Showing papers in "Clinical Chemistry in 1993"

Receiver-operating characteristic (ROC) plots: a fundamental evaluation tool in clinical medicine.

Abstract: The clinical performance of a laboratory test can be described in terms of diagnostic accuracy, or the ability to correctly classify subjects into clinically relevant subgroups. Diagnostic accuracy refers to the quality of the information provided by the classification device and should be distinguished from the usefulness, or actual practical value, of the information. Receiver-operating characteristic (ROC) plots provide a pure index of accuracy by demonstrating the limits of a test's ability to discriminate between alternative states of health over the complete spectrum of operating conditions. Furthermore, ROC plots occupy a central or unifying position in the process of assessing and using diagnostic tools. Once the plot is generated, a user can readily go on to many other activities such as performing quantitative ROC analysis and comparisons of tests, using likelihood ratio to revise the probability of disease in individual subjects, selecting decision thresholds, using logistic-regression analysis, using discriminant-function analysis, or incorporating the tool into a clinical strategy by using decision analysis.

Total homocysteine in plasma or serum: methods and clinical applications.

Abstract: Total homocysteine is defined as the sum of all homocysteine species in plasma/serum, including free and protein-bound forms. In the present review, we compare and evaluate several techniques for the determination of total homocysteine. Because these assays include the conversion of all forms into a single species by reduction, the redistribution between free and protein-bound homocysteine through disulfide interchange does not affect the results, and total homocysteine can be measured in stored samples. Total homocysteine in whole blood increases at room temperature because of a continuous production and release of homocysteine from blood cells, but artificial increase is low if the blood sample is centrifuged within 1 h of collection or placed on ice. Different methods correlate well, and values between 5 and 15 mumol/L in fasting subjects are considered normal. Total homocysteine in serum/plasma is increased markedly in patients with cobalamin or folate deficiency, and decreases only when they are treated with the deficient vitamin. Total homocysteine is therefore of value for the diagnosis and follow-up of these deficiency states and may compensate for weaknesses of the traditional laboratory tests. In addition, total homocysteine is an independent risk factor for premature cardiovascular diseases. These disorders justify introduction of the total homocysteine assay in the routine clinical chemistry laboratory.

Radioimmunoassay for the pyridinoline cross-linked carboxy-terminal telopeptide of type I collagen: a new serum marker of bone collagen degradation.

Abstract: We developed a radioimmunoassay (RIA) for the carboxy-terminal telopeptides of type I collagen (ICTP), cross-linked with the helical domain of another type I collagen molecule, after isolation from human femoral bone. The cross-linked peptide was liberated by digesting insoluble, denatured bone collagen either with bacterial collagenase or with trypsin, and purified by two successive reversed-phase separations on HPLC, with monitoring of pyridinoline-specific fluorescence. The purity of the peptide was verified by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and its origin in the type I collagen fibers was determined by amino-terminal amino acid sequencing. Polyclonal antibodies and a separation reagent containing second antibody and polyethylene glycol are used in the RIA. An immunologically identical, somewhat larger antigen is present in human serum; its concentration increases in multiple myeloma and in rheumatoid arthritis. The ICTP antigen seems to be cleared from the circulation by the kidneys, because glomerular filtration rates that are two-thirds of normal or less are associated with increased circulating ICTP concentrations. The CVs of the method are between 3% and 8% for a wide range of concentrations. The analysis of 40 serum samples can be completed in 4 h.

Optimized steps in fluorometric determination of thiobarbituric acid-reactive substances in serum: importance of extraction pH and influence of sample preservation and storage.

Abstract: A simple, reliable, and reproducible fluorometric method for measuring thiobarbituric acid-reactive substances (TBARS) in serum is proposed, based on the reaction between malondialdehyde (MDA) and thiobarbituric acid. Formation of TBARS was complete at pH 2.4-2.6, but extraction with n-butanol proved complete only at lower pH, i.e., 1.6-1.7. Analytical recoveries of MDA added to serum were 94%-101%; within- and between-run CVs were 2.4-3.6% and 4.6-5.5%; and the detection limit for TBARS in serum was 0.10 mumol/L. Optimized conditions included: (a) collection of either serum or heparinized plasma, (b) preservation from in vitro autoxidation by glutathione and EDTA, and (c) storage at -20 degrees C up to 35 days. The mean (+/- SD) TBARS concentration in 47 healthy adults was 1.01 (0.21) mumol/L; no sex-related difference was observed. Higher concentrations were measured in patients with renal insufficiency undergoing hemodialysis and in patients with insulin-dependent diabetes, chronic pancreatitis, or liver cirrhosis.

