion of Thromboxane 
B.sub.2 in Patients with Venous Thromboembolic Disease," Chest 85:329-335 
(1984)), and in renal and cardiac transplant rejection (Foegh et al.. 
"Lipid Mediators in Organ Transplantation," Transplant. Proc. 18, suppl. 
4: (1986)), the urinary excretion of immunoreactive-TXB.sub.2 
(i-TXB.sub.2) which includes 2,3 dinor TXB.sub.2, is significantly 
increased. 
For illustration, in Foegh et al., Lancet, supra report that not only may 
urinary i-TXB.sub.2 be a predictor of clinical renal allograft rejection, 
it may also be a used in the early diagnosis of venous thrombosis. Klotz 
et al., Chest, supra, studied the use of urinary i-TXB.sub.2 as an 
indication of platelet activation and as a possible adjunct for diagnosing 
acute thromboembolic disease. 
Coronary artery thrombosis, which may lead to acute myocardial infarction, 
is another thrombotic event where an increase in thromboxane formation 
might be anticipated. Attempts to measure plasma values are not entirely 
satisfactory owing to the large potential for sampling artefacts. 
(Granstrom et al., "The Thromboxanes," in Prostaglandins and Related 
Substances, (C. Pace-Asciak and E. Granstrom eds.), Elsevier, Amsterdam 
(1983), p. 45) Urinary i-TXB.sub.2 determinations provides a means of 
avoiding such artefacts but whether infarction is associated with a rise 
in urinary i-TXB.sub.2 is not known. 
SUMMARY OF THE INVENTION 
This invention is directed to the use of thromboxane B.sub.2 or a 
metabolite of TXB.sub.2 or TXA.sub.2 as an early indicator for coronary 
artery thrombosis (acute myocardial infarction), and assays for detecting 
thromboxane B.sub.2 or a metabolite of TXB.sub.2 or TXA.sub.2.

DETAILED DESCRIPTION OF THE INVENTION 
Thromboxane B.sub.2 has been shown to be of use for the diagnosis of renal 
allograft rejection (M. L. Foegh et al., "Urine i-TXB.sub.2 in Renal 
Allograft rejection," Lancet ii:431 (Aug. 81)) and deep venous thrombosis 
(T. A. Klotz et al., "Urinary Excretion of Thromboxane B.sub.2 in Patients 
with Venous Thromboembolic Disease," Chest 85(3):329 (Mar. 84)). In Klotz 
et al., the authors also tested patients with coronary thrombosis, but 
were unable to show any significant increase in thromboxane levels in such 
patients. The accepted explanation for this has been that the thrombus 
developed in that case are rather small compared to the thrombus that are 
developed in venous thromboembolic diseases. 
The fact that Klotz et al. were unable to show any difference between 
thromboxane B.sub.2 levels in patients suffering from myocardial 
infarction or angina pectoris indicated that thromboxane could not be used 
as an early indicator for infarction, and consequently the onset of 
treatment with the proper thrombolytic agent had to be postponed, which 
entailed an increased risk for the patient. 
It is believed that approximately 50% of those patients who die from an 
infarction could be saved if an early indication would allow for an early 
onset of the proper treatment. Especially for people who are susceptible 
to thrombic events, this assay described and claimed herein is of great 
importance. 
This invention is therefore directed to an assay for measuring for the 
presence of thromboxane B.sub.2 or a metabolite of TXB.sub.2 or TXA.sub.2 
in a sample from a patient suspected of having coronary artery thrombosis 
or a myocardial infarction. As used herein the measurement of thromboxane 
B.sub.2 may also include the measurement of metabolites of thromboxane 
B.sub.2, such as 2,3-dinor TXB.sub.2 or 11-dehydro-TXB.sub.2, and other 
known metabolites. This invention is also meant to include other 
metabolites of TXA.sub.2 which can be measured. In this invention, the 
measurement of thromboxane B.sub.2 is also meant to mean the measurement 
one or more of the metabolites of thromboxane B.sub.2 alone, such as the 
measurement of 2,3-dinor TXB.sub.2 or 11-dehydro-TXB.sub.2, or the 
measurement of one or more of the metabolites of thromboxane A.sub.2 
alone. 
