bluuebunny/crossref_metadata_2025 huggingface dataset:
Negligible Interference by Spironolactone and Prednisone in Digoxin Radioimmunoassay
Authors: Ravel Richard
Published: January 2020

Spironolactone and prednisone reportedly cause measurable values for apparent digoxin, even when none is present. Effects of these medications were evaluated with 125l-labeled digoxin kits from five different manufacturers. Values obtained for apparent digoxin were either negligible or not sufficiently great to be clinically significant, regardless of kit methodology or manufacturer.
An Example of Lyophilized Protein-Based Materials Not Simulating Patient Sera
Authors: Steele Bernard W , Koehler Donald F , Blaszkowski Thomas P , Azar Miguel
Published: January 2020

Use of reconstituted lyophilized protein-based materials in the clinical laboratory is partly based on the assumption that these materials adequately simulate patients' sera. We examined several of these materials and found that certain ones do not have the same adsorbancies at 340 and 380 nm as do most sera. The implication of this is examined with respect to glucose determination by the hexokinase method on a dual-wavelength blank-subtraction instrument.
Fluorometric Assay for N-Acetylprocainamide
Authors: Matusik Edward , Gibson Thomas P
Published: January 2020

We describe a simple, rapid fluorometric assay for separate quantitative analysis of procainamide and N-acetylprocainamide in mixtures. The effective linear range (fluorescence vs. concentration) in serum is 0.1 to 10.0 mg/liter, regardless of the ratio (by weight) of the two drugs from 1:10 to 10:1. Analytical recoveries by the extraction method used were 100.0 ± 3.0% and 98.0 ± 4.0%, respectively. For determination of either compound, the maximum coefficient of variation was 10%.
Immunofluorescence Method for Detecting Anti-Myocardial Antibodies, and Its Use in Diagnosing Heart Disease
Authors: Twomey Stanley L , Bernett George E
Published: January 2020

Demonstration of autoimmune antibodies to myocardial tissue enables one to detect and assess cardiac disease long after abnormalities in serum enzyme activities are no longer measurable. We describe an indirect immunofluorescence procedure in which cryostat sections of rat heart (ventricle) and Evan's Blue counterstaining are used to detect anti-myocardial antibodies. Sera from patients with myocardial infarct or some other cardiac diseases reveal a distinct fluorescent staining of the sarcolemmal membrane. In contrast, sera from patients with systemic lupus erythematosis demonstrate nuclear plus diffuse staining and sera from myasthenia gravis patients show a characteristic striated staining pattern. The role of anti-myocardial antibodies in cardiac disease is discussed briefly.
Gas-Chromatographic Analysis for Cocaine in Human Plasma, with Use of a Nitrogen Detector
Authors: Jatlow Peter I , Bailey David N
Published: January 2020

We describe a procedure for measuring the concentration of cocaine in plasma by gas chromatography, with use of a nitrogen detector. The procedure was tested on patients receiving intranasal cocaine for topical anesthesia. The propyl ester of benzoylecgonine is used as an internal standard. The lower limit of sensitivity of the procedure is 5-10 µg of cocaine per liter of plasma. The coefficients of variation are 5.3% and 3.0% for concentrations of 50 and 100 µg/liter, respectively.
Rapid Kinetic Measurement of Lactate in Plasma with a Centrifugal Analyzer
Authors: Pesce Michael A , Bodourian Selma H , Nicholson John F
Published: January 2020

In this method, blood is collected in ammonium heparinized microhematocrit tubes and lactate is directly determined in the plasma, separated within 15 min from the erythrocytes. Lactate is assayed by mixing 10 µl of sample with NAD+ and lactate dehydrogenase in tris(hydroxymethyl)aminomethane hydrazine buffer. The rate of increase in absorbance of the NADH formed, measured at 340 nm, is proportional to lactate concentration. The assay is complete in 4 min and absorbance is linearly related to concentration from 0.625 to 15 mmol/ liter. Analytical recoveries of lactate added to plasma averaged 104% (range, 91-116%). Results compared well for plasma samples analyzed by this method with the CentrifiChem and the Du Pont aca.
Accurate Estimation of Standard Deviations for Quantitative Methods Used in Clinical Chemistry
Authors: Burnett Robert W
Published: January 2020

Although the standard deviation is the most widely used measure of the precision of quantitative methods, there is a need to re-examine the conditions necessary to obtain a meaningful estimate of this quantity. The importance of the material to be sampled, the sample size, the calculation of confidence intervals, and the segregation of outliers are discussed.
A Systematic Approach to Enzyme Assay Optimization, Illustrated by Aminotransferase Assays
Authors: London Jack W , Shaw Leslie M , Fetterolf Donald , Garfinkel David
Published: January 2020

We have developed a systematic approach to optimization of reagent concentrations for assays of alanine aminotransferase and aspartate aminotransferase: (a) Michaelis constants describing the initial-velocity kinetics of the coupled enzyme reactions were evaluated by a nonlinear least-squares fit of the appropriate equation to measured enzyme activities. Activities of more than 50 normal and pathological sera were measured at 30 °C. (b) These kinetic equations are used to calculate the set of reagent amino- and keto-acid concentrations that all yield a selected fraction of the theoretical maximum enzyme velocity. An optimal pair is determined by defining an additional criterion, such as minimal reagent cost or minimal concentration to Km ratio. (c) The optimum amounts of reagent NADH and coupling enzyme, being a function of desired pre-incubation and measurement intervals, maximum aminotransferase activity to be measured, and endogenous keto-acid concentration, are determined by computer simulation. An approximate relationship and an exact method for computing assay lag time are presented, along with experimentally measured endogenous keto-acid concentrations in serum. All procedures may be applied to other enzyme assays if appropriately modified.
Fluorometric Screening Method for Detecting Benzodiazepines in Blood and Urine
Authors: Valentour James C , Monforte Joseph R , Lorenzo Beverly , Sunshine Irving
Published: January 2020

