bluuebunny/crossref_metadata_2025 huggingface dataset:
Immunochemical determination of human immunoglobulins with a centrifugal analyzer.
Authors: Finley P R , Williams R J , Byers J M
Published: January 2020

We used an unmodified centrifugal analyzer (Aminco "Roto-Chem II") to measure human immunoglobulins IgG, IgA, and IgM in diluted sera, with diluted commercial monospecific antisera. Turbidimetric endpoint readings at 340 nm were taken at 4 min for IgG and 12 min for IgA and IgM, although multiple timed-interval readings were printed so that the kinetics of the reaction could be observed. Polyethylene glycol was used to enhance the antigen--antibody reaction. A wide range of concentrations can be measured: for IgG, 0.1--24 g/liter; for IgA and IgM, 50--4000 mg/liter. Correlation with radial immunodiffusion and automated immunoprecipitin techniques was satisfactory. Precision, accuracy, linearity, and sensitivity were quite acceptable. Day-to-day precision ranged from 2.5% to 5.2%, depending on sample concentration. The technique is more precise than radial immunodiffusion, and is rapid, simple, and reliable.
Derivatives of 1-naphthyl ethylenediamine dihydrochloride for standardization of direct-bilirubin assays done with the Technicon "SMAC".
Authors: Furda J , Morgenstern S , Snyder L R
Published: January 2020

The Jendrassik--Groff assay for direct bilirubin was adapted for analysis rates of 150/h on the Technicon "SMAC" continuous-flow analyzer. This requires development of a standard that is compatible with the other 19 channels on this analyzer. N-1-Naphthyl ethylenediamine dihydrochloride has been used for standardization of direct bilirubin assays, but we found it to be unsuitable because values for potassium are falsely elevated when potassium is determined with a valinomycin ion-selective electrode. This interference can be eliminated by alkylating the aliphatic amine group in the standard. The resulting compounds undergo the coupling reaction in the same way as the original compound and function equally well as standards for the direct bilirubin reaction. The only limitation of these analogs is their decreased solubility at physiological pH in some cases. Thus only certain alkyl groups are suitable.
Chromatography on Florisil in the quantitative estimation of urinary and other porphyrins.
Authors: Schwartz S , Stephenson B , Sarkar D
Published: January 2020

We describe the use of Florisil chromatography to purify and fractionate porphyrins into uro-, copro-, and protoporphyrins before their measurement by quantitative fluorometry. Porphyrinogens in fresh urine may be rapidly oxidized to porphyrins by chloranil, or slowly in weakly alkaline solution, before the sample is applied to the Florisil column. Aliquots of the separate ammoniacal eluates may be lyophilized for additional fractionation by direct thin-layer chromatographic analysis if desired. The behavior of delta-aminolevulinic acid, porphobilinogen, and other pyrrole compounds in this system is also described.
Equilibrium binding studies of insulin antibodies in diabetic subjects.
Authors: Baxter R C , Yue D K , Turtle J R
Published: January 2020

Equilibrium binding of insulin to human insulin antibodies from diabetic subjects has been studied by two methods. Results obtained by using a nondissociating gel filtration assay differed from those obtained by a conventional competitive binding method. Both methods yield data consistent with two classes of insulin binding site, having association constants of approximately 10(9) and 10(7) liter/mol. The principal differences are the higher association constant and lower number of binding sites for the high-affinity binding component, observed with the gel filtration method. In the conventional competitive binding assay, damaged radioligand, dissociation of the binding complex, or limitations of ligand availability may be responsible for the erroneous results. Data from nine diabetic patients indicate that insulin resistance and stability of diabetes cannot be explained by the binding properties of insulin antibodies alone.
Alkaline phosphatase. I. Kinetics and inhibition by levamisole of purified isoenzymes from humans.
Authors: Van Belle H
Published: January 2020

I studied the kinetics and sensitivity toward inhibition by levamisole and R 8231 of the most important human alkaline phosphatase isoenzymes. N-Ethylaminoethanol proved superior to the now widely used diethanolamine buffer, especially for the enzymes from the intestine and placenta, behaving as an uncompetitive activator. The optimum pH largely depends on the substrate concentration. The addition of Mg2+ has no effect on the activities. The meaning of Km-values for alkaline phosphatases is questioned. Isoenzymes from human liver, bone, kidney, and spleen are strongly inhibited by levamisole or R 8231 at concentrations that barely affect the enzymes from intestine or placenta. The inhibition is stereospecific, uncompetitive, and not changed by Mg2+. Inhibition is counteracted by increasing concentrations of N-ethylaminoethanol. The mechanism of inhibition is suggested to be formation of a complex with the phosphoenzyme.
Analysis of variation in automated determination of sodium, potassium and calcium ions in human serum.
Authors: Bokelund H
Published: January 2020

The concentration of sodium ion (Na+), potassium ion (K+) and calcium ion (Ca2+) in patient sera were assayed simultaneously on two separate flame photometers and then evaluated by analysis of variance (ANOVA). Two different experimental designs were devised in order to separate the error components assignable to pipetting, dilution, and instrumental measurement. Within-series variability was compared with the between-series variability. Similar statistical treatment of results from quality control samples gave estimates of variances between days, between dilutions, between instruments, and between channels, as well as the interaction terms and instrumental measurement errors. The total (overall) coefficients of variation (CV) were 1.15, 1.60, and 1.38% for Na+, K+, and Ca2+, respectively. The adequacy of the quality-control system to predict expected variation in patient specimens was verified by comparing errors from patient-sample duplicates and from control samples.
Catabolism of plasma enzymes, as studied with 125I-labeled lactate dehydrogenase-1 in the rabbit.
Authors: Wilkinson J H , Qureshi A R
Published: January 2020

