bluuebunny/crossref_metadata_2025 huggingface dataset:
Correlation of Selected Serum Constituents: 1. Inter-Individual Variation and Analytical Error
Authors: Winkel Per , Statland Bernard E , Bokelund Henning , Johnson Eugene A
Published: January 2020

The intra-subject correlations of three clinically meaningful combinations of serum constituents—(a) potassium, calcium, and albumin; (b) urea, creatinine, and uric acid; and (c) aspartate aminotransferase, lactate dehydrogenase, and alkaline phosphatase—were studied in 11 healthy men. Duplicate serum samples were obtained at 800 h, 1100 h, and 1400 h on five different days. All assays were performed on the AutoChemist Multichannel Analyzer. Correlation coefficients differed significantly among the subjects for the following six pairs of serum constituents: urea and creatinine, urea and uric acid, creatinine and uric acid, aspartate aminotransferase and lactate dehydrogenase, aspartate aminotransferase and alkaline phosphatase, and lactate dehydrogenase and alkaline phosphatase. Nonbiological positive correlation between analytical errors (i.e., errors of two different assays performed on the same specimen) was demonstrated for two of the pairs: potassium and calcium, and aspartate aminotransferase and lactate dehydrogenase. The error correlations of these two pairs of constituents comprised a significant component of the observed intra-subject correlations. Probable reasons for these analytical error correlations are discussed
Simple, Highly Selective Screening Method for Barbiturates in Urine
Authors: Pehr František
Published: January 2020

I present a new, simple colorimetric method for detecting and estimating barbiturates in urine. After the barbiturates are extracted with ether, an aliquot of the washed ether phase is added to the color reagent (a bivalent mercury/dithizone chelate in chloroform). On addition of diluted pyridine and shaking, a pinkish-violet color appears if a barbiturate is present. The overall sensitivity of the above method was evaluated by probit analysis in the case of sodium phenobarbital. The concentration of sodium phenobarbital in urine detectable at least 99% of the time was 6.72 mg/liter, with 95% confidence limits of 5.37 to 10.36 mg/liter. Sodium phenobarbital, 10 mg/liter, can be detected in the presence of phenytoin (50 mg/liter), glutethimide (100 mg/liter), or bemegride (100 mg/liter). The whole procedure requires <10 min. An excretion study illustrates application of the procedure
Reaction-Rate Assay of Serum Sorbitol Dehydrogenase Activity at 37 °C
Authors: Rose Clive I , Henderson Arthur R
Published: January 2020

The sorbitol dehydrogenase (EC 1.1.1.14) activity of human serum and liver was investigated at 37 °C. Various buffers were examined but tris(hydroxymethyl)aminomethane HCl in a final concentration of 90 mmol/liter (pH 6.6, at 37 °C) was the one with which activity was the greatest. The enzyme is inhibited by its substrate, D-fructose, and activity was greatest with a substrate concentration (in the final assay mixture) of 500 mmol/ liter and an NADH concentration of 247 µmol/liter. The specimen volume was 100 µl. The enzyme from liver and serum was shown to have similar Km values and activation energies, and we conclude that the liver enzyme was unchanged on passing into the extrahepatic space. Normal values for this assay are 0-2.6 U/liter (2.5 and 97.5 percentiles)
Specificity of Oxidation of Bile-Salt Hydroxyl Groups by Crude Extracts of Pseudomonas Testosteroni (ATCC 11996) Used in Determining Bile Salts
Authors: Beher William T , Lin Grace J
Published: January 2020

Recent experimental evidence suggests that, presumably as a result of mutation, crude extracts of Pseudomonas testosteroni (ATCC 11996) now contain amounts of 7α-and 12α-hydroxysteroid dehydrogenate activity that invalidate the results of bile-salt determinations. To confirm or deny this, we studied the specificity of hydroxysteroid dehydrogenase contained in crude extracts of currently available samples of this bacterium in the oxidation of bile-salt hydroxyl groups. The dehydrogenases in these extracts specifically oxidized 3α-hydroxyl groups of cholate, chenodeoxycholate, and deoxycholate anions. No significant amount of 12α-or 7α-hydroxysteroid dehydrogenase activity was detected. Currently available crude extracts of Pseudomonas testosteroni (ATCC 11996) are therefore suitable for bile-salt quantification
Simple, Sensitive Method for Measuring Plasmin and Plasminogen Activity in Plasma
Authors: Uete Tetsuo , Shimano Noriko , Morikawa Mamiko
Published: January 2020

We describe a sensitive, simple, and rapid method for measuring plasmin and plasminogen in plasma, with fibrinogen as substrate. The assay can be done within 1 h. Plasma is diluted 20-fold with buffer and 0.1 ml (5 µl of original plasma) is incubated for 5 min at 37 °C, with or without 400 units of streptokinase. Then 2.0 ml of fibrinogen solution is added, the mixture again incubated (37 °C, 5 min), and the reaction stopped. The amount of tyrosine liberated from the fibrinogen is measured and is related to activity. This method is suitable for routine clinical work if the same batch of fibrinogen is used
Fluorometric Determination of "Albumin-Titratable Bilirubin" in the Jaundiced Neonate
Authors: McCluskey Susan B , Storey G N B , Brown G K , More D G , O’Sullivan W J
Published: January 2020