Enzyme immunoassay for intact human insulin in serum or plasma.

Abstract: We describe an enzyme-linked two-site immunoassay for quantitation of intact insulin in human serum and plasma. The method uses two murine monoclonal antibodies that bind to two different epitopes on the insulin molecule. The immunoassay is specific. Human proinsulin is not bound by the antibodies, and the binding of partially processed proinsulin intermediates is believed to be of minor clinical importance. The relative response of human, bovine, and porcine insulin is 1, 1, and 3, respectively. The assay is sensitive (detection limit 5 pmol/L), accurate (101% recovery with 50 pmol/L insulin added to samples, 95% with 100 pmol/L, and 89% with 300 pmol/L), and fast (results within 3 h), and has a high analytical capacity (done in microtiter plates). The working assay range selected is 5-600 pmol/L, corresponding to a clinically useful range. Because of its specificity, this two-site immunoassay gives results that are lower than those obtained by using a competitive radioimmunoassay, both in normal individuals and in non-insulin-dependent diabetics.

Homocysteine and other thiols in plasma and urine: automated determination and sample stability.

Abstract: We have developed a modified version of our fully automated column-switching HPLC method for determining total plasma homocysteine based on single-column (reversed-phase) separation. Homocysteine, cysteine, and cysteinylglycine in plasma (total concentrations), acid-precipitated plasma (non-protein-bound concentrations), and urine can be determined. The derivatization and chromatography were performed automatically by a sample processor. The successful separation of all thiol species (within 15 min) was accomplished by accurate adjustment of the pH of the mobile phase to 3.65 (plasma) or 3.50 (acid-precipitated plasma, urine). Maximal fluorescence yield of cysteine, cysteinylglycine, and, to a lesser degree, homocysteine was dependent on optimal concentrations of EDTA and dithioerythritol during reduction (with NaBH4) and derivatization (with monobromobimane). The method is sensitive (detection limit approximately 0.05 pmol) and has a high degree of precision (CV < 5%). The sample output is approximately 70 samples in 24 h. Serum and heparin plasma can also be analyzed. Hemolysis up to approximately 2.0 g/L of hemoglobin did not interfere with the analytical recovery of homocysteine or cysteine. Collection of blood, separation of plasma from whole blood, and acid precipitation must be standardized to obtain reproducible thiol results. Our modifications and the standardization of blood-sampling procedures have substantially improved the method and broadened its applications.

Determination of vitamin D status by radioimmunoassay with an 125I-labeled tracer.

Abstract: We report here the first radioimmunoassay for a vitamin D metabolite utilizing a radioiodinated tracer. Antibodies were generated in a goat immunized with the vitamin D analog 23, 24, 25, 26, 27-pentanor-C(22)-carboxylic acid of vitamin D, coupled directly with bovine serum albumin. The 125I-labeled tracer was prepared by reacting a 3-amino-propyl derivative of vitamin D-C(22)-amide with Bolton-Hunter reagent. The primary antiserum, used at a 15,000-fold final dilution, cross-reacted equally with all cholecalciferol and ergocalciferol metabolites tested except 1,25-dihydroxycalciferol metabolites and the parent calciferols; the antiserum did not cross-react with dihydrotachysterol. Calibrators were prepared in vitamin D-stripped human serum. 25-Hydroxycholecalciferol was quantitatively extracted from serum or plasma (50 microL) with acetonitrile. The assay consists of a 90-min incubation at room temperature with primary antiserum, followed by a 20-min incubation with a second antiserum and separation of bound from free fractions by centrifugation. The detection limit of the assay was 2.8 micrograms/L for 25-hydroxycholecalciferol. Results with the present assay compared well with those from a liquid-chromatographic procedure involving specific ultraviolet detection of 25-hydroxycalciferol in plasma.

Rare earth cryptates and homogeneous fluoroimmunoassays with human sera.

Abstract: The rare earth cryptates are used as long-lived fluorescent labels. They are formed by inclusion of a europium ion in the intramolecular cavity of a macropolycyclic ligand containing bipyridine groups as light absorbers. The use of fluoride ions in the measuring medium allows a total shielding of the label fluorescence. Here, I have established the conditions under which the signal of these cryptates can be amplified through the use of nonradiative energy transfer, and applied these conditions to homogeneous immunoassays by using allophycocyanin as the acceptor. Because only a low proportion of the cryptate label is involved in the transfer amplification, and because allophycocyanin emission occurs between the europium emission lines, time-resolved measurement of europium and allophycocyanin emission allows real-time correction of the optical properties of the assay medium. These features have allowed the development of a rapid homogeneous immunoassay of prolactin that can detect as little as 0.3 microgram/L (3rd International Standard 84/500).

