bluuebunny/crossref_metadata_2025 huggingface dataset:
Serum and Erythrocyte Argininosuccinate Lyase Assay by NADH Fluorescence Generated from Formed Fumarate
Authors: Sherwin John E , Natelson Samuel
Published: January 2020

Measurement of argininosuccinase (I; EC 4.3.2.1) activity is useful in following the course of disease in hepatitis and in screening for the genetic defect, argininosuccinic aciduria. Methodology is proposed for a novel procedure for the determination of I in serum and erythrocytes. In the procedure, fumarate, generated in the reaction, is assayed by conversion to malate with fumarase, determining the malate enzymatically with malate dehydrogenase, and estimating the NADH formed spectrofluorometrically. By this procedure, the enzyme activity in serum from normal individuals is less than 11 µmol/liter of serum/per hour. The normal range for this enzyme's activity in erythrocytes is 1120 to 2420 µmol/liter of erythrocytes/per hour. The correlation coefficient between results by this method and by the colorimetric method, which measures the arginine generated in the reaction, is +0.97 for serum and +0.98 for erythrocytes. The proposed procedure has a relatively low initial blank, requires less serum, and is completed faster.
Evidence Supporting a Proposed Mechanism Explaining the Inverse Relationship between Guanidinoacetate and Guanidinosuccinate in Human Urine
Authors: Koller Anthony , Comess Jay D , Natelson Samuel
Published: January 2020

A proposed mechanism [Clin. Chem. 19, 668 (1973)] for the inverse relationship between guanidinoacetate (I) and guanidinosuccinate (II) in human urine is explored. The mechanism proposes that canavaninosuccinate (III) may be reduced to form homoserine and II or, alternatively, that the III may be acted upon by a lyase to form canavanine and fumarate. The canavanine would then proceed to transamidinate to glycine to form I. This study demontrates for the first time that lyase activity for converting III to canavanine and fumarate exists in human liver and kidney extracts. Transamidination from canavanine to glycine to form I is also readily accomplished with human tissue. Reductive cleavage of III to II and homoserine has been demonstrated before [Clin. Chem. 15, 397 (1969)]. The optimum pH for the lyase reaction is 6.5, for the reductive cleavage it is 8.7. In following the course of the lyase reaction, we developed a technique whereby the fumarate formed was hydrated with fumarase (EC 4.2.1.2) and then dehydrogenated with malate dehydrogenase (EC 1.1.1.37). The changes in absorbance of NADH formed in the reaction were then measured and used to determine the amount of fumarate formed, as a measure of lyase activity. Canavanino-succinate lyase activity follows pseudo-first-order reaction kinetics. The Michaelis constant of this lyase was 6.16 x 10-4 mol/liter, for argininosuccinate lyase 9.74 x 10-4 mol/liter. These data suggest that the binding affinity for III to the enzyme is greater than that for argininosuccinate. Glycine added to the reaction acts as an activator, probably because it removes the canavanine from the reaction mixture. On the other hand, arginine acts as an inhibitor of Ill-lyase. Other substances tested, such as canavanine, fumarate, and argininosuccinate had no effect on the reaction kinetics.
pH Measurement Problems Affecting Assay of Carcinoembryonic Antigen
Authors: Ladenson Jack H , Bell C Elliott
Published: January 2020

Measurement with combination pH electrodes of the pH of the dilute buffers used in a commercial kit (CEA-Roche) for assay of carcinoembryonic antigen resulted in pH values 0.1 to 0.3 unit lower than pH values measured on an electrode system with a capillary junction. If the pH values of these buffers were adjusted, based on such measurements, an error in the assay of 0.2 to 0.6 ng/ml in the 1.5-3.0 ng/ml range would result. We recommend that the pH of dialyzed samples and of the working ethylenediaminetetraacetate and ammonium acetate-acetic acid buffers be monitored with pH electrodes that have a capillary junction between sample and saturated KCl, as is true of most blood-pH instruments. We also recommend use of a 1 mol/liter rather than 2.5 mol/liter stock ammonium acetate-acetic acid buffer, because of the closer similarity of the pH of buffers at this molarity to those at 0.01 mol/liter.
Lipoprotein Electrophoresis Should Be Discontinued as a Routine Procedure
Authors: Iammarino R M
Published: January 2020

Reviewing our five-year experience with more than 1000 lipid profiles (assays for cholesterol and triglycerides In plasma of fasting patients and lipoprotein electrophoresis of their plasma lipoproteins), I conclude that lipoprotein electrophoresis is superfluous for routine clinical laboratory use, being generally redundant to the other parts of the profile. This conclusion is also prompted in part by recent information that changes our conceptions of lipoprotein metabolism and the clinical expressions of genetic hyperlipoproteinemia. A different approach to lipid profiling is suggested.
Interlaboratory Comparison of Enzymatic Methods for Serum Glucose Determination
Authors: Gochman Nathan , Ryan William T , Sterling Rex E , Widdowson Graham M
Published: January 2020

This report describes an interlaboratory comparison of enzymatic serum glucose methods as currently applied in several instrumental adaptations. Four spectrophotometric, hexokinase-based methods used with the Du Pont "aca," the Abbott "Bichromatic Analyzer," the Aminco "Rotochem," and the Technicon "AutoAnalyzer II" were compared with glucose oxidase-based methods as used with the Beckman "Glucose Analyzer" and the "AutoAnalyzer II." We analyzed both normal samples and samples that contained potential interfering substances or were otherwise abnormal. Although all methods were satisfactorily precise, methodological bias was noted in several cases, particularly with abnormal specimens. The hexokinase-based methods were generally more variable. The most consistent data were obtained from the two glucose oxidase methods and the Du Pont aca. Results of this study comprises part of the background information required for development of an accurate glucose reference method under the auspices of the AACC Standards Committee.
Electrophoresis on Cellulose Acetate and Chromatography on DEAE-Sephadex A-50 Compared in the Estimation of Creatine Kinase Isoenzymes
Authors: Yasmineh Walid G , Hanson Naomi Quast
Published: January 2020

