Showing papers in "Clinical Chemistry in 1983"

A peroxidase-coupled method for the colorimetric determination of serum triglycerides.

Abstract: We describe an enzymatic method for rapid, precise measurement of serum triglycerides with use of sample:reagent ratios as large as 1:200. Hydrolysis of triglycerides is catalyzed by lipase to produce glycerol and free fatty acids. The glycerol generated is then phosphorylated by adenosine 5'-triphosphate in the presence of glycerol kinase. Oxidation of the resulting glycerol 3-phosphate to produce hydrogen peroxide is catalyzed by L-alpha-glycerophosphate oxidase. An intense red chromogen is produced by the peroxidase-catalyzed coupling of 4-aminoantipyrene and sodium 2-hydroxy-3,5-dichlorobenzenesulfonate with hydrogen peroxide. This sensitive chromogen system not only permits use of unusually small sample volumes, it also facilitates a linear response to serum triglyceride concentrations up to at least 10 g/L while displaying good Ringbom (measure of accuracy) characteristics.

Reagent for the enzymatic determination of serum total cholesterol with improved lipolytic efficiency.

Abstract: We describe a sensitive method for quantifying the extent of cholesterol ester cleavage during enzymatic assay of total cholesterol in serum. Lipids are extracted from the assay mixture with chloroform/methanol (1/1 by vol), concentrated, then quantified by "high-performance" thin-layer chromatography. Although with conventional enzymatic reagents for determination of serum total cholesterol the hydrolysis of the cholesterol esters may be incomplete, a new enzymatic cholesterol reagent (Monotest Cholesterol, High Performance, Boehringer Mannheim) gives virtually complete cholesterol ester cleavage (i.e., greater than or equal to 99.5%). Use of this reagent with its improved lipolytic efficiency yields results for serum total cholesterol that are identical to those measured with a candidate reference procedure involving alkaline cholesterol ester saponification.

Simultaneous determination of retinol and alpha-tocopherol in serum or plasma by liquid chromatography.

Abstract: Submitters: George L. Catignani, Department of Food Science, North Carolina State University, Raleigh, NC 27650 John G. Bieri, Nutritional Biochemistry Section, Laboratory of Nutrition and Endocrinology, National Institute of Arthritis, Metabolism, and Digestive Diseases, Bethesda, MD 20014 Evaluators: William J. Driskell and Mark M. Bashor, NutriLional Biochemistry Branch, Clinical Chemistry Division, Center for Environmental Health, Centers for Disease Control, Atlanta, GA 30333 Charles P. Turley and Marge A. Brewster, Metabolic Lab., Arkansas Childrens Hospital, Little Rock, AR 72201

Radioimmunometric assay for a monoclonal antibody-defined tumor marker, CA 19-9.

Abstract: We describe a solid-phase radioimmunometric sandwich assay for a new tumor marker defined by a monoclonal antibody (19-9). This antibody reacts with a carbohydrate antigenic determinant (CA 19-9) found at low concentrations in sera from healthy individuals but frequently increased in sera from patients with adenocarcinomas. The assay is sensitive and simple to perform. It requires duplicate 100-microL samples and may be performed in 6 h. The concentration of CA 19-9 in samples is determined by reference to a standard curve, which is essentially linear from 0 to 120 arbitrary units/mL. The average CV is approximately 10% in the range of 5.8 to 120 units/mL. The minimum detectable dose is 1.4 units/mL and analytical recovery of CA 19-9 is 97.6 to 100.6%. The average concentration of CA 19-9 in sera from 1020 healthy individuals was 8.4 (SD 7.4) units/mL; only 0.6% of such sera had concentrations greater than 37 units/mL. The assay has high specificity (98.5%), even among patients with benign diseases, and has high sensitivity (up to 79%) for patients with gastrointestinal adenocarcinomas, especially those of the pancreas.

Quantification of high-density-lipoprotein cholesterol by precipitation with phosphotungstic acid/MgCl2.

