For appropriate pain medication monitoring, the analytical method must be sensitive enough to detect the prescribed medication and metabolites at a sufficiently low concentration to recognize ...compliance, even with a low-dose prescription. The method must also provide excellent selectivity to identify simultaneously present drugs even with similar chemical structures. The analytical method should uncover common illicit drugs/nonprescribed medications. Traditional immunoassays cannot satisfy these criteria, but liquid chromatography tandem mass spectrometry can. It requires expensive instrumentation, careful test design, and extensive validation and produces a large amount of data that must be interpreted according to the clinical context.
Chronic pain is often treated with narcotic analgesics. The most commonly used narcotic analgesics are the opiates (natural or modified compounds of the poppy plant) or opioids (synthetic chemicals ...that act on opiate receptors). While opiates and opioids are excellent analgesics, they can also have significant side effects that include respiratory depression, coma, or death. Tolerance, physical dependence, and addiction (psychological dependence) are other severe side effects of opioid use. Patients who develop dependence or addiction often times abuse other, non-opioid narcotics and may trade their prescription medication for illegal street drugs (called "diversion"). In order to minimize side effects, detect possible multidrug abuse and prove diversion, simultaneous monitoring of numerous prescription and illicit drugs is required. The method described in this chapter is for the quantitative measurement of 43 different drugs in urine. The panel includes narcotic pain medications, benzodiazepines, NIDA drugs, and other, commonly abused medications. The analytes of interests are injected in the presence of deuterated internal standards to correct for possible extraction inefficiencies, ion suppression, or other interferences. The sample is prepared by adding dilution buffer with the deuterated internal standards to the sample, followed by reversed-phase, gradient HPLC separation on a Phenyl-Hexyl column using water and methanol as mobile phases. Detection of the analytes of interest is done by isotope-dilution mass spectrometry on a triple-quadrupole tandem mass spectrometer following electrospray ionization in the positive mode. Mass spectrometric (MS) data are collected in the scheduled MRM (sMRM) mode. Two MRM transitions are monitored for each analyte and one MRM transition is monitored for each IS. Quantitation of the unknown analytes is achieved by comparing the peak area ratios of the analytes to that of the internal standards and reading the unknown concentration from a seven-point calibration curve.
In the failing human heart myofibrillar calcium sensitivity of tension development is greater and maximal myofibrillar ATPase activity is less than in the normal heart. Phosphorylation of the cardiac ...troponin I (cTnI)-specific NH2-terminus decreases myofilament sensitivity to calcium, while phosphorylation of other cTnI sites decreases maximal myofibrillar ATPase activity.
We examined cTnI phosphorylation in left ventricular myocardium collected from failing hearts at the time of transplant (n=20) and normal hearts from trauma victims (n=24). The relative amounts of actin, tropomyosin, and TnI did not differ between failing and normal myocardium. Using Western blot analysis with a monoclonal antibody (MAb) that recognizes the striated muscle TnI isoforms, we confirmed that the adult human heart expresses only cTnI. A cTnI-specific MAb recognized two bands of cTnI, designated cTnI1 and cTnI2, while a MAb whose epitope is located in the cTnI-specific NH2-terminus recognized only cTnI1. Alkaline phosphatase decreased the relative amount of cTnl1, while protein kinase A and protein kinase C increased cTnI1. The percentage of cTnI made up of cTnI1, the phosphorylated form of TnI, is greater in the normal than the failing human heart (P<.00).
This phosphorylation difference could underlie the reported greater myofibrillar calcium sensitivity of failing myocardium. The functional consequence of this difference may be an adaptive or maladaptive response to the lower and longer calcium concentration transient of the failing heart, eg, enhancing force development or producing ventricular diastolic dysfunction.
Perioperative myocardial infarction is the most common cause of morbidity and mortality in patients who have had noncardiac surgery, but its diagnosis can be difficult. The present study was designed ...to determine whether the measurement of serum levels of cardiac troponin I, a highly sensitive and specific marker for cardiac injury, would help establish the diagnosis of myocardial infarction.