Coronary artery thrombosis is generally a physiologic event resulting from 
a thrombus in the coronary artery. Coronary artery thrombosis can lead to 
acute myocardial infarction which is defined as an area of necrosis in the 
heart tissue resulting from obstruction of the local circulation by a 
thrombus or embolus. Pain is frequently associated with myocardial 
infarction, however, other heart ailments are also associated with pain 
but not with thrombus, such as angina. 
The assay of this invention involves the measurement of thromboxane B.sub.2 
using methods known in the art, such as gas chromatography/mass 
spectrography (GC/MS) or radioimmunoassay (RIA). Other immunoassays may 
also be used including immunometric assays, such as forward sandwich 
immunoassays, reverse sandwich immunoassays, and simultaneous assays, and 
competitive assays. 
With the immunometric assays, and the radioimmunoassay, a high titer 
antibody is used that is specific against TXB.sub.2, or a metabolite of 
TXB.sub.2, such as 2,3-dinor TXB.sub.2 or 11-dehydro-TXB.sub.2, or a 
metabolite of TXA.sub.2. The antibody can be either monoclonal antibody or 
polyclonal antibody. 
The sample to be tested for coronary artery thrombosis may any biological 
sample in which TXB.sub.2 can be measured. Typically the sample will be 
blood, plasma, serum, or urine. 
The urine sample collected from the patients may be extracted or purified 
prior to the assay. If an immunometric or RIA is performed, the 
thromboxane in the urine sample may be measured by direct assay without 
extraction. Typically, however, the urine will be extracted either by 
centrifugation alone, or the sample can be collected and mixed with 
indomethacin at 10-20 ug/ml urine and then centrifuged. The indomethacin 
does not interfere with the assay of i-TXB.sub.2. If a GC/MS assay is 
used, the urine sample is first purified using known means in the art. 
The immunoassays in the method of the invention are ideally suited for 
preparation of a kit. Such a kit may comprise a carrier means being 
compartmentalized to receive in close confinement one or more container 
means such as vials, tubes, and the like, each of said container means 
comprising the separate elements of the immunoassay. For example, there 
may be a container means containing the first antibody immobilized on a 
solid phase support, and further container means containing detectably 
labeled titrating antibodies either lyophilized or in solution. 
Further container means may contain standard solutions comprising serial 
dilutions of thromboxane B.sub.2 to be detected. The standard solutions 
may be used to prepare a standard curve with the concentration of the 
thromboxane B.sub.2 plotted on the abscissa and the detection signal on 
the ordinate. The results obtained from a urine sample containing 
thromboxane B.sub.2 may be interpolated from such a plot to give the 
concentration. 
In another embodiment of the kit, there may be a container means containing 
a dipstick which comprises a thromboxane B.sub.2 antibodies immobilized to 
a defined area of the solid phase support. Further container means may 
contain a common titrating antibody which is specific for the thromboxane 
B.sub.2 antibody. Further container means may contain standard solutions 
of thromboxane B.sub.2 to be detected. The standard solutions of the 
thromboxane B.sub.2 may be used to provide a standard reference dipstick 
for comparison with the sample dipstick. 
Thus, this invention is directed to the use of thromboxane B.sub.2, and/or 
its metabolites, and/or the metabolites of thromboxane A.sub.2 as an early 
indicator for coronary artery thrombosis prior to acute myocardial 
infarction. Further, this invention is directed to the use of thromboxane 
B.sub.2, and/or its metabolites, and/or the metabolites of thromboxane 
A.sub.2 in detecting acute myocardial infarction. For example, the assay, 
in kit form, may be performed by a patient in order to monitor his or her 
situation. Such assays will put the patient and/or physician in a better 
position to select a suitable treatment, such as by prescribing 
thrombolytic agents, at an early stage, and preferably prior to the 
infarction. Nonsteroidal anti-inflammatory drugs, such as aspirin, inhibit 
the cyclooxygenase enzyme such that thromboxane A.sub.2 is not formed from 
the metabolism of arachidonic acid. Thus, the use of aspirin must be 
documented to have an accurate measurement of thromboxane according to 
this invention. If nonsteroidal anti-inflammatory drugs have been used by 
a patient prior to the assay of this invention, then repeat testing is 
generally warranted. 