Abstract: We evaluated the use of a modified phosphotungstic acid/MgCl2 precipitation procedure for the precipitation of apolipoprotein B-containing lipoproteins. Precipitation of these lipoproteins [very-low- and low-density lipoproteins, and lipoprotein (a)] is complete, with negligible coprecipitation of high-density lipoprotein subfractions (HDL1, HDL2, HDL3), even in hypertriglyceridemic sera. In comparison with ultracentrifugation, the precipitation method yields, on the average, values that are 0.17 mmol/L lower for cholesterol values but almost identical for apolipoprotein A-I and phosphatidylcholine. Looking for delta 3,5-cholestadiene formed from cholesterol in the precipitation residue, we used "high-performance" liquid chromatography and "high-performance" thin-layer chromatography and found none.

Time-resolved fluorometer for lanthanide chelates--a new generation of nonisotopic immunoassays.

Abstract: Pulsed-light time-resolved fluorometry of lanthanide chelates has proved to be very sensitive for use with nonisotopic immunoassays. We describe a manually operated fluorometer with a conventional xenon flashtube. Sensitivity for 1-s determinations is similar to that of radioisotopic methods.

Hormones in saliva: mode of entry and consequent implications for clinical interpretation.

Abstract: Assay of hormones in saliva would be more convenient than assay in blood, but there is no information on the route by which hormones enter saliva, information that would provide insight into the clinical value of such assays. We have examined the mode of entry of various hormones into saliva. The results suggest that unconjugated steroids enter saliva by diffusing through the cells of the salivary glands and that their concentration in saliva does not depend on the rate of saliva production. Conjugated steroids enter saliva via "ultrafiltration" through the tight junctions between the acinar cells, and their concentration in saliva is highly flow-rate dependent. Thyroxin and choriogonadotropin enter saliva via the ultrafiltration route or by contamination of the saliva by plasma or gingival fluid. We conclude that the salivary concentration of unconjugated steroids may usefully reflect the concentration of free (nonprotein-bound) steroids in plasma. Conversely, the concentration of conjugated steroids, thyroxin, and protein hormones such as choriogonadotropin in saliva probably does not reflect their concentration in plasma in any clinically useful way.

Acridinium esters as high-specific-activity labels in immunoassay.

Abstract: A chemiluminescent acridinium ester has been synthesized that reacts spontaneously with proteins to yield stable, immunoreactive derivatives of high specific activity. The compound has been used to prepare chemiluminescent monoclonal antibodies to human alpha 1-fetoprotein having average incorporation ratios as great as 2.8 mol of label per mole of antibody, which corresponds to a detection limit of approximately 8 X 10(-19) mol. These antibodies have been used in the preliminary development of a two-site immunochemiluminometric assay for human alpha 1-fetoprotein, which requires only a 30-min incubation and a quantification time of 5 s per sample.

A reference method for measurement of alkaline phosphatase activity in human serum.

Abstract: We present an official AACC reference method for the measurement of alkaline phosphatase, the culmination of optimization experiments conducted by a group of independent laboratories. The details of this method and evaluation of factors affecting the measurement are described. A metal ion buffer has been incorporated that maintains optimal and constant concentrations of zinc(II) and magnesium(II) ions. Final reaction conditions are: pH (30 degrees C), 10.40 +/- 0.05; 2-amino-2-methyl-1-propanol buffer, 0.35 mol/L; 4-nitrophenyl phosphate, 16.0 mmol/L; magnesium acetate, 2.0 mmol/L; zinc sulfate, 1.0 mmol/L; and N-(2-hydroxyethyl)ethylenediaminetriacetic acid, 2.0 mmol/L.

On the calculation of a reference change for comparing two consecutive measurements.

Abstract: We describe a statistical method for calculating a "reference change," defined as that difference between two consecutive test results in an individual that is statistically significant in a given proportion of all similar persons. By allowing for variation in within-person variances, this procedure computes a reference change that is more specific (i.e., less prone to false positives) than that obtained directly from the distribution of observed differences between measurements. Moreover, the method may easily be extended to a test for trend in three successive measurements. The method has been applied to semi-annual measurements of serum calcium and alkaline phosphatase in 698 men and women enrolled in a large health-maintenance program. We believe that these ideas may also be usefully applied to successive laboratory tests in carefully defined patient populations--but this introduces special problems, which are discussed briefly.