Circulating enzymes may be inactivated in the plasma and the inactive breakdown products may be hydrolyzed in the lumen of the small intestine. Evidence for this mechanism was based upon previous studies with 125I-labeled lactate dehydrogenase-5, and here similar studies with radioiodinated lactate dehydrogenase-1 are reported, to determine whether this isoenzyme is similarly catabolized. The pure rabbit enzyme was labeled with 125I by use of lactoperoxidase and hydrogen peroxide (the labeled enzyme had 80-85% of the original catalytic activity). After its intravenous injection into rabbits, plasma enzyme activity and radioactivity disappeared during the first 4 h at similar fast rates, apparently because of distribution of the injected enzyme throughout the extracellular fluid. During a second phase (30-h), catalytic activity disappeared significantly faster than radioactivity, suggesting inactivation of the enzyme in either the plasma or a compartment in close proximity to it, or both. Enzyme activity then remained constant while plasma radioactivity continued to decrease at a slower, exponential rate, apparently owing to removal of breakdown products. In no case did tissue radioactivity, studied 6 h after injection, approach that of plasma. We therefore conclude that removal of the enzyme protein or its breakdown products is a passive process. Appreciable radioactivity was detected in the intestinal contents, a finding which suggests that removal via the small intestine is an important route for the removal of inactivated enzyme products from the circulation. Less than 3% of the injected radioactivity appeared in the feces during the first three days; urinary excretion accounted for about 67% during the same period, about 60% of which consisted of radio-iodinated amino-acids, the remainder of iodide. Free mono- and di-iodotyrosines were among the products excreted. These appear to originate from absorption of the products of further breakdown of the enzyme molecule in the intestine.
Use of a cholesterol-rich bovine lipoprotein to enhance cholesterol concentrations in the preparation of serum control materials.
Authors: Proksch G J , Bonderman D P
Published: January 2020

We describe a process for preparing a cholesterol-rich lipoprotein extract from bovine serum. The material is used to enhance cholestrol concentration up to 3.5 g/liter in a clear control material from human serum. The optical clarity of the enriched serum is only slightly affected by lyophilization and reconstitution. The lipoprotein additive neither produces any evidnet interference in the measurement of the commonly analyzed consitutents nor affects the stability of components after lyophilization and reconstitution.
Micromethod for analysis for chloride in physiological fluids.
Authors: Schosinsky K H , Chavarria M , Lehmann H P
Published: January 2020

A micromethod for the analysis for chloride, based on the chemical precipitation of silver chloride by radial diffusion through agar gel containing silver nitrate, is described. The method is simple to run, requires little or no instrumentation, and requires only 10 mul of sample. Results by coulometric titration (Buchler Cotlove Chloridometer) correlated well for serum (r = 0.961), urine (r = 0.997), cerebrospinal fluid (r = 0.991), and sweat (r = 0.998). Other halide ions or protein do not interfere. Precision studies gave a within-day reproducibility (CV) of 1.3% and a day-to-day variability of 2.1% for a serum sample averaging 115 mmol/liter.
Measurement of glucose in plasma, with use of immobilized glucose oxidase and peroxidase.
Authors: Kiang S W , Kuan J W , Kuan S S , Guilbault G G
Published: January 2020

We describe a novel fluorometric technique for simple, rapid, and economical assay of glucose by use of immobilized glucose oxidase and peroxidase. A cylindrical magnetic stirrer was specially designed to hold the immobilized enzymes firmly and to allow the reaction minzymic transformation quickly. Blood plasma can be assayed directly with no pretreatment. Ascrobic acid, uric acid, and creatinine in concenctrations of 0.25, 0.25, and 0.2 g/liter, respectively, did not interfere. The linearity of the assay was extended to 4.0 g of glucose concentrations. Results by our technique and by the otoluidine or hexokinase methods agreed well. The immobilized enzymes are stable for several months and can be used for several hundred highly accurate and reproducible assays.
Improved continuous-flow method for detemination of total serum hexosamines.
Authors: Sobocinski P Z , Canterbury W J , Jurgens K H
Published: January 2020

We describe an automated determination of serum hexosamine by the Elson-Morgan reaction, together with reagent modifications that minimiz interference from amino acids and sugars present in acid hydrolysates of sera. We used a novel 15-min autoclave procedure for the acid hydrolysis of sera before analysis to facilitate the detemination as compared to the 4-h hydrolysis used in the conventional manual method. Results correlated well (r = 0.906) with those obtained by the corresponding manual method.
Stability and precision of a new ampuled quality-control system for pH and blood-gas measurements.
Authors: Komjathy Z L , Mathies J C , Parker J A , Schreiber H A
Published: January 2020