We report a fluorometric technique for determination of albumin-titratable bilirubin in the jaundiced neonate. Although bilirubin alone has very little native fluorescence, considerable emission is observed in the presence of albumin under acid conditions. Analysis of the plasma sample alone and in the presence of excess human serum albumin solution appears to reflect the bilirubin tightly bound to albumin and the total serum bilirubin, respectively. The difference between these two values has been designated as "albumin-titratable bilirubin." Where the concentration of albumin-titratable bilirubin is considerable, a typical saturation effect is observed. In samples where the circulating bilirubin is strongly bound to endogenous albumin, no change in fluorescence is seen when exogenous albumin is added. Results correlate well with the clinical picture
Therapeutic Monitoring of Anticonvulsant Drugs: Gas-Chromatographic Simultaneous Determination of Primidone, Phenylethylmalonamide, Carbamazepine, and Diphenylhydantoin
Authors: Least Charles J , Johnson George F , Solomon Harvey M
Published: January 2020

We describe a sensitive and precise gas-chromatographic method in which benzylmalonate methylester monoamide is used as the internal standard for the simultaneous determination of primidone, phenylethylmalonamide, carbamazepine, and diphenylhydantoin. The trimethylsilyl derivatives of the anticonvulsants are well separated from each other and from normal serum constituents. The lower limit of detection for each drug is 0.5 mg/liter when 1 ml of serum is analyzed. Withinrun precision (CV), established by analysis of 10 replicates, was as follows: primidone (5.4 mg/liter), 2.6%; phenylethylmalonamide (5.5 mg/liter), 1.6%; diphenylhydantoin (6.6 mg/liter), 3.8%; and carbamazepine (10.4 mg/liter), 3.2%. Fifty specimens were analyzed for primidone and 35 for diphenylhydantoin by a standard gas-chromatographic method involving on-column methylation and by the procedure we have developed. The mean value observed for primidone with the on-column alkylation procedure was 9.3 mg/liter and with our procedure was 9.6 mg/liter. When values for our assay were regressed against values for the standard method, the slope of the least-squares line was 0.936, the intercept was 1.00 mg/liter, and r was 0.939. The mean values observed for diphenylhydantoin by on-column methylation and with our procedure were both 12.6 mg/liter. When values for our assay were regressed against the standard method, the slope of the leastsquares line was 0.944, the intercept was 0.3 mg/liter, and r was 0.988
Measurement of Brain-Specific Creatine Kinase Isoenzyme Activity in Serum
Authors: Nealon Daniel A , Henderson Arthur R
Published: January 2020

We further modified a diethylaminoethyl-cellulose column-chromatographic system [Clin. Chem. 21, 392 (1975)] so that it will separate all three serum creatine kinase isoenzymes. The modified system can be used to detect brain-specific creatine kinase isoenzyme in serum after brain surgery
Colorimetric Measurement of Ornithine Carbamoyl Transferase Activity in Plasma, and Results for a Supposedly Healthy Population
Authors: Bagrel Alain , Museur Gérard , Siest Gérard
Published: January 2020

Determination of ornithine carbamoyl transferase (EC 2.1.3.3) activity in plasma is important for detection of liver diseases. The assay established in this paper has been made optimum. A blank is needed containing both substrates, carbamoyl phosphate and ornithine. We used a new colorimetric assay, based on a complex with a phosphoferric-antipyrine reagent and diacetyl monoxime, to measure the citrulline formed. The highly sensitive assay permits low activities to be determined accurately. Values for blood plasma from 425 supposedly healthy people, varied from 0 to 16 U/liter (95th percentile), and 27% of this population showed an activity of less than 2 µmol of citrulline formed per minute per liter, 2 U/liter being the limit of the method’s sensitivity.
Evaluation of Kinetic Light Scattering as an Approach to the Measurement of Specific Proteins with the Centrifugal Analyzer. I. Methodology
Authors: Buffone Gregory J , Savory John , Cross R E , Hammond J E
Published: January 2020

A laser-modified centrifugal analyzer was used to develop kinetic light scattering methods for the measurement of human immunoglobulins IgG, IgA, and IgM. Comparison of equilibrium light scattering methods with kinetic procedures for IgG and IgA demonstrate equivalent precision and relative accuracy. However, quantitative results obtained by equilibrium and kinetics methods for IgM were found to differ significantly. Recovery studies performed with purified IgM have shown that both methods can yield quantitative results in the normal range. Our observations demonstrate that the technique offers a viable and in some respects a superior alternative to other methods currently used in the clinical laboratory.
Evaluation of Kinetic Light Scattering as an Approach to the Measurement of Specific Proteins with the Centrifugal Analyzer. II. Theoretical Considerations
Authors: Buffone Gregory J , Savory John , Hermans J
Published: January 2020

Development of centrifugal analyzers capable of making light-scattering measurements has prompted the investigation of the application of this type of instrumentation to the measurement of specific proteins. Investigation of antigen/antibody complex dimension and the kinetics of the IgG/anti-IgG reaction as followed by light scattering are examined in relation to improved analytical methodology. The effect of nonspecific protein on the reaction rate has been investigated for IgM, and data are presented that provide insight into the mechanism of the effect of periodic mixing.
Evaluation of Trinder's Glucose Oxidase Method for Measuring Glucose in Serum and Urine
Authors: Lott John A , Turner Kathie
Published: January 2020

Trinder's method for glucose has nearly all the attributes of an ideal automated colorimetric glucose oxidase procedure. The chemicals used in the color reaction with peroxidase are readily available, the solutions are stable and can be prepared by the user, the method is highly specific and largely free of interferences, the sensitivity can be adjusted by the user to cover a wide range of glucose concentrations, and the reagents are not hazardous. We found very good agreement between results by this method and by the hexokinase and Beckman Glucose Analyzer methods. The method has been modified and adapted to the AutoAnalyzer I and SMA 6/60 (Technicon) with manifolds that give very little interaction between specimens. A study of the method by the simplex technique revealed that the glucose oxidase activity in the reagent is the most critical variable.
Scatchard Plot and Heterogeneity in Binding Affinity of Labeled and Unlabeled Ligand
Authors: Hollemans H J G , Bertina R M
Published: January 2020