We have compared two methods for separating the isoenzymes of creatine kinase in serum and measuring their activity: chromatography on diethylaminoethyl-. Sephadex A-50, with both continuous and discontinuous gradient elution, and electrophoresis on cellulose acetate. Results by continuous and discontinuous gradient elution correlated well both for tissue extracts and sera. Electrophoresis on cellulose acetate is evidently less sensitive than the chromatographic method for detecting and measuring low activities of the isoenzyme designated MB. We describe how the isoenzymes may be separated by discontinuous gradient elution from microcolumns of the Sephadex and their activity rapidly quantitated by the Rosalki method [J. Lab. Clin. Med. 69, 696 (1967)], with a centrifugal analyzer.
Separation of Creatine Kinase Isoenzymes in Serum by Ion-Exchange Column Chromatography (Mercer's Method, Modified to Increase Sensitivity)
Authors: Nealon Daniel A , Henderson Arthur R
Published: January 2020

We describe our experience with Mercer's method [Clin. Chem. 20, 36 (1974)] for separating isoenzymes of creatine kinase (EC 2.7.3.2) in serum by ion-exchange chromatography. By using diethylaminoethyl cellulose rather than diethylaminoethyl Sephadex in the column and by changing the ionic strength of the eluting buffer, we could detect a significant proportion of the MB isoenzyme of serum creatine kinase at normal activities of creatine kinase in serum, even in the absence of cardiac disease.
Improved Biuret Procedure for Routine Determination of Urinary Total Proteins in Clinical Proteinuria
Authors: Rice Eugene W
Published: January 2020

This communication describes and evaluates an improved routine methodology for quantitating clinical proteinuria. Based on investigations of Piscator and of Savory et al., a modified Tsuchiya's reagent (ethanolic HCl-phosphotungstic acid) is used to precipitate proteins at 56 °C, followed by biuret spectrophotometry at 540 nm. The accuracy of the proposed procedure was assessed by comparisons with results obtained by using an ultrafiltration membrane that retains solutes with an average molecular weight in excess of 10 000 for separating of urinary proteins before they are measured with the biuret reaction. Precision of the method (coefficient of variation) is typically 2-3%.
Use of Nucleic Acid Hybridization for Specific Detection of Submicrogram Quantities of DNA, and Its Application to Human Plasma
Authors: Steinman Charles R
Published: January 2020

A technique is described for using radiolabeled RNA complementary to human DNA as a probe for the specific identification of submicrogram concentrations of human DNA by formation of RNA-DNA hybrids. An example is given of its application to the semiquantitation of human DNA in human plasma, a substance that is ordinarily difficult to examine because materials are present that interfere with the usual colorimetric or fluorometric assays. An example is also given of the use of an analogous approach to analyzing rabbit serum for circulating bacterial DNA. Unique to the hybridization technique is a degree of specificity sufficient to identify specific base sequences and hence the origin of the DNA being detected, a point that may be important in the examination of circulating DNA reported to occur in patients with systemic lupus erythematosis. This technique may also be of value in clarifying the presently conflicting data regarding the occurrence of free DNA in the normal human circulation.
Lipase-Triggered Kinetic Assay of Serum Triglycerides
Authors: Bucolo Giovanni , McCroskey Ralph , Whittaker Nadine
Published: January 2020

We describe a kinetic method for assay of serum or plasma triglycerides, by use of an enzymatic hydrolysis and reaction sequence already described [Clin. Chem. 19, 476 (1973)]. The reaction is triggered by addition of lipase, at a time when free glycerol, or pyruvate (or both) are no longer present. In this method, therefore, there is no need for a blank glycerol assay. In the procedure, reagents are used that are available commercially in the form of stable, dry powders; the method for the preparation of the reagents has been changed to achieve improved stability and performance. Stability and recovery of added triglycerides are satisfactory.
pH Dependence of the Binding of Folates to Milk Binder in Radioassay of Folates
Authors: Givas Joan K , Gutcho Sidney
Published: January 2020

We compare the binding of folic acid and N-5-methyltetrahydrofolic acid to milk binder in the pH range 7.4 to 10.1. At pH 7.4 the relative affinities are quite disparate, with folic acid showing the greater affinity for milk binder. As the pH is increased from 7.4 to 9.3, the difference in affinities becomes smaller, and at pH 9.3 the affinities are nearly the same. As the pH is Increased from 9.3 to 10.1, the relative affinities again begin to differ, with N-5-methyltetrahydrofolic acid displaying the greater affinity. These results indicate that at a pH of 9.3 the more stable folic acid may be used as the standard in radioassay of endogenous folates.
Micro-Scale Blood Lead Determinations in Screening: Evaluation of Factors Affecting Results
Authors: Marcus Martin , Hollander Melvin , Lucas Robert E , Pfeiffer Norman C
Published: January 2020