We describe an evaluation of the in-use stability and short-term precision of a three-level ampuled quality-control system for monitoring pH, pCO2, and pO2 measurements on clinical blood-gas analyzers. In three hospital laboratories, 324 such ampuls were opened and allowed to stand with their contents exposed to atmospheric conditions for accurately timed intervals up to 240 s. Contents were then analyzed for pH, pCO2, and pO2. Student's t-test was used to evaluate the significance of differences observed in recoveries after time exposure. At a signifcance level of P less than or equal to 0.05, the only significant changes observed throughout the first minute of exposure were average pO2 increases of 180 Pa (1.4 mmHg) (+ 1.4%) and 230 Pa (1.7 mmHg) (+ 2.9%) at levels of 13.4 and 7.7 kPa kPa (101 and 58 mmHg), respectively. The ampuled system was found to be stable precise convenient, and suitable for use in the routine laboratory.
Ion chromatography: a new technique for clinical chemistry.
Authors: Anderson C
Published: January 2020

Ion chromatography is a new analytical technique in which the ions to be analyzed are separated on a special pellicular ion-exchange column. Once separated, the effluent from the separating column passes into a second ion-exchange column, which removes the eluent or buffer ions from the moving phase without removing the sample ions, which are detected and measured by electrical conductivity on a background of low conductance. Trace quantities can be so determined. This technique has been applied to four biological fluids: blood serum, tissue extract, urine, and cerebrospinal fluid. It is shown to be a practical analytical tool, particularly for ions having no chromophores, such as sodium, ammonium, potassium, magensium, calcium, chloride, nitrite, phosphate, and sulfate.
A propagation of error analysis of the enzyme activity expression. A model for determining the total system random error of a kinetic enzyme analyzer.
Authors: Tiffany T O , Thayer P C , Coelho C M , Manning G B
Published: January 2020

We present a total system error evaluation of random error, based on a propagation of error analysis of the expression for the calculation of enzyme activity. A simple expression is derived that contains terms for photometric error, timing uncertainty, temperature-control error, sample and reagent volume errors, and pathlength error. This error expression was developed in general to provide a simple means of evaluating the magnitude of random error in an analytical system and in particular to provide an error evaluation protocol for the assessment of the error components in a prototype Miniature Centrifugal Analyzer system. Individual system components of error are measured. These measured error components are combined in the error expressiion to predict performance. Enzyme activity measurements are made to correlate with the projected error data. In conclusion, it is demonstrated that this is one method for permitting the clinical chemist and the instrument manufacturer to establish reasonable error limits.
Multicomponent analysis in clinical chemistry by use of rapid scanning fluorescence spectroscopy.
Authors: Warner I M , Callis J B , Davidson E R , Christian G D
Published: January 2020

To be useful in the clinical laboratory, multicomponent fluorescence analysis requires both the rapid measurement of the fluorescence intensity at a variety of excitation and emission wavelengths and the unambiguous reduction of the data by efficient algorithms. The Video Fluorometer, which exploits the multi-channel capability of a low-light-level television sensor to simultaneously acquire excitation and emission spectra, can meet the first requirement. For example, a complete set of emission and excitation spectra for perylene can be obtained in less than 2 s at concentrations of 10(-10) mol/liter. To meet the second need, we present two types of data-reduction strategies: (a)a least-squares fit to the data, with use of the spectra of previously determined compounds likely to be present; and (b)a determination of the eigenvalues and eigenvectors fo the fluorescence matrix, from which the number of components and the possible spectra of each can be estimated. Computer simulations of the least-squares fitting algorithms show that five strongly overlapping components can be determined in the presence of noise with an accuracy of better than 5%. Also, a fluorescent sample containing two species with very similar but unknown spectral properties can be resolved to obtain the spectrum of each.
Centrifugal system for affinity chromatography with eluate monitoring.
Authors: Shumate S E , Scott C D
Published: January 2020

A prototype centrifugal system has been developed that permits parallel photometric monitoring of eluate streams from a radial array of chromatographic columns. The modular rotor design consists of a discoidal center insert for eluent and sample apportionment, the chromatographic columns, and flow-through cuvetts-all mounted on an aluminum base plate. A common sample is introduced simultaneously to each column; a single eluent stream is used for all columns. The goal is to assay simultaneously for eight to 16 serum proteins separated by affinity chromatography from a single sample. The system is here exemplified by the use of immunosorbents (consisting of antibodies to human immunoglobulins covalently bound to alumina particles) to allow simultaneous determinations of IgG AND IgM from a single human serum sample in less than 16 min.
Reversed-phase liquid-chromatographic analysis of hemodialysate from uremic patients.
Authors: Senftleber F C , Halline A G , Veening H , Dayton D A
Published: January 2020

With reversed-phase high-performance liquid chromatography we effected rapid and efficient separation of metabolites present in hemodialysate fluid from uremic patients on the artificial kidney. With an aqueous sodium acetate/methanol gradient as mobile phase, more than 50 ultraviolet-absorbing constituents were resolved in a 70-min chromatogram of a 100-mul sample of dialysate. Many of the metabolites could be detected in concentrations below 0.1 mg/liter. From results of ultraviolet spectrophotometric analysis and the gas- and liquid-chromatographic properties of standards, half the observed chromatographic peaks have been characterized or identified. The method has been shown to be more than 10-fold faster than comparable separations by ion-exchange techniques.
Comparison of estimates of long-term analytical variation derived from subject samples and control serum.
Authors: Williams G Z , Harris E K , Widdowson G M
Published: January 2020

