Showing papers in "Clinical Chemistry in 1994"

Role of serotonin in the pathophysiology of depression: focus on the serotonin transporter.

Abstract: Considerable evidence has accrued in the last two decades to support the hypothesis that alterations in serotonergic neuronal function in the central nervous system occur in patients with major depression. These findings include the following: (a) reduced cerebrospinal fluid (CSF) concentrations of 5-hydroxyindoleacetic acid (5-HIAA), the major metabolite of serotonin (5-HT) in drug-free depressed patients; (b) reduced concentrations of 5-HT and 5-HIAA in postmortem brain tissue of depressed and (or) suicidal patients; (c) decreased plasma tryptophan concentrations in depressed patients and a profound relapse in remitted depressed patients who have responded to a serotonergic antidepressant when brain tryptophan availability is reduced; (d) in general, all clinically efficacious antidepressants augment 5-HT neurotransmission following chronic treatment; (e) clinically efficacious antidepressant action by all inhibitors of 5-HT uptake; (f) increases in the density of 5-HT2 binding sites in postmortem brain tissue of depressed patients and suicide victims, as well as in platelets of drug-free depressed patients; (g) decreased number of 5-HT transporter (determined with [3H]imipramine or [3H]paroxetine) binding sites in postmortem brain tissue of suicide victims and depressed patients and in platelets of drug-free depressed patients. In our studies, this reduction in platelet 5-HT transporter binding is not due to prior antidepressant treatment of hypercortisolemia and is not observed in mania, Alzheimer disease, schizophrenia, panic disorder, fibromyalgia, or atypical depression. In a pilot study, this deficit predicted treatment response to an experimental antidepressant. These findings support the hypothesis that alterations in 5-HT neurons play a role in the pathophysiology of depression.

Serum cystatin C, determined by a rapid, automated particle-enhanced turbidimetric method, is a better marker than serum creatinine for glomerular filtration rate.

Abstract: We describe a fully automated particle-enhanced turbidimetric assay for cystatin C in undiluted serum and EDTA-plasma. The throughput is 90 samples per hour and urgent samples can be analyzed in 7 min. The assay range (0.4-14.1 mg/L) covers the concentration range in health and disease. The within- and between-run imprecision is 0.9% and 2.2%, respectively. Analytical recovery of additions of recombinant cystatin C averaged 98%. Rheumatoid factors (< or = 323,000 IU/L), bilirubin (< or = 150 mumol/L), hemoglobin (< or = 1.2 g/L), and triglycerides (< or = 8.5 mmol/L) do not interfere in the assay. In view of the superior (by ROC analysis) diagnostic accuracy of serum concentrations of cystatin C for reduced glomerular filtration rate (GFR) in comparison with creatinine, cystatin C seems an attractive alternative to creatinine for estimation of GFR.

Association of low serum concentration of bilirubin with increased risk of coronary artery disease.

Abstract: We examined serum bilirubin and various liver-function enzymes as possible risk factors for angiographically documented coronary artery disease (CAD). The studies involved a "training" set of 619 men for whom complete data on all risk factors considered were available, and a "test" set of 258 men for whom some risk factor data were not available. In both study groups, the liver enzymes were not related to CAD; however, In[total bilirubin] was inversely and statistically significantly related to the presence of CAD, both univariately and multivariately after adjustment for the established risk factors of age, total cholesterol, high-density lipoprotein cholesterol, smoking history, and systolic blood pressure. A 50% decrease in total bilirubin was associated with a 47% increase in the odds of being in a more severe CAD category. Our data suggest that serum bilirubin is an inverse and independent risk factor for CAD, with an association equivalent in degree to that of systolic blood pressure.

Limit of detection (LQD)/limit of quantitation (LOQ): comparison of the empirical and the statistical methods exemplified with GC-MS assays of abused drugs.

Abstract: The limit of detection (LOD) for any analytical procedure, the point at which analysis is just feasible, may be determined by a statistical approach based on measuring replicate blank (negative) samples or by an empirical approach, consisting of measuring progressively more dilute concentrations of analyte. The limit of quantitation (LOQ), or concentration at which quantitative results can be reported with a high degree of confidence, may likewise be determined by either approach. We used both methods to determine LOD and LOQ for forensic gas chromatographic-mass spectrometric (GC-MS) analyses of abused drugs. The statistically determined LOD and LOQ values for these assays underestimated the LOD because of the large imprecision associated with blank measurements and the inability of blank samples to meet typical GC-MS acceptance criteria. The empirical method provided much more realistic LOD values, supported by reasonable experimental data, and are 0.5-0.03 the magnitude of the corresponding statistical LODs. The empirical LODs and LOQs are identical for these GC-MS assays. The observations made here about the LOD/LOQ for specific forensic GC-MS procedures are generally applicable to any type of analysis.