Having generally described the invention, further understanding can be 
obtained by reference to certain specific examples which are provided 
herein for purposes of illustration only and are not intended to be 
limiting unless otherwise specified. 
EXAMPLES 
Immunoreactive thromboxane B.sub.2 (i-TXB.sub.2) was measured by 
radio-immunoassay (RIA) in urine collected over eight hours on the day of 
admission in 25 patients who were admitted with the diagnosis of 
myocardial infarction. In 16 of the patients, myocardial infarction was 
confirmed by ECG and plasma enzymes. Another patient presented with 
pulmonary embolism and the remaining eight patients had angina pectoris. A 
further eight hour urine collection was obtained 24 hours later from 
eleven of the sixteen patients with myocardial infarction. In these eleven 
patients myocardial infarction was associated with five fold higher urine 
i-TXB.sub.2 (2.72 .+-.0.48 ng/ml) at the day of admission when compared to 
patients admitted under the same diagnosis but found to have angina only 
(0.51.+-.0.08 ng/ml, p&lt;0.001). In patients with myocardial infarction the 
urine i-TXB.sub.2 values were reduced 24 hours later (1.58.+-.0.27 ng/ml, 
p &lt;0.01). One patient was followed with urine i-TXB.sub.2 from three days 
prior to diagnosis of myocardial infarction and to one day prior to a 
second infarction. In this patient i-TXB.sub.2 was highest three days 
prior to infarction. We conclude that this early elevation of urine 
i-TXB.sub.2 three days prior to diagnosis of infarction and the increased 
i-TXB.sub.2 in patients with myocardial infarction when compared to 
patients with angina suggest thromboxane is probably released from 
activated platelets prior to infarction. Thus, the measurement of urine 
i-TXB.sub.2 is of value in the differential diagnosis between coronary 
artery thrombosis and early myocardial infarction on the one hand and 
angina pectoris not caused by thrombotic events on the other hand. 
PATIENTS AND METHODS 
Twenty-five patients aged 45 to 86 years old (average 63) were admitted to 
the intensive cardiac care unit at the Herley Hospital with anginal pain 
and possible myocardial infarction. Urine were collected during an 
eight-hour period for urine i-TXB.sub.2 determination by RIA. Sixteen 
patients had myocardial infarction diagnosed by electrocardiogram and 
serum enzymes (lactic dehydrogenase and aspartate transferase). Eleven of 
the 16 patients had in addition i-TXB.sub.2 determined in an eight-hour 
urine sample which was collected 24 hours later. One patient had pulmonary 
embolism, and the remaining patients had angina but not myocardial 
infarction. Two patients from the group with myocardial infarction were 
excluded due to extremely low urine i-TXB.sub.2 concentrations in the 
range which occurs following ingestion of aspirin (Fitzgerald et al., 
"Endogenous biosynthesis of prostacyclin and thromboxane and platelet 
function during chronic administration of aspirin in man," J. Clin. 
Invest. 71:676-688 (1983)), although the medical history did not indicate 
this. At Georgetown University Hospital a renal transplant patient who 
developed myocardial infarction was admitted for pneumonia three months 
after transplantation. He was followed with urinary i-TXB.sub.2 which is a 
routine procedure for the renal transplant patients. 