Rapid diagnosis of phenylketonuria by quantitative analysis for phenylalanine and tyrosine in neonatal blood spots by tandem mass spectrometry.

Abstract: A new method for quantifying specific amino acids in small volumes of plasma and whole blood has been developed. Based on isotope-dilution tandem mass spectrometry, the method takes only a few minutes to perform and requires minimal sample preparation. The accurate assay of both phenylalanine and tyrosine in dried blood spots used for neonatal screening for phenylketonuria in North Carolina successfully differentiated infants who had been classified as normal, affected, and falsely positive by current fluorometric methods. Because the mass-spectrometric method also recognizes other aminoacidemias simultaneously and is capable of automation, it represents a useful development toward a broad-spectrum neonatal screening method.

International Federation of Clinical Chemistry standardization project for measurements of apolipoproteins A-I and B. IV. Comparability of apolipoprotein B values by use of International Reference Material.

Abstract: We performed temporal and thermal stability studies on SP3-07, a liquid-stabilized reference material for apolipoprotein (apo) B, selected during the previous phase of the International Federation of Clinical Chemistry project on standardization of apolipoprotein measurements. Results indicate that SP3-07 stored at -70 degrees C has the long-term stability required for a reference material. We assigned an accuracy-based apo B value of 1.22 g/L to SP3-07, using a nephelometric method that was calibrated with freshly isolated low-density lipoprotein for which the apo B mass value was determined by a standardized sodium dodecyl sulfate-Lowry procedure. Using a common protocol, the study participants transferred the assigned mass value from SP3-07 to the individual calibrators of the analytical systems and measured the apo B concentration of 20 fresh-frozen samples obtained from individual donors and covering a clinically relevant range of apo B values. The among-laboratory CV on these samples, analyzed by 25 analytical systems, ranged from 3.1% to 6.7%. These results demonstrate the lack of matrix effects of SP3-07 and its ability to provide accurate and comparable apo B values in a variety of immunochemical methods. On the basis of the outcome of these studies, the World Health Organization has endorsed SP3-07 as the International Reference Material for Apolipoprotein B.

Prostate-specific antigen: biochemistry, analytical methods, and clinical application.

Abstract: Prostate-specific antigen (PSA) is a glycoprotein produced exclusively by prostatic tissue. PSA's absolute tissue specificity makes it valuable as a forensic marker and, more important, as a tumor marker for prostatic cancer. Prostatic cancer is prevalent in the older male population and is a major cause of death in men. Previously, prostatic acid phosphatase (PAP) was used to help diagnose and monitor the efficacy of therapy for prostate cancer. PAP has now been displaced by PSA, which has greater clinical sensitivity even though it has less clinical specificity. PSA is useful for monitoring therapy, particularly surgical prostatectomy, because complete removal of the prostate gland should result in PSA being undetectable. Measurable PSA after radical prostatectomy indicates residual prostatic tissue or metastasis, and increasing PSA concentrations indicate recurrent disease. PSA is also useful for screening selected populations of patients with symptoms indicative of prostate cancer; its use for general screening is debatable because of its less-than-optimal specificity, the cost of unselected screening, and the lack of evidence that early detection of prostate cancer decreases morbidity and mortality. Distinguishing between patients with prostatic cancer and those with benign prostatic hypertrophy is particularly difficult because of the overlap in PSA values in the two groups. Determining the rate of change in PSA per year from serial measurements or calculating the ratio of PSA per volume of the prostate gland may allow these two groups to be more readily differentiated.

Cardiac-specific immunoenzymometric assay of troponin I in the early phase of acute myocardial infarction

Abstract: The screening by immunoenzymometric assay (IEMA) of 784 monoclonal antibody (MAb) combinations resulted in the selection of an optimal pair of MAbs for measuring human cardiac troponin I (TnI). Using a one-step IEMA described here, we were able to detect TnI within the range of 0.2-20 micrograms/L in 30 min at room temperature. No cross-reactivity was observed with the skeletal isoforms of troponin up to a concentration of 500 micrograms/L. This assay was used to measure cardiac TnI in the plasma of 43 patients with acute myocardial infarction (AMI). TnI was detected relatively early after the onset of chest pain (4.3 +/- 2.1 h, mean +/- SD); the peak occurred after 12.2 +/- 4.6 h in a population that had undergone fibrinolysis. TnI disappearance was generally observed between 5 and 9 days after the onset of chest pain. No cardiac TnI could be detected in 145 healthy donors or in a control group of 6 patients (with skeletal damage or rhabdomyolysis). This assay allows a specific diagnosis of AMI in its early acute phase, with a high diagnostic specificity and sensitivity.