Rapid HPLC determination of total homocysteine and other thiols in serum and plasma: sex differences and correlation with cobalamin and folate concentrations in healthy subjects.

Abstract: High-performance liquid chromatography with fluorescence detection has been utilized for the rapid determination of total homocysteine, cysteine, and cysteinylglycine in human serum and plasma. Our earlier procedure (Anal Biochem 1989;178:208), which used monobromobimane to specifically derivatize thiols, has been extensively modified to allow for rapid processing of samples. As a result, > 80 samples a day can be assayed for total homocysteine, cysteine, and cysteinylglycine. The method is sensitive (lower limit of detection < or = 4 pmol in the assay) and precise (intra- and interassay CV for homocysteine, 3.31% and 4.85%, respectively). Mean total homocysteine concentrations in plasma and serum were significantly different, both from healthy male donors (9.26 and 12.30 mumol/L, respectively; P < 0.001) and healthy female donors (7.85 and 10.34 mumol/L, respectively; P < 0.001). The differences in total homocysteine between sexes were also significant (P = 0.002 for both plasma and serum). Similar differences were found for cysteine and cysteinylglycine. We found a significant inverse correlation between serum cobalamin and total homocysteine in men (P = 0.0102) and women (P = 0.0174). Serum folate also inversely correlated with total homocysteine in both sexes.

Immunoassay for quantifying type I collagen degradation products in urine evaluated.

Abstract: An enzyme-linked immunosorbent assay (ELISA) for measuring type I collagen degradation products in urine < 3 h was evaluated. The measuring range was 0.5-10.5 mg/L with a detection limit of 0.2 mg/L. Within-run and total CVs were 5.3% and 6.6%, respectively. Analytical recovery averaged 100%. The mean (+/- SD) concentrations in urine samples from a healthy premenopausal population (n = 102) were 250 +/- 110 mg/mol creatinine (Cr). A group of healthy postmenopausal women (n = 410) gave a mean value of 416 +/- 189 mg/mol Cr. Values obtained in the ELISA correlated well (r = 0.83) to HPLC values for the established bone resorption marker deoxypyridinoline (n = 214), slightly better than the correlation to hydroxyproline measurements (r = 0.78, n = 421). We conclude that the assay described here presents a useful tool for further elucidating the importance of type I collagen degradation products in urine.

Comparable detection of acute myocardial infarction by creatine kinase MB isoenzyme and cardiac troponin I.

Abstract: Although measurement of cardiac troponin I (cTnI) is, in some situations, more specific for detection of cardiac injury than is measurement of the MB isoenzyme of creatine kinase (MBCK), its sensitivity and specificity relative to MBCK for detection of myocardial infarction has not been established. Accordingly, we studied prospectively 199 consecutive patients admitted to the coronary care unit. Values of MBCK and cTnI mass were determined in all samples. Of the 188 patients admitted with a suspicion of acute myocardial ischemia, 89 were diagnosed as having an acute myocardial infarction on the basis of the patterns of MBCK values. Eighty-six of these patients also had increased cTnI (concordance, 96.6%); three did not. Of the patients diagnosed as without infarction, five with unstable angina and symptoms in the day(s) prior to admission had increased cTnI, for a cTnI specificity of 94.9%. Receiver operating characteristic curve analysis indicated that cTnI and MBCK had statistically indistinguishable diagnostic accuracies for the detection of acute myocardial infarction.

PCR in a silicon microstructure.

Abstract: Devices for performing polymerase chain reactions (PCR) have been developed for use with photolithographed silicon. Microchambers capable of holding between 5.0 and 10 microL of PCR reagents were constructed by etching specific areas of rectangular silicon chips (17 x 15 mm), which were then capped with Pyrex glass. These silicon devices (PCRChips), which were etched to depths of 40-80 microns, permitted free flow of fluids in the microchannels and microchambers. Access to the microchambers was through holes in the silicon. Thermal cycling of the PCR reagents was achieved by placing the disposable PCRChip in a small holder containing a computer-controlled Peltier heater-cooler. Successful amplification was demonstrated by electrophoresis of products in agarose gel containing ethidium bromide, and the migration of the product was compared with that obtained in a commercially available thermal cycler. The thermal characteristics of the silicon, coupled with the high surface area to volume ratio in the new devices, are particularly advantageous features for amplification by PCR.