In saturation analysis the Scatchard plot is a generally accepted method for calculation of the affinity constant, K, and the molar concentration, q, of the binder. However, in a system where the K's for the labeled and unlabeled ligand are unequal, a nonlinear plot can be obtained from which incorrect values for K and q may be calculated. This paper mathematically explains how the plot may deviate and under which conditions there will be a maximum in the curve. When the binding sites are homogeneous, the coordinates of this maximum can be used to calculate K and q. A general mathematical expression is derived on the basis of which a linear curve can be constructed for calculation of q and K, which is valid even when affinity for the labeled and unlabeled ligand is not identical.
Fluorometric Assay of Serum Acid or Alkaline Phosphatase, Either in Solution or on a Semisolid Surface
Authors: Rietz Bernd , Guilbault George G
Published: January 2020

We describe enzymatic fluorometric methods for determining activities of serum alkaline phosphatase and of serum acid phosphatase in solution and on silicone rubber pads. 4-Methylumbelliferone phosphate is used as substrate, in either tris(hydroxymethyl)aminomethane or citrate buffer. In solution, the reaction is measured at 37 °C in a 3-ml Pyrex cuvette. Measurements on the pads are also made at 37 °C, after establishing a stable substrate film by lyophilizing all reagents on the surface of the pads. Only 20 to 30 µl of substrate solution, 50 µl of buffer solution, and 1 to 10 µl of blood are necessary, making a total volume of 51 to 60 µl for each assay. The rate of appearance of the fluorescent 4-methylumbelliferone liberated from 4-methylumbelliferone phosphate by the enzymatic action is measured and equated to enzyme activity. Calibration plots of the change in fluorescence per minute vs. enzyme activity for measurements in solution and on pads show a good proportionality in the range of 30.8 to 633 U/liter for alkaline phosphatase and in the range of 0.265 to 5.3 King— Armstrong units for acid phosphatase, indicating the usefulness of these methods in the clinical laboratory.
Direct Radioimmunoassay for Unconjugated Estriol in Pregnancy Serum, with Use of a Radioiodinated Derivative of Estriol
Authors: Miller Carol A , Fetter Marion C , Boguslaski Robert C
Published: January 2020

We describe a rapid and direct 125I-based radioimmunoassay for quantification of unconjugated (free) estriol in pregnancy serum. Estriol in serum is adsorbed onto a small column of Sephadex, thereby allowing its separation from proteins and interfering materials. A radioiodinated derivative of estriol (6-ketoestriol-6(o-carboxymethyl)oxime-[125I] tyrosine methyl ester) is added to the column, followed by a limiting amount of antiserum. After incubation, the antibody-bound hormone is separated by a buffer wash. The assay exhibits satisfactory recovery and parallelism, and the intraand inter-assay coefficients of variation are <10%. The values obtained by using the assay correlate well (r = 0.97) with those from a comparison method in which solvent extraction and chromatographic purification are used.
Sodium Iodoacetate as an Antiglycolytic Agent in Blood Samples
Authors: Marbach Edward P , McLean Monty , Scharn Marilyn , Jones Tom
Published: January 2020

We evaluated the effect of sodium iodoacetate on glycolysis in a series of randomly selected blood samples from patients. Glucose values for serum and for serum with added sodium fluoride (2.5 g/liter) or sodium iodoacetate (2 g and 0.5 g/liter) were compared at room temperature. Respective declines in glucose values averaged 170, 40, 30, and 30 mg/liter after 24 h. Iodoacetate-preserved (0.5 g/liter) samples showed no visible hemolysis. Results of determinations of urea with urease and of other tests on SMA 12/60 (Technicon) panels were unaffected.
Abnormal Electrophoretic Mobility of a Creatine Kinase MM Isoenzyme
Authors: Velletri Kimberly , Griffiths William C , Diamond Israel
Published: January 2020

We describe the abnormal electrophoretic mobility of an MM isoenzyme of serum creatine kinase on cellulose acetate, a change evidently attributable to complexing of the enzyme with serum β-lipoprotein. This complex formation also may lead to difficulty in interpretation of creatine kinase isoenzyme separation by ion-exchange column chromatography.
Radioimmunoassay of Methaqualone and Its Monohydroxy Metabolites in Urine
Authors: Berman Ann R , McGrath James P , Permisohn Robert C , Cella James A
Published: January 2020

A commercial radioimmunoassay kit was evaluated for efficacy in detecting methaqualone or its metabolites in urine of persons receiving this drug. The drug and its unconjugated 3'- and 4'-monohydroxy metabolites could be detected equally well. The unconjugated α-monohydroxy metabolite was about 80% as reactive and the unconjugated 6-monohydroxy metabolite reacted only very weakly. Quantitation of the conjugated metabolites was less sensitive than of unconjugated. Nineteen urine specimens which reacted positively to radioimmunoassay and which thin-layer chromatography had shown to contain methaqualone and its metabolites were also examined by gas-liquid chromatography. Those specimens that reacted strongly to radioimmunoassay contained high concentrations of the drug or its metabolites. In the specimens examined by gas-liquid chromatography, the apparent concentrations of the metabolites were generally higher than those of the drug itself. Methaqualone in combination with its unconjugated metabolites reacted additively with the radioimmunoassay, resembling the same concentration of parent drug alone. Detection limits were between 10-200 µg/liter.
Comparison of Commercial Kits for Radioimmunoassay: III. Radioassay of Serum Folate
Authors: Kubasik Norman P , Volosin Michael T , Sine Harrison E
Published: January 2020