Evaluation of regression procedures for methods comparison studies.

Abstract: Using simulation, I evaluated five regression procedures that are used for analyzing methods comparison data: ordinary least-squares regression analysis, weighted least-squares regression analysis, the Deming method, a weighted modification of the Deming method, and a rank procedure. I recorded the following performance measures: plus or minus bias of the slope estimate, the root mean squared error of the slope estimate, and correctness of hypothesis testing. I evaluated the unweighted regression procedures by using a simulated comparison of two electrolyte methods; only the Deming method gave unbiased slope estimates. Using the jackknife method, a computer program that estimates the standard error, I showed that hypothesis testing was correct for the Deming method. I used all regression procedures on data from a simulated comparison of two measurement methods with proportional analytical errors dispersed over one decade. Bias of the slope estimates was not a problem for these cases. The weighted least-squares regressionanalysis and the weighted Deming method were most efficient (lowest root mean squared error); the other procedures required 1.6 to 2.2 times as many observations to attain the same precision for the slope estimate. Hypothesis testing was correct by the weighted Deming method with the jackknife principle for standard error computation; the other methods rejected the null hypothesis 1.4 to 4.4 times too frequently. In conclusion, it is preferable to use an alternative to ordinary least-squares regression analysis for methods comparison studies.

Evaluation of a novel point-of-care system, the i-STAT portable clinical analyzer.

Abstract: We evaluated a novel system designed for rapid, point-of-care measurement of sodium, potassium, chloride, urea nitrogen, glucose, and hematocrit. The i-STAT Portable Clinical Analyzer (PCA) system is composed of a hand-held analyzer and disposable cartridges. Sample analysis takes place in the cartridge, which contains a series of thin-film electrodes microfabricated on silicon chips. The PCA was evaluated for precision, accuracy, and utility in emergency department and stat laboratory settings. Precision did not differ significantly between these two locations, the CVs being as follows: sodium, 0.46-0.89%; potassium, 1.06-1.45%; chloride, 0.69-2.76%; urea nitrogen, 2.54-6.12%; and glucose, 4.39-5.19%. The assessment of accuracy was based on comparison of patients' sample values analyzed by the PCA and the Kodak Ektachem 700 (or the Coulter ST for hematocrit). Regression statistics were acceptable for all analytes except chloride, for which the regression data were influenced by the limited range of results. A difference plot of the chloride comparison showed that the bias rarely exceeded 5 mmol/L. Mean hematocrit values significantly differed between the PCA and the Coulter ST, apparently because of different calibration procedures.

Homocysteine and other thiols determined in plasma by HPLC and thiol-specific postcolumn derivatization.

Abstract: We describe a versatile high-performance liquid-chromatographic method for determining homocysteine and other plasma sulfhydryls. Using three different procedures for preparation of plasma, we determined total, free (non-protein-bound), and reduced forms of homocysteine, cysteine, glutathione, cysteinylglycine, and gamma-glutamylcysteine in human plasma. Sample preparation involves disulfide reduction with dithiothreitol and protein precipitation with sulfosalicylic acid. The assay utilizes isocratic reversed-phase ion-pair liquid chromatography at pH 2.4, postcolumn derivatization with 4,4'-dithiodipyridine, and colorimetric detection at 324 nm. The intra-assay precision (CV) of the method for total homocysteine is 1.5%; the interassay precision over 2.5 months is 2.5%. The detection limit for homocysteine is < 50 nmol/L plasma.

Measurement of deoxyribonuclease I activity in human tissues and body fluids by a single radial enzyme-diffusion method

Abstract: In the single radial enzyme-diffusion (SRED) method for assay of deoxyribonuclease I, a precisely measured volume of the enzyme solution is dispensed into a circular well in an agarose gel layer in which DNA and ethidium bromide are uniformly distributed. A circular dark zone is formed as the enzyme diffuses from the well radially into the gel and digests substrate DNA. The diameter of the dark circle of hydrolyzed DNA increases in size with time and correlates linearly with the amount of enzyme applied to the well. Thus, the SRED can be used for quantitation of deoxyribonuclease I with a limit of detection of 2 x 10(-6) unit. This corresponds to 1 pg of purified urine deoxyribonuclease I. We measured the deoxyribonuclease I activity of 17 different human tissues and body fluids from healthy donors. Urine samples showed the greatest activity, 6.0 +/- 2.2 kilo-units/g protein (mean +/- SD). Serum deoxyribonuclease I activity was 4.4 +/- 1.8 units/L.