Retinol, alpha-tocopherol, lutein/zeaxanthin, beta-cryptoxanthin, lycopene, alpha-carotene, trans-beta-carotene, and four retinyl esters in serum determined simultaneously by reversed-phase HPLC with multiwavelength detection.

Abstract: We describe the use of HPLC with multiwavelength detection to measure retinol, alpha-tocopherol, lutein/zeaxanthin, beta-cryptoxanthin, lycopene, alpha-carotene, trans-beta-carotene, beta-carotene, and the linoleate, oleate, palmitate, and stearate esters of retinol in a single 200-microL serum sample. The method is sensitive enough to detect individual retinyl esters in fasting serum from a nonhyperlipidemic population and requires only 12 min for each sample. Serum concentration ranges and means are reported for retinol, alpha-tocopherol, lutein/zeaxanthin, beta-cryptoxanthin, lycopene, alpha-carotene, trans-beta-carotene, and the sum of the retinyl esters from serum analyses of 3480 participants from several different studies.

Interference with clinical laboratory analyses.

Abstract: Interference by endogenous and exogenous substances with assays for clinical analytes is a common problem in laboratory medicine. For this review, we defined interference as "the effect of a substance present in the sample that alters the correct value of the result, usually expressed as concentration or activity, for an analyte." There are four major endogenous compounds that consistently interfere with laboratory results: hemoglobin, bilirubin, lipids, and paraproteins. The major exogenous sources of interference are drugs prescribed for the patient; and there are several excellent compendia of the effect of drugs on clinical laboratory tests. We recommend determining whether the interference is dependent or independent of the analyte for the assay. Further, we propose an approach to the identification and resolution of an interference problem for the clinical laboratory and make recommendations to manufacturers.

Manipulation and flow of biological fluids in straight channels micromachined in silicon

Abstract: Analysis of minute sample volumes is a major analytical challenge that requires an understanding of fluid flow in microstructures. Accordingly, flow dynamics of biological fluids and cell suspensions in straight glass-capped silicon microchannels (40 to 150 microns wide, 20 and 40 microns deep) were studied. We demonstrated that these microstructures are appropriate components for microfluidic analytical devices. Different fluids were easily manipulated in the microchannels, and measurements of flow rate as a function of pressure for whole human blood, serum, plasma, and cell suspensions revealed non-Newtonian behavior. By means of micromachined filters (5 microns) located in channels, blood cells and microparticles were effectively separated from nanoliter-sized samples, clearly indicating the future role of microstructures for a variety of analytical purposes.

New international reference preparation for proteins in human serum (RPPHS).

Abstract: Quality-control surveys in recent years, in various parts of the world, have shown poor between-laboratory agreement for measurements of plasma proteins. Despite the existence of international reference materials distributed by the World Health Organization, standards produced by diagnostics manufacturers and professional organizations differ significantly in their ascribed values. The reasons for this are complex but include poor availability of the primary materials, confusion about their use, and the fact that their turbidity on reconstitution precludes their use in modern optical immunoassays. This unfortunate situation led to an important initiative to produce sufficient quantities of a widely available, optically clear secondary reference material for plasma proteins that could be used worldwide by manufacturers, professional organizations, and laboratories. Here we present an overview on how the laboratory community, including manufacturers, clinical laboratories, professional societies, and regulators, has reached what we consider is a successful conclusion to a difficult problem.

Human urinary arsenic excretion after one-time ingestion of seaweed, crab, and shrimp.

Abstract: We studied chemical speciation of arsenic compounds in urine samples by using HPLC with inductively coupled plasma mass spectrometry detection. We examined urinary arsenic excretion patterns and the arsenic species excreted from nine human subjects who ingested seaweed products and crab (or shrimp). Fast urinary excretion of unchanged arsenobetaine was seen after ingestion of crab and shrimp, which contain arsenobetaine as the major arsenic species. In contrast, the arsenosugars, which comprise the major arsenic species in seaweed, are metabolized and have a longer retention time in the human body. When nine volunteers ingested the commercial seaweed product nori, both the urinary arsenic excretion pattern and the excreted arsenic species varied from individual to individual, and as many as six metabolites could be detected. It seems that arsenosugars are not decomposed by stomach acid and that reactions involving enzymatic and (or) microbial activity in the human body may be responsible for the metabolism of arsenosugars.