Variation in the assays of uniform control serum commonly are assumed to represent day-to-day analytical variation. To test this assumption, we compared the differences between results of serum aliquots assayed immediately for 12 constituents and frozen aliquots accumulated and assayed on a single day with the results of control serum variation from the same period. One aliquot of each weekly sample was stored frozen. Eleven subjects were sampled for 12 weeks. Storage at --20 degrees C for 15 weeks had a mild destructive effect on two enzymes in serum. The control serum data revealed significant linear trends in magnesium (upwards) and alkaline phosphatase (downwards) that substantially increased the respective variances. In the other 10 constituents tested, comparison of variances indicated that long-term (weeks) variation in control serum assays is similar to the difference of variation between aliquots assayed immediately and those frozen and assayed at the same time. For these constituents, this finding justifies the use of control serum to estimate long term analytical variation.
Development and use of analytical systems based on mass spectrometry.
Authors: Horning E C , Carroll D I , Dzidic I , Haegele K D , Lin S , Oertli C U , Stillwell R N
Published: January 2020

Contemporary analytical systems based on mass spectrometry include as components a gas chromatograph, a mass spectrometer, and a computer. The form of operation is usually in electron impact ionization mode for identification and structural studies, and in chemical ionization mode for quantitative analyses. Important stages in the development of these systems included the design of "molecule separators" for the concentration of solutes in the gas phase, the use of mass spectrometers as specific ion detectors, the introduction of chemical ionization techniques, and the development of computer-based operation, data acquisition, and data analysis capabilities. A current line of investigation is concerned with the design and use of systems based on atmospheric pressure ionization. Samples are ionized in a small reaction chamber external to the low-pressure region of a quadrupole mass analyzer. The primary source of electrons is a 63Ni foil or a corona discharge. The ionization process leading to positive ions involves a sequence of ion molecule reactions, usually electrons leads to carrier gas ions leads to reagent ions leads to sample component ions. Negative ions may be formed by direct electron attachment, or by ion molecule reactions that include new types of elimination reactions. The source will accept a variety of gases and solvents. The sample may be introduced in the gas phase without solvents, by probe injection, or in the effluent stream from a gas chromatograph. Samples may be introduced in the liquid phase in solvents by injection after the fashion of gas chromatography or in the effluent stream from a high-performance liquid chromatograph. The novel aspects of atmospheric pressure ionization mass spectrometry lie in its versatility and high sensitivity of detection. Few clinical chemistry laboratories now use these systems. Significant future uses are likely to be in analytical work involving therapeutic drug monitoring and studies of drug metabolism, and in analyses for environmental biohazards including pesticides, herbicides, polyhalobiphenyls, dibenzodioxins, and other toxic compounds.
A list of selected reference materials in clinical chemistry.
Authors: Airall J M
Published: January 2020

This is a compilation of reference materials suggested for scientists considering a laboratory reference collection in clinical chemistry, or for librarians seeking to develop or add to an up-t-o-date library collection. Nine areas are represented: general clinical chemistry; analytical methods, procedures, and techniques; calculations and statistics; the clinical laboratory; drugs and metabolism; enzymology; instrumentation, interpretation of data; and ready reference.
Serum albumin: recent progress in the understanding of its structure and biosynthesis.
Authors: Peters T
Published: January 2020

Major discoveries have been made in the past few years on the structure and mode of biosynthesis of serum albumin. The complete amino acid sequence of this protein has been determined, and its covalent structure shown to be a single peptide chain grouped into a series of nine disulfide-bonded loops. These loops appear to associate into three similar domains. By study of isolated fragments of the molecule it can be demonstrated that the binding of billirubin and the primary binding of long-chain fatty acids are functions of separate domains. The biosynthesis of albumin has been found to involve a precursor form, termed "proalbumin", in which a basic hexapeptide is attached to the amino end of the chain. Similar precursor forms are now known to have a role in the formation of other secreted proteins, but in the case of albumin the purpose of the additional peptide is not clear. Clinical methodology for albumin assay has advanced but little despite--or perhaps in part because of--the increasing use of automation. Hope for improvement is foreseen in the advent of immunochemical procedures and in a better understanding of the specificity of dye-binding reactions.
Comparison of double-antibody radioimmunoassay with Farr-technique radioimmunoassay and double-antibody enzyme immunoassay for alpha-fetoprotein.
Authors: Kirkpatrick A , Wepsic H T , Nakamura R M
Published: January 2020

We describe double-antibody procedures for determining alpha-fetoprotein in human serum. An equilibrium procedure can be done in 24 h with a sensitivity of at least 4 mug/liter and coefficient of variation of 5.5%. There are no interferences from normal human sera or sera with certain commonly seen chemical abnormalities. We also describe and discuss sequential procedures that range in sensitivity from 250 ng to 1mug/liter and require 24-48h incubation. The precise (mid-range)portion of the dose/response curve for sequential procedures can be shifted to higher or lower values by an adjustment of the time of preliminary incubation of antibody with unlabeled antigen. With a 37 degrees C incubation, a sequential procedure can be completed in 7 h. Sensitivity is 1 mug/liter, and coefficient of variation 8.0%. The relative merits of the above assay procedures are discussed. The double-antibody redioimmunoassay is twice as sensitive as the Farr procedure [J. Infect. Dis. 103, 239 (1958)], and it is free of the large and variable nonspecific precipitation that accompanies the precipitation of bound antigen with sodium sulfate solution. Double-antibody radioimmunoassay is superior to enzyme immunoassay in both sensitivity and precision.
Gamma-Glutamyltransferase: kinetic properties and assay conditions when gamma-glutamyl-4-nitroanilide and its 3-carboxy derivative are used as donor substrates.
Authors: Shaw L M , London J W , Fetterolf D , Garfinkel D
Published: January 2020