Serum phospholipases A2 in inflammatory diseases.

Abstract: Phospholipase A2 (EC 3.1.1.4; PLA2) is detected in serum by determination of either the catalytic activity of the enzyme or the concentration of the enzyme protein by immunoassays. The most sensitive methods for determining PLA2 catalytic activity are radiometric assays, with a substrate of synthetic phospholipid (e.g., phosphatidylcholine or phosphatidylethanolamine) containing a 14C- or 3H-labeled fatty acid at the sn-2-position. Membranes of autoclaved Escherichia coli grown in the presence of radioactive oleic acid may also be used as a substrate. The released fatty acids are separated from the unreacted substrate and quantified by liquid scintillation counting. PLA2 catalytic activities are increased in serum in sepsis, acute pancreatitis, peritonitis, multiple injuries, rheumatoid arthritis, and other arthropathies. Immunoassays--radioimmunoassay, enzyme-linked immunosorbent assay, or time-resolved fluoroimmunoassay--are based on the use of either polyclonal or monoclonal antibodies to purified PLA2s. Specific assays have been developed for both pancreatic group I PLA2 (PLA2-I) and nonpancreatic group II PLA2 (PLA2-II). The cellular source of PLA2-I in serum is the pancreatic acinar cell. Increased serum PLA2-I values have been reported in acute pancreatitis, pancreatic cancer, and abdominal trauma. Increased PLA2-II values are found in conditions involving inflammation, e.g., sepsis, infections, acute pancreatitis, various forms of arthritis, cancer, complications of pregnancy, and postoperative states. Good correlations have been found in serum samples between the catalytic activity of PLA2 and the concentration of PLA2-II but not PLA2-I. PLA2-II may represent an acute-phase protein. The cellular source of the PLA2-II in serum is unknown; it is present in large amounts in cartilage and Paneth cells, prostatic gland cells, seminal fluid, lacrimal gland cells, and tears, but cannot be demonstrated by immunohistochemical or immunochemical methods in inflammatory cells.

Determination of metanephrines in plasma by liquid chromatography with electrochemical detection.

Abstract: Metanephrines are O-methylated metabolites of catecholamines. We report the use of liquid chromatography with electrochemical detection to determine plasma concentrations of normetanephrine (NMN) and metanephrine (MN). Plasma NMN and MN in 32 normal volunteers and inpatients were compared with concentrations in 23 patients with pheochromocytoma. Metanephrines were adsorbed from plasma onto a cation-exchange column and eluted with ammoniacal methanol. The dried residue was dissolved in mobile phase and injected onto a reversed-phase column. Recoveries of NMN and MN from 1 mL of plasma averaged 50-70%, and results varied linearly with quantity injected over a range of 0.13-55 pmol. The detection limit was 25 fmol for NMN and 50 fmol for MN. Intra-assay CVs were < 5%. In normal volunteers and inpatients, plasma concentrations of NMN ranged between 0.12 and 0.73 nmol/L (mean 0.38 nmol/L), and MN between 0.06 and 0.63 nmol/L (mean 0.19 nmol/L). Plasma NMN concentrations were increased in all 23 patients with pheochromocytoma (range 1-172 nmol/L), whereas MN concentrations (range 0.10-382 nmol/L) were increased in only 9 patients. The assay method is reliable and sensitive and offers an approach to examine the extraneuronal metabolism of catecholamines. The method may also be useful in the diagnosis of pheochromocytoma.

Carbohydrate-deficient transferrin quantified by HPLC to determine heavy consumption of alcohol.

Abstract: We developed a new fully automated ion-exchange chromatographic method for quantitating carbohydrate-deficient transferrin (CDT) on a Mono Q column. Quantitation relies on the selective absorbance of the iron-transferrin complex at 460 nm. Transferrin isoforms deficient in sialic acid, with pIs 5.7 and 5.9, can easily be separated and quantitated as a percentage of the total transferrin. This method has been applied to samples from teetotalers, occasional drinkers, patients with recent heavy alcohol consumption, and patients during detoxification. The sensitivity of the method was 55% in patients reporting 40-70 g daily ethanol consumption and nearly 100% in heavily intoxicated patients (70-500 g daily consumption). The half-life of the dominating pI 5.7 isoform in this group was 9.5 (+/- 1) days during detoxification. A CDT value > 0.8% is a highly specific marker for alcohol abuse and is greatly superior to other currently available biological markers.