Plasma amino acids determined by liquid chromatography within 17 minutes.

Abstract: We present an HPLC method for the determination of amino acids in plasma. The method is based on automated precolumn derivatization of amino acids with o-phthalaldehyde, separation of the derivatives by reversed-phase chromatography, and quantification by fluorescence detection. Complete separation was achieved within 12 min. Total analysis time, including derivatization, chromatography, and reequilibration of the column, was 17 min. The assay was linear from 5 to 800 mumol/L for all amino acids. Recovery of amino acids added to plasma samples was 96-106%, except for tryptophan (89%). Within-run precision (CV) was 1.8-6.4%, and between-run precision was 2.1-7.2%. The method can be used for determining primary amino acids in plasma and cerebrospinal fluid. The simple sample preparation and short analysis time make the method particularly suitable for routine analysis of large series of samples.

Cardiac troponin-T immunoassay for diagnosis of acute myocardial infarction.

Abstract: We evaluated the analytical and clinical performance of an immunoassay for cardiac troponin T (cTnT). Within-run and total imprecision ranged from 1.6 to 11.3%. The sensitivity and linear range was 0.015 and 13 micrograms/L, respectively. Frozen samples were stable for at least 8 weeks. No interferences were seen with lipids or bilirubin (total and conjugated). Hemoglobin caused a negative bias at concentrations > 4 g/L. Heparinized plasma showed a 6% negative bias compared with serum. The clinical utility of cTnT was compared with that of creatine kinase (CK)-MB (mass assay). The sensitivity of cTnT measurements from 63 patients with acute myocardial infarction (AMI) (cTnT cutoff 0.1 microgram/L) was 60% at 0-3 h, 59% at 3-6 h, 94% at 6-9 h, 90% at 9-12 h, 99% at 12-24 h, 92% at 24-48 h, 83% at 48-72 h, and 100% at 72-96 h. Corresponding results for CK-MB (cutoff 5.0 micrograms/L and 2.5% relative index) were 45%, 64%, 82%, 97%, 87%, 81%, 54%, and 59%. The specificity of the markers from 49 non-AMI patients was 46% and 79% for cTnT and CK-MB, respectively. We show that CK-MB is more specific for diagnosis of AMI, and propose that cTnT is more sensitive to myocardial injury.

Plasma homocyst(e)ine as a risk factor for early familial coronary artery disease.

Abstract: We measured plasma homocyst(e)ine [H(e)] and other coronary risk factors in 266 patients with early coronary artery disease from 170 families in which two or more siblings were affected and in 168 unmatched controls. The mean H(e) concentration adjusted for significant correlates (serum creatinine, uric acid, and low-density lipoprotein cholesterol) was 12.0 mumol/L in proband cases compared with 10.1 mumol/L in controls (P = 0.0001). Many (17.6%) of the proband cases had H(e) concentrations exceeding the 95th percentile for the controls (relative odds = 4.9, P < 0.001). H(e) among cases was bimodally distributed even after adjustment for concentrations of plasma vitamins. Concordant high H(e) was seen in at least 10 (12%) of 85 families with two or more affected siblings. We conclude that a substantial proportion of early familial coronary artery disease is probably related to production of high concentrations of H(e) by one or more major genes.

Understanding the sodium pump and its relevance to disease.

Abstract: Na,K-ATPase (sodium pump; EC 3.6.1.37) is present in the membrane of most eukaryotic cells and controls directly or indirectly many essential cellular functions. Regulation of this enzyme (ion transporter) and its individual isoforms is believed to play a key role in the etiology of some pathological processes. The sodium pump is the only known receptor for the cardiac glycosides. However, endogenous ligands structurally similar to digoxin or ouabain may control the activity of this important molecular complex. Here we review the structure and function of Na,K-ATPase, its expression and distribution in tissues, and its interaction with known ligands such as the cardiac glycosides and other suspected endogenous regulators. Also reviewed are various disorders, including cardiovascular, neurological, renal, and metabolic diseases, purported to involve dysfunction of Na,K-ATPase activity. The escalation in knowledge at the molecular level concerning sodium pump function foreshadows application of this knowledge in the clinical laboratory to identify individuals at risk for Na,K-ATPase-associated diseases.