The Delves micro-scale technique for blood lead analysis is an accurate method for screening capillary blood specimens, obtained by fingerstick, for lead intoxication. Results are affected by the age of cup, loop, and hollow-cathode tube and by the spatial relationship between optical tube and cup. Because the glass in many commercially available capillary tubes (used in specimen collection) contains lead and cannot be decontaminated, a lead-free glass tube must be used. A solution of citric acid in ethanol (20 g/liter) effectively cleanses the puncture site. A double-blind study of 207 specimens gave a mean value of 276.6 ± 105.8 µg/liter (1 SD) for the micro-scale method vs. 273.2 ± 99.0 for a macro-scale method. The mean coefficent of variation for the micro-scale method was 5.75% ± 1.9 (SD). We conclude that values of 480 µg/liter or below are not "toxic" (i.e., are significantly less than 600 µg/liter, the value at which therapy is begun). The method is shown to be satisfactory as a screening procedure and for confirming lead analyses done by other methods.
Ultramicro Analysis for Copper, Cadmium, and Zinc in Human Liver Tissue by Use of Atomic Absorption Spectrophotometry and the Heated Graphite Tube Atomizer
Authors: Evenson Merle A , Anderson C Thomas
Published: January 2020

We describe a method of analysis for copper, cadmium, and zinc in a 15-mg (wet weight) sample of human liver by atomic absorption spectrophotometry. The sample is digested with nitric acid (1.0 mol/liter), evaporated, and dilute HNO3 (10 mmol/liter) added. The reconstituted acid mixture is injected into the graphite tube atomizer for analysis of Cu and Cd and aspirated into the airacetylene flame for measurement of Zn. The absorbance for each metal is suppressed with increasing pH. NaNO3, KNO3, KCl, and NaCl (e.g.) quench the Cd absorbance in acid solutions that contain no protein, but not in the presence of protein. Metal ions added to the predigestion human liver sample at 10% and 100% of the intrinsic metal concentrations were, respectively, 93% and 90% accounted for analytically in the case of Cu, 98% and 102% for Zn, and 101% and 93% for Cd. Analysis of a National Bureau of Standards' Bovine Liver Standard Reference Material yielded results corresponding to 99% (Cu), 112% (Zn), and 91% (Cd) of the mean expected concentrations of these metals. The between-run coefficient of variation for the bovine liver material was 6% for Cu, 9% for Zn, and 10% for Cd. For 16 histologically normal samples of human liver, the mean values were: Cu, 26; Zn, 293; and Cd, 6.0 nanograms of metal per milligram dry weight, in agreement with values published previously. The method can be easily and reliably applied to small samples of liver obtained by closed-needle biopsy.
Effect of the Matrix of the Standard on Results of Atomic Absorption Spectrophotometry of Zinc in Serum
Authors: Momčilović Berlslav , Belonje Bartholomeus , Shah Bhagwan G
Published: January 2020

Zinc in pooled human serum, diluted 10-fold with demineralized water, was determined by flame atomic absorption spectrophotometry. Recoveries of zinc added to the serum were calculated by use of standards prepared in water, physiological saline, synthetic serum, or dilute HNO3 (10 ml/liter). The flow rates were essentially the same (9.1 to 9.3 ml/min) for water, diluted synthetic serum or actual human serum, and diluted nitric acid. The mean apparent percentage recoveries (±SD) were the best (102.6 ± 2.5) when the standards were prepared in nitric acid; the values obtained when standards in water were used were too high (111.5 ± 3.9); those with standards prepared in physiological saline (92.0 ± 3.2) or synthetic serum (92.3 ± 2.5) were too low.
Some Aspects of Specimen Collection and Stability in Trace Element Analysis of Body Fluids
Authors: Anand Vir D , White James M , Nino Hipolito V
Published: January 2020

Analyses for trace elements in biological fluids are uniquely susceptible to extreme errors unless special precautions are taken during collection, storage, and analysis. The integrity of the specimen may be compromised before it is analyzed, by contamination during collection and processing or by attenuation of the analyte concentration during storage. If this happens, determined values are not valid even though the method of analysis is extremely sensitive and highly accurate. Obstacles to obtaining precise and accurate analytical data arising from these factors are discussed. We consider control procedures applicable at all stages for ascertaining the sources of error and eliminating them.
The Prostaglandins in Clinical Medicine. A Developing Role for the Clinical Chemist
Authors: Russell Paul T , Eberie Andrea J , Cheng Hsien C
Published: January 2020

We review the ways that prostaglandins might relate to clinical chemistry. Clinical aspects that are discussed include hypertensive diseases, disorders of the gastrointestinal tract, cancer, inflammation and fever, effects on platelets, the respiratory system and asthma, and reproduction. A survey of procedures for the storage, extraction, separation, and quantitation of the prostaglandins is also presented.
Use of Carbon Dioxide- and Oxygen-Tonometered Phosphate— Bicarbonate—Chloride—Glycerol—Water Mixtures for Calibration and Control of pH, pco2 and pO2 Electrode Systems
Authors: Veefkind A H , Van den Camp R A M , Maas A H J
Published: January 2020