We obtained preoperative measurements of MB creatine kinase, total creatine kinase, and cardiac troponin I, in addition to base-line electrocardiograms and two-dimensional echocardiograms, in 96 patients undergoing vascular surgery and 12 undergoing spinal surgery. Blood samples were obtained every 6 hours for at least the first 36 hours after surgery, and electrocardiograms were obtained daily; a second echocardiogram was obtained approximately three days after surgery. The appearance of a new abnormality in segmental-wall motion on the postoperative echocardiogram (that is, an abnormality that had not been seen on the preoperative echocardiogram) was considered to be indicative of perioperative infarction.
Eight patients who underwent vascular surgery had new abnormalities in segmental-wall motion and received a diagnosis of perioperative infarction. All eight had elevations of cardiac troponin I, and six had elevations of MB creatine kinase. Of the 100 patients without perioperative infarction detected by echocardiography, 19 had elevations of MB creatine kinase, and 1 had a slight elevation of cardiac troponin I.
The measurement of cardiac troponin I is a sensitive and specific method for the diagnosis of perioperative myocardial infarction. It avoids the high incidence of false diagnoses associated with the use of MB creatine kinase as a diagnostic marker.
Levels of MBCK can be increased in patients with skeletal muscle injury or renal failure in the absence of myocardial injury, causing diagnostic confusion. This study was designed to determine ...whether measurement of cardiac troponin I (cTnI), a myocardial regulatory protein with comparable sensitivity to MBCK, has sufficient specificity to clarify the etiology of MBCK elevations in patients with acute or chronic skeletal muscle disease or renal failure.
Of the patients (n = 215) studied, 37 had acute skeletal muscle injury, 10 had chronic muscle disease, nine were marathon runners, and 159 were chronic dialysis patients. Patients were evaluated clinically, by ECG, and by two-dimensional echocardiography. Total creatine kinase (normal, < 170 IU/L) was determined spectrophotometrically, and cTnI (normal, < 3.1 ng/mL) and MBCK (normal, < 6.7 ng/mL) were determined with specific monoclonal antibodies. Values above the upper reference limit were considered "elevated." Elevations of total creatine kinase were common, and elevations of MBCK occurred in 59% of patients with acute muscle injury, 78% of patients with chronic muscle disease and marathon runners, and 3.8% of patients with chronic renal failure. Some of the patients were critically ill; five patients were found to have had myocardial infarctions and one had a myocardial contusion. cTnI was elevated only in these patients.
Elevations of cTnI are highly specific for myocardial injury. Use of cTnI should facilitate distinguishing whether elevations of MBCK are due to myocardial or skeletal muscle injury.
Cardiac troponin T (cTnT), measurement of which has been recommended for diagnosing myocardial infarction, was initially believed to be specific for the heart. However, recent publications have ...reported cTnT in sera of patients without cardiac disease; therefore, we investigated whether cTnT could be found in human skeletal muscle tissues. Using immunohistochemistry, Western blot, and quantitative cTnT ELISA, we assayed human heart (n = 3), normal human skeletal muscle (n = 6), and diseased skeletal muscle samples from patients with polymyositis (PM, n = 13) and Duchenne muscular dystrophy (DMD, n = 6). All heart specimens contained cTnT, but the expression of cTnT in normal skeletal muscle samples varied widely, ranging from no expression (quadriceps femoris) to expression by up to 20% of the muscle fibers (diaphragm). Immunohistochemistry detected cTnT in skeletal muscle of 8 of the PM patients and all of the DMD patients. Mean myofibrillar cTnT concentrations (mg/g myofibrillar protein) were: cardiac = 10.0, normal skeletal = 0.8, PM skeletal = 0.7, and DMD skeletal = 4.37, confirming the results of immunohistochemistry. Western blot analysis also confirmed the expression of cTnT in muscle from DMD patients. These findings provide evidence that cTnT is not 100% cardiac-specific but also is expressed in regenerating (PM and DMD) as well as in normal (nonregenerating) skeletal muscle.