Cadmium: exposure markers as predictors of nephrotoxic effects.

Abstract: Cadmium (Cd) is a cumulative element with a biological half-life of > 10 years in humans. The total amount of Cd accumulated in the liver and in the kidney can be measured in vivo by neutron activation (or x-ray fluorescence), but this technique does not necessarily measure the fraction that is biologically active. At low exposure (i.e., general environmental exposure or moderate occupational exposure), blood Cd is mainly influenced by the last 2 to 3 months of exposure. Under such conditions, the Cd concentration in urine mainly reflects the amount of Cd stored in the body, particularly in the kidney. In Europe and the US, the Cd reference values are usually < 2 nmol/mmol creatinine. Because most of the Cd in urine is probably bound to metallothionein, the changes in the urinary metallothionein concentration parallel those of Cd. The determination of Cd concentration in hair is of limited value because in humans it is difficult to distinguish between externally deposited and endogenous Cd. Fecal Cd is a good indicator of the oral daily intake. The results of several cross-sectional epidemiologic studies of the relation between the prevalence of renal dysfunction and Cd concentration in urine led us to propose a biological limit value for Cd of 5 and 2 nmol/mmol creatine for adult male workers and the general population, respectively.

Simultaneous determination of mercury speciation in biological materials by GC/CVAFS after ethylation and room-temperature precollection.

Abstract: We developed a method for the simultaneous determination of monomethyl mercury (MMHg), inorganic mercury [Hg(II)], and total mercury (THg) in biological materials. A variety of biological materials can be digested in methanolic KOH solution. The MMHg and Hg(II) present are converted to volatile ethyl derivatives, methylethyl mercury and diethyl mercury, by an aqueous-phase ethylation reaction with sodium tetraethylborate. The ethyl derivatives are precollected onto a trapping column at room temperature, in case of disconnection with the separation/detection system, and then thermally desorbed into a packed isothermal gas chromatography (GC) column. Eluted organo-Hg compounds from the GC column are decomposed into Hg0, and detection is completed by cold vapor atomic fluorescence spectrometry (CVAFS). Pure standard solutions can be used for calibration. The sum of MMHg and Hg(II) obtained by this method equals the THg value obtained by digestion with HNO3 and H2SO4, reduction with SnCl2, and single-stage amalgamation/CVAFS for all biological materials studied. Absolute detection limits are 0.6 pg and 1.3 pg of Hg as MMHg and Hg(II), respectively, corresponding to 0.3 ng and 0.6 ng/g (wet) of sample.

Simultaneous measurement of cocaine, cocaethylene, their metabolites, and "crack" pyrolysis products by gas chromatography-mass spectrometry.

Abstract: We developed a sensitive and specific assay for the simultaneous measurement of cocaine, cocaethylene, six of their metabolites, and anhydroecgonine methyl ester, a pyrolysis product, in biological fluids. The assay involves solid-phase extraction columns containing a copolymeric bonded phase for isolation of cocaine analytes, derivatization with N,O-bis(trimethylsilyl)trifluoroacetamide and 10 g/L trimethylchlorosilane, and measurement with gas chromatography-mass spectrometry operating in the selected-ion monitoring mode. Detector responses for analytes were linear over a concentration range of 3.1-1000 micrograms/L. The limits of detection were approximately 1 microgram/L for cocaine, ecgonine methyl ester, and benzoylecgonine and 3-6 micrograms/L for the remaining analytes. Hydrolysis of cocaine and artifact formation of anhydroecogonine methyl ester during extraction and assay was < 1%. Cocaine and its derivatives appear in different proportions in plasma, saliva, and urine according to the biological fluid and time of measurement. Each biological fluid provides unique information on the disposition of cocaine in human subjects.

Selected strategies for improving sensitivity and reliability of immunoassays.

Abstract: Selected recent advances in immunoassay are reviewed. Development has continued on new labels (beta-lactamase, pyrophosphatase, luciferases, photoproteins, pyridopyridazines, europium cryptates, metal carbonyl complexes, porphines, phosphors) and label-detection methods (e.g., chemiluminescence assays, thermometric assays, NADP(+)- and FADP-based coupled assays). Various methods have been explored to increase assay sensitivity, including label amplification via catalyzed reporter deposition (peroxidase label) and immuno-polymerase chain reaction (DNA label). The focus of new immunoassay strategies has been on improved reliability (bispecific antibodies), assay simplification (piezoelectric and surface plasmon resonance immunosensors, phase-modulation fluorescence spectroscopy, polymerized bilayer assemblies), and simultaneous multianalyte testing (e.g., quadruple labeling with lanthanides, one-step test devices for drugs of abuse).