We evaluated six commercial radioassay kits in which tritium or iodine tracers are used to measure serum folate. We assessed the adequacy of the literature supplied with the kits, within-run precision, reagent stability, analytical recovery of added pteroylglutamic acid and N-methyltetrahydrofolic acid, results of analysis of patients' samples, and minimum detection limits. There were serious discrepancies among manufacturers' kits in each of these respects. Such discrepancies could affect interpretation of results and of data on like samples derived from different laboratories that are using different kits. Laboratories are urged to assess carefully the materials and method supplied by a manufacturer before putting a kit to clinical use.
Determination of Serum Thyroxine after Dissociation from Thyroxine-Binding Globulin in Alkaline Solution and Adsorption on Dextran-Coated Charcoal
Authors: Wong William H
Published: January 2020

A competitive protein-binding procedure for determining serum thyroxine is described in which thyroxine is first dissociated from serum thyroxine-binding globulins in an alkaline solution. After binding equilibration, the unbound thyroxine is separated by dextran-coated charcoal. The procedure takes only 100 µl of serum, requires no evaporation, and is sensitive and reproducible. Results by this procedure correlate well with those for a generally accepted method.
Determination of Diphenylhydantoin in Human Serum by Spin Immunoassay
Authors: Montgomery Mark R , Holtzman Jordan L , Leute Richard K , Dewees John S , Bolz Gunner
Published: January 2020

A spin immunoassay for diphenylhydantoin is reported, which appears to give an accurate and precise estimate of serum diphenylhydantoin concentrations, as judged by the disappearance of [14C] diphenylhydantoin from the serum of a rabbIt. The assay also appears to be a reliable technique for routine diphenylhydantoin determinations, as judged from our experience with 28 patients. Serum diphenylhydantoin concentrations in the range of 1.0-50.0 mg/liter are easily determined on a 50-µl sample. Except for primidone, no significant cross reactivity was observed with eight drugs that are commonly used in conjunction with diphenylhydantoin therapy. This fast, simple, and precise method therefore appears to be readily applicable to routine determination of diphenylhydantoin.
Methodologic Differences in Values for M-Proteins in Serum, as Measured by Three Techniques
Authors: Daniels Jerry C , Vyvial Theona M , Levin William C , Ritzmann Stephan E
Published: January 2020

Accurate and reproducible measurement of M-proteins is essential for managing patients with monoclonal gammopathies, but serum protein electrophoresis, radial immunodiffusion, and electroimmunodiffusion yield comparatively divergent results. We have studied these differences and their causes. Sera from cases of IgG-monoclonal gammopathy, IgA-monoclonal gammopathy, and IgM-monoclonal gammopathy were assayed by each of the three techniques. Results indicated intermethod discrepancies as great as fivefold for all proteins studied. For IgG-monoclonal gammopathy, radial immunodiffusion values were uniformly higher; electroimmunodiffusion values were less consistently so. For IgA-monoclonal gammopathy, both radial immunodiffusion and electroimmunodiffusion gave lower results than did serum protein electrophoresis. For IgM-monoclonal gammopathy, results were variable, but values by radial immunodiffusion tended to be higher than, and electroimmunodiffusion comparable to, those for serum protein electrophoresis. The differences were not correlated with protein abundance, serum freshness, immunoglobulin class, light-chain type, ultracentrifugal characteristics, or electrophoretic mobility. Clearly, values for M-protein concentration depend on the techniques used to obtain them. We postulate that subclass differences may contribute to the diversity of radial immunodiffusion results, and that for electroimmunodiffusion the fixed electrophoretic mobility of M-proteins leads to unpredictable results. We conclude that serum protein electrophoresis is the best of the three assay techniques for M-proteins.
Lecithin—Sphingomyelin Ratio in Amniotic Fluid, as Assessed by a Modified Thin-Layer Chromatographic Method in Which a Commercial Pre-Coated Plate Is Used
Authors: Mallikarjuneswara Valyacheri R
Published: January 2020

Commercially available pre-coated thin-layer chromatographic plates consisting of silica gel supported on aluminum sheets were used to determine the lecithinsphingomyelin ratio in samples of amniotic fluid. These lipids were separated in a chloroform-methanol-water (65:25:4 by vol) solvent system (lecithin, RF = 0.33; sphingomyelin, RF = 0.25), and the spots were made visible in 5-15 min by heating to 180 °C after spraying with a solution of copper acetate (30 g/liter) in aqueous phosphoric acid (80 g/liter). The lipids were measured densitometrically after "peeling off" the adsorbent containing the spots by using a transparent cellulose ("Scotch brand") adhesive tape. Virtues of the original Gluck procedure are retained without need to heat the plates to 280 °C after spraying with corrosive aqueous sulfuric acid (698 g/liter) to make the lipids visible. Resuits for synthetic mixtures in concentrations within the physiological range and with a ratio of 0.5 to 3.0 showed an accuracy of 99% ± 7% (SD).
Some Nonparametric Statistical Tests for Quick Evaluation of Clinical Data
Authors: Gindler E Melvin
Published: January 2020

Some rapid statistical tests give (a) rapid answers on how well methods agree and control chart evaluation (sign and run tests) and (b) evaluation of distribution of test results (Tukey's quick test and run test). These tests mainly require counting of data and the use of the given nomograms. An unusual distribution of patient test values—that is, unusual when compared with the generally observed distribution of the data seen in a particular laboratory—may indicate laboratory error, alteration of specimens (as from poor collection and/or storage techniques, such as evaporation), or an unusual patient population.
Effect of Lactate Dehydrogenase Isoenzymes on the Coupled Enzymatic Assay for Alanine Aminotransferase Activity
Authors: Chang Michael M , Chung Tai Wha
Published: January 2020