Kinetic enzymic method for automated determination of total cholesterol in serum.

Abstract: We describe a rapid, kinetic, fixed-time method for determining serum total cholesterol by use of cholesterol esterase, cholesterol oxidase, and the indicator reaction with peroxidase, 4-aminophenazone, and phenol. On addition of the competitive inhibitor 3,4-dichlorophenol the Michaelis constant of cholesterol oxidase is apparently increased, which extends the linear relation between absorbance change and cholesterol concentration to 20.7 to 25.9 mmol/L, depending on the analyzer being used. For calibration, a single standard is used. Total analysis time is in the range of 80 to 210 s. Incubation temperature is 25 degrees C or 37 degrees C. The single-reagent procedure has been adapted to three different centrifugal analyzers and to the Eppendorf ACP 5040 analyzer. It yields precise and accurate results and is insensitive to potential interferences.

Enzymic creatinine assay: a new colorimetric method based on hydrogen peroxide measurement.

Abstract: We describe a new colorimetric method for measuring creatinine in serum and urine. Creatinine hydrolysis is catalyzed by creatinine amidohydrolase, and the creatine so produced is assayed in reactions catalyzed sequentially by creatine amidinohydrolase and sarcosine oxidase in a system that generates hydrogen peroxide. The hydrogen peroxide is measured at 510 nm in a reaction catalyzed by horseradish peroxidase, with 3,5-dichloro-2-hydroxybenzenesulfonic acid/4-aminophenazone as the chromogen. This series of reactions is complete in 30 min at room temperature. A blank sample measurement corrects for endogenous creatine. The standard curve is linear for creatinine concentrations as great as 2.21 mmol/L. Analytical recovery of creatinine in human sera and urine averaged 99.8%. Within-run and between-run precision studies gave CVs of less than or equal to 3.3 and less than or equal to 4.3% for a serum with a creatinine concentration of 69 mumol/L. Results by this method agree well (r greater than 0.99) with those by both the enzymic ultraviolet method of Wahlefeld and the fuller's earth/Jaffe method.

Simple, rapid spectrophotometry of urinary N-acetyl-beta-D-glucosaminidase, with use of a new chromogenic substrate.

Abstract: We have developed a new spectrophotometric assay for urinary N-acetyl-beta-D-glucosaminidase (NAGase) with use of sodio m-cresolsulfonphthaleinyl N-acetyl-beta-D-glucosaminide (MCP-NAG). MCP-NAG was synthesized from acetochloro-glucosamine and m-cresolsulfonphthalein (MCP) in four steps. MCP-NAG reacts well with NAGase (Km = 0.41 mmol/L) and is highly water soluble. The absorption maximum and molar absorptivity of the aglycone MCP are 580 nm and 40 670, respectively. Spectral overlap of interfering substances at 580 nm is almost negligible, so that the urine blank can be omitted from the assay procedure. The high molar absorptivity of MCP gives sufficient analytical sensitivity at a reaction time of 15 min. The correlation between the MCP-NAG method (y) and the fluorimetric method (x) involving 4-methylumbelliferyl N-acetyl-beta-D-glucosaminide is represented by the equation y = 0.995x - 0.669 (r = 0.991). Thus, the present method provides practical advantages over conventional methods, for use in the routine laboratory.

Identification of the serum binding proteins for iron, zinc, cadmium, nickel, and calcium.

Abstract: The binding of five biologically important metals to serum proteins has been studied. After suitable radioactive isotopes were added to serum proteins separated and precipitated by two-dimensional immunoelectrophoresis, the sample plates were exposed to roentgenogram film. 59Fe bound to transferrin alone; 65Zn bound mostly to albumin, but also to another 12 proteins; 109Cd was mostly associated with alpha 2-macroglobulin, but was also present on albumin, immunoglobulins G and A, and prealbumin; 63Ni, added in high concentration, was associated with an alpha 2-mobility protein and albumin; and, finally, 45Ca was mostly bound to albumin, but seven other binding proteins were also identified, with transferrin predominant. The results are not quantitative, but the technique is simple and specific, and the information gained can direct further studies on isolated proteins.