We report a fluorometric method for detecting diazepam, chlordiazepoxide, oxazepam, chlorazepate, and (or) their major metabolites in blood, urine, or gastric contents at low therapeutic concentrations. The drugs are first hydrolyzed to their respective benzophenones and converted to highly fluorescent 9-acridanones. Total benzodiazepines (parent plus metabolites) in blood and gastric contents are semiquantitatively evaluated and compared to results of gas-chromatographic determinations.
Comparison of Human Pancreatic and Parotid Amylase Activities on Different Substrates
Authors: Stiefel Doris J , Keller Patricia J
Published: January 2020

The specific activities of highly purified preparations of human parotid and pancreatic amylase on several different soluble and insoluble starches were compared. The ratio of parotid to pancreatic activity varied with the physical nature of the substrate. With all soluble starches, irrespective of source, parotid amylase exhibited a higher specific activity than did pancreatic amylase; the reverse was true for an insoluble chromogenic starch (Amylose Azure). The variation in enzyme—substrate interaction supports previous indications of configurational differences between the two amylases. The observed organ-specific characteristics according to organ of origin may have value in determining relative parotid and pancreatic amylase activities in body fluids under normal and pathological conditions, thereby helping to clarify their functional and clinical significance.
Standardizing 125I Sources and Determining 125I Counting Efficiencies of Well-Type Gamma Counting Systems
Authors: Horrocks Donald L
Published: January 2020

I describe the technique for determining the absolute decay rate of any 125I source. The problems of varying counting efficiency, varying sample geometry, and extended sources are discussed. By using the calibrated 125I source, the counting efficiency of the given counting system was obtained.
Serum and Tissue Enzyme Activity and Trace-Element Content in Response to Zinc Deficiency in the Pig
Authors: Burch Robert E , Williams Robert V , Hahn Henry K J , Jetton Mary M , Sullivan James F
Published: January 2020

Zinc deficiency is associated with poor growth and development. It has been postulated that a deficiency of the various zinc metalloenzymes results from the lack of dietary zinc. From present study, an examination of some aspects of this postulate, we conclude the following about zinc deficiency in pigs: (1) Of the tissues studied, it results in diminished zinc content only in liver, kidney, and pancreas. (2) It is associated with tissue ab normalities of various trace elements other than zinc. (3) Histochemical enzyme assays are satisfactory for qualitative but not quantitative determination of zinc me talloenzymes. (4) Deficiency of various tissue zinc metalloenzymes is difficult to demonstrate, even in severezinc deficiency. (5) It is associated with decreased activity of hepatic leucine aminopeptidase and ornithine transcarbamylase, and decreased serum cholesterol concentrations. Our findings are consistent with the postulate that alterations in a number of trace elements may occur in the entity called zinc deficiency.
Zinc Deficiency in Sickle Cell Disease
Authors: Prasad Ananda S , Schoomaker Eric B , Ortega Jesus , Brewer George J , Oberleas Donald , Oelshlegel Fred J
Published: January 2020

Clinical similarities between patients with sickle cell anemia and zinc-deficient subjects suggested a secondary zinc deficiency in sickle cell anemia. Zinc was assayed in various biological fluids and tissues by atomic absorption spectrophotometry. Zinc in the plasma, erythrocytes, and hair was decreased and urinary zinc excretion was increased in anemia patients as compared to controls. Erythrocyte zinc and daily urinary zinc excretion were inversely correlated in the anemia patients (r = -.63, P < 0.05), suggesting that hyperzincuria may have caused zinc deficiency in these patients. Carbonic anhydrase, a zinc metalloenzyme, correlated significantly with erythrocyte zinc (r = +0.94, P < 0.001). Plasma RNase activity was significantly greater in anemia subjects than in controls. We administered zinc sulfate, 660 mg per day, orally, to seven men and two women with sickle cell anemia. Two 17-year-old males gained 5 cm and 7 cm in height during 49 and 42 weeks of zinc therapy, respectively. All but one patient gained weight (0.5 kg to 4.1 kg). Five of the males showed increased growth of pubic, axillary, facial, and body hair, and in one a leg ulcer healed in six weeks on zinc and in two others some benefit of zinc therapy on healing of ulcers was noted.
Trace Elements in Hair, as Related to Exposure in Metropolitan New York
Authors: Creason John P , Hinners Thomas A , Bumgarner Joseph E , Pinkerton Cecil
Published: January 2020

Previous studies have revealed that trace element concentrations in hair can reflect exposure in cases of frank poisoning and deficiency. This study reports significant correlations within a single metropolitan area between trace-element content of hair and exposure (as measured by analyses for the corresponding elements in dustfall or housedust) for Ba, Cr, Pb, Hg, Ni, Sn, and V. Age, sex, hair color, and smoking habits were factors included in the statistical evaluation. Several metals increase and decrease together in the hair specimens, in agreement with trends reported for other human tissues.
Direct Determination of Total Serum Cholesterol by Use of Double-Wavelength Spectrophotometry
Authors: Sommers Philip B , Jatlow Peter I , Seligson David
Published: January 2020

