ASSAY DEVELOPMENT
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Figure 1. (click to enlarge) The format of the AspirinWorks competitive assay developed by
Corgenix Medical Co. (Broomfield, CO) and its partners. Standards and samples
are added to wells coated with goat antimouse IgG antibody, followed
by an 11dhTxB2 tracer and 11dhTxB2 monoclonal antibody. After a 2-hour
incubation, the wells are washed and a substrate added for 30 minutes. After
a stop solution is introduced, the plates are read by a microplate reader. Color
intensity is inversely proportional to the amount of 11dhTxB2 in the sample
or standard.
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Current blood-based methods of measuring aspirin effectiveness involve analyzing ex vivo platelet activation. These methods require specialized instrumentation, however, are invasive (they require whole blood), and are subject to exogenous factors such as levels of von Willebrand factor, factor VIII, and hematocrit. This article describes an alternative, a novel enzyme-linked immunoassay (ELISA) that measures a stable urinary analyte of thromboxane A2, namely, 11-dehydro-thromboxane B2 (11dhTxB2).
Demonstrating good analytical performance, this assay requires only ELISA instrumentation commonly found in clinical laboratories. Evidence is presented to show that it offers a precise, noninvasive method for identifying subjects who may not be receiving a beneficial effect from their therapeutic dosage of aspirin.
Aspirin and Cardiovascular Disease
Atherothrombosis of the coronary, cerebrovascular, and peripheral arterial circulation is the world’s leading cause of human morbidity and mortality, and its prevalence is increasing.3 One of the key aspects of atherothrombosis is the activation and aggregation of platelets.4 Antiplatelet therapies thus play a central role in the attempt to manage this disorder. A major opportunity for antiplatelet therapy has been presented by the natural tendency of acetylsalicylic acid (ASA), or aspirin, to inhibit the cyclooxygenase pathway. ASA’s antithrombotic effects have been known for many years.5
Aspirin’s level of effectiveness is well documented. One study found that low-dose aspirin reduced the incidence of cardiovascular events by as much as 25% in patients with arterial vascular disease.6 Similarly, in high-risk vascular patients, aspirin therapy resulted in a 34% reduction in nonfatal myocardial infarction, a 25% decrease in nonfatal stroke, and an 18% decrease in all-cause mortality, according to another recent report.7
Aspirin functions by irreversibly acetylating the platelet cyclooxygenase-1 (COX-1) enzyme, thus inactivating it for the life of the platelet.8 Aspirin inhibits also COX-2, a second cyclooxygenase isoform induced by inflammatory stimuli, but to a much smaller extent than COX-1.8 Low-dose aspirin blocks more than 95% of platelet COX-1 activity.9 This results in a decrease in the production of TxA2.
Aspirin Resistance
Although the mechanism of aspirin’s inhibition of COX-1 is clear, recent reports suggest that not all patients respond to low-dose aspirin to the same degree. The lack of response to ASA has historically been referred to as aspirin resistance, and its prevalence varies depending on the diagnostic method used.10 The wide variation in the determination of prevalence is partly due to the definition of aspirin resistance, ranging from patients who experience an ischemic event while on aspirin (clinical resistance) to those experiencing a lack of reduction in levels of TxA2, platelet activation, or aggregation (biochemical resistance).11
The underlying causes of aspirin resistance, which might better be described as aspirin nonresponsiveness, likely are several, including genetic variations, stress-induced increased turnover of platelets, alternative pathways of platelet activation, other sources of thromboxane production (by COX-2 in monocytes or macrophages, for example), and aspirin bioavailability issues.11
A recent study suggested that patients with coronary artery disease and associated increases in cholesterol, triglyceride, and C-reactive protein levels had a reduced response to aspirin, as measured by both blood-based platelet aggregation methods and methods involving urinary 11dhTxB2.12 Compared with asymptomatic patients, those with coronary artery disease demonstrated significantly higher urinary 11dhTxB2 levels following aspirin ingestion. Regardless of the reason for nonresponsiveness, variability among patients in their response to aspirin is clear.13
Methods of Analysis
Owing to the variability in responsiveness to ASA, a reliable method is needed for analyzing an individual’s particular aspirin response and helping determine whether a change in therapy should be considered. Currently, there are two fundamental approaches to measuring aspirin efficacy: platelet aggregation methods and urinary 11dhTxB2 measurement.14
Platelet aggregation methods come in different forms, but they generally involve collection of patient blood samples and measurement of aggregation in an ex vivo test. Although these tests are relatively quick, they are sensitive to variables that have nothing to do with aspirin’s effectiveness, such as levels of von Willebrand factor, factor VIII, and hematocrit, and the fact that platelets can be activated merely by a variation in temperature.15,16 These variables may explain the results of a recent study that compared two such methods and demonstrated poor agreement between them.17
Additionally, other platelet aggregation methods require specialized instrumentation, which limits the ability of the clinician to run large sample batches in one test procedure. For this and the other reasons just enumerated, a simple urinary test that directly measures a TxA2 metabolite may be a more appropriate means of determining an individual’s response to aspirin.