Calibration of pH, pco2, and po2 electrode systems of modern blood—gas analyzers, designed with one sample cuvet for measurement, is mostly performed separately with buffer solutions and gas mixtures. We describe the development of solutions of known pH, pco2 and po2 for doing such calibrations simultaneously, containing phosphate, bicarbonate, and chloride in glycerol—water mixtures as solvent. A method is suggested for computing the relation between pH and log pCO2 of these solutions in equilibrium with carbon dioxide gas. It is demonstrated that a solution of phosphate (Na2HPO4, KH2PO4, each 25.mmol/liter), bicarbonate (NaHCO3, 30 mmol/ liter), and chloride (NaCI, 30 mmol/ liter) in glycerol-water mixture (3/7 by vol) and equilibrated with CO2 in air (4 vol/100 vol) and CO2 in nitrogen (8 vol/100 vol), respectively, makes possible accurate and simultaneous calibration of the pH, pco2, po2 electrodes of a Corning Model 165 blood—gas analyzer. Similar solutions may also be used for quality—control of blood—gas measurement.
Preparation of a Modified Glucose Oxidase Reagent for the Polarographic Determination of Glucose with the Beckman "Glucose Analyzer"
Authors: Fischl Joseph , Federman Doža , Talmor Nira
Published: January 2020

In this modification, glucose oxidase, 140-180 U/ml, is dissolved in acetate buffer (pH 6):ethanol (96%):glycerol (80:10:10 by vol). Iodine and molybdate are used as catalysts, formaldehyde and mercuric iodide as preservatives, and octanol as an antifoaming agent. The reagent is suitable for use in the Beckman "Glucose Analyzer," it is stable at room temperature for at least six months, it is sensitive, it has a lubricating effect, and its cost is less than a fifth as much as the "Certified" Beckman reagent. It is not suitable for the measurement of glucose in samples containing maltose, cellobiose, gentiobiose, starch, and glycogen.
Improved Radioimmunoassay of Plasma Cortisol
Authors: Donohue John , Sgoutas Demetrios
Published: January 2020

We report a simplified radioimmunoassay procedure for determination of plasma cortisol. The method offers the advantage that tracer, antibody, and assay buffer are added in a single step with the use of semi-automated equipment. Thus, critical pipetting procedures are minimized, and assay time is reduced to 4 h. No prior extraction or chromatographic purification of cortisol is required. The procedure is simple, reliable, and accurate. For either between-assay or within-assay determinations, the coefficients of variation are about 6%. Our results compare well with those obtained from a procedure that included extraction and chromatographic purification of cortisol in several plasma samples. Conditions for optimizing the assay are also discussed.
Procedure for Calibrating the Technicon Colorimeter I
Authors: Black John C , Furman William B
Published: January 2020

We describe a rapid method for calibrating the Technicon AutoAnalyzer colorimeter I. Test solutions of bromphenol blue are recommended for the calibration, in preference to solutions of potassium dichromate, based on considerations of the instrument's working range and of the stray light characteristics of the associated filters.
Gas—Liquid Chromatographic Measurement of Guanidino Acids
Authors: Patel Harini , Cohen Burton D
Published: January 2020

We present a method for separating and measuring guanidino acids by gas—liquid chromatography. Compared to ion-exchange techniques, this system is faster, more sensitive, requires smaller sample volumes, is independent of colorimetric reactions, and permits simultaneous determination of both amino and guanidino acids. N-Trifluoroacetyl-n-butyl esters are formed and then are separated by using a column containing a mixed silicone liquid phase coated on Chromosorb W-HP. Analytical recoveries from plasma ranged from 86 to 112% and were optimal when purification and derivatization were done without prior protein precipitation. Speculations as to the cause of this interference by protein include coprecipitation, protein-binding, and cyclization of the guanidines in the acids used for denaturation. Evidence presented suggests that all three occur: ultrafiltrability is diminished in the presence of protein and nuclear magnetic resonance demonstrates cyclization of some of these esters.
Rapid Separation of Plasma Creatine Kinase Isoenzymes by Batch Adsorption on Glass Beads
Authors: Henry Philip D , Roberts Robert , Sobel Burton E
Published: January 2020

To separate the MM and MB creatine kinase (EC 2.7.3.2) isoenzymes in small plasma samples, we developed a simple, rapid ion-exchange batch-adsorption procedure with "DEAE Glycophase" glass beads (Corning). All separative steps are performed in a single test tube and can be completed within a few minutes. Results of measurements of isoenzymes in plasma samples from patients with acute myocardial infarction were compared to those obtained with an independent quantitative assay method previously reported. Additional measurements were performed on standard plasma samples containing mixtures of MM and MB isoenzyme purified from human myocardium. Results by the two independent assay procedures agreed well, as did measured isoenzyme activities and activities predicted from the amounts of isoenzyme added. Activities in the fractions containing separated isoenzymes were measured by a direct kinetic fluorometric assay. MB activities in normal plasma averaged 1.6 ± 0.28 U/liter (mean ± SD).
Evaluation of the Revised Serum Calcium Procedure Used with the Du Pont Automatic Clinical Analyzer
Authors: Sage Gloria Welt , LeFever David , Henry John Bernard
Published: January 2020

We evaluated a revised serum calcium method (o-cresolphthalein complexone) used with the Du Pont aca. Our results correlated well with those obtained by atomic absorption spectrometry. Within-run and day-to-day coefficients of variation were 0.5 and 1.5%, respectively. Color produced and calcium concentration are linearly related and the relation is not affected by hemolysis, lipemia, or icterus.
Direct Method for Measuring Lipoprotein-X in Serum
Authors: Neubeck Wolfgang , Seidel Dietrich
Published: January 2020

We describe a fast and easy method for routine quantitation of the abnormal lipoprotein, lipoprotein-X. The procedure is based on densitometry of precipitation areas obtained for it after serum electrophoresis in agar gel followed by precipitation with polyanions. The coefficient of variation was less than 3% in one series. Results were linearly related to concentration in the range 0.063 to 6.3 g/liter.
Relative Usefulness of Measuring Polyamines in Serum, Plasma, and Urine as Biochemical Markers of Cancer
Authors: Russell Diane H , Russell Shauna D
Published: January 2020