We describe a simple, accurate method for direct determination of total cholesterol in serum. Systematic investigation of a previously described modified Liebermann— Burchard reagent has indicated the necessity of accounting for both bilirubin interference and decreased specificity owing to exothermia. Double-wavelength spectrophotometry was used to optically null out bilirubin as an interfering factor, whereas adding serum to the cold reagent increases its specificity for the cholesterol color reaction. Comparison of 106 cholesterol values with those obtained by the procedure of Abell et al. [J. Biol. Chem. 195, 357 (1952)] yielded a correlation coefficient greater than 0.99; our inter-run coefficient of variation for pooled laboratory serum was 1.7%.
Calibration of Ion-Selective Electrodes for Use in Biological Fluids
Authors: Mohan M S , Bates Roger G
Published: January 2020

We measured electromotive force at 37 °C with pH, sodium glass, valinomycin, and the Simon calcium electrodes in synthetic electrolyte mixtures simulating serum, in cells both with and without liquid junction. Evidently these electrodes respond in a near-Nernstian manner to changes in concentration of the ion sensed. We suggest that such mixtures may serve as calibrating standards for ion-selective electrodes in clinical analysis. The effect of ionic strength on electrode response can be accounted for almost wholly by alterations in the activity coefficients. The lanthanum fluoride electrode shows promise as a reliable reference in cells without liquid junction.
Quantitation of Free, Total, and Antibody-bound Insulin in Insulin-Treated Diabetics
Authors: Gennaro William D , Van Norman John D
Published: January 2020

We describe a simplified method for measuring free, total, and antibody-bound insulin in insulin-treated patients in whom antibodies to insulin are present. The free, active insulin is extracted from the serum with a polyethylene glycol solution. Total insulin is extracted from the serum with a polyethylene glycol solution after dissociation of the antibody—antigen complex with dilute HCl. Aliquots of the extracts are used in the radioimmunoassay system. The figure for antibody-bound insulin is the difference between the total and free insulin values and reflects the concentration of insulin antibodies present. A commercially available ("Phadebas") radioimmunoassay for immunoreactive insulin was used to quantitate the insulin present in the two extracts. Recovery of added insulin averaged 85% for the free insulin and 87% for the total insulin.
Detection of Cardiac-Specific Creatine Kinase Isoenzyme in Sera with Normal or Slightly Increased Total Creatine Kinase Activity
Authors: Mercer Donald W , Varat Murray A
Published: January 2020

We describe a spectrophotometric kinetic assay for detecting creatine kinase MB isoenzyme activity in the 1 to 10 U/liter range. The MB isoenzyme was isolated [Clin. Chem. 20, 36 (1974)] and assayed (Rosalki method) with an Abbott ABA-100. Good reproducibility was demonstrated for MB isoenzyme activities near 1 U/liter (CV = 2.6%). Sera with normal or slightly increased total creatine kinase activity were evaluated. Sera of 14 patients with acute myocardial infarction contained, per liter, 84 to 236 U of total creatine kinase activity and 4.6 to 28.0 U of isoenzyme MB activity; corresponding ranges for sera from healthy lab technicians and patients with noncardiac disease were 36 to 277 and 0 to 2.6 U. MB isoenzyme activity for infarction patients rose and fell sharply within three days after the infarction. Atypical time-course patterns, MB isoenzyme activity remaining abnormally great for five days, were observed in serum from patients with prolonged atrial fibrillation and congestive heart failure or cardiomyopathy; the BB isoenzyme (1 to 5 U/liter) was also detected in sera of such patients but was absent in sera from infarction patients. Quantification of column-isolated MB by the assay described is rapid, easy, specific, and extremely sensitive for measuring MB in the 1 to 10 U/liter range.
A Versatile Temperature-Controlled Reaction Cuvet
Authors: Busby Michael G , Cottrell David B , Cembrowski George S , Toren E Clifford
Published: January 2020

A thermostatted reaction cuvet, operating under computerized or manual temperature control in the ultraviolet or visible region, is described and evaluated. The cell was designed for use with the automated chemistry system described earlier [Clin. Chem. 19, 1114 (1973)], but can readily be adapted for other applications. The entire 3-ml cuvet assembly warms from ambient temperature to 37 °C in less than 10 min, with ±0.15 °C stability; the long-term stability is ±0.05 °C; temperature recovery time after washing or reagent addition is less than 3 min. The optical pathlength is 10.00 mm and the carryover volume is 18 µl.
Improved Measurement of Erythrocyte Volume Distribution by Aperture-Counter Signal Analysis
Authors: Waterman Cleveland S , Atkinson Earl E , Wilkins Bert , Fischer Craig L , Kimzey Steve L
Published: January 2020