Immunoassay techniques with fluorescent phycobiliprotein conjugates.

Abstract: We describe immunoassay techniques that take advantage of the novel properties of phycobiliprotein fluorescent dyes. These dyes, which can be isolated from a wide variety of algae, exhibit extremely high absorptivities, high quantum efficiencies, and excitation and emission bands across the visible spectrum. These stable, hydrophilic proteins can easily be linked to antibodies by conventional protein cross-linking reagents. One assay technique presented here demonstrates the use of phycobiliproteins in a solid-phase "sandwich" assay, which achieves picomolar sensitivity. In the other technique the unique spectral properties of phycobiliproteins are applied to fluorescence excitation transfer immunoassay.

Some considerations of methodology and standardization of apolipoprotein B immunoassays.

Abstract: Apolipoprotein B, the major protein component of very-low-density (VLDL) and low-density (LDL) lipoproteins in serum, is being widely measured in serum, to help assess risk of cardiovascular disease. Standardization has proven difficult because of the physical and immunochemical heterogeneity of LDL and VLDL, the masking of antigenic determinants of LDL and VLDL, the insolubility of apolipoprotein B, the instability of lipoprotein reference materials, and the multiplicity of analytical methods. Here we have examined the literature and collected current information from lipoprotein scientists to determine the problems--and the ways investigators have attempted to solve them--in preparation, storage, and use of standards and anti-apolipoprotein B sera, analytical procedures and reagents, and the nature of apolipoprotein B in serum. Standardization appears possible with development of stable reference materials that are applicable to the various analytical methods, controlled immunochemical reaction conditions, and establishment of a selected interim reference method for use as a point of reference.

Temperature correction of blood-gas and pH measurements.

Abstract: We critically review formulas for temperature correction of pH, pCO2, and pO2 measurements in whole blood and the clinical usefulness of these formulas. We discuss both the theoretical derivation and experimental verification of temperature-induced changes. We recommend when to use and when not to use these formulas, based upon the clinical interpretation of these assays.

Continuous-flow system for automation of latex immunoassay by particle counting.

Abstract: Latex immunoassay is a nonisotopic method based on agglutination, by protein, of calibrated latex particles coated with a specific antibody The assay has been automated in a simple continuous-flow system by incubating the reaction mixture in a heated mixing coil for 25 min and measuring the agglutination with a cell counter No external shaking of the latex suspension and no additional reagent is required for the agglutination The method can accurately and precisely quantify a wide variety of proteins in plasma and urine, including human ferritin, beta 2-microglobulin, retinol-binding protein, and albumin Depending on the antigen-antibody system, the detection limit ranges from 10(-10) to about 10(-12) mol/L Within- and between-assay CVs are less than 10% In the assay of ferritin, sera are pretreated to eliminate interferences from chylomicrons, complement, and rheumatoid factor

The "HemoQuant" test: a specific and quantitative determination of heme (hemoglobin) in feces and other materials.

Abstract: We describe a new, specific, quantitative method for fecal blood, based on conversion of nonfluorescing heme to fluorescing porphyrins, that obviates serious deficiencies inherent in currently used tests. A two-reagent system is used to determine the two hemoglobin-related fractions that are found in feces. The hot citric acid extract includes only the variable fraction of porphyrins that have been preformed from heme in the intestinal tract; this often is the major fraction. Total hemoglobin is indirectly determined by reaction with heated oxalic acid:FeSO4 reagent, which converts the remaining heme to porphyrin without loss of the preformed porphyrins. A three-step purification procedure eliminates interfering materials. Analytical recovery of added hemoglobin is linearly related to concentration over a several-thousand-fold range. The assay is equally applicable to whole blood or to sub-microgram amounts of hemoglobin in the 8-mg (wet weight) fecal sample tested. Quality control by liquid chromatographic and fluorometric analysis documents fluorescence specificity of the heme-derived porphyrins.

Multi-enzyme membrane electrodes for determination of creatinine and creatine in serum.