Lipase in serum--the elusive enzyme: an overview.

Abstract: Lipase is a glycoprotein with 420-449 amino acid residues and a M(r) of 46,000-56,000 for pancreatic lipase and 32,000-39,000 for serum lipase. Lipase is present in the pancreas, intestines, and a variety of other tissues. The concentration gradient between pancreatic tissue and serum lipase is approximately 20,000-fold. Serine, as part of an Asp-His-Ser triad, is the nucleophilic residue essential for catalysis. Lipase differs from other esterases by the presence of a hydrophobic recognition site. The optimal pH is between 7.5 and 10.0, depending on the reaction condition; the pI for the various forms of the enzyme has been reported as 5.80 and 5.85; 6.4, 6.8, and 7.0; and 7.4 for a purified fraction. Several authors report the presence of two molecular forms in the pancreas and three electrophoretic bands with lipolytic activity. In normal serum two bands have been observed; in pancreatitis as many as four bands have been seen. Lipolytic activity may not always be due to lipase. Assays specific for lipase require a triglyceride as substrate as well as the presence of colipase (a water-soluble and heat-stable protein, essential for lipase action), a secondary bile salt, and Ca2+. The clinical sensitivity of all modern assays is high because of selection of a low decision limit; the clinical specificity varies greatly but can be improved by increasing the cutoff point. Lipase determinations in pancreatitis are superior to amylase determinations. The reasons for the great variability of reports regarding the clinical utility of lipase are discussed, and the clinical utility of lipase determinations is summarized.

FK506 (tacrolimus), a novel immunosuppressant in organ transplantation: clinical, biomedical, and analytical aspects.

Abstract: The macrolide immunosuppressant FK506 (tacrolimus) is a powerful and selective anti-T-lymphocyte agent that was discovered in 1984. This agent, isolated from the fungus Streptomyces tsukubaensis, has a mechanism of action similar to that of cyclosporine. Experimental data were first published in 1987, and clinical trials were started 2 years later in Pittsburgh. The drug has a potent hepatotrophic effect, which could explain its success in liver transplantation. Particularly encouraging results were obtained in liver allograft recipients, suggesting a lower risk/benefit ratio than with other immunosuppressants. However, recent data show that the drug is not devoid of toxicity (mainly nephrotoxicity), which should the percent the need for careful blood monitoring. Several methods of analysis have been described, some satisfactory, others inadequate for routine monitoring. There is still a lack of specific methods to determine routinely the parent drug concentrations in biological fluids for clinical pharmacokinetics purposes. Despite greater experience in therapeutic drug monitoring, the correlation between FK506 concentrations and efficacy or toxicity is still unclear. More investigations are required to better understand and determine the appropriate use of FK506 in organ transplantation and treating autoimmune diseases.

Double-label time-resolved immunofluorometric assay of prostate-specific antigen and of its complex with alpha 1-antichymotrypsin.

Abstract: We have developed a procedure for simultaneous immunofluorometric assay (IFMA) of prostate-specific antigen (PSA) and its complex with alpha 1-antichymotrypsin (ACT). A PSA-specific monoclonal antibody, which captures both free PSA and the PSA-ACT complex, was used as solid-phase antibody. Total PSA immunoreactivity was measured with a Eu(3+)-labeled PSA antibody that reacted with both free PSA and PSA-ACT. PSA-ACT was assayed simultaneously with a Sm(3+)-labeled polyclonal ACT antibody as a tracer. As standard we used pooled serum in which most of the PSA occurred as the PSA-ACT complex. The assay range was 0.03-500 micrograms/L for total PSA and 0.16-450 micrograms/L for PSA-ACT. In comparison with the assay for total PSA, assay of the PSA-ACT/PSA ratio improved the clinical specificity for cancer by reducing the number of false-positive results in prostatic hyperplasia.

Separation of beta-carotene and lycopene geometrical isomers in biological samples.