Blood concentrations of volatile organic compounds in a nonoccupationally exposed US population and in groups with suspected exposure.

Abstract: Exposure to certain volatile organic compounds (VOCs) commonly occurs in industrialized countries. We developed a method for measuring 32 VOCs in 10 mL of whole blood at low concentration. We used this method to determine the internal dose of these compounds in 600 or more people in the US who participated in the Third National Health and Nutrition Examination Survey. From our study results, we established a reference range for these VOCs in the general population of the US. We found detectable concentrations of 1,1,1-trichloroethane, 1,4-dichlorobenzene, 2-butanone, acetone, benzene, chloroform, ethylbenzene, m,p-xylene, styrene, tetrachloroethane, and toluene in most of the blood samples of nonoccupationally exposed persons. The accuracy of VOC evaluations depends on the ability of investigators to make sensitive and reproducible measurements of low concentrations of VOCs and to eliminate all sources of interference and contamination.

Iron-deficiency anemia is associated with high concentrations of transferrin receptor in serum.

Abstract: We evaluated the use of transferrin receptor (TfR) in serum as an index of iron deficiency in 19 patients diagnosed as having iron-deficiency anemia, in 17 patients with anemia of chronic disease, and in a control group of 19 nonanemic patients who underwent elective ocular or nasopharyngeal surgery. The assessment of iron status of the anemic patients was based on the presence of stainable iron on bone marrow examination. In the patients with iron-deficiency anemia, the serum TfR concentration was 5.3 +/- 1.8 mg/L (mean +/- SD), significantly higher than in the control group (1.7 +/- 0.5 mg/L) or in the patients with anemia of chronic disease (1.6 +/- 0.4 mg/L). This study suggests that serum TfR measurement is a reliable index of iron depletion and potentially of importance in the diagnosis of iron-deficiency anemia.

Use of cardiac troponin I to diagnose perioperative myocardial infarction in coronary artery bypass grafting.

Abstract: Cardiac troponin I (cTnI) is a regulatory protein unique to myocardium. We used a cardiospecific 30-min ELISA to measure cTnI in EDTA-plasma samples serially drawn from 28 patients before and after coronary artery bypass grafting (CABG)--26 elective and 2 salvage cases. The cTnI increase in 22 of the elective CABG patients, who did not have perioperative myocardial infarction (not-PMI), reflected the inevitable myocardial damage caused by cannulation and cardioplegic arrest, with peak values of 1.7 +/- 1.0 microgram/L (mean + 2 SD = 3.7 micrograms/L), the peaks occurring on average 8 h (range 4-24) after aortic unclamping. Two of the 22 not-PMI, elective CABG patients showed cTnI peaks > 3.0 micrograms/L (3.9 and 3.4 micrograms/L), indicating more extensive perioperative myocardial damage than the other 20, as confirmed by clinical and electrocardiographic or echocardiographic signs, although creatine kinase isoenzyme MB (CKMB) activity was below our PMI decision limit of 20 U/L (25 degrees C). As classified by electrocardiography, echocardiography, and increased CKMB activity, four of the 26 elective CABG patients did have a PMI. One patient with Q-wave PMI had peak cTnI approximately 30 micrograms/L, and three with non-Q-wave PMI had lower peak values (approximately 5 micrograms/L). The two salvage CABG cases had increased cTnI before surgery. One developed a Q-wave acute myocardial infarction with a 3-h cTnI peak of approximately 35 micrograms/L. We conclude that, after elective CABG, cTnI peaks > 3.7 micrograms/L and concentrations > 3.1 micrograms/L at 12 h or 2.5 micrograms/L at 24 h indicate PMI with high probability.

High-resolution 1H-NMR spectroscopy of blood plasma for metabolic studies.