In aperture counters, particles in fluid suspension flow through a small orifice or aperture, causing a change in the electrical resistance of the aperture. This change is sensed by an external electronic circuit and translated into a voltage pulse, the signal height of which is proportional to the volume of the particle in the aperture. These signal pulses are collated into a spectrum of pulse heights by a multichannel pulse-height analyzer. The channel number (voltage increment) spectrum is proportional to the volume distribution of the particles sensed. A problem is that pulse height not only depends on cell volume, but also on the orientation and shape of the particle sensed and the current density along the path taken by the particle through the aperture. Uneven current density exists, primarily at the aperture entrance and exit, close to the wall. Orientation and shape of particles are altered near the wall by the unbalanced shear forces there. Toward the center of the aperture, the shear forces act so as not to induce continuous change in the orientation of the particles sensed. Thus introduction into the pulse-height spectrum of pulses that do not show a good proportionality to volume is primarily caused by particles that are traveling near the aperture wall. Residence time in the aperture for a particle traveling near the wall will be longer than that for a particle traveling down the center of the aperture, because of the smaller fluid velocity near the wall. Duration of the signal pulse created by a particle traveling near the wall will be correspondingly greater. We discuss an electronic filter to remove from the pulse-height spectrum those pulses that appear to result from particles traveling near the wall and the effect of the filter on the measured signal height and hence the volume distribution of erythrocytes. Use of this technique to characterize erythrocytes by volume distribution is described.
Computerized Multicuvette System for Single Substance and Group Analyses and for the Chromatographic Isolation and Quantitation of Compounds in Complex Mixtures
Authors: Vestergaard Per , Bachman Alvin
Published: January 2020

A new computer-interfaced system of general applicability in the clinical laboratory has been built. The system is constructed around a new type multicuvette. Quantitation is currently by spectrophotometry and is on-line, with use of a mini-computer with output going to an electrostatic printer/plotter. The system can be used for single-substance batch analyses, for group analyses, and for the estimation of profiles of compounds after preliminary high-resolution liquid chromatography tailored to interface with the multicuvette.
Comparison of four kits for enzymatic determination of ethanol in blood.
Authors: Redetzki H M , Dees W L
Published: January 2020

We compared results obtained with four commercially available kits for the enzymatic (alcohol dehydrogenase) determination of ethanol in blood, in a practical test in which standardized blood samples were used. Each of the kits yielded reliable results with acceptable reproducibility. A tendency to record slightly lower values in the intermediate and high alcohol range is most likely related to incomplete (inhomogeneous) deproteinization. Blood samples containing ethanol plus various concentrations of methanol and isopropanol were analyzed to evaluate the specificity of assays. Highly toxic blood concentrations of methanol (1.5 g/liter) increased apparent ethanol values only insignificantly, but even small concentrations of isopropanol (0.5 g/liter) interfered in all kits to different but substantial extents. The specific technical characteristics of the kits, their advantages and disadvantages are discussed. Costs are compared for analysis of small numbers of samples.
Normal limits of urinary excretion of eleven enzymes.
Authors: Maruhn D , Fuchs I , Mues G , Bock K D
Published: January 2020

Urinary excretion of lactate dehydrogenase, hydroxybutyrate dehydrogenase, gamma-glutamyltransferase, alkaline phosphatase, arylsulphatase A, alpha-glucosidase, beta-galactosidase, trehalase, N-acetyl-beta-glucosaminidase, beta-glucuronidase, and leucinearylamidase was studies in a carefully selected group of 100 healthy subjects, 50 women and 50 men. Enzyme activities were assayed in 3-h morning samples after gel filtration of the urine. Activities were related to time volume, and to urinary creatinine concentration. Several transforming functions had to be applied to enzyme output data to obtain an approximation to gaussian frequency distribution. Men showed a significantly higher excretion of gamma-glutamyltransferase, alpha-glucosidase, trehalase, N-acetyl-beta-glucosaminidase,beta-glucuronidase, and leucine arylamidase activity than did women if enzyme activity was related to urinary time volume. Women excreted more lactate dehydrogenase, hydroxybutyrate dehydrogenase, gamma-glutamyltransferase, alkaline phosphatase, alpha-glucosidase, trehalase, and N-acetyl-beta-glucosaminidase activity than did men, if urinary creatinine was used as the basis of reference. Reference intervals were calculated as 2.5 and 97.5 percentiles for both sexes.
Isolation and identification of benzodiazepine drugs and their metabolites in urine by use of Amberlite XAD-2 resin and thin-layer chromatography.
Authors: Sawada H , Hara A , Asano S , Matsumoto Y
Published: January 2020

We used the method described here to detect and identify seven benzodiazepine derivatives--diazepam, chlorodiazepoxide, nitrazepam, cloxazolam, oxazolam, oxazepam, and medazepam--and their metabolites in the urine of rabbits given the seven drugs orally. We column-chromatographed 25-ml samples of urine on Amberlite XAD-2. The drugs and their metabolites in the urine were adsorbed by the resin, irrespective of urinary pH, and upon successive elution with methanol and ethyl acetate/methanol/acetic acid (90/10/0.1 by vol) they could be separated and extracted from the normal components of urine with satisfactory analytical recovery. The conjugated metabolites were then enzymatically hydrolyzed and the hydrolysate was extracted into ethyl acetate and the extract thin-layer chromatographed to detect and identify each drug and each of its metabolites. In an experiment in which the urine of human subjects given 5 mg of nitrazepam orally was analyzed by this method, the metabolites of nitrazepam in the 24-h urine could be identified satisfactorily.
Automated method for determination of mercury in urine.
Authors: Richardson R A
Published: January 2020

I describe a sensitive, specific, automated method for determination of mercury in urine by the "flameless atomic absorption" technique. One can analyze 20 samples per hour, with less than 0.5-ml samples of urine. The detection limit is 0.01 mumol/liter (2mug/liter) and the mean coefficient of variation 12%. The procedure is being used to monitor mercury excretion by workers exposed to mercury.
Clinical usefulness of measuring prothrombin time as a routine admission test.
Authors: Eisenberg J M , Goldfarb S
Published: January 2020