We show an example of the importance of specifying the form of isoenzyme and source of indicator enzymes to be used in coupled enzymatic assays. When we compared H4 (pig heart) and M4 (rabbit muscle) isoenzymes of lactate dehydrogenase for their suitability as indicator enzymes in the assay for alanine aminotransferase activity, we found that about fourfold as much M4 as H4 was required in terms of lactate dehydrogenase activity to reflect accurately equivalent amounts of alanine aminotransferase activity. Moreover, the substrate specificities of the two isoenzymes differed quantitatively.
Examination of Some Factors Affecting Sensitivity and Reproducibility in Radioimmunoassay of Thyrotropin
Authors: Moser Russell J , Hollingsworth Dorothy R
Published: January 2020

Conditions for measuring human thyrotropin by radioimmunoassay have been investigated, to improve the sensitivity and reproducibility of the assay. The ChloramineT method of iodination was used, the reaction time being 20 s. Doubling the iodination reaction volume from 55 to 95 µl did not affect the immunoreactivity. Purification of labeled hormone by use of anion-exchange resin followed by silica (Quso G-32) instead of Sephadex gelfiltration or anion exchange alone yielded a product that was the least (<4%) contaminated with Na125I. Human serum albumin (2.5 g/liter) in phosphate-buffered saline, instead of bovine serum, should be used as diluent for unknowns; within-assay variance was 3% for the former, 62% for the latter. The assay worked equally well for both pregnant and nonpregnant patients, with use of 50 to 100 µl of serum per determination. A five-week-old labeled hormone yielded higher values than did two-week-old material. Use of sequential saturation techniques (tracer added on day 3) resulted in a greater than 50% drop in B/B0 ratio in the standard curve between 0.39 and 50 microunits/ml. Somatotropin, choriomammotropin, and porcine insulin did not cross react in this system. Human follitropin and lutropin did cross react, and this cross reaction could not be prevented with as much as 40 international units of human choriogonadotropin per tube. With a total reaction volume of 0.4 ml before addition of second antibody, 0.2 ml of 10-fold diluted second antibody yielded a standard curve with lower nonspecific binding and higher maximum precipitation than one constructed by using only 20 µl of second antibody. In 29 euthyroid patients the mean thyrotropin value was 5.7 microunits/ml (range 2.8-11); nine hypothyroid patients had a mean of 112 microunits/ml (range 38-267); and 13 hyperthyroid subjects showed suppressed thyrotropin with a mean of 3.1 microunits/ ml (range 2.2-4.5). Following these suggestions, one can expect a more highly purified immunoreactive tracer and a more sensitive assay than is obtained with the procedure from the Pituitary Agency of the National Institutes of Health.
Use of Leuco-Dyes in the Quantitative Colorimetric Microdetermination of Hemoglobin and Other Heme Compounds
Authors: Ahlquist David Alan , Schwartz Samuel
Published: January 2020

Sensitivity, stability, and specificity of the color-producing reaction of hydrogen peroxide with benzidine, leucomalachite green, or o-dianisidine were tested in numerous systems containing hemoglobin and other hemoproteins. Use of urea or low temperature (to -12 °C), or both, was highly beneficial, especially with leuco-malachite green, for which the color reaction was stable, after about 15 min, for longer than 24 h, with a colorless blank. Absorbance was 0.3 at a final hemoglobin concentration of 0.27 mg/liter. Nonspecific color produced by substances such as FeCl3 and ascorbic acid was completely eliminated. Of the three leuco-dyes studied, only benzidine yielded a completely linear calibration curve, but its relative instability and reported carcinogenicity are serious disadvantages. No system tested eliminated completely the known inhibition by plasma of the peroxidase activity of these leuco dyes.
Manual Determination of Intra-Erythrocytic 2,3-Diphosphoglycerate in Whole Blood by Enzymatic Analysis, and Comparison with Ion-Exchange Chromatography
Authors: Detter James C , Gibson Donald F , MacMillan Stuart F , Oas Thomas H
Published: January 2020

A manual modification of an automated method [Atkinson, K. F., Clin. Chem. 18, 1001 (1972)] for enzymatic assay of 2,3-diphosphoglycerate is described, which is suitable for small laboratories. Samples are easily prepared for analysis, and preparations are stable for several months. Virtually all of the color generated in the colorimetric assay is produced by the diphosphoglycerate-catalyzed reaction. The coefficient of variation for multiple samples from a single subject was 2.4%. Delayed preparation of samples, particularly samples from some acidotic subjects, is shown to result in altered results. The enzyme method is compared with analysis by ion-exchange chromatography.
Free Amino Acids in Human Tonsillar Tissue
Authors: Doi Yasuyuki , Kataura Akikatsu
Published: January 2020

Free amino acids in the tonsils of 20 individuals were measured column chromatographically. Those always found in readily detectable amounts included O-phosphoserine, taurine, O-phosphoethanolamine, aspartic acid, hydroxyproline, threonine, serine, glutamic acid, proline, glycine, alanine, α-amino-n-butyric acid, valine, cystine, methionine, isoleucine, leucine, tyrosine, phenylalanine, ornithine, γ-amino-butyric acid, lysine, histidine, and arginine. Results were compared for three clinical pathological groups and for four age groups. Some abnormal values may result from the pathological conditions.
Simultaneous Determination of Serum Cholesterol and Triglycerides after Preliminary Column Chromatography
Authors: Moses Godfrey , Olivero Edwin , Draisey Thomas F
Published: January 2020