Abstract: The analysis of beta-carotene and lycopene, the two predominant carotenoids in human serum and tissues, was extended to the level of geometrical (cis-trans) isomers by using an improved reversed-phase HPLC methodology. We separated five geometrical isomers of beta-carotene and seven of lycopene in human serum and tissues. 13-cis-beta-Carotene was identified as the predominant cisisomer in human serum, contributing about 5% to total beta-carotene. In tissue, however, considerable amounts of 9-cis- and traces of 15-cis-beta-carotene were also detected. In contrast to beta-carotene, the lycopene isomer patterns in human serum and tissues are quite similar.

Monitoring both serum amyloid protein A and C-reactive protein as inflammatory markers in infectious diseases.

Abstract: We examined serum amyloid protein A (SAA) and C-reactive protein (CRP) as inflammatory markers of viral and bacterial infections. Both acute-phase reactants increased in the acute stage and thereafter decreased in the convalescent stage. In viral infections, the mean serum concentrations of SAA during the acute stage were 141 mg/L in infections with adenovirus, 77 mg/L with measles virus, 63 mg/L with influenza virus, 55 mg/L with parainfluenza virus, 31 mg/L with respiratory syncytial virus, and 31 mg/L in aseptic meningitis. The mean serum concentration of CRP was 19 mg/L for adenovirus infection and < 7 mg/L in all other viral infections. The SAA concentrations were 5- to 11-fold greater than the CRP concentrations. Both the SAA and the CRP concentrations were higher in bacterial infections than in viral infections. Changes in the concentrations of serum SAA paralleled those in serum CRP in bacterial infection; during the course of viral infection, however, serum SAA tended to disappear more quickly than CRP did. SAA appears to be a clinically useful marker of inflammation in acute viral infections, with or without significant changes in the CRP concentration.

Apolipoprotein E polymorphism in a healthy Swedish population: variation of allele frequency with age and relation to serum lipid concentrations.

Abstract: We analyzed blood samples from 407 healthy Swedish individuals, 244 men and 163 women, ages 17 to 86 years, for apolipoprotein (apo) E isoforms and serum triglycerides, low-density lipoprotein (LDL), high-density lipoprotein (HDL), and total cholesterol. Parallel genotyping by means of polymerase chain reaction (PCR)-amplified DNA was performed in 200 subjects. Identical results were obtained by genotyping and phenotyping in 95% of all subjects analyzed. The apo E allelic frequencies were 7.8% for epsilon 2, 71.9% for epsilon 3, and 20.3% for epsilon 4. Compared with other Caucasian populations, the present population had a high relative allelic frequency of epsilon 4. The epsilon 4 frequency decreased with increasing age and was significantly lower in individuals > 60 years of age (14.7%). When controlling for age and sex, there were strong correlations between total serum and LDL cholesterol and the various epsilon alleles. The epsilon 4 and epsilon 3 alleles correlated positively with serum cholesterol and the epsilon 4 allele correlated positively with LDL cholesterol. In contrast, HDL cholesterol and serum triglycerides did not show any correlation to the allele types. Thus, the results demonstrate a considerable age variation of the epsilon allele frequency among healthy Swedes and an influence of apo E alleles on serum and LDL cholesterol concentrations.

Multiple forms of prostate-specific antigen in serum: differences in immunorecognition by monoclonal and polyclonal assays.

Abstract: Prostate-specific antigen (PSA) in serum is primarily complexed with alpha 1-antichymotrypsin (alpha 1-ACT). However, 12-15% of prostate cancer (PCa) patients present with the predominant form being uncomplexed (free) PSA (Lilja et al., Clin Chem 1991;37:1618-24). We report that commercial immunoassays demonstrate variations in reactivity, especially to the uncomplexed form. We fractionated and analyzed commercial controls, PSA complexes prepared in vitro, and sera from patients with PCa or benign prostatic hyperplasia, using molecular sieve chromatography and Hybritech Tandem-R, Abbott IMx, and Ciba Corning ACS PSA assays. Peak integration of PCa samples demonstrated ACS:Tandem-R ratios of 1-1.3 for PSA/alpha 1-ACT complex. In contrast, ratios of uncomplexed peaks ranged from 2 to 4, suggesting a greater reactivity of the uncomplexed form in the ACS PSA assay. Discrepancies between assays, when PSA was measured in unfractionated sera, correlated directly with the percentage of the uncomplexed form. In controls, fractionation revealed the presence of uncomplexed PSA only, with ratios of ACS:Tandem-R and IMx:Tandem-R of 3:1 and 1.8:1, respectively. Immunoblots of PCa sera detected uncomplexed PSA (approximately 30 kDa) and PSA complexes of approximately 95 kDa (PSA/alpha 1-ACT) and > 200 kDa, indicative of alpha 2-macroglobulin. Maximal recognition of all forms of PSA may be important for early detection of disease progression.