The urine were collected and mixed with indomethacin, centrifuged and an 
aliquot was stored at -20.degree. C. Urine i-TXB.sub.2 was determined by 
radio-immunoassay as previously described with the widely used antibody of 
Dr. L. Levine of Brandeis University, Mass. (Foegh et al., Lancet 
II:431-434 (1981)). This is a high titer TXB.sub.2 antibody which 
cross-reacts 60% with the major metabolite in urine of TXA.sub.2, namely 
2,3-dinor TXB.sub.2 (Roberts et al., "Metabolism of thromboxane B.sub.2 in 
man: Identification of twenty urinary metabolites," J. Biol. Chem. 
256:8384-8393 (1981)). Urinary determinations made by direct RIA with the 
same antibody as used here, was found to correlate with urinary 2,3-dinor 
TXB.sub.2 as determined by gas chromatography, mass spectrometry in 
patients with deep vein thrombosis and pulmonary embolism (Zipser et al., 
"Urinary thromboxane B.sub.2 and prostaglandin E.sub.2 in the hepatorenal 
syndrome: evidence or increased vasoconstrictor and decreased vasodilator 
factors," Gastroenterology 84:697-703 (1983)). Previous studies have 
demonstrated that urinary i-TXB.sub.2 excretion is influenced by urinary 
flow rate, such that the concentration of i-TXB.sub.2 tends to remain 
constant during different rates of urine flow (Zipser et al., "Regulation 
of urinary thromboxane B.sub.2 in man: influence of urinary flow rate and 
tubular transport," Prostaglandins 87:1228-1232 (1984)), consequently 
urinary i-TXB.sub.2 is expressed as ng/ml, since fluid intake was not 
regulated. 
RESULTS 
The diagnosis of myocardial infarction was based on ECG and plasma enzyme 
changes in all cases. Sixteen of the 25 patients had myocardial 
infarction, but two were excluded due to extremely low urinary i-TXB.sub.2 
(&lt;0.09 ng/ml urine) which was characteristic of aspirin ingestion. A 
statistical significant difference was found between urine i-TXB.sub.2 in 
patients with myocardial infarction (2.72.+-.0.48 ng/ml) and in patients 
with angina without infarction (0.51.+-.0.08 ng/ml; p&lt;0.001). In 
eight-hour urine samples which were collected 24 hours later from the 
group with myocardial infarction, the urinary i-TXB.sub.2 values decreased 
to 1.58 .+-.0.27 ng/ml. These values were still significantly higher 
(p&lt;0.05) than those from patients with angina only (FIG. 1). Three 
patients died due to myocardial infarction; however, the urine i-TXB.sub.2 
level was not higher than in the patients surviving the episode. No 
correlation was found between urine i-TXB.sub.2 and serum lactate 
dehydrogenase or aspartate transferase in the patients with myocardial 
infarction. The medication prior to admission was similar in patients with 
and without myocardial infarction (Table I). During the stay in the 
intensive cardiac care unit the patients continued their usual medication; 
in addition, digoxin and furosemide were initiated in 3 and 5 patients, 
respectively, with myocardial infarction. 
TABLE I 
______________________________________ 
Drugs administered to patients, before 
admission to the cardiac intensive care unit, 
who developed infarction (+M.I.) and those who 
did not (-M.I.). The drugs were continued 
after admission. 
DRUG +M.I. -M.I. 
______________________________________ 
NITROGLYCERINE 2 1 
THIAZIDE 5 1 
FUROSEMIDE 4 4 
DIGOXIN 3 3 
BETA-BLOCKERS 4 1 
Ca.sup.++ BLOCKERS 1 0 
LIDOCAINE 0 1 
INSULIN 2 1 
______________________________________ 
The renal transplant patient admitted with pneumonia who was routinely 
followed with daily urine i-TXB.sub.2 exhibited elevated values three days 
prior to diagnosis of myocardial infarction, after which the values 
decreased. On the day of angina and the diagnosis of myocardial infarction 
by ECG and characteristic enzyme changes, the i-TXB.sub.2 value was 
approximately the same (3.01 ng/ml) as that measured in the patients 
admitted with myocardial infarction (2.72.+-.0.48 ng/ml). The urine 
i-TXB.sub.2 value of this transplant patient continued to decrease after 
the diagnosis of myocardial infarction as seen in the patients with 
myocardial infarction from the Herlev Hospital. A second rise in urine 
i-TXB.sub.2 occurred four days later and the patient died the following 
day from a second myocardial infarction (FIG. 2). 