The kinetics of human serum gamma-glutamyltransferase (EC 2.3.2.2) were investigated, with use of glycylglycine as a gamma-glutamyl acceptor substrate and gamma-glutamyl-4-nitroanilide and its carboxy derivative, gamma-glutamyl-3-carboxy-4-nitroanilide, as donor substrates. The simultaneous occurrence of both gamma-glutamyltransfer and autotransfer was established by descending paper chromatography. Constant-ratio double-reciprocal plots confirm that the enzyme mechanism is nonsequential (ping-pong bi-bi). Inhibition by either donor was not found, and inhibition by glycylglycine was only observed at concentrations above those of clinical interest. Kinetic constants obtained by nonlinear regression analysis of initial velocity data were used to determine reagent substrate concentrations for the assay of this enzyme. An assay with use of 4 mmol of gamma-glutamyl-3-carboxy-4-nitroanilide and 100 mmol of glycylglycine per liter yielded equivalent activities to those by assay with use of 4 mmol of gamma-glutamyl-4-nitroanilide and 40 mmol of glycylglycine per liter. These concentrations of the carboxy donor and glycylglycine are also "cost optimal" and present no procedural problems when used.
A kinetic spectrophotometric assay for rapid determination of acetoacetate in blood.
Authors: Price C P , Llyod B , Alberti G M
Published: January 2020

We describe an automated kinetic assay for acetoacetate in blood. Acetoacetate is enzymatically reduced to D-beta-hydroxybutyrate and the reaction is monitored for 60 s with a reaction-rate analyzer. This technique allows low concentrations of acetoacetate to be measured with good precision and overcomes many of the problems associated with other automated techniques. Our studies on the stability of acetoacetate emphasize the need for care in handling specimens. The use of a reaction-rate analyzer, an item of equipment common to most laboratories, allows for rapid handling of samples in small or large batches, depending on the needs of the laboratory.
Automated kinetic determination of lactate dehydrogenase isoenzymes in serum.
Authors: Bernstein L H
Published: January 2020

A steady-state kinetic method has been revised for measuring lactate dehydrogenase isoenzyme activities, which relates the inhibition of heart-type isoenzyme activity to the overall isoenzyme composition of the enzyme subunits. The method depends on the pH-dependent formation of an inhibitory ternary complex by the heart-type isoenzyme with NAD+ and pyruvate (if the reaction is measured by NADH oxidation). A preincubation step in the previous method is eliminated. The isoenzymes are measured by measuring the reduction of pyruvate in two different concentrations, which favor either the total or fractional activity, depending on the concentrations of pyruvate and the percentage of heart-type subunits. The method has been adapted to a centrifugal analyzer, which has speeded automated isoenzyme determinations, with an accuracy comparable to that for electrophoretic methods.
Clinical pharmacology of anti-epileptic drugs: a summary of current information.
Authors: Leal K W , Troupin A S
Published: January 2020

This compendium represents what we believe to be the most current and reliable pharmacological data on anticonvulsant drugs. The information presented is derived from determinations of the drugs in plasma or serum by gas--liquid chromatography in studies of the efficacy of anti-epileptic agents. We present information on the limitations of therapeutic concentration ranges, half-lives, active and inactive metabolites, and structure/activity relationships of anticonvulsant drugs. This report provides answers to many of the questions clinicians direct to anticonvulsant-monitoring laboratories. Information on other pharmacoloical variables supplements this review in the interest of the clinical investigator.
Value of alkaline phosphatase, 5'-nucleotidase, gamma-glutamyltransferase, and glutamate dehydrogenase activity measurements (single and combined) in serum in diagnosis of metastasis to the liver.
Authors: Kim N K , Yasmineh W G , Freier E F , Goldman A I , Theologides A
Published: January 2020

We assessed, in 98 patients with cancer, the diagnostic value of measuring serum alkaline phosphatase, 5'-nucleotidase, gamma-glutamyltransferase, and glutamate dehydrogenase activities as an aid to detection of liver metastases. All four enzymes showed diagnostic value, but 5'-nucleotidase appeared to have the greatest. It showed the lowest false-positive results (7.4%) with the highest predictive value of a positive test (85.7%) and agreement (81.3%).. gamma-Glutamyltransferase showed the lowest proportion of false-negative results (2.8%), but was the least specific 35% false-positive results). Analysis of various test combinations showed that the best agreement (77.5%) was obtained when the patients were divided into those who had no or only one abnormal test result, and those who had two or more abnormal test results. However, this was not better than the agreement for 5' nucleotidase alone (81.3%). The agreement of 5'-nucleotidase and gamma-glutamyltransferase (i.e., both tests were positive or negative) was excellent (91.4%), but such agreement included only 67% of the patients with liver metastases.
Activity and interference effects in measurement of ionized calcium with ion-selective electrodes.
Authors: Grima J M , Brand M J
Published: January 2020