Breath acetone analyzer: diagnostic tool to monitor dietary fat loss.

Abstract: Acetone, a metabolite of fat catabolism, is produced in excessive amounts in subjects on restricted-calorie weight-loss programs. Breath acetone measurements are useful as a motivational tool during dieting and for monitoring the effectiveness of weight-loss programs. We have developed a simple, easy-to-read method that quantifies the amount of acetone in a defined volume of exhaled breath after trapping the sample in a gas-analyzer column. The concentration of acetone, as measured by the length of a blue color zone in the analyzer column, correlates with results obtained by gas chromatography. Using the breath acetone analyzer to quantify breath acetone concentrations of dieting subjects, we established a correlation between breath acetone concentration and rate of fat loss (slope 52.2 nmol/L per gram per day, intercept 15.3 nmol/L, n = 78, r = 0.81). We also discussed the possibility of using breath acetone in diabetes management.

Ionic binding, net charge, and Donnan effect of human serum albumin as a function of pH.

Abstract: The ionic activities and total molalities of sodium, potassium, calcium, lithium, and chloride in a solution of human serum albumin were measured at different values of pH between 4 and 9. The same quantities were measured simultaneously in a protein-free electrolyte solution in membrane equilibrium with the albumin solution. Taking the residual liquid-junction potential and bias from unselectivity of the electrodes into account, we determined the own, bound, and net charges of albumin. Chloride was amply bound at low pH, and calcium at high pH. The varying charge of ions bound to albumin opposed the effect of acid or base on the net charge. All ions were distributed across the membrane according to the same electric potential difference, which equalled the Donnan potential. The high concordance between observation and theory favors the Donnan theory and furthermore implies that the electrodes are as accurate in a solution with albumin as in a protein-free solution.

Analytical evaluation of i-STAT portable clinical analyzer and use by nonlaboratory health-care professionals

Abstract: We evaluated the performance of the i-STAT Portable Clinical Analyzer, a hand-held instrument that, with its current cartridge, analyzes for electrolytes, urea nitrogen, glucose, and hematocrit in approximately 60 microL of whole blood in approximately 90 s. Accuracy, imprecision, and linearity studies were performed with aqueous controls and standards and by split-sample analysis. Intrarun imprecision (CV) ranged from 0.34% to 3.97%. Total imprecision over a 2-month period ranged from 0.42% to 4.83%, with urea nitrogen and glucose analyses generating the higher values. Patients' results from the Portable Clinical Analyzer correlated well with those obtained for whole blood or plasma by the Nova Stat Profile 5, the Beckman Synchron CX3, or the Technicon H1 Hematology Analyzer, with Sylx values < 0.2 mmol/L for potassium; < 1.5 mmol/L for sodium, glucose, and urea nitrogen; < 2.4 mmol/L for chloride, and < 2.4% for hematocrit. We also ascertained imprecision and accuracy of the system placed in a cardiothoracic intensive-care unit and operated by nurses. There were no significant differences in either the imprecision or accuracy of the system in this setting. We conclude that operator technique is not a factor in the analytical performance of the system and that it can be used by nonlaboratorians with a high degree of confidence that reliable results will be obtained.

Ultrasensitive time-resolved immunofluorometric assay of prostate-specific antigen in serum and preliminary clinical studies.

Abstract: We developed an ultrasensitive method for measuring prostate-specific antigen (PSA) in serum. The assay includes a capture monoclonal anti-PSA antibody coated to microtiter wells, a biotinylated rabbit polyclonal detection antibody, and alkaline phosphatase (ALP)-labeled streptavidin. The activity of ALP is measured with the substrate diflunisal phosphate; the released diflunisal forms highly fluorescent complexes with Tb(3+)-EDTA that are quantified with microsecond time-resolved fluorometry. The assay is precise and accurate and correlates well with the established Hybritech Tandem-PSA kit. Its distinguishing feature is extreme sensitivity (lowest limit of detection is 0.002 micrograms/L or 2 x 10(6) PSA molecules per assay). This is the most sensitive PSA assay reported thus far; we used it to quantify PSA in patients who had undergone radical prostatectomy. Many patients had < 0.01 micrograms/L PSA in their serum. This method could have important clinical applications in postsurgical early detection of relapse or residual prostate cancer, as recently suggested in the literature (Clin Chem 1992;38:1930-2).