A questionnaire indicating the presence of a history or physical findings consistent with liver desease or bleeding disorders was completed by house officers on 301 admissions to a Veterans Administration medical service. Each patient had prothrombin time determined on admission. Only two of 107 patients for whom the prothrombin time was measured as a screening test had an abnormal results and one of these was normal when repeated. Of 73 patients with a history of alcoholism but no other pertient case history or physical examination results, only one had an abnormal prothrombin time. Of the remaining 121 patients, who had a pertinent history or physical examination, 41 had an abnormal admission prothrombin time. We conclude that measurement of prothrombin time adds little additional information to that obtained by history and physical examination in screening for liver disease and coagulation defects unless the patient has specific clinical evidence of liver disease, anticoagulation, or other conditions predisposing to bleeding disorders.
High-pressure liquid-chromatographic determination of 5-(4-hydroxyphenyl)-5-phenylhydantoin in humanurine.
Authors: Kabra P M , Marton L J
Published: January 2020

We describe a sensitive and precise high-pressure liquid-chromatographic method for measurement of total 5-(4-hydroxyphenyl)-5-phenylhydantoin, a metabolite of phenytoin, in urine. An aliquot of urine, containing 5-(4-methylphenyl)-5-phenylhydantoin as an internal standard, is processed and chromatographed. The metabolite and internal standard are identified from their retention times and quantitated from their relative response factors. The metabolite is separated from normal urine constituents and internal standard in less than 8 min. The sensitivity of the method is such that after the usual dose it can be measured in 0.5 ml of urine; the lower limit of detection is 300 ng.
Proficiency testing for blood-gas quality control.
Authors: Delaney C J , Leary E T , Raisys V A , Kenny M A
Published: January 2020

We have conducted a voluntary, community blood-gas proficiency testing program, with use of tonometered human blood, for 32 analyzers located in 16 laboratories. Instruments initially showed inaccuracies as large as -30.8 to +17.3% for po(2), and -14.0 to +42.9% for pco(2), but inaccuracy and imprecision decreased in most laboratories during the program. For a typical 15-week period, mean group precision (CV) was 4.3 To 5.1% for po(2) from 6.92 to 33.3 Pa (52 to 250 mmHg), and 4.0 to 6.9% for pco(2) from 2.0 to 6.8 Pa (15 to 51 mmHg). This program can detect increasing imprecision or inaccuracy caused by analyzer deterioration, and can identify interlaboratory or interinstrument bias and problems not detected by participant quality-control programs. Participants have used the proficiency information in discussing data quality with clinicians, promoting internal control and maintenance programs, and justifying instrument purchases. We believe that proficiency-testing documentation of variability in blood-gas analysis may help to establish realistic patient-care protocols.
Enzymatic, fluorometric assay of alpha-amylase in serum.
Authors: Guilbault G G , Rietz E B
Published: January 2020

An enzymatic, fluorometric method for kinetic assay of serum alpha-amylase (1,4alpha-D-glucan 4-glucanohydrolase, EC 3.2.1.1) is described. A soluble starch is used as substrate, in tris(hydroxymethyl)methylamine buffer. All measurements are made in Pyrex cuvettes at 37 degrees C, with a reaction volume of 1.16 ml. The assay is based on the following reaction sequence: (see article) The rate of appearance of fluorescence of NADH (lambdaex = 365 nm, lambdaem = 460 nm), developed in the indicator reaction (4), is measured and equated to the activity of alpha-amylase in serum. A calibration plot of the change of fluorescence per min vs. enzyme concentration shows a good proportionality in the range of 0.50-5.0 kU/liter.
Spectrophotometric and electrochemical determinations of L(+)-lactate in blood by use of lactate dehydrogenase from yeast.
Authors: Durliat H , Comtat M , Baudras A
Published: January 2020

Lactate can be determined rapidly in blood by spectrophotometric and amperometric (enzyme electrode) procedures based on its oxidation by ferricyanide, the reaction being catalyzed with yeast L(+)-lactate dehydrogenase (cytochrome b2) (EC 1.1.2.3). In the photometric method lactate can be measured in a few minutes, but blood samples must first be deproteinized. In the amperometric procedure no treatment of blood is needed except ferricyanide addition. The enzyme electrode we used has a response time shorter than 1 min when its critical variables are optimized. Preliminary standardization is reduced to minimum operation, because electrode response is proportional to lactate concentration over a wide range (0.1 to 8.0 mol/liter) and many determinations can be done with little cost in enzyme. A simple electrical device ("two-electrode device") is described that is well suited for furture micro-cell construction. Lactate determinations on a series of normal blood samples show no deviation between results by these new methods and the usual ultraviolet spectrophotometric lactate tests.
Creatine kinase in serum: 2. Interference of adenylate kinase with the assay.
Authors: Szasz G , Gerhardt W , Gruber W , Bernt E
Published: January 2020