DISCUSSION 
Urine i-TXB.sub.2 values were significantly elevated five to six fold on 
the day of acute admission of patients subsequently proven to have 
myocardial infarction, when compared to the patients only presenting with 
angina only. The urine i-TXB.sub.2 values subsequently decreased the 
following day and approached the values measured in patients with angina 
only. The data from the renal transplant patient who had two consecutive 
infarcts suggest that urine i-TXB.sub.2 increases prior to the development 
of myocardial infarction. The i-TXB.sub.2 values are elevated at the time 
of diagnosis and decrease over subsequent days as seen with the patients 
at the Herlev Hospital. The case of the renal transplant patient was 
noteworthy in that the second myocardial infarction was also preceded by a 
second increase in urine i-TXB.sub.2. 
In renal transplant patients urinary i-TXB.sub.2 is increased prior to the 
clinical diagnosis of rejection Foegh et al., Lancet, supra.; the 
i-TXB.sub.2 values observed are lower than those seen in the kidney 
transplant patient with myocardial infarction. Moreover, clinically the 
patient was not undergoing a rejection episode by standard criteria. The 
patient was admitted with pneumonia which does not elevate urinary 
i-TXB.sub.2 Foegh et al., Lancet, supra. Finally, urine i-TXB.sub.2 is not 
increased with a decline in kidney function. On the other hand, elevated 
urinary i-TXB.sub.2 of this magnitude may be due to deep vein thrombosis 
(Foegh et al., Lancet, supra.; Klotz et al., Chest, supra.) but this was 
not the case. We conclude that the two elevated urinary i-TXB.sub.2 values 
in this patient were associated with the two infarction episodes. This 
observation strongly indicates that measurement of urine i-TXB.sub.2 is 
capable of detecting on-going coronary thrombosis at a time when damage to 
the myocardium (myocardial infarct) has not yet occurred. 
Plasma TXB.sub.2 is reported to be detectable on the day of myocardial 
infarction in a group of 10 patients Strano et al., Thromb Haemostas 
46:759 (1981). This observation is in accordance with the increase in 
urine i-TXB.sub.2 seen in our patients with myocardial infarction. Thus, 
elevated urinary i-TXB.sub.2 is useful in the differential diagnosis of 
angina and angina associated with coronary artery thrombosis or myocardial 
infarction. The increase in urine i-TXB.sub.2 which occurred 3 days prior 
to coronary thrombosis is similar to the increase previously reported 5 
days prior to diagnosis of deep vein thrombosis Foegh et al., Lancet. 
supra. Furthermore, urine i-TXB.sub.2 values measured prior to coronary 
thrombosis was of the same magnitude as seen prior to deep vein 
thrombosis. This suggests that a general activation of platelets may take 
place. The observation that urine i-TXB.sub.2 is elevated prior to 
diagnosis of myocardial infarction should make intervention more 
effective. 
In summary, patients with myocardial infarction exhibit significantly 
higher values of urine i-TXB.sub.2 than patients with angina. The urinary 
i-TXB.sub.2 decreases rapidly 24 hours after diagnosis. A case history 
suggests that urine i-TXB.sub.2 may increase several days prior to the 
myocardial infarction at the time when the thrombus may be only in cardiac 
formation. Urinary i-TXB.sub.2 may help to differentiate between angina 
alone and angina associated with coronary thrombosis or myocardial 
infarction. 
Although the foregoing invention has been fully described by way of 
illustration and example for purposes of clarity and understanding, it 
will be obvious that certain changes and modifications may be practiced 
within the scope of the invention, as limited only by the scope of the 
appended claims.