Serial samples of plasma and serum were collected in the morning and afternoon from cancer patients, along with 24-h urine specimens. Values for serum and plasma samples taken at the same time from the same patient differed little, suggesting that either procedure is acceptable for polyamine analysis. Increases in concentrations of putrescine and spermidine in serum and plasma correlate well with such increases in 24-h urine samples. Spermidine concentrations in sera were consistently about 10-fold lower than corresponding urine values. Putrescine concentrations were 10to 100-fold different. The variation in putrescine values may be due to its more active metabolism by diamine oxidase, an enzyme known to be present in serum. Concentrations of polyamines in serum and urine increased in response to effective chemotherapy.
Molar Absorptivities of β-NADH and β-NAD at 260 nm
Authors: Hald Erich , Lehmann Paul , Ziegenhorn Joachim
Published: January 2020

To determine the molar absorptivities of β-NADH and β-NAD at 260 nm, we purified the reduced form of the coenzyme by repeated anion-exchange chromatography. A NADH preparation was so obtained for which the 260 nm/340 nm absorbance ratio was 2.265. When calculated with εβ-NADH340 = 6.22 x 103 for β-NADH at 260 nm and 25 °C, a molar absorptivity of 14.1 x 103 liter · mol-1 · cm-1 resulted from this quotient. By use of the lactate dehydrogenase or glycerol-3-phosphate dehydrogenase assay, respectively, and referring to the new absorption coefficient for NADH at 260 nm, the molar absorptivity of β-NAD at 260 nm and 25 °C was established to be 17.4 x 103 liter · mol-1 · cm-1. The values observed are lower than those reported thus far.
Rate of Decrease in Serum Ethchlorvynol Concentrations after Extreme Overdosage—A Case Study
Authors: Pochopien Donald J
Published: January 2020

Ethchlorvynol is a commonly abused sedative hypnotic. I report here a seven-day study of serum ethchlorvynol concentrations in a patient who had received an extreme overdose. These concentrations were correlated with the patient’s response to stimuli. Serum ethchlorvynol concentrations were logarithmically related to time, a fact that can be useful in predicting when a patient’s serum ethchlorvynol concentration will return to a therapeutic concentration. In this case the decrease was about 35% per 24 h.
Comparison of Electrophoresis on Citrate Agar, Cellulose Acetate, or Starch for Hemoglobin Identification
Authors: Hicks Edward J , Hughes Blossie J
Published: January 2020

We compare and discuss three electrophoretic methods for identifying hemoglobins S, A, C, F, and D or G. Electrophoresis on citrate agar gel was more sensitive than electrophoresis on cellulose acetate for detecting hemoglobins S and F, a fundamental consideration in designing cord-blood screening programs for detecting hemoglobin S carriers. Electrophoresis on starch gel is evidently an acceptable method for subtyping hemoglobins AA, CC, AS, SS, AC, and SC, and is more sensitive than cellulose acetate for identifying hemoglobin A1A2. Costs for the citrate agar gel, cellulose acetate, and starch gel procedures are presented.
Practical Applications of Radioimmunoassay Theory. A Simple Procedure Yielding Linear Calibration Curves
Authors: Fernandez Alberto A , Loeb Harold G
Published: January 2020

The fundamental equation describing radioimmunoassays under equilibrium conditions has been recast into a "working equation" in a form more directly applicable to the requirements of the analytical laboratory. Plotting total counts over counts bound vs. ligand concentration, which is conveniently linear over most of its course, is shown readily to yield quantitative data relative to binding site concentration and the equilibrium constant and to provide a means for deriving apparent labeled ligand concentration. Such data are helpful in establishing optimum assay conditions and can serve a continuing quality-control function. The working equation also characterizes the binder and tracer reagents used in the assay. The determination of working-equation parameters has been illustrated for the vitamin B12 assay. Data are presented for seven different assay procedures, involving more than 600 calibration curves and 100 different lots of binding agent and tracer reagent, showing a Consistently High Correlation Coefficient (R> 0.990), between ligand concentration and the response variable.
Erythrocyte Protoporphyrin in the Detection of Iron Deficiency
Authors: McLaren Gordon D , Carpenter John T , Nino Hipolito V
Published: January 2020

Any decrease in the availability of iron for incorporation into the heme moieties of hemoglobin results in an increase in the erythrocyte protoporphyrin concentration. Our aim was to compare protoporphyrin concentrations, determined spectrophotometrically, with body iron stores, as assessed from the amount of iron demonstrable by Prussian blue staining of bone marrow aspirates. The mean protoporphyrin concentration (175 µg/dl) in the erythrocytes of a group of patients with markedly decreased stainable marrow iron or no iron Was Significantly Greater (P < .001) than the mean concentration (76 µg/dl) in a comparable group with adequate bone marrow iron stores, except in the presence of certain interfering conditions. These results suggest that the erythrocyte protoporphyrin test may be a useful addition to the methods now available for assessing disorders of heme synthesis, the most common of which is iron deficiency.
Electrophoretic Method for Assessing the Normal and Pathological Distribution of Alkaline Phosphatase Isoenzymes in Serum
Authors: Lee Lilian M V , Kenny Margaret A
Published: January 2020