Interference of adenylate kinase with Oliver's method [Biochem. J. 61, 116 (1955)] for creatine kinase is usually suppressed by including an adenylate kinase inhibitor, AMP. We studied the kinetics and compared the inhibition capacities of AMP and diadenosine pentaphosphate. Both are competitive inhibitors, AMP being markedly weaker, with a Ki of about 300 mumol/liter for adenylate kinase from erythrocyte, muscle, and liver. AMP also weakly inhibitis creatine kinase. Diadenosine pentaphosphate inhibits erythrocyte and muscle adenylate kinase strongly (Ki about 0.03 mumol/liter), the liver isoenzyme less strongly (Ki about 3 mumol/liter), and has no effect on creatine kinase up to 100 mumol/liter. All three adenylate kinases may be present in a patinet's serum, causing sample blanks to be high in a creatine kinase assay that lacks inhibitors. In acute hepatic damage, liver adenylate kinase activity in serum can be grossly increased. Use of sufficient diadenosine pentaphosphate alone for complete inhibition is relatively expensive. Consequently, we recommend a combination of both inhibitors. Diadenosine pentaphosphate, 10 mumol, combined with 5 mmol of AMP per liter inhibits adenylate kinase from erythrocytes and muscle by 97% and from liver by 95%.
An 125I-labeled cortisol radioimmunoassay in which serum binding protein are enzymatically denatured.
Authors: Hasler M J , Painter K , Niswender G D
Published: January 2020

We report an iodine-125 radioimmunoassay for cortisol in biological fluids, in which interfering binding proteins are enzymatically denatured. An antiserum to cortisol-3-carboxymethyloxime-bovine serum albumin, extremely low cross-reacting with other corticosteroids, was raised in rabbits. A cortisol-3-carboxymethyloxime tyrosine methyl ester derivative was synthesized and labeled with iodine-125 by standard radioiodination techniques. To eliminate the need for extraction and recovery procedures, we digested interfering binding witha proteolytic enzyme, which then was heat-inactivated before adding the labeled derivative and the premixed, preincubated antiserum complex. There was quantitative analytical recovery of esogenous cortisol added to sera from a normal man, a normal woman, and a pregnant woman. Values for the same samples agreed after extraction and chromatographic purification and agreed well with values obtained by other techniques by independent reference laboratories. The five-step assay can be done in 6 h or less.
Low apparent creatine kinase activity and prolonged lag phases in serum of patients with metastatic disease: elimination by treatment of sera with sulfhydryl agents.
Authors: Bruns D E , Morgan W S , Davis J E , Ladenson J H
Published: January 2020

We find lag phases exceeding 20 min in measuring creatine kinase activity, by using the kinetic creatin phosphate leads to creatine assay, in sera of some patients with carcinoma metastatic to the liver. Such long lag phases are accompanied by a decreased apparent enzyme activity. These problems are eliminated by adding sulfhydryl agents to the serum before assay, but not by adding more of such agents to the assay reagent. beta-Mercaptoethanol is supperior to Cleland's reagents, glutathione, and cysteine. The long lag phases could not be explained by inadequate activity of the coupling enzymes, interference with the coupling steps, high proportions of cardiacisoenzymes activity, simple oxidation of the enzyme, low concentrations of albumin, or increased concentrations of glutathione reductase, lactate dehydrogenase, or uric acid. We conclude that the prolonged lag phases reflect inadequate reactivation of the enzyme by sulfhydryl agents under the usual assay conditions. Reactivation before assay can prevent potentially serious negative errors in the assay of creatine kinase.
Error introduced by specimen handling before determination of inorganic phosphate concentrations in plasma and serum.
Authors: Carothers J E , Kurtz N M , Lemann J
Published: January 2020

Values for serum inorganic phosphate (Pi) concentrations in groups of healthy adults vary widely, the coefficient of variation ranging from 10 to 15%. We undertook to determine in 23 healthy adults whether part of this variation could be accounted for by (a) drawing blood in syringes vs. evacuated tubes (b) the time between blood sampling and separation of serum or plasma, and (c) the prevention of clotting. Values were unaffected by a, decreased significantly with time at room temperature between blood sampling and separation of cells in both serum and plasma, and were significantly lower in plasma than in serum. The group coefficient of variation for Pi averaged 13% and was uninfluenced by the blood-processing technique.
Erythrocyte porphyrin analysis in the detection of lead poisoning in children: evaluation of four micromethods.
Authors: Hanna T L , Dietzler D N , Smith C H , Gupta S , Zarkowsky H S
Published: January 2020

We evaluated four procedures for determination of erythrocyte porphyrin: double extraction with ethyl acetate/acetic acid-HCl, single extraction with ethanol, single extraction with acetone, and direct solubilization with detergent-buffer. The ethyl acetate procedure, when used with two portions of HCl, apparently gives complete recovery of porphyrin and is suitable for reference as a comparison method. The ethanol procedure gives a high and consistent recovery and is technically simpler. The acetone procedure gives low and variable recovery of porphyrin, and the detergent-buffer method is subject to serious hemoglobin interference; neither of these two procedures offers any technical advantage. Stability of samples and methods for standardization were explored. A procedure for expressing results in terms of erythrocyte Zn-protoporphyrin content is given. Because of its stability, coproporphyrin is useful as a daily working standard. The ethyl acetate and ethanol methods are about equally efficient for detecting lead intoxication. Because of its simplicity, the ethanol method seems to be the best for use in screening.
High-performance colorimeter with an electronic bubble gate for use in miniaturized continuous-flow analyzers.
Authors: Neeley W E , Wardlaw S C , Yates T , Hollingsworth W G , Swinnen M E
Published: January 2020