We assessed the magnitude of the errors in ionized-calcium measurements resulting from changes in electrolyte composition for three different ion-selective electrodes: the Orion Model 93-20, the Radiometer F2112, and the Simon neutral carrier membrane electrode. We attempted to distinguish between errors arising from changes in calcium ion activity and those due to interferences in the electrode response. Variation in sodium ion concentration over the range 100--180 mmol/liter produces changes in apparent ionized-calcium concentration that are largely attributable to activity effects for the Radiometer and Simon electrodes. The Orion electrode is subject to an additional sodium-interference effect. Apparent ionized calcium concentration measurements are independent of pH for the Radiometer electrode but not for the Orion electrode; the Simon electrode exhibits intermediate pH response, which is probably clinically negligible. Magnesium and potassium ions have little effect on ionized calcium concentration measurements, particularly when these ions are incorporated into calibration standards.
Serum creatinine assay by use of 3,5-dinitrobenzoates: a critique.
Authors: Parekh A C , Sims C
Published: January 2020

Methyl-3,5-dinitrobenzoate and 3,5-dinitrobenzoyl chloride are demonstrated to react with creatinine through their conversion to 3,5-dinitrobenzoate. Analytical recovery of creatinine added to serum is improved by an "acid-supernate" modification, which is also much less susceptible to a combination of known interfering agents than is the picrate procedure. The "neutral-supernate" procedure shows negligible interference by cephalothin as compared to commonly used picrate procedures. Replacement of tetramethylammonium hydroxide by sodium hydroxide was also tested. The interrelationships between dimethyl sulfoxide concentration, recoveries of creatinine, and slopes of correlations (r greater than .992) of various dinitrobenzoate and picrate methods are critically assessed. Because of its precision, greater specificity, and other criteria, we recommend the methyl-3,5-dinitrobenzoate (or 3,5-dinitrobenzoic acid) neutral-supernate method for routine applications.
Detection of endotoxins in human blood and plasma. An improved in-vitro pyrogen test.
Authors: Nandan R , Nakashima C Y , Brown D R
Published: January 2020

We describe an improved in-vitro procedure for detection of endotoxin in human blood and plasma by use of Limulus amoebocyte lysate. Increasing concentrations of Escherichia coli endotoxin added to a constant amount of the lysate cause a proportional increase in protein precipitated by the endotoxin. By measuring the amount of protein precipitated, it was possible to determine the equivalent E. coli endotoxin concentration in unknown samples, when samples were run with E. coli endotoxin standards and negative controls. The E. coli endotoxin, present in human whole blood and platelet-rich plasma, failed to react with the lysate. However, the concentration of endotoxin in whole blood and platelet-rich plasma could be measured with this Limulus test after lysing the platelets to release the endotoxin and subsequently removing the inhibitory proteins by chloroform precipitation. With this procedure it was possible accurately and repeatedly to determine E. coli equivalent endotoxin concentrations as low as 195 ng per liter of whole blood or 49 ng per liter of platelet-rich plasma.
New method for determining thrombin-clottable fibrinogen.
Authors: Frigola A , Angeloni S , Cerqueti A R
Published: January 2020

We describe a new method for determination of thrombin-clottable fibrinogen, which eliminates the systematic error caused by occlusion of other serum proteins in the fibrin clot and reduces the sensitivity to high concentrations of fibrin degradation products. Essentially, the method consists of densitometric quantitation of the fibrin band after a standard electrophoresis run of plasma, thrombin fixation of the fibrinogen, and removal of the non-clotted proteins by washing in saline. The procedure shows good precision and gives results that are accurate, significantly correlate with results for the classical thrombin clotting method (r = 0.92, P less than .001), and are not affected by fibrin degradation product concentrations up to 900 mg/liter. These characteristics make the method especially valuable in establishing fibrogen concentration in patients who are undergoing thrombolytic therapy.
Development, validation, and application of a single-tube radioimmunoassay for cholic and chenodeoxycholic conjugated bile acids in human serum.
Authors: Roda A , Roda E , Aldini R , Festi D , Mazzella G , Sama C , Barbara L
Published: January 2020

A single-tube radioimmunoassay for both cholic acid and chenodeoxycholic acid conjugates in serum is based on the use of [1-(14)C]glycocholic acid and [H-3H]glycochenodeoxycholic acid as tracers. The assay was shown to be specific, sensitive, accurate, and precise (CV = 13% at low concentrations, 5.5% at higher concentrations) when used for serum samples from subjects and patients with increased bile acid in the serum because of liver disease, and results correlate well with those by gas chromatography (r = .99).
Determination of 14CO2 in breath and 14C in stool after oral administration of cholyl-1-[14C]glycine: clinical application.
Authors: Roda A , Roda E , Aldini R , Mazzella G , Festi D , Sama C , Barbara L
Published: January 2020