11-Dehydro-Thromboxane B2
Besides being a popular analgesic, aspirin, owing to its ability to inhibit TxA2 synthesis, its minimal cost, and its proven clinical efficacy in the prevention of ischemic events, is a successful and widely prescribed antiplatelet drug. Direct measure-ment of a patient’s response to aspirin, then, should involve analysis of circulating levels of TxA2. Unfortunately, TxA2 has a very short half-life in the blood.1 This makes it a difficult analyte to measure. TxA2 is metabolized to thromboxane B2 (TxB2), however, which is converted enzymatically into a number of metabolites, including 11dhTxB2 and 11-dehydro-2,3-dinorthromboxane B2 (11dh2,3DTxB2), which are cleared by the kidney and excreted in the urine.18,19 A very stable molecule in urine, 11dhTxB2 is the most abundant urinary metabolite of TxB2.20 It has a relatively long circulating half-life of 45 minutes.19
The usefulness of urinary 11dhTxB2 as a measure of thromboxane production was confirmed in a study of TxB2 metabolism.21 In the experiments, measured urinary levels of 11dhTxB2 provided a more accurate indication of in vivo thromboxane metabolism than TxB2 measured in the blood, the latter method being confounded by technical difficulties encountered in the blood collection process itself. Thus, quantitation of urinary 11dhTxB2 and its truncated form, 11dh2,3DTxB2, may serve as the best means of measuring TxA2 production by platelets and making an accurate determination of in vivo platelet activation.19,22
An important study recently compared aspirin treatment as measured by urinary 11dhTxB2 levels with values gained by means of two different platelet aggregation methods, and related the findings to atherosclerosis risk factors.23 For the investigation, asymptomatic patients at increased risk for coronary heart disease were given 81 mg of ASA per day for two weeks. Meanwhile, researchers obtained urine samples from them and determined 11dhTxB2 levels. Blood samples were drawn in parallel to this, and platelets were analyzed by aggregation methods. Patients were characterized according to the presence or absence of other atherosclerosis risk factors besides their responsiveness to aspirin.
The results of the analytical comparisons indicated that only individuals with nonresponsive urinary 11dhTxB2 levels were associated with a greater cardiovascular risk profile. The platelet aggregation platforms failed to demonstrate this relationship.
The 11dhTxB2Assay
In collaboration with its strategic partners Cayman Chemical Co. (Ann Arbor, MI) and Creative Clinical Concepts (Denver), Corgenix Medical Co. (Broomfield, CO) has developed an ELISA for the detection of 11dhTxB2 in human urine that it calls the AspirinWorks Test Kit. This is a competitive assay that involves microtiter-plate wells coated with goat antimouse IgG antibody (see Figure 1).
The test begins with standards and samples being deposited in the wells, followed by addition of an 11dhTxB2 tracer. This tracer is the 11dhTxB2 molecule conjugated to alkaline phosphatase. Subsequent addition of a mouse monoclonal anti-11dhTxB2 antibody initiates the reaction. During the 2-hour incubation, the endogenous 11dhTxB2 and the 11dhTxB2 tracer compete to bind to the mouse anti-11dhTxB2 antibody, which is captured on the wells by the coated goat antimouse IgG antibody. The wells in the microtiter plate then are washed to remove unbound enzyme. Next, the test technician adds p-nitrophenyl phosphate (pNPP) substrate, which is incubated for 30 minutes. The addition of a stop solution of 0.1-M ethylenediamine– tetraacetic acid (EDTA) terminates the color reaction. The plate then is read at 405 nm in a standard microtiter plate reader.
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Table I. (click to enlarge) Specificity of the 11dhTxB2 monoclonal antibody in detecting thromboxane B2 metabolites and eicosanoids. PGEM and PGFM are prostaglandin E and F metabolites.
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As urinary creatinine values are necessary in order to normalize for urine dilution, spot urine samples may be used. There is no require-ment for a 24-hour collection or a first morning void. The final value is expressed as picograms of 11dhTxB2 per milligram of creatinine.