We describe a simple, reproducible, discontinuous system for polyacrylamide disc gel-electrophoresis, with which the alkaline phosphatase isoenzymes in human serum can be fractionated. No sample preparation is needed. The isoenzymes are classified according to their electrophoretic mobilities (RF values) and quantitated by peak area measurements from spectrophotometric scans. The four alkaline phosphatase isoenzymes usually present in normal sera, in order of descending mobilities (and designated according to principal tissue of origin) are: "fast" liver, "slow" liver, bone, and intestine. Sera of diseased patients show a greater variety of isoenzyme distribution patterns, but the most frequently observed patterns are the same as normal patterns. We conclude that the finding of "fast" liver only is not pathognomonic, as previously reported by others, and that information on relative distributions per se is not diagnostically useful, although information on specific increases in activity is useful. With this system, hepatobiliary disorders can be differentiated from other forms of liver and bone diseases.
Applications of a Vidicon Spectrometer to Analytical Problems in Clinical Chemistry
Authors: Pardue Harry L , McDowell Alan E , Fast Douglas M , Milano Michael J
Published: January 2020

This report discusses characteristics of a custom-designed vidicon spectrometer and evaluates its applicability to several clinical analysis problems. Data show that the vidicon detector response is linear with intensity over about four orders of magnitude and that the uncertainty in absorbance measurements can approach 0.001 absorbance units in the range from 0 to 2 absorbance units. Applications include the enzymatic determination of glucose, the determination of lactate dehydrogenase, and determinations of barbital, chlordiazepoxide, and glutethimide. Capabilities of the instrument system for first-derivative spectroscopy are also discussed. The discussion includes a critical evaluation of the potential advantages and limitations of the concept.
An Analytical System for Rapid Separation of Tissue Nucleotides at Low Pressures on Conventional Anion Exchangers
Authors: Khym Joseph X
Published: January 2020

An analytical anion-exchange procedure has been developed for the rapid separation of acid-soluble nucleotides (the so-called "free" or tissue nucleotides). It permits assay at low pressures (275-415 kPa; 40-60 psi) in less than 1 h on 10-cm columns of Aminex resins (conventional styrene-type anion exchangers) with alkaline citrate solutions as the eluent. Separation variables have been investigated, to determine optimum conditions for the routine analysis of samples containing tissue nucleotides. Also described here is a simple solvent-extraction procedure for removing HClO4 or CCl3CO2H quantitatively from cell extracts that contain acid-soluble nucleotides: they are removed from aqueous acid solutions with a water-insoluble amine dissolved in a water-immiscible solvent.
Stable Isotope Ratiometer-Multiple Ion Detector Unit for Quantitative and Qualitative Stable Isotope Studies by Gas Chromatography-Mass Spectrometry
Authors: Klein Peter D , Haumann Joseph R , Hachey David L
Published: January 2020

We have designed and constructed a stable isotope ratiometer-multipie ion detector unit, which can drive existing gas chromatograph-quadrupole or magnetic sector mass spectrometers to monitor up to six ions in turn. Each of the three pairs of ions can be selected for quantitation; thus three different or successive components can be analyzed in a single gas-chromatographic run. A background subtraction option permits the ion intensity in the absence of sample to be subtracted automatically during sample measurement. Displays of accumulated counts and isotope ratio are updated twice per second during the measurement and can be printed out at its conclusion. All six ions can be monitored in the analog mode by parallel outputs to a multipen recorder. Experience gained in the construction of this prototype indicates that such units could be commercially available for $10 000, or about a third to a sixth of the cost of even an inexpensive computer system.
A Flow-System Multiangle Light-Scattering Instrument for Cell Characterization
Authors: Salzman G C , Crowell J M , Goad C A , Hansen K M , Hiebert R D , LaBauve P M , Martin J C , Ingram M L , Mullaney P F
Published: January 2020

A flow-system cell-analysis instrument is described in which cells from a heterogeneous population are characterized by their light-scatter patterns alone. As the cells pass at high speed through a focused helium/neon laser beam, the scatter pattern from each cell is sampled simultaneously at up to 32 angles between 0° and 30° with respect to the laser beam axis, and the scatter pattern for each cell is transferred to a computer. A mathematical clustering algorithm is used to determine the number of classes into which the cells can be divided, and a linear separation algorithm is used to find the boundaries between the classes. Preliminary results on exfoliated cells from gynecological specimens are presented. This technique may be useful for automated prescreening of gynecological specimens.
Adaptation of a T3-Uptake Test and of Radioimmunoassays for Serum Digoxin, Thyroxine, and Triiodothyronine to an Automated Radioimmunoassay System—"Centria"
Authors: Ertingshausen Gerhard , Shapiro Stephen I , Green Gerald , Zborowski George
Published: January 2020

We report the adaptation of four radioassays to the prototype of an automated radioimmunoassay system ("Centria," Union Carbide). The system consists of three integrated modules: (a) an automated pipettor, which dispenses samples and reagents; (b) the key module, an incubator/separator, in which centrifugal force is used to initiate and terminate multiple radioassay incubations and separations simultaneously; and (c) a gamma-counter/computer, which counts three tubes simultaneously and converts counts into concentration units. Radioimmunoassays for thyroxine, triiodothyronine, and digoxin were developed with use of well-characterized antibodies and of prepackaged Sephadexcontaining columns to separate bound and free radioactive ligand. A triiodothyronine-uptake test in which the same kind of columns were used was also adapted to the instrument. Results for clinical samples compared favorably with those obtained by manual procedures. We report data on correlation between different methods and preliminary data on precision of the prototype system.
Liquid-Chromatographic Analysis for Neutral Carbohydrates in Serum Glycoproteins
Authors: Mrochek John E , Dinsmore Stanley R , Waalkes T Phillip
Published: January 2020