Cardiac troponin-I (cTnI) is not found in sera of patients with skeletal muscle disease in the absence of myocardial injury. It is not known, however, whether trace amounts of cTnI are expressed in ...regenerating human skeletal muscle, as has been observed with creatine kinase MB. Using immunohistochemical and biochemical techniques, we investigated cTnI expression in various human muscle tissues: human heart tissue (n = 5), normal adult skeletal muscle (n = 3), and fetal heart (n = 3) and skeletal muscle (n = 3) obtained, respectively, during heart transplant, from autopsy, or from a tissue bank. Specimens from diagnostic tissue biopsies were used as diseased skeletal muscle: polymyositis (PM), n = 13; Duchenne muscular dystrophy (DMD), n = 6. Frozen sections 8 microns thick were stained immunohistochemically for either cTnI or TnI (cardiac or skeletal) by using monoclonal antibodies (MAb) 2B1.9 (cTnI specific) or 3C5.10 (reactive with all TnI isoforms), respectively. cTnI was measured in tissue homogenates by an immunofluorometric assay. Cardiac muscle was stained by both MAbs. Normal fetal and adult skeletal muscle, and samples from all of the PM and DMD patients, stained only with the nonspecific MAb (3C5.10), confirming the sole presence of skeletal TnI. No cTnI was detectable by immunoassay in any skeletal muscle sample. We conclude that cTnI is not expressed in human skeletal muscle during development or during regenerative muscle disease processes such as PM or DMD.
To improve the specificity of biochemical markers of myocardial infarction (MI), we have developed a double monoclonal "sandwich" enzyme immunoassay to measure cardiac troponin-I (cTnI) in serum. We ...produced eight IgG monoclonal antibodies against human cardiac troponin-I (cTnI) and tested them against human and animal (canine, bovine, and rabbit) troponins. Five antibodies were cardiac-specific; none of the antibodies were species-specific. Two of the five cTnI-specific monoclonal antibodies were utilized in an immunoassay. Standards were made by adding purified human cTnI to affinity-stripped cTnI-free human sera to cover the range 0-100 micrograms/L for cTnI. The dose-response curve was nonlinear but reproducible. Total assay imprecision (CV) varied between 11% and 21%. The upper limit of the reference range (nonparametric 95% interval) was established as 3.1 micrograms/L by measuring cTnI concentration in sera of 159 hospitalized patients without evidence of cardiac disease. Purified human skeletal TnI up to 10,000 micrograms/L did not affect the assay (calculated cross-reactivity < 0.1%). Diagnostic sensitivities of creatine kinase MB isoenzyme (CK-MB) and cTnI were evaluated retrospectively in 49 consecutive patients with proven MI. In the 30 patients for whom sufficient information was available to establish an accurate time course, CK-MB was more sensitive during the first 4 h after the onset of chest pain, but thereafter the sensitivities were similar up to 48 h. However, cTnI is more cardiac-specific than is CK-MB and remains increased longer than does CK-MB.
Cardiac troponin I (cTnI) results vary 100-fold among assays. As a step toward standardization, we examined the performance of 10 candidate reference materials (cRMs) in dilution studies with 13 cTnI ...measurement systems.
Solutions of 10 cTnI cRMs, each characterized by NIST, were shipped to the manufacturers of 13 cTnI measurement systems. Manufacturers used their respective diluents to prepare each cRM in cTnI concentrations of 1, 10, 25, and 50 microg/L. For the purpose of ranking the cRMs, the deviation of each cTnI measurement from the expected response was assessed after normalization with the 10 microg/L cTnI solution. Normalized deviations were examined in five formats. Parameters from linear regression analysis of the measured cTnI vs expected values were also used to rank performance of the cRMs.
The three cRMs demonstrating the best overall rankings were complexes of troponins C, I, and T. The matrices for these three cRMs values differed; one was reconstituted directly from the lyophilized form submitted by the supplier; one was submitted in liquid form, lyophilized at NIST, and subsequently reconstituted; and the third was evaluated in the liquid form received from the supplier. The cRM demonstrating the fourth best performance was a binary complex of troponins C and I supplied in lyophilized form and reconstituted before distribution.
The cRMs demonstrating the best performance characteristics in 13 cTnI analytical systems will be included in subsequent activities of the cTnI Standardization Committee of the AACC.
Myocardial infarction is a rarely reported complication of amphetamine use. We report the case of a healthy 31-year-old man who presented to our emergency department with no clinical evidence of an ...acute coronary event after intravenous injection of amphetamines. However, he subsequently experienced a non–Q-wave anterior wall myocardial infarction associated with the use of amphetamines.
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