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Table II. (click to enlarge) Data from interference studies performed on the AspirinWorks Test Kit. Compounds and metabolites common in the urine of aspirin users were tested in human urine samples at physiologically excessive concentrations, and the 11dhTxB2 values determined were compared with control. The average recovery was 105.9% with reference to the control.
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Analytical Performance
Experiments were conducted on the AspirinWorks Test Kit to determine its analytical performance. Initial testing revealed a limit of detection for 11dhTxB2 in a sample of 222 pg/ml, with a linear detection range of 300–4000 pg/ml. Other parameters measured were interference, precision, linearity, and stability.
At the outset, studies were designed to determine the interfering effect, if any, on the measurement of 11dhTxB2 caused by the presence of high concentrations of certain compounds that may often be found in the urine of aspirin takers. ASA and its metabolite salicylic acid are expected to be in the urine of patients taking aspirin. Acetaminophen is a common antiinflammatory drug. Ascorbic acid is a common dietary supplement, and caffeine is a constituent of coffee, tea, and soft drinks that are routinely consumed. Glucose can be found in urine in certain circumstances, as can hemoglobin. All of these compounds present in many patients’ urine showed no significant interference with the detection of 11dhTxB2 using the assay (see Table II).
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Table III. (click to enlarge) AspirinWorks precision calculations from samples run over
multiple plates and kit lots.
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Figure 2. (click to enlarge) Linearity plot of sample values at various dilutions. Five different
urine samples were run for the linearity study. |
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Figure 3. (click to enlarge) Three 11dhTxB2 controls supplied in the AspirinWorks kit were run
over a period of 12 months and the values plotted to reveal good kit
stability.
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Effects of Aspirin Therapy
The test kit under study is a competitive ELISA that measures 11dhTxB2 levels in human urine to determine aspirin’s effect in individuals. To test this capability, 171 urine samples from three groups of people—those not taking aspirin, those on 81 mg of aspirin a day for 7 days, and those on 325 mg a day for 7 days—were run on the test kit. Eighty-seven of these samples were taken from subjects not on aspirin, 47 from subjects ingesting 81 mg a day, and 37 from individuals taking 325 mg a day. Values were normalized for urinary dilution by dividing by the creatinine concentration.
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Figure 4. (click to enlarge) A box plot comparison of 11dhTxB2 baseline samples with
samples taken from aspirin users. The boxes represent values between the
75th and 25th percentiles, and the horizontal line within each box represents
the median for the group. Dots are sample values falling outside the 90th and
10th-percentile boundaries, which are signified by the bars.
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Figure 5. (click to enlarge) A frequency plot of urinary 11dhTxB2 concentration values comparing
subjects on therapeutic aspirin with those not taking aspirin.
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However, some samples derived from aspirin users also were elevated, reaching levels as high as 3900 pg per milligram of creatinine. This demonstrates again that there is variability in response to aspirin among individuals, and that some portion of the population does not respond effectively to aspirin therapy.
Conclusion
Aspirin continues to be prescribed to millions of people as an aid in the prevention of thrombotic events. However, its effectiveness varies among individuals, suggesting a need for a test to monitor aspirin effect in specific patients. Current techniques for measuring aspirin effectiveness are ex vivo platelet aggregation methods that are susceptible to variables not related to the cyclooxygenase pathway. They are invasive and require specialized instrumentation that makes it difficult to run large numbers of samples at one time. Therefore, there is a clinical need for a noninvasive, simple, and accurate means of confirming aspirin’s effect in individuals on aspirin therapy.
Urinary 11dhTxB2 is a very stable molecule and a good measure of levels of circulating TxA2. The test developed by Corgenix and its partners and described in this article is precise, is effective over a range of urinary dilutions, and provides accurate recovery within the wide concentration range of 300–4000 pg/ml. These performance characteristics suggest that it could be a useful test to determine if aspirin is having the desired effect in an individual patient. Further studies are underway to strengthen the clinical utility of the test.
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F. Jon Geske, PhD, is an
R&D director and project
manager at Corgenix Medical
Co. (Broomfield, CO). He can be reached at
jgeske@corgenix.com.
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Luis R. Lopez, MD, ia the
chief medical officer at
Corgenix Medical Co. He can be reached at
llopez@corgenix.com.
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Ivana J. Muncy is a
research associate at
Corgenix Medical Co. She can be reached at imuncy@corgenix.com.
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Daniel J. Tew is an R&D
scientist at Cayman Chemical
Co. (Ann Arbor, MI). He can be reached at dtew@caymanchem.com.
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