An isopropanolic extract of serum can be made suitable for the simultaneous estimation of cholesterol and triglycerides by passing it through a commercially-available chromatographic column containing activated metallic oxides in which alumina predominates. No centrifugation step nor phase separation is required. Use of the purified extract allows existing methods to be simplified and shortened without loss of reproducibility or stability. Results compare well with those obtained by traditional methods.
Technique for Determining Nickel in Blood by Flameless Atomic Absorption Spectrophotometry
Authors: Zachariasen Hans , Andersen Ivar , Kostol Carl , Barton Richard
Published: January 2020

We describe a technique for determining nickel in serum by flameless atomic absorption, with use of di methylglyoxime. The procedure permits a greater num ber of analyses per technician at a relatively low cost, yet provides acceptable accuracy and reliability. After the sample is evaporated and dry ashed in a muffle fur nace at 560 #{ 176} C for 5 h, the residual salts are dissolved in hydrochloric acid (1 mol/liter). Sodium citrate and di methylglyoxlme are then added for complexation of iron and nickel, respectively. The pH is adjusted to 9 with ammonia and the nickel dimethylglyoxime is extracted with methyl isobutyl ketone. The determination is carried out by injecting 50 µl of the organic phase into the graphite furnace, where the sample is dried at 120 °C and ashed at 1200 °C, respectively.
Influence of Myocardial Infarction on Serum Manganese, Copper, and Zinc Concentrations
Authors: Versieck Jacques , Barbler Fabrice , Speecke Albert , Hoste Julien
Published: January 2020

Reportedly, serum manganese concentrations increase after myocardial infarction, closely correlated with increased serum aspartate aminotransferase activity. However, these conclusions are apparently based on analyses of contaminated samples. Serum manganese concentrations after myocardial infarction have been re-investigated by neutron activation analysis, and no significant increase could be demonstrated. Because serum copper and zinc could be determined simultaneously, analyses for these trace elements are also reported, which confirm the findings of others. After myocardial Infarction a statistically significant (0.02 < P < 0.05) increase in serum copper and a statistically significant (0.001 < P < 0.01) decrease in serum zinc were observed.
Determination of Cadmium in Blood and Urine by Graphite Furnace Atomic Absorption Spectrophotometry
Authors: Perry Elizabeth F , Koirtyohann S Roy , Perry H Mitchell
Published: January 2020

We describe a method for measuring quantities of cadmium in blood, plasma, and urine by using graphite furnace atomic absorption preceded by wet ashing. The method appears to be extremely accurate and reproducible (coefficient of variation is 7% for 2.8 µg/liter concentration), with a detection limit of 2 pg.
Serum Immunoglobulins after Surgical Operation
Authors: Fuller Janet M , Keyser J W
Published: January 2020

We measured immunoglobulins in the sera of 33 patients on days 1, 3, 6, 10, and 17 and three to four weeks after surgical operations (mostly hysterectomy or appendectomy) or (six patients) after spinal injury. In the absence of infection or blood transfusion, IgG usually decreased slightly and transiently after hysterectomy or appendectomy, as did IgA or IgM after hysterectomy. IgD concentrations showed no consistent changes, but in one patient after hysterectomy and with minimal infection IgD concentration decreased sharply, which contrasted with significant and early increases in IgG, IgA, and IgM. IgD concentration was not correlated with type of operation, presence of infection, or changes in the other immunoglobulins. IgE concentrations either did not change or, in some patients, increased or decreased initially, after operation. We conclude that immunoglobulin concentrations in serum are subject to multiple, unpredictable influences after trauma.
Evaluation of the New "System Olli 3000"Kinetic Ultraviolet Analyzer for Measuring Aspartate and Alanine Aminotransferase and Lactate Dehydrogenase Activities in Serum
Authors: Adlercreutz Herman , Peltonen Veikko , Voipio Tauno
Published: January 2020

A new fast kinetic analyzer, System Olli 3000, is evaluated as an instrument for the routine clinical laboratory measurement of the activities of aspartate aminotransferase, alanine aminotransferase, and lactate dehydrogenase in serum. The System Olli 3000 consists of dispensers for simultaneous multiple dispensing of sample and reagents, incubators, vortex-type shakers, and a photometer with quartz fibre optics connected to a computer, allowing cycling measurements of 24 cuvets 24 times in 2 min. An unique slope search algorithm is described. The system shows a high degree of precision and a wide linearity range; activities of at least 10-fold the normal upper limit for all three of these enzymes can be measured without diluting the serum sample. As many as 380 analyses per hour (including calibration and blanks) can be carried out by one technician. For comparison, enzyme measurements were also made with an LKB 8600 Reaction Rate Analyzer and a Pye Unicam SP 8005 spectrophotometer coupled on-line to an IBM 1800 computer. Results obtained with the different instruments correlated well, especially in the region of main interest, i.e., above the normal upper limit. We conclude that the new instrument has many potentialities in kinetic analyses of nonenzymatic constituents in biological fluids.
Radioimmunoassay of Plasma Testosterone, with Use of Polyethylene Glycol to Separate Antibody-Bound and Free Hormone
Authors: Anderson Peter H , Fukushima Kimiko , Schiller Harvey S
Published: January 2020