Twenty patients with intestinal bacterial overgrowth and 20 control subjects were investigated for bile acid deconjugation, by measuring 14CO2 in the breath after cholyl-1-[14C]glycine administration. 14CO2 output/24h was 11.0 +/- 5.2% (mean +/- SD) in controls and 54.2 +/- 14.0% (mean +/- SD) in bacterial-overgrowth patients (P less than .001). 14CO2 excretion rate in 12h, when normalized to 100% of the dose at the 12th hour, gave an even finer discrimination between the two groups (no false responses). 14C in stool, analyzed in 20 malabsorption patients and 20 controls by two different techniques, was 6.6 +/- 4% and 31.38 +/- 21.7% (mean +/- SD), respectively. Results by the two different techniques described here correlated well (r = .99). Bile acid malabsorption was in reasonable agreement (r = .67) with percentage of "chenoid" (chenodeoxycholic acid plus ursodeoxycholic acid) in the stool by gas-liquid chromatography; a poorer correlation was observed when "chenoid" plus "choloid" (cholic acid plus its epimers) were plotted vs. -4C in stool (r = .57, n = 15).
Determination of urinary total phenolic compounds with use of 4-aminoantipyrine: suggested screening test for hyperthyroidism and for catecholamine-producing tumor.
Authors: Yamaguchi Y , Hayashi C
Published: January 2020

We describe a method for determining those urinary total phenolic compounds that are tyrosine analogs or metabolites, such as thyroxine and catecholamines. The urine sample, 4-aminoantipyrine in carbonate-bicarbonate buffer, and potassium ferricyanide solution are mixed and the quinoneimine dye that forms is measured at 500 nm. Some cases of hyperthyroidism, diabetes mellitus, nephrosis, obesity, hypertension, or catecholamine-producing tumor showed above-normal values, so that this determination seems useful as a screening test for these disorders.
A method for serum octanoate in hepatic cirrhosis and hepatic encephalopathy.
Authors: Rabinowitz J L , Staeffen J , Aumonier P , Ballan P , Ferrer J , Terme R , Series C , Myerson R M
Published: January 2020

We describe a new, more efficient, and more reproducible method for determination of octanoate in serum. This method involves ethanol extraction, followed immediately by alkali addition before concentration of the extract. The concentrate is made acidic only before it is to be steam distilled (in a special all-glass apparatus with an alkali trap). The material is acidified again just before separation by gas-liquid chromatography. The yield is 89-107%. When assayed by mass spectrometry, only octanoate was found in the fraction from chromatography. Previous methods yielded only 30-55% of the expected octanoate value and the recovered materials showed impurities by mass spectrometry. Octanoate concentrations were determined in the serum of 24 fasting controls and that of 85 fasting cirrhotic patients, of whom 50 had encephalopathy. Concentrations in arterial and venous blood were significantly higher in cirrhotic patients in coma than in those not in coma, and arterial concentrations were statistically higher than venous concentrations in the cirrhotic patients.
Mechanisms of cellular enzyme release. II. Inhibition of sarcolemmal enzymes by myopathy-inducing agents.
Authors: Verrill H L , Gruemer H D , Baba N
Published: January 2020

A defective membrane mechanism has been suggested [Arch. Neurol. 33, 315 (1976)] for the pathogenesis of Duchenne muscular dystrophy. The characteristic clinical and biological findings, including leakage of cellular enzymes into the serum in the disease, have been duplicated by the imipramine/serotonin rat myopathy model. Sarcolemma was prepared from quadriceps femoris muscles of control and myopathy-affected animals. The activities of sarcolemmal adenosinetriphosphatase and acetylcholinesterase were inhibited in vitro by imipramine and serotonin. The inhibition by imipramine of these sarcolemma-bound enzyme systems decreased the Vmax and increased the Km. This mixed type of inhibition is consistent with an imipramine-induced interference at these enzyme sites and a disruption of lipid-protein interrelations. We hypothesize that such conformational membrane changes might contribute to the leakage of macromolecules such as enzymes from the cell interior.
Urinary 3-methoxy-4-hydroxyphenylacetic (homovanillic) and 3-methoxy-4-hydroxymandelic (vanillylmandelic) acids: gas-liquid chromatographic methods and experience with 13 cases of neuroblastoma.
Authors: Brewster M A , Berry D H , Moriarty M
Published: January 2020

We present a quantitative gas-chromatographic method for determining urinary 3-methoxy-4-hydroxymandelic acid (vanillylmandelic acid) and 3-methoxy-4-hydroxyphenylacetic acid (homovanillic acid). In this rapid technique an internal standard is used and the procedure involves ether extraction and silyl ether formation. Analytical recovery of vanillylmandelic acid averages 87.5% (CV, 0.95%), of homovanillic acid 102.3% (CV, 9.95%). Our data on 34 samples from 13 neuroblastoma patients show that homovanillic acid is more consistently elevated than is vanillylmandelic acid.
Microdetermination of plasma ATP and creatine phosphate concentrations with a luminescence biometer.
Authors: Jabs C M , Ferrell W J , Robb H J
Published: January 2020

We optimized conditions for determination of adenosine-5'-triphosphate (ATP) and creatine phosphate from plasma extracted with ethanol/water (96/4 by vol). The procedures utilize the firefly luciferin/luciferase reaction, the bioluminescence being measured with a Du Pont Biometer. ATP is quantitated directly and creatine phosphate is quantitated by reaction with creatine kinase and ADP, after plasma ATP is removed by incubation with the enzyme apyrase. The method is applied to plasma from humans, rabbits, and rats, and possible clinical applications are discussed.
Improved hydrolysis of urinary 17-hydroxycorticosteroid glucuronides with beta-glucuronidase from Helix pomatia, on adding sodium sulfate.
Authors: Nishikaze O , Kobayashi T
Published: January 2020