Abstract: An enzyme electrode system for the determination of creatinine and creatine was developed by utilizing three enzymes: creatinine amidohydrolase (CA), creatine amidinohydrolase (Cl), and sarcosine oxidase (SO). These enzymes were co-immobilized onto the porous side of a cellulose acetate membrane with asymmetric structure, which has selective permeability to hydrogen peroxide. Two kinds of multi-enzyme electrodes were constructed by combining a polarographic electrode for sensing hydrogen peroxide and an immobilized CA/Cl/SO membrane or Cl/SO membrane for creatinine plus creatine or creatine, respectively. The multi-enzyme electrodes responded linearly up to 100 mg of creatinine and creatine per liter in human serum. Response time was 20 s in the rate method and the detection limit was 1 mg/L. Only 25 microL of serum sample is required. Analytical recoveries, precisions, and correlations with the Jaffe method were excellent, and the multi-enzyme electrodes were sufficiently stable to perform more than 500 assays. No loss of activity of immobilized enzymes was observed after nine months of storage at 4 degrees C in air.

Time-resolved fluoroimmunoassay of human choriogonadotropin.

Abstract: We describe time-resolved fluoroimmunoassay for human choriogonadotropin involving monoclonal antibodies directed against the beta- and alpha-subunits. The latter antibody was labeled with europium, which was measured by counting for 1 s after the immunoreaction was completed. In the solid-phase sandwich assay, both a one-step and two-step procedure were used; the respective measuring ranges were 0.7-135 and 0.7-350 int. units/L, the latter covering a 500-fold dynamic range. The CV within the assay range was between 4 and 8%, depending on the dose. Cross reactivity with lutropin in the one- and two-step procedures was 1.6% and 1.0%, respectively.

Simplified liquid-chromotographic analysis for cyclosporin A, and comparison with radioimmunoassay.

Abstract: We describe a simplified isocratic "high-performance" liquid-chromatographic method for measuring a new immunosuppressive drug, cyclosporin A, in biological fluids with use of its analogs cyclosporin C and cyclosporin D as internal standards. The method is reproducible and accurate and appears to be specific for cyclosporin A; the detection limit is 31 micrograms/L. The chromatographic measurements of the concentration of cyclosporin A in serum of patients receiving the drug were invariably lower than those by radioimmunoassay and the difference became more pronounced the greater the period of time after dosing. Because measurements of cyclosporin A in serum standards were almost identical with both techniques, the differences between the two sets of results for patients' samples suggests that the radioimmunoassay is nonspecific and measures metabolites of cyclosporin A.

Radioenzymatic Assay for Direct Measurement of Plasma Pyridoxal 5'-Phosphate

Abstract: A radioenzymatic assay for measurement of pyridoxal 5'-phosphate (PLP) is described, based on the incubation of L-[3H]tyrosine (10(6) cpm, spec. acty. 1.88 Ci/mol) in the presence of the apoenzyme tyrosine decarboxylase (EC 4.1.1.25) from Streptococcus faecalis and PLP in phosphate buffer (0.1 mol/L, pH 5.5) at 37 degrees C for 60 min. The decarboxylated metabolite formed, [3H]tyramine, was selectively extracted into ethyl acetate, and the tritium radioactivity in the sample was determined by liquid scintillation counting. As little as 0.5 nmol of PLP can be detected per liter. The assay is specific, no cross reactivity having been noted for several compounds closely related to PLP. With this we could directly measure the concentrations of PLP in plasma without prior deproteinization and ether washing of the samples. Using the assay to determine plasma concentrations of PLP in healthy adult populations, we found results that were comparable with previously reported data.

Total bone and liver alkaline phosphatases in plasma: biological variations and reference limits.

Abstract: We have studied factors affecting biological variation in total plasma alkaline phosphatase in a population of 32 329 apparently healthy subjects four years old or older. Quantification of the bone and liver isoenzymes after thermal denaturation made it possible to specify the contributions of each isoenzyme to variations in the total activities. The main factors that modify plasma alkaline phosphatase activity are age, sex, hormonal state (puberty or menopause), and morphometric parameters (height, body weight, or degree of overweight). The bone isoenzyme is mainly responsible for the variations associated with age, sex, and puberty and to some extent with the menopause. Activity of the liver isoenzyme was also altered at the menopause and by certain drugs, such as oral contraceptives and blood-lipid-lowering agents. These data allow us to propose reference limits for total plasma, bone, and liver alkaline phosphatases according to age and sex.