Abstract: Although spin-echo techniques are often used to obtain 1H-NMR spectra of serum or plasma samples, they do not provide reliable quantitative analyses of metabolites. We present a standardized procedure, optimized for sensitivity, for using single-pulse 1H-NMR spectroscopy to analyze deproteinized plasma. The detection limit for various metabolites ranges between 2 and 40 mumol/L. The method allows quantitative analysis of many compounds of interest in studies of inborn errors of metabolism, including betaine and dimethylglycine, which cannot be measured easily with other techniques. For lactate, tyrosine, threonine, and alanine, we obtained results that correlated well with those obtained by established techniques. We also present a library containing resonance positions of 38 compounds occurring in plasma samples in health and disease, including 14 as-yet-unidentified resonances. As an example of the diagnostic power of the technique we show a spectrum of a plasma sample from a patient with 5-oxoprolinuria (pyroglutamic aciduria; McKusick 266130), an enzymatic defect in glutathione biosynthesis.

Assessment of copper nutritional status.

Abstract: Despite increased interest in the role of copper deficiency in clinical problems and an increased understanding of the physiological roles of copper, the diagnosis of a marginal deficiency has not been perfected. The use of non-standardized procedures and the effects of factors other than copper nutriture have impeded identification of the "ideal" indicator of copper nutritional status in adult humans. The specific activity of copper enzymes, or of copper-containing enzymes in blood cells, such as erythrocyte superoxide dismutase and platelet or leukocyte cytochrome c oxidase, may be a better indicator of metabolically active copper stores than the serum concentration of copper or ceruloplasmin, because the enzyme activities are sensitive to changes in copper stores and are not as sensitive to factors not related to copper nutriture. A single index, such as serum copper concentration, is inadequate for assessing the total body copper nutriture of an individual and must be supported by corroborating evidence.

Starburst dendrimers: enhanced performance and flexibility for immunoassays.

Abstract: Starburst dendrimers are novel, water-soluble polymeric materials, with a well-defined composition and structure. In our application, we used dendrimers composed of poly(amidoamine) groups to which we coupled several specific antibodies, to investigate potential formats based on radial partition immunoassay. The coupled antibodies have retained their stability and immunological binding after coupling, both in solution and when immobilized onto a solid support. On the basis of our feasibility studies with model systems, we conclude that immunoassays can be developed with performance equivalent to or better than that in many established systems. By application of a mixture of the dendrimer-coupled antibody and the analyte of interest to the solid phase, we have investigated the performance characteristics of solution-phase immunoassays. Our experiments demonstrate enhanced sensitivity for creatine kinase MB isoenzyme (CKMB), thyrotropin, and myoglobin assays and reduced instrumental analysis time for the CKMB assay.

Urinary and plasma catecholamines and urinary catecholamine metabolites in pheochromocytoma: diagnostic value in 19 cases.

Abstract: We review our data on the measurement of catecholamines and their metabolites in 19 patients with pheochromocytoma. All the assays were specific high-performance liquid chromatographic procedures with electrochemical detection. The assay of fractionated metanephrines was 100% sensitive. Normal values for both urinary norepinephrine and epinephrine were found in two asymptomatic patients with pheochromocytoma. Normal values for 3-methoxy-4-hydroxymandelic acid (VMA) were found in two patients with pure epinephrine-secreting tumors and in one patient with multiple endocrine neoplasia type II. Plasma catecholamines were usually less increased than their urinary counterparts. We recommend the specific measurement of norepinephrine and epinephrine as the initial test for patients with suggestive symptoms, and specific measurement of normetanephrine and metanephrine for patients in whom an adrenal mass is incidentally found. We argue against the use of total metanephrines, total catecholamines, and VMA because of their lack of diagnostic sensitivity.

Simplified isoelectric focusing/immunoblotting determination of apoprotein E phenotype.

Abstract: We developed a rapid, accurate method for phenotyping apoprotein E that can be used for large-scale population studies. In this method, adapted from the method of Kamboh et al. (J Lipid Res 1988;29:1535-43), 10-microL plasma samples are incubated with dithiothreitol and Tween-20 for 15 min and then applied to 5% polyacrylamide gels containing ampholyte (pH 4.5-8) and urea (3 mol/L). After 2 h of isoelectric focusing, the apoprotein E bands are made visible by immunoblotting. Utilizing whole plasma, this method does not require time-consuming ultracentrifugation, delipidation of samples, or dialysis. Small amounts of plasma are required, electrofocusing time is short, and as many as 160 samples can be processed per day. Identification of phenotype is easily accomplished by noting the location and number of protein bands instead of their intensity. Because identification of phenotype is not affected by sialylation, neuraminidase treatment is not necessary. Agreement in identification of 301 individuals from blinded duplicates was 96%, and there was 98% concordance of results for 431 samples that had undergone genetic typing. This method is thus well suited for large-scale population studies.