Sodium sulfate increases the hydrolysis of urinary 17-hydroxycorticosteroid glucuronides with beta-glucuronidase preparations derived from Helix pomatia because it removes the inhibitory activity of urinary high-molecular-weight substances. For maximum hydrolysis of urinary 17-hydroxycorticosteroid glucuronides, the hydrolysis [5 ml of urine, 0.5 ml of 2 mol/liter acetate buffer (pH 5.0)] should be conducted in the presence of sodium sulfate (final concentration: 80 g/liter) with (a) 600 Fishman units of the enzyme per milliliter of urine (18 h at 52 degrees C) or (b) with 1500 units of the enzyme per milliliter of urine (3 h at 57 degrees C). Under conditions a, analytical recovery of steroid glucuronides added to 12 urine samples was 99 +/- 2.1% (96-102%). Values obtained for 20 urine samples with this method were 99 +/- 2.7% (93-104%) as great as those yielded by a method in which 600 units of the enzyme from bovine liver are used together with sodium sulfate (18 h at 48 degrees C).
Use of saliva in therapeutic drug monitoring.
Authors: Horning M G , Brown L , Nowlin J , Lertratanangkoon K , Kellaway P , Zion T E
Published: January 2020

We measured the concentrations of phenobarbital, phenytoin, primidone, ethosuximide, antipyrine, and caffeine in paired samples of saliva and plasma by gas chromatograph-mass spectrometer-computer (GC/MS/COM) and enzyme immunoassay. Mixed saliva was collected for the antipyrine and caffeine studies, parotid saliva for the phenobarbital, primidone, ethosuximide and phenytoin studies. The saliva/plasma (S/P) ratios (by weight) obtained by GC/MS/COM were: phenobarbital, 0.31-0.37; phenytoin, 0.11; ethosuximide, 1.04; antipyrine, 0.83-0.95; caffeine, 0.55. The S/P ratio obtained by enzyme immunoassay were: phenobarbital, 0.32; phenytoin, 0.12; primidone, 0.85. The concentrations of phenytoin, primidone, ethosuximide and antipyrine in saliva correspond to the free fraction of the drug in plasma. When we analyzed samples containing phenobarbital or phenytoin (plasma or saliva) by both techniques, we found that the enzyme immunoassay values were generally higher than GC/MS/COM values, suggesting that the metabolites as well as the parent drug were measured in the immunoassay.
Automated system for infrared spectrometric analysis for total CO2 of plasma contained in capillary tubes.
Authors: Rabinow B E , Geisel A , Webb L E , Natelson S
Published: January 2020

We describe how to assay plasma total CO2 automatically, discretely, and highly specifically by infrared absorption spectrometry. Plasma, 50 mul, is sampled in disposable capillaries and inserted into a block wherein the specimens are protected from exposure to air. A sequencer advances the block and signals a dispenser to flush the sample into a reaction vessel with 2.6 ml fo 50 mmol/liter H2SO4. After 24 s of incubation, the evolved CO2 is swept into a 1-meter infrared gas cell by a stream of nitrogen. A recorder displays the resulting peak in absorbance units. Operation of all valves controlling gas and liquid flow is regulated by the sequencer. In the present version, the time required for analysis of one sample is 65 s. The sample size may be reduced by at least 10-fold and the time sequence accelerated to assay 70-80 specimens per hour.
Two-dimensional immunoelectrophrophoretic pattern of low- and very-low-density lipoproteins, with particular reference to Fredrickson's type III.
Authors: Rerabek J E
Published: January 2020

I report the pattern of low-density lipoproteins (LDL) and very-low-density lipoproteins (VLDL) as revealed by two-dimensional (crossed) immunoelectrophoresis in hyperlipoproteinemia types, II, III, and IV, preliminarily diagnosed by means of a complex lipoprotein profile examination. The higher affinity of Oil Red stain for VLDL- than for LDL-immunoprecipitation zones is peculiar to this lipoprotein class, and crossed immunoelectrophoretograms may thus be helpful for laboratory diagnosis of types IIb and IV in problematic cases. The position and the medium affinity for Oil Red of "broad beta" zones appearing in immunoelectrophoretograms of type III hyperlipoproteinemia are suggested as useful criteria for the diagnosis of this condition. From the structural and staining characteristics of VLDL-immunoprecipitation zones, alteration of both composition and antigenic properties of VLDL in hyperlipoproteinemias IIb-IV is deduced. I present in detail the procedure for crossed immunoelectrophoresis as well as that for the lipoprotein profile examination used in this study.
New, advantageous approach to the direct radioimmunoassay of cortisol.
Authors: Tilden R L
Published: January 2020

Radioimmunoassay of serum cortisol is simplified by a unique solution to the problem of eliminating competition between cortisol-binding globulin and cortisol immunoglobulin. Increasing incubation temperature tends to release cortisol from its carrier protein while favoring the immunoglobulin reaction. We applied this methodology, using reagents supplied as a commercial kit. Serum, 5 mul, is diluted with 125I-containing(hydroxymethyl)aminomethane buffer and incubated at 45 degrees C for 1 h, then aspirated. All standards are constituted in steroid-deprived serum. The clinical validity of the method is supported by comparisons with conventional methods during months of use under routine laboratory conditions. The simplified method also offers improved performance characteristics and, as described, if fully instrumented. The significance of this use of thermodynamic properties is discussed in terms of more general applicability.