A direct radioimmunoassay for aldosterone in plasma.

Abstract: This rapid radioimmunoassay for aldosterone is performed directly on 100 microL of unprocessed plasma, with 125I-labeled aldosterone as the labeled antigen. Our use of steroid-free plasma in preparing the standard curve resulted in an overestimate of aldosterone; this problem was overcome by adding to such plasma a mixture of other steroids to provide a constant steroid/aldosterone ratio. Over a wide range of aldosterone concentrations, results agreed well between the present assay and a routine method involving solvent extraction and paper chromatography (r = 0.85), and sensitivity (20 ng/L) and inter- (10.4%) and intra- (3.9%) assay CVs were better with the present assay. Our assay is especially useful for multiple samples and (or) when only small-volume samples are available.

Liquid-chromatographic assay and identification of mono- and diester conjugates of bilirubin in normal serum.

Abstract: This liquid-chromatographic procedure for determining bilirubin mono- and diester conjugates in normal serum is based on pre-analysis conversion of bilirubin monosugar and disugar conjugates to the corresponding methyl esters by alkaline methanolysis. Here, extracted unconjugated bilirubin, bilirubin monomethyl esters, bilirubin dimethyl ester, and internal standard are separated on a reversed-phase column within 15 min, detected in the effluent at 436 nm, and quantified from their peak areas. Carotenoids do not interfere. Within-day and day-to-day CVs range from 5 to 13%. The smallest concentrations of monoconjugated and diconjugated pigments that are detectable and measurable are about 10 and 20 nmol/L, respectively. Such data are given for sera from 43 healthy adults. Total bilirubin concentrations in serum tended to be lower in women than in men, but the relative amounts of the various bilirubin fractions in sera from men and women were comparable. Analysis of ethyl anthranilate azoderivatives from serum permitted identification of the bilirubin ester conjugates in normal serum as bilirubin 1-O-acyl glucuronides.

"Unbound analog" radioimmunoassays for free thyroxin measure the albumin-bound hormone fraction.

Abstract: We have assessed the influence of albumin-bound thyroxin (T4) on apparent free T4 values obtained by two "unbound analog" free T4 methods (AmerlexR Free T4 and Clinical Assays one-step Free T4). We evaluated sera showing three different albumin anomalies: total hereditary analbuminemia, partially corrected analbuminemia, and familial dysalbuminemic hyperthyroxinemia, where abnormal albumin-binding of analog tracer is associated with high apparent free T4 values by these methods. In hereditary analbuminemia, free T4 was almost undetectable by both assays; in contrast, free T4 by equilibrium dialysis was normal. After addition of T4-free human serum albumin, the apparent free T4 concentration in total hereditary analbuminemia became normal by the analog methods. Immunoprecipitation of [125I]T4 and the unidentified labeled kit analogs by antiserum to human albumin was negligible in untreated total hereditary analbuminemia and approximately twice normal in familial dysalbuminemic hyperthyroxinemia. Therefore, alterations in tracer binding to albumin correlate with the apparent free T4 concentrations obtained by the analog methods. The interactions of the unidentified analog tracers and T4 with albumin are such that these techniques principally reflect the albumin-bound T4 moiety.

Evaluation of the Coomassie Brilliant Blue G-250 method for urinary protein.

Abstract: The Coomassie Brilliant Blue G-250 method for protein in urine has been evaluated for analytical accuracy and clinical applicability. Extremely simple to perform, the test exhibits good precision and sensitivity. The color developed per gram of protein is protein-type dependent, so no single protein standard is completely satisfactory. Color intensity is linearly related to concentration up to 1500 mg/L when used with a manual bichromatic method. Accuracy was clinically acceptable for patients with a variety of protein-losing diseases, and for patients having received renal transplants; however, the method underestimates urinary light-chain proteins. In athletes and premature neonates, we observed increased protein excretion during periods of stress. The upper reference limit for protein excretion in healthy adults is about 120 mg/24 h.