We describe a high-performance colorimeter with an electronic bubble gate for use with miniaturized continuous-flow analyzers. The colorimeter has a flow-through cuvette with optically flat quartz windows that allows a bubbled stream to pass freely without any breakup or retention of bubbles. The fluid volume in the light path is only 1.8 mul. The electronic bubble gate selectively removes that portion of the photodector signal produced by the air bubbles passing through the flow cell and allows that portion of the signal attributable to the fluid segment to pass to the recorder. The colorimeter is easy to use, rugged, inexpensive, and requires minimal adjustments.
Cation-exchange chromatography of guanidine derivatives in plasma of patients with chronic renal failure.
Authors: Perez G , Rey A , Micklus M , Stein I
Published: January 2020

Guanidine derivatives are suspected of contributing to the toxic manifestations of uremia. We describe a method for measurement of guanidine derivatives in 5-ml samples of plasma by liquid chromatography. Concentrations of guanidinosuccinate and guanidinobutyrate in plasma were significantly increased both in undialyzed patients with chronic renal failure (5.54 +/- 0.94 and 17.5 +/- 4.07 mg/liter) and those undergoing maintenance hemodialysis (2.35 +/- 0.41 and 19.4 +/- 3.99 mg/liter) when compared to healthy controls (less than 0.4 and 1.0 +/- 0.3 mg/liter, respectively). Creatine and guanidinoacetate concentrations tended to be higher in hemodialysis patients and lower in the undialyzed group of patients with chronic renal failure. This procedure provides a rapid, sensitive, and accurate method for the study of guanidine metabolism in persons with uremia.
Polarographic method for rapid microdetermination of cholesterol with cholesterol esterase and cholesterol oxidase.
Authors: Noma A , Nakayama K
Published: January 2020

Cholesterol concentrations in serum are enzymatically determined rapidly by use of a polarographic oxygen analyzer with a circuit modified to record simultaneously the amount and rate of oxygen consumption. The final assay system, assessed from the oxygen consumption value that we found to be optimum, consists of 1 ml of sodium phosphate buffer (0.6 mol/liter, pH 7.0) containing NaN3 (10 mg/liter), Triton X-100 surfactant (10 ml/liter), 0.4 U of cholesterol ester hydrolase, and 0.6 U of cholesterol oxidase. Oxygen consumption and cholesterol concentration are linearly related to 8.0 g/liter, and only 10 mul of serum is required. Replicate analyses of pooled serum by the present method demonstrated the following inter-run precision: mean = 1731 mg/liter, SD = 22.3 mg/liter, CV = 1.3%. Bilirubin and ascorbic acid were without effect on the present method, unlike the enzymatic colorimetric methods.
Simple fluorometric determination of aldosterone in urine without use of isotopes or chromatography.
Authors: Whigham W R
Published: January 2020

Aldosterone 18-glucuronide in urine is hydrolyzed by adjusting the pH to 1.0 and allowing the mixture to stand overnight at room temperature. The free aldosterone is then extracted into dichloromethane, which is washed with carbonate to remove acidic compounds and evaporated. The residue is partitioned between a nonpolar organic phase and an aqueous phase, and the aldosterone oxidized to the 13-carboxylic acid derivative with Benedict's qualitative glucose reagent. Neutral compounds are extracted from this oxidation mixture with dichloromethane at pH 7.5, the mixture is acidified, and the oxidized aldosterone extracted into dichloromethane. After washing with pH 3.5 buffer, this extract is evaporated and the oxidized aldosterone determined fluorometrically via a two-stage reaction with sulfuric acid/water (85/15 by vol) and methanol containing ferric chloride.
Direct radioimmunoassay for serum estriol during pregnancy.
Authors: Standefer J
Published: January 2020

I describe an assay for serum estriol during pregnancy, which does not require prior extraction or chromatography of the serum. Fifty microliters of 20-fold diluted serum can be assayed to detect as little as 20 pg of estriol. For a 20 mug/liter serum estriol concentration the CV was 16%; for 100 mug/liter concentration it was 5%. There was no significant interference by serum "binding" proteins. The assay measures unconjugated estriol, estriol conjugated with either sulfate or glucuronide at the 3' position, and a portion of the estriol 16 alpha (beta-D-glucuronide). Serum estriol concentrations increase during gestation from a low at 20 weeks of less than 10 mug/liter to a high of 100-150 mug/liter at term. Serum estriol values correlate well with those for urinary total estrogens and with values for human placental lactogen in serum at various gestational ages.
Normative values of serum immunoglobulins by single radial immunodiffusion: a review.
Authors: Maddison S E , Reimer C B
Published: January 2020

Many previous studies of normative values in adults have suggested that race, age, sex, and environment all have significant effects on the mean values for IgG, IgA, IgM, and IgE in various groups of individuals. Single radial immunodiffusion is the technique most widely used to quantitate immunoglobulins of the three major classes (IgG, IgM, and IgA) in sera. Measurements have been expressed in terms of mass concentration, as percentages of the mean normal adult value, and in arbitrary international units. To improve agreement among laboratories, the WHO has supported the distribution of an International Reference Preparation for the Human Immunoglobulins IgG, IgA, and IgM, and similar (separate) preparations for IgD and IgE.
Rapid radioimmunoassay of total urinary estriol.
Authors: Anderson D W , Goebelsmann U
Published: January 2020