We describe a sensitive, reproducible procedure of analysis for the six neutral carbohydrates in glycoproteins, by high-resolution anion-exchange chromatography. As many as 16 neutral carbohydrates can be separated by elution with a concentration gradient of boric acid (pH 7, 67 to 672 mmol/liter). The carbohydrates are detected with a cerate oxidimetric detector system, which monitors the fluorescence of Ce3+ produced by the reaction of the eluted constituents with Ce4+. Sensitivity to 1 nmol of fucose is demonstrated. Analytical methods and results are presented for mannose, fucose, and galactose in serum glycoproteins of both normal women and those with metastatic breast cancer. We briefly discuss the possibility of separating and analyzing for the three neutral carbohydrates in serum glycoproteins in 4 h by isocratic (constant eluent concentration) elution from a chromatographic column.
Simultaneous determination of five sex hormones in human serum by radioimmunoassay after chromatography on Lipidex-5000.
Authors: Apter D , Jánne O , Karvonen P , Vihko R
Published: January 2020

We describe a method for determination of pregnenolone, progesterone, 17alpha-hydroxyprogesterone, testosterone, and 5alpha-dihydrotestosterone in 1-2 ml of serum from male or female. Using microcolumns of Lipidex-5000 (hydroxyalkoxypropyl Sephadex, 0.5 g) and light petroleum/chloroform (97/3) as the solvent during chromatography, we resolved these five steroids into four fractions, with pregnenolone and 5alpha-dihydrotestosterone eluting together. By use of selected antibodies, the latter two steroids were also determined specifically. Use of microcolumns allowed minimization of solvent volumes and sample transfers. Consequently, blank values for all the five steroids were negligible. Lowest measureable concentrations (in ng/liter) were: pregnenolone 100, progesterone 25, 17alpha-hydroxyprogesterone 50, testosterone 25, and 5alpha-dihydrotestosterone 25. Intra-assay and inter-assay coefficients of variation ranged from 5 to 9% and 10 to 15%, respectively, for the five steroids. Serum concentrations of these steroids are given for women in the follicular and luteal phases of the menstrual cycle and for women on oral contraceptives of the combination type, as well as for normal men.
Organic acids in urine from human newborns.
Authors: Björkman L , McLean C , Steen G
Published: January 2020

The pattern of organic acids in urine from 15 normal newborn infants was investigated by gas chromatography/mass spectrometry of trimethylsilylated derivatives. The urine contains large amounts of succinic, fumaric, 2-ketoglutaric, and 3-hydroxy-3-methylglutaric acids, which are all fairly small components of urine from adults. On the other hand, hippuric acid is a small component in the urine from newborn infants, but a large component later in life. An additional number of differences can be seen. Some previously unrecognized aliphatic dicarboxylic acids were also observed, which are present to some extent in urine from adults.
Measuring picogram amounts of aluminum in biological tissue by flameless atomic absorption analysis of a chelate.
Authors: LeGendre G R , Alfrey A C
Published: January 2020

We describe a flameless atomic absorption method for aluminum determination in ethylenediaminetetraacetate extracts of biological tissues. Picogram quantities can be detected, and the technique appears to be more sensitive than other methods of aluminum determination used for biological samples. The extraction simplifies the method and virtually eliminates contamination with extraneous aluminum during the analysis. The extraction method might be useful for separating various other trace elements from biological tissues before analysis.
Creatine kinase isoenzymes in baboon tissues and organs.
Authors: Yasmineh W G , Pyle R B , Hanson N Q , Hultman B K
Published: January 2020

We determined the activity of the isoenzymes of creatine kinase in homogenates of fresh tissues from six baboons. Twenty-six tissues were removed from each baboon and the isoenzymes in them were separated by chromatography on diethylaminoethyl-Sephadex A-50. Total activity was greatest in skeletal muscle (2600 U/g wet weight) followed by myocardium (800 U/g), brain (180 U/g), and the bladder and tissues of the gastrointestinal tract (about 100 U/g). Ten tissues had total activities of less than 20 U/g: trachea, lung, kidney, thyroid, gall bladder, liver, spleen, pancreas, common bile duct, and adrenal. Skeletal muscle contained only the MM isoenzyme; myocardium contained 81% MM isoenzyme, 18% MB isoenzyme, and 1% BB isoenzyme. Except for thyroid and pancreas, all other tissues contained mainly the BB isoenzyme. Tissues of the gastrointestinal tract contained significant (7 to 17%) MB isoenzyme activity.
Improved separation of creatine kinase cardiac isoenzyme in serum by batch fractionation.
Authors: Morin L G
Published: January 2020

I describe a simple, single-tube batch fractionation procedure for separating MM and MB isoenzymes of creatine kinase on a macroporous strong anion exchanger (AG MP-1, Bio-Rad Laboratories). The isoenzymes can be separated in less than 3 min, with a resulting dilution of the serum with no more than an equal volume of buffer. Without sample concentration or spectrofluorometric measurement, the procedure detects 4 U of MB isoenzyme per liter. Sensitivity is limited by the sensitivity and precision of the method of measurement. The CV for the fractionation can be held to less than 4.0% at 65 U of MB per liter. Current fractionation methods are compared to the proposed procedure. With use of a discrete analyzer (Du Pont aca) the mean MB activity in a population free of heart disease was 3.2 +/- 3.0 U/liter (range, 0 to 8 U/liter). The kinetics and stability of isolated isoenzymes are reported, indicating that advisability of storing or pre-incubating samples with mercaptoethanol.
Enzymatic determinations of cholesterol in high-density-lipoprotein fractions prepared by a precipitation technique.
Authors: Steele B W , Koehler D F , Azar M M , Blaszkowski T P , Kuba K , Dempsey M E
Published: January 2020