We have developed a reliable radioimmunoassay for testosterone in plasma, polyethylene glycol ("Carbowax 6000") being used to separate antibody-bound and free hormone. Testosterone is separated from interfering steroids, notably dihydrotestosterone, by liquid—liquid partition chromatography on infusorial earth (Celite). The assay is sensitive (9 pg for standards), precise, and accurate. The lowest measurable concentration of testosterone is 350 ng/liter for plasma from men and 70 ng/liter for plasma from women. Intraand inter-assay coefficients of variation were 6.9% and 9.7%, respectively, for plasma from men, and 9.6% and 11.8%, respectively, for plasma from women. Our method for separating antibody-bound and free hormone is practical and convenient and may be generally applicable to all radioimmunoassays of steroid hormones in plasma.
Gas—Liquid Chromatography of Phenylalanine and Its Metabolites in Serum and Urine of Various Hyperphenylalaninemic Subjects, Their Relatives, and Controls
Authors: Kitagawa Teruo , Smith Barbara A , Brown Eleanor S
Published: January 2020

Phenylalanine and its metabolites were determined in serum and urine of phenylketonuric subjects and in subjects with milder hyperphenylalaninemia in whom blood phenylalanine concentrations were usually <200 mg/liter. Metabolite concentrations were related to serum phenylalanine, and in hyperphenylalaninemic subjects were between those for treated and untreated phenylketonuric subjects. Phenyllactic and phenylpyruvic acids were excreted by all of the mild hyperphenylalaninemic subjects except for the youngest (one-year-old twins) and the only subject with a serum phenylalanine of <100 mg/liter. Serum and urinary metabolites of heterozygotes of both conditions were similar before and after a phenylalanine load. The similar pattern of metabolites in phenylketonuric and mild hyperphenylalaninemic subjects reinforces the belief that the latter have some phenylalanine hydroxylase activity, and that this is the essential difference between the two groups.
Thermochemical Determination of Glucose in Serum, Plasma, and Whole Blood without Prior Deproteinization
Authors: McGlothlin C David , Jordan Joseph
Published: January 2020

Glucose can be determined by phosphorylatlon in the presence of hexokinase (EC 2.7.1.1) by using Mg2+/ (ATP)2- as the phosphorylating agent. A novel instrumental quantitation method is described, direct injection enthalpimetry, whereby the heat of the enzymatically catalyzed reaction was measured. The reaction was allowed to proceed to virtual completion (99.5+%) in an adiabatic Dewar vessel at 25.00 ± 0.01 °C (range). Samples were sequentially injected into a reaction mixture consisting of hexokinase, ATP, Mg2+, and a tris(hydroxymethyl)aminomethane buffer (pH 8). Single analyses required <2 min for 0-3 g/liter samples. The linear dynamic range was 0.3-10 g/liter, with zero intercept and a precision and accuracy of 2%. Electrical calibration in situ obviated the need for chemical standards, and because protein and color do not interfere, no sample pretreatment was necessary before analysis.
Flash-Heater Ethylation of Some Antiepileptic Drugs
Authors: Friel Patrick , Troupin Allan S
Published: January 2020

We describe a modification of the MacGee method [Anal. Chem. 42, 421 ( 1970)] for rapid determination of phenytoin (diphenylhydantoin) in plasma by gas—liquid chromatography, in which an ethylating reagent is used instead of the more common methylating reagents. With this modification, several N-methylated antiepileptic drugs can be separated from their demethylated metabolites. Our results agree well with those of Butler and Waddell [Neurology 8, 106 ( 1958)], who showed that in patients receiving the N-methylated compounds mephenytoin and mephobarbital, the N-demethylated products, 5-ethyl-5-phenylhydantoin and phenobarbital are found in higher concentrations in plasma than are their respective parent drugs. The method is also useful for routine determinations of phenobarbital, primidone, and phenytoin in serum, cerebrospinal fluid, or saliva.
Simple Manual Procedure for Determination of Serum Triglycerides
Authors: Mendez Jose , Franklin Barry , Gahagan Harry
Published: January 2020

We describe a modified method for determining serum triglycerides (triacylglycerols), which is based on the heptane extraction procedure of Gottfried and Rosenberg [Clin. Chem. 19, 1077 (1973)] with the stable saponification, oxidation, and color development reagents of Neri and Frings [Clin. Chem. 19, 1201 (1973)]. This modified method eliminates one heating step, reduces saponification time to 5 min, absorbances are read at room temperature, and the calibration curve is linear to 3.0 g/liter. A sample comparison between the proposed method and the automated Block and Jarrett [Am. J. Med. Technol. 35, 1 (1969)] procedure showed no significant difference (r = 0.98). The coefficient of variation (47 duplicate samples) for the modified method was 6.3%. Further validation was obtained from analysis of quality-control samples; the proposed method gave equivalent values.
A Radioimmunoassay for Isoniazid
Authors: Schwenk Robert , Kelly Kenneth , Tse Kam S , Sehon Alec H
Published: January 2020

A sensitive radioimmunoassay is described, for measuring isoniazid in therapeutic and subtherapeutic concentrations. This radioimmunoassay was used in an exploratory clinical study to measure, as a function of time, the concentration of this drug in plasma of patients receiving isoniazid therapy.
Colorimetric Enzymatic Determination of Serum Total Carbon Dioxide, as Applied to the Vickers Multichannel 300 Discrete Analyzer
Authors: Norris Kenneth A , Atkinson Alan R , Smith William G
Published: January 2020

Phosphoenolpyruvate carboxylase (EC 4.1.1.31) has been used in developing a simple, inexpensive colorimetric assay for serum total carbon dioxide in an open system. The oxaloacetate formed by the action of the enzyme on bicarbonate and phosphoenolpyruvate is measured by use of the diazonium salt of Fast Violet B. Interferences from bilirubin, pyruvate, and drugs are negligible. Acetoacetate interference is significant only in highly ketotic samples, and a serum blank corrects for it. Serum protein interference is equivalent to 3.3 ± 1.25 mmol of CO2 per liter and hence is sufficiently constant to be corrected for by use of a serum standard or serum blank. The method has been applied to the Vickers M-300 and D-300 systems and within-batch standard deviations of ±0.1 to ±0.6 mmol/liter have been observed. Excellent correlation with orthodox techniques has been obtained.
Simultaneous Separation of Serum Creatine Kinase and Lactate Dehydrogenase Isoenzymes by Ion-Exchange Column Chromatography
Authors: Mercer Donald W
Published: January 2020