We describe a radioimmunoassay for total urinary estriol in pregnancy. A 25-mul aliquot of the urine specimen is acid hydrolyzed, neutralized, and diluted before assay. We use rabbit antisera against estriol-6-(O-carboxymethyl)oxime/bovine serum albumin and ammonium sulfate precipitation at room temperature. Results are unaffected by glucose or methenamine mandelate, a urinary tract antiseptic. Using semi-automatic pipetting equipment, one laboratory technologist can complete 50 assays within 8 h. This technique is both reliable and convenient and should decrease the expense of routine estriol assays.
Profile analysis of blood proteins with a centrifugal analyzer.
Authors: Blom M , Hjorne N
Published: January 2020

We describe a procedure for routine analysis of a profile composed of eight blood proteins with a GEMSAEC centrifugal analyzer. The end-point technique is used and four standards are included in each run in the immunochemical analysis of the individual proteins. Additional computer programs fit the absorbance readings from the samples to the (nonlinear) standard curve and store the results in a patient's file for a final printout, which also presents the patient's data and reference values. The proteins are analyzed in a given order, which allows common blank runs to be used. Antisera are used in suitably high concentrations, so that the "antigen in excess" problem only appears for analysis of IgA and IgM when sera contain high amounts of the M-components of these two classes. The capacity, accuracy, and precision of the method are satisfactory.
Simultaneous gas-chromatographic analysis for phenobarbital, diphenylhydantoin, carbamazepine, and primidone in serum.
Authors: Abraham C V , Joslin H D
Published: January 2020

We describe a simple, sensitive determination of phenobarbital, diphenylhydantoin, carbamazepine, and primidone in serum, by use of gas-liquid chromatography with temperature programming. The methylated derivatives of these anticonvulsants are well resolved, as was 5-(p-methyl-phenyl)-5-phenylhydantoin, the internal standard. The proposed procedure requires only 0.20 ml of serum and can be done in less than 30 min. The lower limit of detection for each of the drugs is 0.5 mg/liter. Analytical recoveries of drug from serum were excellent and peak height and concentration were linearly related up to twice the toxic concentration for serum.
Determination of clomipramine and desmethyl-clomipramine in plasma or urine by the double-radioisotope derivative technique.
Authors: Carnis G , Godbillon J , Metayer J P
Published: January 2020

A double radioisotope derivative method was developed for the determination of clomipramine and desmethyl-clomipramine in plasma or urine. After addition of 14C-labeled clomipramine and desmethyl-clomipramine as internal standards and extractive isolation of both compounds, desmethyl-clomipramine is acetylated with [3H]acetic anhydride. The [3H]acetamide is separated from clomipramine by thin-layer chromatography and its radioactivity is measured. Clomipramine, extracted from the cilica gel, is reacted with trichloroethyl chloroformate. The urethane is saponified and decarboxylated. The resulting desmethyl-clomipramine is acetylated with [3H]acetic anhydride. The [3H]acetamide is purified by thin-layer chromatography and its radioactivity is measured. The sensitivity of the method is 15 mug/liter for clomipramine and 2 mug/liter for desmethyl-clomipramine. Its specificity was made sure by a cross-check with a gas chromatography-mass spectrometry technique.
Simultaneous measurement of phenobarbital, diphenylhydantoin, and primidone in blood by high-pressure liquid chromatography.
Authors: Kabra P M , Gotelli G , Stanfill R , Marton L J
Published: January 2020

We describe a sensitive, precise high-pressure liquid chromatographic method in which 5-(p-methylphenyl)-5-phenylhydantoin is used as the internal standard for simultaneous determination of diphenylhydantoin, phenobarbital, and primidone in whole blood and plasma. These anticonvulsant drugs are well separated from each other and from normal blood constituents in less than 10 min. The lower limit of detection for each drug is 100 ng for primidone, 200 ng for dilantin, and 300 ng for phenobarbital. The eluted drugs were detected by their absorption at 254 nm, and evaluated from their peak heights as compared to internal standard. The method was successfully adapted for pediatric samples (100 to 500 mul of whole blood or plasma). Fifty specimens were analyzed for phenobarbital and diphenylhydantoin and 25 specimens for primidone by a standard gas-chromatographic method and by our liquid-chromatographic method; the resulting correlation coefficient was greater than 0.98.
Results of a nine-laboratory survey of forensic toxicology proficiency.
Authors: Dinovo E C , Gottschalk L A
Published: January 2020

Toxicological determinations are crucial to coroners' or medical examiners' judgments that drugs are significantly involved in a death. However, differences in laboratory procedures, thoroughness of screening, and limits of detection may result in artifactual differences in the toxicological results and the subsequent interpretations of them. To test this possibility, we conducted a toxicology proficiency-testing survey of nine collaborating laboratories. The results for the proficiency samples point out starting interlaboratory differences in accuracy and precision of detection of drugs. These observed variations in toxicological proficiency may introduce a significant source of error in drug-death statistics and in epidemiological deductions based on these statistics.
Primidone analyses: correlation of gas-chromatographic assay with enzyme immunoassay.
Authors: Sun L , Walwick E R
Published: January 2020

Serum specimens from patients on primidone therapy were assayed by two currently available procedures: a commercially available enzyme immunoassay (EMIT) and a published gas-chromatographic procedure. Both procedures were done with commercially available materials and equipment. Results by the two procedures agreed well, which suggests that the two methods could be used interchangeably. For the 94 specimens studied, the correlation coefficient was 0.98, and the least-squares values of slope and intercept were, respectively, 0.97 and 0.51 mg/liter.