An enzymatic method for cholesterol in serum [Clin. Chem. 20, 470 (1974)] was initially found to be unsatisfactory for measuring cholesterol in high-density-lipoprotein fractions prepared by precipitation with Mn2+. A fine precipitate formed in the cuvette and cholesterol values were falsely increased. We describe a simple, convenient method for circumventing these problems. An ethylenediaminetetraacetate solution is used to reconstitute the enzymatic reagent. Cholesterol values by this procedure correlated with those obtained by the Lipid Research Clinic's procedure for the same lipoprotein fraction preparations (regression slope, .998; Y-intercept, 8.9 mg/liter; correlation coefficient, .984; standard error of the estimate, 16.8 mg/liter). Precision of the assay, including the precipitation step, was calculated. The SDwithin day was 9.7 mg/liter and SDoverall was 23.7 mg/liter. Results for total cholesterol with the modified reagent were linearly related to concentrations exceeding 4 g/liter, thereby permitting determination of high-density-lipoproteins and total cholesterol in a single run.
Iterpretation of clinical chemical data with the aid of automatic data processing.
Authors: Young D S
Published: January 2020

I summarize conventional applications of computers in clinical laboratories and discuss the possible role of the computer in providing assistance in interpretation of laboratory data through the use of data bases. The data bases include effects of drugs on the results of laboratory tests, factors influencing references values, and conversion factors for converting data from conventional units to SIunits. Reported diagnostic applications of the computer are discussed with reference to both clinically oriented and laboratory oriented programs.
Determination of serum retinol (vitamin A) by high-speed liquid chromatography.
Authors: De Ruyter M G , De Leenheer A P
Published: January 2020

We report a fast and simple high-speed liquid-chromatographic assay for serum retinol. Only 100 mul of serum is required. The lower detection limit is 50 mug/liter; linearity was demonstrated up to 1.50 mg/liter. On analyzing a serum pool eight times, a CV of 2.5% was obtained. Values by this method are compared with results obtained by a flurometric method [Clin. Chem. 16, 766(1970)].
Gas-chromatographic analysis for 1,2:5,6-dianhydrogalactitol, an anti-neoplastic agent, in plasma.
Authors: Kimura T , Sternson L A , Higuchi T
Published: January 2020

We describe a gas chromatographic method for measuring submicrogram quantities of dianhydrogalactitol, a promising antineoplastic agent (currently undergoing clinical trials in humans), in plasma. The drug is first extracted from blood plasma by saturating the aqueous phase with solid potassium carbonate and extracting with isopropanol/chloroform (9/1 by vol). It is then converted to the corresponding n-butaneboronic ester by reaction at room temperature with butaneboronic acid and chromatographed on an SE-30 (3%) column, with flame ionization detection. Practicality of the method for monitoring drug distribution was demonstrated by administering dianhydrogalactitol in therapeutic doses to a dog and monitoring its concentrations in blood for the next 2 h.
A faster radioimmunoassay of progesterone in plasma, with use of polyethylene glycol as precipitant.
Authors: Schiller H S , Hasselbrack R , Riggs R S , Brammall M A
Published: January 2020

We report a reliable radioimmunoassay for plasma progesterone in which polyethylene glycol ("Carbowax 6000") is used to separate antibody-bound and free hormone. Initially, progesterone is concentrated by putting the sample through a column of LH-20 Sephadex. Procedural losses in each sample are not monitored. We compared the reliability and practicality of this method with that of another method in which procedural losses are monitored for each sample; accuracy and precision were similar. Our method saves technical assay time and scintillation counter space and time without sacrificing reliability.
Reaction of alkaline sodium picrate with creatinine: I. Kinetics and mechanism of formation of the mono-creatinine picric acid complex.
Authors: Vasiliades J
Published: January 2020

Spectrophotometric, kinetic, and nuclear magnetic resonance studies indicate that alkaline sodium picrate and creatinine react to form a 1/1 aduct between picric and creatinine, with a stability constant of log K= 4.26. Kinetic studies indicate that the forward reaction is first order with respect to picric acid, hydroxide, and creatinine concentration. The reverse reaction, the dissociation of the 1/1 complex, shows a complex dependence on hydroxide concentration. The expression for the observed pseudo-first-order rate constant in the presence of excess picric acid is: Kobsd = K1K0[P][OH] +[K2[OH]x. A value of K1K0 = 5.0 (mol/liter)-2s-1 is obtained. For accurate analytical results with this reaction, hydroxide concentration must be maintained at a constant value for both samples and standards.
Microcalorimetric Determination of Glucose in Reference Samples of Serum
Authors: Goldberg Robert N
Published: January 2020

I report a detailed series of microcalorimetric measurement of glucose concentrations in five reference samples of serum. the method utilized (a) calibration in the actual medium of analysis and (b) a correction for interferences owing to the nonspecificity of the enzyme hexokinase. The microcalorimetric results are compared with the tentative results of analyses obtained by spectrophotometric hexokinase/glucose-6-phosphate dehydrogenase method and by isotope-dilution mass spectrometry. In most cases, the microcalorimetric results appear to be in agreement with the results obtained by these latter two method. A discussion of the basis of the mocrocalorimetric measurments is presented.