Lactate dehydrogenase isoenzymes were partially separated by use of a previously described column technique for creatine kinase [Clin. Chem. 20, 36 (1974)]. Extracts of lactate dehydrogenase-rich tissues were used to evaluate column resolution. Samples layered on mini-columns containing DEAE-Sephadex were eluted with Tris-buffered sodium chloride (100 and 200 mmol/ liter). Lactate dehydrogenase activity in column effluents was measured by the Wacker method, and their isoenzyme content was assessed by electrophoresis on polyacrylamide gel. Dehydrogenase isoenzymes 3, 4, and 5 were separated from isoenzymes 1 and 2, and the separation was tissue-specific and reproducible. The electrophoretic technique for isoenzymes 3, 4, and 5 gave values about 20% lower than did the column technique. Sera from 15 healthy laboratory technicians contained total lactate dehydrogenase, isoenzymes 1 and 2, and isoenzymes 3, 4, and 5 in the ranges 94 to 152, 34 to 64, and 38 to 75 U/liter, respectively. Activities of sera from 15 patients with acute myocardial infarction (total lactate dehydrogenase) ranged from 212 to 800 U/liter and lactate dehydrogenase isoenzymes 1 and 2 ranged from 138 to 628 U/liter. Lactate dehydrogenase and creatine kinase isoenzymes were rapidly and easily measured after being simultaneously separated. The procedure is specific and sensitive for following the post-infarct time course of changes in isoenzyme activities.
Interlaboratory Proficiency, Intermethod Comparison, and Calibrator Suitability in Assay of Serum Aspartate Aminotransferase Activity
Authors: Rej Robert , Fasce C F , Vanderlinde Raymond E
Published: January 2020

Sources of variation in assays of aspartate aminotransferase (EC 2.6.1.1) activity were examined in an interlaboratory survey and through an examination of materials used as calibration materials in these assays. Four highly stable lyophilized specimens containing human cytoplasmic enzyme, with activities of 0, 22, 46, and 96 U/liter at 30 °C and optimal substrate concentrations, were assayed by 319 laboratories. Mean values obtained on these specimens by laboratories using 2,4-dinitrophenylhydrazine kits varied among manufacturers and deviated from values expected from this procedure. The average coefficient of variation (CV) with these kits was > 20%. Automated continuous-flow procedures with use of diazonium salt showed the best precision (av CV, < 10%). However, the automated continuous-flow mAlate Dehydrogenase/Nadh Coupled Method Produced An Average Cv > 20%. Results from each of the automated methods were related to a reference malate dehydrogenase/NADH coupled continuous kinetic assay method by temperature relationships alone. Mean values from manual diazonium salt procedures were 1.7-fold greater than similar reference values (av CV was 18%). The higher results were attributed to the use of poorly-defined units and to an artifact caused by chromophore stabilizers in this procedure when aqueous samples are used. The average CV in continuous kinetic methods varied among kit manufacturers, ranging from 6 to 28% for the specimen of highest activity. Variations in results were much larger at 366 nm than at 340 nm. Interassay relationships of these methods are presented. Concentrations of pyruvate in commercially available calibration materials differed between manufacturers, varied in stability, and deviated from the expected concentration. For some colorimetric assays the precision attained on reported absorbance values for the enzyme specimens was of the same order of magnitude as that for pyruvate standards. Other sources of error are revealed by the interlaboratory survey. The value of commercially available sources of enzyme activity as calibration or control materials was assessed by evaluating the following properties: activity at suboptimal concentrations of L-aspartate or 2-oxoglutarate, temperature effects, preincubation lability owing to aspartate and phosphate, pyridoxal phosphate saturation, contamination with glutamate dehydrogenase, and manufacturer's rated activity. These properties are compared to those of human cytoplasmic enzyme in a human serum matrix.
Modification of a Commercially Available Digoxin Radioimmunoassay Kit for Higher Precision
Authors: van der Vijgh W J F
Published: January 2020

The procedure prescribed for the Schwarz/Mann 125Idigoxin kit was modified with regard to pipetting and counting procedure. Incubation time with dextran-coated charcoal was also investigated. The modifications resulted in a significantly higher precision without introduction of a systematic error and the analysis time per eight-tube batch was decreased by 20 min.
High-Speed Quantitative Karyotyping by Flow Microfluorometry
Authors: Gray J W , Carrano A V , Moore D H , Steinmetz L L , Minkler J , Mayall B H , Mendelsohn M L , Van Dilla M A
Published: January 2020

We describe a new flow-system technique for highspeed quantitative karyotyping. Metaphase chromosomes are isolated from cells, stained with a DNA-specific fluorochrome, and individually measured for stain content at rates up to 105/min in a flow microfluorometer. The resulting distribution contains quantitative information about the relative frequency of occurrence and the chromosomal DNA content of the measured chromosomes. Such analyses of Chinese hamster chromosomes are in good agreement with independent results from scanning cytophotometry and with cytogenetic observations of the banded chromosomes. Analyses of two karyotypicaliy different clones of Chinese hamster M3-1 lines provide the quantitative characterization of two stable translocations. Initial data on human chromosomes are presented and agree well with results of scanning cytophotometry and suggest potential clinical applications as preparative and instrumental techniques improve.